key: cord- -amvlm p authors: pauli, eva-k.; schmolke, mirco; wolff, thorsten; viemann, dorothee; roth, johannes; bode, johannes g.; ludwig, stephan title: influenza a virus inhibits type i ifn signaling via nf-κb-dependent induction of socs- expression date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: amvlm p the type i interferon (ifn) system is a first line of defense against viral infections. viruses have developed various mechanisms to counteract this response. so far, the interferon antagonistic activity of influenza a viruses was mainly observed on the level of ifnβ gene induction via action of the viral non-structural protein (ns ). here we present data indicating that influenza a viruses not only suppress ifnβ gene induction but also inhibit type i ifn signaling through a mechanism involving induction of the suppressor of cytokine signaling- (socs- ) protein. our study was based on the observation that in cells that were infected with influenza a virus and subsequently stimulated with ifnα/β, phosphorylation of the signal transducer and activator of transcription protein (stat ) was strongly reduced. this impaired stat activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral ′ triphosphate rna in the cell. socs proteins are potent endogenous inhibitors of janus kinase (jak)/stat signaling. closer examination revealed that socs- but not socs- mrna levels increase in an rna- and nuclear factor kappa b (nf-κb)-dependent but type i ifn-independent manner early in the viral replication cycle. this direct viral induction of socs- mrna and protein expression appears to be relevant for suppression of the antiviral response since in socs- deficient cells a sustained phosphorylation of stat correlated with elevated expression of type i ifn-dependent genes. as a consequence, progeny virus titers were reduced in socs- deficient cells or in cells were socs- expression was knocked-down by sirna. these data provide the first evidence that influenza a viruses suppress type i ifn signaling on the level of jak/stat activation. the inhibitory effect is at least in part due to the induction of socs- gene expression, which results in an impaired antiviral response. influenza a viruses are negative-stranded rna viruses that belong to the family of orthomyxoviruses. the segmented genome of influenza a virus encodes for up to viral proteins. as many other viruses, influenza viruses have evolved strategies to counteract cellular antiviral responses, especially to circumvent the type i ifn system as a first line of defense against the pathogenic invader. among the influenza viral proteins, the ns has been identified as the main type i ifn antagonistic factor. so far two major mechanisms have been described by which ns suppresses the initial expression of ifnb. on the one hand ns inhibits vrnamediated induction of the transcription factors interferon regulatory factor- (irf- ), activating protein- (ap- ) and nf-kb that target the ifnb promoter. this most likely occurs via binding to the rna-sensor retinoic acid inducible gene (rig-i) and inhibition of rig-i-mediated signaling in response to viral rna [ , ] . on the other hand ns inhibits maturation [ , ] and nuclear export of host mrnas [ ] . other functions of the multifunctional protein include block of activation of the dsrnaactivated protein kinase pkr by direct interaction [ ] or activation of the phosphatidylinositol- kinase pi k/akt pathway to prevent premature apoptosis induction [ , ] . while the ns -mediated antagonistic activities of influenza viruses mainly affect the induction of genes such as ifnb, so far no viral suppression of ifn signaling has been described. ifn are among the first molecules synthesized in response to viral infections [ ] . the ifn family includes three classes. type i comprises the well known ifna and ifnb. the only member of type ii ifn is ifnc. type iii ifn comprises ifnl , -l , and -l . all classes of ifn bind to different receptors and are structurally not related [ , ] . type i ifn belong to the key cytokines produced by influenza a virus-infected epithelial cells [ , ] . the antiviral activity of type i ifn is mediated by a set of ifn-induced genes (isgs). binding of ifna/b to its receptor is the initial step in this signaling process, followed by activation of the jak family and subsequent activation of stat proteins [ ] . ligand binding leads to dimerisation of the type i ifn receptor subunits ifnar and ifnar and causes their conformational change. the jak kinase tyk , which is constitutively bound to ifnar , phosphorylates the receptor at tyrosine residues and creates a docking site for stat . subsequently, tyk phosphor-ylates stat at y . at the same time the receptor-bound jak phosphorylates stat at y [ , ] . the phosphorylated transcription factors dimerise and bind to irf- [ ] . the newly formed heterotrimer, called ifnstimulated gene factor (isgf ), translocates into the nucleus and binds to ifn-stimulated response elements (isre), to initiate gene transcription of isgs. treatment of cells with type i ifn upregulates expression of an array of genes including sp , irf- and many others [ ] . among these isgs the , oligoadenylate synthetase (oas ), the mx proteins and the dsrna-activated protein kinase (pkr) are described to directly interfere with viral replication [ ] . both, pkr and the oas / rnasel system are capable of inhibiting cellular and viral translation. ifn-induced jak/stat signaling can be inhibited at different levels by several viral and cellular factors through various mechanisms. the large t-antigen of murine polyomavirus (mpyv) binds to jak and inhibits downstream signaling [ ] , whereas the vp of ebola virus (ebov) binds to karyopherina- thereby blocking nuclear accumulation of stat [ ] . endogenous cellular key regulators, capable of negatively regulating jak/stat-mediated signal transduction, include suppressor of cytokine signaling (socs) proteins, protein tyrosine phosphatases (ptp) and protein inhibitor of activated stats (pias). the family of socs proteins comprises eight members (cytokine-inducible sh domain-containing protein (cis) and socs - ). all members contain a central sh domain, an nterminus of variable length and sequence and a c-terminal amino-acid module called socs box [ ] . the socs box is necessary for recruitment of the ubiquitin transferase system and for stabilization and/or degradation of socs proteins [ ] [ ] [ ] . the n-terminus contains a kinase inhibitory region (kir), which functions as pseudo substrate for the jak [ ] . socs- and socs- differ in their mode of action. for inhibition of the kinase activity of jaks, socs- binds directly to the activation loop of jaks [ ] [ ] [ ] . in contrast, socs- first binds to the receptor [ , ] . induction of socs- gene transcription by viruses was reported for hsv- , hcv [ ] [ ] [ ] and for respiratory viruses, such as sars and rsv [ , ] . the level of induction of socs- by hsv- seems to determine whether infection turns to acute or persistent progression [ ] . for hcv it has been suggested that upregulation of socs- may contribute to the non-responsiveness of hcv patients to ifn therapy [ , [ ] [ ] [ ] . elevated socs- mrna levels during rsv infection were linked to th cell-mediated immune disease as atopic dermatitis and asthma [ , ] . in the present study we show that influenza a virus can be added to the list of viruses that induce socs- expression. the protein functionally interferes with viral replication by providing a virus-supportive ifn-antagonistic activity on the level of type i ifn-signaling that has not been described so far. phosphorylation of stat and stat by members of the jak tyrosine kinase family is a prerequisite for activation of these transcription factors to drive type i ifn-induced gene expression. therefore, we analyzed whether stat phosphorylation patterns are altered in influenza a virus infected cells that were stimulated with ifn at different time points post infection (p.i.). the human alveolar epithelial cell line a was infected with the influenza a virus strain a/puerto-rico/ / (h n ) (pr ) ( figure a ). cells were subsequently stimulated with ifnb at given time-points p.i. and stat phosphorylation was assessed in western blots. both stat and stat were readily phosphorylated upon cytokine stimulation in uninfected cells or in infected cells up to h p.i. ( figure a ). furthermore, virus infection alone resulted in a significant induction of stat phosphorylation - h p.i., presumably caused by virus-induced ifn expression. however, at later time points ( - h p.i.), in a cells both virus-and ifn-induced stat and stat phosphorylation was markedly reduced ( figure a ). similar patterns were observed upon stimulation of cells with ifna or upon infection with other viruses, such as the human influenza virus a/victora/ / (h n ) (data not shown). in addition, this phenomenon could also be detected in other epithelial cells such as the human embryonic kidney cell line hek ( figure e ) or the human umbilical vein endothelial cells (huvec) ( figure s b ). inhibition was not caused by indirect disturbing effects on cellular metabolism or enzyme activities due to ongoing virus replication, since ifnc-induced stat phosphorylation was not affected at all ( figure c ). finally, involvement of any auto-or paracrine action of virus-induced type i ifn could be ruled out, as the inhibitory effect was also observed in vero cells lacking functional type i ifn genes ( figure e ). with regard to the molecular basis of impaired ifna/b-induced stat phosphorylation in infected cells it was striking that the inhibitory effect correlated with the accumulation of viral proteins, as monitored in pb western blots ( figures a and e) . thus, the question arose whether individual expression of viral proteins may result in the interference with stat phosphorylation. out of the viral proteins of pr we choose the nucleoprotein (np), the ns protein, the matrix protein (m ) (figure a ) and the subunits of the viral polymerase, pa, pb and pb ( figure c ), for a representative experiment. these proteins are known to bind to vrna/rnps or to interfere with the rna-mediated innate immune response. for efficient transfection of the expression the type i interferon (ifn) system is one of the most powerful innate defenses against viral pathogens. most rna viruses are sensitive to the action of type i ifn. therefore, these pathogens have evolved strategies to evade this response. for example, influenza viruses express a viral protein, the non-structural protein (ns ), that suppresses production of ifnb by lowering cellular sensitivity to viral nucleic acid as a pathogen pattern. here we present data indicating that influenza a viruses are not only capable of suppressing production of the ifnb gene but also inhibit action of this antiviral cytokine on cells. this occurs by viral induction of a cellular protein, the suppressor of cytokine signaling (socs)- , a potent endogenous inhibitor of ifn signaling. this is a novel mechanism by which influenza viruses inhibit the antiviral response of the host and paves the path to efficient virus replication. this may be especially relevant for influenza viruses that induce high cytokine responses (cytokine burst), such as highly pathogenic avian influenza viruses of the h n subtype. induction of socs- expression would allow efficient replication despite high ifn and cytokine levels. constructs we used the highly susceptible cell line hek that also exhibits impaired ifnb-induced stat phosphorylation at later stages of infection ( figure e ). h post transfection cells were stimulated with ifnb and stat phosphorylation was monitored in western blots (figures a and c) . expression of none of the viral proteins resulted in a significant decrease of ifnb-induced stat or stat phosphorylation (figures a and c ). similar results were obtained in the human bronchial epithelial cell line h when expressing m , ns or np alone or in different combinations (data not shown). thus, we concluded that viral proteins most likely do not play a prominent role as blockers of ifna/b-induced jak/stat signaling. decrease of stat phosphorylation might also be due to the action of virus-induced phosphatases. on the one hand these enzymes may cause direct dephosphorylation of stat proteins. on the other hand phosphatases could act via an indirect mechanism by dephosphorylation and inactivation of jaks resulting in an attenuated phosphorylation of stats. several protein tyrosine phosphatases (ptps) are known to mediate dephosphorylation of both, jaks and stats [ ] . in order to investigate whether influenza a virus activates phosphatases that subsequently target jaks or stats, we treated infected or uninfected a cells with the well-known tyrosine phosphatase inhibitor sodium vanadate [ , ] . uninfected cells or cells infected with pr for h were incubated with increasing amounts of this compound min prior to stimulation with ifnb. this time point of infection was chosen since we observed considerable inhibition of ifn-induced stat phosphorylation in the course of infection ( figures a and e ). increasing concentrations of vanadate lead to a gradual shift of the steady state balance of phosphorylation/dephosphorylation. accordingly, a gradual increase of stat phosphorylation was observed that was similar in both infected and uninfected cells, albeit starting from different basal levels of phospho-stat ( figures a and b ). this is illustrated by an almost identical slope of the regression line in the graphical analysis of the band intensities of the ifnb stimulated samples ( figure b ). if the blockade of ifnb-induced stat phosphorylation would be mediated by specific virus-activated phosphatases, a much steeper slope for vanadate-treated infected cells would be expected. however, the result in figure b indicates that the virus-induced suppression of phosphorylation is not compensated by phosphatase inhibition and consequently no virusactivated phosphatase appears to be involved. in support of these data, phosphatase assays revealed that the overall activity of tyrosine phosphatases in infected cells was not elevated compared to uninfected cells. this is indicated by constant levels of free phosphates released from two different phospho-peptides that represent common tyrosine phosphatase substrates ( figure c ). thus, involvement of phosphatases in influenza virus-induced alteration of stat phosphorylation can be greatly ruled out. phosphorylation of stats in the ifnb signaling cascade may not only be counter-regulated by phosphatases but also by other cellular factors, such as proteins of the suppressors of cytokine signaling (socs) family. action of these proteins is mainly controlled on the level of transcriptional activation. socs proteins are described to have high affinity for jak and stat proteins and to inhibit the transmission of ifna and ifnb induced signaling [ , ] . to examine whether expression of socs genes is induced in influenza virus infected cells, a cells were infected with pr for different time points. subsequently total rna was analyzed for the amount of socs- and socs- mrna by means of quantitative real time-pcr (qrt-pcr). the mrna table for accession numbers of viral genes) using l according to manufacturer's instructions. note that the pol ii constructs in use also give rise to expression of second reading frames in the ns, m and pb genes (ns , m , pb -f ). h post transfection cells were stimulated with human ifnb ( u/ml) for minutes. total protein lysates were subjected to western blot analysis using anti-phospho-stat , anti-phospho-stat , anti-stat antibodies. expression of influenza viral proteins was monitored with antibodies against np, m , ns , pa, pb or pb . (e) hek cells were infected with the human influenza virus pr (h n ) (moi = ) for the indicated time points and were subsequently stimulated for min with either human ifnb at a concentration of u/ml. cell lysates were subjected to western blots as described. (b, d, f) quantification of relative pstat and pstat band intensities in a, c and e using aida software and d densitometry (fuji). doi: . /journal.ppat. .g levels of socs- and socs- differed notably in the time course ( figure a ). while socs- mrna is strongly and transiently elevated in the early phases of infection, socs- gene transcription is only significantly induced h p.i.. elevated socs- mrna levels were also observed in other host cell types, such as huvec starting h p.i. ( figure s a ). although elevation of socs- mrna levels in infected cells was rather transient, there appears to be a robust induction on protein level ( figure c ). first detected at h p.i., socs- protein levels increased and stayed on a high level throughout the observation period. strikingly, expression kinetics of the socs- protein perfectly matched the kinetics of virus-induced inhibition of stat phosphorylation ( figure c ), indicating that both processes are functionally linked. virus mediated socs- gene induction at early stages of infection ( figures a, c and s a) appeared to occur concomitant with an immediate and strong induction of ifnb ( figures b and s d) . this prompted us to analyze whether socs- transcription might be induced due to an auto-or paracrine action of ifnb expressed during infection. a cells were stimulated with ifnb for different time points and socs- gene induction was measured by qrt-pcr ( figure d ). as a control we monitored expression of , oligoadenylate synthetase (oas ) and mxa, genes that are typically induced by ifnb. while oas and mxa mrnas were readily upregulated upon ifnb treatment socs- mrna was not significantly elevated ( figure d ). similar results were obtained from huvec stimulated with ifnb ( figure s e ). to further confirm these results we knocked down the ifnar in a -cells by an sirna approach. although the knock down was efficient and leads to more than % inhibition of ifnb induced stat phosphorylation ( figure e ), the induction of socs- expression was not impaired ( figure f ). socs- levels in the knock down cells were similar compared to wild type cells and even higher than in the vector control ( figure f ). these results are consistent with data gained from previous experiments in vero cells ( figure e ) and indicate that neither induction of socs- mrna nor inhibition of stat phosphorylation is dependent on virus-induced type i ifn expression. since accumulation of viral rna in infected cells is a potent inducer of antiviral gene expression we investigated its ability to induce socs- gene transcription. as a source for viral rna, a cells were infected with influenza a virus for h and total rna from these cells was isolated. rna from uninfected a cells served as a negative control. different amounts of these rnas were used for stimulation of a cells for h (figures a, b and c). transfection of rna from uninfected cells did not result in an increase of socs- or socs- gene transcription ( figure a ) or ifnb induction as a control ( figure b ). however, transfection of rna from virally infected cells led to strongly elevated socs- mrna amounts while socs- mrna is only induced weakly ( figure a ). this dose dependent induction of socs- by stimulation with increasing amounts of rna from infected cells corresponds with a gradual decrease in the ability of this rna to induce or potentiate stat / phosphorylation ( figure c ). in contrast to cellular rna, influenza viral rna carries a triphosphate group at its terminus that was previously shown to be a major pathogen pattern that triggers cellular signaling [ ] . to verify that indeed the viral triphosphate rna in the pool of rnas from infected cells is the major trigger for induction of socs- expression, rna from infected or uninfected cells was treated with phosphatase to remove the triphosphate termini prior to stimulation of a cells ( figure e and f). the dephosphorylated viral rna was only poorly capable to induce socs- ( figure e ) or ifnb ( figure f ) mrna expression. in addition, poly(i:c) was transfected to mimic action of double-stranded (ds) rna ( figure e and f, right bars). however, the dsrna analog showed surprisingly little effects on socs- and ifnb mrna induction. since viral rna is able to induce ifnb gene transcription ( figure b and f) we again wanted to rule out that induction of socs- by viral triphosphate rna is mediated by auto-or paracrine action of de novo synthesized ifnb. in order to do so, cells were stimulated with viral rna after treatment with the protein synthesis inhibitor anisomycin at two different concentrations ( figure g ). socs- mrna was still induced to the same extent in the presence of the protein synthesis inhibitor, providing the ultimate proof that de novo protein synthesis is not required for socs- induction. so far, our data suggest that influenza virus-induced transcriptional upregulation of the socs- gene is not mediated by the . equivalent mrna amounts were normalized to gapdh mrna levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as . to detect socs- protein expression (c) cells were infected for time points indicated or left uninfected. total cell lysate was subjected to western blot analysis using anti-socs- antibody. to allow better comparison of socs- protein expression and stat phosphorylation phospho-stat and stat western blots from figure a are shown again here. (e) to functionally test effective knock down of the ifnar, a wild type, a vector control cells or a cells stably expressing ifnar ii- specific shrna were stimulated with human ifnb ( u/ml) for min. subsequently cells were lysed and levels of phospho-stat were determined by western blotting using specific antibodies. in addition, the relative pstat band intensities were quantified. doi: . /journal.ppat. .g autoregulatory action of type i ifns ( figure d and f) but is directly induced through accumulation of viral rna during infection. this raises the question, which rna-induced signaling pathways are responsible for socs- expression. the mkk/p mitogen activated protein kinase (mapk) pathway [ ] [ ] [ ] as well as the ikb kinase (ikk)/nuclear factor of kb (nf-kb) cascade [ ] [ ] [ ] are both known to be activated by rna or influenza virus infection and to be involved in the control of socs- expression. to assess whether the mkk /p -or the ikk/nf-kb-module is required for socs- gene induction, we generated a cell lines expressing dominant negative forms of either mkk (mkk ala) or ikk (ikk kd) (figure a to d). these mutants have been previously shown to efficiently block p or nf-kb signaling, respectively [ ] [ ] [ ] . to monitor socs- gene induction, wild type, vector or mutant expressing cell lines were infected with pr ( figure a ) or stimulated with rna from virally infected or uninfected a cells ( figure c ). induction of ifnb mrna was monitored as a control ( figure b and d) . while mkk ala expression did not result in significant reduction of socs- in either infected ( figure a ) or rna-stimulated cells ( figure c ), transcription is markedly reduced in ikk kd expressing cell lines. to obtain independent evidence for nf-kb dependence of socs- gene transcription, a wild type cells were incubated with the nf-kb specific inhibitor bay - prior to stimulation with rna from virally infected or uninfected a cells ( figure e ). again, ifnb mrna levels were assessed for control purposes ( figure f ). both, socs- and ifnb mrna levels were strongly reduced in bay - treated cells. this indicates that virus-induced socs- expression strongly depends on ikk and nf-kb activation, while the mkk /p appears not to play a prominent role. to further verify that influenza virus induces socs- via an rna sensory pathway and in an nf-kb dependent manner we infected cells with the influenza a virus mutant deficient for ns (dns ) (figure g and h) . the ns protein is known to block rna dependent signaling and nfkb activation [ ] . accordingly, infection of cells with the mutant virus resulted in a more pronounced and sustained, albeit delayed induction of socs- ( figure g ) if compared to infection with the isogenic wild type, that is a very poor inducer of socs- but still reasonably well induces ifnb. noteworthy, this isogenic wild type strain differs from the pr wild type virus used in the other experiments shown here (see materials and methods for details). to analyze whether nf-kb activation is sufficient for socs- gene induction we stimulated cells with il- b ( figure s a ) or tnfa ( figure s b ) that are both strong activators of the transcription factor. while mrna levels of il- , a strictly nf-kb dependent cytokine, are strongly elevated, socs- gene transcription is not significantly induced. under the assumption that these cytokines do not additionally induce counteracting processes one can conclude that nf-kb is required, yet not sufficient for the induction of socs- . thus viral induction of socs- may require additional factors that are only active in virus-infected cells. furthermore, these results rule out a potential role of virus-induced il- b or tnfa in the induction of socs- . this is supported by the observation that neither expression of il- to further assess a functional role of socs- in virus-induced suppression of stat phosphorylation we analyzed mouse cells with a targeted deletion of the socs- gene [ ] . wild type and socs- deficient mouse embryonic fibroblasts (mef) were infected for different time points with pr . the time of infection was prolonged in comparison to the infection of a cells because the human pr replicates less efficiently in mouse than in human cells. following infection lysates of these cells were assessed for stat phosphorylation ( figure a ). both cell types showed no phosphorylation of stat in the uninfected state. in contrast, infection of socs- knock out cells resulted in strongly elevated phosphorylation of stat in a sustained fashion. to rule out that this stat phosphorylation is due to altered secretion of ifnb or figure . enhanced stat phosphorylation in infected socs- deficient mef correlates with elevated induction of ifnb-stimulated genes. wild type mef and socs- knock out mef were infected with pr (moi = ) for the indicated times. subsequently, cell lysates were analyzed for stat phosphorylation (a). for control of productive virus replication, cell lysates were analyzed for viral protein pb expression. in (e, f, g) wild type and knock out cells were lysed at indicated time-points of infection. subsequently rna was subjected to reverse transcription. cdna was analyzed in quantitative real time pcr to assess mrna amounts of three prototype type i ifn-stimulated genes, sp (e), interferon regulatory factor- (irf- ) (f) and oas (g). equivalent mrna amounts were normalized to gapdh mrna levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as . in (c) wild type mef and knock out mef were infected with pr (moi = ) or left uninfected. supernatants were taken p.i. and used for stimulation of wild type mef for minutes. as control wild type mef were stimulated with u/ml mouse ifnb for minutes. cells were harvested and analyzed for the amount of stat and phospho-stat in western blot analysis by specific antibodies. in (b) and (d) the relative band intensities of phospho-stat of the blots in (a) and (c) were quantified as described. doi: . /journal.ppat. .g other stat -activating cytokines in socs- deficient cells, we performed conditioned medium experiments ( figure c ). mef wild type and mef socs- deficient cells were infected for h and supernatants were subsequently harvested. stimulation of mef wild type cells with these different supernatants for min. revealed no differences in stat phosphorylation, indicating that both infected cell types secrete similar amounts of ifnb and other stat activating cytokines. this is a strong indication that the observed differences in virus-induced stat phosphorylation are directly due to the presence or absence of socs- in wild type and knock out mef, respectively. to answer the question whether enhanced stat phosphorylation in socs- deficient cells would also lead to enhanced expression of isgs, total rna was isolated at different time points p.i. from infected wild type and knock out cells and monitored for induction of sp , irf- and oas ( figure e, f and g ). these genes are described as type i ifn-induced genes [ ] . indeed mrna levels of all three representative isgs were elevated in socs- knock out versus wild type cells at almost every time point during the course of infection. this indicates that enhanced stat phosphorylation and activation in socs- deficient cells results in elevated expression of isgs. the remaining question was, whether the elevated ifn-induced gene response in knock out cells might also affect propagation of influenza a viruses. thus, both wild type and knock out cells were infected with pr ( figure a ) or the strain a/victoria/ / (h n ) ( figure b ). virus titers were assessed at different time points post infection. progeny virus titers from socs- knock out cells were significantly reduced compared to titers from infected wild type cells. to independently confirm these results and to verify that the observed effects are really due to the lack of socs- , we used an sirna approach to specifically knock down socs- mrna in a cells. cells were transfected with nm sirna for h and socs- protein levels were compared to control transfected samples ( figure c, right) . subsequently, cells were infected and progeny virus titers were determined by plaque assay (figure c, left) . similar to the results gained from infected knock out cells, knock down of socs- resulted in decreased virus titers. on the contrary, over-expression of socs- resulted in elevated virus titers ( figure d ) concomitant with an inhibition of ifnb-or virus-induced stat phosphorylation ( figure e) . taken together the data indicate that in the absence of socs- , infection leads to a stronger activation of stat , resulting in enhanced expression of isgs and reduced virus titers. vice versa, over-expression of socs- leads to an inhibition of stat activation and elevated virus titers, probably due to inhibited expression of isgs. this highlights the important role of virus induced socs- to limit the type i ifn-induced antiviral response program. the type i interferon (ifn) system is one of the most powerful innate defenses of vertebrate cells, which limits replication and spread of viral pathogens including avian and human influenza viruses. influenza virus propagation is sensitive to ifn activities and therefore, like other viral pathogens, these viruses do not only induce type i ifn but also antagonize the production and effects of these cytokines at the same time [ ] . for influenza a and b viruses, this is accomplished through their non-structural ns proteins that are structurally related polypeptides of kda (a/ ns ) and kda (b/ns ), which are abundantly expressed in infected cells [ ] . ns proteins predominantly act on the level of ifn gene induction in infected cells by obstructing rig-idependent signaling through interaction with cellular factor(s) and/or sequestration of rnas generated during virus replication [ , , ] . some ns proteins were also described to inhibit the maturation of cellular pre-mrnas raising the possibility that this activity additionally reduces production of ifna/b in infected cells [ , ] . while ns also interferes with the activity of some isgs, such as the dsrna dependent kinase pkr [ , ] , so far no type i ifn antagonistic mechanism was described for influenza viruses that act on the level of ifn signaling rather than gene induction. here we present data, showing that rna-induced expression of socs- in early phases of infection leads to a functional inhibition of ifn-induced stat activation and gene expression. this is a novel mechanism by which influenza virus suppresses the antiviral response of the host and paves the path to efficient virus replication. while it was reported in the literature that expression of socs proteins can be induced upon stimulation with ifn [ ] we could not detect any significant gene induction by ifnb in a cells. instead we observed a significant up-regulation of socs- by viral triphosphate rna, indicating that gene induction occurs via accumulation of vrna during infection. this appears to occur through the rna-mediated activation of the ikk/nf-kb pathway, most likely activated through engagement of the rna sensor rig-i. at a first sight, this might appear controversial since nf-kb activation is among the rig-i-induced signaling responses and ns was reported to inhibit this signaling pathway. however, despite the action of ns it is well known that nf-kb is still significantly activated upon influenza virus infection and many nf-kb and ifnb dependent genes are still expressed. we hypothesized previously that the incomplete inhibition conferred by ns is an indication that the virus exploits the remaining signaling activities for efficient replication [ , , ] . the findings described here, namely nf-kb dependent induction of socs- and limitation of type i ifn signaling responses, provide yet another example how influenza viruses take advantage of nf-kb activity. while the data show that nf-kb is required for viral socs- induction, the factor appears not to be sufficient, since prototype inducers of nf-kb, such as il- b or tnf-a would not induce socs- . thus there seems to be the need of additional virus or rna-induced transcription factors. the most likely candidate would be the constitutively expressed interferon regulatory factor (irf- ), that is known to be simultaneously activated with nf-kb upon virus infection directly via the rig-i rna sensing pathway without the need of type i ifn. furthermore irf- is a factor suppressed by the ns protein. recently it was reported that ifn-induced gene expression responses are potentiated in cells, which lack the nf-kb factors p or p [ ] . although these authors described an inhibitory binding of nf-kb transcription factors to some ifn-induced gene, this mechanism might be cell type dependent since we could not observe similar effects in the cell types used here (data not shown). thus, the underlying molecular mechanisms appear to be not fully clear. it is striking that the effects described for p and p knock out cells in these studies fully correlate with our observations in socs- deficient cells. while in the latter case cells lack the ifnb signaling inhibitor socs- , the p and p knock out cells are deficient for the factors required for socs- induction. thus, given the nf-kb dependent induction of socs- described in the present manuscript, we provide an additional molecular mechanism that may explain the phenomenon described by wei et al. [ ] . first indications for beneficial effects of socs- gene expression on viral replication came from studies using the hcv core protein as a replacement for the influenza a viral ns in the context of infections with a ns deficient influenza virus [ ] . one of the hallmark responses of hcv core expression is a rapid induction of socs- expression. given the role of socs- described here, it was not surprising that hcv core could partially rescue growth of the ns deficient virus [ ] . while this manuscript was in preparation it was demonstrated by pothlichet et al. that influenza a virus-induced socs- and socs- upregulation requires a tlr- -independent, rig-i/ mavs-dependent pathway [ ] . moreover, over-expression of socs- and socs- in infected cells revealed that both molecules inhibit antiviral responses. these studies are perfectly complemented by our findings. here we confirm involvement of rig-i/mavs by showing that triphosphate rna, the ligand for rig-i, is a major inducer of socs- . furthermore, the finding that dsrna is only a weak inducer of socs- is also consistent with the independence from the dsrna sensor tlr- . the only discrepancy of this work and the study of pothlichet et al. is that they show a dependence on the type i ifn receptor. this may be due to the different virus-strains and cell types used. it is well known that the capability of type i ifns to induce socs proteins is strongly cell type specific [ ] . while in some cell types socs- expression appears to be type i ifn dependent (e.g. fetal liver cells) [ ] it is clearly independent of ifn in other cell types [ ] . recently it was shown that socs- is not significantly induced by ifna in a cells [ ] , the major cell type used in our study. evidence that cell type specificities may be the cause of discrepancy is additionally provided by the fact that pothlichet et al. show identical induction kinetics of socs- and socs- . in contrast the kinetics of the two proteins differ clearly in the cells we used, with socs- being induced much earlier than socs- on mrna and protein level. finally, it should be stated that regardless whether socs- is additionally induced by type i ifns at a later stage of infection, it is important that it can be induced earlier and in parallel to ifnb directly by vrna accumulation. this is supported by the finding that ifnb and socs- induction occurs in parallel kinetics ( figure a and b) while ifn-induced genes such as oas and mxa are only up-regulated later in a delayed and more sustained fashion ( figure d ). this makes a qualitative difference since the blocking effect of socs- on ifnb signaling already kicks-in during the first wave of ifnb action. taken together we describe here for the first time that at least some influenza a virus strains are able to suppress type i ifn signaling by a mechanism involving nf-kb dependent activation of socs- expression, which negatively affects stat phosphorylation. this adds a new aspect to our knowledge of the strategies used by influenza a virus to antagonize type i ifn responses. human influenza a/puerto-rico/ / (h n ) (pr ) (giessen variant) and a/victora/ / (h n ) (victoria) were originally taken from the strain collection of the institute of virology, giessen, germany. the human ns deficient influenza virus mutant dns and its isogenic wild type variant were propagated and used as described earlier [ , ] . it should be noted that this isogenic wild type strain as described by garcia-sastre et al. [ ] is different from the pr (giessen variant) used in the other experiments and in many previous studies [ , ] . the supernatant was aspirated and cells were incubated with specific medium containing . % bsa and antibiotics. to score for production of viral plaques the overlay was stained for h using ml neutral red in pbs per well [ ] . to trigger jak/stat signaling cells were stimulated using human ifna/b or c as well as mouse ifnb. for stimulation of a cells or huvecs u/ml human ifna or human ifnb was used. for stimulation of the green monkey epithelial cell line vero or hek cells u/ml human ifnb was applied. ifnc was always used in the concentration of u/ml. mouse embryonic fibroblasts (mef) were incubated with u/ml mouse ifnb. the different ifn were diluted in infection medium. for stimulation after infection, viral supernatants were aspirated and diluted cytokine was incubated for minutes at uc. to investigate the potential of other cytokines to induced socs- gene expression a cells were stimulated with u/ml il b or ng/ml tnfa at uc for times indicated. after stimulation cells were lysed and subjected to immune blotting. to block the activity of phosphatases after infection with influenza virus, sodium vanadate was used. dilutions were prepared using infection medium. sodium vanadate was added to the virus-containing infection medium at the time points indicated. after minutes of incubation ifnb, diluted in infection medium, was added to the medium containing virus and sodium vanadate. the cells were stimulated with ifnb for minutes. incubation with sodium vanadate started min before cells were lysed and subjected to western blotting as described. for conditioned medium experiments wild type and socs- knock out mef were infected with pr (moi = ) for h or left uninfected. supernatants were used for stimulation of mef wild type for minutes. cell lysates were subjected to western blot analysis. to investigate the induction of socs- expression by viral rna, rna isolated from infected or uninfected cells (control) was used. a cells were infected with pr (moi = ) or left mock infected. h post infection rna was isolated using the rneasy mini kit from qiagen according to manufacturer's instructions. to dephosphorylate viral triphosphate rna, calf intestine alkaline phosphatase (ciap) (fermentas) was used. briefly, rna was isolated using trizol according to manufacturer's instructions. for dephosphorylation the reaction mix was set up in a ml volume with mg rna, u ciap and u ribolock rnase inhibitor (fermentas) and was incubated for h at uc. thereafter the rna was isolated using the rneasy mini kit from qiagen. rnas used as control were mock-treated replacing ciap by glycerol. for stimulation, the different rna species and analogues were transfected using lipofectamine (l ) according to manufacturer's instruction (invitrogen). in brief, l was incubated with opti-mem for minutes at room temperature; different amounts of rna were added and incubated for additional minutes. for stimulation of cells with cellular or viral rna ml rna-l mix were added to ml serum-free medium. cells were stimulated for hours and subjected to either western blot analysis or quantitative real time pcr. for silencing socs- mrna, a cells were transfected with nm human socs- sirna h before infection using hiperfect (qiagen) according to manufacturer's instructions. in brief, nm sirna was added to a mixture of d-mem without fcs/antibiotics and hiperfect and incubated for min at room temperature. for transfection ml of this mixture were added to the cells. subsequently cells were subjected to plaque assay analysis or western blot analysis. control sirna was purchased from qiagen. the sequences for the human socs- sirna in use are: human socs- sirna sense -cca aga acc ugc gca ucc adtdt- , human socs- sirna anti-sense -ugg aug cgc agg uuc uug gdtdt- ) (see table for accession number of the human socs- gene). to determine whether tyrosine phosphatases become activated upon infection with influenza virus a phosphatase assay using the tyrosine phosphatase assay system (promega) was performed. a cells were infected for h (moi = ) or left uninfected. cells were harvested in assay buffer ( mm tris-hcl ph . , mm cacl , mm mgcl , . % b-mercapto ethanol), cracked by a single freeze/thaw step at uc and disrupted by ultrasonic pulsing. lysates were precleared from cell debris and residual free phosphates according to the manufacturer's instruction. tyrosine phosphatase activity was measured by enzymatic release of free phosphate of two given pseudosubstrates (phosphorylated peptides representing target sequences for the most common tyrosine phosphatases). quantification was performed in comparison to a given standard according to the manufacturer's instruction. for western blot analysis cells were lysed with ripa [ mm tris/hcl, ph . , mm nacl, % glycerol, . % sds, . % nadoc, % igepal, mm edta, ph . , pyrophosphate mg ml aprotinin; mg ml leupeptin; mm sodium vanadate and mm benzamidine] on ice for a minimum of minutes. supernatants were cleared by centrifugation in a standard tabletop centrifuge (eppendorf) at maximum speed. protein concentration was determined by bradford assay. the phosphorylated and unphosphorylated forms of stat were detected using anti-stat (y ) antibody and anti-stat (bd bioscience). an antibody directed against y of stat was used for detection of the phosphorylated form of stat (upstate). antibodies to detect influenza viral proteins were purchased from serotec (np, m ), santa cruz (pb , pb ). the anti-pa antibody was kindly provided by j. ortin (madrid/ spain). a monoclonal antibody directed against the viral ns was generated at the imv, muenster, germany [ ] . a monoclonal anti-myc-tag antibody to detect myc-m was kindly provided by viktor wixler. imv, muenster, germany. all secondary antibodies were purchased from amersham and diluted : in tbs-t. secondary antibodies were incubated for a minimum of minutes at room temperature. to synthesize cdna from cells, rna was isolated using qiagen rneasy mini kit according to manufacturer's instruction. in brief, cells were lysed in the presence of b-mercaptoethanol and lysates were loaded to a column, washed and eluted in rnase-free water. for reverse transcription mg total rna, . mg oligo dt primer in a total volume of ml were heated for minutes at uc. enzyme mix was prepared ( enzyme buffer (fermentas), water and mm dntps) and pre-warmed at uc for minutes before adding u/ ml revertaid h m-mulv (fermentas). reverse transcription was performed at uc for hour. the enzyme was inactivated at uc for minutes. samples were stored at uc or directly used in quantitative real-time pcr. for analysis of gene expression relative quantification of the dna amount was applied. in order to do that gene expression of the housekeeping gene gapdh was determined. to ascertain changes in expression of the gene of interest the differences between expression of gapdh and the gene of interest was calculated using the ddct method [ ] . for quantitative real time brilliant qpcr sybr green mastermix (stratagene) was used according to manufacturer's instructions. the fragment of interest was amplified in cycles. the following primers were used (see table the pcfg -egz retroviral vector used for transfection [ ] as well as the constructs to express dominant negative mkk (mkk ala) or ikk (ikk kd) have been described earlier [ , ] . the phoenix amphotropic retroviral producer cells (a gift from g. nolan, stanford, ca) [ ] were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum, units/ml penicillin and mg/ml streptomycin. generation of mkk ala or ikkkd expressing producer cells as well as transduction of a cells to stably express these transgenes was performed as previously described [ , ] . figure s infection of huvec results in inhibition of stat phosphorylation and ifnb independent socs- gene transcription. huvec were infected with pr (moi = ) (a, b, d) or stimulated with u/ml ifnb (e) for time points indicated. to assess the mrna levels of socs- (a, e), ifnb (b) and mxa (e) rna was reverse transcribed and cdna was subjected to quantitative real time pcr. equivalent mrna amounts were normalized to endogenous gapdh and calculated as n-fold of untreated cells that were arbitrarily set as . to assess the amount of phosphorylated stat (b) a cells were infected with pr (moi = ) for time points indicated. total cells lysate was subjected to western blot analysis using anti-phospho-stat , anti-stat antibodies. to assess effective viral replication viral ns was detected using an anti-ns antibody. (c) quantification of relative band intensities of (b) using aida software and d densitometry (fuji). figure s il b and tnfa do not affect induction of socs- gene transcription. a wt cells were stimulated with u/ml il b (a), ng/ml tnfa (b) or infected with pr (moi = ) (c) for time points indicated. cells were lysed, and rna was subjected to reverse transcription. cdna was analyzed in quantitative real time pcr to assess mrna amounts of socs- and il (a and b) or il b (c). equivalent mrna amounts were normalized to gapdh mrna levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as . found at: doi: . /journal.ppat. .s ( . mb tif) ifnbeta induction by influenza a virus is mediated by rig-i which is regulated by the viral ns protein inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus the -end-processing factor cpsf is required for the splicing of single-intron pre-mrnas in vivo influenza virus ns protein interacts with the cellular kda subunit of cpsf and inhibits end formation of cellular pre-mrnas binding of the influenza virus ns protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf- translation initiation factor biochemical and genetic evidence for complex formation between the influenza a virus ns protein and the interferon-induced pkr protein kinase influenza a virus ns protein activates the pi k/akt pathway to mediate antiapoptotic signaling responses influenza a virus ns protein binds p beta and activates phosphatidylinositol- -kinase signaling interferons at age : past, current and future impact on biomedicine interferons, interferon-like cytokines, and their receptors ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex activation of ifn-alpha, ifn-gamma, mxa, and ifn regulatory factor genes in influenza a virusinfected human peripheral blood mononuclear cells influenza a virusinduced ifn-alpha/beta and il- synergistically enhance ifn-gamma gene expression in human t cells the jak-stat signaling pathway: input and output integration stats: transcriptional control and biological impact the receptor of the type i interferon family acetylationdependent signal transduction for type i interferon receptor differential gene induction by type i and type ii interferons and their combination interferon signalling network in innate defence the polyoma virus t antigen interferes with interferon-inducible gene expression ebola virus vp binds karyopherin alpha and blocks stat nuclear accumulation suppressors of cytokine signaling and immunity the elongin bc complex interacts with the conserved socs-box motif present in members of the socs, ras, wd- repeat, and ankyrin repeat families vhl-box and socs-box domains determine binding specificity for cul -rbx and cul -rbx modules of ubiquitin ligases the conserved socs box motif in suppressors of cytokine signaling binds to elongins b and c and may couple bound proteins to proteasomal degradation the jak-binding protein jab inhibits janus tyrosine kinase activity through binding in the activation loop a three-dimensional model of suppressor of cytokine signalling (socs- ) three distinct domains of ssi- /socs- /jab protein are required for its suppression of interleukin signaling socs- inhibits il- -induced stat activation by binding through its sh domain to the stat docking site in the il- receptor beta subunit suppressors of cytokine signalling: socs induction of suppressor of cytokine signaling- by herpes simplex virus type confers efficient viral replication induction of suppressor of cytokine signaling- by herpes simplex virus type contributes to inhibition of the interferon signaling pathway ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling- cytokine regulation in sars coronavirus infection compared to other respiratory virus infections respiratory syncytial virus inhibits interferon-alpha-inducible signaling in macrophage-like u cells non-response to antiviral therapy is associated with obesity and increased hepatic expression of suppressor of cytokine signalling (socs- ) in patients with chronic hepatitis c, viral genotype defective hepatic response to interferon and activation of suppressor of cytokine signaling in chronic hepatitis c suppressor of cytokine signaling (socs ) expression and hepatitis c virus-related chronic hepatitis: insulin resistance and response to antiviral therapy socs- regulates onset and maintenance of t(h) -mediated allergic responses role of endogenous inhibitors of cytokine signaling in allergic asthma regulation of jak-stat signalling in the immune system mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate the suppressor of cytokine signaling (socs) and socs but not socs proteins inhibit interferon-mediated antiviral and antiproliferative activities socs- and socs- inhibit ifn-alpha-induced expression of the antiviral proteins , -oas and mxa -triphosphate rna is the ligand for rig-i the mitogen-activated protein (map) kinase p and its upstream activator map kinase kinase are involved in the activation of signal transducer and activator of transcription by hyperosmolarity the mkk / p mitogen-activated protein kinase pathway is capable of inducing socs gene expression and inhibits il- -induced transcription ringing the alarm bells: signalling and apoptosis in influenza virus infected cells dual function of interleukin- beta for the regulation of interleukin- -induced suppressor of cytokine signaling expression ikkalpha, ikkbeta, and nemo/ikkgamma are each required for the nf-kappab-mediated inflammatory response program nf-kappab-dependent induction of tumor necrosis factor-related apoptosisinducing ligand (trail) and fas/fasl is crucial for efficient influenza virus propagation activation of nf-kappa b via the ikappa b kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells multiple signaling pathways regulate nf-kappab-dependent transcription of the monocyte chemoattractant protein- gene in primary endothelial cells type interferons and the virus-host relationship: a lesson in detente il- induces an anti-inflammatory response in the absence of socs in macrophages ns protein of influenza a virus inhibits the function of intracytoplasmic pathogen sensor, rig-i intracellular warfare between human influenza viruses and human cells: the roles of the viral ns protein regulation of a nuclear export signal by an adjacent inhibitory sequence: the effector domain of the influenza virus ns protein binding of the influenza a virus ns protein to pkr mediates the inhibition of its activation by either pact or double-stranded rna suppressors of cytokine signalling (socs) in the immune system influenza viruses and map kinase cascades -novel targets for an antiviral intervention exploited defense: how influenza viruses take advantage of antiviral signaling responses nfkappab negatively regulates interferon-induced gene expression and antiinfluenza activity cutting edge: innate immune response triggered by influenza a virus is negatively regulated by socs and socs through a rig-i/ifnar -dependent pathway interferon-gamma, but not interferon-alpha, induces socs expression in human melanoma cell lines rac and pak are upstream of ikk-epsilon and tbk- in the viral activation of interferon regulatory factor- influenza a virus lacking the ns gene replicates in interferon-deficient systems caspase activation is essential for efficient influenza virus propagation activation of phosphatidylinositol -kinase signaling by the nonstructural ns protein is not conserved among type a and b influenza viruses analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method a expression is stimulated by cd in b cells and rescues wehi cells from anti-igm-induced cell death transcriptional profiling of ikk /nf-kappa b-and p map kinasedependent gene expression in tnf-alpha-stimulated primary human endothelial cells expression vectors and delivery systems we would like to thank akihiko yoshimura, kyushu university, fukuoka, japan, for providing socs / mef via fred schaper, aachen, germany. we also would like to thank viktor wixler, imv, muenster, germany, for providing anti-myc and anti-ns monoclonal antibodies. key: cord- - zh jmc authors: caignard, grégory; komarova, anastassia v.; bouraï, mehdi; mourez, thomas; jacob, yves; jones, louis m.; rozenberg, flore; vabret, astrid; freymuth, françois; tangy, frédéric; vidalain, pierre-olivier title: differential regulation of type i interferon and epidermal growth factor pathways by a human respirovirus virulence factor date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: zh jmc a number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. to understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein c of human parainfluenza virus type (hpiv -c). we found that hpiv -c interacts directly through its c-terminal domain with stat and grb , whereas c proteins from measles or nipah viruses failed to do so. binding to stat explains the previously reported capacity of hpiv -c to block type i interferon signaling, but the interaction with grb was unexpected. this adaptor protein bridges epidermal growth factor (egf) receptor to mapk/erk pathway, a signaling cascade recently found to be involved in airway inflammatory response. we report that either hpiv infection or transient expression of hpiv -c both increase cellular response to egf, as assessed by elk transactivation and phosphorylation levels of erk / , s ribosomal subunit protein s and translation initiation factor e (eif e). furthermore, inhibition of mapk/erk pathway with u prevented viral protein expression in infected cells. altogether, our data provide molecular basis to explain the role of hpiv -c as a virulence factor and determinant of pathogenesis and demonstrate that paramyxoviridae have evolved a single virulence factor to block type i interferon signaling and to boost simultaneous cellular response to growth factors. viruses need to interact with host macromolecules to hijack the cellular machinery and replicate. these interactions are essential for viruses to target endocytic pathways and penetrate host cells, to recruit cellular transcription and/or translation machinery, and to achieve intracellular migration and viral particles assembly. but viruses also encode virulence factors that induce a substantial alteration of host cell functions and genetic programs to increase virus replication and spreading. for example, specific viral factors stimulate survival pathways to prevent apoptosis of infected cells or inhibit cell signaling involved in immune response. among these pathways, ifn-a/b signaling represents a prime target for viruses because of its critical role in the induction of both innate and adaptive antiviral immune responses [ ] . ifn-a/b transduce signals through direct binding to a cell surface receptor composed of two transmembrane subunits, ifnar and ifnar c [ ] . this interaction activates ifnar /ifnar c associated kinases tyk and jak that subsequently phosphorylate stat and stat transcription factors. activated stat and stat , altogether with irf , form the interferon-stimulated gene factor that binds ifn-stimulated response element (isre) promoter sequences to induce a large antiviral gene cluster. as a consequence, most viruses that are pathogenic in vertebrates have evolved virulence factors both to block ifn-a/b expression and signal transduction downstream of ifn-a/b receptor. human parainfluenza virus type (hpiv ) and human parainfluenza virus type (hpiv ) are important human pathogens that belong to respirovirus genus (paramyxoviridae family; [ ] ). these viruses are responsible for upper respiratory tract infections and colds, but often spread to the lower respiratory tract causing bronchitis, bronchiolitis and pneumonia in young children and immuno-compromised patients. hpiv infection is also suspected to exacerbate chronic airway inflammatory diseases like asthma [ ] . sendai virus and bovine parainfluenza virus type (bpiv ) are animal counterparts of hpiv and hpiv that infect mouse and cattle, respectively. respirovirus genome is a single-strand, negativesense rna molecule that encodes six structural proteins (mononegavirales order). while hemagglutinin-neuraminidase (hn) and fusion (f) are membrane glycoproteins associated with the envelop of hpiv particles, the nucleoprotein (n), the phosphoprotein (p) and the viral polymerase (l) form the ribonucleocapsid complex. the matrix protein (m) is at the interface between glycoprotein tails and ribonucleocapsids. the p gene of respirovirus encodes for p but also for a panel of accessory proteins by site-specific editing of p mrna and usage of overlapping open reading frames (orfs). in all respirovirus except hpiv , the co-transcriptional insertion of one g residue at an editing motif midway of p mrna leads to the expression of a chimeric protein called v. the v proteins of bpiv and sendai virus bind mda and suppress double-stranded rna-stimulated ifn-b production, thereby contributing to the virus evasion of host immune response [ ] . surprisingly in hpiv , multiple stop codons localized downstream of the editing site prevent the normal expression of a full-length v protein. as a result, p mrna molecules edited by the addition of one g residue encode for the amino acid (aa)-long n-terminal residues of p followed by only six additional aa (see materials and methods and [ ] ). but p mrna molecules edited by the addition of five g residues encode for d, a protein exhibiting a large and specific c-terminal domain of unknown function ( figure a ). besides co-transcriptional edition, an overlapping orf embedded in the first half of the p mrna allows the expression of a single c protein (hpiv and bpiv ) or a nested set of four proteins called c , c, y and y (sendai virus and hpiv ). the c proteins of sendai virus and hpiv have a high degree of sequence homology and have been studied in details. they are involved in the regulation of viral rna synthesis [ , ] , the inhibition of innate immune response [ ] and potentially contribute to the budding of viral particles [ ] [ ] [ ] . in particular, the c protein of sendai virus both inhibits ifn-b production [ , ] and blocks interferon signaling downstream of ifn-a/b and ifn-c receptors [ ] [ ] [ ] . the c proteins of hpiv and bpiv only share , % of sequence homology with the c proteins of sendai virus and hpiv , but they have also been shown to target interferon expression and signaling [ , ] . although expression of the c protein of hpiv (hpiv -c) is essential to virulence in vitro and in vivo [ ] and explains hpiv ability to block ifn-a/b signaling [ ] , host proteins that bind hpiv -c remain unknown. (a) organization of the gene p of hpiv that encodes for three proteins: p, d and c. whereas conventional transcription and translation lead to the expression of the phosphoprotein p, co-transcriptional insertion of five g residues at the editing site by the virus rna polymerase leads to the expression of a chimeric protein called d. insertion of one g residue can also occur during transcription but two stop codons immediately downstream of the editing site prevent the expression of the protein v that is specific of paramyxoviridae. the protein c is encoded by an overlapping opened reading frame (orf) embedded in p mrna. (b and c) hek- t cells were transfected with expression vectors encoding gst alone or fused to the c proteins of measles virus (mv-c), hpiv (hpiv -c), or nipah virus (nipah-c), and tested for the interaction with endogenous stat (b) or grb (c). total cell lysates were prepared h post transfection (cell lysate; middle and lower panels), and copurifications of endogenous cellular proteins were assayed by pulldown using glutathione-sepharose beads (gst pull-down; upper panel). gst-tagged c proteins were detected by immunoblotting using anti-gst antibody, while endogenous stat and grb were detected with specific monoclonal antibodies. doi: . /journal.ppat. .g respiroviruses are important pathogens responsible for acute respiratory tract infections associated with severe airway inflammation in children, elderly and immunocompromised individuals. their rna genome encodes for structural proteins that compose viral particles, but also for virulence factors that alter cell biology to enhance virus replication and spreading. among them, the c protein plays a critical role by blocking cellular response to type i interferons, the main antiviral cytokines secreted during virus infections. to provide molecular basis to this activity, we found that the c protein of human parainfluenza virus type (hpiv -c), the most frequent human respirovirus, interacts with stat , a key component of type i interferon receptor complex. but hpiv -c was also found to interact with grb , an adaptor molecule involved in cellular response to epidermal growth factor (egf), and to enhance cell response to this cytokine. this pathway increases protein translation, promotes cell survival and contributes to airway inflammation and mucus secretion. thus, our findings show that hpiv -c not only inhibits the antiviral response but also stimulates cellular response to egf, which benefits virus replication and induces an excessive inflammation of airways during infection. in an attempt to answer this question, we performed a yeast two-hybrid screen and we report here the identification of stat and grb as direct interactors of hpiv -c. although binding to stat accounts for hpiv -c ability to block ifn-a/b signaling, the interaction with grb was unexpected. this adaptor protein bridges growth factor receptor tyrosine kinases (rtks), like epidermal growth factor (egf) receptor, to the mitogenactivated protein kinase/extracellular signal-regulated kinase (mapk/erk) pathway. upon engagement by their ligands, rtks autophosphorylate on tyrosine residues to recruit adaptor proteins containing phosphotyrosine binding (ptb) or src homology (sh ) domains like grb [ ] . once associated to rtks by its sh domain, grb recruits the guanine nucleotidereleasing factor son-of-sevenless (sos) to activate ras. downstream events include mapk/erk kinase (mek / ) activation, which in turn phosphorylates erk / . finally, phosphorylated erk / directly or indirectly activates numerous cellular targets including transcription factors (e.g. elk , sap , sap , c-fos, creb, srf) but also cellular factors that control mrna translation like eukaryotic initiation factor e (eif e) or small ribosomal subunit s protein [ , ] . growth factor binding to rtks regulates a multiplicity of cellular processes including proliferation, differentiation and survival. in the respiratory tract, this signaling cascade has been shown to trigger inflammation and mucus secretion by epithelial cells [ ] [ ] [ ] [ ] , suggesting a critical role in innate immunity [ ] . however, excessive activation of this pathway could benefit to virus replication by inhibiting ifn-a/b signaling [ ] and promoting infected cell survival [ ] . altogether, these data provided a rational to investigate the functional impact of hpiv -c expression on ifn-a/b vs egf receptor and mapk/erk signaling pathways. the c protein of hpiv interacts directly with stat and grb to identify cellular targets of hpiv -c, this viral protein was used as bait in the yeast two-hybrid system to screen a human cdna library. the screen was performed at saturation with a -fold coverage of the library ( . + diploids), and positive yeast colonies growing on selective medium were analyzed by pcr and sequencing to identify binding partners of hpiv -c. stat and grb were the main interactors of hpiv -c identified in the screen with and yeast colonies corresponding to these cellular proteins, respectively. in both cases, cdna clones retrieved from the screen corresponded to full-length stat and grb in frame with the gal -ad transactivation domain. to validate these interactions in human cells, gst-tagged hpiv -c was expressed in hek- t cells and purified with glutathion-sepharose beads. as shown in figure b and c, endogenous stat and grb co-purified with hpiv -c. highly divergent c proteins from measles virus (mv-c) and nipah virus (nipah-c) failed to do so, thereby demonstrating the specificity of identified interactions. binding to stat provides molecular basis to the inhibition of ifn-a/b signaling by hpiv -c [ ] , and parallels the interaction previously identified between sendai virus c protein and mouse stat [ ] . altogether, this suggests that stat is a specific cellular interactor of respirovirus c proteins. in contrast, binding to grb is unexpected and suggests a new function for hpiv -c that we decided to investigate. hpiv -c has opposite effects on ifn-a/b and egf signaling pathways the adaptor protein grb plays a critical role in coupling signal from growth factor receptors to mapk/erk signaling pathway. to address the question of hpiv -c interference with this pathway, we used a trans-reporter gene assay that measures elk activation by erk / . in this system, elk transcription factor is fused to the dna binding domain of gal (gal -db) and binds the promoter sequence of a luciferase reporter gene. upon stimulation with a growth factor like egf, elk is activated as assessed by a significant increase in luciferase expression. surprisingly, we observed a -fold enhancement in this cellular response to egf when flag-tagged hpiv -c was expressed in hek- t cells (figure a ). same results were obtained when using hpiv -c without a tag ( -fold enhancement) or tagged with the red fluorescent protein cherry ( -fold enhancement). in contrast to hpiv -c, neither mv-c nor nipah-c enhanced elk activity upon egf stimulation (figure a ) whereas expression levels of hpiv -c, mv-c and nipah-c were similar in this system ( figure f , left panel). elk activity was also enhanced by hpiv -c expression in vero and hela cells as well as beas- b and a , two epithelial cell lines that originate from the respiratory tract, which is the tissue targeted by hpiv in vivo ( table ). the effect of hpiv -c in these different cell lines was highly significant (see p-values in table ) although relatively modest when compared to hek- t cells. this is probably because our reporter system requires the co-transfection of four plasmids and vero, hela, beas- b and a cells are more difficult to transfect than hek- t. in parallel experiments, cellular response to ifn-a/b was monitored using a cis-reporter gene, of which expression is controlled by five isres. as previously reported [ ] , we found that hpiv -c efficiently blocked ifn-a/b signaling ( figure b ) as opposed to what we observed for the egf pathway. again, mv-c or nipah-c was unable to do so. altogether, these results show that hpiv -c enhances the cellular response to egf in addition to its ability to block ifn-a/b signaling. we also determined if similar effects on the egf pathway were observed in infected cells. hek- t cells were infected with hpiv (moi = ) and then transfected with elk activity reporter plasmids. infection of hek- t cells was confirmed by anti-hpiv hemagglutinin-neuraminidase (hpiv -hn) immunostaining and flow cytometry analysis ( figure e ). like hpiv -c alone, hpiv infection enhanced elk activity upon egf stimulation ( figure c ). interestingly, hpiv infection induced a significant level of elk activity in the absence of egf stimulation. this suggests that in addition to hpiv -c interaction with grb , other mechanisms modulate mapk/erk pathway during hpiv infection. finally, to demonstrate that enhancement of elk activation by hpiv -c is completely dependent on erk / activation, hek- t cells were pre-treated with mek / inhibitor u before stimulation with egf. this molecule targets mek / and totally abrogates downstream phosphorylation and activation of erk / [ ] . as shown in figure d , elk activation was blocked by u , whereas hpiv -c expression was maintained ( figure f , right panel). this demonstrates that hpiv -c is acting through erk / stimulation. altogether, these results support a model where hpiv -c interaction with grb enhances cellular response to growth factors as assessed by an increased activation of mapk/ erk pathway. phosphorylation of erk / , eif e and small ribosomal subunit s protein are stimulated by hpiv -c expression or hpiv infection to further document hpiv -c impact on mapk/erk signaling pathway, we compared the kinetic of erk / phosphorylation in hek- t cells expressing hpiv -c or not. cells were transfected hpiv -c impact on ifn-a/b and egf pathways in addition to these three plasmids, cells were co-transfected with an expression vector encoding flag-tagged mv-c, hpiv -c or nipah-c or the corresponding empty vector pci-neo- flag. h after transfection, cells were starved and h later egf was added at a final concentration of ng/ml. after h, relative luciferase activity was determined. (b) hek- t cells were transfected with pisre-luc, a plasmid containing a luciferase gene of which expression is controlled by five isres, and prl-cmv. in addition to these two plasmids, cells were co-transfected with an expression plasmid encoding flag-tagged mv-c, hpiv -c or nipah-c or the corresponding empty vector pci-neo- flag. h after transfection, iu/ml of recombinant ifn-b were added. after h, relative luciferase activity was determined. (c) hek- t cells were infected with hpiv (moi = ) and then transfected with pfa -elk , pgal -uas-luc, prl-cmv vectors. h later, cells were starved during h and stimulated with egf at a final concentration of ng/ml. after h, relative luciferase activity was determined. (d) same experiment as (a) but mm of mek / specific inhibitor u was added as indicated. (a-d) all experiments were achieved in triplicate, and data represent means sd. (e) hek- t cells were infected as in (c), and hpiv -hn expression determined by immunostaining and flow cytometry analysis. (f) hek- t cells were transfected to express flag-tagged mv-c, hpiv -c or nipah-c as described in (a) and (b), and relative expression levels were determined h later by western blot analysis (left panel). in a parallel experiment, hek- t cells were transfected to express hpiv -c and were cultured with or without egf in the presence or absence of u as described in (d). hpiv -c expression level was determined by western blot analysis (right panel). doi: . /journal.ppat. .g with flag-tagged hpiv -c or a control plasmid and h post transfection, they were starved before stimulation with egf. erk / phosphorylation was determined at , and min after stimulation. as illustrated by one representative experiment in figure a , egf stimulation induced erk / phosphorylation in control cells but signal was markedly and reproducibly increased by hpiv -c expression at maximum phosphorylation time point ( . to . fold increase; p = . ; n = ). we then determined the phosphorylation level of two downstream targets of this pathway that are involved in the control of mrna translation, the translation initiation factor eif e and the ribosomal protein s ( figure b ). before egf stimulation, low levels of phosphorylated eif e and s were detectable in mock-treated cells ( figure b and d). hpiv -c expression had virtually no effects on this background. thus, eif e and s phosphorylation levels were determined at different time-points after egf stimulation. because erk / activation precedes eif e and s phosphorylation, maximal phosphorylation occurs at later time points and was determined at min, h, h and h after stimulation. as observed for erk / , phosphorylation levels of eif e and s were enhanced by hpiv -c expression when stimulating the cells with egf. to validate these observations in infected cells, hek- t cells were infected with hpiv (moi = ) and h later, cells were starved for h before stimulation with egf. like hpiv -c expression alone, hpiv infection enhanced erk / phosphorylation at the peak of induction, i.e. min after adding egf to the cells ( figure c ). interestingly, hpiv infection of a cells also enhanced erk / phosphorylation but the induction profile was different. indeed, erk / phosphorylation was not significantly increased at the peak of induction, but the signal was boosted by hpiv infection at late time points ( figure s ). the same profile was observed when eif e and s phosphorylation levels were analyzed in infected hek- t cells. hpiv infection sustained the phosphorylation of these two translation factors at late time points, but showed no increase at the peak of stimulation, i.e. min after adding egf to the cells ( figure d ). this could relate to the fact that hpiv infection also induces low levels of eif e and s phosphorylation in the absence of egf stimulation ( figure d ). this is reminiscent to what was observed for elk ( figure c ), and suggests that hpiv infection induces a basal activation of mapk/erk pathway leading to the constitutive phosphorylation of downstream targets. altogether, these data demonstrate that hpiv infection or hpiv -c expression alone both enhance mapk/erk pathway activation in egf-stimulated cells. several rna viruses require an activated mapk/erk pathway to produce viral components and replicate properly (for review see [ ] ). to test if the same was true for hpiv , cells were treated for h with mapk/erk pathway inhibitor u and infected with hpiv (moi = ). two days after infection, cell surface expression of hpiv -hn was detected by immunostaining and flow cytometry. u completely blocked the expression of hpiv -hn in hpiv -infected cells (figure ), whereas the same inhibitor had no effect when cells were infected with mv ( figure s ). altogether, this demonstrates that mapk/erk signaling is essential for the expression of hpiv proteins and suggests that hpiv manipulates this pathway to increase replication efficiency. the c-terminal region of hpiv -c binds stat and grb to better understand how hpiv -c targets both the ifn-a/b and egf signaling pathways, we characterized the functional domains of hpiv -c that bind stat and grb . to do so, we generated by pcr a full matrix of hpiv -c overlapping fragments and tested their ability to interact with either stat or grb in the yeast two-hybrid system ( figure and ). both forward and reverse primers were designed every nucleotides along hpiv -c sequence and fused to appropriate tails to allow gap-repair recombination with linearized gal -db yeast two-hybrid vector ( figure a ). all possible combinations of forward and reverse primers were used to amplify hpiv -c fragments. finally, corresponding pcr products were transformed in a yeast strain expressing gal -ad fused to either stat or grb , and growth on selective medium was used to detect potential interactions. a (aa)-long peptide encompassing position to located in the c-terminal half of hpiv -c was sufficient to bind stat ( figure b ) or grb ( figure a ). in an iterative process, we then generated a second, a third and a fourth set of hpiv -c fragments corresponding to one-by-one aa deletions ( figure c -e and figure b -d), allowing to further reduce the stat and grb binding motifs to minimal peptides. a aa peptide encompassing residues to of hpiv -c was sufficient to observe the interaction with stat ( figure e ). the binding region to grb was virtually the same, encompassing aa to ( figure d ). the c-terminal region of hpiv -c required to bind stat and grb in the yeast two-hybrid system is highly conserved among respiroviruses ( figure a ) and suspected to fold into a structured coiled-coil domain [ ] . furthermore, virtually the same c-terminal region of sendai virus c protein (aa - ) was previously reported to mediate the interaction with mouse stat [ ] . to further validate our observations performed in the yeast two-hybrid system, we retested by co-affinity purification the ability of hpiv -c fragment encompassing aa - (hpiv -c - ) to interact with stat and grb in hek- t cells. gst-tagged hpiv -c - was expressed together with -flag-tagged stat or grb , and purified with glutathionsepharose beads. full-length hpiv -c and the n-terminal region encompassing aa - (hpiv -c - ) were used as positive and negative controls, respectively. as shown in figure b and c, hpiv -c - interacted with stat and grb , whereas hpiv -c - did not. although hpiv -c - interacted with grb as efficiently as full-length hpiv -c ( figure c ), interaction with stat was weaker suggesting that more residues contribute to the stabilization of this interaction ( figure b ). altogether, these results confirm that aa - of hpiv- include both stat and grb binding sites. as described in figure , hek- t, hela, vero, a or beas- b cells were transfected with pfa -elk , pgal -uas-luc, prl-cmv to measure the activation level of mapk/erk signaling pathway. cells were co-transfected with plasmids encoding flag-tagged mv-c, hpiv -c or nipah-c or the corresponding empty vector pci-neo- flag. h after transfection, cells were starved and stimulated h later with ng/ml of egf. after h, expression of luciferase was quantified. results were normalized so that reporter activity in cells transfected with a control vector equals . experiments were performed in triplicates and data represent means sd. doi: . /journal.ppat. .t hpiv -c impact on ifn-a/b and egf pathways although stat and grb essentially bind to the same region of hpiv -c as demonstrated above, it remained unclear whether these interactions are mutually exclusive. to answer this question, a competition experiment was designed where gst-tagged hpiv -c was co-expressed with stat in the presence or absence of grb ( figure d ). in this setting, grb expression prevents stat copurification together with gst-tagged hpiv -c. this validates our finding that stat and grb interact with the same region of hpiv -c, and demonstrates that stat and grb compete for hpiv -c binding. interestingly, grb interaction with hpiv -c was not affected by stat expression (figure d and data not shown), suggesting that grb has a higher affinity for hpiv -c than stat . both the n-and c-terminal domains of hpiv -c are required for its activity we finally tested if hpiv -c - was able, like full-length hpiv -c, to block ifn-a/b signaling and enhance cellular response to egf stimulation. first, cells were transfected with flag-tagged hpiv -c, hpiv -c - or hpiv -c - together with the ifn-a/b reporter plasmid, and stimulated h later with recombinant ifn-b. reporter gene expression was determined h post transfection and found to be inhibited exclusively by full-length hpiv -c ( figure a ). although this may reflect the weakness of hpiv -c interaction with stat ( figure b ), this also indicates that both the n-terminal and c-terminal regions of hpiv -c are required to block ifn-a/b signaling, even if only the c-terminal region is required for the binding to stat . the same constructs were tested using the elk activity reporter plasmids ( figure b ). again, only full-length hpiv -c was able to enhance elk activation upon egf stimulation whereas full-length hpiv -c and hpiv -c - were expressed at similar levels in transfected cells ( figure b , upper right panel). because grb binding to hpiv -c and hpiv -c - were essentially equivalent in co-affinity purification experiments, we hypothesized that the n-terminal region of hpiv -c was required for its proper subcellular localization. thus, hpiv -c, hpiv -c - and hpiv -c - were expressed in fusion downstream of the red fluorescent protein cherry. as shown in figure c , cherry alone or fused to hpiv -c - localized both in the nucleus and the cytoplasm of transfected cells. in contrast, full-length hpiv -c essentially accumulated at the cellular membrane whereas hpiv -c - was in the nucleus. although we have no explanation for this unexpected localization of hpiv -c - , these observations show that only full-length hpiv -c is able to target the cellular membrane where both ifn-a/b and egf signaling are triggered. paramyxoviridae have evolved various mechanisms to block ifna/b response, in particular signaling downstream ifnar / ifnar c receptor [ , ] . although members of pneumovirinae subfamily have specific genes to encode inhibitors of ifn-a/b signaling pathway, those expressed by other paramyxoviridae (i.e. paramyxovirinae subfamily) are encoded by overlapping reading frames embedded within the gene p. rubulaviruses express v proteins that target stat and/or stat for ubiquitination and degradation, while morbilliviruses and henipaviruses v proteins essentially impair stat / phosphorylation, activation and nuclear translocation. in addition, morbilliviruses and henipaviruses also encode for c proteins of which role in the inhibition of ifna/b response has been a matter of debates [ ] [ ] [ ] [ ] . recent reports showed that morbillivirus c proteins only have a minor role in the inhibition of ifn-a/b signaling [ ] , but are essential to block ifn-a/b induction [ ] . whether henipavirus c proteins can directly block ifn-a/b or promote viral replication through alternative mechanisms is unclear [ ] . in contrast, it has been clearly established that respirovirus c proteins are potent inhibitors of ifn-a/b signaling [ , , [ ] [ ] [ ] . in this report, we show that hpiv -c, but not mv-c or nipah-c, directly interacts with stat and efficiently inhibits ifn-a/b signaling. in addition, we identified a minimal stat binding domain that encompasses aa - of hpiv -c, a region suspected to fold into a coiled coil. interestingly, this conserved domain is localized within the stat binding region shared by all four isoforms of sendai virus c protein [ ] . together, these results confirm the capacity of hpiv -c to block ifn-a/b signaling pathway [ ] , provide molecular basis to this inhibition and clarify the fact that respirovirus c proteins are functionally distinct from morbillivirus and henipavirus c proteins. in addition to stat , we show that hpiv -c interacts directly with grb and enhances mapk/erk signaling downstream of egf receptor (egfr). our data give the first example of a paramyxoviridae protein that contributes to the stimulation of egfr and mapk/erk pathway and provides molecular basis to this activity. this pathway has been known for decades as a prime target of dna tumor viruses and oncogenic retroviruses, and its activation represents an essential step toward carcinogenesis [ , ] . but recent data demonstrate that non-oncogenic rna viruses also activate this signaling cascade to support viral replication and spreading [ ] . whether it is activated upon egfr engagement or other means, mapk/erk pathway regulates a multiplicity of cellular processes including proliferation, differentiation, development, cell survival and inflammation. as a consequence, how the activation of mapk/erk pathway promotes viral replication is a complex question. interestingly, two non-oncogenic rna viruses associated with acute respiratory tract infections have been recently reported to modulate the egfr pathway. both human respiratory syncytial virus (hrsv), a member of paramyxoviridae like hpiv , and a rhinovirus that belongs to picornaviridae family activate egfr and mapk/erk pathway [ , ] . infection of epithelial cells by these viruses stimulates the processing and activation of egfr ligands by membrane matrix metalloproteinase and subsequent engagement of egfr through autocrine/paracrine mechanisms. experiments performed on rhinovirus show that viral replication and tlr engagement by viral rna are both required to activate the egfr and mapk/erk pathway [ ] . in this report, we show that hpiv infection also activates mapk/erk pathway in the absence of external stimuli, a phenomenon that possibly relies on the engagement of pathogen recognition receptors. although hpiv -c alone is unable to activate this pathway, our data suggest that expression of this virulence factor enhances mapk/erk activation above normal level in infected cells, thereby contributing to viral replication and pathogenesis. induction of mapk/erk pathway by rna viruses has numerous consequences on cell biology. first, it results in increased expression of inflammatory factors, in particular cytokines and chemokines that recruit cellular effectors of immunity [ , [ ] [ ] [ ] [ ] [ ] . mapk/erk pathway was also reported to block the antiviral response induced by ifn-a/b, making its activation beneficial to virus replication [ ] . another consequence of mapk/erk pathway activation is the induction of mucin production by infected epithelial cells [ , ] . although mucin expression is a critical innate defense system, excessive production of mucus results in the obstruction of airways and delays the elimination of pathogens. finally, it has been demonstrated in vitro that upon hrsv infection, activation of egfr and mapk/erk pathway sustains viral replication by retarding the death of infected cells [ ] . altogether, these data suggest that although a moderate activation of mapk/erk pathway contributes to the innate response against viruses, an excessive activation leads to deleterious inflammation, inhibition of ifn-a/b response, airway obstruction and infected cell survival figure . identification of a minimal hpiv -c region interacting with grb . fragments of hpiv -c were generated and tested for their ability to interact with grb following the procedure described in figure a . (a) the first iteration identified a grb binding region of aa. after two (b), three (c) and four additional rounds (d), this domain was finally reduced to a minimal grb binding motif of aa. this binding domain, encompassing position to , is contained in the stat binding region of hpiv -c previously identified in figure . doi: . /journal.ppat. .g hpiv -c impact on ifn-a/b and egf pathways [ ] . therefore, it is tempting to speculate that hpiv -c interaction with grb and egfr pathway participates in such deregulation of airway epithelium homeostasis to promote hpiv replication and spreading. a consequence of such perturbations could be an aggravation of chronic inflammatory airway diseases like asthma or chronic obstructive pulmonary disease as already suggested by epidemiological links with paramyxoviridae infections and in vivo models [ , ] . besides its effects on immune response, activation of mapk/ erk pathway has direct consequences on viral replication as assessed by in vitro experiments. it is now well documented that mapk/erk pathway inhibition with u or pd deeply impairs the replication of numerous rna viruses including hrsv ( [ ] ; and for review see [ ] ). similarly, we show in this report that mapk/erk pathway inhibition prevents hpiv protein expression in infected cells as assessed by hpiv -hn detection. in influenza virus infected cells, membrane accumulation of influenza virus hemagglutinin (ha) induces lipid-rafts clustering that leads to mapk/erk pathway activation and nuclear export of viral ribonucleoprotein complexes to achieve a and b) hpiv -c - and hpiv -c - were tested for their ability to modulate ifn-a/b or egf signaling. (a) as described in figure , hek- t cells were transfected with pisre-luc and prl-cmv to determine the activation level of ifn-a/b signaling pathway. cells were co-transfected with expression plasmids encoding flag-tagged full-length hpiv -c (c fl ) or fragments. h after transfection, iu/ml of recombinant ifn-b were added. after h, relative luciferase activity was determined. experiments were performed in triplicates, and data represent means sd. (b) as described in figure , cells were transfected with pfa -elk , pgal -uas-luc and prl-cmv to determine the activation level of mapk/erk signaling pathway. cells were co-transfected with expression plasmids encoding full-length hpiv -c (c fl ) or fragments. h later, cells were starved during h and stimulated with egf at a final concentration of ng/ml. after h, relative luciferase activity was determined. experiments were performed in triplicate, and data represent means sd. as a control, relative expression levels of c fl , c - and c - were determined by western blot analysis (upper right panel). (c) full-length hpiv -c, hpiv -c - or hpiv -c - was expressed in fusion downstream of the red fluorescent protein cherry to determine subcellular localization in hek- t cells. h after transfection, cells were fixed with pfa, permeabilized and labeled with dapi to stain nuclei. single confocal sections show cherrytagged protein fluorescence in red and dapi staining in blue. doi: . /journal.ppat. .g viral particles assembly [ ] [ ] [ ] . because hpiv replication cycle is only cytoplasmic, mechanisms involved are necessarily distinct. a possible link between mapk/erk pathway and hpiv protein expression lies in the fact that among downstream targets of this pathway are essential factors of cellular translational machinery. we show that hpiv -c expression enhances the phosphorylation of s and eif e. the small ribosomal subunit protein s is the major phosphoprotein of eukaryotic ribosomes with five phosphorylation sites (ser , ser , ser , ser , and ser ). two families of serine/threonine kinases phosphorylate s in vitro: s k / and p ribosomal s kinase (rsk). recently it has been shown that mapk/erk signaling pathway activates rsk family members that contribute to s phosphorylation on ser / thereby stimulating cap-dependent translation [ ] . in addition, eif e that interacts with the cap structure and brings translation initiation factors together with the small ribosomal subunit via the scaffold protein eif g, undergoes regulated phosphorylation on ser upon mapk/erk pathway activation. this phosphorylation event is dependent on eif gassociated mapk signal-integrating kinases, mnk and mnk [ ] . eif e is believed to be the least abundant of all initiation factors and therefore considered as a perfect target to regulate protein synthesis. even though there is no direct link between eif e phosphorylation and the enhanced translation observed, the fraction of phosphorylated eif e dramatically increases following treatment of the cells with growth factors, hormones and mitogens. therefore, eif e phosphorylation has been associated with increased translation rates. hpiv mrnas are capped and polyadenylated like their host counterparts. thus, s and eif e phosphorylation together with a high level of viral gene transcription may contribute to a rapid switch toward viral protein synthesis within infected cells. specific biochemical investigations are still required to decipher how hpiv -c can both inhibit ifn-a/b signaling and enhance egfr and mapk/erk pathway. when searching the literature for viral proteins that target grb , we found specific reports on ns a from hepatitis c virus and orf from hepatitis e virus [ , ] . although ns a inhibits mapk/erk activation induced by exogenous egf, orf was described as an activator of mapk/erk pathway like hpiv -c. both ns a and orf exhibit a proline-rich motif (pxxp) to bind the src homology (sh ) domains of grb , but there is no such motif in hpiv -c suggesting that other mechanisms mediate stat and grb binding. interestingly, these two cellular proteins exhibit sh domains. such domains typically bind a phosphorylated tyrosine residue in the context of a longer peptide motif within a target protein. although there is no evidence that hpiv -c becomes phosphorylated, we have tested hpiv -c interaction with mutant stat and grb exhibiting sh domains disabled for the interaction with phosphotyrosine residues. these mutants were not affected for the interaction with hpiv -c (data not shown). this suggests that hpiv -c either binds distinct regions of stat and grb , or interacts with a region of the sh domain that does not involve the phosphotyrosine binding site. finally, our results also show that full-length hpiv -c is required to modulate ifna/b and mapk/erk pathways since aa - that bind stat and grb are unable to do so when expressed alone. full-length hpiv -c was also required to observe a localization at the cell membrane, suggesting a link with its activity. interestingly, the n-terminal residues of sendai virus c protein act as a membrane targeting signal [ ] . but the n-terminal residues of hpiv -c (aa - ) were unable to do so, and sequence analysis did not show any conservation with the c protein of sendai virus. thus, hpiv -c tertiary structure is apparently required to target this protein at the cell membrane. this specific localization could both sequester stat to prevent the stimulation of ifn-target genes and contribute to the aggregation of grb -sos complexes to enhance mapk/erk signaling [ ] . altogether, this suggests that hpiv -c interaction with stat and grb represents a potential target for the development of antiviral molecules against hpiv and possibly other members of respirovirus genus. plasmid dna constructs p-encoding sequence from hpiv wild-type strain (df ) was amplified by rt-pcr (titan one tube; roche applied science) from total rna purified from infected cells (rneasy kit; qiagen). amplification was performed using the following hpiv -p specific primers flanked with gateway cloning sites: -gggga-caactttgtacaaaaaagttggcatggaaagcgatgctaaaaactatc-aaa and -ggggacaactttgtacaagaaagttggttattggcaattatt-gacatcttcattgaac. pcr products were cloned using topo ta cloning kit (invitrogen) into topo vector. a total of clones were analyzed to establish the sequence of hpiv -p (genbank id: eu ). interestingly, clones were not edited, clones were edited by the addition of one g residue, and clones were edited by the addition of g residues. one of the plasmids containing the unedited sequence of hpiv -p was selected and subsequently used as a template to clone hpiv -c. dna sequences encoding full-length hpiv -c or fragments corresponding to aa - or - were amplified by pcr from p(hpiv -p)-topo and cloned by in vitro recombination into pdonr (gateway system; invitrogen) as previously described [ ] . similarly, mv-c was amplified from p(+)mv that contains the full genome of measles virus wild-type strain ichinose (kindly provided by dr. k. takeuchi, [ ] ). nipah-c was amplified from niv-p plasmid (kindly provided by dr. tf. wild; [ ] ). grb coding sequence was amplified from the human spleen cdna library used to perform the yeast two-hybrid screen (invitrogen). the pdonr plasmid containing stat was previously described [ ] . viral or cellular coding sequences were subsequently transferred by in vitro recombination from pdonr into different gateway-compatible destination vectors (see below) following manufacturer's recommendation (lr cloning reaction, invitrogen). to perform yeast two-hybrid experiments, coding sequences were recombined into ppc (invitrogen) to be expressed in fusion downstream of the activation domain of gal (gal -ad) or into pdest to be expressed in fusion downstream of the dna binding domain of gal (gal -db). in mammalian cells, gst-tag and flag-tag fusions were achieved using pdest (invitrogen) or pci-neo- flag vector, respectively [ ] . we also used pci-neo (promega) and pmcherry-c (clontech) to express proteins without a tag or in fusion downstream of cherry, respectively. these two plasmids were made gateway-compatible using the gateway vector conversion system (invitrogen). hek- t, hela and vero cells were maintained in dulbecco's modified eagle's medium (dmem; gibco-invitrogen) containing % fetal bovine serum, penicillin, and streptomycin at uc and % co . a and beas- b cells were maintained in f- k medium (gibco-invitrogen) containing % fetal bovine serum, penicillin, and streptomycin at uc and % co . hpiv (strain c ) was amplified and titrated on vero cells following recommendations of atcc (american type culture collection). recombinant mv-egfp virus used in figure s has been hpiv -c impact on ifn-a/b and egf pathways previously described [ ] . infections were performed for h at uc in optimem (gibco-invitrogen). later on, cells were washed and incubated in fresh culture medium for or h. to detect viral replication, cells were recovered and incubated in pbsparaformaldehyde . % for min. after extensive washing with pbs, cells were permeabilized with pbs-triton . % for min, and then incubated with a monoclonal antibody specific to hpiv -hn (m , abcam). cells were washed and incubated in the presence of an anti-mouse cy -conjugated antibody (jackson immunoresearch). after extensive washing, cellular immunostaining was analyzed using a facscalibur flow cytometer (bd). when specified, cells were pre-treated with mek / specific inhibitor u ( mm final; promega) for h before, during and after infection to study the impact on hpiv infection. to perform co-affinity purification experiments, cloned orfs were transferred from pdonr to pdest expression vector (invitrogen) to achieve gst fusion, and to pci-neo- flag vector [ ] for flag-fusion. cell transfections were performed using lipofectamine (invitrogen). unless specified otherwise, hek- t cells were dispensed in each well of a -well plate, and transfected h later with ng of each plasmid dna per well. two days post transfection, hek- t cells were washed in pbs, then resuspended in lysis buffer ( . % nonidet p- , mm tris-hcl at ph , mm nacl and mm edta) supplemented with complete protease inhibitor cocktail (roche). cell lysates were incubated on ice for min, and then clarified by centrifugation at , g for min. for pull-down analysis, mg of protein extracts were incubated for h at uc with ml of glutathione-sepharose beads (amersham biosciences) to purify gst-tagged proteins. beads were then washed times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer (invitrogen). purified complexes and protein extracts were resolved by sdspolyacrylamide gel electrophoresis (sds-page) on - % nupage bis-tris gels with mops running buffer (invitrogen), and transferred to a nitrocellulose membrane. proteins were detected using standard immunoblotting techniques. flagand gst-tagged proteins were detected with a mouse monoclonal hrp-conjugated anti- flag antibody (m ; sigma-aldrich) and a rabbit polyclonal anti-gst antibody (sigma-aldrich), respectively. specific antibodies were used to detect endogenous stat (clone- ; bd biosciences), grb (clone- ; bd biosciences), phospho-erk / (clone- d ; upstate), erk / (ct; upstate), phospho-eif e (ser ; cell signaling), eif e (cell signaling), phospho-s - (ser / ; cell signaling), s (clone- d ; cell signaling) and hpiv -hn (m ; abcam). secondary anti-mouse and anti-rabbit hrp-conjugated antibodies were from ge-healthcare. densitometric analysis of the gels was performed using a specific module of photoshop cs extended (adobe systems inc.). yeast two-hybrid screening and gap-repair procedure our yeast two-hybrid protocols have been described in details elsewhere [ ] . briefly, pdest plasmid encoding gal -db fused to hpiv -c was transformed in ah yeast strain (clontech), and used to screen by mating a human spleen cdna library cloned in the gal -ad ppc vector (invitrogen) and previously established in y yeast strain (clontech). yeast cells were plated on a selective medium lacking histidine and supplemented with mm -amino-triazole ( -at; sigma-aldrich) to select for interaction-dependent transactivation of his reporter gene. ad-cdnas from [his+] colonies were amplified by pcr and sequenced to identify the host proteins interacting with hpiv -c. the gap-repair procedure was used to map the minimal portion of hpiv -c interacting with stat and grb . as previously described [ ] , both forward and reverse pcr primers were designed along the sequence of hpiv -c and fused to specific tails allowing yeast-based recombination in gal -db two-hybrid vector. matrix combinations of forward and reverse primers were used to amplify fragments of hpiv -c by pcr. ah yeast cells expressing ad-fused stat or grb were co-transformed with ml of each pcr product in the presence ng of linearized pdest vector to achieve recombinatorial cloning by gaprepair. fragments of hpiv -c fused to gal -db were then tested for interaction with ad-stat or ad-grb by plating yeast cells on selective medium lacking histidine and supplemented with mm of -at. luciferase reporter gene assay hek- t, hela or vero cells were plated in -well plates ( cells per well). one day later, cells were transfected with either pisre-luc ( . mg/well; stratagene) or pfa -elk ( . mg/well; stratagene) and pgal -uas-luc plasmids ( . mg/ well; provided by dr. y. jacob) together with prl-cmv reference plasmid ( . mg/well; promega). cells were simultaneously cotransfected with . mg/well of pci-neo- flag, pci-neo or pmcherryc expression vectors encoding viral proteins as specified. h after transfection, cells were stimulated with ifnb (biosource) at iu/ml or starved for h then stimulated with egf (upstate) at ng/ml. h post transfection, cells were lysed, and both firefly and renilla luciferase activities in the lysate were determined using the dual-luciferase reporter assay system (promega). reporter activity was calculated as the ratio of firefly luciferase activity to reference renilla luciferase activity, and normalized so that positive control activity equals . when indicated, cells were treated with u (promega) at mm final concentration upon egf stimulation. subcellular localization of cherry-tagged hpiv -c fl , c - and c -well plates containing coverslips were seeded with hek- t cells ( cells per well). one day later, cells were transfected with pmcherryc expression vector alone or encoding hpiv -c fl , hpiv -c - or hpiv -c - . h after transfection, cells were incubated with pbs-pfa % for min at rt, then treated with pbs-triton . % for min at rt to permeabilize the cells. finally, cells were incubated for min at rt in a pbs-pfa % solution containing dapi ( - -diamidino- -phenylindole) at mg/ml. preparations were mounted using fluoromount-g (southernbiotech), and imaging performed using a sp confocal miscroscope (leica). figure s erk / phosphorylation is enhanced by hpiv infection in a cells. a cells were infected with hpiv (moi = ) and after h, cells were starved for h before stimulation with ng/ml of egf. phosphorylation of erk / was determined by western blot analysis at min, min and h post stimulation. hpiv infection was confirmed by anti-hpiv hemagglutinin-neuraminidase (hpiv -hn) immunoblotting. found at: doi: . /journal.ppat. .s ( . mb tif) hpiv -c impact on ifn-a/b and egf pathways figure s mek / inhibitor u has no effect on mv protein synthesis. hek- t cells were left untreated (a) or treated with mm of u for h (b). then, cells were mocktreated or infected with a recombinant mv strain expressing egfp (moi = ) and cultured with or without u (a and b, respectively). h after infection, egfp expression was quantified by flow cytometry analysis. found at: doi: . /journal.ppat. .s ( . mb tif) pathogenic viruses: smart manipulators of the interferon system how cells respond to interferons ) fields' virology naturally occurring parainfluenza virus infection in adults induces mild exacerbation of asthma associated with increased sputum concentrations of cysteinyl leukotrienes bovine parainfluenza virus type accessory proteins that suppress beta interferon production rna editing in the phosphoprotein gene of the human parainfluenza virus type sendai virus wild-type and mutant c proteins show a direct correlation between l polymerase binding and inhibition of viral rna synthesis paramyxovirus sendai virus c proteins are essential for maintenance of negative-sense rna genome in virus particles sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna aip /alix is a binding partner of sendai virus c protein and facilitates virus budding sendai virus budding in the course of an infection does not require alix and vps a host factors recruitment of alix/aip to the plasma membrane by sendai virus c protein facilitates budding of virus-like particles c and v proteins of sendai virus target signaling pathways leading to irf- activation for the negative regulation of interferon-beta production activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins sendai virus c protein physically associates with stat all four sendai virus c proteins bind stat , but only the larger forms also induce its mono-ubiquitination and degradation importance of the anti-interferon capacity of sendai virus c protein for pathogenicity in mice inhibition of stat phosphorylation by human parainfluenza virus type c protein mutations in the c, d, and v open reading frames of human parainfluenza virus type attenuate replication in rodents and primates paramyxoviridae use distinct virus-specific mechanisms to circumvent the interferon response specificity in signal transduction: from phosphotyrosine-sh domain interactions to complex cellular systems phosphorylation of the cap-binding protein eif e by the mapk-activated protein kinase mnk ras/erk signaling promotes site-specific ribosomal protein s phosphorylation via rsk and stimulates cap-dependent translation epidermal growth factor system regulates mucin production in airways activation of the epidermal growth factor receptor by respiratory syncytial virus results in increased inflammation and delayed apoptosis multiple tlrs activate egfr via a signaling cascade to produce innate immune responses in airway epithelium rhinovirus-induced major airway mucin production involves a novel tlr -egfr-dependent pathway epidermal growth factor receptor-mediated innate immune responses and their roles in airway diseases negative regulation of the alpha interferon-induced antiviral response by the ras/raf/mek pathway identification of a novel inhibitor of mitogen-activated protein kinase kinase rna viruses and the mitogenic raf/mek/erk signal transduction cascade the stat activation process is a crucial target of sendai virus c protein for the blockade of alpha interferon signaling inhibition of interferon induction and signaling by paramyxoviruses the c protein of measles virus inhibits the type i interferon response newcastle disease virus (ndv)-based assay demonstrates interferon-antagonist activity for the ndv v protein and the nipah virus v, w, and c proteins stringent requirement for the c protein of wild-type measles virus for growth both in vitro and in macaques regulation of interferon signaling by the c and v proteins from attenuated and wild-type strains of measles virus the rinderpest virus non-structural c protein blocks the induction of type interferon sendai virus c proteins counteract the interferon-mediated induction of an antiviral state knockout of the sendai virus c gene eliminates the viral ability to prevent the interferonalpha/beta-mediated responses human parainfluenza virus type but not sendai virus replicates in human respiratory cells despite ifn treatment the egfr as a target for viral oncoproteins mapk activation is involved in posttranscriptional regulation of rsv-induced rantes gene expression role of mitogen-activated protein kinases in influenza virus induction of prostaglandin e from arachidonic acid in bronchial epithelial cells ebola virus-like particle-induced activation of nf-kappab and erk signaling in human dendritic cells requires the glycoprotein mucin domain japanese encephalitis virus infection stimulates src tyrosine kinase in neuron/glia spike protein of sars-cov stimulates cyclooxygenase- expression via both calcium-dependent and calcium-independent protein kinase c pathways the causal direction in the association between respiratory syncytial virus hospitalization and asthma persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease erk- / activity is required for efficient rsv infection influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade membrane accumulation of influenza a virus hemagglutinin triggers nuclear export of the viral genome via protein kinase calpha-mediated activation of erk signaling clustering of raftassociated proteins in the external membrane leaflet modulates internal leaflet h-ras diffusion and signaling the orf protein of hepatitis e virus binds to src homology domains and activates mapk ns a, a nonstructural protein of hepatitis c virus, binds growth factor receptor-bound protein adaptor protein in a src homology domain/ligand-dependent manner and perturbs mitogenic signaling targeting of the sendai virus c protein to the plasma membrane via a peptide-only membrane anchor aggregation of membrane proteins by cytosolic cross-linkers: theory and simulation of the lat-grb -sos system measles virus v protein blocks jak -mediated phosphorylation of stat to escape ifn-alpha/beta signaling recovery of pathogenic measles virus from cloned cdna establishment of a nipah virus rescue system human papillomavirus type e oncoprotein represses the transforming growth factor beta signaling pathway by binding to smad a molecularly cloned schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice inhibition of ifn-alpha/beta signaling by two discrete peptides within measles virus v protein that specifically bind stat and stat we would like to thank dr. k. takeuchi and dr. tf. wild for kindly providing p(+)mv and niv-p plasmids, respectively. we thank all members of pf -pasteur genopole sequencing core facility. we thank members of the infection-mapping project i-map for fruitful discussions. we thank dr. m. mesel-lemoine and dr. j. pothlichet for their technical support. we thank miss r. parker for proofreading the manuscript. key: cord- - wo xen authors: gowen, brian b.; ennis, jane; russell, andrew; sefing, eric j.; wong, min-hui; turner, jeffrey title: use of recombinant adenovirus vectored consensus ifn-α to avert severe arenavirus infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: wo xen several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from - %. because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the national institutes of allergy and infectious diseases (niaid) as top priority biodefense category a pathogens. recombinant consensus interferon alpha (cifn-α) is a licensed protein with broad clinical appeal. however, while cifn-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. to address these limitations, we describe the use of def , a replication-deficient adenovirus vector that drives the expression of cifn-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. intranasal administration of def h prior to challenge with pichindé virus (picv) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. a significant protective effect was still observed with a single dosing of def given two weeks prior to picv challenge. def was also efficacious when administered as a treatment to h post-virus exposure. the protective effect of def was largely attributed to the expression of cifn-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal picv challenge. effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. the def technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens. the arenaviridae family of viruses has several members that can cause viral hemorrhagic fever, an acute, often-fatal, viral syndrome characterized by intense fever, malaise, and less frequently, bleeding and neurologic manifestations. case fatality rates of hospitalized patients suffering from arenaviral hemorrhagic fever (ahf) range from - % [ , , , ] . arenaviruses known to cause ahf include junín, machupo, guanarito, sabiá, and chapare in the south american continent, and lassa and lujo in west and southern africa, respectively. primary transmission of the arenaviruses from respective rodent reservoir hosts to humans occurs via exposure to contaminated excreta [ ] . person-to-person transmission can occur through contact with blood or other body fluids during the care and management of infected individuals [ , ] . notably, these viruses are considered a threat to national security and are classified as highest priority pathogens by the niaid [ ] . at present, the treatment of ahf is limited to ribavirin and immune plasma [ , ] . the latter has only been proven to be effective in treating cases of argentine hemorrhagic fever (junín virus infection) within days of disease onset. off-label usage of ribavirin has been shown to be effective in treating lassa fever when therapy was initiated within days of the development of clinical symptoms. however, there are toxicities associated with ribavirin therapy at dosages required for efficacious use, which may contribute to the observed poor patient compliance in completing prescribed treatment regimens [ , ] . very limited case data using ribavirin to treat other ahfs supports the use of emergency protocols [ , , ] , however the utility of ribavirin therapy remains to be seen. interferon alpha (ifn-a) is an effective part of the host innate immune response, which can be manufactured as a recombinant human protein with broad clinical appeal [ ] . consensus (c)ifna, also known as ifn alfacon- and infergen, is a licensed, second generation ifn-a engineered to contain the most frequently occurring amino acids among the nonallelic ifn-a subtypes. previously, we have demonstrated that cifn-a can be used effectively alone, or in combination with ribavirin, to treat pichindé virus (picv) infection in hamsters [ , ] , an experimental model of acute arenaviral disease [ ] . however, while cifn-a has clinical value, its usefulness is hindered by its short half-life and cost to manufacture. there is an initial distributive half-life of minutes and a beta half-life of to hours [ ] . the rapid systemic clearance requires frequent dosing to achieve desired therapeutic levels. consequently, treatment can result in well-documented toxicities which include headache, depression, hair loss, fever, and malaise. in order to combat the rapid degradation, pegylated forms of recombinant ifn-a have been introduced with half-lives that are on the order of days instead of hours, thus reducing the number of injections to once per week [ ] . however, the cost to manufacture peg-ifn-a is exceedingly high, and the pegylation process has been shown to reduce the activity of ifn-a, thereby further increasing the production costs. to circumvent the fast decay of cifn-a, a replicationincompetent, recombinant adenovirus type (rad ) gene delivery platform was designed to drive constitutive expression of the cifna gene from transduced nasal epithelial target cells. this rad cifn-a virus, called def , was first developed in mice and recently shown to be active against yellow fever virus (yfv) infection in hamsters [ , ] . the intranasal (i.n.) inoculation used in the yfv study prevents the host immune system from recognizing the ad vector, thereby bypassing any possible preexisting immunity [ ] . in the present study, we evaluated the use of def administered i.n. for the prevention and treatment of picv infection in hamsters. all animal procedures complied with usda guidelines and were conducted at the aaalac-accredited laboratory animal research center at utah state university under protocol , approved by the utah state university institutional animal care and use committee. female golden syrian hamsters were obtained from charles river laboratories (wilmington, ma) and acclimated for a minimum of days prior to experimentation. they were fed standard hamster chow and tap water ad libitum. animals were approximately - weeks old at the time of virus challenge. picv, strain an , was provided by dr. david gangemi (clemson university, clemson, south carolina). the virus was passaged once through hamsters. virus stocks were prepared from pooled livers harvested from infected hamsters. virus dilutions were made in minimal essential medium (mem), and infectious inoculum was given bilaterally in two intraperitoneal (i.p.) injections of . ml each. the recombinant adenovirus vectored cifn-a (rad -huifn-a; def ) and the rad empty vector (rad ev) control virus were provided by defyrus, inc. (toronto, on, canada) at a concentration of and plaque-forming units (pfu)/ml, respectively. both viruses were prepared in pbs for i.n. instillation in a ml volume. virus titers were assayed using an infectious cell culture assay as previously described [ ] . briefly, a specific volume of liver or spleen homogenate or serum was serially diluted and added to triplicate wells of vero (african green monkey kidney; american type culture collection, manassas, va) cell monolayers in well microplates. the viral cytopathic effect (cpe) was determined to days post-virus inoculation, and the % endpoints were calculated as described [ ] . the assay detection ranges were . to . log % cell culture infectious doses (ccid )/g of liver or spleen and . to . log ccid /ml of serum. in samples presenting with undetectable liver or spleen virus, a value of , . was assigned (, . for serum). conversely, in cases wherein virus exceeded the detection range, a value of. . (. . for serum) was assigned. for statistical analysis, values of . or . log ( . or . for serum) were assigned as needed for samples with undetectable or saturated virus levels, respectively. detection of alt in serum is an indirect method for evaluating liver disease. serum alt levels were measured using the alt (sgpt) reagent set purchased from pointe scientific, inc. (lincoln park, mi) per the manufacturer's recommendations. the reagent volumes were adjusted for analysis on -well microplates. experimental design def dose range titration experiment. hamsters were weighed on the morning prior to the day of infection and grouped (n = for drug treatment groups, for the placebo group) so that the average hamster weight per group across the entire experiment varied by less than grams. varying pfu amounts of def , the rad ev control virus, or saline placebo treatments were administered in a single i.n. dose h prior to challenge with , pfu of picv. five animals from each group were sacrificed on day of infection. serum was collected for assaying alt activity, and virus titers were determined for liver, spleen, and serum samples as described above. the remaining animals ( for the placebo group) were observed days for mortality and weighed individually every days starting on day . sham-infected normal controls (n = ) were included for comparison. extended pre-exposure prophylaxis experiment. the design was similar to the def titration experiment with the following differences. hamsters were weighed on the morning of initial pretreatment (day relative to the infection) and grouped (n = per group). groups were treated once i.n. with pfu of def , rad ev control virus, or saline placebo. treatments were given or days prior to challenge with , pfu of picv. animals were observed for days post-challenge for mortality. post-exposure prophylaxis experiment. the design was similar to the def pre-exposure prophylaxis experiment with the following differences. single dose i.n. treatments with pfu of def or rad ev were administered h prior to, or , , or h after challenge with , pfu of picv. on day postinfection, the surviving animals (including naïve sham-infected controls) were re-challenged. morbidity and mortality were observed out to days after the initial challenge. kaplan-meier survival plots and all statistical evaluations were done using prism (graphpad software, ca). the log-rank test was used for survival analysis. for analyzing differences in viral titers, alt levels, and weight change, a one-way analysis of variance (anova) with newman-keuls post test or the kruskal-wallis (two-tailed) test with the dunn's post test was performed based on gaussian distribution of the data. in the initial trial, hamsters were treated with to pfu of def one day prior to challenge with a lethal dose of picv. pretreatment with the highest dose of pfu of def resulted in % survival, and and pfu doses also significantly protected % and % of hamsters, respectively, from mortality ( figure a) . moreover, the hamster that succumbed in the group, survived days. importantly, only one out of ten hamsters treated with pfu of the control rad ev virus survived the infection; however, there did appear to be a slight delay in the time to death in the hamsters that received the control virus treatment. the weights of the hamsters were measured every days to assess weight gain over the course of the experiment as a marker of well being ( figure b) . notably, from day to day , a time before weight loss due to illness from picv infection would have been expected, hamster weights decreased as the dose of def increased. this would suggest that the higher treatment doses may have resulted in some loss of appetite, probably due to mild illness due to expression of consensus ifn since no overt effects were noticeable when handling the animals. the hamsters that received the pfu dose of def gained weight through day similarly to the animals treated with saline placebo and the normal controls (sham-infected, untreated) ( figure b) . the high-dose of rad ev control virus also resulted in a slight reduction in weight compared to the controls, suggesting that the immune response to the adenoviral vector alone may have caused some malaise in the animals. there was no elevation in serum alt levels on day of infection in samples collected from parallel treated and infected hamsters receiving def (figure a ). eighty percent of the rad ev group and % of the pbs placebo group had elevated levels of alt, reflective of liver disease. interestingly, the and pfu def groups presented with little to no day- virus burden in the serum, liver, or spleen, while the group developed viral titers that were comparable to the rad ev and placebo controls ( figure b-d) . a delay in the development of liver disease in the pfu def treated animals may explain the reduced alt levels. alternatively, saturation of liver virus titers in the low-dose def , rad ev, and placebo groups may have masked a substantial difference between the former and the viral vector and vehicle control groups. we next evaluated the prophylactic window of protection against picv infection using the pfu dose of def . animals were treated one or two weeks prior to challenge with a lethal dose of picv. consistent with the trend observed in initial dose titration study, hamsters treated with the pfu dose of def had significantly reduced weights compared to those that received the rad ev and placebo control treatments ( figure a) . nevertheless, the pretreatment with def seven days before infection was highly protective ( % survival rate; figure b ). notably, the single hamster that failed to survive the challenge succumbed on day , which was several days before the mean time to death measured in both the placebo and rad ev groups. an autopsy to determine the cause of death was not performed. in hamsters treated two-weeks prior to picv challenge, def significantly reduced mortality ( % survival) and extended the time of death in the animals that succumbed ( figure c ). in contrast, uniform lethality was seen with animals that received the rad ev and placebo treatments. of the surviving animals pre-treated with def , one was anorexic at the conclusion of the study on day post-infection. this was reflected by a % weight loss compared to the animals starting weight. it is possible that this hamster, which appeared ill and lethargic, was not able to completely prevent the infection. it was unclear whether it would have ultimately recovered if the observation period had been extended. on both the -day ( figure a , c, e, g) and -day ( figure b , d, f, h) pretreatments, def significantly reduced day- viral loads and liver disease (alt) compared to the controls. the absence of elevated alt levels in the def -treated hamsters may be explained by the - log reduction in liver virus burden ( figure e , f) and a delay in the development of liver disease. although tissue titers were slightly lower when def was given days prior to challenge compared to the day pretreatment, this was not evident with serum viral burden. because most animals had measurable replicating picv ( figure c-h) , it is likely that survivors would have been immunized and protected from subsequent challenge. this may not be the case with hamsters treated with def h prior to challenge since most had no detectable virus titers in spleen, liver, or serum on day of picv infection ( figure b-d) . having observed dramatic protection when administered up to weeks prior to challenge, a final experiment was conducted to determine the therapeutic value of def in the hamster picv infection model. when def was administered or h after challenge, highly significant protection was observed ( figure ). efficacy waned when def treatment was delayed to h postinfection. as anticipated, the treatment given pre-challenge verified previous activity, with all animals surviving challenge. interestingly, there was higher than expected survival with the control rad ev treatments initiated and h post-challenge, suggestive of a slight antiviral effect as the time of treatment was further delayed ( figure ) . the surviving hamsters from this experiment were re-challenged with picv to assess the ability of def to enhance longer-term protection via acquired immunity. with the exception of animals in the h def pretreatment group, and a single animal in the rad ev h group, all animals that were challenged with picv on day of the experiment survived a second challenge figure . def extended pre-exposure prophylaxis protects hamsters from lethal picv challenge. animals were treated i.n. with a single dose pfu of def , the rad ev control virus, or pbs placebo or days prior to picv infection. animal weights were measured two weeks prior to, and at the time of, picv challenge. the effect of -day and -day pretreatments on a) weight change over the two-week period prior to picv challenge and the extended picv prophylaxis efficacy data for the b) -day and c) -day pretreatments are shown. ***p, . compared to respective placebo-treated animals. b p, . , c p, . compared to respective rad ev-treated animals. doi: . /journal.pone. .g on day ( figure ). all six naïve animals that were initially shaminfected succumbed as expected. in animals that were sacrificed on day relative to the first infection, reductions in alt and viral titers were most evident in the groups that received def within h of infection ( figure ) . notably, in the animals treated with the control rad ev, there was an interesting trend that developed with the h post-infection group having the greatest alt levels and viral titers, followed by the , , and h groups. this trend may suggest a low-level immune stimulation in the hamsters relative to the time at which the rad ev was given. the resulting lack of measurable viral replication in the h def group ( figure b-d) is likely insufficient to elicit immunological memory. it is unclear as to why one of the first infection survivors from the h rad ev group ultimately succumbed to the second infection. in the present study, our findings demonstrate that expression of cifn-a following a single i.n. administration of def offers a strong protective effect in hamsters against challenge with picv that included limiting liver disease and inducing an antiviral state that inhibited systemic and tissue viral replication. the lack of significant antiviral activity elicited by the rad ev control virus suggests that the enhanced antiviral response produced by def is largely due to the expression of the cifn-a gene. the weak stimulatory effect seen in of the experiments was not surprising considering the number of host systems that play a role in sensing the adenovirus vector [ ] ; however, the effect was short-lived. in contrast, the enhancement of the host antiviral defenses by def was long-lasting with a -day pre-picv challenge prophylactic window. moreover, def was effective when given - days post-picv infection. these data also suggest that sufficient viral replication may be necessary to elicit an adaptive immune response that confers lasting protective immunity, as, for the most part, only re-challenged animals from the h def pretreatment group succumbed to a second challenge with a lethal picv inoculum. presumably, the robust innate immunity and antiviral state induced by the def pretreatment rapidly controlled the , pfu challenge dose obviating the development of the adaptive immune response and immunological memory. the pathogenic arenaviruses have evolved strategies to suppress and evade the host immune response [ , , , ] , resulting in uncontrolled replication and broad dissemination. however, they appear to be unable to block the induction of ifn stimulated genes via exogenous type i ifn [ ] , which may, in part, explain the success of def and cifn-a treatments [ ] . also essential to the success of def was early intervention prior to significant viral replication and engagement of innate immune suppressive functions. indeed, early induction of a strong type i ifn response is associated with favorable disease outcome in nonhuman primates challenged with lassa virus [ ] . early post-exposure prophylaxis was also required with exogenous cifn-a protein administered by the i.p. route [ , ] . with the multiple strategies that arena and other pathogenic viruses have in place to subdue the ifn-mediated host antiviral response [ ] , the utility of def , recombinant ifn proteins, and ifn inducing agents will depend upon the nature of the ifn pathway blockade and require early administration to be effective post-exposure. notably, with daily cifn-a protein injections of up to mg/kg, significant protection was observed; however, survival rates did not exceed % in those studies employing the same picv hamster model system and virus stock [ , ] . in contrast, def consistently elicited greater protection ( - %). the improved efficacy observed with def may be explained by a combination of factors that includes constitutive expression of fully glycosylated protein and reduced animal stress levels by avoiding daily injections for - days. we hypothesize that with the appropriate dose of def , therapeutic levels of consensus ifn-a can be maintained, effectively eliminating the daily bolus effect produced by i.p. injections. in addition, because cifn-a is produced in genetically engineered escherichia coli, the native glycosylation pattern is lost. conceivably, enhanced immunotherapeutic activity results from fully glycosylated cifn-a expressed from cells transduced with def . previous studies in mice with a related def virus expressing mouse ifn-a (mdef ) have shown the utility of adenovirusbased system to counter viral infections [ , , ] . more recently, in a different hamster model of viral hemorrhagic fever, several of us reported on efficacy of def in mitigating yfv infection and disease [ ] . yfv infection appears to be more sensitive to the effects of def , as a lower dose was able to provide complete protection. taken together with the results of the present study, the experimental animal data support the broad use of def for extended pre-exposure and early post-exposure prophylaxis applications. further investigations using advanced arenavirus models based on challenge of nonhuman primates with pathogenic arenaviruses [ ] are needed to better evaluate the potential of def to prevent severe disease in humans. nonhuman primate models should allow the full spectrum of cifn-a activity not possible in hamsters or guinea pigs. the familiarity of the fda with adenovirus gene delivery technology and approved cifn-a protein support the development of def for clinical use. an important step in the development process is the safety/toxicology testing in rodents, which is presently underway. in our studies, the highest dose of pfu of def administered by the i.n. route appeared to be well-tolerated in hamsters despite evidence of weight loss. they did not appear visibly ill, but clearly the treatment was having some effect that possibly led to reduced food and water consumption consistent with mild toxicity seen with ifn-a therapy. the i.n. delivery route is designed to circumvent pre-existing immunity to adenovirus type in humans [ , ] , and may limit systemic inflammation that could occur by parenteral administration of large numbers of adenovirus particles. ultimately, the production of a shelf-stable, powdered formulation of def for easy i.n. administration and long-term storage would be ideal for stock-piling in the event of the need for mass distribution due to intentional release or (re)emerging disease outbreaks of arena or other viral etiology. nosocomial outbreak of novel arenavirus infection, southern africa treatment of argentine hemorrhagic fever new opportunities for field research on the pathogenesis and treatment of lassa fever venezuelan hemorrhagic fever: clinical and epidemiological studies of cases arenaviridae: the viruses and their replication the counter-bioterrorism research agenda of the national institute of allergy and infectious diseases (niaid) for cdc category a agents efficacy of immune plasma in treatment of argentine haemorrhagic fever and association between treatment and a late neurological syndrome lassa fever. effective therapy with ribavirin review of the literature and proposed guidelines for the use of oral ribavirin as postexposure prophylaxis for lassa fever ribavirin for lassa fever postexposure prophylaxis brief report: treatment of a laboratory-acquired sabia virus infection animals were treated as described in figure and sacrificed on day of infection for analysis of serum (a) alt and (b) virus titer, and (c) liver and (d) spleen viral titers. *p, . , ***p, . compared to rad ev-treated animals treatment of bolivian hemorrhagic fever with intravenous ribavirin interferon-alpha as an immunotherapeutic protein interferon alfacon- protects hamsters from lethal pichinde virus infection combinatorial ribavirin and interferon alfacon- therapy of acute arenaviral disease in hamsters animal models of highly pathogenic rna viral infections: hemorrhagic fever viruses enhanced circulating half-life and antitumor activity of a site-specific pegylated interferonalpha protein therapeutic treatment of yellow fever virus with an adenovirus-vectored interferon, def , in a hamster model pre-and post-exposure protection against western equine encephalitis virus after single inoculation with adenovirus vector expressing interferon alpha nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice vitro and in vivo activities of t- against arenavirus and bunyavirus infections a simple method of estimating fifty percent endpoints recognition of virus infection and innate host responses to viral gene therapy vectors human macrophages, but not dendritic cells, are activated and produce alpha/beta interferons in response to mopeia virus infection differential inhibition of type i interferon induction by arenavirus nucleoproteins short double-stranded rnas with an overhanging ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys z proteins of new world arenaviruses bind rig-i and interfere with type i interferon induction inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus early and strong immune responses are associated with control of viral replication and recovery in lassa virus-infected cynomolgus monkeys type interferons and the virus-host relationship: a lesson in detente singledose intranasal administration with mdef (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal sars-cov balb/c mouse model alpha interferon as an adenovirus-vectored vaccine adjuvant and antiviral in venezuelan equine encephalitis virus infection comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d key: cord- -uvf qzfd authors: kenworthy, rachael; lambert, diana; yang, feng; wang, nan; chen, zihong; zhu, haizhen; zhu, fanxiu; liu, chen; li, kui; tang, hengli title: short-hairpin rnas delivered by lentiviral vector transduction trigger rig-i-mediated ifn activation date: - - journal: nucleic acids res doi: . /nar/gkp sha: doc_id: cord_uid: uvf qzfd activation of the type i interferon (ifn) pathway by small interfering rna (sirna) is a major contributor to the off-target effects of rna interference in mammalian cells. while ifn induction complicates gene function studies, immunostimulation by sirnas may be beneficial in certain therapeutic settings. various forms of sirna, meeting different compositional and structural requirements, have been reported to trigger ifn activation. the consensus is that intracellularly expressed short-hairpin rnas (shrnas) are less prone to ifn activation because they are not detected by the cell-surface receptors. in particular, lentiviral vector-mediated transduction of shrnas has been reported to avoid ifn response. here we identify a shrna that potently activates the ifn pathway in human cells in a sequence- and ′-triphosphate-dependent manner. in addition to suppressing its intended mrna target, expression of the shrna results in dimerization of interferon regulatory factor- , activation of ifn promoters and secretion of biologically active ifns into the extracellular medium. delivery by lentiviral vector transduction did not avoid ifn activation by this and another, unrelated shrna. we also demonstrated that retinoic-acid-inducible gene i, and not melanoma differentiation associated gene or toll-like receptor , is the cytoplasmic sensor for intracellularly expressed shrnas that trigger ifn activation. a specific double-stranded rna (dsrna) structure, $ - bp dsrna with overhangs, plays a critical role in initiating both microrna (mirna)-and small interfering rna (sirna)-mediated gene silencing, as it is the structure recognized by the rna interference (rnai) machinery, the rna-induced silencing complex (risc) ( ) ( ) ( ) . except for preformed sirna duplexes of $ bp, the risc-loaded small rnas are generated by a ribonuclease (rnase) iii-like enzyme that is found in virtually all eukaryotic organisms. this enzyme, aptly named dicer for its ability to cleave a variety of larger (> bp) dsrna molecules into the $ bp dsrna with a characteristic overhang of nt, is a multidomain rna-binding protein and itself a component of risc. the primary sequence of the rnas is not important in risc formation, and rnai can suppress virtually any target as long as rules of sequence complementarities between the small rna and the target rna are satisfied. dsrnas are also a type of pathogen-associated molecular pattern (pamp) that are detected by cellular innate immunity sensors named pattern recognition receptors (prrs) ( ) . the interaction between a pamp and a prr triggers activation of the interferon (ifn) pathway in mammalian cells, which significantly changes the gene-expression profile in the cells and contributes to the well-documented off-target effect of rnai. ifn induction is especially problematic in antiviral studies employing rnai, where the antiviral effect of ifn must be distinguished from that of rnai. typical ifn-inducing structure patterns include dsrna of certain length, single-stranded rna (ssrna) containing -triphosphates ( -ppp), the dsrna analogue polyinosinic-polycytidylic acid (poly i:c), and certain dsdna molecules. these rna patterns are generally believed to possess 'non-self' properties to allow the cell to recognize foreign (often viral) rnas specifically. various forms of sirna duplexes have been reported to trigger ifn induction both in vitro and in vivo ( ) ( ) ( ) ( ) ( ) , probably through the cell surface-and/or endosomeexpressed toll-like receptors (tlrs), including tlr and tlr ( , , ) . short-hairpin rnas (shrnas) expressed from a dna plasmid have also been shown to activate ifn ( ) . the double-stranded form of these rnas is below the size limit of the stem-loop rnas that can be detected by the rna-activated protein kinase (pkr) ( ) and is probably detected by other cytoplasmic prrs. two cytoplasmic rna helicases, retinoic-acid-inducible gene i (rig-i) and melanoma differentiation associated gene (mda ), signal to the ifn-b promoter when activated by specific rna structures ( ) ( ) ( ) . although both prrs signal through the mitochondrial antiviral signaling protein mavs/cardif/visa/ips- ( ) ( ) ( ) ( ) , studies of ligand specificity suggest that rig-i and mda are parallel sensors with overlapping substrates. for example, although both prrs are activated by poly i:c in cell culture systems ( , ( ) ( ) ( ) ( ) ( ) , mda appears to be more important in mediating the poly i:c response in vivo ( , ) . in addition, rig-i can bind and respond to ssrnas bearing -ppp, whereas mda is not activated by -ppp-containing rna ( , ) . finally, several cytosolic sensors for dsdna has been recently reported ( ) ( ) ( ) ( ) ( ) ( ) . nevertheless, current data on what constitutes effective substrates for either prr are incomplete and sometimes controversial. here we report for the first time that shrnas delivered by lentiviral transduction triggered ifn activation and that rig-i and mavs, but not mda or tlr , mediated the ifn activation triggered by intracellularly expressed shrna, which could activate both ifn-a and ifn-b promoters. ifn activation depended on sequence, a -ppp and correct processing of the rna hairpin by dicer; it was independent of promoter choice, presence of blunt ends, route of delivery and rnai potency. gs and lh cells have been described earlier ( , ) . huh- and ft cells were maintained in dmem supplemented with % fbs. we used the following antibodies: anti-cypa (biomol, plymouth meeting, pa, usa); anti-cypb (afenity bioreagents, rockford, il, usa); anti-ku , anti-flag and anti-actin (sigma-aldrich, st louis, mo, usa); anti-ifn stimulate gene (isg) (rockland immunochemicals, gilbertsville, pa, usa); anti-ns a (virogen, watertown, ma, usa) and anti-ns (in-house). gsb and h cells have been described earlier ( ) . poly i : c was purchased from sigma-aldrich, and synthetic hairpin rna was purchased from integrated dna technologies (coralville, ia, usa). synthetic sirna was purchased from ambion (austin, tx, usa). protein contents of cell lysate were quantified with the bio-rad dc protein assay (bio-rad, hercules, ca, usa), and an equal amount of total protein was loaded in each lane. samples for irf- dimerization assay were run on a polyacrylamide gel under non-denaturing conditions ( ) . other samples were denatured and separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (sds-page). proteins were then transferred onto a nitrocellulose membrane and stained with the appropriate antibodies with the snap i.d. tm system (millipore, worcester, ma, usa) according to the manufacturer's instructions. for luciferase assays, cells were seeded to a confluency of %, and for all other assays, cells were seeded to a confluency of %. the next day, transfections of dna plasmids and synthetic rnas were performed with lipofectamine tm (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. plasmids pgl -ifna , pgl -ifnb, prl-tk, pcmv-flag-irf- and pcr . -irf- a have been described earlier ( ) . shrnas were expressed from a human immunodeeciency virus (hiv)-based lentiviral vector ( , ) , and sh-pcaf was constructed on the basis of a previously reported sequence ( ) . plasmid sh-b /h was constructed by cloning of the dna fragment encoding the sh-b rna into psilencer . -h (ambion, austin, tx, usa) according to the manufacturer's instructions. the rig-i and tlr constructs have been described ( , ) . the rig-i c construct encodes flag-tagged, c-terminal aa of human rig-i cloned into a bicistronic expression vector modified from pbicep-cmv- (sigma-aldrich, st louis, mo, usa), in which the cmv promoter was replaced with the elongation-factor- promoter. the mda , mda -c constructs were kindly provided by fujita ( ) . hcv genotype a ns - a protease was expressed from the pcmv- tag- a plasmid (stratagene, la jolla, ca, usa). ft cells were seeded in -well plates and were transfected h later with ng of a shrna expression vector, ng of pgl -ifna or pgl -ifnb, ng of prl-tk and ng of pcr . -irf- a. cells were collected h after transfection. luciferase assays were performed with the dual-glo Õ luciferase assay system reagents (promega, madison, wi) and luminescence quantified with a modulus microplate reader (turner biosystems, sunnyvale, ca, usa). ratios of firefly luciferase (from the pgl vectors) to renilla luciferase (from the prl-tk vector) were calculated, and that of the sh-b sample was normalized to %. sequences of shrna are shown in table . lentiviral vector production and transduction were performed as described earlier ( ) . viral vectors were pelleted by ultracentrifugation at g at c for h and resuspended in a volume of pbs that was % of the original medium volume. the titers of the concentrated vectors were then measured with a p elisa kit (zeptometrix, buffalo, ny, usa). real-time reverse transcription pcr (rt-pcr) was performed as described earlier ( ) . the primers used were oas forward, -agg tgg taa agg gtg gct cc- and oas reverse -aca acc agg tca gcg tca gat- ; rig-i forward -gag gca gag gaa gag caa gag g- and rig-i reverse -cgc ctt cag aca tgg gac gaa g- ; gapdh forward -tca ctg cca ccc aga aga ctg- and gapdh reverse -gga tga cct tgc cca cag c- . the primers for hcv detection were -cgc tca atg cct gga gat ttg- and -gca ctc gca agc acc cta tc- . for flow cytometry, gs cells were fixed h after treatment in a solution of % paraformaldehyde and analyzed with a facscanto flow cytometer (bd biosciences, san jose, ca, usa). mean gfp intensity was plotted, and that of the sh-ntc sample was normalized to %. total rna from transiently transfected ft cells was extracted with rna stat- (tel-test, friendswood, tx, usa) and separated on a . % urea polyacrylamide gel. the transfer of rna onto nitrocellulose membrane and hybridization were performed according to standard molecular biology protocols. the probe for detecting the expression of sh-b and its variants was a synthetic dna oligomer corresponding to the bottom strand of sh-b . radioactive labeling of the probe was performed with an end-labeling protocol with t polynucleotide kinase (ambion, austin, tx, usa). the exposure and detection of the radioactive signal was performed with a typhoon imager (ge healthcare, piscataway, nj, usa) with quantity one software (bio-rad, hercules, ca, usa). a short-hairpin rna directed at cypb induces ifn production in human embryonic kidney cells to investigate the potential role of the cyclophilins (cyps) in hcv replication ( ), we delivered several shrnas directed at mrnas of three cyps into hcv replicon cells by means of a lentiviral vector, using a murine u promoter to drive the expression of the shrna ( figure a ) ( ) . we observed a discrepancy between two anti-cypb shrnas (b and b ) in their relative efficiency in knocking down cypb expression and in suppressing hcv. lentiviral vector sh-b was less efficient in knocking down cypb expression but potently inhibited hcv ns a expression in a human hepatoma cell line containing replicating hcv rna ( figure b , left). viral inhibition was independent of cypb knockdown, as control medium from transfected ft cells that did not contain any lentiviral vector particles, generated by omission of the packaging plasmids during transfection, also inhibited hcv replication ( figure b , right) without affecting cypb expression. the fast kinetics of viral inhibition (complete inhibition with h, data not shown) was also more consistent with ifn than with rnai-based inhibition. the presence of ifn in the lentiviral vector preparation of sh-b was confirmed by strong induction of - -oligoadenylate synthetase (oas ), a classic ifn-induced gene, in both naı¨ve huh- and the hcv replicon cell line (gs ) treated with the medium ( figure c ). in addition, hcv replication in an ifn-resistant hcv replicon cell line (h ), in contrast to that in a wildtype replicon cell line (gsb ) ( ), was not inhibited by the sh-b medium ( figure d ), suggesting the lack of additional viral inhibiting agents in the sh-b medium. expression of sh-b in ft cells also induced dimerization of irf- , confirming the activation of the ifn production pathway in these transfected cells ( figure e ). finally, sh-b was able to activate both ifn-a and ifn-b promoters, although the activation of the ifn-a promoter required coexpression of irf- , which is normally expressed at very low levels in -based cells ( figure f ). these results demonstrate that sh-b is a potent activator of irf- and irf- , master regulators of ifn expression in human cells. we next investigated the role of the different viral/ exogenous rna sensors, rig-i, mda and tlr , in sh-b -triggered ifn production. mammalian expression plasmids encoding each of these proteins, as well as the dominant negative (dn) mutants of rig-i and mda , were transfected into ft cells with shrnas and an ifn-b promoter reporter construct. the signaling to ifn-b promoter and the expression of the prr proteins were then examined h after transfection. in the absence of sensor proteins, the sh-b increased activation of the ifn-b promoter by . -fold ( figure a ). coexpression of mda or tlr did not increase or decrease sh-b 's ability to activate ifn-b promoter relatively to the negative control shrna (sh-ntc), but in the presence of rig-i coexpression, the induction of ifn-b promoter by sh-b was increased to $ -fold. moreover, ectopic expression of a dn mutant of rig-i (rig-i c), but not that of mda (mda -c), completely abrogated ifn promoter activation by sh-b . with the exception of tlr , which required prolonged exposure of the western blot to be detected, the cytoplasmic sensors and their mutants were expressed at comparable levels ( figure b ). moreover, activation of irf- ( figure e ) and ifn promoters ( figure f ) in ft cells, which do not contain a functional tlr signaling pathway ( ) , indicates that tlr plays a negligible role, if any, in ifn induction by sh-b . the combination of sh-b and rig-i produced the highest level of ifn-b promoter activity, which were confirmed by western blotting showing that endogenous isg induction was only detectable in cells cotransfected with sh-b and wild-type rig-i ( figure b ). to confirm further that biologically active ifn was released from these cells, we applied the culture medium of the transfected ft cells to an hcv replicon cell line (gs ) in which ns a-gfp expression is used for monitoring viral rna replication ( ) . hcv replication in this cell line is extremely sensitive to ifn, and the effect of the cytokine can be readily measured as the change in the mean gfp intensity of the treated cells. as shown in figure c , culture medium from sh-b efficiently suppressed hcv replication, resulting in a decrease in figure . a small-hairpin rna directed at cypb induces ifn production in human embryonic kidney cells. (a) sequence of sh-b , which was expressed from a self-inactivating human immunodeficiency virus (hiv) vector with a murine u promoter ( ) . (b) inhibition of hcv expression by culture media of sh-b -transfected ft cells. gs cells were treated with culture supernatant taken from ft cells transfected with various shrna plasmids with (left) or without (right) the packaging plasmids overnight. cells were then cultured in fresh media for an additional days before being lysed for western blotting. (c) oas induction by culture supernatant from ft cells transfected with sh-b . huh and gs cells were treated with culture supernatant from ft cells transfected with either sh-luc or sh-b for h before rna extraction and real-time rt-pcr analysis. oas rna level was normalized to that of gapdh rna. (d) transfected culture media failed to suppress hcv replication in an ifn-resistant cell line. hcv replicon cells were cultured as described earlier ( ) and then treated with the indicated culture medium from transfected ft cells. hcv rna was analyzed with real-time rt-pcr. (e) irf- dimerization in response to sh-b expression. flag-irf- was cotransfected with a shrna into ft cells. cells were lysed h after transfection, and total cell lysate was separated on a polyacrylamide gel under non-denaturing conditions, transferred and stained with an anti-flag antibody. (f) ifn-a and ifn-b promoter activation by sh-b expression. sh-ntc, sh-c (an shrna directed at cypc), or sh-b was cotransfected along with luciferase reporter plasmids with or without irf- . the ratios of firefly luciferase readings to renilla luciferase readings were plotted. the ns a-gfp intensity within h of treatment. cotransfecting wild-type rig-i produced a medium with stronger inhibition, whereas the rig-c drastically suppressed the antiviral effect of the medium. finally, real-time rt-pcr analysis revealed that sh-b , but not the negative control shrna, strongly activated expression of endogenous rig-i, a well-characterized isg whose induction requires paracrine/autocrine action of ifn ( , ) . as expected, poly i : c activated rig-i expression in the same assay ( figure d ). these results, taken together, show that rig-i is the cellular sensor that mediates the ifn induction by sh-b . the majority of the shrnas that we use in the lab do not activate rig-i expression and ifn signaling despite having essentially the same structure as sh-b , so we wanted to determine whether the sequence of sh-b is distinctive enough to trigger the production of ifn. we first tested a synthetic sirna duplex with the same target sequence as sh-b . this sirna (si-b -syn) should resemble the final dicer product of sh-b except for the -ends. the synthetic sirna contains -oh groups, whereas the dicer products probably figure a ) while failing to activate ifn production, as measured by the gfp-hcv assay ( figure b ). to determine whether the sequence of the intact hairpin rna before dicer cleavage is sufficient to trigger ifn, we tested a synthetic shrna (sh-b -syn) that had exactly the same sequence as the predicted intracellular sh-b transcript generated by the u promoter. again, the -end of the synthetic sh-b had a -oh group instead of any phosphate. sh-b -syn behaved similarly to si-b -syn in that it knocked down cypb expression without activating ifn response (figure ) . these results suggest that the -end status of sh-b is important for ifn activation, consistent with the previously finding that a -triphosphate is required for rig-i activation ( , ) . to determine the contribution of the individual residues of the sh-b sequence, we introduced a series of point mutations into the shrna and tested them for ifn induction. we changed the first nucleotide from a to g, c, or t while maintaining base-pairing between nucleotides + and + . these mutant shrnas lacked the ability to activate ifn production (table ) . changing the + nucleotide to g while leaving the + nucleotide intact also abolished ifn activation by the shrna (a /g), as did the reciprocal mutation u /c. the importance of the first nucleotide was further confirmed by the inability of sh-b + to activate ifn. the target of sh-b + was shifted nt downstream on the cypb mrna, producing an shrna starting with a g at the + position. the presence of an a at the + position was not, however, sufficient to render a shrna competent for ifn activation, as replacing the first nucleotide of the sh-ntc with an a did not generate an ifn-inducing shrna (ntc-a and ntc+ ). these results indicate that a protruding/unpaired a at the end of the hairpin or the rna duplex, a potential result of 'breathing' at the end of the dsrna, is not sufficient to trigger ifn induction as previously suggested ( ) . two point mutations located farther into the stem structure of the shrna ( g and b a ) also reduced its ability to induce ifn even though the base-pairing was perfectly maintained in these mutants. finally, replacing the -nt hairpin loop with a -nt loop that had been previously shown to abolish shrna-mediated rnai (loop a mutant) ( ) eliminated sh-b 's ability to induce ifn, suggesting the importance of rna processing in the induction. to determine whether the inability of the mutant shrnas to induce ifn was due to lower expression levels, we performed northern blotting analysis of the shrna expression on the wild-type and two mutants. the mutants a /g and loop a were chosen because their final sirna products have exactly the same sequence as that of the wild-type sh-b and can thus be detected with the same efficiency by the same probe. although sh-a/g and sh-loop a were clearly unable to activate ifn-b promoter ( figure a ), they were both expressed at levels comparable to those of the wild-type sh-b product ( figure b) . interestingly, the final sirna product of sh-loop a was slightly smaller than those of sh-b and sh-a /g, suggesting that cleavage did occur and perhaps occurred one or nt into the stem to compensate for the shorter loop. blunt-ended sirna has been previously reported to be stronger inducers of ifn than the sirnas with overhangs ( ) . indeed, a previously reported ifn-inducing shrna, sh-pcaf (p /creb-binding protein-associated factor), contains a blunt end ( ) and was more potent in activating ifn than sh-b ( figure a ), which is predicted to form an overhang of - ts at each end of the final sirna. we therefore constructed a version of the sh-b that would be blunt at the end that is not processed by dicer by adding two extra as to the -end of the shrna. this modification (blunt sh-b ) did not increase the ability of sh-b to activate ifn-b promoter ( figure a ). we confirmed, in two independent experiments, that ifn induction by sh-pcaf was also mediated by rig-i. first, cotransfection of dn rig-i resulted a -to -fold inhibition of ifn induction by sh-pcaf ( figure b ), whereas wild-type rig-i increased ifn induction by several fold in the same assay. second, when hcv ns - a protease, which cleaves mavs, thereby blocking the rig-i pathway, was coexpressed with either sh-b or sh-pcaf, ifn induction by these shrnas were severely compromised ( figure c ), further substantiating a role of the rig-i and mavs pathway in mediating ifn induction by both the blunt-ended sh-pcaf and the sh-b with overhang. the proper expression of ns - a protease was confirmed by western blotting ( figure d ). to assess the contribution of the promoter choice in ifn activation by intracellular expressed shrna, we expressed sh-b from another commonly used pol iii promoter, the human h promoter. both the original, mu -driven sh-b and the h -driven sh-b activated ifn-b promoter ( figure a ) and resulted in secretion of ifn into the transfected cell-culture media, which in turn suppressed hcv replication ( figure b ). proper expression of the sirna ( figure c ) and the subsequent knockdown of cypb expression ( figure d ) all appeared normal for sh-b expressed from the h promoter plasmid, which has a backbone different from that of our lentiviral vector carrying the mu promoter. these data suggest that ifn induction by sh-b is not restricted to a particular promoter or expression construct. further supporting this conclusion was the observation that the expression cassette by itself, removed and isolated from the lentiviral plasmid by restriction digestion, could also activate ifn production in transfected ft cells (data not shown). to this point, all the ifn induction experiments were done with transient transfection of dna vectors and it was possible that certain features of the double-stranded plasmid dna are responsible for ifn induction. we first tried to address this point by transfecting just the shrnaexpressing cassette, generated either by pcr or restriction enzyme digestion, into ft cells and confirming that these fragments of $ bp were sufficient to trigger ifn induction (supplementary figure s ) . to definitively rule out any contribution by dsdna, we used a lentiviral transduction system which has been suggested to express shrnas that can escape detection by prrs and ifn activation ( ) . we produced lentiviral particles containing shrnas from ft cells using standard . sh-b expressed from an h promoter triggers ifn activation. sh-b expressed from an h promoter was capable of (a) activating ifn-b promoter and (b) triggering ifn production to inhibit hcv replication in gs cells. (c) intracellular levels of u -and h -driven sh-b products. rna extraction and northern blotting were performed as described in figure b . (d) knockdown of cypb expression by sh-b expressed from an h promoter. methods, centrifuged them to separate the vectors from the ifn-containing media, and then used them to infect naı¨ve ft cells ( figure a ). both sh-b and sh-pcaf vectors induced ifn production when delivered as concentrated lentiviral particles, measured both by hcv suppression ( figure b ) and by oas induction ( figure c ) in huh- cells. to rule out the possibility that residual ifn in the concentrated viral particles was responsible for these results, we added u/ml ifn to the negative control vector sample before the concentration step. this preparation, designated sh-ntc*, was not able to trigger ifn production in naı¨ve ft cells, suggesting that the concentration step effectively removed the soluble ifn from the viral particle pellet. proper knockdown of the sirna target of sh-b was confirmed by this route of shrna delivery ( figure d ). to prove definitively that ifn induction by the shrnas was mediated by the lentiviral infection route, we tested the effect of an inhibitor of hiv reverse transcriptase, nevirapine, on ifn induction by sh-b and sh-pcaf. as shown in figure e , inclusion of nevirapine at the time of transduction effectively blocked the ability of both shrnas to induce ifn in the transduced cells, suggesting the importance of the reverse transcription step in the expression of the shrnas delivered by the lentiviruses. to determine whether lentiviral vector-delivered shrna can trigger ifn induction in cells other than ft cells, we transduced a human hepatoma cell line, lh , which has been reported to produce ifn upon viral infection ( ) , and examined ifn induction in these cells. culture medium from lh cells transduced with sh-pcaf contained biologically active ifn, which suppressed hcv replication in gs cells ( figure f ), indicating that the ability of shrnas delivered by lentivirus to induce ifn response was not limited to ft cells. it has been reported that certain chemically synthesized and phage polymerase in vitro transcribed sirnas can non-specifically induce ifn responses and produce offtarget effect via various prrs, including tlrs. however, the induction of ifn response by shrnas and its underlying mechanisms have not been as well studied. the actual number of shrnas that are capable of triggering ifn response will certainly be larger than the few that have been reported in the literature, yet very little is known about the unique characteristics of the select shrnas and the pathway that they use to activate ifn production. the present study identifies rig-i, but not mda or tlr , as the mediator for activation of ifn responses by two shrnas that are distinct in sequence and structure but both capable of ifn induction in human cells. this was demonstrated by induction of irf- dimerization, activation of ifn promoters, induction of endogenous isgs (isg , oas and rig-i), and secretion of ifn, all of which depended on rig-i and its downstream adaptor, mavs. in addition, we show that delivery of these shrnas via lentiviral transduction does not reduce their ifn-inducing capacity, indicating that the ability of lentiviral vector transduction to avoid ifn induction by shrnas, as reported previously ( ), may not be universally applicable to all the shrnas. specific recognition of dsrnas or ssrnas bearing -triphosphates by rig-i is presumably determined mostly by structural features other than the nucleotide sequence of the rna. yet ifn activation by sh-b exhibited a stringent dependence on specific nucleotides at multiple positions of the shrna. an aa dinucleotide at the beginning of the u transcript has previously been suggested to result in aberrant transcription, and preserving a c/g sequence at positions À /+ suggested to avert ifn induction ( ) . we indeed observed a strict requirement for an adenylate at the + position of sh-b for rig-i recognition and ifn activation, but we observed no difference in expression levels or the apparent sizes of the sh-b rnas bearing either an a or a g at the + position. furthermore, mutations introduced elsewhere in the shrna also abolished or diminished sh-b 's ability to activate ifn, suggesting additional sequence requirement for efficient rig-i recognition and ifn triggering. despite these results, because we were not successfully in cloning and sequencing the vectorexpressed sirna, we cannot exclude the possibility that the adenylate at the + position interferes with transcription and that the resultant abnormal transcript contributes to ifn induction. interestingly, the loop a mutant, which contains a predicted loop of nt, generated a sirna duplex inside the cells that is slightly smaller than that of the shrnas with a wild-type hairpin loop, suggesting the processing by dicer into the stem, perhaps fulfilling the requirement of a length of nt for the hairpin loop ( ) . this mutant form of sh-b was not, however, able to trigger ifn activation. despite the abilities of both sh-b and sh-pcaf to activate the rig-i pathway, the two shrnas are unrelated in sequence. two short stretches of sirna sequences, guccuuccaa and ugugu, that have been previously defined as ifn-or cytokine-activating motifs ( , ) are not found in either sh-b or sh-pcaf. any common sequence motifs of ifn-activating shrnas, if any, remain to be defined. the two shrnas also differ in that one is predicted to contain one blunt end and the other two ends with overhangs. these results suggest that, although blunt ends may increase sirna's ability to be recognized by rig-i ( ), they are not required for ifn activation by an endogenously expressed shrna. the best-characterized rna structure motif recognized by rig-i is the -ppp, which is absent from virtually all the cellular rnas as a result of either -capping or internal cleavage before their appearance in the cytoplasm. a synthetic shrna that has the same sequence as sh-b but lacks the -ppp failed to induce ifn, suggesting the -end status of the intracellularly expressed sh-b contributes to ifn activation. whether or not the -end of an shrna is capped has not been investigated. murine u rna does not contain the trimethylguanosine cap that is present on mrnas and other u small nuclear rnas; instead it contains a g-monomethyl phosphate cap at its -end ( ) . capping of heterologous transcripts produced from the mu promoter, however, requires a stem loop at the -end of the transcript and an auauac sequence immediately after ( ) . most shrnas, including sh-b and sh-pcaf, would not meet these requirements and thus should contain unmodified -ppp. similarly, no evidence of a cap structure for h transcripts could be found in the literature. we attempted to express sh-b using a mirna expression cassette and the pol ii promoter ( ) . the primary transcript generated with this construct would be capped at -end by a trimethylguanosine cap and the final sirna duplex would bear a monophosphate at the -ends of both strands because of drosha and dicer cleavage. this version of the sh-b vector was much weaker in its ability to trigger ifn activation. unfortunately the intracellular expression of the rna duplex was also much weaker and barely detectable by northern blotting. in addition, no knockdown of the target cypb mrna was seen with this mirna-based sh-b (data not shown). as a result, whether sh-b , if expressed at higher level from this construct, could effectively activate ifn remains unclear. so far as we know, ours is the first report of ifn activation in the target cells by shrnas delivered by lentiviral transduction. a previous report of ifn induction by lentiviral vector-expressed shrna only examined the ifn generated in the vector-producing cells, which then up-regulated ifn-stimulated genes in the transduced cells ( ) . the distinction is important as lentiviral vectors used in a gene-therapy setting will likely be purified and free of any ifn that has been generated during the vector preparation step, but ifn activation in the target cells would pose a more serious concern. our data suggest the importance of screening shrnas for ifn induction in the transduced cells in vitro before largescale studies. an hiv reverse transcriptase inhibitor efficiently blocked ifn production by both sh-b and sh-pcaf when delivered by transduction, indicating the virion-encapsulated rna was not able to trigger ifn activation. in this respect, it is interesting to note that positive-stranded rna viruses, which produce dsrna intermediates in the cytoplasm during replication ( ) ( ) ( ) ( ) , often replicate in membrane enclosed vesicles ( ) , this sequestration of viral dsrna in membranous structures may shield the rna from the cytoplasmic prrs and contribute to a successful infection. ifn-induction and rnai by shrnas appear to be independent functions of the same rna ( ). our results also showed that ifn-induction by sh-b is independent of its ability to suppress target mrna expression through rnai. on the other hand, it might be possible to screen for duel functional sirnas that confer therapeutic benefits by both rnai and immunostimulation ( ) . for example, sirnas that target either viral genomes or cellular cofactors of the viruses can be screened for their ability to trigger ifn activation in hopes of find 'super sirnas' with increased efficacy against ifn-sensitive viruses. an rna-directed nuclease mediates post-transcriptional gene silencing in drosophila cells single-stranded antisense sirnas guide target rna cleavage in rnai human risc couples microrna biogenesis and posttranscriptional gene silencing pathogen recognition and innate immunity activation of the interferon system by short-interfering rnas small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor interferon induction by sirnas and ssrnas synthesized by phage polymerase sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna induction of an interferon response by rnai vectors in mammalian cells -triphosphate-dependent activation of pkr by rnas with short stem-loops the rna helicase rig-i has an essential function in doublestranded rna-induced innate antiviral responses differential roles of mda and rig-i helicases in the recognition of rna viruses essential role of mda- in type i ifn responses to polyriboinosinic : polyribocytidylic acid and encephalomyocarditis picornavirus ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf visa is an adapter protein required for virus-triggered ifn-beta signaling the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity polyinosinicpolycytidylic acid induces the expression of gro-alpha in beas- b cells ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling expression of ip- /cxcl is upregulated by double-stranded rna in beas- b bronchial epithelial cells rig-i-mediated antiviral responses to single-stranded rna bearing -phosphates aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc aim activates the inflammasome and cell death in response to cytoplasmic dna an orthogonal proteomic-genomic screen identifies aim as a cytoplasmic dna sensor for the inflammasome hin- proteins regulate caspase activation in response to foreign cytoplasmic dna dai (dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway characterization of hepatitis c virus subgenomic replicon resistance to cyclosporine in vitro hepatitis c virus triggers apoptosis of a newly developed hepatoma cell line through antiviral defense system defective jak-stat activation in hepatoma cells is associated with hepatitis c viral ifn-alpha resistance induction of irf- /- kinase and nf-kappab in response to double-stranded rna and virus infection: common and unique pathways a kaposi's sarcoma-associated herpesviral protein inhibits virus-mediated induction of type i interferon by blocking irf- phosphorylation and nuclear accumulation identification of cellular cofactors for human immunodeficiency virus replication via a ribozyme-based genomics approach determinants of interferonstimulated gene induction by rnai vectors gb virus b disrupts rig-i signaling by ns / a-mediated cleavage of the adaptor protein mavs distinct poly(i-c) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro recognition of double-stranded rna and activation of nf-kappab by toll-like receptor effect of cell growth on hepatitis c virus (hcv) replication and a mechanism of cell confluencebased inhibition of hcv rna and protein expression antitumor nk activation induced by the toll-like receptor -ticam- (trif) pathway in myeloid dendritic cells central role of interferon regulatory factor- (irf- ) in controlling retinoic acid inducible gene-i (rig-i) expression a system for stable expression of short interfering rnas in mammalian cells a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells stable expression of shrnas in human cd + progenitor cells can avoid induction of interferon responses to sirnas in vitro gamma-monomethyl phosphate: a cap structure in spliceosomal u small nuclear rna capping of mammalian u small nuclear rna in vitro is directed by a conserved stem-loop and auauac sequence: conversion of a noncapped rna into a capped rna a lentiviral microrna-based system for single-copy polymerase ii-regulated rna interference in mammalian cells visualization of double-stranded rna in cells supporting hepatitis c virus rna replication double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses subcellular localization and membrane topology of the dengue virus type non-structural protein b sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum seeking membranes: positive-strand rna virus replication complexes sirna and isrna: two edges of one sword -triphosphate-sirna: turning gene silencing and rig-i activation against melanoma design of hiv vectors for efficient gene delivery into human hematopoietic cells the authors thank dr andre irsigler and dr jason robotham for technical assistance and dr anne b. thistle for proofreading the manuscript. supplementary data are available at nar online.conflict of interest statement. none declared. key: cord- -vzbh k authors: zhang, weijun; lin, yan; bai, yu; tong, tiegang; wang, qun; liu, nihong; liu, guangliang; xiao, yihong; yang, tao; bu, zhigao; tong, guangzhi; wu, donglai title: identification of cd (+ )cytotoxic t lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in balb/c mice date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: vzbh k twenty-seven nanopeptides derived from the matrix (m) protein of porcine reproductive and respiratory syndrome virus (prrsv) were screened for their ability to elicit a recall interferon-γ (ifn-γ) response from the splenocytes of balb/c mice following dna vaccination and a booster vaccination with recombinant vaccinia virus rwr-prrsv-m. we identified two peptides (amino acid residues k( )fitsrcrl and f( )gymtfvhf) as cd (+ )cytotoxic t lymphocyte (ctl) epitopes. these peptides elicited significant numbers of ifn-γ secreting cells, compared with other m nonapeptides and one irrelevant nonapeptide. bioinformatics analysis showed that the former is an h- k(d)-restricted ctl epitope, and the latter is an h- d(d)-restricted ctl epitope. multiple amino acid sequence alignment among different prrsv m sequences submitted to genbank indicated that these two ctl epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to prrsv. porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine viral pathogens, and has caused significant economic losses to the swine industry worldwide. characterization of field isolates suggested that prrsv are genetically diverse, and this genetic variation increases the difficulty of developing effective vaccines. based on significant sequence differencesprrs viruses are grouped into two distinct genotypes, european isolate (lelystad virus, lv) and north american isolate (vr- ) [ ] prrsv has two major structural proteins, gp and m, encoded by orfs and , respectively. gp , the most important neutralizing antigen of prrsv, has the highest genetic diversity among isolates [ ] . and, recent studies in yorkshire × landrace crossed and outbred pigs, showed that there are two immuno-dominant t-cell epitopes derived from the gp protein: l aalicfvirlaknc and k grlyrwrspvii/vek [ ] . the m protein, which contains highly conserved amino acid sequences, also has very good immunogenicity and is associated with protection against prrsv infection. dna vaccinations have also revealed that m is the most potent inducer of t lymphocyte proliferation [ ] . at present, effective vaccination strategies for the prevention and control of prrsv infection are not available. vaccines based on inactivated prrsv virus have been ineffective at inducing protective immune responses. live-attenuated prrsv vaccines can provide protection against this pathogen, but have been observed to revert to virulence [ ] , restricting the application of this vaccination approach. the rational development of future prrsv vaccines will necessitate a systematic understanding of the protective humoral and cellular immune responses that occur during prrsv infection, and should aim to induce a broad immune responses that accommodates the plasticity of the major antigenic sites. recent research has indicated that cell-mediated immunity may play a very important role in the clearance of prrsv [ ] . major histocompatibility complex (mhc) i restricted cytotoxic t lymphocytes (ctls) can kill virus-infected cells and eliminate potential sources of new virus [ ] . hence, identification of ctl epitopes is crucial in the design of synthetic vaccines, and a number of studies have successfully identified pathogen-derived t cell epitopes [ ] [ ] [ ] [ ] . unlike many other pathogens, there is limited knowledge of the specific prrsv-derived peptides targeted by t-cells. in the current study, we report the identification of ctl epitopes from the prrsv (ch- a strain) m protein in a mouse model. we screened peptides derived from the prrsv m protein for their ability to induce interferon (ifn)-γ in splenocytes harvested from balb/ c mice following dna vaccination and a booster vaccination with recombinant vaccinia virus expressing m protein. the screen identified two peptides that elicited ifn-γ production in cd + cd + splenocytes of vaccinated mice. a multiple amino acid sequence alignment among different prrsv m proteins indicates that these two peptides are strongly conserved across multiple prrsv strains and therefore should be considered for further research. the prrsv ch- a strain, the vaccinia virus wr strain, and the akabane virus obe- strain were part of our laboratory collection. the former was propagated in marc- cells, and the latter two were propagated in bhk- cells, respectively, and these two cell lines were cultured in dmem (invitrogen) supplemented with % of fetal calf serum (fcs, invitrogen) in a humidified °c, % co incubator. total rna was extracted from the prrsv ch- a strain and the spleens of balb/c mice. full length cdnas were amplified based on the complete open reading frames (orfs) of m and ubiquitin (ub) following reverse transcription (genbank accession numbers ay and az ) using the m-f and m-r primers pair or the ub-f and ub-r primer pair ( table ) . the full length cdnas were cloned into the pmd -t vector (takara) and are referred to as pmd-m and pmd-u, respectively. an indirect enzyme-linked immunosorbent assay (ielisa) method was established to evaluate specific antibody against m protein. a truncated m protein ( - aa), which was used as the coating antigen, was expressed and purified using a prokaryotic expression system. the truncated m gene cdna was amplified from pmd-m using the truncated-m-f and truncated-m-r primers (table ) , ligated into pgex- p- (ge healthcare), and subsequently named pgex-m. for protein expression, pgex-m was transformed into e.coli bl (de ) and recombinant protein expression was induced with . mm iptg. the samples were harvested by centrifugation and the pellets were resuspended with phosphate buffered saline (pbs, ph . ). after being analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and western blot with anti-prrsv-m monoclonal antibody (our laboratory collection), the fusion-expressed truncated m protein was purified by gst tag according to the manufacturer's instructions (ge healthcare). finally, the concentration of the purified protein was determined using a bradford kit (bio-rad) according to the manufacturer's instructions. the ub and m genes were fused using splicing by overlapping extension pcr (soe-pcr). the ub and m genes were amplified from pmd-u and pmd-m with the primer pairs ub-f and fusion-m-ub-r and fusion-m-ub-f and m-r, respectively ( table ). the fusion gene product, referred to as ub-m, was amplified from the purified pcr products with ub-f and m-r and inserted into the eukaryocyte expression vector pvax , and was named pvax -ub-m. the transferring vector psc (our laboratory collection), which is composed of the early promoter p . and late promoter p of vaccinia virus, and the lacz and amp genes controlled by the promoter p as the reporter genes, was used in this study. to construct the transferring vector psc -m, the complete m gene was amplified with the psc -m-f and psc -m-r primer pair (table ) and inserted into the psc . the primer p . -f (table ) , derived from promoter p . sequence, was used for directional identification and the positive clones, which were subsequently named psc -m. homologous recombination was performed by lipofectin-mediated co-transfection of the transferring plasmid psc -m and the wr strain vaccinia virus into % confluent tk - cells cultured in mem medium containing μg/ml brdu (sigma). the viruses were collected after the appearance of cytopathic effect (cpe), and recombinant vaccinia virus was purified according to the expression of lacz gene. western blot analysis was performed as described previously [ ] . briefly, a bhk- cell layer was infected with rwr-prrsv-m recombinant vaccinia virus or the vaccinia virus wr strain at a multiplicity of infection (moi) of . . cells were harvested days post-infection, and total cell lysates were prepared with lysis buffer ( mm tris-cl ph . , mm mgcl , . % np , μg/ml dnase i). cell lysates were separated by sds-page and were subsequently transferred to a membrane for western blot analysis using an anti-prrsv-m monoclonal antibody, hrp-conjugated goat anti-mouse igg secondary antibody (bio-rad), and dab substrate. the positive recombinant virus was named rwr-prrsv-m. the expression of m was further confirmed by ifa. briefly, bhk- cells were infected with either rwr-prrsv-m or the vaccinia virus wr strain when the bhk- cells reached - % confluence in a -well plate. m protein expression was evaluated by ifa days post infection using the anti-prrsv-m monoclonal antibody followed by a fitc-conjugated rabbit antimouse igg secondary antibody (sigma). specific fluorescence was observed using a fluorescence microscope (leica dm ire ). the sequences of m were screened for potential h -k d / h -d d /h -l d -mer epitopes using the algorithms from the syfpeithi website [ ] , the hla peptide binding prediction website (bimas) [ ] and pred balb/c [ ] . the peptides (table ) , with highest binding score (bs) as predicted by each algorithm, were synthesized by scilight biotechnology llc (beijing, china) and purified to a purity > % using high performance liquid chromatography (hplc). all animal experiments were performed according to national and institutional guidelines. one hundred and ninety female balb/c mice (vital river laboratory animal technology co., beijing, china) were maintained in isolation cages at the experimental animal center of harbin veterinary research institute (harbin, china). mice were divided into three groups: the pvax -ub-m vaccination group (n = ), the pvax control vaccination group (n = ) and the pbs control vaccinated group (n = ). the plasmid dna used for immunization was purified using the endofree mega plasmid preparation kit (qiagen). the pvax -ub-m and pvax cohorts were intramuscularly (i.m.) vaccinated with μg pvax -ub-m or pvax plasmid dna in μl pbs, respectively. the pbs control group received an i.m. injection of μl pbs. each group was vaccinated four times at -week intervals. to enhance the specific ctl responses to m protein, the mice received an intraperitoneal (i.p.) injection containing . moi of rwr-prrsv-m on day following the fourth dna vaccination. at the same time, five mice from the pvax and pbs vaccination groups were also inoculated intraperitoneally with . moi of rwr-prrsv-m on day following the fourth vaccination in order to compare the specific antibody raised against m protein of different experimental groups following vaccination with rwr-prrsv-m. all procedures were conducted with the protocols approved by experimental animal center of harbin veterinary research institute (hvri) of the chinese academy of agricultural sciences (caas). to investigate the specific antibody response, serum samples was obtained from vaccinated mice days after each dna vaccination and days after boosting with rwr-prrsv-m. an ielisa was performed according to methods described previously [ ] . the purified m protein was used as the coating antigen, the tested sera applied at a : dilution, and an hrp-conjugated goat anti-mouse igg antibody (bio-rad) used as the secondary antibody. the microplates were developed using orthophenylene diamine (opda, amersco) and h o for min, after which the reaction was stopped by the addition of m h so . finally, the optical density (od) was read at nm. an anti-prrsv-m monoclonal antibody was used as a positive control. serum containing antibodies against akabane virus and the sera of control group mice served as negative controls. isolated splenocytes were added to u-bottomed -well plates (corning inc) at cells/well in μl complete rpmi- medium supplemented with % fcs. the cells were then mixed with μl media containing note: the peptides with highest binding score (bs) as predicted by each algorithm are shown, starting with the peptide giving the highest score at the top for each protein. sequences of mers are given from bimas, syfpeithi and pred balb/c predictions, respectively. the n-terminal amino acid position is indicated for epitopes predicted from the whole m protein. a nonamer peptide derived from prssv m protein at μg/ml, or phorbol- -myristate- -acetate (pma ng/ ml) and ionomycin ( ng/ml) as a positive control. cells incubated in medium alone or with a peptide derived from the infectious bronchitis virus (ibv) h strain [ ] were used as negative controls. following a h incubation at °c, um monensin (sigma) was added and the splenocytes were incubated for an additional h at °c before staining. the number of cd + cd + t cells producing ifn-γ on days , and after boosting with rwr-prrsv-m was determined using flow cytometric analysis. ics analysis was performed using a facscalibur flow cytometer (bd) according to the methods described previously [ ] . twenty mice were tested from the group vaccinated with pvax -ub-m, and five mice from the groups vaccinated with pvax and pbs were tested at each time point. the splenocytes from each vaccination group were counted, pooled, and stimulated in vitro with m protein-derived peptides, as detailed in section . . ten thousand cd + t lymphocytes were acquired per sample and the number of ifn-γ-secretion cd + cd + t cells were enumerated. the cd + cd + lymphocytes that expressed ifn-γ following peptide stimulation were considered to be peptide-specific ctls. specific ctl responses were evaluated as the increase in the number of cd + cd + ifn-γ + cells. reagents used include percp-conjugated cd e, pe-conjugated cd a and fitc-conjugated ifn-γ, and bd cytofix/cytoperm™, all purchased from becton, dickinson & co (bd). to further confirm the results of the ics, the other sixty mice from the group vaccinated with pvax -ub-m and thirty mice from the groups vaccinated with pvax and pbs were tested by the ifn-γ elispot assay at three different time points (as detailed in section . ). data are expressed as the number of ifn-γ-secreting cells per two hundred thousand splenocytes. peptide-specific ifn-γ elispot responses were considered to be positive if the response (minus media background) was ≥ fold above the media response and ≥ spot-forming cells (sfc)/ × splenocytes were registered. the ielisa results, the percentage of ifn-γ positive cd + cd + t lymphocytes and the number of spots per × splenocytes were analyzed using the analysis of variance (anova), and a probability value below . was considered significant. as the full-length m protein is difficult to express in vitro, we expressed a truncated version of m that lacks hydrophobic amino acids at the n-terminus. sds-page analysis showed that cells transformed with the pgex-m expression vector produced a large amount of protein with a molecular mass of approximately kda, consistent with the expected molecular weight of the truncated m protein fused with a gst tag (data not shown). western blot analysis using an anti-prrsv-m antibody confirmed the expression and identity of the truncated m protein fused with a gst tag (additional file , fig. s ). the ub proteasome pathway (upp) is the principal mechanism for protein catabolism in the mammalian cytosol and nucleus. in order to enhance the ub-mediated degradation efficiency of m protein, we expressed a ub-m fusion protein in bhk- cells using the eukaryotic expression plasmid pvax -ub-m, in which the ub coding sequence was fused in-frame with the prrsv m coding sequence. following transient transfection of bhk- cells with the pvax -ub-m plasmid, the ub-m fusion protein was expressed and accumulated predominantly in the cytoplasm (additional file , fig. s ). immunization strategies that prime and boost with recombinant dna vectors encoding antigens have been shown to elicit t-cell immunity against hiv in non-human primates [ ] , and more recently, in humans [ ] . therefore, we used a dna vector encoding the prrsv m protein to immunize mice in order to generate and characterize m protein-reactive cd + t cells. the recombinant vaccinia virus rwr-prrsv-m drove expression of an m protein with the expected molecular weight when transfected into bhk- cells (additional file , fig. s ). ifa confirmed expression of m protein following transfection of bhk- cells, and revealed m protein accumulation in the cytoplasm (additional file , fig. s ). m protein-specific serum antibody increased steadily from the second to the fourth dna vaccination with the pvax -ub-m vector which drives m protein expression in vivo (additional file , fig. s ), indicating that dna vaccination elicited m protein-specific immune responses as expected. a subsequent booster vaccination with the recombinant vaccinia virus, rwr-prrsv-m, elicited a further increase in prrsv-specific antibody (p < . , additional file , fig. s ). in contrast, control mice vaccinated with pvax -only or pbs-only did not show significant increases in m protein-specific antibody titers after the booster vaccination with rwr-prrsv-m (p > . , additional file , fig. s ). overall, mice vaccinated with pvax -ub-m generated significantly higher m protein-specific antibody titers compared to mice vaccinated with pvax or pbs (p < . , additional file , fig. s ). these results indicate that rwr-prrsv-m amplifies the protective effects of dna vaccination and reveals the advantage of this priming-boosting strategy. dna vaccination with pvax -ub-m likely drives the differentiation of memory b cells which are subsequently activated by rwr-prrsv-m following the booster immunization, resulting in increased m-specific antibody titers. mice vaccinated with pbs and pvax would not be expected to generate m protein-reactive memory b cells, accounting for a less pronounced increase in m protein-specific antibody titers following rwr-prrsv-m innoculation. in this study we identified potential ctl epitopes in balb/c mice vaccinated with pvax -ub-m and boosted with rwr-prrsv-m. the frequency and number of cd + cd + t cells that produced ifn-γ following stimulation with peptides derived from prssv m protein was enumerated using ics and elispot assays. using these approaches, we identified two peptides from prssv m protein that elicited ifn-γ production from the splenocytes of vaccinated mice. as shown in figure , intracellular ifn-γ staining following stimulation of splenocytes from vaccinated mice with the peptide k fitsrcrl and f gymtfvhf revealed a population of ifn-γ producing cd + t cells comprising - % of the total cd + splenocyte population. in contrast, unstimulated splenocytes and splenocytes exposed to an irrevelant peptide did not contain a population of ifn-γ producing cd + t cells (figure , panel b and c). consistent with the ics data, peptides k fitsrcrl and f gymtfvhf elicited ifn-γ production from splenocytes of vaccinated mice when measured by elispot, whereas unstimulated splenocytes and splenocytes stimulated with an irrelevant peptide did not reveal ifn-γ producing cells (figure ). the k fitsrcrl and f gymtfvhf prssv m protein peptides were identified bioinformatically as h- k d and h- d d restricted ctl epitopes (table ) . specific increases in the number of cells producing ifn-γ following stimulation with the peptides "k fitsrcrl" and "f gymtfvhf" was observed by day after the booster vaccination with rwr-prrsv-m (figure and ) . furthermore, splenocytes from mice vaccinated with pvax -ub-m responded strongly to "k fitsrcrl" and "f gymtfvhf", but not other m protein-derived peptides, at all time points tested following the booster vaccination with rwr-prrsv-m. when the same pattern of reactivity on three different time points after boosting with rwr-prrsv-m within each vaccination group were analyzed statistically using anova analysis, statistically significant differences were noted for the peptides "k fitsrcrl" and "f gymtfvhf" when compared to the other peptides tested among mice vaccinated and boosted with pvax -ub-m and rwr-prrsv-m, respectively (p < . , figure a and b). conversely, and confirming the specificity of these responses, no difference among the stimuli was observed with mock-vaccinated (pvax ) mice and naïve mice (data not shown). it is important to recognize that the ics assay calculates the percentage of ifn-γ + cells among cd + t cells in the spleen ( - %), whereas the elispot assay assesses the number of ifn-γ + cells among all splenocyte cell types ( . %), and cannot definitively assign the production of ifn-γ to a particular cell type. thus, the two assays use different denominators in calculating the frequency of ifn-γ + production by splenocytes. importantly, each method clearly identified k fitsrcrl and f gymtfvhf as the only two peptides from a panel of that elited significant ifn-γ production from the splenocytes of vaccinated mice. in this study we used balb/c mice as a model system to identify ctl epitopes in the m protein of prssv to circumvent limitations derived from shortages of inbred pigs and a paucity of reagents to evaluate porcine immune responses. the identification of prssv ctl epitopes in balb/c mice allow for the future generation of reagents, such as mhc class i: peptide staining reagents, that will enable the in-depth investigations of cd + t cell responses during prssv infection and immuinization. whether these two epitopes can bind the sla class i molecules of pigs remains to be determined. in some cases, specific peptide epitopes are known to be recognized by cytotoxic t cells in different animal species. for instance, the core region of hcv contains an epitope that is recognized by cytotoxic t cells of both mice and humans [ ] . further research on the role of these peptide epitopes in different species is ongoing in our laboratory. in conclusion, we identified peptides "k fitsrcrl" and "f gymtfvhf" from the m protein of prssv as h- k d and h- d d restricted ctl epitopes, respectively. in this study, we also developed a mouse model of prrsv infection and this will undoubtedly contribute to our understanding of the cell-mediated immune responses to prrsv. porcine reproductive and respiratory syndrome virus current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates identification of immunodominant t-cell epitopes present in glycoprotein of the north american genotype of porcine reproductive and respiratory syndrome virus t cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations immunological responses of swine to porcine reproductive and respiratory syndrome virus infection hepatitis b virus immunopathogenesis understanding presentation of viral antigens to cd + t cells in vivo: the key to rational vaccine design identification of an h- d(b)-restricted cd + cytotoxic t lymphocyte epitope in the matrix protein of respiratory syncytial virus characterization of a new h- d(k)-restricted epitope prominent in primary influenza a virus infection dna immunization with c fmdv non-structural protein reveals the presence of an immunodominant cd +, ctl epitope for balb/c mice immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing gp / gp of porcine reproductive and respiratory syndrome virus and swine il- syfpeithi: database for mhc ligands and peptide motifs scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains predbalb/c: a system for the prediction of peptide binding to h d molecules, a haplotype of the balb/c mouse dna vaccination of pigs with open reading frame - of prrs virus localization of a t-cell epitope within the nucleocapsid protein of avian coronavirus a dna/mva-based candidate human immunodeficiency virus vaccine for kenya induces multi-specific t cell responses in rhesus macaques enhanced t-cell immunogenicity of plasmid dna vaccines boosted by recombinant modified vaccinia virus ankara in humans submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution additional file : elisa antibody response in mice after immunization following dna vaccination and a booster vaccination with recombinant vaccinia virus. fig.s . m protein-specific antibody responses in mice immunized with pbs, or pvax or pvax -u-m dna, and boosted with rwr-prrsv-m. serum samples was obtained from vaccinated mice days after each dna vaccination and days after boosting with rwr-prrsv-m, and were evaluated for reactivity to mprotein in an elisa based on coating with the truncated m protein fused with a gst tag. and, the day represents the day of the first dna immunization. statistically significant differences are indicated by "*" or "**" for p-values < . or < . , respectively, as determined by anova.authors' contributions dlw and gll gave me the idea of this study. wjz and yl participated in the design and conducted the majority of the experiments as well as drafted the manuscript. tgt helped revise the manuscript and participated in the first stage of the experiments. yb and qw participated in the prediction of ctl epitopes and analyzed the data. yhx, nhl and ty participated in the sequence alignment. zgb provided the expression system of recombinant vaccinia virus, and gzt provided the prrsv ch- a strain. all the authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - b b p authors: mcwhirter, sarah m.; barbalat, roman; monroe, kathryn m.; fontana, mary f.; hyodo, mamoru; joncker, nathalie t.; ishii, ken j.; akira, shizuo; colonna, marco; chen, zhijian j.; fitzgerald, katherine a.; hayakawa, yoshihiro; vance, russell e. title: a host type i interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-gmp date: - - journal: j exp med doi: . /jem. sha: doc_id: cord_uid: b b p the innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. the cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-gmp) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. in vivo and in vitro studies have previously suggested that c-di-gmp is a potent immunostimulatory compound recognized by mouse and human cells. we provide evidence that c-di-gmp is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. the potency of cytosolic signaling induced by c-di-gmp is comparable to that induced by cytosolic delivery of dna, and both nucleic acids induce a similar transcriptional profile, including triggering of type i interferons and coregulated genes via induction of tbk , irf , nuclear factor κb, and map kinases. however, the cytosolic pathway that senses c-di-gmp appears to be distinct from all known nucleic acid–sensing pathways. our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand. to sense infection, the innate immune system preferentially responds to conserved molecular signatures of microbes that are absent from normal host cells. there is currently great interest in determining what molecular features of pathogens are detected, and how these features are sensed by the innate immune system. a better understanding of the fundamental principles of how immune responses are stimulated is critical for the design of more effective vaccines, adjuvants, and immune therapeutics. one important class of ligands sensed by the innate immune system is nucleic acids. several distinct nucleic acid sensors have been described and have been found to be distributed to distinct subcellular locations and exhibit distinct specificities for different nucleic acid ligands. a common theme is that nucleic acid sensors tend to induce expression of type i ifn genes and, thus, appear to be particularly important for initiating immune responses to viruses. including listeria monocytogenes, mycobacterium tuberculosis, legionella pneumophila, francisella tularensis, group b streptococcus, and brucella abortus, it appears that induction of type i ifn is via a novel cytosolic pathway and is independent of tlrs. it has been suggested that these pathogens trigger the cytosolic dna-sensing pathway, but neither the host sensors nor the bacterial ligands that trigger cytosolic induction of type i ifns by these pathogens have been identified. intriguingly, evidence from l. monocytogenes suggests that bacterial multidrug efflux pumps are required for induction of type i ifn , and raises the possibility that a small molecule substrate of these pumps, rather than dna, is the bacterial trigger of type i ifn gene expression. nucleotide second messengers are critical transmitters of signaling in all living things. camp is used by bacteria and eukaryotes alike, whereas certain nucleotide second messengers are unique to bacteria. for example, guanosine tetraphosphate (ppgpp) is a key regulator of the stringent response in bacteria (srivatsan and wang, ) . another nucleotide second messenger, cyclic-di-gmp (c-di-gmp), is a relatively recently appreciated cyclic ribonucleotide (ross et al., ) synthesized by bacteria from two gtp precursors that are hydrolyzed and ultimately circularized via -to-  monophosphate linkages. the bacterial diguanylate cyclases that synthesize c-di-gmp all contain a characteristic ggdef domain, whereas the phosphodiesterases that specifically degrade c-di-gmp to pgpg contain an eal domain. the ggdef domain, and presumably c-di-gmp, appears to be limited to bacteria (galperin et al., ) and is not found in archaea or eukarya. c-di-gmp appears to play complex roles as a second messenger in most bacterial species, and regulates diverse processes, such as motility, biofilm formation, and virulence gene expression (tamayo et al., ) . the molecular mechanisms by which c-di-gmp regulates these biological processes in bacteria are only beginning to be understood. it appears that c-di-gmp is capable of specific binding to regulatory proteins containing the pilz protein domain (cotter and stibitz, ) . the peld protein of p. aeruginosa is an additional non-pilz-containing c-di-gmp sensor protein (lee et al., ) . several recent reports have suggested that c-di-gmp can stimulate a variety of signaling pathways in mammalian cells in vivo. one early report demonstrated that µm of exogenous c-di-gmp inhibited growth of human colon cancer cells (karaolis et al., a) without toxicity against normal kidney cells. two additional reports have indicated that c-di-gmp can function as an adjuvant. one group demonstrated a highly significant (> -fold; p < . ) induction of anti-clfa igg a titers when mice were vaccinated with clfa and c-di-gmp as compared with vaccination with clfa alone (karaolis et al., a) . another group found a similar induction of anti--gal titers when c-di-gmp was used as an adjuvant (ebensen et al., ) . both groups also found increased t cell responses in mice injected with c-di-gmp. karoalis et al. ( a) also showed that c-di-gmp has immunostimulatory effects in vivo and in vitro on innate cell populations, toll-like receptor (tlr) , , , and are transmembrane nucleic acid receptors that reside in intracellular compartments, where they detect endocytosed or autophagocytosed nucleic acids medzhitov, ) . tlr , , and all require the signaling adaptor myd to initiate downstream signaling, whereas tlr requires the signaling adaptor trif. thus, myd / trif / double-knockout cells are deficient in signaling through all known tlrs that sense nucleic acids (hoebe et al., ; yamamoto et al., ) . interestingly, recent work has established that myd / trif / cells are capable of responding to nucleic acid ligands via cytosolic immunosurveillance pathways. cytosolic rna appears to be sensed by two rna helicase-containing proteins, rig-i and mda (yoneyama et al., ) . interestingly, rig-i and mda do not perform redundant functions and appear to respond to distinct classes of viruses (gitlin et al., ; hornung et al., ; kato et al., ; pichlmair et al., ; . signaling by rig-i and mda requires a common adaptor molecule called mavs (also known as ips- , cardif, or visa; kawai et al., ; meylan et al., ; seth et al., ; xu et al., ; sun et al., ) . mavs recruits the tbk kinase that phosphorylates and activates the irf transcription factor. other transcription factors such as atf /c-jun and nk-b form a coordinated dna-bound complex with irf , and are together required for robust activation of the ifn- promoter (maniatis et al., ; panne, ) . many cell types also appear to respond to the cytosolic presence of dna stetson and medzhitov, a) . the cytosolic response to dna does not require mavs, but does require tbk and irf in most cell types stetson and medzhitov, a; sun et al., ) . recently, a putative cytosolic sensor of dna was identified, and was named dna-dependent activator of ifn regulatory factors (dai; previously known as dlm- or zbp ; takaoka et al., ) . knockdown experiments indicated that dai was involved in sensing cytosolic dna in the l fibroblast-like and raw macrophage-like cell lines (takaoka et al., ; wang et al., ) . dai was expressed in other cell types, such as mouse embryonic fibroblasts (mefs), but was dispensable for the response to cytosolic dna in these cell types, suggesting that additional uncharacterized nucleic acid sensor proteins also exist . indeed, cells from dai knockouts appear to respond normally to cytosolic dna (ishii et al., ) , possibly because of redundancy with other cytosolic dna sensors. although type i ifns are primarily considered to be antiviral cytokines (stetson and medzhitov, b) , there is growing appreciation for their complex role in bacterial infections as well. indeed, it is clear that type i ifns are induced by many, if not all, bacterial pathogens, and can contribute to diverse outcomes in vivo (coers et al., ; o'riordan et al., ; opitz et al., ; stetson and medzhitov, a; henry et al., ; roux et al., ; stanley et al., ; charrel-dennis et al., ) . for several bacterial pathogens, (a) the c-di-gmp compound was chemically synthesized and > % pure by hplc (not depicted), (b) phosphodiesterase treatment of c-di-gmp resulted in a product that is no longer stimulatory ( fig. e) , and (c) macrophages deficient in all tlr signaling and unable to respond to lps still responded to c-di-gmp (see below). thus, these observations suggest that c-di-gmp is sensed in the cytosol, resulting in a potent induction of type i ifn in bone marrow macrophages. we performed microarray experiments to compare the global transcriptional response induced by cytosolic c-di-gmp with that induced by cytosolic dna (fig. f and table s ). whole transcriptome spotted meebo microarrays (verdugo and medrano, ) were used in these experiments. the results indicated that the transcriptional profile of cells stimulated by cytosolic c-di-gmp was very similar to the transcriptional profile of cells stimulated with cytosolic dna. induction of type i ifns and known type i ifn-inducible genes dominated the transcriptional response, but other genes (e.g., il , cd , and cxcl ) were also strongly induced ( fig. f and table s ). although the transcriptional profiles of cells stimulated with c-di-gmp and dna were highly similar, a small number of genes were reproducibly preferentially induced by c-di-gmp as compared with dna (e.g., htra serine peptidase and claudin ; table s ). collectively, these results suggested that similar but not identical downstream signaling pathways are triggered by dna and c-di-gmp. it was previously reported that hek cells stably transfected with various tlrs failed to respond to c-di-gmp (karaolis et al., a) , but the reason for this failure was unclear. for example, hek cells might lack an essential accessory protein. to rule out a role for tlrs in sensing of c-di-gmp, we tested responses in myd / trif / double-knockout macrophages, which are deficient in all tlr signaling (yamamoto et al., ) . both wild-type and myd / trif / macrophages showed a robust induction of ifn- after c-di-gmp stimulation (fig. a) . as expected, the myd / trif / macrophages did not respond to lps (fig. a) . these results indicated that tlr signaling is not required for responsiveness to c-di-gmp, and are consistent with c-di-gmp signaling via a cytosolic surveillance pathway. robust transcription of the ifn- gene requires the coordinate activation of several transcription factors, including irf and nf-b (panne, ) . we therefore expected that the c-di-gmp-induced signaling pathway would also require these factors to induce type i ifn. irf activation requires phosphorylation by the tbk kinase, which leads to irf dimerization and nuclear translocation (fitzgerald et al., ; sharma et al., ; hemmi et al., ; mcwhirter et al., ; perry et al., ) . to address whether tbk is required for activation of type i ifn by c-di-gmp, we tested including monocytes, macrophages, and granulocytes. c-di-gmp induced dendritic cell maturation and led to the production of various cytokines and chemokines, including tnf, il- , ip- , rantes, and cxcr (karaolis et al., a) . however, c-di-gmp did not stimulate induction of type i ifns by plasmacytoid dendritic cells. impressively, preinjection of . mg/kg c-di-gmp protected against subsequent lethal challenge with klebsiella pneumoniae or staphlyococcus aureus (karaolis et al., b; karaolis et al., b) . despite the impressive in vivo biological effects of c-di-gmp, the mechanism by which c-di-gmp stimulates host immunity remains unknown. in this study, we provide evidence that mammalian cells survey their cytosol for the presence of c-di-gmp. we find that c-di-gmp triggers a transcriptional response virtually indistinguishable from the response triggered by cytosolic dna. like the response to dna, the response to c-di-gmp requires the tbk kinase and irf transcription factor. however, the pathway for sensing c-di-gmp can be distinguished from that for sensing dna in certain cell types and, moreover, appears to be distinct from all other known cytosolic sensing pathways. our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand. we hypothesized that c-di-gmp might be sensed by a cytosolic sensor leading to the induction of type i ifns. to test this hypothesis, we compared the ability of overlayed versus transfected c-di-gmp to induce type i ifn in bone marrow macrophages. at the concentrations used (up to µg/ml), overlay of c-di-gmp did not elicit a significant response ( fig. a) , although ifn- production could be detected at higher concentrations ( or µg/ml; not depicted). in contrast, delivery of c-di-gmp to the cytosol by transfection elicited robust activation of ifn- in a dose-dependent manner ( fig. a) . a significant response was detected with a concentration of c-di-gmp as low as . µg/ml (equivalent to . µm). these results suggested that c-di-gmp signals via a cytosolic immunosurveillance pathway. we tested whether compounds related to c-di-gmp were capable of stimulating ifn- expression when delivered to the cytosol. we measured induction of endogenous type i ifn protein by bioassay ( fig. b) , and induction of ifn- and ifn- mrna levels by quantitative rt-pcr ( fig. , c and d). no detectable ifn- production was observed upon transfection with gtp, cgmp, ppgpp (a different bacterial nucleotide second messenger), or pgpg (hydrolyzed c-di-gmp), whereas the cytosolic response to . µg/ml of transfected c-di-gmp was of a similar magnitude to that of . µg/ml of transfected dna (pda:dt) and rna (pi:c), which are known cytosolic inducers of type i ifn. we do not believe that the cytosolic induction of type i ifns in response to c-di-gmp is caused by a contaminant (such as lps) because a role in ifn--mediated antiviral immunity (tenoever et al., ) , and found that ikbke / macrophages responded normally to c-di-gmp (fig. c) . to examine the role of irf in c-di-gmp induction of ifn-, we tested the ability of c-di-gmp to induce signaling in macrophages lacking irf . we observed that irf / macrophages failed to induce ifn- in response to c-di-gmp ( fig. d) . we also tested irf / macrophages and observed a partial requirement for irf by the c-di-gmp pathway (fig. d) . loss of irf is likely compensated for by irf in macrophages. the partial requirement for irf may reflect a c-di-gmp responses in tbk / macrophages (fig. b) . we observed very little ifn- induction by c-di-gmp in cells lacking tbk , suggesting that tbk is required for c-di-gmp signaling. we also observed that tbk is required for the induction of ifn- by pda:dt ( fig. b) , in agreement with a recently published report (miyahira et al., ) . although the data indicate a key role for tbk in the response to c-di-gmp, it is formally possible, albeit unlikely, that tbk protein rather than tbk kinase activity is required for the response. we also tested the requirement for the tbk related kinase ikbke (also called ikk or ikk-i), which plays (jiang et al., ) using recombinant ifn- as a standard. *, p < . as compared with transfected c-di-gmp (student's t test). (b) bone marrow macrophages were transfected with . µg/ml of the indicated molecules (or overlaid with ng/ml lps), and type i ifn was assessed after h by bioassay as in a. *, p < . as compared with unstimulated cells (student's t test). (c) bone marrow macrophages were transfected with . µg/ml of the indicated molecules (or overlaid with ng/ml lps), and transcription of the ifn- gene (ifnb) was assessed by quantitative rt-pcr, with normalization to ribosomal protein rps message. n.d., not detectably induced above background (lipofectamine alone). *, p < . as compared with unstimulated cells (student's t test). (d) as in c, but transcription of the ifn- gene was assessed. n.d., not detectably induced above background (lipofectamine alone). *, p < . as compared with unstimulated cells (student's t test). (e) c-di-gmp, treated or untreated with snake venom phosphodiesterase, was transfected at . µg/ml into b bone marrow macrophages and analyzed after h for ifn- production by bioassay, as in a. *, p < . as compared with c-di-gmp treatment alone (student's t test). results in a-e are representative of at least three independent experiments. data are means ± sd (n = ). (f) bone marrow macrophages were transfected with poly da:dt (dna) or with c-di-gmp. total rna was isolated after h of stimulation. probes were amplified and hybridized to whole-transcriptome spotted meebo microarrays. each dot represents a single gene, and its position is determined by its induction in response to dna (x axis) or c-di-gmp (y axis). most genes lie on the diagonal, indicative of similar induction ratios by both treatments. selected highly induced genes are labeled. two independent microarray experiments gave similar results. transcription factors, as well as phosphorylation of the p , jnk, and erk map kinases, in response to c-di-gmp. activation of irf involves phosphorylation, dimerization, and translocation into the nucleus. we tested whether treatment with c-di-gmp resulted in significant nuclear accumulation of irf . as expected, we found that c-di-gmp resulted in significant translocation of irf to the nucleus (fig. a) . we also tested whether transfected c-di-gmp is capable of activating nf-b. this was particularly important to establish in light of a previous report that exogenous (untransfected) c-di-gmp failed to activate nf-b (karaolis et al., a) . there have also been inconsistent reports in the literature as to whether cytosolic dna induces nf-b stetson and medzhitov, a; sun et al., ) . we found that c-di-gmp treatment led to the activation of nf-b, as assessed by using an electromobility gel shift assay, with similar kinetics to that of transfected pi:c and pda:dt ( fig. b) . the probe-bound complex was confirmed to contain nf-b p by supershifting with an anti-p antibody ( fig. b) . we confirmed these results by using a raw role for irf signaling as part of a positive feedback loop involving signaling through the type i ifn receptor (ifnar). indeed, ifnar / macrophages exhibited a diminished response to c-di-gmp (fig. e) . irf and irf did not seem to be required for responsiveness to c-di-gmp (fig. f) . in addition, macrophages deficient in rip , nalp , or doubly deficient in nod / responded normally to c-di-gmp (unpublished data). we validated our results by examining production of a second secreted molecule known to be a target of the tbk -irf signaling axis, the chemokine rantes (ccl ). rantes production was assessed by elisa (fig. s ). as expected, we found that rantes was induced by c-di-gmp in a manner dependent on tbk , irf , and ifnar, but largely or entirely independent of myd /trif, irf , irf , irf , or mavs (fig. s ). our genetic data suggested that the cytosolic responses to dna, rna, and c-di-gmp share a common downstream signaling pathway. if this were true, we would expect to be able to detect biochemical activation of the nf-b and irf myd / trif / mice were transfected with . µg/ml c-di-gmp or pda:dt (dna), or overlayed with ng/ml lps as indicated. after h of stimulation, induction of ifn- mrna was analyzed by quantitative rt-pcr, with normalization to ribosomal protein rps message. n.d., not detectable. (b) as in a, except tnfr / or tnfr / tbk / macrophages were analyzed. tbk deficiency is embryonic lethal, but viability can be rescued on the tnf receptor (tnfr )-deficient background. (c) as in a except ikbke (ikk/ikk-i)-deficient mice were analyzed. (d) as in a, except irf / and irf / macrophages were analyzed. (e) as in a, except macrophages deficient in the type i ifn receptor (ifnar / ) were analyzed. (f) as in a, except irf / and irf / macrophages were analyzed. results are representative of at least three independent experiments. data are means ± sd (n = ). *, p < . ; and **, p < . as compared with wild-type bone marrow macrophages (student's t test) . discrepancy between our results and those of karaolis et al. ( a) , it is more likely that we were able to detect map kinase activation because transfection of c-di-gmp triggers more robust responses than overlayed c-di-gmp, and because we examined later time points. cell type-specific responses to cytosolic dna and c-di-gmp collectively, these results suggest that there are strong similarities in the signaling pathways triggered by the cytosolic presence of c-di-gmp and other nucleic acids, such as dna and rna. however, c-di-gmp is not structurally similar to these nucleic acid ligands. the dna molecules capable of provoking cytosolic responses are double stranded and > deoxyribonucleotides in length stetson and medzhitov, a) . in contrast, c-di-gmp is composed of two guanosine ribonucleotides (not deoxyribonucleotides) in an unusual cyclic conformation. c-di-gmp also lacks features consistent with recognition by rig-i or mda , such as triphosphate, polyuridine, or double strandedness . therefore, we considered whether distinct sensors might be responsible for triggering responses to dna/rna and c-di-gmp, and if so, whether the responses could be distinguished in certain cell types. macrophage cell line stably transfected with an nf-b luciferase reporter. we observed activation of the nf-b luciferase reporter by transfected c-di-gmp (fig. c) . both the gel shift and reporter assays showed that the activation of nf-b by transfected c-di-gmp was not as strong as observed with transfected pda:dt and pi:c. the subtle difference in nf-b induction between cytosolic dna and c-di-gmp may suggest that there are distinct signaling components upstream of nf-b in the two pathways. nonetheless, we concluded that transfected c-di-gmp is capable of activating nf-b. the relatively low levels and late time course of nf-b induction by c-di-gmp (as compared with induction by lps), as well as the fact that we transfected c-di-gmp into cells, may explain why nf-b induction was not previously observed in response to c-di-gmp (karaolis et al., a) . we also tested whether map kinase pathways are activated by c-di-gmp. a previous report found that overlay of c-di-gmp induced erk but not p in human macrophages (karaolis et al., a) . in contrast, we found that cytosolic c-di-gmp stimulated p , erk / , and jnk phosphorylation in mouse bone marrow macrophages transfected with c-di-gmp (fig. d) . although a difference between human and mouse macrophages may explain the data are means ± sd (n = ). *, p < . compared with unstimulated cells (student's t test) . tion, and is downstream of two cytosolic viral rna sensors, rig-i and mda (sun et al., ) . to test if c-di-gmp signals through the cytosolic rna-sensing pathway, we tested c-di-gmp responses in mavs / and mda / macrophages. loss of mavs or mda did not affect the induction of ifn- by c-di-gmp (fig. , a and b; and fig. s c) , suggesting that c-di-gmp does not signal through the cytosolic rna signaling apparatus (gitlin et al., ; kato et al., ; . dai (encoded by the zpb gene) is a recently identified molecule that has been proposed to function as a sensor of cytosolic dna (takaoka et al., ; wang et al., ) . we found that bone marrow-derived macrophages from zbp deficient mice (ishii et al., ) still responded to c-di-gmp (fig. c) . however, as previously reported (ishii et al., ) , zbp -deficient macrophages also responded normally to dna (fig. c) . these results imply that macrophages express at least one cytosolic dna sensor that is distinct from in addition to bone marrow macrophages, we observed induction of ifn- by cytosolic c-di-gmp in a variety of other cell types, including peritoneal macrophages, conventional dendritic cells, l cells, and raw . macrophages (fig. , a-d) . these cells also responded to transfected dna and rna. interestingly, however, we were unable to detect significant induction of type i ifns by c-di-gmp in mefs or in t cells, even though the cytosolic pathways for detecting dna and rna are intact in these cells (fig. , e and f). these results suggest that at least one component of the host signaling pathway responding to c-di-gmp is distinct from that used for responses to cytosolic rna or dna, and is differentially expressed in different cell types. known dna and rna sensing pathways are not required for responses to c-di-gmp mavs (also known as ips- ) is an essential adapter protein in the cytosolic rna pathway leading to type i ifn induc- data are means ± sd (n = ). n.d., not detectable. *, p < . ; and **, p < . as compared with unstimulated cells (student's t test) . and measured their responsiveness to yac- target cells ex vivo. because nk cells respond to in vivo injection of poly i:c, a prototypical ifn inducer, we expected that c-di-gmp might also activate nk cells. indeed, nk cells obtained from b mice injected with c-di-gmp responded to yac- cells, as measured by intracellular cytokine staining for ifn- (fig. b) . however, nk cells obtained from irf / / mice stimulated with c-di-gmp were nonresponsive to yac- target cells. as a negative control, nk cells stimulated ex vivo with pbs did not produce ifn-. these observations indicate that c-di-gmp can stimulate cytokine and nk cell responses in vivo, and these responses require the irf / transcription factors. to obtain a preliminary indication as to whether adaptive immune responses could be stimulated by c-di-gmp, we immunized mice with human serum albumin (hsa) and tested whether coinjected c-di-gmp could function as an adjuvant, as previously reported (ebensen et al., ; dai, and at least one of these uncharacterized dna sensors, or another independent sensor, may respond to c-di-gmp. to validate our findings in vivo, we injected mice intraperitoneally with c-di-gmp ( nmol per mouse) and measured the production of type i ifns in the blood h later. b mice injected with c-di-gmp produced an ifn response to c-di-gmp, and this response was abolished in irf / double-deficient mice (fig. a) . interestingly, single-deficient irf / mice responded to c-di-gmp in vivo (unpublished data), implying a role for irf in responses to c-di-gmp in vivo. to determine whether cytokine induction by c-di-gmp affected cellular responses in vivo, we collected splenic nk cells from mice injected with c-di-gmp figure . c-di-gmp does not stimulate known cytosolic pathways for sensing nucleic acids. (a) mavs / and littermate wild-type macrophages were transfected with . µg/ml c-di-gmp, poly da:dt (pda:dt, dna), or poly i:c (pi:c, rna), and transcription of the ifn- gene (ifnb) was assessed by quantitative rt-pcr, with normalization to ribosomal protein rps message. n.d., not detectable. (b) mda / and wild-type control macrophages were stimulated and analyzed as in a. (c) zbp / (dai knockout) and wild-type control macrophages were stimulated and analyzed as in a. sev, sendai virus. results are representative of at least three independent experiments. data are means ± sd (n = ). *, p < . ; and **, p < . as compared with wild-type bone marrow macrophages (student's t test) . (a and b) b and irf / / mice were injected intraperitoneally with nmol c-di-gmp, and (a) serum ifn was measured by bioassay h later, or (b) h after injection, splenic nk responsiveness to yac- target cells (or pbs control) was measured ex vivo by intracellular staining for ifn-. (c) b and irf / / mice were injected intraperitoneally with hsa ± nmol c-di-gmp, and wk later serum igg specific for hsa was determined by elisa. the experiments in a and b were repeated twice, and the experiment in c was performed once. horizontal bars represent means. **, p < . ; and *, p < . (student's t test) . respond well to c-di-gmp. given the marked chemical dissimilarities among dna, rna, and c-di-gmp, it is perhaps not surprising that these nucleic acids appear to be recognized by different sensors. indeed, we favor the idea that there are multiple cytosolic sensors for nucleic acids leading to induction of type i ifns. the specificities of these various sensors will require further dissection. for example, although dai knockout cells still responded to c-di-gmp, it remains formally possible that dai is just one of several redundant sensors for c-di-gmp. these possibilities can be addressed once additional cytosolic nucleic acid sensors are identified. our results suggest that cytosolic sensing of c-di-gmp is relatively specific. other related small nucleic acid compounds such as gtp, cgmp, ppgpp, and pgpg did not trigger transcriptional induction of type i ifns (fig. ) . in addition, c-di-gmp that was hydrolyzed by snake venom phosphodiesterase did not induce type i ifn (fig. ) . however, our results cannot eliminate the possibility that a metabolite of c-di-gmp, rather than c-di-gmp itself, is the true molecular moiety that is sensed in the cytosol. elimination of this possibility awaits identification of the c-di-gmp sensor and biophysical or crystallographic characterization of its binding to c-di-gmp. our results also cannot eliminate the possibility that c-di-gmp triggers type i ifn expression indirectly, for example, by stimulating the synthesis of a host ligand that functions as the "true" proximal trigger of type i ifn gene expression. it is also possible that c-di-gmp acts "pharmacologically" by disrupting host physiology in a way that results in type i ifn expression. moreover, because the signaling triggered by c-di-gmp and cytosolic dna are very similar, it is possible that the c-di-gmp pathway is a branch off of the dna-sensing pathway rather than a fully independent pathway. however, even if these alternative models are correct, our data indicate that the pathway downstream of c-di-gmp contains at least some novel components and is at least partially independent of known cytosolic or tlr signaling pathways. several bacterial pathogens, including l. monocytogenes, l. pneumophila, f. tularensis, m. tuberculosis, and group b streptococcus, have been reported to induce type i ifns (coers et al., ; o'riordan et al., ; opitz et al., ; stetson and medzhitov, a; henry et al., ; roux et al., ; stanley et al., ; charrel-dennis et al., ) . the mechanism by which these pathogens induce type i ifn resembles that of c-di-gmp: in all cases, type i ifn induction is independent of myd /trif and tlrs, requires tbk and irf , and (with one exception; unpublished data; opitz et al., ) is independent of the cytosolic rna-sensing pathway. it is widely assumed that bacterial dna, perhaps released after bacterial cell lysis, is the relevant ligand that triggers type i ifns in response to pathogens. there are no data that eliminate or confirm this possibility, as sensors required for ifn induction in response to cytosolic bacteria have not yet been reported. however, it is noteworthy that in all cases, induction of type i ifns by cytosolic bacteria requires expression of an auxiliary secretion system, namely the esx- system of m. tuberculosis, the francisella pathogenicity island-encoded a). serum was collected from immunized mice wk after a single intraperitoneal injection. mice immunized with hsa alone did not mount a significant antibody response to hsa. in contrast, mice injected with hsa plus c-di-gmp produced variable but significant titers of hsa-specific igg antibodies, confirming that c-di-gmp can function as an adjuvant in vivo. importantly, our preliminary data indicated that the adjuvant effect of c-di-gmp depended on irf / , as antibody responses in irf / / mice immunized with hsa and c-di-gmp were significantly (p < . ) reduced as compared with immunized b mice (fig. c) . further studies will be required to establish the mechanism by which c-di-gmp functions as an adjuvant, but our results are consistent with a recent report indicating that the irf / kinase, tbk , is required for adaptive immune responses in a dna-vaccine model (ishii et al., ) . it has been well established that innate immune responses are initiated in response to certain microbial ligands that are evolutionarily conserved and that can be distinguished from selfligands. nucleic acids appear to be a favored target of immune recognition, and are sensed by a variety of endosomal and cytosolic sensor proteins. the cyclic dinucleotide c-di-gmp is a bacterial second messenger that exhibits several characteristics that are desirable in an immunostimulatory ligand: it is produced by numerous species of bacteria, it is a critical regulator of bacterial physiology, and it is not similar to host molecules. indeed, several previous reports have demonstrated that c-di-gmp can provoke potent immune responses when injected in vivo into mice (karaolis et al., a; karaolis et al., b; karaolis et al., a; karaolis et al., b) . however, the mechanism by which c-di-gmp stimulates immune responses remained unclear. our data provide evidence that c-di-gmp is sensed by a novel cytosolic immunosurveillance pathway. responsiveness to c-di-gmp is independent of tlrs that monitor the extracellular/endosomal compartments and is strongly potentiated by transfection of c-di-gmp into the host cell cytosol. moreover, c-di-gmp provokes a transcriptional response highly reminiscent of that triggered by the cytosolic presence of dna or rna, and involves activation of tbk- , map kinases, and the irf- and nf-b transcription factors. thus, we propose that c-di-gmp is sensed in the cytosol. how many different cytosolic sensors exist for nucleic acids? our data suggest that sensing of c-di-gmp occurs via a novel cytosolic immunosurveillance pathway. known nucleic acid sensors include mda and rig-i, which sense viral rna and signal via mavs, as well as dai, a putative sensor of dna that signals independently of mavs. based on the finding that most cell types, including mefs, can still respond to dna in the absence of dai (ishii et al., ) , it has been proposed that an additional dna sensor must exist and that mefs express both of these sensors . our results now suggest that there is at least one additional nucleic acid sensor in the cytosol, because we find that mefs do not b. beutler (the scripps research institute, la jolla, ca), and raw-b cells were obtained from g. barton. animal protocols were approved by the university of california, berkeley animal care and use committee. reagents. c-di-gmp was synthesized as previously described (kawai et al., ) , poly i:c was obtained from ge biosciences, pda:dt (poly(da-dt): poly(da-dt)) was purchased from sigma-aldrich, ppgpp (guanosine- , bisdiphosphate) was obtained from trilink biotechnologies, pgpg (diguanosine) was purchased from iba gmbh, gtp and cgmp were obtained from sigma-aldrich, purified lps was purchased from invivogen, and sendai virus was obtained from charles river laboratories. theiler's virus was the gift of m. brahic (stanford university, stanford, ca). cell culture. l , raw . , and mef cell lines were cultured in dmem containing % fbs, glutamine, and penicillin-streptomycin. for bone marrow-derived macrophages, bone marrow cells from femurs and tibias were cultured for d in rpmi media containing % fbs, glutamine, penicillin-streptomycin, and % csf from t cells, with feeding on the fourth day of growth. for conventional dendritic cells (gm-csfdendritic cells), bone marrow cells were cultured for d with rpmi containing % fbs, glutamine, penicillin-streptomycin, -mercaptoethanol, and gm-csf, with fresh media added on the second and fourth days of growth. peritoneal macrophages were elicited by injection of ml % thioglycollate (fluid thioglycollate medium; bd), and were obtained d later by lavage of the peritoneal cavity with rpmi . cell stimulations (transfections). cells were transfected using lipofectamine (lf ; invitrogen) according to the manufacturer's protocol. all nucleic acid stimulants were mixed with lf at a ratio of µl lf / µg nucleic acid, incubated at room temperature for - min, and added to cells at a final concentration of . µg/ml ( -well plates) or µg/ml ( -well plates). for pi:c, mg/ml of the stock solution was heated at °c for min and cooled to room temperature before mixing with lf . transfection experiments were done for h, unless otherwise stated in the figures. for phosphodiesterase treatment of c-di-gmp, . µg/µl c-di-gmp was incubated for h at room temperature in optimem buffer (invitrogen), with mm mgcl and u phosphodiesterase i (ge healthcare). quantitative pcr. stimulated cells were overlayed with rnalater (applied biosystems) and stored. rna was isolated using the rneasy kit (qiagen) according to the manufacturer's protocol, and was treated with rq rnase-free dnase (promega). . µg rna was reverse transcribed with superscript iii (invitrogen). platinum taq dna polymerase (invitrogen) and evagreen dye (biotium) were used for quantitative pcr assays and analyzed with a real-time pcr system (steponeplus; applied biosystems). all gene expression values were normalized to rps (mouse) or sp (human) levels for each sample. the following primer sequences were used: mouse ifnb, (forward) -ataagcagctccagctccaa-  and (reverse) -ctgtctgctggtggagttca- ; mouse ifn , (forward) -tgacctcaaagcctgtgtgatg-  and (reverse) -aag-tatttcctcacagccagcag- ; mouse rps , (forward) -cgcc-attatccccagcaag-  and (reverse) -tgtcgggatccacc-t caatg- ; human ifn, (forward) -aaactcatgagcagtct-gca-  and (reverse) -aggagatcttcagtttcggagg- ; and human sp , (forward) -atccgccagcgccata-  and (reverse) -tcaatgtgcttctgggaatcc- . type i ifn bioassay and luciferase reporter assay. cell-culture supernatants from stimulated cells were overlayed on top of isre-l ifn reporter cells (jiang et al., ) and incubated for - h ( -well plate). the reporter cells were lysed in passive lysis buffer (promega) for min at room temperature, mixed with firefly luciferin substrate (biosynth), and measured on a luminometer (lmaxii ; mds analytical technologies). levels of type i ifn were calculated from a standard curve using recombinant mouse ifn- (r&d systems). secretion system of f. tularensis, the dot/icm system of l. pneumophila, or the mdrm multidrug efflux pump of l. monocytogenes. it is tempting to speculate that a small, bacterially derived molecule such as c-di-gmp could be transported or leak through these secretion systems. it has not yet been possible to test this idea directly because all of these bacterial species encode numerous c-di-gmp synthases, and a strain lacking all c-di-gmp synthesis has not been reported. in any case, interpretation of these experiments would be complicated by the fact that c-di-gmp plays important regulatory roles in bacterial physiology and pathogenesis. nevertheless, our results with synthetic purified c-di-gmp suggest that in addition to dna, c-di-gmp is a candidate for a conserved molecule unique to bacteria that is responsible for triggering transcription of type i ifn genes. in light of a recent report that bacteria appear to be able to synthesize c-di-amp (witte et al., ) , it is interesting to consider whether additional nucleic acids produced specifically by bacteria might also trigger host immunosurveillance pathways. non-nucleic acid small molecules, such as the drug dmxaa, also appear to be able to stimulate the tbk -irf axis (roberts et al., ) . indeed, there may be multiple redundant pathways for cytosolic sensing of bacteria. collectively, the available evidence suggests that host cells may sense a wider array of bacterial ligands than was previously appreciated. our results may have important implications for the design of new adjuvants and vaccines. dna vaccines have attracted considerable enthusiasm as an approach for protecting against a variety of infectious diseases (wang et al., ; yang et al., ) , but there are safety concerns about the insertional mutagenic potential of dna vaccines and/or their potential to trigger pathogenic anti-dna autoimmune antibody responses (schalk et al., ) . the potency of dna vaccines appears to derive from their ability to stimulate the tbk and innate cytosolic dna-sensing pathways . thus, our demonstration that a synthetic nonself non-dna molecule such as c-di-gmp can stimulate an in vitro and in vivo innate and adaptive immune response (fig. ) similar to that induced by dna, without similar autoimmune or mutagenic risks, suggests that c-di-gmp might have valuable application as a small-molecule adjuvant. understanding the molecular basis of c-di-gmp signaling in mammalian cells will be a crucial step toward achieving this aim. mice and cell lines. bone marrow macrophages were derived from various mouse strains, including mavs / (sun et al., ) , mda / (gitlin et al., ) , and zbp / (ishii et al., ) . wild-type c bl/ and tnfr / mice were from the jackson laboratory. myd / /trif / mice were obtained from g. barton (university of california, berkeley, berkeley, ca). irf / , irf / , irf / , and irf / mice were obtained from t. taniguchi (university of tokyo, tokyo, japan). tbk / /tnfr / mice were generated by crossing tbk / mice (provided by w.-c. yeh, university of toronto, toronto, canada) with tnfr / mice. rip / mice were obtained from r. medzhitov (yale university, new haven, ct). nod / /nod / mice were obtained from d. portnoy (university of california, berkeley, berkeley, ca). nalp / mice were obtained from v. dixit (genentech, south san francisco, ca). isre-l ifn reporter cells were obtained from vance is a recipient of a cancer research institute investigator award, and is supported by the hellman family faculty fund and national institutes of health grants ai , ai , and ai . the authors have no conflicting financial interests tlr-independent type i interferon induction in response to an extracellular bacterial pathogen via intracellular recognition of its dna identification of icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for legionella pneumophila intracellular growth c-di-gmp-mediated regulation of virulence and biofilm formation listeria monocytogenes multidrug resistance transporters activate a cytosolic surveillance pathway of innate immunity the bacterial second messenger cyclic digmp exhibits potent adjuvant properties ikkepsilon and tbk are essential components of the irf signaling pathway novel domains of the prokaryotic two-component signal transduction systems essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus the roles of two ib kinase-related kinases in lipopolysaccharide and double stranded rna signaling and viral infection type i interferon signaling is required for activation of the inflammasome during francisella infection identification of lps as a key transducer of myd -independent tir signalling -triphosphate rna is the ligand for rig-i a toll-like receptor-independent antiviral response induced by double-stranded b-form dna tank-binding kinase- delineates innate and adaptive immune responses to dna vaccines cd is required for myd -independent lps signaling nk cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-mhc class i: the rheostat model -cyclic diguanylic acid (c-di-gmp) inhibits basal and growth factor-stimulated human colon cancer cell proliferation the chemotherapeutic agent dmxaa potently and specifically activates the tbk -irf- signaling axis regulation of cellulose synthesis in acetobacter xylinum by cyclic diguanylic acid brucella requires a functional type iv secretion system to elicit innate immune responses in mice differential recognition of double-stranded rna by rig-i-like receptors in antiviral immunity innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna preclinical and clinical safety studies on dna vaccines identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf triggering the interferon antiviral response through an ikk-related pathway control of bacterial transcription, translation and replication by (p) the type i ifn response to infection with mycobacterium tuberculosis requires esx- -mediated secretion and contributes to pathogenesis recognition of cytosolic dna activates an irf -dependent innate immune response type i interferons in host defense the specific and essential role of mavs in antiviral innate immune responses dai (dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response roles of cyclic diguanylate in the regulation of bacterial pathogenesis multiple functions of the ikk-related kinase ikkepsilon in interferon-mediated antiviral immunity comparison of gene coverage of mouse oligonucleotide microarray platforms induction of cd (+) t cell-dependent cd (+) type responses in humans by a malaria dna vaccine regulation of innate immune responses by dai (dlm- /zbp ) and other dna-sensing molecules structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by dna recombination intermediates visa is an adapter protein required for virus-triggered ifn-beta signaling c-di-gmp ( - -cyclic diguanylic acid) inhibits staphylococcus aureus cell-cell interactions and biofilm formation bacterial c-di-gmp is an immunostimulatory molecule cyclic di-gmp stimulates protective innate immunity in bacterial pneumonia differential roles of mda and rig-i helicases in the recognition of rna viruses length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene a new synthetic approach to cyclic bis( → )diguanylic acid ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction distinct tlr-and nlr-mediated transcriptional responses to an intracellular pathogen a cyclic-di-gmp receptor required for bacterial exopolysaccharide production structure and function of the interferon-beta enhanceosome ifn-regulatory factor -dependent gene expression is defective in tbk -deficient mouse embryonic fibroblasts recognition of microorganisms and activation of the immune response cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus tank-binding kinase- plays an important role during in vitro and in vivo type i ifn responses to dna virus infections innate recognition of bacteria by a macrophage cytosolic surveillance pathway legionella pneumophila induces ifnbeta in lung epithelial cells via ips- and irf , which also control bacterial replication the enhanceosome differential requirement for tank-binding kinase- in type i interferon responses to toll-like receptor activation and viral infection rig-i-mediated antiviral responses to single-stranded rna bearing -phosphates role of adaptor trif in the myd -independent toll-like receptor signaling pathway a dna vaccine induces sars coronavirus neutralization and protective immunity in mice the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses accepted: july elisa. levels of rantes protein in supernatants from bone marrow macrophages were measured using the rantes duoset elisa kit (r&d systems) according to the manufacturer's protocol.microarray analysis. macrophage rna from cells ( -well dishes) was isolated using the ambion rnaqueous kit (applied biosystems) and amplified with the ambion amino allyl messageamp ii arna amplification kit (applied biosystems) according to the manufacturer's protocol. microarrays were performed as previously described (leber et al., ) . in brief, spotted microarrays using the meebo -mer oligonucleotide set (illumina) were printed at the university of california, san francisco center for advanced technology. microarray probes were generated by coupling amplified rna to cy dyes. after hybridization, arrays were washed, scanned on a genepix b scanner (mds analytical technologies), and gridded using spotreader software (niles scientific). analysis was performed using the genepix pro and acuity software packages (mds analytical technologies). two independent experiments were performed. microarray data have been deposited in the gene expression omnibus under accession no. gse .cell fractionation, immunoblotting, and gel shift assays. for analysis of map kinase activation, whole-cell extracts were prepared from × bone marrow macrophages stimulated for the times indicated in the figures and lysed in ripa buffer supplemented with mm naf, mm navo , mm -glycerol phosphate, mm pmsf, and × complete protease inhibitor cocktail (roche). for analysis of irf nuclear translocation, nuclear extracts were prepared from × bone marrow macrophages stimulated for the times indicated in the figures using ne-per nuclear extraction reagent (pierce), supplemented with × complete protease inhibitor cocktail, using the manufacturer's protocol. protein levels were normalized using the micro-bca kit (thermo fisher scientific) and separated on % nupage bis-tris gels (invitrogen). proteins were transferred to polyvinylidene fluoride membranes and immunoblotted with various primary antibodies. whole-cell extracts were probed with anti-p map kinase, anti-phospho-p map kinase (thr /tyr ), anti-erk / , anti-phospho-erk / (thr / tyr ), anti-sapk/jnk, and anti-phospho-sapk/jnk(thr /tyr ) antibodies from cell signaling technology. nuclear extracts were probed with anti-irf and anti-lamin b (fl- and m- ; santa cruz biotechnology, inc.). for emsa, nuclear extracts from stimulated bone marrow macrophages were incubated with a p end-labeled nf-b probe ( -gat-cagttgaggggactttcccaggc- ). protein-dna complexes were resolved by native gel electorophoresis and visualized by autoradiography. anti-p (c- ) was obtained from santa cruz biotechnology, inc.in vivo experiments. c bl/ or irf / / mice were injected intraperitoneally with nmol of chemically synthesized c-di-gmp. for analysis of innate immune responses, mice were sacrificed and analyzed - h after injection, as indicated in the figures. serum was collected and tested for ifn as described above. splenic nk cells were assayed ex vivo for production of ifn- in response to yac- target cells, as described previously (joncker et al., ) . for antibody responses, mice were injected with µg of endotoxin-free hsa (sigma-aldrich) mixed with nmol c-di-gmp or saline. wk after injection, mice were bled and serum igg specific for hsa was measured by elisa.online supplemental material. fig. s shows production of rantes in response to c-di-gmp in wild-type, myd / trif / , tnfr / , tbk / tnfr / , mavs / , irf / , irf / , irf / , irf / , and ifnar / bone marrow-derived macrophages. table s contains whole-transcriptome microarray gene expression data for bone marrow-derived macrophages transfected with c-di-gmp and pda:dt. online supplemental material is available at http://www.jem.org/cgi/content/full/jem. /dc .we thank the vance, barton, and portnoy laboratories for discussions, j. leber for advice, and n. arpaia, l. lau, and t. kuo for advice and technical assistance. key: cord- - d rn authors: suthar, mehul s.; ma, daphne y.; thomas, sunil; lund, jennifer m.; zhang, nu; daffis, stephane; rudensky, alexander y.; bevan, michael j.; clark, edward a.; kaja, murali-krishna; diamond, michael s.; gale, michael title: ips- is essential for the control of west nile virus infection and immunity date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d rn the innate immune response is essential for controlling west nile virus (wnv) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. herein, we show that ips- , the central adaptor protein to rig-i-like receptor (rlr) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic wnv. ips- (−/−) mice exhibited increased susceptibility to wnv infection marked by enhanced viral replication and dissemination with early viral entry into the cns. infection of cultured bone-marrow (bm) derived dendritic cells (dcs), macrophages (macs), and primary cortical neurons showed that the ips- -dependent rlr signaling was essential for triggering ifn defenses and controlling virus replication in these key target cells of infection. intriguingly, infected ips- (−/−) mice displayed uncontrolled inflammation that included elevated systemic type i ifn, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory dcs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific cd + t cells and non-specific immune cell proliferation in the periphery and in the cns. this uncontrolled inflammatory response was associated with a lack of regulatory t cell expansion that normally occurs during acute wnv infection. thus, the enhanced inflammatory response in the absence of ips- was coupled with a failure to protect against wnv infection. our data define an innate/adaptive immune interface mediated through ips- -dependent rlr signaling that regulates the quantity, quality, and balance of the immune response to wnv infection. west nile virus (wnv) is a neurotropic flavivirus and is an emerging public health threat. infection with wnv now constitutes the leading cause of mosquito-borne and epidemic encephalitis in humans in the united states [ ] . wnv is enveloped and contains a single strand positive sense rna genome of approximately kb in length that encodes three structural (c, prm/m, and e) and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ). it cycles enzootically between birds and culex mosquitoes, with humans infected as dead-end hosts. wnv infection has been modeled in inbred mice wherein infection and pathogenesis recapitulate many of the features of human infection (reviewed in [ ] ). following subcutaneous inoculation, wnv replicates in dendritic cells (dcs) at the portal of entry and in the draining lymph node. a primary viremia develops and virus spreads to visceral organs including the spleen, where further amplification occurs, leading to central nervous system (cns) dissemination and encephalitis. in humans, wnv causes an acute febrile illness that can progress to severe and sometimes lethal neuroinvasive disease, especially in the elderly and immunocompromised [ ] . however, healthy young adults are also afflicted with severe neurological disease [ , , ] , indicating that virulence can occur independently of immune deficiencies or aging. intracellular innate immune defenses and the actions of type i interferon (ifn) provide a first-line of defense against virus infection and are essential for the control of wnv replication, dissemination, and neurovirulence [ ] . innate antiviral immune defenses are triggered through the recognition of conserved pathogen associated molecular pattern (pamp) motifs within viral products by intracellular pathogen recognition receptor (prr) proteins in infected cells. prr signaling directs downstream activation of latent transcription factors, including nf-kb, interferon regulatory factor (irf)- and irf- , in a cell type-specific manner to induce antiviral response programs that include expression of proinflammatory cytokines, chemokines, type i ifn, and interferon stimulated genes (isgs) [ , , , ] . the isg products induced through autocrine and paracrine actions of ifn confer antiviral activity by limiting virus replication and cell-to-cell virus spread. modulation of ifn signaling has been identified as a virulence feature of pathogenic strains of wnv [ , ] . the rlrs, retinoic acid inducible gene-i (rig-i) and melanoma differentiation antigen (mda ) [ , , , ] , are prrs that play critical roles in triggering immune defenses against rna virus infection, including wnv. rig-i and mda are cytosolic rna helicases that contain an amino terminal tandem caspase activation and recruitment domain (card). upon engaging rna substrates, the rlrs undergo a conformational change and bind to the mitochondrial associated protein, interferon promoter stimulator- (ips- ) through a card-card interaction, leading to ips- -dependent signaling of ifn production and expression of immune response genes [ , ] . rlr signaling and ips- function have an essential role in triggering ifn defenses during wnv infection of mouse embryo fibroblasts (mefs) and human cell lines in vitro. cells lacking either rig-i or mda were attenuated in their ability to generate an effective innate immune response to infection, whereas cells lacking both rig-i and mda or those deficient in ips- alone were unable to respond to infection with wnv and related flaviviruses [ , , , ] . recent studies examined the role of another class of pattern recognition receptors, toll like receptor (tlr) and tlr , and show that these receptors are also important prrs of wnv infection, as they play a role in signaling ifn production and an inflammatory response upon viral ligand recognition [ , , ] . tlr has been shown to contribute to both enhancement and protection of cns inflammation and neurovirulence of wnv in vivo [ , ] , while tlr -dependent signaling was shown to be essential for directing proper immune cell homing to sites of wnv infection during the adaptive immune response in vivo [ ] . type i ifn, a major product of prr signaling, has been shown to link innate and adaptive immune responses. however, the specific prr pathways that mediate this during acute wnv infection have not been delineated nor has the rlr pathway been evaluated in this context. the quantity and quality of the innate and adaptive immune responses after infection must be carefully regulated to avoid aberrant inflammation and immunopathogenesis. regulatory t (t reg ) cells and inflammatory dendritic cell (dc) subsets regulate inflammation during acute virus infection through t cell suppression and by modulating the trafficking and inflammatory cytokine production of immune cells into infected tissues [ , , ] . thus, the level of local and peripheral t reg cells, and the composition of local dc subsets that develop during wnv infection may determine immune control and wnv disease. here, we assessed the role of rlr signaling and ips- in wnv infection and immunity. our studies define ips- as an essential modulator of immunity in vivo and demonstrate that ips- -dependent signaling orchestrates an innate/adaptive immune interface that regulates immune responses to effectively control wnv infection. wnv infection of primary embryonic fibroblasts recovered from rig-i / mice revealed that rig-i was important in eliciting innate antiviral immune defenses early during infection, whereas mda was important for enhancing and sustaining this response [ ] . we further evaluated wnv infection of rig-i / or mda / mice and confirmed that rig-i serves a dominant role among the rlrs for the acute induction of innate immune defenses and protection against wnv infection in vivo (data not shown). since the rlrs signal innate defenses through the ips- adaptor protein [ ] , we also examined the role of ips- in protection against wnv infection upon a sub-lethal virus challenge of wild type and ips- / mice. ips- / mice were highly susceptible to wnv infection and exhibited % mortality with an average survival time (ast) of . days as compared to wild type mice ( . % mortality with an ast of . days; p, . ; fig a) . thus, rig-i and ips- -dependent signaling are essential for protection against wnv infection. to define the role of ips- in controlling wnv in vivo, wild type and ips- / mice were infected subcutaneously (s.c.) with pfu of wn-tx and viral burden within peripheral tissues and the cns was measured over time post-infection (pi). ips- / mice exhibited increased viremia compared to wild type mice ( . fold enhancement at day pi, p, . ) throughout the course of infection (fig b) . similarly, viral loads in the spleen were elevated in the infected ips- / mice (fig c) . wnv infection of ips- / mice displayed an expanded tissue tropism as infectious virus was found in the kidneys, a tissue that is not normally permissive to infection in wild type mice ( fig d) . wnv is typically detected in the cns of wild type mice after s.c. challenge between and days pi [ ] . consistent with this time course, infected wild type mice exhibited detectable viral loads (average viral titer of . pfu/gram of tissue) in the brain by day p.i., although virus was not detected in the spinal cord (fig e and f ). in contrast, wnv spread to the brain ( fig e) and spinal cord of ips- / mice (fig f) by day pi, with viral loads rising through day pi. together these results indicate that ips- , likely west nile virus (wnv) is a mosquito-transmitted rna virus that has emerged in the western hemisphere and is now the leading cause of arboviral encephalitis in the united states. however, the virus/host interface that controls wnv pathogenesis is not well understood. previous studies have established that the innate immune response and interferon (ifn) defenses are essential for controlling virus replication and dissemination. in this study, we assessed the importance of the rig-i like receptor (rlr) signaling pathway in wnv pathogenesis through analysis of mice lacking ips- , the central adaptor molecule of rlr signaling. our studies revealed that ips- is essential for protection against wnv infection and that it regulates processes that control virus replication and triggering of innate immune defenses. we found that ips- plays an important role in establishing adaptive immunity through an innate/adaptive interface that elicits effective antibody responses and controls the expansion of regulatory t cells. thus, rlrs are essential for pathogen recognition of wnv infection and their signaling programs help orchestrate immune response maturation, regulation of inflammation, and immune homeostasis that define the outcome of wnv infection. through rlr signaling of innate immune defenses, limits wnv replication, viremia, and peripheral spread, and is essential for the control of viral invasion of the cns. myeloid cells, including tissue and lymphoid dc and macrophages (mw), are among the first cells to encounter wnv during infection and thus function to restrict the spread of virus to distant tissues and the cns [ ] . to define the role of ips- in controlling virus replication and innate immunity in myeloid cells, we analyzed wnv infection and host responses in primary bone marrow-derived dc and mw recovered from wild type and ips- / mice. dc and mw were infected at an moi of . (relative to viral plaque assay quantification of bhk- cells; see methods) and evaluated for virus replication, ifn induction, and innate immune triggering of isg expression (fig ) . ips- / dcs sustained significantly higher wnv replication at and hours pi compared to wild type infected cells (fig a) . wnv infection of wild type dcs induced ifn-b secretion but this response was completely abolished in ips- / dcs (fig b) . the lack of ifn-b induction in ips- / dcs correlated with a lack of isg expression including rig-i, mda , and stat- ( fig c) . in addition, expression of isg and isg , which are direct irf- target genes [ , ] , were not induced during wnv infection of ips- / dcs (fig c) . moreover, isg , another irf- target gene [ ] , was induced late during infection and to lower levels as compared to isg and isg in wild type, infected dcs. wnv infection of ips- / mw resulted in significantly higher virus replication between and hours pi as compared to infected wild type cells ( fig d) . whereas wild type infected mw expressed ifn-b, this response was completely abolished in ips- / mw (fig f) . we also observed a differential expression of isgs and irf- target genes within wnv-infected mw. rig-i, mda , and stat- were not induced in ips- / mw, whereas, isg , isg , and pkr were expressed at reduced levels and with delayed kinetics. these data establish that ips- -dependent rlr signaling is the major innate immune signaling pathway that controls virus replication in conventional dcs and mw. the rlr signaling pathway triggers the innate immune response to wnv infection in primary cortical neurons neurons represent the target cell of wnv infection in the cns and their death after infection is a key factor in pathogenesis and neurological sequelae [ , ] . to define the role of rlr signaling in restricting virus replication in neurons, primary cortical neurons were generated from wild type and ips- / mice. cells were infected at an moi of . with wn-tx and virus yield, ifn-b induction, and isg expression were evaluated. in the absence of ips- , wnv replicated faster and to higher levels resulting in a . and . -fold (p, . ) increase in viral production at hrs and pi, respectively as compared to infected wild type neuronal cells ( fig a) . this relatively modest virologic effect in neurons compared to that observed in ips- / dc and mw was expected, as ifn-a or -b pre-treatment only inhibits wnv infection in cortical neurons to a maximum of to -fold [ ] , suggesting that the ifn response is comparably less potent in neurons. ifn-b expression was induced to lower levels in ips- / neurons compared to wild type infected neurons at ( -fold, p, . ) and hours pi ( -fold, p, . ) despite the higher levels of virus replication (fig a and b) . expression of isgs, (including rig-i and mda ) and irf- target genes (including isg and isg ) followed this pattern and were dependent on ips- for rapid and high level expression ( fig c) . the presence of ifn-b and isg transcripts in ips- / cells at hrs pi is consistent with the finding that tlr has an independent and subordinate role in triggering innate immune responses in cortical neurons at later time points after wnv infection [ ] . these results demonstrate that the rlr signaling pathway controls virus replication and induces innate immune responses against wnv infection in cortical neurons. neuronal destruction and cns inflammation are enhanced in wnv infected ips- / mice to determine the role of the rlr pathway in protection of neurons against wnv pathogenesis in vivo, we conducted histological analysis of brain tissue from wild type and ips- / mice infected with wn-tx ( fig a) . analysis of brain sections from infected wild type mice revealed little or no inflammation or neuronal damage, with sparse and focal cell infiltrates restricted to the hippocampus and cerebral cortex on day pi. by day pi (a timepoint in wild type mice in which peak virus replication in the cns occurs [ ] ) cellular infiltrates were present in the parenchyma and neuronal destruction was observed throughout the cortex and hippocampus. in contrast, brain sections from infected ips- / mice recovered on day pi displayed extensive injury to neurons in the cortex and granular neurons of the hippocampus. damaged neurons appeared pyknotic with vacuolation, degeneration and cell dropout. somewhat surprisingly, we observed extensive inflammation in the brains from infected ips- / mice within the cortex, hippocampus, and cerebellum (data not shown) displaying prominent perivascular and parenchymal immune cell infiltrates. to evaluate the composition and antigen-specificity of the inflammatory cells within the brains of wnv-infected mice, lymphocytes were isolated from infected brains on day pi and were characterized from pools (n = ) of wild type and ips- / infected mice. brains from ips- / infected mice showed an . fold increase in the total number of immune cells as compared to wild type infected mice (fig b) , and this was associated with an increase in absolute numbers of infiltrating cd + and cd + t cells ( fig c) . among the brain cd + t cells isolated from ips- / mice, there was a remarkable -fold increase in the number of antigen-specific cells that expressed ifn-c after treatment with an immundominant ns b peptide ( fig d) [ , ] . analysis of microglia/mw cells, based on relative surface expression of cd and cd b [ ] , revealed increased numbers of microglial cells (cd + lo /cd b+) and infiltrating macrophages (cd + hi / cd b+) within the brains of infected ips- / mice when compared to wild type mice (fig e) . similar findings were observed in the spinal cords from infected ips- / mice (data not shown). combined with the histological analysis, these results demonstrate that in the absence of ips- , wnv infection induces a strong inflammatory response in the cns. while this response is likely associated with increased viral loads, the failure of this increased inflammatory response to elicit protection or control cns pathology, in the absence of ips- , suggests a role for the rlr signaling pathway as a regulatory program that controls the quality of the inflammatory response to wnv infection. to further characterize how ips- modulates the inflammatory response to wnv infection, we measured levels of systemic type i ifn, proinflammatory cytokines, and chemokines present in the serum of wnv-infected mice at and days pi. paradoxically, a trend towards more rapid induction and increased levels of type i ifn were observed in the serum of ips- / mice compared to wild type mice (fig a) . we note that in this case type i ifn was detected and quantified through a mouse-specific type i ifn bioassay, which does not differentiate between the ifn-a or -b species. this result is consistent with our recent studies showing that serum type i ifn levels accumulate during wnv infection in an irf- -dependent but irf- -independent manner [ , ] . in this case ifn-a species are likely accumulating through a tlr dependent signaling pathway involving plasmacytoid dcs, which do not require the rlr pathway for ifn production [ ] . more recently, town et al. assessed the role of tlr and myd / during wnv infection and found that mice lacking myd produced elevated levels of systemic ifn during wnv infection [ ] . thus, during wnv infection systemic ifn is regulated through rlr-dependent and independent processes. correspondingly, when compared to wild type mice, ips- / infected animals, which show higher viremia (fig b) produced increased serum levels of proinflammatory cytokines and chemokines in response to wnv infection. elevated levels of systemic il- , tnfa, cxcl , and ifn-c were observed at and/or days pi in ips- / mice (fig b) . serum cytokine levels were also compared between uninfected wild type and ips- / mice and showed no differences in basal cytokine expression (data not shown). these results demonstrate that in the absence of ips- , greater proinflammatory cytokine and chemokine responses are induced during wnv infection. altered wnv-specific antibody profiles in ips- / mice wnv-specific antibody responses are essential for suppressing viremia and virus dissemination and limiting lethal wnv infection [ , ] . to determine if a deficiency in ips- modulated the quality and quantity of the humoral immune response, we characterized the antibody profile in sera during wnv infection. in wild type mice, neutralizing virus-specific igm antibodies are typically detectable by day pi with wnv and production of neutralizing virus-specific igg antibodies follow between days and pi [ ] . a time course analysis in wild type and ips- / infected mice showed that between and days pi, wnv-infected ips- / mice exhibited significantly higher levels of virus-specific igm, igg, and igg subclasses as compared to infected wild type mice ( table ) . wnv-specific igg antibodies were detected at low levels on day pi in sera from wild type and ips- / mice. additionally, we observed a , . -fold increase in wnv-specific igg a levels in infected ips- / as compared to wild type mice on day pi and , . -fold increase on day pi. assessment of the virus-specific antibody responses through a prnt assay revealed that neutralization titers in sera from wild type mice increased dramatically between and days pi. sera from ips- / infected mice exhibited a modest increase in neutralization titer to : , despite having much higher levels of virusspecific antibodies. this difference translated into a serum neutralization index that was , -fold lower on day pi in the infected ips- / mice compared to wild type mice. these results demonstrate that the humoral responses in wnv-infected ips- / mice are distinct from responses in wild type infected mice. thus, rlr signaling and ips- actions likely contribute to regulatory processes that govern the levels, igg class switching, and neutralizing capacity of antibodies generated in response to wnv infection. to characterize the immune parameters associating with the dysregulated inflammatory and humoral responses in wnv infected ips- / mice, we analyzed the immune cell composition in draining lymph node and spleen tissues. wild type and ips- / mice were challenged with diluent alone or with wn-tx, and draining popliteal lymph node (dln) and the spleen were harvested at and days pi, respectively. analysis of the popliteal dln provides insight into how ips- modulates the inflammatory response immediately after infection whereas assessment of the spleen elucidates characteristics of the adaptive immune response prior to the infection endpoint. immune cells were isolated from the popliteal dln and were characterized by flow cytometry (fig ) . analysis of cd a dc subsets, which are phenotypically the major antigen presenting cells within lymphoid tissues and are implicated in eliciting virus-specific cd t cell in response to acute wnv infection [ ] , showed that infected wild type and ips- / mice exhibited similar increases in the numbers of cd a + and cd a dcs compared to mock-infected mice (fig a, b) . however, a significant increase (, -fold; p, . ) of a proinflammatory dc subset, characterized as cd c + cd b hi ly c + , was observed within the popliteal dlns of ips- / infected mice (fig c) . this dc subset is monocytederived and typically recruited to sites of acute inflammation where they propagate the inflammatory response [ ] . we found that these dc subsets were not significantly expanded and showed no differences in their recruitment to the dln in either wild type or ips- / infected mice at hours pi (data now shown). thus, as early as hours pi, an elevated cellular inflammatory response is initiated in the ips- / mice. in contrast, similar increases in plasmacytoid dcs were observed within infected wild type and ips- / infected mice (fig d) , demonstrating that an absence of ips- did not directly affect expansion and/or recruitment of this dc subset. within the popliteal dlns, mock-infected ips- / mice compared to wild type mice generally showed elevated numbers of b cells, cd + t cells (p, . ), and cd + t cells (fig e, f, and g) . we further analyzed the lymphocyte composition of the spleen on day after wnv infection (fig ) . gross pathologic analysis revealed that wnv infection of ips- / mice results in massive splenomegaly whereas infection of wild type mice induces only a slight increase in spleen size (fig a) . cell analysis revealed increased numbers of total lymphocytes in the spleens of infected ips- / mice as compared to wild type mice (fig b) . regulatory t (t reg ) cells have recently been shown to contribute to the dampening of inflammation and adaptive immune responses during acute virus infections [ , , ] . moreover, a reduction in the number of circulating t reg in mice leads to enhanced lethality after wnv infection [ ] . a time course analysis of t regs in wild type mice revealed that wnv infection results in a significant increase in the numbers of foxp + t cells as compared to mock-infected mice beginning on day and peaking by day pi (fig c) , indicating the expansion of t regs during acute wnv infection. despite their marked increase in viral load, the infected ips- / mice did not display an increase in the numbers of foxp + t cells at any timepoint analyzed. thus, ips- signaling directly or indirectly impacts t reg proliferation and does so independently of viral load. we also observed that spleens from infected ips- / mice exhibited significantly increased numbers of cd + t cells in comparison to those from infected wild type mice, whereas the expansion of splenic cd + t cells in wild type and ips- / mice were not different (fig d) . the spleens from wnv-infected ips- / mice showed significantly higher numbers ( . -fold; p, . ) of wnv-specific cd + t cells producing ifnc. to further define the phenotype associated with wnv-induced splenomegaly in ips- / mice, we also assessed the numbers of nk cells and neutrophils. spleens from infected ips- / mice contained greater numbers of nk cells (cd cd nk . + cells), nkt cells (cd +/cd +/nk . + cells) and neutrophils (cd b + gr + cells) (fig e) . although wnvinfected wild type mice infected displayed slight increases in the absolute numbers of these specific cell types, a deficiency of ips- resulted in a more marked enhancement of these immune cell populations. in this study, we establish a major role for rlr signaling in protection from wnv pathogenesis, and demonstrate that ips- is critical for the control of wnv infection in vivo. our studies indicate that ips- -dependent rlr signaling functions to establish balanced, effective, and protective innate and adaptive immune responses, and that ips- links adaptive immune regulation with the innate immunity triggered by rlr signaling during wnv infection. in the absence of ips- in vitro, innate immune defense programs of myeloid dcs and macrophages were ablated or severely attenuated. moreover, in vivo analysis of infected ips- / mice showed altered igg and igm antibody responses with diminished virus neutralization activity. the inflammatory response to wnv infection is regulated by ips- -dependent processes such that a deficiency of ips- resulted in elevated proinflammatory cytokine and chemokines and increased numbers of inflammatory dcs, antigen-specific t cells, natural killer cells, and neutrophils in lymphoid organs, and activated macrophage/ microglial cells within the cns. the dysregulated inflammatory response to wnv infection in ips- / mice was associated with a reduction in the numbers of t reg cells and their failure to expand during acute infection. these observations demonstrate the critical role of ips- in mediating rlr signaling of innate antiviral immunity against wnv infection, and reveal novel features of ips- function in regulating immune homeostasis, inflammation, and adaptive immunity to infection. although infection of primary dcs, macrophages, and neuronal cells failed to induce type i ifn upon wnv infection, wnvinfected ips- / mice showed enhanced systemic type i ifn responses. this finding agrees with previous studies that indicate both ips- -dependent and -independent pathways contribute to the systemic type i ifn production in vivo [ , , , ] . most importantly, the enhanced tissue tropism and rapid viral entry into the cns observed in the ips- / mice is not affected by the elevated systemic ifn responses. this suggests that local tissuespecific and intracellular responses triggered by rlr-dependent signaling are more essential for reducing viral burden and dissemination. one possible explanation is that a distinct set of rlr-responsive genes function to control virus replication at the site of infection. this could explain, in part, the elevated levels of virus replication, enhanced tissue tropism and cell-to-cell spread in mice or cells deficient in irf or irf- , each of which are downstream transcription factors of rlr signaling [ , , ] . additionally, it is likely that pdcs, which are specialized dendritic cells for producing systemic type i ifn during a viral infection [ ] , are likely contributing to the ifn responses observed during wnv infection. studies by silva et al. have shown that wnv triggers ifn induction in pdcs through a replication-independent manner [ ] . interestingly, within the dln, we observed similar expansion of pdcs between wild type and ips- / infected mice, yet at the same timepoint ( hours pi), elevated systemic type i ifn responses were observed in ips- / mice. this suggests two possibilities: ) splenic pdcs or circulating pdcs are likely responding to the high levels virus in the serum from the ips- / infected mice to produce ifn at hours pi and/or ) various other cell types that express tlr and/or tlr are responding to wnv infection and contributing to systemic ifn responses. taken together, these studies indicate that rlr signaling and the actions of irf- / are important in triggering ifn production and isg expression to limit wnv replication and spread, and that tlr signaling may impart additional, rlrindependent defenses that regulate immunity against wnv infection. the production of and response to type-i ifn is a major linkage point between innate and adaptive immunity, as ifn-a and ifn-b sustain b cell activation and differentiation [ , , ] , expand antigen-specific cd + t cells [ , ] , cd + t cells [ ] , and activation of nk cells [ ] . one of the most intriguing aspects of this study was the global alteration of the immune response elicited in the ips- / mice, indicating that rlr signaling couples innate immunity with regulation of the adaptive immune response. infection of ips- / mice exhibited increased igm and igg wnv-specific antibodies, enhanced wnv-specific cd +t cell response, and increased expansion of neutrophils, nk cells and nk-t cells. one trivial explanation for these differences is that there is an increased antigen load in the absence of ips- and, as a result, enhanced virus-specific (e.g. cd + t cells, igg and igm antibodies) and nonspecific (e.g. neutrophils, nk cells) responses. however, there are several key findings from this study that argue against these responses simply being attributed to higher antigen load: ( ) in the absence of ips- , the cd and cd t cells, which are protective against wnv infection [ , , , , , , ] , were significantly enhanced in the peripheral and cns compartments but failed protect against infection. one explanation for this observation is that the expansion and migration of cd and cd t cells to different tissues was itself uncontrolled, resulting in t cell-mediated pathology rather than t cell-mediated protection. ( ) while the quantity of virus-specific igm and igg antibody responses were greatly enhanced in the absence of ips- , there was a marked reduction in antibody quality in terms of neutralization capacity. in contrast deficiencies in tlr or myd (data not shown) did not alter virus-specific antibody responses and neutralization capacities. collectively, these findings suggest that rlr-dependent signaling coordinates effective antibody responses during wnv infection through as yet undefined pathway. ( ) while systemic ifn responses provide a link between innate and adaptive immune responses, our studies suggest that the prr signaling pathways (rlr-dependent vsindependent) and levels of ifn production in combination with production other proinflammatory cytokines or chemokines regulate the quantity and quality of the immune response during virus infection. thus, in the absence of ips- signaling, infected conventional dcs or mw, two integral cell types in establishing adaptive immunity, likely do not produce ifn or the normal array and level of proinflammatory cytokines/ chemokines. instead, ifn and other mediators may be strictly produced by infected or bystander cells during wnv infection, occurring with altered kinetics and magnitude, through tlr-dependent pathways, such as tlr and/or tlr [ , ] . ( ) in the absence of ips- , the enhanced expansion of ly c+ ''inflammatory'' dcs failed to limit early wnv replication and dissemination. this inflammatory dc subset migrates to sites of infection, secretes pro-inflammatory cytokines, and promotes cd + t cell expansion during a secondary virus infection, suggesting it sustains the effector t cell response [ ] . our data indicate that ly c+ dc recruitment and/or expansion is governed by ips- -dependent events of rlr signaling. thus, the aberrant recruitment/expansion of these inflammatory dcs may contribute to immunopathogenesis and limit development of an effective immune response to control wnv virus infection. ( ) the lack of t reg expansion during wnv infection correlated with altered ifn levels, increased proinflammatory cytokines and chemokine levels, and an increased number and distribution of antigen-specific cd + t cells. these observations implicate an indirect or direct role for ips- in regulating t reg levels during wnv infection, and provide evidence that links a lack of t reg expansion to immune dysregulation. while their importance in autoimmunity is established [ ] , recent studies have implicated an integral role for t regs in controlling inflammation and adaptive immune responses during acute virus infections [ , , ] . during acute infection t regs function to locally contain and control the immune response with the dual outcome of suppressing viral dissemination while decreasing the likelihood of immune-mediated pathology. in support of this model, infection studies with herpes simplex virus (hsv) and mouse hepatitis virus (mhv), two well established models of viral encephalitis, have demonstrated the importance of t regs in limiting proinflammatory cytokine and chemokine responses to protect the cns and enhance survival [ , ] . recent work also implicates t regs in the control of wnv pathogenesis, wherein peripheral expansion of t regs was associated with asymptomatic infection among wnv-infected blood donors but reduced t reg levels associated with wnv disease [ ] . furthermore, these studies revealed that the conditional depletion of t reg cells in mice results in enhancement of wnv virulence and expansion of antigen-specific cd t cells. interestingly, from our studies, type i ifn does not appear to be the major contributor to t reg expansion during wnv infection, as t regs failed to expand in the ips- / infected mice despite their elevated levels of systemic type ifn. these observations suggest that rlr signaling through ips- provides essential signals that directly or indirectly impart the expansion of t regs during wnv infection. we propose that ips- coordinates an innate/adaptive immune interface wherein ips- -signaling after rlr engagement regulates the quantity, quality, and balance of the subsequent immune response. the integrity of the innate/adaptive immune interface is central to the eliminating virus but also restricting immunopathogenesis and inflammation during infection. rlr signaling is essential for triggering the innate immune response to rna viruses that cause human disease, including the influenza viruses, respiratory syncytial virus and other paramyxoviruses, picornaviruses, reoviruses, flaviviruses, and hepatitis c virus [ , , ] . thus, in addition to wnv, ips- -dependent rlr signaling will likely have a broad impact for the control of inflammation, immune response quality, and viral disease. bhk and l cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), mm l-glutamine, mm sodium pyruvate, antibiotic-antimycotic solution, and nonessential amino acids (complete dmem). wnv strain tx -hc (wn-tx) was isolated by as previously described [ ] . working stocks of wn-tx were generated by a single round of amplification on vero-e (ccl- ; atcc) cells, and supernatants were collected, aliquoted, and stored at uc. virus stocks were titered by a standard plaque assay on bhk cells as previously described [ ] . ips- / (c bl/ sv/ev) and their wild type littermate control mice have been published [ , ] and were obtained as a generous gift from dr. s. akira (osaka university, osaka, japan). mice were genotyped and bred under pathogen-free conditions in the animal facility at the university of washington. experiments were performed with approval from the university of washington institutional animal care and use committee. the methods for mice use and care were performed in accordance with the university of washington institutional animal care and use committee guidelines. age-matched six to twelve week old mice were inoculated subcutaneously (s.c.) in the left rear footpad with pfu of wn-tx in a ml inoculum diluted in hanks balanced salt solution (hbss) supplemented with % heatinactivated fbs. mice were monitored daily for morbidity and mortality. for in vivo virus replication studies, infected mice were euthanized, bled, and perfused with ml of phosphate-buffered saline (pbs). whole brain, spinal cord, kidney, and spleen were removed, weighed, homogenized in ul of pbs, and titered by plaque assay. bone-marrow derived dc and mw were generated as described previously [ ] . briefly, bone marrow cells from wild type and congenic deficient mice were isolated and cultured for days in either rpmi- supplemented with granulocyte-macrophagecolony stimulating factor, and interleukin- (peprotech) to generate myeloid dc or in dmem supplemented with macrophage colony stimulating factor (peprotech) to generate mw. on day , dc or mw were infected with wn-tx at an moi of . and at , , , and hours post-infection (hpi), supernatants were collected for titration of viral burden by plaque assay on bhk cells and levels of ifn-b (described below). cells were collected in parallel for western blot analysis. cortical neurons were isolated from -day-old embryonic mice and cultured as described previously [ ] . on day of culture, neurons were infected with wn-tx at an moi of . and at , , , and hpi, supernatants were collected for virus titration by plaque assay on bhk cells and cells were collected for rna analysis by rt-qpcr (described below). cells were lysed in modified ripa buffer ( mm tris [ph . ], mm nacl, . % sodium deoxycholate, and % triton x- ) supplemented with protease inhibitor cocktail (sigma) and phosphatase inhibitor cocktail ii (calbiochem). protein extracts ( mg) were analyzed by immunoblotting as described previously [ ] . the following primary antibodies were used to probe blots: mouse anti-wnv from the center for disease control; rabbit anti-isg , rabbit anti-isg , rabbit anti-isg , kindly provided by dr. g. sen; mouse anti-pkr from santa cruz; rabbit anti-rig-i and rabbit anti-mda from ibl; mouse anti-tubulin from sigma; and rabbit anti-stat- from cell signaling. secondary antibodies included peroxidase-conjugated goat anti-rabbit, goat anti-mouse, donkey anti-rabbit, and donkey anti-mouse were from jackson immunoresearch. for analysis of viremia, serum was separated (bd microtainer tube sst) and rna was extracted as previously described [ ] . wnv rna copy number was measured by rt-quantitative pcr (rt-qpcr) as previously described [ ] . for cultured cells, total rna was extracted using the rneasy kit (qiagen), dnase treated (ambion) and evaluated for isg , isg , ifn-b, rig-i, and mda rna expression by one-step sybr green rt-qpcr. specific primer sets for isg- , isg- , rig-i, and ifn-b have been described previously [ , ] . primer sets for mda are: -gtggtcgagccagagctgat and -tgtctcatg-ttcgataactcctgaa. ifn-a and -b were measured in sera using a biological assay as previously described [ ] . briefly, l cells were seeded at cells/well in a well plate one day prior to the addition of interferon standards or experimental samples. mouse sera (diluted : in l media) were treated with uv light for minutes to eliminate residual virus. duplicate sera samples were then added to the -well plates in two-fold dilutions along with a murine ifnb standard. the following day, emcv challenge virus was added to the cells in ml/well at an moi of . . twenty-four hours later, cytopathic effect was measured by a blinded scorer and ifn levels in the sera was calculated based on the ifn standard. ifn-b in cell culture supernatants was analyzed using mouse-specific elisa kits from pbl biomedical laboratories according to the manufacturer's protocol. wnv-specific igm, total igg, igg , and igg a levels were determined by an elisa using purified recombinant e protein as previously described [ ] . the neutralization titer of serum antibody was determined by using a previously described plaque reduction neutralization assay [ ] . briefly, sera samples from mock or wn-tx infected mice were diluted in dmem followed by incubation at uc for minutes to inactivate virus and complement factors. sera were further diluted in two-fold increments and incubated with pfu of wn-tx at uc for hour. standard plaque assays were performed on bhk cells and the dilution at which % of plaques were neutralized was determined by comparing the number of plaques formed from wnv-infected sera samples to mock infected sera samples. cytokine/chemokine analysis wnv infected sera were analyzed for the presence and levels of tnf-a, ifn-c, cxcl (ip- ), and il- by a mouse-specific cytokine/chemokine milliplex elisa (millipore). mock-infected or wnv-infected mice were exsanguinated and perfused with pbs, % paraformaldehyde, ph . . brains were embedded in paraffin and -mm sections were prepared and stained with hematoxylin and eosin (h&e) by the uw histology pathology laboratory. sections were analyzed using a nikon eclipse e microscope (uw keck microscope facility). draining lymph nodes from mice were isolated and digested with collagenase (roche) and type i dnase in serum-free rpmi media at uc for minutes with mechanical disruption. cells were then incubated with rpmi media containing % fbs with edta and hepes for minutes at room temperature, pelleted, and resuspended in pbs containing % fbs and . % sodium azide (facs staining buffer). splenocytes were isolated, washed, and re-suspended in rpmi containing % fbs before in vitro stimulation. cells were washed twice before facs staining. for isolation of cns immune cells, mice were euthanized and perfused extensively with pbs to remove residual intravascular leukocytes. brains and spinal cords from mice per experimental group were isolated and pooled. tissues were minced in rpmi media, triturated, and digested with liberase (roche) and type i dnase in serum-free rpmi media at uc for min. immune cells were isolated after gradient centrifugation from a / % percoll interface and washed twice with facs staining buffer. immune cells were stained with antibodies specific to cd c, cd b, b , cd , cd , cd , cd , nk . , gr- , siglec h, and cd (all reagents from ebiosciences). intracellular foxp staining was performed as previously described [ ] . intracellular ifn-c staining was performed on splenocytes and cns immune cells as previous described [ , ] . briefly, lymphocytes were stimulated with mg/ml of the wnv ns b peptide (ssvwnat-tai) for h at uc. cells were washed and stained for cell surface markers followed by permeabilization-fixation using the cytofix-cytoperm kit (bd-pharmingen) and stained with a pacific-blue conjugated ifn-c antibody (ebiosciences) at uc for min, washed and analyzed by flow cytometry. flow cytometry was performed on a bd lsrii machine using bd facsdiva software. cell analysis was performed on flowjo (v. . . ) software. for in vitro studies and immune cell analysis an unpaired student t-test was used to determine statistical differences. for in vivo viral burden analysis, mann-whitney analysis was used to determine statistical differences. kaplan-meier survival curves were analyzed by the log-rank test. a p-value # . was considered significant. all data were analyzed using prism software (graphpad prism ). west nile virus activity-united states pathogensis of west nile virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion virology, pathology, and clinical manifestations of west nile virus disease west nile virus meningoencephalitis in an immunocompetent adolescent severe west nile virus disease in healthy adults emerging viruses in transplantation: there is more to infection after transplant than cmv and ebv evasion and disruption of innate immune signalling by hepatitis c and west nile viruses cell-specific irf- responses protect against west nile virus infection by interferondependent and -independent mechanisms interferon regulatory factor irf- induces the antiviral alpha interferon response and protects against lethal west nile virus infection the host response to west nile virus infection limits viral spread through the activation of the interferon regulatory factor pathway resistance to alpha/beta interferon is a determinant of west nile virus replication fitness and virulence alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival mda- : an interferon-inducible putative rna helicase with double-stranded rnadependent atpase activity and melanoma growth-suppressive properties regulating intracellular anti-viral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity lengthdependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiation-associated gene regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp distinct rig-i and mda signaling by rna viruses in innate immunity west nile virus evades activation of interferon regulatory factor through rig-i-dependent and -independent pathways without antagonizing host defense signaling establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda signaling through ips- differential roles of mda and rig-i helicases in the recognition of rna viruses toll-like receptor has a protective role against west nile virus infection toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis toll-like receptor mitigates lethal west nile encephalitis via interleukin -dependent immune cell infiltration and homing coordination of early protective immunity to viral infection by regulatory t cells natural regulatory t cells in infectious disease monocyte-derived dendritic cells in innate and adaptive immunity card games between virus and host get a new player novel characteristics of the function and induction of murine p family proteins transcriptional profiling of interferon regulatory factor target genes: direct involvement in the regulation of interferon-stimulated genes infection and injury of neurons by west nile encephalitis virus west nile virus infection in the golden hamster (mesocricetus auratus): a model for west nile encephalitis role of cd + t cells in control of west nile virus infection protective capacity and epitope specificity of cd (+) t cells responding to lethal west nile virus infection antigenspecific cytotoxic t lymphocytes protect against lethal west nile virus encephalitis replicon particles of venezuelan equine encephalitis virus as a reductionist murine model for encephalitis cell type-specific involvement of rig-i in antiviral response a critical role for induced igm in the protection against west nile virus infection b cells and antibody play critical roles in the immediate defense of disseminated infection by west nile encephalitis virus batf deficiency reveals a critical role for cd alpha+ dendritic cells in cytotoxic t cell immunity in vivo induction of immune responses to pathogens by conventional dendritic cells role of regulatory t cells in coronavirus-induced acute encephalitis regulatory t cells promote early influx of cd + t cells in the lungs of respiratory syncytial virus-infected mice and diminish immunodominance disparities tregs control the development of symptomatic west nile virus infection in humans and mice production of type i interferons: plasmacytoid dendritic cells and beyond differential activation of human monocyte-derived and plasmacytoid dendritic cells by west nile virus generated in different host cells early b-cell activation after west nile virus infection requires alpha/beta interferon but not antigen receptor signaling type i ifn receptor signals directly stimulate local b cells early following influenza virus infection early type i interferon-mediated signals on b cells specifically enhance antiviral humoral responses type i interferons act directly on cd t cells to allow clonal expansion and memory formation in response to viral infection innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd t cells for clonal expansion and memory formation cutting edge: the direct action of type i ifn on cd t cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection the reciprocal interaction of nk cells with plasmacytoid or myeloid dendritic cells profoundly affects innate resistance functions cd + t cells require perforin to clear west nile virus from infected neurons cd + t cells mediate recovery and immunopathology in west nile virus encephalitis west nile virus-specific cd t cells exhibit direct antiviral cytokine secretion and cytotoxicity and are sufficient for antiviral protection cd + t-cell responses are required for clearance of west nile virus from the central nervous system dendritic cell-induced memory t cell activation in nonlymphoid tissues tregs are regulated by cytokines: implications for autoimmunity essential role of ips- in innate immune responses against rna viruses pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons detection of west nile virus lineages and by real-time pcr innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna differential induction of type i interferon responses in myeloid dendritic cells by mosquito and mammalian-cell-derived alphaviruses we thank kristy szetter and jessica briley for technical help and robert immormino for generation of purified wnv e protein for elisa. key: cord- -p ufea x authors: gao, wei; sun, wenkui; qu, bingqian; cardona, carol j.; powell, kira; wegner, marta; shi, yi; xing, zheng title: distinct regulation of host responses by erk and jnk map kinases in swine macrophages infected with pandemic (h n ) influenza virus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: p ufea x swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (siv). highly virulent siv strains cause mortality of up to %. importantly, pigs have long been considered “mixing vessels” that generate novel influenza viruses with pandemic potential, a constant threat to public health. since its emergence in and subsequent pandemic spread, the pandemic (h n ) (h n pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. pathogenesis in siv or h n pdm-infected pigs remains poorly characterized. proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of siv from pig lungs. in this study, we report a unique pattern of cytokine responses in swine macrophages infected with h n pdm. the roles of mitogen-activated protein (map) kinases in the regulation of the host responses were examined. we found that proinflammatory cytokines il- , il- , il- , and tnf-α were significantly induced and their induction was erk / -dependent. ifn-β and ifn-inducible antiviral mx and ′ ′-oas were sharply induced, but the inductions were effectively abolished when erk / was inhibited. induction of ccl (rantes) was completely inhibited by inhibitors of erk / and jnk / , which appeared also to regulate fasl and tnf-α, critical for apoptosis in pig macrophages. we found that nfκb was activated in h n pdm-infected cells, but the activation was suppressed when erk / was inhibited, indicating there is cross-talk between map kinase and nfκb responses in pig macrophages. our data suggest that map kinase may activate nfκb through the induction of rig- , which leads to the induction of ifn-β in swine macrophages. understanding host responses and their underlying mechanisms may help identify venues for effective control of siv and assist in prevention of future influenza pandemics. swine influenza is an acute respiratory disease caused by swine influenza viruses (siv). the symptoms and signs generally include fever, sneezing, nasal rattles, and respiratory distress in pigs. pigs recover within a few days, but severe signs can develop and mortality can reach up to % when highly virulent strains are involved [ ] or pigs are infected at young ages [ , ] . pigs have long been considered to be the intermediate host of various subtype viruses and ''mixing vessels'' for the evolution and genesis of influenza viruses with pandemic potential because of their susceptibility to swine, avian, and human influenza viruses [ , , ] . this broad susceptibility is due to the presence of both sialic acid (sa) , gal-and sa , -gal receptors present in the respiratory epithelium. three major siv subtypes are prevalent: h n (classical swine h n and avian-like h n ), h n (triple reassortant h n and human-like h n ), and h n [ , , , , , ] . pigs are also susceptible to and show clinical signs when infected with pandemic (h n ) virus (referred to hereafter as h n pdm) [ ] , which emerged in april in north america [ ] , arising at least in part from contemporaneous siv. to date h n pdm has been found in a few swine farms [ , , ] , which further demonstrates a two-way process of both gene and virus trafficking between humans and pigs. though h n pdm has remained antigenically and genetically stable in humans since its emergence, a novel reassortant siv containing a h n pdm-like na and seven other genes from triple-reassortant h n and european ''avian-like'' h n viruses was identified in early [ ] , and that same year h n pdm was shown to be evolving genetically at a faster pace in pigs than it was in humans [ , , ] . effective control of circulating influenza viruses in swine populations is key to reducing consequent genesis of novel pandemic strains that threaten the health of both humans and animals. studies have been conducted to identify proinflammatory cytokines including tnf-a, il- , il- , and ifn-a or ifn-c, which are upregulated in lung or bronchoalveolar secretions in siv-infected pigs [ , , , ] and may be correlated with clinical manifestations. in an alveoli macrophage-depleted pig model, macrophages appeared to be indispensible to effective clearance of siv from lungs. a higher frequency of cytotoxic t, cd t, and treg cells were also detected in infected pig lungs [ ] , which together with the induction of cytokines, contribute to pathogenesis of influenza infection in pigs. exploring the mechanism of regulation of host responses is crucial for understanding the pathogenesis of siv and for controlling swine influenza in pigs. macrophages residing beneath the respiratory epithelium and surrounding alveoli are part of the first line defenses against influenza viruses. during influenza viral replication in bronchial epithelial cells, macrophages are one of the earliest targets to be infected. together with dendritic cells, macrophages coordinate innate immune responses, which subsequently lead to adaptive immunity by initiating antigen presentation and lymphocyte activation. macrophages are indispensable in alveolar host defense and controlling influenza virus in pulmonary organs in pigs [ ] . while protective in launching host antiviral responses and restricting virus spread, induced proinflammatory cytokines and chemokines are also the cause of pathogenicity for the host and may lead to acute respiratory failure (arf), a major cause of death in highly pathogenic h n or h n pdm-infected humans [ ] . needless to say, the roles of macrophages are critical to pathogenicity as well as host protection in siv-infected pigs. however, little is known about the mechanisms of how host responses are regulated in pigs or their macrophages. considering the critical role macrophages play in siv infections, and the threat that h n pdm could further evolve higher virulence in pigs and subsequently infect humans, we were interested in profiling host responses of swine macrophages to h n pdm, and more importantly, in exploring the underlying mechanism of host response regulation including antiviral, proinflammatory responses, and apoptosis in pigs. in this report, we will demonstrate that swine macrophages are susceptible to infection by h n pdm. we will show a unique pattern of proinflammatory cytokine responses to the infection, which are distinctly regulated by swine mitogen-activated protein (map) kinases. we have also observed cross-talk between map kinase and nfkb pathways, and our data indicate that map kinase erk / and jnk / may impact the activation of nfkb through the induction of rig- , leading to ifn-b induction in h n pdm-infected swine macrophages. the d/ cells used in our study are a spontaneouslytransformed line of swine macrophages purchased from atcc (manassas, va) and grown in rpmi medium (invitrogen) containing % fetal bovine serum (fbs). mouse anti-erk and anti-jnk antibodies as well as rabbit anti-phospho erk and antiphospho jnk antibodies (cell signaling), anti-cytochrome c, anti-influenza ns , and alkaline phosphatase (ap)-conjugated anti-rabbit and anti-mouse igg antibodies (santa cruz biotechnology) were obtained from their respective providers. anticleaved caspase antibody was obtained from cell signaling technology, and anti-bak antibody was obtained from emd chemicals. the chemicals purchased from emd chemicals also included inhibitors for map kinases, u (erk / ), sb (p ), and insolution jnk inhibitor ii (jnk / ), and the inhibitors for nfkb and ikk ( -amino- -( -phenoxyphenylethylamino) quinazoline (cat. ) and wedelolactone (cat. ), respectively). a/nanjing/ / (h n ), a pandemic (h n ) virus, was isolated from a swab sample of an outpatient febrile child at the nanjing children's hospital during the pandemic in , nanjing, china. the sampling procedure was performed in accordance with the rules set by the institutional review board at the hospital. the eight genomic segments of this virus have been fully sequenced and the raw data are deposited at genbank under accession numbers jq through jq . the virus was grown in -day-old embryonating chicken eggs; virus allantoic fluid (vaf) was harvested hrs after inoculation, then titrated with standard haemagglutination tests (ha) and plaque assays in mdck cells for ha and infectious viral titers, respectively [ ] . for viral infection, the d/ cells were trypsinized, resuspended in rpmi medium containing % fbs, and plated on -cm tissue culture plates at cells per plate hrs before infection. the cells were infected with h n pdm inocula in vaf at a multiplicity of infection (moi) of . after hr of adsorption, the virus inocula were discarded and ml of serum-free rpmi medium containing tpck-trypsin ( mg/ml, sigma) was added. the cells were incubated at uc and % co for various time points before cell lysates or total rna extraction were prepared. mrna transcript levels of ifn-b, il- b, il- , il- , ccr , ip- , tnf-a, fasl, trail, mx, -oas, retinoic acid-inducible gene i (rig- ), melanoma differentiation-associated antigen (mda- ), and glyceraldehyde- -phosphate dehydrogenase (gapdh) genes were analyzed by a two-step real-time rt-pcr assay as described previously [ ] . mg of total rna, prepared from the cells using the rneasy kit (qiagen), was reverse transcribed with the quantitect reverse transcription kit (qiagen) following the manufacturer's instructions. the sequences of primers used in the study are listed in table . the rt reaction was carried out with the rna after treatment with dnase i at uc for min. real-time pcr was conducted with ml of cdna in a total volume of ml with the iq sybr green supermix (bio-rad) following the manufacturer's instructions. relative expression values were normalized using an internal gapdh control. the fold change of relative gene expression levels was calculated following the formula: (dct of gene dct of gapdh) [ , ] . for each reaction, melting curves were analyzed to determine the specificity of each amplicon. to determine the viral rna level, the total rna from infected cells was reverse transcribed and cdna used for taqman-based real-time pcr (applied biosystems) to measure viral m gene transcripts in the infected cells [ ] . cell lysates were prepared by lysing uninfected and infected d/ cells in % np- lysis buffer containing mm pmsf, % aprotinin, mg leupeptin ul and mm sodium vanadate (sigma) as described previously [ ] . cell lystes were clarified by low speed centrifugation ( g, min at uc) and subjected to sds-page ( to %). proteins were transferred to the immuno-blot pvdf membrane (bio-rad), and western blot analysis was performed following standard protocols [ ] using rabbit or mouse anti-map kinase or phosphor-map kinase antibodies ( : ) in tbst containing % fat-free milk powder for mins incubation at rt. after washes, incubation with apconjugated anti-rabbit or anti-mouse igg antibody for another mins followed. after incubation and thorough washes, bcip/ nbt reagents (sigma) were used for the development of colorimetric signals on the membrane. the membrane was also blotted with a monoclonal anti-actin antibody (santa cruz biotechnology) for input control. for statistical analysis, a two-tailed student's t-test was used to evaluate realtime rt-pcr data. an x analysis was used to evaluate significant differences of the data in two and more groups. the . level of probability (p, . ) was considered statistically significant. to examine the susceptibility of pig macrophages to h n pdm originating from a human host, we infected d/ cells with the a/ nanjing/ / (h n ). typical cytopathic effect (cpe) appeared hrs post infection and the cell monolayer was destroyed hrs post infection (fig. a) . this result demonstrated that h n pdm retains the ability to infect and replicate in swine macrophages, and can reach . pfu/ml as shown in a replicative curve (fig. b) . apoptosis occurred and proceeded through the course of the infection, as we observed cleaved/ activated caspase- as well as the emergence of downstream executioner caspases- , - , and - , which eventually destroyed the infected swine macrophages (fig. c) . clearly, cytochrome c was released into the cytosol (fig. e) , which activated mitochondriamediated intrinsic apoptosis as early as hrs post infection. bak, a pro-apoptotic bcl- family member, was upregulated as detected in the infected cells (fig. d) , and may be involved in the release of cytochrome c from mitochondria in swine macrophages. to elucidate the pathogenesis of h n pdm in pigs, we examined the pattern of cytokine responses in ph n -infected swine macrophages. total rna from infected and uninfected d/ cells collected at different time points post infection (p.i.) were prepared and used for realtime rt-pcr analyses with specific primers to swine cytokines. we found that the levels of proinflammatory cytokines il- and il- were upregulated up to -and -fold at hrs, respectively, and the level of il- continued to rise up to -fold at hrs p.i.. however, the level of il- b remained unchanged throughout the infection ( fig. a) , indicating that il- and il- , as well as tnf-a (fig. b ) as described later, were the main proinflammatory cytokines upregulated. we observed a robust induction of antiviral ifn-b, which rose up to -and , -fold at and hrs p.i., respectively (fig. c ). ifn-inducible antiviral proteins mx and .-oas were induced accordingly up to -and , -fold, respectively, at hrs p.i. (fig. c) . tnf family members were also induced in response to h n pdm infection, which may be attributable to cell death. we found that in pig macrophages the levels of fasl and tnf-a remained undetectable, while tnf-related apoptosis-inducing ligand (trail) seemed to be most abundant before infection, based on ct values from realtime rt-pcr (data not shown). fasl and tnf-a were induced most robustly, but trail was only mildly induced in response to infection (fig. b) . among the induced, the level of tnf-a, critical in both cell death and inflammation, was sharply upregulated up to -and -fold, and fasl up to -and -fold at and hrs p.i., respectively. fasl and tnf-a may play a major role in h n pdm-triggered extrinsic apoptosis. to understand the mechanism of proinflammatory cytokine and tnf family ligand induction in h n pdm-infected swine macrophages, we investigated how map kinases were activated and whether their signaling pathways were involved in the regulation of various cytokines and tnf family ligands in pig immune cells. d/ cells were infected with h n pdm, and cell lysates were prepared at various time points for sds-page and western blot analyses with specific anti-erk / and anti-jnk / antibodies. activated forms of erk and jnk (phospho-erk / and phosphor-jnk / ) were detected by anti-phospho-erk / and anti-phospho-jnk antibodies. as shown in figure a , erk / was basally phosphorylated at a low level before infection, but further phosphorylated between and hrs and thereafter p.i.. phosphorylation and activation of jnk / appeared at hrs and increased to the peak around hrs p.i. (fig. b) . although both erk / and jnk / were activated in response to h n pdm infection in swine macrophages, erk / remained active at basal level even before infection, so did jnk / as shown in some of our experiments (fig. b) . however, our data showed that basal level phosphorylation of both erk / and jnk / remained unchanged in uninfected d/ cells through the period of our infection. in addition to erk / and jnk / , we have also observed the phosphorylation and activation of p map kinase in h n pdm-infected cells (data not shown). to evaluate the role of map kinases in the regulation of proinflammatory cytokine responses in h n pdm-infected swine macrophages, we pre-treated d/ cells with specific inhibitors for erk / , p , and jnk / hr prior to infection. we then infected the cells with the virus and observed how infectioninduced activation of map kinases was affected by inhibition of the respective map kinases. as shown in figure a , d/ cells were pre-treated with inhibitors of erk / (u ), p (sb ), and jnk / (jnk insolution), at concentrations of mm, mm, and mm, respectively. while the phosphorylation of erk / was unaffected by treatment with the p and jnk inhibitors, it was completely abolished at both and hrs p.i. (lines - ) by the erk / inhibitor u (fig. a) . we noted that the basal level phosphorylation of erk / diminished in the presence of u . on the other hand, in light of the p and jnk inhibition with their specific inhibitors, the phosphorylation of erk / appeared to be enhanced (fig. a, lines - ), indicating that a compensatory mechanism may exist among map kinases. we observed a similar response in which a complete suppression of jnk / phosphorylation was observed (lines - ) when the cells were pre-treated with the jnk / inhibitor (fig. b) . however, the phosphorylation of jnk / was not suppressed at all by the inhibitors of erk / and p . we noted that there were double bands for jnk , and a lower band of jnk usually appeared at a later stage of infection ( hrs p.i.). this band was detected mainly by anti-jnk / , but not by anti-phospho-jnk / , indicating that jnk activation was transient and dephosphorylation of jnk occurred at later stages of infection, probably by an uncharacterized map kinase phosphatase (mkp) present in pigs. a basal level phosphorylation of nfkb was also observed in d/ pig macrophages, and was further enhanced upon h n pdm infection, indicating that the nfkb pathway was activated as well in infected pig macrophages (fig. c) . when the cells were pre-treated with specific inhibitors of nfkb ( nm) or ikk ( mm), the phosphorylation/activation of nfkb was effectively decreased or diminished. map kinases and nfkb pathways were activated in h n pdm infected pig macrophages, which could be reversed or inhibited by their specific inhibitors. we used these inhibitors to study the regulation of host responses, which may be controlled by these pathways. to determine how cytokine responses are regulated by individual map kinases, we pre-treated the cells with erk / and jnk / inhibitors, respectively, and measured the induction of the cytokines after infection with realtime rt-pcr. we observed that il- b was barely detected and not induced during h n pdm infection. interestingly, we noticed that il- b was upregulated in the presence of the jnk inhibitor, although no change was observed after the treatment by the erk inhibitor, indicating that il- b could have been induced in swine macrophages infected with h n pdm, but was virtually suppressed by jnk / (fig. a) . we observed that the induction of il- , il- , and il- was completely suppressed in the presence of the erk / inhibitor, which indicates that il- , il- , and il- inductions are all dependent on the erk signaling pathway (fig. b-d) . it is interesting to note that jnk / may play different roles in the induction of il- , il- , and il- based on their responses in the presence of the jnk inhibitor. jnk / may have moderate effects in the induction of il- ( fig. b ), but may be not relevant at all to the induction of either il- or il- ( fig. c-d) . we also noted that ccl (rantes) was strongly regulated by erk / and jnk / in swine immune cells. as shown in figure e , induction of ccl was efficiently blocked in the presence of either erk / or jnk / inhibitors, indicating that ccl is induced by h n pdm infection through erk and jnk signaling pathways. as for antiviral ifn-b, which was robustly induced with h n pdm infection in swine macrophages, erk / appeared to be essential since the induction of its mrna transcripts was virtually abolished in the presence of the erk inhibitor (fig. a ). jnk / may also play a role in ifn-b induction because of its significant decrease at the earlier stage of infection ( hrs p.i.) when d/ cells were pre-treated with the jnk inhibitor. however, erk / seemed to be the primary pathway in the ifn-b induction in swine macrophages. the distinct contributions to the induction of ifn-b by erk / and jnk / were also reflected in the decreased mrna transcript levels of ifn-inducible antiviral proteins, mx and -oas, in the presence of erk and jnk inhibitors, respectively (fig. b-c) , which is in accordance with the suppression of the ifn-b induction by these same compounds in the infected cells. both mx and -oas were suppressed significantly by the erk inhibitor, but only the in contrast to the abundance of trail transcripts, mrna levels of fasl and tnf-a were barely detectable by realtime rt-pcr in swine macrophages (data not shown). however, both fasl and tnf-a were induced profoundly in response to ph n infection ( fig. b and a-c) , while the change of trail was mild. by using inhibitors, we concluded that the induction of fasl and tnf-a are mainly controlled by the erk / and jnk / pathways in pig macrophages. the nfkb pathway could also be critical in host responses, as has been shown in humans and mice infected with influenza a virus. nfkb can be phosphorylated and activated in swine macrophages in response to h n pdm infection ( fig. a and c ), albeit at a later stage. interestingly, when the cells were pre-treated with erk / or jnk / inhibitors, the phosphorylation of nfkb was also suppressed. however, when the cells were pre-treated with the p inhibitor, nfkb phosphorylation decreased much less than with erk / or jnk / inhibitors (fig. a) . this result suggests that a cross-talk may exist between map kinase and nfkb pathways, and that among the map kinases, erk / and jnk / are mainly involved. figure . regulation of swine proinflammatory cytokine gene transcripts by map kinases. d/ cells were pretreated with u and insolution jnk inhibitor, which are inhibitors of erk / and jnk / , respectively, hr before h n pdm infection. total rna was prepared at and hrs post infection for reverse transcription. cdna was used for realtime pcr with specific primers to measure fold changes of cytokine transcripts at different time points. each assay was repeated at least twice. a-e. regulation of il- b, il- , il- , il- , and ccl , respectively, by erk / and jnk / inhibitors. data show mean fold changes plus standard deviation of two or three independent assays. *p, . , student's t-test. doi: . /journal.pone. .g we next examined the expression levels of rig- and mda- , the rlr family members and cytosolic sensors for rna viruses. we found that rig- in particular was significantly induced up to -fold, while mda- was also upregulated up to -fold in infected pig macrophages (fig. b) . we further examined the induction of rig- and mda- and their relevance to map kinases. to do this, we pre-treated the cells with inhibitors of map kinases. as shown in figure c , the induction of rig- was completely abolished by the inhibition of erk / or jnk / inhibitors, and to a much lesser extent, by the p inhibitor, suggesting that the induction of rig- was dependent on erk / and jnk / , but not as much on p . this differentially regulated pattern of rig- induction by erk / , p , and jnk / was similar to the suppression of nfkb phosphorylation/activation by map kinases (fig. a) , suggesting that the induction of rig- was associated with erk / or jnk / activation, but to a much lesser extent with p . since nfkb could be downstream activated by rig- /ips- [ , ] , we postulate that erk / or jnk / may activate nfkb through the activation of rig- /ips- during h n pdm infection in pig macrophages. a similar, albeit less dramatic, induction and suppression of mda- expression was also observed (fig. d) , which indicated that mda- might also be an intermediate adaptor bridging the map kinases erk / and jnk / to the nfkb pathway activation. the cells were pretreated with u , sb , and insolution jnk inhibitor, hr before h n pdm infection. total rna was prepared from infected cells for reverse transcription. cdna was used for realtime pcr with rig- and mda- primers to measure fold changes of rig- and mda- transcripts in treated swine macrophages. each assay was repeated at least twice. data show mean fold change plus standard deviation of two or three independent assays. *p, . , student's t-test. doi: . /journal.pone. .g in the present study, we have demonstrated a pattern of host responses in swine macrophages to h n pdm infection. strong proinflammtory and antiviral cytokine responses including il- , il- , tnf-a, as well as ifn-b, were observed. in contrast, il- b was not induced, and was barely detectable in pig macrophages. this pattern differs from that in bronchoalveolar secretions of siv-infected pigs in which il- b was induced but il- was not [ , , , , ] . the different cell types involved (macrophages and epithelial cells) may account for the difference. it has previously been reported that in human immune cells and patients a weak innate immune response, evidenced by a poor induction of proinflammatory and antiviral cytokines including ifn-b and tnf-a, has been observed in human monocyte-derived dcs and macrophages infected with h n pdm, compared to seasonal h n infection [ ] . highly pathogenic h n viral infection in human macrophages induced higher expression of il- and ccl (rantes) than ph n [ ] , which may explain generally mild clinical disease among h n pdm-infected patients. in human macrophages, similar to our findings, il- b was not detected. map kinase signaling pathways and their roles in the regulation of cytokines and viral replications have not been characterized in influenza-infected pig immune cells. in this study, we found that erk / and jnk / could both be activated in swine macrophages. we noted that erk / was phosphorylated and active at a low level constitutively, which may be important for the rapid physiological responses required upon infection. to elucidate the mechanism that regulates swine host responses, we used specific inhibitors of map kinases to pre-treat macrophages before infection. we determined that the induction of ifn-b, il- , il- , and il- were regulated by erk / , while jnk / may only play a minor or no role in the regulation of these cytokines. as described earlier, il- b was not induced in response to the ph n infection, which could be explained by our data indicating that its induction was in fact efficiently suppressed by jnk / in swine macrophages. this may be the first time that jnk / inhibitory effects on the induction of proinflammatory cytokines have been demonstrated. previous studies found that ifn induction was dependent on the jnk / signaling pathway in epithelial cells infected with influenza virus infection [ ] . however, our data clearly demonstrate that erk / plays a major role in the regulation of ifn-b in pig macrophages, which may indicate that the regulation of ifn differs in different cell types. we noted that basal level activities of both erk / and jnk / were constitutively present in non-infected d/ cells, which may be important in the induction of proinflammatory and antiviral cytokines at the early stages of infection. our data indicate that the induction of il- , il- , il- , ccl- , as well as ifn-b, were apparent at the earliest stages of viral infection even before erk / was further activated. we realized that a transformed monocytic cell line, instead of primary cells, was used in the study, which may compromise the significance of our data. basal level phosphorylation of both erk / and jnk / , which may affect certain cytokine production, would be minimal in primary monocytes. however, specific inhibitors used in the study completely wiped out phosphorylation of both erk / and jnk / (fig. ) . the effect of map kinase phosphorylation and activation on the regulation of affected cytokines as observed in our study with the inhibitors is, therefore, valid, even though the cells were not primary cultures. macrophages appear to die inevitably of apoptosis when infected with influenza virus [ ] . the fas-mediated extrinsic apoptotic pathway is apparently triggered by tnf family ligands. while both fasl and tnf-a were induced vigorously upon the viral infection, induction of trail was rather mild in h n pdminfected swine macrophages. we knew previously that fasl and tnf-a were barely detectable, while the level of trail remained high prior to the infection based on our realtime rt-pcr data (ct) (xing et al., unpublished data). we can therefore presume that h n pdm-induced apoptosis may be mainly attributed to fasl and tnf-a, while pig macrophages could be resistant to trail, since the cells remained intact despite the presence of a high level of trail before infection. furthermore, we were also able to determine that both erk / and jnk / were involved in the induction of fasl, tnf-a, and trail. fasl is also regulated by erk in chicken macrophages infected with an h n avian influenza virus [ ] . both toll-like receptors (tlr) and rna helicases, such as rig- and mda- , are critical to antiviral innate immunity [ , ] . as a cytosolic sensor, rig- binds to dsrna and viral ssrna that contain a -triphosphate not present in host rna, and then is recruited to mitochondrial protein ips via the card domain, leading to activation of nfkb, irf- /- , and induction of ifn [ , , ] . rig- can be induced by viral infection [ ] . in this study, we observed a robust induction of rig- and mda- in h n pdm-infected swine macrophages, which appeared to be suppressed completely by inhibitors of erk / or jnk / , but to be a much lesser extent, by the inhibitor of p . this indicates that the induction of rig- or mda- depends on the activation of erk / and jnk / in pig macrophages. we postulate a mechanism, therefore, that the cross-talk between map kinase and nfkb pathways is through the regulation of rig- and maybe mda- , and that erk / controls the activation of nfkb, leading to the induction of ifn in swine macrophages. characterization of an influenza a virus isolated from pigs during an outbreak of respiratory disease in swine and people during a county fair in the united states pathogenic and antigenic properties of phylogenetically distinct reassortant h n swine influenza viruses cocirculating in the united states protection against a european h n swine influenza virus in pigs previously infected with h n and/or h n subtypes evidence for the natural transmission of influenza a virus from wild ducts to swine and its potential importance for man genetic reassortment between avian and human influenza a viruses in italian pigs influenza-a model of an emerging virus disease the epidemiology and evolution of influenza viruses in pigs antigenic and genetic diversity among swine influenza a h n and h n viruses in europe evolution of swine h n influenza viruses in the united states genetic characterization of novel reassortant h n influenza a viruses isolated from pigs in southeastern china genetic characterization of h n swine influenza a viruses isolated in eastern china genetic and pathobiologic characterization of pandemic h n influenza viruses from a naturally infected swine herd antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans an investigation into human pandemic influenza virus (h n ) on an alberta swine farm evidence of human-to-swine transmission of the pandemic (h n ) influenza virus in south korea reassortment of pandemic h n / influenza a virus in swine pandemic (h n ) : update . world health organization swine influenza h n virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h n influenza virus cytokines and acute phase proteins associated with acute swine influenza infection in pigs porcine innate and adaptative immune responses to influenza and coronavirus infections cytokines in the pathogenesis of influenza alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs pathology of the swine-origin influenza a (h n ) flu replication and plaque assay of influenza virus in an established line of canine kidney cells immune-related gene expression in response to h n low pathogenic avian influenza virus infection in chicken and pekin duck peripheral blood mononuclear cells modulation of the immune responses in chickens by low-pathogenicity avian influenza virus h n genetic and phenotypic characterization of a low-pathogenicity avian influenza h n virus host immune and apoptotic responses to avian influenza virus h n in human tracheobronchial epithelial cells rna recognition and signal transduction by rig-i-like receptors rig-i-like receptors: sensing and responding to rna virus infection pandemic h n influenza a virus induces weak cytokine responses in human macrophages and dendritic cells and is highly sensitive to the antiviral actions of interferons cytokine profiles induced by the novel swine-origin influenza a/h n virus: implications for treatment strategies influenza virus-induced ap- -dependent gene expression requires activation of the jnk signaling pathway roles of the erk mapk in the regulation of proinflammatory and apoptotic responses in chicken macrophages infected with h n avian influenza virus toll-like receptors and rna helicases: two parallel ways to trigger antiviral responses intracellular pattern recognition receptors in the host response -triphosphate rna is the ligand for rig-i recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production rig-imediated antiviral responses to single-stranded rna bearing -phosphates an rna helicase, rhiv - , induced by porcine reproductive and respiratory syndrome virus (prrsv) is mapped on porcine chromosome q we thank members of xing laboratory for their technical assistance in the study and helpful discussions for manuscript preparation. we appreciate excellent editing work performed by sandy shanks. key: cord- -plz ja e authors: dussurget, olivier; bierne, hélène; cossart, pascale title: the bacterial pathogen listeria monocytogenes and the interferon family: type i, type ii and type iii interferons date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: plz ja e interferons (ifns) are secreted proteins of the cytokine family that regulate innate and adaptive immune responses to infection. although the importance of ifns in the antiviral response has long been appreciated, their role in bacterial infections is more complex and is currently a major focus of investigation. this review summarizes our current knowledge of the role of these cytokines in host defense against the bacterial pathogen listeria monocytogenes and highlights recent discoveries on the molecular mechanisms evolved by this intracellular bacterium to subvert ifn responses. listeria monocytogenes is a pathogenic gram-positive bacillus responsible for a foodborne disease in humans and animals called listeriosis (vázquez-boland et al., ) . this highly versatile bacterium can be isolated from multiple sources such as human and animal feces, soil, water, plants and food. as a common contaminant of fruits, vegetables, seafood, meat and cheese, it represents a major economic problem for the food industry. infection usually originates from ingestion of contaminated food (schlech et al., ) and may cause febrile gastroenteritis in otherwise healthy persons (ooi and lorber, ) . in contrast, in immunocompromised individuals it leads to a severe invasive disease, which manifests itself as septicemia, meningitis and encephalitis. in the specific case of pregnant women, infection may cause fetal loss or neonatal bacteremia and meningitis. in the united states, incidence of listeriosis ranged from . to . cases per million population between and (cartwright et al., ) . in france, incidence was . per million between and , and increased risk of listeriosis was noticed in people with underlying diseases, such as chronic lymphocytic leukemia (goulet et al., ) . while relatively rare, listeriosis is among the most deadly foodborne diseases with mortality rates reaching up to % depending on the clinical manifestations (lorber, ) . in addition to the immunological status of the patient, the clinical outcome of the disease depends on the pathogenic potential of the infecting bacteria. l. monocytogenes strains of serovars / a, / b, and b account for % of human cases and serovar b alone is associated to most outbreaks (swaminathan and gernersmidt, ) . the capacity of l. monocytogenes to survive and multiply within the gastrointestinal tract is critical for the initial infection, persistence and transmission. l. monocytogenes is well adapted to this environment and produces multiple factors to compete with microbiota and counteract antimicrobial peptides, acidity, hyperosmolarity, hypoxia, bile and iron deprivation (gahan and hill, ) . crossing of the intestinal epithelium is thought to occur by invasion of enterocytes, in particular goblet cells and m cells of the peyer's patches. invasion of enterocytes requires the specific interaction between the listeria surface protein inla and its cellular receptor e-cadherin (lecuit et al., ; disson et al., ) , which can take place at sites of cell extrusion at the tip and other locations of intestinal villi (pentecost et al., ; nikitas et al., ) . indeed, as recently shown, upon interaction with e-cadherin, listeria preferentially crosses the intestinal barrier by transcytosis through goblet cells (nikitas et al., ) . entry through ileal peyer's patches via m cells does not rely on inla. it has been reported to require listeria invasion protein inlb (chiba et al., ) . after translocation, bacteria reach lymph nodes, the liver and spleen and finally secondary target sites of infection, including the central nervous system and the placenta. a remarkable feature of l. monocytogenes is its capacity to invade non-professional phagocytic cells such as enterocytes, hepatocytes and trophoblast cells. the exceptional repertoire of virulence factors necessary for entry, survival and multiplication has been extensively studied cossart, ) . expression of many virulence genes relies on the transcriptional activator prfa, whose role is pivotal for l. monocytogenes transition from saprophytic to intracellular lifestyle (freitag et al., ; toledo-arana et al., ) . elimination of l. monocytogenes is mostly based on the capacity of the host to mount an efficient cellular immune response to infection (mackaness, ; shi and pamer, ) . in particular, the fate of infection depends on the level of macrophage activation and on listeria ability to counteract bactericidal mechanisms of host cells (shaughnessy and swanson, ; corr and o'neill, ; stavru et al., ) . bacterial escape from the phagosome and avoidance of autophagy for intracytosolic replication and cell-cell spread have been well characterized. they have been shown to depend on five major virulence factors: the secreted pore-forming toxin listeriolysin o (llo) and two phospholipases c (plca and plcb) for vacuolar escape, the surface protein acta for actin-based motility and both acta and a surface protein of the internalin family, inlk, for autophagy evasion (cossart, ; dortet et al., ) . other strategies of immune escape that lead to the modulation of cytokine expression occur through a variety of mechanisms. modifications of l. monocytogenes peptidoglycan by the n-deacetylase pgda and the o-acetyl transferase oata prevent lysozyme-dependent release of microbe-associated molecular patterns (mamps), activation of pathogen-recognition receptors (prrs) and subsequent production of pro-inflammatory cytokines (boneca et al., ; aubry et al., ; rae et al., ) . the toxin llo induces dephosphorylation of histone h and deacetylation of histone h , which correlate with decreased expression of pro-inflammatory genes, such as the chemokine gene cxcl (hamon et al., ) . the secreted internalin inlc inhibits inflammation by interacting with ikk-α, a component of the iκb-kinase complex, which is essential for nf-κb activation and expression of proinflammatory genes (gouin et al., ) . other evasion mechanisms remain to be characterized, such as the control of the expression of il- by the surface internalin inlh (personnic et al., ) . l. monocytogenes also has the capacity to modulate interferon (ifn) production during infection. type i ifn production by infected cells can be controlled by listeria multidrug efflux pumps mdrm and mdrt, via the secretion of the second messenger cyclic-di-amp (crimmins et al., ; woodward et al., ; schwartz et al., ) . synthesis of type iii ifn has also recently been shown to be tuned by listeria nucleomodulin lnta (lebreton et al., ) . our knowledge concerning the role of the ifn cytokine family during listeriosis has rapidly expanded in the last few years and will be the focus of this review. ifns form a family of proteins secreted by many cell types in response to infection. they were originally named for their capacity to interfere with viral proliferation (isaacs and lindemann, ) . this diverse family is composed of three groups of cytokines, namely type i-, type ii-, and type iii-ifns, which are important components of innate immune responses ( table ) . type i-ifns consist of ifn-α, ifn-β, ifn-δ, ifn-ε, ifn-ζ, ifnκ, ifn-ν, ifn-τ, and ifn-ω (levy et al., ) . type ii-ifn is composed of a single cytokine, ifn-γ (pestka et al., ) . type iii-ifns are ifn-λ , ifn-λ , and ifn-λ (formerly il- , il- a, and il- b) and ifn-λ (kotenko, ; prokunina-olsson et al., ) . type i-and type iii-ifns have similar signal transduction systems (see below) and are phylogenetically closer from each other than type ii-ifn (pestka et al., ) . sequence conservation and chromosome location suggest that type i-ifn genes evolved from a single ancestor through duplication. however, the extent of type i-ifn gene diversification varies greatly depending on the species (pestka et al., ) . generally, a single gene encodes the type i-ifn in fish. in contrast, multiple gene duplications and diversification led to the emergence of sub-types of type i-ifns in mammals ( table ) . gene duplication varies also within each sub-type. a single ifn-β gene is found in the human and mouse genomes (decker et al., ; honda et al., ; durbin et al., ) . in contrast ifn-α genes and one pseudogene and ifn-α genes and three pseudogenes are found in the human and mouse genomes, respectively (van pesch et al., ; durbin et al., ) . a single gene encodes type ii-ifn and four genes encode type iii-ifns in human (decker et al., ; levy et al., ) . of note, ifn-λ and ifn-λ are pseudogenes in mice, which prevents the study of these cytokines in this animal model ( table ) (fox et al., ). ifns are important components of the immune system, which generally trigger cellular protective defenses in response to infection or tumor formation. type-ii ifn (ifn-γ) is a paradigm for this, being an important mediator of innate and adaptive immune responses with a key role in clearance of viral and bacterial pathogens and in tumor control. ifn-γ was first described as an antiviral protein (wheelock and sibley, ) , but is now known to exhibit broader biological activities, non-redundant with that of other types of ifns. the crucial role of ifn-γ in immunity to infection is reflected by the phenotype of mice lacking the ifn-γ receptor or the ifn-γ gene, which are highly susceptible to mycobacterium bovis bcg infection (dalton et al., ; kamijo et al., ) . genetic deficiencies resulting in the loss of ifn-γ production or signaling in mice lead to increased susceptibility to infections by other intracellular pathogens, such as l. monocytogenes (see below), salmonella typhimurium and some viruses harty and bevan, ; jouanguy et al., ) . these defects also lead to the loss of tumor control (kaplan et al., ) . patients with deficiencies in the ifn-γ pathway, for instance by mutation in the gene for the ifn-γ receptor , are characterized by severe infections with viruses and intracellular bacteria including l. monocytogenes, salmonella sp. and mycobacteria (jouanguy et al., ; newport et al., ; roesler et al., ; van de vosse et al., ) . ifn-γ mediates macrophage activation, i.e., increased phagocytosis and production of pro-inflammatory cytokines, of microbicidal reactive oxygen and nitrogen species, leading to clearance of intracellular pathogens (schoenborn and wilson, ) . in addition, ifn-γ controls differentiation of t cells in th effector cells, antigen processing and presentation by antigen-presenting cells, which participate to cellular immunity against intracellular pathogens (schroder, ; hu and ivashkiv, ). the immunostimulatory and immunomodulatory properties of ifn-γ have therapeutic implications. indeed, ifn-γ is used in patients with chronic granulomatous disease to reduce infection and mortality, **protein length in amino-acids and protein modifications (g: glycosylation, p: phosphorylation). *** genes and a pseudogene in the human genome, genes and three pseudogenes in the mouse genome. although the clinical benefit has not been demonstrated in all studies (holland, ) . type i-ifns are produced in responses to viruses, many bacteria and parasites. however, in contrast to type ii ifns, these cytokines are not always protective against bacterial infections. indeed, the role of type i-ifns in response to bacterial infection is complex and depends on the microorganism (decker et al., ; monroe et al., ; carrero, ) . they contribute to resistance of the host against infection by extracellular bacteria, such as escherichia coli, helicobacter pylori, streptococcus agalactiae and s. pneumoniae (mancuso et al., ; watanabe et al., ) . in contrast, they are associated with suppression of innate immune responses and increased susceptibility of the host to infection by l. monocytogenes (see below), brucella abortus, chlamydia muridarum, francisella novicida, salmonella enterica, staphylococcus aureus, and yersinia pestis (auerbuch et al., ; carrero et al., ; o'connell et al., ; qiu et al., ; martin et al., ; henry et al., ; de almeida et al., ; patel et al., ; robinson et al., ; archer et al., ) . these different effects on infection are likely linked to the wide range of cellular responses induced by their downstream effectors, the products of ifn-stimulated genes (isgs) (schoggins et al., ) . although type i-ifns have long been known to induce antiviral response in the infected host (isaacs and lindemann, ) , they can also induce apoptosis, autophagy, differentiation and migration, inhibit proliferation as well as angiogenesis and mediate cellular damage, inflammation or autoimmunity (trinchieri, ) . as a result, type i-ifns have a therapeutic potential that can be used to treat tumors and viral infections (pestka, ; heim, ; wilson and brooks, ) , while being detrimental for the host in response to a subset of pathogens. type iii-ifns have been discovered in (kotenko et al., ; sheppard et al., ) and their activities have been less extensively characterized than those of type i-and type ii-ifns. however, several studies suggest that type i and type iii-ifns share common biological activities (levy et al., ; zheng et al., ) . although type iii-ifns respond to different stimuli, use different receptors and are not always expressed by the same cells as type i ifns (see below), engagement of type i-and type iii-ifn receptors leads to similar transcriptional responses. like type i-ifns, type iii-ifns have been involved in antiproliferative and antiviral responses (iversen and paludan, ; mordstein et al., ; durbin et al., ; hamming et al., ) . recently, type iii-ifns have been shown to be induced in response to bacterial pathogens, but their downstream effects are not yet characterized (pietilä et al., ; lebreton et al., ; bierne et al., ) . transcription of ifn genes is induced rapidly in response to microbial infection. type i-ifns can be produced by all cells, while type iii-ifns are secreted by specific cell types, including dendritic and epithelial cells. type i-and type iii-ifns activation is initiated by detection of mamps by prrs such as endosomal transmembrane toll-like receptors and cytosolic receptors (stetson and medzhitov, ; monroe et al., ) . upon recognition of mamps, prrs trigger diverse signaling pathways that involve adaptor proteins and cytosolic or organelle-bound protein scaffolds activating kinases converging to phosphorylation of transcription factors and their subsequent translocation into the nucleus (figure ) . irf , irf , irf , irf , irf , and irf are important for transcription of the ifn-α genes, with irf considered as the master regulator of ifn-α response (honda et al., ; tailor et al., ; levy et al., ) . regulation of the ifnβ gene is more complex. activated irf , irf , ap- , and nf-κb bind to the enhancer/promoter regions of the ifn-β gene and participate to the formation of the enhanceosome, which alters chromatin structure and allows transcription (panne et al., ; panne, ) . in contrast, irfs and nf-κb independently activate transcription of type iii-ifn genes (iversen and paludan, ) . regulation of the ifn-γ gene expression is different from that of type i and type iii-ifn genes. nk cells and nkt cells are effectors of the innate immune response and primary sources of ifn-γ. mature nk and nkt cells quickly react to infection by inducing ifn-γ secretion. upon recognition of ligands expressed on infected cells, nk cell activating-receptors trigger signaling cascades involving adaptor proteins and protein tyrosine kinases leading to activation of ras/sos, plc-γ and mapk pathways and induction of ifn-γ production (schoenborn and wilson, ) . in addition to receptors, il- , il- , il- , il- , and type i-ifns also contribute to induction of ifn-γ production by nk cells (newman and riley, ; schoenborn and wilson, ; marçais et al., ) . similarly, il- and il- induce ifn-γ production by nkt cells (godfrey and berzins, ) . in nk and nkt cells, the ifn-γ gene locus is transcriptionally permissive within accessible chromatin and allows rapid ifn-γ expression upon activation of transcription factors, such as ap- , nf-κb, stat , and t-bet (glimcher et al., ; schoenborn and wilson, ; lazarevic et al., ) . in addition, naive cd and cd t cells can differentiate into th cd effector t cells and cd cytotoxic t lymphocytes capable of ifn-γ secretion (wilson et al., ) . ifn-γ production by cd and cd t cells depends on il- , il- and ifn-γ itself and share many signaling pathways with nk cells. multiple transcription factors act at the ifn-γ promoter, e.g., ap- , atf- /c-jun, c/ebp, eomes, ets- , nfat, nf-κb, runx , stats and t-bet (schoenborn and wilson, ; samten et al., ; wilson et al., ; lazarevic et al., ) . moreover, distal regulatory elements modify the chromatin and remodel the ifn-γ gene locus to facilitate ifn-γ production (wilson et al., ). ifns are rapidly secreted upon infection and then bind to their receptors on the surface of target cells (table ) . type i-ifns bind the ubiquitous ifnar receptor, which consists of two chains, ifnar and ifnar (piehler et al., ) . type iii-ifns bind and signal through a different receptor complex, made of two chains: ifnlr (also known as il- rα) and il r . this receptor is expressed primarily by epithelial cells and hepatocytes (iversen and paludan, ) . thus, the physiological roles of type i-and type iii-ifns are distinct because of the different distribution of their receptors in tissues, type iii-ifns acting predominantly at mucosal surfaces (mordstein et al., ; durbin et al., ) . type i-and type iii-ifns use different receptors but trigger the same jak-stat signal transduction cascade involving tyk , jak , stat , and stat albeit with different kinetics (figure ) (marcello et al., ) . ultimately, stat , stat , and irf form a transcription factor complex, referred to as isgf , which translocates to the nucleus and binds to ifn-stimulated responsive elements (isre) in the promoter of isgs (schindler et al., ) . type ii-ifn, uses a heterodimeric receptor consisting of ifnγr and ifnγr chains, expressed by many cell types (bach et al., ) . ifn-γ activates jak , jak and stat , leading to transcription of genes bearing a γ-activation sequence (gas) in their promoter (figure ) (schindler et al., ) . mamps activate prrs of host cells such as epithelial cells and macrophages (figure ) . infection induces a robust type i-ifn response. in mice, macrophages have been identified as the major source of ifn-β (stockinger et al., ) . in vitro, ifn-β production by bone-marrow-derived murine macrophages has been shown to require bacterial escape from the phagosome and activation of cytosolic surveillance pathways (o'riordan et al., ) . induction of ifn-β depends on the adaptor protein sting and the cytosolic prr ddx , which are activated by bacterial secondary messengers c-di-amp and c-di-gmp and by bacterial dna (woodward et al., ; burdette et al., ; sauer et al., ; parvatiyar et al., ; archer et al., ) . sting is a direct receptor for cyclic-dinucleotides, including the cellular second messenger cyclic gmp-amp (cgamp) which is produced by the cytosolic sensor cgamp synthase (cgas) upon interaction with microbial dna (ablasser et al., ; gao et al., ; wu et al., ; sun et al., ; schoggins et al., ) . interestingly, type i-ifn production requires activation of the rig-i helicase by listeria rna in non-immune cells lacking a functional sting signaling pathway (abdullah et al., ; hagmann et al., ) . another cytosolic prr, the leucine-rich repeat-containing protein lrrfip , has also been implicated in ifn-β production by mouse primary peritoneal macrophages in response to listeria infection, possibly by sensing double stranded dna and rna (yang et al., ) . while production of ifn-β in response to listeria infection is independent from tlrs in bone-marrowderived macrophages (mccaffrey et al., ; stockinger et al., ; o'connell et al., ) , tlr- contributes significantly to ifn-β secretion by peritoneal macrophages, suggesting that specific macrophage populations have evolved different recognition strategies in response to listeria infection (aubry et al., ) . listeria infection has recently been shown to induce type iii-ifn gene expression in cells of epithelial origin, such as intestinal and trophoblast cells and hepatocytes (lebreton et al., ; bierne et al., ) . similar to type i-ifn, type iii-ifn induction is triggered by intracellular listeria . listeria infection also triggers a rapid and robust ifn-γ response. after intravenous infection of mice with l. monocytogenes, nk and t cells are the main sources of ifn-γ (thale and kiderlen, ; bou ghanem et al., ) . ifn-γ producing v δ + -γδ t cells are other murine immune cells induced at an early stage of listeria infection in mice inoculated intraperitoneally (hamada et al., ) . using oral infection of mice, the natural route of infection in permissive hosts, l. monocytogenes has been shown to induce ifn-γ production by intraepithelial lymphocytes of the small intestine (okamoto, ) . more recently, human e-cadherin (hecad) expressing mice, a mouse line permissive for listeria oral infection (lecuit et al., ) , were used to study cells involved in intestinal mucosal immunity. infection induced ifn-γ production in nk cells of the small intestine (reynders et al., ) . the production of ifn-γ by immune cells promotes bacterial clearance and is thus critical in controlling primary l. monocytogenes infections (zenewicz and shen, ) . injection of neutralizing monoclonal anti-ifn-γ antibodies in mice infected intraperitoneally with l. monocytogenes inhibits macrophage activation and increases the mortality rate (buchmeier and schreiber, ) . in addition, resistance of ifn-γ gene or ifn-γ receptor knock-out mice infected intravenously with l. monocytogenes is frontiers in cellular and infection microbiology www.frontiersin.org april | volume | article | severely impaired harty and bevan, ) . recent work using cell-type specific inactivation of stat in mice elegantly demonstrated the key role of ifn-γ and stat in macrophage activation and clearance of listeria (kernbauer et al., ) . interestingly, the role of stat was extremely different after infection of immunized mice. stat signaling in t cells and dendritic cells was critical for adaptive immunity to listeria, while ifn-γ-activated macrophages were not essential anymore once memory cells were produced. upon oral infection of hecad mice with listeria, ifn-γ contributes to the control of bacterial burden in the intestine and of bacterial dissemination to other organs. for instance, blocking ifn-γ with neutralizing antibodies increases listeria load in the small intestine, the mesenteric lymph nodes and in the spleen of mice infected orally (reynders et al., ) . in contrast to ifn-γ, type i-ifn is beneficial to l. monocytogenes. mice lacking type i-ifn receptor or irf are more resistant to listeria intraperitoneal or intravenous infection (auerbuch et al., ; carrero et al., ; o'connell et al., ; garifulin et al., ; jia et al., ) . the role of type i-ifns in increasing host susceptibility could be explained by modulation of components of the immune response involved in controlling bacterial growth such as induction of t cell apoptosis, resulting in greater il- secretion by phagocytic cells, in turn dampening the innate immune response (carrero and unanue, ) , the downregulation of ifn-γr (rayamajhi et al., ; kearney et al., ) , or neutrophil recruitment (brzoza-lewis et al., ) . as shown recently, sting-dependent activation of type i-ifn reduces the adaptive immune response to l. monocytogenes (archer et al., ) . in contrast, recent studies showed that type i-ifns can also play a beneficial role for the host during listeria infection, pointing to the infection route and the timing of type i-ifn production as determinative factors (pontiroli et al., ; kernbauer et al., ) . interestingly, different strains of l. monocytogenes have been shown to vary greatly in their capacity to induce ifn-β (reutterer et al., ; schwartz et al., ) . the lo strain hyperinduces ifn-β (reutterer et al., ) . this strain bears a nonfunctional brta (also named tetr), the transcriptional repressor of the multidrug efflux pump mdrt (schwartz et al., ; yamamoto et al., ) . in listeria, mdrt allows secretion of cdi-amp, which triggers ifn-β. thus, derepression of mdrt in the lo strain promotes ifn-β production. of note, high expression of mdrt in lo correlates with both induction of ifn-β and lower virulence. another listeria multidrug resistance transporter, mdrm, has been involved in the stimulation of ifn-β production, possibly by secreting c-di-amp (crimmins et al., ; woodward et al., ; witte et al., ) . the role of type iii-ifns during listeriosis remains to be determined. since listeria colonizes several tissues of epithelial origins, such as the liver, intestine and placenta, it is tempting to speculate that ifn-λs play a role in the interaction of listeria with epithelia. however, a prerequisite to address this question is the establishment of a new animal model, i.e., a mouse line expressing a human e-cadherin, thus permissive for listeria infection of epithelia (lecuit et al., ) and impaired in type iii-ifn responses, such as il rα knockout mice (mordstein et al., ) . one should keep in mind that the mouse model is not optimal to address the role of type iii-ifn in human listeriosis. indeed, ifn-λ is a pseudogene in mice, while human cells produce this cytokine upon infection with l. monocytogenes. in addition, the type iii-ifn receptor is expressed at very low levels in the mouse liver and the ifn-λ response of the mouse liver is very weak (mordstein et al., ) . in line with this, it has been recently shown that mouse hepatocytes, in contrast to human hepatocytes, are not responsive to ifn-λ (hermant et al., ) . the beneficial or detrimental effects of ifns on listeria infection rely on the functional properties of their downstream effectors. indeed, ifns elicit expression of hundreds of interferonstimulated genes (isgs), which encode proteins involved in a broad range of cellular functions (reviewed in macmicking, ) . however, while about , isgs have been identified so far (rusinova et al., ) , their functions in immunomodulation remain to be characterized. to date, the contribution of interferon-induced proteins on listeria infection has mostly been studied in the context of the ifn-γ pathway. the antilisterial activity of ifn-γ in phagocytic cells involves induction of oxidative and nitrosative defences, via increased expression of enzymes that control production of reactive oxygen and nitrogen species, such as nox /cybb, duox , and inos/nos (macmicking, ). these enzymes play an important role in protecting infected cells against listeria cytoinvasion (myers et al., ; lipinski et al., ) . the assembly of these enzymes requires ifn-γ-inducible guanosine triphosphatases (gtpases) of the gbp (guanylate binding protein) family (boehm et al., ) , which not only participate to oxidative pathways but also regulate autophagy (kim et al., ) . several gbps have been shown to protect cells from listeria infection by coordinating a potent oxidative and vesicular trafficking program (kim et al., ) . ifn-γ also induces the expression of many nuclear genes encoding mitochondrial respiratory chain machinery, via activation of the nuclear receptor errα (estrogen-related receptor α). errα contributes to mitochondrial ros production and efficient clearance of l. monocytogenes (sonoda et al., ) . a family of ifn-γ-induced chemokines (cxcl , cxcl , cxcl ) displays direct antimicrobial activity against l. monocytogenes (cole et al., ) . in dendritic cells, one of the ifn-γ-associated isgs is the immunoregulatory enzyme indoleamine , -dioxygenase (ido), a key enzyme of the tryptophan metabolism. ido is proposed to play a role in the containment of listeria within granulomatous structures, thus avoiding massive t cell activation (popov et al., ) . the function of type i ifn-associated isgs in listeria infection is less documented. zwaferink et al. have observed that upregulation of inos/nos by ifn-β promotes necrotic death of macrophages (zwaferink et al., ) . additionally, several interferon-inducible proteins belong to inflammasomes; thus, type i ifn may potentiate inflammasome activation and cell death by pyroptosis (malireddi and kanneganti, ). yet, the link between these effectors and the observed harmful effects of type i ifns on the host is still unclear. likewise, the role of of interest, a subset of isgs is amongst the most induced genes in the intestinal tissue of gnotobiotic humanized mice infected orally with l. monocytogenes (archambaud et al., ) . however, which type of ifns triggers this response and for which function on the intestinal mucosa remain to be explored. in addition, ifn-independent pathways may contribute to expression of these isgs. listeria has evolved several mechanisms to avoid immune detection and evade ifn responses. it has been demonstrated that deacetylation of listeria peptidoglycan by the deacetylase pgda confers resistance to host lysozyme, thus preventing release of mamps, such as dna, rna and lipopeptides, that trigger ifnβ production (boneca et al., ) . listeria pgda mutants are rapidly killed in murine macrophages, which produce lysozyme, and induce a strong secretion of ifn-β compared to wildtype listeria. the role of pgda is not limited to the control of type i-ifn production as a pgda mutant hyperinduces proinflammatory cytokines as well. modification of peptidoglycan by pgda is an extremely efficient mechanism of immune escape used by listeria, which correlates with its critical role in virulence. remarkably, listeria has evolved a sophisticated strategy to modulate, either negatively or positively, the expression of isgs in epithelial cells, by targeting a chromatin-repressive complex, bahd (bierne et al., ; lebreton et al., lebreton et al., , . indeed, listeria infection promotes, albeit via an unknown mechanism, the targeting of bahd at the promoter of a set of isgs, thereby downregulating type i-and type iii-ifn responses. on the other hand, listeria can produce a nucleomodulin, lnta, which when secreted by intracellular bacteria, enters the nucleus of infected cells, binds bahd and inhibits its function (lebreton et al., , . thus, lnta stimulates ifn responses. consistent with the presence of hdac / in the bahd -associated complex, the level of acetylation of lysine on histone h , which is a mark of active chromatin, increases at the promoters of isgs in the presence of lnta. when, in which host conditions, and how lnta targets bahd specifically at isgs remains an open question. the lnta-mediated stimulation of type iii-ifn responses might support localized pro-bacterial conditions, as was proposed for ifn-i responses. we have an extensive knowledge of the molecular and cellular mechanisms involved in listeria-host interactions. yet, our understanding of the immune response to listeria, and more specifically the role ifns and of their downstream effectors, is far from complete and often relies on studies performed in cultured cells or in mice. however, murine and human listeriosis differ in many aspects (lecuit, ; hoelzer et al., ) . for instance, e-cadherin, the major receptor for listeria in epithelial cells, is not functional for listeria uptake in the mouse. thus, the route of entry of listeria is not strictly the same in mice and humans. moreover, isgs induced in response to infection are not identical in mice and humans. additionally, murine hepatocytes do not respond to type iii-ifns (hermant et al., ) , precluding the study of these ifns during infection by human hepatotropic pathogens, such as l. monocytogenes. altogether, species-specific differences provide limits to the use of mouse models in characterizing ifn pathways engaged during listeria infection in humans, especially in key epithelial organs such as the gut, liver and placenta. it will be important to perform future studies 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expression by beta interferon increases necrotic death of macrophages upon listeria monocytogenes infection conflict of interest statement: the authors declare that the research was con the authors declare no conflict of interest. the authors' work has been supported in part by the institut pasteur, inserm, inra, université paris-diderot, labex ibeid, anr (grant epilis), french ligue nationale contre le cancer, fondation louis-jeantet, fondation le roch, and european research council (advanced grant award to pascale cossart). pascale cossart is an international research scholar of the howard hughes medical institute. key: cord- -lpf lpky authors: chen, yongwen; wu, shengxi; guo, guoning; fei, lei; guo, sheng; yang, chengying; fu, xiaolan; wu, yuzhang title: programmed death (pd)- -deficient mice are extremely sensitive to murine hepatitis virus strain- (mhv- ) infection date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: lpf lpky the inhibitory receptor programmed death- (pd- ) has the capacity to maintain peripheral tolerance and limit immunopathological damage; however, its precise role in fulminant viral hepatitis (fh) has yet to be described. here, we investigated the functional mechanisms of pd- as related to fh pathogenesis induced by the murine hepatitis virus strain- (mhv- ). high levels of pd- -positive cd (+), cd (+) t cells, nk cells and macrophages were observed in liver, spleen, lymph node and thymus tissues following mhv- infection. pd- -deficient mice exhibited significantly higher expression of the effector molecule which initiates fibrinogen deposition, fibrinogen-like protein (fgl ), than did their wild-type (wt) littermates. as a result, more severe tissue damage was produced and mortality rates were higher. fluorescence double-staining revealed that fgl and pd- were not co-expressed on the same cells, while quantitative rt-pcr demonstrated that higher levels of ifn-γ and tnf-α mrna transcription occurred in pd- -deficient mice in response to mhv- infection. conversely, in vivo blockade of ifn-γ and tnf-α led to efficient inhibition of fgl expression, greatly attenuated the development of tissue lesions, and ultimately reduced mortality. thus, the up-regulation of fgl in pd- -deficient mice was determined to be mediated by ifn-γ and tnf-α. taken together, our results suggest that pd- signaling plays an essential role in decreasing the immunopathological damage induced by mhv- and that manipulation of this signal might be a useful strategy for fh immunotherapy. although liver transplantation has emerged as an effective therapeutic approach for treating fulminant virus hepatitis (fh), mortality rates associated with fh remain very high worldwide [ ] . the recent development of a mouse fh model, based upon infection with the murine hepatitis virus strain- (mhv- ), has provided insights into mechanisms underlying the disease pathogenesis and resulted in some novel treatment strategies [ ] . mhv- is a single-stranded, positive-sense rna virus that belongs to the coronaviridae family. in inbred laboratory mice, the virus produces strain-dependent disease profiles that depend on the infection route, age, genetic background, and immune status of the host. for example, balb/c, c bl/ and dba/ mice develop acute fulminant hepatitis, while c h mice develop a mild chronic disease and mice of the a strain exhibit no evidence of hepatitis [ , ] . in contrast to the resistant a strain mice, fh induced by mhv- in susceptible mice is characterized by the presence of sinusoidal thrombosis and hepatocellular necrosis [ , ] . these pathological findings occur concomitantly with expression of fibrinogen-like protein (fgl ), a virus-induced procoagulant molecule in the sinusoidal lining cells in the liver. fgl has the capacity to promote fibrinogen deposition and subsequently directly induce the coagulation cascades by the expression of procoagulant activity (pca) [ ] . thus, up-regulation of fgl is an essential component of the lethal effects of mhv- -induced fh. programmed death (pd)- is an inhibitory receptor expressed on activated t cells, b cells and myeloid cells. pd- -deficient mice (pdcd / ) develop various spontaneous autoimmune diseases, including glomerulonephritis and dilated cardiomyopathy, indicating that this receptor plays a critical role in maintenance of peripheral tolerance [ ] . pd-l (b -h ) and pd-l (b -dc), two immunoregulatory molecules belonging to the b superfamily, were identified as ligands for pd- , engagement of pd- with its ligands mediates negative signaling events via recruitment of phosphatases, such as shp- , and dephosphorylation of some effector molecules involved in downstream t cell receptor (tcr) signaling [ , ] . pd- signaling has also been shown to modulate the balance between antimicrobial immune defense and immune-mediated tissue damage. for example, pd- -deficient mice develop more severe hepatocellular injury than their wild-type (wt) littermates in response to adenovirus infection [ ] . in a herpes simplex virus (hsv) stromal keratitis mouse model, blockade of pd- signaling led to increased hsv- -specific effector cd + t cell expansion, ifn-c production, and exacerbated keratitis [ ] . a functionallysignificant high level of pd- expression has been found to be maintained by exhausted cd + t cells in mice chronically infected with lymphocytic choriomeningitis virus (lcmv), in primates exposed to simian immunodeficiency virus (siv), and in humans suffering from infection with human immunodeficiency virus (hiv), hepatitis b or c virus (hbv or hcv), or human tlymphotropic virus (htlv). however, blockade of the pd- /pd-ls pathway efficiently restored the virus-specific effector functions of the exhausted t cells, and lead to substantially reduced in viral load [ , , , , ] . the pd- signal is also known to play a key role in the chronicity of infections with bacteria (helicobacter pylori and schistosoma mansoni) [ , ] , pathogenic fungus (histoplasma capsulatum) [ ] , and parasitic worms (taenia crassiceps) [ ] . it appears that a number of pathogenic microorganisms exploit the pd- signal in order to evade host immune responses and to establish persistent infection. although the influence of pd- signal activity has been studied in several infection models, there are no data available concerning the role of this pathway in fh. to this end, we used the mhv- induced mouse fh model to demonstrate that pd- signaling acts to limit the immunopathological damage during disease progression. furthermore, our findings suggested that enhanced pd- signaling might represent a useful immunotherapeutic strategy for treating fh. pd- expression has been previously described as being induced on specific cell subsets in response to viral or bacterial infection [ ] . thus, we first determined the status of pd- expression at h after mhv- infection ( pfu) by immunohistochemical techniques. pd- -positive cells were observed in tissues from the thymus, spleen, lymph nodes and liver. cellular expression was localized to the cell membrane and in the cytoplasma while was completely absent from the nuclear compartment. pd- -positive cells were distributed throughout the medulla and cortex of the thymus and lymph nodes. in the spleen, pd- -positive cells were restricted to the germinal center under normal conditions, but extended to the red pulp after infection. in infected liver, more pd- -positive cells were present in the portal and parenchymal areas, as opposed to the relatively low presence of pd- -positive cells in only the portal area in phosphate-buffered saline (pbs) treated-mice (fig. a) . the amount of pd- -positive cells in the different organs of infected and control mice were counted and compared, results showed that the number of positive cells was significantly higher in infected mice (fig. b) . furthermore, facs analysis revealed that pd- expression was enhanced on multiple subsets of immune cells, including the cd + and cd + t cells, nk . + nk cells and cd + macrophages (fig. c) . pd- positive cells were also observed in the lung, heart and kidney, however, the numbers of pd- positive cells in these tissues did not significantly increase in response to mhv- infection (fig. s ). to investigate the potential role of pd- signaling in regulating fh tissue pathology, organs from mhv- infected pd- -deficient (pd- ko) and wt mice were assessed for morphological differences. small and discrete foci of necrosis with sparse polymorphonuclear leukocyte infiltration were observed in liver tissues from pd- -deficient mice after h of infection. in contrast, wt mice exhibited normal liver architecture at this time point. slight liver damage became apparent in wt mice after h of infection, meanwhile, the damaged areas of pd- -deficient mice had enlarged and confluent necrosis had become evident. by h of infection, the damaged region in pd- -deficient mice had extended throughout the entire liver, while wt mice suffered much less damage and up to % of their liver tissue remained normal at this time point ( fig. a) . likewise, higher levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) were observed in serum from pd- -deficient mice after h of infection (fig. b ). more interestingly, pd- -deficient thymus, spleen and lymph node tissues infected with mhv- for h exhibited severely disrupted architecture, loss of cellularity, and the presence of substantial amounts of karyorrhectic/apoptotic cell bodies. the histology of these organs from infected wt mice at h was relatively normal (fig. c ). in conjunction with the apparent tissue necrosis, higher levels of cell apoptosis were also evidenced in the organs from pd- -deficient mice by tunel staining (fig. d ). the architecture of other organs, including the heart, kidney and lung was relatively normal and only rare apoptosis events were observed in these tissues after infection (fig. s ). in all, these results demonstrated that pd- deficiency led to enhanced pathological damage by mhv- in the liver, spleen, lymph node and thymus, where higher levels of pd- -positive cells were found after infection. the earlier and increased organ damage suffered by pd- deficient mice infected with mhv- instigated our monitoring of the mortality rates of pd- -deficient mice and their similarlyinfected ( pfu) wt littermates. as shown in fig. , all of the pd- -deficient mice died within four days after infection, while the principal characteristic of fulminant viral hepatitis (fh) induced by the murine hepatitis virus strain- (mhv- ) is severe hepatocellular necrosis, which is mediated by the fibrinogen-like protein (fgl ), a molecule that has the capacity to promote fibrinogen deposition and activate the coagulation cascades. here, we report that mhv- infection of program death- (pd- )-deficient mice results in tissue damage throughout multiple organs, including the liver, spleen, thymus and lymph nodes. the liver damage, in particular, occurred earlier and was more severe in pd- -deficient mice than in their wild type (wt) littermates. further investigation determined that mhv- infection was associated with high levels of ifn-c and tnf-a in the damaged organs of pd- -deficient mice. conversely, intraperitoneal injection of a combination of anti-ifn-c and anti-tnf-a blocking mabs led to inhibition of fgl expression, greatly attenuated tissue lesions and reduced mortality. our results demonstrate that pd- signaling controls immunopathological damage following mhv- infection, indicating that manipulation of the pd- signal might represent a useful strategy for fh immunotherapy. % of the wt mice survived up to the end of the -day survey period (p = . ). these data indicated that pd- is likely a critical factor that controls mhv- -mediated tissue damage and mortality. to understand the mechanisms of pd- deficiency-mediated tissue damage and mortality, we performed a comparative genome-wide microarray analysis (nimblegen) of genes expressed in liver tissues of pd- -deficient and wt mice after h of mhv- infection. the most notable finding was pronounced upregulation ( . -fold) in the liver of pd- -deficient mice of the fgl transcripts (fig. a) , the protein product of which has been demonstrated to induce lethality of mhv- -induced fh [ ] . in addition, the enhanced fgl expression was confirmed by quantitative (q)pcr, results revealed a . -fold and . -fold higher level was present in liver from wt and pd- -deficient mice, respectively, after h of mhv- infection, as compared to their uninfected controls. moreover, its level in pd- -deficient liver was . -fold higher than that in the wt group at this time point (fig. b) . immunohistochemistry was used to show that fgl -positive cells were present in necrotic liver tissues in pd- deficient mice at h after mhv- injection. the protein expression was found to be enhanced rapidly upon infection, and the highest level occurred at h post-infection. however, occasional fgl -positive cells were detected in the livers of wt mice at h post-mhv- infection and these cells were also present, and slightly enhanced in number, at both the h and h time point (fig. c ). western-blot was used to verify the higher fgl protein level in the livers of pd- -deficient mice, as compared to wt littermates after h of infection (fig. d ). fgl has the capacity to induce fibrinogen deposition, which then activates the coagulation cascades and finally induces procoagulant activity. therefore, the expression of fgl and fibrinogen deposition in damaged liver tissues was measured. dual fluorescent staining evidenced that substantial fibrinogen deposition occurred in the fgl -positive damaged liver tissue (fig. e ). likewise, the level of fibrinogen deposition was more robust in livers from pd- -deficient mice that in livers from wt littermates, at both the h and h time points (fig. f) . to determine whether fgl -mediated pca activity was also involved in inducing damage in the other organs of pd- -deficient mice, the expression of fgl was analyzed in the thymus, spleen and lymph nodes. immunohistochemistry evidenced that fgl positive cells were also present in these organs. in thymus and lymph nodes, fgl -positive cells were detected in both the medulla and cortex. in spleen, however, the positive cells were only found in the red pulp. again, the expression of fgl appeared to be restricted to the cell membrane and cytoplasma. the distribution of fgl -positive cells in pd- -deficient mice had not changed after h of mhv- infection, but the number of positive cells in the examined organs was enhanced significantly and the levels of expression were much stronger (fig. a ). in addition, the transcription of fgl in the spleen of pd- -deficient mice was also significantly increased in response to infection (fig. b) . meanwhile, higher levels of fibrinogen deposition were found in the spleen and lymph node tissues of pd- -deficient mice (fig. c) . moreover, the level of fgl present in serum, as measured by elisa, was found to increase rapidly after infection, and the level in pd- -deficient mice was significantly higher than that in wt littermates (fig. d ). to clarify the source of fgl , fluorescent dual staining was performed on spleen tissues and results demonstrated that fgl was principally associated with cd c-positive dendritic cells (dcs), cd -positive macrophages and cd -positive endothelial cells (fig. e ). all of these results indicated that the absence of pd- signaling can result in enhanced fgl expression, consequently inducing stronger fibrinogen deposition and more severe tissue necrosis in pd- deficient mice following mhv- infection. we further examined whether fgl secretion was regulated by pd- directly or indirectly. fgl /pd- dual fluorescent staining was performed and results indicated that fgl and pd- were not co-expressed on the same cells in the liver, thymus, spleen or lymph nodes (fig. a) . previous studies have shown that the secretion of fgl can be triggered by the pro-inflammatory factors ifn-c and tnf-a [ , ] . on the other hand, the production of ifn-c and tnf-a by activated t cells, nk cells and macrophages can be inhibited by the pd- signal [ ] . therefore, we compared the status of ifnc and tnf-a in pd- -deficient and wt mice in response to mhv- infection. qpcr revealed that the transcription of the ifn-c gene in liver was significantly higher in pd- -deficient mice than in wt mice at h post-mhv- infection (fig. b) . in pd- -deficient spleen, the transcription of both ifn-c and tnf-a was found to be rapidly enhanced upon mhv- exposure (fig. c ). facs analysis indicated that ifn-c secretion from nk cells, but not from cd + t cells, in the liver was much higher in pd- deficient mice at h after mhv- infection (fig. d ). the fact that ifn-c and tnf-a both have the capacity to initiate fgl expression may explain why higher fgl expression was observed in the pd- -deficient mice. to further demonstrate that ifn-c and tnf-a were responsible for the observed fgl up-regulation in mhv- infected pd- deficient mice, pd- -deficient mice were infected with mhv- and simultaneously treated with a combination injection of anti-ifn-c and anti-tnf-a blocking mabs. the expression of fgl was measured by qpcr and protein detected by immunohistochemistry. the transcription of fgl mrna (fig. a ) and its protein levels (fig. b) were completely inhibited by h after injection of anti-ifn-c and anti-tnf-a mabs, as compared to the control rat igg isotype antibodies-treated group. moreover, the tissue necrosis (fig. c ) and liver damage (as indicated by alt and ast levels) (fig. d ) in pd- -deficient mice were also significantly reduced, thus the mhv- -mediated mortality rates were decreased as well (fig. e ). the pd- signaling is best known for its ability to inhibit or dampen the immune response. most of the evidence for this function, however, comes from models of tolerance or chronic infections [ , , , ] . although some studies have indicated that this signal might also participate in regulating acute infections [ , , , ] , its functions in this disease condition are much less clear. here, we used a mouse fh model mediated by mhv- infection to describe the effects of pd- in this disease process. firstly, pd- was found to be significantly up-regulated on t cells, macrophages and nk cells within the thymus, spleen, lymph nodes and liver in response to mhv- infection. to determine the exact role of pd- in the pathogenesis of fh, pd- deficient mice were used to establish an infection model. interestingly, mhv- -induced liver damage in pd- -deficient mice occurred rapidly and the lesion area was much larger than in their wt littermates. we then extended our investigation to the thymus, spleen and lymph nodes, where increased pd- -positive cells were observed post-infection. surprisingly, severe tissue necrosis and substantial apoptosis was observed in these organs of pd- -deficient mice at h after mhv- infection. in contrast, these organs from wt mice exhibited relatively normal histology, a finding in agreement with previously reported results [ ] . taken together, these results suggested that pd- deficiency promoted expansion of the pathological damage from the liver to the lymph organs, including the spleen, lymph node and thymus in this fh model, thereafter, the absence of pd- was associated with higher mortality rates in response to mhv- infection. murine fh induced by mhv- is a recognized and validated model for studying host resistance/susceptibility to human hepatitis virus, and several studies have shown that balb/c or c bl/ mice have an innate susceptibility to the infection [ , ] . fgl has been proposed as a critical mediating factor of lethality in the mhv- -induced fh mice due to the fact that it has the capacity to induce fibrinogen deposition, which in turn activates the coagulation cascades and induces procoagulant activity [ ] . to clarify whether the tissue necrosis we observed in pd- -deficient mice following infection was also mediated by fgl , the expression of fgl was analyzed. results showed that the expression of fgl was principally associated with cd -positive endothelial cells, cd -positive macrophages and cd c-positive dcs. surprisingly, significantly higher levels of fgl were observed after infection in all of pd- -deficient organs, including the liver, thymus, spleen, lymph nodes, and serum than that in those from wt littermates. in addition, increased fibrinogen deposition was observed in the organs of pd- -deficient mice. although we currently have no direct data to evidence that fgl directly mediates the mortality of our pd- -deficient mice, data from other researchers have clearly shown that fgl promoted mouse mortality in response to mhv- infection [ , , , ] . considering this, our results strongly indicate that the mortality of pd- -deficient mice post-mhv- infection is due to the higher level of fgl secretion and increased fibrinogen deposition. indeed, it has been reported that both fgl and pd- are expressed on t cells, macrophages, and dcs, and that targeted deletion of fgl or pd- leads to impaired t cell activity, and these events are related to the development of autoimmune diseases [ , , ] . we here also observed pd- expression as being enhanced on t cells (both cd + and cd + t cells). it was reasonable to propose that the expression of fgl may have been directly regulated by pd- signals. unexpectedly, our fgl /pd- dual staining showed that pd- -positive cells in the liver, thymus, spleen and lymph nodes did not co-express fgl , indicating that the expression of fgl was not directly regulated by pd- . on the other hand, the expression of fgl is believed to be induced by ifn-c and tnf-a [ , ] , while pd- signaling has the capacity to inhibit ifn-c and tnf-a secretion from pd- -positive immune cells [ ] . therefore, we evaluated and compared the status of ifn-c and tnf-a in both pd- -deficient and wt mice. definitively, the transcription of ifn-c and tnf-a genes was rapidly enhanced post-mhv- infection in pd- -deficient mice, as compared to wt controls. in particular, a higher level of ifn-c was observed in nk cells but not in cd + t cells of pd- deficient liver post-mhv- infection, indicating that the pd- signal can inhibit ifn-c secretion from nk cells under such condition. conversely, injection of a the combination of anti-ifnc and anti-tnf-a blocking mabs was able to successfully inhibit fgl mrna transcription and protein expression, resulting in reduced tissue damage and significantly protecting against mhv- -mediated mortality in these mice. these results demonstrated that up-regulation of fgl in pd- -deficient mice after mhv- infection was controlled, at least partially, by ifn-c and tnf-a. recently, the secretion of fgl from naturally occurring cd + foxp + regulatory t cells (tregs) was demonstrated and it was reported that deficiency of treg-produced fgl resulted in increased effector t cell proliferation [ ] . more interestingly, levy and colleagues showed that the frequency of fgl + tregs was higher in lymphoid tissues of mhv- infected mice, and treatment with fgl -specific antibodies reversed mhv- induced liver injury and mortality in vivo. these findings demonstrated that fgl is an important effector cytokine of tregs that contributes to mhv- -induced fh [ ] . pd- signaling has also been described as participating in regulation of treg differentiation and function [ , ] . in our study, we also analyzed the status of foxp + cells in both pd- -deficient and wt controls. however, the number of foxp -positive cells in the liver, spleen, lymph node or thymus was not significantly different between pd- -deficient mice and their wt littermates after h of mhv- infection (fig. s ) . therefore, foxp + cells are unlikely to be involved in the mortality of pd- -deficient mice. however, the functional status of these tregs (for example, the level of fgl secretion) in pd- -deficienct mice requires further investigation, and such studies are in progress in our lab. in conclusion, we have determined that pd- signaling can limit the immunopathological damage induced by mhv- infection in a mouse fh model. our results suggest that enhancing the pd- signal by an immunotherapeutic approach might be a useful treatment for fh. all experiments were approved by and conducted in accordance with the guidelines of the animal care and use committee of the third military medical university. all efforts were made to minimize animals' suffering. mice pd- -ko-n (strain: balb/cj) mice were kindly provided by prof. t. honjo (department of immunology and genomic medicine, kyoto university, japan). the wt control mice were purchased from the animal center of beijing university school of medicine. all mice were maintained in micro-isolator cages and housed in the animal colony at the animal center, third military medical university, standard laboratory chow diet and water was supplied ad libitum. mice were used in experimental analysis at age of six weeks and at an average weight of g (range: , g). mhv- was kindly provided by prof. q. ning (institute of infectious disease, tongji hospital of tongji medical college, wuhan, china). the virus was plaque-purified and then expanded in murine l cells. virus-containing supernatants were collected and stored at - uc until use. mice were intraperitoneally (i.p.) injected with pfu/mouse in a total volume of ml. in some experiments, pd- -deficient mice were infected with mhv- ( pfu) and simultaneously treated with a infection was analyzed by immunohistochemistry. blue color indicates nuclear dapi staining. scale bar = mm. magnification . ns: not significant different. *p, . , ** p, . . doi: . /journal.ppat. .g combination injection of anti-ifn-c ( mg/mouse per day, clone: r - a , ebioscience, san diego, ca, usa) and anti-tnf-a ( mg/mouse per day, clone: mp -xt , ebioscience) mabs, tissues were isolated for hematoxylin and eosin (h&e) staining to detect damage, and for fgl mrna transcription measured by qpcr (see below). serum alt and ast levels were measured by an au automatic biochemistryanalyzer (olympus, japan). in order to monitor the mortality, anti-ifn-c and anti-tnf-a blocking mabs or rat igg control mabs were injected everyday for a total of days. paraffin-embedded tissue blocks were cut into mm slices which were mounted on polylysine-charged glass slides. endogenous peroxidase activity was blocked by exposure to . % h o for min. antigen retrieval was performed in a citrate buffer the expression of pd- on immune cells (cd , cd , nk and macrophages) from different organs was assessed by flow cytometry (facsaria cytometer; becton dickinson, germany). briefly, cell suspensions of liver, spleen, blood and thymus tissues were washed and resuspended in pbs. cells were then incubated for min at room-temperature in the dark using primary antibodies (pe-pd- , fitc-cd , fitc-cd , fitc-nk . and fitc-cd . ebioscience). to analyze the source of ifn-c in the liver, pd- -deficient and wt mice were treated with mhv- ( pfu). after h, liver tissues were isolated and mechanically homogenized, lymphocytes were collected thereafter. cells were then treated with brefeldin a solution (bfa) for h, and fitc-nk . , fitc-cd or pe-ifn-c mabs (ebioscience) were added and the solution incubated for an additional h. for each analysis, cells were evaluated. flow cytometric data were analyzed with cellquest pro software. the microarray experiment was performed under contact by kangcheng co. ltd. (shanghai, china). briefly, total rna was isolated by trizol from liver tissue of pd- -deficient and wt mice treated with pfu mhv- for h. rna concentration was measured on the nd- spectrophotometer (nanodrop, wilmington, de, usa) and quality evaluated by denaturing gel electrophoresis. samples were then amplified and labeled using a nimblegen one-color dna labeling kit and hybridized using the nimblegen hybridization system (roche applied science, shanghai, china). after hybridization and washing, the processed slides were scanned by the axon genepix b microarray scanner. three independent experiments were performed, and for each test and control sample, two hybridizations were carried out by a reverse fluorescent strategy. only genes whose alteration tendency was concordant between both microarray assays were selected as differentially expressed genes. total rna from the liver and spleen of wt and pd- -deficient mice was isolated by trizol (invitrogen, carlsbad, ca, usa), according to the manufacturer's instructions. rna samples were quantitated by measurement of optical density at nm. total mrna ( mg) was reverse-transcribed to cdna using the revertaid h minus first strand cdna synthesis kit (fermentas china, shenzhen city, china), in accordance with the manufacturer's instructions. qpcr was performed to quantitatively analyze the gene transcription levels of fgl , ifn-c and tnf-a genes. the primers for fgl were: sense -tggacaacaaagtgg-caaatct- and anti-sense -tggaacacttgccatc-caaa- . the primers for ifn-c were: sense -tcaagtgg-catagatgtggaag- , and anti-sense -cgcttatg-ttgttgctgatgg- . the primers for tnf-a were: sense -cacgctcttctgtctactgaac- and anti-sense -atctgagtgtgagggtctgg- . the primers for b-actin (internal control) were: sense -cactatcggcaatgag-cggttcc- and anti-sense -cagcactgtgttggca tagaggtc- . the qpcr was performed at uc for s followed by cycles of uc for s, uc for s, and uc for s. the specificity of pcr product was examined by a dissociation curve, and results were analyzed by the ddct method [ ] . the expression of fgl in liver from mhv- infected ( h) pd- -deficient mice or their wt littermates was determined by western-blot; the protocol has been described previously [ ] . the serum fgl level from mice infected with or without mhv- was detected by using the mouse fgl elisa kit (cat: e mu; uscn life science inc., wuhan, china) and following the manufacturer's instructions. all results shown are representative of at least three separate experiments. unpaired student's t-test (two-tailed) or the mann-whitney test was used for comparison of two groups where appropriate. kaplan meier curve with log-rank test (graphpad prism . software) was used to analyze the mortality rate. pvalue , . was considered as statistically significant. its protein expression in the liver, spleen and lymph node from pd- -deficient mice after h of mhv- infection in the presence of ifn-c and tnf-a mabs or rat igg isotype control antibodies was detected by qpcr and immunohistochemistry, respectively. (c) the ifn-c and tnf-a mabs treatment resulted in decreased damage to the liver, spleen, lymph node and thymus after h of mhv- infection. (d) reduced fgl level by ifn-c and tnf-a mabs treatment resulted in reduced liver damage (indicated by ast and alt levels). (e) pd- -deficient mice were infected with mhv- ( pfu) and simultaneously treated with ifn-c and tnf-a blocking mabs (n = ) or rat igg control (n = ), the survival rate was monitored for a total of days. p = . , . was considered significantly different. one representative of three experiments that yielded similar results is shown. magnification . ns: not significantly different. *p, . and **p, . . n = /group. doi: . /journal.ppat. .g figure s the number of foxp -positive cells was not changed significantly in pd- -deficient mice after mhv- infection. foxp -positive cells in the liver, thymus, spleen, and lymph nodes between pd- -deficient and wt mice at h after mhv- infection were detected by immunofluorescence staining (left). statistical analysis of the number of foxp -positive cells in the indicated organs (right). blue color indicates nuclear dapi staining. scale bar = mm. ns: not significantly different. found at: doi: . /journal.ppat. .s ( . mb tif) viral hepatitis in the liver transplant recipient pattern of disease after murine hepatitis virus strain infection correlates with macrophage activation and not viral replication induction of monocyte procoagulant activity by murine hepatitis virus type parallels disease susceptibility in mice acquired immunity of a/j mice to mouse hepatitis virus infection: dependence on interferon gamma synthesis and macrophage sensitivity to interferon gamma the fgl / fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis pd- and its ligands in tolerance and immunity pd- inhibits t-cell receptor induced phosphorylation of the zap /cd zeta signalosome and downstream signaling to pkctheta shp- and shp- associate with immunoreceptor tyrosine-based switch motif of programmed death upon primary human t cell stimulation, but only receptor ligation prevents t cell activation pd- inhibits antiviral immunity at the effector phase in the liver cd ) inhibits the development of herpetic stromal keratitis (hsk restoring function in exhausted cd t cells during chronic viral infection enhancing sivspecific immunity in vivo by pd- blockade upregulation of pd- expression on hiv-specific cd + t cells leads to reversible immune dysfunction dysfunction and functional restoration of hcv-specific cd responses in chronic hepatitis c virus infection programmed death- -induced interleukin- production by monocytes impairs cd (+) t cell activation during hiv infection expression of b -h on gastric epithelial cells: its potential role in regulating t cells during helicobacter pylori infection schistosoma mansoni worms induce anergy of t cells via selective up-regulation of programmed death ligand on macrophages the pd- /pd-l costimulatory pathway critically affects host resistance to the pathogenic fungus histoplasma capsulatum role of the programmed death- pathway in the suppressive activity of alternatively activated macrophages in experimental cysticercosis novel strategies to eliminate persistent viral infections cytokineinduced hepatic apoptosis is dependent on fgl /fibroleukin: the role of sp / sp and stat /pu. composite cis elements intact type immunity and immune-associated coagulative responses in mice lacking ifn gamma-inducible fibrinogen-like protein role of pd- in regulating acute infections pd- expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis recall responses by helpless memory cd + t cells are restricted by the upregulation of pd- pd- on dendritic cells impedes innate immunity against bacterial infection a virus related to that causing hepatitis in mice (mhv) expression of the fgl and its protein product (prothrombinase) in tissues during murine hepatitis virus strain- (mhv- ) infection fulminant hepatic failure in murine hepatitis virus strain infection: tissue-specific expression of a novel fgl prothrombinase the novel cd +cd + regulatory t cell effector molecule fibrinogen-like protein contributes to the outcome of murine fulminant viral hepatitis characterization of human fibroleukin, a fibrinogen-like protein secreted by t lymphocytes targeted deletion of fgl leads to impaired regulatory t cell activity and development of autoimmune glomerulonephritis pd-l negatively regulates cd +cd +foxp + tregs by limiting stat- phosphorylation in patients chronically infected with hcv pd-l regulates the development, maintenance, and function of induced regulatory t cells analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method triptolide inhibits b -h expression on proinflammatory factor activated renal tubular epithelial cells by decreasing nf-kappab transcription we thanks prof. t honjo kindly give us the pd- ko mice. mhv- virus was provided by prof. q ning and she also gave the research some invaluable suggestions. conceived and designed the experiments: yc cy. performed the experiments: yc sw gg lf sg xf. analyzed the data: yc cy yw. wrote the paper: yc yw. key: cord- -f bs r authors: lai, kang yiu; ng, wing yiu george; cheng, fan fanny title: human ebola virus infection in west africa: a review of available therapeutic agents that target different steps of the life cycle of ebola virus date: - - journal: infect dis poverty doi: . / - - - sha: doc_id: cord_uid: f bs r the recent outbreak of the human zaire ebolavirus (ebov) epidemic is spiraling out of control in west africa. human ebov hemorrhagic fever has a case fatality rate of up to %. the ebov is classified as a biosafety level pathogen and is considered a category a agent of bioterrorism by centers for disease control and prevention, with no approved therapies and vaccines available for its treatment apart from supportive care. although several promising therapeutic agents and vaccines against ebov are undergoing the phase i human trial, the current epidemic might be outpacing the speed at which drugs and vaccines can be produced. like all viruses, the ebov largely relies on host cell factors and physiological processes for its entry, replication, and egress. we have reviewed currently available therapeutic agents that have been shown to be effective in suppressing the proliferation of the ebov in cell cultures or animal studies. most of the therapeutic agents in this review are directed against non-mutable targets of the host, which is independent of viral mutation. these medications are approved by the food and drug administration (fda) for the treatment of other diseases. they are available and stockpileable for immediate use. they may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the ebov. electronic supplementary material: the online version of this article (doi: . / - - - ) contains supplementary material, which is available to authorized users. the recent outbreak of the human zaire ebolavirus (ebov) infection starting in west african countries has resulted in , infected patients, as of th of november . a total of , deaths have been reported in six affected countries (guinea, liberia, mali, sierra leone, spain, and the united states of america) and two previously affected countries (nigeria and senegal) [ ] . apart from supportive care, neither a licensed vaccine nor a specific therapy is available for the treatment of the human ebov infection [ ] . the world health organization (who) has considered that it is ethically acceptable to offer unproven interventions that have shown promising results in laboratory and animal models, but have not yet been evaluated for safety and efficacy in humans as potential sources of treatment or prevention [ ] . several promising therapeutic agents have been identified for the treatment and immunization of the ebov. these may include monoclonal antibody (mabs)-based therapies (e.g. zmapp), anti-sense phosphorodiamidate morpholino oligomers (pmo avi- ), lipid nanoparticle small interfering rna (lnp-sirna: tkm-ebola), and an ebov glycoprotein-based vaccine using live-attenuated recombinant vesicular stomatitis virus (rvsv-ebogp) or a chimpanzee adenovirus (rchad-ebogp)-based vector. human trial results of these agents would not be available until next year. moreover, existing supplies of all these experimental medications and vaccines for compassionate use are either extremely limited or exhausted [ ] [ ] [ ] . to combat such an unprecedented global public-health crisis before these experimental agents are available, alternative available interventions that can target different steps in the replication cycle of the ebov should be explored in the management of the human ebov infection as contingency preparation for the international dissemination of the ebov outbreak in west africa. we have reviewed currently available therapeutic agents that have shown to be effective in suppressing the proliferation of the ebov in cell cultures or animal studies. we propose a therapeutic regimen to supplement the current supportive therapy aiming to reduce viral load, the most important factor in the determination of mortality. through viral load suppression, we may be able to prolong a patient's survival in order to provide a better chance for the patient to develop natural immune defense against the ebov. the ebov is an enveloped filamentous rna virus belonging to the family filoviridae. the -kb linear, non-segmented, negative-sense, single-stranded rna genome of the ebov encodes seven structural proteins and two non-structural proteins in the following order within the genome: ′ non-coding region (leader), nucleoprotein (np), virion protein (vp ), vp , glycoproteins (sgp/ssgp/gp , ), vp , vp , rnadependent rna-polymerase protein (l-polymerase), and ′ non-coding region [ ] . the ebov genome encodes one transmembrane protein gp , (gp -gp ) and two secreted non-structural proteins: secretary glycoprotein (sgp) and small soluble glycoprotein (ssgp). a small soluble delta peptide (Δ-peptide) is secreted from ebov-infected cells after the carboxylterminal cleavage of sgp [ ] . gp , is produced through transcriptional rna editing as a precursor for amino acid polyprotein (gp ), which is post-translationally cleaved by furin into two disulfide-linked subunits; a surface subunit, gp ; and a membrane-spanning subunit, gp . gp contains the receptor-binding domain (rbd) for host cell attachment and a mucin-like domain to protect the rbd from humoral and cell-mediated immunity. the rbd responsible for receptor binding, viral entry, and cellular tropism is covered by a heavily glycosylated "glycan cap". the transmembrane gp contains a helical heptad-repeat region, transmembrane anchor, and a -residue cytoplasmic tail. the gp drives fusion of the viral membrane with the endosomal membrane of the target cell. this gp -gp heterodimer then assembles as a trimer on the viral surface. this homotrimeric gp , complex forms the spike on the envelope membrane of the mature viral particles. during processing, gp , are unstable, and an abundant amount of a soluble non-virion form of gp and a scanty amount of gp , are released into the circulation [ ] [ ] [ ] [ ] . the virusassociated gp , and not the other soluble glycoproteins released during the virus infection are responsible for primary target cell activation [ ] . the highly glycosylated mucin-like region of gp is cytotoxic to the host cells [ ] . the shedding of souble gp , -like protein due to cleavage of ebov glycoprotein on the surface of ebov-infected cells by tumor necrosis factor-alpha converting enzyme (tace) can activate non-infected dendritic cells and macrophages to induce cytokine dysregulation and endothelial cell dysfunction [ ] . the gp of the ebov is able to counter the interferon (ifn)-inducible antiviral protein tetherin which restricts the vp -dependent budding of the progeny viral particles from infected cells [ ] [ ] [ ] . the sgp is produced from non-edited mrna species through furin cleavage from a precursor pre-sgp. the sgp shares the n-terminal amino acids with gp , but differs in the carboxyl terminus by amino acids. the sgp is released into the circulation in the form of homodimers in antiparallel orientation [ ] to evade an antibody-associated innate immune response [ , ] . the sgp has an antiinflammatory function and impairs the transmigration and activation of neutrophils [ , ] . while the gp , in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function, sgp protects the endothelial cell against cytokine-induced barrier dysfunction. the sgp constitutes at greater than % of the total gp synthesized during infection. hence, the hypersecretion of the sgp may protect the ebov against host humoral immune defense and the host endothelial cell against cytokine-induced cytotoxicity during the early phase of the ebov infection [ , , ] . Δ-peptide released in ebov-infected cells joins cathepsins and integrins to inhibit further entry of the ebov in a dose-dependent manner to prevent superinfection of ebov-infected cells. Δ-peptide inhibits entry of both marburgviruses and the ebov, indicating that they might interfere with a common pathway used by filoviruses to gain entry into target cells [ ] . the ssgp of a yet undefined function is produced through transcriptional editing and secreted in the form of a disulfide-linked homodimer that is exclusively n-glycosylated. while ssgp appears to share similar structural properties with sgp, it does not appear to have the same anti-inflammatory function as sgp [ , , ] . the ebov, being a rna virus with limited coding capacity, has utilized the host's unique metabolic pathway for its viral entry, replication, and egress. the entry of the ebov into cells is initiated by interaction of the viral gp with host cell surface t-cell immunoglobulin and mucin domain (tim- ) receptors. upon receptor binding, the ebov is internalized into endosomes primarily via macropinocytosis [ ] [ ] [ ] . within the acidified endosome compartment of the host cell, the heavily glycosylated gp is cleaved to a smaller -kda fusogenic form by the low ph-dependent cellular proteases cathepsin l (catl) and b (catb), exposing residues in the receptor binding site. this allows the binding of gp to cholesterol transporter niemann-pick c (npc ), a step in the late endosome phase essential for virus-host membrane fusion and viral entry [ ] [ ] [ ] [ ] . cells where the npc function has been biochemically disrupted or cells lacking npc showed resistance to the ebov infection. cells from subjects with npc disease were resistant to the ebov because of defects in the npc protein [ ] [ ] [ ] [ ] . after complete fusion of the viral and host endosomal membranes via conformational change in gp , viral rna and its associated proteins are released into the host cell cytoplasm [ ] . once inside the cytoplasm of the host cell, the ebov suppresses the innate immune response via vp and vp proteins [ ] , and hijacks transcription and translation for robust genome replication and the production of new virions. the ribonucleoprotein (rnp) complex that mediates transcription and replication of the ebov genome comprises np, vp , vp , and l protein [ ] [ ] [ ] [ ] . vp is essential in the initiation of the ebov transcription, but is not required for viral replication. however, dynamic phosphorylation of vp is an important mechanism to regulate the balance between the transcription and replication processes in the ebov replication cycle [ ] [ ] [ ] . this unique property of vp allows the development of a genetically stable vp deleted ebov vaccine with protective efficacy in the mice and guinea pig models [ ] . the matrix proteins vp and vp associated with the viral lipid coat are important for virus structure and stability. both matrix proteins vp and vp contribute to the regulation of viral genome replication and transcription [ ] and the budding of the virus [ ] [ ] [ ] , an important step prior to viral egress [ , ] . this distinct replication cycle of the ebov serves as an attractive target for the development of therapeutic agents against the ebov (see figure and table ). human ebov hemorrhagic fever, characterized by uncontrolled viral replication together with immune and vascular dysregulation, has a case fatality rate of up to % [ ] . type i alpha/beta interferons (ifn-α/β), encoded by a single ifn-β and homologous ifn-α genes in humans, represent an essential element of host defense against virus infections, including the ebov [ ] . the human ebov infection is associated with robust ifn-α production-with plasma concentrations of ifn-α that greatly ( -to -fold) exceed those observed in other viral infections-but limited ifn-β production [ ] . the upon receptor binding of ebov gp with host tim- receptor, ebov is internalized into endosome via macropinocytosis. within the acidified endosome compartment of the host cell, under the action of the low ph-dependent cellular proteases cathepsins, the receptor binding site of gp to cholesterol transporter niemann-pick c (npc ) is exposed. this results in conformational change in gp , leading to complete fusion of the viral and host endosomal membranes in the late endosome and the release of viral rna and its associated proteins into the host cell cytoplasm. ebov then hijacks transcription and translation for robust genome replication and viral protein production under the action of ribonucleoprotein polymerase complex (rnp polymerase). the accumulation of gp , in the endoplasmic reticulum leads to endoplasmic reticulum overload response (er-overload) which, in turn, induces cytokine dysregulation via the activation of nuclear factor kappa b (nfκb) through the production of reactive oxygen species (ros). new virions are released through atp-dependent budding and egress from host cell membrane. currently available therapeutic agents that target the different steps of the ebov life cycle are described in table . ebov, protected from the host interferon response by its encoded vp and vp proteins [ , [ ] [ ] [ ] , produced a heavy viral load [ ] [ ] [ ] , cytopathic damages [ , , ] , and cytokine dysregulation in humans [ ] [ ] [ ] [ ] . the efficient productive replication of the ebov inside monocyte and macrophages leads to a massive release of proinflammatory cytokines/chemokines and reactive oxygen species (ros) [ , , , , [ ] [ ] [ ] , which in turn leads to diffuse endothelial cell dysfunction [ ] [ ] [ ] [ ] [ ] , disseminated intravascular coagulation [ ] [ ] [ ] , and vasomotor collapse [ ] [ ] [ ] . the infection of the antigen presenting dendritic cells [ ] [ ] [ ] [ ] and profound bystander apoptosis of lymphocytes [ , [ ] [ ] [ ] impairs the development of adaptive immunity [ , ] and ebov-specific cd + t [ ] [ ] [ ] , as well as cd + t cells [ ] that are important for the clearance of, and protection from, the ebov infection. infected monocyte-derived dendritic cells were impaired in the secretion of pro-inflammatory cytokines, the up-regulation of co-stimulatory molecules, and the stimulation of t cells [ ] . numbers of cd + and cd + t cells are substantially reduced in fatal human and nonhuman primate (nhp) infections before death [ , , ] . immune evasion by the glycoproteins of the ebola virus: implications on passive immunization and vaccine development the ebov is able to counteract both humoral and cellmediated immunity through its gp , and sgp [ , ] . the overexpression of mature gp , on the plasma membrane results in the masking of antigenic epitopes on gp , itself and the shielding of mhc-i and integrin β, leading to evasion of antiviral immunity. steric shielding of surface epitopes by the heavily glycosylated gp impairs the recognition and killing of ebov-infected cells by the natural killer and cytotoxic cd + t cell during an acute viral infection. it may also contribute to the persistent infection in the natural reservoir host to perpetuate the spread of the ebov [ ] [ ] [ ] . the sgp can evade host antibody-mediated response through "antigenic subversion" by eliciting non-neutralizing antibodies that cross-react with gp , . thus, the massive secretion of sgp by the ebov may prevent effective neutralization of the virus during an ebov infection and reduce the effectiveness of vaccines that rely upon neutralizing antibody responses against gp , [ , ] . some of the antibodies against gp may lead to enhancement of infectivity of the ebov via interaction with complement component c q, a phenomenon known as the antibody-dependent enhancement. the ebov initiates infection by binding its gp to its specific human receptor sites on the surface of human cells. the interaction of c q enhances binding between the virus-antibody complex and the c q ligands on the cell surface, promoting interaction between the ebov and its receptor. these infectivity-enhancing antibodies were virus species specific and were primarily correlated with immunoglobulin igg a and igm levels, but not with igg levels [ , ] . the presence of infectivity-enhancing antibodies against gp , in the ebov infection raises concerns about the effectiveness of gp-based ebov vaccines, and the use of passive prophylaxis or treatment with gp-based antibodies [ , ] . antibodies against gp of the ebov can be neutralizing, enhancing, or non-neutralizing and non-enhancing. neutralizing antibodies are produced in infection by the ebov at a relatively low frequency [ ] . some anti-ebov antibodies are known to be neutralizing in vitro but not protective in vivo, whereas other antibodies are known to be protective in animal models in vivo, but not neutralizing in vitro [ ] . investigations of anti-gp antibodies against the ebov showed that non-neutralizing antibodies induce interferon-inducible transmembrane proteins (ifitmp) production to restrict entry of ebov. favipiravir suppress viral rna polymerase. inhibit na + /k + -atpase that are important in the budding and egress of encapsulated ebov. ouabain digoxin digitoxin anti-oxidants suppress ros-dependent nfκb activation and cytokine dysregulation induced by gp , -induced er-overload. high dose n-acetylcysteine infusion chloroquine, amiodarone, dronedarone and toremifene administration is associated with an increased risk of qt prolongation and torsades de pointes. verapamil should be avoided in patient with hypotension. recognized gp epitopes in the sgp or non-essential mucin-like domain of gp , while neutralizing antibodies were specific to rbd in gp or conformation-dependent epitopes at the base of the gp , spike where gp meets gp . two neutralizing antibodies (kz and jp k ) against ebov-that recognize conformation-dependent epitopes comprising residues in gp and gp -were identified to have quite distinct mechanisms of neutralization. kz is a human recombinant igg neutralizing antibody derived from a human survivor of a natural ebov infection during the outbreak in kikwit, democratic republic of congo. kz has impaired recognition for the sgp and binding was dependent on the presence of gp residues which are not present in the sgp. kz is able to inhibit cathepsin cleavage of gp , . jp k , a monkey derived neutralizing monoclonal antibody against ebov, recognized the cleaved, fusion-active form of gp [ ] . f is a mice derived monoclonal igg antibody that neutralizes sudan ebov by preventing the conformational changes in gp , required for membrane fusion. both f and kz recognize gp -gp -bridging epitopes at the base of the gp , trimer, indicating that this overlapping epitope may be one of the key sites for neutralization of the ebov, and is thus a target for immunotherapy and a key goal of vaccine design [ ] . antibody subclass may be another important factor in protection against the ebov. igg isotype may offer more effective protection against ebov [ , ] . although fully protecting guinea pigs from infection, kz fails to slow viral replication and protect nhps from the ebov infection [ ] . in contrast, rvsv-ebogp [ ] [ ] [ ] [ ] and rchad-ebogp [ ] [ ] [ ] [ ] based vaccination have demonstrated both prophylactic and post-exposure protection in nhps [ ] . this was previously attributed to the protective action of ebov-specific cd + and cd + t-cell response induced by these vaccines in limiting infection and the inability of kz to completely block all entries of the ebov into cells and its subsequent explosive replication [ ] . rchad-ebogpbased vaccination is able to generate potent humoral and cell-mediated responses. significant antibody titers are detectable at weeks post vaccination [ , ] . cd + cellmediated immunity has been shown to play a critical role in protection against the ebov infection in nhps in rchad-ebogp-based vaccination [ ] . on the other hand, humoral rather than the cell-mediated response contributes to protection against the ebov infection in nhps in rvsv-ebogp-based vaccination [ , ] . candidate vaccines expressing the ebov gp or np protect rodents and nhps from the lethal ebov infection [ ] [ ] [ ] . humoral and cell-mediated immune responses are working together to provide protection against the lethal ebov infection. either response alone may be able to limit virus replication but both arms of the immune response are required to clear the infection [ , ] . vp proteins (vp , vp , vp , and vp ) are poor inducers of cell-mediated immunity and are inaccessible to the protective effect of vp-induced neutralizing antibodies because they are not found on the surface of virions or infected cells [ ] . however, the genetic sites of these internal proteins are susceptible to sirna and pmo interference. tkm-ebola (a sirna targeting l-polymerase, vp , and vp ) can be administered intravenously or subcutaneously in a lyophilized lipid nanoparticle formulation. tkm-ebola offers post-exposure protection against the ebov infection in nhps. the fda has approved an "expanded access" program for the use of tkm-ebola in patients with confirmed or suspected infections [ , ] . anti-sense phosphorodiamidate morpholino oligomers avi- effectively reduce viral load, diminish virallyinduced pathology, and improve survival of nhps with the ebov infection by targeting vp and vp mrna. through judicious placement of positive charges on the drug backbone, the drug is able to bind to a negative charge on the virus even if binding at one or more drugvirus base pairs are lost through mutation. this integration of dual targeting and charge complementation significantly lowers the likelihood of drug resistance through viral mutagenesis [ , ] . currently available therapeutic agents that are effective in targeting the ebov infection in cell or animal studies may include convalescent plasma, favipiravir, chloroquine, amiodarone, dronedarone, verapamil, clomiphene, toremifene, ifn-β, na + /k + exchangers, na + /k + -atpase pump inhibitors, and antioxidants. except for convalescent plasma and favipiravir, most of the therapeutic agents under review are acting against the non-mutable targets of the host cells which participate in the replication cycle of the ebov. they may also have a complementary role to conventional therapy in the management of the current ebov outbreak in west african countries (see table ). the who issued a consensus statement that the use of whole blood therapies and convalescent blood serum needs to be considered as a matter of priority in the recent ebov outbreak in west african countries [ ] . the development of neutralizing antibodies and t-cell responses are important for recovery from the ebov infection [ , ] . patients who are able to mount an immune response to the ebov will begin to recover in seven to ten days and start a period of prolonged convalescence [ ] . in survivors, early and increasing levels of igg, directed mainly against the np and the vp , were followed by the clearance of circulating viral antigen and activation of cytotoxic t cells. in contrast, fatal infection was characterized by impaired humoral responses, with absent specific igg and barely detectable igm [ ] . convalescent blood has been shown to improve survival of ebov-infected patients during the outbreak in kikwit in [ ] . immunity against ebov gp is sufficient to protect individuals against infection, and several vaccines based on ebov gp are under development including recombinant adenovirus, parainfluenza virus, venezuelan equine encephalitis virus, vesicular stomatitis virus, and virus-like particles [ ] . neutralizing human monoclonal antibodies is able to protect mouse and guinea pigs from lethal ebov. however, the protection was achieved only by treatment shortly before or after viral infection [ ] [ ] [ ] . the ebov can rapidly mutate to produce antibody-escape mutants. hence, antibody therapy may require hyperimmune polyclonal serum or a panel of monoclonal antibodies of different epitope specificities to be successful [ , ] . these studies have laid the foundation for subsequent clinical research on the development of monoclonal antibodies [ ] [ ] [ ] [ ] and utilization of a monoclonal antibody cocktail such as mb- [ ] , zmab [ ] , and zmapp [ ] in the treatment of the ebov infection in nhps. it is interesting to note that all three monoclonal antibody cocktails include one antibody that binds to or close to the glycan cap and that two of the three monoclonal antibody cocktails include at least one antibody that binds the gp / gp interface, indicating that these two regions may be especially important in protection against ebov [ ] . the treatment window of monoclonal antibody therapy can be extended by the co-administration of adenovirus-vectored interferon therapy. in a guinea pig model, monoclonal antibodies combined with adenovirus-vectored interferon given three days after infection resulted in % survival, a significant improvement over either treatment alone [ ] . a subsequent study showed that such a combination therapy is capable of saving % of ebov-infected nhps when initiated after the presence of detectable viremia along with symptoms [ ] . ( ) favipiravir (t- ; -fluoro- -hydroxy- pyrazinecarboxamide) favipiravir is a broad-spectrum inhibitor of viral rna polymerase that is able to inhibit the replication of many rna viruses. it is registered in japan for the treatment of influenza virus infection [ , ] . favipiravir is able to suppress the replication of the ebov in cell culture. favipiravir, initiated at day after ebov infection, induced rapid virus clearance, reduced the biochemical parameters of disease severity, and prevented a lethal outcome in % of mice lacking the type i interferon receptor [ ] . oral favipiravir taken twice daily for days is able to give % protection against an aerosol ebov infection in an immune-deficient mice model [ , ] . the survival benefit was suboptimal in nhps. only one of the six animals tested survived. studies using dosages that are two to five times higher and have duration longer than shown in influenza studies are being conducted for the human ebov infection [ ] . bcx , a synthetic adenosine analogue with a viral rna polymerase inhibitor function, is active against the ebov and marburg virus in rodent and cell culture. bcx completely protects nhps from the marburg virus infection when administered as late as hours after infection [ , ] . the antimalarial drug chloroquine is able to increase endosomal ph. an acidic endosomal environment is important for the ph-dependent activation of cysteine proteases catb and catl, the proteases responsible for the cleavage of ebov gp , essential for endosomal virus-host membrane fusion [ , , [ ] [ ] [ ] . however, proteolytic processing of the ebov glycoprotein has been demonstrated to be not critical for ebov replication in cell culture [ ] or nhps [ ] . a recent study using a catb and catl deficient mouse model for the study of the ebov infection demonstrates that catb and catl activity is not absolutely required for ebov replication. the ebov glycoprotein cleavage seems to be mediated through a broader spectrum of proteases making therapeutic approaches targeting limited proteases unlikely to be beneficial to combat ebov infections [ ] . a broad-spectrum small molecule that targets the catl cleavage of the ebov and inhibits the entry of a wide variety of viruses has recently been identified. it has been examined for the potential to develop into a potent broad-spectrum antiviral medication [ ] . multiple cationic amphiphiles including amiodarone, dronedarone, verapamil, clomiphene, and toremifene have been identified as potent inhibitors of the entry of the ebov in an npc -dependent fashion [ , ] . amiodarone used for the treatment of atrial fibrillation and ventricular cardiac arrhythmia can induce lipidosis with features similar to niemann-pick c disease [ ] . amiodarone and dronedarone, having basic pka and high water solubility at acidic ph, accumulates within late endosomal compartments, blocking fluid-phase endocytosis, proteolysis and lipid trafficking, and inducing a niemann-pick c-like phenotype. in contrast to the niemann-pick type-c disease, they are not alleviated by cholesterol removal [ , ] . amiodarone, at concentrations that are routinely reached in human serum during anti-arrhythmic therapy ( . - . μg/ml), is a potent inhibitor of filovirus cell entry through late endosomes (ic . μg/ml for ebov), when induced as a niemann-pick c-like phenotype. significant inhibition is observed in most endothelial and epithelial cells (e.g. macrophage, monocyte, vascular endothelial cell), except for primary hepatocyte and fibroblast. the inhibitory effect of amiodarone on the entry of the ebov was dose-dependent and reversible upon removal of the drug. prolonged exposure to amiodarone will not lead to a compensatory change in the host cell. a similar inhibitory property is observed with the amiodarone-related agent dronedarone and the l-type calcium channel blocker verapamil [ , , , ] . both clomiphene and toremifene have anti-ebov activity in both the vero e (interferon-deficient african green monkey kidney epithelial cells) and hepg (human hepatocellular carcinoma) cell lines. the anti-ebov activity of clomiphene and toremifene is dependent not on its estrogen receptor antagonistic action but upon the ability of both drugs to induce a niemann-pick c-like phenotype to inhibit viral entry at late endosome. clomiphene and toremifene do not disrupt the interaction between primed gp and npc , but mediate the entry block indirectly through npc by targeting other endosomal/lysosomal proteins involved in the cholesterol uptake pathway whose functions may be regulated by npc . clomiphene and toremifene at mg/kg every other day have been shown to result in a % and % survival rate, respectively, in ebov-infected mice compared with % mortality in the control group in an in vivo murine ebov infection model. they are effective in both male and female mice [ , ] . however, the therapeutic dose against ebov cannot be achieved with the oral clomiphene dose used for inducing ovulation in humans [ ] [ ] [ ] . the therapeutic dose against ebov with tolerable side effects can be achieved with toremifene at an oral dose used in the human trial for the treatment of advanced carcinoma of the breast [ ] [ ] [ ] [ ] . toremifene is well absorbed and > . % bound to plasma protein. toremifene undergoes extensive liver metabolism and enterohepatic recirculation. the majority of the toremifene dose is excreted as metabolites in feces. the long terminal half-life of oral toremifene may be due to both plasma protein binding and enterohepatic recirculation [ , ] . interferon-induced transmembrane proteins (ifitms) are expressed basally in the absence of ifn induction in both primary tissues and cell lines [ ] . an ifitm is able to inhibit the entry of viruses to the host cell cytoplasm; permit endocytosis, but prevent subsequent viral fusion; and release viral contents into the cytosol. the human ifitm locus is located on chromosome and composed of four functional genes: ifitm , ifitm , ifitm , and ifitm . ifitm p is a pseudogene. viruses that are restricted by ifitm proteins tend to fuse with host cell membranes in a late endosome or lysosome that precedes the induction of type i ifn in infected cells. viral escape from restriction by ifitm proteins could be more challenging than for antagonizing inhibitory factors that function at later stages of the virus life cycle because the opportunity for de novo synthesis of viral inhibitors is not available. all four human ifitm proteins are induced robustly by both type i and type ii ifns. ifitm is active against multiple viruses, including the ebov and hepatitis c viruses [ ] [ ] [ ] . ifnβ is able to induce interferon-inducible transmembrane protein production to restrict entry of the ebov [ ] . early post-exposure treatment with ifn-β significantly increased survival time of rhesus macaques infected with a lethal dose of the ebov, although ifn-β alone failed to alter the mortality rate. ifn-β treatment was associated with a trend towards lower plasma and tissue viral burden and pro-inflammatory cytokines production [ ] . amiloride and its derivatives are used as potassiumsparing diuretics to treat hypertension and congestive heart failure. apart from inhibiting epithelial na + channel and cellular na + /k + exchangers, these drugs could also affect the function of other less well-defined ionexchangers (na + /ca + and na + /mg + ), and disturb the equilibrium of other ions, such as mn + [ ] [ ] [ ] [ ] . the entry of the ebov into host cells is the first step of infection and a crucial determinant of pathogenicity. upon receptor binding between gp and host tim- receptors, the ebov is internalized into endosomes primarily via the macropinocytic pathway. amiloride is able to inhibit the uptake of many viruses that utilize the macropinocytic pathway for host cell entry [ ] [ ] [ ] [ ] . amiloride at non-cytotoxic dosages leads to potent dosedependent inhibition of the entry and infection of the ebov [ , ] . amiloride can lead to dose-dependent inhibition of rna synthesis. this may be due to a direct blockage of a nucleotide entry tunnel or catalytic site, or due to its effect on the equilibrium of mg + and mn + that are essential co-factors for polymerase activity and nucleotide insertion [ , ] . these novel antiviral mechanisms of amiloride may uncover new targets for drug discovery against the ebov. adenosine triphosphate (atp) is essential in multiple steps in the replication cycle of many viruses. na + /k + -atpase pump is located in the plasma membrane of all animal cells to maintain the cell membrane potential. budding of enveloped viruses is a complex phenomenon that requires concerted actions of many viral and host components. atp may affect multiple steps in the budding process [ ] . atp is required for the assembly and maturation of a number of enveloped viruses such as the influenza virus, vaccinia virus, retrovirus, and herpes simplex virus. the na + /k + -atpase pump inhibitors, ouabain, lanatoside c, strophanthidin, and digoxin are able to inhibit the replication of the influenza virus, newcastle disease virus, and vesicular stomatitis virus through an interferonindependent mechanism [ ] . digoxin and lanatoside c have been shown to inhibit vaccinia virus replication at non-cytotoxic doses [ ] . ouabain has shown antiviral activity against the influenza virus [ ] , herpes simplex virus [ ] , sendai virus [ ] , murine leukemic virus [ ] , cytomegalovirus porcine reproductive and respiratory syndrome virus [ ] , and human cytomegalovirus virus [ ] . one common feature shared by these viruses is that they all possess a lipid envelope. the ebov is an enveloped filamentous rna virus. the secondary matrix protein vp -apart from its role in the evasion of host immune response, nucleocapsid formation, and regulation of replication-has an important role in viral budding and egress. na + /k + -atpase atp a is detected to have a close interaction with vp of ebov during replication. ouabain, at a non-cytotoxic concentration of nm, is able to suppress the replication of the ebov in human mrc- cells [ , ] . among the three cardiac glycosides that may include digoxin, digitoxin, and ouabain, only digoxin is commonly used in clinical practice. ouabain, because of its poor oral availability, is used primarily as a research tool. further research should be conducted to investigate whether digoxin and other na + /k + -atpase inhibitors might play a role in the management of the ebov or other enveloped virus infections. the virus-associated glycoprotein gp , is responsible for the activation of human macrophages [ ] . the highly glycosylated mucin-like region of the gp subunit of gp , is cytotoxic to the host cells [ ] . the mucin-like region in gp leads to an accumulation of gp , at the endoplasmic reticulum, induces endoplasmic reticulum stress [ ] , and activates nuclear factor kappa b (nf-κb) [ ] . mutations of the ebov that lead to an enhanced accumulation of gp , in the endoplasmic reticulum were significantly more cytotoxic than wild-type virus [ ] . in human cells, the accumulation of protein in the endoplasmic reticulum will lead to endoplasmic reticulum overload response (eroverload) which activates nf-κb through the production of ros [ ] . as a major transcription factor for antiviral and immune stimulatory activities, nf-κb is thought to play an important role in the induction of pro-inflammatory molecules, such as interleukin- β (il- β), and tumor necrosis factor α (tnf-α), upon cellular responses against a virus infection [ ] . the cytokine dysregulation of the ebov involves massive ros, nf-κb, tnf-α, and il- β activation [ , ] . the effectiveness of antioxidant therapy for the ebov infection indicates the importance of ros in the pathogenesis of the ebov [ ] . the activation of nf-κb by er-overload is ros-dependent [ ] . nf-κb-induced cytokine dysregulation of novel h n pneumonia has been shown to be suppressible by high-dose n-acetylcysteine (nac) antioxidant therapy at mg/kg continuous infusion daily [ ] . given the poor oral availability of nac in the range of % to % in humans [ ] , a therapeutic dose of nac equivalent to the intravenous route can hardly be delivered by oral preparation. nac is a category b drug for pregnancy and is affordable, with a wide therapeutic window. nac has an established safety profile even in high doses and prolonged use in humans [ ] [ ] [ ] . cytokine dysregulation is a common feature in the ebov infection and is associated with an enhanced mortality [ ] [ ] [ ] [ ] . antiviral medications directed against the mutable viral determinants of the ebov cannot directly prevent cytokine dysregulation. the early endothelial vascular damage characteristic of the ebov infection is not a direct effect of virus-induced cytolysis of endothelial cells, but is due to cytokine dysregulation resulting from massive release of proinflammatory cytokines/chemokines and ros by infected macrophage and monocytes [ ] [ ] [ ] . lymphocytes are resistant to the ebov infection. cytokine dysregulation may also contribute to the diffuse bystander apoptosis of lymphocytes [ , [ ] [ ] [ ] . with the safety profile of nac, if the therapeutic efficacy of a high-dose nac antioxidant therapy to manage ebov-induced cytokine dysregulation is confirmed, it may revamp the future management of the ebov infection. there is a desperate need for a viable treatment regimen in africa to engender hope and encourage people with symptoms and their close contacts to seek medical treatment, so as to limit the spread of the disease. this also helps to recruit and maintain adequate medical staff who are at high risk of contracting the disease. a proposed regimen against the human ebov infection based on available medications and information from in vivo animal testing and in vitro cell culture is attached (see tables and ). this regimen contains a cocktail of currently available medications that can target the different steps in the replication cycle of the ebov aiming to suppress viral proliferation. it has been shown that viral load is major contribution to survival in both human and animal studies [ ] [ ] [ ] ] . through viral load suppression, we may be able to prolong a patient's survival in order to allow the development of natural body immune defense against the ebov. the ebov has undergone a rapid mutation during its spread through humans [ ] [ ] [ ] . the ebov is an rna virus the replication of which is highly error prone with nearly one viral mutation occurring during each cycle of replication. this extremely high mutation rate leads to significant genetic and antigenic diversity that allows the ebov population to evolve resistance to antiviral medications and vaccines [ , ] . a combination therapy has been used in the treatment of rna virus infections, such as the human immunodeficiency virus (hiv) [ , ] and hepatitis c [ , ] to minimize the development of drug resistance. given the broad cell tropism and high replication rate of the ebov due to the potent suppression of both innate and adaptive immune responses of the host, patients with the ebov infection have an extremely high viral load. the selective pressure in the presence of the high mutation rate and viral load during the human ebov infection make the evolution of the ebov viral strains resistant to a single drug inevitable. the currently available medications in the proposed regimen-which is a treatment regimen containing a cocktail of antiviral medications targeting the different steps of the ebov replication in order to achieve maximal suppression of viral replication and to prevent the rapid development of resistance to favipiravir, the only drug in the regimen that is directed against a mutable target of the ebov-has been shown to reduce the replication of the ebov. [ ] [ ] [ ] . the current ebov vaccine (rvsv-ebogp and rchad-ebogp) and therapeutic agents (zmapp, tkm-ebola, pmo avi- , and favipiravir) under development are directed against the mutable targets of the ebov, and their effectiveness is limited by viral mutation. the ebov, being a rna virus with limited coding capacity, has utilized the host's unique metabolic pathway for its viral entry, replication, and egress. most of the therapeutic agents in this current review are directed against nonmutable targets of the host which is independent of viral mutation. these medications are fda-approved for the treatment of other diseases. they are available and stockpileable for immediate use. they may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the ebov. the primary target of the ebov is the mononuclear phagocytic system. the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells during the advanced stage of the disease [ , , ] . the ebov may produce a viral load of up to virions per ml serum in terminally ill patients [ ] . oral amiodarone prophylaxis, by inducing a niemann-pick c-like phenotype in the cells of the mononuclear phagocytic system, may prevent viral entry into these cells during needle stick injury. through protection of the mononuclear system by our prophylaxis and cocktail therapy, we hope to offer a better chance of survival to these patients by allowing them to develop a natural body immune defense against the ebov infection. the liver, containing the largest number of fixed tissue macrophages (kupffer cells), as part of the reticuloendothelial immune defense system of the body, is a major target for the ebov infection [ , ] . the ebov replicates to high titer in the liver [ ] . hepatic apoptosis may play a role in the pathogenesis of the ebov infection [ ] . toremifene is added to the treatment regimen for hepatic protection because amiodarone does not exert inhibitory action against the ebov in hepatocyte. however, both amiodarone and toremifene can increase qtc and the risk of torsades de pointes. therefore electrocardiogram should be carefully monitored if both drugs are to be used. amiodarone, favipiravir, and toremifene are available and stockpileable in oral preparations. these properties are advantageous in outbreak situations and contingency planning of a potential ebov epidemic or pandemic. the avoidance of intravenous administration will prevent needle stick injury in healthcare workers caring for the infected patients. ifn-β may have potential as an adjunctive postexposure therapy for high-risk exposure, such as needle stick injury, by inducing ifitm to limit entry of the ebov. post-exposure ifn-β treatment was associated with a trend towards lower plasma and tissue viral burden and pro-inflammatory cytokines production [ ] . the reduction in viral load and cytokine dysregulation coupled with optimal supportive therapy may improve the chance of survival of the host to allow the development of natural immunity to control the underlying ebov infection. ifitm is active against multiple viruses, including the ebov [ , ] and hepatitis c ml of blood may contain to virions in terminally ill patient. prophylactic amiodarone therapy may protect macrophage, monocyte and endothelial cells immediately from ebov during needle stick injury and accidental exposure and allow time for the consideration of ifn-β, toremifene, favipiravir and convalescent blood serum therapy. amiodarone is unable to protect hepatocyte from ebov infection. both amiodarone and toremifene can increase the risk of qt prolongation and torsades de pointes. the recommended dosage for treatment of human ebov infection may be to times higher than influenza studies. please confirm the recommended dose with the drug company. n-acetylcysteine intravenous infusion at mg/kg/day to control cytokine dysregulation (e.g. add g of intravenous preparation of n-acetylcysteine into each liter of intravenous replacement fluid). [ , , , ] . interferon induced ifitm plays an important role in the treatment of human hcv infection by inhibiting entry of hcv into the host cell [ ] . six million international units (miu) of ifn-β intravenous administration is as effective as a three miu twice-daily regimen for treatment of the hcv infection [ ] , but has lesser side effects that require discontinuation of the medication [ , ] . as the aim of ifn-β therapy in our regimen for post needle stick prophylaxis against the ebov infection is to induce ifitm to limit viral entry, the dose of ifn-β for the post needle stick prophylaxis [ , ] or induction therapy [ , ] for hcv infection in humans is chosen. once infection is fully established, ifn-β are replaced by convalescent blood serum and high-dose nac infusion for providing passive humoral immunity and for the control of ros-dependent nf-κb-induced cytokine dysregulation respectively. the ebov is classified as biosafety level pathogen and is classified by centers for disease control and prevention as a category a agent of bioterrorism with no approved therapies and vaccines for its treatment but carrying a high potential for large-scale dissemination. recent political, economic, military, and religious turbulence around the world raises concerns that the ebov might be used as an agent of bioterrorism [ ] [ ] [ ] . the recent ebov epidemic is spiraling out of control in west africa. the containment measures that worked in the past, such as isolating those who are infected and tracing their contacts, have failed due to an exponential rise in infected patients. although the short-term (threeand six-week) probability of international spread outside the african region is small, the risk of the extension of the outbreak to other african countries followed by international dissemination on a longer time scale is not negligible, indicating that this public health emergency has the potential to grow to extraordinarily destructive dimensions [ , ] . although several promising therapeutic agents and vaccines against the ebov are undergoing the phase i human trial, the current epidemic might be outpacing the speed at which drugs and vaccines can be produced [ ] . to combat such an unprecedented global public-health crisis before these experimental agents are available, alternative available interventions capable of managing the enhanced viral replication and cytokine dysregulation of the human ebov infection should be explored and stockpiled as contingency preparation for the worst-case scenario of an impending human ebov pandemic [ ] . like all viruses, the ebov largely relies on host cell factors and physiological processes for its entry, replication, and egress which, in turn, lead to cytopathic damage, cytokine dysregulation, and death of the host. these non-mutable key steps inside the host may be novel targets for future therapeutic strategies against these rapidly mutating viruses. if the efficacy of amiloride, digoxin, amiodarone, and high-dose nac antioxidant therapy against the human ebov infection is confirmed, the availability and affordability of these stockpileable oral regimen are for those workers who are already on amiodarone prophylaxis with a loading dose of amiodarone mg p.o. twice daily for days followed by maintenance amiodarone mg p.o. daily. electrocardiogram and thyroid function should be monitored. monitor for side effect of thrombocytopenia and proteinuria. intravenous dosage of ifn-β that are used for human hepatitis c virus infection to induce ifitm to limit viral entry. intravenous regimen is for those workers who have not been on amiodarone prophylaxis and agreed for the insertion of a central venous line for drug administration. intravenous amiodarone should be administered via central venous line to avoid phlebitis. the dosage for treatment of frequently recurring ventricular fibrillation and hemodynamically unstable ventricular tachycardia is recommended because it can achieve therapeutic drug level immediately after the first dose of amiodarone. agents make them ideal medications in pandemic situation and in countries with limited resources. they may have a complementary role to other antiviral medications to prevent the emergence of resistant strains. this may also signify a major breakthrough in future management of the ebov infection. additional file : multilingual abstracts in the six official working languages of the united nations. the authors declare that they have no competing interests. authors' contributions kyl and gwyn contributed to the conception, drafting, and writing of the paper. kyl, gwyn and fc revised the draft paper. all authors read and approved the revised paper. world health organization ebola response roadmap situation reports world health organization statement on the who consultation on potential ebola therapies and vaccines world health organization: ethical considerations for use of unregistered interventions for ebola viral 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• no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution key: cord- -rafwlw t authors: hindinger, claudia; bergmann, cornelia c.; hinton, david r.; phares, timothy w.; parra, gabriel i.; hussain, shabbir; savarin, carine; atkinson, roscoe d.; stohlman, stephen a. title: ifn-γ signaling to astrocytes protects from autoimmune mediated neurological disability date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: rafwlw t demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (ms). cells resident within the central nervous system (cns) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. astrocytes, the most abundant cns cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. experimental autoimmune encephalomyelitis (eae) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (ifn-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. inhibition of ifn-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (bbb) integrity or the composition of the acute cns inflammatory response. nevertheless, increased demyelination at peak acute disease in the absence of ifn-γ signaling to astrocytes correlated with sustained clinical symptoms. following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of ifn-γ signaling to astrocytes in neuroprotection. diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. the cns infiltrating leukocyte composition was not altered; however, decreased il- and il- correlated with sustained disease. these data indicate that astrocytes play a critical role in limiting cns autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of ifn-γ receptors. cns resident cells are targets of autoimmune mediated damage but also active participants in disease development, progression and control [ , ] . however, their contributions to neuroprotection and regulation by inflammatory mediators are not well defined. cns insults, including autoimmune disease, initiate rapid astrocyte activation characterized by cellular hypertrophy and increased intermediate filament glial fibrillary acidic protein (gfap) expression [ ] [ ] [ ] . astrocytes form a physical barrier surrounding areas of inflammation initially limiting bystander tissue damage [ , ] . however, this barrier subsequently impedes axonal regeneration contributing to sustained disability [ , , ] . innate and adaptive pro-inflammatory astrocyte functions include production of pro-inflammatory cytokines, reactive oxygen species, chemokines, and matrix metalloproteinases [ ] [ ] [ ] . by contrast, secretion of anti-inflammatory cytokines and scavengers of reactive oxygen species, as well as inhibition of both microglial activation and tumor necrosis factor (tnf) secretion, all support an astrocyte mediated anti-inflammatory function [ ] [ ] [ ] . therefore, astrocyte activation constitutes a ubiquitous, yet heteroge-neous response associated with both promoting and inhibiting cns repair [ ] [ ] [ ] . ms and eae are both associated with t cells secreting ifn-c (th cells) and il- (th cells) which play complex, not fully understood roles in disease initiation and progression [ , ] . in vivo and in vitro evidence indicates that suppression of encephalitogenic t cell proliferation within the cns during eae and activation of anti-inflammatory programs are in part mediated via astrocytes [ , ] . similar to astrocytes, ifn-c mediates both proand anti-inflammatory functions during autoimmune disease [ ] . early ifn-c induced effects are pro-inflammatory; ifn-c facilitates inflammatory cell access, shapes their composition, increases major histocompatibility complex (mhc) expression, contributes to macrophage and microglia activation, and initiates oligodendrocyte death [ ] . similarly, ifn-c mediated protection during eae is also multifaceted [ ] [ ] [ ] [ ] . it acts as a negative regulator of neutrophil accumulation, th cell activation, il- r signaling, protease secretion, and chemokine activity. it also inhibits proinflammatory cytokine secretion via induction of suppressor of cytokine secretion (socs) proteins, facilitates t cell apoptosis and protects oligodendrocytes via inducing endoplasmic reticulum (er) stress responses [ , , , ] . this highlights the critical role of a single mediator in both promoting disease but also limiting inflammatory mediated damage required to initiate repair cascades. based on the gatekeeper functions of astrocytes and the diverse biological effects of ifn-c, we set out to determine how ifn-c signaling specifically to astrocytes influences cns autoimmune disease. the results demonstrate that ifn-c signaling to astrocytes had no profound effects on initial disease progression, but played an essential protective role during the transition from acute to chronic disease. clinical remission induced by ifn-c signaling to astrocytes coincided with reduced demyelination, axonal degeneration, and astrocyte activation. the ifn-c receptor is expressed ubiquitously; however, these data reveal astrocytes as the primary target and mediator of the well established antiinflammatory activity of ifn-c within the cns. to understand the role of ifn-c signaling to astrocytes during the pathogenesis of cns autoimmune disease, eae was induced in transgenic mice expressing a signaling deficient dominant negative ifn-c receptor specifically on astrocytes (gfapcr d mice) [ ] . peripheral activation of self reactive t cells in gfapcr d mice was similar to wt mice (data not shown). this is consistent with both the cns restricted transgene expression in the gfapcr d mice as well as the similar t cell activation following peripheral immunization with a non-self antigen [ ] . following immunization the incidence of disease, initiation of clinical symptoms, and initial symptom progression were unaltered by the inability of astrocytes to respond to ifn-c ( fig. a ; table ). in addition, neither immunized group exhibited clinical symptoms of atypical eae associated with the absence of ifn-c [ ] . however, clinical symptoms in gfapcr d mice began to diverge from wt mice at , day post immunization (p.i.) prior to the peak of clinical disease. in both groups clinical disease peaked at , day p.i., but severity was increased from a score of . in wt mice to . in the gfapcr d group ( fig. a ; table ). gfapcr d and wt mice were compared at the peak of acute disease to determine if ifn-c signaling altered astrocyte activation or cns inflammation. astrocyte hypertrophy and gfap expression ( fig. b) were similar in both groups, indicating no overt effects of ifn-c on initial astrocyte reactivity. furthermore, neither the extent of inflammation nor the anatomic distribution of inflammatory cells was altered (fig. b) . flow cytometry confirmed no difference in the overall extent of cd hi inflammatory cells recruited into the cns ( fig. a) . furthermore, percentages of cd + t cells ( fig. a) , cd + t cells and macrophages within the infiltrates were also similar (fig. ) . in contrast to the association between increased eae severity and neutrophil accumulation in the global absence of ifn-c [ , ] , only a small percentage of neutrophils were identified in the cns of both groups by flow cytometry (fig. ) ; their low presence was confirmed by the inability to identify cells with the characteristic morphology of neutrophils in the brain by histopathology (fig. b) . the absence of increased neutrophils in the cns of gfapcr d mice during acute disease suggested that clinical disease was aggravated by a mechanism distinct from global ifn-c deficiency. in addition to the similar frequency of cd + t cells in the brains of the two groups ( fig. a) , myelin oligodendrocyte glycoprotein (mog) reactive cd + t cells secreting ifn-c were also similar ( fig. b ). by contrast, mog specific cd + t cells secreting il- ( fig. b ) and foxp + regulatory cd + t cells (tregs) were decreased in gfapcr d mice compared to wt mice (fig. c) . reduced th and tregs may be attributed to increased ifn-c [ , ] . alternatively, as ifn-c is protective in eae [ ] [ ] [ ] [ ] , increased disease severity may reflect reduced bioavailable ifn-c due to sequestration of ifn-c binding to the dominant negative receptor. however, measurement of ifn-c in cell free supernatants derived from dissociated brains at the peak of acute disease demonstrated protein levels of . . ng/brain in wt mice versus . . ng/brain in gfapcr d mice (n = ; p, . ). as overall frequencies of mog reactive cd + t cells secreting ifn-c were similar, increased ifn-c in the brains of gfapcr d mice suggested enhanced secretion at the cellular level. although a contribution of nk or cd t cells could not be excluded, these potential sources of ifn-c were unlikely due to their low frequencies (nk , % and cd + t cells , %, see fig. ) and their equivalent frequencies in the wt and gfapcr d mice. furthermore, reduced th cell and treg frequencies were consistent with suppression of these populations due to elevated ifn-c [ , , ] . inflammation in spinal cords from the gfapcr d mice was also similar to wt controls at the peak of clinical symptoms (fig. ) . although numbers and distribution of cd + t cells were also similar ( . . /mm in gfapcr d mice vs. . . / mm in wt mice; fig. s ), spinal cords of gfapcr d mice exhibited a , -fold increase in demyelination (fig. ) . areas of demyelination encompassed . . % of spinal cord white matter in gfapcr d mice versus . . % in wt mice (p# . ). furthermore, the increase in demyelination was associated with a prominent loss of axons in gfapcr d mice compared to controls (fig. ) . despite elevated demyelination and axonal loss in the absence of ifn-c signaling to astrocytes, spinal cords showed no evidence of differential astrocyte activation by either immunohistochemistry (fig. ), or differences in gfap mrna expression during the peak of acute disease (fig. ). although ccl , il- and tnf mrna expression were increased in the spinal cords of the gfapcr d mice, no differences in ifn-c, inos, cxcl or il- mrna were consistent with similar inflammation (fig. ) . these data suggest that the initial disease pathogenesis, reflected by an increased demyelination in spinal cords, but not brain, during ascending paralysis is dampened by ifn-c signaling to astrocytes. subsequent to peak disease severity the clinical symptoms in wt mice began a modest decline by day p.i. (fig. a ). by contrast, gfapcr d mice not only exhibited increased severity of clinical symptoms following day p.i., but the continued disease escalation was associated with increased mortality ( fig. a ; table ). sustained morbidity and significant mortality in gfapcr d mice after day p.i. implied a critical role for ifn-c induced astrocyte signaling in neuroprotection and limiting disability. a similar absence of clinical remission was found in a preliminary experiment comparing eae sjl mice carrying the gfapcr d gene (data not shown). these data suggest that astrocyte responses to ifn-c are protective, irrespective of genetic background. escalating disease in gfapcr d mice coincided with focal areas of intense inflammation in spinal cords, which contrasted with the more diffuse inflammation in wt mice (fig. ). demyelination was also increased with myelin loss encompassing . . % of spinal cord area in gfapcr d mice versus . . % in wt mice (p# . ) at day p.i. (fig. ). the demyelinated areas further exhibited enhanced axonal damage in gfapcr d mice (fig. ) , supporting a correlation between enhanced tissue damage, sustained morbidity and increased mortality ( fig. a ; table ). astrocyte activation associated with areas of myelin loss is a prominent finding in the cns of both patients with ms and rodents with eae. although demyelination was increased in the cns of gfapcr d mice, the extent of astrocyte activation associated with spinal cord white matter lesions was similar in both groups ( fig. ; , gfap + cells/mm ). by contrast, the frequency of activated astrocytes in spinal cord grey matter areas that were not associated with demyelinated lesions, was increased in gfapcr d mice with . . gfap + activated astrocytes/mm versus . . in wt mice (p, . ) (fig. ) . a similar increase in astrocyte activation within grey matter distal to white matter lesions was also detected in gfapcr d sjl mice during chronic eae (data not shown). flow cytometric analysis during chronic disease revealed an , fold increase in cd hi inflammatory cells confirming increased inflammation in the absence of ifn-c signaling to astrocytes (fig. a ). similar to the acute disease, relative percentages of neutrophils, macrophages, cd + and cd + t cells were all similar (fig. ) . furthermore, no differences in expression of activation markers on cns derived cd + t cells, including cd , cd , cd , fas, fasl, icos and cd were evident between the groups (data not shown). the percentages of mog reactive th cells were also not altered in the cns of gfapcr d relative to wt mice (fig. b ) and the decreased percentage of potentially destructive th cells identified during acute disease (fig ) , was also sustained during chronic disease (fig. b ). equivalent expression of il- , il- and tgf-b mrna (fig. c ) suggested that the decrease in th cells was dependent upon an increase in ifn-c and not related to a defect in activation or maintenance signals [ ] . increased inflammation and sustained mhc class ii expression on microglia (fig. a ) further suggested that ifn-c signaling to astrocytes down regulates inflammation universally without altering the composition of the cns infiltrates. this concept is supported by sustained composition of cns infiltrates during inflammation induced astrocyte apoptosis [ ] . the inability to restrain ongoing inflammation during eae in gfapcr d mice was evident at multiple levels. astrocyte activation was sustained consistent with increased expression of gfap mrna. ccl , ccl and cxcl mrna levels were increased (fig. c ). the expression of mrna encoding potentially destructive immune mediators including il- , il- , inos, and tnf were all increased (fig. c ). ifn-c was , -fold higher in the cns of gfapcr d mice (fig. ) . lastly, the level of the antiinflammatory cytokine il- was reduced ( fig. ), suggesting limited availability of il- may be a key in prolonging astrocyte activation and inflammation [ ] . a possible link between reduced il- and the inability of ifn-c signaling to astrocytes is provided by the capacity of ifn-c to induce il- in astrocytes [ ] , thereby promoting il- production. indeed il- in the cns of the gfapcr d mice was significantly reduced compared to wt mice during chronic disease (fig. ). by contrast, il- ( fig. ) , an indirect suppressor of cns inflammation by promoting ifn-c production [ ] , was similar in the cns of both gfapcr d and wt mice. astrocytes derived from gfapcr d and wt mice were stimulated with ifn-c to confirm ifn-c dependent il- secretion by astrocytes [ ] . while ifn-c induced il- secretion by astrocytes from wt mice, il- secretion was significantly reduced in cultures derived from gfapcr d mice (fig. ) . these data support the possibility that ifn-c mediated il- secretion by astrocytes regulates eae effector t cell function, inflammation and tissue destruction via induction of il- ; however, this does not exclude the possibility that the inability of astrocytes to respond to ifn-c could directly or indirectly dysregulate a variety of other immunomodulatory functions [ , , ] . in the eae model of ms, ifn-c functions as a proinflammatory cytokine in the early stages of disease [ , , ], yet it also assumes a prominent anti-inflammatory role during the transition to remission [ , [ ] [ ] [ ] [ ] . protection has been attributed to a variety of potentially interrelated mechanisms including limiting neutrophil accumulation, th cell activation, il- r signaling, matrix metalloproteinase secretion, pro-inflammatory cytokine secretion and chemokine activity; in addition ifn-c facilitates t cell apoptosis and protects oligodendrocytes from death via an er stress response [ , , ] . the activities of astrocytes during autoimmunity also range from pro-to antiinflammatory [ ] [ ] [ ] . however, to what extent a direct action of ifn-c on astrocytes contributes to inhibitory mechanism is unclear. the data herein demonstrate that among the multiple early functions of astrocytes imposed by innate responses are largely pro-inflammatory during eae [ , , , ] . for example, astrocytes contribute to the loss of bbb integrity via secretion of reactive oxygen species, chemokines and pro-inflammatory cytokines [ ] [ ] [ ] . this pro-inflammatory milieu is associated with initial axonal damage prior to accumulation of self reactive t cells [ ] , which further promotes immune mediated damage. blocking the pro-inflammatory transcription factor nf-kb in astrocytes improves recovery during chronic eae [ , ] . supporting an initial pro-inflammatory role of astrocytes dependent on innate, not ifn-c responsiveness, inhibition of ifn-c signaling to astrocytes did not influence eae onset or incidence, initial disease progression, astrocyte activation, or bbb integrity as indicated by similar entry of inflammatory cells into the brain. by contrast, an inflammation dampening effect of ifn-c became evident during the onset of disease remission. astrocytes limit ongoing inflammation and pathogenic processes at several levels. they not only facilitate repair by inhibiting inflammatory cell entry into the cns parenchyma [ , , ] , but also down regulate t cell effector function and proliferation [ , , ] . for example, eae in gfap deficient mice results in more severe and widespread inflammation [ ] . in addition, t cell-astrocyte interactions as well as astrocyte secretion of an unidentified soluble product, distinct from nitric oxide, prostaglandins, or tryptophan metabolism, suppress t cell proliferation [ , ] . t cell proliferation was also not inhibited via defective il- release, despite the suggestion that t cell-astrocyte interactions facilitate secretion of anti-inflammatory cytokines [ , ] . lastly, astrocytes may limit inflammation by triggering apoptosis in t cells [ ] . nevertheless, the role of ifn-c signaling in these potentially anti-inflammatory, protective mechanisms is not clear. elevated ifn-c in the cns of gfapcr d mice during both acute and chronic disease excluded limited ifn-c due to sequestration by the transgenic receptor as a mediator of increased clinical severity and mortality. indeed, enhanced ifn-c production may reflect an attempt to compensate for the loss of ifn-c dependent astrocyte mediated control of inflammation [ ] . sustained expression of pro-inflammatory cytokines, particularly il- and tnf, thus represents a primary mechanism underlying ongoing tissue destruction in gfapcr d mice. il- is known to mediate neurological dysfunction [ ] and astrocytes are the predominant source of il- during cns autoimmune disease. furthermore, its secretion is down regulated by ifn-c induced socs activity [ ] . on the other hand, sustained tnf implicated dysregulated activation of microglia or macrophages via increased ifn-c or il- , as tnf is predominantly secreted by activated cns macrophages and microglia during eae [ , ] . however, the down regulation of microglia activation, including tnf secretion, following interaction with activated astrocytes [ ] questions this notion. while both il- and tnf may thus contribute to sustained pathological changes, the source of tnf remains unclear. similarly, astrocytes are a potential source of the ifn-c induced chemokine cxcl during eae, one of the chemokines controlling t cell recruitment [ ] . however, the increased expression of cxcl coupled with the inability of the astrocytes in the gfapcr d mice to respond to ifn-c suggests altered microglia activation and secretion of cxcl [ ] or activation of cxcl transcription via an independent signaling pathway mediated by tnf or type interferons [ , ] . importantly, pathology further correlated with decreases in both tregs and il- , but not with increased antigen specific th cells, although astrocytes are critical targets of il- [ ] . although it is possible that the increased ifn-c in the cns influenced peripheral activation of antigen specific th cells, previous data demonstrated no evidence for expression of the transgene in peripheral organs, including lymphoid organs [ ] . il- , secreted by a variety of cells types including cd + t cells, cd + t cells and tregs [ ] , inhibits both acute and chronic eae [ , ] . the decrease in this anti-inflammatory cytokine suggests that the induction of il- secretion is a critical aspect of astrocyte responses to ifn-c, thus promoting il- secretion by t cells [ , ] . however, our data is unable to distinguish the contribution of paracrine versus autocrine ifn-c induced signals to astrocytes on il- production, as il- also regulates astrocyte activation [ ] , and can itself be secreted by astrocytes to exert autologous functions [ ] . in addition to mediating an imbalance of protective versus detrimental cytokines, our results demonstrate that ifn-c signaling to astrocytes limits the extent of astrocyte activation during chronic eae, specifically in grey matter. activated astrocytes, especially within and adjacent to areas of demyelination are a prominent feature of white matter plaques associated with both chronic ms and eae [ ] [ ] [ ] . astrocytes protect neurons and oligodendroglia, but also form glial scars which inhibit regeneration after neuronal injury [ , , , ] . the absence of reactive astrocytes increases axonal regeneration after injury [ ] , consistent with the concept that limiting astrogliosis is critical for axonal regeneration after neuronal injury. while the significance of sustained astrocyte activation in grey matter tracks is unclear in our model, it is consistent with increased axonal damage, and suggests possible damage to axons outside the lesions, which may not be manifested by histological analysis. limiting inflammation following either infection or during an autoimmune attack is a prerequisite for the initiation of repair. this is critically important within the cns which has limited regenerative capacity. in summary, our data identify astrocytes as prominent targets underlying ifn-c mediated suppression of chronic cns inflammation [ ] . the data further provide a link between sustained inflammatory responses, enhanced demyelination and axonal degeneration associated with loss of neurological function during eae and chronic progressive ms. although the inability of astrocytes to respond to ifn-c did not alter disease in the brain, engagement of the ifn-c receptor on astrocytes in spinal cord limits demyelination and functions in a neuroprotective capacity. current therapies for ms are primarily focused on antiinflammatory and immunomodulatory approaches and have been partially successful in treating acute episodes. the identification of astrocytes as critical responders and mediators of ifn-c signaling in limiting cns autoimmune disease may provide insights into new approaches to limit long term progression to disability. brains and spinal cords from perfused mice were homogenized separately as previously described [ , ] . homogenates were centrifuged at g for min at uc. supernatants were stored at - uc for cytokine determination (see below). cells were resuspended in % percoll (amersham biosciences, piscataway, nj) and isolated by centrifugation ( g for min at uc) onto % percoll cushions. non-specific binding was inhibited by incubation with anti-cd /cd ( . g ; bd biosciences, san diego, ca) and a % mixture of normal goat, human, mouse and rat serum for min on ice. fitc, pe, percp, and apc conjugates with monoclonal antibodies (mab) used to identify and quantify microglia and inflammatory cells were: cd (gk . ), cd a ( - . ), cd ( -f ), mhc class ii ( g ), ly g ( a ) (bd biosciences), and f / (serotec, raleigh, nc). microglia and inflammatory cells were distinguished based on differential cd expression. cd + t cells were identified as cd hi cd + , cd + t cells as cd hi cd + , macrophages as cd hi f / + and neutrophils as cd hi ly g + mhc class ii -. mog specific induction of cytokines was determined by stimulation of cns cells with mg/ml peptide for h at uc with golgistop (bd biosciences) added for the last h. intracellular cytokines were detected with fitc-anti-ifn-c (clone xmg . ; bd biosciences) and pe-anti-il- (clone tc - h ; bd biosciences). intracellular foxp was detected by staining for cell surface markers, followed by permeabilization with fixation/ permeabilization reagent (ebioscience, san diego, ca) and incubation with pe-labeled anti-foxp (fjk- s; ebioscience). cells were analyzed on a facscalibur flow cytometer (bd biosciences) using cellquest pro software (bd biosciences). data was analyzed using flowjo ( . . ) software (tree star inc., ashland, or). cytokines were determined by elisa using antibody pairs and recombinant cytokine standards from bd bioscience. il- was measured using quantikine mouse il- p immunoassays (r&d systems inc., minneapolis, mn). following anesthesia, mice were perfused with pbs (ph . ). brains and spinal cords were fixed with clark's solution ( % ethanol and % glacial acetic acid), and embedded in paraffin. spinal cords were divided into sections prior to embedding, corresponding to cervical, thoracic and lumbar levels. cross sections ( mm), were stained with either hematoxylin and eosin (h&e) or luxol fast blue (lfb). immunoperoxidase staining was used to identify activated astrocytes with anti-gfap (abcam, cambridge, ma) and axonal integrity with smi- and smi- mab (sternberger monoclonals inc., lutherville, md) followed by visualization using vectastain abc kit (vector laboratories, burlingame, ca) and , -diaminobenzidine (sigma-aldrich). sections from at least separate experiments containing at least mice per group were reviewed in a blinded manner. numbers of gfap + cells in spinal cord were determined in non-overlapping fields ( . mm ) in white matter and gray matter. stained spinal cord sections of all levels on individual glass slides were scanned ( ) and digitally imaged at high resolution with an aperio scanscope (vista, ca). aperio software was used to quantify areas of demyelination within the white matter tracks of each of the sections per individual mouse. for analysis of cd + t cells spinal cords were embedded in tissue-tek o.c.t. (andwin scientific, tryon, nc), flash frozen in liquid nitrogen and stored at uc. blocks were warmed to uc prior to cutting mm sections by cryostat. following fixation with acetone for min at uc, non-specific antibody binding was blocked with cyto q background buster (innovex biosciences, richmond, ca) for min. sections were stained with anti-cd (l t ) antibody (vector laboratories) diluted in cyto q immuno diluent (innovex biosciences) followed by biotinylated rabbit anti-rat, peroxidase abc reagent and visualized with novared substrate (all from vector laboratories). sections were counter stained with hematoxylin to visualize the nuclei. spinal cord sections were scanned ( ) and digitally imaged at high resolution with an aperio scanscope. mixed glial cultures (, % astrocytes) were established from neonatal gfapcr d and wt mice as previously described [ ] . il- secretion was determined h after rifn-c ( ng/ml) stimulation. frozen tissues were homogenized in trizol (invitrogen, carlsbad, ca) and cdna prepared as described [ , ] . quantitative real-time pcr was performed using ml of cdna and sybr green master mix (applied biosystems, foster city, ca) in duplicate on a fast real-time pcr system (applied biosystems). expression levels were normalized to ubiquitin or gapdh using the following formula: statistical significance was determined by two-tailed student's t test. a value of p, . was considered statistically significant. figure s cd + t cell recruitment into the spinal cord during acute eae. cd + t cells were visualized in mm frozen sections of spinal cords from wt and gfapcr d tg mice at day p.i. immunoperoxidase stain (novared chromogen with hematoxylin counterstain). scale bars = microns. (tif) multiple sclerosis: a complicated picture of autoimmunity the immunology of multiple sclerosis astrocytes: biology and pathology astrocytes in the tempest of multiple sclerosis astrocytes-friends or foes in multiple sclerosis leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice reactive astrocytes form scar-like perivascular barriers to leukocytes during adaptive immune inflammation of the cns regeneration beyond the glial scar autoimmune t cell responses in the central nervous system how interferon-gamma keeps autoimmune diseases in check the integrated stress response prevents demyelination by protecting oligodendrocytes against immune-mediated damage intrathecal delivery of ifn-gamma protects c bl/ mice from chronicprogressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes interferon-gamma confers resistance to experimental allergic encephalomyelitis mice with a disrupted ifn-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (eae) ifn-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis cross-regulation of signaling pathways by interferongamma: implications for immune responses and autoimmune diseases socs and socs in the control of cns immunity astrocyte expression of a dominant-negative interferon-gamma receptor regional cns responses to ifn-gamma determine lesion localization patterns during eae pathogenesis empowering t helper cells in autoimmunity gp -dependent astrocyte survival is critical for the control of autoimmune central nervous system inflammation attenuation of astroglial reactivity by interleukin- suppressive effect of il- on encephalitogenic th cells and the effector phase of experimental autoimmune encephalomyelitis early administration of il- suppresses eae through induction of interferon-gamma suppression of autoimmune inflammation of the central nervous system by interleukin secreted by interleukin -stimulated t cells astrocytes are active players in cerebral innate immunity astrocyte-associated axonal damage in pre-onset stages of experimental autoimmune encephalomyelitis inhibition of transcription factor nf-kappab in the central nervous system ameliorates autoimmune encephalomyelitis in mice transgenic inhibition of astroglial nf-kappa b improves functional outcome in experimental autoimmune encephalomyelitis by suppressing chronic central nervous system inflammation reactive astrocytes protect tissue and preserve function after spinal cord injury experimental autoimmune encephalomyelitis in mice lacking glial fibrillary acidic protein is characterized by a more severe clinical course and an infiltrative central nervous system lesion apoptosis of inflammatory cells in immune control of the nervous system: role of glia ifn-gamma shapes immune invasion of the central nervous system via regulation of chemokines neurologic disease induced in transgenic mice by cerebral overexpression of interleukin increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? tnfr signalling is critical for the development of demyelination and the limitation of t-cell responses during immune-mediated cns disease il- production by central nervous system microglia is inhibited by astrocytes chemokines and glial cells: a complex network in the central nervous system tnfa and ifnc synergistically enhance transcriptional activation of cxcl in human airway smooth muscle cells via stat- , nf-kb, and the transcriptional coactivator creb-binding protein concomitant detection of ifna signature and activated monocyte/dendritic cell precursors in the peripheral blood of ifna-treated subjects at early times after repeated local cytokine treatments astrocyterestricted ablation of interleukin- -induced act -mediated signaling ameliorates autoimmune encephalomyelitis regulation and functions of the il- family of cytokines in inflammation and disease il- is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of il- -and il- -deficient and transgenic mice central nervous system expression of il- inhibits autoimmune encephalomyelitis multiple sclerosis: cytokine receptors on oligodendrocytes predict innate regulation axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination distinct regulation of mhc molecule expression on astrocytes and microglia during viral encephalomyelitis we thank bruce trapp for helpful discussions, wenqiang wei, eric barron and ernesto barron for assistance with the histopathology and jeffrey tarcy, kate stenson and maria ramirez for assistance with mouse husbandry and immunological assays. key: cord- -qcjbtflt authors: carrero, javier antonio title: confounding roles for type i interferons during bacterial and viral pathogenesis date: - - journal: international immunology doi: . /intimm/dxt sha: doc_id: cord_uid: qcjbtflt although type i interferons (ifn-i) were initially defined as potent antiviral agents, they can also cause decreased host resistance to some bacterial and viral infections. the many antiviral functions of the ifn-i include direct suppression of viral replication and activation of the immune response against viruses. in addition to their antiviral effects, ifn-i are also protective against several extracellular bacterial infections, in part, by promoting the induction of tnf-α and nitric oxide. in contrast, there is a negative effect of ifn-i on host resistance during chronic infection with lymphocytic choriomeningitis virus (lcmv) and acute infections with intracellular bacteria. in the case of lcmv, chronic ifn-i signaling induces adaptive immune system suppression. blockade of ifn-i signaling removes the suppression and allows cd t-cell- and ifn-γ-mediated resolution of the infection. during acute intracellular bacterial infection, ifn-i suppress innate immunity by at least two defined mechanisms. during francisella infection, ifn-i prevent il- upregulation on γδ t cells and neutrophil recruitment. following listeria infection, ifn-i promote the cell death of macrophages and lymphocytes, which leads to innate immune suppression. these divergent findings for the role of ifn-i on pathogen control emphasize the complexity of the interferons system and force more mechanistic evaluation of its role in pathogenesis. this review evaluates ifn-i during infection with an emphasis on work carried out ifn-i-receptor-deficient mice. the type i interferons (ifn-i) are an extensive family of pleiotropic cytokines that all signal through the ubiquitously expressed ifn-i receptor, termed the 'ifn-α receptor' (ifnar) ( ) . the activity of these cytokines was discovered in the s because of their ability to 'interfere' with viral infection ( ) . molecular cloning techniques and genome sequencing have led to the identification of an extensive number of members of the ifn-i family. in mice, the two best analyzed members of this family are the cluster of ifn-α subtypes and ifn-β molecule. genetic ablation of ifnar (ifnar -/-) in the mouse is sufficient to prevent signaling by all members of the ifn-i family and is the biologically defining activity that groups the ifn-i molecules into one family ( ) . ifnar is composed of two chains (ifnar and ifnar ) that coordinately activate the kinases jak and tyk (tyrosine kinase ) upon ifn-i binding. jak (janus kinase ) and tyk phosphorylate stat and stat that, together with interferon regulatory factor (irf ), form the interferon-stimulated gene factor (isgf ) complex ( ) . isgf binds to interferonstimulated response elements to cause upregulation of over genes. the type ii interferon receptor, ifngr -ifngr , which binds ifn-γ, the only type ii interferon, activates jak and jak , leading to stat phosphorylation and homodimerization ( ) . because of the similarities in signaling, many of the genes that are upregulated by ifn-i are also upregulated by ifn-γ, providing some degree of redundancy between the ifn-i and ifn-γ signaling pathways ( ) . however, there are a large number of genes unique to ifn-i. although many of the ifn-i-specific genes have defined antiviral functions, the role of many remains unresolved. the antiviral activity of ifn-i was initially defined with conditioned supernatant inhibition of viral growth and then with purified cytokines. however, it was the generation of mice deficient in ifnar signaling that permitted examination of infections under more physiological conditions ( ) . this review will focus on the role of ifn-i during murine infection with viral and bacterial pathogens. there is also extensive work on the role ifn-i in the pathogenesis of autoimmunity and cancer, and more recently, in fungal and protozoan infections ( ) ( ) ( ) . although this work has contributed to our understanding of the biological activities of ifn-i, it is beyond the scope of this review. because of the extent of the ifn-i literature, this review will mostly limit itself to experiments that have examined viral and bacterial pathogenesis in ifnar -/mice with a particular emphasis on work that has examined lethality and pathogen burden. four major themes will be covered: first, there is a broad summary of the (generally) protective role of ifn-i in mice infected with viruses. second is the recent finding that ifn-i promote the suppression of adaptive immunity seen during chronic infection with lymphocytic choriomeningitis virus (lcmv). third, there is an examination of the divergent results that have been obtained using different bacterial pathogens. finally, there is a detailed examination of the role of ifn-i during listeria monocytogenes infection. the consensus in the field is that ifnar signaling is protective against most types of viral infection. table summarizes some of the results obtained after infecting wild-type and ifnar -/mice with multiple species and strains of viruses. in most cases, the absence of systemic ifnar signaling by the mouse led to an increase in viral titer, lethality, or both compared with controls. ifn-i is important in handling all major genetic classes of viruses including single-stranded rna (ssrna; +/-stranded), double-stranded rna and double-stranded dna viruses, and acute retroviruses (ssrna-rt). the two exceptions to the strict requirement for ifnar are influenza and dengue virus infections. in the case of influenza and potentially other respiratory viruses, the type iii interferon system (which comprises ifn-λ subtypes and signals using il- r -ifnlr ) plays a dominant role in restricting acute epithelial cell infection, thereby limiting the requirement of ifn-i signaling ( , ) . in dengue, ifnγ-mediated protection is dominant over ifn-i, although the combined ifnar -/-× ifngr -/mice are more susceptible than the ifngr -/mice ( ) . the effects of ifn-i that limit viral infection are extensive, but several aspects are important to consider. ifn-i signaling enhances the susceptibility of virally infected cells to undergo programmed cell death, thereby limiting viral replication ( , ) . dendritic cells (dcs) exposed to ifn-i become activated and secrete proinflammatory cytokines that lead to activation of the adaptive immune response ( ) . nk cells become potent killers of virally infected cells after exposure to ifn-i ( , ) . ifn-i has direct effects on adaptive αβ t cells and sensitizes them to activation via the tcr ( , ) . ifn-i production by plasmacytoid dcs promotes b-cell activation and production of antiviral antibody ( , ) . in general, these effects of ifn-i signaling are beneficial to the host, as they lead to control of viral replication and spread. the importance of host ifn-i signaling is further reinforced by viral evolution. viruses have evolved extensive immune-evasion strategies many of which center around inhibition of the host ifn-i response ( , ) . however, the biological responses to ifn-i do not always lead to beneficial outcomes to the host. in the case of viral infections, the best studied example of the negative role of ifn-i are chronic viral infections, in particular infection with lcmv. a long-standing finding in the lcmv field is that small genetic changes can convert an acutely infective strain of lcmv (armstrong b) into a chronically infective strain (armstrong b clone ; cl ) ( ) . several hallmarks of the negative effects of chronic viral infection have been discovered using this system. mice become chronically infected with lcmv because of t-cell 'exhaustion' that prevents normal clearance ( ) . several factors have been implicated in the suppression of t-cellmediated clearance of chronic lcmv; most salient among them are il- and pd- (programmed cell death ). il- is known to antagonize inflammatory activation on multiple immune cell types and its neutralization prevents chronic infection with lcmv ( ) . pd- , a member of the cd /ctla family of t-cell regulators, is upregulated on exhausted t cells found in chronically infected mice. its ligands, pd- l and pd- l, are broadly expressed and inducible by interferons ( ) . the interaction of pd- with pd-l acts to limit t-cell activity during chronic infection. blockade of the pd- -pd-l interaction using mabs derepresses cd t-cell activity and leads to enhanced adaptive immune responses to lcmv infection ( ) . in two recent publications, the effects of il- and pd- in limiting the response to lcmv infection have been causally linked to ifn-i signaling ( , ) . during the initial stages of infection with cl , the absence of ifn-i signaling allows for an increased viral titer and delayed clearance during the acute phase of the response ( , ) . wild-type mice control the primary infection well but become chronic carriers. ifnar -/mice also become chronic carriers albeit at higher viral loads. the cl strain induces higher levels of ifn-α and ifn-β than the acutely infective armstrong strain ( ) . the major early producer of ifn-i are plasmacytoid dcs that are infected with the virus ( ) . the presence of ifn-i is associated with a prolonged signature of interferon-inducible genes in spleen cells ( ) . despite having higher early titers of lcmv, ifnar -/mice show reduced il- in serum and reduced pd-l expression on myeloid cells. in wild-type mice, blockade of ifnar signaling with neutralizing mabs replicates this effect, leading to reduction of il- and pd-l . furthermore, neutralization of ifnar after the establishment of chronic infection leads to reduced viral burden ( ) . therefore, ifnar plays a major role in the establishment of chronic infection with lcmv and neutralization of ifnar has therapeutic potential for patients harboring chronic viral infections. the response of ifnar -/mice to bacterial infections varies depending on the species and route of infection. table summarizes some of the findings of ifnar -/mice infected with several important pathogenic bacteria. in streptococcus, escherichia coli, and helicobacter infections, ifnar -/mice have higher titers and/or lethality than wild-type controls. during brucella, francisella, salmonella, chlamydia, mycobacterium, or yersinia infections, ifnar -/mice control infection better than wild-type. the ifnar -/mice are more resistant to l. monocytogenes given systemically. a recent report has shown that during oral infection with listeria, ifnar signaling may be protective. based on these initial studies, the simplest conclusion is that ifnar signaling is beneficial during extracellular bacterial infection and detrimental during intracellular bacterial infection. type i ifn signaling provides increased protection during streptoccocus infection by promoting upregulation of tnf-α, ifn-γ and nitric oxide ( ) . this is associated with restriction of bacterial growth. ifnar -/mice have bacteremia, increased systemic titers, and decreased survival. in vitro, streptococcus spp. induce ifn-β production through cell-type-specific signaling pathways ( ) . strepcococcus can trigger ifn-i via the complex of irf , stimulator of interferon genes (sting) and tank-binding kinase (tbk ). in streptococcus-infected macrophages, ifn-β is induced through the irf -sting-tbk complex and this is partially dependent on myd (ifn-i can also be produced in a myd -independent pathway). in contrast, streptococcusinfected dcs induce ifn-β through irf and myd . more mechanistic studies are still required to resolve the molecular triggers and in vivo cellular sources of ifn-i during streptoccocus infection. helicobacter pylori-infected ifnar -/mice have higher titers, but the mechanism of ifn-i action in this infection remains unresolved ( ) . further work needs to be done on the extracellular bacterial infections to determine how ifn-i is protective and what distinguishes ifn-i from ifn-ii in these types of infections. following brucella infection, there is ifnar-dependent upregulation of tnf-related apoptosis-inducing ligand (trail) and splenic apoptosis that is associated with increased susceptibility to infection ( ) . ifnar -/mice also express more ifn-γ and nitric oxide. in the case of salmonella enterica and chlamydia muridarum, ifn-i signaling sensitizes the infected macrophage to undergo cell death ( , ) . prevention of macrophage cell death during s. enterica infection led to decreased bacterial titer. ifnar −/− mice infected with francisella have decreased titers and lethality compared with controls. this is attributed to the inhibitory effect of ifn-i signaling on il- a/f expression ( ) . ifnar -/mice express more il- a/f, have an expansion of il- + γδ t cells and increased neutrophils at the site of infection. finally, treatment with ifn-i agonists such as poly(i:c) also promotes negative outcomes during bacterial infection. in the case of mycobacterium tuberculosis, intranasal delivery of poly(i:c) throughout the course of infection led to increased inflammatory infiltrates and necrosis of lung tissue that was dependent of ifnar signaling ( ) . similar detrimental effects of poly(i:c) treatment are also seen following streptoccocus pneumoniae, staphylococcus aureus and l. monocytogenes infections ( ) (see below). the first example of the detrimental effect of ifn-i during bacterial infection was discovered following l. monocytogenes infection. to date, it remains the best examined system, yielding information on the mechanisms of ifn-i induction, cellular sources and targets of ifn-i, and the nature of biological outcomes. macrophages infected with l. monocytogenes induce expression of ifn-i that is dependent on bacterial expression of the pore-forming toxin listeriolysin o (llo) ( , ) . llo is important for the bacterial egress from the nascent phagosome to the cytosol ( ) . llo alone does not induce strong levels of ifn-i production by the infected macrophage, suggesting that the presence of cytosolic bacteria is the driver of ifn-i production ( ) . this is reinforced by experiments demonstrating that bacillus subtilis expressing llo gain access to the cytosol and also strongly induce ifn-responsive genes. the major molecular driver of ifn-i induction by l. monocytogenes is the cyclic dinucleotide c-di-amp ( ) . the cyclic dinucleotides were initially discovered in bacteria as a second messenger system that also doubles as a pathogen-associated molecular pattern ( ) . interestingly, c-di-amp is actively exported from the bacteria and the induced expression of a c-di-amp synthesizing enzyme (di-adenylate cyclase) increases ifnb gene expression by infected macrophages ( ) . a sensor for cyclic dinucleotides has been identified as the helicase ddx , which recruits sting, tbk and irf (see above) to drive upregulation of ifn-i genes ( ) . splenic macrophages (cd b + cd c -pdca -b -) and tnf/inos-producing dcs (tip-dcs; cd b + cd c + ly c + ) produce ifn-i following l. monocytogenes infection in vivo ( , ) . mice lacking ccr expression, which do not recruit tip-dcs to the spleen, have reduced expression of ifn-α following l. monocytogenes infection ( ) . to date, there is no clear demonstration that a l. monocytogenes-infected myeloid cell population is producing ifn-i in vivo. on the basis of experiments using immunofluorescent colocalization, tip-dcs appear not to be infected ( ) . the work that identified ifn-i production by splenic macrophages did not evaluate the infected status of the cells ( ) . therefore, at this time, the connection between the molecular mechanisms of induction and in vivo cellular sources of ifn-i cannot be definitely established. listeria monocytogenes causes apoptotic cell death of macrophages that is enhanced by ifnar signaling. within h of infection, bone marrow-derived macrophages upregulate ifn-β and phosphorylate stat ( ) . deletion of ifnar on macrophages raises their resistance to l. monocytogenesmediated killing significantly. the death induced by l. monocytogenes is dependent on bacterial expression of llo ( ) . since llo is essential for virulence, it is unclear if it has a direct role in killing the infected macrophage or is only important for allowing egress of the bacteria to the cytosol. ifnar-dependent macrophage death is also found following infection of mice with l. monocytogenes ( ) . a population of tnf-α-producing cd b + macrophages is depleted following infection of wild-type mice. this population is maintained in ifnar -/mice, demonstrating a role for ifn-i in sensitization of macrophage death. it is not known at this time if the macrophages that die in vivo are infected by the bacteria. the most profound ifnar-dependent effect seen in mice infected with l. monocytogenes is the extensive depletion of white-pulp lymphocytes via apoptotic cell death ( ) . in wildtype mice, apoptosis begins in the periarteriolar lymphoid sheath (t-cell area) and extends to the entire white pulp in a dose-dependent manner ( ) . removal of ifnar significantly limits the number of apoptotic profiles and the extent of apoptotic death in any given white pulp ( ) . treatment of t cells with ifn-α sensitizes them to llo-induced apoptosis suggesting that secreted llo may be a killer molecule in vivo ( ) . additionally, ifn-i upregulate trail on nk cells and trail receptor (dr ) on the t cells and macrophages, providing a second potential mechanism for interferon-mediated lymphocyte and macrophage killing ( ) . trail -/mice harbor lower bacterial burdens than wild-type counterparts, have decreased splenic lymphocyte apoptosis, and increased accumulation of myeloid cells in the spleen following l. monocytogenes infection. the reduction in lymphocyte death seen following l. monocytogenes infection is the major reason that ifnar -/mice are more resistant to infection ( ) . several lines of evidence support this conclusion. first, mice deficient in lymphocytes (scid/ rag mice) are highly resistant to l. monocytogenes infection ( , ) . second, mixed bone marrow chimeras that create mice that are ifnar + in all cells except lymphocytes are also resistant to l. monocytogenes infection ( ) . this demonstrates the dominance of ifn-i signaling effects on lymphocytes. third, induction of ifn-i using poly(i:c) increases the susceptibility to infection of wild-type but not scid mice ( , ) . finally, l. monocytogenes infection induces myeloid cell expression of il- that is dependent on ifnar expression by lymphocytes ( ) . the upregulation of il- is a negative regulator of pathogen handling and il- -/mice are more resistant to infection despite having normal lymphocyte apoptosis ( , ) . as an aside, the work on ifnar effects on l. monocytogenes infection was conducted on three different genetic backgrounds with different susceptibilities to infection. in all three strains-c bl/ ( ), s ( ) and balb/c ( )-ifnar signaling was detrimental to the outcome of infection. this demonstrates that ifnar effects are dominant over the genetic susceptibilities of the mouse strains to l. monocytogenes infection. further work needs to be done to determine if this applies to other bacterial infection models. the initial paradigm of the ifn-i system is that it provides antiviral protection that sometimes goes awry in certain autoimmunities. this simplified view has been replaced with a more complex and interesting role for the interferons in regulating immune responses. chronic viral infections are teaching us that while early ifn-i is important in controlling viremia, pathogens that can overcome this initial control benefit from the immune regulation that takes place following long-term interferon induction. the clinical relevance of this can be seen during infection of patients with hiv, where chronic ifn-i leads to trail-mediated t-cell death and poor disease outcome ( ) . future work needs to be done to determine the applicability of ifn-i modulation as a therapeutic to important chronic human viral infections. another important area of research is the interface between viral and bacterial coinfections. the clinical importance of severe bacterial infections occurring after a primary viral infection is well established ( ) . respiratory bacterial infections are more dangerous to patients when they occur following infection with viruses such as influenza and respiratory syncytial virus. this observation has been replicated in mouse models of infection ( , ) . however, the interaction between viral and bacterial infection is not always deleterious. infection with herpesvirus induces prolonged ifn-γ production that leads to protection against infection with l. monocytogenes and yersinia pestis ( ) . the main distinguishing feature between the two potential outcomes (acute versus chronic virus) and (detrimental versus beneficial) appears to center on the balance between ifn-i and ifn-γ effects. this reinforces the need to understand the molecular effects of these cytokines during bacterial infection. in the bacterial world, ifn-i were once believed to be relatively unimportant. this idea was reversed by research on l. monocytogenes. careful examination of additional bacterial infections has demonstrated that both route and tropism of bacterial infections matter in the requirement of ifn-i. future studies will be needed to determine cellular sources, molecular triggers and biological outcomes of ifn-i for many classes of bacterial pathogens. it will be interesting to see if bacteria have evolved mechanisms to manipulate the ifn-i system like some viruses do. recently, it has been shown that the balance of ifn-i and ifn-ii may be important in the outcome of human mycobacterial infections ( ) . reminiscent of chronic lcmv infection, il- is also a key player in the ifn-i-mediated suppression of mycobacterial immunity. future work will be needed to determine if chronic ifn-i production is a common determinant of negative outcomes in infectious diseases. finally, we need a better understanding of how the genes specific for ifn-i lead to different outcomes from their close cousin, ifn-ii. national institute of allergy and infectious diseases (ai ). type i interferons (alpha/beta) in immunity and autoimmunity the jak-stat pathway at twenty mechanisms of type-i-and type-ii-interferon-mediated signalling how cells respond to interferons systematic identification of type i and type ii interferon-induced antiviral factors functional role of type i and type ii interferons in antiviral defense type i interferon: friend or foe? type i interferons and the innate immune response-more than just antiviral cytokines interferons, immunity and cancer immunoediting lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections induction and function of type i and iii interferon in response to viral infection interferon-dependent immunity is essential for resistance to primary dengue virus infection in mice, whereas t-and b-cell-dependent immunity are less critical interferon function is not required for recovery from a secondary poxvirus infection effects of type i interferons on friend retrovirus infection alpha/beta interferons regulate murine gammaherpesvirus type i interferons and infection latent gene expression and reactivation from latency the role of alpha/beta and gamma interferons in development of immunity to influenza a virus in mice the role of interferon in influenza virus tissue tropism critical role for alpha/beta and gamma interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of cytotoxic t cells type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells type i interferons produced by hematopoietic cells protect mice against lethal infection by mammalian reovirus theiler's virus infection of sv mice that lack the interferon alpha/beta or interferon gamma receptors alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival type i interferons in host defense type interferons and the virus-host relationship: a lesson in detente coordinated and distinct roles for ifn-alpha beta, il- , and il- regulation of nk cell responses to viral infection regulation of effector and memory t-cell functions by type i interferon type i interferon-mediated stimulation of t cells by cpg dna plasmacytoid dendritic cells promote rotavirusinduced human and murine b cell responses plasmacytoid dendritic cells induce plasma cell differentiation through type i interferon and interleukin mechanisms of evasion of the type i interferon antiviral response by flaviviruses recent advances in understanding viral evasion of type i interferon molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic t-lymphocyte response and establishment of persistence virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector t cells interleukin- and the interleukin- receptor pd- and its ligands in tolerance and immunity restoring function in exhausted cd t cells during chronic viral infection persistent lcmv infection is controlled by blockade of type i interferon signaling blockade of chronic type i interferon signaling to control persistent lcmv infection myd and sting signaling pathways are required for irf -mediated ifn-β induction in response to brucella abortus infection type i ifns enhance susceptibility to chlamydia muridarum lung infection by enhancing apoptosis of local macrophages type i ifn signaling constrains il- a/f secretion by gammadelta t cells during bacterial infections type i interferon sensitizes lymphocytes to apoptosis and reduces resistance to listeria infection mice lacking the type i interferon receptor are resistant to listeria monocytogenes type i interferon production enhances susceptibility to listeria monocytogenes infection dynamic roles of type i and type ii ifns in early infection with mycobacterium tuberculosis the type i ifn response to infection with mycobacterium tuberculosis requires esx- -mediated secretion and contributes to pathogenesis type i interferon induces necroptosis in macrophages during infection with salmonella enterica serovar typhimurium opposing roles for interferon regulatory factor- (irf- ) and type i interferon signaling during plague type i ifn signaling is crucial for host resistance against different species of pathogenic bacteria nod contributes to mouse host defense against helicobacter pylori via induction of type i ifn and activation of the isgf signaling pathway route of infection determines the impact of type i interferons on innate immunity to listeria monocytogenes type i interferon production induced by streptococcus pyogenes-derived nucleic acids is required for host protection intranasal poly-ic treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population poly i:c enhances susceptibility to secondary pulmonary infections by gram-positive bacteria production of type i ifn sensitizes macrophages to cell death induced by listeria monocytogenes a specific gene expression program triggered by gram-positive bacteria in the cytosol listeriolysin o: a phagosome-specific lysin c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response innate sensing of bacterial cyclic dinucleotides: more than just sting the helicase ddx recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response characterization of the interferon-producing cell in mice infected with listeria monocytogenes a fluorescence reporter model defines "tip-dcs" as the cellular source of interferon β in murine listeriosis tnf/inos-producing dendritic cells mediate innate immune defense against bacterial infection ifn-beta increases listeriolysin o-induced membrane permeabilization and death of macrophages lymphocyte apoptosis during early phase of listeria infection in mice reduced apoptosis and ameliorated listeriosis in trail-null mice mechanisms and immunological effects of apoptosis caused by listeria monocytogenes regulation of macrophage ia expression in mice with severe combined immunodeficiency: induction of ia expression by a t cell-independent mechanism lymphocytes are detrimental during the early innate immune response against listeria monocytogenes both innate and acquired immunity to listeria monocytogenes infection are increased in il- -deficient mice hiv- immunopathogenesis: how good interferon turns bad how do viral infections predispose patients to bacterial infections? type i interferon induction during influenza virus infection increases susceptibility to secondary streptococcus pneumoniae infection by negative regulation of γδ t cells viral infection augments nod / signaling to potentiate lethality associated with secondary bacterial infections herpesvirus latency confers symbiotic protection from bacterial infection type i interferon suppresses type ii interferon-triggered human anti-mycobacterial responses i wish to thank dr emil r. unanue for his support and insightful discussions. the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. key: cord- -c uom f authors: oslund, karen l.; zhou, xu; lee, boram; zhu, lingxiang; duong, trang; shih, robert; baumgarth, nicole; hung, li-yin; wu, reen; chen, yin title: synergistic up-regulation of cxcl by virus and ifn γ in human airway epithelial cells date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: c uom f airway epithelial cells are the first line of defense against viral infections and are instrumental in coordinating the inflammatory response. in this study, we demonstrate the synergistic stimulation of cxcl mrna and protein, a key chemokine responsible for the early immune response to viral infection, following treatment of airway epithelial cells with ifn γ and influenza virus. the synergism also occurred when the cells were treated with ifn γ and a viral replication mimicker (dsrna) both in vitro and in vivo. despite the requirement of type i interferon (ifnar) signaling in dsrna-induced cxcl , the synergism was independent of the ifnar pathway since it wasn’t affected by the addition of a neutralizing ifnar antibody or the complete lack of ifnar expression. furthermore, the same synergistic effect was also observed when a cxcl promoter reporter was examined. although the responsive promoter region contains both isre and nfκb sites, western blot analysis indicated that the combined treatment of ifn γ and dsrna significantly augmented nfκb but not stat activation as compared to the single treatment. therefore, we conclude that ifn γ and dsrna act in concert to potentiate cxcl expression in airway epithelial cells via an nfκb-dependent but ifnar-stat independent pathway and it is at least partly regulated at the transcriptional level. influenza pneumonia remains a major cause of morbidity and mortality worldwide. airway epithelial cells are the first line of defense against viral infections in the lung and are instrumental in coordinating the early inflammation leading to an adaptive immune response. cxcl (ifn gamma inducible kda protein, ip ) is a non-elr cxc chemokine with potent biological effects including monocyte stimulation, natural killer and activated t cell migration, modulation of adhesion molecule expression, inhibition of angiogenesis [ ] as well as antimicrobial effects at high concentrations [ ] . the role of cxcl in viral pneumonia has not been thoroughly characterized but evidence suggests it is important for the migration of nk cells, macrophages, t cells, neutrophils and plasmacytoid dendritic cells into the lung [ , ] . in a mouse model of rsv infection, antibody-mediated neutralization of cxcl resulted in a significant increase in disease symptoms including impaired viral clearance, reduced pulmonary dendritic cell numbers and maturation and a reduction in viral specific cd (+) t cells [ ] . synergistic up-regulation of cxcl has been described in vitro in several cell types and in response to different pro-inflammatory molecules but always in conjunction with ifn c. tnfa and ifn c induce synergistic levels of cxcl in a human endothelial cell line hmec- via an erk dependent pathway [ ] , il- b and ifn c in human intestinal epithelial cell lines [ ] , pdgf and ifn c in blood derived macrophages [ ] , tnfa and ifn c in primary human airway smooth muscle cells and gastric epithelial cells [ , ] , prolactin and ifn c [ ] as well as substance p and ifn c in human keratinocytes [ ] and hyaluronan fragments with ifn c in mouse macrophages [ ] . interestingly, synergistic induction of cxcl has been described with ifn c in conjunction with hiv- in human astrocytes and macrophages and thought to play a role in hiv induced encephalopathy [ , ] . this marked variety of pro-inflammatory molecules that elicit a synergistic response of cxcl in a wide variety of cell types is indicative of a highly conserved and likely biologically important cellular response. however, synergistic induction of cxcl in response to influenza and ifn c in airway epithelial cells has not been previously reported. in this study, we demonstrate synergistic induction of cxcl in well differentiated primary human bronchial epithelial (hbe) cells following influenza virus infection and the treatment with ifn c. we further demonstrate that this synergy was mediated by the interaction between dsrna (an intermediate of viral replication) and ifn c in vitro and in vivo. in the follow-up mechanistic study, this synergism was found to be transcriptionally regulated as demonstrated by a chimeric promoter reporter gene assay. in addition, it was not dependent of the ifnar pathway as neither the neutralizing antibody to ifnar nor the ifnar deficiency affected the synergism. finally, nfkb, but not stat , appeared to mediate this synergism. culture and treatments of primary human bronchial epithelial (hbe) cells human bronchial tissues were purchased from the commercial source (national disease research interchange, philadelphia, pa) as described before [ , ] . no tissues from patients diagnosed with lung-related diseases were used. all those lungs were either autopsy leftovers or were rejected for transplant. they were sent to us with arbitrary numerical code. no identity link to the actual patient can be identified. protease-dissociated hbe cells were plated on transwellh chambers (corning) and were maintained in immersed culture conditions until they reached confluence when they were transferred to an air-liquid interface (ali) culture condition [ , ] . at air-liquid interface, the cells were maintained in a ham's f /dmem ( : ) with the addition of transferrin ( mg/ml), insulin ( mg/ml), cholera toxin ( ng/ml), epidermal growth factor ( ng/ml), dexamethasone ( . mm), bovine hypothalamus extract ( mg/ml), bsa ( . mg/ml) and all-transretinoic acid ( nm). cells were maintained at ali for days and were placed in basal media devoid of the additives, with the exception of retinoic acid, overnight prior to the experiments. recombinant ifn c was purchased from r&d systems and synthetic double stranded (ds) rna (i.e. poly i:c) from emd biosciences. ifn c was used at ng/ml and poly i:c at mg/ ml. a neutralizing antibody to ifnar was obtained from us biologics and used at . mg/ml [ ] . both ikb kinase and jak inhibitors were purchased from emd biosciences and used at mm. for influenza infection, cells were infected with the influenza virus strain mem at hau. for in vivo study, balb/c mice were purchased from charles rivers laboratories and used at - weeks of age for intratracheal instillations. for primary airway epithelial cell cultures, ifnar null mice were a kind gift from dr. nicole baumgarth (ucd) and the wild-type controls mice were c bl/ j. mice were used at - weeks of age. all protocols described were approved by the university of california, davis and university of arizona, which were responsible for the proper care and use of experimental animals. the protocol was performed as described previously [ ] . briefly, at the time of necropsy, the chest and cervical region were exposed. a small puncture was placed in the proximal trachea to allow cannulation with sterile . mm polyethylene tubing (intramedic clay adams) which was secured in place with a . silk suture. a loose suture was placed at the distal end of the trachea just proximal to the carina. the trachea was dissected free and immediately placed in dmem at uc. and each trachea gently inflated with . % protease through the tracheal cannula after tightening the distal suture. dissociated epithelial cells were gently harvested by injecting milliliters of cell culture media through the trachea. mte cells from all tracheas were pooled and re-suspended in cell culture media prior to plating on transwellß chambers (corning) coated with purcolß (advanced biomatrix). mte cells were maintained in submerged conditions until confluent at which time they were kept in air-liquid interface conditions for one week in the presence of nm retinoic acid. for the experiments, mte cells were treated with ng recombinant murine ifn c (r and d systems) and/or mg dsrna for hours. the protocol was performed as described previously [ , ] . briefly, balb/c mice were anesthetized with isoflorane. in dorsal recumbancy, the tongue was gently pulled out, and a pipette tip was gently inserted into their laryngeal region through the oral cavity. mg of dsrna and/or mg of recombinant murine ifn c in ml of lps free pbs was deposited and breathed in. lps free pbs was used as the control. mice were kept in an upright position until recovery from anesthesia. twenty four hours later, mice were euthanized, and lungs were lavaged with ml pbs. all lungs were flash frozen in liquid nitrogen for subsequent rna isolation. total rna was extracted with trizolß reagent (invitrogen) and cdna was generated from an equal amount of rna ( mg per reaction) by moloney's murine leukemia virus-reverse transcriptase (applied biosystems) using random hexamers (invitrogen). sybr green master mix (roche applied science) and the abi ht detection system (applied biosystems) were used following the manufacturer's protocol for real-time pcr analysis. the relative mrna amount of each sample was calculated based on its threshold cycle, ct, in comparison to the ct of the housekeeping gene, beta actin or glyceraldehyde -phosphate dehydrogenase (gapdh). the results are presented as (ct cxcl -ct of housekeeping gene) as fold induction over the control condition. the purity of the amplified product was determined as a single peak of the dissociation curve. throughout the study, there was no observable fluctuation in the ct values of the housekeeping genes from different treatment conditions. primers to human and murine cxcl were purchased from sa biosciences. the primer sequences for human beta actin are as follows: forward: tgtgtccgtcgtggatctga and reverse: cctgcttcaccaccttcttgat. the primer sequences for murine gapdh are as follows: forward: tcctccaccttt-gacgctg and reverse: accaccctgttgctgtagcc. in preparation for collecting conditioned media from primary human airway epithelial cells, the cells were washed twice with sterile pbs. fresh media containing ifn c and/or poly i:c was placed basally with ml of media placed apically. following a hour incubation, the media was harvested, centrifuged to remove any cellular debris and stored at uc. bronchoalveolar lavage fluid was used for the murine cxcl elisa. commercially available elisa kits were used according to the manufacturer's directions (r&d systems). results were normalized to the amount of conditioned media or lavage fluid and expressed as ng/ ml or pg/ml. the construct contains bp upstream of the transcription start site of cxcl was cloned into pgl luciferase reporter vector (promega). the construct was confirmed by dna sequencing. early differentiated hbe cells ( days) were transfected with cxcl promoter-luciferase construct and prl-tk using lipofectamine (invitrogen) according to the manufacturer's specifications. prl-tk was used as the internal control for normalizing transfection efficiency. the empty vector, pgl basic was used as a negative control. in brief, cells were plated onto well plates at - % confluency. the next day, cells were washed twice with opti-mem (invitrogen) before transfection. the cells were incubated at uc with the mixture of dna constructs and lipofectamine in opti-mem for - hours at which time cell culture media was also added to the cells. transfected cultures were treated with ng/ml ifn c and/or mg poly i:c for hours. a dual luciferase reporter assay kit (promega) was used following the manufacturer's protocol. for each transfection, the relative firefly luciferase activity was normalized to the renilla luciferase activity. neutralization of ifnar . mg/ml of a mouse monoclonal antibody to chain of the human alpha, beta, omega interferon receptor (ifnar) was added to culture media of cells for hour prior to the start of the experiment. this antibody has previously been documented to neutralize the extracellular domain of the human type i interferon receptor with high affinity at the dose used in this study [ ] . ifn c and/or poly i:c were added to the culture media following the pre-incubation period and with the ifnra antibody still present. mouse igg was used as the control. following a hour incubation, rna isolation and qpcr were performed as previously described. data are expressed as mean se. experiments were conducted in triplicate with at least three independent cultures. group differences were calculated using an analysis of variance (anova) followed by a bonferroni multiple comparison test. differences were considered significant for p values less than . . as shown in fig. a , well differentiated hbe cells demonstrated significant synergistic induction of cxcl mrna following infection with the mem influenza virus and treatment with ifn c. cxcl induction was also significantly elevated following the combined treatment of mem and ifn c treatment as compared to the treatment with ifn c alone. consistently, potentiation of cxcl protein production by the combined treatments was detected in the cell culture supernatant from hbe cells (fig. b) . apical release of cxcl was enhanced in all treatment conditions with more pronounced levels detected in the combined mem infection and ifn c treatment (fig. b) . interestingly, significant levels of cxcl were also released basally except that they were much lower than the apical secretion. these results demonstrate that influenza virus in combination with ifn c synergistically induce cxcl mrna and protein production from primary human airway epithelial cells. because viral replication and its intermediate-double stranded (ds) rna have been shown to mediate many influenza induced phenotypes. we then examined the possibility if dsrna could synergize with ifn c in the induction of cxcl . as shown in fig. a , hbe cells demonstrated synergistic induction of cxcl mrna in a time dependent manner starting as early as hours after treatment. treatment with ifn c and dsrna resulted in significant synergistic induction of cxcl mrna levels at all the time points. combined treatment for hours resulted in an over fold induction of cxcl mrna. consistently, fig. b demonstrates potentiation of cxcl protein in cell culture supernatant from hbe cells treated for hours. apical release of cxcl was enhanced in all treatment conditions with more pronounced levels detected in the combined ifn c and dsrna treatment. significant levels of cxcl were also released basally, although the levels were much lower than the apical secretions. these results suggest that the synergy between influenza infection and ifn c treatment may be caused by the interaction between dsrna-and ifn c-mediated signaling pathways. to determine whether ifn c and dsrna synergistically induce cxcl in vivo, we performed intra-tracheal delivery of ifn c and dsrna in balb/c mice and examined cxcl mrna and protein levels. as shown in fig. a , dsrna alone induced a significant increase in cxcl mrna, and ifn c and dsrna treatment together synergistically induced cxcl mrna fold in mouse lung. likewise, fig. b demonstrates a significant induction of cxcl protein in bronchoalveolar lavage fluid after treatment with dsrna and synergistic induction following ifn c and dsrna treatment. these results demonstrate synergistic induction of cxcl mrna and protein in vivo following ifn c and dsrna treatment. because dsrna is known to induce the type i interferon pathway, we investigated the involvement of this pathway by using a neutralizing antibody to human ifnar. fig. a demonstrates that the ifnar neutralizing antibody did not affect synergistic induction of cxcl mrna in hbe cells, although it did repress dsrna-induced cxcl . to confirm this result, we isolated mte cells from ifnar null mice, which were completely lack of ifnar expression. fig. b demonstrates that the lack of ifnar did not affect the synergistic induction of cxcl . interestingly, induction of cxcl by dsrna alone was significantly impaired in the mte cells from ifnar null mice as compared to the cells from wild-type mice. taken together, these results demonstrate that synergistic induction of cxcl mrna was independent of the type i interferon receptor in both human and mouse airway epithelial cells. in contrast, dsrna-induced cxcl appeared to depend on the type i interferon receptor, suggesting that the synergy may be mediated via a different pathway. because of the rapid elevation of cxcl by ifn c and/or dsrna ( fig. a) , we tested the possibility if this synergy occurred at transcriptional level by transient transfection of the hbe cells with a cxcl promoter/luciferase chimeric construct containing bp of the upstream regulatory region of cxcl . fig. demonstrates the combined treatment of ifn c and dsrna significantly increased the luciferase reporter gene activity as compared with the treatment of ifn c or dsrna alone. these results support that the synergism occurred at least partially through a direct transcriptional mechanism and the proximal bp region of the cxcl promoter was critical for the induction. because both nfkb and isre sites were present in this cloned cxcl promoter region, we further tested nfkb and stat pathways in this synergism as both pathways have been shown to be responsible for cxcl gene expression. indeed, the specific inhibitor targeting the kinase either upstream of nfkb (ikb kinase) or stat (jak) significantly blocked cxcl induction by ifn c, by dsrna, or by the combined treatment (fig. a) , which confirms the indispensable role of both pathways in . the synergism between ifn c and dsrna(dsr) was independent of ifnar. a) hbe cells were treated with . mg/ml of either an isotype control antibody (black bar) or a monoclonal neutralizing antibody ifnar (grey bar) for one hour prior to the addition of ng/ml ifn c and/ or mg/ml dsrna for hours. rna was isolated followed by qpcr. ns: not significant. $: p, . . isotype control antibody vs. ifnar. b) primary mte cells isolated from either wild-type (black bar) or ifnar deficient (grey bar) mice were grown in vitro and treated with ng/ml ifn c and/or mg/ml dsrna for hours. rna was isolated followed by qpcr. ns: not significant. $: p, . . wild-type vs. ifnar deficient. triplicate wells were used for each experiment and the experiment was repeated at least three times. doi: . /journal.pone. .g regulating cxcl expression. when the signaling pathway was further examined, the treatment of ifn c or dsrna alone was found to induce robust degradation of ikb, a surrogate of the activation of nfkb pathway which is initiated by the reaction catalyzed by ikb kinase. and the combined treatment resulted in much greater ikb degradation as compared to the single treatment (fig. b) . in contrast, despite the activation of stat a and stat b by these treatments, no synergism was observed (fig. b) . therefore, the synergism of ifn c and dsrna on cxcl expression was mediated by nfkb pathway. airway epithelial lining is the first defense against respiratory viral infection. in this study, we seek to understand the modulation of an important chemokine-cxcl by the combined effects of ifn c and viral infection. this is the first report documenting the observation and mechanism underlying the synergism between ifn c and viral infection (or dsrna treatment) in airway epithelial cells. ifn c is a type ii interferon and oftentimes elevated in the context of viral infection. it is generally accepted that direct innate antiviral defense is mediated by type i ifn. indeed, we and others have shown that dsrna (or virus)-induced epithelial derived type i ifn plays critical role in the airway antiviral defense [ , , ] . in the present study, we demonstrate the synergy between dsrna-and ifn c-induced signaling in the regulation of cxcl . interestingly, although dsrna-induced cxcl depended on type i ifn pathway, the synergy between dsrna and ifn c was completely independent of it in both human and mouse epithelial cells. this lack of dependence was not due to the remaining residue of the ifnar functionality in the antibody neutralization assay, because the epithelial cells from ifnar deficient mice used in the fig. a still preserved the synergistic response despite the loss of entire ifnar expression. thus, in the presence of ifn c, dsrna (or virus)-induced signaling is very likely to be altered, which emphasizes the importance of studying the pathway crosstalk as demonstrated in the present study. previous studies have demonstrated that ifn c in conjunction with different pro-inflammatory molecules leads to a synergistic and dramatic induction of cxcl in a variety of cell types including keratinocytes, macrophages, endothelial cells and smooth muscle cells [ , , , ] . several of these studies have demonstrated that the transcription factor, nf-kb, is involved in cxcl induction in a variety of systems [ ] [ ] [ ] . in contrast, very few studies have demonstrated that cxcl induction is dependent on an isre site in the promoter region. kanda and coworkers found substance p and ifn c induced synergism of cxcl in human keratinocytes was dependent on an isre site and two nf-kb sites in the cxcl promoter located to of the transcription start site [ ] . in addition, majumder and co-workers found evidence that ifn c and tnf a act in synergy via p complexes with stat- a binding to this same isre site in the cxcl promoter of human fibrosarcoma lines [ ] . evidence that similar synergism occurs at the transcriptional level in mouse cells has been demonstrated in a small number of in vitro studies [ , ] . using the murine fibroblast nih t cell line, ohmoir and co-workers established that an isre site and one of two nf-kb sites in a bp fragment flanking the transcription start site of the murine cxcl gene were critical for ifn c and tnf a induced synergy [ ] . our study has further extended these studies to the airway epithelial system in the context of viral infection, and the results apparently support the importance of nfkb. figure . nfkb-dependent signaling is responsible for the synergism between ifn c and dsrna (dsr). a) well differentiated primary hbe cells were treated with ng/ml ifn c and/or mg/ml dsrna for hours in the presence or absence of ikb kinase or jak inhibitor. rna was isolated followed by qpcr. solvent control: black bar; ikb kinase inhibitor: grey bar; jak inhibitor: light grey bar. $: p, . . inhibitor treatment vs. solvent control. b) cells were treated with ng/ml ifn c and/or mg/ml dsrna for hours. cellular protein was collected and analyzed by western blot analysis. actin was used as a loading control. pstat : phosphorylated stat . this is the representative image from three replicates. doi: . /journal.pone. .g the present study demonstrates the significant contribution of cxcl from epithelial cells, which is consistent with the emerging role of active defense by these cells in the respiratory viral infection. cxcl secretion by hbe cells appeared to be polarized. the treatment with ifn c, dsrna and combined treatments resulted in a progressively enhanced secretion of cxcl mainly from apical surface. in contrast, the basal secretions of cxcl protein in these cultures were much less. the gradient between the apical and basal compartments may have important implications in vivo leading to enhanced emigration of cxcr positive effector cells (primarily cd + t cells) to the airway epithelial microenvironment. although dsrna has been used extensively as a synthetic dsrna to model viral infections, its stabilized derivatives are also being investigated for clinical use as an immunomodulatory agent for use as vaccine adjuvants in anti-viral treatment and cancer therapies [ ] [ ] [ ] [ ] . while dsrna was used in this study to simulate a viral infection, it is important to note that it, in and of itself, can also be involved in synergistic induction of cxcl in airway epithelial cells. this fact could be an important consideration in the treatment of patients with pre-existing diseases in which ifn c is part of the pathogenesis such as viral infections. in summary, we demonstrate ifn c and the influenza virus synergistically induce cxcl in human airway epithelial cells. this synergism is likely to be mediated by dsrna-induced signaling both in vitro and in vivo, which is independent of the type i interferon receptor pathway. furthermore, we demonstrate the bp proximal region of the cxcl promoter plays a critical role in regulating this synergistic induction. this region of the cxcl promoter contains punitive isre and nfkb transcription factor binding sites. therefore, further study has been carried out to demonstrate the involvement of nfkb, but not stat , in this synergism. this capacity for airway epithelial cells to markedly up-regulate cxcl likely has important consequences for cd + t cell and nk cell migration to the airway epithelial microenvironment following influenza virus infection. the immunobiology of interferongamma inducible protein kd (ip- ): a novel, pleiotropic member of the c-x-c chemokine superfamily cutting edge: ifn-inducible elr-cxc chemokines display defensin-like antimicrobial activity cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd + t cells are important in control of sars-cov infection ip- mediates selective mononuclear cell accumulation and activation in response to intrapulmonary transgenic expression and during adenovirus-induced pulmonary inflammation cxcl /cxcr -mediated responses promote immunity to respiratory syncytial virus infection by augmenting dendritic cell and cd (+) t cell efficacy molecular mechanisms underlying the pro-inflammatory synergistic effect of tumor necrosis factor alpha and interferon gamma in human microvascular endothelium nf-kappab-dependent synergistic regulation of cxcl gene expression by il- beta and ifn-gamma in human intestinal epithelial cell lines pdgf synergistically enhances ifn-gamma-induced expression of cxcl in blood-derived macrophages: implications for hiv dementia regulation of tnf-alpha-and ifn-gamma-induced cxcl expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease ifngamma synergizes with tnf-alpha but not with viable h. pylori in up-regulating cxc chemokine secretion in gastric epithelial cells prolactin enhances interferon-gamma-induced production of cxc ligand (cxcl ), cxcl , and cxcl in human keratinocytes substance p enhances the production of interferon-induced protein of kda by human keratinocytes in synergy with interferon-gamma hyaluronan fragments synergize with interferon-gamma to induce the c-x-c chemokines mig and interferon-inducible protein- in mouse macrophages molecular mechanism(s) involved in the synergistic induction of cxcl by human immunodeficiency virus type tat and interferon-gamma in macrophages proinflammatory cytokines and hiv- synergistically enhance cxcl expression in human astrocytes rhinovirus induces airway epithelial gene expression through double-stranded rna and ifndependent pathways characterization of human mucin b gene expression in airway epithelium and the genomic clone of the aminoterminal and -flanking region identification of a novel subunit of the type i interferon receptor localized to human chromosome stimulation of airway mucin gene expression by interleukin (il)- through il- paracrine/ autocrine loop vanadium pentoxide (v( )o( )) induced mucin production by airway epithelium rhinovirus-induced major airway mucin production involves a novel tlr -egfr-dependent pathway negative control of tlr signaling by ticam down-regulation ) p / stat- alpha-containing complexes play a predominant role in induction of ifn-gamma-inducible protein, kda (ip- ) by ifn-gamma alone or in synergy with tnf-alpha the interferon-stimulated response element and a kappa b site mediate synergistic induction of murine ip- gene transcription by ifn-gamma and tnf-alpha treatment of viral and neoplastic diseases with double-stranded rna derivatives and other new agents a north american brain tumor consortium phase ii study of poly-iclc for adult patients with recurrent anaplastic gliomas synthetic double-stranded rna poly(i:c) combined with mucosal vaccine protects against influenza virus infection antiviral role of toll-like receptor- agonists against seasonal and avian influenza viruses key: cord- - o e authors: bayat, ahmad; burbelo, peter d.; browne, sarah k.; quinlivan, mark; martinez, bianca; holland, steven m.; buvanendran, asokumar; kroin, jeffrey s.; mannes, andrew j.; breuer, judith; cohen, jeffrey i.; iadarola, michael j. title: anti-cytokine autoantibodies in postherpetic neuralgia date: - - journal: j transl med doi: . /s - - - sha: doc_id: cord_uid: o e background: the mechanisms by which varicella zoster virus (vzv) reactivation causes postherpetic neuralgia (phn), a debilitating chronic pain condition, have not been fully elucidated. based on previous studies identifying a causative role for anti-cytokine autoantibodies in patients with opportunistic infections, we explored this possibility in phn. methods: sera from herpes zoster (hz) patients without and with phn (n = and , respectively) were examined for the presence of autoantibodies against multiple cytokines, and other known autoantigens. in addition, a cohort of patients with complex regional pain syndrome or neuropathic pain was tested for autoantibodies against selected cytokines. antibody levels against vzv, epstein barr virus, and herpes simplex virus- were also measured in the hz and phn patients. patient sera with high levels of anti-cytokine autoantibodies were functionally tested for in vitro neutralizing activity. results: six phn subjects demonstrated markedly elevated levels of single, autoantibodies against interferon-α, interferon-γ, gm-csf, or interleukin- . in contrast, the hz and the pain control group showed low or no autoantibodies, respectively, against these four cytokines. further analysis revealed that one phn patient with high levels of anti-interleukin- autoantibodies had a markedly depressed antibody level to vzv, potentially reflecting poor t cell immunity against vzv. in vitro functional testing revealed that three of the five anti-cytokine autoantibody positive phn subjects had neutralizing autoantibodies against interferon-α, gm-csf or interleukin- . in contrast, none of the hz patients without phn had neutralizing autoantibodies. conclusions: these results suggest the possibility that sporadic anti-cytokine autoantibodies in some subjects may cause an autoimmune immunodeficiency syndrome leading to uncontrolled vzv reactivation, nerve damage and subsequent phn. varicella-zoster virus (vzv) is a member of the alphaherpes virus subfamily that causes chickenpox early in life and then remains latent in neurons of sensory ganglia for the lifetime of the individual [ ] . in persons with immune deficiencies and in older individuals with a waning immune system, vzv can reactivate causing herpes zoster (hz), also known as shingles [ ] . hz patients have vesicular, unilateral, painful, dermatomally demarcated skin eruptions that typically resolve in less than month. hz is quite frequent, and approximately one third of the population may experience zoster once in their lifetime [ ] . adults over the age of are particularly susceptible to hz [ ] , implicating a stronger role of acquired factors compared to genetic influences [ ] . one potential consequence of hz is the development of postherpetic neuralgia (phn), defined as pain that lasts for greater than months and which originates in the same area as the hz rash. the frequency of developing phn has been reported to be approximately % after zoster [ , ] . immunocompromised individuals are at higher risk of hz [ , ] , suggesting an important role of immune surveillance in controlling vzv infection. the shingles prevention study showed that vaccination of elderly persons with a live attenuated varicella-zoster virus vaccine reduced the rates of both zoster and phn [ ] . in a followup study, weinberg et al. showed higher cell-mediated immunity at hz onset, which correlated with reduced hz severity and less phn; in contrast humoral immunity, as reflected by antibodies to vzv at onset of hz did not correlate with severity of disease [ ] . these results indicate a critical function of t-cell-mediated immunity for protection against hz and phn. recently, we explored the role of anti-cytokine autoantibodies in several diseases by screening patient sera with a panel of recombinant renilla luciferase cytokine fusion proteins as antigenic probes using the luciferase immunoprecipitation systems (lips) technology [ ] [ ] [ ] . using this approach, thymoma patients with opportunistic infections, including some with disseminated vzv infection, demonstrated autoantibodies against interferon-α (ifn-α), interleukin p (il- p ), and several other cytokines [ ] . high levels of neutralizing anti-ifn-γ autoantibodies were also detected in patients with disseminated nontuberculous mycobacteria and other opportunistic infections, including both with localized and disseminated vzv reactivation [ , ] . based on the late age of onset of phn, we explored in this study whether anti-cytokine and other autoantibodies, might be associated with phn. from screening a cohort of hz patients with and without phn, high levels of autoantibodies against several different cytokines were detected in six phn patients. further analysis revealed that three of the phn patients had neutralizing anti-cytokine autoantibodies. in one phn patient with high level anti-il- autoantibodies, antibody responses against vzv were completely absent. the finding that several patients each harbored single, neutralizing autoantibodies against interferon-α, gm-csf or il- suggests that anti-cytokine immunodeficiency may contribute to development of phn. informed written consent was obtained from all subjects with vzv reactivation in accordance with the human experimentation guidelines of university college london, (east london and the city research ethics committee lrec r&wf / ). a total of hz subjects were studied: without phn (hereafter referred to as hz) and with phn (hereafter referred to as phn). there were subjects with hz who had a dysesthesia, but no phn; using our measured endpoints there were no differences between patients with hz and those with hz and dysesthesia and these two groups were analyzed together. two additional subject groups were studied as controls. first, a small group of healthy blood donor controls (n = ) were used to standardize the assay. second, a phn agematched disease control group (n = ), from patients having either complex regional pain syndrome (crps) or neuropathic pain (np) were tested for selected anticytokine autoantibodies. the crps/np subjects were collected at rush university under an irb approved protocol and with patient written consent. the clinical characteristics of these four different groups of subjects including the age, gender, diagnosis, and the presence of other associated immunodeficiencies, are described in table . the antigen targets used for lips testing have been previously described [ ] [ ] [ ] . an initial autoantibody screen of twenty-four potential autoantigens was performed with a pilot set of phn patients and healthy controls. these included cytokines (gm-csf, il- , ifn-α, il- -p , ifn-γ, ifn-ω, ifn-λ, il- -p , il- , tnf-α, tnf-β, il- and il- α), six known autoantigens (ro , ro , la, rnp-a, sm-d and rnp- ), four neuro-glial proteins, glutamic acid decarboxylase (gad- ), tyrosine hydroxylase, s -β, and aquaporin- , and trim- . based on the results of the pilot study, additional hz patients with and without phn were tested against the most informative targets (ro , ro , ifn-α, ifn-γ, il- , and gm-csf). all the subjects in the cohort also were evaluated for antibodies against blrf of ebv (p capsid), the ge glycoprotein of vzv [ ] and the gg- glycoprotein of hsv- [ ] . for lips autoantibody testing, serum samples were diluted : in assay buffer a ( mm tris, ph . , mm nacl, mm mgcl , % triton x- ), arrayed in deep well microtiter plates, and tested as described [ ] . buffer blanks were used to monitor the performance and background binding activity of the assays. light units (lu) were measured using a berthold luminometer and all lu data were obtained from the average of at least two separate experiments. to evaluate the neutralizing capacity of anti-cytokine autoantibodies, control pbmcs from individual, healthy blood donors, that were distinct from the controls used for antibody analysis, were incubated in the presence of % healthy control or patient sera and left unstimulated or stimulated with the cytokine recognized by the autoantibodies in that particular patient sample. the pbmc were fixed, permeabilized and stained for an increase in phosphorylation of the specific downstream signal transducer and activator of transcription (pstat) molecules by flow cytometry as described previously [ ] . thus, for patients with anti-ifn-α autoantibodies, cells were stimulated with ifn-α ( u/ml) and assessed for ifn-α-induced pstat- in cd + monocytes; those with anti-ifnγautoantibodies were stimulated with ifn-γ ( u/ ml) induced pstat- in cd + monocytes; those with anti-gm-csf autoantibodies for gm-csf ( ng/ ml)-induced pstat- in cd + monocytes; those with anti-il- autoantibodies for il- ( ng/ml) induced pstat- in cd + lymphocytes. antibodies for flow cytometry were purchased from bd biosciences. data were collected using facscanto (bd biosciences) and analyzed using flowjo version . (treestar). using this approach as described [ , , , ] , the magnitude of pstat production due to cytokine stimulation is extremely reproducible and sensitive to incremental changes in the amount of cytokine added and sera was categorized as not neutralizing, partially neutralizing and neutralizing. graphpad prism software (san diego, ca, usa) was used for analyzing the antibody levels in this study. geometric mean antibody levels, expressed as mean log ( ) lu and % confidence intervals (ci), were calculated and presented as antilog values. the nonparametric mann-whitney u statistical test was used for comparison of antibody levels in the control and patient groups. the fischer exact test was used to evaluate the statistical significance of the prevalence of anti-cytokine autoantibodies in the hz and phn subjects. an initial autoantibody screen of twenty-four potential autoantigens was performed with a pilot set of phn patients and healthy controls. seropositivity was observed as follows: % ( / ) for ro and la and % ( / ) for anti ro autoantibodies. antibody levels for these three autoantigens were often -fold higher than the buffer blanks or other seronegative samples. we also observed that four phn patients showed unusually high levels of autoantibodies against single cytokines including ifn-γ, gm-csf and il- . additional weak positive autoantibodies were detected against ifn-ω and aqp- in several healthy controls and phn samples. autoantibodies against rnp- , sm-d , ifn-λ, tnf-α, tnf-β, il- , il- , il- -p , il- -p , s β and gad- were not found in the pilot cohort. based on these findings, an additional subjects with only hz and phn subjects were evaluated with the most informative autoantigens (ifn-α, ifn-γ, gmcsf, il- , ro and ro ) identified in the pilot cohort. in this larger group of samples, ten more of the phn patients were seropositive for anti-cytokine autoantibodies. for analysis and presentation, both the pilot and second set of samples were merged. as shown in fig. a , only three hz patients demonstrated single, seropositive anti-cytokine autoantibodies that were directed against ifn-α, ifn-γ, and gm-csf. in total, the phn subjects contained thirteen seropositive anti-cytokine autoantibodies, which as a group was statistically different than the hz (fischer's exact test p = . ). moreover, an age-matched disease control cohort of crps/np patients (n = ) failed to harbor any statistically significant autoantibody responses against ifn-α, ifn-γ, gm-csf or il- ( fig. ) . particularly relevant was the finding of six phn patients (one against anti-ifn-α, three against ifn-γ, one against gm-csf, and one against il- ) with high levels of autoantibodies greater than , lu that might have potential neutralizing activity against the target (fig. ) . besides cytokine autoantibodies, five phn patients had robust autoantibodies against ro and four phn patients with ro autoantibodies, in which three of the phn patients were co-positive for both ro and ro (data not shown). inspection of the individual patients revealed that only one of the low il- autoantibody seropositive phn subjects was copositive for ro or ro , suggesting that general polyreactivity did not underlie the immunoreactivity seen in anti-cytokine autoantibody patients. lastly, review of the patients' charts in subjects with anticytokine autoantibodies did not document the presence of thymoma, staphylococcus infection, or any other unusual condition to explain these autoantibodies. none of the patients had other known autoimmune diseases associated with anti-cytokine autoantibodies including autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (apeced) or pulmonary alveolar proteinosis (pap). in addition to studying autoantibodies, we evaluated the possibility that the levels of vzv antibodies might be altered between hz and phn. for serologic testing, we measured antibodies against ge, a known viral glycoprotein target of natural and vaccine-mediated immunity [ ] . as shown in fig. a , there were no statistical differences in the ge antibody levels in subjects with hz compared to phn. several hz and phn subjects had very low or no anti-vzv antibodies (fig. a) . however, further inspection of the individual patients with anti-cytokine autoantibodies revealed that one phn patient, # , with high levels of anti-il- autoantibodies was seronegative, having the lowest level of antibodies against vzv in the phn patients (fig. a) . the two other subjects with neutralizing autoantibodies against ifn-α and gm-csf had vzv antibodies in the normal range (fig. a) . to explore whether these blunted antibody responses were unique only to vzv, antibodies against another herpes virus, ebv, using the p capsid protein were measured. the average antibody level to the p capsid of ebv was significantly higher (p = . ) in the phn group compared to the hz group (fig. b ). antibodies to a third herpes virus, hsv- , were too sporadic to be informative (only % of the entire cohort ( / patients) exhibited hsv- seropositivity (data not shown). taken together, viral antibody profiling suggests that, as a group, these patients are not globally immunocompromised yet at a more fine-grained level, patient # , who showed severely blunted vzv antibodies, had high levels of anti-ebv antibodies. based on our previous studies [ , ] , the eight patients in fig. a with high levels of anti-cytokine autoantibodies (> , lu) were selected to test for their in vitro inhibitory capacities to block the activity of the cognate cytokine ( table ) . serum from hz patient # partially inhibited ifnα-induced pstat- , and serum from phn patient # completely prevented ifn-α-induced pstat- ( table ) . for the three patients with high levels of ifn-γ autoantibodies, phn patient # and # did not block ifnγ-induced pstat- whereas serum from the third phn patient, # , was partially blocking ( table ) . phn patient # with anti-gm-csf autoantibodies prevented gm-csf-induced pstat- production, while serum from hz patient # with lower levels of anti-gm-csf autoantibodies did not. testing the phn patient # with the sole anti-il- autoantibodies revealed that serum from this individual prevented il- -induced pstat- . in total, only three phn patients had serum autoantibodies that had complete neutralizing activity against the cognate cytokine. chronic pain conditions are frequently life-long problems that are difficult to treat, in part, because the underlying factors that contribute to human pain disorders are unclear. for phn, the mechanisms that contribute to the transition from zoster to phn remain nebulous. while animal models of phn have been explored they have many limitations in reproducing the multiple factors encountered in the human disease [ ] . thus, clinical research efforts based directly in human subjects are needed to obtain mechanistic insights into phn. based on our earlier work [ , , , ] , the potential contributions of humoral immune processes to hz and phn were investigated with a [ ] . the red square corresponds to patient # (see table ) who is the il- seropositive phn patient who was seronegative for vzv antibodies focus on autoantibodies against cytokines and several known neural and non-neural autoantigens. we further hypothesized that damage to the central or peripheral nervous systems may expose autoantigenic epitopes thereby provoking an autoimmune response [ ] . several reports have appeared examining the presence of autoantigens to peripheral nerve in complex regional pain syndrome [ , ] and to central nervous system proteins in disorders such multiple sclerosis, although results for some antigens are controversial [ ] . in phn, significant shrinkage in the dorsal horn of the spinal cord has been observed that might be attributable to injury and potential autoimmune attack [ ] . using lips, no autoantibodies were detected against two neural antigens, gad- and tyrosine hydroxylase, or two astrocyte antigens s -β and aquaporin- in any of the groups examined. while autoantibodies to four of the six sjögren's/systemic lupus erythematosus antigens tested (rnp-a, rnp- , sm-d , and la), were also seronegative, a few patients exhibited elevated autoantibodies against ro and ro . these data suggest that phn, while a painful, stressful sequelae to an infectious disease process, does not induce an autoimmune state to the proteins tested. previously, the induction of autoantibodies in acute respiratory distress syndrome (ards) and septic shock, conditions characterized by severe inflammation and cytokine storm were observed [ ] . we hypothesized that the autoantibody induction was due to the ongoing systemic inflammation and associated tissue destruction that may mediate a break in tolerance against these self-proteins. in the ards study the levels of autoantibodies were substantially lower than those seen in the anti-cytokine positive patients identified here. this quantitative difference implies that in the phn patients these anti-cytokine autoantibodies may be pathogenic rather than biomarkers for inflammation. moreover, the antiviral humoral response against vzv and other hsvs was similar between the hz and phn patients. with the lips fluidphase immunoassay used, our data indicate that the humoral response to vzv is intact in most subjects with hz and phn. the novel finding of our study was the presence of sporadic anti-cytokine autoantibodies that were associated with phn but not with hz per se. we found six patients with phn who had high levels (> , lu) of anti-cytokine autoantibodies, but only two patients with hz and none with crps/np. in neutralization assays, only three subjects had serum antibodies that blocked cytokine signaling, all of whom had phn. two additional subjects, one with hz and one with phn, had partially neutralizing activities. the anti-ifn-α and anti-gm-csf autoantibodies observed in two phn patients may be associated with inflammation, as previously observed in ards and septic shock [ ] . if the neutralizing levels of autoantibodies against these cytokines were present before or at the time of initial reactivation, then one might expect that they predispose to more severe vzv infection. consistent with this hypothesis, patients with thymoma have both neutralizing anti-cytokine autoantibodies against ifn-α and a predisposition to both localized and disseminated varicella reactivation, raising the possibility that anti-cytokine autoantibodies might contribute to vzv reactivation in some cases [ ] . the potential role of anti-gmcsf autoantibodies in generation of phn is intriguing. up until recently, anti-gmcsf autoantibodies were only thought to cause pulmonary alveolar proteinosis [ ] . however, in the last several years, anti-gmcsf autoantibodies have been found in patients with cryptococcal meningitis [ , ] and cns infections with nocardia bacteria [ ] . in light of the finding of two subjects with vzv reactivation having elevated gmcsf autoantibodies, further investigations of the relationships between gmcsf and vzv infection are warranted. the one phn patient with very high levels of anti-il- autoantibodies (i.e., , , light units) that were neutralizing is potentially instructive. the presence of il- autoantibodies in the general population, based on sampling of over samples [ , ] is quite rare. consistent with a potential role for anti-il- autoantibodies in promoting vzv reactivation, this phn patient lacked antibodies against vzv, but showed normal humoral responses to ebv. it is possible that impaired il- signaling blunts the inflammatory t-cell reaction and impairs adequate signaling to the relevant plasma cells for anti-vzv antibody production. high levels of anti-il- autoantibodies were also noted in a child who developed an acute vzv infection complicated by multiple cellulitis lesions and subcutaneous staphylococcus aureus abscesses [ ] . together these two patients implicate il- autoantibodies in the modulation of primary and reactivation vzv infection. il- is secreted by t cells and macrophages to stimulate the immune response and is known to be increased in the serum of hz [ ] . mechanistically, vzv signals through toll-like receptor- -dependent activation of nf-kappa b to induce an efficient inflammatory response [ ] . low levels of il- may lead to impaired anti-vzv immune responses, causing ineffective containment of vzv, more tissue damage and a stronger "peripheral generator" that then drives stronger central sensitization, all of which can contribute to phn pain. additional studies in more subjects with phn are warranted to examine the possibility that il- and other cytokine autoantibodies contribute to phn. there are several clinical implications of our findings. first, in sporadic cases, anti-cytokine autoantibodies may potentially contribute to phn, highlighting the complex interactions between humoral immunity, t-cell immunity and cytokines. based on the time line for the development of phn from initial vzv reactivation, screening for anti-cytokine autoantibodies is theoretically possible, yet it is not clear how this might impact the course of treatment. the finding that the il- cytokine might be involved in controlling vzv reactivation and thereby might be useful for early stage treatment of immunocompromised individuals warrants further investigation. it is likely that the development of phn is a highly complex, multifactorial process driven by many subtle genetic, environmental and acquired factors. based on the present observations, one such acquired factor is the presence of anti-cytokine autoantibodies, which could act as potential drivers of phn by creating deficits in immune signaling. while the frequency of phn patients harboring anticytokine autoantibodies found in our study was small, it is important to point out that phn is quite common in adults over and these findings would translate into explaining potentially many cases per year. since autoantibodies and decline in immune system function are known to occur more commonly in older subjects [ ] [ ] [ ] , the present observations suggest the likelihood that there may be additional, unrecognized pathogenic autoantibodies against other immune components in older subjects that may increase susceptibility to phn. varicella-zoster the impact of varicella zoster virus: chronic pain herpes zoster disentangling inborn and acquired immunity in human twins diagnosing and managing postherpetic neuralgia management of herpes zoster (shingles) and postherpetic neuralgia a population-based study of the incidence and complication rates of herpes zoster before zoster vaccine introduction predictors of postherpetic neuralgia among patients with herpes zoster: a prospective study a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults varicella-zoster virus-specific immune responses to herpes zoster in elderly 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allergy and infectious diseases, and the vzv research foundation. jb receives funding from the uk national institutes for health research ucl/uclh biomedical research centre. the authors declare no financial interests that may represent a competing interest. the authors declare that they have no competing interests.received: august accepted: october authors' contributions pdb, ajm, jic and mji were involved in study design. ab and pdb were involved in lips testing, data analysis, and manuscript drafting and editing. ab and jk collected and provided the crps/np cohort. skb, bm and smh were involved in the neutralization experiments. mq and jb provided hz and phn patient samples and clinical data. all authors read and approved final manuscript. key: cord- -nph ezii authors: zhu, zixiang; du, xiaoli; li, pengfei; zhang, xiangle; yang, fan; cao, weijun; tian, hong; zhang, keshan; liu, xiangtao; zheng, haixue title: early growth response gene- suppresses foot-and-mouth disease virus replication by enhancing type i interferon pathway signal transduction date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: nph ezii early growth response gene- (egr ) is a multifunctional transcription factor that is implicated in viral infection. in this study, we observed that foot-and-mouth disease virus (fmdv) infection significantly triggered egr expression. overexpression of egr suppressed fmdv replication in porcine cells, and knockdown of egr considerably promoted fmdv replication. a previously reported fmdv mutant virus (with two amino acids mutations in sap domain) that displays a strong type i interferon (ifn) induction activity was used in this study. we found that sap mutant fmdv infection induced a higher expression of egr than wildtype fmdv infection, and also triggered higher ifn-β and ifn-stimulated genes (isgs) expression than wildtype fmdv infection. this implied a link between egr and type i ifn signaling. further study showed that overexpression of egr resulted in sendai virus (sev)-induced ifn-stimulated response element (isre) and nf-κb promoter activation. in addition, the sev-induced isgs expression was impaired in egr knockdown cells. egr upregulation promoted type i ifn signaling activation and suppressed fmdv and seneca valley virus replication. suppression of the transcriptional activity of egr did not affect its antiviral effect against fmdv. this study reveals a new mechanism evolved by egr to enhance type i ifn signaling and suppress fmdv replication. foot-and-mouth disease virus (fmdv) is a non-enveloped virus with positive-sense and singlestranded rna genome. the viral genome is approximately . kb nucleotides in length, including a single large open reading frame that encodes a polyprotein. the polyprotein is subsequently processed by viral proteases during protein synthesis, generating several intermediates and mature proteins (sobrino and domingo, ; grubman and baxt, ) . during co-evolution with the hosts, these viral proteins have acquired many functions to counteract host antiviral responses, cause immunosuppression, and promote viral replication and infection (mason et al., ; rodriguez pulido and saiz, ) . therefore, fmdv causes an acute vesicular disease of infected animals, which is called foot-and-mouth disease (fmd). fmd is a highly contagious disease that can lead to significant economic losses to the local livestock industry (rweyemamu et al., a; paton and taylor, ; zai-xin, ; bouguedour and ripani, ) . the understanding of host-fmdv interaction as well as the involved mechanism contributes to the planning of new strategies for fmd prevention (domingo et al., ; rweyemamu et al., b; rodriguez pulido and saiz, ) . accordingly, many researches on host responses in fmdv-infected cells have to be investigated. early growth response gene- (egr ), also designated zif , is a host transcriptional regulator that expresses rapidly after a number of stimuli like oxygen deprivation, growth factors, cytokines, shear stress and injury (brand et al., ; khachigian et al., ; nishi et al., ; jo et al., ; li et al., ) . egr is involved in diverse biologic functions and a broad variety of host signal transduction cascades that mediates cell growth, survival, differentiation, apoptosis and proliferation (pagel and deindl, ; papanikolaou et al., ) . different pathways have been identified that participate in egr induction and then regulates several biological behaviors. such as, the ras homologue gene family (rho) genes are involved in cell cycle progression, and the rho/rho-kinase pathway has been shown to regulate egr expression (barrientos et al., ; pagel and deindl, ) . as a zinc-finger dna-binding protein, egr also regulates expression of diverse gene families by binding to promoter sequences of target genes (papanikolaou et al., ) . therefore, egr is involved in activation of signal transduction of many pathways. several studies indicate that egr is linked to viral infection and immune response. egr modulates pro-apoptotic pathway and promotes venezuelan equine encephalitis virus (veev) replication (baer et al., ) . knockdown of egr in rhabdomyosarcoma cells decreases enterovirus (ev ) replication (song et al., ) . it seems that egr might play a positive role in these viruses replication. however, egr also appears critical for the initiation of immune response in b cells and t cells. egr plays roles in regulation of the expression of sever cytokines including interleukin- , cd , icam- and tumor necrosis factor genes (skerka et al., ; mcmahon and monroe, ; shin et al., ; cubero and nieto, ) . an sap domain [scaffold-attachment factor (saf)-a/b, apoptotic chromatin-condensation inducer in the nucleus (acinus) and pias (protein inhibitor of activated signal transducer and activator of transcription) domain] previously was identified within the fmdv l pro by de los santos et al. ( ) . mutation of l pro sap domain promotes type i ifn signaling activation and decreases virus growth. in addition, animals inoculated with the fmdv sap mutant display strong neutralizing antibody response and t cell response comparing with infection with wildtype fmdv (de los santos et al., ; diaz-san segundo et al., ) . in this study, a robust egr upregulation was observed in both wildtype and sap mutant fmdv-infected cells comparing with the mock-infected cells by viewing the protein abundance of egr . therefore, we investigated the correlation between fmdv infection and egr , and determined the antiviral role of egr against fmdv. sap mutant fmdv infection induced a higher expression of egr than wildtype fmdv infection. sap mutant fmdv infection also triggered higher ifn-β and ifn-stimulated genes (isgs) expression than wildtype fmdv infection. we also found that overexpression of egr enhanced sendai virus (sev)-induced interferon (ifn)-stimulated response element (isre) activation. sev-induced isgs expression was impaired in egr knockdown cells, which may serve as a link between upregulation of egr and type i ifn signaling. further study showed that egr enhanced tbk phosphorylation during fmdv infection. it indicated that egr upregulation promoted type i ifn signaling activation by enhancing tbk phosphorylation and resulted in decreased fmdv replication. this study reveals a link between egr and innate immune response during fmdv infection. porcine kidney pk- cells, human embryonic kidney t cells (hek t) cells described previously were maintained in dulbecco's modified eagle's medium supplemented with % heat-inactivated fetal bovine serum, u/ml penicillin, and µg/ml streptomycin sulfate. all the cells were cultured at • c under % co . sendai virus (sev), a model rna virus widely used to activate type i ifn signaling in cells, was kindly provide by hongbing shu's laboratory (wuhan university, china) (zhou et al., ; . fmdv strain o/by/cha/ (genbank number: jn ) described previously was used for virus infection (zheng et al., ) . commercial antibodies used in this study include an anti-egr mouse monoclonal antibody (abcam, cambridge, ma, united states), anti-tbk rabbit antibody from cell signaling technology (cst) inc. (beverly, ma, united states), anti-phospho-tbk rabbit antibody (cst), anti-c-myc mouse antibody (santa cruz biotechnology, santa cruz, ca, united states) and anti-β-actin mouse antibody (santa cruz biotechnology). anti-ifn-β and anti-ifn-α antibodies ( nu/ml, pbl biomedical laboratories) and igg isotype antibodies were used in the type i ifn-blocking experiments as previously described (trottier et al., ) . anti-fmdv vp protein polyclonal antibody was previously produced in our laboratory . transfection reagents include opti-mem medium and the lipofectamine that were purchased from invitrogen. poly (i:c) was purchased from invivogen. ifn-β was purchased from pbl biomedical laboratories. the full-length porcine egr cdna fragment was cloned into a pcdna tm . /myc-his(-)a vector (invitrogen) to construct a myc-tagged egr eukaryotic expressing plasmid (myc-egr , including a c-terminal myc tag). the constructed plasmid was analyzed and verified by dna sequencing. a series of plasmids expressing ha-tagged type i ifn pathway-related proteins [including mda , rig-i(card), visa, tbk , irf and irf ], and the ifn-β promoter luciferase reporter plasmids and control plasmid renilla luciferase prl-tk were kindly provided by hongbing shu's laboratory (zhou et al., ; . the plasmids were transfected into cells using opti-mem medium and the lipofectamine (invitrogen) reagent according to the manufacture's protocol. trizol reagent (invitrogen) was used to extract cellular or viral rna following the instruction of the protocol. the firststrand cdna was synthesized by reverse transcription reaction with the extracted rnas as templates. reverse transcription was performed with m-mlv reverse transcriptase (invitrogen) and random hexamer primers (takara) according to the manufacturer's recommendations. the quantification of the cdna was performed by qpcr. the relative amounts of the synthesized cdna was determined as an indicator of the target transcripts. qpcr was carried out using sybr premix ex taq (takara) on a quantstudio real-time pcr instrument (applied biosystems) according to the manufacturer's instructions. the glyceraldehyde- -phosphate dehydrogenase (gapdh) gene was used for normalization in qpcr analysis. relative transcript levels were calculated using − ct method as described previously . all the primers used in this study were listed in table . for western blotting, the cells were collected at the indicated time points and. the lysed cell extracts were resolved by % sds-page and transferred onto a nitrocellulose membrane (pall). the nitrocellulose membrane was then blocked with % skim milk powder in tbst ( mm tris, mm nacl, . % tween ) overnight at • c. the membrane was incubated with primary and secondary antibodies as described previously (zhu et al., ) . the membrane was washed × min, before being protein abundance analysis. the antibody-antigen complexes were visualized using enhanced chemiluminescence detection reagents (thermo). small interfering rna (sirna) was used to knockdown egr protein expression. sirna fragments were chemically synthesized by genepharma company (china). the sequences of the sirnas used in this study include: -ccaugga caacuacccuaatt- (egr sirna- ), -gccuaguga gcaugaccaatt- (egr sirna- ), and -gcuguca ccaacuccuucatt- (egr sirna- ). a non-targeting sirna (nc sirna) was used as a negative control. sirna fragments were transfected into cells using lipofectamine as described previously . forty-eight hours after sirna transfection, cells were used for further experiments. to examine the effect of sirna on egr expression, egr mrna and protein abundance were measured by qpcr and western blotting respectively. hek t cells seeded on -well plates were co-transfected with ng luciferase reporter plasmid with ng internal control renilla luciferase reporter plasmid (to normalize for transfection efficiency) prl-tk (promega), together with the indicated plasmids and/or empty vector controls using lipofectamine according to the manufacture's instruction. to make the cells receive the same amounts of total plasmids, the empty vector plasmids were used in all transfection experiments. as for sevmediated type i ifn signaling pathway activation, the cells were mock-infected or infected with sev ( hau/ml) for h; and the dual luciferase assays were then performed according to the promega dual-luciferase reporter assay system protocol. the relative luciferase activity was expressed as arbitrary units by normalizing firefly to renilla luciferase activity. as for type i ifn pathway adaptor molecules-induced ifn-element activation assay, the hek t cells were co-transfected with the reporter plasmids with the indicated plasmid or vector plasmid for h; and the luciferase activities were measured. all experiments were performed at least in triplicate. the measured values are represented as mean ± sd from three independent experiments. the statistical significance analyses were performed using the student's t-test. data considered significant when * p < . , and highly significant when * * p < . . pk- cells were infected by equal amounts of wildtype or sap mutant fmdv for h as previously described (zhu et al., ) . the expression levels of egr and viral vp protein were detected by western blotting. we observed that egr protein level is significantly upregulated both in wildtype and sap mutant fmdv-infected cells at h postinfection (hpi) ( figure a) . therefore, the correlation between fmdv infection and egr was further investigated. the dynamics of egr in fmdv-infected cells were determined. transcripts of egr were considerably upregulated after fmdv infection and reached to the highest level at hpi. no significant changes were observed in mock-infected cells ( figure b) . egr protein expression was also gradually upregulated as the infection progressed ( figure c ). this indicates that fmdv infection triggers upregulation of egr . to investigate whether egr is an ifn inducible gene, hek t and pk- cells were incubated with ifn-β to induce the expression of ifn inducible genes. the expression of two ifn inducible genes isg and isg was highly induced by incubation of ifn-β. however, the expression of egr was not changed by treatment of ifn-β ( figure d ). this indicated that egr expression was not induced by ifnβ treatment; however, fmdv infection could induce egr expression. to investigate the potential role of egr during fmdv infection, we evaluated the viral replication level in egr overexpressed cells. pk- cells were transfected with different doses of egr expressing plasmids, the cells were incubated with equal amounts of fmdv ( . moi) at h post-transfection (hpt). the viral protein and viral rna expression level was measured at hpi. overexpression of egr significantly suppressed both the viral protein expression and viral rna replication. the viral titers in both vector and egr plasmids ( µg) transfected cells were measured and compared, which showed that fmdv yields were also decreased by overexpression of egr (figure a) . to further confirm the antiviral role of egr during fmdv infection, the sirnas that target egr were designed and evaluated. pk- cells were transfected with the nc sirna or egr sirna for h, the interference efficacy of the sirnas was determined by qpcr analysis. the egr sirna- showed the highest efficacy and was used for egr knockdown assay ( figure b) . pk- cells were transfected with egr sirna- , the cells were infected with fmdv at hpt and incubated for another or h. the expression of egr and fmdv vp protein was detected using western blotting. knockdown of egr considerably increased vp protein expression during fmdv infection (figure c) . the relative fold-change in abundance of fmdv vp protein in fmdv-infected nc sirna or egr sirna cells was determined by densitometric analysis and normalized to β-actin, which confirmed that knockdown of egr enhanced fmdv vp protein expression (figure c , right panel). viral rna detection also suggested that knockdown of egr promoted viral replication ( figure d) . the viral titers were subsequently measured at hpi, which showed that knockdown of egr significantly promoted fmdv propagation ( figure d , right panel). these results suggest the antiviral role of egr against fmdv. both wildtype and sap mutant fmdv infection resulted in egr upregulation, however, sap mutant fmdv resulted in a higher upregulation of egr ( figure a) . previous study indicates sap mutant fmdv infection induces higher expression of ifn-β and isgs than wildtype fmdv infection (de los santos et al., ) . we also investigated the expression state of ifn-β and isgs (isg and mx ) in the cells infected by wildtype or sap mutant fmdv. at hpi, there was ∼ -fold difference in ifn-β transcripts for sap mutant fmdv-infected cells relative to wildtype fmdv-infected cells ( figure a) . a similar pattern was observed for isg and mx , varying from -to -fold higher for sap mutant fmdv compared to wildtype fmdv ( figure a) . these results were similar to the previous results reported by de los santos et al. ( ) . this showed that both egr expression and type i ifn signaling were enhanced in sap mutant fmdvinfected cells. this implied a link between egr and type i ifn pathway. to identify the role of egr on type i ifn signaling, egr is overexpressed in hek t cells, and the sev that is routinely used to induce type i ifns in cell culture was used to activate type i ifn signaling. overexpression of egr significantly promoted sev-induced type i ifn signaling, showing a dosedependent manner (figure b) . the expression of ifn-β, isg and mx in egr overexpressed cells were subsequently evaluated. the results showed that overexpression of egr considerably promoted sev-induced ifn-β, isg and mx expression ( figure c) . the effect of egr on sev-induced nf-κb activation was also evaluated by dual luciferase reporter assay, which also showed that egr positively enhanced nf-κb-mediated transcriptional activity ( figure d) . the role of egr on poly (i:c)-induced type i ifn signaling was further evaluated. overexpression of egr significantly promoted poly (i:c)-induced type i ifn signaling ( figure e) . the expression of poly (i:c)-induced isgs was also measured. the results figure | state of egr in fmdv-infected cells. (a) pk- cells were incubated with equal amounts of sap mutant fmdv or wildtype fmdv for h, the abundance of egr and viral vp protein was detected. (b) pk- cells were infected with wildtype fmdv or mock-infected for , , , , , or h. the transcripts of egr and viral rna were detected by qpcr. (c) pk- cells were infected with wildtype fmdv for , , , , , or h. the expression levels of egr and vp protein were detected by western blotting. (d) hek t or pk- cells were mock-treated or incubated with ifn-β at a concentration of ng/ml for h. the expression levels of isg , isg and egr was measured by qpcr. showed that overexpression of egr considerably promoted poly (i:c)-induced isg and isg expression ( figure f ). sevinduced isgs expression levels in egr knockdown cells were also analyzed. the sirna interference efficacy was also verified in hek t cells ( figure g) . egr was knocked down by transfection of sirna, and the cells were infected by sev at hpt and incubated for h. the expression of ifn-β, isg and mx were measured. the transcript levels of ifn-β, isg and mx remarkably decreased in egr knockdown cells comparing with that in nc sirna cell ( figure h) . these results suggested a positive regulatory role of egr on type i ifn signaling. the type i ifn blocking antibody experiments were also performed. anti-ifn-β and anti-ifn-α antibodies ( nu/ml) was used to block type i ifn signaling in pk- cells, the egr overexpressed cells were treated with ifn antibodies for h and then infected with fmdv. the viral yields were measured at hpi. type i ifn blocking antibodies obviously abrogated the inhibitory effects of egr on fmdv propagation ( figure i) . these results suggested that egr suppressed fmdv replication by enhancing type i ifn signaling. we also evaluated the antiviral role of egr against another picornavirus seneca valley virus (svv) which showed a close relationship with fmdv, and we found that upregulation of egr also enhanced isgs expression (isg and isg ) and suppressed svv replication ( figure j) (b) pk- cells were transfected with nc (negative control) or sirna (egr sirna- , egr sirna- or egr sirna- ) for h. the egr mrna levels were measured by qpcr. (c) schematic diagram of the strategy in egr knockdown experiment and investigation of the viral replication state in egr knockdown cells. pk- cells were transfected with nc sirna or egr sirna- for h. the cells were infected with fmdv for , , or h. viral protein abundance was measured by western blotting. relative fold-change in abundance of vp protein was determined by densitometric analysis using quantity one software (bio-rad) and normalized to β-actin. (d) viral rna levels in fmdv-infected nc sirna or egr sirna- cells at , , and hpi were measured by qpcr. viral yields in fmdv-infected nc sirna or egr sirna- cells at hpi were measured by tcid assay. * p < . was considered as statistically significant and * * p < . was considered as highly significant. i ifn signaling pathway. to screen the potential proteins that were targeted by egr , hek t cells were co-transfected with the myc-vector or myc-tagged egr plasmids and the indicated plasmids expressing rig-i, rig-i(card) (the card domain of rig-i), mda (helicase) (the helicase domain of mda ), visa, tbk , irf and irf , together with isre luciferase reporter plasmid and the internal control plasmid prl-tk. luciferase activity was measured at h after transfection. overexpression of adaptor proteins rig-i, rig-i(card), mda ), visa, tbk , irf or irf all activated the isre luciferase reporter system, and overexpression of mda (helicase) did not activate the isre luciferase reporter system (figure ) . tbk or its upstream proteins (rig-i, mda and visa) mediated type i ifn signaling was significantly enhanced by overexpression of egr . however, overexpression of egr did not promote irf and irf mediated type i ifn signaling (figure ) . irf and irf are the downstream proteins of tbk . therefore, we speculated that tbk or its upstream molecules (rig-i, mda and visa) were the target/targets of egr to enhance type i ifn signal transduction. to investigate whether egr interacted with the adaptors of type i ifn pathway, the coimmunoprecipitation assay was performed by co-transfection of the myc-egr plasmids and the plasmids expressing various ha-tagged adaptors of type i ifn pathway. the transfectants were immunoprecipitated with anti-ha antibodies and subjected to western blotting analysis. no interaction was observed between egr and the adaptors (figure a) . egr enhanced the activation of isre luciferase reporter stimulated by tbk and its upstream molecules. tbk might be a key adaptor to enhance type i ifn signaling. the influence of egr on tbk expression and tbk phosphorylation levels were evaluated in fmdv-infected cells. pk- cells were transfected with µg of myc-egr or its empty vector plasmids. the cells were infected with equal amounts of fmdv at hpt and collected at , , , or hpi. overexpression of egr had no influence on tbk expression during fmdv infection. however, it significantly promoted tbk phosphorylation levels after fmdv infection comparing with that in the empty vector transfected cells (figure b) . the vp was used as an indicator of viral replication. overexpression of egr also resulted in decreased vp abundance ( figure b ). this confirmed that upregulation of egr enhanced type i ifn signaling and suppressed fmdv replication. as a transcription factor, the transcriptional activity is significantly involved in the regulatory function of egr . the transcripts of ifn-β, isg and mx were detected by qpcr. (d) hek t cells were transfected with vector or myc-egr plasmids together with nf-κb luciferase reporter plasmid and prl-tk. the transfected cells were infected with sev, and the luciferase activity was measured by dual luciferase assay. (e) hek t cells were transfected with vector plasmids or myc-egr plasmids and solvent control or poly (i:c) together with isre luciferase reporter plasmid and prl-tk. the luciferase activity was measured by dual luciferase assay. (f) hek t cells were co-transfected with vector plasmids or myc-egr plasmids and solvent control or poly (i:c), the expression of isg and isg expression was measured by qpcr. (g) hek t cells were transfected with nc or egr sirna- for h. the egr transcripts were measured by qpcr. (h) hek t cells were transfected with nc sirna or egr sirna- for h. the cells were then mock-infected or infected with sev for h. the transcripts of ifn-β, isg and mx were detected by qpcr. (i) pk- cells were transfected with equal amounts of vector or egr expressing plasmids for h, the egr -transfected cell were mock-treated or treated with ifn antibodies for h and then infected with fmdv. the viral yields were measured by tcid assay at hpi. (j) pk- cells were transfected with vector plasmids or myc-egr plasmids for h. the transfected cells were infected with svv for h. the viral rna, isg and isg expression levels were detected by qpcr. * p < . was considered as statistically significant and * * p < . was considered as highly significant. to investigate whether the transcriptional activity is related to the antiviral function of egr , znegr , a previous reported dominant-negative mutant of egr that lacks a transcriptional function, was used as an inhibitor of the transcriptional activity of egr (levkovitz and baraban, ) . the myc-tagged znegr expressing plasmid was constructed ( figure a) . pk- cells were transfected with vector, myc-egr plasmids or cotransfected with myc-egr and myc-znegr figure | the target of egr in type i ifn pathway activation. hek t cells were co-transfected with myc-egr or empty vector plasmids and the constructs expressing rig-i, rig-i(card), mda , visa, tbk , irf or irf , together with isre luciferase reporter plasmid and the internal control plasmid prl-tk. dual luciferase activity was determined at hpt. * * p < . was considered as highly significant. plasmids and subjected to fmdv infection. overexpression of egr suppressed fmdv replication, and egr -mediated antiviral effect was not blocked by cotransfection with znegr ( figure b) . we further evaluated the localization of egr in mock-or fmdv-infected cells, and we found fmdv infection did not change the localization of egr compared with that in the mock-infected cells ( figure c) . these data suggest that the transcriptional activity is not involved in the antiviral function of egr . egr , as a multifunctional transcription factor, plays regulatory roles in a variety of cellular responses. in addition, egr shows an anti-tumor function. overexpression of egr decreases tumorigenesis in nude mice and various of human tumor cell lines (huang et al., (huang et al., , . induction of tgfβ and p may lead to the tumor suppressor property of egr (baron et al., ) . p , as a tumor suppressor, has also been implicated in other functions that play important roles in disease and health (fuhrman et al., ). p -dependent antiviral defense has been widely reported (takaoka et al., ; shin-ya et al., ; muñoz-fontela et al., ) . such as, p serves as an antiviral protein during influenza a virus infection by enhancing host innate and adaptive immune responses (muñoz-fontela et al., ) . egr directly induces the transcription of p (liu et al., ) , whether egr is also involved in host antiviral responses remains unknown. in this study, we determined that egr revealed an antiviral function against fmdv, which indicated that egr is implicated in host antiviral response. egr can be upregulated upon viral infection by epstein-barr virus, mouse hepatitis virus (mhv), veev, ev , rabies viruses and japanese encephalitis virus infections (saha and rangarajan, ; cai et al., ; kim et al., ; song et al., ; baer et al., ) . however, egr expression is related to viral pathogenesis during veev, mhv and ev replication. all these studies were performed using mouse or human cells, and most of these viruses can cause central nervous system (cns) diseases. in this study we investigated the function of porcine egr and showed the antiviral role of porcine egr against fmdv. fmdv infection does not cause any cns disease. whether the difference of species or tissue tropism resulted in the different role of egr in different virus infections remain unknown. however, egr has been suggested to participate in ifn-γ-stat pathway in t cells (shin et al., ) . t-bet is a th -specific transcription factor that is directly involved in t cells differentiation (djuretic et al., ) . egr regulates t-bet expression by binding to the promoter of t-bet and induces t-bet transcription (shin et al., ) . t-bet plays a vital role in innate immunity, and lacking of t-bet expression increases host susceptibility to inflammatory disease (garrett et al., ) . this implies a regulatory role of egr in innate immunity. besides, overexpression of egr downregulates nfκb inhibitor (kim et al., ) , which also implies a potential role of egr in innate immunity. in this study, we determined that egr is implicated in innate immunity during fmdv infection. a higher egr expression was observed in sap mutant fmdv-infected cells comparing with that in the wildtype fmdv-infected cells. it has been determined that sap mutant fmdv infection resulted in stronger type i ifn signaling than wildtype fmdv infection (de los santos et al., ) . we found that sap mutant fmdv infection triggered higher expression of ifn-β and isgs than wildtype fmdv infection. this result was similar as the result reported by de los santos et al. ( ) previously. whether the higher expression of egr correlated with the higher expression of ifn-β and isgs was therefore investigated. overexpression of egr significantly activated type i ifn signaling and ifn-β and isgs expression. knockdown of egr considerably impaired sev-induced ifn-β and isgs expression. type i ifn blocking antibodies obviously abrogated the inhibitory effects of egr on fmdv propagation. this suggested egr is implicated in type i ifn pathway activation. a link between egr and type i ifn pathway was reported for the first time. further investigation of egr -mediated enhancive effect showed that egr promoted activation of the type i ifn signaling during fmdv infection. overexpression of egr upregulated tbk phosphorylation during fmdv infection. tbk phosphorylation enhanced type i ifn signaling and strengthened antiviral activity which subsequently suppressed fmdv replication. egr is a transcription factor; however, it does not induce tbk expression. the interaction between egr and various adaptors of type i ifn signaling pathway was not observed by performing coimmunoprecipitation assay. the role of the transcriptional activity of egr for its antiviral function against fmdv was also evaluated. suppression of the transcriptional activity of egr did not affect its antiviral effect. egr might enhance type i ifn signaling independent of its transcriptional activity. how does egr promote tbk phosphorylation is not clear. several phosphatases have been identified as regulator of phosphorylation of tbk (gabhann et al., ; zhao, ) . the regulation of egr on these phosphatases should be studied in future, and the detailed mechanism of egr to promote tbk phosphorylation should be further investigated. in addition, the effect of egr on the adaption of other upstream molecules of tbk should also be exploited. in summary, we present the first investigation of egr in regulation of type i ifn signaling during fmdv infection. we determined that egr showed an antiviral function against fmdv. egr promoted activation of the type i ifn signaling during fmdv infection and resulted in the decreased replication of fmdv. egr suppressed fmdv replication independent of its transcriptional activity. these findings identify an important role of egr in enhancement of type i ifn signaling during fmdv infection. however, the exact mechanism for egr to promote type i ifn signaling should be investigated in future to gain deeper understanding of egr -mediated functions. venezuelan equine encephalitis virus induces apoptosis through the unfolded protein response activation of egr the transcription factor egr is a direct regulator of multiple tumor suppressors including tgfβ , pten, p and fibronectin: egr is a potential target of gene therapy for prostate cancer two novel members of the ablim protein family, ablim- and - , associate with stars and directly bind f-actin review of the foot and mouth disease situation in north africa and the risk of 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suppress foot-and-mouth disease virus replication crucial role for early growth response- in the transcriptional regulation of mir- b in breast cancer physical interaction between p and primary response gene egr- molecular basis of pathogenesis of fmdv the role of early growth response gene (egr- ) in regulation of the immune response transcriptional role of p in interferonmediated antiviral immunity p serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza a virus early growth response- gene mediates up-regulation of epidermal growth factor receptor expression during hypoxia early growth response -a transcription factor in the crossfire of signal transduction cascades a systems approach identifies co-signaling molecules of early growth response transcription factor in immobilization stress developing vaccines against foot-and-mouth disease and some other exotic viral diseases of livestock molecular mechanisms of foot-andmouth disease virus targeting the host antiviral response epidemiological patterns of foot-and-mouth disease worldwide planning for the progressive control of foot-and-mouth disease worldwide common host genes are activated in mouse brain by japanese encephalitis and rabies viruses t-bet expression is regulated by egr -mediated signaling in activated t cells intracellular interferon triggers jak/stat signaling cascade and induces p -dependent antiviral protection a regulatory element in the human interleukin gene promoter is a binding site for the zinc finger proteins sp and egr- foot-and-mouth disease in europe. fmd is economically the most important disease of farm animals. its re-emergence in europe is likely to have consequences that go beyond severe alterations of livestock production and trade early growth response- facilitates enterovirus replication by direct binding to the viral genome rna integration of interferon-|α|/|β| signalling to p responses in tumour suppression and antiviral defence retinoids inhibit measles virus through a type i ifn-dependent bystander effect progress and prospect of the technologies to control foot-andmouth disease and its pathogen characteristics worldwide negative regulation of tbk -mediated antiviral immunity genetic characterization of a new pandemic southeast asia topotype strain of serotype o foot-and-mouth disease virus isolated in china during the erassociated protein zdhhc is a positive regulator of dna virus-triggered, mita/sting-dependent innate immune signaling nonstructural protein of influenza a virus interacts with human guanylate-binding protein to antagonize antiviral activity foot-andmouth disease virus viroporin b antagonizes rig-i mediated antiviral effects by inhibition of its protein expression comparative proteomic analysis of wild-type and sap domain mutant foot-and-mouth disease virus-infected porcine cells identifies the ubiquitin-activating enzyme ube required for virus replication zz, xd, and hz conceived the study and wrote the manuscript. zz, xd, pl, xz, fy, and wc performed the experiments. ht, kz, and xl collected the data, analyzed the data, and revised the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © zhu, du, li, zhang, yang, cao, tian, zhang, liu and zheng. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - n p authors: dargahi, narges; katsara, maria; tselios, theodore; androutsou, maria-eleni; de courten, maximilian; matsoukas, john; apostolopoulos, vasso title: multiple sclerosis: immunopathology and treatment update date: - - journal: brain sci doi: . /brainsci sha: doc_id: cord_uid: n p the treatment of multiple sclerosis (ms) has changed over the last years. all immunotherapeutic drugs target relapsing remitting ms (rrms) and it still remains a medical challenge in ms to develop a treatment for progressive forms. the most common injectable disease-modifying therapies in rrms include β-interferons a or b and glatiramer acetate. however, one of the major challenges of injectable disease-modifying therapies has been poor treatment adherence with approximately % of patients discontinuing the therapy within the first year. herein, we go back to the basics to understand the immunopathophysiology of ms to gain insights in the development of new improved drug treatments. we present current disease-modifying therapies (interferons, glatiramer acetate, dimethyl fumarate, teriflunomide, fingolimod, mitoxantrone), humanized monoclonal antibodies (natalizumab, ofatumumab, ocrelizumab, alemtuzumab, daclizumab) and emerging immune modulating approaches (stem cells, dna vaccines, nanoparticles, altered peptide ligands) for the treatment of ms. in the early s, only a few cases of multiple sclerosis (ms) were reported, which quickly became a common occurrence for admission to neurological wards. today, ms accounts over . million affected individuals with an estimated cost of us$ - billion per annum [ ] . the distribution of ms varies according to geographic location. for example, the further north or south from the equator the higher the prevalence of ms; countries that lie on the equator have extremely low prevalence compared to scotland, norway, and canada. the prevalence of ms has increased progressively over time with / , diagnosed in to / , diagnosed in globally. in fact, in a norwegian cohort over years ( - ) , the prevalence increased from to / , and the incidence increased from . to / , [ ] . it is possible that the increase in prevalence is due to improved diagnostic procedures and reporting and changes in lifestyle (lack of vitamin d and increased smoking) [ ] . ms is commonly diagnosed between years and years of age although it can affect younger and older individuals [ ] , and most commonly affects those with a genetic predisposition (major histocompatibility complex (mhc) class ii phenotype, human leukocyte antigen (hla)-dr and hla-dr most commonly affected). in fact, the incidence of ms is increased -fold in monozygotic information is gathered, and transferred to the rest of the body [ , ] . ms involves main steps, (i) myelin sheath damage resulting in formation of lesions in the cns and (ii) inflammation, which together destroy the neuron tissue [ , ] . in ms, damage of oligodendrocytes and destruction of myelin sheath leads to breakdown of the nerve axon and loss of neuronal function [ ] . demyelination increases the inflammatory activation processes leading to damage of bbb and stimulation of macrophage activation and oxidative stress pathways [ ] . the white matter lesions include myelin breakdown together with infiltration of monocytes, b cells, t cells and dc [ ] . microglia and macrophages are the main innate immune cells present in ms lesions where they either act together with t and b cells, or directly cause neuroinflammatory tissue damage [ ] . cells involved in the inflammatory process include those that are both in the innate and adaptive immune systems and are described below (figure ). nkt cells share properties of both t cells and nk cells and recognize glycolipid antigens presented in complex with the mhc class i-like molecule, cd d. two subsets of nkt cells have been identified (type i, invariant nkt (inkt) cells and type ii, variant nkt (vnkt) cells) and are implicated in the pathogenesis of ms in humans and in the murine model of ms, experimental autoimmune encephalomyelitis (eme). inkt cells express cell surface markers characteristic of activated or memory t cells (cd , cd , cd ) with the majority being cd + as well as markers characteristic of nk cells (nk . or cd , ly ). following activation of inkt cells (via binding to α-galcer-cd d complex) an array of cytokines is secreted that are associated with both pro-and anti-inflammatory immune responses and play a role in both innate and acquired immunity. as such, inkt cells, (i) secrete interleukin (il)- and il- which stimulate cd + t cells to differentiate into anti-inflammatory th cells (il- , il- producers) which inhibit th , th , cd + t cells in the nkt cells share properties of both t cells and nk cells and recognize glycolipid antigens presented in complex with the mhc class i-like molecule, cd d. two subsets of nkt cells have been identified (type i, invariant nkt (inkt) cells and type ii, variant nkt (vnkt) cells) and are implicated in the pathogenesis of ms in humans and in the murine model of ms, experimental autoimmune encephalomyelitis (eme). inkt cells express cell surface markers characteristic of activated or memory t cells (cd , cd , cd ) with the majority being cd + as well as markers characteristic of nk cells (nk . or cd , ly ). following activation of inkt cells (via binding to α-galcer-cd d complex) an array of cytokines is secreted that are associated with both pro-and anti-inflammatory immune responses and play a role in both innate and acquired immunity. as such, inkt cells, (i) secrete interleukin (il)- and il- which stimulate cd + t cells to differentiate into anti-inflammatory th cells (il- , il- producers) which inhibit th , th , cd + t cells in the cns; (ii) secrete il- brain sci. , , of and tumor growth factor (tgf)-beta which stimulate the production of t regulatory (treg) cells (il- , tgf-beta producers) which inhibit th , th and cd + t cells in the cns; and (iii) secrete il- , il- , il- , interferon (ifn)-gamma and gm-csf which activate suppressive myeloid derived suppressor cells (mdcs), dc and macrophages which in turn secrete il- to activate treg cells and suppress th , th and cd + t cells in the cns [ ] . due to the pleiotropic properties of inkt cells, they play a role in protecting the host against pathogens, tumors, autoimmunity and are involved in tissue rejection, ischemia reperfusion injury and obesity related diabetes [ ] ; deficiency or dysfunction of inkt cells has been shown to be linked to the development of autoimmune diseases. indeed, inkt cell numbers are decreased in patients with ms [ ] and are restored in patients in remission [ ] . analysis of inkt cells in ms patients in remission showed a th cytokine profile, suggesting an immunoregulatory effect of inkt cells in ms [ ] . similarly, in the eae mouse model, protection of eae development is associated with high levels of inkt cells and suppression of th and th cells [ ] . interestingly, injections of α-galactosylceramide (α-galcer), and analogues thereof, have potent activities in protecting mice against, cancer, infections, inflammatory conditions and autoimmune disorders. hence, it is possible to develop inkt cell based modulating therapies against ms [ , ] . like inkt cells, variant nkt (vnkt) cells also share properties of both t cells (cd + ) and nk cells (nk . ) and recognize β-linked glycolipid antigens in complex with cd d. they are less common in mice compared to inkt cells but are more abundant in humans. of interest, vnkt cells recognize the self-glycolipid, sulphatide, which is abundantly expressed within the myelin sheath suggesting a role in ms although not yet established [ ] . likewise, vnkt cells recognizing sulphatide self-myelin ligand are present in high levels in mice with eae suggesting their role in disease progression [ ] . mait cells are a subset of t cells of the innate immune system to defend against microbial infections. they are present in the liver, lungs, mucosa and blood and make up to % of cd t cells in healthy individuals; they also support adaptive immune responses in that they have a memory like phenotype [ ] . the mhc class i-like molecule, mri, presents microbial antigens and vitamin b metabolites to mait cells, leading to their activation [ , ] . however, mait cells have also been implicated in autoimmune diseases such as ms, inflammatory bowel disease and rheumatoid arthritis where they are often noted at the site of autoimmune attack. recently, it was reported that in ms, mait cells are highly present at the sites of demyelination and secrete pro-inflammatory th cytokines (ifn-gamma and tnf-alpha) and activate th cells (il- and il- cytokines) [ ] ; the major cytokines in the pathogenesis of chronic inflammatory and autoimmune diseases. in addition, mait cell have been noted in white matter inflammatory lesions [ ] as well as transcription over expression of mr in ms lesions. conversely, it has been reported that mait cells are decreased in blood of patients with rrms [ ] . it is not clear whether mait cells exert a protective or a non-protective role, thus a better understanding of how mait cells are involved in ms and of their interactions would aid in a better understanding of the pathogenesis of ms and development of therapeutic strategies. regulatory t cells (tregs; originally known as suppressor t cells) are a subset of cd + t cells that modulate immunity, maintain tolerance against self-antigens and prevent autoimmunity. tregs are primarily characterized as foxp + cd + cd + and are anti-inflammatory (secrete il- ). one of the first evidence of the role of treg cells in ms was in mouse eae models, where adoptive transfer of treg cells from control mice into mog or plp induced eae mice prevented the onset and progression of eae [ , ] . adoptive transfer of treg cells recovering from eae into mog-induced active eae mice resulted in resolution of eae [ ] . in addition, induction of treg cells by estradiol or by monocytes under glatiramer acetate treatment reduced clinical signs of mog-eae [ , ] . furthermore, injection anti-cd monoclonal antibody in lewis rats results in treg cell expansion and reduction in eae disease severity [ ] . interestingly, injection of anti-cd monoclonal antibody, which blocks the effects of treg cells into c bl/ mice increased susceptibility to eae induction [ ] . in patients with ms however, the frequency of foxp + cd + cd + treg cells does not differ to those in healthy individuals, although the function of such cells are impaired (maturation and migration) [ ] . in addition, mrna and protein levels of foxp are impaired in treg cells of patients with ms especially in rrms and are normalized during spms [ ] . hence, impaired functionality of treg cells is primarily observed in the early stages of ms but not in their chronic stage, suggesting a causative role [ ] . further studies of treg cells in ms may aid in the understanding for why tolerance against self-antigens is broken, leading to disease. however, it is not clear whether the impaired function of treg cells is a direct cause of ms or whether such impairment is a general outcome for all autoimmune disorders. macrophages are divided into m or m based on their pro-or anti-inflammatory cytokine secretion phenotype [ ] . m macrophage phenotype of mice (f / + cd b + cd c + inos + ) and human (cd + cd + cd + cd + ) is induced in the presence of interferon (ifn)-gamma and/or toll-like receptor (tlr) ligands such as lipopolysaccharide (lps). m macrophages are pro-inflammatory and primarily secrete il- , il- , il- , tnf-alpha, inos and mcp- [ ] . in general, they stimulate adaptive immune responses. the m macrophage phenotype of mice (f / + cd c − cd + arg + cd + ) and humans (cd + cd + ) is induced in the presence of il- , il- , il- and arg that blocks inos activity [ ] . m macrophages are anti-inflammatory and primarily secrete il- receptor antagonist, il- , il- , transforming growth factor (tgf)-beta . macrophages play a crucial role in the pathogenesis of ms. in fact, in active demyelinating and early re-myelinating lesions, macrophages are highly present compared to inactive, demyelinated or late re-myelinated lesions [ ] . however, a distinction of m vs m macrophages in human brain tissues is not so clear, with both m macrophages and an intermediate subtype (m /m , cd + cd + ) being present [ ] . like macrophages, microglia cells are divided into m -and m -polarized microglia cells. m microglia cells are pro-inflammatory and express cd , cd , cd and ccr , whereas, m microglia cells are anti-inflammatory and express mannose receptor (cd ) and ccl . in ms brain lesions however, like macrophages, an intermediate microglia phenotype is present expressing cd , cd , cd and ccl but not cd markers [ ] . interestingly, in an eae model it was shown that suppression of ccl decreased m macrophage accumulation in the cns, thus therapies designed to suppress ccl have the potential to decrease demyelination and progression of disease. in addition, in mice m microglia cells have been found to switch to m microglia cells during remyelination, hence m polarization is necessary for efficient remyelination [ ] . indeed, fasudil (a selective rho kinase inhibitor), injected into eae bearing mice shifted m to m macrophages and ameliorated the clinical severity of eae [ ] . cd t cells or t helper (th) cells, recognize short - amino acid peptides presented on the surface of antigen presenting cells (apc) in complex with mhc class ii. cd t cells differentiate into distinct th cells depending on the cytokine secretion profiles [ ] . (i) th cells are pro-inflammatory and produce high levels of il- , il- , tnf-alpha and ifn-gamma; (ii) th cells are anti-inflammatory and secrete il- , il- , il- , il- , il- , il- ; (iii) th cells are pro-inflammatory and secrete high levels of il- a, il- f, il- , il- , il- , il- and low levels of il- and ifn-gamma; (iv) th cells which are a combination of th , th , th phenotype and secrete il- , il- and tnf-alpha and (v) the newest addition to the th subset, th , was identified for its potent secretion of il- . th , th , th cells are key contributors to ms by increasing inflammation within the milieu of the myelin site. th cells and their pro-inflammatory cytokine products are present in high levels within the demyelinating axon and cns lesions of humans and in mog, plp or mbp induced eae in mice. th cells recognize mog, plp and mbp peptide epitopes presented in the context of mhc class ii, hla-drb * (hla-dr , hla-dr ) and hla-drb * (hla-dr ) alleles. as a result cd t cells become activated, cross the blood brain barrier and induce cns autoimmunity. some drug therapeutics target the mhc class ii-peptide-t cell receptor (tcr) complex in an attempt to modulate or divert th responses to therapeutic th responses. indeed, it was recently shown that dimethyl fumarate (dmf) injection in rrms patients reduced th , th and cd t cells and increased th cells; this resulted in high levels of il- and decreased levels of ifn-gamma and il- [ ] . in addition, we have shown that mannan conjugation of self-mbp, plp or mog native peptides or altered peptide ligands, are able to divert th responses to th responses in human pbmc from ms patients, in immunized mouse spleen cells and are able to ameliorate eae in mice [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the role of th cells in ms is not as clear although in mice, il- and th cells induce eae and inflammation and il- knockout mice are protected from developing eae [ ] . th cells play a crucial role in the pathogenesis of ms in both mice and humans by inducing an inflammatory milieu. in fact, il- a is present at high levels in cns lesions, cerebrospinal fluid and in the serum of patients with ms [ ] . th cells express high levels of ccr which binds to the ligand ccl on vascular endothelial cells, enabling their entry through the blood brain barrier where they secrete pro-inflammatory cytokines including il- a. in addition, il- interferes with the remyelination process. of interest, anti-il- a humanized neutralizing monoclonal antibody (ain or secukinumab) injected in patients with ms showed reduction of lesions compared to placebo-treated control subjects [ ] . in addition, th cells are highly present in the peripheral blood and cerebral spinal fluid of patients with active rrms [ ] , and il- mrna and th cells are increased in relapsing ms compared to remitting ms patients [ ] . furthermore, th cells specifically recognize mbp and are resistant to ifn-beta therapy [ ] . il- , a member of the il- /il- cytokine family, is secreted by macrophages, dendritic cells and microglia cells, with pleiotropic roles in immunomodulation being either pro-or anti-inflammatory. il- also stimulates or inhibits t cell differentiation. th cells are induced by il- whereas th , th and treg cells are inhibited by il- . in addition, tr cells a specialized subset of t cells which secrete il- are induced in the presence of il- [ ] . in patients with rrms, circulating plasma il- levels were significantly higher compared to healthy control subjects [ ] . likewise, il- and il- r are elevated in post-mortem ms brain lesions compared to non-ms control brains. macrophages and microglia were identified to be the source of il- and triggering infiltration of cd and cd t cells [ ] . in addition, the effects of il- on microglia cells showed that nitric oxide, tnf-alpha and il- were secreted, promoting th polarization, suggestive that il- enhances microglia neuroinflammation [ ] . hence, suppressing il- may be a strategy to modulate inflammatory responses in patients with ms. , recognize short antigenic - -mer peptide epitopes presented on the surface of apc in complex with mhc class i. in ms there is a genetic association with hla-a [ ] ; hla-a has been shown to reduce the risk of ms in individuals that also express mhc class ii, hla-drb * . the antigen specificity of cd tc cells isolated from patients with ms, has been suggested to be against mog, mbp and plp with cytolytic activity against neuronal cells in vitro [ ] although their pathogenic role in ms is still not clear. more recently other subsets of cd t cells have been identified and are grouped into different subsets based on their cytokine profile. in as such, classical tc cells secrete ifn-gamma, tc secrete il- , tc secrete il- , tc secrete il- , tc secrete il- , tc secrete il- and another subset is characterized by secreting tnf-alpha. in ms, regardless of the stage and activity of disease cd t cells are noted in high numbers, much higher than cd t cells at a ratio of : cd :cd t cells. mhc class i is highly expressed within ms lesions and astrocytes, oligodendrocytes, neurons in addition to the classical apc, dcs and macrophages. in fact, cd t cells are found in great abundance within cns tissues and cerebrospinal fluid of patients with ms. cd t cells present in both acute and chronic ms lesions secrete high levels of il- (classed as, tc cd t cells) [ ] . tc cells secrete il- and tnf-alpha and low ifn-gamma and are negative for granzyme b, perforin and cytolytic activity unlike the classical cd tc cells. in peripheral blood of patients with spms and rrms elevated levels of tc and tc cells are noted as well as a high percentage of tnf-alpha secreting cd t cells [ ] ; tc cells are increased in the remission phase of rrms compared to spms. in addition, higher levels of cd + ifn-gamma + tnf-alpha + il- + t cells in the relapsing phase of rrms compared to remission phase, spms and controls [ ] . it is clear that cd t cells contribute to the pathogenesis of ms, and it is important to understand how such cells escape t cell tolerance and induce cns autoimmunity in order to design and develop new therapeutics against ms. although there is a presence of t cells in ms plaques, b cells also contribute to the pathogenesis of ms where they secrete autoantibodies and cytokines and being apc they activate t cells. in patients with ms the presence of oligoclonal bands (ocb) in cerebrospinal fluid and brain parenchyma is a consistent finding in over % of patients. ocb is a product of clonally expanded b cells and igg synthesis. in ms plaques plasma cells are noted in large numbers where antigen uptake, processing and presentation takes place as well as synthesis of igg. interestingly, over antibodies isolated from cerebrospinal fluid from patients with ms did not react to mbp, plp or mog [ ] but some groups reporting that they bind to intracellular proteins such as, mknk / , fam a, akap a and glial potassium channel kir . , or, against intracellular lipid determinants [ , ] . moreover, anti-mog autoantibodies is a hallmark of childhood ms as well as in some patients with neuromyelitis optical spectrum disorder. it is clear, that abnormal activation of b cells within the cns of patients with ms, suggests that b cells play a role in the pathophysiology of the disease. further studies are required to ascertain whether b cell depletion is able to restore immune function and hence, be used as a therapeutic target against ms. dc are professional apc which process and present antigenic peptide epitopes on their surface in complex with mhc class i or class ii, resulting in cd or cd t cell stimulation respectively. even though ms is generally associated with predominant auto-reactive t cells, emerging evidence indicates that dcs play an important role in the pathophysiology of ms, primarily due to their t cell activating and cytokine secreting properties. following activation of dcs in the periphery, t cells specific to myelin epitopes are activated inducing pro-inflammatory cytokines aiding their entry through the bbb into the cns. in the cns resident apc and t cells are further activated leading to demyelination and motor deficits. in patients with ms, dcs are abundantly present within inflamed lesions, cerebrospinal fluid and in the circulation and produce high levels of tnf-alpha, ifn-gamma and il- [ ] . in addition, the expression of co-stimulatory molecules, cd and cd on dcs are increased in rrms and spms patients, suggesting an activated pro-inflammatory state of dcs, hence their contributing role in the pathogenesis of ms. myeloid-derived suppressor cells (mdsc) are myeloid progenitors, the same lineage to that of macrophages, dc and neutrophils. however, mdsc have strong immunosuppressive properties rather than immune-stimulatory properties as noted with macrophages, dc and neutrophils [ ] . their major role is in tumor development and chronic inflammation having immune suppressive effects [ ] . as such, it was recently shown following mbp - peptide immunization in mice, that mdscs were increased adopting a suppressive phenotype, inhibiting the activation of cd + t cells via arginase- and inducible nitric oxide synthase; such approach inhibited the development of eae in mice [ ] . in addition, mdsc secrete inhibitory enzyme indoleamine , -dioxygenase and th cytokine, il- [ ] . it is not clear whether the number of mdscs are reduced or whether their functionality is altered in patients with ms, leading to the failure of mdscs to suppress autoimmune t cells, as a result of disease progression. the use of ex vivo cultured mdscs could be a viable strategy to develop new improved treatments against ms. the majority of the treatments for ms are long term mainly suppressing the immune system however, such immune-suppressants pose increased risks for infections and cancer [ ] . alternative treatment options involve disease-modifying therapies such as, interferons, glatiramer acetate, monoclonal antibodies and sphingosine- -phosphate receptor modulators (table , figure ). these therapies have dramatically reduced the number of attacks and decreased disease progression. in fact, interferons are effective in the early relapsing phases of ms but not in the advanced phases of the disease [ ] . ultimately, induction of tolerance against self-antigens and re-establishing immune homeostasis can effectively "cure" the disease; such strategies have been the focus of recent research. figure ). these therapies have dramatically reduced the number of attacks and decreased disease progression. in fact, interferons are effective in the early relapsing phases of ms but not in the advanced phases of the disease [ ] . ultimately, induction of tolerance against self-antigens and re-establishing immune homeostasis can effectively "cure" the disease; such strategies have been the focus of recent research. patients with ms who present with a relapse are generally treated with corticosteroids intravenously, plasma exchange or adrenocorticotropic hormone injections [ , ] . although effective in reducing the duration of the relapse and patients recovery faster there are no long-term neuroprotective benefits [ , [ ] [ ] [ ] [ ] . the treatment of ms has been a challenge with treatment options being limited mainly to corticosteroids, the potent alkylating agent cyclophosphamide and potent immunosuppressant methotrexate (table , figure ). however, with the advent of immunomodulatory drugs in mid- s, a big shift was carried to treatment options for the first time [ ] . the first disease-modifying drug for rrms, interferon beta- (ifnβ- ) was the primary key breakthrough for the treatment of ms [ , ] . disease-modifying agents intend to modify the course of the disease rather than improving symptoms. until the approval of the first oral treatment in [ ] , all ms treatments consisted of either intramuscular or subcutaneous injectable drugs. to date, fda approved disease-modifying drugs are available for rrms, and several more agents are in different developmental stages [ , , , , ] . in the last years there has been an evolving trend in novel treatments for ms and the global progress of therapies for ms has been quite promising. in general treatments consist of ampyra ® , aubagio ® , avonex ® , betaseron ® , copaxone ® , extavia ® , gilenya ® , lemtrada ® , novantrone ® , plegridy ® , rebif ® , tecfidera ® and tysabri ® [ ] . such treatment options consist of alemtuzumab (depletes lymphocytes), daclizumab (blocks the cytokine receptor il- ), dimethylfumarate (combines features of immunomodulatory and immunosuppressive actions), fingolimod (modulates the sphingosine-receptor system), natalizumab (inhibits the migration of lymphocytes) and teriflunomide (inhibits activated t and b cells) [ , , ] . examples of current interferons include, schering ag's betaferon/betaseron (ifnβ- b), biogen's avonex (ifnβ- a) and serono/pfizer's rebif (ifnβ- a). in addition, immune modulating agents include, teva's copaxone ® (copolymer glatiramer acetate), amgen/serono's (novantrone ® ; mitoxantrone), azathioprine, cyclophosphamide (endoxan ® ) and natalizumab ® an a -integrin antagonist [ ] [ ] [ ] . disease-modifying agents have commonly been shown to reduce the rate of relapses, reduce mri lesions and stabilize or delay ms disability. the key therapeutic features of disease-modifying drugs are their anti-inflammatory effects in the relapsing phase of ms, although demyelination leading to chronic disability still remains a major hurdle [ , [ ] [ ] [ ] . some studies, however, have shown that early intervention of disease-modifying drugs to patients with rrms can reduce acute disability or death [ , [ ] [ ] [ ] [ ] . in general, disease-modifying drugs main action is by suppressing or altering the immune system. hence, based on this theory that ms is, at least in part, a result of altered or abnormal immune response that results in attack of the myelin sheath. current available drugs and their actions are described below (table , figure ). interferon (ifn) type consist of a group of ifns (ifn-α, -β, -ε, -κ, -τ, -δ, -ζ, -ω, -ν) which help regulate the immune system. ifn-β is primarily produced by fibroblasts but other cells such as nk cells, b cells, t cells, macrophages also secrete ifn-β. ifn-β has anti-viral and anti-tumor activity as well as being effective in reducing the relapse rate in patients with ms [ ] . the mechanism by which ifn-β acts, is that it balances the expression of pro-and anti-inflammatory cytokines in the brain and decreases the number of inflammatory cells crossing the blood brain barrier. as a consequence, there is decreased inflammation of neurons, increases nerve growth factors and improves neuronal survival. moreover, ifn-β reduces th population and il- cytokine which are known to be involved in the immunopathophysiology of ms [ ] . ifn-β injection subcutaneously or intramuscularly to patients with rrms aims to decrease the relapse rate, duration and severity, however, there is lack of efficacy to long-term disability. avonex was approved in , the first fda approved treatment for rrms. to date there are approaches using ifn-β; ifn-β a low dosage (avonex ® ), ifn-β a (rebif ® ) high dosage, and, ifn-β b (betaseron ® , extavia ® ) high dosage. furthermore, pegifn-β- a (plegridy ® ) has polyethylene glycol linked to ifn-β- a allowing it to be active for longer in the body, hence fewer injections are required compared to avonex ® , rebif ® , betaseron ® and extavia ® . the first large scale human clinical trial in patients with rrms using ifn-β was published in and showed that relapse rates were reduced by % in high dose ifn-β b and by % in lower dose compared to placebo group and severity of relapses were also reduced [ ] . subsequent year follow-up data showed that ifn-β a and ifn-β b decreased lesions up to % and reduced the formation of new lesions up to %, however, the study failed to show any reduction in disability progression in patients [ ] . ifns have no direct neuroprotective effects, however, through their direct effect on cd + th cells and altering their profile results in decreased demyelination of neurons, which prevents further neuronal damage [ ] . despite the impact of ifn-β in disease progression in patients with rrms there are limitations in their use, with side effects ranging from local body aches, skin reactions (swelling, redness), fever, myalgia, flu-like symptoms to more serious side effects such as suicidal thoughts, hallucinations, seizures and heart and liver problems [ ] . as a result, many patients have stopped treatment and overall the benefit of using ifns is relatively small. glatiramer acetate (ga) is a synthetic -mer peptide (l-glutamic acid, lysine, alanine, and tyrosine) mimic of mbp, which competes with short antigenic mbp peptides in complex with mhc class ii. initially, ga was designed to induce eae but instead it suppressed eae, which was quickly translated into human trials with ms in order to prevent disease progression, as it bound to mhc class ii and inhibited the activation of encephalitogenic t cells [ ] [ ] [ ] [ ] . ga diverts th cells to th cells that suppress inflammatory responses and activate tregs in the periphery [ ] . in patients, ga significantly reduced disease symptoms and development of new lesions by up to % in rrms, although it showed no improvement in long-term efficacy on progression of disability [ ] . ga injection in patients results in side effects ranging from minor (fever, chills) to more serious (cardiovascular, digestive, muscular, respiratory issues). dimethyl fumarate (bg- ) is a methyl ester of fumaric acid that modulates immune responses and was approved by the fda in . bg- was shown in phase iii clinical trials to reduce relapse rate and increase the time to disability progression in patients with rrms [ ] . bg- reduces the migration of inflammatory cells through the blood brain barrier and activates nuclear factor erythroid -related factor (nrf ) [ ] . nrf regulates anti-oxidative proteins that protect cells against oxidative damage and inflammation. in fact, bg- protects neuronal cells from oxidative stress by increasing glutathione levels and suppressing pro-inflammatory cytokines from splenocytes in vitro [ ] . side effects of bg- include diarrhea, abdominal pain, nausea, abnormal liver enzymes and decreased lymphocyte counts. teriflunomide is an active metabolite of leflunomide (an immunosuppressive disease-modifying drug used for rheumatoid arthritis) which inhibits the enzyme dihydroorotate dehydrogenase [ ] and inhibits the proliferation of b and t cells. in addition, teriflunomide exerts anti-inflammatory properties by inhibiting ifn-gamma producing t cells while il- and il- producing t cells are unaffected [ ] . in ms, oral administration of teriflunomide reduced relapse rates, ms lesions and decreased disability progression [ ] [ ] [ ] [ ] [ ] [ ] . moreover, permanent discontinuation due to side effects was substantially less common in ms patients who received teriflunomide compared to ifn-β- a. side effects include, reduced white blood cell count, alopecia, hepatic effects, nausea, diarrhea, numbness in hand and feet, allergic reactions, breathing issues and increased blood pressure. teriflunomide was approved by the fda in and by ema in for use in patients with rrms. fingolimod was granted fda approval in and was the first oral therapy ( . mg once daily) available for patients with relapsing forms of ms. fingolimod is a sphingosine -phosphate (s p) receptor modulator, which acts as a super agonist of s p receptor causing receptor internalization and leading to reduced infiltration of potentially auto-reactive lymphocytes into the cns, and as such, they remain localized in the lymph nodes [ ] [ ] [ ] . in addition, a secondary beneficial effects of fingolimod is that it targets sip receptors on glia cells in the cns, activating signaling pathways within the cns [ , ] . based on phase iii human clinical trials in patients with rrms (transforms, freedoms and freedoms ii), fingolimod was more effective compared to first line treatment ifnβ- a and placebo, in reducing the frequency of flare-ups (clinical exacerbations), disability progression, mri outcome measures, including brain volume loss and was associated with clearly identified adverse events [ , , ] . more than , patients have been treated with fingolimod in clinical trials and post-marketing settings globally, and the total patient exposure now exceeds , patient-years. side effects include bradycardia (within h after treatment initiation), blurred vision, diarrhea, back pain, headache, cough and vomiting. with reasonable data showing its long-term safety and disease improvement, fingolimod is a great alternative choice for patients with highly active rrms and who prefer the oral treatment option. . . . mitoxantrone (novantrone ® , immunex/amgen, thousand oaks, ca, usa) mitoxantrone is primarily used to treat certain types of cancers, in particular, non-hodgkin's lymphoma, acute myeloid leukemia, breast and prostate cancer. mitoxantrone is a type-ii topoisomerase inhibitor, which disrupts dna synthesis and dna repair of cancer cells, however, normal cells are also affected. it is a potent immune suppressant, suppressing t cells, b cells and macrophages and reduces pro-inflammatory cytokines (ifn-γ, tnf-α, and il- ) [ , ] . in patients with spms, intravenous injection of mg/m mitoxantrone every months up to years resulted in reduced disability progression by % [ , ] . however, several side effects are associated with mitoxantrone which range from nausea, vomiting, hair loss, to, cardiotoxicity, leukemia, infertility, infection, leukopenia and thrombocytopenia [ ] . as a result, its use has significantly been reduced over time. furthermore, due to the risk of cardiotoxicity and leukemia, there is a limit on the cumulative lifetime dose to be administered to patients [ , ] . natalizumab is a humanized monoclonal antibody against the cellular adhesion molecule α integrin. integrins are transmembrane receptors that enable cell-extracellular matrix adhesion activating cell signaling which regulate cell growth, division, survival, differentiation and migration. integrins are expressed on t cells, b cells, monocytes, macrophages, nk cells, dc, neutrophils and eosinophils. interfering or blocking α -integrin affects immune cell migration across the blood brain barrier, thus, by blocking the interaction between α -integrin and vascular endothelial adhesion molecule- , inhibits transendothelial migration to the cns [ ] . natalizumab is administered intravenously once a month [ ] which reduces activated t cells within the cns, resulting in anti-inflammatory responses and hence, neuroprotective effects [ ] . in a phase iii clinical trial natalizumab reduced brain lesions and the rate of disability progression up to months [ , ] . in addition, natalizumab decreased by % of contrast-enhancing lesions, by % of new or expanding t -weighted lesions, and by % in new t -weighted hypointense lesions [ , ] . natalizumab, was approved by the fda in , but was withdrawn due to cases of rare brain infection, progressive multifocal leukoencephalopathy (pml; that usually leads to death or severe disability), but was re-introduced in under a special prescription program. however, by a further cases (or . / ) of pml were reported to be attributed to natalizumab [ ] . despite these reports the fda has not withdrawn natalizumab from the market as the clinical benefits outweigh the risks involved. other side effects include, hepatotoxicity, allergic reactions and increased risks of infection. due to the risks involved with natalizumab, there are reservations over its use as a preferred treatment option. ofatumumab (omb ) is the first fully human type igg kappa (igg κ) monoclonal antibody and is currently licensed for the treatment (of patients with chronic lymphocytic leukemia (intravenously (iv), arzerra ® ). it has also been shown to be beneficial to patients with rheumatoid arthritis, follicular non-hodgkin's lymphoma, diffuse b cell lymphoma and ms. b cells play a role in the pathogenesis of ms. b cells have essential functions in regulating immune response, by activating cd + t-cells and regulating t-cell responses via the secretion of cytokines and antibodies. b cells are present at demyelinating areas and in cerebrospinal fluid of patients with ms [ ] . cd is a marker and present on the cell surface of all b cells. in an attempt to reduce the number of b cells including autoreactive b cells, the use of anti-cd antibodies would conceivably improve ms relapses and progression. in fact, there are several humanized anti-cd antibodies, such as rituximab [ ] , ocrelizumab [ ] and ofatumumab [ ] , which have shown high efficacy in patients with rrms. in , novartis acquired the rights from glaxosmithkline for the development of ofatumumab in oncology and other autoimmune indications. ofatumumab binds to unique novel epitopes on the cd molecule, induces b-cell depletion via complement dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity causing b cell apoptosis [ ] . ofatumumab has demonstrated high efficacy in hematologic malignancies and in rheumatoid arthritis. based on phase ii dosing human clinical studies, ofatumumab demonstrated high efficacy in reducing new mri lesion activity more than % and was well tolerated in patients with ms [ ] . currently, ofatumumab is a few months ago (march ), the fda approved ocrelizumab to be used in ppms, the first drug approved by the fda for this form of ms and phase iv clinical trials were a requirement of the fda to be conducted in order to determine the safety of ocrelizumab in younger patients with ms, ie, risk of cancer and effects on pregnancy (study outcomes due by ); although clinical trials in patients with lupus and rheumatoid arthritis were halted due to high rates of infections and increased risk of progressive multifocal leukoencephalopathy [ ] . in addition, in patients with ms, there was an increased risk of breast cancer ( / females with ms on ocrelizumab compared to / females with ms in other trials) [ ] alemtuzumab is a humanized monoclonal antibody against cd , a cell surface molecule expressed on b and t cells; mature nk cells, plasma cells, neutrophils and importantly, hematological stem cells do not express cd . in phase iii clinical trials in patients with rrms, alemtuzumab showed significantly lower annualized relapse rates and mri measures (gadolinium-enhancing lesions, new or enlarging t lesions and brain atrophy) and were free of clinical disease longer, compared to ifnβ- a [ , ] . alemtuzumab can cause serious side effects including, immune thrombocytopenia, kidney problems, serious infusion problems (trouble breathing, swelling, chest pain, irregular heart beat), certain cancers (blood cancers, thyroid cancer), cytopenia and serious infections. it was approved by the fda in to be used in rrms patients, but due to the frequent and significant adverse events of alemtuzumab, it is generally used in patients with rrms who have used or more ms drugs and have failed to work. daclizumab is a humanized monoclonal antibody against cd , the il- receptor expressed on the surface of t cells. the mechanism by which daclizumab works is that it blocks the il- receptor on t cells, preventing the activation of t cells. it was originally approved by the fda in to prevent acute kidney transplants (together with corticosteroids and cyclosporine) however its use was halted due to low market demand. in recent years its use has re-emerged to treat patients with rrms, it is injected subcutaneously, once a month [ ] . in human clinical trials, daclizumab showed % reduced annualized relapse rates and % lower in the number of new lesions [ ] . the side effects associated with daclizumab are relatively minor compared to other ms drugs, and include infections, skin rashes and liver complications. antigen/peptide specific immunotherapy or using immune cells (i.e., stems cells), aim to restore tolerance while avoiding the use of non-specific immunosuppressive drugs as describe in section , is a promising approach to fight autoimmune diseases including ms. as such, a number of approaches have been utilized. multipotent hematopoietic stem cells (hsc) are cells isolated either from the bone marrow, umbilical cord blood or peripheral blood and are transplanted into the recipient. more commonly used for hematological malignancies (leukemia, multiple myeloma) its application has also expanded into autoimmune diseases. the first report of a bone marrow transplant in in a chronic myelogenous leukemia patient with ms which showed marked improvements in ms brain lesions [ ] quickly led to the use of hsc transplantation (hsct) in ms patients. hsct in patients with active rrms, reduce progression in about % of patients, decrease relapses dramatically and suppresses inflammatory mri activity [ ] . ms patients who have not responded to conventional therapy, who's disease is aggressive with relapsing-remitting course and who are not presenting with high level of disability, are considered appropriate candidates for such treatment [ ] . although the clinical efficacy of hsct long term has not been established. the mechanism by which hsct works is that hsct "reboots" the immune system and thus, prevents inflammation associated with the disease. mesenchymal stem cells (msc) are isolated from an adult's bone marrow, are differentiated in vitro for - weeks and re-injected back into the patient. in recent years a vast amount of research has been conducted in mscs to treat ms with most studies being in mice and eae models, and more recently in human clinical trials. in fact, in a pilot study in advanced ms patients, msc transplantation improved expanded disability scale score with stabilization in / and disease progression in / patients and vision and low contrast sensitivity test showed improvement in / patients with / showing worsening effects [ ] . in a phase ii randomized double-blind, placebo-controlled crossover clinical trial showed lower mean cumulative number of lesions in patients receiving mscs compared to placebo [ ] . no serious adverse events were reported. the mechanism of action of msc includes immunomodulation, neuroprotection and neuroregeneration [ ] . the use of mscs that reduce mri parameters is a new and emerging research focus to develop new improved treatments for ms. bht- , a dna vaccine that encodes the full-length human mbp, was developed with the aim to tolerize patients with ms against mbp [ , , ] . in fact, in patients with rrms or spms who received injections of bht- on weeks , , , with escalating doses of . mg, . mg or mg was reported to be safe and conferred positive changes on brain mri and reduced the number of cd + t cells [ , , ] . in addition, in a retrospective, randomized double blind, phase ii study in ms patients, bht- had no impact on the risk for persistent black holes (axonal loss and disability progression). however, there was a correlation to those who had generated high anti-igm mbp antibodies to reduced risk of persistent black holes [ ] . nanoparticles have extensively been characterized and used as vaccine formulations in pre-clinical models of cancer and infectious diseases [ , ] . polymeric biodegradable lactic-glycolic acid (plga) nanoparticles loaded with mog - peptide together with recombinant il- , were partially endocytozed by dendritic cells, secreted both mog peptide and il- in culture media for several weeks in vitro [ ] . in mice, plga nanoparticles (mog - + il- ) showed significant amelioration of eae and reduction of il- and ifn-gamma secretion by splenic t cells in vitro [ ] . recently, poly(ε-caprolactone) nanoparticles loaded with recombinant human mbp reduced ifn-gamma cytokines, reduced the clinical score and showed only mild histological changes of the myelin sheath [ ] . hence, nanoparticles as a delivery method of self-antigens are a promising tool to treat ms. altered peptide ligands (apl) are peptides closely related to the native (agonist) peptide with defined - substituted amino acid residues which interact with the t cell receptor (tcr) yet retains its binding ability to the mhc [ ] . in phase i/ii clinical trial by neurocrine biosciences inc, used an apl of mbp − , where l-amino acids were changed to d-amino acids at positions , , , (nbi- ) [ ] . however, this mode of apl induced t cell cross reactivity between the apl and the wild-type/agonist mbp − peptide and adverse events in some patients resulted [ ] . a subsequent multi-center double-blinded phase ii clinical trial with nbi- was suspended-th responses were induced (il- , il- ), however, / patients developed immediate-type hypersensitivity, who also generated anti-nbi- antibodies which cross-reacted with native agonist mbp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] peptide [ , ] . national institute of neurological disorders and stroke sponsored trial, cgp , was used in a mri-controlled phase ii clinical trial. cgp , has ala d-amino acids of mbp - peptide at positions , , , (cgp ) of mbp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] peptide, in order to enhance stability [ ] . however, this peptide was poorly tolerated at the dose tested, and the trial had to be discontinued. three patients showed exacerbations to disease of which two were linked to cgp injection with high ifn-gamma and low il- (th -skewing) were secreted by activated cd + t cells. these cd + t cells also cross reacted with the native agonist mbp - peptide [ ] . accordingly, the problems noted with both nbi- and cgp were likely due to inadequate pre-screening of apl effects on the many clonotypes against the targeted epitopes. thus, although the apl was highly effective at blocking or switching some clones, it activated others. thus, further pre-clinical testing is required and new modified peptides need to be designed, or a carrier needs to be used which further changes the resulting immune response. cyclization of peptides increases the stability, since linear peptides are sensitive to proteolytic enzymes. in addition, cyclic peptides are an important intermediate step and a useful template towards the rational design and development of non-peptide mimetics. while mimetic strategy is a challenging perspective it is worth pursuing in particular for mbp epitope-based ms therapy as it is still in its infancy. efforts to design semi-mimetics of mbp - epitope by combining non-natural amino acids as spacers and mbp epitope immunophores (ser, arg, glu, ala, gln), led to substances that were effective to some extent in inducing the onset of eae. cyclic peptides are not only as a step towards non-peptide mimetics but also as putative therapeutics in ms [ ] . structure activity studies of the immunodominant agonist peptide mbp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] showed significantly reduced mechanical pain hypersensitivity compared to cyclic mbp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] peptide. this was associated with reduced t cell and macrophage infiltration to injured nerves of the spinal cord of animals [ ] [ ] [ ] . in addition, these apl decreased cd + t cell line proliferation raised from a patient with ms, increased il- cytokine secretion, bound to hla-dr and were more stable to lysosomal enzymes (cathepsin b, d, h) compared to their linear counterparts [ ] . double mutation of k , p to a , a in either linear or cyclic forms were also shown to be active, with suppression of eae in sjl/j mice, higher th over th cytokines produced, bound to hla-dr , the cyclic forms were more stable to lysosomal enzymes and induced high levels of il- of peripheral blood mononuclear cells from patients with ms [ ] . recently, cyclic native agonist mog peptide was shown to ameliorate clinical and neuropathological features of eae in mice compared to its linear counterpart [ ] . thus, cyclic peptides, which offer greater stability and are able to modulate immune responses, are novel leads for the immunotherapy of many diseases, such as ms [ ] . mannan, a polymannose, isolated from the wall of yeast cells has been shown to bind to the mannose receptor on dendritic cells as well as being a ligand for toll-like receptor [ , ] . mannan conjugated to muc cancer protein induces immune responses in mice and protects mice against tumor challenge. this work was translated into human phase i, ii and pilot iii clinical trials; mannan-muc induces protection against cancer recurrence at years follow-up [ ] [ ] [ ] [ ] . furthermore, ex vivo cultured dendritic cells pulsed with mannan-muc (cvac tm ) and re-injection into patients induces strong cellular and clinical responses in ovarian cancer patients [ , ] . due to the immunomodulatory properties of mannan, its effects as a carrier to ms peptides were determined. mutations of mbp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] peptides stimulated ifn-gamma secreting t cells [ ] . furthermore, mannan conjugated to the immunodominant agonist mog - peptide primes non-pathogenic th and th cells and ameliorates eae in mice [ ] ; a phase i human clinical trial is planned using mannan conjugated to mog peptide later this year. it is clear that, mannan is able to divert immune responses from th to th and is a promising carrier for further studies for the development of immunotherapeutics against ms. dalfampridine (ampyra/fampyra ® , acorda therapeutics) dalfampridine is not intended to delay symptoms or change the course of disease, but rather, to improve motor symptoms such as walking. dalfampridine, is a potassium channel blocker, resulting in improved potassium currents and nerve conductance. dalfampridine is used in patients who have had ms for more than years and it was approved by the fda in . common side effects include nausea, nervousness and dizziness, which are relatively minor compared to other ms drugs. ms is an autoimmune disorder of the cns with an array of immune cells being either activated or suppressed leading to demyelination and disease progression. in addition, genetic predisposition, viral mimicry, vitamin and mineral deficiency, geographical location are also etiological factors that contribute to disease. more recently, citrulination of myelin peptides have been shown to contribute to disease activation [ , ] . a number of treatment options are available to patients with ms, in particular those with active disease, however due to side effects, limited long term effectiveness and 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features of experimental autoimmune encephalomyelitis oxidative/reductive conjugation of mannan to antigen selects for t or t immune responses cell-mediated immune responses to muc fusion protein coupled to mannan pilot phase iii immunotherapy study in early-stage breast cancer patients using oxidized mannan-muc dendritic cell immunotherapy: clinical outcomes antibody and t cell responses of patients with adenocarcinoma immunized with mannan-muc fusion protein up to -year clinical follow-up of a pilot phase iii immunotherapy study in stage ii breast cancer patients using oxidized mannan-muc mannan-muc -pulsed dendritic cell immunotherapy: a phase i trial in patients with adenocarcinoma a phase , single-arm study of an autologous dendritic cell treatment against mucin in patients with advanced epithelial ovarian cancer mannosylated linear and cyclic single amino acid mutant peptides using a small amino acid linker constitute promising candidates against multiple sclerosis design and synthesis of non-peptide mimetics mapping the immunodominant myelin basic protein (mbp - ) epitope to function as t-cell receptor antagonists this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -cgiok ce authors: binjawadagi, basavaraj; dwivedi, varun; manickam, cordelia; ouyang, kang; torrelles, jordi b; renukaradhya, gourapura j title: an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination date: - - journal: int j nanomedicine doi: . /ijn.s sha: doc_id: cord_uid: cgiok ce porcine reproductive and respiratory syndrome (prrs) is an economically devastating respiratory disease of pigs. the disease is caused by the prrs virus (prrsv), an arterivirus which is a highly mutating rna virus. widely used modified live prrsv vaccines have failed to prevent prrs outbreaks and reinfections; moreover, safety of the live virus vaccines is questionable. though poorly immunogenic, inactivated prrsv vaccine is safe. the prrsv infects primarily the lung macrophages. therefore, we attempted to strengthen the immunogenicity of inactivated/killed prrsv vaccine antigens (kag), especially in the pig respiratory system, through: ) entrapping the kag in biodegradable poly(lactic-co-glycolic acid) nanoparticles (np-kag); ) coupling the np-kag with a potent mucosal adjuvant, whole cell lysate of mycobacterium tuberculosis (m. tb wcl); and ) delivering the vaccine formulation twice intranasally to growing pigs. we have previously shown that a single dose of np-kag partially cleared the challenged heterologous prrsv. recently, we reported that np-kag coupled with unentrapped m. tb wcl significantly cleared the viremia of challenged heterologous prrsv. since prrsv is primarily a lung disease, our goal in this study was to investigate lung viral load and various immune correlates of protection at the lung mucosal surfaces and its parenchyma in vaccinated heterologous prrsv-challenged pigs. our results indicated that out of five different vaccine-adjuvant formulations, the combination of np-kag and unentrapped m. tb wcl significantly cleared detectable replicating infective prrsv with a tenfold reduction in viral rna load in the lungs, associated with substantially reduced gross and microscopic lung pathology. immunologically, strong humoral (enhanced virus neutralization titers by high avidity antibodies) and cell-mediated immune responses (augmented population of interferon-γ secreting cd (+) and cd (+) lymphocytes and reduced secretion of immunosuppressive cytokines) in the lungs were observed. in conclusion, combination of np-kag and soluble m. tb wcl elicits broadly cross-protective anti-prrsv immunity in the pig respiratory system. porcine reproductive and respiratory syndrome (prrs) is an economically devastating disease in pigs causing an estimated direct loss of greater than $ million annually to the us pork industry. prrs is caused by prrs virus (prrsv), an enveloped positive-sense rna virus belongs to the family arteriviridae. there are broadly two distinct prrsv genotypes, the european (type i) and the north american (type ii), dovepress dovepress binjawadagi et al which possess a wide range of intra-and intergenotypic, genetic, and antigenic diversity. therefore, developing preventive measures to control prrs outbreaks has been a challenge to the global swine industry. though both modified live virus (mlv) and inactivated prrsv vaccines have been in use since , control of disease outbreaks has remained unsuccessful. live virus vaccines are successful in reducing the clinical disease, but are invariably implicated in spreading the mutated viruses to susceptible pigs. in contrast, available inactivated prrsv vaccines are safe, but they have failed to elicit protective immunity even against homologous infections. in addition, killed vaccine antigens do not undergo intracellular antigen presentation pathways to induce a strong cytotoxic t-cell (ctl) response, which is necessary for clearance of intracellular pathogens like viruses. [ ] [ ] [ ] thus, research aimed at developing better cross-protective inactivated prrsv vaccines is warranted. therefore, several innovative strategies should be adopted to strengthen potency and efficacy of inactivated/killed prrsv vaccine antigens (kag), with respect to suitable methods of viral inactivation and purification, use of potent adjuvants, route, and efficient delivery of vaccine to protect ags from rapid enzymatic degradation in the body. since prrsv infects primarily the pig respiratory tract and the target cells are lung alveolar and interstitial macrophages, induction of strong local mucosal immunity in the respiratory tract is important. the intranasal route of delivery of vaccines to control primary respiratory infections has shown great promise in induction of protective mucosal (ie, local) as well as systemic immunity. , , poly(lactide-co-glycolide) (plga) is a synthetic biodegradable polymer used successfully in particulate delivery of inactivated vaccines. [ ] [ ] [ ] the adjuvant mycobacterium tuberculosis whole cell lysate (m. tb wcl) was shown to augment immunogenicity of both live prrsv vaccine and plga-nanoparticles entrapped with killed prrsv antigens (np-kag) without causing any side effects in pigs [ ] [ ] [ ] [ ] and with other vaccines in rodents, guinea pigs and rabbits. , unlike complete freund's adjuvant (cfa), m. tb wcl is free from water-insoluble toxic cell wall components of the bacterium, , and it is endotoxin free and contains only water-soluble components. therefore, unlike cfa, m. tb wcl does not cause any toxicity or granulomatous lesions at the site of inoculation. previously, we have shown that a single dose of prrsv kag-entrapped in plga ( : ) nanoparticle (np-kag) elicits both mucosal and systemic immune responses. , recently, np-kag coadministered intranasally twice with m. tb wcl induced cross-protective anti-prrsv immune response in blood to a challenged heterologous prrsv, associated with a significant reduction in viremia. in this report, we made use of various types of the lung samples of that recent study to evaluate viral load and local mucosal immunity both at airway surfaces and in the lung parenchyma, and also microscopic lung histopathology in vaccinated, heterologous prrsv-challenged pigs. killed prrsv vaccine antigens (kag) were prepared as described earlier. briefly, north american prototype prrsv strain vr was grown in marc cells, freeze-thawed three times, and the harvested cell culture supernatant was subjected for clarification followed by ultracentrifugation to pellet the virus. resuspended pellet in sterile phosphate-buffered saline (pbs) was subjected to ultraviolet inactivation ( nm for hour) to prepare kag for use in vaccine preparation. for restimulation experiments, similarly prepared kag of the challenge virus, prrsv strain mn , was used. preparation of whole cell lysate of m. tb m. tb was grown in agar medium and wcl was prepared as described previously. briefly, h rv strain of m. tb was grown in oleic acid-albumine-dextrose-catalase enriched h (difco) agar plates (becton, dickinson and company, franklin lakes, nj, usa). the bacterial cells were harvested by centrifugation of colony scrapings at ×g and washed with pbs (ph . ). live bacterial cells were suspended [ g (wet weight)/ml] in pbs containing mm ethylenediaminetetraacetic acid (edta) (becton, dickinson and company), proteinase inhibitors (emd millipore, billerica, ma, usa), dnase and rnase (sigma-aldrich, st louis, mo, usa), and the bacterial cell wall was disrupted by bead beater until . % cell breakage was obtained (confirmed by acid fast staining). the cell lysate was centrifuged at ×g to pellet unbroken cells and insoluble broken cell wall components. the clear supernatant-containing water soluble fraction of the bacterium was harvested and sterilized through . µm low protein binding membrane filter. further, endotoxin levels in every batch of m. tb wcl was confirmed to be less than the acceptable levels (, . µg/mg conventional large white-duroc crossbred - weeks old weaned pigs were procured from a swine herd seronegative for prrsv, porcine respiratory coro navirus, transmissible gastroenteritis virus, and porcine circo virus -specific antibodies. a total of pigs were randomly divided into one of the ten groups (n= pigs/group) and vaccinated with the indicated vaccine formulation, intranasally ( ml/pig), twice at -week intervals ( table ). all the vaccinated pigs were intranasally challenged on postvaccination day with a virulent heterologous north american prrsv (type ii) strain mn ( × median tissue culture infective dose [tcid ]/pig). adjuvant and vaccine were entrapped separately and combined before administering to pigs, and the dose of adjuvant ( mg/dose/pig) and kag were either (low dose) or (high dose) µg/dose/pig of semipurified viral protein containing ∼ . × or . × tcid of killed prrsv, respectively, either entrapped in nps or unentrapped. the vaccine and adjuvant doses were tested to be efficacious in pigs earlier. , pigs were monitored daily for the respiratory symptoms, and rectal temperatures and body weights were recorded every third day postchallenge (pc); animals were euthanized on day pc as per the approved protocol of the institutional animal care and use committee, the ohio state university, and indicated samples were collected during the necropsy. the lungs were examined for gross lesions in all the lobes. the lung samples collected from the right cranial lobe were fixed in % neutral buffered formalin and sections ( µm) were made and stained with hematoxylin and eosin as described previously. the slides were examined by an unbiased certified veterinary pathologist to score prrsvinduced inflammation. collection of bronchoalveolar lavage fluid, preparation of lung homogenate, and isolation of lung mononuclear cells during necropsy, the lungs were harvested and the bronchoalveolar lavage (bal) fluid was collected by washing the airways using - ml of cold pbs containing antibiotics and edta; the harvested fluid was centrifuged at , ×g for minutes at °c and the clarified bal fluid was aliquoted and stored at - °c. the lung homogenate was prepared as described previously. briefly, gram of lung tissue from the right cranial lobe of every pig was collected in ml ice-cold dulbecco's modified eagle's medium and minced and homogenized using a stomacher laboratory blender (seward limited, worthing, west sussex, uk) for minutes, and the clarified supernatant (lung homogenate/lung lysate) was aliquoted and stored at - °c. the lung mononuclear cells (lmncs) from the individual pig lungs were isolated by treating the perfused and minced lung tissue using collagenase and dnase as described previously. estimation of total immunoglobulin (ig) total isotype specific pig ig levels were estimated by elisa as described previously, with a few modifications. briefly, -well enzyme-linked immunosorbent assay (elisa) plates were coated with pretitrated dilution of ( : , ) of goat the assay was performed as previously described. avidity of the prrsv-specific antibodies in the lungs prrsv-specific antibody avidity was determined as described previously, , with a few modifications. briefly, bal fluid ( : ) and lung homogenate ( : ) samples were added to prrsv-ag-coated and -blocked plates. after hours incubation of test samples at room temperature, washed plates ( ×) were treated with serially twofold diluted ammonium thiocyanate (nh cn) ( µl/well) solution at to . mol concentration and incubated at room temperature for minutes. the plates were washed ( ×) and the remaining steps of elisa carried out as described above. for calculation purpose, od value of the test sample from nh cn-untreated ( m) well was considered as % absorbance (contributed by prrsvspecific antigen-antibody interaction), and the od value of a test sample at different molar concentration of nh cn was used to calculate the percent remained antigen-antibody interaction compared to its % absorbance value. estimation of prrsv-specific igg and igg antibody subtypes total prrsv-specific igg and igg subtypes in the lung homogenate samples were analyzed as described previously, with a few modifications. briefly, kag-coated plates were blocked and a serial tenfold diluted lung homogenate was added; mouse antipig igg or igg (abd serotec, raleigh, nc, usa) ( : dilution) secondary antibody was added to detect virus-specific igg or igg subtype, respectively. further, the washed plate was treated with goat antimouse igg-hrp (sigma-aldrich) ( : , dilution). the reaction was developed using tmb ( , ', , '-tetramethylbenzidine) substrate (kpl, gaithersburg, md, usa), stopped using m phosphoric acid, and the plates were read at od nm . for calculation of the prrsv-specific igg and igg antibody levels of test samples, the od values obtained at : dilution was considered. determination of prrsv-specific inter feron gamma (ifn-γ ) secreting cells by enzyme-linked immunospot (elispot) assay the elispot assay was performed as described previously. adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs lated were included as positive and negative controls, respectively. lung homogenates were analyzed for pig cytokines, th (ifn-γ and interleukin [il- ]), proinflammatory (il- ), and immunosuppressive (il- and transforming growth factor beta [tgf-β]) by elisa as described previously. flow-cytometric analyses the phenotypes and frequencies of lymphoid and myeloid cells populations from , events of immunostained lmncs were determined by flow cytometry as described previously. for intracellular ifn-γ staining, monensin (golgiplug, bd biosciences) was added during the last hours of the -hour incubation of lmncs that were unstimulated or stimulated with prrsv mn kag as described above. lmncs were first immunostained using pig lymphocyte-specific monoclonal antibodies (cd ε, cd α, cd α, cd , and tcr n ) conjugated with different fluorochromes. cells were fixed with % paraformaldehyde and permeabilized with a cell-permeabilization buffer ( . % deionized water, % pbs with no ca or mg, % formaldehyde solution, and . % saponin) overnight at °c. cells were washed and stained with a fluorochrome-conjugated antipig ifn-γ or its isotype control monoclonal antibodies (bd biosciences) in . % saponin containing fluorescence activated cell sorting (facs) buffer. immunostained lmncs were acquired using a facs aria ii (bd biosciences) flow cytometer and analyzed using the flowjo software (treestar inc., ashland, or, usa). all the specific immune cell frequencies were presented as the percent of total lymphocytes or myeloid cells. determination of prrsv load, virus-neutralizing antibody titer, and rna copies prrsv titer and virus-neutralizing (vn) titer in bal fluid and lung homogenate samples were analyzed by indirect immunofluorescence assay as previously described. extracted rna was reverse transcribed into complimentary deoxyribonucleic acid (cdna) using quantitect reverse transcription kit (qiagen, venlo, the netherlands). the cdna was subjected to quantitative real-time polymerase chain reaction (qpcr) using primers against prrsv orf in perfecta sybr green fast mix (quanta biosciences, gaithersburg, md, usa) with forward primer (gataac-cacgcatttgtcgtc) and reverse primer (tgccgtt-gttatttggcata). standard curve was generated using serial tenfold dilution of prrsv vr stock virus starting at tcid per µl for viral rna quantification. the data were expressed as the mean ± standard error of mean of three pigs. statistical analyses were performed by one-way analysis of variance followed by tukey's multiple comparison test (or unpaired t-test for figure binjawadagi et al cated that sham or entrapped np particles were spherical with a smooth surface. dynamic light scattering of nps indicated the mean diameter ± standard deviation was ± nm for sham, ± nm for np-kag, and ± nm for np-m. tb wcl, respectively. however, all nps possessed a uniform surface electrostatic potential of - mv. other in vitro studies revealed that the entrapped antigen was pulse-released over a period of several weeks in normal physiological conditions, and was efficiently uptaken by porcine alveolar macrophages. a detailed physical and biological characterization of np-kag vaccine used in this study was published recently. in the serum of all the virus-challenged pigs (groups to ), irrespective of vaccination history at weeks postchallenge, a twofold to threefold increase in total igg amount was observed (data not shown). the total igm and iga amounts in the lung homogenate were comparable in most of the prrsvchallenged pig groups ( figure a and c). however, the levels of igg (both in lung homogenate and bal fluid) ( figure b and e), and igm and iga (in bal fluid) ( figure d and f) in pig groups to were four-to fivefold higher compared to mock enhanced prrsv-specific antibody response in adjuvanted np-kag-vaccinated pigs prrsv-specific iga response was significantly higher both in the bal fluid and lung homogenate of both low ( µg/pig) and high ( µg/pig) doses of adjuvanted np-kag-vaccinated pigs, compared to most of the vaccine trial groups ( figure g and h). in contrast, levels of virusspecific igg were comparable in the bal fluid of all the virus-challenged pig groups ( figure i) . surprisingly, in the lung homogenate, significantly higher levels of virusspecific igg was observed in group and pigs vaccinated with either dose ( figure j ). this suggests that in adjuvanted np-kag-vaccinated pigs (group ), virus-specific iga is the major antibody isotype in the airway surfaces (bal fluid), while both iga and igg isotypes appear to play important roles in the lung parenchyma. the binding strength of heterogeneous antibodies to their cognate ag is defined as avidity. in adjuvanted np-kagvaccinated pigs, increased avidity of virus-specific iga was detected in both bal fluid (both low and high doses) and lung homogenate (only low dose) samples compared to other tested groups (figure a-c) . interestingly, the virus-specific iga avidity in lung homogenate of pigs receiving the highdose vaccine was comparable among all the tested pig groups ( figure d ). comparable levels of avidity of prrsv-specific igg antibody response was observed in lung homogenate of all the tested groups (data not shown). these data suggested that enhanced avidity of prrsv-specific iga antibody persisted for a relatively longer period at the airway surfaces of adjuvanted np-kag-vaccinated pigs. balanced th and th antibody responses in adjuvanted np-kag-vaccinated pigs t helper type (th) -or th -biased immune response is measured by quantifying antigen-specific igg antibody subtypes. in case of pigs, higher levels of igg and igg indicate th and th -biased responses, respectively. our results indicated that in group pigs, comparable levels of both igg and igg subtypes were secreted in lung homogenate ( figure a and c). the ratio of igg :igg , if greater or less than one, indicates th -or th -biased response, respectively. in the lungs of adjuvanted np-kag-vaccinated pigs at day pc a balanced th and th response (ratio close to one) was detected ( figure b and d). in pig groups and , although the ratio was close to one, the detected amount of virus-specific igg and igg subtypes were low ( figure a-d) . enhanced prrsv-neutralizing antibody response in adjuvanted np-kag-vaccinated pigs except group (mock), all the other pigs were challenged using a heterologous prrsv (strain mn ), which is genetically highly divergent (∼ %) compared to the vaccine strain vr . prrsv-specific vn titers were analyzed both in the bal fluid and lung homogenate samples against mn strain, and against another variant type strain, prrsv - - (accession # - ), is genetically distinct from both mn and vr strains. in addition, vn titers were also analyzed against an antigenically highly divergent (∼ %) heterogenotypic (type ) prrsv strain, sd - ( figure e -j). in group pigs, the lung vn titers against mn were significantly higher with mean titers of and with low and high vaccine doses, respectively, compared to other tested groups ( figure e and f). the vn titers against both prrsv - - and sd - strains were significantly greater in group pigs receiving the high-dose vaccine compared to group and group , respectively ( figure h and j). the kag and soluble wcl-vaccinated pigs (group ) also had significantly higher vn titer against only prrsv - - strain compared to group animals ( figure h ). these results indicated that soluble m. tb wcl-adjuvanted np-kag vaccine elicits broadly cross-reactive vn titers. the vn titers observed in bal fluid were low (data not shown). a standard reference for measuring cmi response against prrsv is by determining the frequency of virus-specific iscs by elispot assay. in low-dose vaccinated group pigs, significantly increased iscs in lmncs was detected compared to three other tested groups ( figure a ) and compared to all the tested groups in the high-dose category ( figure g ). the quantity of ifn-γ detected in the lung homogenate was significantly higher in group pigs compared to group (low dose) and groups and (high dose) categories ( figure b and h) . another important th cytokine, il- , was not significantly modulated among the tested pig groups in the high vaccine dose category ( figure j ), but in low-dose groups and , significantly higher levels of il- were detected compared to groups , , and ( figure d ). one of the important proinflammatory cytokines, il- , was significantly reduced in the lungs of all the vaccine trial groups compared to mock-challenged animals ( figure c and i). cytokines il- and tgf-β are immunosuppressive in nature, and they play a vital role in prrsv pathogenesis. , the quantity of tgf-β was significantly reduced in group pigs compared to group ( figure e and k) , and the quantity of il- among all the tested vaccine trial pig groups was comparable ( figure f and l). figure a and b) . the frequencies of intracellular ifn-γ + cells in lmncs either unstimulated or stimulated with mn ags identified the virus-specific memory lymphocyte response. significantly increased prrsvspecific recall ifn-γ response was detected both in cd + and cd + lymphocyte subsets of group pigs vaccinated with a high dose of vaccine ( figure c and d) . these data suggest that both t-helper and ctls were potentially primed in the lungs of only group pigs. when prrsv-specific ifn-γ secreting lymphocyte subsets in only restimulated cells were compared among different vaccine trial groups, only in group animals (both vaccines doses) significantly increased cd + cd -ifn-γ + cells compared to group animals ( figure f and n) , while cd -cd + ifn-γ + cell frequency was significantly enhanced in group pigs were present compared to groups , , and with low vaccine dose and groups to with high vaccine dose ( figure g and o). similarly, cd + cd + ifn-γ + and ifn-γ + γδ t cells were significantly higher in group pigs compared to pigs in groups , , and ( figure h -i, p-q). an increased frequency of activated γδ t cells was detected in group pigs compared to other groups (table a and b) . although there was no significant difference in total natural killer (nk) (cd + ) cell frequency ( figure j and r) , an increase in ifn-γ + nk cell frequency was significant in group pigs compared to other tested groups ( figure k and s) . in addition, macrophage (cd + cd + slaii + ) and dendritic cells (cd + cd c + slaii + ) rich apc populations were significantly higher in group and pigs in the high-dose vaccine category compared to group animals ( figure t and u); in the low-dose category, though a similar trend was detected, the data was not statistically significant ( figure l and m). in the bal fluid of pig groups and (low vaccine dose), detectable replicating prrsv was relatively reduced (but not significant) compared to other vaccine groups ( figure a ); in the same pig groups with the high vaccine dose, detectable replicating virus was absent, and the data was statistically significant compared to pig group ( figure b ). in the lung homogenate of group pigs receiving the low-dose vaccine, detectable replicating virus load was significantly reduced compared to mock-challenged pigs ( figure e) ; detectable virus was absent in the high-dose group pigs, and the data was statistically significant compared to pig groups and ( figure f ). further, prrsv rna copy numbers in bal fluid and lung homogenate were quantified by qpcr. in both the lung sample adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs types of group and pigs in the low-dose vaccine category, reduction (but not significant) in viral rna copy numbers was observed compared to other vaccine trial groups ( figure c and g); and in the same pig groups vaccinated with a high dose, a significant reduction ( - fold) in rna load was detected compared to group animals ( figure d and h). gross lung lesions revealed marked consolidation in pig groups , , and , while the group pig lungs were comparable to mock animals and the group pig lungs were in between mock and the group animals. microscopic lmncs isolated on the day of necropsy were unstimulated or stimulated with killed prrsv mn ags and immunostained using a combination of indicated pig lymphocytespecific cell surface markers followed by intracellular ifn-γ and analyzed by flow cytometry. representative histograms showing stimulated total lymphocytes in lmncs with intracellular ifn-γ + (a and b) . the dotted line: isotype control and solid line: ifn-γ + -specific staining. unstimulated (clear bars) or stimulated (black bars) lmncs with killed prrsv mn ags were analyzed for total ifn-γ + cd -, cd -, and cd -expressing lymphocytes (c-e). only stimulated lmncs were compared for indicated ifn-γ + lymphocyte subsets: cd + cd -ifn-γ + (f and n) ; cd -cd + ifn-γ + (g and o); cd + cd + ifn-γ + (h and p); γδ + ifn-γ + (i and q); and total nk (cd + ) (j and r); cd + ifn-γ + cells (k and s). also immunostained for potential apcs; macrophage-rich (cd + cd + sla-ii + ) (l and t) and dendritic cell-rich (cd + cd c + sla-ii + ) (m and u) populations. each bar indicates the average frequency of indicated cells from three pigs ± standard error of mean. asterisk indicates statistically significant (p, . ) difference between the two indicated pig groups. the unpaired t-test was applied to compare the data of only (c-e); for the other data, one-way anova followed by tukey's t-test was used. figure i ii, iii, and v). these data were consistent with the gross lung lesions, especially in pig groups and ; also, these pig groups had irregular fever with reduced feed intake during first week postchallenge. in contrast, the absence of any clinical prrs disease and gross lung lesions in group pigs was associated with the absence of detectable microscopic lung pathology, and the lung architecture was comparable to mock pigs ( figure i vi) . the other pig group which had moderately reduced lung lesions was group (kag + m. tb wcl) ( figure i iv), suggesting the adjuvant mediated protective immune response in pigs. similar but severe lung pathology was observed in pigs receiving the low vaccine dose in groups , , and (data not shown). potent mucosal vaccines against pathogens which predominantly cause disease at mucosal sites have been proven efficacious, with vaccines against influenza, parainfluenza, adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs respiratory syncytial virus, rotavirus, and hiv/siv. [ ] [ ] [ ] [ ] [ ] since prrsv primarily infects the lungs of pigs, its effective control appears to be possible by induction of strong respiratory mucosal immunity. thus, development of a novel prrsv mucosal vaccine is warranted. further, among the mucosal routes, intranasal delivery of np-based vaccine elicits a higher and longer duration of igg and iga antibody responses compared to rectal, oral, or intramuscular routes. in the lungs, prrsv infects both alveolar and interstitial macrophages, and greater than % of bal cells are macrophages. we investigated anti-prrsv response both in bal cells and lmncs. cd c + apcs are richly present in the lungs in both bal cells and lmncs, but they differ in their antigen presentation potential. apcs present in the lung mucosal surfaces (represented by bal cells) activate only antigen-primed t-cells, while apcs in the lung parenchyma (represented by lmncs) activate both naïve and antigen-primed t-cells. therefore, our study comprehensively investigated overall immune responses and viral load in the lungs of pigs vaccinated and challenged through the intranasal route. a couple of earlier studies showed hypergammaglobulinemia in the serum of prrsv-infected pigs, indicated by a four-to fivefold increase in total igg levels at - weeks postinfection. , consistent with that observation, in our study, an approximately fourfold increase in total igg amounts was observed both in the serum and lung samples (bal fluid and lung homogenate); however, in the lung homogenate of adjuvanted np-kag-vaccinated pigs, the total igg levels were only twofold more compared to mock animals. these data suggest that adjuvanted np-kag has a strong positive influence on humoral response, further indicated by a significant increase in production of anti-prrsv antibodies and virusspecific high avidity vn antibody titers. inactivated vaccines are safe, but suitable adjuvant and delivery system are critical to boost their efficacy. several unsuccessful attempts were made to develop protective killed prrsv vaccines. , a recent study using uv-or bei-inactivated prrsv ( × tcid per dose) coadministered with either freund's incomplete or suvaxyn oil/water adjuvant elicited the vn titer of greater than associated with partial clearance of homologous viral challenge. in that study, cmi response was not investigated. plga is the food and drug administration (fda)-approved agent, and plga-based intranasal delivery of prrsv vaccine was found safe in pigs. , , one of the strategies to augment uptake of particulate antigens by apcs is by increasing its surface hydrophilic nature of the particles by coating with a nonionic surfactant, polaxamer . , efficient th -immunity-inducing adjuvants are critical to promote a strong cmi response to subunit and inactivated virus vaccines. the water-soluble components of m. tb wcl, such as heat shock protein- and pro-glu/ppe, are potent adjuvants. in addition, four other water-soluble components in m. tb wcl, such as short-and long-chain poly-peptidoglycans, acetylated peptidoglycans, and tetrasaccharide-heptapeptide, have potent adjuvant effects comparable to cfa in inducing production of antibodies in rabbits coadministered with an inactivated influenza virus vaccine. plga-np vaccine coadministered with a potent adjuvant elicits a protective immune response. we demonstrated potent adjuvant effects of m. tb wcl to prrs-mlv, [ ] [ ] [ ] , and also to np-kag, with no side effects. our initial studies using a single dose of np-kag elicited partial cross-protective immune response in pigs. , to potentiate the efficacy of np-kag vaccine, in a recent study np-kag was evaluated by coadministering intranasally twice with either entrapped or unentrapped m. tb wcl. our results suggested that combination of np-kag with unentrapped m. tb wcl significantly cleared the challenged heterologous virus from the circulation, supported with strong humoral and cmi responses in the blood. the enhanced t-and b-cell responses in adjuvanted np-kag-vaccinated pigs were attributed to concerted effects of both plga and m. tb wcl. induction of strong local mucosal immunity and clearance of heterologous prrsv from vaccinated pig lungs is important for effective control of prrs. therefore, in this study we investigated the immune responses exclusively both at mucosal surfaces and parenchyma of pig lungs. our results indicated that adjuvanted np-kag (group pigs) did potentiate anti-prrsv immune response in the lungs, as indicated by the following parameters: ) increased prrsv-specific igg and iga response with enhanced antibody avidity and vn titers, and balanced th and th immune responses; ) upregulated secretion of th (il- and ifn-γ) and downregulated immunosuppressive (tgf-β and il- ) cytokines; ) enhanced frequency of iscs and ifn-γ producing cd + , cd + , cd + cd + t cells, γδ + t cells, and nk cells, and expanded frequency of apcs; and most importantly, ) complete clearance of detectable replicating challenged heterologous prrsv and tenfold reduction in viral rna load in the lungs. further, the microscopic lung lesions ( figure i ) strongly supported the observed virus clearance and immunological responses. in our previous study, in pigs immunized with the adjuvanted np-kag, increased frequency of only cd -cd + ifn-γ + cells in restimulated pbmcs was detected, but in stimulated lmncs, increased populations of both cd + cd -ifn-γ + and cd -cd + ifn-γ + cells was observed, perhaps indicating the induction of both t-helper and ctl memory responses in the lungs. like in pbmcs, increased but comparable frequency of other ifn-γ + lymphocyte subsets in both restimulated and unstimulated cells was observed in lmncs of group pigs, suggesting that other t-effector and nk cells were actively secreting ifn-γ in the lungs to prrsv-challenge infection. due to lack of similar data on ifn-γ + lymphocytes in vaccinated pigs isolated prior to challenge, it is difficult to demarcate the vaccine-alone induced response. np-based delivery of vaccine facilitates affinity maturation and activation of b cells, leading to high avidity antibody production and also cmi response. , avidity of prrsvspecific iga antibody isotype was significantly higher in the bal fluid of adjuvanted np-kag-vaccinated pigs. in the lung homogenates of low-dose (but not high-dose) adjuvanted np-kag-vaccinated pigs, high avidity iga response was observed; the reason for this discrepancy could be the time of lung sample collection. overall, our results suggested that virus-specific functional iga response at mucosal tissues (lungs) was enhanced in adjuvanted np-kag-vaccinated pigs. further, our results indicated that in the lung mucosal surfaces, iga isotype appears to play an important role, while in the lung parenchyma both iga and igg isotypes contribute to protective immune response. vn antibodies against putative neutralizing epitopes on prrsv gp and m glycoproteins play an important role in prrsv clearance. in group pigs, high levels of cross-reactive vn titers were detected against a challenged prrsv mn strain, another heterologous type ii viral strain, and most importantly-even against a highly variant heterogenotypic strain (prrsv sd - ), confirming the broadly cross-protective nature of adjuvanted np-kag vaccine formulation. further, prrsv antibody avidity results were positively correlated with vn titers, in agreement with a previous report. typically, killed vaccines elicit a predominantly th response, but np-based vaccines drive either a balanced or th -biased response, required for efficient clearance of virus. in group pigs, enhanced and balanced th -th humoral and cmi responses were detected. a robust cmi response is essential for complete protection against prrsv. a crucial th cytokine, ifn-γ, is produced by nk cells, γδ t cells, cd + and cd + t cells, and cd + cd + t cells. enhanced secretion of ifn-γ and the presence of increased frequencies of ifn-γ + memory cd + and cd + cells in the lungs of adjuvanted np-kag-vaccinated pigs have confirmed the additive effect of plga-mediated delivery and adjuvanticity of m. tb wcl. a possible mechanism of improved cmi response in group pigs was mediated by cross-presentation of plga-entrapped ags to cd + t cells through mhc class i molecules, of dendritic cells and macrophages. thus, our results were consistent with the previous reports on plga np-based vaccines, which elicited strong effector and memory cmi responses. , in addition, increased levels of another important th cytokine, il- , was detected in the lungs of adjuvanted np-kag-vaccinated pigs, associated with reduced production of immunosuppressive cytokines, il- and tgf-β, which play a vital role in prrsv pathogenesis. the γδ t cells are present in high frequency in pigs and are involved in both innate and adaptive immunity at mucosal tissues. enhanced frequency of activated γδ t cells in the lungs of group pigs was observed. significantly reduced production of il- in group pigs indicated the absence of inflammatory response in the lungs of group pigs. importance of immediate availability of unentrapped potent adjuvant to intranasally delivered np-kag vaccine was critical in our study, because inadequate immune response and incomplete viral clearance was observed in other vaccine formulations pigs received. plga-entrapped hepatitis b subunit vaccine coadministered with an encapsulated adjuvant failed to induce strong antibody response, consistent with our results observed in group pigs (np-kag + np-wcl). further, pigs vaccinated with both adjuvant and kag unentrapped formulation (group ) exhibited th -biased and weak ifn-γ response, associated with partial clearance of replicating prrsv. the presence of - fold fewer prrsv rna copies in the lungs of adjuvanted np-kag-vaccinated pigs and the absence of detectable replicating challenged virus is consistent with a previous report, wherein qpcr failed to differentiate infectious and noninfectious virus, and inactivated prrsv is relatively stable in the environment. further, in samples with low levels of prrsv rna copies, cell culture method has failed to detect the replicating virus. plga-based vaccine delivery is shown to dramatically reduce the required vaccine dose by up to times. plga-np-based vaccine delivery is getting global recognition for effective delivery of subunit/inactivated mucosal vaccines, because their size, contents, and cell targeting properties can be engineered. our study has demonstrated that plga-based adjuvanted np-kag vaccine induced strong anti-prrsv immunity both systemically and locally at the lungs. in conclusion, intranasal coadministration of plga-np-entrapped inactivated prrsv vaccine with a potent adjuvant has the potential to induce superior cross-protective international journal of nanomedicine : submit your manuscript | www.dovepress.com adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs immunity in pigs. future studies should aim to fractionate m. tb wcl to identify adjuvant components to np-kag and to identify alternate adjuvants to reduce the cost of vaccine formulation, and perform field trials to validate efficacy of this innovative vaccine delivery approach. our study not only establishes the utility of nanotechnology-based vaccines in large animals, it also envisages its potential application against important human respiratory pathogens. prrs costs industry $ million annually porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology nanotechnology solutions for mucosal immunization role of nanotechnology in pharmaceutical product development vaccine manufacturing: 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virus in pigs during early stage of infection under farm conditions mycobacterium tuberculosis whole cell lysate enhances proliferation of cd positive lymphocytes and nitric oxide secretion in the lungs of live porcine respiratory and reproductive syndrome virus vaccinated pigs towards tailored vaccine delivery: needs, challenges and perspectives vaccine delivery: a matter of size, geometry, kinetics and molecular patterns role of neutralizing antibodies in prrsv protective immunity type /type immunity in infectious diseases international-journal-of-nanomedicine-journal the international journal of nanomedicine is an international, peerreviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field role of sustained antigen release from nanoparticle vaccines in shaping the t cell memory phenotype plga nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses debugging how bacteria manipulate the immune response gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus enhancement of t helper type immune responses against hepatitis b virus core antigen by plga nanoparticle vaccine delivery porcine reproductive and respiratory syndrome virus (porcine arterivirus) porcine reproductive and respiratory syndrome virus: a persistent infection the authors report no conflicts of interest in this work. international journal of nanomedicine : submit your manuscript | www.dovepress.com key: cord- -xs df a authors: tapia, karla; kim, won-keun; sun, yan; mercado-lópez, xiomara; dunay, emily; wise, megan; adu, michael; lópez, carolina b. title: defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: xs df a the innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors irf and nf-κb and the production type i ifns through a mechanism independent of ifn signaling. we demonstrate that these defective viral genomes (dvgs) are generated naturally during respiratory infections in vivo even in mice lacking the type i ifn receptor, and their appearance coincides with the production of cytokines during infections with sendai virus (sev) or influenza virus. remarkably, the hallmark antiviral cytokine ifnβ is only expressed in lung epithelial cells containing dvgs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. together, our data indicate that dvgs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection. the recognition of virus-specific pattern associated molecular patterns (pamps) is a pivotal event in the initiation of the host innate response to infection. in recent years, it has been established that most viral danger signals are derived from oligonucleotide structures exposed during the replication of the viral genomes [ , , , , , ] . however, most viruses produce proteins that antagonize and effectively delay signaling by the primary viral oligonucleotide sensor molecules retinoic acid inducible gene i (rig-i) and melanoma differentiation-associated gene (mda ), allowing the virus to replicate to high titers and produce large amounts of danger signals prior to host intervention [ , ] . it is currently unclear how the host immune response overcomes viral evasion to initiate a protective antiviral response. defective viral genomes (dvgs) arise when the viral polymerase loses processivity during virus replication at high titers, thereby generating truncated versions of the viral genome that contain deletions and/or complementary ends (the later known as copyback or snap-back genomes) [ , ] . dvgs with the ability to interfere with standard virus replication were first described by von magnus in the early s as the genomes of incomplete forms of influenza virus called defective interfering (di) viral particles [ ] . dvgs have been identified in multiple distinct viral families when the viruses are grown in the laboratory at high multiplicity of infection and span a broad range of hosts, from plants to mammals [ ] . importantly, dvgs are found in patients infected with hepatitis a [ ] , hepatitis b [ , ] , hepatitis c [ ] , hiv [ ] , dengue virus [ ] , and influenza virus [ ] . however, the biological role of dvgs in the context of natural infections is not well understood. we and others have shown that stocks of sendai virus (sev) with a high content of copy-back dvgs with interfering activity trigger enhanced production of cytokines in vitro and more potently induce antigen presentation by mouse and human dendritic cells than do virus stocks lacking this kind of dvgs [ , , , , , ] . our group has also demonstrated that in contrast to standard viral genomes, sev copy-back dvgs induce the expression of mda and of a number of other interferonstimulated genes in the absence of type i ifn positive feedback [ , , ] . remarkably, sev copy-back dvgs show this potent in vitro stimulatory activity even in the presence of functional viral encoded antagonists of the host response [ , ] . here, we demonstrate that dvgs that trigger a robust activation of the transcription factors irf and nf-kb accumulate at a high rate in infected cells becoming the main source of viral pamps. these dvgs arise naturally during acute respiratory viral infections in mice and provide essential stimuli for the initiation of the antiviral innate immune response in the lung. these data demonstrate the generation of dvgs in vivo during acute respiratory viral infections and suggest a critical role of these kinds of viral genomes in determining the quality of the host response to infection. sev copy-back dvgs trigger a robust and sustained activation of irf and nf-kb independent of type i ifn feedback to further investigate the cellular mechanisms responsible for the efficient activation of the antiviral response by sev dvgs, we evaluated the phosphorylation of transcription factors that are critical for the expression of type i ifns in cells infected with equivalent amounts of infectious particles of a sev strain cantell stock containing high levels of copy-back dvgs (sev cantell hd) or with sev cantell depleted of dvgs (sev cantell ld). virus stocks were prepared from the same parental virus and their content of dvgs was determined by calculating the ratio of infectious particles to total particles (ratios are specified in the material and methods section). in addition, copy-back dvgs of these stocks were identified by pcr. one predominant copy-back genome was present in cells infected with sev cantell hd (amplicon of bp), while no copy-back defective genome was detected in cells infected with sev cantell ld up to six hours after infection (figs. a and s ). cloning and sequencing of the nt long amplicon confirmed that it corresponded to a previously described sev cantell copy-back dvg of nt in length (dvg- ) [ ] . phosphorylation of irf and of the nf-kb repressor ikba in response to sev cantell hd occurred rapidly and was sustained even in type i ifn receptor ko cells (ifnar / ) ( fig. b and c), while no phosphorylation of irf or ikba was observed for up to ten hours post-infection with sev cantell ld despite equivalent or higher expression of the viral protein np (fig. d) . corresponding with the strong activation of transcription factors, ifnb mrna was expressed in ifnar / cells infected with sev cantell hd (fig. e ). in contrast, type i ifn signaling was required for the cellular response to newcastle disease virus (ndv), an avian virus that only partially inhibits the type i ifn pathway, triggering the expression of type i ifn and other cytokines in the absence of dvgs. to further validate the role of sev copy-back dvgs as triggers of type i ifn-independent antiviral responses, we cloned dvg- under the control of the t polymerase promoter and used this construct to prepare a sev stock containing a single recombinant dvg (rdvg). for this purpose we used sev strain that normally does not produce highly immunostimulatory copy-back dvgs [ ] . equivalent infectious units of sev and sev plus rdvgs had similar levels of total rna (fig. f ) but infection with virus containing rdvgs strongly induced the antiviral response while virus that lacked dvgs did not (fig. g) confirming the dvg immunostimulatory activity. in addition, presence of rdvgs significantly reduced the expression of sev np mrna, demonstrating their strong interfering capacity (fig. g) . notably, mouse embryo fibroblasts lacking the type i ifn receptor expressed ifnb mrna in response to sev containing rdvg (fig. h ) and virus containing rdvgs triggered irf phosphorylation independently of type i ifn feedback (fig. i) , mirroring the response to sev cantell hd. altogether, this evidence conclusively shows that sev copy-back dvgs confer potent immunostimulatory ability to sev stocks, independent of type i ifn feedback. notably, potent ifnb mrna expression in response to sev dvgs was independent of irf , irf , and irf while only partially dependent on irf (fig. s ). this response was maintained in a variety of cell types (fig. s ) . dvgs accumulate at a high rate in infected cells and are a primary source of pathogen associated molecular patterns that trigger rlr signaling to determine whether standard viral genomes and dvg rnas have distinct intrinsic properties that explain their differential immunostimulatory activities, we compared naked rna purified from a stock of sev cantell ld with in vitro transcribed dvg- . rnas were transfected into cells before or after treatment with phosphatase or with rnase a that cleaves of single stranded c and u residues, and/or rnase v that cleaves base paired nucleotides. both genomic rna and dvg rna were susceptible to treatment with phosphatase, as well as to treatment with rnases ( fig. a) , corresponding with the literature that demonstrates a crucial role for -triphosphate-rna in the induction of type i ifns. while transfected dvgs induced stronger expression of ifnb than an equivalent concentration of gsev ( fig. a) , transfection of equivalent molar amounts of genomic and dvg rna resulted in higher immunostimulatory activity of genomic rna compared to dvg rna (fig. b) , demonstrating that sev ld rna can strongly trigger the host response to infection when delivered naked into the cells. paradoxically, cells infected with sev cantell ld alone failed to induce strong type i ifn production even when used at a times higher infectious dose than sev cantell hd (fig. c) . although the amount of gsev rna was significantly higher in cells infected with an moi of of sev cantell ld compared with ten times less sev cantell hd at h post-infection (fig. c) , dvgs were only detected in cells infected with sev cantell hd, confirming a strong correlation between the presence of dvgs in the infected cells and the induction of the host response to infection. to determine whether the amount of total input viral rna affected the immunostimulatory activity of sev cantell ld and hd, we measured the rna content in equivalent infectious doses of these stocks. sev cantell hd had less than two fold higher the amount of total rna than sev cantell ld and total rna levels were equivalent between sev cantell ld and hd when ld was at twice the infectious dose (fig. d) . thus, differences in the net input amount of viral rna cannot explain the more than . fold difference in the expression of ifnb mrna between cells infected with equivalent infectious doses of sev cantell ld and hd. dvgs have an increased rate of replication compared to standard viral genomes due to their shorter size and promoter properties [ ] . to determine whether dvgs replicate faster than gsev, we calculated the rate of replication of gsev and dvgs in cells infected with sev cantell hd. although at an early time point more copies of gsev than dvgs were detected in the cells, dvgs dominated by h post-infection (fig. e ) accumulating at a times faster rate than gsev (fig. f) . these data demonstrate that dvgs rapidly surpass the number of gsev in infected cells, providing large quantities of pathogen associated molecular patterns. in infections with viruses well adapted to the host virusencoded proteins that delay the cellular response allow the virus to replicate to high titers prior to host intervention. the mechanisms overcoming viral evasion of the immune system and leading to the production of the primary antiviral cytokine ifnb are not well established. here, we demonstrate that truncated forms of viral genomes that are generated in situ during virus replication are a primary source of danger signals for the initiation of the host immune response to respiratory viral infections in vivo. defective viral genomes (dvgs) are able to function as triggers of the immune response even in the absence of type i ifn signaling and are strong triggers of the host response to infection while overcoming viral antagonism. supporting previous observations that dvgs from sev stimulate the cellular antiviral response through signaling by rig-i like receptors (rlrs) [ , , ] , the essential rlr adaptor protein mitochondrial antiviral signaling protein (mavs) was required for the activation of the transcription factors irf and nf-kb and for expression of numerous antiviral and pro-inflammatory molecules upon infection with sev cantell hd. in contrast, mavs was not required for the response to herpes simplex virus, which can trigger the host response independently of rlrs (fig. s ). in addition, only dvg rna, but not standard viral genomes, could be amplified from endogenous rig-i and mda complexes immunoprecipitated from infected cells (fig. s c ), supporting published evidence that dvgs bind to rig-i preferentially over the standard viral genomes in infected cells [ ] . as predicted, association of dvgs with rlrs correlated with type i ifn induction, but not with the level of virus replication (fig. s d ). importantly, in addition to the primary role of rig-i in the response to sev dvgs, mda participates in the induction of type i ifn in primary mouse lung fibroblasts infected with sev hd (fig. s e) , similar to what we have observed in dcs [ , ] . overall, these data demonstrate that dvgs are produced in the infected cells at a higher rate than genomic rna and that dvgs are the predominant ligands for both rig-i and mda during sev infection. based on the potent ability of sev stocks containing a high content of copy-back dvgs to induce the host response to infection in vitro [ , , , ] (fig. ) and on our prior reports of strong host responses to dvgs regardless of the presence of functional virus-encoded antagonists [ , ] , we hypothesized that dvgs that arise in situ during viral infections provide essential stimuli to initiate an antiviral immune response. to test this hypothesis, we first determined if sev strains that accumulate copy-back dvgs early in infection induced faster ifnb mrna expression in vitro than viruses with delayed dvg accumulation. for these experiments we used sev preparations that did not show immunostimulatory activity or evidence of copy-back dvg accumulation by h post-infection and all the viruses were used at a multiplicity of infection of . tcid /cell. while standard viral genomes of all the different sev strains used were detected at all tested time points, copy-back dvgs of different sizes were detected starting at h post-infection in cells infected with sev z and at later time points in cells infected with sev , enders, or cantell ld in both murine lung epithelial cells (tc- ) and bone marrow-derived dendritic cells (bmdcs) (fig. a and b and data not shown). sequences of the starred pcr products confirming the amplification of copy-back dvgs are shown in fig. s . remarkably, accumulation of dvgs was directly associated with phosphorylation of irf (fig. c ) and with the expression of ifnb mrna (fig. d ), demonstrating that standard viral genomes alone are not sufficient to initiate this response during infection in vitro and strongly supporting a unique ability of naturally arising dvgs to initiate the cellular antiviral response. to evaluate the impact of dvgs during sev infection in vivo, we infected mice with sev cantell hd or ld. mice infected with sev cantell hd showed diminished morbidity than mice infected [ , , , , , ] . reduced virulence of sev cantell hd was associated with a stronger stimulation of the host antiviral response as shown by the expression of ifnb mrna (fig. c ). to conclusively demonstrate the role of dvgs in diminishing virulence in vivo, we co-infected mice with sev cantell ld and purified viral particles containing dvgs (defective particles; dps). confirming their critical role, dvgs reduced the pathogenicity of sev cantell ld in mice, while uv-inactivated dp particles did not provide significant protection (figs. d-f). interestingly, infection in the presence of dps resulted in reduced expression of sev np protein in the lung at day post-infection, suggesting that in this system, dps reduce virulence by interfering with virus replication. to determine whether immunostimulatory dvgs were generated in situ in the lung during infection, we infected mice with sev cantell ld, and we followed the appearance of copy-back dvgs in the lung by pcr. sev copy-back dvgs were detected in whole lung homogenates at the time of high viral replication (fig. a) . notably, upon infection with sev cantell ld, a copy-back dvg of high molecular weight was detected at day post-infection in the lung, while a dvg of low molecular weight (amplicon of bp) that predominates in the parent stock of sev cantell hd (fig. a) was only detectable at day post-infection. copy-back dvgs also appeared in the lung of mice infected with sev (fig. s ) , showing that dvgs naturally arise during infection in vivo independent on the virus strain. interestingly, accumulation of copy-back dvgs during infection with sev cantell ld was associated with the expression of ifnb and il- mrna in the lung (fig. b) . to determine whether dvgs were necessary for the expression of antiviral cytokines in vivo, we took advantage of ifnb-yfp reporter mice. to demonstrate that yfp expression serves as readout for dvg activity, we first infected bmdcs prepared from ifnb-yfp reporter mice with sev cantell ld alone, or together with increasing doses of purified dps. as shown in fig. c , at h post-infection, yfp was expressed only in the presence of dps and in a dose-dependent manner, and the yfp expression was lost when uv-treated dps were used. dps alone were also able to induce yfp in a dose-dependent manner, albeit at much lower levels than during co-infection with sev. these data agree with our previous reports that demonstrate that the immunostimulatory activity of dps is greatly amplified during dvg replication by the cognate polymerase provided by co-infecting sev [ ] and validate the ifnb-yfp reporter system as a readout for dvg activity. we then infected ifnb-yfp reporter mice with sev cantell ld and analyzed viral genomes in yfp + cells. we focused our analysis on the cd (non-hematopoietic) cellular fraction of the lung as sev replicates predominantly in the lung epithelium [ ] . although full-length viral genomes were detected in both yfp + and yfp cd populations sorted three days after infection, dvgs were only found in yfp + cells (fig. d) , suggesting that the presence of dvgs promotes ifnb production in response to virus infection in vivo. together, these findings show that dvgs are normally generated in situ in the lung during respiratory infection with sev, and that their accumulation is associated with the expression of ifnb in the lung. sev copy-back dvgs are generated in the lung independently of type i ifn signaling to determine whether type i ifns produced early upon infection promoted the generation of dvgs in the lung, we infected wild type or type i ifn receptor deficient mice (ifnar / ) with sev cantell ld and analyzed the lungs at different times post-infection. as shown in fig. e , dvgs accumulated in the lung at a higher rate in mice unable to respond to type i ifns compared with wild type mice, corresponding with the predicted enhanced rate of virus replication in the lack of type i ifn signaling and demonstrating that type i ifns are not required for the generation of sev copy-back dvgs in vivo. to investigate whether the content of dvgs in iav stocks affects virulence similar to sev, we obtained iav strain pr stocks with a high content of dvgs (hd) or lacking dvgs (ld). the stock of iav pr hd produced two predominant dvgs derived from the pa and pb genomic segments in infected cells, while no dvgs were detected in cells infected with iav pr ld (fig. a ) (strategy for iav detection and sequences for the iav dvgs present in infected cells can be found in fig. s ). mice infected with iav pr hd showed reduced morbidity compared to mice infected with iav pr ld (fig. b ) despite similar levels of virus replication (fig. c ). similar to sev cantell hd, reduced morbidity was associated with enhanced ifnb mrna expression in the lung (fig. d) . to determine whether iav dvgs were generated in situ in the infected lung, we tracked their appearance in mice infected with iav pr ld. accumulation of dvgs was clearly observed at day post-infection (fig. e) . representative sequences of starred iav dvgs products are shown in fig. s . similar to sev infection, accumulation of dvgs corresponded with enhanced expression of mrna for ifnb and il- ( fig. f ) despite evidence of reduced genomic viruses at that time point (figs. e and f) . these data demonstrate that dvgs are generated de novo in the lung during infections with iav, and suggest an important role of these types of genomes in promoting the host response to iav in vivo. we have shown that dvgs are naturally generated in the lung during infection with sev and iav and provide primary danger signals for the triggering of the host response to infection. the generation of dvgs during virus growth in tissue culture is a highly conserved phenomenon among viruses of different species and is tempting to speculate that dvgs provide an evolutionary advantage to the virus by contributing to the preservation of both the virus and the host. interestingly, immunostimulatory dvgs result from drastic truncations in the genome of the virus that render it a dead end product unable to persist in the absence of helper virus. it will be relevant to determine how dvgs relate to viral ''quasispecies'' that result from mutations as a consequence of having a viral polymerase with a lower fidelity and processivity [ ] . viral quasispecies have been shown to be essential for viral fitness and virulence [ ] . whether dvgs a tradeoff of this viral polymerase characteristic that enables more rapid virus evolution but makes the virus more vulnerable to innate immune detection remains to be established. our data demonstrate that dvg recognition is not necessary for the response to ndv, while is required for the response to sev. we speculate that the differential dvg requirement may be explained by the poor adaptation of the avian ndv to grow in mice while the murine sev is fully adapted to grow in this species. a critical factor of this adaptation is the activity of the virally encoded v and c proteins that effectively block the induction of type i ifns, as well as the type i ifn-mediated amplification of the type i ifn pathway [ , , , ] . as ndv is adapted to grow in birds, its antagonistic proteins are not fully functional in mammalian cells [ ] allowing unrestricted production and amplification of type i ifns. in contrast, the sev c and v proteins very effectively block the cellular response to sev [ , , , ] and no cellular response is observed unless dvgs are present. interestingly, in the absence of type i ifn feedback (or of the ifn-inducible transcription factor irf ) sev dvgs induce a more potent cellular response compared to ndv, suggesting that dvgs have a unique ability to bypass both virus antagonism and the requirement for irf for strong type i ifn production. we have reported that sev cantell hd, but not ndv, has the ability to induce the expression of the viral sensor mda independently of type i ifn feedback [ ] , and that mda is involved in the recognition of sev dvgs ( [ ] and data in fig. s ). although it is unclear why this newly synthetized mda is less susceptible to inhibition by the sev v protein, preferential binding of dvgs to both rig-i and mda compared to standard sev genomes, together with the availability of high levels of mda , may explain the strong activation of transcription factors and type i ifn expression in response to dvgs, regardless of type i ifn feedback. remarkably, dvgs arise in vivo independently of type i ifn feedback, demonstrating that dvgs do not appear in response to host pressure via type i ifns. notably, viruses containing dvgs have significantly reduced virulence. in a previous study, we reported that a sev strain with a lower propensity to produce dvgs (sev ) persisted longer in the lung than a sev strain able to produce high levels of dvgs (cantell) [ ] . consistently, we observed higher levels of viral np protein at day post-infection in the lung of mice infected with sev cantell ld, compared to mice infected with sev cantell ld plus dps (fig. g) . in additional studies, we have not observed significant differences in the rate of sev-specific t cells in the lung of mice infected with sev ld alone or in the presence of dps (data not shown and [ ] ). although interpretation of this observation is complicated by the reduced amount of sev antigen (np protein) present in infection with sev ld plus dps, we favor the hypothesis that dps diminish virulence by competing for the viral polymerase, thus interfering with the replication of the standard virus, a well-defined characteristic of dvgs. additionally, enhanced production of type i ifns in response to dvgs likely contributes to dampened viral replication. highly immunostimulatory sev dvgs are of the copy-back type. intriguingly, during sev infections in vivo, accumulation of dvgs of high molecular weight preceded the appearance of low molecular weight dvgs, suggesting that the smaller ones may be secondary to longer defective genomic products. copy-back dvgs are not transcribed due to their promoter properties [ ] , thus their stimulatory activity likely derives solely from their genomic composition. iav dvgs are truncated versions of one of the genomic segments that have natural complementarity among their and ends providing the theoretical capacity to form structures similar to copy-back dvgs. notably, it is apparent that both sev genomic and dvg rnas have the potential to induce a host response when delivered naked into the cells. based on our data, we predict that in the context of infection, dvg rna is more available for detection due to their enhanced rate of replication compared to standard viral genomes (fig. ) . interestingly, we have shown that dvgs have the ability to bypass viral-encoded antagonists of the immune response even upon overexpression of viral antagonistic proteins [ ] . it remains to be investigated what is the molecular mechanism behind this dvg property. notably, dvgs of different forms and compositions have been described in the sera of patients chronically infected with a number of different viruses [ , , , , ] and dvgs of various viruses have been shown to promote persistent infections in tissue cultures [ , , , , , , , , ] supporting a role for dvgs in the maintenance of chronic viruses. the role of naturally arising dvgs in promoting virus persistence in vivo remains to be investigated. in summary, we have demonstrated that dvgs arise naturally during an acute respiratory virus infection and that they play a critical role in regulating the virus-host cycle in vivo. importantly, the recognition of dvgs as stimuli for the onset of immunity has multiple practical implications, most directly: (i) dvgs represent novel determinants of virus pathogenesis that could be targeted for therapy, and (ii) dvgs are novel candidate biomarkers to predict the outcome of infections and the rate of virus spread in the population. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the protocol ( ) was approved by the institutional animal care and use committee, university of pennsylvania animal welfare assurance number a - . sev strains cantell, , enders, and z, and influenza a/pr / virus were grown in days hen embryonated eggs (spafas; charles river laboratories). sev cantell was passaged to retain its original high di particle content (hd) or to deplete it of di particles (ld) as we previously described [ ] . in brief, sev , enders, z, and cantell hd were grown in embryonated hen eggs inoculated with , medium tissue culture infectious dose (tcid ) for h. sev cantell hd tcid was calculated by end point dilution in llcmk cells in the presence of trypsin, as described below [ ] . sev cantell hd total particles were calculated by end point dilution of hemagglutination of chicken red blood cells. sev cantell hd stocks had consistently an infectious:total particle ratio of , - , . sev had an infectious:total particle ratio of , . sev enders had an infectious:total particle ratio of , . sev z had an infectious:total particle ratio of , . sev cantell ld was prepared by inoculating embryonated hen eggs with tcid for h. under these conditions only % of the eggs grew virus. allantoic fluid from those eggs was pooled and diluted / for subsequent inoculation into embryonated hen eggs in a total volume of ml. allantoic fluid containing virus ( % of the inoculated eggs) was pooled and tittered as described below. sev cantell ld stocks had consistently an infectious:total particle ratio of , - , . iav strain pr (ld) was grown by inoculating hen embryonated eggs with , tcid obtained directly from infected lung homogenates. allantoic fluid containing the virus was collected h later. egg's allantoic fluid was snap frozen in an ethanol/dryice bath and stored at uc. iav pr containing high dose of di particles (hd) was kindly provided by dr. laurence c. eisenlohr, v.m.d., ph.d (thomas jefferson university). iav hd was grown by inoculating hen embryonated eggs with , pfu of egg-passed virus. eggs were incubated at uc and allantoic fluid containing the virus was collected h later. iav pr hd and ld stocks were originated from the same parent stock but were extensively passaged in the different conditions described. permissive cells were infected with serial : dilutions of lung homogenates or virus stocks in the presence of mg/ml of trypsin to determine the medium tissue culture infectious dose (tcid ). llcmk cells were used for sev titration, while mdck cells were used for iav titration. after h of incubation at uc, ml of supernatant from each well was tested by hemagglutination of chicken red blood cells (rbcs) for the presence of virus particles at the end point dilution. to do this, : dilutions of the cell supernatant were incubated in . % chicken rbcs at uc for min. hemagglutination of rbcs indicated the presence of sev or influenza virus particles. bmdcs were generated as previously described [ ] . detailed procedure can be found in the supplemental information material and methods. bmdcs were infected after days in culture with viruses at an multiplicity of . as we have previously described [ ] . for sev infections, mice were anesthetized with tribromoethanol (avertinh; acros organics) and inoculated in the nostrils with ml of pbs containing or tcid of sev. for iav infections animals were infected intranasally with tcid / mouse in a ml volume. lungs were extracted at different times post-infection, homogenized in . % w/v gelatin-pbs and snap frozen in dry-ice/ethanol for preservation. total rna was extracted from cell lines or lungs with trizol (invitrogen) according to the manufacturer's specifications and total rna was reversed transcribed using the high capacity rna to cdna kit from applied biosystem. for sorted cells, ng of rna were reversed transcribed, for all other experiments - mg of rna were reversed transcribed. cdna was diluted to a concentration of mg/ml and amplified with specific primers in the presence of sybr green (applied biosystem). for the detection of dvgs, isolated total rna was reverse transcribed using superscript iii without rnase h activity, to avoid selfpriming by the dvgs complementary ends and recombinant rnase h (invitrogen) was added later to the samples. for the detection of the standard virus genome, the negative strand of the full-length genome was reverse transcribed with transcriptor first strand cdna synthesis kit (roche). pcr detection for iav was performed using as it has been previously described [ ] . primers and detailed pcr conditions can be found in the supplemental information material and methods. detailed primers and pcr conditions can be seen in the supplemental information material and methods. whole cellular extracts were prepared by lysing of cells in a np- -based lysis buffer containing phosphatase inhibitors, proteinase inhibitors (roche and thermo scientific), and . m edta. the concentration of protein was measured by bradford assay (themo scientific). samples ( mg) were boiled for min and resolved on % bis-tris pre-cast gels (bio-rad). resolved proteins were transferred to a polyvinylidene fluoride (pvdf) membrane (millipore). the membrane was blocked with % nonfat milk and immunoblotted with the indicated antibodies. antirabbit irf , anti-rabbit phospho-irf (ser ), anti-mouse ikba, anti-mouse phospho-ikba (ser / ), and anti-rabbit igg (hrp-conjugated) were purchased from cell signaling. antimouse gapdh was purchased from sigma. anti-mouse igg and anti-mouse igg (hrp-conjugated) were purchased from jackson immunologicals. lumi-light western blotting substrate was used for hrp detection (roche). dp purification was performed as previously described [ ] . in short, allantoic fluid from infected hen eggs was pooled and concentrated by high-speed centrifugation. pellets were suspended in . ml of pbs/ mm edta and incubated overnight at uc in a - % sucrose (fisher) gradient that was prepared using a gradient maker (biocomp). gradients were centrifuged at uc for . h at , rpm and fractions containing low-density viral particles were collected, pelleted, suspended and re-purified using the same procedure. collected low-density fractions were concentrated by centrifugation at uc for h at , rpm. pellets were suspended in pbs, snap frozen, and stored at uc. the content of di particles was determined by calculating the ratio of infectious over non-infectious particles as described above. a nt long product containing the sequence of the t promoter followed by the -nucleotide long copy back dvg from sev cantell, and flanked by the restriction enzymes spei and sapi at the an ends was synthetically synthetized (dna . ) and clone into the psl vector (amersham pharmacia biotech) containing the sequences for the hepatitis delta virus ribozyme and the t polymerase terminator. in order to optimize the transcription of the dvg, g residues were introduced downstream of the t promoter by site-directed mutagenesis (stratagene, ca) using the oligonucleotides ccactagttaa-tacgactcactatagggaccagacaagagtttaagag- and ctcttaaactcttgtctggtccctatagtgag tcgtattaactagtgg- . bsr-t cells were infected with a moi of approximately of partially inactivated sev strain . virus inactivation was performed by exposing diluted virus to uv light ( nm model mrl- , uvp upland, ca) for sec at a distance of inches from the light source. virus inactivation diminished the virus replication rate, while allowing the expression of viral proteins necessary for the replication of dvgs. cells were incubated at uc for h before transfection of mg of vector encoding dvg. transfection was performed with xtremegene transfection reagent (roche) according to manufacturer instructions. cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % bovine serum albumin, % naco , . mg/ml trypsin (worthington) and . % penicillinstreptomycin (invitrogen) and incubated in % co at uc. cells and supernatant containing sev and rdps were harvested after h and ml of the suspension were inoculated in the allantoic cavity of -day embryonated hen eggs (b & e eggs, silver springs, pa). after h allantoic fluid was harvest and ml of undiluted fluid were inoculate in -day embryonated eggs for virus growth and egg inoculation was repeated for three consecutive passages. allantoic fluid from the third passage was quick-frozen in dried ice/ethanol and used for infections. presence of recombinant dvg was confirmed by pcr. no other dvgs were detected. dvg rna was in vitro transcribed (ambion) from the t -dvg plasmid. standard genomic rna was extracted from sev cantell ld stocks. to remove -triphosphates, mg of rna was incubated with u of calf intestinal phosphatase (new england biolabs) for min at uc. to cleave single stranded rna, mg of rna was incubated with ng of rnase a (ambion) for min at room temperature. to cleave double stranded rna, rna was incubated with . u of rnase v (ambion) for min at room temperature. after treatments, rna was purified using trizol or precipitation/inactivation buffer according to the manufacturer's specifications. llc-mk cells were transfected with ng or indicated doses of dvg and genomic rna using lipofectamin (invitrogen). at hours post transfection, the cells were harvested and total rna was isolated using trizol according to the manufacturer's specifications. infected ifnb-yfp cells were collected at h post-infection. reporter mice were sacrificed days post-infection. lungs were collected and dissociated with collagenase (roche), followed by suspension on . m edta and rbc lysis buffer. single cell suspensions were then incubated with cd /cd fcblock for min at c, followed by incubation with biotinylated mouse anti-cd . . washed cells were incubated with anti-biotin microbeads (miltenyi) for min and passed through a magnetic column for negative selection. cd cells were sorted based on yfp expression using a facs vantage se sorter. statistical analyses were performed as indicated in each figure. graphpad prism version . for windows, graphpad software, san diego california usa, www.graphpad.com, was used for analysis. genes ncbi id numbers. tuba b: ; rps : ; ifnb: ; ifnl : ; illb: ; il b: ; tnf: ; il- , . text s supporting materials and methods. the text includes in detail descriptions of the procedures, as well as specific material and methods and references for figures included as supporting information. (docx) preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing -triphosphate rna is the ligand for rig-i lengthdependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiation-associated gene rig-imediated antiviral responses to single-stranded rna bearing -phosphates rig-i detects viral genomic rna during negative-strand rna virus infection recognition of triphosphate by rig-i helicase requires short blunt doublestranded rna as contained in panhandle of negative-strand virus cutting edge: stealth influenza virus replication precedes the initiation of adaptive immunity antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology defective viral particles and viral disease processes the origins of defective interfering particles of the negative-strand rna viruses incomplete forms of influenza virus defective interfering rnas: foes of viruses and friends of virologists detection of defective genomes in hepatitis a virus particles present in clinical specimens hepatitis b defective virus with rearrangements in the pres gene during chronic hbv infection in vivo and in vitro expression of defective hepatitis b virus particles generated by spliced hepatitis b virus rna characterization of hepatitis c virus deletion mutants circulating in chronically infected patients mechanisms associated with the generation of biologically active human immunodeficiency virus type particles from defective proviruses defective interfering viral particles in acute dengue infections sequence analysis of in vivo defective interfering-like rna of influenza a h n pandemic virus the characteristics required for a sendai virus preparation to induce high levels of interferon in human lymphoblastoid cells interferon induction by viruses. xix. vesicular stomatitis virus-new jersey: high multiplicity passages generate interferoninducing, defective-interfering particles activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins mda participates in the detection of paramyxovirus infection and is essential for the early activation of dendritic cells in response to sendai virus defective interfering particles a novel role for viral-defective interfering particles in enhancing dendritic cell maturation differential type i ifn-inducing abilities of wild-type versus vaccine strains of measles virus sendai virus infection induces efficient adaptive immunity independently of type i interferons cytokine-independent upregulation of mda in viral infection sendai virus defective-interfering genomes and the activation of interferon-beta nucleotide sequences that affect replicative and transcriptional efficiencies of sendai virus deletion mutants prevention of death in semliki forest virusinfected mice by administration of defective-interfering semliki forest virus defective interfering viruses: modulators of infection protection of mice from lethal influenza: evidence that defective interfering virus modulates the immune response and not virus multiplication protection of three strains of mice against lethal influenza in vivo by defective interfering virus defective interfering influenza a virus protects in vivo against disease caused by a heterologous influenza b virus defective interfering virus protects elderly mice from influenza melanoma differentiation-associated gene (mda ) is involved in the innate immune response to paramyxoviridae infection in vivo quasispecies diversity determines pathogenesis through cooperative interactions in a viral population the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter ) mda- , but not rig-i, is a common target for paramyxovirus v proteins the paramyxovirus, sendai virus, v protein encodes a luxury function required for viral pathogenesis c and v proteins of sendai virus target signaling pathways leading to irf- activation for the negative regulation of interferon-beta production newcastle disease virus v protein is a determinant of host range restriction the sendai paramyxovirus accessory c proteins inhibit viral genome amplification in a promoter-specific fashion longer and shorter forms of sendai virus c proteins play different roles in modulating the cellular antiviral response persistent infection. i interferon-inducing defective-interfering particles as mediators of cell sparing: possible role in persistent infection by vesicular stomatitis virus role of defective interfering particles of sendai virus in persistent infections comparative study of rabies virus persistence in human and hamster cell lines characterization of west nile virus persistent infections in genetically resistant and susceptible mouse cells. i. generation of defective nonplaquing virus particles characterization of a cell culture persistently infected with the da strain of theiler's murine encephalomyelitis virus persistent infection of some standard cell lines by lymphocytic choriomeningitis virus: transmission of infection by an intracellular agent human cytomegalovirus persistent infection in a human central nervous system cell line: production of a variant virus with different growth characteristics defective interfering particles of human parainfluenza virus type are associated with persistent infection in cell culture establishment of respiratory syncytial virus persistence in cell lines: association with defective interfering particles singlereaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses the authors wish to thank dr. laurence eisenlohr for hd influenza virus, luis muñ oz for technical support, and the flow cytometry and cell sorting resource laboratory and the dna sequencing facility at the university of pennsylvania. we also thank drs. thomas moran, christopher basler, and benjamin tenoever (icahn school of medicine at mount sinai), and marco colonna (washington university) for providing invaluable reagents. conceived and designed the experiments: kt wk cbl. performed the experiments: kt wk xml ys ed ma mw. analyzed the data: kt wk cbl. wrote the paper: kt wk cbl. key: cord- -tsvzg ax authors: fensterl, volker; wetzel, jaime l.; ramachandran, srividya; ogino, tomoaki; stohlman, stephen a.; bergmann, cornelia c.; diamond, michael s.; virgin, herbert w.; sen, ganes c. title: interferon-induced ifit /isg protects mice from lethal vsv neuropathogenesis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: tsvzg ax interferon protects mice from vesicular stomatitis virus (vsv) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (isg) mediate the antiviral effect. a prominent family of isgs is the interferon-induced with tetratricopeptide repeats (ifit) genes comprising three members in mice, ifit /isg , ifit /isg and ifit /isg . intranasal infection with a low dose of vsv is not lethal to wild-type mice and all three ifit genes are induced in the central nervous system of the infected mice. we tested their potential contributions to the observed protection of wild-type mice from vsv pathogenesis, by taking advantage of the newly generated knockout mice lacking either ifit or ifit . we observed that in ifit knockout (ifit (−/−)) mice, intranasal vsv infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. in contrast, wild-type and ifit (−/−) mice were highly protected and survived without developing such disease. however, when vsv was injected intracranially, virus replication and survival were not significantly different between wild-type and ifit (−/−) mice. when administered intranasally, vsv entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and ifit (−/−) mice and induced interferon-β. however, as the infection spread to other regions of the brain, vsv titers rose several hundred folds higher in ifit (−/−) mice as compared to wild-type mice. this was not caused by a broadened cell tropism in the brains of ifit (−/−) mice, where vsv still replicated selectively in neurons. surprisingly, this advantage for vsv replication in the brains of ifit (−/−) mice was not observed in other organs, such as lung and liver. pathogenesis by another neurotropic rna virus, encephalomyocarditis virus, was not enhanced in the brains of ifit (−/−) mice. our study provides a clear demonstration of tissue-, virus- and isg-specific antiviral action of interferon. virus infection of mammals induces the synthesis of type i interferons (ifn), which, in turn, inhibit virus replication. the high susceptibility of type i ifn receptor knockout (ifnar / ) mice to infection by a variety of viruses [ ] [ ] [ ] provides strong evidence for the major role of the ifn system in protecting from viral pathogenesis. in these mice, although ifn is induced by virus infection, it cannot act on target cells. similarly, in genetically altered mice that are defective in ifn production due to the absence of specific pathogen-associated pattern recognition receptors, signaling proteins or specific transcription factors, viral pathogenesis is enhanced [ ] [ ] [ ] . although the critical importance of the ifn system in regulating viral pathogenesis is now well established, in many cases it is still unclear how ifn inhibits the replication and spread of a specific virus in vivo. in this context, activation of different components of the immune system plays a major role in controlling viral diseases that are relatively slow to develop [ ] [ ] [ ] . in contrast, in acute infection by viruses that cause severe pathogenesis and death within a few days after infection, protection is primarily provided by the intrinsic antiviral actions of ifn-induced proteins encoded by the hundreds of ifn-stimulated genes (isgs) [ ] [ ] [ ] , several of which often contribute to the overall effect of ifn against a given virus. our knowledge of the antiviral and the biochemical properties of individual isg products is mostly limited to a few intensively studied examples such as pkr, oas/rnase l or mx [ ] . however, recent systematic investigation of the antiviral functions of the entire family of isgs has started producing exciting new information [ ] . in the above context, we have been investigating the biochemical and biological functions of the members of the ifit family of isgs, which are very strongly induced by ifn. there are three members of this family of genes in mice: ifit /isg , ifit / isg and ifit /isg ; all of the encoded proteins contain multiple tetratricopeptide repeats (tpr), which mediate proteinprotein and protein-rna interactions [ ] . in vitro, p and p , the products of ifit and ifit , respectively, bind to the translation initiation factor eif and inhibit protein synthesis [ ] . the third member, p , the product of ifit , does not share this property [ ] . recently, it has been reported that ifit proteins form a multiprotein complex that can bind to the triphosphorylated end of rnas, an rna-species produced during the replication of some, but not all, viruses [ ] . in vivo, these genes are strongly induced in brains of mice infected with west nile virus (wnv) or lymphocytic choriomeningitis virus (lcmv); surprisingly, different ifit genes are differentially induced in different regions of the brain, suggesting non-redundant functions [ ] . to further explore the antiviral properties of the ifit proteins, we generated ifit knockout (ifit / ) mice and challenged them with different viruses. we observed that ifit / mice were particularly susceptible to a wnv mutant that is defective in its mrna cap -o methylation; the mutant virus killed ifit / mice but not the wild-type (wt) mice [ ] . here, we report on the antiviral properties of the newly generated ifit / mice; these mice, but not ifit / mice, were highly susceptible to neuropathogenesis after intranasal infection with vesicular stomatitis virus (vsv), a negative sense, singlestranded rna rhabdovirus. vsv replication is highly sensitive to the inhibitory action of ifn and is routinely used to assay the antiviral activity of ifn in vitro [ ] . as expected, ifnar / mice are highly susceptible to vsv pathogenesis and the same is true for mice that specifically lack expression of ifnar on the cells of their central nervous system (cns) [ ] . in spite of these observations, little is known about how ifn inhibits vsv replication in vivo. our new results indicate that in the brain, but not in other organs, ifit is a major mediator of ifn's protective effect against vsv. in contrast, ifit could not protect mice from neuropathogenesis caused by encephalomyocarditis virus (emcv), a picornavirus. thus, we have uncovered a virusspecific, tissue-specific and isg-specific antiviral effect of the ifn system. generation of ifit /isg and ifit /isg knockout mice ifit gene knockout (ifit / ) mice were generated by deleting the entire protein-encoding region of the gene, which was achieved by flanking exons and with frt recombinase sites in c bl/ embryonic stem cells and excising the flanked region with flp recombinase ( figure a ). ifit / mice were bred to homozygosity ( figure b) , and deficiency for induced expression of ifit protein was confirmed in lysates of ifn-b-treated primary murine embryonic fibroblasts (mef) ( figure c ). mice deficient for ifit (ifit / ) were derived from c bl/ embryonic stem cells lacking the entire ifit coding region ( figure a ). genotypic homozygosity of the ifit / mice and deficiency for ifit protein induction were confirmed ( figure b and c ). both knockout mouse lines were healthy and fertile. moreover, deletion of one gene within the ifit locus did not alter the pattern of induction of other adjacent gene family members, as compared to wild-type (wt) mice ( figure c ). to determine the impact of ifit on the outcome of viral infections in vivo, we compared susceptibilities of ifit / and wt mice to vsv infection, using ifnar / mice as positive controls of enhanced susceptibility. virus was administered at a low dose [ plaque forming units (pfu)], intranasally, reflecting a natural route of infection for vsv [ ] . as seen previously, % of ifnar / mice rapidly succumbed to vsv infection within days ( figure a , and [ ] ), after suffering symptoms of lethargy. on the other hand, % of wt mice survived, the remaining % succumbed to vsv, and this occurred later, at - days post infection (d.p.i.). in contrast, % of ifit / mice died by d.p.i. (figure a ), with most succumbing by d.p.i.; thus, we observed uniform and more rapidly occurring death of ifit / compared to wt mice after vsv infection. within h before death, both wt and ifit / mice developed neurological signs including ataxia, hind limb paralysis, and hyper-excitability. ifit +/ mice displayed an intermediate survival curve, demonstrating a gene dosage effect ( figure b ). next, the role of a related gene, ifit , in vsv pathogenesis was evaluated by infecting ifit / mice. unlike the results observed with ifit / mice, no statistically significant increase in mortality was observed in ifit / mice ( figure b , % death for wt versus % for ifit / , respectively; p. . ). consistent with this, survival kinetics of ifit / and wt mice were similar. increasing the virus dose by , -fold (to pfu) did not appreciably change the survival curves of wt, ifit / , or ifit / mice ( figure c ). these results demonstrate functional differences between the two closely related proteins encoded by ifit and ifit . the virus-specificity of the antiviral action of ifit was evaluated by infecting ifit / mice with emcv, an unrelated neurovirulent positive-strand rna virus of the picornavirus family ( figure d ). ifnar / mice were highly susceptible to emcv infection with all mice succumbing within d.p.i.; in contrast, wt mice died with a slower kinetics and at a rate of only %. notably, ifit / mice behaved similarly to the wt mice, without enhanced or accelerated mortality ( figure d ). the same conclusion was true for a lower dose of emcv ( figure s ). the survival pattern of emcv-infected ifit / mice also was similar to that of the wt mice ( figure d ). mice of all genotypes either succumbed after developing neurological symptoms, mainly hind limb paralysis, or survived without symptoms. these results demonstrate that the antiviral action of ifit is both virus-and ifitspecific. the uniform penetrance of neuropathogenesis and lethality of vsv-infected ifit / mice, even at a low virus dose, prompted us to examine viral spread along its route from the nasal cavity into the cns ( figure a ). after intranasal administration, vsv infects in mammals, the first line of defense against virus infection is the interferon system. viruses induce synthesis of interferon in the infected cells and its secretion to circulation. interferon acts upon the as yet uninfected cells and protects them from oncoming infection by inducing the synthesis of hundreds of new proteins, many of which interfere with virus replication. vesicular stomatitis virus (vsv), a virus similar to rabies virus, is very sensitive to interferon but it is not known which interferon-induced protein inhibits its replication. here, we have identified a single interferon-induced protein as the protector of mice from death by vsv infection. knocking out the gene encoding this protein, ifit , made mice very vulnerable to neuropathogenesis caused by vsv infection; a related protein, ifit , did not share this property. moreover, ifit failed to protect mice from another neurotropic virus, encephalomyocarditis virus, nor was it necessary for protecting organs other than brain from infection by vsv. our observation that a single ifn-induced protein protects a specific organ from infection by a specific virus revealed an unexpected degree of specificity of the antiviral action of ifn. shared wt mice (n = number of animals used). c, survival of ifit / , ifit / and wt mice after intranasal infection with a higher dose of vsv ( pfu). d, survival of ifit / , ifit / , ifnar / and wt mice after infection with pfu of emcv. in a-d, data are cumulative from at least two independent experiments (exceptions: figure b , ifit +/ mice and figure d , ifit / mice infected in a single experiment). statistical significance of survival differences relative to wt mice is indicated by p-values; n.s., not significant; i.n., intranasal. doi: . /journal.ppat. .g the nasal epithelia including olfactory sensor neurons, which project to the outer layer of the olfactory bulbs (ob) [ ] . this represents the entry step into the cns, which we examined by immunostaining of ob sections. in wt mice, vsv p protein was detected exclusively within the glomeruli of the ob at d.p.i. ( figure b , upper right panel and [ ] ), whereas in ifnar / mice, vsv antigen had spread into deeper layers of the ob ( figure b , lower left panel). in ifit / mice ob, viral antigen was restricted to the glomeruli, as seen in wt mice ( figure b , lower right panel). this similar pattern of viral antigen expression between wt and ifit / mice was reflected in the equivalent levels of viral rna in ob at d.p.i. ( figure c ). in contrast, , times more vsv rna was present in ob of ifnar / mice ( figure c , right panel, p, . ). a comparison of the infectious viral burden between wt and ifit / mice in the ob confirmed these findings: at d.p.i., , pfu/g of vsv was present in both wt and ifit / mice ( figure d , p = . ). however, later in the course of infection, by day , viral ob titers in ifit / mice were not significantly changed, whereas in wt mice average titers of infectious vsv as well as viral rna levels had decreased by , -fold ( figure c and d, both p, . ). these results suggest that vsv initially enters and replicates with similar efficiency in both wt and ifit / ob before spreading into the rest of the brain. the efficiency of vsv replication in the brain, excluding the ob, was examined by quantifying infectious vsv as well as viral rna. early after infection, at d.p.i., virus titers in brains were low (, to pfu/g) and roughly equivalent in wt and ifit / mice ( figure a , p. . ). similarly, viral rna levels at the same figure . ifit does not inhibit vsv entry and replication in olfactory bulbs. a, schematic entry route of vsv into the central nervous system of wt mice after intranasal infection, and vsv spread within brain, as reported in the literature. ob, olfactory bulbs; cx, cortex; mb, midbrain; cb, cerebellum; bs, brain stem; sc, spinal cord. b, vsv p protein in ob of vsv-infected wt, ifit / and ifnar / mice at d.p.i., detected by immunohistofluorescence. c, vsv rna levels in ob of uninfected or vsv-infected wt, ifit / and ifnar / mice at , or d.p.i., plotted as mean+sd on log scale; nd, none detected. d, infectious vsv titers in wt and ifit / ob at and d.p.i.; plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. in c and d, n = - mice per infected group accumulated from three independent experiments; in b, n = mice from two independent experiments. all infections were pfu of vsv administered intranasally. asterisks indicate statistical significance: ** p = . , * p, . ; n.s.: not significant. doi: . /journal.ppat. .g time were low and comparable between wt and ifit / ( figure b , p. . ). however, at the same time, levels of vsv rna ( -fold, p, . ) were much higher in the brains of ifnar / mice ( figure b , right panel). later in the course of infection ( d.p.i.), brains of wt mice accumulated only , -fold more infectious vsv, with occasional clearance of the virus. in contrast, we detected markedly higher vsv titers in the brains of ifit / mice (, -fold higher compared to wt mice, p = . ), reaching , pfu/g ( figure a ); the high virus load likely caused the pronounced lethality. differences in viral rna levels in brains of wt and ifit / mice at d.p.i. correlated well with levels of infectious vsv ( figure b ). to determine whether ifit selectively restricts replication of vsv in particular regions of the brain, we measured viral rna levels in cortex, midbrain, cerebellum and brain stem at d.p.i. in wt mice, vsv rna was present prominently in the cortex, midbrain and brainstem, but not in the cerebellum ( figure c ), which is consistent with published results [ ] . however, in ifit / mice, viral rna was -fold or more (p, . ) abundant in all regions of the brain examined, including the cerebellum. the increase of vsv replication in ifit / brains was not due to a broadened cell tropism of the virus; immunostaining for viral p protein showed exclusive localization to neurons and not other cell types, such as astrocytes ( figure d ). from the above observations, we conclude that after intranasal infection by vsv, ifit protects mice from neuropathogenesis by suppressing replication or spread of the virus in brain neurons. ifit and ifit are induced in vsv-infected regions of ob and brain the protective effect of type i ifn signaling and in particular, ifit , against vsv neuropathogenesis prompted us to confirm its expression in ob and brain of wt mice, and whether it was induced in a type i ifn-dependent manner. in wt ob, ifit , ifit , and ifn-b mrna was induced strongly by d.p.i., and ifit and ifit rna remained abundant until day d.p.i. (figure a ). the induction of these genes was dependent on type i ifn receptor in ob as well as in brain ( figure b and e, and data not shown). ifit suppresses vsv replication in the brain after intranasal infection. a, infectious vsv titers in wt and ifit / brains at and days after intranasal infection, plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. b, vsv rna levels in brains of uninfected or vsv-infected wt, ifit / and ifnar / mice at or d.p.i., plotted as mean+sd on log scale. c, vsv rna levels in different regions of the brains of uninfected or vsv-infected wt and ifit / mice at d.p.i., plotted as mean+sd on log scale. d, vsv p protein in midbrain neurons of ifit / mice at d.p.i.; detection by immunohistofluorescence-labeling of vsv-p (red) and neuron (neun) or astrocyte (gfap) markers (green); in a and b: n = - mice per infected group accumulated from three independent experiments; in c: n = mice per infected group; in d: n = mice per infected group; all infections in a-d were intranasal with pfu of vsv. nd, none detected. brains in a and b were separated from obs assayed in figure d and c, respectively. asterisks indicate statistical significance: *** p# . ; n.s.: not significant. doi: . /journal.ppat. .g furthermore, expression of ifit mrna in wt ob coincided with the presence of detectable levels of the encoded ifit protein ( = p ) at d.p.i. and d.p.i., as seen by immunohistochemistry ( figure c , and data not shown). ifit protein staining was observed in vsv-infected cells within ob glomeruli as well as in surrounding and distant viral antigen-free cells, consistent with a remote ifn-dependent induction of ifit expression ( figure c , arrowheads in magnified images of right panel). ifit and ifn-b mrnas were induced as strongly in ob of ifit / as in wt mice, which correlated well with similar abundance of vsv rna in wt and ifit / ob (figure a compared to figure c ). in brains, at d.p.i., in contrast to ob, induction of ifit and ifn-b mrnas was considerably stronger in ifit / mice compared to wt mice ( figure d , -fold and -fold, respectively, both p, . ). the enhanced gene induction in vsv-infected ifit / mice was not restricted to specific regions of the brain ( figure s ). enhanced cellular gene expression also was observed for several virusinduced cytokine and chemokine genes, as measured by quantitative rt-pcr ( figure s a ). gene expression profiling of brain tissue at day d.p.i., using microarray analysis, revealed that many other genes, including isgs, were also more strongly induced (table s ). these results demonstrated that enhanced virus replication in the brains of ifit / mice led to enhanced type i ifn, other cytokines and isg induction, which nevertheless failed to restrict vsv replication in the absence of ifit . wt mice are as susceptible as ifit / mice to intracranial vsv infection our results from intranasal vsv infection indicated that ifit induction in the brain was mediated by type i ifn that was, in all likelihood, produced by infected cells in the ob ( figure a ). virus replication and resultant ifn induction at d.p.i. were similar in the obs of wt and ifit / mice (figs. c, d and a); presumably, the newly produced ifn diffused into the rest of the brain and induced local ifit expression in the wt mouse brains, prior to the arrival of the infectious virus. if this were the case, one would anticipate that direct infection of the brain, without prior action of ifn produced in infected ob, would minimize the difference between the phenotypes of wt and ifit / mice. to test this idea, we injected a very low dose ( pfu) of vsv intracranially. as hypothesized, wt and ifit / mice were now equally susceptible; almost all mice died by d.p.i. even at this low dose ( figure a ) and there were equally high virus titers and viral rna levels in the brains of mice of both genotypes ( figure b and c). concomitant with virus replication, there was similar induction of ifit and ifn-b ( figure c ) and other cytokines and chemokines ( figure s b ). these results indicate that in the absence of prior induction of ifit by ifn, brain neurons are highly susceptible to vsv infection. unlike the brain, other organs of ifit / mice are not more susceptible to intranasal vsv infection ifnar / mice succumbed within two days after vsv infection without accumulating very high vsv rna levels in the brain ( figure b ). these mice did not develop cns-related signs of disease, but showed severe lethargy before death, suggesting that death was due to efficient replication of the virus in peripheral organs, due to the absence of an otherwise effective type i ifn-mediated antiviral protection of the same organs in wt mice. to test this, we assessed the kinetics of vsv accumulation in brains, livers and lungs of wt, ifnar / and ifit / mice (figure ) . at d.p.i., vsv titers were very high in the liver of ifnar / mice, reaching pfu/g ( figure a ). in contrast, no or little infectious virus was detected in the liver of wt mice at or d.p.i., indicating efficient ifn-dependent suppression of vsv replication; intriguingly, this was also observed in ifit / mice, demonstrating that ifit did not mediate the anti-vsv effects of type i ifn in the liver. in lungs, which directly received a part of the virus inoculum from intranasal inhalation of vsv, the virus also replicated efficiently in ifnar / mice, reaching pfu/g before death ( figure b ). in comparison, lungs of wt and ifit / mice exhibited much lower levels of vsv at and d.p.i. ( , to , -fold lower for wt and ifit / compared to ifnar / mice, all p, . ). by days and d.p.i., the virus was cleared from the lungs of a subset of wt and ifit / mice. in contrast, in brains from the same animals, to -fold higher average titers (p, . ) of vsv accumulated in ifit / compared to wt mice at all time points between and d.p.i. ( figure c ). as expected, in wt mice, both ifit and ifit were induced not only in brains ( figure d ), but also in livers ( figure d ) and lungs ( figure e ); ifn-b was also induced in lungs, but not livers. ifit , ifit and ifn-b mrnas were also induced in the brains of emcv-infected wt mice ( figure s c ). these findings demonstrate an unexpected brain-restricted and virus-restricted function of ifit in the context of the type i ifn-mediated antiviral response to vsv infection. they also indicate that in ifit / mice, other isgs, which presumably protect the peripheral organs of vsv-infected wt mice, are either not induced in neurons or insufficient to protect them. ifns are defined by their antiviral activities. they inhibit the replication of many, if not all, viruses mostly by direct inhibition of replication in the infected cells but also by promoting the ability of immune cells to recognize and eliminate the virus-infected cells [ ] . the direct effects are mediated by isgs, which number in the hundreds, and different isgs are thought to have more potent antiviral activities toward different families of viruses [ ] . however, in most cases, it is not known which isg inhibits the replication of a given virus; the rare exception is the mx-mediated inhibition of influenza viruses, the underlying effect which allowed for the discovery of ifns [ ] . the task of connecting a specific ifn-induced protein to a specific antiviral action is compounded by the fact that often several ifn-induced proteins act in concert to inhibit the same virus at different stages of its life cycle. moreover, a specific ifn-induced protein may be more relevant for inhibiting a virus in one specific cell-type than another. recent systematic investigation of the specific antiviral effects of different isgs has started providing significant insight into this problem [ ] . such findings are complemented by the analyses of the spectra of the antiviral effects of a specific isg or a family of isgs [ ] . we have undertaken an investigation of the ifit family of mouse isgs. the corresponding human proteins are known to have antiviral activities against human papillomavirus (hpv) and hepatitis c virus (hcv), neither of which replicate in mouse cells. the anti-hpv activity of human ifit ( = p ) has been attributed to its ability to bind hpv e protein and to inhibit its helicase activity, which is essential for hpv dna replication [ , ] . the antiviral effect on hcv, on the other hand, is manifested at the level of inhibiting viral protein synthesis as a consequence of the ability of ifit to bind the translation initiation factor eif and inhibit its various actions in translation initiation [ ] . it has been reported recently that the ifit protein can form a complex and bind to rnas with triphosphorylated ends, presumably providing another means to inhibit specific viruses that produce such rnas [ ] . the ifit genes are clustered at a single locus in both human and mouse. in the latter species, two alleles of ifit genes are flanked on two sides by one allele of ifit and one allele of ifit [ ] . to identify their physiological functions, we have separately deleted the entire coding regions of ifit or ifit genes. the ifit / mice exhibited an interesting phenotype in allowing the replication of and resultant pathogenesis by a wnv mutant, which failed to replicate in wt mice [ ] . because this mutant is defective in -o methylation of the cap structure of viral mrnas, its rescue in the ifit / mouse indicates that this antiviral protein recognizes the ends of mrnas, a conclusion that is consistent with the observation that, in vitro, it can bind to rnas having specific structures at the ends [ ] . it remains to be seen whether the proposed property of ifit proteins to recognize ends of rna is connected in any way to their ability to inhibit the functions of eif [ ] , which participates in several steps of translation initiation taking place at or near the ends of mrnas. replication of vsv is highly sensitive to the antiviral activity of ifns, and vsv is widely used to determine the specific activities of ifn preparations quantitatively [ ] . in spite of this strong connection, it is unclear how ifn inhibits vsv replication. an early report indicated that viral primary transcription is inhibited by ifn, but it is not known which ifn-induced protein mediates this inhibition [ ] . the observed sensitivity of vsv replication in vitro is reflected in vivo. ifnar / mice are extremely susceptible to vsv infection; they rapidly die within days after infection and the virus replicates to very high titers in many organs of the infected mice. the extreme sensitivity of ifnar / mice to vsv infection suggests that type i ifn provides the majority, if not all, of the protective innate immune defense. eventually, protection may be facilitated by immune cell-mediated antiviral actions, but this is a slow process that does not appear to function before - days post-infection [ , ] . thus, it is likely that one or more isgs directly inhibit vsv replication in vivo. in this context, it has been reported that mice lacking pkr, a well-studied isg, display higher susceptibility to vsv pathogenesis [ ] . however, detailed investigation of the underlying mechanism revealed that pkr did not execute ifn's antiviral action; rather, it was required for efficient induction of ifn-a/b in the infected mice [ ] . in vivo vsv-infection induces ifn synthesis in many cell types, using either the cytoplasmic rig-i pathway or the endosomal tlr pathway [ , ] ; however, it is unclear how pkr aids this process. our results show that ifit / mice are highly susceptible to intranasal vsv infection and the effect is gene dosage-dependent: ifit +/ mice had an intermediate susceptibility phenotype. infected ifit / mice displayed symptoms of severe neuropathogenesis late after vsv infection accompanied by efficient replication of the virus in many regions of the brain. however, virus replication was restricted to neurons and did not spread to other types of cells in the brain, such as astrocytes. our results are consistent with the hypothesis that prior, ifn-induced, ifit expression in the brain restricts vsv replication. supporting genetic evidence for the requirement of ifn action is provided by the high susceptibility of the ifnar / mice, which possess the functional ifit gene but ifit is not induced by vsv infection because these mice cannot respond to type i ifn. additional evidence comes from a previous study using brain-specific ifnar / mice, which displayed a pattern of susceptibility to intranasal vsv infection similar to that of our ifit / mice [ ] . in our experimental system, the source of the ifn production was figure c ). ifit was also induced at this time in the rest of the brain, without any induction of ifn mrna ( figure d ) suggesting that the source of ifn was the ob. in accord with the well-established concept of ifn action, pre-induction of ifit in neurons, before the onset of infection, was essential for the antiviral effect. in comparison, induction of ifn and ifit that was concomitant with vsv infection failed to have an appreciable antiviral effect, as manifested by robust virus replication at directly infected sites, such as the obs of wt mice infected intranasally ( figure d ) or the brain of wt mice infected intracranially ( figure b ). high mortality of the infected mice correlated with high virus titers in the brains of intranasally infected ifit / mice or intracranially infected wt and ifit / mice. in the intranasally infected ifit / mice, death was not preceded by widespread apoptosis in the brain ( figure s ). however, as expected with high viral loads, ifn and other cytokines and chemokines were strongly induced ( figures d, s and s a) ; consequently, many isgs, except ifit , were also induced (table s ) . pre-induced ifit prevents efficient vsv replication in the brain, most probably by blocking one or more essential step of the viral life cycle including viral entry, uncoating, primary transcription, viral protein synthesis, rna replication, virion assembly or egress. it also might block trans-synaptic spread of the virus, although unlike another rhabdovirus, rabies virus, vsv is not known to depend on transit from neuron to neuron. in this context, it is important to note the observations made by iannacone et al. [ ] using a footpad vsv infection model. they concluded that type i ifn, produced by infected macrophages and plasmacytoid dendritic cells in infected mice, blocked infection of peripheral neurons resulting in lowered infection of the cns and prevention of neuropathogenesis. it is worth noting that in our studies, the absence of ifit did not affect ifn induction by vsv (figures a and c ). further investigation of the biochemical mechanism behind the observed in vivo effect of ifit / is hampered by the absence of a suitable cell culture model of the phenomenon. for example, ifit was not required for mediating the anti-vsv effect of ifn in mouse embryonic fibroblasts ( figure s ), in primary fetal neurons or in ifit -ablated neuroblastoma cells (data not shown), results that are not surprising given the strong tissuespecificity of ifit action observed in vivo (figure ) . specific rnabinding properties of ifit proteins have been recently reported [ ] . following this lead, we examined the rna-binding properties of recombinant murine ifit and ifit using vsv leader rna as the probe in an electrophoretic mobility shift assay: ifit bound rna with a -ppp end but not with a -oh end; however, ifit bound neither ( figure s ). to obtain meaningful leads, future investigation of this kind may require using brain extracts from infected mice to detect protein-viral rna complexes that may contain ifit along with adult neuronspecific proteins. our results revealed several layers of specificity of ifn action, some of which were not anticipated. first, compared to ifit / mice, ifit / mice were much less susceptible to intranasal vsv infection; this was true for both low and high doses of virus. this finding was surprising in view of a recent report on vsv susceptibility of ifit / mice [ ] and the observation that ifit , but not ifit , could bind vsv leader rna in vitro ( figure s ) . the above results demonstrate that different ifit proteins have nonredundant functions in vivo. the second layer of specificity was directed toward the nature of the infecting virus. although both vsv and emcv caused neuroinvasive disease, induced ifn-b, ifit and ifit in the brain and type i ifn action was required for protection against both viruses, ifit was critical only for protection against vsv; the absence of either ifit or ifit did not exacerbate susceptibility to emcv. the third layer of specificity was revealed by the organ-specific action of ifit . in the complete absence of type i ifn action in the ifnar / mice, intranasally infected vsv replicated vigorously not only in brains, but also in livers and lungs ( figure a-c) . in contrast, in ifit / mice, efficient vsv replication was restricted to the brain suggesting that ifit does not act as an anti-vsv isg in the liver or the lung because its absence did not impact virus titers, even though ifit was induced in these organs of infected wt mice ( figure d and e) . the efficient vsv replication in livers and lungs of ifnar / mice, but not wt and ifit / mice, indicates that other isgs must have anti-vsv effects in those organs. further investigation is needed to determine the basis of neuronal specificity of ifit action and the identities of other isgs that inhibit vsv replication in other organs. all animal experiments were performed in strict accordance with all provisions of the animal welfare act, the guide for the care and use of laboratory animals, and the phs policy on humane care and use of laboratory animals. the protocol was approved by the cleveland clinic institutional animal care and use committee (iacuc), phs assurance number a - . all experimental manipulations or intranasal instillations of mice were performed under anesthesia induced by pentobarbital sodium or isofluorane, respectively, and all efforts were made to minimize suffering. all mice used were of c bl/ background and of both sexes; ifit / mice were custom-generated by taconic farms, inc. by flanking exons and of ifit , encompassing the complete protein-encoding region, with frt sites in c bl/ embryonic stem (es) cells, and deleting the flanked region by transfection of flp recombinase. es cell clones were injected into bl/ blastocysts, and heterozygous offspring mice were crossed to homozygosity. ifit / mice were generated from c bl/ es cells lacking the whole coding region of ifit ( ); es cells were obtained from the nih knockout mouse project (komp, allele ifit tm (komp)vlcg ). the same es cell line was independently used to generate mice in another study [ ] . ifnar / mice (lacking ifnar ) were a gift of murali-krishna kaja (emory university, atlanta, ga). congenic wild-type mice were obtained from taconic farms. vesicular stomatitis virus (vsv) indiana was a gift from amiya k. banerjee, lerner research institute, cleveland, ohio. for intranasal infections, between and pfu of vsv in ml of endotoxin-free pbs were inhaled by isofluorane-anesthetized - week-old mice, with pbs-only as control. for intracranial infections, pfu of vsv in ml of endotoxin-free pbs were injected into the brains of - week-old mice, with pbsonly as control. thereafter, mice were monitored daily (twice daily after i.c. injection) for weight loss and symptoms of disease. encephalomyocarditis virus (emcv) k strain was a gift from robert h. silverman, lerner research institute, cleveland, ohio. for intraperitoneal infections, between and pfu of emcv in ml of pbs were injected into the peritoneal cavity of mice. mice were monitored daily for weight loss and symptoms of disease. mice were anesthetized with pentobarbital ( mg/kg) and blood was removed from organs by cardiac perfusion with ml of pbs, followed by perfusion with ml of % paraformaldehyde/pbs for fixation. brains were placed in % paraformaldehyde overnight for complete fixation, submerged in % sucrose/ pbs overnight for cryoprotection, and frozen in o.c.t. compound (sakura finetek usa, torrance, ca, usa). mm sagittal sections were cut at uc in a leica cm cryostat, mounted on coated slides (superfrost plus, fisherbrand, fisher scientific); membranes were permeabilized by . % triton x- /pbs treatment for min. for immunohistochemistry, the envision+ dab kit (dako, carpinteria, ca) was used with antimouse ifit /p [ ] or anti-vsv-p protein (a gift from amiya k. banerjee, lerner research institute, cleveland, ohio) as primary antibodies. for immunohistofluorescence, anti-vsv-p or anti-neun (chemicon intl./millipore, billerica, ma) or anti-gfap (sigma-aldrich, st. louis, mo) were used; labeled brain sections were stained with alexafluor- secondary antibody (invitrogen/molecular probes, carlsbad, ca). for detection of apoptotic cells in brain sections, the deadend fluorometric tunel system (promega) was used according to manufacturer's instructions. all objects were then mounted with vectashield (with dapi, vector labs, burlingame, ca), and examined with a leica drm fluorescence microscope. mice were anesthetized with pentobarbital ( mg/kg) and blood was removed from organs after cardiac perfusion with ml of pbs. brains were separated into olfactory bulbs and the remainder of the brain, snap-frozen in liquid nitrogen (as well as livers and lungs) and rna was extracted using trizol reagent (invitrogen). dnase i treatment (dnafree, applied biosystems/ ambion) and reverse transcription with random hexamers (improm-ii, promega) were performed according to manufacturer's instructions. . ng of rna was used in well-format realtime pcrs in a roche lightcycler ii using applied biosystem's sybr green pcr core reagents. pcr primers for murine isg /ifit , isg /ifit , isg /ifit and s rrna have been published previously [ ] ; primers targeting murine ifnb [ -cttctccgtcatctccataggg- [ ] , with the alternative reverse primer: -cacagccctctccatcaact- ], vsv n rna [ ] or emcv d polymerase genomic region [ ] were described previously. primers for ccl , il b, il , tnf, il b and nos have been described previously [ , ] . average expression levels, relative to s rrna and normalized by use of calibrator samples, were graphed with prism . software. for analysis of different regions of the brain, brains without ob of perfused mice were separated into cortex, cerebellum, brain stem and remaining ''midbrain'', and tissue was submerged into rnalater stabilizing reagent (qiagen) overnight and frozen. rna was then extracted via trizol and further processed and assayed by realtime rt-pcr as described above. for microarray analysis, trizol-extracted and dnase i-treated rna was additionally purified using spin columns (rneasy mini kit, qiagen) before subjection to mrna expression microarray analysis via illumina mouse ref- v beadchip and genomestudio software v . (illumina, inc.); rna hybridization to chips was performed by the lerner research institute genomics core at the cleveland clinic. microarray raw data were deposited in the ncbi gene expression omnibus (geo), accession number gse . for quantification of infectious vsv in organs, mice were anesthetized with pentobarbital ( mg/kg) and blood was removed from organs by cardiac perfusion with ml of pbs. organs were snap-frozen in liquid nitrogen, weighed, pestle/tubehomogenized (kimble/kontes) in ml of pbs per brain or peripheral organ or . ml per pair of olfactory bulbs, and virus was titered in -fold serial dilutions on vero cells by plaque assay. results are expressed as plaque-forming units (pfu) per gram of tissue. for quantification of infectious vsv yields in mef, cells ( /+ifn-b pretreatment as indicated) were infected with vsv inoculum for h, and after another h, cells were freeze/ thawed, and cleared supernatants of lysates were assayed for vsv by plaque assay on vero cells. primary murine embryonic fibroblasts (mefs) were stimulated with u/ml murine ifn-b (pbl, inc., piscataway, nj) for h and lysed in lysis buffer [ mm tris ph . , mm nacl, . % triton x- , mm sodium orthovanadate, mm sodium fluoride, mm sodium pyrophosphate, mm bglycerophosphate and complete edta-free protease inhibitor (roche, indianapolis, in)]. mg of whole cell extract were separated via % sds-page, transferred to pvdf membranes, blocked with % dry milk in tris-buffered saline/ . % tween- overnight and labeled with anti-ifit /p , anti-ifit /p or anti-ifit /p polyclonal rabbit sera [ , ] . single-stranded vsv leader rna (nucleotides - ) was t polymerase-transcribed in presence of [a- p]-ctp, yielding radiolabeled -triphosphorylated (ppp-) rna, followed by alkaline phosphatase treatment for generation of -hydroxyl (ho-) rna. ppp-rna or ho-rna were added to bacterially expressed and purified xhis-tagged ifit or ifit protein in reaction buffer ( mm tris ph . , mm nacl, mm edta, mm dtt, . % triton x- , % glycerol) and incubated for min on ice. reaction products were separated by % native polyacrylamide gel electrophoresis followed by exposure to film. statistical significance of mouse survival differences was calculated by mantel-cox log rank test. to assess significance of differences in gene expressions or virus titers, the two-tailed mann-whitney test was used. all calculations were performed using graphpad prism . software. previously published transcript sequences in the ncbi entrez nucleotide database: ifit , nm_ ; ifit , nm_ ; ifit , nm_ ; ifnb , nm_ ; ifnar , nm_ . figure s survival of wt and ifit / mice after infection with low emcv dose ( pfu). statistical significance of survival differences is indicated by p-value; n.s., not significant. (pdf) figure s enhanced isg and ifn-b induction in intranasally vsv-infected ifit / brain regions. ifn-b-, and ifit / / mrna levels in different regions of brains of uninfected or vsv-infected wt and ifit / mice at d.p.i., plotted as mean+sd. n = mice per infected group; nd, not done. infections were intranasal with pfu of vsv. (pdf) figure s gene induction in brains after vsv or emcv infections. a, mrna levels of select genes in brains (without obs) of uninfected or intranasally vsv-infected wt and ifit / mice at d.p.i., plotted as mean+sd; n = mice per infected group; infection was intranasal with pfu of vsv. b, mrna levels of select genes in brains (without obs) of uninfected or intracranially vsv-infected wt and ifit / mice at h post injection, plotted as mean+sd; n = mice per infected group; infection was intracranial injection with pfu of vsv. c, ifit , ifit , ifn-b and emcv rna levels in brains days after emcv infection ( pfu, n = mice per infected group). (pdf) figure s region-selective induction of apoptosis in brains of intranasally vsv-infected ifit / mice. ifit / mice were i.n. infected with pfu of vsv; at d.p.i., adjacent sections of fixed brains were labeled to detect apoptotic cells (tunel) or vsv p protein (immunohistofluorescence), n = mice; only few regions such as striatum show positive tunel; infected wt brains and uninfected control brains of either genotype did not show appreciable signals, hence data not shown). (pdf) figure s vsv yields from infected wt and ifit / mef. immortalized mef were treated for h with u/ml ifn-b and infected with vsv at moi . hours after infection, virus yields were determined by plaque assay. results are plotted as mean+sd on log scale, representing one of two independent experiments. (pdf) figure s murine ifit protein does not bind ppp-rna. single-stranded radiolabeled vsv leader rnas (nt - ) with either -triphosphorylated or free -hydroxyl-ends (ppp-rna or ho-rna) were in vitro incubated with purified murine ifit ( = p ) or ifit ( = p ) proteins; formation of protein/rna complex was detected by electrophoretic mobility shift assay. (pdf) table s enhanced gene expression in brains incl. obs of intranasally vsv-infected ifit / versus wt mice at d.p.i. wt or ifit / mice were intranasally vsv-infected with pfu, and at or d.p.i., brain (incl. ob) rna expression profiles were obtained by microarray. genes are ranked by their ''fold expression level in ifit / over wt at d.p.i.''. only genes with at least -fold higher expression level in ifit / are included. note: the ifit /isg probe of the illumina mouse ref- chip is defective and therefore the gene is not included in this list. 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deficiency ameliorates viral encephalitis without affecting viral control factors supporting intrathecal humoral responses following viral encephalomyelitis we thank michifumi yamashita and niranjan butchi for sharing technical expertise. key: cord- - s hpakk authors: kwok, hoi-hin; poon, po-ying; fok, siu-ping; ying-kit yue, patrick; mak, nai-ki; chan, michael chi-wai; peiris, joseph sriyal malik; wong, ricky ngok-shun title: anti-inflammatory effects of indirubin derivatives on influenza a virus-infected human pulmonary microvascular endothelial cells date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: s hpakk influenza a virus (iav) poses global threats to human health. acute respiratory distress syndrome and multi-organ dysfunction are major complications in patients with severe influenza infection. this may be explained by the recent studies which highlighted the role of the pulmonary endothelium as the center of innate immune cells recruitment and excessive pro-inflammatory cytokines production. in this report, we examined the potential immunomodulatory effects of two indirubin derivatives, indirubin- ′-( , -dihydroxypropyl)-oximether (e ) and indirubin- ′-oxime (e ), on iav (h n ) infected-human pulmonary microvascular endothelial cells (hpmecs). infection of h n on hpmecs induced a high level of chemokines and cytokines production including ip- , rantes, il- , ifn-β and ifn-γ . post-treatment of e or e could significantly suppress the production of these cytokines. h n infection rapidly triggered the activation of innate immunity through phosphorylation of signaling molecules including mitogen-activated protein kinases (mapks) and signal transducer and activator of transcription (stat) proteins. using specific inhibitors or small-interfering rna, we confirmed that indirubin derivatives can suppress h n -induced cytokines production through mapks and stat signaling pathways. these results underscore the immunomodulatory effects of indirubin derivatives on pulmonary endothelium and its therapeutic potential on iav-infection. scientific reports | : | doi: . /srep manufacturer's instruction. complementary dna was synthesized from dnase-treated total rna using superscript ii first-strand synthesis system (invitrogen). the relative expression of target gene was quantified by real-time rt-pcr using kapa sybr fast abi prism qpcr kit (kapa biosystems, woburn, ma, usa) and detected by a steponeplus real-time pcr system (applied biosystems, foster city, ca, usa). the relative expression of target gene was normalized by the level of glyceraldehyde- -phosphate dehydrogenase (gapdh), and then calculated by the comparative ct method. plaque assay. the virus titers were determined by standard plaque assay on madin-darby canine kidney (mdck) cells. in brief, mdck cells were grown in mem and seeded onto -well plates. diluted cell culture medium from influenza virus-infected hpmecs were added to the confluent mdck cells monolayers for h. then, the inoculum was removed, and a mixture of agarose ( %, w/v) containing l-(tosylamido- -phenyl) ethyl chloromethyl ketone (tpck) ( μ g/ml) was added onto the mdck cells monolayers. after h of incubation, the plate was fixed by formaldehyde ( %, v/v) overnight and then the agarose was discarded. the plaques were counted after staining with crystal violet ( . %, w/v). were prepared by ne-per nuclear and cytoplasmic extraction reagents (thermo scientific, rockford, il, usa) according to the manufacturer's protocol. for extraction of whole-cell lysate, cells were lysed by cytobuster tm protein extraction reagent (novagen, madison, wi, usa) containing protease ( . %, v/v) and phosphatase inhibitor cocktails ( . %, v/v) (calbiochem, san diego, ca, usa). the cell lysate was collected after centrifugation. protein concentration of the sample was determined by the detergent-compatible protein assay (bio-rad, hercules, ca, usa). equal amounts of protein were loaded and separated by % sds-page followed by electroblotting onto nitrocellulose membrane. the membrane was soaked in blocking buffer ( % non-fat milk in tbs-t, w/v) and then incubated with specific primary antibodies overnight at °c and secondary antibody for h at room temperature. immunoreactive bands were visualized using supersignal west pico kit (thermo scientific). in vitro mitogen-activated protein kinases assay. to detect the activity of individual mapks after treatment with iav and indirubin derivatives, the non-radioactive in vitro protein kinase assay kit from cell signaling technology was used. in brief, the sepharose bead-immobilized antibody was used to immunoprecipitate active mapks from an equal amount of total cell lysate ( μ g) overnight. the immunoprecipitate was washed twice with cell lysis buffer and kinase reaction buffer. the immunoprecipitate were then incubated with indirubin derivatives e or e ( μ m) for min before addition of atp. subsequently, kinase reactions using corresponding protein substrate were performed at °c for min. the kinase reaction was stopped with sds loading buffer. phosphorylation of protein substrate was detected by immunoblotting with specific antibody. immunofluorescence microscopy. hpmecs at a density × were seeded onto a glass coverslip in a -well plate. after treatment for the indicated time, cells were fixed with % paraformaldehyde for min at room temperature. cells were incubated with primary antibody ( : dilution rabbit anti-phospho-stat (tyr ) antibody) overnight at °c. the coverslip was washed and then incubated with fitc-conjugated goat anti-rabbit secondary antibody ( : dilution) (invitrogen) for h at room temperature. nuclei were visualized by staining with dapi ( . μ g/ml). the coverslip was washed and mounted on a slide using dako fluorescence mounting medium (carpinteria, ca, usa). fluorescence image was captured by the olympus fluoview fv confocal laser-scanning microscope (tokyo, japan). small interfering rna (sirna) transfection. transfection of sirna was performed using lipofectamine rnaimax (invitrogen). non-targeting-sirna ( nm) was used in parallel with stat -specific sirna ( nm) (ambion, austin, tx, usa). cells plated at % confluence were transfected in opti-mem medium (gibco brl, grand island, ny, usa) for h. after transfection, cells were rinsed with opti-mem prior to further treatment. all results were expressed as mean ± standard derivation (s.d.) of at least independent experiments. statistical significance between groups was determined by one-way anova with tukey's post hoc test. p < . was considered to be statistically significant. influenza a virus h n is a potent inducer of cytokines production in pulmonary endothelial cells. recent studies suggested that lung endothelium is the central regulator of cytokine amplification during influenza a virus infection, while dysregulation of cytokines production may result in systemic inflammation . in this study, we found that the infection of influenza a virus subtype a/quali/hong kong/g / (h n ) on hpmecs induced excessive production of various pro-inflammatory cytokines and chemokines, including ip- ( to examine the immunomodulatory effects of indirubin derivatives, hpmecs were infected with h n for h followed by incubation with indirubin derivatives e or e for another h. we have tested the cytotoxicities of indirubin derivatives in hpmecs prior to the elisa. as shown in fig. was observed at or below μ m in hpmces. next, indirubin derivatives were found to suppress h n -induced ip- ( fig. a) , rantes (fig. b ) and il- ( fig. c ) expression in a concentration-related manner. e significantly inhibited cytokines expression at μ m, a similar inhibitory effect was observed when a higher concentration of e ( μ m) was used. both indirubin derivatives slightly induced and inhibited the basal level of rantes and il- respectively. it may be due to the regulatory effects of indirubins on innate immunity . for . similar to the results of direct virus infection, viral rna stimulated cytokines expressions, and the inhibitory effects of mapks inhibitors were very similar to the experiments in direct infection. this confirmed that the suppression effects of mapks inhibitors on cytokines expression were not due to their potential effects on viral load. as a result, the anti-inflammatory effects of indirubin derivatives are mainly due to its inhibitory effects on different kinases. in contrast, stat -specific sirna had no effects on ip- , rantes and il- production induced by h n infection in hpmecs (fig. d-f) . time-course experiments showed that h n rapidly induced p and jnk phosphorylation in min after addition of the virus, then a second wave of p and jnk phosphorylation were induced at h.p.i. and h.p.i., respectively (fig. a,b) . no activation of erk was found after h n infection. similar to the early phosphorylation of stress-related mapks, stat was phosphorylated early in min after addition of the virus. however, the second wave of stat phosphorylation was found at h.p.i. taken together, these results suggested that h n infection on hpmecs could activate p , jnk and stat signaling pathways rapidly, and the expression of cytokines including ip- , rantes and il- were mainly due to the activation of p and jnk. indirubin derivatives suppress h n -induced cytokines expression through direct inhibition of p and jnk activity. to study the underlying mechanism of the anti-inflammatory effects of indirubin derivatives, hpmecs were treated with indirubin derivatives e or e after h n infection. as shown in fig. , e can significantly reduce h n -induced phosphorylation of p (fig. a) and jnk (fig. b ) at and h.p.i., respectively, and e demonstrated a more potent effect than e . it has been suggested that indirubin and its derivatives are potent inhibitors of various kinases, including mapks. in vitro kinase assay on p and jnk showed that e but not e inhibited p and jnk kinases activity. this action was reflected by the reduced phosphorylation of their direct downstream substrates atf (fig. c ) and c-jun (fig. d) respectively. indirubin derivatives prevent h n -induced ifn-β expression through inhibition of stat phosphorylation and nuclear translocation. though we found no relationship between stat and h n -induced ip- , rantes and il- expressions (fig. d-f) , stat signaling pathway is indispensable for the induction of interferons. we showed that knockdown of stat strongly inhibited h n -induced ifn-β (fig. b ) mrna expression. to elucidate the inhibitory effects of e on stat , western blot analysis was performed. treatment with e or e inhibited h n -induced stat tyrosine phosphorylation (fig. c ). upon activation, stat forms homo-or heterodimers that translocate to the nucleus. hpmecs fractionation of nucleus and cytoplasm was obtained by means of subcellular fractionation followed by western blot analysis. the results showed that h n infection increased phosphorylated stat in the nuclear fraction, while treatment with e significantly reduces the nuclear translocation (fig. d) . similar to the result of western blot analysis, the confocal image also showed that increased fluorescence signal was found in the nucleus after h n infection in hpmecs (fig. e) . treatment with e reduced stat fluorescence signal in the nucleus. the emergence of iav poses a serious global threat to human health. besides regular epidemic outbreaks, severe pandemics like the spanish flu and the more recent swine flu had caused enormous social and economic burden. current treatment of iav infection by m -ion channel inhibitors or na inhibitors emerged high frequency of resistance, and the efficacy and effectiveness of these antiviral drugs are limited by disappointing success rate , so alternative or complementary therapies that modulate the signaling pathways utilized by iav came into focus. in this report, we demonstrated that indirubin derivatives, particularly e is a potent immunomodulatory compound for iav-infection in vitro by inhibiting intracellular signaling pathways in pulmonary endothelial cells. during the early stage of iav infection, innate immune cells are recruited to the site of infection and are associated with an overwhelming production of pro-inflammatory cytokines and chemokines. endothelial cells in the pulmonary vasculature form a barrier between the blood and interstitium. this strategic position suggests that pulmonary endothelial cells are prone to be affected by the cytokines and viral particles released from the iav-infected epithelial cells. a recent study by teijaro et al. identified endothelial cells as the central orchestrator which contribute to the aberrant pro-inflammatory cytokine and chemokine production during early iav infections . concomitant with our in vitro data, we showed that h n virus can efficiently infect hpmecs (fig. a,b) and induce a significant amount of ip- , rantes, il- , ifn-β and ifn-γ . ip- and rantes are the chemoattractants for leukocytes including t cells, nk cells, and granulocytes, while production of il- by endothelial cells initiates infiltration of neutrophils in the early phase of infection . these cytokines have been found histopathologically in the lungs (including epithelial and endothelial cells) of h n infected patients, who showed acute respiratory distress syndrome (ards) , and ards can be characterized by progressive pulmonary endothelial damage. it has been suggested that treatment with antibodies against ip- in h n infected mice can improve the survival rate and reduce acute lung injury . furthermore, suppression of early innate cytokine and chemokine production in the pulmonary endothelium can significantly improve survival of mice infected with lethal h n swine iav . these studies suggested that inhibition of cytokines production of the pulmonary endothelium is an attractive therapeutic strategy against iav-induced cytokine storm. since severe infection of the influenza virus triggers the activation of the innate immune response and sometimes results in the induction to a cytokine storm. in this study, we demonstrated the immunomodulatory effects of indirubin derivatives, particularly e on iav-infected pulmonary endothelial cells. over the past two decades, many studies have identified that indirubin derivatives are potent inhibitors of various kinases, including mapks , , src kinase , glycogen synthase kinase- β (gsk- β ) and cyclin-dependent kinases (cdks) . based on these findings, potential functions of indirubin derivatives have been proposed, including anti-inflammation , [ ] [ ] [ ] , anti-leukemia , antiviral , and angiosuppression , . crystal structure analysis revealed that indirubin can form three hydrogen bonds with the atp-binding pocket of cdks, thereby competitively inhibiting atp binding in the catalytic domain of cdks . the results from our in vitro kinase assay also demonstrated that e is a potent inhibitor of p and jnk (fig. c,d) . the cytokine elisa data also suggest that h n -induced ip- expression of hpmecs was dependent on p and jnk activation, while rantes and il- were controlled by jnk and p , respectively ( fig. a-c) . however, the western blot analysis showed that e could also inhibit phosphorylation of p (fig. a) and jnk (fig. b) , which mean upstream kinases of p and jnk may also be inhibited by e . mapks are important mediators of influenza-induced cytokine expression. in fact, p has been shown to regulate the stability of il- mrna . meanwhile, another study also indicated the critical function of p on ip- during viral infection . furthermore, inhibition of p by specific inhibitor sb in vivo can greatly diminish h n lethal infection . however, the role of jnk in iav infection has not been fully examined. nacken et al. elucidated that influenza viral rna induces jnk phosphorylation in an rig-i dependent manner, but the ns of iav has also an intrinsic jnk activating property . taken together, the potent inhibitory effect of p and jnk signaling pathways by e strongly correlates with its anti-inflammatory function. ifns are pro-inflammatory cytokines crucial for antiviral responses to iav infection. stat and stat are predominant and essential transcription factors of type i and ii interferons signaling pathway, but the role of stat activation after ifn binding remains controversial. undeniably, stat is indispensable for downstream signaling pathway of many other cytokines like il- , vegf or egf . our data showed that h n infection on hpmecs can significantly induce ifn-β and ifn-γ expression. interestingly, stat -specific sirna has no effect on h n -induced il- ( fig. f ) and ifn-γ (fig. b ), but strongly inhibit ifn-β expression level, indicating the involvement of stat in ifn-β induction. the western blot data showed that iav-infection could activate stat in early stage ( min after addition of virus) followed by another activation at h.p.i. (fig. ) . h n was found to upregulate tlr- and myd , which is critical to the induction of ifn-β . in line with this finding, the early activation of stat may function together with the downstream signaling molecules of tlr- , possibly the interferon regulatory factors (irfs), to induce rapid expression of ifn-β mrna at h.p.i. (fig. h) . and then the autocrine effect of ifn-β induces a further amplification of ifn-β expression and result in a cytokine storm. a previous study suggested that e could inhibit stat dimerization and subsequent dna binding . our translocation experiments clearly indicated that e could inhibit stat phosphorylation and nuclear translocation. since the induction of many pro-inflammatory cytokines requires type i interferon signaling, inhibition of ifn-β production by e may blunt the early induction of these cytokines. though ifn-β is a well-known antiviral cytokine, it is also involved in the pathogenesis of influenza infection. in vivo studies focusing on the s p receptor and p pathway also suggested that, even the iav-induced ifns were suppressed by an s p receptor agonist or p inhibitor , the survival rate of the infected mice could still be significantly improved if those strongly induced cytokines were suppressed. in many studies, increased viral load were concomitant of reduced interferons expressions. however, in the present study, viral titer did not further increased in indirubin derivatives treated cells (fig. b ) even ifnβ and ifn-γ were suppressed, the results indicated that suppression of cytokines produced by the infected pulmonary endothelium could reduce iav pathogenicity independent of the viral clearance. in fact, many kinases including cdk and mapks, which are suggested being involved in the influenza replication process are also the target of indirubin derivatives, this might explain the partial antiviral effect of indirubin derivatives in this model. meanwhile the phosphorylation and nuclear translocation of stat leaded to induction of ifn-β . indirubin derivatives particularly e is a potent inhibitor of p and jnk signaling pathways. e could also reduce the phosphorylation and nuclear translocation of stat . by inhibition of these signaling pathways, e could significantly suppress h n -induced cytokine burst in hpmecs. scientific reports | : | doi: . /srep combination therapies coupling with antiviral and immunomodulatory drugs have been investigated intensively . the encouraging results from in vitro and pre-clinical studies have led to an increased interest on this topic. however, to achieve the best clinical outcome, antiviral and immunomodulatory drugs should be administrated at the appropriate time during an infection. further understanding of the immune dynamic could allow us to design an optimum therapy strategy. in this report, we demonstrated for the first time, the potent immunomodulatory effects of indirubin derivatives on pulmonary endothelial cells and their therapeutic potential for iav-infection (fig. ) . as a result, the combinational effects of indirubin derivatives and antiviral drug in animal model warrant further investigation. perspectives on influenza evolution and the role of research structure and assembly of the influenza a virus ribonucleoprotein complex pathogenesis of influenza virus infections: 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cyclin-dependent kinase inhibitors an indirubin derivative, e , exhibits potent angiosuppressive activity automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish the p map kinase pathway signals for cytokine-induced mrna stabilization via map kinase-activated protein kinase and an au-rich region-targeted mechanism innate immune response to h n and h n influenza virus infection in a human lung organ culture model activation of c-jun n-terminal kinase upon influenza a virus (iav) infection is independent of pathogen-related receptors but dependent on amino acid sequence variations of iav ns stats in cancer inflammation and immunity: a leading role for stat human intestinal epithelial cells are susceptible to influenza virus subtype h n this work was supported by the area of excellence scheme of the university grants committee, hong kong sar government (aoe/m- / ) and dr. gilbert hung ginseng laboratory fund. key: cord- -x t pbv authors: kosinska, anna d.; liu, jia; lu, mengji; roggendorf, michael title: therapeutic vaccination and immunomodulation in the treatment of chronic hepatitis b: preclinical studies in the woodchuck date: - - journal: med microbiol immunol doi: . /s - - - sha: doc_id: cord_uid: x t pbv infection with hepatitis b virus (hbv) may lead to subclinical, acute or chronic hepatitis. in the prevaccination era, hbv infections were endemic due to frequent mother to child transmission in large regions of the world. however, there are still estimated million chronic hbv carriers today and ca. , patients die per year due to hbv-related liver diseases. recommended treatment of chronic hepatitis b with interferon-α and/or nucleos(t)ide analogues does not lead to satisfactory results. induction of hbv-specific t cells by therapeutic vaccination or immunomodulation may be an innovative strategy to overcome virus persistence. vaccination with commercially available hbv vaccines in patients with or without therapeutic reduction of viral load did not result in effective immune control of hbv infection, suggesting that combination of antiviral treatment with new formulations of therapeutic vaccines is needed. the woodchuck (marmota monax) and its hbv-like woodchuck hepatitis virus are a useful preclinical animal model for developing new therapeutic approaches in chronic hepadnaviral infections. several innovative approaches combining antiviral treatments using nucleos(t)ide analogues, with prime-boost vaccination using dna vaccines, new hepadnaviral antigens or recombinant adenoviral vectors were tested in the woodchuck model. in this review, we summarize these encouraging results obtained with these therapeutic vaccines. in addition, we present potential innovations in immunostimulatory strategies by blocking the interaction of the inhibitory programmed death receptor with its ligand in this animal model. more than million people worldwide are persistently infected with hepatitis b virus (hbv) and are at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma (hcc) [ ] . an effective and affordable therapy to achieve sustained suppression of hbv replication and remission of liver disease is urgently needed. pegylated interferon-alpha a (ifn-a) is recommended for the treatment of chronic hepatitis b (chb) in the current consensus guidelines of many countries. compared with conventional recombinant ifn-a, however, pegylated ifn-a alone or in combination with nucleoside analogues does not significantly increase the rate of sustained response [ , ] . nucleos(t)ide analogues, such as, entecavir and tenofovir, suppress hbv replication and result in the improvement of liver architecture. however, these agents cannot eradicate hbv genomes from the liver and may further limited by the development increasingly select drug-resistant mutants with prolonged use [ , ] . therapy with additional antiviral drugs targeting other steps in the viral life cycle, in combination with immunomodulatory options, might be more beneficial and effective. more than % of acutely infected adults resolve clinical symptoms and maintain lifelong protective immunity by mounting a vigorous, multi-specific immune response abstract infection with hepatitis b virus (hbv) may lead to subclinical, acute or chronic hepatitis. in the prevaccination era, hbv infections were endemic due to frequent mother to child transmission in large regions of the world. however, there are still estimated million chronic hbv carriers today and ca. , patients die per year due to hbv-related liver diseases. recommended treatment of chronic hepatitis b with interferon-α and/ or nucleos(t)ide analogues does not lead to satisfactory results. induction of hbv-specific t cells by therapeutic vaccination or immunomodulation may be an innovative strategy to overcome virus persistence. vaccination with commercially available hbv vaccines in patients with or without therapeutic reduction of viral load did not result in effective immune control of hbv infection, suggesting that combination of antiviral treatment with new formulations of therapeutic vaccines is needed. the woodchuck (marmota monax) and its hbv-like woodchuck hepatitis virus are a useful preclinical animal model for developing new therapeutic approaches in chronic hepadnaviral infections. several innovative approaches combining antiviral treatments using nucleos(t)ide analogues, with primeboost vaccination using dna vaccines, new hepadnaviral antigens or recombinant adenoviral vectors were tested in the woodchuck model. in this review, we summarize these encouraging results obtained with these therapeutic vaccines. in addition, we present potential innovations this article is part of the special issue "therapeutic vaccination in chronic hepatitis b-approaches, problems and new perspectives". to hbv proteins. by contrast, patients with chronic hepatitis b tend to have delayed, transient or narrowly focused t cell responses [ ] [ ] [ ] . patients who spontaneously recover from hbv infection might experience reactivation of hbv under immunosuppressive treatments. thus, the specific immune responses to hbv remain crucial for the long-term control of hbv infection even after resolution of the acute infection. for chronically infected patients, immunostimulatory and immunomodulatory strategies to boost or to broaden the weak virus-specific t cell response have been proposed to reach an effective control of viral infection. since more than years, numerous clinical trials exploited the conventional prophylactic vaccine based on the hepatitis b surface antigen (hbsag) for therapeutic vaccination (table ) . these studies demonstrated reductions in viremia, seroconversion of the hepatitis b "e" antigen (hbeag) to anti-hbe and hbv-specific t cell responses in some patients after vaccination. however, the antiviral effect was only transient and did not lead to an effective control of the hbv [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a more sophisticated therapeutic vaccination based on hbsag complexed with human anti-hbs was proposed by the group of wen et al. [ ] . immunogenic complexes (ic) stimulate robust t cell responses by increasing uptake of hbsag through fc receptors on antigen-presenting cells (apc) and, therefore, enhance hbsag processing and presentation. it was demonstrated that this vaccine administered to hbeag-positive patients led to decrease of hbv dna in serum and hbeag seroconversion in some subjects [ ] . in a phase ii b clinical trial, hbeag seroconversion was observed in about . % of treated patients. moreover, a moderate decrease in serum hbv dna and hbsag levels was observed after treatment [ , ] . very recently, a large phase iii clinical trial with injections of ic complex failed to show any therapeutic efficacy when compared to the placebo control injected only with alum [ ] . overstimulation with ic-based vaccine did not increase but decreased efficacy of the therapeutic vaccination. these results underline that an appropriate immunization protocol is crucial for the efficacy of therapeutic vaccination. dna vaccines using plasmids expressing viral proteins have gained popularity given their ability to induce strong cellular and humoral immune responses. several phase i clinical studies investigated the therapeutic efficacy of plasmid dna vaccines expressing hbsag in chronic hbv carriers. these studies showed evidence for the safety of hbv dna vaccination (for details see below), but t cell responses were restored or activated at only a low level. furthermore, dna vaccines expressing only hbsag did not result in significant suppression of viremia in chronic carriers of hbv [ , ] . from results of these studies, it can be concluded that the therapeutic vaccination alone is not sufficient to achieve pre-s /pre-s /s, hbcag, polymerase yoon et al. [ ] the control over hbv. high load of virus particles and large amounts of hbsag in the liver and peripheral blood may be responsible for the immune tolerant status in the patients. therefore, pretreatment with nucleos(t)ide analogues has been proposed to achieve better cd t cell response and subsequent therapeutic efficacy after administration of dna vaccines. recently, the results of the trial of this combination therapy have been published. in a large double-blind study, patients were treated effectively with nucleos(t)ide analogues for a median of years resulting in undetectable levels of hbv dna and thereafter randomized into two groups: one received five intramuscular injections of dna vaccine expressing hbsag and one received placebo. nucleos(t)ide analogues were stopped. although this combination therapy was fairly well tolerated, the hbv dna vaccine did not decrease the risk of relapse in hbv-treated patients and did not restore the anti-hbv immune response despite effective viral suppression by analogues [ , ] . during a study in korea, patients randomly received either adefovir (adv) alone or adv in combination with hbsag-expressing dna vaccine. therapeutic vaccination was safe and tolerable in chb patients. vaccine-induced hbv-specific t cell responses and hbeag seroconversion were weaker in korean patients than in caucasian patients [ ] . asian patients, who are generally infected via vertical transmission, may have a higher level of immune tolerance than caucasians who are usually infected later in life. improved vaccines for breaking immune tolerance may be needed to develop effective therapeutic hbv dna vaccines. the aim of a study in vietnam was to evaluate viral suppression following combined treatment with a new vaccine containing all three envelope proteins of hbv (pre-s /pre-s /s) and lamivudine in chb patients. the enhanced suppression of viremia in the combination group was reversed after the discontinuation of vaccine treatment, suggesting that booster doses are required for a sustained viral response. anti-hbs was detected in / vaccine recipients, but only three patients demonstrated hbsag loss, indicating that the vaccine-induced anti-hbs was unable to completely neutralize hbsag in the serum [ ]. the eastern woodchuck (marmota monax) is naturally infected by woodchuck hepatitis virus (whv) which was discovered in [ ] . whv was found to be closely related to hepatitis b virus (hbv) [ ] and classified as the second member of the genus ortho-hepadnavirus, family hepadnaviridae. in contrast to hbv-associated hcc in patients without a preferred integration site of hbv dna, a frequent integration of the whv genome close to the n-myc and c-myc gene has been observed in woodchucks developing hcc [ ] . infections of woodchucks with whv have been shown to be endemic in the mid-atlantic states of the usa, whereas in the state of new york and new england woodchucks are rarely infected with whv. recently, a chinese marmot marmota himalayana was found to be susceptible to whv infection [ ] (fig. this review is focusing on the characterization of woodchuck genes related to innate and adaptive response, the recent development of new tools to determine virusspecific t cell response, therapeutic vaccines, and finally immunostimulatory and immunomodulatory approaches to treat chronic whv infection. these new findings in this preclinical model will help the development of new strategies to treat chronic hbv infection in patients. in recent years, many efforts have been devoted to cloning and characterization of components of the woodchuck immune system. a number of immune function-related genes including cytokines and their receptors, immune cell surface markers and other immune function-related proteins have been cloned and characterized. so far, important woodchuck cytokines and their receptors such as tnf-α, ifn-α, ifn-γ, il- , il- , gmcsf, lymphotoxin (lt)-α and il- r have been cloned and tested for their biological activities [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in patients, ifn has been used in the treatment of chb for many years. therefore, the ifn system has also been characterized in woodchucks. woodchuck ifn-α was shown to reduce whv surface antigen expression in a dose-dependent fashion in whv-infected woodchuck hepatocytes [ ] . the woodchuck ifn-α/β system and their expression in peripheral blood lymphocytes (pbls) from naïve and whv-infected woodchucks have also been studied [ ] . the woodchuck ifn-α genes could be classified into ten subtypes and three pseudotypes. poly(i:c) stimulation on naïve woodchuck pbls could induce ifn-α subtypes one, four and five production, indicating a selective expression of woodchuck ifn-α subtypes. moreover, pbls from chronically whv-infected woodchucks showed a reduced ability to produce woodchuck ifn when stimulated with poly(i:c). the complete or partial sequences of the type i ifn receptors (ifnars) of woodchucks were also obtained and analysed by fan et al. [ ] . ifn-α or ifn-γ stimulation significantly upregulated ifnar expression in primary woodchuck hepatocytes. a decreased ifnar and ifnar expression was observed in woodchucks chronically infected with whv. these data are essential for studying type i ifn-related innate immunity and therapy in hepadnaviral infection in the woodchuck model. il- is a pleiotropic cytokine acting on a variety of immune cells through its cell surface receptor (il- r). it has been suggested to resuscitate antiviral immunity by interfering with il- /il- r pathway. an increased production of il- was observed in patients with chb [ ] , which hints that blockade of il- r might become a feasible therapeutic approach for chb. very recently, jiang et al. [ ] successfully cloned woodchuck il- r and generated antibodies against this molecule. the blockade of woodchuck il- r enhanced the proliferation and degranulation of specific t cells from chronically whv-infected woodchucks in vitro. this work provides a basis for potential therapeutic approaches in chronic hbv infection. important woodchuck immune cell surface molecules which have been cloned so far can be divided into two categories based on their function: molecules involved in innate immunity and molecules involved in adaptive immunity. toll-like receptors (tlrs) are a class of molecules that play a key role in the innate immune system. recent progress in this field revealed that there are significant interactions between the tlr system and pathogens in chronic viral infections [ ] . so far, tlr , tlr , tlr , tlr , tlr and tlr have been successfully cloned in woodchucks [ ] . in a recent study, zhang et al. [ ] showed that tlr ligands induced the activation of nf-κb, pi k/akt and different arms of mapk signalling pathways and the production of pro-inflammatory cytokines in woodchuck hepatocytes. tlr -mediated innate immune responses reduced replication and gene expression of hbv in hepg . . cells and whv in primary woodchuck hepatocytes (see also article from zhang and lu, in this issue). in chronic whv carriers woodchuck model, relatively low levels of tlr expression were found in pbmcs and in liver tissues. tlr expression in pbmcs was inversely correlated with whv dna titres in acute whv infection and in entecavir-treated chronic whv carriers. an effective immune response against viral infections depends on the activation of cd t cells that can clear infection by killing virus-infected cells. therefore, sequence information of woodchuck cd , cd and cd has been used to determine the kinetic of the influx of t cells into the liver during incubation period and acute or chronic whv infection. in week two post-infection, an influx of cd + lymphocytes could be observed and reached higher levels prior and during the recovery phase. the peak level of cd + and cd + t cells coincided with recovery. during transient infection, t cells can accumulate in the liver and reach up to two-thirds of the total number of liver cells [ ] . in the adaptive immune response, cd and ctla- are known to play important roles for the regulation of t cell activation by delivering costimulatory signals. the complete coding regions of woodchuck cd and cytotoxic t-lymphocyte-associated antigen (ctla- ) have been cloned and sequenced [ ] . woodchuck cd showed a similarity of and % to its human and mouse homologues, respectively, according to the deduced amino acid sequences. woodchuck ctla- has a higher similarity of and % to the corresponding human and mouse ctla- molecules, respectively. the strict conservation of critical amino acid residues like cysteine and asparagine residues in woodchuck cd and ctla- suggests that both molecules may structurally resemble their human or mouse homologues. a hexapeptide motif mypppy which has been supposed to be essential for the interaction with cd is present in both woodchuck cd and ctla- [ ] . the advances in sequencing technology provide new tools to characterize genes of the woodchuck immune system in large scale. fletcher et al. [ ] performed the sequencing, assembly and annotation of the woodchuck transcriptome, together with the generation of custom woodchuck microarrays. by using this new platform, they characterized the transcriptional response to persistent whv infection and whv-induced hcc. liu et al. have also performed de novo woodchuck transcriptome assembly by using deep sequencing technology (unpublished data). with the help of this advanced technology, sequence information of important immune genes such as apobec of woodchucks has been revealed. it has been shown that upregulation of apobec led to specific and non-hepatotoxic degradation of nuclear hbv cccdna [ ] . therefore, future cloning and characterizing of apobec in the woodchuck model will evaluate the therapeutic potential for chb. in summary, these efforts on establishing the translational value of the woodchuck model can provide new insight into characterizing immune pathways which may play a role in the persistence of hbv infection. studies in patients underline the important role of hbvspecific t cell response as a leading factor of viral clearance. for many years, the lack of appropriate methods to evaluate antigen-specific t cell responses was the serious limitation of this model. the establishment of the assays for monitoring of cellular immune response in woodchucks is of great importance for a reliable evaluation of therapeutic and immunomodulatory strategies for treatment of chb in the woodchuck model. development of the [ h]-adenine-based proliferation assay enabled to detect the t-helper lymphocyte responses after stimulation of woodchuck pbmcs [ , ] . in addition, several t-helper epitopes within whcag [ , ] were identified in pbmcs from acutely whv-infected animals. significant progress in studying the t cell response of woodchucks was achieved by introduction of the flow cytometric cd a degranulation assay that enables the detection of whv-specific cytotoxic t cells (ctls) in woodchuck pbmcs and splenocytes [ ] . several studies demonstrated that detection of cd a, as a degranulation marker, is a suitable method for determination of antigenspecific cytotoxic t lymphocytes [ , ] . introduction of the immunological tools for studying of the t cell response in woodchucks revealed a significant similarity between the pathogenesis of whv infection in woodchucks and hbv in humans. it was demonstrated that acute self-limiting and resolved whv infections correlate with robust multifunctional t-helper and cytotoxic t cell responses, while whv chronic carriers demonstrate weak or no virus-specific t cell responses against the viral proteins (table ) [ , , ]. moreover, these studies confirmed that the efficient cellular immune response to viral antigens results in liver injury and is necessary for viral clearance. recently described advancements in the characterization and monitoring of the woodchuck immune system during the whv infection made this animal model particularly useful for development of the immunomodulatory approaches in chb. the pioneer investigations with therapeutic vaccines based on whv core [ ] or surface [ , ] reporting that the t cell response to hbv was successfully restored in patients treated with lamivudine. in addition, the quantity of antigen particularly the whv surface antigen (whsag) to which the immune system is exposed can induce different degrees of functional impairment of antiviral t cells, up to physical t cell deletion [ , ] . combination therapy using lamivudine and serumderived whsag vaccination showed no effect on induction of anti-whs antibodies or reduction of whv dna [ ] . our group evaluated the efficacy of the combination therapy in the woodchuck model by combining lamivudine treatment, dna vaccination (three plasmids expressing whsag, whcag and woodchuck ifn-γ) and whsag/ anti-whs immunogenic complexes vaccination [ ] . the triple combination led to a decrease in whv viral load up to . log, in serum whsag up to % and in development of anti-whs antibodies. nevertheless, these effects were not sustained and all parameters reached the baseline levels shortly after withdrawal of lamivudine treatment. in addition, the vaccination did not induce whv-specific t cell responses in the majority of woodchucks, even in animals that exhibited virological responses. later, we modified this protocol by using the more potent antiviral drug entecavir (etv) and increasing the number of the immunizations (with plasmids expressing whsag and whcag from three to six) (lu et al., unpublished results). a significant delay of the rebound of viremia was observed in woodchucks which received additional vaccination, compared to controls treated only with etv. in another study, chronic whv carriers received a treatment of the potent antiviral drug clevudine in combination with an alumadsorbed whsag vaccine. combination treatment resulted in significant and sustained reduction of whv dna loads and whsag concentrations in most treated animals. compared to vaccination alone, combination treatment induced a more robust anti-whs response [ , ] . the results of these studies clearly showed that combination of antiviral treatment and vaccination is more effective in inducing virus-specific t cell responses than therapeutic vaccination alone. nevertheless, the efficacy of these approaches was still too limited when applied for treatment of chb. the vaccination strategies used in some of these studies were even not able to boost a functional antiviral t cell response. a significantly better induction of whcagspecific t cells using more potent vaccines, such as recombinant viral vectors, may be required to achieve sustained antiviral response and viral clearance. recombinant adenoviral vectors (adv) proved to elicit a vigorous and sustained humoral and t cell responses to the transduced antigen [ , ] . adenoviral vectors also act as a natural adjuvants causing dc maturation, enhanced antigen presentation and secretion of antiviral cytokines, such as ifn-α, tnf-α and il- [ ] . however, even single immunization with recombinant adenoviruses may induce immunity, predominantly neutralizing antibodies, against the vector itself. this negative effect of the adenovirusinduced immunity against the vaccine may be overcome by heterologous prime-boost regimen. in particular, subsequent priming immunizations with plasmid dna vaccine followed by a booster vaccination with adv seem to be a very promising strategy. dna prime-adenovirus boost regimen proved to induce more robust and potent immune response in comparison with plasmid dna alone and provided protection against the pathogen challenge in several animal models of infectious diseases [ ] [ ] [ ] (see also article from e. barnes in this issue). recently, our group has investigated whether the heterologous prime-boost immunization strategy using plasmid dna and recombinant adenoviral vectors may improve the efficacy of the therapeutic vaccination in chb in the woodchuck model. a new dna plasmid (pcgwhc) and an adenoviral serotype vector (ad whc) and a chimeric ad displaying ad fibre (ad whc) showing high expression levels of whcag were constructed [ ] . the increased antigen expression was achieved by insertion of an intron sequence in the expression cassette of the vaccines. preliminary results showed that the new vaccines are able to induce strong and sustained whcag-specific t cell response in mice and naïve woodchucks. interestingly, immunization with advs led to rapid and massive production of anti-whs antibodies and as a result resolution of infection after the whv challenge [ ] . the dna prime-adv boost immunization strategy was further used as a therapeutic vaccine against chronic whv infection in combination with antiviral treatment with etv. six chronically whv-infected woodchucks were treated for weeks with etv. starting from week eight, four of the six etv-treated animals received subsequently nine intramuscular immunizations with: dna plasmids expressing whcag (pcgwhc) and whsag (pwhsim), ad whc and ad whc. whsag-and whcag-specific t-helper and cytotoxic t cell responses were detected in all chronic carriers that received immunizations, but not in etv only treated animals. in addition, woodchucks receiving the combination therapy showed a prolonged suppression of whv replication and lower whsag levels compared to controls. excitingly, two of four immunized carriers remained whv dna negative after the end of etv treatment and developed anti-whs antibodies [ ] . these data are encouraging and demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks. persistent hbv infection is associated with functional exhaustion of virus-specific cd t cells [ ] . this defect in virus-specific t cells is one of the primary reasons for the inability of the host to eliminate the persisting pathogen. although it has been shown that nucleoside analogues treatment can induce the recovery of hbv-specific ctl activity in patients [ ] , this effect is only transient [ ] . those findings are consistent with our data obtained from the woodchuck model, in which etv treatment alone only induced either only transient ctl responses [ ] or no responses at all [ ] . therefore, additional strategies that can potently enhance t cell response need to be enroled for the treatment of chb infection. recent studies in chronic virus infection models indicate that the interaction between the inhibitory receptor programmed death- (pd- ) and its ligands plays a critical role in t cell exhaustion [ ] [ ] [ ] [ ] . in chronic hbv infections, upregulation of pd- on virus-specific t cells was observed, and restoration of the t cell function has been achieved by blocking the pd- /pd-ligand (pd-l ) interaction in vitro [ ] . recently, the therapeutic effect of pd- /pd-l blockade has also been investigated for chronic hcv infection in chimpanzees [ ] and in patients [ ] . however, limited effect on restoring t cell function was observed in these studies which used only pd- /pd-l blockade. it has been recently clarified that the proportion of cd t cells expressing pd- and the levels of pd- on virus-specific t cells are strongly correlated with viral load in the plasma [ ] [ ] [ ] . antiretroviral treatment resulted in the dramatic decline of plasma viral load, coincident with a decrease in the pd- expression level on virus-specific cd t cells [ , ] . in line with this, a better restoration of t cell functions upon in vitro anti-pd-l treatment was observed in chronic hbv patients with lower viremia [ ] . therefore, a combination therapy that includes direct antiviral drug and pd-l blockade is a reasonable strategy for the treatment of chronic hbv infection. in line with these findings, zhang et al. [ ] and liu et al. [ ] successfully cloned and characterized the woodchuck pd- /pd-l system in the whv infection woodchuck model. a significant positive correlation between the viral load and the pd- expression on total cd t cells in chronic whv infection was observed. both the proportion of pd- + cd t cells and the levels of pd- expression on cd t cells were significantly higher in the woodchucks with chronic whv infection compared to naïve animals and resolvers. more importantly, during etv treatment of those chronic carriers, a reduction of serum viral load was correlated with a dramatic decrease in the level of pd- expression on cd t cells [ ] . in vitro blockade of woodchuck pd- /pd-l pathway by using a rabbit polyclonal pd-l blocking antibody could partially restore the t cell function in whv-infected woodchucks [ ] . moreover, in vivo blockade of the pd- /pd-l pathway on cd t cells, in combination with nucleoside analogue treatment and dna vaccination, synergistically enhanced the function of virus-specific t cells. the combination therapy potently suppressed whv replication, leading to sustained immunological control of viral infection, anti-whs antibody development and complete viral clearance in some woodchucks [ ] . although similar approaches have been tried in other viruses in the past, such as lcmv, the data presented here may be an advance for the hbv field to new approaches for eliminating the virus itself rather than only suppressing its replication. the woodchuck is a valuable preclinical model for developing new therapeutic approaches in chronic hepadnaviral infections. even though several innovative approaches combining antiviral treatment with nucleoside analogues, dna vaccines and protein vaccines were tested in chronically infected woodchucks, the effectiveness of those strategies was very limited. strategies investigated so far were often hampered by weak t cell responses observed after immunization, suggesting a strong need for alternative strategies to enhance t cell functions during chronic hbv infection. recently, our group published two independent proof-of-concept studies, showing that using a very potent t cell vaccine and blockade of negative signalling in t cells may lead to the resolution of chronic hepatitis b in some woodchucks (table ) . these data are encouraging and implicate the feasibility and usefulness of the immunotherapeutic strategies for the treatment of chronically hbv-infected patients. nevertheless, which factors influence the effect of therapeutic vaccination remains to be further investigated. it has been noticed that satisfactory therapeutic effects could not be documented in the studies using hbsagbased prophylactic vaccines. in the mean time, evidence has supported that hbcag-specific immunity is endowed with antiviral and liver-protecting capacities in chb patients and animal models. with the increasing number of available vaccine formulation, a more crucial question raised recently: what is the optimal combination of these vaccines. obviously, it is necessary to test the mutual influences of different types of vaccines to maximize their effects and avoid the negative interference between the vaccines. also, the question how hbv infection leads to defective immune responses to hbv proteins remains to be investigated. this issue is the key to a more rational design of new therapeutic approaches. figure summarizes the ideas of a potential combination treatment for patients with chronic hepatitis b. the presence of viral components may be a main reason for t cell tolerance in chronic hbv infection. antiviral treatment with nucleoside analogues efficiently reduce hbv replication and release of new virions and may partly restore hbv-specific cd and cd t cell responses, thereby allowing successful therapeutic vaccination. however, hbv proteins are still produced as the transcription of mrnas for the s protein and the core protein on hbv cccdna is not affected by antiviral treatment. even when hbv dna disappears during antiviral treatment, hbsag and hbcag/hbeag are present in the liver or in blood at high levels. it is proposed to reduce hbv protein load by small interfering rnas (sirnas), which lead to the sequence-specific degradation of homologous mrna. using this rna interference (rnai) with chemically synthesized or vector-expressed sirnas, many clinically relevant viruses including the human immunodeficiency virus, hbv and hcv could be inhibited. in in vitro experiments showed that whv transcripts could be degraded by sirnas [ ] . at the same time, the degradation of viral rnas resulted in the activation of multiple pathways of host innate immune responses [ ] . however, future in vivo studies are required to demonstrate the usefulness of this technology. combining gene-silencing approach with nucleoside analogues may further facilitate the stimulation of the immune system by therapeutic vaccines. the epigenetic regulation provides an alternative to interfere with hbv gene expression. hbv minichromosome in hepatocytes is under the complex control of epigenetic mechanisms, and its transcriptional activity could be influenced by methylation, histone acetylation and other mechanisms [ ] . therefore, exploring epigenetic drugs to modify, these regulatory processes may achieve an effective suppression of hbv gene expression and thereby replace antiviral treatment with nucleoside analogues. the stimulation of innate immune responses may contribute to the control of hbv infection. in this special issue, zhang and lu provided a review dedicating to the role of tlr system. interferons and interferon-stimulated genes (isgs) represent still an important part for anti-hbv treatment. a recent review about this aspect described the current progress (pei et al., in press) . recently, the antiviral functions of isgs are under studies. for example, interferon-induced protein with tetratricopeptide repeats and is a cellular factor that was shown to limit 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prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing gag prime-boost vaccination with plasmid dna and a chimeric adenovirus type vector with type fiber induces protective immunity against hiv dna prime-adenovirus boost immunization induces a vigorous and multifunctional t-cell response against hepadnaviral proteins in the mouse and woodchuck model combination of dna prime-adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis b in the woodchuck model t cells and viral persistence: lessons from diverse infections enhancing virus-specific immunity in vivo by combining therapeutic vaccination and pd-l blockade in chronic hepadnaviral infection restoring function in exhausted cd t cells during chronic viral infection pd- blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination pd- :pd-l interactions contribute to the functional suppression of virus-specific cd + t lymphocytes in the liver enhancing siv-specific immunity in vivo by pd- blockade characterization of hepatitis b virus (hbv)-specific t-cell dysfunction in chronic hbv infection immunotherapy of chronic hepatitis c virus infection with antibodies against programmed cell death a randomized, double-blind, placebo-controlled assessment of bms- , a fully human monoclonal antibody to programmed death- (pd- ), in patients with chronic hepatitis c virus infection pd- expression on hiv-specific t cells is associated with t-cell exhaustion and disease progression dysfunction and functional restoration of hcv-specific cd responses in chronic hepatitis c virus infection pd- is a regulator of virus-specific cd + t cell survival in hiv infection antiviral intrahepatic t-cell responses can be restored by blocking programmed death- pathway in chronic hepatitis b the expression of pd- ligands and their involvement in regulation of t cell functions in acute and chronic woodchuck hepatitis virus infection inhibition of woodchuck hepatitis virus gene expression in primary hepatocytes by sirna enhances the cellular gene expression rnai induces innate immunity through multiple cellular signaling pathways regulation of hepatitis b virus replication by epigenetic mechanisms and micrornas interferon-induced proteins with tetratricopeptide repeats and are cellular factors that limit hepatitis b virus replication specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna virology. response to comment on "specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna a pilot study of the cy- t-cell vaccine in subjects chronically infected with hepatitis b virus. the cy t cell vaccine study group clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin- in patients with chronic hepatitis b in vivo immunization by vaccine therapy following virus suppression by lamivudine: a novel approach for treating patients with chronic hepatitis b therapeutic vaccination of chronic hepatitis b patients with virus suppression by antiviral therapy: a randomized, controlled study of co-administration of hbsag/as candidate vaccine and lamivudine the authors thank dr. wolfram gerlich for his critical comments on this manuscript. a number of studies cited in this review were funded by deutsche forschungsgemeinschaft (gk and sfb/trr ). open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. key: cord- -fyztb d authors: young, d. f.; andrejeva, j.; li, x.; inesta-vaquera, f.; dong, c.; cowling, v. h.; goodbourn, s.; randall, r. e. title: human ifit inhibits mrna translation of rubulaviruses but not other members of the paramyxoviridae family date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: fyztb d we have previously shown that ifit is primarily responsible for the antiviral action of interferon (ifn) alpha/beta against parainfluenza virus type (piv ), selectively inhibiting the translation of piv mrnas. here we report that while piv , piv , and mumps virus (muv) are sensitive to ifit , nonrubulavirus members of the paramyxoviridae such as piv , sendai virus (sev), and canine distemper virus (cdv) are resistant. the ifit sensitivity of piv was not rescued by coinfection with an ifit -resistant virus (piv ), demonstrating that piv does not specifically inhibit the antiviral activity of ifit and that the inhibition of piv mrnas is regulated by cis-acting elements. we developed an in vitro translation system using purified human ifit to further investigate the mechanism of action of ifit . while the translations of piv , piv , and muv mrnas were directly inhibited by ifit , the translations of piv , sev, and cdv mrnas were not. using purified human mrna-capping enzymes, we show biochemically that efficient inhibition by ifit is dependent upon a ′ guanosine nucleoside cap (which need not be n methylated) and that this sensitivity is partly abrogated by ′o methylation of the cap ribose. intriguingly, piv m mrna, in contrast to np mrna, remained sensitive to inhibition by ifit following in vitro ′o methylation, suggesting that other structural features of mrnas may influence their sensitivity to ifit . thus, surprisingly, the viral polymerases (which have ′-o-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by ifit . possible biological consequences of this are discussed. importance paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. one factor that will influence disease outcomes is how they interact with innate host cell defenses, including the interferon (ifn) system. here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting ifn-induced antiviral state. strikingly, all the rubulaviruses tested were sensitive to the antiviral action of isg /ifit , while all the other paramyxoviruses tested were resistant. we developed novel in vitro biochemical assays to investigate the mechanism of action of ifit , demonstrating that the mrnas of rubulaviruses can be directly inhibited by ifit and that this is at least partially because their mrnas are not correctly methylated. paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. one factor that will influence disease outcomes is how they interact with innate host cell defenses, including the interferon (ifn) system. here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting ifn-induced antiviral state. strikingly, all the rubulaviruses tested were sensitive to the antiviral action of isg /ifit , while all the other paramyxoviruses tested were resistant. we developed novel in vitro biochemical assays to investigate the mechanism of action of ifit , demonstrating that the mrnas of rubulaviruses can be directly inhibited by ifit and that this is at least partially because their mrnas are not correctly methylated. p aramyxoviruses are a large group of negative-sense singlestranded rna viruses that cause a wide variety of animal and human diseases. the paramyxoviridae family is divided into two subfamilies, the paramyxovirinae and the pneumovirinae subfamilies. the paramyxovirinae are further subdivided into a number of genera, including morbillivirus (e.g., measles virus [mev] and canine distemper virus [cdv] ), respirovirus (e.g., sendai virus [sev] and parainfluenza virus type [piv ]), and rubulavirus (e.g., mumps virus [muv] , piv , and piv ). paramyxoviruses are enveloped viruses; the viral glycoproteins protrude from the outer surface of the envelope and function to attach the viruses to their target cells. on the inner surface of the envelope is the matrix (m) protein, which is required for the structural integrity of the virion. the envelope surrounds a helical nucleocapsid, in which the nucleocapsid protein (np) encapsidates genomic or antigenomic rna. associated with the nucleocapsid is the virally encoded polymerase complex. the viral polymerase both transcribes and replicates the viral genome. viral mrnas are capped and polyadenylated by the viral polymerase (for reviews of the molecular biology of paramyxoviruses, see references and ). despite their limited genetic information, the majority of paramyxoviruses encode small multifunctional accessory proteins that function to aid virus multiplication and block cellular antiviral defense mechanisms; typically, these proteins can block both the production of, and the signaling response to, interferons (ifns) (for reviews, see references , , , , and ). significantly, the mechanisms of action of these multifunctional ifn antagonists differ from one virus to another. undoubtedly, these properties and in general the manner in which paramyxoviruses interact with the ifn system and other innate defense mechanisms are likely to be major factors in determining the type of disease that each virus causes ( ) . the ifn response is an extremely powerful antiviral defense system that, unless counteracted by viruses, will limit their replication to such a degree that they will not cause disease or be efficiently transmitted between susceptible hosts ( , ) . infected cells detect the presence of viruses due to the production by viruses of molecules with molecular signatures (pathogen-associated molecular patterns [pamps]) such as double-stranded rna (dsrna), which activate the ifn induction cascade and result in the secretion of ifn-␣/␤ from infected cells ( , ) . the release of ifn induces an antiviral state in neighboring uninfected cells by upregulating the expression of hundreds of interferon-stimulated genes (isgs), many of which have direct or indirect antiviral activity ( ) . most paramyxoviruses counteract the ifn responses by producing proteins that block ifn induction and/or ifn signaling by a variety of mechanisms ( ) ( ) ( ) ( ) ( ) . furthermore, they tightly control viral transcription and replication, thereby limiting the production of pamps that may activate the ifn response ( , ) . indeed, it is probably mistakes that viruses make during transcription and replication, such as the production of copy-back-defective interfering particles, that activate the ifn response ( - ; reviewed in reference ). nevertheless, the ability of paramyxoviruses to block the ifn response both in tissue culture cells and in vivo is not absolute, and some ifn-␣/␤ will be produced ( , ) . furthermore, ifn-␥, which can also induce an antiviral state in cells, will also be produced by activated subsets of lymphocytes ( ) . therefore, it is inevitable that viruses will infect cells in a preexisting ifn-induced antiviral state, potentially limiting the speed of virus replication and spread. although ifns induce hundreds of isgs, several isgs with direct antiviral activity have been shown to be specific for families or groups of related viruses ( , , ) . with regard to the paramyxoviridae family, we have previously shown that isg /ifit (here referred to as ifit ), which selectively inhibits translation, is the primary effector of the ifninduced antiviral state that limits the replication of the rubulavirus piv ( ) . pretreatment of cells with ifn-␣/␤ inhibits piv protein synthesis but not cellular protein synthesis. this is because ifit selectively inhibits the translation of piv mrnas but does not affect cellular mrnas ( ) . mammalian mrnas have an n- methyl guanosine (m gpppn), termed cap , at their = end that recruits factors involved in rna processing and translation initiation. the first and second nucleosides of mammalian mrnas are also methylated on the = hydroxyl group of the ribose ring, generating cap and cap , respectively. while cap and cap are not required for efficient mrna translation, ifit can inhibit the translation of mrnas that lack cap ( ) ( ) ( ) ( ) . ifit also binds uncapped, =triphosphorylated rna, characteristic of the = ends of the genomic and antigenomic rnas of some rna viruses, as well as those of some viral transcripts ( ) ; for reviews on the mechanism of action of ifit and the ifit family of proteins, see references , , , and . however, recent evidence suggests that there are differences in the mechanisms of action of the murine and human paralog ifit proteins. while murine ifit (ifit b) inhibits the translation of mrnas that lack cap , it has been proposed that human ifit recognizes some other, as-yet-undefined structure near the cap or possibly that = mrna sequences may help define the specificity of inhibition by human ifit ( ) . the rna-capping activity of viral rna polymerases often include =-o-methyltransferases ( =-o-mtases), which modify cap and thus can avoid inhibition by ifit (b), as evidenced by the sensitivity of virus mutants that lack =-o-mtase activity (for reviews, see references and ) . capping and methylation of viral rnas are also important, as such modifications can prevent the activation of rig-i, thereby reducing the amount of ifn produced by virally infected cells (for a review, see reference ). here we have examined the ability of ifit to inhibit the translation of a variety of paramyxovirus mrnas and thus the replication of those viruses. we show that while all rubulaviruses tested were sensitive to ifit , all nonrubulavirus members of the paramyxoviridae tested were insensitive. lack of = o-methylation of rubulavirus mrnas was at least partially responsible for their inhibition by ifit . the possible biological consequences of differences in sensitivity of paramyxoviruses to ifit are discussed. cells, viruses, antibodies, and interferon. a cells and derivatives were grown as monolayers in -cm , -cm , or -cm tissue culture flasks in dulbecco's modified eagle's medium supplemented with % fetal bovine serum at °c. when appropriate, cells were treated with human recombinant interferon (intron a; merck, sharpe and dohme) at , units/ml. viruses used in these studies were piv (strain colindale), piv (strains washington and js and recombinant ⌬c and ⌬d js viruses [ ] ), piv (formerly known as sv ; strains w [ ] and cpi ϩ and cpi Ϫ [ ] ), muv (enders [ ] ), respiratory syncytial virus (rsv) ( ) , sendai virus (strain cantell, free of defective interfering particles), and canine distemper virus (strain mill hill). plaque assays were performed by standard methods in six-well dishes that included . % avicel (fmc biopolymer) in the overlay medium. plaques were visualized by immunostaining by using a pool of monoclonal antibodies or polyclonal antisera specific for the different viruses as described previously ( ) , together with alkaline phosphatase-conjugated secondary antibody by using sigmafast bcip/ nbt as the substrate. preparation of l-[ s]methionine-labeled total-cell extracts and sds-page. infected or uninfected cells that had or had not been pretreated with ifn for h prior to infection were metabolically labeled for h with l-[ s]methionine ( ci/mmol; mp biomedical, usa) at h postinfection (p.i.). after labeling, cells were lysed in disruption buffer, sonicated, and heated for min at °c and then analyzed by gel electrophoresis (sds-page). the gels were fixed, stained, and dried, and resolved bands were visualized by phosphorimager analysis. when appropriate, the same amounts of cell equivalents were run on page. furthermore, the amount of protein in each sample was monitored by staining the polyacrylamide gels (pags) with coomassie brilliant blue. immunofluorescence. cells to be stained for immunofluorescence were grown on -mm-diameter coverslips (mic ; scientific laboratory supplies, united kingdom). cells were stained with specific monoclonal antibodies (mabs), as described in detail elsewhere ( ) . briefly, monolayers were fixed with % formaldehyde- % sucrose in phosphatebuffered saline (pbs) for min at °c, permeabilized with . % nonidet p- - % sucrose in pbs for min at °c, and washed three times in pbs containing % calf serum. piv -and piv -infected cells were detected by indirect immunofluorescence using a secondary goat antimouse ig texas red-conjugated antibody (catalog number ab ; abcam). the primary antibodies were piv -np-a and piv -pe for piv ( ) and , , and for piv ( ) . after staining for immunofluorescence, the monolayers of cells were examined with a nikon microphot-fxa immunofluorescence microscope. rna selection and in vitro translation. rna for in vitro translations was isolated by sedimentation through cscl gradients by a modified method described by leppert et al. ( ) . confluent monolayers of infected cells, grown in -cm flasks, were resuspended in ice-cold lysis buffer ( mm nacl, mm tris-hcl [ph . ], . % np- , protease inhib-itor cocktail [complete mini edta-free, tablet per ml of buffer; roche]) at ϫ to ϫ cells per ml and left on ice for min prior to vortexing for min. nuclei were removed by centrifugation twice at , ϫ g for min at °c. the supernatant (cytoplasmic extract) was collected, made up to mm edta, and layered onto % (wt/wt) cscl in mm tris-hcl (ph . )- mm edta followed by centrifugation at , ϫ g at °c for to h. naked rna (including mrna) forms a pellet at the bottom of the gradient, while viral genomic and antigenomic rnas remain complexed with nucleoprotein and do not enter the % cscl cushion. the supernatant was discarded, and the pellet was resuspended in rnase-free water and adjusted to g/l. selected rna was translated in vitro with a rabbit reticulocyte lysate kit (l ; promega) in the presence of [ s]methionine-cysteine (neg , easytag express protein labeling mix; perkinelmer) using a modification to the manufacturer's instructions: methionine-cysteine-free medium (d ; sigma) was used to provide other amino acids ( l per l reaction mixture). capping and methylation of mrna. human rna guanylyltransferase and =-phosphatase (rngtt), rna guanine- methyltransferase (rnmt), and cap methyltransferase (cmtr ) were synthesized and purified according to the method of gonatopoulos-pournatzis et al. ( ) . as described in that study, the enzymes were all verified as being active by in vitro reactions followed by thin-layer chromatography. capping and methylation reactions were carried out in mm tris-hcl (ph . ), mm kcl, . mm mgcl , mm dithiothreitol (dtt) buffer as follows: l ϫ buffer, l rngtt ( . mg/ml), l rnmt ( . mg/ml), cmtr ( . mg/ml), l sam ( mm), l gtp ( mm), . l rnasin, l rna ( g/l). the reaction mixture was made up to l with h o, including in experiments in which rngtt, rnmt, or cmtr was omitted, and incubated at °c for h. cloning and purification of ifit . ifit was amplified with primer ifit f/ifit xho from the plasmid pgac-ha-ifit , restricted with ncoi and xhoi, and ligated with a modified plou , in which maltose binding protein (mpb) was replaced with sumo, while sali in the mutliple cloning site (mcs) was replaced with xhoi. the primers were as follows: ifit f, ccgccatggctacaaatggtgatgatcatcagg; ifit xho, gcgcctcgagctaaggaccttgtctcacagagtt. the fusion protein his-sumo-(tev)-ifit was expressed in escherichia coli strain rosetta in liters lb-ampicillin-chloramphenicol (lb/ amp/cm). isopropyl-␤-d-thiogalactopyranoside (iptg; . mm) was added when an optical density (od) of . was reached. the expression was carried out at °c overnight. purification was carried out with a routine protocol for his-tagged protein. the binding buffer contained mm tris-hcl (ph . ), . m nacl, and mm imidazole; the washing buffer contained mm imidazole; and protein was eluted with mm imidazole. to remove nonspecifically bound rna, the columns were washed with volumes of . m na hpo - m nacl (ph . ). after desalting into gf buffer ( mm tris-hcl [ph ], mm nacl, % glycerol), the fusion was cleaved with tobacco etch virus (tev) protease ( : ) at room temperature overnight. gel filtration was carried out after passing through ni beads again and addition of mm dtt. the ifit peak was collected and concentrated and had an a /a of . to . . despite the fact that paramyxoviruses encode ifn antagonists that inhibit ifn production and signaling, their ability to block the ifn response is not absolute. thus, they form larger plaques on ifn-incompetent cells than ifn-competent cells ( fig. ) ( ) , showing that during virus replication and spread some ifn is produced and slows the spread of the viruses (see fig. ). in the experiments shown in fig. and below, we used naive a , a /npro, and a /shifit cells; naive a cells can produce and respond to ifn in response to virus infection, and a /npro cells respond to exogenous ifn but cannot produce ifn as they constitutively express npro from bovine viral diarrhea virus (bvdv), which targets irf- for degradation ( ) . furthermore, because irf- is degraded in a /npro cells, they cannot upregulate expression of ifit in an irf- -dependent, ifn-independent manner in direct response to virus infection ( ) . a /shifit cells produce and respond to ifn, but expression of endogenous ifit in response to ifn or viral infection is inhibited due to constitutive expression of small hairpin rna (shrna) to ifit ( ) . we previously showed that ifit is the major cellular protein responsible for the ifn sensitivity of the rubulavirus piv ( ) . to further investigate the role of ifit and the ability of ifn to induce an antiviral state against other paramyxoviruses, we initially tested the ability of piv , piv , and piv to form plaques in a , a /npro, and a /shifit cells. all three viruses induced ifn in a cells as the plaques developed, as observed by the induction of mxa in the uninfected cells surrounding the plaque (fig. a) . as previously observed ( ), piv formed bigger plaques on a /shifit cells than on a cells, but the plaques were not as large as those on a /npro cells (fig. b) . while piv also produced slightly larger plaques on a /shifit than on a cells, the plaques on a /npro cells were obviously bigger (note that the center of mid-to large-sized piv plaques has fallen out of monolayers). piv produced similarly sized plaques on a and a /shifit cells and slightly larger plaques on a /npro cells. these results also support our previous conclusion that in a (and hep ) cells ifit is the primary isg effector to piv ( ) and that the rubulavirus piv is also sensitive to ifit . however, knocking down ifit did not have such a marked effect on piv plaque size as it did for piv . this indicates that there are likely to be additional isgs that play an important role in ifn-mediated inhibition of piv . in contrast, piv (washington strain) produced similarly sized plaques on a and a /shifit cells and only slightly larger plaques on a /npro cells; this suggests that the ifn response is capable of slowing the spread of piv to some degree (but not through the activity of ifit ), but not as dramatically as it does for piv or piv . however, experiments on the js strain of piv showed it to be more sensitive to the antiviral effects of ifn, but this was not because js is sensitive to ifit (data not shown). we next compared the synthesis of viral proteins in cells infected with piv , piv , and piv that had, or had not, been pretreated with ifn prior to infection with piv , piv , and piv . cells were infected at a high multiplicity of infection (moi; to pfu/cell), and the relative levels of np synthesis were visualized by radioactively labeling the cells for h with [ s]methionine at h p.i. (fig. ) . pretreatment of a and a /npro cells with ifn in this assay reduced the expression of the np of piv and piv to barely detectable levels. however, ifn pretreatment had no discernible effect on the expression of the np protein of piv or on the expression of host cell proteins. strikingly, expression of np of piv and piv was largely rescued in ifn-pretreated a /shifit cells, demonstrating that ifit plays a major role in the inhibition of piv and piv protein synthesis observed in a and a /npro cells pretreated with ifn. figure is an exemplar of many similar experiments that we have performed under different conditions (time course, moi, etc.) and that show the same results, namely, that piv and piv are inhibited by ifit while piv is not. having demonstrated that piv and piv are sensitive to ifit while piv is resistant, we tested the sensitivity of other members of the paramyxoviridae family, namely, mumps virus (muv strain enders), sendai virus (sev), and canine distemper virus (cdv). in a set of experiments similar to those illustrated in fig. , a /npro and a /shifit cells were or were not pretreated with ifn prior to infection with high moi with these viruses. the relative levels of np synthesis were visualized by radioactively labeling the cells for h with [ s]methionine at h p.i. (fig. a) . these experiments clearly demonstrated that, as was observed for piv and piv , pretreating a cells with ifn inhibited muv strain enders protein synthesis but knocking down ifit expression could largely restore muv protein synthesis. in contrast, as was observed for piv , although pretreatment of a cells with ifn slightly reduced the expression of sev and cdv protein synthesis, no increase in sev and cdv protein synthesis was observed in a /shifit compared to a cells pretreated with ifn. these results therefore show that muv enders is sensitive to ifit but sev and cdv are not, the weak inhibition of sev and cdv protein synthesis observed in a and a /shifit cells pretreated with ifn presumably being due to the action of other isgs induced by ifn. while muv is sensitive to ifit , it forms pinpoint plaques on a /npro cells only at days p.i. (data not shown), strongly suggesting that there are host cell restrictions other than innate intracellular defense mechanisms on muv replication in a cells ( ) . since in these experiments we used the attenuated enders strains of muv to test whether attenuation may be linked to sensitivity to ifit , we tested a wild-type (wt) isolate of muv-london- (lo- ) for its sensitivity. at the same time, we also tested the sensitivity of another strain of piv , termed cpi ϩ (fig. b) . muv-lo was as sensitive as muv enders, demonstrating that attenuation was not linked to differences in their relative sensitivity to ifit . similarly, piv cpi ϩ was also sensitive to inhibition by ifit . the ifit sensitivity of piv is not rescued by coinfection with an ifit -resistant virus. from these results, it was clear that replication of the nonrubulaviruses piv , sev, and cdv is not inhibited by ifit . to investigate whether piv replication could be rescued by coinfections with an ifit -resistant virus, mixed infections between piv and piv were undertaken. to avoid any possible synergistic effects between piv and piv in dismantling an ifn-induced antiviral state, the cpi Ϫ strain of piv was used in these experiments because, due to mutations in its v protein, it does not block ifn signaling ( ) . a or a /shifit cells were or were not pretreated with ifn for h prior to high-moi ( to pfu/cell) infection with piv , piv , or a mixture of the two viruses (fig. a) . the expression of the np protein of piv was resistant to ifn in both a and a /shifit cells when they were infected with piv alone and when coinfected with piv . in contrast, while the expression of piv np was resistant to ifn in a /shifit cells, its expression was inhibited in a cells, even when the cells were coinfected with piv . immunofluorescence was undertaken to ensure that in these experiments there was no exclusion of one virus by the other (fig. b and c) . these results confirmed that coinfection of piv with piv does not rescue the sensitivity of piv to ifit and strongly suggest that piv does not specifically inhibit the antiviral activity of ifit and that the inhibition of piv np expression is regulated by cis-acting elements. differential inhibition of translation of mrnas of different paramyxoviruses by purified ifit . the data above show that the ifn sensitivity of rubulaviruses is at least in part due to the actions of ifit . since this cellular protein has been shown to inhibit translation in a template-specific manner, we developed an in vitro translation system to study the ability of human ifit to selectively inhibit the translation of rubulavirus mrnas. the gene encoding human ifit was cloned as a sumo fusion protein expressed in escherichia coli, and the recombinant protein was purified (fig. a) . to determine whether the recombinant ifit was able to selectively inhibit piv mrnas, in vitro translation of mrna isolated from mock-and piv -infected cells was carried out in the presence and absence of different concentrations of ifit (fig. b, c, and d) . in the absence of ifit , expression of the np protein (and to a lesser extent the m protein) of piv was clearly visualized in the background of in vitro-translated cellular proteins ( fig. c and d) . increasing concentrations of ifit had no obvious effect on the efficiency of translation of host cell proteins, but in striking contrast, purified ifit selectively inhibited the translation of the np and m proteins of piv in a concentration-dependent manner. having established that the sensitivity of in vitro translation of piv mrna to inhibition by purified ifit correlated with the biological sensitivity of piv to ifit , we next tested the ability of ifit to inhibit the translation of mrna isolated from cells in-fected with other paramyxoviruses (fig. ) . these results clearly demonstrated that translation of (np) mrnas from piv -and from muv-infected cells was inhibited by ifit . in contrast, there was no obvious reduction in the amount of piv np synthesized when increasing amounts of ifit was added to the in vitro translation reaction mixtures. although there was a slight apparent reduction in the amount of sev and cdv np synthesis in the samples in which ifit was added, there was no increase in the inhibition observed by increasing the amount of ifit added to the in vitro translation reaction mixtures, strongly suggesting that the translations of sev and cdv mrnas are also resistant to inhibition by ifit . lack of =-o methylation of the cap structure of muv and piv mrnas is partially responsible for their sensitivity to inhibition by ifit . previous studies have shown that the absence of cap on mrnas renders them sensitive to inhibition to ifit . to investigate whether this was the case for rubulavirus mrnas, we developed an in vitro assay in which purified human mrnamodifying enzymes were used to progressively cap and add different methyl groups to the = ends of mrnas. purified human rna guanylyltransferase and =-phosphatase (rngtt), rna guanine- methyltransferase (rnmt), and cap methyltransferase (cmtr ) were used in these assays. rngtt adds a = guanosine to rnas with =-ppp, while rnmt adds a methyl group to the g of the guanine ring, generating (m g) cap . cmtr adds a methyl group to the = oh position of the adjacent ribose, generating cap . to demonstrate the functionality of this system, we first tested the in vitro translation of luciferase mrna with a =triphosphate group. this rna was efficiently translated in a capindependent manner and was only weakly inhibited by ifit (fig. a, compare lanes and ) . when the luciferase mrna was capped with the addition of =-guanosine by rngtt (generating gppp-mrna), there was a slight decrease in the amount of luciferase made (fig. a, compare lanes and ) . this may have been due to rngtt destabilizing or blocking the translation of gppp-mrnas in the absence of n methylation. however, strikingly, translation of this mrna was completely inhibited by ifit (fig. a , lane ) despite this cap structure lacking n- methylation. as expected, the addition of a methyl group to the n- position of the guanine ring, generating m gpppm n, by rnmt increased the efficiency of translation, but m gppp-luciferase remained completely sensitive to inhibition by ifit (fig. a, lanes and ) . addition of a methyl group to the = oh group of the adjacent ribose, generating cap , by cmtr did not affect the efficiency by which the mrna was translated, but it did clearly reduce the sensitivity of the mrna to inhibition by ifit (fig. a, compare lanes and ) . however, it should be noted that in these experiments, for reasons that are unclear, we were unable to completely restore full translation of the luciferase mrna in the presence of ifit by increasing the amount of cmtr or the length of incubation of the mrna with the enzyme (data not shown). to investigate how similar modifications to the cap of rubulavirus mrnas influenced their inhibition by ifit , we initially used muv mrna in a parallel set of experiments. these results showed that treatment of the muv mrna with rngtt and rnmt did not increase the efficiency of in vitro translation of muv np mrna or its sensitivity to inhibition by ifit (fig. b, lanes to ) , consistent with the viral polymerase adding m gppp-cap at (cap ) to the = end of viral mrnas. however, surprisingly, since rubulavirus polymerases have conserved =-o mtase domains, addition of a methyl group to the =oh group of the adjacent ribose (cap ) by cmtr clearly reduced the sensitivity of the np mrna to inhibition by ifit (fig. b, lanes and ) . as expected, the ifit sensitivity was dependent on the addition of s-adenosyl methionine (sam) to the reaction mixture (fig. c) . similarly, following =o methylation of piv mrna, in vitro translation of piv np became completely resistant to inhibition by ifit (fig. d) . strikingly, in contrast to np, the translation of piv m mrna remained completely sensitive to inhibition by ifit even after =o methylation of piv mrna by cmtr (fig. d and e) ; the basis for this is currently unknown, but we are investigating it further. over the past decade or so, it has become clear that the ways in which paramyxoviruses circumvent innate immune responses, including the ifn response, and differences in the multifunctional nature of their ifn antagonists are likely to influence the types of disease they cause. for example, the viral ifn antagonists within the rubulavirus genus, namely, the v proteins, as well as interacting with common targets such as mda and lgp also have unique properties. the v protein of piv targets stat for degradation, piv targets stat , and muv targets both stat and stat . within the respirovirus and morbillivirus genera, it is a combination of the v and c proteins that counteract innate responses by different molecular mechanisms, and strikingly, although piv encodes a c protein, it does not encode a functional v protein. despite encoding of these powerful ifn antagonists, ifn is produced during virus spread both in tissue culture cells and in vivo, and thus undoubtedly paramyxoviruses will, during the course of an infection, infect cells in a preexisting ifn-induced antiviral state. here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting ifn-induced antiviral state, and we suggest that this may influence the types of diseases caused. strikingly, in contrast to the sensitivity of rubulaviruses to ifit , the other paramyxoviruses that we tested were resistant, strongly suggesting that this might be a distinguishing feature of rubulaviruses, although before this can be firmly concluded the sensitivity of more species of paramyxoviruses to ifit needs to be tested. even within the rubulavirus genus, it appears that there may be differences in how members interact with cells in an ifn-induced antiviral state. in a cells, ifit primarily is responsible for the ifn-induced antiviral state induced to counter piv . however, although piv is sensitive to ifit , there appear to be other isgs that have strong anti-piv activity. this conclusion comes from the observation that while there is a slight increase in the size of piv plaques on a /shifit cells compared to a cells, it is not as obvious as that observed for piv . furthermore, while plaques for piv were smaller on a /shifit cells than on a /npro cells, this difference was not as marked as that observed for piv . muv strain enders is also sensitive to ifit , but there are clearly other major constraints on the growth of muv enders in human cells, as the virus grows extremely poorly in ifn-incompetent human cells but replicates to high titers in vero cells ( ) . it is striking that only rubulaviruses are sensitive to the antiviral activity of human ifit . our data indicate that the inhibition of rubulavirus mrnas was produced by ifit in a cis-linked manner, implying that the restriction is associated with some feature of uncapped =-ppp mrna encoding luciferase synthesized by t polymerase (provided as a control in the promega in vitro translation kit) was translated in vitro in a rabbit reticulocyte lysate in the absence or presence of purified ifit (lanes and ). rngtt was used to add a = guanine cap (lanes and ); then, rnmt was used to methylate the cap at the n position (lanes and ), generating cap , and cmtr was used to methylate the adjacent ribose on the = oh position, generating cap . the modified mrnas were then in vitro translated in the absence (lanes , , and ) or presence (lanes , , and ) of ifit . (b) mrna isolated from muv-infected cells was treated in parallel under the same conditions as those described for panel a. (c and d) mrna isolated from either muv (enders)or piv (w )-infected cells was in vitro translated prior to (lanes and ) or following (lanes and ) modification by cmtr in the presence or absence (lanes and ) of sam. the mrna was also translated in the absence (lanes , , and ) or presence (lanes , and ) of purified ifit . (e) densitometry traces of lanes , , , and of panel d. the numbers at the bottom of the gel indicate the fraction of either the host cell proteins or np proteins made in the in vitro translation mixes in the presence of purified ifit compared to those made in the absence of ifit . the mrna sequence or structure. since ifit can selectively inhibit the translation of mrnas that are incorrectly capped or not methylated at the = oh group of the first ribose, i.e., cap ( , , ) , it was likely that rubulaviruses have a structural motif in their cap, not present or hidden in the mrna of other paramyxoviruses, that is recognized by ifit . to investigate this further, we used purified human enzymes to modify the cap of mrnas. as a control for the activity of the enzymes, we used an uncapped =ppp mrna that encodes luciferase. the =-ppp luciferase mrna translated in a cap-independent manner in vitro using rabbit reticulocyte lysate, and this translation was only weakly inhibited by purified ifit . while addition of a = guanosine nucleoside cap slightly decreased the amount of luciferase synthesized, probably because the enzyme rngtt destabilizes the mrna, addition of the (unmethylated) guanosine nucleoside to the = end of the mrna significantly increased the sensitivity of the mrna to inhibition by ifit . furthermore, although methylation of the guanine ring at position n (m gpppnp-rna) by rnmt increased the efficiency of translation of luciferase mrna, it did not appear to affect the sensitivity of inhibition by ifit . these results are therefore consistent with the observation that human ifit binds with low affinity to =-ppp rna but more avidly to cap rna lacking = o methylation. methylation at position n of the guanine ring has also been reported to increase the affinity of binding of ifit ( ); however, the observation here that gppp-luciferase is inhibited as efficiently as m gppp-luciferase suggests that the methyl group does not play a central role in the inhibition of mrnas by ifit . in contrast, = o-methylation of the first ribose by cmtr to generate cap partially prevented ifit from inhibiting the translation of the cap -modified mrna. however, even by increasing the amount of cmtr and the incubation time, we were unable to completely restore full translational activity of the luciferase mrna. the reasons for this are unclear, but it suggests that other structural features, for example, methylation of the penultimate ribose to generate cap or sequences at the = end of mrnas, may also influence inhibition by ifit , as has been suggested by daugherty et al. ( ) . mrnas isolated from piv -, sev-, and cdv-infected cells were not inhibited by ifit , and neither was the replication of these viruses ( fig. and ). in contrast, = o-methylation of the terminal ribose by cmtr of muv mrnas partially alleviated inhibition of the np mrna by ifit . with regards to piv , our previous studies suggested that piv mrnas were = o-methylated ( ) . furthermore, we never observed complete ifit inhibition of piv np synthesis in vitro, suggesting that at least a proportion of the piv np mrna was correctly capped. however, the fact that treating piv mrnas with cmtr rescued np synthesis in the presence of ifit suggests that a significant proportion of piv mrnas was also not fully methylated. it is also of potential significance that the m mrna of piv appears to be more sensitive than np mrna to inhibition by ifit , and furthermore, translation inhibition of piv m mrna was not rescued by treatment with cmtr . the differences in the relative sensitivity of the np and m mrnas clearly warrant further investigation but may be due to the fact that the viral methyltransferase differentially methylates the viral mrnas (as has been shown for vesicular stomatitis virus [vsv] [ ] ), that cmtr does not recognize the untranslated region (utr) of the piv m mrna, or that inhibition by ifit is influenced by additional structural features present on piv m mrna but not np mrna. regarding the latter point, it is of note that the first three nucleotides of the utrs of np and m differ. furthermore, the or nucleotides downstream of cap are thought to be bound by ifit and may thus modulate ifit -rna interactions ( ) , and some secondary rna structures, e.g., those found at the = end of some alphaviruses, can prevent ifit binding to rna independent of the cap methylation status ( ) . most viruses successfully avoid inhibition by ifit by encoding their own =-o mtase, by cap snatching appropriately capped and =-o methylated structures from cellular mrnas, or by having cap-independent translation with the covalently linked viral protein vpg or a = rna secondary structure that blocks the activity of ifit (reviewed in reference ). indeed, work on virus restriction by ifit has involved primarily the investigation of viruses in which the =-o-mtases have been mutated such that their mrnas do not have a cap structure ( , ( ) ( ) ( ) ( ) . nevertheless, our results show that the viral polymerase of rubulaviruses, unlike other paramyxoviruses, does not fully protect the viral mrnas from inhibition by human ifit . in this regard, it is of interest that although rubulaviruses have the conserved methyltransferase domain in their polymerase, they all have an alanine instead of the first glycine in a gxgxg motif present in the methyltransferase domain of other paramyxoviruses and mononegavirales, which has been shown to affect the efficiency of cap methylation ( ) . most viruses, including other mononegavirales ( ), appear to be naturally resistant to inhibition by ifit . it is therefore intriguing that rubulaviruses have not evolved mechanisms to ensure that their mrnas are correctly capped and methylated or have the appropriate utrs to be resistant to ifit . it is tempting to speculate that there is some unknown biological advantage to being sensitive to ifit . for example, it may help some rubulaviruses (and perhaps hepatitis c virus [ ] , which is also sensitive to ifit ) to establish prolonged or persistent infections. thus, following infection of cells in an ifn-induced antiviral state, ifit restricts piv replication. under such conditions, virus genomes are located in cytoplasmic foci, where, as we have previously suggested, they may remain hidden from intracellular and adaptive immune responses. furthermore, if viral mrna is produced in cells in an ifn-induced antiviral state, then viral protein synthesis will largely be inhibited by ifit , thus reducing the amount of protein that may be processed and presented to cytotoxic t lymphocytes (ctls). eventually, in such cells, however, enough of the virus ifn antagonist, the v protein, will be produced or brought in by infecting virus particles to target stat for proteasomemediated degradation, and the cells will no longer be able to maintain their antiviral state, thus facilitating virus replication ( ) . whether such a scenario occurs in vivo, these and other considerations emphasize that to fully understand the molecular pathogenesis of viruses, it will be necessary to understand the subtleties of how viruses interact with the ifn system and other host cell defense mechanisms. the biology of paramyxoviruses fields virology paramyxovirus activation and inhibition of innate immune responses the regulation of type i interferon production by paramyxoviruses paramyxovirus disruption of interferon signal transduction: status report inhibition of interferon induction and signaling by paramyxoviruses paramyxovirus evasion of innate immunity: diverse strategies for common targets interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures newly described pattern recognition receptors team up against intracellular pathogens pattern recognition of viral nucleic acids by rig-i-like helicases interferon-stimulated genes and their antiviral effector functions plk down-regulates parainfluenza virus gene expression role for the phosphoprotein p subunit of the paramyxovirus polymerase in limiting induction of host cell antiviral responses sendai virus defectiveinterfering genomes and the activation of interferon-beta deep sequencing analysis of defective genomes of parainfluenza virus and their role in interferon induction defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity defective viral genomes: critical danger signals of viral infections parainfluenza virus genomes are located in viral cytoplasmic bodies whilst the virus dismantles the interferon-induced antiviral state of cells virus replication in engineered human cells that do not respond to interferons regulation of interferon-gamma during innate and adaptive immune responses interferon-induced ifit proteins: their role in viral pathogenesis a diverse range of gene products are effectors of the type i interferon antiviral response isg /ifit is primarily responsible for interferon-induced changes to patterns of parainfluenza virus type transcription and protein synthesis inhibition of translation by ifit family members is determined by their ability to interact selectively with the =-terminal regions of cap -, cap -and =ppp-mrnas =-o methylation of the viral mrna cap evades host restriction by ifit family members innate immune restriction and antagonism of viral rna lacking -o methylation ifits: emerging roles as key anti-viral proteins ifit is an antiviral protein that recognizes =-triphosphate rna the isg /ifit gene family evolutionguided functional analyses reveal diverse antiviral specificities encoded by ifit genes in mammals when your cap matters: structural insights into self vs non-self recognition of = rna by immunomodulatory host proteins mutations in the c, d, and v open reading frames of human parainfluenza virus type attenuate replication in rodents and primates multiplication of a myxovirus (sv ) with minimal cytopathic effects and without interference parainfluenza virus surface projections: glycoproteins with haemagglutinin and neuraminidase activities attenuation of virulence with retention of antigenicity of mumps virus after passage in the embryonated egg respiratory syncytial virus: reverse genetics and vaccine strategies heterocellular induction of interferon by negativesense rna viruses intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that icp is associated with progeny virus dna isolation and characterization of monoclonal antibodies to simian virus and their use in revealing antigenic differences between human, canine and simian isolates antigenic analysis of human and bovine parainfluenza virus type strains with monoclonal antibodies plus and minus strand leader rnas in negative strand virus-infected cells ram/fam a is required for mrna cap methylation the npro product of bovine viral diarrhea virus inhibits dna binding by interferon regulatory factor and targets it for proteasomal degradation mumps virus enders strain is sensitive to interferon (ifn) despite encoding a functional ifn antagonist differences in interferon sensitivity and biological properties of two related isolates of simian virus : a model for virus persistence sequestration by ifit impairs translation of =o-unmethylated capped rna structural basis for viral =-ppp-rna recognition by human ifit proteins ribose =-o methylation of the vesicular stomatitis virus mrna cap precedes and facilitates subsequent guanine-n- methylation by the large polymerase protein characterization of transgenic mice with targeted disruption of the catalytic domain of the doublestranded rna-dependent protein kinase, pkr a viral rna structural element alters host recognition of nonself rna ifit inhibits japanese encephalitis virus replication through binding to = capped =-o unmethylated rna attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking =-omethyltransferase activity =-o methylation of the viral mrna cap by west nile virus evades ifit -dependent and -independent mechanisms of host restriction in vivo rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral mrna cap methyltransferase sequence-function analysis of the sendai virus l protein domain vi human and murine ifit proteins do not restrict infection of negative-sense rna viruses of the orthomyxoviridae, bunyaviridae, and filoviridae families isg and ifitm proteins inhibit hepatitis c virus replication catalytic turnover of stat allows piv to dismantle the interferon-induced antiviral state of cells key: cord- - y y authors: shoemaker, jason e.; fukuyama, satoshi; eisfeld, amie j.; zhao, dongming; kawakami, eiryo; sakabe, saori; maemura, tadashi; gorai, takeo; katsura, hiroaki; muramoto, yukiko; watanabe, shinji; watanabe, tokiko; fuji, ken; matsuoka, yukiko; kitano, hiroaki; kawaoka, yoshihiro title: an ultrasensitive mechanism regulates influenza virus-induced inflammation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: y y influenza viruses present major challenges to public health, evident by the influenza pandemic. highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. however, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. we found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin and monocyte chemotactic protein , exhibit ultrasensitive behavior. a systematic exploration of the pathways regulating the inflammatory-associated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the toll-like receptor pathway that regulates stat phosphorylation. this study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. the approach developed here should facilitate the construction of gene regulatory models of other infectious diseases. influenza viruses present major challenges to public health, evident by the influenza pandemic. highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. however, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. we found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin and monocyte chemotactic protein , exhibit ultrasensitive behavior. a systematic exploration of the pathways regulating the inflammatoryassociated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the toll-like receptor pathway that regulates stat phosphorylation. this study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. the approach developed here should facilitate the construction of gene regulatory models of other infectious diseases. invading pathogens induce acute inflammation when molecular signatures are detected by pattern recognition receptors (prrs; e.g., rig-i like receptors [rlrs] and toll-like receptors [tlrs] ) expressed on tissue-resident immune cells and non-immune cell types. prr ligation triggers innate immune responses and leads to the induction of inflammatory and antiviral gene expression, which together function to limit pathogen growth, activate the adaptive immune response, and ultimately resolve the infection [ , ] . precise regulation of prr-mediated signaling is necessary to both avoid inadvertent tissue damage in response to non-pathogenic stimuli, and to prevent immunopathology resulting from excessive expression of inflammatory molecules. in essence, the ideal inflammatory response must exhibit a balance between appropriate activation against a genuine threat and self-limiting behavior once that threat has been controlled. despite its importance in maintaining normal tissue homeostasis and limiting pathogen-associated diseases, the mechanisms underlying the regulation of this balance are poorly understood. influenza a viruses are recognized by both tlrs and rig-i-like receptors (rlrs) [ ] [ ] [ ] [ ] [ ] , and some strains are potent inducers of inflammatory and antiviral gene expression. generally, lung tissues infected with pathogenic isolates exhibit high virus titers and robust inflammatory gene expression, as has been documented in in vivo studies with the spanish influenza virus [ , ] , highly pathogenic h n avian influenza viruses [ ] [ ] [ ] , and the h n pandemic influenza virus [ , ] . in contrast, seasonal influenza viruses typically replicate less efficiently, elicit more restrained inflammatory responses, and are usually not associated with lethal infections. recent evidence has implicated the level of virus replication in infected lung tissues as the primary phenotypic variable driving inflammation and lethal outcomes [ , ] . other data indicate that influenza viruses that exhibit significant differences in pathogenicity stimulate qualitatively similar host responses that differ primarily at the level of magnitude and kinetics [ ] . however, these studies have not revealed the mechanisms that account for the different profile dynamics observed in infections by high and low pathogenic viruses. such information would aid in clarifying not only how some influenza viruses induce lethal disease, but also the general mechanisms that regulate inflammatory balance. to characterize the dynamics of influenza virus-induced inflammation, we developed a novel approach to infer gene regulatory models from dynamic gene expression data. referred to as systems inference microarray analysis, our method builds on current approaches that use co-expression analysis to isolate modules of functional signatures in gene expression data and then extends these methods by fitting the gene expression modules to mathematical equations (models) by using segmented regression analysis. models can be created to look for strain-dependent responses and, unlike traditional differential expression analysis, to predict gene expression under new experimental conditions. by using this method, we set out to determine how influenza viruses that exhibit variable pathogenicity profiles influence the dynamics of the inflammatory response. to characterize the dynamics of the host immune response to specific virus isolates, we infected mice with pfu of three virus isolates with distinct pathogenicity profiles and harvested lung tissues at ). an initial inoculation of pfu was used as previous studies indicated that a high virus dose was needed to invoke different pathologies in h n and ph n -infected mice [ ] . as expected, lung virus titers (virus titers determined by plaque assay and reported in plaque forming units [pfu] per gram lung; fig ) indicated a clear hierarchy of mild, moderate, and severe virus-induced disease. specifically, the h n virus produced the highest lung titers and between days and post-infection, this virus also caused mortality in the animals whose lungs were to be collected on day post-infection (i.e., 'severe' disease). in contrast, all animals infected with the h n or ph n viruses survived the duration of the time course study; however, ph n -infected animals were visibly sicker and exhibited higher lung titers relative to those infected with h n at all time points observed , , , , , , , , , and h and , , and days), and virus titers in lung tissues were determined by plaque assays in mdck cells. error bars illustrate the standard deviation from the mean. the gray boxes at each time point identify significant pairwise differences between the means of the viruses indicated at the right of the figure panel (significance was determined by anova followed by tukey's honestly significantly different test, p < . ). *, three h n -infected mice intended for collection on day succumbed to their infections prior to sample collection. after the first h post-infection (i.e., 'moderate' and 'mild' disease, respectively). histopathological analysis of tissue samples collected on days , , and post-infection (fig ) also showed that h n -infected tissue exhibited the earliest, most severe signs of inflammation and inflammatory immune cell infiltrates followed by ph n -infected tissue, whereas h n -infected tissue showed mild evidence of inflammation and was most similar to tissue from the control mice (mock-infected mice). we next used co-expression analysis to integrate inflammation-associated gene expression differences between influenza-infected and control lungs into a systems level context. we first asked whether the expression of inflammation-associated genes clustered into modules of coexpressed genes. tissues from the same animals that were used to determine virus growth were used to evaluate changes in global lung transcriptional profiles. a total of microarrays were developed (three per time point for h n -infected, ph n -infected, h n -infected and control mice). one microarray was removed after reviewing replicate quality. after filtering transcripts for minimally confident variation (we required at least one time-matched, infected condition compared with mock-infected absolute fold change and a false discovery rate [fdr]-adjusted p-value < . ), the log of the normalized intensity of the retained transcripts ( , ) for all samples were then clustered by using the weighted gene co-expression network analysis (wgcna) algorithm [ ] . in all, distinct co-expression modules were identified (referred to as n , n , etc.; s table provides the module assignments for all transcripts). to identify the biological role of the host response modules, we performed functional enrichment analysis on each gene module by using david [ ] and toppcluster [ ] . because each module was comprised of positively and negatively correlated transcripts, we used the module eigengene (i.e., the first principle component of the gene expression matrix) to divide each module into two submodules containing transcripts that were positively or negatively correlated with the parent module's eigengene, denoted as kme+ and kme-(referred to as module membership), respectively. this procedure allowed us to look for biological processes with similar but opposing dynamic responses to the virus infections. functional enrichment analyses were then applied to each submodule by using two bioformatics platforms to ensure robust results. we identified two submodules (n kme+ and n kme-, referred to as simply n and n in the remainder of the text) that were enriched for inflammatory response and inflammationassociated pathway signatures by using both bioinformatics platforms, and these two modules became the focus of our study (table summarizes the functional enrichment results for the immune and inflammatory related annotations. the complete enrichment results from toppcluster and david are available in s table and s and s files). the n module was uniquely enriched for cytokine activity and type i interferon (ifn) regulating tlr and rlr pathways [ ] , as well as transcriptional signatures associated with ifn-regulated activity (i.e., the transcription factor binding sites [tfbs] of irf , irf , irf , isre, and nf-κb). additionally, n was the only module that exhibited significant enrichment with a compendium of established ifn-stimulated genes ('isgs'; table ; see methods. the list of isgs is available in s file). a more recent study identified ifn stimulated genes in immortalized, human airway epithelial (calu ) cells [ ] . of these, mouse homologs were annotated on the microarrays and of the homolog probes were assigned to the n module (fisher's exact test; pvalue < - ; odds ratio = . ), further associating n interferon-stimulated gene activity. in contrast, the n module was only weakly enriched for some cytokine activity related annotations and not enriched for any of the binding sequences of transcription factors that are members of canonical inflammatory pathways (such as interferons, interferon regulatory factor proteins, or nfκb). instead, it was primarily associated with several annotations related to leukocyte and lymphocyte activity (see summary of toppcluster enrichment results in table ; see s and s files). further analysis with cten [ ] , a platform for associating clustered gene expression data with specific cell types, found n to be highly enriched for genes expressed in macrophages in various cellular states (e.g., bone marrow-derived macrophages exposed to lipopolysaccharide [lps]) ( fig a; additional details available in s table) . the remaining immune-associated submodules (the kme+ n , n , n , and n submodules; described in table ) were enriched for several key immune processes such as antigen presentation, and t cell and natural killer (nk) cell activity, but their further assessment would be beyond our focus and the scope of this study. thus, bioinformatics analyses robustly associated the n module with inflammation, cytokine production, and type i ifn pathway activity-likely activated in resident lung cells-whereas the n module is associated with migration and activation of macrophages in the lung. each module was divided into submodules in which each member gene's expression was positively or negatively correlated with the module's eigengene (denoted kme+ and kme-, respectively). for each module, we provide the number of transcripts assigned to each submodule, the top david annotation clusters for each submodule (parenthesis shows the-log of the average enrichment p value for all annotations in the cluster), the top enriched biological processes determined using toppcluster (parenthesis shows the-log of the fdr-adjusted p value), the enrichment of established transcription factor binding sites in each submodule (tfbs; parenthesis shows the-log of the fdr-adjusted p value; performed using toppcluster), and the enrichment score of a set of ifn-stimulated genes (see methods; enrichment score is the-log of the fdr-adjusted p value). doi: . /journal.ppat. .t a closer examination of the expression dynamics of each of the inflammation response-associated modules revealed patterns of expression that were consistent with the biological roles predicted by our bioinformatics analyses. we used the scaled difference of the module eigengene to characterize the expression of all genes within each module. we subtracted the mean of the eigengene of the control samples from the eigengene of time-matched, virus-infected dynamics of inflammation-associated co-expression modules. panel (a) shows the enrichment scores (-log of the fdr-adjusted p value) of the genetic signatures of cell types in the n module as determined using cten [ ] . data corresponding to macrophage signatures in different cellular states are colored red (see s table for within the inflammation-associated modules (n and n ), the h n virus induced the earliest gene expression changes and the highest peak expression levels, corroborating previous observations that h n viruses are strong inducers of inflammatory and ifn response signaling in vivo [ , , ] . consistent with the prediction that n is involved in detecting virus in infected tissues, the n module eigengene was the most highly correlated with virus titer (pearson pairwise correlation, ρ = . ). module gene expression dynamics further suggested that the n module gene is associated with lymphocyte infiltration. exudate macrophages [ ] and neutrophil [ ] have been identified as factors of severe disease during influenza infection. to associate gene expression dynamics with changes of immune cell counts, a new population of mice were infected with the three influenza viruses, five mice per infection group were sacrificed on days , , and and the changes in the number of macrophages and neutrophils was assessed (see methods). unlike the previous study by brandes, et al. [ ] , strong neutrophil infiltration was not specific to fatal infections but, instead, infiltration of both cell types had a clear hierarchical relationship with the severity of the infection (fig d and e ). the n module eigengene exhibited a lesser correlation to virus titer (ρ = . ), but was tightly correlated to macrophage influx into the lung (ρ = . ; p-value < . student's t-test). of the module identified in the studied, n had the highest correlation to both macrophage and neutrophil influx (the correlation of macrophage and neutrophil influx and all module eigengenes are provided in s table; ρ = . ; pvalue < . ). the n module on the other hand was weakly but significantly correlated with immune cell infiltration (ρ = . [p-value = . ] for neutrophils; ρ = . [p-value = . ] for macrophages), but its eigengene was not the most highly correlated ( other module eigengenes had a greater absolute correlation). these results further associate n with immune cell-specifically macrophage-infiltration, while the sum of the bioinformatics, virus titer correlations and immune cell infiltration evidences associated n with inflammation and type i ifn pathway activity. a further advantage of a network approach is that the functional relevance of genes might be inferred from their positions within the co-expression network [ ] . we used the module membership (the correlation between the gene's expression and the module eigengene, kme) to isolate potential regulators of the n module. among the top intramodular hub genes (i.e., genes with the highest module memberships, see s table) , we found mnda, herc and cd and several interferon regulated, virus replication inhibitory genes such as oas and oas [ ] . herc is involved in ubiquitination [ ] . mnda is significantly up-regulated in monocytes exposed to interferon α [ ] while cd is a transmembrane protein expressed on antigen presenting cells and modulates activation of t cells, b cells and myeloid cells. we also observed that interferon stimulated genes tended to have higher intramodular hub rankings, suggesting a regulatory role for interferon (wilcoxon rank sum test, p-value < - ). we then considered the module membership rankings of transcription factors known to regulate interferon. of the established interferon regulatory factors that are members of the n module (e.g., irf , irf , irf , irf , stat , and stat . nfkb and nfkb were not assigned to n ), irf and irf had the highest module memberships (kme = . and . ; ranking = and , respectively). irf expression was also several orders of magnitude greater than irf (s table) . together, these findings corroborate our bioinformatics analyses by suggesting that n is regulated by interferon, and n expression likely results in enhanced cytopatchic effects and regulation of the lymphocyte immune response. network analysis further suggests that irf may play a regulatory role upstream of interferon transcription. previous studies have suggested that highly pathogenic influenza virus infections induce an irregular or disproportionate inflammatory response relative to seasonal influenza viruses, and that these differences occur early in the host response [ ] . for this reason, we sought to further explore the possibility of isolate-specific or isolate-independent response patterns of the cytokine-associated n module. we wanted to infer mathematical relationships that could describe when inflammatory-associated gene expression occurs and what magnitude of expression is expected. by using the eigengene as a representation of the scaled gene expression dynamics, we attempted to infer simple mathematical models that can be related to common signaling mechanisms. surprisingly, when we plotted the n sde for each isolate against the corresponding virus titer, we observed a consistent profile for all three viruses; regardless of intrinsic virulence, the fold change in n gene expression remained initially low and rapidly increased only after a virus titer of approximately~ pfu/g (of lung) was reached (fig a) . following activation, n gene expression increased as a function of virus concentration at the same apparent rate for all infection conditions, and more complicated dynamics were observed only during the later phase of the infection when virus clearance was observed (i.e., when the virus titers began to decrease). these observations suggest that ifn-regulated (n ) gene expression was induced by an ultrasensitive response mechanism controlled at the level of the virus titer rather than the virus's intrinsic virulence. ultrasensitive responses characterize the dynamics of several signaling pathways that regulate essential and often toxic biological processes such as the cell cycle [ ] and apoptosis [ ] . as shown in fig b, ultrasensitive responses are typified by an attenuated response to low levels of stimulation but a strong response occurs once a threshold level of stimulus is reached. cooperativity [ ] and positive feedback [ ] are two mechanisms that produce ultrasensitive responses. to formalize the hypothesis that the inflammatory gene response follows an ultrasensitive response profile, we selected a segmented linear model (slm, defined in fig c) to be a simplified representation of the ordinary differential equations normally used to model ultrasensitive responses, and we fit the n sde to an slm that was strictly a function of the virus titer. the optimal fit showed a threshold of . ± . pfu/g is required for n module activation to occur, after which the sde's rate of activation (a ) was . ± . log (pfu/g) - with an intercept (b ) of - . (unitless) (see the methods and s fig for additional details) . below this threshold, the model predicted minimal gene expression (a = . ; b = . ). the slm goodness of fit on the training data was an adjusted r = . while an adjusted r = . was observed when the data was fit to a linear model. a davie's test confirmed that a segmented model was a significantly better fit than a linear correlation model (p-value < . e- ). while the h n -infected lung tissue did not exceed an average peak virus titer of . pfu/g (peak titer occurs at hpi in fig a) , we observed increased transcriptional activity in h n -infected mouse lung tissues after this time point, suggesting either that the actual peak virus titer occurred between hpi and the subsequent time point ( hpi), or that the model-predicted threshold was slightly over-approximated. we next sought to validate the threshold model by attempting to predict cytokine-associated gene expression in influenza virus-infected lung when only the virus titers are known. for this, we selected the h n virus, which has previously been associated with an excessive illustration of an ultrasensitive response to increasing stimulus. in our model, the response is the change in inflammatory-associated gene expression and the stimulus is the lung virus titers. a segmented linear model (slm) can approximate key aspects of the ultrasensitive response. in this study, the response is gene expression and the stimulus is the concentration of virus in the lung (c) the mathematical definition of an slm. below some threshold virus titer (thr), the scaled eigengene (se) is approximated by a linear model that is a function of the log of the virus titer, the slope (a ), and the intercept (b ). after the threshold virus titer is reached, the slope and intercept parameters change (a and b ) to describe the se after activation. the parameters were fit to the data shown in panel (a) and used to predict the n se in newly infected mice. (d) shows a comparison of the h n n eigengene from an infection at a dose of pfu (as shown in fig ) and an infection at a dose of pfu. panel (e) compares the predicted scaled eigengene based on the slm versus the measured eigengene behavior in mice infected with pfu of the h n virus. panel (f) shows gene expression changes for selected constituent genes of the n module as a function of virus titer. cytokine response [ ] . we infected mice with pfu of the h n virus (a -fold reduced dose compared with that used in the experiments to fit the model), determined lung virus titers at the same time points used for the initial experiment (s fig), and then evaluated the segmented linear model's ability to predict cytokine-associated gene expression. first, we confirmed that the original transcripts assigned to the n module were again co-expressed, and thus we used the same transcripts originally assigned to the n module to determine the eigengene (see s fig for an analysis of the conservation of the n module between the two experiments). in this experiment, as expected, the peak average virus titer ( . ± . pfu/g) for the pfu dose was delayed compared with that for the pfu dose (s fig, compare to fig ) . moreover, the sde exhibited an -h delay in activation and a -h delay in peak expression compared with the pfu n eigengene (fig d) . importantly, based on the virus titers alone, the fitted segmented linear model accurately predicted n -like sde behavior (r = . for all time points and r = . for time points up to peak expression; fig e and s fig), and this could be further demonstrated at the individual gene level for specific n -associated transcripts (e.g., herc , stat and irf ; fig f) . these observations provide strong evidence that activation of inflammatory-associated gene expression is dictated by a specific virus concentration in infected tissue, and further suggest the novel possibility that the pulmonary innate inflammatory response has a nonlinear, ultrasensitive-like activation profile that promotes tolerance to low concentrations of virus. the dynamics of key inflammatory cytokines are consistent with an ultrasensitive response although transcriptional activation of ifn-stimulated and inflammatory gene expression is a reasonable measure of the effects of inflammation response stimulation, we reasoned that a bona fide ultrasensitive mechanism that regulates this response should be reflected in other aspects of the associated signaling pathway(s). indeed, of the cytokines associated with the n module, -including key inflammatory proteins, such as interleukin (il- ) and monocyte chemoattractant protein- (mcp- )-were significantly correlated with the n module eigengene (pearson's ρ . ; fdr-adjusted p-value < . ; see s table) . in addition, when the protein expression levels of these cytokines were plotted against the corresponding titer data, we observed dynamics similar to that of the inflammatory-associated n gene module. initially, protein expression was low but strongly increased only after the virus titers exceeded the threshold of~ pfu/gram determined in the gene regulation model (fig a) . in contrast, most of the protein levels of the other measured cytokines with transcripts that were not assigned to the n module did not show any obvious relationship to the proposed threshold response (s fig). the only major exceptions were lif, rantes, and il (s fig). lif's gene transcript was not annotated on the arrays whereas il 's transcript was not identified as differentially expressed and therefore was not included in the clustering study. the rantes transcript was included in the clustering study and assigned by the wgcna algorithm to n although the transcript's correlation to the n eigengene suggests it could also have been assigned to the n module (pearson correlation = . and . to the n and n eigengene, respectively). some proteins appeared to conform to the threshold model in ph n -infected, but not h n -or h n -infected, mice. these may be cytokines that have strain-dependent responses and do not conform to the model. overall, changes in n -associated cytokine protein levels in influenza virus-infected mouse lungs were consistent with the proposed virus-titer regulated threshold-mechanism underlying the ifn-mediated response. threshold-like behavior is observed on upstream and downstream components of the ifn signaling pathway. (a) lung tissues from the same infected mice used for the original gene expression analysis were subjected to cytokine array analysis. thirty-two cytokine protein concentrations were measured and log scaled (data points lower than the limit of detection for each protein were set to . to allow scaling), and the average fold change relative to uninfected, time-matched lung samples was determined. the heat map illustrates protein expression values for the cytokines that had transcripts assigned to the n module (a blue-to-yellow scale shows expression levels, as indicated by the color key at the top of the panel; time points in which the change in the cytokine levels were not significant [fdr-adjust p < . ] were set to zero); of these, exhibited expression profiles that were highly significantly correlated with the corresponding transcript and the n module eigengene (pearson's ρ . and fdr < . ; eotaxin and tnf-α were the only two that did not exhibit a significant, direct correlation). non-n cytokines are shown in s (fig b) . for the h n data, significant levels of pstat were observed at hpi which-as noted previously-corresponds to the time immediately after the virus titers in h n -infected mice reached their peak (see previous discussion). on the other hand, significant increases in phosphorylated irf (pirf ) were observed only in ph n and h n infections, whereas significant increases in ifn-β were observed only in the h n infection. changes in the levels of ifn-β and pirf occurred after significant increases in pstat and ifn-α occurred. as such, the change in the percentage of pstat was more closely correlated to the n eigengene than was that of pirf (correlation = . ± . and . ± . respectively), and significant increases in pstat corresponded to time points at which the mean virus titer exceeded the threshold level identified in the gene expression analysis (~ . pfu/g). the greater correlation of ifnα and stat activation to the inflammation-associated, n module's gene expression dynamics and the enrichment of the n module for the irf binding sequence (table ) suggest that the primary driver of the threshold-regulated, inflammatory gene response originates along the irf ! ifn-α ! stat axis (fig c) . our data reveal that the activation of the ifn-associated inflammatory and antiviral response program in influenza-infected mouse lung is characterized by an ultrasensitive response driven by the virus load. the power of the threshold model is illustrated by its ability to accurately predict gene expression in infected mice, and the data further suggest that the molecular basis of threshold behavior originates upstream of ifn-α production. threshold-like and ultrasensitive mechanisms are hypothesized to be necessary for effective management of critical cellular machinery in noisy environments, and are recognized players in the activation of the cell cycle [ ] , mitogen-activated protein kinase signaling [ ] , and apoptosis [ , ] . however, while a role for threshold behaviors have been postulated to be essential for filtering noise or errant signaling in complex biomolecular environments [ ] , our study is the first to directly link threshold-like behavior to the virus-induced innate immune response. the ultrasensitive response observed in this study provides additional insight into the mechanisms that drive severe pathologies during influenza infection. several works have suggested that viral load is a key determinant of pathology [ , ] while other works suggest that highly pathogenic influenza viruses induce early, strong inflammatory responses that are independent of the viral load [ , , ] . recently, it was observed that fatal influenza infections in mice map, with titers surpassing the threshold level identified in the slm indicated in red. (b) another set of mice was infected with pfu of h n , ph n , or h n and lung tissues were harvested ( mice per infection per time point) for immunoblot (stat , phosphorylated stat , irf , and pirf ) or elisa (ifnα, ifn-β) analysis; virus titers were also determined. the heat map indicates the mean percentage of phosphorylated protein (purple) or the mean interferon concentration (pg/ml; green), and virus titers for each time point are indicated below. only significant changes are shown in the heat map (fdr-adjusted pvalue < . relative to mock-infected samples). titers highlighted in red indicate that the threshold level (as determined in the initial analysis) was exceeded. panel (c) shows a schematic of known canonical pathways that regulate ifn production in response to virus infection. black arrows denote induction whereas red lines denote points at which influenza virus is known to impair these pathways. ifn-α is primarily produced by antigen-presenting cells (apcs) whereas ifn-β is produced in virus-infected, non-apc cells. both ifns can induce stat phosphorylation and expression of isgs in responding cells. doi: . /journal.ppat. .g ultrasensitive mechanism regulates influenza-induced inflammation coincide with a strong influx of neutrophils in what the author's describe as a viral load-independent, "feedforward" inflammatory circuit [ ] . the ultrasensitive response suggested by our study consolidates these hypotheses by suggesting that viral load drives cytokine production (and in turn immune cell infiltration) in a nonlinear manner which is capable of producing states of high and low innate immune responses. characterization of key aspects of the inflammatory response, such as the onset and peak inflammatory gene expression, require a high temporal resolution of the virus growth and host response dynamics; an experimental design that was unique to our study. the ultrasensitive response model does not negate the significance of neutrophil infiltration [ ] in determining fatal infections but suggests that viral load drives the high and low innate immune states. the observed threshold may represent the transition to immunopathology; as indicated by the histopathology results (fig ) . moreover, the influenza virus' ns protein is crucial for inhibiting the interferon-mediated antiviral response [ ] . the ns protein of three viruses used in this study have the sumo acceptor site that indicates interferon antagonism capability [ ] [ ] [ ] . additional studies with ns -mutated viruses and other pathogens may better reveal strain-dependencies for the observed thresholding behavior. the ultrasensitive response further suggests that the innate immune response has a limited capacity to respond to influenza virus infection and supports the development of immunomodulatory therapies. interestingly, after the threshold was exceeded, the rate of activation for inflammatory and interferon-associated gene expression (n ) was conserved for a moderately pathogenic and deadly viruses (fig a) . the conserved rate of activation implies that the immune response detects the virus concentration but not the virus growth rate; suggesting the innate immune response is naturally limited in its ability to respond to high growth influenza viruses. additionally, studies in knockout mice indicate that type i ifn-associated pathways are essential for protection during primary infection [ ] and that earlier initiation of these pathways coincides with increased survival in mice infected with highly pathogenic isolates [ ] . in combination with these studies, the findings here suggest a novel means of protecting high risk groups by treating them with compounds that target the molecular mechanisms responsible for the threshold behavior. lowering the threshold required to induce the cytokine response may be a means of providing protection from severe influenza infection. since these compounds would target host proteins, such treatments would be effective against various influenza virus strains. data from the viruses studied here suggest that post-threshold, inflammatory gene expression primarily reflects interferon-regulated tissue damage, but time-course data from additional highly pathogenic viruses are needed to assess the degree to which interferon activity is associated with virus growth suppression. the a/california/ / h n virus (ph n ) was received from the centers for disease control and prevention. the a/kawasaki/utk- / h n virus (h n ) served as a reference for a seasonal influenza, whereas a fatal human isolate, a/vietnam/ / h n virus (h n ), served a highly pathogenic virus. all mouse experience were performed in accordance to the university of tokyo's regulations for animal care and use. these regulations were approved by the animal experiment committee of the institute of medical science, the university of tokyo (approval number: pa - ). the committee acknowledged and deemed acceptable the legal and ethical responsibilities for the animals, as detailed in the fundamental guidelines for proper conduct of animal experiment and related activities in academic research institutions under the jurisdiction of the ministry of education, culture, sports, science and technology, . all experiments with h n viruses were performed in biosafety level containment laboratories at the university of tokyo, which are approved by the ministry of agriculture, forestry, and fisheries, japan. five-week old c bl/ j, female mice were obtained from japan slc. for all experiments, mice were anesthetized with isoflurane and intranasally inoculated with either or pfu of virus. initially, mice were inoculated with pfu of the h n , ph n , or h n virus or mock-infected with pbs (a total of mice). at time points, mice per group were humanely sacrificed and their lungs harvested. the lungs were sectioned and used to assess virus titers (right-upper lobe), cytokine levels (right lower), and the initial gene expression that was used to train the segmented linear model (left-lower). separately, mice were infected with pfu of the h n , sacrificed at the same time points, and lungs sections obtained as described previously to provide the model validation data. the same inoculation method was used for all mice in this study. the numbers of mice used for flow cytometry, western blot, and interferon protein assay experiments are specific in the corresponding sections. for cytokine and chemokine measurements, mouse lungs were treated with the bio-plex cell lysis kit (bio-rad laboratories, hercules, ca) according to the manufacturer's instructions. concentrations of other cytokines were determined with the bio-plex mouse cytokine -plex and -plex panels (bio-rad laboratories) array analysis was performed by using the bio-plex protein array system (bio-rad laboratories). virus titers were determined by plaque assay using mdck cells. mouse lung tissues were placed in rnalater (ambion, ca), an rna stabilization reagent and stored at - °c. all tissues were thawed together and homogenized for minutes at hz using a tissuelyser (qiagen, hilden, germany) as per the manufacturer's instructions. from the homogenized lung tissues, total rna was extracted with the rneasy mini kit (qiagen, hilden, germany) in accordance with the manufacturer's instructions. cy -labeled crna preparations were hybridized onto agilent- whole mouse genome x k microarrays for h at °c. feature extraction software version (agilent technologies) was used for image analysis and data extraction, and takara bio provided whole array quality control metrics. microarray normalization, replicate quality, annotation, and statistical analysis differential expression was assessed by using a linear regression model. by using the limma package [ ] version . . from bioconductor, probe intensities were background corrected by using the "norm-exponential" method, normalized between arrays (using the quantile method), and averaged over unique probes ids. replicate quality was assessed using hierarchical clustering, resulting in the removal of a single array (the array corresponded to a sample collected at hpi in h n -infected mice.) probe intensities were fit to a linear model that compared data from infected samples to time-matched data collected from uninfected mice. probes were annotated by matching to the probe names in the mgug a version . mouse annotation database available from bioconductor. all arrays in this study have been deposited on the geo expression omnibus (gse ). unsigned co-expression networks were constructed by using the blockwisemodules program from the wgcna package version . . [ ] in r. the analysis was performed with several different parameterizations to ensure robust clustering. for the results reported in this text, we removed all probes that did not have a confident fold-change greater than (fdr < . ) for at least one infected-tissue to control-tissue, time-matched comparison. we then clustered the log of the normalized intensities for all microarrays (corresponding the three samples for each time-point for each infected or control population with the exception of data from h n -infected mice at hpi which had two samples). based on the scale-free topology characteristics curve, a power of n = with no reassignment after clustering (reassignthreshold = ) and a maximum cluster size of probes was used. we generally observed that allowing gene reassignment between the modules led to poorer clustering based on the distribution the gene's module memberships (the correlation between a gene and the eigengene of the module to which it had been assigned. s fig illustrates the distribution of the gene kmes for modules n and n ). we then repeated the clustering using different powers (ranging from to ), allowing different cluster sizes, different subsets of the expression data (e.g., clustering data from each infection separately or together), or relaxing the differential expression condition. in all clusterings performed, the two modules discussed in the text were identifiable. fisher exact tests between each clustering run were used to determine whether the initial modules were significantly conserved under different parameterizations. we also considered if the n module would be isolated when using signed versus unsigned network construction. we constructed a signed co-expression network and found that % of the kme+ n genes are again clustered and confirmed that the gene expression dynamics were maintained (see s fig) . toppcluster [ ] and david [ ] were used for gene ontology and pathway enrichment, and toppcluster was also used for transcription factor binding site enrichment analyses. david uses clusters of related annotations constructed from several annotation databases (e.g., pathway and gene ontology annotations) to determine the function of a set of genes and scores the enrichment by averaging the unadjusted p-values (determined by fisher's exact test) of the annotations within the cluster. toppcluster uses hypergeometric tests to determine the enrichment between a set of genes and gene lists contained in categories (databases) detailed in the toppgene suite [ ] . the databases include cis-regulatory motif data [ , ] , referred to as transcription factor binding sites (tfbs) in the text. both tools were used with their default settings and the gene universe was considered to be all annotated mouse genes. for each module, we considered the enrichment of all genes assigned to the module and the kme+ and kmesubsets. generally, the enrichment analysis of the whole module gene set reiterated the enrichment results of the kme+ and kme-subsets albeit with slightly lower but still significant enrichment. since both tools returned similar go and pathway enrichment results, we summarized the functional and pathway enrichment results in table using the results from david. the enrichment of interferon stimulated genes was determined by using a list of interferon stimulated genes from the interferon stimulated gene database [ ] that was downloaded on may , (see s file). for each module, all module genes and the kme+ and kme-subsets were tested for enrichment using fisher's exact test in r. the p values were adjusted to control the false discovery rate. cten [ ] was used to determine enriched cell signatures in select co-expression modules. the enrichment score reported is the-log of the false discovery rate. model fitting and validation was performed in r using the 'segmented' package [ ] . five mice per time point per infection were infected with pfu of the described virus. five uninfected (naïve) mice served as negative controls. whole lungs were collected from mice, and incubated with collagenase d (roche diagnostics; final concentration: μg/ml) and dnase i (worthington; final concentration: u/ml) for minutes at °c. single-cell suspensions were obtained from lungs by grinding tissues through a nylon filter (bd biosciences). red blood cells (rbcs) in a sample were lysed with rbc lysis buffer (sigma). samples were resuspended with pbs containing mm edta and . % bovine serum albumin (bsa), and cell number was determined by using a disposable cell counter (onecell). to block nonspecific binding of antibodies mediated by fc receptors, cells were incubated with purified anti-mouse cd / (fc block, bd biosciences). cells were stained with appropriate combinations of fluorescent antibodies to analyze the population of each immune cell subset. the anti-f / (bm ; ebioscience) antibodies were used. all samples were also incubated with -aminoactinomycin d (via-probe, bd biosciences) for dead cell exclusion. data from labeled cells were acquired on a facsaria ii (bd biosciences) and analyzed with flowjo software version . . (tree star). three mice per time point per infection group were infected with pfu of the described virus. the primary antibodies of mouse anti-stat (phospho tyr ) mab (ab , abcam), rabbit anti-irf (phospho ser ) mab ( , cell signaling), and mouse anti-βactin (a ; sigma-aldrich) were used; the secondary antibodies were hrp-conjugated antimouse igg antibody (ge healthcare) and hrp-conjugated anti-rabbit igg antibody (ge healthcare). mouse lungs were collected and homogenized with ripa buffer (thermo scientific, rockford, il, usa) containing proteinase inhibitor (roche, mannheim, germany) and phosphatase inhibitor cocktails (sigma-aldrich, saint louis, missouri, usa). the lysates were then briefly sonicated and centrifuged. each sample was electrophoresed on sodium dodecylsulfate polyacrylamide gels (bio-rad laboratories, hercules, ca, usa) and transferred to a pvdf membrane (millipore, billerica, ma, usa). the membranes were blocked with blocking one (nacalai tesque, kyoto, japan) for min at room temperature, and then were incubated with the primary antibodies overnight at °c, followed by the secondary antibodies. they were then washed times with pbs plus tween (pbs-t) for min and incubated with secondary hrp-conjugated antibodies (as described above) for min at room temperature, followed by three washes with pbst. specific proteins were detected by using supersignal west femto maximum sensitivity substrate (thermo scientific, rockford, il, usa). photography and quantification of band intensity were conducted with the versadoc imaging system (bio-rad laboratories, hercules, ca, usa). the quantity of target bands from each sample was standardized by their respective β-actin. three mice per time point per infection group were infected with pfu of the described virus. half the lung of each mouse was dissolved in ml of ripa buffer. we measured the interferon-alpha and interferon-beta by using elisa kits (# , # , pbl assay science, nj, usa) according to the manufacturer's instructions. plates were read at an absorbance of nm using a versa max plate reader (moleculardevices, menlo park, ca). additional gene set overlap tests were performed in r with all of the genes annotated on the array as the reference (background) set. statistical tests to compare means within the western blot, flow cytometry, immune cell count and protein assay data sets were performed in r using the 'multcomp' package [ ] . supporting information s fig. the scaled difference of the module eigengene. the eigengene is the first principle component (equivalently the first eigenvector) of a matrix of gene expression data. in clustered data in which all genes are highly correlated (as in the case of wgcna), the eigengene is a scaled approximation of how gene expression changes for all genes in a module (i.e. cluster) across experimental conditions. the eigengene of module n is shown in (a) for the experimental conditions considered in this study ( infection conditions and time points). each point represents data from a single animal in each experimental condition ( animals per condition except for h n -infected animals at hpi which had ; total of samples). while the eigengene illustrates the changes in gene expression of module member genes, it is difficult to interpret the changes with respect to the control data. we therefore scaled the eigengene by subtracting from time-matched data the average of the eigengene from mock-infected animals and then dividing by the highest average eigengene for any experimental condition. the scaled difference of the eigengene (sde) is shown in (b). the sde now represents the fraction of greatest gene expression (i.e., the fraction of the largest log fold change in gene expression observed between all time-matched, infected-to-control samples). for example, the sde peaks in experimental condition (h n -infected animals at hpi), corresponding to the condition in which the log fold change was greatest for n member genes (see fig ) . the sde for ph n -infected animals at day pi (condition ) is~ . . we would expect the log fold change to be approximately half of that observed in h n -infected animals at hpi (see the heatmaps in fig ) . for completeness, we overlay the mean (lines) and the standard deviation to create the segmented linear model to describe the relationship between the n module eigengene and the virus titer data, we selected all titer data that occurred prior to and included the peak of the eigengene and then scaled the data such that the eigengene was bound between [ , ]. the model was then fit to the scaled data. panel (a) shows the time points selected as training data from each infection data set (indicated by the orange dots); and panel (b) shows the number of data points available for different ranges of the virus titer (top) and the segmented model's fit to the training data (bottom). in panel (c), we used the residuals (i.e., the difference between the predicted values and the actual values) to compare the slm's accuracy to that of a simple linear model. the line is the running average and the shaded region is the % confidence interval of the mean. the mean of the residuals of the segmented model was always near zero for the full range of virus titers and, thus, is a better fit than the linear model. two procedures were applied to determine whether the n genes that we identified as co-expressed in the original clustering analysis were also co-expressed in tissue from mice infected with pfu of h n virus. (a) compares the correlation between all , n gene transcripts and the eigengene (referred to as the kme) in the original gene expression data ( pfu infection conditions (referred to as ' pfu')) and the pfu infection condition (referred to as ' pfu'). more than % of the genes exhibited a kme > . . (b) the wgcna algorithm was repeated with all differentially expressed genes (fdr-adjusted p-value < . and fold change > for at least time point) from the pfu h n virus infection, and then the fisher's exact test was used to identify modules enriched for n genes. of the modules in the newly constructed co-expression network, only one module was enriched with the n transcripts. specifically, of the genes originally assigned to n , (benjimini-hochberg-adjusted p-value < . e- ) were differentially expressed and assigned to the same module in the pfu infection condition, as illustrated by the venn diagram. . the h n - pfu eigengene was constructed using the same set of probes assigned to the n module from the pfu infection condition and scaled to between [ , ]; and then the segmented linear model trained to the pfu h n , ph n , and h n data was used to predict h n - pfu scaled eigengene values. panel (a) shows a comparison of the predicted (black) and actual (pink arrows depicting the temporal evolution of the gene response) eigengenes, with error bars illustrating the standard deviation of the eigengene and of the log of the virus titer. panel (b) shows how the prediction residuals are distributed over time. for each time point, individual data points (black points) are shown, as well as the average (red points) and standard deviation (gray bars). the greatest deviations occurred at d and d , which was expected as the model was designed only to predict the onset of gene activation and the peak gene expression (peak of the eigengene). on days and post-infection, both the virus titers and gene expression has already peaked and are declining. panel (c) shows how the prediction residuals are distributed across the spectrum of observed virus titers, as compared to a linear model directly fit to the h n - pfu n eigengene. individual residuals are indicated by points, and the running average and the % confidence intervals are shown by the colored lines and the gray shading, respectively. as for the pfu infection condition, the segmented model performed well across the entire virus titer spectrum, and was significantly better than a linear model fitted directly to the data. (tif) s fig. protein concentrations of non-n module cytokines. as described in the fig leg end, cytokine concentrations were assayed in the lungs of mice infected with pfu of h n , ph n , and h n and compared with those in lung tissues from mock-infected animals. this heat map illustrates protein expression values for non-n -associated cytokines ( cytokine expression profiles are shown; il- (p )( ) was not detected at any time point and is not included), with a blue-to-yellow scale indicating expression levels (see the key to the right of the panel). the module to which the protein's mrna transcript was assigned during clustering is shown on the right hand side of the heat map (proteins whose gene transcripts were not de are labeled 'na'), and the average virus titers are shown below the heat map (red indicates that titers exceeded the threshold concentration predicted by the segmented linear model). while il- and leukemia inhibitory factor (lif) exhibited protein expression patterns consistent with n module behavior, the transcripts mapping to these proteins were not differentially expressed and were excluded from the co-expression network construction. (tif) s fig. virus titer and immunoblot data from an additional infection experiment with pfu of h n , ph n , or h n virus. as described in the fig legend (panel b) , an additional set of mice was infected with pfu of h n , ph n , or h n virus to determine total and phosphorylated levels of transcription factors by means of immunoblotting. here, virus titers (with standard deviation indicated by gray bars) for each infection condition are shown in panel (a), and immunoblot results are shown in panels (b) and (c). for immunoblot analyses, relative protein concentrations were determined by calculating the ratio of the gray intensity of the measured protein (iκbα, total irf , total irf ['irf '], phosphorylated irf [pirf '], total stat ['stat '], and phosphorylated stat ['pstat ']) relative to the gray intensity of actin (i.e., the loading control; referred to as the relative signal intensity, rsi) in each tissue sample. a linear model was used to compare the mean rsi of each protein at each time point to the mean rsi measured in uninfected animals (referred to as 'naïve'), and a significant difference was defined as having a false discovery rate (fdr) < . . the mean rsi of total irf , irf , and iκbα did not significantly deviate from the naïve data, but pstat , pirf and total stat significantly differed from the naïve data at several time points (signifi- the intramodular correlation (kme or correlation between each transcript and the eigengene) can be used to assess clustering quality. we show boxplots to illustrate the distribution of the kmes for all transcripts belonging to the n and n modules. (tif) s fig. applying signed co-expression network analysis also clusters the n kme+ transcripts. the n module presented in this work was identified by developing an unsigned co-expression network and then focusing on the kme-and kme+. therefore, the n kme+ set of genes should also cluster when developing a signed co-expression network. we confirmed this by repeating the wgcna procedure for signed network (power = ) . of the n kme+ transcripts, . % were assigned to the same cluster in the signed co-expression network (the new module is referred to as n signed). furthermore, . % if the n signed transcripts were n kme+ transcripts. we then confirmed that the same eigengene dynamics were observed. the scaled difference of the eigengene (sde) of the n signed module is shown versus time (a) and versus virus titers (b). (tif) s file. each of the kme+ submodules, labeled n , n ,. . .,n were analyzed using david bioinformatics tool to determine enriched biological functions. for each module, we report the top enriched annotation clusters as well as various measurements of enrichment (the bonferroni, benjamini, and the false discovery [fdr] adjusted p value. additional information on the enrichment statisticis can be found at david.abcc.ncifcrf.gov (xls) s file. each of the kme-submodules, labeled n , n ,. . .,n were analyzed using david bioinformatics tool to determine enriched biological functions. for each module, we report the top enriched annotation clusters as well as various measurements of enrichment (the bonferroni, benjamini, and the false discovery [fdr] adjusted p value. additional information on the enrichment statistics can be found at david.abcc.ncifcrf.gov (xls) s file. a list of interferon stimulated genes received from the interferon stimulated gene database. (xlsx) s table. the co-expression module to which each transcript was assigned. for each transcript we provide the entrez gene id, the gene symbol, the module it was assigned to and the correlation between the transcript's expression dynamics and the expression dynamics of its assigned eigengene (kme). module n is the set of transcripts which the algorithm did not identify as co-expressed. (xlsx) s table. enriched annotations identified using toppcluster. each kme+ or kme-submodule was analyzed in toppcluster for enriched domain, go biological process, go molecular function, go cellular component, mouse phenotype, pathway or transcription factor binding site annotations. columns a-d provide information on the annotation, including the category to which the annotation belongs, the database specific annotation id and the full title of the annotation. columns d-at contain the enrichment score for each annotation in each submodule. the enrichment score is the-log of the false discovery rate [fdr]-adjusted p value. a threshold enrichment score of (fdr-adjusted p < . ) was required to be considered as significantly enriched. (xlsx) s table. cten analysis of n module. the probes assigned to module n were analyzed in cten-a platform for identifying the genomic signatures of select cell type in microarray data. the enrichment score reported for each cell type is the-log of the false-discovery adjusted p value. (xlsx) s table. a heatmap of the gene expression of the probes assigned to the n and n modules. arrays were developed from the lungs of mice infected with h n , ph n , or hpai virus at time points. one array from the hpai infected data set was removed due to quality concerns. for each transcript, we provide annotation information (probe name, systematic name, entrez id, gene symbol, and gene name), identify to which module the transcript was assigned, the kme (the pearson correlation coefficient between the transcripts expression and the eigengene of the module it was assigned to), and the log fold change in the transcript's expression across all arrays used the study is illustrated by a heatmap. (xlsx) s table. correlation between the changes macrophage and neutrophil cell counts and each module eigengene. (xlsx) s table. correlation between changes in cytokine protein levels and cytokine gene transcript levels. (xlsx) regulation of toll-like receptor signaling pathways in innate immune responses immune signaling by rig-i-like receptors cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells recognition of single-stranded rna viruses by toll-like receptor differential roles of mda and rig-i helicases in the recognition of rna viruses rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates '-triphosphate rna is the ligand for rig-i aberrant innate immune response in lethal infection of macaques with the influenza virus genomic analysis of increased host immune and cell death responses induced by influenza virus lethal dissemination of h n influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection h n influenza viruses: outbreaks and biological properties fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia in vitro and in vivo characterization of new swine-origin h n influenza viruses integrated network analysis reveals a novel role for the cell cycle in pandemic influenza virus-induced inflammation in macaque lungs h n influenza virus pathogenesis in genetically diverse mice is mediated at the level of viral load viral replication rate regulates clinical outcome and cd t cell responses during highly pathogenic h n influenza virus infection in mice specific mutations in h n mainly impact the magnitude and velocity of the host response in mice wgcna: an r package for weighted correlation network analysis resources: expanded annotation database and novel algorithms to better extract biology from large gene lists toppcluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems snapshot: pathways of antiviral innate immunity pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses cten: a web-based platform for identifying enriched cell types from heterogeneous microarray data lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes integrated clinical, pathologic, virologic, and transcriptomic analysis of h n influenza virus-induced viral pneumonia in the rhesus macaque ccr + monocyte-derived dendritic cells and exudate macrophages produce influenza-induced pulmonary immune pathology and mortality a systems analysis identifies a feedforward inflammatory circuit leading to lethal influenza infection transcriptomic analysis of autistic brain reveals convergent molecular pathology extracellular '- ' oligoadenylate synthetase stimulates rnase l-independent antiviral activity: a novel mechanism of virus-induced innate immunity systematic and quantitative assessment of the ubiquitin-modified proteome the transcriptional landscape of the mammalian genome ultrasensitivity in the regulation of cdc c by cdk bistability in apoptosis: roles of bax, bcl- , and mitochondrial permeability transition pores ultrasensitivity and positive feedback to promote sharp mitotic entry ultrasensitivity in the mitogen-activated protein kinase cascade identifying fragilities in biochemical networks: robust performance analysis of fas signaling-induced apoptosis the ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein d binding influenza a virus lacking the ns gene replicates in interferon-deficient systems identification of the non-structural influenza a viral protein ns a as a bona fide target of the small ubiquitin-like modifier by the use of dicistronic expression constructs modification of nonstructural protein of influenza a virus by sumo sumoylation affects the interferon blocking activity of the influenza a nonstructural protein ns without affecting its stability or cellular localization myd signaling is indispensable for primary influenza a virus infection but dispensable for secondary infection toll-like receptor pre-stimulation protects mice against lethal infection with highly pathogenic influenza viruses linear models and empirical bayes methods for assessing differential expression in microarray experiments toppgene suite for gene list enrichment analysis and candidate gene prioritization systematic discovery of regulatory motifs in human promoters and utrs by comparison of several mammals molecular signatures database (msigdb) . functional classification of interferon-stimulated genes identified using microarrays segmented: an r package to fit regression models with broken-line relationships simultaneous inference in general parametric models we would like to thank susan watson for editing the manuscript. key: cord- -ndz oarf authors: ayithan, natarajan; bradfute, steven b.; anthony, scott m.; stuthman, kelly s.; bavari, sina; bray, mike; ozato, keiko title: virus-like particles activate type i interferon pathways to facilitate post-exposure protection against ebola virus infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ndz oarf ebola virus (ebov) causes a severe hemorrhagic disease with high fatality. virus-like particles (vlps) are a promising vaccine candidate against ebov. we recently showed that vlps protect mice from lethal ebov infection when given before or after viral infection. to elucidate pathways through which vlps confer post-exposure protection, we investigated the role of type i interferon (ifn) signaling. we found that vlps lead to accelerated induction of ifn stimulated genes (isgs) in liver and spleen of wild type mice, but not in ifnar(-/-) mice. accordingly, ebov infected ifnar(-/-) mice, unlike wild type mice succumbed to death even after vlp treatment. the isgs induced in wild type mice included anti-viral proteins and negative feedback factors known to restrict viral replication and excessive inflammatory responses. importantly, proinflammatory cytokine/chemokine expression was much higher in wt mice without vlps than mice treated with vlps. in ebov infected ifnar(-/-) mice, however, uninhibited viral replication and elevated proinflammatory factor expression ensued, irrespective of vlp treatment, supporting the view that type i ifn signaling helps to limit viral replication and attenuate inflammatory responses. further analyses showed that vlp protection requires the transcription factor, irf known to amplify type i ifn signaling in dendritic cells and macrophages, the probable sites of initial ebov infection. together, this study indicates that vlps afford post-exposure protection by promoting expeditious initiation of type i ifn signaling in the host. ebola viruses (ebovs) are enveloped, negative-sense rna filoviruses that can cause a severe hemorrhagic fever in humans and non-human primates (nhps) [ , ] . mouse-adapted ebov causes similar acute disease in mice, offering a useful animal model to study ebov infection [ , ] . ebov infection is characterized by rapid viral replication and dysregulated innate and adaptive immune responses. the disease follows profound suppression of type i ifn signaling and a contrasting excess inflammation that leads to mucosal hemorrhages and multi-organ failure resembling septic shock syndrome [ , ] . virally encoded anti-ifn proteins, vp and vp play major roles in ebov virulence [ , ] . vp blocks type i ifn induction in dendritic cells (dcs) and macrophages, and acts as a virulence factor necessary for a recombinant virus to attain infectivity in the host [ ] [ ] [ ] [ ] . vp , on the other hand, blocks ifn signaling by interfering with ifn activated jak/stat pathways [ ] . lines of evidence support the critical importance of type i ifn signaling in providing resistance against ebov infection; mice deficient in stat , a transcription factor required for ifn induction, or ifnar , encoding the membrane receptor for type i ifns, are susceptible to wild type zaire ebov, against which wild type mice are resistant [ ] [ ] [ ] . a study of sudan ebov infection in humans showed that ifnα levels are significantly higher in surviving patients than those with fatal ebov infection, who had higher levels of proinflammatory cytokines/chemokines such as il- , and mip- β [ , ] . high ifnα production is reported to correlate with increased resistance against ebov in mice as well [ ] . administration of recombinant ifnα or ifnβ confers delayed time-to-death in nhps [ , ] . furthermore, ifnα, used as an adjunct therapy for monoclonal antibody treatment, is shown to enhance protection in nhps [ ] . ebov infection remains a potential threat to public health, which is compounded with the lack of effective prevention or treatment. to overcome this problem, various vaccine candidates have been developed, including various dna constructs, recombinant viruses, vlps, as well as treatment with anti-sense sirna [ ] [ ] [ ] . vlps are subunit-based vaccines, extensively studied for a variety of infectious pathogens [ , ] . vlps prepared from ebov and other filoviruses are composed of the matrix protein (vp ), glycoprotein (gp), and at times nucleoprotein (np) and represent a potentially promising candidate for ebov vaccine. ebov vlps have been shown to confer protection upon rodents and nhps when given prior to infection [ ] [ ] [ ] . in the accompanying paper, we show that post-exposure administration of trivalent vlps protects mice from lethal ebov infection, further crediting the potential of vlps as a possible vaccine [ ] . in that study, we show that vlp protection requires macrophages, dendritic cells (dcs) as well as b and either cd or cd t lymphocytes, indicating that both innate and adaptive immunity are involved in conferring protection. the aim of this study was to further investigate molecular bases of postexposure protection by vlps. based on our previous report that vlps stimulate type i ifn expression in dcs and macrophages, in vitro, we focused on the role of type i ifn signaling, and found that post-exposure vlp treatment leads to accelerated activation of ifn signaling, resulting in early induction of isgs. significantly, vlp stimulated isg induction coincided with the attenuation of proinflammatory cytokine surge in ebov infected mice. the reduced inflammatory responses was attributed to activation of type i ifn signaling, since vlp treated ifnar -/mice were unable to inhibit not only viral replication but proinflammatory responses, and succumbed to death. our results indicate that early type i ifn response is a major mechanism that contributes to vlp mediated protection against ebov infection. ifnar -/-, ifnar +/+ mice of balb/c background and irf -/and irf +/+ mice of c bl/ background were bred in the nichd animal facility and transferred to the facility of the united states army medical research institute of infectious diseases (usamriid) for ebov infection studies. research was conducted in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the guide for the care and use of laboratory animals, national research council, . the facility where this research was conducted is fully accredited by the association for assessment and accreditation of laboratory animal care international. the iacuc committee approving this protocol is the united states army medical research institute of infectious diseases (usamriid) iacuc. animals were monitored at least once daily and their status was evaluated according to an intervention score sheet approved by usamriid iacuc. monitoring increased to three times daily if the animals were given a score of three or four. euthanization was by co inhalation followed by confirmatory cervical dislocation. analgesics and anesthetics were not used in this study and animals were euthanized for humane purposes if they reached a score of five or more, which would be indicated if the animals exhibited ruffled fur, weakness, unresponsiveness, and/or difficulty walking. otherwise, animals were euthanized at the end of the study. vlps were composed of ebov gp, np and vp and were generated in mammalian t cells as reported previously [ ] . vlp preparations used in this study contained < . endotoxin u/mg. mice were infected with~ pfu (~ , ld ) of mouse-adapted ebov via intraperitoneal (i.p.) route [ ] . mice were injected with vlps ( μg) diluted in pbs through i.p. h after ebov infection. morbidity and mortality of ebov infected mice were monitored twice daily for up to days. total rna from liver and spleen of ebov infected mice were extracted by trizol method (invitrogen) and cdna was synthesized from μg total rna by superscript ii reverse transcriptase (invitrogen). qpcr amplification was done with ng cdna in μl sybr green pcr master mix (applied biosystems) with μm of both reverse and forward primers used in the abi prism sequence detection system (applied biosystems). mrna of expression of indicated genes were analyzed as described in detail elsewhere [ ] . the primer pairs were used for ebov gp, '-tgggctgaaaactgctacaatc- ' and '-ctttgtgcacataccggcac- '; nlrp , '-tgctcttcactgctatcaagccct- ' and '-acaagcctttgctccagaccctat- '. all other gene primer sequences were followed from the previous publications [ , ] . transcript levels were normalized with hprt, and expressed as relative expression. statistical analysis was carried out by excel software using two-tail paired student's t test. data represent the mean of at least three independent assay ± sem. a p value < . was considered significant. to assess the role of type i ifn signaling in vlp-mediated protection against ebov infection, we tested ifnar -/mice for protection by vlps. in fig. a , wild type (wt) and ifnar -/mice (both balb/c background, n = ) were injected with μg of vlps h after infection by the mouse adapted (ma) ebov, and the morbidity and mortality were checked daily for the subsequent days. wt mice without vlp injection all died between day and day , whereas % of mice that received vlps survived after ebov infection, confirming that vlps protect mice even when they were given post-infection. in contrast, ifnar -/mice that received vlps all died before or at day as those without vlp injection (fig. a) . these results are in agreement with previous report on early death of ma-ebov infected ifnar -/mice [ ] . ebovs are thought to initially infect dcs and macrophages in liver and spleen, making these tissues the major sites of ebov replication in the mouse, although the virus infects many other organs later [ , ] . to ascertain whether vlps inhibit viral replication, we measured ebov glycoprotein (gp) mrna expression in liver and spleen from wt and ifnar -/mice with or without vlp administration. qrt-pcr analysis in fig. b and c, found that levels of gp mrna rose sharply in ifnar -/mice on day of infection when gp mrna was still at background in wt mice. ifnar -/mice that received vlps also expressed considerable amounts of gp mrna, although levels varied between liver and spleen in the vlp treated group. thus, ebov appeared to replicate faster and to a greater extent in ifnar -/mice than wt mice. it should be noted here that in wt mice, gp mrna levels began to increase rapidly after day , peaking on day to day , and that vlp injection inhibited gp mrna expression by more than half (s fig.) . pfu ma ebov, followed h later by injection i.p. with μg of ebov vlps. one group of ifnar +/+ (wt) mice infected with ebov without vlp served as control. mortality is expressed as percent survival of each group on indicated days. results are a representative of three independent experiments, which gave very similar outcomes. qrt-pcr detection of ebov gp mrna level on day post-ebov infection with or without vlps from liver (b) and spleen (c) of wt or ifnar -/mice. gp transcripts were normalized by hprt and values represent the mean ± sem of duplicate samples from three independent experiments. asterisk denotes significant differences compared to wt controls (*p . , **p . ). these results are in line with the results that ifnar -/and stat -/mice are more susceptible to ebov infection, suggesting the possibility that vlp mediated protection is linked to the activation of type i ifn signaling [ ] [ ] [ ] . however, vlp injection may not have prevented ebov pathogenesis in ifnar -/mice, possibly because the disease manifests more severely in these mice than in wt mice. on the other hand, it has been recently shown that adenovirus based vaccine can protect ifnar -/mice from lethal evob infection presumably through antibody responses, which indicates that ifnar -/mice are not universally vulnerable, and anti-ebov resistance can be attained in some cases [ ] . we recently reported that ebov vlps activate type i ifn transcription in dcs and macrophages in vitro, leading to induction of many isgs in these cells [ ] . here we asked whether vlps stimulate isg induction in vivo. wt mice were infected with ma ebov and received vlps h later, and induction of isg mrnas was tested on days . and . isgs encoding anti-viral proteins were first examined, as they may provide early protection against ebov infection. upper panels in fig. a and b compare induction of anti-viral isgs, ifit , mx , oas a and stat with or without vlp injection in liver and spleen. in this early stage, levels of these isgs were consistently higher in the vlp-injected groups than those without vlps. at later stages of infection, however, the situation reversed, in that mice without vlps had higher levels of isgs, as seen on day (s fig. for complete kinetics) . these results indicate that vlp administration accelerated type i ifn and isg induction, which presumably provide early anti-viral activity, not afforded without vlps. we next tested whether vlps induce other isgs, particularly those with negative regulatory activities. this question was of interest to us, since mice that did not receive vlps expressed higher levels of proinflammatory cytokines and chemokines, which raised the possibility that ifn signaling exerts negative regulatory activity towards proinflammatory responses, perhaps by controlling nf-κb activation [ ] . shown in the lower panels in fig. a and b is induction of irgm , usp , trim and trim . irgm is an ifn inducible gtpase that inhibits lps induced endotoxin shock in mice [ ] . usp is an isg deconjugating factor that negatively regulates tlr signaling and resultant cytokine induction [ ] . trim and trim are members of the tripartite motif family that downregulate tlr induced inflammatory responses [ ] [ ] [ ] [ ] . expression of these isgs was also higher in the vlp injected group than that without vlps both in liver and spleen. similar to anti-viral isgs, expression of these negative regulatory factors changed at the later stage (s fig). these data indicate that vlps accelerate induction of anti-viral and negative regulatory isgs, which may help suppress ebov's anti-ifn antagonism (see discussion). to confirm that vlp induction of isg is dependent on type i ifn signaling, we next tested isg induction in ifnar -/mice. as expected, none of the isgs tested in fig. were induced in ifnar -/mice after vlp treatment or ebov infection (s fig). vlps lower expression of proinflammatory cytokines in ebov infected mice ebov pathophysiology such as severe hemorrhagic symptoms and tissue damage is thought to be associated with dysregulated inflammatory cytokine production [ , ] . given that vlps accelerated induction of negative regulatory isgs, we next evaluated whether vlps modulate expression of proinflammatory genes. in fig. , expression of tnfα, il- and il- β, chemokines such as mcp- (ccl ), mip- α (ccl ), mip- β (ccl ), kc (cxcl ) and inflammasome gene nlrp was measured in ebov infected mice with or without vlps. these genes were all strongly induced upon ebov infection and peaked on day with a gradual decline on days in all cases, their expression was significantly attenuated in the vlp-treated group as compared to the group without vlps. the difference was most dramatic in the early stage on day , where the expression was reduced at least by %. in agreement with these results, we noted that serum levels of some of these proinflammatory cytokines were higher in ebov infected mice that were treated with vlps as compared those without vlps [ ] . these results support the view that limiting superfluous inflammatory responses contribute to vlp mediated protection. ifnar -/mice increasing evidence indicates that type i ifns antagonize inflammatory responses in a variety of settings [ ] [ ] [ ] . in light of the results that vlps stimulate those isgs known to suppress proinflammatory responses, it was of importance to determine whether type i ifn signaling by and of itself affects ebov induction of proinflammatory cytokines and chemokines. results in fig. and s fig compare expression of the above proinflammatory factors in ifnar +/+ and ifnar -/mice infected with ebov. all cytokines and chemokines tested were induced after ebov infection in both strains. importantly, their levels were much higher in ifnar -/mice than ifnar +/+ mice. these results indicate that type i ifn signaling downregulates ebov stimulated induction of proinflammatory factors, possibly through isgs with negative regulatory activities. the above results indicated that type i ifns attenuate proinflammatory responses during ebov infection. to explore whether type i ifns have a similar activity in settings other than ebov infection, we next tested lps and ifnβ induced inflammatory responses in macrophages in vitro. lps activates nf-κb mediated proinflammatory cytokine induction, which can result in endotoxin shock [ ] . as shown in fig. , combined treatment with lps and ifnβ led to hyper induction of tnfα, il- , il- β and a chemokine kc in ifnar -/macrophages as compared to wt cells. lps and ifnβ also induced negative feedback factors, trim and trim , with much lower expression in ifnar -/cells than ifnar +/+ cells. these results support a model in which type i ifns negatively regulate proinflammatory cytokine/chemokine responses at least in some situations. we found that mip- α, mip- β and mcp- were not hyperinduced in ifnar -/cells, suggesting that some proinflammatory genes are regulated not only by type i ifns but other factors (data not shown). alternatively, these differences may reflect variances between in vivo and in vitro conditions. to further define pathways downstream of ifnar activity, important for vlp protection, we directed our attention on irf , a transcription factor expressed in macrophages and dcs [ ] [ ] [ ] . irf is induced by ifns and tlr ligands in a stat dependent manner, and plays a pivotal role in facilitating innate immune responses. although irf is not involved in initial triggering of type i ifn induction, it amplifies ifn transcription in dcs and macrophages [ ] . irf promotes induction of multiple anti-microbial factors and is required for innate resistance against a variety of pathogens [ ] [ ] [ ] . irf stimulates expression of mhc and costimulatory molecules to boost antigen presentation [ , ] . we thus tested whether irf disruption affects vlp-mediated protection against ebov. survival data in fig. a show that approximately % of irf -/mice that received vlps died between day and , which is nearly identical to the mortality curve of wt mice without vlps. as expected, the majority of wt mice that received vlps survived against ebov infection. it is of note that ifnar -/mice died to days earlier than irf -/mice, which may be attributed to the difference in the mouse background. correlating with the lack of protection, ebola gp mrna levels were much higher role of type i ifn in vlp-mediated protection against ebov infection in ebov infected irf -/mice than irf +/+ mice with or without vlps (fig. b) . we next examined whether induction of anti-viral and negative feedback isgs is dependent on irf . data in fig. c illustrate that induction of these isgs was very meager in irf -/mice, in contrast to robust induction in irf +/+ mice. importantly, vlps did not rescue isg induction in irf -/mice. results were similar in liver and spleens ( fig. c and s fig). these results indicate that vlps, upon initial activation of type i ifn cascade, rely subsequently on the activation of downstream pathways represented by irf to confer protection against ebov. to gain insight into the pathways through which vlps confer resistance against ebov infection, we investigated the role of type i ifn signaling in vivo and found that it significantly contributes to vlp-mediated protection. this conclusion is supported by the observation that post-exposure vlp treatment accelerated isg induction in ebov infected mice, leading to reduced viral replication and inflammatory gene expression. further supporting the critical role of type i ifn signaling in the protection, vlps did not induce isgs in ifnar -/mice, and did not protect the mice from lethal ebov infection. these results are consistent with the report that post-exposure ifnβ or ifnα treatment increases protection against ebov infection in nhps [ , , , ] . it is likely that vlps initially stimulated type i ifn genes, which in turn led to early induction of isgs. in line with this notion, we recently showed that exogenous vlps stimulate transcription of ifnα and ifnβ in dcs and macrophages in vitro, an event coupled with immediate and robust isg induction [ ] . it may be reasonable to assume that ifnar -/mice were not protected by vlps primarily because isg induction was absent. however, ifnar -/mice may be susceptible to infection due to additional defects in innate immunity that are a secondary consequence of defective ifn signaling, which obliterates vlps protection. contouring this notion however, it is of note that ifnar -/mice can be protected against ebov by an adenovirus-based vaccine, indicating that ifnar -/mice are not totally without defense [ ] . rather, it is possible that ifnar -/mice are not protected by vlps that rely on isg induction for protection, whereas they are protected by the adenovirus vaccine that depends on antibody response. vlp-induced isgs included anti-viral proteins known to inhibit replication of rna viruses such as ifit , mx and oas a, as well as negative feedback factors that curb excess inflammatory responses, such as irgm , usp , trim and trim . although the question of which antiviral isgs are effective in inhibiting ebov replication awaits further research, it is anticipated that some of anti-viral isgs induced by vlps may interfere with ebov life cycle [ ] . what is the significance of accelerated ifn response in vlp mediated protection? available evidence suggests that vlps may overcome ebov's anti-ifn antagonism. the virally encoded vp and vp disable the entire ifn system in the host; while vp blocks the jak/stat pathway of ifn signaling, vp , an ebov virulence factor, inhibits type i ifn induction in many cell types [ , , , ] . we previously showed that vp inhibits type i ifn induction in murine dcs by premature sumoylation and inactivation of irf [ ] . it is thought that vp and vp have a decisive effect on the subsequent host resistance, since abated ifn signaling would impair proper innate immune responses, leading to deficiency in dc maturation, defective antigen presentation and aberrant inflammation. compromised innate immunity would consequently undermine development of adaptive immunity [ ] (see a model in fig. ) . it is remarkable that in the vlp treated mice, isg induction began early within . to days after ebov infection (which was only . to days after vlp treatment), when little to no isg induction was seen in mice without vlps. the delayed isg induction in ebov infected mice is reminiscent of the reports showing that influenza virus delays isg induction in lung epithelial cells through ns , an influenza anti-ifn protein that is linked to disease pathology [ , ] . an influenza virus strain deficient in ns is shown to induce isgs earlier than wild type virus, although the wild type strain does stimulate isgs later on [ , ] . supporting the view that viral anti-ifn factors stall isg induction, rather than completely abrogate the induction, we also observed isg induction on day and later in mice without vlps. it may be envisaged that vlps trigger ifn activation early on, thereby eluding the activity of the ebov anti-ifn proteins (a model in fig. ) . the most striking observation made in this study is the vlp-dependent suppression of proinflammatory responses. this suppression was a result of type i ifn signaling, as ifnar -/mice expressed higher levels of proinflammatory cytokines and chemokines, observed not only after ebov infection but also by ifnβ and lps stimulation. these results are in accordance with the growing recognition that type i ifns are linked to attenuation of inflammatory responses [ , ] . for example, pinto et al., reported, in the west nile virus infection model that ifnar -/mice express excess proinflammatory cytokines, including those found in this study, as compared to wt mice, which correlated with increased disease pathology. in this system, the overt inflammatory responses were attributed to ifn signaling in macrophages and dcs [ ] . induction of proinflammatory cytokines and chemokines may be negatively regulated by ifn signaling through a series of negative feedback factors [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . irgm , induced by ifn signaling restricts lps induced endotoxin shock without limiting ifnβ expression [ ] . trim and trim inhibit proinflammatory cytokine induction, at least in part by interfering with the nf-κb dependent arm of transcription [ ] [ ] [ ] . in addition, these factors may act by post-transcriptional mechanisms, affecting inflammasome activation [ ] . in this regard, guarda et al. [ ] reported that type i ifns inhibit production of il- by inhibiting activity of the nlrp and nlrp inflammasomes and by il- induction. thus, isgs with negative regulatory activity may preferentially attenuate proinflammatory pathways, while sparing ifn induction pathways. given our earlier observations that vp does not grossly affect nf-κb activation, while strongly inhibiting type i ifn activation, ebov may promote proinflammatory pathways at least in part through vp [ ] . lastly, we show that the transcription factor irf is required for vlp mediated post-exposure protection. our results offer an added mechanistic insight into the pathways through which vlps provide protection. irf is expressed predominantly in macrophages and dcs, and helps to amplify type i ifn gene induction and boosts ifns biological activities [ ] . given that macrophages and dcs are the putative early sites of ebov infection, vlps may exert a major impact on these cells to facilitate early innate immunity, in an irf dependent manner. in conclusion, vlps confer post-exposure protection upon ebov infected mice by rapidly inducing isgs, thereby permitting timely establishment of anti-viral and anti-inflammatory states in the host. vlps may act primarily by relieving ebov's antagonism against type i ifns, resulting in reduced systemic inflammation and subsequent enhancement in acquired immune responses (a model in fig. ). pathogenesis of ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells a compendium of years of epidemiological, clinical, and laboratory studies mouse models for filovirus infections a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete mip- alpha and tnf-alpha and inhibit poly-ic-induced ifn-alpha in vitro how ebola and marburg viruses battle the immune 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signalling and immune regulation the content of this publication does not necessarily reflect the views or policies of the us department of defense or the united states army medical institute of infectious diseases. we thank members of pgd for in depth discussions and critical reading of the manuscript. we also thank ms. monica gupta for breeding ifnar -/mice for this study. key: cord- -idmj d p authors: onabajo, olusegun o.; porter-gill, patricia; paquin, ashley; rao, nina; liu, luyang; tang, wei; brand, nathan; prokunina-olsson, ludmila title: expression of interferon lambda is associated with reduced proliferation and increased cell death in human hepatic cells date: - - journal: j interferon cytokine res doi: . /jir. . sha: doc_id: cord_uid: idmj d p interferon lambda (ifn-λ ) is a novel type-iii interferon that can be generated only in individuals carrying a Δg frame-shift allele of an exonic genetic variant (rs -Δg/tt). the rs -Δg allele is strongly associated with decreased clearance of hepatitis c virus (hcv) infection. here, we further explored the biological function of ifn-λ expressed in human hepatic cells—a hepatoma cell line hepg and fresh primary human hepatocytes (phhs). we performed live confocal imaging, cell death and proliferation assays, mrna expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. not only did we observe significant intracellular retention of ifn-λ but also detected secreted ifn-λ in the culture media of expressing cells. secreted ifn-λ induced strong activation of the interferon-stimulated genes (isgs) in ifn-λ -expressing and surrounding cells in transwell assays. specifically, in phhs, secreted ifn-λ induced expression of the cxcl transcript and a corresponding pro-inflammatory chemokine, ip- . in ifn-λ -expressing hepg cells, we also observed decreased proliferation and increased cell death. all ifn-λ -induced phenotypes—activation of isgs, decreased proliferation, and increased cell death—could be inhibited by an anti-ifn-λ -specific antibody. our study offers new insights into biology of ifn-λ and its possible role in hcv clearance. w ith more than million infected individuals, hepatitis c virus (hcv) infection represents a significant healthcare burden worldwide (mohd hanafiah and others ) . hcv infection is treated with interferon (ifn)-a-based regimens and recently approved ifn-a-free direct-acting antiviral agents (daa) (liang and ghany ) . genome-wide association studies identified a single nucleotide polymorphism rs , located upstream of the ifnl (il b) gene and thus initially referred to as the ''il b marker,'' as one of several genetic variants strongly predictive of hcv clearance (ge and others ; thomas and others ) . further studies showed that rs is located in the intronic region of a novel gene, ifnl , and is in high linkage disequilibrium (ld) with an ifnl exonic frameshift polymorphism rs -tt/dg, initially designated as ss (prokunina-olsson and others ). the rs -dg allele, which creates an open reading frame for a novel human interferon, interferon lambda (ifn-l ), is associated with decreased hcv clearance (prokunina-olsson and others ) [reviewed in o'brien and others ( ) ]. the rs -dg has allele frequency of * % in individuals of african ancestry, * % in europeans, while only %- % in asians (prokunina-olsson and others ). in individuals of african ancestry, rs is more predictive of hcv clearance than rs (prokunina-olsson and others ; aka and others ); while in europeans and asians, these markers are in high ld and thus provide comparable predictive information (prokunina-olsson and others ). a genetic polymorphism rs -c/t, which introduces an amino-acid substitution p s in the ifn-l protein (prokunina-olsson and others ), is associated with reduced biological activity of ifn-l and increased hcv clearance (terczynska-dyla and others ), thereby supporting the critical role of ifn-l in this process. recent clinical trials showed that ifnl variants rs and rs are predictive of treatment efficacy even for daa therapies (fujino and others ; meissner and others a; o'brien and pfeiffer ) and these markers, possibly together with p s, could be used to optimize treatment regimens and duration in resource-limited settings. the functional importance of ifn-l is evidenced by the strong positive selection that favored elimination of ifn-l from human populations (key and others ) . although this selection cannot be explained by any known viral infection, it may reflect antiviral response to some extinct highly deadly infection. previously, we showed that transient transfection of an expression construct that generates ifn-l protein induced interferon signaling, with activation of interferon-stimulated genes (isgs) and generation of antiviral response in hepg , a human hepatoma cell line (prokunina-olsson and others ). however, the function of ifn-l and its role in impaired hcv clearance remained unclear. here, we further explored this question by performing additional functional analyses of ifn-l transiently and stably overexpressed in human hepatic cells-fresh primary hepatocytes and hepg cells. the human hepatoma cell line hepg (atcc hb- ) was purchased from the american tissue culture collection (atcc) and maintained in dulbecco's modified eagle's medium (dmem) supplemented with % heat-inactivated fetal bovine serum (fbs). the custom isre-luc-hepg cell line stably expressing a luciferase reporter under control of the interferon-stimulated response element (isre) was previously described (prokunina-olsson and others ); the cells were maintained in dmem supplemented with % fbs and mg/ml puromycin. fresh primary human hepatocytes (phhs) were purchased from bioreclamation ivt. the cells were received in suspension within h after isolation and were maintained in in-vitrogro hi culture media with torpedo antibiotic mix. the liver donor was a -year-old woman who died of cardiac arrest, and the liver donor was a -year-old man who died of anoxia. both were hcv-free caucasians. on arrival, phhs had % and % viability for donor and , respectively. dna extracted from the phhs was genotyped with a custom taqman assay (prokunina-olsson and others ) for the exonic ifnl genetic variant rs (dg/tt). both donors were homozygous for the rs -tt allele, which is associated with higher hcv clearance (prokunina-olsson and others ). the rs -tt allele introduces a frame shift within the first exon of ifnl gene and eliminates the ifn-l protein. thus, no ifn-l protein could be endogenously produced in these phh samples. the expression constructs (ifnl -halo, p -halo, and control-halo) based on the pfc a vector (promega) were previously described (prokunina-olsson and others ). the control-halo construct generates only a halo-tag protein ( kda), the ifnl -halo construct generates the ifn-l -halo protein ( kda) consisting of the ifn-l protein ( kda) c-terminally fused with the halo-tag protein ( kda), and the p -halo generates a protein of kda, consisting of the halo-tag c-terminally fused with a splicing protein isoform of ifn-l ( aa, kda, designated as p ), which lacks exon and is nonfunctional in induction of interferon response (prokunina-olsson and others ). all constructs have been validated by western blotting with an a-halo antibody (promega) and/or an a-ifn-l antibody (mabf ; emd millipore). the ifnl -halo construct generating the secreted ifn-l -halo protein was created and validated the same way. hepg cells and phhs were transfected for or h with corresponding constructs; hepg cells were transfected using lipofectamine/ltx and opti-mem with standard protocols (life technologies), while phhs were transfected using a nucleofection protocol optimized for primary cells (lonza). briefly, for each transfection, · fresh phhs were resuspended in ml of optimized nucleofection buffer l and transfected with mg of corresponding constructs using d-nucleofector (lonza). immediately after transfection, dead cells were separated by centrifugation ( min, rpm) of the transfection mix overlaid with ml of ficoll (ge healthcare) and ml of supernatant with dead cells was removed. the remaining cells were resuspended in invitro-gro cp media with torpedo antibiotic mix (bioreclamation ivt), plated onto collagen-coated plates (bd biosciences), and incubated overnight. unattached and dead cells were removed h post-transfection by replacing the transfection media with invitrogro hi culture media with torpedo antibiotic mix (bioreclamation ivt). tetracycline-inducible hepg cell lines for ifn-l -gfp, p -gfp, and control-gfp were generated using the tet-on g expression system (clontech). first, a stable hepg cell line expressing the tet-on g transactivator was generated by lentiviral transduction and blasticidin selection ( mg/ml). corresponding cdnas were cloned into a ptreg g plasmid (clontech) to generate tetracyclineinducible ifn-l -gfp, p -gfp, and control-gfp constructs, which were transfected into the stable tet-on g-hepg using lipofectamine/ltx. positive clones were identified by limiting dilution under neomycin selection ( mg/ml) over weeks. expression of corresponding proteins was induced by treatment with mg/ml doxycycline for indicated experiment-specific time periods. hepg cells were transfected with corresponding constructs in -well coverslip chambers ( · cells/well; labtek). after h, transfection media was replaced with live cell imaging solution (life technologies), supplemented with mm glucose. cell-permeant halo-tag ligands (tmr red or oregon green; promega) were added to live cells ( : , for min), and live imaging was performed using a lsm confocal laser scanning microscope (carl zeiss) fitted with a temperature and co -controlled chamber. when applicable, nuclei were visualized using fluorescent hoechst dye (life technologies). for cell death analysis, live cells were imaged using inverted oil lens at · magnification, with s scanning per minute per field of view for h, generating , images per field; fields were imaged in sequence using stage-positioning software. each field of view had at least transfected cells. signs of cell death were scored visually by counting membrane rupture events in the videos representing the -h imaging periods. cell death rates were determined as the percentages of ruptured cells of the total counts of fluorescent cells in each field of view. hepg cells were transfected with corresponding constructs and imaged live as described earlier using oregon green halo-tag ligand. the mobility of the ifn-l -halo and control-halo proteins was evaluated with fluorescence recovery after photobleaching (frap) method (snapp and others ) . briefly, after prebleach scans, a selected region was bleached at % of imaging power with iterations and ms per iteration, with . s per scan. imaging was continued for a total of s. fluorescence intensity was plotted with curve fitting to evaluate fluorescence recovery in the bleached areas over time. half-time fluorescence recovery (t½) and the ratio of immobile fraction were averaged for individually scanned cells for each construct (ifnl -halo and control-halo). recovery data were normalized to unbleached regions inside the cells and adjusted for background fluorescence; data acquisition and analyses were performed using lsm frap software. the stable hepg -isre-luc cells were seeded in -well plates that were either untransfected or transfected with corresponding constructs. after h, the cells were treated for h with the following antibodies: mg/ml of a blocking a-il r antibody (r&d systems), mg/ml of a goat isotype igg control (abcam), a range ( , , , . , or . mg/ml) of a custom rabbit monoclonal a-ifn-l antibody (prokunina-olsson and others ), or mg/ml of a rabbit isotype igg control (cell signaling). recombinant human interferons- ng/ml of custom ifn-l (prokunina-olsson and others ), ifn-a and ifn-b (pbl assay science), or ifn-g (r&d systems) were added to the untransfected cells pretreated with the antibodies. negative controls included nontreated cells, phosphate-buffered saline (pbs) in media, and mock transfection with control-halo constructs. the cells were assayed for luciferase expression of the isre-luc reporter h post-transfection. all transfections and treatments were done in biological replicates. for other experiments, inducible stable hepg cells were seeded into -well plates and protein expression was induced for h. antibodies ( mg/ml of rabbit monoclonal a-ifn-l or mg/ml of rabbit igg control) were added to shared culture media for h. hepg and phhs were transfected with corresponding constructs and seeded onto -well plates. separately, transwell inserts with . mm pores (corning) were seeded with untransfected cells and placed into -well plates with culture media, with insert per well. for phhs, both the plates and transwell inserts were collagen coated (corning). after h, media was replaced and transwell inserts with growing cells were added to the plates containing transfected cells. the pore size of transwell inserts allowed free circulation of the media but not of the cells. for phhs from donor , the transwell experiment was expanded to include antibody treatment. phhs transfected with ifnl -halo, ifnl -halo, and control-halo constructs were incubated with transwell inserts seeded with untransfected phhs. antibodies ( mg/ml of rabbit monoclonal a-ifn-l or mg/ml of rabbit igg control) were added to shared culture media for h. after h of coincubation, cells from both chambers that shared culture media (transfected vs. untransfected transwell cells) were collected separately for rna extraction and stored in rlt buffer; shared culture media was collected for protein analysis and stored at - °c until use. for transient transfections, hepg cells were transfected with corresponding constructs for h and media was changed after overnight incubation. expression of halo-tag in live cells was detected using cell-permeant halo-tag tmr red ligand. for stable hepg cells, expression of proteins was induced for h. in both systems, dead cells were identified using near-ir fixable live/dead marker (life technologies). for analysis of apoptosis, cells were additionally stained for annexin v (bd biosciences). for proliferation assays, the cells that were induced for h were treated with mm -bromo- ¢-deoxyuridine (brdu) for h. dead cells were excluded by removing nonadherent cells, and the remaining cells were stained using the apc brdu flow kit (bd biosciences). multiparametric flow cytometry analysis was performed on facs aria iii (bd biosciences) with flowjo. software (tree star). for viability assays, the protein expression was induced for days in -well plates and viability was evaluated with the multi tox-glo assay (promega). in some experiments, viability assays were performed after protein induction for h, with similar results. quantitative reverse-transcriptase-polymerase chain reaction expression analysis total rna was isolated using an rneasy kit with oncolumn dnase i treatment (qiagen). rna quantity and quality were evaluated by nanodrop (thermo scientific). cdna was prepared from to ng of total rna with the rt first-strand cdna kit and random hexamers with an additional dna-removal step (qiagen). quantitative reverse-transcriptase-polymerase chain reaction (qrt-pcr) mrna expression analysis was performed using sybr green antiviral response qrt-pcr plates (qiagen). the plates included expression assays for target genes, as well as positive, negative, and endogenous normalization controls (supplementary tables s -s ; supplementary data are available online at www.liebertpub.com/jir). a custom expression assay for ifnl (prokunina-olsson and others ) and predesigned taqman expression assays for ifnl (hs _g ) and ifng (hs _m ; life technologies) were not included on the predesigned plates and were used separately (supplementary table s ). the qrt-pcr reactions ( or ml) included expression master mixes (qiagen or life technologies), cdna, and corresponding expression assays; the quantification was performed in - technical replicates on the quantsudio instrument (life technologies). expression was measured in c t values (pcr cycle at detection threshold), which are distributed on log scale. expression was normalized by the geometric means of endogenous controls (actb, b m, gapdh, hprt , rplp ) included on the qrt-pcr plates or of endogenous controls used separately (assay e for gapdh, and assay e for actb; life technologies). differences in expression were calculated according to the relative quantification method, as dc t = c t (control) -c t (target). fold differences between expression of any samples or groups of samples were calculated as (dct -dct ) . heat-map analysis of auto-scaled expression data for a panel of selected isgs was performed with the genex software (multid). protein levels of ip- (encoded by cxcl ) in culture media from hepg and phhs transfected with corresponding constructs were measured with an elisa kit (r&d systems), which has a protein detection range of . - pg/ml. culture media was collected h after transfection media change ( h post-transfection). media samples were diluted : or : , and each sample was measured in technical triplicates. a standard curve was generated using the protein provided with the kit, with a correlation coefficient > . . the plates were analyzed using glomax luminometer (promega). ifn-l was detected with a custom-developed electrochemical elisa on the meso quickplex sq instrument [mesoscale discovery (msd)]. briefly, standard capacity multi-array -well plates (msd) were coated overnight at °c with ml of mg/ml custom monoclonal rabbit a-ifn-l antibody. blocking was performed with bovine serum albumin (msd blocker a) for h at room temperature with rotation at rpm, followed by washes with pbs and . % tween. standards, controls, and experimental samples were prepared using diluent (msd). culture media samples were diluted : and incubated at room temperature for h with rotation at rpm. after additional wash steps, ml of the mg/ml detection antibody [mouse monoclonal a-ifn-l antibody (buffer exchanged into pbs, mabf ; emd millipore)], conjugated with a sulfo-tag (msd) was added to the wells and incubated for h at room temperature with rotation at rpm. after additional washing, the plates were scanned in a · read buffer (msd) on the meso quickplex sq and the results were analyzed with the msd discovery workbench software. purified recombinant ifn-l protein (prokunina-olsson and others ) was used as a standard at concentrations in the range of pg/ml to ng/ml, and the detection range for the standards was determined as pg/ ml to ng/ml. hepg cells were transfected for h with ng of corresponding constructs with/without cotransfection with . ml of the endoplasmic reticulum stress response element (erse)-luc cignal reporter (qiagen). transfections were done in a -well plate, with replicates per transfection. media was changed h post-transfection, and the plate was assayed for luciferase and renilla using the glomax luminometer. data were presented as relative luciferase units, which correspond to luciferase/renilla ratio. the stable hepg -isre-luc cells were transfected with corresponding constructs in -well plates; untransfected cells were treated with human recombinant interferons-ifna ( ng/ml; pbl assay science) or custom ifn-l ( ng/ ml). all experiments were represented by biological replicates. the media was replaced h post-transfection by ml of full culture media with or mm jak inhibitor [active against jak , jak , and jak , # ; emd millipore, in . % dimethyl sulfoxide (dmso)]. for il r blocking experiment, the transfection culture media was replaced by ml of media with mg/ml of an a-il r blocking antibody (mab ; r&d systems) or mg/ml of a goat isotype igg control (abcam). for treatment experiments, cells were pretreated for h with the jak inhibitor or for h with the a-il r or igg control antibodies and then treated with ifn-l ( ng/ml) or ifn-a ( ng/ml). negative controls included untreated cells, cells treated with . % dmso in media, and cells transfected with control-halo construct. the cells were assayed for isre-luc reporter h post-transfection, which corresponds to h posttreatment with jak inhibitor and antibodies. sirna silencing of ifnlr in hepg -isre-luc cells the stable hepg -isre-luc cells were transfected with corresponding constructs in identical -well plates. the cells were co-transfected with pmol/well of a scrambled negative control sirna (am ; ambion) or a set of sirnas against ifnlr (m- - ; thermo scientific). after h, the cells were treated with culture media, ifn-l ( ng/ml), or ifn-a ( ng/ml). after h, the first plate was assayed for the isre-luc reporter on the glomax luminometer. the second plate was used for rna extraction (zymo research) and mrna expression analysis. cdna was synthesized using super script iii reverse transcriptase (life technologies). specific taqman assays for ifnlr (hs _m ) and endogenous control actb (assay e) from life technologies were used for qrt-pcr analysis on the quantstudio instrument (life technologies). expression of ifnlr was normalized to expression of actb and then expression in samples treated with ifnlr sirna was compared with untreated samples (no sirna) or treated with scrambled sirna. all samples were represented by biological replicates. compared with sirna-untreated samples ( %), the expression of ifnlr was decreased to % in si-scr samples and to % in si-ifnlr samples ( supplementary fig. s d ). unless specified, data plotting and statistical analyses were performed with prism (graphpad), p values are for -sided unpaired t-tests. shown are means and standard errors of the mean. previously, by performing western blot analysis, we were unable to detect ifn-l in culture media of hepg cells transiently transfected with an ifn-l -producing construct, even though this transfection resulted in strong activation of interferon signaling (prokunina-olsson and others ). however, ifn-l was detectable at low levels in culture media of transfected cells by western blot analysis after acetone precipitation (hamming and others ) . now, we developed a mesoscale assay (an electrochemical elisa) and were able to detect ifn-l in culture media of transfected hepg cells (fig. a) . treatment of these cells with a custom a-ifn-l antibody decreased the interferon signaling by % (fig. b) , suggesting that ifn-l generated in this experimental system is a secreted biologically active interferon. simultaneously, the a-ifn-l antibody did not affect signaling of the main interferons (ifn-a, ifn-b, ifng, and ifn-l , fig. c ). signaling induced by ifn-l was strongly attenuated by the jak inhibitor and by blocking of the ifn-l family receptors (ifnlr and il r , supplementary fig. s a -c), confirming these canonical components of the jak/stat pathway as essential elements for the ifn-l signaling. we evaluated the biological activity of the secreted ifn-l by its ability to induce expression of a set of isgs (supplementary tables s -s ). we analyzed mrna ex-pression in cells transiently transfected with ifnl -halo or control-halo constructs and in bystander nontransfected cells exposed to media from the corresponding transfected cells in transwell assays (ifnl -trans and halo-trans, fig. a ). in both hepg and phhs, cells transfected with ifnl -halo or exposed to media from those cells (containing secreted ifn-l ) showed strong induction of isgs, such as ddx (rig-i), dhx , ifih (mda ), isg , mx , oas , and stat and chemokines cxcl and cxcl ( fig. a) . expression of cxcl and cxcl was much higher in phhs compared with hepg , highlighting cell-specific differences ( fig. a) . ifn-l was detectable in culture media of ifnl -transfected phhs and hepg cells, but not in corresponding halo-transfected cells (fig. b) . we did not detect expression of other interferons (ifna , ifna , ifnb, ifng, ifnl , ifnl ) in any of the experimental conditions (supplementary table s ). cxcl encodes ifn-g inducible protein (ip- ), which is a chemotactic factor for neutrophils. high levels of ip- have been associated with inflammation and pathogenesis of chronic hcv infection (harvey and others ; lagging and others ) . we measured the levels of ip- in culture media of phhs and hepg transfected with different constructs. in phhs, ip- was detectable in the media from samples transfected with ifnl -halo and ifnl -halo (fig. b) , while it was undetectable in hepg cells transfected with ifnl -halo (data not shown), in accordance with a much lower cxcl mrna expression observed in hepg compared with phhs (supplementary tables s -s ) . importantly, the a-ifn-l antibody added to the shared media strongly decreased mrna expression of isgs, including cxcl , in nonexpressing transwell phhs (fig. a ) and the amount of ip- secreted to the shared media (fig. a, b) . even though some of ifn-l gets secreted, as described earlier, ifn-l was also detected as a cytoplasmic protein by confocal imaging in fixed cells-in hepg cells transiently expressing ifn-l -halo, and in phhs induced to express endogenous ifn-l (prokunina-olsson and others ). we next performed live imaging of intracellular movements of ifn-l -halo and control-halo proteins (supplementary video s ). we also monitored frap (fig. a ). this method does not detect secreted proteins such as ifn-l , but it helps characterize intracellular proteins based on their mobility (fig. b) , which is proportional to the speed of fluorescence recovery (t½, in seconds). the control-halo protein showed very high mobility, with a t½ of . - . s (fig. c and supplementary video s ), while ifn-l -halo showed a much lower mobility, with a t½ of . - . s (fig. c and supplementary video s ). intracellular mobility of proteins could be limited by their attachment to structural elements, involvement in proteinprotein interactions, or confinement within vesicles (lippincott-schwartz and others ; snapp and others ). therefore, we also estimated the fraction of immobile expression of mrna transcripts was quantified using an antiviral response qrt-pcr plate. the cells were transfected with corresponding constructs for h, and transwell cells shared culture media with the corresponding transfected cells for h. all samples were represented by biological replicates. expression of each target assay on the plate was measured using the same amount of cdna; expression was normalized to a geometric mean of endogenous controls included on the plate. the results are presented as dc t values for targets normalized by endogenous controls, on a log scale; less negative dc t values correspond to higher levels of expression. full expression data for hepg cells and phh are available as supplementary tables s and s . protein, which is represented by unrecoverable fluorescence-nearly % of ifn-l -halo ( . % - . %) compared with * % of control-halo protein ( . % - . %) was estimated to be immobile (fig. d) . we conclude that in our experimental system the control-halo was mostly present as a mobile unattached cytoplasmic protein, in agreement with other studies that used halo-protein (huybrechts and others ) . in contrast, we detected ifn-l -halo both as an intracellularly retained protein with limited mobility and as a mobile protein potentially available for secretion or release. while conducting live confocal imaging in transiently transfected hepg cells, we observed dying cells that underwent morphologic changes of swelling, membrane blebbing, and rupture, followed by release of cytoplasmic content (fig. a, b and supplementary videos s -s ), suggesting necrotic-type cell death (fiers and others ; festjens and others ; galluzzi and others ) . cell rupture events over the course of h of imaging were significantly more frequent in ifn-l + compared with control-halo + hepg cells (fig. c) . flow cytometry analysis showed that necrotic cells (defined as annexin v + and live/dead + ) were significantly more common in cells transfected with ifnl -halo compared with p -halo (a nonfunctional protein isoform of ifn-l ), ifnl -halo, and control-halo constructs (fig. d , e). transfection efficiency was comparable for all these constructs (fig. g , but could not be determined for the secreted ifnl -halo), and thus could not explain the observed differences in cell death. we also evaluated cell death in phhs transiently transfected with ifnl -halo or control-halo constructs. in general, in phhs, cell death rates were very high even in untransfected cells and similar for both constructs (data not shown), possibly due to physiologically high baseline cell death rates in primary cells unrelated to the effect of ifn-l . cell death can also be triggered as a result of an endoplasmic reticulum response to unfolded recombinant proteins generated in vitro. previously, we showed that transient expression of ifnl -halo and its splicing forms-p , p , and p -induced the erse-luc reporter in hepg cells (prokunina-olsson and others ). we next repeated this experiment for the erse-luc reporter cotransfected with ifnl -halo or p -halo. transfection with p -halo did not induce cell death (fig. d , e), despite considerable induction of the esre-luc reporter (fig. f ). we conclude that potential activation of the unfolded protein response to in vitro generated ifn-l cannot explain the observed ifn-l -induced cell death. in an effort to define the mechanism of ifn-l -induced cell death, we assayed for ripk -dependent necrosis (necroptosis) and caspase- -dependent cell death (pyroptosis), using specific inhibitors (necrostatin and yvad). however, we saw no evidence of the activation of these pathways after transient transfection with the ifnl -halo construct (data not shown). treatment with zvad, a pan-caspase inhibitor, caused a significant decrease in ifn-l -induced cell death, which is suggestive of apoptosis, but this effect was also ) . graphs represent of independent experiments, n = . (c) cell viability was analyzed with multi tox-glo assay and presented as the percentage of induced to uninduced cells, n = . **p < . , ***p < . , based on t-tests. onabajo et al. observed in controls (data not shown), implying that transfection itself might be contributing to apoptosis in this experimental system. to further explore our observations done in transient expression system, we developed inducible stable hepg cell lines expressing ifn-l and p fused with c-terminal green fluorescent protein (gfp) and a control-gfp cell line, all of which were under control of a tetracycline-inducible promoter. stable clones showed %- % intracellular expression ( fig a) . secreted ifn-l -gfp was detectable in media after h of induction (fig. b ) and was biologically active (fig. c ). to assess cell death, protein expression was induced for h and cells were stained for live/dead + and annexin v + markers. the ifn-l -gfp-expressing cells showed a significant increase in cell death compared with cells expressing p -gfp or control-gfp (fig. a, b) . in addition, cell viability analysis showed that days of induced ifn-l expression resulted in a % reduction in the counts of viable cells compared with uninduced cells, with no change observed for the controls (fig. c) . although we observed increased annexin v + staining in the induced ifn-l -expressing cells (fig. a) suggesting apoptotic cell death, we did not detect any caspase- activation (data not shown), which is expected in apoptosis. we reasoned that cell death might be associated with decreased proliferation, and we evaluated cell proliferation after h of induced ifn-l expression by measuring brdu incorporation. compared with p -gfp and gfp control, induction of ifn-l was associated with a significant reduction of proliferation by % (fig. a, b) . we induced ifn-l expression in the presence or absence of the rabbit monoclonal a-ifn-l antibody, which we previously found to be able to block the ability of ifn-l to induce interferon signaling (fig. ) . the a-ifn-l antibody treatment attenuated ifn-l -induced cell death (fig. a) , while it increased proliferation (fig. b ) and improved cell viability (fig. c ). since the antibody is not expected to enter the cells and can only block the secreted protein, these results indicate that the observed cell death and decreased viability and proliferation are caused by the secreted ifn-l . interestingly, this effect was localized to ifn-l -expressing cells and was undetectable in nonexpressing bystander cells in the transwell assays (supplementary fig. s ) . thus, the effect of ifn-l may be concentration dependent and primarily affecting the ifn-l -expressing cells. fig. . a-ifn-l antibody inhibits ifn-l -induced cell death and proliferation defect. protein expression of ifn-l -gfp, p -gfp, or control-gfp in stable hepg cells was induced with mg/ml doxycycline for h. at h, cells were treated with mg/ ml of rabbit a-ifn-l antibody or mg/ml of rabbit igg control (rigg). cells in different treatment conditions were assessed for cell death (a), cell proliferation (b), and cell viability (c). graphs represent of independent experiments, n = , *p < . , **p < . , ***p < . based on t-tests. we suggest that ifn-l may have at least functions in human hepatic cells-activation of interferon signaling, inhibition of cell proliferation, and induction of cell death. these roles may be overlapping, synergistic, or independent, and some or all of them may be relevant for hcv clearance. activation of isgs by ifn-l was expected based on our previous observations in transiently transfected hepg cells (prokunina-olsson and others ). early activation of a panel of isgs has also been reported in both in vitro hcvinfected and adjacent uninfected phhs (sheahan and others ) . however, we now determined the following about ifn-l -induced isg activation: it is specifically caused by the ifn-l secreted from cells at low but detectable amounts; it has an effect on both primary hepatocytes and hepatoma cells; it similarly affects the expressing and nonexpressing bystander cells; and it can be specifically blocked by the a-ifn-l antibody. anti-proliferative and cell-death-inducing anti-tumor effects are, in general, characteristic of interferons (wang and others ) , including in hepatoma cell lines (murata and others ) . however, we now present the first evidence of these effects caused by ifn-l , the newest addition to the interferon family. while activation of isgs was observed in both the ifn-l -expressing and nonexpressing bystander cells, the decrease of proliferation and induction of cell death were detectable only in the expressing cells. this could mean that these effects require different concentration thresholds. the dose-dependent anti-proliferative effect in hepatic cells has already been shown for ifn-a (lim and others ) , and the same might be true for ifn-l . anti-proliferative activity of ifn-l is isg dependent as it was blocked by the same a-ifn-l antibody that blocks ifn-l induced activation of isgs. some of the isgs induced by ifn-l , including ¢ ¢oas and ip- , are known for their contribution to the anti-proliferative and pro-apoptotic effects of interferons (rysiecki and others ; hassel and others ; aksoy and others ; liu and others ) . at this point, we are unable to determine the mechanism of ifn-l -induced cell death. although we did not detect activation of caspase- by ifn-l , therapeutic anti-tumor mechanisms of interferons have been related to cell growth arrest and induction of caspase- -independent apoptosis via the p pathway (vogelstein and others ; takaoka and others ; , and this pathway should be tested for ifn-l as well. the effect of ifn-l on activation of interferon signaling and cell death might be complex and intertwined, and further studies are warranted. the genetic association between the ability to generate ifn-l (in carriers of the rs -dg and rs -t alleles) and impaired spontaneous and treatment-induced clearance of hcv is well established, but the molecular understanding of this association is still limited. recently, it has also been reported that the same individuals who are more likely to clear the hcv infection, in fact, are significantly more likely to develop liver fibro-proliferative disease (fibrosis), which is associated with increased mortality (eslam and others ) . our new findings regarding ifn-l function may help reconcile these counterintuitive connections. we suggest that the induction of interferon signaling by ifn-l may have both positive and negative implications. the ifn-l -induced isgs might provide some antiviral protection, explaining the lower pretreatment hcv loads in individuals with the rs -dg allele, compared with those who do not carry this allele (uccellini and others ) . on the other hand, the increased pretreatment expression levels of these isgs have been associated with refractoriness to ifn-a therapy (dill and others ), especially in carriers of the ifnl -rs -dg or rs -t alleles (urban and others ; prokunina-olsson and others ) . this could be because ifn-a and ifn-l compete for induction of the same set of isgs, and, once activated by ifn-l , these isgs do not respond to ifn-a. the fact that despite having lower pretreatment hcv levels, individuals with rs -dg allele are more likely to develop chronic hcv and fail ifn-a-based treatment, suggests that either negative effects of ifn-l outweigh its positive effect on lowering hcv viral load or antiviral effects induced by ifn-l are insufficient for complete viral clearance. the ifn-l effect on proliferation and cell death might also be positive and negative. increased cell death has been correlated with inflammation during hcv infection and was suggested as a factor contributing to liver damage (bantel and schulze-osthoff ) . high rates of cell death in ifn-l -expressing cells could amount to substantial cell loss and inflammation, contributing to the development of liver disease. simultaneously, the anti-proliferative effect of ifn-l might limit the tissue remodeling ability that leads to liver fibro-proliferative disease (fibrosis), as has been reported by the largest study on this subject to date (eslam and others ) . individuals who are genetically unable to produce ifn-l or in whom ifn-l is not sufficiently induced by specific stimuli may be more likely to clear the infection, spontaneously or after treatment, especially with ifn-abased therapies. however, they may not have the benefit of an anti-proliferative effect also contributed by ifn-l , and will be more likely to develop fibrosis. since in our system the a-ifn-l antibody efficiently blocked activation of isgs (including ip- ), and ifn-l induced cell death, blocking of ifn-l with therapeutic agents in carriers of the rs -dg allele might be a plausible therapeutic option before or in conjunction with other treatments. blocking of ifn-l might boost mechanisms of endogenous host immunity, which are important for efficient clearance of hcv and prevention of posttreatment relapse (meissner and others b) , and also decrease liver damage due to inflammation and cell death. although the antiviral role of ifn-l has been shown for a number of viruses (hamming and others ; lu and others ) , it remains to be found what other biologically relevant factors can trigger endogenous ifn-l expression. identification of these triggers in hepatic and other human cell types could help explore the role of ifn-l in other relevant clinical conditions. so far, expression of endogenous ifnl has only been detected in liver biopsies from patients infected with hcv, with significant correlations between hcv titers and expression levels of ifnl and isgs (amanzada and others ; konishi and others ; meissner and others b; murakawa and others ). however, ifnl was undetectable in liver biopsies from patients infected with hepatitis b virus or affected by onabajo et al. unrelated liver diseases (amanzada and others ) . in vitro, ifnl expression could be induced in phhs by hcv infection or treatment with polyi:c, but polyi:c treatments failed to induce ifnl expression in hepg cells. the ifnl region is absent in the genomes of mouse and rat, precluding the development of relevant rodent models. we acknowledge the limitations of our experimental system, which is based on expression of ifn-l in human hepatic cells-a hepatoma cell line hepg and phhs. the observed effects might differ from the physiologic conditions in the hcv-infected hepatic cells in the complex whole-organ environment by the magnitude, duration, and efficiency of ifn-l induction. we also did not address the mechanism of intracellular retention and the function of the nonsecreted ifn-l . however, we provide a comprehensive functional analysis of ifn-l , a novel human interferon, and suggest its role in activation of interferon signaling, inhibition of cell proliferation, and induction of cell death in human hepatic cells. association of the ifnl -deltag allele with impaired spontaneous clearance of hepatitis c virus cxcr surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation interferon-lambda (ifnl ) transcript expression in human liver tissue samples apoptosis in hepatitis c virus infection interferon-induced gene expression is a stronger predictor of treatment response than il b genotype in patients with hepatitis c interferon-lambda rs genotype and liver fibrosis in viral and non-viral chronic liver disease necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response more than one way to die: apoptosis, necrosis and reactive oxygen damage predictive value of the ifnl polymorphism on outcome of telaprevir, peginterferon, and ribavirin therapy for older patients with genotype b chronic hepatitis c molecular definitions of cell death subroutines: recommendations of the 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infection antiproliferative effects of interferon alpha on hepatic progenitor cells in vitro and in vivo studying protein dynamics in living cells the emerging role of cxcl in cancer (review) interferon-lambda is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden ifnl -deltag genotype is associated with slower viral clearance in hepatitis c, genotype- patients treated with sofosbuvir and ribavirin endogenous intrahepatic ifns and association with ifn-free hcv treatment outcome global epidemiology of hepatitis c virus infection: new estimates of age-specific antibody to hcv seroprevalence impaired induction of interleukin b and expression of interferon lambda associated with nonresponse to interferon-based therapy in chronic hepatitis c a comparison of the antitumor effects of interferon-alpha and beta on human hepatocellular carcinoma cell lines reply: subgroup differences in response to weeks of ledipasvir/sofosbuvir for chronic hepatitis c ifn-lambda : the paradoxical new member of the interferon lambda family a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus constitutive expression of a ¢, ¢-oligoadenylate synthetase cdna results in increased antiviral activity and growth suppression interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness measuring protein mobility by photobleaching gfp chimeras in living cells integration of interferon-alpha/beta signalling to p responses in tumour suppression and antiviral defence new aspects of ifn-alpha/beta signalling in immunity, oncogenesis and bone metabolism reduced ifnlambda activity is associated with improved hcv clearance and reduced expression of interferon-stimulated genes genetic variation in il b and spontaneous clearance of hepatitis c virus hcv rna levels in a multiethnic cohort of injection drug users: human genetic, viral and demographic associations il b genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis c surfing the p network interferon: current status and future prospects in cancer therapy the work has been supported by the intramural research program ( key: cord- -iy qzq authors: muñoz-gonzález, sara; pérez-simó, marta; colom-cadena, andreu; cabezón, oscar; bohórquez, josé alejandro; rosell, rosa; pérez, lester josué; marco, ignasi; lavín, santiago; domingo, mariano; ganges, llilianne title: classical swine fever virus vs. classical swine fever virus: the superinfection exclusion phenomenon in experimentally infected wild boar date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: iy qzq two groups with three wild boars each were used: group a (animals to ) served as the control, and group b (animals to ) was postnatally persistently infected with the cat strain of csfv (primary virus). the animals, six weeks old and clinically healthy, were inoculated with the virulent strain margarita (secondary virus). for exclusive detection of the margarita strain, a specific qrt-pcr assay was designed, which proved not to have cross-reactivity with the cat strain. the wild boars persistently infected with csfv were protected from superinfection by the virulent csfv margarita strain, as evidenced by the absence of clinical signs and the absence of margarita rna detection in serum, swabs and tissue samples. additionally, in pbmcs, a well-known target for csfv viral replication, only the primary infecting virus rna (cat strain) could be detected, even after the isolation in st cells, demonstrating sie at the tissue level in vivo. furthermore, the data analysis of the margarita qrt-pcr, by means of calculated Δct values, supported that pbmcs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the margarita virus strain, while this virus was able to infect naive pbmcs efficiently. in parallel, ifn-α values were undetectable in the sera from animals in group b after inoculation with the csfv margarita strain. furthermore, these animals were unable to elicit adaptive humoral (no e -specific or neutralising antibodies) or cellular immune responses (in terms of ifn-γ-producing cells) after inoculation with the second virus. finally, a sequence analysis could not detect csfv margarita rna in the samples tested from group b. our results suggested that the sie phenomenon might be involved in the evolution and phylogeny of the virus, as well as in csfv control by vaccination. to the best of our knowledge, this study was one of the first showing efficient suppression of superinfection in animals, especially in the absence of ifn-α, which might be associated with the lack of innate immune mechanisms. members of the pestivirus genus, within the flaviviridae family, account for a variety of diseases in farm animals, the most economically important of which are bovine viral diarrhoea virus (bvdv) and classical swine fever virus (csfv). classical swine fever virus (csfv) is the etiological agent of a highly contagious viral disease of swine affecting domestic pigs and wild boars [ ] , which has caused major losses in stock farming [ , ] . csfv is composed of a lipid envelope, a capsid and a single plus-strand rna genome carrying a single, large open reading frame (orf) flanked by two untranslated regions (utrs). the orf encodes a polyprotein of approximately amino acids, which are processed by cellular and viral proteases in the four structural proteins-c, e rns , e , e -and in the non-structural proteins-n pro , p , ns , ns , ns a, ns b, ns a, and ns b [ ] . recently, it was proved that csfv can generate postnatal persistence by infecting both newborn piglets and wild boars with either low-and/or moderate-virulence strains, respectively. over the six weeks after postnatal infection, most of the infected animals remained clinically healthy, despite persistent high virus titres in the blood, organs and body secretions. importantly, these animals were unable to mount any detectable humoral or cellular immune responses. at necropsy, the most prominent gross pathological lesion was severe thymus atrophy. four weeks after infection, pbmcs from persistently infected seronegative piglets were unresponsive to both specific csfv and non-specific pha stimulation in terms of ifn-γ-producing cells. these results suggested the development of an immunosuppression state in these postnatally persistently infected pigs [ , ] . in addition, it was shown that six-week-old, persistently csfv-infected pigs were unable to elicit specific immune responses following vaccination with a csfv lapinised c-strain vaccine (hclv) [ ] . interestingly, the rna of the vaccinal c-strain was undetectable by specific rt-pcr [ ] in any of the samples analysed after vaccination, including blood, nasal and rectal swabs, or organs throughout the experiment, suggesting a phenomenon of homologous interference, also known as superinfection exclusion (sie), between the high viral load generated by the primary persistent infection and the csfv vaccine strain. the sie phenomenon, defined as the ability of a primary virus infection to interfere with a secondary infection by the same or a closely related virus, has been described in a broad range of virus-host systems, including bacteria, plants, and animals, and in important pathogens of humans, such as rubella virus, human immunodeficiency virus (hiv), and hepatitis c virus (hcv), among others [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . from an evolutionary standpoint, sie might be a conservative strategy, reducing the likelihood of recombination events between related strains [ , , ] , thus determining the stability of viral sequences within the same cell. from a practical standpoint, sie has significant implications for the treatment or prevention of viral infections. in this regard, cross-protection of crops by purposeful infection with milder virus isolates is a widely accepted practice, and it is viewed as an effective and economical antiviral management strategy [ ] . additionally, transplantation of hcv-infected liver grafts has been suggested as a treatment for already infected patients, given that the transplantation of a healthy organ would lead to rapid damage to the newly transplanted liver by the virus of the recipient patient [ , ] . previous studies conducted in cell cultures with bvdv demonstrated that cells acutely infected with this virus were protected from a second infection by a homologous bvdv strain [ ] . additionally, it was shown that csfv is generally noncytopathic, and it readily establishes persistent infections in cell culture. nevertheless, when persistently infected cultures were serially passaged more than times, spontaneous generation of cytopathogenic (cp) csfv variants could occur. the few surviving cells of the cytopathic effect (cpe), although still infected, were also protected from the cpe after superinfection with cp csfv [ ] . both studies supported the ability of pestiviruses to generate sie in cell cultures. thus, along with the availability of a persistent infection model of csfv, in the present study, we sought to assess sie against a highly virulent csfv strain at the organism level in six-week-old wild boars, rendered persistently csfv-infected at birth. our results showed that sie could occur at the systemic level in csfv-infected swine. pk- cells (atcc ccl ) and sk cells [ ] were cultured in dulbecco's modified eagle medium (dmem), supplemented with % foetal bovine serum (fbs), pestivirus-free, at °c in % co . the cells were infected with . tcid /cell in % fbs, and the virus was harvested h later. additionally, st cells (atcc crl ) were cultured in dmem, supplemented with l-glutamine ( %) and % foetal bovine serum (fbs), pestivirus-free at °c in % co . peroxidase-linked assay (pla) [ ] was used for viral titration following the statistical methods described by reed and muench [ ] . the catalonia (cat ) strain used in this study was isolated from the spanish csf epizootic in - [ ] . this isolate belongs to the csfv . genogroup [ ] . the course of infection by this strain was found to be mild [ , ] . finally, the virulent margarita strain, which belongs to the csfv . genogroup [ , , ] , was used. to elucidate the capacity of csfv to generate sie, two groups (a and b), with three male, sixweek-old wild boars in each, were used. these animals were acquired from gestion cinegetica integral sl farm (segovia, spain) and were housed in the experimental isolation facilities in the biosecurity level laboratory of the centre de recerca en sanitat animal (cresa); they were fed a conventional piglet starter diet and pellets until the end of the trial (startrite , kwikstart, and prestarter; sca iberica s.a., zaragoza, spain) and were handled according to previous studies conducted in cresa [ ] . group a (animals to ) was used as controls, and they tested pestivirus-free at the beginning of the study. the second group (group b), housed in an independent isolation unit at the bsl- facility of cresa, (animals to ), were postnatally persistently csfv-infected animals. these animals, which had been intranasally infected in the first h after birth with the csfv cat strain, were viraemic and apparently healthy at six weeks old, although being immunosuppressed, they lacked csfv-specific cellular and humoral responses [ , ] . both groups had an average weight of kg per animal. after a five-day acclimation period, all of the animals were experimentally infected by i.m. injection in the neck [ ] [ ] [ ] with tcid csfv margarita strain. in previous studies, this viral dose caused acute csf and often induced death at - days post-infection (dpi) [ ] . sera and nasal and rectal swabs were collected at , , , and dpi. blood samples for the isolation of pbmcs were obtained at day and at the time of euthanasia. a trained veterinarian recorded the clinical signs daily in a blinded manner [ ] . the clinical signs compatible with csfv infection were anorexia, fever, conjunctivitis, diarrhoea, constipation, cyanosis of the skin, abdominal petechiae, dyspnoea, tremors, locomotive disturbances, reluctant walking, swaying movement of the hindquarters, posterior paresis, convulsions from mild to severe and prostration. particular stress was placed upon the registration of nervous symptoms [ , , , ] . the clinical status of the animals was scored from to [ , , , ] as follows: : no signs; : mild pyrexia; : pyrexia plus mild clinical signs; : mild-to-moderate clinical signs; : moderate clinical signs; : moderate-to-severe clinical signs; and : death. for ethical reasons, the animals were euthanised when the clinical score reached , when exhibiting a fall of the hindquarters, when there was inability to drink or feed, when prostration occurred or when exhibiting moderate nervous disorders. after euthanasia, an exhaustive necropsy was conducted, in which the presence of pathological symptoms in different organs and tissues was evaluated. surviving wild boars were euthanised at dpi, and urine and tissues (spleen, liver, intestine, mesenteric lymph node, prescapular lymph node, bone marrow, medulla oblongata, lung, kidney, thymus and tonsil) were obtained at necropsy. euthanasia was performed according to european directive / /eu, using a pentobarbital overdose of - mg/kg administered via the anterior vena cava. the animal care and procedures were in accordance with the guidelines of the good experimental practices (gep), under the supervision of the ethical and animal welfare committee of the autonomous university of barcelona (uab), and they were approved under number , according to the existing national and european regulations. additionally, the biosafety level of the viruses used in this study was stated as biosecurity level , as approved by the biosafety committee of the uab, with registration assignment ar- - . fifteen representative sequences of the three csfv genogroups were retrieved from genbank and aligned using bioedit [ ] . two primers and probes were designed for specific detection of the margarita strain sequence ( . csfv genogroup) by targeting the ´end of the e gene, as follows: forward primer ( - ), ´-aagattacgaccacaatttacaac- ´; reverse primer ( - ), ´-tcc tactgaccacattaagcg- ´and probe ( - ), ´-ccatcaaggctatctgcacgg- ´. the nucleotide positions were based on the genome sequence of the margarita strain (genbank accession number aj ). the probe was labelled with -fam at the ´end and with bhq at the ´end. the primers and probe were purified by reverse phase hplc. the one-step rt-pcr protocol was undertaken using the commercially available taqman one-step rt-pcr master mix reagents kit (applied biosystems roche). the real-time rt-pcr assay was optimised using a total volume of μl. real-time qrt-pcr was performed using an applied biosystems fast real-time pcr system. the temperature profile was min at °c (reverse transcription), min at °c (inactivation reverse transcriptase/activation taq polymerase), followed by cycles of s at °c (denaturation), s at °c (annealing) and s at °c (elongation). identical temperature profiles were used for all of the real-time rt-pcr runs, and fluorescence values were collected during the annealing step. twenty csfv rna preparations strains were used to determine the specificity and sensitivity of the assay (table ) [ , ] . to exclude the possibility of presence of csfv cat strain rna interfering with the assay sensitivity for the csfv margarita strain rna detection, mixtures from serial rna dilutions from both viral strains were analysed. in addition, mixtures from rna serum samples of group b (prior to the margarita strain inoculation), with samples from group a at days post-infection with the margarita strain, were analysed. initial sample volume of μl to obtain a final volume of μl of rna, which was stored at - °c. the presence of csfv rna in the serum and in nasal and rectal swabs, as well as in tissue samples, was analysed by a generic csfv qrt-pcr [ ] . this test was used in our laboratory for inter-laboratory comparisons of csfv diagnoses, organised by the eu reference laboratory. positive results were considered for threshold cycle values (ct) equal to or less than . samples in which fluorescence was undetectable were considered negative. additionally, the qrt-pcr specific for the margarita strain, designed in this work (described above), was used to distinguish those samples infected with the margarita strain. serum samples were tested with neutralisation peroxidase-linked assay (npla) [ ] , and the titres were expressed as the reciprocal dilution of serum that neutralised tcid of the cat or margarita strain in % of the culture replicates. the detection of e -specific antibodies was performed using a commercial elisa kit (idexx); the samples were considered positive when the blocking percentage was %, following the manufacturer's recommendations. anti-ifn-α monoclonal antibodies (k and k ) and ifn-α recombinant protein (pbl biomedical laboratories, piscataway, new jersey, usa) were used in elisa to detect ifn-α in serum samples at , , and dpi [ , [ ] [ ] [ ] . the cut-off value of the assay was calculated as the average of the optical density of negative controls (blank and negative sera before csfv infection) plus three standard deviations. cytokine concentrations in serum were determined using a regression line built with the optical densities of the cytokine standards used in the tests. elispot assay to detect csfv-specific ifn-γ cells was performed as previously described [ ] , using pbmcs that were obtained at day and at the time of euthanasia. briefly, plates (costar , corning) were coated overnight with μg/ml capture antibody (p g , pharmigen). detection was performed using a biotinylated antibody (p c , pharmigen). a total of x pbmcs/well were plated in triplicate at . multiplicity of infection (moi) of the cat and margarita csfv strains. moreover, the same samples were incubated in the presence of phytohaemagglutinin (pha) ( μg/ml). the controls were incubated in the presence of mock-stimulated wells. the numbers of spots in the media for mock-stimulated wells were considered to constitute the baseline for the calculation of antigen-specific frequencies of ifn-γ-producing cells. samples from animal (group a: margarita acutely infected wild boar; dpi), animal (group b: cat persistently infected wild boar and superinfected with csfv margarita strain; dpi), and a pestivirus-free wild boar (animal before infection), were used to assess sie in pbmcs (fig ) . the pbmcs were isolated from whole blood by centrifugation on ficoll gradients (histopaque- ; sigma). the number and viability of the pbmcs were determined by staining with trypan blue [ ] . a total of x pbmcs/well from each animal were plated in quintuplicate at °c in -well plates with: (i) vehicle; (ii) the cat strain at a . was analysed by generic csfv qrt-pcr [ ] and for the specific margarita strain by qrt-pcr detection assay (see above, section . ). for virus isolation, an established cell line sensitive for specific csfv proliferation, st cells, were cultured at °c in -well plates in triplicate in the presence of each of the collected cell suspensions. after h, the supernatants were removed, and the collected st cells were washed twice and resuspended in μl of sterile pbs. after two cycles of freeze-thaw at - °c, the presence of csfv rna in the st cell samples was analysed by qrt-pcr for csfv [ ] and the margarita strain (see above). in parallel, a st plate similarly inoculated with cell suspensions was used for confirmation by pla [ ] . a delta ct (Δct) for margarita strain rna detection was calculated as the differences between (i) the margarita ct value detected from the isolation of st from groups a or b and (ii) the ct value in st inoculated with margarita-infected naïve pbmc extract, being Δct = ct (a) -ct (b) . the whole protocol was repeated twice, in st and also in sk cells using pbmcs from animals (group a), and (group b) and cells from the naïve animal (number , group a), collected before margarita infection. the e -gene fragment reported by lowings et al. [ ] was amplified by end point rt-pcr [ ] in sera, tonsil, lung and spleen from animals , (group a), and (group b), collected at necropsy. additionally, the viral inoculums used in the experimental infections (cat and margarita strains) were evaluated. the amplification products were checked by electrophoresis on % agarose gel and were directly cleaned with a wizard pcr preps dna purification system (promega, madison, wisconsin, usa). sequencing reactions were conducted under bigdye tm terminator-cycling conditions using an abi xl. forward and reverse sequences obtained from each amplicon were assembled using the contig express application in vector nti software, version (invitrogen). the sequences from the e -gene fragment obtained were aligned to analyse the sequence found in each sample. of the csfv rna strains analysed, the assay detected only the csfv rna from the margarita strain ( . genogroup), while the other csfv rna extractions were negative (table ). this result indicated that the newly developed assay was highly specific for the detection of the csfv margarita strain, and there was no cross-reactivity with the other tested csfv strains from genogroup (including the cat strain). the specificity of the assay was based primarily on mismatches in the probe-binding region but also to some extent on mismatches in primer-binding regions. the sensitivity of the assay was evaluated by testing -fold dilutions of the margarita strain rna. the analytical sensitivity was estimated to be as high as . tcid . the assay had a reaction coefficient (r ) of . (data not shown). positive results were considered for threshold cycle values (ct) equal to or less than . finally, the presence of cat rna strain in the sample containing the margarita strain rna did not affect the assay sensitivity (data not shown). animals persistently infected with the cat strain and inoculated with the virulent margarita strain (group b) showed neither clinical signs of disease nor fever at any time throughout the study, maintaining good health status (fig ) . in contrast, animals from group a, infected with the margarita strain, presented mild clinical signs at dpi that progressed to moderate within - h. at dpi, animal showed a clinical score value of ; however, it was found dead at dpi, with lesions of haemorrhagic diathesis. animals and progressed to dyspnoea, weight loss, swaying movement of the hindquarters, posterior paresis and high fever until dpi, when euthanasia was performed. margarita rna was undetected in the sera from animals in group b, except for animal at dpi with a high ct value (ct . ), considered a low rna viral load ( , ) (fig ) . additionally, csfv margarita strain rna could not be detected in any of the nasal or rectal swabs collected from group b (data not shown). furthermore, in group b, csfv margarita rna was found only in the liver of animal and also in the spleen of animals and , with a low rna viral load. in contrast, all wild boars from group b (csfv persistently infected with cat strain) maintained during the whole trial a high and constant csfv rna load in serum, swabs and organs, when examined by generic csfv q-rt-pcr (table ). in contrast, both qrt-pcrs (generic and specific for margarita strain) were positive in organs and samples collected from animals in group a ( table ). the ct values were positive by the csfv generic qrt-pcr [ ] , in both serum and swab samples, from dpi onwards. ct values for the specific margarita assay were similar to those obtained by the csfv generic qrt-pcr. to evaluate the induction of csfv-specific antibodies, serum samples were analysed at different times after csfv margarita strain infection. the absence of antibody response, in terms of e -specific antibodies and neutralising antibody titres, was found in both csfv acutely and persistently superinfected groups during the entire experiment (data not shown). previously, it was shown that csfv pi animals were unable to elicit an innate immune response, in terms of ifn-α production, against a csfv life-attenuated vaccine [ ] . however, we wondered whether superinfection with a csfv virulent strain would trigger detectable levels of ifn-α in the csfv-superinfected wild boars (group b), given that ifn-α has been largely related to disease severity, as a hallmark of csfv acute infection [ , ] . in the present work, we observed that progression of disease in group a was correlated with an increase in the levels of endogenous ifn-α after infection, as measured by elisa, with values that reached more than u/ml in two of three animals at dpi and dpi (data not shown). in contrast, ifn-α was undetectable in all of the serum samples analysed both before (day ) and after margarita inoculation of csfv catalonia persistently infected pigs (group b) (data not shown). pbmcs from all of the animals were analysed for virus-specific and non-specific ifn-γ responses by elispot assay at and dpi post-margarita strain inoculation. very few ifnγ-producing cells were found upon csfv and pha stimulation of pbmcs from all of the csfv-superinfected animals (group b). these results supported our previous results showing that postnatal infection of piglets with csfv could result in virus persistence due to a lack of b-and t-cell responses (data not shown). it is well known that white blood cells, including the pbmcs, are targets for csfv replication [ , ] . consequently, to examine whether the pbmcs collected from the csfv-superinfected animals (group b) and the acutely infected animals (group a), were permissive (or not) to csfv superinfection, we assayed in vitro inoculation of such samples, with either cat or margarita csfv strains. similarly, pbmc samples were mock-infected. additionally, pbmcs from a naïve animal were used as controls. as was expected, csfv-specific margarita rna was detected in the pbmcs from animals developing the csf acute disease (group a) in both mock and margarita-infected samples. furthermore, pbmcs from group b in vitro inoculated with margarita were also positive for csfv-specific margarita rna detection, but with a high ct value correlated with a lower rna load (table ) . otherwise, pbmcs from group b in vitro mock-infected were negative for csfv-specific margarita rna detection (table ) . following these findings, to decipher whether the detected rna load in group b might correspond to rna traces from the inocula or to the infecting virus, the previously analysed pbmc extracts were inoculated into a st cell line. consistently, the detected rna load notably increased in st after inoculation with the extract from margarita in vitro inoculated-naïve pbmcs; the obtained . Δ ct positive value confirmed the infectivity of the virus recovered from the pbmc samples. in contrast, margarita rna in group b in vitro mock-infected pbmcs remained undetectable even after st inoculation. furthermore, margarita rna load detection in group b in vitro margarita-infected pbmc samples decreased after inoculation of st cells, corresponding to higher ct values than those previously detected directly from pbmc extracts. remarkably, an . Δct value was found in the st cells with margarita in vitro inoculated pbmcs from group b, relative to the value obtained in the st cell extracts from margaritainoculated naïve pbmcs (table ). the whole protocol was repeated twice for animals (group a), and (group b) in both sk and st cells, supporting the results with similar ct values (data not shown). similarly, the cells' positive infection was confirmed by pla testing, although this test cannot differentiate between cat and margarita csfv strains. to detect the presence of csfv rna of both viral strains (cat and/or margarita) in the sera, tonsil, and spleen of animals and (group a) and and (group b), the e -gene fragment reported by lowings et al. [ ] as a phylogenetic marker was amplified by end point rt-pcr [ ] . in all of the samples analysed from animals that developed the csf acute form (group a), the sequence corresponding to the margarita strain (aj ) used as the inoculum was detected. furthermore, the samples analysed from superinfected animals (group b: csfv catalonia persistently infected inoculated with csfv margarita strain) only showed the sequence corresponding to the cat strain [ ] (fig ) . despite its significance, the mechanisms of mutual exclusion by viral variants are far from being completely understood, and the actual knowledge is basically derived from studies at the cellular level in established cell lines [ , , , ] . very few reports have demonstrated the phenomenon of sie at the organism level, and, to our knowledge, these models have been limited to plant viruses, west nile virus (wnv) in mosquitoes, and peking duck hepatitis b virus (dhbv) [ ] [ ] [ ] . in addition, it has not yet been demonstrated in a mammalian host at the systemic level. previous works have reported the capability of csfv to generate postnatally persistent infection in both domestic pigs and wild boars [ , ] . subsequently, it was also shown that postnatally persistently infected pigs were unable to elicit a specific immune response to a csfv live attenuated vaccine and that the viral vaccine rna was undetectable in any of the samples analysed [ ] . against this background, we assessed the capacity of csfv to generate sie in csfv persistently infected swine. for that purpose, csfv persistently infected wild boars were inoculated with a csfv strain that induce acute disease with a higher replication rate [ , ] . because pestiviruses are immunologically and genetically closely related, accurate serological characterisation of csfv isolates is impeded by the extensive cross-reactions observed among pestivirus members and the limited availability of mabs capable of differentiating among different csfv isolates [ , , ] . to differentiate the csfv margarita strain rna from the csfv cat strain rna in the samples from the present study, a specific qrt-pcr for margarita strain rna detection was developed. thus, alongside the model of infection with the margarita strain, the qrt-pcr assay developed allowed for clear discernment of whether there was actually a blockage that prevented susceptibility to infection by the second virus in both the absence of clinical signs and the absence of molecular detection of the superinfecting virus. notwithstanding the high infection rate of the cat strain in persistently infected animals from group b (primary virus infection), good health status was maintained after inoculation with the margarita csfv virulent strain (secondary infection) in the absence of viral detection in sera throughout the study, except in one animal at dpi with a low margarita strain rna load (animal ). despite the important role that neutralising antibodies play in csfv protection [ , ] , complete absence of neutralising antibodies response was found after margarita strain infection in these animals. similarly, absence of an ifn-γ-producing cell response against csfv or pha was also observed. considering the role played by ifn-γ in the control of csfv infection [ , ] and the lack of responsiveness to ifn-γ-producing cells after pha stimulation, the csfv-superinfected animals maintained a immunosuppression state similar to that previously described in postnatal persistent infection [ , ] . previous work has proved how the failure to induce optimal levels of the humoral and cellular responses after csfv infection promoted the spread of the virus and its relationship with disease progression [ , ] . in this regard, the implications of the cellular and neutralising antibody response in clinical protection against the acute form in the csfv-superinfected animals from this study are excluded. furthermore, no superinfecting virus excretion was detected in any of the animals from group b, whilst the high viral load generated by the strain that induced the persistent infection (cat strain or primary infection) was maintained until the end of the trial, supporting our previous results [ ] . in contrast, the csfv margarita strain generated the acute form of the disease in animals from group a, with high margarita rna loads in all of the samples analysed. in addition, the failure of the humoral response in the pigs that developed acute csf was previously described [ ] . in addition to the adaptive immune response, the innate immune response to the virus, as measured by type i ifn-α in the serum, also seemed to be impaired, in terms of ifn-α detection because ifn-α values were undetectable in the sera from postnatally persistently infected wild boars after csfv margarita strain inoculation. at the same time, the progression of the acute disease in group a was correlated with an increase in levels of endogenous ifn-α, as has been previously described [ , , , ] . the absence of an ifn-α response in the cat persistently infected animals after margarita strain inoculation (secondary infection) probably was due to the almost complete lack of margarita strain replication in these animals. otherwise, specific csfv-blockade phenomena for ifn-α might be occurring. efficient viral strategies to escape the type i ifn-induced antiviral mechanisms have been described within pestivirus. in this regard, the viral rna triggers ifn synthesis, and the viral rnase e rns inhibits ifn expression induced by extracellular viral rna [ ] . in addition, the viral protein n pro suppresses type i ifn (ifn-α/β) induction by mediating proteasomal degradation of ifn regulatory factor (irf- ) [ ] [ ] [ ] . for instance, in persistent infection, bvdv maintains "self-tolerance" by avoiding the induction of ifn, without compromising the ifn action against unrelated viruses ("nonself") [ ] . in the case of csfv-infected pigs, it has been recently demonstrated that functional n pro significantly reduced local ifn-α mrna expression responses at local sites of virus replication [ ] . these highly selective "self" models of evasion of the interferon defence system might be key elements in the success of persistent infections and could promote, in addition, the generation of sie phenomena. previous reports have suggested that the availability of mammalian models for sie in vivo is hampered by the interferon response generated against the infecting virus in these species [ , ] . it is noteworthy that csfv postnatally persistently infected swine have shown an immunosuppression state comprising a reduction in interferon responses (types i and ii) [ ] [ ] [ ] . this immunological status might promote the maintenance of a high and constant csfv load, as already described, preventing second viral entry [ ] . nevertheless, further studies would be needed to clarify the molecular mechanisms involved in this phenomenon. at dpi, low levels of margarita rna were detected only in some collected tissues from persistently and superinfected wild boars (group b), principally in animal , in which margarita rna was detected from the spleen and liver, as well as in the serum. however, the level of margarita rna detection was approximately fifteen times less than the acutely infected animals from group a ( table ). the margarita rna levels found in the superinfected animals might be correlated with the low margarita strain viral loads in some macrophages in these tissues [ ] . in contrast, despite pbmcs being a well-known target for csfv viral replication [ ] , after in vitro assay, the presence of csfv margarita rna could not be detected in either the pbmcs or st cell extracts from group b. additionally, the in vitro superinfection of isolated pbmcs failed when they were derived from persistently infected piglets but were clearly positive for assays with cells from naïve animals, as demonstrated by means of calculated Δct values, supporting that pbmcs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the margarita virus strain. these results suggest that sie still occurs at the tissue level (table ). in contrast, the margarita strain rna could not be detected after the sequence analyses of the samples from persistently infected margarita-inoculated animals (group b) nor even in the tonsil, one of the main targets for csfv replication [ , ] . nevertheless, next-generation sequence analyses would be of great interest to analyse these samples in detail, emphasising the spleen and liver tissues that were also positive for rna margarita strain detection after superinfection. altogether, although it is a very complex mechanism, if compared with the acutely infected group a, these results showed that a phenomenon of csfv sie occurred at the systemic level. nevertheless, the colonisation of a multi-cellular host is a complex process during which the viral load can dramatically change in different organs and at different stages of the infection, and not all of the potential target cells are infected in persistently infected animals despite the high viral load generated by the cat csfv strain in persistently infected animals [ , ] . illustrative examples include some of the works performed to demonstrate sie at the cellular level because some cells uninfected by one viral primary infection are subsequently infected by the second viral infection [ , ] . in contrast, the implications of other mechanisms in the host cannot be excluded, and it remains unclear whether the observed phenomenon is really due to a blockage at the level of infection of cells. this was precisely in the case of a citrus tristeza virus (ctv) sie model, wherein a ctv protein (p ) was required to mediate sie at the organism level but that did not appear to be implicated in exclusion at the cellular level [ ] . overall, our results suggested efficient suppression of viral superinfection in a 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proteasomal degradation npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type i interferon induction at local replication sites classical swine fever virus marker vaccine strain cp _e alf: shedding and dissemination studies in boars classical swine fever virus strain "c". how long is it detectable after oral vaccination? j vet med b infect dis vet public health we thank valentí rosell, iván cordón and david solanes for their help in the animal facilities. key: cord- -li sc z authors: ma, jingjiao; wu, rujuan; xu, guanlong; cheng, yuqiang; wang, zhaofei; wang, heng’an; yan, yaxian; li, jinxiang; sun, jianhe title: acetylation at k of the ns protein is important for the replication and virulence of influenza virus date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: li sc z non-structural protein (ns ) of influenza virus is a multifunctional protein that plays an important role in virus replication and virulence. in this study, an acetylation modification was identified at the k residue of the ns protein of h n influenza virus. to further explore the function of the k acetylation modification of the ns protein, a deacetylation-mimic mutation (k r) and a constant acetylation-mimic mutation (k q) were introduced into the ns protein in the background of a/wsn/ h n (wsn), resulting in two mutant viruses (wsn-ns - r and wsn-ns - q). in vitro and mouse studies showed that the deacetylation-mimic mutation k r in the ns protein attenuated the replication and virulence of wsn-ns - r, while the constant acetylation-mimic mutant virus wsn-ns - q showed similar replication and pathogenicity as the wild-type wsn virus (wsn-wt). the results indicated that acetylation at k of the ns protein has an important role in the replication and virulence of influenza virus. to further explore the potential mechanism, the type i interferon (ifn-i) antagonistic activity of the three ns proteins (ns - q, ns - r, and ns -wt) was compared in cells, which showed that the k r mutation significantly attenuated the ifn-β antagonistic activity of the ns protein compared with ns -wt and ns - q. both ns -wt and ns - q inhibited the ifn-β response activated by rig-i card domain, mavs, tbk , and irf more efficiently than the ns - r protein in cells. taken together, the results indicated that acetylation at ns k is important for the ifn antagonistic activity of the ns protein and virulence of the influenza virus. influenza virus non-structural protein (ns ) is a multifunctional protein that is responsible for interacting with cellular factors to antagonize the host antiviral response during viral infection [ ] . the major role of the ns protein is inhibition of both interferon (ifn) and ifn-stimulated proteins by different mechanisms. ns inhibits the transcription of type i ifn by binding the ′ triphosphate viral double-stranded rnas generated during viral replication to prevent the recognition of viral genomic material by host pattern recognition receptors (prrs), including rig-i, dsrna-dependent protein kinase r (pkr), and ′ ′-oligoadenylate synthetase (oas)/rnase l [ ] [ ] [ ] . ns can also interact directly with rig-i in the absence of rna binding to inhibit the conformational change of rig-i required for mavs activation [ ] . moreover, ns is able to interrupt mrna maturation by inhibiting the nuclear export of host mrnas by binding to host poly(a)-binding protein ii (pabpii) and cleavage and polyadenylation specific factor (cpsf ), which are required for host mrna processing, resulting in the accumulation of ifn pre-mrnas in the nucleus of infected cells [ ] . in addition, ns also antagonizes the open access *correspondence: lijinxiang@caas.cn; sunjhe@sjtu.edu.cn shanghai key laboratory of veterinary biotechnology, key laboratory of urban agriculture (south), ministry of agriculture, school of agriculture and biology, shanghai jiao tong university, shanghai , china chengdu national agricultural science and technology center, sichuan, china full list of author information is available at the end of the article ifn signalling response by regulating other host factors, such as phosphoinositide -kinase (pi k) activity, crklike protein (crkl), and the jak-stat signalling pathway [ ] [ ] [ ] [ ] [ ] . the ns protein, typically - aa in length depending on the strain, contains four functional regions: an rna binding domain (rbd, - aa), linker region (lr, - aa), effector domain (ed, - aa), and c-terminal "tail" (ctt, - aa) [ ] . multiple basic amino acids (e.g., r, r, k, and r) in the rbd are important for rna binding activity and suppressing the activation of pkr [ , ] . the ed plays an important role by targeting multiple host factors, such as pkr, cpsf , and p β (pi k), to inhibit antiviral responses and enhance viral replication [ ] . the residue w in the ed domain is important for the dimerization of the ns protein, and the w r substitution impaired ns dimerization and attenuated the virus in vivo [ ] . in addition, residues e, d, and v play important roles in the binding of ns to cleavage and polyadenylation specificity factor (cpsf ), and mutations in those residues weaken the binding of ns to cpsf and impair the ability of the ns protein to shut off host gene expression [ , ] . post-translational modifications, such as phosphorylation, sumoylation, and acetylation, are important for protein function. phosphorylation at t, t, and t of the ns protein are important for interferon antagonistic activity and replication of human influenza virus [ , ] . sumoylation at positions and of ns are crucial for host protein expression shutoff and replication of h n influenza virus [ ] . acetylation is an important post-translational modification that occurs in two forms [ ] . one is the co/post-translational acetylation at the n α -termini of the nascent polypeptide chains [ ] . the other form is acetylation of the ε-amino group of lysine, which was first recognized in histones regulating gene translation [ ] and later was found in non-histone proteins [ ] . the acetylation status is reversible and well balanced by lysine (k) acetyltransferases (kats) and lysine deacetylases (kdacs), which are tightly regulated to perform many cellular functions [ ] . dysfunction of the acetylation machinery can inhibit protein functions and consequently lead to severe diseases [ , ] . acetylation has been found in multiple proteins of influenza viruses. acetylation was identified in the np protein of influenza virus, and deacetylation of the np protein prevented the virus from assembling functional virus particles [ ] . a histone-like sequence (histone mimic) was identified in the ns protein of influenza a h n , which contributes to suppression of the antiviral response [ ] . the n-terminal acetylation of pa-x is required for the host shutoff activity of pa-x and for viral polymerase activity [ , ] . acetylation of pa has been reported to be crucial for polymerase activity, and deacetylated pa protein restricts iav rna transcription and replication. the influenza virus haemagglutinin (ha) has three conserved cysteine residues ( , , and ) at its c terminus serving as acylation sites that are essential for the formation of fusion pores and infectivity [ ] . in the present study, an acetylation modification at k of the ns protein was identified and characterized. the results showed that the deacetylation-mimic mutation k r in the ns protein attenuated the replication and virulence of the virus in vitro and in vivo. ifn-β antagonist assays indicated that the k r mutation attenuated ifn antagonistic ability compared with the ns -wt or ns - q (constant acetylation-mimic) proteins. overall, this study indicated that acetylation at k of the ns protein plays an important role in the replication and virulence of influenza virus. madin-darby canine kidney (mdck) cells were maintained in eagle's minimal essential medium (emem, hyclone, grand island, usa) with % foetal bovine serum (fbs, gibco, grand island, usa), l-glutamine (gibco), and % antibiotic (gibco). human embryonic kidney (hek) t cells and adenocarcinomic human alveolar basal epithelial cells (a cells) were maintained in dulbecco's modified eagle's medium (dmem, hyclone) supplemented with % fbs (gibco), l-glutamine (gibco), and % antibiotic (gibco). the virus strain a/wsn/ h n (wsn), a mouse-adapted human influenza virus, was propagated and titrated in mdck cells. to identify the putative acetylation sites in influenza viral proteins, mass spectrometry was conducted with concentrated influenza virus. briefly, the h n virus was propagated in mdck cells, and then a total of ml of virus stock was prepared. to concentrate the virus, the collected virus was pelleted by centrifugation at g for min to remove the cell debris. clarified virus supernatants were layered on a % (w/v) sucrose cushion and centrifuged at g for h. the virus pellet was suspended in water and subjected to mass spectrometry analysis performed by ptm biolabs llc (hangzhou, china). to generate mutant viruses, the site mutations k r (deacetylation-mimic mutation) and k q (constant acetylation-mimic) were introduced into the reverse genetic plasmid phw -wsn-ns by a commercial site-directed mutagenesis kit (invitrogen, grand island, usa). the mutant viruses were rescued in the background of wsn-h n virus as described previously [ ] , resulting in wsn-ns - r and wsn-ns - q viruses, and all the mutant viruses were verified by sequencing. then, the ns -wt, ns - r, and ns - q genes were amplified and cloned into the pcdna . -flag expression vector (flag-ns -wt, flag-ns - r, and flag-ns - q). the three viruses were inoculated on monolayer mdck and a cells cultured in -well plates with multiplicities of infection (mois) of . and . for each virus, respectively. each time point was set up in triplicate, and then the samples were collected at , , , and hours post-inoculation (hpi). the supernatants were titrated on mdck cells cultured in -well plates following the reed and muench method to calculate tcid /ml. the cells were collected and subjected to western blotting. briefly, the cell lysates were separated on a sodium dodecyl sulfate (sds)-polyacrylamide gel and transferred to a pvdf membrane. the membrane was blocked in pbs containing % skim milk and then incubated with rabbit anti-ns polyclonal antibody (genscript, piscataway, usa) and then with horseradish peroxidase-conjugated secondary anti-mouse antibody (thermo fisher, grand island, usa). the proteins were visualized by using an ecl kit (yeasen, shanghai, china). additionally, to determine the protein expression levels of the three ns expression plasmids, the flag-ns -wt, flag-ns - q, and flag-ns - r plasmids were transfected into t cells. forty-eight hours post-transfection, the cells were collected and subjected to western blotting. forty-eight -week-old female balb/c mice were randomly allocated into four groups, and each group contained mice. three groups were challenged with the indicated viruses, and one group was challenged with pbs as a control. the mice were inoculated with virus intranasally with . tcid of virus in µl solution under slight anaesthesia with co . the mice were monitored for body weight, clinical signs, and survival rate each day until days post-infection (dpi), and they were euthanized if they lost more than % of their original body weight. three mice from each group were euthanized at and dpi. the mouse lungs were collected for viral titration and cytokine analysis. to quantify the cytokine levels of il- β, ifn-β, and tnf-α in mouse lungs, total rna was extracted from lung tissues, reverse-transcribed and subjected to quantitative realtime polymerase chain reaction as described previously [ ] . to detect the ifn-β antagonistic ability of ns proteins, t cells were transfected with the indicated ns expression plasmids ( . μg/well) together with a plasmid expressing firefly luciferase under the control of the ifn-β promoter (pgl-ifn-β-luc, . μg/well), the renilla luciferase expressing plasmid prl-tk ( . μg/ well), and the ifn-β stimulator poly(i:c) ( . μg/well) or a plasmid expressing the active caspase recruitment domain (card) of rig-i (pcdna-rig-i . μg/well), pcdna-mavs ( . μg/well), pcdna-tbk ( . μg/well) or pcdna-irf ( . μg/well) as described previously [ ] . twenty-four hours post-transfection, the cells were lysed and subjected to a dual-luciferase reporter assay kit (promega, madison, usa). mdck cells cultured on glass slides were infected with wsn-ns -k r, wsn-ns -k q, or wsn-wt at an moi of . all cells were fixed with % paraformaldehyde (pfa) and permeabilized with . % triton x- in pbs at , , and hpi. to detect the ns proteins, the fixed cells were incubated with rabbit anti-ns polyclonal antibody (genscript), followed by fitc-conjugated antirabbit igg antibody (yeasen), and then the cells were stained with dapi. all images were obtained on a leica tcs sp confocal microscope (leica microsystems inc., buffalo grove, usa). the animal study was conducted in accordance with the guidelines of the animal care and use committee of shanghai jiao tong university, and the animal study protocols were approved by shanghai jiao tong university (approval no. ). all data were analysed using analysis of variance (two-way anova) in graph-pad prism version . (graphpad software inc., la jolla, usa); a p-value of . or less was considered significant. the mass spectrometry results showed that one acetylation modification was identified at position k of the ns protein of the wsn-wt virus (figure ). to further explore whether k is subtype-specific, we compared the ns amino acid sequences of randomly selected influenza virus strains of each endemic subtype in birds and humans from genbank. most avian h n ( %), h n ( . %), h n ( %), human h n ( . %), and human h n ( . %, isolated before ) viruses contained k in the ns genes, whereas . % of pandemic h n viruses contained r in the ns protein. these data demonstrated that the k residue is relatively conserved in influenza viruses except the pandemic h n . since the ns protein is an important virulence marker and antagonist of host innate immunity, we chose to further explore the influence of acetylated k on virus replication and virulence in this study. to mimic deacetylated lysine, a k r substitution was introduced into the ns protein, since an r substitution prevents acetylation but preserves the positive charge, and a mutant virus containing the ns -k r substitution was generated in the background of wsn virus (wsn-ns - r). moreover, to mimic constantly acetylated lysine at k of the ns protein, a k q substitution that is a known acetylation mimic was introduced into ns , resulting in a mutant virus containing ns -k q (wsn-ns - q). to determine the effect of acetylated k on virus replication, mdck and a cells were infected with wsn-wt or the two mutant virus wsn-ns - r (deacetylation mimic) or wsn-ns - q (constant acetylation mimic) at the indicated mois to obtain multicycle growth curves. all three viruses replicated efficiently in mdck and a cells, and wsn-ns - q and wsn-wt replicated to similar levels at each time point. however, the growth of the deacetylated mutant virus wsn-ns - r was significantly impaired compared with that of the other two viruses in mdck and a cells at and hpi, which indicated that the acetylated k of ns is important for virus replication in vitro at the late stage of infection (figures a and b) . similarly, ns protein levels were detected by western blotting. the results showed that the ns expression levels of wsn-ns - r were lower than those of wsn-wt and wsn-ns - q at different time points in mdck and a cells (figures c and d) . all the mice infected with viruses showed clinical signs such as ruffled fur, depression, and inappetence. the mice infected with constantly acetylated wsn-ns - q displayed more severe clinical signs and started to show mortality earlier than wsn-wt-infected mice. both wsn-ns - q and wsn-wt caused % mortality in infected mice. however, the deacetylated wsn-ns - r virus infection resulted in % mortality, and the mortality was delayed by days compared with other viruses, which indicated that the deacetylation-mimic k r substitution attenuated the wsn-ns - r virus in mice ( figures a and b) . virus titers were slightly lower in the lungs of mice infected with wsn-ns - r than in the other two groups ( figure c) . notably, significantly higher levels of ifn-β, il- β, and tnf-α mrna were detected in wsn-ns - r-infected mice than in the other two groups at dpi but not at dpi ( figures d-f) , which indicated that wsn-ns - r was less efficient at inhibiting the innate immune response than wsn-ns - q and wsn-wt at dpi in mice. to determine whether the k r and k q mutations affect the expression of the ns protein, the expression levels of flag-ns -wt, flag-ns - q, and flag-ns - r in t cells were compared. the three proteins were expressed at similar levels in transfected t cells, which indicated that the k r and k q mutations did not influence protein expression ( figure e) . the major function of the ns protein is inhibition of type i ifn induction, and the acetylated k residue is located in the effector domain of ns , which is important for its ifn antagonistic ability. to determine the effect of the acetylated k residue on ifn suppression by the ns protein, the inhibition of ifn-β promoter activity by flag-ns -wt, flag-ns - q, and flag-ns - r was evaluated. the results showed that flag-ns -wt and acetylation-mimic flag-ns - q suppressed the ifn-β promoter activity stimulated by poly(i:c) ( figure a) ; however, the deacetylation-mimic flag-ns - r protein was significantly less capable of inhibiting the activation of the ifn-β promoter compared with the acetylated ns proteins ( figure a ). this result indicated that the impaired ifn-β antagonistic ability might be responsible for the attenuation of the wsn-ns - q virus in vitro and in vivo. influenza virus infection stimulates type i ifn production by signal transduction from rig-i to tbk to irf . to further explore how the k r mutation attenuated the ifn-β antagonistic ability of the ns protein, we co-transfected ns expression plasmids, an ifn-β reporter plasmid and different type i interferon pathway components, including rig-i card, tbk , and the active form of irf , into t cells. the results showed that ns - q and ns -wt inhibited the ifn-β response stimulated by each component more efficiently than ns - r, which suggested that the acetylated k residue is important for inhibiting the ifn-β response of ns that targets factors downstream of irf or other proteins (figures b-d) . two nuclear localization signals ( - and - ) have been identified in the ns protein, which drive ns to the nucleus during the early stage of infection. to determine whether k r changes the subcellular localization of ns during infection, mdck cells were infected with the three viruses. the ns protein of wsnwt was located in the nucleus and cytoplasm of infected cells at and hpi ( figures a and b) . at the late stage of infection ( hpi), the ns protein was mainly located in the nucleus and perinuclear area of infected cells (figure c ). the ns - q protein was located in both the nucleus and cytoplasm at hpi and hpi, while it was mainly located in the perinuclear area of infected cells at hpi. in contrast, the ns - r protein accumulated mostly in the cytoplasm during the whole infection course. this result indicated that the deacetylation-mimic k r substitution retained ns protein in the cytoplasm of infected cells, suggesting that the acetylated k residue is important for the nuclear localization of the ns protein ( figures a-c ). post-translational modification is important for protein function, stability, cellular localization, and protein-protein interactions. recent studies have shown that posttranslational modifications of viral proteins modulate the virus life cycle, e.g., phosphorylation of influenza viral proteins (ns , m , and np) plays important roles in virus replication [ ] [ ] [ ] [ ] . the ubiquitination of np and m proteins is crucial for viral rna replication and the production of infectious virus particles [ ] . acetylation is an important post-translational modification in eukaryotes, but the occurrence and function of acetylation in influenza viral proteins remain largely unclear. giese et al. reported that acetylation of k, k, and k of np proteins is important for virus polymerase activity and replication [ ] . in the present study, we identified and characterized the acetylation of k in the ns protein, and the deacetylation of k q affected viral replication in cells at and hpi. the expression levels of ns -k q and ns -k r in transfected t cells were similar ( figure e ), while the ns levels in infected mdck and a cells were different ( figures c and d) . this result could be attributed to the deacetylation affecting virus replication, which resulted in low expression of ns in infected cells. moreover, the acetylation of q contributes to the ifn antagonistic ability of the ns protein. the mrna levels of ifn-β, il- β, and tnf-α in mouse lungs of the wsn-ns -k r-infected group were significantly higher than those in the other two groups at dpi, which indicated that ns - r was less efficient at inhibiting the production of innate antiviral cytokines at dpi in mice. the ns protein of influenza virus is a virulence factor that inhibits the antiviral immunity of the infected host, and c-terminal truncation has been widely used as a strategy to generate attenuated virus vaccine candidates [ ] . one mechanism used by ns to inhibit the ifn response is through direct binding and sequestration of rna as well as direct interaction with trim and complex formation with the rna sensor rig-i, resulting in inhibition of the activation of the rig-i card and hence inhibition of irf activation [ ] . the rna binding, rig-i and trim interacting domains are located in the n-terminus ( - aa) of the ns protein. however, the acetylated k is located in the ed domain, and acetylation of k may not affect the rna binding capability of the ns protein. the ns -k r substitution impaired the suppression of ifn promoter activation by poly(i:c), rig- card, tbk- , and irf , which suggested that the ns -k r substitution affected the ifn antagonism of ns either through targeting downstream of irf or a general mechanism that ns uses to inhibit ifn, such as interaction with cpsf , resulting in inhibition of the processing of mrna, including ifn mrna [ ] . notably, the cpsf protein is mainly located in the nucleus and is required for the ′ end processing of all host pre-mrnas. interestingly, the deacetylation-mimic k r substitution retained ns protein in the cytoplasm of infected cells, resulting in a possible impaired interaction between cpsf and the ns protein, subsequently leading to attenuated ifn antagonism. benjamin hale and colleagues found that the a/california/ / (h n ) virus has r in ns , and ns was unable to suppress general host gene expression. nevertheless, the rk substitution in the ns / protein restored its ability to block general gene expression and bind cpsf [ ] . this could explain why attenuation of the ifn antagonistic ability of ns -k r is independent of rig-i card, tbk- , and irf activation. in addition, anastasina et al. [ ] reported that the ns protein binds to cellular dna to block the cellular transcription of ifns and isgs; thus, ns proteins retained in the cytoplasm lose their cellular dna binding function, resulting in impaired ifn-β antagonistic ability. two known nuclear localization sequences (nlss) of ns proteins are located at the - and - positions. the - nls of the ns protein is highly conserved among influenza a virus strains [ ] . the second nls ( - ) is virus strain specific, and the pandemic h n lacks the second nls in the ns protein [ ] . single point mutations, either r a, r a, or k a, completely eliminated importin protein binding, which transports target proteins to the nucleus [ ] . notably, in this study, acetylation of k located outside of the nls affected the cellular localization of ns protein, and the deacetylation-mimic k r substitution blocked the nuclear localization of the ns protein in infected cells; however, the underlying mechanism remains unknown. interestingly, the ns -k residue is relatively conserved in most influenza viruses, except for the pandemic h n . the pandemic h n has r in ns , which causes inefficient general host gene expression shutoff, while r k restores its ability to block general host genes and bind cpsf [ ] . potentially, the pandemic h n virus might use different strategies to overcome the ifn response compared with the other influenza viruses. overall, we identified an acetylation of k of the ns protein of influenza virus, and the acetylation of k plays an important role in the cellular localization, ifn antagonistic ability, replication, and virulence of influenza virus. conformational plasticity of the influenza a virus ns protein inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus rig-i-mediated antiviral responses to single-stranded rna bearing ′-phosphates immunogenicity and protection efficacy of replication-deficient influenza a viruses with altered ns genes structural basis for a novel interaction between the ns protein derived from the influenza virus and rig-i structural basis for suppression of a host antiviral response by influenza a virus influenza virus non-structural protein (ns ) disrupts interferon signaling a site on the influenza a virus ns protein mediates both inhibition of pkr activation and temporal regulation of viral rna synthesis the primary function of rna binding by the influenza a virus ns protein in infected cells: inhibiting the ′- ′ oligo (a) synthetase/rnase l pathway influenza a virus inhibits type i ifn signaling via nf-kappabdependent induction of socs- expression influenza a virus abrogates ifn-gamma response in respiratory epithelial cells by disruption of the jak/stat pathway the multifunctional ns protein of influenza a viruses influenza a virus virulence depends on two amino acids in the n-terminal domain of its ns protein to facilitate inhibition of the rnadependent protein kinase pkr contribution of ns effector domain dimerization to influenza a virus replication and virulence ns protein amino acid changes d n and v i affect interferon responses, thermosensitivity, and virulence of circulating h n human influenza a viruses the k e amino acid substitution in the canine influenza virus h n ns protein restores its ability to inhibit host gene expression roles of the phosphorylation of specific serines and threonines in the ns protein of human influenza a viruses threonine phosphorylation of non-structural protein regulates the replication of influenza a virus by reducing the binding affinity with rig-i modification of nonstructural protein of influenza a virus by sumo years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond proteomics analyses reveal the evolutionary conservation and divergence of n-terminal acetyltransferases from yeast and humans acetylation and methylation of histones and their possible role in the regulation of rna synthesis acetylation and deacetylation of non-histone proteins the world of protein acetylation genetic dissection of histone deacetylase requirement in tumor cells the many roles of histone deacetylases in development and physiology: implications for disease and therapy role of influenza a virus np acetylation on viral growth and replication suppression of the antiviral response by an influenza histone mimic n-terminal acetylation by natb is required for the shutoff activity of influenza a virus pa-x hdac restricts influenza a virus by deacetylation of the rna polymerase pa subunit acylation-mediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity analysis of recombinant h n wild-type and mutant viruses in pigs shows that the q l mutation in ha is important for transmission quantification of murine cytokine mrnas using real time quantitative reverse transcriptase pcr herpes simplex virus ubiquitinspecific protease ul inhibits beta interferon production by deubiquitinating traf mapping the phosphoproteome of influenza a and b viruses by mass spectrometry effects of the s residue of the h n swine influenza virus ns protein on interferon responses and virus replication ubiquitination of the cytoplasmic domain of influenza a virus m protein is crucial for production of infectious virus particles phosphorylation and dephosphorylation of threonine in nucleoprotein is crucial for the replication of influenza a virus attenuation of the virulence of a recombinant influenza virus expressing the naturally truncated ns gene from an h n equine influenza virus in mice inefficient control of host gene expression by the pandemic h n influenza a virus ns protein influenza virus ns protein binds cellular dna to block transcription of antiviral genes nuclear and nucleolar targeting of influenza a virus ns protein: striking differences between different virus subtypes influenza a h n subtype virus ns protein targets into the nucleus and binds primarily via its c-terminal nls /nols to nucleolin and fibrillarin this study was supported by the national natural science authors' contributions jm, jl, and js designed the study; jm was involved in the acquisition of data, analysis, and figure preparation; rw, gx, yc, and zw contributed to some of the laboratory experiments and data analysis; hw and yy helped revise the manuscript; jl and js supervised the study; jm drafted the original paper. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -jne jqf authors: macparland, sonya a.; ma, xue-zhong; chen, limin; khattar, ramzi; cherepanov, vera; selzner, markus; feld, jordan j.; selzner, nazia; mcgilvray, ian d. title: lipopolysaccharide and tumor necrosis factor alpha inhibit interferon signaling in hepatocytes by increasing ubiquitin-like protease (usp ) expression date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: jne jqf inflammation may be maladaptive to the control of viral infection when it impairs interferon (ifn) responses, enhancing viral replication and spread. dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis b virus and hepatitis c virus (hcv). previous studies from our laboratory have shown that expression of an ifn-stimulated gene (isg), ubiquitin-like protease (usp) is upregulated in chronic hcv infection, leading to impaired hepatocyte responses to ifn-α. we examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (tnf-α), lipopolysaccharide (lps), interleukin- (il- ) and il- to upregulate hepatocyte usp expression and blunt the ifn-α response. human hepatoma cells and primary murine hepatocytes were treated with tnf-α/lps/il- /il- and usp , phosphorylated (p)-stat and myxovirus (influenza virus) resistance (mx ) expression was determined. treatment of huh . cells and primary murine hepatocytes with lps and tnf-α, but not il- or il- , led to upregulated usp expression and induced an ifn-α refractory state, which was reversed by usp knockdown. liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. hepatic ischemia/reperfusion injury led to an induction of usp expression in liver tissue and promotion of lymphocytic choriomeningitis replication. these data demonstrate that certain inflammatory stimuli (tnf-α and lps) but not others (il- and il- ) target usp expression and thus inhibit ifn signaling. these findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with usp representing a potential target for intervention in various inflammatory states. importance inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic hcv infection, leading to impaired hepatocyte responses. in this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via usp induction. these findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of usp representing a potential target for intervention in various inflammatory states. i nterferon (ifn) is a key endogenous mediator of viral clearance by the innate immune response. one excellent example of this is hepatitis c virus (hcv) infection of the liver ( ) . interferon signaling drives the expression of multiple interferon-stimulated genes (isgs), which mediate viral clearance. isgs, however, can also be induced by other factors, including inflammatory stimuli such as tumor necrosis factor alpha (tnf-␣) ( ) . hcv infection, which induces chronic inflammation of the liver, is associated with high serum tnf-␣ ( , ) . interestingly, anti-tnf-␣ treatment has been shown to lead to an improved virologic response to ifn-␣/ribavirin antiviral therapy for hcv ( ) , while other data show safety of combined treatment but no effect on hcv viral loads of hcv treatment-naive individuals ( ) . the purpose of the present study is to define an important causal link between inflammation and the host hepatic innate immune response, with broad viral relevance. our recent work in the host innate immune response to chronic hcv infection suggested an association between hepatocytes and the liver resident immune cells that drive liver inflam-mation ( ) ( ) ( ) . there is a dichotomous response to chronic hcv infection in the liver, and this response predicts who will and who will not respond to exogenous ifn-␣ treatment. in patients that do not respond to therapy with ifn/ribavirin, hepatocytes have strong preactivation of the ifn-response, with high expression of a subset of isgs ( ) ( ) ( ) . in patients exhibiting a sustained virologic response on therapy with ifn/ribavirin, tissue macrophages show a high expression of isgs ( , ) . the expression of isgs in macrophages or hepatocytes is more predictive of treatment outcome than il b polymorphisms ( ) ; this finding suggests a link between hepatic inflammatory cell activation and viral clearance. liver kupffer cell inflammatory responses are induced by and play a role in the control and clearance of multiple viral infections of the liver ( , ) . although the mechanisms underlying these patterns are undoubtedly complex, the association between liver macrophages and hepatocytes raises the question of how the hepatocytes react to the cytokines produced by the macrophages (and other inflammatory cells). if isg expression in hepatocytes reflects a response to inflammatory stimuli, then how the liver responds to a viral infection will be modulated by the inflammatory milieu. in hcv infection, we have found that the isg /usp pathway is an important regulatory pathway. both isg and usp are isgs that are strongly upregulated in the livers of ifn treatment-resistant patients ( ) ( ) ( ) . isg is a ubiquitin-like protein that is strongly upregulated by ifn and conjugates to multiple cellular proteins ( , ) . usp is an isg -specific protease that is also upregulated by ifn-␣ ( , ) . both isg and usp have been suggested to blunt type ifn signaling ( , ( ) ( ) ( ) . in addition to its ability to remove isg from its conjugated proteins, usp has been shown to bind to the type ifn receptor and blunt ifn signaling ( ) . upregulation of usp may represent a negative-feedback loop counteracting the effects of type ifn. furthermore, knockdown of usp increases both isg induction and anti-hcv activity of ifn-␣ ( ) , and data have shown that ifn-␣ treatment given to mice in vivo increases hepatic usp and blunts the effect of a subsequent dose of ifn-␣ ( ) . the mechanisms controlling usp expression in the liver are poorly understood but will have relevance to understanding innate immune mechanisms in chronic viral infection. although the majority of work on usp has centered on its upregulation by type ifn, interferon stimulated genes have multiple upregulatory stimuli other than ifn-␣ ( , ) . for example, inflammatory stimuli, e.g., endotoxin and lipopolysaccharide (lps), have been shown to upregulate usp in peritoneal exudate macrophages ( ) . if similar stimuli lead to increased usp expression in hepatocytes, then this pathway could represent a novel link between inflammatory and innate immune responses in the liver. the present study is based on the hypothesis that liver inflammation will directly impact hepatocyte expression of usp and therefore will impact ifn signaling. this link will have relevance to multiple diseases, since inflammatory stimuli such as tnf-␣ and lps have been shown to play roles in many liver diseases, including viral hepatitis ( ) ( ) ( ) . the link may also help to explain our observations in chronic hcv infection, since chronic hcv infection is characterized by increased serum tnf-␣ ( , ), increased hepatocyte usp , and impaired ifn responsiveness ( ) . quite apart from hcv, the importance of the link between hepatic inflammation and innate immune response lies in the downstream effects of the impairment of innate immunity. we speculate that by blocking ifn-␣ signaling, usp expression may lead to an enhanced susceptibility to infection with interferon-sensitive viruses and enhanced viral proliferation. support for this notion is found in a mouse study in which expression of usp in macrophages led to lower ifn responsiveness, leading to locally restricted replication of vsv ( ) . in the present study, treatment of hepatic cells with lps and tnf-␣, but not il- or il- , led to upregulated usp expression in hepatocytes. the enhanced usp expression was associated with decreased ifn-␣-stimulated expression of p-stat and isgs, a phenomenon reversed by usp knockdown. as an in vivo correlate of our in vitro findings, experimentally induced hepatic ischemia/reperfusion injury induced usp mrna expression in liver and enhanced lymphocytic choriomeningitis (lcmv) replication, an effect not seen in usp Ϫ/Ϫ mice. these data demonstrate that certain inflammatory stimuli (tnf-␣ and lps), as well as ischemic injury, but not other cytokines (il- and il- ) can lead to enhanced hepatocyte usp expression and thereby inhibit ifn signaling. these findings lend new knowledge to our understanding of how inflammation can modulate hepatic innate immune responses, with usp representing a potential target for intervention to reverse any proviral effect of inflammation. phorylated-stat (p-stat- ; cell signaling technology, boston, ma), rabbit polyclonal anti-stat- (santa cruz biotechnology), goat polyclonal anti-ube antibody (santa cruz biotechnology), or mouse monoclonal anti-actin (sigma) antibodies, followed by anti-mouse or anti-rabbit igg conjugated to horseradish peroxidase (calbiochem, billerica, ma). an enhanced chemiluminescence detection kit (amersham pharmacia biotech, uppsala, sweden) was used to determine the levels of protein expression. flow cytometric quantification of usp expression in huh . cells. usp expression on huh . cells treated with lps or tnf was quantified by using flow cytometry as previously described ( ) . briefly, cells were fixed, permeabilized, and stained with mouse anti-human usp polyclonal antibody (abnova) or a relevant isotype-matched control antibody. staining was detected with a secondary goat anti-mouse igg labeled with fluorescein isothiocyanate (fitc; santa cruz biotechnology). the cells were acquired using a facscalibur cytometer (bd biosciences, san jose, ca). a minimum of , events were collected. the resulting data were analyzed using flowjo software (tree star, inc.). the experiments were repeated four times. sirna targeting murine usp and ube l. usp small interfering rna (sirna) is a pool of three target-specific -to -nucleotide (nt) sirnas designed to knock down usp gene expression that was obtained from santa cruz biotechnology. the ube l sirna used was a pool of three to five target-specific -to -nt sirnas designed to knockdown mouse ube l gene expression and was obtained from santa cruz. usp and ube l sirna was transfected into ϫ primary murine hepatocytes according to the manufacturer's instructions using the santa cruz sirna reagent system sc- (santa cruz) and as previously described ( ) . inhibition of inflammatory signaling in primary mouse hepatocytes. to inhibit lps-or tnf-␣-stimulated nf-b activation, primary mouse hepatocytes were incubated with the following inhibitors for min prior to h of stimulation with lps or tnf-␣: p , a protein kinase a inhibitor ( ); nsc , a jak inhibitor ( ); p , a protein kinase c inhibitor ( ); pd , a mitogen-activated protein kinase kinase inhibitor ( ); silibinin, an inhibitor of ikk␣ ( ) ; and wortmannin, a phosphatidylinositide -kinase inhibitor ( ) . the concentrations, sources, and main targets of these inhibitors are described in table . after lps or tnf-␣ stimulation, hepatocytes were harvested and usp and il- ␤ (a surrogate of nf-b activation) mrna expression was evaluated as described below. rna isolation and quantitative real-time pcr analysis. huh . cells and primary murine hepatocytes were treated with ifn-␣ ( u/ml), tnf-␣ ( ng/ml), lps ( ng/ml), il- ( ng/ml), or il- ( ng/ ml) over a -h time course, and usp expression was determined by quantitative pcr (qpcr) as previously described ( , ) . briefly, total rna was prepared from cells using the trizol reagent (invitrogen) according to the manufacturer's instructions. the reverse transcription reactions of the extracted rna were performed with a first-strand cdna synthesis kit (amersham pharmacia biotech) according to the manufacturer's directions. first, g of extracted rna was added in a total volume of l of combined cdna reaction reagents with random hexamer oligonucleotides as the first-strand primer in a . -ml reaction tube. samples were heated to °c for min, chilled on ice for min, and incubated at °c for h, followed by a -min incubation at °c. the specific primers for all of the detected genes for the pcrs were based on genbank-published sequences: mus musculus ubiquitin-specific peptidase (usp ; nm_ ) forward primer ( =-tacagcagagagcagcagga) and reverse primer ( =-cacatgtcggagcttgctaa); mus musculus myxovirus (influenza virus) resistance (mx ; nr_ ) forward primer ( =-tctgaggagagccagacaat- =) and reverse primer ( =-actctggtccccaatgacag); mouse hypoxanthine guanine phosphoribosyltransferase (hprt; nm_ ) forward primer ( =-tcagt caacgggggacataaa) and reverse primer ( =-ggggctgtac tgcttaaccag); mus musculus il- ␤ (nm_ ) forward primer ( =-gaaatgccaccttttgacagtg) and reverse primer ( =-tgg atgctctcatcaggacag); homo sapiens ifn-␣ b (ifna b; ay ) forward primer ( =-gcttgggatgagaccctccta) and reverse primer ( =-cccaccccctgtatcacac); homo sapiens ifn-␥ (ifng; nm_ ) forward primer ( =-tcggtaactgacttgaatg tcca) and reverse primer ( =-tcgcttccctgttttagctgc); homo sapiens actin, beta-like (actbl ; nm_ ) forward primer ( =-gtctgccttggtagtggataatg) and reverse primer ( =-tcgagg acgccctatcatgg); homo sapiens ubiquitin specific peptidase (usp ; nm_ ) forward primer ( =-aggagaagcgtccctt tcca) and reverse primer ( =-tggtccttaatcaggttccagag); and homo sapiens myxovirus (influenza virus) resistance- (mx ; nm_ ) forward primer ( =-ggtggtccccagtaatgtgg) and reverse primer ( =-cgtcaagattccgatggtcct). quantitative real-time pcr was performed on an abi prism ht machine (applied biosystems, foster city, ca) with sybr green realtime pcr master mix (applied biosystems) according to the directions provided by the manufacturer. all of the rna samples and controls were assayed in duplicate. real-time pcr conditions were as follows: min at °c, followed by cycles of s at °c and s at °c monitor fluorescence in sybr channel during a °c annealing/extension step. the results were analyzed by using applied biosystems sds . software (applied biosystems). experimental hepatic ischemia/reperfusion injury model. hepatic ischemia/reperfusion injury (hiri) was simulated as before ( ) . partial ( %) hepatic ischemia was induced for min in mice, after which the surgical clamps were removed. control (sham) animals underwent anesthesia and laparotomy alone. animals were euthanized or h after ischemia/reperfusion, the affected liver segments were removed, and target gene expression was determined by qpcr in whole liver tissue. lcmv strain we was propagated in l cells (atcc ccl- ) as previously described ( ) . in additional experiments, after min hiri or sham laparotomy, mice were infected with ϫ pfu of lcmv we by intravenous tail vein injection. the ischemic liver lobes were harvested at day postinfection. snap-frozen liver tissue samples were homogenized in ␣-mem (multicell, usa) supplemented with % fbs, l-glutamine, and u of penicillin/ml plus g of streptomycin/ml using the tissuelyser lt system (qiagen, netherlands). viral titers were examined on mc cells (atcc crl- ) using a focus-forming assay as previously described ( ) . statistical analysis. all data were analyzed using prism version . software (graphpad software, san diego, ca). statistically significant differences in fig. e were calculated by using a two-tailed student t test (prism software). the impact of hiri on lcmv replication (see fig. a and b) was evaluated by one-way analysis of variance with the tukey's post hoc test. usp is induced in hepatocytes by lps and tnf-␣ but not by il- and il- . we have previously shown that usp can modulate the type ifn response ( ) . we sought to determine whether inflammatory stimuli could increase usp expression in hepatocytes. liver tissue inflammation is mediated by both proand anti-inflammatory stimuli, acting through diverse pathways. we therefore compared the effects of three proinflammatory stimuli (tnf-␣, lps, and il- ) and one anti-inflammatory stimulus (il- ), all four signaling through independent receptors and signaling cascades ( , ) . in a -h time course experiment, usp mrna expression was measured after stimulation with lps ( ng/ml), tnf-␣ ( ng/ml), il- ( ng/ml), or il- ( ng/ml). usp mrna expression was induced by tnf-␣ and lps but not by il- or il- ( fig. a) . meanwhile, neither tnf-␣ nor lps treatments had a marked effect on mx mrna expression (fig. b) , indicating that the effects we observe are not due to a generalized upregulation of isgs or to type ifn signaling. tnf-␣ and lps treatment also led to augmented usp protein expression by western blotting (fig. c ) and by intracellular staining (fig. d to f). treatment of huh . cells with tnf-␣ or lps induced expression of usp in . % Ϯ . % and . % Ϯ . %, respectively, compared to untreated controls in which usp expression was . % Ϯ . % (fig. fi) . usp induction was also demonstrated with a significant shift in the mean fluorescence intensity of usp staining after tnf-␣ or lps simulations (fig. fii ). in these assays, the degree of protein expression did not correlate completely with mrna expression, a finding that is consistent with many genes in the liver ( ) in that the degree of mrna induction was higher than the observed usp protein expression. however, the functional effects we observed in terms of ifn-␣ responses were robust. tnf-␣ and lps block ifn-␣ signaling in huh . hepatoma cells. to determine whether inflammatory stimuli such as tnf-␣ and lps can interfere with type ifn signaling, we sought to determine whether pretreatment of hepatoma cells with tnf-␣ or lps blocked ifn-␣ signaling. huh . cells were treated with tnf-␣ ( ng/ml) or lps ( ng/ml) for h. the cells were subsequently treated with ifn-␣ for h. we chose h to measure mx expression based on previous descriptions of ifn-stimulated mx induction in the literature ( ) and based on our observations with primary mouse hepatocytes. control cells were left untreated, followed by a -h ifn-␣ treatment at h. after the -h ifn-␣ treatment, the cells were harvested, and mx expression was measured by qpcr as a measure of ifn-inducible gene expression. as expected, a -h exposure to ifn-␣ strongly induced mx expression, whereas a -h exposure to tnf-␣ and lps did not (fig. , columns , , and ). both tnf-␣ and lps pretreatment inhibited the effect of h of exposure to ifn-␣ (fig. , columns , , and ), indicating that ifn-␣ signaling was impaired in the presence of these inflammatory stimuli. having shown that certain inflammatory stimuli both increase hepatocyte usp and blunt ifn-␣ signaling, we next sought to determine whether the blunting of ifn-␣ signaling is mediated via increased usp . we addressed this question by selective knockdown of usp mrna. thus, huh . cells transfected with usp sirna or control sirna were treated with lps ( ng/ml), tnf-␣ ( ng/ml), and/or ifn-␣ ( iu/ml) for h or pretreated with lps ( ng/ml) or tnf-␣ ( ng/ml) for h and then either left untreated or treated with ifn-␣ ( iu/ml) for h (a -h time point was selected for optimal expression of pstat in huh . cells). the expression of pstat , stat , and isg conjugates (as a marker of usp knockdown), usp , and actin was detected by western blotting. as seen in fig. a , usp expression was largely knocked down in cells transfected with usp sirna. ifn-␣-induced phosphorylation of stat- was inhibited by pretreatment (for h) with tnf-␣ or lps (fig. a , sirna control lanes f and h versus lane d), but this effect is reversed by knockdown of usp (fig. a , sirna usp , lanes f and h versus lane d). of note, usp knockdown did not increase pstat in response to ifn-␣ before h; these findings are consistent with previous observations ( ) . we next wanted to confirm whether our findings from hepatoma cells would hold true in primary mouse hepatocytes. with this in mind, primary murine hepatocytes from usp ϩ/ϩ mice were isolated, transfected with usp sirna or control sirna, and then exposed to ifn-␣ ( iu/ml), lps ( ng/ml), or tnf-␣ ( ng/ml) for h. after a washing step, the cells were treated with ifn-␣ for an additional h. mx isg mrna expression was measured by qpcr (normalized to expression of the hprt housekeeping gene) as an index of downstream ifn-␣ effect. ifn-␣, lps, and tnf-␣ treatment blocked the effect of the final dose of ifn-␣ (fig. b, columns , , , and ) . however, knockdown of usp reversed this effect and augmented the effect of ifn-␣ (fig. b, columns , , , and ) . the data from this experiment are consistent with those obtained with human huh . cells (data not shown). thus, the ability of tnf-␣ and lps to block ifn signaling is seen in both primary and immortalized hepatocytes. as noted earlier, usp has dual roles: it both strips isg from its target proteins and impairs type ifn signaling independent of its protease activity ( ) . to determine whether the usp -dependent blunting of hepatocyte ifn signaling was due to its ability to strip isg from its conjugates, we blocked the process of isgylation by knockdown of the isg e enzyme, ube l. murine hepatocyte ube l was knocked down via transfection with sirna specific for ube l. usp ϩ/ϩ murine hepatocytes transfected with ube l sirna or control sirna were stimulated with ifn-␣ ( iu/ml), lps ( ng/ml), or tnf-␣ ( ng/ml) for h and then treated with an additional dose of ifn-␣ for h. knockdown was measured in the hepatocytes transfected with ube l sirna by western blotting probing for ube l expression and isg conjugate formation (fig. a) . next, to examine the effect of this knockdown on isg induction, wild-type murine hepatocytes transfected with ube l sirna or control sirna were stimulated with tnf-␣ ( ng/ml) and lps ( ng/ml) for h and then restimulated with ifn-␣ ( iu/ ml) for h, at which time mx isg mrna expression was measured by qpcr. as seen in fig. a , ube l knockdown was achieved, resulting in a decrease in isg conjugates. however, as seen in fig. b , we observed that neither the presence nor the relative absence of isg conjugation impacted the ability of tnf-␣ and lps to block ifn-␣ signaling since there was no change in isg expression after h of ifn-␣ treatment when usp ϩ/ϩ hepatocytes transfected with anti-ube l sirna were compared to hepatocytes transfected with irrelevant sirna (fig. b , sirna control, columns , , and , and ube l sirna, columns , , and ). these results are consistent with there being no role for isgylation (and, thus, for the ability of usp to strip isg from its protein conjugates) in the ability of tnf-␣ and lps to block type ifn signaling in hepatocytes, and this finding is in agreement with previous data showing that usp blocks ifn signaling independent of its enzymatic activity ( ) . we next sought to determine whether tnf-␣ and lps could induce usp expression in hepatocytes via the induction of ifn-␣. as observed in fig. a , stat phosphorylation shows distinct activation profiles with ifn-␣ stimulation compared to tnf-␣ or lps stimulation (fig. a , sirna control and sirna usp , lanes b and c compared to lane d). although these data strongly suggest that the effect of lps and tnf-␣ stimulation are a result of direct induction of isgs and not secondary to the induction of ifn-␣, we wanted to confirm whether lps and tnf-␣ could induce type or type ifn (ifn-␣ or ifn-␥) in primary murine hepatocyte. thus, primary murine hepatocytes were treated with ifn-␣ ( u/ml), lps ( ng/ml), or tnf-␣ ( ng/ml) for , , , or h; they were then lysed and assessed for ifn-␣, ifn-␥, and usp expression by qpcr. we observed that neither ifn-␣, lps, nor tnf-␣ induce much, if any, hepatocyte expression of ifn-␣ or ifn-␥, although the same doses induce strong expression of usp (fig. ) . thus, the induction of isgs by lps and tnf-␣ is very unlikely to reflect the induction of hepatocyte type or type ifn, which suggests that the observed usp induction is not due to type or type ifn secretion and autocrine stimulation of the ifn-␣ receptor. experimental hepatic ischemia/reperfusion induces usp expression and enhances lcmv replication. we then assessed whether tissue-wide hepatic inflammatory stress increases liver usp expression, as an in vivo confirmation of our in vitro findings. this was approached by inducing partial ( %) hepatic ischemia/reperfusion injury (hiri) for min in mice, euthanizing the animals at or h (time points relevant to our in vitro time course) and measuring induction of usp by qpcr. we chose hiri as a very well-characterized inflammatory stress, known to be driven both by tnf-␣ and lps ( ) . as seen in fig. a , hiri alone induced usp mrna expression in whole livers by Ͼ fold that of untreated animals. thus, in vivo liver inflammation leads to increased usp expression at the organ level. after determining that hiri induces usp expression, we next wanted to examine the impact of hiri on viral control. thus, we induced % hiri for min prior to lcmv we infection of usp ϩ/ϩ and usp Ϫ/Ϫ mice. as seen in fig. b , hiri led to a significant increase in lcmv viral titers in usp ϩ/ϩ mice, an effect that was not observed in usp Ϫ/Ϫ mice. having shown that lps/ tnf-␣ stimulation increases hepatocyte usp expression, we then sought to determine whether we could pharmacologically inhibit the induction of usp by lps and tnf-␣. we focused on small-molecule agents that have been linked to the nf-b signaling pathway, because of the central role of this pathway in inflammatory activation in response to a large number of inflammatory mediators, including lps and tnf-␣ ( ) . we were particularly interested in inhibitors, such as silibinin, that in addition have been linked to clinical suppression of hepatic viral production (in the case of silibinin and hcv) ( , ) . thus, we preincubated primary mouse hepatocytes for min with various inhibitors of lps and tnf-␣ signaling and measured their impact on usp induction, while simultaneously measuring expression of proinflammatory cytokine il- ␤ mrna, since nf-b is also known to promote il- ␤ transcription ( , ) . as seen in fig. a and as table , tnf-␣ induced expression of usp was potently downregulated by all inhibitors tested, and this inhibition coincided with impaired il- ␤ mrna expression (fig. b) . meanwhile, only two inhibitors potently (Ͼ %) inhibited usp expression as well as il- ␤ mrna expression in response to lps stimulation; nsc (an inhibitor of jak ) and silibinin (an inhibitor of ikk␣) (fig. a and b and table ). these data suggest that the induction of usp by tnf-␣ and lps, and pos-sibly other inflammatory stimuli, is promoted by nf-〉 signaling and that hepatocyte usp expression in particular-compared to il- ␤-may be an attractive target for pharmacologic manipulation in the setting of liver inflammation. in this study we examined the role of various inflammatory stimuli in the induction of usp and the downstream establishment inflammatory stimuli, we examined the induction of stat- phosphorylation in huh . cells with or without usp knockdown (a) and the expression of isg mrna (mx ) in primary mouse hepatocytes and the ability of usp knockdown to restore isg induction after lps and tnf-␣ stimulation (b). (a) huh . cells were transfected with anti-usp sirna or control irrelevant sirna. huh . cells were then pretreated with lps ( ng/ml), tnf-␣ ( ng/ml), or ifn-␣ ( u/ml) or left untreated for h and then exposed to ifn-␣ or left untreated for an additional h. as controls, huh . cells were treated with lps, tnf-␣, and ifn-␣ for h only. the expression of usp , pstat , stat , and isg conjugates and actin proteins was measured by western blotting. (b) primary murine hepatocytes from usp ϩ/ϩ mice were isolated, transfected with anti-usp sirna or control irrelevant sirna, and pretreated with ifn-␣ ( iu/ml), lps ( ng/ml), or tnf-␣ ( ng/ml) for h (ifn-␣ pre, lps pre, or tnf-␣ pre, respectively). after being washed, the cells were cultured in the presence or absence of ifn-␣ for h (ifn-␣ final). mx isg mrna expression was measured by qpcr and normalized to the expression of the hprt housekeeping gene. the data were generated from pooled triplicate experiments analyzed in duplicate. error bars represent the sem for duplicate pcrs. of an ifn-␣ refractory state. we used multiple methods to show that certain inflammatory stimuli, including lps and tnf-␣ are able to induce the expression of usp , which results in downstream downregulation of ifn-␣-induced isg expression. the role of usp in the ifn-␣ refractory state has been previously demonstrated by human and mouse usp knockdown studies ( , ( ) ( ) ( ) . other inflammatory stimuli, including il- and il- , did not induce usp or impair expression of isgs, including usp . in vivo, hepatic inflammatory stress (ischemia/reperfusion injury) led to increased hepatic usp gene expression that was associated with poor control of lcmv infection. thus, the hepatic inflammatory milieu, contributed to by individual inflammatory cytokines and stimuli, modulates usp expression. these results demonstrate one mechanism by which liver inflammation directly impacts the hepatocellular innate immune response. usp is known to be induced by multiple inflammatory stimuli in macrophages ( ) and lymphocytes ( ) . our findings in hepatocytes are consistent with work done by other groups in immune cells. for example, lps treatment of a murine macro- phage cell line upregulates usp in an irf -dependent manner ( ) . the cytokine specificity of our results is also consistent with finding that il- alone is not able to induce usp in murine t cells ( ) . only when t cells were treated with il- and another proinflammatory cytokine, such as transforming growth factor ␤, il- ␤, and il- , was usp expression induced ( ) . these data point out that usp can be induced by inflammatory stimuli in multiple cell types in the absence of ifn. usp is therefore well positioned to act as the mediator of "cross talk" between innate immunity and inflammatory responses. in the present study, we show that increased usp expression following exposure of hepatocytes by inflammatory stimuli blunts the ifn response. the binding of usp with the ifn-␣ receptor has been shown to inhibit the interaction of stat- with the ifn-␣ receptor and thus block downstream ifn signaling ( , ) . our data are consistent with this mechanism, since the blunting of hepatocyte ifn signaling after exposure to inflammatory stimuli is independent of usp -mediated removal of isg from its target proteins. the degree of tnf-␣ and lps-induced ifn-␣ refractoriness was not changed by ube l knockdown, although isgylation was considerably reduced. however, other mechanisms may also be at play. recent work has demonstrated that usp has the ability to deubiquitinate the transforming growth factor-activated kinase (tak ) complexes required for nf-b activation in t cells and that overexpression of usp leads to decreased nuclear activation and impaired formation of tak complexes ( ) . usp -mediated nf-b inhibition may be of importance not only for t cell adaptive immunity but also for liver inflammation. the role of tak in innate and adaptive immunity has been previously demonstrated ( ) , and studies have also shown that tak deletion interferes with hepatocyte homeostasis and leads to hepatic injury ( ) . thus, increased hepatocyte usp in response to inflammatory stimuli may constitute a negative-feedback cycle with relevance to multiple aspects of the liver's response to infection. our in vivo experiments demonstrated that inflammation resulting from ischemia/reperfusion injury induces usp expression, which coincides with diminished lcmv control in mouse livers. the general finding that liver inflammation promotes lcmv production is in agreement with work showing that polymicrobial sepsis, characterized by high tnf-␣ and inflammation, leads to increased susceptibility to lcmv infection ( ) . in our present study, hepatic ischemia/reperfusion injury did not enhance lcmv production in usp knockout mice. these data are intended as proof-of-concept but do suggest that the role of usp in hepatic viral infection and inflammation deserves further investigation. our in vitro and in vivo findings suggest a new model for how inflammation alters hepatic innate immune responses. in this model, liver inflammation leads to increased hepatocyte usp , which in turn makes the liver more susceptible to infections targeting the hepatocyte, such as hcv. this mechanism may help to explain the clinical observation that hcv infection of a transplanted, hcv-naive liver graft (that has gone through an ischemia/reperfusion cycle) is more aggressive than the original infection and that the severity of the reinfection correlates with the severity of the ischemia/reperfusion injury suffered during the transplant ( , ) . however, we have also found that usp is necessary but not sufficient on its own to induce an ifn-␣ refractory state ( ) . with this in mind, we hypothesize that the innate immune response and its ability to control ifn-sensitive viruses will depend on cumulative effect of multiple intrahepatic signals, including the induction of usp . if these results are to be translated into a clinical setting, then one approach is to target usp induction pharmacologically. using multiple inhibitors of tnf-␣/lps signaling, we found that two inhibitors, silibinin (an inhibitor of ikk␣) and nsc (a jak inhibitor) possess the ability to inhibit tnf-␣ and lps-induced usp expression and proinflammatory effects. these findings raise the possibility of pharmacologically targeting usp expression during inflammatory events to prevent the establishment of an ifn-␣ refractory state. previously, inhibition of jak signaling led to a protection of mouse livers from ischemic insult ( ) . as well, silibinin has been shown to reduce of hcv liver graft reinfection ( ) and enhance hcv clearance in ifn-␣ nonresponders ( ) via multiple mechanisms, including by direct inhibition of hcv ns b rna polymerase ( ) . usp modulation may be one mechanism by which silibinin exerts its anti-hcv effects. the present study focuses on a relatively small number of inflammatory stimuli; while tnf-␣ and lps are important to a large number of liver diseases, they are far from being the only drivers of the hepatic inflammatory response and tissue levels of usp are likely influenced by other stimuli as well. for example, we previously demonstrated that increased usp expression in the liver is and enhances lcmv replication. to examine the in vivo effect of an inflammatory stimulus on usp expression and viral replication, a hepatic ischemia/reperfusion model was used. (a) partial ( %) hepatic ischemia was induced for min, after which the portal vascular clamp was removed. control animals (sham) underwent anesthesia and a laparotomy alone. animals were euthanized and h after ischemia/reperfusion (i/r), and the affected liver segments were removed. usp mrna expression was determined by qpcr in whole liver tissue and normalized to hypoxanthine-guanine phosphoribosyltransferase (hprt) expression. data are expressed as means Ϯ the sem with n ϭ to mice/group. a p value of Ͻ . was considered significant. (b) after min of hiri or sham laparotomy, mice were infected with ϫ pfu of lcmv we. liver lobes were harvested at day postinfection. viral titers were examined on mc cells using a focus-forming assay. data are expressed as means Ϯ the sem with n ϭ to mice/group. a p value of Ͻ . was considered significant. inhibition of nf-b activation impairs lps-and tnf-␣-stimulated usp induction. to investigate the link between lps/tnf-␣ stimulation and usp induction, inhibitors of nf-b activation were added to primary mouse hepatocytes for min prior to stimulation with ng of tnf-␣/ml or ng of lps/ml for h (the inhibitor names, targets, and concentrations used are given in table ). inhibitor-treated cells were also left unstimulated as a control. as a control for usp induction, hepatocytes were stimulated with u of ifn-␣/ml for h. usp (a) and il- ␤ (b) mrna expression levels were evaluated by pcr and normalized to the hprt housekeeping gene expression prior to determining the fold increase versus mock-treated samples. the data were generated from pooled triplicate experiments analyzed in duplicate. error bars represent the sem for duplicate pcrs. predictive of patients with chronic hcv infection who will not respond to ifn-based anti-hcv therapy ( ) . although we found that tnf-␣ hepatic mrna expression was increased in treatment nonresponders, it was relatively more increased in treatment responders ( ) . we suspect that whereas increased tnf-␣ does contribute to usp expression, there are other stimuli, for example, lps and perhaps the recently described interferon-lambda ( ) , that also modulate hepatic usp expression. we propose that the ultimate usp expression is due to the liver's coordinate response to multiple stimuli. the finding that hepatic usp expression is modulated by inflammatory stimuli is a new paradigm for the interaction of the liver inflammatory microenvironment and viral infection. taken together, these data may suggest that usp represents a good target for 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to interferon therapy silencing of usp potentiates the antiviral activity of interferon against hepatitis c virus infection ubp is a novel regulator of interferon signaling independent of its isg isopeptidase activity usp -based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response gene induction pathways mediated by distinct irfs during viral infection lipopolysaccharide activates the expression of isg -specific protease ubp via interferon regulatory factor tumor necrosis factor-alpha in liver ischemia/reperfusion injury the role of intestinal endotoxin in liver injury: a long and evolving history viral and host factors induce macrophage activation and loss of toll-like receptor tolerance in chronic hcv infection enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus complete replication of hepatitis c virus in cell culture tnf-alpha-induced sphingosine -phosphate inhibits apoptosis through a phosphatidylinositol -kinase/ akt pathway in human hepatocytes oncostatin m is a potent inducer of hepcidin, the iron regulatory hormone lipopolysaccharide, immune activation, and liver abnormalities in hiv/hepatitis b virus (hbv)-coinfected individuals receiving hbv-active combination antiretroviral therapy protein interferon-stimulated gene conjugation delays but does not overcome coronavirus proliferation in a model of fulminant hepatitis hepatitis c virus persisting after clinically apparent sustained virological response to antiviral therapy retains infectivity in vitro protein-kinase inhibitor-( - )-amide peptide analogs with standard and nonstandard amino-acid substitutions for phenylalanine- : inhibition of campdependent protein-kinase bone marrow-derived myofibroblasts promote colon tumorigenesis through the il- /jak /stat pathway nonmuscle myosin is regulated during smooth muscle contraction pd- is a specific inhibitor of the activation of mitogen-activated protein-kinase kinase in-vitro and in-vivo silibinin inhibits constitutive and tnf alpha-induced activation of nf-b and sensitizes human prostate carcinoma du cells to tnf alpha-induced apoptosis wortmannin is a potent phosphatidylinositol -kinase inhibitor: the role of phosphatidylinositol , , -trisphosphate in neutrophil responses protective strategies against ischemic injury of the liver interleukin- determines viral clearance or persistence in vivo quantification of lymphocytic choriomeningitis virus with an immunological focus assay in -well or -well plates dubbing down innate immunity malignant pirates of the immune system a comparison of selected mrna and protein abundances in human liver kinetic differences in the induction of interferon stimulated genes by interferon-alpha and interleukin b are altered by infection with hepatitis c virus nf-b in the liver-linking injury, fibrosis, and hepatocellular carcinoma successful prevention of hepatitis c virus (hcv) liver graft reinfection by silibinin mono-therapy differential in vitro effects of intravenous versus oral formulations of silibinin on the hcv life cycle and inflammation dexamethasone inhibits il- ␤ gene expression in lps-stimulated raw . cells by blocking nf-b/rel and ap- activation nf-b regulates il- ␤ transcription through a consensus nf-b binding site and a nonconsensus cre-like site usp inhibits nf-b and nfat activation during th differentiation by deubiquitinating the tak -tab complex essential function for the kinase tak in innate and adaptive immune responses disruption of tak in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis polymicrobial sepsis increases susceptibility to chronic viral infection and exacerbates cd ϩ t cell exhaustion recipient age affects long-term outcome and hepatitis c recurrence in old donor livers following transplantation prolonged rewarming time during allograft implantation predisposes to recurrent hepatitis c infection after liver transplantation the ubiquitin specific protease usp is necessary but not sufficient for a hepatocyte ifn refractory state: variable roles in type i and type iii ifn responsiveness blockade of janus kinase- signaling ameliorates mouse liver damage due to ischemia and reperfusion silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus we thank dong er zhang for the gift of the usp Ϫ/Ϫ mice. s.a.m. thanks the casl/cihr hepatology fellowship program and the national cihr research training program in hepatitis c for financial support. key: cord- -co s x authors: thukral, akanksha; ross, kathleen; hansen, chungyi; phanse, yashdeep; narasimhan, balaji; steinberg, howard; talaat, adel m. title: a single dose polyanhydride-based nanovaccine against paratuberculosis infection date: - - journal: npj vaccines doi: . /s - - -y sha: doc_id: cord_uid: co s x mycobacterium avium subsp. paratuberculosis (m. paratuberculosis) causes johne’s disease in ruminants and is characterized by chronic gastroenteritis leading to heavy economic losses to the dairy industry worldwide. the currently available vaccine (inactivated bacterin in oil base) is not effective in preventing pathogen shedding and is rarely used to control johne’s disease in dairy herds. to develop a better vaccine that can prevent the spread of johne’s disease, we utilized polyanhydride nanoparticles (pan) to encapsulate mycobacterial antigens composed of whole cell lysate (pan-lysate) and culture filtrate (pan-cf) of m. paratuberculosis. these nanoparticle-based vaccines (i.e., nanovaccines) were well tolerated in mice causing no inflammatory lesions at the site of injection. immunological assays demonstrated a substantial increase in the levels of antigen-specific t cell responses post-vaccination in the pan-cf vaccinated group as indicated by high percentages of triple cytokine (ifn-γ, il- , tnf-α) producing cd (+) t cells. following challenge, animals vaccinated with pan-cf continued to produce significant levels of double (ifn-γ, tnf-α) and single cytokine (ifn-γ) secreting cd (+) t cells compared with animals vaccinated with an inactivated vaccine. a significant reduction in bacterial load was observed in multiple organs of animals vaccinated with pan-cf, which is a clear indication of protection. overall, the use of polyanhydride nanovaccines resulted in development of protective and sustained immunity against johne’s disease, an approach that could be applied to counter other intracellular pathogens. m. paratuberculosis is the causative pathogen of johne's disease (jd) characterized by chronic gastroenteritis, diarrhea, weight loss and low milk yield in ruminants. while jd is a worldwide problem, its prevalence in the united states is estimated to be > % in dairy herds, causing a combined loss of $ - million to the us dairy industry. , the financial losses are incurred due to premature culling of infected animals, decreased milk production, and increased somatic cell infiltration in milk. , jd is a slowly progressing disease and can infect - % of the herd before becoming symptomatic in a single animal. , currently there is no treatment for jd and controlling the disease progression by culling the infected animals is very expensive, whereas vaccination offers a reasonable alternative. mycopar® (boehringer ingelheim) is an oil suspended, heat killed, whole cell vaccine licensed in the united states. however, mycopar® fails to completely protect against jd , and can cause severe inflammatory lesions at the site of injection. it also poses a health risk to vaccinators due to accidental inoculation, which leads to a chronic inflammatory reaction that potentially requires surgical intervention. given the challenges to control jd with the current vaccine, we directed our efforts to develop a more effective and safe vaccine against jd using polyanhydride nanoparticles (pan). an ideal vaccine should elicit a robust immune response without causing untoward reactions in the vaccinee or risk to the vaccinator. another important aspect of vaccine development against mycobacterial infection is its capability to elicit a polyfunctional t cell response with simultaneous production of pro-inflammatory cytokines by t cells. , to elicit robust immunity, antigens are often formulated with adjuvants to prolong their release and enhance their protective immunity. in this study, we used whole cell lysate and culture filtrate proteins encapsulated in biodegradable polyanhydride nanoparticles (adjuvant) that provide sustained release of m. paratuberculosis antigens by surface erosion. pan-based vaccines (i.e., nanovaccines) have been shown to impart long lasting protective immunity against several infectious diseases including influenza, pneumonic plague, respiratory syncytial virus, and pneumonia, using pathogen-specific protein antigens. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the amphiphilicity of the pan chemistry provides antigen stability and the copolymer composition enables sustained release of the encapsulated immunogens. , [ ] [ ] [ ] [ ] [ ] the small size (~ nm) and large surface area of the nanoparticles allows them to carry antigens across cellular membranes and deliver them to their targets. [ ] [ ] [ ] in addition, their molecular chemistry and size has pathogen-mimicking characteristics, allowing pan to be engulfed by, persist within, and subsequently stimulate antigen presenting cells (apcs). , polyanhydride particles on their own exhibit adjuvant-like properties by activating apcs - and inducing both humoral and cell-mediated immune responses; , [ ] [ ] [ ] formulating them with immune-stimulatory antigens results in protective immunity. , finally, these particles have been shown to be safe and induce less inflammation at the administration site compared with traditional adjuvants such as alum and incomplete freund's adjuvant. , m. paratuberculosis whole cell lysate and culture filtrate proteins have been shown to exhibit immunogenic properties and have previously been evaluated as a potential vaccine. , therefore, we utilized m. paratuberculosis antigens together with pan to formulate nanovaccines that can elicit robust and sustainable protective immune responses. in this study, a single, subcutaneous dose of nanovaccine in c bl/ mice was evaluated for protection against m. paratuberculosis jtc- challenge in comparison to both inactivated and live vaccine candidates. the live vaccine candidate lipn, developed previously by our group, was constructed by knockout of a fatty acid lipase/esterase gene lipn from m. paratuberculosis k . this gene was significantly upregulated in m. paratuberculosis shed in the cow feces, as revealed by transcriptional profiling. also, lipn mutant was analyzed and found to be attenuated in mice as indicated by reduced histopathological lesions and colonization of the liver. its protective efficacy has been observed in goats challenged by virulent m. paratuberculosis strain. the study was conducted in two phases, viz: trial i and trial ii. in the trial i studies, the focus was on the safety of the nanovaccine formulations while in the trial ii studies, the focus was on the efficacy of nanovaccine formulations (fig. ) . scanning electron photomicrographs of m. paratuberculosis lysateencapsulated (pan-lysate) and culture-filtrate (pan-cf)-encapsulated polyanhydride nanoparticles showed similar spherical morphology and size as blank (i.e., empty) nanoparticles, indicating that antigen encapsulation did not change the average diameter, which was ca. nm (fig. ) . the encapsulation efficiency of the lysate into the nanoparticles was . ± . % and that of the culture filtrate was . ± . % and . wt% of the protein content of the lysate or culture filtrate (cf) was encapsulated into the particles. to evaluate nanovaccine safety, we monitored immunized mice on a daily basis. animals vaccinated with mycopar® gradually developed an abscess at the injection site which progressed and persisted throughout the study (supplemental fig. ) . on the other hand, no lesions were observed in nanovaccine-immunized and live attenuated (lipn) vaccine immunized animals. at weeks post-vaccination (wpv) and before any challenge, histopathology of vaccinated mice groups demonstrated lymphoid depletion in the spleens of animals immunized with the commercial vaccine, while minimal to moderate lymphocytic infiltration and granulomatous inflammation was observed in the livers of the rest of vaccinated animals regardless of formulation, which is indicative of induced immunity. no pathology was observed in the negative control group (pbsvaccinated mice). pre-challenge immune responses for trial i (i.e., safety study), the t cell response was evaluated at weeks post-vaccination by performing ifn-γ elisa on spleen derived lymphocytes (described in methods) (supplemental fig. ). the mycopar-vaccinated animals showed significantly higher ifn-γ levels than the rest of the groups (***p < . ) (supplemental fig. a ). for trial ii (i.e., efficacy study), spleen derived lymphocytes were stained with various antibody markers and analyzed using flow cytometry. we assessed antigen specific, polyfunctional t cell responses by multi-parametric flow-cytometry. in this analysis, lipn vaccinated mice showed significantly higher percentage of double cytokine (ifn-γ,tnf-α) and single cytokine (ifn-γ) producing cd + t cells as well cd + t cells in comparison with both pbs and mycopar® vaccinated animals ( fig. a, b) . interestingly, the percentage of triple cytokine producing (ifn-γ, il- , tnf-α) cd + t cells was significantly higher in mice immunized with pan-cf when compared with pbs and mycopar®. also, the pan-cf vaccinated animals exhibited significantly higher double (ifn-γ, il- ) cytokine secreting cd + t cells in comparison with pbs vaccinated mice (fig. b) . of note was the breadth of the polyfunctional cd + t cell response observed from mice immunized with pan-cf. in contrast to all the other treatment groups where the majority of the cd + t cells was dominated by ifn-γ secreting single positive cells, pan-cf vaccinated mice showed a broader profile of triple, double and single cytokine secreting cd + t cells (fig. b) . post-challenge immune responses to evaluate t cell response in trial i/safety study ifn-γ elisa was performed and result of which depicted no significant differences in ifn-γ levels among the groups (supplemental fig. b ) while at weeks post challenge mycopar and pan-lysate vaccinated animals showed significantly higher ifn-γ levels as compared with control animals given pbs (supplemental fig. c ). t cell responses for the trial ii/vaccine efficacy study were evaluated by flow cytometry for which spleens from vaccinated mice were collected at and weeks post-challenge (wpc). at wpc, multiparametric flow cytometry analysis indicated that mice immunized with pan-cf elicited a significantly higher percent of antigen specific double cytokine (ifn-γ, tnf-α + ) and single cytokine (ifn-γ) producing cd + t cells compared with non-vaccinated and mycopar® vaccinated mice (fig. ). in addition, pan-cf and mycopar®-vaccinated animals also displayed low levels of triple cytokine secreting cd + t cells. similar to the pre-challenge cd + t immune response, mice immunized with pan-cf showed a pre-challenge immune response specific to lysate of m. paratuberculosis. c bl/ mice (n = ) were immunized with various vaccine groups and wpv, five mice from each group were euthanized. spleens were harvested; lymphocytes were isolated and stimulated with the m. paratuberculosis lysate for h. cells were then stained for cd + (a) and cd + (b) cell surface markers and intracellular cytokines. the total percentage of t cells secreting particular cytokines are indicated below each pie chart (denoted by t = number). the error bars show the standard error of the mean for five individually analyzed mice. * indicates p < . ; ** indicates p < . . * denotes comparison with pbs while # denotes comparison with mycopar®. results were expressed as the increase in the percentage of the cells with positive staining relative to that of an unstimulated sample stained with the same antibody. early cellular responses in vaccine groups following challenge with a wild type strain of m. paratuberculosis. six to eight week-old c bl/ mice were immunized with various vaccine candidates. at wpv they were challenged with m. paratuberculosis jtc- and euthanized weeks later ( wpc). the lymphocytes were isolated from the spleens and stimulated with whole cell lysate of m. paratuberculosis for h. cells were then stained for cd + (a) and cd + (b) cell surface markers and intracellular cytokines and were measured by flow cytometry. the total percentage of t cells secreting particular cytokines are indicated below each pie chart (denoted by t = number). the error bars show the standard error of the mean for five individually analyzed mice. * indicates p < . ; ** indicates p < . . * denotes comparison with pbs while # denotes comparison with mycopar®. results were expressed as the increase in the percentage of the cells with positive staining relative to that of an unstimulated sample stained with the same antibody. broader profile of cytokine secreting cells at wpc (see pie chart in fig. ). the cumulative percentage of cd + t cells that were either triple, double or single cytokine secretors was also higher in animals vaccinated with pan-cf (total percentage of cells secreting cytokine; t = . ) in contrast to that in animals vaccinated with the other formulations, indicating the robustness of the induced cd + t cell response. also, animals receiving pan-cf + lysate showed significantly higher levels of double cytokine secreting (ifn-γ, il- ) cd + t cells in comparison with animals that received pbs and significantly higher levels of double cytokine secreting (ifn-γ, il- ) cd + t cells in comparison with animals receiving both pbs and mycopar®. at wpc, the percentages of mycobacterial antigen-specific double positive cd + t and cd + t cells (ifn-γ + il- + ) were significantly higher in pan-cf vaccinated mice compared with pbs-vaccinated mice (supplemental fig. ). protection against challenge with virulent strains of m. paratuberculosis in spite of the fact that safety was the main goal of the trial i studies, we were able to evaluate the protective efficacy of each vaccine candidate at wpc when bacterial colonization remained similar in organs of all vaccinated groups (supplemental fig. ). at wpc, the bacterial load was significantly lower in the spleens and mesenteric lymph nodes of mycopar and pan-lysate and lysate vaccinated groups in comparison with non-vaccinated group. livers of mycopar and pan-lysate vaccine group showed significant reduction in comparison with pbs group (supplemental fig. ). to better evaluate protection offered by each vaccine formulation in trial ii/efficacy study against recent isolates of m. paratuberculosis, we quantified the level of bacterial tissue colonization following a challenge with m. paratuberculosis jtc , a clinical isolate of the bovine origin. as expected, mice that received pbs had high levels of bacterial load in all the tissues studied (liver, spleen, intestine and mesenteric lymph node) at wpc (fig. ). all the vaccinated mice showed significantly lower bacterial burden in the liver in comparison with pbs-treated mice (fig. a) . bacterial load was significantly lower in the spleens of all vaccine groups (including mycopar®, lipn, and pan-lysate) with a two-log reduction observed in the spleens of animals vaccinated with pan-cf in comparison with the load in the spleens of animals that received pbs. the pan-cf immunized mice also showed significant reduction in bacterial load burden compared with mycopar® vaccinated mice (fig. b) . interestingly, mycopar® did not provide any protection in terms of a reduced bacterial load in the small intestine (fig. c ) compared with the pbs-treated animals. in contrast, mice vaccinated with lipn mutant, pan-cf + lysate and pan-cf had significantly lower mycobacterial colonization levels in the small intestine. all mouse groups displayed a reduction in bacterial colonization of the mesenteric lymph nodes compared with the pbs control (fig. c, d) . at wpc, no significant differences were observed in the bacterial colonization in the mouse tissues among any of the vaccine groups, including pbs (supplemental fig. ). to analyze the level of tissue damage induced by challenge with the wild type m. paratuberculosis jtc strain, we performed histopathology of the main body organs in all vaccine groups. at wpc, all animals administered pbs had granulomatous inflammation in the liver while only % of the animals receiving the pan-lysate, pan-cf + lysate, or lipn vaccine exhibited minimal to mild pathology (table ; fig. ). the granulomatous lesions in livers involved variable size aggregates of lymphocytes with macrophages visible in some lesions. in the mycopar® vaccinated group, % of the animals had minimal to moderate granulomatous inflammation in the liver (table ) . at wpc, granulomatous and lymphocytic inflammation were larger in size and involved more sections of the liver in all groups with no significant differences among vaccine groups. despite many challenges and shortcomings, vaccination against map is still considered to be the most efective strategy to curb johne's disease. the commercially available vaccines, such as mycopar®, gudair®, and silirum®, are comprised of whole inactivated map and provide moderate protection at best. , the limited benefits provided by these vaccines are overshadowed by their drawbacks, which include granulomatous reaction at the injection site and poor protection against bacterial tissue colonization and shedding. , in this report, we utilized the polyanhydride nanovaccine platform technology to design a safer and more efficacious vaccine aginst jd. previous studies have shown that pans can encapuslate a diverse array of biologics including subunit proteins, peptides, and antibiotics. , here we demonstrate that polyanhydride nanoparticles can encapsulate complex payloads such as m. paratuberculosis proteins (from whole cell lysates and culture filtrate) and induce effective immune responses in mice indicating that the immunogenicity of the encapsulated cargo is intact. this novel delivery approach to inactivated vaccine improve their efficacy but maintain their safety profile. the strategy of using m. paratuberculosis proteins in both encapsulated and soluble form enables primed (provided by the soluble protein) and sustained (provided by the nanoparticleencapsulated protein) immune responses. , , consistent with previous studies that examined the safety of inactivated vaccines (e.g., mycopar®), , we observed abscess-like lesions at the inoculation site in several mice vaccinated with mycopar® (supplemental fig. ) . in contrast, no adverse injection site reactions were observed in pan-vaccinated mice, clearly indicating their excellent safety profile. these results add to the body of literature on the minimal reacotogenicity of polyahydride nanovaccines, as demonstrated previously. , although oral infection and mucosal immunizations against m. paratuberculosis could be more benificial in the target host (cattle), we selected parental infection and subcutaenous injection of mice to test vaccine formulas using an entery level model for vaccine testing against paratuberculosis. for both the murine and bovine models of paratuberculosis, cd + and cd + effector t cells play a crucial role in eliciting protective cell mediated immunity against mycobacterial infection. , effector t cells producing multiple pro-inflammatory cytokines such as ifnγ, tnf-α, and il- have been shown to be associated with protection against various intracellular pathogens including m. tuberculosis. , even though the exact mechanism(s) of polyfunctional t cell mediated protection are still not clear, it has been shown that two or more cytokines can work synergistically to control infection, as in the case of a closely related mycobacterium, m. tuberculosis and leishmania spp. indeed, pan-cf vaccinated (prechallenged) mice exhibited polyfunctional cd + t cells (ifn-γ+, il- +, tnfα+) but showed superior protection (significantly lower bacterial burden) compared with both nonvaccinated (all organs) and mycopar (only spleen) vaccinated groups. we hypothesize that protection can be attributed to a robust polyfunctional cd + t cell response, especially for the nanovaccine formulas. recently, nano-peptide based adjuvant enhanced bcg primed immune response by induction of a robust polyfunctional cd + t cells. overall, the presented analyses (figs , ) clearly indicated robust induction of polyfunctional t cell responses in mice immunized by the nanovaccines as depicted by higher triple and double cytokine producing cd + t cells when compared with non-vaccinated (pbs) and mycopar®-vaccinated mice. it is noteworthy to mention here that for m. tuberculosis vaccines, polyfunctional t cells were mainly cd + unlike the predominantly cd + population observed in animals vaccinated with the pan-cf group. this is likely induced by the inclusion of polyanhydride nanoparticles in the pan-cf group, consistent with previous observations. , the nanoparticle chemistry used in this study (i.e., : cpteg: cph) was rationally selected based on previous reports showing its potency as an adjuvant as exhibited by robust induction of cellular immune responses. similarly, in this study pan-cf vaccinated mice at pre-challenge not only induced a polyfunctional cd + t cell response but also had more breadth as indicated by induction of more types of cytokine secreting cells. the robust polyfunctional t cell response observed in pan-cf vaccinated animals for wpc may be attributed to sustained release of antigen from polyanhydride particles. remarkably, pan-cf + lysate was able to produce double cytokine (ifn-γ + il- + ) cd + t cells significantly higher than pbs and mycopar® and exhibited lower bacterial burden in all the mice tissues as compared with non-vaccinated mice (fig. ). most importantly, pan-cf vaccinated mice had the lowest bacterial burden in three out of the four tissues evaluated. moreover, despite this significant reduction was not maintained at wpc, the level of polyfunctional t cells remained robust at this prolonged time. this indicates that several other paramters (beyond the scope of present study), such as th- induction, tissue homing properties of t cells, memory and effector phenotype could also play critical roles. , these parameters deserve more attention, especially as we advance vaccine testing to ruminant models. as expected, histological lesions post-challenge correlated with protection based on bacterial burden in body organs. for example, granulomatous lesions that are typical of mycobacterial infection were seen in the liver of pbs and mycopar®-vaccinated animals in higher frequency than in the lipn or pan-vaccinated animals ( table ) , consistent with the levels of mycobacterial colonization. similar to other vaccine candidates (lipn mutant), pan-based vaccines were able to significantly lower m. paratuberculosis levels in the liver, spleen and lymph nodes but did not prevent dissemination of infection. approaches focused on developing significant mucosal immunity at the main site of m. paratuberculosis entry (intestine) could definitely reduce organ dissemination of the infection. overall, the nanovaccines and lipn mutant were able to impart superior protective imunity against m. paratuberculosis challenge in mice compared with a commercial vaccine, mycopar®. further, in contrast to mycopar®, nanovaccines were also well tolerated and did not induce any adverse reactions at the site of injection. both these features makes pan nanovaccines ideal for further testing in larger animals such as goats and cattle following mucosal immunization and oral infections, to memic natural infection. although promising, nanovaccines have room for improvement before becoming suitable for field applications. for example, methods to improve the encapsulation efficiency of complex protein mixtures like whole cell lysate into pan are highly desirable and if developed, could improve vaccine production and facilitate immunization. such strategies could open the door for the development of more effective vaccines targeting other intracellular pathogens such as m. tuberculosis. for safety experiments, m. paratuberculosis -k was used to prepare lysate while for efficacy experiments, m. paratuberculosis strain jtc- was used to derive lysate and culture-filtrate (cf) proteins for vaccine formulation. both isolates belong to c-type of m. paratuberculosis with almost identical genomes. the same strains were used for the animal challenge studies, as described below. the strains were grown in middlebrooks h broth (difco, sparks, md) supplemented with . % glycerol (v/v) and % (v/v) adc (albumin, dextrose, catalase) or tween and mg/ml mycobactin j (allied monitor, fayette, mo) in a shaking incubator at °c until it reached the log phase (od = . - . ). the m. paratuberculosis field isolate, jtc- , was sub-cultured in watson reid medium, filter (nalgene). further, it was size fractionated by ultrafiltration (corning ultra spin columns, mwco) and the filtered volume retained on the membrane was dialyzed twice against mm phosphate buffered saline (pbs) (ph . ). the concentrated culture filtrate proteins were quantified using bicinchoninic acid kit (pierce) and stored at − °c. to obtain the lysate, the bacterial cell pellet was resuspended in protein lysis buffer ( mm tris-cl, mm nacl, mm mgcl , mm pmsf, complete ultra protease inhibitor cocktail, ph . ) and placed in microcentrifuge tubes containing . -mm zirconia/glass beads. tubes were shaken in the mini bead-beater cell disrupter for four s pulses followed by -min incubation on ice. cellular debris and beads were pelleted by centrifugation at × g for min. the supernatant was quantified for protein using bicinchoninic acid kit (thermo fisher scientific, rockford, il) and stored at − °c. diacids of , -bis(p-carboxyphenoxy)- , -dioxaoctane (cpteg) and , -bis (p-carboxyphenoxy)hexane (cph) were synthesized as described in detail. , next, melt polycondensation was used to synthesize : cpteg:cph copolymer. the purity and molecular weight of the copolymer were verified using h nuclear magnetic resonance spectroscopy (vxr mhz, varian, palo alto, ca) before proceeding to nanoparticle synthesis. nanoparticles were synthesized using solid-oil-oil double emulsion nanoprecipitation. briefly, m. paratuberculosis lysate and culture filtrate proteins were dialyzed to nanopure water by using k mwco spin-x® uf concentrators (corning, corning, ny) and lyophilized overnight. the : cpteg:cph polymer ( mg/ml) containing . wt% proteins was dissolved in methylene chloride. the solution was sonicated for s to ensure uniform distribution of the protein throughout the solution. particles were precipitated by pouring the solution into chilled pentane ( : methylene chloride:pentane) and collected via vacuum filtration. nanoparticle size and morphology were characterized via scanning electron microscopy (fei quanta , fei, hillsboro, or). the encapsulation efficiency was determined by incubating the nanoparticles in pbs at °c. the released protein was quantified via a micro-bicinchoninic assay (pierce) and compared with the amount of protein theoretically encapsulated. the final nanovaccine formulation was a combination of free protein and nanoparticle-encapsulated protein. a total of mg of nanoparticles encapsulating µg protein was suspended in μl pbs containing µg free protein per mouse. the mixture was sonicated for s on ice to disperse any nanoparticle aggregates prior to administration. female c bl/ mice ( - weeks of age) were obtained from taconic inc. and maintained in bio-safety level- containment. all procedures were in compliance with institutional animal care and use committee, university of wisconsin, madison. experiments were run in two trials ( table , n = / group) with trials i and ii comprising of four and six vaccine groups, respectively. animals were vaccinated subcutaneously as per the experimental design shown in table . in all experiments, five mice from each group were sacrificed at weeks post-vaccination. the remaining animals in each group were challenged with cfu of m. paratuberculosis jtc- in μl of pbs, injected intraperitoneally (ip). the subcutaneous vaccination regime and intraperitoneal challenge model has been successfully employed before in mouse models and the resulting data translated well to ruminant systems such as in goats. , , the dose was confirmed by plating the serially diluted challenge inoculums on h plates. mice were monitored daily for adverse reaction(s) from vaccination and for the progression of infection. at weeks (trial i), weeks (trials i and ii) and weeks (trial ii) post-challenge, mice (n = ) were sacrificed and the liver, spleen, small intestine and mesenteric lymph nodes were harvested from each sacrificed mouse in order to quantify the bacterial burden. organs were homogenized in ml pbs and undiluted and -fold serial diluted samples were plated onto antibiotic free and selective media (hygromycin μg/ml) to differentiate between lipn and challenge strain. when the selective media were not used, organs were plated onto standard h plates supplemented with adc, mycobactin-j, and vancomycin ( mg/ml), amphotericin b ( mg/ml), and nalidixic acid ( mg/ml) to reduce bacterial and fungal contamination. finally, tissue sections were collected for histopathology and stained with hematoxylin and eosin. slides were scored by a trained pathologist blinded to the samples. the animal experimental design is shown in fig. . spleens from five animals/group were aseptically harvested and placed in rpmi (corning, manassas, va) supplemented with % fbs (atlanta biological, lawrenceville, ga), % l-glutamine (gibco, grand island, ny), % penicillin-streptomycin (mediatech, inc. manassas, va) and % nonessential amino acids (gibco). spleens were pressed against the wire mesh screens to isolate splenocytes. the cells were washed with rpmi and resuspended in - ml of rbc lysis buffer (tris buffered ammonium chloride) for min, washed, and resuspended in rpmi with % fbs. cells were counted using trypan blue dye to assess viability. a total of cells/ well were seeded into -well round bottom plates and stimulated with µg/ml of whole cell lysate of m. paratuberculosis k (trial i) or m. paratuberculosis jtc- (trial ii) and u/ml il- (bd biosciences, san jose, ca). a control of unstimulated cells from each sample was also plated and treated with μl of media and u/ml il- . plates were incubated for h at °c, % co followed by addition of golgi-plug to each well and incubated for an additional h. cells were harvested by centrifugation and stained with immune markers to be analyzed by flow cytometry and the supernatant was used to detect ifn-γ by elisa. supernatant from the stimulated splenocytes was collected and tested for ifn-γ levels using mouse ifn-γ elisa max tm deluxe kit (biolegend, san diego, ca) following the manufacturer's instructions. in brief, -well plates (maxisorp-immuno plates; nunc) were coated overnight with capture antibody (monoclonal capture antibody specific for mouse ifn-γ) at : dilutions in the coating buffer at °c. plates were washed with pbst ( mm nacl, . mm kcl, . mm na hpo , . mm kh po , ph . , . % (v/v) tween ), blocked with μl of assay diluent, and incubated on a shaking plate for h at room temperature. plates were washed five times with the pbst and μl of sample and standards were added to the appropriate wells and incubated for h on the shaking plate. plates were washed with pbst and μl of detection antibody (biotinylated rat monoclonal anti-mouse antibody) was added and incubated for h at room temperature. after three more washes, μl of avidin-horse radish peroxidase conjugated solution was added to each well and incubated for min on the shaking incubator. after a last few washes, μl of freshly made ', , , ' -tetramethylbenzidine (tmb) substrate solution was added to the wells and incubated for min in the dark. the reaction was stopped by adding μl of stop solution ( n h so ). the plates were read at a wavelength of nm and analyzed with softmax pro software (molecular devices, sunnyvale, ca). splenocytes from five individual mice per group were counted and × cells/well were plated in -well plates. stimulated cells were harvested by centrifuging at × g for min at °c. supernatants were removed and a. thukral et al. cells were washed with pbs twice. fixable viability dye efluor (ebioscience, san diego, ca) was diluted in pbs ( / ) and added to each well except unstained control, incubated for min in dark at - °c and cd /cd receptors were blocked with fc block (bd pharmingen, san diego, ca). cell surfaces were stained with cocktail of buv conjugated anti-cd antibody, clone gk . (bd pharmingen); buv conjugated anti-cd , clone - . (bd pharmingen); bv conjugated anti-cd , clone pc (biolegend) and incubated for min in dark at °c. cells were washed with cold facs buffer twice and resuspended in μl of fixation/permeabilization working solution (foxp staining buffer set, ebioscience, san diego, ca). after h incubation, cells were washed with x permeabilization buffer and stained intracellularly with apc conjugated anti-ifn-γ, clone xmg . (bd pharmingen); pe conjugated anti-il- (bd pharmingen); pecy conjugated anti-tnfα, clone mp -xt (ebioscience); and alexa fluor conjugated anti-foxp , clone fjk s (ebioscience). cells were analyzed using bd facscalibur and data were analyzed using flowjo software (flowjo, llc, ashland, or). results were expressed as the increase in the percentage of the cells with positive staining relative to that of an unstimulated sample stained with the same antibody. statistical analysis was performed using graphpad prism (la jolla, ca). data were analyzed using one-way anova followed by tukey's multiple comparison. results with p < . or better were considered significant. all research reported here was conducted in accordance with all relevant guidelines and procedures, and that the work was approved by the university of wisconsin-madison. further information on research design is available in the nature research reporting summary linked to this article. effect of paratuberculosis on culling, 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this report or the accompanied supplemental tables and figures.received: february ; accepted: january ; a.m.t. perceived the original idea and supervised the whole project. b.n. is the inventor of the p.a.n. delivery system and supervised the nanovaccine synthesis and charcaterization. a.t., k.r., c.h. and y.p. conducted all of the experiments while h.s. was responsible for the histology analysis. all authors contributed equally to the writing and editing of the paper. dr. adel m. talaat has an ownership interest in pan genome systems, inc, which is working in the area of animal vaccine development. also, dr. yashdeep phanse is currently employed by the same company. supplementary information is available for this paper at https://doi.org/ . / s - - -y.correspondence and requests for materials should be addressed to a.m.t. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -nrq ncdf authors: mlera, luwanika; melik, wessam; bloom, marshall e. title: the role of viral persistence in flavivirus biology date: - - journal: pathogens and disease doi: . / - x. sha: doc_id: cord_uid: nrq ncdf in nature, vector-borne flaviviruses are persistently cycled between either the tick or mosquito vector and small mammals such as rodents, skunks, and swine. these viruses account for considerable human morbidity and mortality worldwide. increasing and substantial evidence of viral persistence in humans, which includes the isolation of rna by rt-pcr and infectious virus by culture, continues to be reported. viral persistence can also be established in vitro in various human, animal, arachnid and insect cell lines in culture. although some research has focused on the potential roles of defective virus particles, evasion of the immune response through the manipulation of autophagy and/or apoptosis, the precise mechanism of flavivirus persistence is still not well understood. we propose additional research for further understanding of how viral persistence is established in different systems. avenues for additional studies include determining if the multifunctional flavivirus protein ns has a role in viral persistence, the development of relevant animal models of viral persistence as well as investigating the host responses that allow vector borne flavivirus replication without detrimental effects on infected cells. such studies might shed more light on the viral-host relationships, and could be used to unravel the mechanisms for establishment of persistence. defining mechanisms of viral persistence will be critical for understanding vector-borne flavivirus infections. these viral infections account for considerable human morbidity and mortality worldwide. furthermore, incidence is increasing and infections are being appreciated in previously nonendemic geographic locations. prominent vector-borne flaviviruses (vbfvs) associated with significant human infections include both tick-borne and mosquito-borne agents. the tick-borne flaviviruses (tbfvs) are exemplified by the tick-borne encephalitis virus (tbev) sero-complex group, omsk hemorrhagic fever virus, kyasanur forest disease virus, alkhurma virus, powassan virus (powv), and deer tick virus (dtv), the latter two occurring in the united states (holbrook et al., ; brackney et al., b; ebel, ) . the mosquito-borne flaviviruses (mbfvs) are perhaps better known and include yellow fever virus (yfv), west nile virus (wnv), japanese encephalitis virus (jev), and dengue virus (denv) serotypes - . infections with all of these viruses can lead to severe disease, prolonged debilitating neurological sequelae, hemorrhagic fever, and/ or death in some cases (solomon et al., ; glass et al., ; haglund & gunther, ; madden, ; van gerpen, ; carson et al., ) . viral persistence is a hallmark of the ecology of vbfvs. both tbfvs and mbfvs are cycled between arthropod and vertebrate hosts (figs and ) , and in many cases, they are maintained without deleterious effects on the hosts. in nature, tbfvs, such as powv and tbev, alternately infect small vertebrates such as rodents, hares, some carnivores, and a range of hard-bodied (ixodid) ticks, although recent evidence suggests that the soft-bodied ornithodoros (argasid) ticks can also support tbfv (rajagopalan et al., ; charrel et al., ) . similarly, mbfvs, such as wnv and jev, primarily alternate in nature between small mammals, birds, and mosquitoes ( fig. ). in addition, there is evidence that mbfv and tbfv persistence also occurs in humans, and persistence in cell culture is well documented (poidinger et al., ; lancaster et al., ; bugrysheva et al., ; farfan-ale et al., ; murray et al., ) . although the hosts for the vbfvs are highly varied, the genomic and structural organization of the viruses themselves is remarkably similar. all flaviviruses are spherical, enveloped particles that contain a genome of (+) ssrna measuring approximately kb. the genome also functions as mrna and encodes a single polyprotein that is cleaved into proteins (table ) . reasonable progress has been made toward understanding how these viruses replicate (chambers et al., ; mandl, ; miorin et al., ; tuplin et al., ; pierson & diamond, ; brinton, ; pierson & kielian, ) . we present a simplified overview of the general aspects of a vbfv replication cycle in fig. . the individual proteins are three structural proteins: capsid (c), precursor membrane/mem- fig. flavivirus maintenance and transmission cycle in ticks and vertebrate hosts. ticks are crucial for viral persistence as they remain infected once they acquire viral infection. infected ticks are capable of transmitting tbfvs to other ticks when they feed in close proximity on the same animal, as well as the different stages of the tick life cycle. tbfvs persist also in a cycle between small mammals (e.g. rodents) and the ticks that feed on them. large mammals and humans tend to be incidental, dead-end hosts. fig. a representation of a mosquito-borne flavivirus amplification and transmission cycle. wnv is cycled between the mosquito and avian hosts that play an amplification and maintenance role. swine are important amplifying hosts for jev, and mosquitoes that acquire blood meals on infected pigs can become infected and transmit the virus. similar to tbfvs, mbfv infection in humans and large animals, such as horses, is accidental. the flavivirus proteins, untranslated regions (utrs), and their known functions the utr contains conserved rna stem loops (sl), cis-elements located upstream and downstream of the aug region (uar and dar, respectively), and complementary sequence (cs) sponsoring cyclization of the genome by interacting with the utr to form a 'panhandle' (alvarez et al., ; villordo & gamarnik, ; friebe & harris, ; friebe et al., ) . the 'panhandle' structure is essential for certain steps in the flavivirus replication cycle such as replication/translation and viral assembly (gritsun & gould, b ). sl - or (sl-a and sl-b) is located within the utr, whereas the sl - is extended and situated in the n-terminal of the open reading frame orf (alvarez et al., ; lodeiro et al., ; villordo & gamarnik, ; friebe & harris, ; friebe et al., ) . the sl structure is essential for viral replication and acts as a promoter which is targeted initially by ns and then delivered to the end via cyclization (filomatori et al., ; zhang et al., ; lodeiro et al., ) although there is low nucleotide conservation between flaviviruses and different cs homology (hahn et al., ) , the utrs of tbfvs share the same genomic organization as the mbfvs (kofler et al., ) structural proteins capsid (c) kda, aa cytosol/nucleus the capsid protein is a dimeric alpha-helical (jones et al., ) protein and assembles into an icosahedral structure, measuring nm in diameter, which initiates encapsidation of the associated genomic rna in virus-induced membrane invaginations of the er (welsch et al., ). the c protein is also a pro-apoptotic factor in various cell lines (yang et al., (yang et al., , oh et al., ; netsawang et al., ; morchang et al., ) . c of jev was reported to limit viral neurovirulence (mori et al., ) the c-encoding nucleotide sequence of tbev contains conserved rna structures that function as replication enhancer elements (tuplin et al., ) † precursor membrane (prm) - , kda, aa er lumen during virion assembly, the prm forms a heterodimer with the e protein and acts as a chaperone for correct e protein folding (kuhn et al., ; lorenz et al., ; zhang et al., ) . in the final step of virion maturation in the trans-golgi network prior, to viral release, the precursor is cleaved by furin and only the c-terminal region (m) is retained in the viral membrane (heinz et al., ; elshuber & mandl, ; lindenbach et al., ) . prm protein of jev also limits viral neurovirulence (kim et al., ) † (bressanelli et al., ) . the glycoprotein forms 'head to tail' homodimers that completely cover the surface of the mature icosahedral virions. it is also responsible for receptor binding, which leads to virus internalization by clathrin-mediated endocytosis (lindenbach et al., ) . the e protein contains three b-sheets domains (di-diii): dii includes the membrane fusion peptide (beasley & barrett, ; bressanelli et al., ; sukupolvi-petty et al., ) ; diii is associated with receptor binding and is also a target for several neutralizing monoclonal antibodies. crystal structures of the e protein from several flaviviruses all show similar secondary structure and domain organization (modis et al., (modis et al., , zhang et al., ; kanai et al., ; nybakken et al., ) . the e protein is important in determining neuroinvasiveness and neurovirulence for several mbfvs (cecilia & gould, ; hiramatsu et al., ; ni & barrett, ; sanchez & ruiz, ; chambers et al., ; beasley et al., ; lee et al., lee et al., , bordignon et al., ) . for wnv, jev, and denv, mutations in the e protein (located in the lateral surface of diii and the base of dii) affect fusion and receptor binding with target cells (lee et al., ) the e protein was first described in and later has been extensively defined and crystalized for many other flaviviruses (rey et al., ) . tbfv e protein is important for determining neurovirulence and neuroinvasiveness (holzmann et al., ; mcminn, ; beasley et al., ; mandl, ; engel et al., ) nonstructural proteins ns - / - kda/ aa er lumen/cytosol/ secreted low-resolution structural studies found the ns structure to be an open barrel configuration with d symmetry measuring nm by . nm and with a central cavity approximately . nm in diameter (gutsche et al., ; edeling et al., ) . all flavivirus ns genes share a conserved degree of homology. ns is found at different cellular locations such as vesicular compartments associated with the cell membrane or on the cell surface and is also secreted as an extracellular † westaway et al., ; lindenbach et al., ) . each subunit forms a homodimer located in the er lumen, and it has been shown to colocalize with viral dsrna. secreted and cell surface associated ns is immunogenic and induces an antibody response that can serve as an important diagnosis marker (falgout et al., ; avirutnan et al., ; sun et al., ) . ns is also implicated in immune evasion functions as it activates human complement and induces host vascular leakage by specific inhibition of the classical and lectin pathways of complement activation. this occurs through a direct interaction with complement components c and c s (avirutnan et al., ) . denv, wnv, and yfv ns are shown to limit c b deposition and c convertase activity by enhancing cleavage of c through the recruitment of the complement-specific protease c s (avirutnan et al., ) . ns a (blitvich et al., (blitvich et al., , (assenberg et al., ). ns is a highly conserved and multifunctional protein: the protein has protease activity in one domain, rna-stimulated ntpase activity and rna triphosphate activity (rtpase) in the other domain (chambers et al., ; valle & falgout, ; lescar et al., ) . ns protein is an essential member of the rc and is activated in association with ns to bind the genomic rna sl prior to replication (chen et al., ; mackenzie et al., ; lindenbach et al., ) . beside its essential role in the viral replication cycle, ns can also induce apoptosis (shafee & abubakar, ; ramanathan et al., ; yang et al., ; yiang et al., ) . ns is involved in the inhibition of the transcriptional factor ap signaling pathway, which also directly affects apoptosis (lin et al., ) . studies on neurovirulence associated with denv- ns have shown that mutations in ns (leu ser) enhance the ability of replication in cns causing leptomeningitis and encephalitis in mice (bordignon et al., ) . this mutation was also shown to induce cell death in denv- -infected cells (duarte dos santos et al., ) . the ns protein can be targeted by antiviral drugs such as ivermectin, ribavirin as inhibitors of the atpase activity of helicases (mastrangelo et al., ) the (lindenbach et al., ) . its interaction with ns is essential for viral rna replication as ns a acts as a docking site (bartenschlager et al., ; miller et al., ) . ns a interacts with the polypyrimidine tract-binding protein (ptb), with the latter being implicated in translation, transcription, and viral processes for various viruses (bieleski et al., ; florez et al., ; shi & lai, ) including the role in regulation of hcv replication (tischendorf et al., ; domitrovich et al., ; aizaki et al., ; chang & luo, ) . ns a-ptb plays an indirect role in stabilization of the viral genome dsrna intermediates by binding to host cell ptb protein in a similar manner as stabilizing wnv rna synthesis by the elongation factor alpha (davis et al., ; jiang et al., ) . ns a together with ns b and ns is also responsible for inducing the rearrangement of the host er membrane leading to the formation of virus-induced membranous spherules and vesicles encasing the dsrna and rc; this is thought to minimize exposure of the replicating rna to innate immune sensors such as retinoic acid-inducible gene-i, rig-i roosendaal et al., ; miller et al., ) . the mature ns a can also induce the pi k-dependent autophagy signaling, which in turn lead to protection from camptothecin-and staurosporine-induced cell death (mclean et al., ) . the role of ns a in the up-regulation of the autophagy and er rearrangement makes the protein a potential target for antiviral drug development † k - , kda, aa complete cleavage of the vbfv polyprotein generates an er-spanning k peptide located between ns a and ns b (lin et al., ; roosendaal et al., ; miller et al., ) . the k peptide functions as a signal peptide for ns b and might play a role in enhancing viral replication by conferring resistance to lycorine, a suppressor of flavivirus rna synthesis (zou et al., ) † miller et al., ) . ns b appears also to be involved in er rearrangement and modification during viral replication (westaway et al., ) . the ns b protein of wnv, jev, and denv inhibits type i interferon (ifn-a/b) response by blocking the phosphorylation of stat (muñoz-jord an et al., , lin et al., ; liu et al., ; evans & seeger, ) . the same is true for kunv and wnv strain ny , which blocks the activation of stat / stat and its translocation to the nucleus (liu et al., ) . these events disrupt the host immune responses by blocking the ifnalpha/beta/gamma pathways † ns - , kda, aa cytosol/er/nucleus ns is the largest and most conserved among the vbfv proteins. ns primarily functions as the rna-dependent rna polymerase (rdrp) through its c-terminal domain (lindenbach et al., ) . the ns has been crystalized and contains an n-terminal domain with s-adenosyl methionine methyltransferase activity (mtase), and possibly guanylyltransferase (gtase) for rna capping (egloff et al., ; malet et al., ; yap et al., ) . ns is also involved in activating other viral proteins, such as ns ntpase and rtpase activity in a dose-dependent manner. denv ns contains both nuclear localization signal (nls) and nuclear export signal (nes): these facilitate its importation into the nucleus by both importin b and the importin a/b complex, and an additional function of the signals could be enhancement of virus replication and hyperphosphorylation of ns (johansson et al., ; brooks et al., ) . for wnv, ns is exported from the nucleus in a chromosome region maintenance (crm )-dependent manner (pryor et al., ; rawlinson et al., ). ns is involved in different cellular pathways and has a crucial role in the escape from, or blocking, the interferonalpha/beta (ifn-alpha/beta) signaling pathway. denv ns inhibits host tyk and stat phosphorylation and prevents the activation of jak-stat signaling pathway (mazzon et al., ) in addition to the viral rdrp functions of ns described for the mbfvs, the tbfv ns was the first to be implicated in disrupting innate immune signaling. to suppress critical host responses, lgtv ns interacts with ifnar and ifngr , two ifn receptor subunits, and antagonizes ifndependent responses by suppressing jak-stat signal transduction (best et al., ; park et al., ) . cellular responses targeted at restricting viral replication occur via lysosomal degradation of ns , ns b, and ns . the ns of lgtv and tbev interacts with trim -alpha, an ifn-inducible protein, and induces the degradation (taylor et al., ) . the cellular antiviral state is also compromised by tbev ns interaction with the scaffold protein scribble (werme et al., ) . this complex blocks the phosphorylation of stat and disrupts the jak-stat signaling pathway the extreme terminal region contains secondary structures and rna elements important for cyclization of the genome and virus viability. the mbfv utr can be divided into three regions (markoff, ; liu et al., ): a variable region directly upstream of the stop codon; a second region that is relatively conserved in nucleotide sequences containing hairpin motifs; and a highly conserved core element contains stable sl structures and cyclization motif (villordo & gamarnik, ) the utr of tbfvs exhibits significant heterogeneity in length and sequence even among closely related strains. the tbfv utr can also be divided into a variable region part and an extremely conserved core element (gritsun et al., ) , where the length variability of the variable region has been suggested to be related to the number of laboratory passages (mandl et al., ; mandl, ) . a hypothesis interprets the biological importance of the variable region that influences the replication/translation dynamics in mammalian and tick cells (elvang et al., ) . the conserved core element contains highly essential sequential and structural motifs defined as sl - for viral maintenance (mandl et al., ; pletnev, ) . the pentanucleotide (gagag) motif exposed on the sl is strictly conserved among all the flavivirus genomes . other important elements present in the conserved region include the homopurine box, a homopyrimidine box, and cyclization sequences (mandl, ; gritsun & gould, a, b) *nucleotide numbers are related to the strain neudoerfl of tbev. † shared function is presumed for both mosquito-and tick-borne flaviviruses. in many cases, no specific studies have been carried out in tbfv. brane (prm/m), and envelope (e), and seven nonstructural proteins: ns , ns a, ns b, ns , ns a, ns b, and ns (chambers et al., ; ryan et al., ; bollati et al., ) . the defined functions of these proteins are presented in table . however, the precise function of some of the proteins remains to be elucidated. furthermore, it should be noted that the bulk of the studies have been carried out in mbfv systems, and it is possible that the functions may not be identical in tbfv. recognizing that the vbfvs cycle between vertebrate and arthropod hosts and that both viral and host factors are likely to be involved, it is highly probable that viral persistence is exceedingly complex. the purpose of this review is to evaluate the current literature on flavivirus persistence as well as to suggest ideas for additional research into this interesting and important area. humans are inadvertent targets of vbfv infection (figs and ), and infection is often associated with debilitating, acute neurological syndromes or hemorrhagic fever. however, there are several lines of evidence suggesting flavivirus persistence in humans (murray et al., ; gibney et al., ; baty et al., ) . the establishment of flavivirus persistence in humans seems to be mainly associated with encephalitic flaviviruses (diamond, ) . however, yfv and denv persistence has been described in eukaryotic cells in culture and in successive generations of aedes mosquitoes (lodge et al., ; takasaki et al., ; farfan-ale et al., ) . yfv and denv commonly cause a hemorrhagic illness, jaundice, and dengue shock syndrome, and encephalitis is atypical and extremely rare , and delivered to endosomes, where a ph-dependent fusion of the particles with the endosome membrane occurs (c). subsequent to uncoating, the single-stranded, positive-sense rna genome (d) migrates to the endoplasmic reticulum (er) and is translated (e) as a polyprotein traversing the er membrane several times (f). the polyprotein is cleaved into the viral proteins by viral and cellular proteases, although prm and e remain covalently attached. through the agency of several viral nonstructural proteins and cellular proteins, there is a proliferation of er-derived membranes and the formation of spherules that maintain a pore-like connection to the cytoplasm (g); the viral genome is replicated within these spherules by the viral proteins comprising the replication complex. by an as yet uncharacterized mechanism, progeny genomes are delivered to adjacent er membranes where the capsid protein mediates assembly and inclusion of prm-e into immature virions (h). the immature virions transit the golgi membrane system (i), and as a mild ph change occurs, the cellular enzyme furin cleaves the prm-e linkage (j), allowing the virus particle to assume its final mature stage prior to release from the cell (k). defined roles for the individual viral proteins are enumerated in table . (tomori, ; gulati & maheshwari, ; varatharaj, ) , but persistence cannot be excluded. human infection with the encephalitic viruses almost always occurs following a bite from infected ticks or mosquitoes (figs and ) . generally, illness tends to be biphasic. the first phase is characterized by a flu-like illness with symptoms such as headache, arthralgia, and malaise. the second phase may present with neurological symptoms, such as mild meningitis to severe encephalitis. survivors of encephalitis may develop persistent neurological sequelae, suggesting neurological tissue damage and/ or viral persistence. neurological tissue damage, such as loss of neurons, has been reported for wnv infection (guarner et al., ) . long-term morbidity following tbev infection is known to occur frequently (haglund et al., ; haglund & gunther, ) . a study conducted in sweden reported chronic postencephalitic syndrome in % of patients who had been infected with tbev (haglund et al., ) . in up to % of patients who develop encephalitis due to wnv infection, neurological and/or neuropsychological symptoms continue to be reported for up to years following the infection (murray et al., ; sadek et al., ) . the most definitive evidence for viral persistence would be the isolation of viable virus or the demonstration of viral antigens or rna long after the acute illness. several reports describe the isolation of infectious vbfvs from persistently infected humans. for example, the siberian tbev strain za was isolated from an individual who had harbored the virus for years (gritsun et al., ) . he later died following years of a progressive form of tbev encephalitis (gritsun et al., ) . in addition, isolation of jev from the cerebrospinal fluid was possible for more than weeks following infection in % of individuals that had developed encephalitis (ravi et al., ) . jev persistence was also demonstrated in peripheral mononuclear cells in infected children in northern india (sharma et al., ) . in these children, virus could be isolated by culture - months after acute infection (sharma et al., ) . furthermore, wnv has been transmitted to patients who had received blood transfusions or organ transplants from asymptomatic donors (pealer et al., ; montgomery et al., ; cdc, ) , suggesting viral persistence in the donors as well as stored blood for transfusion. long-term presence of viral nucleic acid is another indicator for persistent infection; however, study results are inconsistent. viral rna can be readily sought using molecular biology techniques, such as rt-pcr and transcription-mediated amplification (tma) (s anchez-seco et al., ; muñoz-jord an et al., ; patel et al., ) . using tma, wnv rna was detectable in the blood of donors for up to days following the index donation prince et al., ) . using rt-pcr, wnv rna could also be detected in urine of persistently infected individuals for up to . years following the acute phase of infection (murray et al., ) , although virus could not be isolated from the rna-positive urine samples. in contrast, wnv rna could not be detected after years in urine in another study reported a year later (gibney et al., ) . baty and colleagues also failed to detect wnv rna by rt-pcr, but were able to detect viral rna using tma (baty et al., ) . similarly for jev, viral rna could not be detected by rt-pcr in the individuals who had anti-jev igm antibodies (zhao et al., ) . in the study by gibney et al. ( ) , wnv persistence could not be excluded. even though the results in these studies are somewhat variable, it seems reasonable to conclude that the presence of viral rna can also be taken as an indicator of persistent infection. viral serology may be an additional surrogate measure for vbfv persistence. in general, infection or vaccination leads to a sterilizing immunity, but long-term persistence of anti-vbfv igm antibodies has often been assumed to indicate continued exposure to viral antigens or virus particles (ravi et al., ; stiasny et al., ) . an exception to this is, of course, denv where antiviral antibody plays a key role in pathogenesis (martina et al., ; pierson, ) . igm antibodies induced by flaviviruses, such as tbev and wnv, are known to persist in serum and csf for months or more (kapoora et al., ; stiasny et al., ) . indeed, igm antibodies against tbev persisted for up to months (stiasny et al., ) . furthermore, anti-wnv igm antibodies persist in previously exposed blood donors for up months prince et al., ) . however, igm antibody persistence does not necessarily correlate with infectious virus persistence. for example, persistent igm antibodies against tbev can be detected in some cases following vaccination without active viral infection (rendi-wagner et al., ; stiasny et al., ) . for wnv infection, igm antibodies against the nonstructural protein ns cannot be used to distinguish between recent/active infection from past infection (prince et al., ) . thus, serology may be a less useful marker for viral persistence. a major consideration in vbfv in humans would be identification of sites of viral persistence. as flavivirus persistence seems to be mainly associated with the neurotropic and encephalitogenic viruses, the central nervous system may well be the preferred site for viral persistence. furthermore, this may account for why patients who recover from encephalitis often have prolonged neurological symptoms. however, other studies by murray and colleagues postulated kidneys as a preferred site for the establishment of persistent flavivirus infections following the detection of wnv rna in urine (murray et al., ) . certainly, additional work is required to define the true incidence, the biology, and the pathogenic potential in humans of persistent vbfv infections. arthropod hosts play crucial roles in the biology of both tbfv and mbfv and contribute to persistent infection. in nature, ticks are the important arachnid reservoirs of tbfvs. the persistence of the tbfvs in arachnids is well established, and the role of ticks as long-term reservoirs and vectors for the viruses is clear. estimates suggest that ticks transmit about % of the known flaviviruses. tbfvs are capable of infecting about tick species, but the ixodid ticks, ixodes scapularis and ixodes ricinus, account for nearly all transmission of tbev to humans. ixodes cookei may also harbor powv and dtv, while the soft-bodied onithodoros tick transmits alkhurma virus (main et al., ; charrel et al., ) . to transmit virus, these ticks need only min of attachment to the host (crowder et al., ) . the virus is present in the tick salivary glands, and saliva constituents enhance infectivity (nuttall & labuda, ; girard et al., ) . a recent publication revealed that a selected subset of salivary gland genes is expressed when infected ticks feed on na€ ıve mice (mcnally et al., ) . ticks acquire the virus while feeding on infected rodents, typically by a process called 'cofeeding' (fig. ; labuda et al., a, b) , and the virus persists throughout several life stages. ticks at the larvae, nymph, and adult life stages can become infected by feeding on infected animals (horizontal transmission) or can transmit virus vertically across instars (transstadial transmission) and through the eggs (transovarial transmission; fig. a ; labuda et al., a labuda et al., , nuttall & labuda, ) . indeed, results of a carefully controlled study in our laboratory show that transstadial transmission seems to be very high (mitzel et al., ) . the blood meal remains in the midgut for long periods of time, allowing infection of the epithelial cells lining the midgut with subsequent translocation into the hemocoel (nuttall & labuda, ) . infection of the midgut cells may be facilitated by the heterophagous nature of ticks (i.e. blood meal digestion is principally an intracellular process). in the open circulatory system, tissues and organs are bathed in hemolymph, which acts as a medium for transporting nutrients, hormones, and immune effector molecules. therefore, the hemolymph likely serves as a viral dissemination medium. tbe virus was found in the esophagus and subesophageal ganglion in dermacentor marginatus larvae and in columnar epidermal cells of dermacentor reticulatus nymphs (nuttall & labuda, ) . in d. reticulatus nymphs, tbe virus was demonstrated in epidermal cells and in vacuoles in the region of golgi complexes of salivary gland cells (nosek et al., ) . the precise mechanisms by which tbfvs traverse various tissues in the tick and reach the salivary glands remain to be fully elucidated. prevalence surveys indicate that . - % of ixodid ticks carry the virus in europe, but prevalence of up to % has been reported in russia (ustinova et al., ) . in north-central usa, up to . % of i. scapularis ticks are infected by powassan or dtv (brackney et al., b; dupuis et al., ) . interestingly, in a study carried out in chicago, i. scapularis was found to infest . % of wild birds, which figure prominently in the mbfv cycle ( fig. b; hamer et al., ) . however, identification of tbfv was not attempted in this study. in brief, tbfv persistence in ticks is well established, and the essential role of ticks in the biology of these viruses is not in doubt. as is the case for tbfvs, the role of mosquitoes in the maintenance of the mbfv cycle is also well characterized. mosquitoes are thought to account for transmitting approximately % of more than known flaviviruses (gould, ) . diverse mosquito species can be infected by mbfvs. however, the two major mosquito vectors are aedes aegypti (e.g. transmission of yfv and denv) and culex species (e.g. transmission of wnv and jev). adult female mosquitoes become infected when they obtain blood meals from flavivirus-infected animals, and virus replication in the mosquito has been well described (girard et al., ; mcgee et al., ; colpitts et al., ) . girard et al. ( ) used immunohistochemistry to demonstrate that wnv infects epithelial cells of the culex pipiens midgut and that viral antigen can be detected as early as day postinfection. viral antigen staining becomes more intense in the cells of the midgut over time until day to following infection (girard et al., ) . using denv- , virus titer in the midgut peaks to about pfu ml À by days postinfection, but declines to about . pfu ml À by day (s anchez- vargas et al., ). dissemination of virus into various tissues occurs at various time points, and the amount of antigen also varies. similar to tbfvs in ticks, the hemolymph serves as a vehicle for viral dissemination. however, virus also spreads in a cell-cell fashion to the muscle of the posterior midgut from to days postinfection (girard et al., ) . studies with denv- showed that infected mosquitoes mount an rna interference (rnai)-mediated antiviral response, but impairing the vector rnai resulted in increased viral replication (s anchez- vargas et al., ) . this mechanism could be associated with observations of flavivirus-related rna that persists as dna in mosquitoes (crochu et al., ) . as mosquitoes are such efficient vectors for the transmission of the mbfvs, these reports suggest that the viruses have evolved a mechanism of evading the host response to persist in the vector. mosquitoes also acquire and deliver virus horizontally during blood meals and are competent to transmit vertically to progeny by transovarial passage (rosen et al., ) . wnv can persist overwinter in mosquitoes that hibernate in cold months. cool temperatures also facilitate persistence of flaviviruses in adult female mosquitoes. for example, st. louis encephalitis virus (slev) survived for more than days of winter in culex quinquefasciatus (kramer & ebel, ) . similarly, jev and wnv have been transmitted by mosquitoes that carried the viruses at cold temperatures when exposed to temperature increases equal to ambient levels (kramer & ebel, ) . however, at low temperature, mosquitoes are less likely to acquire new infections (colpitts et al., ) . at °c, low wnv dissemination to the legs of the c. pipiens was observed, and rapid viral dissemination occurred at higher temperatures (colpitts et al., ) . furthermore, higher temperatures increase vector population growth rate and the rate of viral evolution in the mosquito (girard et al., ) . the prevalence and maintenance of wnv across landscapes is mediated by environmental factors, such as local effects of agriculture on vector and host communities (crowder et al., ) . results of investigations carried out in the states of oregon and washington showed that the prevalence of wnv in both c. pipiens and culex tarsalis was similar at . % or . %, respectively (crowder et al., ) . however, a study conducted in stratford, connecticut, showed that the c. pipiens was the most dominant mosquito captured in this wnv focus area (anderson et al., ) . in the captured mosquitoes, more than % of wnv isolations were from the same species, whereas culex salinarius accounted for between % and % (anderson et al., ) . in a similar study conducted in mexico, c. quinquefasciatus was more common at . % and mbfv rna was detected in % of the pools (farfan-ale et al., ) . it is evident that mosquitoes are important as viral hosts and vectors. furthermore, it can be concluded that the mbfvs have evolved mechanisms of evading the host innate responses to persist in the mbfvs. the principal vertebrate hosts for vbfvs are small mammals, marsupials, and birds (fig. ) , but the viruses can also infect reptiles (mackenzie et al., (mackenzie et al., , steinman et al., ; jacobson et al., ; root et al., ; marschang, ) . in most instances, larger animals, such as cervids, goats, and sheep, are incidental hosts. the outbreak of wnv in humans in new york is thought to be associated with wnv infection in birds (strausbaugh et al., ) . the mosquito-borne wesselsbron virus could also be cycled between mosquitoes and birds, but not much is known about the vertebrate host(s). thus, in nature, numerous species are susceptible to vbfvs and might serve as reservoirs or secondary amplifying hosts. in this section, we will survey the literature and the three potential markers for viral persistence: isolation of virus, identification of viral rna or protein, and viral serology. small mammals, particularly rodents, are the principal vertebrate hosts and reservoirs for tbfvs (mansfield et al., ; dobler et al., ) . in europe, the yellow-necked mouse (apodemus flavicollis) and bank vole (myodes glareolus) are implicated as the most common hosts, and they develop sufficient viremia to infect ticks that feed on them (weidmann et al., ; dobler et al., ; knap et al., ) . in various wild rodent species captured in brandenburg, germany, an average tbev infection rate of % was reported (achazi et al., ) . in the captured rodents, tbev rna was detected by rt-pcr in the brains and spleens. in north america, deer mice (peromyscus), squirrels, and the striped skunk (mephitis mephitis) are important reservoirs of powv and dtv (main et al., ; telford et al., ) . based on serological studies, a . % dtv prevalence in the red-backed voles (myodes rutilus) was found in siberia and alaska (deardorff et al., ) . dtv in new mexico was serologically prevalent in peromyscus truei and peromyscus maniculatus at . % and . %, respectively (deardorff et al., ) . therefore, it seems likely that persistent infection of these small mammals occurs. deer may stabilize and maintain tbfvs at levels that are important for transmission (pugliese et al., ; carpi et al., ; mcgee et al., ; dobler et al., ) . while deer are important sources of blood meals for ticks, they develop low virus titer and are therefore not competent in transmission of tbfvs. however, in a broader sense, deer are important in viral persistence because they help sustain tick populations (cagnacci et al., ) . the situation for domesticated animals is also less clear than for rodents. dogs, horses, and monkeys can be infected with tbfvs, but case reports in veterinary practice are relatively infrequent (jaenson et al., ) . large domesticated animals, such as goats, sheep, and cattle, become viremic for a short while and develop antibodies following infection with tbev, but do not show any specific clinical signs of illness (mansfield et al., ; klaus et al., ) . however, transmission of tbev via milk from these domesticated animals (fig. ) has been documented, suggesting that virus may persist following the acute viremia in at least some instances (vereta et al., ; caini et al., ; hudopisk et al., ) . birds and primates are considered to be the primary reservoirs of mbfvs. other mammals are generally accidental hosts, but this may not always be the case, and when viremic, these domestic animals are able to infect mosquitoes. for jev, the major amplifying hosts are birds and pigs, which attain high levels of viremia. they provide a source of infection for the mosquito species that subsequently transmit jev to humans (hukkanen et al., ; van den hurk et al., ) . jev control has been achieved through vaccination of pigs and humans in korea, japan, and taiwan (igarashi, ) while horses are considered dead-end hosts of jev infection due to a very low level of viremia. in geographic regions where pig populations are low, swine may not be important for zoonotic transmission of jev. herons and egrets are important jev-amplifying hosts and source of infection for mosquito species that transmit jev to humans (nemeth et al., ) . wild-caught pigeons were shown to have antibodies that persist for up to months. it is not clear whether the antibody persistence is associated with viral persistence. although various bird species do play a prominent role in jev infection, evidence of birds as a source of persistent infection is less certain. in some instances, birds can acquire jev viremia and then fail to develop or lose neutralizing antibodies. birds are also excellent amplifying hosts for wnv, and migratory birds can move the viruses to different areas because they become viremic for several days. near % mortality is associated with natural or experimental wnv infection in birds, such as american crows (corvus brachyrhynchos), blue jays (cyanocitta cristata), and greater sage-grouse (centrocercus urophasianus; steele et al., ; komar et al., ; clark et al., ) , but some birds are able to carry the virus for a longer time before they develop antibodies or succumb to disease (mckenzie & goulet, ) . for instance, a combined % of house sparrows that were either naturally or experimentally infected with wnv tested positive for wnv rna by rt-pcr (wheeler et al., ) . in the same study, wheeler et al. ( ) showed that % and % of wnv-infected house sparrows and finches, respectively, seroconverted following exposure to wnv. birds that develop neutralizing anti-wnv antibodies are protected from reinfection (nemeth et al., ), suggesting total virus clearance in seroconverted birds, and antibodies against wnv have been found in wild birds, suggesting exposure to wnv. early demonstration of wnv persistence was described in blue-gray pigeons, from which virus was isolated up to days postinfection, and viral antigen could be detected in liver tissue for up to days (ruiz et al., ) . american robins (tardus migratorius) are known to be competent reservoirs of wnv and slev (kilpatrick et al., ) . wnv persistence was also demonstrated by the detection of viral rna in the presence of antibody for up to weeks in spleens of naturally infected house sparrows and finches (wheeler et al., ) . taken together, it seems that there is decent evidence for persistent infection by wnv in some bird species. however, many details about the biology of persistence remain to be studied. antibodies to various flaviviruses, such as slev, powv, jev, and wnv, have been identified in chelonians, snakes, and crocodiles in different geographic locations (steinman et al., ; marschang, ) , and it is known that alligators and crocodiles can be infected with wnv. however, there are no reports suggesting that reptiles host persistent mbfv infection. in summary, it is apparent that various animals and birds can sponsor persistent vbfv infection. nevertheless, the precise role that these persistent infections play in larger scheme of viral maintenance merits additional study. there are several animal models of persistent mosquito-borne flavivirus infection, including wnv and slev (charlier et al., ; kimura et al., ) . as shown in table , mice and hamsters have been the main study models. in a nonhuman primate study, wnv was isolated from central nervous system tissues more than months post-intra-cerebral inoculation , suggesting that these species might also be relevant system. persistent wnv has also been studied experimentally in the golden hamster. the clinical outcomes of wnv infection in hamsters vary and depend on animal age and immune competence, viral dose, and route of infection. wnv infection can lead to asymptomatic illness, encephalitis, severe paralysis, and acute death (xiao et al., ; morrey et al., ; tesh et al., ) . adult golden hamsters infected with wnv develop chronic infection, characterized by shedding of virus in urine for up to months (xiao et al., ; ding et al., ; tonry et al., ) . the chronically infected animals exhibit antigens in tubular epithelial cells, interstitial cells, and macrophages of distal renal tubules . interestingly, na€ ıve hamsters inoculated intraperitoneally with wnv-containing urine from persistently infected hamster do not develop clinical disease, but the hamsters become viremic and develop antibody responses (ding et al., ) . this suggests possible viral genetic changes, which may facilitate persistent infection, although other explanations cannot be totally excluded. hamsters have also been used as experimental models to study the persistence of slev and are among the natural vertebrate hosts of the banzi flavivirus, a member of the yfv group of flaviviruses (grard et al., ) . golden hamsters infected with slev did not develop clinical signs of illness, but they developed viruria and antibodies against slev at days postinfection (siirin et al., ) . (appler et al., ; pierson & diamond, ) : wnv rna persisted in a pantropic manner in % of infected mice for up to months. infectious virus could be isolated in % of mice for up to months. c h mice survival rate was lower and % when compared to the survival rate of b mice, which was % macaque rhesus (pogodina et al., ) : virus persisted in asymptomatic animals for ½ months and could be isolated from cerebellum, cerebral subcortical ganglia, lymph nodes, and kidneys golden hamster (mesocricetus auratus; tesh et al., ; tonry et al., ) : chronic renal infection with persistent shedding of virus for up to months. virus could be recovered by culture, and genotypic and phenotypic changes were identified house sparrow (passer domesticus; nemeth et al., ) : infectious virus persisted in tissues for up to days, but was not detectable in sera after days. wnv rna persisted in tissues for up to days slev golden hamster (siirin et al., ) : infected animals remained asymptomatic, but virus could be cocultivated in various organs for up to days jev swiss albino mice (mathur et al., a, b) : persistence was demonstrated by reactivation in % of congenitally infected pups. in adult mice, viral persistence was shown to last longer ( weeks) in pregnant mice compared with weeks in nonpregnant mice tick tbev macaque rhesus (pogodina, ; pogodina et al., pogodina et al., , : monkeys recovered from encephalitis and virus persisted for at least days. in asymptomatic animals, virus persisted for days liv* immunosuppressed guinea pigs (zlotnik et al., ) : liv was lethal in young animals, but older animals acquire a nonapparent infection with viral replication in the brain and spleen powv deer mouse (peromyscus leucopus; telford et al., ) : not a well-characterized model, but adult mice appear to survive infection *louping ill virus infects sheep. hamsters that become persistently infected with slev shed virus in their urine for up to days postinfection. the shedding of virus in the urine of infected hamsters also suggests that the kidneys could be an organ preferred for viral persistence. c bl/ j mice maintained a persistent wnv infection in the face of robust neutralizing antibody levels for more than months (appler et al., ; stewart et al., ) . in this study, infectious virus was recovered in the skin, and viral rna was identified in the skin, as well as the spinal cord and brain (appler et al., ) . in contrast, wnv persistence was uncommon in kidneys and rare in the heart (appler et al., ) . however, in another study in which c bl/ j mice were infected with wnv derived from the urine of persistently infected hamsters, the kidney was found to be the preferred organ for viral persistence (saxena et al., ) . the hamster-derived wnv was also found to be highly attenuated in both neuroinvasiveness and neurovirulence in infected mice (saxena et al., ) . these results also suggest that the virus acquires phenotypic changes to be able to persist. while the inbred laboratory mouse models are patently useful for understanding some aspects of flavivirus persistence, very few natural rodent hosts have been established as experimental animal models. evidence of exposure to wnv has been reported in rodent species, such as rats (rattus), bank voles (m. glareolus), and deer mice (peromyscus; molnar et al., ; root et al., ; docherty et al., ; gomez et al., ) . however, infectious virus was only identified in the bank vole, whereas antibodies were detected in all of the species (molnar et al., ; root et al., ) . although persistence in mammals obviously plays a significant role in tbfv infections, little attention has been devoted to experimental studies of this aspect of tbfv biology. while disease or therapy models have been established for tbfvs (kreil et al., ; holbrook et al., ; hayasaka et al., ; palus et al., ) , none of these specifically investigated viral persistence. experiments to determine the susceptibility of several wild and domesticated mammals to powv showed that viremia lasted for - days in the goat, pig, skunk, red fox, and gray fox (yiang et al., ) . in another study, adult deer mice (peromoyscus leucopus) survived challenge with powv without apparent illness, but evidence of viral persistence was not sought (telford et al., ) . development of suitable experimental models for persistent tbfv infection would clearly provide useful information about this aspect of these important pathogens. the previous sections have looked at flavivirus persistence in humans, animals, and arthropod vectors, as well as some relevant animal models. in this section, we will survey information related to the initiation and maintenance of persistence. when mammalian cell cultures are acutely infected with tbfv, a legion of general cellular defense and antiviral systems are triggered, as are specific factors designed to limit or restrict virus reproduction. some of these include type i interferon (ifn-a/ifn-b), type iii interferon (ifn-k), mitochondrial activated signaling, and the induction of inflammatory factors, such as interleukins (tam & messner, ; madden, ; van gerpen, ) . for example, the ifn-induced tripartite motif protein, trim a, has been shown to restrict tbev replication by degrading ns (taylor et al., ) . the unimpeded deployment of these antiviral factors and systems would lead to cell death. cell death is thought to be mediated primarily through apoptosis (r u zek et al., ) , and programmed cell death induced by various tbfvs has been described in neurons, epithelial cells, hepatocytes, kupffer cells, and neuroblastoma cells (ramanathan et al., ) . impeding or evading the antiviral response is one characteristic of vbfvs, which plays an important role in viral persistence. during flavivirus infection, ifn production is induced within hours, but viral rna replication complexes are enclosed in vesicles, which may offer protection from recognition by pathogen recognition receptors, thus delaying ifn production (welsch et al., ; gillespie et al., ; overby et al., ; offerdahl et al., ; ye et al., ) . in addition, some vbfvs can directly affect ifn secretion by inhibiting ifn gene transcription, suppressing ifn signaling or impairing the functions of interferon-stimulated genes (best et al., ; robertson et al., ; ye et al., ) . the humoral and cell-mediated immune responses are also prone to inhibition by vbfvs. studies with wnv in c bl/ mice suggest that wnv-specific antibodies correlate with decreased spread to the cns (diamond, ) . antibody escape mutants could also evade the t cell recognition (diamond, ) , but a precise role of these in viral persistence is yet to be defined. the e protein is thought to be responsible for provoking the cytopathic effect (isaeva et al., ; prikhod'ko et al., ) . however, the ns protease and ns b-ns protease precursor (table ) are also known to induce apoptosis by binding to caspase (shafee & abubakar, ; ramanathan et al., ; safronetz et al., ) . in addition, ns a has also been implicated in causing ifn-independent cytopathic effect (chang et al., ; melian et al., ) . observations in our laboratory (l. mlera, d. offerdahl & m.e. bloom, unpublished results) and those of others (lancaster et al., ) indicate that tbfv infection in mammalian cell culture is characterized by an acute phase, which kills most infected cells; however, a small number of cells survive the initial cytolytic phase, and the culture is repopulated with cells almost all of which are infected. clearly, vbfv induces an acutely cytopathic infection in mammalian cells, and thus, the development of a persistent infection implies that cell death factors must be evaded or modulated, although the precise mechanisms are still obscure. the implication of specific viral proteins, domains, or sequences that combat cytolytic cell death has been rather limited, but any viral determinants that limit cell death in the face of acute infection might also enhance the initiation of persistent infection. for example, viral ns a (table ) was shown to induce phosphatidylinositol -kinase (pi k)-dependent autophagy and thereby leading to protection from cell death (mclean et al., ) . the manipulation of apoptosis by jev, which activates pi k in infected cells, was also reported (lee et al., ) . jev triggers apoptosis during the late stages of infection, and the activation of pi k is thought to provide protection from early cell death. limited replication implies a low level of viral protein expression and may favor viral persistence. over the years, significant attention has been focused on defective interfering (di) virus particles, which limit replication of the wild-type virus (schmaljohn & blair, ; debnath et al., ; blitvich et al., ) . the di particles represent truncated genomes that can be replicated and encapsidated and compete with wild-type viral particles when they infect cells. in vesicular stomatitis virus (vsv), di particles are thought to modulate virulence (cave et al., ) . however, the role of vbfv di particles in persistence is not completely certain. for tbev, the c protein is reported to tolerate internal deletions ranging from to amino acids, and the deletions seem to favor attenuation and immunogenicity (kofler et al., ) . di particles of vbfvs, such as murray valley encephalitis (mve), tbev sofjin strain, and wnv, are known to generate truncated ns proteins, following infection at high multiplicity of infection (debnath et al., ; poidinger et al., ; chen et al., ; bugrysheva et al., ) . persistent infection of vero cells with mve was associated with a truncated ns , whereas this form of ns was not noted in the acute infection (brinton, ; lancaster et al., ) . the truncated ns in mve virus was a result of the presence of di rna, which contained a large internal deletion (lancaster et al., ) . in this case, mve di particles reduced the wild-type mve titer by - % (poidinger et al., ) . however, a causal role for the mve di particles in maintaining persistent infection was not conclusively demonstrated. studies of the far eastern sofjin strain of the tbev complex identified a -kda truncated ns in both acutely and persistently infected human kidney rh cells (bugrysheva et al., ) . for wnv, naturally occurring di particles interfere with transmission in mosquitoes and minimally impact pathogenesis in mice (pesko et al., ) . furthermore, truncated denv rna species, suggesting the presence of di particles, have been identified in acute human infections (li et al., ) , but have not been described in other acute vbfv infections in humans. similarly, banzi virus di particles seem to have been generated in resistant c h/rv mice (smith, ) . although di particles and the truncated ns may be frequently observed in persistent infections, it is not at all clear that they are independently sufficient for the establishment and maintenance of a persistent infection in vitro or in vivo. stable expression of truncated ns failed to render persistent infection with jev, suggesting that truncated ns is a consequence rather than a cause for viral persistence (liao et al., ) . the role of di particles merits further investigation, but the role of other aspects of virus biology should also be scrutinized. restricted expression of the envelope (e) protein may also favor development of persistence. in kn cells that were persistently infected with jev, the e protein was found to be expressed at markedly low levels compared with acutely infected cells (feng et al., ) . in these studies, the expression of ns was found to be unchanged in the acute and persistent phases of jev infection (feng et al., ) . as the e protein is important for pathogenesis and immunity (cdc, ), low-level expression could result in immune tolerance and contribute toward viral persistence. in addition to low expression levels, mutations in the e protein of jev, yfv, and wnv might also play a role in vbfv persistence (ding et al., ; farfan-ale et al., ) . a e?k mutation in the e protein of jev and wnv was shown to inhibit cell-cell spread of the virus and to contribute toward the development of a small-plaque phenotype (carson et al., ) . while this mutation leads to viral attenuation (carson et al., ) and is key in the attenuation of jev sa - - vaccine (monath et al., ) , further elucidation of mutations that may play a role in persistence is required. observations that hamsters and mice infected with mbfvs obtained from urine of other mbfv-infected animals do not suffer severe disease and become persistently infected indicate that the virus is attenuated (rosen et al., ; ding et al., ) . a number of amino acid-changing mutations in c, e, ns , ns a, ns b, and ns were reportedly associated with persistence of wnv in serially passaged hamsters (s anchez- vargas et al., ) . these mutations are thought to have attenuated the virus (s anchez- vargas et al., ) . however, the same mutations have not been reported by other groups, suggesting that the mutations may not be specific to development or maintenance of viral persistence. mechanisms not directly involving viral proteins may also play a significant role in persistence. for instance, jev delays the exposure of dsrna to innate sensors and inhibits phosphorylation of irf via noncoding viral short flaviviral rna (espada-murao & morita, ; chang et al., ) . similarly, wnv delays recognition by pathogen recognition receptors by activating irf in a rig-i-dependent manner without antagonizing the host ifn response (fredericksen & gale, ) . the mechanism of evasion is currently not clear, but membrane-bound vesicles that enclose the rig-i-activating dsrna of tbev have been described (overby et al., ) . as the delayed recognition of viral pathogen-associated molecular patterns is only temporary, these manipulations are probably just among the suite of mechanisms deployed for the establishment of viral persistence. the specific viral genes and how they manipulate apoptosis at a cellular level needs further examination. specific host genes that may contribute toward the development of flavivirus persistence in the vertebrate host have not been defined, but there are some suggestions. for instance, the overexpression of the proto-oncogene bcl- was reported to prevent apoptosis and promote persistence in jev-infected bhk and cho cells (liao et al., ) , suggesting that the control of apoptosis is likely to be implicated. in addition, some inbred laboratory mice strains can carry the - -oligoadenylate synthetase gene, flv r , which confers flavivirus resistance (urosevic et al., ) . the animals are productively infected with flaviviruses, but produce low virus titer (brinton & perelygin, ; barkhash et al., ) . mice that are susceptible to flavivirus infection carry the flv s allele. the flv r -like allele has also been characterized in wild mice and could be a partial explanation of flavivirus persistence in rodents in nature (urosevic et al., ) . additional vertebrate genes, apart from the flv, could play a role in varied susceptibilities of different mouse strains to flavivirus infection. this was suggested from recent observations that tbev-infected balbc mice were moderately resistant, sts mice are highly resistant, whereas the balbc/sts recombinant mice were highly sensitive to infection (palus et al., ) . immunosuppression may also be a potentiating factor for the establishment of flavivirus persistence in animal hosts. for example, wnv persistence was demonstrated by the detection of wnv rna and immunohistochemistry in brain tissue of an immunosuppressed -year-old man months after the initial diagnosis (penn et al., ) . the follicular b cell lymphoma, from which he had suffered, may have facilitated, or aggravated, the persistence of wnv. in mice, transient immunosuppression with cyclophosphamide leads to wnv recrudescence (appler et al., ) , an observation suggesting that some aspects of the immune system operate to restrict wnv replication during persistence. however, the situation is complicated because wnv is able to persist for up to months in the face of a robust humoral immune response in c bl/ mice (appler et al., ) . furthermore, wnv persistence was reported in the brains of cd + t cell-deficient rodents (shrestha & diamond, ) , but the cd + t cell deficiency did not affect the antibody response in this mouse model. knowledge about specific immune system components that could facilitate or control viral persistence remains to be characterized. infection by vbfvs of arthropod cells has not received the same degree of study; however, acute infection is not accompanied by the same cytopathic response observed in mammalian cells. in addition, very little is known about how or in what tissues tbfvs persist in the ticks, but the viruses likely evolved mechanisms of modulating or evading the tick immune system over the millenia (robertson et al., ) . persistent infection of the i. scapularis cell line ise- (munderloh et al., ) was readily established and was noncytopathic (offerdahl et al., ) . detailed studies of persistent tbfv infection in arthropod cell lines using contemporary techniques and methods are certain to yield useful and interesting information. the mechanism(s) of how mbfvs persist in mosquitoes also remains a suitable topic for investigation. slev persists in the midgut of c. pipiens for hours before infecting the midgut epithelium (whitfield et al., ; brackney et al., a) , but this is a short time. interestingly, approximately two-thirds of flavivirus-related sequences were reportedly detected as integrated dsdna form in laboratory-bred and wild aedes mosquitoes (crochu et al., ) , although detection of a complete flavivirus genome was not reported. furthermore, the finding of partial flavivirus-like sequences in dna form is not clear. clearly, research into the biology of flavivirus persistence in mosquitoes and ticks has been limited and is worthy of extensive additional research. in summary, it should be apparent that both viral and host factors play a role in the initiation and maintenance of persistent infection at both the cellular and organismal levels. in addition, successful persistence in nature almost certainly depends upon ecological and environmental forces, but these latter factors are wholly beyond the scope of this limited review. multiple rna and dna viruses are known to establish persistence in culture as well as in humans and animals. consideration of several may provide insight, if not direct parallels, useful in the study of biology of persistent vbfv infections. the hepacivirus genus, containing hepatitis c virus (hcv) species, belongs to the flaviviridae family, together with the flavivirus genus under which vbfvs are classified. despite the virus-specific cytotoxic t lymphocytes and antibodies, persistent hcv infections, which are established with high efficiency, are known to occur in humans and animals, such as chimpanzees (main et al., ; caini et al., ; mcnally et al., ) . hcv persistence is associated with various strategies, such as the high genetic variability that facilitates passive immune evasion. in vivo, hcv fails to activate cd + t cells, leading to exhaustion of cd + t cells. at cellular level, hcv can block interferon induction by blocking rig-i and mitochondrial antiviral signaling (mavs) using its ns -ns a protease, which cleaves the ifn promoter-stimulator (gould, ; muñoz-jord an et al., ; baril et al., ; hudopisk et al., ; perera-lecoin et al., ) . hantavirus infections are another interesting example of viral persistence. hantaviruses are segmented, rna viruses that cause lifelong infections in their reservoir rodent hosts, despite high levels of neutralizing antibodies (botten et al., ; meyer & schmaljohn, ; easterbrook & klein, ) . pathogen recognition receptors, such as rig-i and tlr , are not elevated in the lungs of infected rats, suggesting that evasion of viral recognition may contribute toward the establishment of a persistent infection. perhaps, the reason for noninduction of rig-i is the fact that hantaviruses do not produce detectable amounts of dsrna (wang et al., ) . ifns, such as ifn-b, ifn-k, mxa, and pro-inflammatory chemokines, cytokines, and transcription factor genes are elevated midway in the infection followed by a down-regulation that favors the expression of tgf-b (schountz et al., ) . a continued up-regulation of the cytokines could be detrimental to the cell, and the virus would fail to persist. results of a recently established animal model also show that host-adapted snv achieves prolonged and disseminated infection, with no disease in hamsters (safronetz et al., ) . therefore, hantaviruses may have evolved mechanisms of manipulating target genes, which establishes persistence when induced. one of the best-studied models of persistent rna virus infection is the rodent-borne arenavirus, lymphocytic choriomeningitis virus (lcmv). in lcmv clone (cl ), a glutamine at position in the glycoprotein (lysine in the arm b strain of lcmv) is important for persistence, but its precise function is unknown (salvato et al., ; moskophidis et al., ) . the mechanisms of lcmv persistence are linked to the down-regulation of mhc and costimulatory molecules, inflammatory cytokines, as well as virus-induced production of immunosuppressive cytokines (ng et al., ) . the inability of cd -deficient mice to control lcmv infection over time was reported to be a result of cd and cd responses (boettler et al., ) . in addition, ox also has a role in the establishment of persistent lmcv infection (boettler et al., ) . a recent report showed that ifn blockade of type i ifn signaling results in a cd t cell-dependent clearance of lcmv cl (teijaro et al., ) . this is an interesting observation considering that ifn induction is part of the antiviral response and that its induction can lead to apoptosis. however, the precise mechanism of persistence is not completely clear (easterbrook & klein, ) , but it also demonstrates 'clever' viral manipulation of the host system to establish persistence. coxsackievirus infections are another system from which lessons might be drawn. coxsackievirus b has been implicated toward the development of certain chronic muscle diseases, such as chronic inflammatory myopathy (lodge et al., ; tam & messner, ) . coxsackievirus persistence in cell culture can take two forms: ( ) an incurable steady state characterized by nonlytic virus infection and ( ) an antiviral-curable carrier culture system (brinton, ; pierson & kielian, ) . while mechanisms of coxsackievirus persistence are not completely understood, down-regulation of the coxsackievirus receptor (car) has been suggested to play a role in viral persistence (varatharaj, ) . down-regulation of car in coxsackievirus-infected hl- cells occurs rapidly from % following three passages to % at passage (varatharaj, ) . the importance of car down-regulation is emphasized by the fact that in vitro car knockout results in reduced viral replication as well as virus-induced cell lysis (tomori, ; gulati & maheshwari, ) . these selected examples simply highlight the diversity of possible mechanisms that the vbfv might harness in initiating and maintaining persistence and provide concepts that might be useful to investigate. in the preceding sections, we surveyed a substantial literature with relevance to various aspects of flavivirus persistence. elucidation of how flaviviruses persist in humans could help toward the development of therapeutic interventions that could alleviate morbidity and budgetary burdens associated with neurological sequelae. despite the current knowledge, a relative dearth of knowledge still exists, and additional research is merited on these significant human pathogens. for instance, the definition of specific viral proteins and cellular factors and their interactions in the establishment and maintenance of persistent infection is very limited. as noted, for flaviviruses to persist in infected cells in culture or in vivo, specific host defenses need to be evaded or controlled. the role of ns as an interferon antagonist (especially in tbfvs) has been established (best et al., ) , but its ifn antagonism in the context of vbfv persistence has not been fully explored. this is also true for mbfv ns b, which impairs ifn-a/b induction via jak/stat signaling (muñoz-jord an et al., ) . furthermore, mutations in ns a of kunjin virus result in increased ifn levels, suggesting an ifn antagonistic role of ns a (liu et al., ) . intriguing is the fact that vbfvs antagonize ifn even in the cells that will eventually die in the acute phase of infection. as ifn antagonism seems to be important for hcv, interrogating the role of these vbfv proteins to ascertain their role in the establishment of viral persistence will be critical. the specific mammalian host immune responses that are evaded or controlled also need to be identified precisely. although overexpression of bcl- in bhk and cho cells resulted in the inhibition of apoptosis and jev persistence, a direct viral effect on bcl- was not determined (liao et al., ) . it would be useful to understand which, and how, vbfv proteins interact with the bcl- pathway. indeed, other host pathways could be involved and need to be elucidated further. the exact correlates of persistent flavivirus infection in the arachnid or insect vector host also need to be determined. the biology of ticks and that of mammals is completely different. for ixodid tick vectors, identifying genetic or biological targets that could play a role in viral persistence is imperative. these vector mechanisms might find application, when known, in efforts to control natural flavivirus persistence cycles in ticks. the mechanism(s) could involve viral genetic changes, such as specific mutations that may render the virus less detectable in infected cells. an important question for transmission and viral evolution dynamics is why arthropod vectors do not appear to be killed by viral infection. the down-regulation of the car, as a coevolutionary development that favors persistence, is intriguing (fechner et al., ; pinkert et al., ) . for flaviviruses, various receptors have been identified as possible virus-entry mechanisms (smit et al., ; perera-lecoin et al., ) . investigations into whether there is a down-regulation of flavivirus receptors, as in coxsackievirus persistence, could be useful in defining flavivirus persistence mechanism (s). finally, the establishment of relevant animal models of vbfv persistence will also be crucial for understanding the dynamics of viral persistence and host responses. animals that serve as the natural host reservoirs will be key in developing these models. some models have been established, but may have failed to answer ecologically important question. thus, future work will combine 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induces p -mediated apoptosis via the sequestration of hdm to the nucleolus japanese encephalitis virus ns b-ns protease induces caspase activation and mitochondria-mediated apoptosis in human medulloblastoma cells crystal structure of the dengue virus rna-dependent rna polymerase catalytic domain at -angstrom resolution immune evasion strategies of flaviviruses the ns protease and helicase domains of japanese encephalitis virus trigger cell death via caspase dependent and independent pathways structures of immature flavivirus particles west nile virus genome cyclization and rna replication require two pairs of long-distance rna interactions japanese encephalitis virus rna not detected in urine the persistence of louping ill virus in immunosuppressed guinea-pigs a single-amino acid substitution in west nile virus k peptide between ns a and ns b confers resistance to lycorine, a flavivirus inhibitor this work was supported by the intramural research program, national institute of allergy and infectious diseases, national institutes of health (nih). we wish to acknowledge the able assistance of anita mora and heather murphy for graphic support. danielle offerdahl and jennifer lam provided assistance in manuscript preparation. key: cord- -e ciil authors: feng, min; dai, manman; xie, tingting; li, zhenhui; shi, meiqing; zhang, xiquan title: innate immune responses in alv-j infected chicks and chickens with hemangioma in vivo date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: e ciil avian leukosis virus subgroup j (alv-j) infection can cause tumors and immunosuppression. since the precise mechanism of the innate immune response induced by alv-j is unknown, we investigated the antiviral innate immune responses induced by alv-j in chicks and chickens that had developed tumors. spleen levels of interleukin- (il- ), il- , il- β, and interferon-β (ifn-β) were not significantly different between the infected chick groups and the control groups from day post hatch to days post hatch. however, il- , il- β, and ifn-β protein levels in the three clinical samples with hemangiomas were dramatically increased compared to the healthy samples. in addition, the anti-inflammatory cytokine il- increased sharply in two of three clinical samples. we also found a more than -fold up-regulation of isg - mrna at day post infection (d.p.i.) and a twofold up-regulation of zc hav mrna at d.p.i. however, there were no statistical differences in isg - and zc hav mrna expression levels in the tumorigenesis phase. alv-j infection induced a significant increase of toll-like receptor (tlr- ) at d.p.i. and dramatically increased the mrna levels of melanoma differentiation-associated gene (mda ) in the tumorigenesis phase. moreover, the protein levels of interferon regulatory factor (irf- ) and signal transducer and activator of transcription (stat ) were decreased in chickens with tumors. these results suggest that alv-j was primarily recognized by chicken tlr and mda at early and late in vivo infection stages, respectively. alv-j strain scau-hn did not induce any significant antiviral innate immune response in week old chicks. however, interferon-stimulated genes were not induced normally during the late phase of alv-j infection due to a reduction of irf and stat expression. avian leukosis virus (alv) is a member of the α-retrovirus genus of retroviridae, causing neoplastic disease, immunosuppression, and reproduction problems in the poultry industry worldwide. alv strains are divided into subgroups from a to j based on their viral envelope composition, host range, and cross-neutralization patterns (payne et al., ; barnard et al., ) avian leukosis virus subgroup (alv-j) isolates have been obtained from broiler breeders and laying chickens in most parts of china (cui et al., ) and these infections result in serious economic losses in commercial layer flocks and local chicken breeds (sun and cui, ; gao et al., ) . avian leukosis virus subgroup j can induce the formation of different types of tumors such as haemangiomas and myelocytomas and immunosuppression due to alv-j infection also increases susceptibility to other avian diseases (abolnik and wandrag, ). in the current study we analyzed the transcriptional level of selected immune-response genes we had previously identified from whole-transcriptome profiling of alv-j-induced tumors in chicken spleen samples (li et al., ) . these genes included those with known anti-viral properties such as the single copy mx gene, isg - as well as the toll-like receptor tlr- and other cytokine-related genes. we investigated the effects of alv-j infection on the in vivo mrna and protein expression of related immune-response genes and cytokines during the early and late phases of infection. our findings extend our current understanding of the host-response mechanism to exogenous alv infection. all animal research projects were sanctioned by the south china agriculture university institutional animal care and use committee. all animal procedures were performed according to the regulations and guidelines established by this committee and international standards for animal welfare. the alv-j strain scau-hn was kindly provided by dr. weisheng cao, south china agricultural university. infection with this virus leads to haemangioma development. thirty-six -day-old specific-pathogen-free (spf) white leghorn chickens were hatched from eggs (guangdong da hua nong animal health products co., ltd) and housed in isolator cages. three -day-old sick yellow chickens with neoplasms (designated sc , sc , and sc ) and three -day-old normal yellow chickens (designated nc , nc , nc ) were collected from the same flock from a farm in guangdong province, china. a total of -day-old spf chickens were randomly assigned to two groups: an scau-hn -infected group and control group, each with eighteen chickens per group. chickens were inoculated intraperitoneally at a dose of . ml ( . tcid / . ml) of strain scau-hn . the control group was injected with dmem media alone. spleens were aseptically collected from three infected chickens and three control chickens at , , , , , and d.p.i., respectively. plasma samples were aseptically collected and centrifuged at • c at rpm for min to isolate leukocytes. these were stored as viral stocks at − • c. spleens and livers from clinical samples were either stored at − • c or fixed in % buffered formalin until use. detection of spf chicken infected with alv-j rna was extracted from the spleens of spf chickens (fastagen, shanghai, china) and reverse transcribed into cdna (thermo-fisher scientific, usa) using commercial kits. rt-pcr analyses was employed to detect alv-j via specific primers (smith et al., ) . clinical sample detection, histopathology, and immunohistochemical staining dna was extracted from the spleens of the clinic samples using a commercial kit (omega, usa). specific primers were employed to differentiate between alv-j, alv-a/b, and other suspected viruses including marek's disease virus (mdv) and reticuloendotheliosis virus (rev) . according to a previously described method (li et al., ) , chicken plasma was used for virus isolation by inoculation into df cells. df- cells are only susceptible to exogenous alv virions (maas et al., ) . infected df cell culture supernatants from clinical samples were tested for alv group-specific antigen (p ) using the alv antigen test kit r (idexx, usa) according to the manufacturer's instructions. alv-j infection was further confirmed by immunofluorescence using standard techniques (venugopal et al., ) . infected cell images were collected using nis-elements br analysis software (nikon). liver tissue samples fixed in % buffered formalin were stained with hemotoxylin and eosin (he) and examined histopathologically (cheng et al., ) . immunohistochemical staining was used for further diagnoses with je monoclonal antibody (kindly provided by dr. kun qian, yangzhou university). binding of the je antibody was detected using anti-mouse-hrp (zhongshan goldenbridge, beijing, china). in our previous study, the transcriptome profiles of alv-j-induced tumors in spleen samples compared to healthy spleen samples from white recessive plymouth rock chickens were used to identify the genes related to alv-j invasion (li et al., ) . in this study, the related genes of innate immune transcriptional responses were analyzed according to the transcriptome profiles. expression of related genes of innate immunity were analyzed by quantitative real-time polymerase chain reaction (qrt-pcr). total rna was extracted from frozen spleens of spf chickens and clinical samples using the rnafast kit (fastagen), followed by cdna synthesis of mrna with the revertaid first strand cdna synthesis kit (thermo-fisher scientific) according to the manufacturer's instructions. qrt-pcr was performed on a biorad cfx real-time detection system using itaq tm universal sybr r green supermix kit reagents (biorad, ca, usa) according to the manufacturer's specifications. primers used for qrt-pcr were designed using the ncbi primer blast program and were based on published target sequences ( table ) . data analyses were performed using the − ct method (livak and schmittgen, ). spleen homogenates of spf and clinical chicken samples were used to detect cytokine protein levels. spleen tissues were rinsed in ice-cold pbs to remove excess blood thoroughly and weighed before homogenization. homogenized them in ml of pbs with a steel ball using tissuelyser ii (qiagen, germany). the resulting suspension was subjected to two freeze-thaw cycles to further break the cell membranes. the homogenates were centrifugated for min at × g and the supernatant was removed and was assayed immediately. enzyme-linked immunosorbent assay (elisa) kits for chicken interferon-β (ifn-β), interleukin- β (il- β), il- , and il- determination were obtained from wuhan uscn (cloud-clone corp. china). the elisa experiments were performed according to the manufacturer's specifications. tissue homogenates were prepared from spleens of chickens with tumors and normal chickens as above described. the lysates were collected and incubated on ice for min and then cleared by centrifugation at , g for min at • c. total protein content was determined with a bca protein assay kit (life technologies, usa). total protein ( µg) was resolved by % sds-page and transferred onto nitrocellulose membranes (whatman, maidstone, uk). membranes were blocked with % http://www.ncbi.nlm.nih.gov/tools/primer-blast/ w/v skim milk for h at • c, and then incubated overnight at • c with mouse anti-gapdh antibody (beyotime inst biotech, shanghai, china), rabbit anti-interferon regulatory factor (irf- ) and rabbit anti-signal transducer and activator of transcription (stat ) antibodies (lsbio, seattle, wa, usa). after three rinses with pbs tween (pbst) buffer, the membranes were incubated at • c for h with anti-rabbit-hrp or anti mouse-hrp (zhongshan goldenbridge, beijing, china) that had been diluted in pbst. membranes were washed three times with pbst and signals were detected using an ecl kit (zhongshan goldenbridge, beijing, china). statistical comparisons were made by graphpad prism (graphpad software inc., san diego, ca, usa) and statistical significance was represented by p values of > . , < . , . , or . . to determine whether the spf chickens were successfully infected, we measured gene expression levels of alv-j-specific genes from chickens infected with the scau-hn virus strain. rt-pcr tests of spleen samples from spf chickens at - d.p.i. using alv-j-specific primers were all positive ( figure a , panel ). alv-j could not be detected in the control animals (data not shown). to verify that the clinical tumors were induced by alv-j and no other oncogenic viruses, we used additional methods for pathogen detection. the presence of hemangiomas is a characteristic of alv-j infection. these tumors were evident on the skin of the joints and digits, in the ocular region and in the livers of birds from the same flock ( figure b , panels - ). histologically, the tumors were typical cavernous hemangiomas with malignant vessel hyperplasia visible as a closely packed meshwork of blood vessels. microscopically, the tumor cells were relatively uniform large myeloid cells and lymphoid cell hyperplasia was observed in liver ( figure c , panel ). bluish blisters were also observed and some of these lesions were associated with continuous bleeding. we also observed a significant up-regulation of viral gp expression in tumor cells from alv-j-infected livers using immunohistochemical staining with the je monoclonal antibody (figure c, panel ) . pcr tests on the genomic dna of the three sick chicken spleens using alv-j-specific primers were all positive (figure a, panel ) . we detected no related viral infections as judged by the absence of amplicons using primers specific to alv-a, alv-b, mdv, and rev ( figure a , panels , , , ). the pcr tests of three normal chickens from the same flock were negative using all the listed primers ( figure a , panels , , , ). we could also identify the presence of virion-encoded p from the sick, but not the healthy chickens (data not shown). furthermore, immunofluorescence of infected cells using an alv-j-specific monoclonal antibody were all positive ( figure d , panels - ) and the uninfected control completely lacked any according to the transcriptome profiles of alv-j-induced tumor spleen samples and healthy spleen samples from white (recessive) plymouth rock chickens in our previous experiments (li et al., ) , we analyzed the transcriptional level of related innate immune genes. as a guide, we compared healthy spleens to alv-j-infected spleen samples that possessed tumors and found a general decreasing trend except for tnfaip which increased slightly (figure a) . genes that were decreased two to eightfold included interferon-stimulated genes such as the single copy antiviral gene mx, ifnα-stimulated genes and (isg - , isg - ) and zc hav (zinc finger ccch-type antiviral protein ) (figure a) . considering this data, we examined whether alv-j induces or inhibits innate immune host responses during both early and late phases of infection. avian leukosis virus subgroup j infection in the early stages resulted in a more than -fold up-regulation of isg mrna at d.p.i. (p < . ) and greater than twofold up-regulation of zc hav mrna at d.p.i. (p < . ). however, alv-j late infection exhibited no statistical differences in isg - and zc hav mrna expression in the tumorigenesis phase (p > . ) ( figure b ). avian leukosis virus subgroup j also induced a significant increase of tlr- at d.p.i. (p < . ) as well as melanoma differentiation-associated gene (mda ) in the tumorigenesis phase (p < . ) (figure c) . these results suggest that alv-j was primarily recognized by chicken tlr and induced isg - expression at d.p.i. chicken mda was the main alv-jsensing pattern-recognition receptor during the late infection phase in vivo. to further explore the differences on innate immune responses between the early and late phases of alv-j infection, we measured cytokine levels in spleen homogenates comparing healthy chickens to chickens with alv-j infection. interestingly, il- , il- , il- β, and ifn-β showed no significant differences between the groups of spf chicks from to d.p.i. (p > . ) ( figure a) . however, clinical chickens with neoplasms showed il- , il- β and ifn-β levels that were significantly altered within the clinical group ( figure b ). in addition, the anti-inflammatory cytokine il- was also increased sharply in two of three clinical samples with neoplasms ( figure b) . taken together these results indicated that alv-j early infection induced no obvious antiviral innate immunity responses in chicks sampled from to d.p.i. however, this was not the case for late infections and there were significant increases in type i ifn, pro-inflammatory cytokines as well as il- . the jak-stat pathway as well as irf- are key regulators of viral immune responses. therefore we measured expression levels of irf and stat via western blotting. compared with the control uninfected chickens, irf and stat levels were decreased in the tumor samples. this was especially true with chicken sc that also displayed liver tumors (figures a,b and see figure a ). innate immunity plays a dominant role in antiviral responses of chickens at - days of age due to the incomplete structural organization of their secondary immune organs (mast and goddeeris, ) . alv transmission primarily occurs at hatching or in the st weeks of life (witter and fadly, ) . accordingly, we deliberately studied the innate immune response of chicks within a week of hatching in response to alv-j infection. our findings showed that cytokine levels were not significantly different between the infected chick group and the control group from to d.p.i. (p > . ). even so, il- , il- , and ifn-β levels were all increased immediately after infection at d.p.i. (figure a) . it was also at this time-point that isg - and tlr expression levels were dramatically increased. hence, day of infection plays a pivotal role in innate immune responses of chicks to some degree. retroviruses can selectively trigger an array of innate immune responses through various pattern recognition receptors (prrs) (van montfoort et al., ) . however, the nature of the exact innate sensors that detect alv-j had remained elusive until now. recognition of hiv- by tlr does not require retroviral replication, and only requires attachment and endocytosis (van montfoort et al., ) . our results showed that alv-j induced a significant increase of tlr- at d.p.i., so we speculated that alv-j was primarily recognized by chicken tlr at d.p.i. the cytoplasmic sensor rig-i can serve as a sensor for hiv genomic rna (van montfoort et al., ) and chickens lack rig-i, but the mda can partially compensate to generate an interferon response (magor et al., ) . as host mrnas, retroviral genomic rnas are capped and polyadenylated (solis et al., ) . during the tumor phase in vivo, alv-j had been integrated into the host genome (li et al., ) . alv-j induced a significant increase of mrna expression of mda in the tumorigenesis phase (p < . ), we speculated that mda was the main sensing receptor during the late phase in vivo and this is consistent with previous reports (hang et al., ) . since il- and ifn-β can induce the expression of ifn-stimulated genes by activating the jak-stat pathway (hoffmann et al., ; wang and zhang, ) , we speculated that alv-j was primarily recognized by chicken tlr . this would lead to regulation of isg - expression via jak-stat pathway activation at d.p.i. however, taken together, the early antiviral innate immune response was too weak to resist alv-j invasion as evidenced by the lack of obvious cytokine expression. in fact, the spf chicks were susceptible to alv-j within - days post hatch. during late infection stages, the secretion levels of il- , il- β, and ifn-β in the three clinical samples with neoplasms had significantly increased. of note was the anti-inflammatory cytokine (il- ) that possessed immunosuppressive effects (sabat et al., ) , and this cytokine was sharply increased in the two of three clinical samples. in other words, alv-j late infection caused significant immune responses including increasing type i ifn, pro-inflammatory as well as anti-inflammatory cytokines. however, there were no statistical differences in isg - and zc hav mrna expression in the tumorigenesis phase. we speculated that functional isgs cannot be induced in the late infection phase. as a tumor suppressor, irf expression is decreased in a variety of human cancers (connett et al., ; wang et al., ) . irf can also exert antiviral activity by inducing interferonstimulated gene expression directly (stirnweiss et al., ) . our previous study demonstrated that irf expression was decreased as a target of mir- b in the white (recessive) plymouth rock chickens with alv-j infection (li et al., ) . in that study we also showed that the antiviral activity of irf was inhibited during the late phase of alv-j infection. there are a number of reports concerning jak-stat pathway inhibition caused by reducing stat expression or by inhibiting its phosphorylation (precious et al., ; audsley and moseley, ) . our previous transcriptome results and the western blotting data presented here demonstrate that stat levels are decreased in chickens with tumors induced by alv-j infection. we hypothesize that alv-j escapes through inhibition of the host antiviral immune response by modulating the jak-stat signaling pathway. additional studies concerning this hypothesis are currently being conducted. in summary, the present study demonstrates that the alv-j strain scau-hn produces an almost undetectable antiviral innate immune response in week old chicks. cytokines were induced in the yellow chickens with tumors caused by alv-j infection. however, ifn-stimulated gene expression was not induced normally during the late phase of alv-j infection. this is most likely the result of a reduction in irf and stat expression. mf participated in the design of the study, performed the experiments, collected and analyzed data, and drafted the manuscript. md performed western blot assay. tx and zl helped with the animal experiment. ms and xz participated in the design and coordination of the study. all authors read and approved the final manuscript. avian gyrovirus and avirulent newcastle disease virus coinfection in a chicken flock with neurologic symptoms and high mortalities paramyxovirus evasion of innate immunity: diverse strategies for common targets avian sarcoma and leukosis virus-receptor interactions: from classical genetics to novel insights into virus-cell membrane fusion tumors associated with avian leukosis virus subgroup j in layer hens during interferon regulatory factor (irf- ) and irf- expression in breast cancer tissue 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for spontaneous haemangiomas in layer chickens in china upregulation of chicken tlr , tlr and myd in heterophils and monocyte-derived macrophages stimulated with eimeria tenella in vitro key: cord- - xf o oy authors: sung, pil soo; shin, eui-cheol; yoon, seung kew title: interferon response in hepatitis c virus (hcv) infection: lessons from cell culture systems of hcv infection date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: xf o oy hepatitis c virus (hcv) is a positive-stranded rna virus that infects approximately – million people worldwide. in , the first hcv infection system in cell culture was established using clone jfh- , which was isolated from a japanese patient with fulminant hcv infection. jfh- replicates efficiently in hepatoma cells and infectious virion particles are released into the culture supernatant. the development of cell culture-derived hcv (hcvcc) systems has allowed us to understand how hosts respond to hcv infection and how hcv evades host responses. although the mechanisms underlying the different outcomes of hcv infection are not fully understood, innate immune responses seem to have a critical impact on the outcome of hcv infection, as demonstrated by the prognostic value of ifn-λ gene polymorphisms among patients with chronic hcv infection. herein, we review recent research on interferon response in hcv infection, particularly studies using hcvcc infection systems. hepatitis c virus (hcv) is a positive-stranded rna virus in the family flaviviridae, and it is estimated - million people are infected with hcv worldwide [ ] . acute hcv infection is spontaneously cured in %- % of patients, but the majority of infected patients fail to clear the virus and develop chronic persistent infection [ ] [ ] [ ] . in addition to a combination regimen of pegylated interferon (ifn)-α and ribavirin, direct acting antiviral drugs (daas) against hcv have been developed, and a high rate of sustained virological response (svr) has been achieved by using these antiviral drugs [ ] . however, the high cost of these drugs results in limited access in developing nations where the disease burden is high; therefore, there is still a need for the development of a prophylactic vaccine. until now, the pathogenesis of hcv infection has not been clearly elucidated yet. importantly, the detailed mechanism of innate immune activation by hcv and its implications for viral persistence and treatment response have not been clearly explained. therefore, understanding hcv-host interactions and immune responses are important novel therapeutics with a higher barrier to viral resistance can be developed [ ] . however, there is no established small animal model for the study of the entire life cycle of hcv infection and immunopathogenesis [ ] . severe combined immunodeficiency mice grafted with human hepatocytes are the only small animals that can be infected with hcv, although they cannot exert adaptive immune responses [ ] . recently, genetically-humanized mouse models are being developed to recapitulate the entire life cycle of hcv [ , ] , but these models have restricted replication of hcv, limiting their utility. as an experimental tool, development of cell culture-derived hcv (hcvcc) systems has dramatically facilitated hcv research over the last years. here, we review recent advances in the research on innate immune response in hcv infection and focus primarily on interferon response of host cells. it was not until , more than years after the discovery of hcv [ ] , that the first efficient cell culture model of hcv became available. the identification of a clinical isolate (genotype a) that replicates efficiently in huh- hepatoma cells [ ] made the first cell culture system possible. this isolate was obtained from a japanese patient with fulminant hcv infection and was called jfh- [ ] [ ] [ ] . viral particles produced by the transfection of huh- cells with in vitro transcribed jfh- rna could infect naïve cells in cell culture and the liver of chimpanzees in vivo [ ] . the hcv virion particles derived from the cell culture system were named "hcvcc" [ ] . until now, only jfh- spontaneously replicates in huh- cells without adaptive mutations and releases infectious virus particles [ , ] . after the discovery of jfh- -based hcvcc system, other hcv cell culture systems with various genotypes were established. for genotype cell culture systems, j cc (genotype a) [ ] and j cc/dh cc/dh cc (genotype b) [ , ] were developed. they replicated and propagated efficiently in huh- . cells, although they had adaptive mutations to facilitate their replication [ , ] . the first genotype a strain, h -s, replicated and released infectious particles in huh- cells and immortalized human hepatocytes, although the amount of released virus was lower than jfh- [ , ] . the con (genotype b) cell culture system was also reported, but a very low level of replication has also limited its utility [ ] . recently, a new cell culture system of genotype a was developed. the tn genome with eight mutations (tncc) [ ] and h c recombinant harboring mutations (h ccc) replicated and spread efficiently in huh- . cells [ ] . recently, a cell culture system for infectious genotype a was also established by introducing adaptive mutations into the s strain [ ] . hcvcc system has some limitations that should be considered. the most important limitation is the restricted availability of genotypes established in cell culture models. currently, hcvcc systems for genotypes , , and are unavailable. for genotypes and , only specific patient clones have been propagated in cell culture systems. it should be noted, however, that a new host factor, sec l was recently reported to enable replication of non-adapted hcv in hepatoma cells [ ] . new cell culture system utilizing sec l -expressing hepatoma cells may overcome the limited availability of hcvcc system. another limitation of the current hcvcc system is the non-polarized nature of huh- -based cells [ , ] . hepatocytes are highly polarized in the liver and cell-to-cell transmission takes an important part in the spread of hcv, but the current hcvcc system does not reflect the viral spread occurring in the infected liver. in addition, huh- cells are not fully differentiated [ ] and, thus, have a defect in activation of the innate immune response by hcvcc infection [ ] . in primary human hepatocytes (phhs), replication and virus production by hcvcc infection have been reported [ ] , but it is difficult to obtain phhs for experimental use. immortalized human hepatocyte was reported to support hcv genome replication, virus assembly, and robust ifn response against the virus [ , [ ] [ ] [ ] and, thus, can be used as an alternative. differentiated hepatocyte-like cells (dhcs) induced from pluripotent stem cells have also been used for hcvcc infection [ ] [ ] [ ] . dhcs were found to mount an efficient innate immune response after hcvcc infection, including the production of chemokines and type iii ifns [ ] . recently, dhcs from adipose tissue-derived human mesenchymal stem cells (at-hmscs) were used for hcvcc infection [ ] , and the entry and replication of hcvcc were found to occur efficiently in dhcs from at-hmscs. hcvcc infection systems provide a unique opportunity to study innate immune responses to hcv infection. here, we focus mainly on recent advances in the study of interferon response in hcv infection. in hcv-infected cells, viral rna is sensed by retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated protein (mda- ) in the cytoplasm and toll-like receptor (tlr ) in the endosome, which leads to downstream signaling that results in the induction of type iii and i ifns and other inflammatory cytokines [ , [ ] [ ] [ ] [ ] . among these receptors, a role of mda- in hcv sensing has remained controversial for several years, and it was recently proven that mda- also participates in hcv sensing in the cytoplasm using hcvcc infection systems [ , , ] . intracellular signals from rig-i, mda- , and tlr are transmitted via mitochondrial antiviral signaling protein (mavs) and toll/il- receptor domain-containing adaptor inducing ifn- (trif), respectively, which leads to the interferon regulatory factor- (irf- )-dependent induction of ifns and nf-κb activation in hcv-infected cells [ , ] . similar to other viruses, hcv uses several mechanisms to interfere with the induction of ifns, particularly ns / a protease. ns / a cleaves mavs, which leads to the impairment of signaling and ifn production in response to hcv rna [ ] . mavs cleavage by ns / a has also been confirmed in hcv-infected liver tissue [ ] . ns a also contributes to immune evasion from the host. ifn-γ expression is inhibited in ns a-transgenic mice after adenoviral challenge, meaning that ns a plays an important role toward establishment of chronic hcv infection [ ] . despite hcv interference with the induction of ifns, ifns are endogenously produced by hcv-infected cells [ , , [ ] [ ] [ ] . both genotype a [ , , [ ] [ ] [ ] and genotype a [ ] were reported to activate intracellular interferon signaling pathways. ifns are currently classified into three major classes: type i, type ii, and type iii. among them, type i and iii ifns are considered innate immune response ifns. among type i ifns, there are ifn-αs, in addition to ifn-β, ifn-ω, ifn-ε, and ifn-κ [ ] . ifn-λs (ifn- or il- ; - or il- a; and - or il- b) are a new family of ifns that have been designated as type iii ifns. since the discovery of ifn-λs in , their functions have been considered to overlap with type i ifns because signaling via the ifn- receptor is similar to that via the ifn-α/β receptor. after binding to their receptors, type i and iii ifns initiate a signaling cascade through the janus kinase (jak)-signal transducer and activator of transcription (stat) pathways. the cellular actions are then mediated by the induction of interferon-stimulated genes (isgs) that have antiviral and/or immunomodulatory activity [ , ] . recently, it was demonstrated that ifn-s are major ifns produced by hcv-infected cells [ , [ ] [ ] [ ] . ifn-s activate the same jak-stat pathway as type i ifns [ ] [ ] [ ] , thereby inducing a similar set of isgs. although the exact source of ifn-s in hcv-infected liver remains to be clarified, it seems that the production of ifn-s by hcv-infected hepatocytes results in the expression of isgs, presumably through autocrine and/or paracrine signaling via the ifn-λ receptor [ , [ ] [ ] [ ] . although hcv interferes with the induction of ifns, continuous isg up-regulation in hcv-infected liver has been demonstrated in chimpanzee models [ , ] and hcv-infected patients [ , ] . interestingly, hcv rna and isg mrna are detected simultaneously in hepatocytes from patients with chronic hcv infection [ ] . this finding suggests that hcv infection potently stimulates the production of endogenous ifns, which leads to isg up-regulation in infected liver [ ] , and that hcv survives under the isg up-regulation perhaps due to the protein kinase r (pkr)-mediated suppression of isg protein translation [ , ] . as a rapid response to type iii and i ifns, ifn stimulated gene factor (isgf ), which consists of tyrosine-phosphorylated stat (py-stat ), tyrosine-phosphorylated stat (py-stat ), and irf , mediates the induction of numerous isgs, including stat , stat , and irf themselves [ ] . recently, it was demonstrated that prolonged induction of a set of isgs is mediated by unphosphorylated isgf (u-isgf ), which is composed of unphosphorylated stat (u-stat ), unphosphorylated stat (u-stat ), and irf [ , ] . the u-isgf level is increased by sustained exposure to ifns, and u-isgf leads to enhanced expression of a set of isgs (u-isgf -downstream isgs, u-isgs) [ ] . in other words, there appear to be two phases of isg expression following type iii or i ifn stimulation. the initial rapid response is driven by the classical phosphorylated form of isgf , which is followed by a second, more prolonged response driven by u-isgf [ ] . in line with this report, we recently demonstrated that endogenous production of type iii and i ifns by hcv infection increases the levels of u-isgf , composed of u-stat , u-stat , and irf proteins [ ] . using hcvcc infection systems with immune-competent liver cells such as phhs and tlr -transfected huh cells, we demonstrated that u-isgf induces the expression of u-isgs [ ] . as mentioned above, isgs induced by endogenous type iii or i ifns are up-regulated in hcv-infected liver [ , , [ ] [ ] [ ] . representative isgs that are maintained at high levels of expression include isg , ifi , ifi , mx , and oas- [ , , [ ] [ ] [ ] . these isgs are regulated not only by isgf but also by u-isgf , and they are mainly antiviral [ , ] . we recently found that increased levels of u-stat , u-stat , and irf are able to inhibit hcv rna replication without exogenous ifn treatment [ ] . this finding suggests that the sustained expression of isgs by u-isgf has antiviral activity against hcv in the infected liver but is insufficient to clear the virus. previously, it was demonstrated that patients with high levels of isgs in their liver at baseline respond poorly to combined therapy with pegylated ifn- and ribavirin [ , , [ ] [ ] [ ] [ ] [ ] . moreover, it has been shown that increased isg expression at baseline is a stronger predictor of a poor response to pegylated ifn-/ribavirin therapy than is the il b genotype [ ] . some reports have emphasized usp as a critical factor conferring unresponsiveness to exogenous ifn-α treatment by suppressing intracellular signaling [ ] [ ] [ ] [ ] . however, the mechanism underlying the increase and maintenance of usp protein levels in hcv-infected liver has not been clearly elucidated. recently, we found that prolonged exposure to ifn- up-regulates u-isgf and u-isgs, including isg , and that isg causes the refractoriness to exogenous ifn- treatment by stabilizing usp protein [ ] . in , the gene ifnl was first described [ ] . ifnl expression is influenced by a germline dinucleotide frameshift variant located in exon of ifnl [ ] . the ifnl -∆g allele generates the full-length ifn-λ protein, whereas the ifnl -tt allele does not create ifn-λ due to a premature stop [ ] . the ifnl -∆g allele is associated with a poor response to pegylated ifn-α/ribavirin therapy [ , ] , and a recent study concluded that the ifnl -∆g/tt genotype is the primary polymorphism underlying poor treatment response in hcv-infected patients [ ] . another study showed that ifnl -∆g genotype is associated with high levels of isgs and that hepatic levels of isg in chronic hepatitis c are strongly associated with ifn-λ expression, suggesting that ifn-λ contributes to induction of isgs in hcv-infected liver [ ] . forced expression of ifnl gene up-regulates isgs in phhs and hepg cells [ , ] , and has antiviral effects against hcv [ ] . recombinant ifn-λ protein activates the jak-stat pathway through binding to the ifn-λ receptor [ ] , evokes similar gene expression pattern to ifn-λ [ ] . future study using hcvcc infection systems will explain the mechanism of ifn-λ induction in hcv-infected cells and the effects of endogenous ifn-λ on both isg induction and the response to exogenous ifn-α treatment. after hcv infection, the expression of some genes is down-regulated, and the expression of those genes tends to be further down-regulated in the liver of non-responders to ifn treatment [ , ] . one of the genes down-regulated in hcv-infected liver is dual specificity phosphatase (dusp ), a mitogen-activated protein kinase phosphatase (mkp) that de-phosphorylates mitogen-activated protein kinases (mapks) [ ] . we demonstrated that silencing dusp expression inhibits hcv replication in hcvcc-infected cells and hcv replicon cells by up-regulating antiviral isgs [ ] . dusp silencing enhances the nuclear translocation of stat and causes the induction of isgs [ ] . although the detailed mechanism of dusp down-regulation in hcv-infected liver remains to be elucidated, this serves as an example of how hosts regulate the expression of isgs and restrict viral infection while bypassing endogenous ifn production. in virus-infected cells, viral peptides are processed and loaded onto major histocompatibility complex (mhc) class i molecules and presented to viral peptide-specific cd + cytotoxic t cells [ ] . recently, we demonstrated using an hcvcc system that ifn-induced up-regulation of mhc class i molecules is attenuated by hcv infection [ ] . hcv rna activates pkr, which phosphorylates the translation initiation factor eif α to block the translation of proteins, including isgs [ ] and mhc class i [ ] . the attenuated expression of mhc class i by hcv infection causes a reduction of the effector functions of hcv-specific cd + t cells [ ] . before our study, several studies had investigated the effect of hcv proteins on mhc class i expression with conflicting results. the expression of mhc class i was not affected by overexpression of hcv proteins in one study [ ] , whereas another study showedup-regulation of mhc class i expression by the hcv core [ ] . by using an hcvcc model, we were able to evaluate the effect of the whole life cycle of hcv infection on mhc class i expression, and we demonstrated the attenuation of ifn-induced mhc class i expression by hcvcc infection. the isolation of the jfh- clone and the establishment of hcvcc infection systems made it possible to perform various studies on host-virus interactions and innate immune responses against hcv infection. now is the era of daas, and hcvcc systems have greatly contributed to the advent of the daa era. however, much remains to be resolved. above all, the precise mechanism of interferon response and its paradoxical contribution to viral persistence should be elucidated. novel cell culture models that closely mimic host responses with various clinical strains are the prerequisite for understanding the pathogenesis of hcv infection and clarifying the mechanism of viral persistence. epidemiology and natural history of hcv infection hepatitis c virus-induced hepatocellular carcinoma hepatitis c virus and hepatocarcinogenesis cd (+) t-cell responses in acute hepatitis c virus infection current and future therapies for hepatitis c virus infection antiviral resistance and direct-acting antiviral agents for hcv animal models for the study of hepatitis c virus infection and related liver disease hepatitis c virus replication in mice with chimeric human livers completion of the entire hepatitis c virus life cycle in genetically humanized mice murine models of hepatitis c: what can we look forward to? isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome efficient replication of the genotype a hepatitis c virus subgenomic replicon complete replication 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using human induced pluripotent stem cells productive hepatitis c virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation microrna- a modulates hcv infection in differentiated hepatocyte-like cells from adipose tissue-derived mesenchymal stem cells mda plays a critical role in interferon response during hepatitis c virus infection control of temporal activation of hepatitis c virus-induced interferon response by domain of nonstructural protein a regulation of hepatic innate immunity by hepatitis c virus immune responses to hcv and other hepatitis viruses eftud is a novel innate immune regulator restricting hepatitis c virus infection through the rig-i/mda pathway cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system inhibition of intrahepatic gamma interferon production by hepatitis c virus nonstructural protein a in transgenic mice hepatitis c virus induces interferon-lambda and interferon-stimulated genes in primary liver cultures il- is the dominant type iii interferon produced by hepatocytes during acute hepatitis c virus infection hcv infection induces a unique hepatic innate immune response associated with robust production of type iii interferons interferons at age : past, current and future impact on biomedicine ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il b inhibits hepatitis c virus replication through the jak-stat pathway interleukin- uses a type interferon-like program to promote antiviral responses in human hepatocytes genomic analysis of the host response to hepatitis c virus infection stealth and cunning: hepatitis b and hepatitis c viruses interferon-induced gene expression is a stronger predictor of treatment response than il b genotype in patients with hepatitis c interferon signaling and treatment outcome in chronic hepatitis c simultaneous detection of hepatitis c virus and interferon stimulated gene expression in infected human liver hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation hepatitis c virus attenuates interferon-induced major histocompatibility complex class i expression and decreases cd + t cell effector functions ifnβ-dependent increases in stat , stat , and irf mediate resistance to viruses and dna damage unphosphorylated stat prolongs the expression of interferon-induced immune regulatory genes roles of unphosphorylated isgf in hcv infection and interferon responsiveness hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis c viral infection cell-type specific gene expression signature in liver underlies response to interferon therapy in chronic hepatitis c infection hepatic gene expression during treatment with peginterferon and ribavirin: identifying molecular pathways for treatment response hepatic isg expression is associated with genetic variation in interleukin b and the outcome of ifn therapy for chronic hepatitis c protein isgylation modulates the jak-stat signaling pathway silencing of usp potentiates the antiviral activity of interferon against hepatitis c virus infection interferon induces long-lasting refractoriness of jak-stat signaling in the mouse liver through induction of usp /ubp interferon-β and interferon-λ signaling is not affected by interferon-induced refractoriness to interferon-α in vivo a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus ifn-λ : the paradoxical new member of the interferon lambda family comparison of functional variants in ifnl and ifnl for association with hcv clearance hepatic expression levels of interferons and interferon-stimulated genes in patients with chronic hepatitis c: a phenotype-genotype correlation study prokunina-olsson, l. expression of interferon λ is associated with reduced proliferation and increased cell death in human hepatic cells interferon-lambda is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden interferon λ signals via the ifnλ receptor to regulate antiviral activity against hcv and coronaviruses transcriptome analysis reveals a classical interferon signature induced by ifnlambda in human primary cells suppression of dual specificity phosphatase i expression inhibits hepatitis c virus replication dual-specificity phosphatases: critical regulators with diverse cellular targets mhc class i antigen presentation: learning from viral evasion strategies expression of hepatitis c virus proteins does not interfere with major histocompatibility complex class i processing and presentation in vitro upregulation of major histocompatibility complex class i on liver cells by hepatitis c virus core protein via p and tap impairs natural killer cell cytotoxicity the authors declare no conflict of interest. key: cord- -rjrw d authors: chen, jidang; ly, hinh title: immunosuppression by viral n proteins date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: rjrw d nan arenaviruses and coronaviruses (cov) are enveloped rna viruses. the nucleoproteins (n) of these viruses (termed nucleocapsid in cov) play a key role in the formation of the viral ribonucleoprotein (rnp) complexes and in causing type i interferon (ifn) suppression. the primary function of the n proteins of cov, such as the severe acute respiratory syndrome coronavirus (sars-cov), and of arenaviruses, such as lassa virus that can cause severe and lethal hemorrhagic fever infections in humans, is to package the viral single stranded genomic rna(s) into rnp complexes [ , ] . another important function of these proteins is to suppress type i interferons (ifns) in infected cells, the molecular mechanisms of which have only recently been elucidated and will be discussed in this article. the host innate immune system presents a significant barrier and defense against viruses. the host pattern recognition receptors (prrs), including retinoic acid-inducible gene i (rig-i), melanoma differentiationassociated protein (mda ) and laboratory of genetics and physiology protein (lgp ), can specifically recognize virus-specific components, such as rna, dna or glycoproteins. following virus recognition by the prrs, important cellular signaling pathways are activated to lead to production of cytokines, chemokines and type i ifns, which play a critical role in the eradication of the virus. arenaviruses and cov both have evolved unique mechanisms to evade recognition by prrs, which lead to type ifn inhibition. immune stimulatory dsrna generated during virus replication is important for rig-i recognition and subsequently mediating type-i ifn production. we have previously discovered that arenaviruses encode a '- 'exoribonuclease function in the c-terminal domain of the n protein (np) that acts as a type i ifn-antagonist [ ] . arenavirus np exoribonuclease, a rnase member of the deddh superfamily, degrades dsrna which is the important signal for rig-i activation. we have shown that recombinant arenaviruses carrying np rnase-defective mutations induce strong ifn responses to inhibit virus replication early in infection in vivo through the activation of the rig-i pathway [ ] . coronaviruses are the only other family of viruses known to encode the deddh exoribonuclease in the n-terminal domain of their non-structural protein (nsp ), which has been attributed to its important proofreading role during viral rna genome replication [ ] . it has recently been shown that nsp exoribonuclease can also inhibit type i ifn production [ ] . recombinant viruses with mutations in one of the zinc-finger motifs of the nsp exoribonuclease domain of the transmissible gastroenteritis coronavirus (tgev) were found to only mildly affect genome replication and transcription, but they appeared to result in less dsrna accumulation in virus-infected cells. the reduction in dsrna levels correlates with a decrease in the levels of type i ifns and of some representative interferon-stimulated genes (isgs). taken together, coronaviral nsp exoribonuclease, similar to that of the arenaviral np exoribonuclease, has the potential to degrade immune stimulatory dsrna during virus replication and thereby suppress type i ifn production. the n protein of cov has recently been found to employ a different way to mediate suppression of type ifn production. hu et al. [ ] showed that the c-terminus of the sars-cov n protein could bind to the spry domain of the cellular trim e ubiquitin ligase. this tripartite motif protein (trim ) e ligase plays important role in post-translational modification of the n terminal caspase recruitment domains (cards) of rig-i by ubiquitination [ ] . the interaction between the c-terminus of n protein of sars-cov with the spry domain of the trim e ubiquitin ligase interferes with the association between trim and rig-i and thereby inhibiting trim -mediated rig-i ubiquitination and activation and type ifn production [ ] . similarly, gack et al. have reported that influenza a virus (iav) contains a novel domain in its ns protein that can also block trim multimerization and rig-i card domain ubiquitination via the interaction between iav ns protein with the coiled-coil domain of trim [ ] . taken together, the molecular mechanism of inhibition of type ifn production via rig-i's ubiquitination by direct protein-protein interaction appears to have been exploited by different viruses that include but are not necessarily limited to sarv-cov and iav. therefore, an in-depth understanding of the conserved molecular mechanisms of innate immune evasion by different viruses may lead to the development of novel broad-spectrum antivirals against many medically significant human viruses. this is an open-access article distributed under the terms of the creative commons attribution license . (cc by . ), which permits unrestricted use, distribution, and reproduction in any medium key: cord- - xauocti authors: huang, chung-guei; lee, li-ang; wu, yi-cheng; hsiao, mei-jen; horng, jim-tong; kuo, rei-lin; huang, chih-heng; lin, ya-chu; tsao, kuo-chien; chen, min-chi; chen, tse-ching; shih, shin-ru title: a pilot study on primary cultures of human respiratory tract epithelial cells to predict patients’ responses to h n infection date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: xauocti avian influenza a(h n ) virus infections frequently lead to acute respiratory distress syndrome and death in humans. we aimed to investigate whether primary cultures of human respiratory tract epithelial cells are helpful to understand h n virus pathogenesis and tissue tropism, and to evaluate how patient-related characteristics can affect the host's response to infection. normal human bronchial epithelial cells (isolated from two different donors) and primary epithelial cells (harvested from patients undergoing airway surgery) were experimentally infected with h n and/or h n pdm for h. after virus infection, the culture media were collected for viral rna quantitation and cytokine detection. both h n and h n pdm viruses replicated and induced a cytokine response differently for each donor in the normal human bronchial epithelial model. h n replicated equivalently in epithelial cells harvested from the inferior turbinate and paranasal sinus, and those from the larynx and bronchus, at h post-infection. viral rna quantity at h was significantly higher in patients aged – years than in patients aged ≥ years; however, no effects of sex, medical comorbidities, and obesity were noted. h n -infected cultured cells released multiple cytokines within h. levels of interleukin- β, interleukin- , interleukin- , interferon-γ, and tumor necrosis factor-α were associated differently with patient-related characteristics (such as age, sex, obesity, and medical comorbidities). in the era of precision medicine, these findings illustrate the potential utility of this primary culture approach to predict a host's response to h n infection or to future infection by newly emerging viral infections, and to dissect viral pathogenesis. avian influenza a(h n ) virus infections are a serious public health threat. h n infections were initially reported in china in march [ ] . the clinical presentation of h n infections varies with the individual; however, fever and cough generally represent the core symptoms [ ] . serious complications include pneumonia, acute respiratory distress syndrome, and death. although the first outbreak has subsided, four subsequent seasonal epidemics were observed in china [ ] . the severe illness of h n infections and the presence of natural reservoirs represent significant concerns. in this context, the identification of vulnerable subjects is paramount to the prevention of spread. h n transmission generally occurs from poultry to humans, and the closure of live poultry markets has been an effective control strategy [ ] . however, evidence also indicates that: ) h n viruses are transmissible from ferrets in a direct contact setting [ ] [ ] [ ] ; ) they can bind to both human and avian receptors [ ] ; ) they can attach to the epithelium in both the upper and lower respiratory tracts of humans [ ] ; and ) they efficiently propagate in human alveolar tissue [ ] . all of these features are important factors for human-to-human transmission. fortunately, h n viruses do not appear to transmit easily from person to person. however, there are many more factors that are important in the adaptation to sustained inter-human transmission. in this scenario, primary cultures of human respiratory tract epithelial cells would be invaluable to understand h n virus tissue tropism and pathogenesis, as well as to evaluate how patient-related characteristics can modulate the host's response to infection. notably, old age (≥ years), male sex, medical comorbidities, and obesity have been previously identified as risk factors for the development of severe disease [ , [ ] [ ] [ ] [ ] [ ] ]. an age-and sex-based analysis of patients infected by the h n virus showed that elderly men are most commonly affected, whereas no deaths were observed in elderly women. age-dependent changes in the airway epithelium mean that older patients may be more prone to respiratory tract infections [ ] and the development of symptomatic h n disease [ ] . moreover, the presence of comorbidities is the only independent risk factor for acute respiratory distress syndrome in h n -infected individuals [ ] . in the most recent epidemic episode in china (during september to december (n = )), distributions of age, sex, and severe illness remained unchanged compared with other epidemics [ ] . nevertheless, the mechanisms by which age, sex, medical comorbidity, and obesity can influence h n virus replication kinetics remain unclear. however, numerous studies have shown that increased levels of specific proinflammatory cytokines and chemokines are robust predictors of morbidity and mortality in h n infected patients [ , , [ ] [ ] [ ] [ ] . h n pdm virus is a swine-origin influenza virus that can transmit efficiently among humans. although the clinical severity of h n pdm was milder than that of avian h n virus, there were many severe h n pdm patients because of the widespread transmission of the virus in the human population in [ ] . compared with h n virus infections, young age (≤ years) and female sex were identified as risk factors for severe h n pdm infection [ ] . respiratory epithelial cell culture systems have been used to investigate the interaction between influenza viruses, as well as other viruses, and their host, [ , [ ] [ ] [ ] [ ] . while these studies have recognized many important characteristics that can help to understand the pathogenesis of influenza viruses, few studies have investigated personto-person differences in virus replication and the influenzarelated cytokines and chemokines of respiratory epithelial cell cultures. recently, mindaye et al. [ ] found different protein expression levels of pro-viral and antiviral factors among normal human bronchial epithelial (nhbe) cells isolated from three different donors, and suggested that this model may provide a way to identify individuals or population groups who are susceptible to severe influenza disease. in the present study, we addressed the hypothesis that respiratory epithelial cells from different human donors would vary in their response to influenza virus infections. to determine the impact of age on cellular response after influenza virus infection, commercial nhbe cells cultured from -year-old and -year-old donors were infected with both h n pdm virus (a/california/ / [ ] ) and avian h n virus (a/taiwan/ -cgmh/ [ ] ), and viral growth kinetics and the cytokine response were compared. we further explored how different donors' characteristics (i.e., age, sex, medical comorbidities, and obesity) could influence both virus replication kinetics and the cytokine response to experimental h n infections. human respiratory tract primary epithelial cells were cultured from patients undergoing upper or lower airway surgery and experimentally infected with h n . the limited amount of primary epithelial cells available from each patient meant that only h n virus infection was conducted. in consideration of biosafety issues, the viral rna quantity was determined to represent changes in viral titer at each time point. figure a demonstrates that the viral www.impactjournals.com/oncotarget figure : changes in viral rna quantity (panel a) and core cytokine levels (panels b-g) in influenza virus infection can induce a cytokine storm, and tumor necrosis factor (tnf), interferon (ifn), interleukin (il)- , il- , and monocyte chemotactic protein- are key cytokine storm mediators [ ] . an earlyonset cytokine storm is associated with h n -related mortality [ , ] ; therefore, we specifically focused on the six cytokines that were found to most consistently predict death in previous studies (i.e., il- β, il- , il- , ifn-γ, interferon gamma-induced protein (ip- ), and tnf-α). such molecules are also produced by human airway epithelial cells [ ] [ ] [ ] [ ] [ ] , and are termed "core cytokines" in the present study. in cases in which the cytokine concentration was undetectable, we assumed a value of . pg/ml for statistical purposes, in accordance with the recommendation of guo et al. [ ] . figure b shows that the il- β levels in the culture media of h n virus-infected -year-old nhbe cells were significantly higher than those of the mock and h n pdm virusinfected -year-old nhbe cells at and h p.i. the il- β levels of the h n virus-infected -yearold nhbe cells were significantly higher than those of mock and h n pdm virus-infected -year-old nhbe cells at h p.i. figure c reveals that the il- levels in the culture media were not related to virus type or the donor's age. in figure d , the il- level of the culture medium of h n -infected -year-old nhbe cells was significantly higher than those of the mock and h n pdm infected cells. figure e shows that the ifn-γ level of h n -infected nhbe cells (either -year-old or -yearold) was significantly higher than those of the mock and h n pdm infected cells. figure f demonstrates that levels of ip- in h n -infected -year-old nhbe cells were significantly higher than the others at , , and h p.i., whereas those of h n and h n pdm virus-infected -year-old nhbe culture media were significantly higher than that of mock at h p.i. figure g demonstrates that the levels of tnf-α of h n virus-infected nhbe cells obtained from -year-old and -year-old donors were significantly higher than the others at h p.i. airway tissue specimens were collected from patients ( table ). there were males ( %) and eight females ( %), with a mean age of . ± . years. patients were further dichotomized into "aged - years (n = [ %])" and "aged ≥ years (n = [ %])" groups. seven ( %) patients had at least one medical comorbidity (hypertension [n = ], diabetes mellitus [n = ] and asthma [n = ]); and nine ( %) were obese (body mass index ≥ kg/m [ ] ). the anatomical sites from which the primary epithelial cell cultures were obtained were as follows: inferior turbinate (n = [ %]), paranasal sinus (n = [ %]), larynx (n = [ %]), and bronchus (n = [ %]). there were relatively fewer numbers of paranasal sinus-and larynxderived cultures; therefore, we combined the inferior turbinate and paranasal sinus-derived cultures as "upper anatomical location" (n = [ %]) and larynx-and bronchus-derived cultures as "lower anatomical location" (n = [ %]) for statistical evaluation. spearman's rank correlation coefficients revealed that a lower anatomical location was significantly inversely associated with male sex (r = - . , p < . ). age ≥ years, medical comorbidity, and obesity were not significantly associated with the other patient characteristics (all p > . ). the epithelial origin (≥ %) of cultured cells was confirmed by immunofluorescence staining using an anti-cytokeratin antibody. when all cultures were considered in combination, viral rna quantities increased significantly at both and h p.i. compared with the baseline, followed by a plateau between and h after infection (figure a ), as suggested by generalized estimating equations (gees), with or without adjustment for anatomical location, age, sex, medical comorbidities, and obesity. gees can account for possible correlations in repeated measures over time and are suitable to explore the differences among different time www.impactjournals.com/oncotarget points [ ] . the percentage change in viral rna quantity between and h was also significant (table ). with regard to virus tropism, viral rna quantities were significantly higher in epithelial cells obtained from the upper anatomical locations than from the lower anatomical locations, without adjustment (p = . ); however, the difference lost significance after adjustment for age, sex, medical comorbidities, and obesity (p = . ; figure b ). viral rna quantities in cells explanted from patients aged ≥ years were significantly lower than those measured in patients aged - years, with or without adjustment for anatomical location, sex, medical comorbidities, and obesity (unadjusted p = . , adjusted p = . ; figure c ). in contrast, no significant sexrelated differences in viral rna quantities were evident ( figure d ). the viral rna quantities in primary epithelial cells from patients with or without medical comorbidities did not differ significantly ( figure e) . similarly, the impact of obesity on viral rna quantities was not evident ( figure f ). the results of the cytokine analysis showed that the concentrations of all six cytokines in virus-infected culture supernatants increased significantly from to h p.i. ( table ). the largest increase was observed for ip- . ip- mediates both necrotic inflammation [ ] and lung injury [ ] ; therefore, our primary culture model supports the hypothesis that a cytokine storm could mediate airway tissue necrosis during the early stages of h n infection. viral rna quantities were positively correlated with the concentrations of ip- and tnf-α at both and h p.i. (table ) . notably, the concentrations of il- , which were previously reported to be increased in the plasma of h n -infected patients during the first week p.i. [ ] , were not significantly associated with viral rna quantities using this primary culture model. the associations between the observed increases in viral rna quantities over time (between and h p.i.) and core cytokine levels were then analyzed in relation to patient-related characteristics (table ) . obesity was positively correlated with increases in viral rna finally, we graphically summarized the changes in core cytokine levels over time in relation to the anatomical sites of the explant and patient-related characteristics. at both and h p.i., the levels of il- and ifn-γ in virusinfected culture supernatants were significantly higher in the upper anatomical location group than in the lower anatomical location group, after adjustment for patientrelated characteristics ( figure a ). the levels of il- β and il- at and h p.i. were significantly higher in patients aged - years, whereas the levels of il- were significantly higher in patients aged ≥ years ( figure b ). male sex was significantly associated with higher levels of il- β, il- , il- , and ifn-γ at h p.i., as well as il- , and ifn-γ at h p.i. (figure c ). the presence of medical comorbidities was not significantly associated with cytokine levels at and h p.i.; however, changes in the il- level were significantly associated with medical comorbidities after adjustment for anatomical location and other patient-related characteristics ( figure d ). except for a change in il- level from to h p.i., obesity was not significantly associated with cytokine levels after adjustment, ( figure e ). in line with the observations obtained in mouse and ferret models [ ] [ ] [ ] ] , the h n virus is not only able to penetrate the human respiratory epithelia [ ] , but also can successfully replicate in ex vivo tissues. however, neither the mouse nor the ferret model can mimic the severity of h n infection in humans. in the present study, our objectives were to discover whether viral replication and cytokine responses of nhbe cells from two donors of different ages are distinct after h n and h n pdm virus infection, and to validate whether these changes are different after h n virus infection using primary epithelial cells from the respiratory tracts of donors with various patient-related characteristics. in the second model, we used a primary culture approach to possibly retain the effects of patient-related characteristics. a standard operation procedure was used to culture all primary epithelial cells to maintain comparable culture conditions. we found that a donor's age might have an effect on viral rna quantities (h n and h n pdm) and cytokine levels (il- β, il- , ifn-γ, ip- , and tnf-α) of the commercial nhbe culture supernatants, and certain patient-related characteristics could modulate viral replication and the cytokine response (e.g. age ≥ years could decrease viral rna quantity and the levels of il- β and il- , and increase the il- level; male sex increased the levels of il- β, il- , il- , and ifn-γ; medical comorbidity increased the il- level; and obesity abbreviations: fdr, false discovery rate; ifn, interferon; il, interleukin; ip- , ifn-γ-induced protein; tnf, tumor necrosis factor. a indicates a statistically significant fdr when the benjamini and hochberg approach for multiple testing correction was applied. increased the il- level at h p.i., and decreased the il- level at h p.i., increased the changes in viral rna quantity, and decreased the changes in il- β and ip- ; figure ) for h n infection in human respiratory tract primary epithelial cells. in addition, we also found that viral replication was equivalent in cells obtained from the upper and lower anatomical locations. specifically, our data suggested that the level of il- is markedly dependent on age, sex, medical comorbidities, and obesity. in general, the levels of several cytokines were found to increase significantly from to h p.i. in nhbe and primary epithelial cell models (table & table ). explant and patients' age, sex, medical comorbidities, and obesity (panels a-e) . a p < . , b p < . , and c p < . , generalized estimating equations in which age, sex, medical comorbidity, obesity, and/or anatomical location were included as confounding variables. among them, il- β and ifn-γ, widely recognized as early markers of h n infection [ , , ] , are involved in the modulation of the inflammatory response [ ] and apoptosis [ ] . high systemic levels of ifn-γ and il- are associated with neutrophil activation [ ] and can predict the need for inpatient care in h n -infected patients [ ] . one of the main findings of this study was that the h n virus showed equivalent and efficient replication in epithelial cells derived from both the upper and lower anatomical locations. this finding indicates that avian influenza a(h n ) virus a/taiwan/ / [ ] , similar to avian influenza a(h n ) virus a/anhui/ / [ ] , can bind to both α , -linked and α , -linked sialic acids of the upper and lower respiratory tract epithelial cells. intriguingly, infections of the upper airways were associated with a marked release of il- , ifn-γ, and tnf-α within h p.i. these observations suggested that the h n virus infection induces a site-specific, amplified, local innate immune response [ ] . notably, chan et al. [ ] found that h n virus infection did not enhance the mrna expression of il- in peripheral blood monocyte-derived macrophages. these findings suggested that il- released by h n -infected epithelial cells of the respiratory tract could be an important clue to the development of severe disease. we demonstrated that the h n virus replicated more slowly and induced lower levels of il- β and il- , accompanied by a more pronounced increase in il- levels, in cell cultures obtained from patients aged > years compared with those from patients aged - years. these observations may be attributable to "immunosenescence," i.e. an age-related decline in immune function accompanied by the dysfunction of chemokine signaling pathways [ ] . in contrast, younger subjects are more likely to survive after h n infection [ , [ ] [ ] [ ] [ ] [ ] . it is possible that the high viral replication and ) the individual cytokine response. the h n virus can replicate equivalently in the epithelial cells obtained from the upper and lower anatomical locations, which may be negatively affected by age (≥ years). after h n infection, the anatomical location and patientrelated characteristics are significantly positively, negatively, or miscellaneously associated with elevated levels of certain core cytokines. occurring in their respiratory tract can act as a reservoir [ , ] . by contrast, advanced age and increased il- levels have been shown to be significant predictors of mortality [ , , [ ] [ ] [ ] [ ] [ ] ] . male sex was significantly associated with higher levels of il- β, il- , il- , and ifn-γ between and h p.i. these findings might explain why men are at a higher risk of more severe disease. in our study, cells from patients with medical comorbidities produced higher il- levels after experimental infection compared with those from otherwise healthy individuals, which might explain the absence of an association between medical comorbidities and the severity of h n infections after adjustment for age and sex [ ] . obesity was significantly associated with a higher increase in viral rna quantities; a higher level of il- at h and a lower level at h p.i.; and less pronounced increases in il- β and ip- . our data indicated that cells derived from obese individuals have subtle but widespread changes in viral replication and cytokine response following experimental h n infection. furthermore, adiposity has been shown to be a critical factor in the age-associated lethal cytokine storm in an animal model [ ] ; nevertheless, the corresponding clinical effect might be modest. some limitations of our study merit comment. first, we used a primary epithelial culture model. consequently, we cannot exclude the possibility that our findings related to h n viral replication and its related cytokine response might be different in vivo. second, because of the limited amount of primary epithelial cells, we did not include control virus infections in the primary culture model of explanted cells to verify the viral response. however, we confirmed that the h n virus could replicate and induce a cytokine response differently from h n pdm virus in the nhbe experiment. moreover, avian influenza a(h n ) virus a/anhui/ / could attach moderately or abundantly to both the upper and lower respiratory tract, and could be replicated in primary cultures of the nasopharynx and bronchus [ ] . nevertheless, the primary epithelial cells used in the present study seemed to reflect the properties of virus infection in patients, where the virus interacts with primary epithelial cells. finally, the relatively small sample size precluded the analysis of a greater number of host characteristics that could affect virus replication kinetics and cytokine response. further studies are needed to confirm and expand our findings. in conclusion, we describe primary epithelial cellular models that might be useful to clarify the influence of patient-related characteristics on h n viral replication and the associated cytokine response. interestingly, il- β, il- , il- , ifn-γ, and tnf-α were the most differentially influenced by grouped host-specific features. our results require independent replication in clinical studies before the widespread use of our primary cellular model can be recommended. however, our platform is less costly and labor-intensive than the use of animal models to mimic h n virus infection. our preliminary results using a primary cellular model have revealed many interesting differences among patient responses, and might represent a potential tool to identify individuals more prone to developing severe h n infections. ethical approval for human tissue explants and the collection of demographic and clinical data was granted by the institutional review board at the chang gung medical foundation, taoyuan, taiwan. ( - b). all the procedures described in the study complied with the principles of the declaration of helsinki and the standards of good clinical practice. written informed consent was obtained from all participants. all experimental procedures were conducted in biosafety level facilities by personnel wearing biosafety level personal protective equipment. between march , and april , , we enrolled patients (n = ) who were scheduled for upper or lower airway surgery at the linkou chang gung memorial hospital (taoyuan city, taiwan). inclusion criteria were as follows: ) age > years; ) need for upper airway surgery (for chronic hypertrophic rhinitis, chronic rhinosinusitis, or laryngeal cancer) or lung surgery (for lung cancer; with no prior history of radiation or chemotherapy); and ) the existence of clinical records detailing the presence of medical comorbidities. patients were excluded if they experienced respiratory tract viral infections within the two weeks preceding surgery, because some influenza viruses (such as h n ) can trigger hypercytokinemia in the early stage and this effect can last for up to two weeks [ , ] . patients were also excluded if they had a known history of chronic viral infections (e.g., human immunodeficiency virus, hepatitis b virus, or hepatitis c virus), and/or were unwilling to participate. ( - yearold and -year-old female caucasians) were purchased from lonza (lonza walkersville, inc., walkersville, md, usa) and maintained in bronchial epithelial growth media (lonza walkersville, inc., walkersville, md, usa). cells at the third passage were grown into a confluent monolayer for subsequent infection experiments. airway tissue specimens were collected from the following anatomical sites: ) inferior turbinate, ) paranasal sinus, ) larynx, and ) bronchus. immediately after harvesting, tissues were submerged in appropriate volumes of ice-cold earl's balanced salt solution to preserve cell viability. epithelial cells were expanded in parallel using standard methods [ ] [ ] [ ] . after rinsing and removal of excess tissue, specimens were minced into cubes ( - -mm ) and transferred onto a six-well transwell cell culture system. each transwell was coated with collagen ( μg/ml), fibronectin ( μg/ml), and fetal bovine serum ( μg/ml) and allowed to adhere. culture medium (dulbecco's modified eagle's medium/ ham f , % antibiotic/antimycotic, % fetal bovine serum) was added basolaterally to the co-culture dishes, and the plates were then incubated at °c, with % co in a humidified atmosphere. the culture solution was changed every two days. when the epithelial cells grew around the explant to cover a - cm area (approximately - weeks), tissue explants were transferred onto new scratched and coated plates. when confluent monolayers were observed, epithelial cells in the transwells were infected with the h n virus. for fixation, % paraformaldehyde was applied to the cell slides at room temperature for min, followed by three washes with phosphate-buffered saline (pbs). cells were permeabilized with pbs containing . % triton x- (sigma-aldrich co. llc, saint louis, mo, usa) twice for min at °c. the cells were then incubated with % bovine serum albumin (bionovas biotechnology co., ltd., toronto, ontario, canada) in pbs for min. slides were then incubated at °c for h with anti-cytokeratin antibodies (abcam plc., cambridge, uk) diluted : with blocking solution (final concentration μg/ml), followed by three washes with pbs. fluorescein isothiocyanateconjugated affinipure goat anti-mouse igg + igm (h+l; jackson immunoresearch laboratories inc., west grove, pa, usa), diluted : to a final concentration of μg/ ml, was applied as the secondary antibody and incubated at °c for min, followed by three washes with pbs. for nuclear staining, slides were incubated with ′, -diamidino- -phenylindole (life technologies, grand island, ny, usa) for min at room temperature, followed by three washes with pbs. finally, the slide was dipped into pbs containing evans blue for background counterstaining to show the localization of the cells. we used influenza a/taiwan/ -cgmh/ (tw / h n ) [ ] and a/california/ / (h n pdm) [ ] in this study. all of the h n infection-related procedures were conducted in biosafety level facilities. a/taiwan/ -cgmh/ is genetically close to the a/anhui/ / strain [ ] . the viral rna quantities were determined by a plaque assay on madin-darby canine kidney epithelial cells. experimental infections of human respiratory tract primary epithelial cells (collected from different anatomical sites, as outlined above) were performed in triplicate. cells ( × cells/well) were initially exposed to serum-free bronchial epithelial cell basal medium (lonza walkersville, inc.) containing . % trypsin, and subsequently infected with the h n virus at a multiplicity of infection of . at °c, % co . mock-infected cells served as a negative control. after infection, cells were washed five times with hanks' balanced salt solution to remove unbound viruses. at each predetermined time-point (see "variables of interest"), virus-infected culture supernatants were collected and stored at − °c until immediately before analysis. all procedures were conducted in biosafety level facilities by personnel wearing biosafety level personal protective equipment. quantitative real-time reverse transcription polymerase chain reaction (qrt-pcr) was used for h n virus load quantitation. viral rna was extracted from culture supernatants using a qiaamp viral rna mini kit (qiagen inc., valencia, ca, usa) and subjected to qrt-pcr. we used the flua primers and probe originally developed by the world health organization collaborating centre in beijing, china ( ). the nucleotide sequences were as follows: forward primer, ′-gaccratcctgtcacctctgac- ′; reverse primer, ′-agggcattytggacaaakcgtcta- ′; probe, ′-fam-tgcagtcctcgctcactgggcacg-bhq - ′. in brief, μl of supernatant was mixed with μl of lysis buffer ( μl for up to × cells). the volume of extracted rna was adjusted to μl with rnase-free water. negative controls consisted of sterile water and were subjected to all the preparation steps in parallel with the extracted samples. a realtime ready rna virus master kit (roche diagnostics, gmbh mannheim, germany) was used for amplification. to set up the amplification reaction, μl of template was added to each reaction tube; the working concentrations for the primers and probe were and μm, respectively. the final volume for the qrt-pcr was μl. reverse transcription and amplification were carried out in a onestep reaction on a bio-rad pcr system (cfx ; bio-rad laboratories, hercules, ca, usa). the conditions for qrt-pcr were as follows: °c for min, °c for min; followed by cycles at °c for s and °c for s. the qrt-pcr was repeated for samples with a ct value > . pcr products were cloned into a plasmid to generate positive controls. for quantification, plasmid dnas at six different concentrations, from copies/μl to copies/μl, were run in parallel with all the samples. the concentrations of six core cytokines in virusinfected culture supernatants were determined using the bio-plex pro human cytokine -plex panel (bio-rad laboratories). specifically, the following analytes were included for statistical analysis: il- β, il- , il- , ifn-γ, ip- , and tnf-α. samples were initially incubated on antibody-coupled beads for min to allow binding, followed by incubation with detection antibodies for min. conjugates were treated with streptavidin for min, washed on a bio-plex pro ii wash station, resuspended, vortexed, and quantified by fluorescence. all data were analyzed using a bio-rad bio-plex luminex instrument. standard curves (log (x) -linear (y)) were generated with the bio-rad bio-plex manager v . software. triplicate measurements of all samples were performed. viral rna quantities and cytokine levels in the h n -infected culture supernatants at h and h p.i. served as the main variables of interest. the differences in viral rna quantities and cytokine levels at h p.i. were expected to be less significant than those at h p.i.; therefore, a more conservative power analysis was performed, based on the variables of interest at h p.i. the priori sample size of the model of primary cultures of human respiratory tract epithelial cells was estimated using outcome effects (viral rna quantities and cytokine levels) based on a pilot study of the model of nhbe cells (mean . [standard deviation . ] and mean . [standard deviation . ] for "aged years" and "aged years", respectively), and a twotailed mann-whitney u test. to reach % power with an effect size of . , a type i error of . , and an allocation ratio . , we obtained a minimum sample size of ( and four patients for "aged - years" and "aged ≥ years", respectively) for comparison of ip- levels at h p.i. viral rna quantities and cytokine levels were log-transformed (log ) to improve the normality of the distribution. to avoid gaps in the assay results and inaccuracies in the estimates of concentrations, a value of . pg/ml was entered in the data set when an analyte was undetectable [ ] . means and standard errors of the mean were used to summarize continuous variables. the viral rna quantities and cytokines were compared using the wilcoxon signed rank test, the kruskal-wallis test, or the mann-whitney u test, as appropriate. at selected time-points, viral rna quantities and cytokine levels were compared according to both the anatomical locations of the explant and patient-related characteristics of interest using gee models [ ] , which were employed to determine whether changes in virus and cytokine levels were significantly different at specific time-points after adjusting for anatomical location and patient-related characteristics, or between different anatomical locations and patient-related characteristics after adjustment for other confounding variables. the use of gee allowed the monitoring of viral rna quantities and cytokine level as a function of time, anatomical location, age, sex, medical comorbidities, and obesity. the anatomical locations of the explants were dichotomized for the purpose of analysis in the upper (inferior turbinate and paranasal sinus) versus lower (larynx and bronchus) respiratory tract. spearman's rank correlation coefficients were used to analyze the associations between patient-related characteristics, viral rna quantities, and cytokine levels. we applied benjamini and hochberg's approach to control the false discovery rate. all statistical calculations were performed with the g * power . . . software (heinrich-heine university, dusseldorf, germany), the ibm spss (version ; international business machines corp.), and the graphpad prism for windows (version . ; graphpad software, inc.) software packages. a p value < . (twotailed) was considered statistically significant. the study was approved by the institutional review board at the chang gung medical foundation, taoyuan, taiwan. 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increased human adaptation compared with avian h n virus th and th hypercytokinemia as early host response signature in severe pandemic influenza influenza virus-induced lung injury: pathogenesis and implications for treatment immunosenescence-related transcriptomic and immunologic changes in older individuals following influenza vaccination how to interpret the transmissibility of novel influenza a(h n ): an analysis of initial epidemiological data of human cases from china pathogenesis of influenza-induced acute respiratory distress syndrome adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice direct association between pharyngeal viral secretion and host cytokine response in severe pandemic influenza the authors thank mrs. hsin-ju wang and mr. jung-chin li for their expert technical assistance. www.impactjournals.com/oncotarget the authors declare no conflicts of interest. this study was financially supported by grants from the ministry of science and technology, taiwan, roc ( - -b- - & - -b- - to lal) and the chang gung medical foundation, taiwan, roc (cmrpg g to cgh; cmrpd d , d , & d to srs). key: cord- - ao chx authors: kim, seong bum; choi, jin young; uyangaa, erdenebileg; patil, ajit mahadev; hossain, ferdaus mohd altaf; hur, jin; park, sang-youel; lee, john-hwa; kim, koanhoi; eo, seong kug title: blockage of indoleamine , -dioxygenase regulates japanese encephalitis via enhancement of type i/ii ifn innate and adaptive t-cell responses date: - - journal: j neuroinflammation doi: . /s - - - sha: doc_id: cord_uid: ao chx background: japanese encephalitis (je), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic je virus (jev). indoleamine , -dioxygenase (ido) has been identified as an enzyme associated with immunoregulatory function. although the regulatory role of ido in viral replication has been postulated, the in vivo role of ido activity has not been fully addressed in neurotropic virus-caused encephalitis. methods: mice in which ido activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. neuroinflammation was evaluated by central nervous system (cns) infiltration of leukocytes and cytokine expression. ido expression, viral burden, jev-specific t-cell, and type i/ii interferon (ifn-i/ii) innate responses were also analyzed. results: elevated expression of ido activity in myeloid and neuron cells of the lymphoid and cns tissues was closely associated with clinical signs of je. furthermore, inhibition of ido activity enhanced resistance to je, reduced the viral burden in lymphoid and cns tissues, and resulted in early and increased cns infiltration by ly- c(hi) monocytes, nk, cd (+), and cd (+) t-cells. je amelioration in ido-ablated mice was also associated with enhanced nk and jev-specific t-cell responses. more interestingly, ido ablation induced rapid enhancement of type i ifn (ifn-i) innate responses in cd c(+) dendritic cells (dcs), including conventional and plasmacytoid dcs, following jev infection. this enhanced ifn-i innate response in ido-ablated cd c(+) dcs was coupled with strong induction of prrs (rig-i, mda ), transcription factors (irf , stat ), and antiviral isg genes (mx , mx , isg , isg , isg ). ido ablation also enhanced the ifn-i innate response in neuron cells, which may delay the spread of virus in the cns. finally, we identified that ido ablation in myeloid cells derived from hematopoietic stem cells (hscs) dominantly contributed to je amelioration and that hsc-derived leukocytes played a key role in the enhanced ifn-i innate responses in the ido-ablated environment. conclusions: inhibition of ido activity ameliorated je via enhancement of antiviral ifn-i/ii innate and adaptive t-cell responses and increased cns infiltration of peripheral leukocytes. therefore, our data provide valuable insight into the use of ido inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses. japanese encephalitis (je) is an acute zoonotic, mosquitoborne disease caused by je virus (jev), a single-stranded, positive-sense rna (~ kb, monopartite, linear) virus belonging to the family flaviviridae and the genus flavivirus [ ] . infection with neurotropic flaviviruses of the je serotype, which include je, murray valley encephalitis, st. louis encephalitis, and west nile virus (wnv), results in debilitating neurological disorders in a significant proportion of clinical cases [ , ] . je is a leading cause of viral encephalitis manifested by extensive neuroinflammation in the central nervous system (cns) and disruption of the blood-brain barrier (bbb). in humans, the clinical presentation of jev infection ranges from mild febrile illness to severe meningoencephalitis [ ] . due to rapid changes in climate and demography, vector-transmitted je poses an increasing threat to global health and welfare with nearly , cases reported annually [ ] [ ] [ ] . the incubation period of je ranges from to days, and most jev infections in endemic regions manifest as mild febrile, subclinical disease which leads to protective adaptive immune responses [ ] . however, approximately - % of je cases, mostly in infants, are lethal and % of cases result in permanent neuropsychiatric sequelae [ ] . thus, je is considered more fatal than encephalitis caused by wnv infection, which has a fatality rate of - % ( deaths/ , symptomatic infections) [ , ] . currently, more than % of the world's population inhabits je endemic areas, such as eastern and southern asia, and the virus is spreading to previously unaffected regions, including indonesia, pakistan, and northern australia [ , ] . however, despite the importance of je, little is known regarding potential therapeutic strategies for regulating je progression. indoleamine , -dioxygenase (ido) has been identified as an enzyme associated with powerful immunoregulatory function, likely derived from its enzymatic activity, which leads to catabolism of the essential amino acid l-tryptophan (l-trp) [ ] [ ] [ ] [ ] [ ] [ ] . therefore, ido-mediated depletion of l -trp and the resulting metabolites (l-kynurenine, l-kyn) induces an immunosuppressive environment through provoking tolerogenicity of antigenpresenting cells (apcs), t-cell anergy, and immune cell death [ , ] . ido can be induced in a variety of cell types, including dendritic cells (dcs) [ ] , macrophages [ ] , and epithelial cells [ ] . these cell types play an important role in controlling viral replication and facilitating antigenspecific adaptive immune responses [ , ] . in various tissues, ido activity has been induced by several cytokines after viral infection, and its enzymatic activity can be blocked using the pharmacological competitive inhibitor, -methyl-d,l-tryptophan ( -mt) [ ] . thus, inhibition of ido with the competitive inhibitor -mt may be a promising strategy for enhancing immune responses in various viral infection models, including human immunodeficiency virus (hiv) and influenza virus [ , ] . also, ido ablation was shown to suppress viral replication through the upregulation of type i interferon (ifn-i) production in a retrovirus-infected murine model [ ] . although the regulatory role of ido in viral replication has been reported in a few studies using an in vivo viral infection model, the in vivo role of ido activity is not fully understood in viral encephalitis caused by infection with neurotropic viruses such as jev and wnv. the molecular pathogenesis of viral encephalitis, including je, remains unclear. however, je is considered an immunopathological disease because cns invasion by jev drives the stimulation of microglia/glia and infiltrating leukocytes, leading to indirect death of neuron cells via the uncontrolled secretion of pro-inflammatory cytokines (such as il- and tnf-α) and soluble mediators [ , ] . furthermore, jev infects and kills neuron cells directly in the cns. this complicated progression of je after viral infection in the host prompted us to explore the role of ido in je progression. here, we found that ido expression in myeloid cells and in neurons of the lymphoid and cns tissues was closely associated with clinical signs of je. furthermore, the inhibition of ido activity using genetic ablation or -mt provided enhanced resistance to je, along with a reduced viral burden and the early and increased cns infiltration of myeloid and lymphoid leukocytes. je amelioration was also associated with enhanced nk and antigen-specific t-cell responses. more interestingly, the rapid enhancement of ifn-i innate immune responses in cd c + dcs (conventional and plasmacytoid dcs) and neuron cells likely contributed to the protective role of ido ablation in je. therefore, our data suggest that the inhibition of ido with pharmacological inhibitors could be a promising therapeutic strategy for regulating je progression. all animal experiments described in the present study were conducted at chonbuk national university according to the guidelines set by the institutional animal care and use committees (iacuc) of chonbuk national university and were pre-approved by the ethical committee for animal experiments of chonbuk national university (permission code - ). the animal research protocol used in this study followed the guidelines set up by the nationally recognized korea association for laboratory animal sciences (kalas). all experimental protocols requiring biosafety were approved by institutional biosafety committees (ibc) of chonbuk national university. c bl/ (h- b ) mice ( - weeks old) were purchased from samtako (o-san, korea). ido (h- b ) knockout (ko) mice were obtained from the jackson laboratory (bar harbor, me, usa). all mice were genotyped and bred in the animal facilities of chonbuk national university. jev beijing- strain was obtained from green cross research institute (suwon, korea) and propagated in the mosquito cell line, c / , using dmem supplemented with % fbs, penicillin ( u/ml), and streptomycin ( u/ml) [ ] . the c / cultures were infected with jev beijing- at a multiplicity of infection (moi) of . and were incubated in a humidified co incubator for h at °c. after absorption, the inoculum was removed, and ml of a maintenance medium containing % fbs was added. approximately - days post-infection (dpi), cultures of host cells showing an - % cytopathic effect were harvested. the virus stocks were titrated using either a conventional plaque assay or a focus-forming assay and were stored in aliquots at − °c until use. the mabs used for flow cytometric analyses and other experiments were obtained from ebioscience (san diego, ca, usa) or r&d systems (minneapolis, mn, usa): fluorescein isothiocyanate (fitc)-conjugated anti-cd (rm - ), cd ( - . ), ly- g ( a ), b (ra - b ); phycoerythrin (pe)-conjugated anti-mouse-cd b (m / ), foxp (fjk- s), cd (mr ); peridinin chlorophyll protein complex (percp)-conjugated anti-ly- c (hk . ); pe-cyanine dye (cy . )-conjugated anti-mouse ifn-γ (xmg . ); pecyanine dye (cy )-conjugated anti-mouse nk . (pk ), il- (trfk ); and allophycocyanin (apc)-conjugated antimouse-cd ε ( - c ), cd (pc . ), cd b (dx ), ly- g (gr- ), tnf-α (mp -xt ), il- (bvd - g ), and il- a (ebio b ). the peptides of i-a b -restricted epitopes (jev ns - [tfvvdgpetkecpd] and ns - [wcfdgprtnail]) and h- d b -restricted epitopes (jev ns b - [savwnstta]) were chemically synthesized at peptron inc. (daejeon, korea). jev-specific primers for the detection of viral rna (jev , - , forward, ′-ccc tca gaa ccg tct cgg aa- ′ and jev , - , reverse, ′-cta ttc cca ggt gtc aat atg ctg t- ′) and primers specific for cytokines, ifn-i (ifnα/β), and rlrs, irfs, isgs (table ) were synthesized at bioneer corp. (daejeon, korea) and used for pcr amplification of target genes. quantitative real-time rt-pcr for viral burden and cytokine expression viral burden and the expression of cytokines (il- , tnf-α, and ifn-α/β), cc chemokines, and ido in inflammatory and lymphoid tissues were determined by conducting quantitative sybr green-based real-time rt-pcr (real-time qrt-pcr). mice were infected intraperitoneally (i.p.) with jev ( . × plaque-forming unit (pfu)) and tissues, including brain, spinal cord, and spleen, were harvested at and dpi following extensive cardiac perfusion with hank's balanced salt solution (hbss). total rna was extracted from tissues using easy-blue (intron, inc., daejeon, korea) and subjected to real-time qrt-pcr using a cfx real-time pcr detection system (bio-rad laboratories, hercules, ca, usa). following reverse transcription of total rna with the high-capacity cdna reverse transcription kits (applied biosystems, foster, ca, usa), the reaction mixture contained μl of template cdna, μl of × sybr premix ex taq, and nm primers, yielding a final volume of μl. the reactions were denatured at °c for s and then subjected to cycles of °c for s and °c for s. after the reaction cycle was complete, the temperature was increased from to °c at a rate of . °c/ s, and the fluorescence was measured every s to construct a melting curve. a control sample that contained no template dna was run with each assay, and all determinations were performed at least in duplicates to ensure reproducibility. the authenticity of the amplified product was determined by melting curve analysis. all data were analyzed using the bio-rad cfx manager, version . analysis software (bio-rad laboratories). viral burden was expressed by the copy number of viral rna per microgram of total rna, after calculating the absolute copy number of viral rna in comparison with the standard cdna template of viral rna. the levels of l-kynurenine in the sera and brain homogenates following jev infection were measured via hplc after deproteination using a c reverse phase column [ ] . the levels of l-kynurenine in the sera and brain homogenates were expressed as micromolars and picomoles per milligram tissue, respectively. levels of l-kynurenine were used for an in vivo index of ido enzyme activity. mice infected with jev were perfused with ml of hbss at or dpi via cardiac puncture of the left ventricle. brains were then harvested and homogenized by gently pressing them through a -mesh tissue sieve, after which they were digested with μg/ml collagenase type iv (worthington biochem, freehold, nj, usa), . μg/ml trypsin inhibitor nα-p-tosyl-l-lysine chloromethyl ketone, μg/ml dnase i (amresco, solon, oh, usa), and mm hepe in hbss for h at °c, under shaking conditions. cells were separated using an optiprep density gradient ( / / %) with centrifugation at ×g for min (axis-shield, oslo, norway), after which cells were collected from the to the % interface and washed twice with pbs. cells were counted and stained for cd b, ly g, ly c, cd ε, cd , cd α, dx , and nk . with directly conjugated antibodies (ebioscience) for min at °c. finally, the cells were fixed with % formaldehyde. data collection and analysis were performed with a facscalibur flow cytometer (becton dickson medical systems sharon, ma, usa) and flowjo (tree star, san carlos, ca, usa) software. jev-specific cd + and cd + t-cell responses were determined by intracellular cd [ , ] , ifn-γ, and tnf-α staining in response to stimulation with jev epitope peptides. surviving mice infected with . × pfu jev were sacrificed at dpi, and splenocytes were prepared. the erythrocytes were depleted by treating single-cell suspensions with ammonium chloride-containing tris buffer (nh cl-tris) for min at °c. the splenocytes were cultured in -well culture plates ( × cells/well) in the presence of synthetic peptide epitopes (ns - , ns - , and ns b - ) for and h, in order to observe cd + and cd + t-cell responses, respectively. monensin at a concentration of μm was added to antigenstimulated cells h before harvest. cells were washed twice with pbs and surface-stained with fitc-anti-cd or cd antibodies for min at °c, and then washed twice with pbs containing monensin. after fixation, the cells were washed twice with permeabilization buffer (ebioscience) and stained with pe cy . -anti-ifn-γ or apc-anti-tnf-α in permeabilization buffer for min at room temperature. intracellular cd was detected by the addition of cd mab to culture media during peptide stimulation. finally, the cells were washed twice with pbs and fixed using fixation buffer. sample analysis was performed with a facscalibur flow cytometer (becton dickson medical systems) and flowjo (tree star) software. intracellular staining for analysis of cd + th , th , and treg cells to monitor cd + th subsets, mice were infected i.p. with . × pfu of jev, sacrificed at dpi, and splenocytes were prepared. splenocytes were then cultured in -well culture plates ( cells/well) with pma/ionomycin (th and th ) in the presence of monensin ( μm) for h at °c. the stimulated cells were washed twice with pbs and surface stained with fitc-anti-cd for min at °c and then washed twice with pbs containing monensin. after fixation, the cells were washed twice with permeabilization buffer (ebioscience) and stained with percp-anti-ifn-γ and apc-anti-il- α in permeabilization buffer for min at room temperature. finally, the cells were washed twice with pbs and fixed using fixation buffer. to monitor treg cells, splenocytes were stained by surface staining for fitc-anti-cd markers for min on ice and then fixed with fixation/permeabilization concentrate buffer (ebioscience) for h at °c. after fixation, the cells were washed twice with permeabilization buffer (ebioscience) and stained with pe-anti-foxp in permeabilization buffer for min at room temperature. the sample analysis was performed using a facscalibur flow cytometer. bone marrow-derived conventional dcs (bmdcs), plasmacytoid dcs (pdcs), and macrophages (bmdms) were prepared from bm cells from bl/ and ido ko mice, as described earlier with some modifications [ ] . briefly, for bmdcs and pdcs, bm cells ( × cells/ml) from femurs and tibiae were cultured in rpmi supplemented with gm-csf ( ng/ml) plus il- ( ng/ml) and flt -l ( ng/ ml), respectively. on day , another ml of fresh complete medium containing gm-csf plus il- and flt -l was added, and half of the medium was changed on day . on day , non-adherent and loosely adherent dcs were harvested by vigorous pipetting. cells were then characterized by flow cytometric analysis, which revealed that the culture generally consisted of > % cd c + cells ( % cd c + cd b + and % cd c + cd α + ), and > % cd c + pdca- + b + . bmdms were prepared by culturing bone marrow cells in dmem supplemented with % l cell-conditioned medium (lccm) as a source of macrophage colony-stimulating factor (m-csf). on day , another ml of fresh complete medium containing % lccm was added, and half of the medium was changed on day . the cultured cells were harvested following an -day incubation and were analyzed by flow cytometry. the prepared bmdms were composed of > % f / + cells that consisted of . % f / + cd b + and~ % f / + cd c + cells. prepared bmdcs, pdcs, and bmdms were infected with jev at mois of . , . , and for analysis of viral replication and moi for evaluating cytokine expression. primary microglia cells were recovered from the brains of fetal bl/ and ido ko mice ( - days old), as described previously [ ] . primary microglia cells recovered from the brains via trypsin + edta digestion were initially seeded on poly-d-lysine/laminin-coated plates in dmem containing % fbs and mechanically isolated from glia cells by a brief duration of shaking ( rpm, h). primary microglia cells were infected with jev at mois of . and for analysis of viral replication and moi for the evaluation of cytokine, ifn-i, rlr, and isg gene expression. primary cortical neurons were prepared from -day-old embryos, as described previously [ ] . cortical neurons were seeded in -well poly-d-lysine/laminin-coated plates in dmem containing % fbs and % horse serum for h, and then cultured for days with neurobasal medium containing b supplement and l-glutamine (invitrogen, carlsbad, ca, usa). primary cortical neurons were infected with jev at mois of . and . for analysis of viral replication and . moi for the evaluation of cytokine expression. generation of bm chimeric mice and determination of serum ifn-β bl/ mice ( weeks old) and ido ko mice were irradiated with one dose of rads. within h, recipient mice were reconstituted with donor bm cells derived from bl/ and ido ko mice. the recipient mice were given sulfamethoxazole and trimethoprim in their drinking water for days after irradiation. mice were infected with jev - weeks after irradiation. a commercial elisa kit (pbl biomedical laboratories, piscataway, nj, usa) was used to measure levels of secreted ifn-β protein in sera according to the manufacturer's protocol. all data were expressed as the average ± standard deviation, and statistically significant differences between groups were analyzed by unpaired two-tailed student's t tests for ex vivo experiments and immune cell analyses or anova and post hoc test for multiple comparisons of the mean. the statistical significance of viral burden was evaluated using the mann-whitney test or unpaired two-tailed student's t test. kaplan-meier survival curves were analyzed with the log-rank test. a p value of ≤ . was considered significant. all data were analyzed using prism software (graphpad prism , san diego, ca, usa). to determine whether ido expression changes during je progression, several tissues obtained from jevinfected bl/ mice were used to evaluate ido mrna expression at the early stage of infection (from to dpi) prior to the presentation of neurological disorders. as shown in fig. a , jev infection induced no apparent changes in the expression of ido in the examined tissues, including lymphoid (spleen, mesenteric ln, bone marrow), extraneural (liver), and cns tissues (brain, spinal cord) prior to the onset of neurological disorders. in general, infected mice showed clinical signs starting with generalized piloerection, paresis, and rigidity, which then progressed to severe neurological signs, such as postural imbalance, ataxia, and generalized tonic-clonic seizure, by to dpi. thus, we were interested in testing whether ido expression varies between mice showing clinical signs, such as paralysis, and mice displaying no clinical signs. to this end, mice were divided into two populations showing either paralysis or no paralysis dpi, the point at which approximately - % of infected mice showed neurological disorders, and the expression of ido mrna was evaluated in several tissues. interestingly, enhanced induction of ido expression was observed only in lymphoid tissues (spleen and bone marrow) and the cns (brain and spinal cord) of mice showing paralysis compared to mice showing no paralysis (fig. b) . however, other extraneural tissues, including mesenteric ln and liver, showed no apparent increases in expression of ido in paralyzed mice. ido expression at the protein level was also confirmed by western blotting using total lysates derived from several tissues. in agreement with the enhanced induction of ido mrna expression in paralyzed mice, mice showing paralysis displayed an apparent increase in the expression of ido protein in lymphoid tissues (spleen, bone marrow) and neural tissues (brain and spinal cord) (fig. c) . dcs and macrophages in peripheral tissues are the primary target cells of jev, and neuron cells support jev replication after the virus gains access into the cns. also, microglia, tissue-resident macrophages in the cns are believed to regulate neuroinflammation caused by various insults. therefore, we evaluated ido expression in primary myeloid-derived dcs, pdcs, macrophages, microglia, and primary cortical neuron cells after jev infection. conventional dcs (bmdcs), pdcs, macrophages (bmdms), microglia, and neuron cells displayed different dynamic patterns of ido expression, depending on the time after jev infection (fig. d) . pdcs showed the most rapid expression of ido, with levels peaking at h pi, whereas bmdcs displayed a delayed expression pattern with the levels peaking at h pi. ido was also expressed in macrophages with gradual and moderate increases in levels up to h pi, and microglia showed basal levels of ido expression up to h pi and increased levels thereafter. neuron cells showed a gradual increase in ido expression with approximately fivefold higher levels at h pi compared to levels in the mockinfected group. this result indicates that the primary target cells in the periphery and cns tissues can actively express ido with different intrinsic patterns in response to jev infection. collectively, these results indicate that ido expression in myeloid cells and neurons of the lymphoid and cns tissues is closely associated with the clinical signs of je. notably, ido expression in the neural tissues (brain and spinal cord) was likely to be coupled with neurological disorders such as paralysis. blockage of ido provides enhanced resistance to je because ido expression in lymphoid and neural tissues was closely associated with je progression, we were interested in investigating the role of ido during je progression. bl/ mice infected with jev showed gradually elevated release of the tryptophan catabolite l-kynurenine by cells expressing functional ido enzyme activity in the sera and brain homogenates relative to basal levels in ido ko mice (fig. a) , which suggests that ido expression increases during je progression. also, we examined and compared the susceptibility of ido-deficient (ko) mice to je caused by neurotropic jev infection with that of wild-type bl/ mice (fig. b) . the ablation of ido resulted in a markedly increased survival rate of % in ido ko mice vs. % in bl/ mice after jev infection ( . × pfu) (left curve in fig. b) , thereby signifying significantly enhanced resistance to je. likewise, ido ko mice showed delayed signs of neurological disorder starting - dpi compared to bl/ mice, which displayed signs of neurological disorder sooner and in a higher proportion of animals (middle graph in fig. b) , and ido ablation resulted in less change in body weight (right graph in fig. b) . these results indicate that , and liver (liv), , , and days following jev infection. b comparison of ido expression between aparalytic and paralytic hosts. ido expression was determined by real-time qrt-pcr using total rna extracted from several tissues in mice showing a neurological disorder such as paralysis (paralytic) and mice showing no paralytic symptoms (aparalytic) dpi. c detection of ido protein during je progression. ido protein levels in several tissues of jev-infected mice were evaluated by western blot using a monoclonal antibody specific for ido. differences in ido protein levels between aparalytic and paralytic hosts were evaluated after mice were divided into groups showing either aparalytic (ap) or paralytic symptoms (p) dpi. d ido expression in primary myeloid cells, microglia, and cortical neurons after jev infection. bone marrow-derived dcs (bmdcs), plasmacytoid dcs (pdcs), macrophages (bmdms), microglia, and primary cortical neurons were infected with jev ( moi for bmdcs, pdcs, bmdms, and microglia; . moi for neurons) and ido expression was determined by real-time qrt-pcr at the indicated time points. the levels of ido expression were expressed as fold relative to mock-infected cells ( h). *p < . ; **p < . ; ***p < . compared to levels in the indicated groups ido ablation ameliorates je progression. furthermore, we tested the effect of the ido-specific inhibitor -methyl-[d]tryptophan ( -mt) on je progression. as suggested by our other results, oral treatment with -mt resulted in reduced mortality, delayed neurological disorder, and less change in body weight compared to untreated bl/ mice (fig. c) . therefore, this result strengthens our finding that ido ablation provides enhanced resistance to je. to better understand je progression in ido-ablated mice, we examined viral burden in peripheral lymphoid and cns tissues after jev infection. ido ko mice were observed to contain less amount of virus, with around a tenfold decrease in the spleen and brain compared to levels in bl/ mice (fig. d) . ultimately, these results suggest that ido ablation ameliorates je progression by regulating the viral burden in peripheral lymphoid and cns tissues. cns infiltration by cd b + ly- c hi monocytes is a hallmark of cns inflammation caused by neurotropic viral infection [ ] . these cells migrate into the infected brain, where they differentiate into dc, macrophage, and microglia populations [ ] [ ] [ ] [ ] . although the potential contribution of cd b + ly- c hi monocytes to neuroinflammation remains controversial, cns infiltration by cd b + ly- c hi monocytes is believed to support their protective role during lethal neuroinflammation [ ] [ ] [ ] [ ] . therefore, in order to better understand the amelioration of je in ido ko mice, we examined cns infiltration by leukocytes, including cd b + ly- c hi monocytes, during je progression. wild-type bl/ and ido ko mice contained comparable levels of cd b + ly- c hi monocytes and cd b + ly- g hi granulocytes in the brain before jev infection. however, the frequency of cd b + ly- c hi monocytes gradually increased in the cns of ido ko mice during je progression (fig. a) . also, cns infiltration by cd b + ly- g hi granulocytes was transiently increased in ido ko mice dpi, after which the ly- g hi granulocyte frequency was comparable in both bl/ and ido ko mice dpi. similarly, the accumulated total number of cns-infiltrated ly- c hi monocytes and ly- g hi granulocytes was increased in ido ko mice, compared to those of bl/ mice (fig. b) . furthermore, because cd + th , cd + , and nk cells may play beneficial roles in controlling je progression [ ] [ ] [ ] [ ] , we examined cns infiltration of nk, cd + , and cd + tcells. as with cns infiltration by ly- c hi monocytes, cns infiltration by nk, cd + , and cd + t-cells was observed to gradually increase in ido ko mice compared to levels observed in bl/ mice (fig. c, d) . notably, cd + t-cells showed marked infiltration with threefold increased levels in the cns of ido ko mice. with respect to cns inflammation, the expression of cytokines and chemokines within the cns may be required to fully explain encephalitis, because encephalitis caused by neurotropic viruses is indirectly derived from cns degeneration caused by robust immunological responses, such as the uncontrolled secretion of cytokines, including tnf-α, and the resultant activation of microglia and astrocytes [ , ] . therefore, we examined the expression of tnf-α and cc chemokines in the cns. our results revealed that the expression of cc chemokines was comparable in both bl/ and ido ko mice, except that the expression levels of tnf-α and ccl were modestly decreased in ido ko mice (fig. e) . this result implies that cc chemokine expression may not play a role in increased cns infiltration by ly- c hi monocytes, nk, cd + , and cd + t-cells. collectively, these results suggest that ido ablation allows for early and increased cns infiltration by myeloid and lymphoid leukocytes, which may mediate the early control of viral replication in the cns. antiviral innate nk cell activation is believed to play an important role in regulating je progression through the control and clearance of jev in extraneural and neural tissues [ ] . therefore, in order to characterize the immunological parameters associated with control of jev replication in ido ko mice, we examined and compared nk cell responses in both bl/ and ido ko mice. because jev was administered intraperitoneally, analysis of the spleen provides insight into how ido modulates the innate immune and inflammatory responses during the early phase of infection. analysis of splenic nk cells revealed that bl/ mice exhibited a reduction in cd − nk . + dx + nk cells following jev infection (fig. a) , as (see figure on previous page.) fig. blockage of ido enhances resistance to je and reduces viral burden. a levels of l-kynurenine in the sera and brain of bl/ and ido ko mice. following jev infection, levels of l-kynurenine in the sera and brain homogenates were estimated by hplc at different time points. b susceptibility of idoablated mice to je. bl/ and ido ko mice ( to weeks old, n = - ) were inoculated i.p. with jev ( . × pfu) and examined over days for their survival. c enhanced resistance to je by ido inhibition. bl/ mice were infected i.p. with jev and administered an ido inhibitor ( -mt, and mg per mouse) every day. left graph, curve showing survival rates; middle graph, proportion of mice showing neurological disorders during je progression every h from to dpi; right graph, changes in body weight. changes in body weight were expressed as the mean percentage ± sd of body weight relative to the time of challenge. d viral burden in lymphoid and inflammatory tissues during je. viral burden in the spleen and brain of infected mice was assessed by real-time qrt-pcr at the indicated time points post-infection. the viral rna load was expressed as viral rna copy number per microgram of total rna (n = - ). *p < . ; **p < . ; ***p < . compared with levels in the indicated groups or in bl/ control mice if wild-type mice previously showed decreased number of nk cells [ ] . however, ido ko mice showed less of a reduction in the number of cd − nk . + dx + nk cells. consequently, an increase in the total number of splenic nk cells was detected in ido ko mice compared to bl/ mice. moreover, when the activation of nk cells was evaluated by the production of ifn-γ and granzyme b from nk cells, the frequency and the total number of cd − nk . + dx + nk cells producing ifn-γ and granyzme b were apparently increased in ido ko mice (fig. b, c) . therefore, this result indicates that increased activation of nk cells plays a role in the early control of jev replication, thereby resulting in the amelioration of je progression. in addition to nk cells, adaptive immune responses specific for jev antigen are required for the regulation of je progression through peripheral control of jev replication [ ] [ ] [ ] [ ] . furthermore, ido may in some cases affect antigen-specific antibody responses [ , ] , which contribute to the control of jev dissemination and replication in the brain. our data revealed that ido ablation induced no significant changes in serum igm and igg specific for jev antigen (fig. a) . because ido is known to suppress t-cellmediated adaptive immune responses in a range of clinically relevant syndromes, including autoimmune, allergic, and infectious diseases [ , ] , we also evaluated cd + and cd + t-cell responses specific for jev antigen in surviving bl/ and ido-ablated mice dpi. ido ablation resulted in moderately increased jev-specific cd + t-cell responses when cd + t-cell responses were evaluated by intracellular cd , ifn-γ, and tnf-α staining in response to stimulation with two epitope-peptides (ns - and ns - ) derived from jev (fig. b) . consistent with this finding, the total number of cd + t-cells producing ifn-γ or tnf-α in response to jev epitope stimulation was higher in idoablated mice (fig. c) . furthermore, somewhat interestingly, ido-ablated mice displayed markedly increased cd + tcell responses with three-to fivefold higher levels, compared to bl/ mice (fig. d) , and consistently contained a higher total number of jev-specific cd + t-cells producing the frequency and number of ly- c hi monocytes and ly- g hi granulocytes in the cns. the frequency (a) and total number (b) of ly- c hi monocytes and ly- g hi granulocytes in the cns were determined by flow cytometric analyses and dpi using vigorous heart perfusion. values presented in the representative dot plots denote the average percentage of the indicated population after gating on cd b + cells (n = - ). c, d accumulated number of nk cells, cd + , and cd + t-cells in the cns. total accumulated number of nk cells (cd − nk . + dx + ), cd + (cd + cd + ), and cd + (cd + cd + ) t-cells in the cns were enumerated by flow cytometric analysis and dpi. e the expression of tnf-α and cc chemokines in the cns. the expression levels of tnf-α and cc chemokines were determined by real-time qrt-pcr using total rna extracted from brain tissue dpi. data show the average ± sd of the indicated cell populations derived from at least five mice per group. *p < . ; **p < . ; ***p < . compared with levels in the indicated groups ifn-γ or tnf-α in response to a jev cd + t-cell epitope (ns b - ) (fig. e) . these results indicate that the increased cd + and cd + t-cell responses generated in ido-ablated mice could contribute to the control of je progression during the late stage of infection. cd + cd + foxp + treg cells may contribute to controlling lethal neuroinflammation caused by neurotropic viruses [ ] . in contrast, il- + cd + th cells play a critical role in autoimmune and virus-caused immunopathologic diseases by facilitating pathologic consequences through neutrophil recruitment [ , ] . the generation of cd + cd + foxp + tregs is reciprocally linked with il- + cd + th and ifn-γ + cd + th cells [ , ] . because ido may regulate the equilibrium of cd + foxp + tregs and il- + cd + th cells in inflammatory diseases [ , ] , we examined the generation of each cd + th subset, including cd + foxp + tregs, il- + cd + th , and ifn-γ + cd + th cells, early during je progression. our results revealed that ido-ablated mice showed no significant alterations in the frequency or number of cd + cd + foxp + tregs, compared to wild-type bl/ mice (fig. a, b) . in contrast, ifn-γ + cd + th cells were detected at higher levels in ido-ablated mice, and the frequency and number of il- + cd + th cells were not changed by ido ablation (fig. c, d) . therefore, this result suggests that the increase in ifn-γ + cd + th cells in ido-ablated mice contributes in part to the control of je progression during the early stage of infection. myeloid cells, including dcs and macrophages, are the primary target cells of jev infection in the peripheral tissues and function to regulate the spread of virus to distant tissues such as the cns [ ] . furthermore, myeloid cells can produce ifn-i via prr recognition upon jev infection, which plays a crucial role in controlling viral replication [ , ] . additionally, ido ablation appears to regulate viral replication via ifn-i production [ ] . because viral loads at the periphery in ido-ablated mice were lower than in wild-type bl/ mice, we evaluated the contribution of dc subsets (conventional and plasmacytoid) and macrophages to the ifn-i innate immune responses caused by jev infection in the idoablated environment. in order to assess the role of ido in inducing the ifn-i innate response of jev-infected dc subsets and macrophages, bmdcs, pdcs, and macrophages (bmdms) prepared from ido-ablated mice were infected with jev and used to evaluate viral replication and the induction of ifn-i and pro-inflammatory cytokines. interestingly, ido-ablated bmdcs and pdcs, but not bmdms, showed significantly lower jev replication (fig. a-c) . notably, bmdcs derived from ido-ablated mice exhibited impaired jev replication throughout the examination period compared to replication in cells from wild-type bl/ mice. furthermore, the inhibition of jev replication in bmdcs and pdcs derived from idoablated mice was closely associated with enhanced expression of ifn-i (ifn-α/β) following jev infection (fig. d) . ido-ablated bmdcs and pdcs, but not bmdms, showed rapid induction of ifn-α/β in response to jev infection compared to levels measured in cells from wild-type bl/ mice. however, it was curious that pdcs showed less apparent regulation of viral replication than bmdcs, despite pdcs inducing stronger ifn-i innate responses. presumably, this discrepancy is related to cell-intrinsic properties or other mechanisms involved in regulating viral replication. in addition, rapid and increased induction of il- and tnf-α mrnas was observed upon jev infection in ido-ablated bmdcs (fig. e) . collectively, these results imply that rapid and increased ifn-i innate responses in cd c + dc subsets (conventional dcs and pdcs) may contribute to the early control of viral replication in the absence of ido. to further characterize ifn-i innate responses in jev-infected dc subsets derived from ido-ablated mice, we also measured the induction levels of isg genes. we specifically focused on pattern recogni- ). our results revealed that bmdcs and pdcs derived from ido-ablated mice showed rapid and enhanced expression of the stat , oas , mx , mx , and isg genes with slightly different patterns at h pi, whereas bmdms derived from ido-ablated mice showed either no alteration or a slight decrease in the expression of prrs, ifr and irf , and isg genes (fig. ) . also, it was interesting that bmdcs and pdcs derived from ido-ablated mice displayed early enhanced induction of prrs (rig-i and mda ) and their transcription factor (irf ) at h pi. this indicates that rapid and increased expression of isg genes in jev-infected bmdcs and pdcs follows the inhibition of jev replication via enhanced expression of ifn-α/β. collectively, ido ablation appears to provide rapid and increased responses of ifn-i innate immunity in myeloid-derived dcs and pdcs upon jev infection, thereby contributing to the amelioration of je through early control of viral replication. microglia cells are cns-resident macrophages that play an important role in regulating the neuroinflammation caused by sterile and non-sterile insults [ ] . bystander damage caused by pro-inflammatory mediators released from microglia likely contributes to the exacerbation of je progression. furthermore, because ido-ablated microglia are considered to show stronger inflammatory responses after the virus gains access to the cns, we examined the innate immune responses of primary microglia cells derived from the brain of fetal bl/ and ido ko mice in response to jev infection. our results revealed that primary microglia recovered from the brain of ido-ablated mice showed similar innate immune responses to jev infection as shown in macrophages derived from bm cells of ido ko mice. ido-ablated microglia were observed to allow jev replication with moderately but not significantly lower levels compared to those of bl/ mice (fig. a) . consistent with this, ido-ablated microglia displayed no apparent enhancement of ifn-i innate responses when the expression of ifn-α/β and isg genes (isg , isg , isg ) was examined (fig. b, c) . also, no significant differences in the expression of rlrs and transcription factors irf and irf were observed between microglia derived from ido ko and bl/ mice (fig. d, e) , and both idoablated and wild-type microglia showed comparable expression of pro-inflammatory cytokines tnf-α and il- in response to jev infection (fig. f ) . therefore, these results indicate that microglia could not provide dominant contribution to the regulation of jev (see figure on previous page.) fig. ido ablation enhances cd + and cd + t-cell responses specific for jev antigen. a jev e-specific igm and igg response. sera were collected from surviving mice dpi and used in elisa to detect igm and igg levels specific for the jev e protein. the data show the average ± sd of the jev e-specific igm and igg levels derived from surviving mice (n = - ). b, c cd + t-cell response specific for jev antigen. the splenocytes prepared from surviving mice (n = - ) were stimulated with the jev epitope peptides of cd + t-cells (ns - and ns - ) for h. the frequency (b) and absolute number (c) of cd + t-cells specific for the jev epitope peptides were determined by intracellular cd , ifn-γ, or tnf-α staining, combined with surface cd staining. d, e cd + t-cell response specific for jev antigen. the frequency (d) and absolute number (e) of cd + t-cells specific for the jev epitope peptide (ns b - ) were determined by intracellular ifn-γ or tnf-α staining after an -h stimulation with peptide. values in the representative dot plots denote the average percentage of the indicated cell population, and the bar charts show the average ± sd of the values derived from at least four mice per group. *p < . ; **p < . ; ***p < . compared with levels in the indicated groups dissemination and je progression in the cns under an ido-ablated environment. neurons are the main target cell of jev replication within the cns, and their death is a key factor in the pathogenesis and neurological sequelae of jev [ ] . furthermore, neuron cells have also been shown to produce antiviral ifn-i in response to viral infection and are consequently involved in controlling viral replication in the cns [ , ] . therefore, we examined viral replication and ifn-i innate immune responses in primary cortical neuron cells generated from wild-type bl/ and ido-ablated mice after jev infection. consistent with the results obtained from bmdcs and pdcs, primary cortical neurons derived from ido-ablated mice showed transiently reduced jev replication (fig. a) . this transient reduction of jev replication in ido-ablated neurons was associated with significantly increased induction of ifn-i (ifn-α/β), relative to the levels measured in cells from wild-type bl/ mice (fig. b) . also, it seemed that the expression of antiviral isg genes (isg , isg , isg ) in ido-ablated neurons followed ifn-i innate responses and the transient reduction in viral replication (fig. c) , whereas the expression levels of transcription factors (irf , irf ), but not prrs (rig-i, mda ), were decreased by jev infection (fig. d, e) . therefore, these results suggest that the ablation of ido could provide enhanced ifn-i innate immune responses in neuron cells to regulate the spread of jev in the cns. the frequency and number of cd + foxp + tregs in the spleen of ido-ablated mice. the frequency (a) and absolute number (b) of cd + foxp + treg cells in the spleen of wild-type and ido ko mice were determined by flow cytometric analysis dpi. the values in the representative dot plots show the average percentage of cd + foxp + cells in cd + t-cells; the bar charts denote the average number ± sd of cd + foxp + tregs in the spleen derived from at least four mice per group. c, d the frequency and number of ifnγ + cd + th and il- + cd + th cells in the spleen of ido-ablated mice. the frequency and number of ifn-γ + cd + th and il- + cd + th cells were determined by intracellular cytokine staining of the splenocytes prepared from wild-type and ido ko mice dpi in response to pma + ionomycin stimulation. the values in the representative dot plots show the average percentage of the indicated cell populations after gating on cd + t-cells; the bar charts denote the average number ± sd of ifn-γ + cd + th and il- + cd + th cells in the spleen derived from at least four mice per group. **p < . compared with levels in the indicated groups ido ablation in hsc-derived leukocytes alleviates je via enhancement of the ifn-i innate response our results support the idea that ido ablation ameliorates je progression by provoking potent antiviral ifn-i innate responses in myeloid-derived dcs and pdcs at the periphery. therefore, we were interested in confirming whether myeloid cells derived from hematopoietic stem cells (hscs) play a dominant role in regulating je progression in ido-ablated mice via the induction of enhanced ifn-i innate responses. to this end, we used a bm chimeric model of wild-type bl/ and ido-ablated mice. in support of our hypothesis, our results revealed that myeloid cells derived from hscs played a dominant role in conferring amelioration of je in the ido-ablated environment. specifically, wild-type bl/ recipients of ido ko bm donor cells (ko-wt) showed enhanced resistance to je, compared to the ido ko recipients of wild-type bl/ bm donor cells (wt-ko) and wild-type bl/ recipients of wild-type bl/ bm donor cells (wt-wt) (fig. a) . furthermore, ko-wt and ko-ko bm chimeric models showed less change in body weight after jev infection compared to the other bm chimeric models (fig. b) . also, potent and rapid ifn-i innate immune responses were observed in ko-wt and ko-ko bm chimeric models compared to those observed in the wt-wt bm chimeric model, as evaluated by the determination of serum ifn-β (fig. c) . this result indicates that myeloid cells derived from hscs of ido-ablated mice play a dominant role in the ifn-i innate response in ido ko hosts. collectively, it appears that the ablation of ido in myeloid cells derived from hscs has an important role in ameliorating je by inducing a potent and rapid ifn-i innate immune response. in this study, ido expression from myeloid and neuron cells located in extraneural and neural tissues during je progression appears to be closely associated with clinical signs such as neurological disorders. furthermore, we demonstrated that inhibition of ido activity through genetic ablation or use of an ido inhibitor ( -mt) enhanced the resistance to je progression induced by jev infection. in our mechanistic studies on the role of ido in regulating je fig. the expression of rlr, irf, and isg genes in bmdcs, bmdms, and pdcs following jev infection. bmdcs, bmdms, and pdcs recovered from wt and ido ko mice were infected with jev at a moi of and used to analyze the induction of irf, isg, and rlr genes at and h pi. the expression of each irf, isg, and rlr gene was normalized to that of β-actin after determining the mrna levels by real-time qrt-pcr and displayed as the average of at least four independent samples, according to the indicated color on a log scale (see figure on previous page.) fig. ifn-i innate immune responses of ido-ablated myeloid-derived cells after jev infection. primary bone marrow-derived conventional dcs (bmdcs), plasmacytoid dcs (pdcs), and macrophages (bmdms) recovered from bl/ and ido ko mice were infected with jev at mois of . , . , and for viral replication and for cytokine expression. jev replication and the expression of cytokines and ifn-α/β were evaluated by real-time qrt-pcr using extracted total rna. a-c jev replication in bmdcs, bmdms, and pdcs. d, e ifn-α/β, il- , and tnf-α expression in bmdcs, pdcs, and bmdms. the bar charts show the average ± sd of the values derived from bmdcs, bmdms, and pdcs assayed in quadruplicates. *p < . ; **p < . ; p < . compared with levels in the indicated groups progression, ido-ablated mice showed early and increased cns infiltration by myeloid cells (ly- c hi monocytes and ly- g hi granulocytes) and lymphoid cells (cd − nk . + dx + nk, cd + , and cd + t-cells), which were associated with a reduced viral burden in the cns. ido ablation increased the activity of cytolytic effector cells following jev infection, due to the enhancement of ifn-γ or granzyme b-producing nk cells and jev-specific cd + t-cell responses in ido-ablated mice. in addition, the enhanced production of ifn-γ from cd + th cells in response to jev antigen might contribute to early viral clearance from extraneural and neural tissues of ido-ablated mice. also, somewhat intriguingly, our data revealed that potent and rapid ifn-i innate immune responses in cd c + dcs and neuron cells following jev infection could effectively regulate the spread of jev in ido-ablated mice. this finding was supported by the results that ido ablation in myeloid cells derived from hscs played a dominant role in ameliorating je and that ido-ablated hsc-derived leukocytes were major contributors to ifn-i innate responses in the host. collectively, our data suggest that the ablation of ido activity with specific inhibitors, such as -mt, could be a valuable therapeutic tool for the treatment of je. ido is considered a negative regulator of the immune system because ido activity in apcs, including dcs and macrophages, is a potent mechanism for inducing immunosuppression and tolerance. therefore, it is probable that the attenuation of immunosuppressive ido activity leads to enhanced innate and adaptive immune responses for viral clearance. indeed, the inhibition of ido activity with a specific inhibitor resulted in enhanced cd + th type and cd + t-cell responses specific to the influenza virus [ ] . furthermore, the inhibition of ido activity reportedly modifies the immunodominance hierarchy by increasing the number of cd + t-cells specific to a subdominant epitope derived from the influenza virus. the ablation of ido also suppresses viral replication in retrovirus-infected mice through ifn-i production [ ] . these findings strongly support our data that ido ablation ameliorates je progression through enhanced type i/ ii ifn innate and adaptive immune responses. although the in vivo role of ido after viral infection has been investigated in a few studies using murine infection models, the regulatory role of ido in neuroinflammation caused by neurotropic viruses, such as jev, has not been addressed to date. to the best of our knowledge, our findings provide the first evidence that ido inhibition could regulate the in vivo progression of viral encephalitis caused by neurotropic viruses such as jev and wnv. the molecular pathogenesis of je remains unclear, but je is considered an immunopathological disease since uncontrolled over-activation of microglia/glia and infiltrated leukocytes after cns invasion of jev drives neurological disorders [ , ] . although cns infiltration by peripheral innate and adaptive immune cells appears to play a critical role in host defense against infections with neurotropic viruses such as jev, control of cns infiltration is also required because excessive or inappropriate cns infiltration can cause profound damage. therefore, the outcome of je pathogenesis appears to depend on the complicated interplay between virus-induced tissue damage and host immune response against jev antigens. cns infiltration by cd b + ly- c hi monocytes is a hallmark of neuroinflammation caused by neurotropic viruses. these cd b + ly- c hi monocytes migrate into the inflamed cns, where they differentiate into dcs, macrophages, and microglia to regulate neuroinflammation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although conflicting roles have been postulated for ly- c hi monocytes in cns inflammation, cns infiltration by cd b + ly- c hi monocytes is essential in the control of neuroinflammation caused by neurotropic viruses, which supports their protective role during cns inflammation [ ] [ ] [ ] [ ] . notably, the differentiation state of ly- c hi monocytes that infiltrate into the cns appears to affect the progression of neuroinflammation caused by various insults [ ] [ ] [ ] . supportively, ido ablation was observed to lead to enhanced cns infiltration of cd b + ly- c hi monocytes, which may contribute to a beneficial outcome in je. furthermore, early and increased infiltration of nk, cd + , and cd + t-cells in the cns of ido-ablated mice appears to play a role in reducing the viral burden, because infiltration of nk, cd + th , and cd + t-cells producing ifn-γ is required for early clearance of the virus from the cns [ ] [ ] [ ] [ ] . after peripheral introduction of jev via mosquito bites, jev initially replicates in its primary target cells, including dcs and macrophages, at the periphery and subsequently gains entry into the cns through the bbb. presumably, jev-specific cd + th and cd + t-cell responses generated in the peripheral lymphoid tissues of ido-ablated mice would likely provide more effective control of viral replication, thereby reducing the amount of virus supplied from the periphery. in support of this notion, our data revealed that the viral burden in the extraneural and neural tissues of ido-ablated mice were lower than those in wild-type bl/ mice. the depletion or adoptive transfer of specific cell populations of nk and cd + t-cells was previously reported to provide no significant contribution to host survival and viral clearance [ ] . furthermore, co-transfer of immune cd + and cd + tcells, but not individual transfer of either t-cell subpopulation, significantly contributed to a favorable je outcome and promoted host survival [ ] . these facts suggest the possibility that enhancement of broad immunity, including nk, cd + th , and cytotoxic cd + t-cells, in idoablated mice accounts for the effective regulation of early viral clearance in extraneural and neural tissues, thereby providing protection against je progression without tissue injury. however, ido ablation did not result in any significant change in the humoral immune responses specific for jev antigen. the effects of ido on the humoral immune response appear to be variable, depending on the experimental model used [ , ] . because b-cell-deficient (μmt) mice uniformly succumbed to viral encephalitis caused by flaviviruses in a previous study, one obvious conclusion is that an early and sustained humoral immune response is absolutely essential to surviving infection with jev and other related flaviviruses [ ] . this notion suggests a limitation that ido inhibition may not provide the perfect protection against je progression due to the lack of substantial enhancement in the humoral response against jev antigen. thus, further studies on how to enhance the humoral immune response are needed in the context of ido inhibition. the requirement of ifn-γ in the recovery from infections with flaviviruses has been shown to be variable. ifn-γ provides an early protective immune response against a virulent north american isolate of wnv [ ] and mouseadapted strains of dengue virus [ , ] , whereas ifn-γ is dispensable in the control of infection with less virulent strains of wnv [ ] or of yellow fever virus [ , ] . additionally, ifn-γ shows only a modest protective role against murray valley encephalitis [ ] . similarly, in the je model, fig. ido ablation in hsc-derived leukocytes alleviates je via enhancement of the ifn-i innate response. bm cells from wt or ido ko mice were grafted to lethally irradiated wt or ido ko recipient mice, which were then infected with jev ( . × pfu). a susceptibility of ido ko bm chimeric models to je. infected recipient mice (n = ) were examined over days to determine the survival rate. b changes in body weight. data are expressed as the average percentage of body weight relative to the time of challenge. c systemic ifn-β levels in an ido ko bm chimera. the amount of serum ifn-β was determined by elisa at the indicated time points. *p < . ; **p < . compared with levels in wt-wt bm chimera h pi. #p < . ; ##p < . compared with levels in wt-wt bm chimera h pi il- -mediated induction of ifn-γ has been reported to result in suppressed protective immunity [ ] , whereas ifn-γ was associated with a beneficial effect on the outcome of je [ ] . our data favor the latter result, showing a beneficial role of ifn-γ in je progression, because ido ablation markedly increased ifn-γ-producing nk, cd + th , and cd + t-cells. ifn-γ is believed to play diverse roles in infectious diseases, including the activation and polarization of cd + th cells, upregulation of fas in infected target cells, upregulation of mhc i and ii-restricted ag-presentation pathways, macrophage activation, and direct antiviral activity that overlaps with activities triggered by ifn-i [ ] . also, ifn-γ produced from cd + th and cd + t-cells in ido-ablated mice is believed to induce the maturation of ly- c hi monocytes, which may contribute to better je outcomes [ , ] . however, ifn-γ-producing nk cells do not appear to significantly contribute to host survival, because nk-cell-depleted mice showed no change in viral burden or survival [ ] . furthermore, flaviviruses including wnv exhibit immune escape from nk cell attack via the upregulation of mhc-i in infected cells [ ] . in contrast, antigen-specific cd + t-cell cytolytic activity on infected target cells is thought to play a crucial role in disease recovery, given that depletion of cd + t-cells resulted in an increased viral burden in the cns [ ] . supportively, our data revealed that ido ablation provided marked enhancement of cd + t-cells producing ifn-γ and tnf-α upon jev ag stimulation. in addition, ido ablation induced an increase in cd + th cells producing ifn-γ at the early stage, but il- + cd + th and cd + foxp + tregs were not altered by the ablation of ido. collectively, these findings suggest that ifn-γ produced from cd + th and cd + t-cells play an important role in regulating je progression in ido-ablated mice. the role of ido in antiviral activity has been postulated in different in vitro experiments. for example, ido activity in astrocytes is induced by the tlr ligand polyinosinicpolycytidylic acid, which is involved in antiviral function via the exhaustion of the essential amino acid l-trp in the environment [ ] . this finding is inconsistent with our data, but it is notable that this finding was derived from an in vitro model. the intriguing result in the present study was that ido-ablated cd c + dcs, including conventional and plasmacytoid dcs, showed potent and rapid ifn-i innate immune responses to jev infection. also, ko-ko and ko-wt bm chimeric models showed systemic ifn-β production at levels higher than in wt-wt and wt-ko bm chimeric models. this supports the notion that myeloid cells derived from hscs, including cd c + dcs, may dominantly contribute to the rapid ifn-i innate immune response. this result is strengthened by the finding that ido ko and -mt-treated mice showed induction of a markedly increased ifn-i innate response in the spleen following retrovirus infection [ ] . conventional and plasmacytoid dcs exhibited potent and rapid ifn-i innate responses with different dynamic patterns in the expression of prrs, transcription factors, and isg genes following jev infection. these different ifn-i innate responses in both types of cd c + dcs are thought to be derived from the intrinsic properties of the specific cell type. although we did not investigate the detailed mechanism by which idoablated cd c + dcs displayed a potent ifn-i response to jev infection, our data suggest the involvement of prrs (rig-i, mda ) and transcription factor irf because the expression levels of rig-i, mda , and irf- were rapidly enhanced in ido-ablated cd c + dcs. also, considering that only a small fraction ( - %) of myeloid-derived cells are infected by jev [ ] , uninfected myeloid-derived cells are thought to contribute substantially to the induction of antiviral isgs through early stimulation of the stat transcription factor which induces isg , isg , and isg [ ] . also, it is worth noting that primary cortical neuron cells derived from ido-ablated mice showed potent ifn-i innate responses resulting in delayed replication of jev. although the induction patterns of prrs and their transcription factors in neuron cells differed from those of cd c + dcs following jev infection, the induction of isgs in ido-ablated neurons definitely followed potent ifn-i innate immune responses, such as ifn-αβ production. however, because cortical neurons are relatively insensitive to the antiviral effects of ifn-i [ ] , the regulation of viral replication in primary cortical neurons by ifn-i innate responses may ultimately show some limitations. indeed, our data indicating that primary cortical neuron cells derived from ido ko mice showed transient inhibition of viral replication support this speculation. therefore, further studies are needed to define the mechanistic and functional intermediates that link ido to the regulation of ifn-i innate immune responses in different cell types. je pathogenesis in the murine model may be affected by the route of administration, dosage, and strain of the virus, and the virus propagation conditions used [ ] . ido was expressed with different dynamic patterns in conventional and plasmacytoid dcs, macrophages, and neuron cells upon jev infection. thus, it is thought that je pathogenesis may also be affected by how the innate responses of specific cell types are exhibited after jev infection. rapid ifn-i/ii innate immune responses are considered more crucial in the control of je progression than adaptive tcell responses, which take time to develop. considering that ido ablation provided early and enhanced ifn-i/ii innate responses and subsequent adaptive t-cell response in the host, the inhibition of ido may be a valuable tool for regulating je progression. the inhibition of ido ameliorates je progression via rapid enhancements 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cross-presentation of cd alpha + cd c + dendritic cells by japanese encephalitis virus in a tlr / myd signal pathway-dependent manner pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons challenges in the discovery of indoleamine , -dioxygenase (ido ) inhibitors research progress of indoleamine , -dioxygenase inhibitors this study was supported by a national research foundation of korea (nrf) grant funded by the korean government (misp) ( r a a ). the funder had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript. cns infiltration by ly- c hi monocytes. also, ido ablation may accelerate viral clearance from the host at a later stage via the enhancement of jev-specific ifn-γ-producing tcell responses. therefore, these results suggest that inhibition of ido activity by specific inhibitors [ , ] may be a promising tool for therapeutic and prophylactic strategies against je. the authors declare that they have no competing interests.authors' contributions sbk and jyc conceived the study and discussed the data with ske. eu, amp, and fmah conceived the study and contributed to the experimental design. jh, syp, jhl, and kk contributed to the reagent/materials/analysis and tools and provided critical conceptual guidance. sbk and ske wrote the paper with contributions from all authors. all of the authors reviewed and approved the final version of the manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -v w wi c authors: rahmatpanah, farah; agrawal, sudhanshu; jaiswal, natasha; nguyen, hannah m.; mcclelland, michael; agrawal, anshu title: airway epithelial cells prime plasmacytoid dendritic cells to respond to pathogens via secretion of growth factors date: - - journal: mucosal immunol doi: . /s - - - sha: doc_id: cord_uid: v w wi c plasmacytoid dendritic cells (pdcs) are critical for defense against respiratory viruses because of their propensity to secrete high levels of type i interferons (ifn). the functions of pdcs in the lung can be influenced by airway epithelial cells. we examined the effect of human primary bronchial epithelial cells (pbecs) on pdc functions by performing rna-sequencing of pdcs after co-culture with air liquid interface differentiated pbecs. functional analysis revealed that pdcs co-cultured with pbecs displayed upregulation of type i ifn production and response genes. upregulated transcripts included those encoding cytosolic sensors of dna, zbp- ,irf- , and nfkb as well as genes involved in amplification of the ifn response, such as ifnar , jak/stat, isg . in keeping with the rna-seq data, we observe increased secretion of type i ifn and other cytokines in response to influenza in pdcs co-cultured with pbecs. the pdcs also primed th responses in t cells. the enhanced response of pdcs co-cultured with pbecs was due to the action of growth factors, gmcsf, gcsf, and vegf, which were secreted by pbecs on differentiation. these data highlight possible mechanisms to enhance the production of type-i ifn in the airways, which is critical for host defense against respiratory infections. plasmacytoid dendritic cells (pdcs) are a subset of dendritic cells, which are found in circulation and other organs. they are distinguished by their capacity to produce copious amounts of type i interferons (ifn) , via stimulation of toll like receptor and with nucleic acids or viruses. because of this property, they play a crucial role in fighting viral infections. , however, the cytokine secretion by pdcs is not restricted to ifns as they can also secrete other cytokines and chemokines including il- , il- , cxcl , cxcl , ccl , and ccl . similar to conventional myeloid dcs, they also express mhc class ii and costimulatory molecules and migrate to prime t cell responses. , production of type i ifns along with il- by pdcs primes cd + t cells and ifn-γ secreting th cd + t cells. pdcs can also induce t regulatory cells via induction expression of ido. , in addition, type i ifn production by pdcs enhances b cell activation, plasma cell generation and antibody secretion. tnf-related apoptosis inducing ligand (trail) and granzyme b serve as immunoregulatory factors that endow pdcs with the capacity to kill tumor cells, induce apoptosis of infected cd + t cells and suppress t cell proliferation. , pdcs also play an important role in pulmonary immune responses. pdcs in the lung have been reported to be immature, expressing low levels of mhc ii and costimulatory molecules but high levels of pdl- . these characteristics make them poor inducers of t cells and that is why they are thought to play an immune-regulatory role in the lung. depletion of pdcs in a mouse model of asthma enhanced the inflammation against harmless antigens. similarly, in acute lung injury (ali) and acute respiratory distress syndrome (ards), activated pdcs are protective and have been shown to prevent recruitment of pro-inflammatory monocytes into the lung. in contrast, pdcs were reported to be key drivers of immune-inflammatory cascade during asthma exacerbations via boosting of th responses. thus, pdcs can be inflammatory or tolerogenic depending on the context. activated pdcs are major drivers of protection against respiratory infections such as influenza and rsv by virtue of production of type i ifns and other cytokines. the functions of lung dc are influenced by many soluble factors such as growth factors, chemokines, and cytokines that are released from different cells within the lung. these soluble factors regulate the intensity and duration of immune response by stimulating or inhibiting activation and function of dc. for example, previous studies from our group and others have demonstrated that pbecs enhance the inflammatory and immune-surveillance capacity of mdcs. [ ] [ ] [ ] however, their effect on pdcs is not elucidated. this knowledge is essential for manipulation of pdc response to respiratory pathogens. here we examined the changes introduced in human pdcs by primary bronchial epithelial cells at gene and functional level. peripheral blood samples were obtained from healthy volunteers ( - years) , approved by the institutional review board of the university of california (irvine, ca, usa). written, informed consent was obtained. obtained from lonza inc. (basel, switzerland) and were differentiated at the ali on transwell plates as described. briefly, × pbecs per insert were seeded into -well transwell plate with apical chamber coated with the rat tail collagen (bd biosciences, san jose, ca, usa). cells were seeded in µl b-ali™ growth medium onto the collagen coated trans well inserts; µl of b-ali™ growth medium was added to the basal chamber of wells containing the inserts. on day after seeding, once the monolayer of pbec was confluent, media were removed from the apical chamber and allowed for air-liquid differentiation. the media in the bottom chamber were changed every two days. approximately days post-differentiation, pbecs were tested for the presence of cilia by staining for beta-tubulin and mucus secretion (data not shown). isolation and culture of human plasmacytoid (pdcs) with pbecs for rna sequencing pdcs were purified from the peripheral blood mononuclear cells (pbmcs) of young subjects by negative selection using a dc enrichment kit (stemcell sep, vancouver). enriched dc population was subsequently sorted using facsaria (bd biosciences) to obtain a pure pdc population. purity was above %. (cd +/cd c−) pdcs from different donors were added to the bottom chamber of the transwell with ali-differentiated pbecs for h. subsequently, the pdcs were collected for rna-seq and other studies. pdcs cultured in pbec medium without pbecs were used as controls. rna isolation from pdcs total rna was isolated from cultured pdc cells from four individuals using ffpe rna/dna purification plus kit (cat # , norgen biotek corp) with minor modifications. this kit is suitable for challenging samples such as formalin fixed paraffin embedded tissue in, which the recovery of quality rna is difficult. we applied this kit to isolate rna from pdcs cells since the numbers of cells were small. the rna quality and quantity was assessed using agilent bioanalyzer and qubit fluorimeter respectively. high yield and quality rna was obtained. the illumina truseq rna access library prep kit was used to study gene expression profiling in stimulated and unstimulated pdcs. the access method of illumina is based on sequence specific capture of coding rna. this method is suitable for wide range of rna quality and low rna input. briefly, ng of rna was used for library preparation. the total rna is fragmented into small pieces using divalent cation at o c. then cdna is synthesized using the cleaved rna fragments using random priming during the first and the second synthesis. sequencing adaptors are ligated to the dscdna fragments. the quality of libraries was assessed after the first pcr amplification. the coding regions of the transcriptome are then captured from the library using sequence specific probes to create the final libraries (as described by the manufacturer). samples were assayed after the second pcr amplification on the agilent bioanalyzer. qualification assay (qpcr) was performed using kapa sybr fast library quantification kit (illumina) universal qpcr mix ref # . samples were sequenced at × cycle at the uci high throughput genomic core facility (https://biochemghtfuci.wordpress.com/). sequencing reads were aligned to genome reference files from ensembl using strand ngs . (http://www.strand-ngs.com/) sequencing analysis package. approximately - % of mapped reads were aligned to protein coding regions as shown by the pie chart plot (supplementary fig. ). reads were filtered on duplicates to remove pcr artifacts. reads with better mapping quality and base average were retained (we set the number of duplicated reads to be permitted at " ") (excel supplementary data). deduplicated sequencing reads for each sample was quantified for gene expression, which is the measurement of the association of reads to genes and exons "raw reads". to normalize the raw reads we used reads per kilo million (rpkm) method in strand ngs program. we measured the variations in ′ to ′ end coverage along each transcript using gene body coverage analysis in strand ngs. uniform transcript coverage was observed among all samples. pooled analysis was performed using the audic claverie test (ac), which pools together raw counts across four pdcs and pdcs co-cultured with epithelial cells, separately, and tests the differences between them based on a poisson assumption on distribution of counts. multiple testing correction: benjamini hochberg fdr of . and the maximum p value cut off . was used (excel supplementary data). the genes of ac test were ranked based on p value adjusted (fdr corrected) and cut off was set at p adj < . to select for statistically significant differentially expressed genes between the pdcs co-cultured with epithelial cells and pdcs. the methods used for rna isolation and rna seq library construction worked with samples with small number of cells. for co-culture experiments purified pdcs were cultured without or with pbec differentiated at ali. after overnight co-culture, pdcs from both groups were collected and put together with purified cd t cells at a ratio of : (pdc:cd t) in serum free aim v medium (invitrogen) for days. subsequently, the cells were collected and stained with specific antibodies for cd , cd and foxp (rnd systems). gated cd cells were analyzed for the expression of cd and foxp using flow jo. supernatant collected was assayed for ifn-γ and il- by elisa. for co-culture experiments purified pdcs were cultured without or with pbec differentiated at ali. after overnight co-culture, pdcs from both groups were collected and stimulated with influenza (heat killed influenza a strain a/pr/ / -(flu, charles river, north franklin, ct) at µg/ml) for h. culture supernatants were collected h post stimulation and run on multiplex kit. the specific kit used was milliplex human -plex cytokine panel (cat# hcytmag- k-px ), which contains the following analytes: gm-csf, g-csf, ifnγ, il- α, il- rα, il- β, il- , il- , il- , il- , il- , il- , il- , il- , il- p , il- p , il , il , il- , mcp- , tnfα, tnfβ, eotaxin, type i ifn, ip- , mip- α, mip- β, egf, vegf, and rantes. the procedure followed was according to manufacturer's protocol. briefly, supernatant was mixed with premixed beads ( cytokines) overnight and after incubation with detection antibodies and streptavidin-pe for h each, the plate was run on magpix to identify specific cytokines. culture of pdcs with pbecs/growth factors purified pdcs were cultured with and without gmcsf ( ng/ml), g-csf ( ng/ml) or vegf ( ng/ml), il- ( ng/ml), il- ( ng/ml), mcp- ( ng/ml) (rnd systems) or a cocktail of these in various combinations and stimulated with influenza for h. supernatant collected was assayed for inflammatory mediators with multiplex as described above. culture of pdcs with blocking antibodies pdcs cultured without or with pbec were stimulated with influenza in the presence or absence of specific blocking antibodies against gmcsf (invitrogen), gcsf, vegf (rnd systems) or a mixture of the three antibodies at µg/ml. after h supernatant was collected and assayed for type i ifn, tnf-α, il- β, il- using multiplex. percent inhibition in the antibody treated groups was calculated compared to control group without antibody treatment. pbecs from three different subjects were used for this experiment. pbecs were allowed to differentiate at ali. the conditioned basal airway epithelial cells prime plasmacytoid dendritic cells to respond to. . . f rahmatpanah et al. medium in the bottom chamber was collected once the aec differentiation was complete. pbec differentiation media alone was used as control. multiplex detection of factors (same as above) was performed to determine the factors secreted by pbecs using milliplex. statistical analysis for cell culture experiments was performed using graphpad prism (graphpad inc., san diego, ca, usa). one way anova followed by tukeys multiple comparison test was used for the analysis. a p-value of < . was considered statistically significant. the effect of pbecs on pdcs gene expression changes was investigated using the transwell co-culture system. to investigate the effect of aecs in the form of pbecs on pdcs, the pbecs were cultured at ali till differentiation was achieved. pdcs purified from the blood were then added to the basal side of the monolayer to model the airways. , an aliquot of the pdcs was also cultured without pbecs but with the media. twenty-four later pdcs were collected and rna was extracted. pdcs from four different individuals were used. the description is provided in supplementary table . the viability was comparable between pdcs cultured with and without pbecs (data not shown). because of the low quantity of rna, we used the access method of library construction from illumina, which is based on sequence specific capture of coding rna. this method is suitable for wide range of rna quality and low rna input. paired end rna sequencing (rna-seq) was performed to determine the gene expression differences between pdc and pdcs co-cultured with epithelial cells. we observed a uniform reads coverage in the gene body for all rna seq data plot. we did not observe very large increases or decreases in transcript abundance, which was to be expected because pdcs were exposed to pbecs and there was no stimulation with a pathogen. pdcs in the airways are reported to induce tolerance via the expression of ido. , we found increased expression of ido- (fig. a) in the pdcs cultured with pbecs compared to pdc alone. in addition, the expression of costimulatory molecules, cd and cd was decreased. however, expression of other costimulatory molecules including, cd , ox , icos, icam was upregulated. the expression of the inhibitory costimulatory molecule, pdl- was also decreased. the pdcs therefore displayed a mixed phenotype in terms of costimulatory molecules. we performed functional analysis to confirm whether the pdcs were tolerized by pbecs. pdcs exposed to pbecs induced significantly increased levels of cd + cd + foxp + t regulatory cells to controls (p = . ), fig. b, c) . the secretion of ifn-γ was significantly decreased (p = . ) while il- was increased (p = . ) in the pdcs exposed epithelial cell group compared to control. these data suggest that pbecs enhance the tolerogenic capacity of pdcs at homeostasis. rna-seq data indicates pbecs enhance the production and response of type i ifn in pdcs pdcs in the airways are also important for host defense against infections because of their capacity to secrete high levels of type i ifns. our results indicate that the major pathways altered in pdcs in response to pbecs are the type i ifn related pathways such as the type i ifn signaling, interferon stimulated gene (isg ), cytosolic sensors of dna as well as jak-stat (fig. a-d) . in the type i ifn pathway, the expression of ifnar as well as jak-stat genes such as jak , stat , stat , which signify activation, are upregulated while socs -and , which inhibit the pathway, are downregulated ( fig. a-d) . furthermore pathways involved in the interferon response such as isg also displayed alterations after co-culture (fig. b) . among the innate sensors, the expression of cytosolic dna sensor, zbp- was upregulated on pdcs after co-culture with apcs (fig. c) . the expression of irf- and nfkb , which are required for signaling via this receptor, was also upregulated. in contrast, the negative regulators of the pathway, nfkb a, ikbkb are downregulated. interestingly, the majority of the genes in the prostaglandin pathway which is a major negative regulator of ifn production were downregulated (fig. e) . in addition to ifn related pathways, g-protein coupled receptor (gpcr) signaling pathway also displayed major changes in pdcs co-cultured with pbecs compared to pdcs (fig. f) . several of the chemokine receptor genes, such as ccr , ccr , ccr , ccr , cx cr , cxcr were upregulated indicating an activated state of pdcs. genes in the il- pathway also displayed changes (fig. g) . il- is known to promote pdc activation and survival. in summary, rna seq data suggests that the capacity of pdcs to produce and amplify type i ifn production is upregulated in pdcs after co-culture with pbecs. pbecs enhance the cytokine and chemokine response of pdcs to influenza to confirm the rna-seq data we performed functional assays to determine the alterations in the capacity of pdcs to produce type i ifn after exposure to pbecs. briefly, pdcs co-cultured with/without pbecs differentiated at ali were stimulated with influenza and cytokines and chemokines were assayed in the supernatants. conditioned pbec medium was used as control. pdcs exposed to pbecs secreted significantly higher levels of type i ifns (p = . ) on stimulation with influenza as compared to pdcs cultured alone (fig. a) , which is in keeping with the rna-seq data. tnf-α secretion was also significantly increased (p = . ) in influenza stimulated aec-exposed pdcs as compared to unexposed pdc (fig. a) . secretion of il- α (p = . ) and il- β (p = . ) was also increased significantly in pdcs co-cultured with pbecs with and without stimulation with influenza (fig. a) . no type i ifn, tnf-α, il- α, and il- β was detected in the conditioned medium. secretion of other mediators including anti-inflammatory cytokine, il- was below the limit of detection (data not shown). these data indicate that pbecs enhance the inflammatory response of pdcs to pathogens as suggested by the rna-seq data. to confirm that the aec-exposed pdcs were indeed primed to induce enhanced responses to influenza, the pdcs co-cultured with/without pbecs and influenza were cultured with purified t cells for five days and secretion of t cell specific cytokines was determined by multiplex. culture of t cells with pdc exposed to pbecs and stimulated with influenza induced significantly higher level of il- (p = . ) as compared to pdcs stimulated with influenza without aec exposure (fig. b) . enhanced il- secretion indicated increased proliferation. in keeping with the increased proliferation, the levels of tnf-α (p = . ) and ifn-γ (p = . ) secretion by these t cells was also significantly enhanced (fig. b) . other cytokines such as il- , il- , il- , il- were not induced by influenza primed pdcs and were comparable between the t cells cultured with pdc with and without pbecs (data not shown). these results suggest that pbecs enhance the inflammatory response of pdcs to influenza, which leads to increased induction of th responses. mechanisms used by pbecs to prime pdcs, a multiplex analysis was performed to determine the soluble factors in the conditioned medium from pbecs differentiated at the ali. the medium for differentiation was used as control. remarkably, out of mediators tested (see methods) we observed only six factors, which were significantly upregulated in the medium of ali differentiated pbecs (fig. ) . differentiated pbecs secreted significant levels of growth factors g-csf (p = . ), gmcsf (p = . ) and vegf (p = . ). secretion of cytokines, il- (p = . ) as well as chemokines, cxcl- (il- , p < . ), ccl- (p = . ) was also upregulated. ali differentiated pbecs might be priming the pdcs via secretion of the growth factors and the cytokines, chemokines. to identify the specific mechanism that leads to pdc priming in presence of pbecs, the effect of the factors found to be significantly enhanced in differentiated pbecs were explored. initial experiments were performed with three cocktails, growth factor and cytokine, chemokine cocktail (gmcsf, vegf, gcsf, il- , il- , ccl- ), growth factor cocktail (gmcsf, vegf and gcsf) and cytokine, chemokine cocktail (il- , il- , ccl- ). purified pdcs were cultured in the presence or absence of these cocktails and activated with influenza. culture of pdcs with combination cocktail as well as growth factor cocktail enhanced the secretion of type i ifn while the cytokine, chemokine cocktail had no significant effect (supplementary fig. ). further experiments were then done with growth factors. we tested the individual effects of the growth factors to determine whether this was due to a single factor or a synergistic effect of all factors. both gcsf (p = . ), gmcsf (p = . ) led to increase in type i ifn secretion by pdcs in the response to influenza (fig. a) . the increase by vegf was not significant (p = . ). the secretion of type i ifn was maximal when a cocktail of all three growth factors was used indicating a synergistic effect. both gmcsf and gcsf were also able to induce significantly increased (p = . -gmcsf; p = . -gcsf; fig. a ) levels of tnf-α. tnf-α was also significantly increased in the cocktail (p = . ). il- β displayed no significant increase with the growth factors as well as the cocktail suggesting that the growth factors do not induce il- β in pdcs. to further confirm the action of the growth factors, experiments using blocking antibodies against gmcsf, vegf and gcsf as well as a combination of all three antibodies were performed. pdcs cultured in the presence of pbec conditioned medium were stimulated with influenza in the presence of the antibodies and percent inhibition of type i ifn, tnf-α and il- β secretion by pdcs was calculated. as is evident from fig. b conditioned media and stimulated with influenza (data not shown). these data suggest that though gmcsf and gcsf can enhance the induction of type i ifn and tnf-α in pdcs, the synergistic action of all three factors is really what is responsible for the increase of these cytokines observed in pdc cocultured with pbecs. type i ifn secretion by pdcs plays a critical role in fighting viral infection at the respiratory mucosa. here we demonstrate for the first time that the capacity of pdcs to secrete type i ifn is enhanced by growth factors secreted by pbecs. previous studies suggest that pdcs may play a tolerogenic role in the respiratory mucosa via induction of t regulatory cells. , a very recent study in mice by lynch et al. has demonstrated that depletion of pdcs early in life enhanced features of asthma such as airway hyper-responsiveness, allergic inflammation and airway remodeling due to an impaired ability to expand tregs and maintain tolerance in the airways. our data indicates that exposure to pbecs induces tolerance in pdcs at homeostasis but concomitantly also primes pdcs to enhance their response to infections (figs. , ) . the induction of tregs by pdcs is enhanced which could be due to the increased expression of ido. the factors which lead to the increase in tolerogenic capacity of pdcs remain to be explored. in keeping with the activation genes in rna-seq, we also observed increased secretion of type i ifn from influenza stimulated pdcs co-cultured with pbecs. the expression of tlr and zbp was upregulated. zbp has recently been shown to be the innate sensor of influenza virus. it signals via irf and nfkb both of, which were upregulated in pdcs exposed to pbecs. the rna-seq data did not display significant changes in pathways known to induce ifn secretion in pdcs such as the irf . however, increased nfkb related genes may play a role in tlrdependent ifn production. , interestingly, pge has been reported to be a negative regulator of type i ifn secretion by human dcs upon stimulation with tlrs and prostaglandin pathway was downregulated in pdcs exposed to pbecs (fig. e) . the expression of another gene, fcɛriγ/fce a is significantly downregulated in pdcs co-cultured with pbecs and it has been demonstrated to inhibit tlr mediated type i ifn production in human pdcs via itam-mediated signals. similarly socs , a key negative regulator of myd -dependent type i ifn signaling , is also downregulated in pbecs exposed pdcs. in addition to negative regulators, pdcs co-cultured with also pbecs displayed increased expression of genes involved in type-i-ifn signaling. type i ifn can act both in an autocrine and a paracrine manner. it can amplify its own secretion via autocrine actions. increased expression of ifnar would enhance the autocrine action of ifns. therefore, pbecs seem to prime pdcs to enhance type i ifn secretion. this is supported by the increase in the genes of the isg pathway. isg is not only an interferon response pathway but is also reported to bind to irf- and enhance ifn responses. , furthermore, isg pathway can also affect important signaling pathways such as ifn, nfκb, and jnk pathways, all of, which are upregulated in pdcs after exposure to pbecs. together the gene expression changes suggest that pbecs enhance the capacity of pdcs to produce type i ifn via inhibiting the negative regulators as well as by enhancing the expression of molecules, which can amplify the production of type i ifns. we had observed similar priming of mdcs by pbecs earlier. , even though rna-seq data suggests that pdcs cocultured with pbecs express type i ifn at homeostasis, we were unable to detect type i ifn in our supernatants without stimulation with influenza. this could be due to lack of sensitivity of the elisa which may not be able to detect the various type i ifn subtypes. it is also possible that the type i ifn is bound to the receptor and therefore not detectable. high level of type i ifn secretion in the absence of infection is not desirable since its upregulation under such conditions is usually associated with autoimmunity. the tolerogenic nature of pdcs at homeostasis may therefore prevent secretion of the type i ifn to prevent inflammation. it is also possible that the changes visible at gene level are not transcribed to proteins as it is well established that only about % of genes are transcribed to proteins. this is especially true for cytokines. we also find that growth factors, gmcsf, gcsf, and vegf secreted by pbecs help prime pdc responses to influenza (fig. ) . interestingly, these factors signal via the jak-stat pathway, which displays significant upregulation in the rna-seq data (fig. d) . some of the growth factors have already been reported to modulate dc function. for example, knockdown of gcsf receptor in dcs has been reported to reduce the expression of at least proinflammatory cytokines and antigen presenting molecules, while the expression of pdl and pdl was increased. gmcsf is also considered as a dc activation agent and has been demonstrated to enhance the ifn-α production from pdcs in lupus patients. moreover, gmcsf secretion by kidney epithelial cells has also been reported to enhance maturation and type i ifn production in pdcs. additionally, gmcsf has been shown to synergize with tlr-dependent microbial stimuli to upregulate of proinflammatory cytokines. interestingly, bdca- , which functions as a marker for pdcs is a receptor on endothelial and tumor cells for vascular endothelial growth factor (vegf-a). studies examining the effect of vegf on lung dc subtypes in vegf-transgenic mice have reported an increase in lung mdc and pdc populations. an increase in inflammatory responses of mdc was also reported in the same study. the effect of vegf on pdc responses has not been examined. our results indicate a possible synergistic effect of vegf with other growth factors on type i ifn production by pdcs. the present study only examines effect of soluble factors from pbecs on pdc functions. information regarding cell to cell contact between pbecs and pdc is missing which can also play an important role in modulation the functions of pdcs. this is because existing technology only allows the differentiation of pbecs at ali on a transwell insert which prevents contact with pdcs. development of systems which can allow pbec differentiation on surfaces which are capable of providing cell to cell contact would be very valuable in studying such interactions. in summary, rna-seq data indicates that exposure of pdcs to pbecs both tolerizes and activates pdcs. pbecs exposed pdcs induce increased levels of tregs at homeostasis. however, the pdcs are also primed by pbecs to respond to infections. in keeping with the rna-seq data, we observe increased secretion of type i ifn and other cytokines in response to influenza in pdcs cocultured with pbecs. the pdcs also primed th responses in t cells. the enhanced response of pdcs co-cultured with pbecs was due to the action of 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cytokines and costimulatory molecules in dendritic cells systematic cytokine receptor profiling reveals gm-csf as a novel tlr-independent activator of human plasmacytoid predendritic cells regulation of the interferon-alpha production induced by rna-containing immune complexes in plasmacytoid dendritic cells human plasmacytoid dendritic cells acquire phagocytic capacity by tlr ligation in the presence of soluble factors produced by renal epithelial cells cutting edge: granulocyte-macrophage colony-stimulating factor is the major cd +t cell-derived licensing factor for dendritic cell activation bdca- , bdca- , and bdca- : three markers for distinct subsets of dendritic cells in human peripheral blood lung vascular endothelial growth factor expression induces local myeloid dendritic cell activation this study was supported by grant from the nih ag (to aa), and from the national center for research resources and the national center for advancing translational sciences # ul tr . the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. we are grateful to icts uc irvine for providing the blood samples. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -jv o authors: marcos-villar, laura; díaz-colunga, juan; sandoval, juan; zamarreño, noelia; landeras-bueno, sara; esteller, manel; falcón, ana; nieto, amelia title: epigenetic control of influenza virus: role of h k methylation in interferon-induced antiviral response date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: jv o influenza virus stablishes a network of virus-host functional interactions, which depends on chromatin dynamic and therefore on epigenetic modifications. using an unbiased search, we analyzed the epigenetic changes at dna methylation and post-translational histone modification levels induced by the infection. dna methylation was unaltered, while we found a general decrease on histone acetylation, which correlates with transcriptional inactivation and may cooperate with the impairment of cellular transcription that causes influenza virus infection. a particular increase in h k methylation was observed and the use of an inhibitor of the specific h k methylase, dot l enzyme, or its silencing, increased influenza virus replication. the antiviral response was reduced in conditions of dot l downregulation, since decreased nuclear translocation of nf-kb complex, and ifn-β, mx and isg expression was detected. the data suggested a control of antiviral signaling by methylation of h k and consequently, influenza virus replication was unaffected in ifn pathway-compromised, dot l-inhibited cells. h k methylation also controlled replication of another potent interferon-inducing virus such as vesicular stomatitis virus, but did not modify amplification of respiratory syncytial virus that poorly induces interferon signaling. epigenetic methylation of h k might have an important role in controlling interferon-induced signaling against viral pathogens. chromatin is a dynamic structure that adapts itself to alter the spatial arrangement of genetic information and thus meet the changing demands of cell functions; it has a key role in the control of gene expression. epigenetic modifications of chromatin regulate heritable and reversible gene expression without altering the dna sequence, and can also modify chromatin accessibility. dna methylation and post-translational histone acetylation and methylation are major mechanisms responsible for epigenetic regulation of gene expression . dna methylation typically represses gene transcription when takes place in the promoters of regulated genes . histone acetylation opens the condensed chromatin structure by reducing dna affinity for histones; this enables the dna to uncoil from nucleosomes so that transcription factors and rna polymerase can access the dna and increase transcription . histone methylation can increase or decrease gene transcription, depending on which amino acids in the histones are modified and how many methyl groups are added to specific residues . influenza virus is an rna virus whose genome consists of eight single-stranded rna segments with negative polarity. the virus uses a very uncommon transcription mechanism. the heterotrimeric viral polymerase synthesizes capped, polyadenylated viral mrnas through an initiation process; it uses short-capped oligonucleotides as primers, which are scavenged by a viral endonuclease from newly synthesized host rna polymerase ii transcripts , . the viral transcription strategy thus requires continuous cellular transcription for viral mrna infected cells. to identify epigenetic changes induced by influenza virus infection in the cell transcription machinery, we first analyzed dna methylation levels. human a lung epithelial cells were mock-infected or infected at high multiplicity of infection (m.o.i.; pfu/ml) with a hypervirulent a/pr / / (pr hv) strain [ ] [ ] [ ] [ ] (fig. a) . at h post infection (hpi), dna was isolated and methylation profiles evaluated in triplicate using the k infinium dna methylation beadchip. correlation analysis of the , valid probes showed an extremely strong relationship between samples (pearson correlation coefficient from r = . to r = . ; fig. b ). to identify specific differentially methylated candidates, we performed a parametric analysis to compare average beta values from infected with mock-infected cells, selecting those with differences in methylation levels > (− . >delta> . ) and a standard deviation value < % (desvest< . ). we found no significant variations, even after analysis of selected cpg sites from infection-associated genes (not shown), which indicated that global methylation of dna does not change during influenza virus infection in this system. histone fractions were purified from the same samples used for the study of dna methylation and mass spectrometry (ms) analysis were performed. we used unbiased analysis with the nanolc-ms platform to identify and quantify influenza virus-induced epigenetic changes in histone post-translational modifications (ptm). three biological replicates were analyzed and quantitative analyses completed (see methods). we considered a mascot score threshold of as high-confidence peptide identifications, and all peptides methylated/acetylated on lysine (above or below this threshold) were validated manually to assure correct tandem mass spectra matches. only ptms that were present in at least two replicas are presented (fig. c) and it has to be pointed out that all modified and unmodified peptides were identified reliably more than once. results in infected cells showed a reduction in lysine acetylation of histone and . this decrease would impair host cell expression, in accordance with the role of acetylation in opening the condensed chromatin and activating transcription . non-methylated h k and non-acetylated h k levels increased moderately during infection; since h k methylation and h k acetylation are hallmarks of transcription elongation , , the increased levels of these unmodified residues would also contribute to transcriptional inactivation of host cells. histones have a large proportion of basic residues. this property impedes detection of specific residues, probably due to the presence of another lysine(s) that trypsin proteolysis would render as peptides too small to be identified reliably by ms-based techniques. lysines not detected by ms include h k and h k , whose ptm are recognized as histone marks that control cell transcription. h k trimethylation is a common modification in active chromatin , whereas h k methylation is linked to transcriptional repression , . decreased h k me levels have been observed during influenza virus infection ; here we evaluated possible changes in methylated h k and in acetylated h k , as the latter is a conventional histone marker of active chromatin . total extracts of a mock-infected or pr hv-infected cells at high m.o.i. were collected at various times post-infection and levels of acetylated h k and dimethylated h k were tested by western blot. the results indicated that at late times after infection, influenza virus triggers a decrease in h k ac and an increase in h k me (fig. d ), which could contribute to a general decrease in host cell transcription activity. in contrast to this general effect on histone modifications, which seems to impair transcription, we detected a clear increase in methylation of lysine of histone , especially monomethylation (fig. c ). this result was unanticipated, since studies in saccharomyces cerevisiae and in humans, h k methylation levels correlated clearly with transcriptional activity . as methylation of this lysine gave the highest score in ms analysis, we evaluated this increase by two additional methods. we purified histones from cells mock-or pr hv-infected at high m.o.i.; at hpi, h k me levels were evaluated by western blot analysis (fig. e ) and a quantitative colorimetric method based on anti-h k me antibody detection (epigentek) (fig. f ). both techniques verified the h k methylation increase observed in proteomic analysis. these results indicate that epigenetic modifications induced by influenza virus infection mainly target the histone component of host cell chromatin, with h k residue methylation the most frequently modified. that methylation of infected cell dna is unaffected indicates, that in these conditions, influenza virus does not permanently injure the host cell, but the epigenetic modifications may be transitory. methyltransferases modify different lysines, only one known methyltransferase, dot l, mono-, di-and trimethylates h k ; the inhibitor epz- (epz) specifically inhibits dot l. to confirm this ability to inhibit h k methylation in our system, we incubated a cells with the inhibitor and used western blot to analyze h k me accumulation at various times after epz addition. treatment with or μm epz substantially decreased h k methylation at - h post-epz addition, without affecting h k me levels (fig. s a ). the effect of dot l inhibition on cell viability was analyzed by mtt assay (see methods and ); at these doses, epz did not notably affect cell viability, even at h after treatment (fig. s b) . to determine whether h k methylation alters influenza virus infection, we inhibited dot l and analyzed its effect on production of infectious particles. a cells were plated alone or with dot l inhibitor ( h), then infected at low m.o.i. with the pr hv strain, with another laboratory-passaged influenza strain, a/wsn/ (wsn) ( fig. a ) or with one of two natural pandemic isolates, a/california/ / (cal ) and a/ california/ / (cal ) (fig. b ) and analyzed production of infectious particles in each condition. dot l inhibition caused an increase in viral replication, higher in cells infected with the natural isolates, which suggests a general role of h k methylation in control of the influenza virus life cycle. to confirm the effect of dot l activity on influenza virus replication, we tested the effect of dot l knock-down on production of infective particles. for rnai-mediated dot l silencing experiments, we used lentiviruses expressing shrna specific for dot l (shdot l , shdot l ) or a control that expresses irrelevant shrna (shtm) , . treatment of a cells with the dot l inhibitor or expression of the dot l-shrnas reduced h k me levels compared with shtm, measured by western blot analysis (fig. a ) and quantitative colorimetric method (fig. b ). dot l silencers decreased h k methylation in infected cells in a lesser extent than epz treatment. to analyze the relevance of dot l in virus replication, we transduced a cells with lentiviruses expressing the dot l silencers or control shrna ( days), followed by infection with the pr hv ( role of h k methylation in antiviral responses. the above results suggested that modification of lysine of histone may mediate the antiviral response; its demethylation could decrease the host response against the pathogen allowing higher production of infectious particles. in agreement with this proposal we did not find significant reduction of viral titer in cells infected at high m.o.i where ifn signaling does not play a major role, in the presence of the dot l inhibitor (data not shown). these data suggested that h k methylation would affect host response in conditions close to natural infections that take place in the epithelium of the respiratory tract at low multiplicity of infection and trigger an efficient ifn induction. nf-κb is a protein complex that controls dna transcription, cytokine production and cell survival. nf-κb is involved in cell responses to stimuli such as viral antigens and has a key role in regulating the innate immune response to infection . nf-κb has been shown to be activated upon accumulation of influenza virus rna . in response to specific stimuli, nf-κb translocates from cytoplasm to the nucleus, where it binds promoters of regulated genes, activates transcription of the interferon pathway , and stimulates isg transcription. type i interferon, which includes the ifnα and β subtypes, is induced during influenza virus infection and has an essential role in host defense against the virus by activating expression of a large number of isg . to analyze the effect of dot l methylase in nf-κb activation and ifn signaling, we evaluated nf-κb subcellular distribution in cells treated with tumor necrosis factor (tnf)-α ( ng/ml, min), or infected with influenza virus ( pfu/ml, hpi), alone or with dot l inhibitor ( h). after tnf-α binding to the receptor, the inhibitory protein iκbα, which normally binds nf-κb and inhibits its translocation, is phosphorylated; this is followed by ubiquitination and degradation, releasing nf-κb, which is then translocated to the nucleus . nf-κb distribution was monitored using antibodies to the p subunit of the nf-κb complex and anti-lamin a/c antibodies to identify the nuclear envelope; influenza np detection was used to monitor infection. we analyzed > cells to quantitate nuclear and cytosolic nf-κb distribution (fig. ) . tnf-α addition produced a significant increase in nuclear import of nf-κb, and dot l inhibitor treatment significantly decreased its nuclear translocation (fig. a) . influenza virus infection similarly induced nf-κb nuclear import, and inhibition of h k methylation reduced its nuclear translocation (fig. b) . subcellular distribution of nf-κb was additionally examined in cells previously transfected with lentiviruses that express the specific dot l silencers or the control ( days). using the conditions described above, the corresponding cells were treated with tnf-α, or infected with pr hv influenza virus. in agreement with the results obtained using the dot l inhibitor, the dot l silencers decreased the nuclear translocation of nf-κb both, in tnf-α treated cells (fig. a ) or in influenza virus infected cells (fig. b) , whereas the expression of the control silencer did not change the subcellular distribution of nf-κb. together these results indicate that methylation of h k modulates the pathway involved in the nuclear translocation of nk-κb. as dot l inhibition impaired nf-κb activation, we examined its effect on steps that follow its nuclear translocation, that is, ifn and isg rna production. a cells were left untreated (control), treated with u/ml ifnαβ (ifn) or were infected with pr hv strain ( pfu/cell) (pr hv), alone or with dot l inhibitor ( h). after h or h of ifn treatment or virus infection, rna was extracted and used for qpcr detection. at h, we found a weak increase on ifnβ, ifn-stimulated gene (isg ) and interferon-induced protein mx (mx ) rna levels after ifnαβ addition or influenza virus infection, and dot l inhibitor treatment did not significantly decreased their accumulation (fig. b,c) . at h, we observed an increase in ifnβ, isg and mx rna levels and a reduction of these rnas when cells were pretreated with the h k methylase inhibitor (fig. b,c) . the significant reduction in nf-κb nuclear translocation and decreased ifnβ, isg and mx rna expression after inhibition of dot l both, support a role for h k methylation in modulating the antiviral response. given the role of h k methylation in the control of ifn signaling, we analyzed the effect of dot l inhibitor on influenza virus replication in cells with normal or deficient ifn responses. we used pr hv and cal strains at low m.o.i. to infect mdck, mdck v or mdck npro cells, untreated or treated with dot l inhibitor. mdck v cells express v protein, which targets stat and for degradation , and mdck npro cells express npro protein, which targets irf- for polyubiquitination and subsequent degradation ; ifn, but not isg, is thus induced in mdck v cells, and neither ifn nor isg are induced in mdck npro cells. we analyzed viral titers at different hpi and observed that the increased infectious particle production in mdck cells treated with the inhibitor and infected with pr hv or cal (fig. a and b) was reduced or lost in 'ifn-compromised' cells treated with dot l inhibitor. the effect of influenza virus infection on h k methylation in mdck cells with normal and deficient response to ifn was checked by colorimetric assays. influenza virus infection increased h k methylation levels in normal and interferon deficient cells, likewise in a cells (fig. s a) . since h k methylation does not affect influenza virus replication in cells with impaired ifn signaling, we analyzed the effect of dot l inhibitor in subsequent stages of viral infection. we evaluated ifnβ, isg and mx rna amounts after influenza virus infection ( pfu/cell) in ifn-competent or -deficient cells, untreated or treated with dot l inhibitor. influenza virus infection increased ifnβ, isg and mx production in mdck control cells, and inhibitor treatment reduced their accumulation (fig. c, top) . infection of mdck v cells increased ifnβ production, which was reduced by dot l inhibitor, whereas accumulation of isg was not observed, in accordance with the need for stat signaling for their induction (fig. c, center) . infection of mdck npro cells did not increase ifnβ, isg or mx rnas, independently of the presence of dot l inhibitor (fig. c, bottom) , which coincided with the inability of these cells to produce ifn and isg. in addition, total extracts of mdck, mdck v and mdck npro cells infected with pr hv at m.o.i for h were used for western blot analysis to detect irf- , stat , mx and isg to confirm the differential expression of these proteins in normal or ifn-deficient cells (fig. s b) . these results indicate that the interferon antiviral response to influenza virus infection is at least partially regulated by h k methylation via canonical type i ifn signaling mediated by the stat pathway at the level of ifn activation and response. these results indicated the ability of dot l methylase to control interferon signaling pathways. to evaluate whether h k methylase has a broader role in the control of virus multiplication, we tested the effect of dot l inhibitor and the lentiviruses expressing the dot l silencers on the replication of two distinct rna viruses, respiratory syncytial virus (rsv) and vesicular stomatitis virus (vsv). like influenza virus, rsv is a respiratory virus, and is the most common cause of viral lower respiratory tract infections in infants and children . although ifnβ appears to restrict viral replication in lung epithelial cells, it is generally agreed that rsv is relatively resistant to ifnαβ, effects, and ifn has not been detected in natural infection . vsv can infect insects, cattle, horses and pigs and is a potent ifn inducer both in cell culture and animal models . human epithelial a cells, untreated or treated with dot l inhibitor ( h) (fig. a,c) , or transduced with lentiviruses expressing the dot l silencers or control shrna ( days) (fig. b,d) , were infected at low m.o.i. with rsv ( pfu/ml) (fig. a,b) or vsv ( − pfu/ml) (fig. c,d) ; at various times post-infection, cell supernatants were obtained for viral titration (see methods). no changes were observed in rsv replication in the presence of the inhibitor or the dot l silencers, whereas infectious particle production clearly increased in vsv-infected cells treated with dot l inhibitor or when dot l was down-regulated through the expression of the specific silencers. we also evaluated the expression of viral and isg proteins (isg and mxa) during infection with rsv (fig. b, right) , and vsv (fig. d, right) . the corresponding viral proteins were detected and we observed a lack of ifn-stimulated gene induction in rsv-infected cells, while infection with vsv efficiently induced ifn response in our system. these results support a role for h k methylation in the control of ifn signaling, and a potential general dot l methylase function in regulating pathogen infection controlled by the ifn pathway. viruses are obligate intracellular parasites that completely subordinate host cell metabolism. although influenza virus does not integrate into the genome of the infected cell, the virus efficiently switches off expression of host cell genes , a result of a complex interplay of virus-induced activities closely coordinated to reduce the host response to eliminate the viral infection. consequently, during infection there is a network of viral-and host-induced modifications of cellular gene expression with a close dependence of chromatin-based functions and therefore of chromatin dynamic. accordingly, specific interactions between chromatin remodelers of the chd family and influenza virus proteins have been described such as the association of chd with the non-structural protein ns or chd and chd with the viral polymerase complex , , . in spite of this association, little effort has been made to identify epigenetic changes in chromatin induced by the infection. few alterations in dna methylation have been reported during influenza virus infection [ ] [ ] [ ] . among them changes in promoter methylation levels of some proinflammatory cytokines and interleukine genes when using the high virulence h n strain have been described. infection of human respiratory epithelial cells with pr hv strain did not show variations on dna methylation (fig. b) , however it is possible that changes in promoter methylation of specific genes and their subsequent inactivation, take place in response to particular virulent influenza strains and/or in different systems. previous studies showed that influenza virus infection modulates ptm of several isg. chromatin immunoprecipitation assays and antibodies that recognize canonical marks of transcription activation such as h k me or of inactivation such as h k me showed addition or removal of these ptm in several isg; modifications depended on strain virulence when h n virus was compared with the h n pandemic strain . this study emphasized the importance of epigenetic control in the antiviral response elicited by influenza virus infection; nonetheless, they focused on the search for specific canonical transcription activation or repression marks of histones that correlate with up-or down-transcriptional regulation of the corresponding genes and do not provide an overview of all possible changes to chromatin in response to the viral infection. for a broad overview of chromatin modifications in influenza virus-infected cells, we used an unbiased search for global changes in chromatin epigenetics at the dna and histone levels. we observed a general decrease on histone acetylation in h and h lysine residues after infection, as well as increased levels of unmodified h k , h k and dimethylated h k (fig. c and d) . in addition, previous studies showed reduction of h k me ; one of the hallmark of active chromatin . histone acetylation has a key role opening the condensed chromatin, resulting in charge neutralization and a more relaxed, open, and transcriptionally active chromatin structure . decreased acetylated histones, trimethylated h k and increased non-methylated h k and dimethylated h k , all associate with transcriptional inactivation [ ] [ ] [ ] [ ] . these epigenetic changes would impair host cell expression, in accordance with the transcription inactivation of the host cell that occurs during infection and constitutes one of the major mechanisms triggered by the virus to inactivate host transcription machinery and consequently to decrease the antiviral response. unexpectedly, changes in methylation of lysine of histone were the most prominent. lysine is located within the globular domain of histone h and is mono-, di-, and trimethylated by dot l, which modifies this lysine exclusively . h k methylation is increased in actively transcribing genes and appears to have a role in cell cycle regulation and dna damage response . dot l has been studied particularly in the modulation of mixed-lineage leukemia (mll)-related leukemogenesis, as mll fusion target gene expression depends specifically on the functional dot l gene ; indeed, inhibition of dot l activity is currently in clinical trials. the role of h k methylation in viral infection, is poorly characterized. infection of human primary fibroblasts with human cytomegalovirus (hcmv), produces a marked increase in h k me levels and downregulation of dot l expression results in a decreased viral growth . the genome of hcmv is a double-stranded dna molecule that is maintained as episome during infection. the viral dna lacks histones when encapsidated in the virion however, upon infection the viral genome is transported to the nucleus, where it becomes associated with host cell histones. it has been speculated that dot l directly mediates replication of the hcv genome, in agreement with the chromatinization of its dna genome . the function of h k methylation modulating influenza virus replication, suggest a very different mechanism, since influenza virus is an rna virus that does not integrate in the host chromatin and its genome lacks histones. in physiological conditions where antiviral signaling is induced, decreased h k methylation clearly enhanced viral replication (figs and ) . moreover, nuclear translocation of nf-κb (figs and ) and accumulation of ifnβ and isgs (isg and mx ) decreased in influenza virus-infected cells with dot l downregulated (fig. ) . influenza virus and vesicular stomatitis virus induce a strong antiviral immune response characterized by robust production of antiviral type i interferons. during infection, double-stranded rna molecules are produced, which are recognized by the rig-i helicase . after activation, rig-i recruits various tnf receptor-associated factors, which trigger phosphorylation and activation of the iκκ complex, leading to iκbα phosphorylation and degradation to allow nfκb nuclear translocation . nf-κb and ifn regulatory factor direct expression of type i ifn, which promote transcription of a variety of genes that further limit viral replication , . we observed a marked increase on replication of influenza virus and vsv, both potent inducers of ifn, in cells with low levels of methylated h k (figs , and ). this effect is lost when cells deficient on ifn signaling are infected with influenza virus (fig. ) . together the data indicate that demethylation of h k would decrease antiviral ifn signaling and therefore, viruses may develop different strategies attempting to control epigenetic modification of h k that elicit the anti-pathogen response. methylation of lysine of histone might have a general role in controlling the host response of pathogens that are interferon inducers. epz was purchased from novagen, human tumor necrosis factor α from sigma-aldrich, and recombinant interferon αβ (pbl - universal type i ifn) was provided by s. guerra. lentiviral particle production and cell transduction. lentiviral particles were produced in hek t cells by cotransfection of plasmids pspax and pmd .g with each of the plko-based shrna vectors, as described , . supernatants were collected to h post-transfection, filtered through a . μm filter, and used to transduce the corresponding cells. as the lentiviral vectors confer puromycin resistance, the minimum amount of supernatant necessary to confer % resistance to puromycin ( μg/ml) was used. silencing was tested by western blotting or colorimetric assays, normally at days post-transduction. western blot was performed as described . to detect influenza virus proteins, the following antibodies were used: for pa, monoclonal antibodies (mab) and ( : ; ; for pb , mab ( : ; , and for pb , rabbit polyclonal antibody ( : ; ; for β-tubulin, mouse anti-β-tubulin mab ( : ; sigma). to analyze histones, we used h k ac ab ( : ; abcam) and h k me ( : ; active motif). polyclonal antibodies to h k me d e , h k me c d , h # and h d h ( : ) were from cell signaling. for vsv detection, a monoclonal antibody mix against g, n and m viral proteins provided by m. esteban was used. for rsv detection, a monoclonal antibody against np protein provided by j.a. melero was used. colorimetric determination of h k me . the iquik global di-methyl histone h k quantification kit (colorimetric) from epigentek was used for elisa-like measurement of total h k me amounts, following the supplier's protocol. proteomic analysis of histone modifications. enzymatic digestion and itraq- plex labeling. the enriched histone fraction ( μg) for each condition, prepared following the epigentek protocol, was precipitated by the methanol/chloroform method, reconstituted in m urea/ m thiourea/ mm teab buffer (triethylammonium bicarbonate, ph . ), reduced with mm tris( -carboxyethyl) phosphine (tcep, sciex), and alkylated with mm methyl methanethiosulfonate (mmts, pierce); this was followed by trypsin (sigma-aldrich) proteolysis at a : enzyme:protein ratio. the tryptic peptides were labeled using the itraq- plex isobaric mass tagging kit (sciex) according to manufacturer's instructions (mock, tag- ; pr lv, tag- , pr hv, tag- ). samples were pooled, dried and desalted on a sep-pak c cartridge (waters). peptide fractions were subjected to lc-ms/ms analysis using a nano liquid chromatography system (eksigent technologies nanolc ultra d plus) coupled to a high speed triple tof mass spectrometer (sciex) with a nanoelectrospray ion source. samples were injected on a c pepmap trap column ( μm, μm i.d. x cm; thermo scientific) at μl/min, in . % formic acid in water, and the trap column was switched on-line to a c nanoacquity beh analytical column ( . μm, Å, μm i.d. x cm, waters), equilibrated in mobile phase a ( . % formic acid in water), and peptide was eluted in a min linear gradient from - % b ( . % formic acid in acetonitrile) at nl/min. the mass spectrometer was operated in data-dependent acquisition mode. for tof scans, accumulation time was set to ms, and up to precursor ions were monitored per cycle. data analysis. ms and ms/ms data obtained were converted to mgf files, which were also searched against a homo sapiens protein database containing protein-coding genes (including reversed entries to calculate false discovery rate; fdr) using the mascot server v. . (matrix science). search parameters were set as follows: enzyme, trypsin; allowed missed cleavages, ; fixed modifications, beta-methylthiolation of cysteine and itraq- plex (n-term and k); variable modifications, oxidation of methionine, acetylation (k and n-term) and methylation and dimethylation (k). peptide mass tolerance was set to ± ppm for precursors and . da for fragment masses. the confidence interval for protein identification was set to ≥ % (p < . ) and only peptides with an individual ion score above the % fdr threshold were considered correctly identified. dna methylation. isolated dna was processed according to the manufacturer's protocols for illumina infinium assay, as described . data filtering. from the initial , cpg, we excluded all probes with detection p-values > . ( cpg removed). we eliminated cpg containing single-nucleotide polymorphisms (snp) located within bp of the target cpg (snp ) and internal controls (ch and rs), resulting in cpg removed. a total of , valid probes were included in the final analysis. methylation score was represented as β-values, average for each probe was calculated and β-value differences were used for analysis. confocal immunofluorescence microscopy. cells were fixed in % paraformaldehyde ( min, room temperature) and stored in pbs. for immunofluorescence, cells were permeabilized ( min) in pbs containing % triton x- and incubated with primary antibodies diluted in pbs/ % bsa (w/v) as follows: rat anti-np ( : ;, anti-p ab ( : ; abcam) and anti-lamin a/c ( )( : ; sc- , santa cruz) to label nuclear envelope. confocal microscopy was performed with a leica tcs sp laser scanning system. images of × pixels and an eight-bit grayscale depth were acquired sequentially every . - . μm using las af version . . software (leica) and analyzed using las af and metamorph premier version . . image analysis software (molecular devices). p nuclear translocation was quantified by counting at least cells/condition, and the ratio of relative p intensity in nucleus and cytoplasm of each cell was calculated. qrt-pcr analysis. for rna extraction, cell pellets were resuspended in ml trizol reagent (invitrogen) and rna was purified as recommended by the manufacturer. rna was digested with rnase-free dnase ( u/ mg; h, °c), extracted with phenol-chloroform-isoamyl alcohol and ethanol-precipitated. for reverse transcription, we used the high-capacity cdna rt kit (applied biosystems). pcr were performed in -well pcr plates using sybr green pcr master mix (applied biosystems). pcr were carried out in a prism sequence detection system (applied biosystems). the cycle threshold (ct) was determined with analytical software (sds; applied biosystems). serial dilutions of cdna were used to ensure amplification. chromatin remodeling, 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transcription and replication we are indebted to j. ortin, p. gastaminza and u. garaigorta for critique of the manuscript. the technical assistance of s. ciordia and r. navajas from the proteomics facility (cnb-csic, proteored isciii) is greatly appreciated. we thank c. mark for editorial assistance. lmv was supported by ciber de enfermedades respiratorias. this work was funded by the spanish ministry of economy and competitivness, plan nacional de investigacion científica, desarrollo e innovacion tecnologica (bfu - -r, (aei/feder)) and ciber de enfermedades respiratorias. js is a miguel servet researcher at isciii (ms / ). supplementary information accompanies this paper at https://doi.org/ . /s - - - .competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - pzqj authors: terasaki, kaori; ramirez, sydney i.; makino, shinji title: mechanistic insight into the host transcription inhibition function of rift valley fever virus nss and its importance in virulence date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: pzqj rift valley fever virus (rvfv), a member of the genus phlebovirus within the family bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the african continent and middle east. rvfv nss protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. these include suppression of general transcription, inhibition of ifn-β promoter induction and degradation of double-stranded rna-dependent protein kinase r. although each of these biological functions of nss are considered important for countering the antiviral response in the host, the individual contributions of these functions towards rvfv virulence remains unclear. to examine this, we generated two rvfv mp- strain-derived mutant viruses. each carried mutations in nss that specifically targeted its general transcription inhibition function without affecting its ability to degrade pkr and inhibit ifn-β promoter induction, through its interaction with sin -associated protein , a part of the repressor complex at the ifn-β promoter. using these mutant viruses, we have dissected the transcription inhibition function of nss and examined its importance in rvfv virulence. both nss mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with mp- . it has been reported that nss suppresses general transcription by interfering with the formation of the transcription factor iih complex, through the degradation of the p subunit and sequestration of the p subunit. our study results lead us to suggest that the ability of nss to induce p degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p may not play a significant role in this function. importantly, rvfv mp- -nss mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice, compared with its parental virus mp- , highlighting the contribution of nss-mediated general transcription inhibition towards rvfv virulence. rift valley fever virus (rvfv), a member of the genus phlebovirus within the family bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the african continent and middle east. rvfv nss protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. these include suppression of general transcription, inhibition of ifn-β promoter induction and degradation of doublestranded rna-dependent protein kinase r. although each of these biological functions of nss are considered important for countering the antiviral response in the host, the individual contributions of these functions towards rvfv virulence remains unclear. to examine this, we generated two rvfv mp- strain-derived mutant viruses. each carried mutations in nss that specifically targeted its general transcription inhibition function without affecting its ability to degrade pkr and inhibit ifn-β promoter induction, through its interaction with sin -associated protein , a part of the repressor complex at the ifn-β promoter. using these mutant viruses, we have dissected the transcription inhibition function of nss and examined its importance in rvfv virulence. both nss mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with mp- . it has been reported that nss suppresses general transcription by interfering with the formation of the transcription factor iih complex, through the degradation of the p subunit and sequestration of the p subunit. our study results lead us to suggest that the ability of nss to induce p degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p may not play a significant role in this function. importantly, rvfv mp- -nss mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice, rift valley fever virus (rvfv) is the pathogen causing rift valley fever, which affects both humans and domestic ruminants, primarily in countries of the african continent and middle east. the virus is an arbovirus and circulates between mosquito vectors and ruminants in endemic areas. rvfv causes high mortality rates in young ruminants and a high rate of abortions in pregnant ruminants [ ] . humans are infected with the virus either by mosquito bite or by direct contact with materials of infected animals. the majority of patients show influenzalike symptoms but few develop hemorrhagic fever, neurological symptoms, and ocular disease [ ] . due to its major impact on public health, rvfv is classified as a category a priority pathogen by the national institute of allergy and infectious diseases. currently there is no approved vaccine available for humans and animals in non-endemic areas. rvfv belongs to the family bunyaviridae, genus phlebovirus. rvfv is an enveloped virus and carries segmented rna genomes, the l, m and s segments, which are of negative or ambisense polarity. the l segment encodes l protein, a viral rna-dependent rna polymerase. m rna encodes kda protein, nsm protein, gn protein and gc protein, the latter two of which are major envelope glycoproteins and generated by co-translational cleavage of precursor gn/gc polyprotein. kda protein is dispensable for virus replication [ ] , whereas it plays important roles in virus dissemination in mosquitoes [ , ] . nsm is a viral anti-apoptotic protein [ , ] and also is important for efficient virus replication in macrophage cell lines [ ] . s rna expresses a nucleocapsid (n) protein and a nonstructural protein nss by using an ambisense coding strategy. the n protein encapsidates the viral rna and forms a ribonucleocapsid complex with l protein [ ] . rvfv nss protein is a phosphoprotein with an apparent molecular weight of kda and is localized in both the cytoplasm and nucleus [ ] . in the nucleus, nss forms filament-like structures by self-dimerization through its c-terminal domain [ ] . rvfv nss protein is a major virus virulence factor and has various important biological functions, which are important for countering the host antiviral response. one of the nss functions is suppression of ifn-β mrna transcription. nss binds to sin -associated protein (sap ), a subunit of a corepressor complex, and maintains ifn-β promoter in a transcriptionally silent state, leading to suppression of ifn-β mrna transcription [ ] . in addition to the specific inhibition of ifn-β transcription, nss suppresses general transcription; it has been proposed that nss exerts suppression of general transcription by interacting with subunits of transcription factor ii h (tfiih) complex, p and p [ , ] . a recent study showed that the nss-mediated general transcription inhibition contributes to the inhibition of ifn-β mrna transcription [ ] . although nss-mediated transcription suppression has been considered to be important in suppressing the host antiviral response, the exact effects of the nss-mediated general transcription inhibition on virus virulence have not been defined. nss promotes the degradation of doublestranded rna-dependent protein kinase r (pkr), an antiviral ifn-stimulated gene product, through a proteasome pathway to prevent phosphorylation of eif -α triggered by rvfv infection [ ] [ ] [ ] [ ] . furthermore, nss contributes to cellular stress responses such as an increase in reactive oxygen species, activation of dna damage signaling, cell cycle arrest, and activation of the p signaling pathway [ ] [ ] [ ] [ ] . although how nss induces the cellular stresses remains largely unknown, these stress responses may contribute to rvfv-induced cell death. to elucidate the mechanisms of the different functions of nss, it would be of great value to characterize a series of nss mutants, each of which specifically lacks one of these nss functions but retains other functions. however, introducing any short in-frame deletion in the rvfv nss resulted in loss of all functions [ ] , suggesting to us that the nss protein structure is important for its biological activity. hence, it has been challenging to generate nss mutants that lack a specific biological function but retain its other functions. the lack of these nss mutants has prevented a detailed mechanistic analysis of each biological function of the nss. in this study, we generated two rvfv mutants, each carrying mutations in nss, that specifically targeted its general transcription inhibition function, to delineate the mechanism of its inhibition by nss. furthermore, we examined the importance of the nss-mediated inhibition of general transcription on virus-induced cytotoxicity and tested its role in rvfv virulence by using a young mouse model. all mouse studies were performed in facilities accredited by the association for assessment and accreditation of laboratory animal care in accordance with the recommendations in the guide for the care and use of laboratory animals (institute of laboratory animal resources, national research council, national academy of sciences, ). the animal protocol (protocol number, a) was approved by the institutional animal care and use committee of the university of texas medical branch. a standard recombinant pcr method, in which pprot -s encoding antiviral-sense s rna [ ] served as a template, was used to generate pprot -s carrying mutations in nss coding region. the nss coding region of pprot -s was amplified by using primers carrying mfe i or a not i site. after digestion with mfe i and not i, the pcr fragment was cloned into eco ri and the not i site of a pcaggs plasmid, resulting in the plasmids expressing nss protein. for expression of human fbxo isoform (fbxo / ) [ ] , intracellular rnas of mrc- cells were subjected to cdna synthesis, and the fbxo / gene was amplified with primers carrying mfe i or the not i site. after addition of an n-terminal v tag, the pcr product was cloned into ecor i and the not i site of pcaggs. for expression of human sap , pcr product encoding human sap with n-terminal v tag was cloned into ecor i and the xho i site of pcaggs. all of the constructs were confirmed by sequencing. bsr-t / cells which stably express t rna polymerase [ ] were maintained in glasgow's minimal essential medium (mem) (lonza) containing % fetal bovine serum (fbs), % tryptose phosphate broth, mem amino acids solution and geneticin ( mg/ml). vero e cells were maintained in dulbecco's modified mem (gibco) containing % fbs. mrc- cells were maintained in eagle's mem (emem) (gibco) containing % fbs, mem non-essential amino acids solution (gibco), and % sodium pyruvate (sigma). hela cells were maintained in emem containing % fbs. mp- is a highly attenuated rvfv strain obtained after serial passages of an rvfv zh strain in the presence of -fluorouracil [ ] . a recombinant mp- strain and other mp- -derived mutants were rescued from cdnas as described previously [ ] , except that bsr-t / cells were used in place of bhk/t - cells. titers of the rescued viruses were determined by using a plaque assay [ ] . for the virus carrying r h/ m k mutations in nss, passage (p ) virus obtained from plasmid-transfected bsr-t / cells were serially diluted and inoculated into veroe cells. the highest titer of p virus was selected and used for the study. anti-pkr rabbit polyclonal antibody, anti-flag tag mouse monoclonal antibody, anti-flag tag rabbit monoclonal antibody, and anti-v tag rabbit monoclonal antibody were purchased from cell signaling technology. anti-gtf h (p ) mouse monoclonal antibody, anti-gtf h (p ) mouse polyclonal antibody, and anti-xpd mouse monoclonal antibody were purchased from abcam. anti-tfiih p (n- ) goat polyclonal antibody, anti-β-actin goat polyclonal antibody and horseradish peroxidase (hrp)-labeled anti-mouse, anti-goat, and anti-rabbit secondary antibodies were purchased from santa cruz biotechnology. anti-gst-n rabbit polyclonal antibody was generated by inoculating a rabbit with gst-n fusion protein (the entire n protein was fused with the c terminus of gst protein) followed by affinity purification of the serum [ ] . anti-mp- mouse serum was provided by dr. robert b. tesh at the university of texas medical branch. the monoclonal antibody h k k d k (h k), which is against major histocompatibility complex class i antigen, was obtained from dr. paul gottlieb at the university of texas at austin. cells were washed with phosphate-buffered saline (pbs) and suspended in sds polyacrylamide gel electrophoresis (sds-page) sample buffer. samples were boiled for - min and subjected to sds-page. proteins were electroblotted onto polyvinylidene difluoride membranes (immune blot: bio rad). after blocking with skim milk, the membranes were incubated with the primary antibody for h at room temperature and with the secondary antibody for h at room temperature. the proteins on the membrane were detected by using an ecl western blotting detection reagent (ge healthcare life sciences) or ecl plus western blotting substrate (pierce). vero e cells were infected with either mp- or its mutants at a multiplicity of infection (m.o. i.) of . at h post infection (p.i.), the culture media was replaced with methionine/cysteinefree medium. after starvation for min, the infected cells were labeled with μci/ml of s-methionine/cysteine ( , ci/mmol; mp biomedicals) for h. the radiolabeled cells were suspended in x sds-page sample buffer, resolved by sds-page and visualized by coomassie blue staining or autoradiography. the click-it rna alexa fluor imaging kit was purchased from thermo fisher scientific. vero e cells were infected with recombinant viruses at an m.o.i. of and incubated with mm of -ethynyl uridine ( eu) for h at h p.i. cellular rna was stained with alexa fluor, -coupled azide for min by following the manufacturer's protocol. for a negative control, mock-infected cells were treated with μg/ml of actinomycin d (actd) for min prior to eu treatment and for h during eu treatment. after the click reaction, rvfv n protein was stained with anti-gst-n rabbit polyclonal antibody followed by alexa fluor conjugated anti-rabbit antibody. images were captured on a zeiss axiophot fluorescence microscope with a x magnification lens and processed with the imagej software [ ] . for flow cytometry analysis, the infected cells were detached from the dish by accumax (innovative cell technologies) after the eu treatment and suspended in culture media. after washing with pbs containing % bovine serum albumin (bsa), cells were fixed with % formaldehyde/pbs for min at room temperature and blocked with blocking buffer (pbs containing . % saponin and % bsa) for min on ice. cellular rna was stained by alexa fluor -coupled azide for min in the presence of . % saponin and % bsa. after washing with the blocking buffer, rvfv n protein was stained by anti-gst-n rabbit polyclonal antibody for min on ice followed by alexa fluor conjugated anti-rabbit antibody. the cells were washed with pbs containing % bsa, passed through a cell strainer (bd falcon), and analyzed on an lsrii fortessa (bd biosciences). single cells were gated based on their forward scatter and side scatter profile. more than , count of the gated single cells were analyzed for each experiment. confluent vero e cells grown in -well plates were either mock infected or with mutant virus at an m.o.i. of . cell viability was determined by using viral toxglo (promega), which measures cellular atp. at various times p.i., atp detection reagent was added to each well. after incubation for min, luminescence was measured by spectramax m e (molecular devices). total rnas were extracted by using trizol reagent (invitrogen) and subjected to northern blot analysis as described previously [ ] . strand-specific digoxigenin-labeled rna probes and a digoxigenin (dig) system (roche) were used to detect rna. the -nucleotide-long, cell-derived, and dig-labeled ifn-β riboprobe [ ] was used for ifn-β mrna detection. cells cultured on chamber slides were fixed in % paraformaldehyde for min. the fixed cells were permeabilized with . % tritonx- for min and blocked with % bsa in pbs for min. after the blocking, the cells were stained with primary antibody diluted with the blocking solution, followed by incubation with alexa fluor or -conjugated secondary antibodies (molecular probes). images were captured by a zeiss axiophot fluorescence microscopy and processed with imagej software [ ] . to detect the interaction between nss and p , we infected hela cells with mp- -nss-flag, which expresses nss with the c-terminal flag tag or its mutant viruses at an m.o.i. of . at h p.i., the cells were lysed in lysis buffer [ mm tris-hcl, ph . , . % np- , mm nacl, mm edta, protease inhibitors (sigma), and u/ml benzonase (sigma)]. after cycles of freeze-thaw, the cell lysate was cleared by centrifugation at °c and , x g for h and incubated with dynabeads protein g (life technologies) conjugated with anti-gtf h (p ) mouse monoclonal antibody or h k antibody according to the manufacturer's protocol. precipitates were washed with the lysis buffer with . % tween and analyzed by western blotting using the anti-p goat polyclonal, and anti-p mouse monoclonal and anti-flag mouse monoclonal antibodies. to detect the interaction between nss and sap , we transfected hela cells with pcaggs-v -sap which encodes human sap carrying an n-terminus v tag by using fugene hd transfection reagent (promega). at h post transfection, the cells were infected with mp- -nss-flag or its mutant viruses. the cells were lysed with lysis buffer ( mm tris-hcl, ph . , mm nacl, % np- , mm edta, and protease inhibitor cocktail) [ ] at h p.i. the cell lysate was cleared by centrifuge at , x g and °c for min and incubated with anti-v -tag mab-magnetic beads (mbl international) according to the manufacturer's protocol. precipitates were washed with the lysis buffer containing mm nacl and analyzed by western blotting by using anti-v tag rabbit monoclonal and anti-flag mouse monoclonal antibodies. for detection of interaction between nss and p , hela cells were infected with mp- -nss-flag or its mutant viruses at an m.o.i. of . at h p.i., the cells were lysed in lysis buffer [ mm tris-hcl, ph . , . % tritonx- , mm nacl, mm edta, protease inhibitors (sigma), and u/ml benzonase (sigma)] [ ] . after cycles of freeze-thaw, the cell lysate was cleared by centrifugation at °c and , x g for h and incubated with dynabeads protein g conjugated with anti-gtf h (p ) mouse monoclonal antibody according to the manufacturer's protocol. precipitates were analyzed by western blotting using the anti-p mouse monoclonal and anti-flag mouse monoclonal antibodies. to detect the interaction between nss and fbxo, we transfected hela cells with pcaggs-v -fbxo / , which encodes fbxo / carrying an n-terminus v tag, by using fugene hd transfection reagent. at h post transfection, the cells were infected with mp- -nss-flag or its mutant viruses and lysed with lysis buffer ( mm tris-hcl, ph . , . % np- , % glycerol, mm nacl, . mm mgcl , and protease inhibitor cocktail) [ ] at h p.i. the cell lysate was cleared by centrifugation at , x g and °c for min and incubated with anti-v -tag mab-magnetic beads (mbl international) according to the manufacturer's protocol. precipitates were washed with the lysis buffer and analyzed by western blotting by using the anti-v tag rabbit monoclonal and anti-flag mouse monoclonal antibodies. hela cells prepared in -well plates were transfected with . μg of the pcaggs-based plasmid encoding either nss or mutant nss or an empty vector along with . μg of prl-sv (promega) which expresses renilla luciferase under control of an sv promotor. renilla luciferase activities in the transfected cells were measured by using a renilla luciferase assay system (promega) at h post transfection. eighteen-day-old cd- mice were intraperitoneally inoculated with pfu of mp- , mp- -m k or mp- -r h/m k or with hank's balanced salt solution (hbss). the animals were observed for survival for days post inoculation. we previously generated and characterized an rvfv mp- strain-derived mutant virus, which is deficient for efficient virus genome co-packaging due to a large deletion in the ' untranslated region of m rna segment [ ] . to obtain virus variants, carrying mutations that can compensate for this deficiency, we serially passaged this mutant virus in type i ifn-incompetent vero e cells; the virus inoculum, used for each passage, was diluted times and the released viruses were harvested at days p.i. the titer of the mutant virus progressively increased with each passage; at passage , the virus titer was . x pfu/ml, which was logs higher than the initial titer of . x pfu/ml prior to the serial passage. we isolated plaque-cloned viruses from passage level , and had a large deletion in the nss gene in the s segment, whereas plaque-cloned viruses retained the full-length nss gene. we determined the full genome sequence of clones that retained the full-length nss gene and found that all of the isolated viruses had several mutations in the l, m, and s segments. table shows mutations found in the nss genes of plaque-cloned viruses and the uncloned passage virus. all five plaque-cloned viruses and the passage virus had an amino-acid substitution at position , including m k or m t mutations. two plaque-cloned viruses and the passage virus had a d g substitution. other mutations found in plaque-cloned viruses were r h and a e. although k n mutation was found in the uncloned passage virus sample, this mutation was absent in the plaque-cloned viruses. to test whether the mutations affect nss functions, we generated mp- -based mutant viruses, each carrying r h, r h+m k (nss-r h/m k), a e, d g, d g+m t, m k (nss-m k), or m t mutations in the nss, by using a reverse genetics system [ ] . accumulations of nss protein in veroe cells infected with mp- carrying the d g mutation or the d g+m t mutations were significantly lower than those in mp- infected cells (fig ) , indicating that the d g mutation affected efficient nss accumulation. due to the poor accumulation of nss, we excluded the mp- mutant carrying the d g mutation and that carrying the d g+m t mutation from subsequent analysis. in cells infected with the other mutants, levels of nss protein accumulation were similar to that in mp- infected cells. in this study, we refer to mp- nss as wild type nss (wt nss). to evaluate host transcriptional shut-off activities of these nss mutants, levels of rna synthesis in virus-infected veroe cells were measured by labeling with eu from h to h p.i. mock-infected cells alone, as well as those treated with actd, and mp- -infected cells and cells infected with mp- Δnss, which lacks the nss gene [ ] , served as controls. eu-labeled rna, which was detected as a fluorescence signal, was mainly observed in the nucleus but not in the cytoplasm, indicating that viral rna synthesis, which occurs exclusively in the cytoplasm, was not very active at h post infection. as shown in fig a, strong signals of fluorescent-labeled rna were observed in the nucleus of mock-infected cells and mp- Δnssinfected cells, demonstrating active host rna synthesis. in contrast, actd-treated cells showed very weak fluorescent signals, demonstrating actd-mediated host transcriptional shut-off. mp- -infected cells showed weaker fluorescent signals in the nucleus, compared with those in mp- Δnss-infected cells, demonstrating an inhibition of host transcription by nss. fluorescent intensity observed in cells infected with virus carrying mutation m t, r h or a e in nss was similar to that in mp- -infected cells, suggesting that host transcription was still inhibited by these nss mutants. in contrast, cells infected with mp- carrying nss-r h/ we next used flow cytometry analysis to quantitatively measure the levels of eu-labeled rnas in mp- -m k-infected cells and in mp- -r h/m k-infected cells. we used the same controls as described above. fig b shows the results using density plots, wherein eu incorporation and n protein levels are shown on the x and y axes, respectively. in mockinfected and actd-treated samples, most of the cells were found in quadrant (q ) and q , respectively, demonstrating active host transcription in the former, but not in the latter ( fig b-a and b-b) . in all infected samples, . - . % of cells were rvfv n protein positive (q +q ), demonstrating that most of the cells were infected. the level of eu-labeled rna in mp- Δnss-infected cells was similar to that in mock-infected cells, demonstrating similar rna synthesis activity in these two samples (fig b-a and b-f ). mp- -infected cells showed lower levels of eu-labeled rna compared with those in mp- Δnss infected cells, demonstrating its lower rna transcription activity (fig b-c and b-f ). mp- -r h/m kinfected and mp- Δnss-infected cells showed similar density plot patterns, suggesting to us that host transcription inhibition did not occur in mp- -r h/m k-infected cells (fig b- e and b-f). mp- -m k-infected cells showed lower levels of eu-labeled rna compared with those in mp- Δnss-infected cells (fig b-d and b-f) . however, the percentage of mp- -m k-infected cells with low transcription activity was . % (q /q +q ), while in the mp- samples, it was . %. these results suggested that mp- -m k replication suppressed host general transcription, and yet the inhibitory activity of mp- -m k was weaker than that of mp- . we next examined the effect of differences in the transcription inhibitory activities of the mutant viruses on global protein synthesis by incorporation of s-methionine/cysteine into newly synthesizing proteins in mock-infected cells and in cells infected with mp- , mp- Δnss, mp- -m k, or mp- -r h/m k (fig c) . consistent with our previous study [ ] , mp- replication, but not mp- Δnss replication, suppressed host protein synthesis. mp- -r h/m k replication did not inhibit host protein synthesis, while mp- -m k replication moderately inhibited host protein synthesis. taken together, these analyses showed that nss-r h/m k lost the general transcription suppression activity, leading to efficient host protein synthesis in infected cells, while nss-m k exerted inefficient general transcription suppression activity, causing moderate levels of host protein synthesis inhibition in infected cells. analysis of replication kinetics of mp- , mp- Δnss, mp- -r h/m k and mp- -m k showed that all viruses replicated efficiently with similar replication kinetics in vero e cells (fig a) . mp- formed clear plaques in vero e cells, whereas mp- -r h/m k and mp- -m k formed turbid-type plaques like mp- Δnss (fig b) , indicating that the r h/ m k mutation and the m k mutation in nss affected virus-induced cytotoxicity. cell viability assays showed that mp- replication caused a % reduction in cell viability as compared with mock-infected cells at h p.i. (fig c) , whereas mp- Δnss-infected cells and mock-infected cells showed similar cell viabilities throughout the course of the experiments. mp- -r h/m k-infected cells and mp- -m k-infected cells showed an % and % decrease in cell viability, respectively, as compared with that in mock-infected cells at h p.i., demonstrating that both of the mutant viruses were less cytotoxic than mp- . mp- , mp- -m k and mp- -r h/m k had similar replication kinetics, regardless of their differential ability to induce cytotoxicity (fig ) , implying that virus-induced cytotoxicity did not have a significant impact on rvfv replication kinetics in vero e cells. because others reported that mp- replication induces nss-dependent p stabilization, which contributes to virus-induced cell death [ ] , we next examined stabilization of p in cells infected with mp- and other mutant viruses (fig d) . amounts of p protein significantly increased in mp- -infected cells, but not in mp- Δnss-infected cells, confirming the nss-dependent p stabilization in the infected cells. accumulation of p also occurred in mp- -m k-infected cells, whereas the accumulation levels were lower than those in nss interacts with pkr and triggers degradation of the pkr by a proteasome pathway [ ] [ ] [ ] [ ] . to investigate whether the nss mutants retain the ability for pkr degradation, we compared the total amount of pkr in virus-infected cells. similar levels of reduction in the amounts of pkr occurred in cells infected with mp- , mp- -m k or mp- -r h/ k, suggesting that nss-r h/m k and nss-m k promoted pkr degradation as efficiently as wt nss (fig ) . le may et al. proposed that interaction of nss with sap interferes with the recruitment of co-activator protein cbp at activation sites on the ifn-β promotor, leading to the inhibition of ifn-β mrna transcription [ ] . to investigate the ability of mp- -r h/m k or mp- -m k to suppress ifn-β mrna transcription, we examined the levels of ifn-β mrna in mrc- cells infected with mp- , mp- -Δnss, mp- -m k or mp- -r h/ k at various times p.i. (fig a) . mp- Δnss replication induced robust ifn-β mrna transcription, whereas ifn-β mrna was not detectable in mp- -or mp- -m k-infected cells. in contrast, low levels of ifn-β mrna accumulation occurred in mp- -r h/m k-infected cells, demonstrating that the nss-r h/m k was unable to efficiently inhibit ifn-β mrna transcription. mp- -m k replicated as efficiently as did mp- in mrc- cells, whereas the virus titer of mp- Δnss was significantly lower than that of mp- at h p.i. (fig b) , demonstrating the efficient inhibition of ifn-β production in the former, but not in the latter. although mp- -r h/m k replicated better than mp- Δnss, it replicated less efficiently mp- -r h/m k-flag). cells, transiently expressing a v -tagged sap , were infected with these viruses, and the co-localization of the mutated nss protein with sap was examined by fluorescence microscopy analysis. like wt nss, both nss mutants formed filament-like structures in the nucleus (fig c) . the expressed sap was mainly observed in the nucleus and was co-localized with wt nss and both mutated nss in the filaments. we also performed co-immunoprecipitation analysis to examine nss-sap interaction. as a negative control, v -tagged venus was expressed in place of v -tagged sap . anti-v antibody co-precipitated wt nss and both nss mutants along with v -tagged sap , while the amounts of nss-m k that were co-immunoprecipitated with sap were lower than those of wt nss ( fig d) . anti-v antibody did not co-precipitate wt nss along with v -tagged venus (fig d) . these data demonstrate that both nss mutants bound to sap in infected cells. taken together, these data indicated that nss-r h/m k was unable to completely block ifn-β transcription despite its ability to bind to sap . both nss mutants retained some nss functions, including degradation of pkr and sap binding, and yet these nss mutants and wt nss showed different levels of general transcriptional suppression activities in virus-infected cells. transcriptional suppression activities of the mutated nss were also examined in cells transiently expressing nss along with renilla luciferase from co-transfected plasmids. luciferase activity was strongly inhibited in the cells coexpressing wt nss and renilla luciferase (fig a) . consistent with the data obtained from infected cells (fig ) , nss-r h/m k expression did not inhibit luciferase activity, whereas nss-m k expression moderately inhibited the luciferase activity. western blot analysis of nuclear and cytoplasmic fractions from virus-infected cells showed that like wt nss, nss-r h/m and nss-m k were also distributed in both the nucleus and the cytoplasm (s fig) , demonstrating that these mutations did not affect the subcellular localization of nss. these data prompted us to further examine the interplay between these nss mutants and a form of host transcription machinery, tfiih, as other studies have shown an association of tfiih with nss in the nss-induced host transcription shut-off. rvfv nss binds to p , a subunit of tfiih, and interferes with the formation of the tfiih complex by inhibiting the subsequent interaction of p and xpd [ ] . the reduction in the abundance of xpd and p also occurs upon rvfv infection, although the mechanisms that govern the reduction of these proteins are unclear [ ] . in addition, nss binds to both p , a tfiih subunit, and fbxo , a component of e ubiquitin ligase, which leads to p degradation [ , ] . we first examined the abundance of tfiih components, including p , p and xpd, in infected cells. substantial reduction of p abundance and moderate reductions in the abundances of p and xpd occurred in cells infected with mp- , or mp- -m k at h p.i. (fig b, left panels) . in contrast, there was no substantial reduction in the abundance of these tfiih components in cells infected with mp- Δns or mp- -r h/m k. the same results were obtained when viruses carrying flag-tagged nss were used. to exclude the possibility that different transcription suppression activities of these viruses affected the results, we repeated the experiments in the presence of actd and obtained similar results (fig b, right panels) . next, we tested the interaction of mutated nss with p and p . cells infected with mp- or its mutants, all of which carried flag-tagged nss, were subjected to co-immunoprecipitation analysis using anti-p antibody (fig c) . anti-p antibody co-immunoprecipitated wt nss and both mutant nss along with p , whereas control anti-h k antibody precipitated neither p nor nss. these data demonstrated that both nss mutants bound to p . to examine the interaction of p and nss, cells were infected with the viruses as described above, and cell extracts were prepared at h p.i., when p was still detectable in mp- -nss-flag-infected cells (fig d) . anti-p antibody, but not anti-h k antibody, co-immunoprecipitated wt nss and both mutant nss along with p , demonstrating binding of the mutant nss proteins to p . we noted that the amounts of nss-m k that were co-immunoprecipitated with p and with p were lower than those of wt nss, implying that the binding efficiencies of nss-m k for p and for p were lower than those for wt nss. fbxo is an interactor of rvfv nss that is engaged in the degradation of p ; nss interacts with the full-length fbxo protein (fbxo / ) as well as with a shorter splice variant of the fbxo that lacks the c-terminal acidic domain and poly(r) region [ ] . because nss-r h/m k did not induce p degradation regardless of its ability to bind to p (fig d) , we suspected that nss-r h/m k would not interact with fbox . to test this possibility, cells transiently expressing the v -tagged fbxo / were mock infected or infected with mp- -nss-flag, mp- -m k-flag, or mp- -r /m k-flag. at h p.i., cell extracts were prepared and subjected to co-immunoprecipitation analysis by using anti-v antibody ( fig e) . all of the nss proteins, including nss-r /m k, were co-immunoprecipitated with fbxo / . table summarizes interactions of the nss mutants with p , p and fbxo . nss is a major virulence factor of rvfv [ ] . the nss-mediated inhibition of type i ifn production is thought to contribute to the virulence of the virus, yet it remains unclear whether the general transcription suppression function of nss contributes to this virulence. we examined the importance of the nss-mediated transcription inhibition in rvfv virulence by using a young mouse model. although mp- is known as an attenuated strain, the intraperitoneal inoculation of pfu of mp- into -day-old cd mice resulted in the death of % of the mice within days p.i. (fig ) . under the same experimental conditions, none of the mp- -r h/m k-inoculated mice and % of the mp- -m k-inoculated mice died. no obvious clinical signs, including neurological symptoms, were observed. these data demonstrated that the mp- -r h/m k lacked virulence, and mp- -m k was less virulent than mp- in this young mouse model. in this study, we demonstrated that the m k and r h/m k mutations in nss differentially reduced its inhibitory activity on host transcription without affecting its ability to inhibit ifn-β transcription, through its interaction with sap , and induced pkr degradation. nss-r h/m k, which completely lacked the activity to inhibit general transcription, correspondingly lost the ability to promote the degradation of p . unexpectedly, nss-r h/ m k was able to interact with fbox and p (fig ) . these data possibly suggest that the fbxo -nss-p interaction may not be sufficient to trigger p degradation. nss-m k exhibited a partially reduced activity to inhibit general transcription. the results of co-immunoprecipitation assays showed that a reduced amount of nss-m k co-precipitated with sap , p and p , compared with that in wt nss (figs and ). however, the slightly impaired ability of nss-m k to inhibit general transcription could not be solely attributed to the lower binding efficiency of nss-m k to p because nss-m k efficiently suppressed ifn-β transcription and induced p degradation, despite its lower efficiency of binding to sap and p . moreover, nss-r h/m k, which lacked transcription suppression activity, efficiently interacted with p ( fig ) . these data bring into question the importance of nss-p interaction for its host transcriptional shut-off function. one possibility is that the nss-p interaction may only make a modest contribution towards its transcription inhibition activity and possibly could have additional, as yet unidentified, biological function(s). a second possibility is that only wt nss, but not the mutated nss, is able to interfere with the formation of an active tfiih complex. nss competes with xpd for binding to p , resulting in inhibition of the tfiih complex formation [ ] . although both nss-m k and nss-r h/m k retained the ability to bind to p , it is possible that the binding of these mutated nss to p did not exclude the binding of xpd. the r h single mutation did not affect the ability of nss to inhibit transcription (fig ) . although the m k mutation is in close proximity to the Ωxav motif located at the c-terminal region of nss, which is essential for p degradation [ ] , mp- -m k still retained the ability to degrade p (fig ) . however, the r h/m k double mutant lost the ability to induce p degradation. these results imply that the combined mutations, r h and m k, induced an unfavorable structural alteration in nss, which abolished its function to degrade p . mp- -r h/m k replication induced low levels of ifn-β mrna (fig ) , indicating that nss-r h/m k was not able to block ifn-β mrna synthesis as efficiently as wt nss despite its ability to interact with sap (fig ) . it has been reported that nss-induced p degradation contributes to the inhibition of ifn-β production [ ] . accordingly, our data suggested that mp- -r h/m k was unable to completely block ifn-β mrna synthesis due to a lack of ability to promote the degradation of p (fig ) . although mp- -r h/m k replication induced ifn-β mrna synthesis, the level of the ifn-β mrna was significantly lower than that in mp- Δnss-infected cells (fig ) , suggesting the importance of interaction of nss with sap for ifn-β inhibition. taken together, the data shown here and those of others [ , ] strongly imply that nss-sap interaction and nss-induced general host transcriptional suppression function are both necessary for efficient inhibition of ifn-β mrna transcription. mp- Δnss or mp- encoding a reporter gene in place of the nss gene causes less prominent cytopathic effects than does mp- , demonstrating the contribution of the nss towards the induction of cytotoxicity [ ] . mp- replication induces nss-dependent p stabilization, which contributes to virus-induced cell death [ ] , and yet it was unclear which function of the nss contributed to the induction of the p -mediated cytotoxicity. as shown in fig , mp- -infected cells showed the lowest cell viability, followed in order by mp- -m k, which moderately suppressed host general transcription, and mp- -r h/m k, which did not suppress host transcription. hence, there was a correlation between the strength of nss-mediated, host transcriptional shut-off activity and cell viability in the infected cells (table ) . likewise, accumulation of p was the highest in mp- -infected cells, followed in order by that in mp- -m k-infected cells and mp- -r h/m k-infected cells, suggesting to us that p stabilization also correlates with the transcription inhibition activities of the different nss mutants. these results indicate that the nss-mediated host transcriptional shut-off triggered p stabilization, leading to p -mediated cell death. although mp- is an attenuated rvfv strain, % of -day-old cd mice died after intraperitoneal inoculation with pfu within days p.i. (fig ) . as the immune system is not yet fully developed in young mice, it likely failed to prevent systemic infection by mp- . consistent with this notion, intraperitoneal inoculation of mp- into severe combined immune deficiency mice also caused % mortality [ ] . mp- -r h/m k carrying nss that lacks the host transcription inhibition function was completely attenuated in -day-old cd mice (fig ) . mp- -m k carrying nss with an impaired ability to inhibit transcription also exhibited reduced virulence when compared to that in its parental virus mp- . these data highlight the role of the host transcription inhibition function of nss in rvfv virulence. type i ifn has been shown to play a critical role in protecting the host from rvfv-induced disease in animal models [ ] . notably, both the nss mutants, carrying either the m k single mutation or the r h/m k double mutation, retained the ability to bind to sap , the factor that is targeted by nss to inhibit ifn-β mrna transcription. however, the induction of ifn-β mrna synthesis was not completely blocked in mp- -r h/m k-infected mrc- cells, most probably due to the lack of inhibition of host transcription in these cells expressing the mutated nss. these data suggest the possibility that the inability of mp- -r h/m k to completely block the production of ifn-β in infected mice could have contributed towards its attenuation. in addition, the production of other antiviral and/or proinflammatory cytokines could have also contributed towards the attenuated phenotype of both of these mutant viruses, carrying nss with an impaired ability to block host transcription. it is also possible that the lower levels of virus-induced cytocidal effects might have contributed to the lower virulence of these nss mutant viruses, as both mutant viruses caused less severe cytopathic effects and cytotoxicity than did mp- in cultured cells (fig ) . we believe that experiments using rvfv mutants, carrying the m k single mutation and the r h/m k double mutation, in animal models would yield valuable information about the role of nss-mediated host transcription inhibition in regulating host cytokine responses and its impact on the pathogenesis of rvfv. mp- is an attenuated live vaccine candidate, but it still harbors residual virulence in a young mouse model. although further studies are required to test the immunogenicity and protective efficacy of mp- -m k and mp- -r h/m k, there is a potential for developing these mp- -derived nss mutant viruses as safer live attenuated rvfv vaccine candidates. we found that the serial passage of an mp- -derived mutant virus having a large deletion in the ' untranslated region of m rna segment [ ] in vero e cells resulted in accumulation of variant viruses that were able to replicate better than the original mutant virus. most of viruses in passage had a large internal deletion in the nss gene, which may mean their nss proteins are biologically inactive. others have reported that rvfv carrying large deletions in the nss gene start accumulating from the th serial passage in bhk cells of the rvfv p strain, which is defective in ifn-α/β signaling [ ] . in our experiment, the large deletions in the nss gene were detected after the th serial passage of the virus. although the cell lines used in the studies were different, the deletion of the nss gene as early as after passages implied that there was a selective pressure to remove the nss gene from the virus genome during the passaging of the mutant virus. the full-length nss gene of the uncloned passage viruses had m k, m t, k n and d g mutations (table ). in the cells infected with mp- carrying nss with a d g mutation, the accumulation of nss was poor (fig ) , indicating that the d g mutation affects the efficient accumulation of nss. as two out of the five plaque-cloned viruses (clones and , table ) also had the d g mutation in nss, this possibly affected its accumulation in infected cells. the remaining three clones carried nss with a m k or r h/m k mutation. our results showed that these mutations in nss partially or completely abolished its host transcription inhibition function without affecting its ability to interact with sap to inhibit ifn-β mrna synthesis. these data implied that the host transcription function of nss was unfavorable for the mutant virus, carrying a deletion in the ' utr, to replicate well in veroe cells. mp- -m k and mp- -r h/m k replication induced low cytotoxicity due to its lower inhibitory activity on host transcription (figs and ) . we suspect that the lack of nss-induced host transcription suppression created a favorable cellular environment for the mutant virus, thereby allowing the emergence and accumulation of mutant viruses that lacked this function. further studies are required to delineate the importance of nss-mediated host transcription inhibition in the virus life cycle. with mp- -nss-flag or its mutants, at an m.o.i. of and at h p.i., the cells were lysed using the lysis buffer ( mm tris-hcl, ph . , mm kcl, . mm mgcl , . % triton x- , protease inhibitor cocktail) followed by incubation on ice for min. after centrifugation at , x g for min, the resulting supernatant was collected as the cytoplasmic fraction (c). the pellet from this centrifugation was washed once with the lysis buffer (without triton x- ), suspended in x sds sample buffer and denoted as the nuclear fraction (n). the subcellular fractions were analyzed by western blotting using anti-flag, anti-hsp and anti-lamin a antibodies. (tif) breaking the chain: rift valley fever virus control via livestock vaccination the pathogenesis of rift valley fever nsm and -kilodalton proteins of rift valley fever virus are nonessential for viral replication in cell culture deletion of the nsm virulence gene of rift valley fever virus inhibits virus replication in and dissemination from the midgut of aedes aegypti mosquitoes the rift valley fever accessory proteins nsm and p /nsm-gn are distinct determinants of virus propagation in vertebrate and invertebrate hosts nsm protein of rift valley fever virus suppresses virusinduced apoptosis the c-terminal region of rift valley fever virus nsm protein targets the protein to the mitochondrial outer membrane and exerts antiapoptotic function recent advances in the molecular and cellular biology of bunyaviruses the rift valley fever virus nonstructural protein nss is phosphorylated at serine residues located in casein kinase ii consensus motifs in the carboxy-terminus the carboxy-terminal acidic domain of rift valley fever virus nss protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein a sap complex inhibits ifnbeta expression in rift valley fever virus infected cells tfiih transcription factor, a target for the rift valley hemorrhagic fever virus nss protein of rift valley fever virus promotes posttranslational downregulation of the tfiih subunit p virulence factor nss of rift valley fever virus recruits the f-box protein fbxo to degrade subunit p of general transcription factor tfiih rift valley fever virus nss protein promotes post-transcriptional downregulation of protein kinase pkr and inhibits eif alpha phosphorylation nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase protein kinase r degradation is essential for rift valley fever virus infection and is regulated by skp -cul -f-box (scf) fbxw -nss e ligase nss virulence factor of rift valley fever virus engages the f-box proteins fbxw and beta-trcp to degrade the antiviral protein kinase pkr reactive oxygen species activate nfkappab (p ) and p and induce apoptosis in rvfv infected liver cells induction of dna damage signaling upon rift valley fever virus infection results in cell cycle arrest and increased viral replication p activation following rift valley fever virus infection contributes to cell death and viral production nonstructural nss protein of rift valley fever virus interacts with pericentromeric dna sequences of the host cell, inducing chromosome cohesion and segregation defects functional analysis of rift valley fever virus nss encoding a partial truncation rescue of infectious rift valley fever virus entirely from cdna, analysis of virus lacking the nss gene, and expression of a foreign gene generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns is not essential for virus replication in tissue culture, and the human rsv leader region acts as a functional brsv genome promoter mutagen-directed attenuation of rift valley fever virus as a method for vaccine development nih image to imagej: years of image analysis rift valley fever virus nonstructural protein nss promotes viral rna replication and transcription in a minigenome system severe acute respiratory syndrome coronavirus nsp suppresses host gene expression, including that of type i interferon, in infected cells mechanism of tripartite rna genome packaging in rift valley fever virus characterization of clone , a naturally attenuated avirulent isolate of rift valley fever virus, which is altered in the small segment a Ωxav motif in the rift valley fever virus nss protein is essential for degrading p , forming nuclear filaments and virulence recombinant rift valley fever vaccines induce protective levels of antibody in baboons and resistance to lethal challenge in mice interplay between the virus and host in rift valley fever pathogenesis host alternation is necessary to maintain the genome stability of rift valley fever virus we thank robert tesh for anti-mp antibody; paul gottlieb for the monoclonal antibody h k k d k ; mark griffin (flow cytometry and cell sorting core, the university of texas medical branch) for support with the flow cytometry analyses; and krishna narayanan for critical reading of the manuscript. conceptualization: kt sm. key: cord- -p h p bm authors: lindqvist, richard; upadhyay, arunkumar; Överby, anna k. title: tick-borne flaviviruses and the type i interferon response date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: p h p bm flaviviruses are globally distributed pathogens causing millions of human infections every year. flaviviruses are arthropod-borne viruses and are mainly transmitted by either ticks or mosquitoes. mosquito-borne flaviviruses and their interactions with the innate immune response have been well-studied and reviewed extensively, thus this review will discuss tick-borne flaviviruses and their interactions with the host innate immune response. tick-borne flaviviruses (tbfv), flaviviridae family, includes many pathogens causing severe human disease, ranging from mild fever to encephalitis and hemorrhagic fever. there are more than viruses in the genus flavivirus, and they are transmitted by arthropods such as mosquitoes (dengue virus (denv), japanese encephalitis virus (jev) and west nile virus (wnv), yellow fever virus (yfv), and zika virus (zikv) and ticks (tick-borne encephalitis virus (tbev), langat virus (lgtv), kyasanur forest disease virus (kfdv), omsk hemorrhagic fever virus (ohfv), powassan virus (powv), and louping-ill virus (liv)) [ ] [ ] [ ] [ ] [ ] . among the tbfvs, tbev, powv, and liv are encephalitic, whereas ohfv and kfdv are hemorrhagic viruses. there are general features that distinguish tbfv from mosquito-borne. one such feature is that tick-borne viruses tend to persist in certain stable foci whereas mosquito-borne viruses may suddenly emerge and they can rapidly spread to new areas and continents causing large epidemics [ ] [ ] [ ] [ ] [ ] [ ] . mosquito-borne flaviviruses are transmitted horizontally from mosquito to vertebrate to mosquito, and for some viruses, humans act as the amplifying host [ ] [ ] [ ] . mosquitoes have a short life compared to ticks, as it can take several years for a tick to develop from egg to adult [ ] . ticks also only take one blood meal at each of their life stages, larvae, nymph, and adult, this means that tick-borne viruses need to be maintained in infected ticks during a very long time [ ] . thus, replication in ticks needs to be lower compared to mosquitoes, which have higher viral turnover and a faster generation cycle, this also contributes to the high mutation rate in mosquito-borne flaviviruses, and relative stable genomes of tbfvs [ , ] . infection of tick-borne flaviviruses usually cause a short viremia in humans and larger vertebrates. this route of transmission has been described as less important in tick-borne flavivirus infection [ ] , instead tbev has been shown to be transmitted by viremic rodents and among co-feeding ticks without viremia [ ] . humans act as dead-end hosts since they do not produce high enough viremia to transmit the virus to new ticks. according to phylogenetic differences, tbev has been divided into three different subtypes, european, siberian, and far eastern. the european subtype is mainly transmitted by ixodes ricinus, whereas the siberian and far eastern subtypes are primarily transmitted by ixodes persulcatus [ , ] . tbev is found in central, eastern, and northern europe and asia ( figure ) and correlates with the presence of infected ticks. ixodes ricinus is found throughout europe, whereas ixodes persulcatus is found in eastern europe in the west, and china and japan in the east [ ] . tbev is considered one of the most important arboviruses in central and eastern european countries and in russia, with about , estimated human cases annually [ ] . in fact, over the last decade there has been an approximately % increase in the number of tbe cases in europe [ ] , and tbev is currently spreading into new regions in france, sweden, norway, and italy [ ] [ ] [ ] [ ] . this increase is thought to be due to growth in population and spread of ticks, which is promoted by factors including climate change, social and political change, and changes in the land use [ , ] . the increased expansion in europe also poses an increased risk for the population engaged in outdoor activities. tbev is a zoonotic disease and the natural cycle of tbev is dependent and maintained in a complex cycle involving ticks as the vector and reservoir of the virus and small rodents as hosts for ticks [ ] . humans are not part of the natural transmission cycle of tbev and are the incidental host when infected by a bite from an infected tick [ ] . transmission through consumption of unpasteurized milk has also been reported for tbev [ , ] , as well as transmission via solid organ transplant [ ] . during the tick bite, the virus is inoculated into the skin of the vertebrate host. the initial replication is believed to occur locally in the dendritic cells. this is followed by infection of the draining lymph nodes, resulting in the primary viremia and subsequent infection of the peripheral tissues, where further replication maintains the viremia for several days [ ] [ ] [ ] [ ] . the disease course of tbev is biphasic; the initial phase is characterized by flu-like symptoms and is followed by a second phase involving cns infection, with meningitis, encephalitis, or meningoencephalitis [ ] [ ] [ ] . the mortality rate of tbev varies from to % depending on the subtype, in which the european tbev subtype has shown lower mortality rates compared to the siberian and far eastern [ , , ] . among the patients that experience neuroinvasive tbev infection, approximately - % of the survivors suffer from long lasting neurological sequelae [ , ] . no antivirals are available for treatment of tbev infection but there is an effective vaccine [ , ] . lgtv, which is closely related to tbev, is found in south east asia and russia [ ] . lgtv has not been associated with human disease under natural infections although it shares % sequence identity with tbev [ ] . because of its avirulence in humans and close similarity to tbev, lgtv is often used as a model virus for tbev under biosafety level- conditions. powv is found in russia and north america, and is the only tbfv present in america ( figure ) [ ] . it is transmitted by ixodes scapularis, ixodes cookei, and several other ixodes tick species, to small and medium size mammals, whereas humans are accidental dead-end hosts. milk-borne powv transmission might also be possible since powv virus has been found to be secreted in milk under experimental settings [ ] . although not much is known about powv pathogenesis, recent studies in mice have found that tick saliva was important to enhance powv transmission and the outcome of disease [ ] . furthermore, it has been demonstrated that powv infects macrophages and fibroblasts in the skin, shortly after the tick bite, also, other unidentified cells were shown to be infected [ ] . interestingly, macrophages were found to be the primary target for powv in the spleen [ ] , and in the cns, which is the main target site for powv infection, neurons have been shown to be the primary target for powv in mice and humans [ , ] . during the last years there has been an increase of powv in the usa with approximately reported cases [ , ] . the recent rise in incidence could be due to increased surveillance and diagnosis of powv, or it may represent a true emergence of the disease in endemic areas, or both [ ] . the incubation period ranges from week to month. the symptoms of powv infection may include fever, headache, vomiting, weakness, confusion, seizures, and memory loss with a case fatality rate of % [ ] . approximately half of the survivors experience permanent neurological symptoms, such as recurrent headaches, muscle wasting, and memory problems (https://www.cdc.gov). there are no antiviral treatments or vaccines available against powv. liv is mainly distributed in the uk and ireland, but it has also been detected in sheep in norway and on the bornholm island in denmark ( figure ). liv is most commonly known as pathogen of sheep and red grouse although humans can also get infected [ , ] . animals develop a febrile disease, which can progress to fatal encephalitis [ ] . like tbev, the vector of liv is the tick species ixodes ricinus [ ] . ticks transmit the virus to animals, however, natural exposure to humans is rare [ ] . instead humans that are exposed to infected animals such as veterinarians, farmers, butchers, and abattoir workers, as well as laboratory scientists have acquired liv infection [ ] [ ] [ ] [ ] . in between the years of and , human cases of liv was described [ ] . in humans, liv causes a disease that closely resembles tbev, with initial flu-like symptoms which can progress to severe neurological disease. distinct but closely related viruses are also found in spain (spanish sheep encephalomyelitis virus), turkey (turkish sheep encephalitis virus), and greece (greek goat encephalitis virus) [ ] . ohfv distribution is restricted to western siberia ( figure ) [ ] . the main vector of ohfv is the meadow tick, dermacentor reticulatus, which can also transmit the virus to humans. however, humans are mainly infected after contact with infected muskrats (ondatra zibethicus) which are very sensitive to the infection and often succumb to the infection [ ] . muskrats develop high viremia which can last for several weeks. human infection occurs through contact with urine, feces, and blood [ ] . secretion of ohfv in unpasteurized goat milk has been reported but no milk-borne outbreaks have been observed [ ] . the exact number of annual cases are uncertain because of misdiagnoses and unreported cases, but cases were reported between and [ ] . ohfv may cause a biphasic disease; the initial phase is characterized by high fever, bleeding from the nose, mouth, and uterus. thirty to fifty percent of the cases experience a second phase characterized by high fever and reappearance of the symptoms from the initial phase. case fatality rates range from . to . % [ ] . no antiviral treatments are available against ohfv, instead treatment is focused on supportive care to minimize hemorrhage and other complications [ ] . kfdv is only found in india (figure ), although a similar genetic variant, alkhurma hemorrhagic fever virus (ahfv) has been detected in saudi arabia [ , ] . kfdv is mainly transmitted by ticks belonging to the genus hemaphysalis but other tick genera have also been shown to be able to transmit kfdv [ , ] . during hemorrhagic kfdv infection, the initial phase is similar to tbev infection with fever and flu-like symptoms, but it may also include bleeding from the nose, mouth or gastrointestinal tract [ , ] . the second phase, which is experienced by - % of the cases, includes neurological symptoms such as headache, mental disturbance, and tremor, however, no evidence of meninges or encephalitis have been found [ ] . there are about - reported annual cases of kfdv [ ] , with case fatality rates ranging from to % [ , ] . in a kfdv vaccine was developed, although it provided some protection, due to low efficacy, the number of cases still increased from to [ ] [ ] [ ] . treatment of kfdv is limited to supportive care [ ] . tbfvs are enveloped viruses around nm in diameter. the envelope carries two surface proteins, the envelope (e) protein and the membrane (m) protein. the latter is derived from a precursor protein, prm. the nucleocapsid (nc) lies inside the viral envelope and consists of multiple copies of capsid (c) protein and the viral genome. the tbfv genomes are single stranded, positive-sense rna of approximately , nucleotides. it has a -cap with a single open reading frame (orf). the orf is flanked by and untranslated regions (utrs). the viral protein is encoded by the orf as a single polyprotein, which is co-and post-translationally cleaved by cellular and viral proteases into individual viral proteins (three structural and seven non-structural). the polyprotein is arranged in the order -c-prm-e-ns -ns a-ns b-ns -ns a-ns b-ns - [ , ] . the first step of virus replication starts when the virus binds to its receptor and is taken up into the cell by receptor-mediated endocytosis. the attachment is mediated by viral e protein and entry receptor on the host cell. the entry receptor for tbfvs has not been identified, but attachment to heparan sulphate and glycosaminoglycan, which are present in abundance on many cell types of both vertebrates and ticks, is predicted to play a role during binding and entry [ ] . once the virus has entered the cell, it is transported to endosomes, where the acidic environment of these vesicles leads to reorganization and conformational change of the e protein, resulting in the fusion of the viral and endosomal membrane and the release of the viral capsid into the cytoplasm [ ] [ ] [ ] . the viral rna (vrna) also functions as mrna, associating with ribosomes to produce the polyprotein. the transmembrane domain of the viral protein is recognized as a signal peptide and recruits the vrna/ribosomes/nascent polypeptide complex to the er membrane, where it is co-translationally translocated into the er membrane [ , ] . the nascent polypeptide is then processed by the cellular and viral proteases into structural and non-structural (ns) viral proteins. some of the viral proteins such as ns b, ns a, and ns b integrate and alter the membrane of the er to form a membrane vesicular structure, with a small pore connecting the interior of the vesicle to the cytoplasm [ ] [ ] [ ] [ ] [ ] . these vesicles are the site for the formation of replication complexes (rc) and rna replication [ , , ] . following rna replication, genomic rna is proposed to exit the vesicle through the vesicular pore and is packaged by c proteins into the nc on the cytoplasmic side of the er membrane [ , , ] . during the process of budding of nc from the cytoplasm into the er, it acquires the lipid envelope along with e and prm proteins, which are associated with the er membrane. the mechanism that ensures efficient incorporation of nc into the er membrane with the e and prm protein is poorly understood. following budding into the er lumen, the immature virions are transported through the cellular secretory pathways in a copi and copii dependent manner [ ] to the extracellular medium. the immature virion has heterodimers of e and prm that completely cover the lipid bilayer to form a spiky proteinaceous coat. during the transport through the golgi, the e protein is glycosylated [ ] . the acidic environment of the golgi induces conformational changes in the e and prm proteins, which exposes a cleavage site on prm. cleavage of prm by the cellular protease furin results in the formation of a mature virion [ , ] . mature virions are then released by exocytosis, which completes the viral life cycle [ ] [ ] [ ] (figure ). in arthropods, such as mosquitos and ticks, rna interferences (rnai) are the most important antiviral defense and tick cells have been shown to mount rnai responses against lgtv and tbev [ ] . proteins involved in the rnai mediated response such as argonaute (ago ), ago and dicer (dcr) were subsequently identified as inhibitors of lgtv [ ] . furthermore rna-seq and mass spectrometric analysis revealed that when challenged by tbev infection, tick cells upregulated genes involved in immunity and metabolism, whereas genes involved in cellular stress were downregulated [ ] . using gene silencing approaches, this study confirmed the antiviral effect of ago and dcr in tick cells. furthermore, novel antiviral genes such as complement factor h, heat shock protein (hsp) and and trypsin was found to inhibit lgtv in tick cells [ ] . taken together, studies so far have identified activation and antiviral actions of the rnai mediated response in tick cells as well as other inhibitory proteins such as hsp , hsp , and trypsin. the innate immune response provides the first line of defense against viral infections. this defense includes physical and chemical barriers such as the skin and mucous membranes and the acidity of the stomach. they serve as an initial barrier that protects the host from infection. once these barriers have been breached, the innate immune response relies on a set of germline-encoded receptors, known as pathogen recognition receptors (prrs), which recognize pathogen specific molecular patterns that are sensed as a danger signal by the host cell [ , ] . engagement of prrs results in the activation of several defense mechanisms including, production of interferon (ifn), induction of phagocytosis, cytokines, chemokines, antimicrobial peptides, antiviral proteins, as well as activation of leukocytes and t-cells. furthermore, the innate immune response harbors cellular components, such as dendritic cells, macrophages, and natural killer (nk) cells [ ] . in isaacs and lindenmann discovered ifn as a secreted factor that interfered with viral replication [ ] . in the early pioneer work on ifn in the fifties and sixties, tbev served as a model system, and tbev was shown to induce ifn after infection and was also sensitive to pretreatment of ifns [ , [ ] [ ] [ ] [ ] . pretreatment of ifn was also found to inhibit kfdv, ohfv, and powv titers in a cells (adenocarcinomic human alveolar basal epithelial cells) [ ] , thus ifn pretreatment induced broad spectrum inhibition of tbfvs. ifn has also been found to be strongly induced in the brains of powv infected peromyscus lecuopus, which are known as a natural host of the virus [ ] . similarly, ifn was found to be induced in sheep infected with liv [ ] , and ifn-stimulated genes were induced in brains of kfdv infected mice, indicating an activated ifn response [ ] . three distinct classes of ifn, type i (ifn α and β), type ii (ifnγ), and type iii ifn (ifnλ) have been described. the expression of the ifnλ-receptors and ifnγ are restricted, but the type i ifns can be expressed by most cell types and their receptor, the interferon-α/β receptor (ifnar) is expressed on all nucleated cells [ ] . there are different type i ifns in humans; these cytokines are produced by cells upon recognition of pathogen-associated molecular patterns, which are foreign to the host cell [ , ] , and mediates antiviral activity by autocrine and paracrine signaling through ifnar [ , ] . signaling through ifnar induces an antiviral state by the expression of hundreds of interferon stimulated genes (isgs) [ , ] . the host cell detects invading pathogens through prrs, which recognize foreign molecular patterns that are generated during infection. the particular prr involved in the recognition of the pathogen depends on the infecting virus [ ] . in flavivirus infection, the most important prrs are toll-like receptor (tlr) , tlr , tlr , retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein- (mda- ). tlr recognizes double stranded (ds) rna, which is formed as an intermediate during flavivirus replication [ , ] . tlr and , which are located in the endosomes, recognize single stranded (ss) rnas, such as the genomic rna of flaviviruses [ , [ ] [ ] [ ] . rig-i and mda- recognize viral rna in the cytoplasm of infected cells; rig-i recognizes short dsrna and triphosphorylated and diphosphorylated ssrna, whereas mda- recognizes long dsrna [ ] [ ] [ ] . upon recognition, the different prr will recruit distinct adaptor molecules. tlr recruits tir-domain-containing adapter-inducing interferon-β (trif) [ ] , tlr and recruit myeloid differentiation primary response (myd ) [ ] , whereas the rig-i-like helicases, rig-i and mda- , recruit interferon-beta promoter stimulator- (ips- ) (also known as mitochondrial antiviral-signaling protein (mavs), virus-induced signaling adapter (visa) and card adapter-inducing ifn-beta (cardif)) [ ] [ ] [ ] [ ] . ligation of prrs and downstream recruitment of the adapter molecules result in the activation of transcription factors nf-κb and interferon regulatory factor (irf) and irf , which translocate to the nucleus and induces the expression and subsequent secretion of type i ifn [ ] . although the innate antiviral response has been well-studied in mosquito-borne flavivirus infection, the responses to tbfvs are less investigated, and most studies have used tbev which therefore will be the main focus of the remainder of this review. in tbev infection, ifnβ induction has been shown to correlate with amount of intracellular viral rna, and the ips- pathway has been shown to protect mice from lethal lgtv infection and prolong survival after tbev infection [ , ] . absence of ips- resulted in a lower systemic ifnα response, which correlated with higher viral replication in the peripheral tissues [ ] . furthermore, ips- was shown to be of particular importance for the ifn production locally within the brain, where ips- −/− mice had lower induction of ifnβ within the olfactory bulb despite higher viral burdens [ ] . furthermore, ifnβ induction was shown to be completely dependent on ips- and irf in mouse embryonic fibroblasts and viral recognition through the ips- -pathway was shown to be dependent on rig-i and not mda- in human osteosarcoma cells (u os) [ , ] . interestingly, rig-i has been shown to co-localize with stress granules (sg) during tbev infection [ ] . sg contains ribonucleoprotein aggregates with translationally stalled mrnas, s ribosomes, and several rna-binding proteins. sg function to prevent the generation of defective proteins [ ] . sg are induced during tbev infection and sg components tia- /tiar was found to bind viral rna and inhibit viral translation [ ] . little is known about the role of the tlr / -myd pathway in tick-borne flavivirus infection; however, tlr was shown to suppress lgtv replication within neurons of the brain although it did not affect pathogenesis [ ] . what we are aware of; no animal experiments have shown any significance of the tlr -trif pathway in tick-borne flavivirus infection. however, several studies have looked at the prevalence of polymorphisms in the tlr gene in tbe patients [ ] [ ] [ ] [ ] . in particular, one polymorphism has been investigated the t allele in rs . this polymorphism has been shown to reduce tlr signaling by about % [ ] . although the data regarding tlr is somewhat conflicting, grygorczuk et al. hypothesized that a functional tlr facilitates the onset of neurological disease [ ] by supporting the penetration through the blood brain barrier, but has a protective effect during the established cns infection [ ] . the differences between studies might also be connected to the different subtypes of the tbev strain and the genetic background of the studied populations [ ] . taken together, studies so far have demonstrated the importance of the rig-i-like-ips- pathway in tick-borne flavivirus infection, whereas the role of the tlr pathways remains unclear. after secretion, type i ifn signals in an autocrine and paracrine manner by binding to the heterodimeric ifnar, which consists of two subunits, ifnar and ifnar [ ] . these subunits are associated with janus activated kinases (jak) localized in the cytoplasm; ifnar interacts with tyrosine kinase- (tyk ), whereas ifnar interacts with jak . ligation of ifnar results in activation of tyk and jak , which subsequently phosphorylate signal transducers and activators of transcription (stat)- and stat- . phosphorylated stat- and stat- then form a heterodimer, which associates with irf to form ifn-stimulated gene factor- (isgf ) that binds to the ifn-stimulated response element (isre) in the promoter of many isgs to enhance the transcription of several hundreds of ifn-stimulated genes [ , , ] . together, these isgs act to coordinate an antiviral response that is able to inhibit almost any step in the viral life cycle [ ] . the ifn response in vivo in mice is very important to protect mice from lethal infection with lgtv. mice lacking ifnar all succumbed within days of infection, whereas % of wt mice ( - -week-old) survived the infection. interestingly, all ifnβ −/− mice survived the infection, demonstrating that the ifnαs can compensate for loss of ifnβ in lgtv infection. furthermore, ifnar was shown to be a critical determinant of lgtv tropism as lgtv rna was found in all organs in the absence of ifnar, whereas in wt, only low viral burdens can be detected in the olfactory bulb [ , , ] . using transgenic mice, it was further shown that the ifn response was needed both in the peripheral tissue, as well as locally in the cns, in order to clear lgtv infection [ , ] . within the cns, lgtv mainly infected neurons [ , ] , which is also the case in lethal tbev infection of humans [ ] . although neurons remain the main target cell of tbev, astrogliosis have been shown in post mortem human brains [ , ] . in vitro studies on primary cortical astrocytes show a fast up regulation and secretion of ifn after tbev infection, which is able to protect neighboring astrocytes and neurons from infection already and h post infection, respectively [ ] . astrocytes have been shown to be resistant to tbev-induced cytopathic effects [ , ] , and it was later shown that ifnar expression protected the astrocytes from the virus-induced cytopathic effects [ ] . pretreatment of cells with ifns strongly inhibits growth of most tbfvs in cell culture [ , , [ ] [ ] [ ] [ ] and this is due to the upregulation and concerted actions of several hundreds of isgs. only a few isgs have been identified to play a role in tbev and lgtv infection [ , , , , ] . one of them, the - -oligoadenylate synthetase ( - -oas) (oas) is activated by double-stranded rna, leading to the oas protein polymerization into - -linked oligoadenylates ( - as) [ , ] . these - as activate rnase l, resulting in the degradation of viral rna [ ] . several polymorphisms in the oas genes have been shown to correlate with severe forms of tbe in patients [ ] . also, the murine isoform oas b, which is often lacking in inbred mice strains, confers strain dependent resistance against neurovirulence from far eastern tbev [ ] . two other isgs which have been shown to be antivirally active against tbev are virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) and tripartite motif- α (trim α). the trim proteins are a family of proteins able to mediate antiviral activity against many different viruses [ ] . the rodent specific trim α was identified to interact with lgtv ns in a yeast two-hybrid screen. trim α expression inhibited viral infection of lgtv and tbev by mediating lysosomal dependent degradation of ns . interestingly this mechanism was quite specific to tick-borne flaviviruses as the mosquito borne wnv was not inhibited by trim α nor did trim α interact with ns of wnv [ ] . viperin is highly conserved in evolution and was first identified as an ifn-inducible protein with antiviral activity against human cytomegalovirus in [ ] . over the last years, viperin has gained lot of attention and was shown to exhibit broad antiviral activity [ ] . viperin is also known as one of the most highly upregulated genes after viral infection [ , ] . within the family of flaviviridae, viperin has been shown to inhibit several members, such as wnv [ ] , denv [ ] , zikv [ ] , hepatitis c virus [ ] , and tbev [ , , , ] . viperin is an iron sulphur protein with three domains; a n-terminus amphipathic alpha-helix which mediates the intracellular localization to the er, a s-adenosylmethionine (sam) radical domain [ , , ] homologous to a family of proteins that use sam as a cofactor [ ] , and a highly conserved c-terminal domain, important for iron sulphur (fe/s) maturation [ , ] . although viperin is able to inhibit several different viruses, the mechanism of action, the important motif in viperin and the step of viral life cycle inhibited differs for different viruses [ ] . viperin interacts with many viral and host factors for its antiviral function. tbev replication is strongly inhibited by viperin, our research shows that viperin has no effect on the binding or entry of tbev. however, viperin targets genome replication, packaging, and release of tbev (figure ) [ , , ] . viperin specifically targets the plus-sense rna synthesis with no significant effect on the negative-sense rna of tbev during genomic replication [ ] . viperin's fe/s maturation is dependent on ciao [ , ] . the fe/s cluster and a functional sam domain of viperin is essential to inhibit the synthesis of the plus-sense rna of tbev [ ] . however, the target for the radical sam activity important for the antiviral activity of tbev is currently unknown. the structure of viperin was recently characterized [ ] , and based on similarities to other radical sam enzymes, several different hypothesis have been put forward regarding the substrate of viperin and its antiviral activity [ ] [ ] [ ] . however, none of these have shown importance in the context of mammalian viral infection. although the exact mode of action against tbev is not well understood, recent data indicates that viperin interacts with several viral proteins; both structural prm and e and non-structural ns a, ns b, and ns . these interactions lead to a viperin-ns dependent degradation of viral proteins. the degradation of tbev ns was shown to be proteasome dependent [ ] (figure ) . interestingly, ifn treatment induces an increase of tbev capsid particles and this effect was found to be dependent on viperin. viperin mediated this effect by interacting, via its n-terminus, with the cellular protein golgi brefeldin a resistant guanine nucleotide exchange factor (gbf ) [ ] . gbf is a key protein in the cellular secretory pathway and essential in the life cycle of many rna viruses, which utilize vesicular trafficking in their replication cycle and assembly process [ ] [ ] [ ] [ ] (figure ). viperin targets the ns protein for proteasomal degradation which inhibits the synthesis of + strand rna. furthermore, ns interacts with e, ns a, and ns b and these proteins are degraded by viperin in a ns -dependent manner. viperin also interferes with particle assembly by inducing secretion of c particles in a copii-dependent manner, independent of copi. viperin mediates this effect by interacting and sequestering gbf . red arrow = secretory pathway, blue arrow = "?" vesicular transport via unknown pathway. although viperin has been demonstrated to be antiviral active against many different viruses in vitro, few studies have investigated viperin's role in vivo. in tbfv infection, viperin was shown to control lgtv dissemination and replication in the brain after intraperitoneal administration, and viperin promoted survival after intracranial infection [ ] . interestingly, viperin has been shown to be expressed in the brain at the basal state [ ] , with high basal expression in primary astrocytes [ , ] . viperin's role within the brain during neurotropic lgtv infection was further mapped to certain brain regions, as viperin inhibited viral replication in the olfactory bulb and cerebrum, but not in the cerebellum or brainstem. this correlates very well with tbev infection since viperin strongly inhibited tbev replication in primary neurons and astrocytes from the cerebrum, but not in granular cell neurons isolated from the cerebellum. interestingly, cortical neurons were completely dependent on viperin for ifn-mediated inhibition of tbev, whereas in astrocytes, in which the ifn-mediated antiviral activities are strongly dependent on viperin, other isgs could partly compensate for loss of viperin [ ] . taken together, viperin has been shown to be an isg that strongly inhibits tick-borne flaviviruses in vivo and in vitro. this strong antiviral effect on tbev is mediated by targeting the virus at multiple steps of the life cycle. interestingly, viperin targeting the synthesis of plus-sense rna is dependent on the sam domain or the c-terminal domain, while the n-terminal domain of viperin is responsible for interfering with virus assembly and release. in order to establish an infection, a pathogen needs to overcome or evade the innate immune response. to breach the very first line of defense, the skin-tbfvs use the tick to deliver the virus through the skin via tick saliva. furthermore, tick saliva contains immunomodulatory compounds that enhance viral transmission and dissemination [ , ] . although there are several mechanisms in which mosquito-borne flaviviruses actively suppress the induction of type i ifn, no such mechanism has been identified in tick-borne flaviviruses so far [ ] . instead, tbev, like other flaviviruses, utilizes a passive evasion mechanism in which the virus hides its dsrna intermediates in vesicular structures inside the er membranes, and thus delaying the recognition by the cytosolic rig-i like receptors and subsequent irf phosphorylation and ifn induction ( figure a ) [ , , ] . the most conserved inhibition of the type i ifn system within the flaviviridae family is the antagonism of ifnar signaling carried out by ns ; this mechanism is conserved between several mosquito and tick-borne flaviviruses [ , [ ] [ ] [ ] [ ] [ ] . in lgtv infection, ns was shown to inhibit the jak-stat pathway and ns was shown to interact with the ifnar receptor [ ] . similarly, it was shown that ns of tbev interacts with scribble (hscrib) which mediates ns localization to the plasma membrane and this interaction enables ns to inhibit type i and type ii ifn mediated jak-stat signaling [ ] . knockdown of hscrib altered ns cellular localization and reversed the inhibition of the jak-stat signaling [ ] . further studies revealed that ns of tbev inhibited the cell surface expression of ifnar by binding to prolidase (pepd) [ ] . pepd is a peptidase that is needed for ifnar maturation and subsequent cell surface expression. ns binding of pepd prevented maturation of complex n-linked oligosaccharides on ifnar , which in turn disrupted its surface expression ( figure b ) [ , ] . in kfdv infection, ifn treatment failed to reduce viral titers when added after infection, this effect was also found to be mediated by the ns proteins antagonism [ , ] . during tbfv infection, a subgenomic noncoding rna is formed, called subgenomic flavivirus rna (sfrna) [ , , ] . it is produced as a product of incomplete degradation of genomic viral rna by cellular - exoribonuclease xrn [ ] . the ability to produce sfrna in wnv was shown to be needed for efficient viral growth in vitro and for pathogenicity in mice [ ] . interestingly, denv sfrna was found to bind to trim to inhibit rig-i-induced type i interferon expression in huh- cells [ ] . furthermore, denv and wnv sfrna was found to suppress the rnai response in both mammalian and insect cells [ ] . similarly, in tbev infection, sfrna has been demonstrated to inhibit the antiviral rnai response in tick cells [ ] . pepd is needed for maturation and subsequent transport of ifnar to the plasma membrane. ifnar and ifnar heterodimer on plasma membrane can be activated by ifnα/β which leads to the signaling cascade and phosphorylation and translocation of stat / -irf into the nucleus and upregulation of isgs. right panel: ns interferes with ifn signaling. ns protein interacts with pepd thus preventing ifnar plasma membrane localization (red t). ns also prevents stat phosphorylation (red t). arrows indicate protein transport. even though recent studies have shed light on the role of innate immunity during tick-borne flavivirus infection, much remains unknown. for example, the role of the tlr-trif and tlr-myd pathway in pathogenesis and viral recognition. furthermore, most studies were performed using lgtv and tbev, and although they are closely related to powv, liv, kfdv, and ohfv, their interactions with the innate immune response might differ. although ifn has been shown to strongly control viral tropism and pathogenesis of tick-borne flaviviruses, few antiviral isgs have been identified. viperin has been shown to be the most important isg in cortical neurons, however, other isgs that target tbev expressed in astrocytes and granular cell neurons are yet to be identified and very little is known about the early response against the hemorrhagic tbfvs. we also know very little about how the innate immune response regulates the neuroinvasion of neurotropic tbfv, and the specific interactions between the tick vector and the different viruses. emergence and spreading potential of zika virus dengue 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effectors of the type i interferon antiviral response interferon-stimulated genes: roles in viral pathogenesis cell-type-and region-specific restriction of neurotropic flavivirus infection by viperin visualization of central european tick-borne encephalitis infection in fatal human cases contribution to the histology of tick-borne encephalitis infection and injury of human astrocytes by tick-borne encephalitis virus tick-borne encephalitis virus infects rat astrocytes but does not affect their viability analysis of tick-borne encephalitis virus-induced host responses in human cells of neuronal origin and interferon-mediated protection trim alpha, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral rna polymerase inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns as an interferon antagonist viperin is an iron-sulfur protein that inhibits genome synthesis of tick-borne encephalitis virus via radical sam domain activity viperin restricts zika virus and tick-borne encephalitis virus replication by targeting ns for proteasomal degradation the human , -oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties the nature of the catalytic domain of - -oligoadenylate synthetases antiviral actions of interferons variability in the - -oligoadenylate synthetase gene cluster is associated with human predisposition to tick-borne encephalitis virus-induced disease susceptibility to flavivirus-specific antiviral response of oas b affects the neurovirulence of the far-eastern subtype of tick-borne encephalitis virus trim family proteins: retroviral restriction and antiviral defence viperin (cig ), an ifn-inducible antiviral protein directly induced by human cytomegalovirus the role of viperin in the innate antiviral response differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded rna viruses the interferon-inducible gene viperin restricts west nile virus pathogenesis viperin is induced following dengue virus type- (denv- ) infection and has anti-viral actions requiring the c-terminal end of viperin viperin is an important host restriction factor in control of zika virus infection the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein a viperin: a multifunctional, interferon-inducible protein that regulates virus replication the interferon inducible gene: viperin structural characterization reveals that viperin is a radical s-adenosyl-l-methionine (sam) enzyme cellular requirements for iron-sulfur cluster insertion into the antiviral radical sam protein viperin structural studies of viperin, an antiviral radical sam enzyme a unifying view of the broad-spectrum antiviral activity of rsad (viperin) based on its radical-sam chemistry a b -dependent radical sam enzyme involved in oxetanocin a biosynthesis quantitative proteomic analysis of host-virus interactions reveals a role for golgi brefeldin a resistance factor (gbf ) in dengue infection identification of class ii adp-ribosylation factors as cellular factors required for hepatitis c virus replication a kinome-wide small interfering rna screen identifies proviral and antiviral host factors in severe acute respiratory syndrome coronavirus replication, including double-stranded rna-activated protein kinase and early secretory pathway proteins role of the gtpase rab b in ebolavirus particle formation enhancement of tick-borne encephalitis virus transmission by tick salivary gland extracts tick salivary compounds: their role in modulation of host defences and pathogen transmission innate immune evasion mediated by flaviviridae non-structural proteins tick-borne encephalitis virus ns associates with membrane protein scribble and impairs interferon-stimulated jak-stat signalling the many faces of the flavivirus ns protein in antagonism of type i interferon signaling the generation of a reverse genetics system for kyasanur forest disease virus and the ability to antagonize the induction of the antiviral state in vitro identification of residues critical for the interferon antagonist function of langat virus ns reveals a role for the rna-dependent rna polymerase domain flavivirus antagonism of type i interferon signaling reveals prolidase as a regulator of ifnar surface expression limited effects of type i interferons on kyasanur forest disease virus in cell culture a highly structured, nuclease-resistant, noncoding rna produced by flaviviruses is required for pathogenicity noncoding subgenomic flavivirus rna: multiple functions in west nile virus pathogenesis and modulation of host responses dengue subgenomic rna binds trim to inhibit interferon expression for epidemiological fitness noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells we would like to acknowledge yong-dae gwon for constructing figure , and harry tracy for critically reading the final version of the manuscript. the authors declare no conflict of interest. key: cord- -so xl authors: ebert, gregor; paradkar, prasad n.; londrigan, sarah l. title: virology downunder, a meeting commentary from the lorne infection and immunity conference, australia date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: so xl the aim of this article is to summarise the virology content presented at the th lorne infection and immunity conference, australia, in february . the broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting. the lorne infection and immunity conference is one of five scientific meetings held during each month of february in seaside town of lorne, on the great ocean road in victoria (australia). the specific aim of the meeting is to bring together basic, clinical and translational researchers -those who examine microbes and their impact on the innate or adaptive immune response, researchers who study the mechanisms that regulate immune responses, and those who apply this knowledge to preventing and treating infectious and inflammatory diseases. was the th lorne infection and immunity conference, convened by heidi drummer (burnet institute, melbourne, australia) and paul hertzog (hudson institute of medical research, melbourne, australia). the broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting. the 'infection and inflammation' session of the meeting was opened with a well-received presentation by linfa wang (duke-nus medical school, singapore) entitled 'holy immune balance, batman', about the highly adapted immune response of bats to viral infections. bats have been characterized as an important reservoir of various zoonotic viruses including nipah, sars, mers, marburg and ebola viruses [ ] , but remarkably are able to live asymptomatically with otherwise potentially lethal viruses [ ] . wang and colleagues are interested in understanding the underlying immune mediated regulatory mechanisms that facilitate a highly effective balance between viral defense and tolerance in bats as viral hosts. during their evolutionary adaption to effective flight, bats not only developed elevated levels of basal alertness reflected in increased metabolic heart rate and body temperature, they also seem to have evolved a highly adapted immune defense against viral infections [ ] . remarkably, bats have increased tolerance to viral infections by exhibiting higher basal levels of innate defence regulators but also a dampened innate immune response upon infection, substitutional to increased responsiveness to viral pathogens [ ] . the bat innate immune response appears to be 'pre-activated' with higher basal levels of type i interferon expression, in contrast to humans, who are very quick responders to viral infections, but require a lot more dampening of their immune signals afterwards to get back to basal levels. current research also shows the absence of any aim mediated inflammasome activation [ ] , dampened nlrp mediated inflammasome activation [ ] and dampened sting activation [ ] in bats upon infection, which are all mediators of a robust type i interferon response. overall, wang et al. demonstrated that bats' response to stress in form of viral infections is more targeted and thus potentially more effective by numerous adaptions and modifications of the innate immune system. in 'viruses and their hosts' session, vinod sundaramoorthy (csiro-australian animal health laboratories) discussed a novel defence mechanism in neurons against rabies virus (rabv). rabv is a neurotropic virus, which causes tens of thousands of deaths every year, despite available vaccines [ ] . endemic dog rabies results in an ongoing risk to humans in many resource-limited countries, whereas rabies in wildlife is important in north america and europe [ ] . requirement for an uninterrupted vaccine cold chain and the high cost of the immunoglobulin component of rabies prophylaxis therapies substantiate the unmet need for novel rabv-specific antivirals [ ] . furthermore, the pathogenesis of rabv relating to viral replication in neurons is not fully understood [ ] . although most of the infection with rabv manifests as the 'furious' form, where virus silently spreads through the neuronal axon without any damage, in % of cases it can cause axonal damage in peripheral neurons leading to paralysis [ ] . using a new in vitro microfluidics model for studying synaptically connected neurons, sudaramoorthy investigated the pathogenesis of different strains of rabies virus, showing distinct mechanisms at play in determining disease outcomes for each strain. future efforts to define a key molecular determinant in the viral replication pathway in neurons may provide a novel therapeutic target. in , an association between zika virus (zikv) infection during pregnancy as a cause of microcephaly and other congenital abnormalities in the developing fetus and newborn, was made [ ] . as such, there has been a plethora of recent zikv research focused on understanding the pathogenesis of disease, as well as immunity to the virus and the development of vaccines and effective therapeutics. novel findings pertaining to all of these areas were presented throughout the meeting. developments in understanding the structure of zikv particles was showcased by shee-mei lok (duke-nus, singapore). lok and colleagues have previously solved a thermally stable . Å resolution cryo-electron microscopy structure of zikv [ ] , and were able to now show high resolution structures of zikv at various stages of viral assembly. specifically, the organisation of the envelope protein of the virus and changes during assembly and maturation were presented. previously, lok's lab has also published structures of mature and immature dengue virus, providing insight into the viral maturation process [ ] . the research from lok and colleagues will have future implications in designing novel therapeutics and vaccines for zikv. efforts to develop a zikv vaccine using virus like particles were presented by julio carrera, working with researchers at the monash university and the university of melbourne. in the 'pathogenesis and prevention of infection' session, rosa coldbeck-shackley working with michael beard at the university of adelaide, australia, and also colleagues at the hudson institute, presented findings on the importance of interferon-epsilon (ifn-ɛ) in the innate immune response to zikv infection. ifn-ɛ is a novel type i ifn, encoded within the type i ifn locus in mice and humans, whose function has recently been characterized [ ] . like other type i ifns, it acts via ifn-α receptors and , activating interferon stimulated genes (isgs). however, ifn-ɛ is preferentially expressed by epithelial cells of the female reproductive tract in both mice and humans, and in contrast to viral induced type i ifn expression, ifn-ɛ is hormonally regulated. other than mosquito-borne transmission, zikv is also sexually transmitted [ ] . this makes the research presented by coldbeck-shackley significant, and concurs with previous studies where ifn-ɛ-deficient mice were more susceptible to infection with sexually transmitted pathogens [ ] . thus, ifn-ε appears to be a potent antipathogen and immunoregulatory cytokine that may be important in combating sexually transmitted infections that represent a major global health and socioeconomic burden. also in the 'pathogenesis and prevention of infection' session, allison abendroth (university of sydney) presented 'disarming the killer: targeting of natural killer cells by varicella zoster virus'. varicella zoster virus (vzv), is known to infect numerous immune cell types such as t-cells and dendritic cells [ ] . moreover, the virus is able to modulate and manipulate innate and adaptive immune responses to infection to its replicative benefit. vzv infection of dendritic cells dampens type i ifn responses [ ] and leads to evasion of cd t cell recognition via downregulation of mhc class i expression [ ] . in addition, vzv mediated delay in immune responses to infection facilitates establishment of initial primary infection and lifelong latency in neurons. most recently, abendroth and colleagues found that vzv also productively infects natural killer (nk) cells and that nk cells effectively transmit infection to other permissive cell types [ ] . the group is now trying to understand, how nk cells, a cell type that normally demonstrates very effective direct and indirect antiviral capacity, is manipulated by vzv infection, leading to impaired cytotoxicity and cytokine responses upon infection and facilitating infection and spread of the virus. led by the observation that patients with impaired nk cell functionality are highly susceptible to severe and life threatening vzv infection, abendroth et al. found limited nk cell activation and efficacy upon vzv infection of target cells and characterised differential modulation of ligands normally recognised by activated nk cells [ ] . the exact underlying mechanisms, how vzv infection prevents the release of proinflammatory cytokines during infection of nk cells to impair their general function and whether correlations can be drawn to vzv infection of monocytes and macrophages [ ] , is currently under their investigation, abendroth stated. a key highlight at the conclusion of the meeting was a presentation from the victorian infection and immunity network young investigator prize winner, simone park (the university of melbourne). park, working alongside thomas gebhardt and laura mackay, has recently published seminal research investigating skin tissue-resident memory t cells (trm cells). specifically, park has demonstrated how these cells contribute to antiviral immune memory in peripheral tissues, using a herpes simplex model of infection [ ] . using an epicutaneous melanoma model, park and colleagues also demonstrate that trm cells play protective role in tumor surveillance [ ] , which has important implications for advancing anticancer immunotherapies. the organisers are looking forward to celebrating the th lorne infection and immunity meeting in and invite all researchers with an interest in infectious and inflammatory diseases and associated immune responses to participate. for more information: http://www.lor neinfectionimmunity.org/. viruses in bats and potential spillover to animals and humans bats and viruses: friend or foe? comparative analysis of bat genomes provides insight into the evolution of flight and immunity the egyptian rousette genome reveals unexpected features of bat antiviral immunity unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing dampened nlrp -mediated inflammation in bats and implications for a special viral reservoir host dampened sting-dependent interferon activation in bats rabies molecular virology, diagnosis, prevention and treatment status of antiviral therapeutics against rabies virus and related emerging lyssaviruses immunological aspects of rabies: a literature review human rabies: neuropathogenesis, diagnosis, and management increase in reported prevalence of microcephaly in infants born to women living in areas with confirmed zika virus transmission during the first trimester of pregnancy -brazil structure of the thermally stable zika virus immature and mature dengue serotype virus structures provide insight into the maturation process interferon-epsilon protects the female reproductive tract from viral and bacterial infection leparc-goffart i. evidence of sexual transmission of zika virus varicella-zoster virus infection of human dendritic cells and transmission to t cells: implications for virus dissemination in the host impact of varicella-zoster virus on dendritic cell subsets in human skin during natural infection varicella-zoster virus productively infects mature dendritic cells and alters their immune function varicella zoster virus productively infects human natural killer cells and manipulates phenotype varicella -zoster virus and herpes simplex virus differentially modulate nkg d ligand expression during productive infection infection and functional modulation of human monocytes and macrophages by varicella-zoster virus local proliferation maintains a stable pool of tissue-resident memory t cells after antiviral recall responses tissue-resident memory cd (+) t cells promote melanoma-immune equilibrium in skin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions ge, pp and sl all contributed equally in the writing of this manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -rodqdtfj authors: montaner-tarbes, sergio; del portillo, hernando a.; montoya, maría; fraile, lorenzo title: key gaps in the knowledge of the porcine respiratory reproductive syndrome virus (prrsv) date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: rodqdtfj the porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine diseases in the world. it is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from to % in the most extreme cases of emergent highly pathogenic strains. prrsv displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. in this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). as nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. thus, representing a novel vaccination approach against this devastating disease. the porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine diseases in the world. it is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from to % in the most extreme cases of emergent highly pathogenic strains. prrsv displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. in this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). as nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. thus, representing a novel vaccination approach against this devastating disease. keywords: porcine reproductive and respiratory syndrome virus, prrsv, virus biology, immunology, vaccinology, extracellular vesicles economic impact prrsv is responsible for respiratory disease in weaned and growing pigs, as well as reproductive failures in sows. it is considered one of the most important swine diseases worldwide, with an economic impact estimated at $ million in losses every year to u.s. producers, representing an increase of . % in the last years ( , ) . in europe, the situation is similar and economic disease models have been carried out to determine the economic burden in the best and worst case scenario combining reproductive failure and respiratory disease, estimating annual losses from a median of e , , if the farm was slightly affected during nursing and fattening, to a median of e , if a farm of , sows is severely affected in all productive phases ( ) . nevertheless, there is scarce of information about the economic impact of this disease as a consequence of multiple factors (vaccination, treatment, respiratory symptoms, reproductive failure, and other prrsv-related diseases) making a difficult task to quantify exactly this parameter under field conditions. thus, the exact economic impact of prrsv remains a key gap in the knowledge for this disease. the porcine reproductive and respiratory syndrome virus (prrsv) was first isolated in the early s in europe and north america ( , ) . it is an enveloped single-stranded positivesense rna virus of the family arteriviridae, genus porarterivirus according to the international committee of taxonomy of viruses ( ) . presently, there are four distinct species included in this genus (porarterivirus), prrsv- and prrsv- (with - % variation in nucleotide sequences), along with other two viruses that do not affect pigs (lactate dehydrogenaseelevating virus and rat arterivirus ) ( ) . the genome size of prrsv is about kb with open reading frames (orfs), with replicase genes located at the ′ -end followed by the genes encoding structural proteins toward the ′ -end ( ) . the majority of the genome (∼ - %) encodes non-structural figure | genome structure and mature viral particle of prrsv virus. (a) non-structural proteins are located in the ′ end of the genome, codifying for two different polyproteins pp a and pp ab that are cleaved into at least nsps (nsp to nsp and nsp α and nsp β, and nsp α, and nsp β). structural proteins located near the ′ end, are associated to the viral envelope and rna packaging. (b) prrsv mature viral particle, composed of a lipid bilayer envelop with viral receptor glycoproteins involved on infection and cell internalization. single stranded positive rna is associated with nucleocapsid protein in the internal layer of the virus. proteins involved in replication (orf a and orf ab), whereas orfs - encodes structural proteins (n, m, gp -gp , e) ( figures a,b) ( ) . using orf in molecular epidemiological studies, an enormous genetic variability has been described ( ) . yet, data on whole genome sequencing is scarce and constitute another important gap in the knowledge of this virus and its evolution (box ). prrsv replicase genes consist of two orfs, orf a and orf b, which occupy the ′ proximal three-quarters of the genome ( figure a) . both are expressed from the viral genome, with expression of orf b depending on a conserved ribosomal frameshifting mechanism. subsequently, extensive proteolytic processing of the resulting pp a and pp ab polyproteins yields at least functional non-structural proteins (nsps), specifically nsp to nsp , with both the nsp and nsp parts being subject to internal cleavage (giving origin to nsp α and nsp β, and nsp α, and nsp β, respectively), most of which assemble into a membrane-associated replication and transcription complex ( ) . recently, a programmed ribosomal frameshift encoding an alternative orf that generates two extra proteins, nsp tf and nsp n, was discovered in prrsv and other arteriviruses ( , ). these nsps, described for prrsv, have proven to box | gaps in knowledge in prrsv. be necessary and sufficient for the induction of membrane modifications resembling those found in infected cells ( ) . most importantly, all positive rna viruses seem to induce one of two basic morphotypes of membrane modifications: invaginations or double-membrane vesicles. prrsv also has a set of structural proteins, including a small non-glycosylated protein and a set of glycosylated ones: gp ab, gp , gp , gp , and gp a, m and n proteins ( ) . however, nsp , traditionally classified as a non-structural protein, has been found to be incorporated in multiple isoforms within the viral envelope (ovarian tumor domain protease region, hypervariable region and c-terminal region) ( ) , giving new insights into the structure of this virus ( figure b) . first, the nucleocapsid protein (n), as one of the most important parts of the mature viral particle, has been deeply characterized on prrsv, finding important features shared in most nonsegmented rna viruses. the n protein consists of amino acids for genotype and amino acids for genotype . the viral envelope glycoproteins (gp to gp ) are the first interactors with host cell receptors to initiate infection and are exposed to the immune system when viral particles are in blood and lymphoid tissue circulation (figure ). there is also another protein that contribute to virion structure, m protein, that is required during viral entry to interact with heparan sulfate cell receptor on macrophages. later, gp is thought to bind to sialoadhesin and virus internalization and uncoating is triggered by a formation of a viral heterotrimer (gp a, gp , and gp ) with scavenger receptor cd (figure ) ( , ) . gp is the most abundant glycoprotein. first, it interacts with two cell entry mediators, heparan sulfate glycosaminoglycans and sialoadhesin/cd ( , ) to favor viral entry and then possibly with the n protein and its mhc-like domain to carry n-viral rna complex to the budding site (figure ) . gp , gp , and gp are protected with glycan shields, like most prrsv membrane proteins, to avoid antibody recognition and neutralization. gp has two glycosylation sites, gp have seven and gp have four, figure | interactions between viral proteins and cell receptors for virus attachment, entry, uncoating and release of genetic ssrna to cell cytoplasm. blocking cd , cd tetraspanin or vimentin seems to inhibit viral replication or infection in the host cell, but reduced replication or no effect is seen when receptors such as heparan-sulfate or siglec- are blocked, demonstrating that some viral proteins and cell receptors are indispensable in terms of production of infectious viral progeny and dissemination in the host. three of which are directly related to virus survival, causing lethal damage in virus production when more than two of these sites are mutated ( ) (figure ). viral replication starts by interaction of viral glycoproteins with different cellular receptors (figure ) ( ) . cd and cd play a main role during infection, uncoating of the viral particle, activation of clathrin-mediated endocytosis and release of viral genome in the cytoplasm ( ) . cd has been defined as the main receptor for viral infection by evaluating the effect of prrsv on cd knockout pigs, where there is complete resistance to infection ( ) . cysteine-rich domain in this receptor seems to be necessary to establish interactions with prrsv- species, since its deletion by crispr/cas system (exon of the gene encoding this region) implies protection for a large panel of these viruses demonstrated by in vitro challenge of edited-pig macrophages and in vivo experiments with srcr animals ( ) ( ) ( ) . more important, edited pigs show no side effects when kept under standard husbandry conditions and cd seems to maintain its biological function (hemoglobin-haptoglobin scavenger) regardless the lacking cysteine-rich domain, nevertheless, other unknown functions could be impaired by this modification. in conclusion, gene-edited pigs lacking srcr region of cd could be an important asset to confront prrsv epidemics with the final goal of eradication. cd seems to be related only to co-interactions with sialic acid in the virion surface, however, knockout pigs for either exon , , or of cd were not protected from infection and viral load as well as antibody responses were similar to heterozygous (cd +/− ) or wild type pigs (cd +/+ ) ( ) . the former experiments suggested that other unknown mechanisms could be involved in prrsv infection such as other receptors, new unknown susceptible cell types different from macrophages or possible leaking of cd expression in the knockout model. other molecules are also involved in viral entry, such as cd ( ) and vimentin ( ) ; blocking of any of these four molecules (cd , cd , cd , and vimentin) had an effect on viral infection, either on internalization or complete inhibition of viral replication ( ) . after cell entry, prrsv causes a series of intracellular modifications to complete its replication cycle, which includes rearrangements of intracellular membrane organelles to generate the replication complex. these include the formation of perinuclear double membrane vesicles apparently derived from endoplasmic reticulum, synthesis of genomic rna (grna), transcription of segmented rna (sgrna) and expression of viral proteins ( , ) . at late stages of replication, the mature virions accumulate in the intracellular membrane compartments and they are then released into the extracellular space through exocytosis ( ) . a non-classical spread pathway has been detected in several viruses including prrsv where virus dissemination is mediated by cell to cell nanotubules ( ) . it was reported that almost all prrsv proteins interact with myosin and actin (especially f-actin and myosin iia) where nanotubules connected cells allowing the movement of structural proteins and rna, infecting naïve cells in a non-classical way even in the presence of neutralizing antibodies in the cell media. in addition, this non-classical pathway demonstrated that prrsv cell entry receptors were not necessary to establish infection, as nonpermissive cells became infected when were contacted by infected cells via nanotubes. this spreading strategy has been proposed as a mechanism to facilitate infection either by surfing of viral particles between adjacent cell membranes or as a receptor-independent mechanism for infection ( ) ; importantly, has been reported for other viruses such as hiv- where nanotube number on macrophages increases after infection ( ) and herpesvirus transmission between bovine fibroblasts ( ) . interestingly, although several viral proteins were detected in nanotubules (nsp β, nsp , nsp tf, nsp , nsp , and nsp , gp and n), gp was detected in only a few nanotubes. in particular, the role of gp in this nonclassical spread pathway is not fully understood and it will be interesting to further evaluate gp interaction with other cellular components to elucidate the reason why gp is not transported to new recipient naïve cells. altogether these data indicate that prrsv has evolved different pathways to spread even though, in vivo, the virus shows narrow cell tropism for monocytes and macrophages ( , ) (box ). the innate immune response is the first system any given pathogen encounters, specially to prevent viral replication and invasion into mucosal tissues (respiratory tract in the case of prrsv) and, importantly, to initiate the strong adaptive immune response to fight against intracellular infectious agents ( ). type i interferons (ifn α/β) comprise one of the most potent mechanisms against invading viruses in the first stages of infection, triggering an array of ifn-stimulated genes (isg) ( ) . generally speaking, all nucleated cells have the ability to produce ifn α/β, but plasmacytoid dc (pdc) are the most potent producers of this family of cytokines ( ) . prrsv has evolved a set of mechanisms for suppressing ifn α/β in vivo, maintaining low expression levels of this cytokines on infected pigs ( ) during almost all time-course of infection shortly after transient elevation in the lungs ( ) . suppression of ifn α/β also takes place in vitro in prrsv infected marc- and porcine alveolar macrophages ( , , ) . further studies have shown that ifn type i suppression is a major strategy of prrsv to modulate host antiviral defense. in fact, several viral proteins have been identified as ifn antagonists (nsp α, nsp β, nsp , nsp , nsp , and n) ( , ( ) ( ) ( ) . as an example for n protein, upon dsrna stimulation, ifn-β production was shown to decrease proportionally with increasing levels of n expression and additionally it was found to downregulate ifn-dependent gene production by dsrna interfering with dsrna-induced phosphorylation and nuclear translocation of irf ( ) . among prrsv non-structural proteins with type i ifn modulation capacity, nsp has been considered as the strongest antagonist of ifn-β production by acting on interferon regulatory factor (irf ) phosphorylation and nuclear translocation. almost all nsps, excepting nsp , have been related to the perinuclear region, associated with intracellular membranes, supposedly derived from the endoplasmic reticulum (er), which are modified into vesicular double-membrane structures with which the viral replication and transcription complex (rtc) is thought to be associated with ( , , ) . nsp translocates to the nucleus during the first hours of infection, where it is capable of inhibiting irf association with creb-binding protein (cbp), promoting cbp degradation by a proteasome-dependent mechanism, without which the transcription enhanceosome may not assemble the transcription machinery for the interferon expression ( , ) . recently, post-transcription protein expression of ifn β was shown to be regulated by prrsv by means of upregulating cellular mirna in porcine alveolar macrophages ( ) nsp is the largest (mature) prrsv protein and contains at least four distinct domains: the n-terminal cp/otu domain, a central hypervariable region, a putative transmembrane domain, and a c-terminal region of unknown function that is rich in conserved cysteine residues. this protein is unique in the context of prrsv due to its genetic heterogeneity, its participation in diverse roles supporting the viral replication cycle, and its packaging within the prrsv virion ( , ) . previous studies suggest that nsp has different roles related to immune evasion mechanisms. it has been determined that nsp otu domain (thiol-dependent deubiquitinating domain) inhibits the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) by interfering with the polyubiquitination process of ikbα (nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor) and, subsequently, preventing the degradation of the ikbα protein ( ) . moreover, viable deletion mutants in nsp , when infecting cells, caused a downregulation of cytokines (il- β and tnf-α) mrna expression, in comparison with that of parental virus, suggesting that certain regions of nsp might contribute to the induction of a virus-specific host immune response and that deletion of such a region could produce a more virulent virus ( ) . there are several isoforms of nsp , sharing a consistent core set between viral strains, which are integrated into mature virion at the final stage of replication (figure b) , although some of them could be strain-specific. inclusion of nsp within the prrsv virion suggests that it may function in previously unknown roles related to extracellular function, entry, or immediate-early viral replication events ( ) . truncated forms of nsp have also been identified, named nsp tf and nsp n, with apparent roles in modulation of immune evasion. when deletion mutants for those forms were used to infect cells, there was a significant change in gene expression, a strong activation of those involved in cytokine-cytokine receptor interaction, tnf signaling, toll-like receptor signaling, nodlike receptor signaling, nf-κb signaling, rig-i-like receptor signaling, chemokine signaling, jak-stat signaling, cytosolic dna-sensing, and nk cell mediated cytotoxicity ( ) , suggesting that an active role (direct or indirect) is played by these truncated forms in modulating host cells innate immune response, making prrsv infectious cycle more complicated than it was initially thought. nsp , is a nidovirus conserved endoribonuclease with an uridylate-specific endonuclease (nendou). it has been demonstrated in vitro that overexpression of nsp enhanced viral titter ( ) . moreover, nsp antagonizes type i ifn, specifically ifnβ production, activated by the retinoic acid inducible gene like receptor, showing substrate specificity toward mitochondrial antiviral signaling proteins (mavs) and rig-i (transcripts and proteins), and demonstrating that this activity was associated to the endoribonuclease activity of this protein in which transfection mutant viruses were unable to degrade mavs mrna and impair ifnβ production ( ) . another mechanism whereby this protein limits antiviral response is related to inflammasome and synthesis of il- β, due to its important role in both the innate and adaptive immune response and in pathological mechanisms. it has been shown that prrsv could activate nlrp inflammasome in early stages of infection but induce host's immunosuppression later as measured by determining the levels of pro-il- β and procaspase- mrna and the mature il- β protein in porcine alveolar macrophages (pam) ( ) . it is not surprising that nsp also interacts with the rna-silencing complex (risc), as it has been demonstrated in vitro in a marc- cell line that this protein and nsp α are responsible for inhibiting risc and downregulating argonaute- protein expression increasing viral titter significantly, which demonstrates a direct relationship between this silencing complex and viral replication at least in vitro ( ) . other non-structural proteins have been studied but there is an important gap on information about in vivo and in vitro functions and interaction in signaling pathways. additionally, the enormous variation among strains makes it difficult to characterize all protein variants and interactions with cell systems (macrophages, dendritic cells "dcs, " monocytes and others) (box ) . recently, a body of evidence associates host genetics with different outcomes following prrsv infection in the respiratory and reproductive form of the disease ( ) ( ) ( ) ( ) ( ) . although pathways and mechanisms involved in specific disease-resistance traits have not yet been fully characterized, it is clear that the genetic variation in disease resilience is polygenic, regulating aspects of both innate resistance and acquired immunity ( ) . in connection with innate response, the average daily gain (adg) after prrsv infection was associated with a single genomic region in chromosome (ssc ) which is best represented by the snp tag marker wur, located in the ′ non-coding region of the interferon-inducible guanylate-binding protein (gbp ) gene ( ) . the pig genetic resistance to prrsv infection has been historically overlooked in prrsv research probably generating a confounding factor in immune response studies. a key gap in the knowledge of prrsv is linked the pig genetic variability after prrsv infection with the enormous variability of the virus itself (box ) . in pigs, prrsv replicates in cells belonging to the innate immune system. pams are the primary cells to be infected in the lungs as well as other cells of the monocyte/macrophage lineage, which later could disseminate the virus to other tissues or support replication to release viral particles into the bloodstream ( ) (figure ) . moreover, prrsv is thought to be able to infect professional antigen presenting cells such as dcs and monocyte derived dendritic cells, (modc) impairing their normal antigen presentation ability by inducing apoptosis, down-regulating the expression of ifn-α, mhc class i, mhc class ii, cd b/c and cd , upregulating the expression of il- and inducing minimal th cytokine secretion ( ) ( ) ( ) ( ) . nevertheless, new evidence suggest by in vivo and in vitro experiments that specifically lung cdc , cdc , and modcs are not infected by prrsv- viruses from subtypes and and one possible explanation is the lower expression of cd and cd in those dc subtypes, associating previous results of infection in dcs to culture conditions of monocytes in vitro that could cause a sensibilization to infection by certain strains as lena ( ) . in addition, these findings were also tested in tonsil cdc and tracheal cdc and cdc observing that those cell populations are not infected by prrsv virus ( , ) . moreover, a new type of pam has been characterized and named porcine intravascular macrophages (pim) due to its association to endothelial lung capillaries and not to the alveoli, presenting strong capacity to phagocytised bloodrelated particles ( ) . importantly, when infected pim cells gave similar results of viral load to those derived from infected pam, but significantly upregulates of tnfα and non-significantly il- and il- expression after infection when compared to normal alveolar macrophages, indicating that these cells have an important pro-inflammatory role during prrsv infection in the lungs ( ) . new interactions between cells and the virus need to be further explored to unravel possible immunological features that leads to correlates of protection. recently, it has been shown that a domain within nsp α is able to stimulate the secretion of cd , which in turn inhibits modc function in vitro, impairing the ability of modc to stimulate t cell proliferation ( ) . production of ifn α/β and the mechanisms for cell activation by pdc are severely suppressed during prrsv infection, although these cells are not permissive to prrsv infection ( , ) . however, this phenomenon is strain dependent, as other prrsv strains are able to stimulate pdc for ifn α/β production in large quantities ( ) . again, there is an enormous variability between prrsv strains in relation with their effect on antigen presenting cells which prevent scientists from finding common mechanisms. it might be of interest to link this key gap of knowledge for prrsv with host genetics (box ) . moreover, in prrsvinfected cells, n is abundantly expressed benefiting from the discontinuous transcription mechanism ( ) . this protein is also distributed in the nucleus, induced by two nuclear localization signals called cryptic nls or nls- and functional nls or nls- (positions - and - , respectively) ( ). the effect of n protein has been examined in pams and modcs using transfection, finding a significant upregulation of il- gene expression. natural killer (nk) cells constitute another powerful arm of the innate immune system against prrsv, particularly when considering the high percentage of circulating nk cells in pigs ( ) . the cytotoxic function of nk cells is reduced in prrsv infected pigs from day after infection up to - weeks ( , , ) . initial studies using in vitro systems demonstrated that the stimulation of porcine nk cells with proinflammatory cytokines (il- and il- ) was capable of activating nk cells and inducing them to express high levels of ifn-γ and perforins to cause lysis of infected cells, but a different scenario appears if cells are evaluated post-infection, indicating that a virus such as prrsv is capable of impairing nk cell cytotoxicity ( ) . in vitro, the nk cytotoxicity against prrsv-infected pams was decreased and degranulation of nk cells inhibited ( ) . in vivo, the immune response is the same as that observed in vitro, with some studies reporting that approximately half of viremic pigs had a reduction > % in nk cell-mediated cytotoxicity and enhanced secretion of il- , il- , and il- and reduced frequency of cytotoxic t-cells (cd − cd + t) and double positive t cells (cd + cd + t) and upregulated frequency regulatory t-cells (tregs) ( ). innate immune responses against prrsv are obstructed by different mechanisms as are adaptive responses. the modest and delayed b cell mediated neutralizing antibody response is one of the main characteristics associated to prrsv acquired immune responses. even though prrsv specific antibodies appear early at - days post-infection, the efficacy of those antibodies remains unclear. neutralizing antibodies take longer, appearing nearly month after infection ( ) . however, passive transfer of these neutralizing antibodies conferred almost full protection in a prrsv reproductive model ( % of offspring alive after challenging pregnant sows with high neutralizing antibody titter). nevertheless, in another experiment using the reproductive model, when the presence of prrsv was examined after the transfer of neutralizing antibodies, lungs, tonsils, buffy coat cells, and peripheral lymph nodes contained replicating prrsv similar to infected controls, although pigs were apparently protected against infection. in summary, passive transfer of high neutralizing antibody titter conferred protection to gilts and offspring (not detectable viremia), but did not eliminate the presence of viral particles in peripheral tissues nor transmission to animals they were in contact with ( ) ( ) ( ) . curiously, the role of neutralizing antibodies in the protection against the respiratory form of the disease is a key gap of knowledge for prrsv. this point is critical to define precisely targets for improved vaccines based on the humoral immune response against this virus (box ). n protein is involved in several mechanisms for immune evasion and is also one of the most immunogenic structural proteins ( ) . antibodies against n appear early during acute infection, together with those against m and gp proteins, but are non-neutralizing and could be involved in antibody dependent enhancement ( , ) . there are other "antibody-related mechanisms" that do not necessarily involve neutralizing activity. antibody-dependent cell-mediated cytotoxicity (adcc), antibody-dependent complement-mediated cytotoxicity (cdc) and antibodydependent complement mediated virolysis (adcv) have been examined in the context of prrsv, although none of these mechanisms were evident during infection or have not been deeply investigated on in vitro and in vivo models of this virus ( ) . it is important to note that neutralizing antibodies appear late in prrsv infection and other immune mechanisms (cellular or antibody mediated immune response) might be acting to suppress viral replication in blood, causing the virus to be isolated in lymphoid tissues and maintaining suboptimal replication that will finally end in viral clearance. for type prrsv- it has been demonstrated that immunization of pigs with ectodomain peptides from gp /m complex did not induce neutralizing antibodies ( ) although those ectodomain-specific antibodies generated were capable of binding virus. an important feature that makes difficult to validate the location of neutralizing epitopes is the number of glycosylations in or around it. for prrsv- strains, up to glycosylations may be found in, or flanking the gp neutralizing epitope that is located between amino acids - ( ) , whereas for prrsv- strains there are four potential glycosylation sites ( ) . when tested, prrsv with mutations in gp glycosylation sites (either at n or in the hypervariable region, upstream the neutralizing epitope) enhanced immunogenicity with increased concentration of antibodies directed to this epitope - fold higher compared with those induced by the wild type strains ( ) . same results were obtained when administering another deglycosylation mutant (double deglycosylation in the putative glycosylation moieties on gp ) twice, which conferred better protection against homologous challenge ( ) . in addition, when this protein is expressed early during infection, it stimulates production of early neutralizing antibodies and ifn-β, two main antiviral mechanisms, demonstrating its role in induction of self-protection mechanisms from the host ( ) . available data about neutralizing antibodies induced by this protein are controversial, which may be due to the high variation among prrsv strains ( ) and, as previously commented, the host genetics. orf is also complemented by a small frameshift of the subgenomic mrna called orf a, encoding a type i membrane protein consisting primarily of alpha helix with a membrane-spanning domain (called gp a) that is incorporated into virions as a very minor component, playing a role in viral replication, as mutation in the initiation codon or premature termination related to expression for this protein leads to non-efficient viral replication and lower titter ( , ) . this protein is capable of eliciting specific antibody immune response in natural infections and after immunizations, although those are not neutralizing neither protective in a challenge trial after infection, making difficult to define the role of this particular small protein in the whole immune response and viral clearance of prrsv infection ( ) . in summary, the role of humoral immunity remains elusive in prrsv infection (neutralizing and non-neutralizing antibodies) and a better characterization will be required to overcome this relevant gap of knowledge (box ) . treg typically increase in number in chronic viral diseases to prevent a persistent inflammatory response and pathological damage associated to viral infections. conversely, tregs are described as key contributors in modulating the host immune response to viral infection. this cell population is an important component in regulating the magnitude of the immune response to infection (in viruses such as hiv and hcv), thus preventing excessive inflammation and tissue damage. however, they can also be inappropriately induced by viruses to switch the balance of the immune response in favor of maintaining viral replication ( ) . in prrsv, the role of tregs remains unclear and appears to be a consequence of il- induction of some strains as early as days post infection ( ) . in some experiments, in vitro infected dcs with prrsv- exhibited an unbalanced ability to stimulate t cell immune responses in a strain-dependent manner, but no tregs were detected, at least in vitro, as measured by expression of cd and foxp markers ( ) . when using prrsv- strains, the case seems to be different, as the virus was capable of stimulating il- production with concomitant generation of tregs ( ) which was associated to nucleocapsid protein expression in the in vitro system. this group also suggested that il- production and treg could be related to impaired gamma interferon (ifn-γ) production and altered development of protective t-cell response by inhibiting t-cell proliferation as seen in the early stage of infection with viruses such as hcv. vaccine strains currently in use in the united states do not provide adequate heterologous protection, one possible explanation could lay on their inability to induce an adequate ifn-γ response due to their ability to stimulate tregs, at least in vitro ( ). structural conformation, but not nuclear localization, of the expressed n protein was suggested as essential for the ability to induce il- that, in consequence, causes induction of tregs as measured by markers cd +cd +foxp + ( ) . it should be noted that when the role of the nuclear localization signal was evaluated using deletion mutants, results suggested that nls- was not essential for virus survival, although pigs developed a significantly shorter duration of viremia and higher neutralizing antibodies than those of wild-type prrsv-infected pigs ( ) . the role of tregs cells in the immune response against prrsv is a key gap of knowledge in order to develop more efficacious prrsv vaccines (box ) . moreover, reports have highlighted the impact of prrsv infection on thymic cellularity mainly as a loss of cd + /cd + cells in the thymus of prrsv-infected pigs. acute lymphopenia, thymic atrophy, and lymphadenopathy associated with the presence of prrsv antigen in the thymus are some of the mechanisms whereby prrsv suppresses the immune response. in addition, presence of prrsv antigens in the thymus could also induce tolerance and presents a mechanism that could explain the presence of tregs during prrsv infection ( ) . nevertheless, the picture is not complete and basic knowledge about the effect of prrsv on cell development in the thymus would be of great interest to understand the effect of this viruses in the host. prrsv immunology thus remains an unsolved puzzle due to complex interactions between different viral strains and the host. similar immune responses could be the key feature of this virus, such as persistence viremia, a strong inhibition of innate cytokines (ifn-α/β, tnf-α, il- β, ifn-γ), dysregulation of nk cell function (cytotoxicity and degranulation), rapid induction of non-neutralizing antibodies, delayed appearance of neutralizing antibody, late and low cd + t-cell response, and induction of regulatory t cells (tregs) ( ) . as a whole, neutralizing antibodies and prrsv-specific ifn-γ secreting cells do not fully depict the immune effector functions related to protective immunity, as the viral targets related to them are unknown. as a consequence, correlates of protection remain elusive for this infection due to the laborious work in vitro and in vivo and the enormous genetic diversity that causes confusion and makes it difficult to predict how immune responses against one isolate or strain could be applied to another in a cross-protective immune prediction model ( , ) . without any doubt, the most important gap of knowledge for prrsv is the lack of correlates of protection that makes extremely difficult to have robust models to check vaccines efficacy against this disease (box ). since the beginning of prrsv outbreaks in europe and the usa, the development of efficacious prrsv vaccines has been a challenge. classical approaches are not working properly for several reasons: viral mutation can lead to more pathogenic strains, there is a lack of knowledge on how the porcine immune system interacts with all prrsv proteins, and most importantly, there is no robust parameter (surrogate marker) that can be unequivocally linked with viral clearance. thus, there is no relationship between complete homologous or heterologous protection and classic immunological parameters such as an increase/decrease in particular cell population ( ) , ifnγ production, neutralizing antibodies ( ), non-neutralizing antibodies and clinical outcome ( ) . in addition, highly divergent strains make it more difficult to develop a universal vaccine for this virus ( ) . several different vaccines against prrsv have reached the market and have been reviewed recently ( ) . most of these vaccines rely upon modified live virus (porcilis prrs from merck, ingelvac prrsflex eu from boehringer ingelheim, amervac-prrs from hypra, pyrsvac- from syva) against prrsv- , as well as some to control prrsv- (fostera prrs from zoetis, ingelvac prrs mlv/ingelvac prrsatp from boehringer ingelheim). there is also evidence that most mlv vaccines of both prrsv- and prrsv- species elicit specific humoral and cell-mediated immune (cmi) responses, as they confer protection to homologous parental strains and partial protection to heterologous strains. although it is possible to control some prrsv outbreaks by use of mlv in combination with good practices, there are major safety issues such as a high mutation rate leading to reversion to virulence and recombination among vaccine and wild type strains. cases have been reported in which new viruses have been introduced as a consequence of mlv vaccines. for example, nucleotide sequence identities of atypical danish isolates were between . and . % with the vaccine virus respprrs and . - . % with vr , which is the parental virus to the vaccine virus, supporting the conclusion that the introduction of prrsv- in denmark was due to the spread of vaccine virus ( ) . in china a recombination event was reported in which a prrsv variant with nucleotide deletions and insertions in the non-structural protein (nsp ) gene also showed a possible recombination event between a mlv strain and a prototype chinese field strain ( ) . current inactivated vaccine approaches are not highly effective since elicited immune responses are not enough to prevent spreading of the virus. however, this type of vaccine can augment anamnestic virus neutralizing antibodies and virusspecific ifn-γ responses following a wild-type virus infection or prrsv-mlv vaccination which can contribute to viral clearance ( , ) . thus, the combination of modified live vaccines with inactivated ones can be a reasonable approach to control the disease under field conditions ( ) but unfortunately, there is no robust data comparing this approach with other options available on the market. on the other hand, most inactivated vaccines are not approved for use in the united states due to the poor efficacy showed in challenge trials ( ) as measured by production of prrsv specific neutralizing and non-neutralizing antibodies and low cellular immune responses leading to their failure in the porcine market. according to the centre for food security and public health of iowa state university, only box | exosomes and therapeutic applications in prrsv. frontiers in veterinary science | www.frontiersin.org "biosuis prrs inact eu+am" is approved to be used in the us. however, new strategies are being evaluated to overcome these problems ( ) , including nanoparticle entrapped antigens ( ) ( ) ( ) ( ) , plant based approaches ( ) or vectored vaccines ( ) . several attempts have been made to use structural proteins to develop vaccines against prrsv because they are specific targets of neutralizing antibodies. for this reason, one may hypothesize that antibodies against those proteins could be the main key to inhibit viral replication and spread as it is common for many viruses. approaches such as vlps combining different structural proteins have been tested ( ) ( ) ( ) , finding that anamnestic response is possible (boosted igg and ifn-γ producing cells) in previously vaccinated or infected pigs but not in the prechallenge period. these structural proteins are able to prime the immune system, but no reduction of viremia was observed after challenge ( ) . those results suggest that other viral proteins may be targeted to induce a protective response in pigs. a plausible explanation for this finding may be based on the presence of few neutralizing epitopes in their sequences, most of which are located in variable regions of the proteins, to the phenomena of glycan shielding for epitopes and to the high variability observed between prrsv virus strains. again, a critical gap of knowledge for prrsv is to precisely characterize common epitopes that are present in all prrsv strains. epitopes responsible for generating an efficient immune response eliciting cross-protective immunity remained elusive. taken together, this evidence points to the need for new vaccination approaches that comply with a pathogen free strategy, capable of eliciting effective cellular and antibody responses with mid to long term protection against homologous strains and preferable to heterologous challenge as well. extracellular vesicles(evs) are gaining increased scientific attention as novel vaccines against infectious diseases, including animal diseases of veterinary importance by its capacity of self-antigen presentation, activation of host cell and antibody immune responses and more important, to induce protection in lethal challenge trials ( - ) (box ). in the case of prrsv, artificial micrornas (amirna) were initially synthetized to try suppressing expression of sialoadhesin (sn) or cd by recombinant adenoviral vectors to be contained in exosomes, causing a subexpression of sn and cd at mrna and protein level, and reducing viral titter when porcine macrophages were pre-treated with amirna thus providing new evidence supporting the hypothesis that evs can also serve as an efficient small rna transfer vehicle for pig cells ( ) . more recently, prrsv viral proteins associated to extracellular vesicles (evs) in the size range of exosomes, were reported ( ) . moreover, a targeted-pig trial using evs from sera of infected pigs who had overcome the disease, demonstrated that evs are capable of inducing specific ifn-γ secreting cells after a prime-boost strategy, are safe, free-of-virus and can differentiate infected from vaccinated animals ( ) , moreover, it was demonstrated that those evs contained antigenic viral proteins recognized by pig immune sera and not by the pre-immune one. of interest, however, a recent article indicated that prrsv derived evs are capable of transmitting the virus from one cell to another ( ) . whether these discrepancies are due to in vivo vs. in vitro experimental work and methods applied to isolate evs from serum samples or culture supernatant, remains to be determined. evs have also been explored as novel control strategies in other viral diseases. for example, in respiratory syncytial virus infection, evs are released with a selected modified cargo when compared with uninfected epithelial cells. when analyzed in detail, several viral proteins and diverse species of rna were detected and capable of activating innate immune responses through induction of cytokine and chemokine release ( ) . similar scenarios of viral proteins exported in evs have been observed and extensively reviewed for hiv/hcv/htlv- ( ), ebv ( ) , and other viral diseases. moreover, viral products of various origin and size including ebola virus vp , vp , and np, influenza virus np, crimean-congo haemorrhagic fever np, west nile virus ns , and hepatitis c virus ns , when fused with nef c-terminal domain through dna vectors, were directed to the evs membrane or packaged into them and remained stable after fusion. more importantly, when injected in mice, dna vectors expressing the diverse fusion products elicited a well detectable antigen-specific cd + t cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells, proving its promising results as a cytotoxic t lymphocyte vaccine ( ) . prrsv is a complex disease and several gaps in the knowledge of its economic impact, biology and evolution, genetic polymorphism, mechanism of viral infections, elicitation of protective immune responses and novel control strategies, have been reviewed here (box ). since the late 's, different approaches have permitted to examine more closely this virus allowing the discovery of new features of the complex replication cycle, the identification of proteins and nucleic acids playing a role together with extracellular vesicles and nanotubules in facilitating spreading, and a better understanding of immune evasion (non-neutralizing antibodies, glycan shielding, mutation, recombination events, among others) to further vaccine development. presently available prrsv vaccines have many limitations in terms of heterologous protection, but some efforts 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cells exosomes and their role in the life cycle and pathogenesis of rna viruses pathogenic role of exosomes in epstein-barr virus (ebv)-associated cancers an exosome-based vaccine platform imparts cytotoxic t lymphocyte immunity against viral antigens we are particularly grateful to christa helwig for english editorial assistance. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer sg declared a past supervisory role with one of the authors mm to the handling editor.copyright © montaner-tarbes, del portillo, montoya and fraile. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - rzzos authors: klotz, daniela; gerhauser, ingo title: interferon-stimulated genes—mediators of the innate immune response during canine distemper virus infection date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: rzzos the demyelinating canine distemper virus (cdv)-leukoencephalitis represents a translational animal model for multiple sclerosis. the present study investigated the expression of type i interferon (ifn-i) pathway members in cdv-induced cerebellar lesions to gain an insight into their role in lesion development. gene expression of manually selected genes in acute, subacute and chronic lesions was analyzed using pre-existing microarray data. interferon regulatory factor (irf) , irf , signal transducer and activator of transcription (stat) , stat , mx protein, protein kinase r (pkr), ′- ′-oligoadenylate synthetase (oas) and interferon-stimulated gene (isg) expression were also evaluated using immunohistochemistry. cellular origin of stat , stat , mx and pkr were determined using immunofluorescence. cdv infection caused an increased expression of the antiviral effector proteins mx, pkr, oas and isg , which probably contributed to a restricted viral replication, particularly in neurons and oligodendrocytes. this increase might be partly mediated by irf-dependent pathways due to the lack of changes in ifn-i levels and absence of stat in astrocytes. nevertheless, activated microglia/macrophages showed a strong expression of stat , stat and mx proteins in later stages of the disease, indicating a strong activation of the ifn-i signaling cascade, which might be involved in the aggravation of bystander demyelination. canine distemper virus (cdv) is an enveloped, single-stranded, negative-sense rna virus [ , ] , which can infect terrestrial and aquatic carnivores via the oropharyngeal route [ , ] . the clinical course of the disease depends on the cdv strain as well as the immune status and age of the infected host [ , ] . cdv-infected animals can either trigger a strong humoral and cellular immune response between the th and th day after infection and recover or, in the case of a weak immune response, die or develop a persistent infection [ , ] . clinically, canine distemper is predominantly characterized by catarrhal respiratory and enteric disease signs, but a systemic form affecting also the central nervous system (cns) and unusual manifestations including the so-called "hard pad" disease can occur [ ] . the clinical disease of canine distemper is similar in pathogenesis and symptoms to measles virus infection in humans [ ] . neurologic manifestations of canine distemper can be caused by gray and/or white matter disease [ ] [ ] [ ] [ ] . the rarer polioencephalitis is divided into "old dog encephalitis", "post-vaccination distemper encephalitis" and "inclusion body polioencephalitis" [ , ] . however, the nervous form usually manifests as leukoencephalitis within the cerebellum [ ] . the lesions can be the original microarray analysis performed by ulrich et al. ( ) revealed differentially expressed probe sets and the dominating change was an up-regulation of genes related to the innate and the humoral immune response [ ] . of manually selected genes of the ifn-i pathway, % ( / ) were up-regulated in a relevant manner (significant fold change > . ) at least in one group of cdv-induced lesions, and gene expression usually peaked in the subacute stage ( figure , table , table s ). toll-like receptor (tlr) , tlr and tlr (fold change up to . , . and . ) were moderately increased, whereas pkr and mda were highly up-regulated (fold change up to . and . ) . only a mild increase in irf , irf and nuclear factor of kappa light polypeptide gene enhancer in b-cells (nfkb) gene expression (fold change < ) was found, whereas irf and irf were strongly induced (fold change up to . and . ) . no transcriptional changes were found in ifn-i gene expression and ifnar genes were only mildly increased (fold change < ) in the subacute stage of cdv-infection. similarly, most signal transducers including jak , socs and tyk did not show significant transcriptional changes. nevertheless, stat , stat and irf were moderately up-regulated (fold change up to . , . and . ) . the classical isgs were strongly induced by cdv-infection with particularly high levels for isg , ifi l, isg , oas and mx (fold change up to . , . , . , . and . ). additionally, several mhc class i and class ii genes were highly up-regulated (fold change up to . and . ). an overview of fold changes for selected interferon-dependent genes is given in figure , table and table s . the original microarray analysis performed by ulrich et al. ( ) revealed differentially expressed probe sets and the dominating change was an up-regulation of genes related to the innate and the humoral immune response [ ] . of manually selected genes of the ifn-i pathway, % ( / ) were up-regulated in a relevant manner (significant fold change > . ) at least in one group of cdv-induced lesions, and gene expression usually peaked in the subacute stage ( figure , table , table s ). toll-like receptor (tlr) , tlr and tlr (fold change up to . , . and . ) were moderately increased, whereas pkr and mda were highly up-regulated (fold change up to . and . ) . only a mild increase in irf , irf and nuclear factor of kappa light polypeptide gene enhancer in b-cells (nfkb) gene expression (fold change < ) was found, whereas irf and irf were strongly induced (fold change up to . and . ) . no transcriptional changes were found in ifn-i gene expression and ifnar genes were only mildly increased (fold change < ) in the subacute stage of cdv-infection. similarly, most signal transducers including jak , socs and tyk did not show significant transcriptional changes. nevertheless, stat , stat and irf were moderately up-regulated (fold change up to . , . and . and . ). an overview of fold changes for selected interferon-dependent genes is given (nfκb, irf , irf , irf , irf ) . these transcription factors translocate into the nucleus and induce ifn-i expression. right side: receptor binding of ifn-i activates the jak-stat signaling pathway leading to the formation of stat /stat heterodimers in the cytoplasm. these heterodimers form a complex with irf , termed interferon-stimulated gene factor (isgf ). in the nucleus, isgf induces the transcription of interferon-stimulated genes including isg , mx , mx , pkr, irf , oas , oas and oasl. irf also acts in a positive feedback loop to stimulate ifn-i expression. columns behind the gene symbols display fold changes in three different stages of cdv-infection (acute; subacute; chronic). fold changes are shown on a logarithmic scale. (nfκb, irf , irf , irf , irf ) . these transcription factors translocate into the nucleus and induce ifn-i expression. right side: receptor binding of ifn-i activates the jak-stat signaling pathway leading to the formation of stat /stat heterodimers in the cytoplasm. these heterodimers form a complex with irf , termed interferon-stimulated gene factor (isgf ). in the nucleus, isgf induces the transcription of interferon-stimulated genes including isg , mx , mx , pkr, irf , oas , oas and oasl. irf also acts in a positive feedback loop to stimulate ifn-i expression. columns behind the gene symbols display fold changes in three different stages of cdv-infection (acute; subacute; chronic). fold changes are shown on a logarithmic scale. lesions ( ) found in cerebellar tissue of cdv-infected dogs were analyzed using he-and lfb-stainings and cdv immunohistochemistry. in lesions, cdv antigen changes but not histological ones were detected (group ). twenty-six acute lesions were characterized by white matter vacuolation and absence of demyelination (group ). forty-eight subacute lesions showed demyelination in the lfb-staining but no inflammatory cell infiltration in the he-staining (group ). forty chronic lesions demonstrated advanced demyelination, perivascular cuffing and lymphocytes in the parenchyma (group ). in addition, areas were randomly selected in the cerebellar white matter of six control dogs for evaluation (group ). representative pictures of the investigated lesions are shown in figure in general, immunohistochemical analysis detected an increase in the protein expression of all investigated ifn-i pathway members in cdv-induced white matter lesions (figures and , table , figure s ). in general, immunohistochemical analysis detected an increase in the protein expression of all investigated ifn-i pathway members in cdv-induced white matter lesions (figures and , table , figure s ). a statistically significant up-regulation of irf protein expression was only found in group lesions compared to controls (p < . ). endothelial cells, neurons and glial cells were positive for irf in all investigated groups. in addition, perivascular lymphocytes present in group expressed irf . irf protein was not expressed in perivascular lymphocytes but in glial cells and neurons in infected and control groups. there was a prominent staining in the granular cell layer and a slight staining of purkinje cells. the irf protein expression was significantly up-regulated in acute (p = . ), subacute (p < . ) and chronic lesions (p < . ) and continuously increased during lesion progression. stat protein expression was increased at all stages of cdv-induced leukoencephalitis (group : p = . ; groups - : p < . ). in cdv-infected cerebellar tissue, stat proteins were mainly detected in glial cells, whereas only a few neurons and endothelial cells expressed stat and perivascular cells were mainly negative. in control tissue, only single glial cells, as well as some neurons and endothelial cells, slightly expressed stat . stat protein expression was significantly elevated only in chronic lesions (group : p = . ). perivascular lymphocytes were negative for stat , whereas neurons, especially purkinje cells and glia cells, expressed stat with a strong intensity in cdv-infected dogs and a weak intensity in control dogs. isg , mx and pkr proteins were significantly increased at all investigated stages of cdv-induced leukoencephalitis (p < . ). perivascular lymphocytes expressed mx and pkr proteins but not isg . mx proteins were also detected in endothelial cells, neurons and glial cells of cdv-infected dogs. in control tissue, only some neurons and endothelial cells showed a mild mx expression. furthermore, glial cells and neurons, especially purkinje cells and some endothelial cells, were positive for pkr in cdv-infected dogs. in control animals, pkr proteins were only detected in neurons, especially purkinje cells. isg proteins were detected in neurons, endothelial cells and glial cells of cdv-infected dogs, whereas control animals only showed a weak isg protein expression in neurons and endothelial cells. a significant up-regulation of oas proteins was found in acute (p = . ), subacute (p < . ) and chronic lesions (p = . ) but not in cdv-infected tissue without histological lesions (group ; p = . ). in cdv-infected dogs, oas proteins were detected in perivascular lymphocytes, endothelial cells, glial cells and neurons. in control animals, only neurons and endothelial cells expressed oas proteins. control dogs; group are foci with cdv antigen expression but without histological lesions; group are acute cdv lesions; group are subacute foci without inflammation; group are chronic cdv lesions with inflammation. shown are the percentages of the immunopositive area using box plots in a logarithmic scale and significant differences between the cdv-infected groups - and the control group based on kruskal-wallis-tests and dunn's multiple comparison tests. * p < . ; ** p < . ; *** p < . . ++ ++ ++ ++ +++ + +++ ++ + - +++ - + - ++ - endothelial cells +++ +++ (+) (+) + (+) - - ++ (+) ++ (+) + + (+) - spearman's rank correlation coefficients demonstrated a moderate correlation between cdv antigen and stat , isg , mx and pkr protein expression (table ) . cdv antigen correlated only weakly with irf and stat and did not correlate with irf protein expression. irf also correlated weakly with stat , oas and pkr. moreover, there was a weak to moderate correlation between stat , isg , mx, oas and pkr, a moderate correlation between irf and isg , and a strong correlation between isg and mx (table ) . double staining with cellular markers revealed a stat expression in nogoa + oligodendrocytes, gfap + astrocytes and iba + microglia/macrophages, which was limited to histological lesions. stat was detected in nogoa + oligodendrocytes located in white matter lesions and also in normal-appearing white matter. stat was also found in activated iba + microglia/macrophages (gitter cells), which were present in subacute and chronic white matter lesions. mx was expressed by intralesional gfap + astrocytes, nogoa + oligodendrocytes and iba + microglia/macrophages. pkr was also found in intralesional gfap + astrocytes and some nogoa + oligodendrocytes but not in iba + microglia/macrophages ( figure ). immunofluorescence double-staining was also used to determine the infection status of cells expressing stat , stat and mx. a low number of cdv-infected cells expressed stat and mx. however, cdv-infected cells expressing stat were nearly absent ( figure ). the present study gives an overview of the protein expression of selected ifn-i pathway members and compares it with existing microarray data. previous studies often investigated isg expression in cell cultures, whereas studies on the protein expression of isgs in a naturally occurring animal disease are rare [ ] [ ] [ ] [ ] [ ] . irf plays, together with the closely related irf , an important role in the ifn-i signaling cascade and the control of viral infections [ ] . ysebrant de lendonck et al. ( ) reported that irf is constitutively expressed in most tissues and cell types [ ] . similarly, in the present study, irf was expressed in most cell types present in cdv-induced cerebellar lesions as well as in control tissue (endothelial cells, neurons, glial cells and perivascular inflammatory cells). this strong constitutive expression of irf might also explain that the present microarray analysis and immunohistochemical investigation did not detect changes in the gene or protein expression of this transcription factor in acute, subacute or chronic cerebellar lesions. nonetheless, there was a significant increase in irf protein expression in cdv-infected areas lacking histological lesions (group ) compared to controls demonstrating an early activation of ifn-i signaling in the disease process. irf interacts with other transcription factors, coactivators and repressors including other irfs and nf-κb and thereby regulates t cell differentiation into th , th and th cells as well as induces apoptosis during viral infections [ ] . thus, irf most likely modulates adaptive immune responses at all stages of cdv-induced cerebellar lesions due to its action as a signaling platform, which irf can fulfill without changes in expression. unlike irf , irf is reported to be expressed only at low levels in most cell types and has to be continuously produced due to its short half-life time of . - h [ , ] . nevertheless, irf protein was present in neurons of the granular layer and individual glial cells, even in control cerebellum, showing low constitutive expression of this transcription factor in the canine cns. oasl knockout mice studies showed that oasl inhibits irf translation and thereby negatively the present study gives an overview of the protein expression of selected ifn-i pathway members and compares it with existing microarray data. previous studies often investigated isg expression in cell cultures, whereas studies on the protein expression of isgs in a naturally occurring animal disease are rare [ ] [ ] [ ] [ ] [ ] . irf plays, together with the closely related irf , an important role in the ifn-i signaling cascade and the control of viral infections [ ] . ysebrant de lendonck et al. ( ) reported that irf is constitutively expressed in most tissues and cell types [ ] . similarly, in the present study, irf was expressed in most cell types present in cdv-induced cerebellar lesions as well as in control tissue (endothelial cells, neurons, glial cells and perivascular inflammatory cells). this strong constitutive expression of irf might also explain that the present microarray analysis and immunohistochemical investigation did not detect changes in the gene or protein expression of this transcription factor in acute, subacute or chronic cerebellar lesions. nonetheless, there was a significant increase in irf protein expression in cdv-infected areas lacking histological lesions (group ) compared to controls demonstrating an early activation of ifn-i signaling in the disease process. irf interacts with other transcription factors, coactivators and repressors including other irfs and nf-κb and thereby regulates t cell differentiation into th , th and th cells as well as induces apoptosis during viral infections [ ] . thus, irf most likely modulates adaptive immune responses at all stages of cdv-induced cerebellar lesions due to its action as a signaling platform, which irf can fulfill without changes in expression. unlike irf , irf is reported to be expressed only at low levels in most cell types and has to be continuously produced due to its short half-life time of . - h [ , ] . nevertheless, irf protein was present in neurons of the granular layer and individual glial cells, even in control cerebellum, showing low constitutive expression of this transcription factor in the canine cns. oasl -knockout mice studies showed that oasl inhibits irf translation and thereby negatively regulates ifn-i production during acute and chronic viral infections [ , ] . the present study detected a strong increase in oasl mrna transcripts in acute, subacute and chronic lesions, which might affect irf expression. nonetheless, there was a strong up-regulation in irf gene and protein expression in cerebellar lesions, which was most pronounced at later stages of cdv-induced leukoencephalitis. this continuous increase in irf expression is probably caused by the positive feedback mechanism of irf and indicates that the ifn-i signaling pathway contributes to lesion progression [ , [ ] [ ] [ ] . stat proteins can form stat /stat heterodimers and stat /stat homodimers to induce transcription of isgs and pro-inflammatory cytokines after ifn-i and ifnγ stimulation, respectively [ ] . svitek et al. ( ) demonstrated that cdv has to block stat signaling for immune evasion in ferrets, whereas inhibition of stat does not contribute to cdv virulence [ ] . the present study demonstrated a strong increase in stat mrna transcripts and proteins in the cdv-infected cerebellum (groups - ), whereas control animals expressed only minimal amounts of stat . stat proteins were constitutively expressed in cdv-infected and control tissue and only weakly up-regulated in a chronic lesion, but microarray analysis revealed a moderate increase in stat mrna transcripts also in acute and subacute lesions. however, the antibody used to detect stat only binds to phosphorylated and thus activated proteins (p /p ), whereas the anti-stat antibody recognizes activated and non-activated proteins. this difference explains the detection of less prominent changes in stat compared to stat protein expression using immunohistochemistry. interestingly, both transcription factors were found in neurons, oligodendrocytes and activated microglia/macrophages, whereas astrocytes, endothelial cells and inflammatory cells only expressed stat but not stat . this observation suggests that the canonical ifn-i pathway with stat /stat heterodimer formation only plays a minor role in the latter cells and non-canonical jak-stat signaling via stat /stat homodimers or other pathways might mediate isg expression [ ] . moreover, the present analysis of the previously published microarray did not detect an increase in ifn-i expression in the cdv-infected cerebellum, which is most likely caused by the non-structural v protein of cdv interfering with mda -dependent virus detection [ ] . consequently, the ifn-i response and the down-stream jak-stat signaling cascade are blocked during canine distemper encephalitis. nevertheless, the present study demonstrated a strong increase in isg gene and protein expression, which seems to be mediated by ifn-i-independent pathways. similarly, a recent microarray study of mice infected by theiler's murine encephalomyelitis virus (tmev) described a strong isg expression in demyelinating spinal cord lesions despite low ifn-i levels [ ] . isg expression can directly be induced by irf (formerly called isgf ), which is a transcription factor involved in anti-viral and anti-bacterial immune responses, t cell and nk cell differentiation, mhc class i and ii expression and induction of apoptosis [ , , ] . correspondingly, the present canine study demonstrated a strong up-regulation of irf and several mhc genes at all stages of cdv-induced cerebellar lesions. dla- was particularly increased in acute and subacute distemper lesions (fold change up to , ). most importantly, this highly polymorphic mhc class i protein can present peptides derived from cdv hemagglutinin, large polymerase, matrix, nucleocapsid, and v proteins [ , ] . irf is also described as inducing isg transcription in the absence of ifn-i [ ] . consequently, irf -and irf -dependent pathways presumably mediate a major part of isg expression in the cdv-infected cerebellum, counteracting common viral strategies, which modulate or inhibit the ifn-i response [ , ] . isg showed the strongest increase of all investigated genes, with a fold change of almost in subacute lesions. in addition, a weak isg immunoreactivity was detected in neurons and endothelial cells of uninfected canine cerebella, confirming previous descriptions of a low constitutive isg expression [ ] . isg represents an ubiquitin-like protein, which binds to over cellular proteins involved in immune regulation, including members of the ifn-i, nf-κb and jnk pathways [ , ] . this process known as isgylation can activate or inhibit the respective signaling cascades in a species-specific manner [ ] . murine isg targets rig , which reduces type i interferon promoter activity and nf-κb responses [ ] . in contrast, human isg can bind to irf preventing its degradation by the proteasome and thereby increasing the ifn-i response [ ] . nevertheless, humans born with inactivating isg mutations are highly susceptible to mycobacterial and autoinflammatory diseases but not to viruses, this underlining the complexity of isg -mediated effects [ , ] . the susceptibility of isg -deficient patients to environmental mycobacteria is probably related to the cytokine-like functions of secreted isg , which acts in synergy with il to induce ifnγ production by t cells or nk cells, thus enhancing the antimycobacterial activity of macrophages [ , , , ] . only a few studies have investigated the functions of isg in dogs, indicating an antiviral activity of this protein in canine cells, which might be mediated by binding isg to viral proteins interfering with viral replication [ , , , ] . ifnγ can be found in the csf of dogs with chronic but not early lesions [ ] [ ] [ ] . consequently, highly up-regulated isg levels in cdv-induced cerebellar lesions most likely contribute to viral elimination and might modulate the canine immune system. pkr can be activated by double-stranded rna and isgylation, which results in autophosphorylation and subsequently in phosphorylation of the eukaryotic translation initiation factor alpha (eif α), thereby down-regulating cellular and viral protein synthesis [ ] [ ] [ ] [ ] . in addition, the activation of this constitutively expressed virus sensor protein can induce apoptosis, thus ensuring the eradication of infected cells from the tissue [ , ] . the present study demonstrated a strong increase in the expression of phosphorylated, hence activated pkr in the cdv-infected cerebellum. this protein was detected in neurons, intralesional astrocytes and oligodendrocytes as well as perivascular lymphocytes but not in microglia/macrophages. similarly, tmev-infected mice showed a strong expression of activated pkr proteins in spinal cord white matter lesions [ ] . nevertheless, these proteins were predominantly expressed by intralesional gitter cells and to a lesser extent by perivascular immune cells, oligodendrocytes and neurons but not by astrocytes in tmev-infected mice. similarly, in mice suffering from experimental allergic encephalomyelitis, another mouse model of human ms, activated pkr proteins were found in oligodendrocytes and neurons as well as in intralesional t cells and macrophages [ ] . these observations suggest a so far undescribed species-specific difference in pkr expression and/or activation of murine and canine astrocytes and microglia/macrophages. similar to pkr, oas proteins are constitutively expressed in many tissues (spleen, lung, liver, thymus, small intestine, brain) and bind double-stranded rna resulting in their activation [ ] [ ] [ ] . activated oas proteins then induce the formation of - -oligoadenylates from atp molecules, which trigger the ubiquitously expressed endoribonuclease rnase l [ ] . this enzyme can degrade cellular and viral rna and thereby inhibit viral protein synthesis [ ] [ ] [ ] . the production of small rna cleavage products by rnase l again initiates ifn-i production, creating a positive feedback mechanism in antiviral defense [ , ] . rnase l has also been described as a potent inducer of apoptosis in fibroblasts [ ] . in addition, kristiansen et al. ( ) suggested that oas proteins produced by virus-infected cells can be released into the extracellular space, where it acts as an rnase l-independent paracrine antiviral agent to protect neighboring cells from infection [ ] . the present study revealed a strong increase in oas gene and protein expression in acute, subacute and chronic cerebellar lesions, which most likely contributes to the restriction of viral replication. oas proteins were detected in neurons and endothelial cells as well as in intralesional glial cells and perivascular inflammatory cells. previous studies demonstrated that the oas-rnase l pathway is particularly important in murine macrophages and inhibits the replication of picornaviridae including tmev [ , [ ] [ ] [ ] [ ] [ ] . interestingly, the oas activity in canine serum is -to -fold higher than in cats, rabbits and guinea pigs, indicating a prominent role of oas proteins in the canine humoral immune system [ ] . however, the exact role of rnase l-dependent and independent functions of oas proteins in the pathogenesis of cdv-induced leukoencephalitis remains to be determined. the present study showed a significant increase in the expression of mx proteins during all stages of canine distemper leukoencephalitis, which was even found in cdv-infected areas without histological lesions. the presence of mx proteins in neurons and endothelial cells of the normal canine brain demonstrates a low but constitutive expression of this antiviral protein, whose expression was described to be strictly dependent on ifn-i [ ] . similarly, porter et al. ( ) found a strong mx expression in astrocytes, macrophages/microglia and neurons in cdv-infected brain tissue [ ] . endothelial cells were also described to be immunopositive for mx by porter et al. ( ) , but this staining was interpreted as non-specific [ ] . however, strong expression of mx proteins in canine distemper lesions most likely impedes viral replication and, similar to increased isg , pkr and oas levels, contributes to the typical decrease in viral antigen observed during the progression of the disease [ , , ] . moreover, the low number of cdv-infected cells expressing stat , stat and mx indicates that the expression of these type i ifn pathway members is not directly induced by virus infection. the strong expression of stat , stat and downstream isg proteins in oligodendrocytes and neurons is probably related to their well-known restricted cdv infection, which is characterized by the presence of cdv nucleic acid sequences and lack of viral antigen [ ] . in contrast, astrocytes did not express stat proteins, which favors viral replication in these cells due to the blocking of the canonical jak-stat pathway. the cell-type specific differences in the activation of the ifn-i pathway might also explain the fact that up to % of all cdv-infected cells within acute lesions are astrocytes [ ] , whereas oligodendrocytes hardly express cdv protein [ ] . however, the restriction of virus replication in neurons and oligodendrocytes does not prevent axonal pathology and massive down-regulation of myelin gene expression (oligodendrocyte dystrophy) [ , ] . both processes contribute to the initiation of the demyelination process, whereas later stages of demyelination are mainly mediated by so-called bystander mechanisms [ , , , ] . this bystander demyelination results from the activation of macrophages/microglia, which is characterized by an increased phagocytic activity, oxygen radical production and expression of mhc class ii molecules and the release of myelin damaging enzymes such as matrix-metalloproteinases (mmps) [ , , [ ] [ ] [ ] . the progressive development of demyelination is driven by a strong continuous expression of il , il , il , il and tnfα promoting innate and th -biased immune responses [ , , , [ ] [ ] [ ] . interestingly, recent studies demonstrated that the ifn-i pathway is critically involved in the differentiation and activation of immune cells as well as the expression of mmps and pro-inflammatory cytokines [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ifn-i can modulate the phenotype of microglia, regulate phagocytosis and affect blood-brain barrier integrity [ ] . irf is required for nk cell development and differentiation of cd + t cells promotes the differentiation of th cells via transcriptional control of il p , inhibits tnfα-induced mmp expression and contributes to the activation of the nlrp inflammasome [ , , , ] . irf stimulates the transcription of il , il and mmps through interaction with c-jun and the ap- promoter site [ ] [ ] [ ] . irf regulates the expression of il through stabilization of il mrna and induces the transcription of cxcl , a chemokine for macrophages, t cells and nk cells [ ] . moreover, studies of monogenic autoinflammatory cns diseases demonstrated that they can be caused by an uncontrolled up-regulation of ifn-i signaling and jak inhibition may be a successful therapeutic strategy in patients with these type i interferonopathies [ ] [ ] [ ] . consequently, the strong activation of the ifn-i signaling pathway in infiltrating immune and resident cns cells including activated microglia/macrophages most likely contributes to immunopathological mechanisms aggravating cdv-triggered demyelination. this study was conducted in accordance with the german animal welfare act. all animals used in this study were dead at the time of submission for necropsy in order to investigate the causes of death and disease. consequently, the authors confirm that this study does not contain data obtained from animal experiments and no animals were infected or sacrificed for the purpose of this retrospective pathological case-control study. moreover, all dog owners provided written consent for the dogs' tissues to be collected and used for research purposes. paraffin-embedded cerebellar tissue of naturally cdv-infected dogs and six control dogs without any cns lesion was collected from the archive of the department of pathology of the veterinary university of hannover, germany and used for histological analysis and immunostainings. anamnestic data of the investigated dogs are summarized in table s . serial two-micrometer paraffin sections were stained with hematoxylin and eosin (he) or luxol fast blue (lfb) or used for immunohistochemistry. the lesions in the cdv-infected dogs were categorized with he-and lfb-staining as previously described [ , , ] . briefly, randomly selected areas in the cerebellar white matter of dogs without any lesions were used as controls (group ). white matter lesions of cdv-infected dogs were divided into four groups. group ("antigen without lesion") included areas with antigen expression but with no lesions in he-or lfb-stainings. group lesions ("acute") were characterized by vacuolation in the he staining and absence of demyelination in the lfb-staining. group ("subacute") included lesions with demyelination but with no inflammatory infiltrates in the he-staining. group ("chronic") contained demyelinated lesions with variable numbers of inflammatory cells in the perivascular area or diffusely distributed ones in the parenchyma. an miame-compliant expression raw data set of a previously published microarray study upon cdv-induced demyelinating leukoencephalitis performed on genechip canine genome . arrays (affymetrix inc., santa clara, usa) was accessed via the arrayexpress database (accession number: e-mexp- ; http://www.ebi.ac.uk/arrayexpress) [ ] . rna used for the original microarray study was isolated from frozen cerebellar specimens of control dogs and cdv-infected dogs, which were grouped using a classification system similar to the present study (control; acute; subacute; chronic) [ ] . fold changes in this data set were calculated as the ratio of the inverse-transformed arithmetic means of the log -transformed expression values between the respective groups. downregulations are given as negative reciprocal values [ ] . in order to perform a bottom-up analysis of transcriptional changes in cdv-induced lesions, candidate genes including pattern recognition receptors (prrs), ten ifn regulatory factors, eight type i/ii ifns, five type i/ii/iii receptors, signal transducers, ifn-dependent antiviral effectors and mhc class i/ii genes were manually extracted from peer-reviewed published literature (table s ) [ , , , , ] . if necessary, murine and human genes were converted into orthologous canine gene symbols using the madgene web tool (http: //cardioserve.nantes.inserm.fr/madtools/madgene/) [ ] . the expression values of these genes were evaluated for significant differences between the respective groups employing independent pairwise mann-whitney u tests (see statistical analysis). for immunohistochemistry, sections were dewaxed, rehydrated and incubated in ethanol with . % hydrogen peroxide for min to block endogenous peroxidase. except for stat , sections were pretreated by boiling them in mm sodium citrate buffer (ph ) for min in a microwave oven ( w). sections were blocked with phosphate-buffered saline (pbs) containing % goat serum for min at room temperature and incubated with rabbit polyclonal anti-phosphorylated stat (p /p , sc- , santa cruz biotechnology inc., santa cruz, ca, usa, : ), anti-oas (sc- , santa cruz, : ) and anti-isg (sc- , santa cruz, : ), rabbit monoclonal anti-irf (ab , abcam inc., cambridge, ma, usa, : ), anti-irf (ab , abcam, : ) and anti-phosphorylated pkr (phospho t , ab , abcam, : ) as well as mouse monoclonal anti-cdv (d ; kindly provided by prof. dr. a. zurbriggen, university of bern, bern, switzerland, : ), anti-stat (sc- , santa cruz, : ) and anti-mx (m , kindly provided by prof. dr. haller and pd. dr. kochs, university medical center freiburg, freiburg, germany, : ) antibodies overnight at • c. sections incubated with normal rabbit serum (r , sigma-aldrich chemie gmbh, munich, germany) or igg-containing ascites of balb/c mice instead of primary antibodies served as negative controls. biotinylated goat-anti-rabbit igg (ba- ) or goat-anti-mouse igg (ba- ), diluted : (vector laboratories inc., burlingame, ca, usa), were used as secondary antibodies. immunolabeling was visualized by using the avidin-biotin-peroxidase complex method (pk , vector laboratories) and the chromogen , -diaminobenzidine-tetrahydrochloride with . % hydrogen peroxide. finally, sections were slightly counterstained with mayer's hematoxylin. every immunohistochemically stained slide was photographed with a digital microscope (hs all-in-one fluorescence microscope bz- generation ii, biorevo, keyence deutschland gmbh, neu-isenburg, germany) and afterwards analyzed using analysis software (analysis . software package, soft imaging system gmbh, münster, germany). percentage of immunopositive areas are shown as box-and-whisker plots with median, minimum and maximum in a logarithmic scale. to determine the cellular origin of selected members of the jak-stat signaling pathway (stat , stat ) and downstream isgs (mx and pkr), immunofluorescence double-labeling was performed using markers for oligodendrocytes (nogoa), astrocytes (gfap) and microglia/macrophages (iba ). moreover, double-staining was used to investigate the infection status of cells expressing stat , stat and mx. immunofluorescence was performed on selected sections of subacute and chronic lesions due to the strong expression of the investigated markers in the microarray analysis and immunohistochemical evaluation. dewaxing, rehydration, pretreatment and blocking of non-specific binding with % goat serum or % horse serum was performed as described for immunohistochemistry without blocking the endogenous peroxidase. sections were co-incubated with antibodies directed against phosphorylated stat (sc- ; : ), stat (sc- ; : ), mx (m ; : ) or phosphorylated pkr (ab ; : ) and antibodies directed against gfap (goat polyclonal, ab , abcam, : or rabbit polyclonal, dakocytomation gmbh, hamburg, germany, : ), nogoa (c- , mouse monoclonal, sc- , santa cruz, : or rabbit polyclonal, millipore/chemicon, catalog-no. ab p, : ), iba (mouse monoclonal, ab , abcam, : or rabbit polyclonal, thermofisher scientific, schwerte, germany, catalog-no. pa - , : ) or cdv (d , mouse monoclonal, : or # , kindly provided by c. Örvell, karolinska university hospital, stockholm, sweden, rabbit polyclonal, : ) overnight at • c. negative controls were included in accordance with the immunohistochemical investigation. subsequently, sections were incubated with cy -and cy -conjugated secondary antibodies (goat anti-rabbit, - - ; goat anti-mouse, - - or - - ; donkey anti-goat, - - ; jackson immunoresearch europe ltd., cambridge, uk; goat anti-rabbit, a- ; invitrogen/thermo fisher; donkey anti-rabbit, ab ; abcam; : ) for min at room temperature in the dark. nuclei were visualized by . % bisbenzimide (h , sigma-aldrich) and sections were mounted with mounting medium (shandon immu-mount, thermofisher scientific, catalog-no. , usa). sections were photographed with a digital microscope (hs all-in-one fluorescence microscope bz- generation ii, biorevo, keyence deutschland gmbh, neu-isenburg, germany). microarray data were analyzed with non-parametric mann-whitney u tests (prism , graphpad software ltd., la jolla, ca, usa), comparing each group of cdv-infected dogs with controls. immunohistochemically data were analyzed using kruskal-wallis tests and dunn's multiple comparison tests comparing groups - (cdv-infected) with group (control). spearman's rank coefficients were calculated to evaluate the correlation of cdv antigen and isg protein expression. statistical significance was designated as p < . . the ifn-i pathway is activated in the early stages of cdv-induced leukoencephalitis, leading to the expression of various isgs, which restrict viral replication particularly, in neurons and oligodendrocytes. in contrast, the lack of stat expression in astrocytes and subsequent suppression of canonical jak-stat signaling might explain their high infection rate in cerebellar lesions. the up-regulation of isgs seems to be partly mediated by irf -and irf -dependent pathways counteracting the inhibition of the ifn-i response by cdv. however, the strong activation of the ifn-i signaling cascade, especially in activated microglia/macrophages, probably contributes to immune-mediated mechanisms of demyelination in later stages of the disease. these results encourage further research to elucidate the regulation of ifn-i signaling and its contribution to demyelinating cns diseases in dogs and humans in order to develop novel treatment strategies targeting specific ifn-i pathway members. the authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences new aspects of the pathogenesis of canine distemper leukoencephalitis demyelination in canine distemper virus infection: a review pathogenesis and immunopathology of systemic and nervous canine distemper virale infektionen von welpen und junghunden unter berücksichtigung der staupevirusinfektion abundant expression of viral nucleoprotein mrna and restricted 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regulatory factors to inhibit activation of the type i ifn response: implications for treatment of autoimmune disorders synoviocyte innate immune responses: ii. pivotal role of ifn regulatory factor poly(i:c) induces expressions of mmp- , - , and - through various signaling pathways including irf in human skin fibroblasts therapeutic approaches to type i interferonopathies the type i interferonopathies type i interferon-mediated monogenic autoinflammation: the type i interferonopathies, a conceptual overview up-regulation of major histocompatibility complex class ii antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis the yin and yang of viruses and interferons interferon-stimulated genes: roles in viral pathogenesis madgene: retrieval and processing of gene identifier lists for the analysis of heterogeneous microarray datasets the authors wish to thank bettina buck, caroline schütz, kerstin schöne and petra grünig for their excellent technical assistance. furthermore, we would like to thank a. zurbriggen (university of bern, switzerland) and c. Örvell (karolinska university hospital, stockholm, sweden) for providing the antibodies against cdv and o. haller and g. kochs (university medical center freiburg, germany) for providing the antibody against mx. key: cord- -ejgzfzd authors: vogel, wolfgang title: treatment of chronic hepatitis c patients not responding to combination therapy with ribavirin and interferon α — hype or hope? date: journal: wien klin wochenschr doi: . /bf sha: doc_id: cord_uid: ejgzfzd nan infection with the hepatitis c virus (hcv) is still a major cause of chronic liver disease resulting in need for liver transplantation and of hepatocellular carcinoma in the western world. the prevalence of infection is believed to be . % in central europe with figures as high as % reported in endemic areas such as egypt. historically, the major route of infection was poor medical practice. currently, intravenous drug abuse probably accounts for the majority of new infections. our observations suggest that about new infected patients will be diagnosed in austria annually. if all patients were treated with the best regimen available, based on a markov model, the number of decompensated cirrhotic patients could be reduced by almost two-thirds [ ] . interferon-a (ifn) was introduced as therapy in the late eighties of the last century and since then has proved to be the mainstay of treatment. the sustained virological response rates (svr), defined as pcr negative months after the end of therapy, which is considered as proof of final elimination of the virus, were initially dismal. therapy prolongation up to months and administration of ribavirin, a nucleoside analogon with non-specific anti-hcv effects, to ifn improved svr from % to %. treatment with pegylated interferons in combination with ribavirin results in > % svr after months of therapy in genotype patients and> % after months of therapy in genotype and patients. despite this dramatic improvement of therapy results, for to % of the treated patients, we will have to look for new treatment options. a particularly difficult group of patients are nonresponders to ifn therapy. these patients typically will remain hcv-rna positive during the whole period of therapy. patients achieving hcv-rna negativity during treatment, but becoming positive again during the treatment phase or after the end of therapy are defined as break-through or relapse patients. on the basis of prospective studies, patients not becoming negative after weeks of therapy or whose hcv-rna titre failed to drop by at least two log, have a likelihood of achieving svr of only . %, whereas patients with a significant fall in hcv-rna have a % likelihood of svr [ ] . re-treatment of these patients employing pegylated ifn in combination with ribavirin will result in svr only in approximately % of the patients. patients pretreated only with ifn monotherapy, however, have a % probability of svr when retreated with ifn-ribavirin combination therapy. relapsers or partial responders to treatment with conven-tional ifn plus ribavirin are believed to have a more favourable prognosis, but prospective controlled studies are lacking. factors associated with non-response are genotype or , high serum hcv-rna concentration at baseline, cirrhosis, current alcohol abuse, race, dose reduction and non-compliance. only a few of these factors are correctable. a svr of % upon re-treatment in patients infected with genotype or and in patients with base-line hcv-rna concentration of < . million iv/ml can be achieved. the search for more efficacious treatment options has brought amantadine (ama) to the forefront. in a pilot study of patients with chronic hepatitis c who failed treatment with ifn monotherapy and subsequently treated with ama was carried out [ ] . in this study a reduction of necro-inflammation and a decrease of transaminase activity in % of patients were observed. four out of patients cleared the virus and achieved svr. however, this favourable effect of ama could be reproduced in subsequent trials neither in non-responders to ifn nor in naive patients [ , ] . the observed reduction in transaminase levels is reminiscent of the effects of ribavirin, which improves liver biochemistry but has no effect on viral replication. amantadine, a tricyclic amine, has antiviral activity against toga, myxo, arena, flavi and corona viruses [ ] . known mechanisms of action include an early step in viral replication and interaction with the influenza a viral matrix protein [ ] . indirect assessment of the effect of ama on hcv protease, helicase, atpase, rna polymerase, and hcv internal ribosomal entry site (ires) translation has been performed by in vitro biochemical assays [ ] . although no inhibition was observed, adenylyl cyclase associated protein (cap) and ires reporter genes were suppressed at higher levels probably by non-specific inhibition of translation. on the basis of these results it was concluded that ama has no direct specific inhibitory effect on hcv replication. in the hcv replicon system, ifn induces a dose-dependent inhibition of hcv rna levels, while ama and ribavirin had no effect. a competent immune response is mandatory for the efficient clearance of the virus. ama can more effectively suppress the hcv specific proliferative response of pbmcs than ifn [ ] . in summary, ama has some weak anti-inflammatory properties without direct anti-viral effects. the lack of specific anti-viral drugs encouraged a number of large randomized clinical trials, which, however, had conflicting results. a comparative study in which wolfgang vogel naive patients were investigated for assessing the effectiveness of combination therapy ifn and ama on the one hand, and of ifn plus placebo, on the other, demonstrated the former to be significantly superior. in this german study, % of patients had svr compared to % in the monotherapy arm after weeks of treatment [ ] . a similar study performed in italy on a cohort of naive patients who were administered slightly higher doses of ifn, mu t.i.w, reported svr in % and % of patients on the combined vs. monotherapy, respectively. the ama dose of x mg daily was identical in both studies [ ] . two further studies, one from italy and another from the uk of a total of naive patients confirmed svrs of % versus % similar to those reported in the german study. in all these studies ama had no effect on the safety profile of ifn treatment. another approach to increase the efficacy of ifn treatment would be to improve the initial virological response by induction therapy with high-dose ifn. the initial decline in viral load predicts the outcome of therapy as patients with svr are characterized by a greater than % drop in viral titre within weeks of initiation of treatment. to test this concept a prospective randomized trial was performed by the austrian hepatitis study group [ ] . in this pivotal study of naive patients receiving mu ifn induction followed by ifn-ribavirin combination therapy, genotype patients had a significantly better svr of % versus % with induction therapy than without. however, no difference was observed in genotype patients. the issue of three-drug combination therapy was first studied by brillanti [ ] . in a randomised prospective trial patients with chronic hepatitis c not responding to a previous course with inf were either treated with mu ifn on alternate days in combination with ribavirin ( - mg daily) or additionally with ama ( mg daily) for months. an encouraging % of the patients on triple therapy achieved svr but only % of the patients on dual therapy. however, a german study of nonresponders found no difference between the two treatment regimens [ ] . in a number of further smaller studies of non-responders to ifn monotherapy, no benefit of adding ama to ifn-ribavirin combination therapy could be demonstrated, either with or without induction therapy [ ] . the difference in the studies is difficult to explain, aspects to consider are definition of non-response, patient selection with respect to genotype and stage of liver fibrosis. the interesting question of whether the addition of pegylated interferons to the combination of ribavirin and ama can improve svr in non-responders is still a matter of ongoing studies. preliminary results are promising. in this issue of the journal, stauber and the austrian hepatitis group present the findings of a prospective trial in the difficult-to-treat group of patients who have failed previous therapy with standard ifn and ribavirin [ ] . the study included non-responders, relapsers after a standard treatment period and patients, who had experienced a break-through of disease after an initial response while on therapy. eighty percent of the patients were infected with genotype and % were cirrhotics. the novel approach was to combine an induction period of daily ifn therapy for weeks with standard dose ama and ribavirin. interestingly, % of patients were negative at week of therapy whereas only % had svr. relapsers and break-through patients had a higher svr than non-responders, but these differences did not reach statistical difference. the tolerability of the triple therapy was again not different from that reported for the dual therapy in previous trials. although the authors felt that the slightly higher svr of % compared to % in other trials was somewhat disappointing, the findings of the study offer some hope for this group of patients and raises interesting points. the surprisingly high response rate after weeks of therapy suggests that modification of ifn dosing is efficacious in non-responders. approximately % of the responders were lost during treatment phase and another % during follow-up. because of the trial design it is difficult to separate clearly the effect of the induction phase and the effect of ama. but it is obvious that the gain in responders during the early phase of the trial was lost in the long run. this implies that ama is not capable of maintaining the early advantage by boosting the immune-response and increasing the clearance rate of infected hepatocytes. the conclusion therefore would be that this kind of patients could benefit from either longer therapy or higher ifn dose or both. however, the question of the extent to which addition of ama can increase the response rate still remains open. with the advent of more efficacious pegylated ifn, studies of longer treatment periods with and without ama are warranted. in summary, the question if ama offers additional benefit in the treatment of non-responders to combination therapy is still open. this inexpensive and well-tolerated drug holds some promises which need further evaluation. the current outlook for new treatment options is poor, and as the new designer drugs to specifically inhibit viral replication enter phase ii clinical trials, the likelihood that ifn with all its limitations and side effects will remain the mainstay of therapy for the foreseeable future is very high. so researchers are challenged to expand the existing treatment options for improved results, particularly in the difficult-to-treat group of patients. projecting future complications of chronic hepatitis c in the united states monitoring of viral levelsduringtherapy of hepatitis c treatment of chronic hepatitis c with amantadine treatment of chronic hepatitis c with amantadine treatment of chronichepatitis c with amantadine hydrochloride in patients who had not responded to previous treatment with interferon-alpha and! or ribavirin antiviral drugs: current state of the art amantadine and rimantadine haveno direct inhibitory effects against hepatitis c viral protease, heli-vogel, treatment of chronic hepatitis c patients case, atpase, polymerase, and internal ribosomal entry site-mediated translation in vitro effect of amantadine and interferon alpha- a on hepatitis c virus markers in cultured peripheral blood mononuclear cells from hepatitis c virus-infected patients randomized, double-blind, placebo-controlled trial of interferon alfa a with and without amantadine as initial treatment for chronic hepatitis c a randomized trial of amantadine and interferon versus interferon alone as initial treatment for chronic hepatitis c combination of interferon induction therapy and ribavirin in chronic hepatitis c triple antiviral therapy as a new option for patients with interferon non-responsive chronic hepatitis c randomized, placebo-controlled, double-blind trial with interferon-alpha with and without amantadine sulfate in primary ifn-alpha non-responders with chronic hepatitis c amantadine for chronic hepatitis c: a magic bullet or yet another dead duck? retreatment of patients with chronic hepatitis c not responding to interferonlribavirin combination therapy with daily interferon plus ribavirin plus amantadine key: cord- -lchdm xr authors: liu, yang; king, nicholas; kesson, alison; blanden, robert v.; müllbacher, arno title: flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro date: - - journal: j neuroimmunol doi: . / - ( ) - sha: doc_id: cord_uid: lchdm xr west nile virus (wnv) infection of astrocytes can up-regulate their expression of both class i and class ii major histocompatibility complex (mhc) antigens as determined by flow cytometry with monoclonal antibodies specific for class i and class ii mhc antigens. the up-regulation of class i mhc antigen expression could be partly caused by interferon secreted after wnv infection because the synthetic interferon inducer polyinosinic-polycytidylic acid (poly i : c) has similar effects. in contrast the up-regulation of class ii mhc antigen expression was not induced by poly i : c. the increased mhc antigen expression by wnv infection had significant effects on t cell recognition. thus, wnv and influenza virus a/wsn double-infected astrocytes but not astrocytes infected by a/wsn alone were lysed by influenza virus-immune cytotoxic t cells. similarly, wnv-infected astrocytes were better stimulators than normal astrocytes for a class ii mhc-reactive t cell line, both in terms of t cell proliferation and interleukin release. it is well established that the major histocompatibility complex (mhc) antigens form an essential part of antigenic structures recognised by t iymphocytes (reviewed by zinkernagel and in the case of experimental allergic encephalitis of rats, where transfer of syngeneic t cells specific for myelin basic protein (mbp) results in full expression of the disease (lipton et al., ; tourtellotte et al., ; kreth et al., ; booss et al., ; wekerle et al., ) . the paradox has been partly resolved by findings which indicate that at least some cells from the cns, such as astrocytes, ofigodendrocytes, brain endothelial cells and neurons can be induced to express class i and/or class ii mhc antigens by lymphokines such as "t-interferon (t-ifn) and by certain viral infections such as murine hepatitis virus and theiler's murine encephalomyelitis virus (hirsch et al., ; massa et al., ; suzumura et al., ; rodriguez et al., ) . additional data suggested that such elevation of the mhc antigens on astrocytes and/or brain endothelial cells can potentiate the antigen-presenting capacity of these cells male et al., ) . thus, the induction of expression of mhc antigens on the cells of the cns could be an important regulatory mechanism in the initiation, amplification and targeting of the local immune response. the etiology of a number of neurological disease, most notably, multiple sclerosis, still remains a matter of debate. the preferred hypotheses involve an autoimmune phenomenon in which cellular and/or humoral immune responses are directed against constituents of myelin. however, epidemiological data implicate some kind of acquired factor, most likely an infection, in the etiology of multiple sclerosis (waksman et al., ) . in recent years, evidence has accumulated that viral infections can result in the breakdown of immunological tolerance. thus both autoreactive b cells (haspel et al., a, b) and autoreactive t cells (pfizenmaier et al., ; komatsu et al., ) have been observed during viral infection. in this context it would be of interest to study the possible effect of neurotropic viral infection on the local immune response in the cns. flaviviruses are one group of viruses which are responsible for a number of neurological diseases (monath, ) . in this study, we address the effect of west nile virus (wnv) infection on astrocytes in vitro. west nile virus sarafend strain (wnv) was obtained from dr. i.d. marshall and grown either in vero cells (wnv-v) or in new-born mouse brain (wnv-b). the stocks were prepared and titrated as described (reed and muench, ; taylor and marshall, ) . influenza virus a/wsn was prepared by a standard method (yap and ada, ) . mice were bred under pathogen-free conditions at the animal breeding establishment of the john curtin school of medical research. the strains used were: cba/h (h- k), balb/c (h- d), b .t( r) (kqiqdd), b .aqr (kqlkdd). newborn mice were used at or days of age and adult female mice at - weeks. astrocytes were prepared in essentially the same way as has been described (mccarthy and de vellis, ) . briefly, -to -day-old cba/h mice were anesthetized at - °c for min. after careful removal of the meninges, the brains were broken down by pipetting and were then subjected to treatment with trypsin/dnase i solution ( . ~ trypsin and . ~ dnase i, w/v) for min, shaken occasionally. the action of trypsin was stopped by adding equal volumes of neural medium (dulbecco's minimum essential medium (dmem) supplemented with l-arginine mg/l, l-asparagine mg/l, folic acid mg/l, v-glucose g/l, and non-essential amino acids ~ v/v (flow laboratories, . the suspe-sion was centrifuged and the pellet was dispersed. these cells were cultures in cm nunclon plastic flasks at a density of /ml. at day , the cells were confluent and the medium was changed. one day later, the flasks were shaken for - h ( °c, rpm). the medium was then sucked off and the cultures were washed with puck's saline, trypsinized into single-cell suspensions and cultured again. the secondary cultures were tested by indirect immunofluorescence microscopy with rabbit anti-human glial fibrillary acidic protein (gfap) serum (dako, code a ) and shown to he more than % gfap positive. the secondary cultures were used in this study. after astrocytes were grown to confluency (approximately l s cell/well in linbro -well plastic iissue culture plates), the medium was sucked off and west nile viruses ( plaque-forming units (pfu)/cell of wnv-b and pfu/cell wnv-v, respectively) were added in /li and incubated at o c for i h. at different times after inoculation, cells were harvested and frozen at - ° c. free virus was obtained by freeze-thawing plus ultrasonication as described (taylor and marshall, ) . the protocol is essentially the same as described by de clercz ( ) . briefly, astrocytes were grown in -well tissue culture trays until confluency; the cultures were then exposed to poly i:c (sigma, /lg/ml) for h and washed with dmem medium times. fresh neural medium was added and the cultures were further incubated for h. the supernatants were harvested and ifn activity determined. ifn was assayed by inhibition of viral rna synthesis in l cells as described by morris et al. ( ) . the titer of ifn was defined as reciprocal of the final dilution that gave % inhibition of semliki forest virus rna synthesis. / of : dilution of supernatants from h wnv-b-infected astrocytes were incubated with an equal volume of medium or units of rabbit antiserum to mouse ifn (a +/]) (lee biomedical research, cat. no. ) at °c for h. the ifn activity of these supernatants was titered as described above. spleen cells from b ~t( r) mice (kqlqd d) were stimulated with those from b .aqr (kqlkd d) as described (king et al., ) for three passages. at the fourth passage, an enriched medium for t cell lines (dmem supplemented with x - m -mercaptoethanol, ~ fetal calf serum (fcs) and % concanavalin a (cona)-activated spleen cell supernatant (cs) prepared as described by sinickas et al ( a) ) was added together with allogeneic stimulators. generation of secondary cytotoxic t cells against influenza virus a/wsn and primary allo-reactive cytotoxic t cells has been described in detail (yap and ada, ; king et al., ) . the method for the l target system has been described (yap and ada, ) . wnv-v-infected or uninfected astrocytes were cultured in flat-bottomed tissue culture plates at °c for h. these cultures were then reinfected with influenza virus a/wsn ( eid~o/cell) and labeled with cr by adding /~ medium containing x eids /well influenza virus a/wsn and : dilution of na slcr and incuba~,a at °c for h. the rest of the assay was carried out in the same way as that for l targets. the proliferation of the h- ik-reactive t cell line was determined by [ h]methyl-thymidine (amersham) incorporation as has been described (sinickas et al., b) . both spleen cells and astrocytes were used as stimulators. after growth to confluency in -well tissue culture plates (approximately × cells/well), the astrocytes were irradiated with r from a e°co v-ray source. /~g/ml of indomethacin was added to the enriched medium of t cell lines and /tl of this medium per well was added together with x cells of an h- k-reactive t cell line. h later, . ;tci/well of the [ h]methyl-thymidine was added. after another h, the cultures were harvested onto glass fiber filter strips and the incorporation of label into dna was determined by scintillation counting. wnv-infected or mock-infected astrocytes grown to confluency in -well plates ( x / well) were irradiated with r and were used as stimulators of × t cells in . ml dmem medium per well supplemented with - m mercaptoethanol, #g/ml indomethacin and % fcs. h later the supernatants were collected and their interleukin activity was tested, as described by lafferty et al. ( ) . it is now known that this assay can measure interleukin- (il- ) and/or imerleukin- (il- ) activity (severinson et al., ) . moneclonal antibodies (mcabs) specific for d k, i-a k and i-a a antigens were obtained from the supernatants of cultures of hybridoma clones - - s, - . . . , and mk-d (oi et al., ; ozato et al., ; kappler et al., ) (atcc nos. hb- , t b- and hb- ), respectively, obtained from the american type culture collection. the supernatants were concentrated -fold on an american p membrane. in adoition, an mcab specific for k k (clone - . ) was purchased from becton-dickinson. rabbit anti-mouse immunoglobulin (ramig) was raised in a new zealand white rabbit, separated from whole serum using a protein a-sepharose column (pharmacia) and conjugated to fluorescein-isothiocyanate (fitc) by standard methods (( oding, ) . astrocvtes ~ ere infected with wnv or treated with recombinant ~-ifn ( u/ml) or poly i : c for h. s:,ngle-cell suspensions of astrocytes were obtained from the culture vessel by gentle trypsinization. the cells were counted and viability, as measured by trypan blue dye exclusion, was invariably > ~. all subsequent steps of the labeling procedure were carried out at ° c. samples of × l s astrocytes were incubated in /l of anti-mhc antibody for rain. after the first antibody incubation the cells were centrifuged through a bed of pl fcs, the supernatant removed, and the astrocytes resuspended in /~ of ramig-fitc for a further min at a dilution giving saturation labeling (determined by prior titration). following this incubation, the cells were centrifuged through an fcs bed and resuspended in # dmem for flow cytometry. fluorescence was measured using a facs iv (beckton-dicldnson) equipped with an argon ion laser set at the standard excitation wavelength of nm for fitc. emitted fluorescence between and nm was measured. × cells were analyzed from each labeled sample. in no experiment did any of the astrocyte sample populations show changes detectable by lowangle (cell size) or right-angle (membrane configuration) scatter analysis on the facs. thus we presume that differences in the mhc fluorescence distributions between astrocyte groups reflect a ~:hange in the cell surface mhc antigen concentration. astrocytes grown to confluency in -well tissue culture plates were infected with either pfu/cell of wnv-b or pfu/cell of wnv-v, and viral titers at different times after infection were determined. titers dropped about -fold within h (table ) . however, a clear increase in viral titers was observed by h. the titers reached a plateau at h after infection and remained at a similar level for at least week. further experiments revealed that viral antigens became detectable after h by immunofluorescence microscopy and by h - ~ of the astrocytes were expressing detectable viral antigen. the proportion of viral antigen-positive cells remained at a similar level for at least weeks. these data show that wnv can productively infect some cells in astrocyte populations. astrocyte cultures, either infected with wnv or treated with poly i:c, were tested for their ability to produce ifn. high titers of ifn were detected in the supernatant of astrocytes infected by wnv-b or wnv-v, or treated by poly i : c, all for h ( table ). the ifn titers were -fold . . . . . . . . . . . . • west nile virus prepared from mouse brain. b west nile virus prepared from verb cell line. lower in the supernatant of poly l:c-treated astrocytes than that of wnv-infected astrocytes. furthermore, the ifn produced in wnv-b-infected astrocytes can be neutralized by antibody specific for ifn (a + ~ ), hence the ifn produced is a-and/or p.ifn (fig. ) . the expression of class i mhc antigens on cba/h astrocytes was investigated by flow cytometry. the single-cell suspensions were stained with mcabs specific either for h- d k or h- k k. after the astrocytes were infected by wnv-v for h, the expression of h- k ~ as determined by peak fluorescence increased by more than -fold compared to mock-infected astrocytes (fig. a) . the binding of the mcab to the h- k k was specific as an isotype control mcab specific for h- -a d (hb- ) did not bind significantly to either normal or wnv-infected astrocytes. similar enhancement was observed in h- d k expression (fig. b) . as both wnv-b and wnv-v are potent ifn inducers (table ) , the question arose as to whether the elevation in mhc antigen expression was due *.o the ifn produced. to investigate this question a synthetic ifn inducer, poly i:c was used. as shown in fig. c , poly i : c treatment can significantly enhance the expression of class i mhc antigens, which admits the possibility that the enhanced expression after wnv infection is at least partly the result of virus-induced ifn. as class i mhc antigens are the major restric- in each case astrocytes were treated for h. virus-immune tc (table ). in contrast, astrocytes infected by influenza virus or wnv alone were not lysed by influenza virus-immune tc more than normal astrocytes. the activity of the tc was confirmed as *.hey lysed influenza virus-infected l (h- t) cells, but not mock-infected l cells. this result revealed that wnv infection enhances mhc-restricted virus-specific tc recognition on astrocytes. lysis of wnv-infected astrocytes by balb/c anti-cba/h alloreactive t cells was significantly higher than that of normal astrocytes while comparable to the lysis of y-ifn-treated astrocytes (fig. ) , hence wnv infection enhances ulloreactive tc recognition on astrocytes. the expression of cell surface cl~s ii mhc antigens on cba/h astrocytes was also determined by flow cytometry. astrocytes were labeled with h- i-ak-specifi¢ mcab tib ; control cells were treated with hb- , an mcab specific for h- -a d. normal cba/h (h- k) astrocytes express little or no detectable class ii mhc antigens, as their binding to tib (h- -ak-speeific) is not significantly higher than to hb- (h- i-a dspecific) (fig. a) . however, after the astrocytes were infected by wnv-b for h, a distinct increase in expression of h- -a k antigen was detected fig. b) , although the amount of h- i-a k expressed on wnv-infected astrocytes was not as , and astrocytes treated with "g-ifn (c). c: as for b, but normal astrocytes (a) and astrocytes treated with poly : c (b). d: as above but astroq,'tes infected by wnv-v. ast.~ocytes were subject to pre-treatment with wnv or ifn or poly i : c for h before labeling. much as that on y-ifn-treated astrocytes. the specificity of the labe~ng was ascertained in two ways: (a) hb- (anti-h- i-a °) mcab dad not bind wnv.infected cba/h (h- k) astrocytes (fig. a) ; (b) as hb- is of the igg a subclass, while tib~ is igg b, another igg b mcab specific for murine cd was tested and shown not to bind either wnv-infected or normal astrocytes (data not shown). it seems unlikely that material other than wnv in wnv-b (e.g. murine ifn) is responsible for the up-regulation of class ii mhc antigen, as wnv-v that was passaged times in veto cells (a permanent cell line originating from monkey kidney that does not secrete murine ifn) also consistently up-regulated class ii mhc antigen, though to a lesser extent than wnv-b in this particular experiment (fig. d) . furthermore, poly i :c, which "nduced ifn production and enhanced class i mhc antigen expression in astrocyte cultures (fig. c , table ) dad not induce class ii mhc ant/gen expression (fig. c) . the mechanism of the induction of class ii mhc antigen is at present unknown. an alloreactive b .t( r) anti-b .aqr t cell line specific for h- i k antigens was used to de-table termine if the wnv-induced expression of class ii mhc antigens on astrocytes can be recognised by t cells. the t cell line was induced to proliferate by spleen cells from mice bearing h- i k antigens, e.g. b .aqr and cba/h mice, but not by antologous b .t( r) spleen cells (table , expt. ). wnv-infected cba/h astrocytes also stimulated the proliferation of the h- ik-reactive t cell line (table , expt. ). the stimulation was comparable to cba/h spleen cells; mock-infected astrocytes had no stimulation effect. to test whether the induced stimulation effect of astrocytes was due to induced ¢lass ii mhc antigens on their surface, the h- i-ak-specific mcab tib was added. tib blocked the proliferation of the h- k-specific cell line stimulated by either b .aqr spleen cells or by wnv-v-infected cba/h astrocytes ( wnv-infected and uninfected astrocytes were also compared for their ability to stimulate il- and/or il- release from the h- ik-specific t cell line described above. wnv-infected cba/h astrocytes stimulated significant release of il- and/or l- , whereas uninfected cba/h astrocytes were, much poorer stimulators (fig. ) . the quantity of il- and/or il- released was about -fold higher with wnv-infected astrocytes than with normed astrocytes. specificity of the stimula- wnv-infected cba/h astrocytes stimulated significantly il- and/or il- release from the primary h- ik-reactive t cell population, while co!s the normal astrocytes induced essentially no il- and il. release (fig. ). since it was established that mhc antigens formed an important part of the antigenic struc- ture recognised by t lymphocytes (zinkernagel and doherty, ; schwartz, ) , there "has been great interest in the regulation of mhc antigen expression on potentjal antigen-presenting cells. it is well documented that different virus infections may either inhibit or enhance the expression of mhc antigens, notably, infection by adenovirus (f~i~lbo et al., ) , herpes simplex virus type (jennings et al, ) , ectromeiia virus (gardner et al., ) and measles virus (rager-zisman et al., ) can down-regulate class i mhc antigen expression of their host cells and may make the latter less susceptible to cytotoxic t cells. on the other hand, murine hepatitis virus (massa et al., ; suzumura et al., ), epstein-barr virus (mccune et al., , moloney murine leukemia virus (flyer et ai., ) and theiler's murine encephalomyelitis virus (rodriguez et al., ) can up-regulate class i and/or class ii mhc antigen expression. our results described here revealed that flavivirus infection can up-regulate the e:~pression of cell surface class i and class i i~,~hc antigens. it has been docuraented that interferon can up-regulate class ! and ii mhc antigen. all three classes of ifn l~own (a, t, ~,) have been reported to enhance ,class i mhc antigen expression, while only ~,-ifn can up-regulate the expression of class ii mhc ~mtigens ovong et al., ) . in our experiment, any treat~ent that can induce ifn production, e.,g. wi, v-b and wnv-v infection or poly i : c treatmen~,, can up-regulate class i mhc antigens; it is possible ~hat the up-regulation of class i mhc antigen expression by wnv infection is at least patti , the result of ifn produced after virus infection. in contrast, poly i:c treatment of astrocytes, which induces good ifn production and increases class i mhc antigen expression equal to or more than wnv infection, fails to induce class ii mhc antigen expression. this finding together with the data that ifn produced in wnv-b-infected astrocyte culture can be neutralized by anti-(a + fl)-ifn antibody strongly suggests that the induction of class ii mhc antigen by wnv does not depend on ifn produced. addition of antibodies that neutralize y-ifn to wnv-infected astrocytes culture would help to answer the question if free v-ifn was involved in class ii mhc antigen induction. however, the question as to what extent virus replication per se and induced interferon actually contribute to enhanced class i mhc expression is more problematic. some obviol:s approaches used by others (massa et al., ) , i.e. use of ultraviolet-inactivated virus and neutralizing mcab to the virus cannot provide definile information, because it is not known to what extent uv inactivation will inhibit viral protein and nucleic acid synthesis and the mechanism of viral neutralization is at present not well understood. furtherraore, addition of ifn antibody may not help to exclude the role of ifn due to the autocrine activity without the need of secretion (sanceau et al., ) . it seems unlikely that the binding of h- i-a k-speo_p._c mcab to wnv-infected but not normal astrocytes of cba/h(h- k) was due to the molecular mimicry by a viral product of an epitope on the i k molecule, because an mcab specific for h- -a d can bind to wnv-infected astrocytes from balb/c(h- d) mice but cannot bind to wnv-infected astrocytes from cba/h(h- k) mice; wnv can. repucate productively in astrocytes from both strains of mice (data not shown). a number of studies have shown that treatment which up-regulates the expression of cell surface class i mhc antigens increases the susceptibility of cells to lysis by appropriately sensitised tc cells, probably due to recognition by low-affinity clones that could not exhibit cytotoxic activity without the elevation of mhc antigen expression (shimoukevitz et al, ) . infection by viruses that down-regulate cell surface mhc antigen expression, on the other hand, results in reduced susceptibility of the infected cells to lysis by virus-specific cytotoxic t cells. thus it has l~een shown that herpes simplex virus type infection, which down-regulates cell surface mhc expression, reduces the susceptibility of an sv -and hsv -double-infectezl targets to lysis by sv specific tc cells, as compared to target cells infected with sv alone (jennings et al., ) . adenovirus type infection inhibits the cell surface expression of class i mhc antigen via the e protein. this phenomenon results in less efficient recognition by adenovirus-specific tc cells of target cells infected by this wild-type virus compared to target cells infected by an e deletion mutant which has no effect on host mhc antigen expression (miillbacher and bellett, personal communication) and could be responsible for the tumorigenicity of the wild-type virus (eager et al., ) . a moloney murine leukemia virus that increases cell surface class i mhc antigen expression was also reported to increase the susceptibility of host cells to lysis by alloreactive tc cells (flyer et al., ) . however, no attempt was made to address the effect of up-regulated mhc antigen expression on the recognition of mhc-restricted, antigen-specific t ells. in this paper, we compared wnv.infected astrocytes with uninfected controls for their ability to act as targets of influenza virus-specific tc cells and as stimulators for an ik-reactive t cell line. the results showed that wnv infection has significant effects on t cell recognition of astrocytes. 'ilms, after infection by influenza virus, normal astrocytes are poor targets of influenza virus-specific tc, while doubly infected (with wnv and influenza) astrocytes can be well recognized by influenza-specific tc. wnv-infected astrocytes from cba/h mice can also stimulate ik-reactive t cells significantly better than the normal astrocytes, in terms of both t cell proliferation and interleukin release. the latter function was also enhanced in populations of primary anti-h- i k t cells. these findings are particularly interesting because astrocytes are a large population of cells in the cns, presumably important both for the physiology and immunology of the cns (fontana e~ al., ) . elevated t cell recognition of astroeytes could have an important effect on the organism. these findings also raise interesting questions regarding self tolerance. if t cell tolerance to self antigens is determined quantitatively (blanden et al., ) the up-regulation of mhc antigens in the cns could possibly break t cell self tolerance in the cns. this may explain the autoimmune/virai etiology of some neurological diseases. quantitative considerations of t cell activation and self-tolerance immunohistological analysis of t lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis interferon induction by polynucleotides, modified polynucleotides, and polycarboxylates expression of histocompatibility antigens h-k. -d, and -l is reduced in adenovirus- -transformed mouse cells and is restored by interferon ~ ) astrocytes as antigen-presenting cells. i. induction of la antigen expression on astrocytes t cells via in-anune interferon and its effect on antigen presentation retrovirus induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic t lymphocytes the endothelium-astrocyte immune control system cell mediated cytotoxicity against ectromelia virus-infected target cells conjugation of antibodies with fluorochromes: modrficat!ous to the standard methods viral induced autoimmnnity: monoclonai antibb.lies that react with endocrine tissues multiple organ-re.~ctive monoclonal autoantibodies expression of la antigens by cultured estrocytes treated with y-interferon quantitative variation in la antigen expression plays a central role in immune regulation effect of herpes simplex virus type and on surface expression of class ! major histecompatibility complex antigens on infected cells antigen-inducible, h- -restricted interleukin- -producing t cell hybridomas lack of independent antigen and h- re. cognition relationship between surface h- concentration, size of different target cells and lysis by cytotoxic t cells clones of cytotoxic lymphocytes can recognize uninfected cells in a primary response against influenza virus immunohistochemical identification of t lyn~lphocytes in the central nervous system of patients with multiple selerosis and subacute sclerusilag panencephalitis an improved assay for interleukin- (lymphocyte growth factor) produced by mitogen activated lymphocytes theiler's virus-induced demyelination: prevention by immunosuppression preparation of separate astre~ljial and ollgodendroglial cell cultures from rat cerebral tissue enhanced representation of hl-a antigens on human lymphocytes after mitogenesis induced by phytohemagglutinin of epstein-barr virus antigen presentation in brain: mhc induction on brain end~ thdium and astrocytes compared viral particles induce la antigen expression on astrocytes pathobiology of flaviviruses infection of cultured mnrine brain cells by semliki forest virus: effect of interferon-o.~ on viral replication, viral antigen display, major histocompafibility complex antigen display and lysis by cytotoxie t lymphocytes properties of mo~clonal antibodies to mouse ig allotypes, h- and ia antigens hybridoma cell lines secreting monoclonal antibodies to mouse h- and la antigens adenoviruses of subgenera b, c, d, and e modulate cell-surface expression of major histccompatibility complex class i antigens temporary pr~-~,ence of self-reactive cytotoxic t iymphocytes during murine lymphocytic choriomeningitis decreased expression of h- antigens following acute measles virus infection a simple method of estimating fifty percent end points hlunone response geue products (la antigens) on gliai and endothelial cells in virus induced demyelination intracellular v-interferon triggers an antiviral state in transformed l cells immune response (it) gene of the murine major histocompatibility complex interlenkin (iggi induction factor): a multifunctional lymphokin acting also on t cells clonal analysis of cytolytic t lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for ly- / function l~e cytotoxic response to murine cytomegalovirus. i. parameters in vivo the cytotoxic response to murine cytomegalovirus. ii. in vitro requirement for generation of cytotoxlc t cells coronavirus infection induces h. antigen expression on oligodendrocytes and astrocytes adaption studies with ross river virus: laboratory mice and cell cultures multiple sclerosis: the blood-braln barrier and the measurement of de novo central nervous system of ig synthesis viral etiology of multiple sclerosis: where does the truth fie'? t lyraphocyte auto-inununity in experimental autoimmune encephalomyelitis dist~bution and quantitation of hla-abc and dr (ia) antigens on human kidney and other tissues inducible expression of h- and la antigens on brain cells interferon-v induces the expression of h- and la antigen on brain cells cytotoxic t cells specific for influenza virus infected target cells mhc-restricted cytotoxic t cells: studies on the role of polymorphic major transplantation antigens determining t cell restrictionspecificity, function and responsiveness we thank boehringer ingelheim for the kind gift of recombinant -ifn and barb geary for typing the manuscript. part of the work was carried out when one of us (y.l.) was a recipient of a world health organisation fellowship. key: cord- - z yeb authors: nan title: abstracts des . internistenkongresses date: journal: med klin (munich) doi: . /s - - -y sha: doc_id: cord_uid: z yeb nan so far, platelets were regarded static cells without further movement once they were adherent to fibrinogen or other extracellular matrices. migration of adherent platelets has not been discussed by the scientific audience so far. methods and results: -dimensional gel-electrophoresis of platelets revealed that fibrinogen-adherent platelets synthesize or modify multiple proteins. most of these proteins include cytoskeletal proteins and proteins of the migration apparatus. we concluded that adherent platelets might be able to migrate on the surface of an extracellular matrix or endothelium. to demonstrate platelet migration we exposed fibrinogen-coated coverslips with adherent platelets to continuous flow under high shear rates as in the arterial vascular system. all non-adherent platelets were removed from the experiment. under continuous flow we observed shear stress induced mechanotaxis of human platelets some of which started to actively migrate along the direction of flow. migrating platelets were still tightly adherent to the extracellular matrix and would have otherwise been swept away by the continuous flow. we found, that these migrating platelets underwent cytoskeletal rearrangement and formed pseudopodia. to support that our observation is an active cellular process, we blocked cellular signal transduction on pi -kinase level with ly /wortmannin. after exposure to ly /wortmannin, we could no longer observe platelet migration under flow conditions. we subsequently examined chemokine-directed migration of platelets inlcuding sdf , rantes and ena- . we created a chemokine source constantly releasing chemokines and thereby generating a gradient. as for the sdf (stromal cell derived factor- ; μg/ml) source we observed that about % of platelets adherent around this artificial source started to move towards the sdf- source. platelets were actively migrating for several hours in this in-vitro setting. during migration platelets polarized and formed pseudopodia with modification of several proteins of the migratory aparatus under sdf exposure. when the sdf- source was removed, platelets migrated without a specific direction. also with blockade of the cxcr receptor, the specific receptor for sdf- , directed platelet migration could no longer be observed. we are currently performing inhibitory experiments on different levels of cell signalling to characterize the migratory apparatus of platelets. the role of other cytokines associated with cell migration including rantes and ena- will be investigated. we apply immunofluorescence, western-blot, -dimensional gel electrophoresis and mass spectrometry to differentiate the migratory apparatus of migrating cells. conclusion: platelets that adhere to fibrinogen are not irreversibly fixed on matrix surface. they are able to move into a specific direction guided by shear stress induced mechanotaxis or directed along a chemokine gradient . by these mechanisms, platelets might be able to migrate into a platelet clot to further stabilize the hemostatic plug. moreover, platelet migration may be a physiological mechanism to precisely cover denuded vascular lesions and further play a role in the recruitment of platelets to the site of vascular lesions or inflammation. influence of genetic variation in four obesity candidate genes on body mass index and metabolism j. aberle , p. peitsmeyer , n. a. beck , f. u. beil . medizinische klinik, hamburg objective: it is believed that is in most cases obesity derives from an obesogenic environment on the basis of a genetic predisposition. a large number of genes and genetic variations have been associated with obesity. research methods and procedures: in a cohort of caucasion men and women with a body mass index (bmi) of kg/m² or more we investigated four candidate genes at baseline and following a months low fat caloric restriction diet by polymerase chain restriction and restriction digestion: the g/a variant of the cannabinoid receptor (cb ), the p t polymorphism in fatty acid amide hydrolase (faah), the l p variation in neuropeptide y (npy) and the - c>g polymorphism in resistin. results: comparing groups according to genotype for each gene separately revealed a significantly greater reduction of body weight and bmi in carriers of at least on mutant allele in cb as well as a greater reduction in triglycerides in resistin mutant allele carriers. we furthermore investigated gene-to-gene interaction: adding faah mutant alleles to carriers of at least one mutant allele in cb , or resistin significantly influenced triglycerides, total-or ldl-cholesterol levels respectively. combination of npy n-allele with either faah t-allele or cb a-allele as well resulted in an increase of triglyceride levels. discussion: our results support the hypothesis of obesity as a complex genetic disorder with several genes involved in the development of an obese phenotype and obesity related comorbidities. rt-pcr, die konzentrationen sezernierter proteine mittels immunoassay quantifiziert. ergebnisse: die adiponektinstimulation von rasf resultierte in veränderungen der genexpression sowohl auf mrna-als auch proteinebene. unter diesen regulierten genen waren proinflammatorische zytokine ( schlussfolgerung: adiponektin nimmt einfluss auf rasf, indem u.a. die expression und sekretion von proinflammatorischen und prodestruktiven/matrixabbauenden mediatoren modifiziert werden. diese erkenntnisse könnten dabei helfen, die pathophysiologischen mechanismen in der ra besser zu verstehen und potentielle zielmoleküle zur therapeutischen intervention zu identifizieren. in dieser globalen studie wurde der nutzen einer verlängerten thromboseprophylaxe nach totalem hüftgelenkersatz untersucht. es wurde die kurzzeit-thromboseprophylaxe mit enoxaparin mit einer verlängerten thromboseprophylaxe von bis zu wochen mit einem neuen, oralen, direkten faktor-xa- . bei studienbeginn und nach tagen wurde die wundfläche vermessen und eine mm große biopsie aus der wunde entnommen, aus der mit hilfe der real-time pcr (taqman ® ) die mmp-mrna bestimmt wurde. die wundflüssigkeit wurde mit hilfe der gaze täglich gesammelt und mittels zymographie aufgearbeitet. ergebnisse: die expression von mmp- mrna zeigte keine veränderungen in beiden gruppen. die mmp- und - -konzentrationen in der wundflüssigkeit unterschieden sich nicht signifikant in beiden gruppen zu beginn der studie. allerdings wurde eine signifikante reduktion der aktiven form von mmp- zwischen dem . und . behandlungstag in der pi-gruppe verzeichnet (p= , ). die ergebnisse von mmp- pro zeigten in der therapiegruppe ebenfalls eine sinkende tendenz ( % reduktion zu % anstieg in der kontrollgruppe) erreichten aber nicht das signifikanzniveau. außerdem zeigte sich eine reduktion der wundgröße in der pi-gruppe von % im vergleich zur kontrollgruppe (p= , ). schlussfolgerung: es traten keine veränderung der mmp-mrna-expression unter pi-therapie auf. allerdings fanden wir eine signifikante reduktion der biologisch aktiven form von mmp- in der wundflüssigkeit. die lokale behandlung mit einem pi beeinflusst also primär nicht die expression von proteasen, sondern scheint die wundflüssigkeit so zu modulieren, dass eine geringere aktivität von mmps auf der wundoberfläche zu finden ist und durch eine geringere proteolytische aktivität eine verbesserte wundheilung eintritt. einleitung einer antiretroviralen therapie in zwei ländlichen regionen in rwanda (n= ), fibrose in mehreren regionen (n= ). nur die gruppe, die baseline keine fibrose aufwies zeigte unter dreijähriger ert eine normalisierung der wanddicke (von , ± , auf , ± , mm; p< , ) und eine weitgehende funktionelle normalisierung der regionalen herzfunktion (strain rate radial: von , ± , s- auf , ± , s- , p= , ; vergleichskollektiv , ± , s- ). die anderen beiden gruppen zeigten zwar in hinblick auf die wandstärken einen positiven effekt, hinsichtlich der herzfunktion konnten sie jedoch bei bereits deutlich erniedrigten funktionswerten zum baseline-zeitpunkt lediglich stabilisiert werden (reduktion der wandstärke nach jahren ert: gruppe wenig fibrose= mm; gruppe viel fibrose= mm) schlussfolgerung: die enzymersatztherapie ist eine effektive langzeitbehandlung bei patienten mit fabry kardiomyopathie. hierbei fällt insbesondere auf, dass eine rechtzeitige therapie, bevor sich eine myokardiale fibrose entwickelt hat, indiziert ist. klinikgestützte poststationäre mobile gesundheitsservices; "hospital-to home®" -im poststationären versorgungsmanagement chirurgischer patienten adult-onset still´s disease (aosd) represents a rare systemic inflammatory disorder with an estimated incidence between and cases per million inhabitants in western europe. due to this small incidence only few data on diagnosis and therapy are available. the etiology of aosd remains elusive. currently the hypothesis of an exacerbated immune response based on dysregulation of cytokine mediated signalling cascades is favoured among scientists. according to resent results the proinflammatoy cytokine interleukin (il- ) seems to play a central role in pathogenesis of the more common juvenile as well as adult-onset still´s disease. in view of that, we report about a fast and sustained response of a year old woman with aosd under first-line treatment with the il- receptor antagonist anakinra. the fast mode of action compared to conventional dmards and tnf inhibitors paired with an acceptable safety profile had prompted us to initiate a primary il- receptor blocking therapy. the observed rapid clinical response (cessation of fever within hours) and normalization of highly elevated acute phase laboratory parameters within a few days encourage a first line use of anakinra in aosd-patients. longer observation periods and larger series of patients will be needed to answer open questions like optimal length of anakinra-treatment, long-term outcome and complications. cardiovascular complications in patients with diabetes mellitus are associated with increased oxidative stress in vascular tissue. a key event for no synthase (nos) uncoupling involves downregulation of bh synthesizing enzyme gtpcyclohydrolase i (gch-i). we here investigated the effects of at -receptor block ade with chronic telmisartan therapy on gch-i expression, nos uncoupling and endothelial function in streptozotocin (stz)-induced diabetes mellitus (type i). diabetes was induced by single i.v. injection of stz ( mg/kg, weeks) in male wistar rats ( g). telmisartan ( mg/kg/d) therapy did not improve blood glucose and body weight. aorta from diabetic animals had vascular dysfunction as revealed by isometric tension studies. ros produced by nadph oxidase, mitochondria and xanthine oxidase were increased in the diabetic group. nadph oxidase subunits mrna and protein expression was increased in response to stz. importantly, the expression of the gch-i and enos activating ser phosphorylation was decreased by stz. therapy with telmisartan normalized all these parameters almost completely. the results of the present studies demonstrate for the first time that at receptor treatment of diabetic animals with telmisartan is able to prevent downregulation of the important bh synthesizing enzyme gch-i, which prevents enos uncoupling and an activation of cardiovascular superoxide sources. der einfluss der sauren sphingomyelinase auf die expression von matrix-metalloproteinase- in intestinalen epithelzellen und fibroblasten background: the calcineurin (cn)/nf-at signaling cascade takes a crucial role during t-cell activation and the development of myocardial hypertrophy. in addition to nfat, the phosphatase cn is also translocated into the nucleus. for nuclear import, specific carrier proteins, so-called importins, recognize nuclear localization sequences (nlss) in the sequence of the respective target proteins. we developed a synthetic import blocking peptide (ibp) which is identical to the nls sequence of cn. ibp prevented the interaction between cn and its importin and subsequently inhibited nuclear import of cn. methods and results: according to immunohistochemical studies both the subcellular localization of cn, the inhibitory effect of ibp and also the specificity of the blocking peptide were demonstrated. while the inhibitory effect within the t-cell activation is analyzed by [ h]-thymidine incorporation ( ± , vs. ± [%]), the impact on angiotensin ii stimulated cardiomyocytes was examined on the transcriptional (nfat reporter assay; ± vs. ± [%]) and on the translational level ([ h]-leucine incorporation; ± vs. ± [%]). by using ibp, the expression of hypertrophy markers, e.g. myosin heavy chain β, was suppressed, the cell size was not increased. interestingly, phosphatase activity was not effected (cna assay, biomol). the translocation of diverse transcription factors, like nfat, nfκb or erk , was not effected, which illustrated the specifity of ibp. the results could be confirmed in a pilot animal study for heart failure. discussion: ibp prevents the nuclear translocation of calcineurin both in t-cells and in cardiomyocytes. the inhibitory activity is specific for calcineurin und does not affect the cn phosphatase activity. ibp has also an effect in vivo, where it has been demonstrated to suppress the development of myocardial hypertrophy in an animal model. in summary, ibp suppresses t-cell activation und prevents the development of myocardial hypertrophy. proteasome inhibitors suppress angiogenesis by altering endothelial vegfr- expression the ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells that controls a wide range of cellular regulatory proteins, including transcription factors and cell cycle regulatory proteins. recent evidence established the importance of the proteasome also in tumor development, showing anti-tumor and anti-angiogenic actions by selective inhibitors in vivo. as signaling via the vegfr pathway is critical for angiogenic responses to occur, we explored whether anti-angiogenic effects via proteasome inhibition were mediated in part through diminished endothelial vegfr expression. our studies show that different proteasome inhibitors (mg , alln and lactacystin) all blocked vegfr expression in a time-and concentration-dependent manner, which was paralleled by respective inhibition of capillary like structure formation and endothelial cell migration. in contrast, tie- or vegfr- expression was not significantly affected by proteasome inhibitor treatment. the suppressive effects on vegfr expression were neither conveyed by increased shedding nor by shortened protein half-life, suggesting that transcriptional mechanisms accounted for the observed effects. in line with this conclusion, proteasome inhibition significantly suppressed vegfr mrna accumulation. in addition, inhibitor treatment considerably decreased transcriptional activity of '-deletional vegfr promoter gene constructs. proteasome inhibition-mediated repression was conveyed by a gc-rich region, harboring one consensus sp binding site. subsequent emsa analyses demonstrated diminished constitutive sp -dependent dna binding in response to proteasome inhibition. hence, vegfr- expression may constitute a critical molecular target of proteasome inhibitors that mediates their anti-angiogenic effects in vivo. loss of ifn-γ inducibility of the mhc class ii antigen processing pathway in head and neck cancer: evidence for posttranscriptional as well as epigenetic regulation abnormalities of the major histocompatibility complex (mhc) antigens by tumor cells impair the cellular immune response and promote tumor evasion from immune surveillance. so far, studies analyzing the mhc class ii expression levels in hnc are limited. therefore, we investigated the constitutive and interferon (ifn)-γ-regulated expression profiles of mhc class ii apm in various hnc cell lines and also analyzed the mhc class ii expression in hnc lesions. hnc cell lines analyzed in vitro lacked constitutive mhc class ii surface expression. despite the ifn-γ-mediated induction at the mrna level, six out of ten cell lines did not show any relevant mhc class ii surface expression. this phenomenon might be attributed to a posttranscriptional dysregulation of specific mhc class ii apm components. one cell line displayed a loss of ifn-γ induced ciita-expression that corresponded to impaired mhc class ii surface expression, which could be linked to hypermethylation of the ifn-γ-responsive ciita-promoter iv. in vivo, immunohistochemistry analyses of patients revealed that about % of hnc tissues exhibit a negative or only marginally positive staining, whereas % displayed a heterogenous or highly positive mhc class ii surface expression. there was no statistical correlation between tumor differentiation and the mhc class ii expression in hnc lesions. taken together these results suggest a high frequency of mhc class ii abnormalities in hnc in vitro and in vivo, which could occur at different steps of the antigen processing pathway. this information may have a significant impact on practical and clinical aspects of tumor immunotherapeutic strategies. ciita versus ifn-γ induced mhc class ii expression in head and neck cancer cells effective and safe reduction of blood pressue by the combination of amlodipine /valsartan mg in patients with hypertension and metabolic risk factors not controlled by amlodipine mg or felodipine mga subanalysis of the express-m trial introduction: atrial fibrillation (af) is the most common cardiac arrhythmia and frequently occurs in patients with coronary heart disease. prophylactic oral anticoagulation (oac) is indicated in patients with af and at intermediate or high risk for thrombembolic events. however, an anticoagulant regimen has not been standardized for patients with af after coronary stent implantation, which may require additional dual antiplatelet therapy. methods: we identified retrospectively patients with af who underwent pci in our department from - . we compared baseline variables and incidence of a combined endpoint (stroke, mi, death, bleeding) in patients receiving clopidogrel and aspirin (n= , group ), versus patients receiving the combination of clopidogrel and aspirin with low molecular weight heparin (lmwh) (n= , group ), versus patients receiving the combination of clopidogrel and aspirin with oac (n= , group ) at discharge. results: the groups were not relevantly different regarding classical risk factors, however, patients discharged with triple therapy including oac seemed to be at ( ) higher risk: patients in group had decreased left ventricular ejection fraction and increased inflammatory state as measured by plasma fibrinogen. in a median follow up of , years (range from months to . years) severe bleeding events, myocardial infarctions, strokes, and cardiovascular deaths occurred. kaplan meyer analyses showed that the occurrence of severe events was not significantly different (p= . ). conclusion: in our analyses no treatment regimen seemed to be superior. however, in the follow-up period incidence of myocardial infarctions and strokes were high in patients with solely clopidogrel and aspirin treatment, and incidence of cardiovascular deaths was high in patients receiving the combination of clopidogrel and aspirin with lmwh. the number of patients receiving the combination of clopidogrel and aspirin with oac (triple therapy) was low, however, only one severe event was found in the follow-up period in this group. the current data underline the importance for prospective trials investigating the optimal anticoagulant-/antiplatelet treatment in patients with af after coronary stent implantation. valvuloplastie der kalzifizierten aortenstenose: neue erfolge durch weiterentwicklung der herkömmlichen methode background: we have shown that assessment of cardiac output (co) by the inert gas rebreathing method provides reliable measurements as compared to magnetic resonance tomography (mrt) in patients with sinus rhythm. during atrial fibrillation, however, co measurement based on the determination of stroke volume may be impaired by heart rate variation, potentially leading to divergent results. the present study therefore aimed to compare co measurements by inert gas rebreathing to mrt in patients with atrial fibrillation. methods: in a prospective case-control study, we included consecutive patients with atrial fibrillation undergoing cardiac mrt, and control patients pair-matched for gender, co by mrt, height and weight from a collective of patients with sinus rhythm undergoing mrt. the co of reclining patients was determined by inert gas rebreathing with dinitric oxide and sulfur hexafluoride (innocor, innovision, odense dk) immediately before or after the mrt. the data determined by mrt for co served as reference value comparing the methods by means of bland-altman analysis. the study collective covered a wide range of patients ( to years, median , men in the atrial fibrillation group vs. to , median in the sinus rhythm group). bland-altman analysis showed a good correspondence of the two methods for co with an average deviation of . ± . l/min in the atrial fibrillation group vs. . ± . l/min in the sinus rhythm group. no statistically significant difference could be found between the two groups (p = . , mann-whitney-test). conclusion: co measurements by mrt and by the rebreathing method with dinitric oxide and sulfur hexafluoride provide closely related results even in the presence of atrial fibrillation. the rebreathing method therefore allows an easy and reliable non-invasive determination of the co in this patient collective. the importance of the method for diagnosing and treating cardiac diseases remains to be assessed by further studies. when analyzing the plasma lipid profile for each polymorphism, we found the following differences in subjects with gallstones: significantly higher triglyceride and cholesterol levels in carriers of the common abcb allele and significantly lower hdl-cholesterol concentrations in carriers of the common rs allele. however, differences were also detected in controls with regard to cholesterol and total hdl-cholesterol concentrations. our results do not support a link between common abcb and abcb polymorphisms, lithogenic dyslipidemia and gallstone risk. evaluation der prävalenz schlafbezogener atemstörungen bei patienten mit vorhofflimmern und unauffälliger linksventrikulärer pumpfunktion mittels kardiorespiratorischer polygraphie hintergrund: in erregbaren herzzellen sind spannungsgesteuerte natriumkanäle für die auslösung und weiterleitung von aktionspotenzialen verantwortlich. natriumkanäle bestehen aus einer die pore bildenden α-untereinheit und zusätzlichen β-untereinheiten. verschiedene isoformen der α-untereinheiten haben ganz bestimmte entwicklungs-und lokalisationsmuster im herzmuskel, und sie besitzen spezifische pharmakologische und funktionelle eigenschaften. das kugelfischgift, tetrodotoxin (ttx), ein spezifischer natriumkanalblocker, inhibiert konzentrationsabhängig verschiedene α-isoformen. die kardiale isoform nav . wird durch mikromolekulare konzentrationen blockiert (ttxresistent), während neuronale ttx-sensitive isoformen bereits durch konzentrationen im nanomolaren bereich inhibiert werden. die α-untereinheiten sind mit zusätzlichen β-untereinheiten assoziiert. methoden und resultate: humanes vorhofmyokard wurde mittels der patchclamp-technik, kontraktilitätsmessungen, immunhistochemie und western blot untersucht. wir konnten zeigen, dass nicht nur kardiale ttx-resistente (ttxr) natriumkanäle am menschlichen herzen vorkommen, sondern ebenfalls neuronale ttx-sensitive (ttxs) isoformen exprimiert werden. ttxs-natriumkanäle tragen zu % zum gesamtnatriumstrom bei, während die restlichen % des stroms von ttxr-kanälen übernommen werden. funktionelle einschränkungen durch die blockade von neuronalen natriumkanälen konnten in kontraktilitätsmessungen ebenfalls nachgewiesen werden. immunhistochemische experimente bestätigten isoformenspezifische lokalisation der α-untereinheiten nav . bis . sowie aller bekannten β-untereinheiten ( - ). schlussfolgerung: im humanen herzen kommen neben kardialen auch neuronale natriumkanäle vor. sie tragen funktionell % zum gesamtnatriumstrom im menschlichen herzen bei und haben ebenfalls einen einfluss auf die kontraktilität. p /p -mapk-aktivierung in hypokontraktilen gefäßen zirrhotischer ratten: geänderte mechanismen der aktivierung jedoch gleichzeitig die g-protein abhängige aktivierung der p /p mitogenactivated protein kinase (mapk). die g-protein abhängige p /p aktivierung durch den angiotensin-ii typ rezeptor (at r) erfolgt dagegen über protein-kinase c. methoden: leberzirrhose bei ratten wurde durch gallengangsligatur (bdl) induziert. isolierte aorten wurden in vitro mit bzw. ohne angiotensin-ii und verschiedenen inhibitoren inkubiert. protein-expressionen und -phosphorylierungen wurden über western-blot analyse untersucht. die interaktion zwischen dem at r und b-arrestin wurde in co-immunopräzipitations-experimenten untersucht. ergebnisse: trotz ausbleibender angiotensin-ii induzierter aktivierung der g-protein abhängigen kontraktilen signalwege in aorten von bdl ratten kommt es in aorten zirrhotischer und nicht-zirrhotischer ratten zu einer angiotensin-ii stimulierten aktivierung der p /p mapk. diese angiotensin-ii stimulierte erk-aktivierung ist in aorten von bdl ratten, nicht jedoch der nicht-zirrhotischen kontrollen, resistent gegenüber hemmung der protein-kinase c und a. gleichzeitig führte die stimulation mit angiotensin-ii zur bindung von b-arrestin an den at r, die bei bdl ratten verstärkt ist. diskussion: bei leberzirrhose kommt es in extrahepatischen gefäßen zu Änderungen in der kopplung von vasokonstriktor-rezeptoren an ihre signalwege. anstatt g-protein abhängiger signalwege unter normalen umständen (z.b. kontraktions-kaskaden) werden bei leberzirrhose b-arrestin -abhängige signalwege aktiviert. the prediabetic and diabetic in vivo modification of circulating low density lipoproteins decreases their potential to stimulate adrenal steroidogenesis context: biochemical modification of low density lipoprotein (ldl) has been implicated in the pathogenesis of impaired glucose tolerance (igt) and type diabetes mellitus (dm ) and its related micro-and macrovascular disease. objective: since ldl serves as a major cholesterol source for adrenal steroidogenesis we investigated whether oxidative and glycoxidative modifications of ldl isolated from igt and dm subjects influence aldosterone and cortisol release from human adrenocortical cells. in a cross-sectional study ldl were isolated from subjects with normal glucose tolerance (ngt), subjects with igt, and patients with dm . individual oxidation and glycoxidation characteristics of ldl apolipoprotein b- were assessed by gas chromatography-mass spectrometry (gc-ms) analysis. human adrenocortical cells (nci-h r) were incubated for h with μg/ml of isolated ldl and after removal of supernatants the cells were further stimulated for next h with angiotensin ii. aldosterone and cortisol release were then quantified in the supernatants after h and h, respectively. results: circulating ldl from igt and dm subjects were substantially more oxidized and glycoxidized than the ldl samples from ngt subjects. each of the five measured oxidation/glycoxidation markers was significantly positively associated with glycemic control, measured as hba c. ldl stimulated aldosterone and cortisol release from adrenocortical cells. however, hormone secretion was inversely related to the degree of ldl modification. conclusion: prediabetic and diabetic biochemical ldl modifications may promote impaired adrenocortical aldosterone and cortisol synthesis. die modulation der interaktion von nephrin und β-arrestin durch protein-kinase c alpha steuert die nephrin-vermittelte endozytose und signaltransduktion zielsetzung: hepatische sternzellen (hscs) sind die primären fibrogenen zellen in der fibrotischen leber. selektive apoptose von hscs stellt einen neuen antifibrotischen therapieansatz dar. wir haben kürzlich gezeigt, dass das endocannabinoid -arachidonoylglycerol ( -ag) die proliferation von hscs blockieren sowie apoptose induzieren kann. hepatozyten sind jedoch gegenüber diesen mechanismen resistent. der exakte molekulare mechanismus dieser selektiven zelltod-induktion in hscs ist noch nicht geklärt. ziel dieser arbeit war es, eine mögliche rolle von cox- im differentiellen zelltod-verhalten primärer hscs und hepatozyten durch -ag-metabolisierung in pro-apoptotischen prostaglandin-d -glycerolester (pgd -ge) zu überprüfen. methoden: primäre hepatozyten und hscs wurden durch collagenaseperfusion aus lebern gesunder ratten isoliert. -ag-oder pgd -ge-induzierte apoptose wurde mittels ldh-assay und western blot für caspase -und parp-cleavage analysiert. bildung von reaktiven sauerstoffspezies (ros) wurde durch dcfda-fluoreszenz ermittelt. cox- -expression wurde mittels western blot bestimmt. ergebnisse: hscs zeigten eine signifikante cox- -proteinexpression, wohingegen hepatocyten keine wesentliche cox- -expression aufwiesen. behandlung von primären in kultur aktivierten hscs mit -ag induzierte einen dosisabhängigen zelltod ( % nach h bei μm), begleitet von parp-und caspase -cleavage. pgd -ge induzierte in primären hscs ebenfalls dosisabhän-gig apoptose. dagegen verursachte -ag in primären hepatozyten sogar bis zu konzentrationen von μm keinen zelltod, pgd -ge jedoch führte ab konzentrationen von μm zum zelltod. -ag und pgd -ge führten zur bildung von ros in hscs, was auf gemeinsame ros-abhängige apoptose-signalwege hinweist. schlussfolgerung: hscs und hepatozyten weisen einen signifikanten unterschied in der expression von cox- auf, was eine unterschiedliche metabolisierung des endocannabinoids -ag bedingt. die produktion von pgd -ge durch cox- in hscs trägt möglicherweise zur disparaten empfindlichkeit von hepatozyten und hscs gegenüber -ag-induzierter apoptose bei. die rolle des hypoxie-induzierbaren-faktors a in der strahlentherapieresistenz von humanen adenokarzinomzellen der lunge (a ) zielsetzung: hypoxie hat einen entscheidenden einfluss auf die chemo-und strahlentherapieresistenz von soliden tumoren und somit auf die prognose von tumorerkrankungen. die hypoxie-induzierbaren-faktoren (hif- a und hif- a) sind transkriptionsfaktoren, die über spezifische zielgenaktivierung adaptive prozesse in den hypoxischen tumorarealen steuern. in dieser studie wurde die strahlensensibilität der adenokarzinomzelllinie der lunge (a ) unter dem einfluss von hif- und hif- untersucht. methoden: im ersten schritt wurde die auswirkung von bestrahlung gy ( x, x, x) auf die a zellen untersucht (zellzählung, kolonie-assay, immunzytofluoreszenz: caspase- , kerngrößen, multinuklei). unter normoxischen und hypoxischen bedingungen wurde die hif- a und hif- a expression mittels western blot und pcr mit und ohne radiatio untersucht. die strahlentherapieresistenz unter inhibition von hif (sirna) wurde in vitro (kolonie-assay, zellzählung) und in vivo (tumor growth delay assay) untersucht. ergebnisse: bestrahlung mit gy führte zu einer reduktion der proliferation von a zellen ohne beeinflussung der apoptose. gleichzeitig änderte sich die kernmorphologie mit einem anstieg der kerngröße und vermehrten multinuklei unter fraktionierter bestrahlung. dies konnte auf einen anstieg der "seneszenten" zellen zurückgeführt werden. ein direkter einfluss einmaliger radiatio auf die expression von hif- oder hif- wurde nicht beobachtet. allerdings konnte über die inhibition von hif- a mittels sirna eine erhöhung der strahlensensibilität in den a zellen in vitro erreicht werden. dies wurde in vivo mittels tumor growth delay assay bestätigt. schlussfolgerung: die aktivierung von hif- a in hypoxischen tumorarealen hat einen entscheidenden einfluss auf die sensibilität von a zellen gegenüber bestrahlung. conditional overexpression of neuronal nitric oxide synthase is cardioprotective in ischemia-reperfusion introduction: we previously demonstrated that conditional overexpression of the neuronal nitric oxide synthase (nnos, nos ) inhibited l-type ca + -channels. we now hypothesize that nnos overexpression has an impact on myocardial contractility and acts cardioprotective after ischemia-reperfusion. we assessed cardiac function in the newly established transgenic mouse model with conditional, myocardial nnos overexpression. nos-activity ( ± . vs. ± μm/sec, n= , p< . ) was significantly enhanced after nnos overexpression. co-immunoprecipitation experiments indicated interaction of nnos with sr ca + atpase and additionally with l-type ca + -channels in nnos overexpressing animals. ica,l (reduction of ± rel.%, n= , p< . ) as well as intracellular ca + -transients and fractional shortening in cardiomyocytes were clearly impaired in nnos overexpressing mice ( . ± . f/f vs. . ± . f/f , n= , p< . and . ± . % vs. . ± . %, n= , p< . ). in vivo examinations of the nnos overexpressing mice showed a decrease of +dp/dtmax (reduction for ± %, n= , p< . ) as well as a reduced ejection fraction ( ± % vs. ± %, n= , p< . ). ischemia-reperfusion experiments showed a cardioprotective effect of nnos overexpression ( min post-ischemia, lvdp ± in non-induced animals vs. ± mmhg in nnos overexpressing animals, n= , p< . ). discussion: in summary, we demonstrated that under basline conditions, conditional transgenic overexpression of nnos resulted in a mild reduction of myocardial contractility, mainly due to inhibition of the l-type ca + -channel. in contrast, under pathophysiological conditions (i.e. ischemia-reperfusion) nnos overexpression acts cardioprotective. these effects might be caused by a reduction of myocardial ca + -overload after reperfusion. hintergrund und ziele: die pathogenese der erhöhten apoptose in der hepatitis c virus (hcv)-infizierten leber ist weitestgehend unbekannt. für zahlreiche hcv proteine ist eine modulation von zellulären apoptosemechanismen beschrieben worden. trail, der tnf-related apoptosis-inducing ligand, wurde kürzlich als zytotoxisch für hcv-infizierte hepatozyten beschrieben. um die zugrunde liegenden molekularen mechanismen einer potentiellen rolle von trail bei der hcv-induzierten hepatitis und der viralen clearance zu studieren, untersuchten wir die effekte der hcv replikation auf die trail-induzierte apoptose in humanen hepatomazellen. methoden: huh . zellen wurden mit dem in zellkultur infektiösen hcv stamm jfh- sowie den mutanten jfh- /Δe e , jfh- /gnd und sgr-jfh- hcv rna transfiziert bzw. infiziert. apoptose wurde durch bestimmung von parp, caspase- und - spaltprodukten sowie mittels tunel-reaktion untersucht. ergebnisse: transfektion von huh . zellen mit replikations-kompetenter jfh- , nicht jedoch mit replikations-defizienter jfh- /gnd rna, führte zu einer apoptose von huh . zellen, nachgewiesen mittels parp spaltung und positiver tunel reaktion. die verstärkte trail apoptose war in mit jfh- /Δe e und sgr-jfh- subgenomischer hcv rna transfizierten huh . zellen ähnlich stark wie beim vollständigen jfh- virus, was auf eine unabhängigkeit der apoptose-sensibilisierung von strukturproteinen sowie die notwendigkeit der viralen replikation hindeutet. untersuchungen zur signaltransduktion der jfh- -bedingten apoptose-sensibilisierung zeigen eine deutliche abhängigkeit von caspase- aktivierung sowie eine jfh- -induzierte suppression von bcl- und bcl-xl. dies deutet auf eine zentrale bedeutung des mitochondrialen signaltransduktionsweges bei der jfh- -induzierten apoptose hin. schlussfolgerung: unsere daten zeigen eine apoptosesensibilisierung von huh . hepatomazellen durch hcv replikation, welche unabhängig von hcv strukturproteinen und wahrscheinlich mitochondrial beding ist. diese ergebnisse geben wichtige hinweise für die pathogenese der hepatitis c und die mechanismen der viralen clearance. bier, jedoch nicht ethanol stimuliert in vitro die enzymsekretion der pankreasazinuszelllinie ar - j und frisch isolierter pankreasazinuszellen der ratte a. gerloff , m. v. singer , p. feick ii. medizinische klinik, universitätsklinikum mannheim, mannheim hintergrund: da der pankreasazinuszelle in der frühen phase der entwicklung der alkoholischen pankreatitis eine wichtige rolle zugesprochen wird, wurde die wirkung von alkohol (ethanol) auf die funktion dieser zelle bereits seit jahren ( ) intensiv untersucht. allerdings wird alkohol meist in form von schmackhaften getränken konsumiert, die viele nichtalkoholische inhaltsstoffe enthalten, welche ebenfalls einen pathophysiologischen einfluss auf die funktion des pankreas haben können. ziel: vergleich der wirkung von bier und adäquaten ethanollösungen auf die proteinsekretion und signaltransduktion der pankreasazinuszelllinie ar - j (ratte) und frisch isolierter ratten-azinuszellen. methoden: ar - j-zellen wurden für h mit dexamethason differenziert und frisch isolierte azinuszellen durch collagenase-verdau des pankreas von spraque-dawley-ratten gewonnen. nach inkubation der zellen für min. wurde die amylasefreisetzung als maß der proteinsekretion mit hilfe eines kommerziellen testkits bestimmt. ergebnisse: die inkubation von ar - j-zellen mit bier ( - % (v/v)) bewirkte eine dosisabhängige stimulation der basalen amylasesekretion. reiner ethanol in vergleichbarer konzentration wie im bier hatte keinen effekt auf die amylasefreisetzung. die bestimmung von ldh nach h inkubation der ar - j-zellen zeigte, dass die bierinduzierte amylasefreisetzung nicht auf einer membranschädigung beruhte. die verwendung selektiver inhibitoren und des ca-indikators fura- /am ergab, dass die bierinduzierte amylasesekretion vorwiegend durch die aktivierung von phospholipase c und der anschließenden kalzium-freisetzung aus intrazellulären speichern vermittelt wird. der stimulatorische effekt von bier auf die proteinsekretion war in frisch isolierten azinuszellen reproduzierbar. schlussfolgerung: unsere daten zeigen, dass der effekt von bier auf die sekretion von pankreasazinuszellen auf nichtalkoholische inhaltsstoffe zurückzuführen ist. diese inhaltsstoffe müssen bei der untersuchung von alkoholinduzierten pathologischen und funktionellen veränderungen berücksichtigt werden. pharmakokinetik und pharmakodynamik einer fckw-freien, fixen kombination aus beclometason-dipropionat und formoterol (- , , , und - , mm hg) erreichte keine statistische signifikanz. bei den mit liraglutid behandelten patienten zeigte sich im vergleich zu placebo eine senkung der triglyceride (adjustiert) von - % ( , mg, p= , ), - % ( , mg, p= , ) und - % ( , mg, p= , ) . es gab keine klinisch relevanten und konsistenten unterschiede bezüglich der gesamtcholesterin-, hdl-, ldl-, apob-, il- -, tnf-α-, leptin-und adiponectin-konzentrationen zwischen den liraglutid-behandlungsgruppen und placebo, eine signifikante reduktion des ldl-cholesterins wurde unter placebo im vergleich zu den aktiven behandlungs-gruppen beobachtet, das galt aber nicht für apob, bei dem lediglich in der , mg behandlungsgruppe ein unterschied zu verzeichnen war. es konnte ein ausgeprägter effekt von liraglutid auf die pai- -konzentrationen mit reduktionen von - % ( , mg, p= , ), - % ( , mg, p= , ) bzw. - % ( , mg, p= , ) gegenüber placebo beobachtet werden. das galt auch für die bnp-konzentrationen mit dosisabhängigen senkungen von - % (p= , ), - % (p= , ) und - % (p= , ) gegenüber placebo für die entsprechenden liraglutid-behandlungsgruppen. die beobachteten dosisabhängigen senkungen des crp gegenüber placebo (- %, - % und - %) erreichten keine statistische signifikanz. schlussfolgerung: die behandlung mit liraglutid war mit einer blutdrucksenkung und einer abnahme der triglycerid-, pai- -und bnp-konzentrationen verbunden, die beobachteten effekte müssen in langzeitstudien bestätigt werden und bedürfen weiterer untersuchungen. objectives: the majority of treated hypertensive patients do not achieve target blood pressure levels < / mmhg. one key reason is inadequate adherence with the prescribed drug regimen. dosing regimens are either not executed as prescribed (non compliance) or patients stop taking the medication (non persistence). it has been demonstrated that drug adherence with angiotensin receptor antagonists like valsartan is superior in comparison to other drug classes. the present study was designed to evaluate whether drug adherence could be further improved by the use of supportive tools. design and methods: centers were randomized to provide pharmacological treatment with or without a set of supportive measures (e.g. structured physicianpatient interaction, printed information about hypertension, reminder stickers, pill box with alarm, home blood pressure measurement). newly diagnosed patients or patients with stage hypertension (blood pressure at baseline . ± . / . ± . mmhg) who had not been treated for at least year were included in this trial. all patients entered the -week treatment phase with valsartan mg. titration to valsartan mg/hctz . mg was allowed if necessary. drug adherence was assessed by electronic monitors (medication event monitoring system -mems). results: patients treated with a valsartan-based therapy in combination with supportive measures demonstrated lower rates of treatment discontinuation. per-sistence at the end of the months observation period was % in the control patients and % in patients receiving supportive measures. in addition, better execution of the dosing regimen (compliance) could be observed in patients receiving supportive measures but this effect tended to fade with time. bp control improved more in patients with the supportive measures. conclusions: drug adherence can be improved with the use of supportive measures. in this study, while the effect on compliance decreased over time, the effect of chosen supportive measures on persistence seems to be long lasting. randomized, double blind parallel group study to evaluate the reduction of the urinary albumin/ creatinine ratio by valsartan plus lisinopril versus lisinopril or valsartan alone in hypertensive patients with microalbuminuria -the valeria trial objectives: microalbuminuria is known as an independent predictor for stroke, myocardial infarction and death and has a higher prevalence in hypertensive subjects than in the general population. intensified inhibition of the renin-angiotensin-aldosterone system seems to be an efficient treatment option and might be achieved by dual blockage with arbs and aceis. it was the aim of the study to compare the efficiency and safety of a combination therapy comprising valsartan and lisinopril with valsartan and lisinopril monotherapy in patients with hypertension and microalbuminuria. methods: this was a randomized, double blind parallel group study. patients with hypertension (mean sitting diastolic blood pressure = mmhg and < mmhg) and microalbumiuria (urinary albumin/creatinine ratio (uacr) = . mg/mmol and = . mg/mmol for men and uacr = . mg/mmol and = . mg/mmol for women in the first morning urine sample on at least two of three visits) were eligible for participation. after a wash-out/placebo-run-in phase of max. weeks, patient were randomized to treatment ( : : ) with either lisinopril - mg (lis), valsartan - mg (val) or the combination of valsartan/linisopril / - / mg (com) for weeks. results: median uacr at baseline in the com, val, and lis group was . mg/mmol, . mg/mmol, and . mg/mmol, respectively. at baseline, systolic/ diastolic bp for com, val, and lis was . / . mmhg, . / . mmhg, and . / . mmhg. treatment with com, val, and lis resulted in an uacr reduction of %, %, and % (median) after weeks of treatment. the reduction achieved with com was significantly greater than with lis (p= . ). normalization of microalbuminuria (uacr < . mg/mmol for men and < . mg for women) with com, val, and lis was achieved in %, % and % of the patients (p= . for com vs. lis). blood pressure differences between the groups were not statistically significant. all treatments were well tolerated. conclusion: in patients with hypertension and microalbuminuria the combination of valsartan and lisinopril provided a significantly better reduction of uacr and doubled the rate of patients with normalized uacr compared to lisinopril alone. analyse der mikrozirkulation der oberen extremitäten unter ldl-apherese the baseline ppg of the rebleeder (group b) was . +- . mmhg before tips and , +- . mmhg after tips insertion, giving an overall ppg reduction of , %. patients who underwent tips for variceal bleeding never had a bleeding episode again after tips (group a) . the baseline ppg of these patients was . +- . mmhg before tips and , +- , mmhg after tips, giving an overall ppg reduction of , % statistical analysis showed that the ppg difference between , mmhg in rebleeder (group b) and mmhg in non-rebleeder (group a) was statistically significant (p-value < . ). patients with refractory ascites never suffered from an episode of bleeding after tips independently from their initial ppg reduction. conclusion: patients who underwent tips for recurrent variceal bleeding do have a significant higher risk for rebleeding with an initial ppg reduction to , mmhg whereas those patients in which the ppg reduction to mmhg can be achieved a rebleeding episode is unlikely. we conclude that a ppg reduction to at least mmhg should be aimed to prevent further bleeding episodes. these findings differ from previous reports in which ppg of less than mmhg is thought to prevent rebleeding. ergebnisse: tgr männchen jedoch nicht tgr-weibchen entwickeln eine albuminurie (tgr-c: mg/ h) und glomerulosklerose, die durch flutamid zu > % unterdrückt wird (tgr-fl: mg/ h). testosterongabe induziert in tgr-weibchen jedoch nicht in wt-weibchen albuminurie (tgr-ovt: mg/ h, wt-ovt: . mg/ h) und glomerulosklerose (schadensindex-tgr-ovt: , wt-ovt: , ). ovariektomie induziert keine albuminurie. testosteron stimuliert in tgr-und wt-weibchen das renale wachstum und die glomeruläre angiotensinogenexpression, das glomeruläre wachstum wird jedoch nur in tgr-weibchen erhöht. androgenrezeptoren werden im glomerulus geschlechtsunabhängig exprimiert. schlussfolgerung: at rezeptorüberexpression in podozyten transgener ratten induziert albuminurie und glomerulosklerose. testosteron ist ein entscheidender kofaktor, der möglicherweise über eine direkte stimulation der glomerulären angii bildung wirkt. hohe prävalenz der ass-non-responder bei acvb-patienten in den multiplen testanalysen hintergrund und zielsetzung: in der therapie der hiv-infektion sind wechselwirkungen und unverträglichkeit der mittlerweile zahlreich zur verfügung stehenden antiretoviralen medikamente ein grosses problem. mehr als % der antiretroviralen medikamente und begleitenden antiinfektiva gegen opportunistische infektionen weisen positiv geladene reste (kationen) auf. die ausscheidung organischer kationen über die leber und niere erfolgt u.a. über die organischen kationentransporter (oct) und , die eine breite substratspezifität aufweisen. ziel der studie war die charakterisierung kationischer antiretroviraler medikamente und antiinfektiva als inhibitoren und substrate von oct / . methoden: hek- zellen wurden stabil transfiziert mit humanem oct und kloniert im eukaryotischen expressionsvektor pcdna . die ic -werte wurden durch messungen der spezifischen aufnahme von h-gelabeltem -methyl- -phenylpyridinium (mpp+) bestimmt. die quantitative bestimmung der spezifischen substrate erfolgte mittels liquid chromatography electrospray-ionizationtandem mass spectrometry (lc-esi-ms/ms). km und vmax wurden mit der michaelis menten gleichung bestimmt. ergebnisse: pentamidine (ic (hoct ) = . ( ) μmol/l (mittelwert (standardfehler); (oct ) . ( )), nelfinavir ( ( ); ( )), ritonavir ( ( ); ( )), lamivudine ( ( ); ( )), trimethoprim ( ( ); ( )), zalcitabine ( ( ); ( )), saquinavir ( ( ); ( )) und indinavir ( ( ); ( )) zeigten eine signifikante hemmung von oct / . lamivudine und zalcitabine sind substrate von oct / (lamivudine: oct (vmax= . ( . ) nmol/mg/min; km= ( ) μmol/l) oct (vmax= . ( . ); km= ( )) zalcitabine: oct (vmax= . ( . ); km= ( ); oct (vmax= . ( . ); km= ( ) ergebnisse: der endogene ligand s p, der selektive s p -rezeptor agonist sew und der unselektive s p-rezeptor agonist fty transaktivieren den ebenfalls bereits als vermittler protektiver intrazellulärer signalwege beschriebenen egf-rezeptor. damit assoziert ist die aktivierung von akt in ratten-kardiomyozyten. im rattenmyokard verbessert bei applikation beginnend mit reperfusion fty , nicht jedoch der selektive s p rezeptor agonist sew die erholung der mechanischen funktion signifikant. in humanen myokardpräparaten zeigt fty ex vivo einen analogen effekt bei applikation unter reperfusion. zusammenfassung: stimulation von s p und s p -rezeptoren induziert die aktivierung von akt in kardiomyozyten. eine verbesserung der erholung der mechanischen funktion lässt sich nur über stimulation des s p rezeptors unter reperfusion erzielen. s p-rezeptoren scheinen daher ein klinisch interessanter angriffspunkt für eine behandlung im sinne einer pharmakologischen postkonditionierung. systemic effects of glucose content in pd fluids on life span and neuronal function in c. elegans background: glucose content in peritoneal dialysis fluids has a decisive role in the formation of glucose degradation products (gdps) during sterilization and is thus associated with the degradation of peritoneal membrane integrity. absorption of gdps from the peritoneal cavity in peritoneal dialysis patients results in rising plasma age levels. one precursor in age formation is methylglyoxal, which is metabolized by glyoxalase- . to further investigate systemic effects of glucose content in pd fluids on life span and neuronal function, wild type and glyoxalase- overexpressing c. elegans were used as model system. homologies to % of all human genes are preserved in c. elegans. thus, this model system allows not only performance of life span assays, but also analysis of neuronal function. methods: c. elegans were cultivated under normal conditions and in the presence of . % and % glucose derived from peritoneal dialysis fluids to perform life span assays. simultaneously locomotive patterns as parameter of neuronal function and neuronal gfp expression were evaluated for each group. results: wild type c. elegans showed a significant reduction of life span of % under the influence of glucose , % and of % using % glucose. in contrast, glyoxalase- overexpression led to an increased resistance towards glucose expo-sure with no significant reduction of life span under . % or % glucose. additionally, with glucose exposure an impairment of neuronal function was observed in wild type c. elegans displaying severe changes in locomotive patterns, which were not present in transgenic c. elegans. neuronal gfp expression in wild type c. elegans declined significantly with glucose exposure without a dose dependant effect. conclusion:. glucose derived from pd fluids has a significant impact on life span and neuronal impairment in c. elegans. whether potential systemic toxicity and neurotoxicity in pd patients can be attributed to pd fluids has to be further evaluated. common pathways in endogenous major depression and depression during ifn-α therapy for chronic hepatitis c background: a major complication of combination therapy with pegylated interferon-alpha (ifn-α) and ribavirin in patients with chronic hepatitis c is the induction of depressive side effects. methods: to elucidate the underlying pathophysiological mechanisms, a total of caucasian patients with histologically proven chronic hepatitis c were treated with standard combination therapy consisting of pegylated ifn-α a and ribavirin. the transcriptional profile h before and h after the first injection of ifn-α was analysed using human genomic microarrays (affymetrix, hg u a) and quantitative real-time rt-pcr. class prediction analysis was performed to identify genes which are differentially regulated in patients with or without ifninduced depression. furthermore, pbmc from patients hospitalized for se vere major depression and controls were cultivated in vitro with pegylated ifn-α a to validate the data in this cohort. results: / hcv patients ( %) developed clinically relevant ifn-induced depression. using class prediction analysis, the development of depression could be predicted with % accuracy by genes including dynlt , gch , tor b, disc , mef a and st gal that were previously described to be relevant for recurrent major depression or neuronal development in the brain. in accordance with this data, increased endogenous ifn-production and selective hyper-responsiveness of these genes to ifn stimulation were observed in hcvnegative patients with severe major depression. conclusions: these data suggest that selective hyper-responsiveness to exogenous (ifn therapy) or endogenous (endogenous depression) type i ifns may lead to the development of depressive symptoms. these data could lead to the discovery of novel therapeutic approaches to treat ifn-induced and major endogenous depression. renal toxicity of glucose degradation products in peritoneal dialysis purpose (zielsetzung): in peritoneal dialysis (pd) residual renal function contributes to improved patient survival and quality of life. glucose degradation products (gdp) impair not only the peritoneal membrane, but also appear in the systemic circulation with the potential for organ toxicity. here we show that in a model of advanced renal failure, gdp affect the structure and function of the remnant kidney. sprague-dawley rats were randomly assigned to a twostage subtotal nephrectomized (snx) or sham operation and were left untreated for weeks. the snx+gdp group received chemically defined gdp intravenously for weeks; the snx and the sham-operated rats remained without gdp. the complete follow-up for all groups was weeks post-operatively. we analyzed renal damage using a semiquantitative score for glomerulosclerosis and tubulointerstitial damage as well as for immunohistochemical analyses. the snx+gdp rats developed significantly more albuminuria ( . ± . mg/ h vs. snx . ± . mg/ h; p ≤ . ) and showed a significantly higher score of glomerulosclerosis index ( . ± . vs. . ± . ; p ≤ . ) and tubulointerstitial damage index ( . ± . vs. . ± . ; p ≤ . ). in the snx+gdp group the expression of carboxymethyllysin and methylglyoxal was significantly higher in the tubulointerstitium and the glomeruli compared to the snx rats. caspase staining and tunel assay were more pronounced in the tubulointerstitium and the glomeruli of the snx+gdp group. in snx+gdp animals, the expression of the slit diaphragm protein nephrin, was significantly lower compared to snx and sham operated animals. in subtotally nephrectomized rats, administration of gdp increased albuminuria, indices of glomerular and tubulointerstitial damage significantly, specifically with a perturbation of podocytes. it is likely that gdp-free pd solutions maintain and stabilize residual renal function in pd. non-parenchymal liver cells (wu et al., hepatology, in press ). therefore, the aim of this study was to investigate whether hbv has the ability to suppress tlr-induced antiviral responses in parenchymal and non-parenchymal liver cells. we have isolated murine kc by counterflow elutriation as well as murine lsec by anti-lsec microbeads. in addition, primary murine hepatocytes were isolated by a two-step perfusion method. npcs and hepatocytes were cultivated in the presence or absence of hbsag, hbeag, hbv virions or supernatants from hbv-producing hbv-met cells, and were stimulated by tlr - ligands. supernatants were collected and tested for antiviral cytokines by viral protection assay or co-cultured with differentiated hbv-met cells. primary hepatocytes, undifferentiated hbv-met cells and highly differentiated hbv-met cells were stimulated by tlr - ligands. results: pretreatment of hepatocytes and npcs with hbv-met cell supernatants (contain hbsag, hbeag and virions), hbsag, hbeag and hbv virions almost completely abrogated tlr and tlr induced antiviral activity which correlated with suppressed isgs induction by poly i:c and lps on the transcriptional level. tlr - stimulation had no direct antiviral effect on hbv-met cells in contrast to primary hepatocytes. in addition, expression of pro-inflammatory cytokines (tnf-a, il- ) after tlr - , - , - , - and - stimulation were significantly reduced in highly differentiated hbv-met cells compared with undifferentiated hbv-met cells. our data indicate that integrated hbv can downregulate the tlr-mediated activation of hepatocytes. furthermore, hbv can almost completely abrogate the antiviral activity of hepatocytes and npcs induced by tlr and tlr stimulation. this has major implications for the interaction between hbv and the immune system. schimäre im pankreas: ein glukagon-produzierendes neuroendokrines karzinom assoziiert mit hypoglykämien conclusions: dbe-ercp is an alternative method for diagnostic as well as therapeutic interventions in the biliary as well pancreatic system in the operated patient. however, it should be limited to selected patients, e.g. with contraindications for ptc, as it is a time consuming as well as a cost intensive procedure. undifferenziertes embryonales sarkom der leber -eine seltene primäre leberneoplasie beim erwachsenen patients with metabolic syndrome (ms) and type diabetes are at increased risk for coronary artery disease. lipoprotein metabolism is characterized by elevated triglycerides (tg), low hdl cholesterol (hdl-c) and a predominance of atherogenic dense ldl (dldl). this is also denoted as atherogenic lipoprotein phenotype (alp). methods: multicenter, randomized, open-label cross-over study investigating the effect of combination therapy of fluvastatin/fenofibrate ( / mg) (ff) on the ldl-subfraction distribution compared to combination therapy of simvastatin/ ezetimibe ( / mg) (se) in patients with ms and type diabetes. at baseline and after weeks of combination therapies ldl-subfractions were measured by endpoint gradient ultracentrifugation. results: patients were randomized, completed the study. blood lipid samples of patients could be analysed. out of patients ( %) showed a profile dominated by dldl. in these patients, tg were elevated and hdl-c was lower compared to non dldl patients. in all patients reduction of total and ldl cholesterol by se was stronger than by ff. the increase of hdl-c was stronger with se in the non dldl group, whereas in the dldl group there was no difference between treatments. in non dldl patients there was no difference with regard to tg reduction and no effect on calculated ldl-radius. however, in the dldl group ff was more efficient in reducing tg (p= . between treatments) and se reduced ldl radius even further, whereas ff increased ldl radius to larger ldl particles (p< . between treatments). conclusions: simvastatin/ezetimibe combination is more efficient in reducing total and ldl cholesterol. this is also true for hdl-c increase in patients without dldl. however, in ms patients with dldl fluvastatin/fenofibrate were more efficient in reducing tg and increasing ldl radius. overall, the number of patients with aes and myalgia was . % hintergrund: bnp und nt-probnp sind molekulare biomarker, welche ebenso für die diagnostik und prognose der chronischen herzinsuffizienz genutzt werden können wie für die optimierung einer medikamentösen therapie und das langzeit-management dieser erkrankung. erstmalig untersuchten wir den einfluss eines strukturierten, multi-modalen, stationären interventionsprogramms -wie es in deutschland in vielen kardiovaskulären rehabilitationskliniken angeboten wird -auf den zeitlichen verlauf der nt-probnp-werte während und sechs monate nach einer solchen intervention. methodik: diese studie wurde in sechs deutschen kardiovaskulären rehabilitationszentren durchgeführt mit folgenden vier interventionsmodalitäten: ( ) optimierung der medikamentösen therapie, ( ) erziehung zu einem und durchführung eines krankheits-bezogenen ausdauertrainings-programms, ( ) intensive information über art und verlauf der erkrankung und erziehung zu einem adäquaten ernährungsverhalten, und ( ) teilnahme an den physischen und psychischen entspannungsübungen. zur steigerung der motivation und dokumentation wurden speziell für diese studie entwickelte patienten-tagebücher ausgegeben, welche den gesamten sechsmonatigen studienverlauf abdeckten. nt-probnp-analysen erfolgten am anfang (r ), während (r ) und am ende der stationären rehabilitationsperiode (r ), ebenso -auf ambulanter basis -nach drei monaten (r ) und sechs monaten r ) nach entlassung. zum vergleich wurde eine gleichaltrige kontrollgruppe von patienten herangezogen, welche nicht an einem solchen intensiven programm teilnahmen, sondern in einer herzinsuffizienz-ambulanz medizinisch betreut wurden. hypothese: wir erwarteten einen positiven effekt unseres interventions-programms auf die nt-probnp-spiegel während des stationären aufenthaltes, und entweder eine konstanz dieser werte nach der entlassung oder -auf grund von mangelhafter compliance -sogar einen wiedereinstieg der werte bis zu sechs monaten nach entlassung. ergebnisse: komplette nt-probnp-werte lagen für patienten vor, welche an einer chronischen herzinsuffizienz im stadium nyha ii -iii litten, eine klinisch relevante dekompensation erlitten hatten und eine lvef = % aufwiesen: der medianwert der nt-probnp-spiegel betrug pg/ml am anfang (r ), war geringfügig aber nicht signifikant reduziert auf (r ) und pg/dl (r ) während des stationären aufenthaltes, fiel jedoch signifikant und klinisch relevant auf pg/ml nach drei monaten (r ; p< , ) und auf pg/ml nach sechs monaten ab (r ; p< , ). der medianwert der kontrollgruppe änderte sich nicht während der sechsmonatigen beobachtungsperiode ( pg/ml und pg/ml; ns). schlussfolgerungen: im gegensatz zu unserer hypothese hat eine modernes, strukturiertes, stationäres rehabilitationsprogramm keinen kurzzeit-effekt auf die nt-probnp-spiegel innerhalb von wochen, sondern vielmehr einen signifikanten und größenordnungsmäßig klinisch relevanten langzeit-effekt innerhalb von monaten. dies könnte in der tatsache begründet liegen, dass die molekularen und strukturellen umbauprozesse des linken ventrikels im sinne eines umgekehrten "left ventricular remodelling" entsprechend zeit benötigen. da die nt-probnp-spiegel mit der prognose der erkrankung assoziiert sind, kann indirekt geschlossen werden, dass unsere stationäre rehabilitationsstrategie langfristig positive effekte auf hospitalisierungsrate und Überlebensprognose hat. cancer fatigue und gestörte ruhe/aktivitätsregulation bei rezidivfreien mammakarzinom-patientinnen, ergebnisse einer prospektiven studie r= , , p= , ) . vergleichbare beziehungen bestanden zwischen dem pap und ahi (r= , , p= , ), dem ai (r= , , p= , ) und dem zai (r= , , p= , ). das hzv war bei patienten mit csa ( , ± , l/ min/m ) signifikant niedriger als bei osa ( , ± , l/min/m ) oder patienten ohne sas ( , ± , l/min/m ). das hzv korrelierte negativ mit dem zai (r= - , , p= , ), dem ai (r= - , , p= , ) und dem ahi (r= - , , p< , ). bei pat mit einer osa waren solche beziehungen nicht nachweisbar. die vorliegenden befunde unterstützen die these, wonach das auftreten einer zsa ein parameter für die schwere einer hi sein kann und erhöhte pa-und pcw-drücke über pulmonal-vagale j-rezeptoren zur hyperventilation und damit entstehung und unterhaltung einer zsa beitragen können. der quotient aus serum-natrium/ urin-natrium-konzentrationen zu den serum-kalium/ urin-kalium-konzentrationen (sus:pup) im screening auf einen primären aldosteronismus hintergrund: das herzzeitvolumen (hzv) ist ein wichtiger parameter in der diagnostik und therapie kardialer erkrankungen. die aktuellen standardmethoden zur bestimmung des hzv sind jedoch entweder invasiv (rechtsherzkatheter, picco) oder technisch aufwändig bzw. teuer (magnetresonanztomographie, mrt). die bisherigen nicht-invasiven methoden zur messung des hzv mittels rückatmung von kohlendioxid oder thorakaler bioimpedanz sind zwar einfach durchzuführen, weisen aber methodisch bedingte ungenauigkeiten auf. ziel der vorliegenden prospektiven studie war es daher, eine neue methode zur bestimmung des hzv mittels cw-doppler ultraschall (cwd) zu evaluieren. methodik: bei konsekutiven patienten wurde unmittelbar vor oder nach einer kardialen mrt das herzzeitvolumen (hzv) mittels cwd im liegen bestimmt (uscom, uscom ltd, sydney australia). als referenzwerte dienten die in der mrt bestimmten werte, die mit dem arithmetischen mittel aus zwei aufeinanderfolgenden cwd messungen verglichen wurden. der statistische vergleich der methoden erfolgte mittels bland-altman-analyse. ergebnisse: das patientenkollektiv bestand aus männern (alter - jahre, median jahre) und frauen (alter - jahre, median jahre). insgesamt konnte bei von konsekutiven patienten im untersuchungszeitraum das hzv und sv mittels cwd bestimmt werden, bei patienten ( %) konnte kein ausreichendes ultraschallsignal empfangen werden. das hzv mittels mrt lag bei , +/- , l/min (mittelwert +/-sd, minimum , l/min, maximum , l/min), das hzv mittels cwd bei , +/- , l/min (minimum , l/min, maximum , l/min). die bland-altman-analyse ergab eine gute Übereinstimmung der beiden methoden für das hzv von , +/- , l/min (mittlere abweichung +/-sd) und für die bestimmung des schlagvolumens von , +/- ml. die methode zeigte eine gute reproduzierbarkeit mit einer mittleren abweichung von , +/- , l/min für das hzv und , +/- , ml für das sv. schlussfolgerung: der cw-doppler ultraschall erlaubt eine zuverlässige nichtinvasive bestimmung des hzv mit guter reproduzierbarkeit. der zukünftige stellenwert der methode in der diagnostik und therapiesteuerung muss durch weitere untersuchungen belegt werden. nichtinvasive bestimmung des herzzeitvolumens mittels inertgas-rückatmung und cw-doppler-ultraschall -sind die ergebnisse vergleichbar? bei patienten ( %) war die messung mittels cwd nicht möglich, bei patienten ( %) konnte die igr-messung nicht durchgeführt werden. das hzv mittels igr lag bei , +/- , l/min (mittelwert+/-sd, , bis , l/min) und bei , +/- , l/min ( , bis , l/min) mittels cwd. die bland-altman-analyse ergab eine gute Übereinstimmung für das hzv von , +/- , l/min (mittlere abweichung+/-sd) und für das sv von +/- ml. es zeigte sich eine gute reproduzierbarkeit mit einer mittleren abweichung von , +/- , l/min für das hzv mittels igr bzw. , +/- , l/min mittels cwd. schlussfolgerung: die bestimmung des hzv und sv mittels igr und cwd ergab eine gute Übereinstimmung und reproduzierbarkeit, so dass die methoden als vergleichbar einzuschätzen sind. zur identifikation des optimalen patientenkollektives für jede der beiden methoden sind weitere untersuchungen zur erarbeitung eines score-systems geplant. validierung einer einfach anzuwendenden neuartigen oszillometrischen methode zur bestimmung der pulswellen-geschwindigkeit obesity in humans is mainly due to high-fat intake and associated with an increased incidence of glomerular injury that is associated with low-grade inflammation and increased production of reactive oxygen species (ros). lauric acid (c : ), a major constituent of coconut oil, has anti-inflammatory activities, however its effects on pro-inflammatory gene expression in the kidney during obesity are unknown. the aim of the study was to quantify and compare renal cortical gene expression of pro-and antioxidant enzymes and icam- in c bl/ j mice fed with standard chow diet (sd), or diets rich in animal fat (afd) or vegetable fat (lauric acid, vfd) for weeks. changes in body weight and glucose tolerance (ip ggt) were also determined. both high-fat diets caused weight gain and glucose intolerance to a similar degree (n.s., afd vs. vfd, both p< . vs. sd). expression levels of proinflammatory genes (no synthase , nos ; catalytic subunit of the phagocytic nadph oxidase, nox ; intracellular adhesion molecule- , icam- ) were more than -fold lower compared to antioxidant genes (gluthatione peroxidase, gpx and cu/zn superoxide dismutase, sod ) and the regulatory subunit of the nadph oxidase complex p phox. only vfd-treatment reduced renal expression of p phox. treatment with afd but not with vfd increased mrna expression of nos , nox , and icam- . vfd, but not afd treatment was associated with upregulation of the antioxidant genes sod and gpx. these data suggest that fat diets rich in lauric acid, in contrast to diets containing animal fats, have distinct and possibly beneficial effects on expression of enzymes regulating cellular redox state in the kidney. contractility in the carotid artery obesity is a risk factor for diseases such as diabetes mellitus and atherosclerosis and has been associated with increased carotid artery intima thickness in obese patients. leptin has been implicated in formation of reactive oxygen species (ros), and functional leptin deficiency or impairment of leptin receptor activity result in obesity. endothelin- (et- ), ros and reduced no bioactivity are involved in vascular changes in obesity and diabetes. the aim of this study was to investigate the role of ros in et- mediated vasoreactivity in the carotid artery of obese leptin-deficient ob/ob and lean c bl/ j wild-type mice. rings were preincubated for minutes with the no synthase inhibitor, l-nitro-argininemethylester (l-name, μmol/l) to block etb receptor-mediated no release. rings were exposed to et- ( . - nmol/l) in the presence or absence of the ros antioxidant epck- (a combination of vitamin c and vitamin e, . mg/ml). endothelium-dependent relaxation to acetylcholine (ach) was in-vestigated in the presence of non-specific cyclooxygenase (cox) inhibitiors. maximal et- -mediated contraction in control mice was similar to obese mice. antioxidant treatment with epc-k reduced et- -induced contractions in control (from . ± . % to . ± . %, p< . ), but not in obese mice ( . ± . % vs. . ± . %, n.s.) endothelium-dependent relaxation to ach remained unchanged in both groups. in conclusion, these results indicate that ros contribute to et- -induced contractions in the carotid artery of healthy mice, an effect that disappears during obesity. leptin may modulate contraction to et- in the carotid artery by mechanisms that are independent of ros. high dietary intake is a major cause of obesity, a risk factor for the development of hypertension, diabetes, and atherosclerosis. the aim of this study was to investigate whether high dietary fat intake-induced vascular changes are reversible upon dietary fat restriction. body weight, glucose tolerance and vascular reactivity were measured in c bl/ j mice, which were fed either with standard chow or with high-fat diet (hfd, % kcal from fat) for months or with hfd for months followed by fat restriction for months. vascular reactivity was analyzed in the carotid artery in response to the α-adrenergic receptor agonist phenylephrine (pe; x - m), and to acetylcholine (ach; - - - m) in the presence or absence of ng-nitro-l-arginine methyl ester (l-name; x - m), a nitric oxide (no) synthase inhibitor. high-fat diet increased body weight and impaired glucose tolerance (p< . vs. control). vascular contractions in response to pe ( x - m) and ach ( - m) were increased both in the absence or presence of no (p< . vs. control), while the endothelium-dependent relaxation was unaffected. in contrast, dietary fat restriction normalized the body weight similar to control animals and also the relative insulin resistance (p< . vs. hfd). in parallel, vascular contraction to ach was attenuated in the absence of l-name (p< . vs. hfd). unexpectedly, dietary fat restriction did not reverse pe-and ach-mediated contraction in the absence of no induced by hfd. in conclusion, intake of high dietary fat has a "memory" effect on vascular reactivity, which cannot completely be reversed even upon dietary fat restriction. this may contribute to an increased risk of vascular injury seen in patients even after normalization of body weight and insulin resistance. die phagozytotische aufnahme von hcv-infizierten apoptose-körperchen durch hsc führt zur induktion der fibrogenese a minority of neuroendocrine tumors (net) present functional syndromes by uncontrolled secretion of peptide hormones or messengers. peptide receptor radiotherapy (prrt) and transcatheter arterial chemoembolisation (tace) have demonstrated efficacy as monotherapy in the treatment of functional nets. prrt has a slower and longer acting mechanism of action whereas tace can be immediately effective. however, there are no reports that both therapies have been applied in combination. we treated three consecutive patients with severe functional syndromes caused by hepatic metastasis of nets by sequential prrt and tace. the first patient (female years) suffered from a pancreatic net metastasized into the liver. severe hypercalcemia with levels of . mmol/l developed leading to fatique and somnolence despite medical treatment.the patient received one course of mci y- dota-tate prrt and days later, tace was performed at the right liver lobe due to persistent hypercalcemia. after days, calcium levels were normalized and the patients was able to walk with support. after demission, the patients died of progressive disease weeks later. the second patient (male, years) presented with somnolence and hypoglycaemia down to mmol/l by disseminated insulinoma of the pancreas. the patient received two courses of prrt and three coursed of tace involving both liver lobes. he tolerated the combined treatment well and is off intravenous glucose since the first tace. the third patient (male, years) presented with severe hypoglycaemia due to hyperinsulinemia caused by disseminated net of unknown origin with prominent liver metastases in both liver lobes. initially, the patient was treated by tace followed by courses of prrt with -lu. the treatment was well tolerated, the patient is off intravenous glucose. a second tace was intended but was not be performed due to remission of the liver metastases. initial experience with combined treatment of prrt and tace demonstrated that hepatic failure did not evolve in our patients and that the treatment was well tolerated. however, prognosis remains poor due to disseminated and progressive disease. die zahl zirkulierender endothelialer progenitorzellen steigt mit zunehmendem schweregrad der peripheren arteriellen verschlusskrankheit these data indicate that the key mirna-processing enzymes dicer and drosha and consequently the overall expression of mirnas are downregulated during neointima formation in vivo. moreover, the augmented proliferative-and attenuated apoptotic response observed following knock down of drosha and dicer in vsmc implicate an important role of these enzymes and of mirnas for vsmc function during the development of vascular proliferative disease. ( ) hochaktives endokrines organ. das fettgewebe produziert nämlich zahlreiche adipokinine, welche möglicherweise einen autokrinen und parakrinen einfluss auf den fettstoffwechsel, sowie eine endokrine wirkung auf andere organe haben. die entdeckung solcher faktoren eröffnet die frage, ob die adipokinine die gesuchte verbindung zwischen adipositas und den damit assoziierten erkrankungen sein könnten. im diesen zusammenhang haben wir untersucht, ob das fettgewebe die herzfunktion direkt mittels sekretion von kardioaktiven faktoren beeinflusst. wir isolierten deshalb adipozyten aus humanem fettgewebe und untersuchten anschließend den effekt ihrer sekretionsprodukte auf kardiomyozyten in zellkultur sowie mittels langendorff system auf isoliert-perfundierte rattenherzen. wie wir feststellen konnten, hatten die sekretionsprodukte der adipozyten die kontraktion der kardiomyozyten durch eine verminderung des intrazellullären kalziumstroms stark gehemmt. durch erhitzen oder trypsin-behandlung wurden die kardiodepressiven faktoren inaktiv. mittels filtration nach molekulargewicht konnten die kardiodepressiven faktoren zwischen und kd isolierten werden. auf ähnliche weise führte die perfusion von isoliertem herz mit adipozytenfaktoren zu einer starken reduktion der kraftentwicklung sowie zu einer reduktion des koronarflusses durch eine kontraktion der koronararterien. zusammenfassend, die ermittelten daten lassen auf einen bislang unbekannten, negativen einfluss des fettgewebes auf die herzkontraktion schließen. dies geschieht einerseits direkt durch die verminderung des intrazellullären kalziumstroms in die kardiomyozyten, andererseits indirekt durch die reduktion des koronarflusses. diese daten liefern somit eine neue erklärung für den zusammenhang zwischen adipositas und herzinsuffizienz. a comparison of serum fractalkine in patients with coronary heart disease, insulin dependent diabetes, and healthy controls background: atherosclerosis is a multifactorial process that involves inflammation of the vessel wall arising from interactions of leukocytes with vascular endothelial and other local cells. these interactions are regulated by cytokines, including chemokines, as well as adhesion molecules. the chemokine fractalkine (fkn) and its receptor, cx cr , have emerged as interesting intermediaries in atherosclerosis. fkn is a unique chemokine because it exists in soluble and membrane-anchored forms. membrane-bound fkn contributes to adhesion of cx cr -expressing leukocytes, thus providing a novel pathway for leukocyte activation. soluble fkn has leukocyte chemotactic activity. therefore, serum fractalkine levels may indicate the inflammatory situation in chd patients. we investigated the serum concentrations of fkn in non-diabetic patients with coronary heart disease (chd), and insulin dependent diabetics (iddm) in patients attending for rehabilitation in the curschmann-clinic, timmendorfer strand. serum fkn was also determined in healthy controls. fkn was analyzed by elisa. results: soluble fkn was slightly, but not significantly increased in patients with chd in comparison to healthy controls (mean ± standard deviation: ± pg/ml in chd-patients versus ± pg/ml in controls, p= . ). surprisingly, soluble fkn was decreased in insulin dependent diabetics (mean±sd ± pg/ml thus, iddm versus controls p= . , and iddm versus chd p= . ). conclusion: previous studies described that soluble fkn is increased in chd patients compared with healthy controls and that membrane-bound fkn was increased in human carotid arteries and animal models. the decreased soluble fkn in insulin dependent diabetics compared with chd-patients and controls in this study may partly be explained by the quiescent disease state predominant in our rehabilitation patients. hintergrund: neben der jeweiligen bilddokumentation und dem deskriptiven befund des interventionalisten exsistieren keine objektiven daten zur veränderung der hämodynamik während peripheren arteriellen gefässinterventionen und dem unmittelbaren hämodynamischen erfolg nach der intervention. versuche der etablierung eines monitorings durch dopplerultraschall scheiterten in der vergangenheit offensichtlich u.a. an der aufwändigen fixierung der dopplersonden. methode: wir verwenden zur kodomo die doppler x-plore sonde ( , mhz, firma medi-stim, deisenhofen), welche durch einen saugnapf mit ultraschallgeldepot über einer zuvor dopplersonographisch detektierten fussarterie fixiert wird. es können so während der intervention kontinuierliche dopplersignale dokumentiert werden. ergebnisse: phänomene die mittels kodomo bei infrainguinalen eingriffen periinterventionell erfasst werden können sind: verbesserung des dopplerflusses postinterventionell, artefakte durch kontrastmittel, periphere embolien, veränderungen des dopplersignals während der angioplastie. beispielhaft wird ein fall mit filiformer superficialisstenose links demonstriert (abb. ). schlussfolgerung: kodomo gibt dem interventionalisten über die bildgebung hinausgehende hämodynamische zusatzinformationen und ist ein verfahren zur unmittelbaren dokumentation des hämodynamischen erfolges der interventionellen therapie. es spart in konsequenter anwendung möglicherweise kontrast-mittel, durchleuchtungs-und untersuchungszeit. durch den artefiziellen gefäßverschluss zum zeitpunkt der angioplastie kann der kollateralfluss der zielläsion zukünftig gemessen werden. einfluss von shunt-verkalkungen auf das Überleben von hämodialysepatienten introduction: two-dimensional strain ( ds) is a novel method to measure strain from standard two-dimensional echocardiographic images. strain imaging has been proposed as a sensitive tool for the assessment of the left ventricular myocardial function. the aim of our study was to characterize global and regional function abnormalities using this technique in patients (pts.) with constrictive pericarditis (cp) and restrictive cardiomyopathy (rcm). methods: we studied consecutive patients (pts.) with heart failure of either proven pericardial (cp) or myocardial origin (rcm; biopsy proven cardiac amy-loidosis). global longitudinal strain (gls) and regional peak systolic strain (pss) was assessed by ds in the apical four-chamber-view using a dedicated software package (vivid , ge healthcare). all pts. underwent a complete echocardiographic and hemodynamic assessment. results: out of the pts. (mean age: ± years) had cp, and rcm. the thickness of the interventricular septum (ivsd) was significant increased in pts. with rcm ( ± vs. ± mm). mean gls was - . ± . % in the cp-group and - . ± . % in the rcmgroup (p< . ). pts. with rcm showed a significant decreased longitudinal pss in the septal segments (basal: - . ± . % vs. - . ± . %, p< . , mid: - . ± . % vs. - . ± . %, p< . ; apical: - . ± . % vs. - . ± . %; p< . ) and a decreased pss in the lateral segments (basal: - . ± . % vs. - . ± . %, n.s.; mid: - . ± . % vs. - . ± . %, n.s.; apical: - . ± . % vs. - . ± . %, n.s.). conclusion: two-dimensional strain is a simple and rapid method to measure gls and pss. this technique might be used as new helpful tool for the differentiation between pts. with rcm and cp, and for the detection of early regional myocardial dysfunction before the onset of congestive heart failure (chf) in patients with cardiac amyloidosis. nichtinvasive koronare plaquedifferenzierung: mehrschicht-computertomographie validiert mit intravaskulärem ultraschall Über die klassischen risiken hinaus sind auch lokale faktoren an der initiation und progression von atherosklerotischen ablagerungen beteiligt. diese faktoren beinhalten multiple variablen wie wechselwirkungen zwischen gewebe und flüssigkeiten, wandspannung und flussgeschwindigkeit . die direkte messung dieser faktoren ist in-vivo nur im tierversuch möglich. entsprechende parameter können jedoch über den einsatz moderner bildgebungstechniken (cardio-cta) unter verwendung von flusssimulationen (computational fluid dynamics=cfd) berechnet werden. das ziel dieser studie war daher ) die durchführbarkeit der in-vivo cfd-berechnung an humanen koronararterien zu demonstrieren und ) die ergebnisse mit radiofrequenzmessungen im intravaskulären ultraschall (ivus) zu korrelieren. methoden und ergebnisse: zehn patienten mit suspektem koronarem befund wurden prospektiv in die studie einbezogen. diese erhielten innerhalb von vier wochen eine ct-gestützte koronarangiographie (dual source slice ct) und eine invasive koronarangiographie. bei diesen patienten wurde die intravaskuläre ultraschalluntersuchung in allen drei hauptstammgefäßen durchgeführt, die erhebung der radiofrequenzdaten geschah simultan. mit hilfe der axialen cta-schnitte wurde ein gittermodell der jeweiligen gefäße erstellt. dieses konnte genutzt werde um die gewebeflüssigkeitinteraktionen, flussgeschwindigkeit und wandspannungsverhältnisse zu simulieren. die berechnung der cfd-parameter konnte in / herzkranzarterien erfolgreich durchgeführt werden. siebzehn koronararterien wurden zusätzlich durch ivus beurteilt. die wandspannung der gefäße korrelierte umgekehrt zu vorhandenen durch ivus ermittelte plaques(p< , ). ein zusammenhang zwischen den erhobenen cfd-daten und den radiofrequenzdaten der gewebe konnte bisher nicht nachgewiesen werden. zusammenfassung: die ergebnisse dieser studie demonstriert die möglichkeit gewebeflüssigkeitsinteraktionen in menschlichen koronararterien, mit hilfe von modernen computertomographischer bildgebung, nichtinvasiv abzuschätzen. die bedeutung dieser zusätzlichen informationen muss prospektiv weiter untersucht werden. welchen einfluss hat die myokardiale fibrose und funktion bei patienten mit hochgradiger aortenklappenstenose auf den klinischen langzeitverlauf nach aortenklappenersatzoperation? ziel dieser studie ist es, die entwicklung des linksventrikulären remodellings sowie der regionalen myokardialen funktion bei patienten mit hochgradiger aortenklappenstenose sowohl vor als auch im verlauf nach aortenklappenersatzoperation zu untersuchen. methodik: bei patienten mit hochgradiger aortenklappenstenose wurde sowohl präoperativ als auch monate postoperativ eine konventionelle echokardiographie und eine strain-rate-imaging studie mit bestimmung der longitudinalen systolischen spitzen-strain-rate durchgeführt. zur beurteilung der myokardialen fibrose wurde eine magnetresonanz tomographie (mrt) mittels gadolinium late enhancement technik durchgeführt und eine biopsie aus dem linksventrikulären septum intraoperativ entnommen. ergebnisse: die patienten wurden entsprechend der nyha-klasse monate nach der operation in drei gruppen aufgeteilt. gruppe (gutes outcome=nyha-klasse i; n= ), gruppe (mittleres outcome, nyha-klasse ii; n= ), gruppe (=schlechtes outcome, nyha -klasse iii bzw. iv; n= ). präoperativ zeigten sich bei allen drei gruppen eine vergleichbare ejektions-fraktion (gruppe = ± %; gruppe = ± %; gruppe = ± %). wohingegen bei der longitudinalen strain rate signifikant höhere werte in gruppe als in gruppe gemessen werden konnten (gruppe = , ± , s - ; gruppe = , ± , s - ; gruppe = , ± , s - ). interessanterweise stieg die longitudinale strain rate erst mit der abnahme der wandstärken nach monaten an. außerdem konnte ein signifikant höherer grad an interstitieller fibrose mittels biopsie-score (gruppe = , ± , ; gruppe = , ± , ; gruppe = , ± , ) sowie auch ein häufigeres late enhancement in der mrt bei patienten mit schlechtem klinischem outcome nachgewiesen werden (segmente mit fibrose: gruppe = , ± , ; gruppe = , ± , ; gruppe = , ± , ). zusammenfassung: diese daten lassen vermuten, dass bei patienten mit hochgradiger aortenklappenstenose sowohl eine myokardiale fibrose als auch eine reduzierte regionale myokardiale funktion präoperativ entscheidene hinweise auf den klinischen langzeit-verlauf nach aortenklappenersatzoperation geben können. indikationen und limitationen der ultraschall-elastographie bei patienten mit erkrankungen des pankreas vor dem hintergrund immer wirksamerer, aber auch sehr kostenintensiver behandlungsoptionen einerseits und dem bewusstsein der früh einsetzenden strukturellen schädigung im arthritischen prozess ("window of opportunity") andererseits, ist ein monitoring des therapieansprechens bei patienten mit rheumatoider arthritis von besonderer bedeutung. eine exakte darstellung des entzündlichen substrates gelingt besonders mit der sonographie, wodurch therapieversager frühzeitig identifiziert werden können. die arthrosonographie erlaubt eine sehr gute differenzierung zwischen exsudativen und proliferativen synovialisveränderungen sowie die beurteilung der perfusion durch die doppler-verfahren. voraussetzung für eine sonographische beurteilung des verlaufes des arthritischen prozesses ist eine quantifizierung der entzündungsaktivität. in anlehnung an den von scheel und kollegen publizierten synovitisscore, wurden patienten mit aktiver rheumatoider arthritis, die durch eine biologikatherapie (tnf-inhibitoren, rituximab, abatacept) in klinische remission gebracht werden konnten (das : < , ), standardisiert sonographisch nachuntersucht. bei der mehrheit der ra-patienten konnte trotz klinischer remission durch die sonographischen nachuntersuchungen und monate nach initiierung der biologikatherapie eine persistierende entzündungaktivität nachgewiesen werden. während eine deutliche therapiebedingte abnahme der hyperperfusion zu beobachten war, konnte nur eine geringgradige abnahme der synovialen proliferationen und ergussbildung festgestellt werden. diese ergebnisse zeigen, dass bei der mehrzahl der ra-patienten trotz klinischer remission eine sonographisch nachweisbare snovitis persistiert, was möglicherweise die ursache für den radiologischen krankheitsprogress bei einem teil dieser patienten darstellt. zielsetzung/aims: granulocyte-colony stimulating factor (g-csf) was shown to improve cardiac function after myocardial infarction (mi). recently, we histologically demonstrated enhanced arteriogenesis and reduced infarct size by g-csf treatment after mi in mice. in this study, we non-invasively, repetitively, and quantitatively investigated g-csf effects on perfusion by a pinhole single-photon emission computed tomography (spect) system after mi in mice. methoden/methods: mi was induced by lad ligation in wt mice (c bl/ j). g-csf ( μg/kg; n= ) or saline (n= ) was daily injected for days. extent of left ventricular (lv) perfusion defects was determined with a [ mtc]-sestamibi triple headed pinhole spect system at days (baseline) and days after lad occlusion. polar maps were normalized by mean of a standardized reference region of interest (roi) in the septal region ( %). best threshold value for identifying infarcted areas was determined by comparing perfusion defects with the histological infarct sizes in these animals. defect size was indicated as% of lv myocardium. primary endpoint was change of defect size from baseline to days after mi. ergebnisse/results: best threshold for identifying infarcted areas compared to histology was less than % of the septal reference roi. mean infarct size was similar in saline ( , % ± , ) and g-csf group ( , % ± , ) at baseline. at day after mi, a slight difference (p= , ) was found between saline ( , ± , ) and g-csf ( , % ± , ) groups. however, change of defect size was significantly different (p= , ) between g-csf (- , % ± , ) and control animals (- , ± , ) . schlussfolgerung/conclusions: this is the first study, demonstrating the suitability of triple headed pinhole spect system for repetitive infarct size measurement in mice, offering a new non-invasive imaging technique for cardiovascular therapy monitoring. in this context, we showed that g-csf administration significantly reduces lv perfusion defects indicating positive effects on ventricular remodelling after mi in mice. endoscopy in patients with acute leukemia after intensive chemotherapy conclusions: endoscopy can be performed relatively safely in patients who received myelosuppressive chemotherapy. the procedure may induce fever and bacteremia. the percentage of patients in whom endoscopic hemostasis was applicable and effective was low. we recommend conservative treatment first. endoscopy should be reserved for patients who are unstable or refractory. impact of pregnancy on prevalence of goitre and nodular thyroid disease in women living in a region of borderline iodine deficiency purpose: an interplay of genetic, epigenetic and environmental factors contributes to thyroid disease. in a cross-sectional study we aimed to determine the actual influence of parity on goitre and nodular thyroid disease (ntd) in women living in a region with borderline iodine deficiency. methods: thyroid ultrasonography ( . mhz; merck thyromobil) was performed by the same investigator in women living in thuringia and saxony. furthermore, age and bmi were documented and all women were asked about the number of previous pregnancies, family history of thyroid disease and past or present smoking. results: goitre prevalence was . %. solitary thyroid nodules were detected in . %, multiple nodules in . % of the study population. age was positively correlated with goitre prevalence and ntd (due to multiple but not solitary nodules). no association was found between parity and goitre prevalence. in contrast, a significant increase in both, solitary ( . %) and multinodular thyroid disease ( . %) was observed in women with at least one pregnancy compared to nullipara ( . % and . %, respectively). bmi in women with goitre ( . kg/ m ) was significantly higher than in women without ( . kg/m ). in addition, a significant correlation was detected between bmi and presence of multinodular disease ( . kg/m ). , % of women with goitre reported a positive family history for thyroid disease, as opposed to % of women with normal size thyroid gland. neither goitre nor ntd were associated with a present or past history of smoking. conclusion: ntd and/or goitre are present in up to % of woman in an area with borderline iodine deficiency. besides age, bmi and family history, parity is positively correlated with presence of ntd, whereas smoking was not associated with goitre/ntd. defizite in der medizinischen versorgung von patienten mit nebennierenkarzinom in deutschland kardiologie, universität heidelberg, heidelberg; koordinierungszentrum für klinische studien leipzig, universität leipzig, leipzig; kardiologie, universität würzburg, würzburg; institut für herzkreislaufforschung, dortmund; kardiologie, universität marburg, marburg; kardiologie, berlin; institut für frauenspezifische gesundheitsforschung, deutsches herzzentrum berlin, berlin; kardiologie, universität göttingen, göttingen; kardiologie, essen einleitung: Ältere patienten und frauen sind in großen klinischen studien zur chronischen herzinsuffizienz regelhaft unterrepräsentiert. die vorliegende arbeit analysiert daher die geschlechts-und altersabhängige leitlinien-Ädhärenz im rahmen der erhebungen des kompetenznetzwerkes herzinsuffizienz. methodik: wir evaluierten die datensätze aller patienten mit systolischer herzinsuffizienz (lvef echokardiographisch ≤ %) die zwischen / und / in das kompetenznetzwerk herzinsuffizienz eingeschlossen wurden: n= ; mittleres alter , ± , jahre; , % frauen. die nyha-angepasste, leitliniengerechte verschreibung der pharmakotherapie (=adhärenz) wurde über die einnahmehäufigkeit der herzinsuffizienzmedikation unter berücksichtigung von kontraindikationen erfasst. der guideline-adherence-indicator (gai) wurde für betablocker, ace-hemmer/at -blocker und aldosteron-rezeptorblocker berechnet (=gai- ), sowie für die zusätzliche indikation für diuretikum und glykosid (=gai- ). ergebnisse: der gai- betrug in den nyha klassen i/ii/iii/iv / / / % und der gai- / / / % (p für trend jeweils < . ). die therapie-adhärenz (gai- und gai- ) war bei männern höher als bei frauen (p= . und p< . ). in der multivariablen ordinalen regression waren zwar alter (or pro dekade , , p= , ; und or , , p< , ) und nyha stadium iii-iv (or , und , , p jeweils < , ), nicht jedoch das geschlecht (p= , und p= , ) prädiktoren des gai- und gai- . zusammenfassung: aktuelle daten des kompetenznetz herzinsuffizienz zeigen bei der therapie der chronischen herzinsuffizienz im vergleich zu früheren studien in deutschland hinsichtlich der verordneten substanzklassen eine klinisch hoch relevante zunahme der leitlinien-adhärenz. in zukünftigen studien wird zu klären sein, ob auch leitliniengerechte dosierungen in der klinischen praxis sinnvoll umsetzbar sind. effekt von nt-probnp purpose: the endothelial specific angiopoietin (ang)-tie ligand-receptor system has a key role in regulating vascular integrity and quiescence. the antagonistic ligands ang- and ang- control the expression of endothelial adhesion molecules and intercellular tight junctions. the role of ang- in anca-associated vasculitis (aav) has not been investigated yet. methods: we measured serum ang- in healthy controls and patients with aav ( patients with active aav at initial presentation and during follow-up, patients in stable long-term remission, patients prior to systemic relapse, and patients with active "limited" wg (ent). the disease activity was monitored by the birmingham vasculitis activity score (bvas) and the enumeration of circulating endothelial cells (cecs). for statistical analysis we used one-way anova with dunn´s correction, friedmann and wilcoxon tests, spearman´s rho test and linear regression. results: ang- was elevated in active aav (mean . ± . ng/ml sem) compared to controls ( . ± . p< . ). most notably, ang- was also elevated in archival serum samples of patients in long-term remission just before systemic vasculitic relapse ( . ± . ng/ml p< . ). in contrast, ang- was normal in patients with stable remission of aav ( . ± . ng/ml and "limited" granulomatous ent disease ( . ± . ng/ml). ang- was elevated at initial presentation ( . ± . ng/ml sem) and declined rapidly after immunosuppessive therapy at month ( . ± . ng/ml), - months ( . ± . ng/ml) and months ( . ± . ng/ml). linear regression analysis demonstrated a strong association of ang- with bvas (rs = . p< . ) and cecs and (rs = . p< . ). these results indicate that ang- might be a useful early marker to discriminate systemic vasculitic activity in aav from limited granulomatous ent disease and remission. moreover, ang- might also be a mediator of endothelial inflammation and detachment. further investigation of the ang/tie-system in vasculitis is crucial since pharmacologic inhibitors of ang- will become available shortly. warfarin-induzierte kalzifikation in mäuseneine neues modell zur untersuchung der interaktion von verkalkungsinhibitoren aim of the study: although hypercholesterolemia, a predominant risk factor of coronary heart disease, is related to cholesterol metabolism, the association between cholesterol metabolism and coronary heart disease is not well known. therefore, this study investigates effects of cholesterol metabolism on coronary heart disease and family history of cardiovascular diseases. methods: in addition to conventional coronary risk factors (age, sex, bmi, arterial hypertension, diabetes mellitus, smoking, family history of cardiovascular diseases, plasma cholesterol) campesterol and sitosterol (indicators of cholesterol absorption) and lathosterol (indicator of cholesterol synthesis) were determined in consecutive men and women with severe aortic stenosis. on coronary angiograms prior to aortic valve replacement extend of coronary heart disease was determined ( , , , -vessel disease). furthermore, a semiquantitative score of the extend of coronary atherosclerotic lesions was determined. results: in patients with a positive history of cardiovascular diseases the ratio of campesterol to lathosterol was significantly increased (p< . ). there was a significant increase in campesterol to lathosterol ratio in plasma with increasing extend of coronary heart disease ( , , , -vessel disease; p< . ). furthermore, there was a positive correlation of coronary vessel score with the ratio of campesterol to lathosterol in plasma (r= . ; p< . ) and in aortic valve cusps (r= . ; p< . ), indicating that enhanced absorption and reduced synthesis is related to the extend of coronary heart disease. logistic regression analysis revealed that of all coronary risk factors tested the ratio of campesterol to lathosterol was the sole, significant predictor of coronary heart disease in this subset of patients (p< . ). the results of this study suggest that in patients with severe aortic stenosis elevated ratios of campesterol to lathosterol are directly related to concomitant coronary heart disease and may serve as a predictor for coronary heart disease in humans. bei postmenopausalen frauen und bei männern mit rheumatoider arthritis und glukokortikoid-therapie besteht zu über prozent die indikation für eine bisphosphonat-therapie nach dvo-leitlinie hintergrund: ein geringes ansprechen auf clopidogrel nach koronarer stentimplantation ist assoziiert mit einem erhöhten risiko für das auftreten von stentthrombosen. die cytochrom p (cyp) isoenzyme cyp c and cyp a / sind bei der bioaktivierung von clopidogrel beteiligt. das ziel der vorliegenden studie ist es, den zusammenhang zwischen dem ansprechen auf clopidogrel und genetischer varianten der cyp isoenzyme bei patienten mit symptomatischer koronarer herzerkrankung zu untersuchen. methoden und ergebnisse: genotypisierungen für cyp c (* , * , * ), cyp a * b und cyp a * polymorphismen wurden bei konsekutiv eingeschlossenen patienten (n= ), die einer koronare stentimplantation aufgrund einer symptomatischen koronaren herzerkrankung erhielten, durchgeführt. die adenosin diphosphat (adp)-induzierte thrombozytenaggregation wurde frühestens stunden nach erstgabe von mg clopidogrel gemesen. träger der cyp c * variante zeigten eine signifikant höhere residuelle thrombozytenaggregation (rta) (chi-quadrat ; p< . ). die übrigen untersuchten polymorphismen zeigten keinen signifikanten einfluß auf die rta. auf der grundlage des predict-score zur vorhersage einer erhöhten rta wurden der cyp c * genotyp und zuvor identifizierte nicht-genetische einflussvariablen (alter, akutes koronarsyndrom, typ diabetes, reduzierte linksventrikuläre funktion, und niereninsuffizienz) untersucht. die logistische regressionsanalyse ergab eine signifikante korrelation der nicht-genetischen risikofaktoren (chi-quadrat , ; p= , ) und des cyp c * polymorphismus (chi-quadrat , ; p< , ) mit einer erhöhten rta, und einen kumulative assoziation zwischen rta und der kombination aus genetischen und nicht-genetischen faktoren (chi-quadrat , ; p < , ). diskussion: träger von zumindest einem cyp c * allel haben ein ungefähr -faches risiko, eine erhöhte rta zu entwickeln. genotypisierung der funktionsverlust-variante cyp c * könnte zu einer verbesserten prädiktion des ansprechens auf die antithrombozytäre therapie mit clopidogrel beitragen. the shape of the glucose curve during an oral glucose tolerance test correlates with changes in glucose tolerance m. reimann , j. li , s. bornstein , j. schulze , p. schwarz medical clinic iii, endocrinology, medical faculty carl gustav carus, technical university dresden, dresden; sächsische landesärztekammer, dresden introduction and objectives: the shape of the glucose curve during an oral glucose tolerance test (ogtt) can be categorized in monophasic and biphasic. the latter has been associated with normal glucose tolerance. the aim of the study was to explore the association between the shape of the glucose curve and changes of glucose tolerance. research design and methods: ogtt data from subjects with different stages of glucose tolerance were analyzed at baseline and three years follow up. the shape of the glucose curve at baseline was classified as monophasic, biphasic and unclassified. the shape index was calculated as the difference between glucose at min and at min and treated as continuous variable in correlation analyses. in the biphasic group, there was a significantly higher proportion of subjects with normal glucose tolerance and a lower proportion of subjects with impaired glucose tolerance and type diabetes. subjects with a biphasic shape had a significant lower bmi and a better profile of carbohydrate metabolism. the shape index correlated significantly with changes of plasma glucose at baseline and at min and insulinauc after adjustment for glucose tolerance state. the prevalence ratio for disease regression was significantly higher in subjects with biphasic glucose curve shape. the shape index gives additional information regarding the stage of glucose tolerance beyond the who classification. a biphasic shape is associated with improvements in plasma glucose and may predict regression of disease irrespective of glucose tolerance. die residuelle aggregation unter dualer thrombozytenhemmung beeinflusst die inzidenz schwerer kardiovaskulärer ereignisse unabhängig vom einsatz medikamentenbeschichteter stents kritisch kranke patienten weisen häufig eine charakteristische konstellation des thyreotropen regelkreises auf, die als non-thyroidal-illness-syndrom (ntis) bezeichnet wird und mit einer erhöhten morbidität und mortalität verbunden ist. trotz langjähriger forschung zu details dieses komplexes sind wichtige fragen noch immer offen. so ist bislang noch unbekannt, inwiefern vorbestehende schilddrüsenerkrankungen zur entstehung und ausprägung eines ntis beitragen. im rahmen der prospektiven aqua-fontis-studie untersuchten wir daher patienten, die auf einer internistischen, einer chirurgischen und einer herzchirurgischen intensivstation des universitätsklinikums bergmannsheil versorgt wurden, bezüglich des auftretens von autoantikörpern gegen thyreoglobulin (tgak), schilddrüsen-peroxidase (tpo-ak) oder tsh-rezeptoren (trak). die antikörpertiter, die wir stunden nach aufnahme auf die intensivstation bestimmten, korrelierten wir mit parametern der schilddrüsenhomöostase, der dauer des stationären aufenthaltes und dem Überleben der patienten. Über % der untersuchten patienten wiesen negative oder intermediäre tgakoder tpo-ak-titer auf, weniger als ein prozent zeigten positive antikörpertiter. ausnahmslos alle untersuchten patienten waren trak-negativ. die höhe sämtlicher antikörpertiter korrelierte weder mit dem auftreten einer thyreotropen insuffizienz noch mit der dauer des stationären aufenthaltes oder der intensivbehandlung und auch nicht mit dem Überleben der betroffenen patienten. auch in der subgruppe der überlebenden patienten korrelierten die antikörpertiter nicht mit der dauer der behandlung. es kann daher festgestellt werden, dass bei kritisch kranken personen schilddrüsenautoantikörper mit annähernd der gleichen prävalenz wie bei einer normalpopulation auftreten. darüber hinaus haben autoimmunthyreopathien keinen stellenwert in der prognose von patienten mit ntis. typ- -diabetes mittels ekg identification of markers for prediction of the clinical course remains a major challenge in the management of diabetic nephropathy. we established a proteomics approach for identification of diabetic nephropathy related biomarkers in urine. we used seldi-tof mass spectrometry and sax protein arrays to compare protein profiles from urine of four defined patient groups. samples from patients with type diabetes (dm) (n = ) without nephropathy and without microalbuminuria (dm-wnp), dm patients with macro-or microalbuminuria (dm-np) (n = ), patients with proteinuria due to non-diabetic renal disease (n = ), and healthy controls (n = ) were analysed. anionic exchange, reversephase fractionation, gel electrophoresis, and mass spectrometry were used to isolate and identify proteins with high discriminatory power. a protein with m/z (p < . ) was strongly released in the urine of healthy controls, patients with proteinuria due to non-diabetic disease, and dm-wnp in contrast to dm-np-patients. a m/z protein (p < . ) was selectively excreted in the urine of dm-np patients, whereas the protein with m/z (p < . ) was significantly excreted by patients with proteinuria and dm-np. the m/z and m/z mass peaks were identified as beta- -microglobulin and uba , an ubiquitin ribosomal fusion protein respectively. the protein with m/z was identified as a processed form of ubiquitin. moreover the ubiquitin degradation assay confirmed the potential role of a urinary protease whose absence was specific for diabetic nephropathy. the release of high amounts of uba in urine of dm-np patients could serve as a diagnostic marker. the identification of the protease and longitudinal studies in larger patient groups will determine the usefulness of the short form of ubiquitin as a marker for predicting the clinical course and the potential role of the protease in the pathophysiology of diabetic renal involvement. rolle von freien fettsäuren und fettsäuretransportproteinexpression in der apoptosevermittelten pathogenese der fettlebererkrankung extravascular lung water index (elwi) has been demonstrated to predict mortality and to correlate to pao /fio -ratio and compliance in patients with sepsis and ards. however, with an increasing number of obese patients, there is the question which body weight should be used for indexation of extravascular lung charité -universitätsmedizin berlin, campus virchow-klinikum, berlin; koordinierungszentrum für klinische studien würzburg therefore it was the aim, to investigate the correlation of elwi to pao / fio -ratio and oxygenation index (mean airway pressure* /pao ) using different weight parameters for indexation. methods: in patients of a medical icu with a body mass index > kg/m , measurements of extravascular lung water were performed using the picco system elwi correlated to the functional parameters with high significance (p< . ) independently of the the index used: correlation to pao /fio -ratio conclusions: .) in obese patients, extravascular lung water and its indices adjusted to abw, pbw, ibw and adpw significantly (p< . ) correlate to pao / fio -ratio and oxygenation index. .) the highest correlation to pao /fio -ratio was found using adbw unsere fallsammlung zeigt den facettenreichtum seltener pilzinfektionen. weitere zentren sind eingeladen, ihre fälle unter www.fungiscope.net zu registrieren ps , ps , ps ps , ps , ps ps , ps , ps ps , ps , ps , ps ps trinkmann, frederik . . ps , ps ps van den elsen antithrombozytäre effekte von ace-hemmern und at -blockern: modifizierte ex-vivo-plättchenaggregation bei kardiovaskulären patienten a. viktor , i. tuleta , m. steinmetz , g. bauriedel , g. nickenig , d. skowasch abteilung für kardiologie und pulmologie, zentrum für innere medizin, stuttgart; innere medizin (kardiologie/pneumologie), universitätsklinikum bonn, medizinische klinik ii, bonn; innere medizin (kardiologie), klinikum meiningen, medizinische klinik i, meiningen hintergrund: ace-hemmer und at -rezeptorblocker (arbs) sind eckpfeiler in der therapie kardiovaskulärer patienten. in mehreren randomisierten und plazebo-kontrollierten studien konnte eine signifikante verminderung von kardiovaskulärer mortalität, myokardinfarkt-und schlaganfallrate nachgewiesen werden. vor diesem hintergrund untersucht diese studie mögliche antithrombozytäre effekte von ace-hemmern und arbs; vergleichskollektive sind patienten mit ass/clopidogrel und unbehandelte patienten. methoden: proben von insgesamt kardiovaskulären patienten wurden mittels vollblutaggregometrie analysiert. dabei war die agonisten-induzierte plättchenaggregation (adp, kollagen) durch die zunahme der impedanz (ohm) quantifiziert. die daten wurden mit vorliegen bzw. fehlen der medikation korreliert. ergebnisse: als zentraler befund war die plättchenaggregation bei studienteilnehmern mit ace-hemmern, arbs, ass und clopidogrel vermindert. die kollagen-induzierte plättchenaggregation wurde unter ace-hemmern ( , %, p< , ) und arbs ( , %, p< , ) signifikant reduziert; unter therapie ass ( , %, p< , ) bzw. ass/clopidogrel ( , %, p= , ) nahm sie ebenfalls ab. nach adp-induktion war die plättchenaggregation unter therapie mit ace-hemmern ( , %, p= , ) und arbs ( , %, p= , ) signifikant reduziert; unter ass ( , %, p= , ) und ass/clopidogrel ( , %, p= , ) zeigte sich ebenfalls eine reduktion. eine zusätzliche verlaufsbeobachtung nach neueinstellung mit dem wirkstoff valsartan zeigte nach tagen eine signifikant reduzierte kollagen-induzierte plättchenaggregation ( %, p= , ). eine in vitro-messreihe mit der rohsubstanz valsartan konnte für äquivalente therapeutische dosen keine signifikante beeinflussung zeigen. folgerung: die therapie mit arbs und ace-hemmern führt zu einer signifikanten verminderung der plättchenaggregation ex vivo. das antithrombotische potential der arbs und ace-hemmer könnte für die reduktion der konsekutiven thrombosen und der progression atherosklerotischer organleiden mitverantwortlich sein und so die reduktion kardiovaskulärer endpunkte zumindest miterklären. kombination einer dendritischen zellvakzine mit gemcitabin im murinen pankreaskarzinom:charakterisierung der immunantwort und wirksamkeit m. dauer with or without the tlr- ligand cpg before (prophylactic) or after (therapeutic) tumor induction. cd + t cell responses were analyzed in peripheral blood. antigen uptake, activation and cytokine production of dendritic cells (dc) in lymph nodes was analyzed by flow cytometry. data: vaccine draining lymph nodes increased in size and cellularity. the iscom vaccine was effectively taken up by dc, resulting in upregulation of activation markers, production of il- and potent t cell stimulation. key: cord- -r wkx ml authors: jacobs, sophie; wavreil, fanny; schepens, bert; gad, hans henrik; hartmann, rune; rocha-pereira, joana; neyts, johan; saelens, xavier; michiels, thomas title: species specificity of type iii interferon activity and development of a sensitive luciferase-based bioassay for quantitation of mouse interferon-λ date: - - journal: j interferon cytokine res doi: . /jir. . sha: doc_id: cord_uid: r wkx ml the type iii interferon (ifn-λ) family includes ifn-λ subtypes in man. in the mouse, only the genes coding for ifn-λ and -λ are present. unlike mouse and human type i ifns (ifn-α/β), which exhibit strong species specificity, type iii ifns were reported to act in a cross-specific manner. we reexamined the cross-specificity and observed that mouse and human ifn-λ exhibit some species specificity, although much less than type i ifns. mouse ifn-λ displayed clear species specificity, being -fold less active in human cells than the closely related mouse ifn-λ . this specificity likely depends on amino acids in α helices a and f that diverged from other ifn-λ sequences. human ifn-λ , in contrast, retained high activity in mouse cells. we next developed a firefly luciferase-based reporter cell line, named fawa-λ-luc, to detect ifn-λ in biological fluids with high specificity and sensitivity. fawa-λ-luc cells, derived from mouse epithelial cells that are responsive to ifn-λ, were made nonresponsive to type i ifns by inactivation of the ifnar gene and strongly responsive to ifn-λ by overexpression of the mouse ifnlr . this bioassay was as sensitive as a commercially available enzyme-linked immunosorbent assay in detecting mouse ifn-λ in cell culture supernatant, as well as in serum and bronchoalveolar lavage samples of virus-infected mice. the assay also enabled the sensitive detection of human ifn-λ activity, including that of the divergent ifn-λ with a bias, however, due to variable activity of ifn-λ subtypes. t ype i and iii interferons (ifns) are typically produced in response to viral infection, and these cytokines induce an antiviral state in target cells (isaacs and lindenmann ; kotenko and others ; sheppard and others ) . type i ifns consist of ifn-a subtypes and ifn-b, -o (human) , and -z (mouse) -e and -k. the type iii family of ifns in humans comprises functional genes that express ifn-l (il- ), ifn-l (il- a), and ifn-l (il- b) (kotenko and others ; sheppard and others ) . a fourth ifn-l subtype (ifn-l ) is present in a part of the human population, depending on a dinucleotide frameshift polymorphism upstream of the ifnl gene, which creates or disrupts the open reading frame (orf) encoding ifn-l (prokunina-olsson and others ) . in the mouse, ifnl is a pseudogene, and only ifn-l and ifn-l are expressed. unlike mouse and human type i ifns that exhibit strongly species-specific activity (veomett and veomett ) , type iii ifns were reported to act on cell types from both origins (lasfar and others ; hermant and others ) . the type i ifn family members signal through a unique heterodimeric receptor (ifnar), composed of the ifnar and ifnar subunits. type iii ifns engage a distinct receptor (ifnlr), composed of chains: the ifn-l-specific ifnlr , and il rb which is shared by other il -related cytokines (kotenko and others ; sheppard and others ) . while ifnar is ubiquitously expressed, ifnlr is expressed by a restricted range of cell types and mostly acts at mucosal surfaces. epithelial cells are well-established targets of ifn-l in vivo (sommereyns and others ) , although some immune cells such as neutrophils and dendritic cells have been characterized as ifn-l responders (koltsida and others ; blazek and others ; broggi and others ; espinosa and others ) . although type i and type iii ifns act on distinct receptors, they activate a similar jak-stat transduction pathway, leading to the phosphorylation of stat and stat that associate with irf to form the isgf complex. type i and type iii ifn signaling leads to the transcription of an overlapping set of ifn-stimulated genes (isgs) (dumoutier and others ; ank and others ) . type i ifn in biological samples can be measured by a variety of bioassays, but efficient techniques for type iii ifn quantification are largely lacking. enzyme-linked immunosorbent assay (elisa) for ifn-l detection is time and cost intensive and fails to detect ifn-l . conventional cytopathic effect reduction bioassays to quantify antiviral activity as a proxy for the presence of ifn require the manipulation of infectious virus. luciferase reporter cells that have previously been used for recombinant human ifn-l detection were still responsive to type i ifn and would not allow specific ifn-l detection from biological samples (uze and monneron ) . in this work, we reexamined the species specificity of ifn-l activity, and we developed a very sensitive luciferase-based bioassay specific for type iii ifn detection. the lkr cell line (kind gift from guido bommer, de duve institute, brussels, belgium) is derived from lung adenocarcinoma tissues from a k-rasla mouse ( johnson and others ) . a cells (atcc) were kindly provided by pierre coulie, balb/ t fibroblasts (aaronson and todaro ) those cells, and derivatives, were maintained in dulbecco's modified eagle's medium (dmem) (lonza, vervier, belgium) containing . g/l glucose, supplemented with % fetal calf serum (fcs) (sigma-aldrich, overijse, belgium). african green monkey kidney (vero) cells (atcc) were cultured in dmem supplemented with % fcs, nonessential amino-acid, l-glutamine, and sodium pyruvate. bhk- cells (atcc) were cultured in glasgow's minimum essential medium (gibco; thermo fisher scientific, asse, belgium) supplemented with % newborn calf serum and . g/l tryptose phosphate broth. all media were supplemented with u/ml penicillin and mg/ml streptomycin (lonza). expression vectors used in this study are listed in table . plasmids psj and psj are pcdna derivatives (invitrogen; thermo fisher scientific) used for mouse ifn-l and ifn-l expression in t cells. these plasmids were constructed by cloning the orf encoding ifn-l (psj ) and between the bamhi and xbai sites of the vector. coding sequences were polymerase chain reaction (pcr)-amplified from liver cdna samples of a c bl/ mouse infected with influenza turh (h /n ) strain (hermant and others ) , using the following primers: forward (fw): bamhi-agei-kozak-mifn-l / , ¢-aaa agg atc cac cgg tgc cac cat gct cct cct gct gtt gcc tct g- ¢; reverse (rev): mifn-l / -stop-xbai, ¢-aaa atc tag att atc aga cac act ggt ctc cay tgg c- ¢. the sequence of psj matches the genomic ifnl sequence but diverges from a few nucleotides from that of the previously constructed pcs plasmid (sommereyns and others ) . both plasmids encode functional ifn-l . pph was obtained by pcr-cloning the ifna orf from hela-m cell cdna, between the ecori and noti sites of pcdna (fw: ecori-kozak-ifna , ¢-aaa aga att cac cat ggc ctt gac ctt tgc ttt- ¢; rev: ifna - noti, ¢-aaa agc ggc cgc tca ttc ctt act tct taa act tt- ¢). pmk is a lentiviral vector, derived from ptm , that carries the mouse mx gene promoter driving the expression of the firefly luciferase gene. it is derived from pcclsin. cppt.hpgk.gfp.pre (follenzi and others ) in which a cloning polysite was first inserted to replace the coding sequences, yielding ptm . the firefly luciferase gene from pgl . (promega) was inserted between the xhoi and xbai sites of the vector, and the mx promoter was then cloned from pbsk-mx , a gift from peter staeheli (freiburg university, freiburg, germany), as a bamhi/bsabi fragment. the lentiviral vector psj , used for expression of the mouse ifn-l receptor, was obtained by cloning the ifnlr orf between the bamhi and xbai sites of ptm , a lentiviral vector allowing the coexpression of the cloned gene and of mcherry (hermant and others ) . the ifnlr orf sequence was subcloned in this plasmid from pcr . -licr , kindly provided by laure dumoutier (de duve institute, brussels, belgium). gene inactivation was done with px plasmid (pspcas n- a-gfp) coding for the cas nickase and green fluorescent protein (gfp) (ran and others a). the single guide rnas (sgrnas) were designed using the mit clustered regularly interspaced short palindromic repeats (crispr) design tool website (http://crispr.mit.edu). the selected sgrna pair (sgrna -fw: ¢-cac cgt caa att ctg gcg gct caa g- ¢; rev: ¢-aaa cct tga gcc gcc aga att tga c- ¢; sgrna -fw: ¢-cac cga gac cac ata aac gtg acg a- ¢; rev: ¢-aaa ctc gtc acg ttt atg tgg tct c- ¢) was targeting exon of the mouse ifnar gene (genbank: y . ) and exhibited no expected off-target cleavage site. the sgrnas were cloned into px to form the plasmids psj and psj . these constructs were cotransfected in lkr cells, using transit Ò -lt transfection reagent (mirus bio llc, madison), according to the manufacturer's instructions. after h, gfp-positive cells were sorted by fluorescence activated cell sorting (facs aria iii; bd biosciences) and cloned in -well plates. clones were screened for loss of type i ifn response with an antiviral assay. genome editing of the targeted exon was further confirmed by sequencing (genewiz). isg expression was measured by reverse transcriptionquantitative pcr. ifnar -knockout (ko) and wild-type (wt) lkr cells were treated with u/ml mouse ifn-aa, pg/ml mouse ifn-l , or control supernatant (mock) for h before rna extraction. total rna extraction, reverse transcription, and sybr green quantitative pcr for mrna encoding mouse b-actin, oasl , and usp were performed as previously described (paul and michiels ) . for infection analysis, cells were dissociated with trypsin-edta and suspended in phosphate-buffered saline (pbs) containing % of filtered fcs and . % of paraformalde-hyde. data acquisition was done with an lsrfortessa flow cytometer (bd biosciences) using facsdiva software. data were analyzed using flowjo . . . the rate of infection was defined as the percentage of mcherry-positive cells h postinfection with . pfu/cell tm . for cell sorting, transduced cells were suspended in pbs containing % fcs and mm edta. mcherry-or gfppositive cells were cloned at cell per well in -well plates using the facs aria iii (bd biosciences). immunoblotting stat , p-stat , and b-actin were detected by western blot using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing % acrylamide and run in a tris-glycine buffer. blots were probed with anti-stat polyclonal (sc- ; santa cruz biotechnology, heidelberg, germany), p-stat monoclonal (# ; cell signaling technology, leiden, the netherlands), and anti-b-actin monoclonal (a ; sigma-aldrich) antibodies. mouse ifn-aa, ifn-b, ifn-l , ifn-l , and human ifn-a were produced as described previously from t cells transfected with pcdna -ifn-aa, pcdna -ifn-b (van pesch and others ), pcdna -ifn-l (psj ), and pcdna -ifn-l (psj ). human ifn-a was produced similarly, using pcdna -ifn-a (pph ). supernatant collected from t cells transfected in parallel with the empty pcdna vector was used for control treatment (mock) of cells and diluted as for ifns. recombinant mouse ifn-l ( -ml- ) and human ifn-l ( -il- ), ifn-l ( -il- ), and ifn-l ( -il- ) were purchased from r&d (r&d systems, minneapolis). recombinant human ifn-l was produced as described (hamming and others ) . mouse (ref - ) and human (ref - ) ifn-g were purchased from pe-protech (london, united kingdom). genbank accession numbers for mouse and human ifn-l are listed in table . ifns were diluted in culture medium for cell treatment or in reagent diluent for elisa. mouse ifn-l and ifn-l were quantified by elisa. mouse ifn-aa and human ifn-a antiviral activities were quantified, as described previously, by a cytopathic effect reduction assay in mouse balb- t and human hela-m cells, respectively, using mengovirus (van pesch and michiels ) . similar assays were conducted in human a cells and mouse lkr cells for quantification of ifn-l cross-species activity. ifn- l was quantified by a cytopathic effect reduction assay in a cells, using recombinant human ifn-l as a standard, which was reported to have a similar specific activity (hamming and others ). mouse ifn-l / measurement by elisa was performed using the mouse ifn-l / duoset (r&d systems), according to the manufacturer's protocol. mouse sera were diluted -to -fold, and bronchoalveolar lavage fluids (balfs) were diluted -fold. when required (in case of samples derived from infected cells or mice), infectious virus present in biological samples was ultraviolet light (uv)-inactivated. for uv inactivation, . mm thick fluid samples were exposed at fixed uv dose ( . - . - - j/cm ) with the uv irradiation system biolink (vilber lourmat, eberhardzell, germany), either in -( ml) or in -well ( ml) plate. uv-exposed samples were titrated on bhk- cells by a standard plaque assay to confirm virus inactivation. to confirm that the uv irradiation procedure that was established for picornaviruses effectively inactivates respiratory syncytial virus (rsv), samples of ml containing . · pfu of rsv were exposed to j/cm with the uv irradiation system bio-link at room temperature in a -well plate (uv irradiated samples). as controls similar samples of ml containing . · pfu of rsv were incubated at room temperature in a -well plate (untreated samples). the titers of replicating rsv in the uv irradiated and untreated samples were determined by plaque assay using vero cells. cells were seeded at , cells per well in -well plates for cell supernatant and recombinant ifn analysis or at , cells per well in -well plates for mouse serum and bal analysis. forty-eight hours after seeding, cells were treated with ifn-l / or mock-treated or incubated with uv-treated mouse serum ( -and -fold dilutions) or bal ( -fold dilutions). samples were diluted in culture medium. recombinant mouse ifn-l provided in the elisa kit was used as a standard. firefly luciferase activity was measured h after treatment using the luciferase assay system (promega). twenty microliters and -ml lysis buffer were used in -and -well plates, respectively; ml of the lysate was mixed with ml of substrate for detection. luminescence was measured with a glomax / luminometer (promega). the limit of detection (lod) for each test was established based on the mean of mock-treated samples, plus standard deviations, in all assays. tm is a theiler's murine encephalomyelitis virus (tmev) (strain da) derivative that carries a capsid adapted to infect l cells and the orf encoding the mcherry fluorescent protein cloned as a xbai/bsiwi fragment to replace codons - of the leader protein coding region, in the pkj vector ( jnaoui and michiels ) . mengovirus (a strain of encephalomyocarditis virus) used for ifn bio-assay is an attenuated variant that has been described previously (hermant and others ) . those viruses were quantified by plaque assay on bhk cells. rsv propagation and enrichment were performed as described in schepens and others (schepens and others ) . mouse experiments were conducted according to the national (belgian law / / and / / , belgian royal decree / / ) and european (eu directives / /eu, / /eeg) animal regulations. animal protocols were approved by the ethics committee of ghent university (permit no. la , approval id - ). all efforts were made to avoid or ameliorate suffering of animals. specific pathogen-free female balb/c mice at the age of - weeks were purchased from charles river. the mice were housed in a specific-pathogen-free temperature controlled environment with -h light/ -h dark cycles and given water and food ad libitum. mice were used at weeks of age after adaptation in the animal room for week. for challenge, the mice were sedated with isoflurane and infected intranasally with · pfu of human rsv. mock infection of the control group was performed with hank's balanced salt solution. five days postinfection, bal was isolated under anesthesia with an intraperitoneal injection of avertin ( . % in pbs). a gauge cannula was inserted into the trachea, and cells were collected by washing the airway lumen twice with . ml pbs containing . mm edta. after removing the cells by centrifugation ( min at g), the balf was stored at - °c. serum samples from norovirus-infected and plasmidelectroinjected mice were reused from a previously published experiment (rocha-pereira and others ) to minimize animal experimentation. statistical analysis was performed using prism software (graphpad software, san diego, ca). [***p < . , **p < . , *p < . , and ns no statistically significant difference (p ‡ . )]. unlike mouse and human type i ifns (ifn-a/b) that exhibit strong species specificity, type iii ifns were reported to act in a cross-species manner. to confirm whether a mouse cellbased bioassay would permit the detection of ifn-l subtypes from different mammalian species, the species specificity of mouse and human type iii ifns was reexamined. crossreactive antiviral activity was systematically compared in a cytopathic effect reduction assay, using epithelial cell lines from mouse (lkr ) and human (a ) origin. as expected, type i human (ifn-a ) and mouse (ifn-aa) ifns were extremely species-specific in the antiviral assay, with an activity difference higher than , -fold when applied to mouse and human cells. ifn-l exhibited some level of species specificity although much less than type i ifns (fig. a) . mouse ifn-l displayed the most pronounced species specificity. for a quantity that yielded the same antiviral activity in mouse cells, ifn-l was times less active in human cells than the closely related mouse ifn-l . human ifn-l , ifnl- and, to a lesser extent, ifnl- exhibited significant antiviral effect on mouse epithelial cells. as ifn-l has been shown to have a specific activity similar to that of human ifn-l in human cells, it was quantified accordingly using the antiviral assay (hamming and others ) . remarkably, human ifn-l displayed much more antiviral activity on mouse cells than equivalent amounts of human ifn-l . accordingly, treatment of lkr cells with concentrations of human ifn-l and ifn-l that yielded equivalent antiviral activities in human cells resulted in clear stat phosphorylation after ifn-l but not after ifn-l treatment (fig. b) . in line with their epithelial origin, mouse lkr cells respond to ifn-l. to ensure a selective detection of type iii ifns, the gene coding for the ifnar subunit of the type i ifn receptor was inactivated using the double nickase crispr/crispr-associated (cas) technology (ran and others b). an lkr -ifnar -ko clone was selected for the absence of response to type i ifn and intact response to type iii ifn. sequencing of the targeted exon revealed frameshift mutations in both ifnar alleles, resulting in premature stop codon appearance. in contrast to ifn-l treatment, ifn-a treatment of this clone failed to induce the expression of the ifn-stimulated genes, oasl ( fig. a) and usp (fig. b) , and to protect against infection with tm , a tmev derivative expressing mcherry (fig. c) . to develop ifn-l reporter cells, the lkr -ifnar -ko clone was transduced with a lentiviral vector encoding the firefly luciferase gene under the control of the mx pro-moter. among the clones tested for luciferase activity induction after ifn-l treatment, the best one displayed not more than -fold luciferase activity induction. to try to boost the reporter gene responsiveness to ifn-l, this clone was transduced with sj , a lentiviral vector that coexpresses the mouse ifnlr and mcherry. transduced cells were sorted, based on mcherry expression, and cloned. a selected clone, named ''fawa-l-luc,'' responded vigorously to ifn-l . this clone displayed increased constitutive stat expression and strong stat phosphorylation after ifn-l but not after ifn-a treatment (fig. d) . a significant luciferase activity increase was measured in fawa-lluc cells as soon as h after treatment with pg/ml (quantified by elisa) of mouse ifn-l . maximal induction was reached within h and lasted up to h posttreatment (fig. a) . the sensitivity of the fawa-l-luc assay was compared to that of an available elisa test for mouse ifn-l. mouse ifn-l and - produced by transfected t cells and recombinant mouse ifn-l were quantified in parallel by elisa and the fawa-l-luc assay. in the elisa, responses were linear between , and . pg/ml, the experimental lod of this test (fig. b ). in the fawa-l-luc assay, linearity was reached in a narrower concentration range ( . - . pg/ml for mouse ifn-l ; . - pg/ml for mouse ifn-l ), but a higher sensitivity was observed with the detection limit being below . pg/ml and . pg/ml, for ifn-l and - , respectively (fig. c, d) . based on elisa quantification, mouse ifn-l bioactivity in this assay was -fold higher than that of mouse ifn-l . importantly, after up to passages, the intensity of the luciferase signal in the fawa-l-luc cells tended to decrease, but the sensitivity of the bioassay was preserved because table showing the relative antiviral activity of mouse and human ifn-l on mouse lkr and human a cells. species specificity index was calculated as the ratio between the relative antiviral activity on cells of homologous to nonhomologous species (eg, for mouse ifn: relative activity in mouse cells/relative activity in human cells). relative activities in a given cell line were calculated as the ifn dilutions (starting from ng/ml) that yielded similar antiviral activities. *due to the low amount of ifn-l available, the initial concentration of this ifn was estimated by comparison with human ifn-l antiviral activity that was reported to have a similar specific activity. (b) western blot showing stat phosphorylation in mouse lkr cells min after treatment with control medium (mock) or ifn-l. concentrations of human (hu) ifn-l ( pg/ml) and ifn-l that yielded the same antiviral activity on human a cells were used. as a control, mouse ifn-l was used at a concentration ( . ng/ml) showing equivalent antiviral activity as human ifn-l on mouse cells. results are representative of experiments. the background activity also decreased with high passage numbers. to confirm the specificity of fawa-l-luc cells for ifn-l, we also evaluated any possible luciferase induction after treatment with type i and type ii (ie, ifn-g) ifns. as expected, no signal was observed after treatment with up to , iu/ml of either mouse ifn-aa or ifn-b. human ifn-g also failed to induce a detectable luminescent signal in the fawa-l-luc cells in the tested concentration range ( - ng/ml). the lod for mouse ifn-g was . ng/ml, a concentration at least -fold higher than what is observed in mouse fluids in infectious contexts (pomeroy and others ; claser and others ) . ifn-g is thus not expected to interfere with ifn-l detection in biological samples. we tested the sensitivity of our bioassay for human ifn-l detection. responses were analyzed individually for all human ifn-l subtypes (fig. a-d) . as their cross-species activity differed, different detection sensitivities were reached with our mouse cell-based reporter assay: . pg/ml for ifn-l , pg/ml for ifn-l , . pg/ml for ifn-l , and . pg/ml for ifn-l . the bioassay turned out to be extremely sensitive for human ifn-l detection. it is, however, worth noting that the recombinant ifn-l stock concentration was determined by comparison of its activity with that of a human ifn-l standard. when testing biological samples, potential occurrence of infectious virus may interfere with the viability of reporter cells. it is therefore important to inactivate infectious viruses in such samples. as ifn-l had previously been shown to be acid labile (kugel and others ) , uv inactivation of the virus was preferred. we thus tested whether virucidal uv doses would affect ifn-l activity. to this end, tm virus, mouse ifn-l , and mouse ifn-l were irradiated with increasing uv doses. mock-and uv-exposed samples were then analyzed with the bioassay for mouse ifn-l and ifn-l detection and by plaque assay to determine viral titers. a j/cm uv exposure was sufficient to ensure a virus titer reduction of more than , -fold ( . · pfu to < pfu). at this uv dose, mouse ifn-l activity was not significantly reduced and mouse ifn-l showed a bioactivity reduction of less than % (fig. a) . similarly, ifn-l and ifn-l bioactivities were reduced by %, while human ifn-l and ifnl- were slightly more sensitive to irradiation, with about -fold activity decrease (fig. b) . we then compared the sensitivities of the fawa-l-luc and of the elisa assays to detect ifn-l in mouse serum and bal samples. ifn-l was first quantified in the serum of mice that were either injected intramuscularly with an ifn-l expressing plasmid (pcs ), thus expected to produce circulating ifn-l, or infected with mouse norovirus (rocha-pereira and others ). a preliminary test revealed that the minimal serum dilution that did not interfere with spiked-in ifn-l detection was fold for the bioassay and -fold for the elisa (fig. c, d) with both the fawa-l-luc and elisa assays. ifn-l was clearly detected at day and in the serum of mice that were electroinjected with the ifn-l expressing plasmid, as well as at day postnorovirus infection (fig. a, b) . at day after a single ifn-l injection, the cytokine was detectable in out of mice by elisa and in out of mice by the bioassay. the detection limit was higher for the elisa ( pg/ml) than for the bioassay ( . pg/ml), showing the higher sensitivity of the luciferase-based bioassay (fig. a, b) . next, we compared ifn-l detection by the fawa-l-luc assay and by elisa in balf of balb/c mice that were mock infected or infected with rsv. balf and lungs were collected days postinfection. plaque assay revealed that balf of all infected mice contained between . · and . · pfu/ ml of live replicating virus. the used uv irradiation procedure proved to readily inactivate rsv (rsv titer reduction of more than , -fold; . · pfu to < pfu). five-fold diluted control balf did not modify ifn-l / detection in any of the assays. using -fold dilutions of the balf as in the bioassay, we failed to detect any ifn-l by elisa (fig. c ). in contrast (fig. d) , ifn-l was detected by fawal-luc assay at a very low concentration in the balf of the rsv infected mice ( / ) and not detected in the control group. percentage of ifn-l activity (mean and sd) remaining after uv treatment (n = ) at j/cm . ifn activity was measured in fawa-l-luc cells for ifn concentrations that yielded equivalent luciferase activities ( , rlu) before uv treatment ( pg/ml mifn-l and mifn-l , pg/ml human ifn-l , ng/ml huifn-l , pg/ml huifn-l , and . pg/ml huifn-l ). (c, d) influence of serum dilution on ifn-l detection by fawa-l-luc cells (c) and elisa (d). (c) fawa-l-luc cells were treated in triplicate with fixed doses of and pg/ml mifn-l supernatant and with -fold serial dilutions ( - -fold) of control mouse serum. (d) pg/ml recombinant mifn-l was mixed with -fold serial dilutions of control mouse serum ( . - -fold) before detection by elisa. (a-c) reporter cells were exposed to ifn for h before luciferase assay. student's t-test: */**/***denotes a significant difference in signal compared to no uv exposure (a) or the absence of serum (c). uv, ultraviolet light. we conclude that the fawa-l-luc-based assay described in this study allows to quantify ifn-l from biological samples in a highly sensitive way. we reexamined the species specificity of ifn-l and highlighted a previously overlooked difference in bioactivity between mouse and human type iii ifns. mouse ifn-l displayed a surprising species specificity, with a -fold difference in relative antiviral activity when applied to lkr (mouse) and a (human) cells. this contrasted strongly with the closely related mouse ifn-l ( % amino acid sequence identity), which displayed little species specificity and was times more active in human cells than mouse ifn-l . in contrast, human ifn-l displayed a strikingly strong activity on mouse cells compared to human ifn-l . although ifn-l is not expressed in mouse and rat, it is highly conserved in many mammalian species (key and others ) where it was shown to be cross-reactive. the nonhuman ifn-l orthologs were reported to activate ifnlr in human cells, sometimes with a higher efficacy than human ifn-l (eg, dog ifn-l ) (paquin and others ). the mouse cell response to human ifn-l is thus in line with the ability of ifn-l to be cross-reactive among mammalian species, although we do not have any physiological explanation for this phenomenon. ifn-l proteins follow the typical class ii cytokine structure, consisting of secondary structure elements (a-f) (pestka and others ) . helix a is involved in both ifnlr and il rb binding, while helix d binds il rb and helix f binds ifnlr (gad and others ). among amino acid residues that have been characterized as crucial for ifnlr binding and activation (gad and others ), a few divergences are observed between sequences from mouse and human ifn-l (fig. ) . notably, the more species-specific mouse ifn-l has amino acid residues in a helices a and f that diverge from the other ifn-l sequences, including the related mouse ifn-l . in helix a, gly uniquely replaces the asp residue present in the other sequences. in helix f, residues are unique to mouse ifn-l : asp (ala / asp), gln and bioassay (b) in the serum of ag mice or days after electroinjection of mouse ifn-l (mifn-l ) expressing (pcs ) or empty (pcdna ) plasmids, days after injection of pcs , or days after infection with mouse norovirus. ifnl detection in the serum by elisa was performed without uv exposure to keep maximal sensitivity. (c, d) ifn-l / detection by elisa (c) and bioassay (d) in the bronchoalveolar lavage of balb/c mice, days postinfection with rsv, compared to control mice (mock). balf were uv-exposed before testing. (a-d) the horizontal dotted line represents the lod. mann-whitney: */**indicates a significant difference compared to pcdna group at days or (a, b) or compared to mock (d). balf, bronchoalveolar lavage fluid; rsv, respiratory syncytial virus. (arg / gln), and leu (thr / leu). future site-directed mutagenesis studies could help defining the key residues involved in the species specificity. we designed a highly sensitive bioassay named ''fawa-lluc'' based on luciferase reporter cells, specific for ifn-l detection and quantification. the bioassay is based on a cell line that is naturally responsive to ifn-l in which the type i ifn receptor gene was inactivated. overexpression of the mouse ifnlr receptor in those cells led to increased induction of the reporter gene after ifn-l treatment. unlike previously described bioassays, such as hl- fibroblasts stably transfected with the human ifnlr (uze and monneron ), fawa-l-luc cells permit the selective detection of type iii ifn because they lack the type i ifn receptor. they also fail to respond to ifn-g concentrations that are reached in infected mouse serum. this novel bioassay is efficient in measuring ifn-l from mouse bal and serum. ifn-l activity was hardly affected by uv exposure at virucidal doses, thus allowing detection in infected biological fluids. the high sensitivity of the assay permits sparing biological material as -fold more diluted serum samples could be used for bioassay detection ( - -fold dilution) compared to the elisa ( - -fold). when cell toxicity of the tested samples is suspected, cell viability may independently be confirmed with nonlytic viability assays such as resazurin-based tests, which are compatible with luciferase detection. importantly, our bioassay offers a physiologic analysis, attesting of the functionality of the ifn-l present in the samples, irrespective of specific activity. the bioassay also allows to detect human ifn-l. given the divergent cross-species activity and the higher uv lability of the human type iii ifns, the bioassay might be used for individual subtype analysis, but would not fit for their detection in complex biological samples. finally, it offers a sensitive detection of the divergent ifn-l , for which no efficient commercial test is available. development of t -like lines from balb-c mouse embryo cultures: transformation susceptibility to sv lambda interferon (ifn-lambda), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo ifn-lambda resolves inflammation via suppression of neutrophil infiltration and il- beta production ifn-lambda suppresses intestinal inflammation by non-translational regulation of neutrophil function host resistance to plasmodiuminduced acute immune pathology is regulated by interleukin- receptor signaling basis for regulated rna cleavage by functional analysis of rnase l and ire p role of the interleukin (il)- receptor tyrosine residues for antiviral and antiproliferative activity of il- /interferon-lambda : similarities with type i interferon signaling type iii interferon is a critical regulator of innate antifungal immunity gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by hiv- pol sequences the structure of human interferon lambda and what it has taught us interferon lambda signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses ifn-l sequence alignments. sequences of human and mouse ifn-l regions implicated in receptor binding were aligned. key amino acid residues involved in receptor activation that differs between mouse and human ifn-l and ifn-l sequences are indicated in bold in mouse sequences. residues unique to mouse ifn-l in helices a and f are indicated in bold red. *indicates identical amino acids between human ifn-l and ifn-l human but not mouse hepatocytes respond to interferon-lambda in vivo ifnepsilon is constitutively expressed by cells of the reproductive tract and is inefficiently secreted by fibroblasts and cell lines virus interference. i. the interferon adaptation of theiler's virus to l cells: mutations in the putative receptor binding site on the capsid map to neutralization sites and modulate viral persistence somatic activation of the k-ras oncogene causes early onset lung cancer in mice selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda (ifnl ) il- a (ifn-lambda ) modulates lung dc function to promote th immune skewing and suppress allergic airway disease ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex novel nonviral bioassays for mouse type i and type iii interferon characterization of the mouse ifn-lambda ligand-receptor system: ifn-lambdas exhibit antitumor activity against b melanoma comparative functional analysis of mammalian ifn-lambda orthologs cardiovirus leader proteins are functionally interchangeable and have evolved to adapt to virus replication fitness interferons, interferonlike cytokines, and their receptors role of interferon-gamma in murine cytomegalovirus infection a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus double nicking by rna-guided crispr cas for enhanced genome editing specificity genome engineering using the crispr-cas system interferon lambda (ifn-lambda) efficiently blocks norovirus transmission in a mouse model protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein il- , il- and their class ii cytokine receptor il- r ifnlambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo il- and il- : newcomers to the interferon family characterization of the murine alpha interferon gene family characterization of interferonalpha , a novel constitutive murine interferon-alpha subtype species specificity of interferon action: maintenance and establishment of the antiviral state in the presence of a heterospecific nucleus the authors are grateful to nicolas dauguet for his help in flow cytometry (ludwig institute for cancer research). work was supported by the eos joint programme of fonds de la recherche scientifique-fnrs and fonds wetenschapellijk onderzoek-vlaanderen-fwo (eos id: ), by interuniversitary attraction poles program initiated by the belgian science policy office (iap-p / belvir), by actions de recherche concertées (arc), and by a pdr grant (t. . ) of fnrs to t.m., and b.s. is a doctoral assistant at the department of biomedical molecular biology of ghent university. no competing financial interests exist. key: cord- -utsvsja authors: uematsu, takayuki; iizasa, ei’ichi; kobayashi, noritada; yoshida, hiroki; hara, hiromitsu title: loss of card -mediated innate activation attenuates severe influenza pneumonia without compromising host viral immunity date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: utsvsja influenza virus (ifv) infection is a common cause of severe viral pneumonia associated with acute respiratory distress syndrome (ards), which is difficult to control with general immunosuppressive therapy including corticosteroids due to the unfavorable effect on viral replication. studies have suggested that the excessive activation of the innate immunity by ifv is responsible for severe pathologies. in this study, we focused on card , a signaling adaptor known to regulate innate immune activation through multiple innate sensor proteins, and investigated its role in anti-ifv defense and lung pathogenesis in a mouse model recapitulating severe influenza pneumonia with ards. we found that influenza pneumonia was dramatically attenuated in card -deficient mice, which showed improved mortality with reduced inflammatory cytokines and chemokines in the infected lungs. however, viral clearance, type-i interferon production, and the development of anti-viral b and t cell immunity were not compromised by card deficiency. syk or card -deficient dcs but not macrophages showed impaired cytokine but not type-i interferon production in response to ifv in vitro, indicating a possible role for the syk-card pathway in dcs in excessive inflammation of ifv-infected lungs. therefore, inhibition of this pathway is an ideal therapeutic target for severe influenza pneumonia without affecting viral clearance. the host's innate immune system including macrophages and dendritic cells (dcs) to secrete inflammatory cytokines/chemokines, such as il- , tnf, cxcl and cxcl , , which play crucial roles in the pathogenesis of ards , [ ] [ ] [ ] . therefore, inhibition of cytokine/chemokine production via targeting the host's innate immune system may lead to the development of effective treatment options for ards. caspase recruitment domain family member (card ) is an adaptor protein that delivers nf-κ b activating signals through multiple innate sensor proteins, such as a wide array of c-type lectin (clr) and immunoglobulin (ig)-superfamily receptors that are coupled to immunoreceptor tyrosine-based activation motifs (itams) [ ] [ ] [ ] , as well as cytoplasmic rna sensors, such as rig-i and mda , and the dna sensor rad , . several lines of evidence have implied a role for card -mediated innate activation pathways in influenza pathogenesis and immunity. the clr dendritic cell-specific icam- grabbing non-integrin (dc-sign), which has a cytoplasmic itam-like sequence, was shown to bind influenza virus a , . additionally, card was found to be required for the production of inflammatory cytokines after infection with rna viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, by regulating nf-κ b signaling through rig-i and mda . however, the roles of card in influenza pneumonia, as well as in protection against ifv are yet to be elucidated. in this study, we demonstrate a role for card -mediated innate activation in influenza pathogenesis and immunity. our results showed that the card pathway was involved in fatal influenza pneumonia mediated by inflammatory cytokine/chemokine production, whereas it was dispensable for type-i interferon production as well as the development of anti-viral acquired immunity. therefore, inhibition of this pathway may represent an ideal target for the treatment of severe influenza pneumonia without affecting viral clearance. adapted ifv a/pr/ / strain (pr ) shows similar lung pathology to human ards - , we intratracheally infected wild-type (wt: c bl/ ) and card -/mice with a lethal dose ( pfu/mouse) of pr to determine whether card -mediated innate immune responses contributed to severe influenza pneumonia. we observed that pr -infected wt mice appeared visibly ill with ruffled fur and reduced oral intake during the to days after infection, whereas card -/mice appeared more active than wt mice. consistent with their activity, the final survival rate up to day after infection was dramatically improved in card -/mice (~ %) as compared with wt mice (~ %; fig. a ). histopathological analysis of ifv-infected lungs on days and revealed that lung inflammation, which was the most obvious on day in wt mice, was much less severe in card -/mice (fig. b) . given that inflammatory cytokines/chemokines were linked to lung damage in severe influenza pneumonia [ ] [ ] [ ] [ ] [ ] [ ] , we analyzed infiltrated cells and cytokine/chemokine levels in bronchoalveolar lavage fluid (balf) collected from the ifv-infected lungs of wt and card -/mice. consistent with the pathological data, the number of cd + balf cells were significantly fewer in the lungs of card −/− mice than in those of wt mice on day , with a marked decrease in the numbers of t cells and neutrophils ( fig. c ) that were the two main infiltrated subpopulations at this time point (supplementary fig. s ). however, the numbers of b cells, nk cells, macrophages, cdcs, and pdcs were not significantly affected by the card deficiency. the balf levels of inflammatory cytokines and chemokines, i.e., il- , tnf-α , ccl /mip- α , cxcl /kc, and cxcl /ip- , which have been reported to contribute to lung pathology , , , peaked on day and were considerably lower in the card −/− mice than in the wt mice (fig. d ). however, no reduction in cytokine/chemokine levels was evident on day , indicating that a card -mediated innate response controls cytokine/chemokine production at an early time point after ifv infection and influences subsequent inflammatory cell recruitment and lung pathology at a later time point. collectively, the loss of card attenuated severe influenza pneumonia and improved host mortality. card deficiency does not compromise anti-viral protective immunity. activation of innate immunity in response to ifv regulates anti-viral protective immunity . to evaluate the impact of card deficiency on anti-ifv protection, we first analyzed the viral burden in the ifv-infected lungs of wt and card -/mice. notably, we found that card deficiency did not increase virus titers and the amount of viral rna in the lungs, but instead significantly accelerated viral clearance in the later stage (day ) of infection ( fig. a) . type-i interferons (ifn-α /β ) are the primary factors that induce host resistance to ifv , . in contrast to cytokine/chemokine production, card deficiency did not significantly affect ifn-α /β production in ifv-infected lungs (fig. b) . the type ii interferon ifn-γ is not essential for viral clearance , but its presence during ifv infection ameliorates the severity of inflammation and lung injury . ifn-γ production was significantly more in the lungs of card −/− mice than in those of wt mice on day but not on day (fig. b) ; thus, this increase might contribute to the improvement of lung pathology in card −/− mice. moreover, the expression of ip- encoded by an ifn-responsive gene was significantly impaired in card −/− mice on day (fig. d) , albeit no significant reduction was seen in either type i or type ii interferons, suggesting that synergistic signals of interferons and various cytokines/chemokines are required for the higher expression of ip- at an early phase of infection. next, we examined the induction of virus-specific cd + t cells using the h- d b tetramer coupled with a viral nuclear protein (np)-derived peptide (np - ) , . the percentages of np-specific cd + t cells in the draining lymph nodes of the lungs on day were comparable between wt and card −/− mice (fig. c) . to evaluate the impact of card deficiency on acquired immunity to ifv, wt and card −/− mice were immunized with a sub-lethal dose ( / of ld ) of the pr strain and rechallenged with a high lethal dose ( ld ) of pr after weeks. we found that card deficiency did not alter viral clearance after the challenge (fig. d ). next, we assessed the induction of humoral and cellular acquired immunity to ifv in card -/mice. we re-infected wt and card -/mice on day after their first infection (representing the second infection) and analyzed the production of virus-specific antibodies as well as the development of virus-specific cd + t cells on day after the second infection. we found that the card deficiency affected neither the production of virus-specific igg in the serum nor virus-specific iga in the lung mucosa (fig. e) . additionally, card deficiency did not affect the ifn-γ response by splenic cd + t cells specific to a np antigen epitope np - (fig. f) . collectively, the card pathway is dispensable for the induction of protective immunity against both the primary and secondary ifv infections, and its deficiency even improves primary viral clearance upon lethal-dose infections probably because of improvement in lung damage and elevated ifn-γ production. card deficiency impairs inflammatory cytokine, but not type-i interferon, production by dcs. because macrophages and dcs are known to be an early source of inflammatory cytokines and type-i interferons in pulmonary ifv infection , , we examined whether card was required for their production by myeloid cells in response to ifv. thioglycolate-induced peritoneal macrophages (tg-mfs), bone marrow-derived macrophages (bmmfs), alveolar macrophages (amfs), conventional dcs (cdcs), or flt ligand-induced plasmacytoid dcs (flt l-dcs) prepared from wt or card -/mice were brought into contact with pr in vitro and the production of il- , tnf-α , and ifn-α /β was measured. il- and tnf-α produced by tg-mfs (fig. a) or bmmfs (fig. b ) and those mrnas produced by amfs (fig. c ) in response to pr were not affected by card deficiency. as previously reported , , card was required for a cytokine response to ox-zymosan through dectin- -syk but dispensable for the response to lps through tlr -myd in cdcs ( supplementary fig. s a ). we found that card -/-cdcs (fig. d ) or flt l-dcs (fig. e ) produced significantly lower levels of il- and tnf-α than wt cells in response to pr . in contrast, ifn-α /β production was not compromised in card -/-cdcs or flt l-dcs. these findings suggest that card deficiency primarily affected dcs but not mfs regarding the production of inflammatory cytokines but not ifn-α /β in response to ifv. it has been reported that the card -ips- pathway mediates the cytokine response to rna viruses through rlhs , while card has been found to transmit signals from myeloid itam-coupled receptors via syk [ ] [ ] [ ] . thus, we evaluated the contributions of these pathways. ips- -deficient (ips- -/-) cdcs and myd -deficient (myd -/-) flt l-dcs produced significantly lower levels ifn-α /β than wt cells in response to pr (fig. d ,e), consistent with previous findings showing that the ifn-α /β response to rna viruses is mediated mainly through rig-i/ips- in cdcs, and through tlr /myd in pdcs , . in addition, il- and tnf-α production was impaired in ips- -/-cdcs and myd -/-flt l-dcs. lower levels of cytokine production were also observed in myd -/-cdcs, likely due to impaired activation through several tlrs (i.e. tlr , , and ) that sense virus nucleic acids , . we found that inducible syk-deficient (syk del/del ) cdcs and flt l-dcs produced significantly lower levels of inflammatory cytokines than wt cells similar to the responses of card -/-cdcs and flt l-dcs, whereas the syk deficiency did not affect cytokine production in response to lps (supplementary fig. s a ). consistent with these findings, treatment of wt cdcs (fig. a ) and flt l-dcs (fig. b) with the syk inhibitor bay - reduced the production of inflammatory cytokines but not type-i interferons in response to pr in a dose-dependent manner. the treatment with bay - reduced the cytokine response to ox-zymosan but not to lps, indicating that the effect of the inhibitor on cytokine expression depends on the stimulus but cannot be attributed to its general effect on cytokine gene expression ( supplementary fig. s b ). overall, these results suggest that the attenuation of influenza pneumonia by card deficiency was likely attributable to the reduced cytokine/chemokine production by pulmonary dcs through the syk-card pathway in response to ifv. we have shown that card -mediated activation of the innate immune system exacerbates influenza pneumonia in mice. card deficiency resulted in the reduction of inflammatory cytokine/chemokines and the infiltration of inflammatory cells in ifv-infected lungs, resulting in improved mouse mortality rates. given the pathological similarity between the mouse intratracheal infection model and influenza-associated ards in human - , we postulate that the card pathway contributes to the exacerbation of human influenza pneumonia. although the syk-card -mediated innate immune balf were analyzed on day and after the second infection. (f) ifv-specific adaptive t cell response. splenocytes from wt and card -/mice (n = per group) days after infection as in (e) were stimulated in vitro with ifv nucleoprotein peptide (np - ) for days. the culture supernatants were analyzed for ifn-γ production. data are presented as mean ± sd of triplicates. data are representative of three independent experiments. *p < . by student's t-test. scientific reports | : | doi: . /srep response is crucial for anti-fungal acquired immunity [ ] [ ] [ ] [ ] , our data showed that card was dispensable for anti-ifv immunity, as demonstrated by the findings that card deficiency did not alter viral burden, the elevation of ifn-α /β , or the induction of anti-viral adaptive t and b cell responses in the ifv-infected mice. thus, other innate mechanisms independent of the card pathway may play a more dominant role in protective immunity against ifv. innate immune sensors for ifv and its downstream signaling pathway have been well illustrated , , . in our study, card deficiency resulted in a selective reduction in inflammatory cytokines, but not ifn-α /β production, by both cdcs and flt l-dcs in response to ifv. this pattern of impairment was observed in ips- -/-cdcs, and was consistent with results from previous reports indicating that card selectively regulates the cytokine response through rig-i-ips-i in cdcs . additionally, we found that syk del/del and syk inhibitor-treated cdcs showed the same pattern of impairment as card -/or ips- -/-cdcs. on the other hand, in flt l-dcs known to release large amounts of inflammatory cytokines and type-i interferons upon recognition of ifv , , ips- deficiency affected neither cytokine or type-i interferon responses. however, syk deficiency in flt l-dcs, similar to that in cdcs, resulted in a selective reduction in inflammatory cytokines but not ifn-α /β . thus, it is likely that the syk-card pathway controls detrimental cytokine production by pulmonary dcs upon acute influenza infection. alternatively, because card deficiency impairs cytokine production by dcs following stimulation with several tlr ligands , it may be important to examine whether the syk-card pathway is involved in the tlr -meditated response to ifv. the involvement of the syk-card pathway implies the presence of itam-coupled receptors that sense ifv and stimulate cytokine production by dcs. it has been reported that an interaction between dc-sign, which possesses an itam-like "hemitam" motif, and the carbohydrate of ifv hemagglutinin induces maturation of dcs and promotes endocytosis of the virus , indicating the signal-activating capacity of dc-sign upon virus binding. however, it is unclear whether the dc-sign hemitam is capable of transmitting sufficient signals to produce inflammatory cytokines. with regard to other viruses, recognition of the dengue virus by the dap -associated clr clec a results in increased vascular permeability due to the overproduction of tnf-α , resulting in fatal outcomes . however, its ability to recognize the ifv remains unknown. thus, it is worth examining whether these clrs are involved in the cytokine response to ifv. it is also conceivable that damage-associated molecular patterns (damps), generated due to lung damage by ifv infection, may be ligands for some itam-coupled receptors. indeed, the fcrγ -associated clr mincle was shown to recognize sap , a component of the ribonucloprotein released from dead cells . in conclusion, card -mediated innate immune activation in pulmonary dcs acts to exacerbate severe influenza pneumonia, but is dispensable for host protection against ifv. thus, inhibiting the card pathway may represent a promising therapeutic target for the control of pivp without affecting viral clearance. mice. card -/-, syk flox/flox , ips- -/and myd -/mice have been previously described , [ ] [ ] [ ] . these mice were backcrossed at least times onto c bl/ mice. c bl/ mice were purchased from clea japan, inc. (tokyo, japan). the animals were housed in specific pathogen-free conditions. all experiments were approved by the institutional animal care and use committee for kitasato university medical center and animals were treated in accordance with the regulations for animal experiments in kitasato university. all surgeries were performed under ketamine hydrochloride/xylazine anesthesia, and all efforts were made to minimize suffering. antibodies and reagents. fluorescein isothiocyanate (fitc)-conjugated anti-f / (clone bm ), fitc-conjugated anti-cd r/b (clone ra - b ), fitc-conjugated cd (clone d ), phycoerythrin (pe)-conjugated anti-ly- g (clone a ), pe-conjugated anti-siglech (clone ), biotin-conjugated anti-nk . (clone pk ), biotin-conjugated anti-bst /pdca- (clone ), peridinin chlorophyll protein/cy . (percp/cy . )-conjugated anti-cd (clone -f ), phycoerythrin-cy (pc )-conjugated anti-cd ε (clone - c ) and pc -conjugated anti-cd c (clone n ) monoclonal antibodies were purchased from biolegend (san diego, ca). fitc-conjugated anti-cd (clone kt ) monoclonal antibodies, pe-conjugated h- d b tetramer coupled with a viral np-derived asnenmetm peptide (np - : asnenmetm), h- d b -restricted influenza np - peptide were purchased from mbl (nagoya, japan). syk inhibitor iv (bay - ) was purchased from merck millipore (billerica, ma). lps from escherichia coli :b was purchased from sigma-aldrich (st. louis, mo). naclo-oxidized zymosan was prepared as described . ifv infection in mice. c bl/ and card -/mice were anesthetized and infected with plaque forming units (pfu) (unless otherwise indicated) of a mouse-adapted ifv (a/pr/ / strain: h n isotype, kindly provided by the kitasato institute, tokyo, japan) by intratracheal administration as described - . histology. whole lungs were collected at , , and days after ifv infection. paraffin embedding and hematoxylin and eosin staining of tissues were performed using standard methodologies. . bal was carried out as described previously , . in brief, tracheas of mice were cannulated with . -mm diameter polyethylene catheters. lungs were instilled with ml of pre-warmed pbs containing mm edta, followed by the retrieval of lavage fluid aliquots. cells in the bal fluid (balf) were counted after red blood cell lysis and subjected to flow cytometric analysis. the supernatants of the balf were subjected to multi cytokine/chemokine expression analysis and cytokine elisa. cytomix cytokine bead assay (bender medsystems, vienna, austria), with the exception of ifn-β , which was measured by a verikine tm mouse interferon-β elisa kit (pbl interferonsource, piscataway, nj). cell culture supernatants from splenocytes were assayed using a specific elisa kit for ifn-γ (biolegend). cell culture supernatants from tg-mfs, bmmfs, cdcs, and flt l-dcs were assayed using specific elisa kits for il- , tnf-α (biolegend), ifn-α and ifn-β (pbl interferonsource). all measurements were performed in triplicate. for quantitative pcr (qpcr) analysis for cytokine mrnas, total rna was extracted from cells with reliaprep rna cell miniprep system (promega, madison, wi) and cdna was synthesized with primescript rt reagent kit (takara bio, shiga, japan) according to the manufacturer's instructions. qpcr was performed with the applied biosystems ht fast real time pcr system (life technologies). primer sequences were as follows: mouse il , forword, ′-cca ctt cac aag tcg gag gct ta - ′, and reverse, ′-gca agt gca tca tcg ttg ttc ata c - ′; and tnf forword, ′-gtt cta tgg ccc aga ccc tca c - ′, and reverse, ′-ggc acc act agt tgg ttg tct ttg - ′. viral copy numbers. mice were euthanized by intraperitoneal administration of sodium pentobarbital at , , or days after ifv infection. lung tissues were homogenized using a gentlemacs dissociator (miltenyi biotec) and rna was extracted with an isogen ii rna extraction kit (nippon gene, tokyo, japan). reverse transcription was conducted with the uni- primer ( ′-agc aaa agc agg - ′) and qpcr was preformed with primers specific for np (forward: ′-gat tgg tgg aat tgg acg at - ′; reverse: ′-aga gca cca ttc tct cta tt - ′) using the applied biosystems ht fast real time pcr system (life technologies, carlsbad, ca). the standard calibration curve for qpcr was obtained by stepwise dilution of the cloned np gene fragment with a known copy number. ifv rechallenge. wt and card -/mice were left uninfected or infected intratracheally with sublethal dose ( pfu/mouse: / ld ) of pr . twenty-eight days after the first infection, mice were challenged with a high lethal dose ( pfu/mouse: ld ) of pr . two days postchallenge, virus titers in the lungs were measured in a plaque assay in mdck cells. antigen-specific b cell responses. b cell-mediated humoral responses were measured as virion-specific immunoglobulin production by elisa, as previously described . briefly, -well elisa plates (corning) were coated with ultrasonicated influenza virion (a/pr/ / strain) at × pfu/ ml in a carbonate buffer (ph . ), and incubated overnight at °c. plates were then washed with pbs containing . % tween (wako pure chemical industries). serum and balf collected from mice at day after the secondary infection were serially diluted with pbs/tween containing % skim milk, applied onto the virion-coated plates, and incubated for h at room temperature. after washing, goat anti-mouse total igg or iga conjugated to horseradish peroxidase (jackson immunoresearch, baltimore pike, pa) was applied and incubated for h at room temperature. after washing, the plates were stained with a tmb substrate set (biolegend). the reaction was terminated with m h so (wako pure chemical industries) and the absorbance was measured. antigen-specific t cell responses. ifv-specific t cell responses were measured as viral np-specific ifn-γ secretion by splenocytes, as described previously , . briefly, × splenocytes extracted from mice -days after the secondary infection were seeded on -well cell culture plates (corning) and then stimulated with μ g/ml of np - peptides. after days of culture, supernatants were collected and analyzed for ifn-γ production by elisa (biolegend). tg-mfs were prepared as described . amfs were isolated from balf of wt or card -/mice. bmmfs, cdcs, or flt l-dcs were prepared by culturing bone marrow cells for - days with rpmi medium (wako pure chemical industries) supplemented with % fetal bovine serum (life technologies) and antibiotics ( iu/ml penicillin and μ g/ml streptomycin; sigma-aldrich) containing m-csf ( ng/ml, peprotech, rocky hill, nj), gm-csf ( ng/ml, peprotech) or human flt -ligand ( ng/ml, peprotech), respectively. the flt l-dcs derived from the culture contain constantly - % of pdca- + siglech + plasmacytoid dcs (pdcs) irrespective of deficiency in syk, card , ips- or myd . for syk deletion in vitro, cultured cells derived from syk flox/flox mice were incubated with . μ m of the active metabolite of tamoxifen, -hydroxytamoxifen (sigma-aldrich) for d. for stimulation of mfs/dcs of, × cells were seeded on -well culture plates (corning) and incubated overnight, followed by replacement of μ l of serum-free medium containing pfu (multiplicity of infection [m.o.i] = ) of pr . after h of incubation, unabsorbed viruses were removed and the cells were incubated for a further h in serum-containing medium. dcs were also stimulated with lps ( ng/ml) or naclo-oxidized zymosan ( μ g/ml) for h in serum-containing medium. the culture supernatants were assayed for il- , tnf-α , ifn-α , and ifn-β by elisa. for stimulation of amfs, × cells were seeded on -well culture plate (corning), stimulated as above with pfu (m.o.i = ) of pr for h, and total rnas were extracted for qpcr analysis for cytokine expression. for treatment of cells with a chemical syk inhibitor, wt dcs were incubated with . or μ m bay - for h prior to virus stimulation. cell viability. cell viability was determined by the mts assay using the celltiter aqueousone solution cell proliferation assay kit (promega). in brief, μ l of mts solution was added to cells in -well plates after in vitro ifv stimulation and the plates were incubated for h at °c, then the absorbance at nm was measured using a benchmark microplate reader (bio-rad, hercules, ca). statistical analysis. survival curves were generated by the kaplan-meier method, and statistical analyses were performed using the log-rank test. the statistical significance was assessed by student's t-tests. a p value < . was considered significant. avian influenza a (h n ) infection in humans the genesis of a pandemic influenza virus characterization of the reconstructed spanish influenza pandemic virus aberrant innate immune response in lethal infection of macaques with the influenza virus h n and pandemic influenza virus infection results in 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pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? systemic cytokine responses in patients with influenza-associated encephalopathy card controls a non-tlr signalling pathway for innate anti-fungal immunity the adaptor protein card is essential for the activation of myeloid cells through itam-associated and toll-like receptors card versus carma in innate and adaptive immunity recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production rad -card interactions link cytosolic dna sensing to il- β production dc-sign and immunoregulation dc-sign mediates avian h n influenza virus infection in cis and in trans critical role of il- receptor-associated kinase-m in regulating chemokine-dependent deleterious inflammation in murine influenza pneumonia innate and adaptive immune responses to viral infection and vaccination interferons, interferon-like cytokines, and their receptors the role of alpha/beta and gamma interferons in development of immunity to influenza a virus in mice production of interferon-gamma by influenza hemagglutinin-specific cd effector t cells influences the development of pulmonary immunopathology the epitopes of influenza nucleoprotein recognized by cytotoxic t lymphocytes can be defined with short synthetic peptides a previously unrecognized h- d(b)-restricted peptide prominent in the primary influenza a virus-specific cd (+ ) t-cell response is much less apparent following secondary challenge differential role of tlr-and rlr-signaling in the immune responses to influenza a virus infection and vaccination plasmacytoid dendritic cells delineate immunogenicity of influenza vaccine subtypes detrimental contribution of the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells dectin- is required for host defense against pneumocystis carinii but not against candida albicans dectin- is required for beta-glucan recognition and control of fungal infection c-type lectin mincle is an activating receptor for pathogenic fungus dectin- recognition of alpha-mannans and induction of th cell differentiation is essential for host defense against candida albicans pathogen recognition and innate immunity pattern recognition receptors and inflammation binding of dc-sign to the hemagglutinin of influenza a viruses supports virus replication in dc-sign expressing cells clec a is critical for dengue-virus-induced lethal disease mincle is an itam-coupled activating receptor that senses damaged cells syk-dependent signaling pathways in neutrophils and macrophages are indispensable in the pathogenesis of anti-collagen antibody-induced arthritis ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction targeted disruption of the myd gene results in loss of il- -and il- -mediated function beta-glucan derived from zymosan acts as an adjuvant for collagen-induced arthritis immunokinetics in severe pneumonia due to influenza virus and bacteria coinfection in mice universal primer set for the full-length amplification of all influenza a viruses interleukin- is critical in the pathogenesis of influenza a virus-induced acute lung injury we thank yoshiyuki miyazaki, mako nakaya, fumika mi-ichi, masanori yamazaki, naoko ozaki, sayoi sato, shinsuke yasukawa, mio kubota, honglian tong, takashi fukuyama, taiga yamazaki, tomoko fujita and rui yamamura for helpful discussions and technical support. we also thank dr. yumiko imai and dr. keiji kuba (akita university) for helpful discussions, and dr. shinobu suzuki (nippon boehringer ingelheim co., ltd) and dr. shizuo akira (osaka university) for kindly providing the . the funders had no role in the study design, data collection or analysis, the decision to publish, or the preparation of the manuscript. key: cord- - v yfa authors: zisman, david a.; keane, michael p.; belperio, john a.; strieter, robert m.; lynch, joseph p. title: pulmonary fibrosis date: journal: fibrosis research doi: . / - - - : sha: doc_id: cord_uid: v yfa idiopathic pulmonary fibrosis (ipf) is a chronic fibrosing lung disease limited to the lungs and associated with the histologic appearance of usual interstitial pneumonia (uip) on surgical lung biopsy. the estimated prevalence in the united states is between , and , cases, and evidence suggests that the prevalence is increasing for ipf. risk factors associated with pulmonary fibrosis include smoking, environmental exposures, gastroesophageal reflux disease, commonly prescribed drugs, diabetes mellitus, infectious agents, and genetic factors. the diagnosis requires a careful history and physical examination, characteristic physiological and radiological studies, and, in some cases, a surgical lung biopsy. the natural history of ipf is not known, but evidence supports the concept of a continuum of idiopathic interstitial pneumonias that may overlap in time. most patients with ipf succumb to respiratory failure, cardiovascular disease, lung cancer, pulmonary embolism, infection, and other health problems. the median survival time for patients with ipf is less than yr. factors that predict poor outcome include older age, male gender, severe dyspnea, history of cigarette smoking, severe loss of lung function, appearance and severity of fibrosis on radiological studies, lack of response to therapy, and prominent fibroblastic foci on histopathologic evaluation. conventional therapy (corticosteroids, azathioprine, cyclophosphamide) provides only marginal benefit. lung transplantation should be considered for patients with ipf refractory to medical therapy. in light of the poor prognosis and lack of response to available anti-inflammatory therapy, alternative approaches to therapy are being pursued. emerging strategies to treat patients with ipf include agents that inhibit epithelial injury or enhance repair, anticytokine approaches, agents that inhibit fibroblast proliferation or induce fibroblast apoptosis, and other novel approaches. the diagnosis and management of idiopathic interstitial pneumonias (iips) remains a challenge to the clinician. recently, there have been substantial changes in our understanding and approach to theses diseases. with greater comprehension of the clinical relevance of the different histopathological subgroups that make up the idiopathic interstitial pneumonias, the term idiopathic pulmonary fibrosis (ipf) is now reserved to patients with idiopathic usual interstitial pneumonia (uip) on surgical lung biopsy. the following review will provide an updated discussion of the epidemiology, risk factors, diagnosis, natural history, morbidity and mortality, prognosis, and conventional therapy of ipf, as well as emerging strategies for the treatment of patients with this disease. the true prevalence of ipf, also known as cryptogenic fibrosing alveolitis (cfa), is unknown. despite the poor quality of the data and the changes in diagnostic criteria and classification, there is evidence suggesting that ipf is increasing ( ). there are approx to . cases of ipf per , in the general population ( - ). the prevalence increases with older age, history of smoking, and male gender ( , , ) . in a population-based study in bernalillo county, new mexico, the prevalence for adults from age to yr was . per , , but surpassed per , for individuals older than yr ( ). the prevalence of ipf is higher in men ( . cases per , ) than in women ( . cases per , ) ( ). the estimated prevalence in the united states is between , and , cases ( ). the incidence of ipf is estimated at . cases per , per year for males and . cases per , per year for females ( ). vital statistics figures are limited and incomplete. in , there were , hospitalizations and deaths in the united states owing to pulmonary fibrosis (compared with , hospitalizations owing to chronic obstructive pulmonary disease and asthma). in japan, the mortality rate for ipf per , population was estimated to be . in men and . in women, with an overall rate of . in both sexes ( ). in the united kingdom, the annual number of deaths from ipf increased twofold between and . the age-adjusted rate of pulmonary fibrosis among deceased in the united states increased from . per , in to . per , in in males, and from . per , in to . per , in in females ( ). pulmonary fibrosis listed as a cause of death increased from % in to % in . in the united states, the age-adjusted mortality rates are highest in the western and southwestern states and lowest in the midwest and northeast. in the united kingdom, highest mortality rates are found in industrialized areas of england and wales ( , ). in case-control studies, smoking has been identified as a possible risk factor for ipf with an odds ratio (or) of . ( % confidence interval [ci] . - . ) for ever-smoking, and . ( % ci . - . ) for former-smokers ( , , ) . smokers of to pack-years have an or of . ( % ci . - . ) for pulmonary fibrosis ( ). in a recent study in japan, the adjusted odds ratio for cigarette smoking was estimated at . ( % ci . - . ) ( ). interestingly, three studies reported improved survival among current or former smokers with uip compared to never-smokers ( ) ( ) ( ) . however, others found no such effect ( ) ( ) ( ) . it is possible that the apparent protective effect of cigarette smoking may relate to the following: lead time bias; alterations in the balance of proteinases and antiproteinases that would influence net deposition of extracellular matrix in the lung; or inhibitory effects of cigarette smoke on lung fibroblast proliferation and chemotaxis ( , ). instillation of acid in several animal models results in aspiration-induced lung injury and pulmonary fibrosis ( ) . clinical data suggest that a high percentage of patients with ipf have clinically silent gastroesophageal reflux disease (gerd) ( , ) . small tracheobronchial aspirations of gastric acid may play a role in the pathogenesis of ipf; however, a causal relationship has not been established ( , ) . investigators at the university of washington medical center are conducting an ongoing prospective study of patients with ipf. interim results indicate that the prevalence of ipf patients with gerd is %, with only % of patients reporting symptoms ( ) . however, the perpherial pattern of uip in the lung would be unusual if gerd was truly the cause, as compared with an association. in one case-control study, ipf was associated with exposure to antidepressants with an or of . . - . ] ). these associations were independent of smoking and occupational dust exposure. the authors concluded that exposure to antidepressants may be responsible for approx % of cases of ipf seen in their population. no significant association was noted between ipf and the other drug groups tested (anticonvulsants, β-blockers, antibiotics, and nonsteroidal anti-inflammatory drugs [nsaids]) ( ). in a recent study, clinical and demographic data were extracted from medical records of consecutive patients with ipf admitted to a japanese hospital. ipf was associated with diabetes mellitus (dm) with and or of . ( % ci . - . ). the authors concluded that dm might be a risk factor for ipf ( ). the etiology of ipf is unknown, but environmental factors may play a causative role. metal and wood dust environments may be important risk factors for pulmonary fibrosis. in one study, metal dust exposure was identified as a risk factor with an odds ratio (or) of . ( % ci . - . ) , and wood dust exposure with an or of . ( % ci . - . ). in that study, metal and wood dust exposure may have caused up to % and % of pulmonary fibrosis cases, respectively. dust containing brass, lead, cobalt, aluminum, zinc, cadmium, mercury, and pine dusts were associated with pulmonary fibrosis ( ). certain occupations may predispose to pulmonary fibrosis. in one study, farming was identified as a potential risk factor with and or of . ( % ci . - . ) and exposure to livestock was associated with an or of . ( % ci . - . ). hairdressing was linked to pulmonary fibrosis with an or of . ( % ci . - . ), metal dust exposure with an or of . ( % ci . - . ), raising birds with an or of . ( % ci . - . ), stone cutting/polishing with an or of . ( % ci . - . ), and vegetable/animal dust exposure with an or of . ( % ci . - . ) ( ). a number of viruses have been associated with pulmonary fibrosis, but true cause-effect relationships remain unproven. a serological survey found an association between active epstein-barr virus (ebv) infection and ipf ( ). egan and colleagues reported immunohistochemical evidence of ebv-productive cycle antigens in type ii alveolar epithelial cells in ipf ( ). subsequently, these investigators detected ebv dna by polymerase chain reaction (pcr) in the lung tissue of patients with ipf ( ). further study demonstrated that productive ebv replication is common in ipf and it is not associated with immunosuppressive therapy ( ). tang in a recent study, human t-lymphtropic virus type i (htvl-i) positive ipf patients had more affected lung parenchyma, demonstrated traction bronchiectasis with honeycomb change, and exhibited increased levels of specific cytokines that correlated with activated t-cells in the bronchoalveolar lavage fluid (balf). these findings suggested that htlv-i infection might contribute to the development of ipf via activation of t-cells ( ). the latent nature together with episodic reactivation of many of these viruses may provide a scenario for the concept of "multiple hits" host defense followed by repair that these patients may experience during the course of their disease. the genetics of familial ipf have not been elucidated. an autosomal dominant trait with variable penetrance may account for approx % of cases; there is no clear mode of transmission in the remaining % ( , ). investigators have linked ipf to an increase in mz phenotype for α -antitrypsin inhibition on chromosome ( - ). using a candidate gene approach, researchers identified surfactant protein c gene mutations in large familial pulmonary fibrosis kindred, including adults with uip and children with nonspecific interstitial pneumonia (nsip) ( ). in a separate study, selman and co-workers demonstrated that surfactant protein a and b genetic variants predispose to ipf ( ). genetic polymorphisms for interleukin- receptor antagonist (il- ra) and tumor necrosis factor (tfn)-α appear to be important in determining risk ( ). in contrast, transforming growth factor (tgf)-β polymorphisms do not predispose to ipf, but these polymorphisms may affect the course of the disease ( ). it is unknown what proportion of ipf is familial, but it is estimated that . to . % of cases have a genetic basis ( ). thirty-eight families affected by pulmonary fibrosis have been identified and are currently under active investigation. the familial aggregation in those families is consistent with a genetic basis in at least a subset of patients with ipf ( ). ipf is a specific form of chronic fibrosing interstitial pneumonia limited to the lungs and associated with the histological pattern of uip on surgical lung biopsy ( ). many earlier studies included various other idiopathic interstitial pneumonias under the term idiopathic pulmonary fibrosis, but the clinical term ipf is now reserved to patients with idiopathic uip. the diagnostic approach to any patient with diffuse lung disease must include a thorough history and physical examination with attention to symptoms or signs suggestive of a connective tissue disease, occupational or environmental exposures, use of fibrogenic drugs, and family history of pulmonary fibrosis. patient age at disease onset is generally between and yr of age and ipf is more common in males than females. ipf typically presents insidiously, with gradual onset of a nonproductive cough and dyspnea ( ). patients are often treated for other conditions such as congestive heart failure, "walking pneumonia," bronchitis, or asthma before the diagnosis is made. the physical examination in most patients (> %) reveals fine bibasilar inspiratory crackles ("velcro rales"), and clubbing is noted in up to % of patients ( , ). signs of right heart failure are evident in advanced cases ( ). laboratory abnormalities are mild and nonspecific. one early study described "autoimmune factors" in blood of patients ( of whom had autoimmune-associated diseases) among patients with diffuse fibrosing alveolitis ( ). a positive rheumatoid factor occurred in of patients with unexplained pulmonary fibrosis and positive antinuclear factor in the serum (ana) ( ). a later study examined serum specimens from patients with ipf and compared them with specimens from age-and sex-matched controls; anas were present in % of patients with ipf and in % of the control subjects ( ). positive circulating anas or rheumatoid factor occur in % to % of patients with ipf, but titers are rarely high ( , , , ). an elevated erythrocyte sedimentation rate, lactate dehydrogenase, or hypergammaglobulinemia may be found in patients with ipf, but are nondiagnostic ( ). serological findings do not correlate with extent or severity of disease, and have no prognostic value ( , ). in the absence of symptoms of connective tissue disease, the presence of autoantibodies does not imply an underlying systemic disorder. recently, investigators reported the occurrence of low fasting triglyceride and high free fatty acid levels in patients with pulmonary fibrosis. because insulin-like growth factor (igf)-i is known to lower triglycerides and increase free fatty acids, the authors hypothesized that the reported increased production of igf-i in patients with ipf may explain such findings ( ). classic chest radiographic findings in ipf include a basal predominant reticular, or reticulonodular, pattern associated with decreased lung volumes, and in later stages, cystic areas representing honeycomb (hc) lung ( , - ). when a "confident" diagnosis of ipf is made on the basis of the chest radiograph, it is correct in to % of cases ( , , ). most patients with ipf will have an abnormal chest radiograph but, rarely, patients may present with a normal plain film ( ). it is important to review all previous chest films to assess the rate of change in disease activity. in addition, radiographs are indicated if clinical deterioration occurs in order to identify superimposed infection or malignancy ( , ). pleural effusions, upper lobe predominant disease, airbronchograms, or prominent lymphadenopathy should suggest an alternative diagnosis. typical high-resolution chest computed tomography (hrct) features of ipf/uip include patchy, predominantly peripheral, subpleural, and symmetrical bibasilar honeycombing, reticular abnormalities, and limited "groundglass" opacities (ggo) ( [ ] [ ] [ ] [ ] . several studies have shown that experienced radiologists can make a "confident" diagnosis of uip with specificity greater than %, provided ct features are typical ( , [ ] [ ] [ ] [ ] . although a characteristic hrct is highly specific for ipf/uip, "typical" hrct identifies only to % of patients with histological uip; therefore, a surgical lung biopsy (slb) is recommended when clinical and radiological information result in an uncertain diagnosis ( , , , ). one study evaluated the proficiency of physicians with expertise in interstitial lung diseases to identify accurately the hrct scans from patients with biopsy-proven ipf/uip. when these investigators made a "confident" diagnosis of ipf based on hrct scan and clinical data, they were right in more than % of the cases. however, more than half of the patients with ipf had an uncertain diagnosis on the basis of hrct and clinical assessment ( ). in a subsequent analysis of these data, investigators identified hrct features associated with a pathological diagnosis of uip. on multivariate analysis, lower lobe honeycombing (or, . ), and upper-lung irregular lines (or, . ) were the only independent predictors of uip. when they combined those two factors, a diagnosis of uip was established with a sensitivity of %, a specificity of %, and a positive predictive value of % ( ). interestingly, in that study, adenopathy was observed in % and % of patients with uip and without uip, respectively. this finding suggests that patients with ipf generate a marked lymphoproliferative response to an unknown antigen or antigens; we believe that this observation requires further investigation as it may provide important clues to further our understanding of the pathogenesis the disease. in a separate study, two radiologists independently assessed ct scans from a cohort of patients with either uip. ct features were "typical" for uip in only % patients, and all of them had histological uip on slb. typical ct features of uip are associated with advanced, latestage disease. among patients with earlier phases of uip, ct features may be atypical ( ) or indeterminate ( ). extensive ggo is not a major feature of uip, and suggests an alternative diagnosis such as desquamative interstitial pneumonia (dip), nsip, lymphocytic interstitial pneumonia (lip), cryptogenic organizing pneumonia (cop), hypersensitivity pneumonia (hp), or pulmonary alveolar proteinosis (pap) ( ). in contrast, honeycomb change is a cardinal feature of uip, and is rare in other iips ( , ). pulmonary function tests (pfts) characteristically reveal a restrictive ventilatory defect with impaired gas exchange; however, smokers may have preserved lung volumes or airflow obstruction in the initial stages of the disease ( , , ). impairments in gas exchange (i.e., carbon monoxide diffusing capasity [dl co ]) and oxygenation may be evident early in the course of the disease, even when spirometry and lung volumes are normal ( ). the most appropriate and simple tests are vital capacity and dl co ; these are most useful for assessing the extent and monitoring the progression of the disease ( ). cardiopulmonary exercise testing (cpet) demonstrates hypoxemia, widened a-a o gradient, submaximal exercise endurance, reduced oxygen consumption (vo ), high respiratory frequency, low tidal volume (v t ) breathing pattern, increased dead space (v d /v t ), increased minute ventilation for the level of vo , and a low o pulse ( - ). arterial desaturation and abnormal widening of a-a o gradient with exercise may be elicited with relatively simple tests, such as the -min walk test ( , ). balf may play a role in the diagnosis of inorganic dust diseases, suspected malignancy, infections, some hematological disorders, drug-induced diseases, pulmonary alveolar proteinosis, langerhans' cell histiocytosis, and alveolar hemorrhage ( ). balf is useful in research studies but of limited clinical application when evaluating iips ( ). increases in polymorphonuclear leukocytes, eosinophils, mast cells, alveolar macrophages, and countless cytokines are noted in balf from patients with ipf/uip; lymphocyte numbers are usually normal ( ). balf neutrophilia is present in to % of patients with ipf/cfa ( , ), but does not predict prognosis or therapeutic responsiveness. elevations in balf eosinophils were associated with more severe clinical impairment ( , ), but balf eosinophil counts do not correlate consistently with prognosis ( , ). by contrast, the presence of balf lymphocytosis, found in fewer than % of cases, was associated with a greater responsiveness to corticosteroid therapy, a more cellular biopsy, and less honeycombing ( , ). data compiled from two studies documented favorable responses to corticosteroids in of patients exhibiting balf lymphocytosis, but in only of without lymphocytosis ( , ). because the studies citing balf lymphocytosis in steroid-responsive patients with ipf ( ) or cfa ( ) antedated the description of nsip, it is possible that balf lymphocytosis reflects disorders distinct from uip (such as cellular nsip). nevertheless, in a recent study, researchers hypothesized that balf findings may distinguish between uip and nsip; balf total and differential cell counts were not different between the two groups, and in neither group, were balf findings predictive of survival or changes in lung function ( ). the american thoracic society (ats) and the european respiratory society (ers) in collaboration with american college of chest physicians (accp) published an international consensus statement on the diagnosis and treatment of ipf. this statement stated that the definite diagnosis of ipf requires an slb showing the uip pattern. however, an slb is not recommended in patients with suspected ipf in whom the clinical or radiographic information are stereotypical of ipf/uip. it has been suggested that, in the absence of an slb, the presence of all four major diagnostic criteria and at least three minor criteria increases the likelihood of an accurate diagnosis of ipf/uip ( ). however, these criteria have not been prospectively validated. an slb, preferably by video-assisted thoracoscopy (vats), is recommended in patients with suspected ipf in whom the clinical or radiographic information are not typical of ipf/uip ( ). given the patchy and heterogeneous nature of the uip lesion, a large piece of lung tissue is required and tbbx are used mainly to rule out other disorders that mimic ipf. however, emerging data suggest that tbbx findings may be more useful in diagnosing uip than previously recognized; characteristic histological features of uip (interstitial fibrosis with fibroblastic foci [ff] and/or hc change) can be appreciated even in a small sample obtained by tbbx; these findings in a patient with characteristic clinical and radiographic features may indicate a diagnosis of uip. however, these findings require prospective validation ( ). significant advances have been made in our understanding of the idiopathic interstitial pneumonias (iips). the most important advancement has been the greater appreciation of the clinical relevance of the different histopathological subgroups that make up the iips. until recently, inflammatory or fibrotic lung disorders of unknown etiology were "lumped" under the term ipf ( , , ). however, with the advent of vats lung biopsies and the greater availability and quality of surgical specimens, pathologists have recognized the heterogeneous nature of these disorders and described specific histopathological patterns that predict response to therapy and survival ( , , ). ipf/uip is the most common of the iips, comprised of to % of the cases ( , , , ). the uip lesion is characterized by temporal and geographic heterogeneity, with areas of old scar and hc change, admixed with granulation tissue and normal lung; the lesion has predilection for the subpleural and basilar regions of the lung, there is scant inflammation, and prominent aggregates of fibroblast and myofibroblasts, so-called "fibroblastic foci," which actively secreting extracellular matrix ( , , ). additional features include smooth muscle hypertrophy, metaplasia and hyperplasia of type ii pneumocytes, destroyed and disrupted alveolar architecture, traction bronchiectasis and bronchioloectasis, and secondary pulmonary hypertension changes ( , , ). other categories of iip that must be distinguished from ipf/uip include nsip, dip, respiratory bronchiolitis-associated interstitial lung disease (rbild), acute interstitial pneumonia (aip), cop, and lip ( ). nsip is observed in approx % of patients with iip ( ). this provisional category is used to describe a temporally homogeneous lesion with varying degrees of inflammation and fibrosis with favorable response to therapy and prognosis. nsip can be subdivided into nsip-cellular and nsip-fibrotic varieties depending on the degree of inflammation and fibrosis present in the surgical specimen ( ). this subclassification provides important prognostic information; patients with idiopathic nsip, cellular pattern have a better -and -yr survival than those with idiopathic nsip, fibrosing pattern ( % vs % and % vs %, respectively) ( ). in many patients, however, histological overlap between uip and nsip is evident. flaherty and co-workers reviewed slbs from patients with iip who had multiple lobes biopsied and reported histopathological variability between lobes in % of patients. importantly, in that study, uip in at least one lobe defined prognosis ( ). in a later study, the pathological findings in biopsy and subsequent explant specimens from patients with uip were reviewed to refine histological criteria and to assess the relationship between uip and nsip. the important new finding was that nsip-like areas were present in the majority of uip patients ( %) in both biopsy and explants specimens, and in some, these areas were extensive, making accurate diagnosis of uip difficult (cases were misdiagnosed as nsip). the most useful feature for diagnosing uip in difficult cases is the presence of a distinct "patchwork" or variegated pattern of parenchymal involvement ( ). rbild and dip comprise approx % of iips ( ) . these entities are thought to be smoking-related diseases and, like nsip, tend to be responsive to anti-inflammatory therapy. this is not surprising, as pathologically, these lesions are characterized by varying degrees of intra-alveolar and/or peribronchial pigmented macrophage infiltration with scant or no fibrosis. aip is characterized by active fibrosis consisting of proliferating fibroblasts and myofibroblasts with minimal collagen deposition resembling the organizing stage of diffuse alveolar damage (dad) ( ). finally within the iips, some include cop (previously termed bronchiolitis obliterans organizing pneumonia, or boop), and lip. both are relatively steroid-responsive lesions; pathologically, cop is characterized by the presence of intra-alveolar plugs of granulation tissue, and lip by lymphocytic infiltration of the alveolar walls. it should be noted, however, that many experts argue that lip should not be included within the iips because it is considered as lymphoproliferative disorder which, in turn, is rarely idiopathic and is mostly observed in association with infections (e.g., hiv) or collagen vascular diseases (cvds) ( ). the natural history of the pathogenesis of ipf is not known. the description of temporal heterogeneity for the histopathological entity of uip would suggest that this process occurs over a significant period of time within the same low-power microscopic field of the lung. moreover, the description supports the notion that the lesions are not homogeneous for both the timing of the orginal injury and the subsequent response to the injury and repair. in addition, the histopathological entity of uip is not unique to ipf, and can be found in patients with connective tissue diseases, end-stage asbestosis, and end-stage hypersensitivity pneumonia. therefore, uip may represent an end-stage of a "process," not the beginning, intermediate, and end-stage of a disease. the only insight into the natural history of this process comes from two recent studies that suggest the potential concept of a continuum of iips that may overlap in time. in a study of patients whom had multiple lobes biopsied, histological variability was evident in % of the patients. patients concordant for uip were older ( ± yr) than those discordant for uip ( ± yr) or with fibrotic nsip ( ± yr) or cellular nsip ( ± yr), suggesting that nsip may be an early lesion that progresses with time to uip ( ). in a separate study, investigators found discordant histological diagnoses between lobes in % of the patients. nsip-like reactions were evident in % of patients with uip, suggesting that nsip may evolve into uip ( ). in a genetic study, two different histopathological patterns of interstitial pneumonia were found to exist in members of a family who shared protein c gene mutations: adults with uip and children with nsip; this supports the notion that nsip may be a precursor lesion to uip ( ). it has been suggested that most patients with ipf progress in a relentless and insidious manner with a median survival of less than yr ( ). this concept was challenged in a recent study of subcutaneous interferon (ifn)-γ b ( μg thrice weekly) in patients with mild to moderate ipf (forced vital capacity [fvc] > % and dl co > %), where a trend toward lower mortality was seen in ifn-γ b-treated patients compared with placebo-treated patients ( ). interestingly, there were no significant differences in lung function or gas exchange between ifn-γ b-treated and placebo-treated patients at wk of follow-up. in both study groups, % of patients remained stable, % deteriorated, and the rest improved, suggesting that most patients with mild to moderate ipf remain stable for at least yr on no specific therapy. the socalled "ipf exacerbations" may explain the trend toward lower mortality seen in this study, but this requires further study. ipf exacerbations may be defined as an accelerated deterioration of ipf in the absence of apparent infectious agents and heart failure ( , ). it is often a terminal event, with features of dad or organizing pneumonia on lung biopsy or autopsy ( , ). this syndrome is indistinguishable from idiopathic aip ( ), and is similar to acute respiratory distress syndrome (ards). the factors responsible for this accelerated phase of ipf are unknown, but viral infections, high concentrations of oxygen, or drug reactions are plausible etiological factors ( ). only one study has addressed mechanism of mortality of patients with ipf. panos and colleagues ( ) found that most patients with ipf succumb to respiratory failure ( %), cardiovascular disease ( %), lung cancer ( - %), pulmonary embolism ( %), infection ( %), or other health problems ( %) ( ). although infection would appear unlikely as a cause of mortality in ipf patients, clinically we can only determine the micro-organism cause of community acquired pneumonia in % of all patients. therefore, we do not know the true incidence and prevalence of infection as a cause of mortality in patients with ipf, and perhaps a significant portion of the respiratory failure mortalities had infectious etiologies. with regard to cardiovascular disease, congestive heart failure and coronary artery disease (cad) account for % of deaths ( ). patients with ipf appear to be at increased risk of developing cad. in a cross-sectional study of patients referred for lung transplantation, fibrotic lung diseases were associated with an increased prevalence of cad compared with nonfibrotic diseases after adjustment for traditional risk factors (or . ; % ci, . - . ); the authors theorized that the fibroproliferative process may influence cells beyond the pulmonary compartment, and that mediator molecules produced in these disorders might promote atherogenesis ( ). pulmonary arterial hypertension occurs in % of patients with advanced ipf and its presence correlates with a vital capacity (vc) below % of predicted or a dl co under % of predicted ( ). left ventricular (lv) dysfunction occurs in less than % of patients and it is mostly ( %) a result of coexisting right heart failure. other causes of lv dysfunction include ischemic and hypertensive heart disease ( , ). six to % of patients with ipf develop bronchogenic carcinoma. lung cancer in patients with ipf typically presents as a peripheral squamous cell carcinoma in older male smokers ( ). predisposing factors include squamous metaplasia, atypical epithelial cells, or occupational exposures ( , , ) . hubbard and colleagues, in a population-based cohort in the united kingdom, studied patients with ipf and controls. the risk ratio for ipf and lung cancer was . ( % ci . - . ). importantly, adjusting for previous smoking had little effect on this ratio, suggesting that ipf is an independent risk factor for lung cancer ( ). pulmonary embolism occurs in approx to % of patients; inactivity, heart failure, bronchogenic carcinoma, and possibly corticosteroid therapy predispose patients to thrombosis ( ). pulmonary infection causes to % of deaths in patients with ipf; immunosuppressive therapy, traction bronchiectasis, and possibly gerd are predisposing factors ( ). pneumothorax occurs in up to % of patients with ipf and tends to be less responsive to tube thoracostomy, often necessitating surgical intervention ( ). corticosteroids (cs) can cause a myriad of side effects including myopathy, peptic ulcer disease, cataracts, osteoporosis, compression fractures, fluid and electrolyte abnormalities, adrenal insufficiency, and infection ( , ) . in a cross-sectional study in patients with asthma, chronic obstructive pulmonary disease, or "alveolitis" taking oral corticosteroids (n = ) vs controls (n = ), the or for bone fractures was . ( % ci . - . [vertebral fracture or , hip fracture or , and ribs or sternum fracture or . ]). patients tak-ing corticosteroids experienced more cataracts, used more antacids, had more muscle weakness, back pain, bruising, and oral candidiasis, and had fewer teeth compared with controls ( ). one prospective study included patients with ipf; received mg/d of prednisone for mo and received mg/d for mo followed by mg/d for mo. patients were monitored monthly for steroid-related side effects. all patients experienced at least one side effect. common side effects included insomnia ( %), cushingoid change ( %), weight gain ( %), irritability ( %), infection ( %), blurred vision ( %), abdominal bloating ( %), glucose intolerance ( %), and fractures or avascular necrosis ( %) ( ) . cytotoxic agents (cyclophosphamide [cp], azathioprine [aza]) can cause infections, bone marrow suppression, hepatitis, hemorrhagic cystitis (cp) and/ or malignancies ( , ) . in a prospective uncontrolled study, patients with biopsy-proven uip who were unresponsive or intolerant to cs therapy were treated with oral cp ( - mg/kg/d) for mo. nearly two-thirds of patients reported adverse effects, and % of patients discontinued therapy because of intolerable side effects. common side effects related to cp include nausea/ vomiting ( %), anorexia ( %), cytopenias ( %), weight loss ( %), alopecia ( %), infection (herpes zoster) ( %), and ovarian failure ( %) ( ). several factors have been shown to predict poor outcome in ipf. these include older age at presentation, male gender, severe dyspnea at presentation, history of cigarette smoking, severe loss of lung function, severity of reticular opacities or honeycomb change on hrct, characteristic hrct appearance, lack of response to conventional therapy, and histopathological findings showing prominent ff ( , , , , ) . investigators from the university of michigan evaluated the impact of histological diagnosis, baseline clinical, physiological, and radiographical factors on survival in patients with suspected iip. the presence of histological uip was the most important risk factor for mortality (risk ratio [rr] of . [ % ci . - ]), followed by the presence of honeycombing on hrct, a radiographic feature that was shown to be a good surrogate for histological uip (sensitivity of %, and a specificity of %) ( ). the same group evaluated the impact of hrct appearance on survival in patients with iip. patients with histological uip and stereotypical hrct appearance of uip had a shorter survival (median survival . yr) when compared with patients with histological uip and indeterminate hrct scans (median survival . yr) ( ). three studies have shown that a higher hrct-fibrosis score identify patients with worse prognosis. gay and colleagues did not find any measure of pulmo-nary function to be predictive of survival, but did find both the hrct-fibrosis score and the pathological fibrosis score to be useful in predicting survival ( ) . similarly, investigators from the united kingdom found that baseline percent-predicted dl co and hrct-fibrosis score were independent predictors of mortality ( ) . japanese investigators demonstrated that the baseline hc score and the rate of hc progression were both predictive of worse survival in patients with ipf ( ). histopathological findings showing prominent ff identify patients with poor outcome. nicholson and associates retrospectively studied ( ) patients with ipf/uip and analyzed the prognostic significance of four specific microscopic features of uip. multivariate analysis revealed that increasing ff and mononuclear cell infiltrate scores were associated with worsening lung function. higher profusion of ff and a lower dl co were independent predictors of mortality ( ). these results supported the findings by king and co-workers, who also demonstrated that an increase in the number of ff correlated highly with mortality in patients with ipf/uip ( ). in a separate study, expert pathologists reviewed slb from patients with idiopathic or cvd-associated uip and assigned a score for ff. patients with idiopathic uip had more ff and worse survival compared with patients with cvd-associated uip ( ). a number of composite scoring systems have been developed with which to predict survival in ipf. king and co-workers studied patients with ipf/ uip and derived a clinical-radiological-physiological (crp) scoring system using clinical (age, smoking status, clubbing), radiographical (extent of interstitial opacities, presence of pulmonary hypertension on chest radiographs), and physiological parameters (reduced lung volume, abnormal gas exchange during maximal exercise). these investigators demonstrated that the crp score correlated with important histopathological findings and was helpful in predicting survival in patients with ipf. a second abbreviated crp scoring system that excluded pa o during maximal exercise was inferior in predicting survival ( , ) . similarly, investigators from the brompton hospital devised a composite physiological index (cpi) using radiographical and physiological information that predicted mortality more accurately than individual pft in patients with ipf ( ). even though these composite scoring systems are accurate in their predictive ability, they are expensive and cumbersome to generate in clinical practice. with this in mind, the prognostic value of oxygen desaturation during a -min walking test was evaluated in patients with ipf. desaturation defined as a fall in oxygen saturation to % or less during the min walk test identified patients with higher mortality compared with patients who did not desaturate. the -yr survival rate of ipf patients who desaturated to that level was . % compared with . % in patients who did not desaturate ( ). predicting survival in ipf has been centered on baseline radiographical, pathological, and/or physiological testing. recently, researchers have focused on the association of serial changes in pulmonary function or radiographical features and prognosis. one study determined that a decrease in fvc (> % from baseline) during the initial mo of follow-up was associated with increased mortality (hazard ratio . ; ci . - . ) ( ). in a separate study, investigators concluded that at and mo of follow-up, serial pulmonary function trends (change in dl co , fvc, forced expiratory volume in s [fev ], and the cpi) provided important prognostic information in ipf ( ). a third study showed that assessment of changes in clinical and physiological variables (dyspnea score, total lung capacity, thoracic gas volume, fvc, fev , dl co , p o , oxygen saturation, and alveolar-arterial oxygen gradient) at and mo provide clinicians with more accurate prognostic information than baseline values alone ( ). serum markers and nuclear medicine testing may have a predictive role in ipf. greene in a prospective study, investigators analyzed the usefulness of inhaled mlabeled diethylenetriamine penta-acetic acid ([ m] tc-dtpa) aerosol clearance and survival in a cohort of patients with uip. multiple stepwise cox regression analysis identified fast clearance as an independent predictor of mortality ( ). conventional therapy (cs, aza, or cp) for ipf provides only marginal benefit. unfortunately, in many studies, diagnoses were not based on the findings of lung biopsies or were not classified by current pathological criteria; thus, there is uncertainty as to the nature of the disease being treated. two recent meta-analyses searched two large databases for randomized controlled trials (rct) and controlled clinical trials (cct) using cs or non-cs agents in patients with histological uip or who fulfilled all ats criteria for ipf; the authors could not find rcts or ccts evaluating cs alone in ipf and concluded that there are scant good-quality data regarding the efficacy of non-cs agents in ipf ( , ) . the following is a brief discussion of anti-inflammatory (conventional) therapy in the treatment of ipf. cs were the mainstay of therapy for more than four decades, but are of unproven efficacy, and are associated with significant toxicities ( , , ) . early studies of patients with ipf/cfa cited response rates of to % with cs (alone or combined with immunosuppressive agents), but complete or sustained remissions were rare ( , - ). more importantly, many responders likely had iips other than uip (e.g., nsip or rbild/dip). in recent studies, response rates to cs among patients with histological evidence for uip are low ( - %) ( , , , , ) . large retrospective studies of patients with ipf showed no survival benefit with cs ( ; , , ) . in one retrospective study from england, survival was worse among ipf patients treated with cs or cp, although this likely reflects a selection bias ( ). given the potential severe toxicities associated with cs ( , ), recent international consensus statements argue that high-dose cs should not be used to treat ipf ( , ) . however, because anecdotal responses to cs are occasionally noted in patients with ipf/uip ( ), these statements acknowledge that selected patients with clinical or physiological impairment or worsening pfts should be treated ( , ). both statements ( , ) advocate an individualized approach to treating ipf/uip. among patients requiring treatment, both statements recommend combining therapy with either oral aza or cp plus low-dose prednisone or prednisolone ( . mg/kg [lean body weight per d] for wk, then . mg/kg for wk, then . mg/kg). this represents a substantial departure from earlier regimens advocating high-dose prednisone (e.g., ≥ mg/kg/d for ≥ - wk) ( , , ) . combined therapy should be continued for mo in the absence of adverse effects. treatment should be continued beyond or mo or later time points only if patients improve or remain stable. it should be emphasized that these recommendations ( , ) reflect expert opinion, but have not been validated in clinical trials. we believe cs should not be given to patients at high risk for adverse effects (e.g., age > yr, osteoporosis, dm, extreme obesity, and so on). two prospective studies evaluated aza for ipf ( , ) . in both studies, aza was combined with prednisone. in the first study, patients with progressive ipf were initially treated with prednisone alone for mo ( ). at that point, aza ( mg/kg/d) was added and both agents were continued for an additional mo or longer. twelve patients ( %) responded. the independent effect of aza was difficult to assess, because all patients received prednisone concomitantly. in a second, double-blind trial, raghu and associates compared the effect of aza plus prednisone on lung function with that of prednisone alone in previously untreated patients with ipf (the study population may have included patients with iip other than uip). forty-three percent of patients randomly assigned to aza plus prednisone died during the -yr follow-up period, compared with % of patients randomly assigned to prednisone alone. the difference became statistically significant only after adjustment for age (p = . ) ( ). two randomized trials evaluated cp for ipf ( , ) . in one -mo trial, patients with "mid-course" ipf were randomized to prednisone alone (n = ); prednisone plus oral cp ( . mg/kg/d) (n = ); or cp alone (n = ) ( ) . mean balf neutrophil counts declined in the cohort receiving cp, but pfts did not change in any group. johnson and colleagues compared the effect of prednisolone alone with that of prednisolone plus cp on breathlessness, radiographic appearance, and lung function in patients with ipf (the study population included patients with cvd and with iip other than uip). initial improvement occurred in of the patients in the prednisolone-only group and in of the patients in the cp plus prednisolone group. however, at mo, only of the patients in the prednisolone-only group remained improved, and only of the patients in the cp-prednisolone group remained improved. life-table analysis suggested better survival in patients in the cp-prednisolone group, but this was not statistically significant ( ). in a prospective uncontrolled study, zisman and associates studied the efficacy of cp in patients with biopsyproven uip who were unresponsive or intolerant to cs therapy. only patient improved; remained stable, and deteriorated. nearly two-thirds of the patients developed drug-related side effects and one half of the patients discontinued therapy due to intolerable side effects ( ). intermittent, intravenous "pulse" cp, administered every to wk, has been tried for ipf refractory to cs in nonrandomized studies, but benefit was not convincing ( - ). lung transplantation should be considered for patients with ipf refractory to medical therapy ( , ) . two-year survival following single lung transplant (slt) ranges from to %; -yr survival is to % ( - ) . in one study, lung transplantation reduced the risk of death by % ( ) . in addition, patients surviving lung transplantation appear to achieve considerable improvement in most dimensions of health-related quality of life ( ) ( ) ( ) . unfortunately, owing to a shortage of donor organs, waiting time may be prolonged (up to - yr) and many patients with ipf die while awaiting transplantation ( , ) . one study evaluated baseline pft and hrct fibrosis scores and the relationship to -yr survival in patients with ipf younger than yr of age; the optimal points on the receiving operator characteristics (roc) curves for discriminating between survivors and nonsurvivors corresponded to a combination of dl co of % predicted with hrct-fibrosis score of . ( ) . in a separate study, investigators reviewed all transplant referrals for iip that were listed for lung transplantation at their center. the aim of the study was to determine a parameter that would discriminate between patients who survived and patients who died awaiting transplantation. the severity of hypoxemia at rest was the only significant difference between both groups ( ). unless contraindications exist, patients with severe functional impairment (e.g., fvc < % predicted, dl co < % predicted), oxygen dependency, and a deteriorating course refractory to medical therapy should be listed promptly for transplantation ( , ). historically, the fibrotic process in ipf has been thought to be preceded by a chronic inflammatory process that injures the lung and modulates fibrogenesis ( ). conventional management of ipf has been primarily based on the notion that suppressing inflammation may prevent progression to fibrosis. evidence against the notion that inflammation plays an important role in the pathogenesis of ipf comes from the lack of correlation of most markers of inflammation with disease stage or outcome, and the recognition that inflammation is not a prominent histopathological finding in uip ( , ) . additionally, emerging evidence suggests that inflammation is not required for the development of a fibrotic response ( ). in light of the poor prognosis and lack of response to available anti-inflammatory therapy, alternative approaches to the treatment of ipf are being pursued. the following is a brief discussion of antifibrotic therapy and other promising agents in the treatment of ipf. colchicine is an alkaloid derivative of the plant colchicum autumnale, which has been used in acute attacks of gout. it is known to bind microtubular proteins necessary for intracellular trafficking and cellular mitosis, thus adversely affecting secretion of proteins from cells and cellular proliferation ( , ) . its antifibrotic activity was described following the discovery that colchicine inhibits secretion of collagen and other important growth factors necessary for fibroblast proliferation ( ). however, further studies ( ) with or without additional therapeutic agents, such as steroids, failed to document efficacy of colchicine in the treatment of human pulmonary fibrosis. it is thus not currently recommended for use in therapy of ipf. the d-isomer of penicillamine has been extensively studied in animal models of fibrosis, in which it has been shown to prevent accumulation of collagen in the lung by interrupting cross-linking of collagen molecules ( ). this observation has led to its use in treating fibrotic lung disease associated with systemic sclerosis with good results ( ). however, its efficacy in the treatment of ipf has been disappointing ( ), and it is known to have toxic and significant adverse effects ( ). thus it is currently not recommended as therapy for ipf. pirfenidone is a novel agent with broad-spectrum antifibrotic activity. numerous in vitro and animal model studies have demonstrated its effectiveness as an antifibrotic agent. in vitro studies have shown that pirfenidone significantly reduced mrna levels of type i and type iii collagen, and may act at the transcriptional or translational level of collagen synthesis ( ). in vivo, it has been shown to inhibit tgf-β -induced collagen synthesis, decrease extracellular matrix deposition, and suppress the overexpression of tgf-β in the bleomycin model of pulmonary fibrosis ( , ) . because ipf is becoming increasingly recognized as primarily a fibrotic process, the potential role of pirfenidone as a therapeutic agent is being explored. in a prospective open-label study, raghu and colleagues ( ) treated ( biopsy-proven) consecutive patients with pirfenidone who were either unwilling to receive or unresponsive to conventional therapy. survival rates of % at yr and % at yr compared favorably with historical controls. in addition, % of patients discontinued prednisone therapy and the remaining % were able to reduce their daily dose. all patients treated with immunosuppressive therapy tolerated discontinuation of the drug. interestingly, patients whose lung function had deteriorated before enrollment appeared to stabilize after beginning pirfenidone. side effects were relatively common, with patients reporting nausea ( %), fatigue ( %), and photosensitivity ( %). despite these encouraging observations, the results of this study are difficult to interpret owing to the lack of appropriate controls, incomplete pre-entry and follow-up pulmonary function test data, a small study population, and a bias associated with survivorship effect (pulmonary function data of patients who died were not included in the analysis, and this could have biased the results). furthermore, the observed steroid-and immunosuppressive-sparing effects of pirfenidone may have simply reflected lack of efficacy of conventional therapy rather than a true effect of pirfenidone. in a second open-label trial, japanese investigators evaluated oral pirfenidone in eight patients with ipf and two with diffuse lung disease associated with systemic sclerosis; after yr of therapy, there was no change in chest radiographic scores and arterial oxygen tension; the drug was well tolerated ( ). early treatment with pirfenidone appears to slow the progression of pulmonary fibrosis in patients with hermansky-pudlak syndrome ( ). a randomized-controlled trial focusing on early treatment is warranted to test the efficacy and safety of this agent in ipf. ifns play an integral role in the regulation of fibroblast proliferation and collagen synthesis, but the mechanism by which they exert their effect is not clearly understood. recent observations have shown that ifn-γ has antiproliferative, immunomodulatory, and antifibrotic effects ( ), and thus may play a crucial role in the pathogenesis of ipf. ifn-γ decreases collagen content in the bleomycin model of lung fibrosis by inhibiting tgf-β transcription and subsequent procollagen mrna production ( ). in addition, ifn-γ inhibits fibroblast proliferation in cultures derived from normal and fibrotic human lung, making an argument that ifn-γ may have therapeutic applications ( ). lower levels of ifn-γ have been found in patients with ipf compared with patients with less fibrotic diseases such as pulmonary sarcoidosis ( ). kuroki and associates ( ) measured levels of type iii collagen in patients with progressive pulmonary fibrosis and found an inverse correlation with ifn-γ levels, particularly in patients with ipf. these studies suggest that patients with ipf may have a defect in ifn-γ production or function, which predisposes them to develop fibrosis following injury. however, the potential for developing fibrosis is not likely to be dependent on one factor; rather it is likely the result of a complex interplay of fibrotic mediators, differential gene expression, and feedback mechanisms. a study by shaw and colleagues ( ) showed that alveolar macrophages from patients with interstitial lung disease had increased production of platelet-derived growth factor, which is a potent mitogen for fibroblasts. this increase in platelet-derived growth factor was upregulated following treatment with ifn-γ, suggesting that ifn-γ may act to potentiate fibrosis in certain cellular environments. clearly, there is a complex regulatory mechanism in place with regard to whether fibrosis occurs or not, and conflicting in vitro studies must be interpreted with caution. a randomized, prospectively controlled trial was conducted in patients with ipf comparing ifn-γ b and low-dose prednisolone with prednisolone alone for mo ( ) . the results were remarkable in that patients with progressive pulmonary fibrosis treated with ifn-γ b plus lowdose prednisolone demonstrated improvement in pulmonary function, whereas those who received prednisolone alone experienced further decline in pulmo-nary function. the authors showed that all patients treated had almost undetectable levels of ifn-γ mrna, and increased levels of both tgf-β and connective tissue growth factor mrna in lung tissue. furthermore, after treatment with ifn-γ b, transcription of tgf-β and connective tissue growth factor were both significantly decreased. several concerns have been raised regarding the findings reported by ziesche and co-workers ( ), particularly the unexpectedly good results with ifn-γ b. to address some of the issues raised, an outside panel of experts reanalyzed the study data by reviewing each patient's lung function studies, ct scans, and slbs to assess the clinical course and diagnosis of ipf according to the international consensus statement ( , ) . fifteen of patients had either definite (n = ) or probable (n = ) ipf. the panel reanalyzed treatment response using published criteria and eliminated the patients who definitely did not have ipf. patients treated with ifn-γ b plus low-dose prednisolone demonstrated either stability or improvement in pulmonary function and gas exchange after yr of treatment, whereas treatment with prednisolone alone was associated with no improvement in all patients ( ) . the observed benefit of ifn-γ b on lung function has not been reproduced in subsequent studies. in one retrospective uncontrolled observation of patients with ipf treated with ifn-γ b, only one patient experienced objective improvement, discontinued therapy (owing to lack of perceived benefit), and died after mo of therapy ( ). in a separate study of five patients with ipf treated with ifn-γ b, only one patient improved, two discontinued treatment owing to adverse effects and decline in lung function, and one died after mo of therapy ( ). in a recent prospective, randomized, placebo-controlled, double-blind, multicenter phase iii clinical trial of subcutaneous ifn-γ b ( μg thrice weekly) in patients with mild to moderate idiopathic pulmonary fibrosis (fvc > % and dl co > %), a trend toward lower mortality was seen in ifn-γ b-treated patients compared with placebo-treated patients. however, there were no significant differences in lung function or gas exchange between ifn-γ -treated and placebo-treated patients after wk of therapy ( ). a prospective controlled multinational trial is planned to verify the possible survival benefit observed with ifn-γ b therapy in ipf. ifn-β is used for the treatment of chronic hepatitis c and multiple sclerosis. in vitro ifn-β a has been shown to reduce fibroblast proliferation ( ), inhibit collagen production by fibroblasts ( ), increase collagenase mrna ( ), decrease pro-collagen mrna ( ), and increase collagenase activity ( ). a multicenter randomized, double-blind clinical trial examining the efficacy of ifnβ- a was recently completed. patients were randomized into four groups: placebo or ifnβ- a at , , or μg intramuscularly twice per week for a minimum of mo and up to . yr. preliminary results suggest that ifnβ- a lacks significant efficacy ( ). with a dismal response to existing therapy and its accompanied toxicity, the search for additional therapies has intensified in the past decade. the following is an overview of other therapeutic approaches, most of which are undergoing investigation and have not been adequately studied in humans. the prior discussion has centered on fibroblast proliferation and collagen deposition as two areas of therapeutic intervention. it is becoming more apparent that the fibrotic process has multiple pathogenetic mechanisms. one of the fundamental hypotheses of pulmonary fibrosis involves an imbalanced response to injury, in which the capacity of the alveolar epithelium to repair itself is compromised, ultimately leading to fibrosis. some investigators propose that the alveolar epithelium itself has antifibrotic properties, and that chronic loss of alveolar epithelium leads to an environment conducive to the development of fibrosis ( ). support for this notion exists in the fact that induction of apoptosis of alveolar epithelium has been shown to occur following administration of bleomycin ( ). therapies that either inhibit epithelial injury or enhance repair may limit the fibrotic response. in this regard, captopril, an angiotensin-converting enzyme inhibitor widely used in clinical practice, may have a role in the treatment of ipf. in vitro, captopril inhibits fibroblast proliferation, and in models of bleomycin-induced pulmonary fibrosis, it has been shown to reduce alveolar epithelial cell apoptosis and fibroproliferation. in addition, captopril abrogates fas-induced apoptosis in human alveolar epithelial cells ( , ) . there is currently an ongoing clinical trial at the national institute of respiratory diseases in mexico testing the efficacy of captopril in patients with ipf ( ). another agent that may protect the alveolar epithelium is keratinocyte growth factor (kgf). this class of growth factor stimulates type ii cell proliferation with no direct effects on fibroblasts ( ). keratinocyte growth factor increases surfactant protein gene expression and sodium/potassium adenosine triphosphatase, factors that may protect the alveolar epithelium ( ). in vivo, kgf has been shown to protect animals from injury and subsequent development of fibrosis caused by a variety of insults ( ). there is evidence that an exaggerated oxidant stress may play a role in the pathogenesis of pulmonary fibrosis by injuring the alveolar epithelial cells. this oxidant burden is thought to be a consequence of both increased levels of reactive oxygen species and a defective antioxidant response. a major protector of oxidant-induced injury of the alveolar epithelium is glutathione, which has been shown to be deficient in the balf of patients with ipf ( ). moreover, in vitro studies have shown that n-acetylcysteine (nac), a precursor for glutathione synthesis, may augment the antioxidant defense system and protect the alveolar epithelium from free radical-induced injury ( ). in vivo, hagiwara and associates ( ) reported a significant inhibition of bleomycininduced lung fibrosis in mice following aerosolized nac during the early inflammatory phase of injury. whether this effect was secondary to nac inhibition of cellular inflammation, or its role as a scavenger of reactive oxygen species, is not clear. german investigators evaluated oral nac as a strategy to augment lung glutathione levels in patients with biopsy-proven ipf. following therapy with nac, glutathione levels in balf were significantly increased compared with pretreatment levels ( ). in a separate study, behr and colleagues ( ) prospectively studied patients with ipf and assessed the redox balance of the lung and changes in lung function following high-dose nac therapy for wk. they reported an increase in the total and reduced form of glutathione concentration in the balf, and significant improvement in pulmonary function. the authors suggest that nac may be considered as an adjunct in the treatment of ipf. currently, there is a clinical trial in europe to evaluate the potential benefits of nac in ipf. as mechanisms of fibrosis at the cellular and molecular level become elucidated, their application to the development of novel therapeutic strategies appears promising. given the temporal heterogeneity of the uip lesion, early histopathological abnormalities may be present even in patients with advanced ipf. if early cytokine release is relevant to the initiation of this pathogenic response, then the targeting of early cytokines such as tnf-α should be considered. tnf-α appears to be upregulated soon after bleomycin-induced injury and has been implicated in a variety of inflammatory processes ( ). sime and colleagues ( ) showed that transient overexpression of tnf-α in rat lung led to fibrosis associated with concomitant tgf-β expression and proliferation of myofibroblasts ( ). furthermore, upregulation of tnf-α expression has been shown to occur in inbred murine strains that are sensitive to bleomycin-induced lung fibrosis, with similar expression being absent in resistant strains ( ). in addition, ortiz and colleagues ( ) showed that tnf receptor-deficient mice did not develop pulmonary fibrosis following exposure to bleomycin despite increased tnf expression. studies of human lung biopsy specimens of patients with ipf have shown an upregulation of tnf-α mrna and protein ( ). these observations along with other studies in animal models demonstrating abrogation of pulmonary fibrosis following treatment with soluble tnf receptors suggest that tnf-α may play an important role in the pathogenesis of pulmonary fibrosis ( ). several agents that can block tnf-α are now available for human use ( ). there is currently an ongoing clinical trial evaluating the safety and efficacy of etanercept, a tnf-α receptor antagonist, in patients with ipf. the expression of tnf-α is inhibited by certain cytokines such as il- , which is produced by a variety of cells including t-helper (th) cells, monocytes, and alveolar macrophages ( , ) . it is conceivable that il- may be useful in blunting the action of increased tnf-α observed following bleomycin-induced lung injury, therefore possibly inhibiting progression to fibrosis. arai and colleagues ( ) investigated the possible inhibitory effects of il- by introducing the il- gene into mice exposed to bleomycin and found that bleomycin-induced pulmonary fibrosis was suppressed. these results suggest that treatment with il- during the early inflammatory phases of lung injury may be promising and requires further investigation. concerns regarding the role of this agent in treating pulmonary fibrosis exist in that il- is a type- th (th ) cytokine that could suppress ifn-γ expression and promote fibrogenesis ( ). a clinical trial to study the effect of this cytokine is now underway in the united states. the realization that th cell subsets could be categorized on the basis of cytokine profiles has helped clarify our understanding of chronic cell-mediated immune responses. the type- (th ) cytokines include ifn-γ, il- , il- , il- , and th cytokines include il- , il- , il- , and il- . analysis of subset populations of th cells within the interstitium of patients with ipf reveal a predominantly th -type pattern of cytokine production, suggesting that alterations in t-cell subpopulations of th and th cells and their associated pattern of cytokine production may contribute to progression of ipf ( ) . supporting evidence comes from studies demonstrating that ifn-γ (a th cytokine) has profound antifibrotic effects in ipf possibly because it shifts the balance away from a th -dependent profibrotic environment. thus, it seems reasonable to target therapy to correct the th imbalance by either favoring a th phenotype or abrogating the predominant th response (e.g., administration of il- to promote ifn-γ expression, or inhibition of il- , il- , and so on). tgf-β is a critical cytokine for the promotion of fibrosis. in bleomycininduced pulmonary fibrosis, passive immunization with neutralizing antibod-ies against tgf-β reduces collagen deposition ( ) . in addition, the overexpression of tgf-β results in a fibrogenic response resembling uip (i.e., abundant ff) ( ) . in patients with ipf, increased expression of tgf-β is localized to bronchiolar epithelial cells, epithelial cells of hc cysts, and hyperplastic type ii pneumocytes ( ) . it is possible that therapy with neutralizing antibodies against tgf-β , or utilization of a tgf-β inhibitor such as decorin, may become useful in the treatment of ipf ( ) . tgf-β signals through a receptor that activates transcription factors smad and smad promoting tgf-β gene transcription. interestingly, ifn-γ inhibits the activation of smad and induces the expression of smad , an antagonistic molecule that inhibits tgf-β expression. smad can be produced in the laboratory and may become a useful molecule in the treatment of ipf ( ) . monocyte chemoattractant protein (mcp)- is a member of the c-c subfamily of chemokines involved in monocyte/macrophage mediated inflammation ( , ) . in addition, mcp- has been shown to stimulate pulmonary fibroblasts, tgf-β synthesis, and collagen production ( ) . analysis of serum, balf, and lung biopsy specimens from patients with ipf reveal increased levels of this chemokine ( , , ) . furthermore, serum mcp- levels correlate with clinical course in patients with interstitial lung disease ( ) . further investigation into the clinical significance of mcp- and its contribution into the pathogenesis of ipf are necessary, and may provide the groundwork for novel therapies. another potential mediator of fibrosis produced in a variety of cells in the lung is the vasoactive peptide endothelin (et) - ( , ) . first thought to be primarily a vasoactive agent, et- has been shown to stimulate fibroblast proliferation, activate monocytes, induce collagen production, and regulate cytokine production ( ). mutsaers and colleagues ( ) revealed that et- levels are augmented following administration of bleomycin with increased localization of the agent in areas of fibrosis. with increases in et- synthesis following tnf-α and tgf-β stimulation, one may speculate that et- may play an important role in the cascade of events leading to pulmonary fibrosis ( , ) . additional support for its role in pulmonary fibrosis comes from studies in animals in which fibrosis was attenuated following treatment with bosentan, an et receptor antagonist ( ). et- has also been associated with pulmonary fibrosis in humans. in a study examining the expression of et- in patients with interstitial lung fibrosis, giaid and associates ( ) found a striking expression of et- in lung tissue that correlated with parameters of disease activity in patients with ipf. there is currently an ongoing clinical trial evaluating the safety and efficacy of bosentan in patients with ipf. some have hypothesized that inducing fibroblast apoptosis may curb progression of fibrosis. lovastatin is a pharmacological agent widely used in the treatment of hypercholesterolemia that inhibits -hydroxy- -methylglutarylcoenzyme a, therefore affecting many cellular functions essential for normal cell homeostasis including proliferation and cell survival. tan and associates showed that clinically achievable concentrations of lovastatin induced apoptosis of human lung fibroblasts in vitro, and in vivo reduced granulation tissue formation and induced fibroblast apoptosis in a guinea pig wound model of fibroproliferation ( ) . with its known safety profile and potential antifibrotic effect, lovastatin is an attractive candidate in the treatment of ipf. suramin is a sulfonated napthylurea that has been used to treat onchocerciasis, acquired immunodeficiency virus, and prostate cancer. in vitro, suramin antagonizes the effects of a number of growth factors that promote fibrogenesis such as tgf-β, insulin-like growth factor- , platelet-derived growth factor, epidermal-like growth factor, and fibroblast growth factor. in vivo, suramin has been shown to delay wound healing ( ). relaxin is a protein secreted by the gravid uterus responsible for remodeling of the interpubic ligament and cervix during the later phases of pregnancy. relaxin inhibits the tgf-β-mediated overexpression of extracellular matrix, stimulates the expression of collagenases by lung fibroblasts in vitro, and has been shown to block bleomycin-induced fibrosis in mice ( ). the eicosanoids are potential candidates for therapeutic intervention. the prostaglandin pge is a potent inhibitor of fibroblast proliferation and extracellular matrix deposition and may ameliorate the fibrotic process in ipf ( ). indomethacin, an inhibitor of cyclo-oxygenase, has been shown to decrease bleomycin-induced pulmonary fibrosis in an animal model but to our knowledge, it has not been evaluated in human ipf ( ). the profibrotic leukotriene b has been shown to be increased in balf and lung tissue of patients with ipf ( ). inhibition of leukotriene production may be an effective adjuvant therapy, and drugs are now available to block leukotriene synthesis. gene-specific antisense therapy against proteins known to be important in human lung fibroblast proliferation may become an effective approach in treating ipf patients. in vitro, chen and associates showed that gene-specific oligonucleotides (oligos) against c-ki-ras substantially inhibited the proliferation of human fibroblasts ( ). beractan is a natural bovine lung extract containing phospholipids, neutral lipids, fatty acids, and surfactant-associated proteins. in vitro, beractan pro-voked fibroblast apoptosis, induced collagenase- expression, and decreased type i collagen ( ). pulmonary fibrosis can be complicated by pulmonary hypertension limiting exercise tolerance and survival. german investigators performed a randomized-controlled, open-label trial in individuals with pulmonary hypertension secondary to pulmonary fibrosis. they compared oral sildenafil with inhaled nitric oxide and infused epoprostenol. a single dose of sildenafil reduced pulmonary vascular resistance by nearly one-third and increased the mean arterial blood oxygen tension by mmhg. the drug was well tolerated with no adverse effects on ventilation-perfusion matching ( ). a clinical trial evaluating sildenafil in patients with ipf and pulmonary hypertension will be conducted soon. in recent years, significant advances have been made in our understanding and management of ipf. however, in order to further our knowledge and make significant progress in the care of these patients, it is critical that we improve our understanding of the natural history and pathogenesis of ipf. in addition, we need to pursue novel imaging and diagnostic technologies to improve earlier diagnosis and we must also educate primary care physicians and pulmonologists to refer patients early to 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endothelin- in bleomycin-induced pulmonary fibrosis and the effect of an endothelin receptor antagonist lovastatin induces fibroblast apoptosis in vitro and in vivo. a possible therapy for fibroproliferative disorders sildenafil for treatment of lung fibrosis and pulmonary hypertension: a randomised controlled trial key: cord- -y fcw dj authors: kalodimou, georgia; veit, svenja; jany, sylvia; kalinke, ulrich; broder, christopher c.; sutter, gerd; volz, asisa title: a soluble version of nipah virus glycoprotein g delivered by vaccinia virus mva activates specific cd and cd t cells in mice date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: y fcw dj nipah virus (niv) is an emerging zoonotic virus that is transmitted by bats to humans and to pigs, causing severe respiratory disease and often fatal encephalitis. antibodies directed against the niv-glycoprotein (g) protein are known to play a major role in clearing niv infection and in providing vaccine-induced protective immunity. more recently, t cells have been also shown to be involved in recovery from niv infection. so far, relatively little is known about the role of t cell responses and the antigenic targets of niv-g that are recognized by cd t cells. in this study, niv-g protein served as the target immunogen to activate niv-specific cellular immune responses. modified vaccinia virus ankara (mva), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for the generation of mva–niv-g candidate vaccines expressing different versions of recombinant niv-g. overlapping peptides covering the entire niv-g protein were used to identify major histocompatibility complex class i/ii-restricted t cell responses in type i interferon receptor-deficient (ifnar−/−) mice after vaccination with the mva–niv-g candidate vaccines. we have identified an h -b-restricted nonamer peptide epitope with cd t cell antigenicity and a h -b mer with cd t cell antigenicity in the niv-g protein. the identification of this epitope and the availability of the mva–niv-g candidate vaccines will help to evaluate niv-g-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of niv-g infection. of note, a soluble version of niv-g was advantageous in activating niv-g-specific cellular immune responses using these peptides. nipah virus (niv) is an emerging zoonotic pathogen of global concern that was ranked recently by the world health organization (who) as a high-priority pathogen. niv is a negative-sense, single-stranded rna virus that is a member of the genus henipavirus (family paramyxoviridae). niv was first identified during a large outbreak affecting humans and pigs in malaysia and singapore in [ ] . from onwards, seasonal outbreaks are observed almost annually in bangladesh and sporadically hohenpeißenberg, germany) and had access to food and water ad libitum. all experiments were approved by the government of upper bavaria, munich germany and were performed in compliance with the german animal welfare act ( . vet- .vet_ - - , . . ). primary chicken embryo fibroblasts (cef) were isolated from -day-old spf chicken embryos (valo, cuxhaven, germany) and grown in minimum essential medium (mem) (sigma-aldrich, taufkirchen, germany) supplemented with % heat-inactivated fetal bovine serum (fbs) (sigma-aldrich), % penicillin-streptomycin (sigma-aldrich), and % mem nonessential amino acid solution (sigma-aldrich). human hela cells (atcc ccl- ) were maintained in mem supplemented with % heat-inactivated fbs (sigma-aldrich) and the above antibiotics. df- cells (atcc crl- ) were grown in vle dulbecco's modified eagle's medium (dmem) (merck, darmstadt, germany) supplemented with % heat-inactivated fbs (sigma-aldrich), % penicillin-streptomycin (sigma-aldrich), % mem nonessential amino acid solution (sigma-aldrich), and % hepes solution (sigma-aldrich). cells were maintained at • c in a humidified % co atmosphere. the cdna that encoded for the entire amino acid sequence of the niv-g protein (nipah virus isolate ummc , genbank accession number ay . ) was modified in silico by introducing silent codon alterations to remove termination signals of vaccinia virus early transcription (tttttnt) and g/c nucleotide runs. for construction of the soluble form of niv-g protein (nivsg), the cytoplasmic and transmembrane domains were deleted and an internal leader sequence and amino acid linker sequences were added as described previously [ , ] . for vaccinia virus (vacv)-specific transcriptional regulation, we placed the nivsg gene sequences under control of the synthetic vacv early/late promoter (pmh ) [ ] and used the strong natural early vacv promoter pvgf [ ] [ ] [ ] for expression of niv-g sequences. the cdnas encoding for niv-g or nivsg including the cleavage sites for the restriction endonucleases xhol and apal were generated by dna synthesis (genewiz, leipzig, germany). cdna sequences were cloned into the mva transfer plasmid plw- [ ] , which directs insertion of heterologous sequences to a site between the open reading frames (orf) of the essential viral genes, mva r and mva l. recombinant mva viruses expressing the nivsg and niv-g proteins were generated as described previously [ , ] . to summarize, cef cells at - % confluence were infected with nonrecombinant mva (clonal isolate mva f ) at a multiplicity of infection (moi) of . and transfected with vector plasmid dna containing nivsg or niv-g gene sequences using x-tremegene™ hp dna transfection reagent (roche diagnostics, penzberg, germany). after h incubation, cells were harvested, and the recombinant viruses mva-nivsg and mva-niv-g were clonally isolated by screening for co-expression of the fluorescent protein marker gfp and plaque passaging several times. resultant vector virus isolates were quality controlled using standard protocols [ ] . polymerase chain reaction (pcr) analysis of genomic viral dna served to confirm the genetic identity and genomic stability of the mva vector viruses. the replicative capacity of the recombinant mva-niv viruses compared with nonrecombinant mva was tested by multi-step growth experiments in cef and human hela cells. to generate vaccine preparations, recombinant mva-niv viruses were amplified in cef, purified by ultracentrifugation through % sucrose cushions, and reconstituted in mm tris-hcl buffer, ph . , to make stock preparations [ ] . cef and hela cells were infected with recombinant mva-nivsg and mva-niv-g viruses at a moi of . cells infected with mva (moi ) and inoculation medium alone (mock) were used as controls. cell lysates were prepared or culture supernatants were collected at various time points after infection and stored at − • c. cell-associated and secreted proteins were resolved by sodium dodecyl sulfate (sds)-polyacrylamide ( %) gel electrophoresis (sds-page), and proteins were transferred onto nitrocellulose membranes by wet electroblotting. to investigate the glycosylation pattern, proteins were pretreated using the protein deglycosylation mix ii kit (new england biolabs, ipswich, ma, usa) according to the manufacturer's instructions prior to sds page. membranes were blocked with blocking buffer, which consisted of pbs containing % non-fat dried milk powder (carl roth, karlsruhe, germany) and . % v/v tween (sigma-aldrich) for one hour at room temperature. membranes were then probed with the primary antibody (polyclonal mouse anti-niv-g ( : ) or polyclonal rabbit anti-niv-g ( : )) diluted in blocking buffer overnight at • c. membranes were washed three times with pbs containing . % tween (pbs/t) and probed with goat anti-mouse igg conjugated to horseradish peroxidase (hrp) ( : ; agilent dako, glostrup, denmark) or goat anti-rabbit igg hrp ( : ; cell signaling technology, leiden, the netherlands). membranes were washed again with pbs/t and were developed using supersignal ® west dura extended duration substrate (thermo fisher scientific, planegg, gemany). blots were visualized using a microchemi . imager (dnr bio-imaging systems, neve yamin, israel). confluent monolayers of hela cells were infected with recombinant mva-nivsg or mva-niv-g viruses at moi . . controls included mva (moi . ) and inoculation medium alone (mock). after infection, cells were incubated for h in a • c incubator. cells were fixed with % paraformaldehyde (pfa) (sigma-aldrich) and on some occasions, permeabilized with . % triton x- (sigma-aldrich). cells were blocked in blocking buffer containing % bovine serum albumin (bsa) (sigma-aldrich). cells were stained with polyclonal rabbit anti-niv-g diluted : , in pbs containing . % bsa (pbs/bsa). cells were washed with pbs and stained with the secondary antibody goat anti-mouse igg alexa fluor (af) ( : ; thermo fisher scientific) diluted in pbs/bsa buffer. fluorescence was visualized using the keyence bz-x microscope (keyence, osaka, japan). mice were immunized with pfu in µl vaccine buffer ( mm tris and mm nacl, ph . ) of recombinant mva-nivsg, recombinant mva-niv-g or mva or vaccine buffer as a mock vaccine control via the intraperitoneal or intramuscular routes. mice received either one (prime) or two (prime-boost) immunizations over a day interval. for t cell analysis, mice were euthanized days after immunization. blood was collected on days , , and . coagulated blood was centrifuged at × g for min in minicollect vials (greiner bio-one, alphen aan den rijn, the netherlands) in order to separate serum, which was subsequently stored at − • c until further use. antigen-specific igg responses induced by immunization with the vaccine candidates were analyzed by enzyme-linked immunosorbent assay (elisa) using purified soluble recombinant niv glycoprotein g expressed in mammalian hek cells. flat bottom -well elisa plates (nunc™ maxisorp™ plates, thermo scientific) were coated with ng/well recombinant protein ( µl volume) and incubated overnight at • c. plates were washed three times with µl/well pbs/t. plates were blocked with blocking buffer containing % bovine serum albumin (sigma-aldrich) and . m sucrose (sigma-aldrich) in pbs for h at • c. plates were then washed with pbs/t as described above. sera were serially diluted in pbs containing % bsa (pbs/bsa) three-fold down the plate, starting at a dilution of : ( µl volume/well) and incubated for h at • c. after washing, plates were incubated with µl/well goat anti-mouse igg conjugated hrp ( : ; agilent dako, denmark) diluted in pbs/bsa for h at • c. plates were then washed with pbs/t as described earlier. then, µl/well , , , -tetramethylbenzidine (tmb) liquid substrate system for elisa (sigma-aldrich) was added, and plates were incubated until a color change was observed. the reaction was stopped by the addition of µl/well stop reagent for tmb substrate ( nm, sigma-aldrich). the absorbance was measured on an elisa plate reader at nm with a nm reference wavelength. total igg titers were calculated from the inflection point of the titration curve as logarithms of the reciprocal. the protein sequence for niv-g was obtained from the uniprot database (id: q ih ). using an in silico approach, we identified individual synthetic peptides that spanned the external domain niv-g protein, starting from the third amino acid of the n-terminal side to the c terminus (amino acids - ). our peptide library consisted of mer peptides that overlapped by mer. all peptides were synthesized by thermo fisher scientific as crude material (< % purity) on a - mg scale. the two-dimensional peptide pool matrix system was used for screening [ , ] . briefly, peptides were organized into two-dimensional matrix peptide pools (h -h and v -v ) containing - peptides. for mapping of potential cd t cell epitopes in positive mer peptides, every possible sequence of peptides - mer in length was determined. theoretical peptides were then analyzed for binding to the mouse major histocompatibility complex (mhc) class i allele h -b using the syfpeithi database. the peptides of each amino acid sequence length were synthesized and tested. for identification of cd t cell epitopes, mhc class ii binding predictions were performed on mer peptides found within positive peptide pools identified by ifn-γ enzyme-linked immunospot assay (elispot). using the mhc class ii binding, t cell epitope prediction resource of the immune epitope database (iedb, https://www.iedb.org/), peptides restricted to mouse mhc class ii allele h -iab were analyzed using "iedb recommended" prediction method [ ] . the most promising candidates were then chosen for further experimental epitope prediction studies. all peptides were dissolved to a concentration of mg/ml in pbs, aliquoted, and stored at − • c until use. t cell analysis by elispot was performed as described previously [ ] . briefly, spleens were collected from mice days after the final immunization. single-cell suspensions were prepared by teasing spleens through a µm cell strainer (falcon ® corning, corning, ny, usa). red blood cells (rbc) were removed using red cell lysis buffer (sigma-aldrich), and cells were washed and resuspended in rpmi- , which consisted of rpmi- (sigma-aldrich) containing % heat-inactivated fbs (sigma-aldrich) and % penicillin-streptomycin (sigma-aldrich). for experiments that required cd and cd t cell purification, splenocytes were incubated with mouse cd and cd microbeads (miltenyi biotec, bergisch gladbach, germany) and processed by negative selection using the quadromacs separator (miltenyi biotec). ifn-γ-producing cells were measured by ifn-γ elispot assay using the mouse ifn-γ elispotplus kit (mabtech, stockholm, sweden) as described in the manufacturer's protocol. in summary, × splenocytes were seeded onto -well flat bottom plates ( µl/well) (sarstedt, nümbrecht, germany), and µl/well peptide pools, subpools, or individual peptides were added (each peptide diluted to µg/ml in rpmi- ). after mixing, the splenocyte/peptide mixtures were transferred onto plates precoated with ifn-γ detection antibody and incubated for h at • c. nonstimulated cells were used as a mock control, and the positive controls cultures were treated with phorbol myristate acetate (pma) and ionomycin (both from sigma-aldrich) or vaccinia virus (vacv)-specific cd t cell epitope, b r - (tsykfesv) [ ] . after incubating, plates were processed as described in the kit manufacturer's protocol (mabtech). spots were counted and analyzed using the automated elispot plate reader (a. el. vis eli.scan and a. el. vis elispot analysis software, hannover, germany). splenocytes were diluted to × cells/ml in rpmi- , and µl/well ( × cells) was added onto a -well u-bottom plate. then, µl/well peptide diluted to µg/ml in rpmi- was added to give a final peptide concentration of µg/ml. the vacv cd t cell epitope b r - (final concentration of µg/ml) was used as a positive control along with pma ( ng/ml) plus ionomycin ( ng/ml). pbs diluted in rpmi- was used as a mock stimulated control. after plating, cells were incubated for h at • c. then, µl/well x brefeldin a, a golgi inhibitor that was prepared by diluting x brefeldin a (biolegend, san diego, ca, usa) in rmpi- , was added to give a final dilution of x brefeldin a. cells were then incubated for an additional h at • c. after incubating, plates were centrifuged ( g for min), and the supernatant was removed. samples were filtered through µm nylon mesh (sefar pty ltd., huntingwood, nsw, australia) into ml round bottom facs tubes (sarstedt). single-color controls were prepared for each facs analysis using onecomp ebeads™ compensation beads (ebioscience, thermo fisher scientific) for fluorophore-conjugated antibodies and cells for the viability dye zombie aqua. data acquisition was performed by macsquant vyb flow analyser (miltenyi biotec), and data was analyzed using flowjo (flowjo llc, bd life sciences, ashland, or, usa). data were analyzed using graphpad prism version . (graphpad software inc., san diego, ca, usa) and were expressed as mean ± standard error of the mean (sem). statistical analysis was performed using the unpaired, two-tailed t-test to compare two groups and one-way anova to compare three or more groups. the threshold for statistical significance was p < . . to generate the recombinant mva viruses delivering niv-g antigens, we used the gene from niv malaysia (isolate ummc , genbank accession number ay . ) and generated codon-optimized gene sequences encoding a full-length glycoprotein g (niv-g) or a soluble external domain g protein (nivsg). these synthetic gene sequences were placed under the transcriptional control of the vaccinia virus-specific promoters pvgf or pmh and introduced into the mva genome by homologous recombination targeting the intergenic site between the essential mva genes and l. the clonal isolation of the recombinant viruses mva-nivsg and mva-niv-g was facilitated by the co-production of the green fluorescent reporter protein (gfp), as previously described [ ] . the final recombinant viruses containing the niv-g gene sequences (mva-niv-g and mva-nivsg) were obtained after removal of the gfp reporter gene by intragenomic homologous recombination ( figure s a , marker gene deletion). to verify the identity of the desired modification, we performed the standard quality control experiments as described previously [ ] . pcr analysis of viral genomic dna confirmed the proper insertion of the recombinant gene sequences at the target site in the genome of mva (figure s b-e) and the genetic characteristics and stability of the recombinant mva viruses. we assessed the growth behavior of the recombinant viruses mva-nivsg and mva-niv-g in multi-step growth analyses in human hela cells and primary cef, which are routinely used for amplification of recombinant mva in vaccine manufacturing ( figure s f ). mva-nivsg and mva-niv-g efficiently replicated in cef and demonstrated an increase of infectivity titers that was comparable to that obtained with nonrecombinant mva. however, mva-niv-g and mva-nivsg did not productively grow in human hela cells, confirming that they had retained the characteristic replication deficiency of mva in cells of mammalian origin. these findings corroborated the expected mva phenotype and confirmed that the recombinant viruses could be handled under laboratory conditions of biosafety level . of note, we originally generated another recombinant mva virus using the synthetic early-late promoter pmh for transcriptional control of recombinant gene expression and production of the full-length niv-g protein. however, upon growth testing, this candidate virus failed to reach levels of infectious progeny in cef to be eligible for large-scale amplification as needed for vaccine production processes. our vaccine candidates, mva-nivsg and mva-niv-g, should produce recombinant niv glycoprotein g in its full-length form (niv-g) and, in parallel, the soluble form (nivsg). niv-g is a amino acid long protein consisting of an n-terminal internal domain, a transmembrane domain, and a c-terminal external domain ( figure a ). for nivsg protein, an internal leader sequence and three amino acid linkers have replaced the internal and transmembrane domains ( figure a ), as described previously [ , ] . we assessed the correct expression and studied the cellular localization of the niv-g and nivsg by immunofluorescence microscopy of mva-niv-infected cells immunolabeled with anti-niv-g antibody, followed by a fluorescently labelled secondary antibody. cell nuclei were stained with dapi ( nm). as anticipated, we observed different patterns of green fluorescence, with varying cellular localizations depending on the mva-niv construct. green fluorescence, specific for niv-g, was identified in permeabilized and nonpermeabilized cells infected with mva-niv-g ( figure b ), but not in mva-infected or mock control cells. this data confirmed that the recombinant full-length niv-g protein encoded by mva-niv-g was indeed anchored on the cell surface. in contrast, the niv-g-specific staining in cells infected with mva-nivsg virus appeared to be predominantly located within the cells and was readily detected after permeabilization. as anticipated, we failed to detect nivsg in considerable amounts without permeabilization, confirming that nivsg was not expressed on the cell surface ( figure b) . to further investigate the synthesis of niv-g proteins after infection with mva-nivsg and mva-niv-g, respectively, total proteins from infected cef and hela cell cultures were analyzed by western blot using a niv-g-specific antibody. total cell lysates or culture supernatants obtained from cef and hela cultures infected with recombinant mva virus were separated by sds-page and immunoblotted. we specifically detected a protein with an estimated molecular mass of approximately - kda in lysates from cef cells and hela cells infected either with mva-niv-g or mva-nivsg ( figure c ). in the cell lysates, the glycoprotein was first detectable at h post-infection. since the nivsg gene encoding sequences were modified to result in the secreted soluble version (sg), we performed additional western blot experiments to determine whether the protein still maintained glycosylation sites comparable to wild-type niv-g. lysates and supernatants obtained from cultures of df- cells infected with mva-nivsg were treated with enzymes that deglycosylate proteins and analyzed by western blotting. enzyme treatment resulted in a reduction in the molecular mass of recombinant nivsg protein from - kda to - kda ( figure d ), matching the expected size of unmodified g protein. this suggested that recombinant nivsg has retained the normal glycosylation pattern of wild-type niv-g. to assess the immunogenicity of the recombinant mva-niv-g/nivsg candidate vaccines, we vaccinated ifnar−/− mice with pfu via the intramuscular route at days and . serum samples were tested for niv-g-binding igg antibodies by elisa days after the first immunization (prime) and days after the second immunization (prime-boost) (figure ). even a single application of the mva-niv-g vaccines induced abundant levels of niv-g-specific igg antibodies in the mice. after booster immunization, all vaccinated animals produced even higher levels of circulating niv-g-specific antibodies, with the antibody titers increasing by approximately ten-fold. currently, there is only limited information available on niv-specific t cell immunity. in order to characterize the niv-g-specific t cell response induced by our candidate vaccines, we first aimed to identify niv-g polyprotein-specific t cell epitopes. ifnar−/− mice on a c bl/ background (mhc i = h -db/h -kb (h -b) and mhc ii = h -iab) were immunized twice with the mva-nivsg candidate vaccine via the intraperitoneal (i.p.) route, and splenocytes were prepared days after the final inoculation, cd and cd t cells were purified and restimulated with overlapping mer peptides corresponding to the niv-g protein. we pooled overlapping peptides using a twodimensional peptide matrix system ( figure a ) and screened for their ability to induce ifn-γ by elispot. ifn-γ production above background levels was observed after stimulation with out of the peptide pools tested ( figure b ). cd t cells from mice immunized with mva-nivsg, but not the mva and mock groups, showed elevated numbers of ifn-γ spot-forming counts (sfc) after stimulation with peptide pools v and h ( figure b ). in the next step, the peptides within the v and h peptide pools were used to characterize the t cell epitope specificities in more detail. for this, pools v (v a and v b) and h (h a and h b) were subdivided into subpools containing - peptides each. since two mer peptides were shared between the pools, # (g - = ytrstdnqavikdal) and # (g - = tdnqavikdalqgiq), these peptides were also tested separately. for this experiment, we used an identical immunization protocol and splenic cd t cell purification procedure. cd t cells from these mice were restimulated with the above subpools and individual peptides # and # . stimulation with the subpools h a and v a resulted in ifn-γ production above background levels in the mva-nivsg group with ifn-γ sfc mean ± sem values of ± and ± ifn-γ sfc/ splenocytes, respectively ( figure c ). subpools h b and v b, however, did not stimulate cd t cells in the mva-nivsg group. stimulation of cd t cells with individual peptides # and # , which were present in subpools h a and v a, resulted in different outcomes. ifn-γ production above the background was observed in cultures stimulated with # (mean = ± sfc/ cells), but not # , in the mva-nivsg group. these data suggested that the mer peptide # contained peptide sequences that stimulated niv-g-specific cd t cells. niv-g or niv-sg currently, there is only limited information available on niv-specific t cell immunity. in order to characterize the niv-g-specific t cell response induced by our candidate vaccines, we first aimed to identify niv-g polyprotein-specific t cell epitopes. ifnar−/− mice on a c bl/ background (mhc i = h -db/h -kb (h -b) and mhc ii = h -iab) were immunized twice with the mva-nivsg candidate vaccine via the intraperitoneal (i.p.) route, and splenocytes were prepared days after the final inoculation, cd and cd t cells were purified and restimulated with overlapping mer peptides corresponding to the niv-g protein. we pooled overlapping peptides using a two-dimensional peptide matrix system ( figure a ) and screened for their ability to induce ifn-γ by elispot. ifn-γ production above background levels was observed after stimulation with out of the peptide pools tested ( figure b ). cd t cells from mice immunized with mva-nivsg, but not the mva and mock groups, showed elevated numbers of ifn-γ spot-forming counts (sfc) after stimulation with peptide pools v and h ( figure b ). in the next step, the peptides within the v and h peptide pools were used to characterize the t cell epitope specificities in more detail. for this, pools v (v a and v b) and h (h a and h b) were subdivided into subpools containing - peptides each. since two mer peptides were shared between the pools, # (g - = ytrstdnqavikdal) and # (g - = tdnqavikdalqgiq), these peptides were also tested separately. for this experiment, we used an identical immunization protocol and splenic cd t cell purification procedure. cd t cells from these mice were restimulated with the above subpools and individual peptides # and # . stimulation with the subpools h a and v a resulted in ifn-γ production above background levels in the mva-nivsg group with ifn-γ sfc mean ± sem values of ± and ± ifn-γ sfc/ splenocytes, respectively ( figure c ). subpools h b and v b, however, did not stimulate cd t cells in the mva-nivsg group. stimulation of cd t cells with individual peptides # and # , which were present in subpools h a and v a, resulted in different outcomes. ifn-γ production above the background was observed in cultures stimulated with # (mean = ± sfc/ cells), but not # , in the mva-nivsg group. these data suggested that the mer peptide # contained peptide sequences that stimulated niv-g-specific cd t cells. graphs shows ifn-γ sfc (spot-forming counts) of stimulated cd t cells. differences between groups were analyzed by one-way anova. asterisks represent statistically significant overall differences for a specific peptide subpool or individual peptide. * p < . . to map the potential cd t cell epitope within the niv-g protein in more detail, we dissected the mer peptide # into every possible - mer sequence ( figure a ). the sequences were then analyzed for h -b binding computationally using the syfpeithi database, and the four best of each amino acid length were chosen (table ) . to test these peptides, we immunized ifnar−/− mice twice with mva-nivsg via the i.p. route and analyzed total splenocyte activation by elispot assay. of the sixteen - mer overlapping peptides tested, eight resulted in the stimulation of measurable ifn-γ in the mva-nivsg group ( figure b ). the positive peptides included three mer in length ( . , . , and . ), two mer in length ( . and . ), two mer in length ( . and . ), and one mer in length ( . ) ( figure b ). the mean ifn-γ sfc value was lower for cells stimulated with peptide . relative to peptide . . the mean values for the mva-nivsg group were ± ifn-γ sfc/ cells for peptide . and ± ifn-γ sfc/ for peptide . . comparative analysis of the positive peptide sequences demonstrated that the sequence of peptide . (rstdnqavi) was present in all positive - mer peptides (table ). in addition, the sequence of peptide . (stdnqavi) was present in the eight positive peptides. to characterize the induction of ifn-γ sfc by these peptides in more detail, ifnar−/− mice were vaccinated twice via the i.m. route, a commonly used route for human vaccinations, and in vitro stimulated splenocytes were analyzed by elispot assay. for this experiment, we chose the most promising peptides of each amino acid sequence length (peptides . , . , . , . , . , and . ). the six peptides tested significantly stimulated the activation of ifn-γ in the mva-nivsg group relative to the mva and pbs controls ( figure c ). mean sfc values, however, did not show statistically significant variations between each individual peptide. importantly, the mean sfc value for peptide . was again nonsignificantly elevated relative to peptide . (mean ± sem = ± and ± ifn-γ sfc/ cells respectively). consequently, we chose peptide . (rstdnqavi) (mean = ± ifn-γ sfc/ cells) as a potential h -b-restricted epitope candidate of niv-g ( table ). the alignment of peptide . to the full sequence of niv-g is shown in figure s . ( . , . , . , . , . , . ) . differences between groups were analyzed by one-way anova. asterisks represent statistically significant overall differences for a specific peptide. * p < . , ** p < . . peptide chosen as the most promising h -b-restricted peptide of niv-g; peptide chosen as the most promising h -iab-restricted peptide of niv-g. rows in bold indicate promising niv-g-specific t cell epitopes. to comparatively evaluate the activation of niv-g-specific immunity after i.m. vaccination with mva-nivsg and mva-niv-g using a standard dose of pfu, we stimulated splenocytes with peptide . ( table ) and analyzed cytokine production by ifn-γ elispot assay and ifn-γ plus tnf-α ics. comparisons between the two candidate vaccines showed that the mean sfc values were significantly higher for the mva-nivsg group relative to the mva-niv-g group ( figure a and figure s a ). the means of the mva-nivsg group were ± ifn-γ sfc/ cells, and the means of the mva-niv-g group were ± ifn-γ sfc/ cells ( figure c ). ifn-γ ics data showed that both the frequency and absolute number of ifn-γ+ cd t cells were significantly higher than the control background levels ( figure b ). our peptides did not induce ifn-γ production by cd t cells ( figure s b ), indicating that they indeed stimulated antigen-specific cd t cells only. when we compared our two candidate vaccines, we found that the percentage and absolute number of ifn-γ+ cd t cells was significantly higher in the mva-nivsg group. the mean frequencies of ifn-γ+ cd t cells were . % ± % for the mva-nivsg group and . % ± . % for the mva-niv-g group. mean absolute number of ifn-γ+ cd t cells were in the range of ± cells/ splenocytes and ± cells/ splenocytes for the mva-nivsg and mva-niv-g groups, respectively. taken together, our elispot and ifn-γ ics data indicate that mva-nivsg activates higher numbers of peptide-specific cd t cells than mva-niv-g. when we analyzed for coproduction of ifn-γ and tnf-α, we found that the majority of niv-g peptide-specific cd t cells from mva-nivsg and mva-nivg immunized mice were double positive for the cytokines (ifn-γ + tnf-α+) ( figure s a-c) . after showing that prime-boost immunization with our two vaccine candidates generated robust niv-g-specific cd t cell responses, we tested their immunogenicity after a single vaccination. ifnar−/− mice were vaccinated once with mva-nivsg, mva-niv-g, mva, or mock via the i.m. route and analyzed by elispot and ifn-γ + tnf-α ics as described before. our elispot results overall showed elevated sfc counts relative to the background levels of our mva and mock controls ( figure c ). statistically significant differences between the mva-nivsg and mva-niv-g were detected, with mean sfc values of ± ifn-γ sfc/ cells and ± ifn-γ sfc/ for them, respectively. facs analysis showed that the frequency and absolute number of ifn-γ+ cd t cells was higher in the experimental groups relative to the mva and mock controls ( figure d ). the mean percentage of ifn-γ + cd t cells was . % ± . % for the mva-niv-g groups, whereas for the mva-nivsg group, the mean percentage range for the peptides was . % ± . %. while mva-niv-g prime immunization induced a low frequency of ifn-γ+ cells, the mva-nivsg group showed ifn-γ+tnf-α+ secreting t cells after a single vaccination ( figure s d-f) , although percentages were lower than what was observed after prime-boost immunization ( figure s a-c) . this indicates that prime immunization with mva-nivsg was sufficient to generate a sizeable population of polyfunctional antigen-specific cd t cells. table ) and measured by elispot assay and ifn-γ ics plus facs analysis. (a,b) antigen-specific cd t cell response induced by prime-boost immunization. (a) ifn-γ sfc for stimulated splenocytes measured by elispot assay. (b) ifn-γ production by stimulated splenic cd t cell measured by ics and facs analysis. graphs show frequency and absolute number (per splenocytes) of antigen-specific ifn-γ+ cd t cells. (c,d) antigen-specific cd t cell response induced by prime immunization. (c) ifn-γ sfc for stimulated splenocytes measured by elispot assay. (d) frequency and absolute number (per splenocytes) of antigen-specific cd t cells measured by ifn-γ ics plus facs analysis. differences between individual groups were analyzed by one-way anova and tukey post-hoc test. asterisks represent statistically significant differences between two groups. * p < . , ** p < . , *** p < . . initially, we also analyzed purified cd t cell cultures from mva-nivsg immunized mice for their ability to stimulate ifn-γ by elispot using a two-dimensional pooled-peptide matrix system ( figure a , section . . ). in contrast to cd t cell-enriched splenocytes, the ifn-γ sfc signals in these cultures were lower (figures b and a) . in order to determine definitively which peptide pools were above background levels, we selected a cut off value of ifn-γ sfc/ cells ( figure a , grey line). mean ifn-γ sfc values above background were observed in out of the pools (pools h , h , v , v , v , v , v , and v ) ( figure a ). in order to identify potential cd t cell epitopes of h -iab, we performed a computational analysis of the mer peptides in the five most positive peptide pools measured by elispot assay (pools h , h , v , v , and v ). using mhc ii binding predictions obtained from the iedb online resource, we found two promising peptides. these peptides were # (lfmtnvwtppnpntv) and # (nvwtppnpntvyhcs) ( table ). next, we used ifn-γ ics to identify directly antigen-specific cd t cells after stimulation with these peptides. for this, splenocytes from mice that had been vaccinated with mva-niv-g or mva-nivsg were stimulated with peptides # and # and analyzed by flow cytometry. a small population of peptide-specific ifn-γ+ cd t cells was observed in the mva-nivsg and mva-niv-g groups ( figure b ). due to low frequencies of ifn-γ-producing cells, we chose a cut off value of . % to differentiate between positive signals and background. overall, cd t cells from the mva-nivsg group had a frequency of ifn-γ+ above the cut off relative to mva-niv-g group ( figure c ). the mean percentage of ifn-γ+ cd t cells for the two peptides was . - . % and . - . % for mva-nivsg and mva-niv-g, respectively. moreover, the frequency and absolute number of ifn-γ-producing cd t cells in peptide # stimulated cultures was significantly higher in the mva-nivsg group when compared with the mva-niv-g group (mean = ± and ± cells/ splenocytes respectively). for peptide # , mva-nivsg vaccinated mice showed a significantly elevated frequency of ifn-γ+ cd t cells only. in conclusion, our data indicate that peptide # (nvwtppnpntvyhcs) is a promising h -iab-restricted cd t cell epitope candidate of niv-g. the alignment of the peptide to the full amino acid sequence of wild-type niv-g is shown in figure s . the continuous threat of suddenly emerging niv outbreaks, particularly in bangladesh and india, demonstrate the need for countermeasure approaches ready to use in an immediate public health response. at present, there are no licensed niv vaccines for use in humans available. the existence of a niv candidate vaccine should significantly reduce the risk of infection and transmission of the virus in the case of an outbreak scenario. there are some experimental niv vaccines that have already been tested in different preclinical animal models. the major focus of these approaches was to evaluate the immunogenicity and efficacy in the context of niv challenge infection. in those studies, efficacy has been mostly associated with the generation of niv-specific antibodies, and immune monitoring is mainly relying on the detection of virus-neutralizing antibodies [ , , ] . however, there is relatively little known about the induction and the relevance of niv-specific cellular immune responses. in that context, the availability of appropriate tools to investigate the role of t cells in niv-specific immunity is an important prerequisite in the development of new vaccines and therapeutics. thus, it will be indispensable to monitor in animal models the contribution of virus-specific t cells to protective immunity but also to potential niv antigen-specific immune pathology. here, we identified a major histocompatibility complex (mhc) haplotype h -b-restricted peptide epitope in the niv-g protein by stimulating t cells from mva−nivsg vaccinated ifnar−/− mice with a two-dimensional ( d) matrix pool of overlapping peptides. ifnar−/− mice have been already established as a valuable preclinical animal model for niv infection with a ld of × pfu after intraperitoneal challenge infection [ ] . moreover, in previous studies, we have already successfully demonstrated that interferon type i receptor knockout mice (ifnar−/−) [ ] can be readily used as animal models to study the immunogenicity and protective capacity of mva immunization [ , ] . here, we wished to specifically assess the ability of mva-delivered niv-g antigen to induce the activation of cellular immune responses in mice. in general, the envelope g protein is known as the well-conserved attachment glycoprotein for both hev and niv. in a previous study, a recombinant adeno-associated virus vaccine expressing a full-length niv-g protein protected hamsters in an niv infection model [ ] . in another approach, a soluble version of hev-g has been engineered and showed an even more advantageous efficacy when tested in different preclinical animal models [ , , ] . using hevsg, a monoclonal antibody m . was derived and has already been successfully tested as a therapeutic approach in humans. to further enhance henipavirus g protein-induced immunogenicity, we also designed and tested a soluble version of the niv-g protein (nivsg) similar to the hevsg antigen used for the generation of m . . to comparatively evaluate the immunogenicity of the nivsg protein, we also generated an mva expressing full-length g. the recombinant viruses mva-niv-g and mva-nivsg produced stable amounts of niv-g antigen upon in vitro infection of human cells, indicating the unimpaired expression of the target gene under transcriptional control of the synthetic vaccinia virus-specific early-late promoter pmh or the strong natural early promoter pvgf. in the case of mva-nivsg, removal of the transmembrane domain and cytoplasmic tail resulted in the secretion of the niv-g from mva-infected cells and accumulation also in the supernatant of cell cultures, as demonstrated in western blot analysis and immunostaining. a similar result was obtained with hevsg, as expressed by conventional recombinant vacv [ ] . in contrast, the full-length mva-produced niv-g protein was not released in the supernatant, indicating the stable presentation on the cell surface through the transmembrane domains. another important aspect of proper protein expression, folding and conformational stability is influenced by the n-glycans. recent studies indicated that niv-g n-glycans reduce fusion efficiency because removal of some n-glycans caused cell-cell hyperfusogenicity and increased viral entry [ ] . glycosidase treatment of full-length mva-produced niv-g resulted in a polypeptide of kda, corresponding to the molecular mass predicted from the niv g gene encoding sequences. the glycosidase treatment of the nivsg also indicated the presence of all the n-glycans sites within the soluble version of the glycoprotein. a first in vivo evaluation in mice revealed that treatment with the mva-niv-g and mva-nivsg candidate vaccines resulted in the induction of similar levels of g-binding serum antibodies, confirming the immunogenicity of both antigen versions [ ] . in that context, the presence of binding antibodies seems to play a substantial role in the blocking of niv entry, since the mechanism of niv neutralization is complex and involves more antigenic sites than those required for simple receptor binding [ ] . however, more recent studies in different animal models suggest that cellular immune responses are also involved in mediating protection against niv infection [ , ] . this observation is further supported by studies in a pig model for niv infection showing substantial activation of cd t cells after oral infection with niv [ ] . in line with these preclinical data, humans surviving niv infection [ ] showed significant levels of proliferating (ki- +) cd t cells, indicating the presence of acute effector cells. these data emphasize that in addition to the humoral immune responses, t cells could be associated with recovery from niv infection. in a more recent study, stroh and coworkers confirmed the activation of niv-specific cd t cells in mice after vaccination with niv-like particles. these data further highlight that t cells may play a critical role in niv infection [ ] . another hypothesis is that niv-specific t cells could be involved in potential immunopathologies. in this context, data from infections with other neurotropic viruses, for example, west nile virus, demonstrated that antigen-specific t cells can open the blood brain barrier and contribute to virus infections of the brain [ ] [ ] [ ] [ ] [ ] . thus, to allow for more detailed studies characterizing t cells in niv-associated immunity or pathogenesis, it is essential to identify niv-g peptide epitopes allowing for the specific mhc-restricted antigen presentation and the activation of niv-specific t cells. we identified a nonamer epitope niv-g- . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (rstdnqavi). analysis of this peptide sequence showed that niv-g . - could be functionally conserved in hendra virus, but not cedar virus (another recognized henipavirus), g antigens ( figure s ). structural and functional analyses reveal promiscuous and species-specific use of ephrin receptors by cedar virus [ ] . this potential epitope will support a more detailed experimental characterization of t cells induced by niv infection in the mouse model and their contribution for pathogenesis and protection. in this study, we found that mva-nivsg vaccination induced significantly higher amounts of niv-g epitope-specific cd t cells compared with the mva-niv-g candidate vaccine. this was a somewhat surprising observation as the immunizations with both antigens had elicited very comparable levels of g-specific antibodies. it is tempting to speculate that nivsg, as a soluble antigen, can trigger enhanced t cell responses because it is available to two different pathways of antigen presentation. on the one hand, nivsg as intracellularly synthetized antigen is endogenously processed and presented via mhc-i on the cell surface to activate cd t cells. in addition, the nivsg is secreted in high amounts from the mva-nivsg-infected cells, and such extracellular antigen can efficiently fuel the cross-presentation pathway and thereby induce elevated cd and cd t cell immune responses [ , ] . interestingly, the mva-nivsg candidate vaccine also proved to rapidly induce niv-g epitope-specific cd t cells after single-dose application. these data are of relevance, since the epidemiology of the more recent niv outbreaks demonstrated that a potential vaccine candidate should rapidly protect. importantly, an h b-restricted epitope has been identified in ifnar−/− mice, which serve as an established small animal model for niv infection [ ] . in addition, we showed the induction of niv-g-specific cd t cells upon prime-boost immunization in the ifnar−/− with the mva-niv vaccines and using peptides for in vitro stimulation, as identified by using mhc ii binding predictions obtained from the iedb online resource (www.iedb.org). this data goes well along with the hypothesis that cd t cell responses are also significantly elevated upon infection [ ] . again, mva-nivsg vaccination results in more efficient activation of cd t cell responses. further experiments will be needed to characterize the contribution of niv g-specific cd t cells for niv infection in more detail. taken together, our findings showed the activation of niv-g-specific t cells in ifnar−/− mice following vaccination with mva-based candidate vaccines. we confirmed the identification of potential h -b-restricted niv-g cd and cd t cell peptide epitopes. in this study we also demonstrated that an mva-nivsg candidate vaccine may have superior immunogenicity, resulting in niv-specific antibodies and t cells in ifnar−/− mice. these data emphasize the promise of future studies in this animal model further evaluating the role of niv-specific t cells activated by the g- . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and g- - peptide epitopes, in both vaccine-induced protection and potential contribution to niv-induced pathologies. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : generation and characterization of recombinant mva-nivsg and mva-niv-g, figure s : ifn-γ production by cd and cd t cells stimulated by the niv-g peptide . , figure s : amino acid alignment of h -b-and h -iab-restricted peptides, figure s : quality of activated cd t cells from mice immunized with recombinant mva expressing niv-g. author contributions: g.k., s.v., c.c.b., g.s. and a.v. conceived and designed the experiments; g.k., s.v., s.j. and a.v. performed the experiments; g.k., s.v., u.k., c.c.b., g.s. and a.v. analyzed the data; g.k., g.s. and a.v. wrote the paper. all authors have read and agreed to the published version of the manuscript. nipah virus: a recently emergent deadly paramyxovirus adaptive immune responses in 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for hendra virus and nipah virus ephrinb is the entry receptor for nipah virus, an emergent deadly paramyxovirus two key residues in ephrinb are critical for its use as an alternative receptor for nipah virus quantitative analysis of nipah virus proteins released as virus-like particles reveals central role for the matrix protein functional studies of host-specific ephrin-b ligands as henipavirus receptors potent neutralization of hendra and nipah viruses by human monoclonal antibodies therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody pathogenic differences between nipah virus bangladesh and malaysia strains in primates: implications for antibody therapy. sci a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection nipah virus: vaccination and passive protection studies in a hamster model recombinant nipah virus vaccines protect pigs against challenge single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal nipah virus disease single-dose live-attenuated nipah virus vaccines confer complete protection by eliciting antibodies directed against surface glycoproteins single-dose replication-defective vsv-based nipah virus vaccines provide protection from lethal challenge in syrian hamsters single-dose live-attenuated vesicular stomatitis virus-based vaccine protects african green monkeys from nipah virus disease peri-exposure protection against nipah virus disease using a single-dose recombinant vesicular stomatitis virus-based vaccine rabies-based vaccine induces potent immune responses against nipah virus recombinant measles virus vaccine expressing the nipah virus glycoprotein protects against lethal nipah virus challenge a single-dose chadox -vectored vaccine provides complete protection against nipah bangladesh and malaysia in syrian golden hamsters protection against henipavirus infection by use of recombinant adeno-associated virus-vector vaccines hendra virus and nipah virus animal vaccines receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble g glycoprotein of hendra virus site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of hendra virus a recombinant subunit vaccine formulation protects against lethal nipah virus challenge in cats vaccination of ferrets with a recombinant g glycoprotein subunit vaccine provides protection against nipah virus disease for over months a hendra virus g glycoprotein subunit vaccine protects african green monkeys from nipah virus challenge protection against henipaviruses in swine requires both, cell-mediated and humoral immune response chapter five-modified vaccinia virus ankara: history, value in basic research, and current perspectives for vaccine development functional role of type i and type ii interferons in antiviral defense biochemical, conformational, and immunogenic analysis of soluble trimeric forms of henipavirus fusion glycoproteins development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus infection in an animal model kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep rna sequencing myristoylation increases the cd +t-cell response to a gfp prototype antigen delivered by modified vaccinia virus ankara elucidating and minimizing the loss by recombinant vaccinia virus of human immunodeficiency virus gene expression resulting from spontaneous mutations and positive selection easy and efficient protocols for working with recombinant vaccinia virus mva cells responding to the middle east respiratory syndrome coronavirus nucleocapsid protein delivered by vaccinia virus mva in mice pooled-peptide epitope mapping strategies are efficient and highly sensitive: an evaluation of methods for identifying human t cell epitope specificities in large-scale hiv vaccine efficacy trials iedb-ar: immune epitope database-analysis resource in identification of poxvirus cd + t cell determinants to enable rational design and characterization of smallpox vaccines a vlp-based vaccine provides complete protection against nipah virus challenge following multiple-dose or single-dose vaccination schedules in a hamster model hendra virus vaccine, a one health approach to protecting horse, human, and environmental health rapid expansion of cd + t cells in wild-type and type i interferon receptor-deficient mice correlates with protection after low-dose emergency immunization with modified vaccinia virus ankara critical role of perforin-dependent cd + t cell immunity for rapid protective vaccination in a murine model for human smallpox a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge n-glycans on the nipah virus attachment glycoprotein modulate fusion and viral entry as they protect against antibody neutralization antibodies to henipavirus or henipa-like viruses in domestic pigs in ghana neutralization assays for differential henipavirus serology using bio-plex protein array systems henipavirus-like particles induce a cd t cell response in c bl/ mice pathways exploited by flaviviruses to counteract the blood-brain barrier and invade the central nervous system toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis west nile virus-induced disruption of the blood-brain barrier in mice is characterized by the degradation of the junctional complex proteins and increase in multiple matrix metalloproteinases nile virus: crossing the blood-brain barrier cd + t cells mediate recovery and immunopathology in west nile virus encephalitis structural and functional analyses reveal promiscuous and species specific use of ephrin receptors by cedar virus cross-priming of cytotoxic t cells dictates antigen requisites for modified vaccinia virus ankara vector vaccines distinct pathways of antigen uptake and intracellular routing in cd and cd t cell activation pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -vsvc g authors: georgiades, jerzy a.; fleischmann, w. robert title: oral application of cytokines date: journal: biotherapy doi: . /bf sha: doc_id: cord_uid: vsvc g a number of different laboratories reported on studies with orally administered interferons and cytokines. their observations extend previous observations which showed that orally administered interferons and cytokines can exert both local and systemic effects. as difficult as it may be to understand how orally administered interferons and cytokines may exert both effects, the increasing number of laboratories that demonstrate biological effects with orally administered cytokines suggests that serious consideration be given to the possibility that orally administered interferons and cytokines can indeed exert effects. they also raise the possibility that these effects may have biological relevance for the treatment of human disease. moreover, they may indicate that the nasal/oral region is a window on the environment. it is most important, however, to assure that these experiments are performed with special care to avoid presenting preliminary data that is not properly controlled. it is essential to carry out these studies with sufficient animals or patients to ascertain their significance; and to plan the studies as double-blind evaluations to avoid misinterpretations when subjective tests are used. nevertheless, the overall data presented give one the impression of an area that should be pursued. oral or nasal administration of interferons has been shown in a number of studies to have a protective effect against viral infections. the majority of the studies suggested that orally or nasally administered interferons exerted their antiviral activity locally [cummins and hutcheson, ; cummins and rosenquist, ; greenberg et al., ; hayden et al., ; higgins etal., ; merigan etal., ; schaferetal., ; smith et al., ; turner et al., ] . more recently, a number of studies have suggested that the orally administered interferons might be exerting their antiviral activity through a systemic effect [cummins et al., ; hutchinson and cummins, ; koech et al., ] . further studies have extended the original observations with the antiviral activity of orally administered interferons to include demonstrations of immunoregulatory and antiparasitic activities of orally administered interferons [cummins and hutcheson, ; fleischmann et al., ; young et al., ] . additional studies have further extended the observations with oral administration of interferons to include immunoregulatory and antibacterial activities mediated by oral administration of other cytokines [baqar et al., ; koren and fleischmann, ] . the concept that oral administration of interferons could have a systemic effect has been a difficult one to evaluate. it is difficult to understand how orally administered interferons, particularly ph sensitive ifn- could survive passage through the acidic and/or peptidase rich environment of the stomach and intestinal tract to trigger a systemic effect. one concern involves the lack of parallel controls in many of the studies. a further concern involves questions about the biological relevance of the often relatively modest effects obtained with orally administered interferons. other concerns relate to the lack of universality in the observation of biological effects of orally administered interferons [sperber et al., ; witt et al., ] . the efficacy of the interferon therapy may depend upon the form of disease (acute or persistent), the daily dosage of interferon , and the type of interferon preparation (whether it is made from natural or recombinant sources) . furthermore, the type of disease and the duration of therapy may influence the final response to treatment . these concerns are real and appropriate. it will be attendant upon the researchers investigating the oral administration of cytokines to address all of them effectively. the oral administration of cytokines was the topic of a number of presentations at a recent workshop presented in conjunction with the annual meeting of the international society for interferon and cytokine research in budapest, hungary. the mechanism by which orally administered ifn-a might exert an antiviral effect was probed in vitro in studies with cultured primary human buccal epithelial cells [smith etal., ] . david chi and his colleagues used florescence studies to show that ifn-a treatment significantly increased the number of hla-dr positive cells. a trend toward greater expression of hla-dr by hla-dr positive cells was also observed. the indication that ifn-a causes an upregulation of hla-dr expression by human buccal epithelial cells and peripheral white blood cells suggests that orally administered ifn-a may exert an antiviral activity against local viral infections. this anfiviral activity may be medicated at least in part by a greater degree of recognition of viral infected cells in the context of hla-dr by the host immune system. the mechanism by which orally administered ifn-a might exert antimicrobial action was examined in studies reported by kunihiro ohashi and his colleagues . it was found that the saliva of healthy volunteers contained il-la and il- . established epithelial cell lines were also found to produce il-la and il- spontaneously. the possible immunological roles of ifn-a in combination with these lymphokines were evaluated in vitro. il- a, used at a level equivalent to that in the saliva, and ifn-c~ were examined for cooperative effects on activation of neutrophil mediated anticandidal action. they were found to have an additive effect. the production of il- was found to be enhanced by treatment of established epithelial cell lines by ifn-c~ treatment. the ifn-a was also found to partially restore il- production in'a monocyte cell line that had been latently infected with human immunodeficiency virus- (hiv- ) . finally, the ifn-a treatment of detroit (a pharyngeal cancer cell line) and kb cells (oral cancer cell line) caused enhanced binding to a human t cell line, suggesting that ifn-a caused an enhanced recognition of the tumors by t cells. in this regard, the investigators showed an enhanced expression of icam- , cd , and cd wb on some oral-mucosal epithelial cells treated with ifn-a in vitro. taken together, the work provides some in vitro evidence to support the concepts that in vivo oral administration of ifn-a may (a) locally activate neutrophils in the buccal cavity to exhibit a greater antimicrobial action, (b) affect local lymphokine production by epithelial cells in contact with the ifn-a, and (c) increase cell surface antigens in mucosal epithelial cells making them more sensitive to tumor surveillance mechanisms. mary tompkins reported on the effects of intranasally administered ifn-a on lymphoid cell phenotype and function in lymph nodes and the spleen [tonkonogy et al., ] . she and her colleagues showed that lymphocytes isolated from periglandular lymph nodes responded to in vitro stimulation with immobilized anti-cd activated t cells to produce -fold more ifn- than control mice. lymphocytes from superficial cervical lymph nodes of oral ifn-a-treated mice produced -fold more ifn- than those from control mice. no differences in ifn- production were detected for lymphocytes from more distal lymphoid organs such as axillary lymph nodes, mesenteric lymph nodes, peyer's patches, or spleens from ifn-a and control mice. effects of orally administered ifn-a on the expression of cell surface markers of lymphocytes in the lymphoid tissue were also measured. no differences in proportion of expressing cells or degree of expression of cell surface cd , cd , mhc class ii or immunoglobulin were seen in the lymphocytes from any of the lymphoid tissues from ifn-a treated and control mice. the results indicate that intranasal administration of ifn-a establishes a greater responsiveness of lymphocytes in periglandular and superficial lymph nodes to ifn- induction by exposure to anti-cd . this greater responsiveness occurs in the absence of a change in phenotype of the lymphocytes. further, the results suggested that the effect of intranasal administration of ifn-a and ifn- production may be primarily a local effect, since it diminishes with increasing distance from the site of ifn-a administration. a paper presented by robert fleischmann summarized his published work on the myelosuppressive effects of orally administered interferons in mice [fleischmann et al., [fleischmann et al., , koren and fleischmann, ] . the presentation reviewed work showing that oral administration of each of three interferons (ifn-a, ifn-/ and ifn- ) exerted a dose dependent suppressive effect on peripheral white blood cell counts [fleischmann et al., ] . moreover, the peripheral white blood cell suppression was shown to be reflective of a suppression of bone marrow function by the orally administered interferons [koren and fleischmann, ] . of importance in addressing the concerns about the biological relevance of orally administered interferons, the magnitudes of the peripheral white blood cell and bone marrow suppressions induced by oral interferons were equivalent to those induced by subcutaneous or intraperitoneal administration of the interferons. studies on the kinetics of establishment of peripheral white blood cell and bone marrow suppression indicated that the suppressive effects developed more slowly for orally administered interferons ( days for peripheral white blood cell suppression and days for bone marrow suppression) than for injected interferons ( day and days, respectively). finally, the mechanisms of establishment of peripheral white blood cell and bone marrow suppression by orally administered interferons and injected interferons were examined [fleischmann et al., ] . in contrast to observations with injected interferons, the systemic activities of orally administered interferons were not blocked by the presence of circulating antibody. further, the peripheral white blood cell suppressive effects of orally administered interferons were shown to be able to be adoptively transferred by inoculation of white blood cells to otherwise untreated mice. these studies show in a mouse model that orally administered interferons are as potent as injected interferons in suppressing both the peripheral white blood cell counts and the bone marrow. moreover, they indicate that the mechanism by which orally administered interferons exert these systemic effect, is different from that of injected interferons. in a study reported earlier [georgiades et al., ] , georgiades and colleagues found that the antiviral state could be transferred from the oral cavity of mice treated with molfn-a or rat rifn- to peripheral blood lymphocytes and spleen cells. they demonstrated that white blood cells of mice treated with - iu and . - iu ifn-~, could transfer antiviral resistance in vitro to fetal fibroblasts. such a transfer apparently happened by direct cell to cell contact when the spleen cells from mice orally treated with interferon were cocultured with fetal fibroblasts in the absence of interferon as previously described [blalock and baron, ] . the phenomenon appeared within hrs after initiation of oral interferon treatment and, with continued oral interferon treatment, persisted through at least days. neither thymus cells nor plasma of orally interferon- treated mice had the ability to transfer the antiviral effect. taken together, these and other controlled experiments suggest that interferon may be absorbed quickly in the oral cavity by cells of mucosa, somehow activating leukocytes present in mucosal tissue resulting in the activation of leukocytes in the lymphatic system and in the peripheral circulation. a paper presented by srecko koren reported parallel observations for il- [koren and fleischmann, ] . oral administration of il- was shown to exert systemic effects by suppressing both the peripheral white blood cell counts and the bone marrow in a dose dependent manner. moreover, the potency of orally administered il- was equivalent to that of injected il- . taken together with the results of fleischmann, these observations suggest that the induction of systemic effects by orally administered cytokines may be a general phenomenon. taking together all of the above information, it appears that not only ifn-a but also several other interferons and cytokines introduced to the mucosal membrane of the oral cavity can transfer signals through the mucous membrane and induce systemic responses that can be measured by a variety of techniques. this evidence about systemic effects was confirmed by clinical studies carried out on patients with chronic active hepatitis b, and c and on patients with aids as discussed below. a paper presented by shahida baqar reported on the effects of orally administered il- , il- and il- on gut mucosal immunity to campylobacter jejuni [baqar et al., ; b aqar et al., ] . she and her colleagues showed that oral administration of il- resulted in a log reduction (relative to control mice) in the amount of c. jejuni excreted in the feces within hours after administration of the c. jejuni. oral administration of il- showed a similar reduction though the time course for the reduction was delayed relative to il- . no reduction in c. jejuni was observed with oral administration of il- . these results indicate that oral administration of il- and il- can reduce the initial colonization by c. jejuni. the duration of the effect of the three lymphokines was probed by rechallenging mice that had been treated with oral administration of il- , il- or il- to establish a recolonization with c. jejuni. the results indicate that, for all three lymphokines, the initial level of recolonization was logs lower than in control animals; however, the recolonization ultimately progresses to the level of the control for il- and il- treated animals. only il- treated animals showed a durable suppression of c. jejuni colonization. specific iga antibody response to c. jejuni was also measured for mice treated orally with the three lymphokines. il- treated mice, but not il- or il- treated mice, showed enhanced levels of c. jejuni-specific iga antibodies in the intestine and in the blood. in further experiments on the effects of orally administered lymphokines on iga production, il- was evaluated for its potential as an oral mucosal adjuvant for vaccination with formalin-killed c. jejuni. a three-fold enhancement in siga was observed relative to control mice. this enhancement in slga activity was biologically relevant since it led to an enhanced rate of clearance of c. jejuni from the intestine and a reduced number of c. jejuni that were present in the feces. oral administration of ifn-a has previously been reported to be efficacious in the treatment of feline leukemia [cummins et al., ] . thomas toth reported on a double-blind study evaluating the effects of orally administered il- or ifn-a on various hematological variables in feline leukemia virus-positive cats [toth et al., ] . the hematological parameters measured included total red blood cell counts, total white blood cell counts, differential white blood cell counts, total hemoglobin, mean corpuscular hemoglobin, mean corpuscular-hemoglobin concentration, mean corpuscular volume and hematocrit. oral treatment with il- was given times (days , and ) at dosage levels ( and units/treatment). the hematological variables were monitored on days and . orally administered il- affected only one hematological variable as it reduced the mean corpuscular hemoglobin concentration to a significant level by day in cats treated with units il- . by day the mean corpuscular hemoglobin concentration returned to the control level. oral treatment with ifn-a was given times (days , , , , , , and ) at dosage levels ( . , . and . iu/treatment). the hematological variables were monitored on days , , , and . oral administration of ifn-a caused significant and dose dependent elevations in total red blood cell count, total hemoglobin, and hematocrit on days , and . interestingly, with . iu/treatment, monocyte counts were also significantly elevated on these days. the other hematological variables were at the control level. these results indicate that oral administration of il- and ifn-a can affect several hematological variables, suggesting that these biological response modifiers cause systemic effects. the biological importance of these systemic effects and their relevance for the control of the feline leukemia virus infection are as yet unknown. finally, miklos degre [ ] reported on his studies on the effect of oral administration of two biological response modifiers on the course of an enteric bacterial infection in mice [degre, ] . he found that orally administered tumor necrosis factor did not have an effect on the course of infection. however, orally administered ifn-a reduced the mortality of infected mice relative to controls. the degree of mortality with orally administered ifn-a was the same as that observed for intraperitoneally administered ifn-c~. the results suggest that oral administration of ifn-a may exert a protective effect against enteric bacterial pathogens. race horses suffer from inflammatory airway disease that impairs their ability to perform on the race track. bonnie moore presented the results of a double-blind, placebo controlled study on the efficacy of oral ifna in the treatment of inflammatory airway disease . horses were randomly assigned ( horses/group) into a placebo group and three ifn-a dosage groups ( , and iu/day for days). fifteen days after initiation of therapy, horses were evaluated for the degree of inflammatory airway disease by semi-quantitative endoscopic examination score and by cytologic evaluation of bronchoalveolar lavage fluid. oral administration of and iu of ifn-a resulted in a reduction in the endoscopic examination score as well as a reduction in the number of white blood cells in the bronchoalveolar lavage fluid. administration of iu of ifn-a was not as efficacious. the relative percentages of the various lymphocyte subpopulations was unaffected by the ifn-a treatments. the results suggest that orally administered ifn-a may have a beneficial effect on inflammatory airway disease in horses. previously published work suggested that oral administration of ifn-a may be useful as a treatment of patients with aids [hutchinson and cummins, ; koech et al., ] . two presentations discussed work evaluating the efficacy of orally administered ifna against hiv, while one presentation evaluated the efficacy of orally administered ifn-a against feline leukemia virus. musabbir mian reported on a study in poland of patients with aids or arc (aids related complex) who had been given low dose oral treatment with ifn-a [babiuch and mian, ] . this report represented the results of studies initiated four years earlier. part of these studies were already published [babiuch et al., ; georgiades and babiuch, ] and found confirmation by others [jordan, ] . it was reported that most patients with initial cd + cell counts above became asymptomatic, gained weight, had improved karnofsky performance scores and had either increased or stable cd + cell counts and total lymphocyte counts. patients with initial cd + cell counts below did not respond to oral ifn-o~. martin cummins reported on a double-blind, placebo controlled study in zambia involving patients divided randomly and equally into three treatment groups: group , placebo; group , iu/day ifn-a administered on an alternate weekly basis with placebo for weeks; and, group , iu/day ifn-a administered continuously for weeks [mukunyandela et al., ] . the study showed that oral ifna treatment had a significant effect in reducing hivrelated signs and infections. both groups and had a significantly greater resolution of pre-existing hivrelated skin infections than the placebo treated group . group had a greatly reduced incidence of new, mucocutaneous hsv infections ( %) compared to the placebo treated group ( %), with group having an intermediate value ( %). interestingly, patients in the alternate ifn-a/placebo group (but not the continuous ifn-a group ) showed a reduction in global symptom score and small, but significant, increases in absolute and mean cd + counts compared with placebo treated group . taken together, these studies provide significant support for the concept that orally administered ifn-a may have beneficial effects for aids patients, provided they are treated in the early stage of disease. as a caution, however, several negative studies have been reported showing a lack of efficacy of orally administered ifn-a in the treatment of hiv infected patients [hulton et al., ; sperber et al., ] . these negative observations may relate to the limited period of oral ifn-a treatment employed in these studies or to the use of recombinant as opposed to natural ifn-a. while it is not now possible to be certain of the anti hiv effects of oral ifn-a in humans, several studies provide additional support. the observation that aids patients with baseline cd + cell counts below cells/mm responded poorly to oral ifn-a therapy was in agreement with reports of kaiser et al. [ ] in germany, hutton et al. [ ] in canada, and sperber et al. [ ] in the usa. all of these studies reported similar findings: patients having < cd + cells/mm did not respond very well to ifn-a therapy. however, patients with baseline cd + cell counts > cells/mm survived longer than control patients and required much less medical attention than patients in the control group [babiuch and mian, ; georgiades and babiuch, ] . these observations were confirmed by a report from california where physicians had similar experiences with hiv-l-infected patients [jordan, ] . interestingly, the beneficial effects of ifn-a therapy may not be constant through the course of ifna therapy. babiuch reported that, from time to time, patients may apparently lose their sensitivity to oral ifn-a therapy [babiuch and mian, ; georgiades and babiuch, ] . this apparent loss of sensitivity to ifn-a therapy (or deterioration of clinical status) can be reversed by slightly increasing the dose of ifn-c~ [babiuch and mian, ] . these observations may represent clinical manifestations of a phenomenon described in vitro by yamamoto and colleagues [yamamoto et al., ] . these investigators examined the effects of ifn-a using long-term culture of hiv- -infected white blood cells. they observed a transient loss of sensitivity of the white blood cells to ifn-a. the transient loss was observed twice during a day culture period. further studies will be needed to determine if hiv- resistance observed in vivo and in vitro occur by the same mechanism. small scale trials with hepatitis b patients with chronic active hepatitis have previously suggested that oral administration of ifn-a might be of therapeutic benefit . similar beneficial results were seen in a small scale trial with six hepatitis c patients with chronic active hepatitis who were treated with oral ifn-a . zielinska reported on the long-term follow-up of hepatitis b patients with chronic active hepatitis who were treated with oral ifn-a [zielinska et al., ] . the diagnosis of chronic active hepatitis was based on clinical and biochemical signs of active disease. oral ifn-a was given daily at a dose of - iu/day, depending on the body weight of the patients, for an average of months. seroconversion from hbeag to antihbe was observed in of patients. the median time to seroconversion was . weeks. in addition, two patients seroconverted from hbsag to antihbs. in all patients who seroconverted to antihbe, hbv dna was not found to be present in serum. also, biochemical markers associated with liver function returned to the normal range. further, of the patients who did not seroconvert to antihbe were negative for the presence of hbv dna in the serum. the remaining patients had serum hbv dna levels that were significantly lower than at the initiation of oral ifn-a treatment. taken together, these observations suggest that oral ifn-a may be an effective therapy for control of chronic active hepatitis. the annual meeting of the international society for interferon and cytokine research (isicr- ) held in budapest provided, for the first time, compelling evidence that oral doses of cytokines modulate immune functions and increase resistance to infectious disease. studies were presented by investigators from a number of different laboratories. clinical data, especially in humans, indicates that ifn, given though the oral mucosa, is capable of inducing systemic reactions and may have potential as a new therapeutic method. a number of different laboratories reported on studies with orally administered interferons and cytokines. their observations extend previous observations which showed that orally administered interferons and cytokines can exert both local and systemic effects. as difficult as it may be to understand how orally administered interferons and cytokines may exert both effects, the increasing number of laboratories that demonstrate biological effects with orally administered cytokines suggests that serious consideration be given to the possibility that orally administered interferons and cytokines can indeed exert effects. they also raise the possibility that these effects may have biological relevance for the treatment of human disease. moreover, they may indicate that the nasal/oral region is a window on the environment. it is most important, however, to assure that these experiments are performed with special care to avoid presenting preliminary data that is not properly controlled. it is essential to carry out these studies with sufficient animals or patients to ascertain their significance; and to plan the studies as double-blind evaluations to avoid misinterpretations when subjective tests are used. nevertheless, the overall data presented give one the impression of an area that should be pursued. an interim report on the effect of natural human interferon alpha (ifna) lozenges in patients seropositive for the human immunodeficiency virus type (hiv- ) oral mucosal administration of natural human alpha-interferon in hiv- seropositive patients modulation of mucosal immunity against campylobacter jejuni by orally administered cytokines modulation of mucosal immunity against campylobacter jejuni by orally administered cytokines. workshop presentation interferon-induced transfer of viral resistance between animal cells the effect of low dose oral human interferon alpha therapy on diarrhea in veal calves oral therapy with human interferon alpha in calves experimentally injected with infectious bovine rhinotrachetis virus effect of interferon on feedlot cattle low dosage of interferon to enhance vaccine efficiency in feedlot calves protection of calves against rhinovirus infection by nasal secretion interferon induced by infectious bovine rhinotracheitis virus oral use of human alpha interferon in cats interferons and other eytokines in infectious diseases: molecular mechanisms and clinical value. review speech modulation of peripheral leukocyte counts in mice by oral administration of interferons orally administered interferons exert their wbc suppressive effects via a novel mechanism results of the prolonged use of natural human interferon alpha (nhuifna) lozenges in the treatment of human immune deficiency virus infection natural human interferon alpha (nhulfna) given orally has different effects on patients with distinct forms of chronic viral hepatitis b (chbv) transfer of antiviral resistance by spleen and blood cells of mice receiving low oral doses of ifn alpha or gamma antiviral activity of intranasally applied human leukocyte interferon prevention of natural colds by contact prophylaxis with intranasal alpha -interferon intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers randomized, placebocontrolled double-blind study of low-dose oral interferon-a in hiv- antibody positive patients low-dose oral interferon in patient with aids three open-label studies of oral interferon alpha in the treatment of hiv disease low-dose oral natural human interferon-a in patients with hiv- infections: a double-blind randomized, placebo-controlled trial low dose oral alpha-interferon therapy for patients seropositive for the human immunodeficiency virus type- (h/v- ) modulation of peripheral leukocyte counts and bone marrow function in mice by oral administration ofinterleukin- administered interferons suppress bone marrow function inhibition of respiratory virus infection by locally applied interferon oral administration of interferon-alpha for treatment of inflammatory airway disease in standard-bred racehorses treatment of symptomatic hiv- infected patients with low dose oral natural human interferon alpha effects of interferon-a on a reduced release of interleukin- from latenfly hiv- infected monocytic cell line u cells interferon alpha (ifna) in antimicrobial mechanisms in the oral cavity: possibility of oral-mucosal use of ifna to treat fungal infections in the oral cavity interferon administered orally: protection of neonatal mice from lethal virus challenge intranasally administered alpha/beta interferon prevents extension of mouse hepatitis virus strain jhm into the brains of balb/cbyj mice effect of ifn a on hla-dr expression in human buccal epithelial ceils. workshop presentation low-dose oral recombinant interferon-alpha a in patients with hiv- infection: a blinded pilot study effect of intranasal administration of interferon-alpha on lymphoid cell phenotype and function. workshop presentation effects of very-low-dose oral cytokine treatment on hematological values in feline leukemia positive cens prevention of experimental coronavirus colds with intranasal alpha- b interferon absence of biological effect of orally administered interferon-bser inhibition of human immunodeficiency virus type replication by human interferons alpha, beta, and gamma low-dose oral administration of human interferon alpha can control the development of theileria parva infection in cattle treatment of fourteen chronic active hbsag+, l-ibeag+ hepatitis patients with low dose natural human interferon alpha administered orally treatment of six patients with chronic active hcv hepatitis with low dose natural human interferon alpha administered orally long-term follow-up of chronic active hepatitis b patients treated with low dose natural human interferon alpha administered orally key: cord- -eq zhtd authors: nan title: abstracts of oral presentations and posters date: journal: ann hematol doi: . /bf sha: doc_id: cord_uid: eq zhtd nan since the blood cells are primarily concerned with host defense, the introduction of the csfs as therapeutic agents offers the opportunity to develop unique therapeutic strategies designed to enhance overall host defense, particularly with relevance to cancer and aids. administration of csfs is associated with profound changes in cellular function, and treatment strategies will need to consider the potential deleterious effects of heightened host cell activity and potential effects on nonhematopoietic cells. memorial sloan-kettering cancer center, new york, ny , usa a cytokine the majority of hemopoietic stem cells in the steady state marrow are dormant in the cell cycle. using serial observation (mapping) of blast cell colony formation from bone marrow cells of mice that have been treated with a high dose -fluorouracil ( -fu), we have identified a number of cytokines that appear to control the cell cycling of the primitive hemopoietic progenitors. the early-acting cytokines may be divided into three groups. the first group would consist of il- , gm-csf, and il- . the second group consists of il- , g-csf, il- , il- , and lif/dia. the third group consists of steel factor (sf). according to our studies in culture, cytokines in each group can interact with those in other groups to initiate cell division in the cell cycle dormant primitive progenitors. while studies using retrovirally-labeled murine stem cells demonstrated unequivocally the presence of lymphohemopoietic progenitors that are capable of producing both lymphoid and myeloid progenies, it has not been possible to identify and quantitate these progenitors in culture. recently, we have developed a two-step methylcellulose culture method to quantitate murine lymphohemopoietic progenitors that are capable of producing myeloid cells and and pre-b-cells. after establishing the primary culture system initially with medium conditioned by pokeweed mitogen stimulated spleen cells, we characterized combinations of cytokines that are able to maintain the b-lymphoid potentials of the primary colonies. we observed that two-factor combinations including sf such as sf plus il- , sf plus g-csf, sf plus il- , sf plus il- were effective in maintaining the proliferation of b-cell progenitors. somewhat less effectively il- -based combinations such as il- plus il- and il- plus il- also supported the b-cell potentials of the the primary colonies. interestingly, il- -based combinations were unable to maintain the b-cell potentials of the primary colonies even though the cells in myeloid lineages proliferated strongly. we also found that addition of il- to an effective two-factor combination such as sf plus il- inhibit the b-cell potentials of the primary colonies. our cell culture for the murine lymphohemopoietic progenitors may provide an important tool for studying the mechanisms regulating the early process of lymphopoiesis. the survival and proliferation of haemopoietic stem cells, induced by growth factors, occurs concomitantly with differentiation and developmeat of the stem cells and their progeny, into mature blood cells. are the growth factors simply permissive for these processes? or do they have an inductive role to play in lineage commitment of stem cells and subsequent maturation into phenotypically mature cells? from various experiments, it is clear that the outcome of the response (that is the types of mature cells produced) is a reflection of the range of growth factors to which the cells are exposed-suggesting that combinations of growth factors may well influence the choice of lineage options taken by multipotent cells. the problem, of course, is that in all systems studied to date, no detailed examination has been made of cell death, and that even in growth factor combinations where, for example, no erythropoiesis is found, it is possible that erythroid progenitors are being produced as a consequence of differentiation of the multipoteni cells but that these cells are then dying due to lack of availability of the appropriate survival (growth factor) stimulus. we have recently been able to circumvent some of these problems and have shown that, orovided the cells receive a survival stimulus, differentiation can occur hi the absence of added growth factors and also that proliferation it not a prerequisite for acquisition of a mature cell phenotype. in other words, the growth factors may act primarily as survival and mitogenic stimuli and not as "inducers" of differentiation. expression of p bcr/abl by transfection converts interleukin- (il- )-dependent cell lines to factor independence and transforms immature hematopoietic cells in vitro. we tested the hypothesis that p bcr/abl may induce factor-independence by constitutively activating signal transduction pathways which are normally regulated by il- /gm-csf. in both the il- -dependent murine myeloid cell line, dcl , and the il- /gm-csf-dependent human line, mo e, p bcr/abl induces rapid factor-independence despite continuous growth in il- -containing medium. one-and two-dimensional antiphosphotyrosine immunoblotting showed that most proteins tyrosine phosphorylated by p bcr/abl are different that those phosphorylated in response to il- . several signaling molecules have been found to be activated or phosphorylated by both il- /gm-csf and p bcr/abl, including raf- , map kinase, shc, vav, and probably pi k. other signal transducing proteins were found to be phosphorylated only by pz bcr/abl (p rasgap, two rasgap associated proteins, and c-fes), or only by . in order to better define the biochemical activities of p bcr/abl which lead to mitogenesis, a series of cell lines were constructed in which the functional expression of pz bcr/abl was inducible. the uninduced cell lines had a wild-type phenotype while the induced cell lines displayed markedly reduced apoptosis in the absence of growth factor, and some were hyper responsive to growth factors. the phenotypes of these cell lines have been stable in culture, and the lines should be useful to define biochemical activities of p obcr/abl which are important for mitogenesis. r.e. donahue, m.r. kirby, p.d. lawman, s.e. sellers, s.w. kessler, and m.j.p. lawman a novel factor has been purified to homogeneity from a cell line derived from a human mixed germ cell tumor. by western blot analysis, using a polyclonal rabbit antibody raised to the purified native protein, scpf was found to be expressed both as a kd secreted and as a kd membrane-bound protein. to further evaluate scpfs' ability to support cd + cell growth in culture, scpf was used in short-and long-term cultures using immunoselected cd + cells. for short-term culture studies, cd + cel~s were evaluated prior to and subsequent to a six day exposure to either media alone or media supplemented with il- , scpf, or il- +scpf. the greatest expansion of cd + cells was in those expressing od . compared to pre-culture, cultures maintained in scpf, il- , or il- +scpf had, respectively, a . , . , or . -fold increase in cd +cd + numbers. there was also a consistent increase in the ratio of large cd + + cells to small cd +cd + cells. presumptively, this change represents an increase in the number of cells in either the g /m or s phase of the cell cycle. of the cytokine combinations evaluated, only the combination of il- + scpf led to a t . -fold expansion of cd + cd -cells above baseline values. by themselves, scpf and il- led to a . and . -fold reduction in cd +cd -numbers. when compared, however, to the number of cd + cd -cells present in media alone after the days in culture, scpf, il- , and il- + scpf hat respectively a . , . , and . -fold greater number of cd +cd -cells. in long-term culture assays in the absence of scpf, the cultures deteriorated rapidly and were test by day . in the presence of scpf, cell numbers were maintained over the initial - days in culture, with proliferation becoming evident days post-culture. at day , some of the cells were removed from culture media containing scpf and replatad in culture media alone. after an additional days in culture the cells that were no longer exposed to the scpf had differentiated. interestingly, the cells that were cultured in scpf continued to proliferate. after days in culture, these cells were predominantly cd +, cd +, cd +, cd +, cd +, and hi.a-dr +, and failed to express thy t, cd , cd , cd , od , or cd . karyotypic analysis demonstrated that these cells hat multiple chromosomal aberrations, civin, a. gewirtz, p. rockwell, l. witte we have cloned the cdna for stk- (stem cell tyrosine kinase ), a human growth factor receptor tyrosine kinase, and investigated its expression in bone marrow, leukemias, and leukemic derived cell lines. stk- expression is restricted to the cd + fraction of normal human bone marrow, the fraction containing all of the hematopoietic stem cell activity of marrow. experiments in which cd + cells grown on irradiated bone marrow stromal feeder layers were exposed to stk- antisense oligonucleotides resulted in inhibition of colony formation. stk- is also expressed in most cases of aml, b lineage all, and t cell all. a number of hematopoietic tissue culture cell lines which express stk- have been identified, including kmt , kgla, kg , ml- , hl- , nalm- , and reh. ml- cells stop growing and differentiate after exposure to phorbol esters and other agents. stk- expression is completely shut off by this treatment. antipeptide antibody generated against several regions of stk- identifies a doublet of proteins of kd and kd, probably corresponding to different degrees of glycosylation, in several of the cell lines. these data imply a possible role for the stk- receptor in the normal proliferation of hematopoietic stem cells and the abnormal proliferation of leukemic cells. further antisense experiments and the isolation of the growth factor for this receptor will be necessary to fully understand its role in hematopoiesis and leukemia. murine long term repopulation and double transplantation assays have clearly demonstrated irreversible damage to early stem cells by repeated doses of alkylating agents or anti-metabolites. the ability to protect from the short term effects of chemotherapy-induced myelosuppression by administration of hematopoietic growth factors has obscured the potential problem of long term stem cell insufficiency. indeed in murine models involving repeated cyclophosphamide administration, csf administration has been reported to protentiate stem cell damage. the development of techniques for isolation of human hematopoietic stem cells in a cd + lin -re fraction of marrow and blood, together with long term culture-initiating assays on marrow stroma or long term ex vivo expansion with cytokine combinations, permits quantitative analysis of human stem cell proliferation potential. it is becoming apparent that extensive chemotherapy treatment gravely compromises the population of primitive hematopoietic stem cells as reflected in their impaired capacity to peripheralize and to be represented in the blood cd + population following csf treatment with or without cytoxan. five strategies are currently under evaluation: ) upfront harvesting of marrow and/or elicited peripheral blood prior to onset of chemotherapy with subsequent "rescue" following chemotherapy; )fine tuning of cytokine and chemotherapy administration to take advantage of "rebound quiescence" of stem cells; )administration of negative regulators to suppress stem cell proliferation. transforming growth factor , macrophage inflammatory protein e and tumor necrosis factor have all proved protective in preclinical models. ) utilize cytokines, eg il- , that protect stem cells by increasing drug enzymatic inactivation, decreasing drug influx and/or increasing drug efflux, and inducing dna repair or decreasing dna damage. ) ut ze gene therapy to introduce into hematopoietic stem cells drug resistance genes such as mutated dihidrofolate reductase that confers methotrexate resistance or enhance the expression of the multi-drug resisting gene (mdr) expression. interleukin- : a novel hematopoietic cytokine possessing multiple biological activities in vitro and in vivo. j.p. leonard and s.j. goldman, genetics institute, cambridge, ma. interleukin- is a multifunctional hematopoietic cytokine which was originally identified in the conditioned medium from an il- stimulated primate stromal cell line. the human cdna was subsequently cloned from a fetal fibroblast cell line enabling the expression and purification of the human protein. the in vitro biological activities of rhll- result predominantly from synergistic interactions with other growth factors. in combination with other cytokines, rht[,- has been shown to support the formation of primitive human blast ceil coionies from bone marrow, promote erythroid burst formation and stimulate both early and late stages of megakaryocyte proliferation and differentiation. in addition, rhll- alone directly increased the size and ploidy of enriched megakaryocytes. although rhll- has no inherent cell growth factor activity, rhll- has been shown to stimulate immunoglobulin producing b cells both in vitro and in vivo. rhll-i is biologically active in mice, rats, dogs and primates when administered as a single agent in vivo. the predominant effect of rhll- in naive animals was on cells of the megakaryocyte lineage, increasing the number of bone marrow megakaryocyte progenitors, stimulating megakaryocyte endoreplication and increasing peripheral platelet counts in a dose dependent fashion. in a variety of murine rtiodels of myelosuppression, the effects of rhtl- were multitineage, stimulating the recovery of megakaryocyte, erythroid, and granulocyte and macrophage progenitors in the bone marrow, rhll-i administration reduced the platelet and hematocrit nadirs and the overall duration and severity of thrombocytopenia and anemia in these models. in a murine bone marrow transplant model, rhll- also accelerated neutrophil recovery. the results from ongoing preclinical studies continue to confirm the broad spectrum of biological activities possessed by rhil- in vitro and suggest this cytokine may be an effective agent in the treatment of myelosuppression associated with cancer chemotherapy and bone marrow transplantation. preclinical biology of human il- t.l. nagabhushan, s.k. narula and m.i. siegel human il- (hull- ) is a amino acid polypeptide synthesized by a number of different cell types. it is a pleiotropic factor with both immunosuppressive and immunostimulatory activity. recombinant hull- expressed in cho cells is not glycosylated, and when expressed in e. coil the protein retains the biological activity of the cho-derived product. in the presence of antigen presenting cells, hull- inhibits cytokine synthesis in t cells. hull- downregulates ,f-ifn and il- induced mhc class ii antigen expression on monocytesmacrophages. in contrast, it has no effect on class ii expression on purified tonsillar and peripheral b cells. hull- also inhibits the synthesis of il- % il- /l il- , il- , tnf-m gm-csf and g-csf at the protein and rna levels in monocytes activated by lps or lps and .r the y-ifn or gm-csf induced phagocytosis of opsonized yeast particles by human peripheral blood-derived macrophages and granulocytes is downregulated by hull- . il- induced ige synthesis by pbmnc is inhibited by hull- . the protein also potentiates a strong lak activity in human pbmnc. these results will be discussed along with some early in vivo biology in rodents. a monomeric pentapeptide (peedck) inhibits murine hematopoiesis in vitro and in vivo while its dimer counterpart (peedck): formed through a disulphide bridge has a stimulatory effect in the same systems. stable peptides of the rimer have been made by substitution of the disulphide bridge with a dimethylene bridge. in vitro the dimer seems to have no direct effect on gm-cfc or on purified lin-scal + cells in the hpp-cfc assay ( cells per culture, - % pe). the monomer does not affect gm-cfcs, but inhibits approximately % of the purified hpp-cfcs. the dimer stimulates human or mouse stromal cells to produce m-csf and possibly other cytokines which augment colony formation in vitro and also activate human pbl as measured by an increased expression of cd lb. in ex vivo experiments it was found that the dimer increases and the monomer decreases cell cycle rate of cfu-gm and cfu-s in the bone marrow. intraperitoneal injection of the dimer ( to ng/kg) into mice, led to increased progenitor cell number in the bone marrow and also increased survival of mice given a lethal dose of cyclophosphamide ( mg/kg). at present receptors of the peptides have not been identified. these studies have shown that the dimer has an indirect effect on hematopoiesis as a stimulator of cytokine production, while the monomer seems to act both as an antagonist of dimer action and is also able to directly inhibit early myeloid progenitor cells. the possibility that these two compounds have therapeutic efficacy in diseases involving a myelosuppressed bone marrow is indicated. there is evidence that basic fibroblast growth factor (bfgf) plays a role in the regulation of normal blood cell proliferation and differentiation. basic fgf is produced by and is a potent mitogen for human slromal cells. it is found in megakaryocytes and cells of the granulocyte lineage, in vivo, and it enhances megakaryopoiesis and myelopoiesis in human long term bone marrow cultures. it stimulates progenitor cell growth and augments the proliferation of progenitor cells when added in conjunction with other hematopoietic growth factors. in addition, it counteracts the suppressive effects of transforming growth factor beta. basic fgf also synergizes with stem cell growth factor to augment granulocyte macrophage colony stimulating factor stimulated progenitor cell growth. based on the observations in normal hematopoiesis, the role of bfgf in malignant bematopoiesis is presently an area of active investigation. scf is one of the earliest-acting hematopoietic growth factors. pre-clinical studies have demonstrated its multi-lineage hematopoietic effects. we have conducted a phase i trial of scf and report now on the first patients (pts) with stage iiib (n= ) or iv (n= ) breast cancer. the study was designed to evaluate the toierabllity and biologic effects of scf. pts received scf prechemo (cycle o) and following cycles - of c/a chemotherapy. cohorts of lots were randomized in a : ratio to receive either scf (n= ) by subcutaneous injection at dosages of i , , and so/~g/kg/day for days or no scf as a parallel c/a control group (n= ). various physiologic, biochemical, pharmacokinetic, and hematologic parameters were studied. bone marrow (bm) and peripheral blood (pb) progenitors were assayed. scf administration was associated with pb progenitor mobilization in cycle at all dose levels. absolute neutrophil counts demonstrated modest doserelated increases over baseline ranging from median values of % ( pg/kg/d) to % (so/jg/kg/d). no reproducible effects were seen on red blood cells or platelets. the principal adverse events were derrnatologic in nature. they included mild to moderate reactions at the injection site in all pts, and moderate to severe reactions distant to the site in i pts (primarily at higher doses). at pg/kg/d, / pts experienced dose-limiting respiratory symptoms including cough, hoarseness, and laryngospasrn. because of scfs known mast cell effects, prophylaxis with h /h blockers and bronchodilators is being evaluated. no pts developed antibodies to scf. evaluation of effects following chemotherapy is ongoing. we conclude that scf is an active hematopoietin capable of stimulating production of bm and pb progenitor cells as well as peripheral neutrophils. g. trinchieri nksf/il- is a heterodimeric cytokine produced by monocytemacrophages, b cells and other accessory cells in response to various stimuli including bacteria and bacterial products. nksf/il- acts on t cells and nk cells inducing cytokine production, proliferation, and enhancement of cytotoxic activity. nksf/il- is a particularly efficient inducer of ifn- production, acting alone or in synergy with other ifn- inducers such as il- , antigens, anti-cd or anti-cd antibodies, mitogens, and phorbol diesters. nksf/il- appears to play a major role in regulation of natural resistance: when produced by monocyte-macrophages, it directly activates the cytotoxic activity of nk cells and induces both nk and t cells to produce ifn- and other cytokines with important effects on activation of phagocytic cells. the important role of nksf/il- in response to bacterial products is clearly demonstrated by the ability of anti-nksf/il- antibodies to inhibit in vivo in mice the ifn-y production induced by lps ii~ a toxic shock model. nksf/il- is also an important factor in the regulation of adaptive immune response, by inducing the differentiation and growth of t helper cells type (th- ) and by preventing the differentiation of il- producing th- cells. a possibly obligatory role of nksf/il- for th- cell differentiation can be demonstrated in vitro in the human lymphocyte response to allergens or bacteria-derived antigens and both in vitro and in vivo in the murine system, e.g. in the immune response to leishmania infection. production of nksf/il- by accessory ceils is stimulated by ifn- , a product of th- cells and suppressed by il- or il- , products of th- cells. these results suggest that nksf/il- represents an important link between natural resistance and adaptive immunity and is at the center of a cytokine network that regulates the equilibrium between cellular and humoral immunity. work with alpha-interferon in cml started at mdacc in and has evolved over the years to allow us interpretation of response rate in a large number of patients with relatively long follow up. newly diagnosed patients (<i year from diagnosis) were treated with a starting dose of mu/m of interferon. of such patients followed in different studies, we noted a % complete hematologic remission; % cytogenetic response, and % complete and partial cytogenetic response (< % phi). in long-term analysis of patients, achieved cytogenetic response ( %) of which ( %) were complete; ( %) were partial, and ( %). were minor ( %) of the cytogenetic responses are durable. of major interest is the fact that % of the complete cytogenetic remissions are durable ( of ) but only a minority of the partial and minor cytogenetic responses are durable ( % and %, respectively). this clearly set our goal on improving the incidence of complete eytogenetic remission. median survival ranges between - months. however, of particular interest is the excellent survival rate of the complete cytogenetic responders ( of alive at time of follow-up). current studies are focusing on idealizing combination therapies and will be discussed. cml is a paradigm of tumor progression; therefore, we were looking for characteristics associated with disease progression and identified evidence of autocrine growth factor loop formation with disease progression and the overproduction of il-ib. finally, we are also focusing on the study of minimal residual disease in patients with complete cytogenetic remissions and have identified residual ph i progenitor cells in the majority of these patients, including patients with unmaintained remission. the nature of such a remission will be discussed as well. the mxa-protein: a new and excellent marker for endogenously produced type-i-ifn d.jakschies , k.u.nolte , r.zachoval , h.deicher , and p.v.wussow the human mxa-protein is a recently identified human ifn-induced protein, which is synthezised by peripheral blood cells specifically and dose-dependently upon stimulation with type-i-interferons both in vitro and in vivo. mononuclear cells (mnc) both from healthy persons and cancer patients (n= ) are usually (in the absence of a virus infection) mx-negative. patients starting an ifn-a-therapy become mxa-positive (n= ) within hours and remain positive throughout their therapy. furthermore, two groups of patients are known to have frequently measurable serum ifn-titer: sle-patients and aids-patients (n= ) studied expressed significant mxa-levels in their mnc. while all patients with measurable ifn-titer had mxa-levels above u/ . mnc, also the vast majority of serum ifn-negative patients possessed detectable mxa-levels indicating that also these patients produce endogenous ifn, although due to its rapid clearence from the circulation it may not be measurable in serum. in addition, patients with acute hepatitis were investigated for the mxa-protein. surprhingly large difference in the endogenous tfnproduction were observed. only in the first two weeks after the onset of clinical symptoms hepatitis a patients (n= ) had both high mxaprotein and - -a-synthetases levels, but not thereafter. the levels of both ifn-induced activities reached concentrations found in cancer patients treated with - -mill. iu rifn-c~ per dose. in acute hepatitis b and c (n= ;n= ) only marginally elevated mxa and - -as levels were found. in summary, the mxa-protein is a new and excellent marker for endogenously produced type-i-ifns. m.e. rybak interleukin (il- ) is a cytokine with pleiotropic immunomodulatory activities. these actions include b cell stimulation and co-stimulation, enhancement of il- mediated generation of cytotoxic t cells, increase in the expression of hl-a class ii antigens by monocytes and the inhibition of the production of a number of monokines including il- , il- and tnf~. human il- has been cloned and expressed in e. coli. the mature protein has amino acids, mw , d. studies of rhuil- in malignancy were prompted by i__nn vitro data demonstrating the ability of il- to inhibit the proliferation of chronic myelomonocytic cells, non-hodgkin's lymphoma and myeloma cells in culture, and to inhibit the il- induced proliferation of chronic lymphocytic leukemia. inn vivo models utilizing murine il- demonstrate an immune response to tumors (e.g lung, sarcoma and melanoma) following systemic administration of il- or the administration of tumor cells transfected with the il- gene. initial phase i trials of rhuil- have been completed in patients. forty-seven patients received daily subcutaneous (s.c.) rhuil- , . - . ~g/kg. side effects seen in ~ % of patients included headache, fatigue, fevers (< ~ myalgias and reversible elevation of sgot, sgpt and alkaline phosphatase. nausea, pedal and periorbital edema, and rhinitis were also seen. the mtd for rhuil- was approximately ~g/kg/d s.c. activity has been seen in hodgkin's disease, non-small cell lung cancer, chronic lymphocytic leukemia, and multiple myeloma. current studies include phase ii investigations in advanced non-small cell lung cancer, refractory hodgkin's disease, and relapsed multiple myeloma. an update on these studies will be presented. this lecture will review the evidence concerning the possibility that sustained overstimulation by growth factors might provoke or accelerate myeloid leukemia development. the regulatory factors likely to be relevant are the four colony stimulating factors although a possible role for scl and il- probably also needs consideration. in general, prolonged overstimulation by growth factors in the mouse either using transgenlc mice or engraftment of hemopoietic cells engineered to produce growth factors leads to hyperplasia but not leukemia. in contrast insertion of csf genes into immortalized, but non-leukemic cell lines leads to prompt leukemic transformation. if fdc-pi cells are engrafted in preirradiated animals, these can undergo delayed leukemic transformation often by activation of csf genes by inserted ~ particles. normal cells can be transformed to leukemic cells by retroviral insertion of a combination of a csf gene with fox . , the apparent function of the hox . being to dysregulate the process of self-generation in early hemopoietic precursor cells. thus two distinct changes appear to be necessary for leukemic transformation -abnormal self-generation and an acquired capacity for autocrine growth factor production. all the above studies involve autocrine growth factor production but we have recently completed a study indicating that sustained stimulation by exogenous growth factors can also accelerate leukemic transformation. this study involved fdc-pi cells engrafted in gm-csf transgenic mice. again the actual transformation process often involved rearrangement of csf genes with autocrine csf production. the latter experiments raise the potential clinical hazard of prolonged csf treatment in myelodysplastic patients but this possibly should not be overinterpreted. since granulocyte (g-csf) and granulocyte macrophage (gm-csf) colony stimulating factors were first introduced in to clinical trials in , there have been a vast number of clinical trials demonstrating the utility of these agents in reducing nentropenia, and therefore infection in patient groups ranging from congenital nentropenia to bone marrow transplantation. the discovery that a large number of progenitors are released into the peripheral blood by these agents either alone or after chemotherapy was not one of the major indications predicted in . the rapid reconstitution of both nentrophils and platelet counts when these peripheral blood progenitors (pbpc) are used as autologous rescue after myeloblative treatment, is encouraging many centres to use this type of rescue instead of abmt. interesting studies are at present indicating that these pbpcs can be expanded in vitro prior to clinical use. attention is now moving to the stimulation of platelet productions with il- , il- , and l- . there is increasing evidence from experimental haematologn/ that combinations of hgfs give optimal stimulation often of more primitive cells than previously thought. clinical studies are now being performed, but the logistic problem of testing all the possible comb'mations is daunting. as the use of haemopoietic growth factors successfully protecta the patients against haematological problems, other organs such as the gi tract, become dose limiting, often keeping the patient in hospital after haemopoietic recovery. repeated high dose treatments also deplete the haemopoietic stem cells. great interest is now being found in protecting the normal haemopoietic and other organ stem cells from the effects of radiochemotherapy. this may be possible using molecules such as mip alpha or tgf beta. other workers have used the timing of the use of stimniatory hgfs to induce quiescence of the haemopoietic stem cells. the optimum use of hgfs would probably be to in sequence, prime the haemopoietic progenitors, then protect them from radiochemotherapy, followed by rapid expansion under the influence of probably a mixture of hgfs. crc deft of medical ontology, christie hospital, wilmslow road, withington, manchester, m bx. il- is the first cytokine available for human use which was predicted to have both a activity on early hematopoietic progenitor cells, as well as specific action leading an increase in the number of circulating platelets. phase i/ii clinical trials have confirmed these predictions and have demonstrated, in addition, moderate activity on the late myeloid cell lineage leading to increased numbers of circulating neulrophils and eosinophils, events predicted from monkey studies, but not from in vitro and rodent studies. phase i studies provided a clear definition of the maximum tolerated dose ( #g/kg daily iv or s.c.), since at /lg/kg an unacceptable frequency of severe headache, flushing and tissue swelling were observed. at doses between . - #g/kg, depending on the bone marrow reserve, cytotoxic chemotherapy and malignant disease, il- prevented or reduced the severity of neulropenia and thrombocytopenia, reduced the number of platelet transfusions needed and allowed adherence to myelotoxic chemotherapy. in combination with other cytokines, such as gm-csf or g-csf, enhanced myeloid responses were seen including increased numbers of peripheral blood progenitor cells. these observations have led to now ongoing phase iii clinical trials using il- in conjunction with cytoxic chemotherapy in several malignancies and in conjunction with autologous bone marrow transplantation. there appears little doubt at present that il- will be an important contribution to management of the patients with oncologic or hematologic diseases. extensive phase ii studies have shown a reduction in the severity and duration of neutropania when filgrastim (r-methug-csf) is administered soon after chemotherapy. two phase iii studies demonstrated that filgrastim, given to small cell lung cancer patients from the day after chemotherapy, reduced the incidence of febrile neutropenic episodes by approximately % (crawford et al, ; trillet-lenoir et al, ) . these studies administered filgrastim from the day after chemotherapy, and they suggest that filgrastim is highly effective when used as an adjunct to chemotherapy to prevent fever and neutropenia. in a recent study (maher el al, in press), pilgrastim administration was delayed until after the onset of fever and antibiotic therapy. patients enrolled in lhis study were a median of days after their last dose of chemotherapy. it was found that this later commencement of filgrastim could significantly accelerate neutrophil recovery, produce a shorter duration of febrile neutropenia, and decrease the risk of prolonged hospitalization; however, maximal benefit appears to be derived from adjunctive use of filgrastim prior to the onset of fever and neutropenia. from in vitro and animal studies it is known that il- is a stimulator of thrombopoiesis, a stimulatory factor for t and b cells, that it stimulates hepatocytus and can have antitumor activity. an ongoing phase i-ii study with rhll- before and after chemotherapy (ct) is performed. patients (pts) with breast cancer or non-small cell lung cancer receive e. coil derived rhll- (sandoz, basle) in doses of . , , . , and #g/kg/d, with > pts per dose step. before ct rhll- is given dl iv and d - sc. ct (mitoxantrone mg/m and thiotepa mg/m , iv dl) starts d , q wks. after ct rhll- is given d - se in all cycles (c). before ct pts were treated. rml~ related side effects were: fever who gri-ii ( %), headache ( %), myalgia ( %), local erythema ( %) and starting at . #g nausea ( %), increase of liver enzymes who gri-ii ( %) and anemia ( %). symptoms were controllable with acetaminophen (n= ) and antiemetics (n= ). upto #g them was a dose response related platelet increase compared to dl (r= . , p< . ), with highest count after rml- cessation. at . /~g max. increase was . + . (se), at ~g . + . %. on d a leukocyte increase compared to dl was observed starting at . #g upto /~g (dl . + . , d . + . , p< . ) mainly due to neutrophil increase (p< . ). at d increase of lymphocytes occurred compared to dl (dl . + . , d . + . , p< . ). there was a reversible hemoglobin reduction especially at and i ,gg, starting d with a max. decrease at dg ( /zg . + . g/l, #g . + . g/l), at d initial values were almost reached without intervention. a dose related acute phase response was observed, with max. value of crp (mg/l) • at . /zg and • at /tg (r= . , p< . ), and max. value of serum amyloid a (rag/l) at . /zg of • and at #g + (r= . , p< . ). after ct c (no c= ) were evaluable upto dose #g, in these e rhll- related side effects were: fever ( %), headache ( %), myalgia ( %), local erythema ( %), nausea ( %) and increase in liver enzymes ( %). uptu c there was no difference in platelet nadir, moment and duration of platelets < x /l between . - /~g and . - #g doses. the same was observed for leukocytes. in conclusion, rml- upto #g/kg/d has a acceptable toxicity profile and results, before ct, in dose dependent platelet and acute phase proteins increase. after ct, evaluable now for the first cycle, there is a faster platelet recovery at higher dose steps. university hospital groningen, division of medical oncology, department of internal medicine, oostersingel , ez gronlngen, the netherlands. g-csf in acute lymphoblastic leukemia. d.hoelzer, o.ottmann rhg-csf given in acute lymphoblastic leukemia (all) after induction/consolidation therapy or allogeneic/autologous bmt can shorten the recovery time of neutrophils by - days, with reductions of the infection rate in some studies. granulocytopenia is the major dose limiting toxicity not only after but also during chemotherapy. for example, severe neutropenia occurs in ~ of patients during the intensive wk induction regimen of the german all trials. therefore, the effect of parallel administration of g-csf (filgrastim) and chemotherapy was explored, rhg-csf ( ~g/kg) was given concurrently with cyclophosphamide, arac, -mercaptopurine, prednisone and prophylactic cranial irradiation, rhg-csf was continued until neutrophil counts exceeded /~i, but for at least seven days following the last dose of chemotherapy. in a pilot study of pts, the median duration of granulocytopenia < /~i was days compared to days in a historical control group. when rhg-csf was applied throughout the entire wk induction treatment (scherrer et al) in pts, the cr rate was %, the tim~ of nm,trophil reeoverv to > ~o~l~l after the end of chemotherapy was reduced from , days to , days, and the infection rate was lower in the g-csf treated patients. from these studies it was concluded that the concomitant administration of rhg-csf and intensive chemotherapy in adult all is leasable, well tolerated and without major side effects; in particular, there was no evidence of prolonged neutropenia due to stimulation of hemopoietic stem cells by g-csf and their subsequent elimination by chemotherapy. the clinical effects of rhg-csf given concomitantly to chemotherapy require confirmation in a randomized trial. in an ongoing randomized multicenter trial~ an interim analysis of pts revealed a highly significant reduction of the total duration of severe neutropenia (< /~l) in the patients receiving g-csf. further analysis of the potential clinical benefits of this treatment modality is in progress. we performed a phase h trial to assess the ability of g-csf -mobilized pbpc to rapidly and completely restore hemopeiesis after high dose chemotherapy in the absence of bone marrow infusions, with selection for pbpc-only infusions based on yield of granulocyte -macrophage colony -forming cells (gm-cfc) after g-csf treatment. twenty-nine adults with acute lymphoblastic leukemia, non-hodgkin's lymphoma, or hodgkin's disease who were eligible for autologons bone marrow transplantation were treated. g-csf was given at or ixg/kg/d for - d and mononuclear cells collected by apheresis on days , , or , , . yield of pbpc was assessed by assay for granulocyte-macrophage colony forming cells (gm-cfc). high yield was defined as total gm-cfc collected > x /kg. high dose bnsulfan ( mg/kg/d x d) an& cyclophosphamide ( mg/kgtd x (: ) were administered and hemopoiedc cells infused• and recovery of hemopoiesis mouitored. progenitor cell yield was high in of patients. patients given infusions of g-csf-mobilized pbpc, but without bone marrow infusion, experienced recovery of hemopoiesis in all cases. no patient given pbpc alone required bone marrow infusion in up to months of follow up. kinetics of recovery of both the platelet and neutrophil counts were more rapid in the high yield (pbpc-alone) group than in the low yield group (pbpc pins bone marrow). pintelels recovered to > x / at a median of days and neutrophils to > . x / at days in the high yield group, compared with days and days in the low yield group (p= . and . respectively). fewer platelet transfusions were required in the high yield group (median packs per patient vs. , p--- . ). we conclude that in patients with a high yield of pbpc after g-csf therapy, infusion of g-csf-mobilized pbpc results in rapid and sustained restoration of hemopoiesis. use of g-csf mobilised pbpc to support multiple cycles of hdc for treatment of high-risk stage ii and iii breast carcinoma is now being examined in a phase i study pursued by our group. our data on pbpc moblisation without chemotherapy allows consideration of g-csf-mobilised pbpc for haemopoietie ceil allotmnsplantation and for gene therapy trials. our approach for high-dose (hd) chemotherapy is to first treat patients eligible for dose intensification with a standard dose chemotherapy (vip: vp = etoposide: mg/m , ifosfamide: g/m , cis-platinum: mg/m ) followed by the application of colony stimulating factors (g-csf, gm-csf or il- + gm-csf) in order to combine a regimen with broad anti-tumor activity with the recruitment of peripheral blood progenitor cells (pbpcs). pbpcs are reinfused into the patients after high dose intensification chemotherapy (according to underlying disorder: hd-vip, vic-e or beam). highest numbers of pbpc were recruited by the sequential administration of _- + gm-csf (median of cd + cells/iji and . total progenitors/ml, including multilineage as well as megakaryocytic progenitors). csf-mobilized pbpcs include lineage negative cd + cells, -hc resistant precursors as well as long term culture initiating cells (ltc-ic). so far, a total of patients were supported with pbpos and csfs following hd-chemotherapy. the period of severe neutropenia (< //ji) as well as thrombocytopenia (< . o//ji) was reduced to a median of or days respectively. in order to decrease the number of potentially contaminating tumor cells in the leukapheresis preparations, we have started to positively select cd + cells by an avidin-biotin immunoadsorption column (provided by r. berenson, cellpro, usa). in all these patients, recovery data were comparable to the patients who received unseparated cells. to provide sufficient numbers of pbpcs for repetitive use or in patients with low progenitor cell yield as well as to possibly avoid leukapheresis, we investigated the ability of hematopoietic growth factor combinations to expand pbpcs ex vivo. chemotherapy + g-csf recruited cd + cells from patients were enriched by immunoadsorption (purity: . -+ . ) and cultured in suspension. a combination of stem cell factor, erythropoietin, interleukin- , il- , and il- was identified as the optimal combination for the expansion of clonogenic progenitors. proliferation peaked at day with a mean -fold increase (range - ) of clonogenic cells. interferon-gamma synergized with the -factor combination, whereas the addition of gm-csf or g-csf decreased the number of clonogenic cells. large-scale expansion of cd + cells in auto-iogous plasma supplemented with the -factor combination resulted in an equivalent expansion. our data indicate the feasibility of large-scale pro+genitor cell expapsion in cancer patients, starting from small numbers of cd ceils. given the pressures on health care resources new health technologies need to be assessed to establish whether their benefits justify their costs. g-csf is an important new technology that has profound impacts on cancer chemotherapy and patients outcome. as clinical studies show, the main economic benefits of g-csf come from * a significant reduction of hospitalisation during chemotherapy, * reduced expenditure on inpatient and outpatient i.v. and oral antibiotic treatment, * and reduced platelet and rbc transfusions. in addition, it is expected that a therapy with g-csf during cancer chemotherapy shows intangible and non-monetary benefits: * increase in the rate of treatment as intended (time, number of cycles) * increase of quality of life of patients * reduction of drop-outs * ability to intensify dosage recently coducted economic cost-cost studies show that the cost of managing neutropenia and infection and the cost of chemotherapy are lower for patients receiving g-csf than those who do not. future studies have to focus mainly on the quality-of-life cost-effectiveness of g-csf. the aim of this study is to evaluate the effects of recombinant human erythropoietin (rhu epo) on hematopoietic regeneration after allogeneic or autologous bone marrow transplantation. patients were randomized to receive either u rhu epo/kg body weight or placebo as continuous intravenous infusion from day after bmt until independence from erythrocyte transfusions for days or until day . the randomization was performed per each center and stratified according allogeneic or autologous bmt and major abo-blood group incompatibility. at the time of the planned interim analysis with patients treated, the time to erythrocyte transfusion independence after allogeneic bmt was shorter in group a (n = ) than in group b (n = ). after autologous bmt no difference between group a (n = ) and b (n = ) could be detected so far concerning time to transfusion independence or the number of transfusions applied, considering either erythrocyte or thrombocyte transfusions. there were no major differences in side effects between groups a and b. as of october , the study was finished with a total of patients. since rcc patients have been screened for entry into clinical trials and entered onto surveillance as first management policy until symptoms or serial radiology indicated tumour progression. + ( %) have demonstrated unexplained "spontaneous" cr+pr(median duration mths), and ( %) "stable" disease(median mths). none of not nephrectomised regressed, while cr+pr was % of nephrectomised at diagnosis of metastasis, % of developing metastases - mths after nephrectomy and % of developing metastases more than mths after nephrectomy. it was % of with lung only verses % of with other sites. possible evidence for relief of potentially immunosupressive influences have been demonstrated in of patients demonstrating unexplained "spontanous regression. study of hla class i and ii antigen abnormally on these tumours and their influence on tumour infiltrating lymphocytes will be reviewed. patients seen to progress on surveillance were entered into treatment trials, the remainder being too old, sick or having bone or brain metastases needing radiotherapy. + ( %) achieved cr+pr. + of ( %) of patients with lung only verses + of ( . %) of those with other sites of metastasis. although these results suggest that no harm has come from a period of preliminary surveillance, the fact that the therapeutic benefits including durable complete remission from therapy are confined almost entirely to the good risk small volume asymptomatic patients makes it difficult to justify a policy of surveillance for such patients. auto bmt, as alternative of allo bmt, lack{ immunotherapical effect delivered by allo graft. however immune reconstiturion after auto bmt shows features recalling the post allo setting. futhermore in vitro and in vivo in rll is able to stimulate the proliferation and the lyric functions of nk and t cells, major effectors of this gvl effect. for these reasons from , we have so far treated patients with acute leukemia (aml- ;all: ) in cr with auto bmt followed with rll . all conditioning regimen included tbi. median rime between diagnosis and bmt was r~. mths ( - mths).arll was started as soon as platelets reach x ~/ and anl . x ~/ . rll was given in cycles of days (c ) and days (c to c ), starting on d ,d ,d ,d ,d ~. rll was given as a continuous infusion at a median of m iu/m~/d ( m- m). no patient died of rll related toxicity. platelet toxicity was obvious during the starting cycle but did not impair the long term hematological recovery. immune stimulation was major and intense for both nk and t cells and both lak and nk activity (all p < . ). long term infusion conducts to privilegiate nk stimulation. with a median follow-up of mths, relapse and survival probabilities are respectively % and % fo all and % and % for aml comparying favorably with historical control ; these data invited us to set up a randomised study in cr aml and all after auto bmt. started in in france, italy and england, pts have been so far included. we have shown that local, inhalative il- application in combination with low dose systemic il- and ifna is highly effective and has low toxicity (j urol , , ) . here we report about longterm follow-up from to month of an outpatient schedule in patients with pulmonary metastasis of renal cell carcinoma. treatment protocol based on time daily inhalation of il- ( x . i j) combined with low dose i.v. il- ( x u/ days every weeks) in patients or low dose il- s.c. injection of . u per day in patients. all patients received x s.c. ifna three times weekly. median treatment duration was month (range - month). toxicity of il- inhalation was low, no fever, no vasculary leakage. even after up to month of continuous inhalative treatment no evidence for irreversible pulmonary damage due to il- induced immunomodulation occured in patients. in pulmonary metastases complete response ( month), partial response ( , , , , + , , , , ) and stable disease ( , , , + , , , +) were achieved. of patients responded with nonpulmonary metastasis. overall tumor response considering both, pulmonary and nonpulmonary metastases was complete response, partial responses stable diseases, mixed responses and progressive disease. while il- per inhalation had to be stopped because of grade ii toxicity in patient only, ifna systemically had to be stopped in of and il- i.v. cycles in of patients. patients are alive with median actual survival of all patients of . month which seems to be prolonged compared to risk factors. inhalafive treatment with il- combined with low dose systemic cytokines represents a highly valuable model for the low toxicity of lccal il- application, resulting in an effective long term treatment schedule with long term responses. recent clinical trials for the biological therapy of solid tumors have used recombinant human cytokines in combination with conventional chemotherapy. in patients with metastatic renal cell carcinoma, we established a -drug combination of subcutaneous recombinant human interferon-u (ifn-u), interleukin- (il- ) and -fluorouracil ( -fu) as outpatient regimen. treatment consisted of eight weeks each of ifn-~ ( million u/m' x per week sq) combined sequentially with il- ( - million iu/m' x per week sq for four weeks) and -fu ( mg/m iv weekly for four weeks). among consecutive patients treated, there were complete ( . %) and ( . %) partial responders, with an overall objective response rate of . % ( % confidence interval, - %). median response duration was calculated at + months, and no relapse has occurred among complete responders. systemic toxicity was mild to moderate, with no severe -fu related mucositis or diarrhea. there were no dose limiting adverse effects of sq il- and no toxic deaths. in summary, this outpatient biochemotherapy was as effective as the most aggressive inpatient iv il- regimen; it appeared to significantly improve the therapeutic index in patients with metastatic renal cell carcinoma. medizinische hochschule, d- hannover, germany metastatic melanoma: immunotherapy with ifna and il- , dtic/ifna as second line treatment, ifne(/il- + dtic or cddp. u keilholz, c scheibenbogen, w tilgen, d maclachlan, p brossart, th m hler, w hunstein. this abstract summarises our -year experience in the treatment of metastatic melanoma with sequential or combined chemo-immunotherapy. patients with progressing metastatic melanoma have been treated with ifna and il- . mill u/sqm ifn(~ were given s.c. on days - , and a new decrescendo regimen of il- was used: lmg/sqm/ hours, followed by mg/sqm/ hours, and mg/sqm/ hours, and . mg/sqrrv hours x . the current response rate is % ( cr, pr, mr/sd, pd). patients not responding to ifnedll- (sd and pd) were eligible for subsequent chemotherapy with dtic, mg/sqm day , followed by ifna, mill u/day - . the response rate for this second line regimen is % ( cr, pr, sd, pd, n= ). using this sequential approach, the overall response rate in this cohort is %, and the median survival is months. in preparation of a randomized trial comparing chemo-immunotherapy and immunotherapy a pilot study was performed. patients not responding to the standard ifnedll- regimen received a single dose of dtic, mg/sqm (n= ) or cddp, mg/sqm (n= ) on day one, followed by ifn~il- according to the identical protocol as previously without chemotherapy. in the case of cddp, grade nephrotoxicity was observed in / patients. pharmacokinetics of il- were not influenced by previous chemotherapy, except in the patients with cddp-associated nephrotoxicity. induction of secondary mediators (tnfa, ifn'~, neopterin, scd ) by il- was not diminished by previous chemotherapy. patients unresponsive to immunotherapy alone showed tumor shrinkage upon chemo-immunotherapy. conclusion~: with initial immunotherapy followed in nonresponders by chemotherapy a response rate above % is achieved. combined chemoimmunotherapy is feasible, and the immunologic response to il- is not diminished by previous chemotherapy. a randomised trial is necessary to determine, whether combined chemo-immunotherapy is superior to immunotherapy alone. inflammation is characterized by an accumulation of leucocytes at the site of injury. it hab been well established that the disease associated lesions are caused by a plethora of soluble mediators released by both the infiltrated leukocytes as well by tissue cells, including many degrading enzymes, lipid mediators, reactive oxygen species, or cytokines. among those, cytokines play a crucial role in the inflammatory process as their release appears to represent the initial response which mediates the secretion of subsequent effector molecules responsible for the pathophysiological effects (e.g. changes of vascular resistance) in an autocrine or paracrine fashion. this holds true for the acute inflammation during which circulating granulocytes or monocytes are activated directly by a noxious agent; an example being the pivotal function of tumor necrosis factor (tnf) in septic shock. chronic inflammatory diseases are initiated and perpetuated by immune reactions. cytokines represent an important link between immune reactions and the recruitment and activation of blood borne infiltrating inflammatory ceils. thus interferon r and tumor necrosis factor , secreted by activated t lymphocytes, are potent stimulators of mononuclear phagocytes. the activated macrophages themselves secrete cytokines such as interleukin-t (il-t), tnf a or interleukin- (il- ) which have been termed collectively as inflammatory cytokines, il- and tnf again in an autocrine or paracrine way upreguiate the synthesis of the ultimate pathogenic mediators in macrophages-e.g. the secretion of degrading enzymes as in rheumatoid arthritis-. equally important they induce functional changes in vascular and autochthonous tissue cells which are thus recruited to contribute to the inflammatory lesions. il- , and similarly the lymphocyte derived rantes, chemotactically attracts to the site of lesion and activates granulocytes, which then participate in the inflammatory reaction. while this rapidly recruits circulating phagocytic cells, the secretion of colony stimulating factors by activated t lymphocytes and macrophages also increases the pool of granulocytes and monocytes by stimulating their synthesis in the bone marrow. in turn il- an tnf a synthesized by monocytes (and also some tissue cells) are coactivators of t lymphocytes, and thus contribute to al local immune reaction. besides this general role of cytokines in inflammation their involvement in specific situations becomes increasingly apparent, an important example being the allergic inflammation. cytokines synthesized by t lymphocytes such as interleukin- or interleukin- not only regulate immunogtobulin synthesis of b lymphocytes, including ige, but equally important modulate the functions of mast cells or eosinophilic leukocytes to become effective effector cells of allergic reactions. the detailed functions of cytokines in different acute or chronic inflammatory diseases will be discussed in the subsequent contributions. clinical results e holier, r hintermeier-knabe, b ertl, hj kolb, m kaul, u behrends, s thierfelder, w wilmanns experimental as well as clinical studies suggest dual involvement of proinflammatory cytokines as tnfalpha and ill in complications and gvhd following allogeneic bmt: nonspecific activation of recpient tissues during pretransp~ant conditioning results in early release of tnfa which strongly enhances donor t cell activation; in the effector phase, t cell stimulation is amplified by ifngamma and lps mediated release of tnf and ill. the role of these mediators as critical effectors of gvhd related tissue damage could be confirmed by application of cytokine antagonists early after murine bmt: both, anti-tnfa and ill-receptor-antagonist (illra, as shown by j ferrara, boston) resulted in a > % reduction of gvhd related mortality in mice, while pentoxifylline proved to be ineffective. in human bmt, phase l/ll studies analyzing anti-tnfa and illra in refractory gvhd report substantial improvement in - % of patients, though reoccurrence of symptoms after cessation of treatment in most patients indicates a late effector function of cytokines at least in advanced gvhd. contribution of tnfa to recipient related induction of gvhd is further suggested by a recent phase / study performed in our institution as prophylactic application of anti-tnfa during pretransplant conditioning suppressed occurrence of early gvhd in high risk patients as compared to historical controls. though these data indicate a future role of a variety of cytokine antagonists in management of clinical complications of bmt their impact on long term survival has to be evaluated and compared to classical strategies (e.g.the use of corticosteroids and t-cell-manipulation) in randomized studies. the cytokines il- and tnf play central roles in inflammatory responses which can lead to tissue injury, naturally occurring antagonists such as soluble cytokine receptors or receptor antagonists are also produced during inflammatory responses. these soluble receptors and antagonists may act as a buffer system to reduce and limit tissue injury induced by cytokines. however, in some disease states this naturally occurring buffer system may not be sufficient to inhibit the detrimental actions of an inflammatory response. responses to il- are mediated via the type receptor (il- r, type i). the type ii receptor (il- r, type ii) has never been shown to signal, but instead appears to be shed as an il- antagonist. both recombinant soluble il- r, type i an il- r, type ii are capable of binding il- and inhibiting responses in vitro. in a phase i clinical study evaluating soluble l- r (type ) in modifying cutaneous allergic responses, il- r was a potent inhibitor of allergen induced latephase inflammation in the skin, with a high safety profile. responses to tnf are mediated via the ps or pt tnf receptor. soluble forms of both the p and tnfr occur naturally and are increased in many inflammatory states. dimeric constructs of the p tnf have been engineered to form a soluble fc fusion protein with two monomeric tnfr extraceuular portions contained on a truncated ig heavy chain (tnfr:fc). the tnfr:fc possesses higher affinity than a monomeric soluble receptor and the ig-like fc structure imparts a longer half life. animal studies have shown that soluble il- r, type i and tnfr:fc are effective antagonists of inflammation. in animal models for arthritis and pulmonary inflammation, il-ir and tnfr:fc reduced inflammatory celt infiltration and tissue damage. both molecules are currently in clinical trials and hold promise for treatment of autoimmune and allergic diseases such as rheumatoid arthritis and asthma. recently, evidence was raised that pentoxifylline (pof} is able to suppress the synthesis of tumor necrosis factor-alpha [tnf} in cell cultures, in vivo, and to protect experimental animals against endotoxin shock: it was found that pof selectively inhibits the formation of tnf but not interleukin- (il- ]. we could confirm these pharmacological effects of pof in humans under controlled conditions of endotoxlnemia. ten healthy volunteers recieved a bolus injection of endotoxin [ ng of lps of s.a.e.], which caused a transient increase of circulating tnf and il- . weeks later the voluntecrs were again injected with endotoxin and pof was also infused. due to pof administration, there was no rise in circulating tnf, whereas il- levels rose in parauel with body temperature, comparable to those seen in the first part of the study. treatment of allograft transplant recipients with the murine anti-cd monoclonal antibody okt leads to an acute cytokine release syndrome. especially tnf seems to play a pivotal role in the pathophysiology of the okt first-dose reaction. pretreatment of patients with pof prior to okt administration was able to reduce the endogetlous tnf formation significantly as compared to controls and, thus, prevents severe clinical side effects, whereas il- formation and febrile response were not affected. severe pulmonary tuberculosis is associated with a chronic cytokine release syndrome [elevated levels of circulating tnf and il- ]. in patients pof treatment inhibited chronic tnf release selectively, and, thus, reduced tnf-dependent caehexia without affecting chronic il- formation and related symptoms, such as fever and night sweat. in conclusion, we suggest that pof may improve therapeutic strategies in cases of acute and chronic cytoklne release syndromes. tumor necrosis factor (tnf) plays a central role in the maintenance of the inflammatory events in rheumatoid arthritis. we evaluated the expression of p and p tnf receptors (tnfr) and of tnf-a and tnf-i~ on the surface of synovial fluid mononuclear cells in patients with rheumatoid arthritis (ra) (n = ), spondylarthropathy (spa) (n = ), and traumatic effusions (n= ). synovial t-lymphocytes from ra patients express in all cases the p tnfr on the cell-membrane, in / cases also a weak expression of the p tnfr is detectable, both mrnas can be detected by polymerase chain reaction (pcr). synovial macrophages also express the p tnfr and low amounts of the p tnfr. patients with active ra also have circulating p tnfr positive t-lymphocytes in their blood. high concentrations of soluble tnfr (stnfr) are found in the joint effusions of ra patients: up to ng/ml of p stnfr and ~jp to ng/ml p stnfr. significantly lower stnfr levels are found in spa effusions. both receptors are also more elevated in the serum of ra patients ( . _+ . ng/ml p stnfr and . + . ng/ml p stnfr) as compared to spa patients ( . _+ . ng/ml p stnfr and . + . ng/ml p stnfr, p < . ). tnf-a could be detected in the synovial fluid of ra patients (up to pg/ml), but not in the serum. the soluble tnfr are biologically active and neutralize the effects of tnf-a in a cytotoxicity assay. the high levels of soluble tnfr in the inflammatory effusions may reflect tnf neutralizing activity in an environment where enhanced tnf synthesis has occured. we have generated several anti-sense-tnf-a oligonucleotides (as-tnf), in order to down-regulate tnf biosynthesis at the mrna level. with as-tnf- we could achieve more than % inhibition of tnf secretion in pha-stimulated peripheral blood or synovial fluid lymphocytes. the effects of as-tnf- on the expression of tnfr and on the synthesis of other cytokines are currently being investigated. to investigate the effects of g-csf (r-methug-csf, aragon, thousand oaks, ca) on neutrophil production, blood distribution, survival and function, daily subcutaneous injections of g-csf were administered to healthy, young (y) ( - years) and healthy elderly (o) ( - years) subjects for days at three dose levels, mcg (n= ), mcg (n= ), and mcg (n = ). daily cbcs and serial measurements of neutrophil oxidative activity by chemiluminescence were made. in addition, blood neutrophil kinetics were determined (day ), transit time of the marrow neutrophil post mitotic pool (ntt) (days to ), neutrophil migration to skin chambers (days and ), and blood colony forming cells (cfu-gm) (day and ), as well as routine marrow morphology studies (days , and ) were performed. baseline neutrophil counts were similar in the y and o subjects and counts increased similarly for the two age groups, with peak counts of . --+- . for the mcg dose. g-csf significantly shortened the ntt from . - . days (control) to . - . days ( mcg) and . - . days ( mcg) (p< . ). a concomitant of the shortened ntt was a dose dependent increase in the chemiluminescence responses, reflecting higher myeloperoxidase activity. the shortened ntt was also reflected by a decreased proportion of marrow cells in the post mitotic pool (metas, bands and pmns) and apparently lengthened blood half life of the circulating pmns. neutrophil migration to cutaneous inflammatory sites was also decreased in a dose dependent fashion. comparison of the age groups showed the only significant difference to be in the mobilization of blood colony forming cells with blood cfu-gm increasing fold in the young versus fold in the elderly over days ( mcg). no significant toxicities were observed in these normal subjects. these studies demonstrate the dose dependent stimulation of neutrophil production with g-csf administration, which is not affected by the age of the subjects. the neutrophils released into the peripheral blood have enhanced oxidative responses but somewhat reduced migratory capacities, probably reflecting the accelerated production and early release of the developing neutrophils. these changes are remarkably similar to changes in neutrophil production and function observed with bacterial infections in hematological normal individuals. the systemic regulation of the host response during acute inflammatory states remains poorly understood. among the regulatory systems that are likely to play a role in controlling host responses to bacterial infection is the neuroendocrine axis. the pituitary for example, is ideally situated to integrate central and peripheral stimuli and among other effects initiates the systemic increase in glucocorticoid production that accompanies host stress responses. we studied the secretory response of the murine pituitary cell line att- in vitro and whole pituitaries in vivo after endotoxin (lps) stimulation. we identified macrophage migration inhibitory factor (mif), a previously described t-cell cytokine, as a major secreted protein of att- cells that were stimulated by sub-nanogram amounts of lps. to study the expression of pituitary-derived mif in vivo, balb/c mice were injected ip with sub-lethal amounts of lps. pituitaries and serum were collected at intervals and pituitary mif mrna and pituitary and serum mif protein were measured. pituitary mif mrna specifically increased with time and reached a plateau - hr after lps challenge. mif protein, which is present constitutively in the pituitary, decreased within hr. determination of serum mif in normal, athymic and hypophysectomized balb/c mice indicated that the pituitary is an important source of serum mif that appears in the post-acute phase ( - hr), whereas t cell mif contributes primarily to the hyper-acute rise in serum mif ( hr). these data suggest that mif plays a central role in the systemic response to endotoxin and that pituitary mif is likely to reflect the interplay of diverse neurohumoral stimuli that regulate acute and chronic inflammation. pretreatment of mice or rats with granulocyte colony-stimulating factor (g-csf) protected against endotoxin-induced lethal shock or against endotoxin-induced liver injury in galactosamine-sensitized animals. in the animals protected by such pretreatment, the systemic tumor necrosis factor r (tnf) bioactivity was significantly suppressed. various macrophage populations taken ex vivo from g-csf-pretreated animals showed an attenuated tnf release following lps stimulation. however, g-csf had no such effects on macrophages when directly added in vitro to the cells. these findings show that g-csf protects against septic shock via tnf suppression in a way requiring the participation of additional circulating cells or factors. pretreatment of rodents with granulocyte macrophage colony stimulating factor (gm-csf) greatly enhanced the susceptibility of the animals to endotoxin and led to a tremendous increase of the systemic tnf found following an lps challenge. an anti-gm-csf antibody significantly protected animals against septic organ failure. considerable amounts of endogenous gm-csf are released following endotoxin chaitenge with a maximum at h. the enhancement of lps-inducible tnf release from rnacrophages takes also place in vitro. it is concluded that gm-csf is a pivotal mediator of sepsis as well as a directly acting enhancer of lpsinducible tnf release. we have previously demonstrated that protein production and mrna expression of gm-csf, g-csf, and il- were decreased in activated mnc from umbilical cord (c) compared with adult (a) peripheral blood (cairo, et al, pediatr res : , , and : , ) . rhg-csf + recombinant rat stem cell factor (rrscf) administration in newborn rats has, however, resulted in a significant induction of neutrophilia, an increase in bone marrow post-mitotic pool, and is synergistic with antibiotics during experimental group b streptococcal sepsis (cairo, et al, blood : , , and : , . to assess the toxicity and efficacy of rhg-csf in newborns with presumed sepsis, nb < days ( - wks) with a diagnosis of presumed sepsis were randomized to either placebo (p), . , . , or . pg/kg q hrs, . or . /lg/kg q hrs of iv rhg-csf x days. cbc, differential, and platelet counts were obtained at time , , , , , and hrs. tibial bone marrow aspirates were performed at hrs and bone marrow nsp, npp, cfu-gm, and cfu-gemm were determined. serum for g-csf levels was obtained before and , , , , , , and hrs after rhg-csf dosing and measured by a sandwich elisa assay. rhg-csf induced significant neutrophilia at hrs vs. placebo ( + %) following both . ( + %, p< . ) and . ijg/kg/d ( -+ %, p< . ). significant neutrophilia continued at and hrs at both . (p< . and < . ) and h'g/kg/d (p< . s and < . ) respectively. rhg-csf also significantly induced a dosedependent increase in the bm post-mitotic pool (p vs. ijg/kg/d) ( +- . vs. + . %) (p< . ). t /z of g-csf in the nb was . _+ . hrs (r= - . ) with peak g-csf levels occurring within hrs. to date there has been no evidence of acute or chronic toxicity secondary to rhg-csf. future studies are required to determine the clinical implications of the biological efficacy of rhg-csf in newborns with presumed sepsis. children's hospital of orange county, orange, california usa depenbrock, t. block, h. vogelsang, ch. fellbaum, j. rastetter, a.-r. hanauske tgf-po is known to function both as inhibitory and stimulatory factor depending on the type of cell line investigated. the purpose of our study was to determine the effects of tgf-~ and tgf- (concentrations: . - - ng/ml) on soft agar colony formation of freshly explanted i~uman tumors in vitro. of specimens, had to be excluded from further analyses ( confirmed benign, bacterial/fungal contamination). of the remaining tumors showed evaluable growth in control capillaries ( %). at ng/ml, tgf-po inhibited colony formation (_< . x control) in specimens ( %): / renal, / non hodgkin's lymphoma, / breast, / ovarian, / melanoma (median: . x control, range: . - . ). at ng/ml, tgf-e,= showed inhibition of colony formation in specimens ( %) with a similar spectrum of activity (median: . x control, range . - . ). stimulation (colony formation _> . x control) was observed in only / specimens. combination of tgf-po with epidermal growth factor (egf) reversed the inhibitory activity in / specimens ( %). combination of tgf-p~ with platelet derived growth factor (pdgf) reversed the inhibitory activity in / specimens ( %). similar results were observed for tgf z. we conclude that tgf-i~ and tgf-i~ inhibit a subgroup of freshly explanted clonogenic tumor cells. their activity, however, appears to be modulated by other growth factors. a korfel. z yon marschall. d ol~erbera. e thiel. and we berdel mip-i~ is a member of a family of proinflammatory cytokines produced by activated macrophages which has been shown to be a negative regulator of early hematopoietic stem cell progenitors. our group investigates the interactions between hematopoietic cytokines and non-hematopoietic malignant cells. here, we describe results testing rhmip-lc~ (rh stem cell inhibitor, sci; kindly provided by dr. wolpe, genetics institute, cambridge, ma, usa) on the clonal growth of different human non-hematopoietic tumor cell lines in vitro. cell lines tested included the following histologies: gtioblastoma x, neuroblastoma lx, head and neck carcinoma x, lung carcinoma lx, colorectal carcinoma x, gastric carcinoma lx, pancreatic carcinoma l x, breast carcinoma l x, bladder carcinoma lx, prostate carcinoma lx, choriocarcinoma lx, ovary carcinoma lx, osteosarcoma lx, melanoma x. mip- ( , , , ng/ml) was tested in human tumor cloning assays (htca) in mixtures of methylcellulose and agar. htca has previously been shown to detect positive and negative growth control by cytokines. plating efficacy of the cells in the controls was > % (median %, range . - . %) in this series of experiments: tumor cells were continuously exposed to the cytokine for the complete assay period. clonal growth of none of the celt tines was significantly and reproducibly stimulated or inhibited by mip-lm since mip-lc~ may enter clinical trials for indications such as protecting hematopoietic stem cells from damage caused by cytotoxic chemotherapy in tumor patients, further experiments should be performed in vitro and in vivo. dfg be / - klinikum steglitz, freie universitaet berlin, berlin, germany clinical studies have demonstrated the activity of single hematopoietic growth factors (hgf) in restoring bone marrow function after chemotherapy. these studies have prompted clinical trials using multiple growth factors to promote the maturation of precursor cells at various stages of differentiation. however, hgf also have the capability to stimulate growth of non-hematopoietic tumor ceils at least in long-term cell cultures. we have assessed the growth-modulating activity of combinations of various hgfs on freshly explanted human tumor colony forming units in vitro. a total of tumor specimens were exposed for - days to il- , gm-csf, g-csf, scf (a~l at final concentrations of ng/ml) and il- (final concentration: ng/ml) or combinations of these hgfs using a capillary soft agar cloning system. specimens ( %) showed evaluable growth in control capillaries. stimulation of colony growth was observed in / tests ( . %) with / ( %) specimens expressing sensitivity to individual hgfs, / ( %) to combinations of two hgf, and / ( %) to combinations of more than two hgfs. inhibition of colony growth was observed in a total of / tests ( . %) with / ( %) specimens expressing marginal sensitivity to individual hgfs, / ( %) to combinations of two hgf, and / ( %) to combinations of more than two hgfs. for inhibitory effects, median colony survival for combinations > hgfs was . x control (range . - . ). for stimulatory effects, median colony survival for combinations > hgfs was . x control (range: . - . ). our data indicate that combinations of hgf will not substantially alter the pattern of clonal proliferation of the majority of freshly explanted tumor cells in vitro. however, growth modulation may occur in a minority of tumors. klinikum rechts der isar der technischen universit&t m nchen, ismaninger str. , m nchen supported by grants w / /ha- - from the deutsche krebshilfe and . . from the withe~m-sander stiftung is granulocyte colony-stimulating factor an angiogenic factor in human glioblastoma? s. corbacioglu, k. welte and t. pietsch granulocyte-colony stimulating factor (g-csf) belongs to a family of glycoproteins winch regulate growth, differentiation and function of hematopoietic ceils of the myelomonocytic lineage. in addition, g-csf induces migration and proliferation of endothelial cells in vitro. to investigate the effects of g-csf on vascularization of glioblastoma in vivo we transplanted the human glioblastoma cell line u- mg which is capable of producing g-csf in high amounts. after s.c. inocculation of these cells into the backs of athymic mice, they developped highly vascularized solid tumors. in order to block the effect of g-csf directly or to inhibit the production of g-csf by tumor cells neutrolizing monoclonal antibody (mab) a against g-csf or dexamethasone were injected intravenously. at -day intervals the tumor volumes were measured. after seven days the mice were sacrificed and the tumors were explanted. blood was collected for differential blood counts and serum was tested for g-csf. fresh frozen sections of the tumors were specifically stained for capillaries using bandeiraea (griffoina) simplicifolia lectin i isolectin b (bsl b ). morphometric studies of the stained sections were performed in order to quantitate the vascularization of the tumors. the differential blood counts showed significantly increased neutropinls due to the effect of human g-csf produced by the glioblastoma cells. tins effect was inhibited by anti-g-csf mabs or dexamethasone. however neither g-csf mabs nor dexamethasone could inhibit tumm growth compared untreated tumor bearing mice. dexamethasone significantly decreased the tumor vascularization whereas anfi-g-csf mabs did not have any effects on tumor vascularization. these findings suggest that g-csf is not an essential angiogenic factor in vivo. pediatric hematology/oncology, medical school hannover, konstanty-gutschow-str. , harmover , germany michael martin, torsten spencker, karen welch*, and andreas strasser* the spgm cell line was established from a transplantable mouse progranulocytic/promacrophage tumor (d hrsen u et al. , leukemia : - ) . extensive phenotyping of this mouse progenitor line revealed the properties of a typical cd positive pre-b cell, spgm being positive for pb , b (cd r), and the pre-b cell immunoglobulin receptor complex, comprising p-heavy chains, xs-and vpree-surrogate light chains and the igma and igl~ co-receptor molecules. southern blot analysis revealed clonal rearrangement of the p-heavy chain locus and germline light chain loci. interestingly, spgm readily formed blast cell, macrophage and occasional granulocytic colonies in soft agar in the presence of interleukin (il- ). in suspension cultures il- also induced macrophage differentiation, the cells becoming larger, adherent and independent of -mercaptoethanol in the culture medium. il- induced an initial burst of proliferation during differentiation, accompanied by loss of self renewal capacity, subsequently followed by a decrease and cessation of proliferation. the earliest changes were detectable at hours by northern blot analysis. il- treatment increased mac mrna, induced cfms mrna (m-csf receptor) and down regulated mrna for p, x , vpree, and mbl. after to days the cells morphologically, phenotypically and functionally resembled macrophages, expressing strongly mac and f / , and phagocytosing latex beads (martin met al. j.immunol. in press). thus, il- induced the cd positive pre- cell line spgm to switch its differentiation program in a coordinate fashion from a pre-b cell to a macrophage. igf is known to be mitogenic for a variety of cell lines in vitro. we have studied the effects of insulin-like-growth-factor i [igf-i] and insulin-like-growth-factor ii [igf-ii] on freshly explanted human tumors using a capillary soft agar cloning system. specimens had to be excluded from further analyses ( confirmed benign, bacterial/fungal contamination). / specimens ( %) showed adequate growth in controls ( renal, breast, lymphoma, colorectal, miscellaneous) with a median colony formation of . colonies/capillary (range: . - . ). at final concentrations between lr m and . m, igf-i significantly stimulated colony formation (_> . x control) in / evaluable specimens ( %) with a median of . x control (range: . - . ) and inhibited colony formation ( _< . x control) in / specimens ( %, median: . x control, range: . - . ). igf-ii stimulated / specimens ( %, median: . x control, range: . - . ) and inhibited / specimens ( %, median: . x control, range: . - . ). the optimal concentration for stimulation was found to be -~ m for igf-i and igf-ii. of specimens not significantly stimulated by either igf-i ( g m) or epidermal growth factor (egf, ~ m), ( %) were significantly stimulated by the combination of the two factors. ( %) specimen was stimulated by a combination of igf-ii ( -~ m) and egf. we conclude that igf modulates the clonogenic growth of a subgroup of freshly explanted human cancer cells in vitro. the myeloid growth factors g-csf and gm-csf are increasingly introduced in therapy trials in neutropenic disorders and in mds. in a series of therapy trials . % up to . % of patients treated with gm-csf displayed a stimulation of blast cells (antin , estey , ganser , vadhan-raj . recently, we could demonstrate a growth advantage for monosomy -cells in gm-csf containing bone marrow cultures (haase, ) which is supported by results from others (andreeff ). now, further data are available corroborating an association of monosomy and leukemic blast stimulation by myeloid growth factors. in a cytogenetic follow-up study of patients with mds under gm-csf (in preparation) we could observe a cytogenetic, clinical, and cytologic progression in a patient with monosomy within days. in a patient with kostman's syndrome, receiving g-csf, we performed sequential cytogenetic analyses. the patient's disease progressed to mds and finally to aml. he initially had a mosaic karyotypeof normal and monosomy cells but displayed a rapid karyotype evolution with supersession of normal cells and gain of additional abnormalities. a recent publication from a japanese group adds further information to a possible association of monosomy with stimulation of leukemia in % of neutropenic children treated with g-csf (kojima, ) . besides the need for further cytogenetic follow-up studies in growth factor therapy trials, data are accumulating that monosomy is a risk factor in gm-csf and g-csf therapy. ii- is a cytokine with multilineage activity and stimulates proliferation of immature hemopoietic progenitors (yang, ) . recently, il- has been introduced in clinical trials in mds (dunbar, ; ganser, ) . as in gm-csf therapy one major concern attributes to an il- mediated stimulation of leukemic cells, since a rise in blast counts has been observed in two of patients reported (ganser, ) . however, as yet it is not known whether the blasts stimulated belong to the normal or leukemic population. we performed cytogenetic analyses of bone marrow cultures with and without addition of i ng/ml il- (behringwerke, marburg). the influence of this cytokine on the clonal compostion in patients ( anll, mds) with mosaic karyotypes was examined. in all patients independent from diagnosis and chromosome abnormality the normal cell population was promoted by il- with different intensity. individual data are outlined on the the effect of stem cell factor (scf) on peripheral blood b-cll-celis were studied in vitro by bromodeoxyuridine/propidiumiodide (brdu/pi) cell-cycle-analysis. peripheral blood cells from patients with b-cll were examined with cd-markcrs and prepared as follows: after ficoll centrifugation and lysis of monocytes by leucine-methyl-ester (lme) t-lymphocytes were depleted with a cd monoclonal antibody and magnetic cell sorting. the cells were grown in serum free culture medium (cg-medium) containing #mol/l brdu, and . ng/ml up to ng/ml scf. controls were grown without cytokines. samples were drawn repeatedly at , , , , and hours. cell-cycle-analysis was performed after double dna staining with propidiumiodide and anti-brdu-antibodies and determinated by flow cytometry. minimal alterations were observed with t-cell depletion, the b-cll cells from patients were stimulated by scf during the first hours reaching a maximum of - . % compared with the controls after hours in cultures containing ng/ml scf. on the other hand, cultures without t-cell depletion showed no effect for scf. we conclude that scf has only a minimal stimulatory, early effect in inducing the proliferation of b-cll cells. stem cell factor (scf) is known to promote proliferation of hematopoieticprogenitors and mast cells.however, the spectrum ot its biological effects is not completely understood. since scf may be able to accelerate hemopoietic recovery after chemotherapy or in other situations where severe immunosuppression is present, we were interested in its effects on t cells. thus, we have studied the influence of human recombinant scf on t cell proliferation invitro. when cultured for days in serum-f~ee medium, h-thymidine incorporation of unstimulated peripheral blood mononuclear ee ells s (pmnc) resulted in -+ epm without growth factors, +_ clam with .- ( ng/ml), +_ cpm with scf (lon~/ml), and _+ c.m with both . and scf (n=fi~ addition of scf to one way" mixed lymphocyte cultures (mlc) increased thymidine incorporation by % ( - %); addition of scf plus id increased thymidine incorporation by % ( - %; id alone %, n= ). after depletion of monocytes and the majority of b cells from pmnc, the proliferation of the remaining eeu fraction, which consisted mainly of t cells, was not enhanced by id , scf or il- + scf. we conclude that scf can promote proliferation of unstimtdated or allostimulated t cells in the presence of id . since this effect requires monoeytes or other accessory ceils, a direct influence of scf on t cells does not become evident from our data. it is, however, still unclear whether ltb acts in this regard directly or indirectly by stimulating the release of chemotactic and inflammatory cytokines. here we report that ltb induces synthesis of interleukin (il)- by human blood monocytee through transcriptional activation of the il- gene. we furthermore demonstrate that this process involves activation of the transcription factor nf-rb and, to a lesser extent, of nf-il , white the activity of the transcriptior~ factor ap- , shown to otherwise confer il- inducibility, appeared to be unaffected by ltb . involvement of nf-~:b and nf-il in induction of il- transcripts by monocytes was demonstrated using deleted forms of the il- promoter. activation of the il- promoter by ltb was not only associated with accumulation of the respective transcripts but resulted in synthesis of functional il- protein as well. in addition, ltb mediated transcactivation of a heterologous promoter construct containing the nf-~:b or the nf-il enhancer, but not the ap- enhancer. the signalling events mediating this effect appeared to involve the release of h , since ltb failed to induce nf-rb or nf-il in the presence of the scavenger of h , n-acetyi-l-cysteine. both interleukin- (il- ) and granulocyte-macrophage colony-stimulating factor (gm-csf) have been previously identified to induce rapid phosphorylation of the map-kinase (blood : (blood : , . however, little is known about signalling events initiated by il- /gm-csf which occur downstream of the map-kinase. map-kinase has been shown to phosphorylate the ap- transcription factor and to activate two kinases designated insulin-stimulated protein kinase- (ispk- ) and mapkapkinase . we here report that il- and gm-csf induce mapkap-kinase activity in the human megakaryoblastic leukemia cell line mo e and phos-phorylate the human small heat shock protein hsp on serine residues. in contrast, neutrophils failed to phosphorylate hsp upon il- , while gm-csf induced hsp phosphorylation in a similar range as observed in mo e cells suggesting that mapkap-kinase mediated hsp activation occurs independently of proliferation. hsp phosphorylation is dose-dependent and occurs as early as minutes following exposure to il- or gm-csf. moreover, hsp phosphorylation is inhibited by tyrosine kinase inhibitors such as genistein or herbimycin a. in addition, we show that protein tyrosine phosphatase and protein phosphatase a (ppa ) interfere with the ability of il- or gm-csf to induce serine phosphorylation of hsp . taken together, our findings indicate that tyrosine phosphorylation of map-kinase is a prerequisite for serine phosphorylation of hsp mediated by mapkap-kinase . hsp is localized in the nucleus and has been linked to the cellular stress response. its precise function, however, is largely unknown. our data identify hsp as a target of il- /gm-csf stimulation via map-kinase and mapkap-kinase . further-more, our results indicate that hsp may also exert phosphorylation-activation functions involved in growth signalling pathways in unstressed cells. in recent years it has been shown that the mechanism of betalactam antibiotic-induced agranulocytosis involves a direct inhibition of the replicative dna polymerases alpha, delta, and epsilon. in this report we examine, as a representative of these antibiotics, the effect of freshly prepared ceftazidime (cef) and degradation products of cef on myelopoiesis. we investigated freshly dissolved cef and cef incubated for hours at + (cefd) on the production of myeloid cells in the supernatant (sn cells) and colony stimulating activity (csa) by the stroma. one week after drug treatment of the mltbmc a significan~ dosedependent inhibition of the myeloid cell production (xl ~ and csa (assessed by cfu-gm assay) occured, as summarized in the following we here report that human lung fibroblasts respond to x-ray treatment (xrt) with release of interleukin (il)- . synthesis of il- upon ionizing radiation is preceded by an increase of il- transcript levels resulting from transcriptional activation of the il- gene. analysis of deleted fragments of the il- promoter indicated that transcriptional activation of the il- promoter was due to enhanced binding activity of the transcription factor nuclear factor (nf)-~b. although activation protein (ap)-i did not participate in the rapid induction of the il- promoter, its binding activity was also enhanced by xrt. in contrast to binding kinetics observed with nf-~:b, ap- binding following xrt was biphasic with the second peak being dependent on de novo protein synthesis. in contrast, however, nf-il- activity was not enhanced by xrt in fibroblasts. the introduction of both the nf-~b-and the ap- recognition sequence, conferad inducibility by xrt to a heterolgous promoter, with reporter gene activity being maximal hours or hours following xrt, respectively. sequential acitvation of two distinct transcription factors might thus contribute to synchronize transcriptional activation of different genes participating in the x-ray (xr) response. on the basis of study of functional and morphological characteristic of bone marrow stromal tissue of human fetuses, - week gestation, and of adults aged - years, and in experiments on animals, the role of the sinusoidal endothelium, reticular, fat and endosteal cells in hemopoiesis regulation has been concretely defined. the endothelium of sinuses forms the histo-hematic barrier "bone marrow-blood', ensures the wanscellular migration of stem cells and mature blood cells, releases hemopoiesis-regulating factors and is involved in the erythroid cell maturation. bone marrow reticular cells participate in the formation of intramedallar supporting frame-work, regulate the transvasal and intramedullar cell migration, form the extracellular matrix, produce humoral regulators of hemopoiesis, effect the cell differentiation by way ot their intercellular contacts with hemopoietic precursors and give origin to adipocytes, lntramedullar adipocytes present an energetic depot of hemopoietic and stromal tissues and in the stage of preadipocytes they effect, by means of contacts, the granuloeyte development. the endosteal cells of bone marrow are the source of intramedullar stromal tissue, they participate in the anchorage of the hemopoietic stem cells and form the microenvironment of the latter, regulate the endosteal ca ion levels and might be a possible source of hemopoietic tissue (population of cells of the residual embryonal mesenchyma). the thesis on the mechanism causative of the impaired regulation of precursor proliferation and differentiation in hematologic diseases is proposed. the self-renewal capacity of cfu-s (spleen colony-forming unit) following the treatment of (cbaxc b )f female mice with recombinant human granutocyte colony-sffmulating factor (rhg-csf) was investigated. the possibility of synergism between erythropoietin (epo) and rhg-csf in blood cfu-s mobilization was also studied. during the investigation the following observations were made: and mkg/kg/day injection of rhg-csf expended the number of cfu-s- in circulation -and -fold, accordingly. the self-maintenance potential of peripheral blood cfu-s-i did not change significantly. the treatment of mice with mkg/kg/day of rhg=csf resulted in two fold increase of spleen cellularity and fold augmentation of cfu-s- number, without noticeable changes in their self-renewal capacity. moderate changes in cfu-s- number were observed after epo administration in spleen and peripheral blood, however no significant synergistic effect of epo with both doses of rhg-csf was detected. the multifold increase of cfu-s- number in peripheral blood following rhg-csf administration with no reduction in their self-maintenance potential suggests that mobilized with rhg-csf blood stem cells provide an appropriate source for reconstitution of the hematopoietic system. we used the brdu-incorporation method to show the effects of l- ( , , u/ml),il- ( . , , ng/ml) and il- ( u/nil) plus il- ( ng/ml) on b-cll-ceiis. after ficollseparation, lysis of the erythrocytes (nh ci) and lysis of monocytes ( - eucin-methyl-ester), cells were divided into two groups. group was cultured in a serum free medium (+brdu +cytokine) without any t-cell depletion. group was cultured in a serum free medium (+brdu +cytokine) after t-cell (cd +) elimination by the macs (magnetic activated cell sorting). samples were taken after h, h, h, l h and h. after staining with anti-brdu fitc and propidiumiodide (pi) proliferation was measured by flow cytomemy (facs scan). . p=proliferation n=no effect i=inhibition conclusions: i[,- shows a proliferative effect on b-cll-cells independent of t-cells. il- shows heterogeneous effects. by itself it has most often no effect on proliferation, but sometimes it inhibits or increases the prolifoation. this effect does not seem to depend on t-cells. it could depend on the dosage or some unknown patients' characteristics. further on il- inhibits ,- induced proliferation in nearly all cases independent of t-cells. ii-i and tnf are inflammatory cytokines with overlapping biological activities. human vascular endothelial cells express ii-i and tnf receptors and respond to ii-i and tnf stimulation by elaboration of colony stimulating factors such as g-csf and gm-csf.however quantitative data are required in order to evaluate the contribution of ii-i and tnf to the activation of inflammatory hemopoietic cells such as granulocytes and macrophages by csfs. therefore we quantified the production of g-csf and gm-csf in endothelial cell cultures subsequent to treatment with il-l~ ( .l- u/ml) and tnf( - u/ml)or ll- in combination with tnf.a dosedependent stimulation of g-csf and gm-csf secretion was found following ll-i and tnf treatment of endothelial cells.ll-i was a more potent inducer of g-csf secretion than was tnf using approximately equipotent doses of il-i ( u/ml) and tnf ( u/ml) regarding the induction of gm-csf.in addition significant snperadditive stimulation of g-csf and gm-csf production was found with a low dose of il- ( iu/ml) and a saturating dose of tnf( oou/ml) in combination.however the costimulatory effect of ii-l(iu/ml) was significantly more pronounced with g-csf than with respect to gm-csf.in summary the differential modulation of g-csf and gm-csf production ~n endothelial cells by ii- and tnf may indicate 'a disparate role of ii-i and tnf in vascular inflammatory processes and atherosclerosis regarding recruitment and activation of inflammatory leukocytes. cytokines are known to be involved in the pathophysiology of graft versus host disease (ghvd) and to effect lymphohematopoetic progenitor cell growth after bone marrow transplantation. the use of t-cell depleted marrow in human transplantation is associated with suppression of gvhd but decreased rates of engraftment. we have shown in rodent models that uvb irradiation (uvb) of donor inoculum inhibits gvhd while preserving engraftment. to determine the effects of uvb on eytokine production by cells associated with gvhd, human marrow mononuclear cells isolated by ficoll density gradient were uvb-irradiated at doses of - j/m and then stimulated with pha/lps or allogeneic cells. elisa assays were used to measure the production of gm-csf, il- , lif, il-lbeta, il- , il- , tnf-alpha, and lfn-gamma by stimulated cells . two week methylcellulose cultures were used to determine viability of cfu-gm, bfu-e and cfu-gemm progenitor cells after uvb. all results were compared to non-uvb-irradiated marrow and to marrow depleted by soybean agglutination and sheep erythrocyte rosetting. progressively increasing doses of uvb produced progressively decreasing levels of all cytokines except il- , which remained unchanged. t-cell depleted marrow produced decreased levels of all cytokines except il- . uvb at j/m resulted in higher levels of gm-csf and il- as compared to t-cell depleted marrow. this same dose of uvb essentially preserved cfu-gm, bfu-e, and cfu-gemm colonies. we conclude that uvb may inhibit the cytokine cascade active in gvhd while preserving progenitor cell growth at uvb jim . uvb may serve as gvhd prophylaxis in clinical marrow transplantation and in vivo studies on non-human primates are in preparation. the ability to migrate is fundamental for the acquisition of invasive properties by tumor cells. a tumor derived cytokine was identified by its ability to induce direct and random migration via a receptor mediating signal pathway, i.e. autocrine motility factor (amf). we identified the receptor for amf (hamf-r) and found ~at hamf-r plays a role in invasiveness and metastasis in human bladder carcinomas. we investigated the expression pattern of hamf-r in fresh frozen bladder cancer specimens by immunofluorescence technique on the translation level and found a strong correlation (p< . ) to tumor stage and grade. furthermore we have shown that patients who were found to be hamf-r positive have an increased risk for early tumor progression (p< . ). a large number of substances from the working place and in the general environment such as quartz and coal mine dusts are causing silicosis and leading to lung fibrosis. alveolar macrophages are the primary target for the noxious effect of quartz dust. quartz dust incubated human macrophages release in vitro a cytokine, which stimulates cell proliferation of human lung fibroblasts (wistar ). in recent studies we found that beside fibroblasts also epithelial cells of the alveolar unit, such as pneumoeyte type ii cells (a- ) respond to the cytokine with strong proliferation. supernatants of quartz dust exposed macrophages were concentrated by ultrafiltration and thereafter fractionated by gelfiltration (sephadex g , pharmacia). biological activity of the factor eluted within a molecular range of - kd. furthermore, the factor was purified by anion exchange chromatography (q-sepharose). fractions revealing biological activity were further analysed by sds-gel eleetrophoresis (page) and showed two protein bands with apparently molecular masses of and kd,respectively. after addition of the supernatant initially spindle shaped pneumocytes were detected, followed by epithelial-like cells when cell proliferation progressed. induction of spindle-shaped pneumocyte type ii cells could also be seen after addition of pure tumor necrosis factora. however, in this case no cell proliferation was observed. we assume that beside the cytokine, which is responsible for the induction of cell proliferation tnf~ is present in supernatant. differences in a variety of immunologic parameters. in peripheral blood however differences to healthy persons have hardly been described. in this investigation we compared serum levels and concentrations of interferon- (fin-y) and il-l--ct in stimulated samples of whole blood. ixl whole blood of healthy controls (ref) and patients with sareoidosis, without treatment (pot) and under corticoid medication (put), was incubated in medium at ~ and % co . con a was used for stimulation of ifn-y and lps for il- --m concentrations were determined with an elisa. results: without stimulation no measatalg, e amounts of ifn--y could be found. after stimulation ref showed median coneentrations of ng/ml, pob ng/ml and put ng/ml. the difference between ref and put respectively pot was statistically significant. il-l-m without lps the differences were not significant. under stimulation pot had si~ificantly higher values ( pg/ml) compared to ref ( pg/ml) and also to put ( pg/ml). in conclusion we were able to demonstrate that in conlrast to serum levels stimulation of peripheral whole blood reveals significant differences in concentrations of ifn--y and il- --ct between patients with sarcoidosis and healthy references. we established a modified polymerase chain reaction protocol for the detection and semiquantitative assessment of mrna-transcdpt levels for il- , il- , il- , il- , ifn- , tnf-cl, gm-csf, tgf- and il- -receptor-c{ (il- r). the method was shown to distinguish -fold differences in template concentration after rounds each of amplification in the range from to cycles; the lower threshold of sensitivity was at copies per pcr-reaction. reproducibility was > % for a positive result after rounds of amplification; it decreased to % after rounds. this corresponded to the detection of , and template copies, respectively, per pcr-reaction. human peripheral blood mononuclear cells (pbmc) and tumor cell lines were evaluated for mrna-expression with or without stimulation and these results were compared to secretion of the corresponding cytokine. for pbmc, constitutive mrna-expression was found positive for tnf-o~, ifn- , il- , il- , tgf- and il- r, whereas detectable expression of il- , il- and gm-csf was induced only after stimulation. using ~-actin as an intemal standard, the pcr could demonstrate relative differences in cytokine transcripts after stimulation with (a) ]u/ml il- , (b) % lymphocyte-culture conditioned media (ccm) and (c) pma ( ng/ml) plus a ( ng/ml). for il- transcripts no detectable expression was found without stimulus or after addition of il- , whereas ccm resulted in a , -and pmna in a , -fold increase. other mrnatranscripts increased t -fold (tnf-(~) up to , -fold (gm-csf) with or without differences between the stimulating agents. the cell lines caki- (renal cell carcinoma) and daudi (burkitt lymphoma) also expressed comparable levels for cytokine transcripts, with a strong induction after stimulation with pmna . the relative ifn~ mrnacontent in caki- increased from to , , for gm-csf from to , . the influence of different cytostatics on il- production by peripheral blood mononuclear cells (pbmc) was studied. pbmc were pre-incubated with or without phytohemagglutinin p (pha-p), then pulse exposed during hr to different cytostatics in their therapeutical concentrations, washed three times and incubated additionally during - hrs. then the supernatants were collected and il- biological activity was measured in mttmodified b -cells biological assay. though significant individual variations of the il- production were found, all the studied drugs can be separated on severa) groups: ) adriamycine and methotrexate induced late increase of il- production ( - hrs ); ) cytarabine strongly increased the early as well as the late il- production ; ) the pretreatment with etoposide and rubomycine suppressed subsequent production of il- by pbmc during - hrs, the late production was increased; ) in contrast, the cyclophosphamide pretreatment stimulated the early production and strongly suppressed the late one. the changes of il- production by pbmc was not due to the cellular death. the pha-p stimulated pbmc produced usually more il- that unstimulated cells did. these data suggest that the cytostatics possess the different effects on il- production by pbmc that could be important in the therapy of malignancies. recent studies implicating a deficiency of interleukins in several diseases have underlined the importance of measuring in vitro the dna synthesis and the cytokine production (il- , il- , il- , tnfalpha) in the same cell system. previously had found that normal peripheral blood mononuclear cells (mnc) of patients suffering from high-malignant non-hodgkin lymphomas showed a decreased capability to proliferate after mitogenic stimulation (pha, con a, pwm). here we have studied the dna synthesis and the production of different cytokines (il- , il- , il- , tgf-i~ and tnf-alpha) by pokeweed mitogen (pwm) stimulated mnc from healthy control subjects and from patients with nhl. the il- production of pwm-stimulated mnc of patients with nhl was found to be significantly decreased, wheras the il- , il- and tnf-alpha release were not changed significantly. these data showed a good correlation with the reduced capability of mnc from patients with nhl to proliferate after mitogenic stimulation. the multifunctional cytokine transforming growth factor-ii (tgf-i ) is known to inhibit the dna synthesis, as well as the il- production of mitogenstimulated mnc. however, tgf-i~ release was not significantly changed in call culture supernatants from patients with nhl in comparison to healthy controls. we conclude that the suppressed dna synthesis and _- production of mnc from patients with nhl is not the consequence of a deceased tgf- level secreted by these cells. is a co~on problem after chemotherapy and requires supportive care until normal hemopoiesis has recovered. to study the importance of endogenously produced cytokines for regeneration of bone marrow progenitors we measured serum levels of g-csf and ii- . blood samples were obtained before and hours after chemotherapy, during the stage of leukopenia (< /ul) and recovery (> /ul)of leucocyte counts. in / patients we found a more than -fold increase in serum g-csf levels at the stage of leukopenia. highest amounts were observed in two patients with lethal outcome. there was no correlation between thrombocytopenia and levels of g-csf or il- . serum g-csf (pg/ml, mean, range) before (leuko> ) after (< /ul) chemotherapy aml dav ( -i ,n= ) ( - ,n= ) all 'h~izer" ( - ,n= ) ( we measured different hematopoietic cytokines as g-csf, gm-csf and il- in amniotic fluid and cord blood to ctearify their physiological and palhophysiological role in fetal and neonatal development amniotic fluid was available from the th to the st week of gestation (n= ), cord blood from the th to the nd week of gestation (n- ). activity levels of cytokines were determined by stimulation of the ceii lines nfs- , d , and tf-i. which are responding specifically to g-csf, il- , and gm-csf. specificity has been proved by neutralizing antibodies. calculation of cytokine levels was done by standards of recombinant growth factors. sensivity for g-csf and il- : pg/ml for gm-csf: oo pg/ml. in amniotic fluid g-csf tanged from to i .ooo pg/m[ and il- from to . pg/ml, whereas gm-csf was not detectable. in cord blood g-csf was between and . pg/ml and [l- between and pg/mh in most of the samples (qo%~ gm-csf was beyond the sensivity limit. in % ( of cases) g-csf levels were elevated over pg/ml and associated with amnion infection syndrome, while green amnioflc fluid alone during delivery did not stimulate the production of g-csf, the levels of the hematopoietic cytokines showed no influence by the gastational age. identical twins without maternal infection showed the same g-csf levels. inflammation of the amniotic membrane and maternal sepsis is associated with elevated g-csf levels in cord blood. gm-csf can normally not be detected in cord btcod and amniotic fluid. detectable levels of gm-csf in cord blood and amniotio fluid maybe give a hint for a pathological situation during pregnancy. total number of children with all, boys and girls, aged from to years were included to the study. tnf production was studied acc. the method based on growth inhibition of sensitive to tnf l mice fibroblasts, ill- production ace. method based on inhibition of autologous rosette formation by thymoeyteg of cba mouse and l- production ace. to conventional el sa genzyme-test. twenty five healthy children served as the control group. it was found that in children with all during the whole period of therapy the il- and il- production, was significantly lower than that observed in the control group of healthy children (p . ). the tnf production in all children before therapy was lower in comparison with the control group values. during cytostafic therapy was higher and grew up above the normal limits after cessation of the therapy. il- production grew up after the end of the therapy but never reached the value of the control group. efs at ruth in all children with il- production < /~ before therapy was higher than that in children with il- < /~ (efs % v %). the il- production seems to be a good prognostic marker. (pts) with active autoimmune disorders as well as with malignant lymphomas. in addition, cd and its soluble form (scd ) is thought to be involved in the regulation of b-cell proliferation. therefore, we examined scd , scd , and scd in pts with b-cell chronic lymphocytic leukemia (b-cll) in order to assess their role as indicators of disease activity. fifty-five pts with b-cll were studied so far. staging was performed according to the classification systems of rat and binet, respectively. serum samples were freshly stored in liquid nitrogen until further processing. levels of scd , scd , and scd were measured by a sandwich elisa technique using commercially available assays (biermann, germany). advancing rat stages were associated with a progressive increase of all the three serum factors (scds: rat + u/mt vs rat iv + u/ml, scd : rat - - u/ml vs rat iv __. u/ml; scd : rat + u/ml vs rat iv +_ u/ml). this progression was also evident when binet's classification was applied. occurrence of b-symptoms was associated with high levels of scd (p< . ), whereas scd and scd were found also to be increased but without statistical significance. high levels of all the three factors strongly correlated with a lymphocyte doubling time < months, a lymphocyte count > . /tzl, and with the presence of hepato-and splenomegaly. interestingly, occurrence of bulky lymph nodes (i.e. at least one nodule of > cm in diameter) was linked with high levels of scd only (p< . ). in summary, ( ) progressive serum levels of scd , scd , and scd correlate with advancing stages of disease in b-cll. ( ) b-symptoms were associated with high levels of scd . ( ) we found scd to be the more sensitive marker of the total tumor load than scd and scd . thus, scd may be useful in monitoring pts with b-cll. cytosine arabinoside (ara-c) is one of the most active single agenls in the treatment of acute myeloid leukemia (aml). its cytotoxic activity mainly depends on its phosphorylation to ara-ctp and on its incorporation into the dna. based on recent in vitro studies showing that hematopoietic growth factors like gm-csf and il- enhance the cytotoxicity of ara-c on clonogenic leukemic cells, the gm-csf priming concept is currently explored in clinical phase ii and iii studies. in an ongoing study at the universities of muenster and goettingen gm-csf ( /~g/m /d) is started hrs before induction chemotherapy (tad /ham) until recovery of blood ceil counts. this study provided a means to assess the effect of gm-csf on the intraceunlar ara-c metabolism in vivo in pts with aml. enzyme activities of deoxycytidine kinase (dck), thymidine kinase (tk), dcoxycytidine deaminase (dcd), dna polymerase (pol) and dna polymerase alpha (pola) were determined before therapy, hrs after the administration of gm-csf and hrs after the administration of ara-c. in addition, ara-c incorporation into the dna was measured after hrs ara-c administration. enhancement of enzyme activity was observed in / , / , / , / and / cases for pola, pol, tk, dck and dcd, respectively. increases ranged from - % for pola (median %), - % for pol (median %), - % for tk (median %), - % for dck (median %) and - % ( %) for dcd. inadequate blast cell reduction after tad (> % blast cells on day or ) was associated with significantly higher dcd blast cell activities compared to the dcd activity values obtained for pts with adequate blast cell clearance (median values: . vs . nmol/min x mg, p< . ). cases with dcd activities < nmol/min x mg showed significantly higher ara-c incorporation into the dna compared to pts with dcd activities > nmol/min x mg ( . vs . ng/ cells). furthermore, inadequate blast cell clearance was associated with lower ara-c incorporation into the dna (median . vs . ng/ cells) and lower pola activities (median . vs . pmol/min x mg). in pts we investigated simultaneously the effect of gm-csf pretreatment on ara-c metabolism in vitro. enzyme activities of pola, tk and pol correlated significantly in vivo and in vitro (rp~ rtk=o. , rp~ p< . ). these data demonstrate that gm-csf enl/ances dna synthesizing enzyme activities in vivo and in vitro. furthermore, these data suggest that gm-csf might improve the therapeutic response to induction chemotherapy by increasing dna polymerase alpha activity and thereby increasing the ara-c incorporation into the dna. the effects of interferon-alpha (ifn-alpha), interferon-beta / lnterleukin- (il- ) and interferon-gamma (ifn-gamma) in inducing megakaryocytic differentiation of blast cells from acute megakaryoblastic leukaemia (amegl) patient determined by the increase in cd and cim b expressions using monoelonal antibodies in apaap technique were investigated in liquid suspension culture. after six days of culture, the percentage of cd and cd b positive cells increased in control cultures from , % and , % on day to , i , % and , • , %, respectively. the addition of ifn-alpha significantly increased the number of cimi and cim b positive cells by about two to three fold compared to control cultures, p < , and by about four to six fold compared to day , p < , . similarly, ifn-beta /il- induced a significant increase in cd and dc b positive cells. on the other hand, ifn-gamma failed to increase the number of cim and cd b positive cells in comparison to control cultures on day and instead induced a significant increase in the number of monocytes/macrophages from only , _+ , % in control cultures to , + , %, , + , %, , • , % and , • , % in , , and units/nil ifn-gamma-treated cultures, respectively, p < , . the present results suggest that megakaryocytic differentiation of blast cells in amegl could be induced by ifn-alpha and il- and support a clinical role for il- in the treatment of amegl patients. also, the present results showed that monocytic differentiation of blast cells in amegl could be induced by ifn-gamma~ supporting the multipotent stem or progenitor cell origin of the amegl subtype of acute myeloid leukaemia. a monoclonal antibody-based elisa and bioassay were used to measure leukemia inhibitory factor (lif) protein levels, activity and the functional role of lif in superuatants of cultured stromal cells derived from patients with acute and chronic myelogenous leukemia, myelodysplastic syndrome, and hairy cell leukemia and from normal controls. we demonstrate that biologically active lif protein is constitutively produced and secreted by coltured bone marrow stromal cells from all subjects studied. in addition, adherent-layer conditioned-media lif protein levels were significantly elevated in samples from patients with all hematologic malignancies studied as compared to samples from normal controls. confluent adherent layers exposed for hours to interleukin (il) or tumor necrosis factor- (tnf-a) showed a significant increase in lif protein levels, whereas exposure to il- (sterling drug inc., great valley, pa) resulted in a dose-dependent decrease in lif levels. . (i. - . ) . ( . - . ) . interestingly, neutralizing antibody against lif caused a % reduction in normal progenitor proliferation derived from the superuatant but not from the adherent layers, and this effect was reversible by the addition of recombinant lif protein. we conclude that (i) biologically active lif protein is constitutively produced by adherent layers from normal donors, (ii) tnf-ct and il- increase and il- decreases adherent layers lif protein levels, (iii) the steady state levels of lif protein produced by adherent layers from leukemic patients is significantly elevated, and (iv) lif may participate in the interaction between adherent layers and hematopoietic progenitors to maintain normal hematopoietic colony growth. it is well known that bone marrow stromal cells have crucial impact on haemopoietic cell proliferation. little is known about stromal humoral factors leukemic cell proliferation. the aim of this study is to evaluate the effect of stromal cell conditioned media (sccm) on the h-thymidine uptake by normal and leukemic target cells. patients with aml were studied treated with " + " based regimens. long term bone marrow cultures were established in non-leukemic and leukemic patients (before and during treatment). target cells for sccm were normal haemopoietic cells and leukemic blasts. the results are the comparison of the effects of leukemic and non-leukemic stromal cells. a part of the patients revealed high stimulative activity upon non-leukemic cells (+ + %, p< . ) and inhibited proliferation of leukemic cells (- • %, p< . ) this group entered complete remission. sccm of another group of patients inhibited proliferation of nonleukemic cells (- _+ %, p< . ) and stimulated blast cell proliferation (+ + %, p< . ). the magnitude of the figures was even more profound later: during treatment, bone marrow aplasia, recovery. this group of patients failed to achieve remission. it seems that stromal cells has significant on impact on restoration of normal or leukemic hemopoiesis after chemotherapy. peripheral b-cll-cells from patients were investigated upon the proliferating effect of ifn~, tnf~ or combination of both in serum free culture medium (cg-medium). blood cells were drawn from patients and lymphocytes separated by ficoll hypaque and monocyte-lysis (leucinemethyl-ester incubation). t-cells were depleted using a, cd mab and macs (magnetic activated cell separat r, miitenyi biotec). at each step heterogeniety of the population was controlled by facs analysis with different mab (cd , , , o, , , , ) . the homogeneous population (contamination less than %) was co-incubated with both cytokines ( . - ng/ml) and bromodeoxyuridine (brdu) in cgmedium. after , , , and t hours cells were harvested an analysed for brdu-incorporation into the genome. ifnu and tnfc~ measurings ( in total) were almost similar: patients were non-responder and showed no stimulatory effect on cells; patients showed an inhibitory effect; cells from patients were responding upon cytokine cultivation. the combination of both ifn~ and tnf~ produced in these cells an additive effect ( out of ). best results could be observed when the control population (without cytokine) was minimal proliferating compared to no proliferation. a high correlation was observed between cytokine response and pre-treatment: without glucocorticoid treatment of patients prior to measurements the influence of cytokines on resting b-cll-cells was significantly higher (with methyl-prednisolone %, without we-treatment % were responders). functional defects of the monocyte/macrophage system probably contribute to the increased rate of severe infections in patients with myelodysplastic syndromes (mds). therapeutic trials with hematopoietic growth factors (hpgf) have resulted in substantial improvement of cytopenia, especially neutropenia. however, little is known about the alterations of the monocyte/macrophage system during these therapeutic interventions. it therefore was the aim of the present study to analyze the capacity of monocytes/macrophages from mds patients prior to and after hpgf therapy to secrete il- ~, tnfe, il- , and il- upon in vitro stimulation with lipopolysacharide (lps). sixteen patients were studied: t had a refractory anemia, had a refractory anemia with excess of blast cells. prior to therapy, the capacity of adherent monocytes/macrophages to secrete il- ~, tnfc~, il- , and il was significantly reduced by - percent as compared to normal controls. on the other hand, oxygen radical release was normal in mds patients tested. treatment with gm-csf ( - /~g/m /d sq x - ; n= ), il- ( - /~g/m /d sq x - ; n= ), and g-csf ( - fg/kg/d sq x in combination with all-trans retinoic acid; n= ) normalized the potenital of monocytes/macrophages to secrete il-ti~, tnf~, and il- . il- secretion was only improved by il- or gm-csf dosages _> /~g/mz. oxygen radical release was significantly stimulated by both gm-csf and il- . these results indicate that treatment of mds patients with gm-csf, il- , and g-csf (the latter in combination with all-trans retinoic acid) can restore deficient monocyte/macrophage secretory function to normal. depletion of cd positive t ceils has been used in human patients for prevention of gvhd. we studied depletion of cd + cells from canine marrow for induction of gvh-tolerance across a complete dla-haplotype difference. prompt engraftment and fatal gvhd occurs in a littermate combination of dla-homozygous donors and dla-heterozygous recipients when undepleted marrow is given. aiiogeneic marrow depleted with a crossreactive antibody to human cd and immunomagnetic beads was given to dogs. one dog died with haemorrhage on day due to thrombocytopenia, dogs showed complete hematopoietic recovery. dogs became tolerant chimeras and one dog died with gvhd due incomplete depletion. chimerism was mixed early after transplantation, became complete later and is still complete in / dogs after - years. dogs received cd depleted marrow grafts and loijg/kg/d s.c. r-canine g-csf starting on day after transplantation. although all dogs had fast recovery of granulocytes, dogs receiving . x mnc/kg died of marrow aplasia on days and without recovery of thrombopoiesis. two dogs receiving x mnc/kg had sustained engraftment with delayed recovery of thrombocytes compared to dogs without g-csf. facs analysis of depleted marrow showed complete depletion of cd + cells but about % of cd + cells; cfu-c growth and nk-activity was retained after depletion. cd depleted marrow inhibited the generation of cytotoxic cells. these experiments indicate that qualitative t-cell depletion is effective, since cd recognizes only subpopulations of t-cells. the application of r-canine g-csf enhanced the recovery of granulocytes but led to graft failure in dogs receiving a low number of marrow cells. gsf-inst. fdr immunologic, marchioninistr. , mqnchen supported by the wilhelm-sander foundation we wished to analyse the factors which may affect the yield of pbpc (cfu-gm) to be collected by leukapheresis following high-dose cyclophosphamide (hd-cyc: g/m ). we retropectively studied the following criteria in patients with high-risk mm of which received gm-csf (sandoz sa/ schering-plough) after hd-cyc: time from diagnosis to hd-cyc, number of chemotherapy cycles (ctc) (fermand, ) , b microglobulin, bone marrow plasma cell count before hd-cyc, administration of gm-csf after hd-cyc, "slow" or "fast" rate of platelet and wbc recovery (jagarmath, schwartzenberg, ), "poor" or "good" mobilization ofpbpc (jagannath, ), differential wbc count between "day x ~ and "day x-l" during haematopoietic recovery. each variable was studied as continuous (regression analysis) and discontinuous (t-or chisquare tests). when the differential wbc count was < wbc/ial, % of the leukapheresis procedures performed on day x yielded more than x cfu-gm vs % when it was _> /tjl (p< . ). the infusion of gm-csf was asaocaated with a higher yield of cfu-gm (bidt, ). the patients with "good ~ pbpc mobilization (> x cfu-gm in >_ leukapheresis) could all be transplanted with pbpc alone (vs % of those with ~poor" mobilization). they had a shorter duration of aplasia after transplantation than the other patients (p< . ). the "fast" wbc recoverers had a higher yield of cfu-gm than the other patients (p< . ). when only the patients who did not receive gm-csf after hd-cyc were considered, a higher yield of cfu-gm was achieved in patients who underwent < crc as compared to those who underwent > ctc before hd-cyc (p<o, ). no other criteria had a significant prognosis value. these criteria will help to define which patients may need the collection of bm or the infusion of gm-csf (i) to collect pbpc and (ii) after transplantation. haemopoietic cells released into the circulation by g-csf (lenograstim) alone or in combination with intense chemotherapy: assessment of progenitors and stem cells. i.baumann*, ng testa*, mehm van hoef ~, c.lange*, e.de wynter*, a.howell $, tm dexter*. we report results of a phase i/ii recombinant human granulocyte colony stimulating factor trial for dose intensification in advanced breast cancer. sixteen patients were studied. seven patients underwent the phase i g-csf trial before entering the phase ii dose intensification study plus chemotherapy. nine patients were entered into the phase ii study with four cycles of dose escalating chemotherapy. the release of progenitor cells into the circulation was determined by clonogenic assays. two stage long term bone marrow cultures were used to assess the repopulating capacity and therefore the stem cell quality of the mobilised cells. normal bone marrow from allogeneic transplant donors (with informed consent) were used as controls. we found equivalent total numbers of progenitors in both cycle and cycle of g-csf plus chemotherapy peaking at day ia- , respectively. in contrast, we found significant differences between the progenitor cells of cycle and generated from circulating haemopoietic cells when seeded onto preestablished irradiated bone marrow stroma. this indicates that peripheral blood stem collection should be performed in an early stage of chemotherapy due to an increased damage of the stem cell pool with increased levels of chemotherapy. the capacity of haemopoietic cells released by g-csf to repopulate bone marrow stroma was equivalent to normal bone marrow as recently reported. in autologous bone marrow transplantation (abmt) acute phase responses usually occur between day and day following abmt, and increases in bilirubin levels are observed in early endothelial complications such as endothelial leakage syndrome and veno-occlusive disease (vod). il- is the central cytokine modulating acute phase responses. therefore we performed a prospective study analyzing il- , tnf-u, sil- r and g-csf levels in serum samples of patients undergoing abmt ( all, aml, hodgkins disease, non hodgkins lymphoma, ewing sarcoma). patients received g-csf post abmt, patients were transplanted without g-csf. during leukopenia < /gl g-csf levels of _+ pg/ml ( - pg/ml) were measured in patients not receiving g-csf. during regeneration, when leukocyte counts exceeded . /tal, the endogenous g-csf levels declined to the normal range (< pg/ml). severe transplantation associated complications were defined as vod or interstitial pneumonia. il- levels increased from +_ pg/ml before therapy to • pg/ml during febrile leukopenia (p<. ). patients with complicated abmt had significantly higher il- levels ( -+ pg/ml n= vs • pg/ml n= ) (p<. ). sil- r-levels increased from + u/ml before abmt to _+ u/ml during leukopenia. in patients receiving g-csf sil- r-levels further increased to _+ u/ml during regeneration, while in patients not receiving g-csf the sil- r-levels were • pg/ml (p<. ). tnf-a levels remained unchanged during the course of abmt; there was no correlation with febrile neutropenia or severe complications. these data suggest a direct feedback regulation of endogenous g-csf release by increases in peripheral granulocyte counts. monitoring of il- serum levels during abmt may give insights into pathophysiology of abmt associated complications resulting from activation of monocytes and endothelial cells. we have previously reported that the rate of hematopoietic recovery following autologons blood stem cell transplantation (absct) could be influenced either by the type of conditioning regimen or by the underlying disease. we also reported that peripheral blood stem cell (pbsc) growth was sensitive to the stimulation of allogeneic irradiated stmmal layers. thus we undertook a study to examine the quality of the bone marrow (bm) raiemenvironment of patients who had undergone absct for either malignant lymphoma (ml) or multiple m~,eloma (mm) after either myeloablative chemotherapy or tbi conditmning regimens. thirteen patients with ml and eight patients with mm underwent absct using cells collected during recovery phase after intensive chemotherapy. we used the long term culture system (ltc) to investigate the quahty of the bm microenvimnment from patients (pts) with ml and patients with mm. all bm were collected after absct. twenty-two ltc established with bm from patients with ml were investigated; pts received myeloablative chemotherapy (cbv) as conditmning regimen and out of ioof these pts developed a complete confluent stmmal layer. three of the ml pts received tbi and / of these pts developed a complete confluent stromal layer. eight ltc were established with bm from patients with mm, who had att received" till" prior to absct. three ~f the mm pts de~ a complete confluent stromal layer. we evaluated the cfu-gm production by the stmmal layer from all patients. the type of disease (ml vs mm at d p= , and di p= , ) , the conditioning regime used prior to absct o'b! vs cbv at d p= , and di p= , ) , the presence or absence of a complete, confluent stromal layer (at d p= , and d p= , ) did not influence the production of cfu-gm by bm stromal layers from autografted patients. nor was there a correlation between the development of a confluent stromal layer and the duration between ltc and absct or the date of the cytapheresis as compared with the date of diagnosis or the number of naeleated cells and cfu-gm used to engraft the patients. interestingly, when we examined the ability of confluent and non confluent stmmal layer to support hematopoiesis, we observed no difference in the total cumulative production of nucleated cells or cfu-gm, suggesting that the quality of the bm stromal miemenvironment after absct cannot explain the differences seen in hematopoetic reconstitution. the present study was designed to estimate the immunologic and hematopoietic reconstitution after several consecutive chemotherapy courses (ctx). patients selection: all (n = / ctx-cycles), aml (n = / ), nhl (n = / ), m. hodgkin (n = / ), and non-seminomatous germ cell tumor pts. (nsgct, n = / ). ctx was performed according to the current treatment protocols used at our institution. the nhl and nsgct pts. received g-csf before ctx for mobilization of stem cells for apheresis. g-csf application ( "pg/kg) after ctx was used in all nhl, all and nsgct and aml pts. the collected pbsc were reinfused after each following ctx cycle to all nhl and nsgct pts. the cd , cd , cd , cd , cd , cd , cd and hla-dr positive cells in peripheral blood were analyzed, using dual color fluorescence and flow cytometry. cfu-gm from peripheral blood mnc's were used as control for the clonogenic capacity of cd * cells. samples were obtained before, immediately after ctx and several times after the wbc's reached > lxlog/l. a positive correlation of the rising and decreasing subpopulation counts within the mnc's were noticed (r=. -. ), however the cd * were in inversed ratio to the cd § cells (r =-. ). the percentages of cd * and especially of the cd + cells showed an increment immediately after ctx, where the proportions of cd " and cd § cells tended to fall. there was also a correlation between cd " and cd § cells (r=. , p<. ) and between cd § ceils and cfu-gm growth (r= . , p<. ). an increased clonogenity was associated with low numbers of cd * cells: cd ~: cfu-gm : before ctx; : immediately after ctx; : during the regeneration phase. the same phenomenon could be seen by intensively pretreated compared to less intensively pretreated patients. the hematopoietic reconstitution parameters (median) were as follows in the pbsc-rescued vs non-rescued pts: platelet transfusions - vs (p<. ), rbc transfusions - vs , days with platelets < // - vs (p<. ), duration of neutropenia with wbc< //ji - vs , days with fever - vs . . the augmentation of cd § cells correlated with rising numbers of mnc's and especially of cd § but not with cd *. the correlation between cd " cells and cfu-gm in peripheral blood was convincing. dose-escalated cytotoxic therapy with stem cell support may be considered for patients with stage ii multiple myeloma, because of the poor median survival of only . years with conventional treatment. a threshold quantity of x cd + eells/kg bw is necessary for a rapid and sustained engr~ent following myeloablative conditioning therapy. since june six patients (median age: years, range - ) with mm received pg g-csf/kg bw (neupogan r, amgen) so. daily at the time of best response with conventional treatment. the content of cd + cells in the peripheral blood was monitored by facs each day. leukapheresis were started when a detectable population of cd + cells appeared. in of steady-state leukaphereses, more than , xl ~ cd +cells were harvested. after the therapy with high dose eyclophosphamide ( pts g/m , pt g/m ) plus g-csf more than , xl cd + cells were collected in of leukapheresis. to date two pts have undergone myeloablative conditoning therapy with hyperfractioned total body irradiation ( , gy) and melphalan ( mg/mz). one patient received mg/sqm melphalan as eonditoning therapy. after the reinfusion of the g-csf-mobilized pbsc, a rapid engraftment was achieved with median time of days ( range - ) to reach , x / neutrophils and days (range - ) for . x / platelets. no hematopoietic growth factors were given post-transplantation. in this pilot study, high dose eyclophosphamide and g-csf is an effective method for harvesting pbsc. evaluation of the mobilization of hematopoietic stem cells during steady-state hematopoiesis using higher doses of g-csf is planned. were treated with mtx, ifo, ara-c, prednisolone and escalating doses of vp . pts. (n = ) with relapsed or advanced non-seminomatous germ cell tumors were treated with cisplatinum, escalating doses of vp and ifo. the protocol design was similar: g-csf before ctx ( x pg/kg/die s.c.) with pbsc-aphereses at days to (nhl) or days , (nsgct} followed by to ctx-courses. cytaphereses were also performed after ctx when the total wbc's recovered above lxl ~ the ctx-cycles were followed by reinfusion of the previously collected pbsc (n = ) and application of g-csf ( pg/kg/d.; n = ) up to the last day of the subsequent stem cell collection. the cd § cells, the clonogenic peripheral blood progenitor cells (cfu-gm & bfu-e) and light density cell (ldc) counts were determined in cytapheresis samples. the ctx/g-csf courses contributed to substantially higher progenitor cell amounts than g-csf alone (n= / ; p< , ), without a difference in the collected ldc ( . • vs . • ldc; . • vs . • ~ cd § cells; . + . vs . • . x cfu-gm; . • . vs . • . x bfu-e/kg/apheresis; n = / ; mean + sem), but with approximately two times lower clonogenity. two or three leukaphereses were enough to rescue ctx-courses with a minimal dose of x cd + cells/kg/patient. the optimal time to initiate pbsc-collection after ctx in the studied patient group was proved to be at the l't to ~h day after reaching leukocyte > pl. the original total leukocyte, light density cell (ldc) and platelet counts in the peripheral blood at start of leukapheresis played an essential role for the efficiency of the procedure (eff), as shown by regression analysis: eff to total leukocyte count correlation was r =- . (p< .ot); eff to ldc-count r =- . (p<o. ); and eff to platelet count r =- . (p= . ). the results of pbsc retransfusion on hematopoietic recovery were as follows: days to reach leukocytes> pl and days -platelets> ooo pl; duration of neutropenia was days; and days to become platelet transfusion-independent (median). total number of children, girls and boys, with neoplasia aged from - years were treated with gm-csf-leucomax sandoz and g-csf filgrastim hoffmann-la roche, during severe myelosupression occuring after intensive polychemotherapy. in children gm-csf was applied twice after consequent courses of chemotherapy. one child received gm-csf four times after chemotherapy courses. twenty children with malignancies served as historical control group. gm-csf was given at dose #g/kg, g-csf - #g/kg daily s.c. duration of therapy ranged from - days with median days. after cytokines therapy increase of mean and median numbers of total wbs, neutrophils, monocytes and eosinophils was observed. the median time to hematopoietic recovery was shorter in the group of children treated with cytokines when compared with the control group. ( v days). in of children signs of infection disappeared even before granulocyte count increase. also shorter median time of febrile days, v days, in comparison with the control group was observed. no serious side effects during cytokines therapy were noticed. only in one patient local erythema in injection place was observed. in two children transient retrosternal pain was seen. our results showed that gm-csf and g-csf administered in children with neoplasia after chemotherapy shortens the period of neutropenia and infection duration. for both hodgkin's disease and non-hodgkin's lymphoma the outcome of chemotherapy has been shown to correlate closely with the dose intensity of treatment. however, dose intensification is limited most often by severe myelosuppression with the subsequent risk of fever and infections. we performed a clinical trial in patients with hodgkin's disease or non-hodgkin's lymphoma to evaluate whether r-methug-csf could facilitate the safe and timely administration of an intensive chemotherapy regimen. patients who developed neutropenia _< . x /l for more than two days and / or fever _> . ~ and / or signs of infection after a cycle of chemotherapy (ceboppnim protocol administered at intervals of days), as well as patients in which chemotherapy had to be delayed due to an anc _< . x /l on day , were eligible for treatment with r-methug-csf. in the subsequent cycles r-methug-csf was given subcutaneously at a dose of #g/kg/d from day to . of patients were evaluable, one patient had received only day of r-methug-csf treatment. of the evaluable patients experienced neutropenia with an anc of less than . x /l during the chemotherapy course preceeding r-methug-csf treatment, whereas only patients had ancs _< . x , l after the subsequent therapy with r-methug-csf (p< . ).overall analysis showed that the duration of anc nadirs < . x /l was on average . days in cycles without r-methug-csf compared to . days in cycles with r-methug-csf treatment. the administration of chemotherapy had to be delayed only for . days (mean value) during cycles with r-methug-csf. side effects probably related to r-methug-csf, were moderate muscle and joint pain in patients and chills in one patient. in general, r-methug-csf was well tolerated. under this treatment regimen patients reached complete remission, patients reached partial remission and one patient had stable disease. one patient was treated adjuvantly after gastrectomy. in conclusion, r-methug-csf allowed the safe and timely administration of this intensive chemotherapy regimen and reduced myelosuppression for patients with hodgkin's disease and non-hodgkin's lymphoma. based on previous studies (cancer res. : , , blood, : , we know that many patients (pts) cannot receive ct consisting of carboplatin (cbdca) mg/m and cyclophosphamide (cyclo) mg/m for oc every wks without hematopoietic growth factor support. the desirable dose of rhil- based on a phase i/ii study in this setting was or gg/kg/day. a study was designed to determine whether rhil- would allow ct administration every wks with pts treated to date. cyclo was administered mg/m and cbdca was dose adjusted to creatinine clearance: - ml/min: mg/m , - : mg/m , - : mg/m , > : mg/m . a total of cycles (c) was administered. rhil- ( or /zg/kg/d) was given sc d - in each c. at and #g doses are ( c) and ( c) pts evaluable for toxicity and i ( c) and ( c) pts for efficacy. side effects were fever and headache controllable with acetaminophen. at #g rhll- in three c ( pts) and at #g in six c ( pts) urticaria occurred. in episodes dyspnea and/or oedema was observed. this reaction only occurred during c ~ and was controlled with antihistamine and prednisolone. ct could be administered every wks in / c ( / c at /~g, and in / c at /zg (ns)), every wks in / c and > wks in / c. no platelet transfusions were required. thus, in % of c it was possible to give a ct dose intensification of %. if full dose ct were to be given every wks it would have been possible to administer in % of c in time. conclusion: with rhil- ct dose intensification of % is possible by reducing ct intervals, while no platelet transfusions were required with rhil- . dept. of medical oncology. university hospital groningeu. oostersingel , ez groningen. the netherlands. k. mempel, a. reiter, e. yakisan, e. odenwald, m. pfetsch, g. schwab, h. riehm, k. welte. in the multicenter trial all-bfm , we have initiated a phase iii study of rhg-csf in children with high risk all. high risk (hr) patients are characterized by at least one of three criteria: . prednisone poor response ( /mm absolute blasts number in the blood at day after days' exposure to prednisone), . failure to achieve complete remission at day of induction therapy, and . t( ; ). the primary objective was to test whether rhg-csf reduces the incidence of febrile neutropenic episodes. the second objective was to examine whether rhg-csf administration allows closer adherence to planned dosing schedule and to determine the overall response to chemotherapy. hr-all pts are randomized to receive either cycles of chemotherapy (hrg i) or cycles of chemotherapy (day - ) followed by rhg-csf (day - ; hrg li). up to date, pts have been treated according to this protocol (hrg i: pts, hrg i : pts). in hrg ii, rhg-csf is well tolerated without g-csf related adverse events. in each arm, one pt relapsed. the incidence of neutropenia was % in hrg i and % in hrg i . more importantly the incidence of febrile neutropenia was % in hrg i and % in hrg ii. these data demonstrate that rhg-csf allows for reduction of the incidence of febrile neutropenia in hr-all-patients. the patient has experienced complete resolution of stomatitis, fever and malaise. the administration of g-csf in patient with idiopathic neutropenia significantly increased the absolute ueutrophil counts (p < . ). g-csf was effective in reducing the severity of neutropenia and infectious complications in our patient. hansen f., stenbygaard l. and skovsgaard t. twenty patients with recurrent metastatic breast cancer treated with high-dose myelosuppressive antjr eoplastic drugs (cyclophosphamide , g/m or epirubicin mg/m both q . weeks) as first or second line chemotherapy were randomized in a prostective study to gm-csf (n= ) microg/ kg/dag for ten days after cessation of chemotherapy or control (n= ). compared to the control-group highly significant reduction in granulocyte nadir duration (two days ( - ) with gm-csf vs. seven ( - ) days) and severity (wbc . x /i with gm-csf vs. . x i /i) was found. no difference in frequency of neutropen fever or antibiotic use could be observed. even though the patients treated with gm-csf at random were more heavily pretreated with chemotherapy, there was a surprisingly higher responserate in these patients as compared to the control-group, namely % vs. %, resp. no severe side effects were seen, but presumably due to gm-csf one patient developed an allergic type i reaction and one patient developed a possible pericardia[ exudation. both were fully reversible after cessation of gm-csf treatment. keywords: gm-scf, chemotherapy, breast cancer. twelve adult patients with chronic neutropenia, including patients with idiopathic sporadic neutropenia, with idiopathic familial neutropenia and with cyclic neatropenia have been treated with rhu-met-g-csf (amgen, thousand oaks, usa). treatment has been started in all patients with #g/kg/d sc once daily. doses have been modified according to wbc. all patients had a rapid increase of absolute neutrophil counts. data are shown for idiopathic neutropenia (base line, after , and weeks). doses required ranged from . to #g/kg/d. treatment has been continuously given up to three years in patients with severe preceeding infections. the clinical efficacy of the treatment was excellent with abrogation of significant infections. one patient with idiopathic i sporadic neutropenia recovered after days of treatment with an anc of "~ ~o o > /# after stop of g-csf. in a patient with familial cyclic neutropenia cycle length shortened from to days. in another patient with acquired idiopathic cyclic neutropenia the cycle length of days remained constant but re t w w w the nadir of anc rose from to /# . this patient was taken off therapy because of urticaria related to g-csf on day . there were no further significant adverse events. no loss of effectivity was observed during long-term treatment. we conclude that g-csf is safe for long-term treatment of idiopathic neutropenie with severe preceeding infections. as response to treatment is quic, it may also be an effective interventional treatment in acute infections in these patients. cytokines and growth factors are widely used to promote growth and proliferation ofbematologic cell populations. improvement or wonnu healing by stimulation with g-csf has been relxn'ted in patients suffering from kostmann syndrome, felty syndrome or from neutro-~ nia due to chemotherapy. e report on two patients with mds/ra (ha, female, age ; sh, male age ); duration of disease was months and years, respectively. ha was admitted for neutropenia (neuu'ophils: . - . g/l), epistaxis and a growing ulcerous wound in the pubic area (diameter mm) already pretreated with antibiotics for days. surgery was not possible due to poor heart condition and thrombocytopenia refractory to donor platelets. mu g-csf were administered subcutaneously daily for days resulting in neutrophil counts of . g/ and effective wound granulation and epithelialisation. the patient died of cardiac failure on day . sh was adnutted for infected hematoma of the left thigh. subcutaneous infection progressed due to severe neutropenia (neula-ophils < . g/l). incision and resection (ulcus diameter ram with deep invasion into the fascia) was performed. days later the defect measured x mm, reaching the knee joint, and the patient underwent a second surgical interventaon. enterococcus, staphylococcus epidermidis and bacterium xerosis could be cultured from direct swabs. therapy with g-csf at a dose of mu s.c. was started on day . neutrophils reached . g/i and g-csf was reduced to every other day. complete wound healing without any further surgical intervention was achieved by day and sh was dismissed. after discontinuation of g-csf the patient is well and has normal differential blood counts. we conclude that g-csf is successful in promotion of wound healing in mds patients due to enhancement of neutrophil production. leukemic conversion of mds during g-csf was not observed. we report on a year old male patient presenting in / with moderate thrombocytopenia~ transfusion dependent macrocytic anemia and normal., wbc. trephine biopsy showed hyperceuular marrow without fibrosis wzm trilineage dysplasia (mds/ra). cytogenetic analysis was ,xy. in / vasculilas ~as diagnosed. from / to / three cycles of gm-csf ( #g/m s.c. - days) were administered, resulting in both transient leukocytosis and increased platelet counts_ bone marrow aspirations showed dysplasia but no blast proliferation. in / vasculitis progressed, splenomegaly and hemolytic anemia developed requiring prednisolon. in / high dose erythropoietin was started ( iu/kg i.v. twice weekly) and continued till / . there was no change clinically, bone marrow smears and cytogenetics. in / the patient complained of pain in the lumbosacral region and neuralgia in both legs developed; a ct scan was negative. both pain and neurological symptoms (paraplegia and sensibility disorders) progressed. act scan and an mrt showed an intraspinal tumor (d -d ). "although severe thrombocytopenia refractory to high dose i.v. immnnoglobulin and platelet support (hla class i & lymphocytotoxic antibodies) developed, therapeutic laminectomy was performed in / , but only a part of the tumor could be resected. histologically the tumor consisted only of erythropoiesis with dysplasia without excess of blasts. wound healing was without complicatmns. after surgery gamma irradiation and therapy with ifnc~ ( #g s.c. times/week) were performed. the patient recovered totally from neurological disorders and is still alive but iransfusion dependent because of severe cytopenia. we conclude: intraspinal extramedullary hematopeiesis is a rare symptom in mds. althoughthis infiltration was diagnosed months after gm-csf a_ad hd-epo therapy, it could be induced by cytokine therapy. ( monoclonal antlbodles elther' with or without gam crosslinking. in addition, we added the cytokines. the phenotypic change of expression of fcy receptors was measured. ~o production and calcium flux using the dihydrorhodamine (dhr) and fluo- am methods, resepectively. there were no changes in expression of fcy receptors, but a significant enhancement of pmn activation via fc receptors by all three cytokines. we observed an increase of h production . fold by g-csf, fold by gm-csf and . fold by il- . a fcyriii-b specific monoclonal antibody fgr pmn (id ), which was alone unable to mobilize ca i+, together with all three cytokines was a potent stimulator. the effects of gm-csf and g-csf were calcium independent, in contrast, il- also enhanced calcium mobilization significantly. in summary, all three cytokines potentiate the fcy receptor activation of pmns and therefore play a significant role in inflammatory granulocyte activation as in leukocytoclastic vasculitis. g-csf is a hematopoetic growth factor required for proliferation and differentiation of hematopoeitic progenitor cells. it is now being successfully used to overcome neutropenias of various etiologies. recently, we demonstrated that rhg-csf induced neutrophils from patients with severe congenital neutropenia showed altered surface marker expression (upregulation of fcyri (cd } and cd and downregulation of fcvriilicd )) as well as decreased chemotaxis towards a variety of chemoattractants including fmlp. to separate the effects of the underlying disease from those of the rhg-csf therapy, we investigated neutrophils from patients receiving cytotoxic chemotherapy (n = ) and healthy adults (n = ) after application of rhg-csf. results: neutrophils from patients receiving daily application of rhg-csf (neupogen ~ #g sc.) were studied ex vivo one day before, three times during and - days after cessation of rhg-csf treatment. expression of fcvri, cd and cd (measured by flow cytometry) increased during therapy reaching a maximum at - days after initiation of rhg-csf therapy, whereas expression of fcvrll! decreased to a minimum after - days. chemotaxis of neutrophils under agarose towards fmlp was also reduced during therapy. investigation of surface marker expression and chemotaxis - days after cessation of rhg-csf revealed return to levels before therapy. to exclude the possibility that the observed alterations were caused by the underlying disease or chemotherapy, five healthy adults were treated with a single dose of rhg-csf (neupogen', pg, sc.). a continuous upregulation of fcvri and cd starting h after application with a maximum after hours (fc~l) and hours (cd ) and a downmodulation of fcrriii reaching a minimum at hours was observed. chemotaxis towards fmlp decreased to h after application and returned to normal after h, whereas expression of fcrri, cd and f%riii showed baseline values after hours. conclusions: the results obtained from the healthy test subjects clearly demonstrate that neither the malignant disease nor chemotherapy, but rhg-csf induced the profound alterations of fcr receptor and cd expression and chemotaxis in neutrophils in vivo. the continuous character of the surface marker alterations without appearance of subpopulations and the increase in cd expression suggests that preactivation rather than immaturity of rhg-csf induced neutrophils alone might be responsible for the observed phenomena. fraunhofer institute ita, nikolai-fuchs-sral e , w- hannover severe congenital neutropenia (scn) is a disorder of myelopoiesis characterized by a maturation arrest on the level of promyelocytes with absolute neutrophil counts below /pt in the peril~heral blood. in this study we investigated the expression of receptors for the granulocyte colony-stimulating factor (g-csf) on neutrophils from patients with scn during g-csf therapy. the normal g-csf receptor expression on neutrophils is in the range of - receptors per cell. neutrophils from scn patients express increased numbers of receptors in the range of - receptors per cell. the dissociation constant of the binding of g-csf to the g-csf receptor is not altered as compared to healthy donors. in contrast neutrophils from patients suffering from cyclic neutropenia express normal g-csf receptor numbers ( - receptors per ceil). in addition, we have compared the g-csf receptor cdna of neutrophils from healthy donors and scn patients using the polymerase-chain-reaction technique. we could not detect any major alterations in the g-csf receptor cdna in scn patients. preliminary cdna sequencing data also did not reveal any point mutation. from this data we conclude that there is no defect in g-csf receptor expression and no alteration in the sequence of the g-csf receptor mrna in scn. pediatric hematology and konstanty-gutschow-str. , d- hannover oncology, medical school hannover, severe congenital neutropenia is a disorder of myelopoiesis characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmenn's-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median anc below / ~ (cyclic neutropenia). we have treated patients with scn and patients with cyclic neutropenia. thirty of patients with scn and all patients with cyclic neutropenia responded to rhg-csf treatment with an increase of the median anc to above /ixl. the doses needed to achieve and maintain the response varied between . and i~g/kg/d. long-term treatment did not exhaust the myelopoiesis: the mean anc remained stable up to years of treatment. the increase in anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment episodes, and reduction of hospitalizations. no severe bacterial infections occured in any of the rhg-csf responders during long-term treatment. severe adverse events, most likely associated with the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the benefical effects of rhg-csf treatment in severe congenital neutropenia patients. fifty-two patients (pts) (median age - years) with philadelphia chromosome positive (ph +) chronic myeloid leukemia (cml) have been treated with ifn ( x units/m ) within six months of diagnosis (median . months (mths), range - mths). we divided the pts into three groups according to sokars classification: low risk group (n = ), intermediate risk group (n = ) and high risk group (n = ). forty-three pts acheived a complete hematological response (chr) as defined by the houston criteria. the cytological response was evaluable in pts: pts ( . %) demonstrated a partial or major eytogenetical response (more than % ph i negative metaphases). the hematological and cytogenetical responses were influenced bsr the risk factors, os the percentage of chr and cytogenetical responses was higher for the pts from the low or intermediate risk groups ( % and % respectively) than for the high risk group ( % and %). transformation occurred in four pts who did not demonstrate a cytogenetical response. the estimated chance of surviving at three years was % for the overall population. toxicity was mild but ifn had to be internpted in four pts for cardiac (n = ), liver (n = t) or neurological (n = ) tocxic effects. these results confirm that ifn is a very effective treatment for cml. the effect of rhg-csf on platelets was studied in healthy volunteers with the thrombometer, a specially developed device which is described in detail. additionally, conventional aggregation tests were performed low doses of rhg-csf enhance functional platelet activity, as shown by significant acceleration of the occlusion of the thrombometer channel. similar results were found in conventional aggregation tests using collagen for induction. with g-csf concentrations of , and , ng/ml the time of response was significantly accelerated and the maximum response was observed in a higher proportion ofplatelets. however, the second phase of aggregation induced by epinephrine was significantly inhibited by , ng/ml g-csf. the expression of cd , cd and cd on platelets' surface was determined in ten patients before and niter administration of g-csf (facscan flow cytometer).quality controls were done by calculating the events positive for cd and cd , which were expressed in nearly t % of the platelets without being changed by the cytokine. the expression of cd in the platelets' surface however was significantly enhanced indicating a depletion of the c~-granules. no platelet aggregation was observed. cd expressed on thrombocytes' or damaged endothel cells' membrane is a receptor for macrophages. this property facilitates rapid adhesion of leucocytes to endothelium at regions of tissue injury as well as platetetleucocyte interactions at areas of inflammation and hemorrhage.-in contrast cd can also have an antiinflammatory function because exposure of tnfa-activated neutrophils to plasmatic cd inhibits their cdl -dependent adhesion to resting endothelium and superoxide production. ifn a has a unique activity in cml leading to complete and partial remissions in - % of the patients. to improve these results, we are currently treating patients with ph'+ cml with a combination of cytosine-arabiuoside at a maximum dose of mg/m z sc on days per week and ifn c~- b. ifn u- b is started at a dose of muim sc dally and escalated to the maximum tolerated dose. patients ( male, female, median age years) have been entered into the trial. patients have been pretreated with other regimen for a median time of months. patients are without pretreatment. the treatment has been well tolerated. besides the ifn a related side-effects some patients experienced gastrointestinal toxicity with nausea and vomiting after prolonged ara-c application. the median observation time in the study is now months and patients are still entered. up to now complete hematologic remissions have been achieved in patients, and partial ones in patients. the rate of complete hematologic remissions was higher in patients without pretreatment ( %) compared to patients who have been pretreated ( %). five partial eytogenetic remissions have been observed and minor reductions in the ph'+ cell done. all cytogenetic responses have been found in patients without pretreatment. we conclude that a combination of cytosine-arabinoside and ifn u- b is well tolerated in patients with cml. early results are encouraging. longer follow up times are necessary to evaluate whether combination therpy will give superior results compared to a txeatment with ifn a alone. during rlfn-~ therapy a minority of patients develops high-titered antibodies neutralizing the injected rlfn-a . the rlfn-c~-andbodies from six of such patients, who lost their clinical response to rlfn-c~ and showed a relapse of their leukemia ( cml, hcl) despite continuous rlfn-c~ a-therapy, as well as ifn-c~-specific antibodies from two patients with systemic lupus erythematusus (sle) were characterized. the anti-ifn activity was purified by sequential protein g -and rifn-c~ affinity chromatography and was found to consist only of igg-antibodies. these antibodies were further tested for their capacity to neutralize the antiviral and antiproliferative activity of various rifns-c~-subtypes. all six sera tested showed a common pattern of neutralization (mdbk-vsv bioassay) distinct from the sleantibodies. all six neutralized rifn-c~a and rifn-ak consistently with a higher titer against aa. three of the six sera neutralized aa, ak, ~c, ~c/j and m, but not m, m and some other subtypes. therefore, from the structure of the c/jl-hybrid, it seems that one epitope recognized by these three sera is at the nh -terminal half of the molecule. in contrast, the sle associated antibed]es neutralized the antiproliferative and antiviral activity of every subtype tested. these data indicate that the therapy-induced antibodies against rifn-c~ recognize very selected epitopes on the rifn-cd-molecule suggesting that only a part of the rifn-~ molecule is immunogenic. in vitro experiments indicate higly synergistic effects of combining ifn with cytostatic drugs such as anthracylines (a). in a phase i/ii-study patients (pts) with pro$ressive inoperable hcc were treated with e mg/m z weekly x and ifn mio iu/m s.c. x weekly for weeks, followed by one week off treatment. in case of at least no change (nc) and tolerable toxicity the therapy was continued. escalation of e in steps of mg/m z per cycle was attempted. pts characteristics: median age years ( - ); male , female pts. pretreated with a pts. toxicity and treatment: total number of cycles ; median ( - ) per pt. worst toxicity per pt (who): wbc ~ %, ~ %; platelets ~ %, ~ %; diarrhoea ~ %, ~ ~%; n~isea/vumitinq ~ %; ~ %; ifn related fever (maximal ~ in %. ifn-related wasting syndrome pts, no severe organ toxicity. divisione di ematologia -ospedale san camillo -roma from november to october six patients with acute leukaemia, who achieved their first complete remission with standard chemotherapy followed by autologous bone marrow transplantation (abmt), were consecutively treated with r.interferon alfa- a (ra-ifn). patients ( all and anll) were from ii to years old, of them ( anll and all) were reinfused with autologous bone marrow purged with asta-z i mcg/ml/ x cells and in the remaining all patients immunomagnetic purging was employed. conditioning regimens were bucy in patients and cy-tbi in the others, ra-ifn started at median time of months ( - ) after abmt when complete consolitated hemopoietic recovery occurred. the ra-ifn dose was . iu/sqm times a week for years. none of the patients presented significant toxicity and only short suspensions occurred for fever or alt level increase. the incidence of infectious complications were particularly low compared with other autotransplanted groups of patients who received similar antinfectious prophilaxis. one case six months after abmt and days after acyclovir prophilaxis interruption presented a mild herpes-zoster complication which required new acyclovir therapy and resolved i days later. the amount of these patients is extremely low because the study was early interrupted to start a new protocol including il- ; but the long duration of the good continous complete remission ( / years after abmt) in all these unselected and consecutively treated patients is very interesting. surgical procedures may be associated with an inceased risk of tumor spreading due to surgical mobilisation of the tumor and transient postoperative immunosuppression. recurrences may result either from early growth of micrometastases already present at the time of surgery or from the seeding of malignant cells shed during operative manipulation of the tumor. imrnunomodulators have been proposed to correct the immunological impairement induced by surgical procedures. from / to / , patients with advanced stage cancer underwent surgical resection with peri-operative interferon-alpha administration. patients received interferon alpha- a (roferon-a), by daily subcutaneous injection for fourteen days, starting on three days before surgery. incremental doses were , , , , and x iu for , , , , and patients respectively. peripheral blood lymphocyte (pbl) subset numbers were assessed using flow cytometric analysis the day before injection (d- ), before surgery (d-i), at the day and . absolute numbers of total t cell (cd +) and nk cell (cd +, cd -) were determined, as well as auxiliary t cell (cd +), activated t cell (cd +, dr+), and b cell (cd +) counts. short-term cytotoxicity of pbmc against k and daudi target celts in a -hour standard chromium release assay were determined. no w.h.o. grade iv toxicity were observed. a significant post-operative fall of the total pbl count, of cd +; cd +; cd +; cd - + occured from d- to d . the decrease were not significant for cd +dr+. values were not significantly different, between d- and d , only for cd dr+ and cd - +. cytotoxicity against k and daudi target ceils increased significantly from d- to d-l, and from d-i to d , with a significant fall of cytotoxicity against daudi from d- to d . peri-operative interferon alpha administration is well tolerated even at the . iu doses. in spite of treatment and increasing cytotoxicity activity, we observed a post-operative fail for the majority of the imunological parameters. further studies are necessary to compared with a control group, with more patients treated with . tiiu daily. patients: six patients have been treated (med. age yrs.; - yrs.) four patients had acute myeloid leukemia (aml m : , m : , m eo: ). two patients had acute lymphocytic leukemia (both t-all). all patients had manifest disease with more than % blasts in the bone marrow. patients were selected not to have rapidly progressive diesease allowing the application of the cytokine. treatment and toxicities: ifn a was given for a median of ( - ) days at a dose of - mu sc daily. the following toxicities _>grade who were observed: fever , gpt , pulmonal , infection ( pneumonia), pains . the patients with aml ware transsusion dependent for platelets and erythrocytes. no significant additional bleeding was observed. results: one aml patients had stable disease and three had disease progression. of the all patients one had disease stabilization and one progression. conclusions: ifn a is well tolerated in patients with refractory aml and all if they ere in a relatively stable condition. the effectivity of ifn a- b as a single agent is poor in patients with refractory aml or all. freund et al. eds, springer-verlag, ) have confn'med the wide range of clinical usefulness of ifn alphabased therapy in cancer patients (pts). we present in this paper a retrospective analyses of cases with advanced neoplasias treated between - by a sequential administration of ifn alpha and standard chemotherapy. there were w, m, aged - y, with solid cancers pats and lymphomas pts. the treatment consisted of a sequential association of ifn alpha (also with ra all-trans) and cht plus tamoxifen (for appropriate cancers). the ifn schedule was of monthly series, one series consisting of million iu/d for consecutive days. cht, appropriate for each primary cancer also administered in monthly series. the results are grouped according to the status of the disease (subsets of pts) at the onset of ifn-based therapy, and refer especially to long term survival ( - y +). for minimal residual disease (mrd) from cases there were cr ( / breast ca, / cr melanomas, each sts and rcc / rc, / gastric and head and neck cancers together, /t lymphoma. for progressive disease, pre-lfn-based therapy there were pts, and post ifn-cht treatment there were sd and pd. in of failure pts association of ifn alpha and bropirimine (ifn-inducer) appeared an unusual good response. conclusions: ) ifn alpha-based therapy is a very useful one especially in mrd. ) the therapy must be individualized for selected subsets of pts and for each patient day to day. authors have used inf alpha b in cases of haematological malignancies for three years. the number of cases not too high, and non fo cml cases was so called "early" cml. our cases are: cml, non-hodgkin malignant lymphoma, myeloma multiplex, essential thrombocythemia. the tnf alpha b was used as monotherapy in the cases of low grade non-hodgkin malignant lymphoma, and essential thrombocythemia, and was combined with chemotherapy in the other cases. on the basis of our initial results, we recommend the inf alpha b in the treatment of haematological malignancies in suitably selected cases. department of haematology and oncology, hospital of ministry of interior, vftrosligeti fasor - the influence of low oral doses of human leukocyte derived interferon alpha on the immune system of chronically hbv infected patients with depressed immunological response. low oral doses &the interferon were given to a group &seven children with limphoblastic acute leucaemia in the state of remission, chronically hbv infected. interferon alpha was produced by hayashibara biochemical laboratories inc. okayama, japan, in tablets of iu and iu respectively. immunological response was checked by measuring population and subpopulations of limphocytes, level ofimmunoglobulin and complement c fraction. a distinct stimulation of cellular immune response was observed: the fraction of activated limphocytes t increased significantly, index cd /cd became normal and population of limphocytes b increased gradually. there was no influence of interferon treatment on immunoglobulin and complement c fraction serum level. the interferon treatment improved the patients' general condition and shortened the period of intoxication after cytostatic treatment. no side effects were observed. none of the children eliminated the hb virus during the six months treatment. cytokines play an important role in activating the immune system against malignant cells. one of these cytokines, il- has entered clinical phase i trials because of its immunoregulatory potency. in the present study we report that rhll- has direct antiproliferative effects on some human lung cancer cell lines in vitro as measured by a human tumor cloning assay (htca). this activity could be abolished by neutralizing antibody against rhll- . the biological response of the tumor cells to the cytokine is correlated with expression of receptors for bll- on both the mrna level and the protein level. the most responsive cell line ccl secretes il- after being incubated with rhll- . on the other hand, neutralizing antibodies against il- showed no influence on the growth modulatory efficacy of rhil- in this cell line. furthermore, ccl does not show detectable production ot il- , tnf-~ or ifn-y alter incubation with rhll- . thus, the response to rhll- is not mediated through autoeriee production of these cytokines triggered by rhll- . in a next series of experiments the ceillines were xenotransplanted to balb/c nu/nu mice. subsequently, the mice were treated for > days with twice , mg/m rhll- (rhll- was a kind gift from dr. urdal, immunex, seattle, usa) or control vehicle subcutaneously per day. treatment with rhll- yielded a significant inhibition of tumor growth versus control in the responsive cell lines ccl and htb , but no therapeutic effect in the non-responsive cell lines. plasma levels of rhtl- were sufficient for in vitro growth-inhibition in the responsive lines. histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of il- . we conclude that rhll- has direct antiproliferative effects on the growth of some human lung tumor cell lines in vitro and in vivo which together with its regulatory effects on various effector cell populations makes this cytokine an interesting candidate for further investigation in experimental cancer treatment. the combination of systemic chemotherapy and immunotherapy comprising interleukin- and alpha-interferon leads to significant tumor regressions in patients with advanced malignant melanoma. in contrast to chemotherapy by itself, the combination produces a significantly extended remission duration in the majority of treatment responders. we conducted phase ii studies.to assess the potentially additive or synergistic effects of chemotherapy and immunotherapy in metastatic malignant melanoma patients: the first study comprised two cycles of carboplatin ( mg/m ) and dacarbazine ( mg/m~); the sesond study included up to four cycles of cisplatin ( mg/m x days), dacarbazine ( mg/m x days), bcnu ( mg/m , cycle i+ ) and tamoxifen ( mg daily). chemotherapy was followed by up to cycles of a -week immunotherapy comprising interleukin- ( - million iu/m ~ sc x weekly) and alpha-interferon ( - million u/m sc x weekly). among evaluable patients in study i, there were ( %cr, %pr) objective responders; median remission duration was + months for complete, and + months for partial responders. chemotherapy intensification in study ii lead to an increased response rate of % ( out of patients). in both studies, the progression free interval was significantly extended when compared to patients who received chemotherapy, only (historic controls). the role of immunotherapy as maintenance in patients with advanced metastatic malignant melanoma is currently being evaluated in a prospective randomized trial. integrin receptors play a crucial role in cell-cell and cell-matrix adhesive function, and thus are supposed to influence invasion and metastasis. very little is known about the impact of interleukins on integrin regulation in tumor cell lines. therefore, we investigated the expression of of and i integrin subunits on well (ht ) and poorly differentiated (sw ) human colon cancer cell lines using a panel of specific monoclonal antibodies and cdna probes. ht and sw expressed similarly high levels of ~ , a , ~ gt, and f~ subunits on the cell surface. no a , ~ , and ~ was detected on either cell line. while a was not expressed on ht , sw showed higher levels of the laminin receptor ~ . the poorly differentiated cell line sw was resistant to il- , whereas ht was sensitive. treatment with il- induced a decrease in ~ , a , '~ , ~v, l~l, and integrin expression. however, ~ subunit was markedly upregulated. in contrast to il- , there was no evidence that il-lfi could modulate integrin expression on these celt lines. the function of integrin receptors was assessed by measuring adhesion to collagen, laminin, vitronectin, and fibronectin. il- significantly increased the adhesion of ht to fibronectin, while attachment to collagen, laminin, and vitronectin remained unchanged. these results suggest differential integrin expression pattern on well and poorly differentiated tumor cell lines. we provide evidence that integrin expression may be selectively regulated by il- , but not by il-ib. furthermore, il- can alter adhesive behavior of tumor cells. since il- is currently studied in clinical trials, the metastatic potential of malignant tumors should be monitored thoroughly. immunotherapy with ifncz and il- is an active regimen in malignant melanoma and has shown response rates of - %. in previous studies no prognostic parameters for response could be identified. patients with progressive metastatic melanoma have been enrolled in various immunotherapy trials including ifno~ and high dose il- since with an overall response rate of %. patients with mm treated in our phase ii trials could be analysed to identify possible prognostic parameters for response. patients were divided into three groups: responder ( cr/ pr), stable disease ( sd/ mr), and nonresponder ( pd). all patients had measurable tumor, a karnofsky index of > %, no cns metastasis, and no severe cardiorespiratory or renal disease. we examined the following pretreatment parameters for prognostic relevance of response: age, sex, performance status, time from diagnosis to onset of first metastases/ to begin of immunotherapy, tumor toad, number of metastatic sites, organ sites of metastases, ldh, ap, esr, and hla-type. of these several variables were found to significantly correlate with response: tumor load (p= . ), number of metastatic sites (p= . ), serum ldh (p= . ) and ap (p= . ). tumor load, ldh and ap are no independent parameters. while time from diagnosis to onset of first metastasis is of no prognostic significance for response, the time between first diagnosis and begin of immunotherapy, usually reflecting metastatic disease necessetating systemic treatment, significantly correlates with probability of response (p= . ). since several hla class i alleles have been shown to function as restriction elements for recognition of melanoma cells by specififc t cells in vitro, namely a , a , b , and cw , we compared the frequency of these hla antigens between responder and non-responder. we found a , b and cw to be increased in responder vs. non-responder. our results indicate that in patients with mm tumor load, number of metastatic sites, ldh, and time from diagnosis to begin of immunotherapy are prognostic parameters for response to immunotherapy. these parameters may be useful to determine patients with good and poor risk for response to immunotherapy and are of relevance for stratification in randomized clinical trials. dept of medicine, university of heidelberg, hospitalstr. , heidelberg, germany surgery of metastatic melanoma following successful il- based immunotherapy. u keilholz , e stoelben , c scheibenbogen , hd saeger , k neumann , w hunstein surgery of advanced metastatic melanoma is of limited value and usually not recommended. immunotherapy using high dose il- is effective in a substantial proportion of patients, however, the duration of responses is limited, and benefits in survival are not yet proven. this evaluation was done to determine the value ot resection of residual tumor lesions following successful immunotherapy. patients with progressive metastatic melanoma have been enrolled in various immunotherapy trials including ifna and high dose il- since . patients showed evidence of antitumor response ( cr, pr, mr/sd). in patients responding to immunotherapy, residual lesions were resected, whenever technically possible and patients agreed to surgery ( patients). of the responding patients without surgery relapsed, the median time to progression was months (range - ), almost all initial relapses occured locally, patients died so far. of patients who underwent surgery ( pr, mr/sd) were converted into cr by surgery. of the patients disease-free after surgery relapsed, locally ( , , and months after surgery), and one cns months after surgery. patients are still free of recurrence ( +, +, +, +, +, +, + months after surgery) and of are still alive. in the patient with cns relapse complete resection of this lesion was again possible, and there was no evidence of recurrence for months after this second surgery. histology revealed vital tumor cells in almost all resected specimens, however in of patients profound necrosis of the tumor tissue was observed. of special importance is the observation that patients with minor response or sd according to imaging procedures were found to have an almost complete response histologically. interestingly, almost all metastases resected after immunotherapy had developed a fibrous capsule. surgical reevaluation and resection of residual lesions should be considered in patients with partial response after immunotherapy, and in selected cases also with stable disease. this approach offers the chance for extended disease free survival, and may be curative in certain patients. t-cell-receptor (tcr) vcz~ usage of tumorinfiitrating t-cells in primary, regressing and progressing melanoma metastases following [mmunotherapy with ifn~ and il- : evidence for a specific t-cell response. mshler, t., willhauck, m., scheibenbogen, c., pawlita, m.#, bludau, h.#, brossart. p.. keilholz. u. the identification and characterization of immunological effector cells mediating tumor regression in immunotherapy with il is of great interest for understanding and further development of this therapeutic approach. tumor infiltrating lymphocytes specific for autologous tumor cells can be expanded from certain melanoma tissues. t cells recognizing the same antigen use a limited tcr repertoire with a certain vc~ and [ variable region, determining the specifity of their receptor. we therefore analyzed t-cell receptor v-regio,q distribution in tumor tissue from melanoma patients prior to and following immunotherapy with il- . we used a highly sensitive rna-pcr method. after rna-extraction from tissue and subsequent cdna-synthesis semiquantitative pcr with different primers for all known vc~-and vii-t-cell receptor gene families ( vc~ and v~) was pedormed. tumor tissue samples were analysed including samples of primary malignant melanoma and tumor samples of three patients after immunotherapy. the results were compared to control tissues (peripheral blood, unatfected skin, and liver tissue). the analysis of primary malignant melanoma tissue showed a weak overexpression of different v[ -families. preferential usage of different tcr-v~,genes was more obvious in tumor tissue of patients alter immunotherapy. of special interest is a patient with a mixed response to immunotherapy with progressing and regressing skin metastases, in the regressing lesion we could demonstrate a predominant usage of tcr-v[~ -gene almost lacking in the progressing lesion. this suggests a role of v[ -expressing t-cells in mediating tumor regression in this patient. cloning and sequenzing analysis are currently performed to assess wether this represents a true clonal t-cell proliferation. recently a highly sensitive assay combining reverse transcription and polymerase chain reaction (rt/pcr) to assess for melanoma cells in peripheral blood has been developed. the detection of tyrosinase mrna, a tissue spezific enzyme in melanocytes and melanoma cells in peripheral blood indicates the presence of melanoma cells, we used rt/pcr assay to determine malignant melanoma ceils in peripheral blood of patients with malignant melanoma in different stages of disease. in none of patients with stage i (localised tumor) but in of patients in stage ii (regional lymph node metastases) tyrosinase transcripts were detected. tyrosinase mrna was found in all patients with distant metastases (stage iii). this method may be helpful to define a group of patients at high risk for development of hematogenous metastases, that would be a possible target group to explore adjuvant treatment strategies. we then examined blood samples and bone marrow aspirates of patients with metastatic malignant melanoma for presence of melanoma ceils prior to and after therapy with ifn-a and il- . patients showed antitumor response to immunotherapy: complete remissions (cr) and ? partial remissions (pr r. hilse, m.meffert, j.grosse, h.kirchner, h.poliwoda, and j.atzpodien we investigated the use of pcr for a semiquantitative estimation of cytokine expression patterns in pbmc before and after administration of il- to patients with advanced renal cell carcinoma or malignant melanoma, mrna of cytokines was measured using a modified polymerase chain reaction protocol, which could detect -fold differences in mrna-contents of stimulated pbmc in vitro. weekly rna-samples of patients receiving a total of treatment cycles were examined for long term changes, in patients frequent samples were taken immediately after tl- -administration for transcript-kinetics, mrnaexpression for il- , il- , il- , ifn-)', tnf-c~, gm-csf, tgf-~ and il- receptor-c~ was clearly detectable in most of the samples, including four healthy donors. however, our method could not detect significant changes in transcript-levels of pbmc during days following injection of (a) mio.lu or (b) x mioju dl- daily. this was in marked contrast to cytokine secretion assayed by elisa. thus, serum il- peaked - hours after administration followed by secondary cytokines with a peak - hours later. increases for tnf-m ifn- , il- and il- r serum levels were significant (p< , ) with the highest response found for il- , increasing -(a) and -fold (b) at day , or -/ -fold at day . comparing normal individuals to patients, only small differences in constitutive cytokine expression were seen (< -fold) with no distinct pattern. during therapy, changes could be seen for all cytokines except for il- and tgf-i . in one patient, a -fold increase for il- , tnf-c~ and ifn- transcripts was observed during week of the second treatmentcycle, other changes were approximately -fold. abt. h&matologie und onkologie, medizinische hochschule hannover, d- hannover , germany regional immunotherapy: perfusion of liver metastases with lak cells u. keilholz, c. scheibenbogen, m. brado, w. tilgen, and w. hunstein a regional approach of adoptive immunotberapy with interleukin- and lymphokine activated killer cells for the treatment of liver metastases is reported. the treatment consists of continuous infusion of interleukin- i.v. or into the splenic artery, and transfer of ex vivo generated lymplaokine activated killer cells into the portal vein or the hepatic artery. patients with malign ant melanoma, with renal cell carcinoma, and with thyroid carcinoma have been treated. all had progressive liver metastases. trafficking studies using indium-oxine labelled cells revealed that > % of the lak cells remained in the liver after regional adoptive transfer. in patients with liver metastases of cutaneous melanoma, cr ( and + months), pr ( + months, converted to cr by surgery), sd ( and months), ad pd were observed. the lesion in the patient with pr was resectable after two cycles of treatment, and histology revealed almost completely necrotic tumor tissue surrounded by a dense fibrous capsule. no responses were observed in patients with liver metastases of ocular melanoma, suggesting an immunologic difference between these two melanoma subtypes. pr ( months) and sd ( months) were achieved in patients with renal cell carcinoma, and sd ( months) in the patient with thyroid carcinoma. evidence for the crucial role regional cell transfer is provided by the observation in a patient with an anatomic variation of hepatic blood supply in whom we achieved complete and durable tumor regression. in this case anti-tumor responses were only observed in anatomic areas of the liver which were perfused with lak cells. depts. of internal medicine, diagnostic radiology, and dermatology, hospitalstrage , heidelberg, germany in a randomized phase ii study we evaluated the response and side effects of a combined administration of interleukin- (il- ) and interferon-alpha b (ifn-alpha b) versus interferon-gamma (ifn-gamma) in patients with metastatic renal cell cancer. patients in group a received subcutaneous (sc.) meg ifn-gamma once a week. in group b patients were treated with a (sc.) combination therapy of ill- ( x iu/m in week and , x' iu/m in week , , and , twice a day for days) and ifn-alpha b ( x u/m ) over weeks once a day times a week. up to now patients were treated, patients in each group. toxicity of ifn-gamma treatment was absent. the therapy with il- and ifn-alpha b led to sideeffects grade (who): fever, chivering, fatigue and weight-loss. treatment were withheld in %, follow up after months ( - months) showed stable disease in patients and progression inl patients in group a in group b there were complete remissions, partial remission and patients with progressive disease. although the combination therapy showed % objective response (p< . , fisher-test) no significant improvement on survival was seen (p = . logrank test). patients with locally advanced renal carcinoma are at high risk of relapse after initial radical surgery. we initiated a clinical phase ii trial using autologous tumor vaccines for the surgical adjuvant therapy of renal cancer patients. seventy-two patients (pts) ( female, male; median age, yrs; range, - yrs) with locally advanced renal carcinoma (pt b- pn or ptxni- m ) received autologous newcastle disease virus modified and lethally irradiated tumor vaccines in combination with . million iu of il- and . million u of ifn-~ , once weekly over consecutive weeks. toxicity was very mild with transient flu-like symptoms. among evaluable patients, there were relapses ( pts, pt ani- ; pts, pt bn ); the median relapse-free survival was + months with a range from to + months; survival probability in this vaccine treated cohort was significantly better than in all historic controls. using western blot analyses, we could demonstrate a vaccine specific in-vivo b-cell response in all patients receiving ndv tumor vaccine. a subset of peripheral blood natural killer (nk) cells has been found to exhibit high density surface expression of the nk associated cd antigen; it has been suggested that these nk cells respond to lower concentrations of il- when compared to the majority of nk cells expressing cell surface cd at low density. we evaluated density of the cd antigen on circulating nk cells of patients with advanced renal cell carcinoma by flow cytometry. patients received a combination of low-dose subcutaneous recombinant interleukin- (ril- ) at million iu/m /day on days and , followed by . million iu/m /day, days per week, over consecutive weeks, in combination with recombinant ~-interferon (rifn-~) at million iu/m , three times weekly. antigen density of cd before therapy was found . -fold higher (p< . ) in patients who subsequently achieved a complete or partial tumor remission (n= ) when compared with patients who presented with progressive disease on therapy (n=ll). after a -week treatment cycle, nk cells of treatment responders expressed significantly ( .l-fold; p< . ) more cd antigens than nk cells in nonresponding patients. these results suggested a potential role of both pre-and posttreatment nk antigen density levels as a biologic correlate to treatment response in tumor patients receiving low-dose ril- and rifn-a. intravesical immunotherapy against superficial bladder tumor recurrences and carcinoma in situ is a recognized and highly effective regimen in urology. to further clarify the mode of action of this approach, the local immune response of patients was investigated: the cytokines il- , il- , and tnf were determined in the urine before and after intravesical instillation by elisas and biological assays. furthermore, bladder biopsies taken before and after the treatment course were analysed by means of immunohistology for the presence of mononuclear cell subsets. the results show a significant increase of urinary cytokines with a maximum - hours after the instillation of bcg which returned to baseline values within hours. this intense locai immune activation was further reflected by the accumulation of activated mononuclear cells, predominated by t cells as demonstrated with bladder biopsies. the local t-helper/t-suppressor cell ratio shifted towards the t-helper subset. these changes persisted for more than year after the initial treatment course. in conclusion, this local immune response may be associated with the therapeutic success of bcg. further analyses will dissect the role of each factor with regard to antitumor cytotoxicity against bladder carcinoma. enhancement of therapeutic effect of intarleukin- (il- ) by association with cyclephcsphamide (cy) was studied on el-/+ lymphoma maintained in ascitic form in syngeneic c bl/ (h- b) mice and lymphoreticulosarcoma (spontaneous origin) maintained in solid form in syngeneic cba (h- k) mice. immunotherapy with il- (obtained by in vitro stimulation of el- lymphoma cells with phorbol myristate-acetate) was applied hours after transplantation of el- lymphcma and i days after transplantation of lym phoreticulosarcoma be administration i.p. (intratumorel) in el- bearing mice and s.c.(peritumoral) in lymphoreticulosarcoma bearing mice for three consecutive days. cyclophosphamide was administered at a dose of mg/kg i.p. six hours before the immune treatment with il- . the results obtained demonstrated that the prolongation of the survival rate expressed by the median survival time (mst) and the percentage of increasing llfe span (ils) of the groups treated with il- associated with cy was significantly higher than that of the groups which receveid a single treatment with il- or cy. enhancement of therapeutic effects of il- in association with cy on lymphoreticulosarcema was revealed by inhibition of tumor growth with a marked regression in the vol~tme of the established tumors and even resorption in some cases. we conclude that the antitumoral effect of il- treatment was enhancemented by association with c a well known cytoreductive drug which selectively removed t-suppressor l~mphocytes from the tumor bearers. this could be conside red as an alternative to immune-or chemotherapy in cancer. to investigate the toxicity and clinical efficacy of aerosolized nil- (biotest) patients presenting with advanced malignancy were entered into a phase i trial. patients suffered from metastasizing renal cell carcinoma, patients from advanced bronchial carcinoma. at start the patients received either , or u nil applied as a single dose. if no adverse events were observed, treatment was continued with the same dose times daily for six weeks. in addition to standard invetigations detailed evaluation of the respiratory function was performed once weekly. soluble interleukin receptor serum levels and the effect on numbers and/or phenotype of lymphocytes in the bronchoalveolar lavage fluid were measured for assessment of biological response to inhalative nil- treatment. treatment with aerosolized nil- was well tolerated. most prominent toxicity appeared to be resistant cough in all patients treated with x u/d. no febrile reactions or other constitutional side effects were observed. a dose-dependent increase of the numbers of memory t lymphocytes, macrophages and eosinophil granulocytes could be demonstrated in bal fluid. in addition, the treatment resulted an increased expression of adhesion molecules on lymphocytes. patient suffering from renal cell carcinoma achieved a partial remission after weeks of treatment with x u/d. we conclude that treatment with aerosolized nil- is biologically active and well tolerated and should be further tested in clinical phase ii trials. divisions of hematology and pulmology of the iiird department of internal medicine, medical center of the johannes gutenberg university, w- mainz, department of urology, univ. hospital eppendorf, hamburg. soluble interleukin- receptors (sil- r) exert a potential role in immunoregulation. we investigated the ex vivo effects of sil- r on several interleukin- (il- )-dependent activation events. proliferation of the il- -dependent mouse cell line ctll- and isolated human pbmc stimulated with recombinant il- (ril- ) was suppressed by sil- r added to the culture medium in a dose-dependent way. preincubation of sil- r with ril- did not enhance this suppression. cytotoxicity of ril- -stimulated human pbmc against the human cell lines k and daudi was correlated inversely to the concentration of sil- r in the culture medium during ril- stimulation. sil- r concentrations higher than . pm produced a significant decrease in cytotoxicity (p< . ). light microscopy of il- -stimulated pbmc revealed no signs of cellular activation when high dosages of sil- r had been added. the effect of different sil- r concentrations added to cultured human pbmc on secondary il- and sil- r production was tested by elisa. initial supply with high sil- r dosages yielded weak increase and subsequent slow reduction of il- levels. in contrast, strong secondary il- production followed by rapid clearance was observed when low sil- r concentrations had been added. endogenous shedding of sil- r in response to ril- was abrogated by the initial exogenous addition of high amounts of sil- r whereas low exogenous addition of sil- r was followed by a continuing endogenous production of sil- r after five days of culture. our studies may lead to a better understanding of il- -related immunoregulation in the preclinical and clinical settings. we investigated the effect of interferons (ifn) on expression of il- and other cytokines regulating inflammatory responses in various cellular models in vitro and in vivo. in peripheral blood mononuclear cells (pbmnc) of healthy individuals il- gene expression which was upregulated in vitro was significantly reduced in presence of ifn-o~. in dose titration experiments a reduction of the il- protein was detected at ifn concentrations as low as u/ml. in cml patients with constitutive expression of il- a reduction of il- mrna expression was seen after therapeutic administration of ifn-a. by contrast, in lps stimulated granulocytes ifn failed to inhibit il- expression in vitro. we investigated the mechanism of il- inhibition in the thp- cell line more in detail. nuclear run on assays and rna decay analysis in presence of acinomycin d suggested that the effect was regulated predominant/y at a posttranscriptional level. de novo protein synthesis was not required since the inhibitory effect was also detected in presence of cycioheximide. in addition to il- expression we studied the effect of ifn-o~ on the synthesis of il- , tnf, il- and il- ra in pbmnc and bone marrow stromal cell cultures. these experiments revealed an antagonistic effect of il- action by ifn x at two levels which was most striking in bone marrow stromal cells. expression of il- mrna was downregulated whereas the production of il- ra was enhanced by ifn-c~. in contrast expression of il- and tnf was enhanced by ifn. we conclude that ifn-o~ differentially regulates proinflammatory cytokines. the inhibition of il- and il- action suggest an antiinflammatory role of type i ifns. in a phase i clinical trial of recombinant human interleukin- (il- ), patients were entered to receive daily subcutaneous injections of il- over days followed by a two week observation period and another weeks of daily il- injections. doses varied between . and pg/kg body weight. patients were evaluable for studying immune functions. at all dose levels il- administration led to a marked increase in serum levels of c reactive protein and complement factor c . natural killer (nk) cell activity was reduced at doses exceeding pg/kg. similarly lymphokine activated killer (lak) cell activity induced by in vitro culture over days in the presence of u/ml interleukin- (il- ) was suppressed at and p.g/kg, as was the proliferative response to il- in vitro. however no changes were observed in the proliferation induced by phytohaemagglutinin, pokeweed mitogen or fixed staphylococcus aureus. there were no changes in peripheral blood lymphocyte subpopulations as measured by cd and cd , nor in the expression of hla dr. serum levels of immunoglobulins iga, igm and igg remained unaffected by il- treatment. in contrast we found consistent elevations in levels of ige all over the dose range. we conclude that il- inhibits nk and lak activity in vivo which may be of interest in future studies with cytokine combinations and that the role of il- in ige related diseases might be more important than previously thought. one of the most potent stimulatory agents for the induction of cytokines in myeloid cells is the bacterial ceuwallproduct lps (liopopolysaccharide or endotoxin). in the bloodstream it forms a complex with lbp (lps binding protein) and is recognized by effector cells via the cdi receptor. here we report on studies performed with human peritoneal macrophages that were stimulated in vitro with lps and a synthetic lps homologue in the presence and absence of serum. as revealed by elisa-based analysis of the cellsupernatants, strong, serum-dependent resonses were seen for tnf-, il- , il- and g-csf production, while unstimulated cells produced basically only il- . repeated stimulation of the cells with lps resulted in adaptation that was different for certain groups of cytokines. the "adapted" ceils produced much less tnf and il- while il-i and g-csf was superinduced. stimulation of the cells with the lipid a anolog mrl showed a similar picture, given that mrl had to be used in higher concentration. "adapatation" of the cells with mrl also resulted in an "adapted" response to a challenge with a high dose of lps so that it might be useful as a therapeutic agent for preventing the septic shock syndrome, e.g. northern blot analysis showed that the differentiated response of the cells towards a low dose lps stimulation regarding cytokine production after an lps challenge occured on transcriptional level, as mrna levels were regulated accordingly. these results give evidence that two different pathways for lps dependent stimulation of myeloid cells for cytokineproduction exist and experiments to further elucidate this phenomenon and possibly discover cdi independent and dependent pathways are underway. the neutrophil-activating peptide (nap- ), a member of the "intercrine"-family of chemotactic and reparative host defense cytokines, represents one of several n-terminally truncated cleavage products that originate from platelet-derived -thromboglobulin through proteolytic processing. here we present evidence that there exists also a naturally occurring c-terminally truncated form of nap- that is about four times more potent in eliciting neutrophil degranulation than the original cytokine. the novel molecule was detected in concentrates of culture supernatants from peripheral blood mononuclear ceils and could be separated from authentic nap- by several steps of column chromatography. according to amino acid sequence analysis it had a n-terminus identical to nap- , whereas electrophoretic analyses indicated a lower molecular weight as well as a higher isoelectric point. immunochemical analyses performed with epitope-characterized antibodies raised against nap- c-terminal synthetic peptides identified limited truncation at the c-terminus of the variant molecule. comparison of reactivity patterns of these antibodies in western blots as well as in a nap- biologic assay (pmn degranulation assay) confirmed that the variant nap- was truncated by at least one and by maximally three residues. thus, there is for the first time evidence that proteolytic processing at the n-terminus is not necessarily the only mechanism regulating the formation of neutrophilactivating peptides, but that modification at the c-terminus may assist in the fine-adjustment of biological activity. cytokines like interleukin- -beta (ill), interleukin- (il ), interleukin- (il ) and tumor-necrosis-factor-alpha (tnf) are involved in the pathogenesis of fever and infection. however, intra-and inter-individual values differ considerably and there is only limited data on early.cytokine serum levels and their evolution in febrile neutropenic patients. therefore. we measured cytokine levels in adults with chemotherapy-induced aplasia and fever. concentrations of ill. il and tnf were determined by irma. il by elisa in specimens per patient. ill and tnf were elevated in of patients with peak values of pg/ml and pg/ml, respectively. il and il were elevated in all patients with a maximum of . pg/ml (median pg/ml, range - pg/ml) for il and . oo pg/ml (median pg/ml, range - . ) for il . both cytokines showed a high correlation (r= . ). the individual il concentration-time curve closely paralleled the temperature curve. in of patients il was elevated before onset of fever and peaked at or one hour before the temperature maximum in seven cases. so also in the cytopenie patient lacking a main source of cytokine producing cells and cytokine target cells consistently high il and il serum levels can be detected very early in the course of fever and infection. the biological and clinical significance of this cytokine response and its regulation mechanisms remain to be determined. interleukin (il- ) and the neutrophil-activating peptide (nap- ) are two closely related members of the "intercrine" family of host defense cytokines. the expression of at least two different receptor classes for il- on human neutrophils (pmn) exhibiting similar affinities has been demonstrated recently. using iodinated ligands we could directly demonstrate that si-nap- specifically bound to pmn with two different affinities, characterized by kd-values of about . nm and . nm, respectively. cold -residue il- competed with iodinated nap- for binding to the high affinity site(s) with practically equivalent efficacy, while it was significantly more effective in displacing %nap- from its low affinity site(s) than was cold nap- itself. as new findings, unlabeled il- could completely displace ~ -nap- and vice versa, indicating that there are no distinct binding sites for either cytokine on pmn. in contrast to il- , nap- did not induce pmn degranulation at concentrations ( nm), engaging solely its high affinity site. however, short-term priming of pmn with the same amount of nap- dramatically down-regulated degranulation inducible by higher concentrations of nap- as a secondary stimulus. the il- -induced secondary response was also diminished, but to lower extents. these phenomena correlated with the rapid downregulation and internalization of nap- high affinity binding sites from the cell surface. thus, our data provide direct evidence for a regulatory function of nap- at very low concentrations, obviously occurring through the modulation of nap- and il- receptor expression on pmn. determination of cytokine plasma levels possesses many promising features concerning monitoring and studying of an array of different diseases including febrile reactions. detailed analysis of the role of these factors is of crucial importance for the understanding of the complex cytokine network. investigation of cytokine blood levels however is complicated by their short half-fife in circulation, the presence of soluble inhibitors and the ill-defined beginning of fever. looking for a suitable in-vivo-model allowing a sequential and well-defined analysis of cytokine plasma levels we chose the acute toxicity after intravenous amphotericin b (am b) application consisting of fever, chills and hypotension. these side-effects were reported to be mediated by release of pro-inflammatory cytokines such as tnf a and interleuldn (il- ). in order to compare mutual interations and different temporal patterns of liberation we determined a panel of cytokines including tnf a, s-tnfreceptor (s-tnf-r), interleukin- , interleukin- -receptor-antagonist (il- -ra), intefleukin- and interleukin- from patients suffering from acute leukemia and fungai infections. serial edta-plasma samples were obtained before and up to hours after start of am b infusion. samples were immediately centrifuged and stored at - ~ c until analysis by elisa (medgenix and r&d systems). patients experiencing adverse reactions showed tnf c~ peak plasma levels - minutes after starting am b infusion reaching maximum concentrations of pg/ml. concentrations declined m base levels within the following - hours. il- as welt as il- concentrations showed similar, but delayed changes of plasma concentration. compared to tnf a s-tnf-rlevels peaked about minutes later with a prolonged decrease to basal concentrations. in contrast to tnf c~ no circulating il-i- could be detected, while il- -ra demonstrated up to -fold increases in concentration. we conclude that this model of a drug induced acute-phase-reaction offers wide possibilities for studying the behaviour of inflammatory cytokines and their inhibitors by means of plasma level determination. inflammatory processes following severe trauma were found to be associated with an abnormal high secretion of inflammatory cytokines. these cytokines are discussed to be involved in neutrophil activation associated with the release of high amounts of destructive lysosomal proteases into the extracellular space. the task of our investigations was to evaluate the possible regulation of the degranulation of neutrophils by the immunostimulatory cytokine il- and the immunosuppressive factor tgf-i.~. we analysed the concentration of the cqantltrypsin-complex of the lysosomal protease elastase as markers for the degranulation of neutrophils as well as the levels of il- and tgf-i~ in the plasma of patients with multiple trauma or after severe surgeries. the time courses of the plasma levels of il- and the elastase-inhibitor-complex were found to be highly correlated, suggesting a possible regulatory role of this cytokine on the neutrophil degranulation. however the plasma concentrations of tgf-~, were not significantly altered in comparsion to the control group. in additional experiments, the effect of both cytokines on the degranulation of healthy donors was investigated in vitro. pathological high concentrations of rh~l- up to ulml (as detected in several probes from the surgical area) were found to be capable to induce a significant degranulation of the azurophilic granules ( , _+ , % of the total cellular enzyme content) under serum free conditions as detected by measurement of elastase release by elisa technique and enzymatic methods. in contrast to this, the degranulation of neutrophils was found to be uneffected by tgf-i~. in conclusion, these data suggest that the inflammatory cytokine il- may contribute to the activation of neutrophil granulocytes in acute inflammatory processes following severe irauma, whereas the immunosupresive factor tgf-fil seems to have no direct regulatory effects beside the described chemotactic effects on neutrophils. we determined serum concentrations of soluble tumor necrosis factor receptor (stnf-rs) in hiv infected individuals. eighty-five percent of these had increased serum concentrations of stnf-r type i (p ) (stnf-r ) and % had increased stnf-r type ii (p ) (stnf-r ). the extent of the increase of stnf-r was greater in more advanced hiv infection (p= . ) as it was measured by dividing the individuals into two groups according to the median of the cd + t cell count, stnf-r- did not differ between these two groups. a strong correlation was found between stnf-r and the soluble immune activation markers b -microglobulin (rs= . , p< . ) and urinary neopterin rs= . , p< . ), and a less strong correlation with interferon gamma (rs= . , p= . ). the correlations observed for stnf-r were also significant but were always weaker than that for stnf-r . a weak inverse correlation was found between the number of cd + t cells and stnf-r (rs=- . , p= . ), no such correlation was observed with stnf-r . our findings suggest that increased concentrations of serum stnf-rs in hiv infection are linked to immune activation where synergistic action of interferongamma and the tnf-alpha system are likely to play an important role. to answer the question whether il- is of hematogenous origin, immunohistochemistry and in situ hybridization with il- specific s labeled rna probes was established to study the frequencies of il- expressing peripheral blood mononuclear cells. a two color immunofluorescence assay with antibodies to cd and il- was utilized to correlate il- mrna expression and il- protein production in monocytes of patients and control individuals. - % of circulating monocytes spontaneously expressed il- mrna compared to % in normal individuals. a strong correlation of il- protein production and il- mrna was found in monocytes of patients and controls. semiquantification of il- mrna and il- i mrna by pcr demonstrated that about to fold higher amounts of mrna were found in untreated patients compared to normal individuals. in contrast, we did not find evidence for excessive synthesis of tnfa mrna. the overproduction of il- and il- in the absence of detectable tnfn mrna establishes a specific cytokine pattern in circulating monocytes of pmr and gca patients. we analyzed the il- mrna expression in biopsy specimens from gca patients applying in situ hybridization. autoradiographs were analyzed by visual examination and by using an image-analysis system. tissue infiltrating macrophages expressed il- mrna, however, the proportion of il- mrna + cd t was lower than in the peripheral blood. also, immunohistochemistry with an il- specific antibody demonstrated that only a small fraction of macrophages produced this cytokine. these data suggest that in pmr and gca, circulating monocytes are activated and produce il- and il- . tissue infiltration of macrophages is not accompanied by a local activation, in contrast, cytokine production is downregulated. our objective was to evaluate the prognostic value of p protein levels in patients with advanced hivinfection during cytokine therapy (ifn or ifn/combination). methods: p serum levels were measured every two months by elisa in hiv-infected patients during a period of to months (median , ) and compared with clinical stage (wr), disease progression and response to therapy. patients suffered from an infection or an inflammation disease during therapy and from kaposi sarcoma (ks). results: in patients with stable disease constant p levels (• ng/ml) were observed. changes of disease correlated with distinct increasing levels of p (in patients with acute infection and/or inflammation disease, in patients with progress of ks). after start of azt-therapy, chemotherapy and antibiotic/ antimycotic therapy p level decreased. the highest amount of p (up to ng/ml) was found in phases with opportunistic infections. the lowest amount of p ( ng/ml) was observed in a clinically stable patient without an evident progress of disease. conclusions: here, we detected increased levels of p in hiv-i infected patients under ifn-therapy during acute opportunistic infection. protein p may be a useful marker for the activation of the immunesystem and a prognostic marker during the course of ifn-therapy. previous studies have shown that the diacetylated synthetic pentapeptide splenopentin (dac-sp- ) accelerates hemopoietic recovery following sublethal irradiation and nutologons bone marrow transplantation in mice, but the effects of the peptide on human bone marrow cells were still unknown. in this study, human granulocyte-macrophage (rhgm-csf), macrophage (rhm-csf), and granulocyte colonystimulating factor (rhg-csf) as well as interleukin-l~ (rhil-l~) and interleukin- (rhll- ) were compared for their stimulatory activity on human granulocytemacrophnge colony-forming cells (cfc-gm) alone and in combination with dac-sp- . after depletion of accessory cells from bone marrow mononuclear cells (bmmnc) in semi-liquid cultures the combination of rhgm-csf plus splenopentin stimulated the growth of cfc-gm/-m in dose-depended manner. furthermore, similar effects were seen in combination of dac-sp- plus rhil-la and and rhil- , but not in rhg-csf and rhm-csf plus splenopentin. in unseparated as well as in bmmnc enriched for cd + cells comparable stimulatory effects of sp- alone were missed. additionally, in bmmnc enriched for cd +/cd + population, the preculture with dac-sp- for l days enhanced the ability of rhgm-csf, rhil-lct and rhll- to induce colony formation from this cell source. however, in all these combinations, mainly differentiation to macrophage lineage has been observed. pheuotypie analysis of precultured bmmnc with splenopentin leads to the suggestion that this compound may recruit a cell population being more sensitive to gm-csf, il-la and il- , because the percentage of cd + cells decreased rapidly, whereas the expression of hla-dr + was enhanced. therefore, this potentially interresting molecule might be a candidate as a therapeutic adjuvant with hemopoietic growth factors. although, the examination of gm-csf, il-lcc and . the role of t-lymphocyte subsets in the development of aplastie anaemia (aa) remains poorly understood. therefore we analysed by cytophomotry the contribution and proportion of lymphocyte subpopulationa in the peripheral blood (pb) and bone maxrow (ibm of patients with aa before, after weeks of treatment and additionally after weeks of therapy with anti-lymphocyte globulin (alg), methylprednisolon, and eyelosporine a (csa). for double labeling immtmofluorescence studies mouoelonal antibodies directed against the following specifities were used: tcrct~-, tcr~ , tcs , cd , cd , cd , cds, cd , cd , cd , and hla-dr. hi patients with aa a sigmficant decrease of cd positive t-cells was observed after weeks of therapy (pb , . , ; bm , • as compared with normal controls (pb , • bm , + , ). before therapy, the cd /cd ratio in pi and lqm did not differ ~om the ratio in the control population; however a reversed ratio (< ) was present in pb and bm after and weeks of therapy. the number of activated t-cells defined by the antigens cd , hla-dr and cd were low or in the normal range,and did not fu~er decrease during therapy in contrast to the non-activated t-cells. ? -t-cells were significantly decreased betbre and after weeks of therapy as compared with healthy controls. however, the proportion of the ~/~-subpopulation ~tcsi was markedly increased betbre (pb +_ , ; bm +_ , i) after weeks (pb , + , ; bm , +_ , ) and esspeciauy after weeks of therapy (pb ,.t~l . ) as compared with that in normal subjects (pb _+ , ; bm , _+_ , ). these data indicate that tcs -t-celis are a t-cell population not affected by treatment with alg, methylpredrtisolone and csa . ftwther studies have to show whether tiffs subpopulafion has an effect on the hematopoiesis of patients with aa. il- j , il- , tnf-i , g-csf and other cytokines are characterised to have proline in the second position of the n-terminal peptide sequence. from this they could be potential substrates of the dipeptidyl peptidase iv (dp iv). using the method of capillary electrophoresis, here we show that purified soluble dp iv is capable of hydrolysing oligopeptides with sequences analogous to the n-terminal part of human il-lb, il- , tnf-b and mouse il- up to a length of amino acids. furthermore, it could be demonstrated that hydrolysis rates are negatively correlated the with chain length of the oligopeptides. glycosylation of threonine in the third position of the il- hexapeptide sequence has no effect on the hydrolysis rate of this peptide by the dp iv. in contrast to these results, no degradation was found in the case of rll-lp, rll- , natural il- , and rg-csf, using up to -fold higher dpiv concentrations than in the experiments with oligopeptides. after incubation with both, dp iv and aminopeptidase n, also no cytokine degradation was found. possible explanations for this results will be discussed. hemopd~'etic growth factors are now being tested in several institutions, in an effort to reduce the duration of neutropenia after bone marrow trasnplantation (bmt). in the past years, we have conducted or participated to several double blind multicentric studies using gm-csf and g-csf (schering-plough/sandoz, behring/hoechst and roche lab.) in patients (pts) with nhl. we wish to summarize these studies and also report on our own observations. -gm-csf post-abmt: double blind international studies and the french national trial that we conducted (blood (blood , , (blood - have clearly demonstrated the gm-csf infusion post-abmt significantly accelerates neutrophil recovery by to days, both in pts receiving unpurged marrow or marrow purged by mafosfamide. the duration of hospitalization is reduced by days with a possible cost benefit. -in pts with documented cmv infection requiring dhpg treatment, myelosuppression was not seen in those who received gm-csf and dhpg concomitantly while in contrast a severe neutropenia developped in those who received dhpg after administration and discontinuation of gm-csf (lancet, , . - pts. in our institution received gm-csf in a compassionate use for delayed enraftment or engraftment failure after autologous (abmt : ) or allogeneic bone marrow transplantation (bmt : ). the pretransplant regiment included total body irradiation (tbi) in and consisted of high dose polychemotherapy only in . interestingly pts were transplanted for acute myelocytic leukemia (aml), a disease in which the presence of receptors to gm-csf has been detected in vitro in about % of cases, pts hat nhl, and all. of the abmt, pts received marrow heavily treated in vitro by mafosfamide and marrow purged by long term marrow culture (ltc). in (aml , all , nhl ), granulopoietic recovery occured within days ( - ). this included a pt with refractory all who received ltc marrow, developped cytomegalovirus (cmv) infection and had not engraftment by day : in this pt the absolute nucleated count (anc) peaked to . /i, days later. an additional pt with aml had not engrafted by day when gm-csf was administered for days with no efficacy. infusion of back up marrow (which has never been effective in our past experience) combined with gm-csf and cyclosporine a was follwoed by sustained engraftment occuring days later. dt (aml) had a minor and transient response and failed. additional pts in the context of entgraftment failure have since received in various sequences gm-csf and cyclosporine a + back up marrow after no response to gm-csf. have recovered sufficient hemopdfesis strongly suggesting a role of cycloporine a in engraftment failure (manuscript in prepration). we conclude that gm-csf is now the first line treatment for poor engraftment and/or engraftment failure. we do not freeze any longer back up marrow in pts autografted after pretransplant regimens not containing tbi. in pts with engraftment failure post tbi, who do not respond to first line gm-csf, we suggest that the combination of back up marrow and cyclosporine a on top of gm-csf should be further evaluated. -we administered gm-csf at a dose of #g/m /day to pts with resistant nhl and bone marrow involvement after the beam myeloablative regimen; reconstitution occurred within the same delays than observed after abmt in . we propose that gm-csf may replace abmt in highly selected cases of non-hodgkin's lymphoma with progressive disease and bone marrow involvement (lancet , , ) . -we have transplanted since september nhl pts with cd purified stem cells followed by infusion of gm-csf. successful engraftment was obtained in all with recovery to . x pmn/i by day ( - ) and to x plts/l by day ( - ). the expansion of cd + cells in short term liquid culture with cytokines (including gm and g-csf) is another approach toward transplantation with total abrogation of neutropenia. overall the introduction of hemopoietic growth factors in transplant units has considerably changed the situation in several aspects. further studies will combine gm-csf and g-csf to other cytokines. dept. of hematology, bone marrow transplant unit -h pital st-antoine, paris, france alter a pre-phase of vcr . rag/@ (maximum rag) i.v. days , and pradnisolone rng/n~ p.o. days - the high-dose chemotherapy consisted of prednisolone rag/n? p.o. days - , ifosfamide days - , methotrexate , mg/m day as a hour infusion, cytosine-arabinoside , mg/m i.v. days + , and etopeside i.v. days + . etoposide has been escalated from mg/rn to rng/ nf at the present time. the dose of itostamide is currently escalated from , rag/ rlf to , mg/rtf and finally , mg/rn as a continuous hour infusion. the high-dose chemotherapy is repeated for a maximum of four times. during the prephase pg/kg fiigrastim (recombinant g-csf) are given twice daily. apheresis of apbsc is done on days - . apbsc are reinfused after the high-dose chemotherapy and fiigrastim is given at a dose of p.g/kg. with wbc rising to > , /p.i aphereses are performed repeatedly. the pre-phase for induction of pbsc was excellently tolerated and a median of we report here the effects of long-term g-csf subcutaneous administration in patients (congenital n= , cyclic n= , idiopathic n= ) treated for - years. a sustained anc response was seen in / congenital patients, in / cyclic patients, and in / idiopathic patients. the g-csf doses needed to maintain these responses ranged between and #g/kg/d. the anc responses were associated with a significant decrease in the incidence of severe infections and the need for intravenous antibiotics. g-csf has been well tolerated in the majority of patients and resulted in a dramatic improvement in the quality of life. the adverse events noted included: osteopenia (n= ), vasculitis (n= ), mesangioproliferative glomerulonephritis (n= ), and the development of mds/leukemia (n= ). these adverse events are most likely associated with the underlying disease and not caused by g-csf treatment we have evaluated recombinant human granulocyte-macrophage colonystimulating factor (rhgm-csf) (sandoz-schering/plough) as an adjunct for cop-blam in the primary treatment of high grade malignant non-hodgkin's lymphomas (nhl). patients (n = , stage ii-lv, age - years), were randomized to rhgm-csf ( #g) or placebo for days s.c. following chemotherapy. efficacy was analyzed for patients receiving at least % of study medication (n = ). the frequency of clinical relevant infection was reduced by rhgm-csf ( vs infections, vs patients, p = . ) with a cumulative probability of remaining infection free in % vs % (p = . , log rank test at days). periods of neutropenia (p _< . in / courses), days with fever ( . vs . , p = . ) and days of hospitalization for infection ( . vs . days, p = . ) were significantly reduced. complete response (cr) rates, assessed by prognostic risk patients (pts) entered an outpatient protocol designed to test the tolerance, and the clinical and biological effects of extended administration of sc low-dose il- following high dose chemotherapy with autologous bone marrow rescue (bmt). two il- regimens were used. protocol a consisted of a once daily dose administered in -day cycles of millions iu/m z sc ril- (ru kindly provided by roussel-uclaf) every other week. the pts were treated at home and the treatment was scheduled for months (i.e. cycles for months) in the absence of major disease progression or sideeffects. protocol b consisted of a once daily dose of millions iu/m sc il in days/cycles for consecutive weeks (i.e. cycles for weeks). il- was started to days following bmt. blood lymphocyte subsets and nk/lak cytotoxic activity were determined monthly. pts received regimen a and pts were given regimen b. in both regimens, inflammatory skin reactions were the main side effect, leading to interruption of treatment in case. no capillary leak was observed. flue like syndrom was occasionny observed in protocol b. hematological parameters were not adversely affected by il- . at the lowest dose levels (regimen a), long term administration of il- did not produce any changes of blood lymphocyte subsets. on the other hand, the administration of millions iu/m il- resulted in a significant increase of cd +, cd + cells and cd -cd + ceils, and nk/lak cytotoxic activity of fresh pbls. this study confirms the feasability of long-term administration of sc lowdose il following autologous abmt. a dose of millions iu/m /d resulted in detectable activation of circulating lymphocytes. further studies are needed to assess the clinical impact of prolonged low-dose il- in this clinical setting. aim: to study the incidence of infectious episodes (ie) in a single centre series of autologous bmt patients who received il- in an attempt to prevent relapse. patients. methods: il- was given as a oontinuous i.v. infusion through a hickman catheter with a portable pump: . iu/m /d for the first week and . iu/m /day thereafter during weeks. il- was started when full neutrophil engraftment was achieved and platelet counts remained stable over . platelets/mm (median: day + . range: - ).median age was years ( - ). were mate. bmt was performed for acute lymphoblastic leukemia ( ), lymphoma ( ), and myeloma ( ). results: il- planned therapy was completed in patients ( early relapse, refusal) in a median of days ( - ). median intensity dose of il- was . x iu/m /day ( . x - . x ). infectious episodes who precised admission to the hospital were observed in patients. ol these infections were microbiologically documented: % of isolations corresponded to gram negative and % to gram positive. first ie was detected at a median of days ( - ) after the starting of il- . median cumulative tl- dose until first infectious episode was x iu/m ( . x ~' - x loe). catheter were removed in of them. in our experience, low dose continuous i.v. administration ol il- is associated with a high incidence of infectious episodes. sr (sanofi recherche) is a cho-derived r-il , glycosylated on the threonine in position . its specific activity is identical to native il . dose escalation studies were performed using sr either as iv bolus or iv continuous infusion in advanced-stage cancer patients. treatment schedule consisted in three -day cycles with -day rest in between. pts received iv bolus q - hrs at dose levels ( ; ; ; millions iu/m-'/bolus) and pts received iv continuous infusion at dose levels ( . ; . ; ; ; millions iu/m /day). mtd were found to be millions iu/m q hrs/day and millions iu/m /day for bolus and continuous iv infusion respectively. toxieities were similar in nature to those described under treatment with non-glycosylated rll- . dlt were mainly related to capillary leak. pr (nhl) was obtained. a significant rise of t-cell and nk cell subsets as well as nk/lak cytotoxicity in blood was observed at day . stimulation was already maximum for the lowest dose levels. at . millions iu/mz/day, the mean number of cd + cd + cells increased from to /mm -~ on day . similarly, cd + cells increased from to /mm , and cytotoxicity of fresh pbl against k increased from % to % (e/t ratio = : ). in vivo, sr appears to exhibit effects similar to those observed with higher doses (x - fold) of non glycosylated rll . although intravesical therapy with bacillus calmette-gutrin (bcg) against superficial bladder cancer recurrences and carcinoma in situ is highly effective, its mode of action is still unclear. the bladder tumor cell lines bt-a, bt-b (grade transitional cell carcinoma), sbc , and sbc (grade transitional cell carcinoma) are nearly resistant to natural killer cell activity in vitro. we could demonstrate that these cell lines are susceptible to lymphokine-activated killer cells generated by interleukin (il)- or interferon (ifn)-% we have now investigated whether bcg itself is able to activate a lak cell-like reaction against bladder tumor cells. our results show that activation of pbmc with viable bcg is a potent way of generating cytotoxic effector cells against bladder tumor cells. in contrast, killing of the nk and lak cell-sensitive k cell line was not enhanced by bcg-induced pbmc. because of their different target cell pattern and their, compared to lak ceils, distinct way of activation these cytotoxic effector cells were termed "bcg-activated killer (bak) cells". antibodies neutralizing ifn-t activity blocked the induction of bak cell cytotoxicity, indicating that this cytokine is playing a crucial role during this process. with respect to the phenotype of bak cells, our data show that the effector cells belong to the cd +/cd § lymphocyte subset. depletion of either cd + or cd + cells from bcg-induced pbmc led to a decrease of cytotoxicity against bladder tumor cells. furthermore, positively selected cd + cells could maintain the level of cytotoxicity exerted by bcg-induced pbmc whereas the depletion of cd § cells from this population also eliminated the cytotoxic effect. a direct involvement of cd + cells or macrophages in the killing of bladder tumor cells was not observed. in patients with superficial bladder cancer lak and bak cells might play an important role in the maintenance of the relapse free state. in this report we summarize our experience with high-dose therapy and peripheral blood stem cell (pbsc) autografting in advanced malignant lymphoma. since may , patients ( male / female) were included into this study. the median age was years (range - ). patients had hodgkin's disease and non-hodgkjn's lymphoma ( low-grade / highgrade nhl). four patients received recombinant g-csf (filgrastim) ( i~g/m / day s.c.) during steady-state hematopoiesis, while in the remaining patients recombinant g-csf (filgrastim) was started hours after conventional chemotherapy. for all patients, a target quantity of . x /kg total nucleated cells (tnc) was reached. a wide interindividual range with respect to the level of circulating hematopoietic progenitor cells ( -fold for cfu-gm/ml and fold for cd + cells/id) was observed. with a median number of leukaphereses (range - ), a median of . x cfu-gm/kg bw and . x cd + celts/kg bw could be harvested, respectively. high-dose therapy consisted of either tbi ( . gy, hyper-fractionated)/cyclophosphamide ( mg/kg) or the beam-protocol. with the exception of patient who died of a respiratory distress syndrome days following autografting, transplant-related toxicity was moderate. the low toxicity is reflected by the kinetics of hematological reconstitution: (median) days for . x / pmn and days for x ~/i platelets. platelet reconstitution was closely related to the number of cd + cells reinfused. twenty-six patients are evaluable for response: patients ( %) relapsed after a median of months (range - ) posttransplant. the remaining patients are alive in remission with a median follow-up of months (range - ). in summary, recombinant g-csf (filgrastim) is highly efficient in mobilizing pbsc capable of restoring hematopoiesis after myeloablative conditioning therapy. the quantity of cd + cells harvested was inversely related to the amount of previous cytotoxic chemotherapy. therefore, high-dose therapy with stem cell support should be considered as an upfront treatment modality in poor prognosis nhl patients.department of internal medicine, university of heidelberg, heidelberg, germany. many of the in vivo effects of the haemopoietic cell growth factors may well have been anticipated based on their known ability to stimulate proliferation and development of multipotent and lineage-restricted progenitor ceils in vitro. what was not predicted from these in vitro studies, however, was the observed ability of at least some of these growth factors to mobillse large numbers of haemopoietic progenitor cells from the bone marrow into the peripheral blood. this was an added "oonus" effect of growth factors and one that is now being widely exploited in a variety of clinical situations.initially these mobilised pbpc were used, in combination with bone marrow cells, to facilitate the more rapid recovery of myeloid cells following transfer into patients receiving high dose cytotoxic therapy. but when experimental studies showed that the mobilised pbpc also contained primitive (repopulating) stem cells -this encouraged the use of pbpc alone as a source of cells for transplantation. in most of these studies however, collection of the pbpc was performed over several cycles of apheresis -and this was somewhat limiting to their widespread use.because of this, our approach has been to define the optimal growth factor regimen for mobillsation of pbpc such that apheresis is not required, i.e. to use whole blood and then to identify groups of patients who may benefit from receiving these cells either for autologous or for allogeneic marrow recenstitution. using a sensitive immunohistochemical technique, . -> . logs of breast cancer cell depletion was documented in the positively-selected fractions of marrow ( patients) and -> logs from the pbpc fractions ( patients), in whom tumor was initially detected. to date no tumor has been detected in the cd + pbpc fractions. the engraftment rates for cohorts and were significantly faster than those of the other cohorts. the preliminary data suggest that since cd + pbpcs alone are capable of restoring hematopoiesis following high-dose therapy, a marrow fraction may no longer be needed for this purpose. longer follow-up will be required to assess the ultimate therapeutic effect of the entire treatment program. the principal morbidity and mortality of high dose chemotherapy with autologous bone marrow support (abmt) relates to the infectious complications which occurs during - week aplasia until the marrow autografi recovers. progenitor cells can be mobilized into the peripheral blood compartment by hematopoietic growth factors, alone or used after chemotherapy. we describe four trials using cytokine-mobilized peripheral blood progenitor cells (pbpc). in the first trial, pbpc collected after gm-csf administration are used to augment marrow. reconstitution of tririneage marrow function occurred quickly, resulting in short hospital stays and fewer platelet transfusions. in a second study, gm-csf/chemotherapy-mobilized pbpc were used as the sole hematopoietic support during high dose chemotherapy. granulocyte and platelet reconstitution was rapid. time to hematopoietie recovery, transfusion requirements and duration of hospital stay were all significantly improved for the patients receiving pbpc compared with similar patients receiving marrow alone. however, some patients had poor platelet engraftment. two recent trials were designed to explore multiple high-dose therapy. in the third trial, pbpc with and without marrow made it feasible to deliver two sequential cycles of high dose therapy. the fourth trial utilizes pbpc in addition to cytokines to deriver four cycles of dose-intensive therapy utilizing doses of chemotherapy that could not be given with cytokine support alone. pbpc appears to make multiple course combination high dose therapy feasible, is particularly useful to support platelets, and may enhance the safety, tolerance and cost of high dose therapy. severe neutropenia and functjonal defects of neutrophils are an increasing problem in treating advanced hiv infected p. neutropenia may result from bone marrow failure due to hiv by itself, or maybe.secondary to drug toxicity (mainly zidovudine, ganciclovir, chemotherapy) or bone marrow infiltration (mycobacterial infection, neoplasms). p. with severe neutropenia are at a higher risk of developing bacterial infections. since both gm-csf and g-csf have been-successfully used in cancer to optimalize dose intensity and to prevent neutropenia and secondary infections in p. receiving myelotoxic agents, clinical trials have recently been conducted in arc and aids p. with neutropenia to determine their potential benefits and toxicity profile. our previous experience in treating p. with non hodgkin's lymphoma (nhl) with a -cycle third generation regimen (promnce-cytabom), with gm-csf given at alternate cycles, clearly demonstrated that doses of chemotherapy following the adjunction of gm-csf were significantly higher than doses given without prior gm-csf administration. gm-csf was largely well tolerated in those p., with no sign of active viral replication as measured by hiv- p antigenemia. we have also performed an open clinical trial in p. with severe neuttopenia to assess the efficacy and tolerance of gm-csf with a three weekly subcutaneous administration in p. who initially responded to a daily dose. out the enrolled p. (median duration : days) showed a rise of the absolute neutrophil count (anc) above the target value of /mm after a median period of day at mcg/kg/d. only / p. were given antiretrovirals while other concomitant drugs included mainly ganciclovir ( ) and pyrimethamine ( ). of the p. who entered the maintenance phase at weekly doses, ( %) showed a sustained increase of the anc > , while in the remaining p, a new episode of neutropenia was observed.in contrast to p. with nhl, adverse reactions were commonly observed, mostly fever ( %) and flu-like symptoms ( %). furthermore among the p. without antiretroviral therapy who were p ag negative at baseline, a detectable p antigenemia was found in p. these preliminary data on this original schedule of gm-csl = administration was effective in preserving anc levels at > in % of the responders. such an approach could enhance p.'s compliance and have a direct impact on the cost/benefit evaluation of such therapy. in addition, antiretrovirals should always be associated with gm-csf to reduce the increased risk of viral replication. attempts to further improving the curative treatment for aml have to overcome the problem of residual disease represented by leukemic cells surviving in a therapy resistant state. a novel approach aims at recruiting those cells into a sensitive state by the use of gm-csf before and together (priming) with chemotherapy. in vitro, the recruitment of leukemic blasts to colony forming cells in presence of gm-csf (blood : , ) is documented by numerous reports. given to patients with newly diagnosed aml gm-csf priming decreased the proportion of leukemic cells in go and increased cells in sensitive cycle phases within - h (blood : , ) . a similar priming with preinfusion of gm-csf for a variable period of - days before chemotherapy started resulted in significantly inferior outcome and more persistent leukemias than in historical controls suggesting protective effects of gm-csf (blood : , . in a first study we showed that gm-csf following chemotherapy in high risk aml effectively reduced both neutrophii recovery and early mortality and had no adverse impact on leukemic regrowth and remission duration (blood : (blood : , . in current randomized study in patients with newly diagnosed aml we give gm-csf from h before chemotherapy and then on to neutrophil recovery which is repeated in each of the initial treatment courses and is compared to chemotherapy alone. / years from study start and after entering patients (median age , range - years) this update in the gm-csf group and controls shows % and % cr, and persistent leukemias and a clearance of day b.m. blasts to < % in % and in %, and remission duration shows a trend in favour of the gm-csf group. in addition to recruitment, other effects of gm-csf like enhancement of ara-c cytotoxicity (leukemia : , ; ch. router et al. leukemia in press) may contribute to this strategy. thus, gm-csf appears not to antagonize antileukemic chemotherapy. whether gm-csf priming and longterm administration ultimately improves the cure rate in aml should be shown some later from the multiple course strategy used in this study.medizinische universit~itsldinika (h~imatologie/onkologie), albert-schweitzer-str. , d- mfinster in this multicenter trial was evaluated, whether rhu gm-csf given concomitant and after chemotherapy (ct) can improve the outcome of aml patients by increasing the cytotoxic effect of ct and by reducing the rate of infectious complications. induction and early consolidation therapy included cytarabine (ara-c, mg/m , day - civi), daunorubicin ( mg/m , iv, day - ,) and etoposide ( mg/m , day - , h iv infusion) with reduced dosages in the second induction and early consolidation course. late consolidation included one cycle with high-dose ara-c ( g/m , doses) and daunorubicin ( mg/m , day - ) for patients aged years and younger, whereas patients over years received a reduced dose of ara-c ( . g/m , doses). patients were randomized after the first induction course to receive either rhu gm-csf (e. coil, pg/m /day, s.c.) or placebo starting hours prior to the second induction and the subsequent courses and given throughout chemotherapy until absolute neutrophil count had recovered > /pl. eighty out of patients (median age = years) included into the study could be randomized to receive either gm-csf (n = , median age = years) or placebo (n = , median age = years). out of patients ( %) achieved a complete remission (cr). patients ( . %) were treatment failures. two patients died and in one patient the response to therapy was not evaluable. there was no statistically significant difference in cr rate between patients of the gm-csf ( %) and placebo group ( %) nor between patients aged years or less and those over years old. the proportion of relapse free survival at a median follow up of months is % in the gm-csf versus % in the placebo group (p = . ). the proportion of survival of the gm-csf group at months is % versus % in the placebo group (p = . ). gm-csf did not significantly shorten the period of critical neutropenia and prolonged the period of critical thrombocytopenia especially in patients aged over years. the overall incidence of infectious complications as well as the non-hematological toxicity was similiar in boths groups. thus gm-csf therapy is feasible in aml therapy while its influence on remission quality and survival remains still open.dept. int. med. iii, university of uim, uim/d, frg.al key: cord- -n pu yeu authors: rogers, meredith c.; miranda-katz, margot; zhang, yu; oury, tim d.; uccellini, melissa b.; garcía-sastre, adolfo; williams, john v. title: stat limits host species specificity of human metapneumovirus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: n pu yeu the host tropism of viral infection is determined by a variety of factors, from cell surface receptors to innate immune signaling. many viruses encode proteins that interfere with host innate immune recognition in order to promote infection. stat is divergent between species and therefore has a role in species restriction of some viruses. to understand the role of stat in human metapneumovirus (hmpv) infection of human and murine tissues, we first infected stat (−/−) mice and found that hmpv could be serially passaged in stat (−/−), but not wt, mice. we then used in vitro methods to show that hmpv inhibits expression of both stat and stat in human and primate cells, but not in mouse cells. transfection of the murine form of stat into stat -deficient human cells conferred resistance to stat inhibition. finally, we sought to understand the in vivo role of stat by infecting hstat knock-in mice with hmpv, and found that mice had increased weight loss, inhibition of type i interferon signaling, and a th -polarized cytokine profile compared to wt mice. these results indicate that stat is a target of hmpv in human infection, while the murine version of stat restricts tropism of hmpv for murine cells and tissue. human metapneumovirus (hmpv) is a negative sense single-stranded rna virus and a member of the pneumoviridae family with its closest related human pathogen respiratory syncytial virus (rsv) [ ] . hmpv is a leading cause of severe respiratory illness in children and adults [ ] [ ] [ ] [ ] [ ] , and causes significant morbidity and mortality in immunocompromised hosts [ , [ ] [ ] [ ] . even though virtually all humans are infected with hmpv by the age of years [ , ] , reinfection with hmpv is common, which can lead to particularly poor outcomes in immunocompromised and elderly patients [ ] [ ] [ ] [ ] [ ] [ ] . due to the wide prevalence and burden of severe disease in multiple risk groups, it is essential to better understand the virus and host factors that are involved in promoting and restricting hmpv infection. many viruses encode proteins that interfere with interferon (ifn) signaling or inhibition of ifn stimulated genes (isgs) [ ] . pneumoviruses and the related paramyxoviruses antagonize host ifn signaling, specifically stat (signal transduction and activator of transcription) and/or stat . paramyxoviruses utilize alternate transcripts of the phosphoprotein [ ] , while rsv encodes the nonstructural proteins ns and ns that suppress stat /stat signaling [ ] . despite being related to rsv and paramyxoviruses, none of the nine proteins encoded by the hmpv genome have homology with known pneumovirus or paramyxovirus inhibitors of stat or stat [ , ] . in spite of this, hmpv was shown to inhibit phosphorylation of stat in cell lines and primary human epithelial cells [ ] . recent work in our lab identified the hmpv small hydrophobic (sh) protein as necessary and sufficient to inhibit phosphorylation of stat [ ] . others found no inhibition of stat by hmpv; however, these experiments used a relatively low multiplicity of infection (moi) in cell culture [ , ] . other groups have also shown roles for sh [ , ] as well as the hmpv proteins g and m - [ ] [ ] [ ] in the inhibition of innate immunity. viruses have tropism for cell types, tissues, and host species, which explains why natural infections only cause certain symptoms in certain species. tropism can be caused by a host cell lacking a viral entry receptor, or by the virus' ability to circumvent host immunity only in certain cells or species. mouse models for hmpv have been developed, but mice are only semi-permissive for hmpv, requiring a high viral inoculum for productive in vivo infection ( [ ] and unpublished observations), which may be due to species differences in the innate immune proteins antagonized by the virus. for example, human and mouse stat have~ % amino acid similarity, while human and mouse stat are relatively divergent, with only~ % similarity [ , ] . as a consequence of this sequence divergence, murine stat restricts the related human viruses rsv, hpiv , and hpiv in cell culture [ , ] . we sought to test the hypothesis that hmpv may similarly be restricted by stat in a species-specific manner. in this study, we used in vitro and in vivo approaches to determine the effect of hmpv on human and murine stat / . we found that hmpv antagonized expression and nuclear localization of both stat and stat in primate cells but not murine cells. transfection of stat -deficient u a cells with hstat or mstat revealed that suppression of both hstat and hstat were prevented by mstat . in vivo, hmpv infection of hstat knock-in (hstat ki) mice led to greater weight loss, inhibition of interferon stimulated genes, and a th -skewed cytokine profile compared to wt mice. these data indicate that mstat restricts hmpv's ability to inhibit both stat and stat and suggest that productive infection of humans by hmpv is partly due to inhibition of hstat antiviral signaling. the following cell lines were used: beas b (atcc crl- ), vero e (atcc ccl- ), nih/ t (atcc crl- ), u a (ecacc) (atcc: manassas, va, usa; ecacc: salisbury, uk). cmt / (ecacc) and u a (ecacc) cells were purchased from sigma (st. louis, mo, usa). all cell lines were maintained, infected, and transfected in dmem supplemented with % fbs. puno -hstat and puno -mstat plasmids were purchased from invivogen (san diego, ca, usa). hmpv clinical strain tn/ - (subtype a ) was grown in llc-mk cells (atcc ccl- ) and virus titers measured by plaque assay in llc-mk cells as previously described [ ] . for cell experiments, cells were inoculated with hmpv strain tn/ - at an moi of - in a -well plate. mock-infected cells were inoculated with media or llc-mk cell lysate, which had an equivalent effect on stat and stat protein levels and phosphorylation [ ] . then, - h post-infection, cells were treated with u/ml human ifnα (alpha a) (pbl; piscataway, nj, usa) for human and primate cells, or u/ml murine ifnβ (pbl) for mouse cells for - min. after treatment, media were aspirated from the tissue culture dish and cells were lysed in ripa buffer (thermofisher; waltham, ma, usa) for western blotting or fixed in % paraformaldehyde for immunofluorescence. transfections were performed using lipofectamine (life technologies; carlsbad, ca, usa) following the manufacturer's protocol with some exceptions: for each well in the -well plates, . µl lipofectamine was diluted into . µl opti-mem (not supplemented) and was mixed with µg plasmid in . µl opti-mem for a total volume of~ µl. this was incubated for min before being added to wells. fifty percent of media was replaced after h. then, - h post-transfection, cells were treated with ifnα/β as above and lysed for western blotting. cells were lysed in ice-cold ripa buffer (thermo scientific, waltham, ma, usa) containing halt protease and phosphatase inhibitors (thermo scientific). samples were centrifuged at , × g for min to pellet debris, and supernatant was used for protein analysis. total protein from cell lysate was quantified by bca assay (thermo scientific) and protein was normalized between samples. a total of - ug of protein was loaded into each well for each western blot experiment. after total protein quantification, samples were normalized and more concentrated samples were diluted in ripa buffer to achieve equal concentrations across samples. samples were diluted in × lds sample buffer (invitrogen; carlsbad, ca, usa) and × sample reducing agent (invitrogen: carlsbad, ca, usa) and boiled for min at • c. proteins were separated on a - % bis-tris polyacrylamide gel before transfer to a pvdf membrane. membranes were blocked in % bsa in tris-buffered saline with . % tween- (tbs-t) or % nonfat dry milk in tbs-t. primary antibodies against stat (cell signaling technologies (cst, danvers, ma, usa), d k y), pstat (cst, d ), stat (cst, d j l), pstat (for human, cst d p p; for mouse (polyclonal), emd millipore (burlington, ma, usa)), actin (hrp conjugated, abcam (cambridge, uk), and gfp (invitrogen, a- )) were used in a : dilution (or a : , dilution for actin) overnight with rocking at • c. after tbs-t wash, hrp-conjugated secondary antibodies against rabbit or mouse were added in % bsa-tbs-t or % milk-tbst for h. blots were washed with tbs-t and put in tbs until imaging. western blots were developed using west femto (thermo scientific) and imaged on a chemidoc xrs+ (biorad, hercules, ca, usa). band quantification was performed using image lab v . (biorad). after infection and ifn treatment, cell supernatant was removed from beas b cells and cells were fixed with % paraformaldehyde, then permeabilized with % methanol at − • c. cells were blocked with % goat serum and . % triton-x in pbs. primary and secondary antibodies were diluted in % bsa and . % triton-x in pbs. dapi ( µg/ml) was added to distinguish nuclei. antibodies used were stat (cst, d k y) stat (cst, d j l), hmpv anti-fusion protein g [ ] , secondary alexa fluor -conjugated anti-human and alexa fluor -conjugated anti-rabbit (invitrogen). all fluorescent images were equally color enhanced (increased brightness and contrast) for better visibility in publication. for quantification of nuclear stat or stat signal, original (non-color enhanced) fluorescent images were analyzed using fiji imagej [ ] [ ] [ ] . nuclei were selected from the dapi viruses , , of channel, then the mean intensity was measured in the stat or stat channel. for quantification of cytosolic protein, the cytosol was selected from the transmitted light image and the mean intensity was measured in the stat or stat channel. c bl/ mice were obtained from jackson laboratories (bar harbor, me, usa). stat −/− and stat −/− mice were from dr. david levy (new york university; new york, ny, usa) [ ] and dr. christian schindler (columbia university; new york, ny, usa) [ ] , courtesy of dr. john alcorn. hstat ki mice were from adolfo garcia-sastre (icahn school of medicine at mount sinai; new york, ny, usa) [ ] . male and female - -week-old mice were used for all experiments, with control groups age and sex matched. all mice were bred and maintained in specific pathogen free conditions in accordance with the institutional animal care and use committee of university of pittsburgh (protocol # , approved march ). for all animal experiments, mice were anesthetized with ketamine-xylazine (mouse passaging experiments) or isofluorane (hstat ki experiments) and intranasally (for mouse passaging) or intratracheally (for hstat ki) infected with - × pfu/ml hmpv tn - , or lung homogenate from a previously infected mouse, in a -µl volume. for serial passage, mice were euthanized at day post-inoculation. lungs were harvested and homogenized in - ml % opti-mem in a glass tenbroeck homogenizer as previously described [ ] . lung homogenates were clarified by centrifugation at rpm ( × g) for min. clarified lung homogenate was aliquoted into cryovials and snap-frozen in an ethanol dry ice bath before storage at − • c. for serial passage, clarified lung homogenate was pooled from - mice before inoculation into recipient mice. the left lobe of the lung was inflated and fixed in formalin, then subsequently sectioned and stained with h&e, with groups combined into a single slide. all fields of each h&e-stained slide were scanned at × magnification, and each field was scored as follows: : normal lung tissue, : % to % of tissue area with inflammation, : - % of tissue area with inflammation, : - % of tissue area with inflammation, or : - % of tissue area with inflammation [ ] . the frequency of each inflammation score was combined by group. inflammation scores were combined as follows to allow for statistical analysis by fisher's exact test: scores of - were categorized as mild/moderate inflammation and scores of - were categorized as severe inflammation. lung homogenate from the whole lung was frozen at − • c until use for quantitative rt-pcr (qpcr). rna was extracted from the lung homogenate with the purelink rna mini kit (thermo fischer scientific, waltham, ma, usa) according to manufacturer instructions and stored at − • c until further use. five microliters of extracted rna were used for qrt-pcr in -µl reaction mixtures on an abi steponeplus real-time pcr system (thermo fisher scientific) using the agpath-id one-step rt-pcr kit (thermo fisher scientific). taqman primers and probes were used according to manufacturer's instructions (all thermo fisher scientific). all values were normalized to the housekeeping gene hprt, and fold change in chemokine was measured in infected groups compared to mock-infected controls using the ∆∆ cycle threshold method. lung homogenate from the whole lung was used for cytokine analysis by cytokine & chemokine -plex mouse procartaplex panel a (invitrogen) according to manufacturer's instructions. data were analyzed using prism version . (graphpad software; san diego, ca, usa). a one-tailed student's t test was used to analyze fold change from western blots. fisher's exact test was used to analyze differences in histology inflammation scores. a two-tailed unpaired student's t test was used to analyze comparisons between two groups. for comparisons between multiple groups, a one-or two-way anova was performed. error bars on graphs represent sd. some respiratory viruses, including influenza, sars, and mers, have been serially passaged in mice to generate mouse-adapted virus strains, which have provided important tools for studying viral and host determinants of disease and tropism [ ] [ ] [ ] [ ] [ ] . we initially sought to generate mouse-adapted hmpv by serially passaging infected lung homogenate directly into a recipient mouse. however, viral titers in lung homogenate of infected c bl/ mice were not sufficiently high enough to productively infect a recipient mouse, despite repeated attempts, and serial passage in rag- −/− or ifnar −/− mice was also unsuccessful [ ] . however, hmpv replicates to significantly higher titer in ifnar −/− mice [ ] , indicating that type interferon signaling restricts hmpv in mice in vivo. we therefore inoculated stat −/− mice with hmpv strain tn/ - and found that the virus grew to significantly higher titer in stat −/− mice compared to stat −/− or wt mice ( figure a ). hmpv could be passaged mouse to mouse at low level detectable by qpcr in stat -/mice, but we chose to pursue stat based on our data showing higher replication in stat −/− mice and the fact that the stat protein is less conserved between human and mouse compared to stat . we subsequently inoculated stat −/− and wt mice with clarified lung homogenate from infected stat −/− and wt mice, respectively, and found that hmpv could be serially passaged mouse to mouse in stat −/− but not wt mice ( figure b ). these data show that serial passage of hmpv is restricted by stat in vivo, indicating that this protein and/or the ifn signaling pathway in general is a barrier to murine infection by hmpv. viruses , , of inoculated stat −/− and wt mice with clarified lung homogenate from infected stat −/− and wt mice, respectively, and found that hmpv could be serially passaged mouse to mouse in stat −/− but not wt mice ( figure b) . these data show that serial passage of hmpv is restricted by stat in vivo, indicating that this protein and/or the ifn signaling pathway in general is a barrier to murine infection by hmpv. since stat appeared to be important in restricting hmpv infection of mice, we next explored how hmpv infection affects stat and stat in human cells. previously, it had been shown by us and by others that hmpv can specifically inhibit stat phosphorylation and expression [ , ] . to further understand the effects of hmpv on stat and stat , we used a human bronchoepithelial cell line, beas b, to perform imaging studies of stat and stat in the presence or absence of hmpv. after infection with hmpv for h, beas b cells were treated with ifn to induce phosphorylation and nuclear translocation of stat and stat . after treatment with ifn, stat and stat translocated to the nucleus in mock-infected cells, whereas nuclear import was inhibited in hmpv-infected cells but not in uninfected cells in the same dish ( figure ). these data indicate that hmpv infection inhibits nuclear translocation of the stat / heterodimer in vitro, suggesting that antagonism of stat / may be a key step to promote hmpv infection. hmpv grew to significantly higher titer in stat −/− mice than in wt mice, leading us to hypothesize that hmpv inhibits stat and stat in primate cells but fails to inhibit these in murine cells. to test this hypothesis, we infected both primate and murine cell lines with hmpv and measured expression as well as phosphorylation of stat and stat after ifn treatment. we found that hmpv infection of veroe cells (primate cells that are ifn-responsive but cannot produce endogenous ifn) led to decreased total and phosphorylated stat and stat (figure ) . the decreased levels of pstat and pstat appeared to be driven by the decreased protein abundance of total stat and stat in vero cells, as quantified by the relative fold change in phosphorylated compared to total stat proteins. in contrast, when murine cell lines cmt / (c bl/ lung adenocarcinoma) and nih/ t (murine fibroblast, deficient in some steps of ifn signaling [ ] ) were infected with hmpv, we saw increased total and phosphorylated stat and stat (figure ) . these data indicate that hmpv antagonizes stat and stat in primate cells but fails to achieve this inhibition when introduced to murine cells. cells. to test this hypothesis, we infected both primate and murine cell lines with hmpv and measured expression as well as phosphorylation of stat and stat after ifn treatment. we found that hmpv infection of veroe cells (primate cells that are ifn-responsive but cannot produce endogenous ifn) led to decreased total and phosphorylated stat and stat (figure ). the decreased levels of pstat and pstat appeared to be driven by the decreased protein abundance of total stat and stat in vero cells, as quantified by the relative fold change in phosphorylated compared to total stat proteins. in contrast, when murine cell lines cmt / (c bl/ lung adenocarcinoma) and nih/ t (murine fibroblast, deficient in some steps of ifn signaling [ ] ) were infected with hmpv, we saw increased total and phosphorylated stat and stat (figure ) . these data indicate that hmpv antagonizes stat and stat in primate cells but fails to achieve this inhibition when introduced to murine cells. studies in hpiv and hpiv showed that degradation of stat or stat , respectively, required the expression of both stat and stat [ ] . to understand whether hmpv inhibition of stat and stat is dependent on expression of both proteins, we used u a and u a cells, which are specifically deficient in stat and stat , respectively [ ] . we found that hmpv infection of stat -deficient u a cells led to reduction of stat and pstat in a dose-dependent manner with increasing moi (figure a,c) . hmpv infection of stat -deficient u a cells also reduced stat and pstat , but only at a moi of ( figure b,d) . at moi = in u a cells, hmpv did not inhibit stat expression or phosphorylation. these data indicate that hmpv has the capacity to target stat and stat independently of each other; however, antagonism of stat by hmpv appears to be a more efficient process than stat inhibition. viruses , , x for peer review of studies in hpiv and hpiv showed that degradation of stat or stat , respectively, required the expression of both stat and stat [ ] . to understand whether hmpv inhibition of stat and stat is dependent on expression of both proteins, we used u a and u a cells, which are specifically deficient in stat and stat , respectively [ ] . we found that hmpv infection of stat -deficient u a cells led to reduction of stat and pstat in a dose-dependent manner with increasing moi (figure a,c) . hmpv infection of stat -deficient u a cells also reduced stat and pstat , but only at a moi of ( figure b,d) . at moi = in u a cells, hmpv did not inhibit stat expression or phosphorylation. these data indicate that hmpv has the capacity to target stat and stat independently of each other; however, antagonism of stat by hmpv appears to be a more efficient process than stat inhibition. we next asked whether the species-specific stat inhibition was specifically due to differences in the human and murine forms of stat , or whether hmpv fails to inhibit some other step in the innate immune response in murine cells. u a cells were transfected with either human or murine stat (hstat and mstat , respectively), infected with hmpv, treated with ifn, and analyzed for stat / expression and phosphorylation. we found that cells transfected with mstat were more resistant to inhibition of stat and than those transfected with hstat (figure ). at an moi of , a significant reduction of stat expression was seen in hmpv-infected cells that had been transfected with hstat , but not those transfected with mstat . however, with an increased moi of , reduced stat protein levels were seen regardless of the transfection type ( figure d ). interestingly, we found that even though stat was not required for stat inhibition (figure ) , the presence of mstat in u a cells constrained inhibition of stat by hmpv ( figure b,c) . overall, these data suggest that species-specific differences in stat limit the inhibition of both stat proteins by hmpv. to establish whether hmpv antagonizes the human form of stat compared to murine stat in vivo, we used mice engineered to express human stat in place of murine stat (hstat ki) [ ] . we infected hstat ki and wt mice with either a low ( × pfu/ml) or high ( × pfu/ml) inoculum of hmpv. at both low and high inoculum, hstat ki mice lost significantly more weight compared to wt controls ( figure a ). this correlated with a greater degree of lung inflammation ( figure b) , where hstat ki mice had a significantly increased frequency of inflammation scores that were categorized as severe (scores of or , p = . ). the weight loss and increased inflammation was not associated with higher viral titers in infected hstat ki mice; in contrast, at day five of lower inoculum infection, hstat ki mice had modestly lower viral burden compared to wt mice, though this was not replicated at higher titer ( figure c ). these data suggest that, in vivo, stat antagonism worsens disease severity but does not contribute to increased virus replication in mice. viruses , , x for peer review of to establish whether hmpv antagonizes the human form of stat compared to murine stat in vivo, we used mice engineered to express human stat in place of murine stat (hstat ki) [ ] . we infected hstat ki and wt mice with either a low ( × pfu/ml) or high ( × pfu/ml) inoculum of hmpv. at both low and high inoculum, hstat ki mice lost significantly more weight compared to wt controls ( figure a ). this correlated with a greater degree of lung inflammation ( figure b) , where hstat ki mice had a significantly increased frequency of inflammation scores that were categorized as severe (scores of or , p = . ). the weight loss and increased inflammation was not associated with higher viral titers in infected hstat ki mice; in contrast, at day five of lower inoculum infection, hstat ki mice had modestly lower viral burden compared to wt mice, though this was not replicated at higher titer ( figure c ). these data suggest that, in vivo, stat antagonism worsens disease severity but does not contribute to increased virus replication in mice. figure . hstat ki mice have greater disease severity and inhibition of isgs compared to wt mice after infection by hmpv, despite no increase in virus titer. hstat ki mice and c bl/ mice were intratracheally inoculated with hmpv at a low ( × pfu/ml) or high ( × pfu/ml) titer and weighed daily over the course of infection (a). (b) lung specimens were taken at day five postinoculation with hmpv at low titer, stained with h&e, and scored by a pathologist. histology images were captured at ×. histological scoring was calculated by percentage of inflammation per field of view, with scores of , , (mild/moderate) representing , - , and - %, and or (severe) representing - or - %, respectively. the difference between wt and hstat ki mice in the frequency of mild/moderate and severe inflammation scores was statistically significant by fisher's to better understand the ability of hmpv to antagonize interferon signaling in vivo, we quantified levels of ifnα and the isgs mx and socs in hstat ki and wt mice after hmpv infection. ifnα is produced via a feedback amplification loop of isgs during the type i ifn response [ ] , while mx and socs were selected for their roles as known isgs that have differential functions in the type i ifn response. we found that mrna expression of mx and socs were reduced in hstat ki mice compared to wt mice at h post-hmpv infection ( figure d) . additionally, protein level of ifnα declined in hstat ki mice compared to wt mice during hmpv infection ( figure e ). overall, these data indicate that hmpv is indeed able to antagonize type i ifn signaling when mstat is replaced by hstat in vivo, though this did not lead to increased virus replication. we hypothesized that the increased weight loss and inflammation despite slightly decreased viral titer in hstat ki mice was due to aberrant stat signaling in these mice. to better understand the global consequences of substituting human stat into mice in vivo, we performed multiplex cytokine analysis on lung homogenates of hmpv-infected hstat ki and wt mice at day five after high-dose inoculation, as day five represents a time point that bridges innate and adaptive immunity. overall, hstat ki mice had reduced expression of the characteristic th cytokines ifnγ and tnfα ( figure a ) and adopted a th -skewed cytokine profile demonstrated by increased concentrations of il- , il- , il- , eotaxin, and il- ( figure b ). altogether, our data suggest that the substitution of hstat for murine stat leads to skewing of both the innate and the adaptive immune response to hmpv. to better understand the ability of hmpv to antagonize interferon signaling in vivo, we quantified levels of ifnα and the isgs mx and socs in hstat ki and wt mice after hmpv infection. ifnα is produced via a feedback amplification loop of isgs during the type i ifn response [ ] , while mx and socs were selected for their roles as known isgs that have differential functions in the type i ifn response. we found that mrna expression of mx and socs were reduced in hstat ki mice compared to wt mice at h post-hmpv infection ( figure d) . additionally, protein level of ifnα declined in hstat ki mice compared to wt mice during hmpv infection ( figure e ). overall, these data indicate that hmpv is indeed able to antagonize type i ifn signaling when mstat is replaced by hstat in vivo, though this did not lead to increased virus replication. we hypothesized that the increased weight loss and inflammation despite slightly decreased viral titer in hstat ki mice was due to aberrant stat signaling in these mice. to better understand the global consequences of substituting human stat into mice in vivo, we performed multiplex cytokine analysis on lung homogenates of hmpv-infected hstat ki and wt mice at day five after high-dose inoculation, as day five represents a time point that bridges innate and adaptive immunity. overall, hstat ki mice had reduced expression of the characteristic th cytokines ifnγ and tnfα ( figure a ) and adopted a th -skewed cytokine profile demonstrated by increased concentrations of il- , il- , il- , eotaxin, and il- ( figure b ). altogether, our data suggest that the substitution of hstat for murine stat leads to skewing of both the innate and the adaptive immune response to hmpv. figure . hmpv-infected hstat ki mice adopt a th -skewed cytokine profile. hstat ki mice and c bl/ mice were intratracheally inoculated with hmpv at × pfu/ml. at day five post-infection, lungs were homogenized and cytokines were measured by multiplex cytokine assay. (a) measurement of prototypical th cytokines ifnγ and tnfα in lung homogenates of hstat ki and c bl/ mice. (b) concentration of th -related cytokines il- , il- , il- , eotaxin, and il- in murine lung homogenates. * p < . , ** p < . , unpaired t test. n = /group, one experiment. animal models are essential tools for studying infections by human viruses. the differences in host responses between humans and animals, both in vitro and in vivo, help reveal important mechanisms of how viruses interact with the immune system. we showed that hmpv can be serially passaged in stat -deficient mice, whereas serial passage in wt mice was not possible, suggesting a role for stat in mediating in vivo restriction. hmpv inhibited expression and nuclear localization of stat and stat in primate airway epithelial cells, while the virus failed to inhibit either stat in murine cells. stat inhibition occurred in a hstat -dependent manner, as transfection of mstat into human cells reduced stat and stat antagonism. furthermore, the knock-in of hstat into mice enabled hmpv to inhibit type i ifn signaling and alter the cytokine profile of infected mice. collectively, these data suggest that stat in mice represents a significant barrier to murine infection by hmpv. we observed that a relatively high moi of hmpv (three or more) was needed to inhibit stat , whereas inhibition of stat occurred at a moi of one. these effects at different mois suggest that stat inhibition by hmpv is more efficient than stat inhibition. hmpv proteins could have a higher affinity for stat compared to stat , or perhaps stat inhibition is achieved with a viral protein that is transcribed at a lower abundance. further research to elucidate which hmpv protein is responsible for antagonizing stat will increase our understanding of how hmpv inhibits different steps of the innate immune response. a limitation of the in vitro component of this work is that cell lines may have inherent differences in the strength of their innate immune signaling, in addition to the differences in the efficiency of infection and transfection between cell lines. previous work in our lab showed that autocrine and paracrine effects of ifn signaling on neighboring uninfected cells could confound the effects of hmpv infection on stat expression and phosphorylation [ ] . here, we attempted to control for this effect in primate cells by using vero e cells, which cannot produce endogenous ifn. however, no equivalent murine cell line currently exists, though we opted to use the murine nih/ t cell line for experiments in figure , as this line has some known impairments in endogenous ifn signaling [ , ] . several prior studies have described species-specific differences in stat inhibition by the flaviviruses dengue, zika, and yellow fever viruses [ ] [ ] [ ] . consequently, hstat ki mice infected with mouse-adapted zika virus exhibited increased viral replication, broader tissue spread, and enhanced transplacental spread [ ] . in contrast, we found that despite the clear preference for inhibition of hstat over mstat in vitro, hmpv-infected hstat ki mice had similar virus replication in the lung compared to wt mice. nevertheless, the presence of hstat resulted in reduced expression of ifnα and mx in these mice, consistent with inhibition of stat / signaling, although not to a sufficient level to result in a significant increase in viral replication in lung. interestingly, hstat ki mice had worse disease than wt mice when infected with hmpv, as measured by weight loss and lung inflammation. this phenotype was associated with an altered cytokine profile that favored th cytokines over th . since ifnα was significantly reduced in hstat ki compared to wt mice, it is likely that either direct or indirect inhibition of stat by hmpv drove the th phenotype in this study. ifns and il- have opposing roles in the immune response, with ifns promoting an antiviral th response and il- driving a th response characterized by asthma and allergy. this response is driven in part by ifn-mediated inhibition of il- via increased socs expression [ ] ; our data here are consistent with this previous work, as we saw increased il- and other th cytokines while ifns and socs were reduced (figures and ) . due to the dramatic skewing of the th /th axis in the presence of hstat , we hypothesize that antagonism of stat by hmpv in human infection may contribute to the association of hmpv and asthma in susceptible individuals [ ] [ ] [ ] . in addition, stat has immunoregulatory functions that may have been disrupted in the hstat ki mice [ ] [ ] [ ] . overall, these data indicate that hmpv targets expression of both stat and stat in a host-specific manner. future studies to determine the specific hmpv protein(s) and molecular interactions involved in stat inhibition will reveal mechanisms of productive infection and pathogenesis in humans. while mouse models of human diseases have strengths and limitations, these data highlight how the innate immune response to hmpv in mice may be profoundly different than it is during natural infection of humans, an important consideration for the development of future therapeutics and vaccines. taxonomy of the order mononegavirales: update human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children the role of human metapneumovirus in upper respiratory tract infections in children: a -year experience virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility human metapneumovirus (hmpv) infection in immunocompromised children rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections 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transcriptional activity human metapneumovirus glycoprotein g inhibits innate immune responses human metapneumovirus glycoprotein g inhibits tlr -dependent signaling in monocyte-derived dendritic cells human metapneumovirus m - protein inhibits innate immune response in monocyte-derived dendritic cells the cotton rat (sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity murine stat is uncharacteristically divergent stat : a shape-shifting anti-viral super stat respiratory syncytial virus nonstructural proteins ns and ns mediate inhibition of stat expression and alpha/beta interferon responsiveness stat acts as a host range determinant for species-specific paramyxovirus interferon antagonism and simian virus replication unpublished observations a broadly neutralizing human monoclonal antibody exhibits in vivo efficacy against both human metapneumovirus and respiratory syncytial virus imagej : imagej for the next generation of scientific image data an open-source platform for biological-image analysis image to imagej: years of image analysis targeted disruption of the mouse stat gene results in compromised innate immunity to viral disease immune response in stat knockout mice an immunocompetent mouse model of zika virus infection il- restrains il- to limit lung pathology characteristics following pulmonary infection with francisella tularensis live vaccine strain a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice mouse-adapted mers coronavirus causes lethal lung disease in human dpp knockin mice adaptation of pandemic h n influenza viruses in mice studies on the mechanism of adaptation of influenza virus to mice the pathogenesis of infections of the mouse caused by virulent and avirulent variants of an influenza virus role of type i interferon signaling in human metapneumovirus pathogenesis and control of viral replication a mouse cell line, which is unprotected by interferon against lytic virus infection, lacks ribonuclease f activity selective stat protein degradation induced by paramyxoviruses requires both stat and stat but is independent of alpha/beta interferon signal transduction use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway interferon-inducible antiviral effectors the cgas-sting signaling pathway is required for the innate immune response against ectromelia virus mouse stat restricts early dengue virus replication zika virus targets human stat to inhibit type i interferon signaling host-specific ns ubiquitination determines yellow fever virus tropism interferons inhibit activation of stat by interleukin in human monocytes by inducing socs- gene expression human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults human metapneumovirus infection in children hospitalized for wheezing stat is an essential adaptor in usp -mediated suppression of type i interferon signaling selective loss of type i interferon-induced stat activation caused by a minisatellite insertion in mouse stat the combination of ifn beta and tnf induces an antiviral and immunoregulatory program via non-canonical pathways involving stat and irf the hstat ki mice used for this study were produced by the mouse genetics and gene targeting center of research excellence (core), which is supported by the icahn school of medicine at mount sinai and a cancer center support grant ( p ca ) from the national cancer institute/national institutes of health. committee for glaxosmithkline, neither activity involved with this publication. key: cord- -rapinodd authors: vidovic, maria; sparacio, shaun m.; elovitz, michal; benveniste, etty n. title: induction and regulation of class ii major histocompatibility complex mrna expression in astrocytes by interferon-γ and tumor necrosis factor-α date: - - journal: j neuroimmunol doi: . / - ( ) -t sha: doc_id: cord_uid: rapinodd astrocytes can function as antigen-presenting cells (apc) upon expression of class ii major histocompatibility complex (mhc) antigens, which are induced by interferon-γ (ifn-γ). previous data from this laboratory had shown that the cytokine tumor necrosis factor-α (tnf-α) enhances ifn-γ-mediated class ii antigen expression on astrocytes. we have now investigated the effect of ifn-γ and tnf-α on class ii mhc mrna expression in astrocytes using northern blot analysis. astrocytes do not constitutively express mrna for class ii mhc. kinetic analysis of class ii mhc mrna expression in ifn-γ-treated cells demonstrated an h time lag, which was followed by an increase over the next h. optimal expression of class ii mrna was detected after a h incubation with ifn-γ. this level of expression was further enhanced by the simultaneous addition of ifn-γ and tnf-α to the astrocytes, while tnf-α alone had no effect on class ii mrna expression. tnf-α does not act by increasing the stability of ifn-γ-induced class ii mrna, indicating its action is not at that specific level of post-transcriptional control. furthermore, astrocyte class ii mrna expression was inhibited when cycloheximide (chx) was added together with ifn-γ or ifn-γ/tnf-α, and when chx was added up to h after treatment with ifn-γ or ifn-γ/tnf-α. these results indicate that astrocyte class ii mrna expression is mediated by newly synthesized proteins induced by ifn-γ and/or ifn-γ/tnf-α. the expression of class ii antigens on astrocytes, and cytokine modulation of their expression, may be important in the initiation and perpetuation of intracerebral immune responses. the non-neuronal cells of the central nervous system (cns) are made up of the macroglia (astrocytes, oligodendrocytes and ependymal cells) and the microglia. collectively, these glial cells perform a variety of active roles during development of the brain (rakic, ; silver and sapiro, ) and subsequently in the maintenance of normal cns physiology (hertz, ; janzer and raft, ) . recent work has suggested that glial cells such as astrocytes and microglia may be involved in immunological events occurring in the brain. the astrocyte can be stimulated to secrete a number of immunoregulatory molecules, including interleukin- (il-i) (fontana et al., ) , interleukin- (il- ) (frei et al., ) , interleukin- (il- ) (frei et al., ; benveniste eta[., ) , prostaglandins (fontana et al., ) , leukotriene (llartung et al., ) , tumor necrosis factor-c~ (tnf-co (robbins et al., ; lieberman et al., ; chung and benveniste, (i) and ifn-c~/, (tedeschi et al., ) . the microglia can also be stimulated to secrete l- (giulian et al., ) , il- (frei et al., ) and tnf-a (frei et al., ) , thus providing the cns with numerous endogenous sources of cytokines necessary, for immunological response. more importantly, the astrocyte and microglia can function as antigen-presenting cells (apc) in the cns (fierz et al., ; frei et al., ) . these cells are able to internalize, process, express and present antigen to encephalitogenic t cells (fontana et al., ) . however, such a function is only possible upon expression of class i major histocompatibility complex (mhc) molecules. indeed, astrocytes can be induced to express class ii antigens both in the cns and in vitro, following exposure to interferon- (ifn-t) (wong et al.. : fierz et al., or virus (massa et al., ) . mhc-encoded class molecules are heterodimerit glycoproteins which have a central role in the regulation of immune responses (benacerraf, ) . the expression of class ii antigens is primarily restricted to b cells, monocytes/macrophages and dendritic cells (hammerling et al., ) , although certain non-lymphoid cells can be induced to express class ii upon exposure to ifnq,, and function as apc. these include pancreatic beta cells (markmann et al.. ) , keratinocytes (gaspari et al., ) , brain endothelial cells (mc-carron et al., ) , and most pertinent to this study, astrocytes (fontana et al., ) . abnormal control in the level of expression of class i genes, and aberrant expression in cells normally class ii negative have been implicated in autoimmune phenomena. because of the importance of class ii mhc antigens, many studies have been directed toward understanding the regulatory mechanisms involved in class ii mhc gene expression. it is generally accepted that induction of class ii gene expression by ifn-y occurs at the transcriptional level (basta et al., ; blanar et al., : fertsch-ruggio et al., : rosa and fel-ious, : amaldi et al., , and that transacting factors interacting with cis-acting dna regulatory elements are involved in the transcriptional regulation of class ii mhc expression (accolla et al., " salter et al., sherman et al., sherman et al., , blanar et al., , ama[di et al., celada et al., ) . these trans-acting regulatory factors have been postulated to function positively or negatively, and to be expressed ubiquitously, or in a tissue-or stage-specific manner. although ifnq, is considered the primary inducer of class ii antigens, there is evidence for other cytokines contributing to class i expression. we have previously shown that tnf-~ enhances ifnq,-induced class ii antigen expression on astrocytes, and that this is a synergistic interaction as tnf-a alone has no effect on class i expression (benveniste et al., ) . the present study was undertaken to extend these previous findings, and to examine, at the molecular level, the effect of fn-y and tnf-a on astrocyte class ii gene expression. we report that astrocytes express class ii mrna h after treatment with ifn- ¢ or fn-t/tni'-~,, indicating a long lag period between exposure to the cytokines and initiation of class ii gene expression. tnf-~ does not act to stabilize ifnq,-induced class ii mrna, suggesting it may act at other levels of post-transcriptional control or at the transcriptional level. furthermore, the expression of class ii mhc mrna was completely inhibited by cycloheximide (chx), suggesting a role for newly synthesized proteins in astrocyte class ii mhc expression. as astrocytes can be stimulated to secrete tnf-a (robbins et al., ; lieberman et al., ; chung and benveniste, ) , and express high affinity tnf-a receptors (benveniste et al., ) , tnf-a can act in an autocrine fashion to enhance class ii gene expression in astrocytes. by modulating class ii gene expression and thereby stimulating the apc function of astrocytes, ifn-y and tnf-a in concert may play a pivotal role in the regulation of intracerebral immune responses. rat recombinant ifn-y (specific activity: x u/mg) was purchased from amgen biologicais (thousand oaks, ca, u.s.a.), and human recombinant tnf-a (specific activity: . x u/mg) was the generous gift of genentech (south san francisco, ca, u.s.a.). monoclonal antibody to glial fibrillary acidic protein (gfap) was obtained from boeringher mannheim (indianapolis, in, u.s.a.), and monoclonal antibody to rat class i mhc antigens (clone ox- ) was from accurate corporation (westbury, ny, u.s.a.). second antibody was affinity-purified goat anti-mouse lg conjugated to fluorescein-isothiocyanate (fitc) from southern biotechnology (birmingham, al, u.s.a.). cycloheximide and actinomycin-d were purchased from sigma chemical company (st. louis, mo, u.s.a.) . primary glial cell cultures were established from neonatal rat cerebra by a modification of the mccarthy and de vellis technique ( ) as previously described (benveniste and merrill, ) . meninges were removed from rat brains prior to glial cell dissociation and culture. culture medium (cm) was duibecco's modified essential medium (dmem), high glucose formula supplemented with glucose to a final concentration of g/l, mm glutamine, . mm non-essential amino acid mixture, . % gentamicin, and % fetal bovine serum (hyclone, logan, ut, u.s.a.). after days in primary culture, oligodendrocytes were separated from the glial cultures by mechanical dislodging, and the astrocytes were obtained by trypsinization ( . % trypsin/ . % edta) and replated at a density of - x cells/ mm tissue culture plate and allowed to adhere for at least h. the cells were counted using trypan blue; cell viability was - %. the astrocytes were monitored for purity by immunofluorescence, and by non-specific esterase staining for contaminating microglia as previously described (benveniste and merrill, ) . the primary astrocytes were plated ( . x ) on mm glass coverslips, incubated in culture medium for days, washed twice with phosphate-buffered saline (pbs), and fixed for s in cold acetone. the cells were then stained for gfap, an intracellular antigen unique to astrocytes (bignami et al., ) , using a monoclonal antibody to gfap ( : ) for min at room temperature, followed by a min incubation with goat anti-mouse ig/fitc ( : ). the coverslips were then mounted in % glycerol, and visualized by fluorescent microscopy. astrocyte cultures were routinely > % positive for gfap, and less than % of the cells were microglia based on their positive staining for non-specific esterase. total cellular rna was isolated from confluent monolayers of astrocytes that were incubated for various intervals ( - h) without or with ifn-y and/or tnf-a. in some experiments, the protein synthesis inhibitor, chx ( #g/ml) or the rna synthesis inhibitor, actinomycin d ( #g/ml), were added to the cytokine-treated astrocytes for - h. rna isolation followed the procedure of chomczynski ( ) . the cells were collected, washed times with cold pbs, and pelleted. rna was extracted with guanidinium isothiocyanate and phenol, and precipitated with ethanol. samples ( ~g) of total cellular rna were denatured with formaldehyde for rain at °c, and rna was size fractionated by electrophoresis through a . % agarose gel" containing ethidium bromide for visualization of s and s ribosomal rna bands. the visualization of rna bands was useful for assessing the integrity of the rna and for varifying the amount of rna loaded. the rna was then transferred to nitrocellulose paper in x standard saline citrate (ssc) ( m naci and . m sodium citrate) at °c. after the transfer, the nitrocellulose paper was air-dried and the rna cross-linked in a uv stratalinker oven. prehybridization was performed at °( in a solution containing % (v/v) formamide, x ssc, × denhardt's solution, p,g/ml of denatured salmon sperm dna, and . % sodium dodecyl sulfate (sds) for - h. hybridization was carried out at ( ` for h in prehybridization solution containing % dextran sulfate, . mm na phosphate buffer and denatured ? p-labeled murine class i e-e~ cdna probe ( x w' cpm/ml). the blots were then washed in x ssc (twice for min) at room temperature, followed by x ssc containing . % sds (twice for min) at °c and finally in . x ssc for rain at °c. the blots were dried between whatman filter paper and exposed to kodak x-omat ar film plus intensifying screens at - °c. the autoradiographs were quantitated by scanning densitometry with a bio-rad model video densitometer. filters were stripped to remove bound class mhc probe, and rehybridized with a second control probe, cyclophilin. a edna probe (peacll) specific for mouse class ii e-a (mathis et al., ) was the generous gift of dr. jerold woodward, university of kentucky. the . kb ecori insert was isolated, and labeled with [et-s p]deoxyctp using an amersham nick translation kit according to the manufacturer's instructions. a specific activity of . - x () ~ cpm/~g dna was routinely attained. a edna probe for rat cyclophilin (plb ) (danielson et al., ) was the generous gift of dr. jim douglass, the oregon health sciences university. primary rat astrocytes were resuspended in dmem containing % fetal bovine serum (fbs), and plated at - x ~ cells/well into -well ( mm) plates (costar, cambridge, ma, u.s.a.). the plates were incubated overnight to allow recovery of the cells from trypsinization and to assure adherence of the astrocytes. after h the original medium was aspirated off and fresh serum-free medium ( ml) was added to the wells. triplicate wells of primary rat astrocytes were treated with u/ml of recombinant rat ifn-y and/or ng/ml of recombinant human tnf-a for various incubation periods ( days). at each time point, the cells were trypsinized and stained for class it antigens, as previously described (benveniste et al., ) . briefly. astrocytes were incubated with ~ of ox- monoclonal antibody for rain in the cold. washed times with pbs containing . % fbs and . % azide (pbs-fbs-azide), and then incubated with /tl of goat anti-mouse ig-fit(" ( : ) for another rain in the cold. after washing times with pbs-fbs-azide, the cells were fixed in a final volume of ~ of % paraformaldehydc and analyzed on the facstar (becton-dickinson, mountain view, ca, u.s.a.) for class ii antigen expression. negative controls were incubated with /tl of pbs-fbs-azide in place of first antibody, or with an irrelevant monoclonal antibody of the same isotype. the gate window of forward-angle light scatter lay between channels and : the gate window for log of green fifc fluorescence lay between channels and . ten thousand cells were analyzed for each sample. the level of class i mhc mrna was examined in astrocytes following treatment for various times with ifn-y, tnf-a or a combination of the two cytokines. to determine the steady-state level of mrna for class ii, northern blot analysis was performed using a edna probe for murine class ii genes (e-a), with total rna isolated from cultured astrocytes. as seen in fig. , a . kb class ii mhc mrna transcript was present in ifn-~, treated astrocytes (lanes and ) and absent in untreated cells (lanes and ) . class it m}tc mrna expression was more pronounced when the cells were cultured with ifn-y in serum-free medium (sfm) (fig. , lane ) as opposed to serum-containing medium (fig. , lane ) , thus, all the subsequent experiments were con- in primary rat astrocytes. northern blot of rna from astrocytes that were incubated in serum containing media (lanes and ) or serum-free medium (sfm) (lanes and ) without (lanes and ) or with if'n-), ( u/ml) (lanes and ) for h. total rna was extracted and size fractionated by gel electrophoresis. hybridization was performed with a cdna probe (e-a) specific for a murine class i mhc gene. the blot was then exposed at - °c for h to kodak x-omat ar film plus two intensifying screens, kb, kilobases. ducted in sfm. optimal expression of class ii mrna was detected when cells were stimulated with - u/rnl of ifn-'r (data not shown). some variability in the concentration of ifn-t required for induction of class ii mrna was noted, and this variability was dependent on the lot of ifn- used. therefore, it was necessary to do a dose-response study for each lot of ifn-t used. for this study, u/ml of ifn-t was sufficient for maximal expression of class ii mrna. the optimal time required for class ii mrna expression following treatment of astrocytes with ifn-t is illustrated in fig. . astrocytes were incubated in sfm without or with ifn-~, for , or h prior to harvesting. a low level of class ii mhc mrna was detected at h following treatment with ifn-~,, with maximal expression detected after a h incubation with ifn-t. there was a . -fold increase in class ii mhc mrna expression from to h, and a slight reduction at h. we have previously shown that the level of class ii protein expression, based on fluorescenceactivated cell sorting (facs) analysis, was enhanced when the cells were treated with both ifn-t and tnf-a (benveniste et al., ) . similarly, in this present study, the incubation of as- kb fig. . kinetic analysis of ifn-y treatment on astrocyte class ii mhc mrna expression. astrocytes were cultured in sfm without (lanes , , and ) or with ifn-'r (lanes , , and ) for h (lanes and ) , h (lanes and ) or h (lanes and ). total rna was extracted and analyzed for class mrna by northern blot hybridization method. the blot was exposed to kodak x-omat ar film plus two intensifying ~reens at - °c for h. trocytes with both ifn-y and tnf-a resulted in an enhanced expression of class ii mrna compared to ifn- alone (fig. ) . optimal enhancement of class ii mrna was demonstrated using tnf-a at ng/ml (fig. , lane ) , which correlates with the concentration of tnf-a used for synergistic induction of class ii mhc protein (benveniste et al., ) . a . -fold increase in class ii mrna expression in the presence of ng/ml of tnf-a was detected, compared to ifn-y alone. as expected, tnf-a alone did not induce mrna for class ii antigens (data not shown). class ii mhc mrna expression induced by ifn-y/tnf-a was also enhanced when experi- kb , for h. rna was isolated for analysis by northern blot hybridization method. the blot was probed with labeled e-a cdna, and exposed at - °c for h to kodak x-omat ar film plus two intensifying screens. lg ments were performed in sfm (data not shown), indicating that a serum component(s) has a slight inhibitory effect on class i mrna expression. in other cell types, a lag phase of approximately - h precedes the appearance of class ii mrna induced by ifn- (basta et al,, ; blanar et al., , rosa and fellous, ; amaldi et al., ) . we performed a more indepth analysis of the kinetics of induction of class ii mrna by ifn- and ifn- /tnf-a in astrocytes. analysis of mrna was performed at different times after induction with ifn-y (fig. , lanes , , , and ) and ifn-y/tnf-a (fig. , lanes , , , and ). no class ii mrna was detected until h following treatment with ifn- , with maximal exvression detected h after exposure to ifn-y. similarly, class mrna was not detected until h in astrocytes that were stimulated with ifn- /tnf-a; however, the intensity of the rna signal was increased in the presence of both cytokines, as expected. thus, there was an h time lag before class ii mrna was detected in astrocytes. at early time points ( and h), mrna doublets are seen which ultimately merge into a diffuse, and ) . and h (lanes and ). astrocytes in sfm alone (lane ). the blot was exposed to kodak x-omat ar film plus two intensifying screens at - o c for days. more intense . kb band at h. this may be due to multiple transcription initiation sites described for the e-a gene (mathis et al., ) . in addition, a larger mrna species of . kb is seen at h. the significance of this band is unknown at this time. results for the induction of class ii mhc antigen expression and mrna accumulation are summarized in fig. . tnf-a increases ifn-v-induced class ii expression by increasing levels of mrna for the class ii molecule. however, it is not known whether tnf-a acts by increasing transcription or by stabilizing the mrna. experiments were conducted to assess class ii mrna stability in the presence of ifn- or ifn-y/tnf-a. class ii mrna was induced in astrocytes with either ifn-"/or ifn--//tnf-a for h, then actinomycin d (a transcription inhibitor) was added for various times ( , , , , , and h). total cellular rna was isolated and analyzed by northern blotting. preliminary results indicated that a decrease in class ii mrna was not detected until h of actinomycin d treatment (data not shown). subsequent experiments were performed utilizing rna extracted after , and h of actinomycin d treatment. as shown in fig. actinomycin d treatment, tnf-a did not appreciably affect the stability of e-a mrna compared to the stability of ifn-y-induced e-a mrna. in fact, it appears that tnf-a contributes to an accelerated destabilization of class ii mrna. the approximate half-life of e-a mrna in the presence of ifn-v/tnf-a was h, compared to greater than h in the presence of ifn-v alone. these same blots were reprobed for cyclophilin mrna to demonstrate that the integrity and quantity of rna loaded in each lane was similar (data not shown). these data indicate that tnf-a does not act by mrna stabilization to enhance if/q-v-induced class ii expression. the h delay in class ii mrna expression after ifn-v or ifn-v/tnf-a stimulation of as- trocytes suggests that signal transmission initiated by these cytokines involves a number of intermediary steps, possibly the expression of newly synthesized gene products. to test this, we examined whether protein synthesis was required for induction of class i mrna by ifn-v and ifny/tnf-a. chx, an inhibitor of protein synthesis, was added to astrocytes at a concentration ( /.tg/rnl) that inhibited protein synthesis by more than %, while still maintaining cell viability (data not shown). astrocytes were cultured for h in the presence of ifn-y, ifn-y/tnf-a, chx alone, ifn-y plus chx, ifn-y/tnf-a plus chx, rna extracted, and then analyzed. fig. demonstrates the effect of chx on the induction of class ii mrna by ifn-v and tnf-a. no mrna for class ii was detected in cells treated with chx alone, ifn-y plus chx, or ifny/tnf-a plus chx (fig. , lanes , , and ). inhibition of protein synthesis completely abolished the induction of class ii mrna by ifn-y and ifn-y/tnf-ct. however, there was no inhibition of cyclophilin mrna expression (fig. b) , and no alteration in the pattern of ethidium bromide staining of rna in all the samples treated with chx alone or chx plus the cytokines (fig. c) , indicating that chx did not cause a generalized inhibition of mrna expression in astrocytes. cyclophilin was used as a control for these experiments as rna levels do not change upon treatment with ifn-v or ifn-v/tnf-a. that newly synthesized protein(s) is required for the induction of the class ii mhc gene in astrocytes treated with ifn-y or ifn-y/tnf-a was suggested by results in fig. . the duration of protein synthesis required to allow expression of the class ii mhc gene in astrocytes was examined in cells that were pretreated with ifn-y or ifn- /tnf-a for different lengths of time prior to the addition of chx. class ii mhc mrna was measured h after the treatments were started. as shown in fig. a , when chx was added simultaneously with ifn-y/tnf-a or - h after ifn-y/tnf-a treatment, there was no detectable expression of class ii mhc mrna. however, when astrocytes were incubated with ifn- however, in samples that were treated for h with ifn-y/tnf-c~ before ctlx was added, there was still a % reduction in the expression of class ii mhc signal compared to the positive control of fn-y/tnf-a alone (fig. a, lane ) , suggesting that continuous synthesis of protein is required for optimal expression of the class mhc gene. chx treatment had no effect on the expression of cyclophilin rna (fig. b) . similar results were seen when astrocytes were incubated with ifn-t and chx, except that the expression of class ii mhc mrna was detected only after cells were incubated with ifn- for h prior to the addition for h. total rna was extracted, northern blot hybridization performed and the blot was exposed to kodak x-omat ar film plus two intensifying screens at - °c for days. autoradiograph of class ii mrna (a). autoradiograph for cyclophilin mrna was obtained by stripping class i probe and rehybridizing with a second probe to detect cyclophilin mrna expression (b). photograph of the original gel stained with ethidium bronude to show that there was no alteration in the quantity or the quality of rna in all the samples treated with chx (c). ~q /tnf-a for h prior to addition of chx, and class ii rna measured h later, a low level of class ii rna was detected. the increase in the level of class i mhc mrna detected parallels the increase in the amount of time the cells were treated with ifn-t/tnf-a before the addition of chx, i.e., the longer the treatment with ifn- /tnf-a before the addition of chx, the stronger the mrna signal. these results, therefore, suggest that protein synthesis, initiated within h of the cells encountering ifn-t/tnf-a, is critical for subsequent class ii mhc mrna expression. the blot was exposed at - ° c for days to kodak x-omat ar film plus two intensifying screens (a). autoradiograph for cyclophilin mrna obtained by stripping class ii probe and rehybridizing with a second probe to detect cyclophilin mrna (b). photograph of the original gel stained with ethidium bromide (c). of chx (data not shown), indicating that h of protein synthesis was critical for ifn-~-induced class ii mrna expression. in this study we have shown that primary neonatal rat astrocytes, upon stimulation with ifn-~,, express mrna transcripts for class ii mhc genes, and that tnf-a enhances the expression of ifn-~,-induced class ii mrna. these results support previous findings that ifn-~, and tnf-a synergize in the induction of class ii mhc protein expression in rat astrocytes (benveniste et al., ) . kinetic analysis demonstrated that class ii mrna was first detected after h of treatment with ifn-y, followed by an increase in mrna expression over the next h. when astrocytes were treated with ifn-~, and tnf-a simultaneously, the kinetics of class ii mrna expression did not change; however, the overall amount of steady-state class ii mrna was increased. optimal expression of class ii mrna was detected h after incubation with ifn- , alone or ifn-~,/tnf-a. although the predominant forms of gene regulation occur at the transcriptional level, a number of control mechanisms can act on rna once its transcription has been initiated. post-transcriptional regulatory mechanisms include ( ) changes in mrna stability, ( ) alternative rna splicing, ( ) poly a addition, and ( ) control of translational initiation. in our experiments, tnf-a did not increase the stability of ifn- ,-induced class ii mrna, indicating that tnf-a did not act at that level of post-transcriptional control. preliminary results from our laboratory suggest that the increase in class ii mrna occurs primarily by an increase in transcription of the e-a gene since nuclear run-on assays detected no transcription of the class ii genes without induction by ifn-y, and enhanced transcription in the presence of ifn-~, plus tnf-a. further experimentation is necessary to determine conclusively if tnf-a acts solely at the transcriptional level, or whether both transcriptional and post-transcriptional events result in increased class ii mhc mrna and protein. the time required for the appearance of class ii mhc mrna following treatment with ifn-~, or ifn-~,/tnf-a ( h) suggests that cytokine signal transmission is complex and may involve a number of intermediary steps. we examined whether protein synthesis was required for ifn-), or ifn-"t/tnf-a-induced expression of astrocyte class ii genes. the expression of class ii mrna was completely inhibited when chx was included with ifn-~, and ifn-'t/tnf-~ treatment, indicating that newly synthesized protein is required for astrocyte class ii mhc gene expression. a minimum of h of active protein synthesis was required for subsequent ifn-t/tnf-a-induced class ii mrna expression, while h was required for subsequent ifn-), expression. however, in experiments where chx was added h after treatment with ifn-), or ifn-),/tnf-~, there was still a % and % reduction, respectively, in the expression of class ii mrna compared to astrocytes incubated with the cytokines alone. this indicates that the synthesis of novel proteins is required continuously for optimal class ii gene expression in astrocytes. other studies have shown that protein synthesis was required for up to h after ifn-~, was added to murine p d cells to detect an increase in the level of i-aa (boettger et al., ) , while in peritoneal mouse macrophages, a % decrease in i-at~ mrna levels was observed even when chx was added after h of ifn-'rtreatment (fertsch et al., ) . in contrast, celada et al. ( ) demonstrated that protein synthesis was only required for rain after murine macrophages were treated with ifn-), for an increase in i-aft mrna to be detected. thus, different cell types have varying requirements for active protein synthesis to express class ii mrna in response to ifn-),. other reports on ifn-t-induced expression of class i genes have indicated that protein synthesis is not required. induction of dra mrna in the human glioblastoma cell line u -mg (basta et al., ) , dermal fibroblasts (collins et al., ) and i-aa in murine wehi- cells (woodward et al., ) , occurs in the absence of protein synthesis. this suggests that the expression of class ii mrna in these cells is mediated by pre-existing trans-acting factors that are triggered by ifn-'t (woodward et ai., ) . it is also important to note that primary astrocytes (this study) and glioblastoma cells (basta et al., ) differ in their requirements for protein synthesis for class expression, illustrating fundamental differences between normal astrocytes and transformed glial cells. tnf-a may be an important enhancer of class i expression in the cns as it can function in an autocrine fashion on the astrocyte. in addition to responding to tnf-a and expressing specific high affinity receptors for this factor (benveniste et al., ) , astrocytes can also secrete tnf-a (robbins et al., ; sawada et al., ; chung and benveniste, ) . more importantly, ifn-t primes the astrocyte to produce tnf-a (chung and benveniste, ) , thus ifn-t can influence both tnf-a and class ii gene expression in the astrocyte. although class ii expression on astrocytes has been conclusively demonstrated in vitro, in vivo studies have generated conflicting results. direct injection of ifn-t into the brains of mice induced class ii antigens on astrocytes, indicating that astrocytes have the potential to express these antigens in vivo (wong et al., ) . many laboratories have examined whether astrocytes express class i antigens in a variety of immune-mediated disease states to better understand the possible role of the astrocyte as a local apc. traugott et al. ( ) demonstrated class ii expression on astrocytes in active chronic multiple sclerosis (ms) lesions, and then confirmed these studies by performing double-staining for both class ii and gfap (traugott and lebon, ) . a study by hofman et al. ( ) also identified class ll-posirive astrocytes in ms brain by double-staining. rodriguez et al. ( ) have studied class i expression on glial cells in an animal model of cns demyelination induced by theiler's virus. in susceptible strains of mice (bio.s and bio.asr ), the majority of class ii-positive glial cells had morphological characteristics of astrocytes, while uninfected mice or resistant strains (bio.s, ( r)) were class ii negative. in sjl mice with acute or chronic relapsing experimental allergic encephalomyelitis (eae), an animal model for ms, some class ii-positive ceils were identified as astrocytes (sakai et al., ) . however, other studies investigating the eae model in lewis rats failed to detect class ii-positive astrocytes in the brain (hickey et al., : matsumoto et al., vass et al., ) . thesc conflicting results may bc due solely to technical problems involved with antigen fixation and staining methodologies, or may indicate that the ability of astrocytes to function as apc in vivo may only bc relevant in certain diseases or specific stages of disease. another possibility may be the loss of class ii-positive astrocytes by class ii mhc-restricted t cell-mediated cytotoxicity as shown by sun and wekerle ( ) . the disease eae appears to be strain-specific as brown-norway rats and balb/c or c bl/ mice are resistant, whereas lewis rats and sjl mice are susceptible (linthicum and frelinger, ) . recent studies have demonstrated that astrocytes derived from susceptible strains express much higher levels of class ii antigen upon treatment with either ifn-~, or virus compared to astrocytes prepared from eae-resistant strains (massa et al., a, b) . this hyperinduction of class ii in eae-susceptible animals was astrocyte specific as both peritoneal macrophages and microglial cells of susceptible and resistant strains showed identical patterns for class ii induction. this differential expression of class ii on astrocytes in response to ifn-t compared to microglia suggests that regulation of class i expression on astrocytes may correlate with antigen-presenting capacity and ultimately, disease development in the cns. we have begun, at the molecular level, to dissect the regulatory mechanisms utilized by primary rat astrocytes for class i mhc gene expression. future studies will focus on the regulation of gene expression at the transcriptional level, and ifn-t/tnf-a-induced trans-acting regulatory factors required for class ii gene expression. reactivation by a trans-acting factor of human mhc-ia gene expression in interspecies hybrids between an ia-negative human b cell variant and an la-positive mouse b cell lymphoma induction of hla class ii genes by ifn-y is transcriptional and requires a trans-acting protein identification of an interferon-y response region ' of the human histocompatibility leukocyte antigen dret chain gene which is active in human glioblastoma multiform lines detailed delineation of an interferon-y-responsive element important in human hla-dra gene expression in a glioblastoma multiform line role of mhc gene products in immune regulation stimulation of oligodendroglial proliferation and maturation by interleukin- tumor necrosis factor-a enhances interferon-y mediated class i! antigen expression on astrocytes induction and regulation of interleukin- gene expression in rat astrocytes localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence a ( ) transcriptional activation of hla-dra by interferon y requires a trans-acting protein cycloheximide, an inhibitor of protein synthesis, prevents y-interferon-induced expression of class ii mrna in a macrophage cell line lnterferon-y activates multiple pathways to regulate the expression of the genes for major histocompatibility class ii i-a#, tumor necrosis factor and complement component c in mouse macrophages single step method of rna isolation by acid guanidinium thiocyanate-phenolchloroform extraction tumor necrosis fac- tor-alpha production by astrocytes: induction by lipopolysaccharide, interferon-gamma and interleukin- recombinant human tumor necrosis factor increases mrna levels and surface expression of hla-a,b antigens in vascular endothelial cells and dermal fibroblasts in vitro plbl : a cdna clone of the rat mrna encoding cyclophilin induction of macrophage la antigen expression by rlfn-y and down-regulation by ifn-a/fl and dexamethasone are mediated by changes in steady-state levels of la mrna induction of macrophage la antigen expression by rlfngamma and down regulation by ifn-alpha/beta and dexamethasone are regulated transcriptionally astrocytes as antigen presenting cells, t. induction of la antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation production of prostaglandin e and an interleukin-l-like factor by cultured astrocytes and c glioma cells astrocytes present myelin basic protein to encephalitogenic t-cell lines astrocytes of the brain synthesize interleukin- - ike factors antigen presentation and tumor cytotoxicity by interferon-y-treated microglial cells on the cellular source and function of interleukin- produced in the central nervous system in viral diseases class ii mhc-bearing keratinocytes induce antigen-specific unresponsiveness in hapten-specific th clones interleukin- of the central nervous system is produced by ameboid microglia tissue distribution of la antigens: la on spermatozoa, macrophages and epidermal cells primary rat astroglial cultures can generate leukotriene b functional interactions between astrocytes and neurons expression of la molecules by astrocytes during acute experimental allergic encephalomyelitis in the lewis rat immunoregulatory molecules and il- receptors identified in multiple sclerosis brain astrocytes induce bloodbrain barrier properties in endothelial cells production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus acute autoimmune encephalomyelitis in mice. i . susceptibility is controlled by the combination of h- and histamine sensitization genes antigen presenting function of class !i mhc expressing pancreatic beta cells viral particles induce la antigen expression on astrocytes inducibility of la antigen on astrocyte by murine coronavirus jhm is rat strain dependent hypersensitivity of la antigen on astrocytes correlates with strain-specific susceptibility to experimental autoimmune encephalomyelitis the murine e-a immune response gene lmmunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to la-positive cells with dendritic morphology presentation of myelin basic protein by murine cerebral vascular endothelial cells preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissues neuron-glial relationship during granule cell migration in developing cerebellar cortex. a golgi and electromicroscopic study in maccacus rhesus production of cytotoxic factor for oligodendroeytcs by stimulated astrocytes immune response gene products (la antigens) on glial and endothelial cells in virus-induced demvelination regulation of hla-dr gene by ifn-~,. transcriptional and post-transcriptional control ) la expression in chronic relapsing experimental allergic encephalomyelitis induced by long-term cultured t cell lines in mice evidence for two trans-acting genes regulating hla class ii antigen expression pr(xluction of tumor necrosis factor-alpha by microgila and astrocytes in culture upstream dna sequences required for tissue-specific expression of the hla-dra gene class ii box consensus sequences in the hla-dra gene: transcriptional function and interaction with nuclear proteins axonal guidance during development of the optic nerve: the role of pigmented epithelia and other intrinsic factors ) la-restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes astrocytes produce interferon that enhances the expression of h- antigens on a subpopulation of brain cells interferon-'), and la antigen are present on astrocytes in active chronic multiple sclerosis lesions on the presence of la-positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation the distribution of la antigen in the lesions of rat acute experimental allergic encephalomyelitis inducible expression of h- and la antigens on brain cells mhc class ii transcription in different mouse cell types: differential requirement for protein synthesis between b cells and macrophages this work was funded in part by grants rg -a- and rg -a- from the national multiple sclerosis society (e.n.b) and grant bns- from the national science foundation (e.n.b). we acknowledge the support of the university of alabama at birmingham flow cytometry core facility (am ).we thank mr. keith berry for facs analysis, and ii yup chung, j. gavin norris and john r. bethea for helpful discussions. key: cord- -fz ucwwm authors: freundt, eric c.; drappier, melissa; michiels, thomas title: innate immune detection of cardioviruses and viral disruption of interferon signaling date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: fz ucwwm cardioviruses are members of the picornaviridae family and infect a variety of mammals, from mice to humans. replication of cardioviruses produces double stranded rna that is detected by helicases in the rig-i-like receptor family and leads to a signaling cascade to produce type i interferon. like other viruses within picornaviridae, however, cardioviruses have evolved several mechanisms to inhibit interferon production. in this review, we summarize recent findings that have uncovered several proteins enabling efficient detection of cardiovirus dsrna and discuss which cell types may be most important for interferon production in vivo. additionally, we describe how cardiovirus proteins l, c and l(∗) disrupt interferon production and antagonize the antiviral activity of interferon effector molecules. picornaviridae is an important family of single-stranded, positive-polarity rna viruses that includes > genera with over species (zell et al., ) . within picornaviridae, the genus cardiovirus includes encephalomyocarditis virus (emcv), theiler's murine encephalomyelitis virus (tmev) and saffold viruses (safv). although emcv has been described as a potential zoonotic agent, safvs are the only cardioviruses known to regularly infect humans, with the vast majority of people showing evidence of infection (zoll et al., ; carocci and bakkali-kassimi, ) . emcv has been found to infect over host species and contains one serotype, mengo virus, which was isolated in in the mengo district of uganda (dick et al., ) . tmev was discovered in by max theiler and is found in wild mice and rats worldwide. tmev can cause different diseases, depending on the virus strain and host genetics, ranging from fatal encephalitis to a chronic demyelinating disease that has served as a model for multiple sclerosis (brahic et al., ) . the genome of cardioviruses is approximately . - . kb and contains and untranslated regions (figure ) . translation of the genome gives rise to a polyprotein that is cleaved by the c protease, leading to the production of proteins. two additional proteins, l * and b * , are expressed from alternate open reading frames. l * is only expressed by tmev and is important for infection of macrophages, persistence of the virus in mice and inhibiting rnase l (van eyll and michiels, ; sorgeloos et al., ) , b * results from a frameshifting mechanism conserved in cardioviruses that regulates the ratio of structural and non-structural proteins translated over time. protein b * itself is only thought to be important for replication of emcv, as mutants that abolish its expression had a small plaque phenotype. b * in tmev and safv is unlikely to act as a protein as it is predicted to encode a peptide of amino acids (loughran et al., ) . in this review, we focus on the ways in which cardioviruses trigger the innate immune response and the efficient mechanisms that they have evolved to suppress these signaling pathways. we consider which cell types may be most important for production of interferon (ifn) in vivo, and also describe how cardioviruses disrupt the functions of interferon effectors. double-stranded rna (dsrna) is a necessary product of picornavirus replication, as positive-stranded genome is copied to produce a negative-stranded, full-length template, which is in turn used to produce additional genomes. dsrna is recognized by several sensor proteins within the cell and triggers a signal transduction pathway that results in transcription of the type i ifn (ifn-α/β) genes, as well as ifn-λ. in the endosome, dsrna is detected by toll-like receptor (tlr ), which signals through the adaptor protein trif to activate ifn regulatory factor (irf- ) and nuclear factor kappa b (nf-κb) (yamamoto et al., ) . cytoplasmic dsrna is detected by the rig-i-like receptor (rlr) family of proteins, which includes rig-i (retinoic acid-induced gene i) and mda (melanoma differentiation-associated gene ). upon recognition of dsrna, these proteins undergo a conformational change that exposes n-terminal caspase activation and recruitment domains (cards). the rlrs are then capable of stimulating the mitochondrial antiviral signaling (mavs) protein, also known as ips- , cardif, and visa, which in turn activates tank-binding kinase- (tbk ), inducible i-κb kinase (ikk-ε) and irf- , which then translocates to the nucleus to facilitate transcription of ifn genes (reviewed in gebhardt et al., ) . although rig-i and mda both detect dsrna within the cytosol, their functions are non-redundant. rig-i recognizes relatively short dsrna species (< kb) with ppp or pp, which are produced in certain virus infections (hornung et al., ; pichlmair et al., ) . in contrast, mda recognizes long dsrna, which is present during picornavirus replication (kato et al., ; pichlmair et al., ) . thus, while rig-i is activated during infection with flaviviruses, paramyxoviruses, influenza, and others, mda is responsible for detection of picornaviruses (gitlin et al., ; kato et al., ; pichlmair et al., ; wang et al., ; feng et al., ) . the importance of mda for control of cardioviruses was demonstrated in mda -deficient mice, which failed to control emcv infection and did not efficiently produce ifn (gitlin et al., ; kato et al., ) . in addition to rig-like helicases described above, which activate the mavs pathway, another ifn-inducible rna helicase, moloney leukemia virus homolog (mov ) was reported to enhance ifn induction (cuevas et al., ) . mov expression in hek cells restricted emcv replication. interestingly, mov acts through irf- activation, in a rlr and mavs-independent way and signals require ikk-ε but not tbk . such mavs-independent pathways are likely not critical for global ifn production in emcv infected mice, given the major impact of mda or mavs deficiency in mice, but they may be important in specific cell types or in conditions where the other pathways may be less potent. laboratory of genetics and physiology (lgp ), also known as dhx , is also a member of the rlr family but lacks a card domain (yoneyama et al., ) . given its structural similarity and lack of a card domain, lgp was initially thought to negatively regulate dsrna recognition by rig-i, as its overexpression limited ifn induction by sendai virus and newcastle disease virus (rothenfusser et al., ) . however, although the negative effect of lgp on rig-i remained controversial, later studies have established that lgp acts as a co-activator of mda . mice deficient for lgp were impaired in responding to rna ligands for mda or to emcv infection (venkataraman et al., ; satoh et al., ) . lgp was also shown to increase the rate of mda interaction with rna and downstream signaling by facilitating the formation of numerous, shorter mda filaments (bruns et al., ) . thus, it appears that lgp can act as both a positive and negative regulator of rlr signaling, with the outcome likely dependent on the concentration of lgp (bruns and horvath, ) . however, recombinant mda was directly activated as measured by an atp hydrolysis assay by the replicative form of dsrna coxsackievirus b , showing that lgp is not essential for activation of mda in vitro by dsrna (feng et al., ) . both lgp and mda are important for detecting cardiovirus replication. mefs deficient for either protein produce lower amounts of ifn-β when infected with emcv (deddouche et al., ) . intriguingly, lgp may enhance activation of mda during emcv infection by binding to rna complementary to the leader (l) gene and forming a complex with mda . this rna sequence from l was also shown to be a potent activator of mda in the absence of virus infection (deddouche et al., ) . although dsrna can bind and activate recombinant mda in the absence of other proteins, it is likely that additional partners are required for efficient activation of mda in vivo. for example, mda is phosphorylated in resting cells, and requires dephosporylation by pp α/γ (wies et al., ; takashima et al., ) . additional proteins that participate in recognition of cardiovirus dsrna have recently been described, including a study showing that tar rna binding protein (trbp) interacts with lgp in a yeast two-hybrid screen (komuro et al., ) . lgp was found to interact with trbp in co-immunoprecipitation experiments and depletion of trbp by sirna reduced interferon production induced by tmev and emcv. moreover, trbp enhanced ifn induction by tmev and emcv when overexpressed. depletion of trbp did not affect induction of ifn by sendai virus, which is recognized by rig-i. this study establishes that trbp participates in detection of cardiovirus dsrna by lgp /mda but the mechanism and its importance in vivo remain to be elucidated. as trbp is a component of the rnai machinery (chendrimada et al., ; haase et al., ) , other molecules involved in rnai were assessed for their role in dsrna detection and protein activator of pkr (pact) was found to also participate in activation of ifn signaling by cardioviruses. when pact was depleted by sirna, interferon production was decreased during infection of both tmev and emcv. overexpression of pact also increased ifn activation when lgp was co-expressed with mda . intriguingly, single-stranded tmev genome enhanced the association of lgp and pact, which suggests that a secondary structure in the tmev genome might facilitate this interaction (miyamoto and komuro, ) . in a separate report, pact was shown to be required for induction of ifn by emcv but not sendai virus. this study also demonstrated that pact and mda were recruited to dsrna (poly(i:c)) but not single stranded rna, and that pact expression increased the amount of mda oligomerization (lui et al., ) . both trbp and pact have also been reported to bind to double stranded rna-dependent protein kinase (pkr), and pact can bind rig-i (park et al., ; patel and sen, ; kok et al., ) . at the present time, it is not clear if these interactions are important for mediating recognition of cardioviruses. yet another partner in detecting dsrna in cardiovirus infection was recently uncovered. a cdna screen to identify genes involved in regulating ifn signaling revealed that dhx expression increased transcription of an ifn-β reporter plasmid in response to high molecular weight (hmw) poly(i:c) (zhu et al., ) . depletion of dhx resulted in decreased phosphorylation of tbk and irf- in response to hmw poly(i:c) and emcv as well as decreased production of ifn-β. the authors also show that dhx binds to mda but not mavs or rig-i, and binding could only be detected when mda was activated by emcv or hmw poly(i:c). mechanistically, this study demonstrated that dhx mediated rna binding of mda by interacting with mda through its n-terminus and rna through its dexd and helicase domains, and that dhx promotes formation of mda filaments, which are required for activation (zhu et al., ) . dhx was also independently described to interact with rig-i (sugimoto et al., ) , although it may be of greater importance for activation of mda (zhu et al., ) . together, these studies show that multiple proteins facilitate recognition of dsrna by mda during cardiovirus infection (figure ). thus far, these proteins include lgp , dhx , pact and trbp. additional helicases are also likely to participate in rlr-dsrna complex formation but their activities remain to be clarified (oshiumi et al., ) . moreover, recent discoveries have identified a novel role for pkr in recognition of dsrna and activation of mda , which will be discussed below. as the number of proteins mediating mda activation grows, so does the number of questions about how this pathway functions. for example, what role does each of the proteins play and how do they work together mechanistically to activate mda ? do they play non-redundant roles, or do they function in the same way but in different cell types? resolving these questions will allow for deeper understanding of the first line of immune defense against rna viruses. upon recognition of dsrna, pkr controls virus infection by phosphorylating eukaryotic initiation factor (eif α), which inhibits translation (farrell et al., ) . in this way, an infected cell can suppress production of viral proteins. phosphorylation of eif α also leads to stress granule formation and induces autophagy, and both pathways are commonly observed during virus infection (paul and munz, ; poblete-duran et al., ) . not surprisingly, many viruses have evolved strategies to inhibit the activation or function of pkr (reviewed in garcia et al., ) , as evidenced by the fact that pkr-deficiency did not modify the survival time of emcv infected mice (yang et al., ) . in addition to its role in inhibiting translation, recent evidence has emerged to show that pkr participates in production of ifn. for example, pkr appears to be important for nuclear translocation of irf- following mda activation (pham et al., ) . pkr was found to bind to mda and this interaction was not disrupted by nuclease treatment, indicating that binding does not depend on the presence of rna. in pkr-deficient cells, emcv infection failed to induce irf- translocation to the nucleus. moreover, a constitutively active mutant of pkr induced ifn through mavs. the effect of pkr on induction of ifn required its catalytic activity but did not depend on phosphorylation of eif α (pham et al., ) . in a separate report, pkr was shown to be important for normal processing of ifn-β mrna, suggesting that pkr may function at multiple points in the ifn pathway (schulz et al., ) . additionally, activation of pkr by hmw poly(i:c) was shown to be inhibited in cells depleted of mda and mavs, suggesting that mavs influences activation of pkr. moreover, mavs and pkr were found to interact in co-ip experiments, and the interaction depended on the card domain in mavs and the dsrna binding domain of pkr (zhang et al., ) . together, these studies clearly indicate a role for pkr in mda -dependent ifn induction, although several mechanisms may be involved, which require clarification. the role of pkr in ifn production after cardiovirus infection remains to be resolved. in one study that examined mengo virus infection, knockdown of pkr led to decreased induction of ifn-β in hela cells, suggesting that pkr may play a role in the mda pathway during cardiovirus infection (langereis et al., ) . this observation fits with the model proposed by onomoto et al. ( ) , which was based on influenza virus infection and suggests that pkr triggers the formation of "antiviral stress granules" that serve as a recruitment platform for dsrna and rig-like helicases, thereby enhancing ifn production. pkr is, however, not essential for ifn production in cardiovirus-infected cells because mengo virus possessing a deletion in the zinc-finger of l, which abrogates its functions as an interferon antagonist, was found to induce interferon in the cells lacking pkr and rnase l (feng et al., ) . cardioviruses may also inhibit pkr through activity of the l protein, as stress granule formation was prevented by the l protein of mengo, tmev, and safv- during virus infection (borghese and michiels, ) . however, direct inhibition of pkr by l remains to be demonstrated. during infection, viral dsrna can also be released into the extracellular milieu, either non-specifically during lysis of infected cells or possibly intentionally by exocytosis to trigger innate immunity by uninfected cells. dsrna can then be endocytosed by neighboring cells and infiltrating immune cells and lead to ifn production. tlr recognizes dsrna in endosomes and signals through trif to activate irf- and nf-κb. the importance of tlr in context of cardiovirus infection may depend on the model of infection. for example, mice deficient for myd or tlr were not significantly more susceptible than wild-type mice to emcv infection . in a separate report, however, tlr -deficient mice had higher viral loads in the liver and heart and were more susceptible to infection (hardarson et al., ) . also, a study that evaluated the role of tlr in controlling a strain of emcv with tropism for β cells of the pancreas found that tlr protected mice from a fatal infection and that tlr -deficent mice produced less ifnβ early in infection ( and h post-infection). however, this deficiency was transient and mice lacking tlr produced levels of ifn-β equivalent to wild-type animals at h post-infection. in the same experiments, the authors demonstrated that mice lacking mda succumbed to the infection more rapidly than tlr -deficient mice and produced less ifn-β at h postinfection (mccartney et al., ) . finally, a recent report using intracerebral inoculation of the gdvii strain of tmev evaluated the importance of these molecules for control of virus replication and induction of ifn. trif-/-, myd -/-, and mice lacking both trif and myd showed wild-type levels of ifn induction, while mavs-deficient animals were slightly but significantly impaired. when trif, myd and mavs were all depleted, however, mice were unable to induce ifn. only mice lacking myd and trif, or mice lacking myd , trif and mavs showed increased titers of gdvii in the brain (pfefferkorn et al., ) . thus, both tlr and rlrs contribute to controlling virus replication in vivo, although the relative importance of these pathways may depend on the virus and route of inoculation. surprisingly, mda is also responsible for the vast majority of ifn produced from extracellular dsrna in vivo. mice deficient for mda produced substantially less ifn when administered polyi:c, whereas tlr -deficient mice responded like wild-type (gitlin et al., ) . these results raise the question of how dsrna taken up through endocytosis could gain access to the cytoplasm. the mechanism by which dsrna could be internalized and then access the cytoplasmic rlrs has been unresolved until a recent discovery identified sidt , the mammalian ortholog of the sid- dsrna transporter in caenorhabditis elegans, as a transporter of dsrna from the endosome to the cytoplasm. sidt was shown to be important for mediating detection of dsrna in the context of emcv infection in vivo. mice that were deficient for sidt failed to control replication, produced less ifn-β, and succumbed to infection (nguyen et al., ) . these data suggest that a crucial pathway for innate signaling in emcv infection is release of viral rna into the extracellular milieu, endocytosis, and subsequent transfer of viral rna to the cytoplasm to access mda . two proteins encoded by cardioviruses were shown to counteract ifn production in infected cells: protease c, which is responsible for the processing of the virus-encoded polyprotein, and the leader protein (l), which corresponds to the n-terminal peptide of the polyprotein. c is a cysteine proteinase with a trypsin-like serine protease fold, responsible for most cleavages occuring during the maturation of the viral polyprotein (pelham, ) . like c proteases of other picornaviruses that were shown to target critical factors involved in ifn induction such as rig-i (barral et al., ) , emcv c was reported to cleave traf family member-associated nf-kb activator (tank) in infected cells, thus disrupting the complex involving tbk , ikke and irf and limiting type i ifn production (huang et al., ) . likewise, emcv c was shown to target mov , an rna helicase that acts in a mavs-independent way, as a possible innate immune evasion mechanism (cuevas et al., ; figure ). l is a small, multifunctional protein of - amino acids expressed by all cardioviruses (figure ). l contains an n-terminal zinc finger motif (cys-his-cys-cys), an acidic domain, a serine/threonine rich domain, and a c-terminal theilo domain, which is present in safv and tmev but absent in emcv. l was shown to be dispensable for replication of tmev in cell culture but its loss inhibits spread in cells that have a functional interferon response, like l cells, and also impairs the viral persistence in vivo (van pesch et al., ) . l deletions in figure | interferon antagonism by the cardiovirus c protease. the c protease is responsible for cleavage of the cardiovirus polyprotein produced from translation of the genome. in addition, the cardiovirus c protease cleaves host proteins, such as tank and mov , to prevent the cell from producing ifn. frontiers in microbiology | www.frontiersin.org emcv prevent the virus from shutting off host protein synthesis and enable interferon production (zoll et al., (zoll et al., , . mengo virus containing a mutation in the zinc finger of l failed to inhibit ifn synthesis and its replication was inhibited during low moi infections in vitro. in mice lacking the ifn α/β receptor, the mutant virus behaved as wild-type, but in wild-type mice, replication of the l mutant virus was impaired and it failed to cause disease, demonstrating that activity of l is important for pathogenesis in vivo (hato et al., ) . mutations in the zinc finger domain or the theilo domain of tmev or safv l inhibit its ability to antagonize interferon signaling (ricour et al., ) . a critical step in production of ifn following detection of viral replication by mda and other molecules is nuclear translocation of irf- and nf-κb. these proteins enable transcription of the ifn genes to produce mrna, which must then be exported from the nucleus for translation. since picornaviruses do not replicate within the nucleus, many viruses within this family disrupt nucleocytoplasmic trafficking, which results in inhibition of ifn production and translocation of nuclear proteins to the cytosol to benefit viral replication (reviewed in yarbrough et al., ; flather and semler, ) . the mechanism of how l interferes with ifn production may be due to its abilities to disrupt nucleocytoplasmic trafficking, activation of irf- , and assembly of stress granules in infected cells (figure ) . each of these activities will be explored below. the l protein of cardioviruses perturbs the function of the nuclear pore complex (npc) (porter et al., ) . in mammals, the npc consists of approximately different proteins, called nucleoporins (nups) and enables transit across the nuclear membrane (gorlich and kutay, ; wente and rout, ) . while small molecules and ions are able to diffuse through the npc, molecules larger than approximately - kda require active transport, which is regulated by transport receptors called karyopherins (yarbrough et al., ) . transport into the nucleus requires a short amino acid motif, called a nuclear localization sequence (nls) that can interact with either the α or β subtypes of karyopherins, depending on the sequence of the protein's nls. binding and dissociation of nls-containing proteins by karyopherins is also regulated by small gtpase ran. in the cytosol, ran is bound to gdp and can bind cargo proteins. once in the nucleus, however, ran is converted to the gtp bound form by the ran guanine nucleotide exchange factor (rangef) and dissociates from cargo. export then requires a nuclear export sequence (nes) that binds to karyopherins bound to rangtp, and dissociation of this complex occurs in the cytoplasm when a ran gtpase-activating protein (rangap) hydrolyzes gtp to gdp. in this way, the rangdp/gtp gradient regulates directional transport into and out of the nucleus. localization of l to the nucleus depends on expression of a, which contains a nls in its c-terminus (groppo et al., ) . upon nuclear localization, l interacts with ran with high affinity and a is displaced as the binding sites for a and ran partially overlap (petty et al., ) . l from emcv, tmev, and safv induce hyper-phosphorylation of nups including nup and nup (ricour et al., ; ciomperlik et al., ) , likely by recruiting and activating a kinase, which may be facilitated by l binding of exportins crm and cas (ciomperlik et al., ) . chemical inhibition of erk and p was able to block l-mediated hyper-phosphorylation of nups (porter et al., ) . additionally, l of emcv is phosphorylated by casein kinase (ck ) and this phosphorylation is required for nup phosphorylation, although ck did not phosphorylate l of safv or tmev . it is possible, although it remains to be shown, that these kinases also play a role in inhibition of nucleocytoplasmic trafficking by l of tmev and safv. in addition to its role in disrupting nucleocytoplasmic trafficking, tmev and mengo l prevent production of type i ifn in infected cells by interfering with irf- dimerization and tmev l also prevents export of mrna from the nucleus (delhaye et al., ; ricour et al., ) . for both tmev and mengo virus, dimerization of irf- was impaired despite the protein having been phosphorylated. inactivation of irf- occurs despite reports that it accumulates in the nucleus of infected cells (delhaye et al., ) . these data suggest that dsrna is detected in cardiovirus infected cells leading to activation of mavs and downstream kinases, but that irf- is unable to induce ifn transcription. stress granules can form in cells during virus infection and often result from inhibition of translation following phosphorylation of eif α by pkr (white and lloyd, ) . the l protein of mengo, tmev, and safv- inhibits stress granule formation during infection and ectopic expression of l was able to prevent thapsigargin-and arsenite-induced stress granules (borghese and michiels, ) . stress granules formed during infection with viruses containing deletions in the zinc-finger domain or a mutation in the theilo domain of l, indicating that these motifs are also important for inhibition of stress granules (borghese and michiels, ) . while l inhibits nucleocytoplasmic trafficking, stress granule formation, and possibly pkr activation, it has not been possible to uncouple these events using l mutants. when one function of l is disrupted, all functions are simultaneously impaired. therefore, it remains possible that l inhibits ifn production by blocking pkr activation, by interfering with irf- dimerization or nucleocytoplasmic trafficking, or through a combination of these mechanisms (figure ) . the importance of these antiviral pathways in controlling infection is underscored by the variety of mechanisms that viruses have evolved to prevent their activity. for example, the l protein of foot-and-mouth disease virus (fmdv), a picornavirus in the genus aphthovirus, has proteolytic activity whereas the l protein of cardioviruses does not. despite the major differences in these proteins, they all still function to inhibit induction of ifn. fmdv l pro can cleave eif g (devaney et al., ; kirchweger et al., ; guarne et al., ) and therefore reduce translation of cellular mrnas, and can also perturb ifn transcription by cleaving nf-κb (de los santos et al., . however, in the once in the nucleus, l interacts with high affinity with ran gtpase, thus displacing a. the l-ran complex would activate a kinase and trigger nucleoporin hyper-phosphorylation, thereby leading to nuclear pore complex dismantling and to nucleocytoplasmic trafficking perturbation. on the other hand, l may inhibit pkr, thus preventing translation arrest through eif α phosphorylation and therefore block assembly of stress granules. inhibition of ifn gene transcription by l may result from irf- trafficking perturbation and/or from the absence of pkr-enhanced dsrna detection by mda . context of a chimeric mengo virus infection, fmdv l pro was less effective at inhibiting ifn induction in vitro and in vivo (hato et al., ) . similar convergent evolution is apparent when considering the a protein of picornaviruses. whereas a functions as a protease for most picornaviruses and cleaves mediators of type i interferon signaling, this activity is not present in cardioviruses. nevertheless, l still targets some of these same molecules for inactivation (agol and gmyl, ) . additionally, both a of enteroviruses and l of cardioviruses inhibit stress granule assembly (yang et al., ) . the functions of l appear to be sufficiently important to the virus so that it maintains high levels of l expression throughout infection. emcv and tmev undergo a frameshift during translation later in infection by a binding to a stem-loop structure in the genome (napthine et al., ) . this frameshift decreases expression of non-structural proteins bc- abcd by - % (finch et al., ) . a follow up study using metabolic labeling estimated the frameshifting to be - % efficient (ling and firth, ) . this mechanism may allow for cardioviruses, and perhaps other picornaviruses, to increase the translation of structural proteins later in infection. due to its position in the genome, however, l expression would remain high throughout infection despite it not having a structural role for virus assembly. thus, it may be important for cardioviruses to express sufficient levels of l to counteract the immune response throughout the replication cycle. with effective ways to inhibit the production of interferon during infection, control of cardioviruses likely depends on nearby uninfected cells to produce interferon. intriguingly, these pathways seem to also depend on mda , although tlr may also be important in certain cell types such as plasmacytoid dendritic cells (hornung et al., ) . as discussed, recent data suggest a model where viral dsrna is released, endocytosed, and then the rna is translocated to the cytosol where it is detected by mda . in the cns, astrocytes appear to be the primary producers of ifn-β for several neurotropic viruses that preferentially infect neurons, such as tmev and la crosse virus (kallfass et al., ; pfefferkorn et al., ) . using transgenic mice that expressed firefly luciferase under the control of the ifn-β promoter restricted to different cell types, the authors were able to determine that % of ifn-β production during a neurotropic tmev infection was from astrocytes, whereas only % was from neurons, which are the primary target of infection. in mice lacking mavs, ifn-β production was slightly but significantly reduced, suggesting that the rlr pathway is active during infection but may not be the only pathway activated by tmev in astrocytes. intriguingly, mice deficient for myd and trif did not show a significant decrease in ifn-β induction, although induction of ifn-β was completely abrogated in mice deficient for mavs, myd and trif. therefore, it appears that both rlr and tlr signaling are important for ifn-β production after tmev infection of the cns. astrocytes were also shown to be primary producers of ifn during infection with rabies virus and vesicular stomatitis virus. in the case of rabies virus, astrocytes are stimulated to produce ifn by an abortive infection. that the virus is unable to replicate fully may prevent expression of viral interferon antagonists and allow for robust production of ifn. how viral replication is prevented in these cells will be important to uncover and may lead to novel insights about viral control in vivo. it is likely that abortive infection of astrocytes occurs during infection by tmev. however, this remains to be demonstrated and viral rna may well be encountered by other means. mda is critical for induction of ifn against cardioviruses in the periphery as well. ex vivo, cells such as macrophages, conventional dendritic cells and fibroblasts depend on mavs for production of ifn in response to dsrna (sun et al., ) . similarly, mda was shown to be essential in these cells for type i ifn production after emcv infection, in contrast to pdcs which induce ifn production in a tlr-dependent fashion (gitlin et al., ; kato et al., ) . after emcv infection of mice, some ifn is produced through tlrs, likely by pdcs, but most ifn was produced by mda activation (gitlin et al., ; kato et al., ) . levels of ifn-i were strongly decreased in the serum of mda -deficient mice infected by emcv. whereas mda expression strongly influenced survival in response to infection, the effect of myd depletion had a modest effect and loss of trif or rig-i did not affect survival . thus, mda is essential for controlling emcv infection in the periphery. interferon secreted by infected cells binds to its receptor on surrounding cells, activating a signaling cascade that leads to expression of hundreds of interferon-stimulated genes (isgs). two of these isgs, pkr and oligoadenylate synthetases (oas) are part of the best-characterized interferon effector pathways. as described earlier, pkr is likely antagonized by the l protein, as l inhibits pkr-induced stress granule assembly. moreover, a recent study reported increased sumo conjugation figure | inhibition of rnase l activation by l * tmev l * binds rnase l ankyrin repeats and (numbered) through a direct protein-protein interaction, thereby preventing association of - a with rnase l monomers and the consequent dimerization and activation of the enzyme. of pkr in emcv-infected cells, which dampens pkr activation and promotes caspase-dependent pkr degradation (maarifi et al., ) . oligoadenylate synthetases are enzymes responsible for rnase l activation. cardioviruses have evolved two strategies to interfere with the oas-rnase l pathway. in an infected cell, oas are activated by dsrna and produce - oligoadenylates ( - a). binding of two - a molecules to the ankyrin domain of the latent endoribonuclease rnase l triggers its dimerization and activation (figure ) . active rnase l then cleaves viral and cellular ssrna leading to decreased viral replication and ultimately to apoptosis of the cell. interestingly, rna fragments generated by rnase l can amplify ifn production in a rig-i, mda and mavs-dependent way (malathi et al., ) . rnase l targets both viral and cellular mrna but is also predicted to cleave the genome of ssrna viruses, as reported for emcv (li et al., ) . in addition to -phosphodiesterases and phosphatases that tightly regulate the system by degrading - a within minutes of their synthesis, rnase l activity can be negatively regulated by the rnase l inhibitor (rli/abce) (bisbal et al., ) . rli/abce expression is induced by emcv and correlates with rnase l inhibition (martinand et al., ) . accordingly, overexpression of rli/abce inhibited the action of ifn against emcv (bisbal et al., ) . rnase l inhibition by emcv-induced rli is, however, partial as rnase l antiviral activity against emcv was demonstrated in vitro using dominant negative rnase l and oas overexpression (chebath et al., ; zhou et al., ) and in vivo, in rnase l-deficient mice, which presented increased emcv infection and mortality compared to wild-type mice (zhou et al., ) . the l * protein of tmev was found to potently inhibit rnase l through a direct protein-protein interaction (sorgeloos et al., ) . mechanistically, l * binds to rnase l ankyrin repeats and , thereby preventing - a binding to the enzyme and further activation steps (drappier et al., ; figure ). in wild-type macrophages, replication of l * -mutant was significantly impaired as compared to that of the wild-type virus (sorgeloos et al., ) . in contrast, l * -mutant and wildtype viruses replicated to the same level in rnase l-deficient primary peritoneal macrophages. moreover, l * was shown to be active in vivo in the context of mhv chimeric viruses; l * could substitute for another viral rnase l inhibitor, namely the ns phosphodiesterase of mhv, in the liver of infected mice (drappier et al., ) . the fact that the virus devotes one of its proteins to rnase l antagonism highlights the importance of this antiviral pathway against tmev. interestingly rnase l inhibition by l * is highly species-specific; l * of a mouse tmev strain inhibits mouse rnase l but not its orthologs from other species including rat (sorgeloos et al., ; drappier et al., ) . accordingly, l * of a rat tmev strain inhibits rat but not mouse rnase l. theiler's murine encephalomyelitis virus is the only cardiovirus expressing l * , and thus the only cardiovirus known to directly inhibit rnase l. this could stem from its tropism for macrophages, which are the main tmev target during the chronic phase of infection and in which the oas-rnase l system is particularly active (zhao et al., ) . however, macrophages were reported to play important roles in emcv pathogenesis, including for viral replication and dissemination in piglets (papaioannou et al., ) and as reservoir cells for emcv persistence in rats (psalla et al., ) . since emcv is sensitive to rnase l activity, it is possible that another emcv protein will have developed some rnase l antagonistic activity, which might be identified using the appropriate host-pathogen context. interactions between safvs and rnase l have yet to be described, but it is also likely that these viruses have evolved ways of inhibiting this pathway. given the many ways that picornaviruses inhibit interferon production and signaling in infected cells, it is not surprising that the most important producers of ifn would be uninfected or abortively infected cells. indeed, cardioviruses efficiently block ifn in infected cells but loss of mda in mice causes them to be more susceptible to virus infection. these data indicate that detection of cytoplasmic dsrna by mda occurs in cells that are not productively infected (gitlin et al., ) and the recent finding that sidt mediates this process opens many new exciting areas of research (nguyen et al., ) . which molecules might be important for release of viral rna? is there a role for exosomes in this process? how might these pathways be stimulated pharmacologically? preventing release of viral rna and subsequent detection by uninfected cells may represent selective pressure favoring non-lytic release. given the exquisite genetic malleability in response to natural selection displayed by viruses, it is likely that viruses will have evolved mechanisms of inhibiting detection of viral rna by uninfected cells, perhaps by restricting dsrna release or by secreting proteins that inhibit rna transport into uninfected cells. it will be exciting to see how discoveries unfold in this area of research. several recent studies involving cardioviruses have revealed a more complicated picture regarding initial detection of replicating rna and induction of ifn. while it is clear that the helicases lgp , dhx , pact and trbp work in concert with mda for detection of dsrna, it remains to be determined how these molecules coordinate and interact and whether they function in a cell-type specific manner. it will also be important to resolve the way in which pkr functions to activate ifn signaling. future studies in this area will likely have broad relevance for innate detection of viruses. finally, the mechanisms by which l disrupts nucleocytoplasmic trafficking, stress granule formation, and interferon production clearly require further clarification. mutational analysis of l has revealed that these activities are tightly coupled, suggesting that the l interacts with protein(s) that can serve as a common node in each of these pathways. as ifn signaling and stress granules are important for a variety of viral pathogens, answers to these questions may provide broadly relevant insight into host-pathogen interactions. ef, md, and tm wrote the manuscript and approved its final version. ef was supported by a david delo research grant. md was supported by action de recherches concertée (arc). research in the tm lab was supported by the belgian fund for medical research (frsm, pdr # t. . ) and by eos joint program of fonds de la recherche scientifique -fnrs and fonds wetenschapelijk onderzoek -vlaanderen -fwo (eos id: ). viral security proteins: counteracting host defences rig-i is cleaved during picornavirus infection encephalomyocarditis virus leader is phosphorylated by ck and syk as a requirement for subsequent phosphorylation of cellular nucleoporins cloning and characterization of a rnase l inhibitor. a new component of the interferon-regulated - a pathway the leader protein of cardioviruses inhibits stress granule assembly the genetics of the persistent infection and demyelinating disease caused by theiler's virus lgp synergy with mda in rlrmediated rna recognition and antiviral signaling the innate immune sensor lgp activates antiviral signaling by regulating mda -rna interaction and filament assembly the encephalomyocarditis virus constitutive expression of ( '- ') oligo a synthetase confers resistance to picornavirus infection trbp recruits the dicer complex to ago for microrna processing and gene silencing three cardiovirus leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways cardiovirus leader proteins bind exportins: implications for virus replication and nucleocytoplasmic trafficking inhibition mov provides antiviral activity against rna viruses by enhancing rig-i-mavs-independent ifn induction the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response degradation of nuclear factor kappa b during foot-and-mouth disease virus infection a conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function identification of an lgp -associated mda agonist in picornavirus-infected cells the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins leader protein of foot-and-mouth disease virus is required for cleavage of the p component of the cap-binding protein complex mengo encephalomyelitis; a hitherto unknown virus affecting man a novel mechanism of rnase l inhibition: theiler's virus l * protein prevents - a from binding to rnase l phosphorylation of initiation factor elf- and the control of reticulocyte protein synthesis mda detects the double-stranded rna replicative form in picornavirus-infected cells characterization of ribosomal frameshifting in theiler's murine encephalomyelitis virus picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positivestrand rna virus the dsrna protein kinase pkr: virus and cell control discrimination of self and non-self ribonucleic acids essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus transport between the cell nucleus and the cytoplasm mutational analysis of the emcv a protein identifies a nuclear localization signal and an eif e binding site structure of the foot-and-mouth disease virus leader protease: a papainlike fold adapted for self-processing and eif g recognition trbp, a regulator of cellular pkr and hiv- virus expression, interacts with dicer and functions in rna silencing toll-like receptor is an essential component of the innate stress response in virus-induced cardiac injury the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor differential ifn-alpha/beta production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus '-triphosphate rna is the ligand for rig-i encephalomyocarditis virus c protease attenuates type i interferon production through disrupting the tank-tbk -ikkepsilon-irf complex visualizing production of beta interferon by astrocytes and microglia in brain of la crosse virus-infected mice length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene differential roles of mda and rig-i helicases in the recognition of rna viruses foot-and-mouth disease virus leader proteinase: purification of the lb form and determination of its cleavage site on eif- gamma the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response the tar-rna binding protein is required for immunoresponses triggered by cardiovirus infection mda localizes to stress granules, but this localization is not required for the induction of type i interferon rnase l mediates the antiviral effect of interferon through a selective reduction in viral rna during encephalomyocarditis virus infection an analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators ribosomal frameshifting into an overlapping gene in the b-encoding region of the cardiovirus genome pact facilitates rna-induced activation of mda by promoting mda oligomerization differential effects of sumo and sumo on pkr activation and stability small self-rna generated by rnase l amplifies antiviral innate immunity rnase l inhibitor is induced during human immunodeficiency virus type infection and down regulates the - a/rnase l pathway in human t cells rna sensor-induced type i ifn prevents diabetes caused by a beta cell-tropic virus in mice pact is required for mda -mediated immunoresponses triggered by cardiovirus infection via interaction with lgp protein-directed ribosomal frameshifting temporally regulates gene expression sidt transports extracellular dsrna into the cytoplasm for innate immune recognition critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity accessory factors of cytoplasmic viral rna sensors required for antiviral innate immune response pathogenesis of encephalomyocarditis virus (emcv) infection in piglets during the viraemia phase: a histopathological, immunohistochemical and virological study tar rna-binding protein is an inhibitor of the interferon-induced protein kinase pkr pact, a protein activator of the interferoninduced protein kinase, pkr autophagy and mammalian viruses: roles in immune response, viral replication, and beyond translation of encephalomyocarditis virus rna in vitro yields an active proteolytic processing enzyme binding interactions between the encephalomyocarditis virus leader and protein a abortively infected astrocytes appear to represent the main source of interferon beta in the virus-infected brain pkr transduces mda -dependent signals for type i ifn induction rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates activation of mda requires higher-order rna structures generated during virus infection who regulates whom? an overview of rna granules and viral infections a picornavirus protein interacts with ran-gtpase and disrupts nucleocytoplasmic transport nucleoporin phosphorylation triggered by the encephalomyocarditis virus leader protein is mediated by mitogen-activated protein kinases pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats inhibition of mrna export and dimerization of interferon regulatory factor by theiler's virus leader protein the rna helicase lgp inhibits tlrindependent sensing of viral replication by retinoic acid-inducible gene-i lgp is a positive regulator of rig-i-and mda -mediated antiviral responses protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity evasion of antiviral innate immunity by theiler's virus l * protein through direct inhibition of rnase l helicase proteins dhx and rig-i cosense cytosolic nucleic acids in the human airway system the specific and essential role of mavs in antiviral innate immune responses riok -mediated phosphorylation of mda interferes with its assembly and attenuates the innate immune response influence of the theiler's virus l * protein on macrophage infection, viral persistence, and neurovirulence the leader protein of theiler's virus inhibits immediate-early alpha/beta interferon production loss of dexd/h box rna helicase lgp manifests disparate antiviral responses mda and mavs mediate type i interferon responses to coxsackie b virus the nuclear pore complex and nuclear transport regulation of stress granules in virus systems dephosphorylation of the rna sensors rig-i and mda by the phosphatase pp is essential for innate immune signaling role of adaptor trif in the myd -independent toll-like receptor signaling pathway picornavirus a protease regulates stress granule formation to facilitate viral translation deficient signaling in mice devoid of double-stranded rna-dependent protein kinase viral subversion of nucleocytoplasmic trafficking shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity ictv virus taxonomy profile: picornaviridae ips- plays an essential role in dsrna-induced stress granule formation by interacting with pkr and promoting its activation antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology interferon action and apoptosis are defective in mice devoid of ' , '-oligoadenylate-dependent rnase l impact of rnase l overexpression on viral and cellular growth and death dhx functions as an rna co-sensor for mda -mediated emcv-specific antiviral immunity saffold virus, a human theiler'slike cardiovirus, is ubiquitous and causes infection early in life mengovirus leader is involved in the inhibition of host cell protein synthesis the mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of nf-kappa b key: cord- -n xv l authors: plötz, frans b.; vreugdenhil, harriet a.; slutsky, arthur s.; zijlstra, jitske; heijnen, cobi j.; van vught, hans title: mechanical ventilation alters the immune response in children without lung pathology date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: n xv l objective: this study was undertaken to examine the hypothesis that mechanical ventilation in association with anesthesia would alter the cytokine profile in infants without preexisting lung pathology. design and setting: prospective observational study in pediatric intensive care unit in a university hospital. patients: twelve infants who were subjected to an uncomplicated diagnostic cardiac catheterization procedure were studied. all subjects were ventilated with a volume control mode, . fio( ), cmh( )o peep, and ml/kg tidal volume. volatile (servoflurane) anesthetics were given. measurements and results: tracheal aspirates and blood samples were obtained before and after h of mechanical ventilation. in tracheal aspirates and in supernatants of stimulated whole-blood cultures cytokine concentrations were measured. in the tracheal aspirates the immune balance was characterized by a proinflammatory response pattern, with a significant increase in tnf-α and il- concentrations; concentrations of anti-inflammatory mediators remained very low. the functional capacity of peripheral blood leukocytes to produce inf-γ, tnf-α, and il- in vitro was significantly decreased. this was accompanied by a significant decrease in the killing activity of natural killer cells. conclusions: two hours of servoflurane and mechanical ventilation using a tidal volume of ml/kg is associated with remarkable changes in the immune response in infants without preexisting lung pathology undergoing cardiac catheterization. in the lungs the immune balance favors a proinflammatory response pattern without detectable concentrations of anti-inflammatory mediators. the th immune response by peripheral blood leukocytes was decreased. the observed change in th /th balance in favor of th cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. blood leukocytes to produce inf-γ, tnf-α, and il- in vitro was significantly decreased. this was accompanied by a significant decrease in the killing activity of natural killer cells. conclusions: two hours of servoflurane and mechanical ventilation using a tidal volume of ml/kg is associated with remarkable changes in the immune response in infants without preexisting lung pathology undergoing cardiac catheterization. in the lungs the immune balance favors a proinflammatory response pattern without detectable concentrations of antiinflammatory mediators. the th immune response by peripheral blood leukocytes was decreased. the observed change in th /th balance in favor of th cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. it has become clear that alterations in the immune balance may prevent an appropriate and effective response to various stimuli [ , ] . cd + t-cells can be divided functionally into th and th cells based on their cytokine profiles [ ] . th cells secrete interferon (ifn) γ while th cells secrete interleukin (il) , il- , il- , and il- . macrophages secrete proinflammatory and anti-inflammatory cytokines such as il- β, tumor necro-sis factor (tnf) α, il- , and il- . for example, an alteration in the th /th balance, resulting in a th dominance, is thought to contribute to enhanced pulmonary disease in respiratory syncytial virus bronchiolitis [ ] . on the other hand, new evidence indicates that a disturbance of the balance between proinflammatory mediators and anti-inflammatory mediators may initiate or amplify the inflammatory response in patients with the acute respiratory distress syndrome (ards) [ , ] . for example, the ratio of il- β to il- receptor antagonist is markedly elevated in patients with ards, favoring the unopposed proinflammatory activity of il- β. the observation that low intrapulmonary concentrations of il- and il- receptor antagonist at the onset of ards are associated with a poor outcome suggests that a lack of inhibitory cytokines is correlated with a poor prognosis. it has also been suggested that mechanical ventilation produces alterations in the immune balance [ ] . experimental studies have demonstrated that mechanical ventilation results in an inflammatory reaction in the lungs and that the degree of inflammation depends on the ventilatory strategy and mode [ , , , , ] . this inflammatory reaction may not be limited to the lungs but may initiate or propagate multiple system organ failure [ , , ] . a possible explanation for the spillover of inflammatory mediators as a result of mechanical ventilation is loss of compartmentalization [ ] . the important concept of compartmentalization refers to the fact that the inflammatory response remains compartmentalized in the area of the body were it is produced [ , ] . haitsma et al. [ ] have shown in rats that injurious ventilatory strategies, although not conclusive, disturb the compartmentalization of the early cytokine response in both the lung and the systemic circulation [ ] . infants who undergo cardiac catheterization may have multiple risk factors that may affect the inflammatory milieu in their lungs, including mechanical ventilation, exposure to anesthetic agents, and the stress of the procedure. the present study was designed to examine the hypothesis that mechanical ventilation in association with anesthesia would alter the cytokine profile in the lungs, and/or systemic circulation, of patients without preexisting lung pathology. the study included children (median age . years, range - ) who were undergoing a diagnostic cardiac catheterization procedure. the children had a history of a congenital heart disease, some of whom had been (partially) corrected: atrial-ventricular septal defect, transposition of the great arteries [ ] , aortic valve insufficiency, ventricular septal defect [ ] , tetralogy of fallot, coarctation of aorta, tricuspid atresia, pulmonary atresia [ ] , double outlet right ventricle. patients with a history of allergic or respiratory diseases, known chromosomal or immunological disorders, and patients recently hospitalized or mechanically ventilated were excluded. all subjects were intubated and ventilated with a volume control mode and a fractional inspiratory oxygen of . , a maximum peak inspiratory pressure of . ± . cm h o, a mean positive end-expiratory pressure (peep) of . ± . cm h o, and a mean tidal volume of . ± . ml/kg (measured body weight). the end-tidal co was maintained between - mmhg. if peep, inspiratory oxygen concentration, or tidal volume needed to be adjusted to maintain an adequate oxygenation or to maintain normocapnia, the patient was excluded from the study. heart rate and blood pressure of the individual patients remained constant during the procedure. all patients received servoflurane ( . %) anesthetic during the procedure. the study was approved by the medical ethics committee, and parents gave informed consent. tracheal aspirates and blood samples were obtained immediately after intubation, before the start of mechanical ventilation, and after h of mechanical ventilation. tracheal aspirates were obtained as previously described [ ] . the suction catheter was rinsed with . ml sterile normal saline and added to the suction trap. the aspirate was placed immediately on ice. thereafter % dithiothreitol ( %; µl per ml aspirate) was added, and the samples were centrifuged at rpm for min. supernatants were stored at - °c until analysis. blood samples were drawn from a venous catheter. heparinized blood was diluted : in rpmi- medium (roswell park memorial institute life technologies, grand island, n.y., usa), and whole-blood cultures were set up. the whole-blood culture stimulated with lipopolysaccharide (lps) is a suitable ex vivo method to study monocyte cytokine production under conditions in which many of the physiologically relevant cellular interactions remain intact [ , ] . to induce lymphocyte cytokine production (il- , ifn-γ) anti-cd , and anti-cd , ( : ) plus anti-cd ( : ) monoclonal antibodies (clb, amsterdam, the netherlands) were added, and cultures were incubated for h at °c in % co in air. all cultures were performed in quadruplicate. to induce the production of monocyte il- , il- , tnf-α, lps (difco laboratories, detroit, mich., usa) ( ng/ml) was added to the diluted blood samples and cultures were incubated for h at °c in % co in air. to induce monocyte il- production lps ( ng/ml) was added, and cultures were incubated for h at °c in % co in air. to induce monocyte il- production lps ( ng/ml) and ifn-γ ( ng/ml) were added, and cultures were incubated for h at °c in % co . addition of ifn-γ results in a more optimal il- response in the presence of lps. cytokine assays tnf-α, il- , il- , il- , il- , il- , and ifn-γ were measured via enzyme-linked immunosorbent assay (clb). the detection limit was - pg/ml for tnf-α, . pg/ml for il- , pg/ml for il- , - pg/ml for il- , - pg/ml for il- , pg/ml for il- , and - pg/ml for ifn-γ. when cytokines were not detectable, the minimum detectable level was used in the calculations. the composition of peripheral leukocytes was determined by analyzing the forward-sideward scatter. for lymphocyte subset analysis, whole blood was incubated with conjugated monoclonal antibodies under saturating conditions specific for cd , cd , cd , and cd / (simultest, becton and dickinson, mountain view, calif., usa). subsequently, red blood cells were lysed and samples were analyzed with a flow cytometer (facs-star+, becton and dickinson). the difference between negative and positive fluorescence was determined by measuring unstained cells and cells stained with an irrelevant isotype control body. natural killer cell activity natural killer cell (nk) cell activity was analyzed by determining the capacity of peripheral blood cells to kill cr-labeled k target cells as described previously [ ] . cortisol was measured by a chemiluminescence immunoassay performed on the fully automated advia centaur immunoanalyzer (bayer, leverkusen, germany). all values were expressed as mean ±sd and were analyzed by the nonparametric wilcoxon signed-rank test. differences were considered significant at the level of p< . . the concentrations of tnf-α in the supernatant of the tracheal aspirates increased significantly h after me-chanical ventilation (p= . ; fig. ). there was a trend towards an increase in il- levels (p= . ; fig. ). il- concentrations showed high interindividual variation both before and after mechanical ventilation. the concentrations of the anti-inflammatory cytokines il- and ifn-γ remained unchanged just above the detection level (fig. ). the capacity of lymphocytes to produce cytokines was determined in whole-blood cultures stimulated with anti-cd /cd [ , ] . after h of mechanical ventilation a significant decrease in ifn-γ production was observed in the cultured supernatants (p= . ), but no significant changes in il- concentrations were observed (fig. ) . the capacity of monocytes to produce cytokines was determined in whole-blood cultures stimulated with lps. after h of mechanical ventilation there was a decrease in the production of proinflammatory cytokines il- (p< . ) and tnf-α by peripheral blood monocytes (p< . ; fig. ). il- concentrations showed high interindividual variation before and after mechanical ventilation. the amount of il- and il- produced by monocytes was unaltered in all patients as a result of h of mechanical ventilation (fig. ) . we observed significant changes in the cellular composition of the whole-blood samples. there was a increase in the percentage of granulocytes (p< . ) and a decrease in the percentage of lymphocytes (p< . ; table ). the percentage of cd and cd increased slightly but significantly (p< . ). the percentage of cd / tended to decrease (table ) . as a result of h of mechanical ventilation the killing capacity of nk cells to lyse k target cells decreased significantly (p< . ). the mean percentage of activity decreased from . ± . to . ± . . this remarkable decrease in killing capacity of nk cells cannot be explained by a decrease in the total numbers of nk cells (table ) . the major finding of the present study is that exposing infants with normal lung function to h of volatile anesthetics, mechanical ventilation, and cardiac catheterization is associated with remarkable changes in immune responses. we observed a proinflammatory response in the lungs with a significant increase in tnf-α, while antiinflammatory cytokine concentrations in tracheal aspirates remained virtually unchanged, just above the detection level. in addition, the functional capacity of peripheral blood leukocytes to produce proinflammatory cytokines in vitro was significantly decreased, in particular ifn-γ, tnf-α, and il- . this was accompanied by a significant decrease in the activity of nk cells. this indicates that this procedure is associated with a change in the th /th balance with a decreased th immune response. a major question from our study is which aspect of the total procedure consisting of exposure to volatile anesthetics, ventilation, and catheterization is responsible for the observed changes in the inflammatory response of our patients. a recent review article summarized the effect of anesthetic agents on the immune response and concluded that there is little evidence to support the concept of clinically relevant immune modulation by anesthetics during major surgery [ ] . no clinical study has examined the effect of servoflurane on the immune response in infants and young children. experimental studies have shown that during mechanical ventilation of uninjured lungs several volatile anesthetics may augment gene expression of proinflammatory cytokines in rat alveolar macrophages [ ] . however, servoflurane was not associated with a significant increase in gene expression of proinflammatory cytokines or with concentrations of tnf-α in the lavage fluid of these rats over that with mechanical ventilation alone [ ] . kotani et al. [ ] demonstrated in mechanically ventilated adult patients that intravenous propofol or volatile isoflurane produced a similar increase in gene expression of all proinflammatory cytokines on alveolar macrophages. this is remarkable since the route of administration of these anesthetics are completely different. one would have expected that by directly acting on alveolar macrophages the volatile anesthetic -isoflurane -would induce faster and probably more pronounced gene expression. it therefore remains questionable whether these clinical observed effects are all attributable to general anesthesia. any effect of anesthesia is likely to be overwhelmed by the neuroendocrine stress response during major sur-gery [ ] . however, in our study the response of the hypothalamo-pituitary-adrenal axis to the catheterization procedure is probably negligible. serum cortisol levels measured before and after mechanical ventilation were similar. other factors such as hemorrhage, hypotension, ischemia/reperfusion, and blood transfusion, which may affect immune competence during major surgery, were negligible in our study. thus the catheterization procedure can therefore not considered to be major surgery. we are therefore left with the possibility that the changes in the immune response in our study were the result of mechanical ventilation, although we are aware that definitive conclusions cannot be made. several experimental studies have reported that injurious ventilatory strategies increase tnf-α mrna expression and lung lavage levels of tnf-α protein [ , , ] . in these studies tidal volumes were very high ( ml/kg), and/or there was preexisting lung injury. pretreatment with intratracheal instillation of anti-tnf-α antibodies improved oxygenation, reduced infiltration of leukocytes, and ameliorated pathological findings [ ] . the results of the experimental studies clearly demonstrated that tnf-α plays a pivotal role in initiating an inflammatory cascade induced by mechanical ventilation. the lung macrophage may be the critical mechanosensor cell capable of producing tnf-α in response to stretching mechanical forces [ ], although there is evidence that the pulmonary epithelium may also be a key player in this regard [ ] . it is remarkable, however, that such a significant proinflammatory response was observed with the ventilatory strategy we adopted. our patients had normal lungs, and a tidal volume of ml/kg should not cause overdistention, since the patients would not have the marked heterogeneities in pulmonary compliance that exist in patients with ards [ , ] . this is supported by the observation that peak inspiratory pressures remained low ( . ± . cmh o) throughout the -h period. to our knowledge, only one other study has examined the effect of mechanical ventilation on release of cytokines in patients with normal lung function [ ] . wrigge et al. [ ] observed that after h of mechanical ventilation plasma levels of pro-and anti-inflammatory mediators remained low and did not differ from baseline. unfortunately, the local production of cytokines in the lung was not measured. the observed effects in our study may be explained by a two-hit hypothesis in which any one factor by itself does not induce an effect, but a combination of factors act synergistically to cause the changes in immune response, i.e., mechanical ventilation and volatile anesthetics. it remains speculative what causes the onset of the peripheral immune response. one of the mechanisms could be that tnf-α produced locally in the lung causes leukocyte redistribution from the systemic circulation into the alveolar space [ , ] . mechanical ventilation may recruit t cell subsets with distinctive properties with respect to homing and trafficking into inflamed sites [ ] . we observed that the functional capacity of peripheral blood leukocytes to produce proinflammatory cytokines in vitro was significantly decreased, in particular inf-γ, tnf-α, and il- . ifn-γ is associated with a th response, which is considered to be beneficial in terms of an appropriate and effective response to various stimuli, including trauma, infection, and perhaps mechanical ventilation [ ] . ifn-γ is also important in stimulating the cytolytic activity of nk cells and cd + cytotoxic t lymphocytes. the decrease in ifn-γ production was also accompanied by a significant decrease in the killing activity of nk cells. the altered th /th balance in favor of th cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. in conclusion, h of servoflurane and mechanical ventilation with a tidal volume of ml/kg is associated with remarkable changes in the immune response in infants without preexisting lung pathology undergoing cardiac catheterization. in the lungs a proinflammatory response pattern dominates without detectable concentrations of anti-inflammatory mediators. we observed a decrease in the th immune response by peripheral blood leukocytes. the altered th /th balance in favor of th cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. further studies possibly using different anesthetic agents, different operative procedures, and different ventilatory strategies are needed to establish the mechanisms and clinical relevance of our findings. cytokines and the acute respiratory distress syndrome (ards): a question of balance dominance of t-helper -type cytokines after severe injury human th and th subsets: doubt no more anti-il- treatment at immunization modulates cytokine expression, reduces illness, and increases cytotoxic t lymphocyte activity in mice challenged with respiratory syncytial virus the acute respiratory distress syndrome ventilator-induced lung inflammation: is it always harmful? injurious ventilatory strategies increase cytokines and c-fos m-rna expression in an isolated rat lung model role of high-frequency ventilation in surfactant-depleted lung injury as measured by granulocytes inflammatory chemical mediators during conventional ventilation and during high frequency oscillatory ventilation ventilator pattern influences neutrophil influx and activation in atelectasis-prone rabbit lung intraalveolar expression of tumor necrosis factor-alpha gene during conventional and high-frequency ventilation multiple system organ failure: is mechanical ventilation a contributing factor? effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized clinical trial mechanical ventilation as a mediator of multisystem organ failure in acute respiratory distress syndrome compartmentalized lung cytokine release in response to intravascular and alveolar endotoxin challenge loss of compartmentalization of alveolar tumor necrosis factor after lung injury ventilator-induced lung injury measurement of interleukin in bronchoalveolar lavage from preterm ventilated infants a convenient whole blood culture system for studying the regulation of tumor necrosis factor release by bacterial lipopolysaccharide monocyte il- production during respiratory syncytial virus bronchiolitis is associated with recurrent wheezing in a one year follow-up study the authors thank the pediatric cardiologists and cardioanesthetists for their technical assistance. key: cord- -tlvgyzft authors: chan, kok fei; carolan, louise a; korenkov, daniil; druce, julian; mccaw, james; reading, patrick c; barr, ian g; laurie, karen l title: investigating viral interference between influenza a virus and human respiratory syncytial virus in a ferret model of infection date: - - journal: j infect dis doi: . /infdis/jiy sha: doc_id: cord_uid: tlvgyzft epidemiological studies have observed that the seasonal peak incidence of influenza virus infection is sometimes separate from the peak incidence of human respiratory syncytial virus (hrsv) infection, with the peak incidence of hrsv infection delayed. this is proposed to be due to viral interference, whereby infection with one virus prevents or delays infection with a different virus. we investigated viral interference between hrsv and pandemic influenza a(h n ) virus (a[h n ]pdm ) in the ferret model. infection with a(h n )pdm prevented subsequent infection with hrsv. infection with hrsv reduced morbidity attributed to infection with a(h n )pdm but not infection, even when an increased inoculum dose of hrsv was used. notably, infection with a(h n )pdm induced higher levels of proinflammatory cytokines, chemokines, and immune mediators in the ferret than hrsv. minimal cross-reactive serological responses or interferon γ–expressing cells were induced by either virus ≥ days after infection. these data indicate that antigen-independent mechanisms may drive viral interference between unrelated respiratory viruses that can limit subsequent infection or disease. viral interference is a phenomenon whereby infection with one virus limits or delays infection with a second virus. it has been described in human epidemiological studies observing viral epidemic peaks [ ] [ ] [ ] [ ] , vaccine efficacy studies [ ] , studies assessing virus infections in clinical samples [ ] [ ] [ ] , animal studies [ ] [ ] [ ] [ ] [ ] and in vitro infectivity studies [ ] . viral interference has been observed between a range of viruses, including between arboviruses, such as yellow fever and dengue virus [ ] ; between different respiratory viruses [ , , ] ; and between influenza viruses of different types [ ] and subtypes/lineages [ , ] . at a population level, respiratory virus infections may display distinct epidemic peaks. observational studies from the netherlands, france, and hong kong showed that emergence of pandemic influenza a(h n ) virus (a[h n ]pdm ) delayed infections with human respiratory syncytial virus (hrsv) [ , , ] . influenza a virus infections also interrupted peak incidences of hrsv infections in japan during - [ ] and in the netherlands during - [ ] . negative associations between respiratory viruses have been reported when analyzing the proportion of coinfections with different respiratory viruses, using swab specimens from patients [ , , ] . a(h n )pdm was least likely to be detected with any of the other respiratory viruses tested, including hrsv, in samples from all age groups [ , ] . taken together, these data suggest that interference may occur between a(h n )pdm and hrsv. the ferret provides an ideal model of human influenza because animals can be directly infected with virus without adaptation and display similar disease symptoms to those in humans [ , ] . historically, the ferret has also been used to study hrsv infection [ ] [ ] [ ] , with recent studies assessing the pathogenesis, immunity, and transmission of hrsv [ , ] . clinical symptoms are mild in ferrets infected with hrsv strains described to date [ , ] . previously, we used the ferret model to demonstrate that viral interference can occur following infection with human influenza a and b viruses and will prevent, delay, or limit subsequent infection with an influenza virus of a different type, subtype, or lineage [ , ] . notably, this effect depends on the virus combinations and the order and timing of sequential infections [ , , ] . we have established complementary influenza viral dynamics models that explain these observations via the innate immune response [ ] and cross-reactive adaptive immune responses [ ] . ecological data suggest that infection with a(h n )pdm can prevent or delay infection with hrsv. using our ferret models of influenza and hrsv, we have systematically investigated this hypothesis. adult ferrets were housed at the peter doherty institute for infection and immunity bioresources facility. experiments were conducted with approval from the university of melbourne microbiology and immunology animal ethics committee, in accordance with the australian national health and medical research council code of practice for the care and use of animals for scientific purposes. all ferrets were seronegative for antibodies to currently circulating influenza viruses and hrsv (long and a strains) before use in experiments. a/tasmania/ / (a[h n ]pdm ) virus was passaged allantoically in embryonated hen's eggs and stored at − °c. the infectious influenza virus titer was measured by a % tissue culture infectious dose (tcid ) assay [ ] , read by hemagglutination with turkey red blood cells. hrsv long and a strains were passaged [ ] . infectious hrsv titers were determined by plaque assay [ ] . ferrets were infected intranasally with . tcid a(h n ) pdm in µl and plaque-forming units (pfu) of long hrsv or pfu of long or a hrsv in µl and monitored [ , ] . ferrets were housed in pairs, by infection group. nasal wash specimens were collected and stored [ ] . on the day of collection, viral rna was extracted from -µl nasal wash specimens for quantitative polymerase chain reaction (qpcr) analysis. blood samples were obtained from ferrets before primary virus infection and immediately before and days after challenge, and serum was isolated. the proportional change in weight was calculated as the percentage difference from the weight on the day of challenge. four microliters of viral rna [ ] was assayed by rt-qpcr with a(h n )pdm hemagglutinin-specific primers/probes from the cdc influenza virus rt-qpcr influenza a (h /h /h pdm ) subtyping panel, obtained from the influenza reagent resource (available at: http://www.influenzareagentresource.org/) and hrsv n-specific primers/probes [ ] . copy numbers for a(h n ) pdm viral rna were calculated relative to plasmid phw -a/ tasmania/ / hemagglutinin; copy numbers for rsv rna were calculated relative to a hrsv rna standard [ ] . mrna was isolated from nasal wash samples [ ] . mrna expression of cytokines, chemokines, and housekeeping genes was quantified by qpcr [ , ] . infectious hrsv in nasal wash samples was measured using the vs assay [ ] . interferon γ (ifn-γ) enzyme-linked immunospot (elispot) assay ifn-γ-producing cells were detected by a ferret ifn-γ elispotplus assay (mabtech). single cell suspensions were prepared from ferret retropharyngeal lymph nodes [ ] . a total of × lymph node cells were cultured with or without live influenza virus, hrsv, or µg/ml concanavalin a (sigma) for hours at o c in % co [ ] . titers of antibodies to a/tasmania/ / were measured using hi assays [ , ] . titers were expressed as the reciprocal of the highest dilution of serum for which hemagglutination was prevented. geometric mean titers (gmts) were calculated, with undetectable titers expressed as having a value of " ." seroconversion was defined as a titer of ≥ at the end of the experiment and at least a -fold rise from baseline. titers of antibodies that neutralize hrsv long and a were measured using vs mn assays [ ] . seroconversion was defined as a titer ≥ at the end of the experiment and an increase of at least -fold from the baseline titer. antibodies that bind to the f glycoprotein of hrsv were detected by an elisa [ ] . viral kinetics were assessed in viral rna from nasal wash specimens. for a(h n )pdm , > copies of hemagglutinin/ µl of nasal wash were positively correlated with replicating virus, based on the tcid assay [ ] and the level of infectious virus as measured by transmission in ferrets [ ] . for hrsv, . copies of n/ µl of nasal wash corresponded to a % chance of a sample being positive by the virospot assay, as determined using a probit regression model (supplementary figure ) . accordingly, samples were considered to be infectious for hrsv when the amount of viral rna exceeded . copies/ μl nasal wash and infectious for a(h n )pdm when viral rna exceeded copies/ µl of nasal wash for at least measurement. clinical signs (ie, weight loss and fever) were assessed daily, and seroconversion was measured days after challenge. statistical analysis was conducted using prism, version . g, unless otherwise indicated and is described in the figure legends. ferrets were first infected with a(h n )pdm virus then challenged with hrsv , , or days later, or vice versa ( figure a ). the intervals between inoculations spanned the times of peak titer and clearance of both virus infections [ , ] and induction of humoral immunity ( figure a ). primary infection with a(h n )pdm prevented subsequent infection with hrsv in of ferrets when primary infection and challenge were separated by days. shedding of hrsv was minimal in the single ferret infected, compared with control animals ( figure b and c) . no ferrets in this group seroconverted to hrsv (figure bi and bii ). primary infection with a(h n )pdm prevented infection with hrsv in of ferrets when infections were separated by days ( figure d ). ferrets that did not shed virus did not seroconvert (figure bi and bii), while ferrets that shed virus seroconverted to hrsv (figure bi and bii). prior infection with a(h n )pdm did not prevent infection with hrsv days later ( figure e ), with all ferrets showing a similar pattern of virus shedding ( figure b ) and antibody titers to control animals that received hrsv alone (figure bi and bii). the kinetics of hrsv shedding was examined in animals not protected from hrsv challenge. the peak of hrsv shedding was delayed in ferrets infected with a(h n )pdm followed by hrsv as compared to control animals infected with hrsv alone (median, vs days; p = . by the mann-whitney test; figure ci ). there was no change in the duration of virus shedding ( figure cii ). clinical signs following hrsv challenge were minimal (supplementary figure ) , consistent with our previous study [ ] . all ferrets, except control ferret infected with hrsv, maintained or gained weight (supplementary figure figure biv ). the median duration of a(h n )pdm shedding was increased in ferrets infected with hrsv followed by a(h n )pdm as compared to control animals infected with a(h n )pdm alone ( vs days; p = . by the mann-whitney test; figure civ ). there was no change in the peak day of shedding (figure ciii ). prior infection with hrsv did reduce disease following infection with a(h n )pdm . the mean maximum weight loss (±sd) among control ferrets infected with a(h n )pdm was . % ± . % (supplementary figure a and d) . the mean maximum weight loss (±sd) for ferrets in all test groups (n = ) was . % ± . % (supplementary figure b , c, and e). thus, prior infection with hrsv significantly reduced morbidity, as measured by weight loss, after challenge with a(h n ) pdm (p = . by the mann-whitney test). no fever was detected following a(h n )pdm infection (supplementary figure f -j). ferrets were infected ( ) with an increased viral dose of the same hrsv strain, long, or ( ) with an alternate hrsv strain, a (also at an increased viral dose), then challenged with a(h n ) pdm days later. a is a laboratory-adapted strain that is shed at similar levels to long in ferrets and transmits between cohoused animals [ ] . infection of ferrets with a -fold higher inoculum (ie, pfu) of hrsv long led to a small increase in virus shedding on days - after infection, compared with animals infected with pfu of hrsv long, although these differences were not significant (supplementary figure ) . primary infection with pfu hrsv long or a did not prevent infection with a(h n )pdm when infections were separated by days (figure ). all animals seroconverted to a(h n )pdm at similar levels ( figure bv and bvi). most ferrets lost weight after a(h n )pdm infection. the mean maximum weight loss (±sd) among ferrets infected with a(h n )pdm alone was . % ± . %, whereas the mean maximum weight loss (±sd) for ferrets (n = ) that received primary infection with hrsv prior to a(h n )pdm challenge was . % ± . % (p = . by the mann-whitney test; supplementary figure a -c). there was no difference in fever (supplementary figure d -f) and no change to the kinetics of infection between animals that received a prior hrsv infection, compared with those that did not (data not shown). inflammation induced by viral infection may contribute to viral interference [ , ] . we investigated the localized immune response following infection with hrsv or a(h n )pdm . nasal wash specimens were collected early (day ) and later (day / ) after infection, because the pattern of inflammatory mediators changes throughout h(h n )pdm [ ] and hrsv [ ] infections. expression of influenza virus matrix (m) mrna was highest on day after infection, whereas expression of hrsv nucleoprotein (n) mrna was highest on day ( figure a ). two days after infection, animals infected with a(h n )pdm had significantly higher levels of interferon β (ifn-β), granzyme b, ifn-γ, interleukin (il- ), monocyte chemoattractant protein (mcp- ), and tumor necrosis factor α (tnf-α) mrna as compared to animals infected with hrsv ( figure d, g-i, n , and o). expression of ifn-α and granzyme a mrna was increased but not significantly (ifn-α, p = . ; granzyme a, p = . ; figure e and f). on day / after infection, levels of granzyme closed circles and open circles indicate animals that did or did not, respectively, shed detectable challenge virus, as determined by quantitative reverse-transcription polymerase chain reaction analysis of viral rna from nasal wash (nw) samples. for statistical analysis, titers or fold changes were compared between test and control groups, using -way kruskal-wallis analysis of variance with the dunn multiple comparison test. *p < . and **p < . . c, the kinetics of shedding was analyzed for all ferrets that shed challenge virus in figures and . data from ferrets obtained at the -day, -day, and -day intervals were pooled into the test group. the number of days from challenge inoculation to the peak level of challenge virus shedding (ci and ciii) and the number of days the challenge virus was shed (cii and civ) was determined for each ferret in the indicated groups. horizontal lines indicate median values. the number of days of virus shedding were compared between test and control groups, using the mann-whitney test. *p < . and **p < . . a, granzyme b, ifn-γ, interleukin , and mcp- mrna were significantly higher in animals infected with a(h n )pdm as compared to those infected with hrsv ( figure f -h, m, and n). there was significant increase in expression of interleukin (il- ) mrna days after infection and of interleukin β, il- , and il- mrnas / days after infection in ferrets infected with hrsv as compared to a(h n )pdm ( figure c, i, and j ). this suggests a localized inflammatory response was induced after hrsv infection, which coincided with the increase in hrsv virus replication ( figure a ). to directly compare the magnitude of expression of cytokines and chemokines induced by both virus infections, we assessed mrna expression on the day after infection at which the level of virus shedding was highest (ie, day for a(h n ) pdm and day for hrsv). when assessed at these times, an equivalent fold change in mrna expression was observed for rsv n and influenza virus m ( figure a ). infection with a(h n )pdm induced significantly higher levels of ifn-β (p = . ; figure d ), il- (p = . ; figure i ), interleukin p (p = . ; figure l ), mcp- (p = . ; figure n ), and tnf-α (p = . ; figure o ) mrna expression in ferrets, compared with hrsv. these data suggests there is increased inflammation in nasal tissues of animals infected with a(h n )pdm as compared to hrsv. there was no difference in expression of any cytokines or chemokines between ferrets infected with or pfu of hrsv long (data not shown). we have demonstrated that cross-reactive ifn-γ cellular responses can be detected between influenza b virus lineages and may contribute to viral interference [ ] . thus, we assessed whether cellular immunity induced by infection with a(h n ) pdm showed any cross-reactivity to hrsv. whereas retropharyngeal lymph node cells from a(h n )pdm -infected ferrets were restimulated with a(h n )pdm ( figure b ), few cells produced ifn-γ when stimulated with hrsv ( figure a ). lymph node cells from hrsv-infected ferrets were restimulated with hrsv in vitro, although at much lower levels ( figure a ), and were not restimulated by a(h n )pdm ( figures b) . responses to concanavalin a were similar for all ferrets regardless of infection ( figure b ). moreover, there was limited serological cross-reactivity. animals infected with a(h n )pdm had high levels of influenza virus-specific neutralizing antibodies ( figure c ), yet minimal total serum or neutralizing antibodies to hrsv ( figure d and e) . similarly, infection with hrsv induced total serum and neutralizing antibodies to hrsv but few antibodies that were reactive with a(h n )pdm ( figure c-e) . discussion we have demonstrated that infection with a(h n )pdm can prevent infection and replication of hrsv in a ferret model of human disease for up to days. infection with hrsv did not prevent subsequent infection with a(h n )pdm ; rather, animals were coinfected, albeit with reduced morbidity. infection with a(h n )pdm leads to increased levels of proinflammatory cytokines in the respiratory tract as compared to infection with hrsv. overall, these data support the ecological observation that viral interference induced by a(h n )pdm infection delayed infection with hrsv in the winter of - . infection with a(h n )pdm induced higher expression of mcp- , il- , type i ifns, tnf-α, ifn-γ, and granzyme a/b mrnas as compared to hrsv infection. mcp- and tnf-α regulate the migration of macrophages/monocytes and natural killer (nk) cells into the respiratory tract. macrophages produce mcp- , tnf-α, and il- ; thus, upregulation of these genes suggests an influx of macrophages and nk cells into the respiratory tissues [ ] . nk cells produce ifn-γ, which activates macrophages and neutrophils and promotes t-cell proliferation and killing of virus-infected cells [ ] . because cytotoxic t lymphocytes and nk cells also produce granzymes a/b, increased expression of ifn-γ and granzyme a/b mrnas on day after infection suggests recruitment/activation of these cells to the site of infection. il- and type i ifns are produced by respiratory epithelial cells, monocytes/macrophages, and dendritic cells [ , ] . il- is a proinflammatory cytokine, whereas type i ifns induce an antiviral state that may also limit replication and spread of hrsv [ , ] it would be useful to explore the cellular infiltrate following a(h n )pdm and hrsv infections to gain further insight . ferrets were infected with plaque-forming units (pfu) of hrsv strain long or . % tissue culture infectious doses of a(h n )pdm (n = ferrets/virus). nasal wash specimens were collected after challenge from ferrets on days and after infection, and mrna was assayed for the indicated genes, using quantitative polymerase chain reaction (qpcr) assays. for each graph, qpcr data are expressed as fold changes relative to values for nasal wash specimens from uninfected animals and normalized to atf and gapdh housekeeping genes. in panel a, expression of n is shown for hrsv-infected ferrets, and expression of m is shown for influenza virus-infected ferrets. for statistical analyses, inflammatory mediators were compared between ( ) hrsv-infected and a(h n )pdm -infected animals sampled on day after infection, ( ) hrsv-infected and a(h n )pdm -infected animals sampled on day / after infection, and ( ) a(h n )pdm -infected animals sampled on day and hrsv-infected animals sampled on day after infection. fold changes were compared between viruses, using the mann-whitney u test. ifn, interferon; il- , interleukin ; il- , interleukin ; il- , interleukin ; il- , interleukin ; il- p , interleukin p ; il- , interleukin ; mcp- , monocyte chemoattractant protein ; tnf-α, tumor necrosis factor α. *p < . , **p < . , ***p < . , and ****p < . . into potential differences in the level and cellular composition present in local inflammation. infection with a(h n ) pdm induced a -fold higher cellular ifn-γ recall response as compared to infection with hrsv in our study. because there was no significant difference in ifn-γ responses to concanavalin a between the groups, this observation was not due to a difference in overall t-cell numbers but, instead, was due to an increase in the reactivation of a(h n )pdm -specific cells. taken together, these data suggest that infection with a(h n )pdm induces a robust cytokine and chemokine response that strongly stimulates the adaptive and memory immune responses. conversely, infection with hrsv elicited a weaker and more limited cytokine and chemokine response that led to a reduced antigen-specific cellular response. however, it is possible that hrsv may not infect the ferret respiratory tract as efficiently as a(h n )pdm does, and this could result in reduced inflammatory responses. yet, infected animals seroconverted at titers consistent for sterilizing immunity, indicating a productive infection (data not shown) [ ] . furthermore, increasing the inoculum of hrsv did not significantly affect the pattern or amount of virus shedding nor the expression of inflammatory mediators, suggesting that the hrsv level was already maximal in this ferret model. notably, increased expression of inflammatory mediators following infection with influenza virus as compared to hrsv has been observed in studies assessing human clinical samples and in vitro airway epithelial cell cultures [ ] [ ] [ ] [ ] . what is the mechanism of viral interference induced by a(h n )pdm ? the increased antiviral state and inflammation observed after a(h n )pdm infection has the potential to prevent subsequent infection or delay shedding of hrsv, as was observed here. both viruses predominantly infect ciliated airway epithelial cells, and we have shown that a(h n )pdm and hrsv long replicated in the upper and lower respiratory tracts of ferrets [ , ] . infection with a(h n )pdm can also prevent infection with an influenza b/yamagata virus [ ] . there are minimal shared epitopes between influenza a and b viruses [ ] , and we showed that minimal cross-reactive ifn-γ-producing cells were induced between hrsv and influenza virus. these data suggest that short-lived mechanisms drive this effect, as no effect was detected in ferrets after one week or, as shown by others, in mice, when infections were separated by days [ ] . the timing of interference indicate that interactions between different viruses may also be important. it is possible that different mechanisms act on different virus combinations. gene expression analysis of early markers of the immune response (cona; b) . the number of interferon γ (ifn-γ)-producing cells was determined by an enzyme-linked immunospot (elispot) assay. c-e, sera were tested for antibodies to a(h n )pdm , by a hemagglutination inhibition (hi) assay (c); for total serum antibody binding to hrsv f protein, by an enzyme-linked immunosorbent assay (elisa; d); and for neutralizing serum antibody to hrsv long or a , by a virospot microneutralization (mn) assay (e). data were obtained from ferrets per group. of respiratory epithelium infected with the virus strains used in these studies may provide further insight. it is notable that infection with hrsv reduced morbidity induced by a(h n )pdm infection. although virus loads were not decreased in nasal wash specimens, virus shedding may be reduced in the lower respiratory tract, limiting clinical disease. il- mrna expression was elevated in nasal wash samples of ferrets infected with hrsv as compared to a(h n ) pdm . increased il- expression has been associated with milder disease in ferrets infected with pathogenic influenza virus strains, potentially mediated by rapid recruitment of neutrophils, which assist in clearing virus [ ] . analysis of the lung influenza virus loads in animals that have been infected with hrsv prior to challenge with a(h n )pdm would be of interest. epidemiological data reported in france described a - week delay in the peak incidence of hrsv infections following the emergence of the a(h n )pdm , compared with previous years [ ] . similarly, a delay of - weeks of the expected peak of hrsv was reported following an early influenza a season in the netherlands. [ ] . these population-level observations of viral interference arise from the interplay between ( ) immunodynamics (ie, host-level viral interference), ( ) heterogeneity between hosts (ie, differences in immunity to virus strains between individuals), and ( ) transmission dynamics (ie, within or between different age groups) [ ] . for influenza, these processes have been investigated in some detail. others have demonstrated that a short period (ie, days rather than weeks) of viral interference at the host level may result in substantial separation between epidemic waves at the population level [ ] . our results provide the first hostlevel immunodynamic evidence in support of these processes driving the epidemiological interactions observed previously in europe [ , ] . our study has limitations. we used a circulating strain of a(h n )pdm from early and laboratory strains of hrsv, long and a . the long and a strains induce consistent infections and disease in ferrets, with characterized cytokine profiles [ ] . use of a circulating clinical isolate of hrsv may provide more-realistic data but was not available for these experiments. interference between influenza virus and hrsv has been reported in epidemiological studies in various years [ , ] , suggesting that influenza viruses other than a(h n )pdm may also prevent/limit infection and replication of hrsv. there are currently no licensed vaccines for hrsv. identification of host-and/or virus-encoded factors that contribute to viral interference provides a platform to facilitate development of novel prophylactic or therapeutic strategies to prevent or ameliorate respiratory infections, such as hrsv infection, that have a significant burden in the community, especially among young children. impact of the influenza a(h n ) pandemic wave on the pattern of hibernal respiratory virus epidemics the clinical features of respiratory syncytial virus: lower respiratory tract infection after upper respiratory tract infection due 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weber, friedemann title: nss protein of sandfly fever sicilian phlebovirus counteracts interferon (ifn) induction by masking the dna-binding domain of ifn regulatory factor date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: g sandfly fever sicilian virus (sfsv) is one of the most widespread and frequently identified members of the genus phlebovirus (order bunyavirales, family phenuiviridae) infecting humans. being transmitted by phlebotomus sandflies, sfsv causes a self-limiting, acute, often incapacitating febrile disease (“sandfly fever,” “pappataci fever,” or “dog disease”) that has been known since at least the beginning of the th century. we show that, similarly to other pathogenic phleboviruses, sfsv suppresses the induction of the antiviral type i interferon (ifn) system in an nss-dependent manner. sfsv nss interfered with the tbk -interferon regulatory factor (irf ) branch of the rig-i signaling pathway but not with nf-κb activation. consistently, we identified irf as a host interactor of sfsv nss. in contrast to irf , neither the ifn master regulator irf nor any of the related transcription factors irf , irf , and irf were bound by sfsv nss. in spite of this specificity for irf , nss did not inhibit its phosphorylation, dimerization, or nuclear accumulation, and the interaction was independent of the irf activation or multimerization state. in further studies, we identified the dna-binding domain of irf (amino acids to ) as sufficient for nss binding and found that sfsv nss prevented the association of activated irf with the ifn-β promoter. thus, unlike highly virulent phleboviruses, which either destroy antiviral host factors or sequester whole signaling chains into inactive aggregates, sfsv modulates type i ifn induction by directly masking the dna-binding domain of irf . importance phleboviruses are receiving increased attention due to the constant discovery of new species and the ongoing spread of long-known members of the genus. outbreaks of sandfly fever were reported in the th century, during world war i, and during world war ii. currently, sfsv is recognized as one of the most widespread phleboviruses, exhibiting high seroprevalence rates in humans and domestic animals and causing a self-limiting but incapacitating disease predominantly in immunologically naive troops and travelers. we show how the nonstructural nss protein of sfsv counteracts the upregulation of the antiviral interferon (ifn) system. sfsv nss specifically inhibits promoter binding by ifn transcription factor (irf ), a molecular strategy which is unique among phleboviruses and, to our knowledge, among human pathogenic rna viruses in general. this irf -specific and stoichiometric mechanism, greatly distinct from the ones exhibited by the highly virulent phleboviruses, correlates with the intermediate level of pathogenicity of sfsv. keywords dna-binding domain, irf , nss, sandfly fever sicilian virus, interferon beta promoter, interferon induction m embers of the genus phlebovirus (order bunyavirales, family phenuiviridae) are present worldwide and gain increasing attention as vector-borne agents of disease ( ) . in addition to prominent, recently emerged phleboviruses such as severe fever with thrombocytopenia syndrome virus (sftsv) in asia and heartland virus (hrtv) in north america ( ) , there are long-known members, such as rift valley fever virus (rvfv), punta toro virus (ptv), toscana virus (tosv), and sandfly fever sicilian virus (sfsv), that are often reemerging or spreading into new geographical areas ( ) . in addition to these highly virulent (sftsv, hrtv, and rvfv) and intermediately virulent (tosv and sfsv) human pathogens, rapid progress in high-throughput sequencing enabled the identification of novel phleboviruses for which the disease potential is either recognized (e.g., sandfly fever turkey virus [ ] and adria virus [ ]) or not yet clarified (e.g., massilia virus [ ] , aguacate virus [ ] , and dashli virus [ ] ). infection by sfsv and related sandfly fever viruses, all transmitted by phlebotomine sandflies, typically presents as an acute febrile disease with abrupt onset, often developing into incapacitating myalgia, headaches, malaise, leukocytopenia, or ocular or gastrointestinal symptoms ( , ) . an outbreak of this so-called "sandfly fever," "pappataci fever," or "dog disease" during the sicilian campaign of world war ii in enabled albert sabin to isolate sfsv from infected soldiers ( ) . sfsv later proved to be one of the most widespread phleboviruses; it is present across the entire mediterranean basin, in portugal, in the middle east inclusive of the arabian peninsula, in sudan, in ethiopia, and in somalia and in locations as distant as india and bangladesh ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in regions of endemicity, seroprevalence can reach levels of up to % in humans and close to % in dogs and other domestic animals, including cattle ( , , , ) . hence, sandfly fever viruses are recognized as a significant public health threat, predominantly for immunologically naive groups such as soldiers or travelers ( ) ( ) ( ) ( ) . nonetheless, little is known about the molecular interplay of sfsv and sfsv-like viruses with the host organism. like all phleboviruses, sfsv contains a tripartite single-stranded rna genome ( , ) . while the large (l) genome segment and the medium (m) genome segment encode the viral polymerase (pol) l and the glycoproteins, respectively, in a negative orientation, the small (s) segment codes for the nucleocapsid protein n and the nonstructural protein nss in an ambisense manner. the genomic rna segments are packaged into ribonucleoproteins (rnps) by the nucleocapsid n protein and the l polymerase and are transcribed and replicated in the cytoplasm ( ) . due to complementarity of the = and = termini, the three rnp-packaged genome segments have the capacity to anneal to a so-called "panhandle." this rna structure, with its short double-stranded region and =-triphosphate moiety, is an activator of the cytoplasmic rna helicase rig-i, an important virus sensor of the antiviral type i interferon (ifn) system ( ) . ligand-bound rig-i signals via the adaptor mitochondrial antiviral-signaling protein (mavs) and the kinases tbk /ib kinase (ikk) to eventually activate the ubiquitously expressed transcription factor interferon regulatory factor (irf ) ( ) . the latter thereby becomes phosphorylated, dimerizes, and accumulates in the nucleus, where, together with nf-b and atf- /c-jun, it transactivates the ifn-␤ promoter to kick off a first wave of ifn secretion ( ) . autocrine and paracrine action of ifn-␤ then triggers the upregulation of irf , which amplifies and diversifies the initial irf -driven ifn response by inducing both the ifnb gene and multiple ifna genes ( ) ( ) ( ) . simultaneously, it induces the transcription of ifn-stimulated genes (isgs), several of them with demonstrated antiphleboviral activity ( ) . phleboviruses counteract the induction of the ifn response by means of their nss protein ( , ) . the best-characterized nss, namely, that of rvfv, allows the full rig-i signaling cascade to reach the point of irf binding to the ifn-␤ promoter but then abrogates host gene expression by targeted sequestration and deletion of general transcription factors, as well as by the recruitment of corepressors and induction of an mrna export block ( ) ( ) ( ) ( ) ( ) ( ) . in the case of tosv, in contrast, the nss protein causes proteasomal degradation of rig-i ( ) , and for sftsv, the nss sequesters multiple factors of the signaling cascade into cytoplasmic aggregates ( ) ( ) ( ) ( ) . for many phleboviruses, including the sandfly-borne sfsv, however, the mechanism of nss action is unclear. we and others previously found that the nss of sfsv, expressed by a recombinant rvfv, was able to block transcription of the ifnb gene ( , ) . here, we investigated the molecular mechanism and identified irf as a functional target. sfsv nss inhibits ifn induction. sfsv nss expressed by recombinant rvfv was previously shown to inhibit the upregulation of the ifnb gene ( , ) . accordingly, infection with parental sfsv strain sabin resulted in only limited upregulation of ifn-␤ mrna, as measured by reverse transcriptase quantitative pcr (rt-qpcr) (fig. a) . as controls, we used rvfv strain mp (expressing a functional rvfv nss) and clone (expressing an internally deleted rvfv nss) in parallel ( ) , which suppressed and activated ifn induction, respectively, in the expected manner. unlike rvfv, neither a natural nor a recombinant nss-deficient strain is available for sfsv. in order to abort nss function, we designed a pool of four small interfering rnas (sirnas) that specifically target the nss gene sequence. the efficiency of the sirnas was tested by cotransfection of an expression plasmid for ϫflag-tagged sfsv nss and either the nss-targeting sirna pool or a control sirna. the specific sirnas caused a significant reduction of sfsv nss rna levels in rt-pcr and a complete loss of the flag signal in immunoblot analysis, while the control sirna had no effect (fig. b) . in contrast, rna and protein levels of the ϫflag-tagged nss of ptv-a were not affected, confirming the specificity of the sirna pool for sfsv nss. we then combined transfection of the nss-specific sirna pool with infection by either sfsv or rvfv mp- , followed by rt-qpcr analysis. of note, in infected cells the sirna pool as well as the pcr primers can target not only the nss transcript but also the entire s genome segment. therefore, we could not determine whether only the nss mrna was affected by the sirnas or whether the viral genome was also affected. however, due to encapsidation of the genome, we expect a certain level of protection, which in turn would result in an underestimation of sirna effects on nss transcripts. in any case, a substantial depletion of nss sequence-containing rna species (fold reduction, . Ϯ . ) was observed (fig. c) . moreover, in the presence of the nss-specific sirnas, sfsv infection upregulated the amounts of ifn-␤ transcripts (fold increase, . Ϯ . ) (fig. d) , despite the fact that virus replication (measured via analysis of l segment levels) was diminished (fold reduction, . Ϯ . ) (fig. e) . for rvfv mp , in contrast, the sfsv nss-specific sirnas affected neither the ifn-␤ mrna levels (fig. c ) nor the accumulation of its s segment (fig. f) . the same applied to clone , tosv, and the closely related sandfly fever turkey virus (data not shown), demonstrating both the specificity of the sirna pool and its effect on the induction of ifn-␤ by sfsv. furthermore, no intrinsic ifn-stimulatory activity of the sirna pool was observed in the mock samples (fig. c) . taking into consideration the opposing effects of the sirna on ifnb induction and on sfsv replication, a normalized fold induction of . Ϯ . was calculated for ifnb, compared to . Ϯ . for rvfv (fig. g, right column) . of note, the impairment of sfsv replication by the nss-specific sirna was far less pronounced in ifn-incompetent vero b cells (data now shown), indicating that it was largely mediated by the antiviral ifn system rather than by interference with the integrity of the genomic s segment. in summary, sirna knockdown of sfsv nss resulted in simultaneous upregulation of ifn induction and downregulation of sfsv replication in ifn-competent cells, reminiscent of the behavior of nss-deficient phleboviruses. together with the data from recombinant nss-expressing rvfv ( , ) , this validates the identification of sfsv nss as an ifn induction antagonist. sfsv nss acts in a nondegradative manner. many pathogenic phleboviruses are known to counteract the ifn response by diminishing the levels of key host factors ( ) . the nss of rvfv induces proteasomal degradation of cellular proteins such as tfiih-p (to block ifn induction) and protein kinase r (pkr) (to prevent the antiviral action of ifn) ( , , ( ) ( ) ( ) . the nss of tosv was also shown to cause pkr degradation and to block ifn induction by decreasing rig-i levels ( , ) . we investigated whether the nss protein of sfsv might execute a similar form of degradative activity on host proteins. as controls, we employed tosv nss and rvfv nss, and we also included the so far hpi, and analyzed by rt-qpcr analysis for ifnb (n ϭ ; mean Ϯ sd). (b) a cells were cotransfected with expression constructs for ϫflag-tagged sfsv or ptv-a nss and nontargeting control sirna or sfsv nss-specific sirna. samples were subjected to rt-pcr analysis (upper panels) and immunoblotting using anti-flag and anti-tubulin antibodies (lower panel) h after transfection. to exclude amplification of nss sequences from plasmid dna, a duplicate set of reactions was performed without the reverse transcription step (no rt). (c to f) a cells were pretransfected with control or sfsv nss-targeting sirna and infected with sfsv or rvfv mp at an moi of . rna was isolated hpi for rt-qpcr analysis for nss-containing rna (c), ifnb (d), the l segment of sfsv (e), and the s segment of rvfv mp (n ϭ ; means Ϯ sd) (f). (g) summary of the relative fold induction data depicted in panels c to f, normalized to the mock sample pretreated with control sirna as well as the fold induction of ifnb in sinss-treated cells over sictrl-treated cells that occurred in a manner independent of the viral burden (means Ϯ sd). n.a., not applicable. little-investigated ptv nss, which is known to inhibit host cell transcription ( ) . for ptv, there are two distinct strains, namely, adames (ptv-a) and balliett (ptv-b), which strongly and weakly suppress ifn induction, respectively ( , ) . to directly compare the degradative capacities of the nss proteins of rvfv, tosv, sfsv, ptv-a, and ptv-b, we infected a cells with recombinant rvfv encoding the respective nss genes and monitored the intracellular levels of the known phleboviral targets tfiih-p , pkr, and rig-i, as well as of the central rig-i signaling factors mavs, tbk , and irf . as shown in fig a, levels of tfiih-p were reduced only by rvfv nss. moreover, and in agreement with previous studies ( , , ) , pkr levels were decreased upon expression of the nss of rvfv and tosv but not by those of sfsv and ptv. rig-i levels were left unchanged by the nss of rvfv or ptv-a, strongly decreased by the nss of tosv, and upregulated after infection with the recombinant rvfv expressing nss of sfsv (weakly) or ptv-b (strongly). in fact, in the presence of ptv-b nss the upregulation of rig-i was indistinguishable from the level seen with the nss-deficient control virus rzhΔnss. the levels of mavs, tbk , and irf were not affected by any of the nss proteins. these results were confirmed in cells infected with the parental sfsv strain sabin (fig. b) . thus, the nss proteins of sfsv and ptv do not degrade the host targets of other phleboviruses. sfsv nss inhibits the irf branch of ifn induction. for our further investigations, we focused on the nss of sfsv but also included those of rvfv (as a well-characterized control) and ptv. to interrogate their activity on ifn induction, we performed luciferase reporter assays. human hek cells were transfected with increasing amounts of expression plasmids encoding the respective nss proteins, along with a reporter construct harboring the firefly luciferase (ff-luc) gene under the control of the ifn-␤ promoter and a constitutively expressing renilla luciferase (r-luc) plasmid for normalization. activation of the ifn-␤ promoter was stimulated by cotransfection of a mavs cdna plasmid. as expected, overexpression of mavs strongly activated the ifn-␤ promoter, which was undisturbed by increasing doses of the n terminus of the human mxa protein (Δmx [ ] ) which was used as a negative control (fig. a) . expression of the nss proteins of rvfv, sfsv, and ptv-a, in contrast, suppressed the promoter in a dose-dependent manner. ptv-b nss showed only a partial effect in response to large plasmid amounts, in line with previous observations ( ) . the ifn-␤ promoter contains several positive regulatory domains (prds), among which prdi binds transcription factors of the interferon regulatory factor (irf) family and prdii binds nf-b ( , ) . reporter assays showed that the inhibitory effect of sfsv nss on the prdi promoter element was comparable to that seen with the full ifn-␤ promoter but that prdii activity was inhibited only weakly ( fig. b and c). this is in contrast to the nss of ptv-a, which, like the rvfv nss, inhibited the two prd reporters indiscriminately. as similar results were obtained when tbk was used for stimulation instead of mavs (data not shown), we concluded that sfsv nss specifically targets the irf branch of ifn induction at the level of tbk or further downstream, whereas ptv-a nss blocks ifn induction in a broad manner, as shown previously ( ) . sfsv nss interacts with irf in a highly specific manner. previously, we took part in a large proteomics screen to identify host cell interactors of viral ifn antagonists that included sfsv nss ( ) . the sfsv nss cdna, equipped with the sequence for a c-terminal tandem affinity purification (tap) tag, was inserted into recombinant rvfv to replace the rvfv nss gene (rrvfvΔnss::nss sfsv -ctap). t cells were infected with this recombinant virus, tandem affinity purification was performed, and protein complexes were analyzed by liquid chromatography-mass spectrometry (lc-ms). strikingly, irf was among the host cell interactors of sfsv nss, which is compatible with the results of our reporter assays. in order to test the data obtained by mass spectrometry, , as well as stimulation-dependent firefly luciferase (ff-luc) and constitutively active renilla luciferase reporters. firefly luciferase was under the control of (a) the entire ifn-␤ promoter (n ϭ ; means Ϯ sd), (b) irf-driven prdi (n ϭ ; means Ϯsd), or (c) nf-b-driven prdii (n ϭ ; means Ϯ sd). cell lysates were harvested h after transfection for dual-luciferase assays. firefly reporter activities were normalized to the renilla reporter activities, and the positive controls were set to % prior to calculating means and sd across biological replicates. we performed pulldown analyses. an enhanced green fluorescent protein (egfp)-irf fusion protein was coexpressed with the recombinant ϫflag-tagged nss of sfsv, rvfv, ptv-a, or ptv-b or with the negative-control Δmx. cell lysates were then subjected to immunoprecipitation using a plate coated with a nanobody directed against gfp. the nss proteins of rvfv and ptv-a negatively affected the coexpression of egfp-irf ( fig. and data not shown), but egfp-irf was enriched in all gfp precipitates nonetheless. sfsv nss clearly coprecipitated with egfp-irf but not with egfp alone. in contrast, neither of the other phleboviral nss proteins interacted with egfp-irf . similar results were also observed in an inverse setting; i.e., sfsv nss was able to pull down egfp-irf (or hemagglutinin-irf [ha-irf ]), while ptv-a and Δmx were not (data not shown). this confirms our earlier mass spectrometry data ( ) and demonstrates that sfsv nss is unique among the tested phleboviral proteins in its interaction with irf . we extended our assays to include other members of the irf family. irf is the family member most closely related to irf in both sequence and function ( ) . however, sfsv nss did not coprecipitate with egfp-irf (fig. a) . likewise, egfp-irf , egfp-irf , and egfp-irf did not interact with sfsv nss (fig. b) . hence, we conclude that sfsv nss selectively targets the immediate early-acting ifn transcription factor irf . sfsv nss does not inhibit irf activation. in uninfected cells, irf localizes predominantly to the cytoplasm. upon activation, irf becomes phosphorylated by tbk /ikk, dimerizes, and accumulates in the nucleus, where it associates with the transcriptional cofactors cbp and p ( , ( ) ( ) ( ) ( ) . transiently expressed sfsv nss, on the other hand, localized diffusely to both the cytoplasm and the nucleoplasm (data not shown), suggesting that it could interfere with irf activation or function at any level. we thus simultaneously investigated the three classic hallmarks of irf activation in sfsv-infected cells. first, immunoblot analysis showed that irf phosphorylation was affected neither in sfsv-infected cells (fig. a ) nor in cells infected with a recombinant rvfv expressing sfsv nss (fig. b) . the latter experiment also demonstrated that ptv nss was acting downstream of irf phosphorylation. also, irf dimerization (fig. c ) and virus-triggered accumulation in the nucleus (fig. d) were not impaired by sfsv infection. thus, sfsv-like rvfv, which was used as a control ( )-was not preventing phosphorylation, dimerization, or nuclear localization of irf . we tested the impact of sfsv nss on specific irf mutants. irf ( d) is constitutively active and dimerized due to phosphomimetic aspartate residues that replace five serine and threonine phosphorylation sites in the region from amino acid (aa) to aa ( , ) . sfsv nss was able to inhibit both ifn induction and prd i activation by irf ( d) (fig. a and b) , just like the nss of ptv-a, which was used in parallel. sfsv nss, however, was additionally able to pull down irf ( d) (fig. c ). sfsv nss also interacted with irf mutants that are deficient in dimerization, namely, irf (s a/s a) ( ) (fig. d ) as well as irf (s a/s a-r a/r a) and irf (s a/s a-r a/ h a/h a), further derivatives with additional mutations of essential arginine and histidine residues within the dimerization interface (data not shown). in summary, these experiments demonstrated that sfsv nss inhibits a molecular step that takes place after the nuclear importation of activated irf but prior to irf -driven transcription and that the interaction interface on irf is accessible in both the inactive and the active states. sfsv nss interacts with the dna-binding domain of irf . irf possesses an n-terminal dna-binding domain (dbd; aa to ) ( ) which also contains the bipartite nuclear localization signal (nls; k /r and r /k ) ( , ) , followed by an activation domain comprising the nuclear export signal (nes; aa to ) ( , ), a proline-rich domain (pro; aa to ), an irf association domain (iad; aa to ) ( ) , and a serine-rich domain (sr; aa to ) that is phosphorylated upon activation ( ) (fig. a ). crystal structures of the c-terminal portion of irf (aa / to ) indicate that irf phosphorylation induces a marked conformational change in the iad, resulting in the exposure of residues that facilitate dimerization and the interaction with cbp/p ( , , ) . we employed systematic deletion analysis to map the irf domain that is bound by sfsv nss. as a first step, we cut gfp-tagged irf into two halves at position . as shown in fig. b , only the n-terminal part, ranging from aa to , was able to pull down nss. we then removed the remaining domains from this fragment one by one in the c-to n-terminal direction. in this way, we found that the n-terminal dbd alone (aa to ) was sufficient for binding sfsv nss (fig. c) . unfortunately, fine mapping by further c-terminal deletions was inconclusive, as were our attempts to map the corresponding irf -interacting region within sfsv nss (data not shown). sfsv nss prevents irf from binding to the ifn promoter. we hypothesized that sfsv nss might interfere with the promoter-binding activity of irf . to investigate this, we established an assay in which we used biotinylated ifn-␤ promoter oligonucleotides to pull down mavs-activated irf via the use of streptavidin-coated magnetic beads. egfp-irf and mavs were coexpressed in hek cells either on their own or together with increasing doses of ϫflag-tagged sfsv nss or the negative-control Δmx. as observable in the input samples, overexpressed mavs induced the phosphorylation and dimerization of egfp-irf , as expected (fig. , left panels, and data not shown). the presence of sfsv nss did not affect irf activation, confirming our observations of sfsv-infected cells. analyzing the precipitated proteins (fig. , right panels), we detected activated egfp-irf but not egfp, indicating specific binding to the ifn-␤ promoter oligonucleotide. furthermore, no protein precipitation was observed when empty beads without the biotinylated oligonucleotide were used (data not shown). the sequence specificity of egfp-irf binding was confirmed by the addition of an excess of nonbiotinylated ifn-␤ promoter oligonucleotide, which strongly diminished egfp-irf binding, whereas a scrambled control oligonucleotide had no such effect. importantly, coexpression of sfsv nss reduced the amount of promoter-bound egfp-irf in a dose-dependent manner, but the control protein Δmx had no influence. of note, sfsv nss did not coprecipitate with the promoter oligonu- cleotide, indicating the absence of intrinsic or indirect dna-binding activity. thus, we conclude that sfsv nss stoichiometrically impairs the binding of irf to the ifn-␤ promoter by covering essential amino acid residues within the dbd. sfsv, first isolated in ( ), is one of the geographically most widespread members of the genus phlebovirus, with high seroprevalence rates in regions of endemicity ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . despite the long-standing association with an acute incapacitating disease, little is known about the interaction of sfsv with the host cell. also, there is no and total plasmid adjusted to equal levels with empty vector. firefly activities were normalized to those of renilla, and the stimulation control was set to % (n ϭ ; means Ϯ sd). (b) a dual-luciferase assay was performed in parallel with a prdi-responsive firefly luciferase reporter (n ϭ ; means Ϯ sd). (c and d) interaction with irf mutants. (c) ϫflag-tagged sfsv nss or ptv-a nss was coexpressed with irf ( d) in hek cells. cell lysates were then subjected to immunoprecipitation using an antibody against flag that was covalently coupled to magnetic beads beforehand. (d) gfp-irf (s / a), egfp-irf , or egfp, as well as ϫflag-tagged sfsv nss, was obtained by transient transfection of hek cells. immunoprecipitation was performed via the use of gfp. established animal model ( , ) , prompting earlier researchers to fall back on experiments with human volunteers ( ) . we found that the induction of ifn-␤ in sfsv-infected cells is inhibited by nss, although sfsv does not destroy any of the key antiviral host factors that other dipteran-borne phleboviruses attack. rather, sfsv nss binds to the dbd of irf , thus prohibiting ifn-␤ promoter activation. curiously, none of the other irf family members, including the master regulator irf ( ), are targeted, indicating high specificity. although a significant role in the generation of a full ifn response has been attributed to the ifn-inducible irf ( ), the constitutively expressed irf is indispensable for the induction of a first wave of ifn-␤ expression from virus-infected cells and the subsequent upregulation of irf expression ( , ) . hence, irf knockout mice exhibit substantially increased susceptibility to viral infection ( , ) . irf activation is the target of a number of virulence factors ( ), e.g., human papillomavirus (hpv ) e ( ), the v protein of paramyxoviruses ( ), herpes simplex virus (hsv- ) vp ( ), and rotavirus nsp ( ) . however, in contrast to sfsv nss, these virulence factors affect phosphorylation, dimerization, nuclear accumulation, or the expression level of irf . sfsv nss interacted with nonactivated and constitutively active (and dimerized) as well as dimerization-incompetent irf , suggesting an ability to target irf both before and after it becomes activated. moreover, this interaction pattern pointed to a region of irf that is accessible independently of its activation and dimerization state. domain mapping consistently revealed that the n-terminal dbd alone was sufficient for binding of sfsv nss. taking the data together, including the interference with sfsv nss at a late stage in the signaling pathway on the one hand and the domain mapping on the other hand, a mechanism involving the sequestration of the dbd by sfsv nss from the ifn-␤ promoter was strongly implied, and its presence was confirmed by a promoter binding assay. the n-terminal dna-binding domain of interferon regulatory factors is about amino acid residues long and displays a conserved architecture consisting of three ␣ helices, four ␤ sheets, and three loops (l to l ) in the order ␣ -␤ -␤ -l -␣ -l -␣ -␤ -l -␤ ( ). as sfsv nss (i) interferes with the promoter binding activity of irf but (ii) does not interact with other irf family members, one could speculate that it targets amino acid residues that are involved in dna binding but that are not conserved within the irf family. irf residues l , r , and r are both nonconserved and involved in specific dna promoter binding ( ) . however, r and r are also part of the bipartite irf nls. since sfsv nss does not interfere with the nuclear importation of irf , these residues are less likely to mediate the interaction with sfsv nss. that leaves dna binding residue l (situated in loop l ) as well as less-conserved strands ␤ and ␤ and loops l and l as the most probable candidate binding sites for sfsv nss. among the other viral proteins known to target irf , only us (also icp ) of herpes simplex virus and np of human bocavirus have been described to employ a similar mechanism ( , ) . like sfsv, both these viruses target the irf dbd and disrupt promoter binding, but whether this is restricted to irf or also true for any other member of the irf family was not addressed. kaposi's sarcoma-associated herpesvirus proteins k-bzip and lana- also prevent the binding of activated irf to its cognate promoter sites but do so by occupying the promoter sites themselves ( , ) , which we did not observe for sfsv nss. in addition to these dna viruses, bovine viral diarrhea virus interferes with promoter binding and then induces the degradation of nuclear irf via its npro protein ( , ) . a direct interaction between npro and irf could not be demonstrated, however. hence, to our knowledge sfsv nss seems to be the only virulence factor from an rna virus which acts by directly masking the dbd of irf to prevent promoter binding and ifn induction. given the remarkable diversity of the phleboviral nss proteins with respect to sequences, subcellular localizations, and molecular mechanisms, it is tempting to speculate on a correlation between the specific anti-ifn strategy of a given nss protein and the degree of virulence of the respective phlebovirus. the nss of highly virulent rvfv uses multiple strategies, mostly based on proteasomal degradation, to globally and rapidly blunt host gene expression at the transcriptional and posttranscriptional levels ( , ) . the nss of the highly virulent sftsv abrogates ifn induction by sequestering several key signaling components, including rig-i and tbk , into cytoplasmic aggregates ( ) ( ) ( ) ( ) . the intermediately pathogenic tosv acts by degrading rig-i itself ( ) , but its nss seems to be degraded along with its host target, cutting down its inhibitory efficiency ( ) . the nss of the apathogenic uukuniemi virus (uukv), in contrast, does not significantly inhibit ifn induction ( , ) . how does the intermediately pathogenic sfsv fit into this picture? on the one hand, by masking the dbd to sterically hinder irf from binding the ifn-␤ promoter, sfsv nss blocks irf independently of its conformation or activation state. on the other hand, however, this stoichiometric mechanism requires nss to accumulate to levels that are sufficient for sequestering the cellular pool of irf , which, during the early phase of infection, outnumbers nss. moreover, sfsv nss inhibition does not include irf , the master regulator of innate immunity ( ) . thus, sfsv nss fail to impair ifn induction in cells where upregulation of irf took place before infection or in cells with physiologically high basic levels of irf , such as plasmacytoid dendritic cells ( ) . in other words, the stoichiometric and irf -specific nature of the anti-ifn induction strategy makes sfsv nss a modulator rather than a full antagonist of ifn induction. this places sfsv between tosv and uukv with regard to both anti-ifn strategy (rig-i degradation versus weak ifn antagonism) and virulence (fever and meningitis/encephalitis versus no disease). curiously, ptv-a does not seem to quite fit the picture; while its nss protein seems to act as a global host transcription inhibitor, infection of humans has so far been associated only with febrile symptoms. in rodent models, such as mouse and hamster, however, ptv-a and chimeric rvfvs that express ptv-a nss are also highly virulent ( , , ) , suggesting that ptv may be an outlier with respect to humans but not other mammals. thus, the demonstration that the intermediately virulent sfsv specifically targets irf in a highly specific and stoichiometric (i.e. nondestructive) manner supports our hypothesis that the molecular strategy employed by the nss protein can correlate with the degree of virulence of the parental phlebovirus, although other factors, e.g., cell tropism, rna polymerase activity, species-specific host protein interactions, and escape from adaptive immunity, are of course equally important. cells, viruses, and plasmids. a , bhk- , hek , hek t, vero b , and vero e cells were cultured in dulbecco's minimal essential medium (dmem) and ccm medium (dmem with addition of . mg/liter l-alanine, . g/liter glycine, mg/liter l-glutamic acid, mg/liter l-proline, . mg/liter biotin, mg/liter hypoxanthine, and . g/liter sodium bicarbonate) supplemented with % fetal calf serum (fcs), mm glutamine, u/ml penicillin, and g/ml streptomycin. the sabin strain of sfsv was obtained from the world reference center for emerging viruses and arboviruses (wrceva) and propagated in vero b cells. attenuated rvfv strains mp and clone were propagated in bhk- cells. recombinant rvfv strains rzh , rzh Δnss, rzh Δnss::nsssfsv, and rzh Δnss::nsstosv have been described previously ( , , ) . rzh Δnss::nssptv-a, rzh Δnss:: ptv-b, and rzh Δnss::nsssfsv-ctap were generated using a polymerase i (pol i)/pol ii-based rescue system as described for the other recombinant rvfv strains ( , , ) . in brief, nss coding sequences for ptv-a and ptv-b nss (genbank accession no. ef and ef , respectively) were obtained by gene synthesis (mr. gene) and inserted into modified s-segment rescue plasmid phh _rvfv_vn_tcs. the reading frame of sfsv nss was amplified from cdna of infected cells and inserted into rescue plasmid phh _rvfv_vn_mcs_ctap, which contains a c-terminal tag for tandem affinity purification (tap). primer sequences are available on request. the resulting plasmids were transfected together with l-and m-segment rescue plasmids phh _rvfv_vl and phh _rvfv_vm, respectively, as well as helper plasmids pi. _rvfv_l and pi. _rvfv_n into cocultures of hek t and bhk- cells. recombinant rvfv strains were harvested days after transfection, propagated in vero e cells, and characterized by rt-pcr and sequencing of the n-and nss-coding regions. titers of all virus strains were determined on vero e cells via plaque assay. both the cell lines and the virus stocks were routinely tested for mycoplasma contamination. to generate constructs encoding ϫflag-tagged nss of sfsv (genbank accession no. ef . ), ptv-a, or ptv-b, the viral open reading frames were amplified from cdna (sfsv) or synthesized dna (ptv-a and ptv-b) and inserted into pi. by ligation-dependent cloning via the use of = bamhi and = xhoi restriction sites. primer sequences are available on request. pi. -nssrvfv- ϫflag and pi. - ϫflag-Δmx were described before ( ) . firefly luciferase reporter constructs p- luc, p- c bluc, and p- a luc ( ) were kindly donated by takashi fujita, and prl-sv was purchased from promega. expression plasmids for human tbk ( ) and irf ( d) ( ) were kindly provided by john hiscott, for human mavs by shizuo akira ( ) , and for full-length pegfp-c -irf ( ) dual-luciferase assay. hek cells seeded into -well plates ( . ϫ per well) were transfected the following day with firefly and renilla luciferase reporter constructs ( ng each), as well as expression constructs for mavs ( ng) and nss proteins or the control protein Δmx ( . ng, ng, and ng) via the use of transit-lt (mirus bio llc). the total plasmid dna amounts were adjusted to equal levels with empty vector pi. . cells were processed h after transfection, and luciferase activities were measured with a dual-luciferase reporter assay system (promega) according to the manufacturer's recommendations. firefly luciferase activities were normalized to those of renilla luciferase, and the stimulated control samples were set to % within each biological replicate. means and standard deviations (sd) were calculated across the indicated number of biological replicate data sets. proteomics. as described previously ( , ) , approximately ϫ hek t cells were infected with the recombinant rvfv strain expressing tap-tagged sfsv nss (rzh Δnss::nsssfsv-ctap) at an moi of . the cells were washed with and scraped off in prechilled pbs at h postinfection (hpi). the cell pellet was snap-frozen in liquid nitrogen, lysed in tap buffer ( mm tris-hcl [ph . ], mm nacl, . % np- , % glycerol) supplemented with protease and phosphatase inhibitors, snap-frozen again, and stored at Ϫ °c until further processing. tap purification was performed by sequential pulldowns using streptavidin agarose and ha-agarose beads. bound protein complexes were eventually eluted in laemmli buffer and subjected to one-dimensional sds-page prior to trypsin digestion and peptide analysis by liquid chromatography-tandem mass spectrometry (lc-ms/ms), which was described in detail elsewhere ( ) . coimmunoprecipitation. hek cells ( . ؋ per -cm-diameter dish) were transfected with expression plasmids ( g each) via the calcium phosphate method. cells were washed twice in pbs the following day and lysed in prechilled lysis buffer ( mm tris-hcl [ph . ], mm nacl, % igepal- ) freshly supplemented with protease (roche) (complete, edta-free) and phosphatase inhibitors (phosphatase inhibitor cocktail set ii; calbiochem). finally, cell debris was removed by centrifugation ( , ϫ g, min, °c), and the supernatants were used for further processing. for immunoprecipitation via the use of gfp, supernatants were applied to prewashed wells of a gfp-multitrap (chromotek) and incubated at °c for to min under conditions of mild shaking. wells were washed extensively with lysis buffer and bound proteins eluted for min with preheated laemmli buffer under conditions of strong agitation. for immunoprecipitation via the use of flag, magnetic beads ( - d; invitrogen) were covalently coupled with flag m antibody (f ; sigma) overnight and processed according to the manufacturer's recommendations. lysates were then added to the coupled beads followed by incubation under conditions of rotation at °c for h. after extensive washing, bound proteins were eluted by boiling in laemmli buffer at °c for min. to map the binding region within irf , constructs comprising a t promoter, the open reading frames (orf) of the respective truncated irf mutants fused to egfp, a stop codon, and a poly(a) stretch were assembled via pcr (primer sequences available on request) and purified via gel extraction (omega bio-tek) and dna precipitation. the respective proteins were then produced by coupled in vitro transcription-translation using rabbit reticulocyte lysate (l ; promega) and added to lysate of hek cells transiently expressing sfsv nss. immunoprecipitation via gfp was performed according to the aforementioned protocol. irf dimerization assay. a cells infected with sfsv were lysed as described above and then processed as described before ( ) . in brief, % native polyacrylamide gels were prerun at ma for min in native running buffer ( mm tris, mm glycine, ph . ), with % deoxycholate added to the cathode buffer. samples were supplemented with native loading buffer ( mm tris-hcl [ph . ], % glycerol, % deoxycholate, . % bromophenol blue), run at ma for the desired duration, and finally transferred to pvdf membranes via semidry blotting. immunofluorescence assay. a cells were seeded onto glass coverslips ( ϫ per wells) day prior to infection at an moi of . the cells were washed with pbs at hpi and fixed overnight in pbs- % paraformaldehyde (pfa) at °c. the coverslips were then washed with pbs, and the cells were permeabilized with pbs- . % triton x- , washed again, and blocked in pbs- % fcs. staining with primary antibodies diluted in blocking buffer (irf fl- , : ; sfsv, : , ) was performed for h in a humid chamber. afterward, the coverslips were washed with pbs and incubated with secondary antibodies (alexa fluor donkey anti-mouse [a ] and alexa fluor donkey anti-rabbit [a ]; thermo fisher scientific) (both : ) and =, -diamidino- -phenylindole (dapi) ( . g/ml) for min in a humid chamber. samples were washed again in pbs, rinsed in demineralized water, and mounted on microscopic slides using fluorsave reagent (calbiochem). confocal microscopy was performed using a leica sp microscope and the accompanying software. promoter binding assay. biotinylated dna covering the irf -responsive positive regulatory domains within the human ifn-␤ promoter and the downstream sequence as a linker (genbank accession no. ef . ) was ordered as complementary single dna strands (sense, =-gac ata gga aaa ctg aaa ggg aga agt gaa agt ggg aaa ttc ctc tga ata gag aga gga cca tct cat ata aat agg cca tac cca tgg aga aag gac att-biotin- =; antisense, =-aat gtc ctt tct cca tgg gta tgg cct att tat atg aga tgg tcc tct ctc tat tca gag gaa ttt ccc act ttc act tct ccc ttt cag ttt tcc tat gtc- = ) and subsequently annealed by initial denaturation at °c for min, followed by slow cooling ( °c/min) to room temperature ( ) . the double-stranded biotinylated oligonucleotide ( pmol per sample) was bound to streptavidin-coated magnetic beads (dynabeads m- streptavidin; invitrogen) ( l per sample) according to the manufacturer's instructions. hek cells seeded into wells ( . ϫ per well) were transfected with plasmids coding for egfp-irf or for egfp ( ng), mavs ( ng), ϫflag-tagged sfsv nss, or Δmx ( , , or ng) and empty vector (to adjust plasmid amounts) via the use of transit-lt and lysed in the presence of protease and phosphatase inhibitors as we are grateful to besim berisha for excellent technical assistance. we are indebted to patricia aguilar and robert tesh from the world reference center for emerging viruses and arboviruses (wrceva) for providing the sabin strain of sfsv along with mouse immune ascites fluid and to alejandro brun for providing anti-rvfv antiserum. expression and luciferase reporter constructs were kind gifts from john hiscott, takashi fujita, luis martinez-sobrido, adolfo garcia-sastre, and shizuo akira. work in our laboratories was supported by the deutsche forschungsgemeinschaft (grant sfb to f.w. and grants pi / , pi / , trr , and trr to a.p.), by an erc starting grant (erc-stg ivip no. ) to a.p., and by the bundesministerium für bildung und forschung (infect-era; grants "escential" and "erase" to f.w. and a.p., respectively). emerging phleboviruses a new phlebovirus associated with severe febrile illness in missouri phleboviruses and the type i interferon response characterization of a sandfly fever sicilian virus isolated during a sandfly fever epidemic in turkey novel phlebovirus in febrile child massilia virus, a novel phlebovirus (bunyaviridae) isolated from sandflies in the mediterranean aguacate virus, a new antigenic complex of the genus phlebovirus (family bunyaviridae) isolation and sequencing of dashli virus, a novel sicilian-like virus in sandflies from iran; genetic and phylogenetic evidence for the creation of one novel species 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factor, a target for the rift valley hemorrhagic fever virus a sap complex inhibits ifn-beta expression in rift valley fever virus infected cells toscana virus nss protein inhibits the induction of type i interferon by interacting with rig-i suppression of type i and type iii ifn signalling by nss protein of severe fever with thrombocytopenia syndrome virus through inhibition of stat phosphorylation and activation viral suppression of innate immunity via spatial isolation of tbk /ikkepsilon from mitochondrial antiviral platform hijacking of rig-i signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type i interferon responses evasion of antiviral immunity through sequestering of tbk /ikk epsilon/irf into viral inclusion bodies nss protein of rift valley fever virus induces the specific degradation of the double-stranded rnadependent protein kinase characterization of rift valley fever virus mp- strain encoding nss of punta toro virus or sandfly 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association of irf- with ifn-beta promoter human bocavirus np inhibits ifn-beta production by blocking association of ifn regulatory factor with ifnb promoter kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen inhibits interferon (ifn) beta expression by competing with ifn regulatory factor- for binding to ifnb promoter binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferon-responsive factor elements modulates antiviral gene expression inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor -dependent mechanism the npro product of bovine viral diarrhea virus inhibits dna binding by interferon regulatory factor and targets it for proteasomal degradation truncation of the c-terminal region of toscana virus nss protein is critical for interferon-beta antagonism and protein stability differential antagonism of human innate immune responses by tickborne phlebovirus nonstructural proteins generation of mutant uukuniemi viruses lacking the nonstructural protein nss by reverse genetics indicates that nss is a weak interferon antagonist plasmacytoid dendritic cells: recent progress and open questions pathogenesis of a phleboviral infection (punta toro virus) in golden syrian hamsters attenuation of pathogenic rift valley fever virus strain through the chimeric s-segment encoding sandfly fever phlebovirus nss or a dominant-negative pkr toscana virus induces interferon although its nss protein reveals antagonistic activity t rna polymerasedependent and -independent systems for cdna-based rescue of rift valley fever virus triggering the interferon antiviral response through an ikk-related pathway ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction the ebola virus vp protein inhibits activation of interferon regulatory factor rapid detection of important human pathogenic phleboviruses interferon priming enables cells to partially overturn the sars coronavirus-induced block in innate immune activation high-throughput assay for determining specificity and affinity of protein-dna binding interactions key: cord- -c snhymz authors: mauerhoff, thekla; pujol-borrell, ricardo; mirakian, rita; bottazzo, gian franco title: differential expression and regulation of major histocompatibility complex (mhc) products in neural and glial cells of the human fetal brain date: - - journal: j neuroimmunol doi: . / - ( ) - sha: doc_id: cord_uid: c snhymz the cells of the central nervous system (cns) have the peculiarity of physiologically expressing very low levels of hla molecules. in multiple sclerosis (ms), however, as in endocrine autoimmune diseases, there is a marked increase of hla expression in the tissue (i.e. the plaques) and this is attributable not only to infiltrating cells but also to the astrocytes. to gain an insight into the regulation of hla in the different cell types in the cns and to compare it to that observed in the endocrine organs, we have studied the effect of the lympho/monokines interferon (ifn)-α and -γ, tumour necrosis factor (tnf)-α, and interleukin (il)- and other agents on this aspect of the biology of human fetal brain cells in culture. a two-colour immunofluorescence technique which combines antibodies to diverse cns cell markers and monoclonal antibodies (moabs) to the non-polymorphic region of hla molecules was used throughout this study. in control cultures, only astrocytes expressed mhc class i, but after incubation with either ifn-γ or tnf-α oligodendrocytes acquired class i expression. surprisingly, astrocytes became spontaneously class ii positive in culture and this was greatly enhanced by ifn-γ. other agents such as il- , epidermal growth factor, phorbolmyristate acetate and lectins had no effect. the expression of hla molecules in the cells of the cns both in basal conditions and in response to lymphokines is therefore selective and highly heterogenous, thus reflecting their intrinsic biological diversity. these findings may help to explain the features of the immunopathology of ms and also of latent viral infections of neural cells. the level of cell surface expression of class i and class ii products of the major histocompatibility complex (mhc) (hla in humans) varies widely among different cell types and it seems to be determined not only by their ontogenic lineage but to be also influenced by the direct action of several inducers and modulators. of the latter, the best known are lymphokines and in particular those belonging to the interferon (ifn) family (review in pujol-borrell and . recently, tumour necrosis factor (tnf-a) and lymphotoxin (lt or tnf-fl) , produced by macrophages and t lymphocytes respectively, have also been found to induce and/or enhance hla molecule expression in certain systems (collins et al., ; pfizenmaier et al., ; . our previous work has clearly indicated that human endocrine cells have different sensitivities to the in vitro effect of lympho/monokines with regard to the induction of hla class ii expression. thus viable thyrocytes are easily inducible following incubation with mitogens (pujol-borrell et al., ) or ifn- , , but pancreatic islet cells are much more resistant to the elicitation of this phenomenon when exposed to the same or other putative modulators . in fact, using islet cell cultures, it was only the combination of tnf or lt with ifn-'t which ultimately produced the first positive results . these latter studies were prompted by the interest generated by the hypothesis that endocrine cells inappropriately expressing hla class ii could present their own surface autoantigens to helper t cells and in this way generate an autoirnmune response . this postulation was based on the observation that thyroid cells have the capacity for de novo expression of class ii molecules (pujol-borrell et al., ) , and the finding that in thyroid glands affected by autoimmune disorders, the follicular cells strongly expressed class ii ). since the above hypothesis was proposed, the occurrence of inappropriate class ii expression has been documented in the target organs of many organ-specific autoimmune diseases (review in bottazzo et al., ) and the ability of class ii-positive thyroid cells to present antigen to t cells has been experimentally demonstrated (londei et al., (londei et al., , . the identification of class ii-positive astrocytes in the active 'plaques' of patients with multiple sclerosis (ms) has attracted a great deal of interest and has led to the suggestion that astrocytes inappropriately expressing class ii molecules in vivo could play a role relevant to the pathogenesis of ms (traugott et al., ) , a hypothesis which is somewhat parallel to that originally proposed by us . as an extension of our interests, we report here the results obtained in studies on the inducibility of hla molecules on human fetal brain cells in culture and their differential sensitivity to the action of a variety of stimuli known to be potent modulators of hla expression in other cell types. tissue was obtained from fetal brain specimens (provided by the medical research council tissue bank, royal marsden hospital, london, u.k.), after legal terminations carried out by aspiration between and weeks of gestation. the experimental protocol described here was approved by the university college/middlesex hospital ethical committee. the meninges and visible vessels were carefully removed from the brain, tissue was washed with hanks' balanced salt solution containing % bovine serum albumin (bss-bsa), minced with scissors and forceps and digested for min at °c in a solution which contained . % trypsin (sigma, london, u.k.), and . % dnase (dnase , sigma, london, u.k.) in dulbecco's modified eagle's medium (dmem, gibco, paisley, scotland, u.k.). digestion was stopped by the addition of % fetal calf serum (fcs, gibco). the undigested tissue was further disrupted by repeated pipetting. the digest was passed through a #m nylon mesh to remove large fragments, pelleted by centrifugation at rpm for min and resuspended in 'complete medium'. this consisted of % dmem, % f (flow, irvine, scotland, u.k.), % fcs, glucose mg/ ml, glutamine mm, gentamicin gg/ml, and penicillin gg/ml. cells were counted and viability, as assessed by differential staining with acridine orange/ethidium bromide under a uv microscope, was always close to %. aliquots of cells were plated on glass coverslips ( mm diameter), placed in -multiwell plates (nunc, kamstrud, denmark) and 'complete medium' added to a final volume of . ml/well. cultures were kept at °c in a % co humidified incubator and left undisturbed for days. in some instances small fragments of brain tissue were directly snap frozen and stored at - °c until used for immunofluorescence studies on cryostat sections. given the complexity of the architecture of the brain tissue and the heterogeneity of cell types present in primary cultures prepared from it, a two-colour ifl technique was employed throughout the study combining monoclonal antibodies (moabs) to hla products with monoclonal or polyclonal antibodies to different cell markers (review in . this ensured the precise identification of positive and negative cells under observation. cryostat sections and monolayer cultures were stained similarly following the same protocols. for the identification of hla class i and ii molecules the following staining protocols were applied (for the specificity and source of the antibody used, see table ). hla class l st layer: moab w / ; nd layer: tritc-conjugated goat anti-mouse igg; rd layer and th layer: either rabbit anti-glial fibrillary acidic protein (astrocytes) or rabbit anti-factor viii (endothelial cells) followed by fitclabelled goat anti-rabbit ig or moab (oligodendrocytes) followed by fitc goat anti-mouse igm. hla class . (using igg moabs to class ii) st layer: moab mid (class ii), tu (dq specific), b / (dp specific) or vic-y (class ii invariant chain specific); nd layer: tritc-labelled goat anti-mouse igg; rd layer and th layer: either rabbit anti-glial fibrillary acidic protein (gfap) followed by fitc-labelled goat anti-rabbit ig or moab followed by fitc-labelled goat anti-mouse igm. hla class ii. (using rfdr igm moab to class ii) st layer: moab rfdr (class ii); nd layer: fitc goat anti-mouse igm; rd layer: moabs gc (oligodendrocytes), (astrocytes), or rt. (neurons); th layer: tritc-labelled goat anti-mouse igg. moabs were used at a final concentration of /~g/ml. unstimulated control cultures were always stained in parallel with treated cultures. in addition, nonspecific binding was ruled out by staining parallel stimulated cultures with an unrelated moab of the same ig class. undesirable cross-reactivities among reagents derived from different species were assessed by omitting one of the layers in turn. to determine hla membrane expression, viable cultures were incubated with the corresponding moabs, stained by the appropriate conjugate and then, prior to counterstaining with antibodies to cell-specific cytoplasmic antigens, cultures were fixed with methane/acetone ( : , v/v) for min. when the second antibody was recolmizing membrane antigens distinct from hla molecules, the fixation step was done at the end of the entire procedure. for the detection of cytoplasmic class i, class ii and class ii invariant chain the cultures were fixed prior to the staining. all preparations were examined with a x oil immersion objective using a zeiss iii uv photomicroscope equipped with epi-illumination and phase contrast. fluorescence intensity in both membrane and cytoplasm was evaluated in - cells per culture except in the case of oligodendrocytes where, given their small number, only fewer cells (around ) could be evaluated. ifl intensity was arbitrarily scored from negative to +. table lists the various biological and chemical compounds used to induce hla product expression on human fetal brain cells in culture. • this compound was tested only for its effect on astrocyte mhc expression. in sections from three different human fetuses stained for neurofilaments with the moab rt. , the soma of neurons was clearly visible among the complex network of fibres, while the antiserum to glial fibrillary acidic protein (anti-gfap) produced a very intricate reticular pattern which was denser around the vessels. in successive sections stained for class i, the only cells clearly positive were the endothelial cells lining the capillaries, these identified by antibodies to factor viii. in spite of careful and intensive search, no cells positive for class ii products were detected in the parenchyma or in the vessels of the brain when sections were stained with the appropriate moabs. cell attachment occurred as early as h by the time most preparations were processed, between days and , cultures showed a complex organization similar to that described by dickson et at. ( ) . cells with long processes identified as neurons (rt. +) and oligodendrocytes ( +) (see fig. la-d) laid on a carpet of astrocytes (gfap +) (fig. b ) (raft et at., ) . the long processes of the neurons were arranged in dearly distinct bundles forming bridges among clusters of cells which seemed to serve as orga~i jng centres. immature ofigodendrocytes were present in smaller numbers compared to neurons and were often located in the periphery of these clusters, their intricate branching processes completing the complex three-dimensional network in which all the cells were included. the number of neurons decreased during the period of culture but even after days these cells were quite numerous in the monolayers. fibronectinpositive cells (fibroblasts) presumably derived from contaminating meningeal cells, were very scarce in the initial days and, although their number increased with time, they never overgrew the other cell types even after weeks in culture. attempts were not made to identify microglial cells because we lacked specific reagents to recognize them. endothelial cells were not present in our monolayers, probably due to their inability to grow on glass surfaces (zetter, ) . (b) basal expression of hla molecules. class i molecules were consistently detected throughout the culture period on astrocytes but not on oligodendrocytes or neurons ( fig. a and b) . class ii molecules were seen in less than % of the cells in the culture up to day . however, astrocytes (identified by gfap-positive staining) became gradually class ii positive, and by day , around % of them showed a clear and bright staining ( fig. c and d, time-course in fig. ). this 'spontaneous' class ii expression was observed in both the protoplasmic and the fibrous type of astrocytes (identified by their morphology under phase contrast and confirmed by gfap + staining) and also in the astrocyte subtype recently defined by the moab (dickson et at., ) . astrocyte class ii expression included also hla-dp and dq subregion products and did not depend on the gestational age of the fetal brain. passive absorption of class ii molecules to the membrane of the astrocytes could be excluded by the concomitant detection of the non-secretory class ii invariant chain in the cytoplasm of these cells when they were stained with moab vic-y . the other cell types (neurons and oligodendrocytes) remained negative throughout the culture period (up to days). for the reasons mentioned above, microghal or endothelial cells could not be detected. table were tested during the initial days of culture when the percentage of spontaneously positive astrocytes was less than %, as to minimize the interference of the spontaneous expression of class ii in astrocytes in the reading of the preparations (see above). among the biological and chemical compounds used (table ) , only ifn-a, ifn- and tnf-a had a clear effect on hla expression in the different cell types and the results are summarized in table . neurons and oligodendrocytes both = astrocytes did not express class ii in the early stages of culture, but an increasing percentage were found spontaneously positive after days in culture (see text and fig. ) . lacked class i expression in basal conditions but, while the former remained always negative, oligodendrocytes acquired a clear membrane staining for class i after culture with either ifn-t or tnf-a (fig. ) . ifn-a and ifn-t both produced an increase in class i expression in astrocytes and fibroblasts. none of the mediators or chemicals tested were able to induce class ii expression in neurons or oligodendrocytes, and this also applies to the combination of ifn-t and tnf-a. ifn-~, was able to produce a dramatic increase in the number of astrocytes positive for membrane and cytoplasmic class ii and days after the addition of u/ml to the cultures, all gfap-positive cells were stained for class ii. this effect was dose related (see fig. and legend) and was also detectable with the moab vic-y which reacts with the cytoplasmic invariant chain of class ii products. no apparent synergism was observed between ifn-~ and tnf-a in the induction of class ii in astrocytes (fig. ). it has previously been reported that primary cultures of human fetal brain tissue can be a useful substrate for studying the expression of cell type-specific antigens, e.g. gangliosides, tetanus toxin receptors (dickson et al., (dickson et al., , , and the present data confirm that they can also be successfully employed to investigate the expression and modulation of hla molecules. our findings that neurons do not express hla class i or class ii products in basal conditions are in agreement with previous reports using a similar culture system of murine origin (wong et al., ; dubois et al., ; suzumura et al., ; tedeschi et al., ) . neither ifn- nor tnf-a separately or in combination were able to induce class i or class ii expression in human fetal neurons. with regard to class i expression our results differ from those reported by wong et al. ( ) who showed that ifn-y can induce class i in up to % of mouse neurons, and from a similar report by lampson et al. ( lampson et al. ( , ) on a human neuroblastoma cell line. stages of development, species differences and neoplastic transformation could account for these apparent discrepancies. referring to class ii molecules, our results agree with the generally accepted concept that cells of the neuronal lineage are refractory to the effect of available modulators, alone or in combinations, on the levels of hla class ii expression regardless of the species employed (wong et ai., ) . the inability of ifn-t to induce class i or class ii in neurons contrasts with the strong positive modulation it produces on most cell types including terminally differentiated human cells such as thyrocytes and melanocytes (houghton et al., ) . the biological meaning and molecular basis for this differential regulation are at present unknown. the potential of oligodendrocytes to express hla products has been extensively studied, especially as these cells seem to be the target of the postulated autoimmune response in ms and experimental allergic encephalomyelitis (eae). our finding that human oligodendrocytes are constitutively negative for both class i and class ii molecules but express class i after incubation with ifn-t is in agreement with numerous previous studies on cultures of murine brain cells and suggests the presence of receptors for ifn-t on the surface of these cells (wong et al., ; suzumura et al., ; tedeschi et al., ) . more interesting is the observation that tnf-a can also induce class i expression in oligodendrocytes from human fetal brain. reports of a similar action of tnf-a on other cell types (collins et al., ) have suggested that this may be one of the mechanisms through which this mediator exerts its anti-tumour action in vivo (pfizenmaier et ai., ) . neither ifn-t or tnf-a alone or the combination of the two were able, however, to induce class ii expression in oligodendrocytes in our system. this is in contrast with our recent findings which showed that human pancreatic islet cells, another cell type unresponsive to ifn-t alone, could be induced to express class ii products by the synergistic action of ifn-t and tnf-a or -fl . there is no consensus in the literature on whether adult human oligodendrocytes can express class ii molecules following incubation with ifn-t. lysak et al. ( ) , like us, did not find class ii expression in stimulated primary cultures while kim ( ) and kim et al. ( ) reported - % of class ii-positive oligodendrocytes (although only in half of the culture preparations); the reason for these differences remains at present unclear. regulation of hla expression in astrocytes has also been extensively studied both in rodent (hirsch et al., ; wong et al., ; dubois et al., ; massa et al., ) and in human systems (shen et al., ; takiguchi et al., ) , again due to its potential importance in the pathogenesis of ms. astrocytes expressing class ii are present in the 'active' plaques in the brain of ms patients (traugott et al., ) and, most importantly, it has been shown that they can present antigens to t cell clones in vitro (fierz et al., ) . the postulate has accordingly been made that by presenting myelin basic protein (mbp) or related antigens to autoreactive t lymphocytes, class ii-positive astrocytes may initiate and possibly perpetuate an autoimmune recognition to mbp which subsequently leads to the demyelination process in active ms (fontana, ; sun and wekerle, ; takiguchi et al., ) . in short-term cultures we found that astrocytes express class i but not class ii molecules and this is in accordance with previous work (fierz et al., ) . however, we were surprised by the arousal of a population of astrocytes positive for class ii after - days while these cells were growing in culture. these results may substantiate the report by kim ( ) which showed that in cultures of human adult brain - % of the astrocytes were class ii positive at day . similar findings were described on a human fetal astrocyte cell line, but the spontaneous class ii expression disappeared after subsequent passages (pulver et al., ) . this phenomenon seems to be a unique characteristic of the astrocytes since fibroblasts and other cell types present in our cultures remained negative, thus arguing against the possibility that it is mediated by ifns or other non-specific soluble factors generated during the culture period or by the medium employed, as previously suggested (pulver et al., ) . this spontaneous induction of class ii expression indicates that in vivo the expression of class ii by astrocytes should be constantly suppressed by still unidentified humoral or locally produced mediators. alternatively, it is also possible that the abundant debris which our cultures initially contained could have stimulated phagocytosis by the astrocytes and this then triggered the expression of class ii, as part of the general activation process undergoing in these cells. in this context, it is of interest to recall that it has recently been shown that the phagocytosis of coronavirus particles induced class ii expression in rat astrocytes (massa et al., ) . as expected, ifn-a and ifn- , both produced an increase in class i expression in astrocytes. the studies of class ii modulation on astrocytes were carried out at the beginning of the culture period while the level of spontaneous expression was still low and in this way we were able to reproduce previous work which has shown that ifn- is indeed able to induce a rapid increase in the percentage of class ii-positive human fetal astrocytes (pulver et al., ) as well as enhance the intensity of the staining (fig. ) . microglial cells were not easy to identify in our monolayers and this was most probably due to the early gestational age of the fetal tissue. however, even if these cells were present in small amounts, our lack of any specific marker made their identification, solely on morphological ground, very difficult. in summary, we have presented for the first time data on a large number of human fetal brains both in sections and in primary cultures indicating that the regulation of the expression of hla products in human fetal brain cells varies widely among the different cell types. there is a gradation in both constitutive expression and inducibility which ranges from neurons, which lack hla products completely and are refractory to all inducers and modulators tested so far, to astrocytes which in culture become spontaneously positive for class ii and are highly responsive to ifn-y. this heterogeneity of hla regulation among the brain cells is reminiscent of that observed for class ii in the human pancreas where ductal/exocrine cells are easily induced to express class ii while islet endocrine cells require a two-med~tor signal . considering the analogies between the different cell populations which form these two complex organs it seems that the cells with a high or intermediate turnover (ductal cells, astrocytes) are more easily induced to express hla molecules than those with a low turnover (e.g. islet cells) or no turnover (e.g. neurons). one may speculate on the biological significance of this differential regulation. one possibility is that the lack of inducibility of class ii expression may constitute one of the mechanisms which maintain tolerance to autoantigens present in highly specialized cells cowing, ) . on the other hand, 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factor increases mrna levels and surface expression of hla, a, b, c antigens in vascular endothelial cells and dermal fibroblasts in vitro does t cell restriction to ia limit the need for self-tolerance? cell surface antigens of human foetal brain and dorsal root ganglion cells in tissue culture identification of cell surface antigens present exclusively on a subpopulation of astrocytes in human foetal brain cultures cellular distribution of antigen and galactocerebroside in primary cultures of human foetal spinal cord expression of major histocompatibility complex antigens in neonate rat primary mixed glial cultures monoclonal antibody to a plasma membrane antigen of neurons astrocytes as antigen presenting cells. i. induction of la antigen expression on astrocytes by t cells via immune interferon and its effects on antigen presentation astrocytes present myelin basic protein to encephalitogenic t cell lines aberrant expression of hla-dr antigen on thyrocyte in graves' disease: relevance for autoimmunity expression of la antigens by cultured astrocytes with gamma interferon surface antigens of melanoma and melanocytes. specificity of induction of ia antigens by human gamma-interferon antigen expression by glial cells grown in culture expression of ia antigens on the surface of human oligodendrocytes and astrocytes in culture weak hla and beta -microglobulin expression of neuronal cell lines can be modulated by interferon monoclonal antibody analysis of mhc expression in human brain biopsies: tissue ranging from 'histologically normal' to that showing different levels of glial tumour involvement epithelial ceils expressing aberrant mhc class li determinants can express antigen to cloned human t lymphocytes human t cell clones from autoimmune thyroid glands: specific recognition of autologous thyroid cells cultured human oligodendrocytes and rat schwann cells do not have immune response gene associated antigen (la) on their surface viral particles induce la antigen expression on astrocytes tumour necrosis factor enhanced hla-a, b, c and hla-dr gene expression in human tumour cell inappropriate class ii expression in endocrine cells. is it a primary event lectin induced expression of dr-antigens on human cultured follicular thyroid ceils differential expression and regulation of mhc products in the endocrine and exocrine cells of the human pancreas hla class li induction in human islet cells by ifn-gamma plus tnf or lt cultured human fetal astrocytes can be induced by interferon-y to express hla-dr cell-type specific markers for distinguishing and studying neurons and the major class of ghal cells in culture cellular and antigenic properties of cultured normal and fetal brain and ghoma cells monoclonal antibodies (o to ) to oligodendrocyte cell surface. an immunocytochemical study in the central nervous system la restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes expression of h antigen on oligodendrocytes is induced by soluble factors from concanavalin a activated t cells induction of antigen presentation ability in purified cultures of astroglia by interferon-gamma response of glioma cells to gamma-interferon: increase in class ii mrna, protein and mlr stimulating ability astrocytes produce interferon that enhances the expression of h- antigens on a sub-population of brain cells laboratory investigation of autoimmune endocrine diseases interferon-gamma induces hla-dr expression by thyroid epithelium on the presence of ia positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation inducible expression of the h and ia antigens on brain cells interferon-gamma induces the expression of h- and ia antigens on brain cells the endothelial cells of large and small blood vessels we are greatly indebted to all the scientists and commercial firms cited in the text and tables for their generous donations of monoclonal antibodies and other reagents and to the medical research council tissue bank, royal marsden hospital, london for the supply of fetal brain tissue. we thank our colleagues dr. a. belfiore and dr. i. todd for advice and help during the development of this work, professors deborah doniach and ivan roitt for their constant support, and dr. m.l. cuzner and dr. j.g. dickson for very useful discussion. we would also like to thank marian pine for her help in the preparation of the manuscript. key: cord- -yyzbv ys authors: arslan, mehboob; yang, xin; santhakumar, diwakar; liu, xingjian; hu, xiaoyuan; munir, muhammad; li, yinü; zhang, zhifang title: dynamic expression of interferon lambda regulated genes in primary fibroblasts and immune organs of the chicken date: - - journal: genes (basel) doi: . /genes sha: doc_id: cord_uid: yyzbv ys interferons (ifns) are pleiotropic cytokines that establish a first line of defense against viral infections in vertebrates. several types of ifn have been identified; however, limited information is available in poultry, especially using live animal experimental models. ifn-lambda (ifn-λ) has recently been shown to exert a significant antiviral impact against viral pathogens in mammals. in order to investigate the in vivo potential of chicken ifn-λ (chifn-λ) as a regulator of innate immunity, and potential antiviral therapeutics, we profiled the transcriptome of chifn-λ-stimulated chicken immune organs (in vivo) and compared it with primary chicken embryo fibroblasts (in vitro). employing the baculovirus expression vector system (bevs), recombinant chifn-λ (rchifn-λ ) was produced and its biological activities were demonstrated. the rchifnλ induced a great array of ifn-regulated genes in primary chicken fibroblast cells. the transcriptional profiling using rna-seq and subsequent bioinformatics analysis (gene ontology, differential expressed genes, and keggs analysis) of the bursa of fabricious and the thymus demonstrated an upregulation of crucial immune genes (viperin, ikkb, ccl , il β, and ap ) as well as the antiviral signaling pathways. interestingly, this experimental approach revealed contrasting evidence of the antiviral potential of chifn-λ in both in vivo and in vitro models. taken together, our data signifies the potential of chifn-λ as a potent antiviral cytokine and highlights its future possible use as an antiviral therapeutic in poultry. viral pathogens pose significant threats to the poultry industry around the globe. this necessitates the development of novel and alternative antiviral therapies to contain the impacts of pathogens. avian influenza viruses (aivs) are a particular threat, which cause severe damage to the poultry industry, especially in developing countries where huge monetary losses are incurred [ , ] . public health is also threatened by aivs, owing to their zoonotic importance. active preventive strategies would minimize the risk of viral transmission to humans and also benefit the poultry industry. interferons (ifns) are pleiotropic functional cytokines with antiviral, antitumor, and natural immune-boosting effects. ifns play a significant role in eliciting an antiviral state in vertebrates [ ] . ifns are broadly categorized into three distinct types based on their molecular structure, receptor specificity, and induction pathway [ ] . type i ifns include ifn-α, ifn-β, ifn-ε, ifn-κ, and ifnω, and all signal via common cell surface receptors (ifnαr- ) and (ifnαr- ), which are situated on a broad range of cells [ ] . type ii ifns consist of ifn-γ, which is activated through highly specific ligand interactions with distinct ifn-γ receptors (ifn-γr ) and (ifn-γr ). the third family of ifns consists of ifn lambda, which interacts with a heterodimeric receptor complex (il- rα and il- β). ifn-λ was first discovered in mammals and subdivided into ifn-λ (also known as il- ), ifn-λ (il- a), ifn-λ (il- b), and ifn-λ [ ] . ifns are crucial in an innate immune response, as their expression and antiviral potential is dependent on their cognate receptor interaction in a particular system [ ] . in chickens, type i ifns primarily interact in fibroblasts, whereas epithelial cells (gastrointestinal and respiratory tract) are the primary site for the actions of type iii ifns [ ] . despite morphological diversity, ifns share integrated, interconnected, and a precisely coordinated cascade in immunity pathways [ ] . ligand recognition and interaction by ifn receptors results in rapid activation of janus kinase/signal transducers and activators of transcription (jak-stat pathway). this leads to phosphorylation of stat and stat , activation of interferon stimulated gene factor (isgf ), binding of ifn-stimulated response elements (isres), and expression of ifn stimulated genes (isgs) [ ] . once expressed, these isgs demonstrate an essential role in the antiviral response. it is evident from published data that ifns upregulate identical sets of isgs, which in turn express antiviral proteins. ifn-induced transmembrane protein (ifitms), viperin and myxovirus resistance protein (mx) are some of the potent antiviral proteins expressed in response to viral infections [ ] . once expressed, these isgs control viral replication, which provides an antiviral atmosphere to limit viral propagation in infected cells. compared to the mammalian ifn-λ repertoire (ifn-λ , ifn-λ , ifn-λ , and ifn-λ ), chicken ifn-λ is the sole member in birds and demonstrates structural identity with human ifn-λ . ifn-λ is chiefly involved in protection against viral infection of the respiratory and gastrointestinal tract epithelia (aiv, ndv, ibv), and due to the distribution of il- rα in epithelium-rich organs, ifn-λ demonstrates significant potential to limit viral propagation [ ] . while most of the current studies in chickens are mainly focused on type i and type ii ifns, we investigated the potential of type iii ifns in innate and adaptive immunity. previously, it was established that chifn-α presented a significant delay in the propagation of rous sarcoma virus and confirmed in vivo [ ] . it was also revealed that chifn-α treatment ameliorates infection progression in experimental chickens with highly pathogenic influenza a virus (hpaiv) subtype h n [ ] . compared to type i ifns, chifn-λ has also been shown to elicit moderate antiviral response in both the chicken macrophage cell line hd and primary chicken embryo fibroblasts (cef) [ ] . another published study demonstrated that cefs treated with recombinant chifn-λ induced isgs in a temporal fashion [ ] . however, the antiviral potential of chifn-λ in live animals (e.g., chickens) has not yet been investigated, which could provide evidence for the potential of chifn-λ in animals per se. to investigate the impact of exogenous chifn-λ on the innate immune system in chickens, we first expressed chifn-λ in a silkworm bioreactor platform utilizing a baculovirus expression vector system (bevs) [ ] . compared to the autographa californica nucleopolyhedrovirus (acmnpv)-sf cell expression system, the bombyx mori nucleopolyhedrovirus (bmnpv)-silkworm system possesses greater post-translational modifications and enhanced expression efficiency [ , ] . comparative transcriptomic profiling revealed the key mechanisms, signaling pathways, and expression patterns of genes involved in interferon-induced immunity. our results highlight the dynamics of chifn-λ roles in chicken innate immunity. bm cells (bombyx mori-derived cell line) were cultured and maintained at • c with % fetal bovine serum (fbs, gibco, usa) in tc (insect cell culture medium) (applichem, darmstadt, germany) as per the published literature [ ] . for co-transfection, bm cells were cultured at a constant density of × cells per well in six well plates for hours with tc media containing fbs. tc media without fbs was used to wash the cells twice and a mixture of transfection and co-transfection was introduced to cells. between - h post-transfection, fbs was introduced to the cell culture media. for viral amplification and expression, cells were infected with a multiplicity of infection (moi) of . for - h. the ensembl chicken genome database (ftp://ftp.ensembl.org/pub/release- /fasta/gallus_ gallus/dna/) was extensively screened for homologues of chifn-λ by employing the blast algorithm (http://www.ncbi.nlm.nih.gov/blast/). a stretch of sequences demonstrating high sequence identity was identified and characterized. ] were acquired from the national center for biotechnology information (ncbi) and aligned using the clustalw program, and phylogenetic analysis was performed using the neighbor-joining method with bootstrap n = in mega software (version ). amino acid sequences of ifn-λ from multiple species were aligned using the clustalw algorithm. the espript . (http://espript.ibcp.fr/espript/cgi-bin/espript.cgi) was utilized to analyze the sequences. in our previous study, we developed a novel defective-rescue recombinant bombyx mori bacmid (rebmbac) expression system [ ] . we used this in-house built and developed system to express chifn-λ. the rebmbac-silkworm expression system was employed to construct chifn-λ (interferon lambda- [gallus gallus]; sequence id: xp_ . ; length: ). briefly, in order to enhance expression efficiency by codon optimization, chifn-λ genes were optimized for expression in the silkworm (bombyx mori) and synthesized by genscript company (china). plasmid-containing orf + with gene of interest (chifn-λ) and pph as a promoter was co-transfected with rebmbac in the bm cell line. recombinant virus containing the chifn-λ gene was harvested - days post co-transfection. expression product was acquired after - days of silkworm/pupae infection. the plaque assay was performed to evaluate the recombination efficiency [ ] . luciferase assay kit (promega, usa) was employed to analyze expression quantity of luciferase in µg of protein lysate. the bradford method was used to measure the amount of protein [ ] . antiviral activity of chifn-λ was assayed in the gfp-reduction assay using recombinant vesicular stomatitis virus (vsv-gfp) [ ] . cefs were prepared from - days old specific pathogen free (spf) chicken eggs and maintained in cell culture flasks [ ] . after hours, cefs were stimulated with chifn-λ and cells were harvested after hours post treatment, snap frozen, and stored at − • c for further processing. all experiments were performed in triplicate. the present study was conducted in accordance with animal ethics guidelines and approval was given by the beijing administration office of laboratory animals, china. a total of newly hatched spf chicks were obtained from beijing arbor acre company ltd., p.r. china. chicks were reared in cages (n = birds/cage) and placed in six cages in a temperature-controlled environment at the biotechnology research institute, chinese academy of agricultural sciences (caas), p.r. china. birds were offered standard commercial feed obtained from cp group ltd., p.r. china. unrestricted access to water was provided via nipple drinker lines and ad libitum feed was offered. a treatment group of -day old chicks were injected daily with chifn-λ ( , iu/kg body weight) ( iu/ml). phosphate buffer saline (pbs) was injected intramuscularly to the control group. the bursa of fabricious and thymus were obtained by euthanizing the chickens at five days post-treatment. tissue samples were rapidly collected, snap-frozen in liquid nitrogen, and stored at − • c for further processing. total rna was extracted from virus-infected or mock-treated cefs (in triplicates), as per manufacturer's guidelines [ ] . similarly, a total of five immune organs (bursa of fabricious and thymus) were pooled (in duplicates) from randomly selected chicken from each virus-or mock-infected group. total rna extraction was performed as per manufacturer's instructions [ ] . extracted rna quality was analyzed by employing % agarose gel and rna integrity was assured using rna nano assay kit from bioanalyzer system (agilent technologies, ca, usa). extracted samples were sent to novogene beijing for sequencing. samples were sequenced using hiseq x ten (ilumina) and pe platforms. rna-seq generated from cef, bursa of fabricious and thymus samples of chicken (both chifnλ-treated and control groups) are presented in supplementary table s . reads were mapped to the reference genome database (ftp://ftp.ensembl.org/pub/release- /fasta/gallus_gallus/dna/). individually mapped reads for each sample were assembled by stringtie (v . . b) using a reference-based approach. featurecountsv . . -p was utilized to estimate read numbers mapped to each gene. fragments per kilo base of transcript sequence per million base pairs sequenced (fpkm) of each gene was analyzed on the basis of length of gene and read count mapped to this gene. differential expression analysis was accomplished by employing deseq r package ( . . ). using benjamini and hochberg's approach, p-values were adjusted for controlling false discovery rate (fdr). genes with (p < . , |log fold change|> ) observed by deseq were designated as differentially expressed. for differentially expressed genes, both gene ontology (go) enrichment analysis and kyoto encyclopedia of genes and genomes (kegg) pathway enrichment was conducted using the clusterprofiler r package. go terms with adjusted p-values < . were considered as significantly enriched (http://www.genome.jp/kegg/). using the chicken ifn gene as a query, we constructed the phylogenetic tree by employing the neighbor joining method (bootstrap n = ). this demonstrates the relationship of chifnλ with its mammalian orthologues by illustrating that chifn-λ is distinct in its evolution. furthermore, this revealed the contrasting consensus sequence from databases including ensembl and genbank. chifn-λ encodes a putative protein of amino acids and further demonstrates typical characteristics of type iii ifns. a pairwise blast analysis demonstrated that chifn-λ shares %, %, %, % and % sequence similarity with recently characterized pig, mouse, human, cattle, and frog ifn-λ, respectively. based on amino acid homology, conserved amino acids among distinct avian and mammalian ifn-λ are identified. taken together, this comparative characterization further shows that chifn-λ shares characteristic features of type iii ifns (supplementary figure s a ,b). in order to construct chifn-λ, we employed a bevs study. in order to determine the expression efficiency, we used a luciferase reporter gene for quality control as we described previously [ ] . the luciferase gene was acquired from pgl -basic vector by employing bglii/xbai digestion and insertion into the bamhi/xbai-digested pvl vector to construct pvl -luc vector. a combination of pvl -luc and rebmbac dna was co-transfected in bm cells (figure ) . a viral plaque assay was used to determine a suitable virus strain with which to express luciferase. supernatant from bm cells containing recombinant bmnpv (rebm-luc) was harvested five days post-transfection before inoculation into silkworms. after four to five days, protein was harvested from silkworms and µg protein from lysed larval haemolymph was subjected to luciferase assays. luminescence detected from silkworm larval haemolymph was approximately . ± . × relative light units (rlu), compared to - rlu from luc-negative virus-infected samples. pcr amplification (qpcr) further verified and validated the chifn-λ gene expression in bevs (supplementary figure s c ). in order to construct chifn-λ, we employed a bevs study. in order to determine the expression efficiency, we used a luciferase reporter gene for quality control as we described previously [ ] . the luciferase gene was acquired from pgl -basic vector by employing bglii/xbai digestion and insertion into the bamhi/xbai-digested pvl vector to construct pvl -luc vector. a combination of pvl -luc and rebmbac dna was co-transfected in bm cells (figure ) . a viral plaque assay was used to determine a suitable virus strain with which to express luciferase. supernatant from bm cells containing recombinant bmnpv (rebm-luc) was harvested five days post-transfection before inoculation into silkworms. after four to five days, protein was harvested from silkworms and μg protein from lysed larval haemolymph was subjected to luciferase assays. luminescence detected from silkworm larval haemolymph was approximately . ± . × relative light units (rlu), compared to - rlu from luc-negative virus-infected samples. pcr amplification (qpcr) further verified and validated the chifn-λ gene expression in bevs (supplementary figure s c) . in order to investigate the possible biological, cellular, and molecular mechanisms involved in the cascade of interferon-induced immunity, we performed transcriptomic analysis on chicken embryo fibroblasts and organs of live chickens. transcriptomes from the bursa of fabricious and thymus (most important immune organs in chicken) were compared with the control group to identify differentially expressed genes (degs) among all groups. experimentation started at day post-hatch as this is a phase of rapid growth and development, and we hoped to achieve biologically active transcriptional changes. the differences in degs observed in the present study control cellular architecture, immune function, metabolic pathway, and muscular function. it has previously been established that huifn-λ signals via il- and il- r exhibit typei-like antiviral potential [ ] . protection from simian foamy virus (sfv) and avian influenza (ai) augments the antiviral functioning and further postulates its diverse antiviral potential against avian pathogens. in this context, we stimulated chickens with silkworm-expressed chifn-λ and profiled the gene expression in immune organs (thymus and bursa) and compared it with that in primary chicken fibroblasts using rna-seq. an overall low isg expression was noticed in chifn-λstimulated cef; out of , genes, were degs ( upregulated and downregulated) (p< . , │ log fold change │ > ) (figure a) . although cef do not possess receptors for ifn-λ, slight temporal expression of degs in response to chifn-λ treatment signifies its antiviral potential in primary cells. next, we monitored the gene expression in the thymus and bursa. between the chifn-λ-treated and non-treated thymus, a total of , genes were expressed. among them, genes were degs, in which genes were upregulated and genes were downregulated (figure b) . in the bursa of fabricious, out of , genes were differentially expressed ( upregulated and in order to investigate the possible biological, cellular, and molecular mechanisms involved in the cascade of interferon-induced immunity, we performed transcriptomic analysis on chicken embryo fibroblasts and organs of live chickens. transcriptomes from the bursa of fabricious and thymus (most important immune organs in chicken) were compared with the control group to identify differentially expressed genes (degs) among all groups. experimentation started at day post-hatch as this is a phase of rapid growth and development, and we hoped to achieve biologically active transcriptional changes. the differences in degs observed in the present study control cellular architecture, immune function, metabolic pathway, and muscular function. it has previously been established that huifn-λ signals via il- and il- r exhibit typei-like antiviral potential [ ] . protection from simian foamy virus (sfv) and avian influenza (ai) augments the antiviral functioning and further postulates its diverse antiviral potential against avian pathogens. in this context, we stimulated chickens with silkworm-expressed chifn-λ and profiled the gene expression in immune organs (thymus and bursa) and compared it with that in primary chicken fibroblasts using rna-seq. an overall low isg expression was noticed in chifn-λ-stimulated cef; out of , genes, were degs ( upregulated and downregulated) (p < . , |log fold change|> ) (figure a ). although cef do not possess receptors for ifn-λ, slight temporal expression of degs in response to chifn-λ treatment signifies its antiviral potential in primary cells. pathways [ ] (figure ) . due to the induction of a distinct subset of genes, a lower level of antiviral activity is observed as compared to type-i ifns. it is speculated that the activation of chifn-λ is similar to type-i ifns but they are diverse in functional capability. the chifn-λ have particular significance in viral infections of epithelial origin, where they are optimally active by eliciting a broad antiviral state. using conventional approaches, we have confirmed the expression of selected genes as shown in supplementary figure s a and s b. next, we monitored the gene expression in the thymus and bursa. between the chifn-λ-treated and non-treated thymus, a total of , genes were expressed. among them, genes were degs, in which genes were upregulated and genes were downregulated ( figure b ). in the bursa of fabricious, out of , genes were differentially expressed ( upregulated and downregulated) ( figure c ). interestingly, a relatively low number of genes overlapped among these three groups ( figure d ). in order to confirm the expression of degs, we used a conventional approach (qpcr) and show (supplementary figure s a,b) a scenario corresponding to the rna-seq data. on the basis of abundance and fold change, degs were further characterized (supplementary table s ). cumulatively, a significant upregulation of crucial cytokine and chemokine genes (il -β, ccl , ccl , and cx cl ) was observed. these are broadly involved in antiviral response, apoptosis, cellular proliferation and differentiation, cytokine-cytokine receptor interaction and inflammation pathways [ ] (figure ). due to the induction of a distinct subset of genes, a lower level of antiviral activity is observed as compared to type-i ifns. it is speculated that the activation of chifn-λ is similar to type-i ifns but they are diverse in functional capability. the chifn-λ have particular significance in viral infections of epithelial origin, where they are optimally active by eliciting a broad antiviral state. using conventional approaches, we have confirmed the expression of selected genes as shown in supplementary figure s a degs were further analyzed for go terms and the kegg pathway by utilizing deseq [ ] . of go terms associated with chifn-λ-treated cef, go terms were significant (p < . ) ( figure a ). in the bursa, among biological processes, we observed the wnt signaling pathway (wif /camk a), cytokine-cytokine receptor interactions (tnfsf ), the apelin signaling pathway (ryr /myl ), and the significant antiviral pathway (novel gene) in cellular components (figure b) . in the thymus, out of go terms, we observed significant, and in the bursa, out of go terms, were significant (p < . ). in order to understand the biological functions associated with degs, we further analyzed the data in three distinct categories, including biological processes (bp), cellular components (cc), and molecular functions (mf) (figure c) . degs were further analyzed for go terms and the kegg pathway by utilizing deseq [ ] . of go terms associated with chifn-λ-treated cef, go terms were significant (p < . ) ( figure a ). in the bursa, among biological processes, we observed the wnt signaling pathway (wif /camk a), cytokine-cytokine receptor interactions (tnfsf ), the apelin signaling pathway (ryr /myl ), and the significant antiviral pathway (novel gene) in cellular components ( figure b ). in the thymus, out of go terms, we observed significant, and in the bursa, out of go terms, were significant (p < . ). in order to understand the biological functions associated with degs, we further analyzed the data in three distinct categories, including biological processes (bp), cellular components (cc), and molecular functions (mf) ( figure c ). further to gene ontology and differential expression, we investigated kegg pathway enrichment. in cefs, significant enrichment was seen in pathways including the mapk signaling pathway ( further to gene ontology and differential expression, we investigated kegg pathway enrichment. in cefs, significant enrichment was seen in pathways including the mapk signaling pathway (fos/il b/fosb), the toll-like receptor signaling pathway (fosb, il l , il b, fos, ccl ), influenza a (il l /il b/ccl ), cytokine-cytokine receptor interactions (ccl /il l /il b/cx cr /ccl ), salmonella infection (fosb/il l /il b/fos), the nod-like receptor signaling pathway (il l /il b/ccl ), and herpes simplex infection (fosb/il b/fos/ccl ) ( figure a ). in bursa, wnt signaling (wif ), the apelin signaling pathway (ryr ), and the calcium signaling pathway (ryr ) were significantly observed ( figure b ). for the thymus, the nod-like receptor signaling pathway (plcb /mapk ), the mapk signaling pathway (srf/mapk ), influenza a (rsad /mapk ), and mapk (salmonella, toll-like, herpes simplex infection) were observed ( figure c ). collectively, apoptosis (jun/birc /ctsc/actg ), rna degradation (eno /btg /c d), the tca cycle (mdh /idh a), the p signaling pathway (perp /ccnb ), biosynthesis of amino acid (eno /idh a), influenza a (rsad /jun/actg ), and the toll-like receptor signaling pathway (jun) were among the most significant. here, we present the first comprehensive report on cloning and expression of chifn-λ by employing bevs and demonstrate that it is biologically active in both cef (in vitro) and live chickens here, we present the first comprehensive report on cloning and expression of chifn-λ by employing bevs and demonstrate that it is biologically active in both cef (in vitro) and live chickens (in vivo). the identification of this potentially significant ifn among the ifn family advances fundamental aspects and functionality of chifn-λ in avian type-iii ifns. it is evident from the data that this ifn, like human interferon lambda (huifn-λ) , demonstrates similar type-i ifn-like properties. however, a distinct pattern of expression of isgs in chifn-λ contrasts it from other type-i ifns. knowledge regarding ifns is fundamental as rapid outbreaks of viral pathogens cause huge economic losses to the poultry industry every year. the present study investigates the isgs and signaling pathways associated with avian immunity and will bring new horizons to target problematic viral pathogens, e.g., aivs, circulating within the poultry industry. interferon lambda is a biologically active type-iii interferon which primarily acts on epithelial tissues [ ] . studies have demonstrated the antiviral potential of ifn-λ against highly pathogenic avian influenza by eliciting a broad antiviral state [ ] . ifn-λ is structurally peculiar as it possesses five exonic regions located on chromosome , contrary to type-i ifns, which are intronless and situated on the z sex chromosome in chicken [ , ] . this is in agreement with human ifn-λ subfamily which are anatomically identical by possessing five exonic regions on chromosome of the human genome [ ] . furthermore, % of amino acids are identical between huifn-λii and chifn-λ, which signifies the similarity of these two ifns. however, unlike mammals, only one member exists in chicken (chifn-λ). this is in agreement with the other types of chicken ifns, which have fewer members compared to mammalian ifns [ ] . reduced expression of isgs in response to chifn-λ in our experiment demonstrates the fact that cefs are optimally less receptive to ifn-λ, which is in agreement with published reports [ ] . one study revealed that chifn-λ can actively inhibit the viral replication of ai in primary embryonic tracheal organ cultures and clec- (chicken lung cell line). it is further postulated that with treatment of chifn-λ, isgs are expressed significantly, especially mx gene, which is primarily expressed in epithelial rich organs (i.e., trachea, lungs, and intestine) was also observed in the present study [ ] . furthermore, studies have also revealed that a high degree of cell type specificity in receptor-ligand interactions make avian ifns distinct from mammalian ifns. recently, it has been established that chicken ifn-λ inhibits low pathogenic influenza virus replication in cefs; however, as compared to chifn-γ and chifn-β, higher doses are required to induce isgs and maintain the strong antiviral state in the cells [ ] . go and kegg analysis of each experimental group demonstrated overlapping biological functions. an important gene involved in the host response of infected samples is rsad , also termed viperin, which is one of the potent interferon stimulated genes (isgs) responsible for eliciting a broad antiviral state against a variety of viral and bacterial pathogens [ ] . in mammals, it is highly expressed in response to invading viral infections [ ] . elevated expression of viperin in chifn-λ-treated organs further augments the expression of isgs in response to injected ifn in vivo. viperin was upregulated in response to chifn-λ treatment, which is symbolic for all isgs. ifn-inducible transmembrane protein- (ifitm- ) is one of the potent isgs expressed in response to either type of ifn and plays an antiviral role by blocking cytoplasmic entry [ ] . it is further demonstrated that ifitm alters membrane fluidity, hence producing curvature in the outer leaflets of the membrane or by interfering with intracellular cholesterol homeostasis [ , ] . significant upregulation of ifitm in the chifn-λ-treated thymus augments the temporal expression of isgs in response to ifn treatment. further studies are needed to investigate the possible future role of chifn-λ as a potent and novel therapeutic in the poultry industry. although the immune response elicited by type iii ifns is still not very clear, in the present study we also found some novel genes involved in the cascade of the avian immune response. furthermore, in vitro exposure of cef to chifn-λ demonstrated a rapid surge of pro inflammatory cytokines. considering their vital role in immune pathways, cytokine gene expression is widely employed as an indicator for the immune response. we did observe some genes that were previously illustrated in publications; one such example is chemokine (c-c motif) ligand (ccl , ensgalt ) [ ] . chemokines are secreted chemotactic cytokines that play a fundamental role in the recruitment and migration of lymphoid and myeloid cells in target tissues, and hence govern the avian immune response [ ] . ccl is a chemokine secreted by monocytes that is capable of activating macrophages and t lymphocytes [ ] . ccl , like its mammalian orthologue, is responsible for recruiting lymphoid cells and is involved in the early immune response in chickens [ ] . likewise, ccl , ccl , and ccl were also upregulated in cef and are chiefly involved in the innate avian immune response. the present study describes the transcriptomic analysis of differential gene expression following exposure to chifn-λ and the resultant pro-inflammatory response in both cef and chicken tissues. this response ostensibly is due to rapid and sustained signaling via cell surface receptors and a surge of chemokines and cytokines, which in turn create an antiviral environment. a contrasting feature of the present study is the upregulation of the toll-like receptor (tlr) signaling pathway in all three treatment groups, where it is evident that numerous genes are upregulated in tlr mediated cytotoxicity. tlr , a unique chicken receptor expressed on the surface of fibroblasts, heterophils, and macrophages, shares % sequence identity with tlr [ ] . it is evident from experimentation that tlr is a broad spectrum tlr that has the capability to recognize heat stable components of both gram-positive and gram-negative bacteria, cpg oligonucleotides, lipopolysaccharide (lps), and tripalmitoylated lipopeptide [ ] . tlr , an avian-specific tlr, plays a significant role in avian immune responses against bacterial and viral pathogens. recently, it has been demonstrated that diacylated lipopeptide from mycoplasma synoviae activated tlr and regulated innate immune responses [ ] . similarly, significant upregulation of tlr , observed in the present study, highlights a possible role of chifn-λ against mycoplasma infections in chicken. however, it warrants future studies to delineate the molecular processes. it has also been established by repeated experimentation that chifn-λ has been seen to cause delay in viral excretion and the spread of highly pathogenic avian influenza (hpai) h n [ ] . it is evident that in mammals, ifn-λ elicits a protective antiviral response toward ai, whereas ifn-λ plays a minor role in lung epithelia [ ] . similarly, in the respiratory tract of chickens, not all mucosal cells are responsive to chifn-λ. therefore, treatments can only delay, but do not significantly support the complete removal of viral loads of h n or halt the virus crossing the epithelial barrier [ ] . however, for low-pathogenic avian influenza (lpai), it is evident that chifn-λ has demonstrated significant antiviral activities [ ] . recent reports revealed another contrasting feature of ifn-λ, where it significantly elicited strong antiviral potential on intestinal epithelial cells to control murine rotaviruses [ , ] . it will be fascinating to investigate in the future whether the same antiviral phenomena occurs, and chifn-λ might also demonstrate epitheliotropism like rotaviruses and halt viral pathogens of the gastrointestinal tract in chickens. nuclear factor kappa-b (nf-kb) is the most significant, evolutionarily conserved, pleiotropic, inducible transcription factor responsible for regulating genetic expression in a variety of fundamental processes, including apoptosis, growth, immune response, inflammation, stress response, etc. [ ] . notably, the upregulation of nf-kb in response to chifn-λ treatment on cef signifies their potent role in the immune response. activator protein (ap- ) is a transcription factor complex highly responsive for cytokine signaling and growth promotion [ ] . formed through noncovalent dimerization between the fos and jun family of nuclear oncogenes, this complex activates ap- -dependent genes, hence controlling cell proliferation, differentiation, and apoptosis [ ] . consistent with these observations, our study demonstrated that many genes associated with this pathway were upregulated. this finding suggests a link between ap and the transcriptional cascade associated with recombinant interferon treatment. overall, transcriptomic analysis revealed significant upregulation of fos and jun in cef and bursa, and thymus of chicken. the innate immune response is a highly complex, precise, interconnected, and integrated response that relies on many factors. the genes investigated in our study control direct protein interactions and are significantly involved in the avian innate immunity cascade. however, further validation of a broad set of immunity-related genes will also be required to elucidate the mechanism of interferon-induced immunity. a more comprehensive study including a larger set of immune genes and multiple recombinant ifns, which will correlate their integrated role, will enable researchers to provide comprehensive insight into the avian innate response. other future studies involving backyard poultry to assess whether similar patterns of innate immunity prevail in indigenous breeds in response to chifnλ are also important and will further develop our understanding of avian immunity. in the current study, we employed rna-seq to illustrate vital transcriptomes involved in the cascade of avian biology and observed divergent results in recombinant interferon-treated chickens compared to a control group chickens. our data suggest that significant antiviral, cell cycle regulators, and biologically active genes are expressed in response to administered chicken ifn. functional characterization of these vital genes warrants further investigation to determine the future possible role for recombinant chicken ifn in the poultry industry. the authors declare no conflict of interest. phylogeography and evolutionary history of reassortant h n viruses with potential human health implications h n avian influenza virus in korea: evolution and vaccination avian interferons and their antiviral effectors characterization and transcriptional analysis of the mouse chromosome cytokine receptor gene cluster ifn-λs mediate antiviral protection through a distinct class ii cytokine receptor complex lambda interferon (ifn-λ), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against 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than just antiviral cytokines a genomic analysis of chicken cytokines and chemokines characterisation of chicken zap the antiviral response. microbes infect palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm ifitm proteins restrict viral membrane hemifusion the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry marek's disease virus-induced immunosuppression: array analysis of chicken immune response gene expression profiling cloning, expression and functional characterization of chicken ccr and its ligand ccl unique chemotactic response profile and specific expression of chemokine receptors ccr and ccr by cd + cd + regulatory t cells identification, mapping, and phylogenetic analysis of three novel chicken cc chemokines unique features of chicken toll-like receptors expression of the avian-specific toll-like receptor in chicken heterophils is mediated by gram-negative and gram-positive bacteria, but not tlr agonists diacylated lipopeptide from mycoplasma synoviae mediates tlr induced innate immune responses ifn-λ: a new spotlight in innate immunity against influenza virus infection differential responses of innate immunity triggered by different subtypes of influenza a viruses in human and avian hosts type iii interferon gene expression in response to influenza virus infection in chicken and duck embryonic fibroblasts distinct roles of type i and type iii interferons in intestinal immunity to homologous and heterologous rotavirus infections interferon-λ and interleukin act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection interferons and viral infections innate immune sensing of dna viruses neuronal activity-dependent local activation of dendritic unfolded protein response promotes expression of brain-derived neurotrophic factor in cell soma key: cord- -qote nx authors: vassão, r. c.; sant' anna, o. a.; pereira, c. a. title: a genetic analysis of macrophage activation and specific antibodies in relation to the resistance of heterogeneous mouse populations to mhv infection date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: qote nx the genetically selected high antibody responder mice (h(iii)) are susceptible and the low antibody responder mice (l(iii)) are resistant to the experimental infection with mouse hepatitis virus (mhv ). the mortality rates of the f( ) hybrids and of the f( ) segregants showed the codominance of the susceptible and resistant characters. the direct individual intrapopulation correlation between the induction of antiviral state in macrophages activated by ifn gamma and the resistance to the virus infection, showed that an antiviral state could be induced in resistant mouse macrophages, whereas in susceptible mouse macrophages no restriction of virus replication could be observed. a direct inter- and intrapopulation correlation of pre-existing antibody titres against mhv with the mortality and a direct interpopulation correlation of those titres with the mean survival time of susceptible animals was shown. the data indicate, among the mechanisms of resistance against the virus infection, a role of ifn gamma macrophage-activation and of antibodies against mhv which may delay the mean survival time in susceptible animals. the mouse hepatitis virus (mhv) strains of coronavirus are responsible for well-known epizootics of enteritis that occur endemically in most mouse colonies. most of the animals in these colonies were found to have antibodies against different types of mhv, such as mhv , which have been isolated from a variety of mouse strains under diverse conditions [ , , , , ] . the mhv was isolated by dick et al. [ ] and has been used as a model of viral infection in which resistance varies according to the genetic background of the mouse strain [- , , , , , ] . the resistance pattern of mouse strains has been shown to be not directly linked to the presence of antibodies in sera of animals from contaminated colonies [ ] . the virus replication in target cells, the antiviral state induced by interferon (ifn) and the expression of a monokine with procoagulant activity (pca) have been implicated in the resistance/susceptibility of the genetic homogeneous or heterogeneous mouse populations to the mhv infection [t, , , , , , , . we have shown that resistance to mhv infection can be a consequence of a t-cell-dependent mechanism, in which the production of ifn gamma and the sensitivity of macrophages to ifn gamma play an essential role [ , , ] . the genetically defined high (h) and low (l) responder selected mice and their segregants and hybrids have been proved to be a useful tool for studying the intervention of multiple alleles in the polygenic control of infectious diseases such as salmonella typhimurium and rabies virus infection [ , ] . moreover, the analysis performed in the f heterogeneous population allows the elucidation of the major specific and nonspecific traits implicated in resistance/susceptibility to a given pathogen, as well as that of the environmental and genetic factors that play in these characters. mice selected for high (h) and low (l) responsiveness, were obtained by bidirectional selective breeding, and the effect of the polygenic regulation of responsiveness to selection antigens is essentially multi-specific, i.e., selected genes regulate the antibody response to many complex immunogens unrelated to those used during the selective breeding, the high or low states resulting from distinct mechanisms, including the regulatory role of macrophages [ ] . the hm and lm mice, although showing no mhv in their tissues, were chronically infected by a mhv strain, and had in their sera distinct levels of antibodies against mhv . h m mice were shown to be fully susceptible and lm mice fully resistant to the experimental infection with mhv . one of the mechanisms suggested to be involved in the resistance against this virus infection was the ability of macrophages to develop an antiviral state following ifn gamma activation [ , . the present study was undertaken in an attempt to investigate whether the sensitivity ofmacrophages to the induction of an antiviral state and the antibody responsiveness characters, correlated individually with the resistance to mhv and were influenced at least partially by the same genetic control. therefore the investigation was directed towards a co-inherited expression of the two traits, aiming to elucidate the complexity of the biological factors that intervene in resistance to infection. high (hm) and low (lm) antibody responder mice from selection ill [ ] , from the laboratorio de imunogenetica, instituto butantan, were used in the experiments. mice of both sexes were analysed at months of age. interline reciprocal crosses of (h m x ln~)f hybrids, f segregants and backcrosses (bchh~ and bclh , were analysed as well. virus mhv originally obtained from dr. j. l. virelizier, institut pasteur, paris, france, was cloned by limiting dilution. one plaque was selected and amplified on l cells to serve as the inoculum for future stocks - ] to limit spontaneous mutations. the stocks were always titrated by plaque assay on l cells as previously described [ ] . aliquots containing x l s plaque forming units per milliliter (pfu/ml) were stored at - °c and used in all experiments. for the study of the resistance to virus infection, the animals were subcutaneously inoculated with pfu of mhv , and mortality was recorded daily for days. the peritoneal exudates were collected by peritoneal lavage with ml of rpmi, and centrifugated at g for minutes. the peritoneal exudate cells were re-suspended at a concentration of x cells/ml of rpmi containing % of fetal calf serum (fcs) and cultured on -well plates ( gl/well). the cells were incubated for h at °c in % co and washed three times with medium after vigorous shaking to remove nonadherent cells. ninety percent of the cells were macrophages as determined by their ability to take up zymosan particles. peritoneal macrophages were treated with u/ml of murine recombinant ifn gamma (holland biotechnology, leiden, holland). twenty-four hours later, activated or nonactivated cultures were infected with mhv at a multiplicity of infection (m.o.i.) of . , in order to study the inhibition of mhv replication. the supernatants of cell cultures were collected at h after infection and tested for the virus titre by plaque assay [ ] . for the study of the individual correlation between in vivo resistance and in vitro inhibition of virus replication by ifn gamma, peritoneal macrophages from hm, lm and f segregants were collected without sacrificing the animals, which were infected with mhv a week later. the macrophages were treated with ifn gamma, infected with mhv and the supernatants titrated, as described above. mice from the colony were bled from the retro-orbital venous plexus and the individual anti-mhv antibody titres in their sera, reported as log , were expressed by the rec]procal of the highest serum dilution producing a % inhibition of cytopathic effect induced by mhv on l cells [ ] . the results in table show the resistance/susceptibility of mice to m h v infection, and indicate that the macrophages from lm or resistant f or f mice were sensitive to the induction of an a n t i -m h v state by i f n gamma. however, under the same conditions, no a n t i -m h v effect could be induced in macrophages from the susceptible hin parental line nor from the susceptible r.c. vass~o et al. cultured macrophages were individually collected, treated for h with ifn gamma ( u/mt) before infection with . m.o.i, of mhv . virus titres were determined in the supernatants collected h after the infection. one week later, the same animals were infected subcutaneously with p f u of mhv , observed for days and the percent (~o) of mortality recorded. the mhv titres are the average +_ standard deviation fi or f mice. the in vitro treatment with i f n gamma, which induced an anti-mhv effect only in resistant mouse macrophages, correlated with the in vivo resistance observed after mhv infection. figure shows the linear regression analysis of individual values of the induction of antiviral state in macrophages of resistant and susceptible f phenotypes, indicating a significant direct correlation between the resistance and the induction of an antiviral state in the macrophages. the figure shows the linear regression analysis of the correlation between the mean survival time (mst) and the antibody titres against mhv in groups which exhibited mortality. it indicates a significant direct correlation (r = . , p < . ) between the mean survival time of susceptible mice infected with mhv and the anti-mhv antibody titres. the genetic studies based on the segregation analysis of resistant or susceptible inbred mouse lines to mhv infection showed that these characters are under the control of two major loci [ , ] . more recently, a comparative study performed in genetically homogeneous and heterogeneous mouse populations confirmed these results, and demonstrated the codominance of the susceptibility and resistance characters. the same genes were shown to be present in the isogenic balb/c (susceptible) and a/j (resistant) lines, as well as in genetically selected hh~ (susceptible) and l m (resistant) antibody responder mice [ ] . the use of hm and lm mice made it possible to analyse the relationship between distinct traits in segregation studies, and demonstrated a direct interpopulation correlation between antibody production to unrelated antigen and mortality to mhv infection [ ] . the present studies go deep into the intra-and interpopulation analysis of the correlation between resistance to infection and antiviral macrophage action induced by ifn gamma or specific anti-mhv antibody production. by studying, individually, the mortality of the parental mouse lines, their f~ hybrids, f segregants and backcrosses, the anti-mhv activity of their macrophages after ifn gamma activation, as well as the titre of antibodies against mhv , we were able to show: i) a direct individual correlation between the resistance and the ability of macrophages to partially inhibit mhv replication after ifn gamma activation (table and fig. ) , ii) an inter-and intrapopulation inverse correlation between the mortality and the antibody titres against mhv (fig. ) , iii) an interpopulation direct correlation between the mean survival time of hn~, f~, f and bchm susceptible animals and the antibody titres against mhv (fig. ) . the genetic analysis performed in this study, demonstrates that the resistance to mhv experimental infection and the ability of ifn gammaactivated macrophages from resistant mice to display an antiviral activity, are submitted to at least partially common genetic control. this brings further support to our previous suggestion that one of the main mechanisms involved in the resistance of mice to the mhv infection is based on the ability of their macrophages to restrict the virus replication upon ifn gamma activation [ , . although the participation of antibodies in the resistance to mhv infection remains unclear, the data here shown indicate their possible role in resistance mechanisms and in prolongating the mean survival time of susceptible animals. adult mouse hepatocytes in primary monotayer culture express genetic resistance to mouse hepatitis virus type a comparative study of resistance to mhv infection in genetically homogeneous and heterogeneous mouse populations a virus related to that causing hepatitis in mice (mhv) susceptibility/resistance in mouse hepatitis virus strain 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gamma synthesis and macrophage sensitivity to interferon gamma in vivo depletion of interferon gamma leads to susceptibility of a/j mice to mouse hepatitis virus infection temperature sensitive mutants of mouse hepatitis virus type (mhv ): isolation, biochemical and genetic characterization virus specificity of the antiviral state induced by ifn gamma correlates with resistance to mhv infection gennari m (t ) rabies virus resistance of heterogeneous mouse populations to mhv infection immunity in genetically selected high and low responder lines of mice interaction between mouse hepatitis viruses and primary cultures of kupffer and endothelial liver cells from resistant and susceptible inbred mouse strains induction of natural killer cells and interferon during mouse hepatitis virus infection of resistant and susceptible inbred mouse strains mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection in mice salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection selective breeding of mice for antibody responsiveness to flagellar and somatic antigens of salmonellae isolation of a routine hepatitis virus from swiss mice treated with antilymphocyte serum resistance of genetically selected mice to mhv infection is not dependent on the h release by macrophages role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. v. protective role in mouse hepatitis virus type infection of susceptible and resistant strains of mice we thank d. mouton for critically reading the manuscript, f. souberbielle for editorial assistance and m.l. silva and a.c. barbosa for technical assistance. this work was supported in part by grants from the fapesp and cnpq. c.a. pereira and o.a. sant'anna are recipients of cnpq research fellowships, key: cord- -o ai r authors: wang, wei; xiong, liang; wang, pengyun; wang, fubing; ma, qingfeng title: major vault protein plays important roles in viral infection date: - - journal: iubmb life doi: . /iub. sha: doc_id: cord_uid: o ai r viral replication and related protein expression inside the host cells, and host antiviral immune responses can lead to the occurrence of diverse diseases. with the outbreak of viral infection, a large number of newly diagnosed and died patients infected with various viruses are still reported every year. viral infection has already been one of the major global public health issues and lead to huge economic and social burdens. studying of viral pathogenesis is a very important way to find methods for prevention, diagnosis, and cure of viral infection; more evidence has confirmed that major vault protein (mvp) is closely associated with viral infection and pathogenesis, and this review is intended to provide a broad relationship between viruses and mvp to stimulate the interest of related researchers. viruses are acellular that cannot naturally reproduce outside of the living host cells and only assemble themselves depending on the host cellular metabolism. virion, known as the complete viral particle, consists of nucleic acid surrounded by capsid, which is enveloped with lipids in some viruses. virion is less than nm in diameter, and its self-assembly is very fast, viral replication inside of the host cells may manipulate and damage the host cells, and the antiviral immune response of the host can damage tissue simultaneously. under the effort of viral toxicity and host immunity, the host is prone to get many kinds of diseases. hepatitis b virus (hbv) and hepatitis c virus (hcv) can cause chronic infection, which can lead to liver cirrhosis and subsequently develop hepatocarcinoma, the patients with viral hepatitis serve as reservoirs of infectious virus. some viruses, including hepatitis a virus (hav), human enterovirus, ebola virus, sars virus, and avian influenza, can cause an outbreak of epidemic infection. [ ] [ ] [ ] [ ] the typical antibiotics are not effective of antiviral infection, antigenic drift of viruses can make effective treatments ineffective, and treatment of viral infection is still one of challenges for humanity. abbreviations: aids, acquired immunodeficiency syndrome; atf, activating transcription factors; c/ebpβ, ccaat-enhancer-binding protein β; egf, endothelial growth factor; eif a, eukaryotic initiation factor a; erk, extracellular signal-related kinase; irf , interferon regulatory factor ; mapk, mitogen-activated protein kinase; mda , melanoma differentiation-associated protein ; mdm, monocytederived macrophages; mvp, major vault protein; myd , myeloid differentiation primary response ; nf-kb, nuclear factor kappa-lightchain-enhancer of activated b cells; pbmc, peripheral blood mononuclear cells; pkm , pyruvate kinase isozyme m ; prrs, pattern recognition receptors; pten, phosphatase and tensin homolog deleted on chromosome ; srsfs, serine/arginine-rich splicing factors; stat- , signal transducer and activator of transcription- . recent studies have shown that many host-encoded proteins are associated with viruses: heat shock protein is incorporated into the virions of human immunodeficiency virus type (hiv- ) ; serine/arginine-rich splicing factors (srsfs) are related to viral replication, srsf promotes anogenital tumorigenesis by maintaining the stability of e e mrnas of human papillomavirus (hpv ), which is the pathogen of anogenital cancer; hiv- replication is increased by srsf , srsf , and srsf within the host cells ; host-encoded proteins are presented in influenza virions ; mvp is involved in antiviral immune response ; and the study of hostencoded proteins in relation to viruses contributes to finding novel targets for antiviral drugs. vaults, the large ribonucleoprotein particles, are composed with mvp, poly (adp-ribose) polymerase, telomerase-associated protein- (tep ), and one or more noncoding rna. , the human mvp, encoded by mvp gene that is located in chromosome p . , is highly conserved during evolution , and predominant component of vaults. [ ] [ ] [ ] [ ] the expression of mvp is very strong and widespread, the mvp is mainly located in the cytoplasm and associated with the cytoskeleton, and a small amount is localized at or around the nuclear membrane and the nuclear pore complex. , current studies have confirmed that mvps are associated with multidrug resistance in treatment of non-small lung cancer, human colon cancer, and mesial temporal lobe epilepsy with hippocampal sclerosis. mvp/vaults play important roles in several signal transduction pathways, suppress c-jun-mediated ap- transactivation by associating with cop , participate the phosphoinositide -kinase pathway by interacting with endogenous phosphatase and tensin homolog deleted on chromosome (pten) with the help of ca + modulation, act as a signaling scaffold protein of extracellular signal-related kinase (erk)/mitogen-activated protein kinase (mapk) pathway by interacting with src in response to endothelial growth factor (egf), and affect the jak-stat signaling pathway by responding and interfering the interferon (ifn)-gamma-mediated stat signals. growing evidences also confirmed that mvp is closely associated with other multiple cellular processes, such as nuclear-cytoplasmic transport, malignant transformation, senescence/ aging, autophagy, and innate immunity. interestingly, mvp has been linked to several types of viral infectious diseases as well as to virus-mediated immune responses. , here, we focus on the roles of mvp in the intracellular viral replication and host immune responses. the innate immune response, including the production of ifn- , is the first barrier of eliminating invaded pathogens early. in host cells, tlrs, rig- (rig-i-like receptor dsrna helicase enzyme), and mda (melanoma differentiation-associated protein ) act as pattern recognition receptors (prrs), ifn-stimulated proteins, and sensors for viral infection. [ ] [ ] [ ] the interferon regulatory factor (irf ) plays the master transcriptional role in viral infection-induced ifn production and immune responses, [ ] [ ] [ ] activates ifn-β production mediated by myd (myeloid differentiation primary response )independent rig- /mda pathway, also activates ifn-α production mediated by the myd -dependent tlrs pathway. [ ] [ ] [ ] the ifn- inhibits viral replication (including hcv, influenza a virus [iav], and hiv) by the production of ifn-stimulated effective proteins. , , after host cells or tissues are infected by hcv, prrs of host cells recognize stimulation signals of products of hcv processing, the interaction between prrs and stimulation signals activates ikba kinase to phosphorylate ikbα, which is associated with nf-kb protein complex in the cytoplasm, phosphorylated ikbα is released from nf-kb complex and degraded by ubiquitin-proteasome pathway, free nf-kb complex translocates to the nucleus, and subsequently activates mvp transcription under coactivators including hcv protein ns a. hcv infection also induces mvp expression through the sp signal pathways, and the infection of vesicular stomatitis virus (vsv), iav, and enterovirus (ev ) has the same effect with hcv infection. inducible mvp is helpful for the nuclear translocation of irf and nf-kb, and performs antiviral activity by promoting endogenous ifn- production and expression of the ifn-stimulated genes. the production of ifn is the critical step in an innate immune response, and mvp plays strong antiviral activity in an ifn- -dependent manner. with the advent of effectively prophylactic vaccines and antiviral drugs, hbv infection remains a global public health problem, , an estimated million people with chronic hbv infection are hbv carriers, deadly complications of hbv chronic infection (including cirrhosis and hepatocellular carcinoma) result in approximately , deaths per year, and hbv infection brings heavy economic pressure for individuals and heavy social burden for the world. as a type of pathogen, hbv causes host cells to produce ifns to increase protective defense of host immune system, ifns play important roles of antivirus by regulating the host immune system, and have been used to treat some cancers and hbv infection. , hbv virus infection leads to the production of type ifns by two main pathways. toll-like receptors / (tlr / ) recognize viral nucleotides and glycolipids and recruit the adaptor protein trif (tir-domaincontaining adapter-inducing ifn-β), trif interacts with traf (tumor necrosis factor [tnf] receptor-associated factor ) to activate nf-kb (nuclear factor kappa-lightchain enhancer of activated b cells), and activated nf-kb provokes ifnb production. , another pathway is triggered by tlr / and tlr , tlrs recognized viral nucleotides in the endosome recruit myd , in turn recruit irak / (interleukin- receptor-associated kinase / ) to the complex and interact with traf (tnf receptorassociated factor ), , and then activate irf / (ifn regulatory factor / ) to induce ifnα expression. mvp is a virus-induced protein, and the level of mvp in peripheral blood mononuclear cells (pbmcs), sera, and liver tissue derived from patients with chronic hepatitis b (chb) is higher than healthy individuals; mvp expression is also increased in hbv stable expression cell lines (hepg . . and huh . ) and hbv-infected hepatocarcinoma cell lines (hepg and huh ). during hbv infection, tlrs recruit and activate myd , which interacts with irak / , irf / , and traf to form a complex, the middle domain (aa - ) of mvp can interact with myd , high expressed mvp joins the myd -mediated complex by interacting with myd to promote ifn- production through translocation of irf and nf-kb from the cytoplasm to the nucleus. , however, hbsag and hbeag competitively bind the myd -binding region of mvp and suppress the ifn- production by disrupting mvp/myd interaction; the ifn- increment effect induced by mvp is counterattacked through hbeag and hbsag binding to mvp (figure ). evidence suggests that hbv has other strategies to suppress the host immune response. hbv polymerase (pol) may inhibit ifn-ɑ-induced myd induction, hbeag suppresses tlr-induced ifn-β, hbsag can block the irf- mediated ifn-ɑ production pathway, and multiple mechanisms lead to hbv immune escape. when the host is attacked by harmful pathogens including viral infection, one of protective immune response is inflammation to eliminate damage. ifn to interfere viral replication, interleukin (il- ) acted as a proinflammatory cytokine, and interleukin (il- ) served as a chemokine for neutrophils and monocytes are important mediators of immune response, and activation of il- and il- gene expression is regulated by transcription factors. , activator protein (ap- ), composed of proteins belonging to c-fos, c-jun, activating transcription factors (atf) and maf families, is a heterodimeric complex and acts as a transcription factor. the function of ap- complex is heavily dependent on the c-fos and c-jun subunits, ap- complex binds dna at ap- specific sites at the promoter and enhancer regions of target genes and increases target gene expression, , and researchers had confirmed that the ap- complex is involved in il- and il- regulation. , ccaatenhancer-binding protein β (c/ebpβ) is a member of the c/ebp transcription factor family, the gene of c/ebpβ can be translated into three polypeptides: the kda and kda liver-enriched transcriptional activating proteins (laps), and the -kda liver-enriched transcriptional inhibitory protein (lip). c/ebp proteins interact with certain gene promoters containing ccaat box motif, then recruit co-activators to promote gene expression. the promoters of il- and il- consists of the ccaat box motif region, wherein c/ebpβ can bind and affect il- and il- expression. f i g u r e hbsag and hbeag weaken the effect of mvp on promoting ifn production in order to restrict the spread of infected virus, some activated transcription factors contribute to the production of inflammatory-related cytokines and chemokines. iav, as a kind of negative single-stranded rna viruses (ssrna), produces replicative intermediate double-stranded rna (dsrna) in the infected cells, dsrna and the synthetic dsrna analog polyinosinic-polycytidylic acid (poly[i:c]) are recognized by tlr , , then activate the tlr -ifn production pathway to robustly express type i ifns. , mvp, as a regulator in the proinflammatory response and an effector in ifn signaling pathway, increases to against viral replication during viral infection. mvp has been proven to be a nuclear-cytoplasmic transport protein and interacts with c-fos of the ap- complex components and c/ebpβ-laps. the interaction promotes the ap- complex and c/ebpβ-laps translocation from the cytoplasm to nucleus and follows to activate the il- and il- expression by the ap- complex and c/ebpβ-laps binding to the il and il promoters, and mvp plays a synergistic role in the expression of il- and il- . the expression of mvp, il- , and il- increases simultaneously in iavinfected a or dsrna-stimulated pbmcs, and the expression of il- and il- is impaired in mvp knockdown cells and knockout mice ; mvp plays a pivotal role in virus-triggered proinflammatory response by mediating the ap- and c/ebpβ signaling pathways. the model of mvp functions for proinflammatory response is summarized in figure . hepatitis e virus (hev), belonged to the genus hepevirus, is classified as a positive-strand rna virus ([+] ssrna virus), and hev infection is an important public health problem. hev is mostly transmitted via the fecal-oral route in developing countries under poor sanitary conditions, , and often spread in many countries by food borne, blood transfusion, , and zoonotic origin. hev can cause chronic infection in immunosuppressed patients, pegylated ifn-alpha- b is used in the treatments for chronic hepatitis e (che) virus infection in liver transplant patients, pegylated ifn-alpha- a is used in the treatments for che virus infection in a hemodialysis patient, and ribavirin as monotherapy may be effective in the treatment for che virus infection in solid-organ transplant patients. silvestrol is a natural cyclopenta(b)benzofuran and acts as an inhibitor of the eukaryotic initiation factor a (eif a) via hindering translation initiation from the capped and -utr of mrnas. the hev is a (+) ssrna virus containing -cap and -utr structure, the released hev particles from persistently hev-infected a cells treated with silvestrol are robustly reduced, which are caused by the decrease of the intracellular hev capsid protein. silvestrol also affect the expression and localization of antiviral host factor mvp, the mvp amount of the cytoplasm is reduced after treating with silvestrol in hev-infected cells, and the mvp transfers from the cytoplasm to the perinuclear area that affects mvp-mediated ifn production. the translation of mvp is highly activated to play an antiviral role by hev infection; however, the change of translation and cytoplasmic localization affected by the silvestrol treatment counteracts part of antiviral effect, mvp plays a complex interplay between the anti-hev replication and the effect of treating with silvestrol for hev infection. the infection of hiv is the pathogenesis of acquired immunodeficiency syndrome (aids) and one of major global public health issues. hiv infected immune cells, including monocytes, lymphocytes, and macrophages, act as stable rival reservoirs, and are main barrier to f i g u r e mvp plays a pivotal role in the proinflammatory response caused by (−) ssrna viral infection eradicate virus by antiviral therapy. the level of cystatin b, a cysteine protease inhibitor, is higher in blood monocyte-derived macrophages (mdm) than in placental macrophages, which are more resistant to hiv- infection than mdm. , the expression of cystatin b is upregulated in hiv- -infected mdm, and cystatin b promotes hiv- replication by interacting with pyruvate kinase isozyme m (pkm ), which is associated with the cocaine enhancement of hiv- replication. in hiv-infected mdm, upregulated cystatin b interacts with mvp and signal transducer and activator of transcription- (stat- ). mvp, as an ifn-responsive protein, directly inhibits tyrosine phosphorylation of stat- to weaken ifn-induced antiviral response by interfering the jak/stat signal pathway, then promote hiv replication. cystatin b directly interacts and decreases tyrosine phosphorylation of stat- , and inhibits ifn-β response and stat- translocation from the cytoplasm to nucleus to reduce jak/stat signal pathway activity, and ultimately promote hiv replication. under the cooperation of the cystatin b and mvp, hiv replication is activated by the damage of jak/stat signal pathway activity mediated by the low tyrosine phosphorylation of stat- . mvp is involved in the diversely cellular processes, including multiresistant cancers, - signal transmission pathways, [ ] [ ] [ ] [ ] and immune response associated with viral infection and treatment. , , , , viruses with divergent virulence and spreadways can cause diverse human diseases with different types and degrees of damage, as a response of viral infection, studies have confirmed that mvp is enhanced in diverse viral infection, including hbv, hcv, hiv, iav and vsv, and so on. the infection of (−) ssrna viruses (including hcv, vsv, iav, and ev ) or dsrna stimulation activates proinflammatory response by inducing the expression of mvp, il- , and il- , enhanced mvp further increase the expression of il- and il- by translocating transregulatory elements (ap- protein complex and c/ebpβ-laps) to the nucleus, and lipopolysaccharide synthesized during viral replication also activates the tlr signaling pathway to induce cytokines, chemokines, and ifn- against iav replication ; however, the value of mvp in the diagnosis, treatment, and prognosis of viral infection remains unclear and additional studies are still required. hbsag and hbeag compete to bind with mvp, facilitate hbv replication and survival by attenuating the effect of mvp-induced ifn, and ifn and nucleotide analogs (nas) are used for the treatment of patients infected with hbv, the stage of liver diseases is important in guiding antiviral therapy ; however, the effect of mvp on the severity of liver disease and the efficacy of different treatments is unclear. silvestrol, as a potent antiviral compound, inhibits hev assembly by interfering hev capsid protein translation, but deactivates the antiviral effect of mvp by translocating mvp to the perinuclear membrane ; cystatin b, as a cysteine protease inhibitor, increases hiv replication by interacting with mvp and pkm to inhibit ifn response and tyrosine phosphorylation of stat- . mvp plays an opposite role in hiv infection by comparing with iva and hbv infection, weakens the antiviral efficacy of silvestrol in the treatment of hev infection, and additional studies are necessary to clarify the role of mvp more clearly in viral infection. i would like to thank my collaborators for their kind help to organize the thoughts and concepts. synthetic viruses: a new opportunity to understand and prevent viral disease significance of hbv dna by pcr over serological markers of hbv in acute and chronic patients. 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authors declare no potential conflict of interest.orcid pengyun wang https://orcid.org/ - - - fubing wang https://orcid.org/ - - - qingfeng ma https://orcid.org/ - - - key: cord- -i on authors: nan title: abstracts dgrh-kongress date: - - journal: z rheumatol doi: . /s - - - sha: doc_id: cord_uid: i on nan im namen der dgrh, der dgorh und der gkjr begrüßen wir sie ganz herzlich zu unserem diesjährigen kongress visualisierung therapeutischer effekte von vasodilatantien beim sekundären raynaud-syndrom mittels fluoreszenzoptischer bildgebung di. stellenwert der gelenksonographie bezüglich diagnose, behandlung und therapiekontrolle der bursitis intermetatarsalis -einer häufig übersehenen differenzialdiagnose. fünf fallbeispiele wegen der deutlich eingeschränkten nierenfunktion konnten therapeutisch keine nsar angewandt werden. wir haben mit × , mg colchicin täglich behandelt. die anfänglich schwerkranke bettlägerige patientin konnte innerhalb von h mobilisiert werden. um eine abschließende sicherung der diagnose einer uratinduzierten sakroiliitis erreichen zu können ist die patientin mit einem dual-energy-ct (dect) untersucht worden. ergebnisse. mit dieser methode konnten gichttophi in beiden sakroiliakalgelenken dargestellt werden, ebenso an beiden mtp -gelenken. schlussfolgerung. aktuell liegen bisher noch keine weiteren berichte vor, dass diese methode auch für die diagnostik einer gicht im bereich der sakroiliakalregion zuverlässige ergebnisse liefern kann. zudem zeigt dieser krankheitsverlauf, dass sich die gicht durchaus primär im bereich des achsenskeletts manifestieren kann und nicht in erster linie an den peripheren gelenken zu entsprechenden beschwerden führen muss. a. glimm , s. werner , s. ohrndorf , c. schwenke , g. schmittat , g. burmester einleitung. typische pathologische veränderung bei der rheumatoiden arthritis (ra) ist die synovialitis. auch bei der osteoarthrose (oa) lassen sich entzündliche veränderungen der gelenke finden. diese können mittels fluoreszenzoptischer bildgebung (foi) und dem gelenkultraschall (us) sichtbar gemacht werden. ziel der studie: vergleich der foi mit dem us bei patienten mit ra und oa. methoden. es wurde bei patienten ( ra, oa) die foi beider hände sowie die us des handgelenks (hg) und der fingergelenke (mcp, pip, dip) der klinisch beschwerdeführenden hand von dorsal und palmar sowohl im b-bild (b-us) als auch mit power-doppler (pd-us) durchgeführt. synovialitis und tenosynovitis im us sowie die intensität des fluoreszenzsignals im bereich der gelenke in der foi wurden qualitativ als auch semiquantitativ nach standardisierten verfahren für den primavistamode (pvm) und drei verschiedene phasen (p - ; [ ] ) bewertet. in der statistischen analyse wurden anschließend sensitivitäten und spezifitäten für die foi bei der ra und oa getrennt für synovialitis und tenosynovitis, dorsal und palmar jeweils für b-us und pd-us als referenzmethode berechnet. ergebnisse. in abhängigkeit von der betrachteten phase zeigen sich für die ra und oa moderate sensitivitäten und spezifitäten. für die ra wurden in der phase des foi die höchsten sensitivitäten mit % für b-us und % für pd-us berechnet. auch bei der oa ergaben sich die höchsten sensitivitäten in der phase des foi mit % für b-us und % für pd-us als referenzmethode. die höchsten spezifitäten für beide diagnosen wurden in der foi in phase erreicht. hierbei lag die spezifität bei der ra für b-us bei % und für pd-us bei %. der höchste spezifitätswert bei der oa sowohl für b-us als pd-us war % (. tab. background. pet is a nuclear imaging technique that depicts functional processes within the body with high sensitivity by detecting annihilation radiation from radioactive decay of a positron-emitting radionuclide that was labeled to a biologically active molecule (tracer) and introduced into the body. f-fluoride ( f) can be used for pet as a bone-seeking agent reflecting bone perfusion and remodeling. we inaugurated a pilot study with simultaneous pet/mr to examine whether addition of pet provides different and additional information in comparison to mri in axspa patients. methods. eleven axspa patients, median age y, disease duration range . - y, mean basdai . , were examined by pet/ -tesla mri minutes after injection of a mean dose of mbq of f using a integrated whole-body pet/mr scanner (siemens biograph mmr®). t-mris were scored blinded to patient's clinical characteristics by two readers ( rheumatologist and radiologist/nuclear medicine specialist) using the berlin mri score and also by recording inflammatory lesions on a vertebral edge (ve) level. in a second step pet/mris were read blindly by the same readers also based on the ve involvement of individual vertebral bodies. results. the procedure was successful in all patients. the resulting mean effective radiation dose per patient was . msv. co-registration of pet/mri fusion images was highly accurate, allowing a precise comparison of mri and pet. in the direct comparison of the mri and pet signal the two readers saw consistent signals in almost % of the sites studied. however, there were areas where signals differed, e.g. within existing syndesmophytes where pet signal was increased but conventional mris showed no signal, or the sternum area and lateral or posterior spinal elements such as facets and spinous processes. conclusion. the new technique of integrated pet/mri provides similar imaging signals as conventional mri. however, we observed differences between the two modalities in areas with less inflammatory activity but where bone metabolism seemed to be active or in areas with blurred resolution on conventional mri. the possibility that pet detects osteoblastic activity in areas where no inflammatory signal is detected with mri seems to be of interest. einleitung. sensitiven bildgebenden verfahren wie der hochauflösenden arthrosonographie kommen bei der detektion initial entzündlicher veränderungen im rahmen der frühdiagnostik der psoriasisarthritis (psa) eine große bedeutung zu. die vorliegende prospektive studie untersucht die diagnostische und prognostische wertigkeit der sonographischen befunde im vergleich zur klinischen untersuchung auf ebene einzelner gelenke bei früher psoriasis-arthritis (psa). methoden. rekrutierung von patienten mit therapienaiver früher psa. sonographie von gelenken mit semiquantitativer graduierung (grad - ) von b-bild (gsus) und power-doppler-aktivität (pdus; baseline, monate). klinische parameter: anzahl druckschmerzhafter und geschwollener gelenke (tjc , sjc ), visuelle analogskala, das -crp, health assessment questionnaire haq. für jede followup-visite erfolgte eine kategorisierung des klinischen ansprechens nach eular-response-kriterien und der für die psa validierten minimal-disease-activity(mda)-kriterien (coates et al.). ergebnisse. baseline patienten, nach monate patienten, erkrankungsdauer ( ± , monate). patienten ohne therapie ( ), mit nsar ( ), steroid i.a. ( ) , dmards ( ), biologicals ( ). bei diagnosestellung zeigte sich eine signifikante korrelation zwischen dem us synovitis score und folgenden klinischen parametern: tjc (r= , ), scj (r= , ), das -crp (r= , ). nach monaten zeigte sich eine gute korrelation zwischen der relativen veränderung des us synovitis scores und der relativen veränderung folgender klinischer parameter: tjc (r= , ), haq (r= , ), pasi (r= , ), das -crp (r= , ). zu baseline waren von gelenken sonographisch auffällig, davon zeigten kein klinisches korrelat (subklinisch). nach monaten zeigten % der initial subklinischen gelenke einen unveränderten befund, % waren sonographisch nicht mehr auffällig und % wurden klinisch manifest. bei den klinischen respondern war der rückgang deutlicher ausgeprägt. schlussfolgerung. der ultraschall-synovitis-score korreliert mit klinischen aktivitätsparametern sowohl zum zeitpunkt der diagnosestellung als auch im krankheitsverlauf unter immunsuppressiver therapie. die subklinischen veränderungen bilden sich unter immunsuppressiver therapie zu einem großen teil zurück, deutlicher bei klinischen respondern. ein geringer anteil der initial subklinischen gelenke wird im verlauf klinisch manifest, in höherem maße bei klinischen non-respondern. t. diekhoff , k. hermann charité -universitätsmedizin berlin, radiologie, berlin einleitung. die gicht ist mit einer prävalenz von , - , % insbesondere in den industrieländern eine häufige erkrankungen, die mit gelenkschmerzen einhergeht. bei typischer symptomatik und laborkonstellation ist die diagnose der arthritis urica oft einfach zu stellen, ein atypisches beschwerdebild kann jedoch gelegentlich die abgrenzung zu anderen erkrankungen erschweren. besonders die kalziumpyrophosphat-kristallarthropathie (cppd oder pseudogicht), die selbst mit sehr variabler symptomatik auftreten kann, ist eine relevante differenzialdiagnose, besonders bei älteren patienten. methoden. mit der dual-energy-computertomographie (de-ct) steht ein modernes, innovatives verfahren zur verfügung, das eine detektion von harnsäurehaltigen weichteilverkalkungen ermöglicht und darüber hinaus eine sichere abgrenzung zu kalziumhaltigen verkalkungen gewährleisten kann. das prinzip der de-ct ist relativ simpel und seit längerem bekannt: die messung des untersuchungsvolumens mit zwei unterschiedlichen röhrenspannungen macht es möglich, einen schwächungskoeffizienten zu errechnen, der spezifisch für das untersuchte material ist. allerdings ermöglichten erst moderne cts mit zwei röntgenröhren die klinische anwendung. in jüngster zeit werden jedoch anstrengungen unternommen, die de-ct auch für ein-röhren-systeme verfügbar zu machen. ergebnisse. mit der de-ct können gichttophi sicher vom knochen aber auch von anderen verkalkungen getrennt und zum beispiel farblich kodiert dargestellt werden. im gegensatz zum konventionellen röntgenbild verspricht die de-ct jedoch nicht nur eine höhere sensitivität für tophöse veränderungen, sondern als schnittbildverfahren auch eine bessere abgrenzung und einordnung von anderen morphologischen veränderungen wie zum beispiel von erosionen. schlussfolgerung. dieser vortrag fasst die vor-und nachteile der de-ct in der detektion und abgrenzung von weichteilverkalkungen bei kristallarthropathien zusammen und gibt darüber hinaus einen ausblick auf zukünftige entwicklungen in diesem gebiet. background. anionic glycosaminoglycans interact with a variety of soluble and membrane bound molecules. chondroitin sulfate was shown to have anti-inflammatory properties but its role in arthritis is controversial. methods. we have analyzed the effect of chondroitin sulfate on collagen induced arthritis starting treatment before and after induction of arthritis and in mice with established arthritis. results. in all of these settings chondroitin sulfate significantly reduced the severity of arthritis. it prevented joint destruction, diminished the inflammatory infiltrate and reduced proinflammatory cytokines in joints and plasma. splenocytes restimulated with collagen produced less il- and more il- and il- . the beneficial effects of chondroitin sulfate were transient and closely correlated to the suppression of the collagen-specific humoral immune response. chondroitin sulfate, but not other glycosaminoglycans induced a direct btk and syk-dependent proliferation of b cells and markedly expanded the number of plasma cells in the spleen. in immunized mice chondroitin sulfate reduced the number of antigen specific plasma cells in the bone marrow and was able to suppress established humoral immune responses. conclusion. displacement of disease inducing plasma cells from the bone marrow might contribute to the beneficial effects of chondroitin sulfate and could be an attractive strategy to suppress antibody mediated autoimmunity. background. in rheumatoid arthritis a functional deterioration of the hpa-axis in form of inadequately low secretion of glucocorticoids in relation to severity of inflammation can be detected. the reasons for this phenomenon are not known. the purpose of this study was to find possible reasons responsible for adrenal insufficiency during arthritis. methods. da rats were immunized with type ii collagen in incomplete freund adjuvant to induce arthritis. plasma corticosterone was evaluated by ria and plasma acth by elisa. adrenal cholesterol was quan-titatively studied by sudan-iii staining and scavenger receptor class bi (sr-bi, the hdl receptor) by immunohistochemistry. fluorescent nbd-cholesterol uptake kinetics were analysed by flow cytometry. ultrastructural morphology of adrenocortical mitochondria and lipid droplets was studied by electron microscopy. results. initially increased corticosterone and acth levels were reduced to baseline levels in the later phase of the disease. serum levels of corticosterone relative to il- β were markedly lower in arthritic than control animals (inadequacy). cholesterol storage in adrenocortical cells and expression of sr-bi did not differ between immunized and control rats. however, number of impaired mitochondria largely increased during the course of arthritis (maximum on day ), and this was paralleled by reduced numbers of activated cholesterol droplets (inhomogenous droplets relevant for generation of glucocorticoids). in addition, number of normal mitochondria positively correlated with serum corticosterone levels. conclusion. this first study on adrenal reasons for inadequate glucocorticoid secretion in arthritis demonstrated impaired mitochondria and altered cholesterol breakdown paralleled by low corticosterone levels in relation to ongoing inflammation. justus-liebig universität gießen, kerckhoff-klinik gmbh, rheumatologie u. klinische immunologie, osteologie, physikalische therapie, bad nauheim, agaplesion markus krankenhaus, akademisches lehrkrankenhaus der johann wolfgang goethe-universität, klinik für orthopädie und unfallchirurgie, frankfurt/main, universitätsklinikum gießen und marburg, orthopädische klinik, labor für experimentelle orthopädie, gießen, universitätsklinikum gießen und marburg, orthopädie und orthopädische chirurgie, gießen, universitätsklinikum erlangen, medizinische klinik , rheumatologie und immunologie, erlangen background. obesity is a risk factor in osteoarthritis (oa), but there is limited information about the interaction between bone formation and adipose tissue-derived factors, the so-called adipokines. adipokines such as adiponectin, resistin or visfatin are associated with the pathogenesis of rheumatoid arthritis (ra) and oa. adipokines are produced also by other cell types than adipocytes in ra and oa joints, for example osteoblasts, osteoclasts or chondrocytes. however, in contrast to their joint-destructive role in ra, their role in oa joint remodeling is unclear. therefore, adipokine expression in osteophyte development and bone forming cells as well as their effect on these cells was analyzed. methods. osteophytes and bone were obtained from oa patients during joint replacement surgery. serial sections of bone tissue were stained (masson trichrome, trap) and scored from grade one (no ossification, mainly connective tissue and cartilage) to five (ossified, mineralized osteophyte, < % connective tissue, ossified remodeling zones). immunohistochemistry against alkaline phosphatase, collagen-type ii, adiponectin, resistin, and visfatin was performed. oa osteoblasts were stimulated with adiponectin and measurements of il- , il- and mcp- were performed in cell culture supernatants. results. adiponectin, resistin and visfatin were detectable in osteoblasts and all osteophyte grades. in non-ossified osteophytes (grade ), especially adiponectin and to a lower extend resistin and visfatin were localized in connective tissue fibroblasts. in ossified osteophytes (grade - ), resistin, visfatin and to a lower extend adiponectin protein expression was co-localized with osteoblasts. resistin and visfatin were expressed by osteoclasts. visfatin was found in chondrocytes of all osteophyte grades ( % of chondrocytes) and adiponectin was detectable in blood vessels. osteoblast stimulation with adiponectin increased the release of the inflammatory mediators il- ( . -fold), il- ( . -fold), and mcp- ( . -fold). zeitschrift für rheumatologie suppl · | conclusion. the expression of adiponectin and visfatin expression in osteophyte connective tissue and cartilage suggests their involvement in early osteophyte formation. resistin and visfatin expression by osteoblasts and osteoclasts in ossified osteophytes indicates a role in bone remodeling of osteophytes at later stages. osteoblasts respond to adiponectin stimulation with the release of inflammatory mediators. therefore, adipokines are most likely involved in osteophyte formation at different stages affecting different cell types of bone remodeling. free fatty acids contribute to promotion of arthritis k. frommer , a. schäffler , s. rehart , a. sachs , u. müller-ladner , e. neumann justus-liebig universität gießen, kerckhoff-klinik gmbh, rheumatologie u. klinische immunologie, osteologie, physikalische therapie, bad nauheim, universitätsklinikum regensburg, klinik und poliklinik für innere medizin i, regensburg, agaplesion markus krankenhaus, akademisches lehrkrankenhaus der johann wolfgang goethe-universität, klinik für orthopädie und unfallchirurgie, frankfurt/main background. obesity is a known risk factor for several arthritic diseases and mechanical stress has been shown not to be the only factor. due to increased levels of free fatty acids (ffa) in obese compared to nonobese individuals and due to the involvement of ffa in inflammatory cardiovascular and metabolic diseases, we hypothesized that ffa play a role in the promotion of arthritic diseases. therefore, we therefore investigated the effect of ffa on various effector cells of arthritis. methods. rheumatoid (ra) synovial fibroblasts (sf), osteoarthritis (oa) sf, psoriatic arthritis (psa) sf, human primary chondrocytes (hch), human osteoblasts (ob), human macrovascular (huvec) and microvascular (hbdmec) endothelial cells were stimulated in vitro with different ffa within their physiological range of concentrations. immunoassays were used to quantify ffa-induced protein secretion. sulfosuccinimidyl oleate sodium (sso) was used to inhibit fatty acid translocase (fat). results. ffa dose-dependently increased the secretion of the proinflammatory factors (il- , il and mcp- ) as well as matrix-degrading enzymes (mmp- and mmp- ) in rasf (e.g. for lauric acid [ µm] with rasf/il- : . -fold increase; il : . fold increase; mcp- : . fold increase; pro-mmp : . -fold increase; mmp- : . fold increase). saturated and unsaturated ffa had similar effects on rasf. however, saturated ffa induced strong secretion of il- in chondrocytes, while unsaturated ffa only had a weaker effect on this cell type. at µm, both saturated and unsaturated ffa significantly increased il- secretion by osteoblasts to a similar degree as for sf. a high concentration of ffa ( µm) significantly induced il- secretion in huvec and hbdmec, whereas a low concentration of ffa ( µm) did not have a significant effect (p> . ) on human endothelial cells. blocking ffa transport into rasf by using sso almost completely abolished the effect of palmitic acid on il- secretion. conclusion. ffa are not only metabolic substrates but can also directly contribute to articular inflammation and degradation mediated by various effector cells of arthritis. our data also show that ffa transport into the cell is required for ffa-induced effects in sf. background. chronically inflamed tissues in ra are characterized by local hypoxia and enhanced angiogenesis. the hypoxia inducible factor (hif)- and (hif)- serve as key regulators of adaptation to hypoxia thereby promoting both angiogenesis and metabolic adaptation of endothelial cells. to investigate the impact of hif- /hif- on the angiogenic and metabolic transcriptome under hypoxia ( % o ) versus normoxia ( % o ) we performed a knockdown of either hif- α or hif- α in human microvascular endothelial cells (hmec). methods. specific knockdown of either hif- α or hif- α was achieved using shrna-technology. angiogenic and metabolic transcriptome of hmecs was studied by performing an agilent human whole genome microarray under normoxia vs hypoxia. significantly regulated genes were allocated to angiogenic and metabolic processes using panther database. results. in comparison to normoxia the incubation of untransduced hmecs under hypoxia resulted in regulated angiogenesis related genes and regulated cellular metabolism related genes. in both hif- α and hif- α knockdown cells, hypoxia was still capable of inducing a differential gene expression pattern, but with a much less pronounced effect compared to control cells. analysis of angiogenesis related processes (vegf-pathway, hif-activation, egfr-pathway) showed that % of the differentially expressed genes are controlled by both hif- and hif- . another % of the regulated genes are controlled by hif- . the remaining % of regulated genes are under control of hif- . the differentially regulated genes involved in the cellular metabolism (atpsynthesis, glycolysis, tca-cycle) were found to be to % controlled by both hif- and hif- . the remaining % are dependent on the presence of hif- . conclusion. hif- and hif- are both key regulators of the adaptation of endothelial cells towards hypoxia with overlapping functions. however, they do differ in their capacity to regulate cellular energy metabolism and angiogenesis. this leads us to conclude that hif- affects angiogenesis via indirect effects on cellular energy metabolism as indicated by the regulation of metabolic transcriptome to one fifth. hif- does more influence angiogenesis directly via regulating the synthesis of proangiogenic factors (as has been previously shown).these findings provide new insights into the divergent regulation of angiogenesis in inflamed (hypoxic) tissues by hif- and hif- and are, therefore, considered to be of clinical relevance in ra. background. membrane bound glucocorticoid receptors (mgr) play a pivotal role in pathogenesis of chronic inflammatory diseases as indicated by clinical observations. patients with sle show high frequencies of mgr positive monocytes, sometimes even higher than found in patients with active ra. with increasing glucocorticoid dosages expression of mgr on monocytes of sle-patients is downregulated, suggesting a negative feedback loop to control glucocorticoid action. these receptors represent an effective target for diagnosis and monitoring of different inflammatory diseases, but a feasible detection method is still necessary. objectives. we compare two methods of high-sensitive immunofluorescence staining -the well established liposome procedure with the commercialized faser-technique. methods. hek t cells were cultured for h with/without µg/ml brefeldin a in a humidified incubator at °c. human cd positive t cells and cd positive monocytes were isolated via magnetic-activated cell sorting and subsequently cultured in rpmi . monocytes were incubated for h with/without µg/ml lps. for liposome based highsensitivity immunofluorescence staining cells were incubated with the monoclonal (digoxigenin conjugated) anti-gr antibody, followed by incubation with anti-digoxigenin/anti-biotin matrix. subsequently biotinylated cy liposomes were added. faser technique was performed as described by the manufacturer (miltenyi biotec). dead cells were excluded by adding pi before cell acquisition, using a bd facs calibur flow cytometer. the acquired data were analyzed using flowjo . . software. results. the human mgr, which cannot be reliably detected with conventional staining methods, is detectable with the liposome procedure as well as with the commercialized faser-apc technique. furthermore, the faser-apc-procedure is more sensitive ( . % vs . %) and more specific ( . % vs. . %) compared to the liposome technique. additionally, minor changes of mgr expression can also be demonstrated with the faser technique. the faser procedure shows technical advantages: the commercially available faser-apc-kit is performed according to a standarized protocol and is less time consuming compared to the liposome procedure. conclusion. the human mgr is easily detectable with the commercialized faser kits, which represent an alternative due to a consistent quality and a standardized production. this method facilitates the analysis of the role that mgr play in the pathogenesis of chronic inflammatory diseases and perhaps provoke new insights in glucocorticoid therapy. background. in previous studies we detected th-positive, catecholamine-producing cells in inflamed hypoxic synovial tissue. therefore, the aim of our study was to investigate the influence of hypoxia induced catecholamines on inflammatory responses in arthritis. methods. synovial cells of rheumatoid arthritis (ra) and osteoarthritis (oa) patients were isolated and cultivated under normoxia or hypoxia with/without stimulating enzyme cofactors of th and inhibitors of th. expression of th and release of cytokines and catecholamines was analyzed. the effect of th+ cells was tested by adoptive transfer into dba/ mice with collagen type ii-induced arthritis (cia). th+ cells were generated from mesenchymal stem cells by defined dopaminergic factors. results. hypoxia increased th protein expression and catecholamine synthesis and decreased release of tnf in oa/ra synovial cells compared to normoxic conditions. this inhibitory effect on tnf was reversed by th inhibition with alpha-methyl-para-tyrosine (αmpt). incubation with specific th cofactors (tetrahydrobiopterin and fe +) increased hypoxia-induced inhibition of tnf, which was also reversed by αmpt. adoptive transfer of th+ cells reduced cia in mice, and hydroxydopamine, which depletes th+ cells, reversed this effect. conclusion. in summary, this study presents that th-dependent catecholamine synthesis exhibits anti-inflammatory effects in human ra synovial cells in vitro, which can be augmented under hypoxic condi-tions. in addition, the anti-inflammatory effect of th+ cells has been presented the first time in experimental arthritis in mice. background. previously, we demonstrated that long-lived plasma cells contribute to the pathogenesis of antibody-mediated diseases and should therefore be considered as a promising therapeutic target in systemic lupus erythematous (sle). in bone marrow stromal cells expressing the chemokine cxcl organize these niches that provide for the plasma cell survival. cxcl is the ligand of cxcr expressed on plasma cells. in this study we investigated the contribution of cxcl -cxcr interaction to the longevity of plasma cells in the murine model of lupus. methods. plasma cells purified from spleens of nzb/w mice were incubated with the cxcr blocker amd and then adoptively transferred to immunodeficient rag −/− mice. after days we analyzed the number of plasma cells in bone marrow. furthermore, ova immunized nzb/w mice were treated intraperitoneally with amd after boost; anti-ova secreting plasma cells in bone marrow were checked on day and after boost. the effect of plasma cell depletion was investigated in nzb/w mice using amd alone or combined with bortebomib for two weeks. results. two weeks after adoptive transfer the number of plasma cells treated with amd was lowered by % in bone. after secondary immunization with ova the amd treatment resulted in a significant reduction of anti-ova secreting plasma cells in bone marrow by % on day and by % on day . after days the number of mhc class ii negative anti-ova secreting plasma cells significantly decreased by % in bone marrow of treated mice. amd efficiently depleted plasma cells including long-lived. after two weeks treatment, total plasma cell number was decreased by % in spleen and % in bone marrow; long-lived plasma cells were reduced by % in spleen and % in bone marrow. the combination of bortezomib with amd in nzb/w significantly enhanced the depletion of long-lived plasma cells compared to monotherapy. conclusion. cxcr blockade with amd can reduce the homing of plasma cells to the bone marrow and the survival of long-lived plasma cells. the combination of bortezomib with amd shows synergistic effects on plasma cell depletion. the findings highlight the importance of the cxcr -cxcl axis for the plasma cell niche. zeitschrift für rheumatologie suppl · | er. tnfr expression defines synovial tissue infiltrating cd + t cells in patients with rheumatoid arthritis k background. one hallmark of rheumatoid arthritis (ra) is the infiltration of the synovial membrane by cd + t cells. it has previously been shown that infiltrating cd + t cells differ from non-infiltrating ones in their increased expression of tnfr . furthermore, tnfr is expressed on a fraction of circulating cd + t cells from ra patients, but not from healthy controls. aim of the study was the characterization of tnfr + cd + t cells in patients with rheumatoid arthritis. methods. peripheral tnfr + cd + t cells from ra patients were analyzed by flow cytometry. the expression of naive and memory t cell markers (cd ra and cd ro), markers for t cell activation (cd , cd and cd ) and of icam- as well as the frequencies of the positive cells were determined. to identify the t helper cell signature of tnfr + cd + t cells, intracellular staining of the th , th and th master transcription factors t-bet, gata- and ror-γt, respectively, was performed. results. peripheral tnfr + cd + t cells have neither a preferential naive nor a memory phenotype, but showed higher expression of the activation markers cd , cd and cd than tnfr -cd + t cells. tnfr + cd + t cells express higher frequencies of the t-bet and rorγt than tnfr -cd + t cells. there is no difference in gata- expression between tnfr positive and negative cd + t cells. functionally, it has been shown that the cytokine tnf acts as chemokine to attract cd + t cells to the rheumatoid joints. beside this direct effect of tnf, there are known indirect effects of tnf including the upregulation of cell adhesion molecules like icam- . therefore, icam- expression of migrating tnfr + t cells was investigated. the results show, that migrating tnfr + t cells recovered from synovial tissue are more frequently icam- positive than non-migrating ones. conclusion. tnfr + expression characterizes cd + t cells functionally capable of infiltrating the rheumatoid synovium in an icam- dependent manner. the results show, that tnfr expression defines a pathogenic subset of activated cd + t cells with th and/or th signature in patients with rheumatoid arthritis. hypoxia increased the production of interleukin- β in lps-primed human monocytes n background. monocytes are major players in the innate immune system and are recruited to sites of inflammation, where the environmental conditions vary extremely compared to the interstitium under physiological conditions. for example, in rheumatoid arthritis the inflamed joints are severely hypoxic. this decreased oxygen level could be a triggering factor for the activation and survival of monocytes. aim of the study was to analyze the influence of hypoxia on lipopolysaccharide (lps)-induced cytokine production in primary human monocytes methods. immunomagnetically separated monocytes from the blood of healthy donors were cultured for h under hypoxic conditions ( % oxygen). results. cytokine measurement in the supernatant with elisa showed increased concentrations of interleukin- β ( . ng/ml vs. . ng/ ml, p= . ) and interleukin- ( . ng/ml vs. . ng/ml, p= . ), but not of tnf, after hypoxia and lps-stimulation. cleavage of the il- β proform to its active form is dependent on the assembly of the inflammasome and the recruitment of caspase- followed by their activation. when inflammasome assembly was blocked with high extracellular k+-buffer or by inhibiting intracellular ca-signalling with the ca-chelator bapta-am, hypoxia induced il- β release was abrogated. similarly, il- β release after culture under hypoxia was also abolished in monocytic thp -cells, which are genetically made deficient for the inflammasome components nlrp and asc. one activating signal for the inflammasome was shown to be the release of reactive oxygen species (ros), since mitochondrial ros staining with mitosox revealed an increased mitochondrial ros release under hypoxic conditions. accordingly, the induction of mitochondrial ros through decoupling of the electron transport chain with rotenone also triggered an increase of il- β release under normoxic conditions. analysis of blood monocyts from ra patients showed no difference in lps and hypoxia induced il- β release compared to healthy controls ( . ng/ml vs. . ng/ml). conclusion. this study shows, that hypoxia leads to the activation of the inflammasome, the recruitment of caspase- and the subsequent cleavage and release of interleukin- β in human primary monocytes. intracellular calcium mobilization and mitochondrial ros production were shown to be essential mechanisms triggering inflammasome assembly. background. cell-derived membrane-coated microparticles have been identified as important mediators in intercellular communication. during the process of apoptosis, dying cells start to dynamically release microparticles. polymorphonuclear neutrophils are the most abundant type of leukocytes, representing - % of all white blood cells. due to their very short lifespan, they are the source of massive amounts of apoptotic cell-derived microparticles (admps). while the interaction between neutrophils and t lymphocytes has been focus of extensive research, the influence of neutrophil-derived microparticles on t cells has not been analysed yet. in this study, we investigated the effect of membrane-coated microparticles released by apoptotic neutrophils on different t helper cell subsets. methods. different cd + t cell subtypes were sorted according to the expression of cd , cd , cd ra and cd ro and co-cultured with admps or apoptotic cell remnants purified from uv-irradiated neutrophils isolated from the peripheral blood of healthy donors. t cells were stimulated by okt and anti-cd antibodies and cell proliferation was measured by h-thymidine incorporation or pkh -staining. secretion of cytokines was quantified by elisa. results. admps released by neutrophils selectively suppressed the proliferation of cd +cd -cd + tc in a dose-dependant manner and prevented the upregulation of cd on the t cell surface, while maintaining the expression of cd . the secretion of tumor necrosis factoralpha (tnfα) by t cells stimulated in the presence of admps was significantly reduced. interestingly, in contrast to admps, the apoptotic cell remnants of neutrophils exerted no effect on t cells. the suppressive effect of admps could be completely abrogated by the addition of interleukin(il)- or il- or by the presence of cd +cd +cd + t cells. conclusion. neutrophil admps suppress the proliferation of cd +cd -cd + t cells under conditions of limiting il- and il- concentrations. this could represent an important mechanism to prevent inappropriate activation and expansion of resting t helper cells in the absence of sufficient stimulation and cytokine production. t. alexander background. recent reports have shown dysregulated micrornas in murine lupus models, among them increased expression of mirna- , which has been demonstrated to target the transcription factor foxo in activated cd + t cells. the loss of foxo activity in t cells is associated with spontaneous t cell activation, clonal expansion and autoantibody production, all of which are present in systemic lupus erythematosus (sle). methods. expression levels of microrna- and foxo were analyzed with rt-pcr in magnetic purified peripheral blood cd + t cells from patients with sle and healthy controls (hc). multicolor flow cytometry was performed to analyze cd + t cell expression for ccr , cd ra, ki- , foxp , the interleukin- receptor-α and phosphorylated stat- a (pstat ). analysis of serum il- levels was performed with elisa in sle patients and hc (r&d systems). results. mirna- was significantly upregulated in cd + t cells from sle patients compared to hc (median expression . × e- vs. . × e- , p= . ) while foxo mrna levels were decreased, yet without reaching statistical significance. analysis of ki- expression revealed an increased percentage of proliferating cd + t cells in sle ( . % vs . %, p= . ). overall, cd + t cellular proliferation in sle was associated with increased frequencies of cd ra-ccr -effector memory t cells and enhanced basal pstat levels (median mfi . vs . , p= . ), suggesting a recent stimulation with common gamma chain(γc)-signaling cytokines. in this regard, tcons from sle samples displayed decreased expression levels for the foxo target gene cd (mfi vs. , p= . ) and serum il- levels were significantly higher in sle compared to hc ( . pg/ml vs. . pg/ml, p= . ). conclusion. mir- expression has been shown to be dependent on stat activation and to promote clonal expansion of activated cd + t cells. our data suggest that enhanced il r/stat signaling mediates induction of mir expression, which in turn promotes the proliferation of tcons in sle. the relative contribution of il r/mir- /foxo axis on the enhanced proliferative capacity of sle tcons remains elusive and merit further investigation. collectively, our data provide new insights in the pathophysiology of t cell hyperactivity in sle and identifies mir- as a candidate target for future therapeutic approaches. background. cell activation and apoptotic cell death leads to the formation of membrane-coated vesicles (mcvs). mcvs have previously been identified as mediators of cell-to-cell communication and carriers of microrna. an impaired clearance of apoptotic debris caused by an increased rate of apoptosis or a defect in phagocytic-cell clearance has been observed in sle patients. in this study, we analyzed the microrna content of activated and apoptotic lymphocytes and their corresponding mcvs from both normal healthy donors (nhds) and sle patients. further we investigated the immunomodulatory effect of mcv uptake by monocytes. methods. microrna content of activated and apoptotic lymphocytes and corresponding mcvs of nhds and sle patients were compared in an agilent microrna array and validated by qpcr. apoptosis was induced by uvb-irradiation. mir- expression in monocytes after uv-mcvs engulfment was determined by qpcr. expression of mir- target protein tab- was analyzed by western blot. results. mir- * levels were decreased after apoptosis induction in lymphocytes and apoptotic mcvs compared to their viable correlates. mir- , mir- a and mir- b were decreased in apoptotic lymphocytes compared to viable ones but increased or not significantly changed in apoptotic mcvs compared to viable mcvs, indicating a directional transport of microrna into mcvs. mir- a was expressed at higher levels in viable sle lymphocytes and mcvs compared to nhds. mir- b expression was decreased in uv-lymphocytes and uv-mcvs of sle patients. functional assays confirmed higher mir- levels and consecutively decreased target protein levels in monocytes after engulfment of uv-mcvs. conclusion. within this study we could show an unequal distribution of distinct microrna into mcvs released by activated or apoptotic lymphocytes. further the microrna content was regulated in whole apoptotic cells after uvb-irradiation. this suggests a directional transport rather than a random distribution. thus, cells regulate their microrna as well as the microrna content within released mcvs. we could show a microrna and protein expression change in phagocytes after mcv engulfment. hence, our results suggest mcvs could serve as a transport vehicle for microrna to mediate cell-to-cell communication and influence intracellular processes in phagocytes. disturbances of this system might contribute to the pathogenesis of sle. results. we found in the spleens of nzb mice -times higher numbers of long-lived plasma cells and megakaryocytes compared to wildtype, in nzw mice equal numbers and in nzb/w mice numbers between those for nzb and nzw or wildtype. moreover, in the spleen a fraction of plasma cells clustered around megakaryocytes. we also detected a missense mutation in the c-mpl gene of nzb mice leading to an amino acid replacement within the essential tpo-binding site. upon tpo stimulation of splenocyte and bone marrow cultures nzb cultures responded significantly stronger resulting in the double amount of megakaryocytes compared to nzw cultures. conclusion. in summary, our data indicate that augmented megakaryopoiesis enables the accumulation of a greater number of autoreactive plasma cells in lupus prone nzb/w mice. thus, we assume that enhanced megakaryopoiesis and higher megakaryocyte numbers are contributing to the development and/or pathogenesis of sle. background. baff is a cytokine important for the stimulation and survival of autoreactive b cells and therefore might play a role in several autoimmune diseases, e.g. autoimmune arthritis. in psoriasis arthritis, baff correlates with disease activity and testosterone, but only in male patients, suggesting a role for sex hormones in the regulation of baff. therefore, we wanted to determine if baff production in rheumatoid arthritis and osteoarthritis fibroblasts was regulated by neuroendocrine mediators. methods. fibroblasts were isolated from synovial tissue of ra (n= ) and oa (n= ) patients and cultured in vitro under different conditions. baff was determined by elisa. results. isolated fibroblasts were cultured in the presence or absence of interferon-gamma (ifn-γ), il- , lipopolysaccharide (lps), tumor necrosis factor (tnf), cpg, poly i:c, and cortisol in different combinations for and hours to determine the optimal stimulation strategy for induction of baff production (measured by elisa in supernatants) in fibroblasts. ifn-γ best induced baff in ra and oa fibroblasts. ifn-γ-induced baff production in fibroblasts was decreased by dihydrotestosterone in a concentration dependent manner. the effect was specifically inhibited by nilutamid, a testosterone receptor antagonist. furthermore, stimulation of beta-adrenoceptor increased, whereas stimulation of alpha-adrenoceptors did not change inf-γ-induced baff in synovial fibroblasts. in general the effects were more pronounced in ra as compared to oa fibroblasts. conclusion. taken together, inf-γ-induced baff production in synovial fibroblasts is decreased by testosterone and increased by betaadrenergic stimuli. therefore, neuroendocrine regulation of inflammation in the inflamed joint might be in part mediated by regulating baff production in synovial fibroblasts. a. grützkau , c. kyogoku , b. smiljanovic , j. grün , r. biesen , t. alexander , f. hiepe , a. radbruch , t. häupl deutsches rheuma-forschungszentrum (drfz), berlin, charité -universitätsmedizin berlin, medizinische klinik mit schwerpunkt rheumatologie und klinische immunologie, berlin background. gene expression profiling experiments using peripheral blood mononuclear cells (pbmcs) revealed a crucial role of type i interferon (ifn) in the pathogenesis of systemic lupus erythematosus (sle). however, it is almost unknown how particular leukocyte subsets contribute to the overall type i ifn signature described for pbmcs. furthermore, a detailed analysis of how ifn signatures differ in autoimmune disease from that observed after viral infection is missing so far. therefore, we compared expression levels of ifn signature genes in peripheral cd + t helper cells and monocyte (mo) subsets isolated from patients with sle, healthy donors (nd) and nd vaccinated against yellow fever by global gene expression profiling. methods. peripheral blood from patients with sle and nd were recruited. same nd were examined before and after immunization by yellow fever vaccine. after sorting cells, isolated rna were applied to affymetrix human genome u plus . array. data analysis was done using bioretis database, genesis software and ingenuity pathway analysis (ipa). results. comparing gene expression profiles of yellow fever immunized individuals and active sle patients it was possible to identify a "common" and an "autoimmune-specific" ifn signature. although major ifn signature genes were commonly expressed in cd + t cells and mo of patients with sle and immunized nd, expression magnitudes of them were higher in patients with sle compared to immunized nd. in sle, in addition to the typical "viral-induced" ifn signature, genes that are involved in apoptosis signaling, antiviral pkr signaling, fcγ receptor-mediated phagocytosis and il- -/il- -/il- -mediated jak/ stat signaling pathways were identified by ipa. conclusion. this study demonstrated that ifn signature in autoimmunity and that in viral infection are quite different in the number of ifn-related genes activated and their expression magnitudes. autoimmunity is characterized by a much stronger expression of ifn signature genes and is obviously modulated by a separate set of co-regulated genes defining the "autoimmune-specific" ifn signature. in summary, "common" and "autoimmune-specific" ifn signature genes are of potential interest as clinical biomarkers in sle diagnostics to differentiate between a disease flare and a viral infection. of peripheral blood lymphocytes (pbl). there is currently no data available about nk cells in gpa. the aim of this study was to evaluate the presence of nk cells in gpa granulomas and their proportions in pbl as a basis for a potential role in gpa. methods. paraffin sections of granulomas of gpa, sarcoidosis and tuberculosis patients were stained with a cd monoclonal antibody. nk cell (cd -cd +) proportions of pbl in gpa patients and healthy controls (hc) were analysed by facs analysis. clinical data was extracted from medical records. results. contrary to granulomas from tuberculosis and sarcoidosis which showed a considerable infiltration by cd positive cells, there was not a single cd positive cell in granulomas from gpa patients. therefore, the tissue destructive character of gpa granulomas is associated with a lack of nk cells. gpa patients with inactive disease [birmingham vasculitis activity score (bvas) = , n= ] possessed a significantly higher nk cell proportion in pbl (mean ± standard deviation: . ± . %) than both gpa patients with active disease (bvas> , n= , mean= . ± . %) (p= . ) and hc (n= , mean= . - . %, p= . ). thus, clinical remission is accompanied by an increase in the nk cell proportion in pbl. interestingly, patients with inactive disease that had "normal" nk cell proportions of less than % of pbl (n= ) showed a more severe disease course than those with more than % of pbl. conclusion. nk cells might, therefore, be helpful to limit granulomatous inflammation. whether nk cell proportion in pbl might be a useful biomarker in gpa, e.g. as predictor for relapses, will be further evaluated in our future studies. v. gerl background. plasmacytoid dendritic cells (pdcs) are considered a crucial element in sle pathogenesis due to their potency to produce high levels of ifn-α. this innate immunological function of pdcs is lost by terminal differentiation into a professional antigen-presenting cell (pdc-derived dc), thereby upregulating costimulatory molecules and downregulating innate characteristics, e.g. bdca- and ifn-α expression. pdc-derived dcs have not been described in vivo yet, probably due to the fact that they lose their specific markers during differentiation. furthermore, pdcs can differentiate into myeloid dcs by various stimuli. in sle, where low expression of bdca- is commonly seen, this differentiation could be relevant and point to such a lineage switch as well as to an activated state of pdcs. aim. to characterize pdc subsets of differentiation/activation in human peripheral blood and to study their impact on autoimmune inflammation in sle. methods. -color-flowcytometric analyses were performed on whole blood of healthy donors and sle patients. pdcs were identified by cd -/cd -/cd -/cd high//bdca- +/hla-dr+ expression and characterized for cd c, bdca- and the macrophage-associated siglec- , expressed on monocytes of active sle patients in an ifn-α dependent manner. cd and cd expression were measured in parallel. results. we found a small subpopulation of siglec- expressing pdcs in human peripheral blood. compared to siglec- negative pdcs, siglec- positive pdcs express significantly lower bdca- and cd , higher hla- dr background. agonistic autoantibodies against the angiotensin ii receptor type (at r) and the endothelin receptor type a (etar) have been identified in patients suffering from systemic sclerosis (ssc). here we examined the expression of at r and etar in human immune cells and pathological effects mediated through these receptors by corresponding autoantibodies (aabs). methods. at r and etar protein expression on peripheral blood mononuclear cells (pbmcs) from healthy individuals and ssc patients was analyzed using flow cytometry, mrna expression was examined by real-time pcr in pbmcs from healthy donors. in addition, pbmcs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin g (igg) fractions from ssc patients positive for at rand etar-aabs, and with igg from healthy donors serving as control. alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, elisa, and chemotaxis assays, respectively. results were correlated with characteristics/clinical findings of the igg donors. results. both at r and etar were expressed on human peripheral lymphocytes and monocytes. protein expression of both receptors was decreased in ssc patients when compared to healthy donors and correlated negatively with disease duration. in addition, igg fractions of ssc patients induced t cell migration in an anti-at r and anti-etar aab level-dependent manner. moreover, igg of ssc patients was capable of stimulating pbmcs to produce more il- and ccl than igg of healthy donors. all effects could be significantly abrogated by the application of selective at r and etar antagonists. statistical analysis revealed a negative correlation between ssc igg-induced il- concentrations and disease duration, between ssc igg-induced ccl concentrations and time since onset of lung fibrosis as well as an association of ccl concentrations with vascular complications of the corresponding ssc igg donors. conclusion. we demonstrated the expression of both, at r and etar, on human peripheral t cells, b cells and monocytes and found signs for a chronic receptor activation in ssc patients. the inflammatory and profibrotic effects upon aab stimulation in vitro, and their associations with clinical findings suggest a role for autoantibody-mediated activation of immune cells mediated through the at r and etar in the pathogenesis or even the onset of the disease. the bioenergetic role of hif- and hif- during angiogenesis of human microvascular endothelial cells background. hypoxia and angiogenesis are features of inflamed and injured tissues. the transcription factors hypoxia inducible factor (hif)- and (hif)- regulate the cellular and metabolic responses to reduced oxygen tensions thereby promoting angiogenesis with implications on the pathogenesis of ra. we investigated the effects of a knockdown of either hif- α or hif- α in human microvascular endothelial cells (hmec) on angiogenesis and bioenergetics under hypoxia ( % o ) versus normoxia ( % o ). methods. specific knockdown of either hif- α or hif- α was conducted by shrna-technology. to assess angiogenesis of hmecs both tubuli and node formation under hypoxia versus normoxia were investigated. expression of hypoxia driven genes involved in the metabolic response to hypoxia (gapdh/pgk/glut /ldha) was quantified by realtime rt-pcr. the bioenergetic status of the cells was quantified via atp/adp measurements. results. knockdown of hif- α/hif- α resulted in a loss of hypoxia induced angiogenesis. focusing on bioenergetic aspects, we found hypoxia to significantly induce pgk, ldha and gapdh in control cells. knockdown of hif- α and hif- α, respectively, did not affect the hypoxic induction of pgk and ldha. in hif- α and hif- α knockdown-cells, hypoxia was still capable of inducing gapdh, with a less pronounced effect in hif- α knockdown-cells. hypoxia did not significantly up-regulate glut , neither in control nor in hif- α or hif- α knockdown-cells. the knockdown of hif- α resulted in significantly decreased expression of glut under hypoxia. we also found the atp/ adp ratio to be similar in control, hif- α and hif- α knockdown-cells under normoxia. under hypoxic conditions hif- α knockdown-cells showed significantly reduced atp/adp ratios -indicating that less atp is available -compared to hif- α knockdown-cells. conclusion. hif- α and hif- α are both key regulators of angiogenesis. however, they do differ in their potency to regulate cellular energy metabolism. this leads us to conclude that hif- α does directly influence angiogenesis via regulating the synthesis of proangiogenic factors (as previously shown), whereas hif- α affects angiogenesis via effects on cellular energy metabolism as indicated by the reduced expression of gapdh and the diminished atp/adp ratio. these findings provide new insights into regulation of angiogenesis in inflamed (hypoxic) tissues and are, therefore, considered to be of clinical relevance in ra. low baseline complement levels, autoantibody persistence and delayed thymic reactivation are risk factors for development of relapses after hematopoietic stem cell transplantation for refractory sle background. our previous research has provided the evidence that an autoreactive immune system can be "reset" into a healthy, tolerant state by immunoablative treatment to eradicate pathogenic effector cells, followed by transplantation of hematopoietic progenitor cells (hsct). nevertheless, disease flares may occur in a subset of these patients posttransplantation. here, we longitudinally analyzed the immune reconstitution of these patients to identify markers for favorable long-term responses. methods. since , patients with refractory sle received a cd +selected autologous stem cell transplantation after immunoablation with antithymocyte-globulin (atg) and cyclophosphamide as part of a monocentric phase i/ii clinical trial. autoantibody titers were evaluated with elisa, peripheral t-and b lymphocyte subsets immunophenotyped using multicolor flow cytometry. results. clinical remission (sledai ≤ ) could be achieved in all patients, despite immunosuppressive drug withdrawal, associated with disappearance of anti-dsdna antibodies and marked reduction of protective antibodies in serum. unfortunately, two patients died due to transplant-related infections. from the remaining eight patients, five patients are in long-term clinical remission for up to years after hsct, while three patients suffered a relapse of sle at , and months post-transplantation, respectively. patients with early relapses (≤ months) had decreased baseline complement levels, showed persistence of antinuclear antibodies (ana), less significant reduction in protective antibody levels and had slower repopulation of cd + cd ra+ thymic-derived cd + t cells after hsct (< /µl at months) when compared to long-term responders. in addition, flow cytometric analyses revealed an expansion of circulating plasmablasts and increased coexpression of siglec- on monocytes (as surrogate marker for type-i interferon signature), preceding the clinical flares by ~ months. conclusion. low baseline complement levels, persistence of ana and delayed thymic reactivity post-transplantation could be identified as risk factors for development of lupus flares after hsct. since atg-mediated cell lysis is complement-dependent, we conclude that low serum complement is directly associated with incomplete depletion of immunologic memory cells in these patients, which provides a rationale for complement substitution before immunoablation. moreover, lupus flares may be predicted individually by flow cytometry with plasmablast expansion and recurrence of type-i interferon signature. background. systemic lupus erythematosus (sle) is a chronic autoimmune disease characterized by the generation of pathogenic antibodies directed against a variety of autoantigens. we have previously shown that long-lived autoreactive plasma cells can contribute to chronicity and refractoriness of sle. our study is aimed to develop new methods for depletion of long-lived plasma cells in nzb/w mice, a model of sle. methods. we studied different treatment protocols on plasma cell survival: irradiation-based and more selective depletive treatments. - week-old nzb/w f mice were exposed to three different irradiation doses ( , , and gy in two splitted doses with a -h interval). the following protocols were also investigated: ) two bortezomib (bz) injections ( , mg/kg, i.v.) combined with anti-mouse cd ( mg/kg, i.v.), ) three bortezomib injections combined with anti-mouse cd , ) three bortezomib injections combined with anti-lfa- and anti-vla- antibodies (affecting directly the plasma cell niche; µg, i.p.) in a -d interval, plus anti-mouse cd and anti-b ( µg, i.v.). the plasma cells were analyzed in spleen and bone marrow by facs and elispot. results. the frequency of remaining plasma cells in bone marrow after , and gy irradiation were , and , % respectively, and in spleen were almost , and , %. short-term treatments with agents that affect plasma cells (bortezomib, anti-lfa plus anti-vla ) effectively deplete plasma cells including long-lived plasma cells in spleen and bone marrow of nzb/w mice. because of the b cell hyperactivity in nzb/w mice, we observe a rapid regeneration of autoreactive plasma cells in spleen and bone marrow. therefore, plasma cell depletion protocols were combined with b cell depletion. especially, the combination of plasma cell targeting with bortezomib, anti-lfa and anti-vla with b cell targeting (anti-cd plus anti-b ) interrupted the repopulation of autoreactive plasma cells in spleen and bone marrow. conclusion. very high doses of irradiation result in effective depletion of long-lived plasma cells but lower doses not. depletion of long-lived plasma cells can be achieved by the proteasome inhibitor bortezomib and by targeting both adhesion molecules lfa and vla . the combination with b cell depletion is needed to prevent regeneration of autoreactive plasma cells. varicella-zoster-virus(vzv)-specific lymphocytes and igg antibody avidity in patients with juvenile idiopathic arthritis or rheumatoid arthritis background. varicella zoster virus (vzv) is a herpes virus that establishes a life-long latent infection with risk of reactivation (shingles) particularly in immunosuppressed patients with autoimmune disorders. patients with rheumatoid (ra) or juvenile idiopathic arthritis (jia) have a high risk for disseminating varicella zoster virus (vzv) infection or herpes zoster. this study was aimed to investigate the humoral and cellular immune response to vzv including assessment of igg-anti-vzv avidity and vzv-specific reactivity of lymphocytes in ra (n= ) or jia patients (n= ) on different treatments, including biologic agents, such as anti-tumor-necrosis-factor(tnf)-alpha or anti-interleukin- (il- ) receptor inhibition (tocilizumab), compared to healthy adults (ha) and children (hc). methods. igg-anti-vzv concentrations and avidities were quantified by an adapted elisa. vzv-specific interferon-gamma-producing lymphocytes (spot forming units, sfu/ , , cells) were analyzed by elispot. results. no significant differences in the vzv-igg concentrations or avidities were found between the groups. however, lower igg-anti-vzv concentrations were found in tocilizumab-treated ra compared to ha and ra without biologic agents. ra showed lower median sfu ( / , , cells) than ha ( / , , cells), with lowest sfu in adalimumab-treated ra ( / , , cells). sfu were not altered in tocilizumab-treated ra and after incubation with anti-il- in vitro. no differences regarding igg-anti-vzv concentrations, rai and cellular reactivity were found between jia and hc. conclusion. our study demonstrated that ra and jia patients are still able to maintain humoral and cellular immune responses to vzv despite immunosuppressive therapy or biologic agents. in ra, the role of lower cellular reactivity for risk of herpes zoster has to be considered for recommendations on vaccination. cmv-specific cd + t cells from ra patients contribute to autoimmune disease zeitschrift für rheumatologie suppl · | . increased frequency of lir- (also called cd j or ilt ) on cd + t cells has been associated with autoimmune disease. furthermore, it has been shown that latent cytomegalovirus (cmv) infection contributes to the expansion of cd − t cells. hence we were interested in the influence of cmv infection on the lir expression on t cells in ra patients. methods. we were interested in the role of lir + t cells in ra patients, which potentially contribute to the autoreactive t cell pool, especially in cmv+ patients. therefore, we investigated the expression and function of lir- on cd + t cells in peripheral blood mononuclear cells (pbmc) from patients with rheumatoid arthritis by flow cytometry and cytotoxicity assay. results. flow cytometry analysis revealed higher frequencies of lir- + cd + t cells in cmv seropositive ra (n= , mean%: . ) compared to cmv+ hd (n= , mean%: . , p= . ). using hla-a* /cmvpp dextramers we analyzed cmv-specific cd + t cells. patients with ra had higher frequencies of cvm specific cd + t cells (n= ; mean%: . ) compared to healthy individuals (n= ; mean%: . , p= . ). phenotypically, cmv-specific cd + t cells are mainly cd negative and express lir- . analysis of the cytolytic potential by cd a expression revealed higher numbers of cd a+cd + t cells in ra patients (n= , mean%: , ) compared to healthy donors (n= , mean%: , ). importantly, we found a significant correlation (p= . ) of high numbers of cd +lir- + t cells with high disease activity score (das ) in ra patients without immunosuppressive treatment (n= , r= , ) . tab. . conclusion. this is the first demonstration of significantly increased frequencies of lir- +cd + t cells and of cmv-specific cd + t cells in patients with rheumatoid arthritis. these cells are characterized by a terminally differentiated phenotype. the higher cytolytic potential of cmv-specific t cells likely can be attributed to their function in containing latent cmv infection and to prevent cmv disease, but might potentially contribute to disease severity in ra patients. background. systemic lupus erythematosus (sle) is an autoimmune disease characterized by an acquired il- deficiency, which leads to a homeostatic imbalance between regulatory t cells (treg) and effector t cells (tcon; humrich et al. ). we recently demonstrated that treg homeostasis in lymphoid organs of diseased (nzbxnzw) f mice can be restored by treatment with recombinant il- (il- ) resulting in an amelioration of kidney disease. the aim of this study was to investigate the impact of il- therapy on intrarenal foxp + treg and kidney infiltrating conventional cd + t cells (tcon) in the (nzbxnzw) f mouse model of lupus nephritis. methods. (nzbxnzw) f mice with active nephritis were treated with recombinant il- either for a short period of days or for a longer period of days in total. absolute numbers, phenotype and proliferation of kidney infiltrating cd + t cell subsets were determined by flow cytometry at different time points. results. short-term il- treatment resulted in an enhanced proliferation and increased numbers and frequencies of intrarenal cd +foxp + treg compared to untreated control mice. on the other hand, long term il- treatment did not result in a persistent expansion of the intrarenal foxp + treg population. however, total numbers of kidney infiltrating cd + tcon with a memory/effector phenotpye were diminished and cd + tcon showed markedly reduced signs of cellular activation. conclusion. our data indicate that short term il- treatment is able to expand the size of the intrarenal treg pool. in contrast, long term il- treatment decreases the numbers of kidney infiltrating memory/ effector t cells and reduces cellular hyperactivity suggesting that treg suppress the activation and expansion of infiltrating tcon. these results may in part explain the amelioration of disease induced by treatment with il- and underline the important role of intrarenal treg for the suppression of kidney disease in lupus mice. these results also provide additional important rationales for an il- based immunotherapy of human disease. from transcriptome to protein biomarkers in ra: joint compartment and monocytes outperform serum and whole blood background. a main challenge in disease-management of ra is to establish objective criteria relevant for diagnosis and therapeutic stratification of patients. this study focused on global approaches in dissecting inflammation in ra including transcriptome analyses of synovial tissue and blood monocytes and proteome analyses of synovial fluid and serum. methods. gene-expression profiles from synovial tissues and blood monocytes of ra and osteoarthritis (oa) patients were generated by affymtetrix arrays. elisa and multiplex immunoassays were used for validation of candidate markers at the protein level in synovial fluid (sf) from ra and oa patients and in serum from the same group of patients and healthy donors. results. transcriptome analyses of synovial tissues from ra and oa revealed more than differentially expressed genes. to avoid difficulties in sampling synovial tissue and to avoid fluctuation in cellular composition of various cell types in blood, the transcriptome analyses from peripheral blood was focused on a specific cell population. monocytes were selected as the favourable cell type involved in the production of cytokines, which are often considered as therapeutic targets in ra. comparisons between ra and oa monocytes disclosed differential expression of more than genes. in total, genes that were up-regulated in synovial tissues and/or monocytes were used for validation at the protein level as potential biomarkers for ra. among these biomarkers, chemokines (cxcl , ccl , ip ), adhesion molecules (vcam , icam , e-and p-selectins), proteolytic enzymes (mmp , a at), and the shedding form of cell surface molecules (cd , cd ) background. idiopathic membranous nephropathy (imn) is a common cause of nephrotic syndrome in adults and has recently been identified as an autoimmune-mediated disease [ ] . autoantibodies directed towards the m-type phospholipase a receptor (pla r) are fairly specific for idiopathic mn and only found to a small percentage in sera from patients with secondary mn [ ] . the outcome of patients with imn is quite diverse: about one third of patients have spontaneous remission, another third progress to require dialysis and the last third continue to have proteinuria without progression to renal failure. we performed serological profiles of imn patients in order to compare antibody profiles to antibody frequencies found in the normal healthy population and to hopefully identify factors that help to predict disease course in imn. methods. serum samples of patients with imn were assayed for a variety of autoantibodies by elisa, addressable laser bead immunoassay (albia) and to dsdna by crithidia luciliae assay. results. the prevalence of autoantibodies found in our imn cohort is summarized in tab. . anti-pla r antibodies were found in about % of imn patients whereas the frequency of other antibodies was mostly below %. the one exception is anti-dfs that was found in . % of imn patients. conclusion. the prevalence of anti-pla r positive patients in our imn cohort matches what has been previously described [ ] . the frequency of the other antibodies that we determined is comparable to what has been reported in the normal healthy population. it is important to note that anti-dfs antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases (sard; [ ] ) whereas anti-ro reactivity is often regarded as a marker for sard. the absence of anti-ro and the high prevalence of anti-dfs confirms that imn is a rather organ specific autoimmune disease. background. activity and the quality of movement belong to the most fundamental diagnostic parameters for neurobehavioural analysis but in the past it has been difficult to include this information into pre-clinical murine disease models. here we tested the applicability of a radiofrequency identification (rfid) based automated tracking system in the experimental murine model of ovalbumin induced arthritis. methods. c bl/ mice were immunized twice with cationized ovalbumin in freund's complete adjuvant and onset of arthritis was induced two weeks after the last immunization by direct injection of cationized ovalbumin into the knee joint of the right hind leg. severity of arthritis was assessed through measurement of joint swelling and evaluation of histological changes. additionally mice were implanted with a rfid transponder and throughout the experiment their activity level was monitored by an id-grid sensor plate placed underneath the homecage. results. the joint inflammation in the ovalbumin induced arthritis model showed a quantifiable impact on the activity levels of the mice. our experiments could also show that movement activity correlates with disease severity as evaluated by clinical and immunological parameters. in the past employing behavioral methods was often limiting by group size, observation time and reproducibility and the stress of handling and new surroundings made results difficult to interpret. in our experiments a rfid-based automated tracking system allowed us to monitor individual activity long-term without removal of the mice from their homecage environment. this allowed for the correlation of clinical parameters to behavioral factors and adds another level of analysis to an established murine model. progranulin antibodies in a wide spectrum of autoimmune diseases results. autoantibodies against progranulin, a secreted and direct inhibitor of tnf-α receptors & were frequently identified in primary vasculitides. in detail, progranulin-antibodies were found during the course of disease in giant cell arteritis/polymyalgia rheumatica ( / ), takayasu's arteritis ( / ), classical panarteritis nodosa ( / ), behcet's disease ( / ), in granulomatosis with polyangiitis ( / ), churg-strauss syndrome ( / ) and in microscopic polyangiitis ( / ). in extended screenings progranulin-antibodies were also frequently detected in autoimmune connective tissue disorders, in rheumatoid and psoriatic arthritis and in inflammatory bowel disorders. in contrast progranulin-antibodies were only detected rarely in healthy controls ( / ), patients with obesity ( / ), residents of nursing homes ( / ), not in patients with cutaneously limited psoriasis ( / ), not in patients undergone sepsis ( / ), and not in patients with melanoma ( / ). a significant association of progranulin-antibodies with active disease states in granulomatosis with polyangiitis suggested a pro-inflammatory activity of progranulin-antibodies. this was supported by an observed neutralizing effect of progranulin-antibodies on the levels of circulating progranulin in elisa and western-blot. moreover, functional assays revealed, that progranulin-antibody containing sera render wehi-s cells far more sensitive to effects of administrated tnf-α, providing evidence for the suspected pro-inflammatory effect of progranulin-antibodies. conclusion. progranulin-antibodies occur in a widespread spectrum of autoimmune diseases and have a pro-inflammatory effect by neutralizing the physiologic tnf-blocker progranulin. background. flow cytometry (fcm) is widely used in research for molecular characterization at single cell level. conventional analysis is a semiautomated process of user defined gating and investigation in -d projections. for multiple parameter analysis with hundreds of marker combinations, this manual process is most limiting and impedes high throughput analysis. therefore, we developed a new algorithm for automated and standardized analysis of multiplex fcm data. methods. automation included asinh-transformation of data, cell grouping, population detection and population feature extraction. for grouping of cells, an unbiased unsupervised model based t-mixture approach with expectation maximization (em)-iteration was applied. populations were identified by meta-clustering of several experiments according to position and extension of cell-clusters in multi-dimensional space and by including a general procrustes analysis (gpa) step. for validation, peripheral leukocytes from healthy donors and patients with rheumatoid arthritis (ra) were prepared by hypoosmotic erythrocyte lysis and stained with different sets of lineage-specific antibodies. in parallel, different leukocyte samples were depleted of one of these populations by magnetic beads. qualitative and quantitative characteristics of major populations were compared with conventional manual analysis. results. whole blood leukocytes stained simultaneously with up to markers were correctly distinguished in all major populations including granulocytes, t cells and their subpopulations, monocytes, b cells, and nk-cells. the result was comparable to the "gold standard" of manual evaluation by an expert. the new technology is able to detect subclusters and to characterize so far neglected smaller populations based on the new parameters generated. automated clustering did not require fluorescence compensation of data. cell-grouping is applicable even for large fcm datasets of at least parameters and more than million events. comparing the cell-clusters between ra and healthy controls, differences were detectable in several cell (sub-)populations, stable enough to perform correct classification into controls and disease. conclusion. this new approach reveals promising results for automated and time-saving analysis of large datasets from multiplex fcm. the algorithm avoids operator-induced bias, is able to detect unexpected sub-clusters and to characterize so far neglected populations. this may reveal not only new markers for disease activity but also for therapeutic stratification. background. lasp- localizes at focal adhesions, along stress fibres and leading edges of migrating cells regulating metastatic dissemination of different tumors. since rasf have been implicated in the spreading of disease by leaving cartilage destruction sites, migrating via the bloodstream and re-initiating the destructive process at distant articular cartilage surfaces, the underlying mechanisms are of special interest. therefore, we investigated the role of lasp- in sf migration and its effects on ra. methods. to identify lasp- expression and its sub-cellular distribution in human sf as well as in hind paws of wt and htnftg mice, we performed western blots and immunofluorescence. the migration of sfs derived from wt, lasp- -/-, htnftg and lasp -/-/htnftg mice was studied in a modified scratch assay as well as in live cell imaging studies. furthermore, a transmigration assay using sf from all four genotypes and murine endothelioma cells (bend. ) as an endothelial barrier was carried out. sf transmigration under inflammatory conditions was also evaluated by tnf-alpha stimulation of the endothelial cells in vitro. results. lasp- expression was up regulated in rasf and in sf from htnftg mice compared to healthy controls and was found at structures of cell adhesion and invasion. the scratch assay as well as the live cell imaging studies showed a significantly reduced migration of lasp- ( ng/ml) was applied. in parallel, il- stimulation significantly amplified the expression of anti-apoptotic bcl- in sle treg but not tcon. conclusion. in analogy to our previous findings in lupus-prone mice, treg from sle patients show the classical hallmarks of il- deficiency with loss of cd expression and a homeostatic imbalance between treg and tcon. these findings could be associated with a reduced il- expression by cd + t cells in sle patients. on the other hand, low-dose il- stimulation in vitro could restore these defects, underlying the potential of il- as a novel therapeutic option in sle. the glucocorticoid-dependent modulation of immune-mediated inflammatory arthritis by osteoblasts in mice is t cell independent background. at present, the role of adiponectin in rheumatoid arthritis is still controversial. there is some evidence indicating anti-inflammatory effects, for example adiponectin reduces the tnf release by macrophages. in contrast to its anti-inflammatory role, adiponectin also exerts pro-inflammatory effects locally in joints, inducing for example pro-inflammatory factors and matrix-degrading enzymes in ra synovial fibroblasts. moreover, our immunohistochemical analysis of ra bone tissue showed a co-localization of adiponectin with key cells of bone remodelling (osteoblasts, osteoclasts). however, the role of adiponectin in bone remodelling of ra still needs to be defined. in this study, we therefore focussed on adiponectin and its immunomodulatory properties on ra osteoblasts and osteoclasts. methods. human osteoblasts and osteoclasts were isolated from bone tissue and blood samples of ra patients. immunocytochemistry and rt-pcr were used to analyze the expression of adiponectin and its receptors in osteoblasts and osteoclasts. osteoblasts and osteoclasts were treated with adiponectin ( µg/ml). adiponectin-mediated effects on the cytokine expression in osteoblasts and osteoclasts were analyzed using elisa. results. the expression of adiponectin and its receptors (adipor , adipor , and paqr ) by cultured ra osteoblasts and osteoclasts could be confirmed on translational and transcriptional level. stimulation of primary ra osteoblasts and osteoclasts with adiponectin resulted in an alteration of cytokine release. osteoblasts showed a time-and dose-dependent increase in il- production. furthermore, adiponectin induced the secretion of il- and gro-alpha and significantly increased the il- and mcp- production (il- : -fold, p= . ; mcp- : -fold, p= . ). stimulation with adiponectin resulted in an increase in il- production in pre-osteoclasts ( -fold) but not in osteoclasts. the secretion of il- was increased in pre-osteoclasts ( -fold) and osteoclasts ( -fold). the results of the present study confirm the pro-inflammatory potential of adiponectin in ra. the cytokines released after adiponectin treatment by osteoblasts and osteoclasts promote osteoclastogenesis or the migratory potential of osteoclasts and monocytes. together with the finding that adiponectin is present in the bone compartment of ra suggest an involvement of adiponectin in articular destruction. acknowledgement: funded by the german research society (spp , immu-nobone, ne / - ). novel mechanisms of glucocorticoid therapy in arthritis anti-inflammatory acting glucocorticoids (gcs) are an important component of rheumatoid arthritis (ra) therapy. but their beneficial usefulness, especially in ra therapy, is hampered by severe side effects like glucocorticoid-induced osteoporosis (gio). until now the molecular mechanisms underlying the beneficial and side effects of gc therapy are poorly understood. gcs exert their actions via the glucocorticoid receptor (gr) that alters gene expression by either binding as a dimer to gc response elements in the promoter region of target genes or by interacting with and thus interfering with other transcription factors. for a long time gr dimerization was considered as the molecular base of side effects. interference of pro-inflammatory transcription factors, such as ap- and nf-κb by the gr monomer was believed to contribute to the therapeutic effects of gcs. in a model of gio we previously showed that unexpectedly interaction of the gr monomer with ap- , but not nf-kb in osteoblasts is decisive for bone loss (cell metabolism : ). in contrast, in antigen-induced arthritis (aia), we could demonstrate that gcs act in the acute inflammation of ra via the dimerized gr. particularly, gc therapy suppressed th and th cell derived pro-inflammatory cytokines in a dimerization dependent manner. furthermore th , rather than th cells seem to be the most crucial targets for an efficient gc therapy since il- -/-mice were resistant to gc therapy whereas ifnγ-/-mice responded as efficient as wild type mice to steroid treatment (pnas : ). in a more chronic arthritis model, the k/bxn serum transfer induced arthritis, we demonstrate now that, unexpectedly, dimerization of the gr in non-hematopoietic cells also contributes to the anti-inflammatory effect of gcs. thus, for immunosuppression of arthritis the gr is required in distinct cell types. taken together, for anti-inflammatory actions the gr dimerization dependent gene regulation is decisive in ra, whereas gio depends on the suppression of ap- dependent gene expression. intriguingly for anti-inflammatory activities of gcs immune and non-immune cells are involved. our approaches give new insights into gc action on arthritis and bone that can be translated into new concepts for anti-inflammatory therapies preventing gio. background. new bone formation and ankylosis are a hallmark of ankylosing spondylitis (as). the impact of cytokines and different mechanisms of new bone formation (endochondral vs. membranous) to syndesmophyte formation and joint ankylosis in as are still poorly understood. in order to analyze cartilage hypertrophy -as a potentially important element of endochondral bone formation -and to assess the possible influence of cytokines, we performed an immunohistochemi-cal study of the hyaline articular cartilage of facet joints of as patients in comparison to autopsy controls and patients with osteoarthritis (oa). methods. the cytokines interleukin(il)- , il- and il- , as well as the marker of cartilage hypertrophy runt-related transcription factor (runx ), matrix metalloproteinase (mmp ) and collagen type (col ) were determined in facet joints from patients with as (undergone correction surgery of rigid hyperkyphosis), oa patients (undergone surgery of the lumbar spine, because of neurological deficits) and controls (autopsies without spinal diseases). immunohistochemistry was performed and the entire cartilage area was analyzed for the frequency of positively stained chondrocytes. background. immunization with glucose- -phosphate isomerase (g pi) induces arthritis in susceptible strains of mice. depletion of regulatory t cells (tregs) prior to immunization switches the usually acute, self-limiting course to a non-remitting, destructive arthritis. this provides a possibility to study molecular switches for the transition from acute, self-limiting to chronic, destructive arthritis within one mouse model. to examine the role of fibroblast-like synoviocytes (fls), which are known to modulate immune responses via the production of pro-and anti-inflammatory mediators, the phenotype and function of fls from mice with either acute, self-limiting or non-remitting, destructive arthritis was studied. methods. fls from dba/ mice that developed either the acute or the chronic form of arthritis were isolated from joints over a time course of days. to investigate the phenotype of fls elisa studies as well as zymography have been performed. for the functional clarification of those cells the matrix-associated transepithelial resistance invasion (matrin) assay and a cartilage attachment assay have been used. furthermore, fls have been transferred in vivo into the knee joints of immunodeficient mice and the joints have been scored histologically. results. fls from treg-depleted mice produced significantly more cytokines (e.g. interleukin (il- )) upon stimulation with other cytokines, growth factors and tlr ligands. this increased susceptibility to cytokine stimulation in chronic animals compared to acute ones is observable throughout the disease course ( days). furthermore, the secretion and activity of matrix metalloproteases (mmps) was enhanced in the fls from chronic mice compared to samples from acute ones. additional functional differences include the collagen-destructive potential and the potential to attach and eventually invade wild type cartilage. here, fls from treg-depleted chronic arthritic mice showed a higher invasive and destructive potential. ultimately, fls from treg-depleted mice were able to destroy cartilage in immunodeficient mice. conclusion. our results are compatible with the hypothesis that uninhibited inflammation in the early phase of treg-depleted mice causes the acquisition of an autonomously aggressive phenotype of synoviocytes which contribute to the switch from acute to chronic arthritis even in the absence of late support from t and b lymphocytes. collagen-induced arthritis modulates reactivity to sympathetic neurotransmitter stimuli during osteoclastogenesis of bone marrow-derived macrophages from da rats background. osteoclast(oc)-mediated bone destruction contributes to increased disease burden in rheumatoid arthritis. simultaneously, changes in synovial tissue innervation occur, leading to a reduction in catecholaminergic nerve fibres. studies on sweat gland innervation revealed that catecholaminergic fibres are capable of phenotypic transition to cholinergic nerves. the sympathetic neurotransmitters norepinephrine (ne) and acetylcholine (ach) affect osteoclastogenesis oppositely prompting us to study osteoclastogenesis at different phases of collagen-induced arthritis (cia) in an altered neurotransmitter microenvironment. methods. for induction of experimental arthritis, da rats were immunized with bovine collagen type ii while controls received isotonic nacl solution. to generate oc, bone marrow-derived macrophages (bmm) were isolated and differentiated with recombinant m-csf and rank ligand. the influence of ne and ach stimulation on osteoclast differentiation and activity was compared between arthritic and control animals at the acute ( days post immunization, pi) and the chronic ( days pi) disease state. as the nicotinic α ach receptor subunit is involved in the cholinergic anti-inflammatory reflex, we also applied a specific agonist, arr- . additionally, the gene expression profile for ne and ach neurotransmitter receptors was analyzed. results. ach stimulation generated significantly more osteoclasts in controls ( days pi). arr- mediated effects were similar to ach. ne decreased osteoclastogenesis via β-adrenoceptors and enhanced via α-adrenoceptor stimulation. cells from arthritic animals were less affected by ne and ach stimulation.oc from arthritic animals showed tendentially decreased activity in an enzymatic cathepsin k activity assay. ach and arr- stimulation decreased cathepsin k activity days pi, but the effect disappeared days pi, representing the chronic arthritis state. ne stimulation significantly inhibited enzyme activity days pi, but has little effect under chronic conditions. the receptor gene expression profile changed in the time course of arthritis. days past immunization muscarinic ach receptors m and m were significantly upregulated whereas after days adrenoceptors α d and α b were significantly downregulated. conclusion. we conclude that cia differentially modulates neurotransmitter influence during oc differentiation and activation but the underlying processes remain still unknown. the observed time pointdependent changes in neurotransmitter receptor gene expression may constitute a regulatory mechanism to counteract alterations in the local neurotransmitter composition. background. the generation of memory t lymphocytes allows effective and fast immune responses during antigen re-challenge and represents a hallmark of adaptive immunity. previous work from our group has demonstrated that murine memory cd + t cells reside in specific bone marrow niches and are characterized by the high expression of cd and ly- c. these cells were designated as resting in the context of gene expression and proliferation. here, we aimed to phenotypically and functionally characterize human memory t lymphocytes in peripheral blood and bone marrow of healthy individuals. methods. mononuclear cells were isolated from paired blood and bm samples from individuals undergoing hip replacement surgery. phenotypic analysis and cytokine profile of distinct memory t cell subsets were assessed by flow cytometry. proliferation and cell cycle status were analyzed using ki- and propidium iodide (pi) staining, respectively. results. distinct populations of cd -expressing cd +cd ra-and cd +cd ra-t cells were detected in bone marrow but not in the periphery. ccr and cd l expression was reduced on bone marrow cd +cd +cd ra-and cd +cd +cd ra-t cells compared to their cd -counterparts in bone marrow and peripheral blood. cell cycle analysis and ki- expression levels demonstrated the nonproliferative state of bone marrow memory t cells. furthermore, bone marrow resident memory t cells showed reactivity against various pathogenic agents, such as tetanus, measles and cmv. conclusion. we have identified a population of cd -expressing cd +cd ra-and cd +cd ra-t cells in the bone marrow. despite cd expression, which is generally regarded as early activation marker, the cells were resting in terms of proliferation. bone marrow cd + memory t cells have downregulated ccr and cd l indicating reduced homing capacity to secondary lymphoid organs. our data underline the role of the bone marrow as a major reservoir for resting memory t lymphocytes. therapeutic methods exerted an influence on satisfaction and future expectations in patients with rheumatoid arthritis (ra). methods. when visiting their rheumatologist, patients with ra were asked to complete a questionnaire at home after the consultation and then return it to an independent opinion research centre, where the data was collected and analysed. the form comprised various areas, namely demography, aspects of the diagnosis, medical care, therapeutic measures, and the illness in a personal context. results. patients ( females/ males) from the whole of austria with a particular emphasis on lower austria, upper austria and tyrol completed the questionnaire (of distributed), resulting in a response rate of %. % of the patients lived in settlements of under , inhabitants; a further % in settlements of under , inhabitants.. the rheumatologist attended could be reached within one hour for % of the patients and within minutes for %. in slightly fewer than % of the respondents the diagnosis was made within three months, in % within six months. in %, the diagnosis was made by a rheumatologist. after experiencing the first symptoms, % contacted their general practitioner. a high degree of satisfaction appears to originate from the information supplied by the rheumatologist attended. most patients felt they were involved in decisions regarding their therapy. % of the respondents were employed prior to their illness; as a consequence of the disease % had to leave their jobs. conclusion. the majority of the respondents came from rural areas. the correct diagnosis was made within six months for almost half of the patients questioned. most patients felt well informed by their rheumatologists and involved in therapeutic decision-making. , combinational therapy of synthetic dmards ( . ; . - . ), and biological-monotherapy ( . ; - ). all of these differed significantly on later observation periods. comparing the prescriptions separately by sequential treatments there were no differences in retention rates for the individual dmard classes. regarding retention, in the first treatment synthetic dmards showed the longest retention, but from the second on this was the case for tnfi-combinational treatment and non-tnfi-biologicals (. abb. ) conclusion. traditional dmards are the starting point of therapy and mtx is the anchor drug for the first and all subsequent courses. already at the second sequential course, the combination therapies of mtx+tnfi become numerically more relevant, and their retention is better than mtx. therapie der rheumatoiden arthritis im letzten jahrzehnt -was hat sich verändert? abb. | ev. anzahl eingeschlossener patienten nach einschlussjahren und therapie sowie zeitlichen entwicklungen im das und der eular-response cal features of ra (erosive arthritis with classical radiological features) plus specific laboratory markers (ccp-antibodies). the third patient, a -year-old female presented initially with features of ra and sle simultaneously (alopecia, subcutaneous nodules, leucopenia, positive ccp-antibodies, high titres of ana and dna-antibodies). the fourth patient, a -year-old patient presented with severe polyarthritis in the upper and lower limbs, subcutaneous nodules, fever and cervical lymphadenopathy, she had high titres of ccp-antibodies ( u/ml by a normal range of less than iu/ml), ana of (normal less than ), and dna of iu/ml (normal less than iu/ml). conclusion. the take home massage of this presentation is to be aware of rhupus if the sle patient develops erosive arthritis or subcutaneous nodules, or if the ra patient develops features of sle like leucopenia, active urine sediment, or clinically significant serositis. rhupus seems to be a distinctive entity and should be kept in mind while dealing with patients having ra or sle as it can affect the treatment and outcome. vorgeschichte. bei der abklärung eines akuten thoraxschmerzes sind auch seltene ursachen, so zum beispiel der einbezug thorakaler organe in eine entzündliche systemerkrankung, zu bedenken. anhand eines besonderen falles möchten wir den weg zur diagnose bei einem schwer kranken notfallpatienten zeigen. leitsymptome bei krankheitsmanifestation. ein -jähriger mann wird bereits zum dritten mal mit akuten thoraxschmerzen in die notaufnahme aufgenommen, jeweils war eine kardiale ischämie ausgeschlossen und ambulante diagnostik empfohlen worden. nach der schmerzcharakteristik, der vorgeschichte von rezidivierenden thoraxschmerzen und entsprechenden risikofaktoren wird bei massiver symptomatik jetzt von einem akuten koronarsyndrom ausgegangen und die invasive diagnostik durchgeführt. eine akute koronare ischämie kann jedoch ausgeschlossen werden. fieber, hohe entzündungszeichen, hinfälligkeit und gewichtsverlust von mehr als kg in den letzten wochen lassen dann eine infektbedingte oder tumorbedingte ursache vermuten, abdomensonographie und röntgen-thorax sowie ausführliches labor samt immunologie führen nicht weiter. wegweisende weitere organbefunde finden sich nicht. die behandlung mit antibiotika hat keinerlei effekt auf klinik oder entzündungsparameter. nach tagen wird der krankheitsverlauf kritisch evaluiert, eine nichtinfektiöse ursache der beschwerden wird in betracht gezogen, eine transösophageale echokardiographie wird zum ausschluss einer endokarditis und zur beurteilung der aorta (leitsymptome thoraxschmerzen und fieber!) durchgeführt. dabei zeigen sich die klappen sämtlich unverdächtig, die aorta ascendens weist eine massive echoarme wandverdickung auf. zum sicheren ausschluss eines intramuralen hämatoms wird sofort eine ct der thorakalen aorta durchgeführt, die eine ausgeprägte zirkuläre wandverbreiterung ohne hinweise auf dissektion zeigt. diagnose. riesenzellarteriitis mit aortitis. therapie. steroide, einleitung einer remissionsinduzierenden therapie mit cyclophosphamid boli. weiterer verlauf. innerhalb von tagen ist der patient beschwerdefrei, die entzündungsparameter sind halbiert, der bettlägerige patient lässt sich mobilisieren und verlässt nach tagen die klinik. bei der diffe-renzialdiagnose des akuten thoraxschmerzes sollten eine unklare entzündungsserologie und eine b-symptomatik frühzeitig an eine aortitis denken lassen. in diesem fall fand sich bereits in tee und ct ein ausgeprägter befund, der sich am ehesten durch den langen verlauf vor diagnosestellung erklären lässt. einleitung. die progressive familiär intrahepatische cholestase (pfic) gehört zu einer heterogenen gruppe seltener, autosomal rezessiv vererbter erkrankungen der gallensäurenexkretion mit intrahepatischer cholestase, hohem risiko der leberzirrhose bereits im kindesalter und hepatozellulärem karzinom. das auftreten eines sle bei pfic ist bisher in der literatur nicht beschrieben. methoden. wir berichten über eine jetzt -jährige patientin mit genetisch gesicherter pfic- (defekt der atp abhängigen gallensalz-exportpumpe bsep), biliostomaanlage im kindesalter, therapie mit udca und kontinuierlicher hepatologischer betreuung. / manifestierte sich ein sle mit az-minderung, florider polyarthritis, polyserositis, splenomegalie, anämie und leukozytopenie. die ana waren mit > : homogen erhöht, die dns-ak im elisa mit u/ml, ena und antiphospholipid antikörper waren negativ. eine steroidtherapie wurde mit prednisolon mg/tag begonnen. bei guter verträglichkeit und stabilen cholestaseparametern wurde die therapie um chloroquin mit mg an tagen der woche ergänzt. die betreuung wurde interdisziplinär fortgesetzt. ergebnisse. unter der steroidtherapie waren gelenkbeschwerden und serositis gebessert, das crp, die leukozytopenie und anämie normalisiert. die dns-antikörper fielen auf u/ml, c und c stiegen um %. es persistierten lediglich physiotherapeutisch behandelte muskuläre schmerzen und schonatmung nach pleuritis. bei steroidreduktion unter mg traten die gelenkbeschwerden wieder auf, die dns-antikörper stiegen auf u/ml und c /c fielen um %, das crp blieb normal. die steroiddosis wurde auf mg/tag und die chloroquindosis auf × mg/woche erhöht. neue organkomplikationen im rahmen des sle sind nicht aufgetreten. die gallensäuren waren mit µmol/l (norm < ) im jahr (letzte messung vor manifestation des sle) deutlich erhöht, bei manifestation des sle mit µmol/l bereits deutlich gebessert und unter hoch dosierter steroidtherapie mit , µmol/l nach woche sowie , µmol/l nach monat normalisiert. unter prednisolon mg/tag stiegen sie bei inaktiviertem sle auf µmol/l nach monaten an, bei steroidreduktion unter mg auf µmol/l. nach erneuter steroiddosiserhöhung auf mg prednisolon/tag fielen sie auf µmol/l ab. schlussfolgerung. die pfic- schützt nicht vor der manifestation eines sle. eine behandlung mit steroiden und chloroquin ist auch bei pfic sicher und wirksam. der floride sle und die höher dosierte steroidtherapie senken trotz bestehenden genetischen defekts unabhängig voneinander und synergistisch die gallensäurenspiegel im serum bei genetischer störung der atp-abhängigen sekretionspumpe bsep. augenentzündungen. eine ausgeprägte b-symptomatik mit fieber, nachtschweiß und abgeschlagenheit seit ca. zwei jahren hatte sich jeweils unter der therapie der hörstürze verflüchtigt. die mr-angiographie der aortenwand zeigte den typischen befund eines ausgeprägten wandenhancements der thorakalen aortenwand sowie der supraaortalen gefäße, duplexsonographisch fand sich eine zirkuläre wandverdickung der proximalen und mittleren acc beidseits ohne relevante stenosen. bei fehlendem nachweis zerebraler vaskulitischer oder embolischer veränderungen im kraniellen mrt und bei normalem eeg wurde der cerebrale krampfanfall als gelegenheitsanfall dd im rahmen der hochaktiven grunderkrankung interpretiert. diagnose. takayasu-arteriitis mit assoziiertem cogan-syndrom und rezidivierender polychondritis. therapie. steroidstoß, darunter rasche reduktion der entzündungszeichen und promptes ansprechen der b-symptomatik, beginn einer steroidsparenden therapie mit mtx. bei anhaltender krankheitsaktivität und hohem steroidbedarf wird der patient aktuell mit tocilizumab behandelt. schlussfolgerung. der präsentierte fall zeigt, dass mittels neuer bildgebender verfahren gelegentlich eine großgefäßvaskulitis detektiert werden kann, obwohl das klinische bild (hier: kopfklinik) eine kleingefäßvaskulitis vermuten lässt. das cogan-syndrom ist häufig mit einer großgefäßvaskulitis assoziiert, in diesem fall auch mit einer polychondritis. umgekehrt erweitert sich unser wissen um das befallsmuster der großgefäßvaskulitiden, die zwar überwiegend große, aber auch mittelgroße und kleine gefäße einbeziehen können. in unserem zentrum ist dies der zweite fall einer takayasu-arteriitis mit assoziiertem cogan-syndrom in einem kollektiv von fällen. diagnostik. im rahmen einer vorstellung in unserer rheumatologischen ambulanz zur abklärung eines möglichen sjögren-syndroms, konnte in der lungenfunktionsdiagnostik eine leichte restriktion und Überblähung festgestellt werden. in einem auswärtig durchgeführten mrt der halsweichteile zeigten sich die glandula parotis und submandibularis beidseits inhomogen und kräftig kontrastiert, ebenfalls mehrere grenzwertig große lymphknoten. aufgrund der erneut pathologischen lungenfunktion und dem verdacht auf eine lungenbeteiligung bei sjögren-syndrom erfolgte ein ct-thorax. hier zeigte sich eine zysti-sche rarefizierung vor allem der zentralen lungenanteile, differentialdiagnostisch mit einer langerhans-zell-histiozytose vereinbar. auch erschien der diabetes insipidus passend zur histiozytose. die immunologischen untersuchungen waren unauffällig, insbesondere konnten keine ssa-/ssb-antikörper oder eine hypergammaglobulinämie nachgewiesen werden. somit erschien ein sjögren-syndrom unwahrscheinlich. zur weiteren abklärung erfolgte eine bronchoskopie, in den biopsaten konnte immunhistochemisch eine langerhans-zell-histiozytose nachgewiesen werden. in einer pathologischen nachuntersuchung auswärtiger parotis-biopsate bestätigte sich diese, zudem konnte eine mutation des braf-proto-onkogens nachgewiesen werden. im abschließend durchgeführten pet-ct konnte eine vermehrte kontrastmittelaufnahme des hypophysenstiels, eine vermehrte stoffwechselaktivität der parotis beidseits sowie kutan axillär links diagnostiziert werden. abschließend lag somit eine langerhans-zell-histiozytose mit befall von hypophyse, lunge, parotis, haut, lymphknoten sowie einem fraglichen befall des darms vor. therapie. in absprache mit der adulten langerhans-zell-histiozytose studiengruppe erfolgt nun eine therapie mit cytarabin. weiterer verlauf. der weitere verlauf nach geplantem therapiebeginn bleibt abzuwarten. einleitung. die anti-gbm-erkrankung (goodpasture-syndrom) ist eine prototypische autoimmunerkrankung mit ernster prognose, wenn sie als "pulmorenales syndrom" mit der trias rapid-progressive glomerulonephritis, alveoläre hämorrhagie und nachweis von autoantikörpern gegen glomeruläre basalmembranen (anti-gbm-ak) auftritt. Über weniger aggressive verläufe ist wenig bekannt. in der literatur wird die bedeutung der frühen diagnosestellung für die prognose der patienten betont. wir berichten über eine -jährige patientin, die sich mit abgeschlagenheit, müdigkeit und blutbeimengungen im sputum vorstellt. sie betreibt einen nikotinabusus. methoden. wir sehen eine blasse patientin (hb , mmol/l, normochrom, normozytär), die keine weiteren auffälligen befunde in der körperlichen untersuchung zeigt. die apparative diagnostik mittels endoskopie und sonographie ergibt keine befunde, die die anämie erklären können; in der bronchoskpie zeigt sich das bild einer alveolären hämorrhagie ohne aktive blutungszeichen. neben der ausgeprägten anämie bestehen eine milde proteinurie mit mg/d sowie eine geringe erythrozyturie. die immunologische diagnostik ergibt gering erhöhte anti-gbm-ak, negative ana und anca. die nierenbiopsie zeigt eine rapid-progressive glomerulonephritis mit halbmondbildung in einem glomerulum, zusätzlich können lineare igg-ablagerungen in der immunfluorenz dargestellt werden. es ist ein glomerulum in der biopsie betroffen, die anderen glomeruli sind unauffällig. es bestehen einzelnen erythrozytenzylinder, diffuse entzündungszeichen lassen sich nicht nachweisen. die nachträglich durchgeführte immunfluoreszenz in der lungenbiopsie bestätigt den befund linearer igg-ablagerungen. ergebnisse. in unserem fall sehen wir eine junge patientin in einem relativ guten allgemeinzustand mit gering erhöhten anti-gbm-antikörpern und einer gering ausgeprägten nierenschädigung mit einem befallenen glomerulum in der biopsie. es wird angesichts des jungen alters der patientin und der geringen ausprägung der erkrankung eine therapie mit cyclophosphamid nach dem "euro-lupus-protokoll" von f. hossiau eingeleitet. im verlauf steigt der hb auf , mmol/l, die alveoläre hämorrhagie sistiert und die proteinurie ist rückläufig. schlussfolgerung. das goodpasture-syndrom mit einer inzidenz von , - , / mio. einwohner und jahr ist eine sehr seltene autoimmunerkrankung mit ernster prognose. möglicherweise spielen umweltfaktoren (hier: nikotinabusus) eine rolle für die manifestation der erkrankung. interessant ist, dass die erstbeschreibung im rahmen einer influenza-epidemie -wie auch in diesem jahr bei unserer patientinerfolgte. Über die therapie gibt es wenige informationen. die meisten empfehlungen gibt es zum pulmorenalen-syndrom, über weniger aggressiv verlaufende manifestationen gibt es nur wenige informationen. einleitung. wir berichten über einen -jährigen raucher mit akut aufgetretener polyarthritis und neu aufgetretenem raynaud-phänomen. auffallend war die diskrepanz zwischen nur geringer systemischer entzündungskonstellation und einer hochaktiven polyarthritis mit schwerem raynaud-phänomen. im verlauf kam es zu einer spontanen thrombophlebitis der v. cephalica. methoden. pathologische werte: crp maximal , mg/dl (norm < , ), zellzahl im gelenkpunktat . leukozyten/µl. im normbereich lagen: bsg mm/ h, ana, ena, ds-dns-ak, rheumafaktoren, anca, kälteagglutinine, kryoglobuline, tumorsuche initial ohne befund (ct-thorax, bronchoskopie, ct abdomen und becken, coloskopie, gastroskopie, skelettszintigraphie), fdg pet-ct: zwei suspekte lymphknoten rechts cervical sowie suspekte rechte tonsille. ergebnisse. selbst unter mg prednisolon weiterhin hochaktive polyarthritis. nach unauffälliger initialer tumorsuche veranlassten wir ein fdg-pet ct mit nachweis zweier suspekter lymphknoten rechts cervical sowie einer suspekten rechten tonsille. die biopsie der klinisch sich nur in der palpation diskret induriert darstellenden region ergab ein gering differenziertes, gering verhornendes plattenepithel mit %iger sequenzhomologie mit hpv typ . nach resektion des tumors und radiatio sistierten sowohl die polyarthritis als auch das raynaud-phänomen ohne weiteres rezidiv auch nach absetzen der glukokortikoide. schlussfolgerung. eine akut auftretende hochaktive polyarthritis mit hohem glukokortikoidbedarf in kombination mit einem raynaud-syndrom, auffallend niedriger systemischer entzündungsaktivität und fehlendem autoantikörpernachweis sollte insbesondere bei einem langjährigen raucher anlass zu einer intensiven tumorsuche geben. ein fdg-pet-ct kann bei okkulten tumoren zielführend sein. bemerkenswert ist hier der nachweis von hpv- im tumor als weiterer risikofaktor neben dem zigarettenrauch. methoden. bei der klinischen untersuchung fielen ein beidseits positives menell-zeichen und deutlicher klopfschmerz über dem lumbosakralen Übergang auf. labordiagnostisch konnte ein erhöhtes c-reaktives protein und ein positiver hla-b nachgewiesen werden. der bath ankylosing disease activity (basdai) index betrug , . die beckenübersichtsaufnahme zeigte eine definitive bilaterale sakroiliitis grad gemäß den modifizierten new-york-kriterien. der befund der kontrastmittelunterstützten mrt der iliosakralgelenke bewies das vorliegen einer bilateralen floriden sakroiliitis. bei gesicherter hla-b positiver ankylosierender spondylitis wurde die indikation zur einleitung einer biologikatherapie mit einem tnfα-inhibitor gestellt. während der abklärung von kontraindikationen wurde in der konventionellen röntgen-thorax-aufnahme eine rundliche, glatt begrenzte zystische läsion mit flüssigkeitsspiegeln im rechten mittellappen entdeckt. die weitere abklärung mittels nativer thorax-ct, bronchoskopie und biopsieentnahme bestätigte den verdacht auf eine bronchogene zyste. die erregerdiagnostik in der bronchoalveolären lavage zeigte lediglich eine kontamination mit der residenten standortflora. ergebnisse. in anbetracht der geplanten immunsuppressiven therapie, die mit einem erhöhten infektionsrisiko einhergeht, wurde die bronchogene zyste im september operativ entfernt. zur linderung der beschwerden erhielt die patientin eine schmerztherapie mit nsar. als sich die patientin sechs monate später erneut zur einleitung der biologikatherapie vorstellte, berichtete sie, dass die schmerzen etwa einen monat nach der operativen resektion praktisch verschwunden seien. die klinische untersuchung war unauffällig, der basdai-index lag bei , . auch das zur verlaufskontrolle angefertigte mrt der isg zeigte im vergleich zur voruntersuchung einen deutlichen rückgang der floridität. schlussfolgerung. es handelt sich um den ersten fall einer kompletten klinischen und radiologischen remission einer hla-b -positiven ankylosierenden spondylitis nach operativer entfernung einer bronchogenen zyste als potenziellen entzündlichen fokus. bei der systemischen verlaufsform der erkrankung treten neben kutanen erscheinungen zusätzlich muskuloskeletale, hämatopoetische ( - %), renale (ca. %), kardiale, cerebrale und pulmonale ( - %) manifestationen auf. eine polyserositis ( - %) ist häufig. mit - % werden im sle-schub abdominelle schmerzen beschrieben. nur in seltenen fällen (amerika , %, asien , - , % aller sle-patienten) kommt es zum bild einer lupusassoziierten mesenterialen vaskulitis (lmv). die Ätiologie der lmv ist weitestgehend unklar, eine genetische präsidsposition sowie auslösende faktoren (bakterielle darminfektionen, medikamente wie nsar, phosphodiesterasehemmer) werden diskutiert. pathogenetisch wird eine mesenteriale ischämie durch eine mikroangiopathie (arteriolen, venolen) bei inflammatorischer immunkomplexpräzipitation sowie thrombembolischen ereignissen angenommen. radiologisch/sonographisch zeigt sich ein segmentales darmwandödem mit darmdilatation. endoskopisch dominieren oberflächliche ulzerationen, perifokale hämorrhagien bis hin zur gangrän. eine erhöhte perforationsneigung wird beschrieben. mikroskopische befunde zeigen eine fibrinoide nekrose subseröser gefäße mit leukozytoklasie der gefäßwand bzw. ein submuköses Ödem mit nur diskreter invasion mononukleärer zellen. die prognose der lmv scheint abhängig von genetischer prädisposition, raschem beginn einer immunsuppression sowie restriktivem einsatz operativer interventionen und wird je nach literaturquelle mit einer letalität bis zu % angegeben. background. we report a patient with tma in the context of sle treated successfully with the c inhibitor eculizumab. the patient had sle with lupus nephritis (ln). before she developed tma with renal failure and neurologic manifestations, she was treated with various immunosuppressive regimens for mucocutaneous and musculoskeletal manifestations and later for ln. the diagnosis of atypical hemolytic uremic syndrome (ahus) was made based on the presence of coombs-negative hemolytic anemia, thrombocytopenia, renal failure, seizures due to cerebral ischemia and signs of tma in the renal biopsy. plasma exchange and hemodialysis were started immediately and could stabilize her condition. six weeks after the beginning of plasmapheresis but still severely compromised renal function and thrombocytopenia, complement inhibition with eculizumab became a therapeutic option. after the first infusions, renal function, anemia and thrombocyte counts markedly improved. dialysis could be stopped. extensive genetic testing of mutations associated with the overactivation of the alternative complement pathway was negative. after months, when the patient was still in remission, eculizumab infusion intervals were widened and it was finally stopped after months of treatment. since then, renal function remained stable with nearly normal glomerular filtration rates. background. il- signaling plays an important role in inflammation but is restricted by different regulatory mechanisms. these mechanisms include the decreased availability of gp , the signal transducing chain of the il receptor, on the cell surface. the aim of this study was to determine whether the inflammatory environment in the arthritic joint has an impact on monocytic gp surface expression and the extent to which regulatory processes in the synovial fluid (sf) can be transferred to an in vitro model. flow cytometry and live cell imaging were used to measure the cell surface expression and internalization of gp . stat phosphorylation was monitored by flow cytometry and western blotting. results. the level of cell surface gp expression on sf monocytes was reduced compared to peripheral blood (pb) monocytes from patients with juvenile idiopathic arthritis (jia). this reduction could be reproduced by stimulating pb monocytes from healthy donors with sf and was dependent on p mapk. the induction of p by il- β in pb monocytes interfered with il- signaling due to the reduced cell surface expression of gp . the results suggest that p -mediated pro-inflammatory stimuli induce the downregulation of gp on monocytes and thus restrict gp mediated signal transduction. this regulatory mechanism could be relevant in the inflamed joints of patients with jia. kr. sjia patient characteristics of those who successfully discontinued corticosteroids during canakinumab treatment: secondary analysis from a pivotal phase trial background. interleukin- β (il- β) is a key driver in the pathogenesis of systemic juvenile idiopathic arthritis (sjia). canakinumab (can), a selective fully human anti-il- β monoclonal antibody, has been shown to be efficacious in the treatment of sjia [ ] . corticosteroids (cs) are a mainstay of therapy for sjia, however due to the well-known long-term side effects, reduction of cs dosage is desirable. objectives. to assess patient features associated with cs discontinuation during can therapy. methods. patients ( - years of age) with active sjia received s.c. can ( mg/kg to mg max) every four weeks during the maximum week cs-tapering phase [ ] . cs tapering was to be initiated when at least an adapted acr was achieved and no fever. a -year-old boy presented with nocturnal tingling paresthesia affecting his feet and his calves. no excessive leg movements were noted at night. within a few months, his symptoms worsened. the paresthesia occurred both during the day and at night. moreover, the paresthesia came to be triggered by merely standing up. affecting a sharply demarcated area not corresponding to dermatomes, symptoms resolved promptly with movement. the paresthesia was associated with local skin erubescence in spots that slowly began spreading all over the affected area. symptoms did not occur while the patient was seated. mild painless swelling around both of the ankles was noticed in the evenings. approximately one and a half years after the initial manifestation, painful triphasic color changes of all fingers and toes triggered by cold or stress occurred. the family history was positive only for psoriasis. extensive laboratory studies excluded inflammatory and hematological conditions as well as occlusive arterial diseases known to be associated with secondary raynaud's phenomenon. polyneuropathy and other neurological disorders were excluded as well. inflammatory joint disease suspected from the initial imaging with magnetic resonance of the feet and ankles was not confirmed by repeated investigations and scintigraphy. the only consistent abnormality was a reduced pulse amplitude corresponding to vasospasm, which was revealed by photoplethysmography of toe vessels. additionally, paradoxical amplitude reduction after application of nitroglycerine was seen in finger vessels. placing his hands or feet in cold water did not trigger raynaud's phenomenon. initial treatment with non-steroid anti-inflammatory drugs, topical isosorbiddinitrate and local steroid instillation (suspicion of inflammation of tibialis posterior tendon) was ineffective. systemic therapy with the calcium channel blocker amlodipine was initiated. the initial dosing of mg ( . mg/kg/day) was slowly increased to mg ( . mg/kg/day) which lead to complete resolution of the patient's ailments. after three years of pharmacotherapy and . years in remission, a weaning off the treatment is planned. based on the patient's positive response to calcium channel blocker, we conclude that the lower-leg paresthesia was of vascular origin and can be considered an atypical presentation of raynaud's phenomenon. background. the initial treatments of choice for jdm are high-dose corticosteroids and methotrexate. however, no consensus exists about second line therapeutic options in refractory or recurrent cases. results. we present a -year-old boy who was diagnosed with jdm due to severe proximal muscle weakness, dysphagia, a heliotrope rash, gottron's sign, nail teleangiectasia and a characteristic muscle biopsy. creatine kinase levels were within normal range and no antinuclear antibodies were present. over a period of seven years, the patient was treated with high-dose corticosteroids, methotrexate, intravenous immunoglobulins, oral steroids, mycophenolate mofetil, rituximab and infliximab. despite all treatment efforts, skin and muscle inflammation persisted and the boy developed severe subcutaneous calcifications, rendering him wheelchair-bound. as il- production correlates with disease activity in adult and juvenile dm, treatment with tocilizumab ( mg/kg every weeks) was initiated, leading to a complete resolution of skin inflammation within months. within months of treatment, the disease activity score (das) decreased from to (out of ), the childhood myositis assessment scale (cmas) increased from to (out of ) and the kendall manual muscle test (mmt) increased from to (out of ). in daily life the wheelchair was no longer necessary. treatment was well tolerated but accompanied by a moderate increase in liver transaminase activities. interestingly, therapy with rituximab was associated with a decline in igm levels only, whereas igg and iga stayed markedly elevated. in contrast, following initiation of tocilizumab treatment, igg levels rapidly declined to normal range, emphasizing the role of the humoral immune system in the pathogenesis of dm. conclusion. taken together, treatment of a severely affected jdm patient with tocilizumab was safe, well tolerated and led to a significant improvement in disease activity. further investigations of il- -blocking agents as a treatment option in otherwise therapy-resistant jdm patients are warranted. functional capacity of jia patients with an initial adjustment to an anti-tnf-alpha therapy background. thirty three percent of patients with polyarticular jia are treated with biologics [ ] . despite substantial improvement achieved by anti-tnf-α treatment according to disease activity [ ] patients have joint-specific impairments. this factor should be considered when analyzing the functional effects on joint limitations while performing daily activities. methods. in a prospective study on polyarticular jia patients treated with anti-tnf-α therapies plus functional therapies we study the longitudinal effects on joint function. the measurements include -d gait analysis (eight vicon f cameras, omg, london, balance control (s -check, tst, großhoeflein), pedobarography (emed plate ( sensors/ cm², novel, munich), daily activity assessment (step watch, orthocare innovations, ok usa), acr pedi and joint mobility testing. we here present the cross-sectional data of the first patients ( . ± . yrs, . ± . cm, . ± . kg, pain-level: . ± . / vas, active joints: . ± . , chaq: . ± . ). results. preliminary results demonstrate that the ability to walk is slightly limited. patients have a reduced push off power generation within the ankle joint (patients: . ± . w/kg; healthy controls: . ± . w/ kg). further on they show limited sensory motor control and stability in comparison to patients with an inactive disease status while performing balance tests (patients: sensory index: . ± . , stability-index: . ± . , patients with inactive status: sensory-index: . ± . , stability index: . ± . (p< . ). note: lower indices values are better results. conclusion. it is one of the first studies which show functional joint-specific deficits during every day activities in patients who receive an initial anti-tnfα-therapy. the limited stability and motor control might be due to limited joint integrity in the ankle joint. this is supported by the impaired push off function while walking. the next study step will show possible effects of the anti-tnfα-therapy. background. the role of sport as a therapeutic tool in treating patients with jia is becoming more important recently [ ] . effects of exercise therapy are reviewed beneath others by takken et al. [ ] . they state that short-term effects look promising but the effects of long-term studies remain unknown. methods. the preventive mobility workout (pmw) is a whole body home-exercise-therapy ( min each day) for patients with an inactive diseases status. it counteracts the deficits which were observed during functional studies in the past [ ] . it consists of exercises for muscular strengthening (squads: hamstring to quadriceps ratio), hamstring flexibility (lift and raise), core stability (prone bridge -time-to-failure), shoulder griddle mobility (horizontal extension) and ankle joint integrity (mechanical power while walking . for statistical analysis an anova was calculated and the level of statistical significance was set to p< . . results. preliminary results show a group effect of the pmw for the hamstring to quadriceps ratio (h-q-r) for the right side (p< . ) and a tendency for the left side (p= . ). the h-q-r for the right side has changed in the tg and cg from . ± . to . ± . and from . ± . to . ± . , respectively. it has changed for the left side in the tg and the cg from . ± . to . ± . and from . ± . to . ± . , respectively. all other parameters regarding flexibility or joint integrity show low or no effects. we have re-tested n= out of so far and the pmw training show little training-effects. the preliminary results might be a reasonable proof for long-term effects. a possible reason for the little effects might be that patients are supposed to train every day but the training diaries show that they exercise approximately three times a week. the authors would like to thank the "deutsche kinder-rheumastiftung" for supporting the study. conclusion. we will validate these proposed definitions prospectively in a jia associated uveitis cohort. based on the results, we will weight these measures to develop an overall scoring system. background. minute walk is a primary outcome measure in studies in pulmonary hypertension. currently we have a two of sets of data [ , ] regarding test results in the minute walk test ( mwt) in healthy children with a large span in the norm values in the different age groups. aim of the study was to establish norm values for healthy german children for the minute walk test. methods. the team of an occupational therapist and a study nurse were visiting schools. permission from the parents was give before the test. always just probands from one class were invited to participate. the test were performed according the international guidelines [ ] . the demographic data of the probands were collected and the parents filled out a short survey regarding the physical activity and the health condition. children with chronic diseases, which decrease the stamina were excluded. up till now probands participated from the age to years. of them were female. the mean minute walk continuously increased with age (. tab background. juvenile idiopathic arthritis (jia) is the most common chronic disease in pediatric rheumatology which often results in foot impairments [ ] . patients with jia are reported to have smaller pressure loads underneath the foot while walking [ ] . the aim of the study was to analyze the peak plantar pressure distribution of a well described cohort of jia patients with an active symmetrical ankle joint arthritis and no history of foot involvement. [ ] ) wurden in bezug auf therapie und outcome anhand der wallace-kriterien beurteilt: "active disease" (ad), "inactive disease" (id), "clinical remission under medication" (crm), "clinical remission off medication" (crom; [ ] background. familial mediterranean fever (fmf) is one of the most common autoinflammatory diseases (aid). pathogenomic relevant mutations in the mefv gene show autosomal recessive inheritance, but co-dominant mutations have been described. we aimed to evaluate correlations between ethnic origin, phenotype and genotype for fmf patients in the german aid-net-registry. methods. we used two common scoring systems modified for children (mor et al., pras et al.) to assess disease severity in fmf patients of the aid-net-registry. for the five most frequent mutations, we tested for a correlation of the genotype with the phenotype, mean crp and ethnic origin, respectively. furthermore, we evaluated the applicability of the two severity scores for children. results. among the patients, we detected a total of pyrin mutations and different sequence variants, including one new mutation (p.gly asp). the five most frequent alterations were p.met val ( %, n= ), p.met lle ( %, n= ), p.val ala ( %, n= ), p.glu gln ( %, n= ) and p.met ile ( . %, n= ). ethnic origin could be determined in cases; the prevailing ancestry was turkish ( %, n= ), % (n= ) were lebanese. p.met val in homozygous form ( %, n= ) was correlated with a more severe disease activity, based on the score by mor, as well as with a higher mean crp ( mg/l, n= , mg/l, n= ) compared to patients without this mutation (p= . and p< . , respectively). the score suggested by pras did not yield a significant genotype-phenotype correlation; indeed, the two scoring systems were inconsistent with each other (κ< . ). although a typical distribution of mutations in different ethnic populations was obvious, this trend was not statistically significant, probably due to the divergent number of cases. conclusion. the homozygous p.met val substitution was associated with a more severe disease activity. there was no origin-genotype correlation in this fmf population. the well-known severity scores for children (mor, pras) are inconsistent. the aid-net is working on a new scoring system. . all patients with rp should be investigated by capillaroscopy. capillaroscopy will be classified into "normal", "aspecific changes" or "scleroderma pattern". . all patients who have additional symptoms pointing to a definite connective tissue disease should be evaluated according to disease specific guidelines. . ana-negative and capillaroscopy-negative patients should be followed-up at least every months. . ana positive patients without disease-specific antibodies and with negative capillaroscopy findings should be followed-up at least every months. . ana and disease-specific antibody positive patients should have organ specific evaluation according to symptoms, examination and relevant to that particular disease e.g. patients who are ana and scl- positive may need organ specific evaluation for jssc as per the juvenile systemic sclerosis inception cohort protocol (www.juvenilescleroderma.com). . ana-positive patients, who have no disease specific antibody but have positive capillaroscopy results, should be followed-up at least every months. . ana-negative patients with positive capillaroscopy result should be followed-up at least every months. . the group could not reach an agreement regarding treatment, due to a lack of data for the paediatric age group. the group agreed that implementation of adult recommendations conclusion. the group made a suggestion for a standard of good clinical practice for rp in children. our aim is that this will facilitate a large multicentre prospective follow-up study of children with rp. background. chronic non-bacterial osteomyelitis (cno) is an inflammatory disorder of the skeletal system of unknown etiology. long-term follow-up and response to treatment data have rarely been reported. the aim of the study was to characterize the clinical, radiological, histological and laboratory data at juvenile cno onset, and to analyze the long term treatment response. methods. the course of disease of juvenile patients with non-bacterial inflammatory bone lesions was evaluated retrospectively. clinical, radiological, histological and laboratory data were assessed at disease onset and for a median time of disease of months. results. the mean age at disease onset was . years, the mean time between the first symptoms and the diagnosis of cno was months. % of the patients had multifocal bone lesions. biopsy was performed in patients. only when bone biopsy was taken within months of symptom onset, cellular infiltrates could be observed. at later time points, fibrosis, hyperostosis and bone edema predominated. the initial treatment consisted of non-steroidal anti-inflammatory drugs (nsaids). % of the patients required second line therapy consisting of sulfasalazine and short term oral corticosteroids, % of the patients required bisphosphonates or tnf-blocking agents. the number of clinical lesions decreased to % within . months and reached . % after months of treatment. the number of radiological lesions, however, declined to only . % after months of treatment. in detail analysis of the tre-atment response revealed that initiation of sulfasalazine treatment in nsaid non-responders led to a significant and sustained decline of the clinical, as well as the radiological number of lesions. conclusion. the rapid clinical improvement in cno, following initiation of therapy with nsaids, is not accompanied by a likewise decrease of the number of radiological lesions. treatment with sulfasalazine is effective in childhood cno. background. exercise has a wide variety of beneficial health effects. it stimulates bone formation and maintains bone strength as well as decreases the risk of falls. moreover, exercise at regular intervals is also assumed to positively affect immune functions. conversely, in more than % of the astronauts during/after space flight and under simulated weightlessness immune functions are suppressed. to assess the effects of simulated weightlessness during the nd berlin bedrest study (bbr- ) on immunological parameters. furthermore, to compare the effects of two different exercise performances (resistive vibration exercise and resistance exercise without vibration). methods. physically and mentally healthy male volunteers ( - y) experienced days of six degree head down tilt bed rest. they were randomized to groups: resistive vibration exercise (n= ), resistance exercise without vibration (n= ), inactive controls (n= ). blood samples were taken days before bed rest, on day and after beginning of bed rest. composition of immune cells was analyzed by flow cytometry. cytokines and neuroendocrinologic parameters were analyzed by a multiplex suspension array/ elisa in plasma. general changes over time were identified by paired t-test, exercise-dependent effects by -group repeated measurements anova. results. for all cases pooled, the number of granulocytes (p< . ), nkt cells (p< . ) and hematopoietic stem cells (p< . ) increased during the study; the concentrations of dhea (p< . ) and eotaxin (p< . ) decreased. different impacts of the specific types of exercise on the change over time were shown for lymphocytes, nk cells, nkt cells, tcell subpopulations and the concentrations of ip- and rantes. conclusion. we found immobilization/simulated weightlessness to significantly impact immune cell populations, and cytokine and neuroendocrine factor concentrations. exercise was able to specifically influence immunologic parameters. interestingly, these changes resemble those found during the aging process. background. novel therapies have made remission and low disease activity (lda) achievable goals in ra. we assessed the impact of treatment with subcutaneous abatacept or adalimumab on these goals and on functional and radiographic outcomes in ample (abatacept versus adalimumab comparison in biologic-naïve ra subjects with background methotrexate), the first head-to-head trial of biologics in ra patients with inadequate response to mtx (mtx-ir). methods. ample is a -year, phase iiib, randomized, investigator-blinded study. biologic-naïve ra patients with mtx-ir were randomized to receive mg abatacept weekly or mg adalimumab biweekly, combined with a stable dose of mtx [ ] and radiographic non-progression (defined as change in modified total sharp score ≤ . ) were analysed in patients achieving or not achieving remission or lda at days or . results. baseline clinical characteristics of abatacept and adalimumab treatment groups were balanced, as was clinical, functional and radiographic efficacy and safety at day [ ] . the proportions of patients meeting each of the remission criteria or lda at day were similar for both groups, but significantly more patients achieved das (crp) remission compared to cdai, sdai or rapid remission, and the smallest proportion achieved boolean remission. compared to remission, a higher proportion of patients achieved lda. across all definitions of remission or lda, > % of the patients achieving remission at days and were haq responders at day . more than % of patients achieving remission or lda at days and were radiographic nonprogressors at day . improvement in physical function and radiographic outcomes were consistent between the two treatment groups in both remission and lda populations (. tab. ). conclusion. through year, patients treated with subcutaneous abatacept or adalimumab in ample achieved comparable rates of remission and lda. similar improvements in physical function and radiographic outcomes were observed. these data help to illustrate the relationship between remission, lda and functional and radiographic outcomes independent of treatment with subcutaneous abatacept or adalimumab. background. previous small studies suggest that responses to some immunizations may be attenuated by intravenous abatacept but remain clinically meaningful [ , ] . we investigated the magnitude of response to pneumococcal and influenza vaccination in a larger number of patients receiving subcutaneous (sc) abatacept therapy. the objective of the study was to evaluate the antibody response to the standard -valent pneumococcal polysaccharide vaccine and the - seasonal influenza trivalent vaccine in adult patients with ra on sc abatacept and background dmards. these multicentre, open-label sub-studies of the -valent pneumococcal polysaccharide vaccine and seasonal influenza vaccine enrolled patients in the acquire (pneumococcal and influenza) or attune (pneumococcal) studies. patients were enrolled at any point during their sc abatacept treatment cycle after completion of ≥ months' abatacept treatment. all patients received fixed-dose sc abatacept mg/week with background dmards. a pre-vaccination blood sample was collected and vaccines administered, while continuing background sc abatacept and dmards. after ± days, a post-vacci- background. in real life, dosage increases are common with biologic agents [ ] . intravenous abatacept is administered by patient body weight ( mg/kg) and weeks after the first infusion and every weeks the-reafter [ ] totalling infusions over the first months. no adjustments to this schedule are recommended. abatacept retention rates, efficacy and safety over months in action (abatacept in routine clinical practice) have been reported previously [ , ] this study was designed to assess adherence to recommended dosing of abatacept over the first months in action. methods. action is an ongoing, -year, international, non-interventional, prospective cohort of ra patients treated with intravenous abatacept. all patients on abatacept treatment for ≥ months, and with infusion data available at initiation and at months, were considered in this analysis. good adherence was defined as correct dose by patient body weight and number of actual-to-recommended infusions within the range - % (i.e. - infusions). results. in total, / ( . %) patients received abatacept ≥ months and had the infusion data available. most had established ra and failed ≥ anti-tnf agent ( . %). of patients with body weight data available at initiation, . % received the recommended initial dose, . % a lower dose and . % a higher dose than recommended. good adherence to the abatacept treatment schedule was found in / ( . %) patients. over months, . % of patients received infusions, . % received infusions and . % had infusions. change in dosage over time was assessed in / patients with data available at both time points. the majority of patients ( . %) maintained the recommended dosage. / ( . %) patients received abatacept at the recommended dose for body weight and at the recommended treatment schedule over months. conclusion. in the real-world action study, adherence to the recommended abatacept treatment regimen over months was good. few patients received changes in dose and/or frequency of administration over this time period. background. in rheumatoid arthritis (ra), synovial fibroblasts (sf) secrete large amounts of il- , il- and matrix metalloproteinases (mmps) which are crucial for cartilage destruction. rasfs are sensitive to the action of cannabinoids and they express cannabinoid receptors type i and ii (cb and cb ), the vanilloid receptor (trpv ) as well as endocannabinoid degrading enzymes. cannabinoids are regarded as antiinflammatory and since anandamide (aea) is found in ra synovial fluid we investigated how this endocannabinoid affects adhesion, proliferation, and production of inflammatory mediators of rasf. methods. adhesion was assessed by the xcelligence system. proliferation was quantified by the amount of incorporated fluorescent dye into cellular dna. mmp- and cytokines were detected by elisa. in oasf, aea dose-dependently decreased the il- β induced production of mmp- (by %) in a trpv -mediated manner. il- and il- levels were only weakly modulated. in rasf however, aea decreased il- β induced production of il- ( %), il- ( %) and mmp- ( %). the effects of aea were not inhibited by cb , cb or gpr antagonists but were blocked by the trpv antagonist capsazepine. the inhibitory capacity of aea was enhanced by cyclooxygenase- inhibition in rasfs and oasfs, but was unaltered or even slightly reduced by faah inhibition. aea was even more potent in reducing above mentioned mediators when rasfs but not oasfs were incubated under hypoxic conditions and treated with tnf. furthermore aea increased adhesion of oasfs and rasfs to fibronectin. adhesion was modulated by cb , gpr , and trpv antagonists. combined faah and cyclooxygenase- blocked the stimulatory effect of aea on adhesion. proliferation was decreased by aea in rasfs and oasfs via a cyclooxygenase- but not via cb , cb or trpv dependent mechanism. conclusion. in conclusion, aea promotes an antiinflammatory phenotype of rasfs and oasfs by activating trpv . cb , trpv , and gpr act in concert to modulate adhesion of sfs and this is highly dependent on the intracellular concentration of aea. additionally, cyclooxygenase- metabolites of aea exert their anti-proliferative effects independent of cb and cb . fc. it has been reported that lower levels of czp, compared to ada or ifx, are transferred from treated mothers to the neonate [ ] . this discrepancy may be due to active transport of antibodies across the placenta thought to be mediated by the neonatal fc receptor (fcrn). however, anti-tnf binding to fcrn, and fcrn-mediated transcytosis have not been studied. the objective of this study is to quantify binding of czp, ifx, ada and eta to fcrn and to measure fcrn-mediated transcytosis. a biacore™ assay was used to determine binding of czp, ada and ifx to human fcrn. anti-tnfs were passed over an fcrn-coated chip .mdck-ii cells transfected with human fcrn were used to measure fcrn mediated transcytosis. the anti-tnfs and the control antibody (p ), which possessed a fc modified to prevent binding to fcrn, were biotinylated to allow visualization. the amount of each anti-tnf transcytosed across the cell layer over hours was measured by msd assay. results. ifx ( nm) and ada ( nm) had high binding affinity to fcrn while the binding affinity of eta to fcrn was - -fold lower ( nm). in contrast, czp did not bind to the fcrn with any measurable affinity. the levels of transcytosis seen with ifx and ada were . ng/ml and . ng/ml, respectively (mean of experiments). transcytosis of eta ( . ng/ml) was lower than that of ada and ifx. in contrast, the level of czp transcytosis was significantly lower, at . ng/ml, than that observed with the other anti-tnfs and comparable to the control p ( . ng/ml). conclusion. czp didn't bind to fcrn and thus no fcrn-mediated czp transcytosis was detected. in contrast, ada and ifx had a relatively high binding affinity to fcrn and were actively transcytosed. eta showed lower binding affinity and transcytosis, but fcrn-mediated transport could still be measured. these results explain the previously observed active transport of anti-tnfs across the placenta seen in patients treated with ifx and ada, whereas only low levels were observed with czp [ ]. background. anti-cyclic citrullinated peptide (ccp) status was reported previously as predictive of abatacept response [ ] . predictors of retention with abatacept have not been published previously. this study was designed to identify predictors of abatacept retention after failing ≥ biologic agent. in routine clinical practice) is an ongoing, -year, international, non-interventional, prospective cohort including patients with ra treated with intravenous abatacept [ , ] . patients from canada, germany, greece and italy, where patient numbers were sufficient to explore between-country effects, were included. at data cut-off (february ), all patients had -year follow-up (interim analysis). abatacept discontinuations were reported by the investigator at any time point during follow-up. socio-demographics, disease characteristics and medical history at abatacept initiation, and previous and concomitant treatments were deemed potential predictive variables. clinically relevant variables and those with p≤ . (univariate analysis) were entered into a multivariate cox proportional-hazards regression model, adjusted for clustered data from one investigator. using backwards selection, variables with p≤ . were retained in the final model. . there were no interactions or effects of c-reactive protein level, rheumatoid factor status, type of previous anti-tnf failure, infection at initiation and abatacept monotherapy. sensitivity analysis, including all variables significant in univariate analysis, was consistent. conclusion. in this first report of real-world predictors of abatacept patient retention, anti-ccp positivity and failing < prior anti-tnf agents were associated with higher retention. differences in retention between some countries may reflect specificities in healthcare systems and populations. abatacept, a biologic agent with no contraindications or special warnings for cardiac comorbidity, seems to be a good option for these patients. weekly subcutaneous abatacept confers comparable onset of treatment response and magnitude of efficacy improvement over months when administered with or without an intravenous abatacept loading dose zeitschrift für rheumatologie suppl · | methods. patients from the intent-to-treat populations of the acqui-re [ ] and ample [ ] studies randomized to subcutaneous abatacept plus mtx were included. all patients received fixed-dose subcutaneous abatacept mg/week; in acquire but not ample, patients also received an intravenous loading dose (~ mg/kg based on weight range) on day . for this post-hoc analysis, assessments included acr and haq-di response (improvement ≥ . ) over months, with patients who discontinued considered non-responders. mean changes from baseline over months in das (crp) were assessed in patients with das > . at baseline (last observation carried forward) to account for differences in baseline disease activity between the two studies. results. all patients were biologic naïve at baseline, with mean disease duration of . and . years, das (crp) . and . , and haq-di . and . in acquire and ample, respectively. efficacy was compared throughout the study. for patients treated with subcutaneous abatacept with and without an intravenous loading dose, acr response rates were similar (. tab. ). haq-di response rates were also similar with and without the intravenous loading dose (. tab. ). for the overall populations, mean (standard deviation [sd]) changes from baseline to day in das were − . ( . ) and − . ( . ) in acquire and ample, respectively. for patients with baseline das > . , mean (sd) changes in das from baseline to day were − . ( . ) and − . ( . ) in acquire and ample, respectively. conclusion. time to onset and magnitude of acr and haq-di responses and das improvements were generally similar with subcutaneous abatacept with or without intravenous loading in patients with ra and an inadequate response to mtx. the findings from this posthoc analysis suggest that subcutaneous abatacept can be given effectively without an intravenous abatacept loading dose. background. ra is associated with pain and impairment of physical function, significantly impacting a patient's health-related quality of life (hrqol) and ability to perform daily activities. patient-reported outcomes (pros) related to hrqol and daily activity have become an essential part of assessment in ra. we continue to report here comparative findings from pros assessed with subcutaneous abatacept or adalimumab on background mtx in the first head-to-head study, ample. we compared changes in pros at year in patients with ra treated with abatacept or adalimumab, both on background mtx. methods. ample is a phase iiib, randomized, investigator-blinded study of months' duration. biologic-naïve patients with active ra and inadequate response to mtx were randomized to either mg abatacept weekly or mg adalimumab biweekly in combination with mtx. pros evaluated through day included: hrqol, assessed using short form- (sf- ; including physical and mental component summary subscores [pcs and mcs]); activity limitation over the previous days, using the activity limitation questionnaire (alq; [ ] ); productivity, using the work productivity and activity impairment questionnaire for ra [ ] ; physical and psychosocial independence, captured using items from haq, sf- score; and alq [ ] . other pros previously reported from ample include: patient pain, patient global assessment, fatigue, and physical function [ ] . all efficacy analyses were done using the intent-to-treat population, which included all patients who were randomized and received at least one dose of study drug. baseline characteristics were analysed descriptively and changes in pros from baseline were assessed using ancova. results. baseline demographic and clinical characteristics of the abatacept and adalimumab treatment arms were similar. improvements in all domains of the sf- , including pcs and mcs observed at day parameter baseline woche woche itt-gesamt das , mw ± sd , ± , (n= ) , ± , (n= ) , ± , (n= ) vas da pat., mw ± sd , ± , (n= ) , ± , (n= ) , ± , (n= ) sjc , mw ± sd , ± , (n= ) , ± , (n= ) , ± , (n= ) vas schmerz, mw ± sd zielsetzung der ole-studie beinhaltete die beurteilung der verträglichkeit und der wirksamkeit von czp. die retentionsraten sowie die wirksamkeit wurden bis woche und die verträglichkeitsdaten bis woche beobachtet. in die verträglichkeitsanalyse wurden alle pat einbezogen, die in die ole-studie eintraten und czp erhielten (n= ; n= kombitherapie; n= monotherapie), einschließlich der plazebo/czp-patienten, die die ausgangsstudien erfolgreich abgeschlossen/abgebrochen haben. bezüglich der wirksamkeit wurden folgende analysen vorgenommen: ) czp pat, die die ausgangsstudien erfolgreich beendet haben und zu irgendeinem zeitpunkt während der ausgangsstudien oder ole-studie andere dmards eingenommen haben (n= ; kombitherapie completer); ) czp pat, die die fast ward studie erfolgreich beendet haben und zu keinem zeitpunkt andere dmards eingenommen haben (n= ; monotherapie completer). ergebnisse. verteilung und häufigkeit der unerwünschten ereignisse (ue), einschließlich der reaktionen an der injektionsstelle (ereignisse/ patientenjahre: monotherapie , , kombitherapie , ) und der schwerwiegenden unerwünschten ereignisse (sue) waren mit dem vergleichbar, was bisher für czp berichtet wurde (. tab. ) . das auftreten von schwerwiegenden infektionen (si) und malignitätsraten war niedrig. es wurden todesfälle berichtet: kardiovaskuläre ereignis-se, infektionen, unfall und tumorerkrankung. die retentionsraten der pat, die die ausgangsstudien erfolgreich beendet haben, waren zur w in der czp monotherapie-( / , %) und der czp kombitherapie-gruppe ( / ; %) vergleichbar. der durchschnittliche das - (crp)-wert und dessen abweichung vom baseline-wert der ausgangsstudien zum zeitpunkt des eintrittes in die ole-studie und nach w der monotherapie-completer und der kombitherapie-completer, sowie die zugehörigen haq-werte sind in . tab. dargestellt. schlussfolgerung. vorliegende ole-studie konnte das günstige risiko-nutzen-profil der czp-monotherapie bestätigen. die langzeitwirksamkeitsdaten zeigten keine unterschiede zwischen pat, die czp als monotherapie erhielten und pat, die czp in kombination mit anderen dmards erhielten. background. rheumatoid arthritis (ra) is the most common disease of joints that non-or deficiently treated leads to functional loss and premature cardiovascular death within years. but nearly % of the ra patients fail to treatment with tnfα-inhibitor (tnfi) indicating a switch to rituximab (rtx). the urgency of personalized promising treatment in time presupposes predictive parameter. rheumatoid factor (rf) and anti-citrullinated protein antibodies (acpas; especially accp) are shown to be better diagnostic than less theranostic biomarkers. in that context we investigated the role of antibody subtypes against mutated citrullinated vimentin (amcv) that determine response outcome in rtx-treatment. methods. a cohort of only amcv igg positive ra patients was tested for amcv subtype igm and iga (additionally for rf igg, igm, iga and accp igg) by elisa at baseline (after failure to first approach with tnfi) and at week (after first rtx cycle). responders were characterized by a difference in their das of ≥ . (eular good-response) between baseline and week . the cohort comprises responders (rr) and non-responders to rtx (nrr). results. amcv igg, igm and iga showed higher treatment related decreases compared to rf and accp ig subtypes and additionally even diverged in both groups depending on response outcome: especially amcv iga exhibited a higher mean titer decline of rr by % at lo- wer baseline titers ( . to . u/ml) and a mean titer increase of nrr by nearly % at higher baseline titers ( . to . u/ml). at baseline rr displayed relatively more negative iga titers ( %; n= / ) than nrr, who in return showed more iga positive titers ( %; n= / ). amcv iga positive patients were more likely to show positively for rf iga ( %) and igm ( %), what could be inversely detected for iga negative patients with seronegativity of rf iga ( %) and igm ( %). conclusion. amcv immunoglobulin subtypes showed treatment dependent changes contrary to already known antibodies (accp). especially amcv iga reflects response outcome: amcv iga negativity at baseline and decreasing titers during treatment are predictive for good eular-response to rtx. einleitung. im rahmen der abklärung eines unter tocilizumab-therapie aufgetretenen arzneimittelexanthems erfolgte die bestimmung von c c und c -beide parameter waren erniedrigt. bei recht geringer und nur kurz andauernder ausprägung des exanthems wurde die therapie komplikationslos fortgeführt. die komplementfaktoren wurden im verlauf bestimmt und blieben erniedrigt. im weiteren verlauf erfolgte die konsekutive messung bei weiteren patienten. methoden. nephelometrische bestimmung von c c und c im serum vor und während der therapie mit tocilizumab (jeweils vor der nach wochen anstehenden infusion) bei patienten mit gesicherter rheumatoider arthritis (rf+, ccp+). ergebnisse. c c-und c -komplement wurden bei konsekutiven patienten mit rheumatoider arthritis vor und unter tocilizumab-therapie bestimmt. bei allen patienten fielen sowohl c c, als auch c unter der therapie mit tocilizumab ( mg/kg kg) ab. / patienten hatten eine c c-erniedrigung (bestimmter wert unterhalb des laborinternen normbereichs). / patienten hatten eine c -erniedrigung (bestimmter wert unterhalb des laborinternen normbereichs). drei patientinnen entwickelten unter der therapie ein exanthem, davon hatten eine komplementerniedrigung. keine "offensichtlich" erhöhte infektneigung in abhängigkeit von komplementspiegeln. bei verlängerten infusionsintervallen aufgrund von infekten zeigte sich, dass der effekt von tocilizumab auf die komplementspiegel reversibel ist. durch blockade des il- -rezeptors tocilizumab kann ein erworbener komplementmangel induziert werden. Ähnliche daten wurden im rahmen einer pilotstudie an sle-patienten erhoben, die mit tocilizumab behandelt wurden. der effekt ist bei der rheumatoiden arthritis nicht vorbeschrieben. der genaue umfang des komplementmangels ist bisher nicht untersucht (andere bestandteile der kaskade?) wurde in der sle-studie ausführlicher untersucht. da die verschiedenen komplementbestandteile erniedrigt waren wurde auf eine synthesestörung und nicht auf einen gesteigerten verbrauch geschlossen, was auch in dieser kohorte der fall zu sein scheint. der erworbene komplementmangel könnte einen teil der infektiösen komplikationen unter der therapie erklären. eine korrelation ist aber aufgrund der geringen fallzahl nicht möglich. schlussfolgerung. anhand dieser studie konnte eine exzellente korrelation zwischen den parametern der dxr und des bx verifiziert werden. mittels der neu entwickelten voll digitalisierten bx-technik ist somit eine quantifizierung der periartikulären demineralisation möglich und als surrogatparameter der radiologischen progression bei einer ra eingesetzt werden. jahre, die ra bestand im median seit , jahren. , % der patienten waren mit tnf-alpha-blockern vortherapiert, , % ausschließlich mit dmards. der mittlere das lag zur baseline bei , . zur woche zeigten , % der patienten eine das remission (< , ) und , % bzw. , % der patienten ein gutes bzw. moderates ansprechen gemäß eular-kriterien. Über den beobachtungszeitraum stieg der anteil der tcz-monotherapiepatienten von , % auf , %. die mtx-komedikation sank im gleichen zeitraum um , %. , % der patienten, die tcz zunächst zusammen mit einem dmard erhalten haben, konnten dieses absetzen. tcz zeigte in der mono-und kombinationstherapie eine vergleichbare wirksamkeit: , % bzw. , % der patienten erreichten eine cdai remission (≤ , ). der anteil von patienten ohne glucocorticoid(gc)-begleittherapie stieg über den beobachtungszeitraum um , % auf , % an, der anteil mit einer tagesdosis ≤ mg auf , %. bei , % war eine reduktion der gc-dosis möglich, nur bei , % war eine erhöhung notwendig. bei , % der patienten, die zur bl mit gc behandelt wurden, konnten diese komplett abgesetzt werden. die mittlere gc-tagesdosis verringerte sich kontinuierlich von , (bl) auf , mg/d (w ). schlussfolgerung. diese interimsanalyse der nichtinterventionellen studie ichiban zeigt bei den ersten patienten mit mittelschwerer bis schwerer ra über die bisherige beobachtungsdauer von wochen deutliche verbesserungen der aktivitätsparameter, sowie eine reduktionen der begleitenden dmard-therapien und des bedarfs von glucocorticoiden unter behandlung mit tcz. vergleichbar mit den kontrollierten studien ist die tcz-monotherapie auch unter praxisbedingungen der kombination mit dmards ebenbürtig. diese anhaltende wirksamkeit wird erstmals in rheumatologischen praxisdaten für den langzeitverlauf von , jahren gezeigt. zeitschrift für rheumatologie suppl · | einleitung. die arteriosklerose (as) steht als häufigste todesursache im besonderen fokus der medizinischen forschung. neuere erkenntnisse weisen auf einen starken zusammenhang zwischen parametern der systemischen entzündung und der pathogenese der as hin. patienten mit rheumatoider arthritis (ra) haben daher ein stark erhöhtes kardiovaskuläres risiko. ziel: untersuchung des zusammenhangs zwischen verschiedenen ra-krankheitsspezifischen risikofaktoren und dem auftreten einer arteriosklerose bei ra-patienten. methoden. ra-patienten, davon % weiblich, ± , jahre alt, wurden hinsichtlich der krankheitsaktivität (krankheitsdauer , ± , ; das , ± , ; serum-crp , ± , mg/dl,; anti-ccp-antikörper , ± , u/ml, radiologisches stadium , ± , ; davon , % mit erosionen), sowie klassischer kardiovaskulärer risikofaktoren der as erfasst, welche durch den score-wert (systematic coronary risk evaluation) zusammengefasst wurden. zur as darstellung wurde eine carotis-duplexsonographie mittels eines mhz-schallkopfes (ge vivid pro) durchgeführt. die mittlere intima-media-dicke (imd) der a. carotis communis wurde durch ein softwaregestütztes messverfahren ermittelt. ergebnisse. plaques waren bei patienten ( %) nachweisbar. diese korrelierten mit einer erosiven form der ra (p= , ), einer längeren krankheitsdauer (p= , ) und höheren anti-ccp-antikörpern (p= , ). die mittlere imd betrug , ± , mm. je ausgeprägter die radiologischen veränderungen sind, umso höher war die wahrscheinlichkeit der plagues (p= , ). mittels altersadjustierter partieller korrelationsanalyse wurde der das als altersunabhängiger einflussfaktor auf die imd ermittelt (p= , ). mittel-und hochgradige stenosen zeigten sich bei fünf ra-patienten ( , %), welche ausnahmslos eine erosive verlaufsform aufwiesen. normalbefunde stehen in zusammenhang mit einem crp-wert unter mg/dl (p= , ). auch die traditionellen kardiovaskulären risikofaktoren haben signifikanten einfluss auf as. der score-wert erwies sich als äußerst verlässlicher prädiktor für plaques (p< , ), imd-verdickung (p= , ) und stenosen (p< , ). durch elimination der traditionellen risikofaktoren mittels partieller score-adjustierter korrelationsanalyse bestätigte sich erneut die assoziation von pathologischen ultraschallbefunden mit dem das (p= , ). schlussfolgerung. die erhebung klassischer risikofaktoren bei ra-patienten ist unerlässlich. die nutzung des score-werts als screening-parameter ist besonders effektiv. zusätzlich sollten parameter der krankheitsaktivität von ra zum management von arteriosklerose herangezogen werden. besonders aussagekräftig hierfür sind der das , ein erosiver krankheitsverlauf, die crp-werte und die erkrankungsdauer background. apremilast, an oral small molecule specific inhibitor of phosphodiesterase- , works intracellularly to modulate inflammatory mediators. the palace - trials compared the efficacy and safety of apremilast vs placebo in patients with active psa despite prior dmards and/or biologics. the overall safety and tolerability of apremilast was assessed in a pooled analysis of the palace , and placebo-controlled phases. methods. safety data was pooled from phase , randomized, placebo-controlled studies; patients with active psa despite prior dmards and/or biologics were randomized : : to placebo, apremilast mg bid (apr ), or apremilast mg bid (apr ) stratified by baseline dmard use. at week , patients with < % reduction in swollen and tender joint counts were required to be re-randomized (early escape) to apr or apr (placebo group) or remained on initial apremilast dose through week . stable concurrent dmard therapy was allowed (mtx, sulfasalazine, leflunomide, or combination). the analysis comprises data from the placebo-controlled periods (weeks - ). results. patients were randomized to placebo (n= ), apr (n= ), or apr (n= ) and included in the safety population. baseline demographic and disease characteristics and prior/concurrent therapy were comparable across treatment groups; . % had prior biologic exposure. adverse events (aes) occurred in . % (placebo), . % (apr ), and . % (apr ) of patients. aes occurring in ≥ % of any treatment group were diarrhea, nausea, headache, and urti (. tab. ); most occurred within the first weeks of treatment and nearly half resolved within weeks. of patients with these aes, most ( - %) were mild or moderate. rates of discontinuation due to aes were low: . % (placebo), . % (apr ), and . % (apr ). serious aes occurred in . % (placebo), . % (apr ), and . % (apr ) of patients. one death occurred (apr ) due to multiorgan failure not suspected to be treatment-related. no cases of serious systemic opportunistic infections, lymphoma, vasculitis, or reactivation/de novo tb were reported. there were no clinically meaningful differences between apremilast and placebo in terms of major cardiovascular aes, changes in blood pressure, malignancies, or effects on laboratory measurements. conclusion. apremilast was generally well-tolerated with no new safety concerns identified compared with the known profile. the aim of the current study was to investigate the relationship between worsening of functional status, clinical disease parameters and radiographic spinal progression over two years in patients with early axial spondyloarthritis (axspa). methods. in total, patients with early axspa ( with as and symptom duration ≤ years, and with non-radiographic axspa (nr-ax-spa) and symptom duration ≤ years) from the german spondyloarthritis inception cohort (gespic) were included in the current analysis based on the availability of radiographic data and data on the functional status at baseline and after years of follow-up. spinal radiographs were scored according to the modified stoke ankylosing spondylitis spinal score (msasss). functional status was assessed by the bath ankylosing spondylitis functional index (basfi), and clinical disease activity by the bath ankylosing spondylitis disease activity index (basdai). results. basfi worsening in ≥ point after years (n= , . %) was significantly associated only with higher basdai worsening over years in comparison to those without functional worsening: . ± . vs - . ± . , p< . . basfi worsening by ≥ points (n= , %) was, however, associated not only with basdai change ( . ± . vs - . ± . , p< . ), but also with a higher rate of radiographic spinal progression measured by the proportion of patients with msasss worsening by ≥ units ( . % vs. . % in patients without basfi worsening, p= . ), or with new syndesmophyte formation ( . % vs. . %, p= . ). importantly, in the multivariate analysis both basdai increase and progression of structural damage in the spine remained statistically significantly associated with basfi worsening. no other disease-related parameters (e.g. sex, hla-b positivity, symptom duration etc) were found to be significantly associated with basfi worsening over two years. conclusion. in this prospective study we could demonstrate that only factors were significantly associated with worse functional outcome over two years in patients with early axspa: ) increase of disease activity and ) progression of structural damage. elevated serum vascular endothelial growth factor is highly predictive for radiographic spinal progression in patients with axial spondyloarthritis who are at high risk for progression background. vascular endothelial growth factor (vegf) is an essential mediator of the endochondral ossification and, therefore, might play a pathogenetic role in the process of syndesmophyte formation in axial spondyloarthritis (axspa). the aim of the study was to investigate the role of serum vegf as a predictor of radiographic spinal progression in patients with axspa. methods. altogether patients with definite axspa [ with ankylosing spondylitis (as) and with non-radiographic axspa] from the german spondyloarthritis inception cohort (gespic) were included in the current study. radiographic spinal progression was defined as ) worsening of the modified stoke ankylosing spondylitis spine score (msasss) by ≥ units after years, and ) development of a new syndesmophyte or progression of existing syndesmophytes after years. serum vegf levels were detected at baseline. results. mean baseline vegf values were significantly higher in patients with msasss worsening by ≥ units after years (n= ) as compared to those without progression ( ± vs. ± pg/ml, respectively, p= . ) and in patients with syndesmophyte formation/ progression (n= ) as compared again to those without progression ( ± vs. ± pg/ml, respectively, p= . ). area under the curve (auc) was . , p= . for the msasss worsening ≥ units and . , p= . for syndesmophyte formation/progression. importantly, the performance of vegf as a predictor of radiographic spinal progression was clearly in patients who were already at high risk for such a progression due to the presence of syndesmophytes at baseline (n= ): auc was . , p= . , and . , p= . , respectively. vegf serum level of > pg/ml in high-risk patients had a sensitivity of %, a specificity of %, and an odds ratio (or)= . ( %ci . - . ) as a predictor of msasss worsening by ≥ units over years. the same serum level of results. immediately after the second session of plasmapheresis, therapy with infliximab mg/kg resumed. after weeks of hospitalization with repeated administration of infliximab had good dynamics (bas-dai . , asdas (crp) = . , basfi . ), significantly reduced pain in the joints and spine, stiffness, increased mobility in the joints and spine. the treatment continued: holding plasmapheresis followed by infliximab. after infusions patient experienced a good effect -basdai . , asdas (crp)= . , basfi . . conclusion. plasmapheresis in some patients could be effective by reducing activity and dealing with secondary tnf inhibitors failure, since this procedure deletes the macromolecular blood proteins, including tnf-, igg antibodies, and circulating immune complexes results. there were still signs of osteitis in sacroiliac joints in patients at week , in patient the mri-determined sacroiliitis has resolved completely. the patient has improved clinically and fulfilled asas improvement criteria. there was a minor decrease in sparcc sacroiliitis score (from to ) in patients at week , indicating reduction of inflammation in sacroiliac joints. sparcc sacroiliitis score stayed the same in the remained patient. conclusion. rituximab may be of some benefit in decreasing mri-evident sacroiliitis in patients with highly active as, even in patients in whom tnf-α inhibitors have failed. background. despite the differences in the pathogenesis of ra and as, neck pain is a frequent clinical symptom in both diseases. we evaluated the correlation between subjective reports of neck pain and objective signs of inflammation as assessed by f bone marrow edema (bme) on mri in ra and as patients. methods. stir-mri of the cervical spine of patients ( ra, as) were included. mris were scored by two blinded readers using a recently published mri scoring system, with quantification of the extension of bme in the atlantoaxial region, corpus, facet joints and processus spinosus of all cervical vertebrae, ranging from - points. the presence or absence of degenerative changes was also recorded. conclusion. the majority of patients with ra and as had objective signs of bme but also degenerative changes on mri at different cervical locations. assessment of bme in the atlantoaxial region is important in clinical practice, in addition to degenerative changes, since its presence seems to influence the intensity of neck pain reported by these patients. x. baraliakos , having mri data at w . of these , ten were treated with secukinumab and with placebo in the core study. mris were rescored for this study. asspimri-a scores and the occurrence of vertebral edges (ve) inflammatory and fatty lesions were evaluated by an independent blinded reader. results. all pts completed this exploratory mri substudy. in pts receiving × mg/kg secukinumab followed by × mg/kg (n= ) secukinumab, spinal inflammation was reduced compared to bl at w -similar to the results of the core study -and this reduction was sustained up to w (abb. ). also in pts who had initially received placebo switching to secukinumab at w , mri inflammation at w was reduced. of the ves evaluated, the proportion of ves with inflammatory lesions was reduced from . % (n= ) at bl to . % (n= ) at w and . % (n= ) at w . in contrast, the proportion of fatty lesions at bl ( . %, n= ) remained largely unchanged at w ( . %, n= ) and w ( . %, n= ). secukinumab reduced mri inflammation at w and w . conclusion. mri analysis suggests that the il- a inhibitor secukinumab can reduce spinal inflammation and this effect may be sustained for up to years. unlike reports with tnf blockers, secukinumab appeared to leave the proportion of fatty lesions unchanged. the potential impact of these preliminary findings on radiographic progression under secukinumab therapy will be studied in larger trials. schlussfolgerung. es zeigen sich keine signifikanten unterschiede in der krankheitsaktivität der beiden gruppen (vor einleitung der ada-therapie nach dmard-und nach anti-tnf-versagen). auch bei der patientengruppe mit mehreren vortherapien mit tnf-inhibitoren können keine signifikanten unterschiede in der ausprägung der erkrankung nachgewiesen werden. im trend wurde ein früheres einsetzen der haut-und gelenkmanifestation sowie eine stärkere systemische entzündungsreaktion in den patienten mit vorheriger tnf-therapie festgestellt werden, während die dauer der erkrankung und der bmi mit den charakteristika der patienten mit ausschließlicher dmard-vortherapie vergleichbar sind. long-term ( -week) results of a phase , randomized, controlled trial of apremilast, an oral phosphodiesterase inhibitor, in patients with psoriatic arthritis (palace ) background. apremilast, an oral phosphodiesterase inhibitor, works intracellulary to modulate a network of pro-and anti-inflammatory mediators. the palace study assessed the efficacy and safety of apremilast in patients with active psoriatic arthritis (psa) despite prior dmards and/or biologics. methods. patients were randomized : : to placebo, apremilast mg bid (apr ), or apremilast mg bid (apr ). at week , patients with < % reduction from baseline in swollen/tender joint counts were required to be re-randomized (early escape) to apr or apr (placebo group), or remained on their initial apremilast dose. at week , all remaining placebo patients were re-randomized to apr or apr through week . results. patients were randomized. at week , significantly more apr ( . %; p= . ) and apr patients ( . %; p< . ) achieved an acr vs placebo ( . %). at week , all patients had a minimum weeks of apremilast exposure. response to apremilast was generally maintained over the treatment period. at week , acr was achieved by . % (apr ) and . % (apr ) of patients (table) . exposure-adjusted incidence rates for adverse events (aes), severe aes, and serious aes were comparable between - and - weeks. the proportion of patients remaining on apremilast to week who first reported the most common gi disturbances (e.g., diarrhea, nausea, and vomiting) after week was low (ranging from . - % for apr and - . % for apr ). there were no clinically meaningful laboratory findings with exposure up to weeks. no deaths beyond the previously reported in the - week period were observed in the - week period. no safety signals with respect to major cardiac events, malignancies, and opportunistic infections were observed, consistent with the - week period. no cases of lymphoma, tuberculosis, or tuberculosis reactivations were reported for the -week period (. tab. ). conclusion. apremilast administered to patients with psa beyond weeks continued to demonstrate meaningful clinical response. for patients who completed weeks of the study, acr response rates up to % were observed. apremilast continued to be well tolerated with an acceptable longer-term safety profile. methods. to identify how conventional cd + and cd + t cells and regulatory t cells are recruited into the inflamed kidneys in ln, serum and urine samples of sle patients were analyzed for chemokines using multiplex assays. based on the assay's results a group of corresponding chemokine receptors (ccr - , cxcr and cxcr ) was chosen, whose frequencies on urinary t cells were subsequently determined in patients with acute ln by flowcytometry. results. chemokines (ccl , ccl , ccl , ccl , ccl , ccl , ccl , ccl , cxcl , cxcl , cxcl and cx cl ) were significantly elevated in the urine of patients with active ln when compared to the control group. the other chemokines (ccl , ccl , ccl , cxcl , cxcl ) and cxcl showed no significant differences between the groups. ccr and cxcr were the most prominent receptors on both urinary cd + and cd + t cells, although cd + t cells also expressed high amounts of ccr and ccr . however, when compared to t cells in the blood, urinary cd + t cells showed significantly higher expression of all examined chemokine receptors but ccr while urinary cd + t cells only had higher expression of ccr and ccr . the chemokine receptor expression on cd +foxp +cd -regulatory t cells (treg) differed from conventional cd + t cells as well. treg expressed significantly more ccr and significantly less cxcr . conclusion. ccr and cxcr are the primary receptors in the mechanism of recruiting t cells into the inflamed kidney. key chemokines are ccl , ccl , ccl and ccl as well as cxcl and cxcl . however, at least for cd + t cells, there are secondary pathways of recruitment involving ccr /ccl and ccr /ccl . also, treg recruitment seems to rely more on ccr than that of conventional cd + t cells. methods. observe is a multicenter, retrospective medical chart review study. rheumatologists from german academic and non academic centers who treat > sle patients annually and have > years of practice experience were randomly recruited. physicians identified consecutively all their adult sle patients who had received belimumab as part of usual-care. index date was the first belimumab infusion date. the primary outcome was the change in overall sle disease manifestations months after index date based on physician judgment. the overall response rates as well as reasons for early treatment discontinuation within months were assessed. changes in formal disease area indices, e.g. selena-sledai if available and changes in oral steroid dose are also reported. results. previous analyses from us patients treated with belimumab have described significant clinical improvement across relevant organ systems based on clinical judgment and formal disease activity indices and marked reductions in corticosteroid use in patients that received at least infusions of belimumab. the current study is the first description of patient characteristics and outcomes after months of therapy with belimumab outside of the us. it is also the first time overall responder rates and reasons for discontinuation with belimumab have been described in a real world setting. the study provides insights into the effectiveness and safety of belimumab in an ex-us clinical setting. larger, prospective observational studies are needed to confirm the results. commercial support grant disclosure: research funded glaxosmithkline. background. toll-like receptor (tlr- ) signaling is considered to play an important role in b cell hyperreactivity in sle. b cells from slepatients express significantly more tlr- than those from healthy donors (hd), especially if patients have positive dsdna-antibodies and high disease activity. tlr- stimulation of b cells is tightly linked to their differentiation into plasma blasts and memory cells. the objective of this study was to analyze in a comprehensive manner the effect of tlr- signaling on cytokine production by b cells from sle-patients, in comparison to b cells from hd, and in relation with disease activity. methods. b cells from sle-patients and hd were stimulated in vitro using cpg for hours, and culture supernatants were then tested for cytokines and chemokines (bio-plex). the cytokine responses were compared between both groups. in addition, within sle patients, the patterns of cytokines produced by b cells were compared with indices of disease activity. results. cpg-stimulation significantly increased cytokine production ( out of parameters; p< . ) compared to baseline. striking increases were found for il- ra ( ± pg/ml), il- ( ± pg/ml), il ( ± pg/ml) and ip- ( ± pg/ml; p< . ). there was no significant difference between both groups. remarkably, production of il- , il- , il- , il- p , il , il- , il- a, eotaxin, basic fgf, g-csf, gm-csf, ifn-γ, ip- , mip- α, and vegf correlated inversely with the sledai (p< . ) and even more (additionally il- β, il- ra, mip- β and tnf-α) with anti-dsdna antibody titers. the frequency of cd + memory b cells showed a positive correlation between the production of ip- and tnf-α in sle, whereas the levels of il- β, il- , mip- α, and mip- β showed a positive correlation with cd + b cells in hd. conclusion. the current data indicate hitherto unknown perturbations of cytokine/chemokine production by b cells in active sle. the inverse correlation of cytokines/chemokines produced by b cells from sle patients with sledai and anti-dsdna titer suggests that the known enhanced b cell proliferation and differentiation upon tlr -stimulation possibly diminishes cytokine production. background. several cytokines, including ifn-γ, il- , il- , and il- have been implicated in the pathophysiology of autoimmune disease. il- , a potent inducer of ifn-γ, enhances th responses that are thought to be synergistic and dependent on il- . we tested the hypothesis that intra-renal il- mediates kidney and systemic disease in mrl-faslpr mice. methods. by constructing il- p /il- -/-mrl-faslpr mice and using an ex-vivo gene transfer to deliver il- intra-renally, we determined that il- , independent of il- and/or il- , incites kidney disease in mrl-faslpr mice. moreover, we provide the novel finding that local intra-renal il- mediates systemic disease (lung pathology, systemic auto-abs). results. thus, our data indicate that il- is a potential therapeutic target for immune mediated kidney and systemic disease in mrl-faslpr mice. using a caspase- inhibitor, that inhibits the release of active il- and il- β, we successfully treated kidney (improved renal function, pathology) and systemic disease (skin lesions, lymphadenopathy, and splenomegaly) in mrl-fas lpr mice, while administration of an il- receptor antagonist did not influence disease progression. probing further we found that inhibition of il- activation results in an amelioration of lupusnephritis by a reduction of intra-renal infiltrating leukocytes (macrophages and t cells) and reduced activation of these leukocyte populations. moreover, caspase- inhibition resulted in decreased inf-y and il- production, indicating an altered balance of th and th cell responses in this model. conclusion. taken together, our findings indicate that il- , independent of il- β, il- and/or il- , is the major mediator of kidney and systemic disease mrl-faslpr mice. therefore, caspase- inhibition is a potential therapeutic target for autoimmune disease in the mrl-faslpr mice. background. in the treatment of giant cell arteritis (gca) glucocorticoid-related adverse effects occur frequently, particularly in patients with relapsing disease. a -year-old woman presented with a month history of fever, chills, arthralgias and cephalgias and markedly elevated serum inflammatory markers. whereas further evaluation including ultrasound of the temporal arteries was unremarkable, a positron emission tomography-computed tomography (pet-ct) demonstrated an intense fluorodeoxyglucose uptake of the aorta, the subclavian, carotid and femoral arteries. gca was diagnosed and treatment with high dose prednisone was begun. results. because of disease flares at prednisone dosages below mg/ day and the occurrence of vertebral fractures, cyclophosphamide and methotrexate (mtx) were added as glucocorticoid-sparing agents. as these treatments had to be stopped because of intolerance and mtxpneumonitis, respectively, we started tcz infusions ( mg/kg body weight). the clinical status rapidly improved. after infusions of tcz follow-up pet scan showed resolution of the previously seen uptake and we were able to taper the daily dose of prednisone to mg. treatment was well tolerated. however, the patient developed mild hyperthyroidism with a rapid rise of the initially normal levels of anti-thyroid peroxidase and anti-thyroid antibodies, anti-tsh receptor antibodies remained normal. thyroid function normalized and the antibody-levels fell without further treatment in the following months. in conclusion, this case demonstrates the successful treatment of a patient with relapsing giant cell arteritis with tcz. for the first time, we report the occurrence of a transient autoimmune thyreoiditis possibly induced by tcz. klinik für pädiatrie mit schwerpunkt pneumologie und immunologie, sektion rheumatologie, berlin, vestische kinder-und jugendklinik der universität witten/herdecke deutsches zentrum für kinder-und jugendrheumatologie organización médica de investigación arthr care res ar&t in press sp division of rheumatology diagnosesicherung: a + b) mr-morphologisch myositistypische veränderungen (os) histologie + c) generalisierte myalgien und laborchemisch dtl. elevierter ck, sowie positivem nachweis von ana und jo- -ak, pulmonales ct mit diffusen milchglasinfiltraten, in bronchoalveolärer lavage neutrophile alveolitis. ergebnisse. vormedikationen: a) glukocorticoidmonotherapie, mtx-monotherapie, mtx in kombination mit etanercept, cyclophosphamidboli, und zuletzt intravenöse immunglobuline (ivig) in kombination mit mycophenolatmofetil . b) mtx-monotherapie, mtx in kombination mit glukokortikoiden, cyclophosphamidboli, intermittierend intravenöse immunglobuline, cyclophosphamid per os (fau-ci). c) cyclophosphamidboli jeweils gutes ansprechen des ck-wertes auf jeweilige rituximabgaben mit ebenfalls ansprechen des klinischen bildes mit guter regredienz des aus myalgien resultierenden schmerzniveaus. im fall a keine beatmung mehr notwendig. im fall von c) auch gute regredienz subjektiver dyspnoesymptomatik und besserung wichtiger lungenfunktionsparameter, regredienz ctmorphologischer milchglasinfiltrate, im verlauf fehlender nachweis neutrophilie in bal. weitere rituximabgaben bei a, b und c im verlauf zum remissionserhalt nach jeweiligem klinischem befund production of cytokines by b cells in response to tlr stimulation inversely correlates with disease activity in sle-patients berlin zeitschrift für rheumatologie suppl · | das muskuloskeletale system, eines der am häufigsten betroffenen organsysteme bei sle (bei - % der sle-patienten). das ziel dieser analyse war es um diejenigen parameter zu identifizieren, die zu diesem effekt beigetragen hatten, wurde jeder der einzel-parameter zur untersuchung und symptom-erfassung innerhalb des muskuloskeletalen bilag-organsystems analysiert. die post-hoc-analyse umfasste nur patienten, bei denen ein parameter zu studienbeginn als vorhanden gewertet wurde, und jeder parameter erforderte ≥ patienten-beobachtungen pro kohorte um einen vergleich zu erstellen dadurch wurde die zahl der patienten mit einer initialen beteiligung des muskuloskeletalen systems aufgedeckt, die eine in woche auflösung der manifestation aufwiesen auch im selena-sledai-score war die rate der verbesserung bei dem arthritis-parameter in der belimumab-gruppe mit mg/ kg ( , %; n= ) und mg/kg ( , %; n= ) signifikant höher als die daten weisen darauf hin, dass mg/kg belimumab effektiv auf muskuloskeletale organmanifestationen sind akzeptiert als posterbeitrag auf dem eular klinische forschergruppe für rheumatologie (kfr), freiburg i. br., universitätsklinikum ulm, klinik für dermatologie und allergologie die physikalische therapie (pt) ist ein wesentlicher bestandteil der medizinischen versorgung von ssc-patienten patientenregister des dnss erfasst prospektiv, jährlich klinische verlaufsdaten zur organbeteiligung und therapie von patienten mit systemischer sklerodermie. die mittels freitext erfassten angaben zur verordneten pt wurden ausgewertet hivamat n= ( , %) und hylase n= ( , %) anwendung. die anzahl der verfahren, die die patienten zeitgleich erhielten, variierte zwischen mind. und max. . Über % der patienten erhielten anwendungen gleichzeitig. insgesamt wurden therapiearten genannt. , % der patienten mit gelenkkontrakturen zeigten nach einem jahr physikalischer therapie eine signifikante verbesserung der symptomatik (p= , ) gegenüber den patienten die keine physikalische therapie erhielten. nach drei jahren waren es , % der patienten (p= , ). bei den patienten mit muskelschwäche zeigten % der patienten eine signifikante symptomverbesserung (p= , ) dieser studie kann erstmals gezeigt werden, dass pt-symptome wie gelenkkontrakturen und muskelschwäche bei ssc-patienten signifikant verbessern kann. dennoch erhält weniger als die hälfte der ssc-patienten eine physikalische therapie punkten zur kontrolle einer mmf-therapie in der klinischen praxis zu untersuchen bei patienten ( -mal sle, je -mal systemische sklerose, sharp-syndrom und primäres sjögren-syndrom) die mmf erhielten, wurde , und min nach einnahme von mmf die mpa-konzentrationen im serum per hplc bestimmt. die mpa-auc wurde durch die mathematische methode der bayes %) und in der standarddosis von g/tag bei von patienten ( %) eine mpa-auc von > µg.h/ml. bei zwei patienten wurde nach der messung die dosis adjustiert: eine patientin mit einem sle mit diffus-proliferativer lupusnephritis hatte trotz einer mmf-dosis von g/tag nur eine mpa-auc von , µg.h/ ml. die dosis wurde daraufhin auf g/tag erhöht. der mpa-auc stieg danach auf max. , µg.h/ml und die krankheitsaktivität nahm ab (sledai von auf , proteinurie von auf mg/ h und prednisondosis von auf mg/tag) pharmakokinetic study of mycophenolate mofetil in patients with systemic lupus erythematosus and design of bayesian estimator using limited sympling strategies mycophenolic acid area under the curve correlates with disease activity in pupus patients treated with mycophenolate mofetil colony stimulating factor- (csf- ) -neuer aktivitätsmarker der lupusnephritis? brigham and wome's hospital, boston, renal division the authors would like to thank pfizer for supporting the study. furthermore the authors would like to thank the "deutsche kinder-rheumastiftung". einleitung. bei patienten mit früher axialer spondyloarthritis (spa) mit einer krankheitsdauer von< jahren und nachweis von akut-entzündlichen veränderungen in der ganzkörper-magnetresonanztomographie (mrt) in der wirbelsäule und/oder den sakroiliakalgelenken (sig) zu baseline [ ] untersuchten wir die langzeit-effektivität über vier jahre. methoden. in der esther-studie wurden patienten mit etanercept (eta, n= ) vs. sulfasalazin (n= ) behandelt [ ] . ab dem zweiten studienjahr wurden alle patienten mit eta behandelt (einige patienten unterbrachen zwischenzeitlich die therapie (n= ) zur untersuchung der biologika-freien remission und wurden dann (erneut) mit eta behandelt) [ ] . klinische, laborchemische und mrt-daten der patienten, die zu den jeweiligen studienzeitpunkten vorhanden waren, wurden im vierten studienjahr analysiert (as-observed-analyse). ergebnisse. von patienten, die zu baseline eingeschlossen wurden, erreichten , % das ende von jahr (n= ). in der gesamtgruppe zeigte sich ein gutes bis sehr gutes ansprechen, wobei etwa % eine asas partielle remission und etwa - % eine asdas inaktive erkrankung erreichten (. tab. ). der anteil der patienten mit normalem crp ("crp-remission") stieg von , % zu screening auf , % zu woche , während der anteil der patienten mit negativem mrt ("mrt-remission" definiert als fehlen akut-entzündlicher veränderungen in den sig und der wirbelsäule gemäß beider scorer) auf von % auf , % anstieg. , % der patienten zu woche waren sowohl in asas-remission, im status einer asdas inaktiven erkrankung als auch in mrt-remission. das ansprechen nach vier jahren war sehr ähnlich in den gruppen unabhängig davon, ob im ersten jahr sulfasalazin gegen wurde oder die therapie im jahr unterbrochen worden war (ergebnisse werden nicht gezeigt).schlussfolgerung. es zeigte sich ein konstantes und anhaltendes ansprechen bei patienten mit früher axialer spa, die mit etanercept behandelt wurden. das ansprechen scheint besser zu als bei patienten mit etablierter ankylosierender spondylitsi mit einer langen krankheitsdauer (> jahren; [ ] ). einleitung. in einer -wöchigen placebokontrollierten studie mit -wöchiger offener verlängerung bei patienten mit aktiver nichtröntgenologischer axialer spondyloarthritis (nr-axspa) wies adalimumab eine gute effektivität auf [ ] . bei patienten, bei denen es zum wiederauftreten der krankheitsaktivität nach absetzen des medikaments in woche kam, wurde die therapie wiederbegonnen ziel der studie war es, die langzeiteffektivität nach wiederaufnahme der therapie von adalimumab nach stopp über jahre zu evaluieren. methoden. bei ursprünglich in die studie eingeschlossenen patienten wurde die therapie nach wochen beendet und patienten ( % männlich, mittleres alter jahre, range - , mittlere krankheitsdauer vor therapiebeginn jahre, range - , % positiv für hla-b ) hatten, definiert durch erreichen eines % ansprechens gemäß der assessments in spondyloarthritis society-kriterien (asas ), gut auf die therapie angesprochen. bei wiederauftreten von krankheitsaktivität (definiert durch nicht mehr erreichen von asas ) wurde adalimumab mg alle wochen über jahre (woche r ) weitergeführt. die asas kriterien und der bath ankylosing spondylitis disease activity index (basdai) wurden in form einer completer-analyse berechnet. ergebnisse. der patienten mussten wiederbehandelt werden: / ( %) erreichten jahr , / ( %) erreichten jahr und / ( %) der patienten erreichten jahr der wiederbehandlung. nach jahren wiederbehandlung mit adalimumab erreichten / ( %) wieder asas und / ( %) erreichten partielle remission gemäß der asas-kriterien. nach jahren erreichten / ( %) und nach jahren / ( %) asas . asas partielle remission wurde nach jahren von / ( %) und nach jahren von / ( %) patienten erreicht. in der completer analyse fiel der mittlere basdai von , ± , zum zeitpunkt der wiederbehandlung auf , ± , im jahr (p< , ), , ± , (p= , ) im jahr und auf , ± , (p= , ) im jahr der wiederbehandlung ab. schlussfolgerung. in dieser gruppe von patienten mit aktiver nr-axspa, die ein gutes therapieansprechen über wochen mit adalimumab erreicht hatten und die bei wiederauftreten von krankheitsaktivität nach stopp der therapie in woche weiterbehandelt werden mussten, sprach die mehrheit der patienten, die in der studie verblieben, gut und anhaltend auf das fortsetzen der therapie an. tab. | sp- langzeit-effektivität über jahre etanercept-therapie bei patienten mit früher axialer spondyloarthritis. daten zu baseline (bl), jahr (w ), jahr (w ), jahr (w ) und jahr (w ). daten background. secukinumab (ain ) is a new fully human monoclonal antibody (mab) targeting il- a for the treatment of inflammatory diseases. administration of mabs can be associated with immunogenicity via the induction of anti-drug antibodies (adas). adas can lead to unwanted clinical consequences, such as loss of exposure, loss of efficacy due to altered pharmacokinetics and/or functional neutralization and, in the worst case, anaphylactic reaction and immune complex diseases. the assessment of ada formation is therefore a critical component in the assessment of biotherapeutic safety. methods. the immunogenicity assessment strategy for secukinumab follows a three-tiered approach. first, samples are analyzed for presence of ada in a screening assay which takes a % false-positive rate into account. in a second step, screening assay positive samples are tested in a confirmatory assay that identifies true positive responses. finally, true immunogenicity-positive samples are quasi-quantified via titration. a biacore-based assay was used during the early stages of the secukinumab program, and an msd-based bridging assay was applied during the later stages of the program. in addition, pharmacokinetics and clinical efficacy as well as safety data are also evaluated. samples to assess immunogenicity were obtained from individual subjects encompassing clinical studies in different indications during treatment and during follow-up. dosing regimens included single doses such as mg subcutaneously in psoriasis patients as well as multiple × mg/kg doses intravenously in ms patients over a six-month period.results. none of the subjects tested for immunogenicity developed sustained adas. in total, subjects met the definition of treatment-related, transient positive immunogenicity showing low ada titers. none of these subjects had evidence of loss of efficacy, deviating pk behavior or reported anaphylactic reaction or immune complex disease.conclusion. based on the available data, secukinumab appears to carry a low risk of immunogenicity. in the very few transient immunogenicitypositive patients identified so far, there has been no indication of altered pharmacokinetics or loss of efficacy, and no adverse event that could be linked to immunogenicity has been detected. more data from the ongoing phase studies are required to strengthen this encouraging finding in a larger patient population. risikofaktoren für eine aa-amyloidose bei entzündlich-rheumatischen erkrankungen und bei der idiopathischen aa-amyloidose methods. we report a case of an -year-old woman suffering from ulceration and signs of infection of the ulnar aspect of the right forearm due to subcutaneous calcification in association with crest syndrome.results. this case presents an unusual case of extensive subcutaneous calcification in crest syndrome requiring surgical excision due to secondary ulceration, inflammation and infection. while a surgical approach has already been described for calcification in different connective tissue diseases, only scant data of massive subcutaneous calcification related to a forearm in crest syndrome followed by surgical excision exist. conclusion. in crest syndrome, extensive subcutaneous calcification related to the forearm can occur. surgical excision followed by primary wound closure can lead to an excellent postoperative result. background. the whole blood interferon signature (wbifns) is measured in several clinical trials studying inhibitors of interferon alpha (ifn-α) in sle, but failed repeatedly -in contrast to the less sensitive ifnα -to reflect longitudinal changes in lupus activity and to guide dosage finding of rontalizumab. therefore, better ifn biomarkers reflecting disease activity over time and individual response to the inhibition of ifnα are needed to optimize the risk-benefit ratio of ifn-inhibitors. here, we show that the highly sensitive monocyte restricted ifnα response protein siglec- , also known as sialoadhesin or cd , is a useful biomarker to monitor longitudinal changes in disease activity of sle patients. methods. ifn-α and siglec- were measured by delfia and flow cytometry, respectively, in accurately characterized lupus patients over a period of up to months (overall visits). changes of biomarker and changes of disease activity (bilag ) were correlated using spearman rank test (srt). disease courses of selected sle patients were plotted to demonstrate in detail the relations of ifn-biomarkers with disease activity, sle medication and clinical manifestations. background. a -year-old woman was admitted because of sudden attack of convulsion and somnolence situation with positive canca and myeloperoxidase antibodies. cerebral magnetic resonance imaging (mri) showed thickening and marked progression of the dura-meningeal enhancement and edematous changes at pre and post central gyrus left side. based on these findings, it was diagnosed as hypertrophic cranial pachymeningitis related to anca-associated vascultis as unusual presentation. there was only temporarily und partial responce to a -month therapy with cyclophosphamide mg i.v and oral glucocorticosteroids . taking into consideration the severe, life-threatening course of the disease in the case of our patient, the decision was made to use rituximab, a chimeric, monoclonal igg antibody directed against cd , leads to destruction of b cells via complement mediated lysis and antibody dependent cellular cytotoxicity. the first administration of the medication was performed according to the pattern for rheumatoid arthritis patients treated with rituximab, i.e. infusions for mg in -day intervals in combined therapy with glucocorticosteroids. a follow-up mri at months after start with rituximab showed significant regression of the meningeal pathology at temporo-occipatel aspects (pachymeningitis) and completely resolution of edematous changes at pre and post central gyres. the complete clinical remission was achieved by introducing rituximab. conclusion. rituximab seems to be successful therapie for the induction and maintenance of remission in patients with anca-associated vasculitis (aav) with cns involvement (hypertrophic cranial pachymeningitis ) , who had previously failed to respond to standard treatment with cyclophosphamide and steroids and a range of alternative treatments [ , ] . antikörperdiagnostik. mit prednisolon-therapie ( mg/kgkg, mg/ tag) und zusätzlich methotrexat mg wöchentlich war keine anhaltende normalisierung der entzündungsserologie zu erzielen. infliximab ( mg/kg) -wöchentlich i.v. erbrachte nur kurzzeitig eine normalisierung der entzündungswerte, dann trotz weiterer infusionen einen erneuten anstieg der bsg bis auf mm, crp mg/l. nach umstieg auf tocilizumab ( mg / mg/kgkg) alle wochen konnte nach wochen eine bislang anhaltende normalisierung der entzündungsserologie erzielt werden (crp , mg/l, bsg mm . std.). der allgemeinzustand der patientin besserte sich deutlich, der hb-wert normalisierte sich auf , mmol/l. in der kontrastverstärkten sonographie fand sich ein abfall in der kontrastmittelaufnahme der a. carotis communis. die maximale intima-media-dicke reduzierte sich bislang auf , mm. schlussfolgerung. die bisherige standardtherapie der takayasu-arteriitis mit prednisolon und mtx führte auch im vorliegenden fall nicht zur remission. für infliximab fanden wir ein frühzeitiges therapieversagen des sonst erfolgreich beschriebenen ansatzes einer tnf-α-blockade bei riesenzellarteriitis. dennoch gelang mit tocilizumab eine bislang über monate andauernde klinische, sonomorphologische und serologische remissionsinduktion bei monatlicher fortführung der il- -blockierenden therapie. background. cd is the prototypic nk receptor that is also expressed on a unique population of effector cd + cells. these cd -expressing t cells are expanded in rheumatoid arthritis patients and had features of senescent cells. nkg d is another nk receptor over expressed on effector cd + cells in aav patients. cd + as well as nkg d + t cells seem to be involved in tissue injury as they are capable of mediating tcr-independent immune activation. it is hypothesized that il- is able to up regulate the expression of nk cell receptors. interleukin- (il- ) is a proinflammatory cytokine that is over expressed in aav and is linked to the expansion of cd + effector memory t cells (tem). in aav in remission a persistent expansion of these cd + effector memory t cells has been observed. in the present study we assessed the expression cd on cd + t cells of aav and if expression of these molecules was influenced by il- . methods. the distribution of cd + tem and the proportion of cd +cd + t cells and nkg d+ cd + t cells were analysed in aav-patients and hcs by facs. in vitro effects of il- on the expansion of cd + tem and up regulation of cytotoxic markers were assessed in the same way. in addition il- serum levels were measured in patients and hc by elisa. results. we observed an increased proportion of circulating cd +cd + t cells in aav as well as nkg d+ cd + t cells in patients in remission compared to hc ( . vs . p< . and vs . p< . ). % to % of these cells were cd + effector memory t cells. the percentages of the cd +cd + t cells and nkg d+ cd + t cells were constant over time. we also observed elevated il- serum levels in patients in remission compared to hc (p= . ). in vitro stimulation of pbmcs with il- increased not only the proportion of cd + memory cells (cd ro+) but also the expression of cd and nkg d on these cells. conclusion. the driving force behind the persistent expansion of a cytotoxic subset of cd + effector memory t cells expressing cd and nkg d+ and being tcr -independent is likely the increased il- expression in aav patients . ergebnisse. unabhängig von regime der remissionsinduktion und der primären erhaltungstherapie lag am ende der nachbeobachtungsperiode bei % der patienten eine renale remission vor ( %; % pr). % hatten eine persistierende proteinurie von > , g/tag bei stabiler nierenfunktion, % eine persistierende niereninsuffizienz mit erhöhtem kreatinin bei inaktivem sle, bei % wurden eine persistierende aktive ln und/oder renale rezidive beobachtet. vier patienten verstarben. patienten mit langzeit-cr waren gekennzeichnet durch einen niedrigeren tubulointerstitiellen chronizitätsindex in der initialen nierenbiopsie ( , ± , vs. , ± , ; p= , ), eine hochsignifikant geringere proteinurie nach cyc-pulsen ( , ± , vs. , ± , g/tag; p= , ) und niedrigere dsdna-ak ( ± vs. ± u/ml; p< , ) zum zeitpunkt des beginns der erhaltungstherapie. eine proteinurie von < , g/tag nach pulsen cyc zeigte eine sensitivität von % und eine spezifität von % für eine langzeit-cr. schlussfolgerung. eine proteinurie von < . g/tag nach remissionsinduktion mit pulsen cyc sowie ein geringer tubulointerstitieller chronizitätsindex in der nierenbiopsie sind prädiktoren einer anhaltenden kompletten renalen remission bei ln. background. to evaluate and compare clinical efficacy of three biomarkers for interferon activity (measured directly and indirectly) and six traditional biomarkers to indicate current disease activity in sle. methods. ifn-α (delfia), ip- (elisa) and siglec- (flow cytometry) was measured in accurately characterized lupus patients and compared to serum titres of anti-dsdna (elisa and ria), anti-dsdna-ncx elisa, anti-nuc elisa, c and c . disease activity was evaluated using bilag- and a modified sledai- (msle-dai- k). additionally, clinically quiescent patients were monitored for flares over the course of days. results. increased levels of ifn-α, ip- and siglec- were found in %, % and % of active sle patients. ifnα (r= . ; p< . ) and siglec- (r= . ; p< . ) correlated better with bilag- than ip- (r= . ; p= . ), farr assay (r= . ; p= . ), anti-dsdna-ncx elisa (r= . ; p= . ), anti-dsdna elisa (r= . ; p= . ), anti-nuc elisa (r= . ; p= . ), c (r=- . ; p< . ) and c (r=− . ; p= . ). predictors of sle flares were disease duration ≤ months, mild clinical activity (in contrast to no activity), complement c ≤ mg/dl and ifn-α ≥ pg/ml, while only lymphocyte count and age were independent predictors in multivariate analysis. conclusion. ifn-α, ip- and siglec- emerged as beneficial biomarkers for disease activity in lupus patients. therefore, implementation of ifn biomarkers in standard lupus diagnostics should be reappraised, especially in view of emerging anti-ifn-directed therapies. . ) and carried significantly more often other antibodies ( . %; p< . ), which were separated into u rnp-( . %), ro-( . %), pmscl-( . %) antibodies, followed by . % with rheumatoid factors, . % with la-, . % with dsdna-and . % with jo- -and . % with ku-antibodies. the kaplan-meier analysis of the onset of organ involvement revealed a clear inclined position of overlap patients between patients suffering from lcssc and dcssc, especially regarding lung fibrosis and heart involvement. patients suffering from pah, oesophagus involvement and kidney involvement, overlap and lcssc patients showed nearly similar curve progression (log rank < . ). furthermore musculoskeletal involvement was significantly more frequent and more progressive in patients with overlap disease, followed by patients with dcssc and lcssc (log rank < . ). conclusion. these data support the current concept, that ssc-overlap syndromes should be regarded as a separate ssc subset, distinct from lcssc and dcssc, due to a different course of the disease, different proportional distribution of specific autoantibodies and skin/organ involvement. methoden. patienten mit gpa ( mit aktiver und mit in remission befindlicher gpa) wurden durchflusszytometrisch analysiert und mit gesunden verglichen. eine färbung für cd , cd , cd , igd, iga, cd , mhcii, wurde mittels flowjo-software analysiert. die statistische auswertung erfolgte mit "graph pad prism" und p-werte< , wurden als signifikant angesehen. die studie wurde von der ethik-kommission der charité genehmigt. ergebnisse. deutliche unterschiede (p= , ) wurden sowohl für die absolute zahl als auch die frequenz der plasmazellen im peripheren blut der patienten mit gpa mit krankheitsaktivität ( , ± , /µl) im vergleich zu denen mit einem bvas von ( , ± , /µl) oder gesunden ( , ± , /µl) gefunden, ähnlich wie bei sle. bei patienten mit gpa ist außerdem eine signifikante erhöhte anzahl der plasmazellen igapositiv (p= , ). die anzahl der plasmazellen sowie die frequenz der plasmazellen an den b-zellen im blut korrelieren mit dem bvas (r= , ; p< , ). interessanterweise zeigte sich keine expansion der doppelt negativen memory-zellen, die zum beispiel beim sle beschrieben ist. für die naiven b-zellen fand sich ebenfalls ein signifikanter unterschied zwischen patienten mit aktiver erkrankung im vergleich zu gesunden. bei den t-zellen fanden sich nur diskrete veränderungen. schlussfolgerung. die anzahl der plasmazellen ist bei patienten mit aktiver gpa deutlich erhöht, was eine rolle von plasmazell-vermittelten mechanismen in der pathogenese nahelegt. ein großteil dieser plasmazellen ist iga-positiv, diese könnten eine rolle bei der hno-beteiligung spielen. key: cord- -pnw ytj authors: corredor, juan c; redding, nicole; bloté, karen; robbins, stephen m; senger, donna l; bell, john c; beaudry, paul title: n-myc expression enhances the oncolytic effects of vesicular stomatitis virus in human neuroblastoma cells date: - - journal: mol ther oncolytics doi: . /mto. . sha: doc_id: cord_uid: pnw ytj n-myc oncogene amplification is associated but not present in all cases of high-risk neuroblastoma (nb). since oncogene expression could often modulate sensitivity to oncolytic viruses, we wanted to examine if n-myc expression status would determine virotherapy efficacy to high-risk nb. we showed that induction of exogenous n-myc in a non-n-myc-amplified cell line background (tet- n) increased susceptibility to oncolytic vesicular stomatitis virus (mutant vsvΔm ) and alleviated the type i ifn-induced antiviral state. cells with basal n-myc, on the other hand, were less susceptible to virus-induced oncolysis and established a robust ifn-mediated antiviral state. the same effects were also observed in nb cell lines with and without n-myc amplification. microarray analysis showed that n-myc overexpression in tet- n cells downregulated ifn-stimulated genes (isgs) with known antiviral functions. furthermore, virus infection caused significant changes in global gene expression in tet- n cells overexpressing n-myc. such changes involved isgs with various functions. therefore, the present study showed that augmented susceptibility to vsvΔm by n-myc at least involves downregulation of isgs with antiviral functions and alleviation of the ifn-stimulated antiviral state. our studies suggest the potential utility of n-myc amplification/overexpression as a predictive biomarker of virotherapy response for high-risk nb using ifn-sensitive oncolytic viruses. neuroblastoma (nb) is the most common cancer in the first years of life, and the most common solid tumor of childhood. patients are risk-stratified using a combination of clinical, pathological, and molecular characteristics. the survival of patients with high-risk disease has not improved and remains less than %. historically, standard therapy for high-risk disease includes chemotherapy, surgery, radiation, and bone marrow transplant, which appear to provide some control of disease progression, but is complicated by significant morbidity and mortality. , innovative approaches such as gd- antibody-mediated immune therapy have demonstrated the first improvements in survival for high-risk nb patients in over two decades, though mechanisms limiting its efficacy still occur. therefore, novel approaches to this disease are necessary. viral oncolysis is a novel approach to nb that has shown promise in various preclinical cancer models. , despite their promise as therapeutics, oncolytic viruses (ovs) face application hurdles due to our incomplete understanding of the role of the tumor microenviroment and antiviral immune responses on virotherapy. in general, ovs can selectively kill tumor cells while leaving normal cells intact. they achieve this by exploiting the same cellular defects that promote tumor growth. one of such defects is the type i interferon (ifn) signaling, which sensitizes tumor cells to ifn-sensitive ovs such as vesicular stomatitis virus (vsv) and newcastle disease virus. [ ] [ ] [ ] in this study, we used vsv based on its known efficacy as a potent oncolytic agent to several tumor types. [ ] [ ] [ ] the deletion of a single amino acid of the m-protein (vsvΔm ) increases safety by restricting its infection to cancer cells with defects in type i ifn response. , however, tumors with functional type i ifn signaling can hamper its clinical application. n-myc amplification, although not present in all cases, is the best-characterized aberrant genetic alteration associated with poor prognosis in high-risk nb. the mechanisms whereby myc proteins (c-myc, n-myc and l-myc) sensitize cancer cells to ovs remain unexplored. previous studies have shown that some c-myc-amplified cancer cell lines are highly susceptible to vsv-induced cell killing. though not studied in the context of oncolytic virotherapy, c-myc negatively regulates type i ifn signaling through stat- , which is one of the mechanisms of pathogenesis in burkitt's lymphoma and uveal melanoma. , since oncogenic expression often correlates with increased susceptibility of cancer cells to ovs [ ] [ ] [ ] and the effects of n-myc on virotherapy are unknown, we reasoned that n-myc overexpression, due to amplification, could be a clinically important biomarker of virotherapy efficacy to high-risk nb. we showed that n-myc-amplified nb cell lines and a non-n-myc-amplified cell line (tet- n) induced since vsv infection induces the expression of type i ifns, we hypothesized that n-myc-driven regulation of these cytokines promoted differential susceptibility of all nb cells to virus-induced cell killing. as positive controls for these assays, cells were treated with polyinosinic-polycytidylic acid (poly i:c) for hours. poly i:c is a strong inducer of type i ifns when delivered intracellularly (transfection) or extracellularly (in cell culture medium). intracellular and extracellular poly i:c mainly activate the retinoic acid-inducible gene (rig-i) and toll-like receptor- (tlr- ) signaling, respectively, for the expression of type i ifns. cells were either infected at moi of . or treated with poly i:c (intracellular and extracellular) and cells and cell culture supernatants were harvested at indicated time points. secreted ifn-β was detected and quantified by elisa while rt-pcr was used to detect the transcription of ifns-α and -β. detection and quantitation of secreted ifn-β was performed at , , and hpi. this cytokine was best detected at hpi and thus our analysis was focused at this time point (figure a) . ifn-β levels increased in the tet- cells in response to infection, except for tet- c (dox -). however, the increased levels of this cytokine did not differ significantly to basal levels (p > . ), except for, surprisingly, tet- c (dox +). ifn-β levels increased upon stimulation with poly i:c, either intracellular or extracellular ( figure b ). interestingly, the increased levels of this cytokine in tet- n (high n-myc) did not significantly differ to basal levels (p > . ). in contrast, endogenous levels of ifn-β significantly increased in tet- n (low n-myc) and tet- c (dox -/+) upon poly i:c treatment (p < . ). ifn-β levels in virus-infected and poly i:c-treated tet- n (high n-myc) did not differ significantly (p > . ). in contrast, levels of this cytokine in poly i:c-treated tet- n (low n-myc) and tet- c (dox -/+) were significantly higher than levels observed in virus-infected cells (p < . ). these observations collectively suggest that, regardless of n-myc expression status, virus infection does not seem to induce enough levels of ifn-β to stimulate the antiviral state in cells, while n-myc seems to have negative effects in the expression of this cytokine when stimulated with poly i:c. at the transcriptional level, significant increases of ifn-β was observed upon infection and intracellular poly i:c. interestingly, ifn-β transcript levels were lower in tet- n (high n-myc) than those in tet- n (low n-myc) (supplementary figure s a ). transcription of ifn-α was detected upon infection and stimulation with extracellular poly i:c in tet- n (high and low n-myc) but not in tet- c cells (dox-/+). transcription of type i ifns were also detected in all nb cells upon infection and treatment with poly i:c but their levels were cell-type specific regardless of n-myc amplification status (supplementary figure s b) . type i ifn signaling through jak/stat ifn-α/β secreted by virus infected-or poly i:c-treated cells interacts with the ifn (α, β, and ε) receptor (ifnar ) to activate the jak/ stat pathway. activation of this pathway results in phosphorylation of stat- and stat- at tyrosine and , which allows them to heterodimerize and translocate into the nucleus to associate with irf- and form the isgf- complex. isgf- activates promoters for the expression of several ifn-stimulated genes (isgs). the collective contribution of isgs renders cells in the antiviral state. we therefore studied the ifn signaling through jak/stat by analyzing the expression of the isgf- complex and activation of stats and by phosphorylation. we analyzed the expression of rig-i as readout for the induction of the jak/stat signaling. cells were infected at moi of . and harvested at indicated time points before cytopathic effects were apparent, and components of the isgf- complex were analyzed by western blot. as controls for these assays, cells were treated with exogenous ifn-β ( u/ml) and extracellular poly i:c. intracellular poly i:c was not included in the analysis due to its poor induction of the jak/stat signaling in tet- cells, based on stat- and rig-i expression (supplementary figure s ) . basal levels of stats and and their phosphorylated forms, before infection, were similar between tet- n (high n-myc) and tet- n (low n-myc). levels of irf- , on the other hand, were slightly lower in tet- n (high n-myc) (figure c ). virus infections in tet- cells did not induce significant changes in the levels of the isgf- figure effects of n-myc overexpression on vsvΔm spread and oncolysis. human-derived neuroblastoma (nb) cell lines consisting of n-myc-amplified cells (imr- , irm- and lan- ) and non-n-myc-amplified cells (sk-n-sh, sk-n-as, and sh-ep) were seeded in mm cell culture dishes. the number of cells for each cell line was calculated to achieve - % confluency and the multiplicity of infection (moi) ( . ) was calculated accordingly. after infection with vsvΔm (moi of . ) for hour at rt, cells were washed five times with serum-free medium followed by incubation in dulbecco's modified eagle's medium containing % fetal bovine serum. aliquots of culture supernatants were collected at indicated time points for virus titration in bhk cells and generation of multi-step growth curves. virus production was normalized and titers were expressed as numbers of pfu/ × cells (y axis) (a). to examine the virus-induced killing rates, the analyzed cells were seeded in -well plates and infected at moi of . . viability was determined by alamar blue at indicated time points (b). before infection, tet- cells were seeded in mm cell culture dishes and treated or not with μg/ml doxycycline (dox) for hours. subsequently, cells were infected with vsvΔm (moi of . ) and cell culture supernatants were collected at indicated time points for virus titration in bhk cells and generation of multi-step growth curves. virus titers were expressed as numbers of pfu/ × cells (y axis) (c,d). to study the differential cell killing kinetics, tet- n and tet- c cells were seeded in -well plates, treated or not with doxycycline for hours and infected with vsvΔm at mois of . (e,f). viability was determined with alamar blue at indicated time points. error bars indicate mean ± standard deviation of the mean from three replicates. dox (-), no doxycycline added. dox (+), doxycycline added. members. levels of phospho-stat- (y ) slightly increased upon infection in tet- n (low and high n-myc) at hpi. however, at and hpi, its levels slightly increased and decreased in tet- n (high n-myc) and tet- n (low n-myc), respectively. these observations suggest that vsvΔm does not induce robust changes in the jak/stat signaling in tet- cells, probably due to insufficient levels of ifn-β expression. treatments with ifn-β or extracellular poly i:c upregulated the expression of irf- , rig-i and stat- and significantly increased the levels of phospho-stat- (y and s ) and stat- (y ) in tet- cells regardless of n-myc expression status ( figure c) . furthermore, the level of these proteins were similar between cells with low and high n-myc. relative to tet- n (low n-myc) and tet- c (dox-/+), rig-i levels were slightly lower in tet- n (high n-myc) after treatment with ifn-β or poly i:c (figure c ). these observations suggest that the jak/stat signaling is functional regardless of n-myc expression status. consistent to the observations in tet- cells, virus infection did not cause significant changes in the expression of stats and and their phosphorylated forms in the analyzed nb cells (figure d ). levels of irf- slightly increased in imr- (n-myc-amplified) and sh-ep (non-n-myc-amplified) in response to infection. treatment of all cells with ifn-β upregulated the expression of isgf- members and rig-i, though their expression and phosphorylation status of stats and varied among cells (figure d ). consistent to our observations in the tet- system, the upregulation of these isgs by ifn-β suggests a functional jak/stat signaling regardless of cell type and n-myc amplification/overexpression status. effects of n-myc on the establishment of the antiviral state since the jak/stat signaling is functional in all cells, we hypothesized that treatment with ifn-β prior to virus infection would induce the antiviral state in all nb cells. cells were either treated or not with poly i:c and ifn-β ( u/ml) for and hours, respectively, followed by virus infection (moi of ) at indicated time points. as expected, infection of control cells resulted in cell death. interestingly, treatment of tet- n (high n-myc) with exogenous ifn-β induced a weak antiviral state insufficient to protect cells from virus-induced cell killing during the course of the experiment (figure a ). in contrast, tet- n (low n-myc) and tet- c (dox -/+) established a robust antiviral state that fully protected cells from virus-induced cell killing (figure a to further investigate the negative effects of n-myc expression on the establishment of the antiviral state, we studied the antiviral activity of conditioned media from poly i:c-treated tet- cells. we also included conditioned media from tet- cells infected with vsvΔm at hpi, which was uv treated for minutes to inactivate virus. sk-n-as cells (non-n-myc-amplified) were used as indicators due to their ability to establish a robust antiviral state as demonstrated in this work and by others. since extracellular poly i:c figure effects of n-myc on the establishment of the antiviral state. tet- n and tet- c cells were either pretreated or not with μg/ml doxycycline (dox +/-) and incubated for hours. cells were then treated with either u/ml ifn-β (a,b) or extracellular poly i:c (c,d) for and hours, respectively, followed by virus infection at multiplicity of infection (moi) of . viability was determined with alamar blue at indicated time points. nb cell lines were transfected with . ng (in a -well plate format) or treated with μg/ml poly i:c for hours. alternatively, cells were treated with exogenous ifn-β ( u/ml) for hours. cells were subsequently infected with vsvΔm at moi of (e,f). viability was determined with alamar blue. error bars correspond to mean ± standard deviation of the mean from three replicates. dox (-), no doxycycline added. dox (+), doxycycline added. microarray analysis and identification of isgs since n-myc levels correlated to the differential susceptibility to virus-induced cell killing, we then sought to perform microarray assays in tet- n cells to study the n-myc-driven global changes in gene expression in response to infection. for these assays, we used vsvΔm -gfp to monitor virus infection based on gfp expression. upon optimization, we found that infections at moi of . for hours were the most suitable for microarray analysis for the following reasons: (i) earlier and later time points showed limited infection to tet- n (low n-myc) and extensive cytopathic effects in tet- n (high n-myc), respectively; (ii) infections, based on gfp expression, reached between - % of cells with limited cytopathic effects in tet- n (high n-myc) (not shown); and (iii) infections with rna viruses at high mois are known to interfere with the microarray analysis. comparisons were made as follows: group , tet- n (low n-myc, infected/uninfected); group , tet- n (high n-myc, infected/uninfected); and group , tet- n controls (high n-myc/low n-myc). figure illustrates the comparison groups and the numbers of identified probe sets (numbers in black). a total of and probe sets were identified in tet- n (low n-myc) and tet- n (high n-myc), respectively, in response to infection. these data suggest that the higher number of changes in tet- n (high n-myc) in response to infection seems to correlate to the increased susceptibility to virus-induced cell killing. some of the identified probe sets corresponded to isgs, which were identified by the interferome database v . several isgs are known for their antiviral functions and to be induced by ifn-dependent or independent mechanisms in response to virus infection. we therefore focused our analysis on these genes. a total of , , and probe sets for isgs were identified exclusively in groups , , and , respectively. , , and isgs were common between groups and , and , and and , respectively. interestingly, most identified isgs in all groups were downregulated ( figure ). a total of out of isgs ( %) were downregulated when n-myc was overexpressed (figure ) . some of these genes had known antiviral functions such as il- , aim , ifnar , stat- , irf- , and ifitm ( table ) . as shown in figure c , basal irf- was slightly lower in tet- n (high n-myc) than tet- n (low n-myc), which is consistent with the microarray data. other isgs that are negative regulators of ifn signaling with potential proviral functions were also found downregulated such as nmi and ifi- (table ). these observations suggest that n-myc-driven downregulation of isgs with antiviral functions may further sensitize cells to virus-induced cell killing and contribute to the alleviation of the ifn-induced antiviral state. n-myc-independent regulation of isgs upon infection infection of cells with vsvΔm up and downregulated the expression of and genes, respectively, regardless of n-myc expression status ( figure and table ). fold changes in the transcription of these genes were slightly higher in tet- n (high n-myc) except for rap a. interestingly, transcription of rap a, also known as krev , had the highest fold change in response to infection: . -and -fold in tet- n (high n-myc) and tet- n (low n-myc), respectively. though its role on virus replication is unclear, rap a has been shown to reverse the proliferative signal of polyomavirus middle t-antigen in transformed rat cells. we are currently investigating the role of rap a on vsv replication and oncolysis. virus-mediated regulation of isgs in tet- n (low n-myc) eight probe sets for isgs were exclusively identified in tet- n (low n-myc) in response to infection (figure and table ). though proand antiviral functions for these isgs have been reported, their contribution to vsv infection is unknown. table , and supplementary table s ). from these probe sets, and were up-and downregulated, respectively. most of the identified isgs have unknown roles on virus replication. table from all probe sets, ddit- /chop was the most highly upregulated isg in response to infection ( . -fold), suggesting its possible implication on differential susceptibility to vsvΔm determined by n-myc. ddit- /chop, known for its pro-or antiviral functions, , is a transcription factor upregulated during cellular stress including glucose deprivation, amino acid starvation, endoplasmic reticulum (er) stress, and virus infections. the direct role of ddit- /chop on vsv replication is yet to be determined. to validate our findings, cells were infected with vsvΔm at moi of . for , , and hours. as controls for ddit- /chop expression, cells were either treated or not with μg/ml tunicamycin (an er poison) for and hours. upon virus infection, ddit- /chop protein was detected at hpi in tet- n (high n-myc) while undetected in tet- n (low n-myc) and tet- c (dox -/+). tunicamycin treatment robustly induced ddit- / chop in all cells regardless of n-myc expression status (figure a) . we further validated our findings in the analyzed nb cell lines. at the protein level, ddit- /chop could not be detected despite several attempts. at the transcriptional level, ddit- /chop in response to infection varied among the analyzed nb cells regardless of n-myc expression status (figure b ). for example, slight upregulation of ddit- /chop was observed in lan- (n-myc-amplified) and sk-n-as (non-n-myc-amplified) while no changes were observed in imr- (n-myc-amplified) and sh-ep (non-n-myc-amplified). these findings suggest that the link between n-myc and ddit- /chop oncolytic viruses (ovs) are promising new therapies for many cancer types including high-risk nb. however, their application in the clinic could be hampered due to the complexity of the tumor microenviroment and the presence of host immune responses to ovs. it is now understood that the interaction of cancer cells and stroma contribute to cancer progression, metastasis, resistance to chemo drugs, recurrence after treatment, , and resistance to virotherapy. due to the highly heterogeneous nature of nb, it is not surprising if some subpopulations of tumor cells with differences in permissiveness to virus infection or functional ifn signaling would hamper the virotherapy efficacy. we performed in vitro assays to study n-myc expression, virus susceptibility, and innate immunity in the context of virotherapy for nb. in this study, we used tet- n cells, which have been a powerful system to study n-myc in nb biology. this cell system allows the overexpression of n-myc at will by adding and removing tetracycline from the cell culture medium (tet-off system). however, limitations of this system include the use of tetracycline, known to alter the cellular gene expression and thus confound microarray analysis, and the lack of tumorigenic capacity of tet- n (low n-myc) to perform in vivo studies in mice. tetracycline seemed to have had a major effect on ifn-β expression in tet- c (dox +) in response to infection, though the expression of this cytokine by poly i:c treatment, virus replication, virus-induced cell killing and establishment of the antiviral state did not seem to have been affected by this antibiotic. furthermore, sk-n-as cells were not protected by conditioned media from infected tet- c (dox +) cells (not shown) suggesting limited effects of doxycycline in virus-induced cell killing. although cautious interpretation of data from the tet-system (on and off) is paramount, many of the microarray hits from tet- n cells (arrayepress, accession number e-mexp- ) have been validated. , some validated hits such as c-myc and some wnt signaling pathway genes were consistent with our microarray data. although we did not include tet- c in our microarray analysis and the effects of tetracycline could not be ruled out, validation of some hits (stat- , irf- , c-myc and ifitm ) by semiquantitative rt-pcr in both tet- n and tet- c confirmed our microarray analysis (not shown). little is known about the role of myc oncoprotein family on susceptibility of cancer cells to ovs. some cell lines transformed with c-myc are susceptible to vsv in vivo, but its role on promoting virus-induced oncolysis in such transformed cells remains unknown. in this study, we found that n-myc did not cause significant changes in the levels of virus replication, either in the analyzed nb or tet- cells. however, n-myc-amplified cells and tet- n (high n-myc) had increased susceptibility to virusinduced cell killing and failed to establish a robust antiviral state induced by exogenous ifn or poly i:c (either extracellular or intracellular). virus infection did not cause serious changes in the jak/ stat signaling, probably due to insufficient levels of secreted ifn-β. treatments with exogenous ifn-β or poly i:c, on the other hand, caused significant changes in the expression and phosphorylation levels of these proteins regardless of n-myc expression status. overall, our western blot and microarray data suggest that n-myc overexpression does not affect the ifn signaling through jak/stat pathway directly but rather the expression of some isgs stimulated by this pathway. therefore, these findings suggest that the increased susceptibility of nb cells to vsvΔm and alleviation of type i ifn-induced antiviral state by n-myc at least involves the downregulation of isgs with antiviral functions. our findings contrast previous studies showing the negative effects of c-myc on ifn signaling through jak/stat pathway in the context of pathogenesis of burkitt's lymphoma and uveal melanoma. , those studies demonstrated that isgf- protein members and ifn signaling were downregulated by c-myc in a dosedependent manner. our western blot analyses, on the other hand, showed no drastic differences in the levels of isgf- proteins and activation of jak/stat signaling through phosphorylation of stats and when n-myc was overexpressed (figure c ). we and others have observed elevated levels of c-myc in non-n-myc-amplified nb cells, and its expression significantly decreases when n-myc is overexpressed (not shown). despite the elevated c-myc levels in these cells, treatments with ifn-β or poly i:c established a robust antiviral state. therefore, our observations and those previously reported for c-myc suggest differential functions of myc proteins on ifn signaling and, perhaps, susceptibility to virus infection and establishment of the antiviral state. relative to tet- n (low n-myc), significant changes in global gene expression were observed in tet- n (high n-myc). though only one time point was examined ( hpi), differential changes in global gene expression driven by n-myc are likely to occur during the whole replication cycle and virus spread. most of the identified isgs have unknown antiviral functions, but it is highly likely that the collective contribution of isgs and non-isgs further sensitizes cells to virus infection and alleviates the ifn-stimulated antiviral state. interestingly, ddit- /chop, known for its proapoptotic function, was the most highly upregulated isg in tet- n (high n-myc) in response to infection. this finding, confirmed by western blot, suggested a possible link between n-myc and ddit- /chop. however, the expression of ddit- /chop in response to infection was cell line-dependent regardless of n-myc expression status. since nb is a highly heterogeneous malignancy, it would not be surprising if this link were restricted to the tet- cell system or, perhaps, other nb cell populations. the same seems to be true for the link between n-myc and type i ifn transcription in response to infection and poly i:c treatment (supplementary figure s b) . though our studies with the tet- cell system and nb cell lines suggest a link between n-myc and the observed effects on virus susceptibility and establishment of the ifn-induced antiviral state, such link may not be true for all nb cell populations. studies on the frequency of these links in cells from various clinical specimens would help determine the utility of n-myc as biomarker for virotherapy response. our microarray data consisted on several hits that we classified between isgs and non-isgs. non-isgs consisted on genes that were not found in the interferome database. due to the high number of identified isgs in our microarray data, known antiviral functions in some of these genes and the differential establishment of the antiviral state by exogenous ifn-β or poly i:c, we decided to focus our analysis on these genes. however, the observed differences in virus replication and virus-induced cell killing can also be attributed to non-isgs with various cellular functions (e.g., cell cycle progression, activation/regulation of signaling pathways, enzymatic and apoptotic functions, etc). we are currently investigating the role of some non-isgs on virus replication. in summary, we show that n-myc-amplified cells and cells induced to overexpress n-myc were more susceptible to virus-induced cell killing and failed to establish a robust antiviral state by type i ifn. high n-myc levels correlated with increases in susceptibility to virus-induced cell killing and changes in global gene expression. our data suggest that n-myc-driven downregulation of isgs with antiviral functions may be some of the mechanisms promoting differential virus-induced cell killing. these studies suggest that n-myc amplification, a hallmark of high-risk neuroblastoma, can be a potential biomarker of vsvΔm response, while virotherapy efficacy in high-risk disease with non-n-myc amplification may be suboptimal. therefore, oncolytic virotherapy for high-risk nb with no n-myc amplification would probably require sensitization of tumors with chemo drugs prior to infection. development of syngeneic models with isogenic cell lines expressing low and high n-myc levels would provide insights into functional relationships between tumor and stroma, which may determine the virotherapy efficacy in vivo. viruses and infections vsvΔm , a genetically modified vsv strain derived from the indiana serotype of vsv, was propagated on bhk cells. this mutant virus has a deletion of methionine in the m protein and insertion of an extra cistron encoding green fluorescent protein (gfp) between the g and l sequences. cells were infected at indicated multiplicity of infections (mois) and cell culture supernatants were collected at various time points for virus titration in bhk cells. virus growth curves (one-and multi-step) and alamar blue assays (invitrogen, frederik, md) were carried out to study virus replication and spread and cell viability, respectively. tet- n cell line (derived from sh-ep) harbors a repressible control system to regulate the expression of exogenous n-myc at will. n-myc overexpression was induced by doxycycline withdrawal from culture medium for hours. tet- control cells (tet- c) harbor the same repressible system except that exogenous n-myc gene is not present. n-myc expression was assessed by rt-pcr and western blot. type i interferon (ifn) expression and ifn signaling through jak/ stat cells treated or not with doxycycline were infected with vsvΔm at moi of . for , , and hpi or treated with intracellular or extracellular polyinosinic-polycytidylic acid (poly i:c; sigma-aldrich, st louis, mo): ng/well in a -well plate format and μg/ml, respectively, for hours. transfections with poly i:c above ng/well had toxic effects for tet- n (low n-myc). cell culture supernatants were harvested, pulsed to remove cell debris, and stored at − °c until ready to use. detection and quantitation of secreted ifn-β were carried out using the verikine human interferon beta kit (r&d systems, minneapolis, mn) according to the manufacturer's instructions. for transcriptional analysis, rna was extracted (qiagen) followed by rt-pcr. degenerate primers were used to detect the ifn-α subtypes and specific primers to detect ifn-β (forward, ′-tggcaattgaatgggaggct- ′; and reverse gtctcattccagccagtgct) and n-myc (forward, ′-tccaccagcagcacaactatg; and reverse, ′-gtcta gcaagtccgagcgtgt ′). as controls for these assays, cells were treated with either intracellular (transfection) or extracellular poly i:c in reduced serum medium (life technologies). concentrations of intracellular and extracellular poly i:c were optimized for all cells. except for sk-n-as, nb cells were treated with - μg/ml extracellular poly i:c. sk-n-as were treated with . μ/ml extracellular poly i:c because of toxicity beyond this concentration. poly i:c transfections were performed in a six-well plate format. n-myc-amplified cells were transfected with μg poly i:c using lipofectamin (invitrogen, carlsbad, ca). non-n-myc-amplified cells were transfected with . μg poly i:c, as higher amounts had toxic effects on these cells. tet- cells were incubated with extracellular ( μg/ml in reduced serum medium) or intracellular poly i:c ( . μg in six-well plate format) for hours. transfections beyond . μg poly i:c had toxic effects for tet- n (low n-myc). to monitor transfection efficiency, cells were transfected with various amounts of pll . carrying the gfp gene (addgene, cambridge, ma). for analysis of the jak/stat signaling and n-myc detection, cells either treated or not with doxycycline were seeded at × cells per well in six-well plates and incubated at °c in % co . cells either infected or not were washed with ice-cold pbs, collected by scraping and lysed in μl of lysis buffer ( mmol/l tris ph . , mmol/l nacl, % glycerol, % np , . % leupeptin, . % aprotinin, and . % sodium orthovanadate) for minutes followed by sonication (three times, -second pulse, % amplitude) and centrifugation at °c. all protein preparations were quantified (bca protein assay, biolynx, brockville, on, canada), run in % polyacrilmyde gels and transferred onto nitrocellulose membranes. membranes were incubated with monoclonal antibodies to n-myc (emd millipore, darmstadt, germany), stats and , phospho-stat- y (abcam, cambridge, ma), actin (emd millipore), rig-i (novous biologicals, littleton, co); and polyclonal antibodies to irf- , phospho-stat- (s ) and phospho-stat- (y ) (abcam). goat anti-mouse and -rabbit igg-hrp (santa cruz biotechnology, dallas, tx) were used as secondary antibodies. cells treated or not with doxycycline were seeded in -well plates ( × cells/well) for hours followed by pretreatment with u/ml of exogenous human ifn-β (pbl interferon source, piscataway, nj) for hours. cells were then infected with vsvΔm at moi of and viability determined with alamar blue assay (invitrogen, frederik, md) according to the manufacturer's instructions. all experiments were done in triplicate. phase-contrast and fluorescent images of cells were taken using a carl zeiss inverted microscope (axiovert m) mounted with a carl zeiss digital camera (axiocam mrc, lake success, ny). conditioned media protection assays tet- n and tet c treated or not with μg/ml doxycycline were cultured in cm dishes and incubated for hours. cell culture supernatants were removed and replaced with μg/ml poly i:c in reduced serum media (life technologies). cells were incubated for another hours. supernatants were removed and cells were washed four times with serum-free medium (dmem, life technologies) to eliminate any remaining poly i:c. fresh serum-free dmem was added onto cells followed by incubation for another jc corredor et al. molecular therapy -oncolytics ( ) official journal of the american society of gene & cell therapy hours. supernatants were harvested, centrifuged and either four times concentrated or not. fbs was added ( % final concentration) and supernatants were used as conditioned media. for protection assays, sk-n-as was cultured in conditioned media or regular dmem medium ( % fbs) overnight followed by infection (moi of for hours) and viability assays with alamar blue (life technologies). before use, conditioned media from virus-infected cells ( hpi, moi of . ) were uv-irradiated for minutes to inactivate virus. uv-irradiated conditioned media were tested in imr- cells to monitor virus inactivation. global gene expression analysis by microarray assays microarray assays using affymetrix genechip human prime view was carried out. cells either treated or not with doxycycline were infected at an moi of . for hours, time by which around - % of cells expressed gfp encoded by vsvΔm . rna was extracted with rneasy plus mini kit (qiagen). to assess rna quality, rna integrity number (rin) was measured with agilent rna nanochip on bioanalyzer (agilent technologies, santa clara, ca). the quantity was measured using nanodrop (nanodrop technologies, wilmington, de). a total of ng of rna for each sample with a rin higher than was labeled with ' ivt express kit (ambion) and hybridized to affymetrix genechip human primeview arrays at °c for hours. arrays were stained and washed using affymetrix genechip fluidics_ following manufacturer's protocol and scanned using the affymetrix genechip scanner g system. for data analysis, affymetrix genechip array data files were generated using genechip command console software (agcc) and statistical analysis was carried out using partek genomics suite . (partek incorporated). analysis of variance, p < . , was applied as statistically significant. fold changes of ≥ . and ≤− . with p < . were included in the analysis. isgs were identified using the interferon database v . differential expression of ddit- /chop cells were infected at moi of . for , , and hours. as controls, cells were treated with μg/ml tunicamycin (sigma-aldrich) for and hours. cells were harvested for rna and protein extractions to analyze ddit- /chop expression at the transcriptional (semiquantitative rt-pcr) and protein levels, respectively. rt-pcr for ddit- /chop was carried out with forward ( ′ ttgcctttctcttcggacact ′) and reverse ( ′ gctagctgtgccactttcc ′) specific primers. mouse monoclonal antibody to ddit /chop (abcam) was used for western blots. aggressive surgical therapy and radiotherapy for patients with high-risk neuroblastoma treated with rapid sequence tandem transplant tandem high-dose therapy in rapid sequence for children with high-risk neuroblastoma anti-gd antibody with gm-csf, interleukin- , and isotretinoin for neuroblastoma neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus complete regression of human neuroblastoma xenografts in athymic mice after local newcastle disease virus therapy recent progress in the battle between oncolytic viruses and tumours vsv-tumor selective replication and protein translation tumor selective replication of newcastle disease virus: association with defects of tumor cells in antiviral defence interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures propagation, purification, and in vivo testing of oncolytic vesicular stomatitis virus strains vesicular stomatitis virus has extensive oncolytic activity against human sarcomas: rare resistance is overcome by blocking interferon pathways rvsv(m delta )-m is an effective and safe oncolytic virus for cancer therapy vsv strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents expression of the apoptosis-suppressing protein bcl- , in neuroblastoma is associated with unfavorable histology and n-myc amplification association of multiple copies of the n-myc oncogene with rapid progression of neuroblastomas oncolytic activity of vesicular stomatitis virus is effective against tumors exhibiting aberrant p , ras, or myc function and involves the induction of apoptosis c-myc activation impairs the nf-kappab and the interferon response: implications for the pathogenesis of burkitt's lymphoma the relation between c-myc expression and interferon sensitivity in uveal melanoma infection of human cancer cells with myxoma virus requires akt activation via interaction with a viral ankyrin-repeat host range factor targeted and armed oncolytic poxviruses: a novel multimechanistic therapeutic class for cancer connecting reovirus oncolysis and ras signaling neuroblastoma: biology and molecular and chromosomal pathology human cell culture phenotypic diversification in human neuroblastoma cells: expression of distinct neural crest lineages conditional expression of n-myc in human neuroblastoma cells increases expression of alphaprothymosin and ornithine decarboxylase and accelerates progression into s-phase early after mitogenic stimulation of quiescent cells rig-i mediates nonsegmented negative-sense rna virus-induced inflammatory immune responses of primary human astrocytes astrocytes recognize intracellular polyinosinic-polycytidylic acid via mda- tyrosine-phosphorylated stat and stat plus a -kda protein all contact dna in forming interferon-stimulated-gene factor differential toll-like receptor (tlr ) expression and apoptotic response to tlr agonist in human neuroblastoma cells improved microarray gene expression profiling of virus-infected cells after removal of viral rna interferome v . : an updated database of annotated interferon-regulated genes membrane perturbation elicits an irf -dependent, interferon-independent antiviral response reversion of middle t antigen-transformed rat- cells by krev- : implications for the role of p c-ras in polyomavirus-mediated transformation hepatitis e virus orf protein activates the pro-apoptotic gene chop and anti-apoptotic heat shock proteins upregulation of chop/ gadd during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway roles of chop/gadd in endoplasmic reticulum stress targeting pediatric cancer stem cells with oncolytic virotherapy tumor microenvironment promotes cancer progression, metastasis, and therapeutic resistance reactive stroma as a predictor of biochemical-free recurrence in prostate cancer induction of antiviral genes by the tumor microenvironment confers resistance to virotherapy tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research inhibition of focal adhesion kinase decreases tumor growth in human neuroblastoma p is a direct transcriptional target of mycn in neuroblastoma suppression of myc by high expression of nmyc in human neuroblastoma cells wnt signaling and its downstream target n-myc regulate basal progenitors in the developing neocortex resistance to oncolytic myxoma virus therapy in nf (-/-)/trp (-/-) syngeneic mouse glioma models is independent of anti-viral type-i interferon analysis of antiviral response in human epithelial cells infected with hepatitis e virus systematic identification of type i and type ii interferon-induced antiviral factors ifitm proteins-cellular inhibitors of viral entry a diverse range of gene products are effectors of the type i interferon antiviral response targeted inflammation during oncolytic virus therapy severely compromises tumor blood flow stimulation of the liver x receptor pathway inhibits hiv- replication via induction of atp-binding cassette transporter a role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles interferon-inducible protein ifi negatively regulates rig-i antiviral signaling and supports vesicular stomatitis virus replication the mitochondrial antiviral protein mavs associates with nlrp and regulates its inflammasome activity negative regulation of nmi on virus-triggered type i ifn production by targeting irf a critical role of n-myc and stat interactor (nmi) in foot-and-mouth disease virus (fmdv) c-induced apoptosis functional role of type i and type ii interferons in antiviral defense adenovirus e orf protein downregulates myc expression through interaction with the pp a-b subunit cytokeratin is expressed in cells infected with respiratory syncytial virus via nf-kappab activation and is associated with the formation of cytopathic syncytia both the structure and dna binding function of the barrier-to-autointegration factor contribute to reconstitution of hiv type integration in vitro osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells assembly of infectious hepatitis c virus particles bip and multiple dnaj molecular chaperones in the endoplasmic reticulum are required for efficient simian virus infection identification of ptch requirement for influenza virus using random homozygous gene perturbation rapamycin-resistant mtorc kinase activity is required for herpesvirus replication age-dependent resistance to lethal alphavirus encephalitis in mice: analysis of gene expression in the central nervous system and identification of a novel interferon-inducible protective gene, mouse isg trim proteins and the innate immune response to viruses reovirus-induced apoptosis is mediated by trail modulation of the interferon antiviral response by the tbk / ikki adaptor protein tank the role of brca in transcriptional regulation and cell cycle control reticulon binds the c protein of enterovirus and is required for viral replication anti-hiv- activity of trim trim is a negative regulator of mda -mediated type i interferon production this work is licensed under a creative commons attribution-noncommercial-noderivs . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material the authors declare no conflict of interest. the laboratory of p.b. is supported by kids cancer care foundation of alberta and collaborative research and innovation opportunities (crio). the authors want to thank manfred schwab (deutches krebsforschungszentrum, division b tumor genetics) for providing the tet- n and tet- c cells. the authors also want to thank xiuling wang at the southern alberta cancer research institute microarray and genomics facility for her technical support and data interpretation, douglas j. mahoney for his critical review of the manuscript, mana alsheri for providing technical support, and Éva nagy for allowing the performance of some experiments in her laboratory. key: cord- - vpp xdl authors: schlee, m.; barchet, w.; hornung, v.; hartmann, g. title: beyond double-stranded rna-type i ifn induction by prna and other viral nucleic acids date: journal: interferon: the th anniversary doi: . / - - - - _ sha: doc_id: cord_uid: vpp xdl production of type i ifn is the key response to viral infection. since the discovery of type i ifns in , long double-stranded rna formed during replication of many viruses was thought to be responsible for type i ifn induction, and for decades double-stranded rna-activated protein kinase (pkr) was thought to be the receptor. recently, this picture has dramatically changed. it now became evident that not pkr but two members of the toll-like receptor (tlr) family, tlr and tlr , and two cytosolic helicases, rig-i and mda- , are responsible for the majority of type i ifns induced upon recognition of viral nucleic acids. in this review, we focus on the molecular mechanisms by which those innate immune receptors detect viral infection. based on the recent progress in the field, we now know that tlr , tlr , and rig-i do not require long double-stranded rna for type i ifn induction. type i ifns (ifn-α isoforms and ifn-β) are regarded as the dominant mediators of antiviral defense in vertebrates. since their initial discovery half a century ago as acid-stable, soluble factors "interfering" with viral proliferation in cultured cells (isaacs and lindenmann ; nagano and kojima ) , intense research has focused on type i ifn receptor signaling and the plethora of type i ifn-mediated effects (theofilopoulos et al. ) . for the host, an intact type i ifn response is critical for the survival of many viral infections (gresser et al. ; muller et al. ) . sensing of viral replication has been proposed to be responsible for triggering the production of type i ifns by infected host cells. however, the specific host immune receptors and their respective molecular ligands remained elusive until very recently. moreover, to mount an appropriate antiviral response, the innate immune system must distinguish viruses from bacteria, fungi, and multicellular parasites. charles janeway was the first to propose that the detection of highly conserved pathogen-associated molecular patterns (pamps) may be mastered by a limited number of germline-encoded pattern recognition receptors (prrs) (janeway ) . a few years later, the first experimental evidence of such a receptor came from the fruit fly (lemaitre et al. ) . shortly afterwards, a member of the family of toll-like receptors (tlr), the mammalian homolog of drosophila toll, was demonstrated to be responsible for detecting lipopolysaccharides (lps), a characteristic component of the cell walls of gram-negative bacteria (medzhitov et al. ; poltorak et al. ). this observation was confirmed by the subsequent generation of tlr -deficient mice (hoshino et al. ) . parasites, bacteria, and fungi rely on a multitude of molecules that are distant in evolutionary terms from the mammalian organism, and are thus readily discernible as non-self by members of the toll-like receptor (tlr) and nodlike receptor (nlr) families (reviewed in meylan et al. ) . in sharp contrast, all components of viruses are produced within the infected host cell, and therefore lack distinguishable non-self molecular patterns. nevertheless, viruses are promptly recognized by the innate immune system and elicit pronounced antiviral type i interferon and cytokine responses. shortly after the discovery of type i interferons, it was proposed that viral nucleic acids could be stimulating the type i ifn response (isaacs et al. ) . many viruses synthesize double-stranded rna (dsrna) during their replication cycle (baltimore et al. ; montagnier and sanders ) , whereas dsrna was thought to be absent in uninfected cells. therefore dsrna formed during viral infection was postulated to be the molecular signature of viral infection. in support of this hypothesis, the enzymatically generated double-stranded rna polynucleotide polyinosinic:polycytidylic acid (poly i:c) was found to be a potent inducer of type i ifn (field et al. ) . although the authors carefully emphasized that all other double-stranded polynucleotides were inactive, the notion that long viral double-stranded rna elicits type i ifn became commonplace, and poly i:c has been used as an interferon-inducing mimic of viral dsrna ever since. in early attempts to uncover the inducers of interferon and of other mediators of antiviral activity, ifn-α and poly i:c-treated or reticulocyte extracts were analyzed. chromatographic separation of lysates revealed proteins that were increased by preincubation with ifn-α, and whose enzymatic activity depended on the presence of dsrna (usually poly i:c) (farrell et al. ; hovanessian et al. ; zilberstein et al. ) . two proteins, interferon inducible double-stranded rna-activated protein kinase (pkr) and the ′, ′ oligoadenylate synthetase (oas) could be affinity purified using poly i:c-cellulose (farrell et al. ; hovanessian et al. ) . both activated pkr and oas were found to block translation of viral rna by distinct mechanisms. in the presence of poly i:c, oas catalyzes the synthesis of ′, ′ oligomers of adenosine ( - as) (hovanessian et al. ; zilberstein et al. ) , which activate rnase l (farrell et al. ) . rnase l in turn degrades single-stranded viral and cellular rnas (farrell et al. ) in a sequence-independent manner (minks et al. ) . consequently rnase l-deficient mice displayed a reduced antiviral activity of ifn-α, as well as impaired apoptosis (zhou et al. ) . in contrast, the serine threonine kinase pkr was found to more specifically block the translation of viral rna (farrell et al. ) by phosphorylation of the eukaryotic translation initiation factor eif a. besides its function in limiting translation of viral protein, pkr was also reported to activate nf-κb (kumar et al. ) . pkr was therefore proposed as a key receptor mediating virus-and dsrna-induced production of type i interferons (kumar et al. ). however, these findings remained controversial, as other studies that examined pkr-deficient mice and cells (chu et al. ; iordanov et al. ; maggi et al. ; smith et al. ; yang et al. ) found no defects in the induction of interferon in response to poly i:c or viral infection that could not be overcome with type i ifn pretreatment. further analysis revealed that pkr is not only activated by poly i:c but is able to interact with dsrna as short as bp. however, at least bp are required to activate pkr kinase activity (manche et al. ) . in another study (zheng and bevilacqua ) , recombinant pkr could also be activated by rna oligonucleotides containing a -bp dsrna stem loop in combination with a more than bp-long single-stranded rna part at the ′ or ′ end. all these studies question the often quoted requirement of a dsrna molecule longer than bp; furthermore, it became evident that the translational shut-down by pkr is not linked to the induction of type i ifn synthesis and secretion. the finding that pkr -/cells still produce type i ifns spurred further research on receptors capable of recognizing long double-stranded rna. such investigations led to a member of the toll-like receptor (tlr) family, tlr , which was proposed to bind to long dsrna and to induce ifn-β (alexopoulou et al. ) . tlrs are transmembrane receptors that were shown to recognize a variety of conserved pathogenassociated molecular patterns (pamps) of bacterial, fungal, and parasitic origin. the study of alexopoulou et al. was the first to demonstrate a role for tlrs in the recognition of viruses. tlr was found to be the receptor for unmethylated cpg motifs in dna (hemmi et al ) ; however, cpg-dna at first was thought to be characteristic for bacterial dna, and the role of tlr in detecting dna viruses was only proposed later (krug et al. a (krug et al. , b tabeta et al. ) . upon engagement with their specific ligands, tlrs trigger signaling pathways that lead to the activation of nf-κb and irfs (signaling of tlrs reviewed in moynagh ) . tlr was found to induce type i ifns upon poly i:c stimulation by activation of the kinase tbk , which phosphorylates the transcription factor irf , resulting in the induction of ifn-β (doyle et al. ; fitzgerald et al. ; sharma et al. ) . another group reported that tlr is activated by ssrna (kariko et al. b ); however, tlr -deficient mice and mice deficient in the signaling adapter trif (gitlin et al. ; kato et al. ) still responded to poly i:c. moreover, dendritic cells derived from tlr -deficient mice were still stimulated by dsrna transfected into the cytosol (diebold et al. ). unlike tumor cell lines, which were examined in early studies on type i ifn and dsrna, primary immune cells such as peripheral blood mononuclear cells (pbmcs) express a wide spectrum of functional tlrs. different immune cell subsets express distinct patterns of tlrs (hornung et al. ) . plasmacytoid dendritic cells (pdcs) (reviewed in colonna et al. ) are the major producers of early type i ifn production upon viral infection. pdcs express tlr and tlr but not tlr . both tlr and tlr are located in the endosomal compartment and signal via the adaptor molecules myd , irak , and traf , leading to activation of irf and the induction of type i interferons as reviewed by moynagh ( ) . in addition, recent studies show that traf plays a crucial role in the myd -dependent signaling cascade (hacker et al. ; oganesyan et al. ) . single-stranded dna and the small antiviral compound r had been shown to induce ifn in pdcs dependent on tlr and tlr , respectively (hemmi et al. (hemmi et al. , jurk et al. ; krug et al. b; rothenfusser et al. ) . tlr detects unmethylated so-called cpg motifs in single-stranded dna (hemmi et al. ) . different classes of synthetic cpg oligodeoxynucleotides (odn) were developed based on the distinct effects on the two tlr -expressing immune cell types: pdcs and b cells (hartmann et al. ; hartmann and krieg ; krug et al. a) . in contrast to tlr , both tlr and depend on the signaling adapter myd . accordingly, pdcs derived from tlr -or myd -deficient mice are unable to produce type i ifn in response to dna viruses such as herpes simplex viruses (hsv) and murine cytomegalovirus (mcmv) (krug et al. a (krug et al. , b lund et al. ; tabeta et al. ) . while tlr was responsible for detecting viral dna, tlr was shown to recognize rna: tlr detects synthetic short ( - bases) single-stranded rna (diebold et al. ; heil et al. ) and short interfering double-stranded rna (sirna) (hornung et al. ; judge et al. ; sioud ; reviewed in schlee et al. ). the amount of type i interferon induction was dependent on the rna sequence. ironically, hornung and colleagues came across a very potent type i interferon inducing small rna sequence core motif ( ′-guccuucaa- ′) in the attempt to knock down the interferon inducer tlr in pdcs using the sirna technology (hornung et al. ) . it was demonstrated that these sirnas induce systemic immune activation in mice, and that the immunological activity required tlr . of note, the same sirna did not induce type i interferon in immortalized human embryonic kidney cells (hek ), which produced type i interferon in response to poly i:c. in subsequent studies, similar findings were reported by judge et al. ( ) (identifying a core motif ′-ugugu- ′) and sioud ( ) . in all three studies, transfection with cationic lipids (e.g., dotap, lipofectamine) or cationic polymers (e.g., pei, polyethylenimine) was essential for the immunological activity of sirna. the same applies for the immunological activity of single-stranded rna (diebold et al. ; heil et al. ; scheel et al. ). while for short rna oligonucleotides the immunological activity is clearly sequence dependent (hornung et al. ; judge et al. ) , for long rna molecules such as mrna, sequence specificity of immunological activity is less prominent (scheel et al. ) . this raises the question of how the immune system is able to distinguish between self and non-self (for example viral) rna. this question was addressed recently by kariko and colleagues ( ) who showed that human mitochondrial rna, when transfected into monocyte-derived dendritic cells, provoked secretion of tnf-α at similar quantities compared to total rna isolated from escherichia coli . in contrast, rna of other cellular compartments showed no immunological activity. the authors proposed that mammalian rna is masked by naturally occurring nucleoside modifications that are expected to be similar in closely related species. according to this concept, mitochondrial rna is stimulatory since it resembles bacterial rather than mammalian rna. in healthy cells, mitochondrial rna will not be released. in contrast to other self-rna, mitochondrial rna never enters the cytosolic compartment. as a consequence, mitochondrial rna under healthy conditions is not detected by cytosolic mechanisms of detection. only if the cell is lysed can mitochondrial rna enter the endosomal compartment of immune cells via phagocytosis. indeed, the stimulatory effect of in vitro rna transcripts composed of unmodified nucleotides in their study could be abrogated by incorporation of modified nucleosides such as pseudouridine, -methylcytidine, n -methyladenosine, inosine, and n methylguanosine. in order to examine modification sensitivity of different tlrs, hek cells expressing tlr , tlr , tlr , or tlr were transfected with rna containing modified nucleosides. transfection of unmodified rnas stimulated il- production (sensitive readout for immunoactivation of hek cells) in hek cells overexpressing tlr , , and . interestingly, rna recognition by tlr , tlr , and tlr is suppressed by the presence of different types of modified nucleotides within the rna ligand. tlr was the least sensitive receptor with regard to suppression by nucleoside modifications. furthermore, the authors showed that in monocyte-derived dendritic cells, %- % of modified nucleosides were sufficient to inhibit tnf-α secretion by %- %. together, these results show that rna modification contributes to the distinction of self versus non-self rna by the immune system. further insight into the properties that render rna molecules stimulatory to the immune system is driven by sirna technology. based on studies by tuschl and colleagues (elbashir et al. ) , sirna is now used worldwide as a robust tool for target-specific gene silencing in cell lines and human primary cells. however, depending on the mode of synthesis and the sequences used to generate sirna, also nonspecific, so-called nonspecific off-target effects of sirnas were observed. to overcome limitations with the transfection of synthetic sirna, vectorbased (e.g., lentiviral) expression systems for the introduction of short hairpin sirnas (shrna) mimicking sirnas were developed (brummelkamp et al. ; harborth et al. ; paddison et al. ) . the most commonly used shrna expression system consists of a rna-polymerase iii dependent promoter driving the expression of two complementary -to -bp rna sequences linked by a short loop of - nt. the resulting transcript is exported to the cytoplasm and processed by dicer. lentiviral vectors haboring the pol iii-shrna expression cassette (li et al. ; rubinson et al. ; tiscornia et al. ) allow rnai-mediated gene silencing via sirna in cells that are otherwise difficult to transfect. sequence specificity of gene silencing by such shrna was questioned by bridge et al. (bridge et al. ) , who demonstrated that infection of human lung fibroblasts with pol iii-shrna containing lentivirus directed against the gene morf l not only silenced morf l but also stimulated interferoninducible genes such as ′, ′-oas, an indicator of type i interferon. the ifninducing effect was dependent on the sequence and the dose of the vector; seven of shrnas targeting different genes exhibited ifn induction. in contrast, transfection of synthetic sirna with the same putative ifn-inducing sequences led to sequence-specific silencing without triggering an ifn response. northern blot analysis of shrna showed that the majority of shrna transcripts were correctly processed to nt transcripts. the authors speculated that remaining unprocessed transcripts could be detected by cytosolic rna sensing receptors. in the follow-up paper, the group of iggo (pebernard and iggo ) , further correlated the u promoter sequence with oas induction. this study revealed that the region between - (the end of the promoter) and + (the start of rna transcript) is crucial for the immune stimulatory effect, which was lost when they used the endogenous human sequence (ccga). further mutations leading to a partial mismatch in the shrna (predicted to create a -bp duplex) suggested that stimulation required more than a -bp duplex. william´s group (sledz et al. ) described the induction of ifn target genes by transfection of synthetic sirnas into a human glioblastoma cell line (t g) or a renal carcinoma cell line (rcc). when comparing the two studies from bridge and colleagues and from sledz and colleagues, it is important to note that different cell lines (bridge, human lung fibroblasts; sledz, rcc and t g) and different ways of sirna generation (bridge, synthetic sirnas and shrnas; sledz, synthetic sirna and t -phage-polymerase sirna) were used. using mouse embryonic fibroblasts (mefs) with different gene deficiencies related to the ifn response system, sledz et al. proposed that pkr was the interferon-inducing receptor for sirnas. later, the same group postulated a different sirna receptor (rig-i, see below) in t g cells (marques et al. ) . of note, in the two studies published by bridge et al. ( ) and sledz et al. ( ) , type i interferon was not analyzed at the protein level. kariko et al. ( a) suggested that tlr was responsible for the induction of type i ifn by sirna. these data are based on keratinocyte (hacat) and hek cells, which responded to synthetic sirna but not to the singlestranded components (ssrna) by secretion of low amounts of ifn-β that was comparable to stimulation with poly i:c. overexpression of tlr in hek cells resulted in fourfold higher induction of type i ifn secretion in response to transfected sirna. however, overexpression of nf-κb-inducing receptors such as tlr may also contribute indirectly to the enhanced type i ifn response induced by sirna, for example by upregulating ifn-inducible cytosolic rna receptors. for example, tlr overexpressing hek cells secrete more il- than empty vector or tlr overexpressing hek cells (kariko et al. ) ; consequently, such studies do not necessarily provide evidence for a direct interaction between sirna and tlr . kim et al. ( ) showed that the induction of type i ifn by sirna depended on the use of t -rna polymerase (t rnap) for sirna generation. in contrast to bridge et al. ( ) and sledz et al. ( ) , in the study by kim and colleagues, type i ifn was measured at the protein level, which is less sensitive than measuring ifn-dependent responses on the transcriptional level and thus underscores the magnitude of the ifn response they reported. in their study, kim and colleagues examined sirnas targeting the early icp gene of hsv- . only t rnap-derived transcripts but not synthetic sirna elicited a potent antiviral activity when transfected into hek cells. the same antiviral activity was observed by transfection of t transcripts with unrelated sequences. analysis of supernatants revealed the presence of substantial amounts of ifn-α and ifn-β protein. these results were reproduced in hela cells, as well as k , cem, and jurkat cells. it is well known that unlike capped mammalian mrna, the ′ ends of t transcripts harbor a triphosphate gtp-nucleotide. treatment of t transcripts with rnase t (with the ′ end p-ggg removed, which was single-stranded in their case) and alkaline phosphatase was sufficient to completely abrogate interferon -inducing activity. additional experiments using t and sp phage rna polymerases demonstrated similar induction of type i ifn. the examination of multiple cell lines by kim et al. ( ) pointed to a powerful ubiquitously expressed sensor for short triphosphate rna. yoneyama and colleagues identified the interferon-inducing cytoplasmic dexd/ h box rna helicase rig-i, containing a caspase recruitment domain (card) (yoneyama et al. ) . expression of the card domain sensitized cells to activate the transcription factor irf , leading to the induction of the ifn-β promoter. later on it was shown that this pathway involves the irf kinase tbk , which is activated by the newly characterized adaptor protein ips- , also known as cardif, mavs, or visa (kawai et al. ; meylan et al. ; seth et al. ; xu et al. ; reviewed in sen and sarkar ) . in overexpression experiments, rig-i was shown to bind poly i:c. however, overexpression of a dominant negative mutant of rig-i impaired irf activation by newcastle disease virus (ndv), a negative-strand rna virus, while irf activation by poly i:c was not inhibited. subsequent studies with rig-i -/mice and mefs showed no defect in the response to poly i:c. hornung and colleagues ( ) demonstrated that rig-i detects in vitro transcribed rna. rna with a triphosphate at the ′ end (now termed prna), which is generated during in vitro transcription, was identified to be the ligand for rig-i. the minimal length of prna was nucleotides. the activity of prna was independent of double-strand formation. both exogenous prna transfected into the cell and endogenously formed prna (expression of t rna polymerase) activated rig-i. genomic rna prepared from a negative-strand rna virus and rna prepared from virus-infected cells, but not rna from noninfected cells, triggered a potent ifn-α response in a ′-triphosphate-dependent manner. binding studies of rig-i and prna revealed a direct molecular interaction. the ′ capping or incorporation of modified nucleotides such as pseudouridine, -thiouridine, and ′-o-methylated uridine in place of uridine in short prna strongly diminished ifn-α induction. in a parallel study, pichlmair et al. ( ) attributed the inhibitory effect of the influenza virus protein ns to its binding and inhibition of the rig-i triphosphate rna complex. these results provide evidence that uncapped unmodified prna is detected by rig-i in the cytosol of eukaryotic cells. of note, all primer-independent rna transcripts in a normal uninfected cell initially contain a ′-triphosphate end. however, most if not all self-rna species entering the cytosol lack a free ′triphosphate end. before self-rna leaves the nucleus, rna is further processed, which applies to rna transcripts of all three dna-dependent rna polymerases (pol) in eukaryotes. pol i transcribes a large polycistronic precursor of ribosomal rna (rrna) that contains the sequences for the mature rrnas ( , . s, - s rrna), two external transcribed spacers, and two internal transcribed spacers. this primary transcript is subjected to endo-and exonucleolytic processing steps to produce the mature rrnas. the net result of this maturation process is a monophosphate group at the ′ end of all pol i transcribed rrnas (fromont-racine et al. ) . messenger rnas (mrnas) and small nuclear rnas (snrnas), which are transcribed by pol ii, receive a -methyl guanosine group that is attached to the ′-triphosphate of the nascent rna by a process called capping (shatkin and manley ) . thus, upon export into the cytoplasm, no free triphosphate groups are found in pol ii transcripts. all mature trnas (pol iii) have a ′-monophosphate (xiao et al. ) , as it is likely to apply to s rrna. u rna receives a γ-monomethylphosphate cap structure following transcription. however, sl rna (pol iii) has a triphosphate at the ′ end, and is present at high copy numbers in the cytosol. therefore, the presence or absence of a ′ triphosphate might not be the only structural feature of rna responsible for the distinction of self and viral rna. it is well known that eukaryotic rna undergoes significant modifications to its nucleosides and its ribose backbone. among all nucleoside modifications, pseudouridinylation is one of the most common post-transcriptional modifications of rna that appears to be universal among rrnas and small stable rnas such as splicing small nuclear rnas (snrnas), trnas, and small nucleolar rnas (snornas). however, the frequency and location of pseudouridinylated nucleotides vary phylogenetically. intriguingly, eukaryotes contain far more nucleoside modifications within their rna species. human ribosomal rna, for example, the major constituent of cellular rna, contains ten times more pseudouridine and times more -o-methylated nucleosides than e. coli rrna (rozenski et al. ) . the same applies to eukaryotic trnas, the most heavily modified subgroup of rna with up to % of modified nucleosides. the host machinery that guides nucleoside modifications and ′-o-methylation of the ribose backbone is located in the nucleolus, and consists of rna-protein complexes containing snornas and several associated proteins (snornps) (decatur and fournier ) . information on nucleolus-specific nucleoside modifications or ribose ′-o-methylation of viral rna genomes is limited. since most rna viruses do not replicate in the nucleus and modification is tightly confined to the sequence and structure of their target, extensive modification of viral rna seems unlikely. altogether, post-transcriptional modifications of eukaryotic rna such as ′ processing or capping, as well as nucleoside modifications or ribose backbone methylation, provide the molecular basis for the distinction of self-rna generated in the nucleus from viral rna of cytosolic origin containing ′-triphosphate ( prna). the mrnas of viruses infecting eukaryotic cells also commonly contain -methyl guanosine cap-structures at their ′ ends and poly(a) tails at their ′ends (furuichi and shatkin ) . some viruses make use of the host transcription machinery to acquire caps and poly(a) tails. rna viruses that do not rely on the host transcriptional machinery produce their own capping enzymes or utilize other mechanisms such as snatching the ′terminal regions of host mrnas. despite these adaptations of viruses to the host transcriptional system, viral rna synthesis leads to transient cytosolic rna intermediates with an uncapped ′-triphosphate end. with notable exceptions such as the picornavirus family (see below), viral rna-dependent rna polymerases (rdrp) initiate polymerase activity de novo, without a specific primer (kao et al. ) . as a consequence, these rdrp-dependent transcripts start with an uncapped ′-triphosphate. this has been studied in great detail for the replication of positive-strand rna viruses of the family of flaviviridae (including the genera flavivirus , pestivirus , and hepacivirus ); members of all of these virus genera were reported as being recognized via rig-i (honda et al. ; kato et al. ; sumpter et al. ) . segmented nsv rely on a cap-snatched primer for mrna transcription, yet initiate genomic and the complementary antigenomic rna replication by a primer-independent de novo mechanism resulting in a ′-triphosphateinitiated transcript (honda et al. ; neumann et al. ) . nsv with a nonsegmented genome (order mononegavirales), including the paramyxoviruses and rhabdoviruses, initiate both replication and transcription de novo leading to ′-triphosphate rna in the cytosol. both the full-length replication products, vrna and crna, and a short leader rna, which is abundantly synthesized during initiation of transcription, maintain their ′triphosphate (colonno and banerjee ; whelan et al. ), while the virus-encoded mrna transcripts are further modified at their ′ ends by capping and cap methylation. consequently, genomic rna from nsvs per se is expected to trigger an ifn-response without the need for replication and presumed dsrna formation. consistent with this notion, not only live virus but also rna purified from nsv virions (vsv) has been shown to trigger strong type i interferon responses depending on rig-i ). hornung and colleagues confirmed and extended these observations by demonstrating that dephosphorylation of the viral rna isolates completely abolished the ifn-response, thereby indicating that the ′-triphosphate moiety is strictly required for recognition . a notable exception are the viruses in the picornavirus-like supergroup (picornavirus, potyvirus, comovirus, calicivirus, and other viruses), which exclusively employs a protein known as viral genome-linked protein (vpg) as a primer for both positive-and negative-strand rna production. this protein primer is part of the precursor rdrp and is cleaved off as elongation of the initial complex occurs, usually to become a ′-genome-linked protein (lee et al. ) . thus during the life-cycle of picornaviruses uncapped, triphosphorylated ′ ends are absent. consequently, based on our studies, rig-i is expected to be involved in the detection of flaviviridae and nsv but not picornaviruses. this is confirmed in a recent study . a number of studies suggested that the helicases mda- and rig-i recognize dsrna (andrejeva et al. ; rothenfusser et al. ; yoneyama et al. ) . the results in the work of hornung and colleagues ( ) demonstrated that double-strand formation of rna is not required for rig-i-rna interaction, and that dsrna is not sufficient for rig-i activation. these results further demonstrate that mda- is not involved in ′-triphosphate rna recognition. although there is convincing evidence that mda- is activated by the long dsrna mimic poly i:c, activation of mda- by natural long dsrna is still controversial . taken together, tlr is so far the only receptor that induces type i ifn upon binding of the natural molecule long dsrna, but the contribution of tlr to type i ifn induction and viral clearance in vivo seems to be weak (rudd et al. ) . there is good evidence that short dsrna such as sirna generated by dicermediated cleavage of long dsrna does not elicit a type i ifn response in nonimmune cells (elbashir et al. ; hornung et al. ; kim et al. ) . a recent study suggests that the two-nucleotide overhang at the ′ end of dicer cleavage products are essential for the lack of immunorecognition of short dsrna (marques et al. ) . the same study proposed that synthetic blunt-end short dsrna is recognized via rig-i. the conclusion that rig-i is the receptor for blunt end short dsrna is based on experiments using rig-i overexpression and using anti-rig-i sirna (short dsrna with two-nucleotide ′overhangs) on top of stimulation with blunt end short dsrna stimulation. rig-i-deficient cells have not been examined in this study. this experimental design does not provide clear-cut evidence for the primary involvement of rig-i in type i ifn induction by blunt-end short dsrna. furthermore, in the study by hornung and colleagues, ′ triphosphate blunt-end rna and ′ triphosphate -nt overhang rna showed identical rig-i ligand activity, suggesting that the molecular feature -nt overhang does not inhibit rig-i-mediated recognition ). mda- is structurally related to rig-i, as it also contains two card domains and a helicase domain. mda- was originally identified as a type i ifn-inducible molecule mediating cell cycle arrest and apoptosis in melanoma cells (hence the name melanoma differentiation antigen ) (kang et al. (kang et al. , kovacsovics et al. ) . a first indication of a role for mda- in virus recognition came from the observation that a paramyxoviral protein that mediated immune evasion bound to mda- (andrejeva et al. ). in overexpression experiments, mda- was shown to bind poly i:c, and enhanced the interferon response to poly i:c as well as several viruses. conversely, sirna mediated knock-down blocked type i ifn induction in response to these stimuli . mda- was then shown to play an essential role in the detection of picornaviruses such as encephalomyocarditis virus (emcv) or theiler's virus (gitlin et al. ; kato et al. ). in addition, mice deficient in mda- were found to be highly susceptible to emcv. although the nature of the natural rna ligand that engages mda- has so far remained obscure, a surprising observation was that cells derived from mda- -deficient mice, as well as mda- -/mice stimulated in vivo were found unable to mount a type i ifn response to poly i:c, establishing mda- , rather than the several other receptors that bind, or have been shown to be activated by poly i:c, as the dominant receptor mediating the interferon response to poly i:c (gitlin et al. ; kato et al. ). however, the natural viral ligand for mda- has not yet been identified. in addition to rig-i and mda- , another cytosolic receptor may exist for detecting dna. until recently tlr was the only innate sensor for detecting microbial dna. recent studies indicate that dna is detected in the cytosol independently of tlr (okabe et al. ; stetson and medzhitov ) , but the receptor has not been identified yet. the cytosolic receptor mediating recognition of b-form dna, unlike rig-i and mda- , signals independently of ips- . as discussed in the previous sections, immune and nonimmune cell types express characteristic patterns of nucleic acid receptors (table ) . for example, melchjorsen and colleagues reported that activation of innate defense against a paramyxovirus is mediated by rig-i, tlr , and tlr in a cell-type-specific manner (melchjorsen et al. ) . they found that nonimmune cells relied entirely on rna recognition through rig-i for activation of an antiviral response. in contrast, immune cells such as myeloid cells utilized tlr and tlr . unlike modifications that have been shown to prevent detection by the receptors indicated and that are frequently found in mammalian nucleic acids in nonimmune cells, rna sensing in paramyxovirus-infected myeloid cells was independent of rig-i, tlr , and pkr. kato and colleagues also found celltype specific involvement of rig-i in antiviral immune response. in their study type i ifn induction in both fibroblasts and myeloid dendritic cells was rig-i-dependent, while type i ifn induction in pdc was rig-i-independent . it is important to note that the mechanisms used for rna sensing may not only be cell-type-dependent but may also depend on the type of virus and its strategy to enter the target cell and to evade immune recognition. in contrast, recognition of synthetic rna or of rna transcribed from vector systems is more predictable because there is no immune evasion and because the mode of delivery is known. of note, the use of cationic lipids and polycationes leads to both endosomal and cytosolic delivery (almofti et al. ; boussif et al. ) and thus both tlr-and rig-i-mediated rna sensing is triggered, provided these receptors are expressed in the cell type examined, and the appropriate rna ligand is delivered. of note, subcellular localization of tlr is cell-type-specific (matsumoto et al. ) : in fibroblasts, tlr is located on the cell surface, and the tlr -mediated activity can be blocked by anti-tlr antibodies. in myeloid dendritic cells, tlr is found in the cytosolic compartment. a more detailed analysis in tlr -transfected b cells revealed that tlr is detectable in multivesicular bodies, a subcellular compartment situated in the endocytic trafficking pathway (matsumoto et al. ) . bacteria, fungi, or cellular parasites are recognized via conserved molecules typical for the respective type of pathogen. in contrast, all virus components are formed within the infected host cell; consequently, a virus-specific detection system is more difficult to achieve. it is now evident that host cells are equipped to detect viral nucleic acids. for viral infection in vivo, the following picture is evolving: large parts of the early type i ifn response upon viral infection are due to tlr and tlr expressed in pdcs; in fact, pdcs are the only considerable source of tlr -and tlr -induced type i ifn production upon viral infection. the major advantages of this pdc response are that the presence of viral particles is sufficient for recognition, that viral infection of cells is not required for detection, and that viruses are recognized before viral proteins have a chance to mediate immune evasion. this first wave of type i ifn production plays an important role in limiting viral spread by pdc-derived direct antiviral mechanisms early on, and by sensitizing yet uninfected cells for cytosolic recognition of viral nucleic acid via strong upregulation of the two cytosolic helicases, rig-i and mda- . these two cytosolic receptors are then responsible for the second and prolonged wave of type i ifn production and for the induction of apoptosis of virally infected cells. for all four receptors, distinction of self from viral nucleic acid is based on a combination of localization and molecular structure. in this sophisticated system of virus detection, the following situations signal viral danger: . appearance of unmodified rna in the endosomal compartment of pdcs . appearance of dna containing unmethylated cpg motifs in the endosomal compartment of pdcs . unmodified rna with a triphosphate group at the ′ end ( prna) in the cytosol of any cell type . dna in the cytosol of any cell type it is still unclear whether long dsrna in the cytosol is sufficient to elicit an antiviral response via one of the receptors known to date. although poly i:c is a ligand for mda- , long double-stranded rna seems insufficient as a ligand, and the natural ligand still needs to be identified. in addition to rnadetecting receptors, the cytosolic receptor for dna may add new perspectives in therapeutic viral mimicry. with regard to viruses that perform inside the nucleus such as hbv and hiv, uncovering molecular mechanisms of sensing viral nucleic acids in the nucleus appears on the radar of scientific challenges. recognition of double-stranded rna and activation of nf-kappab by toll-like receptor cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter virus-specific double-stranded rna in poliovirus-infected cells dendritic cells respond to influenza virus through tlr -and pkrindependent pathways a versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine induction of an interferon response by rnai vectors in mammalian cells a system for stable expression of short interfering rnas in mammalian cells control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon jnk and ikkbeta are required for activating the innate response to viral infection plasmacytoid dendritic cells in immunity complete nucleotide sequence of the leader rna synthesized in vitro by vesicular stomatitis virus rna-guided nucleotide modification of ribosomal and other rnas innate antiviral responses by means of tlr -mediated recognition of single-stranded rna viral infection switches non-plasmacytoid dendritic cells into high interferon producers irf mediates a tlr /tlr -specific antiviral gene program duplexes of -nucleotide rnas mediate rna interference in cultured mammalian cells interferon-beta is required for interferon-alpha production in mouse fibroblasts interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded rna inducers of interferon and host resistance. ii. multistranded synthetic polynucleotide complexes lps-tlr signaling to irf- / and nf-kappab involves the toll adapters tram and trif ribosome assembly in eukaryotes viral and cellular mrna capping: past and prospects essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus role of interferon in the pathogenesis of virus diseases in mice as demonstrated by the use of antiinterferon serum. i. rapid evolution of encephalomyocarditis virus infection specificity in toll-like receptor signalling through distinct effector functions of traf and traf sequence, chemical, and structural variation of small interfering rnas and short hairpin rnas and the effect on mammalian gene silencing rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells mechanism and function of a newly identified cpg dna motif in human primary b cells species-specific recognition of single-stranded rna via tolllike receptor and small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway a toll-like receptor recognizes bacterial dna identification of the ′ terminal structure of influenza virus genome rna by a newly developed enzymatic method role of a transductional-transcriptional processor complex involving myd and irf- in toll-like receptor signaling ′-triphosphate rna is the ligand for rig-i sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr quantitative expression of toll-like receptor - mrna in cellular subsets of human peripheral blood mononuclear cells and sensitivity to cpg oligodeoxynucleotides cutting edge: toll-like receptor (tlr )-deficient mice are hyporesponsive to lipopolysaccharide: evidence for tlr as the lps gene product synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells activation of nf-kappab by doublestranded rna (dsrna) in the absence of protein kinase r and rnase l demonstrates the existence of two separate dsrna-triggered antiviral programs foreign nucleic acids as the stimulus to make interferon virus interference. i. the interferon approaching the asymptote? evolution and revolution in immunology sequencedependent stimulation of the mammalian innate immune response by synthetic sirna human tlr or tlr independently confer responsiveness to the antiviral compound r- expression analysis and genomic characterization of human melanoma differentiation associated gene- , mda- : a novel type i interferon-responsive apoptosisinducing gene mda- : an interferon-inducible putative rna helicase with double-stranded rna-dependent atpase activity and melanoma growth-suppressive properties de novo initiation of viral rna-dependent rna synthesis small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna cell type-specific involvement of rig-i in antiviral response differential roles of mda and rig-i helicases in the recognition of rna viruses interferon-alpha induction through tolllike receptors involves a direct interaction of irf with myd and traf ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction interferon induction by sirnas and ssrnas synthesized by phage polymerase overexpression of helicard, a card-containing helicase cleaved during apoptosis, accelerates dna degradation tlr -dependent recognition of mcmv by ipc and dc generates coordinated cytokine responses that activate antiviral nk cell function herpes simplex virus type activates murine natural interferon-producing cells through toll-like receptor identification of cpg oligonucleotide sequences with high induction of ifn-alpha/beta in plasmacytoid dendritic cells toll-like receptor expression reveals cpg dna as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with cd ligand to induce high amounts of il- double-stranded rnadependent protein kinase activates transcription factor nf-kappa b by phosphorylating i kappa b a protein covalently linked to poliovirus genome rna the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults inhibition of hiv- infection by lentiviral vectors expressing pol iiipromoted anti-hiv rnas toll-like receptor -mediated recognition of herpes simplex virus- by plasmacytoid dendritic cells recognition of single-stranded rna viruses by toll-like receptor potential role of pkr in double-stranded rna-induced macrophage activation interactions between doublestranded rna regulators and the protein kinase dai differential viral induction of distinct interferonalpha genes by positive feedback through interferon regulatory factor- a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells subcellular localization of toll-like receptor in human dendritic cells a human homologue of the drosophila toll protein signals activation of adaptive immunity activation of innate defense against a paramyxovirus is mediated by rig-i and tlr and tlr in a cell-type-specific manner cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus structural requirements of doublestranded rna for the activation of ' , '-oligo(a) polymerase and protein kinase of interferon-treated hela cells replicative form of encephalomyocarditis virus ribonucleic acid tlr signalling and activation of irfs: revisiting old friends from the nf-kappab pathway functional role of type i and type ii interferons in antiviral defense inhibition of vaccinia infection by a liquid factor in tissues infected by homologous virus orthomyxovirus replication, transcription, and polyadenylation critical role of traf in the toll-like receptor-dependent and -independent antiviral response toll-like receptor-independent gene induction program activated by mammalian dna escaped from apoptotic dna degradation short hairpin rnas (shrnas) induce sequence-specific silencing in mammalian cells determinants of interferon-stimulated gene induction by rnai vectors rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene the rna helicase lgp inhibits tlrindependent sensing of viral replication by retinoic acid-inducible gene-i plasmacytoid dendritic cells: the key to cpg the rna modification database: update a lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by rna interference deletion of tlr alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection positive feedback regulation of type i ifn genes by the ifn-inducible transcription factor irf- toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mrna sirna and isrna: two edges of one sword hitching rig to action identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf triggering the interferon antiviral response through an ikk-related pathway the ends of the affair: capping and polyadenylation induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded sirnas is sequence-dependent and requires endosomal localization activation of the interferon system by short-interfering rnas irf and irf phosphorylation in virus-infected cells does not require double-stranded rna-dependent protein kinase r or ikappa b kinase but is blocked by vaccinia virus e l protein recognition of cytosolic dna activates an irf -dependent innate immune response regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i toll-like receptors and as essential components of innate immune defense against mouse cytomegalovirus infection type i interferons (alpha/ beta) in immunity and autoimmunity a general method for gene knockdown in mice by using lentiviral vectors expressing small interfering rna transcription and replication of nonsegmented negative-strand rna viruses eukaryotic ribonuclease p: a plurality of ribonucleoprotein enzymes visa is an adapter protein required for virus-triggered ifn-beta signaling deficient signaling in mice devoid of double-stranded rnadependent protein kinase shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses activation of the protein kinase pkr by short double-stranded rnas with single-stranded tails interferon action and apoptosis are defective in mice devoid of ' , '-oligoadenylate-dependent rnase l isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase key: cord- -rxzgfk k authors: a. lucchiari, maria; modolell, manuel; eichmann, klaus; a. pereira, carlos title: in vivo depletion of interferon-gamma leads to susceptibility of a/j mice to mouse hepatitis virus infection date: - - journal: immunobiology doi: . /s - ( ) - sha: doc_id: cord_uid: rxzgfk k the possible role of interferon-gamma (ifn-γ) in the resistance of a/j mice to mhv infection was investigated. monoclonal antibodies specific for ifn-γ, cd and cd molecules were administered in vivo to deplete selectively the ifn-y synthesized or the appropriate subset of t cells. the animals were then infected with mhv and the course of infection was followed by studying different parameters, such as, the mortality, the virus growth in the tissues and the ifn-γ synthesis in sera and peritoneal exudates. after mhv infection, a full resistance of control a/j mice was observed, in contrast to the high mortality rate observed among the depleted animals, where higher virus titers were found in different tissues. the ifn-γ synthesis in sera and peritoneal exudates of depleted mice, after mhv infection, drastically decreased when compared to that detected in control mice. the data presented are consistent with the hypothesis that ifn-γ plays an essential role in the resistance of a/j mice to mhv infection. the ai] mice have been reported to be resistant following mhv infection, since they develop a mild disease which disappears to days later ( , ) . the mechanisms suggested to be involved in the resistance include the antiviral state induced by ifn, the virus replication in target cells and the expression of a mono kine that demonstrates pro coagulant activity ( ) ( ) ( ) ( ) ( ) ( ) . we have recently shown that the resistance of ai] mice this work was supported in part by grants from the funda"ao de amparo a pesquisa do estado de sao paulo (fapesp) and conselho nacional de pesquisa (cnpq). m.a.l was a recipient of the deutscher akademischer austauschdienst (daad) during part of this research. abbreviations; mhv = mouse hepatitis virus ; ifn = interferon; fes = fetal calf serum; pfu = plaque-forming units; ip = intraperitoneally; ab = antibodies; pbs = phosphate buffer solution against our strain of mhv is acquired after immunization and the mechanism involved is dependent on the ifn -y synthesis and the macrophage sensitivity to ifn-y ( ). cd and cds t lymphocytes are essential for the development of protective immunity to several infections ( ) ( ) ( ) . one of the functions of these cells is the production of ifn-y, a major t celllymphokine, which can also be produced by nk cells. ifn-y activates macrophages, as assessed by the modulation of antiviral state ( ), the increased expression of class ii major histocompatibility complex gene products ( ) , the enhancement of phagocytosis ( ) , the induction of production of reactive oxigen intermediates ( ) and tumor necrosis factor ( ) . in the experiments described here, with the aim to investigate the in vivo participation of ifn-y in the development of resistance to mhv infection, mice have been depleted in vivo of the ifn-y synthesized after infection or the cd and cds subsets of t cells, using monoclonal antibodies. the data show a direct evidence that the in vivo neutralization of synthesized ifn-y during the infection led to susceptibility to mhv and that mice depleted of cd or cds t lymphocytes were unable to synthesize the high levels of ifn-y that were found in the controls after mhv infection, and died of acute hepatitis. these results are further support to the already proposed crucial role of ifn -y in the expression of resistance of ai] mice to mhv ( , , ) . six-week-old mice of the inbred ai] strain obtained from the institut pasteur, paris, france were bred in our mouse colony. the animals were periodically tested for the preexisting corona viruses or antibodies against mhv following procedures already described ( ). mhv was cultivated and titrated by plaque assay on l cells at dc as previously described ( ) . aliquots containing x plaque-forming units per ml (pfu/ml) were stored at - dc and used in all experiments. for the determination of mhv titer in tissues, the animals were sacrificed, and the peritoneal exudates and livers were obtained. these were ground, resuspended in ml of rpmi medium with % fetal calf serum (fcs) (gibco ltd, paisley, scotland), penicillin ( u/ml) plus streptomycin ( [tg/ml) and the virus titrated on l cells. the peritoneal exudates were collected by peritoneal lavage with ml of medium, centrifuged at x g for min and the virus in the supernatants titrated on l cells. the mhv titer obtained were expressed as pfu per milliliter of peritoneal exudate (pfulml), or pfu per gram of liver (pfu/g). cells producing rat monoclonal antibodies against mouse ifn-y (r - a ) was kindly donated by g. l. spitalny, trudeau institute, ny, usa ( ) and cells producing rat monoclonal antibodies specific for cd and cd determinants (yts . and yts . . respectively) were a gift of s. cobbold and h. waldmann, cambridge university, cambridge, u.k. ( ) . the antibodies were purified from tissue culture supernatants by affinity chromatography on protein a-sepharose (pharmacia, uppsala, sweden). the immunoglobulin concentration was determined by spectrophotometric measurement at nm. they were concentrated by dialysis and sterilized by filtration. the activity of the antibodies against ifn-y or cd and cd was tested, respectively, by neutralization of cytopathic effect or inhibition of t cell proliferation and cell binding by cytofluorometric assay. sera of normal rats in a pool were used as a source of normal rat antibodies. groups of ai] mice were immunized once by intraperitoneal (ip) injection of pfu of mhv inactivated by ultraviolet (uv) radiation. after a period of days they were injected ip with normal rat antibodies (controls) or purified antibodies against ifn-y, cd or cd in pbs. groups of mice were injected ip with ~g of purified antibodys at day - , - , , , , , and pfu of mhv at day o. the mice were maintained under sterile conditions for the duration of the experiment and the t cell depletion, mortality, virus growth in the tissues and ifn synthesis determined. a cytopathic effect reduction test technique using monolayers of l cells and encephalomyocarditis virus, described in detail in previous papers ( , ) , was used as an ifn assay. for characterization of ifn-a/~ and ifn-y antibodies to mouse ifn-a/~, produced in rabbits, and monoclonal antibodies to recombinant mouse ifn-y (holland biotechnology, leiden, holland) with activity of x and x neutralizing units per mg, respectively, were always used. one unit was defined as the amount of antibodies sufficient for neutralizing one unit of ifn. these antibodies showed no cross-reactivity. as shown in table , the treatment of ai] mice with antibodies against ifn-y or cd or cds t lymphocytes led to a high susceptibility to mhv infection. control a/j mice were fully resistant to the infection and the animals showed only a mild disease that disappeared days later. in contrast, the treated animals had a period of disease characterized by ruffled fur, hunching, loss of weight, diarrhea and general lassitude, that started earlier and remained for a longer period. most of the animals died of acute hepatitis to s days after the infection. it can be clearly seen in figure previous work on mhv infection postulated that both resistance gene( s) controlling the degree of viral replication in target cells and the intact immune response are required for resistance ( , ) . we have shown that a nutritionally induced hypercholesterolemia in resistant ai] mice caused susceptibility to mhv infection, and that the inhibition of the host resistance was a consequence of an impairment of kupffer cell functions, such as the sensitivity to the induction of an anti-mhv state by ifn ( ) . recently, we have proposed that the mechanism involved in the ai] mice resistance to mhv is dependent on the t cells activity and rely on the ifn-y production and the macrophage sensitivity to ifn-y ( , , ) . these mice have macrophages that are very sensitive to ifn -y in order to develop an anti-mhv state and are capable of producing reasonable amounts of ifn-y in the course of the immune response against mhv . thus, soon after initiation of infection, the virus particles could be neutralized and the infection cleared in a few days. alternatively, susceptible balb/c mice have macrophages that are not sensitive to ifn -y in order to develop an anti-mhv state, and despite high concentrations of ifn -y produced during the first days of infection, the macrophages cannot display a restriction of virus multiplication and these animals die to days after infection ( ) . in order to investigate the in vivo role of ifn-y during the mhv infection, we followed a direct approach by treating the ai] mice, previously immunized days before with uv-inactivated mhv , since the resistance is acquired after immunization ( ), with monoclonal antibodies against ifn-y, which has been shown to neutralize different activities of the ifn-y ( ) . since one of the features of cd and cd t lymphocytes is to produce and release ifn-y, it was of interest to investigate whether ai] mice treated with monoclonal antibodies against cd or cd molecules, in such a way that a depletion of cd or cd t lymphocytes occurred, could be rendered susceptible by an inhibition of ifn-y synthesized during the course of the infection by mhv . the data obtained showed that ai] mice treated with anti-ifn-y, anti-cd or anti-cd antibodies became susceptible to mhv , showing a longer period of disease (table ) , and that the treatment induced a neutralization or inhibition of the ifn -y synthesis (fig. ) that normally occurred in control mice during the mhv infection. the effect was due to the treatment with specific antibodies against ifn-y, cd or cd , since mice treated with normal rat antibodies behave like those only infected with mhv , showing that nonspecific effects of control antibodies were not involved. the finding that depletion of either cd or cd t cell subsets showed the same effect on the inhibition of ifn-y synthesis, mainly in the sera, after mhv infection is rather surprising and has been one of the subjects of our present investigations. although, in view of the crucial regulatory function of cd cells and the important role of cds cells as cytotoxic effector cells during a virusspecific immune response, the results cannot unequivocally exclude the interpretation that susceptibility of the immunosuppressed animals may be due to the absence of proper immune effector mechanisms, the lack of clearance of the virus in the peritoneum and liver of ai] treated and infected mice (figure ) , which leads to high rates of mortality (table ) , may be attributed to the lack of ifn-y in the serum or peritoneal exudate of them (fig. ) , which has been shown in vitro to be effective to restrict the mhv growth in target cells such as macrophages ( , , ) . furthermore, our previous observation that the lack of kupffer cells sensitivity to ifn correlated with the susceptibility of hypercholesterolemic ai] mice to mhv ( ) , is in further support to the idea that ifn-y plays a crucial role in the resistance to mhv . beside the direct evidence that ifn -y depleted mice became susceptible to mhv infection, our findings show the crucial intervention of cd and cds lymphocytes by one of their products, the ifn-y, in the development of ai] mice resistance to mhv infection. although the participation of other factors linked to the generation of the immune response can not be excluded, these results are in further support to the involvement of ifn-y in the mechanism of resistance against mhv infection. interactions between mouse hepatitis viruses and primary cultures of kupffer and endothelial liver cells from resistant and susceptible inbred mouse strain role of macrophages and interferon in natural resistance to mouse hepatitis virus infections adult mouse hepatocytes in primary monolayer culture express genetic resistance to mouse hepatitis virus type susceptibilitylresistance to mouse hepatitis virus strain and macrophage procoagulant activity are linked and controlled by two non-h- linked genes immunopathology of mouse hepatitis virus type infection. . role of humoral and cell mediated immunity in resistance mechanisms a major role of macrophage activation by interferon gamma during mouse hepatitis virus type infection. . genetically dependent resistance a major role of macrophage activation by interferon gamma during mouse hepatitis virus type infection. ii role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. v. protective role in mouse hepatitis virus type infection of susceptible and resistant strains of mice acquired immunity dependence of a/j mice resistance to mouse hepatitis virus infection: dependence on interferon gamma synthesis and macrophage sensitivity to interferon gamma induction of maloney murine sarcoma virus tolerance in adult mice by anti-cd monoclonal antibody treatment roles of cd -and cd -bearing t lymphocytes in the immune response to erythrocytic stages of plasmodium chabaudi effective clearance of mouse hepatitis virus from the central nervous system requires both cd + and cd + t cells differential regulation of class ii mhc determinants on macrophages by ifn gamma and il- induction of crisis forms in the human malaria parasite plasmodium falciparum by gamma interferon activated, monocyte macrophages identification of interferon gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity activation of mouse peritoneal macrophages in vitro and in vivo by interferon gamma induction of natural killer cells and interferon during mouse hepatitis virus infection of resistant and susceptible inbred mouse strains monoclonal antibody to murine gamma interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities therapy with monoclonal antibodies by elimination of t-cell subsets in vivo inhibition of mouse hepatitis virus type multiplication in activated kupffer cells. braz genetically determined resistance to mouse hepatitis virus type is expressed in hematopoietic donor cells in radiation chimeras increased susceptibility of mice to mhv infection induced by hypercholesterolemic diet: impairment of kupffer cell function laboratorio de imunologia viral, a v. dr. vital, brasil key: cord- -yh zi ee authors: weiss, r.c.; oostrom-ram, t. title: effect of recombinant human interferon-alpha in vitro and in vivo on mitogen-induced lymphocyte blastogenesis in cats() date: - - journal: vet immunol immunopathol doi: . / - ( ) -m sha: doc_id: cord_uid: yh zi ee the effect of recombinant human interferon-alpha (rhuifn-α) in vitro and in vivo on mitogen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. pre-incubation of isolated feline peripheral blood lymphocytes (pbl) in vitro with either ( ) or ( ) international units (u) of rhuifn-α for h significantly suppressed (p< . and . , respectively) blastogenic responses to the phytomitogens concanavalin a (con a) and pokeweed mitogen (pwm). lower doses of ifn (range, – (− ) u/ml) neither suppressed nor enhanced mitogenesis. in the absence of phytomitogens, incubation of pbl with ( )– ( ) u (p< . ) or u (p< . ) of rhuifn-α/ml resulted in a significant decrease in incorporation of [methyl-( )h] thymidine into newly synthesized cellular dna. cultures of pbl exposed continuously for days to rhuifn-α doses of ( ) u/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of ifn apparently were independent of any direct cytotoxic effects. to investigate the dose-response effects of rhuifn-α in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on consecutive days before and then h after each cat was inoculated intramuscularly with either a high dose ( ( ) u/kg), moderate dose ( ( ) u/kg), or a relatively low dose ( ( ) u/kg) of rhuifn-α. cats inoculated with ( ) u ofrhuifn-α/kg had significantly reduced (p= . ) blastogenic responses to con a at h postinoculation compared to preinoculation values; mean pwm responses were also decreased, but this effect was not statistically significant. in contrast, inoculation of cats with either ( ) or ( ) u of rhuifn-α/kg significantly enhanced (p= . or . , respectively) con a-induced blastogenesis and had no discernible effect on pwm responses. these findings suggest that very high doses of rhuifn-α given parenterally may be associated with suppression of certain t-cell responses in cats; conversely, much lower doses may be immunoenhancing. gen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. pre-incubation of isolated feline peripheral blood lymphocytes (pbl) in vitro with either or international units (u) of rhuifn-o~ for h significantly suppressed (p < . and . , respectively) blastogenic responses to the phytomitogens concanavalin a (con a) and pokeweed mitogen (pwm). lower doses of ifn (range, - - u/ml) neither suppressed nor enhanced mitogenesis. in the absence of phytomitogens, incubation of pbl with - u (p < . ) or u (p< . ) of rhuifn-o~/ml resulted in a significant decrease in incorporation of [methyl- h] thymidine into newly synthesized cellular dna. cultures of pbl exposed continuously for days to rhuifn-o~ doses of u/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of ifn apparently were independent of any direct cytotoxic effects. to investigate the dose-response effects of rhuifn-~ in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on consecutive days before and then h after each cat was inoculated intramuscularly with either a high dose ( u/kg), moderate dose ( u/kg), or a relatively low dose ( l s u/kg) of rhuifn-ol. cats inoculated with u of rhuifn-~fkg had significantly reduced (p = . ) blastogenic responses to con a at h postinoculation compared to preinoculation values; mean pwm responses were also decreased, but this effect was not statistically significant. in contrast, inoculation of cats with either or u of rhuifn-o~/kg significantly enhanced (p-- . or . , respectively) con a-induced blastogenesis and had no discernible effect on pwm responses. these findings suggest that very high doses of rhuifn-o! given parenterally may be associated with suppression of certain t-cell responses in cats; conversely, much lower doses may be immunoenhancing. introduction interferons (ifn) are a group of cellular proteins that inhibit viral replication, modulate immune responses, inhibit normal cellular division and have antitumor activity (borden and ball, ) . there are several classes of ifn ( a, fl and y) which differ on the basis of their antigenic, biological and physic ochemical properties (mannering and deloria, ) . because ifn (particularly human ifn-c~ ) has antiviral and immunomodulatory activities that cross species lines (pallikoff et al., ; desmyter et al., ; gresser et al., ; krakowka et al., ) and because recombinant dna-produced human ifn is now available in relatively large quantity, the use of human ifn in veterinary medicine has increased. human ifn-c~ has been used clinically in cattle afflicted with respiratory diseases (roney et al., ; cummins and hutcheson, ) , in cats infected with either feline leukemia virus (felv) or feline infectious peritonitis virus (fipv) , and in dogs infected with canine parvovirus (dr. j. cummins, unpublished data, ) . dosages of ifn used clinically in domestic animals have been empirical and extrapolated largely from studies in persons or mice using interferon preparations that can differ in purity and potency. unfortunately, studies of the immunological effects of homologous or heterologous ifn in domestic animals have been lacking; the effects of human ifn-c~ in vitro on canine immune responses, however, have recently been reported (krakowka et al., ) . our interest in studying ifn is related to its application clinically as an antiviral and immunomodulating drug in cats with viral diseases. the effects of heterologous ifns on feline immune responses in general are not known. resistance to diseases such as fip is associated with effective cell-mediated immunity (cmi) (pedersen and floyd, ; ; cats infected with other viruses like felv may have severely impaired t-cell responses (rojko and olsen, ) . obviously, an understanding of the relationship between ifn dosage and modulation of cmi is necessary prior to recommending ifn as treatment for viral diseases where stimulation of cmi is required. mitogen-induced lymphocyte blastogenesis, which is a widely used in vitro assay for evaluation of cmi (oppenheim and schechter, ) , has been used previously in cats to assess alterations in cmi induced by immunomodulating agents such as cyclosporin (gregory et al., ) . in the studies reported here, the effects of doses of human ifn-c~ in vitro and in vivo on lymphocyte blastogenesis in normal cats was investigated. the cats used in these studies were healthy, - -month-old specific-path-ogen-free (spf) males and females, weighing approximately . kg. the cats were purchased from a commercial breeder (liberty laboratories, liberty corners, nj ) and were felv test-negative (by elisa) and feline coronavirus antibody-negative prior to the studies. the cats were housed separately in cages located in the scott-ritchey animal isolation facility and were tested and cared for according to humane standards as set forth in the "guide for the care and use of laboratory animals" (publication no. - , national institutes of health, bethesda, md). all experimental protocols were approved by an independent animal welfare committee prior to the studies. the recombinant dna-derived (bgl-ii restriction endonuclease-specified) human leukocyte (alpha) hybrid {subtypes a/d) ifn (rhuifn-a), lot no. ro- - , was kindly supplied by dr. richard cordts, hoffman laroche, nutley, nj. the ifn was stored lyophilized (at a concentration of x international units (u)/vial) at - °c prior to use. the ifn was diluted before use in hank's balanced salt solution (hbss; ph . ) and was sterilefiltered through a . ]~m cellulose acetate membrane (nalge co, rochester, ny ). the biological activity of the rhuifn-c~ (expressed in u/ml of antiviral activity) was determined in the manufacturer's laboratory, using an international reference standard for human leukocyte ifn. cats were lightly anesthetized with an intramuscular injection of ketamine hydrochloride (vetalar; parke, davis & co, detroit, mi) and blood collected by jugular venipuncture into glass syringes containing heparin ( units/ml of blood). peripheral blood lymphocytes (pbl) were isolated and prepared as previously described (cockerell et al., ; tham et al., ) . briefly, pbl were separated by ficoll-diatrizoate (histopaque- ; sigma chemical co, st louis, mo) gradient separation and the interface mononuclear cells collected and washed in hbss. the washed cells were resuspended to x viable cells/ml in tissue culture growth medium (gm) consisting of rpmi- (gibco, grand island, ny) supplemented with % heat-inactivated fetal bovine serum, u of penicillin/ml and /lg of streptomycin/ml along with . mm l-glutamine and mm hepes. for the in vitro ifn studies, pbl ( . x cells, . ml) were pipetted into wells of sterile -well flat-bottom microtitration plates (corning glass works, corning, ny) and then incubated with gm containing varying amounts of rhuifn-a or gm only ( . ml/well) for h at °c in humidified air containing % co . for the in vivo studies, preincubation of pbl with ifn was omitted. the cell suspensions were then cultured h with either concanavalin a (con a) ( . ttg/well) or pokeweed mitogen (pwm) ( . ttg/well), or they were not treated with mitogens ( replicates/treatment). previous titrations in feline pbl indicated that the concentrations of mitogen used were optimal for our assay. to each well, . /~ci ( . ml ) of [methyl- h ]thymidine (specific activity . ci/mmol) (dupont, nen research products, boston, ma) was added for the last h of incubation. the cultures were then stored at - ° c until harvested. cells were harvested using a semi-automatic multiple cell microharvester (bellco glass, vineland, nj) so that the cellular proteins were collected onto glass fiber filter paper strips (bellco). the paper strips were dried at °c for min and the filter discs transferred to scintillation vials into which ml of toluene base cocktail (scinti verse ii; fischer scientific, fairlawn, nj) was added. the incorporation of [methyl- h]thymidine into newly synthesized cellular dna was quantitated in a liquid scintillation spectrometer (lkb-wallac oy, turku, finland) using channels ratio method of quench correction. the net counts per minute (c.p.m.) of a total of wells for each variable were averaged to obtain the mean c.p.m. the stimulation index (s.i.) was determined by dividing the mean c.p.m, of the mitogen-stimulated (or rhuifn-~-stimulated only) cells by the mean c.p.m, of unstimulated (media control) cells. stock solutions of ifn, thymidine and fetal calf serum were prepared from the same lots, respectively, prior to the studies and only fresh reagents were used. to determine the potential cytotoxic effect of rhuifn-c~ on feline pbl, lymphocytes were isolated from eight normal cats as described before and were exposed to varying amounts of rhuifn-c~ continuously for several days. briefly, freshly isolated pbl ( . × cells, . ml) were cultured with gm ( . ml/ well) containing rhuifn-c~ (ranges, - . u/ml) or gm only for h at °c in -well microtitration plates ( replicates/dose). the nonadherent cells were aspirated from a total of wells for each ifn dose; the number of viable cells, determined by trypan blue dye exclusion, were counted in duplicate using a neubauer hemocytometer. results were expressed as mean viable cell density (number of viable cells/ml) in rhuifn-c~-treated or medium control cultures. a study was designed to evaluate the effects of giving varying amounts of rhuifn-c~ parenterally on lymphocyte blastogenesis in cats. cats were randomly assigned to one of three experimental groups and were treated as follows: a high-dose ifn group (n= cats) received a single injection i.m. of rhuifn-~ at a dosage of u/kg; a moderate-dose ifn group (n= cats) likewise received u of rhuifn-c~/kg; and a low-dose ifn group (n= cats) similarly received u of rhuifn-c~/kg. three additional cats were inoculated similarly with hbss (diluent controls). cats were evaluated by lymphocyte blastogenesis in response to phytomitogens at h postinoculation. in order to minimize normal diurnal variation in test results, all cats were evaluated by lymphocyte blastogenesis at -h intervals on successive days preceding inoculation and then h postinoculation. for each group of cats, a single preinoculation mean con a or pwm response ( -day average) was determined and then compared statistically against the corresponding postinoculation group mean. each group of cats was evaluated separately on alternate weeks. the one-tailed student's t test was used to determine statistical significance between groups. all data analysis was performed using a computerized statistical analysis program (abstat; anderson-bell, canon city, co). a conservative number of degrees of freedom was used (i.e., one for each animal rather than one for each well). p values of ~< . were considered significant. incubation of normal feline pbl with doses of rhuifn-a in vitro suppressed lymphocyte proliferative responses to con a and pwm (table ) . suppression of blastogenesis was dose-dependent; significant inhibition occurred at relatively high in vitro doses of rhuifn-c~ ( or u/ml). doses of u of rhuifn-c~/ml also decreased mean con a or pwm responses, but this effect was not statistically significant. lower doses of rhuifn-a ( . - - u/ml) neither suppressed nor enhanced mitogenesis. incorporation of [methyl- h ] thymidine into newly synthesized cellular dna in the absence of phytomitogens was significantly inhibited in feline pbl cultures exposed to rhuifn-~ doses of - . u/ml compared to untreated pbl (table ) . mean thymidine incorporation (c.p.m.) in cultures treated with - or with . u/ml was approximately % or %, respectively, of the thymidine incorporation measured in untreated cells. varying amounts of rhuifn-c~ ( - . u/ml) were added to pbl cultures to determine whether direct cytotoxic effects from the ifn itself may have contributed to the dose-related suppression of blastogenesis observed in vitro. differences in mean viable cell density between untreated pbl cultures and those treated with ifn, however, were not observed. (fig. ) . to determine the effects of varying amounts of rhuifn-a in vivo on mitogen-induced blastogenesis, cats in each of three groups were given a single injection of either a high ( u/kg), moderate ( u/kg), or relatively low dose ( u/kg) of rhuifn-~ and then evaluated h later. to minimize normal diurnal variation in responses, a -day average of daily mean responses was determined preinoculation for each group. day-to-day variation in blastogenic responses of individual cats evaluated on consecutive days of the same week was not significant (p > . , n--- ; data not shown). cats inoculated with a single high dose of rhuifn-~ had a significant suppression (p= . ) in con a-induced lymphocyte blastogenesis h postinoculation compared to preinoculation values ( fig. a) . mean pwm responses were also decreased, but this effect was not statistically significant. in contrast to the cats inoculated with a high dose of rhuifn-~, cats given or u of rhuifn-c~/kg had significantly enhanced (p= . or . , respectively) blastogenic responses to con a; pwm responses, however, were unaffected ( fig. b and c ) . inoculation of cats with hbss alone had no significant effect on mitogen-induced blastogenesis h postinoculation (data not shown). the effects of ifn in vitro or in vivo on the immune system are manyfold and sometimes apparently contradictory. overall, it appears that ifn can either stimulate or suppress various arms of the immune response, depending on the timing of administration and dosage (epstein, ) . ifn in general seems to inhibit immunologic responses when given prior to an immunogen but enhances responses if given some days later (white and fenner, ) . moreover, high doses of ifn can produce opposite effects or annul the responses obtained with much lower doses. for example, very low doses of ifn-c~ in vitro or in vivo may stimulate development of antibody-forming spleen cells in mice, whereas high doses are immunosuppressive (braun and levy, ; epstein, ) . the addition of low doses of ifn ( or units/ml) in the primary mixed lymphocyte reaction increases the cytotoxic response in mice severalfold, but larger doses ( units/ml ) depress the response (fradelizi and gresser, ) . similarly, low doses of ifn can enhance lymphocyte blastogenesis in mice or persons, whereas large doses are suppressive (miorner et al., ; taylor-papadimitriou, ) . depending on the timing of administration, extremely low doses of ifn-~, fl ( × -lo u) in mice significantly increase the number of antibody-secreting cells in spleen and strongly stimulate cytotoxic activities of allospecific t-cells (daurat et al., ) . the results-of this study showed that very high doses of rhuifn-~ either in vitro or in vivo suppressed mitogen-induced lymphocyte proliferative responses in cats. the rhuifn-~ also directly inhibited the in vitro incorporation of [methyl- h ]thymidine into newly synthesized cellular dna of unstimulated pbl and at much lower ifn concentrations than those required to suppress mitogen-induced blastogenesis. the inhibitory effects of various types of ifn on dna synthesis and lymphocyte blastogenesis after mitogenic stimulation have been described previously in different species, including mice, cattle, and persons (lindahl-magnusson et al., ; blomgren et al., ; bielefeldt-ohman and babiuk, ; kim et al., ; roth and frank, ). ifn has also been shown to directly inhibit thymidine incorporation into the dna of normal cells, including both lymphoid and epithelial cells, in the absence of mitogenic stimulation (brouty-boye and tovey, ; stadler et al., ; roth and frank, ) . although the suppressive mechanism (s) associated with rhuifn-c~ on dna synthesis and lymphocyte blastogenesis were not investigated in our study, it is possible that high doses of ifn in vitro enhanced lectin-binding on lymphocytes and stimulated suppressor cell activities. ifn can markedly enhance the binding of lectins to lymphocyte membranes (miorner et al., ) , and suppressor cells are activated by high doses of mitogen, particularly con a (piguet et al., ) . the ifn-mediated suppression of lectin-induced blastogenesis observed when feline pbl were preincubated h with relatively high doses of rhuifna was also seen h after cats were inoculated i.m. with very high doses ( u/kg) of rhuifn-a. significant decreases in blastogenic responses to t-cell mitogens (con a) in particular were observed after parenteral administration of high-dose rhuifn-a. although responses to pwm (which acts both as a b-and t-cell mitogen) (heegaard and muller, ) were diminished, this effect was not statistically significant, suggesting that t-cell responses perhaps were somewhat more sensitive to the suppressive actions of high doses of ifn in vivo. curiously, lower parenteral doses of rhuifn-a ( - u/kg) significantly enhanced the blastogenic responses of pbl after stimulation with t-cell mitogens (this phenomenon, however, was not observed when low doses of ifn were incubated in vitro with pbl). an inverse relationship between ifn dose and lymphocyte proliferative responses similar to that which we observed in cats has been documented previously in vitro and in vivo in mice and persons (miorner et al., ; taylor-papadimitriou, ; kim, ) . seemingly, a mechanism associated with this phenomenon may have been ifninduced activation of suppressor cells. bielefeldt-ohmann and babiuk ( ) reported that in vitro treatment of bovine pbl with recombinant bovine ifngamma (rboifn- ) induced suppressor cells which may have competed with interleukin (il)- ; moreover, the suppression in lymphocyte blastogenesis after in vivo administration of high doses of rboifn- was found to be reversible by addition of human il- to the lymphocyte cultures. lower amounts of ifn, however, may actually enhance the cellular immune response by selectively blocking suppressor pathways (knop et al., ) . in support of this theory, daurat et al. ( ) demonstrated enhanced cytotoxic activities of allospecific t-cells and enhanced cytotoxic responses of nk cells in mice inoculated several times with ifn-a, fl at dosages as low as . or × -lo u. the inverse dose-response effects of ifn, particularly its biologic activity at very low pharmacologic doses, has suggested a predominantly hormone-like action of ifn as a homeostatic regulator of immune functions (daurat et al., ) . undoubtedly, an understanding of the dose-response effects of ifn on normal feline immune responses is imperative when considering ifn therapy in cats with disease. additional in vitro and in vivo studies on the effects of ifn on t-and also b-cell responses in cats will be required so that specific recommendations concerning the use of ifn in feline viral or other diseases rationally can be established. alteration of some leukocyte functions following in vivo and in vitro exposure to recombinant bovine alpha-and gamma-interferon effect of human leukocyte interferon on the response of lymphocytes to mitogenic stimuli in vitro interferon: biochemical, cell growth inhibitory, and immunologic effects interferon preparations as modifiers of the immune response inhibition by interferon of thymidine uptake in chemostat cultures of l cells phytomitogen-and antigeninduced blast transformation of feline lymphocytes low dosage of interferon to enhance vaccine efficiency in feedlot calves oral use of human alpha interferon in cats immunomodulatory activity of low doses of interferon o~, fl in mice human interferon that crosses species lines the effects of interferons on the immune response in vitro and in vivo interferon inhibits the generation of allospecific suppressor tlymphocytes response to isoantigens and mitogens in the cat: effects of cyclosporin a pronounced antiviral activity of human interferon on bovine and porcine cells lectins and the immune system interferon inhibition of il- -mediated lymphocyte proliferation inhibition of the t-suppressor circuit of delayed-type hypersensitivity by interferon the effects of human interferon-alpha upon in vitro canine immune responses interferon inhibits dna synthesis induced in mouse lymphocyte suspensions by phytohaemagglutinin or by allogeneic cells the pharmacology and toxicology of the interferons: an overview regulation of mitogen-induced lymphocyte dna synthesis by human interferon of different origins manual of clinical immunology tissue specificity of interferons prepared in various tissue cultures experimental studies with three new strains of feline infectious virus: fipv-ucd , fipv-ucd , and fipv-ucd induction or suppression of b cell proliferation and differentiation by phytohemagglutinin or concanavalin a in mouse spleen cell cultures the immunobiology of the feline leukemia virus effect of human leukocyte interferon on prevention of infectious bovine rhinotracheitis virus infection recombinant bovine interferon-y as an immunomodulator in dexamethasone-treated and non-treated cattle effect of recombinant alpha a-interferon on dna synthesis and differentiation of human keratinocytes in vitro effects of interferons on cell growth and function optimal conditions for in vitro blastogenesis of feline peripheral blood lymphocytes evaluation of immunity to feline infectious peritonitis in cats with cutaneous viral-induced delayed hypersensitivity effect of interferon or propionibacterium acnes on the course of experimentally induced feline infectious peritonitis medical virology ( rd edn research program. the authors thank ms. lewanne french and dr. mitzi martinez for their assistance. key: cord- -zggk x q authors: lindemans, caroline a.; kimpen, jan l. l. title: the immune response to viral lower respiratory tract infection date: journal: hot topics in infection and immunity in children ii doi: . / - - - _ sha: doc_id: cord_uid: zggk x q viruses are responsible for the majority of respiratory infections in childhood,causing considerable morbidity and mortality. it is estimated that in the united states approximately $ million per year is spent on medical costs for respiratory syncytial virus (rsv) related disease alone (paramore et al., ). viruses cause a variety of respiratory diseases in children from the common cold to life-threatening pneumonia and bronchiolitis. the host reacts to a viral infection with a combination of innate and adaptive immune mechanisms, usually resulting in the clearance of the virus and clinical recovery. however, there is an accumulating evidence for a number of viral infections that the host immune response actually enhances disease in the course of clearing virus from the infected organs. interestingly, the effectiveness of the immune response seems to be dependent on the age and probably genetic background of the child. this has important implications for treatment as well as vaccine development. viruses are responsible for the majority of respiratory infections in childhood, causing considerable morbidity and mortality. it is estimated that in the united states approximately $ million per year is spent on medical costs for respiratory syncytial virus (rsv) related disease alone (paramore et al., ) . viruses cause a variety of respiratory diseases in children from the common cold to life-threatening pneumonia and bronchiolitis. the host reacts to a viral infection with a combination of innate and adaptive immune mechanisms, usually resulting in the clearance of the virus and clinical recovery. however, there is an accumulating evidence for a number of viral infections that the host immune response actually enhances disease in the course of clearing virus from the infected organs. interestingly, the effectiveness of the immune response seems to be dependent on the age and probably genetic background of the child. this has important implications for treatment as well as vaccine development. viral infections play an important role in both childhood and adult asthma. they might be instrumental in the inception of asthma and are associated with the majority of exacerbations in asthmatic individuals (johnston et al., ; bont et al., ) . in respect to the role of viruses in the pathogenesis of acute and chronic airway disease in children, it is of utmost importance that we gain a proper understanding of the underlying mechanisms involved in order to design effective therapeutic and preventive strategies. although viral respiratory tract infections are considered to be mainly pediatric diseases, there is an increasing acknowledgement of their pathogenic potential in the immunocompromised host of all age groups and in the elderly. causative agent is not identified. when an upper respiratory tract infection in an infant progresses to lower respiratory tract disease, bronchiolitis and pneumonia are most common. both disease entities are hard to differentiate and no clinically relevant differences with regard to outcome have been identified (van woensel et al., ) . rsv belongs to the paramyxoviridae family and the genus of pneumovirus. it is an enveloped unsegmented single-stranded rna-virus of which two subtypes are known (a and b) . a clear relationship between subtype and disease severity has not been established (kneyber et al., ) . since rsv infection does not lead to complete immunity, reinfection is common. immaturity of the immune system during initial infection seems to be the main cause of incomplete memory-response although an as yet undefined mechanism of partial immune evasion by rsv cannot be ruled out . recently, it was suggested that rsv could cause persistent infection or latency (dakhama et al., ; schwarze et al., ) , although the significance of this is not clear. rsv affects % of infants in the first year of life, and by the age of two nearly all children have been infected. (figure . ) it is likely that a specific balance and timeframe of changes in air temperature and humidity are responsible for the well-defined yearly winter outbreaks of rsv (stensballe et al., ) . in most infants as well as in older children and adults, rsv is the cause of upper respiratory tract infection with mild symptoms. however, in the very young, the infection spreads to the lower respiratory tract in approximately % of cases. one to three percent of infants develop bronchiolitis or pneumonia requiring hospitalization, with a considerable number requiring mechanical ventilatory support. apart from the obvious respiratory symptoms, very young children frequently present with atypical symptoms such as impaired feeding, vomiting, lethargy, and apnea (kneyber et al., ) . several risk factors for more severe disease have been identified, including age less than weeks, prematurity, pre-existent cardiorespiratory disease and immunological impairment (bont and kimpen, ) . respiratory symptoms are directly related to airway pathology. necrosis of the airway epithelium is a key phenomenon resulting in sloughing of the epithelial cells. together with a dramatic influx of inflammatory cells into the airways and increased mucus production, this leads to the formation of copious secretions that block the small airways. mucosal edema and bronchospasm through irritation of subepithelial nerve endings further compromise airway diameter. although antibiotics are prescribed for up to % of children with lower respiratory tract infections, proof of bacterial superinfection is only found in a minority of patients and the role of this event in the pathogenesis of severe disease remains controversial (purcell and fergie, ; bloomfield et al., ) . on the other hand, it has been demonstrated that rsv infection of airway epithelial cells in vitro enhances adherence of s. pneumoniae (hament et al., ) . the influenza virus belongs to the orthomyxoviridae family and is an enveloped segmented single-stranded rna (ssrna) virus (table . ). the structural proteins of the virion are encoded by separate gene segments and include three viral rna polymerases, nucleoprotein, matrix, and the hemagglutinin (ha) and neuraminidase (na) surface glycoproteins. on the basis of their nucleocapsid and matrix protein antigens, the influenza viruses are divided into three distinct immunological types (a, b, and c). although all three influenza viruses cause respiratory disease in humans, only a and b are known to cause epidemics. induction of a memory-response results in long-lasting immunity and it is the antigenic variation that is responsible for frequent reinfection with the virus. the most antigenic variation is seen in the virus that infects both animals and humans, influenza a. fourteen subtypes of hemagglutinin (h -h ) and nine types of neuraminidase (n -n ) are circulating in nature. the segmentation of the genome makes exchange of genetic material between subtypes possible, resulting in the structural changes observed in influenza, which has caused pandemics in the past. when two different subtypes of influenza a virus infect the same cell, major changes (antigenic shifts) can occur through rearrangement of genetic segments from both infecting viruses. minor changes (antigenic drifts) in na and/or ha proteins occur through accumulation of point mutations, and provide a mechanism for the virus to escape protective antibodies and cause respiratory symptoms every year. influenza virus epidemics are difficult to separate in time from rsv epidemics, and the diseases caused by both viruses can also be difficult to differentiate (zambon et al., ) . (figure . ) compared to other viruses, morbidity caused by influenza is high in all age groups. children with influenza infection of the respiratory tract are more likely to present with fever. infants aged less than months and older children with an impaired immune system, or other serious health problems, have a higher risk of hospitalization and mortality. subclinical infections with influenza in children are common, suggesting children can be an important reservoir and source of transmission. yearly updated vaccines are available and effective, and recently it has been proposed to extend the current recommendations to children less than years of age, children with recurrent acute otitis media or respiratory tract infections, and healthy children attending day-care centers or elementary schools (principi and esposito, ). adenoviruses, belonging to the adenoviridae family and the genus mastadenovirus, are a group of dna-viruses of which at least serotypes are known. for lower respiratory disease, subtypes , , , , , , and are most important. it is an icosahedral capsid virus with extruding fiber proteins, which are required for viral entry to epithelial cells (howitt et al., ) . although human adenoviruses are ubiquitous, and cause primary infection in the first year of life, there is geographical variation in the distribution of serotypes and in the association of serotypes with different age groups. in europe, adenovirus is the cause of infection in approximately % of hospitalized patients with viral lower respiratory tract disease. however, in some south american and asian countries, adenovirus is the second most prevalent pathogen for acute lower respiratory tract infection in children after rsv (carballal et al., ) . although adenovirus infections in general occur the whole year round, respiratory adenovirus infections are most common during late winter, spring, and early summer. adenovirus type , acquired by inhalation, has been associated with more severe lower respiratory tract disease (larranaga et al., ) . subtype-specific immunity occurs. however, some types are capable of establishing persistent asymptomatic infections in tonsils, adenoids, and intestines of infected hosts, and shedding can occur for months or years. children under -years old. though it is the causative agent of similar disease entities, hospitalizations occur four times less frequently than for rsv infections (hall, ) . two subtypes are clinically important respiratory pathogens in children, piv- and piv- . piv- is the main cause of croup in - -year olds, while piv- is responsible for parainfluenza bronchiolitis in children under -months old. piv- only causes mild upper respiratory tract infections. because of acute narrowing of the subglottic region of the larynx, moderate to severe croup may require emergency management with systemic or inhaled corticosteroids, which are effective in improving stridor in a few hours (cetinkaya et al., ) . the hallmark cytopathic effect of acute infection with piv- is comparable to that of rsv with extensive cell fusion resulting in syncytium formation. for fusion to occur, two piv glycoproteins are required, including the hemagglutinin-neuraminidase (hn) glycoprotein interacting with host cell sialic acid receptor and the viral fusion (f) glycoprotein. rhinovirus infections account for the largest number of respiratory tract infections in children. however, rhinovirus infections produce mild symptoms compared to rsv, piv, and influenzavirus. most of the symptoms caused by rhinovirus are confined to the upper respiratory tract. although present in the community the whole year round, rhinovirus infections peak at the onset of fall, which is probably related to schools starting after summer break. rhinoviruses can cause severe lower respiratory tract infection (guittet et al., ; papadopoulos, ) and in immunocompromised patients, life-threatening pneumonia. rhinovirus is a positive-stranded rna-virus belonging to the picornavirus family and over serotypes exist, making it difficult to develop an effective vaccine. rhinovirus subtypes have been divided into a major and minor group with respect to the receptor used for cell entry (table . ). major group rhinoviruses use epithelial intracellular adhesion molecule l (icam- ) for cell entry, while minor group viruses bind to the low-density lipoprotein (ldl) receptor. during rhinovirus infection, a predominant granulocyte and monocyte recruitments are observed. while specific antibody production occurs, it is probably not required for viral clearance, although neutralizing antibodies can provide some temporary protection against rhinovirus reinfection (van kempen et al., ) . icam- blocking antibodies have also been utilized and have been shown to decrease inflammation in vitro. there are, however, indications that rhinovirus can adapt to this with changes in receptor usage (reischl et al., ). in , a new respiratory virus was identified in the netherlands causing infections similar to rsv in children (van den hoogen et al., ) . the reported incidence rate of human metapneumovirus (hmpv) infection in children with acute respiratory symptoms varies between % and %, of which three-quarters occur in children less than -year old (williams et al., ) . by the age of years, approximately % of children have developed antibodies to hmpv. hmpv very much resembles rsv in its clinical spectrum, varying from coryza to bronchiolitis and pneumonia. however, hmpv is less likely to cause pneumonia than rsv and influenza virus. children with hmpv infection present less frequently with atypical symptoms such as vomiting, and on physical examination, rales and wheezing are found less often. co-infection with rsv and hmpv does occur and has been suggested to result in more severe disease (greensill et al., ) . there is some evidence that secondary hmpv infection occurs frequently in childhood, probably accompanied only by mild symptoms (ebihara et al., ) . several investigators have found chemokine profiles during acute infection to be different in children with hmpv infections compared to those with rsv infections, with higher interleukin- (il- ) and lower rantes concentrations in hmpv patients. however in another study, inflammatory cytokine (il- , tnf-␣, il- ␤) levels in respiratory secretions were -fold lower than in children infected with rsv (jartti et al., ; laham et al., ) . tthe physiological relevance of these observations remains unclear. the outbreak of severe acute respiratory distress syndrome (sars), which started in late in east asia, and spread throughout the world during that winter, was found to affect mainly health-care workers and close contacts of diseased individuals. sars, which was proved to be caused by a new coronavirus, induces an atypical pneumonia with fever, dry cough, and shortness of breath. many adults also suffer from myalgia, dizziness, chills, and rigors. it was concluded from postmortem examinations that sars-pathology is primarily caused by immunological damage to the lungs. the interstitial space of the lungs was mainly filled with mononuclear infiltrates and there was diffuse hemorrhage on the lung surface. sars coronavirus (sars-cov) spreads mainly via the respiratory route, the epithelial cell being its primary target cell. as for other coronaviruses, the spike proteins, s for cell entry and s for fusion, also seem to be important for sars entry of host cells, despite the fact that sars is only - % homologous to other coronaviruses. very recently, angiotensine converting enzyme (ace ) was identified as the host cell receptor for sars-cov (li et al., ) and surface expression of ace on alveolar epithelial cells was demonstrated (hamming et al., ) . transmission of sars-cov occurs by droplets and most cases have occurred through close contact exposure. however, recently evidence of airborne transmission has emerged (yu et al., ) . although many individuals were infected in the initial weeks of the epidemic and the disease spread rapidly over several countries, the numbers of infected children stayed relatively low in all regions (Ͻ %). furthermore, children tend to develop less severe disease. after an incubation period of - days, similar to adults, infected children developed symptoms of a mild upper respiratory tract infection, clinically indistinguishable from other common colds. none of the pediatric sars cases in hong kong turned out to be fatal and only one adolescent required mechanical ventilation (leung et al., ) . adolescents are more likely to develop severe disease, as observed in adult sars patients (leung et al., ) . a sore throat and a high initial and peak peripheral blood neutrophil count were found to be independent risk factors for severe disease in children with a laboratory-confirmed sars infection. furthermore, children seemed to spread the disease less easily to others and there have appeared no reports in the literature demonstrating transmission from children to other individuals. many children with laboratory-confirmed sars do not meet the who criteria for diagnosis of sars. as children seem to have a much milder clinical course, the term "sars" may not represent the disease in children very well. the role of the innate immune system in viral lower respiratory tract infection has not been studied intensively until recently. studies have focused on the adaptive immunity with the goal of developing a vaccine, for example, for rsv. understanding the mechanisms underlying primary and recurrent viral infection has attracted increased attention. the immunological response against viral invasion of the lower respiratory tract comprises both adaptive and innate immune response mechanisms with both beneficial as well as detrimental characteristics. the innate response occurs in the early phase of the infection and increasing evidence suggests that these early events determine disease course and possibly even long-term outcome (garofalo and haeberle, ; tasker et al., ) . for the rest of the discussion on immunological phenomena, focus will be on rsv as a prototype. epithelial cells are key regulators of the innate immune response against viral infections (garofalo and haeberle, ) , producing a number of inflammatory mediators in response to rsv infection. these include cytokines (interleukin- , - , tumor necrosis factor (tnf)-␣), several chemokines (il- , macrophage inflammatory protein (mip)- ␣, monocyte chemotactic protein (mcp- ), rantes), type-l interferon (ifn-␣/␤), and growth factors (gm-csf, g-csf). epithelial-derived levels of chemokines correlate with disease severity (bont et al., ; smyth et al., ) . surfactant proteins produced by epithelial cells (sp-a and sp-d) may also play a role as opsonins for viruses and bacteria. thus, epithelial cells provide a potential mechanism for serum-independent phagocytosis. many of these mediators are induced both at the level of secretion and transcription. interestingly, some mediators (e.g., il- and rantes) are also upregulated by inactive forms of the virus (harrison et al., ) . rsv uptake by immune and non-immune cells is a receptor-mediated process. experiments with blocking antibodies against g-protein revealed inhibition of binding of rsv to epithelial cells. the fractalkine receptor, also known as the cx cr chemokine receptor, is involved in g-protein-mediated uptake by epithelial cells . other receptors may very well be involved in uptake by dendritic cells, macrophages, and other cells of the innate immune system (harris and werling, ) , and toll-like receptors (tlrs), especially tlr- , are being investigated as possible candidates for mediating viral uptake (haeberle et al., a, b; monick et al., ) . several groups have demonstrated activation of the transcription factor nf-kb in rsv-infected epithelial cells (tian et al., ) . many of the exhibited effects observed in epithelial cells can be explained by activation of nf-kb. several cytokines associated with rsv infection have nf-kb binding sites in their promoter or enhancer regions (bitko et al., ) . epithelial nf-kb activation has also been observed in other viral infections, including parainfluenza, influenza a, and rhinovirus (pahl and baeuerle, ; kim et al., ; bose et al., ) . nf-kb could be an exciting target for therapy development and experiments in which balb/c mice were treated with perflubron have confirmed this concept. perflubron has already been shown to be effective in clinical trials of patients with respiratory distress syndrome because of its physical characteristics. besides the beneficial physical effect in improvement of gas exchange and of lung compliance, this agent was found to have anti-inflammatory effects. rsv-infected balb/c mice treated with perflubron intranasally showed a reduction in cellular inflammatory infiltrates and decreased chemokine expression in the lung tissue. both the anti-inflammatory effects were directly linked to interference of perflubron with nf-kb-mediated transcription (haeberle et al., a, b) . the chemokines produced by epithelial cells attract t-cells, neutrophils, monocytes, and possibly eosinophils to the respiratory tract. besides induction of secreted products, epithelial cells upregulate expression of adhesion molecules for neutrophils on their surface, allowing neutrophils to adhere firmly to infected cells (wang and forsyth, ) . furthermore, neutrophils are the dominant cell type found in bronchoalveolar lavage (bal) fluid of rsv patients (everard et al., . however, their role in fighting viral infection is not as well established as in bacterial infections. pathological studies of lungs of rsv-infected calves have shown a major influx of neutrophils in the infected airway mucosa, observed earlier than any other cell type involved. furthermore, neutrophils are the dominant cell type found in bronchoalveolar lavage (bal) fluid of rsv patients (everard et al., " several chemokines and cytokines involved in neutrophil activation have been associated with rsv lower respiratory tract infections. recently, local neutrophil il- production has been linked to rsv bronchiolitis (mcnamara et al., ) . as shown by wang et al., a major increase in epithelial damage occurs, when rsvinfected epithelial cells are co-cultured with neutrophils (wang and forsyth, ) . this is suggestive of a detrimental role for neutrophil-induced immunopathology in lower respiratory tract infections. rantes and mip- ␣ are produced by the epithelium in response to rsv infection and these chemoattractants recruit eosinophils to the inflammatory site. the analogy between clinical features of virus-induced wheezing illnesses and asthma has made eosinophils an attractive subject for studies aimed at improving understanding of rsv pathogenesis. however, mainly because of their absence in bal of rsv patients, their involvement remains controversial. however, eosinophil-derived cationic protein (ecp) has been linked to bronchiolitis and postbronchiolitic wheezing pifferi et al., ; dimova-yaneva et al., ) . in vitro, eosinophils have also been shown to be susceptible to rsv. eosinophil priming, superoxide production, and degranulation were induced by incubation with rsv kimpen et al., ; olszewska-pazdrak et al., ; tachibana et al., ) . rosenberg and domachowske ( ) have suggested a beneficial role for eosinophils in rsv bronchiolitis. they identified antiviral properties for the eosinophil based on ribonuclease activity of eosinophil-derived neurotoxin (edn) and ecp. this enzymatic activity leads to destruction of extracellular ssrna virions and delayed replication both in vitro and in vivo. recently, there has also been great interest in the involvement of macrophages and dendritic cells in rsv pathogenesis. these cells were already appreciated for their role in antigen presentation, at which dendritic cells are by far superior. macrophages express phagocyte activity, which may be of importance in clearance of infected epithelial cellular debris. fascinating new players in host defense against viruses are pattern recognition receptors. toll-like receptor- (tlr- ) and cd , both present in a complex on these cells, have been found to interact with rsv and receptor-binding results in triggering of the innate immune system. tlr- has been shown to activate nf-kb in macrophages of rsv-infected mice (haeberle et al., a, b) and tlr- -deficient mice have impaired nk-cell and cd ϩ cell trafficking and delayed viral clearance . furthermore, intracellular pattern recognition receptors tlr- and - , may be involved in recognizing doubleand single-stranded rna (dsrna/ssrna), respectively (akira and hemmi, ; lund et al., ) . dsrna is produced during replication of rna-viruses and is a potent inducer of ifn-␣/␤. all human cells can produce ifn-␣/␤ in response to viral infection, while only t-cells and nk-cells produce ifn-␥. dsrna also activates dsrna-dependent protein kinase r (pkr) and nf-kb via distinct pathways. transcription of pkr is under control of ifn-␣/␤. pkr controls enzymes directly involved in protein synthesis, thereby inhibiting cellular and viral protein translation. ifn-␣/␤-deficient mice as well as pkrϪ/Ϫ mice are extremely sensitive to influenza infection (balachandran et al., ) . several viruses, including rsv, have evolved mechanisms to escape the interferon system, which will be discussed below. respiratory epithelial cells are the principal host cells for viral pathogens in lower respiratory tract disease. the degree of replication and the mechanism of spread along the epithelial layer depend on the virus family characteristics. through the fusion (f) protein, rsv is capable of syncytium formation, which allows it to replicate and spread relatively undetected by the immune system for a relatively long period. the virus itself is directly responsible for cytopathology and viral envelope proteins are expressed on the surface of infected epithelial cells. dendritic cells, lining the basal membrane of the respiratory epithelium encounter rsv, pick up viral antigens and migrate to mediastinal lymph nodes where viral antigen is presented to naïve cd ϩ t-cells. antigen presentation and co-stimulatory molecule expression lead to maturation to the t-helper phenotype. this then induces b-cell proliferation with the production of specific antibodies as well as proliferation of virus-specific cytotoxic cd -cells. cellular responses are responsible for controlling and terminating acute infection with rsv. in primary infections, the adaptive cellular immune response develops within days. these cd ϩ cells can recognize and eliminate virus-infected epithelial cells resulting in perforin-mediated cytotoxity. epithelial cells are nonprofessional antigen presenting cells (apc) expressing mhc class l on the surface (garofalo et al., ) . when infected, epithelial cells present viral antigen in association with mhc class l molecules. mhc class l restricted antigen presentation to cd ϩ cells, among other factors, may determine the strength of the cytotoxic response. in cd -deficient mice, there is delayed viral clearance; however, these mice also exhibit decreased disease severity (graham et al., ) . therefore, it is conceivable that cd ϩ t-cells are crucial in viral clearance while a surplus of cytotoxicity may result in pulmonary injury. in humans, a cytotoxic t-cell response is elicited against all viral proteins, except the g-(attachment)-protein, which is required for cell entry (bangham et al., ; hacking and hull, ) . it is suggested that a defective response against g-protein is directly associated with enhanced disease. however, g-protein can induce a cd ϩ response in mice, which is associated with th -cytokine production and eosinophilia both during primary and secondary infection (openshaw, ) . the immune response to the f-protein is dominated by ifn-␥ production and subsequent polarization toward a th -type cellular response, and therefore it has been postulated that responses to the other viral proteins can modulate the strong th response to g-protein (graham et al., ) . a stronger th -response seems to induce a more rapid viral clearance and milder disease (bont et al., ; legg et al., ) . besides activated t-cells, nkcells also produce considerable amounts of ifn-␥ (hussell and openshaw, ) . ifn-␥ has important antiviral effects and provides a link between adaptive and innate immune system. it can induce expression of tnf-related apoptosis inducing ligand (trail) on immune cells, which has the potential to trigger apoptosis of virus-infected cells (sedger et al., ) . in vitro findings suggest that rsv-infected cells in vivo are susceptible to killing by immune cells through the trail pathway (kotelkin et al., ) . nk-cells are also thought to play a role in activating cd ϩ cells, further modulating the degree of cytotoxicity (hussell and openshaw, ) . in summary, in rsv lower respiratory tract infections, cytotoxic cd ϩ t-cells are involved in viral clearance while the humoral response is required for the protection against reinfection. however, as has been discussed before, memory is incomplete and repeated infections with rsv are common. both igm and igg as well as secretory iga against rsv are formed in infants, and a more vigorous antibody response seems to be protective against rsv infections (meurman et al., ; welliver et al., ). rsv infections are most severe in the youngest age group, which is the least mature in terms of immunity to infections. relative deficiencies in both innate and antigen-specific immunity in infancy have been characterized. these include delayed trafficking of immune cells, less-efficient antigen presentation by dendritic cells, and impaired production of ifn-␥ by t-cells in response to antigen presentation (bont and kimpen, ) . the fetus derives maternal igg-antibodies via the placenta fairly late in gestation. this partly explains why prematurity is an important risk factor for severe disease caused by rsv, as well as the physiological characteristics of the small airways. antibody titers produced by infants are relatively low compared to older children. trials with humanized monoclonal antibodies against rsv-f-protein have shown a % reduction in rsv lower respiratory tract-related hospitalizations in this highrisk group for severe disease (impact study group, ). the cytokine milieu at the time of infection is another factor possibly contributing to the occurrence of severe rsv bronchiolitis especially in the youngest age group. at birth, there is skewing toward a th -phenotype and rsv bronchiolitis was long thought to be a th -type disease. this role of th -skewing is an attractive concept, because it provides some explanation for the association between rsv bronchiolitis and the development of asthma. asthma and allergy have long been acknowledged to beth -mediated conditions. however, convincing evidence that primary rsv infections are mediated by th cytokines is lacking. dendritic cells are thought to have an important function in skewing the th /th -ratio. viruses may be important in maturation of dendritic cells, which can then drive differentiation of naive t-cells into either a th -or a th -phenotype. the role of regulatory t-cells that suppress both th and th differentiation has not been studied in rsv bronchiolitis so far. gene polymorphism studies have been undertaken to identify a genetic background to explain the individual susceptibility to rsv lower respiratory tract infection. several polymorphisms situated in genes relevant for the adaptive and innate immunity have been found to correlate with occurrence of rsv infection. polymorphisms of interleukin- , il- r, and its receptor, have been associated with rsv bronchiolitis, which is consistent with the th -hypothesis (choi et al., ; hoebee et al., ) . very recently, a polymorphism of the gene coding for interleukin- (il- ) was found as well (hoebee et al., ) . this is particularly interesting since il- is a cytokine produced by t-regulatory cells and monocytes, thought to be primarily involved in development of allergy. gene polymorphisms involved in innate immunity include surfactant proteins spa and d (lahti et al., ) , the chemokine il- (hull et al., ) , tlr- (tal et al., ) , and the chemokine receptor for rantes and mip- ␣, ccr (hull et al., ) . immunocompromised patients have a higher risk of developing severe disease from viral respiratory tract infections. in particular, the presence of defects in cellular immunity result in an increased duration of viral shedding and enhanced risk of developing severe disease. most cellular immunodeficiencies are iatrogenic in nature. an important cause is intensive immunosuppressive treatment. the number of pediatric patients undergoing organ or stem-cell transplantation is increasing and high doses of chemotherapeutic and immunosuppressive agents are often used in the pre-and posttransplant regimens. immunosuppressive drugs are used in cancer treatment regimens and for a number of inflammatory conditions. community acquired respiratory viruses such as rsv, rhinovirus, adenovirus, influenza a, influenza b, and the parainfluenza group are frequent causes of respiratory disease in these patients (soldatou and davies, ) . adenovirus infections have a particularly high risk of adverse outcome, mortality rates are high, and no effective treatment exists. the presence of lower respiratory tract infection and infection in the pre-engraftment phase of hsct is believed to have a particularly poor prognosis (khushalani et al., ) . the risk of severe disease is higher during allogenic hsct than autologous hsct. besides causing increased morbidity and mortality, respiratory tract infections are associated with a greater risk of delayed engraftment (abdallah et al., ) . in solid organ transplant patients, respiratory virus infections are also associated with a higher incidence of rejection (wendt, ) . prolonged shedding of respiratory viruses for weeks or months has been documented in hiv-infected adults and children. this has important implications for infection control in medical facilities. in addition, respiratory viral infection may result in increased hiv replication and, theoretically, hiv disease progression (king, ) . in hiv-infected children, rsv infections are less limited by season (madhi et al., ) . however, generally, the course of rsv infections in hiv patients is not more severe, unless there is profound lymphopenia or pre-existing lung disease (soldatou and davies, ) . other viruses may also cause respiratory complications in the immunocompromised patients. in particular herpesviruses, such as cytomegalovirus (cmv) and varicella zoster virus, can cause severe pneumonia. with a cmv-negative donor and a cmv-positive recipient, there is an especially high risk of reactivation which may lead to severe disease. this reactivation also occurs with epstein barr virus (ebv), human herpes virus (hhv)- , - , and - , although these are much less frequent causative agents of pneumonia. the innate immune defense to viral respiratory tract infections consists of the mucosal layer, type interferons, activated phagocytes, and nk-cells. the impact of primary defects in the innate immune defense has not been well documented. phagocyte defects are primarily related to a higher incidence of bacterial infections. one indication that impaired phagocyte function also leads to increased severity of respiratory viral infection can be derived from a report of severe abnormalities on lung-ct-scans of rsv patients with phagocyte defects (uzel et al., ) . interferon-gamma receptor deficiency, which may have implications for both the adaptive and innate immune system, has also been associated with increased susceptibility to viral respiratory pathogens (dorman et al., ) . chronic lung disease also increases susceptibility to respiratory viruses (meert et al., ; griffin et al., ) . premature patients with bronchopulmonary dysplasia are candidates for rsv-immunoprophylaxis because of their increased risk of developing severe lower respiratory tract infections. in children with cystic fibrosis (cf), % are already hospitalized with respiratory virus infection in their first year of life. furthermore, there is a correlation between viral infections in infancy and disease progression. infants with cf suffering from a respiratory virus infection are at significant risk for lower respiratory tract disease, hospitalization, and deterioration in lung function that persists months after the acute illness (hiatt et al., ) . cf infants were found to be four times more likely to develop an lrti compared with controls. it has been shown that cfderived airway epithelial cells allow a higher degree of piv replication and have an increased production of pro-inflammatory cytokines (zheng et al., ) . cfderived epithelial cells are also unable to express no-synthase , which results in a decrease production of nitric oxide (no), which has antiviral capacity, reducing effects on replication. furthermore, in cf-cells there is no viral induction of Ј Јoligoadenylate synthetase (oas), an enzyme that is normally induced by dsrna and ifn-␥. oas is involved in inhibition of cellular protein synthesis, thereby inhibiting viral replication. respiratory viruses can be isolated from the secretions of approximately % of children and of more than half of adults during asthma exacerbations (johnston et al., ; lemanske, ) . recently, copd exacerbations have also been attributed to viral infection by rhinovirus, rsv, and piv (seemungal and wedzicha, ) . the underlying mechanisms for this are, however, still a matter of debate. from experimental rhinovirus (rv) infections in humans, it has been shown that rv infection causes increased bronchoconstriction in atopic non-asthmatic and asthmatic individuals, while symptoms in normal individuals are relatively mild. this implies that induction of a wheezing episode requires both rv infection and a preexisting tendency to develop allergic or asthmatic disease (message and johnston, ) . rv-specific t-cell responses can be activated by either serotype-specific or shared viral epitopes. cross-reactivity between rv-subtypes could result in vigorous t-cell responses and may amplify allergic inflammation. other proposed mechanisms linking viral infections to asthma exacerbation are epithelial dysregulation, airway remodeling, the immune response to virus, and alterations of neural responses (message and johnston, ; gern, ) . upregulation of icam- -expression, which is the entry receptor for major group rhinoviruses, has been found in susceptible individuals. this may be one mechanism predisposing atopic individuals to rv-induced exacerbations. rhinovirus can induce a number of inflammatory mediators (kinins, arachidonic acid) and cytokines (e.g., il- , il- , ifn-␣/␤, gm-csf, tnf-␣) that can further enhance inflammation. th cytokines seem to have a general antiviral effect while a predominant th -cytokine response leads to enhanced disease, failure to clear the virus, and amplification of allergic inflammation (message and johnston, ) . eosinophil numbers were found to be increased in bronchial biopsies from both healthy and asthmatic human volunteers after experimental rhinovirus infection. this cell type is associated with allergic inflammation in the lung. in allergic rhinitis patients, the increased level of eosinophils in bal even persisted for weeks. these data suggest a potential role for eosinophils in virus-induced asthma, which can be either pathogenic or protective. virus-induced exacerbations of asthma tend to be resistant to treatment with corticosteroids and may require a different therapeutic approach. in vitro, blocking icam- has been tried with positive results, which may be of particular relevance to rhinovirus infections. the possibility of other immunomodulating drugs is being investigated and may be of significant benefit to future asthma treatment. a causal relationship between viral respiratory tract infections and asthma exacerbations is generally acknowledged. however, the suggestion that respiratory virus infection is a causal determinant in the development of asthma is highly controversial. according to the hygiene hypothesis, viral infection would be expected to have an inhibitory effect on the development of asthma (an allergy), and this is supported by a study from matricardi et al. ( ) , showing an inverse relation between hepatitis a seropositivity and atopy among soldiers. the hygiene hypothesis is based on the theory that the immune system is directed toward a more th -skewed immune response with each viral infection. however, this hypothesis is not supported by the observation that rsv infections, severe enough to cause bronchiolitis, are significantly associated with a higher incidence of asthma up to the age of - years (stein et al., ; sigurs et al., ) . these data convincingly show a link between rsv bronchiolitis and recurrent wheezing in childhood. in a recent study, wheezing following rsv lower respiratory tract infection was found to develop independent of atopy . it may, however, be true that the transmission route, the organs involved, and exposure to microbial products may be important in determining the final effect of a virus infection on the development of asthma and allergy (gern and busse, ) . the link between rsv infection and atopy is even less clear than the one with recurrent wheezing and asthma, at least in humans. animal studies have yielded conflicting results. one group found that rsv infection in mice enhances subsequent allergic inflammation (schwarze et al., ) , while others reported a decrease in allergic sensitization and bhr after rsv infection (peebles et al., ) . no proof exists that severe rsv infections are associated with atopy that persists into adulthood (peebles, ) . a key question is whether the association with the development of asthma is merely an expression of increased susceptibility to both asthma and rsv-induced lower respiratory tract infections or whether true causality is involved. the prevailing theory on this subject involves maturation effects of the th /th -balance. the system shifts from a th -polarization in fetal life, which is an optimal environment for the placenta, to a more balanced th /th -phenotype in adulthood. most viruses are known to induce a th cytokine response (ifn-␥). this theory states that when infections occur early in infancy, there is a reduced ability to react with an appropriate antiviral th -response. low ifn-␥ production may result in spread of the virus to the lower respiratory tract. this is in agreement with findings that in children with severe rsv lower respiratory tract infections, lower amounts of ifn-␥ are produced . the dynamics of this shift toward a more balanced th /th immune response may differ between individuals. both environmental factors and genetic make-up may contribute to a slower maturation of th competence in some individuals. respiratory virus infections in infancy and an atopic sensitization to aero-allergens, both of which are related to th -skewed responses and intermittent wheeze, may than synergistically result in persistent wheeze (holt and sly, ) . it is likely that more links between atopic sensitization and respiratory infections exist, while preventive rsv-ivig treatment of children results in a decreased sensitization to aeroallergens as well. rsv-prophylaxis may therefore have a long-term benefit in the development of persistent wheeze (piedimonte and simoes, ; wenzel et al., ) . another theory linking viral infections in childhood to the development of asthma involves the pathologic effects of viral lower respiratory tract infections on airway physiology. wall thickening with consequent increased resistance may predispose the airway to more infections and thus influence bronchial hyperreactivity (bardin et al., ) . however, it may also be true that small airways predispose to both asthma and airway symptoms from viral infections. remodeling of the submucosal neural networks by rsv, as observed by piedimonte et al., is also proposed to result in increased responsiveness to airway irritants (piedimonte, ) . walter et al. ( ) have proposed that paramyxoviral infection has the ability not only to induce acute hyperresponsiveness, but also to result in long-lasting changes in airway behavior. from mouse-studies with a piv (sev), it has been concluded that viruses cause long-term effects in epithelial cells, associated with airway reactivity and goblet cell hyperplasia. long-term effects are induced by the virus in the acute phase, and later on, the presence of virus is no longer required for the persistence of symptoms. it is speculated that primary paramyxoviral infection within the proper genetic background may result in chronic dysfunction of epithelial cell behavior. their results have indicated that different mechanisms are responsible for the induction of the acute and the chronic response (walter et al., ) . several theories on the induction of asthma have thus been proposed. the current view is that virus infection modulates the development of an asthmatic phenotype in a susceptible host. the relationship is, therefore, not purely causal but certainly requires an intrinsic vulnerability. the effectiveness of respiratory virus infections in the host depends partly on the ability to evade the immune system (figure . ). while several viral evasion mechanisms have evolved, not all have been intensively studied in respiratory viruses. viral entry into host cells is one of the first obstacles viruses have to overcome. since the cell membrane is in principle impermeable to macromolecules, viruses caroline a. lindemans and jan l. l. kimpen must first have an effective method to attach to the cell membrane. some viruses bind putative cell surface receptors that do not simply play a role in viral attachment, but also allow viral entry by inducing endocytosis. for some viral pathogens, such as rhinovirus, these receptors have been identified (icam- and ldl-r), while for others, such as rsv, the receptor that is used for cellular entry has not been unequivocally defined. the cx cr -chemokine receptor, also known as fractalkine-receptor, may be involved and tlrs have also been proposed to play a role. many enveloped viruses have glycosylated proteins, which not only bind to cellular receptors, but also have additional functions as membrane fusion factors, or receptor-destroying enzymatic activity. membrane fusion factors such as the rsv fprotein also allow cell-to-cell transmission of virus, which keeps it relatively hidden from the cellular immune system (smith and helenius, ) . escaping the "interferon signaling system" is one of the common mechanisms most viruses have acquired. as mentioned earlier in this chapter, both ifn-␣/␤ and ifn-␥ have potent antiviral properties. both types of interferon regulate transcription of a variety of target genes through activation of interferon inducible transcription factors. ifn-stimulated genes encode a variety of cellular enzymes, including pkr and Ј Ј-oligoadenylate synthetase, both involved in inhibition of viral protein synthesis. furthermore, interferons induce cellular apoptosis and upregulate mhc expression, targeting cells for cd ϩ t-cell-mediated cytotoxicity. additionally, ifn-␥ activates the adaptive cellular immune system. rsv infection leads to an increase in ifn-␣/␤, but does not induce ifn-␥ from mononuclear and nk-cells as efficiently. despite the fact that interferons are known for their antiviral properties, intranasal administration of either lfn-␣/␤ or ifn-␥ in the airway does not lead to reduction of symptoms of viral respiratory infections (ramaswamy et al., ) . this is suggestive of a viral mechanism to evade the host's interferon response. the paramyxoviridae family (figure . ), responsible for a large part of children's respiratory infections, consists of two subfamilies based on the structural differences in the gene encoding for the polymerase complex (p-protein). the paramyxovirinae subfamily members are able to block interferon-mediated promoter activity. these paramyxovirinae, to which parainfluenza-, measles-, and mumps virus belong to, have a p-gene that encodes for additional proteins besides the p-protein, the v-proteins. it is these v-proteins that have been found to be responsible for evading the interferon signaling pathways in this group of viruses. ifnmediated transcription is predominantly mediated by signal transducers and activators of transcription (stat). v-proteins have the ability to block interferon-mediated signaling by targeting stats for proteosomal degradation (horvath, ) . in contrast, rsv, belonging to the pneumovirinae subfamily, fails to inhibit ifn-induced promoter activity. the pneumovirinae consist of only one genus, the pneumovirus, which also includes hmpv. in this subfamily, p-genes only encode for the p-protein and therefore rsv cannot block interferon-mediated signaling (young et al., ) . however, recently it was demonstrated that, although rsv does not inhibit interferon-induced promoter activity, rsv replication is still resistant to ifn treatment of infected cells. apparently, an alternative mechanism to circumvent the interferon antiviral response exists. this has been attributed to additional proteins, characteristic of these pneumoviruses (spann et al., ) . these are nonstructural proteins (ns and ns ) that have no homologs in the paramyxovirinae. however, the underlying molecular pathway has not yet been elucidated. rsv infection of epithelial has been shown to lead to an upregulation of trail-receptor expression on these cells (kotelkin et al., ) . apoptosis of infected cells is an effective way to eliminate intracellular pathogens without damage to the surrounding tissue (figure . ) . however, several respiratory viruses have developed mechanisms to inhibit apoptosis. it has been demonstrated that rsv is able to effectively inhibit apoptosis of epithelial cells in vitro, in accordance with the limited pathology induced by rsv in epithelial cells during the first few days of the infection (thomas et al., ) . eventually, necrosis is observed when mature viral particles are released from the cells, after - days. furthermore, experiments with both adult and cord blood monocytes have shown a prolonged longevity of cells, when cultured in the presence of rsv (krilov et al., ) . of all respiratory viruses, viral evasion techniques of adenoviruses have been studied most intensively. it appears that approximately a third of the adenovirus genome is devoted to counteracting innate and adaptive immune defenses (burgert et al., ) . adenoviruses encode the protein e a that blocks interferon-induced gene transcription. through the va-rna protein that blocks activation of pkr, they also interfere with the antiviral enzymes that are synthesized under interferon control. additionally, adenoviruses have developed several mechanisms to inhibit both constitutive and death receptor induced apoptosis (figure . ) . the e b/ k protein inhibits p -mediated apoptosis, e / k 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immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic paramyxoviridae use distinct virusspecific mechanisms to circumvent the interferon response evidence of airborne transmission of the severe acute respiratory syndrome virus contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study impaired innate host defense causes susceptibility to respiratory virus infections in cystic fibrosis is likely to be crucial to the improvement of immunotherapies for prevention and treatment of viral respiratory tract infections. key: cord- -z vv u authors: nielsen, claus h.; bendtzen, klaus title: immunoregulation by naturally occurring and disease-associated autoantibodies: binding to cytokines and their role in regulation of t-cell responses date: - - journal: naturally occurring antibodies (nabs) doi: . / - - - - _ sha: doc_id: cord_uid: z vv u the role of naturally occurring autoantibodies (nabs) in homeostasis and in disease manifestations is poorly understood. in the present chapter, we review how nabs may interfere with the cytokine network and how nabs, through formation of complement-activating immune complexes with soluble self-antigens, may promote the uptake and presentation of self-molecules by antigen-presenting cells. both naturally occurring and disease-associated autoantibodies against a variety of cytokines have been reported, including nabs against interleukin (il)- α, il- , il- , il- , granulocyte-macrophage colony-stimulating factor, interferon (ifn)-α, ifn-β, ifn-γ, macrophage chemotactic protein- and il- . nabs against a variety of other self-antigens have also been reported, and using thyroglobulin as an example we discuss how nabs are capable of promoting uptake of immune complexes via complement receptors and fc-receptors on antigen-presenting cells and thereby regulate t-cell activity. knowledge of the influence of nabs against cytokines on immune homeostasis is likely to have wide-ranging implications both in understanding pathogenesis and in treatment of many immunoinflammatory disorders, including a number of autoimmune and autoinflammatory diseases. naturally occurring antibodies play an essential role in our defense against invading microorganisms by directly neutralizing viruses and bacteria, by activating the complement system and by enhancement of phagocytosis, as described elsewhere in this book and in reference . in contrast, naturally occurring autoantibodies (nabs) are characterized by binding to self-molecules, and their primary function may involve clearance of senescent cells and metabolic waste products, but they also appear to play important roles in immunoinflammatory processes. [ ] [ ] [ ] [ ] [ ] in the present chapter, we shall focus on the immunomodulatory effects that self-reactive nabs may have as a result of interactions with cytokines or formation of complement-activating immune complexes (ics) with other soluble self-antigens and targeting these self-antigens to b cells and presumably other antigen-presenting cells (apcs). nabs have been shown to bind a variety of self-molecules, ranging from cytokines and other plasma proteins to tissue-specific antigens, structural proteins, metabolic enzymes, heat shock proteins and dna (tables and ). some nabs, including those reacting with various cytokines and thyroglobulin (tg), are relatively specific, while others, e.g., igm anti-dna antibodies of all species, tend to be polyreactive. nabs exist as igm, iga and igg isotypes. the occurrence of the igg isotype among nabs is indicative of t-cell involvement in shaping of the autoreactive b-cell repertoire. [ ] [ ] [ ] igm nabs are already synthesized in newborns, and different babies produce igm nabs to a similar set of self-molecules. several reports have documented the presence of autoantibodies against cytokines both in healthy individuals (nabs) and in patients with various immunoinflammatory disorders (disease-associated autoantibodies, dabs) ( table ). in most cases, the physiological and/or pathological significance of these antibodies is unclear. however, recent evidence suggests that certain anti-cytokine nabs have significant, if not decisive, pathogenic roles in rare disorders (reviewed in refs. , , ) . the biological roles of nabs against cytokines are poorly understood. fab fragments of some of these nabs bind in a saturable and highly specific manner to their respective cytokine. as for example in the cases of nabs to il- , il- and gm-csf. in some cases, these antibodies are found at levels at or above nm, for example in healthy blood donors and apparently without untoward effects. why and how high-affinity nabs to some cytokines and not to others are induced in healthy individuals is unknown. also obscure is whether these in vitro neutralizing antibodies neutralize their respective cytokines in vivo too, or whether they exhibit carrier-or cytokine-regulatory, or even cytokine-protective functions. low levels of igg and igm capable of neutralizing ifn-, ifn-and ifn-have been detected in blood of apparently healthy individuals (as reviewed in refs. , ). igg nabs against ifn-are especially frequent, and are easily detectable in pharmaceutical preparations of normal human igg (ivig). nabs against ifn-have been found in approximately % of healthy caucasians, but the exact frequency is likely to be higher, as these nabs are often difficult to detect in plasma because they are complexed with native ifn-. igg nabs against ifn-are of high avidity and specific, as they do not cross-bind ifn-or other cytokines. nabs to ifn-have been demonstrated in vivo in bioactive form after treatment with ivig. as these nabs neutralize ifn-, ivig therapy suppresses both antiviral and other effects of endogenous ifn-, which may explain some of the many therapeutic effects of ivig. , , , nabs against interleukin- nabs to interleukin (il)- were first found by direct binding of igg from normal individuals to radiolabeled, human, recombinant il- and by igg-mediated competitive interference with il- binding to cellular il- receptors. subsequently, human ivig, cord blood and sera of patients with various immunoinflammatory disorders were found to contain high-avidity autoantibodies that bind to and neutralize il- both in vitro and in vivo. , , , , [ ] [ ] [ ] they bind il- in a saturable fashion and through the fab fragments of the igg isotypes igg , igg and igg . the occurrence of detectable anti-il- igg in sera of healthy individuals vary with age and sex with male preponderance and markedly increased frequency with age (up to % positives among elderly males). , , a human anti-il- autoantibody has been cloned. it is an igg / monoclonal antibody which reacts with il- , but not with il- , the il- -receptor antagonist (il- ra) or several other cytokines. it binds with high affinity (k d » - m), and the presence of somatic mutations in the variable regions suggests antigen-driven affinity maturation. as il- from antigen presenting cells is an important co-activator of t cells, particularly in its membrane-bound form, it is important to note that anti-il- nabs not only neutralize il- in lysates of human blood monocytes, but also membrane-associated il- -activity. igg nabs to il- were first reported in sera of normal individuals. since then, the presence of nabs to il- and similar antibodies in patients with immunoinflammatory and fibrotic diseases has been confirmed (reviewed in refs. , , , ) . high-affinity igg nabs to il- have been detected in up to % of normal danish blood donors with % having titers ranging from to greater than , and . % having exceedingly high titers. the anti-il- nabs bind to il- through their fab fragments and they effectively inhibit binding of il- to il- receptors and, hence, neutralize the bioactivity of il- . nab-positive donors with high antibody titers have no overt signs of pathology even though they are likely to be functionally il- -deficient. nabs to il- are detectable in ivig and are found in bioactive form, binding il- in the circulation following ivig administration. igg nabs against il- have been reported in normal sera and in preparations of pooled normal human igg. , indeed, . % of healthy danish blood donors present with these nabs at such high concentrations and avidity that these blood donors are functionally il- -deficient. these nabs are of the igg isotype and of polyclonal origin. they prevent il- from binding to its receptor thereby neutralizing il- bioactivity. anti-il- nabs are highly specific in that they fail to bind viral forms of il- and other members of the human il- family including il- , il- , il- , il- , il- , il- a, il- b, and il- . using a direct radioligand binding assay, high-affinity nabs to granulocytemacrophage colony-stimulating factor (gm-csf) were reported at very high titers in of , ( . %) apparently healthy blood donors. later, all of tested, apparently healthy japanese individuals were shown to possess low levels of these nabs such that more than % of gm-csf were bound and neutralized by these antibodies. anti-gm-csf nabs have also been found in human ivig. , nabs against xxc-and cc chemokines nabs to various chemokines have also been reported. for example, il- -and macrophage chemotactic protein (mcp)- -containing igg-immune complexes have been demonstrated in sera from healthy individuals, where the chemokines themselves are usually not detected. it is hypothesized that circulating igg nabs to chemokines may play a role as a sink for the spill-over of chemokines produced in local tissues. igg nabs to other cytokines, for example il- , il- , granulocyte colony-stimulating factor (g-csf), nerve growth factor (ngf), leukemia inhibitory factor (lif), tumor necrosis factor (tnf)-, and soluble tnf receptors have also been reported in normal and diseased individuals, while ige antibodies to il- , tnf-, tnf-and various chemokines have been reported in sera of aids patients. some of both isotypes, however, cannot be regarded as nabs in that they bind the relevant mediator(s) with low avidities and in some cases bind only to cytokines denatured by adsorption to nitrocellulose membranes or plastic surfaces (using elisa-technologies). anti-cytokine dabs have been reported in a wide range of pathological disorders, suggesting that pathogenetically obscure diseases may eventually be at least partly explained by the presence of anti-cytokine dabs (reviewed in refs. , , ). anti-type interferon (ifn) dabs, preferentially of igg type, were first reported in patients with varicella-zoster and hepatitis virus infections, in patients with autoimmune and neoplastic diseases, and in a patient with chronic graft-vs.-host disease (reviewed in refs. l , , , , ) . later reports have documented dabs that bind to other ifn species, usually in patients with various infectious diseases or severe immunodeficiencies. neutralizing dabs against ifn-/ / have been demonstrated primarily in patients with thymoma and/or myasthenia gravis. however, patients with thymic malignancy/ myasthenia gravis have also been reported to express dabs to a variety of other cytokines including, il- , il- p and il- p , and il- a. high-titer dabs against ifn- and ifn-have been reported in % of european patients with autoimmune polyendocrinopathy syndrome type i (aps-i). this disease is a result of mutations in the autoimmune regulator gene (aire), which impairs thymic self-tolerance induction in developing t cells. the ensuing autoimmunity particularly targets ectodermal and endocrine tissues, but chronic candidiasis is a frequent and early manifestation. although the underlying immunodeficiency of aps- is unclear, neutralizing anti-ifn dabs and, most recently, anti-il- a and anti-il- dabs appear to be implicated directly in the pathogenesis, as they appear before development of candidiasis in all informative cases. , anti-ifn-dabs have been reported in patients with viral infections and in cerebrospinal fluids from patients with multiple sclerosis and guillain barré syndrome (reviewed in ref. ). circulating dabs to ifn-have also been positively correlated with the severity of both tuberculous and nontuberculous mycobacterial infections (reviewed in ref. ). it is characteristic that patients with extremely high antibody titers had rapidly progressive disease and severe immunodeficiency, most likely due to dab-induced blockade of the crucial macrophage-activating effect of ifn-. although il- is secreted from cells producing the cytokine, the dominant form of il- appears to be the cell-associated precursor form that is found both intracellularly and on the surface of many cell types, including keratinocytes and 'professional' apcs such as macrophages and b cells. on these cells il- is thought to be involved as a juxtacrine co-activator of t cells. il- is usually absent in the circulation, if present then only at low concentrations. during infection and inflammation, however, substantial amounts of il- may be found in the blood, most likely released from dying cells. cell-associated il- is biologically active, and its biological activities are neutralized by antibodies to il- , including igg anti-il- nabs, but not by antibodies against il- . the prevalence of dabs to il- in immunoinflammatory disorders vary considerably. for example, anti-il- dabs have been found more commonly and at higher levels in patients with non-destructive forms of arthritis. progression of joint destruction in patients with rheumatoid arthritis was negatively associated with the occurrence of circulating il- dabs, but patients who seroconverted more than two years after the onset of ra showed the most aggressive development of joint erosion. interestingly, transgenic mice overexpressing the membrane form of human il- in macrophage-like and fibroblast-like synoviocytes develop severe arthritis, correlating with the degree of membrane expression of il- , but not circulating il- . taken together, this suggests that il- and/or the lack of il- dabs play a role in the erosive processes of rheumatoid arthritis. along with dabs to other cytokines, anti-il- dabs have also been demonstrated in patients with juvenile chronic arthritis, thymoma and myasthenia gravis. , dabs against interleukin- there is an increased prevalence of high-avidity igg dabs to il- in patients with rheumatoid arthritis and systemic sclerosis, and the presence of these antibodies signals a poor survival in patients with alcoholic cirrhosis, possibly because of an increased risk for recurrent infections. , , a . -fold increase in anti-il- dab-positivity has recently been reported in type diabetic patients, and mice vaccinated with il- develop obesity and impaired glucose tolerance. these data suggest that an autoimmune reaction against il- may be involved in a subset of type diabetics. anti-gm-csf dabs are of clinical interest not only because of gm-csf's growth-potentiating effect on macrophages and granulocytes, but also because gm-csf appears to be a central mediator affecting bronchial epithelial cells, possibly through its marked effect on eosinophils, both as a chemoattractant, growth promoter and stimulator. in accordance with results obtained in gm-csf knockout mice, anti-gm-csf dabs have been associated with pulmonary alveolar proteinosis (pap), a rare disease in which surfactant lipids and proteins accumulate in pulmonary alveolar macrophages and alveoli, resulting in respiratory insufficiency and failure. recently, isolated anti-gmcsf dabs from a patient with pap were shown to reproduce the pathologic manifestations of the human disease in previously healthy primates. these findings may have therapeutic implications for the potential use of gm-csf not only to treat pap, but also other immunoinflammatory respiratory disorders such as asthma. igg dabs to granulocyte colony-stimulating factor (g-csf) have been demonstrated in felty's syndrome, a relatively rare complication in rheumatoid arthritis, and in some patients suffering from systemic lupus erythematosus (sle) with accompanying neutropenia. igm antibodies were found in neutropenic and normocytic sle patients. interestingly, anti-g-csf antibodies were associated with an exaggerated serum level of g-csf and a low neutrophil count. this may suggest that exposure to high levels of intrinsic g-csf (not known whether bioinactive) may trigger the production of g-csf dabs and, further, that these dabs may have a carrier function in vivo thus slowing the elimination of g-csf from the circulation. using radioimmune and radioreceptor assays, dabs against macrophage-inhibitory protein (mip)- and mip- have been demonstrated in about % of patients, suffering from hiv infection. these antibodies specifically inhibited receptor binding of both chemokines; there was no association between the presence of antibodies and disease stage, or hiv progression rate. igg dabs against il- , il- and transforming growth factor-(tgf-) have recently been demonstrated at relatively high concentrations in up to % of patients with inflammatory bowel diseases using elisa and western blot assays. neutralizing dabs against il- p and il- p have been demonstrated primarily in patients with thymoma and/or myasthenia gravis. indeed, patients with thymic malignancy/myasthenia gravis express dabs to a variety of other cytokines including ifn-/ , ifn-, il- and il- a. , interestingly, among these patients those with opportunistic infections possess multiple anti-cytokine dabs, suggesting that these antibodies may be important in the pathogenesis of infections in patients with thymic malignancy. most patients with autoimmune polyendocrine syndrome type i suffer from chronic mucocutaneous candidiasis, and this immunodeficiency was recently associated with high titers of dabs against il- a, il- f, and/or il- . the dabs against il- a, il- f and il- neutralized these cytokines, but not a host of other cytokines. as these dabs were not found in healthy controls nor in patients with other immunoinflammatory disorders, dabs against il- a, il- f and il- may have a causative relationship with the development of chronic mucocutaneous candidiasis in patients with this polyendocrine syndrome. anti-il- dabs, often complexed with endogenous il- , have been shown to be an important prognostic indicator for the development and outcome of acute lung injury/ acute respiratory distress syndrome (ards). the il- /anti-il- complexes purified from lung edema fluids activate and trigger chemotaxis of neutrophils and regulate neutrophil apoptosis via igg receptor fc riia. these ics promote an inflammatory phenotype of human umbilical vein endothelial cells, and they upregulate the expression of intercellular adhesion molecule (icam)- on the cell surface. lung tissues from patients with ards also express high levels of icam- . hence, il- /anti-il- complexes may contribute to pathogenesis of lung inflammation by inducing activation of endothelial cells through engagement of igg receptors. anti-cytokine tabs may develop in response to prolonged therapies with natural and recombinant-derived human cytokines. it is unclear whether preexisting anti-cytokine nabs play a role in this regard, but they are probably not without importance, considering the high avidity and high titers of some of these nabs (discussed above). in general, however, tabs develop over time as a result of repeated 'inoculations' with cytokines. they may wax and wane, change in affinity, give rise to side-effects, and/or influence the primary intended therapy. the development of neutralizing tabs was first noted in scattered patients undergoing therapies with human fibroblast-derived ifn-and human recombinant ifn-. [ ] [ ] [ ] [ ] this finding received little attention at that time, but the increased use of recombinant human cytokines and cytokine constructs for therapeutic purposes have sharpened the awareness of the clinical importance of anti-cytokine tabs. [ ] [ ] [ ] thus, induction of tabs has now been reported in patients treated with several human recombinant cytokines and growth factors including ifn-, ifn-, ifn-, il- , gm-csf, often resulting in therapeutic failure and in rare cases even in serious side-effects (reviewed in refs. , , ) . while the above-mentioned anti-cytokine nabs are immunoregulatory by nature, there is evidence to suggest that nabs against other self-antigens may also be immunoregulatory, as we shall describe in the following. nabs against a broad variety of self-antigens has been demonstrated ( table ), but their physiological functions, if any, has not been fully elucidated. while some of these nabs have been suggested to play a role in the clearance of senescent cells and metabolic waste products, their role in maintenance (or breakage) of tolerance toward self remains obscure. [ ] [ ] [ ] one mechanism by which nabs against soluble self-antigens may play a regulatory role is by targeting self-antigens to apcs and thereby affecting self-antigen presentation to t cells. it is likely that nabs of igg isotype are capable of directing captured self-antigens to fc -receptor expressing apcs. similarly, nabs of igm or iga isotype may direct self-antigens to b cells and macrophages, expressing the fc / receptor. all these events may promote presentation of self-antigens to autoreactive t cells. one of the self-antigens against which nabs have been most frequently described is thyroglobulin (tg). in a serum-free environment, a subset comprising - % of normal, circulating b cells is capable of binding tg (this subset presumably represents b cells with polyreactive surface immunoglobulins), while tg binds to the entire b-cell population in the presence of autologous serum. the binding can be substantially inhibited by immunoabsorption of tg-reactive autoantibodies from serum or by heat inactivation of serum complement, suggesting that formation of complement-activating ics promoted the binding of tg to the b cells. in accordance with this hypothesis, blockade of complement receptors (cr /cd ) and (cr /cd ) reduced the binding of tg to b cells by > %, and addition of tg to preparations of normal mononuclear cells suspended in sera from healthy individuals lead to increased deposition of c -fragments on b cells. , in accordance with these findings using tg as model self-antigen, thornton et al. showed that normal serum contains nabs with reactivity against the primary antigen keyhole limpet hemocyanin, and that ics formed with these nabs are capable of fixing fragments of complement component (c ) and bind to b cells via cr and cr (fig. b) . , they further demonstrated that expression of the t-cell co-stimulatory molecule cd by b cells required a secondary ligation of ic-associated igg to fc rii. it is widely accepted that b cells bearing specific antigen-receptors efficiently take up and present antigens to t cells (fig. a) . as outlined, non-specific b cells may also engage in antigen presentation provided for the antigen in question is incorporated in complement-opsonized ics and thereby targeted to cr , cr and fc rii (fig. b) . , , , this recruitment of non-specific b cells increases the number of antigen-presenting b cells from a few antigen-specific ones to the entire b-cell population. however, only b cells bearing specific antigen receptors are eventually stimulated by t-helper (th) cells for antibody production. thornton et al. also showed that ic b-containing tg/nab complexes were also taken up by neutrophils, via complement receptors cr and cr (cd b/cd ), indicating that the formation of ics with nabs may also be similarly important for the uptake of self-antigens by myeloid cells (of relevance for dendritic cells). taken together, these findings suggest that antigen presentation is strongly promoted by incorporation of antigens into complement-activating ics with nabs. correspondingly, the presentation of tg on b cells is proportional to the anti-tg nab content of the surrounding serum, and is thus considerably higher in the presence of sera from patients with hashimoto's thyroiditis (ht) or graves' disease (gd) with a high content of anti-tg, as compared with sera from healthy individuals. in analogous studies using myelin basic protein (mbp) as self-antigen, we found that sera from patients with multiple sclerosis (ms) and sera from healthy individuals contained approximately equal concentrations of mbp-reactive igm. upon addition of mbp to normal mononuclear cells suspended in patient or control sera, mbp co-deposited with igm, igg and c -fragments on monocytes. while the deposition of igm and c was approximately similar in patient and control sera, the igg deposition was increased -fold in the presence of ms sera, presumably due to the presence of circulating high-affinity igg dabs in the patients. notably, the deposition of c fragments as well as that of igm and mbp on monocytes was abrogated by disruption of the tertiary structure of mbp by boiling, as would be expected if complex formation depends upon the interaction of antibodies with conformational epitopes on mbp. this, together with co-stimulatory signals mediated through the cd /cd -cd pathway and others, activates the th cells. b) antigen-nonspecific b cells, however, may also contribute to presentation of a given antigen, provided that the antigen is found in complement-activating immune complexes (ics) generated by preexisting nabs and/or dabs. attached to the ics, the final degradation product of complement component (c dg) may bind to complement receptor (cr ) on b cells, which then take up antigen, process it, and present antigen peptides on mhc class ii molecules. c -opsonized nab/dab-containing ics may also be taken up by fc receptor iib (fc riib). this latter type of interaction stimulates the cd -dependent binding to th cells. in the presence of untreated serum, even cd + th cells from healthy individuals respond to a challenge with tg at high concentrations ( g/ml), although tg induces increased responses by th cells (and b cells) from patients with autoimmune thyroid disease. , inactivation of serum complement, immunoabsorption of tg-reactive nabs or disruption of the tertiary structure of tg by boiling.significantly inhibits the tg-induced th cell proliferation and production of t-cell cytokines such as il- and il- . this suggests that th cell responses to self-antigens are strongly influenced by formation of ics between the self-antigens and nabs. presumably, nabs target self-antigens to apcs, as has been shown for polyclonal antibodies to foreign antigens, as well as for dabs to the thyroid self-antigen, thyroid peroxidase (tpo). [ ] [ ] [ ] moreover, recruitment of t cells to the site of infection may also be influenced by nabs and complement. askenase and tsuji demonstrated that t-cell-dependent contact sensitivity responses to hapten and subsequent rises in local ifn-levels were absent in pan b-cell-and antibody-deficient mice, but could be restored by adoptive transfer of purified normal peritoneal b- cells, or by i.v. injection of antigen-specific igm monoclonal antibodies. they concluded that the contact sensitivity response was initiated by the formation of complement-activating ics between hapten and naturally occurring igm antibodies, followed by complement activation and c a-mediated release of vasoactive substances by mast cells, facilitating the recruitment of t cells. it remains to be clarified whether ic-formation with nabs contributes to maintenance of tolerance, or whether it promotes breakage of tolerance. in mononuclear cell cultures from healthy individuals, grown in media containing autologous serum ( % v/v), tg induces immediate production of tnf-and il- by mononuclear cells, followed by an almost exclusive production of il- , a regulatory cytokine with a protective role in autoimmune diseases. , a subset of t cells with a cd ro memory phenotype seems to orchestrate this il- production, suggesting that the tg-driven t-cell response in healthy individuals is protective and contributes to the maintenance of tolerance. by comparison, the foreign antigen tetanus toxoid induces no il- production, but instead a mixed pro-inflammatory th /th -response (il- , ifn-, il- and il- ) under similar conditions. an absolute requirement for an intact tertiary structure of tg and mbp for induction of an il- release by mononuclear cells supports a role for nabs in maintaining tolerance. , nabs and dabs against cytokines and other self-antigens as discussed here, differ from one another in terms of isotype distribution and epitope recognition patterns. for example, in autoimmune thyroid disease most of the anti-tg activity is associated with igg, with only ~ % in the form of igm, whereas tg-reactive nabs are predominantly of igm isotype. nevertheless, as much as . % of igg in ivig preparations for intravenous use are reactive with tg or cytokines such as il- and il- . , , , there is also evidence to suggest that the antibody recognition patterns of nabs differ from those of dabs. for example, the latter are more restricted in idiotypes and less polyreactive than nabs. a certain idiotype, t id, is associated with autoimmune thyroid disease and recognizes one of at least six epitopic clusters on human tg, designated region ii. , dabs derived from sera of patients with hashimoto's thyreoiditis and graves' disease recognize primarily region ii and occasionally another region (region iv). by contrast, nabs frequently react with region v and rarely with region ii. thus, recognition of region v may reflect the normal homeostatic recognition of tg. interestingly, the reactivity against particular epitopes commonly recognized by both nabs and dabs seems to change with aging, without affecting the total igg anti-tg autoreactivity. we have recently demonstrated that nabs to thyroid peroxidase (tpo) show a quantitatively different recognition pattern than dabs from patients with ht. anti-tpo nabs recognize an immunodominant region involving two conformational, overlapping epitopes on tpo, referred to as immunodominant regions a (idr-a) and -b (idr-b). , in ht, approximately % of anti-tpo dabs are directed to the idr-b epitope, while dabs against the idr-a and non-a/non-b regions are approximately equally distributed. tpo-reactive nabs, on the other hand, contain a significantly lower proportion of antibodies to idr-a. interestingly, the propensity to produce autoantibodies directed against the idr-a epitope of tpo seems to be inherited. we recently demonstrated that ht patients and their healthy, monozygotic co-twins had higher proportions of idr-a-reactive anti-tpo antibodies (medians % and %, respectively) than healthy ordinary siblings to ht patients ( %) and euthyroid controls with no family history of ht ( %). these data confirmed the findings by jaume et al. based on family studies that idr-recognition patterns were genetically transmitted. in other words, the propensity to produce certain dab reactivities may be inherited. further studies are required to determine whether this applies to dabs in general. we have reviewed the immonoregulatory role of nabs with special focus on autoantibodies against cytokines and other soluble self-antigens. based on numerous publications, we and others believe that nabs against cytokines and other self-molecules may in many cases contribute to homeostasis, and that dabs may contribute to disease manifestations, in some instances perhaps as causative pathogenetic factors. these diseases likely include both autoimmune and autoinflammatory conditions. moreover, tabs may neutralize the effect of a number of "biologic" drugs and give rise to side-effects control of early viral and bacterial distribution and disease by natural antibodies auto-antibodies and immunological theories: an analytical review naturally occurring autoantibodies to exoplasmic and cryptic regions of band protein, the major integral membrane protein of human red blood cells naturally occurring anti-band- antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes autoantibodies to cytokines -friends or foes? natural autoantibodies: from "horror autotoxicus" to "gnothi seauton natural recognition repertoire and the evolutionary emergence of the combinatorial immune system natural antibodies in childhood: development, individual stability, and injury effect indicate a contribution to immune memory newborn humans manifest autoantibodies to defined self molecules detected by antigen microarray informatics high avidity cytokine autoantibodies in health and disease: pathogenesis and mechanisms anticytokine autoantibodies in infectious diseases: pathogenesis and mechanisms high levels of neutralizing il- autoantibodies in . % of apparently healthy blood donors high-avidity autoantibodies to cytokines natural and induced anti-cytokine antibodies detection of autoantibodies to cytokines increased in vivo antibody activity against interferon a, interleukin- alpha, and interleukin- after high-dose ig therapy neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, interleukin- alpha and interferon-alpha but not other cytokines in human immunoglobulin preparations igg autoantibodies against interleukin a in sera of normal individuals distribution and characterization of autoantibodies to interleukin a in normal human sera effects of human anti-il- alpha autoantibodies on receptor binding and biological activities of il- autoantibodies to il- alpha in sera from umbilical cords, children, and adults, and from patients with juvenile chronic arthritis generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin- alpha specific inhibitor anti-interleukin- antibodies in normal human serum naturally occurring autoantibodies to interleukin- alpha, interleukin- , interleukin- and interferon-alpha a state of acquired il- deficiency in . % of danish blood donors granulocyte/macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects plasma chemokine and chemokine-autoantibody complexes in health and disease interferon-alpha antibodies in autoimmune diseases natural autoantibodies to interferons anti-cytokine autoantibodies in autoimmunity: preponderance of neutralizing autoantibodies against interferon-alpha, interferon-omega and interleukin- in patients with thymoma and/or myasthenia gravis autoantibodies against type i interferons as an additional diagnostic criterion for autoimmune polyendocrine syndrome type i interferon autoantibodies associated with aire deficiency decrease the expression of ifn-stimulated genes chronic mucocutaneous candidiasis in apeced or thymoma patients correlates with autoimmunity to th -associated cytokines autoantibodies against interleukin alpha in rheumatoid arthritis: association with long-term radiographic outcome membrane-associated il- contributes to chronic synovitis and cartilage destruction in human il- alpha transgenic mice anti-interleukin- autoantibodies in plasma are associated with an increased frequency of infections and increased mortality of patients with alcoholic cirrhosis autoantibodies against interleukin- in rheumatoid arthritis interleukin- autoantibodies are involved in the pathogenesis of a subset of type diabetes gm-csf autoantibodies and neutrophil dysfunction in pulmonary alveolar proteinosis patient-derived granulocyte/macrophage colony-stimulating factor autoantibodies reproduce pulmonary alveolar proteinosis in nonhuman primates autoantibodies against granulocyte colony-stimulating factor in felty's syndrome and neutropenic systemic lupus erythematosus low prevalence of antibodies and other plasma factors binding to cc chemokines and il- in hiv-positive patients patients with inflammatory bowel disease may have a transforming growth factor-beta-, interleukin (il)- -or il- -deficient state induced by intrinsic neutralizing antibodies anti-cytokine autoantibodies are associated with opportunistic infection in patients with thymic neoplasia autoantibodies against il- a, il- f, and il- in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type i anti-chemokine autoantibody:chemokine immune complexes activate endothelial cells via igg receptors interferon-neutralizing antibodies in a patient treated with human fibroblast interferon high titer of interferon (ifn)-neutralizing antibody in a patient with glioblastoma treated with ifn-alpha. case report antitumor activity of recombinant-derived interferon alpha in metastatic renal cell carcinoma development of neutralizing and binding antibodies to interferon (ifn) in patients undergoing ifn therapy natural and therapy-induced antibodies to cytokines immunogenicity of recombinant human proteins: causes and consequences critical review: assessment of interferon-beta immunogenicity in multiple sclerosis antibodies against erythropoietin and other protein-based therapeutics: an overview a role for autoantibodies in enhancement of pro-inflammatory cytokine responses to a self-antigen, thyroid peroxidase fc alpha/mu receptor mediates endocytosis of igm-coated microbes natural autoantibodies and complement promote the uptake of a self antigen, human thyroglobulin, by b cells and the proliferation of thyroglobulin-reactive cd + t cells in healthy individuals autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase b cell and cd + t cell proliferation in response to self antigens natural antibody and complement-mediated antigen processing and presentation by b lymphocytes function of c in a humoral response: ic b/c dg bound to an immune complex generated with natural antibody and a primary antigen promotes antigen uptake and the expression of co-stimulatory molecules by all b cells, but only stimulates immunoglobulin synthesis by antigen-specific b cells antigen-specific interaction between t and b cells antigen-bound c b and c b enhance antigen-presenting cell function in activation of human t-cell clones complement opsonization is required for presentation of immune complexes by resting peripheral blood b cells autoantibodies to myelin basic protein (mbp) in healthy individuals and in patients with multiple sclerosis: a role in regulating cytokine responses to mbp self-reactive cd (+) t cells and b cells in the blood in health and autoimmune disease: increased frequency of thyroglobulin-reactive cells in graves' disease antibodies to hepatitis b surface antigen potentiate the response of human t lymphocyte clones to the same antigen presentation by peritoneal macrophages: modulation by antibody-antigen complexes effect of antigen/antibody ratio on macrophage uptake, processing, and presentation to t cells of antigen complexed with polyclonal antibodies b- b cell igm antibody initiates t cell elicitation of contact sensitivity interleukin- and the interleukin- receptor the self-antigen, thyroglobulin, induces antigen-experienced cd t cells from healthy donors to proliferate and promote production of the regulatory cytokine, interleukin- , by monocytes production of interleukin (il)- and il- accompanies t helper cell type (th ) cytokine responses to a major thyroid self-antigen, thyroglobulin, in health and autoimmune thyroid disease subpopulations of thyroid autoantibody secreting lymphocytes in graves' and hashimoto thyroid glands autoantibodies to cytokines in ivig polyreactivity is a property of natural and disease-associated human autoantibodies normal immunoglobulin g (igg) for therapeutic use (intravenous ig) contain antiidiotypic specificities against an immunodominant, disease-associated, cross-reactive idiotype of human anti-thyroglobulin autoantibodies evidence for a restricted idiotypic and epitopic specificity of anti-thyroglobulin autoantibodies in patients with autoimmune thyroiditis antigenic domains on the human thyroglobulin molecule recognized by autoantibodies in patients' sera and by natural autoantibodies isolated from the sera of healthy subjects significance of the recognition of certain antigenic regions on the human thyroglobulin molecule by natural autoantibodies from healthy subjects genetic and epitopic analysis of thyroid peroxidase (tpo) autoantibodies: markers of the human thyroid autoimmune response human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface proportion of antibodies to the a and b immunodominant regions of thyroid peroxidase in graves and hashimoto disease epitope recognition patterns of thyroid peroxidase autoantibodies in healthy individuals and patients with hashimoto's thyroiditis monozygotic twin pairs discordant for hashimoto's thyroiditis share a high proportion of thyroid peroxidase autoantibodies to the immunodominant region a. further evidence for genetic transmission of epitopic "fingerprints thyroid peroxidase autoantibody epitopic 'fingerprints' in juvenile hashimoto's thyroiditis: evidence for conservation over time and in families natural antibodies against tubulin, actin myoglobin, thyroglobulin, fetuin, albumin and transferrin are present in normal human sera, and monoclonal immunoglobulins from multiple myeloma and waldenstrom's macroglobulinemia may express similar antibody specificities naturally occurring antibodies against nine common antigens in human sera. i. detection, isolation and characterization comparative study of natural autoantibodies in the serum and cerebrospinal fluid of normal individuals and patients with multiple sclerosis and other neurological diseases polyreactive antigen-binding b cells are the predominant cell type in the newborn b cell repertoire igm class autoantibodies in human cord serum t-cell autoimmunity in type diabetes mellitus naturally occurring autoantibodies to skeletal proteins from human red blood cells natural polyreactive iga and igm autoantibodies in human colostrum autoantibodies to factor viii antibodies to a conserved region of hla class i molecules, capable of modulating cd t cell-mediated function, are present in pooled normal immunoglobulin for therapeutic use autoantibodies to heat shock protein in the human natural antibody repertoire production and characterization of monoclonal igm autoantibodies specific for the t-cell receptor a monoclonal anti-idiotypic antibody against the antigen-combining site of anti-factor viii autoantibodies defines and idiotope that is recognized by normal human polyspecific immunoglobulins for therapeutic use (ivig) establishment of reference distributions and decision values for thyroid antibodies against thyroid peroxidase (tpoab), thyroglobulin (tgab) and the thyrotropin receptor (trab) b-cell depletion with rituximab in the treatment of autoimmune diseases: graves' ophthalmopathy the latest addition to an expanding family key: cord- -vzkcin l authors: massa, p. t.; dörries, r.; ter meulen, v. title: viral particles induce ia antigen expression on astrocytes date: journal: nature doi: . / a sha: doc_id: cord_uid: vzkcin l nan cally active but which do not bind antibody have been prepared recently; cells transformed by such mutants were not altered morphologically, nor did they show greatly inhibited thymidine incorporation, after injection of antibody • it is assumed that while viral oncogenes function without normal control, their mechanism of action is similar to that of related cellular genes. here we have presented evidence to support this idea in the case of growth factor receptor-like molecules and related oncogenes. it is therefore possible that these data might aid in understanding the way in which cellular genes interact in the control of normal proliferation. our results indicate that some receptor-like oncogenes depend on ras proteins while some cytoplasmic oncogenes do not. there are, of course, numerous oncogenes which we have not yet tested which might behave differently from those described here. with this limitation in mind and on the basis of our present data, we propose that an important class of proliferative signals are received at the cell surface by receptor molecules such as growth factor receptors, and the c-ras proteins are essential in the transfer of these signals to cytoplasmic effectors having serine kinase activity; the effectors then modify target molecules which are directly involved in initiating a proliferative cycle. accordingly, if the cytoplasmic effector were mutated such that it functioned without activation, proliferation would continue independently of c-ras proteins. receptor molecules, on the other hand, would always require c-ras to stimulate proliferation. while our data are consistent with the above scheme, they do not exclude many other possibilities involving multiple metabolic pathways and more complex interactions. for example, we have not reported results with nuclear oncogenes owing to their difficulty in transforming nih t cells. the proposed scheme is primarily attractive because of its similarity to the carefully studied mechanism of signal transduction involving cyclic amp. while it is unlikely that cyclic amp itself regulates proliferation , g-regulatory proteins with enzymatic similarities to c-ras proteins are involved. these regulatory proteins control signal transduction from cell-surface receptors to cytoplasmic serine kinase effector molecules by regulating adenyl cyclase activity . while the present study has examined only one aspect of what is likely to be a highly complex system for regulating proliferation, it does provide a means of functionally comparing separate viral oncogenes. injection of antibody has been used in other studies to characterize the types of molecules responsible for tumour cell proliferation. like nih t cells transformed by mos or raj genes, many tumour cells show no inhibition of proliferation when injected with anti-ras antibody. in this way their proliferation is distinct from that of the normal cell types studied, each of which was efficiently inhibited by the injected antibodyl . this study relied on cell lines and plasmids prepared and characterized by several workers. in addition to those listed above, we thank h. temin, g. thornton, t. papas, s. goff and o. witte for supplying materials. we also thank t. curran and h.-f. kung for critical review of the manuscript and j. hansen for technical assistance. recent studies have shown that y-interferon (ifn-y) induces the expression of ia antigen on astrocytes l , . this observation is of immunological significance because such activated astrocytes can act as antigen-presenting cells, as demonstrated with myelin basic protein for antigen-specific encepbalitogenic t-cell lines • however, the lack of lymphatic drainage in brain and the presence of the so-called blood-brain barrier restricting traffic of cells and macromolecules suggests that ifn-y may not be readily available, at least during the initial phases of viral infections. the question therefore arises as to whether astrocytes can be induced to express ia antigens by other signals directly related to viral infection and possibly independent of ifn-y. in the present report we demonstrate that a neurotropic murine hepatitis virus induces expression of ia antigen on astrocytes in tissue culture without infection, rendering these brain cells competent to participate directly in the immune response to a viral infection. the murine coronaviruses are a group of agents causing acute, subacute or chronic infections in mice or rats accompanied by different disease processes , the jhm strain of this group is neurotropic and has been shown to induce acute or subacute encephalomyelitides which depend on virus as well as host factors • one important factor in the case of subacute encephalomyelitis in lewis rats is the immune response, which is directed not only against the virus but also against brain tissue • as various types of central nervous system (cns) disease are associated with this neurotropic murine coronavirus strain, we chose this virus to define its interaction with rat brain cells in culture with respect to the induction of ia antigen on astrocytes, as a baseline for our study, we analysed the response of lewis primary glial cell cultures consisting of macro phages carrying fc receptors (fc receptor+) and astrocytes expressing glial fibrillary acidic protein (gfap+) (fig, a -c) to recombinant rat ifn-y ( u ml- ; fig. la ). recombinant rat ifn-y induced the expression of ia on numerous cells in the primary cultures, fluorescence-activated cell sorting showed that - % of the cells were induced to express ia compared with the control after h treatment with u ml- ifn-y, reaching a maximum at h, when % of all cells were induced (fig. a) . by immunofluorescence microscopy, ia+ cells also became apparent after h of treatment whereas control cultures showed no ia + cells, double immunofluorescence microscopy revealed that ( , nu ml -' ) infectious jhm virus ( pfu ml -i ) + ultraviolet-inactivated jhm virus ( pfu ml -i ) + jhm virus (glial or dbt cell-derived) +anti-rat ifn-y + jhm virus+a non-neutralizing anti-jhm antibody + j h m virus + a neutralizing anti-jhm antibody + , detectable by immunofluorescence microscopy (induction of ia in at least , - , cells per cm ); -, undetectable by immunofluorescence microscopy (induction of ia in - cells per cm ). dmem, dulbecco's minimal essential medium; fcs, fetal calf serum. the stock preparation of recombinant rat ifn-y contained . x u ml-i and x u per mg protein. polyclonal rabbit antiserum to rat ifn-y, given by dr van der meide, contained . x neutralizing units (nu) ml -' . jhm virus was obtained from tissue culture supernatants of cells infected with wild-type jhm murine coronavirus. virus supernatants were produced from two different sources, primary glial cultures and a cell line permissive for jhm (designated dbt). the amount of virus in the supernatants was determined by titration (as pfu ml-i ) on dbt cells. stock virus from dbt cells numbered x pfu ml -' and from primary glial cultures, x pfu ml -i , when the cytopathic effect reached %. virus preparations were completely inactivated with ultraviolet light ( , fl w cm- ) for min. conditioned supernatants from uninfected cultures served as the control for the virus supernatant preparations. monoclonal antibody directed against the envelope glycoprotein e has been described elsewhere . the neutralizing monoclonal antibody was used at a dilution sufficient to neutralize pfu ml -i . cultures were treated as indicated for days, stained for ia using ox monoclonal antibody, then examined by fluorescence microscopy as described in fig. legend. the total number of cells in lo-day cultures was, on average, cells em - . macrophages as well as a small proportion of the type i astrocyte population were induced to express la ( fig. d-f ). treatment of primary glial cell cultures with either infectious or ultraviolet-inactivated jhm virus also induced la expression by astrocytes in a dose-dependent manner, peaking at plaque-forming units (pfu) ml- (table ) ; higher concentrations had a toxic effect on the cells. jhm virus at pfu ml - gave the maximum response regardless of the source (table ) . immunofluorescence microscopy of cultures treated with ultraviolet-inactivated jhm virus, using a polyclonal rabbit antiserum to jhm virus, confirmed the absence of infected cells throughout the cultures, indicating that there was no jhm virus replication. fluorescence-activated cell sorting showed that - % of ali cells in the cultures became la-positive (fig. ib) . conditioned media from uninfected cultures, diluted correspondingly, had no effect on la expression. in contrast to rat ifn-y, jhm virus induced la primarily in the astrocytic cell population (fig. g-i) , - % of macrophages remaining negative, as determined by double immunofluorescence microscopy. in addition, the kinetics of induction was distinct from that seen with rat ifn-y in that noticeable induction required at least - days of treatment, reaching a peak at - days. double immunofluorescence analysis of gfap and la showed that between and % of the induced cells were astrocytes (fig. g-i) . in cultures treated with infectious jhm virus, the numerous la-positive astrocytes induced were apparently unin- methods. viable lewis primary glial cell cultures were incubated for h with a : dilution of mouse monoclonal ox hybridoma supernatant ( flg ml-' igg) in dmem with % normal horse serum (at °c). cultures were rinsed three times, then incubated with a : dilution of fitc-conjugated rabbit f(ab) anti-mouse immunoglobulin (dakopatts, denmark,) for min. cultures were rinsed with . m phosphate buffer ph . , containing % bovine serum albumin (bsa), removed from the culture dishes and mechanically dissociated by pipette aspiration. mter addition of . flg ml- propidium iodide, cells were analysed immediately. gate window of forward angle light scatter (fals) lay between channels and . gate window for log integral red fluorescence was set for exclusion of non-viable cells stained bright red with propidium iodide. gate window for log integral green fitc fluorescence (ligfl) lay between channels and (abscissa). the number of la-positive cells was computed by integration from channel to for each sample containing , cells. macrophages without virus remained negative; this indicated that the ability of macrophages to express la was positively influenced by jhm virus, but that prostaglandins suppressed expression • astrocytes appeared resistant to such suppression. to determine whether the jhm virus had a direct effect on astrocytes or whether the effect was due to a secondary signal released by macrophages or astrocytes themselves, astrocytic cultures depleted of macrophages were tested for their responsiveness to jhm virus. macrophages were removed by panning of trypsinized primary cultures on hydrophobic plastic; the relatively non-adherent astrocytes remaining in suspension were removed and re-plated. after days of treatment with pfu ml- ultraviolet-inactivated virus, these astrocytes expressed la, just as in cultures containing macrophages. supernatants derived from either pure macrophage cultures or mixed primary cultures, after incubation with inactivated jhm virus, failed to induce la on naive astrocytes. this result indicated that secondary soluble factors were not involved in the induction of ia antigen on astrocytes. the possibility that ia induction was the result of virusinduced interferon synthesis in the cultures was also examined. primary cultures were treated with jhm virus ( pfu ml-t, infectious or ultraviolet-inactivated) for days, after which they na~t~u~r~e~v~o~l~. ~ ~o~ ~ap~r~il~ ~ ~ __________________ letterstonature---------------------------------- ~s~ s fig. a- ). c, phasecontrast photomicrograph. note the numerous lysosomal granules within the cytoplasm of fc+ macrophages. fc+ macrophages were found also to ingest large numbers of zymosan particles and to be nonspecific esterase-positive, both characteristic features of macro phages. d-j, double immunofluorescence and phase-contrast microscopy of one microscopic field of a -day primary glial cell culture treated for h with recombinant rat ifn-y ( u ml-i ). d, two macro phages (arrows) and one astrocyte (arrowhead) labelled for surface la. e, gfap+ astrocytes with characteristic gfap fibrillar staining pattern. not all gfap+ astrocytes are la-positive. the la + macrophages in d are clearly negative for g f ap staining and can be identified by their characteristic micros pikes (d)andlysosomalgranules (arrows in fl. f, phasecontrast photomicrograph. the astrocyte indi-i cated by an arrowhead in d-f is la+. g-i, double immunofluorescence and phase-contrast microscopy of one microscopic field of a lo-day primary glial cell culture treated during the previous days with pfu ml- ultraviolet-inactivated jhm virus. g, two astrocytes expressing cell-surface la (arrowheads). h, gfap+ astrocytes showing a fibrillar staining pattern. the arrowheads in hand i pinpoint the two la+ cells that are indicated by arrowheads in g, showing strong double immunofluorescence of ia and gfap for the same cells. note that the unlabelled gfap+ astrocytes in h are not la-positive in g. i, phase-contrast photomicrograph. the cell labelled with an arrow contains granules typical of macro phages and is negative for la (see g) and gfap (h). the other granule-containing macrophage indicated by the crossed arrow shows only weak expression of la in g at the lower pole of its cell body bearing microspikes. therefore, gfap+ astrocytes are selectively induced to express la while over % of macrophages remain ia-. parallel cultures stained for jhm antigen revealed no infected cells throughout the cultures. methods. primary glial cell cultures were established from newborn ( day postnatal) lewis rat brain as described previousli . six days after plating, at which stage the cultures were treated with ifn-y or jhm virus, three distinct cell populations were present: type i astrocytes, macrophages and a b + precursors to both type ii astrocytes and galactocerebroside-positive oligodendrocytes . staining of fc receptors was achieved by incubating live cultures with a : dilution of normal mouse serum, followed by goat anti-mouse igg conjugated to tritc (zymed, california), at °c (ref. ). after fixation with % formaldehyde and permeabilization with . % triton x-i , gfap filaments were stained using a polyclonal rabbit igg directed against gfap (dakopatts, denmark) diluted : , followed by goat anti-rabbit igg conjugated to fitc (zymed). staining of rat la and gfap was as for fc receptors and gfap. a mouse monoclonal antibody directed against rat la (designated ox ; given by dr d. w. mason) was diluted to f.lg ml- igg from hybridoma supernatant. as a control for the ox monoclonal antibody which is of the igg subclass, two different mouse monoclonal igg antibodies against unrelated antigens were tested and found to be negative. staining of jhm virus antigen was performed as for gfap, using a rabbit igg fraction directed against jhm, diluted to f.lg ml- igg. were challenged with vesicular stomatitis virus (vsv; pfu ml-i ). one day after infection, titrations of vsv released showed no reduction of pfu ml-i compared with the control. in addition, both untreated and jhm virus-treated cultures were totally destroyed by vsv, indicating an absence (or insufficient levels) of interferon(s). in addition, the application of a polyclonal rabbit antiserum to rat ifn-y in conjunction with jhm virus obtained from either infected dbt cells or primary glial cell cultures did not block or reduce ia induction. the concentration of rabbit anti-rat ifn-y used effectively eliminated the la-inducing capacity of u ml-i recombinant rat ifn-y at h post-treatment (table ) . the ia antigen-inducing capacity of jhm appears to depend on direct interaction of jhm virus with the astrocytes. the envelope glycoprotein e would be the most appropriate candidate for such virus-cell interactions as it is responsible for binding of the virus to susceptible cells and for virus-induced cell fusion • to test this notion, cultures were treated with jhm virus in conjunction with a neutralizing monoclonal antibody directed against the e glycoprotein. a non-neutralizing antibody to jhm virus was used as a control. after days of treatment, cultures exposed to non-neutralizing antibody showed a typical pattern of ia induction, whereas the neutralizing antibody to e totally blocked jhm virus induction of la (table ) ; this indicates that virus-cell binding mediated via the e glycoprotein of jhm has an important role in the induction of ia on astrocytes. here, we have described the ability of a neurotropic virus to induce la molecules on astrocytes, and we have presented evidence that jhm virus exerts its effects on astrocytes through direct interaction. this phenomenon is independent of viral replication in astrocytes as ultraviolet-inactivated virus is effective in inducing ia antigen. moreover, a monoclonal antibody to the e glycoprotein which blocks virus binding to susceptible cells , also blocks the induction of ia antigen. these observations suggest that either binding of the virus to the surface of astrocytes through specific cell-surface receptors or phagocytosis of the virus particles initiates a series of events in astrocytes leading to ia expression. the mechanism by which jhm virus induces ia expression is unknown; it is conceivable that viral glycoproteins may act on particular target cells in the induction of la in a similar way to that described for bacterial endotoxin lo • the present results are particularly interesting as ifn-y released by t lymphocytes is thought to be indispensable in the induction of la antigen on certain antigen-presenting cells ll , , including astrocytes l - • astrocytes could be especially effective antigen-presenting cells in the brain owing to their ubiquity and their ability to phagocytose, process and present antigen , in the case of virus invasion of the cns, this cell population may play an important part in mounting an immune response to effectively control the viral infection. on the other hand, high constitutive levels of la expression might carry the risk of inappropriate presentation of self antigens, as is thought to occur in autoimmune processes directed against the thyroid gland\ . this phenomenon may have special relevance to brain antigens and la-expressing astrocytes as the development of immune tolerance to self brain antigens, including myelin, may be hampered by the blood-brain barrier. the induction of la on astrocytes by jhm virus probably has a role in the jhm virusinduced chronic demyelinating disease of lewis rats, which involves induction of myelin basic protein-specific t lymphocytes • the phenomenon reported here may represent a general feature of virus-astrocyte interactions and may have wider implications for human neurotropic viruses and the induction of immunologically mediated chronic demyelinating diseases l . we thank ines tschertner for technical assistance, helga kriesinger for preparing the manuscript, dr d. mason for the ox monoclonal antibody to rat la l , dr van der meide for the recombinant rat ifn-y, dr h. wege for monoclonal antibodies to jhm virus, and dr e. wecker for helpful discussion. this work was supported by hertie-foundation, deutsche forschungsgemeinschaft and phs-nrs a no. f ns - to p.t.m. rna tumor viruses: molecular biology of tumor viroses nd end proc. natn. acad. sci. us.a. proc. natn. acad. sc< us.a proc. natn. acad. sci. us.a. progress in clinical & biological research. experimental allergic encephalomyelitis: a useful modelfor multiple sclerosis in mammals, the immunoglobulin heavy-chain variable region (v u) locus is organized in a linear fashion; individual v u, diversity (d u ), joining (j u ) and constant (c u ) region segments are linked in separate regions!. during somatic development, coding segments flanked by characteristic short recombination signal sequences, separated by intervening sequence regions that may exceed , kilobases (kb), are recombined. combinatorial joining of different segments as well as imprecision in this process contribute to the diversity of the primary antibody response; subsequent mutation further alters functionally rearranged genes. this basic somatic reorganization mechanism is shared by six major families of genes encoding antigen receptors . previously, we have shown that multiple germline genes and mammalian-like recombination signal sequences are associated with the vu gene family of heterodontus franeisei (horned shark), a primitive elasmobranch • studies presented here demonstrate that segmental reorganization involving mammalian-like du and j u segments occurs in the lymphoid tissues of this species. in marked contrast to the mammalian system, we find multiple instances of close linkage (- kb) between individual v u , d u , j u and c u segments. this unique organization may limit combinatorial joining and be a factor in the restricted antibody response of this lower vertebrate , . a heterodontus spleen poly(a)+ messenger rna-complementary dna library was constructed in the vector agtll (ref. ) and screened with a nick-translated probe that complements the entire coding region of hxia, a heterodontus germline v h gene _ multiple positive hybridizing plaques were detected and the structure of one of these, hc- , is illustrated in fig. . regions exhibiting high degrees of nucleotide identity key: cord- -ud mf l authors: li, yingying; zhao, ling; luo, zhaochen; zhang, yachun; lv, lei; zhao, jianqing; sui, baokun; huang, fei; cui, min; fu, zhen f.; zhou, ming title: interferon-λ attenuates rabies virus infection by inducing interferon-stimulated genes and alleviating neurological inflammation date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ud mf l rabies, caused by rabies virus (rabv), is a fatal neurological disease that still causes more than , human deaths each year. type iii interferon ifn-λs are cytokines with type i ifn-like antiviral activities. although ifn-λ can restrict the infection for some viruses, especially intestinal viruses, the inhibitory effect against rabv infection remains undefined. in this study, the function of type iii ifn against rabv infection was investigated. initially, we found that ifn-λ and ifn-λ could inhibit rabv replication in cells. to characterize the role of ifn-λ in rabv infection in a mouse model, recombinant rabvs expressing murine ifn-λ or ifn-λ , termed as rb c-ifnλ or rb c-ifnλ , respectively, were constructed and rescued. it was found that expression of ifn-λ could reduce the pathogenicity of rabv and limit viral spread in the brains by different infection routes. furthermore, expression of ifn-λ could induce the activation of the jak-stat pathway, resulting in the production of interferon-stimulated genes (isgs). it was also found that rrabvs expressing ifn-λ could reduce the production of inflammatory cytokines in primary astrocytes and microgila cells, restrict the opening of the blood-brain barrier (bbb), and prevent excessive infiltration of inflammatory cells into the brain, which could be responsible for the neuronal damage caused by rabv. consistently, ifn-λ was found to maintain the integrity of tight junction (tj) protein zo- of bbb to alleviate neuroinflammation in a transwell model. our study underscores the role of ifn-λ in inhibiting rabv infection, which potentiates ifn-λ as a possible therapeutic agent for the treatment of rabv infection. rabies causes acute incurable encephalitis and is responsible for more than , human deaths each year, thus posing a severe threat to public health [ ] . rabies virus (rabv) has a non-segmented, recombinant rabv strain b c was generated from cvs-b c, which was attenuated from the challenge virus standard (cvs- ) in baby hamster kidney (bhk- ) cells [ ] . the cloned cell line bsr cells were derived from bhk- cells [ ] , which were cultured in dulbecco's modified eagle's medium (dmem) (gibco, grand island, ny, usa) containing % fetal bovine serum (fbs) (gibco, grand island, ny, usa) and % antibiotics (penicillin and streptomycin) (beyotime, wuhan, china). mouse brain capillary endothelial cell line b.end cells (atcc-crl- ) were cultured in dmem containing % fbs and % antibiotics (penicillin and streptomycin). mouse neuroblastoma (na) cells [ ] were maintained in rpmi medium (mediatech, herndon, va, usa) containing % fbs and % antibiotics (penicillin and streptomycin). african green monkey kidney cells (vero, atcc-ccl- ) were cultured in dmem containing % fbs and % antibiotics (penicillin and streptomycin). hek- t cells (atcc-crl- ) were maintained in rpmi medium supplemented with % fbs and % antibiotics (penicillin and streptomycin). na cells or vero cells were seeded into -well plates and cultured for h, and then infected with b c at a multiplicity of infection (moi) of . . at hpi, the cells were treated with or ng/ml of ifn-λ or ifn-λ (bd biosciences, san jose, ca, usa) by addition into the culture medium. at indicated time points after treatment, virus titers in the supernatant were measured. primary mixed glial cell cultures were established as described previously [ ] . briefly, brain tissues from -day-old balb/c mice were dissociated by repeated pipetting and then passed through a -nm nylon mesh (corning, ny, usa). the cells were washed once in cold pbs and cultured in dmem (with high glucose) supplemented with % fbs and % penicillin-streptomycin. the medium was changed on days , , and . on day , the flasks were shaken at rpm for h to remove any non-adherent cells (mainly microglia). the remaining adherent astrocytes were detached with trypsin-edta and then plated again for further experiments. the purity of the isolated astrocytes for further studies was greater than %, which was examined by immunohistochemistry using the anti-gfap antibody. primary microglia cells were prepared from cerebral cortices of -day-old balb/c mice as described previously [ ] . briefly, brain tissues were collected from the mice, and the cortex was dissected and minced in pbs containing . % trypsin for digestion at • c for min with a shake every min. after digestion, the dmem supplemented with % fbs and dnase i were added and incubated for min. then the isolated cells were resuspended with dmem supplemented with % fbs and the cell mass were removed by µm cell sieve filtration. finally, the isolated primary microglia cells were incubated at • c for - days and change the culture medium with fresh dmem containing % fbs at day . the purity of the isolated primary microglia for further studies was greater than %, which was examined by staining the cells with anti-iba antibody using ifa. the rrabv vector pb c was constructed by inserting the genome of cvs-b c into the mammalian expression vector pcdna . as described previously [ ] . a transcription unit containing bsiwi and nhei restriction sites was inserted between the g-and l-coding sequences by deleting the pseudogene. the rb c-ifnλ and rb c-ifnλ cdna clones were generated from pb c as previously described [ ] . briefly, the pb c vector was digested with bsiwi and nhei (neb, ipswich, ma) between the g and l genes. murine ifnλ- / cdnas were prepared by rt-pcr amplification using template rna isolated from vsv-infected mouse lung tissues [ ] . the ifnλ- and ifnλ- genes were then inserted into pb c, generating pb c-ifnλ and pb c-ifnλ , respectively. pcr primers are listed in table . the full length infectious clones (pb c-ifnλ and pb c-ifnλ ) and four helper plasmids (expressing genes n, p, g, and l from the parent virus b c) were separately transfected into bsr cells using superfect transfection reagent (qiagen, valencia, ca, usa) according to procedures described in previous studies [ ] . after incubating for days, the culture medium was harvested and then examined for the presence of rescued rrabvs using fitc-conjugated anti-rabv n antibodies, and the specific fluorescence would be observed under an olympus ix fluorescence microscope if the virus is successfully rescued. table . primers for construction and rescue of recombinant rabies viruses (rabvs) expressing murine ifn-λ. sequence ( - ) bsiwi and nhei sites are underlined. fluorescence morphologies were determined using a direct fluorescent antibody assay as previously described [ ] . na cells were infected at a low moi ( . ) with different rrabvs, overlaid with semisolid medium containing % agar, and incubated at • c for h. the agar was removed and the adhesive cells were stained with fitc-labeled rabv n-specific antibody. twenty fluorescent foci were examined to calculate the number of infected cells per fluorescent focus by using image j software [ ] . virus titers were determined using a direct fluorescent antibody assay as previously described [ ] . briefly, a series of -fold dilutions of the virus were prepared and used to inoculate bsr cells in -well microplates. the inoculations were performed in quadruplicate and then incubated at • c for h. after incubation, the cells were fixed with % ice-cold acetone and stained for h with fitc-conjugated rabv n protein-specific antibodies. antigen-positive foci were observed under an olympus ix fluorescence microscope, and virus titers were calculated and presented as focus-forming units/ml (ffu/ml). elisa was performed to quantify the amount of ifnλ- / in na cell culture supernatants. the assays were performed using commercially available mouse ifnλ- / elisa kits (raybiotech, atlanta, ga, usa), following the manufacturer's instructions. the samples (tissues or cells) were collected on ice and homogenized in trizol (invitrogen). total rna was isolated and used for qrt-pcr. briefly, complementary dna (cdna) was prepared with µg rna as template using a first-strand cdna synthesis kit (toyobo). the thermocycler conditions were used for cdna synthesis ( min at • c, min at • c, and min at • c). each qpcr was conducted in duplicate with approximately ng dnase-treated rna and nm primer pairs, using a one-step sybr green qrt-pcr mix kit (toyobo). primers are listed in table . the following conditions describe real-time pcr amplification ( s at • c; s at • c, s at • c, and s at • c for cycles; s at • c), and the cycle threshold (ct) values were recorded. a standard curve was generated from serially diluted plasmids carrying a rabv n gene and the copy numbers of viral messenger rna of rabv n gene (mrna) and viral genome rna (vrna) were normalized to µg of total rna [ ] . to quantify the level of vrna, the primer vrna-f was used for reverse transcription, while the primer n mrna-r was used for reverse transcription of n-mrna quantification. the ct value was inversely correlated with the mrna concentration, and each ct unit represented a twofold change in the mrna concentration. the mrna levels of chemokines/cytokines and tj proteins were normalized to β-actin mrna levels. the results were expressed as fold change relative to mrna levels detected in mock-infected controls. table . primers for qrt-pcr. sequence ( - ) cell pellets were lysed in ice-cold ripa lysis buffer containing protease inhibitor cocktail (composed of a proprietary mix of aebsf, aprotinin, bestatin, e , leupeptin, and pepstatin a to promote broad spectrum protection against endogenous proteases). the mixture was homogenized and centrifuged at , × g for min at • c. after centrifugation, insoluble material was removed, and total protein concentration in the supernatant was measured using a bca protein assay kit (beyotime, wuhan, china). each sample was subjected to polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane (bio-rad, richmond, ca, usa), and blocked for h at • c with % bovine serum albumin (bsa) in tris-buffered saline with . % tween- (tbst). membranes were then incubated overnight with primary antibodies. after extensive washing with tbst, the membranes were incubated with secondary antibodies. antibody binding was visualized using enhanced chemiluminescence reagents (beyotime). bands were quantified using imagej (nih, bethesda, md, usa). the values represent the relative immunoreactivity of each protein, normalized to the respective loading control. five-week-old female balb/c mice (n = ) were inoculated under isoflurane anesthesia. the groups received the following treatments: (a) inoculated intramuscularly (i.m.) with a µl volume of × ffu; (b) infected intradermally (i.d.) in both ears with a dose of . × ffu in ul dmem; (c) inoculated intranasally (i.n.) in µl of a solution containing ffu of b c, rb c-ifnλ , or rb c-ifnλ or mock-infected with dmem. body weight loss, clinical signs, and survivor numbers were recorded daily for days. the animals were scored for clinical signs as follows: , normal mouse; , disorder movement; , ruffled fur; , trembling and shaking; , paralysis; , dead. all animals were humanely euthanized at the end of the experiment. t cells ( × cells per well) were seeded into -well plates. the cells were transfected with ng of luciferase reporter plasmids (a gift of prof. xiao shaobo from huazhong agricultural university) [ ] , together with pcaggs-ifnλ or pcaggs, using lipofectamine (thermo scientific, shanghai, china). in parallel, ng of prl-tk renilla luciferase reporter plasmid (promega, madison, wi, usa) was transfected to normalize transfection efficiency. twenty-four hours after transfection, the cells were infected with b c, rb c-ifnλ , and rb c-ifnλ for h. luciferase activity in total cell lysates was measured using a dual-specific luciferase reporter assay system (promega, madison, wi, usa). primary astrocytes were mock infected or infected with rrabvs at a moi of . cell supernatants were collected at hpi. inflammatory cytokines (il- β, il- , il- a, ifn-γ, kc, tnf-α, and vegf) were quantified in the mock-and rabv-infected cell supernatants using a quantibody mouse cytokine array kit (raybiotech, norcross, ga, usa), according to the manufacturer's protocol. the array was scanned using a genepix b (molecular devices, axon instruments, silicon valley, ca, usa). data were collected using the genepix pro application at a photomultiplier tube (pmt) gain ranging from to . a gain of generated the optimal standard curve, and the results of this scan were analyzed using q-analyzer for qam-cyt- (raybiotech). a transendothelial permeability assay was conducted as previously described with minor modifications [ ] . b.end cells were cultured on transwell filters (pore size . µm) until reaching % confluence. after treatments, fitc-dextran- ( kda; sigma-aldrich, st. louis, mo, usa) was applied apically at mg/ml for min. samples were then removed from the lower chamber for fluorescence measurements with a fluorimeter (excitation, nm; emission, nm). animals were anesthetized with ether and were perfused by intracardiac injection of phosphate-buffered saline (pbs) as described previously [ ] . mouse brains were fixed with % paraformaldehyde for h at • c and then washed with pbs. brain tissues were harvested and embedded in paraffin for coronal sections. to detect rabv, nonspecific binding was blocked with % goat serum, and then sections were incubated with dapi and fitc-conjugated antibodies against the rabv n protein. to detect inflammatory cells, harvested sections were incubated with primary antibodies against cd at the concentrations indicated in the manufacturer's guidelines, and then incubated with biotinylated secondary antibodies. the sections were observed under an olympus ix fluorescence microscope. for immunofluorescence, b.end cells were seeded on coverslips. after forming a confluent monolayer, they were suspended in medium containing supernatants from infected astrocytes. the cells were subsequently fixed with % paraformaldehyde (pfa), permeabilized with . % triton x- , and then incubated with rabbit anti-zo- polyclonal antibodies. finally, they were incubated with secondary antibody conjugated with alexa fluor , and with dapi for nuclear counterstaining. cells were imaged using a laser confocal microscope (leica, germany). bbb permeability was determined by measuring sodium fluorescein uptake as described previously [ , ] . briefly, µl of mg/ml sodium fluorescein was injected intraperitoneally into each mouse. peripheral blood was collected after circulation for min, and pbs-perfused brains were then harvested. the recovered serum was mixed with an equal volume of % trichloroacetic acid (tca) and then centrifuged for min. the volume of the supernatant was adjusted to µl with m naoh and . % tca. homogenized brain samples in cold . % tca were centrifuged for min at , × g to remove debris. the supernatant was adjusted to µl with addition of m naoh. fluorescence of serum and brain homogenate samples was measured using a spectrophotometer (biotek instruments, vt, usa) with excitation at nm and emission at nm. the amount of sodium fluorescein taken up into brain tissues is calculated as (µg of fluorescence cerebrum, cerebellum, or brain stem/mg of tissue)/(µg of fluorescence sera/ml of blood) to normalize values for blood levels of the dye at the time of tissue collection. data are expressed as fold differences between the amount of tracer in tissues from virus-infected mice and the amount in tissues from the uninfected control. all data were analyzed using graphpad prism (graphpad software, lnc., san diego, ca, usa). for the percent survival tests, kaplan-meier survival curves were analyzed using the log rank test. for the other data, an unpaired two-tailed t-test was used to determine whether differences were statistically significant. data were representative of two independent experiments. for all results, the following notations are used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . . to test whether ifn-λ restricts rabv replication in vitro, na or vero cells were infected with b c strain, and recombinant mouse ifn-λ or ifn-λ at ng/ml and ng/ml were added to treat the rabv infected cells at h post infection (hpi), and the virus titers, the levels of mrna of rabv n gene (n mrna) and viral rna (vrna) were measured at and h after the treatment. in na cells, as shown in figure a -c, the addition of ifn-λ or ifn-λ reduced the virus titers and the levels of n mrna and vrna at both time points. notably, at h after treatment with ng/ml of ifn-λ or ifn-λ , more than -fold decreases of virus titers were observed compared with mock-treated cells. in the vero cells, an ifn-α/β independent cell line, the addition of ifn-λ or ifn-λ reduced the virus titers and the levels of n mrna and vrna at h post treatment as shown in figure d -f. at h post treatment, no significant difference was observed in the virus titers and the levels of n mrna and vrna among all. these results suggest that ifn-λ and ifn-λ inhibits rabv replication in the two cell lines used. to further characterize the role of ifn-λ in rabv infection in the mouse model, recombinant rabvs (rrabvs) expressing murine ifn-λ or ifn-λ , designated as rb c-ifnλ and rb c-ifnλ respectively, were constructed as shown in figure a , and rescued as described previously [ ] . the insertion of both genes into the rabv genome was verified by rt-pcr and sequencing (data not shown). to detect whether the rrabvs could express ifn-λ or ifn-λ , na cells were infected with the rrabvs, and ifn-λ or ifn-λ were determined by elisa. as shown in figure b , elisa results indicate that ifn-λ and ifn-λ were well expressed in rb c-ifnλ and rb c-ifnλ infected cells, respectively, in a dose-dependent manner, whereas those expressed in b c did not. moreover, viral growth curves on bsr, na, and vero cells were depicted. as shown in figure c -e, compared with parent virus b c, more than -fold losses of viral titers were observed in rb c-ifnλ or rb c-ifnλ infected bsr or na cells at and hpi, and in rb c-ifnλ or rb c-ifnλ infected vero cells at all tested time points. additionally, consistent with the results of the growth curve, western blot experiments detecting rabv n protein showed that expression of ifn-λ or ifn-λ by the rrabvs reduced rabv n protein levels in infected na cells at hpi ( figure f) . furthermore, the effects of expression of ifn-λ or ifn-λ on virus spread were measured by observing the fluorescence morphologies of rrabv-infected cells and counting the number of rabv-positive cells using fluorescence microscopy. as a result, the fluorescence foci of cells infected with rb c-ifnλ or rb c-ifnλ were significantly smaller than those infected with b c ( figure g,h) , indicating that expression of ifn-λ suppresses the cell-to-cell spread of rabv. taken together, these results suggest that expression of ifn-λ or ifn-λ inhibits rabv replication and spread in infected cells. ) . the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . to further investigate whether ifn-λ restricts rabv infection in vivo, groups of five-week-old female balb/c mice were mock-infected with dmem or inoculated with b c, rb c-ifnλ , or rb c-ifnλ by different routes. the body weight losses of mice infected with rb c-ifnλ or rb c-ifnλ by the three routes (i.m., i.d., or i.n.) were lower than those infected with b c, as shown in figure a ,d,g. similarly, the clinical scores of mice infected with rb c-ifnλ or rb c-ifnλ by either route were lower than those infected with b c ( figure b,e,h) . consistently, significantly higher percentage of survivor ratios were observed in rb c-ifnλ or rb c-ifnλ infected mice than b c infected mice ( figure c ,f,i). it is worth noting that the most striking enhancement on survival rates among the three infection routes were observed in i.n. route-that % and % of mice i.n. infected with rb c-ifnλ (p = . ) and rb c-ifnλ (p = . ), respectively, survived, while % of b c i.n. infected mice died of rabies within days ( figure i ). all the mice exhibited rabv-related symptoms and death were confirmed by rt-pcr from brain tissues. the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . . to investigate the viral load in the brains of infected mice, balb/c mice were i.n. infected with ffu of rrabvs, or mock-infected with dmem, and viral burdens were analyzed in different parts of the brain by qrt-pcr at , , , and days post infection (dpi). viral copy numbers were extrapolated by quantitating mrnas corresponding to the rabv n gene. as expected, viral copy numbers in the olfactory bulb, cerebrum, cerebellum, and brain stem of rb c-ifnλ or rb c-ifnλ infected mice were significantly lower than those infected with b c ( figure a ). additionally, viral antigen (rabv n protein) in different brain tissues was also detected using immunofluorescence staining. consistent with the qrt-pcr results, almost no (or under detection limit) viral antigens were observed in all the parts of the brains of rb c-ifnλ or rb c-ifnλ infected mice, while an obviously positive fluorescent signal was observed in all parts of the brains of detected mice that were infected with b c ( figure b ). all the above data demonstrate that ifnλ significantly reduces the pathogenicity of rabv in mice. . ifn-λ restricts rabv replication in the mouse brain. five-week-old female balb/c mice were inoculated i.n. with ffu of b c, rb c-ifnλ , rb c-ifnλ , or dmem alone. (a) n mrna copy number was measured in mouse olfactory bulb, cerebrum, cerebellum, and brain stem at , , , and dpi by qrt-pcr (n = ). (b) mouse brain sections (n = ) were stained with fitc-labeled rabv n-specific antibody to detect the distribution of viral antigen in the central nervous system (cns) parenchyma (shown in green); nuclei stained with ', -diamidino- -phenylindole (dapi) are shown in blue. representative images were acquired at × magnification (scale bar = µm). error bars represent the se. the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . as a typical neurotropic virus, rabv mainly infects neurons in the brain. therefore, to define the signaling pathway by which ifn-λ restricts rabv infection, a luciferase reporter assay was carried out in na cells as described in the methods section. the results suggest that expression of ifn-λ or ifn-λ could activate ifnα , ifnβ, and isg -isre, but down-regulated the expression of nuclear factor κb (nf-κb) ( figure a) . furthermore, the mrna levels of ifn-α, ifn-β, stat , interferon-induced protein with tetratricopeptide repeats (ifit ), and ifn-inducible gtpase (iigp ) in different rrabv infected na cells were detected by qrt-pcr. significantly higher levels of ifn-α , ifn-α , ifn-β, stat , ifit , and iigp were detected in the cells infected with rb c-ifnλ or rb c-ifnλ than those in cells infected with b c. meanwhile, the mrna levels of nf-κb (p ) and tnf-α were significantly decreased in na cells infected with rb c-ifnλ or rb c-ifnλ compared with those infected with b c ( figure b) . additionally, expression of isgs (ifit and iigp ) and activation of jak-stat pathway, phosphorylation of stat , were also measured by western blotting assay at hpi, and high levels of phosphorylated stat , ifit and iigp were detected in rb c-ifnλ or rb c-ifnλ infected cell. these data demonstrate that ifn-λ enhances the expression of isgs via activating jak-stat pathway, and thus inhibits rabv replication. figure . ifn-λ activates stat / and enhances the production of isgs. (a) na cells were transfected with plasmids encoding ifn-α , ifn-β, isg -isre or nf-κb firefly luciferase reporter, respectively. after h, cells were left uninfected or were infected for h with different rrabvs. luciferase assays were performed to analyze the promoter activity of isre, ifn-α , ifn-β and nf-κb, respectively. luciferase reporter activity was expressed as fold change that normalized to renilla luciferase activity. (b) na cells were infected with b c, rb c-ifnλ , or rb c-ifnλ at a moi of . at hpi, total rna was isolated and ifn-α , ifn-α , ifn-β, stat , ifit , iigp , nf-κb (p ), and tnf-α mrna levels were analyzed by qrt-pcr. (c) protein levels of stat , ifit , ifit , iigp , and β-actin in different rrabv-infected na cells were measured by western blot at hpi. error bars represent the se (n = ). the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . as is known, astrocytes and microglia cells play an important role in innate immunity and neuroinflammation in the cns. hence, primary astrocytes and microglia cells were isolated as described in the materials and methods section and infected with different rrabvs at a moi of , and the production of several inflammatory cytokines was measured in the infected astrocytes and microglia cells. viral titers in the supernatants of astrocytes or microglia cells incubated with b c, rb c-ifnλ , or rb c-ifnλ were nearly equal ( figure a,b) . the supernatants of astrocytes were then applied to a protein array to quantify the production of specific cytokines, and the mrna levels of gm-csf, il- β, il- , kc, tnf-α, and vegf were quantified in microglia cells by qrt-pcr. as shown in figure c , pro-inflammatory cytokines, including tnf-α, il- , il- a, il- β, chemokine (c-x-c motif) ligand (cxcl /kc), and vascular endothelial growth factor (vegf, which is related to bbb opening), were significantly lower in astrocytes infected with rb c-ifnλ or rb c-ifnλ than those infected with b c. similarly, the mrna levels of gm-csf, il- β, il- , kc, tnf-α, and vegf were significantly decreased in microglia cells infected with rb c-ifnλ or rb c-ifnλ than those infected with b c ( figure d ). together, these results suggest that ifn-λ represses the production of inflammatory cytokines induced by rabv infection. figure . ifn-λ reduces the production of inflammatory cytokines in primary astrocytes and microglia cells. astrocytes and microglia cells isolated from -day-old suckling mice were infected with b c, rb c-ifnλ , or rb c-ifnλ at a moi of , and cell supernatants and lysates were collected at hpi for astrocytes and microglia cells, respectively, for the determination of indicated cytokines. the rrabv titers were measured using a focus-forming assay and are expressed as ffu/ml on astrocytes (a) and microglia cells (b). concentrations of the indicated cytokines in astrocyte supernatants were measured using a cytokine array (c) and microglia cells lysates were measured by qrt-pcr (d). error bars represent the se (n = ). the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . it is possible that the expression of ifn-λ in the cns can subdue the neuroinflammatory response and block the elevation of bbb permeability due to the low expression level of inflammatory cytokines and vegf in rb c-ifnλ or rb c-ifnλ infected astrocytes. meanwhile, the enhancement of bbb permeability that associated with rabv infection is caused by cytokines and the infiltration of inflammatory cells, as demonstrated in a previous study [ ] . to test this hypothesis, six-week-old female balb/c mice were i.c. infected with rrabvs. to exclude the effect of different virus loads on the regulation of bbb permeability and neuroinflammation, the brains of mice infected by different rrabvs were harvested to analyze viral burden by qrt-pcr. at , , and dpi, mrna levels of rabv n were nearly equal in different brain regions of mice infected by b c, rb c-ifnλ , or rb c-ifnλ ( figure a ). sodium fluorescein was injected into the mice via the tail vein for measurement of bbb permeability. at and dpi, no significant difference in the amount of sodium fluorescein uptake was detected among the three groups. at dpi, leakage of sodium fluorescein from the peripheral circulation into the cerebrum, cerebellum, and brain stem in b c-infected mice was significantly higher than those in mice infected with rb c-ifnλ or rb c-ifnλ ( figure b ). moreover, the brain sections were then stained with anti-cd antibody to observe and quantify the infiltration of cd + cells induced by different rrabvs infection. less positive signal was observed in different parts of brain from mice infected with rb c-ifnλ or rb c-ifnλ than those from mice infected with b c ( figure c ). significantly more cd + lymphocytes were found in the cerebral cortex, hippocampus, hypothalamus, cerebellum, and brain stem from the mice infected with b c than those infected with rb c-ifnλ or rb c-ifnλ at dpi ( figure d ). these results indicate that ifn-λ declines the bbb permeability to prevent excessive infiltration of inflammatory cells into the cns during rabv infection. figure . ifn-λ decreases bbb permeability and alleviates neurologic inflammation in the mouse brain. six-week-old female balb/c mice were inoculated i.c. with ffu of rb c, rb c-ifnλ , rb c-ifnλ , or dmem (mock). (a) at , , and dpi, mice (n = ) were euthanized, and the brains were harvested, and viral loads were determined by qrt-pcr. (b) the change in bbb permeability was assessed by measuring the amount of sodium fluorescein uptake in the cerebrum, cerebellum and brain stem using a spectrophotometer after intraperitoneal administration at , , and dpi (n = ). (c) for the assessment of neurologic inflammation, six-week-old female balb/c mice were inoculated i.c. with ffu of b c, rb c-ifnλ , rb c-ifnλ , or dmem (mock). at dpi, mice brains were collected and embedded in paraffin. the sections were then stained with anti-cd antibody to quantify inflammation. the scale bars represent µm (n = ). (d) cd + lymphocytes from at least three different randomly selected areas of each brain region were counted. error bars represent the se. the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . . to explore the mechanism by which ifn-λ reduces bbb permeability, a transwell model was performed. mouse brain capillary endothelial (b.end ) cells were cultured in the upper chamber of a transwell insert, with supporting astrocytes in the lower chamber. the b.end cells were then treated for h with extracts from the supernatants of astrocytes infected with different rrabvs, and the expression of the tj proteins zo- and occludin were then analyzed by qrt-pcr and western blot. both mrna ( figure a ) and protein expression ( figure b ) of zo- in cells treated with supernatants from b c-infected astrocytes was significantly lower than that those incubated with the supernatants from rb c-ifnλ or rb c-ifnλ infected astrocytes. additionally, supernatant-treated cells were also stained with anti-zo- polyclonal antibodies and imaged by a laser confocal microscope to assess the integrity of zo- . the rb c-infected astrocytes supernatants caused dissociation of zo- , while those treated with rb c-ifnλ or rb c-ifnλ infected astrocytes supernatants reduced the effect ( figure c,d) . to further evaluate the integrity of the endothelial monolayer, fitc-dextran , was added to the upper chamber of a transwell insert and the fluorescence in the lower chamber was monitored. as shown in figure e , the leakage of fitc-dextran , was significantly lower when treated with supernatants from rb c-ifnλ or rb c-ifnλ infected astrocytes than that treated with b c infected astrocytes supernatant. these data suggest that ifn-λ maintains the integrity of tj proteins, resulting in the decrease in the bbb permeability during rabv infection. end cells. mouse brain capillary endothelial (b.end ) cells were co-cultured for h with extracts from supernatants of astrocytes that had been mock-infected with dmem or infected with b c, rb c-ifnλ , or rb c-ifnλ at a moi of . expression of zo- and occludin was detected by qrt-pcr (a) and western blot (b). the integrity of tj protein zo- was measured by confocal microscopy (c). relative mean fluorescence intensity for the regions of interest (roi) was determined using image j (d). b.end cells were cultured on transwell inserts and treated with extracts from supernatants of astrocytes that had been mock infected with dmem or infected with b c, rb c-ifnλ , or rb c-ifnλ at a moi of . at hpi, fitc-dextran- was added into the upper insert of the transwell and then the permeation of fitc-dextran- into the lower chamber was measured using a fluorimeter (excitation, nm; emission, nm) (e). error bars represent the se (n = ). the following notations were used to indicate significant differences between groups: *, p < . ; **, p < . ; ***, p < . ; ****, p < . . ifn-λ signaling through the ifnlr receptor on intestinal epithelial cells (iecs) induces antiviral effectors such as isgs via stat /stat /irf -mediated transcription, thereby boosting defenses against intestinal viruses such as rotavirus, reovirus, and norovirus [ ] [ ] [ ] [ ] . the neurotropic west nile virus (wnv) is inhibited in the cns by ifn-λ through a mechanism that modulates endothelial cell tight junction integrity [ ] . in our study, we found that ifn-λ curtails the replication of another neurotropic virus, rabv, in cells. moreover, ifn-λ also reduces rabv pathogenicity in infected mice by different infection routes, especially i.n. route. ifn-λ has been reported to be particularly important for innate pathogen defense at mucosal barriers [ ] , which provides a possible explanation for a better survivor rate in rrabv-expressing ifn-λ-infected mice by i.n. route. in addition, ifn-λ is expressed in a tissue-specific manner and its receptor ifnlr exhibits a much more tissue-specific expression pattern, with a preference for epithelial cells [ , ] . during intranasal challenge with rabv, the expression of ifnlr by nasal epithelial cells is necessary for the responsiveness of the tissue to ifn-λ and the suppression of viral replication in nasal mucosa. therefore, ifn-λ has evolved as a specific factor that can prevent the invasion of some neurotropic viruses through nasal epithelial cells. the ability of ifn-λ to induce isg expression in a targeted set of epithelial cells also suggests that ifn-λ promotes a focused antiviral or immunomodulatory response [ , ] . the main antiviral functions of ifn-λ have been linked to the activation of the stat / signaling pathway [ , ] . activation of stat / is required for optimal transcription of isgs, which establish antiviral defenses. ifn-α/β is also engaged in antiviral processes. cooperation between ifn-α/β and ifn-λ further increases stat / -activation. in our study, isre activity was upregulated by the expression of ifn-λ during rabv infection, indicating that ifn-λ plays a positive role in activating isre, which is involved in the downstream production of isgs [ ] . isgs such as ifit and iigp were reported to restrict rabv replication [ , ] . the two isgs were elevated after the infection of rrabvs expressing ifn-λ in our study, indicating that ifn-λ could activate the stat / signaling pathway during rabv infection, resulting in the downstream production of isgs to restrict rabv infection. encephalitis induced by lab-attenuated rabv is characterized by obvious cns inflammation [ , , ] . it has previously been reported that rabv induced inflammation in microglia cells mainly through p and nf-κb pathways [ ] . the results of luciferase reporter assay in our study implied that ifn-λ expression could suitably suppress the activation of nf-κb. subsequently, production of pro-inflammation cytokines was remarkably decreased, as expected. consistent with these observations, one previous study showed that excessive expression of ifn-λ caused by iav infection, whereas the degradation of iκb and the activation of nf-κb was significantly decreased in the infected cells. in contrast, disruption of ifn-λ signaling pathway resulted in the activation of nf-κb [ ] . on the other hand, ifn-λ that is induced in dcs and macrophages does not augment proinflammatory cytokine production during viral infection [ ] . type i ifn provides antiviral resistance but also induces proinflammatory responses essential for countering infection [ ] . previous study demonstrated that ifn-λ could upregulate suppressor of cytokine signaling (socs ) and socs , which reduce the devastating effects of excessive inflammation [ ] . consistently, in our study, it was found that ifn-λ could decrease the nf-κb activation and further alleviate inflammation via suppression of neutrophil infiltration and proinflammatory cytokines such as tnf-α, il- a, il- α, il- β, il- , il- , and vegf during rabv infection, all of which contribute to bbb breakdown. the role of ifn-λ in nf-κb activation and inflammation production in the context of rabv infection appears to be important, but the detailed mechanism of this process remains to be fully explored. although ifn-λ decreases expression of proinflammatory cytokines, it does not require the activation of inflammation to modulate bbb permeability [ ] . a previous study demonstrated that stat / signaling or protein synthesis is not required for endothelial barrier tightening [ ] . indeed, in our study, ifn-λ signaling was found to maintain the expression of tj protein zo- and protect its integrity, which tightens the endothelial barrier. the tightened barrier also prevents rabv transit across epithelial surfaces, such as the nasal mucosa barrier, which could be another explanation for the strongest attenuation of rabv pathogenicity after i.n. infection. it was reported that approximately kb of dna at gene loci for ifnl and ifnl have nucleotide sequence identity greater than % for the whole genomic region in both human and mouse, indicating that the function of ifn-λ and ifn-λ could be very similar [ ] . consistently, the results in affecting rabv replication and pathogenicity are similar between rb c-ifnλ and rb c-ifnλ in our study. additionally, non-murine cell line vero cells, which are supposed to be an ifn-α/β independent cell line, were also used in our study to exclude the possible involvement of type i ifn during the ifn-λ inhibiting rabv replication. it has been reported that both mouse ifn-λ and ifn-λ were capable of up-regulating mhc class i antigen expression in several human cell lines and induced antiviral protection in mouse b cells or human ht cells infected with vsv [ ] . these data indicate that the mouse ifn-λ could potentially be functional in cells derived from species other than mouse. in conclusion, our data indicate that ifn-λ, either ifn-λ or ifn-λ , can restrict rabv infection by inducing isgs, limiting blood-brain barrier permeability to 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protein involved in rabies virus infection glycoprotein-mediated induction of apoptosis limits the spread of attenuated rabies viruses in the central nervous system of mice attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia suppression of interferon lambda signaling by socs- results in their excessive production during influenza virus infection interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness pathogenic potential of interferon alphabeta in acute influenza infection intracellular protein therapy with socs inhibits inflammation and apoptosis the role of genomic data in the discovery, annotation and evolutionary interpretation of the interferon-lambda family characterization of the mouse ifn-lambda ligand-receptor system: ifn-lambdas exhibit antitumor activity against b melanoma we thank shuaipeng he (laboratory animal center, huazhong agricultural university) for excellent mice management. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -imbpgsub authors: zhang, yun; xu, zhichao; cao, yongchang title: host–virus interaction: how host cells defend against influenza a virus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: imbpgsub influenza a viruses (iavs) are highly contagious pathogens infecting human and numerous animals. the viruses cause millions of infection cases and thousands of deaths every year, thus making iavs a continual threat to global health. upon iav infection, host innate immune system is triggered and activated to restrict virus replication and clear pathogens. subsequently, host adaptive immunity is involved in specific virus clearance. on the other hand, to achieve a successful infection, iavs also apply multiple strategies to avoid be detected and eliminated by the host immunity. in the current review, we present a general description on recent work regarding different host cells and molecules facilitating antiviral defenses against iav infection and how iavs antagonize host immune responses. influenza a virus (iav) can infect a wide range of warm-blooded animals, including birds, pigs, horses, and humans. in humans, the viruses cause respiratory disease and be transmitted by inhalation of virus-containing dust particles or aerosols [ ] . severe iav infection can cause lung inflammation and acute respiratory distress syndrome (ards), which may lead to mortality. thus, causing many influenza epidemics and pandemics, iav has been a threat to public health for decades [ ] . the virus is an enveloped, segmented, negative-strand rna virus, belonging to the orthomyxoviriae family. the eight viral gene segments encode as many as proteins. besides polymerase basic (pb ), pb -n , pb -f , pb , polymerase acid (pa), hemagglutinin (ha), nucleoprotein (np), neuraminidase (na), matrix (m ), matrix (m ), nonstructural protein (ns ) and ns (also known as nuclear export protein, nep), new viral proteins were recently uncovered, such as pb -s [ ] , pa-x (product of ribosomal frameshifting) [ ] , pa-related proteins pa-n and pa-n [ ] , m [ ] , and ns [ ] . ha, na, and m proteins constitute surface of the iav virion, where ha is the most abundant surface protein. according to the genetic and antigenic diversity of the ha and na proteins, iavs were divided into ha and na subtypes. h n and h n subtypes were recently identified in bats [ , ] . ha is a type i glycosylated protein, which is responsible for virus entry to host cell. functional ha protein is a homotrimer structurally composed of a stem region and a globular head region in each monomer. the head region bearing n-acetylneuraminic acid (sialic acid, sa) binding pocket is critical for receptor attachment, and contains most antigenic determinants. the stem region undergoing conformational changes is responsible for low ph-triggered membrane fusion [ ] , and plays an important role in cross protection against heterosubtypic iav infection [ ] . n glycan at this region iavs can infect a broad spectrum of host species, including both wild and domestic birds, as well as many mammalian species. the virus is capable of interspecies transmission to new species. however, no interspecies transmission of the bat iavs has been reported so far [ ] . furthermore, the high frequency of mutations and recombination increases the risk of iav adaptation in humans. besides three pandemic subtypes (h n , h n , and h n ), other subtypes, including h n , h n , h n , h n , n n , and h n could cross the species barrier and cause human infections [ ] [ ] [ ] [ ] . several effect factors are essential in iav host switch events, including the receptor-binding properties of ha [ ] , as well as cellular receptors [ ] [ ] [ ] [ ] . long and his colleagues summarized the role of host factors in iavs adaption to humans, and the review is recommended here for further reading [ ] . noteworthy is the fact that most phylogenetically diverse iavs with different origins could successfully replicate in swine [ ] . since pigs have both sa α- , and sa α- , galactose receptors [ ] , they can serve as a suitable mixing reservoir for both human and avian iavs, thus raising global concern on periodic zoonotic infections. take the emergence of influenza a (h n ) pdm (ph n ) and influenza a (h n ) a/canada/ / strains for instance, both strains are swine-origin iavs and were the consequence of adaption and reassortment of several swine lineages [ , ] . furthermore, some genes of these strains originated from avian iavs [ ] . with the development of gene sequencing technology, machine learning (ml) facilitated with large genomic datasets are used in prediction about sequence changes in newly invaded viruses from other animal hosts, take the "batch-learning self-organizing map (blsom)" method for instance [ ] . ml is also applied in characterization of distinct host tropism protein signatures [ ] , and prediction of amino acid changes for interspecies transmission [ ] . these studies provided measures in identification of potential high-risk strains. in addition, the nucleotides and dinucleotide compositions of viruses play important roles in prediction of viral host species [ ] . combining gene sequencing technology viruses , , of and ml methods, researchers applied large iav genomic datasets to analyze species selection bias of iav mono-/dinucleotide composition and predict human-adaptive swine or avian iavs [ , ] . the application of multi-disciplinary subjects would provide useful information for prediction of pandemic influenza. in general, the life cycle of the iav is generally divided into four steps: virus entry into the host cell, transcription and replication of the viral genome, assembly, and virus budding. though alveolar epithelial cell is the primary target cell for iavs, different iav subtypes have different patterns of viral attachment (pva). for human iavs, alveolar type ii epithelial cells, as well as immune cells such as alveolar macrophages and dendritic cells, are major target cells for an established infection [ , ] . two seasonal iavs and pandemic h n virus, preferred to attach to ciliated epithelial cells and goblet cells in the upper respiratory tract (urt), and avian iavs, take h n for instance, attached seldom to these cells [ ] . in the lower respiratory tract (lrt), human iav h n and h n attached to more cell types than avian iav h n , a highly pathogenic avian iav (hpaiv) strain. however, h n could bind to type ii pneumocytes [ ] . considering the fact that metabolism in the type ii pneumocytes is quite active, infection of hpaivs is more likely to cause severe pneumonia [ ] . other research on low pathogenic avian iavs (lpaivs), which generally do not cause severe pneumonia, showed that these viruses usually attach to human submucosal gland cells, thus can be cleared by the mucus [ ] . iav infection starts from recognition of sa by ha protein, though in vitro research claimed that these n-linked glycans were not essential for virus entry [ ] . the cleavage of ha precursor protein ha into ha (containing receptor binding domain) and ha (containing fusion peptide) in low ph environment during ha transport is critical for virion internalization [ ] . some research showed that type ii transmembrane serine protease such as transmembrane protease serine (tmprss ), human airway trypsin-like protease (hat), transmembrane protease serine (tmprss ), homo sapiens serine protease desc and homo sapiens transmembrane protease, serine (mspl) can cleave human and avian iav ha proteins at an arginine residue [ ] . in addition, for avian iavs, ha of hpaivs can be cleaved by subtilisin-like protease, while that of lpaivs is cleaved by trypsin-like proteases [ ] or thrombin [ ] . therefore, in avian iavs, the cleavage sites are considered to be the major determinants for virus virulence [ ] , and rna folding in the cleavage region could be an important factor for virulence determination [ , ] . proteins in the vrnp complex contain different nuclear localization signals (nlss), thus helping the vrnp complex to enter the host cell nucleus via active transport, take the crm -dependent pathway for instance [ ] . the acidic environment of the endosome also activates m ion channel, hence acidifies the viral core, resulting in entrance of vrnp complex into the host cell [ ] . replication of viral genome does not require a primer but a full-length complementary rna (crna), which is essential for the newly formed vrnp complex. the viral rna polymerases first bind to the end and the end of the segmented viral rna and crna, respectively, then start replication with the help of the cap of host pre-mrnas via a pb -pb -mediated "cap snatching" mechanism [ , ] . the conserved segment-specific nucleotides at the and ends of the viral genome could modulate genome expression and replication during infection [ ] . in addition, dephosphorylation at a specific position of the h n ns protein results in attenuated virus replication [ ] . mature viral mrnas are transported to the cytoplasm by a "daisy-chain" complex and translated subsequently [ , ] . new synthesis of ha occurs on the rough endoplasmic reticulum (er). glycosylation and palmitoylation of the protein are completed later in the golgi [ , ] . after synthesis and maturation of na and m proteins, the trans-golgi network (tgn), together with coat protein i (copi) complex and gtpase rab proteins, transport the newly synthesized ha, na, and m proteins to the apical plasma membrane (pm). these proteins then assemble with viral genomic segments. the virions are finally closed and m and m proteins mediate virion budding from the apical side of the viruses , , of cells [ , [ ] [ ] [ ] [ ] . na protein cleavages the sa residues, which allows the virions to be released from the plasma membrane [ ] . since iav has a relatively small genome, host machinery is required in order to accomplish the viral life cycle. to uncover host dependency factors that are necessary for iav replication, numerous large-scale rna interference (rnai) screens and genome-wide crispr/cas screen were performed [ , [ ] [ ] [ ] [ ] . for instance, son dna binding protein was important for iav virion trafficking in an early infection stage and cdc-like kinase facilitated aiv replication [ ] . usp facilitated viral entry, whereas tnfsf (april) and tnfsf -tnfsf (twepril) helped with viral replication [ ] . using genome-wide crispr/cas screen, several genes of sialic acid biosynthesis and related glycosylation pathways were involved with h n infection [ ] , and wdr , ccdc , and tmem were essential for viral entry and regulation of v-type atpase assembly [ ] . furthermore, single-cell transcriptome sequencing (rna-seq) was applied to explore host-virus interactions, revealing a correlation between defective viral genomes and virus-induced host transcriptional programs [ ] . these data provide valuable information for developing host-targeted therapeutics. host immune system functions immediately after detection of the virus. host mucosal immune system (mis), induced after virus invasion, serves as the first line to prevent iav from adhering to the susceptible cells. in the urt, mucosal response is induced in the naso-associated lymphoid tissues (nalt), while in the lrt, it occurs in bronchus-associated lymphoid tissues (balt). host innate immunity, including phagocytic cells, interferons (ifns), proinflammatory cytokines, etc., applies multiple mechanisms in defending iav infection [ ] . host adaptive immunity, mediated by b lymphocytes and t lymphocytes, together with other immune mechanisms, reacts specifically to neutralize and eliminate the virus. on the other hand, to establish a successful infection, iavs also employ a plethora of strategies to avoid being detected or being cleared by the host immunity. notable strategies include regulation of ifn signaling [ ] , inhibition of cytokine expressions [ , ] , modulation of apoptosis [ ] [ ] [ ] , interference of autophagy [ ] , and effects on antibody production [ ] . the iav-host immunity interaction was summarized by several reviews [ , ] . upon detection of infection, innate effector cells, including natural killer (nk) cells, neutrophils, and dendritic cells (dcs), etc., are recruited to the infected sites. nk cells are large granular lymphocytes, making up % of the resident lymphocytes in the lung. after recruitment from the blood, nk cells interact with dcs and macrophages to secret various cytokines and restrict infection via lysis of the iav-infected cells. the lysis process is mediated by interaction between nk receptors p (most nkp ) and iav ha protein expressed by the infected cell [ , ] . interestingly, liver nk cells other than lung nk cells possessed a memory phenotype to protect mice against subsequent iav infection, though the lung nk cells are important in control of primary iav infection [ ] . however, nk cells are also shown to exacerbate iav pathology, since depletion of nk cells led to increased resistance to high dose h n infection in mice [ , ] . the contribution of nk cells to anti-iav defense in mouse models was later shown to be strain and dose dependent. in addition, the host genetic background also played an important role [ ] . neutrophils are key innate immune cells recruited to infection sites by cellular migration through vascular endothelium. they function in clearance of pathogens via phagocytosis, producing extracellular traps, and degranulation [ ] . in addition, they also regulate adaptive immunity via guiding influenza specific cd + t cells to the infection sites [ ] . the function of dendritic cells (dcs) is to monitor invading pathogens. after iav infection, the conventional dcs migrate from lung to lymph nodes through interaction between ccr and its ligand, and present antigens to t cells [ , ] . one study based on a mouse model showed that, during iav infection, immature and mature dcs were specialized in iav ha processing, since both types of dcs could present one epitope of h n ha (ha amino acids - ), whereas another epitope (ha amino acids - ) could only be processed by mature dcs [ ] . the complex role of dcs in initiation of robust immunity against iav infection is reviewed by waithman and mintern [ ] . t cells and b cells are critical components in adaptive immunity against iav infection. cd + t cells differentiate into cytotoxic t lymphocytes (ctls) and defend iav infection via producing cytokines and effector molecules, and cytotoxic effects (i.e., lysis) of infected cells mediated by mhc class i. cd + t cells target iav-infected epithelial cells through binding with mhc class ii molecules and contribute to b cell activation thus consequently promote antibody production. the activation of t cells and b cells in iav infection will be exposited in section . . the reaction of innate immunity is nonspecific. it is triggered by recognition of pathogen associated molecular patterns (pamps) via host pathogen recognition receptors (prrs). toll-like receptors (tlrs), retinoic acid-inducible gene-i proteins (rig-i), and nod-like receptors are common prrs, the activation of which leads to activation of innate immune signaling and further production of cytokines as well as other antiviral molecules. toll-like receptors are responsible for sensing pathogens at cell membranes, endosomes, and lysosome [ ] . tlr and tlr are shown to be involved in iav detection at endosomes [ ] . tlr recognizes double stranded rna (dsrna) which may be released by cellular stress and cell death [ ] and unidentified rna structures in phagocytosed cells infected with iavs [ ] . in macrophages and dendritic cells, tlr interacted with tir-domain-containing adapter, then activated the serine-threonine kinase iκkε (ikkε) and tank binding kinase (tbk ) to phosphorylate interferon regulatory factor (irf ), the process of which further led to expression of ifn-β [ ] . in addition, an over-reacting tlr activation promoted iav pathogenesis, which could be reduced by a single-stranded oligonucleotide (sson) functioning as a tlr inhibitor, resulting in restrained viral loads both in vitro and in vivo [ ] . tlr recognizes single stranded rna (ssrna). in plasmacytoid dendritic cells (pdcs), after activation of tlr during iav infection, irf or nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) were activated via myeloid differentiation factor (myd ) to induce type i ifns [ ] . in avian macrophages, activation of tlr produced pro-inflammatory molecules such as interleukin (il)- β [ ] . in addition, in mouse models, tlr played an important role in activation of nk cells [ ] . it was also shown to be involved in development of adaptive immunity to prevent iav infection [ , ] . rig-i recognizes ssrnas and transcriptional products of iavs, which triggers activation of the caspase activation and recruitment domains (cards) via dephosphorylation or ubiquitination by e ligases, resulting in activation of transcription factors including irfs and nf-κb [ ] . otub played an essential role in regulation of rig-i [ ] . in addition, melanoma differentiation-associated gene (mda ) was also involved in sensing transcriptional products of iavs in the cytoplasm [ ] . for nod-like receptor family, pyrin domain containing (nlrp ) and nlr apoptosis inhibitory protein were activated after iav infection [ ] . iav m ion channel and pb -f were involved in activation of nlrp inflammasome and stimulate il- β secretion subsequently [ , ] . the role of the nlrp inflammasome in regulation of anti-iav responses is discussed in detail by sarvestani and his colleagues [ ] . delayed oseltamivir and sirolimus combined treatment could suppress nlrp inflammasome mediated secretion of il- β and il- , resulting in attenuation of h n -induced lung injury [ ] . after detecting viral components, transcription factors including nf-κb and irfs are activated, leading to transcription of ifns and pro-inflammatory cytokines. ifns bind to receptors, resulting in upregulation of multiple interferon-stimulated genes (isgs) [ ] . it is well known that type i ifns viruses , , of (ifn-α and ifn-β) and type iii ifns (ifn-λ - ) play critical roles in antiviral responses. mice failed to restrict non-pathogenic iav when both type i and type iii ifn receptors were knocked out [ ] . the expressed ifns consequentially bind to different receptors. type i ifns interact with ifn-α/β receptors (ifnar), whereas type iii ifns interact with ifn-λ receptors (ifnlr). janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [ , ] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [ ] . furthermore, ifn-λs served an important role in programming dcs to direct effective t cell immunity against iav infection [ ] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [ , ] . cholesterol -hydroxylase (ch h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [ ] . guanylate-binding protein (gbp ) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [ ] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [ ] . trim degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [ ] . trim regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [ ] . trim recognized iav pb protein and reduced its polymerase activity [ ] . trim targeted np for ubiquitination and degradation in vitro [ ] . for further reading on other isgs, several reviews regarding ifn responses during iav infection are recommended here [ , ] . a general description of activation of innate immunity and ifn signaling pathway after iav infection is illustrated in figure . signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [ , ] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [ ] . furthermore, ifnλs served an important role in programming dcs to direct effective t cell immunity against iav infection [ ] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [ , ] . cholesterol -hydroxylase (ch h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [ ] . guanylate-binding protein (gbp ) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [ ] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [ ] . trim degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [ ] . trim regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [ ] . trim recognized iav pb protein and reduced its polymerase activity [ ] . trim in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [ ] . the follow-up work showed that poly (adp-ribose) polymerase (parp ) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [ ] . ns is the most important ifns antagonist protein via mechanisms including inhibition of the trim -mediated rig-i ubiquitination, suppression of protein kinase r (pkr), in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [ ] . the follow-up work showed that poly (adp-ribose) polymerase (parp ) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [ ] . ns is the most important ifns antagonist protein via mechanisms including inhibition of the trim -mediated rig-i ubiquitination, suppression of protein kinase r (pkr), phosphorylation of iκb kinases (ikk) α and β in the nf-κb pathway, interruption of the phosphorylation of stat , stat , and stat [ , ] , and degradation of otub [ ] . phosphorylation of ns is crucial for its function of antagonizing ifn-β expression, since dephosphorylation at position and of the protein induced a high level of ifn-β [ ] . non-structural protein pb -f , identified from a+ open reading frame (orf) of pb gene segment [ ] , is multifunctional in deregulation of type i interferon [ , ] . it counteracted rlr-mediated activation of ifn pathway not only by targeting mitochondrial mavs [ , , ] , but also by binding to the dead-box helicase ddx to induce proteasome-dependent degradation [ ] . furthermore, pb -f interacted with mitochondrial tu translation elongation factor (tufm) to mediate formation of autophagosome, thus inducing complete mitophagy, which is critical for mavs degradation [ ] . novel pa-x protein could also modulate innate immune responses. a review regarding the function of ns and pa-x proteins in antagonizing host innate immunity is recommended here [ ] . though autophagy is essential for cellular metabolism and homeostasis, it also plays important roles in innate immune responses against pathogen infection. for cellular homeostasis, the mtor pathway is one of the most conserved autophagic pathways. the mtor complex (mtorc ) negatively regulates the ulk kinase activity, thus affecting the autophagy induction [ ] . c-jun n-terminal protein kinase (jnk ) disrupts the bcl- /beclin- complex through phosphorylation, thus regulating the autophagy induction [ , ] . jnk is also reported to upregulate beclin- expression through phosphorylation of transcription factor c-jun in vitro [ ] . in contrast to the autophagic pathways for cellular metabolism and homeostasis, less is known about autophagosome formation after iav infection [ ] . to restrict infection of multiple viruses including iavs, trim is essential to mediate autophagy via its ring e ligase and adp-ribosylation factor (arf) gtpase activity [ ] . beclin- and tufm-regulated autophagy also inhibited iav replication [ ] . in hela cells and a cells, iav infection activated jnk to induce autophagosome formation and tgf-β-activated kinase might contribute to the process [ , ] . furthermore, autophagy was involved in maintaining memory b cells to counteract iav infection [ ] . iav also utilizes autophagy to complete its life cycle. ns protein is proposed to suppress jnk -mediated autophagy induction [ ] . m could also block autophagosome maturation and mediate microtubule-associated protein light chain (lc )-bound membrane redistribution, thus allowing filamentous budding of iav [ ] [ ] [ ] . circ-gatad a (gata zinc finger domain containing a), induced by iav infection, could inhibit autophagy and promote iav replication [ ] . for a comprehensive reading on iav-induced apoptosis, a review is recommended here [ ] . upon detection of iavs, dcs trigger production of ifns and cytokines, which in turns assist maturation of the dcs into antigen presenting cells (apcs), and initiate t cell immune responses. through the activation of ag-bearing dcs, naïve cd + t cells differentiate into th , th , th , regulatory t cells (treg cells), follicular helper t cells, and killer cells. th and follicular helper t cells are the most abundant cd + t helper cells. they can secret antiviral cytokines, regulate cd + t cell differentiation, promote b cell activation, and maintain immunological memory [ , ] . th cells induced pulmonary pathogenesis and could decrease mortality of iav-infected mice [ , ] . in addition, γδ t cells, expanding in the late stage of iav infection with a t cell receptor (tcr)-independent viruses , , of manner, could efficient eliminate iav-infected airway epithelial cells, resulting in lower viral titers [ ] . new surrogate markers cd d and cd a were used to explore the kinetics of iav-specific cd t cells responses, revealing endogenous cd t cell response to primary iav infection is predominantly composed of t-bet+ cells [ ] . cd + t cells are major components for virus clearance in adaptive immunity. after activated by dcs, cd + t cells undergo rapid expansion, differentiation, and migration to the infected sites. in general, to establish effective primary cytotoxic t lymphocyte (ctl) responses, cd + t cells play an essential role, with a mouse model as an exception [ ] . ctls produce cytotoxic granules containing perforin and granzymes (gra and grb) to induce apoptosis and interrupt iav replication [ ] . in addition, ctls produce cytokines, such as tnf, fasl, and trail, which recruit death receptors to induce apoptosis [ ] . in addition, il deficiency enhanced the th and ctl responses upon iav infection [ ] . furthermore, as cd + cells could last for two years in murine models, iav-specific memory ctls reacted specific to epitopes in conserved iav proteins [ ] . in the nasal epithelia, they could prevent the spread of the virus from the urt to the lung [ ] . to establish memory cd + t cells, autophagy plays an important role [ ] , while the function of cd + t cells in memory ctl responses is "context-dependent". a recent study showed that cd + t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [ ] . grant and her colleagues summarized and discussed the importance of cd + t cell immunity against iavs [ ] , and this review is recommended for further reading. with the help of cd ligand (cd l), cd + cells contribute to b cell activation [ ] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [ ] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blocking iav transmission [ ] . in addition, iav-specific antibody-dependent cell-mediated cytotoxicity (cdcc) also plays a role in cross-protection against iav infection. a general description of adaptive immunity against primary iav infection is illustrated in figure . antigenic shift and drift, resulting in reassorted and mutated ha and/or na, are responsible for aiv escaping from host immunity [ ] [ ] [ ] . furthermore, additional glycosylation on h ha could also induce virus escape from neutralizing antibodies [ ] . apoptosis represents programmed single cell death that occurs in cell physiological remodeling, cell proliferation, or immune response to invading pathogens [ ] . besides prototypical changes, cells undergoing apoptosis can be detected through dna and biochemical assays, take the tunel and in situ end-labeling (isel) techniques for instance. two primary pathways are involved in activation of apoptosis: the intrinsic or mitochondrial pathway, and the extrinsic or death receptor pathway. the intrinsic pathway is also known as "the mitochondrial pathway", which operates in response to various intracellular stress. several factors such as nitric oxide (no), cytochrome c, and second mitochondria-derived activator of caspases (smac) can activate this pathway, and the key player of this pathway is proteins in the bcl- family, which are activated by stress signals and then release apoptotic factors via destabilizing the mitochondrial membrane [ , ] , resulting in release of mitochondrial cytochrome c. cytochrome c then binds to apoptosis protease activating factor- (apaf- ) and forms a complex with pro-caspase (then cleaved into caspase ), the function of which is to cleave its effector pro-caspase [ ] . in addition, smac, localizing in the cytosol, could initiate activation of caspase via blocking the activity of iap [ ] . the extrinsic pathway is regulated by extracellular ligands acting on transmembrane "death receptors": the first apoptosis signal (fas) receptor-fas ligand (fasr/fasl) and the tnf-αtnf receptor (tnfα/tnfr ) [ ] . in the fasr/fasl model, fas ligand binds to its receptor fasr [ ] , forming the death-inducing signaling complex (disc) with pro-caspase , resulting in activation of caspase and downstream activation of other caspases (caspase- , caspase- , and caspase- ) [ ] . in the tnfα/tnfr pathway, tnfr -associated death domain protein (tradd) is activated after binding of tnfα to tnfr , leading to recruitment of fadd and receptor interacting protein (rip) [ ] . fadd then associates with pro-caspase to form the disc, resulting in activation of caspase and apoptosis. could prevent the spread of the virus from the urt to the lung [ ] . to establish memory cd + t cells, autophagy plays an important role [ ] , while the function of cd + t cells in memory ctl responses is "context-dependent". a recent study showed that cd + t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [ ] . grant and her colleagues summarized and discussed the importance of cd + t cell immunity against iavs [ ] , and this review is recommended for further reading. with the help of cd ligand (cd l), cd + cells contribute to b cell activation [ ] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [ ] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blockin during iav infection, viruses modulate host apoptotic responses in a time-dependent manner [ ] . for instance, in order to earn enough time for replication and virion formation, iav inhibited apoptosis via upregulating the anti-apoptotic phophoinositide- -kinase-protein kinase b (pi k-akt) pathway at the beginning of infection. however, in the later phase of infection, the virus suppressed this pathway to upregulate the pro-apoptotic p pathway, thus allowing successful release of virions [ ] . several viral proteins are involved in regulation of host apoptosis. np protein induces host apoptosis to favor viral replication through interaction with ring finger (rnf ) [ ] , apoptotic inhibitor (api ) [ ] , or clusterin [ ] . pb -f also induced apoptosis and promoted viral replication through dysregulating mitochondrial potential [ ] . furthermore, m promoted apoptosis by binding to heat shock protein , thus activating caspase and the subsequent apoptosis [ ] . in addition, ns expression was reported to induce apoptosis in mdck and hela cells [ ] . however, mutant iav lacking the ns gene could induce apoptosis in cultured cells [ ] . the function of ns in inhibiting apoptosis may be explained by its ability to inhibit type i ifn [ , ] . these data demonstrate sophisticated mechanisms of iav in regulating host apoptosis. furthermore, the role of these viral proteins in apoptosis suggests that these proteins may present suitable targets for anti-iav therapies. a comprehensive review on influenza a virus-induced apoptosis discussed by ampomah and lim is recommended here [ ] . in addition, recent in vitro research found that apoptosis was induced at early iav infection stage, while later the cell death pathway was shifted to pyroptosis. the switch process was promoted by the type i ifn-mediated jak-stat signaling pathway through expression of the bcl-xl gene [ ] . during iav infection, multiple immune systems coordinate together to protect the host. accordingly, viruses antagonize the immune system through multiple measures to establish a successful infection. considering the high frequencies in genome mutations and recombination, vaccination is the most effective way to defend against the viruses via inducing cross-protective antibodies and/or enhancing immune responses. several studies in vaccine development have tried to enhance host immune responses. for instance, vaccine candidate containing ha targeted to chemokine receptor (porcine mip α) was shown to enhance t cell responses, resulting in a strong and cross-reactive cellular immunity in vaccinated pigs [ ] . another example is an attempt to intranasally administer a polyanhydride nano vaccine (iav-nanovax), which could promote robust lung-resident germinal center (gc) b cells with lung-localized iav-specific antibody responses as well as lung-resident memory cd + and cd + t cell responses [ ] . for anti-iav drugs, currently, na inhibitors (relenza tm and tamiflu tm ) are applied clinically as anti-influenza drugs [ ] . these drugs inhibit the activity of na by preventing viral budding [ ] . in addition, cap-dependent endonuclease inhibitor (baloxavir marboxil) targeting pa is also applied against influenza a and b virus infection [ ] . our progressing understanding of the iav life cycle of the virus and iav-host interaction could contribute to anti-influenza drug design. since the recognition of ha protein to sa linked glycoproteins is the first step in iav infection, effective blocking of the interaction between viral ha and sa receptor serves as a favorable target in drug design [ , ] . favipiravir, a nucleotide analogue that selectively inhibits the rna-dependent rna polymerase, is licensed in japan to be applied against emerging influenza viruses resistant to other antivirals [ , ] . oleanolic acid (oa), a kind of pentacyclic triterpene natural product, and its analogues, as well as its derivatives, were shown to bind to ha, thus blocking the attachment of iavs to mdck cells [ ] [ ] [ ] . pvf-tet is a peptide-based ha inhibitor, which was shown to sequester ha into amphisome (fusion of late endosome with autophagosome) and protected mice from the lethal iav infection [ ] . new effective drugs targeting the polymerase would be a promising strategy against iav infection, since they would directly reduce or eliminate viral replication. numerous sites, including the cap-binding site [ ] , the endonuclease [ , ] , and pa-pb inter-subunit interface [ ] can serve as potential targeting sites for new drug design. coumarin compounds, including eleutheroside b , isofraxidin, fraxin, esculetin, fraxetin, and scoparone, were investigated for their antiviral and anti-inflammatory activities against influenza virus in vitro [ ] . other candidates, such as naproxen, a non-steroidal anti-inflammatory drug, was shown to target np protein at residues f and y , thus antagonizes the crm -mediated nuclear export of np. it is suggested to have a broad-spectrum anti-influenza activity [ ] . verdinexor (kpt- ), a novel orally bioavailable drug, blocks crm -mediated nuclear export of np and repress nf-κb activation, thus reducing cytokine production and eliminating virus-associated immunopathology [ ] . for further reading on candidate anti-iv therapeutics, a review summarized by davidson is recommended here [ ] . with the increasing knowledge obtained through massive investigations on host immunity against iav infection, promoting host immune responses not limited to antibody enhancement would have good prospects not only for vaccine design, but also for development of novel antiviral agents. author contributions: manuscript preparation, y.z.; revision, z.x.; supervision, y.c.; funding acquisition, y.z. all authors read and approved the final version of the manuscript. funding: this study was supported by the "zhujiang talent program" overseas youth talent introduction program (post-doctoral program) and doctoral initiative project of natural science foundation of guangdong province ( zxxt ). the authors declare that they have no financial and personal relationships with other people or organizations that can influence the work. there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in this review. they do not have any commercial or associative interest that represents conflicts of interest in connection with the work submitted. influenza virus aerosols in the air and their infectiousness influenza: the once and future pandemic identification of a novel viral protein expressed from the pb segment of 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from now and into the future key: cord- - ralcn p authors: schwanke, hella; stempel, markus; brinkmann, melanie m. title: of keeping and tipping the balance: host regulation and viral modulation of irf -dependent ifnb expression date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ralcn p the type i interferon (ifn) response is a principal component of our immune system that allows to counter a viral attack immediately upon viral entry into host cells. upon engagement of aberrantly localised nucleic acids, germline-encoded pattern recognition receptors convey their find via a signalling cascade to prompt kinase-mediated activation of a specific set of five transcription factors. within the nucleus, the coordinated interaction of these dimeric transcription factors with coactivators and the basal rna transcription machinery is required to access the gene encoding the type i ifn ifnβ (ifnb ). virus-induced release of ifnβ then induces the antiviral state of the system and mediates further mechanisms for defence. due to its key role during the induction of the initial ifn response, the activity of the transcription factor interferon regulatory factor (irf ) is tightly regulated by the host and fiercely targeted by viral proteins at all conceivable levels. in this review, we will revisit the steps enabling the trans-activating potential of irf after its activation and the subsequent assembly of the multi-protein complex at the ifnβ enhancer that controls gene expression. further, we will inspect the regulatory mechanisms of these steps imposed by the host cell and present the manifold strategies viruses have evolved to intervene with ifnβ transcription downstream of irf activation in order to secure establishment of a productive infection. the interferon (ifn) system provides mammalian cells with a potent framework to fend off intruding pathogens. interferons are signalling molecules first discovered more than years ago, when virus-infected cells were found to release soluble compounds that could interfere with establishment of virus infection [ ] . since this initial discovery, we have come to understand the pivotal role of interferon signalling for the immune response to invading pathogens, from conveying the very first notice of intrusion to eliciting a well-tailored immune reaction suited to thwart the infection. today, we differentiate three classes of interferons based on the receptor they employ for signal transduction. more than a dozen genes encoding ifnα subtypes and a single ifnb gene give rise to the majority of type i ifns in humans. they are the first messenger molecules released upon detection of a pathogen by infected cells and by bystanders to initiate the intrinsic defence mechanisms and to further involve dedicated cells of the immune system (recently reviewed in [ , ] ). ifnγ, the only type ii ifn, presents in the latent state, a part of the linker and the most c-terminal portion form auto-inhibitory elements (aies). phosphorylation of two serine-rich clusters (c and c ) induces conformational rearrangements of the aies and frees the iad to participate in protein-protein interactions with coactivators and for dimerisation via the phosphorylated plxis motif (p: hydrophilic, x: any amino acid). the protein further contains a nuclear localization signal (nls) and a nuclear exit signal (nes) that enable constitutive shuttling between the cytosol and nucleus. irf and irf are the most closely related members of the irf family with especially high conservation of the aie, enabling formation of functional heterodimers and providing the basis for the shared activation mechanisms as well as the similar mode of action [ ] [ ] [ ] . only in plasmacytoid dendritic cells and macrophages, irf is continuously expressed at high levels and crucial for the control of type i ifn induction following tlr and tlr engagement [ ] . since these immune cells produce the major share of type i ifns in an infected host organism, irf was termed the "master regulator" of ifn expression (reviewed in [ , ] ). in most other cell types, however, irf is expressed at very low levels in absence of stimulation [ ] . in contrast to the ubiquitous irf , participation of irf molecules seems less relevant during the very first response to virus detection at initial viral entry sites, which are usually composed of epithelial or endothelial cells or fibroblasts. nevertheless, irf is an important contributor of the type i ifn response. upon ifnα/β signalling via the interferon-alpha/beta receptor (ifnar), irf expression is highly induced as part of the positive feedback regulation of the antiviral response; in other terms, irf is the product of an ifn-stimulated gene (isg) [ ] . newly synthesized irf undergoes an activation similar to irf and in concert with irf further amplifies transcription of ifnβ. this behaviour can interfere with studies focused on irf activity or modulation thereof in later stages. irf is degraded in the course of its activity, as discussed below (see section . ), while irf , though quickly degraded, is constantly expressed during stimulation [ ] . this shifts the control of gene expression in fibroblasts, epithelial and endothelial cells from irf -mediated regulation upon initial sensing of an infection towards irf -mediated in later phases [ ] [ ] [ ] . knockout experiments in mice have shown that lack consists of an n-terminal dna-binding domain (dbd) linked by a flexible region to the c-terminal irf association domain (iad). in the latent state, a part of the linker and the most c-terminal portion form auto-inhibitory elements (aies). phosphorylation of two serine-rich clusters (c and c ) induces conformational rearrangements of the aies and frees the iad to participate in protein-protein interactions with coactivators and for dimerisation via the phosphorylated plxis motif (p: hydrophilic, x: any amino acid). the protein further contains a nuclear localization signal (nls) and a nuclear exit signal (nes) that enable constitutive shuttling between the cytosol and nucleus. irf and irf are the most closely related members of the irf family with especially high conservation of the aie, enabling formation of functional heterodimers and providing the basis for the shared activation mechanisms as well as the similar mode of action [ ] [ ] [ ] . only in plasmacytoid dendritic cells and macrophages, irf is continuously expressed at high levels and crucial for the control of type i ifn induction following tlr and tlr engagement [ ] . since these immune cells produce the major share of type i ifns in an infected host organism, irf was termed the "master regulator" of ifn expression (reviewed in [ , ] ). in most other cell types, however, irf is expressed at very low levels in absence of stimulation [ ] . in contrast to the ubiquitous irf , participation of irf molecules seems less relevant during the very first response to virus detection at initial viral entry sites, which are usually composed of epithelial or endothelial cells or fibroblasts. nevertheless, irf is an important contributor of the type i ifn response. upon ifnα/β signalling via the interferon-alpha/beta receptor (ifnar), irf expression is highly induced as part of the positive feedback regulation of the antiviral response; in other terms, irf is the product of an ifn-stimulated gene (isg) [ ] . newly synthesized irf undergoes an activation similar to irf and in concert with irf further amplifies transcription of ifnβ. this behaviour can interfere with studies focused on irf activity or modulation thereof in later stages. irf is degraded in the course of its activity, as discussed below (see section . ), while irf , though quickly degraded, is constantly expressed during stimulation [ ] . this shifts the control of gene expression in fibroblasts, epithelial and endothelial cells from irf -mediated regulation upon initial sensing of an infection towards irf -mediated in later phases [ ] [ ] [ ] . knockout experiments in mice have shown that lack of irf delays the immune response, while cells lacking irf respond early but are unable to fend off the infection without the signal amplification [ ] . accordingly, together with the hefty feed-forward amplification of ifnα/β signalling, this feature is essential for the induction of distinct and diverse cytokine subsets by irf , especially of ifnα, that differentiate the reaction and prime cellular immunity [ , ] . for this reason, viruses , , of both irf and irf are crucial for the rapid induction and potent establishment of the antiviral response [ , ] . the crucial role of irf and the posttranslational changes it undergoes upon viral infection were first reported more than years ago: upon stimulation, irf gets phosphorylated and accumulates in the nucleus where it interacts with the coactivators creb-binding protein (cbp)/p to specifically bind to virus-inducible enhancer elements and exerts transcriptional activation of the ifnb gene [ , [ ] [ ] [ ] ( figure ). since these first observations, our knowledge of the mechanism of action of irf has been greatly refined. viruses , , x for peer review of of irf delays the immune response, while cells lacking irf respond early but are unable to fend off the infection without the signal amplification [ ] . accordingly, together with the hefty feed-forward amplification of ifnα/β signalling, this feature is essential for the induction of distinct and diverse cytokine subsets by irf , especially of ifnα, that differentiate the reaction and prime cellular immunity [ , ] . for this reason, both irf and irf are crucial for the rapid induction and potent establishment of the antiviral response [ , ] . the crucial role of irf and the posttranslational changes it undergoes upon viral infection were first reported more than years ago: upon stimulation, irf gets phosphorylated and accumulates in the nucleus where it interacts with the coactivators creb-binding protein (cbp)/p to specifically bind to virus-inducible enhancer elements and exerts transcriptional activation of the ifnb gene [ , [ ] [ ] [ ] (figure ). since these first observations, our knowledge of the mechanism of action of irf has been greatly refined. by rna or dna sensors in the cytosol. activation of retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) like rig-i induces aggregation of the mitochondrial adaptor protein mitochondrial antiviral signalling protein (mavs). detection of dna by the dna sensor cyclic gmp-amp (cgamp) synthase (cgas) activates production of the second messenger cgamp, which in turn induces dimerisation of the adaptor protein stimulator of interferon genes (sting) and its translocation from the endoplasmic reticulum (er) to the golgi apparatus. higher-order structures of the adaptor molecules recruit the kinase tank-binding kinase (tbk ) which leads to their tbk -mediated phosphorylation. irf is recruited to this platform and gets phosphorylated by tbk at key residues in the aie, relieving the auto-inhibition. activated irf heterodimerises and associates with the coactivators cbp/p after translocation into the nucleus, yielding a holocomplex with trans-activation potential. in parallel, the heterodimeric transcription factors p -p (nf-κb) and atf -c-jun (ap- ) are activated and enter the nucleus. first, p -p is recruited to the enhancer element upstream of the ifnb gene, followed by atf -c-jun and the irf -cbp/p holocomplex. the assembled ifnβ enhanceosome promotes recruitment of the basal transcription machinery for the expression of ifnb . hyperphosphorylation is the first step in the activation of irf as a functional transcription factor. in unstimulated cells, the irf protein exists as two forms, an unphosphorylated (i) and a basally phosphorylated (ii) form [ ] . after viral infection, iκb kinase-epsilon (ikkε) and tank-binding kinase (tbk ), two homologs of the inhibitor of nf-κb kinase (ikk), are activated when prrs convey the sensing of virus infection to their adaptor proteins, inducing conformational changes and interactions that lead to the formation of a new interaction surface (reviewed in [ ] ). the kinase binds to this signal-induced adaptor platform and phosphorylates the plxis motif (p: hydrophilic, x: any aa) on the surface of the adaptor proteins [ , ] . irf is then recruited to the platform via its recognition site for the phosphorylated plxis motif and gets further phosphorylated, giving rise to two more protein forms (iii and iv) whose appearance correlates with cbp interaction and ifnβ induction [ , [ ] [ ] [ ] . the serine-and threonine-rich region within the c-terminal aie of irf contains two clusters of potential phosphoacceptor residues: cluster (s /s ) and cluster (s /s -s /t /s ) [ ] . first, tbk phosphorylates cluster residues of monomeric irf , and the additional negative charge induces a reorientation of the aies n-and c-terminal of the iad [ , ] . this unmasks a hydrophobic binding pocket required for further protein interactions and renders the c-terminal tail (ctt) accessible for interactions [ , ] . additionally, the conformational change is relayed to the dbd to enhance the dna-binding affinity. the residues of cluster are functionally largely redundant in terms of phosphorylation-mediated irf activation, though phosphorylation of s seems to predominate in vivo [ , ] . the alternative use of phosphorylation sites could be the reason why some studies found that mutation of irf s to alanine sometimes retained biological function (for example in [ , ] ). second, and induced by the first modification, irf gets further phosphorylated at s which is pivotal for dimerisation [ , ] . consistently, % of irf dimers generated in vitro by incubation with tbk are phosphorylated at both clusters [ ] . in contrast to cluster , added phosphate groups at cluster residues have distinct effects: addition of a phosphate group at s promotes dimerisation of irf and strengthens interaction with the coactivator cbp, while phosphorylation of s negatively affects both interactions [ , , ] . by relief of the auto-inhibition, phosphorylated irf can dimerise and associate with the coactivators cbp/p to form an active holocomplex. irf can also be rendered constitutively active by exchange of the five phosphoacceptor sites in cluster to aspartic or glutamic acid residues (irf - d or - e, respectively), and these mutants display a strong tendency to acquire the cluster phosphorylation and dimerise [ , ] . since the first description of irf dimerisation, it is generally noted as the second step of activation after phosphorylation [ ] . formation of dimers requires homotypic interactions of the iad with a second phosphorylated irf molecule. structural studies revealed that phosphorylation of irf in fact modifies a plxis motif in the ctt, similar to the motif initiating recruitment of irf to the adaptor platform [ ] . enabled by the negative charge at s after phosphorylation, the extended ctt can interact with the plxis-binding surface of a second phosphorylated irf monomer to form a domain-swapped dimer (indicated in figure ). zhao and colleagues further proposed that irf dimerises at the adaptor platform to regain stability, but this was not yet confirmed. due to repulsion caused by the newly acquired negative charges, irf proteins could also dissociate from the adaptor complex before dimerisation and converge subsequently either (i) in the cytoplasm, (ii) after translocation into the nucleus or (iii) after engagement of coactivators during recruitment to the enhancer. further, the phosphorylation-induced rearrangement of the aie unmasks a hydrophobic binding site of irf and enables the interaction with transcriptional coactivators [ , , , ] . association of irf with the histone-modifying lysine acetyltransferases (kats) creb-binding protein (cbp, or kat a) and/or p (kat b) in form of a holocomplex is pivotal for the ability of irf to trans-activate ifnb expression [ , , ] . these coactivators are large proteins that contain several folded domains and additionally a big share of intrinsically disordered regions that allow for specific binding to numerous factors upon interaction [ ] . their flexible structure enables the regulation of gene transcription by integrating the cues from several hundred transcription factors, yielding cbp/p the designation "master" coactivators of transcription. the closely related proteins cbp and p are usually regarded as functionally redundant-thus often referred to in combination as cbp/p -and due to this assumption, studies characterising irf interactions assessed association of one or the other [ ] . however, there is growing evidence that their activity is overlapping but can be distinctly involved in regulation of different pathways, as exemplified by brain development [ , ] . considering that both cbp and p can participate in holocomplex formation with irf but do so with different shares [ , ] , it would be interesting to determine whether engagement of cbp versus p is specific or coincidental. potentially, the contribution of the individual coactivators could change during the different phases of ifnb expression depending on integrated signals. to exercise its function as a transcription factor, it is pivotal that irf can reach the nucleus. in fact, the latent protein already shuttles between the cytoplasm and nucleus in unstimulated conditions driven by its nls and nes. as the influence of the nes seems to prevail the import, the main share of irf molecules localises to the cytoplasm [ ] . the constitutive export of irf was shown to involve exportin (crm ) [ , ] , and the import through nuclear pore complexes involves the importins karyopherin (kpn) subunit α (kpna ), kpna and kpna (or qip ) [ , ] . upon infection, phosphorylation of the aie and accompanying structural rearrangements enable irf to associate with cbp, and this interaction retains the activated holocomplex in the nucleus [ ] . interestingly, in vitro experiments from several groups analysing the interaction between irf and the irf-binding domain (ibid) in the c-terminus of cbp have shown that independently of dimerisation, monomeric irf can interact with cbp once the auto-inhibition is relieved [ , , , ] . moreover, in vitro analyses by chen and colleagues implied that the presence of cbp promotes oligomerisation of irf molecules with the required phosphomimetic mutations that enable dimerisation, while the mutant proteins stayed monomeric without cbp [ ] . in a cellular scenario wherein irf dimerises directly after acquiring the required modifications in the vicinity of the adaptor platform in the cytoplasm, this interaction might not be relevant, though. characterisation of the holocomplex demonstrated that the homodimer of irf is more stable than the holocomplex of irf dimer plus p [ ] , and the association of irf dimers with cbp is stronger than that of monomeric irf [ ] , viruses , , of favouring a model wherein irf first dimerises, and dimers subsequently interact with cbp. however, without determination of the precise temporal succession of interactions, this leaves the possibility that a phosphorylated irf monomer enters the nucleus and interacts with cbp/p before it encounters a second irf molecule to dimerise and swiftly induce a response, as suggested before [ ] . in the end, the coactivator needs to associate with dimeric irf because only this can induce activation of the kat activity that is later required for assembly of the pre-initiation complex, as demonstrated for p [ ] . several groups noted exceptions to the common model of irf activation followed above, drawing attention to the fact that assessment of the conventional signs like dimerisation that reflect the intermediate states is not always sufficient to infer on the downstream biological response [ , , ] . due to these discrepancies we suggest to differentiate between phosphorylated irf that is relieved of the auto-inhibition and can "actively" participate in subsequent steps and irf in the holocomplex that can exercise its trans-activation potential as a transcription factor to "actively" induce ifnb expression. in fact, some of these studies assessed the activity of irf in terms of induction of isg [ , ] , which intermingles the irf -dependent regulation of type i ifns with directly irf -regulated isg expression [ ] . to our knowledge, it remains to be determined whether the form of irf induced for up-regulation of specific isg promoters is the same as the form of irf able to induce ifnb expression. moreover, reports clearly reflect that there are different extents of irf activity, and the protein might not yet be fully activated when initiating a first wave of ifnb expression. instead, irf might acquire modifications and engage in distinct interactions in different waves of the type i ifn response until it is fully activated. in line with this, we noticed several reports referring to "full" activity of irf in later loops of the response, for example enabled by phosphorylation at both clusters of the aie or determined only in the presence of ifnar signalling [ , ] . this aligns with the kinetics of the ifnβ response we will briefly revisit below (see section . ), wherein only a part of the ifnb alleles are engaged in the early response. expression of ifnb is probably the best studied model of induced eukaryotic gene regulation. it exemplifies how factors activated by specific signals cooperate to prompt a defined expression programme, as reviewed before [ , [ ] [ ] [ ] . upon viral stimulation, a multicomponent complex of transcription factors, coactivators and architectural proteins forms at the enhancer sequence upstream of the ifnb gene promoter. this higher-order nucleoprotein complex, termed the ifnβ enhanceosome, works as a molecular switch that turns on upon virus-induced activation of a specific set of transcription factors [ , ] . when completed, the enhanceosome in turn recruits factors with chromatin-modifying activities as well as basal eukaryotic transcription factors to the nearby transcription start site (tss) to initiate gene expression. the assembly is regulated at several levels and highly specific due to an elaborate organisation of transcription factor binding motifs, the required set of activated transcription factors and architectural proteins and finely coordinated interactions of the individual participants as well as of the arising composite structures. from just slightly upstream of the tss of the human ifnb gene, a precisely arranged sequence of cis-acting dna constitutes the key enhancer element that defines ifnb expression. the ifnβ enhancer itself is accessible yet directly flanked by two nucleosomes, one of them covering the tss and a tata box [ ] . this setup strategically restricts the access of the transcription machinery in latent conditions [ , ] . the enhancer can be divided into four positive regulatory domains (prds) in the order iv-iii-i-ii that are discussed in detail in [ ] . of note, however, is the overall architecture that is essential for the precise recognition and following interplay of the array of core transcription factors [ ] . the prd farthest away from the promoter, prdiv, features motifs for binding an ap- heterodimer formed by activating transcription factor (atf ) and c-jun of the basic-region leucine zipper (bzip) family [ , ] . binding of the heterodimer over homodimers is favoured by the intrinsic architecture of the enhancer and important for subsequent interactions [ ] . the last prd in the sequence of the enhancer, prdii, is recognised by a p -p or p -p nf-κb dimer [ ] [ ] [ ] . prdiii and prdi in the middle section feature in total four overlapping irf-binding elements (irf-es) of the consensus sequence -aanngaaa- that form two composite binding sites enabling binding of two irf dimers [ , , , ] . this region was also termed p because these two prds act as a functional unit, an enhanson [ ] . recent in-depth analysis of the binding requirements of different irf members suggests that, in addition to a slight variation of the core gaaa recognition motif, the flanking regions along with the spacing in between the irf-es contribute to specific binding of distinct irf family members, here favouring recruitment of irf and irf [ , ] . the nucleotide sequence of the ifnβ enhancer causes a bent conformation of the dna helix in inactive conditions and in this way drives cooperative binding of the transcription factor heterodimers upon stimulation [ ] . after stimulus-induced activation, the transcription factors translocate into the nucleus. first, the nf-κb dimer p -p is recruited and nucleates assembly [ ] . association of nf-κb is the most limiting factor of enhanceosome assembly, and to optimise recruitment, it is initially targeted to three loci at distinct chromatin regions via alu elements, also termed nf-κb reception centres (nrcs) [ , , ] . the transcription factor thpok (t-helper-inducing poz/krüppel-like factor, also zbtb b) binds cooperatively with nf-κb to these regions, and oligomerisation of thpok mediates interchromosomal interactions of the nrcs with the ifnβ enhancer to deliver nf-κb [ , ] . alongside nf-κb, the architectural dna-binding high mobility group protein i(y) (hmg i(y), also hmga ) binds at two sites to the minor groove in prdii and straightens the dna conformation, thereby supporting interaction of nf-κb [ , [ ] [ ] [ ] [ ] . subsequently, the remaining virus-induced transcription factors are recruited. a second hmg i(y) molecule interacts with atf -c-jun to promote binding of the heterodimer to prdv by straightening the dna helix [ , , ] . additionally, nf-κb mediates capture and binding of the irf -cbp/p holocomplex [ , ] . in vitro, irf displays a surprisingly weak affinity for p , which led to the former model in which irf is the critical irf member in ifnb induction because it freely associates with the enhancer sequence [ ] [ ] [ ] . however, irf was then found to be dispensable for ifnb expression upon prr activation, which is congruent with its expression pattern as an isg [ ] [ ] [ ] . instead, when phosphorylation releases the auto-inhibition of irf and enables interaction with cbp/p , the coactivator strongly increases the affinity of irf for dna and enables association of the complex [ , , , ] . further cooperativity is mediated by the p sequence that ensures concerted binding of atf -c-jun and irf [ , ] and by cbp/p -mediated interactions within the enhanceosome complex [ , ] . clustering of irf target regions with binding motifs for nf-κb or other transcription factors along with low chromatin accessibility in steady-state conditions was recently described as a common feature of irf -dna interactions, implying an obligatory collaborative binding mode of irf that promotes access to chromatin [ ] . at the ifnβ enhancer, a total of four irf / molecules bound to four irf-es in tandem are required to induce gene expression of a luciferase-based ifnβ-reporter plasmid in the commonly used human embryonic kidney cell line hek t [ ] . structural analysis suggests that one irf dimer binds on one side of the duplex, occupying the first and the third irf-e, and the second dimer binds from the opposite side, binding to the second and fourth irf-e [ ] . remarkably, the dbds of the eight transcription factors barely interact with each other despite the great overlap of their binding elements [ ] . instead, the high level of cooperativity between binding events is achieved by the specific order of individual response elements that allows for binding-induced conformational changes of the dna to transmit allosteric effects [ , , , ] . molecular dynamics simulations by pan and nussinov further showed that the combination of overlapping recognition viruses , , of elements with an alternation between consensus and non-consensus motifs optimises the specific interactions by enhancing or restricting binding of neighbouring transcription factors. moreover, the signal integration by cbp/p that interacts with each transcription factor through different domains is essential for the functionality of the enhanceosome [ ] . in addition to the pivotal architectural role of recruiting irf and mediating interactions with the transcription machinery as coactivator, cbp/p also participates in the subsequent chromatin remodelling in its vicinity [ , , ] . as implied above, however, the precise number and identity of coactivator molecules participating in the formation of the active irf -cbp/p holocomplex as well as in the enhanceosome is unknown. potentially, they present a module for further signal integration by participating in dynamic compositions. further, also the subtle modulation by the two hmg i(y) proteins is required for the maximal level of expression [ ] , though their participation in the final assembly is controversial as previously discussed [ ] . in line with the high synergy during assembly, the ifnβ enhanceosome is extraordinarily stable and enables multiple rounds of re-initiation in vitro [ , ] . due to the binding-induced conformational changes of the dna foundation, the fully assembled structure encompasses a straight segment of dna covered by eight specifically bound transcription factors all of which interact with the essential coactivator cbp/p . this final ifnβ enhanceosome presents a single new, continuous activating surface and provides the basis for the association of the basal transcription apparatus. before transcription of the ifnb gene can commence, the chromatin landscape needs to be modified so that the pre-initiation complex can be assembled and access the tss. first, the ifnβ enhanceosome mediates the transient recruitment of a complex of p /cbp-associated factor (pcaf, or kat a) and general control nonderepressible- (gcn or, kat b) to the promoter and induces the remodelling, starting with gcn -mediated acetylation of nucleosomes and hmg i(y) in its vicinity [ , , ] . acetylation of hmg i(y) at k by gcn strengthens the association of nf-κb and thereby further stabilises the enhanceosome [ , ] . next, the modification of the histone tails of nucleosomes at h and h promotes recruitment of the chromatin remodelling brg -or brm-associated factor (baf) complex of the swi/sfn family [ , , , , ] . in parallel, general eukaryotic transcription factors (tfs) tfiia, tfiib, tfiie, tfiih, tfiif and upstream stimulatory activity (usa) cofactors assemble at the promoter along with the rna polymerase ii (rnap ii) holoenzyme [ , , ] . stimulated by the environment rich in acetylated histones, the atpase brahma-related gene (brg or smarca ) of the baf complex induces a conformational change of the nucleosome at the tss [ , ] . now, the tata box at the tss is available for binding by the tata box-binding protein (tbp) and allows association of the tfiid complex [ , ] . notably, tfiid binds here after the rnap ii complex, as opposed to the classical sequence of events in assembly of the pre-initiation complex [ ] . association of tfiid induces a bend of the dna helix so that the nucleosome covering the tata box slides bp downstream of its latent position [ , ] . with this, the arrangement of the pre-initiation complex can be completed, and ifnb transcription can begin [ , ] . notably, while the rapid induction of the type i ifn response is crucial for the host defence, the first round of ifnb expression yields only low levels of ifnβ, as discussed by ford and thanos [ ] . briefly, after stimulation of a cell population, ifnb is initially expressed only by a fraction of cells and from only one allele due to a stochastic phenomenon that indicates a limiting amount of required cellular factors [ ] [ ] [ ] . the initial recruitment of nf-κb to other loci that collect the available transcription factor and transfer it to the required binding sequence highlights the p -p heterodimer as the most limiting factor [ , ] . the weak initial signal of secreted ifnβ induces high-level expression of irf , and newly synthesized irf promotes enhanceosome assembly and ifnb transcription from the remaining allele and in more cells [ ] . finally, the weak initial ifnβ signal is then amplified to eventually induce the cytokine storm of the antiviral response. the expression of ifnb is, at least in murine cells, tightly regulated by different forms of nf-κb dimers in the initial response to infection, as reviewed by balachandran and beg [ ] . at resting conditions, p homodimers associate with the ifnβ promoter region and repress basal transcription in the absence of appropriate stimulation [ , , ] . in addition, a binding site for nf-κb regulatory factor (nrf) overlaps the nf-κb binding element in prdii and negatively affects basal transcription [ , ] . in line with a primary inhibitory effect, knockout of murine p does not affect induction of ifnb expression after virus infection [ ] . however, the p -imposed down-regulation in resting cells is competed with by p -p dimers that are constitutively activated by ikkβ and cycle between the cytoplasm and nucleus [ ] . this low level activity of p supports a basal level of ifnb expression that ensures rapid and robust responsiveness on demand, while the negative regulation by p ensures specificity [ ] . after stimulation, p -p is required to assist in the early recruitment of irf together with cbp/p to support ifnb expression [ , , ] . only at high levels of irf activity, i.e., at the peak of activity later in infection or brought about by expression of the constitutively active irf -s d, p -p is dispensable for ifnb transcription [ ] . subsequent to the initial recruitment, the increasing levels of active irf and irf can fully assume the regulation of ifnβ production, and nf-κb proceeds to regulate expression of pro-inflammatory genes [ ] . the p -p -mediated basal expression of ifnb raises the question how recruitment of the transcription machinery is achieved at resting conditions, considering that irf is missing to participate in a highly cooperative assembly as described above. moreover, since the type i ifn system is not % conserved between mice and humans, and all of these studies were carried out in murine systems, it remains to be confirmed whether the exact same mode applies for the participation of nf-κb in the human system. in addition to the proximal enhancer, dna elements further away from the promoter are involved in ifnb transcription, though again, most studies have thus far focused on the murine system. in non-infected mouse cells, a proximal region of the ifnb locus that contains binding sites for the transcription factor yin yang (yy ) was shown to mediate association with pericentromeric heterochromatin (pch), a feature related to gene silencing [ ] . after infection, the ifnb locus is repositioned away from pch, and this observation correlated with transcriptional activation of the promoter shortly thereafter. josse and colleagues suggested that this effect could be mediated by binding of yy to the proximal promoter. furthermore, yy interacts also in the absence of viral infection in mice with the signal transducer and activator of transcription (stat ), one of the main factors mediating isg expression [ ] , implying important roles of yy proteins at different stages during infection [ ] . in the human cell, yy and yy regulate enhanceosome formation from yy-binding site-containing regions far upstream of the tss of the ifnb gene at − kb and − kb, termed dnase i hypersensitive sites (hs ) and hs , respectively [ ] . yy and yy seem to modulate each other, with yy antagonising the negative effect of yy . in analogy to the murine ifnβ promoter region, this long-range interaction was suggested as requirement for the recruitment of gcn to initiate chromatin remodelling at the ifnβ promoter [ ] [ ] [ ] . a further dna element regulating ifnb transcription is the long-range enhancer l , which was first identified in human cells but is conserved in mice [ ] . l is activated upon viral stimulation and recruits irf and rnap ii dependent on the irf-e within its interferon-stimulated response element (isre). this induces expression of a non-coding rna from the enhancer, and this enhancer rna (erna) in turn supports ifnb expression. however, the mode of action of the erna was not yet determined. the rapid induction of the antiviral state is critical to overcome a nascent infection. while an insufficient response would be beneficial for the pathogen, overshooting activation of the immune system can cause severe damage to the organism. therefore, several mechanisms are implemented by the host organism to regulate irf at all levels, from moderating the potential to activate irf from its latent state, to promoting its participation in formation of the ifnβ enhanceosome, to supporting its activity during ongoing stimulation, through to terminating the response with deliberate timing. factors that contribute to the finely tuned ensemble of modulators can be continuously active, induced or inhibited upon stimulation. a wide array of molecular mechanisms that modulate the biological activity of irf are deployed, including protein-protein interactions, regulatory rnas and common as well as non-canonical posttranslational modifications. to illustrate the abundance of strategies, we will include also proteins expressed predominantly in immune cells. in particular, mechanisms aiming at dampening the activity of irf were usually identified in macrophages, highlighting the importance to keep the response primed but low until the full activation becomes necessary. since posttranslational modifications of irf and especially phosphorylation and ubiquitination were reviewed before [ , ] , we will instead focus on how the host factors engage in the different steps to modulate the biologic activity of irf ( figure ). viruses , , x for peer review of system can cause severe damage to the organism. therefore, several mechanisms are implemented by the host organism to regulate irf at all levels, from moderating the potential to activate irf from its latent state, to promoting its participation in formation of the ifnβ enhanceosome, to supporting its activity during ongoing stimulation, through to terminating the response with deliberate timing. factors that contribute to the finely tuned ensemble of modulators can be continuously active, induced or inhibited upon stimulation. a wide array of molecular mechanisms that modulate the biological activity of irf are deployed, including protein-protein interactions, regulatory rnas and common as well as non-canonical posttranslational modifications. to illustrate the abundance of strategies, we will include also proteins expressed predominantly in immune cells. in particular, mechanisms aiming at dampening the activity of irf were usually identified in macrophages, highlighting the importance to keep the response primed but low until the full activation becomes necessary. since posttranslational modifications of irf and especially phosphorylation and ubiquitination were reviewed before [ , ] , we will instead focus on how the host factors engage in the different steps to modulate the biologic activity of irf ( figure ) . in addition to the essential steps of irf activation delineated above, various protein-protein interactions and additional posttranslational modifications were demonstrated to support the biological activity after induction. before stimulation, for example, protein phosphatase (pp ) removes the phosphate groups at s and s in macrophages and consequently attenuates ifnβ production [ ] . early after tlr-or rlr-mediated stimulation, however, activity of pp is down-regulated to increase the pool of active irf and augment the immune response [ ] . also first identified in murine macrophages, the lysine methyltransferase nuclear receptor-binding set domain (nsd ) targets nuclear irf after viral stimulation and modifies irf by adding a single methyl group at k [ ] . this modification promotes the dissociation of phosphorylated irf from an isoform of pp catalytic subunit γ, thereby hindering the inhibitory effect of pp and maintaining the activated state of phosphorylated irf . similarly, type i ifn up-regulates the expression of the long non-coding rna lnclrrc -as, an antisense transcript to the gene of leucine-rich repeat-containing protein , which supports irf phosphorylation in macrophages by inhibiting an inhibitor [ ] . in the cytosol, lnclrrc -as associates with the protein phosphatase methylesterase (pme- ), which in turn promotes the interaction of pme- and the protein phosphatase a (pp a). pp a is an inhibitor of irf signalling, but lnclrrc -as mediates its demethylation and inactivation to maintain irf activity [ ] [ ] [ ] . additionally, stability of the irf protein is promoted within the loop of the type i ifn response by covalent conjugation of newly synthesised isg to irf on k , k or k mediated by herc (hect and rld domain-containing e ubiquitin protein ligase ) [ , ] . this modification, termed isgylation, disturbs the interaction between irf and peptidyl-prolyl isomerase (pin ), delays the proteolytic degradation of irf and in this way prolongs the immune response [ ] [ ] [ ] . the dual use of irf k for methylation by nsd versus isgylation by herc , both of which contribute to protein stability, hints at a complex network that also modulates the modulators. while inactive irf already shuttles between the cytosol and nucleus in latent conditions, the translocation becomes crucial after its activation in order to exercise the trans-activation activity. just recently, cai and colleagues reported that the ubiquitin specific peptidase (usp ) promotes the antiviral response from the cytoplasm by deubiquitinating importin kpna [ ] . after viral infection, kpna associates with irf for nuclear import, and usp promotes this critical step by stabilizing kpna . importin-mediated translocation is additionally regulated by the widely expressed transcription regulators yes associated protein (yap ), yap and yap : yap / / associate with latent as well as activated irf and block further interactions required for dimerisation or nuclear import [ ] . after virus infection, however, activated ikkε phosphorylates a conserved motif of the yaps, which triggers their lysosomal degradation and reliefs the yap-mediated inhibition. further, an additional phosphate group within the dbd of irf at s is important for the nuclear translocation after viral stimulation [ ] . this modification inhibits the nuclear import, and mass spectrometry revealed that about a fifth ( . %) of irf molecules carry it at latent conditions when exogenously expressed in hek . upon viral stimulation, the dual-specificity protein phosphatase and tensin homolog (pten) removes this phosphate group and consequently the negative regulation of irf [ ] . finally, to keep irf in the nucleus, dna-pk is activated in response to virus infection and phosphorylates irf at t [ ] . this phosphate moiety mediates the nuclear retention and delays proteolysis of the active transcription factor, thereby prolonging the irf -driven response. the association of cbp and irf in the nucleus is supported by the constitutively active enzyme glutaredoxin- (glrx or grx ), which acts in the cytoplasm [ ] . in resting cells, inactive irf is s-glutathionylated and this modification would impede the essential interaction of irf and cbp/p . however, glrx removes this modification after infection and thus supports irf activity. while the deglutathionylation of irf is independent of its phosphorylation or dimerisation state, the accompanying structural changes could be involved in the recruitment of glrx to remove the modification after initial activation events [ ] . within the nucleus, the interaction of irf and cbp is further promoted by a ubiquitously expressed subunit of the endosomal sorting complex required for viruses , , of transport (escrt)-ii [ ] . after virus infection, a fraction of the escrt-ii subunit ell-associated protein of kda (eap , also known as sfn ) localises to the nucleus where it interacts with activated irf and cbp and promotes binding of the holocomplex to target gene promoters. contrasting this, the protein argonaut (ago ), a component of the rna-induced silencing complex, interacts with the iad of irf and inhibits its association with cbp in the nucleus in latent conditions [ ] . upon viral infection, however, ago is exported from the nucleus, and its inhibition is revoked to promote ifnβ production during infection [ ] . a further mechanism of signalling amplification is set into motion by type i ifn-stimulated production of the e like ets transcription factor (elf ). elf is recruited to sting and activated by tbk similar to irf after viral stimulation, though without affecting irf activation itself [ ] . activated elf enters the nucleus and binds to the ifnβ promoter region via ets/irf composite binding elements (eices). dna binding of elf synergises with binding of irf , and thus promotes enhanceosome formation. the critical role of elf in the feed-forward amplification of the murine antiviral immune response was demonstrated in vivo, and notably, it is independent of the signalling in plasmacytoid dendritic cells [ ] . in contrast, a mechanism that due to the predominating expression of both factors in tissues of the immune system applies exclusively to immune cells is the association of irf together with the transcription factor pu. (also spi , spi- proto-oncogene) to the ifnβ promoter region mediated by eice [ ] . li and colleagues proposed that irf and pu. support the rapid induction of transcription by forming a scaffold at the ifnβ enhancer to facilitate recruitment of activated irf [ ] . a striking feature of irf modulation is reflected by mechanisms that are installed not to plainly heighten or terminate the biological activity of irf but to moderate its extent. some of the mechanisms delineated here affect the latent protein, others specifically target activated irf after viral stimulation and still others act on both. macrophages are a prime example in deploying numerous modulators to dampen fluctuations of irf activity until a certain threshold is passed and further to restrain the response once unleashed. for example, the ubiquitin-protein ligase e c (ube c) mediates k -linked ubiquitination of irf and irf , thereby targeting the master transcription regulators in dendritic cells for proteolysis, irrespective of their activation state [ ] . in this way, ube c helps to maintain low amounts of ifn production in resting conditions and additionally restrains the magnitude of the response after stimulation [ , ] . another e ubiquitin ligase, tripartite motif-containing (trim or ro ), was described by two groups to be important for regulation of irf but with opposing findings as already discussed by sin [ ] : higgs and colleagues reported trim -dependent mediation of irf degradation hours after infection [ ] , whereas yang and colleagues found that nine hours after infection, trim interferes with the interaction between irf and pin and in this way prevents pin -mediated ubiquitination and degradation of irf to sustain the immune response [ ] . sin suggested that, in addition to effects potentially imposed by different cell lines, the observation at different times of ongoing infection could have contributed to the contrasting findings [ ] . stability of irf is further modified by addition or removal of small ubiquitin-like modifier (sumo) proteins. in hek cells, endogenous irf is sumoylated at k and k in its dbd, and viral infection slightly increases sumoylation [ ] . the widely expressed human sentrin/sumo-specific protease (senp ) removes these sumo moieties and in this way conditions irf for k -linked ubiquitination at the same residues and subsequent degradation, thereby dampening the antiviral response [ ] . senp targets wild-type irf as well as the constitutively active irf - d mutant, suggesting that the sumoylation initially masks the protein from degradation to prolong its activity but that this modification is constantly countered by senp activity. in addition, stimulus-induced sumoylation of murine irf at k was reported to attenuate the ifnβ response irrespective of the protein's phosphorylation status, though the mediating proteins are unknown [ ] . regulation of auto-inhibition aside, phosphorylation of irf can contribute to the down-regulation of its activity also at subsequent steps. when screening the human kinome for phosphorylation events during nucleic acid sensing, meng and colleagues identified mammalian sterile -like kinase (mst , also stk ) as an inhibitor of ifnβ promoter induction and confirmed the effect of mst in knockout mice [ ] . mst suppresses activation of tbk and ikkε and thereby inhibits the phosphorylation-dependent activation of irf , but it also targets irf directly for deactivation by phosphorylating two sites of irf (t , t ). demonstrating that this effect is independent of the inhibition of the upstream kinases by mst , introduction of a single phosphomimetic mutation, t d, is sufficient to impair the ability of the constitutively active irf - d to dimerise after stimulation [ ] . a further level of negative regulation of ifnβ signalling is imposed by the ubiquitously expressed fas-associated factor (faf ), which interacts with importin (iop , also kpnb ) in resting as well as stimulated cells [ ] . after viral stimulation, irf increasingly associates with ipo for nuclear import, but this is dampened by the faf -ipo interaction which consequently reduces the translocation of phosphorylated irf and the induction of ifnb expression. in the nucleus, formation of the irf /p complex and p -mediated acetylation of irf after virus infection is assisted by bromodomain-containing (brd ) [ ] . generally, interaction of brd with p promotes recruitment of the holocomplex to the ifnβ enhancer and facilitates ifnb transcription. this supporting mechanism seems to come with a time limit, however, as analysis in macrophages revealed that viral stimulation specifically down-regulates brd abundance and thereby terminates its support [ ] . similarly, zhang and colleagues recently reported stimulation-induced non-canonical k -linked ubiquitination at three positions in the dbd of irf (k , k , k ) and demonstrated that this modification is essential for the dna-binding capacity of irf in macrophages and in hek t cells [ ] . as a counter measure, virus-infected cells additionally up-regulate expression of the ovarian tumour domain-containing deubiquitinase (otud ), and otud then removes this ubiquitination from irf , resulting in reduced irf -binding to the ifnβ promoter region at later times [ ] . to allow moderation of enhanceosome assembly after formation of the active irf -cbp/p complex, other dna-binding proteins were reported to interact with motifs contained within the ifnβ enhancer sequence. the first irf-e overlaps with a consensus binding site for an activator of pro-inflammatory responses in macrophages, nfat (nuclear factor of activated t cells ), which is conserved between human and mouse promoter sequences [ ] . nfat constitutively forms dimers that can bind to the ifnβ enhancer region and competes with irf due to the overlapping recognition sequence, resulting in limited recruitment of irf to the enhancer and thus limited promoter induction. in a similar way, the transcription regulator maf bzip transcription factor b (mafb) binds in macrophages to the ifnβ enhancer sequence mediated by ap- -like sites [ ] . in resting conditions, mafb is a weak positive regulator of basal ifnb transcription. upon stimulation and activation of irf , however, it impairs the interaction between the coactivators and antagonises enhanceosome formation. this dual role was suggested to allow the system to deal with fluctuations in irf activity [ ] . during the course of the innate immune response, the signal that started it all has to be switched off again to allow other messengers to refine the defence line. the first mechanism to terminate the irf -dependent response is set into action directly during the activation of irf by phosphorylation of the c-terminal aie [ ] . in addition to releasing the auto-inhibition, the modification represents a signal for degradation of the protein, and so activated irf has a shorter half-life than the latent form [ , ] . in this way, turnover of the activated protein by degradation rather than additional activation of repressors is an integral feature responsible for ending ifnb expression [ ] . several molecules have been reported to mediate ubiquitination of irf in order to induce its depletion after initial stimulation [ ] : pin , foxo [ ] , trim , rbck and trim (see above, section . ) all mediate k -linked ubiquitination and subsequent degradation of activated irf . for example, the peptidyl-prolyl isomerase pin specifically mediates degradation of the activated transcription factor by recognition of a further phosphorylated motif (s phos-p ) irf acquired during stimulation [ ] . additionally, viral infection promotes the nuclear localisation of the e ubiquitin ligase trim , allowing trim to bind phosphorylated irf in the nucleus and promote its k -linked ubiquitination and degradation [ ] . similarly, production of rbcc protein interacting with pkc (rbck ), another e ubiquitin ligase, is induced by viral stimulation and targets irf for ubiquitination [ ] . furthermore, caspase- (casp ) is activated by cytosolic rig-i-dependent signalling and cleaves irf at a recognition motif between dbd and iad ( sqpd ), inducing ubiquitination and degradation of the fragments [ ] . in parallel to the activation of irf , viral stimulation also activates inhibitors of irf . the transcriptional regulator krüppel-like factor (klf ), which is widely expressed in human tissues, increasingly localises to the nucleus after viral infection where it binds to the ifnβ promoter region [ ] . this reduces recruitment of irf and thus inhibits induction of ifnb expression. in immune cells, the lysine acetyltransferase kat was identified by an sirna-screen as a negative regulator of antiviral innate immunity [ ] . viral infection promotes kat -mediated acetylation of irf at k , independent of the phosphorylation and dimerisation status of irf , and this modification interferes with binding of irf to the ifnβ enhancer. a potential effect on assembly of the irf -cbp/p holocomplex was not characterised, however, leaving the exact step of interference to be determined. a further line of down-regulation is introduced by the expression of isgs that negatively modulate irf activity. considering the multitude of induced isgs [ ] , it is not surprising that they target various levels to terminate ifnb transcription. nuclear import is repressed by the increasing interaction of irf with the ifn-inducible dead-box rna helicase ddx , as this interaction competes with the association of irf and ipo [ ] . the irf -dna interaction is inhibited by interaction of newly synthesized cell growth-regulating nucleolar protein lyar (ly antibody-reactive), which is usually low expressed in most cell tissues but induced by the ifnβ response [ ] . lyar interacts specifically with the n-terminal domain of activated irf and in this way interferes with the dna-binding capacity of irf in the irf -cbp/p holocomplex. additionally, a long non-coding rna directly targets the ifnβ promoter region and interferes with transcription factor binding in a unique way [ ] : after rna deep sequencing revealed enhanced transcription of the long non-coding rna lnc-mxa from the mxa locus after viral infection, li and colleagues recently reported its mechanism of action. applying a chromatin isolation by rna purification assay and in vitro pulldown of an ifnβ promoter dsdna fragment with biotin-labelled lnc-mxa, they demonstrated that lnc-mxa forms an rna-dna triplex with the ifnβ promoter region. this changes the structure of the chromatin and interferes with binding of the transcription factors irf and nf-κb to their respective target sequence [ ] . finally, the delayed generation of prdi-binding factor (prdi-bf , or pr/set domain ) mediates recruitment of the histone h lysine methyltransferase g a for epigenetic silencing of ifnb expression [ , ] . the following host factors were additionally reported as negative modulators of irf activity, but it remains to be seen if they constantly inhibit irf or how they are regulated. for instance, calmodulin-like protein (calml or caglp) was identified to negatively modulate irf and independently of the protein's ability to bind calcium ions [ ] . calml interacts with the c-terminal aie of irf , and this interaction is strengthened when irf is phosphorylated. after virus infection, calml thereby impairs dimerisation and nuclear import of irf . the contribution of cellular flip long isoform protein (cflip l ) to regulation of the type i ifn response in several primary cancers was reported by several groups with contradicting outcomes, so gates and colleagues studied the underlying mechanism of action and concluded on an inhibitory function [ ] . when ectopically expressed in hek t cells, cflip l interacts with phosphorylated irf within the nucleus and hinders interaction with cbp and thus with the ifnβ enhancer. cflip l was then confirmed to be highly expressed in several human cancer cell lines and to interact with endogenous irf , mediating reduction of isg expression. as opposed to the major isoform (variant ) of irf that drives the type i ifn response as discussed so far, three of the five described splice-variants of human irf negatively modulate the biological activity of irf , though their regulation remains to be determined. translation of variant (irf -cl) yields a slightly longer protein ( aa) with a unique sequence of aa in the c-terminus that lacks a part of the iad including the aie of irf [ ] . irf -cl constitutively forms homodimers that localize to the cytoplasm. after viral stimulation, ikk-mediated activation induces association of irf -cl with phosphorylated irf , but the heterodimers are retained in the cytoplasm and consequently, the association with this isoform keeps irf from the induction of ifnb expression. in contrast, variant (irf a) lacks the n-terminal part of the functional dbd of irf and is consequently unable to bind to classical irf-es [ , ] . this isoform also heterodimerises with irf variant after stimulation, but in line with the absent dbd, its presence selectively inhibits virus-induced ifnb transcription. interestingly, after virus stimulation, irf a is degraded slower than phosphorylated irf and so the ratio of negative versus positive modulator increases with ongoing infection, potentially contributing to a downregulation of the type i ifn response [ ] . the fourth variant, irf e or irf -nirs, was discovered when marozin and colleagues searched for the reason of the defect in ifnb expression in hepatocellular carcinoma (hcc) cells and discovered that a truncated variant of irf was constitutively expressed in primary cells of hcc or hcc cell lines but not in primary hepatocytes [ ] . irf -nirs is produced when an in-frame exon is aberrantly skipped during splicing, leading to the generation of a protein lacking aa within the β-sandwich core of the iad. in line with this, this isoform is constitutively active and maintains the dna-binding ability. however, due to the compromised iad, irf -nirs seems unable to exercise trans-activation activity and instead competes with irf for the limited irf binding sites in the ifnβ enhancer sequence [ ] . surprisingly, given that association with the coactivator is normally essential for irf to bind to dna motifs, this implies that the interaction surface for cbp/p remains intact despite the missing adjacent structural module. in addition to the human isoforms, one variant of murine irf , mirf- a, was reported as a ubiquitously expressed negative modulator of ifnβ induction in mice [ ] . during the generation of mirf- a, an alternative donor splice site in exon is used and leads to a frameshift with a premature termination codon, yielding a shorter variant ( aa) which differs in the c-terminal region as compared to the major murine variant ( aa). mirf- a freely localises to the nucleus, binds to irf recognition sites and represses promoter activation. not long after the initial steps in the characterisation of the ifnβ enhanceosome were made, also the first viral proteins targeting its assembly were noticed [ , ] . today, a long list of viral factors targeting every step from the stimulation of prrs by nucleic acids, to induction of transcription factor activation, to activity of ifn, through to the stimulation and activity of isgs are known, and the list still grows [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the central role of irf in this pathway makes it an attractive target for viral evasion. strategies applied to interfere with the activation of irf in the cytoplasm include (i) the inhibition of irf expression; (ii) direct antagonism of essential phosphorylation events by targeting the essential kinases, their interaction with irf or dephosphorylating irf (recently joined by herpes simplex virus (hsv)- immediate early protein icp [ ] , and the nucleocapsid protein of peste des petits ruminants virus [ ] ); and (iii) mediating degradation of irf . remarkably, even nuclear phosphorylated irf can specifically be targeted for ubiquitination and proteasome-mediated degradation after activation [ ] . other viral factors aim to evade the type i ifn response more generally by limiting its induction, affecting production of ifn by transcriptional or translational shut-off or dysregulating the processing or trafficking of host mrnas [ , ] . noting that strategies to inhibit irf activation and activity applied by viruses were frequently reviewed for specific virus families as well as in broad summaries [ ] [ ] [ ] [ ] [ ] [ ] , we want to emphasize here the molecular mechanisms viruses deploy to obstruct the function of irf after its trans-activation potential is enabled by phosphorylation (figure ). obstruct the function of irf after its trans-activation potential is enabled by phosphorylation ( figure ). as the activation of irf is a multistep process, in early studies the exact level of viral interference sometimes remained elusive. dimerisation of irf was among the first steps reported to be targeted in order to antagonize its trans-activation activity. the ml protein, a splice variant of the matrix protein of thogoto virus (thov) of the family orthomyxoviridae, blocks the formation of irf homodimers irrespective of the presence of the c-terminal aie and further inhibits association of irf with cbp [ , ] . interestingly, this modulator does not affect nuclear translocation, suggesting that ml does not disable import of irf but renders otherwise activated irf monomers unable to participate in further interactions in the nucleus. the authors ruled out a lasting interaction as the activation of irf is a multistep process, in early studies the exact level of viral interference sometimes remained elusive. dimerisation of irf was among the first steps reported to be targeted in order to antagonize its trans-activation activity. the ml protein, a splice variant of the matrix protein of thogoto virus (thov) of the family orthomyxoviridae, blocks the formation of irf homodimers irrespective of the presence of the c-terminal aie and further inhibits association of irf with cbp [ , ] . interestingly, this modulator does not affect nuclear translocation, suggesting that ml does not disable import of irf but renders otherwise activated irf monomers unable to participate in further interactions in the nucleus. the authors ruled out a lasting interaction between ml and irf that could for example directly compete with binding to cbp/p because they could not detect association of ml and irf by co-immunoprecipitation [ ] . additionally, ml interferes with a later step in the induction of ifnb expression, namely, the cofactor function of tfiib at promoters that require de novo recruitment of rnap ii, which was indicated by the finding that ml also inhibited activity of the constitutively active irf - d mutant [ , ] . the leader (l) protein of the cardioviruses of the picornaviridae also interferes with irf dimerisation. in fact, at first, the l protein of encephalomyocarditis virus (emcv) was reported for its ability to generally disturb trafficking of proteins between the cytoplasm and nucleus [ ] . after infection with a related strain, mengovirus, however, irf still localized to the nucleus. to inhibit type i ifn transcription nonetheless, the mengovirus l protein antagonizes dimerisation of irf downstream of its phosphorylation [ ] . due to the observation of irf translocation without dimerisation, the authors suggested that importin-mediated transport might be a requirement for dimerisation of irf , which would take place within the nucleus, which is in line with nuclear translocation of monomeric irf , as discussed above (see section . ). the l protein from a different cardiovirus species, theiler's murine encephalomyelitis virus (tmev), also interferes with the formation of irf dimers, though it was first identified because it additionally inhibits the nuclear translocation of irf [ , ] . this function depends on the zinc finger motif of l [ ] , but whether l affects preceding phosphorylation of irf as well remains undetermined. the strong impact of l on the antiviral innate immune response is additionally accounted for by the inhibition of mrna export from the nucleus which is achieved by l-mediated phosphorylation of nucleoporin [ ] . in contrast to the yet unclear molecular mechanism of ml and l, an interesting detail of dimerisation antagonism was described for the serine/threonine kinase us of hsv- [ ] . us phosphorylates irf at s , and this hyperphosphorylation interferes with both dimerisation and nuclear translocation of irf . as also activity of irf - d could be inhibited by co-expressed us in a luciferase-based reporter assay, the effect of us seems independent of the activation status. this adds a further phosphate group to the posttranslational modifications that modulate irf activity and raises the question whether the cardiovirus l proteins might also interfere with the formation of irf dimers via small modifications, just as tmev l mediates inhibition of the nuclear pore protein by phosphorylation [ ] . the v protein of simian virus (sv ) was the first reported antagonist of irf translocation [ ] , followed by the multifunctional ns protein of influenza b virus (ibv) [ ] and the l protein of tmev [ ] . again, these early studies did not yet dissect which form of irf is targeted because the role of the essential modifications was not known in detail. later reports of viral antagonists of irf translocation then included an assessment of the activation state of irf , and some were found to specifically target irf after its activation. for example, hsv- initially stimulates activation of irf upon entry but subsequently inhibits irf activity by means of newly synthesized infected cell polypeptide (icp ) [ ] . during the viral replication cycle, a part of the multifunctional icp molecules localises to the cytoplasm and inhibits the translocation of activated irf into the nucleus in order to shorten the ongoing irf -dependent innate immune response. curiously, while this cytosolic function of icp is independent of the activity of its e ubiquitin ligase domain, the functional host proteasome is required for the localisation of icp to the cytoplasm [ ] . the inhibitory function on the innate immune response by the v protein of sendai virus (sev) was first accounted for by the interaction of v with the rna sensor melanoma differentiation-associated protein (mda ) [ ] . however, sev infection is predominantly detected by rig-i [ ] [ ] [ ] , and sev infection still inhibits ifnβ production in mda -knockout mice [ ] . for this reason, the group of sakaguchi continued their studies and discovered that the sev v protein interacts with irf as well as irf - d in the cytosol and inhibits nuclear translocation. in line with this, also the v proteins of the related measles virus (mev) and newcastle disease virus (ndv) of the paramyxoviridae interact with irf and interfere with its trans-activation activity, though mev v seems to target irf after nuclear translocation [ ] . in contrast, the non-structural protein ns of the flavivirus japanese encephalitis virus (jev) suppresses import of irf indirectly by interacting with the nuclear transport proteins kpna and kpna [ ] . this interaction competitively blocks the interaction with the native cargo of the importins, including the transcription factors irf and p . similarly, the ns / a protease of the related hepatitis c virus (hcv) triggers cleavage of importin β (ipob, also kpnb ) and in this way inhibits the transport of irf into the nucleus to interrupt ifnβ production [ ] . once irf has reached the nucleus, it interacts with the coactivator cbp/p to obtain the potential to induce transcription. when studies concerned with the molecular requirements for the induction of type i ifns were still on their way, the critical role of the coactivators for irf activity was underlined by the observation that the antagonistic activity of adenovirus (adv) protein e a on irf -mediated stimulation was dependent on the interaction of e a with cbp [ ] . the inhibition of ifnb transcription by e a could be competed with by overexpression of cbp/p but was not observed for a mutant of e a that was defective in p -binding. mechanistically, these observations implied that e a competed with irf for binding to the essential coactivator [ ] . to interfere with the productive association of irf and cbp/p , the viral antagonists ns of human respiratory syncytial virus (rsv) [ ] , e protein of human papillomavirus (hpv- ) [ , ] and the tegument protein vp of hsv- target both proteins simultaneously [ ] . in contrast, the kinase orf of murine gammaherpesvirus (mhv ) specifically interacts only with activated irf in the nucleus to interfere with the association of coactivators [ ] . this protein is highly conserved, implying similar functions of its homologue in kaposi's sarcoma-associated herpesvirus (kshv), though the conserved kinase activity is not required for this function [ ] . a striking example for the exploitation of structural homology by viruses are the viral homologues of irfs, termed virfs, which are encoded by some herpesviruses to interfere with the activity of host irfs. for details on the interplay of virfs and host irfs and their implications for the viral replication style, please refer to the recent review by myoung and colleagues [ ] . shortly, virf of kshv binds to irf as well as p and in this way obstructs formation of the active holocomplex of irf and cbp/p [ ] [ ] [ ] . additionally, kshv deploys virf to target irf -driven gene expression [ ] . similar to virf , also the virion-associated virf r of the gammaherpesvirus rhesus macaque rhadinovirus (rrv) inhibits ifnb expression by targeting cbp and competing with phosphorylated irf for binding [ ] . as tegument proteins, rrv r and also hsv- vp are released into the host cell alongside the entering virus and can directly take action to interfere with irf activity before a potent type i ifn response can be mounted. another strategy to hinder the participation of the essential coactivator cbp is applied by several rna viruses. the non-structural protein (nsp ) subunits nsp α of murine lactate dehydrogenase-elevating virus (ldv) [ ] , nsp γ of simian haemorrhagic fever virus (shfv) [ ] and nsp α of porcine reproductive and respiratory syndrome virus (prrsv) [ ] [ ] [ ] of the arteriviridae family as well as nsp of porcine epidemic diarrhoea virus (pedv) [ ] of the coronaviridae all suppress ifnb expression by specifically targeting cbp in the nucleus and mediating its proteasome-dependent degradation. this approach implies that these viruses do not require the activity of cbp to express their own genome, which is in line with their cytoplasmic replication. the final aim of irf activation is to enable its binding to recognition motifs within the ifnβ enhancer in order to allow assembly of the ifnβ enhanceosome and induce transcription. again, viral antagonists apply different mechanisms to interfere at this level. the np protein of human bocaparvovirus (bov) and us of hsv- prevent dna binding by interaction with the dbd of irf themselves [ , ] . further, nss of sandfly fever sicilian virus (sfsv) of the bunyaviridae family was recently reported to apply this strategy to prevent association of irf with the ifnβ promoter [ ] . wuerth and colleagues demonstrated that nss specifically interacts with irf via the dbd and by this masking competes for dna binding in a dose-dependent manner. several other strategies were identified in the herpesviridae family: (i) the nuclear share of the hsv- protein icp relocalises irf and cbp/p in the nucleus to special nuclear structures, thereby sequestering them away from their site of activity [ ] . additionally, this mediates deactivation and promotes degradation of irf . the icp variant of bovine herpesvirus (bhv- ) also interacts with p , but in contrast to hsv- , this interaction hijacks the acetyltransferase activity of the coactivator and activates expression from viral promoters [ ] . (ii) the kinase bglf of epstein-bar virus (ebv) directly interacts with irf and phosphorylates several residues between dbd and iad [ ] . without affecting dimerisation or association with cbp, this modification prevents binding of irf to dna. (iii) kshv latency-associated nuclear antigen (lana- ) competes with irf for binding to the prdiii-i region and in this way interferes with stable association of irf to the target dna [ ] . the dna polymerase subunit ul of hcmv was recently reported to act in a similar way [ ] : upon hcmv infection, exogenously expressed ul could be demonstrated to associate with the ifnβ promoter region in a chromatin immunoprecipitation assay and in parallel ul interfered with binding of the central transcription factors irf and p . additionally, ul interacts with both transcription factors irrespective of their phosphorylation status. (iv) the kshv protein k-bzip binds to the ifnβ promoter region itself and prevents binding of irf [ ] , but differently from lana- and ul , k-bzip weakly induces gene expression while it prevents a high and detrimental activation of the type i ifn response. in contrast, the nss protein of rift valley fever virus (rvfv) applies a more indirect approach to inhibit binding of the irf -cbp/p holocomplex [ ] . nss interacts with the host factor sin a-associated protein (sap ), which belongs to the sin a/ncor/hdacs repressor complexes. in turn, sap interacts with the transcription factor yy , and this interaction enables recruitment of nss and sap to the murine ifnβ promoter region. finally, the presence of the nss-sap -yy complex inhibits recruitment of cbp and thus transcriptional activation of ifnb . since the beginning of the detailed characterisation of irf activation more than years ago, we have gained profound insight into the mechanisms enabling the trans-activation activity of a transcription factor with low intrinsic binding affinity for its specific recognition motif and into the intricate network of interactions that allow its participation in the induction of ifnb expression. still, the fundamental principles were mostly characterised in vitro and sometimes biased from prior assumptions, rendering the sequence of events as it occurs in our cells incomplete. to reveal the full dynamics of the events summarised above, experiments with living cells will be required. the many studies reporting factors that affect irf dimerisation and/or translocation and/or trans-activation activity highlight the importance of pinpointing the modulatory mechanism as precisely as possible in future work. at the same time, the diversity of methods applied in the studies summarised here demonstrates that nowadays, dissection of the exact level of intervention with irf activity is within reach. as delineated in this review, the regulation of irf in the context of ifnb expression in itself presents us with a multi-layered circuit of promoting and dampening modulations. the ever-expanding catalogue of host and viral modulators of irf activity not only reflects the key role of this signalling pathway in the mammalian arsenal of antiviral defence mechanisms, but it further alludes to the possibility to use specific mechanisms for medical intervention. directed manipulation of host modulators could present a novel approach to stimulate the host organism to overcome an infection by its own resources or to reduce excessive ifn activity to healthy levels. more information on how to achieve this will surely emerge from the unceasing identification and characterisation of novel irf modulators deployed by host and virus. nevertheless, the multitude of additional genes targeted by irf signifies that we are still just beginning to understand the true complexity of irf fine regulation. in addition to the ongoing characterisation of the direct involvement in the expression of isgs, powerful in silico and sequencing approaches of recent years revealed a growing list of novel targets, including virus-inducible rnas [ , ] . moreover, some of the identified host modulators hint at regulation of irf activity dependent on signalling of other pathways to enable the incorporation of further virus interference. i. the interferon global virus outbreaks: interferons as st responders type i interferons: distinct biological activities and current applications for viral infection the dual nature of type i and type ii interferons shared and distinct functions of type i and type iii interferons interferon-λ orchestrates innate and adaptive mucosal immune responses targeting interferons and their pathways in systemic lupus erythematosus specificity and function of irf family transcription factors: insights from genomics interferon response of chicken embryo fibroblasts to nucleic acids and related compounds inhibition of virus multiplication by foreign nucleic acid 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complex reveals novel dna recognition and cooperative binding to a tandem repeat of core sequences an atomic model of the interferon-beta enhanceosome specific enhancer selection by irf , irf and irf is determined by isre half-sites, and flanking bases, collaborating transcription factors and the chromatin environment in a combinatorial fashion virus infection induces nf-kappab-dependent interchromosomal associations mediating monoallelic ifn-beta gene expression the transcription factor thpok orchestrates stochastic interchromosomal interactions required for ifnb virus-inducible gene expression the high mobility group protein hmg i(y) is required for nf-kappa b-dependent virus induction of the human ifn-beta gene reversal of intrinsic dna bends in the ifn beta gene enhancer by transcription factors and the architectural protein hmg i(y) the mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome the role of hmg i(y) in the assembly and function of the ifn-beta enhanceosome mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements recruitment of cbp/p by the ifn beta enhanceosome is required for synergistic activation of transcription induction of endogenous ifn-alpha and ifn-beta genes by a regulatory transcription factor, irf- evidence for a nuclear factor(s), irf- , mediating induction and silencing properties to human ifn-beta gene regulatory elements critical role of a common transcription factor, irf- , in the regulation of ifn-beta and ifn-inducible genes regulated expression of a gene encoding a nuclear factor, irf- , that specifically binds to ifn-beta gene regulatory elements targeted disruption of irf- or irf- results in abnormal type i ifn gene induction and aberrant lymphocyte development mice devoid of interferon regulatory factor (irf- ) show normal expression of type i interferon genes assembly of a functional beta interferon enhanceosome is dependent on atf- -c-jun heterodimer orientation crystal structure of atf- /c-jun and irf- bound to the interferon-beta enhancer structure of irf- bound to the prdiii-i regulatory element of the human interferon-beta enhancer the role of response elements organization in transcription factor selectivity: the ifn-beta enhanceosome example efficient recruitment of tfiib and cbp-rna polymerase ii holoenzyme by an interferon-beta enhanceosome in vitro virus infection leads to localized hyperacetylation of histones h and h at the ifn-beta promoter deciphering the transcriptional histone acetylation code for a human gene nucleosome sliding via tbp dna binding in vivo eukaryotic transcription turns crystal structure of a yeast tbp/tata-box complex high-density nucleosome occupancy map of human chromosome p - reveals chromatin organization of the type i interferon gene cluster identification of individual interferon-producing cells by in situ hybridization stochastic regulation in early immune response chromosome-specific and noisy ifnb transcription in individual virus-infected human primary dendritic cells defining emerging roles for nf-kappab in antivirus responses: revisiting the interferon-beta enhanceosome paradigm gene repression by coactivator repulsion the specificity of innate immune responses is enforced by repression of interferon response elements by nf-κb p the transcriptional silencer protein nrf: a repressor of nf-kappa b enhancers constitutive silencing of ifn-beta promoter is mediated by nrf (nf-kappab-repressing factor), a nuclear inhibitor of nf-kappab lack of essential role of nf-kappa b p , rela, and crel subunits in virus-induced type ifn expression distinct roles for the nf-kappa b rela subunit during antiviral innate immune responses nf-kappa b rela subunit is crucial for early ifn-beta expression and resistance to rna virus replication association of the interferon-β gene with pericentromeric heterochromatin is dynamically regulated during virus infection through a yy -dependent mechanism yin yang dynamically regulates antiviral innate immune responses during viral infection enhanceosome formation over the beta interferon promoter underlies a remote-control mechanism mediated by yy and yy transcription factor yy binds to the murine beta interferon promoter and regulates its transcriptional capacity with a dual activator/repressor role binding of yy to the proximal region of the murine beta interferon promoter is essential to allow cbp recruitment and k h /k h acetylation on the promoter region after virus infection a novel virus-inducible enhancer of the interferon-β gene with tightly linked promoter and enhancer activities activation and regulation of interferon-β in immune responses post-translational regulation of antiviral innate signaling protein phosphatase pp negatively regulates the toll-like receptor-and rig-i-like receptor-triggered production of type i interferon by inhibiting irf phosphorylation at serines and in macrophage the methyltransferase nsd promotes antiviral innate immunity via direct lysine methylation of irf interferon-inducible cytoplasmic lnclrrc -as promotes antiviral innate responses by strengthening irf phosphorylation recruitment of phosphatase pp a by rack adaptor protein deactivates transcription factor irf and limits type i interferon signaling pp a facilitates porcine reproductive and respiratory syndrome virus replication by deactivating irf and limiting type i interferon production isg enhances the innate antiviral response by inhibition of irf- degradation positive regulation of interferon regulatory factor activation by herc via isg modification negative regulation of interferon-regulatory factor -dependent innate antiviral response by the prolyl isomerase pin yap antagonizes innate antiviral immunity and is targeted for lysosomal degradation through ikkε-mediated phosphorylation the tumor suppressor pten has a critical role in antiviral innate immunity interferon regulatory factor- is an in vivo target of dna-pk s-glutathionylation of irf regulates irf -cbp interaction and activation of the ifn beta pathway pivotal role for the escrt-ii complex subunit eap /snf in irf -dependent innate antiviral defense ago negatively regulates type i interferon signaling pathway by competition binding irf with cbp/p elf is critical for induction of type i interferon and the host antiviral response irf and irf cooperatively regulate rapid interferon-beta induction in human blood monocytes the ubiquitin e ligase raul negatively regulates type i interferon through ubiquitination of the transcription factors irf and irf the e ubiquitin ligase ro negatively regulates ifn-beta production post-pathogen recognition by polyubiquitin-mediated degradation of irf trim is essential to sustain ifn regulatory factor activation during antiviral response senp negatively regulates cellular antiviral response by desumoylating irf and conditioning it for ubiquitination and degradation virus infection triggers sumoylation of irf and irf , leading to the negative regulation of type i interferon gene expression mst shuts off cytosolic antiviral defense through irf phosphorylation fas-associated factor negatively regulates the antiviral immune response by inhibiting translocation of interferon regulatory factor to the nucleus bromodomain protein brd promotes ifnb transcription via enhancing irf /p complex formation and recruitment to ifnb promoter in macrophages otud negatively regulates type i ifn induction by disrupting noncanonical ubiquitination of irf the transcription factor nfat limits infection-induced type i interferon responses the transcription factor mafb antagonizes antiviral responses by blocking recruitment of coactivators to the transcription factor irf involvement of the ikappab kinase (ikk)-related kinases tank-binding kinase /ikki and cullin-based ubiquitin ligases in ifn regulatory factor- degradation negative regulation of interferon-β gene expression during acute and persistent virus infections foxo negatively regulates cellular antiviral response by promoting degradation of irf trim negatively regulates interferon-β production and antiviral response through polyubiquitination and degradation of nuclear irf negative feedback regulation of cellular antiviral signaling by rbck -mediated degradation of irf caspase- -mediated cleavage inhibits irf- protein by facilitating its proteasome-mediated degradation krüppel-like factor negatively regulates cellular antiviral immune response kat selectively inhibits antiviral immunity by acetylating irf a diverse range of gene products are effectors of the type i interferon antiviral response ddx inhibits type i interferon by disrupting assembly of irf -ipo to inhibit irf nucleus import lyar suppresses beta interferon induction by targeting phosphorylated interferon regulatory factor long noncoding rna lnc-mxa inhibits beta interferon transcription by forming rna-dna triplexes at its promoter prdi-bf recruits the histone h methyltransferase g a in transcriptional silencing identification and characterization of a novel repressor of beta-interferon gene expression the ef-hand protein calml suppresses antiviral innate immunity by impairing irf dimerization cflipl interrupts irf -cbp-dna interactions to inhibit irf -driven transcription interferon regulatory factor -cl, an isoform of irf , antagonizes activity of irf functional characterization of interferon regulatory factor a (irf- a), an alternative splice isoform of irf- dual utilization of an acceptor/donor splice site governs the alternative splicing of the irf- gene inhibition of the ifn-beta response in hepatocellular carcinoma by alternative spliced isoform of ifn regulatory factor- characterization of a novel isoform of murine interferon regulatory factor human papillomavirus e oncoprotein binds to interferon regulatory factor- and inhibits its transcriptional activity primary activation of interferon a and interferon b gene transcription by interferon regulatory factor recent advances in understanding viral evasion of type i interferon cellular sensing of viral dna and viral evasion mechanisms early ifn type i response: learning from microbial evasion strategies viral evasion strategies in type i ifn signaling-a summary of recent developments viral dedication to vigorous destruction of interferon receptors on taking the sting out of immune activation cytosolic dna-sensing immune response and viral infection intracellular sensing of viral genomes and viral evasion herpes simplex virus type immediate early protein icp inhibits ifn-β production in mucosal epithelial cells by antagonizing irf activation peste des petits ruminants virus nucleocapsid protein inhibits beta interferon production by interacting with irf to block its activation varicella-zoster virus immediate-early protein orf abrogates the irf -mediated innate immune response through degradation of activated irf viral evasion and subversion of pattern-recognition receptor signalling strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit the interferon antiviral response: from viral invasion to evasion viral suppression of the interferon system interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures ifn regulatory factors and antiviral innate immunity: how viruses can get better ten strategies of interferon evasion by viruses the molecular basis of viral inhibition of irf-and stat-dependent immune responses functional comparison of the two gene products of thogoto virus segment thogoto virus ml protein suppresses irf function the interferon antagonist ml protein of thogoto virus targets general transcription factor iib viral targeting of tfiib impairs de novo polymerase ii recruitment and affects antiviral immunity nucleocytoplasmic traffic disorder induced by cardioviruses the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins inhibition of mrna export and dimerization of interferon regulatory factor by theiler's virus leader protein herpes simplex virus serine/threonine kinase us hyperphosphorylates irf and inhibits beta interferon production recovery of paramyxovirus simian virus with a v protein lacking the conserved cysteine-rich domain: the multifunctional v protein blocks both interferon-beta induction and interferon signaling the n-and c-terminal domains of the ns protein of influenza b virus can independently inhibit irf- and beta interferon promoter activation cellular localization of the herpes simplex virus icp protein dictates its ability to block irf -mediated innate immune responses analysis of interaction of sendai virus v protein and melanoma differentiation-associated gene cell type-specific involvement of rig-i in antiviral response activation of innate defense against a paramyxovirus is mediated by rig-i and tlr and tlr in a cell-type-specific manner differential roles of mda and rig-i helicases in the recognition of rna viruses inhibition of interferon regulatory factor activation by paramyxovirus v protein japanese encephalitis virus ns inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor and nf-κb importin beta targeting by hepatitis c virus ns / a protein restricts irf and nf-kappab signaling of ifnb antiviral response a novel mechanism for the inhibition of interferon regulatory factor- -dependent gene expression by human respiratory syncytial virus ns protein the e protein of human papillomavirus type binds to and inhibits co-activation by cbp and p herpes simplex virus -encoded tegument protein vp abrogates the production of beta interferon (ifn) by inhibiting nf-κb activation and blocking ifn regulatory factor to recruit its coactivator cbp conserved herpesviral kinase promotes viral persistence by inhibiting the irf- -mediated type i interferon response beyond viral interferon regulatory factors: immune evasion strategies functional analysis of human herpesvirus -encoded viral interferon regulatory factor and its association with cellular interferon regulatory factors and p viral interferon regulatory factor of kaposi's sarcoma-associated herpesvirus (human herpesvirus ) binds to, and inhibits transactivation of, creb-binding protein hhv- encoded virf- represses the interferon antiviral response by blocking irf- recruitment of the cbp/p coactivators inhibition of interferon signaling by the kaposi's sarcoma-associated herpesvirus full-length viral interferon regulatory factor protein a rhesus rhadinovirus viral interferon (ifn) regulatory factor is virion associated and inhibits the early ifn antiviral response biogenesis of non-structural protein (nsp ) and nsp -mediated type i interferon modulation in arteriviruses degradation of creb-binding protein and modulation of type i interferon induction by the zinc finger motif of the porcine reproductive and respiratory syndrome virus nsp alpha subunit modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein in marc- and hela cells suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp human bocavirus np inhibits ifn-β production by blocking association of ifn regulatory factor with ifnb promoter hsv- immediate-early protein us inhibits ifn-β production by suppressing association of irf- with ifn-β promoter nss protein of sandfly fever sicilian phlebovirus counteracts interferon (ifn) induction by masking the dna-binding domain of ifn regulatory factor recruitment of activated irf- and cbp/p to herpes simplex virus icp nuclear foci: potential role in blocking ifn-beta induction bovine herpesvirus immediate-early protein (bicp ) interacts with the histone acetyltransferase p , which stimulates productive infection and gc promoter activity epstein-barr virus bglf kinase suppresses the interferon regulatory factor signaling pathway kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen inhibits interferon (ifn) beta expression by competing with ifn regulatory factor- for binding to ifnb promoter human cytomegalovirus dna polymerase subunit ul antagonizes antiviral immune responses by suppressing irf -and nf-kappab-mediated transcription binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferon-responsive factor elements modulates antiviral gene expression a sap complex inhibits ifn-beta expression in rift valley fever virus infected cells we would like to thank friedemann weber for critically reading the manuscript. we would viruses , key: cord- -zwnkrs authors: baker, michelle l.; schountz, tony title: mammalia: chiroptera: immunology of bats date: - - journal: advances in comparative immunology doi: . / - - - - _ sha: doc_id: cord_uid: zwnkrs bats are a large and diverse group comprising approximately % of all living mammalian species. they are the only mammals capable of powered flight and have many unique characteristics, including long lifespans, echolocation, and hibernation, and play key roles in insect control, pollination, and seed dispersal. the role of bats as natural reservoirs of a variety of high-profile viruses that are highly pathogenic in other susceptible species yet cause no clinical disease in bats has led to a resurgence of interest in their immune systems. equally compelling is the urgency to understand the immune mechanisms responsible for the susceptibility of bats to the fungus responsible for white syndrome, which threatens to wipe out a number of species of north american bats. in this chapter we review the current knowledge in the field of bat immunology, focusing on recent highlights and the need for further investigations in this area. bats (order chiroptera) are a diverse group of nocturnal mammals comprising approximately % of all mammalian taxa and consisting of more than species across families (simmons ) . phylogenetic analysis places bats within the superorder laurasiatheria, sister to carnivores (e.g., cats, dogs), ungulates (e.g., horses, cows), and cetaceans (e.g., dolphins) ( fig. ) (tsagkogeorga et al. ) . bats are believed to have diverged from other eutherian mammals approximately million years ago (mya) (lei and dong ) . the traditional classification system divided bats into two suborders: microchiroptera (microbats) and megachiroptera (megabats). microbats are defined by their smaller size ( - cm), the use of echolocation, and the use of hibernation during the winter for many species. megabats consist of the flying foxes (also called fruit bats) and are larger nonecholocating bats (up to . kg with wingspans of . m) belonging to the pteropodidae family. however, more recent phylogenetic analyses based on molecular data have led to a reclassification of bats into the suborders yinpterochiroptera and yangochiroptera. the yinpterochiroptera suborder includes the nonecholocating pteropodidae family (flying foxes) and the echolocating rhinolophoidea family, while the yangochiroptera suborder consists of the remaining echolocating microbats (teeling et al. (teeling et al. , . the two suborders of bats are estimated to have diverged approximately mya (lei and dong ) . although the new classification has strong statistical support, it remains controversial as it suggests that laryngeal echolocation evolved twice in chiroptera, once in yangochiroptera and once in the rhinolophoids (teeling et al. ) . of all the mammals, bats are the most ecologically diverse. they are the only mammals that have evolved powered flight and have adapted to a variety of environments across all continents with the exception of the polar regions. their diets are equally diverse, including fruits, pollen, insects, small vertebrates, and even blood, and they play important roles in the ecosystem through seed dispersal, pollination, and insect control. bats have longer lifespans relative to other mammals, typically living . times longer than mammals of similar size (austad ) . maternal investment is generally high, with most species giving birth to a single pup per year (from tsagkogeorga et al. with permission) and pups averaging approximately % of maternal body weight at birth (barclay and harder ) . curiously, despite their longer lifespans, there is anecdotal evidence that bats are resistant to tumors . the characteristic that has drawn the most attention in recent decades is their role as natural reservoirs for a variety of viruses that are highly pathogenic in other species yet rarely cause clinical disease in bats. this characteristic in particular has led to renewed interest in the immune systems of bats. bats are highly gregarious mammals, with most species living in high-density colonies, providing ideal environments for transmission and maintenance of pathogens within populations. combined with their frequent movement between roosts, transmission of viruses, bacteria, parasites, and fungi could potentially occur readily between individuals and populations, resulting in a situation of constant pathogen exposure. approximately viruses have been detected across different bat species, and many of the viruses identified in bats are highly pathogenic in other species, including humans (moratelli and calisher ) ; however, they likely host many more (anthony et al. ) . examples include high-profile viruses such as the severe acute respiratory syndrome coronavirus (sars-cov), marburg virus, and hendra and nipah paramyxoviruses. these viruses occasionally spill over to other susceptible hosts, causing severe disease and mortality yet causing no disease in bats. the long coevolutionary history of bats and viruses has likely resulted in the establishment of a state of equilibrium, allowing both the viruses and their host to coexist in a disease-free state typical of natural reservoirs. bats also host a variety of other pathogens, including parasites, bacteria, and fungi. unlike viral infections, there are examples of these pathogens causing disease among bats. the fungus that causes white nose syndrome (wns), pseudogymnoascus destructans, has resulted in mass mortalities among a number of north american microbat populations, with some species now threatened with extinction (blehert et al. ). evidence for lower fungal loads consistent with the development of resistance to the fungus have been observed in some bat populations, providing hope that selection on immune genes may lead to the development of resistance or tolerance mechanisms (langwig et al. ) . however, it is unlikely that this will occur rapidly enough for many affected populations. several bacterial infections, including tick-borne spirochaete bacteria, borrelia spp., and some enteric bacteria, have also been associated with pathology in bats (reviewed in brook and dobson, ) . brooks and dobson ( ) presented evidence that bats may have evolved mechanisms to eliminate intracellular pathogens such as viruses at the expense of their ability to eliminate extracellular pathogens (bacteria, parasites, and fungi) and hypothesize that mitochondrial adaptations may play a role. in light of the increasing emergence of infectious diseases and the impacts of pathogens such as wns, deciphering the immune systems of bats has never been more critical and offers potential for identifying novel antiviral therapies and approaches to the conservation of bats threatened by diseases such as wns. fortunately, progress in the area of bat immunology is rapidly advancing as new groups enter the field and advances in technology provide opportunities for more rapid discovery. several reviews that have appeared over the last years have described the various aspects of the immune systems of bats butler et al. ; schountz ; baker and zhou ; schountz et al. ) . in this chapter we provide a broad overview, with a focus on recent highlights in bat immunology and areas for future research. although few studies have examined the histology of bat lymphoid tissues, from an anatomical perspective, bats appear to have the majority of primary and secondary lymphoid organs present in other mammals, including thymus, bone marrow, spleen, and lymph nodes (papenfuss et al. ; zhou et al. b) . bone marrow has been isolated from long bones, including humerus and radius, and from the ribs but appears to be absent in the distal wing bones (papadimitriou et al. ; zhou et al. b) . notably absent, at least in the species that have been examined to date, are peyer's patches, which are generally located in the submucosa and lamina propria of the small intestine. no peyer's patches were present in the horseshoe bat, rhinolophus hildebrandtii, or the common pipistrelle bat, pipistrellus pipistrellus (strobel et al. ; makanya and john ) . the submucosa of the intestine of the horseshoe bat was devoid of lymphoid tissue, with the exception of a few aggregations of lymphoid nodules in the rectal submucosa (makanya and john ) . a range of immune cell types also appear to be present in bats. morphological characteristics have been used to identify lymphocytes, neutrophils, eosinophils, basophils, and macrophages in the brazilian free-tailed bat, tadarida brasilensis (turmelle et al. a) . macrophages and t-and b-cell populations have also been identified in the indian flying fox, pteropus giganteus, based on cellular adherence and scanning electron microscopy (sarkar and chakravarty ) . more recently, the phenotype, morphology, and function of dendritic cells and macrophages have been characterized from bone marrow from the black flying fox, pteropus alecto (zhou et al. b) . cells resembling follicular dendritic cells (fdcs) have also been described in the indian flying fox (sarkar and chakravarty ) . unlike dendritic cells that originate in the bone marrow, fdcs are of mesenchymal origin and are found in primary and secondary lymphoid follicles in b-cell areas of lymphoid tissue. fdcs are essential for high-affinity antibody production and for the development of b-cell memory. they also have the ability to maintain intact antigen for extended periods (van nierop and de groot ; heesters et al. ) . whether they play the same role in bats remains to be determined but presents an interesting possibility for the maintenance of persistent viral infections. the lack of species-specific reagents has often been a hindrance to comparative immunologists. however, bat immunology made a resurgence in an age of rapid advances in species-independent approaches such as next-generation sequencing, proteomics, and gene editing technologies such as crispr/cas . rnaseq studies on tissues and cells from a variety of different species of bats have provided evidence that bats have nearly all of the major components of the immune system that are present in other mammals, including receptors and molecules associated with innate and adaptive immunity and micrornas (papenfuss et al. ; shaw et al. ; cowled et al. ) . rnaseq data from virus-infected bat cells and wnsinfected bat tissues have also offered insights into the genes associated with hostpathogen responses (wynne et al. (wynne et al. , field et al. ) . to date, partial genome sequences of bat species are available in the ncbi database, providing valuable insights into the evolution of immune genes and essential sequence information for the design of primers and the development of reagents essential for studies of the immune responses of bats. the bat k project, which aims to sequence the genomes of the approximately species of bats, will no doubt provide a valuable resource for comparing the immune repertoire of different species of bats (teeling et al. ) . the genomes of bats are condensed compared to other mammals, ranging from . - . gb. smaller genome sizes in both bats and birds have been hypothesized to be associated with the metabolic requirements of flight (kapusta et al. ). a number of genomic regions associated with immunity have been analyzed in detail, in particular in the black flying fox (p. alecto), using a combination of wholegenome data and additional sequencing. these include regions associated with innate, for example, type i interferon (ifn), and adaptive immunity, for example, major histocompatibility class i (mhc-i) and mhc-ii. consistent with the smaller size of the genomes of bats, these regions are also condensed and contain fewer genes compared with the corresponding region from other mammals (ng et al. , zhou et al. a) . for example, the type i ifn locus of the black flying fox is highly condensed and contains fewer ifn genes than any other species sequenced to date (fig. ) . the description of the genomes of two divergent bat species, the australian black flying fox (p. alecto) and david's myotis (mytois davidii), provided the first glimpse into unique genetic signatures within immune pathways of bats, lending support to the idea of inadvertent changes in the immune system associated with the evolution of flight (zhang et al. ). these include changes in the genes associated with dna response/dna repair pathways that are tightly linked with innate immune pathways (fig. ) . the dna damage sensor, dna-dependent protein kinase catalytic subunit (dna-pkcs), which is also part of the cytoplasmic microbial nucleic acid sensing complex, was among the genes that have undergone selection in bats (ferguson et al. ) . accelerated evolution of innate immune genes including nuclear factor-kb (nf-kb) family member rel, ifnar , toll-like receptor (tlr ), ifn stimulated gene (isg ), interleukin- (il- ), and nucleotidebinding oligomerization domain-like receptor (nlr) family, pyrin domain containing (nlrp ) were also observed in the genomes of the two bats, an observation that may be a consequence of the coevolution of bats with viruses (zhang et al. ) . notably absent from the black flying fox and david's myotis genomes is the pyrin and hin domain (pyhin) gene family, which are involved in the recognition of foreign dna (zhang et al. ). this finding was recently confirmed in eight additional bat species across both suborders (ahn et al. ). the family member, absent in melanoma (aim ), is a cytosolic dna sensor and also part of the inflammasome complex that results in the activation of inflammatory cytokines, including il β and il . a second component of the inflammasome, nlrp , has undergone positive selection in the black flying fox and david's myotis, consistent with the possibility that the formation of inflammasomes is impaired in bats, which may in turn dampen the inflammatory response against pathogens (zhang et al. ) . the absence of a number of natural killer (nk) cell receptors from bat rnaseq and genome data sets is also striking (papenfuss et al. ; shaw et al. ; zhang et al. ) . genes that encode mammalian nk cell receptors are located within the leukocyte receptor complex (lrc) and the natural killer complex (nkc) of the genome. the two families have undergone convergent evolution to bind mhc-i molecules for the control of nk cell function. genes within the lrc encode immunoglobulin (ig)-like genes, including the killer cell ig-like receptors (kir), leukocyte ig-like receptors (lilrs), and leukocyte-associated ig-like receptors (lairs). those within the nkc encode lectin-like receptors, including the ly c-type lectin family. the composition of the lrc and nkc varies considerably among species. while most species have expanded either their lrc or nkc gene families, there are exceptions to this rule. in humans and nonhuman primates, the main nk cell receptors are encoded in the lrc and belong to the ig superfamily. rodents and horses have only expanded their ly c-type lectin family of nk receptors (kelley et al. ) . in contrast, cattle appear to have diversified nk genes within both the nkc and lrc regions, whereas domestic dogs and four species of marine carnivores contain single copies of kir and ly genes (hammond et al. ; schwartz et al. ) . in bats, kirs and ly -like receptors appear to be absent from transcriptome and genome data sets from the black flying fox, and only a single pseudogene of ly was identified in the genome of david's myotis bat (papenfuss et al. ; zhang et al. ) . two kirs have been identified in the genome of the big brown bat, eptesicus fuscus, but whether they are functional remains to be determined (guethlein et al. ) . overall, evidence to date is consistent with the contraction of both kir and ly families of receptors in bats. other nk cell coreceptors have been identified in bat genome and rnaseq data sets, hinting at some level of nk cell function in bats. these include the presence of cd and nkg c, which form heterodimers to generate inhibitory signals. the more divergent nkg d, which binds mhc-i chain-related genes, mica/b, and the ul binding proteins (ulbps) in humans (kelley et al. ) , was also detected. coreceptors, including cd , cd , and cd , were also transcribed in the black flying fox (papenfuss et al. ) . the failure to identify a number of nk cell receptors in several bat species supports the hypothesis that bats may have atypical nk cell responses or use different subsets of receptors. the availability of rnaseq and genomic data has also accelerated the characterization of a variety of immune genes and provided opportunities to examine transcription in various tissues and cells. molecular information exists for a variety of mammalian cytokines that have been described in bats including interleukins (il , il , il , il , il ), cytokines (tnfα, tgfβ), and ifns (types i, ii, and iii) (iha et al. ; he et al. he et al. , kepler et al. ; zhou et al. a zhou et al. , a janardhana et al. ; loria-cervera et al. ) . detailed descriptions of pattern recognition receptors, tlrs, and rig-i like helicases have also been reported (iha et al. ; cowled et al. cowled et al. , although only a few studies have examined the nature of ig genes in bats, a few unusual characteristics have already emerged that have been extensively reviewed elsewhere (butler et al. ) . the constant regions of bat igs appear to correspond to the canonical structure and repertoire found in other eutherian mammals. bats transcribe igm, igd, iga, ige, and multiple subclasses of igg (baker et al. ; butler et al. ; wynne et al. ) , although some species do not have ighδ genes and others have only a single ighγ gene gerrard et al. ) . studies of the heavy chain variable (vh) region repertoires of black flying foxes and little brown bats (myotis lucifugus) suggest bats may have the greatest number of vh gene segments among mammals (baker et al. ; bratsch et al. ) . furthermore, evidence from little brown bats indicates that bats may depend more on combinatorial diversity and less on somatic hypermutation . the antigen-binding region of black flying fox vh genes contains amino acids typically associated with lower antigen avidity but greater specificity (baker et al. ) . this, combined with the lack of evidence for somatic hypermutation, is consistent with the possibility that highly specific vh segments are encoded in the genomes of bats because of the long coevolutionary history of bats and viruses. the availability of cell lines from a range of different bat species has provided opportunities to study several aspects of the immune response of bat cells in vitro. this has been particularly useful for studying host-virus interactions. ifn responses of bat cells and cell lines following stimulation with viruses and synthetic tlr ligands, including polyinosinic:polycytidylic acid (polyi:c) and bacterial lipopolysaccharide (lps), have demonstrated that ifn production pathways are functional in bat cells and supernatant from stimulated cells has antiviral activity (stewart et al. ; omatsu et al. ; crameri et al. ; kepler et al. ; zhou et al. b ). significantly, ifnα and ifn signaling molecules, such as ifn regulatory factor (irf ), are constitutively expressed in unstimulated pteropid bat tissues and cells, consistent with the possibility that the innate immune systems of bats are at higher states of activation than other mammals, presumably allowing bats to rapidly respond to microbial infection (zhou et al. , a . the constitutive expression of ifnα has been described in two species of pteropid bats (p. alecto and cynopterus brachyotis) and is a first for any species. curiously, fetal and kidney cell lines from a third pteropid bat species, the egyptian rousette bat (rousettus aegyptiacus), have low constitutive expression of ifnα, indicating that high baseline levels of ifnα may not be a feature of all bat species (kuzmin et al. ) . the downstream signaling events triggered by ifn result in the induction of hundreds of ifn-stimulated genes (isgs), which are responsible for the antiviral state induced by ifns. the profile of isgs in unstimulated bat cells and the kinetics of isg induction following stimulation with ifn also differs from other species. unstimulated cells from the black flying fox have higher levels of isgs compared to human cells. the isg profile of bat cells consists predominantly of a subset not associated with the acute inflammatory responses that often accompany elevated ifn activity (cheon et al. ; zhou et al. a ). stimulation of cells from the black flying fox with ifnα also leads to the induction of novel subsets of isgs, including ribonuclease l (rnasel), that are not known to be induced by ifn to other species and the isg response is elevated for a shorter period of time in bat compared to human cell lines (de la cruz-rivera et al. ; zhang et al. ) ; rnasel is also elevated in bats that die from experimental tacaribe virus infection (gerrard et al. ) . overall, these studies point to differences in the regulation and profile of bat isgs as being central to the ability of bats to tolerate constitutive ifnα expression without pathology. consistent with the nature of the isg response, additional evidence is also accumulating for differences in the activation of other components of the inflammatory immune response in bats. comparison of the inflammatory cytokine production of polyi:c-stimulated cell lines from big brown bats (e. fuscus) and humans have demonstrated that the induction of high levels of proinflammatory cytokines, tnfα and il , occurs in human but not in bat cells (banerjee et al. ). this result again demonstrates that bats may regulate their immune response more tightly compared to other species. experimental infections of bat cells and cell lines have also provided insight into the antiviral response of bats, revealing differences in the responses to different viruses and between cell types. infection of black flying fox splenocytes with the bat paramyxovirus, tioman virus, resulted in the downregulation of type i ifns and the upregulation of type iii ifns, indicating that type iii ifns may play an important role in the ability of bats to coexist with viruses (zhou et al. a ). in contrast, henipavirus infection antagonizes type i and type iii ifn production and signaling in black flying fox cells but only ifn production in human cells (virtue et al. a, b) . the difference in the behavior of bat ifns upon tioman and henipavirus infection may reflect different ifn production mechanisms in splenocytes, which are professional immune cells, and cloned bat cells, which are predominantly fibroblastlike (crameri et al. ). infection of cells from the black flying fox with henipavirus and the egyptian rousette bat with ebola or marburg results in the induction of ifnβ, but curiously no increase in ifnα has been observed, at least at the time points examined in these studies (zhou et al. a; kuzmin et al. ) . as described earlier, p. alecto has high constitutive ifnα, which may account for its low induction, but this does not appear to be the case for the rousette bat. both marburg and ebola viruses, but particularly marburg, induced a potent innate immune response in rousette cells, which was generally stronger than that in human cells. the timing of induction of ifns and isgs in ebola-virus-infected cells was also delayed compared to cells infected with marburg virus (kuzmin et al. ) . the natural reservoir for marburg virus is known to be the rousette bat, but the reservoir for ebola is unknown and believed to be another bat species. the differences in host response of rousette bat cells to the two filoviruses may therefore reflect adaptations associated with the role of this species as a natural reservoir for marburg but not ebola. although isg responses have also been examined following viral infections in vitro, their ability to restrict viral replication has only been examined for a few isgs (de la cruz et al. ; zhou et al. ). the best-characterized isgs include myxovirus resistance (mx) genes and - -oligoadenylate synthetase (oas ). mx proteins are large gtpases that were initially described as inhibitors of influenza viruses and act by detecting viral replication and then trapping viral components. the oas proteins are activated by dsrna leading to the activation of rnase l, which then degrades both cellular and viral rna (sadler and williams ) . mx and oas from the black flying fox have been demonstrated to be highly upregulated by pteropine orthoreovirus nb (prv nb) virus infection, an orthoreovirus carried by pteropid bats . furthermore, bat mx proteins from pteropidae, phyllostomidae, and vespertilionidae demonstrate antiviral activity against ebola and bat influenza-like viruses. however, thogoto virus, a tick-transmitted orthomyxovirus that is not known to infect bats, was not inhibited by bat mx despite the ability of human mx to inhibit thogoto virus replication. evidence for positive selection in two variable and surface-exposed regions of bat mx genes were hypothesized to explain some of the species-specific antiviral activities of these proteins (fuchs et al. ). however, antiviral activity of black flying fox rnasel has been demonstrated against the yellow fever flavivirus, which is carried by mosquitoes, consistent with differences in specificity among different bat isgs (de la cruz et al. ). cell-mediated immune (cmi) responses are controlled by cd + cytotoxic and cd + helper t-lymphocyte populations and result in the killing of virus-infected cells or activation of the antibody and cytokine response. fewer studies have examined cmi in bats. the single type ii ifn, ifnγ, is produced by black flying fox bat cells stimulated with mitogens such as phytohaemagglutinin (pha) and cona, and recombinant bat ifnγ has antiviral activity against semliki forest virus and hev in vitro . at least in vitro, ifnγ from the black flying fox appears to have activity similar to that of ifnγ from other mammals, consistent with its role in the cmi response. curiously, in rousette bat cell lines, ifnγ is induced following infection with marburg virus but not following infection with ebola virus, indicating there may be differences in the cmi response induced by these two closely related viruses (kuzmin et al. ) . a number of earlier studies have described the in vitro responses of pteropid bats and microbats to t-cell mitogens and mixed lymphocyte responses in pteropid bats (mcmurray and thomas ; chakraborty and chakravarty ; chakravarty and paul ; paul and chakravarty ) . although these studies have been relatively crude due to the absence of specific reagents, they have all reported delayed responses compared with those of conventional laboratory animals. the presence of regulatory t cells was implicated in the delay in mitogenic responses of b cells in bats . whether these cells are involved in the delay in t-cell-mediated immune responses observed in bats remains to be determined. more recent studies have used proteomics to functionally characterize black flying fox mhc-i molecules and identify endogenous and viral peptide ligands. peptides derived from bat mhc-i molecules display a relatively broad length distribution, consistent with earlier observations based on sequence information demonstrating relatively large peptide binding grooves in the bat class i molecules wynne et al. ) . furthermore, an unusual preference for a c-terminal proline residue was identified in endogenous and hendra virus (hev)-derived peptides presented by bat mhc-i molecules, consistent with the possibility that differences in antigen presentation or processing may exist in bats ). bats are capable of mounting antibody responses to viruses and model antigens, and the appearance of antibodies appears to follow the same succession as that of other mammals with the early appearance of igm followed by igg (hatten et al. (hatten et al. , chakraborty and chakravarty ; wellehan jr et al. ). although all of the ig isotypes have been detected at the mrna level in a variety of bat tissues, iga protein appears to be present at surprisingly low levels in tissues and secretions from the black flying fox, which may have implications for its role in mucosal immunity in bats ). there are also differences in the time course, quantity, and duration of antibody responses, and questions exist over the protective nature of antibodies in bats (hatten et al. ; mcmurray et al. ; chakraborty and chakravarty ; davis et al. ; wellehan jr et al. ; turmelle et al. b) . responses to antigens such as ϕx bacteriophage and sheep red blood cells have demonstrated that the generation of neutralizing antibodies is delayed in the big brown bat, the pteropid bat, and the indian flying fox (pteropus giganteus) (hatten et al. ; chakraborty and chakravarty ) . isotype switching from igm to igg also appears to be delayed in the big brown bat (hatten et al. ). despite genetic evidence for limited somatic hypermutation in the little brown bat, an increase in antibody affinity as measured by the ability of antibodies to dissociate from ϕx increased following secondary immunization in the big brown bat (hatten et al. ) . measures of cmi in bats have been crude relative to studies in other species and are limited to studies demonstrating t-cell-mediated inflammation to protein antigens such as purified protein derivative (ppd), pha, and bovine serum albumin (bsa). such skin sensitivity tests in two bat species, the common vampire bat (desmodus rotundus) and seba's short-tailed bat (carollia perspicillata), immunized with ppd or bsa revealed delayed responses in both species compared to similar reactions in mice (mcmurray and thomas ) . lack of inflammatory responses have also been reported in most indian flying foxes subjected to skin sensitivity tests using the contact allergen - dinitrofluorobenzene (chakraborty and chakravarty ) . unlike conventional laboratory animals, few "clean" captive colonies of bats exist, and experimental infections often rely on the use of wild caught individuals, which represent a mixed population of unknown age, susceptibility, and prior viral exposure. experimental infections have been performed on a number of species of bats using rabies virus, australian bat lyssavirus (ablv), marburg, hev, nipah virus (niv), japanese b encephalitis (je) virus, and tacaribe virus (tcrv) (williamson et al. (williamson et al. , almeida et al. ; davis et al. ; middleton et al. ; turmelle et al. b; halpin et al. ; cogswell-hawkinson et al. ; paweska et al. ) . although the only immune parameter measured during these studies has been antibody responses, these experiments have provided valuable information on the kinetics of viral infection, the timing and duration of antibody responses and the nature of protective immunity following reinfection. with the exception of rabies virus, ablv and tcrv, bats generally show no clinical signs of disease following infection. neutralizing antibodies to a variety of viruses have been detected in wild caught bats, demonstrating they are capable of mounting an antibody response to viruses (halpin et al. ; lau et al. ; leroy et al. ) . the transfer of maternal antibody to pups occurs in bats, and the decline of maternal antibodies has been examined in captive black flying, variable flying foxes (pteropus hypomelanus), and straw-colored fruit bats (eidolon helvum) baker et al. ). however, whether bats transfer maternal antibody both pre-and postpartum and the isotypes involved is unknown. the interpretation of antibody responses in bats is extremely challenging, and, as described earlier, the nature of antibody responses in bats often differs both qualitatively and quantitatively compared to other species. rabies and ablv are among the only viruses known to result in clinical disease in naturally infected and experimentally infected bats. however, not all bats develop disease, and the mechanisms responsible for differences in disease outcome between individuals are not understood. evidence from experimental infections has demonstrated that even the development of neutralizing antibodies does not always provide protection from reexposure. for example, a group of wild caught bats ( big brown bats, e. fuscus, and mexican free tailed bats, tadarida brasiliensis) challenged by oral-nasal inoculation with rabies virus all developed antirabies neutralizing antibodies within months. rechallenge by intramuscular inoculation months later resulted in an amnestic response in animals, including that developed clinical rabies (davis et al. ). low seroconversion rates have also been reported in big brown bats inoculated with rabies by intramuscular challenge with only of inoculated animals developing antibodies. this study also reported clinical disease following secondary or tertiary infections in bats that had seroconverted following primary inoculation (turmelle et al. b) . similarly, almeida et al. ( ) described the intramuscular challenge of vampire bats (d. rotundus) with rabies virus, of which bats survived. once again, there was no correlation between the level of neutralizing antibody and survival. many bats that developed low or undetectable antibodies, as well as those with high antibody titers, survived infection. infection of gray-headed flying foxes, pteropus poliocephalus, with rabies or ablv results in similar rates of mortality and seroconversion. mccoll et al. ( ) reported clinical signs of disease in three of ten ablv-infected and two of four rabies-infected gray-headed flying foxes, none of which seroconverted prior to euthanasia. five of the ablv-infected survivors seroconverted by dpi, with titers waning by dpi. one of the rabies-infected survivors also seroconverted, but not until dpi (mccoll et al. ) . these studies indicate that antibodies may not provide protection and support a role for other components of the immune response in those animals that survive infection. unlike rabies and ablv infections, clinical disease has not been reported in any bat species either naturally or experimentally infected with a variety of other bat-borne viruses, including hev, niv, marburg, ebola, and je viruses. however, similar to rabies infection, the role of the antibody response in providing protection remains unclear, and many animals survive infection but fail to seroconvert. the henipaviruses hev and niv are carried by pteropid bats. in australia, hev antibodies have been identified in all four species of australian flying foxes (p. alecto, p. poliocephalus, p. scapulatus, and p. conspicillatus) . niv antibodies have been identified in bats from southeast asia and africa. in malaysia, two pteropid species, small flying foxes (p. hypomelanus) and malayan flying foxes (p. vampyrus), are considered to be the reservoir hosts . a number of experimental infections of pteropid bat species have been performed to understand the nature of viral infection in the natural reservoir of these viruses. niv infection of gray-headed flying foxes by subcutaneous injection resulted in the production of neutralizing antibody in all individuals inoculated, but in a separate study, only of malayan flying foxes that were infected by the oral-nasal route produced a neutralizing antibody response (middleton et al. ; halpin et al. ) . both subcutaneous and oral-nasal routes of infection have also been used for hev inoculation of pteropid bats. neutralizing antibody responses were detected in of black flying foxes inoculated oral-nasally with hev (halpin et al. ) . similarly, in gray flying foxes challenged with hev, neutralizing antibodies were detected in two of four bats inoculated by subcutaneous injection and three of the four bats inoculated by the oral-nasal route, with none of the bats displaying clinical signs of disease (williamson et al. ) . a study of four gray-headed flying foxes in late gestation infected subcutaneously with hev also described the presence of neutralizing antibodies in all four bats, and no abnormalities were observed in the fetuses or adults at necropsy (williamson et al. ) . in other mammals, pregnancy results in a bias in the immune response toward humoral immunity and away from cmi, which could be harmful to a fetus (szekeres-bartho ) . whether the nature of the maternal immune response facilitates greater production of antibody in infected bats during pregnancy remains to be investigated. a natural reservoir of marburg virus are the egyptian rousette bats (r. aegyptiacus) (towner et al. ) , and a number of experiments have been performed to study the nature of viral transmission and infection in this species (paweska et al. ; schuh et al. a, b) . marburg virus is capable of horizontal transmission between inoculated and naïve r. aegyptiacus. all inoculated bats seroconverted, with igg antibodies peaking between - dpi. marburg virus antibody titers in both inoculated and in contact bats declined within month following attainment of peak levels and were undetectable after months (schuh et al. a) . a subsequent study revealed that bats rechallenged with marburg virus - months following primary experimental infection developed virus-specific secondary antibody, indicative of the development of long-term protective immunity (schuh et al. b) . clearly, additional work is needed to understand the antibody responses of bats and the nature of antibody-mediated protection against various viruses. given what we have learned about innate immunity, particularly in pteropid bats, it is possible that innate immune mechanisms, such as ifn, reduce viral replication to low levels, delaying the generation and magnitude of an antibody response. evidence for a highly diverse germline repertoire of antibodies and the absence of somatic hypermutation could indicate that bats have evolved a repertoire of antibodies that are highly pathogen specific. such antibodies may provide some level of early protection without reaching the higher titers observed in other species. although no studies have examined the cmi responses of bats to viral infections, the generation of an ifnγ reagent for pteropid bats has been described and will assist in future studies to examine cmi in bats ). wns is caused by a cold-loving (pyrophilic) and keratinophilic fungus (p. destructans) first identified in north american bats in that infects the epidermis and dermis of the muzzle, ears, and wings. since its discovery, it has been detected in six species of north american bats, and infected populations have undergone a decline of up to %, with several species threatened with regional extinction within the next decade. p. destructans infects bats during hibernation, causing them to arouse early, leading to depletion of energy reserves and ultimately leading to a severe inflammatory response and resulting histopathology. the fungus is widely distributed in north america and europe and has recently been found in asia (hoyt et al. ) . although naturally infected european bats also develop histopathological lesions in response to p. destructans, no mass mortality is observed in european or asian bats (zukal et al. ) . similar to the situation with viruses, the long coevolutionary relationship of european and asian bats with p. destructans has presumably led to an equilibrium between the host and pathogen. in the longer term, this may also evolve in north american bats, and evidence of some level of resistance has been reported in some populations (langwig et al. ) . however, the rate of mortality among some bat species is too high to ignore. understanding the hostpathogen relationship and the genes and pathways associated with disease tolerance and resistance will be important for identifying viable treatments and assessing the immune responses of bats to drugs or vaccines. earlier reports describing the immune response of bats during hibernation indicate that, like other hibernating mammals, their immune responses are suppressed during torpor when they are initially infected with p. destructans. for example, hibernating e. fuscus bats maintained at °c fail to generate antibodies in response to infection with je virus (sulkin et al. ). in addition, activation plasma complement against bacteria (escherichia coli, staphylococcus aureus) and fungi (candida albicans) is lower in hibernating little brown bats compared to nonhibernating bats (moore et al. ) . several studies have now begun to examine the host response of bats to p. destructans to determine the level of immune activation that occurs during torpor and after arousal. p. destructans begins to colonize bat skin during hibernation, yet visible signs of inflammation are characteristically absent in torpid animals, and neutrophils and macrophages are absent from sites of pathogen invasion in hibernating bats with wns. in little brown bats, overt skin damage does not occur until - weeks after bats have emerged from hibernation with intense neutrophilic inflammation associated with invasive p. destructans infection (meteyer et al. ) . studies of bats from wns-affected and unaffected sites have also demonstrated significantly higher circulating leukocyte counts in wns-affected bats with elevated body temperatures (above °c). the latter is consistent with the mobilization of cells associated with arousal from torpor and euthermia (moore et al. ) . the absence of neutrophil and t-cell infiltration has been confirmed through rnaseq analysis of wns-infected little brown bat wing tissues during hibernation (field et al. ) . despite the absence of neutrophil invasion, increases in gene expression for inflammatory cytokines have been detected in wing tissues from hibernating wns infected bats compared to hibernating bats not affected by wns. these include il β, il , il c, il , il a, il , and g-csf and chemokines, such as ccl and ccl . hibernating little brown bats exhibiting visible fungal infections elevated levels of transcripts for proinflammatory cytokines, il and tnfα, the anti-inflammatory cytokine il , and the antimicrobial peptide cathelicidin in lung tissue compared to hibernating uninfected bats (rapin et al. ) . overall, these studies are consistent with the induction of an innate antifungal response in wns-infected bats prior to emergence from hibernation followed by infiltration of immune cells and, presumably, activation of adaptive immune responses following arousal. overactivation of the immune response following arousal from torpor, combined with a depletion of energy reserves, appears to be the main cause of mortality. in contrast to p. destructans, bats are known to carry other fungal pathogens, such as histoplasma capsulatum, without disease. h. capsulatum is a pathogenic fungus that causes pulmonary and systemic infections in humans. bats are considered to be the main reservoir of this fungus, and it is commonly found in bat guano (taylor et al. ) . although bats are susceptible to infection, mortality is rare in bats that are inoculated intranasally, which is the most likely route of natural infection. higher mortality rates are observed in bats inoculated intraperitoneally (mcmurray and greer ; greer and mcmurray ) . great fruit eating bats (artibeus lituratus) respond to infection with the generation of complement fixing antibodies by weeks and precipitating antibodies by weeks post infection (mcmurray and greer ) . natural infection rarely results in disease, indicating that, similarly to the situation with most viruses, bats have likely evolved mechanisms to control infection, at least under conditions where they are infected under nontorpid conditions. the field of bat immunology is very much in its infancy, and significant opportunities exist for future research. thanks to advances in technology, such as wholegenome sequencing and rnaseq, considerable progress has been made, in particular with regard to our understanding of the immune system of the black flying fox, p. alecto. however, as bats are a highly diverse group of mammals that have evolved independently for a long period of time, it is possible that different immune mechanisms exist between the two suborders and across species. there is likely much more to be learned from comparative studies across different bat species. comparative genomics of bats have provided important clues to the adaptations that may allow bats to coexist with viruses in the absence of disease. these include evidence for positive selection on a variety of immune genes and differences in the repertoires of nk cell receptors. additional genomic data, including long read assemblies, will be required to resolve highly repetitive regions such as the lrc and nkc to confirm the absence of important receptor families and to resolve other repetitive regions of the bat immunome. a number of genomic regions also remain largely unexplored, partly owing to their repetitive nature. these include b-and t-cell receptor (bcr and tcr) regions. examining the repertoire and diversity of these regions will provide opportunities to examine their functional activities and importance. for example, no information exists on the repertoire of tcrs in bats and the relative importance and roles of αβ and γδ t cells. observations from genomic data sets pave the way to further addressing the role of different components of the immune system in the responses of bats to infection. the mechanisms involved in tcr and bcr diversification also remain unknown. the roles of terminal deoxynucleotidyl transferase (tdt), recombination activating gene (rag), and activation-induced cytidine deaminase (aid) on recombination, somatic hypermutation, gene conversion, and class switching remain to be explored. a number of important differences in the innate immune system have also been identified in bats that are at odds with the responses in humans and other species. in particular, the constitutive activation of ifnα in the black flying fox is striking. in other mammals, constitutive ifn expression can have implications for inflammation and autoimmunity. identifying the mechanisms responsible for the ability of bats to tolerate high levels of ifn in the absence of inflammation has significant potential for identifying novel therapeutics to treat viral diseases in humans and other species. to this end, functional characterization of the different subsets of isgs already identified in both unstimulated and stimulated cells would provide valuable insights into the mechanisms responsible for the control of viral infection in the absence of inflammation. additionally, dissection of the signaling pathways responsible for the control of ifn response will contribute to our understanding of differences in the regulation of ifn in bats compared to other species. as described earlier, a number of functional differences have been identified in the immune system of bats compared to other species. these include the nature of cell-mediated and antibody responses of bats. to advance our understanding of the nature of these responses, appropriate bat-specific reagents will be required. some commercially available human and mouse antibodies generated against highly conserved intracellular proteins are cross reactive with bat proteins and have already proven useful (zhou et al. b) . a handful of bat-specific antibodies have also been generated wynne et al. ) . additional reagents will be necessary to advance the field, including monoclonal antibodies for use in flow cytometry, immunohistochemistry, and elisas, to dissect the roles of different cell types, including b and t cells, dendritic cells, and macrophages. reagents will also be required to examine the responses of various cytokines to examine proinflammatory and anti-inflammatory pathways for comparison to other species and to answer specific questions, including the confirmation of cytokine expression at the protein level (e.g., ifnα to confirm its constitutive expression). recombinant cytokines and growth factors will also be important for examining the responses of cells to cytokine stimulation and the expansion of specific subsets of antigenspecific lymphocytes. lastly, the development of closed breeding colonies of bats will be essential in progressing research into immunity in bats, overcoming the issues associated with wild caught individuals of unknown age and history of infection. renewed interest in bat immunology emerged following the identification of bats as reservoirs for a number of viruses, including sars-cov and ebola, that are highly pathogenic in other species. prior to the emergence of these viruses, few studies had examined any aspect of bat immunology. a number of important observations have already been made through studies of the immune systems of bats, with evidence for adaptations not observed in any other species. significant progress has now been made in the identification of genes and pathways associated with immunity, and one of the recurring themes that is emerging with regard to viral infections is the ability of bats to control inflammatory responses. regulation of the immune system is likely an important mechanism for preventing pathology associated with infection. however, bats are an extraordinarily diverse group of mammals, and the adaptions identified to date may not apply across all bat species. in contrast to the 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transcriptomics reveals hendra virus sensitizes bat cells to trail mediated apoptosis characterization of the antigen processing machinery and endogenous peptide presentation of a bat mhc class i molecule comparative transcriptomics highlights the role of the ap transcription factor in the host response to ebolavirus nipah virus infection in bats (order chiroptera) in peninsular malaysia comparative analysis of bat genomes provides insight into the evolution of flight and immunity ifnar -dependent gene expression profile induced by ifn-α in pteropus alecto bat cells and impact of ifnar knockout on virus infection type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto bat mx and oas , but not pkr are highly induced by bat interferon and viral infection irf in the australian black flying fox, pteropus alecto: evidence for a unique expression pattern and functional conservation contraction of the type i ifn locus and unusual constitutive expression of ifn-α in bats unlocking bat immunology: establishment of pteropus alecto bone marrow-derived dendritic cells and macrophages white-nose syndrome without borders: pseudogymnoascus destructans infection tolerated in europe and palearctic asia but not in key: cord- -az xd p authors: hansbro, nicole g.; horvat, jay c.; wark, peter a.; hansbro, philip m. title: understanding the mechanisms of viral induced asthma: new therapeutic directions date: - - journal: pharmacol ther doi: . /j.pharmthera. . . sha: doc_id: cord_uid: az xd p asthma is a common and debilitating disease that has substantially increased in prevalence in western societies in the last decades. respiratory tract infections by respiratory syncytial virus (rsv) and rhinovirus (rv) are widely implicated as common causes of the induction and exacerbation of asthma. these infections in early life are associated with the induction of wheeze that may progress to the development of asthma. infections may also promote airway inflammation and enhance t helper type lymphocyte (th cell) responses that result in exacerbations of established asthma. the mechanisms of how rsv and rv induce and exacerbate asthma are currently being elucidated by clinical studies, in vitro work with human cells and animal models of disease. this research has led to many potential therapeutic strategies and, although none are yet part of clinical practise, they show much promise for the prevention and treatment of viral disease and subsequent asthma. . introduction asthma is thought to affect at least million people of all ages and ethnic backgrounds worldwide (global strategy for asthma management and prevention, ) . between in and in people are affected in western societies and the prevalence has doubled since (umetsu et al., ; aihw, ) . it is now considered to be an epidemic and results in a massive economic burden to communities. exacerbations are typically caused by exposure to environmental factors to which the individual is allergic. although asthma is clearly recognised as an inflammatory condition, our understanding of the mechanisms of pathogenesis remains rudimentary. clinically asthma is characterised by airway obstruction, wheezing and episodic breathlessness in association with increased sensitivity of the airways to non-specific stimuli (termed airway hyperresponsiveness (ahr)) (bousquet et al., ) . wheezing is a high-pitched whistling or squeaking, which originates from the chest and is made during breathing (michel et al., ) . a predominant feature of disease is the acute-on-chronic infiltration of pro-inflammatory activated cd + th cells and eosinophils into the airways, which are critical regulators of pathogenesis (robinson et al., ; kay, ) . typical pathogenic features include: ige production; airway smooth table important factors released by respiratory epithelium upon viral infection (dakhama et al., a) nitric oxide muscle (asm) and goblet cell hypertrophy/hyperplasia; mucus hypersecretion; eosinophil, neutrophil and mononuclear cell infiltration into submucosal layer of the airways; mast cell and macrophage activation; sloughing of airway epithelial cells; and ahr (foster et al., ; kumar, ; cohn et al., ) . th cells and activated inflammatory cells release a range of mediators that damage the mucosal epithelial lining and promote an exaggerated repair response that leads to airway remodelling and chronic disease. remodelling is the result of structural changes of the epithelium, submucosal layer, asm and vasculature (angiogenesis) (bousquet et al., ; vignola et al., ) . it is thought to be a major contributing factor to the development of ahr, and its progression may lead to fixed airflow obstruction and irreversible loss of lung function (li & wilson, ; vignola et al., ) . thus, airway inflammation is closely linked to ahr and airflow obstruction and recurring inflammatory insults may result in changes that lead to airway remodelling. the mechanisms responsible for the generation of inflammation and remodelling remain poorly understood but may be induced or exacerbated by respiratory viral infection. respiratory infections by rsv, rv, influenza and parainfluenza and metapneumovirus (mpv) have all been implicated in the development of asthma as well as exacerbations. infection with rsv and rv are by far the most widely and commonly associated with bronchiolitis and childhood wheeze and the induction and exacerbation of asthma (papadopoulos et al., a; xepapadaki et al., ) . rsv may cause earlier and more severe exacerbations and is more frequently linked to the induction of asthma whereas rv is the most common cause of exacerbations in later life zhao et al., ) . whether an infection induces disease depends on viral (type (e.g. rsv, rv)), host (genetic susceptibility, age, immune responses) and environmental (allergen exposure, season) factors. initial infection occurs by inhalation and spreads to the lower respiratory tract (lrt). infection is largely restricted to the respiratory epithelium, which induces the release of a wide range of mediators (table ) (dakhama et al., a ) that drive subsequent immune and physiological responses specific for each virus (fig. ) . rsv and rv are the most important causes of lrt infections in infants under years, causing bronchiolitis that results in wheezing, and breathing difficulties, which may in severe cases result in hospitalization (sigurs et al., ; kotaniemi-syrjanen et al., ; henderson et al., ; sigurs et al., ) . asthmatics may be more susceptible to viral infections, which lead to more severe lrt symptoms and are associated with increased hospitalization (corne et al., ) . notably the commonest cause of asthma related-death is respiratory viral infection (mccann & imani, ) . severe bronchiolitis resulting in hospitalization has been shown to be associated in fig. . rsv, rv, asthma and therapy. rsv attaches to and invades the respiratory epithelium through the attachment (g) and fusion (f) proteins. major and minor group rvs bind to icam- and ldlprs on respiratory epithelium, respectively, which induces viral internalisation and upregulation of additional receptors. upon invasion viral proliferation leads to the induction of inflammatory cells and mediators that enhance allergen penetrance and hallmark features of asthma including inflammation, wheezing, airway obstruction and ahr. this may induce the development and exacerbation of asthma. various processes in virus-associated asthma may be targeted therapeutically. some case control studies with a history of recurrent wheeze and a diagnosis of asthma in later childhood (sigurs et al., (sigurs et al., , . rsv is the most widely implicated precipitant but recent studies suggest that rv may also be important, particularly after the age of (heymann et al., ) . methods of detection are constantly been improved which will facilitate the elucidation of the role of these viruses in asthma (pierangeli et al., ) . the mechanisms that underpin virus-induced induction or exacerbation of asthma or the factors responsible for predisposing an individual remain poorly understood. it is unclear whether virus-induced bronchiolitis promotes the development of asthma or if those individuals that suffer more severely from infection are also more susceptible to asthma development as a result of genetic susceptibility (including atopy) or aberrant lung function. understanding the mechanisms of pathogenicity of respiratory viral infections and their association with asthma will be pivotal in the development of prevention and treatment strategies for asthma. indeed it may be possible to identify at risk individuals and delay infection until later in life and to develop novel therapeutic agents or targets. in asthma triggers of chronic inflammatory processes (airway inflammation, mucus hypersecretion) are overzealous responses of the asthmatic immune system to normally innocuous antigens or infections and recurrent stimulation leads to airway remodelling. respiratory viral infections induce immune responses that may have the potential to both initiate and exacerbate asthma. collectively evidence strongly implicates respiratory viral infections in the development of an asthmatic phenotype in children, although a directly causative role has still not been proven. it is possible that either . respiratory infection in early life induces the development of chronic airway inflammation and airway wall remodelling, resulting in persistent wheeze or . that some infants have pre-existing th responses that induces susceptibility to virus-induced wheeze (legg et al., ; stensballe et al., ) . the evidence for virus-induced asthma exacerbations is stronger than for causation and infections are responsible for the majority of acute exacerbations ( % in children, - % in adults) inducing worsened airflow obstruction and symptoms wark et al., ; murray et al., ; tan, ) . to determine if virus infection directly influences disease in acute asthma exacerbations wark et al., recruited adults presenting to emergency with acute asthma and determined that % of these subjects had evidence of a viral infection (wark et al., ) . when compared to subjects with noninfective acute asthma, those with acute asthma and infection had evidence of more severe clinical disease, had a lower mean forced expiratory volume (fev % predicted), were more likely to be admitted to hospital and had a longer median length of stay. an acute neutrophilic infiltrate was observed in induced sputum, with evidence of neutrophil degranulation, unlike the eosinophilic inflammation associated with non-infective asthma (wark et al., ) . in addition lactate dehydrogenase was measured as a marker of asthmatic airway necrosis. those with virus-associated acute asthma had significantly elevated lactate dehydrogenase activity, which correlated closely with the degree of neutrophil influx and airflow obstruction and was an independent predictor of the severity of the acute illness. these results strongly implicate viral infection as a trigger of exacerbations with increased severity and indicate an important role for viruses in modifying inflammation in acute asthma (wark et al., ) . there are many different categories and phenotypes of asthma including mild, moderate and severe as well as clinical, allergic and pathophysiologic phenotypes (wenzel, ) . recently simpson et al., described different inflammatory (neutrophilic, eosinophilic and paucigranulocytic) subtypes of asthma based on the predominant granulocytic cell in induced sputum (simpson et al., ) . the precise roles of respiratory viral infection in the development and exacerbation of the different phenotypes of asthma remain largely unknown, however, there is the potential that viral infection may be particularly important in certain phenotypes. different and customised prevention and treatment strategies may be required for different phenotypes depending on their causes. targeted anti-viral strategies may be more effective in certain asthma phenotypes for example in severe and neutrophilic asthma, where infectious agents may play a substantial role in pathogenesis (wark et al., ) . many factors may be involved in susceptibility to virusinduced asthma particularly virus and host factors. virus infection is the commonest cause of wheeze in children that may lead to the development of asthma (heymann et al., ) . the time of year also plays a role with winter the dominant period for viral infections and wheeze (heymann et al., ) . genetic predisposition may be important and many genes are implicated in susceptibility to asthma including those involved in inflammatory responses, ige regulation, cytokine and chemokine production, airway function and remodelling (umetsu et al., ) . atopy may lead to adverse responses to infection and childhood wheezing is linked to elevated ige and sensitivity to at least one inhaled allergen. however, the genetics of western populations that are experiencing the asthma epidemic remain unchanged and the focus of this review will be on other important factors that are associated with rsvand rv-associated asthma. these include the age of infection and timing of infection relative to allergen exposure, increased innate susceptibility and adaptive immune, cytokine and chemokine responses, as well as environmental conditions and inhaled bacterial endotoxin. rsv is a lipid-enveloped single stranded (ss) negative sense rna pneumovirus and is a member of the paramyxoviridae family. the virus causes the majority of cases of bronchiolitis in n.g. hansbro et al. / pharmacology & therapeutics ( ) to examine effect of different clinical characteristics and treatments on hospitalization of infants for bronchiolitis in outpatient clinic infants ( ) b years presenting with st episode of wheezing retrospective analysis of medical records % of patients with rsv were hospitalized vs % without rsv. children exposed to tobacco smoke hospitalized more often ( %) vs not exposed ( %). treatment with oral corticosteroids associated with fewer hospitalizations in those with family history of asthma/allergic rhinitis ( . % vs %) and without rsv ( . % vs . %). al- shawwa and rao, early life, which may induce wheeze that may develop into asthma and is also a major precipitant of asthma exacerbations. epidemiology -rsv is the most important respiratory pathogen of children under the age of years and primary infections are a common cause of lrt disease (hall, ; henrickson et al., ) . the majority of infants are infected during the first year of life, and the incidence of exposure approaches % by age (parrott et al., ; glezen et al., ; hall et al., ) . these infections are the most frequent cause ( - %) of bronchiolitis and also induce pneumonia and tracheobronchitis ( - %) and there are annual epidemics, primarily in infants (hall, ) . around , children are hospitalized as a result of bronchitis annually in the usa with % less than months and % less than year of age with an estimated cost of $ud million per year (shay et al., ) . hospitalization for bronchiolitis has dramatically increased over the last years (shay et al., ) , which may result from changes in childcare practises or a generalized decrease in th immunity in the population. mortality rates from primary infection are . - . % for healthy or - % for hospitalized children (ruuskanen & ogra, ; chanock et al., ) . most children that contract severe rsv disease have no identifiable risk factors, with the exception of premature birth. infections also cause severe disease in the elderly and immunocompromised and mild upper rt (urt) symptoms (rhinorrhea, nasal blockage, pharyngitis and cough) can occur at any age (falsey & walsh, ; englund et al., ) . re-infection is also common, occurring every - years throughout life usually resulting in mild urt symptoms. importantly this results from the lack of development of long-term resistance to rsv infection by the immune system (bont et al., ) . the majority of symptoms result from the host's immune and inflammatory responses to infection (openshaw, ) and re-infection induces sustained and exacerbated inflammatory reactions. pathogenesis -upon rsv infection and interaction of the virus with the respiratory mucosal surface the viral g protein mediates attachment and the f protein induces the fusion of the viral envelope with the cytoplasmic membrane of the host cell resulting in internalisation (fig. ) . after invasion the viral ssrna is released into the cytoplasm and induces the production of viral rna and proteins that induce inflammatory responses. the rsv proteins and their functions have recently been reviewed by meyer et al. ( ) . the outcome of these inflammatory responses is the development of symptoms of pathogenic infection. typically, rsv infections in humans are restricted to the mucosal epithelial cells of the urt, causing runny nose, nasal congestion and cough (hall et al., ) . during severe rsv infections, the virus spreads to the lrt resulting in more severe symptoms. in vitro studies of human infection, as well as autopsy samples from infants and children with acute rsv infections, show that viral replication in airway epithelial cells, particularly in the superficial layer of the bronchiolar epithelium, as well as types and pneumocytes. infection induces the generation of inflammatory mediators and a mononuclear inflammatory response, plugging of the bronchioles with mucus, cellular debris and fibrin strands, as well as necrosis of the bronchiolar epithelium (piedra et al., ; johnson et al., ) . the lack of cytopathology during infection implicates inflammatory responses as the pivotal driver of rsv-induced disease (zhang et al., ) . inflammatory cells consist mainly of monocytes, t cells and neutrophils and accumulate around bronchial and pulmonary arterioles, airways and parenchyma and are associated with edema, mucus production, wheezing, airway obstruction and ahr . the induction of these disease processes may be involved in the development and exacerbation of asthma. clinical and epidemiological studies have shown that rsv infections are associated with a rapid increase in the incidence of asthma in paediatric and adult populations worldwide (wang & forsyth, ; tan, ) . these infections are also one of the commonest causes of asthma exacerbations. recent epidemiological studies that link rsv with asthma are shown in table and older studies are reviewed in ogra ( ) . rsv infections are the most important risk factor for the development of bronchiolitis leading to recurrent wheezing and respiratory symptoms (decreased lung function, recurrent wheezing, allergic rhinoconjunctivitis). however, it has not yet been conclusively demonstrated that these virus-induced symptoms then progress to the development of asthma. a link between infection, atopy and sensitization to common inhaled allergens (skin prick tests (spts), ige) has also been investigated but the results are inconclusive. asthmatics may be prone to more severe infections, which may be a prognostic indicator of allergic susceptibility. between and % of all cases of childhood hospitalizations for bronchiolitis have been attributed to rsv infections (holberg et al., ) . collectively studies show that rsv-induced bronchiolitis and diseases of the small airways often lead to wheezing which frequently progresses to asthma (reviewed in heymann et al. ( ) ). many studies have reported that up to % of subjects with rsv bronchiolitis suffer from subsequent recurrent wheeze or respiratory symptoms years later (reviewed in ogra ( ) ). an important prospective study by stein et al., showed that children with more than one lrt rsv infection were times more likely to have frequent wheeze by ages and , however the association decreased thereafter and was non-significant by age (stein et al., ) . another smaller prospective study demonstrated that severe rsv bronchiolitis was linked to current wheezing ( %) and reactive airway disease (rad, %) compared to uninfected controls ( % and %, respectively) matched for age, sex family, history and environment (sigurs et al., ) . controlled retrospective and prospective studies indicate a link between rsv and bronchial obstruction and decreased lung function, particularly for children with severe rsv disease that results in hospitalization (reviewed in sigurs ( b) ). indeed bronchiolitis is linked to chronic reductions in lung function for at least years after infection (pullan & hey, ) and children hospitalized with rsv infection before years of age have reduced lung function (but not asthma) years later (korppi et al., b) . furthermore airway obstruction and ahr is increased in those with rsv bronchiolitis compared to controls (sigurs, b) . a nested case-controlled study also showed that rsv-induced hospitalization correlated with wheezing, lrt infections and asthma during first years of life. the correlation decreased with age and was not significant at years but the association held for increased respiratory symptoms and chronic productive cough at - years in alaska native children (singleton et al., ) . in the stein study wheezing subjects were significantly more responsive to bronchodilators, which indicates that reduced lung function results from an abnormality in airway tone (stein et al., ) . rsv bronchiolitis, particularly in early life, is strongly linked to the development of asthma. indeed - % of children with bronchiolitis develop wheezing and asthma ( % versus (vs) %), , . ( % vs %) or even or more years later (sly & hibbert, ; sigurs et al., ; larouch et al., ; sigurs et al., ; ogra, ) . recent studies have found that children hospitalized with rsv have an increased risk of recurrent wheeze up to the age of years, independent of atopy and other asthma risk factors, identifying rsv infection as a potential inducer of asthma. indeed these subjects have significantly more respiratory symptoms, ( % vs % for asthma/recurrent wheezing), sensitization to common allergens ( % vs % spt, % vs % serum ige), airway obstruction and ahr at age years compared with controls (sigurs et al., ) . other recent reports have also shown that rsv infection is associated with concomitant respiratory symptoms (wheezing) and subsequent asthma at years of age and that high rates of rsv are isolated from children with recurrent wheeze or asthma (lazzaro et al., ; lee et al., ) . the association was not confirmed in other longitudinal or cohort studies and may depend on individual airway structure, genetic predisposition and environmental factors (reviewed in martinez ( ) ). there may be a link between atopy, rsv infection and wheezing (sigurs et al., ) and risk factors may include the severity of rsv infection encountered. atopic children have low th responses in cord blood and elevated th responses compared to non-atopics. atopic adults also have th responses to allergens whereas non-atopics have low-level th responses (ogra, ) . elevated expression of th responses may promote susceptibility to rsv infection and may also lead to exaggerated airway inflammation and reduced lung function. however, the link between rsv infections and atopy is controversial and was not found in other studies (stein et al., ; kneyber et al., ) . early rsv infections (first years) may induce allergic sensitization to unrelated antigens in genetically predisposed individuals (sigurs et al., ; forster et al., ) . indeed rsv bronchiolitis in the first year of life leads to increased ige levels ( % vs % in controls) and is the most important risk factor for allergic sensitization and recurrent wheeze ( % vs % controls) (schauer et al., ) . furthermore allergic sensitization and asthma are significantly more common in all age groups in children with a prior rsv bronchiolitis ( % vs % and % vs % of controls, respectively) (sigurs, a ). it appears that the severity of infection may be important and severe infection has been associated with the development of allergic sensitization , or . years after hospitalization (sigurs et al., (sigurs et al., , . mild infection, does not appear to be a risk factor for allergic sensitization (stein et al., ; sigurs et al., ) . however, the data are conflicting and severe infection has been shown not to induce sensitization (at age - years) by other investigators (pullan & hey, ; carlsen et al., ; noble et al., ) . pre-existing th or impaired th responses and asthmatic predisposition may promote susceptibility to acute bronchiolitis and hospitalization for rsv infection (legg et al., ; stensballe et al., ) . furthermore, individuals with damaged airway epithelium or that are otherwise immunologically predisposed may be additionally susceptible to infection enhancing epithelial damage and causing a vicious cycle of disease perpetuation. although rsv infection has been extensively linked with inducing asthma and reduced lung function it is possible that infection is also a marker of susceptibility to allergic and/or infectious respiratory disease. it has been shown that although rsv infection is the greatest risk factor for wheezing and asthma in subjects with a family history of asthma, asthma does not develop in those without infection or a family history (sigurs et al., ; sigurs, ) . this indicates that rsv bronchiolitis is a marker of increased risk of asthma susceptibility. more recently it has been suggested that the host response to infection rather than the nature of the infection itself is the best prognostic indictor for subsequent allergic disease (everard, a (everard, , b . atopy may also be a risk factor for susceptibility to more severe rsv infections and exacerbations, which result in higher rates of mortality and hospitalization (jhawar, ) . a large nested case-controlled study demonstrated that maternal atopic dermatitis and maternal and paternal asthma (rr . and . , respectively) were risk factors for rsv hospitalization in infants b . years (stensballe et al., ) . taken together studies show that rsv infection in early life results in bronchiolitis that leads to wheezing and decreased lung function, which may progress to asthma. these processes may be enhanced in atopic individuals with elevated th responses. moreover there is evidence, although not conclusive, that severe infection may also promote sensitization to allergens, which may further increase the risk of wheeze and asthma. asthmatics may be susceptible to more severe infections, which may be used as an indictor of susceptibility to the development of asthma. experimental rsv infection of humans has been used to investigate the mechanisms of pathogenesis of infectious disease and the association with asthma. these studies have demonstrated that the virus persists in nasal washes for - days (noah & becker, ) and that infection induces bronchiolitis and airway inflammation. this promotes epithelial sloughing, mucus hypersecretion and an increase in viscosity and edema which in turn lead to hyperinflation of the lungs, airflow obstruction, cough and wheeze (holt & sly, a) . symptoms may persist and resolution of tissue damage may take several weeks or results in structural remodelling of the airway wall and airway narrowing (pare et al., ; holt & sly, a) . the mechanisms of rsv pathogenesis that lead to wheezing, ahr, allergy and asthma are still not well understood and many studies have implicated a range of different factors. these include: the age of first infection; type of innate and adaptive responses elicited; mucosal damage and repair involving remodelling (including angiogenesis) and; enhanced neurogenic stimulation leading to asm spasm and bronchoconstriction. according to the hygiene hypothesis (strachan, ) , the th -biased immune system of the newborn must encounter th -inducing agents during childhood in order to develop the ability to mount a th response. however, whether infections in infancy induce beneficial th responses depend upon the type of infection and the mechanisms responsible for these effects have not been characterised. thus the immaturity of the developing immune system during early life and the nature of immune responses to infections may be significant determining factors in the development of persistent wheeze and asthma. whether rsv infection induces immunological and pathological processes that may lead to asthma may depend on the age at which an individual is first infected. the immune phenotype of early life may lead to enhanced viral replication and th -dominated inflammation induced by infection or allergen exposure. increased levels of il- were detected in rsv infected infants less than months old compared to more than months but the converse was true for eotaxin and there was no difference in mip- β or eosinophil cationic protein (ecp) (kristjansson et al., ) . in mothers infection induced high levels of ifn-γ and low levels of il- whereas newborn infants produced - times less ifn-γ and higher levels of il- but at age these levels approached those of their mothers (mbawuike et al., ) . the immune response in early life may be involved in the induction of more severe lrt pathology in response to primary infection but infection of older children is not as severe and involves largely urt symptoms. an additional pathological consequence is that viral infections in early life may generate pulmonary inflammation during the development of the lung, small and large airways and immune, inflammatory and neuronal programing (reviewed in gern et al. ( ) ). this may result in altered pulmonary structure and immune responses leading to enhanced pro-inflammatory and th -biased immune responses that may precipitate deleterious changes in lung structure and function. furthermore, the small size of neonatal bronchioles determines that they may become obstructed more readily, which may result in reduced clearance and confer enhanced severity of pathogenic infection in this age group. the combination of these events may have longterm effects on lung function, chronic respiratory inflammation, remodelling, alveolarization and epithelial dysfunction (gern et al., ) and consequently may promote the development of asthma. however, whether rsv infection induces th or th responses in humans in early life is debateable. another alternative explanation for the association of rsv infection in infancy with asthma is that early life infection induces th memory and therefore cd + ctl responses, which have the potential to induce pathologic immune responses to reinfection. rsv infection induces viral specific cd + cells in infants and the levels of cytotoxic lymphocytes (ctls) are inversely proportional to il- responses. ctl responses are directly linked to protection against infection, ctl memory is initiated upon reinfection and mhc i cd + levels correlate directly with ifn-γ levels (mbawuike et al., ) . immune responses induced to rsv enhance viral clearance but are also implicated in disease pathogenesis. ineffective or aberrant innate and adaptive immune responses against rsv have been widely linked to more severe and recurring infections and the development and exacerbation of asthma in both adults and children (glezen et al., ; hall et al., ) . primary infection, particularly early in life, leads to an incomplete immune response, which does not elicit the development of sustained memory immunity. with respect to allergy rsv infection might only trigger defective immunity in genetically susceptible individuals or that allergic inflammatory and immune responses may promote the influx of virus-specific cells into the airways increasing inflammation and ahr (schwarze et al., c) . elucidation of the pivotal immune responses that are protective against rsv will lead to a better understanding of the processes that result in bronchiolitis, wheezing and progression to asthma. innate responses to rsv infection have not been widely studied but may play an important role in rsv-induced asthma. rsv infection of the respiratory epithelium induces innate cellular and cytokine responses, which have substantial effects on adaptive t cell development and cell-mediated immune responses. neutrophils are the main immune cell in the bronchoalveolar lavage fluid (balf) of patients with severe rsv lrt disease and increased il- levels, which is a potent chemoattractor of neutrophils, are also a prominent feature. macrophages internalise and process virus and present antigens to adaptive immune cells and also release il- and il- . monocytes release il- , - , - , - , platelet-activating factor (paf) and pge on exposure to rsv that further promote proinflammatory responses (schaller et al., ) . eosinophils are also recruited to the airways during primary rsv infection, which may contribute to the development of allergic airways disease (schwarze et al., a) . infected patients also have more plasmacytoid dendritic cells (pdcs) and myeloid dcs (mdcs) in the rt and reduced numbers of these cells circulating in blood (gill et al., ) . this suggests that they are recruited to the lung during infection and may be an important target in rsv vaccine development. understanding the interplays between these different cells types will be crucial in elucidating the effects of infection on the development of asthma. ifn-α is an important type i ifn innate cytokine that is released upon viral infection and induces innate and adaptive cellular responses. blood cultures of asthmatic children and adults release significantly reduced amounts of ifn-α upon rsv infection, which indicates a systemic innate deficiency in asthmatics that may lead to heightened susceptibility to infection (gehlhar et al., ) . . . . . th /th responses. rsv has several t cell epitopes and infection induces cd + and cd + and th and th adaptive cellular responses as well as pro-and anti-inflammatory cytokines and chemokines in humans both in vivo and in vitro (meyer et al., ) . established infections are primarily cleared by a combination of th and th cell responses and the balance between the two may be crucial in determining the outcome of infection, the severity of rsv-induced disease and predisposition to asthma. aberrant responses of either of these subsets may induce pathology. nevertheless most studies suggest that th responses may result in viral clearance and mild symptoms whereas an aberrant bias towards a th phenotype may lead to more intense rsv-induced disease and promote the development of asthma . indeed individuals with elevated th responses are predisposed to reduced viral clearance and more severe disease compared to subjects with only urt infection independently of age or viral load (roman et al., ; aberle et al., ; legg et al., ; gern et al., ) . early life rsv bronchiolitis results in enhanced il- and ifn-γ responses to rsv in later childhood (pala et al., ) and il- is the most important stimulus for th development and ige production. th responses are enhanced during severe disease that develops upon natural infection of rsv-vaccinated infants (kim et al., ; kapikian et al., ) and severe rsv bronchiolitis correlates with elevated humoral th responses, and may be linked with atopy, increased il- :ifn-γ and reduced th (ifn-γ, il- and il- ) responses. rsv infections may also have persistent immunological effects and induce long-term th memory responses during sensitization to inhaled allergens in childhood (holt & sly, a ; van rijt et al., ) . other studies have demonstrated that mild bronchiolitis is associated with a shift towards th responses and is usual in most individuals, and also that there is no increase in th responses in severe bron-chiolitis . these contrasting results may be attributable to differences in the timing of sampling during infection, the lack of definitive detection of rsv or determination of virus load, age and atopic status of individuals. . . . . cytokines and immunomodulatory molecules. cytokines of the th , th or regulatory type are implicated in rsvinduced asthma and the effect of pro-inflammatory cytokines and chemokines released in response to infection may have particularly important roles. th (il- ) cytokines are upregulated in children with acute asthma and children with acute bronchiolitis or il- levels have higher numbers of eosinophils (martinez, ) . these responses may play key roles in the progression from bronchiolitis to asthma, however, the importance of eosinophils and il- in rsv-induced asthma has not yet been confirmed. il- is a th cytokine that promotes the development of th cells, the release of ifn-γ and il- from th and natural killer (nk) cells and suppresses th responses. levels of il- may be reduced in subjects that are more susceptible to infection. non-specifically stimulated whole blood cultures from patients with rsv disease released significantly less il- than controls and il- levels inversely correlated with disease severity (bont et al., b) . il- and ifn-γ responses were suppressed during acute rsv disease but returned to control levels during convalescence and ifn-γ (and il- ) levels were not different in subsequently wheezing infants (bont et al., a) . reduced il- levels may lead to elevated susceptibility to th responses to rsv infection and predisposition to asthma. il- is a regulatory cytokine, which may promote an asthma phenotype by suppressing th cytokine production and antigen presentation promoting enhanced susceptibility to infection and th -dominated responses that induce wheezing and pro-asthmatic responses to subsequent antigen challenge (bont et al., a (bont et al., , b . il- levels did not change during acute rsv disease but increased during convalescence and were significantly higher in subsequent wheezers and those that went on to develop recurrent wheeze and asthma. thus, the enhancement of il- and inhibition of il- production upon rsv infection may suppress immune function and permit more severe infection and disease progression. it has also been suggested that rsv infection may change and enhance the profile of pro-inflammatory cytokine and chemokine release that promotes more severe infection and alters the nature of the t cell response promoting allergy and inflammation. patients with rsv-induced bronchiolitis had elevated levels of mip- α (but not rantes), which correlated with disease severity in nasopharyngeal secretions and rantes, icam- , il- and - and ige in serum (sung et al., ; garofalo et al., garofalo et al., , . furthermore treatment of human epithelial cells with tgf-β, which plays a pivotal role in airway remodelling and asthma, increased rsv replication and tnf-α secretion and p mitogen-activated protein kinase activation. this may contribute to the elevated inflammatory responses in virus-associated asthma (mccann & imani, ). by contrast rsv-induced wheezing does not correlate with il- levels in nasal lavage fluid but is elevated with influenza or rv infection (gern et al., ) . thus it is possible that infection induces regulatory mechanisms that suppress immune responses allowing viral replication resulting in increased inflammatory responses. alternatively infection may induce inflammatory responses that enhance infection and allergic inflammation. this may subsequently promote the induction of regulatory mechanisms to limit inflammation-induced damage of host tissue. . . . . other immune factors. in vivo and in vitro studies have shown that rsv infection of the respiratory epithelium causes the release of other factors that have immune functions including arachidonic acid metabolites and lts, mediators released by eosinophils and chemokines (reviewed in ogra ( )). cell adhesion and homing molecules such as cd b, icam- and e-selectin and antigen-presenting molecules including human leucocyte antigen classes i and ii are also upregulated upon infection. several transcription factors are also activated, which may induce the expression of a range of genes such as nk-il- and nuclear factor (nf)-kb. these factors regulate immunomodulatory mediators (tnf-α, il- β, il- , - , - and gm-csf), adhesion molecules (icam- , vcam- and e-selectin) and chemokines (il- , mip- α, mcp- , eotaxin and rantes) (garofalo et al., ; oh et al., ; john et al., ; makela et al., ; schaller et al., ) . in combination these mediators and molecules induce the influx of inflammatory cells and may contribute to the development of infection-induced inflammatory and immune responses, ahr and asthma. ineffective or aberrant humoral responses may also have a role in rsv-induced asthma. infection induces increases in b cell numbers (roman et al., ) and the production of serum and mucosal igm, iga and igg antibodies. these are important in protection and not disease but occur at lower levels in infants. antibody responses to primary infection are ineffective and involve the production of partially neutralising antibodies against the g and f proteins but these responses are reinforced (especially igg and iga) upon reinfection. rsv-specific ige antibodies are also produced (welliver et al., ) in the majority of children and increased amounts and persistence promote the development of wheezing (ogra, ) . increased vascularity (angiogenesis) surrounding the airway wall is associated with chronic and fatal asthma but also with mild-to-moderate asthma in both children and adults (li & wilson, ; vrugt et al., ; barbato et al., ) , suggesting a pathogenetic role at all stages of asthma development. angiogenesis is regulated by a balance between pro-angiogenic (vascular endothelial growth factor (vegf), basic fibroblast growth factor (bfgf), angiogenins, chemokines) and antiangiogenic (endostatin, canstatin, tumstatin, arresten) factors (cohen, ) . vasodilation of the increased number of blood vessels in response to inflammatory stimuli may lead to edema and inflammation of the bronchial wall (influx of inflammatory cells, including eosinophils and release of mediators), airway narrowing and ahr (black & page, ; wilson, ; salvato, ; nomura et al., ) . elevated levels of vegf, bfgf and angiogenins occur in the airways of asthmatics and correlate with increased vascularity, vessel permeability and ahr (hoshino et al., ; kanazawa et al., ; nomura et al., ; feltis et al., ) . vegf influences vascular permeability through the formation of blood vessel fenestrations and vasiculo-vasculo organelles (dvorak et al., ; esser et al., ; neufeld et al., ) . vegf also stimulates endothelial cell proliferation and migration, matrix remodelling, and vasodilation, as well as inhibiting endothelial cell apoptosis and all of these processes are involved in angiogenesis (neufeld et al., ) . vegf also enhances sensitization of the rt to allergens and promotes th inflammation . rsv infection may contribute to the development of asthma by inducing the production of vegf and angiogenesis. vegf has been detected in nasal washings from rsv infected patients, indicating that infection stimulates vegf production, which may have a role in disease (lee et al., ) . furthermore, features of rsv bronchiolitis and pneumonia such as submucosal, adventitial, and interstitial edema are due to alterations in blood vessel permeability brought about by vegf activity (dvorak et al., ; esser et al., ; neufeld et al., ) . the specific role of vegf in rsv infection has not been intensively investigated. it is released from airway epithelial cells, th cells and mucosal fibroblasts upon infection (lee et al., ) but is also produced by monocytes, macrophages, t cells, keratinocytes, granulocytes, eosinophils and smooth muscle cells (gaudry et al., ; horiuchi & weller, ; neufeld et al., ) . thus a novel mechanism may be involved in rsv-induced asthma whereby the induction of vegf upon infection may lead to the development of angiogenesis that can enhance the inflammatory response. furthermore, rsv-induced secretion of vegf may contribute to exacerbations by increasing vascular permeability, and recurring infections may contribute to cycles of vegf production that promote angiogenesis and remodelling. neurological and immunological interactions that occur as a result of rsv infection have also been linked to the generation of airway inflammation, ahr and rad in children. excitatory non-adrenergic non-cholinergic nerves (nance) release neurotransmitters including substance p that participate in the early phase of the inflammatory response and also have an immunomodulatory role (piedimonte, ) . sloughing of the epithelium during infection may lead to exposure of neurogenic receptors (nk ), which enhances the pro-inflammatory effects of substance p leading to asm spasm and bronchoconstriction and contributing to respiratory symptoms. nerve growth factor participates in neuronal development and has beneficial effects on inflammation, repair and remodelling. however, it has been suggested that these effects may become pathogenetic during rsv infection and allergic inflammation and exacerbate inflammation and ahr (nassenstein et al., ) . the effect of co-infection of with rsv and other viruses has been little studied, however, notably the risk of developing bronchiolitis is times greater in infants with co-infection with rv (papadopoulos et al., a) . animal models of infection and allergic airways disease (aad) have been developed and used extensively to substantially contribute to the understanding of the mechanisms that underpin rsv-induced asthma and exacerbations. in particular rodent (mouse and to a lesser extent rat) and bovine models have been used to elucidate the mechanisms of the associations and to trial therapeutic agents and vaccines. chimpanzees are permissive to human rsv and are the best animal model but their availability and cost limits all but the most advanced clinical tests (whitehead et al., ) . using these models important factors have been identified that may play key roles in rsv-induced asthma and include; age of first infection, timing of infection relative to allergen exposure, induction of asthma, endotoxin exposure, innate factors and adaptive immunity, suppression of immunity, angiogenesis, neural networks and latent infections. the importance of the different viral proteins in infection has also been investigated. the use of animal models enables experimental protocols to be conducted that are not possible in humans. in particular the precise investigation of the different ages of infection, combinations of the timing of infection relative to allergic sensitization and collection of invasive tissue samples can only be achieved in animals. although there are problems with these models of rsv challenge including that rsv does not replicate in mice and does not induce the recruitment of granulocytes that is observed in human disease, such models have been used extensively to gain valuable insights into the mechanisms of rsv-induced disease. in most mouse models rsv infection induces significant acute respiratory inflammation and changes in lung function. high doses ( - plaque forming units (pfu)) induce severe alveolitis and pneumonia and low doses ( - pfu) result in bronchiolitis without these effects (dakhama et al., a) . five days after infection mice develop acute airway obstruction, which correlates with the progression of inflammation and histopathology that is characterised by intense perivascular and peri-bronchial/bronchiolar influx of monocytes/ macrophages and some neutrophils and lymphocytes. these cells also occur in the alveoli during the peak of inflammation but leakage into the airway lumen is absent (jafri et al., ) . infection peaks at days - and resolves between days and and cytokines and chemokines (il- , mips and rantes) are released by the respiratory epithelium and alveolar macro-phages in response to infection. after days nk cells influx into the balf and are replaced between and days by cd + and cd + cells which return to pre-infection levels at days (hussell & openshaw, ) . ctls induced in response to infection contribute to extensive peri-bronchiolar and -alveolar inflammation and are associated with disease symptoms (ostler et al., ) . ifn-γ is important in viral clearance and contributes to pathology and is primarily released by nk cells with levels peaking at day . only low levels of il- and il- are present, however, il- , - , - , - , - and ifn-γ are produced in the lungs within the first few days and their relative levels determines the course of disease (kalina & gershwin, ) . il- plays a pivotal role in the development of infectioninduced ahr in the absence of allergen exposure (makela et al., ) . acute infection develops into chronic disease with features of chronic inflammation (histopathology score) and ahr (in terms of enhanced pause (penh)) after the clearance of virus and up to weeks after infection . penh is a noninvasive method of whole body plethysmography and uses a single exposure to a spasmogen to determine the overall level of ahr in all airways. jafri et al., used penh to show that airway obstruction and ahr remained for and days after infection, respectively, and that ahr correlated with chronic inflammation which persisted but not in alveoli (jafri et al., ) . this supports the concept that rsv disease may induce long-term respiratory changes in children (stein et al., ; sigurs et al., ) . however, these studies need to be confirmed by the measurement of lower airway resistance using invasive methods that precisely measure changes in airway and tissue specific function and employ dose responses to spasmogens. studies using these methods (measurement of respiratory impedance and airway resistance) have shown a lack of longterm effects of rsv infection on lung function (dakhama et al., c; collins et al., ) . infection also induces mucus hypersecretion in the central and peripheral airways in the acute and chronic phases and severe and progressive pneumonia develops with increases in histopathology and chronic inflammatory changes. these processes contribute to airway obstruction (penh) that develops in the acute phase and progresses but does not correlate with viral load in balf, and this agrees with observations made in children (jafri et al., ) . the intensity of inflammation declines over time but remains around airways and vessels. neutralising monoclonal antibodies (mabs) against rsv substantially decrease inflammation and disease severity . using mouse models it has been shown that the age of first infection plays a key role in shaping dominant immune responses later in life (reviewed in hansbro et al. ( ) ) and establishes the subsequent pattern of th cell responses and the nature and severity of ensuing respiratory diseases (culley et al., ; holt & sly, a; horvat et al., ) . primary rsv infection in neonatal mice has the same profile as in adults, however, it is associated with a slower and diminished ifn-γ response and the development and persistence of th cytokine release by cd + t cells. these effects are substantial and can reverse the protective effect of mycobacterial exposure on aad . neonatal rsv infection results in early tnf-α release and has long-term adverse effects on the respiratory system. these effects include the induction of ahr, peri-vascular and -bronchial inflammation and subepithelial fibrosis, which are exacerbated by subsequent allergen exposure and involve persistent il- expression and mucus hypersecretion (you et al., ) . the generation of th responses during immunological development has profound modulatory effects on the balance of subsequent th /th responses and promote a marked increase in the th phenotype in adulthood (chen et al., ; walzl et al., ; culley et al., ) . the neonatal th bias may result from the types of t cells or dendritic cells present, their environment or a combination of these effects (nelson et al., ; goriely et al., ; white et al., ; bartz et al., ) . subsequent re-infection later in life may reinforce aberrant th responses (cytokines (il- ) and cd +/cd + cells) and alter responses to allergens resulting in enhanced inflammation, mucus hypersecretion, ahr and allergy (enhanced weight loss and th cell and eosinophil recruitment to the airways) (chen et al., ; walzl et al., ; culley et al., ; dakhama et al., b) . this suggests that aad of adults primed with an infection as neonates is likely to be caused by factors that promote th responses (culley et al., ; holt & sly, a) . if initial infection is delayed until mice are weeks of age ifn-γ production increases and upon reinfection, although airway inflammation still occurs, there is a subsequent reduction in the severity of disease with no mucus production or ahr (culley et al., ; dakhama et al., b) . thus delaying the age of infection until later in life may be an effective strategy for the prevention of rsv-associated asthma. animal models have been used to determine if rsv can induce the development of asthma by triggering pro-asthmatic immune responses that lead to variable airflow obstruction and airway inflammation. chavez-bueno et al. ( ) demonstrated that rsv induces acute and chronic disease with features of aad independently of allergic sensitization or genetic background in mice. rsv infection of balb/c or c bl/ mice in the absence of allergic sensitization led to similar levels of acute airway inflammation, airflow obstruction and ahr and the degree of airway inflammation correlated with ahr. the immune response was surprisingly similar between the two strains and was characterised by virus-dependent release of ifn-γ. importantly infection and inflammatory responses were not short lived. acute infection developed into persistent infection that correlated with airway inflammation, which were present days after infection. while virus load and airway inflammation were greatest during the acute phase, chronic infection correlated with chronic inflammation and airflow obstruction. thus rsv infection can initiate acute events that result in airflow obstruction and possibly ahr and these changes can persist beyond the initial inflammatory response. other investigators have also shown that rsv infections in early life act synergistically with atopy to drive the development of allergic asthma (holt & sly, b; kusel et al., ) . however, the immunological processes involved in the induction of allergic sensitization by rsv infection are not likely to be the same as those involved in exacerbation of allergic asthma by rsv. the effect of rsv infection on aads may be critically dependent on the relative timing of infection, allergic sensitization and challenge (peebles et al., ; barends et al., barends et al., , . the majority of studies in mice show that rsv augments aad, however, conversely some investigators suggest that rsv infection prevents atopy. indeed rsv induces a strong th and ifn-γ mediated response that may modulate responses to allergens (peebles et al., ; juntti et al., ) . these contrasting observations can be explained as two competing immune responses are occurring simultaneously, which may subtly differ in different protocols. the development of allergy may depend on the phenotype of the immune response to allergens and rsv at the time of exposure. . . . . allergen exposure prior to infection. prior exposure of the airways to allergen independently of allergen type predisposes to increased severity of virus-induced aad in mice and is likely to play a role in humans (peebles et al., ; makela et al., ; kalina & gershwin, ) . infection enhances th cytokine responses, mucus secreting cell hypertrophy, eosinophil influx into the lung and ahr in response to allergen and increases the severity of aad. the potency of the th responses elicited overrides the counterregulatory effects of th responses that are typically induced by infection (randolph et al., ) . . . . . infection prior to allergen exposure. exposure of rsv infected mice or rats to allergens increases inflammation, mucus production and ahr and prolongs rsv replication (peebles et al., ; kalina & gershwin, ; hassantoufighi et al., ) . in particular cd + and cd +, inflammatory responses in the lung are enhanced (lukacs et al., ; barends et al., ; schaller et al., ) . blocking il- during infection reduces chemokine expression and ahr and blocking rantes removes the effects of infection upon later allergen sensitization and challenge (lukacs et al., ) . the absence of the cc chemokine receptor (ccr ) in deficient mice leads to reductions in t cells, il- , eosinophils, mucus and ahr (john et al., ) . these observations suggest that chemokines and their receptors play roles in rsv-induced aberrant responses to allergens and may be important in mobilisation of virus-and allergen-specific t cells and allergic inflammation. it is possible that rsv infection may contribute to the development of allergy by damaging the respiratory mucosa, which exposes apcs and t cells to allergen, which may break tolerance and induce systemic th sensitization. excessive infection-induced respiratory damage may occur in predisposed individuals as a result of defects in t cell mobilisation, activation or activity. if allergen challenge of sensitized mice occurs concomitant with infection the inflammatory response is again enhanced and leads to th responses to rsvand promotes chronic infection (makela et al., ) . il- production leads to eosinophilia, il- to the release of mcp and further th mediated inflammation but il- does not further enhance ahr. if rats are sensitized to extracts of the common household mould aspergillus fumigatus, which induces eosinophilia and th cytokine release, rsv infection before allergen challenge exacerbates the inflammatory response and ahr that is dependent on viral replication. infection causes increased expression of mhc ii on alveolar macrophages, which may be involved in initiating immune responses to allergens. persistence of infection is induced and is related to reduced ifn-γ expression again suggesting that allergic sensitization can affect the progression of rsv infection (kalina & gershwin, ) . treatment of infected a. fumigatus challenged animals with recombinant ifn-γ reduced allergic responses and th and th cytokines but not ahr. this suggests that pathological and physiological factors of disease are independent (hassantoufighi et al., ) . taken together these studies suggest that in general rsv induces increased severity of th -mediated aad and that the development of aad increases the severity and persistence of rsv infection. some reports have suggested that exposure to endotoxin or environmental pollution can affect the progression of rsv infection (gurkan et al., ; monick et al., ) . exposure to such factors alters the relative proportions of cytokines produced upon infection and affects disease progression, however, the cellular and molecular processes involved are not understood. tlr- expression may provide a link between rsv, endotoxin and asthma. tlr- expression is not usual in resting airway epithelium and requires high endotoxin exposure to be upregulated. rsv infection induces increased expression of tlr- and responsiveness to endotoxin and initiates potent inflammatory responses (monick et al., ) . this may be linked to human asthma as endotoxin also induces asthma exacerbations in children with rsv-induced asthma (park et al., ) . animal models have been used extensively to elucidate the host immune responses that are induced by rsv rt infection and how these responses may contribute to the development and exacerbation of asthma. infection may inhibit or modulate the activity of both innate and adaptive (particularly t cell) immune responses during the development of disease, which may play a crucial role in the induction of pathology and aad. primary rsv infection induces innate responses that involve eosinophil and neutrophil influx into the lung that results in ahr and a cytokine response that is dominated by ifn-γ (schwarze et al., b) . eosinophil infiltration is il- but not il- or ifn-γ dependent and is critical for the development of ahr. the innate response (first days) is also characterised by an influx of nk cells producing ifn-γ, which are replaced by adaptive cd + and cd + cells and the release of il- with low levels of il- , - and - (openshaw, ; boelen et al., ; van schaik et al., ; openshaw, ) . t cell responses facilitate viral clearance but also induce host tissue damage and pathology. infection induces innate responses involving tlrs, cytokines, chemokines and dcs that ultimately direct the development of adaptive t cell and antibody responses (durbin & durbin, ; krishnan et al., ) . . . . . tlrs. rsv may use a variety of host cell factors to bind to and enter cells including tlr- , cx r , heparin and caveolin (kalina & gershwin, ) . binding to tlr- initiates the production of il- , - and - β and tnf-α and may be responsible for the initial response to infection. however, the role of tlrs in virus-induced asthma is controversial. some studies report that the innate immune response to rsv infection in mice is dependent on the expression of cd and tlr- (kurt-jones et al., ; haeberle et al., ) , which may interact with the viral f protein (openshaw et al., ) . another report argues the opposite saying there is no significant role for tlr- in infection (ehl et al., ) . tlr- recognises double stranded (ds) rna and is constitutively expressed on respiratory epithelial cells and dcs. tlr- signals independently of myeloid differentiation factor to induce nf-κb activation and the expression of ifnβ activation of tlr- leads to apoptosis and elimination of infected cells and virus. recently it has been shown that tlr- and protein kinase r (pkr) are upregulated in human airway epithelial cells by rsv infection, which enhances epithelial responsiveness by activation of nf-κb and il- and may sensitize these cells to subsequent viral or bacterial infection (groskreutz et al., ) . . . . . chemokines. chemokines are produced by stromal, epithelial and immune cells and regulate immune responses (chemoattraction of leukocytes into the lung), inflammation, mucus production and angiogenesis. although cellular inflammation is often similar in response to different viral infections the types of chemokines and the levels that are released that drive subsequent immune responses differ substantially (schaller et al., ) . primary rsv infections induce the expression of chemokines belonging to the cxc (il- , mip- and ip- ), cc (rantes, eotaxin, mip- α, mcp- and t cell activation gene- ) and c (lymphotactin) families in the lung. mip- α expression is high and mip- α deficient mice have reduced lung inflammation, but rsv titres that are the same as in wildtype mice. thus rsv-associated lung inflammation may be mediated by early production of inflammatory chemokines (haeberle et al., ) . . . . . dcs. dcs are the most important antigen-presenting cell (apc) and take up viral antigens, traffic to local lymph nodes and present antigen to naïve t cells causing their differentiation into effector cells. dcs direct innate and adaptive immune responses to viruses and allergens and are essential for allergic sensitization. different subsets of dcs exist with different functions. mdcs present viral antigens and promote th cell expansion to a great extent than pdcs, which are more important in the development of tolerance (van rijt et al., ) . dc networks are less active in infant animals but can be enhanced and mdcs are increased upon viral infection (holt & sly, b; zuniga et al., ) . it is possible that the reduction of pdcs by conversion into mdcs during early life viral infection may inhibit the development of tolerance and exacerbate allergic responses. the outcome of rsv infection may depend on the nature of the adaptive response and the balance of th and th immunity. primary infection of balb/c mice induces a mixed th /th response with an early burst of ifn-γ release that is important in determining the phenotype of the subsequent response openshaw & tregoning, ) . other adaptive responses involving cd + cells and b cell/antibody responses also play significant roles in responses to rsv. . . . . th cd + t cell responses. rsv infection typically induces a robust th response with elevated levels of ifn-γ and il- in mice. ifn-γ is the archetypal th cytokine and its release and signalling through the ifn-γ receptor during infection are pivotal in controlling the th /th response to infection. these processes are essential in moderating eosinophil migration and ifn-γ and cd + cells are crucial for viral clearance. in the absence of ifn-γ a dominant th response induces eosinophil influx of the lung and ahr (barends et al., ) . by contrast, the absence of il- and il- has little effect . il- does, however, induce th and suppresses th responses, promotes ifn-γ production from nk and cd + cells, reduces il- and il- production from cd + and cd + cells and can prevent but is not essential for inhibiting virus-induced eosinophil influx to the lung (hussell & openshaw, ) . il- does not function through cd + or b cells and exacerbates disease in mice sensitized to allergen (openshaw et al., ) . the removal of cd + or the induction of cd + cells also eliminates eosinophil influx (hussell et al., ) . tnf-α is another th cytokine and is over-produced during viral rt infections, which exacerbates inflammation by promoting neutrophil and eosinophil influx. anti-tnf-α treatment of mice leads to ablation of weight loss and illness without affecting viral clearance and does not induce adverse side-effects, which indicates a potential for use in therapy (hussell et al., ). . . . . th responses. th responses are induced by rsv infection in a variety of animal models or in the absence of ifn-γ , which may contribute to the development of aad. these responses are potent and are similar to th responses observed after allergen exposure of allergic individuals (braciale, ) . rsv-induced ifn-γ and il- responses do not diminish the th response during the development of rsv-induced allergy although the response is even greater in the absence of ifn-γ (barends et al., ) . it is a specific set of t cells, a cd +vβ + subpopulation, that induces a superantigen type response to rsv and is pivotal in inducing th mediated pathology (varga et al., ) . t /st is a surface receptor of the il- family expressed on th but not th cells. t /st was present on a subset of cd + t cells from mice with rsvinduced eosinophilia and t /st mab treatment reduced th but not th pathology (walzl et al., ) . these studies suggest that under some circumstances infection may induce strong th responses but the mechanisms of how this leads to aad are unknown. il- , - , - , - and - are th cytokines that are released during th responses and are likely to be involved in rsv-induced aad. in mice, primary rsv infection results in increased il- levels, however, it's importance in the development of th responses and aad is unclear. il- deficient mice or mice treated with a neutralising anti-il- and immunized with vaccinia virus expressing protein g had no reduction in pulmonary eosinophils or th cytokine secretion. furthermore infection of il- deficient mice resulted in enhanced numbers of eosinophils in the lung and ahr johnson et al., ) . by contrast il- release may be pivotal in rsv-induced th responses and aad. infection of mice results in il- -mediated ahr and eosinophil influx into the lung in association with strong ifn-γ responses. il- but not il- or ifn-γ is the critical mediator of eosinophil influx, ahr and allergy (schwarze et al., a) . il- deficiency leads to a reduction in pulmonary eosinophils and ahr, which can be reversed by replenishment with il- . treatment with anti-very late antigen- prevents the influx of eosinophils into the lubg and ahr in response to rsv infection or il- replenishment. rsv infection induces the expression of il- in mouse pulmonary t cells (hussell et al., ) . the absence of il- inhibits the development of ahr in response to allergen sensitization and challenge. rsv infection overcomes this deficiency and induces eosinophil infiltration of the lung, airway mucus production and ahr, which are associated with increased th responses (makela et al., ) . il- is associated with the development of rsv-induced ahr and may promote the release of other th cytokines (einarsson et al., ) . although il- is required for the development of mucus hypersecretion and ahr in mouse models of aad and after secondary rsv infection of infected neonates (kuperman et al., ; dakhama et al., b) it does not appear to play an important role in the induction of these responses after primary infection . this has not yet been investigated in humans. it is likely that as well as inducing a th phenotype rsv may take advantage of th responses that predominate in asthmatics that are ineffectual against infection. however, surprisingly rsv clearance can occur under th conditions whereby eosinophils take up rsv and inactivate the virus with ecp (soukup & becker, ) . il- increases fasl expression on macrophages and cd + cells and therefore also has an anti-viral effect (ruan et al., ) . cd + t cells target several rsv proteins and are sufficient to clear rsv from infected mice (cannon et al., ; cherrie et al., ) , however, clearance and immunopathology still occurs in cd -deficient mice (graham et al., ) . adoptive transfer of virus-specific cd + ctls which home to the lung eliminates rsv. these cells induce viral clearance through perforin/ granzyme-mediated lysis of virus-infected cells (aung et al., ) . however, perforin (which is also produced by nk cells), cd l and tnf are not necessary but ifn-γ release is crucial for cd + t cell-mediated clearance. rsv was eliminated with unchanged kinetics from perforin deficient mice (aung et al., ; ostler et al., ) , whereas treatment of mice with neutralising antibody to ifn-γ or transfusion of ifn-γ-deficient effector ctls abolished virus control and induced cd + t cellmediated pathology (ostler et al., ) . by contrast, high dose primary infection in ifn-γ deficient mice led to attenuated immunopathology, but only slightly delayed clearance. this suggests that other cells and molecules can partially substitute for ctl-derived ifn-γ driven virus clearance and further implicates ifn-γ as a pivotal immune factor in rsv-induced immunopathology and cd + t cell-mediated control (ostler et al., ) . cd + cells may also suppress the development of virusspecific th -dominated immune responses and eosinophilia although the mechanism of these effects remains unknown. hussell et al., showed that early ifn-γ release by cd + cells in response to rsv infection of mice resulted in suppression of th responses (hussell et al., ) and the suppression of th responses by cd + cells is by other studies (srikiatkhachorn & braciale, a) . interestingly the th -inducing effects of the rsv g protein can be nullified by incorporating a cd + epitope onto the g protein (srikiatkhachorn & braciale, a ). . . . cd +/cd + interactions cd + and cd + t cells are exposed to presented viral antigens in the lymph nodes of the respiratory tract, which induces differentiation, activation and mobilisation of both effector and memory t cells. memory cd + t cells move to the rsv infected rt and proliferate and differentiate into cytokine releasing effector cells and induce their effects in situ (varga et al., (varga et al., , . during differentiation the cells are subject to infectioninduced modulation and it is likely that the cd +/cd + interaction is occurring concurrently. similar interactions may take place during allergen provocation in the rt of asthmatics that have the potential to substantially affect t cell phenotype, an effect that may be enhanced upon infection. thus the complex interactions of cd + and cd + with infectious stimuli and allergens may be pivotally important in the development of rsvinduced aad. these processes may be targeted therapeutically to suppress allergen specific th responses in the lung. differential immune responses are induced by different rsv proteins, which may be important in the development or exacerbation of asthma. protein g vaccination of mice induces cd + but not cd + memory populations and leads to reduced nk cell influx and ifn-γ production. this results in the induction of th responses, in the absence of a ctl response, with il- and il- release by th cells which promotes eosinophilia upon subsequent infection (alwan et al., ; srikiatkhachorn & braciale, a; walzl et al., ; openshaw et al., ) . g protein defective rsv has been used to demonstrate that this protein is crucial in promoting airway inflammation and reduced lung function (schwarze & schauer, ) . f or m protein immunization induces a mixture of nk, th type cd + and mhc i cd + cells, leading to ifn-γ production and reduced disease (alwan et al., ; srikiatkhachorn & braciale, a; openshaw et al., ) . nk cells and the ifn-γ they release regulate the induction and proliferation of cd + cells, which then clear the virus. primary rsv infection induces the expansion of activated m - (h- k d restricted peptide epitope in rsv m protein)-specific cd + t cells in lung. this implies that activation and proliferation of m -specific cd + t cell precursors is normal. taken together these results suggest that natural infection that induces th responses may be dominated by responses to the g protein exacerbates infectious and allergic disease, whereas responses to the f and m proteins may be th mediated and reduce disease. antibody responses may have several roles in rsv-associated aad in mice. rsv infection may induce the development of proallergic ige antibodies and their receptors in the lung which may induce mast cell degranulation and ahr (dakhama et al., ) . the activation of anti-viral protein kinase upon infection may lead to isotype switching of b cells to produce ige (rager et al., ) . other studies have shown that exposure to allergen during acute rsv infection of mice results in the production of antigen specific igg responses, which are characteristic of th immunity (o'donnell & openshaw, ) . moreover non-neutralising antibody produced in response to formalin-inactivated (fi) virus may induce immune complex formation in the lung (openshaw et al., ) , which may be involved in pathogenesis. infants have poor t cell independent antibody responses and produce different types of antibodies compared to adults. early life infection with rsv may fix the nature of antibody production as well as t cell responses to subsequent infection throughout life and promote the development of ahr (openshaw et al., ). studies in mice have suggested that rsv may promote its own infection by suppressing host immunity resulting in enhanced inflammation. the rsv g protein may attenuate innate responses by binding to tnf-α (valarcher & taylor, ) and the induction of cytokine production from monocytes and macrophages through the inhibition of tlr- -nf-κb mediated signalling (polack et al., ) . rsv-specific t cells may play a crucial role in viral clearance and limited evidence suggests that infection may suppress the activation and activity of t cells leading to enhanced viral replication and disease (harcourt et al., ) . during suppression the f protein of rsv may downregulate the activity of t cells and cytokine production including the release of ifn-γ (kondo et al., ; schauer et al., ) . rt infection also suppresses ctl immunity through the rapid loss of virus-specific cd + memory cells and ifn-γ release (chang & braciale, ) , which is mirrored in humans by the lack of the induction of immunological memory. this effect may occur during antigen receptor signalling and varies to different extents in different t cell functions (ctl activity, cytokine release). this may result in reduced ctl activity and allow viral persistence but may also enable the maintenance of cytokine responsiveness facilitating further virus-or allergen-associated inflammation promoting enhanced disease. despite this evidence immune suppression during infection has not yet been observed in humans. these effects may only be present in the lung and are not detectable in the blood (braciale, ) and it is unknown if cd + t cells with this phenotype exist in the rt of humans with severe rsv infection. rsv infection may also attenuate protective immune responses by suppression of type i ifn (ifn-α,β), modulation of dc activity, g protein mimicry of the cx c chemokine, which may inhibit t cell migration and by producing viral variants that are not recognised by neutralising antibodies (tripp, ; meyer et al., ) . the viral elements responsible for dysregulated immune responses are not known. persistent rsv, other viral or bacterial infections, which are local or systemic, or underlying chronic lung disease may promote susceptibility to rsv-induced asthma. emerging evidence from both mouse and human studies suggests that rsv infections may persist at low levels, by mechanisms involving suppression of immunity and avoidance of immune detection and total clearance (seemungal et al., ; . this occurs in mice with functional neutralising antibody and ctl responses even though the virus possesses a cd + epitope and productive infection can be reactivated by depletion of cd + and cd + cells . in mouse models rsv is detectable by culture up to just days after infection but by pcr for up to days independently of genetic background and viral copy number correlates with ahr (tripp, ; chavez-bueno et al., ) . this association has not yet been proven but treatment with neutralising antibody reduces viral numbers and disease severity . however, it is possible that viral detection by pcr under these conditions may represent the persistence of viral debris following the administration of high viral inocula and may not be part of the disease process. it remains unknown whether viral persistence occurs in children following bronchiolitis and the development of wheezing and long-term pulmonary abnormalities. if persistent infection does occur chronic inflammation and altered immune (cytokine/ chemokine) responses may be induced in individuals with rsvinduced aad. thus persistent infection may be important in long-term morbidity and may provide a new therapeutic target. limited mouse and primate studies of acute and chronic asthma also link vegf with pathophysiological features of rsv infection and asthma suzaki et al., ; avdalovic et al., ; lee et al., ) . over-expression of vegf in the lungs of mice to levels found in asthma or during rsv infection, induces angiogenesis, oedema, inflammation, vascular remodelling, mucus cell hyperplasia/metaplasia and ahr . rat models of rsv-induced bronchiolitis have also been developed, where rats rapidly clear the virus in a similar manner to the self-limiting infection of human infants (piedimonte et al., ) . this model has been used to investigate the alteration of neural networks by rsv infection and infection of infant rats has enabled the analysis of the effects of early life infection (king et al., ) . a stronger neurogenic inflammatory response develops in the lrt of infant rats than in adult rats during infection, which may explain why bronchiolitis presents in infants but as an urt infection in older subjects. induction of inflammation involves the upregulation of nk in infected lungs (but not nk that is expressed on asm fibres) that are activated by nance nerves and mediate substance p induced immunomodulation and neurogenic and cellular inflammation that may lead to edema and obstruction (king et al., ; piedimonte, ; tripp et al., ) . nk receptors are also upregulated on t cells in bronchus-associated lymphoid tissue in response to infection, which may then be attracted into the airways and release pro-inflammatory cytokines in response to neurogenic stimulation by airborne irritants. recurrent stimulation may lead to persistent cycles of airway inflammation and obstruction. inhibition of nk or the administration of cgrp prevents rsv-induced ahr and may be potential therapeutic targets (dakhama et al., c) . neurogenic stimulation by rsv infection also induces the release of lts from mast cells that induce mucus secretion (wedde-beer et al., ) . another alternative is that infection-induced damage may expose nerve endings and substance p and neurokinin a may then mediate asm contraction (jacoby, ) . other investigators have shown that rsv infection of rats and ferrets results in increases in cholinergic mediated contraction of asm and reduced inhibitory nance responses (larsen & colasurdo, ) . despite the vast array of literature describing studies of rsv infections in animal models, there is no single model which duplicates the pathological features of disease observed in human infection. the major drawback in studies of rsvinduced disease in mice is that rsv is not a natural mouse pathogen. symptoms induced upon infection are minimal compared to those observed in human infants, the virus has limited replication and infected animals show few if any signs of respiratory illness (domachowske et al., ) . furthermore, large doses of the virus are required and primary infection is rapidly aborted . such discrepancies between human disease and mouse models have severely hampered the investigation of rsv-induced disease. the pneumonia virus of mice (pvm) is the closest genetic relative of rsv and is a natural mouse pathogen (easton et al., ) . unlike rsv, pvm infection of mice reproduces many of the acute inflammatory responses described for rsv infection in humans. pvm replicates rapidly inducing inflammation leading to mucus plugging of the airways and overt signs of disease from urt symptoms to fatal pneumonia, which is dependent on the administered dose easton et al., ) . virus replication is associated with an influx of granulocytes and severe inflammatory bronchiolitis. we have recently established mouse models of rsv-like disease using pvm infection of neonates and adults . importantly neonatally infected mice also exhibit many of the symptoms observed during rsv disease of human infants harrison et al., ; rosenberg et al., ) . these models are now being utilised to more precisely elucidate the host pathogen relationships that result in rsvinduced asthma. the importance of inflammation in inducing disease has been demonstrated and inflammatory responses remain active after the cessation of viral replication. mip- α and its receptor ccr play pivotal roles in these inflammatory responses. a recent study using pvm in wild-type and tlr- deficient mice showed that there is no difference in the clinical, functional, histological and virological parameters investigated indicating that pvm infection is independent of tlr- signalling (faisca et al., ) . anti-viral therapy with ribavirin alone has little effect, however, treatment with ribavirin in combination with the anti-inflammatory agent and the ccr antagonist met-rantes substantially reduces morbidity and mortality following infection (rosenberg et al., ) . bovine rsv, pathogenesis and vaccine development have recently been reviewed (meyer et al., ; valarcher & taylor, ) . bovine rsv is closely related to human rsv and is the most common cause of lrt disease and the major single health problem in calves worldwide, particularly in winter (stott et al., ; valarcher & taylor, ) . indeed - % of epizootic respiratory diseases in the first year of life are attributable to bovine rsv with mortality typically between and % but reaches % in some outbreaks (meyer et al., ) . models of bovine lrt infection have been developed and used to investigate responses to infection and evaluate bovine vaccines. bovine rsv is a natural pathogen and pathogenic infection of cattle shares many similarities with rsv infection in humans (van der poel et al., ) . infection is largely restricted to respiratory epithelial cells but causes little cytotoxicity and pathology is mediated by inflammatory responses to infection (viuff et al., ; valarcher & taylor, ) . these responses also involve innate (neutrophil and macrophage recruitment) and adaptive (pro-inflammatory cytokine and chemokine release) immune responses that result in respiratory damage. the mechanisms of pathology have been investigated using genetic manipulation of the virus and have implicated the g and sh proteins in infection and the f protein and non-structural (ns) proteins in inflammatory responses (reviewed in valarcher & taylor ( ) ). . . . . pathogenesis. bovine rsv is transmitted by direct contact, in airborne droplets or is possibly transferred passively by humans (hall et al., ; mars et al., ) . pathogenic infection of the rt is much more common in calves than adults (stott et al., ) , which results from a lack of specific immunity in naïve animals. maternally-derived antibodies afford some protection but primary infection induces the most effective immunity (kimman et al., ) . infectious disease takes - days to develop and has similar clinical features to humans and may possibly result in persistent infection (valarcher et al., ) . disease of the urt induces mild symptoms of coughing and mucus production. in the lrt bronchiolitis and bronchopneumonia occur in association with edema, wheezing and dyspnea (verhoeff et al., ; belknap, ) . infection of cattle induces inflammation involving the influx of mononuclear cells, neutrophils, cd + (of mixed th /th phenotype) and cd + cells and sometimes eosinophils into peri-bronchial regions along with necrosis and apoptosis of epithelial cells (viuff et al., ; antonis et al., ) . cd + ctls have a major role in the clearance of primary infection in calves and lymphocyte proliferation is attenuated by infection in vitro (keles et al., ; antonis et al., ) , whereas antibody-mediated responses are important in protection against secondary infection. the lumen of the airways become occluded with mucus and inflammatory debris and remodelling events occur that lead to breathing difficulties (kimman et al., ; viuff et al., ) . it is likely that similar immune responses are elicited as in human infection with rsv and involve the induction of nf-kb and the generation of pro-inflammatory cytokines and chemokines. the interaction of viral components (f protein and dsrna) with tlrs is known to drive the development of the immune response in cattle (reviewed in valarcher & taylor ( ) ). as in humans and mice there are age-related differences in immune responses to infection in cattle with reduced protective and enhanced inflammatory responses in younger animals. in response to primary infection younger calves have enhanced fever, virus-specific tnf-α, il- and ifn-γ release from pbmcs and reduced peripheral blood mononuclear and b cells and virus-specific iga and neutralising antibody responses (grell et al., ) . the immunology and pathogenesis of bovine rsv infection has been intensively studied using reverse genetic engineering of the virus, which is less variable that human rsv and the results may be extrapolated to human disease (collins et al., ) . the importance and roles of different bovine rsv proteins in the induction of pathogenesis and immune responses have been elucidated using these genetically manipulated viruses. the g protein of rsv is the major attachment protein and deletion mutants do not replicate in the absence of the g proteins in vivo, although replication is unaffected in vitro (karger et al., ; schmidt et al., ) . bovine viruses expressing only the secreted segment of the g protein also have fold attenuated replication, which is fold higher than mutants missing the entire g protein (teng et al., ) . mutants expressing the membrane-anchored g protein segment have no impairment of replication in the urt but were attenuated at least fold in the lrt and did not induce pathogenic responses in the lung (maher et al., ) . this suggests that both fragments of the g protein are important in infection and pathogenesis with the secreted portion playing a particularly significant role. cleavage of the bovine f protein is required for activation and results in the production of the peptide and tachykinin, virokinin that induces the production of nks, substance p and other neurogenic pro-inflammatory mediators. virokinin itself induces asm contraction and therefore directly contributes to airflow restriction but does not have chemotactic properties (zimmer et al., ) . by contrast cleavage of human rsv does not produce tachykinins (valarcher et al., ) . deletion of various parts of the bovine f protein has shown that it is not necessary for proliferation in vivo but deficient mutants promote substantially reduced airway inflammation and eosinophil influx (valarcher et al., ) . the f protein is responsible for suppressing lymphocyte proliferation (schlender et al., ) , however, there is no effect on the expression of the eosinophil attractants rantes or mip- α in the absence of the f protein suggesting that other mechanisms may be involved in the suppression of eosinophil influx in cattle (valarcher et al., ) . ns proteins, particularly ns , of bovine rsv downregulates the ifn-α/β response involving the attenuation of irf- and stat- (schlender et al., ) , which has subsequently also been shown with human rsv (spann et al., (spann et al., , lo et al., ) . the lack of ns proteins results in highly attenuated replication and a lack of pathogenesis in vitro and in vivo (whitehead et al., ; valarcher et al., ) . the sh protein is not required for replication of bovine rsv in vitro (karger et al., ) but replication is attenuated in the lrt of chimpanzees and calves in vivo (whitehead et al., ; valarcher & taylor, ) . epidemiological studies have widely linked rsv infection to the development of bronchiolitis, wheeze, decreased lung function and possibly asthma in childhood. this association is equivocal later in life but may depend on the severity of the infection. there may also be a role for rsv in the induction of sensitization to allergens and allergy may predispose to more severe infection. enhanced severity of symptoms that result from infection may be a useful prognostic marker for the future development of allergic disease. both human and animal studies have investigated the mechanisms of how infection induces pathogenesis and how this is associated with asthma. the age of first infection is crucial in determining clinical outcomes and early infection may promote the development of a pro-allergic phenotype. this indicates that delaying the age of infection until later in life may be an effective therapeutic strategy. infection induces both innate and adaptive immune responses that promote th immunity and eosinophil influx into the lung that may contribute to the development of ahr. the timing of infection relative to allergen exposure and the immune phenotype of the individual may be significant in determining the effects of infection. an interesting novel concept is that rsv infection may induce the development of angiogenesis, which might exacerbate inflammatory exudation, edema and bronchial obstruction in asthma. other new hypotheses are that rsv may induce immune suppression allowing viral persistence and latent infections that can be reactivated. neural networks also have the potential to be involved through the induction of neurogenic inflammation. rvs are small non-enveloped ssrna viruses of the picornaviridae family and to date over different serotypes have been identified (savolainen et al., ) . epidemiology -rvs are responsible for the majority of cases of urt infectious disease in humans particularly colds and have recently also been implicated as the cause of significant numbers of lrt infections. rv is also the most common respiratory viral pathogen that induces wheeze at all ages (kusel et al., ) . a third of all episodes of acute respiratory illness involve wheezing in both children and adults and rv infections are implicated in times as many cases as other viruses. wheezing may then progress to the development of asthma. the mechanisms of pathogenesis of rv are much less well understood than for rsv. pathogenesis -rvs are divided into major and minor classes based on receptor binding properties. major group rvs bind to icam- while minor group viruses bind to very lowdensity lipoprotein receptors (ldlpr) (fig. ) (dreschers et al., ) . binding of rv to ldlprs induces a conformational change in the capsid that is required for viral uptake. intracellular internalisation occurs through the activation of sphingomyelinase that generates ceramide-rich membrane rafts, which facilitate uptake. rv is not thought to replicate in lrt, however, it is possible that rv can infect the lrt to a certain extent or alternatively that immune responses to urt infection has knock-on effects on inflammation in the lrt. infection of the urt typically causes coryza and pharyngitis and virus can be detected in fluids from the nasopharynx, tonsils and the middle ear (holgate, ) . recent epidemiological studies correlating rv with asthma are shown in table . rv is more strongly linked with exacerbations of asthma although it is emerging that rv may also be important in asthma induction. there is a strong association between rv infection and childhood wheeze and exacerbation of asthma that may lead to hospitalization (el-sahly et al., ; jartti et al., ) . rv is implicated in wheeze in % of children n years of age and - % of adults and is responsible for % of cases of asthma exacerbations nicholson et al., ; atmar et al., ; freymuth et al., ; tan, ) . interestingly the peak of severe asthma exacerbations in children occurs shortly after their return to school after breaks and in adults one week later and this coincides with peaks in rv infections in the spring and fall (longini et al., ) . indeed in the fall rv was present in % and % of children that were asymptomatic or were having asthma exacerbations, respectively . while rsv may be an important factor in early childhood wheeze, the majority of children become infected with rsv, but most do not go on and develop asthma, suggesting that other factors are important in this process. to investigate this lemanske et al. ( ) prospectively recruited a cohort of children ( ) at risk of asthma based on family history and followed them for years to determine the relationship between viral infections and symptoms of asthma, confirming infective episodes and etiology by pcr on nasopharyngeal aspirates. at years non-infective factors such as exposure to environmental tobacco smoke, the presence of asthma in an older sibling and ige-mediated food allergies were associated with persistent wheeze. rsv infections were also linked with an increased risk, however the strongest independent predictor of persistent childhood wheeze was an episode of rv infection associated with wheeze (or = , %). this suggests that rv infection in lemanske et al., infancy is associated with the development of asthma later in life. others agree and have shown that rv infection is the primary risk factor for wheezing in children under years but that atopy is more important thereafter (heymann et al., ) . other recent studies have also indicated that recurrent wheezing in infants is promoted by rv and possibly to a greater extent than rsv (lehtinen et al., ) . a pathophysiological link between rv and asthma is indicated by the tendency of children infected with rv to be older than those infected with rsv ( months vs months) and an increased frequency to present with atopic dermatitis and blood eosinophilia (korppi et al., a) . in the study by korppi et al., children ( ) hospitalized for wheezing before the age of were assessed by bronchoscopy and had a prior median symptomatic period of months. of these children % were found to be asthmatics by the age of years, and % of these were atopic. early predictors of asthma were atopic dermatitis, specific ige and inhalant allergy and rv was common during wheezing ( %). another recent study of infants ( ) under the age of hospitalized with bronchiolitis identified rv and rsv in % and % of cases, respectively (jacques et al., ) . it is unknown if asthmatics are more likely to develop colds, or if they are at a greater risk of more severe colds. corne et al., attempted to investigate this by recruiting couples, one of whom had asthma and determining if the asthmatics were more at risk of developing colds. they found that those with asthma were no more likely to develop colds ( % vs % of controls), but they were substantially more likely to develop lrt symptoms associated with these colds ( % vs %). this implies that viral infections in asthmatics are more likely to increase lrt inflammation (corne et al., ) . other investigators have shown that asthmatic infants hospitalized with wheezing have a greater chance of testing positive for rv than non-asthmatics, which suggests that asthma sufferers may also be more susceptible to infection (xatzipsalti et al., ) . furthermore, rv infection may be more long lasting in asthmatics and may persists for up to weeks after hospitalization for acute asthma exacerbation and rv-induced ahr may also last for several weeks (kling et al., ) . experimental rv infection of human volunteers has enabled the study of the effects of infection on immune responses, lung function and asthma as well as the role of atopy and timing of infection on disease outcomes . infection upon binding of rv to icam- (greve et al., ) not only contributes to pathogenesis but also to the induction of allergic inflammation (canonica et al., ) . rv also induces the upregulation of icam- by activation of nf-κb, which promotes further infection (papi & johnston, ) . rv infects the bronchial epithelial layer and some mononuclear cells and fibroblasts and increases airway inflammation, with changes observed in induced sputum and endobronchial biopsy. the bronchial epithelium of asthmatics is particularly susceptible to the cytotoxic effects of viruses (papadopoulos et al., ; mosser et al., ) . initial infection initiates rv-induced exacerbations and cytotoxicity leads to increases in the penetrance and effects of allergens. . . . . immune responses. in both asthmatics and non-atopic non-asthmatics inflammatory changes involving almost all types of inflammatory cells are observed upon rv infection. in mild to moderate asthma, infection induces increased levels of il- and neutrophils in sputum, promotes the influx of eosinophils, mast cells, cd + and cd + cells into the airway wall and enhances ahr (fraenkel et al., ; grunberg et al., a; van benten et al., ) . de kluijver et al., showed the inflammation upon infection may be less intense in the nonasthmatic, although these differences were small and no direct comparison with asthmatics was made at the time (de kluijver et al., ) . nevertheless reduced ifn-γ responses in subjects with asthma may promote susceptibility to infection and there is an inverse correlation between ifn-γ levels and rv persistence and symptoms in asthmatics . this deficiency may result from either the lack of recruitment of appropriate inflammatory cells or a reduced response from these cells. other differences in immune responses to rv infection have also been observed that may be involved in increased susceptibility of asthmatics to infection or enhanced inflammation. in subjects with acute rv-induced asthma wark et al., found that increased serum ip- levels but not rantes or il- was specifically associated with infection and correlated with the degree of airflow obstruction (wark et al., ) . these subjects also had less bronchodilatory response (increase in fev ) to salbutamol compared to non-infective acute asthma, which correlated with levels of ip- in serum. ip- is a chemokine that binds to cxcr and is important in the recruitment of t cells to infected tissues. increased levels of ip- can activate mast cells and cause their migration. mast cells that are resident in the asm are specific for asthma and also express cxcr . therefore, rv infection of the bronchial epithelium may lead to an early release of ip- that sets off a chain of events that enhance pre-existing asthmatic airway inflammation and promotes the migration and activation of mast cells into the asm thereby worsening bronchoconstriction and reducing the response to bronchodilators. nasal secretions of experimentally infected healthy volunteers contain increased levels of il- β, a pro-inflammatory cytokine (proud et al., ) , while rv infection of subjects with allergic rhinitis or asthma, or in children with virusinduced asthma, resulted in elevated levels of g-csf and il- as well as increased blood and nasal neutrophilia (teran et al., ; gern et al., ) . have demonstrated that rv infection worsens airway inflammation and obstruction and enhances ahr to non-specific bronchoconstrictors in subjects with asthma or atopy. however, despite showing an ability to infect these subjects and recovery of virus, significant exacerbations of disease are not observed (grunberg et al., a) . while treatment with inhaled corticosteroids (icss) effectively reduces ahr and even airway eosinophilia, it has no effect on virus-induced airway inflammation. this suggests that in the acute phase there is a disparity between these features of disease. epidemiological studies also link rv infection with reduced lung function. for example, a recent study detected rv in the respiratory epithelium in % of infants with recurring respiratory symptoms ( , - months) which was associated with abnormal lung function (decreased airways conductance, %) compared with rv negative infants ( %) (malmstrom et al., ) . it is possible that the serotype of rv involved may be important and some strains such as rv are particularly associated with reduced lung function and increased ahr and symptoms of asthma and rhinitis (peebles & hartert, ) . to determine whether atopy influences infection and worsening of ahr, xepadaki et al., studied a group of asthmatic children dividing them by atopy (xepapadaki et al., ) . in both groups ahr increased days after a cold and remained elevated for - weeks. where the groups differed was that atopic children experienced more symptomatic colds and acute exacerbations, leading to a cumulative affect that resulted in the persistence of ahr. therefore, increased susceptibility of atopics to infection was demonstrated and recurring inflammatory insults were implicated as an indirect cause of more severe ahr. . . . . timing of infection. as with rsv the timing of infection relative to allergen exposure may also be important in determining the outcome of rv infection in asthma. the severity of asthma exacerbations increases if infection occurs simultaneously with allergen exposure in asthmatics (green et al., ) . however, infection after allergen exposure has little effect on ahr (de kluijver et al., ) . numerous important studies have used a variety of in vitro cell culture systems to provide valuable insights into the pathogenesis of rv and its association with asthma. specifically the role of cytotoxicity, immune responses, ahr and remodelling involving angiogenesis have all been implicated. importantly it has now become clear that viral infection directly influences lower airway inflammation in asthma and that asthmatics may have a defective innate response in bronchoepithelial cells (becs) to rv. the bec is the first point of contact between infecting viruses and the host and plays an important early role in initiating innate and adaptive immune responses and inflammation (bals & hiemstra, ). there is conflicting evidence surrounding the nature of the cpe of rv infection on epithelial cells. while some studies have found that rv induces considerable cpe albeit with serotype dependent variation (schroth et al., ; papadopoulos et al., ) , others report none or very little cpe in vitro or in vivo (winther et al., (winther et al., , ). the magnitude of immune responses as well as differential regulation of different innate and adaptive components has been implicated in the increased susceptibility of asthmatics to rv and in rv-induced asthma and exacerbations. upon rv infection of asthmatics more intense inflammatory reactions involving mucus production, the influx of neutrophils, eosinophils, activated macrophages, cd + and cd + t cells into the rt and pro-inflammatory mediator release are associated with more severe and persistent lrt involvement and symptoms and delayed repair edwards et al., ; inoue et al., ) . although there are similarities in the cellular immune responses to rsv and rv infection there are significant differences in the levels and types of cytokines and chemokines released and the cell types that produce them. ineffective presentation of viral antigens also occurs in asthmatics, which may impair immunity and lead to asthma symptoms (parry et al., ) . many authors have used epithelial cell culture models to demonstrate that rv induces the release of pro-inflammatory mediators and it is possible that differential regulation of these factors may be important in rv-associated asthma. rv infection promotes the production of oxygen radicals in the bronchial epithelium and leads to the increased expression of nf-κb and enhanced pro-inflammatory responses (contoli et al., ) . the activation of p mitogen-activated protein kinase by rho a has been shown to play an important role in these processes (dumitru et al., ) . rv infection of human cell lines relevant to the rt and asthma has demonstrated that the production of many different pro-inflammatory cytokines, chemokines and molecules is induced including; eotaxins and , il- β, - , - , - , - , ena- , ip- , gm-csf, tnf-α, rantes, mcp- , mip- α and icam- (subauste et al., ; grunberg et al., a; johnston et al., ; yamaya et al., ; gern et al., ; papadopoulos et al., ; suzuki et al., ; papadopoulos et al., ; hosoda et al., ; konno et al., ; gern et al., ; hall et al., ; edwards et al., ) . of particular importance is the increased production of il- , - and - and eotaxin, ip- and rantes, which are released upon rv infection of becs . il- and il- participate in the maturation of b and t cells and eosinophil and macrophage chemotaxis, respectively. rantes and eotaxin are also powerful chemoattractors of eosinophils and drive airway eosinophilic inflammation . il- and ena- and rantes and ip- are potent neutrophil and t cell chemoattractants, respectively . il- is also linked with reduced lung function in experimental rv infection, suggesting that chemokines may be involved in asthma exacerbations (grunberg et al., b) . importantly ip- and rantes are the chemokines released in the greatest quantities from bec infected with rv (wark unpublished). moreover, when becs were treated with dexamethasone ip- was only inhibited at the maximum dose, although were inhibited at all doses. this is in keeping with experimental findings that moderate doses of icss are effective in reducing ahr and infiltration of eosinophils, but do not prevent the accumulation of cytotoxic t cells. therefore rv infection may induce neutrophil and t cell influx that may exacerbate allergic inflammation and which may be refractive to ics treatment. . . . . ifns. the release of type i and iii ifns from becs are important innate responses to infection. they have anti-viral properties and play roles in apoptosis of infected cells and directing adaptive immune responses to clear the virus (takaoka et al., ; wark et al., ) . asthmatics have recently been shown to be particularly susceptible to the development of rv lrt infections as a result of reduced ifn-α responses. this was shown in vitro by demonstrating that peripheral blood mononuclear cells (pbmcs) from both children and adults with asthma responded to rv inoculation with a reduced release of ifn-α (corne et al., ; gehlhar et al., ) . wark et al. , have established a model of acute rv infection of the airway epithelium, using primary becs (pbecs) acquired by bronchoscopy and cultured in vitro. asthmatic pbecs allow a significantly greater replication of rv compared to controls. the innate response of asthmatic pbecs to rv is deficient in the release of ifn-β (wark et al., ) , which promotes susceptibility to infection. this defect directly impairs the ability of the infected host cell to undergo early apoptosis and allows increased virus replication and ultimately cytolysis of infected cells. the defect is the result of altered intracellular signalling and not icam- expression or viral internalisation. the administration of exogenous ifn-β induces apoptosis and suppresses viral replication, which identifies the potential for therapeutic intervention (wark et al., ) . as ifn-β is released mostly by becs, little ifn-β was detected in the balf of individuals experimentally infected with rv. further confirmation of the importance of these pathways was provided when microarray analysis of rv-infected differentiated becs identified genes that were upregulated and most of which are involved in type i ifn pathways. the expression of these genes could be blocked by anti-ifn-β mab or a dsrna inhibitor (chen et al., ) . rv infection of pbecs also induces the release of the novel immune mediator and type iii interferon, ifn-λ, which also has anti-viral effects on rv replication (contoli et al., ) . again this group demonstrated that asthmatic pbecs had a markedly deficient ifn-λ response to infection with rv compared to healthy volunteers. the importance of the early response to infection was confirmed in vivo by experimental rv infection of volunteers; those with the greatest impairment of early release of ifn-λ to rv had significantly increased virus replication, severity of cold symptoms, airway inflammation and a more severe fall in lung function (contoli et al., ) . it has been suggested that defective innate immune responses result in deficient adaptive th responses. in support of this pbmcs from asthmatics have been shown to have deficient ifn-γ responses to rv (wark et al., ) . others have made similar observations and demonstrated time and dose dependent increases in ifn-γ, il- and il- following rv infection of pbmc (papadopoulos et al., b) . cells from normal subjects produced higher levels of ifn-γ and il- , while those from atopic asthmatics predominantly produced il- (papadopoulos et al., b) . . . . . tlrs. tlr- recognises rv and is upregulated upon infection, which leads to nf-κb expression (groskreutz et al., ) . if tlr- is blocked anti-viral responses are suppressed and rv replicates and is released (hewson et al., ) . pkr is released and promotes the generation of pro-inflammatory il- , - and rantes. asthmatics may be deficient in tlr- , which may predispose to reduced immune responses and increased and persistent rv infection that may lead to enhanced viral cytotoxicity (hewson et al., ) . . . . . th responses. th responses that are typically elevated in asthmatics including those involving il- , increase the expression of icam- on airway epithelium, which promotes increased rv infection (bianco et al., ) . icam- may participate in eosinophil and t cell influx into the lrt in asthma. elevated th along with reduced th responses may promote susceptibility to rv infection and more frequent viremia in subjects with acute asthma exacerbations (xatzipsalti et al., ) . . . . . ahr/remodeling. while viral infections initiate lower airway inflammation and asthmatics appear more susceptible these observations do not provide a link that infections increase the severity of ahr or long-term airway remodelling. infection of pbecs clearly will initiate the release of pro-inflammatory mediators that will enhance the recruitment and trafficking of inflammatory cells to the airways. however, it is unknown whether this will affect remodelling, either directly by influencing asm or indirectly by affecting other features of remodelling in chronic asthma. to determine if rv infection could directly influence remodelling, in vitro culture models of pbecs and fibroblasts from subjects with mild or moderate asthma and healthy controls have been employed. fibroblasts have the potential to play a critical role in remodelling and changes in the airway matrix (holgate et al., ) . infection of pbecs and fibroblasts from these subjects does not lead to the release of the known mitogenic factors tgf-β or endothelin- . culture of fibroblasts with media taken from pbecs that had been infected with rv also did not induce the release of these mediators. there was also no increase in the expression of the contractile protein alpha smooth muscle actin, which may be expressed if these cells develop a contractile phenotype and there was no change in cellular morphology. these results suggest that rv infection does not induce the release of proremodelling factors from these cells. however, exposure of fibroblasts to both rv directly and rv conditioned pbec media did promote inflammation with the induction of inflammatory mediators, similar to those observed in becs. this observation agrees with the results of oliver et al., who demonstrated that asthmatic asm cells released significantly more il- upon infection even though the virus does not replicate in these cells (oliver et al., ) . limited data suggest that rv may be involved in the generation of angiogenesis and remodeling. infection induces the release of vegf and fgf and other fibrosis and angiogenesis inducing factors from primary airway epithelial cells, epithelium and fibroblast cell lines and these factors have also been detected in rv-infected nasal secretions (ghildyal et al., ; de silva et al., ; psarras et al., ; volonaki et al., ) . psarras et al., demonstrated that rv infection of becs led to the induction of vegf and that when endothelial cells were treated with the medium of infected becs they began to form tubules and to proliferate. these effects were inhibited by anti-vegf and enhanced when the endothelium was exposed to th cytokines . rv research and the development of therapeutic strategies have been severely hampered by the lack of a small animal model with which to investigate disease pathogenesis and the induction and exacerbation of asthma by rv. mice do not express icam- that can be used as a receptor by major group human rvs and these viruses do not establish infections in mice. minor group rvs, however, can infect airway epithelial cells of mice by binding to ldlprs (dreschers et al., ) . a recent report (newcomb et al., ) , describes a mouse model of minor group rv infection with rv b. virus persists in mouse lungs and induces neutrophilic inflammation in the airways involving mip- expression whereas major group rv does not. this model has since been used to demonstrate that rv infection induces mucin production in vivo and that mucin production in a model of murine aad is also increased by rv infection (bartlett et al., ) . rvs are the most common cause of common colds and urt illness and have recently been implicated in many lrt episodes. rv infections are also the commonest cause of wheezing and asthma exacerbations, particularly in individuals n years of age. rv-induced wheezing may lead to the development of asthma and recent evidence suggests that rv may be the most important risk factor for wheeze and asthma in early life. as is the case with rsv, asthmatics may be more susceptible to rv and are more likely to suffer from more severe rv disease. in vivo and in vitro studies have been used to demonstrate that rv infection exacerbates airway inflammation, obstruction and ahr and atopic status and timing of infection may have important effects. these studies show that the magnitude and phenotype of the immune response to rv has a major impact on asthmatic outcomes. importantly in vitro studies have identified defects in innate (type i and iii ifns) and adaptive responses from cells from asthmatics to rv infection. these defects may promote susceptibility to infection and contribute to the asso-ciation between rv infection and the induction and exacerbation of asthma. in order to prevent virus-induced asthma and exacerbations we require a better understanding of predisposing factors for viral diseases and their association with the causation and exacerbation of asthma. the development of effective anti-viral agents and the conduct of large-scale prospective and intervention trials are also required to assess the potential of such strategies to prevent these diseases (wennergren & kristjansson, ) . in asthmatics rsv and rv infection directly enhanced airway inflammation and worsened airflow obstruction. much of this occurs as a result of the immune responses, which develop rapidly upon infection. for example, responses to naturally occurring colds due to rv infection results in the development of symptoms within h which peak at - days but have resolved within a week (gwaltney et al., ) . the rapid response to infection, the often relatively mild symptoms that occur and brisk resolution determine that it is difficult to devise interventions that will be of substantial clinical benefit. in the case of asthmatics where acute exacerbations are triggered by virus infection the clinical consequences are more severe with increased asthma symptoms, medication use, emergency presentations and hospital admissions (johnston et al., ) . wark et al., have shown that virus-induced acute asthma worsens airway inflammation and the degree of this inflammatory response is directly related to the severity of acute clinical symptoms (wark et al., ) . therefore, effective therapeutic interventions would need to alter the natural history of virus-induced asthma, prevent the worsening of airway inflammation and lead to significant clinical improvement. potential therapeutic strategies to this problem have been discussed but the approaches with the most promise include: prevention of infection from occurring; anti-viral agents that eliminate infection while reducing the host's immune response; treatments that specifically target enhanced inflammatory responses that are induced during virus-induced asthma exacerbations and supplementation of protective anti-viral responses. anti-oxidants and angiogenesis inhibitors may also have beneficial effects (fig. ) . research into the inhibition of rsv and rv infection may have important implications for the prevention of lrt infections and the development and exacerbations of asthma. there are no human vaccines available and further research is required to develop effective anti-viral preventative strategies, specifically anti-viral mabs, passive immunization and vaccines. although the evidence that anti-viral regimes reduce the risk of developing rad later in life is scarce, the use of antivirals to delay rsv infection in particular to later in life may reduce subsequent virus-related illnesses including asthma (culley et al., ) . genetic factors are fixed but investigations of such interventions may be developed to reduce asthma incidence have become areas of intense interest. mabs have demonstrated efficacy in preventing rsv infection and inflammation in animal models and a humanised mab (palivizumab) against the rsv f protein is effective against rsv and wheezing in children and reduces hospitalization in high-risk individuals. studies in mice show that palivizumab reduces viral load, acute disease and long-term lung function abnormalities . in the rat model palivizumab inhibits the invasion of virus particles into the lrt epithelium and the upregulation of the nk receptor. this prevents acute neurogenic lrt inflammation and longterm susceptibility to inflammation that is induced by infection (piedimonte et al., ; piedimonte, ) . a randomised controlled trial with palivizumab in children achieved a % reduction in rsv hospitalization. reductions were observed in children with ( %) and without ( %) chronic lung disease (impact study, ) . a recent large prospective casecontrolled study of at risk pre-term infants showed that palivizumab also protected against recurrent wheezing ( % vs % controls) and physician-diagnosed recurrent wheezing ( % vs % controls) independently of confounding variables (simoes et al., ) . one drawback is that escape mutants have been detected both in vitro and in vivo (zhao et al., ; zhao & sullender, ) , indicating that preventative strategies should be targeted at multiple epitopes, because palivizumab is effective in preventing hospitalization in high-risk groups, optimisation of its pharmacokinetic profile, extension of its half-life and increasing its neutralising abilities would produce an even more efficacious preventative strategy for rsv-associated disease. motavizumab is a derivative of palivizumab and is now in clinical trials. this mab has a fold increase in neutralising abilities in vitro and its use reduces viral titres by fold and inhibits viral replication in the rt compared to palivizumab in rats (wu et al., ) . further studies are required to establish the application of such mabs for the prevention of asthma. other mabs have also been used in mice as treatments of features of rsv-induced disease. rsv infection of mice leads to eosinophil and neutrophil influx into the lung and ahr, which are inhibited by anti-il- (schwarze et al., ) . administration of fi-rsv and anti-il- leads to a decrease in disease features and il- production following challenge, but ifn-γ release is increased (tang & graham, ) . t /st mab treatment reduced th but not th pathology and may also be a good treatment (walzl et al., ) . all of these mabs require further assessment. currently the only option for preventative treatment of rsv is passive immunization, which has been in use for many years. a randomised placebo controlled trial investigated the applicability of rsv immunoglobulin to prevent rsv-associated hospitalization (prevent study, ) . treatment reduced hospitalizations for lrt infections but required monthly administration of high levels of fluids and proteins. in another small study, children with chronic lung disease who were treated with immunoglobulin - years previously had improved lung function, reduced atopy and rad events (wenzel et al., ) . this indicates that prophylaxis may be able to prevent rad in children. studies in rats also show that passive transfer of antibodies against rsv induces protection in the lrt but not the urt and that serum titres of ≥ : are protective whereas titres of ≤ : are not (prince et al., ) . infants who are the most susceptible to virus infections are protected by maternal anti-viral antibodies (largely igg ) that are passively transferred, however these antibodies inhibit the induction of protective responses against primary infection or vaccination. as a result primary or recurrent infections do not induce protective immunity until later in life (henderson et al., ; glezen et al., ) . natural protection against viral disease results from a combination of antibody, cell-mediated and t-helper cell immunity. this combination of responses target the virus before infection of host cells as well as destroying cells that have already been infected with virus. therefore, potentially an ideal vaccine would need to stimulate humoral, cellular and t-helper responses similar to those induced by natural infection. vaccine development has been hampered by the requirement for early-life vaccination, the confounding effects of poor neonatal immune responses, the presence of maternal antibodies, the difficulties in balancing immunogenicity and attenuation and the risk of vaccineinduced disease. mucosal immunization may overcome the immune suppressive effects of maternal antibodies. . . . . fi-vaccines. the widely recognised need for an effective rsv vaccine has also been held back by the adverse events following the implementation of the fi-rsv vaccine. this vaccine was developed in the s but not only was it poorly effective it infamously induced more severe rsv-related disease upon natural infection in clinical trials (kapikian et al., ; kim et al., ) . the vaccine induced high serum antibody titres that were of low neutralising activity, lymphocytes with an enhanced rsv-specific proliferative response and blood eosinophilia . these observations were repeated in mice given the same vaccine or the g protein of rsv. enhanced disease involved eosinophilia of the lung (srikiatkhachorn & braciale, b; tripp et al., ) and aberrant cd + t cells releasing th cytokines (il- , - , - and - ) (waris et al., ; srikiatkhachorn & braciale, b; tripp et al., ) . reduced il- and cd + responses were also detected (waris et al., ; aung et al., ) . fibovine rsv has also been tested in calves and also induces the development of immunopathology, ineffective neutralising antibodies and induced inflammatory responses leading to eosinophil influx into the lung and enhanced ige production upon subsequent infection (gershwin et al., ; antonis et al., ; kalina & gershwin, ) . as is the case with rsv in mice the fi-bovine rsv did not induce long-term t cell memory . thus it has been proposed that vaccines did not possess sufficient neutralising antibody or ctl responses and on subsequent infection the virus was not cleared but induced a potent th response that exacerbated disease. . . . . subunit vaccines. subunit vaccines are not associated with enhancing disease and may have potential applicability. however, the variability of human viruses is an issue that must be taken into account in the development of subunit vaccines. for rsv, vaccination with the f protein confers cross-protective immunity whereas only group-specific immunity is developed when the g protein is used (sullender et al., ; simard et al., ) . intranasal followed by parenteral immunization with the rsv f protein protects against both upper and lower rt infection in mice but may induce pulmonary pathology (murphy et al., ; connors et al., ; walsh, ) . a chimeric parainfluenza viral vaccine expressing the f protein has also been developed and is protective against infectious challenge in primates (tang et al., ) . in a variety of different human studies f protein vaccines have now been shown to be immunogenic and safe in children with or without chronic respiratory disease and pregnant women and reduce the prevalence of infections but not lrt infections (groothuis et al., ; munoz et al., ; piedra et al., ; simoes & carbonell-estrany, ; meyer et al., ) . largescale trials are now required to confirm efficacy. protein g-based subunit vaccines have also been developed but may initially induce detrimental th responses. immune responses involving il- and il- activity following immunization with vaccinia virus expressing rsv-protein g or fi-rsv must be attenuated to modulate protein g-specific responses resulting in severe rsv-induced disease. however, inhibition of il- or - alone has minimal impact on disease (johnson et al., ) . the co-administration of cytokines or th -inducing adjuvants may abolish initial th responses (neuzil et al., ) . other novel subunit vaccines in development include the n protein, other chimeric f and g fusion proteins, synthetic peptides, recombinant proteins, recombinant vaccinia and parainfluenza viruses expressing other viral components and dna vaccines (reviewed in meyer et al. ( ) ). subunit vaccines require co-administration with adjuvants to enhance immunogenicity and induce the most desirable immune response. currently quillaja saponi or its component fractions or cpg oligodeoxynucleotides administered with whole or subunit vaccines induce potent neutralising antibodies and desirable immune responses in mice and reduce disease and infection in calves. this occurs even in the presence of maternal antibodies and particularly when presented as immunostimulating complexes (meyer et al., ) . however the applicability of these approaches to humans remains unknown. . . . . live attenuated vaccines. natural infection does not predispose to enhanced disease upon subsequent infection. therefore, experimental live attenuated viruses are being developed for rsv (karron et al., ) for intranasal administration during the first days of life but the immunology underlying their activity is poorly understood. intranasal delivery would induce systemic and local responses and has the potential to promote protective responses in both the upper and lower airways. infants ( - months) have limited immune responses to viral glycoproteins and only highly attenuated virus strains with minimal adverse side-effects can be used in this age group which are particularly susceptible to vaccine-related illnesses. these agents do however induce protective immunity even in the presence of passively acquired antibodies in mice and chimpanzees, which is dependent on cd + and cd + responses (crowe et al., ) . several cold passaged, temperature sensitive attenuated viruses have been developed that protect against challenge in chimpanzees and induce protective local and systemic immunity in children (crowe et al., ; karron et al., ) . these viruses have since been genetically engineered so that they are sufficiently attenuated for use in infants, although their efficacy against infection has not yet been determined (karron et al., ) . genetic engineering is also currently in use to delete non-essential viral genes to produce other live attenuated vaccines or to insert genes to enhance immune responses, for example the insertion of gm-csf to increase the production of pdcs and macrophages (reviewed in mayer et al. ( ) ). the assessment of attenuated viruses that induce potent protective immune responses with negligible side-effects requires extensive time consuming and expensive clinical trials. novel prime-boost vaccination strategies with live attenuated and subunit vaccines could be used in combination to further enhance protective immunity. . . . . bovine vaccines. the same problems that apply to human rsv vaccines may also occur in the development of vaccines for calves in the farming industry, but the level of risk of trials is more acceptable. vaccine development is also less complex as bovine rsv is less variable than human rsv and there is only major antigenic group (prozzi et al., ) . this has led to the commercialization of several vaccines, which are protective against infection in calves and their development may have applicability to human vaccines. further, cattle may be used as the animal model in preclinical human vaccine studies. however, different bovine models have been used with different concentrations of viral inocula and routes of inoculation with passaged virus that does not induce severe disease. thus results are often not comparable or significant between experimental and control groups (mayer et al., ) . inactivated bovine rsv vaccines have been available for many years and reduce the prevalence and severity of subsequent infection without enhancing disease (west et al., ) . nevertheless, the longevity of induced protection and efficacy in the background of maternal antibodies could be improved. these vaccines are co-administered with cd + and th stimulating adjuvants in order to dampen th responses that may be induced by the vaccine and lead to protective th responses (ellis et al., ) . live attenuated bovine rsv vaccines have been developed by passaging virus in cell culture and have recently been commercialized (vangeel et al., ) . unlike fi-vaccines, live attenuated vaccines induce a primed memory t cell response and reduce pulmonary inflammation upon subsequent infection (miao et al., ) . they are administered parenterally, have equivalent efficacy as inactivated vaccines and induce protective immunity in calves ellis et al., ) . these vaccines are now used in quadrivalent therapies to protect against a number of bovine viruses (salt et al., ) . although effective in cattle inactivated or attenuated bovine rsv are not suitable as vaccines for human rsv as they do not induce protection in chimpanzees . thus there are currently no safe and effective human vaccines for rsvor rvand it is yet to be determined if delay or prevention of infection with neutralising antibodies or vaccination can reduce wheezing, long-term lung function abnormalities and asthma. nevertheless there are many promising approaches and candidates are being used to develop such vaccines, some of which are in human clinical trials to prevent infection, which if successful may be used in attempts to inhibit the development of asthma. the balance of th /th immunity to infection determines the severity of disease where th responses mediate clearance and recovery but th responses induce eosinophilia and more severe symptoms. strategies to augment th responses during vaccination in early life may be useful in preventing the development of virus-induced asthma. recent efforts have concentrated on the development of the combination of subunit vaccines with th inducing adjuvants and on the development of live attenuated vaccines. the most promising approaches for developing vaccines in young infants are the use of live attenuated vaccines and recombinant viruses expressing components of target viruses. there are no effective treatments for rsv/rv-induced disease and currently the optimal therapy for bronchiolitis involves intensive fluid and nutritional support, while the immune response of the subject clears the infection (jhawar, ) . in order to maximize the chances of successful treatment an anti-viral agent would need to be taken early during the course of infection in order to modulate the development of the host's immune response. it would have to be highly effective at controlling infection and the virus would need to have no or at least a limited ability to develop resistance. finally the agent must be acceptable to patients in terms of ease of administration, have a reasonable medication frequency and limited side-effects. currently the agents of most interest are those that inhibit viral attachment, including those that bind to viral capsids, and inhibitors of viral proteases, which are required for viral replication. to cause infection all respiratory viruses need to enter the host's becs and replicate. ninety percent of rvs use icam- as a receptor to enter cells. a soluble icam- molecule (tremacamra) has been developed and tested as a competitive binding inhibitor in randomised controlled trials of experimental rv (rv ) colds, used as either a dry powder or nasal spray (turner et al., ) . treatment results in small reductions in symptoms, virus replication and the development of clinical colds. there were no serious adverse events, but medication had to be taken six times a day and was only effective if used within h of infection. it has not been used as a therapy for acute asthma. the rv outer capsid consists of viral proteins (vp - ) (rossmann et al., ) . several agents have been devised that bind to vp and prevent virus attachment to the host cell. the first agents that were produced, the oxazolinyl isoaxoles, have small clinical effects but unacceptable sideeffects . further developments led to pleconaril, an agent with broad spectrum activity against rv as an oral preparation to be taken twice daily (hayden et al., ) . pleconaril has been assessed in two phase ii clinical trials to determine its efficacy against natural colds (hayden et al., ) . subjects began treatment - . days after cold symptoms commenced. those taking pleconaril had reduced cold symptoms and duration of colds ( . - . days) with few side-effects. an application in the us for fda approval for use in clinical colds was unsuccessful as it was considered that there was a lack of substantial clinical benefit and concerns about the development of resistant rv mutants. at this stage there have been no clinical trials specifically assessing the efficacy of pleconaril in acute asthma. rv relies upon proteases to cleave the viral polyprotein for replication to occur. rupintrivir is a c protease inhibitor that targets this process (dragovich et al., ) and has high-level anti-viral activity against all rvs (dragovich et al., ; binford et al., ) . unfortunately in a natural infection study ruprintrivir failed to improve symptoms or reduce viral load and clinical development was ceased (patick et al., ) . anti-inflammatory treatments have therapeutic potential in virus-associated diseases including asthma by moderating inflammatory responses in response to infection. long-term treatment of asthma with icss controls airway inflammation (juniper et al., ) , improves lung function and symptoms and reduces the risk of acute exacerbations (adams et al., ) . while icss are effective at controlling chronic asthma they do not completely prevent acute exacerbations even in those who achieve good control and are compliant with therapy, with the majority of episodes triggered by viral infection (reddel et al., ) . in an attempt to prevent the recurrence of virusinduced wheeze, infants were treated intermittently with ics or placebo when these symptoms occurred (bisgaard et al., ) . treatment had no clinical effect on acute episodes of wheeze and did not influence whether children went on to develop persistent wheeze (bisgaard et al., ) . another study showed that treatment of acute asthma by increasing ics dose also did not prevent worsening of disease (fitzgerald et al., ) . the incomplete effect of ics in controlling virus-induced asthma was demonstrated best in adult asthmatics using experimental rv infection (grunberg et al., ) . treatment reduced ahr and airway inflammation prior to infection but had no effect on the development of inflammatory changes that were associated with infection (grunberg et al., ) . in a systematic review glucocorticoids were suggested to have little effect in rsv disease (black, ) and they also enhance viral replication and mortality in pvm infection (rosenberg et al., ) . oral corticosteroids have been used to treat acute viral bronchiolitis in children. however, meta-analysis of seven randomised controlled trials found that there was no effect on length of stay or clinical symptoms and complications (patel et al., ) . in asthmatics with persistent symptoms despite ics use, treatment with combination therapy with icss and labas effectively controls chronic symptoms and reduces the frequency of exacerbations (walters et al., ) . it has recently been shown that early treatment of symptoms of worsening asthma with ics/ laba, where the laba has a rapid onset of action and is used to relieve symptoms, does prevent deterioration leading to severe exacerbations (o'byrne et al., ) . while these studies did not identify the cause of the acute triggers it is likely that the majority of these were related to virus infection and the combination of ics/labas appear more effective than icss alone in preventing exacerbations. in vitro studies demonstrate that treatment of airway epithelial cells with ics/labas has synergistic and additive effects in reducing the release of inflammatory mediators . these observations may explain the efficacy of combination therapy and indicate an important role for their use. this may be particularly relevant to children where their value in controlling chronic asthma is not well defined but where ahr is known to persist following viral infection (xepapadaki et al., ) . it remains unknown what effect ics/laba treatments have on specifically preventing or treating virus-induced asthma. it has recently been recognised that macrolide antibiotics in addition to their anti-microbial action have important immune modulating effects and reduce the release of inflammatory mediators from airway epithelial cells (takizawa et al., ) . epithelial cells infected with rv and treated with erythromycin, showed reduced expression of icam- , il- and - (jang et al., ) . initial clinical studies were, however, not promising; using experimental rv infection, treatment of healthy subjects with clarithromycin had little or no effect on the development of cold symptoms or nasal inflammation (abisheganaden et al., ) . however, unlike colds where the host inflammatory response in the airways is relatively mild, in conditions characterised by more intense neutrophilic inflammation, such as cystic fibrosis, macrolides appear to be effective (ferrara et al., ) , which has encouraged their use in asthma. in a small randomised controlled trial of infants with rsv bronchiolitis treatment with clarithromycin reduced systemic inflammation acutely and led to few wheezing episodes in the following months (tahan et al., ) . in a large multicentre study of adults ( ) with asthma, subjects were randomised to receive the ketolide, telithromycin ( mg/d) or placebo (johnston, ) . those treated with telithromycin had a greater reduction in asthma symptom scores, but there was no difference between the groups in terms of improvement in pef rate. the trial did not document the presence of virus infection but the results suggest the effect is due to an ability to reduce inflammation in acute asthma. further studies are required to determine if there is a specific effect on virus-induced acute asthma. type i ifns are known to play crucial roles in defence against viruses by inducing anti-viral proteins such as pkr and rnase l in infected cells, apoptosis preventing virus replication and adaptive immune responses to infection (malmgaard, ) . the recent observations by wark et al., that asthmatic becs are more susceptible to infection with rv, which involves a deficient ifn-β response, suggests that this may be an important therapeutic target to limit the affects of virus-associated acute asthma (wark et al., ) . several trials have also assessed the role of ifn-α treatment ( . × - . × iu/d intranasally) in experimental rv colds in healthy volunteers (scott et al., ; hayden & gwaltney, ) . these studies consistently showed a dose dependent improvement in symptom scores and reduced virus shedding, but treatment was associated with nasal bleeding and a lymphocytic nasal infiltrate. a similar response was observed with recombinant ifn-β (sperber et al., ) . when used to treat natural colds ifn-α reduced the frequency of colds but had at best modest effects on symptoms (monto et al., ) , while serine ifn-β failed to reduce colds or improve symptoms. these modest improvements provide a proof of concept but the clinical benefit is difficult to justify in healthy individuals with mild colds. however, as is the case with the use of macrolides in asthma where the clinical effects of colds are much greater (corne et al., ) , efficacy may be more evident. a single case series exists for the use of ifn-α ( × iu/d) to treat stable but corticosteroid resistant asthmatics (simon et al., ) . treatment resulted in improved lung function and allowed a reduction in oral corticosteroid use. however, side-effects were also common, in the first weeks all subjects experienced a 'flu-like' illness, had nausea and developed headaches. by - months side-effects were less though subject developed leukopenia severe enough to necessitate temporarily ceasing treatment. the benefits of type i ifns now need to be assessed in the context of treatment of virusassociated acute asthma. important issues will need to be resolved including the optimum delivery of medication, whether administration should be to the nose or the airway, how soon treatment needs to commence following the development of cold symptoms and how this may influence airway inflammation and ahr in acute asthma. oxidative stress may plan an important role in asthma but it is unknown whether it is important in virus-induced disease. the anti-oxidant butylated hydroxyanisole has been used in a mouse model of rsv lung infection to reduce rsv-induced oxidative stress, disease symptoms, weight loss and ahr. the effects were mediated by suppression of neutrophil infiltration and cytokine and chemokine release in the lung (castro et al., ) . thus anti-oxidants may prevent rsv-induced inflammation and have long-term beneficial effects, which may ameliorate the consequences of infection in asthma. the use of vegf and angiogenesis inhibitors may have the potential to elucidate the specific roles of these factors in asthma and identify their potential as therapeutic targets. no efficacious vaccines yet exist for the prevention of rsv or rv infections in humans. effective bovine rsv vaccines are available and the formulation of these vaccines may be applicable to the development of human vaccines. combination therapy with icss and labas effectively controls persistent symptoms and numbers of exacerbations. this is the current best treatment option but does not treat the cause of the disease and cannot be used to prevent the development of disease in the first instance. a variety of novel options for the prevention of virus-induced asthma need to be fully assessed for their efficacy and applicability. rsv and rv infection of respiratory epithelium may play an important role in the development and exacerbation of asthma, however the pivotal mechanisms underpinning these relationships remain unresolved. infection is associated with persistent wheeze and decreases in lower lung function and worsens airway inflammation and airflow obstruction acutely in asthma, with other factors contributing to severity. asthmatics are more predisposed to th responses, may be more susceptible to infection and experience more severe lrt symptoms upon infection. the development of more severe disease may identify those people more at risk of developing wheeze and asthma. recurring infections may lead to a cycle of inflammation and repair that worsens airway remodelling. rsv infections are strongly linked to both development and exacerbation of asthma. early-life rsv infections, particularly those that induce severe disease, induce recurrent wheeze and bronchial obstruction and predispose to rad and potentially asthma that persists into later life. it is possible that persistent infection affects the developing lung and immune system and predisposes to recurring rsv infections, ahr, reduced lung function and respiratory symptoms. another alternative is that children with reduced lung function, genetic susceptibility or aberrant immune responses may be at increased risk of infection and that rsv disease is a marker of susceptibility to increased respiratory symptoms. both of these processes may occur under different circumstances. the association with rsv is inconclusive for allergic sensitization, which may involve igemediated allergy and randomised intervention studies are required to confirm this link. the mechanisms involved in rsv-induced asthma include; age of first infection, the timing of infection relative to allergic sensitization, the nature of innate and adaptive immune responses induced upon infection, latent infections and the potential involvement of pathogenetic processes such as angiogenesis and neurogenic inflammation. a combination of human studies and mouse models has been widely used to elucidate the mechanisms of how rsv infection are linked with asthma and novel models of pvm infection may be particularly useful in the future. rvs are widely recognised as the commonest cause of clinically significant rt infections and are strongly implicated in the exacerbation of asthma and more recently in the induction of asthma. the mechanisms of these associations have been investigated in human studies both in vivo and in vitro and have been shown to involve deficient immune responses, which may be different to those associated with rsv, that may be influenced by atopy and reduced lung function, ahr and remodelling. nevertheless more work is needed to elucidate role of rv in lrt diseases. it remains unknown if rv induces the development of wheeze and asthma or if asthmatics are more susceptible to rv infection. recently a novel mouse model has been described which may be used to substantially contribute to our understanding of rv and rv-associated asthma pathogenesis. despite the scope of the problem caused by respiratory viruses in acute asthma no specific vaccines or treatments exist that have been shown to modify the clinical outcome of rsv or rv infection. the optimal approach would be to develop safe and effective prevention strategies, such as efficacious vaccines, which induce immune responses that prevent viral infection. in the absence of a vaccine the different processes involved in the generation of virus-associated asthma and exacerbations could be targeted by specific treatments for individual patients. several therapeutic options are available and more are emerging. studies should focus on how established treatments such as with ics/laba may modify virus-associated acute asthma and assess novel anti-inflammatory strategies including macrolides or the use of ifns either alone or in combination to influence the course of disease. the benefits of these strategies now need to be assessed in the context of treatment of virusassociated acute asthma. important issues will need to be resolved including the optimum delivery of medication, including whether this should be intranasally or directly to the airway. it will be necessary to determine how soon treatment needs to commence following the development of cold symptoms and how this may influence airway inflammation and ahr in acute asthma. in summary, 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effects on cytokine and icam- production exposure of neonates to respiratory syncytial virus is critical in determining subsequent airway response in adults respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology in vivo selection of respiratory syncytial viruses resistant to palivizumab altered eosinophil levels as a result of viral infection in asthma exacerbation in childhood respiratory syncytial virus escape mutant derived in vitro resists palivizumab prophylaxis in cotton rats virokinin, a bioactive peptide of the tachykinin family, is released from the fusion protein of bovine respiratory syncytial virus bone marrow plasmacytoid dendritic cells can differentiate into myeloid dendritic cells upon virus infection key: cord- -b x fn authors: billiau, a. title: the interferon system as a basis for antiviral therapy or prophylaxis date: - - journal: antiviral res doi: . /s - ( ) - sha: doc_id: cord_uid: b x fn nan the interferon system is part of the so-called aspecific defense mechanism of vertebrates against virus infections. the term "aspecific" mainly refers to the fact that the system is operative against all taxonomic classes of viruses. it consists of a set of evolutionary related proteins, interferons, which possess the ability to tune the synthetic biochemistry of the cell so that various processes which are essential for virus replication are impaired. under "normal" conditions the organism synthesizes or releases little if any of the interferons. only under attack from viruses or other forms of biological stress is the interferon system triggered. hence, the two sets of fundamental questions which have governed interferon research : (i) the physiology and biochemistry of induction ( , ) and (ii) the physiology and biochemistry of action ( ) . possessing satisfactory answers to these questions is essential, not only to understand the natural role of the interferon system in virus disease but also to assess possible applications in medicine, in particular the use of interferons or interferoninducing substances as antiviral drugs. a striking feature brought to light by virtually each sector of interferon research is the pleomorphism of the system. thus, each animal species possesses a family of genes for interferon, and in some instances a single gene can give rise to multiple molecular forms. also there are several ways in which production of these interferons can be induced. finally, the antiviral effect is not due to a single cellular modification, blocking the virus at one particular junction in its replicative cycle. rather, virus replication is counteracted at different steps. another feature worth mentioning is that the interferon system is leaky blocking of virus replication or of the effects of viruses on the host is neither complete nor permanent. therefore, if the virus host is placed under the protective influence of interferon, some virus replication still occurs, and as the interferon effect wanes, residual virus can again multiply at full strength unless this would be blocked by some other mechanism (e.g. specific immunity) which has been generated in the. meantime. currently, interferons are defined as endogenous proteins which exert virusnonspecific, antiviral activity at least in homologous cells through cellular metabolic processes involving synthesis of both rna and protein ( ) . one of the main points in the definition is that it excludes all natural proteinaceous factors which inhibit virus infection at extracellular steps, e.g. adsorption. a limitation of this definition is that it would also call "interferon" any protein produced by cells which induces production of a known interferon. for instance, a monokine related to interleukin-i, has been shown to induce production of interferon-b by cells and thereby to exert an antiviral effect. in mammalians three (sero)types of interferon have been described : ifn-a, ifn-b and ifn-y. subtypes, reflecting the presence of multiple, slightly different genes, may occur. thus, in man there are several subtypes of ifn-a (huifn-al, -a , -a , •.. ), but only one ifn-b and -yo types of interferon (a, band y) are distinguished on the basis of reactivity with polyclonal antisera. antiserum against a given type neutralizes biological activity of all its subtypes but does not react with the other types. antigenic differences between the types reflect differences in primary structures. however, some homology in sequence exists between ifn-a and ifn-b. on the other hand, the eequence of ifn-y is completely different from that of either ifn-a or -b. the structural similarities and differences between the ifn types are reflected by the fact that ifn-a and -b, in acting on cells, share a common membrane receptor, while ifn-y uses a different receptor ( ) . interferons for animal experiments or for clinical trials are prepared in various ways. at present it is common and useful to make a distinction between preparations of so-called natural interferons and preparations of recombinant dna-derived interferon (rdd-interferons). among the natural interferon preparations, those most commonly used are as follows. interferon prepared from mouse cell lines infected with newcastle disease virus is a mixture of a and b ( ) . the two types can be separated but this has seldomly if ever been done for preparations destined to perform in vivo experiments. thus, literature data are representative for a mixture of ifn-a and -b. interferon prepared from suspensions of mouse splenocytes incubated in the presence of lymphocyte mitogens (concanavalin a, phytohemagglutinine, staphylococcus enterotoxin, ..• ) contain ifn-y. it should be mentioned that, depending on the degree of purity, these preparations also contain variable numbers and quantities of monokines and lymphokines such as interleukins, colony stimulating factors, etc. hence, all data obtained so far with these preparations represent effects of total lymphokine rather than of ifn-y preparations. the following interferons are currently available for clinical studies in immune interferon is produced from suspensions of blood leukocytes induced with mitogens. the antiviral activity present in these preparations is mainly due to ifn-y. small quantities of ifn-~ and -s may be present. however, the main contaminants are several lymphokines, some of which may indirectly affect viral disease, for instance by modulating the immune system. the prototype of rod-interferon is human ifn-a prepared from e.coli or yeasts containing an adequate expression plasmid into which the interferon genes have been inserted ( ) . in their crude form these preparations do not contain any products of human origin other than the interferons . bacterial products are completely removed by the purification process. the main difference between these products and their natural-source-derived counterparts is that they contain only a single subtype, namely a ' while it is known that different subtypes of huifn-~ may differ in their antiviral potency depending on the cells on which they are tested, it is not yet known whether this has any repercussion for the clinical effects. another currently available rod-interferon is huifn-~ produced in e.coli. this interferon differs from the natural counterpart by the absence of carbohydrate side-chains. although these side-chains do not playa significant role in the effects of the ifn-s on cells, the pharmacokinetic behavior of this rdd-ifn-s was found to be rather different from that of natural fibroblast-derived interferon ( ) . rdd-huifn-s possessing carbohydrate side-chains can be obtained by insertion and expression of the gene in yeasts or mammalian cells. finally, a rdd-huifn-y is available, which is produced from e.coli or mammlian expression systems. rdd-interferons for experiments in animals are becoming available at a much slower pace and in lesser quantities than those necessary for experiments in man. the genes for several animal interferons have been isolated and brought to expression in e.coli, in yeasts or in mammalian cells. this should allow to produce large quantities without great technical difficulty. especially in the case of muifn-y, this will be a great advantage over the cumbersome "natural" production method which depends on the availability of fresh mouse splenocytes. pharmacokinetic studies have been done with human interferons-a and -so in as far as they were done in man, they are of course quite relevant to the design of therapeutic trials. studies with these interferons in animals are of lesser value, especially as it has become clear that comparative studies between interferon-a and -s may yield quite different conclusions depending on the animal species used ( ). one of the key questions is to know whether interferon given by any route is concentrated in some organ. this question has so far not been studied in man although, with current techniques of production and radioactive scanning, it seems quite approachable. that the question is not trivial may be apparent from the observation that human interferon-a and -s, when injected by the intramuscular route, cause comparable increases in nk-cell activity, although blood levels with interferon-a are -to -fold higher ( ) . similarly, studies in mice have revealed that human interferon-a and -s given intraperitoneally, yield different blood levels but quite similar tissue levels (ii). there is evidence that some organs, in particular the brain, are rather difficult to penetrate by interferons. thus, during the initial phases of experimental mengo virus infection in mice, interferon levels in serum being quite high, no interferon was detectable in the brain ( ) . brain interferon became detectable only in later stages of the infection when full-blown virus replication had started in the brain itself. huifn-s on the other hand undergoes rapid uptake, suggesting that desialylation, wherever it may take place, is a first step toward degradation of huifn-s. the kidney was shown to playa key role in that interferons seem to pass easily through the glomerular sieve and to be taken up and degraded by tubular cells. of particular importance are pharmacokinetic studies with rdd-interferons. since these interferons often differ from the natural ones . by , the absence of carbohydrate side-chains it may be expected that their pharmacokinetics will be quantitatively different from those of their natural counterparts. it is not unthinkable that some of the artificial interferons . (e.g. those obtained by sitespecific mutagenesis) may be degraded slowly by the organism, allowing to decrease the doses. however, there is nothing that would lead one to think that site-specific mutagenesis . may enable one to target an interferon to a specific , the main lesson to be learned from these experiments is that interferon therapy is unlikely to achieve protective effects unless started before the major viral replication burst in the organism. in general one can say that this means that therapy has to be started before the first symptoms. as already mentioned, mouse model systems for chronic virus infections are scarce. one example is experimental viral leukemia. it has been known for a long time that continuous interferon administration started before or shortly after infection can significantly delay the development of the disease and can also prolong survival time ( ) . this finding may be expected to receive renewed in- ( , , ) . the question whether interferon therapy may be able to control chronic or recurrent disease at the level of the whole body, is confounded by the fact that much of the symptoms of these chronic virus diseases are not directly due to viral replication or cytopathogenicity but result from immunological and inflammatory reactions. therefore, it is not clear whether alleviation of symptoms by interferon is due to antiviral activity or to interference with secondary effects. thus, in the aforementioned example of friend leukemia in mice, it is not clear whether delay in splenomegaly development is due to reduction in viral load or to antimitotic or immunoregulatory effects of interferon. recurrent herpetic keratitis ( ) can benefit from topical interferon therapy if it is given in conjunction with debridement and/or with some of the older or newer antiherpetics ( ) . acute rhinopharyngitis due to rhinovirus or coronavirus infections can favorably be influenced if rather high doses of huifn-a (but not -s) ( ) are given and if treatment is started early, preferentially before infection. it has been envisaged to use daily instillations of interferon during autumn and winter periods as a means of prophylaxis against common cold. however, it is now being suggested that such long-term instillations cause damage to the nasal mucosa. condyloma accuminatum is amenable to treatment with ointments or gel lies containing interferon ( ) . a problem in interpreting these results is the possible confounding, especially in non-placebo-controlled trials, of the mere effects of hygiene. daily care of the lesions may by itself contribute to regression of the condylomas. however, the interpretation that the effects are largely due, indeed, to the action of the interferon is corroborated by the observation that other manifestations of hpv infections are also amenable to treatment with interferon. thus, skin warts can be made to disappear by repeated injections of fibroblast interferon in the perilesional skin ( ) . beginning or established acute (primary or recurrent) infections. systemic administration of interferons (a-or -type) has been found to favorably affect the course of a number of acute (prima ry or recurrent) virus infections : varicella in imrnunocompromised patients ( ) , zoster in cancer patients ( ) . the practical importance of these observations is rather minor, in that beginning or established hsv and vzv infections can now effectively be treated with nucleoside analogs. prevention of acute infections. systemic treatment with interferon can prevent herpes recurrence after surgical interventions on the trigeminal nerve ganglion (operation for "tic douloureux" ) ( ) . interferon therapy has a l so been considered as a possible means to cope with the increased incidence and severity of viral infections in renal transplant patients ( ) ; it appears that interferon therapy reduces virus-shedding as well as the severity of symptoms of cmv infections. chronic active virus infections . the following chronic active virus infections are currently investigated as pos s ible targets for systemic interferon therapy : persistent infections with hb virus, chronic infections with hpv (warts and wartlike diseases), chronic infection with leukemia viruses, e.g. htlv. the effects of interferon therapy on persistence of hb virus has rather extensively been investigated. in some patients transient and sometimes definitive disappearance of viral infectivity markers, accompanied by clinical improvements, were seen. it is difficult, however, to certify tha t these were not spontaneous cures or placeboeffects ( ) . the need for properly controlled trials, expressed early on by several investigators, has at this time still not been satisfied. therefore it remains impossible to even approximately assess the therapeutic potential of interferon in chronic hepatitis. the situation with chronic hpv infections is quite different. thus it is now generally recognized that juvenile laryngeal papilloma, a rather rare but serious complication of infection with certain hpv types, is quite amenable to control by therapy with various preparations of huifn-a ( ) . the therapy has to be given continuously for months before recurrence of the laryngea l warts is brought under control. arrest of treatment entails recurrence, but it is hoped that suitable low-dose maintenance treatments can be designed, by which protections can be prolonged to the age where the papillomas tend to regress spontaneously. retroviruses have only recently been implicated as etiologic fa c tors in human diseases, namely human t-cell leukemia (htlv-i and -ii) and aids (lav or htlv-iii). interferon therapy has already been investigated as a means to control aids and aids-associated malignancies (kaposi sarcoma), and some encouraging results have been reported in that the symptoms of the disease can in some patients be alleviated ( ) . a cure of aids however, has not been seen. mechanisms of production and action mechanisms of production and action proceedings of symposium on clinical use of interferon key: cord- -rhi hi m authors: wilkes, rebecca p.; hartmann, katrin title: update on antiviral therapies date: - - journal: august's consultations in feline internal medicine, volume doi: . /b - - - - . - sha: doc_id: cord_uid: rhi hi m nan update on antiviral therapies rebecca p. wilkes antiviral chemotherapy use is still relatively uncommon in veterinary medicine. controlled studies evaluating the efficacy of antiviral drugs in cats are lacking, or, if studies have been done, in many cases, the data are insufficient to determine effective dosing for these drugs. with the exception of the recombinant feline interferon (rfeifn)-omega, so far no antiviral drugs are specifically licensed for veterinary medicine, which leaves the veterinary community with the option to use off-label antivirals made for humans to combat viral diseases in feline patients. the goal of research in antiviral chemotherapy is the discovery of antiviral agents that are specific for the inhibition of viral multiplication without affecting normal cell division; however, because viruses are dependent on host cell machinery for replication, drug targets are often nonspecific. this makes antivirals inherently more toxic than antimicrobials are because the antiviral drugs are damaging to not only the virus but also the host cells as well. in addition, agents considered safe for human use are not always safe when administered to cats. antivirals made for systemic use often require host and/ or viral metabolism to be active. therefore, agents designed for use in humans are neither reliably nor predictably metabolized by cats or their viruses. thus antiviral agents should always be tested first in vitro for efficacy and safety, and then followed by pharmacokinetic studies in cats. systemic antivirals often have a relatively narrow safety margin, and special considerations should always be given to patients with reduced hepatic or renal function. well-designed blinded, placebo-controlled studies in client-owned animals should follow studies in laboratory-bred, experimentally infected cats to confirm results in genetically diverse cats. most of the human antivirals are specifically intended for treatment of human immunodeficiency virus (hiv) or human herpesvirus infections. therefore, feline immunodeficiency virus (fiv) and feline herpesvirus type (fhv- ) infections have been the most important indications for antiviral chemotherapy in veterinary medicine. topical antiviral therapy has been mainly used for herpetic ocular disease, but studies have evaluated a systemic antiviral compound (famciclovir) for treatment of multiple clinical syndromes associated with fhv- infections. even though combination antiviral therapy has been successful in slowing disease progression in people with hiv, similar therapy has not been thoroughly evaluated in cats. recent studies have focused on combination therapy and evaluation of additional hiv drugs that have not been previously evaluated in feline cells. it is hoped that expanding the number of drugs that are shown to be effective for fiv will lead to effective combination therapy for feline patients. some additional feline infections that have been the focus of current antiviral studies are feline leukemia virus (felv) infection and feline infectious peritonitis (fip). some of the hiv antivirals, such as raltegravir, are nonspecific, and they display activity against additional retroviruses, including felv, in in vitro studies. identification of the human coronavirus that causes severe acute respiratory syndrome (sars) has led to evaluation of antivirals for treatment of various coronaviruses, including the feline coronaviruses (fcovs) that cause fip, although testing is mainly in in vitro stages. several studies have also evaluated the use of rfeifn-omega for treatment of multiple feline viruses. a review of the literature for antiviral treatment in cats, including current recommendations for drug dosages and use, is given in table - . feline immunodeficiency virus infects lymphocytes, cells of the monocyte-macrophage lineage, and cells of the central nervous system causing a variety of clinical signs (figure - ) . the viral replication cycle of fiv is highly similar to hiv. feline immunodeficiency virus binds to host cells by an initial interaction of the fiv envelope (env) glycoprotein with the cd molecule on the host cell, resulting in subsequent interaction with the co-receptor cxcr on the host cell, followed by viral envelope fusion with the host cell membrane. this allows entry of the viral nucleocapsid into the cytoplasm. the viral rna is released into the cytoplasm and transcribed to complementary dna (cdna) by the reverse transcriptase (rt) enzyme, which is specific to retroviruses. the cdna is subsequently synthesized to double-stranded dna, transported to the nucleus, and integrated into the host genome by another virus-specific enzyme, the integrase. viral messenger ribonucleic acid (mrna) and genomic rna are then transcribed and transported to the cytoplasm. viral proteins are translated and processed by a third virus-specific enzyme, the protease. the immature virion moves to the cell membrane and acquires the viral envelope and glycoproteins and then is finally released from the cells. antiretroviral drugs studied extensively in hiv infection have targeted the three virus-specific enzymes (protease, rt, and integrase), as well as some additional targets, interfering with different steps of the virus replication cycle. as of , approximately compounds are approved by the u.s. food and drug administration (fda) for treatment of different stages of hiv infection. some of these drugs can also be used for fiv, and steps that can be inhibited include: ( ) virus entry into susceptible cells by blocking attachment to the host cell co-receptor cxcr ; ( ) reverse transcription of viral genomic rna; ( ) viral dna integration into host genomes; and ( ) proteolytic processing of precursor viral proteins into mature viral proteins (figure - ). , dose indication close similarities exist between the rt of hiv and fiv, and it has been shown that several rt-targeted antiviral compounds active against hiv are also effective in inhibiting fiv replication in vitro. the rt of hiv is actually the target for three classes of inhibitors: nucleoside rt inhibitors (nrti), nucleotide rt inhibitors (ntrti), and nonnucleoside rt inhibitors (nnrti). nucleoside rt inhibitors and ntrti interact with the catalytic site (the substrate-binding site) of the rt enzyme, whereas nnrti interact with an allosteric site located at a short distance from the catalytic site. for the nrti and ntrti to interact with the substrate-binding site, they need to be phosphorylated. all , and emtricitabine) can be considered as nucleoside analogues, and they act in a similar fashion. after they have been taken up by the cells, they are phosphorylated three times to the active triphosphate form, and they act as competitive inhibitors of the normal deoxynucleoside triphosphate (dntp) substrates, which are used by the cell to make dna. unlike dntp substrates, nrti lack a ′-hydroxyl group on the deoxyribose moiety. once incorporated into the dna chain, the absence of a ′-hydroxyl group, which normally forms the ′-to ′-phosphoester bond with the next nucleic acid, blocks further extension of the dna by rt, resulting in dna chain termination. the analogues cannot be cleaved from the active center and thus block the rt enzyme. nucleoside analogues are not only accepted as false substrates by viral enzymes, but also by cellular enzymes, and infectious diseases mononuclear cells. six of these drugs (abc, ddi, emtricitabine, tc, d t, and azt) had been previously evaluated in feline cells, and three (amdoxovir, racivir, and dexelvucitabine) had not. significant differences among the drugs were not found, but based on the data obtained, amdoxovir, dexelvucitabine, and racivir appear to be options for future studies investigating their potential use in fiv-infected cats. though pharmacological data for cats are not available for these drugs, cytotoxic properties of these compounds suggest they could likely be used in vivo at dosages comparable to that for azt. nucleotide rt inhibitors are distinguished from nrti as they are nucleotide analogues (not nucleoside analogues), which means that they only need two (not three) phosphorylation steps to be converted to their active form. most importantly, they contain a phosphonate group that cannot be cleaved by hydrolases (esterases), which would make it more difficult to cleave off these compounds, once incorporated at the ′-terminal end, compared with their regular nucleotide counterparts. use of these compounds also results in dna chain termination. one of these drugs, cidofovir, is active against virtually all dna viruses, including polyoma-, papilloma-, adeno-, herpes-, and poxviruses. cidofovir has been used for treatment of fhv- (see feline herpesvirus type ). adefovir ( -( -phosphonylmethoxyethyl)adenine [pmea]) has a spectrum of activity that partially overlaps with cidofovir, in that both are active against herpesviruses, but adefovir is also active against hepadnaviruses (hepatitis b) and retroviruses, including fiv and felv. the antiviral activity spectrum of tenofovir (pmpa) is narrower than that of pmea, in that it no longer extends to herpesviruses but is confined to hepadna-and retroviruses. this drug has been tested in vitro against felv (see feline leukemia virus). adefovir has been tested in fiv-infected cats in a -week placebo-controlled, double-blinded, clinical trial; cats received adefovir ( mg/kg subcutaneously [sc] twice weekly) and cats received a placebo. there was no decrease in the proviral or viral loads in treated cats, and the cats developed a progressive, life-threatening anemia. this is a common adverse effect of some nucleotide analogues. adefovir was also tested in combination with the co-receptor inhibitor plerixafor (see co-receptor inhibitors) in the same study, producing the same outcome as seen with use of the adefovir alone. a related drug, (r)- -( -phosphonylmethoxypropyl)- , diaminopurine (pmpdap), has been shown previously to be a potent inhibitor of fiv replication in cell culture and has reduced the viral load in three of four cats experimentally infected with fiv when treated at mg/kg sc three times per week for weeks. there were no changes in the red blood cell counts or hemoglobin values with treatment. a recent study evaluated the efficacy of this drug in a placebocontrolled, double-blind study with a population of cats naturally infected with fiv. no significant differences were found between pmpdap-treated ( mg/kg sc twice weekly for weeks) and placebo-treated cats, although cats treated with pmpdap showed a tendency for improvement this is the major cause of their toxicity. zidovudine is the nrti most studied in cats, including in vivo studies evaluating the clinical response of experimentally and naturally fivinfected cats treated with the drug. zidovudine can increase the cd +/cd + ratio and improve clinical condition scores in fiv-infected cats; however, it can result in adverse effects, such as dose-dependent nonregenerative anemia and neutropenia. , in addition, mutations producing resistance against the drug can develop. , therefore, a study evaluated nine nrti to inhibit fiv replication in feline peripheral blood receptor and co-receptor proteins dna a significant decrease in the provirus load but did not lead to improvement of clinical or immunological variables. a statistical decrease in serum magnesium levels was observed in the treatment group, without clinical consequences. no development of resistance of fiv isolates to plerixafor was found during the treatment period, making it a potential treatment for fiv-infected cats. limited oral bioavailability and short half-life preclude clinical use of plerixafor in hiv infection, , but additional cxcr antagonists are under development and should be tested for efficacy against fiv when available. integrase catalyzes strand transfer ( ′-end joining), which inserts both viral dna ends into a host cell chromosome. integrase inhibitors are used to treat hiv infection. one of the integrase inhibitors (raltegravir) has been shown to be effective for inhibition of felv (see feline leukemia virus). administration of a combination of drugs from different classes, termed highly active antiretroviral therapy (haart), to hiv-infected patients has turned an invariably fatal disease into a chronic but manageable condition. , the goals associated with the use of combinations of three (or more) anti-hiv compounds are: ( ) to obtain synergism among different compounds acting at different molecular targets; ( ) to lower the individual drug dosages to reduce their adverse side effects; and ( ) to diminish the likelihood of development of drug resistance. combination therapy has not been thoroughly investigated for treatment of fiv infection in cats, and use of multiple classes of drugs is more difficult in cats because some of the drug classes that are effective for hiv do not work for fiv. , however, the need for combination antiretroviral therapy for feline patients has been the focus of recent studies. the goal of antiviral therapy should be improvement of the cat's clinical status. this is not always correlated with virus replication, as measured by a plasma viral load. it has been suggested that antiretroviral therapy should be administered to fiv-infected cats in the later stages of the asymptomatic phase of infection, during which the cat does not show clinical signs and the immune system is relatively normal and more likely to respond to treatment. after experimental infection, when the cd +/cd + ratio decreases, viral load increases markedly, and clinical signs of immunosuppression begin to appear. however, the situation in naturally infected cats is different, and the quality of life is not associated with the viral load. therefore, it is debated at which time point antiviral therapy should be started and whether it should be administered to asymptomatic cats. in a recent study, antiretroviral therapy was initiated during the later stages of the asymptomatic phase of infection in naturally infected cats. the cats were defined as being in the later stages of the in their clinical signs and cd +/cd + ratios. mild hematological side effects (slight decline in packed cell volume and hemoglobin values) were seen in the treatment group. compared with other ntrti, pmpdap seems to be slightly less toxic. unlike the nrti and ntrti, nnrti are an active form, with no dependence on intracellular metabolic pathways. nnrti inhibit the rt by binding to the enzyme in a hydrophobic pocket that is located away from its catalytic site. the interaction of the compounds with the rt induces conformational changes that affect the catalytic activities of the enzyme. nonnucleoside rt inhibitors are considered highly specific inhibitors of hiv- , and thus not active against other retroviruses, including fiv. this is due to differences in the structure and/or flexibility of fiv rt that prevent nnrti from interacting with the fiv rt. protease inhibitors are based on the "peptidomimetic" principle, that is, they contain a hydroxyethylene scaffold that mimics the normal peptide linkage (cleaved by the hiv protease) but which itself cannot be cleaved. they thus prevent the hiv protease from carrying out its normal function, which is the proteolytic processing of precursor viral proteins into mature viral proteins. despite similarities between the hiv and fiv proteases, all but one of the currently available hiv protease inhibitors have failed to inhibit the protease of fiv. the one compound of interest, tipranavir, has only been tested against fiv in vitro so far. however, studies have demonstrated that these compounds can be used to inhibit fcov replication (see feline coronavirus). co-receptor inhibitors block viral attachment by binding to receptors on the host cell membrane to obscure the site of interaction of env with the receptor. most of the receptor homologues or antagonists are highly selective for hiv and not useful for veterinary medicine. one exception can be used in cats with fiv infection, the class of bicyclams (e.g., plerixafor). plerixafor ( , ′-[ , -phenylenbismethylene]bis( , , , -tetraazacyclotetradecane)-octachloride dehydrate, [amd ], [ jm ]), is the prototype compound among the bicyclams. bicyclams are dimeric low-molecular weight nonpeptidic compounds that bind selectively to the chemokine receptor cxcr . this is the cell surface co-receptor used by both hiv and fiv for attachment and infection of susceptible cd + lymphocytes, and the amino acid sequences of human and feline cxcr are highly similar. drug binding inhibits attachment of the viral envelope to the host cell. the efficacy of plerixafor against fiv was recently investigated in naturally fiv-infected cats that were treated in a placebo-controlled, double-blind clinical trial. plerixafor was administered at . mg/kg sc every hours. treatment of fiv-infected cats with plerixafor caused infectious diseases agriculture (usda) as a treatment aid for cats infected with fiv or felv. the primary therapeutic effect is activation of progenitor cd t-cells to mature cells, which then produce cytokines, including interleukin (il)- and interferon (ifn). a few studies performed by the manufacturer are highlighted in a review article. the studies suggest reduced virus load, improved clinical signs, and improved hematological parameters with treatment. however, the data for placebocontrolled studies were not shown, and a field study with naturally infected cats lacked a control group. independent placebo-controlled, blinded studies are warranted. additional information about immunomodulators and immunostimulants is provided in the feline herpesvirus type and feline coronavirus sections. feline leukemia virus, like fiv, is a member of the family retroviridae, but unlike fiv, felv is a gammaretrovirus and not a lentivirus. feline leukemia virus causes a wide variety of clinical signs in infected cats (figure - ). structural differences affect the susceptibility of gammaretroviruses to anti-hiv drugs, but the similarities in mechanism of replication suggest that some of these drugs can also inhibit felv. this is true of most nrti. zidovudine effectively inhibits felv replication in vitro, and in vivo in experimental infections. however, in naturally felv-infected cats, it did not reduce plasma virus load, improve immunological and clinical status, increase quality of life, nor prolong life expectancy. its bone marrow toxicity can also cause adverse side effects (e.g., nonregenerative anemia) that are more pronounced in felv-infected cats than in fiv-infected cats. therefore, it is not recommended as a first line of therapy for felv infection. asymptomatic phase of infection when the cd +/cd + ratio reached . , because at this stage of infection, the viral load increased markedly, and clinical signs of immunosuppression began to appear. the ratios were calculated every months for to years prior to initiation of the antiviral therapy, and viral loads of all cats were quantified once a year. the cats were randomly assigned to treatment groups of eight cats each. treatment included combination therapy, but no placebo group was used, and the study was not blinded. the follow-up was performed over year, through clinical evaluation and the determination of viral loads and cd +/cd + ratios. comparisons of pretreatment and post-treatment values from the cats were performed, as well as comparison of values between treatment groups. a combination of two nrti (azt + tc, mg/kg every hours orally [po]) was compared to treatment with azt alone ( mg/kg every hours po). the combination of azt and tc is often used in hiv-infected patients, given that both drugs show a synergistic effect. treatment with azt alone or in combination with tc induced a significant increase in the cd +/ cd + ratio and a significant decrease in viral load within and among groups, with an even greater reduction with combination therapy than with azt alone. only mild side effects, including vomiting in one of eight cats, anorexia in two of eight cats, and anemia in one of eight cats, were seen with this treatment combination, but therapeutic interventions resolved the problems, and treatment did not have to be stopped. however, the lack of a control group and lack of blinding make the results of the study very difficult to interpret. therefore, treatment of asymptomatic fiv-infected cats with antivirals cannot be generally recommended based on the currently available data. an earlier in vivo study was performed in experimentally fiv-infected cats that were treated with a high-dose azt and tc combination ( or mg/kg/day po for each drug). the combination had no anti-fiv activity in these chronically infected cats. severe side effects, which included fever, anorexia, and marked hematologic changes, were observed in some of the cats with such high-dose dual-drug treatment, but the toxic effects were reversed when the dose was lowered to mg/kg every hours. ideally, combination therapy for feline patients will contain at least two to three drugs from at least two different classes, as recommended for human patients. as previously mentioned, pmea (an ntrti) was tested in combination with the co-receptor inhibitor plerixafor; however, because of the toxicity associated with the pmea, this combination cannot be recommended. therefore, use of plerixafor in combination with other ntrti that are less toxic than pmea or compounds of other drug classes are should be further investigated in the future. lymphocyte t-cell immunomodulator (ltci), a protein produced by a bovine-derived thymic stromal epithelial cell line, is conditionally licensed by the united states department of alphaherpesviruses, such as herpes simplex virus (hsv- ). they have been investigated for treatment of fhv- . members of this group of antiviral agents include acyclovir (and its prodrug, valacyclovir), ganciclovir, and penciclovir (and its prodrug, famciclovir). all require three phosphorylation steps for activation. the first of these steps must be catalyzed by the fhv- viral enzyme, thymidine kinase. this makes the drugs less toxic in vivo compared to many of the other antiviral drugs. however, the activity of the thymidine kinase in fhv- is not equivalent to the enzyme of human herpesviruses. the second and third phosphorylation steps must be performed by host enzymes, which are not as effective in cats as they are in humans. this knowledge helps explain why the acyclic nucleoside antiviral agents developed for humans infected with hsv- are not predictably effective when administered to cats infected with fhv- and why pharmacokinetic and efficacy studies are always needed to establish appropriate dosing in cats. acyclovir has been adequately tested in cats for the treatment of fhv- , but it has a relatively low antiviral potency and poor bioavailability. a very high dose is required for effective treatment, which is associated with unacceptable toxicity, with signs related to bone marrow suppression and nephrotoxicity. a prodrug of acyclovir, valacyclovir, was developed for increased bioavailability in humans, but use for fhv- treatment in experimentally infected cats induced fatal renal and hepatic necrosis and bone marrow suppression, and did not reduce viral shedding or clinical disease severity. , therefore, despite its superior pharmacokinetics, valacyclovir should not be used in cats. ganciclovir appears to be at least -fold more effective against fhv- than acyclovir in vitro. ganciclovir is available for systemic as well as topical use in the form of a . % ophthalmic gel formulation in humans. ganciclovir holds promise for feline fhv- infection and currently available formulations warrant safety and efficacy studies in cats. tenofovir, an ntrti used for treatment of hiv, has been shown to be effective against felv in vitro. the anti-felv mechanism of tenofovir is probably similar to what has been described for hiv- . tenofovir is given in the form of a prodrug, which is converted to an acyclic nucleoside phosphate. once converted to the active diphosphate form, tenofovir is incorporated by rt into viral dna, where it acts as a chain terminator to inhibit further elongation of the viral dna. however, in vivo studies in felv-infected cats are lacking. another compound currently used for human hiv therapy, raltegravir, could be considered for the treatment of felv-infected cats. the high degree of conservation across lentiviruses, betaretroviruses, gammaretroviruses, and alpharetroviruses of integrase active sites suggests that felv might be highly sensitive to integrase inhibitors. the mechanism of action against felv is the same as for fiv, inhibition of integration of the viral dsdna that is produced by reverse transcription of the viral rna genome. an in vitro study evaluated the effective % inhibitory concentration (ec ) for felv inhibition of raltegravir in several feline cell lines and found these values are in the range of that observed for hiv and a related gammaretrovirus, xenotropic murine leukemia virus, and are well below the minimal plasma concentrations found in humans. the effective concentration of raltegravir had no appreciable effect on cell viability nor induced apoptosis, suggesting that this could be an effective and safe drug also in vivo. however, raltegravir is partly eliminated as glucuronide, a metabolic pathway that is not very efficient in cats, and it would increase the risk of toxicity resulting from drug accumulation. as of , no in vivo studies have been published. feline herpesvirus type is a member of the subfamily alphaherpesvirinae, order herpesvirales. herpes simplex viruses and and varicella zoster virus are also members of this subfamily, and antivirals developed for the treatment of these human viruses have been used for treatment of fhv- in cats. feline herpesvirus type typically infects epithelial and mucosal surfaces and travels retrograde along sensory axons to establish latency in the trigeminal ganglia. reactivated virus travels down those same axons to infect similar tissues to those that were originally infected, potentially resulting in recurrent or chronic sequelae, including keratitis, conjunctivitis, rhinosinusitis, dermatitis (figure - ) , and potentially blindness. whereas drug combinations have become standard procedure for the treatment of hiv infections, the treatment of other virus infections, including herpesviruses, is routinely based on the use of a single antiviral drug. a group of antiviral drugs known as acyclic nucleoside analogues are used for the systemic treatment of human effective against fhv- . this is potentially an alternative therapy to the use of topical drugs, the majority of which require multiple daily applications. however, an implantable silicone polymer device impregnated with penciclovir has been developed that holds promise for long-term, steadystate subconjunctival delivery of the drug for the treatment of ocular herpetic disease. although herpetic ocular disease is commonly treated with topical antiviral ophthalmic solutions or ointments (including idoxuridine, vidarabine, or trifluridine), these antivirals do not require a virus-specific phosphorylation step for activation. moreover, they damage host cells, specifically resulting in bone marrow suppression. therefore, they should not be used systemically. for good reviews of these topical drugs, see the reports of maggs and gould. cidofovir, a member of the ntrti class of drugs, has been tested for topical treatment of fhv- ocular disease but not for systemic use. it appears to be efficacious topically and is a newer drug (therefore it is included in this section). cidofovir requires the typical two host-mediated phosphorylation steps without virally mediated phosphorylation. its safety when given topically arises from its relatively high affinity for hsv dna polymerase compared with human dna polymerase. it is commercially available only in injectable form in the united states for treatment of a human betaherpesvirus. when applied topically as a . % solution twice daily to cats experimentally infected with fhv- , it led to reduced viral shedding and improvement of clinical disease compared to the placebo group. its efficacy with only twice daily administration (despite being virostatic) is believed to be due to the long tissue half-lives of the metabolites of this drug. there are reports of its experimental topical use in humans and rabbits being associated with stenosis of the nasolacrimal duct, but this has not been shown in cats. the fact that a twice-daily topical treatment is sufficient, whereas all other topical antivirals require application every to hours, makes cidofovir a useful alternative for ocular topical treatment. , , small interfering rna small interfering rnas (sirnas) designed to target the fhv- dna polymerase and glycoprotein d have been used in vitro to induce rna interference in an immortalized cell line and in primary feline corneal epithelial cells to inhibit fhv- replication. rna interference is a posttranscriptional, rna-guided gene-silencing mechanism present in eukaryotes. interference of the fhv- essential genes resulted in reduction of virus replication up to ± %. this type of therapy is intended for topical treatment of chronic herpetic disease. however, a preliminary in vivo study evaluating topical delivery of sirnas to feline corneas was unsuccessful. the lack of delivery was likely the result of sirna dilution and rapid removal by tear film and blinking. studies are ongoing to identify a means of increasing the most promising systemic drug for the treatment of fhv- is famciclovir, a prodrug of the active compound penciclovir, which has been shown to be highly efficacious in inhibiting fhv- replication in vitro. penciclovir is absorbed poorly when given orally, so the oral form famciclovir was developed with increased bioavailability and uptake from the intestinal tract. famciclovir requires di-deacetylation, mainly in the blood, and oxidation by a hepatic aldehyde oxidase for conversion to the active compound penciclovir. unfortunately, hepatic aldehyde oxidase activity is basically absent in cats, which makes the pharmacokinetics of this drug complex and results in lower than expected plasma penciclovir concentrations despite administration of relatively high doses of famciclovir. , despite this, studies evaluating famciclovir in vivo have shown it to be safe and efficacious for use in feline patients. , cats experimentally infected with fhv- and receiving famciclovir mg/kg po three times daily for days had significantly improved outcomes for systemic, ophthalmic, clinicopathologic, virologic, serologic, and histologic variables when compared with placebo-treated cats. treatment was initiated on day zero, the same day the cats were infected. even though this study did not mimic how cats with natural infection would be treated, results from a clinical case study suggested this drug is likely effective for treatment of clinical cases, though it was not blinded and placebo controlled. clinical cases with primary ocular disease, rhinosinusitis, and dermatitis each attributed to fhv- (though not definitively diagnosed), were treated with famciclovir at doses of . mg po once or twice daily for ocular herpetic disease or rhinosinusitis or up to mg po three times daily for dermatitis. famciclovir was well tolerated with each dose and had a positive effect on each clinical condition. a definitive dose rate has not been established for famciclovir. however, penciclovir has no appreciable in vitro effect if present for hours prior to infection, suggesting that famciclovir should be administered more frequently than once every hours to ensure exposure to penciclovir as additional epithelial cells become exposed to viral infection. current pharmacokinetic data suggest that dosing three times daily is required, and mg/kg po three times daily has been suggested for treatment of cats infected with fhv- , based on effective concentrations obtained in in vivo studies , and determination of new in vitro %-inhibitory concentrations. the most commonly reported adverse effects of famciclovir treatment in humans include urticaria, hallucinations, headaches, and confusion (especially in elderly humans), which would likely be more difficult to detect in animals. for these reasons, judicious use of this drug is recommended in client-owned cats, especially those with preexisting hepatic or renal insufficiency. pharmacokinetic studies have also evaluated the concentration of penciclovir in tears, and treatment with an oral dose of mg of famciclovir/kg three times daily achieves a penciclovir concentration at the ocular surface likely to be a bolus and not added to food. any benefit from lysine therapy is likely only possible with daily, lifelong treatment of cats with chronic herpetic disease, rather than use of lysine as a treatment during acute or recrudescent episodes. potentially, daily therapy would reduce episodes of viral recrudescence. however, clinical studies in pet cats are lacking. the cost of this therapy should be weighed against the potential benefits. owners should be made aware that this is only an adjunctive therapy and that administration of antiviral drugs might be necessary to gain better control of signs. polyprenyl immunostimulant (pi) is an immunomodulator that has a conditional license in the united states for treatment of fhv- infection. in blinded, placebo-controlled, experimental challenge studies, pi started on the day of virus exposure significantly reduced the severity and duration of rhinitis and conjunctivitis associated with acute fhv- disease (legendre and kuritz, manuscript in preparation). according to the manufacturer, pi upregulates the innate immune system and modulates the immune response toward a cellular response. this activity was attributed to positive effects associated with treatment of fhv- , which requires a cell-mediated immune response for control. viral titers were not compared between treatment and control groups in the studies, but based on the reduced signs associated with treatment, clinical studies are warranted. feline infectious peritonitis is associated with clinical signs that can affect almost any body system (figure - ) . currently, there is no effective treatment for fip despite its importance as the leading infectious cause of death in young cats. following the discovery that sars is caused by a coronavirus (sars-cov), efforts to find an antiviral drug for coronaviruses increased. a few antiviral agents that target different steps in the replication cycle have been tested against feline fcov. coronavirus spike proteins on the viral envelope initially bind to receptors on the host cell membrane. the spike protein mediates fusion of the viral envelope with host cell membranes. during this process, heptad repeats and (hr and hr ) of the spike protein assemble to form a complex, resulting in a conformational change that is necessary for fusion. peptides have been used as antivirals by inhibiting the hr -hr interaction, thus preventing membrane fusion. the spike protein must be cleaved for entry of the virus into the cytoplasm. feline coronavirus infection is dependent on cathepsin b, a host cysteine protease found within the cell, making this the likely protease responsible for spike protein cleavage. therefore, cathepsin b can serve as a potential target for the development of therapeutic drugs against fcov. following entry into the cell, fcov produce viral polyproteins that are processed into contact time between the corneal cells and sirnas to allow delivery. twice-daily oral l-lysine bolus administration, initiated prior to experimental infection, reduced the severity of conjunctivitis in cats undergoing primary infection. l-lysine bolus administration also reduced viral shedding in latently infected cats experimentally infected with fhv- , following changes in husbandry and housing but not following corticosteroid administration. in vitro, lysine supplementation led to reduction of fhv- replication. arginine exerts a substantial growth-promoting effect on fhv- and is an essential amino acid for viral protein synthesis, and lysine antagonizes this effect. lysine and arginine competitively inhibit transport of each other by using a common transport system, and lysine induces arginase, an enzyme that causes the degradation of arginine. arginine deficiency inhibits synthesis of infectious viral particles and downregulates synthesis of viral proteins. however, unlike the protocol for hsv- -infected humans, owners of cats receiving lysine for fhv- should not be advised to restrict their cat's arginine intake because feeding a diet lacking l-arginine is associated with a severe risk of hyperammonemia and encephalopathy. it has been suggested that the ratio of l-lysine to l-arginine, rather than the concentration of each amino acid, is critical in achieving an inhibitory effect on viral replication. dietary supplementation increases mean plasma concentrations of l-lysine without reducing l-arginine concentrations and has been shown to be safe for use in cats, up to g/kg of diet. supplementation with higher doses has been shown to result in reduced food intake. , despite promising initial in vitro data and in vivo results from experimental studies, current studies question whether viral inhibition with increased lysine concentrations, in the absence of decreased arginine concentrations, can be biologically important. a new study evaluating the effect of various ratios of l-lysine and l-arginine on fhv- dna replication in vitro demonstrated only a modest reduction in viral dna (less than log) at ratios considered difficult to obtain in vivo in healthy cats. a lack of efficacy of l-lysine supplementation has also been demonstrated in vivo in shelter settings. , dietary supplementation was unsuccessful, likely because the cats were anorexic during peak disease and were not ingesting the lysine when they needed it the most. bolus administration was also unsuccessful, likely because of stress associated with the lysine administration. , the stress of bolus administration in shelter situations could negate its effects and even cause transfer of pathogens among cats by shelter workers administering the lysine. however, data do not support dietary supplementation. , unfortunately, no studies to date have been conducted on client-owned cats; however, anecdotal evidence suggests that there is a benefit from administration of lysine in individuals. dosing is mg po twice daily, which should be given as infectious diseases ment to the host cell. the antiviral effects were concentration dependent, and nelfinavir displayed cellular toxicity at higher doses. gna was a better inhibitor of fcov, and when the two agents were added together, a synergistic antiviral effect was produced. the results suggest that the combined use of gna and nelfinavir could have therapeutic potential in the treatment of cats with fip. viral fusion has also been targeted effectively with a synthetic peptide based on the putative hr sequence of fcov. virus replication was significantly inhibited in vitro compared to controls, and the peptide was nontoxic. this peptide was also used in combination with human ifn-alpha. the two displayed a synergistic effect, but the cells were pretreated with ifn prior to infection by the virus. see the section on interferon for further information about interferon treatment for fip. immunomodulators have been considered because fip is an immune-mediated disease. a drug that has shown promise for immunomodulation is pi. this drug has a conditional license in the united states for treatment of fhv- infection. in a case series of three cats, pi was associated with prolonged survival in cats with noneffusive fip. no placebo group was included for comparison, so definitive conclusions about the effectiveness of this drug for treatment of fip cannot be drawn. additional immunostimulants such as immunoregulin (propionibacterium acnes), an inactivated bacterin, and a t-cell receptor peptide (manufactured by imulan biotherapeutics), have been suggested for treatment of fip. these are not antiviral drugs; instead, each of these products is reported to stimulate the immune response toward a cell-mediated response or to reduce an overactive type helper t-cell (th ) response. an imbalance in t-cell versus b-cell immune response has been suggested to contribute to the development of fip. it has been proposed that a strong cell-mediated immune response protects a cat from the development of fip, whereas the production of antibodies is counterproductive, enhancing the uptake and replication of feline infectious peritonitis virus (fipv) in macrophages and contributing to the pathology by producing a type iii hypersensitivity vasculitis. however, this hypothesis has been questioned. therefore, even though the use of these types of drugs for stimulation of a cell-mediated response might seem a logical approach for the treatment of fip, there is currently no data to support their use. an additional non-antiviral drug that has been evaluated for treatment of fip is propentofylline. this drug appears to downregulate proinflammatory cytokines, which in turn can reduce vasculitis. vasculitis, as stated earlier, is responsible for pathology associated with fip. however, in a placebocontrolled, double-blind study in cats with late stage fip, mature proteins by viral-specific proteases, the main protease ( c-like [ cl] protease) and the papain-like protease. because the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is also an attractive target for therapeutic intervention. in an in vitro experiment in crandell-rees feline kidney cells, cl protease inhibitors and cathepsin inhibitors were tested for their ability to inhibit fcov replication. both types of drugs produced effective inhibition with ec values in the nanomolar range and each drug tested was nontoxic to the cells at effective concentrations. the cl protease inhibitors were more effective than the cathepsin inhibitors and when used in combination, these drugs had strong synergic effects. there have not been any in vivo studies with these drugs in cats to date. in one in vitro study, antiviral compounds, including nucleoside analogues used to treat herpesviruses, nrti used for hiv, and protease inhibitors also used for hiv, were tested for their ability to inhibit fcov in cell culture. among the drugs tested, two showed significant inhibition of fcov when compared to the untreated cells. these were nelfinavir and galanthus nivalis agglutinin (gna). nelfinavir is a hiv protease inhibitor that has been shown to target the cl protease of sars-cov by interacting with residues of the protease. the drug was slightly less effective against fcov than against sars-cov, likely because only seven of the corresponding residues of the cl protease of fcov are identical to the sars-cov protease. gna, a carbohydrate-binding agent, exhibits its antiviral effect by binding to coronavirus glycosylated envelope glycoproteins, thereby inhibiting viral attach- ineffective within a few weeks because of the development of neutralizing antibodies that limit its activity. , rhuifn-α can be given orally for a longer period because no antibodies will develop during oral treatment. unlike rhuifn, rfeifnomega, being a feline recombinant product does not induce neutralizing antibodies when administered sc. this means that the high-dose parenteral protocol can be used safely and efficiently even if repeat administration is required. this is an important factor to consider in a condition where management needs to be lifelong. given po, ifns are inactivated by gastric acid and destroyed by trypsin and other proteolytic enzymes in the duodenum and therefore are not absorbed and cannot be detected in the blood after oral administration. direct antiviral effects are unlikely after oral application; however, ifn still seem to have immunomodulatory activity. type i ifns likely bind to mucosal receptors in the oral cavity, stimulating the local lymphoid tissue, leading to cytokine release from lymphatic cells in the oropharyngeal lymphoid tissues, triggering a cascade of immunologic responses that act systemically. interferons have been used for the treatment of feline retrovirus infections. treatment with ifn improved the clinical condition scores of cats infected with felv and fiv, but not because of a reduction in viral load. this suggests that the improved clinical condition seen with treatment is not specific to an antiviral effect, at least not for fiv and felv, but instead is a result of immunomodulation, potentially associated with the innate immune response. , , some clinical signs in fiv-infected cats are caused by immunopathological reactions, such as gingivostomatitis and uveitis. immunomodulation might be the cause of improvement of some clinical signs associated with ifn treatment, probably the result of an effective control over inflammatory cytokines in diseased organs. it has been suggested that a nonspecific stimulation of the immune system with ifn therapy might be contraindicated in fiv-infected cats because it could lead to a rise in viral replication produced by the activation of lymphocytes and macrophages harboring latent infections and therefore accelerate disease progression in these cats, and the use of ifn in hiv-infected humans is controversial. however, use of low-dose oral huifn (natural, not recombinant in this study) in ill fiv-infected cats ( iu per kg on the oral mucosa daily for days on alternating weeks for months, followed by a -month break, and then repetition of the -month treatment) resulted in improvement of clinical signs in a placebo-controlled, doubleblind study. parenteral rfeifn-omega used according to the licensed protocol (table - ) resulted in decreased mortality rates in felv-infected cats, compared with the control group in another placebo-controlled study. , in another study evaluating fiv-and/or felv-infected cats housed in a shelter, hematologic values remained within reference ranges, and there were no biochemical abnormalities there was no statistically significant difference in the survival time, the quality of life, or any clinical or laboratory parameter in cats treated with this drug versus cats receiving a placebo. of the cats in the study, of cats displayed effusion at the start of the study. the drug might be more useful in cats without effusion because it may have a chance to prevent vasculitis and therefore effusions, but studies are lacking. interferons are molecules produced by vertebrate cells in response to viral infections or certain inert substances, such as double-stranded rna, and other microbial agents. there are three types of ifns. type i ifns comprise the largest subfamily and include ifn-α, ifn-β, and ifn-omega. type i ifns are produced by various cell types, such as leukocytes and fibroblasts, in direct response to virus infection. there is only one member of the type ii ifn subfamily, ifn-γ, that is an immunomodulatory cytokine, produced in response to recognition of infected cells by t lymphocytes and natural killer cells of the host's immune system. type iii ifns, which contain three ils, (il- a, il- b, and il- ), are identified. this subfamily also has the ability to interfere with virus replication and has been suggested to be the ancestral antiviral system of vertebrates. interferons are not virucidal; rather, they trigger expression of various antiviral proteins and thus induce an antiviral state within the host cell to limit replication and spread of viruses. further, type i ifns have been shown to potently enhance innate and adaptive immune responses in vivo, through various immunomodulatory effects, such as activation of dendritic cells (dcs), amplification of antibody response, and enhancement of t-cell and natural killer cell cytotoxicity. viruses causing lysis of their target cell are most effectively inhibited by ifn through their antiviral activity in noninfected cells. therefore, ifns have their highest utility in the prophylaxis or early postexposure management for virus infections. given that ifns are not specific for a particular virus, they have been tested for the treatment of multiple feline viruses, including fhv- , fiv, felv, feline calicivirus (fcv), and fcov. two molecules of type i ifns are currently being used for therapy in cats: human recombinant interferon alpha (rhuifn-α), and rfeifn-omega, which is licensed for use in cats and dogs in europe, australia, and some asian countries. ifns are not strictly species-specific in their effects; however, their biologic activity and toleration are greater in cells of genetically related species. in vitro results suggest that rfeifn-omega would likely be more effective than rhuifn-α in vivo, although both ifns have been shown to have therapeutic value in cats. there are two common treatment regimens for use of rhuifn-α in cats: injection of a high dose ( to international unit per kg sc every hours) or oral application of a low dose ( to international unit [iu] per kg every h). when given parenterally to cats, rhuifn-α becomes feline coronavirus shedding was reduced but not significantly, and there was not enough fpv detected in the population to draw any significant conclusions. however, there was no placebo group used for this study, and without a placebo group, it is difficult to determine definitively if the results are due to antiviral effects of ifn or are just consistent with the natural resolution of viral shedding. in a separate study, cats with naturally acquired upper respiratory tract disease housed in a humane society facility were treated with one drop of rfeifn-omega solution ( unit/ml), rhuifn-α solution ( iu/ml), or saline ( . % nacl) solution ( cats/group) in each eye twice daily for days for the treatment of keratoconjunctivitis. there was no statistical difference between the treatment groups and the placebo group with regard to clinical scores or viral shedding (fhv- and fcv), determined by real-time quantitative pcr from oropharyngeal and conjunctival swabs. feline herpesvirus type shedding was lower, though not statistically significant, on day compared with day for all groups (including the placebo group), and clinical scores were significantly decreased on day compared to day , again for all groups including the placebo group. therefore, comparing results between days and in the treated cats without the inclusion of the placebo group would have resulted in a different conclusion for this study. these cats were not infected with felv, and even though the fiv status was unknown for all the cats, the ones that were tested were negative. however, this study highlights the need for a placebo group for accurate evaluation of the effect of ifn therapy on fhv- and fcv shedding and associated disease. oral and sc ifn therapy has been associated with an improvement in oral ulcers and gingivitis and gingivostomatitis in cats infected with fiv, , a condition that is common in cats with fcv infections. feline calicivirus is also associated with chronic gingivostomatitis in cats not infected with fiv or felv, and a study evaluated the efficacy of rfeifnomega ( iu/day for days by topical oromucosal administration) for the treatment of fcv-associated feline chronic gingivostomatitis (fcgs) and caudal stomatitis in fiv-/ felv-negative cats. cats were included in the study if they continued to show persistence of clinical signs of fcgs at least months after periodontal treatment (scaling, subgingival débridement, and polishing), tooth extraction, and weeks of antibiotics with analgesic and anti-inflammatory drugs as needed. twenty-four cats were treated with the ifn, and the effect was compared with a positive control group that received a standard corticosteroid therapy. the results suggested that the ifn therapy was as effective as the corticosteroid treatment for this condition for improvement in clinical signs. feline calicivirus viral loads were not evaluated in this study, and there was no placebo group used for comparison. however, assuming the ifn therapy was the cause of the improvement seen in these cats, the results add to the hypothesis that improvement in oral lesions in fivand/or felv-infected cats likely is associated with the effect of ifn on opportunistic viral infections. differences in the outcome of the different studies could be due to different associated with rfeifn-omega treatment used according to the licensed protocol. these findings suggest that ifn treatment is safe for treatment of fiv-and felv-infected cats, , , , but further studies are required to clearly demonstrate its efficacy against fiv and felv in vivo. a recent study evaluated the use of oral administration of rfeifn-omega for the treatment of symptomatic naturally infected, client-owned fiv-infected cats. the treatment protocol was iu/cat po every hours for consecutive days, administered by the cats' owners. a historical group that was treated sc with the licensed protocol was used as a control for comparison, but no placebo group was included. treatment resulted in significant improvement of clinical scores between pretreatment and post-treatment values, and there was no significant difference between the sc historical control group and the po group, suggesting that po administration of rfeifn-omega could be used effectively as an alternative to the licensed protocol, at a significantly reduced cost. an additional benefit of using ifn therapy for fiv and felv treatment could be the effect of ifn on opportunistic infections by other viruses, including fhv- and fcv. in fact, the effect of ifn on these additional viral infections might be the cause of the improved clinical scores associated with ifn treatment. , both fiv and felv replicate in lymphoid and monocytoid cell subsets and cause immunosuppression. in fiv-infected cats, most of the clinical signs are not directly caused by the fiv itself but are the result of secondary infections, as well as neoplasia. , although felv causes more severe clinical syndromes than fiv does, diseases secondary to immunosuppression account for a large portion of the syndromes seen in felv-infected cats as well. considering that ifn therapy seems to have no effect on fiv and felv virus load but it is immunomodulatory, it would seem advisable to treat retrovirus-infected cats with ifn when they have clinical signs, as they would benefit from its effects in improving their clinical condition. a recent study attempted to evaluate the hypothesis that improvement in clinical scores with ifn treatment in fivand felv-infected cats might be a reflection of reduction in viral shedding of secondary viruses in these cats. sixteen naturally infected fiv-and/or felv-infected cats (seven fiv, six felv, and three coinfected) were followed during rfeifn-omega therapy (used according to the licensed protocol) to monitor clinical signs and to correlate them with excretion of concomitant viruses (fcv, fhv- , fcov, and feline parvovirus [fpv] ). shedding of these viruses was evaluated by real-time quantitative polymerase chain reaction (pcr) (fhv- and fcov) or conventional pcr (fcv and fpv). pretreatment and post-treatment samples were compared. feline calicivirus shedding was detected in of cats on day and not detected on day . the amount of fhv- shedding was significantly reduced in the cats at the end of the study (day ), compared with the beginning. before euthanasia with a mean survival time of days. there was only one long-term survivor (> months) in the rfeifnomega group. interferon treatment might be more effective if started earlier, but this is not of relevance in the treatment of cats with fip in the field. however, ifn therapy might be useful for treatment of cats with chronic fcov shedding, but further studies are required. as previously mentioned, treatment with rfeifn-omega (licensed sc protocol) was associated with a decrease in fcov shedding in fiv-and/ or felv-infected cats; however, the results were not compared with a placebo group. in conclusion, antivirals are still in their infancy for the treatment of feline diseases. however, as new drugs are produced for human viral diseases that can be used for feline patients, and testing of currently available drugs continues, it is hoped that determination of effective protocols for treatment of feline viral diseases will be possible in the future. application methods (e.g., ocular versus oral); however, definitive conclusions cannot be drawn without additional studies that also evaluate viral load in naturally infected cats that are randomized, placebo-controlled, and double-blinded. ifn has also been evaluated for treatment of fip. in a randomized placebo-controlled, double-blinded treatment trial, cats with fip were treated with rfeifn-omega or placebo. in all cats, fip was confirmed by histology and/or immunohistochemical or immunofluorescence staining of fcov antigen in effusion or tissue macrophages. all cats received glucocorticoids, either as dexamethasone in case of effusion ( mg/kg intrathoracic or intraperitoneal injection every hours) or prednisolone ( mg/kg po every hours). cats also received either a placebo or rfeifn-omega at iu/kg sc every hours for days and subsequently once a week. there was no statistically significant difference in the mean survival time of cats treated with rfeifn-omega versus the placebo. cats survived for a period of to days antiviral therapy for feline herpesvirus infections pharmacological inhibition of feline immunodeficiency virus (fiv) anti-hiv drugs: compounds approved within years after the discovery of hiv antiviral efficacy of nine nucleoside reverse transcriptase inhibitors against feline immunodeficiency virus in feline peripheral blood mononuclear cells evaluation of different antiretroviral drug protocols on naturally infected feline immunodeficiency virus (fiv) cats in the late phase of the asymptomatic stage of infection acyclic nucleoside phosphonates: past, present and future bridging chemistry to hiv, hbv, hcv, hpv, adeno-, herpes-, and poxvirus infections: the phosphonate bridge efficacy and adverse effects of the antiviral compound plerixafor in feline immunodeficiency virus-infected cats -phosphonylmethoxypropyl) - , -diaminopurine (pmpdap) in the treatment of feline immunodeficiency virusinfected cats their discovery, development, and use in the treatment of hiv- infection: a review of the last years susceptibility of feline immunodeficiency virus/ human immunodeficiency virus type reverse transcriptase chimeras to non-nucleoside rt inhibitors clinical aspects of feline immunodeficiency and feline leukemia virus infection is azt/ tc therapy effective against fiv infection or immunopathogenesis? lymphocyte-cell immunomodulator (ltci): review of the immunopharmacology of a new veterinary biologic discovery of drugs that possess activity against feline leukemia virus a trial with ′-azido- ′, ′-dideoxythymidine and human interferon-a in cats naturally infected with feline leukaemia virus inhibition of feline leukemia virus replication by the integrase inhibitor raltegravir controlled release delivery of penciclovir via a silicone (med- ) polymer: kinetics of drug delivery and efficacy in preventing primary feline herpesvirus infection in culture a -year journey in search of selective antiviral chemotherapy effects of valacyclovir in cats infected with feline herpesvirus evaluation of orally administered famciclovir in cats experimentally infected with feline herpesvirus type- in vitro cytotoxicity and antiviral efficacy against feline herpesvirus type of famciclovir and its metabolites treatment of feline herpesvirus- associated disease in cats with famciclovir and related drugs pharmacokinetics of famciclovir and penciclovir in tears following oral administration of famciclovir to cats: a pilot study feline herpesvirus- : ocular manifestations, diagnosis and treatment options effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus- infection in cats evaluation of the effects of small interfering rnas on in vitro replication of feline herpesvirus- use of interfering rnas targeted against feline herpesvirus glycoprotein d for inhibition of feline herpesvirus infection of feline kidney cells therapeutic sirna: principles, challenges, and strategies evaluation of delivery agents used for introduction of small interfering rnas into feline corneal cells excess dietary lysine does not cause lysinearginine antagonism in adult cats effects of physiologic concentrations of l-lysine on in vitro replication of feline herpesvirus oral supplementation with l-lysine did not prevent upper respiratory infection in a shelter population of cats potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus c-like protease peptides corresponding to the predicted heptad repeat domain of the feline coronavirus spike protein are potent inhibitors of viral infection synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus effect of polyprenyl immunostimulant on the survival times of three cats with the dry form of feline infectious peritonitis an update on feline infectious peritonitis: virology and immunopathogenesis randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis interferons: signaling, antiviral and viral evasion use of recombinant interferon omega in feline retrovirosis: from theory to practice relevance of feline interferon omega for clinical improvement and reduction of concurrent viral excretion in retrovirus infected cats from a rescue shelter oral recombinant feline interferon-omega as an alternative immune modulation therapy in fiv positive cats: clinical and laboratory evaluation comparative efficacy of a recombinant feline interferon omega in refractory cases of calicivirus-positive cats with caudal stomatitis: a randomised, multi-centre, controlled, doubleblind study in cats lowdose interferon-treatment for feline immunodeficiency virus infection therapeutic effects of recombinant feline interferon-omega on feline leukemia virus (felv)-infected and felv/feline immunodeficiency virus (fiv)-coinfected symptomatic cats effects of topical ocular administration of high doses of human recombinant interferon alpha- b and feline recombinant interferon omega on naturally occurring viral keratoconjunctivitis in cats effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis key: cord- -kczj se authors: yang, bo; zhang, xiaohui; zhang, dajun; hou, jing; xu, guowei; sheng, chaochao; choudhury, sk mohiuddin; zhu, zixiang; li, dan; zhang, keshan; zheng, haixue; liu, xiangtao title: molecular mechanisms of immune escape for foot-and-mouth disease virus date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: kczj se foot-and-mouth disease virus (fmdv) causes a highly contagious vesicular disease in cloven-hoofed livestock that results in severe consequences for international trade, posing a great economic threat to agriculture. the fmdv infection antagonizes the host immune responses via different signaling pathways to achieve immune escape. strategies to escape the cell immune system are key to effective infection and pathogenesis. this review is focused on summarizing the recent advances to understand how the proteins encoded by fmdv antagonize the host innate and adaptive immune responses. foot-and-mouth disease (fmd) is an acute and highly contagious disease affecting the cloven-hoofed animals, such as pigs and cattle. the pathogen that causes fmd is known as fmd virus (fmdv), a single-stranded positive-sense rna virus that is classified into the genus aphthovirus in the family picornaviridae [ ] [ ] [ ] . the pathogen causes vesicular disease of mouth and feet in susceptible animals [ ] . the high mutation rate of the genome of fmdv and the rapid proliferation has led to the rapid evolution of the virus and the formation of seven main serotypes [ ] [ ] [ ] . the antigenic diversity among the serotypes poses challenges to the research of efficient and cross-protective vaccines [ ] . the genome of fmdv contains an open reading frame (orf) that encodes a polyprotein precursor, and it is cleaved into four structural proteins and non-structural proteins by viral autoproteases and host protease [ , ] (figure ). upon infection of the host, a virus will face the attack from the host's immune response. in the long-term battle with the host immune response, the virus has evolved and developed a series of immune escape mechanisms to overcome the killing and inhibition from the host immune system. the mechanism of virus immune escape can be divided into three categories: ( ) enable the virus to avoid the recognition of humoral immune response; ( ) interfere with the function of cellular immune response; ( ) interfere with the host's immune response to the virus [ ] . all these strategies would be exploited by the virus for replication and spreading to other hosts. as a highly contagious and fast-spreading virus, fmdv has multiple ways to evade the killing by the immune system [ ] , which makes it difficult for controlling the virus. viral capsid protein vp and leading protein l pro can inhibit the production of interferon (ifn) and innate immune response by interacting with soluble resistance-related calcium-binding protein (sorcin) or host transcription factor adnp [ , ] . recently, new mechanisms and functions of fmdv proteins inhibiting innate immunity have been discovered. ddx (a kind of rna helicase), participate in rna metabolism and ribosome synthesis is reported to involve in this new mechanism. the interaction between fmdv a and ddx suppresses the host innate immunity by reducing the phosphorylation of irf [ ] . in addition, nucleotide-binding oligomerization domain (nod ), a member of the nucleotide-binding oligomerization domain-like receptor (nlr) family [ ] , activates the nf-κb and ifn-β signaling pathways during fmdv infection and inhibits the replication of fmdv in infected cells [ ] . fmdv b, c, and c pro inhibit the expression of nod protein, which antagonizes the antiviral response [ ] . reportedly, multiple structural and non-structural proteins of fmdv escape the killing of the host immune system. this review summrized the molecular mechanisms of immune evasion caused by fmdv proteins. the present study aimed to fill the gaps of knowledge on fmdv immune evasion mechanism, providing the basis for the prevention and control strategies for fmdv. pathogens , , x for peer review of figure . schematic of the genome and polypeptide processing of fmdv [ , ] . the fmdv genome contains an orf of about kbp, indicated by the shaded rectangle. each region within the orf rectangle represents a single protein. the flank of orf is a long ' untranslated region ( '-utr) and a short '-utr. b covalently binds to the '-end. upon infection of the host, a virus will face the attack from the host's immune response. in the long-term battle with the host immune response, the virus has evolved and developed a series of immune escape mechanisms to overcome the killing and inhibition from the host immune system. the mechanism of virus immune escape can be divided into three categories: ( ) enable the virus to avoid the recognition of humoral immune response; ( ) interfere with the function of cellular immune response; ( ) interfere with the host's immune response to the virus [ ] . all these strategies would be exploited by the virus for replication and spreading to other hosts. as a highly contagious and fast-spreading virus, fmdv has multiple ways to evade the killing by the immune system [ ] , which makes it difficult for controlling the virus. viral capsid protein vp and leading protein l pro can inhibit the production of interferon (ifn) and innate immune response by interacting with soluble resistance-related calcium-binding protein (sorcin) or host transcription factor adnp [ , ] . recently, new mechanisms and functions of fmdv proteins inhibiting innate immunity have been discovered. ddx (a kind of rna helicase), participate in rna metabolism and ribosome synthesis is reported to involve in this new mechanism. the interaction between fmdv a and ddx suppresses the host innate immunity by reducing the phosphorylation of irf [ ] . in addition, nucleotide-binding oligomerization domain (nod ), a figure . schematic of the genome and polypeptide processing of fmdv [ , ] . the fmdv genome contains an orf of about kbp, indicated by the shaded rectangle. each region within the orf rectangle represents a single protein. the flank of orf is a long untranslated region ( -utr) and a short -utr. b covalently binds to the -end. the p structural protein of fmdv was cleaved into three main viral structural proteins vp , vp and vp by c pro protease during the later translation and modification. vp protein was further cleaved into vp and vp proteins by protease c. interestingly, the vp protein is a cleavage precursor of vp and vp [ ] . the last step in the production of mature virions is the cleavage of ab (vp ), which converts residues of the n-terminal into vp and the remaining into vp , although some copies of vp may be retained in the intact virions [ ] . previous studies reported that vp protein of fmdv inhibits the activation of type i ifn signaling pathway by interacting with irf [ ] (figure , table ). however, further studies are essential to assess the combination of vp to irf to restrain the production of ifns. since then, it is reported that vp proteins of fmdv interact with poly (rc) binding protein (pcbp ) to promote the replication of fmdv [ ] (figure , table ). the pcbp can recruit e ligase aip that contains the hect domain into the polyubiquitin and degrades mav [ ] . the vp protein of fmdv suppresses the host's innate immunity by cooperating with pcbp to suppress the activation of ifn-β promoter. vp protein promotes the formation of pcbp -virus-induced signaling adapter (visa) complex, enhances the degradation of visa mediated by pcbp , and promotes the replication of fmdv [ ] . in addition, the vp structural protein of fmdv is necessary for the correct assembly of the virus [ ] . pathogens , , the fmdv capsid protein, vp , was further cleaved into vp and vp proteins by protease c. according to the structure of the virus, the vp protein of fmdv is localized on the inner surface of the capsid [ ] . although vp does not stimulate the production of neutralizing antibodies independently, it contains t and b cell epitopes, which could be recognized by a variety of haplotype mhc molecules and exhibit high immunogenicity [ ] [ ] [ ] . therefore, the combination of fmdv vp and vp could be used as a backup antigen for the development of a universal vaccine [ , ] . in addition, the vp protein plays a crucial role in immunosuppression. the recombinant fmdv vp -vp protein has been reported to have an inhibitory effect on the innate immune function of mouse peritoneal mast cells, putatively mediated by mannose receptor [ ] . furthermore, nucleoside diphosphate kinase (nme ) regulates the function of p to prevent tumor metastasis and progression and inhibit the metastasis of several malignant tumors [ ] [ ] [ ] . the role of nme in viral infection is not yet clarified. recent studies have demonstrated that nme has antiviral activity and enhances p -mediated transcription, while p regulates the expression of many antiviral genes to perform antiviral functions. however, fmdv vp does not directly interact but degrades nme through macroautophagy [ ] [ ] [ ] (figure , table ). this phenomenon promotes the interaction between p and mdm (mdm is a negative regulatory factor of p ), while on the other hand, it enhances the mif-mediated inhibition of p activity, thereby impeding the antiviral response [ ] . autophagy is an ancient and conservative biological process, which exists in almost all eukaryotes. through continuous research, it has been found that autophagy can selectively degrade intracellular redundant or harmful substances [ ] [ ] [ ] , thus affecting the pathogenesis of some diseases. autophagy can also degrade invading microorganisms (such as bacteria, virus, and parasites) [ ] , and is one of the immune mechanisms against pathogenic infection. it has been proved that a variety of viruses can activate autophagy and be swallowed and degraded [ ] . not only that, after virus infection, host cells resist virus infection by releasing inflammatory factors and activating innate and adaptive immune responses, and autophagy also plays an important role in these defense responses [ ] . in the long process of coexistence of virus and host, autophagy pathway has become one of the targets of virus-versus-host immunity. the inhibitory effect of virus on autophagy is also in many ways. for example, some studies have shown that after prrsv infection, the type i microtubule-associated protein light chain (lc -i) is transformed into lc -ii, which activates the autophagy mechanism and leads to the accumulation of autophagosomes by preventing the fusion of autophagosomes and lysosomes. autophagosomes can act as replication sites to enhance prrsv replication [ , ] . vp is one of the structural proteins of fmdv, localized on the surface of the virus. types o, a, and c fmdv vp contain several antigenic sites that present immunological significance [ , [ ] [ ] [ ] . in addition, the amino acid substitution on the vp b-c loop of fmdv type asia not only mediates the significant antigenic diversity but also alters the replication ability and pathogenicity of the virus. for example, the single asp-to-asn substitution at vp position will reduce the virus replication ability and virulence [ ] . however, the exact reasons for this result need to be further studied. a recent study demonstrated that the interaction between fmdv vp and hspb activates the eif s -atf pathway, which in turn, inhibits the akt-mtor pathway, leads to autophagy, and promotes virus replication [ ] (figure , table ). autophagy has been proposed to provide a membrane platform for virus replication complexes or mediate the virus assembly and release [ ] . thus, autophagy plays a crucial role in the replication of fmdv, and the expression of related vp mutants decreases the level of autophagy. therefore, vp -induced autophagy may be one of the mechanisms of fmdv infection. autophagy regulates type i ifn signaling machinery and plays a vital role in antiviral innate immunity [ , ] , and atg -atg conjugate inhibits the production of type i ifn during vsv infection [ ] . thus, it can be speculated that fmdv vp induces autophagy to increase the replication of fmdv, which might be achieved by blocking type i ifn signal. also, the correlation between vp -induced autophagy and host antiviral immunity of fmdv needs to be investigated further. the changes in some sites on the surface of the vp protein of fmdv affects the cellular tropism and adaptability of the virus; for instance, the replacement of (glu to gly) alters the binding characteristics of the virus to cells [ ] . the positively charged lysine residue at the vp site of fmdv a can increase the adaptability of bhk- cells [ ] . furthermore, the change in some sites of vp would also affect the stability and immunogenicity of fmdv. for example, vp h y replacement reduces the acid sensitivity of the capsid of fmdv type asia , makes the h y mutant of fmdv resistant to acid, and enhances the immunogenicity of virions [ ] . the tyrosine at position of vp mutated to phenylalanine (y f) enhanced the thermal stability of the virus. this mutant presented optimal immunogenicity, and neutralizing antibodies could be induced by immunizing guinea pigs [ ] . thus, identifying these specific sites of vp protein in fmdv provides an idea for the preparation of a heat-resistant and immunogenetically superior fmd antigen. the vp protein is the major surface protein on the fmdv and the primary antigen that elicits the neutralizing antibody response. the fmdv vp stimulates the host to produce cd + t cell responses with cross-protection against multiple serotypes of fmdv. therefore, vp protein or its antigenic determinants have become a research hotspot in the development of novel vaccines [ , , ] . fmdv protein aqueous soluble recombinant dna-derived vp (rvp ) binds to integrin induces apoptosis, and fmdv rvp may selectively act as an effective human tumor apoptosis factor by regulating akt signal pathway [ ] . the vp that interacts with host proteins can either enhance or inhibit the production of ifn in cells. first, it was found that vp interacts with soluble resistance-related calcium-binding protein (sorcin), a negative regulator in the innate immune signaling pathway, through yeast two-hybrid and immunoprecipitation experiments. also, vp binds to sorcin and activates the transcription factor stat . stat inhibits the activation of ikk and nf-κb pathway, thus inhibiting the expression of type i ifn and cytokines [ ] (figure , table ). second, the host protein kinases are essential regulators of virus interaction and play a crucial role in virus replication. tpl (tumor progression locus ), a serine/threonine-protein kinase, promotes the activation of ifn-β signaling pathway by increasing the phosphorylation of irf . tpl phosphorylation site thr is vital for promoting irf -induced ifn-β signal activation. vp inhibits the protein expression of tpl phosphorylated at thr , thereby inhibiting the irf -activated ifn-β signaling, while vp reduces the mrna levels of tpl -mediated ifn-β and some isgs ( figure , table ). third, another study proved that vp suppresses the ifn-β signaling pathway at the irf level by inhibiting the irf phosphorylation, dimerization, and nuclear translocation ( figure , table ). another study suggested that the activation of the mitogen-activated protein kinase (mapk) pathway is essential for fmdv replication. fmdv vp interacts with host ribosomal protein sa (rpsa) to continually activate the mapk signal pathway and promote virus replication by inhibiting the rpsa-mediated function [ ] (figure , table ). as a structural protein of fmdv, vp plays a crucial role in virus assembly [ ] . it blocks the ifn signal transduction, promotes the replication of fmdv, and inhibits the host immune response. in addition, vp inhibits the protein and mrna expression of innate immune junction molecule, visa. it interacts with the visa protein to inhibit the formation of visa-regulated complex, thereby inhibiting the dimerization and phosphorylation of irf , inhibiting the expression of antiviral genes induced by ifn-β, and promoting fmdv replication [ ] (figure , table ). previous studies have focussed on the effect of fmdv after treatment of type i ifn. also, it was demonstrated that fmdv vp inhibits the type ii ifn signaling pathway. furthermore, vp interacts with the host protein kinase jak protein and degrades the jak protein through lysosome pathway, inhibits the activation of the jak-stat pathway, and reduces the ifn-induced antiviral gene expression [ ] (figure , table ). during evolution, some host substances can act on viral proteins, inhibit viral replication, and resist infection. reportedly, the fmdv infection stimulates the expression of mir- , which indirectly induces the degradation of fmdv vp protein through the proteasome pathway and strengthens the host immune response to inhibit the replication of fmdv [ ] . moreover, the induction of mir- is earlier than the full activation of nf-κb and irf / [ ] . nonetheless, fmdv infection-stimulated expression of mir- would be a research hotspot in the future. however, the direct goal of mir- has not yet been determined. recently, it has been reported that tank-binding kinase (tbk ) degrades the vp protein of several types of small ribonucleic acid virus, including fmdv, through its kinase and e ubiquitin ligase activity, while p-tbk is highly enriched in the mir- overexpression cells [ ] . thus, mir- may target negative regulatory factors of tbk . it has been reported that single or co-transfection of micrornamir- a- p and mir- a- p in porcine cell lines followed by infection of fmdv resulted in a decrease in viral protein synthesis and virus production. so, mir- a- p and mir- a- p are potential natural biotherapies against foot-and-mouth disease virus [ ] . interaction between vp and hspb activates the eif s -atf pathway, which leads to autophagy and promotes virus replication [ ] . sorcin soluble resistance-related calcium binding protein vp can bind to sorcin to inhibit the activation of ikk and nf-κ b pathway [ ] . tumor progression locus ; a serine/threonine protein kinase vp inhibits the protein expression of tpl phosphorylation site thr , thereby inhibiting the promotion of irf -activated ifn-β signal by tpl . interferon regulatory factor vp suppresses ifn-β signaling pathway at irf level by inhibiting irf phosphorylation, dimerization, and nuclear translocation. ribosomal protein sa vp interacts with rpsa to maintain the activation of mapk signal pathway and promote virus replication [ ] . visa innate immune junction molecule vp inhibits the expression of visa protein mrna, and interacts with visa protein to inhibit the formation of visa-regulated complex, thereby inhibiting the dimerization and phosphorylation of irf [ ] . janus kinase vp can interact with jak protein and degrade jak protein to inhibit the activation of jak-stat pathway [ ] . fmdv l pro , a papain-like protease, is the first translated protein from the fmdv genome and coexists in two forms in vivo and in vitro, lab pro and lb pro [ ] [ ] [ ] . l pro , a key virulence factor of fmdv, suppresses the host immune response and achieves immune escape. l pro can cleave host translation-related proteins or interact with host transcription factors to inhibit the synthesis of antiviral factors. l pro can target cleavage/degradation pattern recognition receptors, the proteins of the interferon pathway, nf-κb pathway, and stress-related pathway. in addition, l pro acts as a deubiquitinase (dub) and deisgylase. first, l pro specifically cleaves the eukaryotic initiation factors (eifs), gi and gii. the loss of integrity of eif gi and eif gii hinders the formation of eukaryotic cellular translation initiation factor f(eif f) complex, while eif f complex significantly affects the cell cap-dependent translation [ ] , thereby preventing the recruitment of capped mrna in host cells and inhibiting the synthesis of antiviral molecules of innate immunity [ ] . fmdv rna starts translation in a cap-independent manner through the internal ribosome entry site (ires) elements [ ] [ ] [ ] . therefore, fmdv can make use of host protein synthesis mechanism to quickly synthesize virus protein and complete virus reproduction. the interaction between l pro and activity-dependent neuroprotective protein (adnp) is crucial in the process of infection and promotes fmdv replication by inhibiting the expression of ifn and ifn stimulated gene (isg) [ ] . however, whether the processing of adnp by l pro is performed directly by l pro or by related enzymes induced or activated by l pro is yet to be deduced. the present study further elucidates the mechanism though which fmdv evades the immune response by interaction with transcription factors. second, fmdv l pro downregulates the expression of nf-κb and irf / , which in turn, interferes with the transcription of ifn-α /β mrna. fmdv l pro induces the degradation of p /rela, the core component of nf-κb, which destroys the integrity of nf-κb and downregulates the transcription of ifn-β in host cells, thus inhibiting the host immune response [ ] . however, the degradation mechanism of p /rela by l pro is still unclear, and the products of p /rela digested by l pro have not been identified. in addition to destroying the integrity of nf-κb, l pro also decreased the expression of irf / , the key factors of virus-triggered ifn-α/β secretion that inhibit the production of dsrna-induced type i ifn [ ] . third, porcine ifn-λ , a type iii ifn, inhibits the replication of fmdv. however, after screening, the lead protease l pro exerts a robust inhibitory effect on the activity of ifn-λ promoter induced by poly(i:c) by inhibiting the rig-i/mda pathway and interfering with the activation of interferon regulatory factors (irfs) and nf-κb. fmdv l pro alone can inhibit the expression of dsrna-induced ifn-λ , suggesting a new mechanism of fmdv antagonizing ifn-λ -mediated innate immune response [ ] . fourth, in addition to its conventional papain-like protease activity, l pro acts as a deubiquitinase (dub) and deisgylase. lb pro has deubiquitination activity, and the deubiquitination functional sites are highly conserved among the seven serotypes of fmdv. these motifs could significantly inhibit the ubiquitination of key molecules of innate immune signaling pathway such as retinoic acid-inducible gene i (rig-i), tank-binding kinase (tbk ), tnf receptor-associated factor (traf ), and traf , thereby inhibiting the innate immune response and achieving immune escape [ ] . lb pro selectively cleaves the c-terminal peptide bond of isg and exposes an easily detected glygly epitope on the substrate of the modifier, which provides a new method for monitoring fmdv [ ] . a new study shows that abolishing/reducing the deisgylase/dub activity of l pro causes viral attenuation independently of its ability to block the expression of ifn and isg mrna [ ] . the latest research shows that l pro 's ability to cleave rlr signaling proteins but not its deubiquitination/deisgylation activity correlates with the reduced ifn-β gene transcription [ ] . fifth, laboratory of genetics and physiology (lgp ), an innate immune sensor promotes the interaction between viral rna and mda , thus producing antiviral signals [ ] . recently, it has been reported that lgp is the biphasic main activator of many innate immune genes that induce the production of ifn by a cascade effect [ ] . however, fmdv l pro cleaves lgp and blocks the effect of lgp -mediated ifn-β induction [ ] . these features represent a new approach of immune escape and provide a basis for in-depth research on the role of lgp in anti-fmdv response and the interaction between mda and lgp -l pro . sixth, recent studies have demonstrated that the stress response was inhibited during fmdv infection. fmdv l pro targets the sg (stress granule) scaffold proteins g bp and g bp to antagonize the formation of sg, a potentially significant antiviral signaling platform [ ] [ ] [ ] [ ] that regulates the integrated stress response [ ] . however, the l pro -mediated sg inhibition mechanism of fmdv might not be the only one in the cells infected with fmdv. although several studies have suggested the antiviral effect of sgs, their exact role as an antiviral signal platform is not yet clarified. therefore, these studies demonstrated that fmdv l pro inhibits the host immune response and promotes the fmdv replication through various mechanisms ( figure , table ). fmdv b protein is a hydrophobic transmembrane viroporin with oligomeric transmembrane pores that can destabilize the integrity of the host cell membrane, disrupt host cell ca + balance, induce host cell autophagy, and promote virion release [ , ] . fmdv b may play an active role in virus immune escape because of its viroporin characteristics. previous studies have shown three pattern recognition receptors, retinoic acid-inducible gene i (rig-i), melanoma differentiation-associated factor (mda ), and lgp that bind to viral rna. of these, rig-i and mda recognize different structures of rna to activate the antiviral signal transduction, and lgp regulates this process [ , , ] . targeted studies have found that rig-i and lgp inhibit the fmdv replication, and lgp significantly inhibits the inflammatory response of fmdv-infected cells. fmdv b protein suppresses the expression of pattern recognition receptors rig-i, mda , and lgp , inhibiting the host antiviral response and promoting fmdv replication. b protein directly interacts, reduces the protein levels, and inhibits the antiviral effect mediated by rig-i, mda , and lgp ( figure , table ). this reduction does not depend on proteasome, lysosome, or autophagy pathway, and the specific molecular mechanism is yet unclear. in addition, the study on whether b reduces the level of other junction molecules in rig-i like receptor (rlr) signaling pathway showed that although fmdv b does not mediate the decline in tbk and irf in the rlr signaling pathway, it inhibits the phosphorylation of tbk and irf , followed by suppression of the expression of type i ifn [ ] [ ] [ ] (figure , table ). the results of yeast two-hybrid screening and immunoprecipitation showed that one of the two host proteins that could interact with fmdv b protein is the eukaryotic translation elongation factor γ (eef g) [ ] . a previous study confirmed that the decrease in eef g affects the synthesis of some membrane proteins necessary for vesicle formation, and its mislocation reduces the synthesis of membrane proteins [ ] . the b protein of fmdv is associated with increased cell membrane integrity and membrane permeability [ ] . therefore, it can be speculated that eef g assists b in producing virus-induced vesicles and inducing cell lysis. however, further studies are required to substantiate these findings. the other host protein is cyclophilin a, which degrades fmdv l pro and a protein that suppresses the innate immune. strikingly, the interaction between b protein and cyclophilin a directly inhibits the degradation of l pro and a protein by cyclophilin a, thereby inhibiting the host immune response [ ] (figure , table ). recent studies have shown that cyclophilin a also promotes the ubiquitination of rig-i, and thus enhances the innate immune response [ ] . however, whether the interaction between b protein and cyclophilin a affects the ubiquitination of rig-i is yet to be elucidated. another study showed that fmdv b protein interacts with nod to reduce the expression of nod protein, for which the b carboxyl-terminal - region was essential, thus inhibiting the activation of nf-κb and ifn-β signaling pathways ( figure , table ). the decrease in nod is not related to the cleavage of eif g, induction of apoptosis or proteasome, nor lysosome or caspase pathways [ ] . fmdv protein c is a highly conserved polypeptide of amino acids (aa) [ , ] . subsequent studies proved that the c protein plays a critical role in virus replication. guanidine hydrochloride, a molecular antagonist of protein c, suppresses the synthesis of viral genetic material in small ribonucleic acid virus-infected cells [ , ] . three host proteins beclin , vimentin, and nod interacting with fmdv c, were screened by yeast two-hybrid assay. beclin is related to the formation of autophagosomes and the fusion of autophagosomes to lysosomes [ , ] . protein c interacts and inactivates beclin , which in turn, inhibits the fusion of autophagosomes containing fmdv and lysosomes, thereby preventing the degradation of the virus [ ] (figure , table ). vimentin plays a role in the lysosomal degradation of proteins and has been shown to be related to autophagosomes [ , ] . in the early stage of fmdv infection, vimentin forms a cage-like structure around c in order to facilitate virus replication. additionally, the expression of the dominant-negative (dn) form of vimentin significantly reduces the replication ability of fmdv. the replication of fmdv requires a complete vimentin network. however, the exact mechanism of governing vimentin cage formation and dissolution remains to be elucidated [ ] . the interaction between fmdv protein c and nod reduces the level of nod protein to help the virus evade the immune response, and the carboxyl-terminal - and - regions of c were essential for the interaction [ ] (figure , table ). strikingly, the interaction between c and beclin or nod evades immune response through different pathways, which contributes to the replication of fmdv. the interaction between c and cellular vimentin is crucial for the replication of fmdv, albeit the specific pathway is not yet clarified. the a protein is a conserved -aa polypeptide of fmdv, larger than other picornaviruses [ ] . fmdv a protein is related to host tropism and virulence, and the deletion of a alters the virus tropism and virulence. reportedly, a single amino acid change in a endowed fmdv with a new adaptive phenotype, and fmdv a protein is associated with membrane correlation and regulation of host protein secretion [ , ] . plaque assay and virus titeration showed that the stable expression of a or ab protein enhances the replication of fmdv. however, the infection level of fmdv decreased after the transient expression of ab protein. these findings suggested that a and ab play a crucial role in the replication of fmdv [ ] . fmdv a has been proved to interacts with cellular protein dctn by the two-hybrid method. dctn is a subunit of the dynactin complex, a cofactor for dynein. dynactin-dynein complexes are related to the transport of intracellular organelles. the overexpression of dctn and the disruption of dynactin-dynein complex significantly reduces the production of fmdv in infected cells. fmdv replication seems to require a complete dynamic protein cell pathway [ ] . in the previous study, fmdv a protein mutantageness study based on reverse genetic technique revealed the effect of amino acid mutation in a protein on the interaction between a protein and dctn was detected by yeast two-hybrid technique. the data show that a could be bound to dctn when amino acid was alanine or leucine, but when amino acid was mutated to proline, which destroy the binding between a and dctn [ ] . interestingly, both the fmdv o/taw/ strain with pldg peptide from - aa on a and the recombinant fmdv o c a virus with deletion of residues - on a lacked the binding site of the host protein dctn . also, the replication rate in primary bovine cells was slower than that of parental virus strains. however, no significant change was detected in the replication rate of either of the two viruses and their parent virus strains in porcine cells [ , ] . thus, it could be speculated that the binding of fmdv a and host dctn might be related to the host tropism of the virus, but the slight difference in dctn among species could be attributed to the range of hosts of fmdv. the recombinant fmdv with pldg residues - on a produced a delayed and mild disease in cattle, suggesting that a-dctn interaction might play a role in the virulence of the bovine virus. in addition, the virus recovered rapidly during infection and regained dctn binding, suggesting that the interaction between fmdv a and dctn is vital for virus replication in cattle [ ] (figure , table ). however, the a-dctn combination needs further exploration. the type i ifn reporting system was utilized to confirm that the a protein inhibits the activation of the ifn-β signaling pathway. further studies demonstrated that a protein interacted with innate immune molecules rig-i, mda , and visa, inhibited the expression of innate immune junction molecules, such as rig-i, mda , and visa, and inhibited the formation of signal transduction complex, thereby escaping the host innate immune system [ ] (figure , table ). the overexpression of the fmdv a inhibited the sendai virus-triggered activation of irf [ ] . a recent study found that the interaction between dead-box helicase (ddx ) and fmdv protein a increases the interaction between ddx and irf and enhances the ability of fmdv a to inhibit irf phosphorylation ( figure , table ). also, fmdv a inhibits the activation of the ifn-β promoter and isre by reducing the phosphorylation of irf and increasing the replication of fmdv. however, the overexpression of ddx cannot significantly reduce the phosphorylation of irf . thus, we speculated that ddx also inhibits the production of ifn through another different pathway, thereby promoting virus replication [ ] . unlike other picornaviruses that encode a single b copy, fmdv encodes three similar but different b proteins ( b , b , and b ), which are ubiquitous in all fmdv isolates [ ] . the effective replication of fmdv in bovine cells requires a full-length a and three vpg ( b). the deletion of the b coding sequence adversely affects the rna replication of fmdv, and the viral activity requires highly conserved b protein [ , ] . although fmdv lacking b and b can also reduce the viral rna synthesis, the growth of the virus on pig-derived cells is only slightly reduced, and the disease of pigs has also been slightly alleviated [ , ] . these studies showed that b is more critical than b and b in maintaining virus rna replication. the efficiency of rna replication is maximal when three b copies coexist, and the absence of b and b might affect the virulence and host range of fmdv. however, the integration of the three proteins in replication needs to be studied further. fmdv b significantly reduces the levels of ifn-β, isg , and il- levels and the activation of ifn-β, nf-κb, and isre promoters induced by poly(i:c), indicating that fmdv b is also a viral escape protein, which inhibits the response of type i ifn in cells. subsequently, b blocked the interaction between rig-i and trim , thus inhibiting the ubiquitination of rig- and the formation of the rig-i-visa complex, which inhibits the ifn signaling pathway [ ] (figure , table ). further studies have shown that fmdv b reduces the expression of type i ifn by inhibiting the visa signaling pathway via interaction with the visa protein ( figure , table ). fmdv c protein has been identified as a protease. it cleaves not only most of the virus precursor proteins [ ] but also the host proteins to block/inhibit the cellular defense mechanism and promote virus replication. fmdv protein c pro cleaves the related host proteins, inhibits the transcription and translation of host cells, or promotes the translation of virus rna, thus promoting virus replication. for example, fmdv protein c pro is related to the cleavage of histone h , as it removes n-terminal amino acid residues from histone h . the amino terminal of h is related to the regulation of chromosomal transcriptional activity in eukaryotic cells. the cleavage of cpro to h inhibits the transcription of host cells and ultimately hinders the translation of host cells [ ] , which is beneficial for the virus to escape antiviral immune response ( figure , table ). in addition, fmdv protein c pro also cleaves the host translation initiation factors, eif g and eif a, which are components of the cap-binding complex eif f, which inhibits the synthesis of host-related antiviral proteins [ ] ( figure , table ). however, it can only partially cleave eif g and eif a but not completely block the translation of host cells [ , ] . unlike the cleavage of eif g by l pro in the early stage of infection, the cleavage of eif g and eif a by fmdv protein cpro requires the accumulation of c protein. hence, it occurs in the later stage of infection cycle, and the cutting site of eif g by c pro is different from that mediated by l pro [ ] . in addition, the cleaving of eif a may be disadvantageous to the virus, and the translation of viral rna requires eif a [ ] . the fmdv protein c pro can also cleave the -kda src-associated substrate during mitosis (sam ), a unique rna-binding protein (figure , table ). truncated sam spreads to the cytoplasm, combines with fmdv rna and attaches to ires to enhance the translation of the virus rna. however, the titer of fmdv was reduced -fold after transfection of sam -targeted sirna molecules, indicating sam might not limit to enhancing virus translation, and sam may play a variety of roles in fmdv infection [ ] . fmdv protein c pro directly or indirectly degrades vital immune molecules, thus inhibiting/blocking the expression of ifn and antiviral genes. fmdv protein c pro can degrade pattern recognition receptors, rig-i and lgp , thus inhibiting the production of antiviral factors and promoting fmdv replication [ , ] ( figure , table ). it also degraded the modulator necessary for innate immune key molecule nemo, the vital regulator of nf-κb, a junction protein necessary for activating nf-κb, and the ifn regulatory factor signaling pathway. this cleavage impaired the activation of irfs and nf-κb and inhibited the expression of downstream antiviral genes ( figure , table ). the cysteine protease activity of c pro is necessary for c pro to cleave nemo [ ] . reportedly, fmdv c suppresses the jak-stat signaling pathway, thus inhibiting the antiviral response induced by ifn. another study found that c protease activity promoted the degradation of kpna , the nuclear localization signal receptor for tyrosine-phosphorylated stat , to block the nuclear translocation of stat /stat to inhibit the jak-stat signaling pathway [ ] (figure , table ). autophagy-associated protein atg -atg is shown to be associated with the replication of fmdv. in the process of fmdv infection, atg -atg upregulates the anti-virus nf-κb and irf signaling pathways, thereby inhibiting the proliferation of fmdv. fmdv protein c pro antagonizes the host antiviral immunity and suppresses autophagy by degrading atg -atg [ ] (figure , table ). furthermore, atg -atg enhances the expression of host protein kinase pkr, a serine-threonine kinase, which can be induced by ifn and activated by double-stranded rna (dsrna). consequently, it blocks the synthesis of cell and virus protein, inhibits the replication of fmdv, and exerts a significant antiviral effect [ ] . moreover, fmdv c pro protein induces pkr degradation through the lysosome pathway and inhibits the pkr-mediated antiviral effect by downregulating the pkr protein. no interaction occurred between fmdv c pro and pkr [ ] (figure , table ). recent studies have shown that c pro decreases the level of nod , thus inhibiting the antiviral effect induced by nod , and the reduction of nod induced by c pro depends on its protease activity ( figure , table ). no interaction occurred between fmdv c pro and nod [ ] . taken together, fmdv c pro exerts its immunosuppressive function and suppresses the innate immunity of the host via a myriad of cascades. can directly or indirectly act on retinoic acid-induced gene i-like receptor (rlr) to inhibit innate immunity. fmdv a and b reduce the expression of junction protein visa at the transcriptional or protein level. l pro , b and a can directly or indirectly target irf to inhibit interferon production. c proteins inhibit jak-stat signaling pathway, thus inhibiting isgs production. l pro and c inhibit the synthesis of antiviral molecules by cutting related factors of host transcription and translation. l pro protein can not only induce apoptosis, but also inhibit host cell apoptosis and promote virus replication, which is achieved by blocking the translation of α-ifn and inhibiting pkr synthesis. c can promote virus replication by regulating autophagy. eif g: eukaryotic initiation factor g; nf-κb: nuclear factor kappa b; irf : interferon regulatory factor ; irf : interferon regulatory factor ; rig-i: retinoic acid inducible gene i; tbk : tank binding kinase i; traf : tnf receptor-associated factor ; traf : tnf receptor-associated factor ; adnp: activity-dependent neuroprotective protein; lgp : laboratory of genetics and physiology ; mda : melanoma differentiation associated factor ; cypa: cyclophilin a; nod : nucleotide-binding oligomerization domain ; dctn : dynactin ; ddx : dead-box helicase ; sam : kda src-associated substrate during mitosis; visa: virus-induced signaling adapter. can directly or indirectly act on retinoic acid-induced gene i-like receptor (rlr) to inhibit innate immunity. fmdv a and b reduce the expression of junction protein visa at the transcriptional or protein level. l pro , b and a can directly or indirectly target irf to inhibit interferon production. c proteins inhibit jak-stat signaling pathway, thus inhibiting isgs production. l pro and c inhibit the synthesis of antiviral molecules by cutting related factors of host transcription and translation. l pro protein can not only induce apoptosis, but also inhibit host cell apoptosis and promote virus replication, which is achieved by blocking the translation of α-ifn and inhibiting pkr synthesis. c can promote virus replication by regulating autophagy. eif g: eukaryotic initiation factor g; nf-κb: nuclear factor kappa b; irf : interferon regulatory factor ; irf : interferon regulatory factor ; rig-i: retinoic acid inducible gene i; tbk : tank binding kinase i; traf : tnf receptor-associated factor ; traf : tnf receptor-associated factor ; adnp: activity-dependent neuroprotective protein; lgp : laboratory of genetics and physiology ; mda : melanoma differentiation associated factor ; cypa: cyclophilin a; nod : nucleotide-binding oligomerization domain ; dctn : dynactin ; ddx : dead-box helicase ; sam : kda src-associated substrate during mitosis; visa: virus-induced signaling adapter. l pro cut eif gi and eif gii, thus preventing the recruitment of capped mrna and inhibiting the synthesis of antiviral molecules [ , ] . nf-κb nuclear factor kappa b l pro induce the degradation of p /rela, which is the core component of nf-κb [ ] . interferon regulatory factor / l pro decreased the expression of irf / to inhibit the production of type i ifn induced by dsrna [ ] . rig-i tbk traf traf retinoic acid inducible gene i; tank binding kinase i; tnf receptor associated factor tnf receptor associated factor l pro can significantly inhibit the ubiquitination of key molecules of innate immune signaling pathway such as rig-i, tbk , traf , and traf [ ] . adnp activity dependent neuroprotective protein l pro and adnp interact to promote the replication of fmdv by inhibiting the expression of ifn and isg [ ] . lgp laboratory of genetics and physiology l pro can cleave lgp and block the effect of lgp -mediated the production of ifn-β [ ] . g bp and g bp stress granule scaffold proteins l pro targets to cleave the sg scaffold proteins g bp and g bp to antagonize the formation of sg [ ] . retinoic acid inducible gene i; melanoma differentiation associated factor ; laboratory of genetics and physiology ; b protein suppress the expression of rig-i, mda and lgp , inhibiting host antiviral response [ , ] . tbk ; irf tank binding kinase i; interferon regulatory factor b suppress the phosphorylation of tbk and irf , and then inhibit the expression of type i interferon [ ] . cypa cyclophilin a the interaction between b protein and cyclophilin a directly inhibits the degradation of l pro and a protein by cyclophilin a [ ] . nod nucleotide-binding oligomerization domain b protein can interact with nod to reduce the protein level of nod , which inhibit the activation of nf-κb and ifn-β signal pathways [ ] . beclin involve in the fusion of autophagosomes to lysosomes c interacts with beclin to induce beclin inactivation, which inhibits the fusion of autophagosomes of containing fmdv and lysosomes [ ] . nod nucleotide-binding oligomerization domain the interaction between fmdv protein c and nod reduces nod at the protein level to help the virus evade immune response [ ] . a protein increases the interaction between ddx to inhibits the activation of ifn-β promoter and isre by reducing the phosphorylation of irf [ ] . retinoic acid inducible gene i; b blocked the interaction between rig-i and trim , thus inhibiting the interferon signal pathway [ ] . visa virus-induced signaling adapter b reduces the expression of type i interferon by inhibiting visa signal pathway by interacting with visa protein. histone h related to the transcription of host cells the cleavage of c pro to h inhibits the transcription of host cells and ultimately hinders the translation of host cells [ ] . eif g and eif a host translation initiation factors c pro is involved in the cleavage of eif g and eif a, thus inhibiting the synthesis of host-related antiviral proteins [ ] . kda src-associated substrate during mitosis c pro can also cleave sam . truncated sam spreads to the cytoplasm and meets the fmdv rna and attaches to the ires to enhance the translation of the virus rna [ ] . retinoic acid inducible gene i; laboratory of genetics and physiology ; protein c pro can degrade rig-i and lgp [ , ] . nemo nf-κb necessary regulator c can also degrade nemo to impaired activation of irfs and nf-κb [ ] . kpna the nuclear localization signal receptor for tyrosine-phosphorylated stat c promoted the degradation of kpna to block the nuclear translocation of stat /stat to inhibit jak-stat signal pathway [ ] . autophagy associated protein fmdv protein c pro antagonizes host antiviral immunity and suppresses autophagy by degrading atg -atg [ ] . pkr a serine-threonine kinase c pro induces pkr degradation and inhibits pkr-mediated antiviral effect by down-regulating pkr protein [ ] . nod nucleotide-binding oligomerization domain c pro induces the reduction of nod , thus inhibiting the antiviral effect induced by nod [ ] . the untranslated region ( utr) of fmdv is about bp, which is larger than that of several small rna virus of the family picornaviridae. it contains s-fragment, poly (c), pseudoknots (pks), the cis-acting replication element (cre) and internal ribosome entry site (ires). studies have shown that the special structure of the untranslated region is essential for the translation and replication of the virus genome [ ] . the first element of the end is the s-fragment with approximately nt long, the sequence can fold and form the stem ring. it is speculated that this structure can block the function of host exonuclease, thereby maintaining the stability and replication of the virus genome [ ] . s fragment is related to the interaction between virus and host protein. some studies have confirmed the direct correlation between the degree of s fragment deletion mutation and the attenuated phenotype. the fmdv mutant with bp deletion in the upper part of the s fragment loop was highly attenuated in vivo [ ] . fmdv with deletion of s fragment induces higher expression of ifn-β and isg mrna, so it is concluded that s fragment of fmdv is necessary for host cell replication and regulation of innate immune response [ ] . downstream of s-fragment is the variable length poly (c) region. studies on the viral genome have shown that a certain threshold length of poly (c) is correlated to the viability of the virus, but there is no evidence that the length of the poly (c) chain is directly related to virulence [ ] . the end of ploy (c) is pseudoknots. some studies have shown that the deletion of -nt in pks reduces the pathogenicity of o/cha/ / strain of fmdv in bovine cells and bovines, and the artificial deletion of bases will reduce the pathogenicity of o/me-sa/panasia strain fmdv in bovine. this deletion occurs naturally in the region of the porcinophilic cathay topotype fmdv genome. it is suggested that the natural absence of pks area may be the reason for the transformation from bovines to pigs as a vector for the transmission of fmdv. it is concluded that the pseudoknots region of fmdv untranslated region is the decisive factor of virus tendency and virulence [ ] . the cis-acting replication element (cre) is a stem-loop structure containing conserved aaaca motifs [ , ] . it has been confirmed that fmdv cre is necessary for genome replication, and cre has been found to be adjacent to ires, which indicates that cre may play a role in coordinating translation and replication, but this still needs further confirmation [ ] . fmdv -utr consists of a nt structural sequence folded into two independent stem loops and a poly (a) tail of variable length [ , ] . related studies have shown that the -utr directly binds to the s-fragment and ires elements of the -utr at different sites, and fmdv -utr affects virus replication and virulence by enhancing the activity of ires elements [ ] . moreover, genetic studies have shown that the recombinant fmdv with missing structural sequence of -utr cannot be recovered [ ] , and it is concluded that the structural region of -utr is essential for the infectivity and replication of fmdv. in addition to the direct rna-rna interaction, - -end bridging, which is also related to protein-protein and protein-rna interactions, it has been found that cellular proteins pcbps and p can directly bind to s-fragment and -utr [ ] . it is inferred that - -end bridging may play an important role in the replication of fmdv. fmdv is a highly contagious virus that infects almost all cloven-hoofed animals, showing vesicles on the foot and mouth, skin erosion on the mucous membranes, fever, weight loss, pacing, and salivation, severely threatening the development of animal husbandry. however, in addition to causing acute infections and diseases, fmdv can be asymptomatic carriers in some cases, which might lead to another outbreak of fmd, making prevention and control challenging and costly. high infectivity, wide geographical distribution, wide host range, short-term immunity without serotype cross-protection, multiple modes of transmission, and persistent infection render the control and eradication of this disease rather difficult. therefore, study the molecular mechanism underlying fmdv evading immunity is imperative for the control of an epidemic situation. the immune system includes innate immunity and acquired immunity, which is a major protective system against the invasion of pathogenic microorganisms, surveillance, and removal of foreign bodies. fmdv suppresses the function of the immune system at the initial stage of infection, such that the virus can proliferate rapidly in the respiratory system and spread to its natural infection site [ ] . in terms of evading the humoral immune system, each serotype of fmdv is prone to antigenic variation, which makes the virus escape from the neutralizing antibodies [ ] . in the aspect of inhibiting cellular immune response, fmdv infection can cause the decrease of host lymphocytes and is accompanied by severe viremia, which will eventually lead to the destruction of t cells and fmdv infection inhibits the function of dendritic cells and weakens the ability of dendritic cells to process them into antigens [ , ] . previous studies have shown that, mhc class i molecule expression on the surface of cells was suppressed at min after fmdv infection, indicating that the cells infected with fmdv will immediately lose the ability to present mhc-i-related viral peptides to t lymphocytes. this mechanism would facilitate the virus escape from the host's cytotoxic immune response. limiting the killing effect mediated by nk cells is also an important mechanism for fmdv to evade the cellular immune response. some studies have shown that the responsiveness of porcine nk cells decreases significantly - days after fmdv infection, and then returns to normal [ ] . strikingly, nk cells isolated from infected pigs could not secrete ifn-γ [ ] . the research on fmdv interference with immune effect and suppression of innate immunity has been widely studied. some proteins of fmdv (l pro , b, a, b, c) can directly or indirectly act on retinoic acid-induced gene i-like receptor (rlr) to inhibit innate immunity [ , [ ] [ ] [ ] ] . fmdv vp , vp , a, and b reduce the expression of junction protein visa at the transcriptional or protein level [ , , ] . fmdv l pro , vp , vp , b, and a can directly or indirectly target irf to inhibit interferon production [ , , , ] . vp and c proteins inhibit jak-stat signaling pathway, thus inhibiting isgs production [ , ] . fmdv proteins l pro and c inhibit the synthesis of antiviral molecules by cutting related factors of host transcription and translation [ , , , , ] . in addition, it is interesting that l pro protein can not only induce apoptosis, but also inhibit host cell apoptosis and promote virus replication, which is achieved by blocking the translation of α-ifn and inhibiting pkr synthesis [ ] . fmdv protein vp and c can promote virus replication by regulating autophagy [ , ] . these mechanisms provide opportunities for rapid transcription and translation of fmdv. in the previous studies on fmdv, hek cells have been widely used in in vitro experiments because of its highly transfected efficiency. however, hek cells are not fmdv susceptible cells and there are species differences between hek cells and fmdv susceptible cells. therefore, the use of hek cells for fmdv-related research has some limitations. in summary, fmdv has evolved a variety of ways to evade the immune response in the long-term combat with the host immune system. although there are many breakthroughs in the research on the immune escape of fmdv, many mechanisms underlying the fmdv-affected host immunity have not yet been elucidated, and the interaction between fmdv protein and host protein need to be explored further. in addition to the interaction between the virus and host protein, exploring the mechanism of synergistic inhibition of immune response by multiple viral proteins is of great significance for the development of specific drugs and new vaccines. previous studies mainly focused on the effect of fmdv with respect to innate immunity. however, there are a few studies on acquired immunity, and these need to be supplemented further. also, persistent infection of fmdv needs to be investigated intensively in the future. it was the goal of the literature study to summarize the current knowledge and to point out future research directions and define the scientific questions that remain to be elucidated to gain better knowledge of immune responses against fmdv and its immune escape mechanisms. foot-and-mouth disease: host range and pathogenesis foot-and-mouth disease foot-and-mouth disease: past, present and future molecular epidemiology of foot-and-mouth disease virus the generation and persistence of genetic variation in foot-and-mouth disease virus foot-and-mouth disease virus evolution: exploring pathways towards virus extinction emergence and selection of rna virus variants: memory and extinction understanding the molecular epidemiology of foot-and-mouth-disease virus molecular basis of pathogenesis of fmdv viral immune evasion: a masterpiece of evolution immune evasion during foot-and-mouth disease virus infection of swine engagement of soluble resistance-related calcium binding protein (sorcin) with foot-and-mouth disease virus (fmdv) vp inhibits type i interferon response in cells interaction between fmdv lpro and transcription factor adnp is required for optimal viral replication ddx cooperates with fmdv a to enhance fmdv replication by inhibiting the phosphorylation of irf activation of innate immune antiviral responses by nod foot-and-mouth disease virus antagonizes nod -mediated antiviral effects by inhibiting nod protein expression the structure of foot-and-mouth disease virus structural protein vp of foot-and-mouth disease virus inhibits type i interferon signaling pathway poly (rc) binding protein interacts with vp and increases the replication of the foot-and-mouth disease virus pcbp mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip dissecting the roles of vp cleavage and rna packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus the structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex construction and immunogenicity of dna vaccine plasmid expressing vp and vp genes of foot-and-mouth disease virus. chin detection and characterization of functional t-cell epitopes on the structural proteins vp , vp , and vp of foot and mouth disease virus o campos molecular design and immunogenicity of a multiple-epitope fmdv antigen and dna vaccination the influence of mhc polymorphism on the selection of t-cell determinants of fmdv in cattle induction of protective immunity by dna vaccination with toxoplasma gondii hsp , hsp and sag genes protein expression profile of mast cells in response to recombinant vp -vp of foot-and-mouth disease virus nm -h suppresses metastasis by inhibiting expression of the lysophosphatidic acid receptor edg cell-permeable nm blocks the maintenance and progression of established pulmonary metastasis implication of metastasis suppressor nm -h in maintaining adherens junctions and limiting the invasive potential of human cancer cells emerging roles of p and other tumour-suppressor genes in immune regulation transcriptional role of p in interferon-mediated antiviral immunity foot-and-mouth disease virus induces lysosomal degradation of nme to impair p -regulated interferon-inducible antiviral genes expression autophagy: more than a nonselective pathway selective autophagy: ubiquitin-mediated recognition and beyond autophagy, immunity, and microbial adaptations autophagy, antiviral immunity, and viral countermeasures autophagy and viruses: adversaries or allies? coronavirus nsp restricts autophagosome expansion porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication identification of neutralizing antigenic sites on vp and vp of type a foot-and-mouth disease virus, defined by neutralization-resistant variants antigenic heterogeneity of a foot-and-mouth disease virus serotype in the field is mediated by very limited sequence variation at several antigenic sites neutralizing epitopes of type o foot-and-mouth disease virus. i. identification and characterization of three functionally independent, conformational sites effects of amino acid substitutions in the vp b-c loop on antigenicity and pathogenicity of serotype asia foot-and-mouth disease virus foot-and-mouth disease virus capsid protein vp activates the cellular eif s -atf pathway and induces autophagy via hspb divergent roles of autophagy in virus infection autophagy in antiviral innate immunity autophagy and innate immunity: triggering, targeting and tuning the atg -atg conjugate associates with innate antiviral immune responses perturbations in the surface structure of a iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity a positively charged lysine residue at vp position allows for the enhanced adaptability of foot-and-mouth disease virus serotype a in bhk- cells single amino acid substitution of vp n d or vp h y confers acid-resistant phenotype of type asia foot-and-mouth disease virus mutation in the vp gene of p - a capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype o enhanced immune response of dna vaccine (vp -pcdna) adsorbed on cationic plg for foot and mouth disease in guinea pigs induction of a cross-reactive cd + t cell response following foot-and-mouth disease virus vaccination vp of foot-and-mouth disease virus induces apoptosis via the akt signaling pathway foot-and-mouth disease virus capsid protein vp interacts with host ribosomal protein sa to maintain activation of the mapk signal pathway and promote virus replication the vp structural protein of foot-and-mouth disease virus inhibits the ifn-β signaling pathway foot-and-mouth disease virus structural protein vp degrades janus kinase to inhibit ifn-gamma signal transduction pathways host microrna mir- suppresses foot-and-mouth disease virus replication by promoting vp degradation and enhancing innate immune response the e ubiquitin ligase tbk mediates the degradation of multiple picornavirus vp proteins by phosphorylation and ubiquitination host microrna- a is antagonistic to the progression of foot-and-mouth disease virus infection two initiation sites for foot-and-mouth disease virus polyprotein in vivo the two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities identification of critical amino acids within the foot-and-mouth disease virus leader protein, a cysteine protease sonenberg, n. eif initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation foot-and-mouth disease virus leader proteinase involvement of c-terminal residues in self-processing and cleavage of eif gi divergent picornavirus ires elements extremely efficient cleavage of eif g by picornaviral proteinases l and a in vitro l protease from foot and mouth disease virus confers eif -independent translation for mrnas bearing picornavirus ires degradation of nuclear factor kappa b during foot-and-mouth disease virus infection foot-and-mouth disease virus leader proteinase inhibits dsrna-induced type i interferon transcription by decreasing interferon regulatory factor / in protein levels foot-and-mouth disease virus (fmdv) leader proteinase negatively regulates the porcine interferon-λ pathway the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase irreversible inactivation of isg by a viral leader protease enables alternative infection detection strategies impairment of the deisgylation activity of foot-and-mouth disease virus lpro causes attenuation in vitro and in vivo dissecting distinct proteolytic activities of fmdv lpro implicates cleavage and degradation of rlr signaling proteins, not its deisgylase/dub activity, in type i interferon suppression lgp synergy with mda in rlr-mediated rna recognition and antiviral signaling pum is a biphasic negative regulator of innate immunity genes by suppressing lgp innate immune sensor lgp is cleaved by the leader protease of foot-and-mouth disease virus stress granules and cell signaling: more than just a passing phase? translation inhibition and stress granules in the antiviral immune response stress granules regulate double-stranded rna-dependent protein kinase activation through a complex containing g bp and caprin the stress granule protein g bp recruits protein kinase r to promote multiple innate immune antiviral responses foot-and-mouth disease virus leader protease cleaves g bp and g bp and inhibits stress granule formation viroporin activity of the foot-and-mouth disease virus non-structural b protein functional analysis of picornavirus b proteins: effects on calcium homeostasis and intracellular protein trafficking differential roles of mda and rig-i helicases in the recognition of rna viruses viral rna detection by rig-i-like receptors foot-and-mouth disease virus viroporin b antagonizes rig-i-mediated antiviral effects by inhibition of its protein expression foot-and-mouth disease virus infection inhibits lgp protein expression to exaggerate inflammatory response and promote viral replication foot-and-mouth disease virus non-structural protein b negatively regulates the rlr-mediated ifn-β induction eef g interaction with foot-and-mouth disease virus nonstructural protein b: identification by yeast two-hybrid system kinectin-dependent assembly of translation elongation factor- complex on endoplasmic reticulum regulates protein synthesis foot-and-mouth disease virus nonstructural protein b interacts with cyclophilin a, modulating virus replication cyclophilin a-regulated ubiquitination is critical for rig-i-mediated antiviral immune responses the picornavirus replication inhibitors hbb and guanidine in the echovirus- system: the significance of viral protein c testing the modularity of the n-terminal amphipathic helix conserved in picornavirus c proteins and hepatitis c ns a protein guanidine-resistant mutants of aphthovirus induce the synthesis of an altered nonstructural polypeptide, p recombination and oligonucleotide analysis of guanidine-resistant foot-and-mouth disease virus mutants targeting beclin for viral subversion of macroautophagy the beclin network regulates autophagy and apoptosis foot-and-mouth disease virus nonstructural protein c interacts with beclin , modulating virus replication a putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. studies with transplanted sendai-viral envelope proteins in htc cells intracellular protein catabolism: evidence for sequestration of proteins into an intermediate-filament fraction before lysosomal degradation foot-and-mouth disease virus modulates cellular vimentin for virus survival role of nonstructural proteins a and b in host rangeand pathogenicity of foot-and-mouth diseasevirus susceptibility to viral infection is enhanced by stable expression of a or ab proteins from foot-and-mouth disease virus interaction of foot-and-mouth disease virus nonstructural protein a with host protein dctn is important for viral virulence in cattle a partial deletion in non-structural protein a can attenuate foot-and-mouth disease virus in cattle evaluation of infectivity and transmission of different asian foot-and-mouth disease viruses in swine foot-and-mouth disease virus non-structural protein a inhibits the interferon-β signaling pathway comparative genomics of foot-and-mouth disease virus vpg gene amplification correlates with infective particle formation in foot-and-mouth disease virus domain disruptions of individual b proteins of foot-and-mouth disease virus do not alter growth in cell culture or virulence in cattle foot-and-mouth disease virus b protein inhibits type i interferon pathway signaling by blocking the interaction of rig-i with trim foot-and-mouth disease virus protease c induces specific proteolytic cleavage of host cell histone h foot-and-mouth disease virus c protease induces cleavage of translation initiation factors eif a and eif g within infected cells cleavage of translation initiation factor ai (eif ai) but not eif aii by foot-and-mouth disease virus c protease: identification of the eif ai cleavage site sequential modification of translation initiation factor eif gi by two different foot-and-mouth disease virus proteases within infected baby hamster kidney cells: identification of the cpro cleavage site dominant negative mutants of mammalian translation initiation factor eif- a define a critical role for eif- f in cap-dependent and cap-independent initiation of translation the nuclear protein sam is cleaved by the fmdv c protease redistributing sam to the cytoplasm during fmdv infection of host cells foot-and-mouth disease virus c protease cleaves nemo to impair innate immune signaling cpro of foot-and-mouth disease virus antagonizes the interferon signaling pathway by blocking stat /stat nuclear translocation foot-and-mouth disease virus infection suppresses autophagy and nf-kb antiviral responses via degradation of atg -atg by cpro regulation of innate immunity through rna structure and the protein kinase pkr foot-and-mouth disease virus induces lysosomal degradation of host protein kinase pkr by c proteinase to facilitate virus replication biological function of foot-and-mouth disease virus non-structural proteins and non-coding elements foot-and-mouth disease virus -terminal s fragment is required for replication and modulation of the innate immune response in host cells infectious foot-and-mouth disease virus derived from a cloned full-length cdna the pseudoknot region of the untranslated region is a determinant of viral tropism and virulence of foot-and-mouth disease virus the rhinovirus type genome contains an internally located rna structure that is required for viral replication identification of an rna hairpin in poliovirus rna that serves as the primary template in the in vitro uridylylation of vpg identification and characterization of a cis-acting replication element (cre) adjacent to the internal ribosome entry site of foot-and-mouth disease virus translation and replication of fmdv rna the end of the foot-and-mouth disease virus genome establishes two distinct long-range rna-rna interactions with the end region enhanced ires activity by the utr element determines the virulence of fmdv isolates deletion or substitution of the aphthovirus ncr abrogates infectivity and virus replication the pathogenesis of foot-and-mouth disease in pigs morphologic and phenotypic characteristics of myocarditis in two pigs infected by foot-and mouth disease virus strains of serotypes o or a. acta veter effect of foot-and-mouth disease virus infection on the frequency, phenotype and function of circulating dendritic cells in cattle loss of plasmacytoid dendritic cell function coincides with lymphopenia and viremia during foot-and-mouth disease virus infection natural killer cell dysfunction during acute infection with foot-and-mouth disease virus cell mediated innate responses of cattle and swine are diverse during foot-and-mouth disease virus (fmdv) infection: a unique landscape of innate immunity discriminating self and non-self by rna: roles for rna structure, misfolding, and modification in regulating the innate immune sensor pkr the authors would like to thank the anonymous editors and reviewers for their valuable comments and suggestions that helped improve the quality of this manuscript. the authors declare no conflict of interest. key: cord- -k edtg authors: nan title: aasld abstracts (pp. a– a) date: - - journal: hepatology doi: . /hep. sha: doc_id: cord_uid: k edtg nan of well-characterized human hepatocyte cell lines could facilitate cell therapies such as htx and bioartificial livers. toward this goal, we have developed a tightly regulated human hepatocyte line. methods human hepatocytes were immortalized with a retroviral vector encoding human telomerase reverse transcriptase (htert) and green fluorescent protein (gfp) cdnas flanked by a pair of loxps. after retroviral transduction, a single cell clone was obtained using flow cytometric cell sorting followed by limiting dilution method. the resultant gfp-positive clones were supertransduced with tamoxifen-inducible cre recombinase expression cassette, mercremer. recovery of the reverted form of htert-immortalized cells was conducted by tamoxifen treatment and subsequent gfp-negative cell sorting with moflo. the expression of hepatocyte markers and albumin secretion was compared before and after tamoxifen treatment. transplantation effect of the reverted cells (lx lo ) into the liver were evaluated in a pig model of liver failure induce by an intravenous administration of d-galactosamine (d-gal). resu ts:a non-tumorigenic human hepatocyte cell line ttnt- -t was established. albumin secretion and gene expression of liver markers were increased in the reverted ttnt- -t cells. transplantation of the reverted cells via the portal vein of pigs treated with d-gal was effective to prolong the survival by providing liver support until spontaneous hepatic regeneration occurred. conclusion: we here demonstrate reversible immortalization of human hepatocytes using tamoxifen-induced crellox self-excision. the resulting cell line would be highly desirable for research and therapeutic applications. acknowledgmenk the work was supported in part by life science project of lst century, japan. background and aim: stem cells play a key-role in tissue homeostasis. haematopoietic stem cells (hscs) could migrate into the liver and transdifferentiate, becoming a "liver stem cell reserve''. we aimed to analyse: human hsc (h-hsc) ability to engraft into the mouse liver and to contribute to its regeneration after a toxic damage. materials and methods: nodiscid mice were so divided: a) chimeric not-damaged mice: mice were submitted to irradiation followed by h-hsc intraperitoneally (i.p.) injection; finally they were killed , and days after; ) chimeric damaged mice: mice were irradiated, injected with h-hscs and, days after, damaged using ally alcohol (aa), i.p.; killing was performed , and days after the damage; c) damaged not-chimeric mice: mice were damaged (with aa injection) and killed , and days after; d) not-chimeric not-damaged mice: mice were killed without any treatment. spleens (s), bone marrows (bm) and livers (l) were tested by flow-cytometry to detect h-hscs and immunohistochemistry (l only) to localize h-hscs and their hepatic derivatives. results: flow-cytometry: group a) presence of low percentage of human cells; groups c) and d) human cells undetectable; group b) presence of low percentage of human cells in bm and s while in the l they represented up to % of whole cellular population (the difference respect to the group a has high statistical significance, being alpha< . %). immunohistochemistry showed the human cell differentiation into parenchymal cells. conclusions: our results suggest that hscs could be an option to improve thie liver regeneration after a toxic injury, supporting the feasibility of bm-derived liver stem cell hypothesis. we have previously demonstrated that cd -mediated immune damage of allogeneic liver parenchymal cells is difficult to regulate due to the activity of "immunoresistant" cd + t cells. in prior studies we demonstrated that short-term interference with cd cd l (but not cd b ) costimulation transiently suppressed cdb-dependent hepatocyte rejection. recently, we have determined that targetintg lfa- alone preferentially suppresses cd dependent versus cd -dependent hepatocyte rejection. in addition, short-term immunotherapy targeting both lfa- and cd lcd l costimulation produced synergistic effects and resulted in longterm suppression of cd -dependent rejection such that hepatocyte survival up to days was achieved in the majority of recipient mice. the purpose of this study was to determine whether targeting both cd lcd l and lfa- mediated signals results in an immunoregulatory state which controls naive cd + t cells. methods: x lo fvbln (halat, hepatocytes, were transplanted into cd ko or c bl .scid (all h- b) mice, and recipients were treated with anti-lfa- mab ( . mg d - ) and anti-cd ol mab (lmg, do, , , ) . hepatocyte survival was monitored by detection of serum halat reporter product by elisa. hepatocyte recipients with longterm survival (> days) were challenged by adoptive transfer of million naive cdb' t cells (isolated from cd ko mice) which are sufficient to initiate rejection of functioning hepatocellular allografts in scid recipients. results: combined treatment with anti-lfa- mab and anti-cd l mab significantly prolonged hepatocyte allograft survival in cd ko mice such that % achieved hepatocyte survival > days posttransplant (mst> days, n= ) in comparison to either agent alone (anti-cd l mab mst= days, n= , p<o.ool; anti-lfa- mab mst= days, n= , p<o.ool). recipient mice induced to achieve hepatocyte survival > days (by short-term treatment with anti-lfa- and anti-cd l mab) received adoptive transfer of million nayve purified cd + t cells. continued survival of longterm hepatocellular allografts despite adoptive transfer of cd + t cells was observed in % ( of ) recipients. in recipients, adoptive transfer of cd + t cells was associated with loss of hepatocellular allograft function by and days following cell transfer. in contrast, control scid mice with functioning hepatocellular allografts which were reconstituted with million naive cd + t cells rapidly rejected hepatocytes with mst of days (n= ). conclusion: targeting of both cd l cd ol and lfa- not only prevents immunoresistant cdb-dependent immune damage of liver parenchymal cells, but also x lo vs. . . x lo and . . x lo vs. . . x , respectively) while at hrs it was . t . x lo vs. . . x (ns) . in the spleen, significantly higher heparanase treated cells were located within the tissue, showing proliferative activity at and hrs post transplantation. already by hrs after transplantation the proliferating index of sec increased from . in controls to in heparanase treated rats (i'<o.o ). conclusions: heparanase increases the long-term presence of transplanted cells within portal radicles, thereby facilitating their engraftment. it has also a significant effect on ec proliferation. disc o s u r e s : yaacov baruch -no relationships to disclose orit goldshmit -no relationships to disclose neta ilan -no relationships to disclose gideon shoshany -no relationships to disclose vladislav tsiperson -no relationships to disclose ella veitzman -no relationships to disclose israel vlodavsky -no relationships to disclose cell activation. arunzazu sanchez, peter n u n , insa s schroeder, valentina m factor, snorri s thorgeirsson, national cancer institute, bethesda, m d substantial evidence suggests that proliferation of hepatic stem cell progenies (referred to as oval cells) is responsible for liver regeneration when the replicative capacity of hepatocytes is impaired. it is well established that activity of the tnf-a , nf-kb, il- , and stat pathways is necessary for the regenerative response of hepatocytes following partial hepatectomy. however, much less is known about the signals mediating oval cell proliferation and differentiation. the aim of the present study was to evaluate the involvement of nf-kbistats signaling pathways in regulation of oval cell activation. high activity of nf-kb was found in regenerating liver after aaflph protocol coinciding with the proliferation of oval cells. urification of the oval cell population by magnetic beads sorting using ov antibody confirmed that the activation of both nf-kb and stat is specific for this cellular compartment. three distinct subpopulations of oval cells were identified based on intensity of ov staining by facs and named as ov dim, medium and bright. real time pcr analysis showed that they represent oval cells at different stages of differentiation along the hepatocytic lineage. ov dim subpopulation was the closest to the hepatic stem cell, as judged by high expression of c-kit. on the contrary, ov bright cells displayed the highest expression of hepatic differentiation markers, ttr, albumin, connexin and hnf- . nf-kb was uniformly activated in the entire oval cell compartment, whereas stat was phosphorylated only in the most differentiated ov bright subpopulation. furthermore, activation of stat tightly correlated with a high proliferative activity in these cells. together, these data provide the first evidence that the transcriptional activity supported by nf-kb and stat is required for oval cell activation. the differential induction of these two transcription factors suggests that they may control different stages of oval cell activation. disclosures: introduction: haematopoietic stem cells (hscs) have been shown to have greater plasticity than previously thought, with studies demonstrating that both human and rodent hscs can differentiate into hepatocytes. our group has also confirmed that human stem cell derived hepatocytes are the result of true differentiation and not the result of cellular fusion (newsome et al, gastroenterology ) . the extent to which endogenous hscs are naturally mobilized and contribute to liver repair is however uncertain. aims ) to establish whether liver injury mobilizes endogenous hscs into the circulation & into the liver. ) to establish if circulating stem cells in liver injury have altered stem cell potential. patients and methods omls peripheral blood was analysed from the following groups (n= ): normal adult controls, patients with alcoholic hepatitis, abstinent chronic alcoholic liver disease and paracetamol-induced liver injury. using a flow cytometry sorting machine, circulating cd positive hscs were quantified as a percentage of the number of mononuclear cells according to the ishage flow cytometry protocol. equal numbers of cd positive cells from each gmup were collected and cultured, to assess their in vitro stem cell properties, as judged by number of colony forming units (cf'us) produced. for patients with alcoholic hepatitis, age, sex, biochemical parameters, maddrey score and outcome (mortality) was recorded. liver biopsies from paracetamol induced liver injury and alcoholic hepatitis patients were stained for cd and c-kit markers to quantitate stem cell numbers (number/ portal triad) and compared with normal adult controls. results .levels of circulating cd positive hscs were significantly elevated in patients with alcoholic hepatitis ( . % . ; p< . ) when compared with all other groups. the corresponding cd positive values for the other groups are as follows: healthy controls ( . % . ); paracetamol liver injury ( . % . ) and abstinent alcoholic cirrhotics ( . % . ).there was no significant difference in mean mononuclear cell count between these groups. .patients surviving their alcoholic hepatitis had significantly higher levels of circulating cd positive stem cells ( . % . ; p<o.o ) when compared with those who had died ( . % . ). the surviving alcoholic hepatitis patients had significantly lower maddrey scores ( . . ; p=o.oos) than those who had died ( . ) whilst no significant difference in sex or age distribution was demonstrated between these two groups. the total mononuclear cell counts were lower in the deceased alcoholic hepatih group ( . xlox l . ) when compared with those that had survived ( . xlox . ). if cd numbers are adjusted for the total mononuclear cell counts then the increase in circulating stem cells between these two groups became even more apparent ( . ? . and . xlox cellsll . ; p< . ). .hscs from alcoholic hepatitis patients produced greater numbers of cfus compared with adult control hscs ( vs. ). .liver biopsies from paracetamol and alcoholic hepatitis patients contained greater numbers of stem cells compared with healthy controls (p< . ). control ( . cellsltriad ?o.l) , alcoholic hepatitis ( cellltriad . ) and paracetamol liver injury ( . cells/ triad . ). stem cells were only identifiedwithin the portal triads and not within the hepatic parenchyma in all of these groups. conclusions . alcoholic hepatitis is associated with increased levels of circulating hscs both in the blood and the liver. . these mobilized cells display in vifro stem cell potential at a level equal to or higher than control hscs. . increased levels of stem cell mobilization are associated with improved clinical outcome. disclosures: background klf is a ubiquitously expressed kruppel like zinc finger transcription factor identified as a tumor suppressor gene in prostate cancer (narla et al, science ) . in addition, klf is frequently lost and/or mutated in hepatocellular carcinomas (tal-kremer, aasld -# ) . klf is also an immediate early gene that is rapidly upregulated in hepatocytes following partial hepatectomy and in hepatic stellate cells after injury. its expression is regulated in a tissuerestricted manner during development (laub et al, mech dev ) . these broad roles yet defined sites of expression suggested a potentially important role of klfg in development. the aim of this project was to examine the role of klf in hepatic differentiation using an embryonic stem (es) cell differentiation system in which undifferentiated cells can be driven along tissue specific lineages under defined stimuli. materials: two targeting constructs were generated in which the second exons of klf were replaced by a neomycinllac z cassette or hygromycin cassette. klf (+/-) es cells were generated by homologous recombination through selection with a klf targeting vector containing the neomy&/lac z cassette. to obtain klf (-/-) es cells, klf (+/-) clones were subjected to the second round of homologous recombination. a l l es cell dones were confirmed to have a normal karyotype after selection. methods and resulk in the undifferentiated state, klfg (-/-)null es cells grew more slowly than wild type es cells, with doubling times up to % longer. following differentiation into embryoid bodies (eb) over days in culture, the number of ebs was reduced by > % compared with wild type ebs. the disparity in growth rate between wt and (-/-) es cells was greater in later stages than in the initial days of differentiation. this was accompanied by prolongation of the epiblast-like stage, associated with markedly delayed expression of endodermal mesodermal and ectodermal markers. next, we examined the capacity of klfg (-/-) ebs to differentiate into hepatocyte like cells by withdrawing leukemia inhibitory factor after two days, then culturing ebs under serum free conditions for days, followed by growth on gelatinized dishes. basic fibroblast growth factor and dexamethasone were added into the medium starting day and day . whereas expression in klfg (+/+) ebs of alpha-fetoprotein and albumin mrna occurred after days of differentiation, their expression was almost absent and significantly delayed in klf (-/-) ebs. an impaired ability of klf (-/-) cells to differentiate along hematopoietic and neuronal lineages was also observed. in conclusion, these data indicate a broad and proximal role of klfg in tissue specific development with specific role in hepatocellular development. cell transplantation has been recognized as a possible future treatment for liver cirrhosis. one of the major questions is which cell will be of greatest usespleen might be a major source of stem cell, if exist, since splenomegaly is a common in cirrhotic patients and even splenectomy is performed for treatment. recent advance in the isolation technique of stem cells based on the efflux of fluorescent dyes hoechst has turned out to be an efficient method to purify stem cells called side population (sp) cells. we utilized this method to isolate stem cells from spleen. we also transfused these sp cells into rat liver treated with carbon tetrachloride (cc ) to see whether these sp cells proliferate and differentiate into both hepatocytes or cholangiocytes. methods. c / j male mice and female nodlscid mice were obtained from nihon crea. egfp transgenic (tg) rat were obtained from nihon slc. splenocytes were prepared by digesting in . % collagenase solution and forcing tissue through sterile mesh. splenocytes were then resuspended at lo cellslml in hanks balanced salt solution (hbss) containing % fbs, mm hepes (hbss+), and ug/ml hoechst w or w/o verapamil and were incubated at degrees centigrade for min. splenocytes were then stained for min. on ice with fitc or pe conjugated various antibodies against sca- , c-kit, thyl. , cd and cd . splenocytes were washed in hbss+ alone times and then in hbss+ with ug/ml propidium iodide. splenocytes were analyzed and sorted on a dual-laser facstar plus flow cytometer. ( excitation: nm, emission: /bp & /bp filter & -nm short-pass dichroic mirror). we also prepared spcells from donor egfp tg rats' spleen as described above. these spleen egfp positive sp cells were directly injected into liver of anesthetized recipient nodlscid mice. the recipient nodlscid mice were injected with ug. cc intrapentoneally every once a week before and after transplantation. immunohistochemical staining were performed to identify engrafted egfp positive cells, liver specific proteins such as cytokeratinl j , afp and albumin. results. initial hst staining of low density splenocytes showed the sp fraction to be readily detectable ( . - %). these cells were verapamil sensitive and could be stained with hst after verapamil treatment. the frequency of each surface markers positive cells were as follows (table and ). two weeks after cell transplantation, donor derived egfp positive cells were engrafted. the some engrafted cells distributed in a cluster indicating donor cell proliferation. hepatic differentiation of engrafted cells were shown by double staining of egfp and rat albumin or cytokeratinld. we observed some of afp positive cells in egfp positive cells, but very few of cytokeratinl positive cells. conclusions. the adult rodent spleen had consistently detectable fraction of sp cells. the surface marker profiles were different from those of bone marrow sp cells. the splenic sp cells could be engrafted in liver and differentiate into hepatocyte like cells. our results suggest that splenic sp cells could be exploited for the repair of damaged liver. backgroundlaim : it has been reported that bone marrow cells (bmcs) can replace liver in a murine model of tyrosinaemia and correct this metabolic disease through the fusion of donor bmcs to recipient hepatocytes. the aim of this study was to test whether or not this replacement is a general phenomenon in other liver injury models. the following three models were tested; ( ) hepatitis b transgenic mouse (tgn(alblhbv)), ( ) albumin-urokinase transgenic mouse (tgn(alblp au)) and ( ) carbon tetrachloride (cc ) treatment model. as the selective liver injury models, irradiated tgn(alb hbv) and tgn(alblp au) were transplanted with lx -lx bmcs from green fluorescent protein (gfp) transgenic mice (tgn(actbegfp)) or from @-galactosidase transgenic mice (tgn(mtnlacz)). as a non-selective liver injury model, irradiated c bl/ mice were transplanted with x bmcs from tgn(actbegfp) or tgn(mtnlacz) followed by the administration of cc ( . mllkg animal weight, twice a week for weeks). a cci protocol without preparative irradiation was also tested, in which c bl mice were first administered ccl, times, then followed by transplantation of tgn(actbegfp) or tgn(mtnlacz) bmcs. after the analysis of the donor cell engraftment in the peripheral blood, these recipient mice were sacrificed and checked for donor-derived hepatocytes at - weeks post-transplantation. sixty liver sections for each animal (containing approximately . ~ ~ hepatocytes), were analyzed for gfp positive cells and the whole livers were inspected for @-galactosidase expression with x-gal cytohistochemistry. however, there were no gfp positive hepatocytes and no gross blue staining of the livers with x-gal in any of the recipient mice. gfp positive cells were only located in sinusoids or associated with larger vessels. they were readily distinguishable from hepatocytes through their morphology and were negative for albumin by immunostaining. in addition, the livers from female animals with gender mismatched bm transplantation were also tested with y chromosome fish analysis, which might be a more sensitive method to detect donor derived cells than transgenic epitope tagging. total of isolated hepatocytes were positive for y chromosome in . ~ ~ hepatocytes analyzed, although it still remains to be elucidated whether these cells arise from spontaneous fusion events or from transdifferentiation. conclusions: these results demonstrate that there is little or no contribution of bmcs to the replacement of injured livers in these models. thus, we do not believe that bm derived cells can generally lead to a cure of liver damage. background &aims: mouse embryonic stem (es) cells are clonal cell lines derived from the inner cell mass of developing blastocysts and have multi-lineage differentiation ability. we previously reported that es cells can be made to differentiate into hepatocytes possessing high metabolic activities by transfection of hepatocyte nuclear factor- beta (hnf- b). in the present study, we investigated the expression of hepatobiliary organic anion transporters and bilirubin uridine diphosphate glucuronosyltransferase (ugtlal) in undifferentiated and differentiating hnf- btransfected es (hnf- b-es) cells. materials & methods: hnf b-es cells were established from a mouse es cell line. undifferentiated hnf- b-es cells were main-tained in gelatin-coated dishes without feeder cells in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs), . mm of z-mercaptoethanol( me), . mm of non-essential amino acids (neaa), mm of sodium pyruvate and ulml of leukemia inhibitory factor (lif). undifferentiated hnf- b-es cells were allowed to differentiate in dmem supplemented with % fbs, . mm -me, . mm neaa, mm sodium pyruvate, and nglml of fibroblast growth factor (fgfz) in the absence of leukemia inhibitory factor (lif). differentiating hnf- b-es cells were collected for rt-pcr analysis on day , and for western blotting on days and . results: the expression of organic anion transporting polypeptide (oatpl), multidrug resistance-associated protein (mrpl), mrp , mrp , and ugtlal was not seen in the undifferentiated hnf- b-es cells by rt-pcr, whereas all were expressed in differentiating hnf- b-es cells. protein expression for oatpl, mrpl, mrp , mrp , and ugtlal was also observed in the differentiating hnf- b-es cells by western blotting. an immunofluorescence examination revealed that oatpl was co-located with desmoplakin, a marker for the basolateral (sinusoidal) membrane, and mrp was co-localized with cd , a marker for the apical (canalicular) membrane, though they were both expressed throughout most of the cell membranes. we have used cdna microarray analysis of clonal murine hepatoblasts to identify genes that are significantly and preferentially expressed in undifferentiated hepatoblasts compared to adult mouse liver. unique stem cell genes were identified by overexpression in undifferentiated hepatoblasts compared to adult liver. genes were identified by these criteria and were designated candidate stem cell genes (cscg). cscg were distributed on all chromosomes of the mouse except chromosomes , and the y chromosome. however, chromosomes , and showed a preferential distribution of cscg compared to the total number of genes mapped on these chromosomes. approximately half of our cscg are commonly expressed in other murine stem cell populations including embryonic stem cells, neural stem cells, mesenchymal stem cells and hematopoetic stem cells, suggesting that hepatoblasts share a molecular signature with other stem cell populations. some of the commonly identified stem cell genes include: sc a , c-myc, nek , phosphodiesterase , nicastrin, smarca and smarca and pias . these genes function in a variety of pathways including ion transport, anion exchange, cell cycle control, chromatin organization, dna binding activity and signal transduction. approximately % of cscg are expressed in early endoderm, fetal liver and/or the pancreatic bud. a search for homology to known proteins indicated that some of these genes contain homology to membrane transporters, zinc finger proteins and spindle apparatus components. this class of genes is of particular interest because they may represent new hepatic lineage markers. the results of this analysis leads us to conclude that a network of pathways some of which are in common with other stem cell populations function together to maintain the liver stem cell phenotype. valentina m factor, aranzazu sanchez, ju-seog lee, tanya n hoang, snom' s thorgeirsson, national cancer institute, bethesda, m d rat liver epithelial cells (rle) were isolated from normal adult rat livers and established as a cell line . rle cells were found to express undifferentiated characteristics resembling adult liver stem cells. we have addressed cellular and molecular mechanisms controlling lineage commitment of hepatic stem cells using rle- . to induce rle differentiation along the hepatocytic lineage, differentiation protocols were developed consisting of a sequential treatment with the demethylating agent -aza-cytidine ( ac), fibroblast growth factors and (fgf), oncostatin m (osm) and hepatocyte growth factor (hgf) in the continuous presence of the synthetic glucocorticoid dexamethasone (dex). these treatments resulted in a remarkable enlargement in cell size and organelle complexity as shown by facs and confocal microscopy. morphological maturation of rle was paralleled by a decrease in cell proliferation. more significantly, rt and real time pcr analysis revealed an induction of hepatocyte specific markers such as tat, ttr, glucose- -phosphatase, and connexin . in addition mrnas encoding liver enriched transcription factors hnf lalp, hnf ru, hnf , and clebp p were notably induced along with hepatocytic differentiation. the pcr results were supported by a cdna microarray analysis. comparison of the gene expression profiles following the treatment protocols reflected an activation of the hepatocytic differentiation program as judged by increased expression of a differentiation-associated gene set (phosphofructokinase, glutathione-s-transferase) and decreased expression of a cell cycle regulated gene set. currently, transplantation studies are performed to identify the potential of the differentiated rle to repopulate and restore the diseased livers. the results indicate that cell lines derived from adult liver stem cells may provide a renewable resource for transplantation. adult liver stem cells (adulis) can be isolated from rodent bone marrow. when cultured under specific conditions in co-culture with isogeneic hepatocytes, adulis fiom days bile duct ligated (bdl) rats are transdifferentiating into a hepatocyte-like lineage and are able to produce urea from ammonia. the aim of the study presented is to describe the hepatocyte specific metabolic capacity of cultured adulis from normal or bdl rats in single or co-culture with isogeneic hepatocytes, with or without interleukin- (il- ). methods: adulis were isolated by a two-step immunoisolation procedure (i.e. beta- -microglobulin negativity and thy- positivity) from rat femoral bone marrow (male wistar rats, g) and cultured on a matrigel layer in -well polystyrene dishes at a cell density of ' cells/cm* in small hepatocyte media. isogeneic hepatocytes were isolated by a two-step collagenase perfusion technique and seeded ( , cells/cm ) on an inlay with a collagen-coated ptfe membrane (poresize: . pm) for co-culture experiments. of note, the culture media was supplemented with % isogeneic serum and dexamethason m) was administered after culture day . il- was added in the corresponding experimental groups ( nglml). after removal of the hepatocyte-inlay (in the co-culture groups) adulis cultures were exposed to . mmol nh ci for hours in dmem and urea formation determined thereafter with a colorimetric assay. cells were harvested in trizol, total rna extracted and after reverse transcription cdna used for real-time pcr determination of s(rm~) content to standardize the metabolic signal for cell number. relative ureagenesis values were compared statistically using jandel scientific. the significance level was set at pc . . results: the amount of adulis isolated from normal rat femoral bone marrow (n=lo) was . . x lo and . . x lo in animals (n= ) after seven days of bdl (p=n.s.). relative urea synthesis in cultures from normal animals was . . in single and . . in co-culture at culture days , , , and . with addition of il- to the culture media urea genesis was determined to be . . in single and . . ' in co-culture. in cell cultures from seven days bdl rats relative urea formation was . . in single and . . in co-culture. with the addition of il- to the culture media values were . . in single and . . in co-culture. addition of il- to the culture media increased the metabolic signal in all cell cultures from normal animals (anova on ranks, p< . ) but not in cultures with cells isolated from bdl animals (p=n.s.). co-culture induced stronger ureagenesis under all culture conditions examined (paired t-test: p< . ). conclusions: to exclude immunological interference from adult immunocompetent bone marrow cells cultures were strictly isogeneic (i.e. adulis, hepatocytes, and serum from the same animal). co-culturing adulis with isogeneic hepatocytes increased ureagenesis in all paired culture experiments. as there is no direct cell contact between hepatocytes and adulis paracrine soluble, so far undetermined, factors must be involved. interestingly by the addition of il- transdifferentiation was inducible in cultures of adulis from normal animals but no additional effect was detectable in the cell cultures from bdl animals. in further studies it will be necessairy to determine if cholestatic serum also induces accelerated and more pronounced hepatocyte specific metabolic capacity in adulis from normal animals. factors responsible for this transdifferentiation should then be isolated from cholestatic serum and might be used to support the failing liver in vivo potentially by activation of the adulis pool in the bone marrow and the liver. disclosures: saji, shinji tamura, yuichi yoshida, shinichi kiso, ayuko iizuka, hitoshi matsumoto, takako kawasaki, yoshihiro kamada, yuji matsuzawa, graduate school of medicine, osaka university, suita, osaka, japan background and aims: recently, the evidence that bone marrow cells (bm cells) have trans-differentiating potential into hepatic lineage cells has been described in animal transplantation experiments and human pathology of bone marrow transplantation recipients. however, the molecular mechanism underlying this phenomenon has remained unclear because of lack of effective culture system. to address this issue, we developed a novel in vitro culture system in which murine bm cells are cultured with growth factors and investigated factors required for the transdifferentiation of em cells into hepatic linage cells. methods and results: ( ) bm cells were isolated from the femora of -to -week-old male c bl/ ] mice. they were cultured in three-dimensional culture system using type i collagen gel and stimulated without or with the growth factors (egf, hgf, hb-egf, osm, bmp- , bfgf, fgf- ; ongiml) for day. to investigate the effect of these growth factors, the gene expression of albumin was examined by quantitative rt-pcr. when bm cells were cultured with hgf, osm, bfgf and fgf- , albumin was induced effectively. among these growth factors, bfgf was found to induce albumin the most effectively in the cultured bm cells. ( ) bm cells were cultured in collagen gel with bfgf ( onglml) for , , days. the gene expression of hepatocyte-specific markers (albumin, cytokeratin , alphal-antitrypsin, glucose phosphates and tyrosine aminotransferase), cholangiocyte-specific marker (cytokeratin ) and liver-enriched transcription factors (hnflalpha, hnf alpha, hnf beta, hnf alpha, gata , gata , clebpalpha, and ciebpbeta) were examined by rt-pcr. upon the stimulation of bfgf, bm cells were found to express cytokeratin , alphal-anitripsin and glucose phosphates, cytokeratin and liver-enriched transcription factors including hnflalpha, hnfjalpha, hnf beta, gata and gata . ( ) bm cells were cultured in collagen gel with bfgf ( ngiml) for days. we assessed expressions of albumin and ck proteins of cultured bm cells by immunohistochemistry. about % cells of them were positively stained for both albumin and cytokeratin . conclusions: we established a novel in vitro culture system of bm cells and demonstrated that basic fibroblast growth factor could induce the trans-differentiation of bm cells into hepatic lineage cells the most effectively. furthermore, this conversion was associated with induction of liver-enriched transcription factors including hepatocyte nuclear factors and gata family proteins. these results indicate these liver-enriched transcription factors are the major components, which induce this trans-differentiation. disclosures: ayuko iizuka -no relationships to disclose yoshihiro kamada -no relationships to disclose takako kawasaki -no relationships to disclose shinichi kiso -no relationships to disclose hitoshi matsumoto -no relationships to disclose yuji matsuzawa -no relationships to disclose yukiko saji -no relationships to disclose shinji tamura -no relationships to disclose yuichi yoshida -no relationships to disclose crtteil, france; nadine martin, hbpital henri mondor, criteil, france; dominique couchie, anne m preaux, yannick laperche, elie ; zafrani, inserm u , cre'teiz, france liver can regenerate from oval precursor cells when the replication of hepatocytes is inhibited. these cells proliferate in the periportal region, migrate in the lobule and differentiate into hepatocytes. stromal-derived factor- (sdf- ) is a chemokine which plays a major role during embryogenesis. since sdf- and its sole receptor cxcr are involved in the differentiation of progenitor cells (e.g. b lymphopoiesis, digestive epithelial cell renewal), the aim of our work was to study the expression of sdf- and cxcr in a model of hepatic regeneration from precursor cells. hepatic regeneration from oval cells was induced by treating rats with acetylaminofluorene followed by partial hepatectomy. rats were sacrificed the day of surgery (day ) and at days , , , and after partial hepatectomy. rare oval cells were observed at day . their number was moderate at day , marked at day and and declined thereafter. oval cells predominated in the periportal region and usually formed canalar structures resembling cholangioles. individual or cholangiole-forming oval cells strongly expressed sdf-i, as demonstrated at the mrna level by in situ hybridization (ish) and at the protein level by irnmunohistochemistry. interlobular bile duct epithelial cells were also positive for sdf- protein but negative for its mrna. cxcr mrna hepatic levels, as assessed by quantitative rt-pcr, paralleled the degree of oval cell proliferation. ish showed marked cxcr mrna expression in individual as well as in cholangiole-forming oval cells. in order to determine which role the sdf- icxcr couple could play in hepatic regeneration from oval cells, rats were treated or not with fucoidan ( mglkg ip, twice daily), a sulfated polysaccharide known to bind to sdf- and to block its biological effects. as compared to untreated animals, oval cell proliferation was markedly decreased in five of the seven fucoidan-treated rats. in conclusion, oval cells express sdf- and its receptor cxcr during hepatic regeneration from precursor cells. our results strongly suggest that the sdf- lcxcr couple acts in an autocrinelparacrine way to stimulate the proliferation of these precursor cells. disclosures: dominique couchie -no relationships to disclose background: bone marrow cells and embryonic stem cells are being used for cell transplantation. although these cells are known to be multipotent and capable to become hepatocyte, several problems have been pointed out, such as cell fusion and teratoma generation. thus we paid attention to side population (sp) cells, those with a verapamil-sensitive ability to efflux hoechst . sp cells have been identified in several tissues. the bone marrow sp phenotype appears to be a common feature of stem cells, but hepatic and splenic sp cells have been less well characterized. aim: we tried to separate and culture sp cells from non-parenchymal liver cells (npcs) and splenocytes, and examined whether they would obtain hepatic phenotype. methods: - -week-old female transgenic rats that ubiquitously express egfp were used to isolate non-parencymal liver cells by the standard collagenase two-step perfusion method, and splenocytes were isolated by forcing the tissue through sterile mesh, then mononuclear cells were separated by ficoll density gradient centrifugation. they were resuspended at lo cellslml in hanks balanced salt solution (hbss) containing % fetal calf serum, mm hepes (hbss+), and uglml hoechst (hst) w or wlo verapamil and were incubated at degrees centigrade for min. cells were washed twice in hbss+ and then resuspended in hbss+ with uglml propidium iodide. cells were analyzed and sorted on a dual-laser facstar plus flow cytometer. rt-pcr was performed with rna extracted from freshly isolated sp cells. isolated sp cells were cultured with male rat primary cultured hepatocytes in collagen gel sandwich. cell morphology was monitored under a phase-contrast microscope and a fluorescence microscope. the expression of albumin, cytokeratin , and cytokeratin was analyzed by immunohistochemistry. fluorescence in situ hybridization (fish) for the sry gene was performed to exclude ceil fusion. results: the rate of npc-sp was . % of isolated npcs. % of npc-sp expressed cd , while % of npc main population (mp) did. freshly isolated sp cells were smaller than mature hepatocytes. they looked like monocytes or lymphocytes. these cells did not express liver specific markers, such as albumin, alpha-fetoprotein, cytokeratin , or cytokeratin . during one-week culture with hepatocytes using collagen gel sandwich method, we could find no change in gfp-positive sp cells, but after further culture, we found several colonies consisting of gfp-positive sp cells, whose shape became polygonal. after -week-culture, most of gfp-positive cells were stained positive for albumin. npc-sp cells died within one week without hepatocyte co-culture. npc-mp cells formed colonies after weeks culture with hepatocytes, but unlike sp cells, they looked like fibroblasts and were negative for albumin. freshly isolated sp cells from splenocytes were also small and monocyte-like, and expressed no liver specific markers. spleen sp cells could culture for more than weeks in the same way as npc-sp cells, and looked like hepatocytes, but did not proliferate. after having been cultured, most of gfp-positive cells were stained positive for albumin and cytekeartin , and negative for cytokeartin ; on the other hand splenic main population cells after weeks' culture expressed those very little. conclusion: we succeeded in culturing liver and spleen sp cells. sp cells which did not express mrna of liver specific markers, such as albumin or cytokeratin , became to express those after the culture with hepatocytes. these results suggest that these sp cells have plasticity and obtain hepatic function. thus, tissue stem cells, especially sp cells, are useful for cell transplantation. spleen sp cell might be a candidate for source of cell transplantation for treatment of liver cirrhosis. miura, takashi goto, ken-ichiro mikami, kunio nakane, kazuo yoneyama, hirokazu nagai, kunihiko terada, toshihiro sugiyama, katsuyuki imai, haruki senoo, sumio watanabe, akita university, akita, japan background epimorphin, a mesenchymal cell surface-associated molecule identified as an epithelial morphogen, is detected on stellate cells in the liver. we have reported that a contact with hepatic stellate cells (hscs) promotes differentiation of hepatic stem-like cells (hslcs). here, we show the involvement of epimorphin in that process.materials and methods: hslcs and hscs were isolated from healthy adult rats using two-step collagenase perfusion and percoll gradient centrifugation, and maintained standard medium, dulbecco's modified eagle's medium with % fetal bovine serum. hslcs were cultured in stellate cell-conditioned medium, co-cultured with hscs to maintain cellcell contact or in the presence of epimorphin. phenotypical and morphologic changes were investigated by rt-pcr and with an electron microscopy, respectively. results: hslcs are polygonal in shape and assume a cobblestone appearance when cultured in standard medium. transmission electron microscopy showed that hslcs , to micro-m in diameter, have a round to ovalshaped nucleus, a few vacuoles, scant organelles and a high nucleuslcytoplasm ratio compared with normal hepatocytes. the phenotypic properties of hslcs did not change as well as their size and shape during this experiment. hslcs characterized by rt-pcr are follows: positive, c-kit, musashi- (a neural stem cell marker), alpha-fetoprotein, cytokeratinl , connexin ; negative, albumin, transferrin, tyrosine aminotransferase, gamma-glutamyl transpeptidase. hslcs cultured in stellate cell-conditioned medium had no phenotypical and morphological changes. hslcs co-cultured with hscs expressed albumin, transferrin, and tyrosine aminotransferase, which were inhibited by an anti-epimorphin antibody. furthermore, epimorphin induced the markers not only for hepatocytes including albumin, transferrin and tyrosine aminotransferase, but also for cholangiocytes including gammaglutamyl transpeptidase in addition to increased expression of connexin and cytokeratinl , with decreased expression of c-kit and musashi- . in addition, clebp beta was enhanced, which has been reported to mediate through morphogenesis by epimorphin. hslcs co-cultured with hscs piled up and subsequent development of bile-canalicui-like structures, which was dramatically inhibited by an anti-epimoirphin antibody. hslcs, close to epimorphin, stated piling up, changed their shape from flat to cuboidal, became rich in mitochondria and rough endoplasmic reticulum and formed bile-canalicui-like structures. conclusions: hslcs have a self-renewing capacity and multilineage differentiation potential. epimorphin is involved in differentiation of hslcs though a contact with hscs. disclosures: takashi goto -no relationships to disclose n order to identify new and differentially expressed genes in fetal rat liver that are specific for epithelial stemlprogenitor cells and genes involved in liver progenitor cell differentiation, we used murine cdna microarrays containing , cdnas, available at the functional genomic facility, aecom. the expression pattern of fetal liver stemlprogenitor cells was studied from embryonic day through birth, days after birth and in adult liver. the driver rnas were isolated from cells adhered to the dish after plating the cell suspension in order to remove the blood cells. reference rna was isolated from the livers of newborn rats. we found that genes present on the cdna microarrays were developmentally regulated. these genes fall in two major hierarchical clusters, according to their pattern of expression. the genes that are down-regulated during fetal liver development were distributed in functional groups and further analyzed. in this study, special attention was paid to genes that were induced in fetal liver but were not expressed (or expressed at a very low level) in adult liver. these genes are of special interest because they can serve as specific markers for identification and for isolation of liver stemlprogenitor cells. in addition, these genes represent links to understanding the fetal liver specific molecular pathways that govern cell proliferation, survival, apoptosis and differentiation. to determine which of the over-expressed genes in - day fetal liver that are down-regulated in adult liver are progenitor cells specific, we searched in the available databases whether the expression of these genes in adult liver was previously reported. seventy genes were further analyzed: the clones of interest were hybridized to radioactive labeled p cdna synthesized from fetal and adult liver rnas. for of the clones, we found that there was little or no expression in adult liver. the expression level of selected clones was analyzed further by quantitative pcr, and they were confirmed as highly induced in fetal hepatoblasts compared to adult liver. half of the clones are ests. the known genes fall in different categories, the major four being: genes related to transcription; signal transduction; morphogenesis, histogenesis and organogenesis; cell adhesion, de-adhesion and migration. some of the known genes over-expressed in fetal liver that are not expressed or expressed at very low level in adult liver are: grblo (aa ), fh (aa ), tnc (aa ), peg (aa ), hey (aa ), enah ((aa ), pkcd (aa ), lox (w ), shcbpl (aa ), magoh (aa ), manba (aa ), klf (aa ), gpc (aa ), pcolce (aa ), ppap c (aa ), nfkb (aa ), adam (aa ), akapl (aa ), tagln (bc . it should be noted, that most of the clones and all those listed here that we have identified as liver progenitor cells specific, are expressed in stem cells of embryonic, hematopoietic, or mesenchymal origin. two of the presented genes encode cell surface proteins: a disintegrin and metalloproteinase domain (adaml ) (meltrin beta), glypican . using in situ hybridization, we are currently verifying whether our putative liver progenitor cell specific genes are expressed in hepatoblasts and in rare progenitor cells that remain in the adult liver. identifying and cloning new genes that are expressed uniquely in liver stemlprogenitor cells will allow us to design a method for isolation of these cells and to study their role in liver development, growth control and regeneration. hepatocyte transplantation has been shown to be an effective treatment for liver diseases including fulminant hepatic failure and metabolic defects in liver function. however, the availability of sufficient numbers of hepatocytes with which to conduct clinical studies limits the wide-spread availability of this therapy. previously, we have shown that human placenta derived stem cells (pdsc) differentiate along neural or hepatic lineages depending on the culture conditions and suggested that these stem cells could be a source of cells for clinical transplantation. here we report on a protocol to further optimize hepatic differentiation of pdsc. a three stage culture system was applied to induce hepatic differentiation. ae cells were propagated in dmem based standard media which contains egf nglml for a week (stage i) and subsequently with growth factors such as fgf- , , , , , oncostatin m, hgf, andlor dexamethasone (dex) and insulin-transferrin-selenium (its) for weeks (stage ). the cells were then changed to media supplemented with different nuclear hormone receptor agonists for week to stimulate hepatic maturation (stage ). total rna samples were examined for hepatocyte specific gene expressions with real-time quantitative pcr (rtq-pcr). additional studies examined hepatic differentiation on different culture substrates including collagen, gelatin, fibronectin, arg-gly-asp-ser, and matrigel %, %, % were also examined. in the three stage differentiation system, % (vlv) matrigel coated plates and dexlits containing media, and phenobarbital (pb) were identified as the best conditions to upregulate liver specific gene expression. the principal human drug-metabolizing enzymes (cytochrome p- , cyp) were also examined by rtq-pcr. cypla , c , d , and a were induced with either rifampicin or pb. in parallel, expression of the relevant liver-enriched transcription factors, hnf- , clebp-a and ciebp-b mrnas were increased under conditions which induced hepatic differentiation. under slightly different conditions, differentiation of pdsc into cells which expressed pax , pdxl and nkx . , insulin and glucagon was observed. flow cytometric analysis showed isolated nayve ae cells contains a population of cells which express the embryonic stem (es) cell markers, ssea- , , tra - , and tra - . furthermore, the placental stem cells formed embryonic body (eb)-like structure when cells were cultured on matrigel. the eb-like structures retained the expression of ssea- , , tra - , and tra - . these data indicate that cells can be isolated from term placenta which express markers of es cells. under appropriate conditions pdsc expand, and differentiate into cells with characteristics of hepatocytes, neurons, or pancreatic cells. we propose that pdsc could provide a new source of cells for clinical transplantation and regenerative medicine. unlike with es cells, there should be no social, ethical or religious objections to the isolation and use of placental-derived stem cells. using the ssh technology, we have identified previously induced clones in day fetal compared to adult liver; of these clones were independent transcripts, of them represented ests and appeared to be new sequences (petkov et al., genomics - , ) . applying the rapid amplification of ' and ' cdna ends to subtracted genes (generace kit, invitrogen), we have obtained the full-length cdnas for two of the subtracted clones. one of them (clone g ) encodes a novel putative serine proteasel subtilase highly expressed in fetal liver and comparatively low in adult. the expression of c mrna was analyzed by northern blots with rna isolated from different rat tissues: fetal and adult liver, isolated day fetal hepatoblasts, brain, bone marrow, kidney, spleen, intestine, colon, stomach and muscle. on northern blot with total rna, a strong signal was detected with fetal liver/ hepatoblasts rna and a weak signal with adult liver rna, which showed that the expression of this mrna is developmentally regulated. to confirm the liver specific expression of g , quantitative rt-pcr was camed out. the results of this analysis confirmed our previous results and revealed also a very low expression of g mrna in the colon. the g cdna is nucleotides in length and codes for a novel protein of amino acids. we were able to identify in the data bases the sequences of a rat contig which contains the complete gene for g . the gene coding for g comprises , bp of the rat genome and includes exons and a long '-utr segment of , nucleotides. the initiation of the translation begins with an aug codon, nucleotides downstream from the '-end of mrna. using specific primers designed from the contig sequences, we obtained clones corresponding to the upstream promoter region and downstream of the gene in the pcr top vector (invitrogen). these two upstream and downstream clones were ligated to the c cdna upstream and downstream sequences, respectively, so that the whole gene with the ' and ' regulatory sequences was obtained in the pcr -top vector. analysis of the structure of g showed that this protein resembles others mammalian subtilases, named convertases described during the last decade: furin, pc / , pc , pc , pace , pc and pc ilpcipc and ski- isip. these proteases are synthesized as proproteins and secreted from the cell. g contains a putative signal sequence for release from the er located between amino acid and (alanine glutamine). however, it does not share the consensus cleavage sequences characteristic for the other convertases: (k,r)-(x)n-(k,r) for processing, release and secretion of the active enzyme form. at present, we do not know the processing site of the proprotein. in situ hybridization experiments showed that this serine protease is expressed in fetal liver hepatoblasts. convertases are secretory proproteins implicated in tissue specific processing of hormones, growth factors, metalloproteinase, extracellular matrix proteins, viral proteins etc. they show tissue specificity and some are implicated in development and differentiation. g is a novel convertase, it is developmentally regulated and most likely functions in processing and activation of growth factorslcytokines or extracellular matrix proteins implicated in morphogenesis. further studies of its promoter region will reveal the control sequences and transcriptional activators and repressors regulating its expression during development. college, london, uk introductioniaims: bone marrow cells (bmcs) can contribute to regeneration of the chronically damaged liver but in human studies and animal models the magnitude of this axis is highly variable. in a murine model of hepatitis b we examined whether this pathway of regeneration is enhanced by inhibiting endogenous hepatocyte regeneration. methods: month old female mice transgenic for hepatitis b surface antigen (hbs-tg) received lethal irradiation and were then transplanted with c b / j male bmcs by tail vein injection. weeks later half the mice were treated with retrorsine, a pyrrolizidine alkaloid, to irreversibly block proliferation of endogenous hepatocytes. mice were sacrificed at and months following retrorsine injections. y chromosome containing hepatocytes were identified using in situ hybridisation and phenotype markers (positivity for cytokeratins / , cytochrome p and glycogen, negative for cd ). results: in the control mice with chronic liver damage there was an increase in y chromosome positive hepatocytes over time, but the proportion remained < % of the total number of hepatocytes. however, . % (corrected count) of y chromosome containing hepatocytes could be found repopulating the livers of the mice that had received retrorsine. conclusions: bmcs contribute to the regeneration of the chronically damaged liver, but under conditions where regeneration of endogenous hepatocytes is possible this is minor. however, when chronic liver damage occurs and regeneration of the endogenous hepatocytes is inhibited, the contribution to liver regeneration from the bone marrow is significantly enhanced. beaujon, paris, france; thierry poynard, groupe hospitalier pitie-salpetriere, paris, france background portal hypertension depends in part on the development of hepatic fibrosis. thus, since fibrotest (ft) is a potential biochemical marker of fibrosis, this test was prospectively used to evaluate the presence and severity of portal hypertension in patients with different liver diseases. methods: consecutive patients ( males; % chronic viral hepatitis c, % hepatitis , % alcoholic liver disease, % transplanted, % miscellaneous) with transjugular liver biopsy had same day measurements done for hemodynamic parameters (gradient, free hepatic, wedged pressures), histological parameters (fibrosis scoring system in stages, necroinflammatory activity grades and modification of architecture in classes), and biochemical parameters via blood sample (ft for fibrosis and actitest for activity). the measurements of histological and biochemical parameters were done blind to any other characteristics. sensitivity (se), specificity (sp), predictive values (npv and ppv), spearman correlation (sc), accuracy (ac), kappa (k) and the area under the roc curves (auroc) were assessed. the main endpoint was the prediction of elevated portal pressure (epp), defined by a gradient of mm hg. the secondary endpoint was the prediction of highly elevated portal pressure (hepp) of mm hg (hepp). results: the mean ft value was . (se . ) for patients without epp, . ( . ) in patients with moderate epp and . ( . ) in patients with hepp (p<o.ol between all groups, dunnett's multicomparison test) (figure ). high aurocs of ft were obtained for the prediction of epp, . ( . ) and hepp . ( . ); a cutoff at . had a % ppv for the presence of epp (se= %, sp= %) and a . cutoff had a % npv for the exclusion of hepp (se= %, sp= %). there was a significant correlation between ft and gradient (sc= . , p<o.ool). the auroc of ft for the histological architectural disturbance (fibrosis or cirrhosis) was very high . , ( . ). there were also excellent ft diagnostic values for stages f f f ac= %; k= . ( . ); sc = . , p<o.ool; auroc= . ( . ). therefore histological staging did not give higher diagnostic values than ft for epp, auroc= . , ( . ) introduction: the prevalence of gastric varices ranges from - % in cirrhotic patients and is associated with high morbidity and mortality rates. conventional endoscopic hemostatic treatment is not effective for gastric variceal bleeding. tips can achieve initial control of bleeding but it is associated with high cost, shunt dysfunction, and increased encephalopathy. cyanoacrylate injection has been shown to be very effective with % hemostasis and low rebleeding rates of - %. in our current series of gastic varix patients at the university of virginia, injection with n-butyl- -cyanoacrylate (histoacryl) has yielded similar results (scaldwell, fda-ide). however, distal embolization after n-butyl- -cyanoaaylate injection has been reported and probably results from "beading" upon polymerization. whether other cyanoacrylates with different physical-chemical properties have less embolic risk with equal or greater efficacy remains to be determined. &to investigate and compare the efficacy and safety of two different cyanoacrylates in the management of gastric varices. meth-ods:polymerization properties of two cyanoacrylates, n-butyl- cyanoacrylate (histoacryl) and octyl-cyanoacrylate (dermabond), were compared in vitro by adding a drop of blood to different concentrations of n-butyl- -cyanoacry atel:ethiodol and octyl-cyanoacry- ate:ethiodol (ethiodol, iodinated poppy seed oil, is radio opaque and delays polymerization). the two cyanoacrylates were then compared for "beading" by injecting each cyanoacrylate solution into a flowing circular system of fresh frozen plasma and then collecting the resultant polymer. lastly, cyanoacrylate injection was tested in an animal model using the femoral vein of a pig to simulate a gastric varix. each femoral vein was injected with n-butyl- -cyanoacrylate and octyl-cyanoacrylate, respectively, and then both veins were ligated and examined for histologic change. results n-butyl- -cyanoacry ate:ethiodol ( :l) and octyl-cyanoacrylate alone had similar polymerization properties as far as initiation of polymerization to formation of a "hard substance"these concentrations were used for subsequent testing. preliminary results from the circular flowing system show that n-butyl- -cyanoacrylate forms a "beaded" and "crystallized" polymer whereas the octyl-cyanoacrylate polymer is more "stringy" and "rubbery". in the animal model, n-butyl- -cyanoacrylate had formed a large, extended thrombus in the vein with an irregular surface that appeared granular. in contrast, octyl-cyanoacrylate appeared uniform throughout and occluded the vein wall with only a s m d thrombus in the center. conclusion octyl-cyanoacrylate appears to be a viable alternative with possible lower embolic risk. vessel occlusion appears to involve a combination of polymer and thrombus formation, although interaction with the dotting cascade has not been adequately investigated. further studies in an animal model of gastric varices are in development to assess the relative safety and efficacy of these and other cyanoacrylates. disclosures: stephen evgeny farber, doron fisher, rami eliakim, nira beck-razi, ahuva angel, ella veikman, irit chermesh, camal yassin, diana gaitini, michael libas, rambam medical center, ha+, israel; soboh soboh, porria hospital, tiberia, israel; yaacov baruch, rambam medical center, haqa, israel background: current guidelines recommend that patients with liver cirrhosis, undergo egd screening every - years for the presence of ev. patients with large ev should be treated with beta-blockers and those with small ev should be rescreened. platelet count and us correlate with high risk patients but are not accurate enough to avoid endoscopy in low risk patients. barium swallow is preferred by most patients but data concerning its sensitivity is not sufficient. to evaluate the accuracy of be to diagnose esophageal varices in patients with compensated cirrhosis and portal hypertension at an early stage. patients and methods: patients with liver cirrhosis where evaluated by egd (japanese general criteria), as a gold standard and be for the presence and size (small, large) of varices. x-rays were taken within weeks of endoscopy, with % barium sulfate (e-z-hd), as suspension adjusted for double contrast studies and barium paste (e-z, %, esophageal cream) for mucous coating. results: patients diagnosed by liver biopsy; % postnecrotic, % cryptogenic, % nash, % alcohol. % were child-pugh's score a and % were child-pugh's r. the correlation between egd and be was very high; r= . , p<o.oool. there were only false negative results with be. overall sensitivity of re was %. all large varices (f -f ) were diagnosed by be. sensitivity declined with f varices to %. the positive predictive value was %. the cost of x-rays was one fourth that of egd. conclusions: re can be used as a non invasive procedure for the screening of patients with varices and compensated liver disease at a minimal cost. using be will allow to avoid universal prophylaxis with beta-blockers. studies dealing with the prevention of rebleeding have shown that hemodynamic response to pharmacological treatment of portal hypertension is adequate when the hepatic venous pressure gradient (hvpg) decreases to ? mmhg or by ? % from baseline. with beta-blockers these targets are achieved in only around % of cases. however, even with such a high rate of poor hemodynamic response, variceal bleeding occurs in few patients when beta-blockers are used for primary prophylaxis (around %). the aim of this study was to assess whether the cut-off value to define response in primary prophylaxis may be defined more accurately and to investigate whether the acute response to beta-blockers may predict evolution (avoiding a second study). methods: a total of cirrhotic patients with large varices and without pre-vious bleeding were investigated. a baseline hemodynamic study was performed in all patients. after initial measurements, propranolol was administered in patients ( . mg/kg i.v.) and measurements were repeated minutes later. subsequently, nadolol was chronically administered to prevent variceal bleeding. a second hemodynamic study was performed to months later in patients. results: during a mean follow-up of months, % of patients had a bleeding episode and % died. there was a significant correlation between acute and chronic hemodynamic response to beta-blockers (r= . , p=o.ool). using roc curve a decrease of hvpg ?lo% from baseline was the better cut-off point to predict bleeding, both for acute and chronic studies. hemodynamic responders (defining response as a decrease of hvpg to ? mmhg or ?lo%), had a significantly lower probability of bleeding (p?o.ol) and of requiring hospital admission because decompensations of cirrhosis, while survival probability was higher, both after acute and chronic beta-blocker administration. the hvpg decreased ? % in % of cases and decreased ?lo% in % (p?o.ool) after acute beta-blocker. these figures were % vs % (p?o.ool) after a chronic administration. conclusions: our results suggest that: ) a decrease of hvfg ?lo% from baseline may be the target to identify hemodynamic responders in primary prophylaxis of variceal bleeding; ) , cute hemodynamic response to beta-blockers may provide an accurate prognostic information on long-term risk of variceal bleeding. ut; william m grosse, corneliu sanda, milton taylor, lndiana university, bloomington, in treatment of chronic hepatitis c patients with type interferon (ifn-alpha) and ribavirin leads to a sustained virologic response in - % of patients. the elimination of serum hcv rna in an individual patient displays biphasic kinetics with the first order attributed to the direct antiviral effects of ifn-alpha and the second order attributed to an immune mediated clearance of infected cells by induction of th cytokines, primarily ifngamma (type ifn). we have postulated that the direct antiviral effects as well as the th response of current therapies may be enhanced by the addition of type ifn (ifn-gamma). to study this hypothesis, we examined the activity of a type ifn, infergen; (ifn-alfaconl) in combination with a type ifn, actimmune; (ifn-gamma l b ) in preclinical models of hcv. these included an infectious flavivirus system (west nile virus) and the hcv replicon. in addition, global transcriptional profiling was examined utilizing the hg-u a genechip; (affymeirix, santa clara, california) system. effective concentration (ec) studies of the l effects of ifn-alfaconl, ifn-gamma l b and the combination against the west nile virus and the hcv replicon are shown below: analysis of synergy for the combination of ifn-alfaconl and ifngamma l b in the hcv replicon using the chau-talalay methodology demonstrated very strong synergistic effects for a range of doses (c < . for all observations). initial statistical analysis (pv<o.ool; fold change . ) of global transcriptional profiling revealed that treating hcv replicon containing cells with ifnalfaconl up-regulated transcripts, while transcripts were significantly down-regulated. ifn-gamma l b treated cells had transcripts up-regulated and down-regulated transcripts. most significant was the finding that several transcripts were up-regu-lated by the combination of ifn-alfaconl and ifn-gamma l b that were not up-regulated by either ifn alone. these transcripts were involved in cell-cycle regulation, antigen presentation, apoptosis and immuno-activation. in addition, several known interferon stimulated genes were highly elevated by the combination when compared to the monotherapy treatments. these results demonstrate synergistic effects of the combination of type and type ifns for the treatment of chronic hepatitis c both on a cellular and molecular level. further study in hcv infected patients is warranted. hcv is leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. clearance of hcv is associated with strong t cell immunity to hcv immunogens including the non-structural ns protein (ns ). dendritic cells (dcs) play a pivotal role in priming rare antigen specific t cells to initiate protective hcv immunity. therefore, dcs are an important target to elicit broader and more robust immune responses against hcv. to target immunogens to dcs, -mer peptides were screened from a phage display peptide library for specific binding to human dcs. a candidate -mer peptide (dc-peptide# ) was identified that bound human monocyte-derived dcs. dc-peptides bind to ligands that are strongly involved in receptor mediated endocytosis. dc-peptides could be linked to hcv ns biochemically or fused genetically with preservation of dc targeting specificity and immunogenicity. dcs pulsed with ns that has been genetically fused to dc peptide augmented t ceil proliferation > fold better thanns alone, and skewed t cells towards thl polarization resulting in elevated levels of ifn-.)i (higher than with ns alone) but not il- or il- . t cells also acquired an activated phenotype (cd +). to test in viva efficacy of ns -dc peptide fusion protein, nod-scid mice were xenotransplanted with hcv-nake t cells, and vaccinated with three separate weekly injections of autolo-gous dcs ( ) pulsed with nothing, ns alone, or the ns -dc pep-tide# fusion construct. vaccination using dcs pulsed with ns -dc peptide# fusion protein significantly increased ns -specific t cell proliferationlactivation and enhanced the expression of ifn-y, and tnf-a compared to ns alone but no detectable levels of il- or il- were observed. these data indicate that targeting hcv subunits to dcs using specific dc-peptides represents a novel vaccine approach that may facilitate targeting of immunogenic antigens to dcs to elicit specific t cell immune responses against ns of hcv. this immunotherapeutic strategy can be implemented against the broad range of immunogenic antigens of various pathogens. it has recently been reported that the administration of eta in the setting of chronic hcv infection enhances ifn-induced viral clearance ( ). the mechanism of these synergistic effects remains to be understood. our hypothesis was that eta enhances antiviral effects of ifn by increasing t cell reactivity to antigens. methods: peripheral blood mononuclear cells (pbmcs) were isolated in cpt tubes from blood of two healthy volunteers. cells were washed and then cultured in complete media -well plates in the absence of any antigen (negative control) and in the presence of immobilized anti-cd antibody (i-cd , nglml, positive control) as a nonspecific t cell stimulant. i-cd stimulated cells were treated immediately with peg ifn alpha- a at two different concentrations consistent with physiologic doses in humans ( nglml or nglml) with or without the addition of eta at two different concentrations that were also consistent with physiologic doses in humans ( . pglml or . pglml). supernatants from each of the previous conditions were collected and assessed by elisa for ifn- secretion as a marker of t cell activation. results: our findings are displayed in the figure below. there was no spontaneous ifn- detected in cells that were not stimulated. ifn-y was detected at a modest level after exposure to i-cd alone, a response that became greater after exposure of cells to peg ifn alone or eta alone. production of ifn- was substantially enhanced in cells that were exposed to a combination of peg ifn and eta compared to those exposed to peg ifn alone or eta alone. conclusion: our findings suggest that eta has a synergistic effect to that of peg ifn in promoting in vitro activation of pbmcs and ifn- production in healthy individuals. increased t cell activation with eta may also suggest that tnfa suppress t cell function and may contribute to refractoriness to inf therapy in hcv patients. the influence of absolute dose and dose in mg per kg body weight of ribavirin on sustained virologic response (svr) in interferonbased combination treatment of chronic hepatitis c as well as the influence of dose reduction is still controversial. here, we address this problem by reanalyzing data of previously untreated patients with chronic hepatitis c from a multicenter trial who were treated with interferon alfa- a plus ribavirin and amantadine or interferon alfa- a plus ribavirin (berg et al, hepatology , , - and completed therapy. thereby, we used a multivariate approach to account for correlations of the dose of ribavirin with body weight and body mass index (bmi) and known predictor variables from this data set which are low baseline hcv rna, high platelet counts, high pretreatment alt, and low y glutamyl transpeptidase (ggt) as well as hcv genotype non- . because per protocol dosage of ribavirin was weight-adjusted ( mg for body weight below kg and mg for body weight kg or more) and body weight was positively correlated with ggt and negatively correlated with platelet counts (spearman rank correlation, p<o.ol% and p= . 'x), respectively, we used a stratification with respect to genotype (gt vs. non- ), ggt, and platelet counts in the analysis of ribavirin dose. when comparing body weight, bmi, the absolute intention to treat (itt) ribavirin dose as well as the itt dose of ribavirin measured in mg per kg body weight and the respective ribavirin doses at the end of treatment (eot), a significant association with svr was found for the eot ribavirin dosage (mg per kg body weight) and bmi (p= . % and p= . %, respectively). for predicting svr, a weight-based ribavirin dosage threshold of . mg/kg was calculated using receiver operating characteristics (roc) curves. the svr rate was % ( ) in patients with a ribarivin dose of more than . mglkg as compared with % ( l ) in patients receiving equal or less than . mglkg ribavirin at eot (odds ratio . ; % ci . - . ; p=o.l%). in conclusion, the analysis of ribavirin dosage motivates new considerations of weight-adjustments of the ribavirin dosage to further increase svr in hcv-infected patients treated with combination therapy. effective and approved for treatment of patients with chronic hepatitis c virus (hcv) infection, the medications have many side effects and frequently require dose reduction or discontinuation. improved adherence to the medical regimen increases sustained response rates. data currently available regarding dose reduction, discontinuation and svr are largely derived from registration trials with experienced hepatologists at academic centers. anecdotal information suggests that dose reduction and discontinuation rates are higher and svr lower in community practice, where the majority of hcv patients are treated. actual dose reduction and discontinuation rates and svr in community practice are unknown. aim: we sought to determine dose reduction and discontinuation rates and svr in community practice in comparison with academic centers within the context of a prospective, randomized, controlled, multi-center trial. methods: patients with chronic hcv (+hcv rna) were evaluated at academic centers and community sites within the context of a randomized trial comparing peg ifn a ( . pglkglwk) + ribavirin ( - mgld) vs. peg ifn a ( . pglkglwk) + ribavirin ( - mg/d). demographic, serological (alt), virological (hcv rna level and genotype) and histological data were obtained. eligible patients were randomized to one treatment group. medications were administered for weeks (hcv gt non- ) or weeks (hcv gt ). tolerability (dose reduction rates, adverse events and discontinuation rates) through week of therapy was determined and comparisons were made between academic (ac) and community-based centers (cc). results: patients have been enrolled, in ac and in cc. patients in ac and in cc remain on therapy within the initial weeks and are not included in this report. genotype, histology, gender and age profiles were similar in the groups. * designates a significant difference p< . . by week , ( %) in ac discontinued medications in comparison to / ( %) in cc". / ( %) discontinuations were due to aefsae in ac in comparison to ( %) in cc. ( %) patients in ac who did not discontinue by wk received peg ifn dose reduction in comparison to / ( %) in cc". / ( %) patients in ac who did not discontinue by week received ribavirin dose reduction in comparison to / ( the clinical outcome in response to combination therapy for treatment of chronic hepatitis c virus (hcv) infection appears to be different for caucasian versus african american patients. combination therapy of ribavirin and interferon alpha- b has generally been found to be more effective for eliminating hcv in caucasians. the present study has been undertaken to further define this difference in response to therapy between caucasians and african americans and to determine whether the difference may be related to a difference in t cell-dependent immune responses to hcv in the two patient populations. to this end peripheral blood mononuclear cells (pbmc) were collected from patients prior to the initiation of combination therapy with ribavirin and pegylated interferon alpha- b, week , and at weeks and weeks after initiation of therapy. the pbmc were stimulated in vitro with recombinant hcv-superoxide dismutase (sod) fusion proteins for ns (sod-c c), core (sod-c - ), ns (sod-c - ), ns (sod-nss), and the relevant recombinant sod controls from yeast or bacteria (all generous gifts from dr. michael houghton, chiron). after four days, h-tymidine was added to the cultures and incubation continued for hours to measure proliferation. the cultures were harvested on day five. supernatants from identical cultures were collected for assay of th , il- and interferon gamma (infy), and t h~, il- and il- , cytokine production. t cell responses to tetanus toxoid and the mitogen phytohemagglutinin (pha) were used as positive controls for t cell response potential. to date patients have entered the study. week data has been collected from patients and normal controls. data have been collected from only patients that have completed the study through week . the results indicate that generally all patients' t cells were stimulated by pha and to lesser extent tetanus toxoid. the major cytokine stimulated by both pha and tetanus toxoid was infy. few of the patients had significant t cell responses to hcv proteins at time measured either as proliferation or cytokine production. over the period of combination therapy, most patients in which serum virus titers were reduced to non detectable levels had significant increases in t cell responses to hcv proteins. although t cell proliferation was varied among the patients, there was generally a marked increase in infy production but little production of other cytokines. responses to hcv proteins were less consistent in those patients that either did not clear the virus or in which there was resurgence of virus after initial clearance. hepatitis c virus (hcv) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer (hcc). the high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for hcv. the hcv ns serine protease is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. intracellular biological processes can be specifically manipulated by anti-viral antibodies. we have recently presented the modulation of hcv ns serine protease transforming activity by non-neutralizing recombinant intracellular antibodies (intrabodies). we herein report the development of a novel bacterial genetic screen for inhibitors of ns serine protease catalysis and its application for the isolation of single-chain antibody-inhibitors. our screen is based on the concerted co-expression of a reporter gene, of recombinant ns and of stabilized single-chain antibodies (scfvs) in e. coli. the reporter system had been constructed by inserting a short peptide corresponding to the ns aib cleavage site of ns into a permissive site of the enzyme p-galactosidase. the resulting cleavable lac gene is carried on a low copy plasmid that also carries the ns coding sequence. the resultant p-galactosidase enzyme when active, conferring a lac' phenotype (blue colonies on indicative x-gal plates) while co-expression of active ns results in loss of / -galactosidase activity (transparent colonies on x-gal plates). the identification of inhibitors, as shown here by isolating ns inhibiting single-chain antibodies is based on the appearance of blue colonies (ns inhibited) on the background of colorless colonies (ns active). our source of inhibitory scfvs was a scfv library we prepared from spleens of ns -immunized mice. once isolated, the inhibitors were validated as genuine and specific ns binders by an elisa and as bone fide ns serine protease inhibitors by an in vitro catalysis assay. these antibodies are also analyzed for intracellular immunization against hcv ns serine protease, as inhibiting intracellular antibodies. single chain ns neutralizing antibodies may emerge as useful clinical reagents for the treatment of infectious diseases and cancer. our genetic screen, although applied here for the isolation of antibody-based inhibitors, should be applicable for the identification of candidate inhibitors from other sources. ntroduction:genetic heterogeneity is an important feature of hepatitis c virus (hcv) genomes with six known genotypes and more than subtypes. genotypes , and are broadly distributed in patients around the world, whereas the other types are more geographically restricted. the peptidomimetic inhibitor biln , optimized to have a high affinity for genotype ns protease, has been reported to reduce viral load in genotype hcv infected individuals. in this study, the ability of biln to inhibit the ns proteases from the other widespread genotypes and was assessed. methods: the ns protease domains and the ns -ns a proteins of genotypes la, lb, ac, b and a were cloned, expressed in e. coli and purified. protease activity was evaluated using fluorogenic depsipeptide substrates derived from the amino acid sequence at the ns a-ns b and ns a-ns b junctions. results: the activity of the ns protease domains revealed no major differences among the various genotypes. for the ns -ns a proteins, the catalytic efficiencies of the non-genotype enzymes, although higher than the ones observed for the corresponding protease domains, were similar to that observed for genotype (within -fold). differences in activity observed among genotypes were mainly related to changes in k,,, values. binding constant (k,) values for biln were similar among non-genotype proteases with k,'s ranging from - nm for the ns -ns a proteins, up to a -fold reduction in affinity when compared to genotype . hepatitis-c-infection (hcv) is associated with an increased incidence of the chronic fatigue syndrome and depressive mood changes. beside immunological changes the modulation of the serotonergic system might be related to these psychiatric syndromes. serotonin concentration, -ht uptake and the activity of the enzyme mao-b in platelets are useful peripheral parameters to monitor serotonergic activity and function in patients with a chronic hepatitis c (chc). method in a prospective controlled study biochemical measures included assays of -ht concentration and -ht uptake activity at a low physiological substrate concentration in platelets as well as mao-b activity of patients with a chronic hepatitis c were compared to age and gender-matched healthy subjects. furthermore the effect of interferon-alpha treatment on serotonergic parameters was evaluated in patients before and during treatment. results: the mean serotonin platelet concentration did not differ between controls and hcv-infected patients. however, -htuptake and the mao-b activity were significant lower in patients with hcv-infection (p<o.ool respectively, t-test, two-tailed). interferon-treatment led to an increase in it reuptake (p=o.ol), to an increase of mao-b activity (p<o.ool) and to a decrease of serotonin concentration (p<o.ol). thus, during hcv-infection serotonin concentration was stabilized by a decrease in re-uptake and reduced metabolism by lowering mao-b activity. treatment with interferon-alpha led to a decompensation of those mechanisms by increasing the mao-b activity and -ht-reuptake, followed by a decreased serotonin concentration. conclusion: our data support the hypothesis of a tryptophan depletion and serotonergic deficit in patients with chronic hcvinfection and interferon-alpha treatment. furthermore the results give evidence for a useful role of serotonin reuptake inhibitors for the treatment of the serotonergic deficiency during chronic hcvinfection and interferon-alpha treatment. chronic background: recent large prospective trials demonstrated that the combination therapy of interferon ( fn)-alphalribavirin significantly increased the ratio of a sustained virological response in patients with chronic hepatitis c in comparison with ifn monotherapy, especially in patients with high hcv-rna titer and genotype lb. however, the mechanism of beneficial effect of ribavirin is still unknown. on the other hand, interleukin (il-m), originally termed ifn-y inducing factor, is one of proinflammatory cytokines, which acts on th- cells and strongly induces intrahepatic nk or nkt cells as well as activated t cells to produce ifn-y. to clarify the effect of ribavirin in combination with ifn, special emphasis on il- , we examined the correlation between serum il- level and anti-viral effect, and compared it with ifn monotherapy in patients with chronic hepatitis c. patients and methods: all patients in this study are biopsy proven chronic hepatitis c with high hcv-rna titer (hcv-rna>loo kcopieslml before therapy) and serotype (genotype l a or lb). forty-two patients were treated with ifn alpha- b ( mu) in combination with ribavirin ( - mg) daily for weeks, following weeks with ifn alpha- b ( mu) times a week (the combination group). another patients who were treated with ifn alpha- b alone between and (before introduction of ribavirin in japan) with the same schedule are used as control (the monotherapy group). serum samples were taken before, weeks after administration and weeks after cessation of therapy. serum il- are examined by enzyme linked immuno-sorbent assay (elisa), using human il- elisa kit (mbl, nagoya, japan). the il- ratio is defined as serum il- level before administration divided by serum il- level weeks after administration. hcv-rnas are quantitatively examined before and weeks after administration. sustained viral responses are confirmed weeks after cessation of administration by rt-pcr. results: there are no differences in the background of patients between the combination group and the monotherapy group. in the combination group, the decline of hcv-rna level highly correlates with the il- ratio in patients with higher viral titer (hcv-rna> kcopieslml before therapy) despite no correlation is observed in patients with lower viral titer (hcv-rna<soo before therapy). similarly, the hcv-rna level weeks after administration controversially correlates with the il- ratio in the higher titer group despite no correlation in the lower titer group. interestingly, serum il- level itself does not correlate with the decline of hcv-rna level in both the higher and lower hcv-rna titer group. on the contrary, in the monotherapy group, the level of up-regulation of serum il- is lower than that of the combination group. furthermore, although the decline of hcv-rna also correlates with the il- ratio in the higher titer group, the level of a correlation coefficient is lower than that of the combination group. however, the sustained viral clearance weeks after cessation of treatment is not correlated with the il- ratio. it suggests that ribavirin enhances the up-regulation of serum il- level in combination with ifn therapy and the effect of ribavirin is critical for the early anti-viral response during therapy, not for sustained viral response. objective: treatment with pegylated interferon alfa and ribavirin has an efficacy in chronic hepatitis c patients with sustained response rates of about %. however, response in genotype patients with high viral load is considerably lower and is not significantly improved by pegylation of interferon. methods: in the present study, the efficacy of cifn daily dosing and induction therapy followed by combination with ribavirin in naive patients with chronic hepatitis c and genotype was evaluated. au patients had histologically proven hepatitis, elevated alt values and were viremic, the average weight was kg. patients were treated with cifn dosages of or ug qd for weeks, followed by or ug qd for weeks. treatment was continued with cifn at ug qd with ribavirin ( . or mglkgld) for another weeks. results: at weeks therapy an undetectable hcv-rna was observed in % and % in the cifn / and / ug groups, respectively. data regarding sustained response rates showed ' and % for cifn / and / ug groups, respectively. due to side effects cifn had to be dose reduced in % and discontinued in % of patients. addition of ribavirin potentiated the effect of consensus interferon in a dose-dependent manner as has been shown for other interferons previously. when viral response rates were related to the initial viral load, in the genotype i low viral load group, sr rates of and % were obtained for the cifn / and / ug groups (diff. n.s.). in contrast, genotype / high viral load patients showed sr rates for the cifn / and cifn / ug groups of and % (diff. sig.), respectively. the viral response rates obtained with daily dosing of cifn were significantly higher than an independently run control arm using cifn at ug tiw with ribavirin irrespective of the initial viral load. four patients ( %) experienced grade i thrombocytopenias, while no grade iv neutropenias or thrombocytopenias were observed. the overall tolerability in the cifn / ug qd arm was comparable to standard therapy with pegylated interferon a b and ribavirin, while the cifn ug arm was less tolerable during the high dosing period. however, the withdrawal rates were not sigruficantly affected by the higher dose of cifn. conclusions: cifn daily dosing l induction therapy in combination with ribavirin thus shows significant response rates with the highest response rates in genotype patients seen to-date. the sustained response rates for genotype i high viral load patients are higher than the ones demonstrated in the peg interferon and ribavirin registration trials. these data suggest that for difficultto-treat genotype / high viral load patients cifn with daily and higher initial dosing may be a worthwhile alternative to standard combination therapy with pegylated interferons. introduction: hcv-induced cirrhosis or hepatocellular carcinoma account for at least a quarter of all patients undergoing liver transplantation. almost % of hcv related liver transplant patients are re-infected with hcv after liver transplantation, often within days. in most of these cases, the recurrent infection leads to chronic hepatitis. these patients are in need of preventive therapy against re-infection of the transplanted liver. antibodies may be used as a stand-alone prophylactic therapy to prevent re-infection following liver transplantation or in cases of accidental exposure (e.g. needle stick injury). in addition, it may be feasible to use monoclonal antibodies in combination with other antivirals to treat chronic hcv patients. purpose: to develop an effective therapy to prevent hcv infection in liver transplant patients. methods: several human monoclonal antibodies were generated from peripheral blood b cells isolated from individuals infected with hcv genotype lb. these antibodies are directed against the hcv envelope protein (e ) of the virus and have high affinity and broad reactivity against different genotypes. the antibodies were shown to neutralize hcv in vitro in a cell-based infection assay and in vivo using the hcv-trimera mouse model. hepex-c, a single human monoclonal antibody, was further developed and studied clinically for safety, tolerability, and potential antiviral activity in phase l a and l b clinical studies in chronic hcv patients. results: in a phase l a study, single doses of . , . , . , , and mg of hepex-c were administered to a total of chronic hcv patients. these patients had varying levels of hcv-rna ( . x lo to x lo iulml) and endogenous anti-e antibodies ( to pglml). doses of hepex-c were well tolerated and no moderate or serious adverse events (sae) were reported. in out of patients, hcv-rna levels were reduced at least by half immediately following infusion and then gradually returned to initial levels. a multidose phase l b study of , , , , and mg of hepex-c in hcv chronic patients was conducted. doses were administered weekly for weeks and then times a week during the fourth week. the patients were followed for an additional months. multiple doses were well tolerated; no drug related saes were reported and no specific pattern of ae noted. out of patients from the through mg cohorts had at least log infusion related reduction in hcv-rna from pre-treatment levels following administration of hepex-c. data from the mg cohort are pending. conclusions: the good safety and efficacy data from this trial provided a basis for a phase a pilot study of the safety and efficacy of hepex-c for the prevention of recurrence in hcvinfected patients who have received a hepatic allograft for endstage liver disease. a multicenter, blinded, placebo controlled study is currently underway in liver transplant patients receiving , , , and mg doses of hepex-c. logic response rates to interferon (ifn) therapy in aa patients w i t h chronic hcv infection have been reported to be lower than that for caucasians (ca), but the effect of therapy on hepatic histology in aa patients remains to be assessed. objective: to evaluate histologic progression of disease in conjunction with anti-viral efficacy and safety in non-hispanic aa hcv genotype patients and non-hispanic ca, hcv genotype patients treated with peginterferon alfa- a ( kd) in combination with ribavirin (rbv). methods an open-label, multicenter study was conducted to evaluate the efficacy and safety of the combination of peginterferon alfa- a ( okd) and rbv in aa patients and in ca patients. all patients were required to have previously untreated chronic hcv genotype infection, elevated alt levels and. a liver biopsy obtained within the months prior to enrollment demonstrating chronic hepatitis but without cirrhosis. patients received peginterferon alfa- a ( okd) pg sc once weekly plus rbv ooo or mg orally based on body weight (< kg or = kg) for weeks, with weeks of treatment-free follow-up. histologic responses based on the knodell hai score were determined from liver biopsies obtained prior to treatment and within weeks of completion of the -week untreated follow-up period. hai activity scores range from - and fibrosis scores range from - . the histology outcome was evaluated for patients with paired biopsies only. improvement in necrointlanunatory activity was defined as point decrease, and improvement in fibrosis as point decrease. the primary efficacy endpoint was sustained virologic response (svr) with undetectable hcv rna (< iulml) at weeks. results: the intent-to-treat population included patients. paired biopsies were available for of the aa patients and of the ca patients. these patients were representative of all patients with respect to knodell hai scores. mean baseline hai activity scores were . . for aa and . & . for ca patients; mean baseline fibrosis scores were . + . and . + . , respectively. the svr rate for all aa patients was l ( %) and for all ca patients, / ( %). for those patients with paired biopsies, svr rates were / ( %) in aa patients and loll ( %) in ca patients. the majority of patients in both groups exhibited improvement in activity scores: % for the aa group and % for the ca group. more importantly, the table beiow shows that the proportion of patients with worsening fibrosis was similar in both groups, while / ( %) of aa patients and only / ( %) of ca patients showed fibrosis improvement. mean changes in fibrosis scores were - . +- . for aa and - . t . for ca patients. among aa patients who had paired biopsies, / ( %) patients who achieved svr had fibrosis improvement; and / ( . %) viral nonresponders exhibited fibrosis improvement. the only patient who had fibrosis improvement among ca was a viral non-responder. conclusions: therapy with peginterferon alfa- a ( okd) plus rbv produced improvement in necroinflanunation and fibrosis in a substantial group of aa patients in this study. although the svr of % in aa with genotype hcv is the highest response to combination therapy yet reported in this population, even patients without an svr achieved a benefit with regard to hepatic histology. higher sustained response rates (svr). however, modification of interferon-alfa by pegylation also reduces biologic potency. delivery of a bio-optimized alpha interferon in an unmodified form (consensus interferon, infergen(r)) by continuous infusion can be expected to provide sustained and constant levels of a fuuy potent protein. we hypothesize that such an approach may potentidy result in greater antiviral activity and improved tolerability by avoiding wide swings in interferon levels. methods:we conducted a phase pilot study to assess the safety, tolerability and viral kinetics of mergen ( fg/day) by continuous infusion using a subcutaneous infusion device (medtronic minimed(r) pump) in combination with ribavirin (loo mg or mg daily). the minimed model micro infusion pump is cleared for use in the treatment of diabetes. hcv rna viral levels were measured at days , , , , , , and then at weeks and . results: among nonresponders treated to date with infergen by continuous infusion with available early viral kinetics data, showed a substantial reduction in hcv viral levels (at least loglo reduction in , and loglo reduction in ). all of these patients were genotype and had less than . loglo decrease with prior treatment while had a . loglo decrease at similar time-points with other combination therapy [either pegylated interferonlrbv ( pts) or interferon-alfa/rbv ( pts)]. no serious adverse events were reported and patients discontinued ( due to moderate interferon-related side effects and due to inability to acclimate to the pump). other patients continuing on study experienced mild adverse events that have been tolerable. conclusions: consensus interferon administered by continuous delivery via the medtronic minimed pump shows early substantial reduction of hcv rna levels among nonresponder patients and shows a good safety and tolerability profile. updated data on a total of patients (including na'ive genotype patients) will be presented. background side effects of interferon-ribavirin combination therapy limit the sustained viral response achievable in a given hcv patient population. furthermore, it has been suggested that higher ribavirin doses and longer treatment duration are required to achieve a sustained response rate in patients with genotype . coupling of ribavirin to hemoglobin offers the potential of a therapeutic with improved safety and efficacy by targeting the delivery of ribavirin to key tissues infected by viral infections such as hcv. these tissues include liver parenchymal cells and macrophages, both of which possess receptors for binding and internalization of hemoglobin-haptoglobin complexes as part of the natural clearance pathway for cell-free hemoglobin. hemoglobin thus serves as a natural drug carrier for targeted therapy of such receptor-bearing tissues. aim: to evaluate the effect of a ribavirinhemoelobin coniueate (rbv-hb. hrc ) in a mouse model of viral hepatitis induced by the coronavirus, mhv- . methods: ribavirin was covalently coupled to highly purified hemoglobin hbao at a mean molar drug ratio of (ribavirin:hemoglobin). the rbv-hb was evaluated for retention of haptoglobin binding and cellular uptake in vitro, as well as the ability to recover bioactive ribavirin following enzymatic cleavage from rbv-hb. the rbv-hb was complexed to haptoglobin prior to administration to mhv- infected balblc mice, and carried one-third of the drug dose that was used for the ribavirin group. the rbv-hbtreated group was compared to control infected untreated and ribavirin-treated groups for survival, clinical behavior, viral titer, biochemistry and liver histopathology. results: rbv-hb was specifically internalized by cultured human and mouse hepatic cells but not by control non-hepatic cells. ribavirin enzymatically cleaved from rbv-hb retained in vitro bioactivity similar to unmodified ribavirin. untreated and mhv- infected mice all developed clinical and biochemical signs of acute viral hepatitis and died by day post infection (altmax iuil). livers recovered from untreated and infected mice showed greater than % necrosis. in contrast, survival was enhanced in both ribavirin and rbv-hb treated and mhv- infected mice with a marked reduction in biochemical (alt iuil) and histologic evidence of hepatic necrosis ( % ) . clinically, rbv-hb treated mice behaved normally at all time points, in contrast to ribavirin treated mice which developed lethargy and abnormal fur texture compatible with drug toxicity or ongoing viral infection. conclusions: in this study, a beneficial effect of rbv-hb was shown on the course of mhv- infection as demonstrated by prolonged sunival, improved clinical behavior, and a reduction in biochemical and histologic disease. the study provides a rationale for additional studies to examine the mechanism for the beneficial effect. ( introduction: the teravic- study targets patients with chronic hepatitis c who do not show a very early viral response at week of therapy, as defined by detectable serum hcv rna (> kjlml, pcr methodology). after weeks of therapy, these patients are expected to achieve a lower rate of sustained viral response (svr) than patients with undetectable hcv rna at week (vr ). models based on viral dynamics and data from pilot studies with conventional interferon (ifn) suggest that a treatment period longer than weeks may lead to a higher svr rate in these difficult-to-treat patients. this trial compares versus weeks of treatment with pegasys(') plus copegus('i in treatmentna'ive patients without vr . methods: this phase , randomized, parallel group, multicenter study is being conducted in spain in accordance with the principles of good clinical practice.(l) after signing the informed consent form, all patients received the study treatment (peginterferon alfa- a [ kd] [pegasys(']] pg once weekly and ribavirin [copegus(')] mg daily mg bid]) for weeks. at this time, viral response (vr) was assessed using the cobas amplicor hcv(') test v . (detection limit hcv rna iulml). patients achieving vr continued therapy for an additional -or -week period, according to their hcv genotype and viral load. patients without vr were randomized :l to continue the study treatment for either an additional weeks (total active treatment duration weeks or an additional weeks (total active treatment duration weeks ). a -week follow-up period is ongoing to determine the svr rate. results: the mean age of the patients was years, the mean weight was . kg, and % were female. the prevalence of hcv genotype or was %. vr occurred in % out of patients, ( % of those with hcv genotype or , % of those with other hcv genotypes). hcv genotype distribution, viral load, age, and body weight were similar in the randomized study groups (group- and group- ). among patients without vr , hcv rna was undetectable in "h in group- and in % in group- at week . the biochemical response rate (br) at week , defined as normal alt levels, was similar as well: % in group- and % in group- . the dropout rate after weeks was % in group- and % in group- . at the end of therapy (eot), hcv rna was undetectable in % of patients with vr . the eot vr rate in group- was % versus y in group- . the rate of br in patients with vr at eot was also higher in group- ( %) than in group- ( %). dropout rates and reasons for dropping out by the eot visit were different between groups: % in group- and % in group- . therapy was well-tolerated and no unexpected adverse events were observed. the prolongation of therapy from to weeks did not cause an increase in the frequency of neutropenia or thrombocytopenia. conclusions: pegasys(') p g weekly plus copegus(') mg bid administered for weeks offers higher rates of vr and br at eot than weeks of treatment in patients with detectable hcv rna four weeks after treatment initiation. the mglday dose of ribavirin used in the trial has been proven to be optimal in patients infected with hcv genotype or , but not in those with genotype who require mglday. this evidence was not established when the trial was initiated. "the teravic- study group also included v. ripollks background isis is an antisense phosphorothioate oligodeoxynucleotide inhibitor of hepatitis c virus (hcv). in a previous phase i study, plasma hcv rna reductions . log were observed in chronic hcv patients treated with isis for weeks. aims: the goals for this phase i study were to evaluate the safety, antiviral efficacy, and pharmacokinetics of isis given for weeks. methods: the study was conducted in noncirrhotic chronic hcv patients. following weeks of thrice weekly intravenous dosing at . mglkg ideal body weight (ibw), patients were treated for an additional weeks at higher doses given either once (group a) or twice weekly (group b). the two doses studied were (initial patientslgroup) and mglkg ibw. results: of patients enrolled into the study, had genotype hcv, had plasma hcv rna levels x lo copieslml, and all but two had previously failed interferon-based therapies. two of the group a and of the group b patients treated at mglkg ibw had plasma hcv rna reductions . log. three in group b had reductions of . - . log. hcv levels remained . log reduced at days after the last isis dose in one of these patients. seventeen patients had transient elevations in alt including all those with hcv rna reductions. peak alt levels ranged - times the upper limit of the normal range (x uln). for those with hcv rna reductions, the alt flares were temporally associated with the reductions. the alt flares were not associated with any clinical signs, symptoms, or sequelae. there were no changes in prothrombin time or albumin level. minor alkaline phosphatase ( . x uln) and bilirubin ( . x uln) elevations occurred in patients. dosing with isis was continued during alt flares, all of which resolved to baseline levels despite continued dosing. three flares, ranging from - x uln, occurred - weeks after the last dose of isis , when presumably little or no drug remained in the liver. similar alt elevations have not been observed in the clinical studies of other phosphorothioate oligodeoxynucleotides. aside from the alt flares, common adverse events in this study were mild to moderate headache, fever, chills, fatigue, nausea, myalgia and other flu-like symptoms. the fever and chills usually occurred several hours after the end of the mglkg infusions and resolved within hours. two serious adverse events that were considered possibly related to isis occurred in the study: one patient had cryoglobulinemic glomerulonephritis and one was hospitalized for precautionary monitoring of an altlastlbilirubin flare. the former event was possibly due to an exacerbation of a pre-existing condition. the isis plasma pharmacokinetics observed in this study were consistent with those seen in the previous phase i study. conclusions: isis appears to have significant antiviral activity in some chronic hcv patients. the plasma hcv rna reductions (up to . log) achieved in this study were in difficult-to-treat patients (i.e., genotype , high virus level, andlor nonresponders to interferon-based therapy). although, alt flares were observed in some patients, the results suggest the flares may not be due to a direct hepatotoxic effect of the drug. other than the alt flares, isis treatment was generally well tolerated. this study established the dose of mglkg ibw twice weekly for further clinical evaluation. a study of isis in combination with peginterferon and ribavirin for patients not achieving an early virologic response during standard peginterferon and ribavirin therapy is in progress. with the long serum half-life of albumin. the objectives of this phase / , open-label, dose escalation study were to evaluate the pharmacokinetics, safety, tolerability, immunogenicity, and pharmacodynamics of albuferon in subjects with hepatitis c virus (hcv) who had previously failed ifna containing regimens. methods: subjects were initially enrolled in sequential dose groups ( - subjects per each group at pg, pg, pg, and pg). within each dose group a minimum of and a maximum of subjects per cohort were enrolled sequentially to receive or subcutaneous (sc) doses of albuferon administered days apart. dose escalation beyond pg included single injections of pg, pg, p g pg and pg. plasma albuferon concentrations and antibody to albuferon were measured by elisa. hcv rna was measured using the amplicor hcv monitor kit (roche). ' ' oligoadenylate synthetase (oas ) mrna levels in whole blood was measured by a research-based taqman pcr assay. results: of the subjects currently enrolled, % were infected with hcv genotype with a mean baseline viral burden of million copies/ml. % of subjects enrolled in these cohorts had previously failed pegylated ifna containing regimens. albuferon pharmacokinetics showed linear increases in auco. and c, , , with mean terminal half-lives of - hours with doses of pg and higher. t, , , occurred between days and . there is an approximately % increase in auc after the second injection in the double injection cohort at pg. albuferon was well tolerated and there were no discontinuations. no subjects developed detectable anti-albuferon antibodies. adverse events were transient and most were mild to moderate. the most common adverse events were injection site erythema ( %), headache ( %), fatigue ( %), mylagia ( %) and arthralgia ( %). the mean reductions in the nadir neutrophil counts in the higher single dose cohorts ranged from - %. there was induction of oasl mrna expression in all cohorts, with approximately -fold increase in median values at day . in the single dose cohorts (= pg), % of subjects showed a maximal . log or greater reduction in hcv viral load during the first two weeks. also, % or greater reductions in alt levels were observed in % of subjects in these single dose cohorts. conclusions: in the ongoing phase study, albuferon demonstrated a favorable safety and immunogenicity profile. the pharmacokinetic profile supports dosing every - wks given its reduced clearance and extended half-life of up to hours. all cohorts showed evidence of biological activity, as demonstrated by oasl induction, with anti-viral activity evident in the higher single dose cohorts. disclosures: v balan -human genome sciences, inc.: investigator t bambury -human genome sciences, inc.: investigator g everson -human genome sciences, inc.: investigator w freimuth -no relationships to disclose h mesghali -no relationships to disclose j murray -no relationships to disclose d nelson -human genome sciences, inc.: investigator l novello -no relationships to disclose b osborn -no relationships to disclose j recta -no relationships to disclose g subramanian -no relationships to disclose m sulkowski -human genome sciences, inc.: investigator j zhong -no relationships to disclose introduction pirfenidone (pfd) is an orally bioavailable pyridone derivative that affects a variety of cytokines, including inhibition of tgfbeta, tnf-alpha, pdgf, and egf. pfd has been shown in clinical studies to improve physiologic parameters in patients with pulmonary fibrosis. we have previously shown that pfd decreased hepatic fibrosis in two different rat models of cirrhosis of hepatology : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) . our objective in this pilot clinical study was to evaluate the safety and preliminary activity of pfd in patients with cirrhosis of varying etiologies. methods all patients had histologic andlor clinical evidence of cirrhosis. pfd was given orally at a dose of mg tid for months. physical examination and labs including alt, ast, bilirubin, albumin and prothrombin time, platelet count were assessed at baseline and on monthly basis. hcv rna levels were measured in patients with chronic hepatitis c (cobas amplicor hcv monitor v . ). liver biopsies were obtained at baseline and after months of treatment and were read independently by two hepatopathologists who were blinded to the biopsy sequence. modified histological activity (hai) index of knodell and ishak fibrosis stage were used to assess changes in necroinflammatory scores and fibrosis stage, respectively. change in steatosis was also assessed. results a total of patients with cirrhosis due to hepatitis c ( ), ethanol (s), amyloidosis (l), autoimmune disease ( ) and budd-chiari syndrome ( ) were included. the mean age was years (range, - ) with males. liver biopsies at end of therapy showed a -point or greater reduction in the ha necroinflammatory score in % of the patients. steatosis decreased in % of the patients, was unchanged in % and worsened in %. while there was no significant reduction in the ishak fibrosis stage, improvement in interstitial fibrosis was noted. evidence of cell regeneration was seen in some patients. hcv rna levels were measured in patients with chronic hepatitis c. at months, patients had a decrease in viral load, patients remained unchanged and patients displayed an increase in viral load compared to baseline. no patient had a sustained virologic response. out of ( %) hcv patients had normalization of alt, out of ( %) had decreased alt values, did not change ( %) and patients showed a modest increase in alt ( %). pfd was well tolerated with the predominant drugrelated adverse events being nausea, photosensitivity rash, and itching occurring in % of the patients and which improved after to months of therapy. conclusions: in this pilot study, treatment of cirrhotic patients with pirfenidone for one year was well tolerated. a significant reduction in necroinflammation ( -point reduction in ha grade) and steatosis was observed in a substantial proportion of patients. a subset of patients with chronic hcv infection showed on-treatment reduction in hcv rna levels. longer treatment duration or a less cirrhotic patient population may be needed to demonstrate effects on fibrosis. these data support conducting clinical studies to evaluate a potential role of i'fd in the treatment of steatosis, or in combination therapy for chronic hepatitis c. this work was supported by grants of marnac, inc and intermune, inc. disclosures: arnulfo alvarez -no relationships to disclose gil arechiga -no relationships to disclose juan armendariz-borunda -no relationships to disclose background: viral kinetic modeling of hcv response to interferon-based therapy provides important insights into factors associated with treatment outcomes. hcv/hiv coinfected patients appear to have lower overall response rates than that observed in monoinfected subjects, but the reason for this is not clear. we speculated that kinetic responses in key parameters would be decreased in coinfected subjects. methods: hcvlhiv coinfected patients and hcv monoinfected patients prospectively matched for treatment, genotype, age (k yrs), gender, race and histology were evaluated. coinfected patients were randomized within the context of a large us. multicenter trial (actg ) to receive pegylated interferon alfa- a + ribavirin vs. interferon alfa- a + ribavirin. quantitative hcv rna (roche cobas amplicor) kinetic testing was performed at , , , , , and hours and at days , . non-linear regression and linear models were evaluated in an effort to best predict response and identify prognostic factors. results: twenty-seven subjects underwent viral kinetic sampling and evaluation. these included twelve hcvlhiv case subjects ( men, women) and matched hcv controls (some patients double-matched). the mean age of coinfected subjects was . years (range, - ). among hiv+ subjects the mean cd + count was cells/mm (range, - ). / had baseline hiv rna copieslml. mean hcv viral load was . log iulml among coinfected vs. . log iulml in controls. % of coinfected subjects had hcv genotype . the remainder were genotype . % of coinfected patients had no detectable virus weeks after completion of weeks of therapy (svr). overall svr in control subjects was . %. efficiency ( e ) of phase response (a) slope at hours, lambda (slope of phase decline) were calculated. efficiency of clearance ( e ) at hours was highly associated with actual viral clearance across all groups (p=o.ool) but a was not. regression analysis failed to demonstrate a relationship between e and baseline cd + count, hcv viral load or genotype. in contrast, # ; was significantly associated with genotype. viral kinetic parameters were not predictive of svr. conclusion: viral kinetic modeling demonstrated that the efficiency of clearance at hours was significantly associated with viral clearance during treatment. coinfection status did not affect key kinetic parameters. peg-ifn was superior to standard interferon in terms of viral clearance efficiency, particularly in the coinfected group. diminished svr in coinfected patients may be related to immune factors that are operative after reduction of viral loads to undetectable levels. the prevalence of chronic hepatitis c (hcv) infection is greater in african-americans (aa) than in caucasian-americans (ca). however, small studies and post-hoc analyses of larger clinical trials have not found racial differences in disease severity at study entry, though indicate that interferon and ribavirin combination therapy is not as efficacious in aa as in ca. it is important to identify disease and patient characteristics that differ between aa and ca in a large study designed to ascertain whether the apparent racial difference in response to therapy can be explained. aim: the study of viral resistance to antiviral therapy in chronic hepatitis c (virahep-c) is investigating reasons for the lack of sustained viral response (svr) to pegylated interferon and ribavirin therapy in previously untreated patients chronically infected with hcv genotype . a key objective is to determine whether previously reported response rate differences between aa and ca exist in a large multi-center cohort. methods: virahep-c will recruit aa and ca from clinical centers in the united states. hypothesizing that aa and ca patients differ by factors that influence svr which could explain racial differences in svr, we compare demographic, clinical, histological, virological and health-related quality of life variables (hrqol) before treatment begins. liver biopsies performed within months of study entry were assessed without knowledge of patient race by a central pathologist. results: based on the first participants recruited, age and sex distributions are similar in the two racial groups with patients averaging years of age and / being male more than % of the cohort has at least a high school education, but aa are more likely to have less than a high school education. body mass indices tend to be higher among aa than among ca (p=. ), though waistto-hip ratios are similar (mean . in each group). there are no racial differences in mode of hcv transmission, estimated duration of hcv infection, or baseline serum hcv rna levels. aa are more likely to have sub-genotype l b (p=. ).the ishak fibrosis score does not differ by race, but severe lobular inflammation is greater in aa patients (p=. ). diabetes and hypertension are more common in aa. the sf- physical component score in ca is similar to the general population and higher than among aa (p=. ). the sf- mental component score is similar in the two racial groups and comparable to the general population. summa-wlconclusion: with few exceptions, pretreatment virological, clinical and liver biopsy findings were generally similar in the two racial groups. differences in response are unlikely to be explained by these factors. lshak flbrosls score (%) severa lobular influnmation (%) hypertension (%) diabetes ( bar-llan university, ramat-gan, israel background: combination therapy with peginterferon alfa and ribavirin for weeks achieves sustained virologic response rates (svr) of - % in patients with chronic hepatitis c. however, the svr rates are highly variable according to baseline (hcv genotype) and on-treatment parameters (initial viral decline). thus, it must be anticipated that current standard therapy recommendations lead to under-treatment in some and over-treatment in other individual patients. objectives: comparison between a dynamically individualized treatment schedule according to the early virologic response versus the standard of care combination therapy with peginterferon alfa- a ( kd) (pegasys @) (peg-ifn) ( pg qw) plus ribavirin (copegus @) (rbv) ( - mg qd) for weeks. the primary aim of the study was to increase svr, while optimizing the available drugs, treatment duration, quality of life and the socio-economical burden of therapy. the secondary aim of the study was to enable a comprehensive analysis of virallhostlimmune correlates of response to treatment (ongoing). methods: all patients (n= ) were initially treated with peg-ifnlrbv for weeks and initial viral kinetics were defined according to centralized serum hcv rna quantifications (cobas amplicor@ hcv monitor v , roche molecular systems) on baseline and days , , , , , , and . after classification into viral response categories at weeks, patients were randomized (n= ) to individualized therapy (arms al, a , b , b , c, d) or continuation of standard therapy (std arm). treatment tailoring included: in rapid viral responders (rvr)discontinuation of rbv (al) or shortening of treatment duration to weeks (a ); in slow partial responders (spr)addition of histamine (bl) or prolongation of treatment to weeks (b ); in flat partial responders (fpr)addition of histamine (c); in null responders (nur)retreatment with high-dose of peg-ifn ( pg qw) plus rbv (d). svr was defined as undetectable serum hcv rna (< iulml) at the end of weeks of untreated follow-up. results: demographic and baseline virologic parameters were similar in the standard and the individualized treatment groups. according to the initial viral decline patients were categorized as rvr ( % for genotype and % for genotype - ), spr ( % and % accordingly), fpr ( % and % accordingly), nur ( % and % accordingly) or unclassified ( %). the overall svr rates for genotype patients were % for the individualized and % for the standard treatment arm (ns), and for genotype - patients % and % respectively (ns). svr rates (by itt analysis) according to hcv genotype and within each initial viral response category and arm are given in the table. conclusion: the overall sustained virologic response rates of % in genotype and % in genotype - patients with chronic hepatitis c treated with peginterferon alfa- a ( kd) in combination with ribavirin support previously presented data. individualized treatment according to initial viral kinetics appears to be clinically feasible, but did not improve the sustained virologic response rate with the drugs and dosages usable at the time of this study. nevertheless, the possibility that in rapid viral responders discontinuation of ribavirin does not decrease the sustained virologic response rate warrants future prospective trials. supported by the european community (qlk - - ), hoffmann la-roche and maxim pharmaceuticals. % % (hepatology ; (suppl) : a). the rectally administered ifn is transferred not into blood but into regional lymphatic system and reaches to the thoracic duct via intraperitoneal lymphatics (pharmaceut. res. ; , ) . therefore, the ifn suppository appears to have different antiviral mechanism from the conventional ifn injection. in this study, we performed a combination of the ifn suppository and ifn injection. the antiviral effect and immune-related markers were examined comparing with the controls (ifn injection therapy). (patients and methods) fourteen patients with chronic hepatitis c were given an ifn suppository and ifn alpha ( - m units) injection daily for weeks. after that, only ifn injection was continued three times a week for weeks. the control group contains patients with chronic hepatitis c, who received ifn alpha ( - m units) injection daily for weeks and were followed by the ifn injection three times a week for weeks. the viral loads were . - kiulml (median kiulml) in the ifn suppository group and . - kiulml (median kiulml) in the ifn injection group. the genotype population (lb/ a,zb) was / and l , respectively. there is no significant difference in the clinical background in the two groups. serum hcv rna was tested every weeks. peripheral blood nk cell activity and cd lcd ratio were investigated before and weeks after the beginning of the therapy. the ifn suppository contains low dose ifn alpha (ball- ). (results) serum hcv rna turned negative in . % of the ifn suppository group and in . % of the ifn injection group (n.s.) after weeks (end point of ifn suppository administration). the hcv rna seronegative rates of the two groups were . % and . % (n.s.) after weeks, respectively. however, while the nk cell activity was decreased in % of the ifn injection-treated patients after weeks, only % of the ifn suppository patients had decreased nk cell activity (p= . , chi-square test). furthermore, whereas serum alt levels were significantly decreased after weeks in the ifn injection group ( . + . iull vs. . + . iull, p= . , paired t test), no change was seen in the ifn suppository group ( . iu/ + . vs. . + . , n.s.). (con-clusion) the conventional ifn therapy decreased nk cell activity and serum alt levels. however, the ifn suppository combination therapy maintained augmented nk cell activity and elevated alt levels. these findings suggest that the ifn suppository continuously activates the hosts' immunity during ifn treatment. we used the ifn suppository only for weeks; however, longer administration may lead to more frequent elimination of hcv the aim of the study was to evaluate the efficacy and tolerability of induction dose pegylated-interferon and ribavirin vs pegylatedinterferon and ribavirin as treatment strategies in relapsers to standard interferon + ribavirin in chronic hepatitis c patients. methods: patients, virological relapsers after a first treatment with standard interferon and ribavirin for at least months, were randomized to receive either pegylayed-interferon alpha- b fglkg qw plus ribavirin - mg qd during weeks then peg-interferon alpha- b pglkg qw plus ribavirin - mg qd during weeks (n= pts) or peg-interferon alpha- b . pglkg qw plus ribavirin - mg qd during weeks (n= pts). efficacy assessments consisted of serum hcv rna level by pcr and serum alt at the end of treatment and after weeks of follow up (week ) and liver fibrosis with metavir score before treatment and at week . safety evaluations included adverse events and laboratory tests. results: patients were not different for baseline characteristics in induction and non induction groups including sex (male % and % ), mean age ( . liver fibrosis metavir score decreased in % of patients in non-induced group and in % of patients in induced group (p= , ) whatever the response ( % in sustained responders and % in non responders or relapsers). conclusion ) combination therapy with pegylated-interferon is efficient in half of relapsers to standard combination therapy. ) the response is similar to the response observed in naive patients. ) weeks induction dose does not increase the virological response. ) we observed a regression of liver fibrosis in % of patients whatever treatment regimen and virological response. backgroundanemia has been shown to be an important factor in impairing hrql in cancer patients receiving chemotherapy, with changes in hb directly correlated with changes in hrql (gabrilove et al., j clin oncol ). since - % of patients with hcv develop anemia as a side effect of combination ifnlrbv therapy (rebetron@ package insert), it is important to assess the relationship between hb and hrql in this population. the objectives of this analysis were to ( ) descriptively correlate changes in hb to changes in hrql in an anemic hcv-infected population; and ( ) analyze the independent relationship of hb to hrql. methods: hrql scores and hb values were obtained from clinical trial data of anemic hcv-infected patients (hb . t- . gldl) who had been receiving ifnlrbv therapy for - weeks (afdhal et al., ddw ) . during the -week double-blind phase, patients were randomized to receive either epoetin alfa , u once weekly or placebo. hrql was measured using the short form- health survey (sf- ), an accepted and validated tool that measures domains of hrql (table l) , and the linear analog scale assessment (lasa), which measures constructs of overall quality of life, energy, and activity. patients were categorized into the following groups based on their change in hb levels between randomization and week : ( ) decrease in hb; ( ) hb increase from to < gldl; and ( ) hb increase gldl. changes in hrql (by domain of each instrument) corresponding to the aforementioned hb categories were summarized (table ) . regression analyses were conducted to evaluate the independent impact of hb change on hrql improvement. the factors included in the regression model (in addition to hb change) were: age, gender, baseline hb, baseline hrql domain, fibrosis status, rbv dose change, duration of hcv therapy, and hcv rna. results: changes in hb values were directly related to changes in hrql for all domains of the sf- and the lasa (table ) . hrql changes were seen in an hb-dependent manner; patients whose hb increased g/dl had higher improvements than patients whose hb increased between to < gldl. similarly, patients whose hb decreased from randomization to week had mean decreases in hrql for most of the domains (table ) and minimal increases for others. regression analysis substantiated that the hb change was a significant independent predictor (p=. to r. ) of hrql change in all subscales of the lasa and the sf- , with the exception of bodily pain and general health. conclusions: similar to cancer patients receiving chemotherapy, improvement in hb is a strong independent predictor of improvement in the hrql of hcv-infected patients. patients on combination ifnlrbv therapy should be monitored and considered for anemia treatment to improve hrql and potentially enhance their adherence to hcv therapy. virological response. the current trial was designed to see if better results could be achieved by retreating with higher doses of peginterferon alfa- b (peg b) + weight-based ribavirin. aim to compare the efficacy, safety and tolerability of three different doses of peg b + weight-based ribavirin among interferonlribavirin non-responders. methods: patients were randomized to yr of treatment with peg b . , . or . mcglkglwk, plus ribavirin - mglkglday. treatment assignment was stratified for sex, race, hcv genotype and histologic fibrosis. treatment was stopped at wk if pcr(+). doses were reduced by % for toxicity; growth factors were not allowed. results: patients: enrollment took place between february and november , with patients recruited from centers; data forms have been received on thus far. enrollment was stopped in the low-dose group after fda approval of higher doses of peg b. the study population is % female, % african-american, % genotype l, and % f / / . efficacy: on-treatment virological response rates were dose-related at wk but less so at wk (see table) . this was partly due to a higher rate of discontinuation after a satisfactory response at wk on the higher dose. on-treatment response rates were lower among african-americans and patients with more advanced fibrosis. sustained response data will be available for most patients by october . 'tolerability: the rate of dose reduction was % on peg b . mcglkglwk, vs. % on . . rates of discontinuation were the same for peg b . and . mcglkglwk: % vs. % overall, and % vs. % for adverse events. the frequencies of subjective adverse events were similar between the two groups. some degree of iieutropenia was observed in % on . mcglkglwk, vs. % on . ; however, only patients in the study discontinued treatment for neutropenia overall. conclusions: ) thirty to percent of interferon/ of high-dose peginterferon alfa-pb + ribavirin m s l d abstracts a ribavirin non-responders achieved initial clearance of viremia on peginterferon alfa- b + ribavirin. ) doubling the peg b dose to . mcglkglwk resulted in a higher viral clearance rate at wk but a similar rate at wk. ) the higher dose of peg b . mcglkglwk was well tolerated, with a higher rate of dose reduction but an identical rate of discontinuation. beliefs about therapy and psychosocial factors may influence the course of treatment and therefore could be important for developing comprehensive medical care plans that improve treatment adherence and optimize response to therapy. objectives: ( ) to describe baseline indices of social support, depression, and self-efficacy (i.e., perceived confidence in the ability to perform health behaviors necessary for successful treatment) of patients with hcv genotype participating in a treatment trial of combination peginterferon and ribavirin therapy. ( ) to assess the degree to which baseline psychosocial measures are interrelated. methods: the sample consisted of the first patients enrolled in the virahep-c study, a multicenter, collaborative clinical trial, designed to examine reasons for non-response to hcv therapy in african american and caucasian patients. using a touch-screen computer, patients completed the -item medical outcomes study social support survey (mos-sss); the -item centers for epidemiological studies depression scale (cesd); questions about self-efficacy, and the sf- quality of life instrument. questionnaires were completed during an -week period prior to initiation of therapy. mean scores were calculated for each of the subscales of the mos-sss (l=supported none of the time, =all the time): a) tangible support (material aid, behavioral assistance); b) affectionate (expressions of lovelaffection); c) emotional-informational (expressions of understandinglencouragement); and d) social interaction (others to have an enjoyable time with). similarly, mean scores were calculated for subscales of the selfefficacy instrument (o=no confidence, = high): a) obtaining help; b) communicating with physicians; and managing c) symptoms, d) depression, e) medications. correlation analysis was used to compare each subscale with the cesd and sf- . results: of the participants in the analysis, % were african american and % were female. on average, perceived social support and self-efficacy beliefs were high at the initiation of treatment. mean scores for the tangible, affectionate, emotional, and social interaction subscales were . (sd= . ), . (sd= . ), . (sd= . ) and . (sd= . ), respectively. for self-efficacy, the mean scores were . (sd= . ) for obtaining help; . (sd= . ) for communicating with physicians; . (sd= . ) for managing symptoms; . (sd= . ) for managing depression; and . (sd= . ) for managing medications. the distribution of social support and self-efficacy scores were similar for both racial groups and genders. depressive symptoms were common in the sample at baseline with % having a score of at least on the cesd and % having a score of at least . in general, the social support and self-efficacy subscales were unrelated to the cesd and to the subscales of the sf- . conclusion: african american and caucasian study participants rate their social support and self-efficacy beliefs high in the weeks prior to initiating combination antiviral therapy for hcv, although depressive symptoms are common. social support and self-efficacy instruments may measure different psychosocial constructs as compared with other instruments used in hcv research like the sf- and cesd. as a result, social support and selfefficacy may represent new and important predictors of adherence and sustained virologic response (svr) in hcv treatment. baseline data from virahep-c will be used to evaluate factors associated with adherence and svr to determine if racial differences exist among these measures. if so, this information can be used to develop new initiatives for educating patients and families prior to initiating hcv therapy. background and aims: interferon( fn) a- blribavirin therapy is an effective anti-viral therapy for the patients with hepatitis c virus (hcv) genotype . however, the response to this therapy is not satisfactory because only about - % of the patients become sustained responder(sr)s. further, the patients receiving ifn a- blribavirin therapy have been frequently withdrawn because of severe adverse effects. the efficacy of ifn therapy is mediated by both genomic type and viral load and to immunological response of the hosts. reportedly, some amino acids were identified to modulate host's immunological response. in order to achieve more higher sr ratio after ifn a - blribavirin therapy and to decrease the number of withdrawn patients, it seems important to improve the nutritional condition and to upregurate potential immunological response by modifying the balance of amino acid. in the present study, we investigated the dynamics of a panel of amino acids during ifna- blribavirin therapy, then analyzed the effects of nutritional condition on the clearance of hcv. materials and methods: seventy patients with chronic hcv infection were included after informed consent. six-million unit of ifna- b in combination with ribavirin ( - mglday) were given during months. during the first weeks of the therapy, ifn a - b was given daily. blood samples were obtained after overnight fasting at zweeks, weeks, and weeks of the therapy and served for the analysis of amino acids. at the same time, peripheral blood mononuclear cells (pbmc) were collected and the number of ifny-and il -producing cells were measured by facs analysis. thllth ratio was calculated as following; thll results: hcv rna was undetectable in %, %, and % of the patients at , , and week of the therapy, respectively. thllth ratio decreased gradually during the therapy. thllth ratio at the end of therapy ( week of the therapy) was significantly smaller than that at the beginning of the therapy ( . . vs . . , p=o.o ). however, there was no significant correlation of thll th level with the rate of hcv eradication. total amino acids (taas) and branched chain amino acids (bcaas) decreased gradually during the therapy. even at week of the therapy, there was a significant decrease in taa and bcaa (p= . and p<o.oool, respectively). fischer ratio (fr) also showed drastic change. it changed from . (at the beginning of the therapy) to . (at week of the therapy), indicating that the balance of amino acid rapidly altered to the similar condition to the patients with liver cirrhosis. of interest, the levels of taa and bcaa at the beginning of the therapy were significantly higher in hcv rna-negative group than in hcv rna-positive group at the end of the therapy (taa: +. vs nmol/ml; p= . , bcaa: vs nmollml; p= . ). although we could not find a significant difference, fr at the beginning of the therapy was larger in hcv rna-negative group than in hcv rna-positive group at the end of the therapy ( . . vs . . , p= . ). analysis of each amino acid showed that there were significant differences in the concentration of citrulline and ornithine, both amino acid are involved in urea cycle, between hcv rna-negative and hcv rna-positive group at the end of the therapy. conclusions: interferon a-zblribavirin therapy induces malnutrition at early stage of the therapy. successful elimination of hcv rna seemed to depend much on nutrition rather than the ratio of thl/th during the therapy, suggesting that better response in interferon a-zblribavirin therapy might be possible by improving the condition of nutrition at the beginning of the therapy. in addition, adequate nutrition support would contribute to the improvement of the prevalence of hcv infection in egypt varies between % and % and approximately % are infected with hcv genotype .to date limited data exist on the effect of antiviral therapy on genotype -infected subjects. the purpose of this study was to assess the effect of interferon (standard or pegylated) in combination with ribavirin in na'ive-to-treatment subjects with chronic hepatitis c who reside in egypt. methods: in a prospective, open-label, randomized clinical trial, egyptian subjects with chronic hepatitis c documented by liver biopsy and detectable hcv rna in serum received interferon alfa b (ifn-a b) mu tiw or pegylated ifn-a b ( mcglweek) in combination with weight-based oral ribavirin ( or mg for both groups). the study design was that if hcv rna was detectable at week , the treatment was stopped but if hcv rna was undetectable at week , treatment was extended for a total of weeks. subjects were observed for an additional weeks and hcv rna status at week determined the response to antiviral therapy. safety laboratory tests were done at regular intervals. the study received irb approval. the hcv rna was done by a pcr technique with a detectability cutoff of -copieslmb results: both randomized groups were similar at baseline without statistical significant difference, ( %) were infected with genotype , mean age was . years ? . years, were men ( %), mean bmi was . kglm , mean inflammatory ha score was / and fibrosis stage . t . . of the genotype- infected individuals, have finished therapy (week ) and had completed the week follow-up (week ). the study will be completed by october . the table shows the intention to treat rates of undetectable hcv rna in genotype- infected subjects. serious adverse events that led to discontinuation of medications were observed in subjects in the standard ifni'rbv group and in llsubjects in the peg ifnirbv, one death was observed in the latter group. expected adverse events were observed in similar rates in both groups. laboratory adverse events were observed between "h and % of subjects receiving either treatment regimen depending on the week of therapy. no growth factors were used in this population. in summary: subjects with chronic hepatitis c and genotype- infection have a similar antiviral response to standard or pegylated interferon in combination with ribavirin. the safety and tolerability of the medications is similar to what has been observed for other hcv genotypes. patients infected with hcv genotype respond effectively to therapy and should be encouraged to undergo antiviral treatment. daily for weeks, followed by times a week for weeks (week ) and followed up for weeks after cessation of therapy (week ). the expression of socs- protein in the liver specimen obtained before the treatment was investigated by immunohistochemistry for socs- . results: patients with the expression of socs- protein in the liver were defined as "socs- positive group". all other patients without staining were classified as "socs- negative group". sixteen patients were socs- negative and patients were socs- positive. we compared baseline characteristics among two groups. socs- positive group had a significant lower rate of svr to ifn therapy than socs- negative group (p= . ). socs- positive group had significantly higher hcv-rna titer than socs- negative group (p= . ). we analyzed factors with ifn svr in univariate logistic regression analysis. not only hcv genotype (p=o.oool) and hcv-rna titer (p< . ), but also the expression of socs- were found to be significant predictors of ifn svr (p = . background: interferon( fn) and ribavirin combination therapy is now a standard regimen for chronic hepatitis c infection and achieves a higher sustained virological response than interferon monotherapy worldwide. but the response rate to patient with genotype l b and high viral load is still not satisfactory. some virological factors have been shown to influence the effect of interferon monotherapy in previous studies. however little is known about those influencing the outcome of combination therapy. to examine the genetic basis of the genotype l b virus resistant to combination therapy, we analyzed full-length nucleotide and deduced amino acid sequences of hcv rna. method: among patients infected with genotype l b and high viral load, patients treated with monotherapy : (group a- nullresponders, relapsers) and patients treated with combination therapy: (group b- nullresponders, relapsers) were studied. the group a consists of naive patients and group b consists of naive patients and patients who failed to previous ifn monotherapy. the group a patients were treated with - miu of ifn, there times a week for months. the group b patients were treated with miu- omiu of ifn three times a week and ribavirin mg- mg per day for months.sera were obtained from the patients at three points: months before the treatment, just before the treatment, and after or during the treatment. complementary dna was reverse-transcribed from total rna extracted from these sera, amplified by polymerase chain reaction and directly sequenced. the mutation rates during natural course of infection and during the treatment were calculated by comparing the nucleotide sequences obtained from the samples just before the treatment and months before the treatment, and just before the treatment and after or during the treatment. the deduced amino acid sequences in different region were also analyzed in each case. result: the mutation rates of natural evolution analyzed in patients were . ~ -~nt isitelyear. the mutation rates of hcv rna from nullresponders during the treatment were almost identical with those before the treatment in both groups a and bcu ( . . vs . . , p=ns and . - . vs . . , p=ns, respectively).in contrast, those from relapsers during the treatment were higher than those before the treatment in both groups ( table. no major differences were observed in the frequency or intensity of saes and aes. conclusion: the combination of peginterferon alfa- a ( kd) (pe-gasysb) and ribavirin (copegusb) was safe and effective in patients with hcv genotype . despite the use of a dose of ribavirin ( mglday) that is lower than the recommended dose for patients infected with g ( mglday), approximately half of the patients in the study achieved an svr. because the study could not be blinded, a bias might have influenced the outcome of the study as suggested by the unequal discontinuation rate. these preliminary data do not support the hypothesis that higher svr rates can be achieved in patients with hcv g l by extending treatment duration. final data will be presented. *itt population, defined as all patients who took at least one dose of study medication and had at least one hcv rna evaluation postbaseline. **safety population, defined as all patients who took at least one dose of the study medication elusive goal, particularly in previously treated patients who failed or relapsed following prior therapy. we hypothesize that response rates in these patients who are re-treated with pegylated interferon and ribavirin will be improved compared to standard interferon preparations with or without ribavirin. the present prospective study is designed to determine the efficacy and safety of treatment with peg-intron (pegylated interferon alfa- b) and ribavirin in a group of patients who failed prior therapy with interferon with or without ribavirin. methods: aatients are uresentlv enrolled in this multi-center study. the first enrolled patie& were treated with . mg/kg o~p e g -intron sc q week, and ribavirin mg po qd, which was standard at the time. the intended duration of treatment is weeks. patients have been trcated for at least weeks and week data is available in patients. adverse events: dose reduction was required in % of pts most commonly due to leukopenia and anemia. permanent discontinuation of therapy was required in % most often due to: depression or anxiety serious adverse events: ( . % of pts) included one patient each with neutropenialmrsa sepsis, optic neuritis with monocular blindness, perirectal abscess, cellulitis and multi-organ failure, cutaneous and pulmonary sarcoidosis, pneumonia, homicidal ideation, and suicide. conclusions: ) overall etr was % and svr was %. ) svr was significant in genotype non- patients and patients who failed or ;elapsed after previous monotherapy ) % a multi-centre, randomised controlled study comparing the combination of interferon and ribavirin with no treatment was undertaken. patients were eligible if they had biopsy proven mild hcv (defined as necro inflammatory scores less than and fibrosis scores less than using the ishak scoring system. interferon was administered at a dose of mu thrice weekly for weeks regardless of viral genotype. ribavirin was administered according to the patient's body weight ( kg mg/day, > kg mgl day in two divided doses). randomisation was stratified according to viral genotype ( and non- ). the primary end point was the virological outcome of treatment with sustained response defined as an undetectable hcv rna pcr months post cessation of therapy. in addition there were a number of secondary aims: . viral kinetics. serum was collected at baseline, days , , and week for quantitative rt pcr to determine hcv viral load. the ability of early phase kinetics and the presence or absence of a log drop at week to predict eventual outcome is assessed in the context of mild disease. . health economics. the collection of resource use data in hcv infected patients who have mild, moderate and cirrhotic disease was performed as an integral part of this study. this data has been incorporated into a markov chain based model to assess likely future costs of the disease and their possible avoidance by treating mild cases. response rates from this trial will form the basis of the model. . quality of life assessments. sf and euroquol data were collected at baseline, weeks and during treatment and at post treatment weeks and in order to compare quality of life between treated patients and controls before during and after therapy. results. patients received treatment and were matched with controls (no treatment). sustained viral response rates will be available at the end of july when the follow up period of the trial is complete and will be included in the presented results. end of treatment results are shown in table . % of patients infected with genotype and % of patients infected with genotype non- achieved end of treatment responses. sustained viral response rates to date are shown in table . % of patients infected with genotype and % of patients infected with genotype non- have achieved sustained viral responses so far. % of patients in the treatment group were unable to complete at least months of therapy due to side effects of medications. there were hospitalisations in the treatment group, of which were related to adverse events. patients with mild hcv have lower response rates to those with more severe histological change on liver biopsy. side effects are common and frequently result in treatment discontinuation, lowering response rates. the decision to treat patients with mild histological change must be weighed against the side effects. deferring treatment until later in the disease process does not prejudice response to therapy and may increase likelihood of success. the hcv ns b polymerase is essential for hcv viral replication and infectivity as demonstrated in a chimpanzee model. the enzyme adopts a unique molecular structure that resembles a "thumb-palm-finger" which has some features different from other known dna and rna polymerases, highlighting the attractiveness for this enzyme as a target of antiviral therapy. from an in-house high throughput screening campaign and the subsequent lead optimisation, we have identified a novel chemical series of substituted thiophene- -carboxylic acids that have good potency against hcv polymerase in vitro. x-ray crystallography studies revealed that these compounds bind to an allosteric site that is - angstrom away from the active site. further studies on selected members of the series confirmed the ability of these compounds to inhibit the sub-genomic replication of hcv in huh- cells. moreover, a representative compound was also found to have good in vitro safety index and favourable in vitro and in vivo metabolic and pharmacokinetic properties. in an open and prospective trial we investigated, if a pre-treatment with citalopram as an ssri can reduce the frequency of ifn-associated depression in hepatitis-c-infected psychiatric risk patients during methadone substitution. patients with a chronic hepatitis c were treated with pegylated interferonalpha b and ribavirin according to body weight. patients without any psychiatric history (group a) were compared to patients during methadone substitution. the methadone substituted patients were separated into two groups: the first group (group b, n = l l ) were only treated with ifn-alpha and ribavirin and the second group (group c, n= ) received a two week pre-treatment with citalopram ( mglday) before combination treatment with ifn-alpa and ribavirin was started. antidepressant treatment was continued over the study period of four months. the hamilton depression rating scale (hamd, -item) was used and major depression defined as > points. patients were followed over the first treatment months. group differences were calculated with anova for parametric and chi -test for non-parametric scales. results: hamd scores at baseline were significantly higher in the psychiatric groups as compared to the controls (p=o.olo dallas, t x background: peg plus rbv is the mainstay of chronic hepatitis c treatment. however, an upper limit in terms of safety and efficacy for dosing with peg has not been evaluated. methods: we studied a group of nake patients using either conventional weight-based dosing of peg; ( . pglkg) or double the dose ( . pglkg) plus rbv + mg/kg/day for weeks. genotype patients were randomized :l and to receive medication accordingly in unblinded fashion, with intent to enroll a total of , nayve patients n, an additional previous nr or r to any type of interferon i rbv in a non randomized arm, using only the . pglkg peg dose. values for hcv rna at week were compared to those obtained at baseline. results: to date, patients n, nr, r have enrolled in the study. patients n, nr, r have reached week . hcv rna results at week are shown in the table below. treatment was well tolerated in most patients. dose reductions were required in % of n . pglkg peg and % of n . pglkg peg patients. side effects were equivalent between the two groups as shown in the table below. serious adverse events (sae's) were less than % and equally observed in both arms of the n patients. none were directly attributed to study drug. there were patients in the wk analysis that discontinued therapy. the reasons these patients were discontinued after reaching wk were side effects ( %), +hcv rna ( %). it is interesting to note that % of the patients that were discontinued had log drop or negative hcv-rna at week and a positive hcv rna was not a reason for their discontinuation. conclusion: .op.glkg peg dosing does not appear to improve wk viral response, but we await wk results and sustained viral response rates. increased side effects as a result of doubling the interferon dose were not observed. more data is needed to verify the long-term efficacy and safety of this dosing regimen for patients with chronic hepatitis c . this study was supported by a grant from integrated therapeutics group a subsidiary of schering plough. background and aim: one of the major adverse effects of the combination therapy for chronic hepatitis c is ribavirin-induced hemolyhc anemia. however, little is known about the mechanism of this anemia. oxidative stress has been suggested as potentially important pathological mechanism in hepatitis c. this burden may cause peroxidation of erythrocyte membrane phospholipid in conspiracy with the accumulation of ribavirin triphosphate in the erythrocyte, which potentially attenuates the mobility of erythrocyte membrane. the aim of this study was to examine the change of fatty acid composition in erythrocyte membrane and the effects of vitamin e and vitamin c on fatty acid composition in combination therapy. methods: thirty-nine patients with chronic hepatitis c were enrolled in this prospective study. they were randomized to receive daily oral vitamin ( mg of vitamin e plus mg of vitamin c) (vitamin group) or none (control group), in addition to injections of million units of interferon-a- b daily for two weeks, followed by thrice-weekly for additional weeks, plus daily oral ribavirin ( or mg) for weeks. blood samples were obtained at , , , , and weeks after initiation of therapy. phospholipid was separated by one-dimensional thin-layer chromatography after extraction of total lipid from the erythrocyte ghosts. following transmethylation, fatty acid methyl esters were quantified by gas chromatograph. a-tocophenol concentrations in erythrocyte were determined by high performance liquid chromatography. plasma thiobarbituric acid reactive substances (tbars) were measured by spectrofluorometer. results seven patients were obliged to suspend or cease receiving therapy because of adverse effects including anemia by weeks after initiation of therapy. twenty-one patients have completed the assigned therapy up to now. among the kinds of fatty acid analyzed, the mean content of n- polyunsaturated fatty acid (pufa) significantly decreased at ( . . mol% vs. in a cross-sectional study we investigated mxa expression in patients with chronic hcv infection(n = ) and in patients with chronic hcv infection receiving ifn-alpha therapy (n = ) as well as in healthy controls (n = ). in a prospective study with chronically infected patients (hcv genotype ) known to be ifn non-responders, we followed mxa gene expression during combination therapy with pegylated ifn-alpha b and ribavirin. results: patients with chronic hcv infection showed higher mxa gene expression levels than healthy controls, indicating that hepatitis c virus induces ifn production. however there was no correlation between mxa gene expression and clinical parameters (viral load, alt, liver histology). patients with chronic hcv infection receiving ifn-alpha had significantly higher mxa levels than patients without treatment or healthy controls. further we addressed the question whether mxa expression in pbmcs during therapy in previous non-responders could be a predictive marker of therapy outcome. mxa expression was clearly induced in all but one patient compared to pretreatment values (median: . fold). importantly, the level and kinetics of mxa expression did not correlate with the response to antiviral therapy. surprisingly, one patient showed an unusually low pre-treatment level of mxa expression and a total lack of mxa induction during ifn therapy, which was associated with complete non-response to therapy. in a neutralization assay, we could detect neutralizing antibodies to ifn-alpha b in the serum of this patient whereas no neutralizing antibodies were found in all others patients. hepatitis c virus (hcv) infection is a common cause of transfusion acquired hepatitis and is today a major health problem throughout the world. the present study reports the prevalence of hepatitis c virus (hcv) infection in general population and in various high-risk groups from the city of hyderabad in south india. a total of out of ( . %) people (both general and risk groups) tested positive for hcv rna by rt-pcr, while anti-hcv antibody positivity, as determined by third generation eia, was found to be . % ( / ). this suggests that a number of cases go unreported, as screening of blood and blood products is done primarily by elisa. among chronic renal failure (crf) patients with history of either renal transplant or hemodialysis, % were infected with hcv alone, . % hbv alone while . % were found to be co-infected with bothe hcv and hbv. our findings implicate these viruses as the major cause of post transplant hepatitis in indian patients with crf and indicate the necessity for immediate implementation of stringent screening procedures for these viral infections. additionally we report here that tattooing and slashing (a cultural practice among one sect of muslims) increase the mode of transmission of hcv infection. in addition, we also report a high incidence of hcv infection ( background in japan, long-term treatment with glycyrrhizin, given as snmc, is used to reduce alt and the risk of hepatocel-glycyrrhizin given as stronger neo-lular carcinoma in patients with chronic hepatitis c. phase / studies confirmed the alt lowering effect of glycyrrhizin therapy in western patients with chronic hepatitis c. the treatment duration in these studies was limited to weeks. alt response was lost during follow-up. aim ) to evaluate which dose frequency is required beyond week to maintain the initial alt response. ) to evaluate the effect of snmc treatment on liver histology. methods: hcv-rna positive patients with alt > x uln, fibrosis stage or necro-inflammation score (ishak's score) who were not eligible for interferon therapy (prior non response, absolute contraindications) could enter this multi-center, randomized, open phase i clinical trial. all patients were treated weeks with six infusions weekly of ml snmc (minophagen pharmaceutical co. ltd., japan) containing mg glycyrrhizin. patients with an alt response at week (defined by a decrease of more than percent of the baseline value or alt . uln) were randomized to continue treatment in three dose frequency groups for a total of weeks. snmc was given times weekly (group l), objectives: it is important to maintain reduced serum alanine aminotransferase (alt) levels in cases with chronic hepatitis c (ch-c) that do not respond to interferon (ifn) and in those with no indication of ifn therapy. we reported previously that dietary restriction of iron intake reduces serum alt levels in such patients. we evaluated ch-c patients treated with iron-restricted diet for two or more consecutive years, mainly focusing on the balance of energy intake, physical examination, and changes in hematological indices of nutrition. methods: twenty-two patients with ch-c (males, ; females, ; mean age, year-old) that consulted our outpatient department were enrolled in this study. the inclusion criteria were as follows: ) elevation of alt levels above the upper normal limit for months or more; ) positive tests for hcv-antibody and hcv-rna; ) absence of other causes of ch (alcoholic liver disease, drug-induced liver injury, hemochromatosis) and negativity for hepatitis b surface antigen and for serum anti-nuclear and anti-mitochondria autoantibodies. twenty cases had received ifn therapy for more than months before the beginning of the study; none of them responded to ifn therapy. dietary prescriptions included iron intake mglday or less, energy intake kcallkglday, protein intake . - . glkglday, and a fat energy fraction of %. nutritional balance was evaluated based on meal records, and instructions was given when necessary. results: the average energy intake before dietary prescription was kcal( . kcallkg)lday, and it was significantly reduced to kcal( . kca lkg)lday (p < om), and then maintained stable at kcallkglday. the average protein intake before dietary prescription was . g ( . glkg)lday and it was reduced to . - . glkglday after the prescription. the average fat intake of . g ( . glkg)lday and the average fat energy fraction of % before the dietary prescription were significantly decreased to . g ( . glkg)lday; p < . and % (p < . ), respectively, after dietary instructions. the fat energy fraction was maintained at a level of % or less. carbohydrate intake did not change remarkably during the observation period, although the carbohydrate energy fraction significantly (p < . ) increased. the average iron intake decreased significantly (p < . ) from . (before) to . , . , . , . , and . mglday , , , and months after die* prescription, respectively. body mass index (bh i) before diet prescription was . on average; bmi had no significant change throughout the course. the body fat percentage was . % on average before the diet instructions, and it significantly decreased after the diet. the average values of aspartate aminotransferase and alt before diet prescription were iull and lull, respectively, and they were significantly reduced to iull and iull, respectively, after months (p < . ). serum iron levels significantly decreased after (p < . ) and (p < . ) months, while unsaturated iron binding capacity tended to increase. the average serum ferritin levels were , , , , nglml before and , , , and months after diet, respectively; there was a significant reduction (p < . ) in the values measured before and after the diet instructions. the average levels of hemoglobin, albumin and cholinesterase did not change significantly during the follow-up period. conclusions: restriction of iron intake is safe and well tolerated for a long period. the results of our present study suggest that decreased dietary intake of iron may constitute an important adjuvant therapy in patients with ch-c. chru, nice, france; c henquell, chru, clermont ferrand, france; c dizrcha, s ughetto, p dechelotte, hotel-dieu, clemont ferrand, france; h lafeuille, chru, clermont fewand, france; g bommelaer, hotel-dieu, clemont fewand, france peginterferon (pegifn) background: hcv co-infection is common among patients with hiv disease. it has been documented that hiv co-infection accelerates the course of liver disease in patients with hcv, and that liver failure is higher in co-infected patients. at this moment, there is no effective treatment for hcv non-responders to interferon therapy. mono-therapy and interferon-ribavirin combination is only effective in - % and - % interferon resistant patients respectively. treatment strategies to slow the progression to cirrhosis and to prevent liver failure in this population are needed. objective: this is a report of a study that examines the efficacy of peg ifn alfa -a (pegasys) vs pegasyslrbv mg in co-infected hcvlhiv patients that have not responded to a previous - months course ifn-alfa. the study also examines the histological benefit of treatment in this population. patients and methods: patients were randomized :l to receive either pegasys mcg weekly x weeks (group ) or pegasys mcg weekly plus mg rbv for weeks (group ). patients that have hcv non-detectable or g decrease from baseline at week (groupl), were added rbv mg. all similar responders (group ) continue treatment for a total of weeks. baseline demographics were similar between both groups. more than % of patients were nonresponders to ifn monotherapy.most patients in both groups ( %) were genotype l. the mean hiv baseline log was . (sd. ) group , vs . (sd. ) group and most patients had baseline cd levels> cells, and were in stable antiretroviral therapy. the majority of patients ( %) are non cirrhotic, with mean grading group . (sd . ), group (sd . ), and mean staging, group . (sd . ), and group . (sd . conclusions: this study is completed and pending some results, that will be available for presentation. sustained virological response(svr) (intention to treat), will be of the order of - %. ( group results are pending). in this study, patients were discontinued because adverse events and were lost to follow up, before week th.svr in patients that completed at least weeks of therapy will be of the order of - %.a significant number of end of treatment responders are relapsing at week th. histology analysis show improvement in both groups of the mean grading and staging after treatment. in responders and relapsers the fpr becomes static or regressive.these results show that pegasys lrbv therapy is effective in this population.the study also suggests that longer duration of therapy ,and higher doses of rbv should be studied in coinfected nonresponders. * -pending results, will be available for presentation. disclosures: josi. rodriguez-orengo -no relationships to disclose maribel rodriguez-torres -roche laboratories: investigator; other: grant to perform study. adverse events by week of therapy were grade to in severity and were therapy related. see table . sixteen patients who continued therapy to week , three dropped out due to adverse events ( anemia, hyperbilirubinemia and related death due to self-overdose). this patient had a history of hypothyroidism and mild untreated depression. at weeks of therapy, patient discontinued treatment due to suicidal thoughts. there was no difference between the ethnic groups regarding the drop out rate due to adverse events. hivfhcv coinfected population had a poor tolerance to pegylated interferon and ribaiirin. this was markedly increased in the first weeks of therapy. the use of high dose pegylated interferon and ribavirin led to a significant drop out rate in comparison to the low dose pegylated interferon. background during liver injury, poorly characterized factors activate quiescent hepatic stellate cells (hsc) to become proliferative, matrix-synthesizing myofibroblasts. activated hsc are the major source of liver collagen and thus, play a key role in the fibrotic response to liver injury.the sns appears to promote fibrosis in injured livers because hepatic fibrosis is increased in the spontaneously hypertensive rat, which has an overactive sns. conversely, prazosin, an adrenoceptor antagonist, inhibits fibrosis in toxin-damaged rat livers. hsc express several neuronal proteins, including glial fibrillary acidic protein (gfap). they also contain synaptic vesicles and receive autonomic fibers. therefore, our hypothesis is that hsc are hepatic neuroglial cells that produce and respond to neurotransmitters in order to become activated during liver injury. methods: hsc were isolated from normal mice and dbh-lmice that cannot produce norepinephrine (ne) due to targeted disruption of the dopamine p-hydroxylase (dbh) gene. lysates of culture-activated hsc were analysed by rt-pcr, immunoblot and hplc to determine if they express adrenoceptors, catecholamine biosynthetic enzymes andlor produce ne. the effect of adrenoceptor antagonists and ne on hsc growth in vitro was also assessed. then in vivo activation of hsc by hepatotoxic diets was evaluated in control and dbh-lmice by comparing numbers of a-smooth muscle actin (asma)+ cells with immunohistochemistry and the induction of tgfp- and collagen a- gene expression by ribonuclease protection analysis of whole liver rna. results: hsc express a-and p-adrenoceptors, tyrosine hydroxylase and dbh. hsc from control, but not dbh-i-, mice release ne. endogenous ne is an autocrine growth factor for hsc because a-and p-adrenoceptor antagonists inhibit proliferation of hsc cultured from control mice. moreover, hsc from dbh-lmice, which cannot make ne, grow poorly in culture and are rescued by addition of ne. exogenous ne also promotes hsc proliferation. inhibitor studies demonstrate that the latter effect is mediated via g-protein coupled adrenoceptors that activate mitogen activated kinases and phosphatidylinositol kinase pathways. hsc activation in response to diet-induced liver injury is inhibited in dbh-imice, as evidenced by reduced hepatic accumulation of asma (+) hsc and inhibited hepatic induction of background & aims: hepatic cirrhosis is six times more prevalent in obese individuals than in the general population, and obesity is one of the risk factors for liver fibrosis in which plasma adiponectin levels are decreased. adiponectin is an adipocytokine, which we previously identified by screening adipose-specific genes in the human cdna project. hepatic stellate cells (hscs) play central roles in liver fibrosis. when they are activated, they undergo transformation to myofibroblast-like cells, then proliferate, migrate, produce transforming growth factor-pl (tgf-pl), and various extracellular matrix proteins, and express a-smooth muscle actin (a-sma). we previously reported that adiponectin suppresses not only the proliferation and migration of hscs, but also the tgf-pl-induced fibrogenic gene expression in hscs. adiponectin could have biological significances in liver fibrosis. in this study, in order to clarify the effect of adiponectin on liver fibrosis in vivo, we tested the role of adiponectin on liver fibrosis using adiponectin-knockout (ko) mice and adenovirus mediated adiponectin expression system. methods: ( ) to investigate the anti-fibrogenic effects of physiological concentrations of adiponectin, male wild type (wt) mice and ko mice were used. mice were each injected with a dose of carbon-tetrachloride (ccl ) ( pllkglbw) intraperitoneally twice a week for weeks to induce liver fibrosis. ( ) to investigate the anti-fibrogenic effects of excessive concentrations of adiponectin, male wt mice were used. mice were injected cc ( pl/kg/bw) intraperitoneally twice a week for weeks. mice were divided into groups. control, received an injection of corn oil only; gr. , mice treated with cc for weeks; gr. , mice treated with cc for weeks after infusion of adenovirus producing adiponectin (adadn); gr. , mice treated with cc for weeks after infusion of adenovirus producing p-galactosidase (adlacz); gr. , mice treated with cc for weeks with adadn infusion at week; gr. , mice treated with cclb for weeks with adlacz infusion at week. results: ( ) ko mice showed extensive liver fibrosis with an enhanced expression of tgf-p and connective tissue growth factor (ctgf) compared to wild type (wt) mice (p< . ). the hydroxyproline content and the numbers of a-sma positive cells in mice liver significantly increased in ko mice. ( ) the fibrosis areas were significantly decreased in gr. and gr. compared to those in gr. and gr. , respectively. the hydroxyproline content in mice liver significantly decreased in gr. and gr. compared to that in gr. and gr. , respectively. moreover, in gr. , the hydroxyproline content was significantly decreased compared to that in -weeks of c c treatment even though ccl, was given for an additional -weeks (total weeks). conclusions: the findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention. disclosures: (ttg) is observed in mature scars and might promote wound repair by protecting the neomatrix from degradation by matrix metalloproteinases (mmps).however, such ecm crosslinking has the potential to hinder the matrix remodelling required for resolution of liver fibrosis. we have explored this hypothesis by examining expression of ttg and its crosslink product epsilon-(gamma-glutamyl) lysine in livers of: a) rats administered cc for weeks ( wk c c cohort)after which liver fibrosis resolves spontaneously after days; b) rats administered cc for weeks ( wk cc cohort) which develop micronodular cirrhosis; c) wk ccl treated rats allowed to recover for days after last cc dose ( wk+ d cohort) which show partial resolution of fibrosis but with persistence of a macronodular cirrhosis. although ttg was identified by immunohistochemistry within and around fibrotic bands in wk ccld cohort, no crosslinks were detectable. however, both ttg and crosslinks were detected in and around mature fibrotic bands in the wk cc cohort and in persisting fibrotic bands in the wk+ d cohort. western blotting of liver homogenates from these two cohorts using antibody against the crosslink revealed a major product of kd which was degradable to lower molecular weight products following incubation of homogenates with bacterial collagenase but was refractory to mmp- . to evaluate if ecm crosslinking in livers correlated with its resistance to mmps, micron cryostat sections of livers from the three cohorts were incubated with purified active forms of mmp- or mmp- for hr at c and any residual ecm was stained with sirius red. ecm of wk cc cohort was effectively degraded by these mmps but that of wk c c and wk+ d cohorts was only minimally degraded. hsc freshly isolated from normal rat or human liver showed no ttg expression whilst hsc activated for - days on plastic substrate expressed ttg protein (by western blotting) and mrna (by rt-pcr). there was ttg in cell lysates and in conditioned media of activated hsc. pure rat tail type i collagen incubated with conditioned media of activated hsc incorporated crosslink antigen, confirming that hsc secreted functional enzyme. however, no crosslink was found in type i collagen incubated with media from hsc which had ttg gene silenced by cell transfection with small inhibitory rna. cultured hsc which had ttg gene silenced had enhanced apoptosis (by acridine orange staining) following serum deprivation in culture. this suggests ttg or ttg activity promotes survival of these cells. we conclude that in persistent, incompletely resolving liver fibrosis there is evidence of ttg mediated crosslinking of the liver ecm and resistance to exogenous mmps. both features are lacking in ecm of fully resolving fibrosis. ecm crosslinking by ttg might limit resolution of cirrhosis. activated hsc are a potential source of ttg following liver injury and, as this protein supports their survival, it might contribute to their persistence in cirrhotic liver. we recently defined multifunctional biochemical properties of the long and frizzled variants and hypothesized that they may be inherent in the -aa-long-specific and the -aa-fz-specific modules, respectively. we thus transfected pcdnas.l-v -his vectors containing variant-specific sequencer in mhat fls hepatoma cells. this cell line stably expresses a truncated hnf protein turning off endogenous expression of liver-specific genes, such as albumin or c . consistently, mhat fls hepatoma cells transfected with an empty vector showed no endogenous c expression. by immunohistochemistry, using variant-specific and tag-specific antibodies and after analysis by confocal microscopy, long was localized in large supranuclear vacuolae suggesting protein storage/maturation in golgi structures or in beaded perinuclear vacuolae suggesting rer cysternae. in contrast, fz was strongly detected in close proximity to the plasma membrane of single cells. clusters of fz-transfected cells showed a strong and dense signal at sites of cell-cell contact, outlining single cells. these findings were confirmed by transfection of prep- vectors containing full-length long or fz forms of human c and confocal microscopy analysis after immunohistochemistry using an antibody directed against an epitope common to all c forms. immunoblot analysis of conditioned media showed that the fulllength long form was secreted into the medium but not fz. the latter was detected as a heavily glycosylated protein (> kd) in the cell layers, predominantly in the triton-x- insoluble pellet after sonication and % sds solubilization, suggesting binding to extracellular matrix proteins. finally, glycosydase analysis of fz and long n-terminal modules showed an important increase in mobility after pngase f plus sialidase a digestion. these data show that, in addition to the previously described plasma (long) and tissue (short) forms of cl , the fz form constitutes the pericellular matrix form, suggesting that the specific modules of c regulate tissue targetting through protein-protein interactions. backgr und:during liver fibrosis, hepatic stellate cells (hsc) acquire an activated phenotype, proliferate and produce an excess of collagens. mycophenolic acid (mpa) is a known immunosuppressive drug. which inhibits the proliferation of b-and t-lymphocytes by inosine mono phosphate dehydrogenase (impdh) inhibition, causing an intracellular guanosine depletion. in addition, mpa has shown to inhibit growth of mesangium cells in the kidney and of skin-and tenon fibroblasts. therefore, we hypothesize that the proliferation of hsc may also be influenced by mpa. in this study we explored whether mpa is able to inhibit the proliferation of primary isolated rat hsc in vitro. furthermore, we studied the in vitro effects, mechanism of cellular uptake, and in vivo pharmacokinetics of mpa coupled to mannose- -phosphate modified human serum albumin (m p-hsa). m p-hsa is a newly developed, hsc-specific drug carrier, homing towards the upregulated m pligf-i receptor on activated hsc. in this way we hope to achieve cell-specific delivery of mpa to the hsc avoiding undesired effects of mpa on the immune system. meth-odslresults: in primary cultures of hsc a dose dependent reduction in the number of brdu-positive nuclei was observed after h incubation with , and nm mpa, as assessed immunohistochemically. coupling of mpa to m p-hsa via a biodegradable ester linker resulted in a conjugate with a maximum drug: carrier ratio of . :l. analysis of the synthesized constructs was performed by hplc, fplc and ms. this conjugate was able to inhibit t -fibroblast growth, as detemined by brdu-incorporation (elisa) in a dose dependent manner, reducing proliferation to . . %, . t . % and . . % of control at , and uglml of conjugate, respectively. when cells were co-incubated with an excess of m p-hsa, a competitor for receptor mediated uptake of the conjugate, the anti-proliferative effect was reduced by yo. the organ distribution of the conjugate, labeled, was evaluated by iv injection of a tracer dose in rats weeks after bile duct ligation (bdl- ). . t- . % of the injected dose accumulated in the liver after min of injection. in contrast, spleen and thymic gland accumulated . f . % of the injected dose. intra-hepatic distribution was assessed in bdl- rats by immuno-histochemical double staining for hsa and specific markers for kupffer cells (ed- ), hsc (desminelgfap) or endothelial cells (his ). m p-hsa-mpa showed a non-parenchymal distribution and co-localization with hsc and kupffer cells. conclusions: mpa is able to inhibit the proliferation of primary isolated rat hsc. this suggests that stellate cells are dependent on intracellular impdh activity to proliferate. coupling of mpa to mcip-hsa, a stellate cell specific drug carrier, resulted in a pharmacologically active construct, able to inhibit fibroblast proliferation in vitro after specific uptake via the m p/igf-ii receptor. the major part of the injected conjugate accumulates in the hsc and kupffer cells of the liver, and avoiding uptake in the major resident organs for band t-lymphocytes. future studies will assess the advantage of this first hsc-selective compound in animals with liver fibrosis. background: platelet-derived growth factor (pdgf) is the most potent stimulator of migration and proliferation of mesenchymal cells. the expression of pdgf-p-receptor is increased during liver fibrosis. our aim was to investigate the effect of p -integrin subunit blockade using the specific non-peptidic inhibitor emd on migration and proliferation of hepatic stellate cells (hsc) and human foreskin fibroblast (hff). materials and methods: cell migration of human hsc and hff was assessed using a scratch assay. scratches of - pm width were made in confluent cell monolayers of cells following h starvation in serum-free medium. after wounding. cells were stimulated with pdgf-bb ( nglml) with or without emd (merck, darmstadt, germany) at increasing concentrations ( - m - - m). cell proliferation was measured as dna synthesis by brdu-incorporation. mitogen-activated protein kinase (erk , p , sapkljnk and akt) phosphorylation was evaluated by phospho-mapk-specific western blotting of cell lysates after min of stimulation. results: stimulation of human hsc cells with pdgf-bb at nglml in the absence of other growth factors resulted in pronounced stimulation of cell migration. pdgf-stimulated migration of human hsc was inhibited dose-dependently between - and - m by emd , with complete abrogation of migration at - m. surprisingly, human skin fibroblast migration was not affected by b -blockage. there was no effect of p integrin inhibition on cell proliferation as measured by brdu-incorporation, neither in human hsc-nor in hff cells. pre-incubation of hsc cells with the p integrin inhibitor at - m did not affect the activation of p mapk, p l mapk or sapkljnk. conclusions: . pdgf-bbinduced migration is strongly b integrin dependent in human hsc, but not in human skin fibroblasts. . in contrast to cell migration, hsc and hff proliferation is p -integrin-independent. . p , p l , sapkljnk and akt mitogen-activated protein kinase signalling pathways are not modified by p -integrin inhibition. . p -integrin antagonist may be an adjuvant approach to treat hepatic fibrosis. epithelial to mesenchymal transition (emt) is defined as a process, in which epithelial cells loose their epithelial characteristics and acquire typical characteristics of fibroblasts. epithelial cells are tightly attached to their neighboring cells via cell adhesion molecules and they adhere with their basal side to their underlying basement membrane matrices, whereas the apical side faces a lumen. in contrast to immobile epithelium, mesenchymal fibroblasts are specifically designed to invade extracellular matrix (ecm). this is reflected by their prominent mesenchymal cytoskeleton and by their lack of a typical polarity. in the adult organism, emt occurs in epithelia in response to injury, potentially as a means to replenish fibroblasts, which are required for repair of injury. in organ fibrosis however, enhanced conversion of epithelium into fibroblasts is considered to contribute to progression of disease, as parenchymal epithelial cells acquire phenotypic and functional properties of activated fibroblasts. recent studies provided increasing evidence that parenchymal epithelium can potentially contribute to activated fibroblasts by emt, as in mouse models of kidney fibrosis % of activated fibroblasts were found to be of tubular epithelium origin. in liver fibrosis, stellate cells are considered the principal source of activated fibroblasts, whereas the role of hepatocytes is considered minor, even though they constitute for more than % of the liver mass. here we provide evidence that the pro-fibrotic growth factors tgf-beta and egf induce expression of fibroblast-markers fibroblast specific protein- (fspl), alpha-smooth muscle actin (alpha-sma) and type i collagen in primary mouse hepatocytes in vitro. furthermore, tgf-beta and egf induce acquisition of fibroblast characteristics, migratory capacity and release of mmp- , suggesting emt of hepatocytes in vitro. additionally, progression of liver fibrosis in a mouse model of tetracarbon chloride-induced liver disease is associated with appearance of fspl positive hepatocytes, indicating emt, which suggests a role of emt in liver fibrosis. in conclusion, our results for the first time provide evidence for an active role of hepatocytes in liver fibrosis by undergoing emt. human studies -serum mcp- levels were significantly elevated in both cfld ( t pglml; p<o.oool) and ba ( , pglml; p<o.oool) vs controls ( pglml). in celd subjects serum mcp- was negatively correlated with the stage of fibrosis (r= conclusion: these results provide strong evidence for a role for mcp- in early fibrogenic events in cholestatic liver injury, ie., ductular proliferation and procollagen q(i) mrna expression, and suggest that serum mcp- may be a very good marker of the onset of cholestatic liver disease. this is the first study to clearly demonstrate elevated excretion of mcp- into bile in obstructive cholestasis, implying a potential role for mcp- in hsc and inflammatory cell recruitment to the biliary tree in cholestatic liver injury. [background and aim] angiotensin i (ang ) is considered to play an important role in hepatic fibrogenesis. however, the molecular mechanism of renin-angiotensin system in hepatic fibrogenesis has not been clarified. we previously reported that ang i up-regulated mrna expression of pro a (i) collagen and tgf-p in isolated rat hepatic stellate cells and an angiotensin converting enzyme inhibitor, lisinopril, reduced carbontetrachroride-induced rat hepatic fibrosis. ang i type i receptor blocker (arb) have shown a good clinical response in patients with hypertension, and a promising preventive effect has been described in rat hepatic fibrosis. recently, the rholrho-kinase has been demonstrated to be involved in the signal transduction pathway of the ang i type i receptor signaling downstream. thus,the aim of this study was to evaluate the role of rholrho-kinase pathway in hepatic fibrogenesis in rat. to that effect, a rat hepatic fibrosis model was produced by chronic administration with cholinedeficient, l-amino acid-defined (cdaa) diet and the effect of an arb and a rho-kinase inhibitor (y ) in this model was investigated. methods: wistar rats (male, weeks-old) were continuously fed the cdaa diet or the control choline supplemented, l-amino-defined (csaa) diet. the rats were treated with mglkg candesartan cilexetil, an arb (a kind gift by takeda chemical induustries, ltd., osaka, japan), or mglkg y , a rho-kinase inhibitor (a generous gift by mitsubishi welpharma co., tokyo, japan), once daily through stomach tube. twelve weeks after the start of administration, the liver was excised and subjected to histological examination for fatty change and fibrosis by hematoxylin and eosin staining. serum alanine aminotransferase (alt) values and hyaluronic acid (ha) levels were also measured. since cdaa diet causes hepatic steatosis and fibrosis via production of free radicals, we also studied the oxidative membrane damage by immunostaining of -hydroxynoneal (hne) adduct. results: histologic examinations showed severe steatosis and fibrosis in the liver of cdaa-fed rats and no significant change in csaa-fed rats. the liver weight of cdaa-fed rats was significantly increased than that of csaa-fed rats. both candesartan cilexetil and y remarkably reduced not only fibrosis, but also severe steatosis and the liver weight of the treated rats was significantly reduced in both treated groups. serum ha levels but not alt values significantly elevated in cdaa-fed rats compared to csaa diet fed group. the increased levels of serum ha were significantly reduced by the treatment with candesartan cilexetil or y . the number of hne-positive hepatocytes increased by cdaa diet and this increase was attenuated in the candesartan cilexetil-or y -treated groups. conclusion: these results suggests that an arb, candesartan cilexetil, and a rho-kinase inhibitor, y , show the same inhibitory effect on hepatic steatosis and fibrosis induced by chronic cdaa diet, and this free radicalinduced hepatic injury is a good therapeutic target of arbs or rho-kinase inhibitors. the rholrho-kinase may be a key enzyme system in the ang i signaling involved in the hepatic fibrosis. in liver cirrhosis, malnutrion and portal congestive enteropathy facilitate bacterial translocation from the intestine to mesenteric lymph nodes and the development of endotoxemia and spontaneous bacterial peritonitis in ascitic patients. igf-i is an anabolic hormone synthesized in the liver, whose levels are reduced in hepatic cirrhosis. igf-i possesses hepatoprotective properties and displays trophic effects on the intestine. here we investigated whether igf-i therapy could in-fluence portal pressure and bacterial translocation in experimental cirrhosis. methods: liver cirrhosis was induced in sprague-dawley rats (n= ) by oral administration of carbon tetrachloride during weeks. after this time, rats were randomized in two groups to receive subcutaneous injection of recombinant human igf-i ( microgllo g body weight in two separated doses per day) (ci-igf, n= ) or vehicle (ci, n= ), for days. eight healthy rats, receiving vehicle were studied in parallel. at the end of treatment period, animals underwent laparotomy to measure portal pressure and to collect blood and tissue samples. endotoxin quantification in blood samples was carried out with a colorimetric limulus amebocyte lysate test. liver and ileum histopathological changes were assessed in tissue sections stained with masson's trichrome, using a numerical scoring system. bacterial translocation was evaluated by culturing blood samples and mesenteric lymph nodes in appropriated culture media. results: treatment with igf-i significantly improved biochemical markers of liver damage, being the alt values significantly lower in the ci-igf group than in ci rats (p< . ). in addition we found that igf-i administration to cirrhotic rats reduced both the hepatic histopathological score ( the rate of progression to cirrhosis varies among individuals infected with hepatitis c virus (hcv) cholesteryl ester transfer protein (cetp) is a plasma glycoprotein involved in lipid metabolism which transfers cholesterol esters from hdl to vldl and ldl. obesity and steatosis are emerging as key factors in the pathogenesis of liver fibrosis in hcv. variability in function or levels of cetp may affect lipid transport and therefore steatosis. the uncommon vallval genotype results in decreased cetp plasma activity and may protect against myocardial infarction. we tested for a relationship between possession of cetp ile val polymorphisms and rate of fibrosis in white european patients chronically infected with hcv. rate of fibrosis was calculated by dividing the fibrosis score (ishak modification of knodell) by infection duration. genotyping was by reverse line blot hybridisation. a significant association was seen between rate of fibrosis and the ile val polymorphism in cetp (p = . (anova)) (figure ) . disease association showed patients possessing the val allele (which is associated with lower cetp activity) were more likely to be slow fibrosers (no cirrhosis for > years) genotype frequency p= . , allele frequency p= . , odds ratio= . . (table ) . possession of the val allele is associated with slow rate of disease progression in hcv. the functionally less active forms of cetp may result in decreased liver steatosis as a result of altered composition or increased levels of hdl, and therefore less fibrosis when a second insult such as infection with hcv is sustained. backgroudslaims: hepatitis c virus (hcv) is a major cause of chronic liver disease worldwide. however, little is known about exact mechanism of fibrogenesis by specific hcv gene or protein because the study of hcv-induced liver fibrosis has been mainly studied in human or animal model. moreover, the dynamically molecular study of hcv role in relation to liver fibrogenesis or immune response has been hampered due to lack of efficient in vitro hcv culture system. in the present study, we investigated whether hcv core protein directly influence on the liver fibrogenesis through stimulation of hepatic stellate cell(hsc) in in vitro or not. methods human and rat hscs were isolated using collagenase perfusion system and density gradient method, and other human hsc line (l ) was purchased from atcc. we established co-culture system of primary hsc and hepg -core stable cell line which hcv-core gene was transfected into hepg cell. co-culture system was divided into two way, mixed co-culture and separated co-culture system. immunocytochemical staining was performed to identify the cytokines such as transfoming growth factor pl (tgf-p ) and a-smooth muscle actin(a-sma) overexpressed from hsc in the liver fibrogenesis. a-sma, tgfpl, transforming growth factorp receptor i (tgforii), collagen type i were also quantitated by western blot analysis. the expression of mmp- and collagen type i in the culture media was measured and analyzed by each zymogram and elisa. results the expression of tgf-p and a-sma was significantly higher in mixed co-culture of hepg -core plus hsc than in hepg plus hsc as negative control. also the markers related fibrosis such as a-sma, tgf-pl, collagen type i and tgfrii, mmp- and collagen type i were highly expressed in separated co-culture of hepg core and hsc conclusions in conclusion, hcv core protein may play a direct role in the fibrogenesis via upregulation of a-sma, tgf-pl, collagen type i and tgfrii, mmp- and collagen type i. further study is needed to clarify exact signal transduction of fibrogenesis by hcv core protein in the co-culture system. background: to current knowledge, transforming growth factor beta (tgf-beta) signaling is mandatory to produce liver fibrosis. various molecular interventions designed to affect the tgf-beta system were successfully used to inhibit fibrogenesis. activated hepatic stellate cells have been considered as a major producer of extracellular matrix proteins in liver injury and fibrosis. in the present study, we wondered whether follistatin, activin inhibitory protein, reduce apoptosis and prevent liver fibrosis and whether activin plays a key role in liver fibrogenesis. methods: wistar male rats weighing around g were injected intraperitoneally with dimethylnitrosamine (dmn) (lopg/g body weight) three times a week for three weeks. either follistatin or saline were also injected intravenously three times a week. on the nd day, blood was collected and biochemical parameters were measured using the standard methods. the liver was either fixed with % bufferedparaformaldehyde for histological examination, or frozen immediately in liquid nitrogen for the rna analysis. tissue sections were either stained with hematoxyline-eosin, masson-trichrome, or subjected to immunohistostaining using antibodies against collagen type iv, alpha-smooth muscle actin (sma), fibronectin, tgf-beta. the mrna expression of activin, tgf-beta, timp were measured by rt-pcr. apoptosis was analyzed by tunel methods. wistar male rats were injected with dmn ( kglg body weight) once and liver tissues were obtained and fixed with % buffered-paraformaldehyde , , , , , , hours and days after injection for immunohistochemical staining. hepatocytes and hepatic stellate cells were isolated by collagenase perfusion method and by collagenase-pronase perfusion method and analyzed activin and tgf-beta mrna expression by rt-pcr. results: % of control rats died, whereas none of follistatintreated rats died within days. the serum level of hyaluronic acid in follistatin-treated rats were significantly reduced. ast and alp levels were also decreased significantly. apoptosis was reduced by follistatin dose-dependently. the expression of tgfbeta, timp, collagen lv and alpha-sma expression were also decreased in follistatin treated rats. we then examined activin expression in liver after dmn treatment. activin expression was observed at the maximum level in hepatocytes hours after dmn treatment. tgf-beta expression was not detectable hours after dmn administration, and it was sfxikingly increased in stellate cells hours after dmn administration but it was not detectable in hepatocytes. activin appeared in hepatic stellate cells as well as in hepatocytes hours after dh n administration. inducible nitric oxide synthase (inos) has been reported to play pivotal roles in the development of various types of liver injury and inos expression may be important in the process of fibrogenesis of nonalcoholic steatohepatitis in obesity. no-induced active substance was shown to activate matrix metalloproteinase. the inos mrna and protein activity have been found to be induce in rats on a high-fat diet. objectives we investigated whether ) induction of nos occurs in liver of mice fed high-fat diet, and ) nos increases matrix metalloproteinase activity and reduces collagen content, thus attenuates liver fibrosis. we compared fatty and fibrotic changes of hepatic tissues in inos-knockout (inos-'-) and wild-type (inos+/+) mice which were fed with high-fat diet for weeks starting from weeks old. marked induction of inos mrna occurred in inos+/' mice, but not in inos-/-mice. immunohistochemically, nitrotyrosine staining which is a footprint of no-induced active substance, showed positive in inos+'+ mice, and negative in inos-'-mice. in histopathologically showed fatty metamorphosis, but did not have any distinction between groups inos+'+ and inos-/-. the extracellular collagen content with azan staining in inos+/+ mice was markedly decreased compared with that in inos-'-mice. in gelatin zymography of matrix metalloproteinases we found that prommp- and prommp- were increased both in inos+'+ and inos-'-mice, but their active form was found only in inos+'+ mice. similar results were obtained from in situ zymography of hepatic tissue. the stronger gelatinolytic activity was found diffusely in hepatic tissues of inos+/+ mice than inos-/-mice. ( xc bl ) were subjected to either sham operation or bile-duct ligation. animals were sacrified two weeks after sur-gery and blood and liver samples obtained. bile duct ligationinduced elevation of serum liver enzymes was similar between wt and atla (-/-) mice. however, the liverlbody weight ratio was greater in wt than in atla (-/-) mice. bile duct ligated wt mice showed severe septa fibrosis, as assessed by trichromic masson and sirius red staining. in contrast, atla (-/-) mice showed minor fibrotic lesions, which were mainly located in peribiliary areas. collagen accumulation, as assessed by morphometric analysis of sirius red staining and hepatic hydroxyproline content, was markedly lower in atla (-/-) mice compared to wt mice. positive sirius red stained area in bile duct ligated wt and atla (-/-) mice were . . and . . %, respectively (p< . ). the increase in hepatic concentration of bioactive tgfb and proinflammatory cytokines (tnfa and illb) was attenuated in atla (-/-) mice compared to wt mice, as assessed by elisa. moreover, immunohistochemistry analysis revealed decreased lipid peroxidation products as well as decreased phosphorylation of c-jun and p - mapk in atla (-/-) mice compared to at (+/+) mice. chronic liver failure stimulates the onset of interstitial liver fibrosis, which eventually results in "capillarization" of the sinusoid, impeding clearance of toxic substances by hepatocyte cells. the goal of this work was to search for the therapeutical effect of adjuvant gene therapy using adenoviral vectors containing cd-nas for human urokinase plasminogen activator and human matrix metalloproteinase- (ad-hupa plus ad mmp- ) on cirrhosis and its relationship with manganese brain accumulation, striatum dopamine and its metabolites in the rat. mn+ is a wellknown neurotoxic metal which has been found accumulated in brain and blood of cirrhotic patients with hepatic encephalopathy. mnt elimination takes place via the hepatobiliary route. methods gr wistar rats underwent bile-duct ligation (bdl) for weeks and concomitantly treated with mglml of mncl in drinking water (bdl/md ). after this point, five animals were sacrificed and serum, liver and brain tissue (striatum) were obtained. of the remaining bdl/mn+ -cirrhotic animals (n= ), were injected with ad-hupa plus ad-mmp- ( x " + . ~ ~' vp/kg respectively) and rats injected with ( . ~ " vp/kg of ad-p-gal). this treatment lasted days. then, biological samples were recollected as before. an additional experimental model represented by cirrhotic rats injured chronically for weeks with cclb were also monitored for their response to this therapy. results seven wk-cc -cirrhotic animals treated with ad-hupa plus ad-mmp- (total . x 'vp/kg) were monitored at , , , , and days after combined gene therapy treatment (n= ). these animals showed improvement in liver fibrosis of up to % as compared with their ad-p-gal (n= ) treated cirrhotic counterparts. these results correlated with hydroxyproline detemiinations. furthermore, survival was determinated in additional cirrhotic animals. cirrhotic rats treated with ad-hupa plus ad-mmp- had a significantly higher probability of survival at days after beginning of treatment as compared with ad-@-gal cirrhotic rats. bdl/mn+ injured rats displayed tremors, rigidity, and gait abnormalities. ten days after treatment with combined therapeutic gene therapy, these symptoms decreased. also, liver fibrosis was evidently less ( %) as compared with ad-p-gal treatment rats. brain tissue (striatum) was recollected days after bdl/mnf animals were injected with either ad-hupa plus ad-mmp- or ad-p-gal alone. dopamine ( . mglgr) was decreased in % in ad-p-gal treated animals, as compared with . mglgr of dopamine found in ad-hupa plus ad-mmp-%treated animals. dyhydroxyphenylacetic acid (dopac a main dopamine metabolite) was as high as in ad-hupa plus ad-mmp-%treated animals ( . mglgr striatum) indicating a higher dopamine turnover in nontherapeutically treated cirrhotic animals. moreover, animals treated with ad-hupa+ad-mmp- showed a % decrease in ascitis and gastric varices as compared with ad-p-gal-treated animals. (gastroenterology , ) . while ppar-a was reported to be involved in induction of antioxidant enzymes expression and activities (life sci : , ). furthermore, in respect of hepatic fibrosis, oxidative stress induces hepatic fibrosis and many reports have demonstrated that several antioxidants can inhibit hepatic fibrosis. therefore, in the present study, we examined whether ppar-a ligands, i.e. wy- , and fenofibrates, can suppress hepatic fibrosis by attenuating oxidative stress in experimental rat model and possess a possibility of therapeutic candidate for hepatic fibrosis. methods: fibrosis was induced in male wistar rats by intraperitoneal administration of thioacetamide (taa) ( mglkg twice weekly for weeks). in the treated groups, ppar-a ligands, wy- , (wy) and fenofibrates, were fed a diet containing . % (wlw), and ppar-y ligands, pioglitazone (pio), were fed containing . % (wlw) all through the experiment. control cirrhotic rats received saline injections for weeks. after killing all rats, histological examinations (he, azan staining, immunohistochemistry of a-sma), serum value of alt and hyaluronic acid, mrna expression of ppars, acyl-coa oxidase (aco), a (i) procollagen, and antioxidant enzymes, such as superoxide dismutase (sod) and catalase. we also determined lipid peroxidation (lpo), glutathione levels, and activities of sod and catalase in the perfused liver. results: semi-quantitative analyses of fibrotic area revealed that wy co-administration with taa reduced to only % of the area of taa-treated rats. there was no significant difference in azan staining of the liver between taatreated rats and taa-treated rats. wy administration could not lower serum alt value in chronic and acute taa injury. these observations suggest that wy fails to modulate the hepatotoxicity of taa. an increased expression of ppar-cy in taatreated rats were observed, but the expression of ppar-a was abolished in taa-and taa-treated rats. wy intensively enhanced mrna levels of aco which was regulated by ppar-a, increase nearly -fold than controls, but the expression of aco diminished in taa-and taa-ppar-a activators, wy- , and fenofibrates, treated rats as well as ppar-a. the mrna levels of a (i) procollagen and tgf-/ l were strongly increased in taa-and taa-administrated rats than in controls, while they were dramatically suppressed in taa-treated rats. the catalase mrna was reduced to % of the controls in taa-and taa-treated rats, however, taatreatment prevented this decrease to % of the control levels. catalase activity increased -fold in taatreated rats than in controls, while it decreased % of the controls in taa-and taa-treated rats. in taa-and taa-treated rats an increase of lpo was observed, but not in ta-atreated rats. we c o n h e d that fenofibrates treatment could reduce hepatic fibrosis to approximately % of taa treatment for weeks by semi-quantitative analysis of fibrotic area. conclusion: our data indicate that war-a activators, not ppar-y, can markedly inhibit hepatic fibrosis in the experimental taa-induced rat cirrhotic model. we suggest that ppar-a and catalase are important in the development of hepatic fibrosis. therefore, we conclude that suppression of hepatic fibrosis by ppar-a activators is probably due to their antioxidant effects via increased activitiy of catalase which reduces hydrogen peroxide, and that fibrates, such as fenofibrates and bezafibrates, might be new candidates for the treatment of hepatic fibrosis. fibrosis is a common end-point in clinical trials of chronic hepatitis c. liver biopsy remains the gold standard for fibrosis evaluation. however, variability in fibrosis distribution within the liver (sampling variability) is a potential limit to liver biopsy. in order to assess the influence of sampling variability on the accuracy of liver fibrosis assessment with biopsy, we measured liver fibrosis on virtual biopsies of increasing length reconstituted from digitalized images of large liver sections using two different methods of fibrosis assessment. method large sections ( cmz) were performed from surgical liver samples with chronic hepatitis c and various degree of fibrosis. measurement of fibrosis on the whole section was considered as the reference value. from the digitalized image of the whole section, virtual biopsies of increasing length ( . to mm) were reconstituted. fibrosis was assessed independently on each individual virtual biopsy using both image analysis and meta-vir score. results were compared to the reference value. results: a total of . virtual biopsies were studied. using image analysis, a strong dispersion of area of fibrosis was observed for virtual biopsies smaller than omm. only % of , mm length virtual biopsies had a measured of area of fibrosis equal to reference value %. this percentage increased progressively with increasing size ( %, %, % for biopsies of mm, omm, loomm length, respectively). using metavir scoring system, % of biopsies of , cm length were correctly categorized according to the score assessed on the whole large section. it increases to % for a , cm size without any substantial benefit for longer biopsies. a same trend was observed whatever the stage of fibrosis. conclusion: sampling variability of fibrosis is a significant limit for fibrosis assessment with liver biopsy. this study suggests that a length of at least mm is necessary to valuably evaluate fibrosis with semiquantitative score. sampling variability become a major limitation for more accurate method such as automated image analysis. (msi- ) , a rna-binding protein, is highly observed in developing central nervous system and thought to be a mammalian neural stemlprogenitor cell marker. we have reported that cells positive for musashi- (msi- ) protein appeared during spontaneous resolution of rat liver cirrhosis and that some msi- -postive cells were also positive for matrix metalloproteinase (mmp)- , possibly implicating active participation of stem cells in the resolution of collagen. though msi- antigen is expected to be expressed during very early stage of differentiation in the cellular lineage of liver, the type of cells appeared in the damaged liver and expressed msi- remain to be elucidated. the present study is designed to characterize the msi- -positive cells in cirrhotic liver from the aspect of differentiation, significance and expected function of stemlprogenitor cells in the liver, especially fibrolytic function. methods: rat liver fibrosislcirrhosis was established by intraperioneal injection of cc twice a week. liver samples were obtained , and days after the last injection of -week cc intoxication. liver tissue samples were immunohistochemically stained for msi- , c-kit, nestin, a-smooth muscle actin (a-sma), ck- and mmp- . gene expression of msi- was also observed by reverse-transcription polymerase chain reaction (rt-pcr). results: neural stemlprogenitor cells, as defined by their expression of msi- andlor nestin, were not observed either in normal rat liver or after weeks of c c administration. rt-pcr analysis revealed very slight signals of msi- mrna at days after discontinuance of -week ccld treatment. on the contrary, remarkable increase of msi- mrna was observed at days after the weeks ccl treatment, then the signals decreased gradually. immunohistochemical analysis showed that a considerable number of cells positive for msi- appeared in the early recovery stage from advanced liver fibrosis, especially at day after the last injection of weeks ccl treatment. expression of msi- in liver tissue was proceeded by and partly overlapped with that of c-kit, a marker of hematopoietic stem cells. some msi- -positive cells observed around perivenular regions were as very small as oval cells, while the size of msi- -positive cells present along the resolving fibrous bands varied and some of them expressed mmp- . some ductal cells were positive for msi- but not for ck- examined in serial sections. msi- positive cells did not express hepatocytes markers such as albumin or afp. cells positive for a-sma and msi- were observed in some portions, but most of msi- expressing cells did not show positive staining for a-sma or desmin. at day , a few msi- -positive cells were observed around the remaining fibrous bands. s-adenosyl-l-methionine represses the fibrosis, constitute a good model for studying the mechanisms responsible for antifibrotic therapy. to test whether administration of s-adenosyl-l-methionine, a precursor of glutathione and an agent shown to prevent liver toxicity in a variety of settings, could repress the activity of the mouse pro-alpha (i) collagen gene in hepatic stellate cells, homozygous transgenic mice harboring the - kb to + bp of the proximal promoter of the mouse alpha (i) collagen gene cloned upstream of the escherichia coli beta-galactosidase reporter gene (lacz) were used. chronic liver injury was induced by injecting intraperitoneally mllkg of body weight of cc ( % vlv in mineral oil) three times per week for weeks. s-adenosyl-l-methionine was administered intraperitoneally at a dose of mglkg of body weight every day for weeks. control groups were given either mineral oil or s-adenosyl-lmethionine alone. hepatocellular damage and protection by sadenosyl-l-methionine was confirmed by measuring serum levels of transaminases; s-adenosyl-l-methionine lowered alt levels in the ccl -treated mice from to ull and ast from to ull (control values in the absence of cc were and ull for alt and ast, respectively). hematoxylin and eosin staining in the cc -treated mice revealed the presence of mallory bodies, lymphocyte infiltration, centrilobular steatosis, and perivenular and pericellular fibrosis, and s-adenosyl-l-methionine minimized the pathology score. masson's trichrome staining showed less collagen deposition in mice treated with cc plus s-adenosyl-lmethionine than in the cc -treated mice. histochemical analysis using x-gal staining allowed for the precise identification of the cell type in which the beta-galactosidase gene was active as driven by the pro alpha (i) collagen promoter. results indicated activation of the pro alpha (i) collagen promoter in mice treated with ccl, and repression of such activation in mice co-treated with s-adenosyl-l-methionine. immunofluorescence analysis of mice injected with cc revealed colocaliiation of alpha-smooth muscle and beta-galactosidase positive cells, suggesting that the activation of the promoter occurred only in hepatic stellate cells. these results suggest that one mechanism by which administration of s-adenosyl-l-methionine, a precursor of glutathione synthesis, could ameliorate liver fibrosis is by decreasing the responsiveness of the promoter of the alpha (i) collagen gene to a profibrogenic stimuli such as ccb. these transgenic mice should prove to be useful in further studies on how s-adenosyl-l-methionine exerts this repression of the alpha (i) collagen gene and perhaps to studies with other liver toxins such as alcohol. background endothelin- (et-i), the most powerful constrictor of the liver vasculature and stimulator of the synthesis, by kupffer cells, of a potent systemic vasodilator, platelet-activating factor, is also implicated in the fibrosis of lung, kidney and liver. thus increased hepatic concentrations of et- and its receptors in human and experimental cirrhosis suggest its major role in the pathology of chronic liver disease and its complications (fibrosis, portal hypertension and systemic hypotension). we investigated whether et- receptor antagonism, after the development of fibrosis and cirrhosis, arrestslreverses the progression of chronic liver disease. methods: chronic liver injury was induced in rats by cc treatment ( of the development of cirrhosis in group (histopathology score of . ? . vs . . , p< . ) and reversal of cirrhosis in group (histopathology score of . . vs , p<o.ol) rats. this was accompanied by reduced a-smooth muscle actin-positive cells (activated stellate cellslmyofibroblasts) in the tak- treated versus saline-treated rats receiving cc . tak- treatment caused significant amelioration of portal hypertension (p< . in both groups), systemic hypotension (p< . in both groups) and liver injury (reduced activities of serum ast, alt and ldh), and improved hepatic synthetic capacity (increased serum albumin concentration) in both groups of rats relative to the vehicle-treated rats. tak- treatment reduced collagen synthesis as evidenced by decreased hepatic hydroxyproline, collagen-a type i mrna expression, and mrna and protein expression of a potent fibrogenic cytokine tgf-p . conclusions: the results emphasize the role of et- in the development of cirrhosis and strongly suggest that blockade of its actions can be a rational therapy for chronic liver disease and its complications. subunits which are reported to be stimulatory. however p and p can form homodimers which have been described as inhibitors and stimulators of gene expression respectively. the aim of the study was to determine the role of the p subunit of nfkb in the progression of fibrosis. methods -chronic liver fibrosis was induced in p knockout and wild type control mice by administering twice weekly ccl injections for weeks. livers and blood samples were harvested at day , , , and after the last ccl injection. results -p knockout mice developed more severe liver disease as measured by serum alt's and peak fibrosis scores. the increased liver injury in p knockout mice was associated with a massive infiltrate of inflammatory cells into the liver compared to the control wild type mice. the majority of the inflammation was neutrophilic as measured by immunohistochemistry, however we also observed an increase in cd positive t cells. taqman quantitative rt-pcr analysis was used to measure collagen i, alpha-smooth muscle actin (asma) and timpl gene expression in the p knockout and wild type mice. all three genes were elevated in the mice laking p subunit of nfkb. immunostaining confirmed an increase in asma positive cells in the livers of p knockout mice compared to wild type controls. conclusion: the p subunit of nfkb may play an important role in protecting against the development of liver fibrosis and reducing liver inflammation in chronic liver injury. gnainsky, volcani center, bet dagan, israel; orna halevy, hebrew university of jerusalem, rehovot, israel; gadi spira, technion -israel institute of technology, haifa, israel; rafael bruck, wolfson medical center, holon, israel; arnon nagler, sheba medical center, tel hashomer, israel; mark pines, volcani center, bet dagan, lsrael introduction halofuginone is an inhibitor of hepatic fibrosis as demonstrated by prevention of dimethylnitrosamine (dmn) and thioacetamide (taa)-induced fibrosis. halofuginone is also responsible for the resolution of advanced fibrosis and increase in cirrhotic liver regeneration. methodslaims in this study atlas microarray technique was applied in vivo in an effort to identify genes involved in the halofuginone-dependent prevention of liver fibrosis. to that end we compared cdna array hybridization of taa-treated rat liver biopsies to biopsies of livers from rats treated with both taa and halofuginone. results from the genes of the array, were differentially expressed. one of the genes up-regulated by halofuginone was identified as protein tyrosine phosphatase of regeneration liver - (prl-l), an immediate early gene known to be induced during liver regeneration. the atlas microarray results were confirmed by northern blot analysis and by in situ hybridization. hepg cells in culture expressed high levels of prl- gene that was down-regulated upon serum starvation. halofuginone increased the prl- gene expression in the serum-starved cells in a dose and time-dependent manner. on the protein level, halofuginone caused translocation of prl- from hepg and huh- nucleus outer membranes to the cytosol as demonstrated by immunofluorescence using prl- antibodies. prl- translocation is known to be associated with alterations in cell cycle. halofuginone caused accumulation of hepg cells in the gym phase as analyzed by flow cytometry suggesting a putative g im arrest. conclusions these results demonstrate the involvement of halofuginone in the hepatocytes cell cycle through prl- gene expression and its cellular translocation. nathalie senant, universitk victor segalen bordeaux , bordeaux, france; genevieve freyburger, hdpital pellegrin, bordeaux, france; toulouse, france; alexis desmouliire, universitk victor segalen bordeaux , bordeaux, france several lines of evidence incriminate the hemostasis system in liver fibrogenesis. first, experimental and human pathological data suggest a role for micro-thrombosis in fibrosis progression. secondly, several hemostasis proteins, such as thrombin, can regulate the expression of fibrogenic mediators through signaling by cell-surface receptors. the aim of this study was thus to evaluate the effect of direct thrombin inhibition on experimental liver fibrosis. fibrosis was induced in rats by administration of cc in olive oil orally times a week for either or weeks. the thrombin antagonist ssr (sanofi-synthelabo research) was given daily orally at mglkg (a dose that has been shown to significantly inhibit thrombin in vivo) starting week after the beginning of cc intoxication. control groups received either the respective solvents or ssr alone. nine animals were used in each group. fibrosis was quantified with histomorphometrical analysis of sirius red-stained liver sections and expressed as a percentage of the surface of the section. we also measured the level of fibrogenic cell activation with quantitation of the area occupied by alpha-smooth muscle actin (asma)-positive cells. the levels of tissue inhibitor of matrix metalloproteinase- (timp-i) transcripts were analyzed by northern blot on total liver rna and expressed as timp-l/gapdh ratios following quantitative measurement with an instant imager. after weeks of cc , the area of fibrosis increased from . % . in controls to . i . % and was slightly but non significantly decreased by ssr ( . lr . %, ns). the area of asma-positive cells was however significantly decreased by ssr from . ? . to . i . % (p = . ). in animals receiving cc for weeks, treatment with ssr resulted in a decrease in fibrosis area from . t . to . t . % (p = . ). similarly, the asma-positive area decreased from . . to . i . % (p = . ). the timp-i/gapdh ratio was decreased by ssr at weeks (from . . to . . , p =om). ssr alone had no effect on the measured parameters at both time points. additionally, it did not alleviate the acute toxicity of cc as shown by the levels of serum transaminases and the area of necrosis that were not significantly modified. altogether, these data provide evidence that thrombin antagonism can reduce the extent of liver fibrosis in this model. ongoing experiments should clarify whether this results from an anticoagulant effect or an inhibition of thrombin signaling effects. center, tel aviv, israel background and aims: proliferation and "activation" of hepatic stellate cells (hscs) and production of collagen are involved in the pathogenesis of liver inflammation and fibrogenesis. ligands of nuclear hormone receptors, such as triiodothyronine (t ) and peroxisome proliferator activator receptor y (ppary) ligands have been shown to affect fibrogenesis. levels of ppary are reduced in activated stellate cells and addition of the iigand increases ppary receptor expression and ameliorates fibrosis. in contrast, t has the opposite effect on liver fibrosis. low circulating t levels reduces liver fibrosis and cirrhosis. in this study, we investigated the in vitro effects of t , ppary ligand and their combination on proliferation and activation of a rat hsc cell line. methods: transforming growth factor (tgf-pi) was used to induce myofibroblast-like phenotype in rat hscs (hsc-t ). activation of thyroid hormone and ppary receptors was induced with t and -deoxy-prostaglandinj( )(pj ), respectively. signal transduction markers and activation marker a-smooth muscle actin were determined using western blotting. collagen i expression was measured by northern blotting. results: both t and pj ( ) inhibit hsc proliferation induced by platelet-derived growth factor and by tgf-p. t inhibits cell proliferation by inducing apoptosis, while pj( ) reduces expression of proliferation marker cyclin d . both "i and pj( ) decrease expression of activation marker a-smooth muscle actin. in addition, t strongly increases both basal and tgfeinduced collagen i expression. by contrast, pj( ) decreases collagen expression and inhibits t -induced collagen production in tgfp-activated hscs. conclusion: both t and pj( ) reduce mitogen-induced proliferation of rat hscs, but have different effects on collagen i expression. these results suggest that prolierationlactivation and collagen i expression are two differently regulated processes in kato, sadakazu aiso, hiromasa lshii, keio university, tokyo, japan connective tissue growth factor (ctgf) is a profibrogenic molecule involved in several human fibrotic disorders including liver fibrosis. in human as well as experimental liver fibrosis, ctgf overexpression, through upregulation of ctgf mrna in hepatic stellate cells (hscs), is associated with the degree of fibrosis. it has been known ctgf is produced in many cell types in the live and exogenous ctgf modulates even the activation of hscs. although alcohol is the major cause of liver fibrosis, the role of ctgf in alcoholic liver fibrogenesis, has been poorly understood. cytochrome p- e (cyp el)-mediated oxidative stress, as a result of chronic and excessive ethanol consumption, has been implicated as a crucial factor in alcoholic liver fibrogenesis. the goal of a series of our studies was to clarify the effect of cyp e mediated ethanol oxidation on ctgf expression. we previously reported the results of the assay using the well-established hepg cell line constitutively expressing cypzei, which was kindly provided by dr. a. cederbaum ( mt. sinai school of medicine, ny). ctgf mrna quantitation assay by a real-time reverse polymerase chain reaction (rt-pcr) showed a significant increase in ctgf mrna levels in ethanol-treated e cells in a time-dependent manner up to hours after initial ethanol treatment. there was nearly a -fold increase in ctgf mrna levels when ethanoltreated e cells were coincubated with phorbol myristate acetate (pma), a reagent which enhances expression of cyp e . ctgf mrna levels were significantly reduced when ethanol-treated e cells were co-incubated with -methylpyrazole( mp), a cyp el inhibitor, or antioxidants, n-acetylcysteine and trolox. in the present study, we visualized the state of oxidative stress in ethanol-treated e cells by using mab f , a newly-established marker for oxidative stress. the mab f , which was generously provided by dr. k uchida (nagoya university, japan), was raised against the acrolein-modified keyhole limpet hemocyanin to verify the protein-bound acrolein in vivo. the staining, procedure was detailed elsewhere. immunohistochemical detection of proteinbound acrolein revealed e cells were stained when treated with ethanol. without ethanol treatment, no staining was observed in ethanol upregulates pro-fibrotic connective cells via cytochrome p- e -mediated e cells even at a -hour incubation period. moderate or no staining was observed when ethanol-treated e cells were coincubated with antioxidants. staining intensity by mab f is associated with ctgf mrna levels in this cell line. collectively, these data suggest ctgf mrna upregulation in e cells seems to be mediated by the state of oxidative stress. our present study first linked cyp el-mediated oxidative stress with ctgf gene regulation, thus suggesting a possible involvement of ctgf in alcoholic liver fiberogenesis. disclosures: sadakazu aiso -no relationships to disclose hiromasa ishii -no relationships to disclose mikio kajihara -no relationships to disclose ( ) human livers with biliary fibrosis. methods: changes in ntp-dase expression in rat liver were determined in normal and -wk bile duct ligated (bdl) or -wk c c rat livers and pflpm using confocal immunofluorescence, immunoblot, and northern blot. changes in ntpdase expression in human liver were determined in de-paraffinized liver biopsy specimens using confocal immunofluorescence. results: as was previously reported, ntp-dase was expressed in pf surrounding intrahepatic bile ducts in normal and sham bdl controls. in bdl rat livers, however, nt-pdase fluorescence was completely lost. no decrease in ntp-dase fluorescence was noted in cc rat livers within portal areas, although an increase in ntpdase fluorescence was seen in central areas. pf from sham bdl control animals expressed nt-pdase and the intermediate filament vimentin. "pf" from bdl animals did not express vimentin but were positive for a-smooth muscle actin (sma), suggesting that they were in fact pmf. most bdl pmf did not express ntpdase ; however, occasional cells were seen with sma and ntpdase in a pen-nuclear compartment. immunoblot analysis of pf revealed a marked decrease in ntpdase detection in bdl cells relative to sham bdl. no change was noted in ccll or c c control cells. northern blot analysis revealed no differences in ntpdase mrna levels in sham bdl, bdl, ccl, control, or cc pf. confocal micrographs of normal human biopsy specimens revealed ntpdase co-localization with vimentin in pf surrounding intrahepatic bile ducts. however, ntpdase fluorescence was either markedly decreased or lost in specimens from patients with primary biliary cirrhosis. no loss of ntpdase fluorescence was seen in biopsy specimens from patients with hepatitis c. conclusions: ntpdase expression is specifically decreased in biliary fibrosis, largely through a posttranslational mechanism. "portal fibroblasts" from bdl rats are in fact portal myofibroblasts, which lose expression of ntpdase . consistent with findings in rats, ntpdase expression is also specifically lost in human biliary fibrosis. work is now underway to determine whether loss of ntpdase in the peri-biliary compartment alters nucleotide signaling in bile duct epithelia. myofibroblasts of bone marrow origin have recently been found in a number of parenchymal organs such as the gut and kidney. we hypothesised that marrow derived myofibroblasts contribute to liver cirrhosis. methods. we sought to analyse the origin of myofibroblasts within cirrhotic liver in two scenarios: three male recipients of female liver transplants who have subsequently developed post transplant hepatitis c cirrhosis and a female recipient of a male bone marrow transplant who later developed hepatitis c induced cirrhosis. through the use of in situ hybridisation for the y chromosome we have been able to track cells of male origin. results. we have detected significant numbers of y chromosome positive cells in fibrotic areas. by combining the in situ hybridisation with immunocytochemistry these cells were found to be positive for vimentin, alpha-smooth muscle actin and negative for cd . these cells were therefore determined to be of a myofibroblast phenotype. in the liver transplant recipients, . % (corrected count . %) of the myofibroblasts were y chromosome positive. in the female recipient of the male bone marrow transplant, . % (corrected count . %) of the myofibroblasts were y chromosome positive. conclusions. within all the livers analysed we found frequent myofibroblasts within and adjacent to the fibrotic bands that are of bone marrow origin indicating an extrahepatic cell contribution to the pathogenesis of human liver cirrhosis. (hsc) is the major fibrogenic cell of the liver and a key target of potential antifibrotic therapies. hepatocyte growth factor (hgf) is antifibrotic in several models of experimental liver fibrosis, but its mechanism of action is uncertain. potential use of hgf as a therapeutic peptide is limited by its size and need for parenteral administration. to overcome these potential limitations, refanlin has been developed as a synthetic small molecule sflhgf mimetic, that has demonstrated antifibrotic properties in renal fibrosis. aim: to study the in vitro and in vivo antifibrotic efficacy of refanlin. methods: serum starved lx cells, an immortalized human hsc line, were treated with mglml of refanlin for hours. proliferation studies using tritiated thymidine incorporation were performed. rna was isolated, and a panel of fibrosis markers (al-smooth muscle actin (asma), collagen i, bpdgf-receptor, , (mmp , , ) , tissue inhibitor of metalloproteinase- , (timp , ), were assessed by real time rt-pcr analysis. cirrhosis was induced in sprague-dawley rats by if thioacetamide (taa) ( mg/kg tiw) for six weeks. in the refanlin treated group, mglkg of refanlin was administered five days per week for six weeks, concurrently with the taa. control rats received ip pbs five days per week for six weeks, along with taa. liver tissue was formalin-fixed and paraffin-embedded, then stained with % picrosirius red and computer morphometric analysis was performed for collagen quantitation using the bio-quanttm software. results: in cultured human stellate cells, there was a % decrease in asma, a % decrease in the expression of tgfb-receptor and mmp , and a % decrease in timp . there was no effect on proliferation. in vivo, fibrosis decreased by one metavir stage in rats treated with c and thioacetamide for weeks. furthermore, c led to a significant decrease in portal pressure compared with non-treated rats with fibrosis. conclusions: refanlin may have antifibrotic efficacy through its inhibition or downregulation of tgfb receptor as well as mmp . in vivo antifibrotic activity of refanlin is associated with reduced portal pressure. these experiments better define the potential targets of hgf's antifibrotic activity. ongoing studies in mechanistically distinct models of fibrosis will broaden our understanding of this agent's effects and point towards therapeutic trials in humans. kanto, aki sato, michiyo inoue, hideki miyatake, mitsuru sakakibara, takayuki yakushijin, chika oki, tetsuo takehara, norio hayashi, osaka university graduate school of medicine, suita, osaka, japan background and aim hepatitis c virus (hcv) infection induces a wide range of chronic liver injuries. impaired thl response against hcv may be involved in hcv persistence, however, its precise mechanism is largely unknown. dendritic cells (dc) are the most potent antigen-presenting cells that play essential roles in initiating as well as regulating anti-virus immune responses. blood dc are grouped as myeloid (mdc) and plasmacytoid dc (pdc). with respect to their functions, mdc produces il- and tnf-a upon stimulation and drives thl response, while pdc releases considerable amount of type-i ifn upon virus infection and mainly skews th . we previously reported that monocytederived dc is functionally impaired (kanto t. et al. j lmrnunol ) , however, the roles of blood dc subsets in the direction of thllth in hcv infection are still elusive. to address these issues, we compared the numbers and functions of mdc and pdc between chronically hcv-infected patients and uninfected donors. subjects and methods sixty-six hcv-positive patients ( chronic hepatitis and asymptomatic carriers) and agematched uninfected donors were enrolled. mdc or pdc was determined by the patterns of lineage markers, hla-dr, cdllc and cd . each type of dc was magnetically separated or sorted under facs vantage and was subjected to the functional analyses. the ability of dc to polarize naive cd t-cells towards thl or th was estimated by the pattern of ifn-ylil.- /il- production from mdc-or pdc-primed cd . results absolute numbers of mdc, pdc and their cd + progenitors are lower in hcv-infected patients than in control subjects, most significantly in those with chronic active hepatitis (mdc, pdc, cd , median, patients vs. controls; . , . , . *. vs. . , . , . /pl, p< . ). both types of dc from patients are impaired in the stimulation of allogeneic cd t-cells and the production of il- p or ifn-a compared with healthy donors. on encounter with nayve cd t-cells, mdc from the patients are less able to drive thl response, while pdc from them prime more il- producing t-cells than those from donors. exogenous il- significantly improved the patient mdcdriven thl response, however, it simultaneously increased il- producers both in mdc and pdc. in contrast, ifn-y or il- stimulated the patient mdc to drive thl without increasing mdc-or pdc-primed il- producing cells. conclusion both types of blood dc in chronic hcv infection are reduced and play decisive roles in the development of an impaired thl and an il- -rich environment, thus favoring hcv persistence. ifn-y or il- is feasible to restore the altered t-cell polarizing ability of blood dc in chronic hepatitis c. hepatitis c virus (hcv) replicates its genome in a membraneassociated complex composed of viral nonstructural proteins and rna as well as altered cytoplasmic membranes and other as yet unidentified host cell components. a specific membrane alteration, designated membranous web, was recently identified as the site of hcv rna replication in huh- cells harboring subgenomic replicons (gosert et al. j virol ; : - ) . here, we describe hcv replicons that allow the direct visualization of hcv replication complexes in living cells. methods: a transposon-mediated random insertion screen followed by selection of viable replicons was performed to identify sites that tolerate insertions in nonstructural protein a (ns a). the green fluorescent protein (gfp) coding sequence was subsequently inserted into selected sites. rna was in vitro transcribed from subgenomic replicon constructs harboring these insertions, followed by electroporation into huh- cells and selection in the presence of g . results: two sites in the c-terminal domain of ns a were found to tolerate the gfp insertion and allowed efficient initiation and maintenance of hcv rna replication. expression of a ns a-gfp fusion protein of the expected molecular mass could be demonstrated by immunoblot, indicating that the gfp was stably retained and that processing of the viral protein occurred normally. more important, expression levels were robust enough to allow direct visualization of this fusion protein by fluorescence microscopy. gfp fluorescence was found in a cytoplasmic reticular pattern and in brightly fluorescing dots, the typical distribution of hcv nonstructural proteins in replicon cells. by confocal laser scanning microscopy, gfp fluorescence was found to colocalize with other hcv nonstructural proteins in the brightly fluorescing dots, suggesting that these represent hcv replication complexes, previously identified as membranous webs. metabolic labeling of the nascent viral rna with brutp and (immuno)electron microscopy studies are underway to further validate these findings. moreover, live cell imaging studies using these replicons have successfully been initiated. conclusion: we have identified sites in ns a that allow insertion of gfp in the context of functional hcv replicons and direct visualization of hcv replication complexes in living cells. these findings have implications for the structure and function of the hcv ns a protein as well as for the architecture of the hcv replication complex. this system will allow to gain a dynamic view of the formation and turnover of hcv replication complexes and may be useful to select for cell populations permissive for hcv rna replication. infection. masahisa jinushi, tetsuo takehara, tomohide tatsumi, takuya miyagi, takahiro suzuki, tatsuya kanto, norio hayashi, osaka university graduate school of medicine, suita, osaka, japan background and aim it has been generally accepted that the pertubation of cellular immunity plays an important role in establishment and persistence of chronicity in hcv infection as well as resistance to ifn therapy. accumulating lines of evidence have unveiled that dendritic cells (dcs), most potent antigen presenting cells, are impaired in patients with hcv infection, which may be involved in disturbance of anti-hcv immune responses. we recently reported that mhc class i-related chain a and b(mical b),ligands of nk activating receptor nkgzd, are induced on dcs upon interferonaand gain their ability to antivate nk cells; their expression is severely impaired in dcs derived from hcv-infected individuals (hcv-dc) ( . immunol. immunol. - immunol. , . in the present study, we evaluated whether various types of cytokines have an effect for inducing micalb expression on hcv-dcs, thus resulting in triggering nk cell activation by dcs. methods dcs from healthy volunteers (n-dcs; n= ) or hcv-dcs (n= ) were generated from monocyte treated with gm-csf ( ulml) and il- ( ulml) for days. after stimulated with various cytokines (ifnalp, ifny, tnfa, il- , il- or il- ) for h, dcs were subjected to flow cytometric analysis of micalb expression. to investigate the involvement of each cytokine in mica/b expression on ifnalp-stimulated dcs, we employed elisa and rt-pcr to examine the cytokine expression (tnfa, il-lp, il- , il- ~ , il- and il- ) in ifnalp-stimulated dcs, and also assessed micalb expression on dcs in the presence of neutralizing antibodies for these cytokines. to further address the stimulatory capacity of dc, allogeneic nk cells were cocultured with n-dcs or hcv-dcs at a ratio of :l for h, and applied for flow cytometry of intracellular ifny expression, as well as cr release assay for the cytolysis of k target cells. in some experiments, masking antibody for nkg d or micalb was added during nkldc cocultures, and then subjected to nk cell function assays described above. results micalb were induced on n-dcs, but not on hcv-dcs, upon iifnalp(% mic positive: n-dc . % vs hcv-dc . %; p<o.ol). in sharp contrast, micalb expression was increased on hcv-dcs at levels similar to n-dcs in the presence of of onglml il- (but not other cytokines) (%mic: n-dcs . % vs hcv-dcs . %). moreover, n-dcs, but not hcv-dcs, was capable of producing il- in response to ifnalp. little differences were detected in n-dcs and hcv-dcs for the production of tnfa, il-lp, l- , il- ~ , or il- . in addition, ifnalp-mediated micalb induction in n-dcs were completely inhibited in the presence of neutralizing antibody of anti-il l il- receptorcu. even much lower doses ( nglml) of il- restored the ability of hcv-dcs to induce micalb and to activate nk cells in response to ifnalp. the ability of hcv-dcs for nk cell activation was largely diminished by adding the masking antibody of nkg d or micalb. conclusion the current study demonstrated that il- acts as an indispensable mediator to induce micalb expression on ifnalp-stimulated dcs, and that hcv-dcs lack the ability to produce il- and to induce micalb expression in response to ifnalp. since defect in il- production is responsible for impaired nk cell activities by hcv-dcs, these findings shed light on the possibility that il- could improve the ifn-mediated anti-viral immune responses in chronic hcv-infected patients. the cellular immune response contributes to viral clearance as well as liver injury in acute and chronic hepatitis c virus (hcv) infection. understanding the relative immunodominance of hcvencoded peptides is important for understanding pathogenesis as well as potential vaccine candidates. we have developed a comprehensive and sensitive method to screen immune responses to all potential hcv epitopes in hcv-exposed individuals. methods: subjects with evidence of spontaneously resolved infection (eia+/hcv rna-neg, n = ) and patients with evidence of chronic hcv infection (mean hcv rna level = . million copieslml; n = , all genotype la) had either leukophoresis or one-unit blood draw. we synthesized peptides ( mer-peptides overlapping by amino acids) that spanned the entire hcv genotype l a polyprotein; responses to pools of peptides were screened directly ex vivo by an ifn-gamma elispot assay. briefly, autologous dendritic cells (dcs) were generated from peripheral blood mononuclear cells selected by plastic adherence then cultured with gmcsf, il- and % hs for d. dcs were pulsed with peptide pools to stimulate bead-purified cd + t cells. the remaining cd + t cells ( . x lo cells/well) were stimulated the same day with the peptide pools; wells with no peptide (or siv peptides) were used as controls. only responses greater mean + sds above controls were considered positive. the frequency and vigor of cd + t cell responses were statistically greater in resolved vs. chronic infection (mean . pools out of ; median in resolved vs. mean . pools; median . in chronics; p =. ). there was an inverse trend between # peptide pools with cd + t cell responses and hcv rna level. as shown in the figure, the hcv peptide pools differentially elicited responses; specifically, resolved infection was associated with greater cd + responses to pools core b, ela, ns protease- , ns helicase- , ns helicase- , ns helicase- , ns helicase- , ns b- , ns a- , ns a- , ns b- . on the average, cd + t cells responded to . pools in resolved infection vs. . pools in chronics (p =.as). cd + t cell responses specifically directed against core , ns helicase- , ns -helicase , ns b- , ns a- were more frequent in resolved as compared to chronic infection. conclusions: comprehensive mapping of hcv-specific immune responses by overlapping pools reveals significant differences in immunogenicity in resolved vs. chronic infection. in particular, regions core b (amino acids - ), ns helicase- (aa - ) and helicase- ( - ), ns b- ( - ), ns a- ( - ) induce both cd + and cd + t cell immunity and thus represent potential vaccine candidates. supported by nih r dk . sud, geoffiey c farrell, priyanka bandara, james g kench, storr liver unit, westmead hospital, westmead, nsw, australia; geoffiey w mccaughan, aw marrow gastroenterology (and liver centre, camperdown ns w, australia; jacob george, storr liver unit, westmead hospital, westmead, ns w, australia introduction: chronic hepatitis c virus (hcv) infection is associated with an increased prevalence of type diabetes mellitus, which is known to accelerate fibrosis in nash. we hypothesized that viral-induced insulin resistance (ir) may be a mechanism for fibrogenesis in chronic hcv infection. methods and results: . to assess the influence of hcv infection on ir independent of any effect of hepatic fibrosis, hcv subjects with minimal (stage ) or no (stage ) fibrosis were compared with healthy volunteers matched by gender, body mass index (bmi) and waistlhip ratio. although the hcv subjects were younger than the healthy volunteers (p= . ), they had significantly higher levels of markers of ir including fasting glucose (p=o.ol), insulin (p=o.o ), cpeptide (p<o.ool) and homa-ir (p=o.o ). . in hcv patients (fibrosis stage to ), e examined the viral and diseaserelated factors (portallperiportal & lobular inflammation, viral genotype) which may be involved in the pathogenesis of ir in a multivariate model, controlling for other demographic and biochemical variables. by multiple linear regression analysis, independent predictors of homa-ir included bmi (p<o.ool), failed previous antiviral treatment (p<o.ool), portal inflammatory grade (p<o.ool) and genotype status (p=o.ol). genotype cases had significantly lower homa-ir than other genotypes at each stage of hepatic fibrosis. the adjusted difference in homa-ir between genotype and non-genotype was - . ( % ci: - . to - . ; p=o.ol). . we then assessed if ir was associated with increased fibrotic severity. by multiple ordinal regression analysis, independent predictors for the stage of fibrosis in the hcv subjects were homa-ir (or . , p<o.ool), portal inflammatory grade (or: . , p<o.ool), past alcohol intake (or . , p<o.ool), age (or . , p < . ), alt (or . , p= . ), platelet count (or . , p<o.ool), and serum cholesterol level (or . , p=o.ool). as cirrhosis is known to cause ir, a separate multivariate analysis was performed for the non-cirrhotic subjects. the independent predictors for fibrosis were unaltered and homa-ir remained in the model. in a subgroup of patients with estimated duration of infection, multiple linear regression analysis showed that homa-ir was independently associated with an increased rate of fibrosis progression (p= . ). conclusion: hcv infection induces insulin resistance irrespective of the severity of liver disease, and this effect appears to be genotype-specific. further, increased insulin resistance contributes to fibrotic progression. disclosures: priyanka bandara -no relationships to disclose geoffrey c farrell -no relationships to disclose jacob george -no relationships to disclose jason m hui -no relationships to disclose james g kench -no relationships to disclose geoffrey w mccaughan -no relationships to disclose background: approximately one half of hepatitis c virus (hcv) infected patients will fail to respond to current treatment regimens, pegylated interferon alfa and ribavirin. modeling of hcv rna decay in response to antiviral therapy can provide insight into viral and host determinants of viral response. aims: pharmacokinetic, pharmacodynamic, and immunologic outcomes were evaluated on co-infected patients: ) to evaluate the relationship between hcv rna decay and peg-ifn alfa b serum concentrations in hivlhcv co-infected patients and ) to evaluate association between hcv-specific immune responses and virologic outcome. methods: co-infected, therapy-nake patients were treated with peg-ifn alfa b . pglkglwk and rbv mglkglday. serum was obtained for hcv rna, hiv- rna and serum peg-ifn alfa b levels at baseline, after the first dose ( , , hours) and on days , , , ; after the second dose ( , , hours) and on days , ; and after the third dose on days , , and . peripheral blood mononuclear cells were obtained at baseline, weekly for the first month, and monthly thereafter. hcv and hiv- rna levels were determined by reverse-transcriptase poly- merase chain reaction and peg-ifn alfa b levels were measured by elisa. cd + cell proliferative responses to hcv antigens (core, ns , ns , ns ) and hiv- gag were performed at baseline, day and , and weeks , , , and . among co-infected patients, are males, the mean age was . years, are caucasian, are african-american and are hispanic. l patients were infected with genotype , / patients with genotype , and / with genotype a. results: pharmacokinetics: to date, patients have been evaluated. / patients were end of treatment responders, but of these were "late responders" (i.e. hcv rna declines after week and is undetectable by week ). data fitting during the first seven days revealed a free virion clearance rate c of . (day-l) and an infected cell clearance rate of . (day-'). the half-lives were . hours and . days, respectively. pharmacodvnamics: viron production was blocked after the first dose of peg-ifn alfa with an average maximum effectiveness (emax ) of %. we estimated that the fifty percent effective concentration (ec ) of peg-ifn alfa b, the theoretical in v i m drug concentration at which the drug efficacy is , was five-fold lower in responders compared with non-responders while the maximum (cmax) and minimum (c,,,in) concentrations did not differ significantly ( table i ). the calculated minimum ( € , , , i n ) and maximum (emax) efficacy were also significantly increased in responders. immunolonic: the mean number of cd +, cd + and cd + cells did not differ significantly at baseline or during treatment between responders and nonresponders. peripheral blood cd + hcv-specific proliferative responses did not differ significantly between responders and nonresponders except that the response to core antigen was increased in non-responders compared with responders at weeks and . hiv gag cd + proliferative responses were stronger than hcv-specific responses at all time points evaluated. conclusions: the serum peg-ifn alfa b concentration necessary for an end of treatment response is fivefold lower in responders compared with non-responders while there are no significant differences in the maximum and minimum peg-ifn alfa b concentrations in responders and nonresponders. these data suggest that host factors responsible for a viral response may be different in responders compared with nonresponders, independent of serum peg-ifn alfa b concentrations. is a significant predictor of liver disease severity in patients who are coinfected with hepatitis c virus (hcv). this accelerated liver disease is presumed to be due, in part, to hn-related immune suppression. hcv quasispecies variability is likely a marker of immune pressure on the virus. hyuotheses: . hcv quasispecies variability is decreased in those with hiv coinfection, and . treatment with highly active antiretroviral therapy (haart) is associated with increased variability, methods: hcv-infected patients with or without hiv were enrolled in a cross-sectional study. patients with genotype infection and a parenteral risk factor for hcv were included. those with injection drug use (idu) were estimated to have acquired hcv years after the onset of idu. alcohol use was determined using standard questionnaires. haart data were collected from the hiv clinic's database. only those with complete records were included. hcvquasispecies variability was determined using clonal frequency analysis of the second envelope gene hypervariable region. quasispecies heterogeneity was characterized according to complexity (the normalized number of unique quasispecies variants within a sample) and diversity (the average genetic distance between individual variants within the same sample). groups were compared using the student t-test for unequal variances, anova and the wilcoxon rank-sum test where appropriate. linear regression was used to assess the independent effects of hiv infection and haart on complexity and diversity. results: to date quasispecies data are available on patients, of whom are hivpositive. as expected, hiv+ subjects had significantly lower cd counts (mean vs cellslul, p<.oool). mean age and hcv duration were significantly lower in the hiv+ group ( . vs . , p=. , and . vs . years, p=.oo , respectively). the groups did not differ with respect to alcohol use over their lifetime or in the months prior to enrollment. quasispecies complexity and diversity are detailed in the table below. while there was no significant difference among groups by anova (p=. ), the hiv+ subjects not on haart had less complexity and diversity than the other two groups, and differences between groups and and groups and were significant (table) . both hiv infection and haart were significantly and independently associated with complexity in a linear regression model; hiv infection predicted decreased complexity (p= . ), while haart predicted increased complexity (p=. ). in addition, higher alcohol use in the prior months was independently associated with decreased complexity (p=. ). hiv infection and haart also were independently associated with decreased and increased diversity, respectively (p=.ool and. ). the cd count did not predict complexity or diversity, and the effect of haart appeared to be independent of the cd count. conclusion: hcv coinfection with hiv is associated with decreased hcv quasispecies complexity and diversity. while this effect is not completely reversed by haart, treatment of hiv is associated with significantly increased hcv complexity and diversity in our sample. furthermore, the effect of haart on hcv quasispecies variability appears to be independent of its effect on the cd count. these findings suggest that the immune pressure on hcv is enhanced in hcvlhn coinfected individuals who are on haart when compared to those who are not on haart. diversity" group (mean .t sd) (mean f sd) in the majority of cases, hepatitis c virus (hcv) becomes chronic and is associated with impaired viral-specific t cell immune responses; the mechanisms underlying viral persistence and lack of protective immunity are poorly understood. considering dendritic cells (dcs) play critical roles in initiating and modulating immune responses, we explored the effect of hcv proteins on dc genetic expression, phenotype, and function. methods: human dendritic cells (dcs) were generated following plastic adherence of monocytes from normal subjects and culture with gm-csf and il- . autologous non-adherent cells were infected with vaccinia constructs expressing various hcv proteins (core, nssa, ns b) or an irrelevant protein / -galactosidase @-gal) as the control. following induction of apoptosis with uv irradiation, these vaccinia-infected cells were co-cultured with dcs. the phenotype, gene expression profiles and functions of the dcs were analyzed. dcs were evaluated after , , and hrs of co-culture as well as after maturation with a cytokine cocktail (tnf-a, il-lp, pge , il- ) for an additional hrs. results: by facs analysis, we found no alteration in expression of co-stimulatory markers (cdso, cd , cd ,) or mhc class i or i molecules. by elispot assay, we found that the recall response of cd t t cells for cmv following pulsing of hcv-dc or control (p-gal)-dc was also not altered. however, between - % of the genes probed in the bd clontech nylon array were differentially regulated. real time pcr has confirmed increased expression of scyc (lymphotactin) in the presence of ns a in normals as well as separate biological replicates. both clontech and affymetrix gene arrays revealed ns a-induced increases in expression of il- , a chemokine with pro-inflammatory properties. functional assays reveal that dcs which had engulfed apoptotic cells containing hcv core, ns a, or ns b demonstrated comparable ability to macropinocytose as measured by uptake of dextran-fitc or albumin-fitc when compared to p-gal-dcs. however, following uptake, the hcv-dcs died at a significantly greater rate than p-gal-dcs (- %). conclusions: taken together, these results demonstrate that following engulfment of apoptotic cells containing hcv proteins, differential gene expression occurs in human dendritic cells, although the expression of mhc and costimulatory molecules and ability to stimulate memory (cmv) responses remain intact. the mechanisms underlying the surprising induction of death of dc-hcv in the macropinocytosis assay is currently under further investigation. institutes of health, bethesdu, md steatosis is a common histological feature of chronic hepatitis c virus (hcv) infection. the cause of hepatic steatosis in hepatitis c remains to be elucidated, but its association with obesity, diabetes and hyperlipidemia suggests an underlying metabolic dysfunction similar to that found in patients with nonalcoholic steatohepatitis (nash). additionally, recent data suggest an association between body mass index (bmi) and the degree of hepatic steatosis and fibrosis in hcv infection. insulin resistance (ir) is a key factor in the pathogenesis of nash and may play a similar role in the steatosis seen in chronic hepatitis c &: to compare the prevalence and severity of hepatic steatosis, visceral adiposity, j r and their relation to disease severity in african americans (aa) vs caucasians (ca) with genotype hcv infection being considered for anti-viral therapy. methods: virahep-c is a prospective study of response rates to peginterferon and ribavirin in treatment-na'ive aa vs ca patients with genotype hcv infection. all patients underwent liver biopsy within months of enrollment. biopsies were read blindly by a central pathologist and scored for inflammation and fibrosis using the histologic activity index [i-iaii and for steatosis ( - +) and features of steatohepatitis (zone ballooning, zone perisinusoidal fibrosis and mallory bodies). waist circumference and waist-hip ratio were measured as determinants of visceral adiposity. ir was determined using fasting glucose and insulin levels based upon the homa method. results: to date, patients have been enrolled and have complete liver biopsy information ( aa and ca). steatosis was present in patients ( %) with similar rates among aa ( %) and ca ( % ca). grades of steatosis were also similar between aa and ca + ( % vs %), + ( % vs %), + ( % vs %) and + ( % vs %) (p = ns). patients with steatosis had significantly higher values for homa index ( . vs . , p<o.ol), total hai score ( . vs . , p<o.oool), hai inflammation score ( . vs . , p<o.oool), waist circumference ( . in vs . inches, p<o.oool) and bmi ( . vs . , p<o.oool). there were no significant differences in amount of alcohol consumption between patients with and without steatosis. spearman's non-parametric correlations were performed to assess associations between the ordinal variables (scores for fat, steatohepatitis ballooning degeneration + perisinusoidal fibrosis + mallory bodies], total hai, inflammation and fibrosis) and one continuous variable (homa index). waist circumference, waist-hip ratio, bmi, homa index, total hai, hai inflammation and hai fibrosis all correlated with both the fat score and steatohepatitis score (p<o.ol). median homa index levels were significantly greater among aa than ca ( . vs . , p< . ), but step-wise logistic regression showed that race was not an independent predictor of steatosis after controlling for alt, ast, waist circumference, waist-hip ratio, bmi and homa index. conclusions: in patients with chronic hepatitis c and genotype , steatosis and steatohepatitis correlate strongly with the presence of insulin resistance and visceral adiposity. race is not an independent predictor of steatosis after controlling for other factors. bulk populations of antigen-presenting dendritic cells (dcs) contain myeloid dcs and plasmacytoid dcs. myeloid dcs polarize t lymphocytes to thl phenotype that produce interferon ( fn)- . plasmacytoid dcs produce abundant amounts of interferon ( fn)-alpha. both ifn-?-producing t cells and adequate amounts of ifn-alpha are essential for viral clearance. in patients with chronic hepatitis c virus (hcv) infection, dcs are infected with hcv, which is presumed to have a role in viral persistence. however the functional implication of this has not been addressed. the aim of this study is to evaluate the functional capabilities of both myeloid and plasmacytoid dcs in patients with chronic hcv infection. myeloid dcs were defined as linage-, cdllc+, hla dr+ and cd -cells, and plasmacytoid dcs as lineage-, hla dr+, cdllcand cd + cells among the peripheral blood mononuclear cells (pbmcs). the frequencies of myeloid and plasmacytoid dcs were measured in patients with chronic hcv infection and agematched control subjects by four-color flow cytometry. to assess the production of ifn-alpha by plasmacytoid dcs, these were cultured with graded doses of herpes simplex virus- ( - plaque-forming unitsldc). the frequencies of plasmacytoid dcs expressing intracellular ifn-alpha among total plasmacytoid dcs were counted by two-color flow cytometry. to evaluate the capacity of myeloid dcs to induce th polarization, dcs were cultured with autologous naive t cells for days. polarization of t cells to thl phenotype was assumed when na'ive t cells expressed intracellular ifn-y. steatosis has been reported to occur in up to % of liver biopsies in patients with chronic hepatitis c virus (hcv) infection.risk factors for steatosis include alcohol abuse and those found in nonalcoholic steatohepatitis: obesity, increasing age, insulin resistance and hypertriglyceridemia. studies have shown an association between genotype and hepatic steatosis in chronic hcv infection, and others have found an association between steatosis and fibrosis stage. previously published studies have been based primarily on cohorts of clinic referral patients and often have not analyzed for confounding variables. we herein report a population-based study of hepatic steatosis in hepatitis c. methods: in a study of alaska nativestamerican indians with chronic hcv infection, patients underwent at least one percutaneous liver biopsy as part of evaluation for treatment. all patients had a positive hcv rna by pcr and were negative for hbsag and hiv. all biopsy slides were reviewed by a pathologist blinded to patient identity, demographic, clinical and biological data. histologic activity was scored using the knodell system and fibrosis using the ishak system. steatosis was graded as , ( so%), ( - %), and (> %). patients were analyzed for a) gender, age, estimated length of infection and bmi at the time of biopsy, b) genotype, current ethanol use, hcv rna within year of biopsy, c) alt within days of biopsy, and d) histologic activity and fibrosis found on biopsy, using univariable and multivariable analysis. results: steatosis was found in / ( %) of liver biopsies; ( %) had grade ; ( %) grade , and ( %) grade . genotype distribution was as follows: genotype ( %), genotype ( %), and genotype ( %). steatosis was found in % ( ) of genotype , % ( / ) of genotype , and % ( / ) of genotype patients (p = . ). grade or was found in % ( / ) of genotype , % ( / ) of genotype , and % ( / ) of genotype patients (p = . ). there was no significant association found between steatosis and gender (p = . ); age at biopsy date (p = . ); alt (p = . ); histologic activity (p = . ), viral load (p = . ) and estimated length of infection (p = . ). there was a significant association of steatosis with bmi (< . ), fibrosis score (p < . ) and current ethanol use (p = . ). bmi occurred in % ( / ) of genotype , % ( / ) of genotype , and % ( / ) of genotype patients (p = . ). after controlling for bmi, there was no significant relationship found between steatosis and genotype. for bmi < , % ( / ) of genotype , % ( / ) of genotype , and %( / ) of genotype patients had steatosis (p = . ). for bmi , % ( ) of genotype , % ( ) of genotype , and % ( / ) of genotype patients had steatosis (p = . ). multivariable analysis was performed on variables (p . in univariable analysis) and genotype. these included age (categories < , - , - , so+), bmi (< , +), current ethanol use (yes, no), ishak fibrosis score ( - , , and estimated length of infection ( , - , - , + years) . a significant association with steatosis was found in variables: ishak fibrosis score (or . , % ci . - . , p = . ), bmi -> (or . , % ci . - . , p = . ), and current ethanol use (or . , % ci . - . , p = . ) after adjustment for hcv infection length, which itself was not statistically significant (p = . ). conclusion: contrary to a number of previously reported studies, this population-based study did not find an association between steatosis and genotype, including genotype in chronic hcv infection after multivariable analysis. we did find that ishak fi-brosis score, bmi and current ethanol use were associated with steatosis after adjustment for hcv infection length, but age and histologic activity were not associated. infection results in necroinflammatory liver disease characterized by the insidious progression of hepatic fibrosis and loss of functioning hepatocyte mass. a cell-mediated immune response with prominent lymphocytic infiltration of liver is thought to play a major role in pathogenesis, although little is known about the molecular mechanism underlying the liver injury associated with this viral infection. on the other hand, non-immune mechanisms have also been proposed as another mechanistic candidate for such sophisticated processes. the recent intensive interest is that the expression of hcv proteins seemingly alters lipid metabolism and transport in the liver in association with the reactive oxygen species (ros) mediated carcinogenesis, although the molecular basis of underlying mechanisms as well as responsible elements of hcv gene for this is yet to be established. thus, the aim of this study was to clarify whether hcv core protein expression directly alters lipid metabolism-relating enzymes to (cause steatosis, especially focusing on nuclear receptors and atp binding cassette transporter proteins expression. methods: . the complementary dna clone of full length hcv core (aa - ) was derived from serum of hcv i b patient by reverse transcription and nested polymerase chain reaction, and hcv-core expression plasmid (pcag-hcvcore) was prepared by a standard procedure. control plasmid was also prepared with p-galactosidase (pcag-lacz). . hepg cells were transfected with pcag-hcvcore or pcag-lacz, thereafter harvested for enzymatic triglycerides (tg) assay, hcv core antigen rneasurement by elisa at h or h. also, lipid metabolism-related enzymes mrna expression was estimated by semi-quantified rt-pcr; mitochondrial and peroxisomal fatty acids ,&oxidation, o-oxidation, abc transporters (mdr , mrp , bsep), microsomal tg transfer protein (mtp), ldl receptor, and nuclear receptors. resultss: . hcv core expression was evidenced at and h at the similar level. . hepatic tg was increased in hcv core expressing cells, along with down-regulation of carnitine palmitoyl transferase a(cptla), a precious protein in liver mitochondrial fatty acid@-oxidation ( % at h, % at ) and up-regulation of acyl-coa oxidase (acol), a precious protein in peroxisomal fatty acidso-oxidation ( % at h). cyp a , a precious protein in microsomal o-oxidation, and mtp, a precious protein in vldl assembly, were not detected or unchanged. superfamily (sf ) helicases and helicase-like proteins share conserved motifs. alignments reveal that several additional conserved motifs are present in the sf helicase encoded by the hepatitis c virus (hcv). the roles of two such motifs are examined here using structure-based site-directed mutagenesis. the first motif (yrgxdv) forms a loop that connects sf helicase motifs and , at the tip of which is arg . when arg is changed to ala, the resulting protein retains a nucleic acid stimulated atpase but cannot unwind rna, unwinds dna poorly, and does not translocate on ssdna. dna and rna stimulate atp hydrolysis catalyzed by r a like the wildtype but the mutant protein binds dna more weakly than wildtype both in the presence and absence of a non-hydrolysable nucleotide analogue. thus, this "arg-clamp" motif anchors the protein on nucleic acid enabling processive unwinding. the second motif (dfsldptf) forms a beta-loop between sf motifs and that connects domains and . when f in this "beta-arm" is changed to ala, the resulting protein is devoid of all activities. when f is changed to ala, the protein retains nucleic acid stimulated at-pase, but unwinds dna and rna poorly. in this case, uncoupling of atp hydrolysis and unwinding is due to the fact that the f a mutant does not release dna upon atp binding like the wildtype. the f a mutant also has a lower melting temperature than the wildtype indicating the hydrophobic pocket formed by the beta-arm and residues in domain stabilizes the protein. data support an inchworm model for helicase action and identify two new potential sites for rational hcv drug design. this work was supported by the aasld liver scholar award from the american liver foundation. tat is an early gene product of hiv- that is essential for replication and viral gene expression. this transactivating protein is secreted by hiv-infected cells and taken up by neighboring cells. tat modulates expression of specific cellular genes and may be a key player in the interactions between hiv and other infections. one of the most common coinfections observed among hiv patients is hepatitis c virus (hcv). if hiv tat has an effect on hcv replication, this could help explain the rapid development of liver disease and cancer among hiv patients. aim this study was designed to test the hypothesis that tat alters hcv replication. methods: soluble tat was added to the media of huh- cells containing a hcv replicon. replicon replication was quantitated using a ribonuclease protection assay. using an ltr-luciferase plasmid to transfect hela cells, we developed a series of controls outlining the relative oxidative state of the tat protein. results exposure to tat protein in the reduced state substantially increased hcv replicon replication. oxidized tat was found to have no significant effect on the replication of the replicon. the increase was dependant on length of exposure to tat and the concentration of tat. present work: further assays seek to define the nature of the effect by tat on hcv replication. conclusion: tat may upregulate hcv replication directly or indirectly, and thereby play a role in modulation of hcv infection and pathogenesis. elucidating the complex interactions between hiv and hcv will be critical in evaluating and developing workable treatments for the increasing number of coinfected hiv/hcv persons with liver disease. acknowledgements: this research was supported by nih grants ai , ai , ai , ca and ca . we have found previously that expression of hepatitis c virus proteins in cell lines or in the liver of transgenic mice inhibits interferon alpha induced signaling through the jak-stat pathway (heim et al., j virol, , : and blindenbacher et al., gastroenterology, , ) . the aim of the present study was to investigate if the same inhibition takes place in livers cells of patients with chronic hepatitis c. from february to april , all patients with chronic hepatitis c referred to the outpatient liver clinic of the university hospital basel were asked for their permission to use part of the liver biopsy for this study. for non-hcv controls, patients that underwent ultrasoundguided liver biopsies of focal lesions (mostly metastasis of carcinomas) were asked for their permission to obtain a biopsy from the normal liver tissue outside the focal lesion. the protocol was approved by the ethical commission of basel. written informed consent was obtained from all patients that agreed to participate in the study. after removal of a to mm long biopsy specimen for routine histopathological workup for grading and staging of the liver disease, the remaining to mm long biopsy cylinders were immediately incubated in pbs or pbs with human interferon alpha (lo iulml) for to minutes at °c. the samples were then used for the preparation of cytoplasmic and nuclear extracts and in some cases for immunofluorescence studies. in a first part, the ex vivo stimulation of biopsy tissue was validated. biopsy samples of patient with different degrees of fibrosis were incubated for , , , and minutes, and the nuclear translocation (a surrogate marker for activation) of statl was visualized by immunofluorescence. we found a transient activation of stat , with a peak after minutes of stimulation with interferon alpha. the signal was shut down in all biopsies after minutes. interferon alpha diffused readily in the biopsy cylinders regardless of the degree of fibrosis. in the second part, consecutive biopsies of patients with chronic hepatitis c and consecutive control biopsies of patients who underwent ultrasound-guided biopsy of focal lesions in otherwise healthy livers (mainly liver metastasis of carcinomas) were used for semiquantitative assessment of interferon alpha induced statl activation using gel shift assays. we found a signhcant inhibition of statl dna binding in patients with chronic hepatitis c compared to controls. as in our previous work with hcv protein expression systems, statl phosphorylation was not impaired in human liver biopsies from patients with hepatitis c. we conclude that interferon induced intracellular signaling can be analyzed semi-quantitatively in human liver biopsies ex vivo by gel shift assays. using this newly validated method, we found that signaling is impaired in liver biopsies from patients with chronic hepatitis c. the block in stat signaling is at the level of dna binding in the nucleus, whereas stat activation at the interferon receptor functions normally. methods we studied the intrahepatic and peripheral hcv-specific cds+ t cell response in a cohort of hla-a positive chronically hcv infected patients by tetramer staining, intracel-m a r ifngamma staining and cfse labeling. in addition, viral amino acid sequences corresponding to the four ctl epitopes used in the study were deduced by nucleotide sequence analysis to assess the potential role of viral escape variants. results ( ) substantial higher numbers of hcv specific cd + t cell responses were detectable in the liver compared to the peripheral blood, suggesting compartmentalization at the site of infection. in addition, intrahepatic cds+ t cells were multispecific whereas peripheral hcv specific cds+ 'i' cell responses were primarily monospecific. these results suggest, e.g., the persistence of hcv-specific t cells at the site of disease or the direct priming of t cells in the liver. ( ) epitope-specific cd + t cells that were detectable in peripheral blood as well as liver were characterized by a good peripheral proliferative capacity after peptide specific stimulation suggesting that proliferation is a prerequisite of peripheral virus-specific cd + t cells to accumulate in the liver. this is further supported by the observation in one patient that the lack of proliferation of virus-specific cd + t cells in the peripheral blood was associated with the absence of the same cds+ t cell response in the liver. ( ) despite the strong accumulation of hcv specific cd + t cells in the liver, the majority of those cells was unable to secrete cytokines after peptide specific stimulation indicating that dysfunction of intrahepatic hcv specific cd + ' i cells might contribute to viral persistence. importantly, ifn gamma producing tetramer positive cd + t cells were only detectable in patients with low viral titers or in patients with high viral titers but with sequence variations in the corresponding viral epitope, suggesting viral escape. the relative contribution of t cell dysfunction versus viral escape to viral persistence is not known. it is important to note, however, that both mechanisms can operate within the same patient. insulin resistance (ir) is a frequent feature in chronic hepatitis c while risk factors of steatosis are body mass index in patients infected with genotype and viral load in those infected with genotype . in patients with chronic hepatitis c, we adressed the following issues: ) is ir the cause or consequence of steatosis and fibrosis ? ) what are the risk factors of ir ; ) does ir play a role (and to what extent) in the occurrence of steatosis ? ) is ir involved in the progression of fibrosis ? therefore, this study was designed to assess relationships between ir, steatosis and fibrosis according to hcv genotypes in non diabetic patients. methods: non-diabetic patients with biopsy proven non-cirrhotic chronic hepatitis c had fasting serum glycemia and insulinemia measurements. ir was evaluated by using homa model assessement. groups of patients were defined according to hcv genotypes ( or ) and degree of steatosis (absent or mild vs moderate to severe). results: the groups were similar in terms of age, sex ratio, bmi and disease duration. prevalence of ir (homa higher than . ) was significantly higher in genotype patients with steatosis than that of other patients ( % vs , and % respectively, p=o.oool). ir was significantly associated with extensive fibrosis in genotype patients (p= . ) but not in genotype patients. among genotype patients, independent parameters associated with ir were age (p= . ) and steatosis (p=o.o ) but not fibrosis. independent risk factors for steatosis in genotype and genotype patients were dr (p=o.o ) and viral load (p= . ) respectively. steatosis was associated with significantly higher fibrosis score whatever the genotype (p=o.o ). among genotype patients, the median progression rate of fibrosis was significantly higher in those having steatosis and ir together than in other patients ( . vs . , p = . ). conclusions : in non-diabetic patients with non-cirrhotic chronic hepatitis c ) steatosis and fibrosis are associated with ir in genotype patients but not in genotype patients, ) among genotype patients, ir mainly depends on age but not fibrosis ) ir is a major risk factor of steatosis in genotype patients, ) the combination of steatosis and insulin resistance is a risk factor of fibrosis progression. these results suggest that ir is not the consequence but rather the cause of steatosis and fibrosis progression. . however, in the first month of life, in of these newborns, hcv rna was only detected in pbmcs and not plasma. in neonates, the sscp band patterns of pbmc-derived viral sequences were different from maternal sequences in serum or pbmc; however, they were identical to hcv rna negative strand amplified from mothers' pbmc. the latter strongly suggests that the infection was transmitted through maternal infected pbmcs. another infant harbored different hcv strains in serum and pbmc; both strains were present in the mother's serum. only of hcv-rna positive children developed hcv antibody at one year. hiv infection was found in out of hcv rna-positive and in out the hcv rna-negative infants (ns). conclusions: we have found that ) hcv strains infecting neonates may be derived from strains residing in maternal pb-mcs. ) hiv+ pregnant women who were hcv rna positive in pbmcs were more likely to transmit hcv to their infants. these data suggest that one mechanism for perinatal transmission of hcv is maternal-fetal transfusion of hcv-infected pbmcs late in pregnancy or during labor and delivery. additionally, hcv infected neonates may have a limited ability to develop hcv antibodies in the first year of life and thus there may be an underestimation of the prevalence of hcv infection by anti-hcv determination among children born to hcv rna positive mothers. (hcc) in this clinical setting is very high. however, the molecular mechanisms of how hcv related proteins may contribute to hcc is not well defined. hcv core protein, a viral structural protein, has been implicated in tumor development. the goal of this study was to characterize the transcriptional regulation induced by core protein expression with respect to generations of a malignant phenotype. meth ds:a cdna fragment encoding hcv core protein of l b genotype was subcloned into the ptre hyg vector and co-transfected with ptet-on vector (clontech) into a human huh hepatoma derived cell line using polyamine (mirus) as a carrier. stably transformed clones were selected by growth in culture medium containing g and hygromycin. clones positive for hcv core protein expression were screened by immunoblotting from cells grown in the presence and absence of tetracycline. one clone with core expression tightly regulated by tetracycline was chosen for further analysis. cell proliferation was evaluated by a modified mtt assay, at , , , and hours after induction of core protein expression. twenty-four hrs after hcv core expression, the cellular transcriptional changes were analyzed by microarray analysis using human genome u array sets (affymetrix). the microarray data were validated by real-time pcr on selected genes. results hcv core protein expression was tightly regulated by the presence of tetracycline and the protein levels were dependent on the amount of the tetracycline present in the culture medium. core protein expressing cells showed significant increase in growth compared to non-expressing cells at , , , and hours after addition of tetracycline indicating a proliferation stimulus provided by core protein. microarray analysis using human gene chip u revealed that of , genes were significantly changed (> fold), with up-and down-regulated. this screen was quite informative since genes involved in regulation of cell proliferation ( genes) and apoptosis ( genes) were changed respectively. conclusions: enhanced growth of liver-derived cells by hcv core protein is due to cellular transcriptional changes induced upon core expression; two major molecular pathway(s) are involved ) those involved in celi growth control and ) those involved in cell survival. such gene regulation by hcv core protein is important to the molecular pathogenesis of hcc daudi-cd cells were infected with pnl - , a t-tropic hiv molecular clone. hours later, uninfected and hiv-infected cells were incubated with either % high titer hcv-positive patient serum, % hcv-negative patient serum, or % fbs (no human serum). infections were continued for , , , and days; cells were harvested, stained with annexin v and propidium iodide, fixed, and apoptosis was measured by flow cytometry. annexin v is a marker of early apoptosis, and propidium iodide indicates cell death either by apoptosis or necrosis. results: six high titer hcv-positive sera and six hcv-negative sera were tested in five separate experiments, and in each case, the percentage of viable cells was higher in the hivihcv-coinfected cells compared to those infected with hiv only. to further elucidate dynamics of the course of coinfection, we did a time course consisting of harvests every eight hours beginning at day (the earliest point at which apoptosis had been observed in prior experiments) and continuing until day plus hours. the results of the five harvests from day plus hours to day plus hours are shown in the figure below. the percentages of viable cells and those undergoing apoptosis are shown as measured by flow cytometry. during this -hour interval the percentage of viable (negative for apoptosis) cells fluctuated very little in untreated cells, or in cells incubated with hcv-positive serum, or even in hn-infected cells incubated with hcv-positive serum. in striking contrast, hiv-infected cells that were incubated with hcv-negative patient serum dropped to only % viability by the end of the hours, compared to % viability in the hiv-infected cells incubated with hcv-positive patient serum. this effect was not observed when apoptosis was induced by either of two chemical inducers of apoptosis in daudi cells: peroxisome proliferator-activated receptor gamma ligand and calpain inhibitor . next, we sought to determine if the effect of apoptosis inhibition was dependent on hcv particles. when h ninfected cells were kept separate in culture from hcv-infected cells by a membrane that prevented virus passage, apoptosis in the hn-infected cells was still inhibited. conclusions: collectively, these results suggest that in our experimental conditions: ) hcv-positive sera, but not hcv-negative sera provide a protective effect to hiv-induced apoptosis in daudi-cd cells, and ) a cellular factor induced by the presence of hcv, rather than hcv particles themselves, is responsible for the inhibition. methods: serum and pbmc samples from an individual with acute hcv-infection (genotype l a ) were collected every two to three months over a time period of months beginning months after infection. pcr amplification of vrna using a set of overlapping primers for the entire hcv genome was performed and pcr-products population sequenced using an abi automated sequencer. cd + t cell responses were defined using an ifngamma elispot assay using an overlapping synthetic peptide set spanning the whole hcv-genome (genotype la). based on the hla-type the complete viral genome was also screened for putative cds+ epitopes using a web based epitope prediction program (syfpeithi). results: elispot screening with the whole hcv-genome peptide set detected only two ex-vivo ifn-gamma responses (ns - and ns b - ), although neither epitope was associated with the development of mutations. however, over the month period of observation we detected a total of mutations throughout the genome ( in el, in e , in ns , in ns , in a ns and in ns ), in addition to multiple changes in the hypervariable regions. interestingly, only five of these mutations resided within known hla-restricted optimal epitopes. however, / mutations ( %) were located within predicted epitopes based on known hla-binding motifs of the subject. in order to begin to define the rate at which these mutations develop an intermediate time point of months was also sequenced. interestingly, / ( %) of the mutations had already occurred by this time. it is likely that at least some of these mutations are the direct result of immune pressure exerted through cd + t lymphocytes. we are currently generating peptide-specific cds+ t cellines against these regions exhibiting viral evolution, providing an opportunity to determine which of these regions are associated with previously undescribed cd + responses. conclusions: hypothesizing that evolving mutations in hcv might be the result of immune pressure by ctl, longitudinal full length sequencing of hcv may represent a powerful tool to define additional hcv-specific cd + t cell responses, especially those exerting substantial selective pressure. using this approach we were able to identify a number of candidate regions where cd + t cell responses may be present and driving viral evolution. determining the extent to which viral escape from cd + responses occurs during hcv infection will elucidate its overall impact in preventing proper control of hcv. indicate that the main open reading frame of hcv contains rna structures in the corelarfp (alternate reading frame protein) and nssb (polymerase) genes. we hypothesized that one or more of these structures are cis-acting replication elements (cre)s. methods. we used custom software, thermodynamic rna folding programs, and classical comparative phylogenetic analysis to build secondary structural models. we then used directed mutagenesis in the subgenomic replicon system to seek stem-loop elements in ns b that are required for viability. the mutations we introduced maintained the sequence of the polymerase protein, i.e., they were silent, synonymous codon substitutions. structural probing was carried out on replicon rna. rnase t and lead (ii), and nuclease v , were used to identify loops and helices, respectively. results. mutations in ns bsl . blocked replication, indicating that this novel structure is an essential cis-acting replication element (cre the aims of this study were: ) to correlate mean alt level with hcv rna by pcr positivity or negativity, ) to compare clinical, demographic and risk factor data as well as viral load and genotype between persons with pnalt, persistently elevated alanine transaminase (pealt) and fluxuating alanine transaminase (fluxalt) who had at least years of data available. methods: for each person in the cohort from whom pcr results were available (n= ), we calculated the mean level of all alts measured over a -year period after enrollment. for a subset of the cohort, we selected persons who met the following criteria: positive pcr tests year apart with the first positive pcr occurring prior to date of the first alt and a minimum of alt levels measured over the subsequent years with a minimum interval of month between alt measurements (n= ). we defined a person as having pnalt or pealt when of alt levels were normal or elevated, respectively, during the -year follow-up period. persons who did not fit into either of the above categories were defined as fluxalt. we reviewed clinical data via chart review, demographic and risk factor data via interviews, as well as data on viral load (performed by branched dna assay version . ) and genotype (restriction polymorphism analysis background: hcv infection rarely presents acutely, but when it does it offers a potential early "window" for effective therapy. it has been suggested that such therapy is efficacious due to enhanced activity of t cell responses, which are most active during acute disease. methods: subjects with acute hcv infection were comprehensively mapped for cd + t cell responses with an interferongamma elispot assay using overlapping peptides, spanning the entire hcv polyprotein. responses were confirmed by establishing peptide-specific t-cell lines and intracellular cytokine staining. the screening for hcv-specific cd + t cell responses was repeated during and after therapy. responses detected in the screening assay were longitudinally studied before, during and after therapy using single peptide elispot ,as well as tetramer assays. we also studied cd proliferative responses over time in some subjects using recombinant hcv proteins. results: cd + t cell responses were detected in / subjects before therapy was initiated and were typically multispecific, with up to epitopes targeted. after the start of therapy, frequencies of hcv-specific cd + t-cells declined following suppression of hcv viremia. therapy also did not increase the breadth of the hcv-specific response as no additional specificities were detected at later timepoints, when elispot screening was repeated. in / subjects viral relapse occurred after therapy was stopped, with subjects being retreated successfully. relapse was not predicted by the presence or absence of hcv-specific cd + t-cell responses. during viral relapse, a vigorous expansion of pre-existing hcv-specific cd + t-cells was observed for most specificities, with single responses reaching almost % of cd + t cells as measured by tetramer staining. these responses were unsuccessful in containing hcv. in the two subjects who were retreated after viral relapse, we observed the identical pattern of declining cd + t cell responses as in the first treatment course. in contrast to cd + t cell responses, cd proliferative responses usually became more vigorous after virus was suppressed on therapy. however, such responses also did not protect from viral relapse. conclusions: these data suggest that although multispecific functional cd + t cell responses may be present during acute disease, this does not predict a successful outcome of antiviral therapy. rather than boosting cd + t cell responses, therapy and viral suppression appear to lead to their attenuation, even though cd + t cell responses may be restored. figure) in the fibrous septum of portal tract in cirrhotic liver. significantly less fltl immunopositivity occurred in nd liver. hepatocytes were negative for fltl. conclusion: the greater de of genes involved in immune activation, fibrosis, cellular proliferation and cell signalling indicated that these processes are more active in the cirrhotic liver that has developed hcc than the cirrhotic liver without hcc development. plgflfltl signalling has an important role in neo-vascular development within organs. the decreased expression of plgf and its receptor flt in cirrhosis with hcc implies that vascular proliferative signals although normally active in cirrhosis itself may be decreased in cirrhotic livers with hcc. prospective analysis of cirrhosis prior to hcc development is needed to indicate whether these findings represent a premalignant phenotype. background: hepatitis c virus (hcv) infection often leads to chronic liver diseases including liver cirrhosis and hepatocellular carcinoma (hcc). at least hcv proteins have been identified, which serve as viral structural and non-structural proteins required for viral replication and virion formation. several viral proteins have been implicated in hcv persistence and the development of hcc. we have previously reported that the ns protein inhibits gene expression f+om various cellular and viral promoters in liver and non-liver derived cells. thus, expression of endogenous cellular proteins was significantly reduced in ns expressing cells. in this regard, the ns protein was recently found to be an inhibitor of the pro-apoptotic cide-b protein and therefore may contribute to hepatic oncogenesis. to understand the molecular basis of repressed gene expression, we constructed successive deletion mutants of the ns protein and tested their effect on luciferase expression driven by cmv promoter. meth-ods: the cdnas encoding the full-length ( - ), n-terminal part ( - ) and internal parts of ns were generated by pcr and cloned into the pcr . vector for expression under control of the cmv promoter. a liver-derived huh- cell line was co-transfected with a plasmid encoding the luciferase reporter gene and plasmid encoding various deletion mutants of the ns gene. inhibition of gene expression was measured by luciferase activity assay at day two after transfection. cell viability was also evaluated as well. results: deletion constructs - , - , - and - exhibited a significant inhibitory effect on luciferase activity comparable to the fulllength ns protein, whereas the deletion constructs - and - lost the inhibitory effect. therefore, the minimal amino acid residues required for the inhibition of gene expression by this viral non-structural protein was mapped to residues - . interestingly, this region partially overlaps with the binding site of the ns to a newly identified cide-b pro-apoptotic protein. il plays an essential role in the antagonism of t helper differentiation and in the induction of antiviral host defence. we postulated that genetically determined attenuation of il production could provide a plausible immunological mechanism able to determine outcome of hcv infection and have investigated a recently described polymorphism in the il b gene. methods: we have extracted genomic dna from whole blood taken from hcv antibody positive patients. patients had chronic hcv with detectable hcv rna and had spontaneously resolved infection, testing hcv rna negative on several occasions. genotyping for a single nucleotide polymorphism (snp) at position (a c) on the ill b gene was performed using polymerase chain reaction and restriction digest. maximal in-vitro il- production by cultured mononuclear cells in response to sac (staph aureus cowan) stimulation was determined in cases by elisa. results: of the hcv rna positive cases ( %) were homozygous for the 'a' allele, ( %) heterozygous 'ac'; and ( %) were homozygous 'cc' whereas of the hcv rna negative cases ( %) were 'aa', ( %) were 'ac'; and ( %) were 'cc'. hcv rna negative cases were significantly more likely to be heterozygous for lac' than hcv rna positive cases (p= . ). of the patients studied for il- production, were genotype 'aa' and 'ac'. were hcv rna positive ( genotype lac' and 'aa') and hcv rna negative ( genotype 'aa' and 'ac'). maximal il- production with sac stimulation was lower in the genotype 'aa' cases (mean units) than in the genotype 'ac' cases (mean units) (p= . ). hcv rna positive cases produced significantly less il- than did hcv rna negative cases (mean units compared to units, p= . ). comparison of hcv rna positive or negative cases only, revealed a trend for higher maximal il- production with genotype 'ac' regardless of hcv outcome. conclusion: cases heterozygous for the variant 'c' allele at position of the il- p gene are significantly more likely to be hcv rna negative than those homozygous for the 'a' allele. recent data found carriage of the variant 'c' allele to be associated with greater il- production capacity and our data from a small number of cases supports this. genetically influenced enhancement of il- production appears to be a factor influencing the outcome of hcv infection. we have previously demonstrated that hepatitis c virus (hcv) core protein expression in huh- hepatoma cells increased reactive oxygen species (ros) derived from mitochondria without inducing apoptosis. the aim of this study was to investigate whether hcv core protein inhibits the rosassociated apoptosis induced by deoxycholic acid (dca). methods:: we measured ros level and -hydroxy- '-deoxyguanosine ( -ohdg) content, and evaluated apoptosis in the presence or absence of dca ( pm), using a human hepatoma-derived cell line with tightly regulated hcv core protein expression under the control of a tet-offrm promoter. cells were incubated with pm of chloromethyl ', '-dichlorodihydrofluorescein diacetate for min for measurement of ros. cellular -ohdg content was quantified with enzyme-linked immunosorbent assay. fragmented nuclei were assessed with ', -diamidino- -phenylindiole staining and dna fragmentation was evaluated by genomic dna laddering. the degree of apoptosis was quantified with enzyme-linked immunosorbent assay. we also examined whether the general caspase inhibitor (zvad-fmk) inhibited the ros-associated apoptosis induced by dca. in some experiments, cells were incubated with wm of ursodeoxycholic acid (udca) in addition to dca. the experiments were repeated or times. results:: strong core expression was detected hours after withdrawal of tetracycline from the culture medium. the expression of core protein increased the basal ros level ( . . -fold, p< . ) and -ohdg content ( . i . -f ld, p< . ) of huh- - cells without inducing apoptosis. dca stimulation produced a . -fold increase in ros content in the presence of core expression and a . -fold increase in the absence of core expression. similarly, the -ohdg content was significantly increased by dca regardless of hcv core expression ( . -fold, p= . for core expression, . -fold, p=o.o for core non-expression). nevertheless, hcv core protein significantly suppressed the ros-associated apoptosis induced by dca (w . ). also apoptosis induced by dca was almost completely inhibited by zvad-fmk. udca significantly decreased the ros (p< . ) and the -ohdg content (p=o.oos) in the core-expressing cells and attenuated dca-induced apoptosis. on-treatment virological response (otr) was defined as complete loss or greater than -fold drop in hcv viremia within - months of therapy by quantitative or qualitative rt-pcr (roche cobas amplicor v . ), based on recommended early virological testing for continued therapy. forty-six patients with at least months of continued antiviral therapy were examined thus far, including with genotype (cl) and with either genotypes or infection (c ). on-treatment response (otr) was achieved in / ( %) patients overall, including % among c and % in c groups. positive and negative control groups included healthy hcv seropositive but nonviremic subjects without history of antiviral therapy ("recovered") and healthy hcv seronegative volunteers, respectively. hcv-specific cd t cell response was examined using recombinant hcv core, ns l , ns and control proteins in standard proliferation and ifn-gamma (ifng) elispot assay at baseline (pre-treatment) and at , andlor months during antiviral therapy. results: hcv-specific cd t cell response was significantly greater in the recovered compared to the chronic patients, consistent with its expected role in natural hcv clearance. among the chronics, c patients (genotype non- ) displayed a baseline t cell response to hcv ns / that was modestly but significantly greater than c patients ( the progression of fibrosis in chronic hepatitis c virus (hcv) infection differs among individuals and determines the ultimate prognosis and thus the need for therapy. the molecular mechanisms associated with the progression of fibrosis are poorly understood. gene expression profiling technologies allow the analysis of gene networks whose expression is associated with specific pathological conditions. we used real-time quantitative rt-pcr assays to compare the mrna expression of selected genes in liver biopsies of controls (n= normal liver samples) and of untreated patients with chronic hcv infection and different stages of fibrosis according to metavir, i.e. stage flal (n= ), f a (n= ), f a (n= ), f a (n=lo), f a ( n = l l ) and f a (n= ). in order to limit the number of pcr experiments, we first studied total mrna pools which were prepared by mixing amounts of individual liver biopsies mrna of each group. this pooled sample analysis allowed the selection of genes displaying significant different expression (> -fold variation) in comparison to "normal livers". the selected genes were then studied at the individual level and their diagnostic performance was assessed using roc curves. the most informative genes were used to construct specific gene expression signatures. we identified several genes specifically involved in various stages of fibrosis. the dysregulated genes mainly encoded extracellular matrix proteases, growth factors, cytokineslchemokines, and ifncu-induced genes. we also observed a statistically significant association between hcv rna amount (as determined with the same real-time quantitative rt-pcr technology) and expression of several genes, mainly ifn-a-induced genes including stat , ifi , mix , oas and gip . understanding the correlates of protective immunity in this setting is an important first step in the development of potential vaccine candidates. methods: two recipients of frozen patellar allograft tissue procured from the same donor developed evidence of acute hcv within months of surgery. in retrospect, the donor was confirmed to be hcv antibody negative but hcv rna positive (genotype la). both patients were enrolled in a prospective study of t cell immunity requiring whole unit blood draw at baseline, , , and months. comprehensive hcv-genomewide analyses were determined by ifn-y elispot responses to overlapping peptide pools that spanned the entire hcv polypeptide (genotype la, aa, total peptides). peptide responses were defined as greater than mean plus sd compared to control wells. results: patient ( yo wf) cleared the virus spontaneously within months; when first evaluated, the pt. demonstrated cd + t cell responses to of ( %) peptide pools, with effector frequencies as high as in , (to ns helicase- , amino acids - ). ifn-y cd + t cells were detected following stimulation with of ( %) pools (highest frequency to pool ns b- , aa - ). remarkably, months later, the repertoire of the hcv-specific cd t t cell response had expanded further, and patient demonstrated responses to of ( %) pools. in contrast, patient ( yo wm) demonstrated persistent viremia ( . million copieslml, bayer assay) and lacked cd + t cell responses to any peptide pools when first evaluated; only one peptide pool (ns -helicase- , aa - ) elicited responses in cd + t cells. despite virologic clearance with antiviral treatment (pegylated interferon and ribavirin), elispot screening failed to reveal emergence of new cd + or cd + t cell responses ( months later). conclusions: comprehensive analyses of hcv-genome-wide cd + and cd + t cell responses reveal significant differences in patients exposed to the same hcv innoculum and correlate with spontaneous clearance versus chronicity. in hcv infection that resolves spontaneously, the repertoire and strength of the hcv-specific immune response may continue to expand in the first year after infection (and may target more than one-third of the hcv polypeptide). in contrast, the profile of t cell responses remains narrow, weak, and constant in the acute infection that becomes chronic, even after successful antiviral therapy. alcohol consumption exacerbates liver injury in chronic hepatitis c and enhanced oxidative stress is one possible pathophysiological mechanism. we have previously developed a hepatoma cell line with conditional, stable expression of hcv core protein and constitutive expression of cyp e . these cells demonstrate dosedependent ethanol toxicity when core protein is expressed. the aims of this study were to determine whether reactive oxygen species (ros) production and mitochondrial dysfunction are responsible for ethanol-induced cytoxicity. methods: huh- cells with conditional expression of core protein and constitutive expression of cyp e (l subclone) were exposed to . micromolar tbooh and/or ethanol ( mm) for up to hrs. cytotoxicity was measured by mtt assay or trypan blue exclusion. ros production was assayed with the oxidation sensitive fluorescent dye dcfda. mitochondria membrane potential was analyzed by flow cytometry using the dye jc- as a probe. apoptosis was detected by cellular dna content analysis with flow cytometry. results: in the presence of . pm tbooh, there was a progressive increase in cell death in huh- cells expressing no heterologous proteins ( %), cypzel only ( l%), core only(l %), or core plus cypzel ( x). addition of mm ethanol to core/ cyp e expressing cells further increased cell death to %. dna content analysis and nuclear morphology showed that this type of cell death represented necrosis and not apoptosis. effects on mitochondrial membrane potential were also examined. under control conditions, only . % of cells had depolarized mitochondria. expression of corelcyp el and exposure to tbooh depolarized mitochondria in % of cells. similar to cell death, ethanol ( mm) addition increased depolarization to % of cells. compared to control cells, core protein increased ros by a factor of . . , and the combination corelcyp el by . . . furthermore, cells expressing corelcyp el ampiified the effect of exogenous tbooh. incubation with tbooh ( . pm) had no effect on ros content of control cells. it approximately doubled the ros content of cells expressing either core or cyp e and it increased ros content of cells expressing both corelcyp el by . . fold. in all cases the increase in ros was completely blocked by the antioxidant n-acetyl cysteine ( mm). the antioxidant also completely blocked both mitochondrial depolarization and cytotoxicity. conclusions: hcv core protein and cyp el synergistically enhance ros production in hepatoma cells, amplify the oxidative stress produced by extracellular ros, and sensitize cells to mitochondrial depolarization and necrotic cell death caused by alcohol. since all these effects are blocked by antioxidants, the formation of ros is likely to be the primary event which subsequently produces mitochondrial dysfunction and cell death. sponse associated with different outcomes of hcv infection may provide important insights into our understanding of hcv pathogenesis. for this purpose, we compared the functional features of hcv-specific cd cells in patients with chronic hepatitis c ( patients, ch), chronic asymptomatic carriers of hcv with persistently normal alt and positive serum hcv-rna ( patients, as) and in subjects with resolved hcv infection, either spontaneously ( subjects, sp) or following anti-viral treatment ( subjects, rt). functional analysis was carried out with highly immunogenic peptides corresponding to ns - , ns - , ns - , ns - , ns - , containing previously identified hla-a restricted epitopes. since the different hcv genotypes were represented in the different groups of patients in different proportions, different sets of peptides designed on genotype , genotype and genotype sequences were synthesized and used to reproduce more closely the sequence of the viruses infecting each individual patient and responsible for in vivo priming of the cds response. the immunological parameters tested were: a) frequency of hcv specific, cd + t cells by tetramer staining, both ex-vivo and after in vitro stimulation with synthetic peptides; b) ifn- production by intracellular cytokine staining; c) cytolytic activity by a standard %hromium release assay. the results of our study indicate that subjects recovered from hcv infection following treatment show lower ex-vivo frequencies of circulating hcv-specific cd cells compared to ch patients but their cells are able to expand and to produce ifn- more efficiently after in-vitro stimulation. indeed, hcv-specific t cell lines were induced in % of rt subjects and in % of ch patients; moreover, ifn- production was induced in % of rt subjects compared to % of ch patients (p< . ). in the group of subjects spontaneously recovered from infection, ex vivo cd frequencies were below the threshold of tetramer detection; however, peptide stimulation in vitro was able to expand tetramer+ cd cells and to induce ifn- production in % and % of the subjects, respectively. these lower levels of reactivity of sr subjects compared to tr patients are likely due to the different time elapsed from recovery, which was approximately years in tr subjects but much longer in sp subjects, who had recovered even decades before the time of immunological analysis. finally, the group of as patients showed the lowest levels of response to hcv, in terms of both frequencies of hcv-specific circulating cd cells and cytokine secretion. in conclusion, the hcv-specific, cd -mediated response has different features in patients with different outcomes of infection, showing a hierarchy of efficiency declining from subjects recovered after treatment who displayed the best responses, to chronic hepatitis patients, subjects recovered spontaneously and asymptomatic hcv-carriers, who appear to be the least responsive as a likely result of a deeper condition of tolerance to hcv. hepatitis c virus (hcv) infection is the most frequent cause of chronic liver disease in western countries and it has been involved in the development of cirrhosis with the risk of hepatocellular carcinoma. cyclooxygenases (cox) are crucial enzymes in the biosynthesis of prostaglandins and cox- , the inducible isoform, has been implicated in inflammation, fibrogenesis and carcinogenesis. to address whether cox- may play a role in hcvrelated hepatic inflammation and fibrosis, we determined the cox- expression pattern in the liver tissue from patients with hcv-induced chronic hepatitis (low histological activity= , high histological activity= ) and with end-stage cirrhosis, searching for correlations between the expression level of this enzyme and the histological activity of liver disease as well as with the intrahepatic expression and activity of metalloproteinases (mmps). we also investigated whether structural and non-structural hcv proteins may promote cox- expression in the human hepatocytederived cell line ccl . western-blot and rt-pcr analysis for cox- demonstrated that cox- expression levels were higher in mild chronic hepatitis (mch, . fold), severe chronic hepatitis (sch, fold) and cirrhosis ( . fold) than in normal liver. moreover, pge levels were also higher in liver samples from patients with sch ( . fold) and in those with cirrhosis ( . fold) than in normal liver. besides other cell types, hepatocellular cox- immunoreactivity was markedly observed in hcv cirrhosis, and although some cox- positive hepatocytes were observed at the edge of hepatic lobules, the majority of them were mainly restricted to the regenerative nodules. in contrast, none or few cox- expressing hepatocytes located in periportal areas were observed in patients with mch and sch, respectively. interestingly, we found a significant correlation between the intrahepatic expression and activity of cox- and mmp- and - in all the histological groups of patients analyzed. cox- mrna, protein and activity was de novo induced in resting ccl cells stably transfected with hcv core and ns a, and this effect was higher ( fold and . fold) when activated with cytokines and phorbol esters, respectively. in conclusion, our results provide evidence that a virus-induced hepatocellular cox- upregulation could mediate important pathogenic events in the course of chronic hcv infection, suggesting that specific cox- inhibitors might be useful for chemoprevention and therapy of hcv-related liver disease. were nahe to hbv. thl responses to hcv proteins ns , ns and core were sought by elispot. as a control thl responses were also sought to hbsag, where results were analysed according to recovery from hbv infection or nayve to hbv. thl responses to each of the proteins was performed before and after depletion of t-regs using anti-cd (> % depletion). results: following treg depletion thl responses were revealed de novo in / and enhanced in patients with ns , and with ns respectively and and with hcv core respectively. the overall increase in the thl responses with t-reg depletion was significant for both hcv core (median increase -fold p = . ) and ns ( - backaround: in both chronic viral hepatitis b and c viral persistence is thought to be due to an inadequate antiviral t cell response, which in turn may be caused by inappropriate priming of t cells by dendritic cells. an important subset of peripheral blood dendritic cells, the plasmacytoid dendritic cell, has been identified to be the major interferon-alpha producing cell in humans. since both hepatitis b and hepatitis c infection can be successfully treated by exogenous interferon-alpha, an impairment of endogenous interferon-alpha producing cells is an attractive hypothesis for the pathogenesis of chronic viral persistence. methods: patients with acute symptomatic ( n = l l ) and chronic hepatitis c (n= ), sustained responders to interferon-alpha therapy (n= ) and patients with acute hepatitis b (n=lo) as well as healthy controls (n= ) were included in this study. plasmacytoid dendritic cells were stained for facs-analysis in peripheral blood mononuclear cells with antibodies against bdca- and cd and by exclusion of lineage marker positive cells (cd , cd , cd , cd ). in addition pdc were stained with various activation and maturation markers (e.g. cdso, cd , cd ). production of interferon-alpha was measured by elisa after stimulation with cpg. results: in acute hepatitis c, ifn-alpha production was about -fold reduced as compared to healthy controls ( pg/ pbmc vs. pg pbmc). this reduction was caused by both a significant reduction in absolute numbers of pdc ( . /~ vs. . /pl, p= . ) as well as by a reduction of ifn-alpha production per cell ( . pg/pdc vs. . pg/pdc; p= . ). in chronic hepatitis c, ifn-alpha production was still reduced by over %, although absolute numbers of pdc were similar to healthy controls. following spontaneous or treatment induced viral clearance both number and ifn-alpha secretion of pdc were not different from healthy controls. importantly, in acute hepatitis b, which runs a self-limited course in the majority of cases, also the ifn-alpha production was reduced ( pg pbmc). this reduction was also caused by both the reduction of absolute pdc count ( . ~ , p=o.l) as well as by the ifn-alpha secretion per pdc ( . pg pdc; p= . ). using a panel of dc activation and maturation markers we did not find any evidence of a more immature or more activated phenotype of pdc in acute hepatitis c. exogenous ifn-alpha administration during acute hepatitis c led to a further reduction of ifn-production by pdc. conclusions: acute viral hepatitis has a dramatic impact on frequency and function of plasmacytoid dendritic cells in the peripheral blood, indicating that pdc play an important role during this phase of viral hepatitis. however, no differences were found between patients with self-limited vs. chronically evolving acute hepatitis c or patients with acute self-limited hepatitis b. future studies have to clarify whether the reduced ifn-alpha secretion by pdc contributes to viral persistence or whether this is rather part of a physiological response to acute viral disease. it has been suggested that hepatocellular steatosis, a frequent histological feature of chronic hepatitis c, is more frequent in hcv genotype infection, and disappearance of steatosis in patients who clear hcv genotype infection after antiviral therapy suggests a possible direct role of this hcv genotype. in order to assess the direct causal role of hcv in steatosis, we studied the relationship between hcv rna load and steatosis according to the hcv genotype. methods: we studied patients with chronic hepatitis c (genotype , n = ; genotype , n = ). the serum hcv rna level was measured at the time of liver biopsy by means of a third-generation "branched dna"-based assay (versant hcv rna . quantitative assay, bayer diagnostics). steatosis was graded as absent, mild (< % of hepatocytes), moderate ( to % of hepatocytes) or marked (> % of hepatocytes). daily alcohol intake during the months prior to liver biopsy, and the body mass index (bmi), were recorded. results: steatosis was more frequent in patients with genotype infection than in those with genotype infection ( . % vs . %, ns). steatosis was significantly more severe in hcy genotype than in genotype infection (moderate or marked in . % vs . %, p=o.oool). in patients infected by genotype , the severity of steatosis was significantly related to hcv rna load but not to alcohol intake or bmi. in contrast, in patients infected by hcv genotype , the severity of steatosis was not related to the hcv rna level but was significantly influenced by alcohol intake and bmi. bivariate analysis demonstrated the effect of the hcv genotype on the association between the severity of steatosis and viral load in genotype -infected patients, and between the severity of steatosis and exogenous metabolic factors in genotype -infected patients. multivariate analysis showed that : (i, in patients infected by hcv genotype , only hcv rna load was independently related to the severity of steatosis (odds ratio (or) = . , % confidence interval (ci): . - . ; p = . ), (ii) in patients infected by hcv genotype , bmi higher than . kglm (or = . , % ci: . - . ; p = . ), alcohol intake exceeding glday (or = . , % ci: . - . ; p = . ), and histological grade (or = . , % ci: . - . ; p = . ) were independently related to the severity of steatosis. conclusion: our results suggest : (i) steatosis is a cytopathic lesion induced by hcv genotype ; (ii) hcv genotype is not steatogenic per se or at the usual in vivo expression levels, and liver steatosis is a feature of associated steatohepatitis in these patients. the effect of hcv genotype sequences and the role of their expression levc s must be tested in vitro, in order to better reflect the in vivo situation. in animal models, steatosis is associated with oxidative stress and lipid peroxidation which is known to promote fibrogenesis. this study was aimed to assess in patients with chronic hepatitis c whether steatosis contributes to fibrosis through oxidative stress. methods: markers of lipid peroxidation and antioxidant status were measured in blood from chronic hepatitis c patients and age and sex matched healthy controls. intrahepatic levels of these markers were also assessed in a subgroup of patients and controls. results: the lipid peroxidation marker malondialdehyde was significantly higher in the blood and in the liver of patients compared to controls (p= . and p=o.o respectively) while the antioxidant glutathione peroxidase was significantly lower (p= . and p = . respectively). the antioxidant superoxide dismutase was significantly higher in the blood and lower in the liver compared to controls (p=o.o and p= . respectively). autoantibodies to soluble liver antigen (sla) are specific for autoimmune hepatitis. the molecular characterization of the sla antigen has allowed the establishment of highly sensitive and specific radioligand assays. to determine serum anti-sla a specific radioligand assay was used. total rna was isolated and reverse transcribed from hepg cells. the cdna encoding sla was amplified by pcr and used as a template to express sla protein eukaryotically in a tnt coupled reticulocyte lysate system (promega corporation, southampton, uk). anti-sla was measured retrospectively in consecutive patients ( girls, median age at transplant months; range - ) with dn-aih, in whom sera were available before transplant, , , , and months post-transplant and at the time of the diagnosis of dn-aih. eight patients had biliary atresia, alagille syndrome, cryptogenic cirrhosis, alpha- -antitrypsin deficiency, druginduced acute liver failure and bsep deficiency. the patient with acute liver failure did not have pre-transplant serum specimen stored. twelve patients had developed smooth muscle andlor antinuclear antibodies, two anti-liver kidney microsomal (one atypical) and two anti-mitochondria antibody. before transplantation, anti-sla was negative in / cases, the two positive patients having cryptogenic cirrhosis and biliary atresia. post transplant, anti-sla remained positive in these two and became positive in further patients. of these, in patients anti-sla became positive at a median of months (range: - ) before the clinical diagnosis of dn-aih. in children anti-sla became detectable as early as months post-surgery. in patients anti-sla levels were highest at the time of the clinical diagnosis of dn-aih. the presence of anti-sla in dn-aih supports the autoimmune nature of this condition; the frequent appearance of anti-sla before the clinical manifestation of the disease makes it a potential predictive marker for development of dn-aih. all patients showed positive response to the therapy, with respect to remarkable release of severe meteorism, active diet, and significant improvement of liver and kidney functions. however, no difference was presented in the markers of electrolytes, blood routine and blood gas analysis before and after the mars, while the effects on serum levels of alanine aminotransferase, aspartate aminotransferase and y-gt were remained uncertain since the alt decreased from ull to ullduring a in conjunction with observations from other centres, our data show that mars treatments resulted in a ramarkable removal of bilirubin, uric acid, bun, cr and ammonia, which could therefore decrease the toxic effects that higher concentrations of these compounds exert on liver and kidney function and could thus contribute to improvements in multiple organ dysfunctions. we found additionally that the higher concentration of serum toxin before detoxication therapy, the more efticacy removal could be achieved which presents mars could be {of therapeutic results even in the very serious cases. it remains questionable whether some of useful substances characted in low molecular weight such as trhlthyrotrophin-releasing factor), gnrh), adh(anti-diuretic hormone and calcitonin will be also removed by albumin dialysis, whether the added synthesis function is necessary for an artificial liver support, and whether this encouraging survival rate applies to long term outcome remains open for further investigation. scoring system has recently been introduced to determine priority for organ allocation in liver transplantation (lt). there is limited available data on resource utilization in the post-meld era.aims: . evaluate the effect of the meld system on lt-associated resource utilization and post-lt survival. . compare the costs of lt in the periods before and after the implementation of meld. methods : patients undergoing lt at our center in the months following meld implementation(cases) were compared to those undergoing lt in the immediately preceding -month period (controzs). the outcome parameters studied were: ) resource utilization: defined as ( there were no significant differences between the two groups in terms of age, sex distribution or presence of hepatocellular carcinoma (hcc). the average meld among cases was . , compared to . in controls (p = . , ns). overall los was similar in the two groups. however, pre-lt los, especially in the intensive care unit (icu), was significantly higher in the pre-meld or control group. the pre-lt cost was also higher in this group(p= . ), translating into significantly higher total costs of lt in the pre-meld period (p=o.ol). the need for post-lt hd, pmv and pressors was no different between the two groups. this data is summarized in table . the meld score was found to be significantly correlated with bile d u d damage is a maior feature in liver graft rejection. ductopenia (dp), defined as loss of more than %-of bile ducts, is a major hallmark of chronic liver graft rejection (cr), which usually leads to graft failure within the first post transplant year. in a previous study we have shown that in patients who developed dp and cr a deficient proliferative response of canals of hering (coh) was present in the preceding biopsies showing acute rejection (ar) when compared with a control group who experienced ar but did not progress to cr. these findings support the postulation that coh represent a regenerative compartment of the biliary unit in the liver. aim of the present studv: to analyze whether preservation of coh might contribute to the reversibility of dp. patients and methods we studied groups of patients. the index group (ig, n= ) developed loss of bile ducts without progression to graft failure due to cr during a follow up of years after transplantation. the second group (crg, n= ) were all cr patients, retransplanted at a median time of months (range - months). reperfusion (rb), week ( -w), month ( -m) biopsies were studied in both groups. in the crg the last biopsies (lb) before retransplantation taken at a median time of days (range - days) were also studied. additionally, in the ig the & ( -y), nd ( -y) and * ( -y) annual biopsies were also included. bile ducts and coh were identified by immunohistology using cytokeratin . ki (mib) was applied to investigate the proliferative activity. the number of bile ducts and coh were counted per portal tract and the number of k + cells was counted as ratio of the total number of cells in these structures. clinical follow-up of the ig group was studied based on liver function tests at similar time points as the biopsies. the ig group showed damaged bile ducts and decreasing numbers of bile ducts in the course of time, leading to dp at a median of years. contrastingly, the crg developed dp at a median time of days, observed in the last biopsies taken before retransplantation. when compared with the crg, the ig showed significantly less proliferative activity in bile ducts at -w (p=o.o ) but not in rb, -m and -y, the latter was compared with the lb of the crg. coh showed an initial increase at -w in the ig, but a progressive decrease in the subsequent biopsies, reaching significant loss at -y (p= . ). in the crg, progressive loss of coh started at -w, leading to a significant loss within year. numbers of coh were consistently lower in the crg at all time points except rb, although not statistically significant. there were also no significant differences in proliferative activity both within the ig in the course of time and when compared with crg. all ig patients continuously showed abnormal liver function tests apart from the bilirubin levels, which were elevated during the first month after transplantation but were normal after -y. at -y, median serum level of alkaline phosphatase was u/l, gamma glutamyl transpherase was ull, bilirubin was micromoll and asatlalat were ull. conclusion: the ig showed a much slower course of loss of bile ducts when compared with crg, leading to graft survival of more than years. a higher proliferative activity in bile ducts at week in the crg did not prevent the progressive development of dplcr in crg. the initial increase of coh at week in the ig might be responsible for the prolonged preservation of coh in the ig. as coh are believed to represent a regenerative compartment of the biliary unit, our findings indicated that prolonged preservation of coh might contribute to the delayed occurrence of dp but apparently not to reversibility of dp. absence of reversibility of dp is supported by the abnormal liver function tests. (ltx) . recurrent hcv poses a significant and possibly increasing threat to graft and patient survival. diagnosis of acute rejection and alterations in immunosuppression (is) may significantly impact the tempo of post-tx hcv. these associations motivated us to re-examine risk factors for early acute rejection (ear) in a large and contemporary cohort of ltx recipients. methods: the study cohort comprised of consecutive adults undergoing primary ltx between / / and / / for chronic liver disease with a minimum of day graft and patient survival. recipients received triple is, typically with steroids, tacrolimus, and mycophenolate mofetil. ear was defined as biopsyproven rejection or treatment for presumed rejection within months of ltx. risk factors for ear were determined by cox proportional hazard methods. spearman rank and phi correlations were used to determine associations between factors. results: men ( %) and women ( %) underwent deceased (n = ; %) or living donor (n = ; %) ltx. the etiologies of chronic liver disease were hcv (n= ; %), aih i pbc / psc (n= ; %), hbv (n= ; %), cryptogenic (n= ; lo%), alcohol (n= ; %), and miscellaneous (n= ; %). our overall incidence of ear was % ( i recipients); recipients ( %) had biopsy-proven rejection while recipients ( %) were treated for rejection without histologic confirmation. cox univariate models showed that hcv, female gender, meld , year compared to year , and lower day - tacrolimus (tac d - ) level were positively associated with ear while asian compared to caucasian ethnicity was negatively associated with ear (table la) . factors such as recipient and donor age, recipient african american or hispanic ethnicities, donor type (deceased or living), other years ( and ) , child's score and class, pre-ltx icu location, and cold and warm ischemia times were not significant risk factors. cox multivariate models showed that hcv diagnosis and female gender remained independent and significant risk factors for ear (table lb) . hbv etiology and asian ethnicity were significantly correlated (phi coefficient . ; p = < . ) and thus, did not remain independent risk factors in multivariate analysis. while lower tacrolimus level tended to predispose to ear, meld and year were no longer associated with ear. lower tacrolimus level was significantly correlated with both higher meld score (spearman rank correlation - . ; p = . ) and later transplant year (spearman rank correlation - . ; p = . ). conclusions: hcv etiology of liver disease is strongly associated with ear after ltx. risk of ear is higher for hcv than etoh, cryptogenic, and hbv etiologies and comparable to aih / psc i pbc etiologies; this effect is independent of gender, ethnicity, year, meld, and post-ltx is. recipients with high meld scores are also at increased risk for ear, at least partly because of lower post-ltx is. it is unclear whether the strong association of hcv etiology with ear is because hcv infection results in an immunologic environment predisposing to ear or whether we are unable to accurately diagnose rejection in the setting of hcv recurrence. aim: although the donor pool has expanded in response to increasing waiting lists the quality of donor livers has suffered as a result. it remains unclear whether such marginal organs can be safely used in high-risk recipients or whether they should be implanted solely into good recipients. we aimed to define recipient selection criteria for implantation of these grafts. methods: a prospectively collected database containing patients who underwent orthotopic liver transplantation between and was analysed. donors were scored using a formula derived by logistic regression that identified covariates (graft steatosis, donor age and cold ischaemia time) that independently correlated with primary graft dysfunction. this enabled them to be stratified into either marginal or non-marginal groups. using logistic regression analysis, recipient factors independently correlated with -year post transplant survival in the marginal group (recipient age, plasma albumin and urea) were built into a mathematical model and each patient was given a score accordingly. patients with scores higher than the defined optimum cut-off value were considered as high-risk and the others with lower scores as low-risk recipients. outcomes were then evaluated in these subpopulations results: marginal and non-marginal donor groups consisted of and patients respectively. the recipient score derived from the multivariate analysis was as follows : score = . x plasma albumin+ . x age group + . x urea, with the recipient albumin level coded as for < . gldl and for . gldl, the recipient age group coded as for > years, as for - years, and as for . mgldl, and as for . mgldl. the roc curve analysis showed that the score had a good discriminating power (area under the curve . , p =. ) and the ideal cut-off value demarcating high-risk from low-risk recipients was . . therefore the recipients were classed as high-risk if they had a plasma albumin level below . gldl with either an age of over or a urea level of above . mgldl or when they had a urea level over . mgldl with an age of over . of the recipients in marginal group, ( %) were classified as high-risk and ( %) were classified as low-risk. there was a huge -year survival difference between high-risk and low-risk recipients ( . % and . %, respectively, p <. background single-center experience with pretransplant use of rabbit anti-human-thymocyte globulin suggests that despite early depletion of lymphocytes, a third of all pediatric liver recipients develop early rejection, while the remainder demonstrate clinical graft adaptation. purposelmethods: to identify potential mechanisms, pediatric liver recipients, median age . years, median followup months, received pre-and post-transplant measurements of . whole blood concentrations of tacrolimus (tac), . interdose changes in mitogen-stimulated t-and b-cell responses (slr), . dendritic cell (dc),and peripheral blood mononuclear cell subsets, and . cd + -suppressor effect on donor antigenpresenting cell types (cd fmonocytes and cd + b-cells). all patients received ratg preconditioning with a total dose of mglkg in two divided doses. the first dose was given before liver transplantation (ltx). maintenance agent was tac without steroids. in patients this was replaced with sirolimus (srl). mitogen-stimulated t and b-cell responses were compared with those seen in a historical population, who had not received ratg pretreatment. results: all measurements were performed at a median interval of days ( - days) after ltx. . in slr, the frequency of t-cells expressing the cytokines ifn-g, tnf-a, and il- decreased with increasing total exposure (auc) to tac in historical controls (n= ). this relationship was markedly dampened in ratg patients (n= ), as suggested by slopes of . , . , and . relating ifn-g, tnf-a and il- on the y-axis to tac auc. . significant decrease in cd absolute counts at week and partial reconstitution at months after ratg pretreatment (mean pretx- vs week- vs month - celllmm ). during this period, monocytes and b-cells remained stable. . dc remained unchanged, while dc frequency increased significantly, from . to . , p= . . . in coculture experiments, purified cd + -subpopulations from two recipients receiving minimal tac doses induced decreased cd expression in donor apc, but increased its expression in hla-mismatched apc. eleven of subjects experienced rejection, while subjects are being maintained on daily (n= ) or every other day (n= ) doses of tac or srl. conclusions: despite lymphocyte depletion, rejection in the setting of t-cell anergy can be explained by relative sparing of antigen-presenting cells, which can recruit alternative effector mediators. the appearance of donor-specific cd + suppressor cells in recipients on low immunosuppression suggests that these conditions may also foster a favorable immunomodulatory response toward the liver allografts. (dropout) . the objective of this study was to evaluate the impact of the hcc-adjusted model for end-stage liver disease (meld) organ allocation scheme on the intention-to-treat outcome. under the meld scheme, patients with hcc meeting the united network for organ sharing (unos) t (single lesion under cm) and t (single lesion between to cm, or to lesions none exceeding cm) criteria were eligible for an initial meld priority score of and , respectively. they were also entitled to an increase in meld score, corresponding to a % increase in mortality, for every months on the waiting list. methods: excluding patients undergoing living-donor liver transplantation, we prospectively evaluated consecutive patients with hcc listed for olt since january . the kaplan-meier probabilities of olt and dropout among patients with hcc listed under the meld system between february and january were compared with patients listed between january and january under the previous system of organ allocation. follow-up in the pre-meld group was censored on february , , when the meld system for organ allocation was implemented by unos. for patients under meld, follow-up was censored on february , , when further refinements of the meld policy for hcc were made. results: the baseline characteristics were not significantly different between the two groups. eighteen of patients ( %) in the meld group and of patients ( %) in the pre-meld group received chemoembolization or various ablation treatments before olt (p= . ). all patients in the meld group met t ( patients) or t criteria ( patients). in the pre-meld group, patients had hcc stage exceeding t but meeting our proposed expanded criteria (single lesion not exceeding . cm or no more than lesions none greater than . cm with total tumor diameter not exceeding cm) by the time of olt. the kaplan-meier cumulative probabilities for olt at , , and . months of longest followup were . %, . % and . %, respectively, in the meld group, versus . %, . %, . %, and . % at , , , and months, respectively, in the pre-meld group (p= . ). under meld, none of the patients with t lesion had received olt. the cumulative probabilities for olt for the patients with t hcc in the meld group were . %, . %, and . %, respectively at , , and . months (p=o.oool versus the pre-meld group). the cumulative probability of dropout was . % at . months of longest followup without olt under meld, whereas the cumulative probability of dropout increased from . % at months to . % at months in the pre-meld era. the difference did not reach statistical significance (p= . ) largely due to low dropout rates in the first months for both groups. among the patients who received olt, of patients in the meld group versus of patients in the pre-meld group had pathologic hcc stage in the explant exceeding t criteria (p= . ). unfavorable histologic tumor features in the explant, including either poorly differentiated grade or microvascular invasion or both, were observed in of patients in the meld group versus of patients in the pre-meld group ( i " . ). the short duration of follow-up under meld precluded comparison of intention-totreat survival or hcc recurrence between the two groups. con-clusion: the hcc-adjusted meld system significantly improved the probability of timely olt, and was not associated with selection of a greater proportion of hcc with unfavorable explant tumor histology. given the low probabilities for dropout in the first months following listing for olt even in the pre-meld era, patients with hcc might have indeed received too high a priority score in the first year under meld. disclosures: nancy l ascher -no relationships to disclose nathan m bass -no relationships to disclose randomized at d to receive maintenance is regimen with ciclosporine microemulsion + prednisone (group ) or without steroids (ciclosporine microemulsion + placebo, group ) after a days blinded oral steroid tapering period. results : patients were recruited and a total of were randomized at d (group = , group = m). there was no difference between the groups for baseline characteristics, proportion of patients with hepatitis c ( . % and . %), or cyclosporine blood levels. the incidence of treated biopsy confirmed acute rejection at months was . % in group and . % in group (p= . ), with a trend for higher incidence of grade rejection ( . % vs . % ; p= . ). this difference was maintained in hcv pos and hcv neg patients. no difference was observed between the groups in terms of adverse events, infections, incidence of hypertension and renal dysfunction. changes from baseline were similar with regards to metabolic parameters. a trend towards a better glucose tolerability was observed, less patients receiving an antidiabetic treatment in the placebo group ( vs ). conclusion : peritransplant immunosuppression with basiliximab, cyclosporine microemulsion, and steroids resulted in excellent low rejection rates in lt patients. early steroid withdrawl strategy at day is not supported by the results of this study, which showed an higher incidence of acute rejection and a trend to more severe acute rejection, only balanced by a trend to a lower need of antidiabetic treatment. to determine if an association exists between meld score and post transplant graft loss within months. methods from / / through / / , , patients underwent liver transplantation, % of whom were followed for a minimum of three months. cox regression analysis was utilized to assess the relationship between meld score and post transplant outcome. transplantation. seventy one percent of patients were positive for autoantibodies: . % were positive for anti-nuclear antibody, . % for anti-smooth muscle antibody and % for anti-liver kidney microsomal antibody. the average alt elevation was (range - ) and ast elevation (range - ). an elevated ggtp was seen in . % of patients. all patients were hepatitis c pcr negative. at the time of diagnosis, patients were being treated with cyclosporine, with f'k , and with steroids in addition to calcineurin inhibitors. after diagnosis, all patients received standard therapy with l-zmg/kg of steroids. in addition, % ( ) began hthioprine and % had their calcineurin inhibitor dose decreased. after a mean of . years of follow-up (range . - . ), % remain steroid dependent and % are off steroids. one patient was re-transplanted for biliary complications and patient required conversion to sirohus. conclusion: diagnosis of de novo am requires a high index of suspicion as auto-antibodies are often negative. steroid therapy, often with the addition of azathioprine, is effective in controlling disease. many patients, however, become steroid dependent in order to remain in remission. background. liver allografts have an improved outcome compared with other solid organ grafts, and rodent studies have suggested that activation-associated lymphocyte death may play a key role. studies from our laboratory have shown increased levels of apoptotic lymphocytes in human liver grafts compared with renal grafts and native liver. although the levels of apoptosis did not differ significantly between those who developed acute rejection (rej) and those who did not (nr), the earliest timepoint studied was days post-olt. we hypothesized that the very early postoperative period (within hours) would reveal a relationship between leukocyte apoptosis and rejection. aims. the aim of this study was to determine the amount of early leukocyte apoptosis and its relationship with the degree of lymphocyte activation, subsequent rejection status and degree of donor cell chimerism. methods. peripheral blood mononuclear cells were isolated from patients undergoing olt and were collected on the day prior, hrs after reperfusion (day ) and hrs post-olt (day ). dna and rna were prepared and real-time pcr used to quantify apoptosis (ligation-mediated pcr), lymphocyte activation (il- , ifn-gamma, il- , cd ligand, using gapdh as an internal standard) and donor cell chimerism (y chromosome dyz or donor-specific drb ). results. the mean level of circulating apoptotic cells in day recipient pbmc was higher than healthy controls ( . % t- . vs . % ? . , ~~ . ) . apoptosis was greater in nr ( . % . ) compared with rej ( . % t- . , p=o.o ). on day the pbmc from nr had increased expression of ifn-gamma (p= . ), il- (p= . ), cd ligand (p= . ) and il- (trend) compared with rej, despite no difference in lymphocyte counts. donor cell chimerism on day did not differ between the groups indicating that this was unlikely to account for increased leukocyte apoptosis in the nr group. interestingly, the level of chimerism hrs postreperfusion (day ) was significantly higher in nr ( . % f . ) compared with rej ( . % t- . , p= . ) and there was a close correlation between chimerism on day and pbmc cytokine expression on day (r= . , p= . ). conclusions. patients who did not experience rejection had a paradoxical increase in markers of lymphocyte activation at day , in association with higher levels of leukocyte apoptosis. this suggests that recipient cell death may have a role in graft acceptance and reduced acute rejection in human olt. the higher donor cell chimerism seen in non-rejectors implicates the passenger leukocytes in the process of heightened cell death. disclosures: andrew clouston -no relationships to disclose wenyi gu -no relationships to disclose julie r jonsson -no relationships to disclose elizabeth e powell -no relationships to disclose daina m vanags -no relationships to disclose amadeo marcos, bridget flynn, paulo fontes, thomas cacciarelli, wallis marsh, michael devera, obaid shakil, noriko murase, anthony demetris, john fun% thomas e stanl, university of pittsburgh, pittsburgh, pa the seminal mechanism of organ engraftment is thought to be immune activation-dependent clonal exhaustion-deletion. the conventional use of heavy immunosuppression may depress the initial step of donor-specific activation to the extent that the treatment is anti-tolerogenic. to avoid this pitfall, we have applied therapeutic principles in management of adults cadaveric liver recipients transplanted between / - / . the treatment principles were, first, host conditioning prior to transplantation, and second, minimum post-transplant immunosuppression. the host conditioning was done with one gram of methylprednisolon and a single infusion of mg of alemtuzumab, completed before liver revascularization. there were males and females in the group with mean age . . years ( - ). main causes of liver disease were hepatitis c ( %), cholestatic liver disease ( %), alcoholic liver disease ( %). daily post-transplant monotherapy with tacrolimus (trough target ng/ml) was started - hours postoperatively (starting tacrolimus dose: . + mg, median ). because of suspected neurotoxicity, patients were switched to cyclosporin. in addition sirolimus monotherapy was substituted for tacrolimus in one patient for management of nephrotoxicity. immune activation diagnosed by liver function tests or by mild or equivocal rejection in liver biopsies frequently was not treated, and tended to resolve spontaneously. clinically and pathologically significant rejection was treated with a bolus of methylprednisolone and/or a mg dose of alemtuzumab. nine patients ( . %) died sepsis; pnf; one coagulation disorder (hypercoagulable state), and one congestive heart failure. of the surviving recipients four experienced rejection during the first two months which was readily reversed with a bolus of methylprednisolon. after demonstrating the absence of immune activation in liver biopsies at days post-transplantation, weaning from monotherapy was initiated in patients (with the aim of completely stopping treatment in selected cases). seven patients experienced one episode of rejection that was reversed with single dose of methylprednisolone and/or infusion of mg of alemtuzumab. presently, patients are on once a day monotherapy, patients on every-other-day, patients on three timedweek, patients on twicelweek and one patient on once-a-week immunosuppressive regimen (latest tacrolimus dose . t . mg, median . ). the patients on cyclosporin and sirolimus are also following the same stepwise weaning pattern. no cases of new onset diabetes, renal failure or hypertension was found in these patients. mean serum creatinine level in the study group at the time of transplantation was . . mgldl and at the latest follow up . . mgldl. cmv infection was seen in % of the patient population but was easily treatable with antivirals. no ptld was seen during the follow-up period. hepatitis c recurrence was seen in over % of the patients. response to anti-hcv therapy in this group, after reduction of immunosuppression and especially during the weaning process was promissing. conclusion: by applying the principles of immunosuppression outlined above, it has been possible to drastically reduce the amount of total immunosuppression relative to any (of our) pre-vious experience with liver transplantation. to see less side effects of chronic and high dose immunosuppressive therapy, and probably to have a chance to get a better response to treatment in our hepatitis c population. the results suggest the possibility of systematically achieving drug-free tolerance after liver transplantation. background:end stage liver disease as a consequence of hepatic sarcoidosis is an uncommon indication for liver transplantation (lt). consequently, there is a paucity of information on the pre-lt findings and postoperative course of individuals transplanted for hepatic sarcoidosis. the purpose of this study was to evaluate our experience with lt for sarcoidosis. methods: cases were identified by review of the mount sinai hospital lt database. patient records were reviewed; including the pathologic analysis of the hepatic explant, to confirm that sarcoidosis was responsible for liver failure. for each case, two control patients with other causes of liver failure matched for age, gender and date of transplant were selected. data collected encompassed a mean follow up period of years (range - years) post-lt. two-tailed student's t-test was used for comparison of continuous data, two-tailed fisher's exact test for comparison of categorical data and log rank test for comparison of survival d,ita. results: hepatic sarcoidosis was the indication for lt in of adult-lt ( . %) performed from september -june . . the mean age at transplant was years and / ( %) were males. the diagnosis of sarcoidosis was established by findings of extensive, non-caseating granulomas in pre-lt biopsy specimens or in the native liver explant. in / cases, sarcoid had been previously diagnosed by biopsies of lung parenchyma or mediastinal lymph node. of patients were ama negative. the sole exception was a patient with an ama titer of : in whom sarcoid was still considered the most likely diagnosis based on liver biopsy findings of granulomas present in both portal and lobular areas and granulomas in a lung biopsy performed -years prior to lt. extrahepatic disease was limited to pulmonary involvement in patients with radiographic findings of either interstitial infiltrates ( ) or interstitial infiltrates and hilar adenopathy ( ). the mean age at transplant and gender distribution were identical in cases and controls. the indications for lt in the control group were: hepatitis c ( ), pbc ( ) and one patient each with hereditary hemochromatosis, cryptogenic cirrhosis, hepatitis b and laennec cirrhosis. no statistically significant differences between the groups were present with respect to pre-lt levels of ast, alt, alkaline phosphatase, total bilirubin, serum creatinine or prothrombin time. cases and controls had a similar prevalence of ascites and anti-hepatitis b core antibodies. in contrast, sarcoid cases were much more likely to have a diagnosis of diabetes mellitus ( % vs. %, p=. ) and less likely to have antibodies to hepatitis c ( % vs. %, p=. ). standard orthotopic lt was performed (two control patients received right trisegment grafts). rates of acute cellular rejection were % in cases and % in controls (p=o. ) with cases experiencing a greater number of acute rejection episodes per patient ( . vs. . episodeslpatient). de novo autoimmune hepatitis developed in one case and one control patient. both de novo autoimmune hepatitis and chronic rejection developed in one case patient. recurrence of hepatic sarcoidosis was diagnosed in two patients at . and . years of follow-up. among cases, the one-year graft and patient survival rates were % and five-year graft and patient survival rates were %. there was no statistically significant difference in five-year graft or patient survival between cases and controls. conclusions: end-stage liver disease as a consequence of sarcoidosis is a rare indication for lt. these patients share many features in common with those transplanted for other indications. despite the small number of patients, a relatively high rate of acute rejection and de novo autoimmune hepatitis was observed in patients with sarcoidosis. recurrence of hepatic sarcoidosis was observed. five-year graft and patient survival rates were comparable to those of patients transplanted for other indications. disclosures: sander florman -no relationships to disclose leona kim schluger -no relationships to disclose kevin m korenblat -no relationships to disclose evan j lipson -no relationships to disclose transplantation. james d eason, ari j cohen, safheesh nair, george loss, ochsner clinic foundation, new orleans, la sirolimus has been used successfully in improving renal function in olt recipients previously treated with tacrolimus and steroids. we report our experience with early sirolimus conversion in steroid-free olt recipients to determine safety and efficacy in patients never treated with steroids. methods: we performed olt over a threeyear period. steroid-free immunosuppression with rabbit atg induction and tacrolimus and mmf was used in of these patients. twenty-five of these steroid-free recipients were converted from tacrolimus to sirolimus within one week to ll months of transplant. results of these patients receiving sirolimus conversion were reviewed. results: renal insufficiency was the reason for conversion in patients, while seven patients were converted because of neurotoxicity. six patients were dialysis-dependent at the time of conversion. one-year patient survival in this high-risk group was % ( / ) compared to % in patients who remained on tacrolimus. the four deaths were in patients converted because of renal failure, three of whom were on dialysis. the incidence of rejection was % in sirolimus patients compared to % in patients who remained on tacrolimus. rejection was treated by the addition of mmf or reintroduction of low-dose tacrolimus. only one patient required steroids to reverse rejection. conclusion: early sirolimus conversion is safe and effective lawal, sandy florman, isabel fiel, ronald gordon, myron schwartz, charles miller, thomas d schiano, the mount sinai medical center, new york, ny introduction: primary non-function (pnf) fter liver transplantation occurs in approximately % of cases and is fatal without timely retransplantation. pnf has been associated with many risk factors, however the etiology remains unknown. some evidence suggests that ultrastructural changes in the liver may be causative. aim: we sought to examine the hepatic ultrastructure of donor allografts in patients with and without pnf using electron microscopy. medical center, over adult orthotopic liver transplants were performed. patients with the classic clinical presentation of pnf requiring retransplant were identified. archived pre-and postreperfusion donor liver biopsies were examined by electron microscopy in patients with pnf and in matched controls. each pnf case was matched by donor age t years, gender, cold ischemic time klhour and the donor's cause of death. all biopsies were blindly reviewed by the same pathologist. particular attention was paid to abnormalities of the mitochondria, endoplasmic reticulum and sinusoidal endothelial cells. in addition, the glycogen content of the cells was also assessed. the recipient age and creatinine, as well as the donor serum peak transaminases and bilirubin were compared.non parametric tests with p values < . were regarded as significant using the spss . program. results: overall, patients with pnf were older ( . t vs. . years, p= . ) and had higher peak alt levels ( ? vs. u/l, p= . ). there was no significant difference in recipient peak serum creatinine, donor peak serum ast, sodium or donor peak serum bilirubin. in all cases, the endoplasmic reticulum and sinusoidal endothelial cells were ultrastructurally normal. the hepatocytes had variable degrees of glycogen pooling and moderate to severe fatty infiltration. in / ( %) pnf cases vs. ly ( %) control had intramitochondrial crystalline inclusions on pre-perfusion biopsy. conclusion: liver allografts from patients with primary non-function have significant mitochondria ultrastructural changes on pre-perfusion biopsies, and may thus have some intrinsic mitochondria abnormalities. introduction: the ideal liver allocation system would allocate donated livers to patients who are most likeiy to die without a transplant, but who have the lowest predicted mortality once they have been transplanted. this mode of allocation assesses the condition of both the recipient and the donor at the time of transplantation. in this study we have constructed a self-organising map (som: this is a neural network or non-linear mode of decision analysis suitable for modelling complex multidimensional relationships) and then validated it by using the som to classify survival in an unrelated population. patients and methods: we have previously described a som consisting of ( recipient and donor) factors (inputs) constructed from ( male; median age years; median meld score ) consecutive primary liver transplants undertaken between / / and / / in the queen elizabeth hospital, birmingham, uk. using a neuron version of this som with three output functions (patient survival at months; year and years) we used the som to classify the post-transplant survival of all primary graft recipients (n= patients) over a year period from a north american centre ( male; median age years; median meld score ). the birmingham (uk) patients were first classified by the som and then the probabilities of survival (p,) at months, year and years calculated by dividing the number of surviving patients by the total number of patients in neuron i. using this som, each of the wisconsin (us) patients was classified to each of the neurons and the distribution of survival probes for each neuron compared between the two populations using the chi squared test. results: there was no significant difference in the survival probabilities of patients in each neuron when the wisconsin population was compared with the birmingham population. thus, the birmingham som was able to classify successfully the survival of the wisconsin patient population at months, year and years following transplantation by using the same donor and recipient factors ( table ) . analysis of the inter-neuronal survival probabilities demonstrated that for month and year survival, neuron was associated with a significantly lower survival probability than neurons and (p= . for months; p= . for months). further, if recipients from neuron received livers from patients classified to neuron by "computer simulated transplantation", a proportion of patients then had a lower predicted survival probability (by som re-distribution to neuron ). this analysis indicates that patient survival post-transplantation is significantly infuenced by both the condition of the recipient and donor factors at the time of transplantation. conclusions: ( )som analysis provides an efficient and automated method for assessing the probability of survival of individual patients in an unrelated population at months, year and years following liver transplantation. ( )the model not only assesses the pre-transplant condition of the patient, but also considers a wide range of donor factors when predicting the probability of survival post-transplant. ( )this unique resource can match a single liver to a population of recipients most likely to die without a transplant whilst ensuring the best possible post-transplant survival for the most suitable recipient ( )som analysis can also assess the probability of survival of individual recipents matched to a range of possible donated livers when they are being considered for transplant programmes. purpose of study: to assess the efficacy of targeted prophylaxis in patients identified to be at highest risk of if after orthotopic liver transplantation. introduction: historically, invasive fungal infection (in) has complicated up to % of cases of orthotopic liver transplantation (olt). retrospective analysis has identified a variety of risk factors associated with a high risk for the subsequent development of ifi. these include patients undergoing retransplantation, transplantation for fulminant hepatic failure (fhf), requiring haemodialysis and prolonged intensive care unit (icu) stay. prophylactic anti-fungal therapy is safe and can reduce the incidence of ifl in olt recipientri. in recent years our unit has moved towards targeting prophylaxis to higher risk transplants. we assessed the efficacy of our policy in reducing the incidence of invasive fungal infection. methods: a retrospective audit was conducted comparing two groups of adult olt recipients over two year time periods, - when targeted prophylaxis was not used (group ) and - when targeted prophylaxis was used (group ). data were collected with respect to risk factors for ifi, anti-fungal prophylaxis use and development of ifi. results: there was no difference in the overall number of risk factors for if associated with olt between the two groups. however, a higher proportion of patients in group had high risk factors for if compared to group (p= . ). there was a significant difference in the use of targeted anti-fungal prophylaxis given to high risk patients in group compared to group ( . % cf . %, p= . ), and there was a significant reduction in the number of ifis in group compared with group ( . % cf . %, conclusion: a policy of targeting anti-fungal prophylaxis to highest risk liver transplant recipients leads to a significant reduction in if this group. there remains a background incidence of infection in low risk or long term recipients in whom prophylaxis is not given. background: only few studies have described risk factors associated with cellular rejection. the diagnosis of cellular rejection requires histology. there are no data evaluating the absence of rejection in a protocol biopsy population. aim: to assess predictive factors associated for the absence of cellular rejection in a protocol liver biopsy population and to evaluate the usefulness of protocol liver biopsies. method consecutive patients transplanted on a data-base at our centre. protocol liver biopsies were performed between and days post transplantation and then when clinically indicated, during the first months. rejection was scored prospectively. the following variables were examined with respect to absence of rejection over months and in the first protocol liver biopsy: a) donor factors: age, gender, race, donorlrecipient gender match, abo match. b) pre-transplantation recipient factors: age, sex, aetiology, ascites, oesophageal varices, tips, encephalopathy grade, renal support, ventilation, total bilirubin, albumin, inr, ast, alt, creatinine, urea, c) graft and surgical factors: surgeon's visual assessment of graft, cold ischaemic time, use of venovenous by-pass, intraoperative blood transfusion and initial maintenance immunosuppression of either cyclosporin or tacrolimus in triple-dual or monotherapy. standard treatment of rejection was g iv daily of methylprednisolone x days. results: absence of rejection over months was in ( %), mild in ( %) and moderatelsevere in ( %). in the first biopsy: absence of rejection in ( %), mild in ( %), moderate in ( %) and severe in ( . %). univariate analysis showed for both first protocol biopsies and months, that variables were significantly correlated with non rejection: pre-transplantation use of renal support (haemodialysis or hemofiltration), (p= . ), cold ischaemic time > hours (p= . ), suboptimal graft visually (p= . ) and blood transfusion <= units cp= . ). no correlation was found between aetiology of liver diseases, initial maintenance immunosuppression. conclusion: a suboptimal graft with prolonged cold ischaemic time in recipients with previous need of renal support seems to be associated with absence of rejection. these data are in contrast with previous reports. these factors may be helpful in identifying patients who do not need to be submitted to protocol liver biopsy. tables and child developed abnormal transaminases and was found to have chronic hepatitis on liver biopsy and was treated with steroids. required adjusting cyclosporine dosage to maintain trough levels in the identified range (range - microgramll as per protocol). summary: there was good correlation between the trough levels taken on occasions in the stable paediatric post liver transplant group of patients. the range of c peak levels were also similar suggesting good bioavailablity conclusion: this preliminary study on long term post transplant recipients suggests that a peak c level is within the range of - microgramll. background hepatocytes and cholangiocytes release substantial amounts of adenosine triphosphate into bile, where it is rapidly degraded by membrane-bound ecto-atpase and '-nucleotidase. this degradation process leads to the generation of adenosine and inorganic phosphate (i?#. whereas adenosine is reabsorbed in a sodium-dependent manner back into hepatocytes, little is known about the fate of biliary pi. in rat, biliary pi concentration is . mm, which is about fold lower than in hepatocytes ( mm) and fold lower than in plasma ( . mm) indicating active reabsorption of pi from bile canaliculi andlor from the biliary tree. aim: the purpose of the present study was to functionally characterize canalicular p, reabsorption in rat liver and to identify the involved p, transport system(s). methods: p, transport was determined in isolated canalicular liver plasma membrane (clpm) vesicles using a rapid filtration technique. identification of putative p, transporters was performed with reverse transcriptase-polymerase chain reaction (rt-pcr) from rat liver total mrna using sodiumlphosphate cotransporter specific primers for napi-iib (gggattgggaaattcatccti ttccaacacaaggttggtca), napi- subtype pit- (catctcggtgggatgtgcitgttgctctctcctccttca) and napi-i subtype pit- (gctctaccattggcttctcglaca-gaggaagtgcctggaga) . on the protein level, phosphate transporter expression was confirmed by western blot analysis in isolated basolateral (blpm) and canalicular (clpm) rat liver plasma membrane vesicles. specific polyclonal antibodies were raised in rabbits against antigenic peptides from mouse napi-iib and human napi-iiilpit- showing > % identity with rat homologues. results: transport studies in isolated clpm vesicles demonstrated sodium-dependent p, uptake (najut > nag pmollmin; k&t > kg pmollmin). initial na+-dependent p, uptake was linear for at least sec and exhibited a clear overshoot indicating transient intravesicular concentration of p, (secondary active p, transport). initial p, uptake rates ( sec) were saturable with increasing p, concentrations and exhibited an apparent k,,, value of - km. furthermore, sodium-dependent p, transport was higher at an acidic (phout = . ; ph,, = . ) as compared to an alkaline extravesicular ph ( . ). in addition, an intravesicular negative membrane potential stimulated sodium-dependent p, transport indicating a na:p, stoichiometry of > . these data are comparable with the transport characteristics of sodiumlphosphate cotransporters napi-iib, napi-iiilpit- and napi-iiilpit- . mrnas of all these three napi's were found to be expressed in rat liver by rt-pcr. however, on the protein level only napi-iib was found to be expressed selectively in clpm, whereas napi-iiilpit- was detected in blpm. no clearcut localization of napi-iiilpit- protein could be obtained. conclusions: the canalicular membrane of rat hepatocytes localizes the sodiumlphosphate cotransporter napi-iib, which can reabsorb p, from primary hepatic bile back into hepatocytes. in contrast, napi-iiilpit- was found to be expressed at the basolat-era hepatocyte plasma membrane. the results indicate that napi-iib regulates p, concentration in bile and may play an important role in the overall p, homeostasis in rat liver backgroundlaims. organic anion transporting polypeptides (oatps) are a family of transport proteins of the basolateral hepatocyte membrane. human oatp (slc a ) is predominantly expressed in hepatocytes and is an uptake system for xenobiotics such as digoxin. the oatp gene promoter possesses an inverted repeat (ir ) element at nt - l- relative to the transcription start site, that binds and is activated by the farnesoid x receptorlretinoid x receptor (fxrlrxr). ligands of fxr include bile salts such as chenodeoxycholic acid (cdca), previously shown to activate the oatp gene. however, because oatp is only a poor bile salt but an efficient xenobiotic transporter, we investigated whether xenobiotics such as rifampicin regulate the oatp gene. rifampicin is a prototypic ligand of the xenobiotic receptor pxr (pregnane x receptor). methods. endogenous oatp mrna levels in caco cells were quantified by real-time pcr. an oatp gene promoter construct containing nucleotides - l + relative to the transcription start site (luc- ) was assayed for reporter activity in transiently transfected cells. the fxr binding site in the oatp gene (ir element) was characterized using the luciferase construct ir -tk-luc that contained a thymidine kinase promoter under the control of the irl element. results. endogenous oatp mrna levels in caco cells were induced fold by cdca ( pmolll). interestingly, incubation of cells with rifampicin ( pmolll) resulted in a . fold increase in oatp mrna levels. to study the mechanism of rifampicinmediated induction of oatp , the promoter sequence was searched for potential pxr binding sites. because the ir element, previously shown to bind the fxrlrxr heterodimer, was the only nuclear receptor binding site found, we hypothesized that the induction by rifampicin could be mediated through the fxr element. we, therefore, studied the effect of cdca and rifampicin on the ir element. in cells cotransfected with the ir -tk-luc construct and expression plasmids coding for fxrlrxr, pxrlrxr or car (constitutive androstane receptor)lrxr, fxrlrxr induced the irl element llfold in the presence of cdca and . fold in the presence of rifampicin, indicating that rifampicin is capable of activating fxr. in contrast, pxrlrxr or carlrxr did not activate the ir element in the presence of cdca or rifampicin. to confirm that an oatp promoter construct is also activated by rifampicin, huh cells cotransfected with the luc- construct and fxrlrxr expression plasmids were incubated with cdca or rifampicin. cdca induced oatp promoter activity -zfold, rifampicin - fold, confirming that rifampicin transactivates the oatp promoter. to investigate whether other xenobiotics also activate the fxr element, huh cells cotransfected with the ir -tk-luc construct and fxrlrxr expression plasmids were incubated with rifampicin, rifamycin, ru , clotrimazol, phenobarbital or pcn. a . fold (rifamycin) to . fold (clotrima- ) induction of the ir element was seen in the presence of these xenobiotics. conclusions. the human oatp gene is induced transcriptionally by xenobiotics that represent prototypic ligands of the xenobiotic receptor pxr. however, activation does not occur through pxr but through activation of fxr bound to the ir element in the oatp gene promoter. the data thus indicate that pxr ligands such as rifampicin and clotrimazol can also activate fxr and thereby induce the fxr-regulated transporter gene oatp . disclosures: may-britt becker -no relationships to disclose michael fried -no relationships to disclose diana jung -no relationships to disclose gerd a kullak-ublick -no relationships to disclose peter j meier -no relationships to disclose epidemiologie de population epi seema sonnad -no relationships to disclose preservation of canals of hering mitigates bile duct loss in liver graft rejection. ivlarius c van den michael angelis -no relationships to disclose jeffery cooper -no relationships to disclose erick edwards -no relationships to disclose richard b freeman jr -no relationships to disclose ann harper -no relationships to disclose abigail mithoefer -no relationships to disclose no relationships to disclose the data was not available. the ca - , cea, and afp levels were obtained using standard commercially available assays results: out of a total of patients with esld fifty-eight patients fulfilled the inclusion and exclusion criteria. of these, thirty-three patients had evidence of ascites and twenty-five did not. the etiology of liver disease in the two groups was similar. the mean levels of ca - , cea and afp levels in patients with ascites were not significantly different from those without ascites (ca - . % furthermore, out of these patients with esld had levels -> ulml, the upper limit of normal in this assay unitslml] vs . . conclusions: ascites does not seem to make an impact on the serum levels of ca - chhaya hasyagar -no relationships to disclose savant mehta -no relationships to disclose severe mitochondrial toxicity after liver transplantation in hiv-hcv coinfected patients liver transplantation (lt) may be the only potentially curative treatment available to these patients at this stage. however its feasibility and benefit has still to be established. severe recurrence of hepatitis c on the liver graft may complicate the post operative course as recently suggested by us and others. mitochondrial toxicity of highly active antiretroviral therapy (haart) may also play an active role on the liver graft of htv-hcv co-infected patients. we aimed to study this complication on the liver graft of hiv-hcv coinfected patients. patients and methods: between he had an history of episode of pancreatitis and haart therapy was azt- tc-nelfinavir. at m post lt, microvesicular steatosis was noted. at m a low content of liver mtdna was found (mtdnalnuclear dna = . ). other patients had microvesicular steatosis respectively at m , m and m post lt. one of these patients had low content of liver mtdna (mtdnalnuclear dna = . ). severe defect of complex iv of the respiratory chain was noted in of these patients. no deletion of mtdna was observed by southern blot or long range pcr conclusion: mitochondrial toxicity on the liver graft may become a major problem during post lt c o m e and could worsen graft lesions related to hcv recurrence on the liver graft in hiv patients daniel azoulay -no relationships to disclose henri bismuth -no relationships to disclose denis castaing -no relationships to disclose duclos-vallee -no relationships to disclose cyrille feray -no relationships to disclose michelle gigou -no relationships to disclose catherine guettier -no relationships to disclose philippe ichai -no relationships to disclose claude jardel -no relationships to disclose anne lombes -no relationships to disclose bruno roche -no relationships to disclose faouzi saliba -no relationships to disclose didier samuel -no relationships to disclose elina teicher -no relationships to disclose daniel vittecoq -no relationships to disclose a identification of sodiumlphosphate cotransporter type iib marek nowicki, usc, los angeks, ca; jorge rakela, tomasz laskus, there is growing evidence that patients with chronic hepatitis c are more likely to have significant changes in their physical and mental well being, commonly manifested as fatigue and depression, than patients with liver disease of other etiology. recently published studies demonstrated also that hcv infection is associated with cognitive dysfunction. hepatitis c virus (hcv) was reported to replicate in monocyteslmacrophages and lymphoid cells. we have recently demonstrated that leukocytes carry hcv across the blood-brain barrier and we found hcv rna in the central nervous system (cns) in some infected patients (j virol , , - ; , - ) . however, biological basis for neurocognitive abnormalities observed in hcv-infected patients remains unclear. aim: to determine the pattern of gene expression in cns in hcv-positive patients as compared to hcv-negative controls. material and methods we analyzed samples of brain tissue obtained at autopsy from hcv-positive patients and hcv-negative control patients. all were men of similar age, none was infected with hiv. all hcv+ patients and out of controls had liver cirrhosis. only deaths (one in each group) were liverrelated. the analysis of gene expression was conducted using three different techniques: differential display (genhunter inc), reverse northern, and microarray analysis (bd atlas plastic miroarray). reverse northern analysis was used for confirmation of differential display findings. analysis of microarray data was done using atlas image . (bd) and cluster . and treeview . (m.eisen; ucb). only those genes that were up or downregulated . times in reverse northern and/or microarray analysis were considered differentially expressed. results: the most striking finding was downregulation of mitochondria] oxidative phosphorylation genes in all hcv-infected patients as compared to controls; impairment of brain oxidative/ energy metabolism has been previously suggested to be the proximate cause of many disorders that impair mentation. another consistent finding in differential display and microarray analysis was downregulation of multiple ribosomal proteins genes and several genes involved in transcription regulation. these could indicate reduced metabolic activities perhaps secondary to deficiencies in oxidative phosphorylation. we also observed upregulation of several genes involved in immune response. there was upregulation of mhc class i and class i and several lymphokine receptors like interferon (alpha, beta), il- , il- , il- as well as lfa- ligand intracellular adhesion molecule icam- and ceacam . furthermore, upregulation of cd o and allograft inflammatory factor- gene (aif- ) suggested the presence of activated microglia cells and/or activated macrophages. conclusions: we found downregulation of oxidative phosphorylation genes and upregulation of genes involved in immune response in brains from hcv-positive patients when compared to hcv negative patients. our findings provide the likely substrate for neuropsychiatric symptoms and cognitive impairment associated with hcv infection. medicine, ehime, japan; emmett v schmidt, raymond t chung, massachusetts general hospital, boston , m a background/aims: we previously reported cell-based hcv replication using a novel binary expression system in which cells were transfected with a t polymerase-driven full length hcv cdna plasmid (pt -flhcv-rz) and infected with vaccinia-t (pnas ). to circumvent vaccinia-induced cytotoxicity, we sought to determine whether replication-defective adenoviral vectors expressing t or cell lines stably transfected with t could support hcv replication. because vaccinia interferes with pkr function, we further sought to define the antiviral activity of interferon-ar (ifn) in these models. methods: hours after transient transfection of cv- and huh cells with pt -flhcv-rz, cells were treated with recombinant replication-defective adenovirus vectors expressing t polymerase (ad-t pol). medium with or without iulml of ifn was changed at day post-infection and every days thereafter. cells were harvested on day , , , , and post-infection. for t -stably transfected cell lines (huh-t ), we transiently transfected with pt -flhcv-rz, then added ifn to selected cells in analogous manner. hcv positive and negative strand were measured by strand specific real-time rt-pcr. hcv core protein expression was assessed by western blotting. results: in the hcv replication system with recombinant adenovirus vectors and with t stable cell lines, no cytotoxicity was observed at days. with pt -flhcv-rz and ad-t'lpol, hcv positive and negative strand rna expression was strongest in the first days post-infection and diminished thereafter, but were expressed throughout the days. in the days after adding ifn, hcv positive strand was significantly decreased ( . . vs. . + . in cv , . t . vs. . ? . in huh , hcv/ gapdh copy ratio, pc . ) than the samples without ifn. sustained expression of hcv rna and ifn inhibitory effect was also observed in t -stable huh-t cell lines. pkr expression was strongly induced by ifn, suggesting that pkr is appropriately induced by ifn. conclusions: we have successfully substituted recombinant adenovirus vectors or t stable cell lines in our cell-based binary hcv replication system. in these systems, ifn inhibits hcv replication, and upregulates the pkr pathway. these improved binary systems are a durable and more authentic model for identification of host cellular processes critical to hcv replication. disclosures:jason blackard -no relationships to disclose raymond t chung -no relationships to disclose yoichi hiasa -no relationships to disclose norio horiike -no relationships to disclose yoshitaka kamegaya -no relationships to disclose morikazu onji -no relationships to disclose emmett v schmidt -no relationships to disclose the meld scoring system for the allocation of cadaveric donor liver for transplantation gives significant priority to cirrhotic patients with a small, t or t , hepatocellular carcinoma. patients on the wait list were given adjusted meld scores equivalent to -month mortality rates of % and % for t and t lesions, respectively based on theoretical tumor doubling times. frequently these patients have compensated cirrhosis and hence their intrinsic meld score is lower, yet they are not candidates for surgical resection secondary to the underlying cirrhosis. to assess whether the additional meld points for hcc is valid, we sought to examine the impact of the meld scoring system on a single center's waiting list one year after implementation.methods: records of all patients undergoing liver transplantation (oltx) at uthsc-san antonio from feb , through feb , were retrospectively reviewed. pathology and radiology reports as well as data from unet were collated for analysis and review. results over a -year time period following implementation of the meld system, patients underwent oltx at our center. of these were excluded from analysis because of the following reasons: status (lo), living-related recipient ( ), pediatric non-tumor ( ). the table below summarizes the pertinent findings. of the remaining patients, ( %) underwent oltx with suspected hcc ( tl, t ).none of the hcc patients had their meld score downgraded because of tumor progression (t -t ). at transplant, patients were found to have extra hepatic disease(one with known hcc and the other unknown) and the cases were aborted. sensitivity of radiological evaluation for the presence of hcc was . % and specificity %. the calculated meld score at the time of listing for oltx and at the time of oltx was significantly lower for those patients thought to have an hcc. exception points for hcc gave those patients a significantly higher average meld score at the time of oltx than patients without hcc. patients wlo hcc had a aupper; true meld of + . at the time of oltx compared to listing as opposed to hcc patients whose a-upper; true meld was - . . during the study period patients (none with suspected hcc) died on the waiting list or were too sick to transplant while new patients were listed compared to dying the prior year with new registrants ( . % vs . %). mean meld score of those dying during the study period was . (median . ) and mean time on the list was days (median days). of those patients dying. had a meld score of . conclusion: meld has effectively prioritized cadaveric donor livers to the sicker patients. however, patients with hcc are given too much priority as evidenced by all of the wait list deaths occurring in patients without hcc and lack of tumor progression (to t ) sufficient to loose meld exception points in those with hcc. likewise, the mean meld score of those dying was less than that given to patients with t lesions( . vs. ). the recent downward adjustments of meld exceptions to and for ti and t lesions, respectively, are reasonable. ongoing analysis will be needed to accurately place hcc patients in the current allocation system. there was no significant difference (p> . ) in weight gain between the sexes, those with a bm > pre-transplant and those with a bmi< pre-transplant or those on prolonged courses (> months) of steroids. weight gain was significantly greater (p< . ) in patients aged over compared to those under and those transplanted for chronic liver disease compared with fulminant liver failure. a pre-transplant bmi> was a strong indicator that the patient would still have a bmi> at years. there was no effect of immunosuppression on weight gain: corticosteroids were discontinued in . % by months and long term use of steroids was not associated with weight gain. weight gain was similar in those on cyclosporine and on tacrolimus.conclusions: a bm > o is common in the liver transplant population, but seems to unrelated to specific immunosuppression and may be more closely linked to lifestyle. most weight gain occurs after the first months, and intervention with dietary and lifestyle advice at this point could be implemented to minimise the long-term morbidity and mortality risks associated with obesity. serum tumor marker estimations are often performed as a part of evaluation for liver transplantation. there is a paucity of information on the effect of esld on the levels of these tumor markers making interpretation difficult one tumor marker, ca- , has been consistently reported to be elevated in patients with ascites from liver disease. purpose:-to compare the levels of serum ca - , cea and afp in patients with esld with or without ascites.-to compare the serum ca - levels in patients with esld vs normal controls methods: all patients referred for liver transplantation between jan and dec at our center were included in the study. men with end stage liver disease (esld) develop a plethora of debilitating symptoms, including severe muscle wasting, that render many of these patients non-suitable for liver transplantation. esld-associated hypogonadism, i.e. testosterone deficiency, may, in part, be responsible for the development and progression of many of these incapacitating symptoms. in general, hypogonadism can be effectively treated with topical testosterone replacement (ttr), which avoids the first pass effect via the hepatic system in contrast to oral anabolic steroids. ttr has been shown to effectively improve muscle mass and overall well being in patients with hiv. however, little is known about the effects of ttr in men with esld. the aim of this study was to determine the potential benefits and safety of ttr in patients with esld and muscle wasting. method:the medical records of liver transplant patients treated for at least months with testosterone gel % ( grams per day) therapy for muscle wasting (mw) from january to march were reviewed. information collected included; demographics, albumin, pre-albumin, transferring, testosterone, estrogen, lh and fsh. drug safety results were also collected and included, acute cellular rejection, cholestasis, malignancy, and patient or graft loss. tumor markers for afp, ca - , cea, psa, prolactin levels and radiographic imaging was reviewed to rule out and potential malignancy. patients not treated with testosterone but with a similar clinical setting were compared (group ). results: thirteen patients were identified with esld and mw, group (n= ), group (n= ). demographics were as follows: (group ) males, females, and mean age years; (group ) males, female, mean age years. disease etiology for group : (hcv (n= ), hcv/hiv (n=l), aih (n=l), cryptogenic (n=l)) group : hcv (n= ), hcvilaennec's (n=l). most patients in group stated a subjective improvement in muscle strength, and overall wellbeing. in group , serum albumin levels increased from day , , ( . / . / . g/dl, respectively) while those in group did not ( . / . / . g/dl) (p= . ). similar results were seen for prealbumin (days , , ) group ( / / mgldl) versus group ( / / mg/dl). in group , / patients received a liver transplant, while remains listed for olt. in contrast, / patients in group were transplanted while / died. two patients in each group were not listed at the time of evaluation due to extreme deconditioning. of those in group , one patient was transplanted and another patient remains listed for olt with an albumin greater than . gldl. this patient presently has no radiographical evidence of ascites. the two patients of group , who were not listed for lt due to extreme deconditioning, died on day and , respectively. there was no rise in the tumor marker elevations in any of the patients. after transplantation, no patients in either group suffered from rejection or graft failure. ascites accumulation, as assessed by abdominal ultrasound imaging, appeared to be less in patients receiving ttr (group ) compared to all living patients in group , who had persistent ascites. one patient of the treatment group developed marked cholestasis days after starting ttr and received a liver transplant days later. prior to starting ttr, this patient underwent weekly large volume paracentesis and suffered from marked deconditioning and recurrent bouts of hepatic encephalopathy while maintaining a meld score of - points over a -month period. conclusion: our preliminary data suggest that ttr increases muscle strength (subjective measures), stimulates albumin synthesis and improves the outcome of olt. thus, ttr (testosterone gel %) appears to be of great benefit in patients with esld and muscle wasting. further studies are needed to determine the efficacy and safety of ttr in patients with esld. percutaneous ethanol injection (pei), radiofrequency tumor ablation (rf), and laser thermal ablation (lta) are percutaneous techniques used in the treatment of unresectable hcc. percutaneous seeding of neoplastic cells along the needle track is a rare complication of these tecniques, and it has been recently suggested to consider with caution these techniques as a "bridge" treatment in cirrhotic patients with hcc candidates to lt. the aim of this study was to verify whether percutaneous ablation treatments represent a risk factor for hcc recurrence in these patients. during the period - , patients (mean age yrs) with hcc complicating liver cirrhosis previously treated with percutaneous ablation treatments underwent lt in italian centers. among the patients, had monofocal hcc, multifocal hcc. in the latter group, had monolobar disease, had bilobar disease. considering the patients with monofocal hcc, were treated with rf (associated with transarterial chemoembolization [tace] in one case), with pei (associated with tace in one case), with rf and pei (associated with tace in one case). among the patients with multifocal hcc, were treated with rf (associated with tace in four cases), with pei (associated with tace in four cases), with lta associated with tace. on the whole, nodules were treated in the patients. furthermore, additional untreated hcc nodules were found at pathological examination of the explanted liver. among the rf-treated nodules, showed total necrosis, partial necrosis, absence of necrosis. among the pei-treated nodules showed total necrosis, partial necrosis, absence of necrosis. the nodules treated with rf and pei showed total necrosis in one case and partial necrosis in the other one while the nodules treated with lta, showed partial necrosis. the mean follow-up period was months (range - months). five patients died during the follow up and the actuarial survival rate was % at one year, % at and years. three patients ( . %) had post-transplant recurrence of hcc which was the cause of death in all cases; the diagnosis of tumor recurrence was made at months from lt (peritoneal carcinomatosis), at months from lt (bone), and at months from lt (abdominal liymphnodes and bone), respectively. the patients had all been treated with pei before lt and no one of them showed recurrence of hcc at the abdominal wall level. according to our results, percutaneous ablation techniques performed before lt do not seem significantly affect both survival and tumor recurrence rate in patients transplanted because of hcc complicating liver cirrhosis. in particular, no cases of recurrence at the abdominal wall level were observed in the overall series and no one case of hcc recurrence was observed in patients treated with rf alone or in combination. background: transjugular intrahepatic portosystemic shunt (tips) is valuable in the management of portal hypertension (phtn) in patients awaiting liver transplantation (olt). recurrent phtn after olt can be refractory to medical management and portosystemic shunting may be considered in rare situations, either as a bridge to retransplantation or as definitive therapy. in this report we review our experience and outcomes with tips after olt. methods: records of primary adult olt recipients, between january and december , were retrospectively reviewed. evidence of refratory post-olt phtn was noted. those who required tips were the subject of this review. demoraphics, indications for olt and tips, evidence of allograft dysfunction and outcomes after tips were described. results: during the study period tips were placed in patients at a mean of . months after olt (range - months). there were males and females, age . l . years. hepatitis c was the primary indication for olt in and primary biliary cirrhosis in . indications for tips included refractory ascites ( ), variceal bleeding ( ) and various degree of associated hepatic vein outflow stenosis ( ). five patients had resolution of phtn and patient with refractory ascites had severe hepatic vein outflow stenosis and associated hepatitis c in the allograft. two patients required re-olt for recurrent hepatitis c. there were deaths: liver failure month after tips done in the setting of allograft dysfunction, months after tips with subsequent re-olt and organ failure, and lung cancer months after tips. bridging fibrosis was present in patients, needed re-olt and died from liver failure while waiting for olt. currently, patients are alive without evidence of phtn , and months after tips. conclusions: -tips effectively and safely controls phtn after olt. -tips in setting of a moderate to severe allograft dysfunction does not abrogate the need for re-olt and can be associated with a high mortality. -timely re-olt should be considered in the presence of fibrosis from recurrent hcv and phtn. -tips has been successful in the setting of mild-moderate hepatic vein outflow stenosis and phtn. introduction: biliary tract complications after liver transplantation (lt) are reported to occur in % to % of patients. the frequency of biliary infections is not well documented. therefore, we prospectivly obtained bile samples during diagnostic and or therapeutic endoscopic retrograde cholangiography (ercp) in all liver transplant patients from to . methods: in patients (mean age . years, range - years) a total of ercp's were performed ( . ercflpatient). all patients had a lumen adapted, end-to-end biliary anastomosis. lt was performed between . months and . years before the intervention. cholangitis was defined as the presence of cholestasis with clinical and biochemical signs of infection not explained otherwise. only in cases of interventions, positive culture results were combined with clinical signs of cholangitis. the follwoing risk factors were identified stenosis of any type, plastic endoprothesis, choledocholithiasis and previous papillotomy. patients with these risk faktors had significantly higher incidence of positive bile cultures ( . % vs %, p<o,ool; chi-square). in addition, they were more often infected with multiresistant bacteria (p< . ). . % were resistant to gentamycin, . % to ceftriaxone. vancomycin ( . % resistance) was the most effective antibiotic. imipenem ( . %), ciprofloxacin ( . %) and amoxicillin ( . %) had intermediate incidence of resistant bacteria. conclusion: in patients with biliary tract complications after lt, biliary infection is common. clinical cholangitis, however, occurs predominantly in those with risk factors. gram-positive bacteria were more commonly demonstrated than gram-negative. cocolonisation with anaerobes andlor candida can occur. ciprofloxacin and amoxicillin seem to be appropriate antibiotics for prophylaxis. selection of an empiric antibiotic therapy for patient after liver transplantation with sings of cholangitis should be directed toward agents with a low incidence of resistance or a combination therapy. disclosures: thomas buratti -no relationships to disclose adolescent health related quality of life after liver transplantation. shikha s sundaram, katie neighbors, lisa amaruso, james sinacore, estella m alonso, northwestern university, chicago, il background: adolescence is an important developmental stage, during which children with chronic diseases begin to assume some of the responsibility for their own medical care. many children who received liver transplants as infants are now reaching adolescence and their unique health perceptions should be considered in providing appropriate healthcare. the aims of this study were to ) quantify the health related quality of life (hrqol) in adolescent liver transplant recipients and compare this to a published healthy population, ) compare parental ratings of adolescent hrqol to adolescent self-reports of hrqol, and ) assess adolescent hrqol and it's relationship to disease specific disability. methods: a cross sectional mailing survey of liver transplant recipients between ages - years, surviving transplant by at least year and receiving care at our center was conducted. primary caregivers of these patients were also recruited. adolescents completed the child health questionnaire, child form (chq-cf ) and primary caregivers the child health questionnaire, parent form (chq-pf ). demographic and medical data was collected using an institutional transplant database and standard demographic survey. a disease specific disability score was calculated using the liver transplant disability scale (ltds). mean scores for chq-cf subscales, including general and mental health, physical and social functioning and self-esteem were calculated. these were compared to published normative values from a healthy population and mean chq-pf subscale scores. results: thirty-seven of surveys mailed were received for a response rate of . %. adolescent respondents had a mean age of . . years, were % female and % caucasian. cadaveric donors accounted for . % of transplants and % of patients were transplanted for biliary atresia. a majority ( . %) of respondents had private insurance and . % indicated mothers were their primary caregivers. after transplantation, adolescents had a mean global health score of . . as compared to mean normative score of . . , p= . . they had higher scores than the normative population for role behavioral, . . vs. . . , p=o.ool and mental health, . z . vs. . . , p= . . all other scores were similar between the groups. significant positive correlations between adolescent and parental scores were observed in relation to physical functioning, (r = . , p< . ), self-esteem (r = . , p < . ) and mental health (r = . , p< . ). the remaining sub-scales lacked statistically significant correlation. an inverse relationship between inpatient hospitalization during the previous months and mean global health score was observed inpatient days had a mean of . ,l- inpatient nights had a mean of . and greater than inpatient nights . . . there were no other significant correlations between subscale scores and ltds scores. conclusion: adolescent liver transplant recipients report their hrqol as equal or better than the normative population. parents may be adequate proxies for some, but not all domains of adolescent hrqol in this population. were quantitated from wild type and fxrnull mice. results:transfection of the moatp -pgl into hepg cells led to a . fold ( . ) increase in reporter gene activity over vector controls. co-transfection with the bile acid receptor fxr and addition of its ligand, chenodeoxyacholic acid (cdca), resulted in a further . ( t . ) -fold increase suggesting fxr-mediated transactivation.fxrlrxr cotransfection and addition of ligands, cdca and -cis retinoic acid decreased transactivation suggesting rxr-mediated antagonism ( jbc, : ,' ). hnf- a also transactivated moatp promoter as shown previously. mutation of ir- sequence in moatp promoter abolished fxr-transactivation. emsa analysis using rat liver nuclear extracts showed specific binding of nuclear proteins to moatp fxre which was supershifted by fxr and rxr antibodies. mrna levels for moatp in fxrnull mice were reduced by . % compared to wild-type mice ( . in wild-type vs . infxr-null mice). conclusion: based on the above data, we suggest that moatp is regulated by bile acids by binding to fxr via ir- in its promoter. , and g a encoded for amino acid changes i f, a t, and v , in the third membrane spanning domain, the second nucleotide binding domain, and the second membrane spanning domain, respectively. the full-length coding regions of the reference mrp and three mutants sequences were inserted into the baculovirus genome. sf insect cells were infected with the baculovirus stocks, and membranes were isolated from the infected cells expressing mrp or the mutants. atpase activity was measured by determining release of inorganic phosphate by a colorimetric assay. atp-dependent transport of h-e g (estradiol-p-( p-d-glucuronide)) or h-e g (estradiol-p-( p-d-glucuronide)) was determined by uptake into inside out membrane vesicles collected on filters and counted by liquid scintillation. results: the total expression levels of mrp and the three mutants in the infected sf cells were comparable by densitometry of western blots of three levels of each protein, indicating total expression in sf cells was not influenced by the mutations. mrp atpase activity was significantly stimulated by the uricosuric probenecid lmm, the cholestatic pm, and the mild choleretic e g mm. although the control and stimulated at-pase activities of the v i mutant were not different from mrpz, the i f mutant had significantly less baseline and less stimulated atpase activities, indicating impaired function. a t had less probenecid-stimulated atpase activity, but activity stimulated by e g and e g was not different from mrp . ursodiol saturably stimulated atpase activity of mrpz by % with km = pm. although the three mutants transported h-e g with affinities not significantly different from mrp (km= pm), the vmax of i f was only . % ( pmollmglmin) and the vmax of v i was % ( pmollmglmin) compared to the vmax of mrpz ( pmollmglmin); the vmax of a t was not different ( pmollmglmin). ursodiol activated transport of h-e g ( nm) with a peak effect of % at pm; higher ursodiol concentrations (up to mm) returned transport to near control values. mrpz-mediated atp-dependent transport of h-e g ( nm) in the presence of ursodiol ( oopm) for the mutants differed from mrp (lo%, %, and % for i f, a t, and v , respectively). conclusions: ursodiol markedly activated mrp transport of e g. although ursodiol has several anticholestatic effects, these data show its direct effect on transport of mrpz substrates, and may suggest activation of mrp as a target in the treatment of cholestatic liver disease. the mrp mutants showed several important differences in activity, including changes in transport vmax, and transport in the presence of ursodiol, and activation of atpase activity by ursodiol, probenecid, and the mrp substrates e g and e g. further characterization of these mrp mutants and others will lead to a better understanding of mrp structure and function and may yield new therapeutic approaches. also, although e g is an established mrp substrate, these data show that its noncholestatic isomer e g is also an mrpz substrate, implicating mrp in the disposition of other estrogen metabolites, which can be interconverted to cholestatic compounds like e g. argentina; nicholas f larusso, mayo medical school, clinic and foundation, rochester, m n previous work from our laboratory is consistent with an important role for aquaporins (aqps), a family of water channel proteins, in canalicular bile secretion principally via agonist-induced exocytic insertion of aqps into the hepatocyte canalicular membrane. to further define the pathways and molecular mechanisms for water movement across hepatocytes, our aim here was to directly assess osmotic water permeability (pf) and activation energy (ea) in basolateral (blmv) and canalicular membrane (cmv) vesicles using stopped-flow spectrophotometry. scattered light intensity was used to follow changes in vesicle volume in response to gradients induced by sucrose solutions of different osmolarities. results: the time course of scattered light for blmv and cmv fit a single-exponential function. in blmv, pf was pm.sec-' ( oc) with an ea of . kcallmol; in cmv, pf was pm.sec-' ( oc) with an ea of . kcallmol. the aqp blocker, dimethylsulfoxide, significantly inhibited the pf of both basolateral ( pm.sec-l; - %) and canalicular ( ? pmsec-'; - %) vesicles consistent with the presence of aqps in both vesicle populations. experimental data for cmv from dibutyryl camp-treated hepatocytes fit best to a double-exponential curve implying the presence of two functionally distinct cmv populations; one population had pf and ea values similar to those of cmv from untreated hepatocytes and the other population had a very high pf ( pm.sec-l, oc) and very low ea ( . kcallmol). dimethylsulfoxide completely inhibited the high pf value in this second vesicle population. in contrast, the pf of blmv was unaltered by camp treatment of hepatocytes. conclusions: our data are consistent with the notion that the canalicular but not the basolateral membrane becomes more permeable to water after a secretory stimulus due to the insertion of aqp molecules. the data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation. disclosures: ariel j caride -no relationships to disclose sergio a gradilone -no relationships to disclose bing q huang -no relationships to disclose key: cord- -s n xij authors: jonsson, m. v.; brun, j. g.; skarstein, k.; jonsson, r. title: germinal centres in primary sjögren's syndrome indicate a certain clinical immunological phenotype date: - - journal: scand j immunol doi: . /j. - . . h.x sha: doc_id: cord_uid: s n xij ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin‐stained paraffin‐embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc‐like lesions were detected in / ( %) of the pss patients. seventy‐two pss patients lacking these structures (gc‐) were randomly selected for comparison. focus score was significantly increased in the gc(+) patients compared to the gc(–) patients (p = . ). in the gc(+) group, . % of the patients presented with anti‐ro/ssa compared to . % in the gc(–) group. anti‐la/ssb was detected in . % of the gc(+) patients compared to . % of the gc(–) patients. sixty‐one percentage of gc(+) patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gc(–) patients (p = . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc(–)). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -x p u authors: alitalo, a.; meri, t.; lankinen, h.; cheng, z.‐z.; jokiranta, s.; seppälä, i.; lahdenne, p.; brooks, c.; hefty, p. s.; akins, d. r.; meri, s. title: lysine‐dependent binding of ospe to the c‐terminus of factor h mediates complement resistance in borrelia burgdorferi date: - - journal: scand j immunol doi: . /j. - . . aj.x sha: doc_id: cord_uid: x p u serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid‐encoded, surface‐exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h‐binding proteins of approximately – kda has been described. the ospe‐related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe‐related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h‐binding regions of ospe‐related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c‐terminal regions of both human and mouse factor h (scrs – ) specifically bind to ospe‐related lipoproteins. we also found fhr‐ , whose c‐terminal scrs – are homologous to scrs – of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i‐v) in ospe that could directly interact with factor h. deleting the c‐terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c‐termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c‐terminal‐binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h‐binding regions were mutated to alanines, we observed that lysines in the factor h‐binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe‐related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c‐termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h‐binding proteins may account for their susceptibility to serum lysis. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -n mlxe p authors: nan title: cis annual meeting: immune deficiency & dysregulation north american conference date: - - journal: j clin immunol doi: . /s - - - sha: doc_id: cord_uid: n mlxe p nan a y.o. female was referred to our clinic with a history of multilineage cytopenias/evans syndrome, a history of idiopathic thrombocytopenic purpura, hemolytic anemia, chronic neutropenia, lymphopenia, and hypogammaglobulinemia treated with ivig. our patient was healthy until she was years old; at that time, she developed joint pain, rash, and bruising. she was found to have evans syndrome with idiopathic thrombocytopenic purpura (itp), neutropenia, and lymphopenia. she was initially diagnosed with lupus and was given steroids. her bone marrow biopsy did not conclude myelokathesis. when she was years old, she remained thrombopenic and was started on high dose of immunoglobulin replacement therapy. in ( years old), she developed polyarthritis in her upper and lower extremities. in ( years old), she had a severe nosebleed, for which she was admitted and treated with amicar twice; her platelets were found to be , k/ul. she received rituximab weekly for weeks resulting in an increase of platelet count to - k/ul. she recently (march ) had a splenectomy to remove her large spleen, and since then, her platelets have rebounded to - k/ul. in , she was placed on long-term immunoglobulin replacement therapy after being hospitalized for bilateral pneumonia for nights requiring iv antibiotics for treatment. in , she developed and was treated for another pneumonia. her family history is characterized by multiple members with autoimmune multilineage cytopenia as well as autoimmune diseases such as multiple sclerosis (mother), thyroiditis and enteropathy. on physical examination, she did not present with any warts and the remainder of her physical examination being unremarkable, except for her scar from her splenectomy and a cervical lymphadenopathy. immunologic evaluations showed igg mg/dl, iga < mg/dl, and igm mg/dl. cbc with differential and lymphocyte screen were as follows (cell/mm ): wbc . x , hemoglobin . g/dl, platelets x ; % neutrophils (anc: ), % lymphocytes, % monocytes, % eosinophils; absolute total t-cell number was ( - cells/mcl), cd + t-cells ( - cells/mcl), cd + t-cells ( - cells/mcl), natural killer cells ( - cells/ mcl), and absolute number of b cells was ( - cells/ mcl). she came to our clinic with her sister, who also had multilineage cytopenia and hypogammaglobulinemia, treated with monthly ivig; and her nephew whom had neutropenia. based on this family presentation all three underwent whole exome sequencing (wes). the patient, the patients sister and the patients nephew were all found to have a variant on cxcr (frameshift mutation on chromosome , p.val fs; refnt: tca; altnt: t). as an important note, the patient had a bone marrow biopsy, which did not conclude myelokathesis. in summary, our patient with trilineage cytopenia and hypogammaglobulinemia, without any warts or myelokathexis, had whim syndrome (warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis), which was discovered by studying her wes. with the identification of her specific diagnosis, this allowed us to discuss the potential future indication of plerifaxor (antagonist of the alpha chemokine receptor cxcr ). and equally important, we discussed family planning and future pregnancies given that the mutation is autosomal-dominant. ( ) submission id# taha al-shaikhly, mbchb , kathleen mohan, arnp , matthew basiaga, do, msce introduction: complement component- (c ) is shared by the classical, lectin and alternative complement activation pathways. c , a major opsonin, facilitates phagocytosis of encapsulated microorganisms. inherited c deficiency is rare and is associated with increased risk of bacterial infections. subjects with connective tissue diseases (ctd) and c nephritic factors can have low and occasionally undetectable c levels, yet they are at an underappreciated infectious risk. we hypothesize that excessive c consumption in secondary complement deficiency disorders (scd) is associated with higher risk of bacterial infections similar to primary complement deficiency disorders (pcd). objectives: to compare the rate of bacterial infections between pcd and scd patients and evaluate the association between c level and bacterial infection risk. methods: we performed a retrospective cohort study. subjects with an undetectable complement activity (ch ) or any of the complement components measured at seattle childrens hospital from - were included in our study. we recorded the number of infections, observation periods, diagnosis (pcd, scd and its underlying etiology), lowest complement component levels, and the immunosuppressive agents used. the date of birth, and date of lowest c level were considered as start points to calculate the observation periods for pcd and scd subjects respectively. infections requiring hospitalization or parenteral antibiotics were categorized as serious bacterial infections (sbis). descriptive analyses were performed to determine medians and ranges for continuous variables. differences in rates of bacterial infection were assessed using the chi-square and kruskal-wallis tests when appropriate. among subjects with ctds, we treated every c measurement as a single observation (n= , ) and studied the association between c concentration and the -day odds of having a sbi. multivariable logistic regression was performed to determine infection risk based on c level while controlling for contributing factors. results: we identified subjects with pcd, and subjects with scd. scd consisted of three subgroups (ctd-related (n= ), nephritic factor-related (n= ), and infection-related (n= )). collectively, ctd subjects had a lower median rate of sbi compared to pcd subjects (p = . ). subjects with ctd and c level < have higher rate of bacterial infection (of any severity) (p = . ) and of sbi (p = . ) when compared to ctd subjects with c >= at the beginning of observation period ( figure ). while controlling for immunosuppression level pediatric resident, baystate medical center faculty advisor, baystate medical center introduction: zap codes for a -amino acid enzyme, zap , a member of the syk-protein tyrosine kinase family that plays an important role in t cell development and activation. zap is phosphorylated at tyrosine kinase residues upon t cell receptor (tcr) stimulation resulting in tcr-mediated signal transduction with src family kinases. zap deficiency results in a rare t+b+ nk+ severe combined immunodeficiency (scid). we report a novel compound heterozygous mutation in zap leading to presumed absent zap function in an infant with a normal trec newborn screen and scid. case description: the patient is a term, fully immunized female, born to non-consanguineous parents who was hospitalized for rsv bronchiolitis at mo. at mo she developed an erythematous, papular rash on her face and extremities, nonresponsive to topical antifungal therapy. at mo she was re-hospitalized with rsv bronchiolitis and subsequently treated with multiple courses of antibiotics for presumed bacterial pneumonia followed by albuterol and oral steroids for possible reactive airways disease. during this course of treatment, her rash resolved. at mo she presented with failure to thrive (wt < . % for age), multifocal pneumonia and respiratory failure requiring intubation. bronchial alveolar lavage confirmed pneumocystis jiroveci pneumonia prompting an immune evaluation. total immunoglobulins were normal for age, however antibody titers to tetanus, diphtheria and streptococcus pneumoniae were absent. lymphocyte enumeration revealed elevated cd t cells and markedly diminished cd t cells, normal b and nk cells. t cell proliferation to mitogens (pha, pwm) and antigens (candida, tetanus) was absent, however t cells proliferated normally to stimulation with pma and ionomycin. trec number was normal by newborn screening, but was std deviations below the mean and would have resulted in a positive screen upon repeat. invitae gene scid panel revealed two variants of unknown significance, c. c>g (p.arg gly) leading to substitution of arg with gly and c. _ dupgcat (p.ile metfs* ) resulting in a premature translational stop signal expected to disrupt the last amino acids of zap protein. parental sequencing revealed these variants to be on opposite chromosomes. the patient was successfully treated for pjp pneumonia and has since successfully engrafted a / matched unrelated donor stem cell transplant. discussion: we report a novel compound heterozygous mutation in zap which we presume led to t+ b+ nk+ scid. our patients clinical presentation of failure to thrive, recurrent lower respiratory tract infections, dermatologic findings and pjp pneumonia are consistent with previously reported cases of zap scid. her paucity of cd t cells, abundance of cd t cells and absent proliferation to mitogens are also consistent with previously described cases of zap . normal proliferation of t cells when bypassing the tcr by stimulating cells with ionomycin and pma confirms a defect in the tcr. we believe this is the second documented case of missed scid by newborn screen in ma since the implementation of trec screening in . pediatric resident (pgy iii), goryeb children's hospital attending physician, pediatric and adult asthma, allergy and immunology, llc introduction: acute otitis media (aom) is one of the most common reasons for antibiotic use in early childhood. we explored the challenges when aom fails traditional therapies and immunologic evaluation does not identify a commonly described immunodeficiency. case description: an eighteen-month-old male presented with episodes of aom and recurrent purulent otorrhea requiring intravenous antibiotics. laboratory evaluation revealed a normal cbc, normal immunoglobulins (igg , iga , igm , ige ) and igg subclasses. lymphocyte subset panel was normal. initial responses to dtap and prevnar boosters were normal, however, there was rapid decline to tetanus and pneumococcal antibody titers. a sub optimal response to haemophilus influenza type b vaccine was noted. although vaccinated twice for mmr, he never mounted mumps specific igg. mitogen response to pha was normal with decreased responses to cona and pokeweed and no detectable tetanus nor candida responses. further investigation revealed decreased non-class and class switched memory b-cells. the patient was recently vaccinated to pcv and at the present time has protective titers. discussion: it has been previously suggested that decreased memory b cells may contribute to decreased antibody responses to select vaccine antigens resulting in recurrent aom in children. our case supports the need to investigate beyond typical immunologic screening for immunodeficiencies. introduction: dna mismatch repair (mmr) system corrects replication errors in newly synthesized dna, and prevent recombination between dna sequences when they were not identical ( ) . msh is a part of mmr genes, ( ) ( ) ( ) . case: a ten-year-old girl presented with fever, brown spots on her skin, hair loss, recurrent pulmonary infections, arthritis on the left hand and right ankle. she has also been followed up with nf ( figure ). there was a first-degree cousin marriage between her parents. physical examination revealed findings of pneumonia and nf. anti-nuclear antibody, anti-ndna, anti-dsdna, anti-histone, anti ro and anti-nucleosome antibodies were positive. in her immunologic assessment showed low igg and iga levels associated with high igm level ( table ). the coexistence of nf, hyper igm syndrome, sle, were considered in the patient. intravenous ig ( mg/kg, every weeks) treatment was started due to hypogammaglobinemia. the frame shift mutation in exon of the msh gene was detected in the boztug's laboratory. in the follow up period, she admitted at years old with back pain. a mass in the left paravertebral area, related to the spinal canal and neural foramina, was detected at the l -l levels in spinal mri. the lymphadenopathy around the liver and hilum and the left parietal bone lesions were developed within two months despite surgical excision of primary mass ( figure ). as a result of pet examination; suvmax was found to be around . in the mass lesion in the paravertebral region and suvmax values did not exceed . in other lymphadenopathy and masses. atypical cellular infiltration suggesting neoplastic events, which were including small-medium size atypical pleomorphic mononuclear cells and t cells. since all these formations did not indicate definite cancer, chemotherapy was not started. interestingly, although chemotherapy was not given, progression stopped, and partial spontaneous regression was observed. discussion: the effect of msh mutations on patients may significantly vary with the inheritance pattern ( ) . leukemias or lymphomas are not common in heterozygote mmr gene defects ( , ) . however, homozygote mutations in mmr genes show a different pattern. wimmer and etzler proposed the new term constitutional mismatch repair-deficiency syndrome (cmmr-d) for patients who have a homozygous mutation in mmr ( ) . cmmr-d characterized by development of childhood cancers, mainly hematological malignancies and/or brain tumors, as well as early-onset colorectal cancers, and neurofibromatosis type ( ) . bi-allelic germline mutations in any of the mmr genes in which msh is involved increases hematological malignancies by % ( , ) . msh mutation has been associated with many cancers since its identification. leukemia, lymphoma, colorectal cancer, endometrial cancer, brain tumors are some of these cancer types ( ) ( ) ( ) ) . msh deficiency is an important disease that can affect different systems at the same time. there is a high risk of malignancy in the cases and therefore they must be closely monitored. this case has also shown that atypical lymphoproliferation may occur in msh homozygous mutant cases. (normal rage: - ) background: advances in inborn errors of human immunity have supported the discovery of new syndromes that are marked by striking features of autoimmunity and immune dysregulation often associated with cytopenias, lymphoproliferation, and a predisposition to reticuloendothelial malignancies leading to evaluation with hematologists/oncologists. moreover, hematologists/oncologists have also seen an increasing use of effector cell-based therapies, checkpoint inhibitors, immunomodulatory and targeted therapies resulting in autoimmunity and hyperinflammatory complications. a working knowledge of clinical immunology could help practicing hematologists/oncologists in the identification and management of these conditions. objectives: to support the advancement of aspho members and the field by facilitating education regarding the best practices in diagnosis and management of immunological disorders. to create a platform for the development of collaborative clinical research in patients with hematological/oncological manifestations of immunological disorders or those requiring hematopoietic stem cell transplantation for a underlying immunological disorder. design/methods the aspho clinical immunology sig was initiated based on collaboration with the clinical immunology society (cis). aspho members who are pediatric hematology/ oncology clinicians, clinical researchers, and trainees are eligible to participate. we have established a steering committee with representatives from across the united states and canada with diverse clinical and research expertise. through regular teleconferences and annual in-person meetings, we have developed a platform to provide our members with a network of immunology resources to ensure a strong foundation of knowledge and tools to conduct clinical care and research pertaining to the diagnosis, evaluation, and treatment of patients with immunological disorders. results we currently support over members within our online community. several educational initiatives have been successfully launched. we have submitted an invited review to pediatric blood and cancer which provides a case-based review of primary immune regulatory disorders. we hosted the first immunology for hematology oncology practice (i-hop) cased-based webinar series. this series features case-based discussions of patients with primary immunodeficiency disorders presented by fellow trainees and mentored by senior clinicians. we will also be hosting an aspho webinar focusing on the laboratory evaluation of primary immunodeficiencies and immune dysregulation syndromes. we have also begun the process of laying the groundwork for clinical research initiatives. conclusion: the aspho clinical immunology sig seeks to serve as a collaborative resource for pediatric hematology/oncology clinicians and researchers. through the development of educational and research initiatives, we envision improving the care of patients with immunological disorders that are often managed by pediatric hematologists/oncologists. moreover, we hope to broaden our understanding and application of clinical immunology within pediatric hematology/oncology. we hope that this successful initiative will serve as a blueprint for the development of future collaborations with other specialty societies and patient groups. autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) is a rare autosomal recessive disease caused by aire gene mutations. clinical diagnosis is established by the presence of at least two components of the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and addisons disease. in europe, the classic presentation is widely recognized and nonendocrine autoimmune manifestations are rarely reported. a recent study of american apeced patients demonstrated a more heterologous presentation, with many nonendocrine manifestations including urticarial eruption, hepatitis, gastritis, intestinal dysfunction, pneumonitis and sjogrens-like syndrome, all uncommon in european reports. within the american cohort, % of patients developed a mean of three non-triad manifestations before reaching the classic triad. finding of aire mutations and high-titer antiifn-autoantibodies is seen in both european and american cohorts. we present the case of two siblings, who demonstrate an apeced-like phenotype with both classical and atypical features. they share the same heterozygous c + _ + delinsct aire mutation. the older, an eight-year-old boy, with history of prematurity, bronchopulmonary dysplasia and onychomadesis in infancy, came to medical attention at months of age due to failure to thrive (ftt), in addition to fevers and urticarial rash lasting months after his mmr vaccine. the fevers resolved with anakinra, which was discontinued two years later due to pneumonia. from age - he developed an alps negative lymphadenopathy which self-resolved. lung issues include chronic cough, initially treated as asthma but with poor bronchodilator response, and frequent lung infections, including - pneumonias per year. at age five evaluation for ftt revealed growth hormone deficiency. two years later he was diagnosed with primary addisons disease. chronic abdominal discomfort, bloating, cyclical constipation/diarrhea, recurrent rashes, dystrophic nails, and sicca symptoms are also present. his sister, age five, shows ftt, but no growth hormone deficiency. at age one, she too developed a fever and rash syndrome lasting months. severe gerd and constipation started in infancy and are ongoing. at age three she developed a transaminitis, initially diagnosed as ebv, but later thought to be autoimmune hepatitis. she has frequent viral respiratory infections, and pneumonia at age two. she has had a chronic cough, with poor bronchodilator response, for most of her life. evaluation of seizure at age three showed normal brain activity. brain mri revealed partial agenesis of the corpus callosum and microgyria. her brother has similar mri findings. both children have had developmental motor delay and poor tone. brain dysgenesis and neurodevelopmental delay has not previously been described in apeced. although there were both typical and atypical symptoms, the history in combination with genetic findings led to further investigation of an apeced-like syndrome. autoantibody testing confirmed high-titer antiifn-autoantibody typical of apeced in both children and hightiter bpifb autoantibodies found almost exclusively in apeced pneumonitis in the brother. whole exome sequencing and copy number variation analyses are underway to further evaluate the patients condition. this case demonstrates the importance of clinical presentation in the evaluation of genetic results and in the guidance of therapeutic management. ( ) submission id# rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties are referred to our center for further evaluation. this study elucidates the demographic and laboratory characteristics of referred infants with transient or persistent idiopathic t cell lymphopenia (tcl) without clearly identified genetic or acquired etiology. methods: a retrospective analysis was performed from september (when trec screening started) through the end of december . descriptive statistics were calculated for demographic and laboratory characteristics. t-test or mann-whitney tests were used to compare laboratory variables. pearson or spearman tests were used to determine correlation between initial trec levels and t cell counts. by definition, the cd +, cd +, and cd + populations of transient tcl patients normalize by age year. results: eighteen infants with transient and with persistent tcl were identified. males comprised . % of the transient and . % of the persistent tcl cohorts. whites comprised . % of the transient and . % of the persistent tcl cohorts. the mean initial trec levels did not differ between the transient and persistent cohorts ( . vs. . trecs/l of blood, p = . ). mean initial absolute counts of cd + ( vs. cells/l, p < . ), cd + ( vs. cells/l, p < . ), and median initial absolute counts of cd + ( vs. cells/l, p = . ), were higher for transient vs persistent cohorts. initial trec level did not correlate with initial cd +, cd +, or cd + absolute counts. the median age of resolution for the transient cohort was . days (range - ). the absolute cd +, cd +, or cd + counts rarely exceeded the reported median values for age, and remained closer or below the th percentile for age up to days of life. the majority of both transient and persistent tcl patients demonstrated unremarkable lymphocyte proliferation to mitogens. conclusion: our centers transient tcl cohort appears to be predominantly male and non-white, whereas the persistent tcl cohort is more evenly distributed by sex but still predominantly non-white. the transient cohort had lower initial trec levels, but higher initial t cell counts. both cohorts appear to have relatively intact in vitro function. introduction: primary immune deficiency disease (pidd) is typically considered a pediatric illness, although advances in treatment and diagnosis are changing this paradigm. currently, data on pidd in older patients are very limited. objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd- codes d , d. . , d . , d . , d . , d . , d . , d . , d . , and d . . a total of records from geographically-diverse clinics were identified and characterized by age, gender, and pidd diagnosis. results: of the pidd patients in the ciic registry, ( %) were between - years of age (see figure) . within this age group, most patients were female (n= , %). the most common diagnoses among patients > years of age included common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d . ; n= , %) and antibody deficiency with near normal immunoglobulins (d . ; n= , %) . in comparison, the registry included ( %) patients aged - years; this age group was predominantly male (n= ; %). the most common icd- codes within the younger cohort were relatively evenly distributed between hereditary hypogammaglobulinemia (d . ), antibody deficiency with near normal immunoglobulins (d . ) , and common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d . ). conclusions: our data suggest that pidd in patients over age may be more prevalent than previously reported. additional research is needed to corroborate these findings, further characterize the nature of pidd in this population, and determine whether there are unique diagnostic and treatment considerations within this demographic. introduction/background: increased susceptibility to invasive infections with neisseria has been well documented in patients with deficiency of terminal complement proteins. the molecular attack complex is constructed with complement components c to c . a deficiency in complement c has been described previously in both african american and south african populations. complement c deficiency is inherited in a co-dominant pattern, with multiple known mutations. we present a case of a -year-old, previously healthy male, who presented with invasive n. meningitides infection. he was found to have a novel mutation noted on genetic sequencing of the complement c gene. objective: we present the case of a -year-old, previously healthy male, who presented with invasive n. meningitides infection. on genetic sequencing, he was found to have three mutations of the complement c gene. two of which have been described previously, and a third novel mutation. methods: a -year-old male with no known history presented to us with a -hour history of emesis. he was found to be febrile, and quickly decompensated, developing septic shock. blood cultures were drawn, and within hours grew n. meningitides. he was treated with broad spectrum antibiotics upon arrival, and subsequently narrowed to ceftriaxone. his hospital course was complicated by disseminated intravascular coagulation, as well as acute tubular necrosis, leading to endstage renal disease for which he is listed for kidney transplant. results: on immunodeficiency evaluation, he was noted to have an undetectable ch (< , reference range - ). complement levels returned with c of . (reference range - ) and c r of . % (reference range - %). complement c function screen returned at % (reference range . - %). all other complement levels were within normal limits. genetic sequencing showed the patient to be compound heterozygous for two of known four variants which have been reported to recur in african patients with complement c deficiency. this included c. del and c. del, which are predicted to result in frameshift and premature protein termination. he was also found to be heterozygous for sequence c g>a, which results in amino acid substitution p.arg lys. this variant is rare, with one large database reporting it in of alleles, and not in a homozygous state. it has not been reported in a case of c complement deficiency previously. conclusions: we present the case of a previously healthy -year-old male with invasive meningococcal disease. he is compound heterozygous for two mutations that have been associated with total complement c deficiency; however, he was found to have subtotal c deficiency. furthermore, he has a third novel mutation of the complement c gene. further investigation is warranted on the significance of this finding and impact on relevance to possible kidney transplant. background: measuring the function of the classical pathway of complement activation is useful in several disease states, including complement deficiency, autoimmune conditions such as systemic lupus erythematosus and certain forms of nephritis. the original method for assessing classical pathway activity was the haemolytic ch method, but this assay can be time consuming and has reagent stability issues due to the use of sheep red blood cells. there can also be high lab-to-lab variability due to differences in the protocols used. here we report the assay characteristics of an automated, commercial, liposome-based assay to measure ch activity. we also compare the results obtained using the traditional haemolytic method with the automated, liposome-based method used on the spaplus turbidimetric analyser. methods: a linearity study was performed based on clsi guideline ep -a. the linear range of the spaplus ch liposome assay was established by analysis of a series of sample dilutions and evaluation of results against pre-defined goals for recovery and %cv. precision was assessed based on clsi guideline ep -a over days. samples with different ch activities ( . - . u/ml) were run in duplicate, with two runs per day using reagent lots and different analysers. interference analysis was performed by spiking haemoglobin, bilirubin, chyle, ascorbic acid or saline (as a control) into samples before measuring the ch activity. for the assay comparison study, sera from routine patient samples were used. samples were collected from chulalongkorn hospital, faculty of medicine, chulalongkorn university, thailand. ch classical pathway activity was assessed using a haemolytic method and also using the liposome based ch assay for use on the spaplus turbidimetric analyser (the binding site ltd., birmingham, uk). c protein concentrations were also available for of these samples. results: the liposome ch assay gives a linear response over the range . - . u/ml, covering the measuring range of the assay ( . - . u/ml) at the standard analyser dilution (neat). the within run, between run and between day %cvs were all . %. the total %cv was . % in all samples. minimal interference was observed with the four common interferents tested. a significant correlation was observed between the two ch methods (p< . , r= . , y= . x± . ), with . % agreement between the methods in determining whether patients were above or below the lower limit of the assay normal range. the individuals in disagreement had normal ch results using the haemolytic method, and low ch values in the liposome assay. of these, c values were available for / , and had c concentrations below the lower limit of the assay normal range. conclusion: the liposome ch assay for use on the spaplus analyser has passed assay development guidelines based on those set out by the clsi for linearity, precision and interference, and there is a strong correlation between this automated assay and the haemolytic ch method used here. five additional patients with low c concentrations were defined as having a low ch using the spaplus liposome method compared to the haemolytic method. ( ) submission id# background/aims: rotavirus vaccine is a live viral vaccine that is part of the routine u.s. childhood immunization schedule. live viral vaccines administered to infants of mothers who received biologic medications during pregnancy can potentially cause vaccine-associated disease. infant death from disseminated mycobacterial infection after vaccination with bacille calmette-guerin (bcg) in infants whose mothers received infliximab during pregnancy has been reported. it is currently recommended that infants born to women who received biologic therapy during pregnancy not receive live viral vaccines, however there is a paucity of information regarding adverse events from live viral vaccines. we report two infants, born to mothers receiving infliximab during pregnancy, who tolerated the complete series of rotavirus vaccine. methods: two infants who received rotavirus vaccine and whose mothers received infliximab (monoclonal antibody against tumor necrosis factor alpha which blocks the inflammatory response) during pregnancy were identified and their charts were reviewed. each mothers chart was assessed for timing of the biologic doses during pregnancy and concurrent immunosuppressant therapy. results: the mother of the first infant had crohn's disease and received infliximab every weeks throughout her pregnancy (final infusion at approximately weeks estimated gestational age [ega] ). she did not take additional immunosuppressive drugs throughout her pregnancy. the infant was born at weeks ega. the infant received rotavirus vaccine at , , and months of age. the infant did not have coexisting medical conditions or recorded hospitalizations during the first year of life. there were no side effects from rotavirus vaccine documented during well child examinations. the childs growth was normal during the first year of life. the mother of the second infant also had crohn's disease and received infliximab infusions every six weeks during pregnancy until weeks ega. additionally, she took mesalamine (anti-inflammatory) daily. the infant was born at weeks ega. the baby had a brief and uncomplicated neonatal intensive care unit stay. she did not have medical conditions diagnosed at the time of birth, or in the first year of life. the child received rotavirus vaccination at , , and months of chronological age, and the infant did not experience documented adverse reactions. the child presented to the emergency department twice in the first year of life: once for thrush at months of age and once for viral gastroenteritis at months of age. the childs growth curve was unremarkable. conclusions: we report two infants, whose mothers received infliximab during pregnancy, who safely tolerated the -dose series of rotavirus vaccination. neither infant in this case series suffered from minor or severe adverse events as a direct consequence of receiving rotavirus vaccine. this suggests that administration of rotavirus vaccine may be safe in infants whose mothers received biologic therapy. introduction: combined immunodeficiencies (cids) can arise from partial loss of function variants in recognized scid genes, which can lead to relative lymphopenia with poorly functioning and oligoclonal t cells. cids have been most commonly associated with variants of the rag genes, but other genes are also implicated. clinical symptoms may be less severe, and the onset generally is delayed, compared to typical scid presentations. case report: a -year-old female presented with a history of recurrent and progressively worsening infections involving multiple microorganisms and organs, starting in infancy and requiring frequent hospitalizations. bacterial or viral infections included rhinosinusitis, otitis media, herpetic stomatitis, dental abscesses, pneumonias, pulmonary mycobacterial abscesses, cmv hepatitis, urinary tract infections, dermal abscesses, and groin hidradenitis. fungal and yeast infections included cryptococcal meningitis, oral thrush, dermatophytosis of the face, osteomyelitis of a finger, and onychomycosis. laboratory tests in showed: mildly low t cell counts ( /ul) with a reversed ratio of cd /cd t cells ( . ); almost absent b cells ( /ul) ; and low nk cell counts ( /ul). cd + t cells were mostly of the memory phenotype ( %). t cell development showed low counts of th cells. t-cell stimulation tests demonstrated poor proliferation responses (< %) to concanavalin a, tetanus toxoid, and candida albicans, with near-normal responses to pokeweed (> %) and pha (> %). she had low ig levels (iga , igm , ige < ), except for igg ( mg /ml; due to replacement since early childhood). limited genetic evaluation at age showed a heterozygous variant in the rag gene (g. t>c, c. t>c, p.met thr; nm_ . ). discussion: loss of function variants in rag or rag genes are known to cause a t-b-nk+ type scid. more than missense variants have been reported for rag , with disease-associated variants predominantly in zinc binding regions. the rag missense variant in our patient also lies within the zinc binding region (amino acids - ). the variant is rare (mean allele frequency . in gnomad) and has been identified in at least one other individual with scid (t-, b cell-, nk+). although classified as a variant of unknown significance, occurrence in at least two individuals with deficiencies of t and b cells-within a functionally important rag domainsupports an interpretation that the variant may be pathogenic. most patients with cid with rag variants are either homozygous for a poorly functional allele or have one nonunfucitonal and a second, poorly functional allele. we detected only a single potentially pathogenic allele. our patient has decreased nk cells in addition to t and b cell defects. further genetic studies including whole exome sequencing, are planned to identify further variants in rag or other relevant genes. rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties in new york were referred to northwell health for further evaluation after abnormal newborn screens. the demographic and immune parameters of infants with transient t cell lymphopenia (ttcl) without clearly identified genetic or acquired etiology are described. tcl is considered transient if the lymphopenia resolves by months of age. similar data from the following infants with low lymphocytes (fill) program of the united states immunodeficiency network (usidnet) are presented. methods: a retrospective analysis of two separate patient cohorts with ttcl are described. cohorts include patients referred to a single center, northwell health, in ny from september to december and at usidnet using data tracked by fill from june to july . results: out of , referrals at northwell, infants with ttcl were identified. infants were predominantly male ( . %) and non-caucasian ( . %). out of fill participants, infants with ttcl were identified. infants were predominantly male ( . %) and non-caucasian ( . %). initial laboratory parameters for the northwell versus fill cohorts are summarized: a) median trec levels: . vs. . trec/l of blood; b) median absolute cd + count: vs. cells/l; c) median cd + count: vs. . cells/l; d) median absolute cd + count: . vs. . cells/l. initial naïve cd + t cell information was available for northwell and fill infants (median %). mitogen proliferation studies were performed in ( . %) northwell and ( . %) fill infants with % of these northwell and % of these fill infants demonstrating normal proliferation. genetic testing, such as targeted genetic panels or chromosomal microarrays (cma), was performed in northwell and fill infants. no genetic or chromosomal aberrations were identified. whole exome sequencing (wes) was not performed in either cohort. of ( . %) northwell and of ( . %) fill infants did not receive the initial rotavirus vaccine. no fill infants were vaccinated but no adverse effects were reported in of ( . %) northwell infants who received the first rotavirus dose. of these, of ( . %) had normal mitogen proliferation while ( . %) had decreased proliferation to phytohemagglutinin. conclusions: identifying biomarkers for ttcl and developing evidencebased guidelines for the diagnosis and management of ttcl are important knowledge gaps. this descriptive study is limited by small sample size and the constraints of registry-based research. although there appear to be differences between these cohorts, our findings suggest that ttcl may disproportionately affect different segments of the population. ttcl infants with normal mitogen proliferation may be able to tolerate rotavirus vaccination. thus, routinely checking proliferation studies in all ttcl infants may help risk stratify these patients and minimize vaccinerelated adverse events. currently, there is insufficient evidence to recommend more extensive genetic testing such as genetic panels, cma, or wes. systematically collecting information about patient characteristics and outcomes, as well as encouraging increased participation in registries such as fill, may help address these shortcomings. background: systemic lupus erythematosus (sle) is a chronic, inflammatory disease that affects multiple organs. the measurement of anti-dsdna antibodies (abs) is a gold standard serological test used in the diagnosis and monitoring of sle, with higher serum levels associated with worse prognosis. however, not all anti-dsdna abs are pathogenic, and some patients have consistently high levels with low disease activity. one mechanism suggested for the pathogenicity of these antibodies is complement activation. here we describe an assay to measure the c q binding activities of anti-dsdna abs in sle patients. materials & methods: the concentration of anti-dsdna abs was determined using the quantalite dsdna elisa kit (inova) as per the manufacturers instructions. in order to determine the c q binding capacity of bound abs, samples were added to the pre-coated plate and incubated. bound anti-dsdna ab/c q complexes were then detected using a biotinylated anti-c q antibody ( ng/ml) and streptavidin peroxidase ( mg/ml). normal reference ranges were developed in serum samples from healthy controls, and upper limits of these normal ranges were used as cut-offs. the dsdna abs and c q binding capacity of bound abs was then assessed in sle patients, and compared to other markers and the sle disease activity index (sledai) score. results are displayed as absorbance at nm (au). results and conclusions: the th percentile ranges for anti-dsdna abs ( . - . au) and c q binding activities ( . - . au) were developed from the measurements generated in healthy serum samples. sle patients with an increased anti dsdna ab concentration (> . au) were then separated into those with low (< . au) and high (> . au) c q binding activities. patients whose dsdna abs had high c q binding activity were found to have significantly higher sledai scores (mean . vs . ) . serum c q concentration, serum dsdna abs (measured by another method) and serum c and c concentrations were not significantly different between the two groups. this assay suggests that dsdna abs from sle patients differ in their ability to bind complement, and that high complement binding activity of these antibodies may be linked to a more active form of disease. x-linked lymphoproliferative (xlp) is a primary immunodeficiency, caused by signaling lymphocyte activation molecule (slam)-associated protein (sap) deficiency. patients with xlp have severe immune dysregulation, usually triggered by ebv infection, leading to fulminant infectious mononucleosis, dysgammaglobulinemia and lymphoproliferation. without hematopoietic stem cell transplant (hsct) fatality is reportedly % by age . we report the natural history of xlp in a patient, and describe the lessons learned. our patient was healthy and developed normally until -years of age, when he developed progressive respiratory symptoms. lung biopsy revealed mature lymphoplasmacytic infiltrate in the alveolar septa, consistent with lymphoid interstitial pneumonia (lip). he received corticosteroids and cyclophosphamide with significant improvement. at age , he developed severe infectious mononucleosis (fever, hepatosplenomegaly, lymphadenopathy, lymphocytosis). he had a protracted clinical course, but eventually recovered and seroconverted to a typical convalescent pattern. he subsequently developed hypogammaglobulinemia, and was started on intravenous immunoglobulin (ivig). during the same year, his -year-old brother developed lip, and subsequently hemophagocytic lymphohistocytosis (hlh) and died within months from overwhelming candidiasis. unfortunately, his youngest brother (age ) then developed lip and died months later from a massive gastrointestinal bleed. both siblings were treated with corticosteroids and cyclophosphamide; they did not have detectable ebv infection. at age years, our patient experienced recurrent strokes and was found to have biopsy-proven cns vasculitis. he was treated with interferon-and recovered with residual left sided weakness, but was lost to follow-up. he continued on ivig, with no other immunomodulatory agents for several decades. he had progressive lung disease and recurrent seizures controlled with anti-epileptics. at age , he developed sudden vision change, headache and right-sided weakness, followed by a seizure. mri of the brain revealed small bilateral areas of acute infarction suggestive of a central embolic event, however, no primary thrombus was identified. he did not receive any immunosuppression but was anti-coagulated. eventually he was discharged home with resolution of weakness to his baseline. the patient was referred to our clinic after discharge and we re-evaluated him after years. immune profiles at the time showed therapeutic igg troughs, low/undetectable igm/a/e, normal t/b/nk-cell counts, normal spontaneous, but decreased antibody-dependent nk cytotoxicity, % sap protein expression (on cd +cd +, cd -cd + and cd + cd + cells), and deletion on the x chromosome encompassing the sh d a gene which encodes sap. his mother was a carrier of the same deletion. his functional status excluded the option of hsct. a year later, he had rapid deterioration with recurrent lung infections, liver failure, and thrombocytopenia. bone marrow biopsy revealed hodgkins lymphoma. he declined chemotherapy and died few days after diagnosis. our case represents a rare patient with xlp surviving to the fifth decade without hsct, particularly having experienced mononucleosis and non-ebv related cns vasculitis. our patient survived decades longer than his brothers (who most likely shared the same genetic defect) without evidence of somatic reversion ( % sap expression in cd +cd +) to explain his milder clinical phenotype. this case may help in understanding the natural history of xlp, and confirms that prognosis remains poor without hsct. haematology and oncology, chu de québec ctla- is a major negative regulator of immune responses, and ctla- haploinsufficiency has been identified as a monogenic cause of primary immunodeficiency in patients presenting with a common variable immunodeficiency (cvid) phenotype with autoimmunity. here we present the case of pb, a -year-old man who had been followed by the immunology service of our center for years. a diagnosis of cvid had first been made when the patient presented with atypical transverse myelitis, low immunoglobulin levels, and lymphopenia. over the years, his clinical picture was dominated by various forms of autoimmunity, namely inflammatory demyelinating disorder of the central nervous system, autoimmune haemolytic anemia, immune thrombocytopenia, cryptogenic organizing pneumonia, rheumatoidlike polyarthritis, chronic liver transaminitis with biopsy-proven moderate fibrosis, and lymphocytic colitis with malabsorption. immunoglobulin replacement therapy was started at diagnosis, and autoimmunity was sequentially treated with methotrexate, interferon beta -a, cyclophosphamide, mycophenolate mofetil, rituximab, and finally a combination of low-dose prednisone and sirolimus, with stabilization of his neurological condition, the most debilitating complication of his immune dysregulation syndrome. bone marrow transplant had been offered, but declined by the patient due to perceived good quality of life compared to transplant-associated risks. the patient was later referred to our hematology ward in july of for septic shock complicating febrile neutropenia, which was part of a twomonth, gradual-onset pancytopenia. the diagnosis of immune-mediated aplastic anemia soon became apparent, as demonstrated by a bone marrow biopsy performed in a peripheral center two days prior to admission. the underlying pneumonia and thereafter biopsy-induced staphylococcus aureus iliac osteomyelitis and soft-tissue abscess were treated with broad-spectrum antibiotics as well as multiple surgical interventions. the patient was started on eltrombopag, high-dose corticosteroids and cyclosporin a, the latter promptly switched to tacrolimus due to liver enzymes disturbances, all of which resulted in no significant hematologic response despite over seven weeks of treatment (with concurrent treatment of complicating infection, upper gastrointestinal bleeding, and intensive-care-unite myopathy). during that time, genetic confirmation of ctla- haploinsufficiency was received, and the patient was thereafter started on abatacept on day of current hospitalization. administration of equine anti-thymocyte was initially foregone because of perceived infectious risk in the setting of poor iliac wound healing and superimposed adenovirus viremia; however, given the lack of response, it was given on days through of hospitalization. haematologic response began on day of hospitalization with a steady rise in alllineage myelopoiesis up to a complete neutrophil response, platelet near-complete response as well as resolution of transfusion needs by day . while waiting for a well-matched bone marrow donor, isolated platelet decrease was observed and attributed to multiple factors, including low-grade thrombotic microangiopathy, inflammatory consumption and drug-related thrombocytopenia, but the patient remained well. to our knowledge, our patients presentation is one of the most severe manifestation of ctla- haploinsufficiency to have responded to targeted therapy with abatacept, as a bridge to hematopoietic stem cell transplantation, with resolution of both immune and infectious complications, showing that genetic diagnosis is helpful in optimizing the management of presumed cvid patients. hospital de octubre health research institute (i+ ), madrid, spain, dept. of immunology, university hospital octubre. madrid. spain background: xlf/cernnunos deficiency is a rare primary immunodeficiency classified within the dna repair defects. these patients present severe growth retardation, microcephaly, lymphopenia and increased cellular sensitivity to ionizing radiation. here, we describe two unrelated cases with the same nonsense mutation in the nhej gene showing significant differences in clinical presentation and immunological profile but a similar dna repair defect. methods: missense nhej mutation was identified by targeted next-generation sequencing with an in-house designed panel of genes. for foci experiments, primary skin fibroblasts were irradiated with ionizing irradiation ( cs) or treated with mm etoposide for hour. after irradiation, the cells were seeded at a density of x cells/ml in t flasks in triplicate. to evaluate cell sensitivity to gamma-ir ( and gy),adherent cells were trypsinized and counted days later. pbmcs from patient and healthy controls were irradiated with gy, fixed and stained for cd , cd and phospho-histone h ax. mean fluorescence intensities (mfi) of gamma-h ax were evaluated on gated cd + lymphocytes. results:we report two patients harboring the same homozygous mutation in cernunnos/xlf/nhej gene. strikingly, their clinical phenotype ranges from severe combined immunodeficiency to isolated thrombocytopenia followed until escolar age (table ) . they harbour the same c. c>t mutation in nhej gene but different immunologic features (table ) . p presented with mild t lymphopenia, hypersensitivity and nhej repair defect, typical for patients with xlf/nhej defects. on the other hand, p presented a more severe phenotype (t-b-) , however hypersensitivity and nhej repair defect was similar to p .of note, p has survived into the first decade of live. both patients are alive and well after hsct. discussion: usually the repair defect in these disorders is assessed by immunofluorescence assays of irradiation-induced gamma-h ax foci using skin fibroblasts. a high throughput, sensitive and reliable assay to quantify gamma-h ax foci in pbmcs isolated from blood samples would be a valuable tool to diagnose these patients and perform hsct early. flow cytometry (fc) can be applied as a rapid diagnostic tool for dna repair disorders. patients with the same homozygous mutation (p.r x) in nhej gene have been previously reported. two patients died at . and years while another of the patients is already years old and is alive (without hsct). however,none of these patients presented severe t lymphopenia as it has been observed in our first patient. conclusions: the assignment of a timely and accurate diagnosis is of paramount importance in the management of patients with defects in dna repair. in the era of nbs an abnormal trec assay should be followed by ngs approach as cernunnos deficiency may present early in life as scid,as other rs-scid defects. since genetic diagnosis takes time,functional radiosensitivity assays in peripheral blood may lead to the correct diagnosis and avoid exposure to alkylating agents during the conditioning regimen prior to genetic diagnosis. it would also be helpful in cancer patients to individualize and to guide the dosing of ionizing radiation (ir) and/or genotoxic agents to avoid accumulation of cells with genomic instability that could accelerate cancer development. figure ). her skin lesions also significantly improved after starting the medication ( figure ). her hospitalizations were complicated by fluid overload and hypertension. both fluid overload and hypertension resolved prior to discharge. she remains on mg prednisone daily, cetirizine, ranitidine, cromolyn and benadryl and hydroxyzine prn. to our knowledge, this is the youngest patient successfully treated with midostaurin and she is doing very well on therapy with no apparent side effects. she has had resolution of many of her systemic mastocytosis symptoms including skin lesions, axillary mass and improvement in her diarrhea and growth as well as objective improvements in her tryptase levels. case report: a two-year-old male presented to the hospital with a painful, non-pruritic facial and groin rash. the rash started one week prior to presentation. he had no associated fevers. his history was remarkable for failure to thrive (ftt) and chronic bilateral leg pain with antalgic gait. over the preceding months, he had been diagnosed with hand-foot-mouth disease and varicella. he had also had recurrent cervical lymphadenopathy (lad) for greater than one year requiring incision and drainage. gram stain and gomori methenamine-silver nitrate stain (gms) were negative and pathology showed only acute and chronic inflammation with areas of necrosis. his family history was negative for autoimmune disease or immunodeficiency. infectious exposure history was significant for an incarcerated father with unknown tuberculosis status and history of living in a shelter. on physical examination, the patient was well appearing with multiple erythematous papules, with superficial erosions and scabbing on the face (figure ), lower abdomen, genital area, buttocks and proximal lower extremities. he had large, firm, non-tender submandibular lymph nodes. he also had small palpable axillary and inguinal lymph nodes bilaterally. his laboratory workup revealed normal white blood cell and platelet counts, but microcytic anemia, an erythrocyte sedimentation rate of mm/hr, and c-reactive protein of . mg/dl. full body magnetic resonance imaging (mri) revealed bilateral cervical, supraclavicular, right hilar and inguinal lymphadenopathy and a patchy right upper lobe consolidation with at least one small area of cavitation ( figure ) and an adjacent smaller area of ring enhancement. it also revealed three small nonspecific hypodense foci within the right lobe of the liver and borderline splenomegaly. given these findings, there was concern for granulomatous diseases. the patient underwent a liver biopsy ( figure ) which showed non-specific evidence of necrotizing granulomatous disease. microbiological cultures and stains for bacteria, acid-fast bacilli and fungi were negative. his infectious work-up was negative for hsv, tuberculosis, hiv, syphilis, histoplasmosis, and toxoplasmosis. superficial bacterial cultures from the face and groin grew mixed gram positive and negative organisms, including methicillin-susceptible staphylococcus aureus (mssa). his immunologic workup revealed borderline elevated iga and igg with normal igm, normal t,b, nk-cell counts and pneumococcal and tetanus titers. a dihydrorhodamine (dhr) flow cytometric test was positive, consistent with a diagnosis of chronic granulomatous disease (cgd). genetic testing confirmed x-linked disease. he was treated with acyclovir and ceftriaxone with resolution of his rash. conclusion: we present a case of a two-year-old male with newly diagnosed x-linked cgd. though he had been seen by multiple healthcare providers for recurrent lymphadenopathy over the preceding year, he had no other history of recurrent viral or bacterial infections or significant family history that might implicate a primary immunodeficiency. at time of presentation, he had diffuse rash which could have caused his palpable lymphadenopathy on exam. a high index of suspicion for cgd in the setting of recurrent lad and ftt prompted sending the dhr, which led to the diagnosis. chronic granulomatous disease (cgd) is an inherited primary immunodeficiency (pid) which results in both inflammatory response dysregulation and an increase in susceptibility to certain bacterial and fungal infections. without curative treatment such as a bone marrow transplant, it remains a chronic disease with daily medication management, intermittent treatment and life-long surveillance. in general, chronic disease involves physical, psychological and social effects which can affect the patients quality of life. although some research has been done on how pid affects quality of life, there is little research in the united states about how cgd affects patients quality of life. to examine the effect of cgd on patients quality of life, as a part of a voluntary research protocol examining the natural history of immune deficiencies, we administered the who qol-bref instrument to adult cgd patients enrolled on a nih irb approved protocol and seen in the infectious disease clinic at the national institutes of health (nih) over a five-month period. the who qol-bref is comprised of items, which measure the following broad domains: physical health, psychological health, social relationships and environment. each item is rated on point likert scale. it has been validated cross culturally and has been widely field tested. the survey was interview administered to patients ( males, females) with genetically confirmed cgd. the age range was - years old (mean age . years) with a distribution of % x-linked cgd and % autosomal recessive cgd. results have been obtained and will be presented. rationale: common variable immunodeficiency (cvid) is the most common primary immunodeficiency with an estimated prevalence of : , . we aimed to analyze the clinical presentations and their associated comorbidities amongst cvid patients in usa. methods: data on , cvid patients reported in the united states immunodeficiency network (usidnet) from to were analyzed based on clinical, immunological and genetic factors. univariate analysis with spearman rank coefficients was done to analyze correlations between disease outcomes. observed survival was estimated using the kaplan-meier method. results: among the patients, ( . %) were female and ( . %) were male. median age at diagnosis was years [mean (sd), . ( . ); range, - ; iqr, - ] with median age of onset of years (mean (sd), . ( . ) ; range, - ; iqr, . females showed a longer delay in diagnosis ( . vs. . years, p= . ). higher body mass index (bmi) linearly correlated with the age of diagnosis (r= . ). in survival analysis, a -year delay in age at diagnosis increased the risk of death by . % (hr: . , % ci: . - . , p= . ). conclusions: our study suggests a longer delay in diagnosis in female subjects and a strong association with diagnosis of cvid in patients with higher bmi. females may have a longer period without symptoms leading to a diagnostic delay. gender-based and disparities-based inquiry into these trends may need additional study. the physical well-being of those with primary immunodeficiency (pi) and the physical maladies of those with pi are well-documented. since the s, advances in identification and treatment of pi has for many led to lives where the physical infections of these groups of diseases are manageable. however, not as well understood are the emotional and mental health aspects of living with pi. as part of a larger survey project the idf national patient survey, this study aims to quantify any potential mental health issues or challenges faced by adults with pi. our hypothesis-those with pi, suffer from statistically higher rates of depression when compared to the u.s. general population. the idf national patient survey was a nationally distributed, unincentivized, mail-based survey of , persons in the idf patient database identified as being either adults with pi or the parent/caretaker of a child with pi. the questionnaire comprised approximately main questions about pi as well as the validated sf- v , brief fatigue inventory and the patient health questionnaire- (phq- ) instruments. additional questions asked about current use of prescription medications for anxiety, depression, stress and pain. for the purpose of this study, only adult respondents with pi are included as the basis for analysis. the two-item patient health questionnaire (phq- ) meets the criteria for general screening of depression suggested by the u.s. preventive services task force. scored on a scale of - , a score of three or higher is suggested as the cut-point for depressive screening. according to a ahrq study that utilized meps data, , of the , ( %) respondents scored three or greater. in our survey of the ( %) adults scored three or greater ( <. .) overall, those in our survey scored lower on the sf- v mcs scale when compared to the u.s. population ( . v. . , p<. ) . further, adults with pi who scored three or higher on the phq- had an average mcs of . . those who met the phq threshold in our survey were also more likely to report moderate to severe limitations in normal activities as a result of emotional problems than those that fell below the threshold ( % versus %, p <. ). not surprisingly, those that met the phq threshold reported much higher use of prescription medications for anxiety, depression, stress ( % versus % below threshold, p <. ) as well as a higher reported use of prescription pain medications ( % versus % below threshold, p <. ). though moderate to severe fatigue was reported by % of those below threshold, % of those with phq scores at threshold reported experiencing moderate to severe fatigue (p <. ). health care providers should consider including the phq- in the overall health assessments of their patients with pi. those scoring three or higher should be referred to the appropriate professional for further evaluation. (lek et al., ) . the w l is a semi-conservative amino acid substitution, which may impact secondary protein structure. in-silico analyses supported a deleterious effect, located within the sh domain, which is a critical functional domain (chandesris et al., ; koskela et al., ) . it was thus determined that this variant is likely pathogenic. the patients prophylactic treatment was optimized with tmp-smx ( mg- mg) twice daily for prevention of infections. she was also started on hibiclens (chlorhexidine) baths once per week. she was referred to pulmonology for optimization of pulmonary health in the setting of bronchiectasis and mild decline in dlco. she was advised to followup on a yearly basis to the primary immunodeficiency clinic to assess for recurrent infections and for changes in pulmonary health. finally, targeted testing and clinical evaluation of both of the patients parents was recommended to determine if w l was inherited or arose de novo. the pathogenic role of the w l missense change would be further supported if it had occurred de novo or if it segregates with the disease in the family. uploaded file(s) uploads pulmonary function testing results.pdf j clin immunol ( ) (suppl ):s -s s introduction: lipopolysaccharide-responsive and beige-like anchor protein (lrba) deficiency is a rare autosomal recessive disease of the immune systems characterized by hypogammaglobulinemia and decreased ctla expression on t regulatory cell (t regs) due to defective intracellular trafficking of ctla . previous in vitro study has shown a significant increase of ctla expression on lrba deficient t cells after overnight culture with chloroquine, an older anti-malarial agent. this effect is likely due to increasing lysosomal ph. however, there is no evidence of such effect in human subjects after administration of weight appropriate doses anti-malarial agents. we are presenting a set of siblings with lrba deficiency who had ctla expression measured before and four weeks after starting hydroxychloroquine. case reports: case is a -year-old east-indian boy with autoimmune thyroiditis, type diabetes mellitus (dm), short stature, autoimmune cytopenias, and lymphadenopathy. he was referred to immunology clinic at years of age for suspicion of autoimmune lymphoproliferative disorder. primary immunodeficiency genetic panel was sent which revealed a homozygous mutation in lrba gene (c. _ del). this novel variant resulted in a frameshift and created a premature stop codon amino acids downstream from this location which may lead to absent or abnormal protein. lung ct scan showed interstitial lung disease. lung biopsy showed interstitial nodular and diffuse lymphoid proliferation. this diagnosis led to the testing of his sister (case ) given her history of autoimmune illnesses and the family history of consanguinity. case is a now -year-old girl with type dm, autoimmune thyroiditis, lymphadenopathy, psoriatic arthritis, and seizures. her lung imaging showed pulmonary nodules without interstitial lung disease. both cases received hydroxychloroquine while waiting for insurance approval of abatacept. ctla expression on tregs was measured prior to and four weeks after starting hydroxychloroquine treatment. at baseline, . % of case s cd cells were treg (foxp +ve, cd hi) and . % of them expressed ctla- (in contrast to . % tregs in the healthy control) with mean fluorescence intensity (mfi) of . this ratio and mfi did not change after weeks of hydroxychloroquine treatment ( mg/kg/day). soluble interleukin- receptor levels were measured: case had a baseline level of pg/ml, which decreased to pg/ml after weeks of hydroxychloroquine treatment. for case : . % of her cd + t cells were found to be foxp +cd hi and . % of these tregs expressed ctla- . this ratio increased by % after one month of hydroxychloroquine. increase in mfi was also noted from to . case had a drop in soluble interleukin- receptor level from pg/ml to pg/ml after treatment. conclusion: in contrast to the previous in vitro assays, we did not find a significant increase in ctla expression on t regulatory cells in vivo after weeks of mg/kg/day hydroxychloroquine. interestingly, soluble il- receptor levels improved dramatically with hydroxychloroquine. ( ) submission id# human nf-kappab defect results in defective intrinsic b-cell differentiation, function and class switching introduction/background: autosomal dominant heterozygous mutations in nfkb (encoding for the protein nf-kb ) have been identified in the etiology of a form of primary immunodeficiency disorder that presents with hypogammaglobulinemia, defects in b-cell maturation, endocrinopathy, and autoimmune manifestations. in humans, the effects of altered nf-kb and mechanisms of immune system impairment have not been fully delineated. objectives: to understand the mechanism of the antibody deficiency in patients with hypomorphic mutations in nfkb (c. dela; p.lys serfs* ) by evaluating b-lymphocyte proliferation, differentiation, function, and gene expression. methods: immunophenotyping of primary b-cells from subjects with mutant nfkb was completed by flow cytometry. proliferation of b-cells was assessed by cfse stimulation of primary cd + b-cells from healthy and nfkb mutant subjects. differentiation of healthy and affected naïve b-cells (cd -cd -) into plasmablasts (cd +cd +) following stimulation was assessed by flow cytometry. the supernatant from these cells were assayed for iga, igg and igm production by elisa. to study the defect in class-switch recombination, naïve b-cells and ebvtransformed b-cells from affected and healthy individuals were stimulated and expression of the aicda gene was quantified by qpcr. in parallel experiments, ebv b-cells from wildtype and nfnb mutant individuals were stimulated and aid (activationinduced cytidine deaminase) protein levels were determined by western blot. results: patients with hypomorphic mutations in nfkb (c. dela) had low memory b-cell (cd + cd + igd-igm+) and class-switched memory b-cell (cd + cd + igd-igm-) numbers. in vitro, primary bcells from these patients demonstrated a % reduction in proliferation and cell division in response to cd l and il- (p = . ). compared to healthy naïve b-cells, mutant naïve b-cells had a significant reduction in plasmablast differentiation (p = . ) and secreted significantly lower levels of immunoglobulins in response to cd l and il- stimulation. mutant naïve b-cells and mutant ebv b-cells failed to increase aicda expression and aid protein levels in response to cd l and il- stimulation. conclusions: our studies demonstrate that a hypomorphic nfkb mutation in humans affects intrinsic b-cell proliferation and differentiation. the mutation impairs transcription of the aicda gene that encodes aid, a key protein involved in b-cell class-switch recombination. the nfkb gene defect also impairs immunoglobulin production, as seen in common variable immunodeficiency-like cases. these studies provide unique translational insights into physiological activities of nf-kb in downstream immunologic outputs in humans, expanding those suggested by experimental observations in mice. background: few studies have evaluated the quality of life (qol) and patient reported outcomes of primary immunodeficiency disease (pidd) patients, and no studies have assessed medical provider perceptions of their pidd patients qol, neurocognition, physical well-being and psychosocial health. understanding provider beliefs regarding patient reported outcomes is essential to improving clinical management of pidds. here we report our pidd medical provider survey results. methods: providers were contacted via email with the assistance of the clinical immunology society. participants completed adult and/or pediatric-based likert scale survey questions via a secure online survey service. in addition to demographic information, survey questions assessed provider perceptions of patients overall qol and their impression of the impact of disease or its associated treatment on mental health, physical well-being, neurocognition, social relationships and school/work performance. clinicians were expected to make their assessments based on their pidd patient cohort as a whole rather than on specific diagnoses or patients. given the small sample size, a p-value < . was considered statistically significant; repeated measures anova and paired t-test analyses were used. results: study participants (n= ) were primarily from the united states ( %), born between - ( %) , and trained in allergy/ immunology ( %). % of survey takers practiced within an academic center, % were female and % cared for children with % of providers concurrently caring for adults. there was a statistically significant difference (p= . ) in the perceived overall qol of pediatric versus adult pidd patients with % of providers feeling as though their pediatric patients had a good qol while only % believed their adult patients had a good qol. clinicians believed adult pidd individuals had more difficulties related to associated co-morbidities rather than their actual pidd compared to pediatric pidd patients (p= . ). providers felt that the neurocognition and school performance of children were more often negatively affected by a pidd than the neurocognition and work performance of immunodeficient adults (p= . ). clinicians believe children with pidd more frequently had difficulties related to their concentration than memory (p< . ). % of those who care for pidd adults believe their patients work performance or daily mental functioning is at times negatively impacted. anxiety symptoms and social relationships were viewed as being more negatively impacted by a pidd diagnosis or treatment than anger or depressive symptoms in both children and adults (p< . ). % of pediatric clinicians feel their pidd patients experience anxiety symptoms often or almost always. of physical health parameters, energy, rather than mobility or pain, was deemed to be more deleteriously influenced by an immunodeficiency in adult and pediatric patients (p< . ). conclusions: our results show that medical providers perceive the overall qol of pediatric pidd patients to be superior to that of adults with pidd, but most clinicians feel a diagnosis or associated treatment regimen for pidd can negatively impact the physical well-being, psychosocial health, school/work performance and neurocognition of both children and adults. [cbm] complex is a critical signalling adaptor that regulates lymphocyte activation, proliferation, survival, and metabolism. primary immunodeficiencies affecting each component (termed cbmopathies) result in broad clinical manifestations ranging from severe combined immunodeficiency (scid) to lymphoproliferation. we present the laboratory and clinical findings of two canadian first nations patients found to be homozygous for the same novel card mutation (c. c>t; p.r *). results: we have identified an -month-old boy who presented with a severe case of entero/rhinovirus bronchiolitis with interstitial lung disease and a -year-old boy with a history of severe pulmonary infections (including pjp), chronic sinusitis, candidiasis, invasive bacteremia, and severe ileo-colitis and oral ulceration requiring total colectomy. both patients possessed absent tregs, absent memory b cells, and hypogammaglobulinemia. however, only the -month-old had poor t cell proliferation to pha, cona, and cd . both patients were found to be homozygous for the same novel variant of card (c. c>t; p.r *). the mutation rendered card protein expression unstable and it was undetectable by immunoblot. to confirm card deficiency, we stimulated patient b cells with phorbol -myristate acetate (pma) and ionomycin across a time-course and immunoblotted for various signalling proteins in both the nf-b (ikk/, ib, p ) and mapk (mek / , mkk , jnk / , erk / ) pathways as well as various cleavage substrates of the malt paracaspase (relb, cyld, bcl , hoil ). nf-b and jnk activation were completely absent and malt paracapase activity was lost, but surprisingly, mkk (which acts upstream of jnk) was intact. furthermore, co-immunoprecipitation experiments revealed that card was required for optimal malt association with bcl in response to stimulation. conclusions: these two cases highlight the crucial role of card in regulating lymphocyte development, function, and humoral responses. in addition, we have identified the oldest known living individual with card deficiency and he presented uniquely with inflammatory gastrointestinal disease in addition to scid, further adding to the spectrum of phenotypes associated with card -related primary immunodeficiencies. abstract: the usidnet registry began in with an niaid contract with the immune deficiency foundation, which continues today. it aims to provide a resource for clinical and lab research through enrollment of known immunodeficiency patients into a national registry, the usidnet. nih is a major national and international referral center for clinical trials on inborn errors of immunity, or primary immunodeficiency diseases. it is a mechanism for depositing nih data into usidnet. a registry of patient information may help us understand how many people have each disease. the information may improve how we diagnose and treat these conditions. the patient registry is designed to obtain longitudinal data on a large number of patients with primary immunodeficiency diseases who come to nih to participate in research. the data is collected from the nih electronic medical record system, cris and is deposited into a secure registry with restricted and monitored access. all medical information is anonymized for patient privacy. department of biochemistry, emory university, atlanta, ga oas is an intracellular sensor for dsrna that generates the second messenger '- '-oligoadenylate to activate rnase-l as a means of antiviral defense. we describe four patients with a complex early-onset autoinflammatory and immunodeficiency disease caused by heterozygous de novo oas mutations. patients presented early in life with lung inflammation including pulmonary alveolar proteinosis and interstitial lung disease. they had febrile flares with dermatitis specifically with macular, pustule and bullous features often progressing to ulceration. infants had episodes of bloody diarrhea in patients (assoc. with villous blunting and cryptitis in two patients and oesophagitis in one patient). immunoglobulin igm, igg, and iga levels were low while t cell, b cell, and nk cell numbers were generally in the normal range. exome sequencing identified de novo heterozygous oas missense mutations in all patients. one patient had a heterozygous de novo oas mutation p.ala val, with mutant oas protein being expressed in ex vivo generated t cell blasts. in sorted primary patient monocytes and b cells, oas p.ala val was associated with spontaneous rna degradation and apoptosis as determined by rna chip technology and flow cytometry, respectively, while t cells were not affected. monocytes displayed disturbed terminal differentiation and functioning as indicated by reduced gm-csf-r expression and signaling. b-cells display reduced class-switch-recombination. proliferation of allogeneic t-cells was reduced in response to sorted oas mutated monocytes and b-cells. activation of interferon response genes in pbmcs was detected. two further unrelated patients had a heterozygous de novo oas mutation p.cys tyr, which appeared to compromise protein stability in transformed patient fibroblasts and when transfected. cells transfected with this mutant protein had reduced - oligoadenylate synthesis compared to wild type transfected cells. immortalized fibroblast lines demonstrated higher levels of inflammatory cytokines and spontaneous cleavage of rnas. a th patient with the clinical phenotype had a heterozygous de novo oas variant p.val gly, but has yet to have formal validation of the variant. three patients underwent hematopoietic stem cell transplants in an effort to control their diarrhea and skin inflammation. one patient died with ongoing chronic graft versus host disease, while the two others (p.ala val, cys tyr) are alive and reasonably well with a followup of . - years. the untransplanted patient died as a result of respiratory failure. in summary, patients with de novo heterozygous oas mutations have chronic ongoing inflammation of multiple organs. this is at least in part due to spontaneous rna cleavage, apoptosis and production of inflammatory cytokines and type i interferons. this defines a new category of autoinflammatory disorder. introduction: increased susceptibility to infections is the most common complication of chronic granulomatous disease (cgd). hemophagocytic lymphohistiocytosis (hlh) is a severe disorder resulting from hyperinflammation and hypercytokinemia that can lead to multi-organ system dysfunction ( ) characterized by certain criteria: fever, splenomegaly, cytopenias, hypofibrinogenemia or hypertriglyceridemia, hyperferritinemia, increased soluble cd /il- ra, evidence of hemophagocytosis, or decreased/absent nk cell cytotoxicity ( ) . secondary hlh occurs infrequently but often is preceded by smoldering infection in cgd ( , , ) . we present a case of hlh in a -day old male, the youngest reported case with cgd. case: a -day old male with previously diagnosed x-linked cgd, due to known family history, presented with fevers. initial evaluation was unrevealing including chest x-ray, urinalysis, and blood and csf cultures. he was admitted and treated empirically with cefepime. ct demonstrated multiple multifocal nodules of the lungs and spleen. after lung nodule biopsy was performed, antimicrobial therapy was broadened to iv meropenem, voriconazole, and micafungin. despite this, he continued to have fever and developed new onset tachycardia, respiratory distress, and lactic acidosis. further decompensation with vasoactive refractory shock was treated with vasopressors and stress dose hydrocortisone. additional laboratory evaluation revealed rising liver enzymes (ast u/l, alt u/l), cytopenias (hemoglobin g/dl, anc /ul, platelets , /ul), and coagulopathy (fibrinogen - mg/dl). splenomegaly was present on abdominal ultrasound. a diagnosis of evolving hlh was considered and dexamethasone was administered. within hours of clinical decompensation, the patient died of multiorgan failure. subsequent blood cultures returned with gram-negative rods (and ultimately burkholderia cepacia). autopsy confirmed hemophagocytosis within the bone marrow. no mutations were found in genes associated with primary hlh. discussion: patients with cgd are susceptible to infectious complications and auto-inflammation most commonly involving the lungs, gi, and gu systems ( , ) . patients with cgd can be at increased risk of hyperinflammatory syndromes secondary to infections and chronic inflammation. as shown in the included case, hlh can present in infancy and can be deadly. early consideration and directed treatment of hlh is imperative, even in the setting of sepsis malignant proliferation of gamma-delta t cells include hepatosplenic t-cell lymphoma (hstl), primary cutaneous t-cell lymphoma and t-cell large granular lymphocytic leukemia (t-lgl). the former two have often been associated with splenomegaly and cytopenias. however, reactive proliferation of gamma-delta t cells in spleen mimicking malignancy has only been reported once and has a significant risk of misdiagnosis. a -year-old female presented with two years of unintentional weight loss, persistent leukopenia and thrombocytopenia, with leucocytes around - x ^ /l and platelets around x ^ / l. she also had associated macrocytic anemia (hemoglobin= - g/dl) with laboratory evidence of dat (direct anti-globin test) negative hemolysis. physical examination and computed tomography (ct) imaging showed splenomegaly. there was no hepatomegaly or lymphadenopathy. serum liver function test, auto-immune studies, hemolysis and hereditary diseases workup, viral and bacterial serologies were all normal or negative, except for mild hyperbilirubinemia and ldh elevation. bone marrow examination performed four months prior to the splenectomy revealed mildly hypocellular marrow ( %) with trilineage hematopoiesis. flow cytometric analysis and cytogenetics of the bone marrow aspirate and peripheral blood were normal except for small population of large granular lymphocyte and mild low absolute b cell counts in peripheral blood. a laparoscopic splenectomy was performed for diagnostic and therapeutic purposes due to patients worsening luq pain. there was no other treatment given prior to surgery. hours postsplenectomy her leucocytes increased to . and platelets to . her three-month post-splenectomy wbc count and platelet count was . and , respectively. hemoglobin also improved to . . pathology showed red pulp expansion by small lymphocytes (fig. ) and subsequent ihc (immunohistochemistry) was positive for cd ( fig. ) , cd , cd , tia- and negative for cd , cd and cd . cd was difficult to interpret. eber was negative. flow cytometry ( fig. ) showed increased gamma-delta t-cell population ( %) with positive cd , cd and cd and negative cd , cd and cd . molecular studies by pcr didnt reveal any t-cell receptor gamma or beta gene rearrangement. cytogenetics was negative for isochromosome q or any other abnormalities. she was symptom free at months from her splenectomy. the morphology and immuno-phenotype of these gamma-delta t cells show significant overlap with the malignant cells seen in hstl and t-lgl, such as loss or downregulation of cd , cd and cd . awareness of this reactive condition is necessary to prevent making a wrong diagnosis of a malignant disease with a potentially benign, spontaneously resolving disease. additional studies of similar cases is needed in order to establish more definitive criterion to separate benign from malignant processes and delineate the role of gamma-delta t cells. uploaded file(s) uploads fig . flow cytomtery.pptx background: sex steroids in the human thymic environment influence aire expression as well as interactions with its partners, i.e. genes coding for aire interactors. here we investigated the effects of sex steroids on these interactions during minipuberty the surge of sex hormones that occur along the first six months of life -and up to months of life. we employed a network-based approach for investigating aire-interactors gene-gene relationships and how abundantly co-expressed thymic mirnas covariate with those genes. aire-interactors networks allowed the measuring of gender-related differences in gene-gene expression correlation disclosing relevant differences between minipuberty groups. methods: total rna was extracted from thymic surgical explants obtained from male (m) and female (f) infants -aged - months (groups mm and mf, for minipuberty) and - months (group nm and nf, for nonpuberty) and used in dna microarray assays. gene coexpression network (gcn) analyses were performed for aire and its interactors and for mirna-gene coexpression analysis. the set of genes coding for the aire-targeted proteins was previously identified in tecs by abramson et al. (cell : - , ) . aire-interactors networks were obtained for all groups (link strength cut-off for gene-gene > | . | and for mirnagene < - . ). aire expression in mtecs was quantified by immunohistochemistry. these methodologies are described in moreira-filho et al. (sci rep : , ) . results: the mm x mf networks comparison showed that abundantly expressed mirnas are interacting with the different aire interactor genes in both networks. it is interesting to note that network topology were more similar between nm and nf groups, although aire interacts with only one distinct mirna in each network (mir- - p in the nm group or mir- in the nf group). conversely, in the non-puberty networks the sets of mirnas and their interacting genes are distinct for each network. immunohistochemistry analysis revealed a higher percentage of mtec aire positive cells in the minipuberty groups: i.e. there is a significant difference between mm x nm (p = . ) and between mf x nf (p = . ). conclusions minipuberty and genomic mechanisms shape thymic sexual dimorphism along the first months of life. this process does not involve changes in aire expression between genders, but differences in the interactions of aire with its partners that persist throughout the non-puberty period, probably regulated by mirnas and also by genetic and epigenetic factors. introduction: neutrophils are presumed to defend against aspergillus species by releasing reactive oxygen species (ros) and neutrophil extracellular traps (nets) to degrade fungal hyphae. triazole antifungals synergistically enhance neutrophil mediated hyphal degradation. patients with cgd are particularly susceptible to aspergillus species likely due to their inability to create ros and nets, and in severe cases may not be amenable to antifungal therapy alone. objective: we present a case of severe disseminated aspergillosis in a patient with cgd in whom gt served as an important adjunct to antifungal therapy and bridge to transplant. results: a -year-old boy with known cgd, lost to follow up and nonadherent to prophylaxis, presented acutely with right-sided hemiparesis. neuroimaging revealed an embolic left middle cerebral artery infarction and cardiac magnetic resonance imaging showed extensive vegetations involving both right and left ventricles and atria, with an ejection fraction of %. the patient was admitted to intensive care, started on liposomal amphotericin b, meropenem and vancomycin, and underwent debulking of the intracardiac masses on post admission day (pad) . operative findings showed severe constrictive pericarditis with multiple abscesses and intracardiac vegetations. thorough debridement of the vegetations was undertaken, however some deep seated abscesses in the myocardium were not amenable. operative cultures were positive for aspergillus fumigatus. clinical status remained precarious, with ongoing requirement for inotropic and ventilator support. antimicrobial therapy was refined to voriconazole, with amphotericin b remaining on board until therapeutic levels of voriconazole were achieved. as effective neutrophils are integral in the immune response against aspergillus, the decision was made to start granulocyte transfusions to aid in clinical stabilization prior to hsct. interferon gamma infusions were not administered because of the risks of adverse effects and potentially increasing transplant rejection. gts were started on pad , at a dose of approximately x ^ granulocytes, three times a week. the patient tolerated the infusions well, with no allergic or inflammatory response. neutrophil oxidative burst measured one hour post infusion showed . % mean fluorescent intensity, compared to a baseline of % ( figure ). clinical improvement was seen, with inotrope cessation on pad and extubation to bipap on pad . human leukocyte antigen (hla) allosensitizaton was tested on pad , days after the first gt, with no evidence of hla antibodies. a total of gts were given over months, prior to proceeding to a / hla matched related donor transplant (pad ), with two transfusions given before neutrophil engraftment (anc ) on day + . the patient is now stable months post transplant, with no evidence of graft rejection. he remains on chronic suppressive antifungal therapy, to continue until full lymphoid reconstitution. conclusion: gt may be a useful adjunct to antifungal therapy in patients with impaired neutrophil function with severe invasive aspergillosis, and potentially provide a life sustaining bridge to hsct. methods: subjects were enrolled in irb protocol for rvt- . rvt- was implanted into the quadriceps with immunosuppression. results: subject was normal at q . but had hypocalcemia, an asd, pda, and abnormal ears. the subject received a cord blood transplant mismatched at hla-b and hla-c alleles at age months. subsequently mild graft-versus-host disease (gvhd) developed and was treated with antithymocyte globulin, steroids and cyclosporine. donor t cells developed in low numbers. twelve years later, the subject developed epstein barr virus lymphoma and suffered two relapses. while in remission, subject received unmatched rvt- . two weeks after rvt- implantation, the subject developed an adenovirus infection resulting in skin and gut gvhd, presumably from activation of the cord blood t cells. subject was treated with corticosteroids, cyclosporine, cidofovir and infliximab. four years post rvt- , subject is healthy with genetically recipient t cells/mm and % naïve cd t cells. subject was normal at q . but had an asd, pda, hypoparathyroidism, and no t cells at birth. his genetic defect is unknown. subject was treated with a ric myeloablative, allogenic, unrelated, / cord blood transplant, and a subsequent myeloablative, unrelated / cord blood transplant. hematopoietic chimerism was established without t cell development. rvt- expressed the one allele in the recipient that was not expressed by the second cord donor. the post-thymic transplant course included immune thrombocytopenia requiring rituximab and splenectomy and generalized adenopathy for years but no gvhd. he failed weaning of immunoglobulin replacement. three years post rvt- , he has cd , cd , and cd t cells/mm . he is active in school. subject had absent trecs on newborn screening with cd + t cells/ mm. a single mutation in foxn was identified; she has sparse scalp hair. subject received a / matched unrelated umbilical cord transplant. the post-transplant course was complicated by significant morbidity, and no naïve t cell development. rvt- expressed the one allele in the recipient that was not in the cord blood donor. the subject did not develop gvhd, is healthy and at months has naïve cd + t cells. she had resolution of longstanding norovirus and sapovirus gastroenteritis. conclusion: rvt- can improve t cell immunity after poor or failed correction with allogeneic hematopoietic transplants. in subject , gvhd post rvt- was related to an acute viral infection; cord t cells attacked hla mismatches in the recipient. subjects and were given rvt- matched to recipient alleles that were not expressed in the hematopoietic donor. we hypothesize that thymocytes developing in rvt- , if strongly reactive to the recipient-mismatched allele, are deleted by the bonemarrow-donor dendritic cells (that acquire recipient mhc from the recipient-allele-matched thymic epithelial cells) thereby preventing gvhd. rationale: ctla haploinsufficiency is an autosomal dominant immune dysregulation syndrome characterized by variable phenotypes. here we present a young woman diagnosed with evans syndrome and lymphoproliferation as a child, found to have a novel ctla variant as a young adult, and who developed hypogammaglobulinemia and a bacterial endocarditis while stabilized on ctla- replacement therapy. methods: sequencing of genes, including ctla , in primary immunodeficiency panel. results: our patient was diagnosed with evans syndrome at age with manifestations of anemia and thrombocytopenia recalcitrant to treatment over many years with steroids, cyclosporine, and vincristine. bone marrow biopsy reportedly showed normal trilineage maturation and her symptoms responded for a short time to splenectomy at age . symptoms recurred at age when she was also found to have pulmonary reticular opacities, prominent lymph nodes, and elevated b cells. repeat bone marrow and lymph node biopsies at that time were unrevealing. minor responses to treatment with ivig, rituximab, mycophenolate mofetil and gcsf were noted. at age , she developed varicella-related encephalitis shortly after vaccination. with a strong suspicion of an immune dysregulation syndrome, immune evaluation revealed normal immunoglobulins with good vaccine responses, elevated b cell numbers, normal t cell numbers, and normal mitogen proliferation. ctla sequencing revealed a mutation in exon [c. c>a, p.tyr *] causing a premature translational stop signal, which was consistent with previously reported cases of ctla haploinsufficiency. she was started on rapamycin initially for her cytopenias but was then transitioned successfully to abatacept with almost complete resolution of her anemia, neutropenia, and pulmonary opacities. after months of stable control, she developed a precipitous drop in her platelets and was eventually diagnosed with streptococcus viridans endocarditis of her native mitral valve. this responded to antimicrobial therapy, but eventually needed surgical intervention due to ongoing insufficiency. around this time, she was also found to be newly hypogammaglobulinemic, necessitating ongoing igg supplementation therapy. during successful replacement of her mitral valve with a biosynthetic prosthesis, it was noted that her aortic valve also had evidence of previous disease, implicating a prior endocarditis as part of her clinical syndrome as well. conclusions: in this patient, the presentation of recalcitrant cytopenias, lymphadenopathy, elevated b cells, vaccine-induced viral infections and lung findings precipitated concern for immune dysregulation syndromes and allowed for identification of a novel deleterious ctla mutation. in addition to previously reported clinical findings, our patient presents with the first reported case of repeated endocarditis in the setting of ctla insufficiency disease. given the finding in this patient of prior (unrecognized) disease, regularly screening patients with ctla insufficiency for evidence of cardiac affectation may be prudent. clinical research nurse, johns hopkins university background: the relationship between elevated serum alpha fetoprotein (afp) concentration and age, mortality, genotype and neurologic outcome in ataxia telangiectasia (a-t) patients has remained inconclusive over the past decades, leaving afp as a useful marker for disease diagnosis without further clinical significance. objective: to examine the relationship between afp levels and age, mortality, genotype and neurologic outcome using a data set larger than any prior study. methods: we retrospectively collected data on a-t patients at johns hopkins medical center ( - years of age) with both classical (predicted protein null) and variant a-t. this included serum afp measurements ( serial levels in a-t patients, max observations per patient). mixed model compound symmetry covariance was used for statistical analysis to examine the effect of age at visit on afp levels. subgroup analysis by mutation type, mortality, feeding/swallowing scores as a surrogate for neurologic function, x-ray induced in vitro chromosomal breakage and serum transaminase levels were similarly analyzed. results: significant association between age and afp level was found such that for every year increase in age, afp level increases ng/ml (p< . ). subgroup analysis by mutation type found that the patients with missense mutations showed a negative linear relationship be-tween log afp levels and age (r= - . , p= . ). we found greater afp levels in patients who subsequently died, after controlling for age (least square mean afp level in log scale . greater in deceased patients versus living patients, p= . ). we found a significant decline in feeding score by . units (score range - ) per ng/ml afp increase (p= . ) after adjusting for age. there was no significant relationship between afp levels and serum transaminase levels. conclusion: afp increases with age in a-t patients, though this may not apply to patients with missense mutations. there is a statistically significant increase in mortality and worsened swallowing scores with increasing afp levels, but this remains to be proven clinically significant. here we present a pediatric hae patient who had recurrent abdominal attacks in which constipation, secondary to the adhd medication dexmethylphenidate (focalin), appears to be a trigger. of importance, this is the first pediatric patient with hae to be described as having safely undergone a capsule endoscopy for direct visualization of the gastrointestinal tract. this was done to decrease the risks associated with the more invasive procedure of traditional endoscopy and colonoscopy. case presentation: the patient was an -year-old male with hereditary angioedema who presented with day history of diffuse abdominal pain and nausea. in the ed, patient was in no acute distress. abdominal ultrasound showed severe circumferential thickening of the wall of multiple bowel loops and a large amount of simple ascites. x-ray revealed stool in the colon. he was admitted for pain control and hydration. in the next year, he visited the ed five more times for exacerbations of angioedema of his hand, penis, and bowel. each time, he presented he had underlying abdominal pain and constipation. he was seen by gastroenterology and had a workup that was negative for helicobacter pylori, parasites, and other gastrointestinal infections. to further evaluate his abdominal pain, capsule endoscopy was performed and well tolerated. during an admission in january he received a full inpatient bowel cleanout, after which, his angioedema finally improved. of note, he was diagnosed with adhd and started on dexmethylphenidate (focalin) just prior to this period of recurrent angioedema attacks, and he did not have attacks during the summer months when he was off the medication. discussion: abdominal pain is a common complaint in pediatric hospitals, and further workup consists of endoscopy and colonoscopy. this may be easily accomplished in the general population, however, in patients with hae, these procedures carry greater risk and may be avoided, leading to delayed diagnosis and treatment ( , ) . a newer and less commonly used alternative for direct visualization of the gastrointestinal tract is capsule endoscopy. some benefits are that it does not require sedation, is less invasive, and is less likely to be irritating to the mucosa ( ). additionally, since psychological stress may be a trigger for angioedema attacks, the decreased stress associated with a noninvasive procedure such as capsule endoscopy, makes it safer to use ( ) . limitations of capsule endoscopy include dependence on battery life and its inability to biopsy or administer therapy if needed ( ) . hereditary angioedema treatment consists primarily of avoiding triggers and managing acute episodes. in this first case of hae in a pediatric patient where capsule endoscopy was used, the procedure was well tolerated without any complications. recognizing constipation as a trigger and capsule endoscopy as a safe method of direct visualization of the gastrointestinal tract will help others to control and decrease the severity of their hae attacks as well. a year old male with past medical history of common variable immune deficiency (cvid) and related autoimmune complications, including granulomatous-lymphocytic interstitial lung disease (glild), hepatosplenomegaly, leukopenia, and thrombocytopenia tolerated monthly subcutaneous immunoglobulin replacement as outpatient for several years with infrequent infectious complications. four months ago, he was found to have elevated liver enzymes on routine chemistry. a liver biopsy two months later showed pathology consistent with nodular regenerative hyperplasia (nrh) without overt cirrhosis. a hepatic venous pressure gradient (hvpg) of mmhg was found, consistent with portal hypertension. his hepatitis viral markers were negative, he did not drink, and portal venogram was negative for thrombosis. in early october, the patient was admitted to the hospital with anasarca and tense ascites. he underwent a diagnostic and therapeutic large volume paracentesis and was also found to have spontaneous bacterial peritonitis (sbp) and bacteremia with group b streptococcus. the patients course was complicated by polymicrobial peritonitis, vre bacteremia, fungemia, variceal hemorrhage, hepatic encephalopathy, and hepatorenal syndrome. his hepatic complications from portal hypertension were out of proportion to his liver parenchymal disease. transjugular intrahepatic portosystemic shunt (tips) was considered to alleviate portal hypertension but was not feasible due to his degree of encephalopathy. immunosuppressants such as high dose steroids were given while in the hospital with plans to start rituximab to treat patients glild after he had recovered from the acute infections. unfortunately, after two months in the hospital, the patient succumbed to sepsis and progressive liver failure. this case emphasizes the importance of systematic screening and continued vigilance for hepatic complications in patients of cvid as studies have shown that nrh of the liver is present in more than % of cvid patients who undergo a liver biopsy (pmid: ). a cross-sectional study of patients with primary hypogammaglobulinemia and hepatic dysfunction found that histological findings of nrh were present in % of cvid patients and was associated with portal hypertension in % of cases (pmid: ). another study estimated the minimal prevalence of nrh in cvid patients as % (pmid: ), stating that this was likely a gross underestimate as nrh may also be present in patients with normal liver function tests that are not routinely biopsied. therefore, liver enzyme levels may not anticipate the severity of liver involvement. there is currently no treatment for cvid-related liver disease. other causes of non-cirrhotic portal hypertension, including hepatic veno-occlusive disease and budd-chiari syndrome should be ruled out or treated in cvid patients presenting with hepatic disease. in the case of hepatic nrh in cvid patients, early detection could lead to earlier interventions (such as tips prior to hepatic encephalopathy), to mitigate complications. we describe the application of epigenetic quantification of t regulatory (treg) cells in addition to cd +, cd +, cd + t cells, b cells, nk cells, monocytes and neutrophils from as little as μl of fresh, frozen or dried blood. the method yields identical results to flow cytometry from fresh blood samples of a healthy donor cohort, with the advantage of being more sensitive and precise with limited amount of blood and minimal sample preparation (sci transl med ). we have used this method ) to immunophenotype patients with early onset immune regulatory disorders (pird) and primary immune deficiency (pid), and ) to evaluate cell subsets reconstitution early after hematopoietic stem cell transplantation (hsct). patients with immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) and ipex-like pird were evaluated by analyzing the treg-specific demethylated region (tsdr) of the foxp locus in the total of cd + t-cells. despite the dysfunctional foxp mutated protein, ipex patients exhibited elevated treg/cd + cell ratios which seemed to correlate with disease severity. in contrast, most of the patients with ipex-like symptoms without foxp mutations exhibited decreased treg/cd + cell ratios -in line with the possible central pathogenic role of treg function and number in pird. using epigenetic quantification of cd +/b-and nk cells, out of confirmed scid and xla cases were correctly identified within a cohort of newborn dried blood spot (dbs) samples ( % sensitivity, % specificity). the method identified one delayed onset scid as well as a xla case that were missed by combined trec/krec testing. epigenetic immune cell quantification missed one scid case with maternal engraftment that was identified by combined trec/krec testing. abnormally elevated treg/cd + ratio was also detected in a dbs from a newborn who was subsequently confirmed to be affected with ipex syndrome. when applied to serial blood samples during engraftment and reconstitution post-hsct, the epigenetic method allowed identification of the different blood cell subsets, including treg cells, at earlier time points than flow cytometry according to current clinical practice. this opens the way to a better understanding of the correlation between early immune reconstitution events and graft vs. host disease or viral reactivation, earlier than with the current methods, in different types of hsct. these studies underscore the suitability of epigenetic immune cell quantification for accurately measuring multiple immune cell types from limited blood sample sources. we propose this method as uniquely suitable for novel molecular diagnostic applications in settings with limited fresh blood sample or limited cell number, at the point of care as well as for newborn screening. we evaluated a -year-old male with hyperpyrexia, hypertrichosis, conical hypodontia, and a history of illnesses concerning for nemodeficiency syndrome. starting at six months of age, he suffered recurrent episodes of acute otitis media (non-typeable hib and actinobacter iwolffli), pneumonia, and rsv bronchiolitis. whole exome sequencing demonstrated a de novo heterozygous c. g>a (p.r q) mutation in the eda-receptor (edar) gene not present in the parental dna. his physical exam findings and mutation were consistent with hypohidrotic ectodermal dysplasia (hed), a rare genetic condition characterized by abnormal development of skin, teeth, hair, and sweat glands. hed is caused by defects in the ectodysplasin-a (eda)-nfkb signaling pathway but is not typically associated with immune deficiency. consistent with this, immunophenotyping showed normal sub-populations of t-, b-, and nk-cells. immunoglobulin and complement levels were quantitatively appropriate. he had normal mitogen-induced lymphocyte proliferation and normal antibody response to pneumococcal vaccination. nk-cell studies demonstrated robust cytotoxicity. however, nasal mucosa biopsy showed diffuse squamous metaplasia and the absence of ciliated epithelial cells. we hypothesize that recurrent infections in our patient arose from impaired mucociliary clearance due to a ciliary defect. this case raises the possible association between edar variants and ciliary dysfunction. it also underscores the importance of evaluating the immune status of hed patients with recurrent infections which could mimic nemo-deficiency and have broad implications about clinical management. the rapid pace of new gene discovery and phenotype expansion for primary immunodeficiency diseases (pidds) creates challenges for genetic testing and variant interpretation. whereas well-described clinical case reports in published literature have traditionally served as the source of phenotypic data used for variant interpretation, for pidds the causal variants are often private to the patients family and thus the sole source of phenotypic information for a novel genetic variant is frequently the history provided by the clinician on the test requisition form. taking into account such heterogeneous information during variant interpretation requires establishing objective criteria for its inclusion as part of the variant interpretation process. to this end, we adapted our laboratorys preexisting, evidence-based variant classification framework, called sherloc, by developing point-based criteria for the inclusion of clinical information such as a patients phenotype, familial segregation patterns, and whether the variant is inherited or de novo in the patient. as part of this process, we defined clinical criteria for pidd genes. here, we illustrate the application of this method and the importance of integrating clinical information into variant interpretation. between april and october , our commercial diagnostic laboratory performed immunological genetic tests, and information about the patients clinical history was provided in ( %) of these orders. restricting our analysis to just the genes for which case report information is currently used in variant interpretation, these tests revealed variants, ( %) of which were classified as pathogenic or likely pathogenic (p/lp). information from case report descriptions, segregation patterns, and de novo status were applied for %, % and % of p/lp variants, respectively. in ( %) cases, the clinical information provided by the clinician on the test requisition form was used as evidence in the classification of the patients variant as p/lp. ten variants were initially classified as being of uncertain significance and reclassified following receipt of further clinical information or testing of additional relatives. in addition, suspicious variants of uncertain significance were identified in which one or two additional patient case reports would allow for reclassification from uncertain significance to p/lp. these data illustrate the importance of providing good quality clinical information to the genetic testing laboratory both at the time of sample submission and following the receipt of genetic test results. background: cartilage-hair hypoplasia (chh) is a skeletal dysplasia with combined immunodeficiency, variable clinical course and increased risk of malignancy, mostly non-hodgkin lymphoma and basal cell carcinoma. there is a paucity of long-term follow-up data, as well as knowledge on prognostic factors in chh. objective: we conducted a prospective cohort study in finnish patients with chh to describe clinical course and analyze risk factors for adverse outcomes. methods: we recruited finnish patients with chh in - and performed clinical follow-up in - . we obtained health information from finnish national medical databases (covering time period of - ), the finnish cancer registry and the cause-of-death registry of the statistics finland and analyzed all patients' health records. standardized mortality ratios (smrs) were calculated based on the population data. primary outcomes included immunodeficiencyrelated death (from infections, respiratory diseases or malignancies), the development of lymphoma and the development of skin cancer. results: the study cohort included males and females. median age at recruitment was . yrs (range weeks - . yrs) and median duration of follow-up for the surviving patients was . yrs (range . - . yrs). half of the patients ( / , %) had no symptoms of immunodeficiency, while ( %) and ( %) patients manifested symptoms of humoral or combined immunodeficiency respectively, including six cases of late-onset immunodeficiency. in a significant proportion of patients ( / , %), clinical features of immunodeficiency progressed over time. of the patients with non-skin cancer, eight had no preceding symptoms of immunodeficiency. altogether patients had deceased (smr= . , % confidence interval (ci)= . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] including deaths due to pneumonia (n= ), malignancy (n= , smr= , %ci= . - ) and lung disease (n= , smr= , %ci= . . malignancy was diagnosed in / ( %) patients, mostly lymphoma (n= ) and skin cancer (n= ). severe short stature at birth (compared to normal, smr/smr ratio= . , , symptoms of combined immunodeficiency (compared to asymptomatic, smr= ( %ci= . - ) vs smr= . ( %ci= . - . ), hirschsprung disease (odds ratio (or) . , %ci= . - ), pneumonia in the first year of life or recurrently in adulthood (or= . / , , and autoimmunity (or= , %ci= . - ) in adulthood associated with early mortality. in addition, recurrent pneumonia in childhood was associated with the development of lymphoma, while warts and actinic keratosis were associated with the development of skin cancer. birth length standard deviation score correlated significantly with the age at the diagnosis of first malignancy (p= . ), lymphoma (p= . ) and skin cancer (p= . ), demonstrating that patients with shorter birth length developed malignancies at an earlier age. conclusions: patients with chh have high mortality due to infections and malignancies, but also from lung disease. some subjects present with late-onset immunodeficiency or malignancy without preceding symptoms of immune defect, warranting careful follow-up and screening for cancer even in asymptomatic patients. we provide clinicians with the risk factors for adverse outcomes to assist in management decisions. autoimmune lymphoproliferative syndromes (alps and related disorders) are characterized by insufficient apoptosis due to defects in the fas apoptosis pathway. fadd deficiency (omim ) is an autosomal recessive disorder resulting from a mutation in fas-associated protein with death domain (fadd), the adaptor protein involved in fas signaling to caspases and . we present a case of fadd deficiency identified by whole exome sequencing with a novel genetic mutation we describe two brothers with recurrent febrile episodes accompanied by seizures and respiratory compromise. the older sibling initially presented with status epilepticus following the measles mumps rubella vaccination later experiencing similar episodes until his demise at months of age. the younger sibling, who is unvaccinated, presented at months with fever, rash, vomiting, and diarrhea. he developed status epilepticus with respiratory depression that required intubation. he also had enlarged cervical lymph nodes that regressed with antibiotics and steroids. he recovered from that episode but subsequently had a series of similar illnesses with fevers, altered mental status and seizures. with the exception of elevated hhv igg, extensive infectious workup up in all instances was negative. previously described fadd deficiency patients demonstrate an alps like phenotype with increased circulating double negative t cells, lymphocyte apoptosis defects, elevated fas ligand and il , encephalopathy, functional asplenism but no splenomegaly or lymphadenopathy. our patients clinical and laboratory findings were similar. he had normal igg and iga, decreased igm, and lack of isohemagglutinins. absolute cd + count is elevated, with elevated percent of cd + tcr+ cd -cd -. normal mitogen and antigen t lymphocyte stimulation, but with defect in pokeweed induced b cell proliferation. fas ligand and il level are increased (see table ). no hepatosplenomegaly, but howell jolly bodies were detected in peripheral blood indicating functional hyposplenism. whole-exome sequencing revealed two different genetic alterations in the fadd gene: a maternally inherited nonsense mutation predicted to severely truncate the protein and a paternally inherited missense mutation in codon . although this paternal mutation has not been described as pathogenic, a different variant in same nucleotide of fadd has been associated with fadd deficiency (reference ). there are very few cases in the literature of fadd deficiency patients and the overall prognosis is poor compared to classical alps patients, as these patients are at significant risk of deadly sepsis from encapsulated organisms or death from neurologic complications. of the fadd deficiency patients described in the literature, several died prior to years old. while pneumococcal prophylaxis may reduce the risk of sepsis, hematopoietic stem cell transplant has been reported for patients with fadd deficiency (reference ), and is being considered for our patient. rationale: hcuvp is a patient product-introduction program that provides cuvitru® (immune globulin subcutaneous [human], % solution [ig gly]) free of charge for the first infusions to eligible patients with primary immunodeficiency disease (pid). using patient data from this ongoing program, our analysis described the clinical characteristics and infusion parameters of pediatric and adolescent patients who were initiated on ig gly through hcuvp. methods: hcuvp eligibility criteria were: patients aged years old, with a primary icd- -cm code verifying diagnosis of pid, and no current or prior use of ig gly at program initiation. data from patients who received the first ig gly infusion between january , , and september , were included. data from patients receiving infusions after october , were censored. descriptive statistics were calculated for patients demographic and clinical characteristics and prescribed and actual infusion characteristics by age group (< years and years). results: in total, patients who completed all infusions were included in the analysis, of whom were aged < years. among those who previously received immunoglobulin (ig) therapy, a greater percentage of patients aged < years were treated with intravenous ig therapy (n= ; %) compared with adult patients (n= ; %) before initiating ig gly. nine patients aged < years were treatment naïve. the mean infusion volume per site was lower among patients aged < years ( years: . ml; years: . ml; and years: . ml) than among patients aged years ( years: . ml and years: . ml). however, the mean infusion rate per site was similar between patients aged < years ( xmen disease (x-linked immunodeficency with magnesium defect, epstein-barr virus infection and neoplasia) is a primary immune deficiency caused by mutations in magt and characterized by chronic infection with epstein-barr virus (ebv), ebv-driven lymphoma, cd t-cell lymphopenia, and dysgammaglobulinemia. magt gene codifies to magt protein, a mg +-selective transporter, expressed in the human immune system, specifically in the spleen and the thymus. functional studies have established the key role of magt in t cells and natural killer (nk) cell activation. upon cd + t-cell receptor stimulation, magt mediates a transient mg + influx that is necessary for phospholipase c gamma (plcy ) activation, which drives ca + rise and downstream signaling. this mg + influx also regulates cytotoxic functions of nk and cd t cells through nkgd , reason why these patients have impaired cytolytic responses against ebv. eleven male xmen patients have been described. we present the case of a -year old hispanic infant with a pathogenic variant in magt gene that clinically manifested with early pneumocystis jirovecii and cytomegalovirus (cmv) interstitial pneumonia, and ebv chronic infection with good response to intravenous immunoglobulins supplementation without hematopoietic stem cell transplantation or gene therapy. laboratory study highlights low levels of nkg d ligands. the objective of this case report is to broaden the spectrum of clinical presentation of xmen disease, that manifests initially as a combined immune deficiency (cid) and evolved with a favorable course of the disease with intravenous immunoglobulins supplementation therapy and chemoprophylaxis with trimethoprim-sulfamethoxazole. introduction: lysinuric protein intolerance (lpi) is a recessively inherited disorder of the cationic amino acids transporter subunit y+lat caused by variants in the slc a gene. the disease is characterized by protein-rich food intolerance has a heterogeneous presentation. the clinical findings are a result of depletion of lysine, ornithine, and arginine. symptoms can include hyperammonemia, failure to thrive, protein aversion, neurologic disease, and lung disease. there is also evidence that inflammatory manifestations are mediated through upregulation of nfb, il , and tnf that occur independent of intracellular arginine levels and can lead to lifethreatening episodes of hemophagocytic lymphohistiocytosis (hlh). case presentation: a -year-old male presented with history of anxiety, depression, eating disorder, delayed puberty and complex partial seizures. due to poor nutrition and failure to thrive, a gastrostomy tube was placed. following commencement of enteral feeds, he presented with altered mental status, bilateral mydriasis, hyperreflexia, and agitation which lead to a picu admission. ammonia peaked as high as μmol/l and episodes ceased with cessation of enteral feedings. prior to enteral feeds, he had been self-restricting protein in his diet. biochemical testing was consistent with lpi and illumina next-generation sequencing revealed compound heterozygous variants in slc a (p.s lfs* and p.e dfs* ). hyperammonemia resolved quickly with cessation of protein intake and high rate dextrose infusion without the need for ammonia scavenging agents. he was subsequently started on proteinrestricted enteral feeds. at diagnosis he did not have any respiratory symptoms, ct scan of chest showed patchy areas of groundglass opacification that was suggestive of early pulmonary alveolar proteinosis (pap). bronchoalveolar lavage demonstrated foamy, cloudy pink fluid and elevated bronchioalveolar macrophages on cell differential. his clinical course and slc a genotype led to suspicion for smoldering hlh. the findings of elevated ferritin, hypertriglyceridemia, decreased fibrinogen, splenomegaly, elevated il- receptor, decreased nk cell function, along with hemophagocytosis on bone marrow biopsy confirmed the diagnosis. because of his pap and hlh, in addition to dietary modifications, a trial of il- beta inhibition (anakinra) at mg/kg/day was initiated. follow up ct scan of chest months after initiation of anakinra showed complete resolution of pulmonary groundglass opacifications and pap. bone marrow evaluation showed continued hemophagocytosis in spite of the normalization in ferritin, soluble il- receptor, nk function, and triglycerides levels. overall, he is significantly improved on daily anakinra and no longer meets criteria for hlh or pap. discussion: recent data has shown in y+lat models that thp- macrophages and a airway epithelial cells upregulate il and tnf regardless of intracellular arginine content. this suggests that inflammatory manifestations may continue independent of dietary modifications. we present a year old patient with newly diagnosed lpi who was treated dietary modification and anti-il therapy resulting in resolution of hlh and pap. more research is needed to see if long-term il blockade that can consistently control both the immunologic and pulmonary manifestations of lpi and positively impact morbidity and mortality. learning objective: recognize that symptoms of bartonella endocarditis and associated complications can share features of certain immunocompromising conditions. case description: an -year-old caucasian boy with history of repaired pulmonary atresia and aortic root dilation was diagnosed with pancytopenia and splenomegaly during a brief hospitalization for atypical pneumonia. pancytopenia persisted, splenomegaly worsened, and five months after presentation, he developed hypertension and renal insufficiency. he was diagnosed with hypocomplementemic, diffuse sclerosing and crescentic glomerulonephritis and was started on mycophenolate mofetil with improvement in kidney function and stabilization of cytopenias. as part of a comprehensive immune work-up, alps (autoimmune lymphoproliferative syndrome) panel was sent and demonstrated elevated double-negative t (dnt) cells with out of positive immunologic criteria for alps. neither targeted sequencing for alps and alpslike disorders nor whole exome sequencing revealed pathogenic mutations. by age , the patient remained on mycophenolate, but developed failure to thrive, with weight dropping from th percentile to less than rd percentile. he was hospitalized again for low-grade fever, increased work of breathing, left shoulder pain and fatigue and was found to have right lower lobe pneumonia. pancytopenia worsened, and he was started on cefepime and azithromycin without improvement in symptoms. echocardiogram revealed vegetations in his pulmonary conduit and bilateral branch pulmonary arteries, but multiple blood cultures were negative. upon further history, the patient reported contact with kittens. bartonella henselae titers and polymerase chain reaction (pcr) from blood were sent and were both positive. he completed a -week course of gentamicin, -month course of ceftriaxone, and was transitioned to doxycycline and rifabutin. after initiating antimicrobial therapy, his weight and energy significantly improved, his blood bartonella pcr became negative, and his splenomegaly resolved. approximately one year later, the patient underwent pulmonary artery conduit replacement and bartonella pcr testing of the tissue specimen was positive. he has had sustained weight increase, resolution of hypocomplementemia and splenomegaly, decrease in dnt cell frequency from > % to . %, and improvement though not resolution of cytopenias. he currently remains on doxycycline and rifabutin and continues treatment with mycophenolate. discussion: alps is characterized by defective lymphocyte apoptosis and clinical features such as lymphadenopathy, splenomegaly, hepatomegaly, cytopenias, and glomerulonephritis. the hallmark laboratory finding is expansion of dnts. our patient met criteria for a probable alps diagnosis based on the presence of both required criteria (chronic splenomegaly and elevated dnt cells) and secondary additional criteria (typical immunologic findings noted on alps panel). pediatric cases of bartonella henselae endocarditis have been associated with splenomegaly, cytopenias, and glomerulonephritis which mimic many features of monogenic immune dysregulatory disorders. the diagnosis of bartonella endocarditis in our patient therefore raises the question of whether his immunosuppression predisposed him to infection or if his entire clinical presentation can be explained by bartonella endocarditis. physicians taking care of patients with immune dysregulatory disorders should consider bartonella endocarditis in the differential diagnosis of onset or exacerbations of immune dysregulation. rationale: while fever is considered a sign of infection, many individuals with primary immunodeficiency (pi) anecdotally report a lower than normal average body temperature. on immune deficiency foundation (idf) friends and idf pi connect research forum online, pi patients report a diminished fever response even when other signs of infection are present. there is limited knowledge about the average body temperature in persons with pi. however, the implications of missing an infection in those with pi is well established. methods: study investigators partnered with patient investigators to design a prospective cohort study to determine whether body temperature differed between persons living with and without pi. three hundred fifty adults with pi were recruited from idf and one adult household member without pi was also recruited. mckesson digital oral thermometers (model - bgm) were provided and used to record temperatures in all participants three times a day for five consecutive days. descriptive statistics were calculated. median body temperatures were compared between the two cohorts at each time point using mann-whitney test. results: data from households were used for analysis ( . % participation rate). the pi population was largely female ( . %) with a median age of years and largely caucasian population ( . %). the non-pi population was largely male ( . %) with a median age of years and largely caucasian population ( . %). pi diagnoses included cvid ( . %), hypogammaglobulinemia ( . %), igg subclass deficiency ( . %), selective iga deficiency ( . %), specific antibody deficiency ( . %), agammaglobulinemia ( . %), chronic granulomatous disease ( . %), combined immunodeficiency ( . %), and complement deficiency ( . %). a total of individuals with pi ( . %) reported a lower than normal non-sick body temperature, while individuals with pi ( . %) reported a normal (between °f - °f) non-sick body temperature. a total of individuals with pi ( . %) reported absence of fever with infection, while individuals ( . %) reported a normal fever response with infection. the median body temperature was significantly higher for the pi patients in the morning, but not evening or bedtime, reading in of the days (monday: pi = . °f vs. non-pi = . °f, p = . ; tuesday: pi = . °f vs. non-pi = . °f, p = . ; wednesday: pi = . °f vs. non-pi = . °f, p = . ; thursday: pi = . °f vs. non-pi = . °f, p = . ; friday: pi = . °f vs. non-pi = . °f, p= . ). conclusions: despite the limitations of this non-clinical study, individuals with pi are knowledgeable about their conditions and can offer unique insights and direction to researchers. this study demonstrates that collaboration with patient advocacy groups may facilitate patient-centered and patient-driven research with high participation among the target population. introduction: familial mediterranean fever (fmf) is a hereditary condition characterized by recurrent episodes of painful inflammation caused by mutations in the pyrin (mefv) gene. alterations in the mefv gene affect pyrin production leading to recurrent fevers and painful inflammation in the peritoneum, synovium, and pleura. amyloidosis may also develop as a complication. arabic, turkish, armenian, and sephardic jewish populations are most commonly affected. homozygosity for mefv mutations are associated with a more severe course. there is a paucity of information regarding pediatric fmf in the literature. case: we present a case of a -year-old male with minor speech delay diagnosed with compound heterozygous fmf. patient was initially referred due to recurrent fevers and infections. at months of age, he was hospitalized with septic shock requiring intubation secondary to adenovirus. at months of age, the patient began to have recurrent fevers every to weeks, leading to multiple blood draws and courses of antibiotics prior to referral. at , , and months of age, he developed three separate episodes of febrile seizures. a total of - lifetime episodes of acute otitis media occurred prior to bilateral myringotomy tube placement. four episodes of streptococcus pyogenes pharyngitis confirmed by throat culture preceded tonsillectomy. no oral ulcers, joint pain, or abdominal pain were reported. no other infections such as pneumonia, sinusitis, uti, non-viral gastroenteritis, fungal infections, or skin infections were reported. both parents are ashkenazi jewish and a maternal history of early miscarriage was noted. family history was negative for immunodeficiency, malignancy, and autoimmunity. the patients vital signs and physical exam were unremarkable. serology indicated leukocytosis of . k/l with elevated monocytes of cells/l, elevated eosinophils of cells/l, and slightly elevated cd t cell count of cells/l. neutrophil, cd t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus, diphtheria, rubella, streptococcus pneumoniae, and haemophilus influenzae b titers were protective. genetic analysis identified that the patient was compound heterozygous for the e q and v mutations in the mefv gene. family was instructed to keep a fever diary. colchicine . mg once a day was given initially, then increased to . mg once a day for inadequate response. loose stools were observed while patient was maintaining a lactose free diet so he was switched to colchicine . mg bid with resolution of loose stools. apart from two occasions when his colchicine dose was missed, the patient remained afebrile at his follow up visits. conclusion: we present a pediatric case of compound heterozygous fmf (e q and v mefv mutations) in an otherwise healthy -year-old male of ashkenazi jewish background, initially symptomatic at months of age. individuals who are compound heterozygous for the e q and a second mevf mutation are generally symptomatic, although severity cannot be predicted. additional pediatric research on symptomatic heterozygous and compound heterozygous fmf is recommended. natural killer (nk) cells are innate lymphocytes that play a key role in defense against virally-infected cells and in tumor surveillance. nk cells can be divided in two subsets. the majority of nk cells in peripheral blood expressed intermediate levels of cd and are referred to as cd (dim). these nk cells are responsible for nk cell cytotoxicity. a minor population of nk cells express very high expression of cd and are referred to as cd (bright). these nk cells are responsible for cytokine production and are precursors to cd (dim) nk cells. a few immunodeficiencies have been described in which there are abnormal nk cell subsets, such as autosomal dominant gata deficiency where cd (bright) nk cells are absent and irf where there is a paucity of cd (dim) nk cells and relative expansion of cd (bright) nk cells. here we present a patient with an absence in cd (bright) nk cells secondary to cd deficiency. our patient is a -year-old african american female born to non-consanguineous parents. the patients past medical history is significant for chronic lung disease secondary to prematurity, recurrent acute otitis media, failure to thrive and congenital hypothyroidism. family history is significant for an older sister that presented at age with ebv-associated hodgkin lymphoma whose treatment was complicated by chronic activated ebv infection and who ultimately underwent hematopoietic stem cell transplantation (hsct). our patient presented with pancytopenia, fever, lymphadenopathy and splenomegaly. she was found to have ebv viremia with greater than , copies in whole blood by pcr. she was treated with two doses of rituximab followed by etoposide and dexamethasone as a bridge to hsct. whole exome sequencing demonstrated a homozygous mutation in cd . cd is a member of the tumor necrosis factor receptor family and influences the function of t cells, b cells and nk cells. in nk cells, cd is primarily expressed in cd (bright) nk cells. cd deficiency is an autosomal recessive disorder associated with persistent symptomatic ebv viremia, including ebv-driven hemophagocytosis and lymphoma, hypogammaglobulonemia and specific antibody deficiency. our patients immune evaluation prior to initiation of chemotherapy and immunosuppression was notable for very elevated igg, iga and igm. despite hypergammaglobulonemia patient had only out of protective titers against streptococcus pneumoniae. the patient had pan-lymphopenia with appropriate percentages of lymphocyte subsets. assessment of her b cell subsets showed a slight increase in the percentage of transitional b cells/plasmablast and a nearly complete absence of cd -expressing b cells. her nk cell phenotyping demonstrated a complete loss of cd (bright) nk cells with reduced nk cell cytotoxicity, comparable to what has been previously reported in patients with gata deficiency. previous reports of patients with cd deficiency denote normal nk cell numbers with normal to moderately reduced nk cell cytotoxicity, however, cd deficiency causing a specific loss of the cd (bright) nk cell subset has not been previously reported. cd deficiency should be consider in patients with ebv driven disease and abnormal nk cell studies. introduction/background: the transcription factor ikaros is encoded by the ikzf gene and plays a crucial role in lymphopoiesis. somatic, and more recently also germline mutations of ikzf are associated with a hematologic malignancies, most notably b-cell precursor acute lymphoblastic leukemia. germline mutation in ikzf was first reported as a monogenic cause of human disease characterized by marrow failure and immune deficiency in a single neonate in . subsequently, mutations leading to haploinsufficiency were discovered to underlie a proportion of patients with cvid and low b cell numbers, and dominant-negative mutations have been observed to cause more severe combined immune deficiency phenotypes. at this time, there is very little known regarding allogeneic hematopoietic cell transplantation (hct) outcomes for patients with severe dominant-negative ikzf mutations. concerningly, ikaros deficiency has been observed to have a negative impact on graft versus host disease in mouse models. objective: to describe allogeneic stem cell transplant outcomes in patients with the dominant-negative ikaros mutation. methods: we collected transplant data from patients who underwent allogeneic hct at transplant centers around the world. results: patients underwent allogeneic hct using a variety of conditioning regimens. patients received bone marrow (n= ) or cord blood (n= ) grafts from an hla-matched sibling donor (n= ) or single allele hlamismatched unrelated donor (n= ). neutrophil engraftment occurred between day + and + post-transplant. platelet engraftment occurred between day + and + except in one patient who did not have return of normal platelet counts due to underlying liver dysfunction. all patients were documented to have greater than % whole blood donor chimerism at a median of days (range - days) following transplant and maintained > % donor chimerism until last follow-up. only one patient developed grade ii acute gvhd. no patients developed chronic gvhd. one patient died approximately year post transplant related to cryptosporidium cholangitis which existed prior to hct. at the most recent follow up of the surviving patients (range: . - . y), ivig had been discontinued, antimicrobial prophylaxis had been stopped, and patients had received routine vaccinations. they all had excellent performance status. conclusions: allogeneic hct may be a safe option to consider for patients with dominant-negative ikaros mutation as there does not appear to be an increased risk of death or gvhd. moreover, -out-of- of the transplanted patients are alive and well and show no features of the disease. however, because of the limited number of patients evaluated and the retrospective nature of this analysis, our data do not allow firm conclusions to be made, and further studies will be needed to evaluate outcomes in larger cohorts. introduction: when evaluating patients with t-cell lymphopenia, we often are concerned about defects in lymphocyte production and function, especially in the setting of frequent infections. here we outline a case demonstrating t-cell lymphopenia due to increased loss, which should be considered in the differential diagnosis. case report: we report a -year-old male who initially presented with recurrent, right-sided pneumonias requiring frequent hospital admissions including severe episodes necessitating intensive care unit admission. his work up for the pneumonias included a bronchoscopy revealing normal anatomy with minimal inflammation, and a chest ct with mild peribronchial wall thickening. as his pulmonary disease progressed, he developed a persistent, productive cough with expectorated mucous plugs that were plastic-like in appearance. while his pulmonary symptoms responded to steroids, his mucous plug production persisted. sputum cultures were intermittently positive, isolating cryptococcus neoformans and aspergillus niger. he underwent vats and wedge biopsy, concerning for recurrent aspiration. an immunologic evaluation initially demonstrated normal t-and b-cell counts, but serial evaluation of his lymphocyte population demonstrated low cd + cells (ranging - cells/cumm), and low normal cd cells (ranging - cells/cumm) with normal b-and nk-cell numbers. further t-cell evaluation revealed normal ratios of naive and memory p o p u l a t i o n s ( c d c d r a + % , c d c d r o + % , cd cd ra+ %, cd cd ro %), normal trec ( copies per ^ cd cells) and normal thymic emigrants (cd cd cd ra+ : , normal - ), indicative of sufficient thymopoiesis. mitogen and antigen stimulation assays demonstrated normal responses to phytohemagglutin, concanavalin a, and pokeweed mitogen, with a low lymphocyte response to candida. he had normal quantitative immunologlobulins, normal diphtheria, tetanus and streptococcus pneumonia titers. his dihydrorhodamine flow cytometry and fish for chromosome q . deletion were negative. given normal function and thymic output, his immunologic profile was concerning for t-cell loss. our patient was registered with the undiagnosed disease network, and had a second review of his lung biopsy, concerning for plastic bronchitis. subsequent lymphatic imaging demonstrated abnormal lymphatics within the bilateral clavicular space, right greater than left, with questionable partial thoracic duct, explaining his unilateral symptoms. he was diagnosed with plastic bronchitis secondary to abnormal lymphatic drainage, with lymphatic fluid filling his airways and secondary t-cell loss. discussion: plastic bronchitis is a rare and potentially fatal disorder, seen commonly after the fontan procedure for congenital heart disease. this process has resulted in t-cell loss into the airway and subsequent t-cell lymphopenia. in patients with fontan-related protein losing enteropathy, multiple immune abnormalities have been described including reduced immunoglobulins, lymphopenia, and selective cd lymphocyte deficiency. similar findings have been reported in patients with lymphatic malformations. although the impact of t-cell loss on adaptive immunity is not entirely known, there is no indication of increased risk for atypical infections. given his normal mitogen assay, our patient did not start prophylactic antibiotics. he continues to have symptomatic episodes with lymphopenia, but has had no opportunistic infections, and remains stable with an aggressive pulmonary regimen. we conclude by reiterating the importance of considering t-cell loss in patients presenting with lymphopenia, particularly with evidence of normal thymopoeisis and t-cell function. introduction: granulomatous disease (gd) has been described with a variable incidence ( . - . %) in patients with common variable immunodeficiency (cvid). an increase in malignancies has been reported in cvid patient cohorts, particularly for lymphoma, reported in . - . % of the cvid patients depending on the cohorts. prior analysis of a cohort of cvid patients included patients with gd (gd+). in these, there was a suggestion of more cases of lymphoma ( . %) when compared to cases without (gd-) ( . %) although the difference was not statistically significant (p=. ). objectives: compare the frequency of lymphoma in gd+ and gd-patients in the cvid patient cohort from the usidnet registry. methods: we submitted a query to the usidnet registry requesting deidentified data for patients with the diagnosis of cvid, through august . statistical analysis was performed on spss, with comparisons done with pearson chi-square or fisher's exact test, depending on the sample sizes, using an alpha level of . . results: a cohort of cvid patients from the usidnet registry was analyzed. ninety-one patients ( . %) were gd+. overall, patients ( . %) had a malignancy diagnosis, of these ( . %) with lymphoma. lymphoma was present in / gd+ patients ( . %) versus / gdpatients ( . %) (p=. ). overall malignancy was present in / gd+ ( . %) versus / ( . %) (p=. ). discussion: in the cohort of cvid patients from the usidnet registry, we found a frequency of lymphoma of . %, which is in the range of previously described cohorts. the frequency of lymphoma was . % in patients with gd, higher than the . % frequency for gd-patients, but these differences were not statistically significant. our identified frequency of lymphoma in gd+ patients was lower than the one previously identified in the cvid patient cohort, but with similar proportional differences between gd+ and gd-patients. despite no statistical significance, the frequency of lymphoma, as shown here and elsewhere, was higher in cvid patients gd+ than gd-in both studies, with no full understanding of this increased risk of lymphoma. expanding this analysis to larger groups of cvid patients may help to confirm, or deny a more robust association, which may have a meaningful impact in the outcomes of this particular population. introduction: patients with refractory pericarditis have been treated with intravenous immunoglobulin (ivig) or interleukin receptor antagonist (anakinra) with limited and transient benefit. separate or combined therapy with subcutaneous immunoglobulin (scig) and interleukin (il) inhibitor (rilonacept) for refractory pericarditis in a cohort of patients has not been previously described. case descriptions: patients were referred for recurrent pericarditis refractory to traditional therapies at ages ranging from to years. they all had multiple serious sequelae of their pericarditis and abnormal immune parameters including hypogammaglobulinemia, poor responses to vaccines, poor mitogen induced lymphocyte proliferation, and/or b cell lymphopenia. the patients had varied past medical histories and associated conditions. patients were started on ig, with some initiated on ivig, though all were transitioned to hyaluronidase-facilitated scig (hyqvia). patients were then started on either anakinra or rilonacept with patients continuing on rilonacept and remaining on anakinra. all patients had complete or near complete resolution of their pericarditis on dual therapy for greater than year. the markedly elevated il prior to therapy seen in all of the patients normalized post-therapy. some patients had elevated il prior to therapy that also improved post-therapy. patient who has also been diagnosed with familial mediterranean fever (fmf) has stopped both therapies for greater than year with no further episodes of her pericarditis. discussion: patients with recurrent refractory pericarditis and signs of immunodeficiency and autoinflammatory disease on laboratory testing responded to dual therapy with hyqvia and rilonacept or anakinra resulting in resolution of pericarditis. inflammasome and immune abnormalities may be implicated or associated with recurrent pericarditis and may respond to targeted therapies. chief, laboratory of clinical immunology and microbiology, idgs, dir, niaid, nih, bethesda, md, usa hypomorphic recombination activating gene (rag ) mutations result in residual t-and b-cell development in both humans and mice and have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (cid-g/ai). recent studies have shed light on how hypomorphic rag mutations alter the primary repertoire of t and b cells, but less is known about their effect on immune dysregulation in targeted organs. in order to investigate the role of these mutations in determining intestinal disease, we set out to evaluate gut immunity and microbiota interplay in rag mutant hypomorphic mice. we evaluated two mouse models carrying homozygous rag mutations (r q and r w), corresponding to human mutations (r q and r w, respectively) described in patients with cid-g/ai. both mutations fall in the coding flanksensitive region of the rag c-terminal domain. on the basis of aminoacid properties and in vitro studies, the r q mutation has demonstrated a moderate effect on rag protein stability while the r w mutation resulted highly disruptive. analysis of intestinal pathology in rag mutant mice (niaid animal protocol lcim e) revealed different degrees of spontaneous colitis, with the most severe inflammatory infiltrate observed in mice carrying the most disruptive mutation, r w. colonic inflammation was characterized by crypt elongation, epithelial hyperplasia, and an abundant inflammatory infiltrate extending to the colonic lamina propria, with occasional crypt abscesses. a significant increase in activated cd hicd lcd + t cells expressing the gut homing receptor was observed in mesenteric lymph nodes (mlns) of both mutant strains, and was especially prominent in r w mutant mice. additionally, the proportion of mln cd + t regulatory (treg) cells was increased in both mouse models. finally, mln of mutant mice contained a high number of myeloid cells (cd b+ ) along with a decreased number of b + b cells, and these abnormalities were also more prominent in r w than in r q mice. in summary, we have shown that rag mutant hypomorphic mice present with different degrees of inflammatory bowel disease, with the mouse model carrying the most disruptive mutation presenting with the most severe phenotype. we are currently performing studies to evaluate the impact of rag mutations on microbiome composition and diversity in these mouse models of cid-g/ai. background: hypogammaglobulinemia or low serum immunoglobulin g (igg) levels either inherited (primary) or acquired (secondary) is associa t e d w i t h i n c r e a s e d i n f e c t i o n r a t e s . p r i m a r y ( °) hypogammaglobluinemia can be caused by many primary immune deficiencies (pid) including combined variable immune deficiency (cvid), while secondary ( °) hypogammaglobluinemia can be caused by many acquired conditions such as lymphomas, leukemias, or chemotherapies and other immunosuppressive drugs. immunoglobulin replacement therapy (irt) has been the mainstay of treatment in patients with hypogammaglobulinemia by reducing infection through replenishing the quantitative igg. there are other applications of ig therapy such as in autoimmune diseases, where the mechanism of action is thought to be ig mediated immunomodulation. innate immune cells have shown to be involved in such mechanism, but whether irt modulates adaptive immune cells in patients with hypogammaglobulinemia is not well known. hypothesis: irt has an immunomodulatory effect on t-cell function and proliferation in patients with hypogammaglobulinemia. methods: blood from thirty patients with °(n= ) or °(n= ) hypogammaglobulinemia recruited from the immunodeficiency clinic at the ottawa hospital was drawn for peripheral blood mononuclear cell (pbmc) isolation, before starting irt and minimum weeks after starting irt. data regarding igg level, number and type of infections after receiving irt was collected. pbmcs were analyzed using flow cytometry for quantitation of t-cell subset. cultured and anti-cd /cd stimulated pbmc were also analyzed for extracellular and intracellular cytokine production, measured by e l i s a a n d f l o w c y t o m e t r y, r e s p e c t i v e l y. c o m b i n e d cytomegalovirus, epstein-barr virus and influenza virus (cef) peptides were used to study specific t-cell responses. anti-cd / cd stimulated pbmc were used for celltrace t-cell proliferation a s s a y s . d a t a w a s g r o u p e d b a s e d o n n a t u r e o f hypogammaglobulinemia i.e. °or °. results were compared between before and after irt using wilcoxon matched-pairs signed rank test. results: irt was not found to significantly alter proportion of treg, cd +, or cd + t-cell populations or activation state as measured by cd ra/r expression. however, irt was found to significantly increase expression of intracellular ifn-y in cd + and cd + t-cells post-cd /cd stimulation in °(p = . ), but not in °h ypogammaglobulinemia patients. there was no change in extracellular il- and il- cytokine production in both groups. in contrast, cd + tcells in °hypogammaglobulinemia patients showed significantly higher expression of intracellular ifn-y and tnf-a post-cef viral peptide stimulation (p = . ). cd + and cd + t-cell proliferation after cd /cd stimulation was found to be decreased after irt for both groups (p = . & p = . ). conclusions: our results suggest that irt can alter cd + and cd + t-cell function with differential effect in patients with °o r °hypogammaglobulinemia in addition to replenishing serum igg level. more experiments assessing cytotoxicity of t-cells will be conducted to further study t-cell subset function as well as bcell function. these laboratory results will be analyzed for association with clinical outcomes. uploaded file(s) uploads background: severe congenital neutropenia (scn) is a rare immunodeficiency disorder characterised by the extremely low absolute neutrophils count (anc) less than . x /l. the clinical feature of scn is recurrent bacterial infections and the patients the risk of leukemia development. the incidence of scn is estimated to be in individuals. mutations in more than genes have been described causing scn and it is either recessive, dominant or x-linked inheritance. case presentation: we described an years old malaysian girl who presented with recurrent abscesses over the whole part of the body, recurrent oral candidiasis, growth failure and recurrent pneumonias since months old. she also had history of a few episodes of acute tonsillitis, chronic suppurative otitis media and herpes zoster infections. throughout her age, she had persistent neutropenia less than . x /l but in few occasions, her anc elevated up to more than . x /l . she was treated as autoimmune neutropenia, respectively due to few positive results of autoimmunity workout such as antinuclear antibodies (ana) and double stranded dna (dsdna) but eventually later to be negative. later at the age years old, whole exome sequencing was performed and confirmed by sanger s e q u e n c i n g , f o u n d a h e t e r o z y g o u s v a r i a n t i n e l a n e gene(c. g>t; p.gly ter), an autosomal dominant which was described to cause scn. both parents do not carry this mutation, hence, it is a de novo mutation. currently, she had few on and off recurrent infections. despite that, she is relatively well and on prophylaxis antibiotic. conclusion: to our knowledge, we report for the first time a malaysian girl with scn, with confirmed mutational analysis of the elane gene. the delayed diagnosis might be due to the insufficient awareness of the phenotypic presentation of this rare disease. moreover, the genetic analysis is not available in malaysia and need to be done outside of the country. this case demonstrates the importance of the genetic analysis which may help in improving the diagnosis and management of the patient. ( ) submission id# professor of paediatrics and immunology, university college london; great ormond street hospital nhs trust; orchard therapeutics, london, uk background: ada-scid is a rare genetic disorder which causes severe combined immunodeficiency. historically, ada-scid has been treated using enzyme replacement therapy (ert) followed by allogeneic hematopoietic stem cell (hsc) transplant (hsct) from a matched related donor (mrd) or, if none is identified, a non-mrd (matched/mismatched unrelated or mismatched related donor). we developed a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by the short form of the elongation factor- alpha (efs) promoter (efs-ada lv). the drug product (otl- ), composed of autologous hscs transduced ex vivo with the efs-ada lv, was evaluated in a prospective, historically-controlled phase i/ii clinical trial in ada-scid pediatric subjects. we report safety and efficacy at months in ada-scid subjects treated with lentiviral gene therapy (gt) compared to a historical cohort of ada-scid patients treated with hsct. methods: twenty subjects ( male, female; mo . yrs) were treated with gt. autologous cd + hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. busulfan was administered at a single dose ( mg/kg) prior to infusion of otl- . the control group included patients ( . mo . yrs) treated with allogeneic hsct (mrds n= , non-mrds n= ) at great ormond street hospital, uk (n= ) or duke university childrens hospital, usa (n= ) between . results: at months, overall survival (os) and event-free survival (evfs), defined as survival in the absence of ert reinstitution or rescue allogeneic hsct) were statistically significantly higher in the gt group compared with the hsct group (table) . successful engraftment of genetically modified hsc was observed in all gt subjects at months, which persisted over months, based on vector gene marking in granulocytes (median . copies/cell [range . - . ] at months) and peripheral blood mononuclear cells (median . copies/cell [range . - . ] at months), and was associated with increased red blood cell ada enzyme activity and metabolic detoxification from deoxyadenosine nucleotides. over months, none of the gt subjects required peg-ada ert reinstitution and % were able to stop receiving immunoglobin replacement therapy (igrt), whereas % hsct patients required rescue hsct or reinstitution of peg-ada ert, and % were able to stop receiving igrt (table) . nine subjects in the gt group experienced a serious adverse event (sae), most frequently infections and gastrointestinal events; only one was considered treatment-related. in the gt group, there were no events of autoimmunity during the study. due to the autologous nature of the product, there was no incidence of graft vs host disease (gvhd) in the gt group; whereas patients in the hsct group experienced acute gvhd and experienced chronic gvhd events, one of whom died. conclusions: treatment with lentiviral gt for ada-scid is well tolerated and has a favorable benefit-risk profile at months based on sustained gene correction and restoration of immune function, as well as improved os and evfs compared with hsct (mrd or non-mrd) at months. background: ada-scid is a rare genetic disorder that causes severe combined immunodeficiency, with minimal or absent b cell function. prior to, and often after, treatment with allogeneic hematopoietic stem cell (hsc) transplant (hsct) or autologous ex vivo hsc gene therapy (gt), patients are managed with enzyme replacement therapy (ert) and immunoglobulin (ig) replacement therapy (igrt). we evaluated a gt treatment with autologous hscs transduced ex vivo with a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by an internal short form of the elongation factor- alpha (efs) promoter ("efs-ada lv"). at months follow-up, pediatric ada-scid subjects treated with gt were compared to a historical cohort of ada-scid patients treated with hsct. here, we report on b cell reconstitution in these cohorts. methods: twenty subjects ( male, female) aged mo - . yrs received gt. autologous cd + hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. genetically modified cells were administered after conditioning with single dose busulfan ( mg/ kg). the control group included patients aged . mo to . yrs treated with hsct at great ormond street hospital (uk) (n= ) or duke university children's hospital (us) (n= ) between - . the hsct patients received an allogeneic transplant from matched related donors (mrds) (n= ) or non-mrds (n= ). subjects continued to receive igrt post-gt until a clinical decision was made to stop, factoring in b cell reconstitution, general medical condition and seasonal infections. results: by month , in the gt group, % had stopped treatment with igrt compared to % in the hsct group overall. by months and , higher proportions of gt-treated subjects had stopped igrt ( % and %, respectively) compared with mrd hsct patients ( % and %, respectively) and non-mrd hsct patients ( % at both timepoints) (table) . in the gt group, vector gene marking was detectable in peripheral blood mononuclear cells within months and persisted at months post-infusion (median . copies/cell [range . - . ]), suggesting successful gene modification. as evidence of b cell reconstitution, iga and igm levels in peripheral blood sera more than doubled by months, from . mg/dl (range to ) to . mg/dl (range to ) and . mg/dl (range to ) to . mg/dl (range to ), respectively. additionally, antibody response following tetanus vaccination, was evaluated in subjects. all subjects mounted a protective response to the vaccine (median antibody response . iu/ml [range . to . ]), based on a normal threshold of . iu/ml (hammarlund clin infect dis ) and a laboratory reference range ( . to . iu/ml). conclusions: gt with autologous hscs transduced ex vivo with efs-ada lv resulted in b cell reconstitution, as evidenced by doubled iga and igm production at months, cessation of igrt in % of patients by months, and protective specific antibody responses to tetanus vaccine in patients that were evaluated. background: x-linked chronic granulomatous disease (xcgd) results from mutations in cybb encoding the gp phox subunit of phagocyte nadph-oxidase. attempts to treat xcgd with gene therapy (gt) using transduced autologous hematopoietic stem cells (hsc) transduced ex vivo with a gammaretroviral vector have met with limited efficacy due to transient engraftment of gene corrected hscs, gene silencing, and vector insertion-mediated activation of oncogenes leading to myelodysplasia. we developed a novel self-inactivating (sin) lentiviral vector (g xcgd lv) with a chimeric cathepsin g/cfes myeloid-specific promoter driving gp phox expression from a codon optimized cdna. following transplant of g xcgd lv ex vivo transduced autologous hscs into busulfanconditioned xcgd patients, there was long-term restoration of oxidase activity in peripheral blood polymorphonuclear neutrophils (pmn) at months in of severely affected xcgd patients without evidence of genotoxicity. here we present data about the multiple assays used to assess quality and quantity of restoration of pmn oxidase activity. methods: similar trials of gt with g xcgd lv were initiated in the uk (n= , plus compassionate use patient) and usa (n= ). all patients had histories of inflammatory disease and severe, persistent infections (some non-responsive to conventional therapy at time of gt). g-csf plus plerixafor-mobilized cd + hscs were transduced with ex vivo g xcgdlv. subjects received myeloablative conditioning with singleagent busulfan, targeted to net area-under-the-curve of , ng/ml*hr. freshly prepared or cryopreserved quality-tested genetically-modified hsc, manufactured on-site, were administered intravenously. pmn oxidase activity post-gt was assessed by p-nitroblue tetrazolium (nbt) reduction, dihydrorhodamine (dhr) flow cytometry assay, and quantitative ferricytochrome c assay (ferric) measurement of superoxide generation. results: we report results for patients (aged - years) with - . years of follow-up; two additional patients were treated but died within three months of gt from complications deemed related to pre-existing diseaserelated co-morbidities (severe pulmonary disease and anti-platelet antibodies). within month post-gt, oxidase (+) pmn were present in peripheral blood based on nbt testing and dhr flow cytometry. expression of the corrective transgene was confirmed by flow cytometry using antibody detection of gp phox. quantitative biochemical measurements of oxidase activity were also confirmed in some samples using the ferric assay, demonstrating quantitative levels of superoxide production per corrected cell that were within the normal range. functional testing of oxidase burst activity using dhr fluorescent assays was applied serially to follow levels of corrected pmn where oxidase activity per corrected cell also were in the normal range. all patients had > % pmn dhr+ within one month, which remained stable for most patients over the follow-up period ( figure) . follow-up demonstrated sustained stable persistence of - % oxidase burst positive neutrophils in of surviving subjects at months, with restoration to clinically beneficial levels (defined as % of pmn being dhr+) in these patients as of december . conclusion: these results demonstrate corrected pmn function within month in x-cgd patients treated with autologous gt. pmn oxidase activity was sustained at levels which restore biochemical function and provide clinically beneficial levels of immunity for months in / patients. the formulation for igsc % was developed based on the knowledge acquired from the formulation of grifols currently licensed % immune globulin (human), gamunex®-c; however, the protein concentration was increased from % to % to facilitate efficient subcutaneous administration. gamunex-c has an extensive record of safety and tolerability when administered intravenously and subcutaneously for greater than years in diverse patient populations. the igsc % manufacturing process employs the same purification steps as gamunex-c and was demonstrated to be robust and to provide an igg product with the required potency, purity, and quality. the formulation excipient characteristics and compatibility with the drug product have been well established. glycine has been an excipient of intramuscular immune globulin (human) for fifty years and intravenous immune globulin (igiv) for over twenty years. the igsc % formulation has low buffering capacity, and a low ph was selected to achieve a product with low aggregates, low fragments and viscosity suitable for subcutaneous administration. to improve visual clarity, the igsc % formulation contains a small amount of polysorbate (ps ), which is widely used in biopharmaceutical products. subcutaneous administration of the igsc % formulation has been well tolerated in clinical studies. objectives: the goal was to provide the pid population with a new % immunoglobulin liquid product for subcutaneous administration (igsc %). methods: igsc % is manufactured using the current manufacturing process for gamunex-c, followed by an additional concentration step so that the product can be formulated at a higher protein concentration. igsc % and gamunex-c batches were produced at full industrial scale and then subjected to a series of analytical testing including assessment of purity, composition and neutralizing activity. results: the igsc % and gamunex-c manufacturing processes and formulations have preserved the igg integrity, molecular characteristics and potency. the manufacturing processes have eliminated lipids, alcohols, and acetate and coagulation factor impurities, including fxia, which were undetectable by either specific or global methods. the igsc % and gamunex-c batches were % gamma globulin by agarose membrane electrophoresis, and have a subclass distribution similar to normal plasma and acceptable specific antibody content. igsc % was shown to be primarily monomer plus dimer igg ( ± %) with minimal aggregate or fragment, which confirms that appropriately gentle processing conditions were used during the concentration of % igg solutions to % igg. conclusions: igsc % is a highly concentrated igg solution with characteristics comparable to gamunex-c, but with twice the igg concentration in order to facilitate subcutaneous administration with reduced volumes and shorter infusion times. analytical testing demonstrates suitable potency, purity, and neutralizing activity for a number of specific antigens. funding: this study was funded and conducted by grifols, a manufacturer of % immunoglobulin for subcutaneous administration. disclosure: all authors are employees of grifols. frequent respiratory tract infections and seizures cause recurrent hospitalizations in these children and are typically considered a result of neurological impairment and poor airway clearance. evaluation of these patients for immunodeficiency is not a common clinical practice. here we report combined immune deficiency in patients with mds and recurrent respiratory tract infections. case presentation case : a boy with mds was initially referred at age months for an abnormal newborn screen with low t cell receptor excision circles (trec) for severe combined immunodeficiency (scid). initial evaluation revealed moderate cd + and cd + t cell lymphopenia (figure ). initial immunoglobulins levels were normal. he was placed on antiseizure medications. he later developed recurrent and severe respiratory tract infections starting in infancy. at months of age, he developed hypogammaglobulinemia ( figure ). in addition, t cell counts progressively decreased and stayed around cells/ul. immunoglobulin replacement therapy started at months of age. hospitalizations due to respiratory tract infections significantly decreased. case : a -year-old boy with mds had recurrent bacterial and viral respiratory infections which required numerous hospitalizations including intensive care unit stays. newborn screening for scid was negative. he had been on anti-seizure medications. immunologic evaluation at years of age revealed low total cd + cells and cd + t cells (cd +: cells/ul[normal range - cells/ul], cd +: cells/ ul[normal range - cells/ul]), hypogammaglobinemia (igg: mg/dl[normal range - mg/dl]), and non-protective igg levels to tetanus, varicella and pneumococcus serotypes. immunoglobulin replacement therapy started at years of age which resulted in reduced frequency and severity of respiratory infections, and improved quality of life. discussions: t cell lymphopenia and hypogammaglobulinemia were seen in both our cases of miller-dieker syndrome. to our knowledge, immune deficiency has never been reported in mds. one of our cases suggests that low t cell counts may start as early as at birth and may be detected by newborn screening. hypogammaglobulinemia may be primary or secondary due to antiepileptics. both children had reduced frequency and severity of respiratory infections and improved quality of life after immunoglobulin replacement highlighting the importance of screening and early management of immunodeficiency. conclusion: miller-dieker syndrome is likely another syndromic primary immune deficiency disorder. a high index of suspicion with early screening and management of immunodeficiency may be beneficial for children with miller-dieker syndrome. uploaded file(s) uploads this prospective, multi-center, open-label study assessed the pharmacokinetic (pk), safety, and tolerability of immune globulin subcutaneous (human), % caprylate/chromatography purified (igsc %) in subjects with primary immunodeficiency (pi). the objectives were to determine a weekly subcutaneous (sc) dose of igsc % that is noninferior to the intravenous (iv) dose of immune globulin injection (human), % caprylate/chromatography purified (igiv-c %) and to determine the steady state trough igg levels after igsc % and igiv-c % infusions. there were possible phases. if not on a qualifying igg regimen at enrollment, subjects (n= ) were required to enter the run-in phase, receiving igiv-c % to achieve steady-state before entering the iv phase to determine steady-state area-under-the-curve (auc) of iv infusions. subjects with a qualifying igiv-c % regimen ( - mg/kg) (n= ) directly entered the iv phase for steady-state iv pk assessments. upon completion of the iv pk assessments subjects entered the sc phase, receiving weekly doses of igsc % for up to weeks, with steady-state auc determined at the th dose. igsc % was not associated with any reports of serious local infusion site reactions (isrs). the majority of local isrs were mild-to-moderate. igsc % (at a dose conversion factor of . ) provided equivalent exposure to igiv-c % as assessed by steady-state auc - days, with % higher mean igg trough values, lower fluctuations in igg concentrations and the flexibility of at home administration. igsc % was well tolerated with a safety profile comparable to igiv-c %. clinicaltrials.gov identifier: nct disclosure: kecia courtney, elsa mondou, and jiang lin are employees of grifols, a manufacturer of igsc %. grifols is the sponsor of this study. background: in two reports described the deficiency of adenosine deaminase (dada ) as early-onset lacunar strokes, intermittent fevers, livedoid rash, and early onset polyarteritis nodosa (pan). since these first reports, the clinical spectrum has dramatically expanded to include antibody deficiency, liver disease, vasculopathy, pure red cell aplasia, cytopenias, and lymphoproliferative disease. methods: forty-two patients were enrolled in an irb approved study at the nih. sequencing of ada , the gene encoding adenosine deaminase (ada ), was performed in all patients. information was obtained by chart review of all clinical, serologic, and radiographic testing. results: all patients had germline biallelic loss of function mutations in ada , leading to absent or significantly decreased protein expression and function of ada . the cohort comprises females ( %) and males ( %). there were sibling pairs and families with affected individuals. twenty-seven patients had a history of at least one ischemic stroke and experienced a hemorrhagic stroke. the average age at the time of first stroke is . years (range months - years), and the average number of strokes is (range - ). no new strokes have occurred in patients on anti-tnf therapy. skin manifestations occurred in % of patients and include livedo ( %), cutaneous vasculitis resembling pan ( %), and raynauds ( %). hepatomegaly ( %) and splenomegaly ( %) were also notable. portal hypertension was observed in ( %) patients, with patient requiring a spleno-renal shunt for a massive variceal bleed. abdominal mra revealed arteritis and aneurysm in / patients evaluated; patients developed bowel necrosis. peripheral vasculopathy was seen in patients, with one requiring amputation of gangrenous digits. the most common immune abnormality seen in this cohort is hypogammaglobulinemia ( %); patients have low igg, patients have low igm, and patients have low iga. ten of these patients are on immunoglobulin replacement. specific antibody responses to vaccines were inadequate in / patients challenged. lymphocyte phenotyping revealed decreased class-switched memory b cells in / patients ( %) tested. however, there was no relationship between absolute number of class switched memory b cells and hypogammaglobulinemia or infection frequency. hematologic abnormalities include transfusion depended anemia ( %), neutropenia ( %), lymphopenia ( %), and thrombocytopenia ( %). seven patients developed pancytopenia, presented with pure red cell aplasia, and developed aplastic anemia. three patients have undergone bone marrow transplant, with two of those patients requiring a second transplant for graft failure. conclusions: the spectrum of dada has expanded from strokes, intermittent fever, and cutaneous manifestations to include portal and systemic hypertension, immune deficiency, cytopenias, vascular abnormalities, and bone marrow failure. while initiation of anti-tnf therapy improves inflammatory markers, and no new strokes have occurred while on therapy, cytopenias do not seem to improve. bone marrow transplantation should be considered in patients with findings of bone marrow failure, although transplant of our patients has been complicated by immune mediated neutropenia. disease manifestations are heterogenous, making a comprehensive evaluation critical to our understanding of this disease. given the increase in neonatal diagnosis of athymia, clinical care is provided by the referring medical centers prior to rvt- implantation and patients return to the referring centers earlier after rvt- . this creates the need for clear, concise guidelines for the care of these patients. primary goals of pre-transplantation clinical care are ( ) management of pre-existing medical needs such as feeding difficulties, airway obstruction, congenital cardiac defects and developmental disabilities; ( ) management of symptoms related to oligoclonal recipient t cell expansion (autologous gvhd/atypical complete digeorge anomaly) and ( ) prevention of infections. most deaths in the pre and early post-transplantation period are secondary to pre-existing infections. necessary surgical and medical procedures (ie cardiac surgery, hearing aids) should not be delayed. for the first to months after rvt , patients have profoundly low naïve t cell numbers and may require immunosuppression to prevent rejection of rvt- by oligoclonal recipient t cells. immunosuppression needs to be closely monitored and titrated for desired effect while minimizing side effects such as renal toxicity, electrolyte abnormalities and hypertension. t cell counts should be performed every months and are used to guide weaning of immunosuppression. most patients with successful transplants develop greater than /mm naïve t cells by months post rvt- . infection prevention, clinical stability and optimal nutrition are critical for lasting engraftment. clinical guidelines have been developed to address immunosuppression, management of autologous gvhd symptoms (gut, skin and liver), preservation of renal function, and developmental considerations. after the development of naïve t cells, patients should continue to be monitored regularly by an immunologist. patients may develop autoimmune complications such as thyroid disease and transient cytopenias. while risk of complications related to viral infections is greatly decreased after development of naïve t cells, patients with comorbidities (central venous access device dependence, tracheostomy, chronic lung disease) continue to require complex care from multidisciplinary teams. medical conditions associated with athymia but not alleviated by thymus transplantation, such as hypoparathyroidism or cardiac defects, may require lifelong medical care. lastly, patients must be evaluated for readiness for killed and live vaccines. transplant outcomes are influenced by the clinical condition at the time of rvt- implantation and optimization of immunosuppression, nutrition and clinical stability in the first months following rvt- . clinical care that maintains a well-nourished, clinically stable, infection free patient yields the best chance for successful t cell development. guidance documents supporting these goals ensure patients are best prepared to receive rvt- and develop long lasting thymic function. hemophagocytic lymphohistiocytosis (hlh) is a life-threatening disease of immune dysregulation characterized by unchecked inflammatory responses leading to end-organ dysfunction. primary hlh results from inherited mutations that impair capacity for immune regulation whereas secondary hlh arises from inappropriate response to an immune stimulus such as infection, malignancy or autoimmunity. we report a -monthold male who presented with symptoms of hlh as an initial manifestation of congenital disorder of glycosylation (cdg) due to mutations in the gene component of oligomeric golgi complex (cog ) resulting in cog -cdg (cdg-iij). a -month-old male with history of mild motor delay presented with days of fever, vomiting, and diarrhea. initial evaluation identified highly elevated ferritin and triglycerides, transaminitis, coagulopathy, and hyperammonemia. he subsequently developed generalized seizures. liver and bone marrow biopsies demonstrated erythrophagocytosis consistent with hlh. immunologic evaluation was notable for mild hypogammaglobulinemia, neutropenia, thrombocytopenia, and anemia. serum cd levels and nk functional studies were later found to be normal. the patient was initially treated with ammonia-scavenger therapy and fresh frozen plasma (ffp) for coagulopathy with subsequent intravenous immunoglobulin and dexamethasone several days later. within hours after starting ffp, the patients ferritin level declined sharply. hyperammonemia and transaminitis also resolved, and his fever curve improved. additional immunosuppression was considered, but not initiated due to the patients ongoing clinical improvement. over the next months, the patient experienced two further acute episodes of fever, liver dysfunction, coagulopathy, and sepsis physiology. the second episode was successfully treated with ffp, though no clear infectious trigger was identified. the third episode occurred days after routine vaccinations. the patient had prolonged hypotension requiring ionotropic support that resolved after receiving daily ffp, and hypoxia with pleural effusions that resolved after a single treatment with protein c concentrate. as the patient had met / clinical diagnostic criteria for hlh, but also had a history of hyperammonemia, he underwent concurrent biochemical and genetic evaluation for both primary hlh and inborn errors of metabolism. whole exome sequencing identified compound heterozygous mutations in cog , part of an oligomeric protein complex involved in golgi apparatus structure and function. cog mutations have previously been reported in two patients with autosomal recessive cog -cdg (cdg-iij), who were described to have similar clinical symptoms of hypotonia, seizures, coagulopathy, and liver dysfunction, as well as recurrent infections. subsequent immune phenotyping while the patient was healthy was notable for slightly low numbers of nk cells, but normal cd a mobilization and perforin/granzyme b expression in vitro. our patient represents a novel presentation of cdg due to cog defect with associated immune dysfunction manifesting as recurrent episodes of inflammatory crisis with features of hlh. cdg and inborn errors of metabolism should be considered during diagnostic evaluation for patients with hlh symptoms, as cdg patients may develop acute episodes of severe inflammation, in the absence of cellular regulatory defects, for which ffp and protein c concentrate may have therapeutic value. of the deaths with identifiable causes, ( %) were related to infections. the rate of death per person-year was . . the most common autoimmunity-related complication was sweets syndrome, seen in patients ( %) with anti-ifn-g autoantibodies. sixteen of those patients ( %) had recurring sweets syndrome. additionally, patients ( %) developed lymphatic obstruction, which continued to recur in patients ( %). seven patients ( %) in this study did not have anti-ifn-g autoantibodies. the median [iqr] age of autoantibody-negative patients was [ , ] years and patients ( %) were female. none of the autoantibody-negative patients developed new infections during follow-up. at the end of the follow-up period, none of the patients had active/progressive disease and patients ( %) had died. conclusions: ninety-one percent of hiv uninfected thai patients with disseminated ntm infection with or without other opportunistic infections had detectable anti-ifn-g autoantibodies. about one third of patients with autoantibodies to ifn-g had recurrent infections during follow-up. after approximately years of follow-up, % of patients with anti-ifn-g autoantibodies had inactive disease following multi-drug antibiotic therapy while % had active/progressive disease and % had died. patients with anti-ifn-g autoantibodies are at risk for recurrent infections and autoimmunity-related complications. therefore, longterm follow-up is recommended. life-long secondary antibiotic prophylaxis may be required to prevent recurrence of infection in the setting of persistent anti-ifn-g autoantibodies. the study of early t cell development in patients with severe t cell immunodeficiencies is challenging because of the rarity of these diseases, the difficulty to obtain hematopoietic stem cells (hscs), and limitations in the assays to assess in vitro differentiation of hscs to mature t cells. we recently developed a serum-free system that allows faithful analysis of sequential steps of t cell differentiation. in this system, artificial thymic organoids (atos) are generated, based on the d aggregation and culture of a delta-like canonical notch ligand (dll )-expressing stromal cell line (ms -dll ) with cd + cells isolated from bone marrow (bm) samples of normal donors (nd). in this project, we set out to evaluate the possibility of using the ato system to study t cell differentiation in patients carrying t cell defects, in order to define the exact steps of t cell development affected by different genetic defects. using the ato system, we studied in vitro t cell differentiation from cd + cells obtained from patients carrying defects that are intrinsic to hematopoietic cells (rag , rag , ak , il rg) or that affect thymus development (digeorge syndrome, dgs). the ak -deficient patient showed a markedly decreased viability in cd + cells and a very early defect in t cell development, already at the pro-t cell stage. this defect was very similar to that observed in a patient carrying a null il rg mutation who was reported to show autologous reconstitution after unconditioned haploidentical hsc transplantation. in contrast, cd + cells from a patient carrying a missense il rg mutation and with a leaky scid phenotype were capable of differentiating into mature t cells in vitro, although with -fold decreased efficiency as compared to normal donors (nd). interestingly, in the patient carrying the null il rg mutation, we noticed very few cells that could reach full maturation, with an absolute number of cd + tcrab+ cells around -times less than in nd. at variance with pro-t cells (that failed to express the gc protein), these mature t cells did express normal levels of gc, suggesting that they may have derived from residual cd + cells from the bm donor. in addition, cd + cells from the patients carrying rag and rag hypomorphic mutations were able to differentiate to cd +cd + double positive cells, but not to cd +tcrab+ cells. finally, the dgs patient showed a completely normal in vitro t cell differentiation, confirming that t cell deficiency reflected thymic abnormalities. in summary, our data show that the ato system could be extremely useful in determining whether the lack of t cells in patients with unknown gene defects reflect hematopoietic or thymic intrinsic problems, and may therefore provide critical evidence in deciding whether hsc or thymus transplantation is warranted, even without knowing the actual gene defect. introduction: ataxia-telangiectasia (at) is an autosomal recessive disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene, which aids in detection and repair of dna damage. at is characterized by progressive cerebellar ataxia, oculomotor apraxia, choreoathetosis, conjunctival telangiectasias, variable degrees of t-cell lymphopenia (tcl) and immune compromise. patients are at an increased risk for malignancy, particularly leukemia and lymphoma, and are unusually sensitive to ionizing radiation. with the advent of trecbased newborn screening (nbs) for scid, at patients are being recognized with asymptomatic tcl in early infancy. objectives: we present an older child with at and chronic granulomatous lesions and discuss how this may be avoided in individuals with at diagnosed following abnormal nbs. case report: a y/o male was born at term following an uncomplicated twin pregnancy and delivery, prior to institution of scid nbs. he demonstrated mild gross motor and speech delay as an infant and was diagnosed with at at age . he had received all routine immunizations, including live vaccinations. he developed granulomatous skin lesions at age , initially small papules on his cheeks and ears, which subsequently formed large disfiguring plaques on sun-exposed areascheeks, arms and hands (fig ) . following an extensive workup, his lesions were found to be secondary to a mutated vaccine-strain rubella (ra / ) based on bp genotyping, previously described in other immunocompromised individuals [perelygina/sullivan et al. jaci ] . his lesions have been refractory to multiple treatments including nitazoxanide. he is currently on daily oral and topical steroids, tmp/smx and ivig. retrieval of his nbs for trec determination revealed that he would have screened positive [mallot/puck et al. j clin immunol ] . when first measured at age , cd t-cells were low, /ul, with cd /ul and cd /ul. b and nk cell numbers were normal. since april , cases of at were seen at ucsf in infants with non-scid tcl on nbs. these males and female were all born at term and discharged from well-infant nurseries. at was diagnosed at - months of age. their initial trecs ranged from - /ul (normal with perkinelmer enlite kit > ), and all had low t-cells on initial flow cytometry ( - cd /ul, ref range> ) with decreased cd ( - /ul) and cd ( - /ul) t-cells; however naïve t-cells were present, ruling out typical scid and raising concern for non-scid tcl. three infants also demonstrated low b-cells (< - /ul), while nk cells were normal in all. two are currently receiving ivig, one of whom is also on tmp/smx. all have avoided not only rotavirus but also mmr and varicella live vaccinations. conclusions: at is now often diagnosed in infants with low trecs on scid nbs, prior to neurologic manifestations. benefits of early diagnosis include avoidance of live vaccines, including mmr, which led to the debilitating granulomas in our older patient. additionally, patients receive prompt immunologic monitoring and treatment, avoidance of unnecessary radiation, specialty referrals and family genetic counseling. while there is no cure for at, ongoing research may bring neuroprotective treatments in the future. introduction: subcutaneous immune globulin %, ig gly, was well tolerated in the phase / north american study in patients with primary immunodeficiency diseases (pidd). here we assess comorbidities, use of concomitant medications, infusion parameters, and tolerability in advanced age patients ( y) treated with ig gly in the north american study. methods: patients aged years with pidd received weekly ig gly infusions at volumes ml/site and rates ml/h/site for~ . years in the north american study (nct ). the medical history at baseline, medical conditions that were ongoing (defined as comorbid events), use of concomitant medications, adverse events (aes), tolerability, and infusion parameters were assessed by age: in advanced age patients ( y; n= ), adult ( < y; n= ), and pediatric/adolescent patients (< y; n= ). results: the mean number of medical history events at baseline was higher in advanced age patients ( . events/patient; events in patients) versus adult ( . events/patient; events in patients), and pediatric/adolescent patients ( . events/patient; events in patients). of these, the medical conditions that were ongoing at baseline (comorbid events) were also higher in the advanced age patients ( . events/patient; events in patients) versus adult ( . events/ patient; events in patients), and pediatric/adolescent patients ( . events/patient; events in patients). in the advanced age patients, neurological comorbidities ( events) were the most common, followed by those related to eyes, ears, nose, and throat ( events), gastrointestinal ( events), and musculoskeletal comorbidities ( events). concomitant medications were given to treat a preexisting condition in all patients in the advanced age group ( medications in patients). despite the higher mean number of comorbid conditions, infusion parameters in the advanced age patients were comparable to those in the adult age group. median maximum infusion rates and infusion volumes/site were comparable in the advanced age patients ( ml/h/site; . ml/site) and adults ( ml/h/site; ml/site); lower infusion rates and volumes/site were reported in the pediatric/adolescent patients ( . larger infusion volumes and faster infusion rates were not associated with increases in causally related local aes in the advanced age group, consistent with the trends seen in the pediatric/ adolescent and adult patients. conclusions: despite the higher mean number of comorbidities in advanced age patients with pidd, ig gly was infused at relatively high rates and volumes and was well tolerated. introduction: hyqvia (ighy; immunoglobulin infusion % with recombinant human hyaluronidase [rhuph ]) is an immunoglobulin (ig) replacement therapy approved for patients with primary immunodeficiency diseases (pidd) that allows larger infusion volumes, up to ml/site, and has improved ig bioavailability compared with conventional subcutaneous ig products. a post-authorization safety study is being conducted in the united states to acquire long-term safety data on ighy and to assess prescribed administration regimens in routine clinical practice. infusion characteristics and treatment-related adverse events from an interim analysis are reported here. methods: patients aged years with pidd receiving ighy were included in this ongoing, prospective, non-interventional, open-label, uncontrolled, multicenter study. as a part of routine clinical practice, patients are treated with ighy according to standard medical care and their treatment regimen is at the discretion of the treating physician. adverse events (aes) are collected from enrollment to study completion/discontinuation using a subject diary and assessed at every study visit (every months or standard practice). aes are assessed based on seriousness, severity, and causal relatedness to ighy. the presence of anti-rhuph antibody is evaluated on a voluntary basis. treatment preferences for various attributes of ig therapy were assessed annually using a treatment preference questionnaire. results: a total of patients were enrolled at us study sites (data cutoff date: august , ). infusions were self-administered at home ( %) or at the clinical site ( %) most commonly using -week infusion intervals ( . %). the mean maximum ig infusion rate was . ml/h and the mean ig dose was mg/kg bodyweight/ weeks. the mean number of infusion sites used for administration was . and mean infusion duration was . hours. most infusions ( . %) were administered without a rate reduction, interruption, or discontinuation due to aes. there were no serious aes (saes) related to ighy. sixteen patients experienced a causally related non-serious local ae ( . %; . events/patient-year, . events per infusion) and patients experienced a causally related non-serious systemic ae ( . %, . events/patient year, . events per infusion). seven of patients who were tested for anti-rhuph antibody had positive binding antibody test to rhuph (titer : ; maximum titer : at enrollment, : during the study); no neutralizing rhuph antibodies were detected. of the patients who responded to the treatment preference questionnaire at the end of year , the majority ( / [ . %]) preferred to receive their ig therapy at home; . % ( / ) preferred the doctors office; patients preferred treatment at the hospital, had no preference, or indicated other. almost all patients ( / [ . %]) indicated a preference to continue treatment with ighy. conclusion: this interim analysis of patients with pidd treated with ighy in routine clinical practice supports previous observations that ighy is a well-tolerated and preferred therapy with no reports of treatment-related saes or neutralizing anti-rhuph antibodies. background: cartilage hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with variable immunodeficiency. pathogenic defects in rmrp, encoding the untranslated rna subunit of ribonucleoprotein endoribonuclease complex (rmrp), result in reduced mrna and rrna cleavage. rmrp c. a>g is the most common variant, increased in finnish and amish populations. while cellular immunodeficiency is associated with increased morbidity and mortality, there is no established correlation between clinical and immunological phenotype. lymphocyte radiosensitivity has not been described. case: a full-term amish female infant had low trec copies on newborn scid screen. flow cytometry at months-old demonstrated severe t and b cell lymphopenia (cd +t-cells cells/mcl, range: , - , cells/mcl; cd +b-cells cells/mcl, range: - , cells/mcl) with normal nk quantitation (cd / + cells/mcl, range: - , cells/mcl) and cd + memory t-cell expansion ( . %) relative to the naïve subset ( . %). t-cell functional mitogen responses were normal. she was diagnosed with chh with homozygous rmrp c. a>g mutation. lymphocyte subset (t, b and nk cells) radiosensitivity was evaluated by flow cytometric analysis of phosphorylated (p) atm, smc and gamma-h ax after low-dose ( gy) irradiation. an increase in gamma-h ax level was observed in a subset of non-irradiated t cells ( . % v. . % gamma-h ax+) and nk cells ( . % v. . % gamma-h ax+) in the patient, suggestive of a constitutive defect in dna repair. the relative distribution of t, b and nk cells expressing patm, psmc and gamma-h ax at hour postirradiation (ir) was not significantly different from the experimental healthy control (ehc) or pediatric reference range (prr). however, the kinetics of dephosphorylation at hours post-ir was altered with residual gamma-h ax expression in a subset of the patients t cells (delta . %, mode ratio mean fluorescence intensity (mfi)= . ; ehc: delta . %, mode ratio mfi= . ; prr: delta . %, mode ratio mfi= . ). a similar finding was observed in a subset of patient b-cells for gamma-h ax (delta . %, mode ratio mfi= . ; ehc: delta . %, mode ratio mfi= . ; prr: delta . %, mode ratio mfi= . ). the frequency of the patient's lymphocytes with residual gamma-h ax persistence at h post-ir was prominent, with . % t-cells demonstrating persistence of gamma-h ax (compared to . % in the ehc, and . % in the prr), and . % b-cells gamma-h ax+ (compared to . % in the ehc, and . % in the prr). there has been lack of follow-up, but verbal report suggests no significant immunological or infectious concerns at year of age. discussion: lymphocyte radiosensitivity is a novel finding in chh with t and b cell lymphopenia. the ability of rmrp to associate with telomerase reverse transcriptase (tert) and function as an rna-dependent rna polymerase, yielding distinct silencing rna sequences, may underlie radiosensitivity in rmrp mutants. systematic characterization of lymphocyte radiosensitivity and immunological phenotype could provide useful information on whether this could serve as a biomarker for the magnitude or complexity of immunodeficiency. assessment of radiosensitivity has implications in conditioning regimen selection for patients requiring allogeneic hematopoietic cell transplantation. we recommend lymphocyte radiosensitivity assessment in chh infants identified by nbs scid and chh patients with significant immunodeficiency and/or malignancy. novel primary immunodeficiency with lymphoproliferative disease due to biallelic defects in nckap l background: three children from non-consanguineous families and different ethnic backgrounds developed lymphoproliferative disease by years of age. they also had recurrent infections, including pneumonia and bronchiectasis, otitis media, and skin pustules. immune phenotyping revealed low cd + t cell percentages, an accumulation of memory-like cd + t cells, impaired t cell proliferation, and low total nk cell numbers. methods: the affected individuals, unaffected parents, and other unaffected family members underwent exome sequencing. results: all affected cases had rare and bioinformatically damaging biallelic variants, with appropriate familial segregation, in nckap l, which encodes hem . hem is an essential component of the wave regulatory complex (wrc). immunoblotting confirmed destabilization of the wrc in all patients. immunofluorescence microscopy demonstrated defective f-actin and wave localization to immune synapses in nk cells. significant abnormalities were identified in patient lymphocyte and neutrophil migration and morphology, consistent with altered wrc-mediated cytoskeletal dynamics. all patients exhibited impaired inside-out integrin activation. knockdown of hem produced deficient proliferative responses and mtorc -mediated akt activation in control t cells. conclusions: the immunologic and clinical phenotype in the affected individuals recapitulates the phenotype observed in hem -deficient mice. biallelic defects in nckap l therefore result in a novel human primary immunodeficiency disease characterized by lymphoproliferation and susceptibility to infections. background: concurrent existence/significance of immunodeficiency with new onset lymphoproliferative disease remains understudied. just two studies to date have evaluated the prevalence of hypogammaglobulinemia in chronic lymphocytic leukemia (cll) and neither studied prevalence and impact of ige deficiency on outcomes in cll [ , ] . therefore, the objective of this study was to examine the prevalence of hypogammaglobulinemia, examining all isotypes, in newly diagnosed cll patients and to test the hypothesis that patients with hypogammaglobulinemia have a distinct clinical profile and outcome. methods: using the banked sera of newly diagnosed, treatmentnaïve, cll adult patients from the lymphoma molecular epidemiology resource (l-mer), ig (igg, iga, igm and ige) levels were measured. the l-mer was initiated as an observational cohort study of prospectively enrolled newly diagnosed lymphoma patients evaluated at the mayo clinic (rochester, mn) and the university of iowa (iowa city, ia) [ ] . igg/a/m levels were measured using immunoturbidimetric assay whereas the ige level was determined using electrochemiluminescence immunoassay. the associations between ig deficiencies and clinical factors were evaluated with wilcoxon rank sum and chi-squared (fishers exact, where appropriate) tests. cox regression models were used to assess the effects of clinical variables on overall survival (os). time was calculated from biopsy to death due to any cause; patients still alive were censored at last contact. all tests were two-sided and assessed for significance at the % level using sas v . (sas institute, cary, nc). results: the mean age (sd) of the selected cll cohort was . ( . ) years with a male predominance ( . %). . % of the patients were white. with a median follow-up of five years, there were deaths. hypogammaglobulinemia in newly diagnosed, treatmentnaïve cll was common in our cohort with ( . %) patients having a measurable isotype deficiency. the most common ig deficiency was igm ( . %, % ci . - . %), followed by igg ( . %, % ci . - . %), ige ( . %, % ci . - . %) and iga ( . %, % ci . - . %). multiple deficiencies in the same patient were common ( figure ). iga and ige deficiency were associated with higher rai stages (grading system for cll) at presentation (p< . and . respectively) as well as with higher white blood cell counts at presentation (p= . and . respectively). a higher proportion of iga deficient patients needed second treatment during follow-up ( % compared to %, p= . ). when comparing predictors of overall survival, higher rai stage [ - vs , hazard ratio (hr) . , % ci . - . , p= . ] and age (hr . , % ci . - . , p< . ) correlated with worse overall survival. individual immunoglobulin deficiencies did not correlate with overall survival. conclusions: a significant proportion of treatment-naïve patients with cll have underlying ig deficiencies-both in isolation and a combination of different isotypes. a deficiency of iga or ige was associated with severe disease at presentation. the underlying relationship between these two immunologic disorders deserves further study. background: patients with primary immunodeficiency (pid) have an increased risk of developing autoimmune diseases, including rheumatoid arthritis (ra). management of these patients is challenging as immunomodulators can further increase their risk for infections. additionally, patients with ra that undergo therapy with drug modifying antirheumatic drugs (dmards) may develop a secondary immunodeficiency. there are few studies reviewing the characteristics of patients with a pid who later develop ra, and no studies have been reported comparing these patients to those who develop an immunodeficiency after starting dmard therapy for ra. methods: patients were identified as having inflammatory arthritis and a concomitant immunodeficiency (id) at our institution between / / - / / using icd- and codes. manual chart review was performed to confirm and identify the timing of diagnosis of these disorders. patients were excluded if either there was no definitive diagnosis of id or ra (clinically diagnosed by a practicing allergist/immunologist and meeting acr criteria for ra with a score of or higher, respectively), or rituximab was administered prior to diagnosis of id . clinical symptoms, treatment, and laboratory data were extracted. fishers exact test was used to compare the categorical variables between the groups; ttest was used to compare the continuous variables. results: patients met the inclusion criteria. patients were diagnosed with an id and developed ra later in life (group ), and patients were diagnosed with ra and subsequently developed a clinically significant id (group ). the mean ages of diagnosis of id and ra in group patients were . years (sd ± . ) and . years (sd ± . ), respectively. in group , the mean age of diagnosis of ra was . (sd ± . ), compared to . years (sd ± . ) for the diagnosis of id. most patients in both groups were female ( % in group and % in group ). all patients in both groups had a humoral id, including common variable immunodeficiency (cvid) ( % of group patients), specific antibody deficiency (sad) ( % of group and % of group patients), and hypogammaglobulinemia ( % of group and % of group patients). all patients in group were seropositive for rheumatoid factor (rf) or anti-cyclic citrullinated peptide (anti-ccp), whereas only % of patients in group were positive for rf or anti-ccp (table ). most patients in both groups were treated with immunoglobulin replacement therapy. treatment of ra in both groups was similar, but combination dmard therapy was not used in group patients in contrast to group patients. conclusions: our study indicates that even though clinical characteristics and management are similar in patients with coexisting id and ra, rf and anti-ccp are usually negative in patients who develop ra after id, possibly due to impaired antibody production in immunodeficient patients. assistant professor of allergy and immunology, arkansas children's hospital, university of arkansas medical sciences introduction/background: complement deficiencies are relatively rare, comprising less than % of primary immunodeficiencies. they are associated with increased risk for infections with encapsulated organisms and autoimmunity. of all complement deficiencies, the rarest are defects in the alternative complement pathway. properdin deficiency is the most commonly described alternative pathway deficiency, with factor b and factor d deficiency more rarely described. fewer than patients with factor d deficiency have been reported with all reported cases being children of consanguineous parents who succumbed to meningococcal sepsis. objectives: to describe a case of factor d deficiency associated with recurrent respiratory infections with streptococcus pneumoniae pneumonia with associated lung abscess and empyema. methods: retrospective chart review was conducted. laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogen, quantitative serum immunoglobulins, vaccine titers, complement assays and functional evaluation, and genetic evaluation by next generation sequencing. results: a year old marshallese male was transferred from an outside hospital to our facility for further evaluation of worsening pneumonia and was found to have right-sided pleural effusion and pulmonary abscess in the right lower lobe. the abscess was drained and was found to be positive for streptococcus pneumoniae via polymerase chain reaction. he improved after chest tube placement and treatment with intravenous antibiotics. his medical history was significant for recurrent acute otitis media and prior hospitalization out-of-state for pneumonia with empyema secondary to streptococcus pneumoniae, which required chest tube placement and admission to the pediatric intensive care unit at months of age. immunologic work up revealed age-appropriate lymphocyte subpopulations, lymphocyte proliferative responses to mitogens, quantitative immunoglobulin levels, pneumococcal/tetanus/diphtheria titers, and ch complement assay. ah complement assay was decreased to units/ml. complement testing was repeated -with normal ch and ah of units/ml. further evaluation revealed normal levels of factors b, h, i and properdin. factor d level was . mcg/ml, and factor d function was decreased to units/ml, indicating a diagnosis of factor d deficiency. sequencing of the cfd gene revealed a previously undescribed homozygous deletion (c. _ del and p.lys del). the parents were not agreeable to personally undergoing genetic evaluation to determine if this was a de novo mutation. the patient was managed with pneumococcal and meningococcal immunizations, prophylactic amoxicillin and intravenous gamma globulin (ivig) without any further infections. unfortunately, after two ivig infusions, he was lost to follow up. conclusion: factor d deficiency is an extremely rare alternative complement pathway deficiency, described in less than patients. all infections described thus far have been secondary to neisseria meningitidis. this case represents not only a novel mutation in the cfd gene leading to factor d deficiency, but also the first description of a patient with factor d deficiency developing invasive infection secondary to streptococcus pneumoniae. background: viral infections are a significant cause of morbidity and mortality in patients with primary immunodeficiency disorders and following hematopoietic stem cell transplantation. adoptive immunotherapy using virus specific t-cells (vsts) has been shown to prevent and treat viral infections in immunocompromised hosts. human parainfluenza virus- (hpiv ) is a common cause of severe respiratory illness in immunocompromised patients and has no approved antiviral therapies and has not previously been used as a target for t cell therapeutics. introduction: we previously reported that fatigue is increased in common variable immunodeficiency (cvid). however, in previous studies, fatigue was not defined using validated tools. our aim from this study is to identify the prevalence of patient-reported fatigue, using validated questionnaires, and determine the factors predisposing to fatigue in cvid methods: data from cvid who responded to the idf patient national survey a were analyzed. fatigue was measured using the brief fatigue inventory (bfi) questionnaire, which includes seven items to identify fatigue, and measure fatigue severity. a total of patients with cvid and responses to bfi were enrolled. demographics, co-morbidities, immunoglobulin replacement therapy (iggrt) route and dose, co-morbidities, infections, depression, quality of life (qol) (using the sf- v ) and disability were compared between fatigued and non-fatigued. logistic regression was used to identify the significant variables. ebv reactivation without ptld, treated with rituximab. alive and well. j clin immunol ( ) (suppl ):s -s s granulomas are the most significant day-to-day problem for cvid patient management. currently, there are limited options for their treatment and the optimal therapy is unknown. in case reports and small series, infliximab has been reported effective while others found it useless. we here describe a yo white male referred for monthly ivig in august . at age , he developed large areas of erythematous polymorphic plaques in his cheeks, arms and legs. a skin biopsy showed tuberculoid granulomas negative for bacteria, baar and fungi, with infiltrating cd + lymphocytes. a prolonged course of steroids did not improve his skin. he also had multiple pneumonias and bronchiectasis, and oral candidiasis. he received all vaccines, including bcg with no complications. with low immunoglobulins and a poor response to pneumococcal polysaccharides and tetanus toxoid he was diagnosed as cvid and placed on ivig at yo with excellent infectious control since then. at age , his skin lesions persisted and deepened to the bone on his left leg. broad spectrum antibiotics for months were unsuccessful. at yo to yo, skin grafts were performed on his arms, legs and both cheeks. two ulcers persisted on his left leg until august that increased in size, deepened and became erythematous and extremely painful (fig. ) . in september, two new ulcers appeared on his right cheek and right gluteus, respectively. one week later a third ulcer was found on his left calf. on september th, infliximab mg/kg ( mg) was administered. on the second infliximab dose, october th, the pain was completely gone and all ulcers were shrinking, and those ones in the cheek, gluteus and calf almost completely resolved. by the third dose, on november rd the ulcers in his right leg were almost closed (fig. ) . infliximab mg treatment continues every weeks. lab test remained unchanged from till , when his wounds got worsened. (table ) granulomatous disease in cvid is a challenge. both b and t cell directed therapies are encouraged. we add a new case of an infliximab responsive patient to others already reported. over genes have been reported to cause monogenic cvid. a year old girl presented with recurrent pneumonias and a diagnosis of cvid. the parents sought a second opinion. born at weeks gestational age, she was "always smaller and sicker than her friends," and in the prior months she had episodes of pneumonia with fever to f requiring emergency department treatment. two of these were associated with rsv and metapneumovirus, respectively. laboratory evaluation confirmed low levels of igg ( mg/dl) iga ( ) and igm ( ) congenital tuberculosis (ctb) is a rare disease most often associated with maternal genitourinary (gu) tuberculosis (tb) or disseminated tb. due to infertility caused by gu tb, ctb is rarely reported even in endemic countries. infants can acquire tb hematogenously via the placenta or umbilical vein or by fetal aspiration of infected amniotic fluid. presenting symptoms include respiratory distress, fever, hepatosplenomegaly, poor feeding, lethargy, and low birth weight. we report a premature female infant conceived via in vitro fertilization (ivf), who was born to indian immigrant parents at weeks of gestation due to preterm premature rupture of membranes. maternal history was significant for pulmonary tb at years of age. she denied abdominal or gu symptoms. infants nicu course was complicated by opacifications in the right lung and leukocytosis with neutrophil predominance, identified during evaluation of frequent apnea and bradycardia episodes at month of age. clinical improvement was noted after treatment with vancomycin, amikacin and piperacillin-tazobactam; however, leukocytosis of unknown etiology persisted. at . months of age she was discharged to inpatient rehabilitation. at months of age, she was readmitted for fever and respiratory distress. during this admission, an immune evaluation was undertaken due to persistence of symptoms along with unresolved leukocytosis with a peak of , cells/l with neutrophilia to , cells/l, and chest ct evidence of progressive multifocal lung disease worse in the right upper lobe despite empiric treatment with broadspectrum antibiotics. infectious work-up was negative, including acid-fast bacilli testing from bronchoalveolar lavage. due to the pronounced and persistent leukocytosis and neutrophilia, a primary immune defect was suspected. immune evaluation included: normal immunoglobulins (ig) g, a, and e, elevated igm, vaccine-specific antibody titers protective to diphtheria and of streptococcus pneumonia strains, mildly elevated t and b cells, a normal flow cytometry for dihydrorhodamine, myeloperoxidase stain and glucose- -phosphate dehydrogenase level, as well as a peripheral smear with no giant azurophilic granules. her primary immunodeficiency genetic panel was unrevealing. she underwent lung biopsy via video-assisted thoracoscopic surgery, which showed noncaseating granulomas and eventual growth of multi-drug-resistant mycobacterium tuberculosis (mtb). upon treatment with an appropriately adjusted anti-tuberculosis regimen, she showed rapid clinical and laboratory improvement. endometrial samples obtained from mother showed gu tb, confirming the diagnosis of ctb. the slow-growing nature of mtb that resulted in delayed diagnosis, along with the presence of non-caseating granulomas and persistent neutrophilia, prompted an immune work up that was completely normal. this case demonstrates the importance of considering ctb in the differential diagnosis of an infant presenting with severe lung infection, persistent neutrophilia, suboptimal response to broad-spectrum antibiotics and relevant epidemiologic risk factors. furthermore, in the setting of appropriate parental exposures and infertility prompting the use of ivf, maintaining a high level of suspicion of ctb can aid in earlier diagnosis of affected neonates. -year-old caucasian male who initially presented with recurrent otitis media, persistent hsm, lad, and hypogammaglobinemia (igg < mg/dl) at years of age. he was diagnosed with common variable immunodeficiency (cvid) and chronic arthritis when he was and years of age, respectively. subsequently, he developed hepatitis and recurrent pneumonia with mycobacterium avium complex (mac). his arthritis partially responded to anti-tumor necrosis factor (tnf) agents and tofacitinib, but did not respond to anti-interleukin- treatment. a combination of anti-tnf inhibitor, tofacitinib, and low dose prednisone was required to control his arthritis. hypogammaglobulinemia (igg < mg/dl), recurrent otitis media, pneumonia, crohn's disease, celiac disease, lad and failure to thrive at years of age with more recent development of hsm. he required only immunoglobulin replacement therapy. case# is a -year-old caucasian male, the half-brother of case# , who initially presented with recurrent pleural effusion and bilateral pulmonary infiltrates, hsm, lad, abdominal distension and ascites at years of age. a transbronchial lung biopsy revealed chronic eosinophilic pneumonitis. liver biopsy showed increased eosinophils in the sinusoids with diffuse enlargement of hepatocytes, but without hepatitis. colon biopsy revealed minimal colonic eo-sinophilia. his pulmonary infiltrates and pleural effusion responded to prednisone, and he has not required additional treatment for past . years. conclusions: the clinical manifestations of the same genetic variant may be variable and unpredictable even in the same family. stat gof syndrome should be considered in children with multisystem autoimmune diseases, lad, hsm and low switched memory b cells regardless of presence of hypogammaglobulinemia or history of recurrent infections. background: patients with primary immune deficiencies characterized by severe t lymphopenia and/or poor t cell function and patients posthematopoietic cell transplantation are at high risk of severe viral infections. antiviral medications are expensive, not always effective and associated with significant toxicity and/or long-term side effects. as such, there has been increasing interest in the use of donor-derived or thirdparty virus-specific t cells (vsts), and several studies have demonstrated efficacy of vsts generated using various manufacture strategies. however, in depth immunologic and metabolic characterization of vsts has not been reported, limiting correlative investigations into efficacy. methods: ebv-vsts were generated from apheresis t cells collected from healthy donors using three methods: ( ) stimulation and expansion with hla-matched ebv-lymphoblastoid cell lines (lcls) purchased from astarte biologics or sigma-aldrich over a period of weeks, ( ) stimulation with ebv peptivator from miltenyi followed by expansion over - days with different cytokines, and ( ) stimulation with ebv peptivator followed by isolation of activated cells using the ifn-gamma capture system from miltenyi. immunophenotyping by flow cytometry was performed using the miltneyi macsquant analyzer. the nanostring ncounter system was used to measure gene expression for metabolic pathway analysis, and the agilent seahorse xf cell mito stress test system was used to measure mitochondrial respiration. results: ebv-vsts generated using lcls or peptivator plus il- both resulted in a high percentage of cd t cells skewed to the effector memory and terminal effector memory phenotype with high expression of the exhaustion markers pd- , tim- , and lag- . conversely, ebv-vsts generated using peptivator plus il- and il- and the ifn-gamma capture system resulted in a mixed cd and cd t cell population with a high number of central memory t cells and lower percentage of cells positive for pd- , tim- , and lag- . stimulation with peptivator followed by expansion with il- resulted in an intermediate immunophenotype. nanostring results demonstrated upregulation of the glycolytic pathway in ebv-vsts stimulated with peptivator followed by expansion with il- or il- compared to ebv-vsts generated using the other manufacture approaches. the seahorse mito stress test demonstrated that the peptivator plus il- ebv-vsts had a significantly lower spare respiratory capacity than other ebv-vsts and a low extracellular acidification rate despite upregulation of the glycolytic pathway. the peptivator plus il- and il- ebv-vsts had the highest basal oxygen consumption rate, atp-linked respiration, and extracellular acidification rate. conclusions: manufacture of ebv-vsts using the various approaches currently employed clinically results in t cell pools with different immunophenotypes and different metabolic profiles. ebv-vsts stimulated with peptivator followed by expansion in il- and il- and ebv-vsts isolated using the ifn-gamma capture system have immunophenotypes and metabolic phenotypes suggestive of potential greater in vivo persistence, whereas ebv-vsts expanded in il- and il- have characteristics correlated with increased effector function. however, these vsts are more likely to be short-lived and to have impaired metabolic fitness. these phenotypes will enable better correlation with clinical results and suggest combinatorial approaches depending on clinical indication. introduction: majority of patients with primary immunodeficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors for patients. a new . % ig preparation octanorm (octapharma, lachen; tradename cutaquig® in north america) has been developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagam®. objectives: primary outcome was assessment of the efficacy of octanorm in preventing serious bacterial infections. main secondary endpoints included (among others) evaluation of tolerability and safety of octanorm, the number and rate of other infections, number of days missed at work, and use of antibiotics. methods: a prospective, open-label, non-controlled, single-arm phase study involving adult patients with pid was conducted at russian centers. patients treated with at least infusions of ivig prior to enrollment and with igg trough levels . g/l underwent an -week wash-in/wash-out period followed by a week efficacy period. during the study, patients received weekly administrations of octanorm at the same monthly dose as during previous ivig treatment (monthly ivig dose divided by for weekly dose). in total, each patient received scig infusions. results: twenty-four patients completed the study. one patient terminated early (after infusion , during wash-in/wash-out phase; personal reasons). mean age was . years (range - years). fifteen patients ( %) were female and patients ( %) male. no serious bacterial infections were recorded. during the efficacy period a total of non-serious infections was observed in patients. seventeen infections in patients were of mild and infections in patients of moderate intensity. the infection rate per person-year was . . in total patients received infusions of study drug. the average dose of cutaquig® was . g/kg/week. during the entire study, systemic adverse events were reported (including infections). three of these systemic adverse events were rated as related to study drug, all were non-serious. there was no serious or significant adverse event nor was there an adverse event leading to withdrawal. infusion site reactions were reported for % of infusions. serum igg trough levels were nearly constant during the efficacy period. median igg trough levels were . g/l at screening, . g/l at the end of wash-in/wash-out period and . g/l at the termination visit. one patient had a trough level g/l at visits during the efficacy period and the dosing was subsequently adjusted for this patient. during the primary treatment period patients ( . %) used antibiotics in treatment episodes (total of treatment days; range - days) and patients had absences from work or school due to infections (total of days of absence). conclusion: this study demonstrated that the new subcutaneous human normal immunoglobulin . % is well tolerated, safe and effective in adult patients with pid. background: children with chronic granulomatous disease (cgd) are at high risk for fungal infections (especially with aspergillus species) and these infections usually have contiguous site involvement. most patients have pulmonary presentation. infective endocarditis and fungal osteomyelitis of skull are distinctly unusual. we report one such case. case: a -year-old boy, born out of a non-consanguineous marriage, presented with soft tissue swellings of skull for months. his past history was significant with an episode of pneumonia at year and recurrent soft tissue swellings all over the body since ½ years of age. on examination he was wasted, had signs of micronutrient deficiency, rickets, pallor, cervical lymphadenopathy and two abscesses, x cm on right temporo-parietal region and x cm over left frontal region. he was also found to have hyperdynamic precordium with an ejection systolic murmur. investigations revealed hemoglobin g/l; platelet count . x /l; total leukocyte count x /l(n /l /m /e ); elevated c-reactive protein( mg/l) and a raised erythrocyte sedimentation rate( mm sthr). chest x ray revealed cardiomegaly (cardiothoracic ratio %) and d echocardiography showed vegetation of x mm over the anterior mitral leaflet suggestive of infective endocarditis. blood and urine cultures were sterile. culture from pus over the temporo-parietal abscess showed growth of aspergillus fumigatus. human immunodeficiency virus serology was non-reactive. immunoglobulin profile revealed elevated igg . g/l ( . - . g/l) and iga . g/l( . - . g/l); igm was . g/l( . - . g/l). in view of strong suspicion of cgd, nitroblue tetrazolium dye reduction test (nbt) was carried out-it revealed no reduction and dihydrorhodamine (dhr) assay showed a low stimulation index ( . ). flow cytometry for gp phox and gp phox was normal and dhr of mother did not reveal x linked carrier state. contrast enhanced computerized tomography (cect) of head showed osteomyelitis of the calvarial bones. contrast enhanced magnetic resonance imaging (cemri) brain showed heterogeneously enchancing soft tissue lesion in the scalp at right fronto-parietal region and left frontal region with underlying bony destruction suggestive of osteomyelitis. he was given intravenous antimicrobials (ceftriaxone, gentamycin, cloxacillin, voriconazole). after weeks of therapy, he showed resolution of findings on mri brain and a repeat d echocardiography showed significant decrease in size of mitral leaflet vegetation. conclusion: this case highlights a rare presentation of cgd with infective endocarditis and skull osteomyelitis due to aspergillus fumigatus. to the best of our knowledge, this has not been reported previously. background: genetic defect in il r affect cellular immunity, underlie mendelian susceptibility to mycobacterial disease (msmd) and inflammatory bowel disease (ibd) through different pathways. we present for the first time a patient with il- r deficiency from a consanguine family with two different phenotypes. initially diagnosed as crohn's disease prior to the msmd diagnosis. method and material:patient was referred to the clinical immunology and allergy clinic at the at alzahra university hospital for immunological and genetic evaluation . blood samples from patient, his family and healthy donor controls were collected upon informed consent. in this study, we investigated effect of il r mutation in il- /ifnaxis by evaluation of patients whole blood cell response to il- and ifn-, il- r expression in pbmcs and t cell blasts. also wholeexome sequencing has been performed. result and discussion: a years old male from consanguine family , with history of right sub-axillary bcg lymphadenitis, recurrent mouth ulcers , chronic diarrhea in childhood and appendectomy at age of was investigated. based on his clinical presentation abdominal pain, significant weight loss, chronic and bloody diarrhea , endoscopic and pathological findings treatment for crohn's disease (cd) was started at the age of seven . unfortunately, protracted patient's symptoms ends up to resection of his colon and colostomy two years later. he was presented with multi focal osteomyelitis at the age of . although no bacteria was detected in pcr and tissue culture of the bone biopsy and the patient was not responded to antibacterials , he had a dramatic response to empirical anti mycobacterial treatment and his severe bone pain and lesions were healed. even though the bone manifestations were completely controlled, he continuously was under treatment for his gastrointestinal symptoms. genetic analysis was confirmed segregation of homozygous mutation in splice site of exon in il- r . expression of gene was completely abolished in pbmcs of patient and the surface expression of il rb was not detectable in t cell derived pbmcs of the patient compared to healthy control. furthermore, did not response to il stimulation since we could not detect increase of inf-after stimulation with il and bcg. our patient received bcg vaccination at birth and had bcg lymphadenitis as an infant, cd and mycobacterial multifocal osteomyelitis as a child. furthermore there are some evidences which indicate the role of atypical mycobacterial infections as a trigger for cd. conclusion: we reported for the first time contemporary msmd and ibd in years old patient, who had impaired il- signaling and abolished il r expression in pbmcs and t cell blast. however, mycobacterial osteomyelitis is a typical phenotype of msmd patients with deficiency in ifn-r or stat, there were no mycobacterial osteomyelitis reported in il- r deficient patients. background: advanced genetic studies help explain the occurrence of many undiagnosed, rare conditions. recently, nbas variants were identified as a causative basis of recurrent liver failure in infants (infantile liver failure syndrome , ilfs ). the nbas (neuroblastoma amplified sequence) gene encodes a protein involved in golgi to er retrograde transport. nbas functions seem to be broad and loss of function variants in nbas have been associated with multisystem manifestations. case report: a y m old chilean male presented to the er with a three day history of vomiting, diarrhea and one day of fever ( . °f). on examination he was pale, lethargic, and tachycardic. a chemistry profile revealed markedly elevated liver enzymes, increased bilirubin, and coagulopathy, consistent with the acute hepatic failure (alt , ast > , total bilirubin . ( . db), ggt , and inr of . ). he was hospitalized, given vitamin k, and kept on intravenous fluids, ursodiol, and antipyretics. his liver function improved significantly within days of admission (alt was down to , ast , total bilirubin . ). work-up of possible etiologies including autoimmunity and infectious hepatitis was negative. liver sonogram was normal, but liver biopsy was consistent with acute hepatitis with some necrosis. urine organic acid and plasma amino acid screens were not consistent with any inherited metabolic disorders. his parents recalled two previous episodes of liver failure at ages and years. both were preceded with a mild febrile illness and non-specific symptoms including fever, coughing, vomiting, diarrhea, lethargy, and decreased po intake. these subsequently were followed by jaundice and marked elevation of liver enzymes. flu a and adenovirus were identified as causes of febrile illnesses of the two previous episodes. for this admission, adenovirus was found in the respiratory secretions and a mild ebv viremia was also detected. genetic evaluation in chile was reportedly normal. after a literature review we obtained sequencing of nbas which revealed two variants: c. g>t,p.glu * and nbas c. t>g, p.iie ser. both variants have been previously reported in patients with an infantile onset, recurrent liver failure syndrome. his other clinical features include developmental and speech delays, failure to thrive, and facial dysmorphism. he also has a history of recurrent ear infections and has had sets of tympanostomy tubes. further testing was limited due to the lack of insurance coverage. conclusion: nbas deficiency is a newly described syndrome of recurrent acute liver failure that occurs early in life. once individuals have survived to adulthood they do not seem to develop liver failure with illness. typically, liver crisis is triggered by a common childhood febrile illness. the mechanism of disease is thought to be thermal instability of hepatocytes which improves over time in most cases. however, although spontaneous recovery can occur following the crises, each episode can be fatal or result in permanent liver damage required liver transplantation. increased awareness of this disease will lead to the early establishment of the diagnosis. appropriate and timely management of fever at the onset of illness can significantly improve outcome in this potentially fatal disease. associate prof., infectious diseases and tropical medicine research center, isfahan university of medical sciences, isfahan, iran background: pre-eclampsia, a pregnancy-specific complication, has been shown to be associated with cytomegalovirus (cmv) infection. cmv specific t-cell response plays the major role in cmv infection or disease .we explored whether a change in cmv-specific cell-mediated immunity (cmi) is related to the development of preeclampsia. method: cmv-specific cmi was assessed using cmv-quantiferon (qf-cmv) assay in serum from women with pre-eclampsia as well as normal pregnancy controls retrospectively. participants were matched for gestational age individually. proportion of reactive results, mean value of interferon-level produced in mitogen and antigen tubes were compared between the cases and controls via chi-square, wilcoxon rank-sum tests, respectively. odds ratio (or) and confidence interval (ci) were calculated as well. result: no significant differences observed between demographic characteristics of the case and control groups. the qf-cmv assay turned reactive (qf-cmv [+]) in of of patients ( %) vs. of controls ( . %) (p = . ). women with pre-eclampsia had lower mean ifn-levels in antigen tube ( . ± . ) compared with normal pregnancy controls ( . ± . ) (p = . ). there was no statistically significant differences in this value of mitogen tube between cases ( . ± . ) and controls ( . ± . ) (p = . ). women with suppressed cmv-cmi were . times more likely to manifest pre-eclampsia (or= . , % ci: . - . ). this result even strengthened after adjustment for age, gestational age and gravidity (or = . , % ci: . - . ). conclusion: our finding support an association between suppressed cmv specific cmi and pre-eclampsia. introduction: the triad of susceptibility to infections, auto-inflammation, and cancer in a patients personal and family history are always suggestive of an underlying primary immunodeficiency; however, in some cases the diagnosis might be delayed for years. furthermore, the results of immunological and inflammatory evaluation can also be affected by ongoing immunomodulatory therapy initiated by different specialists upon clinical diagnosis. objective: to describe a unique presentation of auto-inflammatory disease with combined immunodeficiency in an adult patient. case presentation: we report here the case of a year old male, who had a long history of infections including recurrent sino-pulmonary bacterial infections starting during childhood, osteomyelitis at years of age, recurrent tonsillitis requiring tonsillectomy at years of age, recurrent cellulitis, an episode of prostatitis with septicaemia, as well as recurrent varicella zoster and warts. the patient was also diagnosed with sclerosing mesentheritis, and reynauds phenomenon, recurrent oral ulcers, arthritis, uveitis, autoimmune thyroiditis, lung fibrosis and suffered repeated episodes of abdominal pain. furthermore, there is a family history of early childhood death, multiple soft tissue cancers, crohns disease, and autoimmune thyroiditis. upon physical examination, the patient had multiple telangiectasia, baseline erythroderma, and flushing. immunological evaluation showed lymphopenia with significant reduction in both circulating b and t cells, however, assessment of humoral immunity revealed low igg and decreased igm with normal iga levels. at the time of the evaluation he had been on low dose daily prednisone ( . mg), colchicine, and methotrexate as immuno-modifying therapy. genetic evaluation revealed a heterozygous mutation in nod as well as compound heterozygous mutations in the mefv gene. discussion: mutations in nod have been described in association with blau syndrome a multisystem auto-inflammatory syndrome which may explain many of the features experienced by our patient. to our surprise next generation sequencing revealed a second aberration in the mefv gene which causes familiar mediterranean fever, another multisystem auto-inflammatory disease, which might lead to the phenotype observed in the patient. conclusion: this is the first report of genetic lesions in two different genes leading to a severe course of auto inflammation. monogenic autoinflammatory syndromes (mais) are a diverse group of disorders characterized by primary over-activation of the innate immune system. induction of the inflammasome complex by innate immune sensors and increased production of il- b are implicated in the pathogenesis of mais. macrophage activation syndrome (mas) is a life-threatening illness defined by acute hyper-inflammation and unopposed cytokine release. it is considered an acquired condition secondary to infection, rheumatoid disease or malignancy. the early therapeutic use of il- b inhibition has profoundly improved the prognosis mas. it has recently been shown that increased free il- levels in the blood are causatively linked to the development of mas. significant overlap in clinical presentation and laboratory markers between patients with mais and mas led us to explore the role of free il- and therapeutic use of il- b inhibition in a patient with cdc mutation. here, we report the case of an months-old female who presented with hydrops fetalis in utero, and later developed failure-to-thrive, splenomegaly, anemia, thrombocytopenia, arthralgias, rashes, frequent febrile episodes and mild facial dysmorphism along with massive increase in crp, esr and ferritin. whole exome sequencing (wes) identified a heterogenous likely pathogenic de novo variant in cell division control protein homolog (cdc ) c. g>a (p.c y). cdc encodes a small rho family gtpase that regulates multiple signaling pathways controlling cell polarity, migration, endocytosis and cell cycle progression. single allele mutations in the cdc gene were recently reported to cause takenouchi-kosaki syndrome manifesting with growth retardation, developmental delay, facial dysmorphism, and thrombocytopenia however systemic autoinflammation has not been described. cdc closely interacts with the wiskott-aldrich syndrome protein but little is known about the mechanism underlying immune abnormalities associated with cdc mutations. our patient had an inflammamosopathy-like syndrome. because of significant clinical overlap to mas, we measured il- , il- , free il- and il- binding protein, all of which were significantly increased. this increase in free il- heightened her risk of developing mas. her il b level was normal, but an increase in il- b is hardly ever detectable in the serum despite playing a critical role in this type of inflammation. indeed, chronic il- b excess in the tissues promotes systemic inflammation and is associated with chronically elevated crp and esr. with this rationale we started the il- receptor antagonist anakinra. within hours from starting anakinra, the parents observed an increase in appetite, resolution of arthralgias and improved mobility. over the course of the following weeks, fever, anemia, thrombocytopenia and rash disappeared, the spleen massively decreased in size and the patient started to meet developmental milestones. crp, esr eventually normalized while ferritin and free il- are still trending down. conclusions: significant increase in free il- and extremely encouraging clinical response to therapy with anakinra in a patient with novel cdc mutation suggests a link between mas and defects in cdc . elucidating the mechanism of inflammasome activation and the drivers of il- increase in mas and mais more broadly may shed light on novel therapeutic targets like the use of human recombinant il- binding protein. j clin immunol ( ) (suppl ):s -s s maintenance; smarcal is enriched in cells that maintain telomeres via the alternative lengthening of telomeres pathway and smarcal decifient cells demonstrate telomere instability with replication fork collapse and increased telomere-associated dna damage. [ , ] telomere analysis of siod patients, including one patient who received a hematopoietic stem cell transplant (hsct) years prior, as well as heterozygous family members revealed significantly shorter telomeres in siod patients compared to heterozygous family members and compared to agematched, healthy controls. methods: peripheral blood mononuclear cells were isolated using a ficoll-hypaque density gradient, cryopreserved, then sent to repeat diagnostics in north vancouver, bc. telomere length measurements were performed at a single-cell level using flow-fluorescence in situ hybridization as previously described. [ ] telomere length was measured in total lymphocytes, naive and memory enriched t cells, b cells, and nk cells and compared to reference samples from age-matched, healthy individuals. results: compared to age-matched healthy controls, three siod individuals had mean telomere lengths (mtls) less than the st percentile for age across all lymphocyte subsets (total lymphocytes, b cells, nk cells, naïve and memory t cells). in comparison, three unaffected family members had normal mtls ( th percentile< x < th percentile) across all subsets, and two unaffected family members had low mtls ( st< x < th percentile) in some subsets. the siod individual who received a matched-sibling hsct years prior, had normal mtl in nk cells ( th < x < th percentile) but low mtls ( st< x < th percentile) for all other subsets. conclusions: these data show that siod patients have significantly impaired telomere lengths across multiple lymphocyte lineages and support a limiting role for smarcal deficiency in telomere maintenance. in comparison, unaffected family members, heterozygous for smarcal mutations, have mean telomere lengths that are normal or slightly low for age. this suggests that abnormally short telomeres are seen in individuals with homozygous but not heterozygous smarcal mutations. for the individual who received a hsct, we do not have pre and post-hsct telomere data, but these results support obtaining pre and post-hsct telomere length analysis in future cases. abnormally short telomeres have been linked to widespread perturbation of gene expression. [ ] we hypothesize that smarcal deficiency, by the effect of stalled forks and shortened telomeres, leads to perturbation in the transcriptome of affected tissues. shortened telomeres may explain the reduced hematopoietic bone marrow production in siod, as bone marrow failure is a cardinal feature of dyskeratosis congenita, a disorder of impaired telomere maintenance. future studies to investigate the role of telomere maintenance in siod include measurement of telomerase activity in polyclonally activated t cells and transcriptome analysis using rna-seq background: yellow fever is a potentially fatal disease for which only supportive treatment is available. vaccination is the primary strategy for prevention of this disease and the vaccine is extremely effective, but there are a few specific populations where it is contraindicated. regarding iga deficiency (the most frequent primary immunodeficiency), current recommendations in the literature are controversial. there are no specific studies in this disease, so case series addressing the safety or possible adverse events after vaccination are essential for decisionmaking during epidemic scenarios, as experienced in brazil in the last years. in this context, this study aimed to describe adverse events after the use of the yellow fever vaccine in iga deficient patients. method: a retrospective cross-sectional study was conducted including iga deficient patients followed at a specialized pediatric outpatient clinic between and . all patients had at least one year of follow-up. immunoglobulin levels, antibody response to vaccines and lymphocyte subset count were evaluated to exclude other immunodeficiencies or the presence of abnormalities that could contraindicate vaccination. demographic data, the presence of infections and comorbidities, use of immunosuppressive medication and adverse events after vaccine administration of the vaccine were described. results: thirty-eight patients with iga deficiency were included in the study and received the vaccine. vaccinated patients had a mean age at the time of the study of . years (sd ± . y). six out of the presented comorbidities: thyroiditis (n= ), type diabetes mellitus (n= ), celiac disease (n= ) and juvenile rheumatoid arthritis (n= ). all patients were atopic and only one had recurrent infections in the last year despite the use of antibiotic prophylaxis. all patients had normal igg and igm levels for their age, positive vaccine responses for measles, rubella and mumps, and age-appropriate lymphocyte subset count. after months of observation, no immediate or late adverse events were reported. among the non-vaccinated patients, only one had a formal contraindication (systemic erythematosus lupus using immunosuppressive therapy). five out of the non-vaccinated patients reported being afraid of receiving the vaccine, still intended to receive it and for other patients data regarding vaccination was unavailable. conclusion: despite the small number of patients, the absence of adverse events in this case series suggests that immunization with yellow fever vaccine may be safe in iga deficient patients, excluded other contraindications. more studies are essential to confirm the safety and help the decision-making process regarding the vaccine administration for iga deficient patients, especially in this yellow fever outbreak scenario. introduction/backround: immunodeficiency, centromeric instability, and facial anomalies syndrome (icf) is a rare group of autosomal recessive disorders involving the triad of hypogammaglobulinemia, centromeric instability, and facial anomalies. the majority of patients have hypo-or agammaglobulinemia, but t cell defects have also been reported. we present the case of a child with icf- who presented with nk deficiency and ultimately developed an ebv-driven malignancy and was successfully treated with bone marrow transplant. methods: whole exome sequencing and nk cell function via -cr cytotoxicity assay and phenotyping via flow cytometry were performed at baylor college of medicine and texas childrens hospital. centromeric banding studies were performed at university of pittsburgh medical center. results: the female patient presented at months of age with cmv pneumonitis and persistent cmv viremia requiring treatment followed by prophylaxis with valgancyclovir. she initially had hypogammaglobulinemia and low t, b, and nk cells; she had normal trecs, lymphocyte mitogen proliferation responses and zap , mhci and mhcii expression. the hypogammaglobulinemia and t-and b-cell lymphopenia resolved within months after initial presentation as she clinically improved from her cmv infection. she was found to have nk cell deficiency on three separate commercially tested samples. whole exome sequencing revealed a homozygous variant in zbtb indicative of icf- syndrome that was confirmed with sanger sequencing (c. _ del, p.q vfs). repeat nk cell studies confirmed impaired function, and phenotyping showed an increase in cd -bright and a decrease in cd -positive cells, suggesting either impaired transition from immature to mature nk cells or impaired survival of mature cells. her karyotype and centromeric banding studies were normal, as were centromeric instability studies. she later developed a memory b-cell defect and presented at months of age with persistent fever, respiratory distress, loss of vaccine titers, hypogammaglobulinemia and low b and t cells. she was found to have ebv viremia and an eber-positive diffuse large b-cell lymphoma in her right lung. due to tenuous clinical status, she received rituximab for treatment of ebv prior to definitive lymphoma diagnosis. she was treated with chemotherapy per protocol anhl , group b (pre-phase with cop, courses and with copadm, and courses and with cym) and her course was complicated by seizures attributed to methotrexate toxicity. she ultimately underwent reduced intensity conditioning with hydroxyurea, alemtuzumab, fludarabine, mephalan, and thiotepa followed by a cd- selected, hla-matched, unrelated donor peripheral blood stem cell transplant. her early post-transplant course was complicated by adeno-, ebv, and cmv viremia, all successfully treated with antivirals and a donor lymphocyte infusion. she is now greater than months posttransplant, off immunosuppression with % donor engraftment, no evidence of organ toxicity or gvhd, and with excellent immune reconstitution. conclusions: this is the first reported case of impaired nk cell function and phenotype and ebv-driven malignancy in a patient with icf- . this case expands the phenotype of icf- and suggests that early bone marrow transplant should be considered in these children. it also demonstrates a novel requirement for zbtb in human nk cell maturation and function. rationale: common variable immunodeficiency (cvid) is a disorder that affects the production of immunoglobulins and is associated with development of autoimmunity. multiple mutations have been described that are associated with cvid, but plcg mutations have only been described in patients with phospholipase c gamma (plc ) associated antibody deficiency and immune dysregulation (plaid) and autoinflammatory plc associated antibody deficiency and immune dysregulation (aplaid). we present a case of a y/o male cvid patient with recurrent upper respiratory tract infections, steroid-dependent autoimmune thrombocytopenia, low b cell count, hepatosplenomegaly, and restrictive lung disease. he was found with a variant of unknown significance at the plcg gene. in contrast to plaid our patient does not exhibit cold urticaria. method: case presentation of a cvid patient followed in our clinics. patients chart and previous laboratories were reviewed. sequence analysis and deletion/duplication cvid panel testing was performed using invitae© discussion: genetic testing has revolutionized the diagnosis of immune deficiencies, but variants of unknown significance are being increasingly reported. in this case, a variant of uncertain significance was identified which replaces threonine for alanine at codon of the plcg protein. this codon is located at the sh domain, which is part of a region that provides auto-inhibitory enzymatic functions. plaid mutations have been identified in sh domain, but it has been known that both sh and sh domains facilitate plcg association with other proteins. studies with deletion of plcg gene have shown functional abnormalities in b cells, natural killer cells and mast cells. to our knowledge, there has not been any previous report of a cvid patient with a variant mutation at the sh domain of the plcg gene without being diagnosed as plaid or aplaid. our patient has immunodeficiency, recurrent upper respiratory tract infections, steroid-dependent recurrent autoimmune thrombocytopenia, rheumatoid arthritis, hepatosplenomegaly, early-osteoporosis and restrictive lung disease. he does not have cold urticaria as seen in plaid, but exhibits autoimmunity not observed in aplaid. conclusion: conclusion: plcg is an important protein in the pathway of b cell development. a novel mutation in the sh domain of the plcg gene may be associated with the cvid phenotype of low b cells and autoimmunity. this could lead to a gain-of-function mutation as seen in plaid but without early-onset cold urticaria. functional studies are required to confirm the significance of this mutation. primary (or familial) hemophagocytic lymphohistiocytosis (hlh) is a rare, life-threatening hyper-inflammatory disease affecting mainly young children. it is caused by mutations in genes involved in the granule-dependent cytotoxic pathway, and is characterized by extreme inflammation and massive tissue infiltration by activated t cells and macrophages. to this day, hematopoietic stem cell transplantation is the only available curative treatment with a transplantrelated mortality of %. thus, the development of new, more efficient anti-inflammatory treatments would be a significant advancement in the treatment of hlh. here, we hypothesize that combination therapies targeting both jak-dependent and independent cytokines will be more effective than either one alone to reduce the lifethreatening symptoms induced by this pathology. using a perforin-deficient mouse model of hlh, we first compared the effect of targeting individual cytokines with blocking antibodies on the progression of the disease. we show that blocking ifng and il- , but not il- , significantly reduces the severity of hlh. targeting the jak-stat signalling pathway with ruxolitinib, a specific inhibitor of jak and jak , downstream of ifng and il- , but not il- , is similarly beneficial. more importantly, combination therapies using ruxolitinib and blocking antibodies to either ifng or il- show synergistic effects, further mitigating the progression of the disease. these results suggest that jak-dependent and independent cytokines drive the pathogenicity of hlh in perforin-deficient mice. it further supports that ruxolitinib, although effective in reducing the symptoms of hlh, should be used in combination with anti-ifng and/or anti-il- antibodies to prevent hlh progression. this is particular relevant since the former were recently approved for the treatment of hlh while the latter (il- binding proteins) are in clinical trials for il- -dependent macrophage activation syndromes. despite the increased risk of opportunistic lung infection in patients with severe t cell dysfunction (e.g. cd l deficiency) and/or severe cd t cell lymphopenia, we are not aware of any reports of disseminated pneumocystis jiroveci infection in non-human immunodeficiency virus (hiv) patients with primary immunodeficiency (pid). we report the first case, to our knowledge, of disseminated pjp in a patient with cvid like/ctla haploinsufficiency. he had been diagnosed with common variable immunodeficiency (cvid) in , approximately eight years prior to being referred to us, and was on intravenous immunoglobulin (ivig). he was also diagnosed with multilineage evans syndrome in . his medical history was also significant for potential granulomatous lymphocytic interstitial lung disease (glild) (lung biopsy in the remote past with interstitial disease), significant splenomegaly ( . cm), severe portal hypertension, nodular liver disease (likely nodular regenerative hyperplasia) complicated by anasarca, history of chronic diarrhea (potential enteropathy), lymphadenopathy s/p biopsy with nodular lymphoid hyperplasia, and a history of multiple pneumonias. in , he had developed disseminated pjp with lung, liver, and bone involvement. the t vertebra pjp invasion was confirmed with a bone biopsy; gomori methenamine silver staining and pcr were performed and concluded pjp. he was treated with trimethoprim sulfamethoxazole (tmp-smx) and steroids, then was continued on tmp-smx prophylaxis. due to his liver damage and his chronic neutropenia, tmp-smx was replaced by atovaquone as a secondary prophylaxis for pjp. his laboratory studies were significant for an absolute neutrophil count of . k/ul, absolute lymphocyte count of . k/ul, hemoglobin of . g/dl, platelets of k/ul, total bilirubin of . t-cell receptor beta chain repertoire analysis showed an oligoclonal distribution. severe combined immunodeficiency panel through ambry genetic testing was negative as was genetic testing for cd l deficiency. given his complex clinical history, whole exome sequencing was obtained and detected an autosomal dominant heterozygous missense mutation (c. g>a) implicated in ctla- haploinsufficiency and previously reported by schwab et al. our patient is currently undergoing therapy with abatacept (ctla- fusion protein), which has been reported to improve glild, splenomegaly and enteropathy in patients with ctla- haploinsufficiency. he is improving on this regimen. he has met with the stem cell transplant team, but at this point of time, due to his abnormal lung function, his liver damage and his significant splenomegaly, he is not a good candidate. defects in the nf-b signaling pathway are implicated in the pathogenesis of several primary immune deficiencies in humans. the clinical features of these conditions vary significantly, reflecting the complexity of the pathway, and its broad role in innate and adaptive immune responses, and the development and differentiation of lymphoid organs. here we report the first case of a human pid caused by a homozygous mutation in nfkbid in a year-old male. he was the second child of consanguineous parents, and was diagnosed with possible cvid at the age of , after recurrent episodes of pneumococcal pneumonia. however the clinical features have evolved over time; he developed severe ebv infection at age , causing hepatitis and pancreatitis. at the age of , he presented with an anca-negative systemic vasculitis, manifesting as pulmonary haemorrhage, and acute necrotizing pauci-immune glomerulonephritis. pulsed methylprednisolone and cyclophosphamide induced an initial remission, however, relapse a year later led to end-stage renal failure. he is now dialysis-dependent, and due to the underlying pid, and chronic cmv viraemia, is not a candidate for renal transplantation. genomic dna was subjected to whole-exome sequencing. variants were filtered using a model of autosomal-recessive inheritance and functional analysis of primary cells was performed. we identified a novel, homozygous, single-base deletion resulting in a frame-shift, and premature stop in nfkbid. nfkbid encodes ibns, a non-classical inhibitor of nf-b signaling. at diagnosis the patient had reduced levels of igg , iga and igm, elevated ige, with absent humoral immune responses to pneumococcal polysaccharide vaccine, and an intact response to tetanus. lymphocyte numbers were initially within normal reference ranges, albeit with an increased proportion of cd +:cd + t cells. however, over time there has been a significant reduction in b cells and cd + t cells. cd + t cells demonstrated a skewing towards a central memory phenotype (cd ro+/ccr +), and cd t cell proliferative responses to pha were comparable to a healthy control. functional analysis of primary cells from the proband revealed a complete absence of bns protein expression, dysregulated nf-b signaling, and elevated pro-inflammatory cytokine production. the patient is currently receiving a trial of targeted therapy to modulate the aberrant immune responses. this novel pid highlights the importance of regulation of nf-b signalling, in orchestrating an appropriate immune response, maintenance of self-tolerance, and protection against viral pathogens. primary immunodeficiency diseases (pid) are a heterogeneous group of conditions with variable clinical features that are frequently associated with significant diagnostic delay. accurate diagnosis has significant therapeutic benefit and may lead to personalized therapies. we established the immunology flagship of melbourne genomics health alliance in australia to determine the clinical utility of genomic sequencing for diagnosis and management of individuals with suspected and confirmed cases of pid. adults and children with suspected or confirmed pid (n= ), autoinflammatory disease (n= ) and hereditary angioedema (hae, n= ) were recruited to the melbourne genomics immunology flagship. whole-exome sequencing (wes) was performed, with targeted gene analysis. variant curation and reporting was performed according to the american council of medical genetics guidelines. overall, wes was diagnostic in % ( / ), confirming a preexisting diagnosis in % ( / ), and offering a new or more specific diagnosis in % ( / ). variants of uncertain significance were identified in a further patients ( %) in genes known to be associated with their clinical diagnosis, that warrant further functional validation. in the hae group, diagnosis was confirmed in only patients ( %), suggesting that wes may not be the appropriate technique for genetic diagnosis in this condition. a higher diagnostic rate was observed for autoinflammatory disorders ( %; / ) compared to pid ( %; / ). of those who received a diagnosis, immediate changes to patient management and treatment occurred for / patients ( %), including hsct for and specific targeted therapy for ( %) individuals. we have demonstrated the utility of wes for accurate diagnosis of complex immune diseases, with the potential to change diagnoses, guide therapeutic intervention and provide opportunities for genetic counseling. further longitudinal analysis will determine clinical outcomes and health economic implications of genomic sequencing for diagnosis and management of immunological conditions in australia. at birth he had neonatal asphyxia and cerebral palsy. at years old he had presented involuntary movements, left paresis, bilateral horizontal nystagmus. at years of age, he had a right nasal obstruction. it was resected by otorhinolist and informed by biopsy: inflammatory polyp and chronic sinusitis. he has had pneumonias, sinusitis and diarrhea. at the age of years, the ataxia telangiectasia was confirmed by sequencing with pcr ( exons, bp) of the atm gene: transition g> a, nucleotide position , codon , affecting splicing. alpha fetoprotein - . u/ml. brain mri, say cerebellar atrophy. he had igg mg / dl - mg / dl, iga . mg / dl, < mg / dl, igm mg / dl - mg / dl, ige . -< iu / ml. subclasses of igg: igg : . g / dl, igg : . gr/dl, low. igg anti hepatitis b , . no seroconversion. hiv negative tcd + lymphocytes: , %, = cells / mm , ltcd +: , % = , cel / mm , ltcd +: , % = , cells / mm , cd / cd : . . for all of the above, common variable immunodeficiency was diagnosed. he receives human immunoglobulin. at , i arrived at this hospital due to fever, respiratory distress and lymphadenopathy in the neck. ct showed ganglionic conglomerate on right side neck. lymph node biopsy: strong tumors with cd and bcl , weak and moderate diffuse pax- ; negativity with cd , cd and cd , and a cell proliferation index with ki of %, diagnosis: diffuse large b cell lymphoma. treated with rituximab and chemotherapy. lymphoma completely remitted. conclusion: the association ataxia telangiectasia and lymphoma is frequent. by contrast, cvid and ataxia telangiectasia are extraordinarily rare. introduction: chronic granulomatous disease (cgd) is a primary immunodeficiency wherein affected patients are susceptible recurrent infections caused by specific bacteria and fungi as a result of defective nadph activity. additionally, inflammatory complications involving the bowel and lungs can cause significant morbidity. currently the only proven permanent cure to cgd remains hematopoietic stem cell transplant. case: a -year-old patient was diagnosed in infancy with x-linked cgd. at age yrs he received a nonmyeloablative peripheral blood stem cell transplant from his / non-carrier sister as previously reported (nejm : , ) . conditioning was cyclophosphamide ( mg/kg) on d- and d- ; daily fludarabine ( mg/m ) on d- through d- ; antithymocyte globulin at mg/kg on d- through d- . posttransplant immunosuppression consisted of cyclosporine on d- through d+ . he received . x cd + peripheral blood stem cells which were t-cell depleted with x add back of cd + cells on day . after days of neutropenia (anc < ) there were signs of engraftment. per protocol, he received donor peripheral-blood lymphocytes containing . x cd + cells/kg on d+ after transplantation. since donor t cells constituted less than percent of his circulating cd + t cells and he had no graft versus-host disease, he received . ¬ cd + cells/kg on d+ . after the discontinuation of cyclosporine, he received a total of three donor-lymphocyte infusions ( . ¬ cd + cells/kg) at -day intervals achieving % t cell and myeloid engraftment at months post-transplant with no acute nor chronic gvhd. at last follow-up years post-transplant ( ) he had % and % lymphoid and myeloid peripheral chimerisms, respectively. the patient and family declined further periodic followup. then, in october he presented with malaise, cough and fevers. he eventually was found to have a large consolidation and a bal grew burkholderia cepacia. his dhr showed % activity and peripheral blood myeloid and lymphoid chimerisms were % and %, respectively. discussion: this late graft failure following peripheral blood transplant occurred following a conditioning regimen which is not the current standard for transplant in cgd. in the case series in which this patients transplant is reported (nejm ), another patients myeloid chimerism fell to % by years post-transplant, remaining stable at that level of chimerism without any serious infections over regular periodic follow up to the present time. current regimens typically include busulfan to enhance engraftment and prevent graft failure. this case reinforces the need for prolonged monitoring of primary immune deficiency patients after transplantation. introduction: with the introduction of severe combined immunodeficiency (scid) newborn screen (nbs) in the state of kansas in , a case of complete digeorge syndrome (dgs) was discovered in an infant born to a diabetic mother with atypical features. this is the first dgs case diagnosed after adding the scid nbs, which emphasizes the need to establish scid nbs in all states. case presentation: the female infant was born via spontaneous vaginal delivery at / weeks to a year old g now p mother. maternal history was significant for chronic hypertension, obesity, insulin dependent type diabetes, anxiety, depression, and scoliosis. the infant was noted to have a left sided abdominal wall defect and hernia, imaging identifying left renal agenesis, and was initially suspicious for vater syndrome. fortunately, the infant's scid nbs revealed low t cell receptor excision circles (trecs). her initial white blood cell count was . with an absolute lymphocyte count of . k/ul. ebv pcr, cmv pcr, and hiv studies were negative. chest imaging discovered absent thymus, abnormal vertebrae with only ribs on the right and ribs on the left, and abnormally formed thoracic vertebrae (t ). echocardiogram detected an atrial septal defect measuring . cm, possible pfo versus secundum asd. endocrinology was consulted for management of labile calcium and phosphorus levels. fish was negative for q . deletion. microarray r evealed a variant of unknown signif icance arr[grch ] p . ( _ )x . sequence analysis of combined and severe immune deficiency genes showed a variant of uncertain significance c. c>a (p.leu met). management and outcome: additional evaluation included: cd ul ( - ul), cd ul ( - ul), cd ul ( - ul), cd ra cells/ul ( - cells/ul), normal cd , and cd / , normal immunoglobulin g level, and normal dihydrorhodamine assay. skeletal survey, ct abdomen and chest, and hla typing were performed in preparation for thymic transplant. discussion: patients with complete dgs, a form of scid found in less than percent of patients with qds, have absent thymus and a t cell count < standard deviations below normal for age (typically < naïve cd + t cells/mm ). in a large series of patients with complete dgs, only percent had an identifiable q . deletion [ ] . infants of a diabetic mother have various genetic and syndromic associations including diabetic embryopathy. [ ] despite the importance of immunological aspects in pregnancy, few studies have reported on the cellular immune modifications of diabetic embryopathy. diabetes during pregnancy may affect the development of the thymus and thus maturation of the immune system in the offspring. [ ] the recent addition of a trec assay to newborn screening can identify such a subset of infants with atypical presentations. scid nbs uses an assay for trecs, a biomarker of t cell development. [ ] [ ] [ ] this initial presentation now places the immunologist in the role of "first responder" with regard to diagnosis and management of these patients, who may present with atypical features. newer genetic and molecular techniques now allow for earlier identification of immune defects in such disorders with life-long clinical concerns. [ ] references: introduction/background: goods syndrome is a rare cause of combined b-and t-cell immunodeficiency occurring in association with a thymoma. affected patents are susceptible to bacterial, fungal, viral, and opportunistic infections. an association with autoimmunity has also been reported. current knowledge of goods syndrome is primarily limited to case reports and small series. objectives: to examine the spectrum of clinical and laboratory features of a major cohort of goods syndrome patients in the us. methods: we conducted a retrospective analysis of patients with goods syndrome in the usidnet registry and the mount sinai hospital (msh) cohort. r e s u l t s : we i d e n t i f i e d p a t i e n t s w i t h t h y m o m a a n d hypogammaglobulinemia (usidnet, n= ; msh, n= ; median age: years; female: %), representing data from patient years. the median age at diagnosis of thymoma and hypogammaglobulinemia were years (range - ), and . years (range - ), respectively. two patients were deceased (at age and years, cause unspecified). all patients had low igg (median mg/dl, range - ). iga and igm were reduced in % and % of patients, respectively. low cd + b cells (median . /mm^ , range - ) were reported in all available records. the absence of cd + b cells was observed up to years postthymectomy. a wide range of additional laboratory abnormalities were identified: low cd + t cells (n= ), low cd + t cells (n= ), low cd / cd ratio (n= ), low nk cells (n= ), and absent peripheral eosinophils (n= ). the most common sites of infections were lower respiratory ( %), upper respiratory ( %), and gastrointestinal ( %). in addition, sepsis ( %), meningoencephalitis ( %), osteomyelitis ( %), and urinary tract infection ( %) were also observed. identifiable infectious agents included: bacteria ( %), virus ( %), fungus ( %), parasites ( %), and protozoa ( %), with opportunistic infections recorded in % of patients. opportunistic infections were significantly associated with absolute cd lymphopenia (p= . , fishers exact test). enterovirus was identified as a previously unreported cause of meningoencephalitis in this population. autoimmune manifestations were reported in % of patients, with a higher prevalence of inflammatory colitis ( %) than previously reported. hashimoto thyroiditis, fibromyositis, and bronchiolitis obliterans organizing pneumonia (n= each) were identified as previously unreported autoimmune/inflammatory conditions in this population. a case of alopecia areata was also observed. additionally, bronchiectasis was recorded in % of patients. all patients were initiated on immunoglobulin replacement, with antibiotics prophylaxis in %, and immunosuppressive medications employed in % of patients post diagnosis of immunodeficiency. conclusion: goods syndrome is a combined immunodeficiency, with a wide range of autoimmunity in a subset of patients. we expanded upon the spectrum of associated infectious and inflammatory complications through a major us cohort. persistent immune dysregulation was observed up to decades post-thymectomy. introduction: primary immunodeficiencies (pids) constitute a large group of rare disorders that affect the immune systems function. some pid patients develop autoimmunity in addition to having increased susceptibility to infections due to their impaired immunity [ ] . ( ) case presentation/ management: a year old caucasian female with history of bipolar disorder, factor v leiden deficiency, anti thrombin deficiency, pulmonary embolism, endometriosis, and seasonal allergies was evaluated for chronic granulomatous disease (cgd) in . the main symptoms were inflammatory breast lesions necessitating surgeries on the right breast, and back, facial, genital, ocular, mouth, and scalp sores. biopsy with cultures of the wounds was positive for corynebacterium, coagulase-negative staphylococcus, enterococcus, bacteroides species, and provatella. neutrophil oxidative burst was ordered by the infectious disease specialist and showed normal and abnormal neutrophil populations, a finding consistent with cgd carrier. patient was started on interferon gamma- b after failing multiple courses of antibiotics. her symptoms were well controlled on interferon gamma- b mcg/ . ml sq every other day, trimethoprim mg tab ( tabs in am and tab in pm), cefixime mg once daily, and topical mupirocin as needed except for her recurrent genital ulcers. cgd can be rarely associated with oral ulcers however there is a limited literature describing associated genital ulcers. according to the international study group diagnostic criteria published in ( ), the patient was diagnosed by a rheumatologist as having behcets disease (bd). there are no pathognomonic laboratory tests in bd; as a result, the diagnosis is made clinically. patient failed a trial of colchicine and was later started on cyclosporine, which resulted in decrease of her mouth and genital ulcers. discussion: bd is a rare disease mostly seen along the silk road. the prevalence has been reported as . (usa) to (in a single village, northern turkey) for inhabitants. ( ) cgd is a primary immunodeficiency caused by defects in any of the five subunits of the nadph oxidase complex responsible for the respiratory burst in phagocytic leukocytes. patients with cgd are at increased risk of life-threatening infections with catalase-positive bacteria and fungi, and inflammatory complications such as cgd colitis. ( ) reports of cgd female carriers with discoid lupus erythematosus, photosensitivity rashes, and other autoimmune phenomena have been published [ , ] ( ) . to the best of our knowledge, this is the first case to report bd in an affected cgd carrier. the treatment of inflammatory disease in patients with cgd poses a difficult balance between therapeutic immunosuppression and the increased risk of severe infection. ( ) . high dose intravenous immunoglobulin, and targeted therapies such as ctla -ig for t cell mediated pathologies, rituximab for b-cell mediated pathologies, and anti-tnf for ibd, may be preferable over the broad immunosuppressive activity of glucocorticoids. in addition, emerging evidence suggests that hematopoietic stem cell transplantation has indication for cases that have been difficult to control using immunosuppression. ( ) given all that, our case emphasizes the need to maintain suspicion for autoimmune disorders / immune dysregulation in patients with pid. introduction: cd -ligand deficiency is an x-linked combined immunodeficiency, characterized by susceptibility to infection, often with associated neutropenia, malignancy, and autoimmunity. central nervous system (cns) manifestations are less commonly reported than respiratory or gastrointestinal complications, but are most often attributed to infection. herein we describe a challenging case of gradual onset episodic memory loss, confusion, and unilateral hemiplegia in a young male with cd ligand deficiency. case presentation: the patient is a -year-old male with cd -ligand deficiency on immunoglobulin replacement therapy presenting with recurrent, episodic altered mental status (ams) and gradual neurocognitive decline. initial neurologic symptoms began at age years, and included fever, nausea, and eyelid fluttering. initial comprehensive infectious workup at this time, including blood and urine cultures, lyme antibody, serum pcr for hsv, cmv, ebv, respiratory viral pcr including atypical viruses, csf studies including culture, lyme eia, pcrs for enterov i r u s , v z v, e b v, c m v, h s v / w e r e u n r e v e a l i n g . electroencephalogram (eeg) and mri displayed generalized slowing and global atrophy, respectively. definitive diagnosis was not made. the patient continued to decline with worsening developmental delay and memory loss. one year later, at age years, he had a recurrent episode of ams with repeat negative infectious workup including blood and urine cultures, respiratory virus pcr including atypical viruses, csf culture including acid fast bacillus and fungi, cryptococcal antigen, viral encephalitis panel by pcr, and serum pcr for ebv and hhv- . eeg at this time showed left hemispheric epileptogenic potential, consistent with seizure activity. his presentation, at age years, was notable for right-sided hemiplegia with facial numbness, dysarthria, nausea, and fever. he was found to have anello virus on pcr of csf, abnormal left temporal region on eeg, and global atrophy with stable, diffuse generalized volume loss on mri. he was diagnosed with occult anello virus-induced encephalitis with hemiplegic migraine and discharged on valproate. discussion: here we present the first reported case of anello virus detected by pcr in a cd -ligand deficient male with neurocognitive manifestations, attributed primarily to hemiplegic migraine. given the anello virus prevalence and relatively avirulent character, it is presumed to be unlikely culprit for encephalitis; however, the significance of this finding is as yet unknown. this case highlights diagnostic challenges in immunodeficiency: infection may go undetected by standard diagnostic techniques; however, the significance of infections identified with advanced techniques may not yet be understood. background: henoch-shönlein purpura (hsp) is an iga-mediated small vessel vasculitis that presents with a tetrad of abdominal pain, arthritis, glomerulonephritis, and purpura. hsp is typically a selflimiting disease of childhood following a viral illness. there is no universal treatment for patients with chronic or recurrent hsp. we report a chronic refractory case of hsp that was successfully treated with a tumor necrosis factor inhibitor (tnfi), etanercept. etanercept functions as recombinant protein that consists of a tnf-alpha receptor ligand-binding region that links to the fc portion of human igg. it is currently approved for use in diseases: juvenile rheumatoid arthritis, rheumatoid arthritis, ankylosing spondylitis, plaque psoriasis, psoriatic arthritis. tnfi are categorized into two broad categories, recombinant receptors (etanercept) and neutralizing antibodies (ex. infliximab and adalimumab). there have been prior case reports of hsp associated with tnfi agents during the treatment of other autoimmune conditions in the adult population. to our knowledge, there have been prior etanercept related hsp reports, one report associated with adalimumab, and one with infliximab. however, there has been no prior report of etanercept use successfully treating chronic refractory hsp. case presentation: a -year-old native american male with year history of chronic hsp, hla-b positive, and enthesitis related arthritis who was initially treated with steroids, sulfasalazine and methotrexate for symptoms of joint pain and purpura. his iga level was mg/dl prior to therapy. despite treatment for one month of steroids, eight months of sulfasalazine exclusively and eight months of methotrexate and sulfasalazine, he continued to have persistent purpura on bilateral extremities without improvement. he was subsequently initiated on etanercept mg weekly and methotrexate was discontinued. approximately one month later, his rash significantly improved. his rash and joint pain recurs when he misses a dose of etanercept. punch biopsies were taken months after initiation of etanercept. the biopsies of a lesion from his left arm showed early leukocytoclastic vasculitis and from his left leg showed weak granular deposition of iga, igm and c within vessel walls. there is controversy whether this is a true iga vasculitis. however, we believe that his clinical presentation and the deposition of iga and c within blood vessel walls seen on biopsy correlates with chronic henoch-shönlein purpura. conclusion: there is no standard treatment of chronic hsp, but there are reports of benefit with nsaid and corticosteroids. per our literature review, there are no prior reports of etanercept use in the treatment of chronic hsp. tnf inhibitor, etanercept should be considered as a treatment for chronic refractory hsp in the pediatric population as it has showed rapid resolution of purpura in this case report. further studies of etanercept in the treatment of chronic hsp should be conducted given the controversial literature of anti-tnf ab induced hsp during the treatment of other autoimmune diseases. although clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers of hfa mutation, we present cases that appear to suggest an increased risk non allergic rhino-sinusitis. case report: we present a year old gentleman with perennial colored rhinorrhea, with facial pressure and tenderness, constant post nasal drip, dry cough and bilateral congestion that had been going on for the past several years. he also had a frequent urge to clear his throat and had frequent episodes of sore throat despite having no history of gerd or lpr. he reported to have multiple sinus infections every year that would progress to pneumonia and eventually require long courses of oral antibiotics. all started in his s intensified in the recent past. he had other siblings; one died in his s due to liver complications of hh and had a carrier sister and brother with a hx of sino nasal problems exactly similar to the patients. his exam was remarkable for bilateral narrowed nasal passages and moderate edema of the mucosa. his rhinolaryngoscopy showed significant edema and purulent drainage, most notably from bilateral middle meati. his skin test was negative. his cbc showed a wbc count of . /ml with % eosinophils and his immunoglobulin panel showed an iga of mg/dl, igg of mg/dl and ige of mg/dl. patient was placed on alkalol sinus rinses and azelastine nasal spray, which he reported to work pretty well. he left for costa rica and is expected to return back with his siblings to a&i clinic in the coming months. discussion: hh is one of the most common inherited disorders in people of northern european descent with an incidence of : and carrier rate of : .. most affected hh patients are homozygous for the mutation designated c y at the hfe gene located at the th chromosome. unlike hereditary hemochromatosis, clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers. hh patients are at risk for a number of infections with bacteria whose virulence is increased in the presence of excess tissue iron. hh is also a risk factor for acute fulminant frs . here the mechanism is postulated to be due to quantitative or qualitative neutrophil defects as this condition is mostly seen in patients with dm, aplastic anemia, and can happen in patients undergoing antineoplastic chemotherapy. no known increased susceptibility for infections through either mechanism is postulated for patients with the heterozygous carrier state. here we present hh carrier patients who present with recurrent rhinosinusitis with no allergen sensitizations and normal ige levels. since most fungal immunity is at the tissue level and is cytokine driven, it can be speculated that increased tissue levels of iron might interfere with mechanisms of innate immunity. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md background: dedicator of cytokinesis (dock ) mutations are associated with a combined immunodeficiency disorder marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. almost all cases can be diagnosed by documentation of the loss of dock protein expression. methods: we describe a -year-old male with a diagnosis of pre-b cell acute lymphoblastic leukemia (all) followed by epstein-barr virus (ebv) associated diffuse large b cell lymphoma (dlbcl). compound heterozygous mutations in dock were documented following the completion of whole exome sequencing (wes). the pathogenicity of the variants was assessed. flow cytometric quantification of intracellular dock protein was completed. dock protein function was assessed by evaluating the morphology of patient lymphocytes when migrating in a d collagen matrix. results: a concern for a primary immunodeficiency was raised due to a history of recurrent otitis media which began at months of age. by years of age, numerous warts were noted on his fingers; however, they were transient for a duration of only years. no atopic features were appreciated. at years of age, a diagnosis of pre-b cell all was made. during all therapy, infectious complications were severe including an intestinal perforation, osteomyelitis, and sepsis. at years of age, still in an ongoing remission from his all, an incidental finding of a lung nodule led to a diagnosis of ebv-associated dlbcl. during therapy, however, infectious complications were again severe including a soft tissue infection and sepsis. wes was performed and compound heterozygous mutations in dock (c. _ del and c. - g>c) were documented. flow cytometric quantification of intracellular dock protein was normal when compared to a normal control. nevertheless, additional functional assessment of dock protein was completed. when migrating through a d collagen matrix, % of the patient lymphocytes studied demonstrated abnormal elongation (stretch ratio > defined by length/width) compared with % of lymphocytes from a normal control. he is being evaluated for hematopoietic stem cell transplant. conclusion: autosomal recessive mutations in dock are a rare cause of a combined immunodeficiency marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. here, pre-b cell all followed by the development of a subsequent malignant neoplasm (ebv-associated dlbcl) led to the discovery of dock deficiency. hence, as our case underscores, for rare instances of high clinical suspicion despite normal dock protein expression, additional functional testing is crucial to make a definitive diagnosis and plan treatment. understanding the spectrum of dock mutants and their phenotypes will improve our understanding of dock deficiency. background: autosomal dominant hyperimmunoglobulin e syndrome (ad-hies) is a rare primary immunodeficiency caused by heterozygous loss-of-function mutations in the signal transducer and activator of transcription (stat ) gene. ad-hies classically characterized by recurrent cold staphylococcal abscesses, pneumonia, eczema, and an elevation of ige level. other additional clinical manifestations of hies have been recognized including skeletal dysplasia (scoliosis, pathologic fractures, delayed dental deciduation), pneumatoceles, coronary-artery aneurysms, brain lesions, and chiari malformations. objective: to describe a unique case of abdominal abscesses in a patient with ad-hies. method: a -year-old female with known ad-hies (c. c>t (p.arg trp)) and a complicated history of early pneumococcal pneumonia and meningococcemia resulting in bilateral amputation below the knees along with loss of several digits, presented for evaluation of skin infection. she had a history of recurrent staphylococcal skin abscesses and presented with inability to use her prostheses due to pain from inflammation around her amputation sites. she underwent imaging and was found to have bilateral extremity abscesses with an associated osteomyelitis of her l tibia (which was found to be mrsa after incision and drainage). while receiving intravenous antibiotics for her osteomyelitis, she developed intractable abdominal pain. imaging showed a thick-walled, multi-septated, paranephric abscess as well as several smaller abscesses scattered throughout her abdomen. she underwent multiple drain placements and drainage of retroperitoneal fluid collections via interventional radiology (ir). purulent fluid from the abdominal abscess drainage grew mrsa. the patient continued to have re-accumulation of abscesses despite multiple drainages. repeat imaging noted increased paranephric abscesses which were not communicating with drains. given lack of response to several ir-placed abdominal drains and to weeks of intravenous antibiotics, she had an open surgical washout with minimal improvement. hospital course was further complicated by development of a left lower lung lobe consolidation and sub-segmental pulmonary embolism necessitating treatment with heparin. finally, after several weeks of escalating antimicrobial therapy and with additional drain placements, the retroperitoneal abscesses started to recede. repeat abdominal imaging several months later while asymptomatic revealed slow but continuing resolution of the abscesses. conclusion: the present case raises awareness of an unusual location for infection in a patient with ad-hies. although the majority of complications of ad-hies are sinopulmonary and skin infections, recalcitrant intra-abdominal abscesses should be considered in the differential of infections in hies. introduction/background: the recent epidemiologic studies have revealed that primary immunodeficiencies (pids) are more common than previously thought. however, there are very few data on epidemiology of pids in korea. objectives: we attempted to estimate the pid epidemiology and disease burden in korea and provide the background information for pid registry for future. methods: to review the previously reported scientific studies, pubmed, koreanmed, google scholar were searched. any studies on pids reported in scientific journal (korean or international) from january to november were searched. both korean and english reports were searched. diagnosis for pid was categorized from group i to group xi according to iuis phenotypic classification. study period was divided into two periods: period from to and period from to , because there was a multicenter study to estimate pid epidemiology from to . in addition, the number of pid patients and the cost for care were estimated among patients who requested reimbursement to health insurance review and assessment service (hira) korea for one year in . results: a total of pid patients were identified in reports. one hundred and ninety-nine patients ( reports) and patients ( reports) were found in period and period , respectively. the pids were reported in patients for immunodeficiencies affecting cellular and humoral immunity, patients for combined immunodeficiency with associated or syndromic features, patients for predominantly antibody deficiencies, patients for diseases of immune dysregulation, patients for congenital defects of phagocyte, patient for defects in intrinsic and innate immunity, patients for auto-inflammatory disorders, patients for complement deficiencies, and none for phenocopies of pid. from hira reimbursement data, the number of pid patients were for combined immunodeficiency, for predominantly antibody deficiency, for common variable immunodeficiency, for functional defect of neutrophils, for immunodeficiency associated with other major defects, for other immunodeficiencies. a total of , pid patients were treated for , days and $ , , was reimbursed in . conclusions: we performed a systematic review on published studies for pid in medical journals and national open data system of hira to estimate the pid disease burden for the first time in korea. to obtain more information on true pid epidemiology and disease burden in korea, a national multicenter study for pid registry is required in the future. micro-thrombocytopenia is one of the most serious challenges for wiskott-aldrich syndrome (was) and x-linked thrombocytopenia (xlt) patients. thrombocytopenia leads to severe, potentially life-threatening, bleeding episodes, which require frequent transfusions and account for % of deaths in patients experiencing was mutations. the gold standard treatment for was patients is hematopoietic stem cell transplantation (hsct) from an hla-identical donor but more recently a number of gene therapy (gt) trials in europe and usa showed promising results. in particular, it has been shown that was patients receiving lentiviral mediated gt, consisting of autologous cd + cells transduced with lentiviral vector encoding the human was gene under the control of the endogenous promoter, in combination with a reduced intensity conditioning regimen, have a significant increase in platelet (plt) counts. even though plt counts do not reach normal levels, treated patients decreased the severity and frequency of bleedings. here, in a cohort of xlt and was patients, fifteen treated with gt, the plt phenotype and function were analyzed by electron microscopy, flow cytometry and proteomic profile. the aim of the project is to assess the presence of plt defects in was untreated patients and the impact of gt treatment on the correction of plt behavior. we demonstrate that plts of untreated was patients have reduced size and abnormal ultrastructure along with hyperactivated phenotype at steady state, showing increased expression of cd p, activated iib integrin and cd l; conversely, activation response to agonist and aggregation capacity are both decreased. analyzing plt samples isolated from treated patients, we found that gt restores plt size and ultrastructure very early after treatment and reduces the hyperactivated phenotype proportionally to was protein (wasp) expression and follow-up length. plts isolated from gt treated patients showed a normal activation response to agonists and restored aggregation capacity in out of analysed patients. by proteomics, various protein pathways were found downregulated in untreated plt samples, mainly involving cytoskeletal-rearrangement proteins, integrins, signal transduction molecules, vesicles-transport proteins; additionally, decreased metabolic capacity were observed. these results are in line with the functional defects observed in plts in terms of activation and aggregation. conversely, the expression of protein-pathways found downregulated in untreated patients is comparable to healthy controls in gt-treated plt samples, reflecting the amelioration of plt phenotype and function. overall, our study highlights the coexistence of multiple defects in the activation and aggregation responses occurring in was patient plts in absence of wasp. gt was able to normalize the plt proteomic profile followed by consequent restoration of plt ultrastructure and phenotype, suggesting gt is responsible for the observed reduction of bleeding episodes in treated patients. introduction: pik cd is an autosomal dominant genetic disorder of the immune system that results in persistent activation of pi k. signaling through pi k is essential for immune cell regulation of metabolism, migration, proliferation and differentiation, leading patient to present with lymphadenopathy, immunodeficiency and senescent t cells. the mutated protein causes t cells to over activate and mature too quickly leading to their death, this over activation also blocks the maturation of b cells. case presentation: a -year-old female with a childhood history of failure to thrive, asthma, chronic rhinitis and common variable immunodeficiency on intravenous immunoglobulin replacement, was seen in immunology clinic to establish care. she reported frequent episodes of pneumonia and bronchitis in her childhood. her family history was significant for family members with leukopenia, but no diagnosed immunodeficiency. patient had son who did not report symptoms concerning for immunodeficiency. physical exam was within normal limits with no lymphadenopathy. laboratory examinations exhibited normal iga ( mg/dl), igg ( mg/dl), and igm ( mg/dl). while flow cytometry showed normal absolute cd ( - cells/ul), cd ( cells/ul), nk cells ( cells/ul), cd ( cells/ul), cd ra ( cells/ul), cd ro ( cells/ul), cd ( cells/ul), and hla-dr ( cells/ul), nonswitched memory cells ( cell/ul) and class-switched memory cells: ( cells/ul). ( - cells/ul). vaccine response was not pursued as patient had been on ivig. genetic testing was pursued, and revealed a mutation in pik cd gene, specifically a mutation in the c. g>a; p.val met variant (rs ). this mutation though seen in databases, is not currently reported in medical literature as associated with this condition. based on these, ct chest was ordered to screen for bronchiectasis, adenopathy and lymphoma. ct showed no cardiopulmonary disease or adenopathy, but did show an incidental adrenal mass which is now being worked up. while the pattern of inheritance of this mutation is autosomal dominant, her son is asymptomatic and testing of her son has not been pursued, though it was advised for her cousins given history of leukopenia. patient has continued on igg replacement therapy. conclusion: recent publication by the clinical immunology society suggests consideration for next generation sequencing when it can affect future family planning or it has treatment and prognostic implications. this case highlights all aspects of the importance of genetic testing as part of the diagnosis of cvid, since it can affect progeny, it offers the possibility of treatment with immune modulating agents and has implications on screening, since patients are at increased risk for malignancies. background: abnormal v(d) j recombination activity in patients with mutations in the recombination-activating genes and (rag / ) results in markedly reduced usage of distal vand j genes at the t cell receptor alpha (tra) locus. mucosa-associated invariant t (mait) cells express a semi-invariant t cell receptor containing the distal trav - gene. mait cells can be identified by flow cytometry using a mab directed against valpha . , which recognizes the product of the trav - gene. by performing high throughput sequencing (hts) of tra rearrangements and flow cytometry, we have confirmed lack of t cells using distal valpha genes in patients with known rag mutations. we now report that flow cytometry with mab against valpha . successfully identified rag deficiency in two patients with an atypical presentation. methods: tra rearrangements were analyzed by hts using gdna from sorted t cell subsets from rag-mutated patients and healthy donors. distal valpha usage was measured in whole blood by flow cytometric analysis with an anti-valpha . antibody. rag mutations were detected by sanger sequencing. patients were enrolled in niaid protocol -i- . results: hts of tra rearrangements revealed lack of distal trav and traj gene usage in patients with rag / mutations. the presence of circulating mait cells in controls and patients with known rag / mutations and various clinical phenotypes was analyzed by flow cytometry using mab against valpha . . we found a virtual lack of valpha . expression in rag mutated patients (< . %) compared to controls ( - %) . we used the valpha . assay to test two patients with unknown immunodeficiency manifesting as skin granulomas and autoimmune cytopenia, and found nearly absent expression ( . % and . %). targeted sequencing of rag / revealed that both patients were compound heterozygous for rag mutations: p.r h/p.c y and p.r w/p.r q, respectively. conclusions: patients with mutations in rag / demonstrate a skewing of their tcralpha repertoire. the reduction in recombinase activity in these patients does not allow for rearrangements of the most distal valpha segments. rapid identification of patients lacking valpha . + t cells by flow cytometry may prompt sanger sequencing and identification of rag / mutations in a matter of days. this assay represents a simple but powerful tool to reduce the cost and time associated with other analysis methods. acknowledgements: supported by dir/niaid/nih. director, centro de inmunología clínica dra.bezrodnik y equipo introduction: the fate of effector t cells is strongly dependent on the expression of bcl- or blimp- , which are inhibited reciprocally through a complex signaling pathway. several studies have shown that bcl- is a key transcription factor for differentiation towards the follicular helper t cells (tfh) lineage able to collaborate with b lymphocytes (bl). on the contrary, the transcription factor blimp- is highly expressed in t lymphocytes th , th and treg, thus regulating the differentiation towards tfh. materials and methods: whole fresh blood and peripheral mononuclear cells from a patient with homozygous mutation in stat b were analysed by flow cytometry. analysis of ctfh (cd +cd ra-cxcr +), ctfh (cxcr +), ctfh (ccr +), ctfh (cxcr -ccr -), naïve bl (lb igm+igd+cd -), memory (mbl) (lb igm+ igd-cd +), switched (mbl-sw) (igd-igm-) and plasmablast (pbc) (cd +cd ++) cells was performed. immunoglobulins were measured in serum. results: the patient with stat b deficiency showed increased values of ctfh ( %) (healthy donors p -p : , - , %) that presented an activated phenotype (icos+ and pd- +) with a skewed to a th profile (ccr +), consistent with her hipergammaglobulinemia and the marked and sustained increase in the switched mbl and pbc subpopulations in peripheral blood over the years. discusion: this immunological phenotype described in the patient with stat b deficiency could explain in part the pathophysiology of the autoimmune disorders. this patient (as well as the other two patients with mutations in stat b previously described by our group), have had chronic hypergammaglobulinemia, autoantibodies and consequently autoimmune processes (psoriasis, hypothyroidism, eczema, alopecia and celiac disease, among others). we believe that the link between this clinical symptomatology and the molecular defect relies in the fact that the absence of stat b promotes a greater expression of bcl- , which generates a bias towards the production of ctfh cells, that give rise to a greater activation of lb, generation of lbm and plasma cells (dysregulation in the cg), events that manifest as hypergammaglobulinemia and autoimmunity. in summary, we provide promising evidence of the mechanisms that lead to autoimmunity in this type of patients that could also be a consequence of the defect in the regulation of gc, highlighting the crucial role of stat b in the humoral immune response and maintenance of the tolerance of the immune system. background/introduction: the term primary immunodeficiencies (pid) encompasses a phenotypically and genetically diverse group of conditions. genetic testing for these conditions can guide treatment, reduce morbidity and mortality, allow for genetic counseling, and identification of additional at-risk family members. however, this testing can be complicated by a number of factors, including pseudogenes, high homology, methodology limitations, and the heterogeneous nature of pids. methods: mayo clinic laboratories launched their first set of nine pid next generation sequencing (ngs) tests approximately one year ago. these tests include one single gene assay for gata deficiency and eight targeted next generation sequencing panels for: atypical hemolytic uremic syndrome (ahus), autoinflammatory disorders, b-cell disorders, monogenic irritable bowel disease (ibd), phagocytic defects, severe combined immunodeficiencies (scid), and severe or cyclic neutropenia. herein we summarize our first year of experience with these ngs tests, with a focus on the eight targeted panel tests. results: from march through november we performed testing for cases. our highest volume of tests was for the ahus panel ( / cases, %). a variant was reported in / cases ( . %). these variants included variants of uncertain significance, likely pathogenic variants and pathogenic variants. the indication with the highest percentage of cases where a variant was reported was scid ( / cases, . %). the number of cases that were considered solved, where the genotype likely explains the patients phenotype, varied widely by indication. twenty cases were found to have a pathogenic or likely pathogenic variant or variants; however / cases were heterozygotes for an autosomal recessive condition and were not considered solved cases. the panel with the highest percentage of solved cases is our scid panel ( / cases, . %). conversely, we have yet to solve an autoinflammatory, irritable bowel disease, or telomere defects case; however % of cases in each of those three panels have had a variant of uncertain significance reported. we hypothesize that one of the reasons for the low detection rate for these three panels is inappropriate test orders. we are also actively looking for ways to update all panels to increase detection rates and clinical utility, for example expanding the gene list of our ibd panel, including large deletion/duplication detection, and including ncf , a difficult gene to capture by ngs, on the phagocytic panel. finally, we present the molecular findings from a number of interesting cases that were solved using our targeted ngs panels. conclusions: the launch of our pid ngs tests in march of has allowed us to aid patients by confirming diagnoses and providing molecular diagnoses that will enable more accurate genetic counseling and risk assessment. we have also uncovered areas for improvement, both on the clinical side: provider education is important to enable better identification of patients who can benefit from molecular genetic testing for pids, and on the laboratory side: introduction of more expanded panels and additional methodologies. the progressive decrease of red blood cells, platelets or neutrophils via a self-directed immune process is jointly termed as autoimmune cytopenias. while autoimmune cytopenias, including autoimmune hemolytic anemia (aiha), immune thrombocytopenic purpura (itp), and autoimmune neutropenia (an), are a common presentation of autoimmunity in the general population, they are particularly frequent and can appear as the first sign in patients with primary immunodeficiencies (pids). possible causes of cytopenia in pids comprise mainly immune dysregulation, bone marrow failure (bmf) and myelodysplasia. our goal is to investigate possible immune mediated mechanisms underlying chronic cytopenia in children in order to achieve an early diagnosis and consequently offer timely and appropriate therapy. we selected patients affected by chronic cytopenia, evaluated with immunophenotyping by flow-cytometry; data were subjected to multivariate analysis by principal component analysis (pca). next generation sequencing (ngs) analysis of genes frequently implicated in pids was performed. among the patients, were affected by bone marrow failure, of which were diagnosed with fanconi anemia and severe congenital neutropenia; were affected by immune-mediated cytopenia and by idiopathic cytopenia. the immunephenotyping showed a typical pattern of cd t cell subpopulations expression in patients compared with healthy donors with an increase of naïve t cells and a reduction of central memory (cm) and effector memory (em) t cells levels. we observed a decrease in total b cells, b switched and b memory cells and an increase in cd low cells. pca showed an overlap between groups, however it revealed a peculiar trend of some single patient, suggesting the pathway involved in immune defect. preliminary results from ngs studies revealed genetic variations in genes previously associated with pids in out of patients investigated. in particular we identify one patient with a mutation in fas, one with a mutation in aire and one with a mutation in ikaros. concerning the remaining patients further studies are ongoing to validate the pathogenicity of the genetic variations. pca is a very effective tool to analyze several parameters at the same time, highlighting patients whose phenotype shows the main peculiarities. the presence of specific lymphocyte subpopulation patterns can be important indicators of immune-mediated cytopenias and helpful signs of specific pids that should promptly be investigated with genetic analysis. the rapid of discovery of novel, monogenic primary immunodeficiencies has been made possible by the broad availability of clinical whole exome sequencing (wes). however, clinical wes has major shortcomings that should be understood by practicing immunologists. focusing on the iuis list of~ monogenic primary immunodeficiency genes, we show here limitations in coverage that could significantly impact clinical interpretation. on the agilent whole exome capture kit, the most common wes platform, there are a number of genes with exons that are poorly covered. specifically, there are at least genes with less than % exonic coverage, with less than % coverage and with less than % coverage (e.g. ikbkb, ncf , taci, unc b and tbx ). beyond this challenging technical issue, there are more subtle issues as well. these include the presence of pseudogenes in at least of our genes (e.g. ak , c qbp, cd , cftr, cr , msn, ncf , ncstn, ikbkg, nhp , pms , pten, rnaseh c, rps, sbds and was), which can make accurate sequencing very challenging. finally, there are many known causative intronic (e.g. btk, ctla- , wasp) and copy number variant mutations (e.g. rag and xiap) as well as large deletions (e.g. dock ) that we cannot expect to be optimally covered using wes. this list of genes requires consideration even with a negative exome and may require additional approaches including whole genome sequencing, sanger sequencing, cnv arrays and/or long-read ngs sequencing. wes is a powerful genomic diagnostic tool, but to avoid missing key diagnostic insights using these alternative approaches may be critical when certain genes are in the differential diagnosis. going forward, as pid phenotypes continue to broaden, these issues remain fundamentally important even if these genes are not obviously implicated in a given clinical phenotype. more physicians are utilizing targeted genetic panels to reach a definitive diagnosis for their patients with immunodeficiency. however, this increase in testing also has led to the discovery of many more variants of uncertain significance (vus) in the genes tested. these findings can often leave the patient and the physician with more questions than answers. we present a patient with recurrent infections found to have multiple variants of uncertain significance in several genes associated with primary immunodeficiency. a -year-old female who was diagnosed with crohns disease at age after intestinal perforation and jejunal resection experienced two discrete episodes of epstein barr virus (ebv) meningoencephalitis and septic shock. the first episode was diagnosed when patient had fever and altered mental status and occurred prior to her crohns disease diagnosis and the second episode was complicated with altered mental status, disseminated intravascular coagulation (dic) and hypotension requiring picu admission. aside from these two major infections, the family denied any other infections requiring antibiotics in the last years and reported a remote history of repeated streptococcal pharyngitis that have not recurred. immunology was consulted at the time of the second episode of meningoencephalitis and work up was mainly unremarkable with normal immunoglobulins, adequate vaccine response to hib, tetanus, diphtheria, rubella, measles and pneumococcus ( out of protective titers). she had normal t cell numbers with slightly decreased natural killer numbers for age. neutrophil studies showed normal dihydrorhodamine (dhr) analysis, glucose- -phosphate dehydrogenase levels and myeloperoxidase (mpo) stain. commercial testing of her toll like receptors ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) showed normal function. invitae primary immunodeficiency panel demonstrated a heterozygous variant in nod (c . c>t; p.arg trp) as well as heterozygous variants of uncertain significance in il r (c. g>t; p.ser ile) and tlr (c. c>g; p.leu val). the patients nod variant is known to be associated with an increased risk for crohns disease. even with our patients presentation with recurrent severe viral infections and ibd, it is not immediately clear how these genetic results explain the pathology. innate immune defects probably contribute to her presentation and it is currently unclear if and how the combination of multiple genetic variants has left her immunologically vulnerable. we use this case to demonstrate that even when genetic testing does not elucidate a clearcut diagnosis of primary immunodeficiency, it can still provide helpful insight into a patients underlying immune phenotype. introduction: xiap deficiency is a rare primary immune deficiency characterized by hemophagocytic lymphohistiocytosis, recurrent fever and inflammatory syndromes, inflammatory bowel disease, hypogammaglobulinemia, recurrent infections, and other manifestations. loss of xiap results in abnormal tnf receptor signaling and nlrp inflammasome actvity which leads to dysregulated production of il- beta and il- . we hypothesized that suppressing the nlrp inflammasome with either targeted deletion or pharmacologic inhibition would suppress abnormal production and secretion of inflammatory il- beta and il- . methods: bone marrow derived macrophages (bmdms) from control, xiap-deficient, and xiap and nlrp double knock-out mice were derived with week of culture in l -cell conditioned media. bmdms were stimulated with a variety of tlr agonists or tnf-alpha, with or without a variety of inhibitors including the nlrp inhibitor mcc , the cathepsin b inhibitor ca- , and quercetin, which is a natural flavonoid (antioxidant) found in many fruits and vegetables, and available as a nutritional supplement. il- beta, il- , and tnf-alpha were measured in supernatants by elisa, and cell death was evaluated by flow cytometry using pi exclusion. results: as expected, bmdms from xiap deficient mice had markedly increased tlr-agonist-or tnf-alpha-induced il- beta production compared to normal bmdms. genetic deletion of nlrp and the pretreatment of cells with the nlrp inhibitor mcc greatly reduced abnormal il- beta production; residual production of il- beta could be inhibited by caspase- inhibition. pre-treatment of cells with the cathepsin b inhibitor ca- also decreased cytokine production but was toxic at higher concentrations. quercetin reliably abrogated il- beta, and also il- . quercetin was found to inhibit priming of the nlrp inflammasome (decreased upregulation of pro-il beta and nlrp ) and also decreased tnf-alpha secretion following tlr agonist stimulation. conclusion: quercetin suppresses the nlrp inflammasome and may be a promising therapeutic option for patients with xiap deficiency. it prevents il- beta and il- secretion. it is a particularly appealing option given that it is a naturally occurring antioxidant, has a great safety profile, and is readily available as a nutritional supplement. human studies are needed. recently, single cell rna sequencing (scrnaseq) analysis in mice has disclosed an unexpected complexity of thymic stromal cells, and medullary thymic epithelial cells (mtecs) in particular. however, the developmental origin, hierarchy, and function of these subpopulations remain ill-defined. moreover, although cortical tecs (ctecs) are thought to represent a more homogeneous population, their characterization has been largely restricted to the adult thymus. we have previously shown that impaired lymphostromal cross-talk in the thymus of patients with combined immunodeficiency (and of corresponding mouse models) is associated with abnormalities of thymic architecture and tec maturation. here, we sought to compare tec distribution and gene expression in wild-type (wt) and in mice carrying rag hypomorphic mutations observed in patients with combined immune deficiency and immune dysregulation. methods: multi-color flow cytometry and scrnaseq were used to analyze composition and distribution of ctec and mtec subpopulations in wt and rag mutant mice at various weeks of age (niaid animal protocol: lcim- e). results: we observed that rag mutant mice have an excess of ctecs, and that their mtec compartment is predominantly represented by cells with high levels of mhc class ii (mhc-ii) expression, recapitulating the phenotype of neonatal wt thymi. while mhc-iihi mtecs are thought to represent a minor fraction of mtecs in adult wt mice and include mature aire+ cells, a relative abundance of mhc-iihi mtecs is observed also at neonatal age, where they are thought to represent immature mtecs. to define more precisely tec maturation, we performed scrnaseq on sorted cd -epcam+ cells, and identified and distinct clusters of tecs in wt and rag mutant mice, respectively. a large proportion of cells in rag mutant mice could be ascribed to the ctec compartment, confirming our previous flow cytometry and histopathology results. furthermore, scrnaseq analysis also disclosed a different distribution of mtec subsets in wt and rag mutant mice. to address the hypothesis that this difference in ctec and mtec abundance and subset distribution may reflect different maturation stages in tec development in wt and rag mutant mice, we will perform lineage tracing and transplantation experiments, and we will also extend tec scrnaseq analysis to wt and mutant mice of embryonic and neonatal age. in parallel, to evaluate the contribution of thymocyte maturation in shaping the stromal populations, scrnaseq will be performed on thymocytes. conclusions: we have further refined the complexity of tecs, and shown that impaired development of t cells in combined immune deficiency (as exemplified by rag mutant mice) has profound effects on the composition and maturation of tecs and may thus contribute to abnormalities of immune tolerance that are often associated with these conditions. the advent of next-generation sequencing (ngs), with the development of whole-exome sequencing (wes) in particular, has allowed the identification of unknown genetic lesions for many diseases and the implementation of specific therapeutic strategies. primary immunodeficiencies (pids) are a group of rare diseases which have benefited from ngs, with the discovery and molecular characterization of previously genetically undefined diseases and the identification of novel molecules involved in the regulation of the immune system. pids are often associated with autoimmune disease due to the dysregulation of the immune system as a whole. the clinical phenotypes are heterogeneous and often overlapping. while a monogenic cause of disease has been identified in a most subsets of patients, the recent application of whole-genome sequencing has found that a polygenic cause is likely. our aim is to investigate the genetic background of patients with immunedysregulations and autoimmunity and to evaluate the possible pathogenicity of the identified gene variants through extensive functional studies. we select patients with sign of immunedysregulation and autoimmunity, extended immunophenotyping and next-generation sequencing (ngs) analysis of genes frequently implicated in pids was performed. in six of them we identify a single gene as responsible of the clinical feature. in particular, we identify two patients with gain of function mutation in stat , one patient with a mutation in ctla , one patient with an activating pik cd mutation, one with a rag mutation and one with a fas mutation. in most of them variants in multiple genes have been detected. interestingly, we find that some genes are recurrently mutated in more then one patient such as was, dock , casp , casp , nfatc and fcgr a. further studies are ongoing to validate the effect of the variations identified. our results strongly suggest that the old hypothesis, based on a single gene mutation as a cause of illness, should be revised in favor of the concept that "is the sum that causes the effect" and that a different point of view on pids now seems inevitable. physician, omni allergy, immunology, and asthma introduction/background: immunoglobulin replacement therapy (igrt) may be optimized to reduce the severity and incidence of infections and potentially delay or abrogate the development of pulmonary complications of primary immune deficiencies. pulmonary complications including bronchiectasis are common in common variable immune deficiency (cvid) and contribute significantly to morbidity and mortality in these patients. it remains unclear whether continued obstructive bronchial changes are a result of repeated respiratory infections, associated inflammation and immune dysregulation, or simply lung-damage that is irreversible by the time therapy is initiated. it has also been suggested that under-treatment in addition to the diagnostic delay may contribute to the development of bronchiectasis in patients with pid. lower serum igg levels with any given dose of immunoglobulin replacement therapy have been demonstrated in patients with bronchiectasis compared to those pid patients without this complication. in addition, earlier studies have shown that greater doses of ig ( mg/kg/ month) may reduce the frequency and duration of infections and help prevent or slow progression of chronic lung disease. objective: to evaluate the prevalence of bronchiectasis in a cohort of patients with a diagnosis of cvid and identify associated ig dosing patterns and clinical outcomes. methods: data were analyzed from the ideal (immunoglobulin, diagnosis, evaluation, and key learnings) patient registry. this is a prospective, longitudinal registry study of patients receiving ig replacement therapy in the home or ambulatory infusion suite with one national home infusion provider. nursing and pharmacy standard of care forms were collected, and dose, infection rate, and prevalence of bronchiectasis were evaluated in patients with a diagnosis of cvid (icd- codes: d . , d . ) results: there were patients in the registry with cvid, ( . %) of which bronchiectasis was also observed. seventy-nine percent (n= ) of the study population was female, and % (n= ) of the cases of bronchiectasis were observed in females. the mean age of the patients with concurrent bronchiectasis was ± . at start of care compared to ± . in those without this observed bronchial obstruction. most bronchiectasis patients (n= ) received igrt subcutaneously every week with a mean dose of . ± . mg/kg/wk. the mean dose of ig in the remaining patients receiving ig intravenously was . ± . mg/kg/month. the average annual rate of infection in ivig and scig patients with bronchiectasis was . ± . and . ± . , respectively, however many were serious bacterial infections. at time of analysis, of the bronchiectasis patients remained active in the registry and had withdrawn. reasons for withdrawal included stopping igrt due to the following: patient decision (n= ), physician decision (n= ) insurance change (n= ), and patient expired (n= ). conclusions: there were documented cases of bronchiectasis in our cohort of cvid registry patients, and dosing patterns aligned with standard doses despite the presence of bronchial obstruction. further studies are necessary to assess evolution of lung damage with respect to ig dosing in patients with cvid. background: activated phosphoinositide -kinase syndrome type (apds ) is a combined immunodeficiency resulting from gain-offunction (gof) mutations in pik cd, the gene encoding the catalytic subunit of phosphoinositide -kinase (pi k). this form of pid is characterized by recurrent respiratory tract infections, susceptibility to herpes virus infections, impaired antibody responses, lymphoproliferation and autoimmunity. previous studies showed that patients with apds have b cell defects that contribute to the clinical phenotype. furthermore, these patients display t cell abnormalities, including increased numbers of memory t cells and t follicular helper cells (tfh), reduction of naïve t cells and impaired t regulatory cell (treg) function. whether these t cell abnormalities are also associated with perturbations of t cell repertoire in unknown. objective: we aimed to investigate the effects of increased pi k signaling on the t-cell repertoire of patients with apds. methods: high throughput sequencing was used to study composition and diversity of t-cell receptor (tra) and t-cell receptor (trb) repertoire in sorted treg, tfh, conventional cd + (tconv), and cd + t cells from patients with pik cd gof mutations and healthy controls. results: treg cells of patients with apds show restriction of tra and trb repertoire diversity, and increased clonality. no repertoire restriction was detected in tfh, tconv, and cd + t cells from the same patients. however, the trb repertoire of treg and cd + cells was enriched for the presence of hydrophobic amino acids in position and of the cdr , a biomarker of self-reactivity. conclusion: these data demonstrate that the t-cell repertoire of patients with apds is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. furthermore, our result support the notion that the pi k pathway is a key regulator of treg cell development and homeostasis in humans. j clin immunol ( ) (suppl ):s -s s ( ), iii. predominantly antibody deficiencies ( ), i. immunodeficiencies affecting cellular and humoral immunity ( ), vii. auto-inflammatory disorders ( ), ix. phenocopies of pid ( ) . two non related cases of ataxia-telangiectasia and one case of schimke syndrome (smarcal compound heterozygous mutation) were diagnosed in the last year. we observed a wide range of age (we evaluate adult and pediatric population) with a male:female ratio close to : immunodeficiency, immune dysregulation, and systemic autoimmunity. clinical diagnosis of these disorders is complicated by overlapping phenotypes. in april , a -gene next generation sequencing (ngs) panel inclusive of copy number variation analysis was launched by a commercial laboratory to facilitate clinical diagnosis of primary immunodeficiency (pid), monogenic autoimmunity and autoinflammatory disorders. we assessed the outcomes of genetic testing utilizing this panel on a cohort of pediatric patients with immunohematologic phenotypes evaluated at our tertiary care center during an -month period ( / / - / / ). eligible subjects were evaluated by at least two of three providers from a multidisciplinary pediatric hematology-immunology team, including a hematology physician, immunology physician and a geneticist or genetic counselor. twenty-three patients met inclusion criteria; ( %) were caucasian, ( %) were male with an average age of . years. the two most common phenotypic diagnoses included cytopenias, single-or multilineage (leukopenia, neutropenia, anemia, thrombocytopenia) primarily attributed to autoimmune causes or hypogammaglobulinemia. five ( %) were given a definitive genetic diagnosis as a result of panel testing, though in two of these cases, the causative mutations were listed as variants of uncertain significance (vus). diagnoses included common variable immunodeficiency due to a pathogenic variant in nfkb , stat multiorgan autoimmunity due to gain-of-function mutation, and familial cold autoinflammatory syndrome due to a pathogenic mutation in nlrp . biallelic dnmt b vus were found in a patient whose phenotype and further laboratory studies (including karyotype) were consistent with immunodeficiency-centromeric instability, facial anomalies syndrome. further, a stat vus was identified in a patient with multiorgan autoimmunity and his father with hypothyroidism; studies from an outside research laboratory were consistent with gain-of-function with this variant (private communication). an additional three patients had vus identified that were suspected to be related to their phenotype, prompting eligibility for research studies. four ( %) patients had increased risk alleles in nod , conferring an increased risk of crohns disease. three ( %) patients had pathogenic or likely pathogenic carrier findings warranting genetic counseling. in addition, vus (an average of per patient) thought to be unrelated to phenotype were identified, necessitating further investigation and counseling. the use of an ngs panel in a cohort of pediatric patients with immunohematologic disorders led to a definitive diagnosis in % of previously undiagnosed patients and prompted further research investigation in several more. genetic testing also led to the identification of clinically significant carrier findings, risk alleles and vus unrelated to phenotype, necessitating genetic counseling. our experience illustrates the value of genetic testing for diagnosis of immunohematologic disorders, and the importance of multidisciplinary care, including genetic counseling, for the proper evaluation and management of these patients. background: allogeneic hematopoietic cell transplantation (allohct) is curative for primary immune deficiencies (pid). however, many patients lack a fully-matched unaffected sibling, or may have an unknown underlying genetic defect, rendering it undesirable to use related donors. many pid patients have significant comorbidities at the time they are referred to allohct, precluding the use of myeloablative conditioning. the use of alternative donors with reduced-intensity conditioning (ric) has historically led to increased rates of graft failure, graft-versus-host disease (gvhd), and transplant-related mortality (trm). posttransplantation cyclophosphamide (ptcy) as gvhd prophylaxis immunomodulates the graft through the preferential sparing of regulatory t cells and hematopoietic stem cells from its cytotoxic effects, thus allowing for robust donor engraftment that overcomes the hla barrier while effectively preventing severe acute and chronic gvhd. we report the outcomes of two institutions using a ric allohct regimen with alternative donors and ptcy in patients with pid. design: we transplanted pid patients (table ) using alternative donors and ric, either serotherapy-free (n= ) or alemtuzumab-based (n= ). all patients received ptcy for gvhd prophylaxis on days + and + , either alone (n= ), or combined with mycophenolate mofetil and either sirolimus (n= ) or tacrolimus (n= ). donors included haploidentical family members (n= ), matched unrelated (n= ), and mismatched unrelated (n= ). stem cell source was t cell-replete bone marrow (n= ) or peripheral blood stem cells (n= ). results: the median follow-up is months (range . - years). at months, overall survival is %, and event-free survival (defined as alive without graft failure) is %. the median days of neutrophil and platelet engraftment are (range - ) and (range - ), respectively. there were patients who developed acute gvhd, grade (n= ) or grade (n= ), and there were no cases of grade or agvhd. seven of eight patients treated with systemic corticosteroids responded, and one was corticosteroid-dependent, then responded to second-line therapy. one patient developed skin-only chronic gvhd, which responded to corticosteroids and puva light therapy. five patients developed graft failure, either primary (n= ) or secondary (n= ), and four were successfully re-transplanted and remain engrafted. one patient with secondary graft failure had autologous recovery and has not required a second allohct given some durable infection control gained during initial engraftment. there were three deaths prior to day due to infection, and one death at . years secondary to presumed overdose. in ongoing follow-up of engrafted survivors (n= ), evidence of phenotype reversal has been demonstrated in all patients, with complete or ongoing resolution of some or all of their underlying disease manifestations, including infection, transfusion-dependence, autoimmunity, malignancy, and/or immune dysregulation. discussion: we have observed high rates of engraftment, low rates and severity of acute and chronic gvhd, and low trm in patients with pid transplanted using alternative donors, ric, and ptcy-based gvhd prophylaxis. ric allohct with ptcy shows promise for curing pid, and its use minimizes toxicity and widely expands the donor pool, thus allowing us to offer this curative therapy to many more patients with pid. chronic granulomatous disease (cgd) is a primary immune disorder that involves mutations in the nicotinamide adenine dinucleotides (nadph) oxidase complex (deffert, cachat, & krause, ) . two-third of cgd cases are caused by loss-of-function mutations in the cybb gene that encodes the gp pox subunit of the nadph. the increased in patients' life expectancy thanks to progress in diagnosis and management has underlined the burden of inflammatory manifestations occurring independently of infectious agents (dunogue et al., ; marciano et al., ) . cgd patients develop inflammatory granulomatous disorders, notably colitis, as a consequence of a dysregulated inflammasome activation. the treatment of inflammatory manifestations remains challenging, as it can be associated with an increased risk of infections. thus, understanding the pathophysiological mechanism of auto-inflammation in cgd could help improve the therapeutic arsenal for the management of these manifestations. to reveal the precise pathophysiological mechanism of auto-inflammation in cgd, we have developed a cellular model that reproduces the cgd phenotype in phagocytic cell. through crispr-cas gene-editing we generated a thp- c e l l l i n e h a r b o r i n g t h e p r e v i o u s l y d e s c r i b e d mu t a t i o n c. _ delccginsggt (p.tyr ter) in the cybb gene responsible for gp phox knock-out by early termination of translation. this cell line recapitulates the phenotype of cgd phagocytes: (i) decreased h o production (ii) and enhanced inflammatory responses after pma stimulation as evidenced by increased il- , il- and tnfa secretion levels (kuijpers & lutter, ) . these features were rescued by complementation through lentiviral transduction of a wild type cybb gene. this new model will help us to investigate the auto-inflammation reported in cgd patients and also to propose new therapeutic targets of inflammatory manifestations in this disorder. interleukin- (il- ) driven responses. children with irak- deficiency are predisposed to recurrent and invasive infections secondary to streptococcus pneumoniae, staphylococcus aureus and other pyogenic bacteria with high mortality rates in early childhood. the frequency and severity of infections is thought to decrease with age due to the acquisition of humoral immunity and immunologic memory, however due to the rarity of the disease, the natural history of this condition beyond early childhood is not well described. objectives: we present three unrelated irak- deficient patients with persistent chronic rhinosinusitis with nasal polyposis that developed in childhood. cases: patient is a y/o male with compound heterozygous mutations in irak (p.g afs* /c. - g>t) with a history of recurrent s. pneumoniae osteomyelitis (left hip at age and left knee at age ) and c. septicum sepsis at age following acute bowel perforation. additionally, he experienced recurrent aom during infancy and recurrent uti since age . despite prophylactic antibiotics and ivig, he has had recurrent polymicrobial (mrsa, s. pneumoniae, h. influenzae, p. aeruginosa, a. fumigatus) rhinosinusitis with nasal polyposis since age refractory to medical management requiring surgical intervention and prolonged courses of iv antibiotics. patient is an y/o female with homozygous deletions (exons - ) in irak with a history of ruptured appendicitis complicated by pseudomonas abscess and bacteremia at age , culturenegative sepsis with septic arthritis and osteomyelitis of the right leg at age , and septic shock secondary to mssa bacteremia complicated by rhabdomyolysis and dic at age . she has a history of chronic rhinosinusitis, and despite ivig and prophylactic antibiotics, she developed polymicrobial (h. influenzae, b. fragilis) rhinosinusitis with associated nasal polyposis pending surgical management. patient is a y/o female with homozygous mutations in irak (q x/q x on exon ) with a history of s. pneumoniae meningitis at months, m. catarrhalis epiglottitis and neck cellulitis at months, rsv bronchiolitis at months, enterococcus bacteremia at months, s. pneumoniae sepsis at age and streptococcus lymphadenitis at age . despite ivig and prophylactic antibiotics, she developed recurrent polymicrobial (h. influenzae, b. fragilis, mssa, v. cholera, p. aeruginosa, a. fumigatus) rhinosinusitis refractory to medical management requiring surgical intervention and iv antibiotics. conclusions: in our centers experience, irak- deficient patients continue to suffer from infectious complications, most prominently recurrent polymicrobial sinus infections beyond early childhood. the consistent presence of sinonasal polyps in these children is unusual, as it is not typically found in uncomplicated pediatric chronic rhinosinusitis. these infections have occurred despite antimicrobial prophylaxis and ivig, highlighting the role of irak- in sinopulmonary epithelium. additionally, the infectious organisms identified in our patient cohort are not commonly associated with irak- deficiency. further study of chronic rhinosinusitis and nasal polyposis in a larger cohort of irak- deficient patients and other innate immunodeficiencies may help identify pathways for targeted treatment of these patients. introduction: chronic granulomatous disease (cgd) is an inherited phagocytic defect associated with inability to clear catalase positive organisms. infections in patients with cgd are severe and recalcitrant. commonest infections are pulmonary followed by soft tissue infections and suppurative lymphadenitis. osteomyelitis is an uncommon infection in patients with cgd. it poses several diagnostic and therapeutic challenge. we herein report our experience of osteomyelitis in cgd over the last years. material and methods: review of records was carried out to describe the profile of osteomyelitis in cohort of patients with cgd at pediatric immunodeficiency clinic, advanced pediatrics centre, postgraduate institute of medical education and research, chandigarh, india. the diagnosis of cgd was based on nitroblue tetrazolium dye reduction test (nbt) and dihydrorhodamine reduction (dhr) assay. results: of the patients with cgd, ( . %) had osteomyelitis ( males and females; age range - years). most patients had their first episode of serious infection in early childhood (mean age: . years). stimulation index (si) of dhr assay ranged from to . . mutational analysis was done in / patients ( x-linked; autosomal recessive). site of involvement was variable ribs- ; vertebrae- ; radius- ; skull- ; tibia- . aspergillus fumigatus was the most common isolate ( %; / ); others had aspergillus flavus, aspergillus terreus and serratia marcescens each. all patients with rib osteomyelitis had concurrent pneumonia, and fungus was isolated in all of them (aspergillus fumigatus- , aspergillus flavus- , zygomyces spp.- ). antifungals (intravenous amphotericin b) were given for a duration of - weeks and were followed by oral voriconazole in therapeutic doses for to months in majority of them. debridement and resection of ribs was required in one patient, while other patients were managed conservatively. out of patients, ( %) succumbed to pneumonia and respiratory failure. conclusion: osteomyelitis in the context of cgd is usually caused by aspergillus spp. involvement of ribs and vertebra usually occurs with the contiguous spread of infection from the lungs. therapy often requires prolonged duration of anti-microbials, and may require surgical debridement in addition to it. a -year-old woman with history of hypogammaglobulinemia and acute liver failure a -year-old woman with a -month history of nausea, vomiting, and abdominal pain was admitted to an outside hospital with new onset of jaundice and anasarca. liver biopsy was thought most consistent with alcoholic steatohepatitis, and she was discharged with counseling on alcohol cessation and medical management of liver disease. she presented to our facility for a second opinion. over the following days, she developed further rise in direct hyperbilirubinemia up to . mg/dl, new coagulopathy with an inr . and hypoalbuminemia to . mg/ dl in the absence of ongoing alcohol consumption. liver sonography revealed course echotexture and patent vessels. pcrs directed against multiple hepatotropic viruses were negative and copper studies were normal. due to a history of moderate alcohol consumption, she was started on high-dose corticosteroids due to a presumptive diagnosis of alcoholic hepatitis. additional history raised concern for a possible primary immunodeficiency, including idiopathic thrombocytopenic purpura at years of age, multiple episodes of sinusitis treated with antibiotics and sinus surgery, one episode of suspected bacterial pneumonia, and one hospitalization for influenza a during which she developed neutropenia. in her s, she developed refractory genital warts, prompting infectious diseases evaluation. initial immune evaluation had revealed low immunoglobulins (iga < mg/dl, igg mg/dl, igm mg/dl) with very low responses to tetanus and diphtheria, despite a recent booster dose, and b and t cell lymphopenia (cd + cells/μl, cd + cells/μl, cd + v, cd + cells/μl, cd / + cells/μl); antigen and mitogen proliferation were not assessed. intravenous immunoglobulin replacement was initiated but discontinued by the patient due to infusion-related adverse effects, and she was lost to follow up until she presented with liver failure. both parents were deceased from cardiovascular disease in their s and she had no siblings. she had limited knowledge of family history but no known immune diseases. due to suspicion for genetic etiology of immune disorder and liver disease, we performed next-generation sequencing of a panel of over genes implicated in primary immune deficiencies. patient was heterozygous for a nucleotide substation (c. + g>a) within a splice site at the exon /intron boundary of the nfkb gene. during the hospitalization, immunoglobulin replacement and trimethoprim-sulfamethoxazole prophylaxis were initiated. an attempt was made to refer the patient for additional immunological evaluation and transplantation evaluation but unfortunately, she developed worsening liver failure and multiple complications, including extended-spectrum beta-lactamase (esbl)-producing e. coli bacteremia, hypotension requiring vasopressors and extensive bowel ischemia, and died in the hospital. in summary, this case highlights both the risk of diagnostic delay in adult patients presenting with a primary immune deficiency and potential for genetic testing to clarify the diagnosis. while the particular genetic change has not been described, other splice site and predicted loss-offunction mutations have been reported as pathogenic in this gene, which have been implicated in autosomal dominant common variable immunodeficiency. this case further expands on the genetic causes and spectrum of disease associated with changes in the nfkb gene. introduction: malnutrition and micronutrient deficiency are underrecognized causes of acquired immunodeficiency in adults, and may occur even in patients with high body mass index (bmi). methods: a -year-old woman with a medical history significant for one remote urinary tract infection presented to the emergency department after sudden onset of severe right flank pain. the pain was accompanied by urinary frequency and not relieved by ibuprofen; she denied fevers or chills. she was diagnosed with pyelonephritis and discharged on ciprofloxacin, which was later changed to trimethoprim-sulfamethoxazole after her culture grew resistant e. coli. her pain continued despite treatment, prompting her to return to the hospital three days later. upon presentation, she was afebrile with blood pressure of / mmhg and heart rate of bpm. her body mass index was . kg/m^ . her physical exam was otherwise notable for right costovertebral angle tenderness. laboratory studies revealed a leukocyte count of , /ul with % neutrophils; alkaline phosphatase of units/l and albumin of . g/dl, but otherwise normal liver function tests; normal lactic acid; and urinalysis with , wbc/hpf, rbc/hpf, moderate bacteria, and the presence of wbc clumps. ct scan of the abdomen and pelvis demonstrated an obstructing mm right renal stone with hydronephrosis and a right renal abscess contiguous with a right-sided hepatic abscess measuring . x . x . cm. she was treated with ceftriaxone and metronidazole, and underwent imaging-guided drainage of the abscesses. abscess cultures again grew resistant e. coli. she was discharged from the hospital with drains in place and a plan to continue trimethoprim-sulfamethoxazole until definitive management of her nephrolithiasis with ureteroscopy and lithotripsy. discussion: there remained the question of how an ostensibly immunocompetent patient had developed such severe intraabdominal infection with little systemic inflammatory response (e.g. no fever and only mild leukocytosis). a hiv antibody screen was negative. on further interview, she described a lb intentional weight loss over the preceding years, accomplished by dietary restriction to less than calories per day. nutritional assays revealed prealbumin, vitamin c, and vitamin b levels below the threshold of detection. she had low-normal b and b . out of concern for an acquired immunodeficiency resulting from malnutrition with micronutrient deficiency, balanced nutrition was discussed with the patient who agreed to liberalize her diet. background: the past decade has brought dozens of new mendelian disorders of immunity. yet, the genetic contribution(s) to diverse disorders of the immune system remain largely unelucidated. the majority of research participants referred to the national institute of allergy and infectious diseases (niaid) for what may be a mendelian disorder evade molecular diagnosis. making progress in this area requires a coordinated, systematic, and transparent approach to clinical genomics research which leverages the unique environment at the national institutes of health clinical center (nih cc). methods/design: this study is designed to systematically apply exome sequencing and related technologies with clinical grade interpretation and reporting to niaid research participants at the nih cc under a single protocol in order to facilitate research and clinical genetics care across niaid. we are recruiting approximately participants per year from approximately intramural clinical investigators. we generate genomic data, collect standardized phenotyping and report clinical interpretation in the medical record, all while providing linked genetic counseling. results: to date, we consented participants, we sent out samples for exome sequencing and samples underwent copy number variant analysis. we have completed analysis for families ( individuals) and finalized and resulted cases. here we present a case series illustrating some of our findings. case : a year-old female was referred to niaid for neonatal onset multisystem inflammatory disease (nomid). developmental delay and mild intellectual disability were appreciated on clinical evaluation. exome sequencing detected a mosaic novel likely pathogenic variant in nlrp . chromosomal microarray analysis (cma) showed ã mb interstitial deletion of chromosome previously associated with developmental delay and intellectual disability. case : a year-old ukrainian male was referred to niaid for the clinical diagnosis of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced). exome sequencing and cma did not detect pathogenic variants in aire, but did find a de novo variant in fam b. defects in fam b are associated with poikiloderma with tendon contractures, myopathy, and pulmonary fibrosis (poiktmp). the clinical features of the patient were consistent with poikmp. case : a -year-old man had a history of brain, liver and kidney nocardiosis, disseminated mac infection, prostate cancer and lymphoma. family history was significant for prostate cancer. exome sequencing showed a heterozygous pathogenic variant in brca , associated with susceptibility to breast-ovarian, male breast, pancreatic and prostate cancer. conclusion: this case series illustrates that multiple diagnoses, unexpected diagnoses, secondary genomic findings, and data sharing helped identify variants in candidate genes. process standardization supports data integrity and efficiency while accommodating the need for investigator flexibility and providing tailored patient care. rationale: activated pi kinase delta syndrome (apds) is a primary immunodeficiency caused by dominant mutations that increase activity of phosphoinositide- -kinase (pi k). the catalytic subunit p is mainly expressed in cells of the hematopoietic system, primarily lymphocytes and myeloid cells, and mutations affect both b-and t-cells. we sought to further evaluate the role of the t-cell receptor (tcr) repertoire in immune dysregulation and the pathogenesis of autoimmunity and lymphoproliferation in patients with apds. methods: we evaluated the tcr repertoire in the peripheral blood in patients with pik cd mutations and compared these to the peripheral tcr repertoire in patients with common variable immunodeficiency (cvid) and healthy controls to investigate the role of the tcr in disease. the tcr repertoire in affected tissue of patients with pik cd mutations was also evaluated (tissue included lymph nodes for both patients, in addition to gastrointestinal tract and lung tissue in one patient). a fixed number of tcrs were subsampled ( , for blood and , for tissue) and diversity was calculated using the gini and shannon indexes. results: using the shannon and gini diversity indexes, the tcr repertoire in patients with pik cd mutations had less diversity/ increased clonality as compared to healthy controls and those with cvid ( figure ). for the two apds patients with biopsy tissue available for analysis, the diversity of the tcrs in tissue was increased as compared to the peripheral blood tcr repertoire ( figure ). conclusions: pi k plays an important role in the development and function of both b-and t-cells. patients with apds were found to have decreased tcr repertoire diversity in the circulating t-cell compartment compared to healthy controls and other cvid patients. the increased tcr diversity in the affected tissues compared to peripheral blood implicates the pi k/akt signaling pathway with t-cell trafficking and tissue immune homeostasis, and suggests this pathway may play a role in the development of inflammatory and lymphoproliferative complications in these patients. gain-of-function mutations in pi kd result in a human primary immunodeficiency, named apds (activated pi k-delta syndrome), characterized by lymphopenia, lymphoproliferation, respiratory infections and inefficient responses to vaccination. however, what promotes these immune disturbances at the cellular and molecular level remains unknown. we have recently published a mouse model that recapitulates major features of this disease and used this model and patient samples to probe how hyperactive pi kd fosters aberrant humoral immunity. we found that mutant pi kd alters the intrinsic function of t and b cells, leading to icos-independent increases in t follicular helper (tfh) and germinal center (gc) b cells, disorganized gcs, and poor class-switched antigen-specific responses to immunization. these phenotypes were associated with increased phosphorylation of akt and s in t and b cells, and lower threshold of activation, with altered regulation of foxo and bcl family members. moreover, b cells showed enhanced responsiveness and proliferation to both antigens and innate stimuli, accompanied by reduced cell death. strikingly, aberrant responses were accompanied by increased reactivity to gut bacteria, and a broad increase in autoantibodies that were dependent on commensal microbial stimulation, as demonstrated by striking reduction of self-reactivity upon antibiotic treatment in mutant mice. we now have further examined b cell function in these mice and demonstrate that altered foxo plays a major role in disruption of both b and t cell function. we further provide evidence for altered activation of metabolic pathways in b cells, compared to wt cells, that may contribute to the dysregulated b cell reactivity. our findings suggest that proper pi kd regulation is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome. this research was supported in part by the intramural research program of the nih, nhgri and niaid. autoimmune cytopenias are seen in a significant proportion of patients with immunodeficiencies affecting antibody production. previous b-cell maturation studies using fluorescence-activated cell sorting (facs) have associated various phenotypes of primary immunodeficiency diseases affecting antibody production with differing levels of b-cell differentiation. in this study we analyzed the peripheral b-cell compartment of patients with a hypogammaglobulinemia and > % b-cells with and without a history of autoimmune cytopenias. b-cells were isolated from peripheral blood using monoclonal anti-cd and these cells were gated to identify the proportion of memory b cell (cd +cd + ), igm+ memory b (cd +igm+), marginal zone b-cells (igm+ igd+), isotype-switched memory b-cells (cd +igm-igd-) and transitional cells (igmhicd hi). pid patients with a history of aic had decreased proportions of total cd + b-cell ( . % vs . %; p= . ) and igm memory b cells ( . % vs . %; p = . ). conversely, the proportion of marginal zone b-cells was increased in this group ( . % vs . %; p = . ). consistent with previous reporting, the proportion of isotype-switched memory b-cells was significantly lower in the aic group ( . % vs . %; p = . ). statistically significant inter-group difference was not seen within the transitional b-cell subset. our data suggest that maturation arrest of marginal zone (cd +igm+ igd+) b-cells may be implicated in the development of autoimmune cytopenias in humoral immunodeficiency. ( ) submission id# taissa de matos. kasahara , sudhir gupta, md phd student, state university of rio de janeiro and university of californis irvine professor, university of california at irvine, irvine, ca, usa introduction/background: common variable immunodeficiency (cvid) is the most frequent form of primary hypogammaglobulinemia with decreased serum igg and iga levels and variable levels of igm in adults. in addition to decreased serum immunoglobulins, - % of cvid patients present autoimmune manifestations. the mechanisms that lead to a breakdown of selftolerance in cvid are not completely understood. however some differences in b and t cells subsets and autoreactive b and t cells can be detected. elevated expression of surface igd and downregulation of igm receptor are hallmarks of anergic naïve b cells that contain autoreactive receptors in human peripheral blood. moreover, memory b cells that have class switched to igd and present an igd+igm-phenotype are also highly reactive to self-antigens in healthy individuals. the role of these autoreactive naïve and memory b cells in the immunopathogenesis of cvid has not been evaluated. here we investigated the frequency of cd -and cd + b cells expressing igd and igm in peripheral blood of cvid patients. methods: peripheral blood mononuclear cells (pbmc) from cvid patients (n= ) and health subjects (n= ) were separated by ficollhypaque and incubated with anti-human cd -percp, cd -fitc, igd-bv and igm-apc to identify different subsets of b cells by flow cytometry. cd +cd -igd+igm-and cd +cd -igd+ igm+ b cells were sorted, loaded with cfse and cultured with cpg and ant-cd for days to evaluate the proliferation. results: among the compartment of cd -b cells, cvid patients showed an increased frequency of igd+igm+ cells and a lower frequency of igd-igm-cells as compared to control group. no differences were observed in the frequency of igd+igm-cells in cd -b cells between cvid patients and controls. in contrast, in the compartment of cd + b cells, cvid patients showed an increased frequency of igd+igm-, igd+ igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. when the patients were divided in two groups based on autoimmune manifestations, the group with autoimmune disease showed an increased frequency of igd+igm+ and igd-igm+ cells in cd -b cells when compared to the control groups. both patient groups showed an increased frequency of igd+igm-, igd+igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. regarding the proliferation, naïve b cells from cvid patients showed a reduced proliferative capacity in response to in vitro stimulation as compared with naïve b cells from health subjects. conclusion: our results suggest that the increase of cd +igd+igm-b cells can be related to the susceptibility of autoimmunity in cvid patients. introduction: immunoglobulin g -related disease (igg -rd) is a group of immune-mediated conditions where tissues are affected with dense lymphoplasmacytic infiltrations with a predominance of igg -positive plasma cells and storiform fibrosis, usually in the setting of elevated serum concentrations of igg . common presentations include autoimmune pancreatitis, sclerosing cholangitis, retroperitoneal fibrosis, salivary gland disease, and orbital disease, among others. symptoms of asthma or allergy are present in approximately percent of patients and they typically exhibit a good initial therapeutic response to glucocorticoids. case presentation: a -year-old female with a history of gastroparesis, cutaneous lupus erythematosus and suspected autoimmune pancreatitis was referred to allergy/immunology clinic for evaluation of elevated igg . she reported a -year history of recurrent abdominal pain attributed to recurrent pancreatitis based on previous mild lipase elevations. prior endoscopic ultrasound (eus) of the pancreas revealed edema. there was concern for gallstone pancreatitis but ercp followed by cholecystectomy, biliary and pancreatic sphincterotomy had no change in her symptoms. in , she was noted to have a positive ana and high serum igg , per patient (values from osh records could not be obtained). symptoms improved with a course of steroids, hence suspicion for autoimmune pancreatitis. in she developed a rash on her arms and face. biopsies of the affected areas revealed cutaneous lupus erythematosus on the arms and a basal cell carcinoma on the face, which was excised. ana was only : at that time. at the visit, she complained of severe allergic rhinitis, joint pains, as well as a malar rash, which responded to intermittent courses of prednisone by prior providers. laboratories obtained at initial visit were significant for thrombocytopenia ( thou/cu mm), positive lupus anticoagulant ( sec) and elevated igg ( mg/dl; normal range - mg/dl). c , c , c q, ana, anti-double stranded dna, anti-smith antibodies, antiphospholipid panel, upep and spep were all unremarkable. ct chest and abdomen were also normal. given the patient's history of cutaneous lupus erythematosus, plaquenil was started as a steroid sparing agent. eus of the pancreas with possible biopsy was ordered in an attempt to obtain a histopathologic diagnosis of igg -rd. conclusion: this case exhibits the association between elevated igg , pancreatitis of unknown origin, allergic rhinitis, and cutaneous lupus erythematosus, highlighting the value of identifying a pathologic connection between seemingly unrelated disorders in patients with elevated igg , as they may be manifestations of igg -rd. in order to make the diagnosis, histopathologic findings showcasing lymphoplasmacytic tissue infiltration consisting mainly of igg -positive plasma cells and small lymphocytes is essential. the majority of patients respond to glucocorticoids, and while the duration of response is variable, most patients flare during or after glucocorticoids are tapered, as noted in this patient. rituximab has been shown to be effective in some patients and will be considered in this patient if symptoms persist. ( ) submission id# rationale: pnp deficiency is an autosomal recessive disorder due to defective purine metabolism leading to severe combined immunodeficiency (scid) and neurological deterioration. newborn screening utilizing t-cell receptor excision circle (trec) assay can detect affected patients before complications arise. herein, we describe an infant initially identified by newborn screening with pnp deficiency and congenital cmv, a previously unreported presentation. methods: cmv quantitative pcr (qpcr) was performed by nebraska medicine, pnp enzyme activity by duke and genetic sequencing by invitae. results: a small for gestational age (sga) male infant was reported to have an abnormal trec assay on day of life (dol) . he was hospitalized for further evaluation. initial studies revealed profound lymphopenia, normal lymphocyte proliferation to mitogens and no evidence of maternal engraftment. additionally on dol , he had cmv viremia and viruria; thus with sga, failed unilateral hearing screen and head ultrasound with bilateral parenchymal calcifications, congenital cmv was suspected. pnp enzyme activity was abnormal. cmv treatment was initiated with ganciclovir on dol . foscarnet was added on dol . cmv qpcr levels decreased below the limit of detection by dol . genetic testing found a pathogenic homozygous mutation in pnp (c. - g>a). the infant has a / hla-matched, unaffected, cmv positive sibling and will proceed to hematopoietic stem cell transplantation. conclusions: to our knowledge, this is the first reported case of pnp deficiency identified through newborn screening. this novel case of congenital cmv and pnp deficiency highlights the importance of cmv screening and need for treatment strategies for congenital cmv in scid. despite a dramatic increase in the use of next generation sequencing over the last decade, the majority of the more than million identified human genomic variants do not have well-established clinical implications. progress is being made on this complex challenge through multiple approaches, including data sharing. to maximize our understanding of genomic data, platforms that enable effective and responsible data-sharing are essential. this means that genotypic and phenotypic data must be findable, accessible, interoperable, and reusable under conditions that are ethical and transparent. to highlight innovations in data-sharing and their potential to advance discovery, we present three data-sharing mechanisms. for each platform, we will present a case highlighting its key functionality and discuss opportunities and challenges that may arise as each platform is scaled up. ( .) genomic research integration system (gris) is a collaborationengendering web application that facilitates the identification of genetic variants associated with rare immunological disorders. users can access integrated and standardized phenotypic and genomic data that is analyzable within the platform. gris enables systematic and automated capturing, and links patient data from disconnected systems and paperbased records. standardized annotations allow for the comparison of data from different clinical studies. the main goal of this tool is discoverability of other affected individuals enrolled in separate protocols within the niaid intramural research program. this internal database was used to find a second family with a rare variant in a candidate gene. ( .) the genomic ascertainment cohort (tgac) is a resource that aims to improve our understanding of the phenotypic consequence of genetic variation by providing access to aggregate, de-identified genomic data from large nih intramural and related cohorts. participants have provided informed consent to be re-contacted for additional phenotyping in the future. the main goal of this tool is to enable further study of the clinical consequence of variants in a large, unbiased cohort of patients ascertained for many indications. this database was used to investigate findings in participants with previously published pathogenic variants in genes associated with primary immune deficiency based on medical record review. ( .) clingen is dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for precision medicine and research. through the sharing of genetic and health data, clingen seeks to answer whether a given gene is associated with a disease (clinical validity)?; whether a given variant is causative (pathogenicity)?; and whether the information is actionable (clinical utility)? this resource is meant to convene disease-and gene-specific expert groups to curate the medical literature on mendelian disease to better define gene-disease and variant-disease relationships using many lines of evidence. this resource was used to clarify clinical validity of disease-gene assertions. together these efforts help create a clinical research ecosystem that maximizes the value of clinical research data and ultimately improves patient care. this research was supported by the intramural research program of the nih, niaid. introduction: according to the population reference bureau, the number of elderly americans, defined as age and older, is projected to more than double from million to million by , rising from % to % of the total population. the impact of immunodeficiency in this important segment of the population remains understudied. methods: the usidnet registry was queried to obtain demographic, clinical data of elderly patients defined as age and older. descriptive analyses were performed on the data. results: participants ( . %) were eligible out of total registry participants. the median age of the cohort was years and predominantly female ( . %) and white ( . %) with a median bmi of . ± . .the majority ( . %) of subjects were living. humoral deficiencies comprised the majority of diagnoses ( . %), with common variable immune deficiency being the most frequent ( . %). of the remaining non-humoral diagnoses, immune dysregulation ( . %) and immunodeficiency with myelodysplasia ( . %) were the most frequent. the majority ( . %) of subjects reported having received immunoglobulin replacement therapy (igrt) at some point, with . % reporting via iv route. of the infections that occurred in this cohort, sinopulmonary infections were the most commonly reported, specifically sinusitis ( . %), pneumonia ( . %), upper respiratory infection ( . %), and otitis media ( . %). in this cohort, autoimmune, cardiovascular, and granulomatous complications were reported . the number of patients with malignancy was , with some patients diagnosed with multiple malignant disorders. of the reported malignancies, the majority ( . %) were solid tumors. conclusions: compared to the age-matched non-immunodeficiency united states population, this cohort had more females . % (usidnet) versus . % (us population) and fewer whites . % (usidnet) vs . % (us population. humoral immunodeficiencies, specifically cvid, were most common diagnoses, similar to other age groups of immunodeficiency patients. majority of these patients have received igrt, with approximately half via iv route. this cohort reported living with a variety of non-infectious complications, including autoimmunity and malignancies. more research which specifically focuses on elderly patients with immunodeficiency is needed. clinical microbiologist and infectious disease physician, university of calgary x-linked agammaglobulinemia (xla) is a primary immunodeficiency caused by mutations in the bruton tyrosine kinase gene which leads to b cell maturation failure and defective antibody production. this puts patients at risk of recurrent sinopulmonary infections, gastrointestinal infections, and recurrent skin infections including infections caused by helicobacter sp. helicobacter sp are gram negative bacilli commonly found in the gastrointestinal tract of various animals. helicobacter sp. have been linked with gastritis most notably helicobacter pylori causing gastric ulcers in humans. helicobacter sp. has been found in rare cases to cause disseminated infections including pyodermic gangrenosum and cellulitis notably in patients with agammaglobulinemia. infections caused by helicobacter bilis are challenging to diagnosis due to difficulties with culturing the pathogen as well as poor guidelines for antimicrobial management. case report: the patient was diagnosed with x-linked agammaglobulinemia at the age of months with a history of recurrent sinusitis and was started on ivig q weeks. despite regular ivig, he developed bronchiectasis. at years of age in , he developed a chronic rash around his left knee resembling erythema nodosum. by , he had developed a left knee effusion associated with left sided calf pain. his knee pain was found to improve during courses of ciprofloxacin to treat recurrent lung infections. given case report data of h. pylori causing erythema nodosum in patients with agammaglobulinemia, he was treated empirically for an h. pylori infections with no improvement. in he was found to have progressive cellulitis with pyomyositis of the left leg. a skin biopsy of a calf nodule was found to be culture negative but s pcr was positive for h. bilis. he was started on treatment with ertapenem and levofloxacin with subsequent resolution of his rash. his left ankle pain progressed and by late and was found to have possible osteomyelitis of the left ankle on mri. in he was found to be bacteremic with h bilis. due to progressive symptoms with significant impact on function and rising inflammatory markers despite months of antimicrobial treatment, doxycycline and flagyl were added leading to clinical improvement and normalization of his inflammatory markers. he was continued on oral doxycycline and flagyl for months for a chronic osteomyelitis. discussion: h. bilis is a slow growing pathogen which is challenging to culture in the laboratory often requiring special agar plates and prolonged incubation. in patients with agammaglobulinemia and associated chronic skin infections or erythema nodosuma, h bilis should be suspected as a possible pathogen. due to challenges with culturing, s pcr or amplification of the s ribosomal subunit should be considered to try to identify the pathogen. there are poorly delineated clinical antimicrobial breakpoints to help guide therapy with minimal evidence. case reports suggest prolonged therapy with aminoglycosides and penicillin. other studies have successfully treated patients with a carbapenem, azithromycin and levofloxacin. in the absence of sensitivity data, prolonged treatment ( months) should be considered with a combination of antimicrobials. patients should be followed closely as recurrent infections are not uncommon. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md introduction: dock deficiency is a combined immunodeficiency characterized by eczema, recurrent sinopulmonary infections, viral skin infections, malignancy and early mortality. in recent years, liver disease and vasculopathy have been increasingly recognized as a complication of dock deficiency. we clinically characterized our cohort of dock deficient patients, with a specific focus on these newly identified areas of disease involvement. methods: chart reviews were performed on patients seen at nih with genetic and clinical diagnosis of dock deficiency. patients were all enrolled on irb approved niaid protocols. results: we identified patients from families with dock deficiency in our nih cohort, ranging in age from - years. of the families, had homozygous mutations. of the patients, food allergy was diagnosed in ( %), eczema in ( %), and asthma in ( %). chronic or recurrent viral skin infections were seen in / ( %). chronic ebv viremia by pcr positivity was seen in / patients ( %); only patients were known to be ebv immune without viremia. cmv viremia was infrequent. sinopulmonary infections were common, with bronchiectasis occurring in / ( %) with available imaging. liver disease was diagnosed in ( %), with having biliary tract abnormalities on imaging and stool positive for cryptosporidia; most patients with cryptosporidia were without diarrhea. the incidence of cryptosporidia is likely under-represented due to more recent availability of sensitive assays for cryptosporidia detection. other liver abnormalities included fatty liver, metastatic disease from malignancy and medication related hepatitis. vasculopathy, predominantly of the aorta and cerebral arteries, was diagnosed in , with patients in the last years being prospectively imaged. autoimmunity was rare ( %) including autoimmune cytopenias and hypothyroidism. of with follow-up are alive ( %) with age range - years. of the living patients, ( %) have had a hsct. causes of deaths include malignancy ( ), infection ( ) , and hsct complications ( ) . long-term follow-up of patients with hsct (up to years) has revealed resolution of the infection susceptibility and eczema, no new cancers, and stabilization of vasculopathy. conclusions: in addition to the well described manifestations of dock deficiency including eczema, allergy, recurrent sinopulmonary infections, skin viral infections and malignancy, our cohort revealed a relatively high incidence of liver disease, frequently associated with stool positivity for cryptosporidia, as well as vasculopathy. both of these clinical manifestations should be considered during preparation for hsct as they may affect management through transplant. autoimmunity has likely been over-estimated in prior descriptions of dock deficiency. long-term follow-up after hsct is needed to determine the prognosis from the vasculopathy, liver disease, and malignancy risk. ( ) submission id# yasuhiro yamazaki , stefano volpi , luigi d. notarangelo introduction/background: extl (exostosin like glycosyltransferase ) is an exostosin family member which initiates heparan sulfate (hs) chain biosynthesis and elongation. we have reported homozygous extl hypomorphic mutation (r w) as a cause of immunoosseous-dysplasia syndrome. fourteen patients who have extl homozygous mutation were reported so far. eight of them manifested t cell lymphopenia, and presented with severe combined immunodeficiency (scid) or omenn syndrome. using patient-derived induced pluripotent stem cells (ipscs) as a model, we have previously reported that extl mutations affect differentiation to thymic epithelial progenitor cells as well as expansion of hematopoietic progenitor cells. consistent with the latter, previous studies have suggested that mutations in other genes involved in hs biosynthesis affect hematopoietic stem cell (hsc) differentiation. however, the exact mechanisms by which extl mutations affect hematopoiesis are not known. objectives: we tried to clarify gene expression difference in hscs derived from wild-type, extl hypomorphic and extl knock-out (ko) human ipscs. methods: the control bj ipsc line was engineered by crispr/cas gene targeting. extl ko ipscs were obtained which carried compound heterozygous extl mutations (c. _ inst; c. _ insgatattt). hsc differentiation was induced using the stemdiff hematopoietic kit (stemcell technologies). bulk rna from each ips cells and each differentiated cd +cd +cd + was analyzed by rna sequencing. results: as compared to control ipscs, patient-derived cells showed slightly lower capacity to generate cd +cd +cd + cells. on the other hand, extl ko cells showed no differentiation into cd + cd +cd + cells. gene set enrichment analysis showed enriched expression of genes involved in hematopoietic progenitor cell differentiation, regulation of hemopoiesis, and positive regulation of hemopoiesis in both control and patient-derived cd +cd +cd + cells compared to parental ipscs. moreover, these gene sets were more abundantly enriched in control than in patient-derived cd +cd +cd + cells. the gene set of response to type i interferon was significantly enriched in control versus patient-derived cd +cd +cd + cells. conclusions: these results confirm that extl plays an important role for hsc homeostasis in human cells. because type interferons play a role in hsc proliferation, the decreased type i interferon signature may account for the reduced number of hscs that we have previously reported upon in vitro differentiation of extl -mutated versus control-derived ipscs. this study was supported by the division of intramural research, niaid, nih, under protocol -i-n . a case of autoinflammatory syndrome with osteoporosis and specific antibody deficiency autoinflammatory syndromes are inherited disorders with an exaggerated inflammatory response with no specific trigger. the clinical phenotypes of variants of autoinflammatory syndromes may overlap. we report a case of a year old male with prior diagnosis of specific antibody deficiency, periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (pfapa) syndrome, arthralgia and moderate atopic dermatitis. he was diagnosed at years of age with specific antibody deficiency based on persistently low pneumococcal titers against repeat immunizations. due to recurrent infections, he was placed on immunoglobulin replacement therapy (igrt) at years of age. igrt was discontinued at years of age due to full resolution in infections and patient demonstrated robust response to immunizations. patient had lifelong history of recurrent fevers (every weeks) associated with pharyngitis and aphthous ulcers consistent with diagnosis of pfapa. as he became older these episodes became less frequent. last episode of fever was over a year ago. the father had similar symptoms of recurrent fevers and oral ulcers as a child but currently remains asymptomatic. paternal grandfather died of kidney disease. patient has been generally in good health until recent year with intermittent abdominal pain, arthralgia and several long bone fractures with no history of prior trauma. a bone density scan revealed osteopenia and osteoporosis with a z score of - . of lumbar spine, - . of left femoral neck, - . of left hip. given history of familial autoinflammatory disease, and antibody deficiency genetic testing was obtained which identified a pathogenic heterozygous variant of taci and mefv c. g>a (p.met lle). taci mutation has been linked to antibody deficiency syndromes. genetic study for family members is pending. the mefv gene is associated with autosomal recessive familial mediterranean fever (fmf) and has been reported in autosomal dominant fmf as well. fmf is characterized by recurrent episodes of fever associated with serositis, arthralgia, and arthritis. patients with fmf have elevation in acute phase reactants during attacks with most returning to normal levels during the episode-free periods. multiple studies have shown that patient with fmf have lower bone mineral density and zscores than the general population. inflammation in fmf is thought to be mediated by several different cytokines (il- , il- , il- , il- , il- , il- , il- and tnf-). these same cytokines play a role in osteoclast activity and bone resorption. it has been suggested chronic inflammation during acute attacks and subclinical inflammation during the disease-free period lead to bone loss and osteoporosis. regular use of colchicine, the main treatment for fmf, may slow down osteoporosis. beside careful monitoring of clinical and laboratory phenotype, genetic evaluation is an important step in distinguishing between overlapping entities and can prevent complication and promote targeted intervention. a year old previously healthy boy was referred for periodic fever/ pfapa and mosquito bite hypersensitivity. eight weeks earlier he developed fever to f, mouth sores and exudative tonsillitis; a rapid strep screen was negative. one week later he developed moderate cervical lymphadenopathy and had a positive ebv early antigen antibody.. one month later he had several severe local reactions to mosquito bites. each manifested - cm of erythema and induration with a + cm bullae which left an ulcer after rupture and healed with a hypopigmented scar. the bites were accompanied by fever to f for days. one febrile episode was treated with low dose prednisolone for presumed pfapa, and the fever resolved within hours. his past history was positive for nasal allergy and mild asthma. his parents are not related: mom is of european-indonesian and dad european-african (creole ancestry. testing prior to this visit showed normal igg, iga and igm, elevated ige ( , u/l) and normal cbc. lymphocyte subsets revealed cd + % ( /mcl), cd + % ( /mcl), cd + % ( / mcl), cd + % ( /mcl), nk cells % ( /mcl). on examination he appeared well with height at th%ile and weight at th%ile. there was no lymphadenopathy, hepatosplenomegaly or inflammed skin lesions; there was a cm round scar on the right plantar surface at the site of a prior mosquito bite. laboratory studies confirmed nk lymphocytosis % ( /mcl) and elevated ige ( , u/l). lymphoproliferation to mitogens, cd /cd , cmvand hsv were normal, but absent to tetanus and candida antigens. ebv antibodies reflected past infection (vca-igg+, vca-igm-, ebna+); quantitative ebv pcr was > , , copies/ml whole blood. nk cytotoxicity and cd a expression were decreased. bone marrow nk analysis suggested conality. the patient was diagnosed with "hypersensitivity to mosquito bites with ebv-associated t-/ nk lymphoproliferation." this disorder represents a subset of chronic active ebv (caebv) that is rarely seen outside of east asia. the lack of organomegaly or lymphadenopathy with hyper-ige and nk lymphocytosis and decreased nk function support the likelihood that nk cells are the target of ebv infection in this patient. this diagnosis may be a precursor to hemophagocytosis, liver necrosis or lymphoma/leukemia, and the only curative treatment is bone marrow transplantation. the patient's sister is a / hla match. she is seropositive for past ebv infection, and she has no history of extreme reactions to mosquito bites. genetic mutations that cause familial hemophagocytic lymphohistiocytosis have not been reported in caebv, and to the best of our knowledge familial cases of this disorder have not been identified. the response to bmt in this patient is pending. introduction/background: a number of case reports have described symptomatic hypogammaglobulinemia following administration of anti-epileptic drugs (aeds), specifically lamotrigine, carbamazepine, and levetiracetam. the mechanism by which symptomatic hypogammaglobulinemia develops is unclear. we evaluated the prevalence and the clinical significance of hypogammaglobulinemia associated with use of these aeds. objectives: our aim was to characterize the prevalence of aed-induced hypogammaglobulinemia, identify specific aeds associated with hypogammaglobulinemia, and characterize the timeline to development of hypogammaglobulinemia after initiation of therapy. methods: a retrospective, multicenter, electronic medical record review spanning years identified patients with hypogammaglobulinemia who were on aed therapy (lamotrigine, carbamazepine, or levetiracetam). patients were excluded if they had a pre-existing primary immunodeficiency (pid), malignancy, protein-losing enteropathy, or significant proteinuria. patients on chronic immunosuppressive therapy, those without laboratory criteria for hypogammaglobulinemia, or those on one of the aeds for less than one month were also excluded. results: of the cases reviewed, patients met our inclusion criteria. the median age was ; % were adults, % were female, and % were white. lamotrigine was implicated in / of the cases, carbamazepine in / , and levetiracetam in / . tetanus and pneumococcal titers were available for / patients. of those patients, / had protective titers to both per report with responses to > % of the serotypes. only one patient reported severe, recurrent infections while the remaining four had little to no symptoms. interestingly, the patient with severe infections did have protective titers. of the five laboratory proven hypogammaglobulinemia patients, one died of an infection, two have continued on the medication due to refractory seizures responsive only to these medications, and two are currently being tapered off of their aed. conclusion: while it appears that aed-induced hypogammaglobulinemia is quite rare, it should be considered in a patient without other secondary causes of hypogammaglobulinemia on aed therapy. many antiepileptics downregulate nfkb signaling suggestive that patients who develop symptomatic hypogammaglobulinemia may have hypomorphic mutations in the nfkb signaling pathway. ( ) submission id# autoimmune lymphoproliferative syndrome (alps) results from defective apoptosis of lymphocytes mediated through the fas/fas ligand (fasl) pathway. the hallmark lab finding is an expansion of t cells that express the alpha/beta t cell receptor, but lack both cd and cd (double negative t cells) in the setting of normal or elevated lymphocyte counts. patients present with chronic, nonmalignant, noninfectious lymphadenopathy or splenomegaly. for definitive diagnosis, patients need to have ( ) a pathogenic mutation in fas, fas ligand or caspase or ( ) a defective fas-induced lymphocyte apoptosis. we describe a probable case of alps with heterozygous mutation in fas c. a>g(p.his arg), a variant that has not been previously reported (his lymphocyte apoptosis assay is pending). unique to this case is the patients castleman disease-like features on pathology. a year-old male referred from hematology clinic presented with an year history of chronic lymphadenopathy, splenomegaly, anemia, and no underlying diagnosis. malignancy had previously been excluded by bone marrow aspirate and biopsy years prior. however, he had a right sided lymph node that had increased in size for the past months. he was otherwise asymptomatic. a lymph node biopsy years prior was reportedly normal. his exam demonstrated significant bilateral lymphadenopathy, greater on right, with an approximately x cm mobile right neck mass. he had splenomegaly palpated cm down and across to midline. he was therefore admitted for excisional lymph node biopsy to evaluate for possible malignancy and labs were sent to evaluate for alps. labs were supportive of alps. he had elevated t cell receptor alpha beta double negative t cells (tcr a/b dntcs) in blood ( . %). b level was elevated (> pg/ml). plasma soluble fasl level was elevated ( pg/ml). interleukin- (il- ) and il- levels were elevated ( and pg/ml respectively). he had multilineage cytopenias: anemia with hgb of . g/dl and neutropenia (absolute neutrophil count of k/ul). he had hypergammaglobulinemia with an igg level of mg/dl. broad infectious work-up was negative, including hiv, quantiferon, cocci, bartonella, toxoplasma, coxiella burnetii, ebv pcr and, cmv igm. lymph node biopsy showed no evidence of malignancy. immunostains and flow cytometry showed the presence of expanded tcr a/b dntcs in the lymph node, consistent with alps. interestingly, lymph node histology showed morphologic features typical of plasma cell variant castleman disease. numerous castlemanlike follicles showed typical regressive changes with onion-skinning morphology. paracortical hyperplasia with sheets of plasma cells was noted. there was negative staining for hhv (a well-known cause of plasma cell variant castleman disease). the diagnosis of idiopathic multicentric hhv -negative castleman disease was excluded by definition in the setting of alps, per evidence-based consensus criteria published in . in addition, our patient did not show any symptoms typically associated with it, such as fever, night sweats, weight loss, weakness or fatigue. should his fas-induced lymphocyte apoptosis be defective (in separate assays), this would confirm his alps-fas diagnosis and we would start the patient on sirolimus. head of immunology unit, children' s hospital ricardo gutierrez introduction: slc a gene encodes the proto-couple folate transporter (pcft), which supports intestinal folate uptake, and participates in folate transport into the central nervous system. slc a mutations cause pcft defects, resulting in low folate levels in serum and cerebrospinal fluid. hereditary folate malabsorption (hfm) is a rare, autosomal recessive disorder with pcft deficiency resulting in cerebral folate deficiency. most of the patients present megaloblastic anaemia, moderate pancytopenia in the first few months of life, failure to thrive, diarrhoea and/or later onset neurological symptoms including seizures and developmental delay. i m m u n o d e f i c i e n c y i n h f m c a n m a n i f e s t i t s e l f w i t h hypogammaglobulinemia with normal t-cell function. b-cell precursor compartment seems to be particularly vulnerable to folate deficiency in some hfm patients. this immunodeficiency can be restored with specific treatment with folic acid. aim: to describe a female patient with a homozygous pathological variation in the slc a gene. results: a months old girl, born of non-consanguineous parents. she started at months old with diarrhoea due to rotavirus, low weight and bicytopenia with normal bone marrow aspiration. she presented low levels of folic acid . ng/ml (nv . - . ng/ml) at first thought due to secondary to malnutrition. treatment with folic acid supplementation was administrated, improving platelets counts. at months old she presented steatorrhea with severe perianal panniculitis which required surgical treatment. no germs were rescued after a skin biopsy. moreover, she suffered from a respiratory infection due to picornavirus with two episodes of pneumothorax which required intensive care. at that moment ivig treatment was administered due to hypogammaglobulinemia and clinical severity. chronic diarrhoea worsened with bloody depositions. three rectal ulcers were found in the gut biopsy. bowel inflammatory disease was suspected and mesalazine administration was started with weight improvement. furthermore, at months old she presented status epilepticus, with pathological eeg and normal mri; one of them related to a cmv infection, successfully treated. in the immunological evaluation igg and iga were low with normal igm and igd. the protein-antibody response was not evaluated. she presented normal lymphocyte and t cells extended populations, t cells proliferation assay, dhr, treg cells, complement, cd a expression, alpha-fetoprotein, without autoantibodies a molecular panel testing was done by ngs and a homozygous variant in slc a gene was found, causing impaired intestinal folate absorption. conclusion: hfm should be considered in the diagnosis of patients with cytopenias and hypogammaglobulinemia in order to provide specific treatment. hfm has wide clinical manifestations, not only with megaloblastic anaemia and neurological impairment but also with gastrointestinal and skin manifestations. with folate treatment, clinical and immunological defects can be normalized. introduction: multifocal epithelial hyperplasia (meh), or hecks disease, is a rare, benign infection of the mucosa caused by human papilloma virus (hpv). clinically, meh manifests as numerous painless, soft, sessile papules or plaques, and typically occurs in the labial, lingual, and buccal mucosa. meh lesions are usually associated with hpv types and , and seen more commonly in patients of caribbean or central/south american descent. prior studies in adults have shown that tumor necrosis factor alpha (tnf) promotes hpv, and may influence duration of hpv infection. case: we present a five-year-old full term male of haitian descent referred for assessment of multiple flesh colored, papular lesions on the buccal and labial mucosa that had persisted and quantitatively increased over one year, although some lesions regressed. he had no pain or difficulty eating. medical history significant for one seizure; negative for infection. no family history of infection, immunodeficiency, consanguinity, or miscarriage. head and neck examination failed to reveal cervical lymphadenopathy, masses, or hypertrophy in the salivary glands. intraoral examination revealed multiple papular nodules, mostly flat although some were corrugated. the greatest concentration was noted on the lower left labial surface extending to the mucosal vermillion interface, not involving the vermillion or commissure region. lesions extended into the mandibular vestibule and the left buccal mucosa. no other lesions were noted on extremities, genitalia, or any other visualized mucosal surface. based on history and exam, he was diagnosed with meh. white blood cell count, neutrophils, lymphocytes, cd and cd t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus and streptococcus pneumoniae titers were protective. cytomegalovirus igg and igm were negative. epstein-barr virus igg was positive, igm and early antigen ab negative. serology was significant for elevated tnf ( pg/ml; reference range < pg/ml) while interferon gamma and interleukins , , , , , , , , , and were normal, as was il- receptor cd . one month after the initial visit, lesions were stable and unchanged. nine-valent hpv vaccination was considered, but not administered. conclusions: meh is a rare but benign disease caused by hpv. awareness of the disease and its course is important to prevent unnecessary expanded immunodeficiency work-up and possible procedures to eliminate lesions. although mucosal immunity can be site specific, especially with hpv, our understanding of t-cell cytokine and chemokine responses to hpv in cervical and laryngeal lesions may be instructive. the mechanism which allows hpv persistence in meh is not characterized, but it likely is due to increased viral persistence and an inability for the host immune response to successfully induce viral latency and successful containment. elevated tnf levels, with normal levels of il- , il- , il- , il- , may correlate with decreased clearance of hpv and prolonged duration of meh. it remains unclear if viral persistence is the cause of, or the sequela of, increased tnf. longitudinal monitoring of cytokine (tnf, il- , il- , il- , il- ) and chemokine (ccl , ccl , ccl , ccl , ccl , and ccl ) serum concentrations may be useful biomarkers for disease resolution. introduction: autosomal dominant hyper ige (jobs) syndrome is a rare primary immunodeficiency characterized by eczema and sinopulmonary infections as well as musculoskeletal and vascular complications. as in all chronic illnesses, patient education is an ongoing need. in the rare disease population, patient education is especially important as patients must be able to explain their unique healthcare concerns in a variety of medical settings. we focused on ad-hies, due to our relatively large cohort of patients, the frequent lack of classic signs of illness often impairing diagnosis of severe infection, and the diverse nonimmunologic clinical features of this disease. objectives: we aimed to increase understanding of the clinical manifestations of ad-hies to promote earlier recognition of symptoms and to increase self-efficacy for symptom management in the adult hies population. methods: adult patients were asked to participate in a patient education project. demographic information was collected from participants. they also completed a -item multiple choice test about symptom recognition in ad-hies and promis self-efficacy for managing symptoms, an item validated survey. then, patient education handouts that focused on pulmonary symptoms, eczema, bone health, and cardiovascular complications were reviewed with the participant. six weeks later, participants were asked to repeat the -item test and the self-efficacy survey. the demographic information, test, and self-efficacy were collected anonymously. results: participants provided demographic information, completed the test and the self-efficacy survey. of the participants, were male and were female. participants ranged in age from to years. / ( %) reported looking for information about ad-hies using search engines and most patients ( %) report that they have been given information about ad-hies from a doctor. / ( %) participants identified pulmonary symptoms as the symptom that concerns them most and / ( %) participants identified more than one symptom of concern. participants returned the second test and second survey. the mean test score increased from . to . with / participants achieving a score of / or higher. the self-efficacy scores were unchanged with a mean score of . before reviewing the patient education handouts and . after. conclusions: participant feedback to this project was generally positive. ad-hies patients are seeking information and an educational intervention can improve their understanding of disease. self-efficacy results were mixed and unchanged overall, but suggest that ad-hies patients manage symptoms as well as other patients with chronic illnesses. patient education should continue at each encounter. this project can be expanded to include more topics, pediatric patients, and other rare disease populations. funded by the nci contract no. introduction: bcl b plays an important role in the development and maintenance of the immune system and the central nervous system. expression of bcl b represses nk and myeloid factors while inducing t cell lineage genes in thymocytes at the dn stage. conditional loss of bcl b expression in murine thymocytes leads to t cell deficiency while complete knockout of bcl b was fatal within a few days of birth. recently, specific heterozygous bcl b mutations have been reported in individuals with global development delay. however, only of these cases, both carrying heterozygous missense variants, had low trec values with other cases having frequent infections. little is known regarding the impact of bcl b on human nk and t cell function. methods: we identified a novel heterozygous truncating mutation in bcl b in an infant who was first detected by trec newborn screening. she subsequently developed severe autoimmune hemolytic anemia at the age of months. we used standard immunoblotting and flow cytometry methods to assess protein expression and the impact of this bcl b mutant on t cell and nk cell development and function. results: the patient has a novel single base-pair deletion in the bcl b gene, which is predicted to produce a truncated protein with the loss of of zinc finger domains in bcl b. immunoblotting of t cell blast lysates revealed a reduced bcl b expression in the patient consistent with the heterozygous defect in bcl b but also generated a novel band with a smaller molecular weight that we postulate represents the truncated protein product. while mitogen responses to cona and pha were normal, both cd + and cd + t cell counts were decreased, especially cd + naïve and cd +cd + naïve t cells, suggesting reduced thymic output. the function of th cells was skewed with reduced il- production but increased ifn levels after pma and ionomycin stimulation. moreover, t regulatory cell counts were below normal range. nk cell counts were normal but these were mostly cd bright nk cells. of the few cd dim nk cells that presented, approximately half did not express cd , the fc receptor for adcc. perforin was only present in cd expressing nk cells. as such, anti-cd stimulation understandably led to low but not defective nk cell degranulation. function after stimulation with k cells was normal when controlled for nk cell counts. conclusion: we report a novel bcl b truncating mutation with a leaky scid phenotype that manifested with t-cell lymphopenia and autoimmunity. lowered thymic-derived naïve t and regulatory t cells, skewed th cytokine response, and incomplete nk cell development suggests that bcl b is important for the development and differentiation of multiple lymphocyte lineages. introduction: chronic diarrhea is one of the most common gastrointestinal complaints in patients with common variable immune deficiency (cvid) and can lead to life-threatening complications such as malabsorption and malnutrition. chronic diarrhea in cvid could be caused by infections, an inflammatory bowel disease-like picture, as well as malignancy. giardia lamblia is one of the most common parasites causing diarrhea in cvid (up to %), and can be refractory in these patients, leading to villous atrophy, weight loss, and failure to thrive. case report: a -year-old female with a history of cvid presents with chronic diarrhea and significant weight loss. her cvid was diagnosed by hypogammaglobulinemia (low levels of igg, igm, and iga), inadequate responses to protein and polysaccharide-based vaccines, decreased memory b cells (cd +cd + . %), and recurrent sinopulmonary infections. she was started on immune globulin replacement therapy and had significant improvement in her rate of infections. four years before her presentation to our center, she developed chronic, severe diarrhea. work up revealed giardia lamblia infection on endoscopy and colonoscopy. biopsy showed intraepithelial lymphocytes, villous blunting, and atrophic gastritis with rare plasma cells concerning for non-infectious enteropathy related to her cvid, in addition to the high burden of giardia organisms. she was initially treated with metronidazole for several weeks. however, her diarrhea did not improve, and she developed significant peripheral neuropathy leading to lower extremity weakness and limited mobility. her diarrhea persisted and was associated with approximately a -pound weight loss. repeat endoscopy and colonoscopy two years later showed persistent high burden giardiasis of the small intestine, as well as reactive lymphocytic infiltrates and atrophic gastritis. she was treated with nitazoxanide but continued to have diarrhea, and her stool continued to show trophozoites. given the significant inflammation and the lack of response to multiple antiparasitic agents, she was referred to our center for further evaluation. she was started on oral budesonide ( mg daily) and oral immune globulin ( grams weekly for weeks). with this regimen, she had significant improvement in her diarrhea with a -pound weight gain. repeat colonoscopy showed considerable improvement in inflammation and resolution of her giardia infection, though her stool antigen continues to be positive. conclusions: persistent diarrhea in our patient is most likely due to a combination of cvid enteropathy and giardiasis. a prolonged course of metronidazole and later nitazoxanide did not control her diarrhea and led to significant side effects. switching to an immunomodulatory approach significantly decreased the inflammation in her bowel and may even have helped to reduce the burden of giardia in the gut. targeting both underlying bowel inflammation as well as active infection in cvid patients with chronic diarrhea might be needed to control symptoms. introduction: sphingosine- -phosphate (s p) is a lipid chemoattractant that is critical for lymphocyte egress from lymphoid organs. following a s p concentration gradient maintained by s p lyase ubiquitously expressed in tissues, lymphocytes within lymphoid organs are drawn to efferent lymph and blood unless their s p receptor is internalized or downregulated. owing to diminished degradation of not only s p, but also other sphingoid bases, deleterious mutations in sgpl (encoding s p lyase) perturb sphingolipid catabolism in numerous tissues. correspondingly, human s p lyase deficiency results in multiorgan dysfunction including kidney, skin, endocrine gland, and neurologic impairment alongside expected lymphopenia. although severe t cell lymphopenia (< cells/microliter) rivaling that of severe combined immunodeficiency (scid) can be seen in patients with s p lyase deficiency, no such patients have been identified by newborn screening of t cell receptor excision circle (trec) counts, which are a surrogate measure of effective t cell production. herein, we describe an infant boy with an undetectable trec count at birth who was found to have two novel, biallelic sgpl mutations resulting in s p lyase deficiency. case description: a -day-old boy with a preceding history of fetal hydrops is born at a gestational age of weeks and presents with renal failure, anasarca, and respiratory failure. trec analysis of a dried blood spot obtained at hours of life reveals zero copies/microliter. subsequent peripheral blood studies show profound lymphopenia, with diminished cd + t ( /microliter; cd +, cd +), cd + b ( /microliter), and cd / + natural killer ( /microliter) cell counts. recent thymic emigrants are reduced ( . % of cd + t cells are cd ra+cd +), as is the ratio of naïve-to-memory cd + t cells ( % cd ra+, % cd ro+). expedited whole genome sequencing identifies two novel variants in sgpl a paternally inherited splice site variant (c. + t>c) predicted to impact a canonical splice donor site, and a maternally inherited missense change (c. g>a; p.cys tyr) located in a well-established functional domain of s p. in addition to nephrotic syndrome and lymphopenia, the patient displays evidence of adrenal insufficiency and has increased plasma levels of sphingoid bases and ceramides. before further analyses could be pursued, the infant dies at days of age due to ongoing complications of renal failure and eventual cardiorespiratory failure. summary: we report the first case of s p lyase deficiency identified by newborn trec screening for scid. as sgpl is not included in most commercially-available, scid-tailored gene panels, s p lyase deficiency would be missed by conventional genetic testing. therefore, analysis for variants in sgpl should be considered in neonates with low-to-undetectable trec counts, nephrotic syndrome, and other suggestive sequelae. w a r t s , hypogammaglobulinemia, recurrent infections, and myelokathexis) is a rare autosomal dominant primary immunodeficiency. it is caused by a defect in the gene encoding the chemokine receptor cxcr . this receptor, along with the associated ligand cxcl , regulates leukocyte migration. we present the case of a -year-old female, who presented after she self-identified the signature signs of whim syndrome in herself and multiple family members. objectives: we present the case of a -year-old female who presented with a history of recurrent warts, leukopenia of unknown cause, and recurrent infections as a child. as a child, she experienced multiple ear and sinus infections, along recurrent warts on her upper and lower extremities that have persisted to this day. furthermore, during a routine examination when she was -years-old, she had a complete blood count drawn significant for leukopenia. no further workup was undertaken at that time. when continued leukopenia was noted at the age of , referral to a hematologist and a bone marrow biopsy was completed. bone marrow was significant for myelokathexis with borderline hypercellular marrow for patient age ( % cellularity), and normal cell line quantity. a trial of neupoegen was undertaken, without significant improvement. her family history is significant for father and brother with both leukopenia and recurrent warts. results: genetic analysis showed a heterozygous pathogenic variant in the cxcr gene, c. _ dup (p.ser phe fs* ). recent complete blood count was significant for a total wbc count of . k/ul, with a differential consisting of % neutrophils and % lymphocytes. lymphocyte subsets were significant for quantitatively low cd +, cd + and cd + subsets, with normal numbers of cd + and nk cells. immunoglobulin levels revealed an igg of mg/dl, iga of mg/ dl, and igm of mg/dl; igg anti-diphtheria and tetanus titers were protective, however, none of the s. pneumoniae serotype titers were > . ug/ml. mitogen (pha, cona and pwm) and antigen (candida and tetanus) stimulation of lymphocytes were normal for all stimuli. conclusions: we present the case of a -year-old female with a history of recurrent infections, warts, and myelokathexis. on genetic analysis, she is noted to have a pathogenic mutation of the cxcr gene. the substitution of a phenylalanine for a serine decreases one of the seven serine phosphorylation sites in the carboxy tail of the molecule that occurs upon binding to its ligand, cxcl (sdf ). additionally, the variation generates a premature stop condon terminating the remainder of the carboxy terminal amino acids including ser - , known to have a role in carboxy terminial beta-arrestin binding. failure to generate adequate beta-arrestin binding sites leads to prolonged cxcr cxcl interaction resulting in myelokathexis. background: lacking protective antibodies, patients with primary antibody deficiencies (pad) suffer from frequent respiratory infections leading to chronic pulmonary damage. macrolides prophylaxis has been proven effective to successfully manage chronic lung diseases as cystic fibrosis, bronchiectasis, copd. we conducted a trial to evaluate the efficacy and safety of orally low-dose azithromycin prophylaxis when added to the usual care in pad patients. methods: a -year, phase ii, prospective, multicenter, randomized, double-blind, placebo-controlled trial on pad patients (age - years) with chronic infection-related pulmonary disease. patients received azithromycin mg or placebo once daily three-times a week for months. the primary outcome was the decrease of annual episodes of respiratory exacerbations. secondary endpoints included: time to the first exacerbation, number of hospitalizations, additional doses of antibiotics, health related quality of life measures, and safety. results: forty-four patients received azithromycin and patients received placebo. the mean number of exacerbations was · per patientyear ( %ci · - · ) in the azithromycin arm, and · ( %ci · - · ) in the placebo arm (p= · ). in the azithromycin group the hr for having an acute exacerbation was · ( %ci , - · , p= , ) and the hr for hospitalization was . ( %ci , - · ) (p= · ). the rate of additional antibiotic treatment per patient-year was · ( %ci · - · ) in the intervention and · ( %ci · - · ) in placebo groups (p= · ). improvement in hrqofl was observed in intervention group. azithromycins safety prole was comparable with placebo. conclusion: in pad with respiratory exacerbation, azithromycin prophylaxis led to reduction of exacerbation episodes, of additional courses of antibiotics, and of risk of hospitalization. given the deleterious effects of respiratory diseases adding azithromycin to pad treatment should be considered as a valuable option. background: the autosomal-dominant hyper-ige syndrome (hies), is a primary immunodeficiency caused by mutations in signal transducer and activator of transcription (stat ) that leads to defective th immunity. adverse reactions following -valent pneumococcal polysaccharide vaccine (ppsv ) have been reported in % of stat -hies patients, including severe local reactions that appear to be specific to this vaccine. case report: we present the case of a six-year-old girl, second child of nonconsanguineous healthy parents, that developed an extensive inflammatory skin reaction at the vaccination site following a single dose of ppsv . the vaccine was prescribed due to history of recurrent respiratory tract infections and an incomplete vaccine calendar with no previously administered pneumococcal vaccines. the reaction began after hours with local erythema and edema at vaccination site, expanding in hours to a phlyctenular lesion with no well-defined borders. within the first weeks, it progressively evolved to a deep necrotic lesion that required surgical debridement. the subsequent skin defect required surgical repair with a split-thickness skin graft from her right thigh as the donor site. the complete wound healing process took about months, leaving a large scar ( figure) . the patient had a longstanding history of recurrent infections with multiple hospitalizations including severe neonatal pneumonia that required respiratory support, a colon perforation with secondary peritonitis and septic shock that required a hemicolectomy at months of age, recurrent oral candidiasis, recurrent pneumonias of different lobes, recurrent acute otitis media, a cervical phlegmon, three episodes of dental abscess and multiple kidney abscesses due to gram-negative bacteria treated with intravenous antibiotics and surgical drainage. family history is notable for an older sibling that died due to sudden infant death syndrome. the patients mother has large and wide nose suggestive of stat -hies phenotype, but no history of infections. immunological work up showed mild eosinophilia ( cells/ mm ), elevated ige ( mg/dl), normal igg, iga, igm and lymphocyte subsets (cd , cd , cd , cd , cd ). peripheral th cells were markedly decreased ( . % vs. . % of normal control). specific pneumococcal antibodies evaluated month after psv revealed / serotypes in protective levels. high resolution thorax ct showed multilobar bronchiectasis. echocardiogram and total spine x-rays were normal. stat -hies was suspected with a national institutes of health score of . a novel heterozygous missense variant in stat affecting the src homology (sh ) domain (p.lys glu) was found by next-generation panel sequencing. a variant in the same position (p.lys met) has been previously reported in a hies patient (clinvar). currently, she is on monthly ivig and prophylactic antibiotics (cotrimoxazole, azithromycin and fluconazole). conclusions: the case presented raises awareness on the risk of severe local adverse reactions to ppsv in stat -hies patients. the etiology of such reactions is unclear and warrants further study. the benefits and risks of immunizing stat -hies patients with ppsv should be weighed carefully by medical providers. abstract (max words) introduction: dock deficiency is a rare primary immunodeficiency characterized by susceptibility to viral infections, atopic eczema, defective t-cell activation and th differentiation, impaired eosinophil homeostasis and dysregulation of ige. to date, there are no reported cases from malaysia. objective: we aimed to describe the clinical, immunological profile and mutational analysis of three siblings of consanguineous parents, presented with hyper-ige and lymphopenia between the years and , which were solved by mutational analysis of the second and third siblings. methods: clinical data and investigation results were collated from the medical record. scoring of the symptoms and physical examination findings using nih score was performed. t, b, nk lymphocyte subsets and serum igg, iga, igm, total ige quantification, lymphocyte proliferation test and pneumococcal specific antibody response were performed. mutational analyses were performed in freiburg, germany. result: three siblings presented at different time points over a -year span with raised ige levels, recurrent infections, eczema, hypereosinophilia and bronchiectasis. the nih scores for hyper-ige syndrome (hies) ranged from . we also documented two serious infections in the siblings, which were disseminated cryptococcus neoformans and salmonella sp. immunological results showed t-cell lymphopenia, defective t-cell proliferation, decreased igm, raised ige, hyper-eosinophilia and defective pneumococcal antibody responses present but not in all siblings. we identified a large deletion in dock starting from exon - in of the siblings from mutational analysis performed. we will proceed with next generation sequencing and dock protein assay in malaysia to further characterize the defect. conclusion: our on-going study is the first description of dock in a family from malaysia. the diagnosis of dock should be suspected in cases with raised ige levels, recurrent infections and lymphopenia, despite no warts infection in the history. this study emphasized the importance of international research collaboration and networking in solving complicated cases. the index patient presented at the age of years with increased susceptibility to lower airway and gastrointestinal infections (hospital admissions x/year until puberty). she suffered from mumps and varicella disease despite immunization, as well as from recurrent local, partially destructive hsv infections. she was diagnosed with common variable immunodeficiency (cvid) at age and started on immunoglobulin replacement therapy. following a hypoglycemic seizure at age , the patient was diagnosed with isolated acth insufficiency with secondary adrenal insufficiency requiring hormone substitution. during and following her first pregnancy at age , she suffered from recurrent bronchopneumonias including pneumocystis jirovecii infection, resulting in bronchiectases documented on chest ct at age . currently, chronic lung disease is severely limiting her quality of life (table ) . her daughter was noticed to be hypogammaglobulinemic soon after birth and failed to develop antibody responses to inactivated vaccines. she was started on immunoglobulin replacement therapy. she has not suffered from severe lower airway infections, but developed alopecia totalis at age and nail dystrophy. w h o l e e x o m e s e q u e n c i n g r e v e a l e d a h e t e r o z y g o u s c. _ insacccgag (p.lys profster , nm_ ) mutation in exon of nfkb in both mother and daughter. this monoallelic loss-of function frameshift mutation was not found in gnomad, gvs washington or clinvar databases. as previously published, a monoallelic mutation in this c-terminal domain leads to impaired phosphorylation and subsequent reduced nuclear translocation of the nfkb /p active form. pediatricians and internal specialists need to be aware of the combination of hypogammaglobulinemia, acth deficiency, immune dysregulation and ectodermal dysplasia which is unusual for cvid -possibly indicating nfkb deficiency. this clinical syndrome may overlap with symptoms and signs found in both apeced/ aire (ar) and eda-id/nfkbia (ad) deficiencies. besides ig and hormone replacement therapy, curative treatment with hematopoietic stem cell transplantation is a therapeutic option for patients with nfkb deficiency, although the experience is limited. table introduction: the modes of immunoglobulin (ig) administration for primary immunodeficiency diseases (pidd) differ in pharmacokinetics, infusion parameters, and tolerability. during consecutive clinical studies, a cohort of patients with pidd experienced all modes of administration with the same ig % product in sequence from intravenous (iv) to subcutaneous (sc), then to hyaluronidase-facilitated sc (ighy), providing a unique opportunity to assess each administration modality within the same patient cohort treated and observed at the same sites. here we report the rates of infections stratified by igg trough levels, and the rates of adverse events (aes) with the modes of ig administration (ivig, scig, ighy) within this patient cohort. design and methods: this analysis included patients with pidd aged years who participated in clinical studies: in study (nct ) patients received ivig % every weeks followed by weekly scig %; in study (nct ), patients were treated with ighy every weeks; in study (nct ; extension of study ), patients continued with the same ighy dose. to assess a potential association between the administration route at comparable igg trough levels and the infection rate, igg trough levels were categorized as < mg/ dl, < mg/dl, < mg/dl, < mg/dl, < mg/dl and mg/dl. periods where patients had trough levels within these strata were assessed, and the infection frequency was calculated. the time periods for this analysis were months for ivig and months each for ighy and scig % ( . years) treatments. in order to account for differences in the frequency of administration, rates of systemic and local aes were assessed as aes/patient-year for each mode of therapy. results: for igg trough levels of < mg/dl, the associated annual infection rates were lower or similar for ighy than scig ( the treatment involves the control of infections and immune dysregulation with chemotherapeutic regimens followed by definitive treatment with hematopoietic stem cell transplant (hsct). aim: to describe a female patient with a pathogenic variation in stx with normal cd a expression. results: she was a years old female, the th daughter of nonconsanguineous parents, without relevant personal or family records. she was admitted due to a prolonged febrile syndrome, lymphoproliferation, pancytopenia and hepatitis, with hhv rescued in bone marrow and blood. gancyclovir treatment started with good response. she was admitted one month later with similar clinical symptoms with relapsed hhv infection. furthermore, hemophagocytosis was found in the bone marrow and evaluation of nk cell cytotoxicity demonstrated slightly reduced cytotoxic activity. functional studies for primary fhl were performed: perforin expression and cd a surface expression were normal. she fulfilled criteria of fhl, and treatment with gancyclovir and steroids was administered. despite this treatment, she persisted with activated macrophagic parameters, and started with hlh treatment protocol. she improved the clinical symptoms and laboratory parameters, but persisted with hhv low viremia. three months later, when immunosupression was decreased, she was readmitted with similar clinical manifestations and added neurological symptoms (facial paralysis, abnormal movements and sleep tendency). cerebral spinal fluid was pathological with hhv positive rescue. immunosupresive treatment was adjusted, but hhv copies in blood increased markedly. foscarnet treatment was administered and immunosupression was suspended for days in order to control viral infection. unfortunately the patient died days later. although specific functional tests were normal, sequencing of stx gene by ngs revealed a homozygous variation in c. _ deltgcc, which is a previously reported mutation responsible for fhl. conclusion: despite the fact that cd a was normal, the strong clinical and laboratory results must keep the fhl diagnosis in mind and intensive treatment should be early administered; in order to give the patient the opportunity to achieve the curative treatment. objectives: to report and characterize the clinical course of a patient with apeced and specific antibody deficiency. methods: retrospective chart review was performed. the patient was enrolled in niaid irb-approved protocol -i- . results: the patient is a year-old-girl with apeced caused by homozygous aire c. _ del , who manifested cmc, hypoparathyroidism, adrenal insufficiency, sjogrens-like syndrome, autoimmune hepatitis, intestinal dysfunction and autoimmune pneumonitis. she suffered from recurrent sinusitis and severe pneumonias requiring hospitalization and administration of intravenous antibiotics several times per year. at age , she presented to our institution with fever and cough, a computed tomography (ct) of the chest revealed bilateral pulmonary infiltrates and bronchiectasis. bronchoscopy showed mucopurulent secretions in the bilateral lower lobes with culture of the bronchoalveolar lavage fluid growing streptococcus pneumoniae. further evaluation for an underlying disorder such as primary ciliary dyskinesia and cystic fibrosis including exome sequencing and sweat chloride testing was unrevealing. quantitative immunoglobulins were normal. despite prior vaccination, specific antibody testing showed negative rubeola igg and protective levels (> . mcg/ml) to only of pneumococcal serotypes. lymphocyte enumeration showed normal b cell subsets. as approximately % of apeced patients may experience asplenia, splenic ultrasound was performed confirming the presence of a cm spleen and peripheral blood smear did not reveal howell-jolly bodies. serotyping of the s. pneumoniae isolate confirmed serotype f, which is part of the -valent vaccine. follow up vaccine challenge with the valent pneumococcal polysaccharide vaccine showed an inadequate response. hence, she was started on monthly immunoglobulin replacement and over the following years she has experienced a single methicillin sensitive staphylococcus aureus pneumonia. she has missed very few school days and other parameters including linear growth have improved, she is now along the fifth percentile for height and along the tenth percentile for weight. although she continues to experience intermittent cough she remains active participating in sports without limitation. conclusions: we report the evaluation, treatment and outcome of a patient with apeced complicated by autoimmune pneumonitis and specific antibody deficiency. as infectious susceptibility of apeced classically pertains to the signature infectious disease, cmc, patients with invasive or recurrent infections should be evaluated for underlying immune deficiency. investigation should include assessment for asplenia, quantitative immunoglobulins and specific antibodies with response to antigens. in patients with predominate respiratory symptoms, autoimmune pneumonitis should be evaluated given the near % prevalence of pneumonitis observed in american apeced patients. acknowledgements: supported by dir/niaid/nih introduction: autoinflammatory diseases are genetically heterogeneous disorders of innate immunity characterized by recurrent fever, rash, and/ or serositis, which generally are considered distinct from autoimmune diseases. we report a case of a patient with lupus-like disease and a mutation of nucleotide-binding oligomerization domain-containing protein (nod r w, yao syndrome) suggestive of an overlap between autoinflammatory and autoimmunity processes. case presentation: a -year-old man was evaluated for recurrent pleural effusions, morning stiffness, erythematous rashes, and fever up to °c. history was notable for hashimotos thyroiditis and multiple admissions for presumed pneumonia with recurrent bilateral lung infiltrates and pleural effusions. transbronchial biopsy showed nonspecific pneumonitis and organizing pneumonia. antinuclear and anti-dsdna antibodies were positive. he received prednisone for presumed lupus pneumonitis leading to improvement. prednisone was tapered and hydroxychloroquine was started, but his fevers, pleuritic pain and pleural effusion reoccurred. genetic testing revealed a nod sequent variant (r w) associated with autoinflammatory disease. hydroxychloroquine was stopped and colchicine was added to his regimen, allowing prednisone to be tapered without recurrence of symptoms. further immunological testing revealed increased signaling through the type i interferon receptor (interferon signature). conclusion: although this patient had several clinical (serositis, arthralgia) and immunological (antinuclear and anti-dsdna antibodies, interferon signature) manifestations of lupus, his clinical presentation also was consistent with yao syndrome. in retrospect, he had been having recurrent inflammatory symptoms for many years. recent studies in both mice and humans suggest that inflammasome activation and il- production are involved in the pathogenesis of lupus. this case provides further support for the idea that lupus and hashimotos thyroiditis, prototypical autoimmune diseases, may have overlapping autoinflammatory features. background: the implementation of severe combined immunodeficiency (scid) newborn screening by trec assay has played a pivotal role in identifying these patients early in life. the screen has also led to the identification of infants with other immunologic abnormalities, of which the clinical implications have been unclear and there are limited data on their outcomes. objective: to review immunologic and genetic outcomes of infants referred to an immunology service of a tertiary care center with abnormal newborn scid screens. methods: we retrospectively reviewed charts of infants with positive scid screen from july to november . we excluded patients who had positive screen at < weeks corrected gestational age. we classified outcomes into groups including scid, non-scid t-cell lymphopenia (nscid-tcl) and normal t-cell count. idiopathic t-cell lymphopenia was defined as nscid-tcl (cd + < , cells/mcl) with negative chromosome microarray and negative whole exome sequencing/or genetic panel (either genedx® scid panel or invitae® primary immunodeficiency panel). results: of infants, % were male, % were caucasian, and % were african-american. fifty-four % and % of infants were identified by illinois and missouri screens, respectively. the mean age at initial evaluation was days ( - days). % of infants had a normal tcell count (n= ) or normal repeat newborn screen (n= ), % had nscid-tcl, including mild (cd + , - , cells/mcl, n= ) and moderate (cd + - , cells/mcl, n= ) tcl, and % had scid (n= ), leaky scid (n= ) or complete digeorge (n= ). genetic etiologies of nscid-tcl included q deletion (n= ), trisomy (n= ), and mutations of tbx (n= ), foxn (n= ), and cd e (n= ). three of these infants had novel variants at the time of diagnosis. secondary causes of tcl were identified in infant (thoracic infantile fibrosarcoma). one infant had idiopathic tcl. eighteen infants with nscid-tcl were followed clinically without complete genetic testing performed. for scid, mutations were found in jak (n= ), ada (n= ), il rg (n= ), and rag (n= ). the patient with leaky scid had negative whole exome sequencing. all patients with scid and leaky scid underwent hematopoietic stem cell transplantation at a median age of weeks ( weeks - months), with successful engraftment in all but patient. of idiopathic and nscid-tcl cases followed clinically, had at least one follow-up visit at median age months ( . months . years) and the majority had improved or stable lymphocyte count without serious infections requiring intravenous antibiotics, though had a hospitalization for rsv infection. the mysm patient died after cord blood transplant from unclear etiology. our study had limitations. half of infants with nscid-tcl did not have a complete genetic workup, and only a fifth of patients with nscid-tcl were inpatients, potentially explaining the relatively low number of infants with secondary lymphopenia. conclusions: in our cohort, one-fourth of infants with abnormal scid screen had nscid-tcl. although the majority of nscid-tcl did well, approximately one-third of them had underlying genetic abnormalities associated with their t-cell lymphopenia. ( ) submission id# introduction: accumulation of intracellular adenosine and deoxyadenosine nucleotides (daxp) due to adenosine deaminase deficiency results in profound lymphopenia and severe combined immunodeficiency. left untreated this form of scid is uniformly fatal. while allogeneic hematopoietic cell transplant (hct) and autologous gene corrected stem cell therapy (gt) are potential cures for ada-scid , initiating enzyme replacement therapy (ert) immediately upon diagnosis regardless of definitive treatment is standard of care. hct and gt are not therapeutic options for all ada-scid patients and ert offers immediate therapeutic intervention for these patients leading to partial immune reconstitution, and durable survival in most patients treated. adagen (pegademase), approved by the fda in in the usa, is a pegylated bovine ada (nada) with the enzyme harvested from bovine intestines. this unsustainable production process led to the development of a recombinant enzyme source based on the bovine protein sequence and an improved pegylated linker by using succinimidyl carbamate (revcovitm-(elapegademase-lvlr). methods: a phase ii/iii clinical trial was performed at us sites under institutional irb approval. eligible ada-scid subjects were stable on adagen and without complicating underlying conditions. demographics, medical history, lymphocyte counts, immunoglobulin levels, trough plasma ada activity and rbc daxp measurements were collected. patients were treated with adagen as a single, weekly im dose adjusted to achieve a trough plasma ada activity of > mmol/hr/l and rbc daxp < . mmol/l (protocol target levels). once patients had achieved this level ( - weeks), a seven-day pk on adagen was done and the patients were transitioned to revcovi based on the formula for enzyme equivalent activity of mg revcovi = units adagen. after weeks on revcovi, trough ada and daxp were assessed and a seven-day pharmacokinetic study was conducted at week . patients were assessed periodically for clinical and laboratory values and evaluation of the study endpoints was done at week . subjects subsequently continued on revcovi and were assessed periodically. results: six patients, ages - entered the trial with initial adagen dosing at . - . u/kg/wk (see table ). adagen dosing was adjusted to target endpoints of ada trough activity (> mmol/hr/l) and rbc daxp (< . mml/l). patients transitioned to weekly revcovi using the aforementioned conversion formula at doses of . - . mg/kg/wk. the spectrum of clinical manifestations range from infections to autoimmunity and inflammation among patients with hypomorphic recombination gene and (rag / ) pathogenic variants. auto-antibodies targeting cytokines ifn-alpha, ifn-omega and il- were reported in a large proportion of these patients and their occurrence often coincides with viral infections. we report the time of emergence and relative frequency of anti-cytokine antibodies in children and adults, and their persistence among patients with hypomorphic rag deficiency. antibodies were measured from plasma samples of patients by enzyme linked immunoassay (elisa). our rag cohort includes patients with rag (n= , %) and rag deficiency (n= , %). antibodies targeting ifn-alpha ( %) were most common followed by il- and ifn-omega ( % each). two asymptomatic patients who were detected by newborn screening for scid and received hematopoietic stem cell transplantation had no detectable anti-cytokine antibodies. in the cohort of young children (ages mo- years, n= ), all patients had detectable antibodies to ifn-alpha, prior history of severe viral infection and subsequently developed autoimmune cytopenias. other anti-cytokine antibodies were less common (ifn-omega %, il- %). similarly, children between - yo age (n= ) also had high fraction of anti-ifn-alpha antibodies ( %) with prior history of infections ( %) and continued to have other anticytokine antibodies less commonly (ifn-omega %, il- %). in the adult cohort (n= , ages - years) the frequency of anti-ifnalpha anti-cytokine antibodies were lower ( %,) and il- and ifnomega ( % each) continued to persist. three adult patients had anticytokine (ifn-alpha, ifn-omega and il- ) antibodies tested at multiple timepoints and elevated titers persisted up to years. our data demonstrates that anti-cytokine antibodies, especially those targeting ifn are frequent and emerge early in life in association with viral infections in patients with rag deficiency. a lower fraction of adult patients have detectable anti-cytokine antibodies, and maintain these over several years. anti-ifn-alpha may serve as a useful biomarker for identifying partial rag deficiency among young and adult patients with history of viral infections and autoimmune cytopenias. the role of these antibodies to cytokines is yet to be determined but a specific signature of these antibodies may help to identify an underlying immunodeficiency and initiate early definitive treatment with bone marrow transplantation. anti-cytokine antibodies appear to be a novel tool in evaluation of autoimmune diseases including rag deficiency. introduction: norovirus is one of the most common pathogens causing gastroenteritis in immunocompromised patients, often leading to chronic infection, causing villous atrophy, malabsorption, weight loss, organ failure, need for parenteral nutrition, and death. norovirus treatment in immunocompromised patients is challenging. oral immunoglobulin (poig) has been used to treat norovirus gastroenteritis with variable success. our aim in this study was to determine the outcomes of treating norovirus gastroenteritis in immunocompromised patients methods: electronic medical records were reviewed for patients with norovirus infection confirmed by rt-pcr since january . our initial cohort was focused on patients with primary immunodeficiency (pid), lung, and liver transplant. data on demographics, immunological phenotype, treatment with poig, the number of bowel movements (bm), and virus clearance were collected. descriptive statistical methods were used to describe treatment outcomes. further analysis of patients immunophenotype, immunosuppression medications, and co-morbid illnesses is underway. results: twenty-six immunocompromised patients ( norovirus infection episodes, as one patient had reinfection) were analyzed twelve females, age range months- years. twelve patients had pid diagnosis ( common variable immunodeficiency, severe combined immunodeficiency, x-linked agammaglobulinemia, wiskott-aldrich syndrome, digeorge syndrome, hyper-igm, stat gain-of-function, nemo and lymphopenia in a patient with trisomy ), patients were status-post liver transplant, and two patients were status-post lung transplant. of patients were on ig replacement therapy at the time of the norovirus infection. the average number of bm/day in all patients was . (range - ) . eight patients received poig ( - mg/kg) weekly for a duration from - weeks. three of those received additional nitazoxanide and received ribavirin. / patients in the poig group were receiving total parenteral nutrition (tpn), and / on no treatment group received tpn. the average number of bm/day in poig before treatment was . (range - ), and . (range - ) in those who did not receive any treatment. of ( %) on poig vs. of ( %) in the no treatment group cleared the virus. the average number of weeks to return to baseline bm was . (range - ) in the poig group vs. . (range days- weeks) in the no treatment group. of on poig continued to have chronic diarrhea that is still ongoing. conclusion: despite anecdotal reports suggesting successful use of poig in immunocompromised patients, our data did not show a significant decrease in stool output in patients treated with poig, compared to no treatment. however, poig led to a higher rate of virus clearance. a study with larger sample size might be warranted to identify the patients who benefit from poig in the context of norovirus infection and ensure the appropriate use of ig products, especially given the concerns for the national shortage of ig products. chief medical officer, novimmune sa primary hemophagocytic lymphohistiocytosis (phlh) is a life-threatening, immune regulatory disorder characterized by immune hyperactivation that is driven by high production of interferon (ifn)-. patients with hlh typically develop fever, splenomegaly, cytopenias and coagulopathy. until recently, there have been no fda approved treatments for hlh, and standard dexamethasone/etoposide-based treatment has not evolved significantly in + years. emapalumab-lzsg (ni- ) is a fully human, anti-ifn-monoclonal antibody that neutralizes ifn-and which was recently approved (november ) by the fda for the treatment of adult and pediatric (newborn and older) patients with phlh with refractory, recurrent, or progressive disease or intolerance with conventional hlh therapy. results of the pivotal trial supporting this approval are presented herein. methods: this open-label pivotal study (nct ) includes patients years with a diagnosis of phlh and active disease. data presented were from patients, of whom had failed conventional hlh therapy prior to study entry. the initial emapalumab-lzsg dose was mg/kg given intravenously every - days. subsequent doses could be increased up to mg/kg based on the evolution of response parameters. dexamethasone was administered concomitantly at to mg/m /day and could be tapered during the study. treatment duration was weeks, with possible shortening to a minimum of weeks, or extension up to the time of allogeneic hematopoietic stem cell transplantation (hsct). the primary efficacy endpoint was the overall response rate (orr) at end of treatment, assessed by pre-defined objective parameters, including normalization or at least % improvement from baseline of fever, splenomegaly, cytopenias, hyperferritinemia, fibrinogen, d-dimer, central nervous system (cns) abnormalities, and with no sustained worsening of scd serum levels. the primary analysis used an exact binomial test to evaluate the null hypothesis that orr be % at a one-sided . significance level. patients were eligible to enter an extension phase for follow-up after completing the main study (nct ). the data cut-off applied is july . results: patient characteristics are summarized in table and efficacy is summarized in table . disease at study entry was consistent with the broad spectrum of phlh abnormalities. over % of patients had signs and/or symptoms of cns disease. orr was significantly higher than the pre-specified null hypothesis of %, meeting the primary endpoint. the response rate based on investigators clinical judgement was . %. emapalumab-lzsg infusions were in general well tolerated, with mild to moderate infusion-related reactions reported in % of patients. the observed safety events (pre-hsct conditioning) mostly included hlh manifestations, infections or toxicities due to other administered drugs. infections caused by pathogens potentially favored by ifn-neutralization occurred in patient during emapalumab-lzsg treatment (disseminated histoplasmosis), and resolved with appropriate treatment. no off-target effects were observed. conclusions: treatment with emapalumab-lzsg was able to control hlh activity with a favorable safety and tolerability profile in a very fragile population. the majority of patients proceeded to hsct with favorable outcomes. our results indicate that emapalumab-lzsg should be considered as a new therapeutic option in phlh thanks to its targeted mode of action. results: a total of genes were differentially expressed between t cells of qds patients (n= ) and healthy controls (n= ) (log fold change range (- . , . )).when these genes were tested for pathway enrichment, the top pathways in t lymphocytes based on their p value included communication between innate and adaptive immune cells, cross talk between dendritic cells and natural killer cells, allograft rejection signaling, dendritic cell maturation, and b cell receptor signaling. the top biological processes with differential expression included immune response, inflammatory response, apoptotic process, interferon gamma mediated signaling pathway, nucleosome assembly, defense response to virus, lipopolysaccharide mediated signaling pathway, positive regulation of nf-kappa b import into nucleus, type i interferon signaling pathway, and neutrophil chemotaxis genes. we compared gene expression between qds participants with low t cell counts (n= ) and qds participants with normal t cell counts (n= ) and found genes that were differentially expressed (q< . ) (log fold change range (- . , . ) patient began experiencing recurrent high fevers and developed splenomegaly. elevated transaminases and concern for lymphoproliferative disease prompted a splenectomy and liver biopsy. both the spleen and liver biopsy were positive for ebv but were negative for malignancy. bone marrow biopsy was unrevealing. genetic testing identified a pathogenic variant in xiap/ birc ( c>t), and the patient was treated with high dose oral steroids resulting in an improvement in symptoms. subsequently, therapy with anakinra was started and steroids were tapered. during the steroid taper, he noticed a change in the vision of his left eye consistent with naion, as well as worsening of his colitis. there was loss of the inferior visual field and fundoscopic exam was significant for left optic disc swelling. oct noted superior retinal nerve fiber layer thinning. oral steroids were restarted with improvement in optic disc swelling, but without improvement or change in vision. as of his most recent exam, the patient has persistent bilateral inferior visual field defects with segmental optic nerve atrophy typical of naion. he has continued therapy with anakinra, and subsequently tapered off of prednisone; though he remains on a physiologic dose of hydrocortisone. conclusions: this case demonstrates an unreported ocular manifestation in a patient with xiap deficiency, which clinically appeared sensitive to immunomodulation. our patient is an unusual candidate for naion due to his young age, the average age of onset being the mid to late s, and lack of vascular risk factors. we hypothesize that his hyper-inflammatory condition contributed to irreversible vascular damage in the optic nerve head, resulting in naion. therefore, it may be useful to consider the involvement of systemic inflammatory and immune dysregulatory conditions when treating patients with atypical naion. additionally, naion should be considered in patients with xiap deficiency and sudden unilateral vision loss. the importance of de novo mutations in causing severe sporadic immune disease is well described, yet significance of such a variation in less severe and later onset of immune disease is poorly investigated. whole exome sequencing has been a powerful tool to resolve and explain the genetic basis of novel syndromes in immune related diseases. however, proving causation can be complicated due to low number of the affected individuals. we performed whole exome sequencing in a cohort of patients with noncongenital immune defects, along with detailed cellular biochemical phenotyping. we report and describe a novel non-congenital combined immune deficiency arising from a de novo gain-offunction mutation in ikbkb(c. g>a). this gene encodes ikk , and activates canonical nfkb signalling. cellular and biochemical studies of the proband revealed that ikk v i results in enhanced nf-kb signaling, as well as t and b cell functional defects. ikk v is a highly-conserved residue, and to prove causation, we generated a crispr/cas mouse model that carry the precise orthologous missense mutation. we show that mice and humans carrying this missense mutation exhibits remarkably similar cellular and biochemical phenotypes. dysregulation in patients. total rna isolated from cryopreserved peripheral blood mononuclear cells was reverse transcribed to generate cdna. we selected four known gata transcriptional targets, gata , gata , tal and zfpm (encoding fog ) and used droplet digital pcr to quantify transcript levels normalized to the low-expressing gene tbp . we used samples from individuals with wild-type gata (wt), known gata mutation patients (mut) and two individuals suspected of gata deficiency but without identified mutation or allelic imbalance (unk , unk ). transcript analysis revealed significantly decreased transcript levels of gata , gata and tal in mut pbmcs compared to wt. most wt samples had higher zfpm transcripts than gata mutated patients however it did not reach statistical significance. strikingly, we were able to use this analysis for two individuals suspected of gata deficiency. in the first case (unk ) a yr old female with primary lymphedema, hypogammaglobulinemia, recurrent infections and possible family history of leukemia was referred for gata testing. no mutation was identified however it was noted that she was homozygous across the gene preventing allelic evaluation. the second patient (unk ), a yr old female, had erethemya nodosa on legs, mycobacteria kansasii and cytopenias. in each of the targets analyzed, transcript levels from unk were lower than the wt samples and in a similar range as the gata mutation samples while unk had a profile consistent with the wt samples. we propose the use of gata targets as surrogate markers in cases where a mutation is not identified and allelic expression analysis is uninformative. are often under-reported and under-recognized. we sought to further understand and evaluate the prevalence, type, and association with serum immunoglobulin e (ige) for cvid patients with atopic manifestations. methods: we performed a retrospective analysis of cvid patients with atopic manifestations in the partners healthcare cvid cohort. we evaluated baseline patient characteristics, atopic diagnoses, and serum ige levels. results: in the partners cvid cohort, the average age was years old (± ) and % female. / ( . %) of patients had a diagnosis of asthma, with the majority of these diagnosed by an allergist ( %) or pulmonologist ( %). eczema/atopic dermatitis was diagnosed in / patients ( %), by either an allergist ( %) or a dermatologist ( %). allergic rhinitis was diagnosed in / ( . %) with positive skin prick testing in % of these patients. food allergy was diagnosed in patients ( . %). the median cohort serum ige was . iu/ml. the median serum ige was higher in patients with or more atopic complications compared to those with one or less atopic condition ( vs. iu/ml), which was statistically significant (p= . ). conclusions: we report higher rates of atopy than previously described in other cvid cohorts. consistent with previous reports, we find a low median cohort serum ige level in cvid patients compared to the general population. however, we identify a subset of patients with a predisposition towards atopy and higher ige levels within the broader characterization of cvid, and these patients may have a more specific molecular diagnosis that leads to elevated ige and atopic conditions. whole exome sequencing is underway to further evaluate this hypothesis. whim (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a primary immunodeficiency with autosomal dominant inheritance. in most patients, the genetic cause of the disease is a gain-offunction variant in c-x-c chemokine receptor type (cxcr ) that results in arrest of neutrophil migration from the bone marrow. most patients develop hypogammaglobulinemia and early waning of antibody response with vaccination. however, the exact origin of aberrant humoral immunity in whim syndrome patients is yet to be clarified. here we describe a -year-old iraqi female with a heterozygous cxcr p.ser ter variant, which is presented with haemophilus influenzae meningitis, history of tetralogy of fallot, early onset intermittent neutropenia, lymphopenia, recurrent bacterial and viral infections. immunologic evaluation revealed hypogammaglobulinemia, elevated igm level and a lack of protective vaccine titers after tetanus and prevnar vaccinations. a bone marrow biopsy was consistent with myelokathexis. immune phenotyping, functional studies and apoptosis assays were performed on peripheral blood cells by flow cytometry in our whim patient and controls. although we found that all lymphocyte compartments were reduced, naïve cd t helper cells and switched memory b cells were predominantly affected. spontaneous apoptosis was most pronounced in b rather than t cell compartments in whim patients. in addition, naïve b cells easily activated and died upon activation in vitro. cxcl , a ligand of cxcr , induced elevated t helper cell migration and increased actin polymerization in p.ser ter mutant cells. we conclude that intrinsic b cell abnormalities, such as increased rate of apoptosis and altered activation, might be responsible for defective antibody response in whim patients. although most individuals effectively control herpesvirus infections, some suffer from unusually severe and/or recurrent infections requiring anti-viral prophylaxis. a subset of these patients possesses defects in nk cells, innate lymphocytes which recognize and lyse herpesvirus-infected cells; however, the exact genetic etiologies are rarely diagnosed. plcg encodes a signaling protein in nk cell and b cell receptor-mediated signaling. dominant-negative or gain-of-function mutations in plcg cause cold urticaria, antibody deficiency, or autoinflammation. however, loss-of-function mutations and plcg haploinsufficiency have never been reported in human disease. we examined families with autosomal dominant nk cell immunodeficiency with mass cytometry and whole-exome sequencing to identify the cause of disease. we identified two novel heterozygous loss-of-function mutations inplcg that impaired nk cell function, including calcium flux, granule movement, and target killing. although expression of mutant plcg protein in vitro was normal, phosphorylation of both mutants was diminished. in contrast to plaid and aplaid, b cell function remained intact. plcg +/-mice, as well as targeted crispr knock-in mice, also displayed impaired nk cell function with preserved b cell function, phenocopying human plcg haploinsufficiency. we report the first known cases of plcg haploinsufficiency, a clinically and mechanistically distinct syndrome from previously reported mutations. therefore, these families represent a novel disease, highlighting a role for plcg haploinsufficiency in herpesvirus-susceptible patients and expanding the spectrum of plcg -related disease. we pursued genetic diagnosis, which identified bi-allelic frameshift mutations in the rag gene which had not been previously described: c. delg (p.v sfsx ) and c. _ del insaaaagagtg (p.v kfsx ). taken together, his presentation suggested significant immune dysfunction had evolved since transplant leading to extensive pulmonary nontuberculous mycobacterial infection and possible bronchiolitis obliterans. he therefore will undergo a subsequent unconditioned cd + stem cell boost from his sister, the original donor, once he completes mycobacterium abscessus treatment. this case highlights the potential long-term immune dysfunction which may evolve after unconditioned allogeneic stem cell transplant for scid, in which full engraftment in all myeloid and lymphoid compartments is not expected. it also highlights the importance of guideline-driven follow-up of these patients to monitor for said dysfunction, to prevent serious infection and long-term sequelae. somatic hypermutation (shm) in the b cell receptor (bcr) heavy (igh) and light chain genes promotes affinity maturation and also mutation away from self-reactivity, therefore serves as an important peripheral tolerance checkpoint. as an example, unmutated bcr ighv - genes give rise to antibodies that bind to i/i antigen on red blood cells (rbc) and may elicit cold agglutinin disease (cad), a variant of autoimmune hemolytic anemia (aiha). in case of healthy individuals, frequent shms in the i/i binding site of bcr ighv - genes decrease rbc reactivity and cad. patients with primary immunodeficiencies (pid) paradoxically develop autoimmune diseases, including autoimmune cytopenias, especially aiha. it is unclear if impaired shm of bcr, in particular mutation away from i/i binding, is relevant in the development of rbc reactivity and consequently aiha in a pid background. our studies focus on pid patients with hypomorphic recombination activating gene (rag and ), combined immunodeficiency phenotype and history of autoimmunity, in particular aiha (rag cid/ai). we detected increased frequency of unmutated ighv - bcr in memory b cell repertoires of rag-cid/ai patients as well as elevated titer of unmutated ighv - antibodies in the patients' plasma. lower level of shm likely reflect abnormal germinal center (gc) reaction. as rag and heterotetramer primarily shapes the pre-immune t and b cell repertoire, we studied the interaction of follicular helper t cells (tfh) and naive b cells via in vitro co-culture experiment. interestingly, tfh cells from rag cid/ai patients exhibited highly activated phenotype with increased expression of cd l and il- compared to healthy controls and were able to initiate exaggerated response (class switching and shm) of healthy donor naive b cells. on the contrary, in vitro activated naive b cells from rag cid/ai patients showed impaired proliferation, class switching and decreased level of shm with diminished induction of genes involved t cell co-stimulation (cd , il- r) and shm (aicda, repair enzymes) compared to healthy donor naive b cells indicating intrinsic defect in patient b cells. furthermore, b cells from rag cid/ai patients also showed increased apoptosis and accumulation of gamma-h ax foci at steady state indicating reduced cellular fitness. these findings suggest that the development of aiha is a multifactorial process in partial rag deficiency. our studies highlight that impaired germinal center reaction is an important tolerance checkpoint with the inability of patient's b cells to respond to hyperactive tfh cells and introduce proper level of shm. hence, we propose that b cell fitness is compromised which impairs proper gc interaction, shm, including mutation away from self and sustains rbc reactivity in hypomorphic rag deficiency. introduction/background: the forkhead box n (foxn ) transcription factor is an essential regulator of t cell development, affecting the differentiation and expansion of thymic epithelial cells (tecs). autosomal recessive mutations in foxn cause a t-b+nk+ lymphocyte phenotype due to a thymic aplasia in conjunction with alopecia universalis and nail plate dystrophy resulting from keratinocyte dysregulation. this is a classic nude/scid (omim # ) phenotype. we report on the identification of two independent patients, identified through newborn screening with absent trecs and with a t-nk+b+ scid phenotype who presented with a t cell lymphopenia who had compound heterozygous mutations in foxn . notably, these individuals had normal hair and nail beds. objectives: to determine whether distinct compound heterozygous mutations in foxn cause a novel t-nk+b+ phenotype in the absence of a classic nude presentation. neutralizing autoantibodies (autoabs) against cytokines increase the susceptibility for selected infections (e.g. anti-ifn-autoabs for nontuberculous mycobacteria and non-typhoid salmonella, anti-il- -autoabs for mucocutaneous candidiasis and anti-gm-csf-auotabs for infections by cryptococcus, nocardiae and aspergillus spp). however, the role of anti-il- -autoabs is less clear. il- is a key mediator of the acute-phase response and released early in bacterial infections. patients with impaired signaling or affected production of il- are at increased risk for severe bacterial infections. only three patients with high-titer and neutralizing anti-il- -autoabs who suffered from severe infections caused by s. aureus, s. intermedius and e. coli have been described so far. to investigate the prevalence of anti-il- -autoabs in patients with bacterial infections, we investigated a cohort of patients and identified three further patients, all previously healthy, with neutralizing auotabs against il- who hardly developed an acute-phase response. the first patient suffered from life-threatening pneumonia caused by s. pneumonia, the second patient developed a submandibular abscess and septic arthritis caused by s. pyogenes and the third patient suffered from life-threatening pneumonia caused by s. aureus. we also discovered neutralizing anti-il- -autoabs in two adults among a cohort of patients with autoimmune diseases (n = ), in one adolescent among a cohort of obese individuals (n = ) as well as in three mothers of neonates with impaired il- signaling. so far none of the later individuals developed a severe bacterial infection. this suggests that naturally occurring and neutralizing anti-il- -autoabs are a risk factor for severe bacterial infections yet with incomplete penetrance. ( ) submission id# persistent transaminitis in copa syndrome researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city social service intern, immunodeficiencies research unit, national institute of pediatrics pediatrics resident, pediatrics hospital, st century national medical center, mexican institute of social security researcher, data science department, mexican autonomous institute of technology researcher, department of research methodology, national institute of pediatrics background: inborn errors of immunity constitute a heterogeneous group of over individually rare congenital diseases that involve genes coding for proteins of the immune system, and which result in increased susceptibility to infection, inflammation, autoimmunity, allergy and cancer. the complexity of the diagnostic task, and the intrinsic biases and limitations of the human mind, can be aided by computational tools. among the available machine learning approaches, decision tree algorithms select the best node to split based on entropy and information gain; random forests build hundreds or thousands of decision trees randomly (bootstrapping), to improve accuracy and reduce overfitting. aim: to implement a machine learning-assisted clinical decision support system for the diagnosis of inborn errors of immunity (iei). methods: with a local database of patients with suspected iei, we built a decision tree using c . dtc, and a random forest on python (jupyter notebook, scikit, mathplotlib, pandas, numpy). the database was obtained by conducting an electronic search on medsys of patients with the term immunodeficiency in their electronic medical records, and then hand-picking cases in which an iei had been confirmed or ruled out. it consisted of patients, of which had been diagnosed with iei. we first split the dataset randomly into training ( %) and testing ( %) sets. the decision tree was tasked with classifying correctly pid or not. after running the algorithm in the training set, we evaluated in the testing set. the random forest classified all cases by majority vote into nine groups ( to ), according to the iuis pid group. next, we repeated the process on a larger scale with a dataset of , patients from usidnet. accuracy was assessed by out-of-bag (oob) error estimates. results: accuracy was greater than % for the local dataset (pid/ not, groups), and for the usidnet dataset ( groups). we provide a list of decision nodes and a diagnostic route with those questions that achieved a greater information gain and less entropy. this might help clinicians direct their interrogation and diagnostic approach of suspected iei patients. discussion: we built two classification models. decision trees lend themselves more easily to learning and deriving rules of thumb from their sequences. random forests are more robust and better suited for categoric (as opposed to binary) classification. we next want to develop a chatbot that will ask relevant questions in optimal sequence, and extract undiagnosed patients with suspected iei, based on statistical red flags. researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city dna repair defects are inborn errors of immunity that result in increased apoptosis and oncogenesis. dna ligase -deficient patients suffer from a wide range of clinical manifestations since early in life, including: microcephaly, dysmorphic facial features, growth failure, developmental delay, mental retardation; hip dysplasia, and other skeletal malformations; as well as a severe combined immunodeficiency, radiosensitivity and progressive bone marrow failure; or, they may present later in life with hematological neoplasias that respond catastrophically to chemo-and radiotherapy; or, they could be asymptomatic. we describe the clinical, laboratory and genetic features of five mexican patients with lig deficiency, together with a review of other patients available in pubmed medline. four out of five of our patients are dead from lymphoma or bone marrow failure, with severe infection and massive bleeding; the fifth patient is asymptomatic despite a persistent cd + lymphopenia. most patients reported in the literature are microcephalic females with growth failure, sinopulmonary infections, hypogammaglobulinemia, very low b-cells, and radiosensitivity; while bone marrow failure and malignancy may develop at a later age. dysmorphic facial features, congenital hip dysplasia, chronic liver disease, gradual pancytopenia, lymphoma or leukemia, thrombocytopenia and gastrointestinal bleeding have been reported as well. most mutations are compound heterozygous, and all of them are hypomorphic, with two common truncating mutations accounting for the majority of patients. stem-cell transplantation after reduced intensity conditioning regimes may be curative. department of laboratory medicine, clinical centre immunology, allergy and rheumatology division, department of pediatrics, baylor college of medicine, texas children's hospital, houston,texas, usa laboratory of clinical immunology and microbiology, fungal pathogenesis section, national institute of allergy and infectious diseases, department of intramural research, national institute of allergy and infectious diseases (niaid), national institute of health, bethesda maryland, usa card deficiency is an autosomal recessive primary immunodeficiency known to underlay increased fungal infection susceptibility mostly presenting as invasive cns candida infections (in infancy or adulthood) and dermatophyte infections. more recently, a rare card variant (c. + g>c, leading to exon skipping, card del ) showed a significant protective association towards inflammatory bowel disease (ibd) when present in heterozygosity. at the nih we studied an -year-old male patient (p ) born to a non-consanguineous marriage who presented as an infant with recurrent/severe thrush, candida esophagitis, and an episode of tinea pedis; p also has mild hypogammaglobinemia (igg mg/dl at age y). p s gdna was tested by whole exome sequencing and showed a card c. + g>c mutation in homozygous state. segregation analysis and sanger confirmation determined that both parents and p s elder brother carried the same variant in heterozygosity, while his asymptomatic younger brother (p ) was also homozygous. as previously described, this variant caused card exon deletion as determined in p and p s pbmcs by cdna sequencing and by a lower molecular weight card protein by immunoblot evaluation. p and p s pbmcs, as well as the heterozygous parents cells, showed a defective cytokine generation (tnf-, il- , il- and gm-csf) in response to heat killed candida (hkc), but not to lps. while patients pbmcs failed to induce phospho-erk and phospho-p- upon hkc-stimulation but presented an intact response to pma+ionomycin; the parents cells responded normally to both stimuli. moreover, t-cell activation and proliferation was affected in response to hkc but not to pha in both patients, whereas the parents exhibited normal results under the same conditions. when hek cells were transiently transfected with wt or card del vectors together with a trim plasmid (e -ubiquitin ligase, naturally associated to card ), we confirmed that card del failed to bind trim by immunoprecipitation. furthermore, malt , bcl and trim were only co-precipitated by wt card , but no by card del , strongly suggesting trim is an integral part of the card /bcl /malt -cbm-complex. in summary, herein we demonstrate that the card del allele fails to bind trim , and in turn is unable to conform a complete/functional cbm complex. our data also show that card del acts in a dominant negative fashion in terms of cytokine generation (previously reported), but one wt allele seems sufficient to generate normal levels of hkcinduced p-erk and p-p- , as well as t-cell proliferation. while decreased cytokine generation associated with card del in heterozygosity has been described to be sufficient to protect towards ibd, other defective pathways are affected in homozygosity and likely necessary to confer increased susceptibility to fungal infections. altogether these results suggest that card del acts through a gene dosage mechanism that can dissect pathways that associate ibd protection and fungal infection susceptibility. further work is warranted to explore card del role, if any, in b-cell and t-cell biology. professor, endocrinology, university of michigan medical school background: acquired generalized lipodystrophy (agl) syndromes are a heterogeneous group of diseases characterized by selective dysfunction and loss of adipose tissue after birth. this causes ectopic lipid deposition and deficiency of the adipokine leptin, which promotes metabolic dysfunction through impaired glucose handling resulting in insulin-resistant diabetes mellitus, dyslipidemia and steatohepatitis. while the metabolic effects of altered adipokine secretion are known, the molecular mechanism is less clear. many agl cases are suspected to have an autoimmune etiology. effector and regulatory t cells, dendritic cells and macrophages reside in normal adipose tissue. t cells within adipose tissue highly express pd- and regulatory t cells express ctla , which limits immune activation in the adipose tissue under normal circumstances. thus, inhibition of these immune checkpoints may hypothetically cause immune activation, leading to adipocyte dysfunction and autoimmune destruction. we have encountered two cases that raise clinical concern for this process. patient cases: patient is a -year-old female who presented with failure to thrive at months. she was diagnosed with insulin-resistant type diabetes and hypertriglyceridemia at ages and years with progressive subcutaneous fat loss and low leptin levels culminating in a diagnosis of agl. her childhood clinical course was complicated by hypertrophic cardiomyopathy, hepatomegaly, autoimmune hemolytic anemia with massive splenomegaly and severe chronic diarrhea secondary to autoimmune enteropathy. she presented at years with acute liver failure, thrombotic microangiopathy, nephrotic syndrome and progressive kidney insufficiency. evaluation for her multi-faceted autoimmune presentation identified a familial heterozygous pathogenic variant in the ctla gene (c. _ insgttgg,p.ala glyfster ). despite aggressive immune therapies, including ctla -ig (abatacept), her kidney disease and enteropathy have progressed. patient is a -year-old male diagnosed with localized malignant melanoma of the right neck in july . he underwent excisional biopsy and regional lymph node dissection with negative margins. he relapsed in november and underwent a modified radical neck dissection with lymph node positive for disease and received external beam radiation from january-february . additionally, he was started on anti-pd- therapy with the humanized antibody drug pembrolizumab in april but discontinued the drug in february in the setting of toxicities including hypothyroidism. subsequently, he developed up to . % weight loss with progressive loss of subcutaneous fat first in his face, then generalized to the rest of his body. in the ensuing months, imaging with pet-ct demonstrated loss of subcutaneous fat concurrent with elevations in alt and triglyceride levels plus a low leptin level consistent with agl. conclusion: these cases raise concern that inhibition of the immune checkpoints ctla and pd- may facilitate the development of agl. we hypothesize that these defects significantly increase t cell autoimmune activity in the adipose tissue and/or alter t cell metabolism resulting in agl. disorders of immune dysregulation should be considered in the etiology of agl. similarly, patients with either genetic or pharmacologic inhibition of immune checkpoints should be monitored for the development of agl with careful physical exam and periodic monitoring of glucose and triglyceride levels. background: rosai-dorfman disease (rdd; also known as sinus histiocytosis with massive lymphadenopathy) is a rare non-langerhans cell histiocytosis. it is characterized by proliferation and accumulation of activated histiocytes in affected tissues. classically, rdd presents with bilateral, non-tender, and often markedly enlarged cervical lymphadenopathy. case presentation: a -year-old female presented with a -week history of asymptomatic, persistent and bilaterally enlarged cervical lymph nodes. she was otherwise healthy with no significant past medical history. operative excision biopsy of the largest lymph node confirmed the diagnosis of rdd. three months following diagnosis, routine bloodwork revealed that she had developed lymphopenia (lymphocyte count . x /l). between -year and -and-a-half-years post-diagnosis, the patient was hospitalized and treated with intravenous antibiotics for presumed episodes of osteomyelitis and presumed episodes of lymphadenitis. given the recurrent presumed infections and persistent lymphopenia, the patient was referred to immunology for evaluation. she received a full immunologic work-up. lymphocyte immunophenotyping revealed low cd ( cells/mm ) and low cd ( cells/mm ) counts. the rest of her immunologic work-up was within normal limits. approximately -and-a-half-years post-diagnosis, the decision was made to initiate treatment for rdd. she was started on a -week tapering course of prednisone therapy. within -weeks of starting corticosteroid therapy, the lymphadenopathy had diminished, and by -weeks, the lymphopenia completely resolved. at her most recent clinic visit, she had been free of serious infections for more than -years, and her lymphocyte counts had remained stable and within normal limits for over one year. discussion: in the literature, immune system dysfunction has been reported in rdd, with both auto-antibodies and cellular immunodeficiency implicated. in this patient, the persistent lymphopenia and recurrent episodes of presumed infections appeared consistent with an immunodeficiency. given the known association of rdd with immunologic dysfunction, this was certainly a reasonable assumption; however, when these issues resolved following corticosteroid therapy, we questioned whether her clinical presentation could instead represent a manifestation of her underlying rdd. this case highlights the diagnostic challenge of differentiating between an infection and an rdd exacerbation. the episodes of presumed infections were considered probable but not confirmed with microbiologic or histopathologic specimens. the mechanism underlying lymphopenia in rdd is not clear but may involve decreased production, increased destruction, or sequestration of lymphocytes. to our knowledge, this has not been specifically studied in rdd in the past, however lymphopenia has been linked to lymphocyte maldistribution in other diseases. for example, studies have shown that experimentally altering either the surface of the lymphocyte or the environment through which the lymphocyte travels through can cause sequestration of lymphocytes in various lymphoid organs including lymph nodes. conclusion: we describe the case patient with rdd that developed persistent lymphopenia, and multiple episodes of presumed infections resulting in hospitalization and intravenous antibiotic therapy. her lymphopenia resolved and she had sustained remission of rdd following treatment with corticosteroids. we hypothesize that lymphocyte sequestration in enlarged lymph nodes may have resulted in lymphopenia. this, combined with recurrent rdd exacerbations that clinically resemble infections created a presentation that mimicked an immunodeficiency. background: there is an expanding spectrum of immunodeficiency phenotypes linked to dna repair defects, and some patients may not be diagnosed until adulthood. the most well recognized genetic defect linked to dna repair is in the gene, ataxia telangiectasia mutated (atm), which causes ataxia telangiectasia, characterized by combined immunodeficiency, neurodegeneration, radiation sensitivity, and ocular telangiectasias. however, there are several other dna repair defects associated with immunodeficiency, including some syndromic and severe combined immunodeficiency (scid) disorders. objective: we present the case of an adult patient with prolonged history of recurrent infections, facial abnormalities, and autoimmunity who was found to have radiosensitivity suggestive of a dna repair defect. methods: retrospective chart review, immunodeficiency evaluation, flow-based radiosensitivity assay, gene sequencing. results: a -year-old female was referred to our clinic due to a complex history of recurrent infections and immune dysregulation. the patient had a lifelong history of sinopulmonary infections and panhypogammaglobulinemia with low vaccine responses, leading to a diagnosis of common variable immunodeficiency (cvid), necessitating treatment with immunoglobulin replacement. clinical features were also notable for congenital dysmorphia (strabismus, thin and angular face, high arched palate, nasal septal defect, small mouth, missing dentition, clinodactyly, severe equinovarus, and scoliosis). she was subsequently diagnosed with autoimmune features of vasculitides requiring trial of cyclophosphamide, azathioprine, rituximab and belimumab, which was later discontinued due to neutropenia and worsening sinopulmonary and skin infections despite immunoglobulin replacement. in the course of our evaluation she was revealed to have severe b cell lymphopenia ( %), cd naïve t cell lymphopenia, persistent iga and igm deficiency one-year post rituximab therapy, and elevated alpha fetoprotein (afp). radiosensitivity assay revealed decreased atm phosphorylation and elevated levels of h ax -hours after low-dose ( gy) radiation in her lymphocyte subsets (t, b and nk cells) . due to the evidence of radiosensitivity and elevated afp levels, there was concern for an atm or other genetic defects in a dna repair pathway. therefore, a targeted (primary immunodeficiency genes) panel was pursued for genetic testing ( genes, invitae, san francisco). the evaluation did not identify a variant in the atm gene but rather a variant of uncertain significance was identified in the chd gene, in exon , c. g>a (p.gly arg), which may be mosaic. this variant has not been reported in population databases. chd is typically associated with charge syndrome, and while this patient has some dysmorphic features, she is not typical for charge syndrome. currently, studies on copy number variation (cnv) and deep intronic variants in atm are pending. conclusion: dna repair defects may occur in adult patients with a primary diagnosis of cvid. our patient exhibits some phenotypic features of both a chd variant, and atm leading to possible abnormal dna damage responses (ddr). the exact cause of the immune deficiency in our case remains presently unsolved. this case highlights the relevance of both functional studies and genetic evaluation of complex cases of immune dysregulation, for improving our understanding of the phenotypic variability in these immunological disorders. background: womens health issues in patients with immunodeficiency are largely underrepresented in the literature. there are no studies assessing for fertility issues in patients with antibody deficiencies, and there are few sizable studies examining pregnancy and outcomes on progeny in the same cohort. the two largest studies of pregnancy in antibody deficiency, an idf survey and a study of the czech population, provide conflicting data about the safety of pregnancy for these patients. immunoglobulin replacement has been shown to be safe and beneficial in pregnancy for patients with cvid, however, dosing strategies are unguided. we sought to further understand these and other issues associated with fertility and pregnancy in a large cohort of patients with antibody deficiencies. methods: we performed a retrospective chart review of over patients with icd and/or icd codes of cvid or another antibody deficiency from january to december . inclusion criteria also comprised of having reached at least years of age, the beginning of child bearing years. data collected included disease characteristics, comorbidities, laboratory values, and outcomes. this was followed by a phone survey to elucidate data regarding fertility, pregnancy, delivery complications, and outcomes of children. this study was irb approved. results: the current age of women included ranged from to years of age, currently being in childbearing years to being post-menopausal. forty percent of the women had been pregnant, delivering an average of babies per woman who had been pregnant. fertility issues were not a prominent factor for women who never became pregnant. a majority of women who had babies ( %) did not receive a diagnosis of antibody deficiency until after their child bearing years. recurrent upper respiratory tract infections, bacterial sinusitis, and urinary tract infections during pregnancy were common even in those not yet diagnosed with antibody deficiency. immunoglobulin levels and dosing of intravenous and/or subcutaneous replacement were recorded for a subset of patients with recent pregnancies. the data re-enforced that increases in dosing are needed in the third trimester. cord blood igg levels were also recorded for baby and were the same or higher than the mothers most recent igg prior to delivery. it was rare for children of our patients to be diagnosed with antibody deficiency or a related condition, although cvid, hypogammaglobulinemia, combined immunodeficiency, lymphoma, rheumatoid arthritis, and other diagnoses were found. conclusion: this is the largest report of outcomes before, during, and after pregnancy for patients with antibody deficiencies in the united states. this report highlights the importance of closely monitoring women during pregnancy for recurrent infections regardless of whether a diagnosis of antibody deficiency is present. it also highlights that close monitoring of igg levels during pregnancy is necessary for women with antibody deficiencies. backgrounds: autoinflammatory diseases (aids) are a group of disorders with an inborn error of innate immunity, characterized by recurrent episodes of fever and inflammatory attacks. the spectrum of aids is expanding, but no data on clinical presentation and symptom variability exist for the iranian population for timely precise diagnosis. this study aims at establishing the first autoinflammatory registry of an iranian population focusing on the clinical and laboratory features that may help clinicians toward a better understanding and diagnosis of these disorders. methods: clinical and laboratory characteristics of patients who clinically and or genetically diagnosed with aids collected. we used the updated version of classification criteria from the eurofever registry for the clinical diagnosis. results: in our retrospective study, clinical and laboratory characteristics of the participants collected. mean age of disease onset, disease course manifestation, the mean duration of episodes, atypical symptoms, laboratory and imaging studies as well as complications, and response to treatment also reviewed. data resulted in patients of whom were male. their age ranged from to years. out of were genetically diagnosed. familial mediterranean fever (fmf) was the most common clinically and genetically approved diagnosis. there were also patients suspected of nlrp and nod mutations. age at disease onset differed variably and ranged from the neonatal period to adulthood. fever was present in all the participants and the duration of episodes was - days. the frequency of attacks was between to more than per year. some of the common clinical manifestations were as follows: myalgia or fatigue ( %), arthralgia and arthritis ( %), abdominal pain ( %), aphthous stomatitis ( %), chest pain ( %), chronic gastrointestinal symptoms ( %), skin lesion ranging from urticarial rash and severe nodular acne to pyoderma gangrenosum ( %), exudative and or erythematous pharyngitis ( %), consanguineous parents ( %), symptoms of a type of allergy ( %), lymphadenopathy ( %), splenomegaly ( %), increased acute phase reactant ( %), elevated liver function test ( %) . out of of the individuals reported positive family history and in one of the cases, a patient carrying the homozygous mutation in the mefv gene has shown no clinical manifestation. conclusion: this study highlights the most common manifestations of aids in the population of iranian origin and can be used as evidencebased clinical criteria for their diagnosis. background: the term benign ethnic neutropenia (ben) is used to describe patients of african/arabic descent with absolute neutrophil counts (ancs) less than cells/ul in the absence of other causes. historically, race has been used to support the diagnosis of ben, but self-reported race is notoriously imprecise. the duffy null phenotype (fya -/fyb-) is a known molecular cause of ben and may be a more reliable marker of ben than self-reported race. in addition, although the anc is known to be lower in patients with ben, the lower limit of ancs is poorly described. it is important to differentiate patients with ben from primary immunodeficiency diseases (pidd) and to recognize their expected anc values. methods: eligible subjects included patients less than years seen at the university of michigan between january -july . duffy null (fya -/fyb-) patients were identified using electronic medical record search engine (emerse) software and search terms duffy and fyab. potential subjects were identified; patients met inclusion criteria including duffy null status and the absence of other conditions or medications, potentially impacting ancs. unique healthy anc values were recorded from the duffy null patients. age and sex matched controls were identified using emerse software with search terms tonsillectomy, department of anesthesiology and absolute neutrophil count. subjects with conditions or medications that might impact the anc or of african/arabic descent were excluded from the control group. asian and caucasian patients included as controls were presumed to be duffy null given that < % of these populations are expected to be duffy null. control subjects were identified; met inclusion and exclusion criteria. statistical analysis was performed using two-sided two-sample t-test, anova and onesample t-test. results: the median age of the duffy null cases was . years (iqr: . - . ) with . % (n= ) male and all of african or arabic descent. mean anc for duffy null patients was cells/ul (n= , sd= ) while mean anc for controls was cells/ul (n= ; sd= ) with a mean difference between controls and duffy null cases of cells/ul ( % ci: - ; p= . ). the anc levels between duffy null individuals and controls were evaluated by five age categories (p= . for all age categories). however, there was no difference in anc levels between duffy null cases at different age categories (anova, p= . ). ( . %) duffy null cbcs had anc levels in the nonneutropenic range (> cells/ul), ( . %) cbcs had mild neutropenia ( - cells/ul), ( . %) cbcs had moderate neutropenia ( - cells/ul), and ( . %) cbcs had severe neutropenia (< cells/ul). conclusions: although neutropenia can be associated with pidds and is often a sign of a compromised immune system, duffy null patients have a wide range of values that are often much lower than previously appreciated. the degree of neutropenia related to duffy null phenotype appears to persists throughout childhood and young adulthood. in the context of patients of african/arabic descent presenting with asymptomatic neutropenia, duffy null status should be assessed, and ben should be considered in the differential. complications, hypogammaglobulinemia and a unique characteristic of decreased susceptibility to enveloped viral infections. objective: to investigate the role of impaired host n-linked glycosylation on viral susceptibility to ebola virus. methods: to mimic the condition observed on cdg-iib patients, we tested in vitro three proprietary iminosugars (emergentbiosolutions©), uv b, uv , and uv , which act as competitive inhibitors of -glucosidase i and ii. their ability to inhibit the trimming of n-glycans was compared to known n-glycans modifiers as castanospermine, tunicamycin, as well as the bacterial enzyme peptide-nglycosidase f (pngase-f). ebola virus envelope protein gp was chosen as a prototype glycoprotein, as it is heavily glycosylated with nglycosylation sites. hek t cells were seeded at x ^ cells/well in well plate. after h, cells were transfected with pflag-ebolavirus gp by coupling with effectene®. after h, cells were treated with the inhibitors and harvested h after treatment. trimming of n-glycans was evaluated via molecular weight assessment by western-blot. results: all three inhibitors had comparable effectiveness in inhibiting trimming of nglycans from ebola gp glycoprotein compared to castanospermine. a greater molecular weight shift was seen with tunicamycin and pngase f as expected. conclusions: chemical inhibition of the n-linked glycosylation pathway was successfully achieved using three new mogs inhibitors. this approach merits further investigation on potential applications on antiviral therapies. investigator, laboratory of human genetics of infectious diseases, necker branch, inserm u , necker enfants malades hospital, paris, france head, immunodeficiencies research unit, national institute of pediatrics stat gof mutations are associated with infections, autoimmunity and inflammatory manifestations; the rosacea is one of the manifestations described in this disease, however, the etiology rosacea is not clearly established. the characteristics of rosacea are not described in stat gof in the different clinical series. we describe the different characteristics of rosacea in a family with affected members with stat gof. a family with eight members with stat gof mutation were diagnosed through a first affected member affected with tuberculosis and onychomycosis. seven members more had a clinical history of mycobacterial, viral and fungus infections and autoimmunity disease, in all the seven, was documented the same mutation stat gof. in six of these adults patients, we documented rosacea, it started after adolescence, it was localized in the face and/or eyes, was progressive and not ameliorated with medical treatment and caused nose deformity. rosacea has been described previously as a unique manifestation, and the etiology is not clear, an autoimmune hypothesis has been proposed. the fact that is present in patients with stat gof could suggest that have effectively an autoimmune component. physicians face the patients with rosacea must look for other manifestation presents in stat gof mutations. genetic studies in rosacea patients could evidence an new gene defect. introduction: homozygous mutations causing loss of function of the transcription factor forkhead-box n (foxn ) underlie autosomal recessive severe combined immunodeficiency with congenital alopecia and nail dystrophy (nude scid). affected humans, like the scid mouse, have small or absent thymus, absent or severely diminished t cells, alopecia, and nail dystrophy. infants with nude scid have had neonatal lymphopenia and severe, life-threatening infections. studies of heterozygous carriers of foxn mutations are limited, some having been reported with no phenotype or mild disease manifestations, such as nail dystrophy without lymphopenia or recurrent infections. objective: we describe six infants, including two brothers, with t-cell lymphopenia (tcl) following abnormal california newborn screens (nbs) for scid. each had a single heterozygous variant in foxn . case reports: six infants ( female, male) were referred for evaluation after abnormal california nbs for scid (table ) , with t-cell receptor excision circle (trec) counts from undetectable to (normal > ). all infants were well at the time of initial evaluation. five infants with absolute cd t cell counts > cells/ul and cd t cell counts > cells/ul began evaluation as outpatients on home isolation. patient , with undetectable trecs, cd t cell count , and cd t cell count was urgently admitted for inpatient evaluation and management and immediately started on antimicrobial prophylaxis. patient further evaluation was significant for lymphocyte proliferation to mitogens that was initially normal but waned with time, prompting treatment with a paternal haploidentical hematopoietic cell transplant at months of age. patients and developed neutropenia within weeks of birth treated with granulocyte colony stimulating factor (gcsf). patient remains well on gcsf but has had persistent growth failure under continued evaluation. patients , , and remain stable off antimicrobial prophylaxis, but with persistent moderate tcl. as part of an immune evaluation, patients and - had gene panel testing revealing heterozygous variants in foxn . only the variant of patient (presumed shared by patient , his brother) was predicted to be pathogenic; patient had dystrophic nails and sparse hair most evident after years of age, features shared by his mother and his brother, patient . the other patients lack the clinical features of the previously described phenotype of nude scid. their heterozygous foxn variants are of unknown significance; the functional role of these variants in the patients clinical phenotype is unknown. conclusion: six infants with abnormal nbs for scid had lymphopenia and heterozygous variants in foxn . for these infants, variation exists in level of tcl and presence of hair and nail findings. heterozygous variants of unknown significance in foxn have been uncovered in others, including infants with abnormal nbs for scid, highlighting the need for functional studies to address the possible role of each heterozygous foxn variant in congenital lymphopenia and neutropenia. more work is needed before attributing tcl to a novel foxn variant of unknown significance in the absence of family history, abnormal hair or nails, or functional evidence. remains poorly understood. we characterized the intestinal microbiome and metabolome in patients with cgd to determine if intestinal microbiome and metabolomic signatures could distinguish subpopulations of patients with cgd while using the metabolome to add a functional dimension to observed microbiome signatures. methods: clinical metadata and fecal samples were collected crosssectionally from healthy volunteers (hv; n= ) and patients with cgd (n= ). metabolomic profiling and s rrna (v ) sequencing was performed on fecal samples (total samples: ; reads/sample: , to , ; median: , ) . results: samples from patients with cgd had distinct intestinal microbiome signatures and metabolomic profiles depending on genotype, presence of cgd-ibd and specific interventions (e.g. treatment with an elemental diet). notably, samples from patients with active cgd-ibd (compared to samples from patients without a history of cgd-ibd) had significantly different alpha-and betadiversities, and were enriched for enterococcus spp. signal transducer and activator of transcription gain of function (stat -gof) is a primary immunodeysregulatory disease in which a subset of patients have features of autoimmunity and autoinflammation. enteropathy with growth failure and nutrient wasting is a more common feature of immunodysregulation. ruxolitinib is a janus kinase-stat inhibitor that has been shown effective for the treatment of immunodysregulatory features in stat -gof. our patient is a year old male with stat -gof (c. a>g p.h r) with severe total parenteral dependent enteropathy that led to growth failure (weight . kg). treatment with ruxolitinib led to resolution of diarrhea, return of normal diet, and catch up growth. a dose of . mg twice daily was initially started but was decreased to . mg every morning and mg every evening due to elevated transaminases and thrombocytopenia. over the following year the patient thrived gaining . kg with normal every other day stools. despite weight gain, he remained stable on the same dose of ruxolitinib. as he outgrew his dose, he developed an increased frequency of upper respiratory infections (parainfluenza, coronavirus, rhinovirus). one year after initiation of ruxolitinib, he again developed profuse watery diarrhea that was norovirus positive (weight kg, bsa . ). he was placed on bowel rest and ruxolitinib was dose escalated with a goal of mg/m /day. when he reached mg twice daily, enteropathy completely resolved but liver function tests began to rise. he gained weight and began thriving after weeks of therapy. six months later, enteropathy is controlled, and transaminases have remained elevated (alt iu/l, ast iu/ml) but stable. the appropriate dose and pharmacokinetics for ruxolitinib for the treatment of immunodysregulatory symptoms in pediatric patients has not been thoroughly studied. the dose used was extrapolated from data on the use of ruxolitinib in pediatric myelofibrosis. a dose of mg/m /day appears to provide the most benefit with tolerable adverse effects. this dose should be maintained in order to prevent recurrence of disease related manifestations. abstract clathrin-mediated endocytosis (cme) is the major endocytic pathway by which eukaryotic cells internalize cell-surface cargo proteins and extracellular molecules, thereby allowing for a broad range of biological processes, including cell signaling, nutrient and growth factor uptake, and cell fate and differentiation . the fbar domain only proteins and (fcho /fcho ) are involved in the initiation of clathrin coat pit formation. whether fcho and fcho are functionally redundant or have distinct functions is unclear. we report here the first cases of a severe immunodeficiency due to a genetic defect affecting cme. by using whole exome sequencing and genomic analysis of a targeted pid gene panel, we have identified biallelic loss-of-function fcho mutations in five patients from unrelated families of italian (p ), turkish (p , p , and p ) and algerian (p ) origin with severe t cell lymphopenia manifesting as recurrent and severe infections of bacterial, mycobacterial, viral and fungal origin. p developed ebv-associated diffuse large b cell lymphoma. three patients (p -p ) died in childhood, whereas p and p are alive with full donor chimerism at and . years after allogeneic hematopoietic stem cell transplantation, respectively and have cleared pre-transplant infections. patients p , p , and p carried homozygous frameshift mutations predicted to cause premature termination. western-blotting analysis of ha-or flag-tagged fcho constructs showed expression of truncated products in p and p , whereas no protein was detected in p , presumably due to mrna decay. p and p carried homozygous splice-site mutations at the invariant - and + positions, respectively, leading to skipping of exon in p 's fcho cdna. qpcr analysis demonstrated differential expression of the fcho and fcho genes, with the former being predominantly expressed in lymphoid cells, whereas fcho was more abundantly expressed in fibroblasts and k cells. analysis of t cell activation in p (the only patient for whom pre-transplant pbmc were available) revealed reduced t cell proliferation. while tcr internalization in response to cd cross-linking was normal (consistent with recent evidence that tcr internalization occurs through a clathrin-independent pathway), chase experiments demonstrated that transferrin internalization was abolished in activated t cells from p . we had previously reported that a missense mutation in tfrc, encoding transferrin receptor , impairs transferrin internalization and intracellular iron delivery, causing a combined immunodeficiency with defective t cell proliferation. our data identify the first form of severe immunodeficiency due to defects of clathrin-mediated endocytosis, and provide additional evidence in support of the critical role played by iron cellular metabolism in t cell function and homeostasis. natural history of anti-interferon-gamma autoantibody-associated immunodeficiency syndrome in thailand submission id# centralized sequencing initiative at niaid: year therefore, we set out to investigate the pneumococcal-specific responses of igg, igg , iga and igm to prevnar ® in igg subclass deficient (iggscd) patients in this study. pneumococcal responses were measured using the vacczyme pneumococcal capsular polysaccharide igg, igg , iga and igm elisas (the binding site group, birmingham, uk) in control (n= , median age years, range - ) and iggscd patients (n= , median age years, range - ) recruited from the immunodeficiency unit at the karolinska university hospital iga and igm antibodies in response to pcv vaccination was observed weeks post vaccination in iggscd patients (median, . th and . th percentile these median concentrations were lower than those observed in control patients (median, . th and pcv igg mg/l, - however, percentage changes between pre to post vaccination concentrations of igg, igg and iga in response to pcv in iggscd patients were not significantly different to the control patients u/ml vs . u/ml, respectively) iga u/ml and pcv igm u/ml) responders and non-responders of pcv igg iga and igm in response to pcv in iggscd patients were generally lower compared to the control population. these results support the fact that in addition to igg and igg , measurement of iga and igm could also provide useful information for the clinician gain-of-function ikbkb mutation causes human combined immune deficiency submission id# neutralizing anti-il- -autoantibodies are a risk factor for pyogenic bacterial infections national institutes of health, national institutes of allergy and infectious diseases service of immunology and rheumatology, garrahan national pediatric hospital copa mutations impair er-golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis copa syndrome: a novel autosomal dominant immune dysregulatory disease analysis of pulmonary features and treatment approaches in the copa syndrome expanding the phenotype of copa syndrome: a kindred with typical and atypical features the forest and the trees: machine learning to classify cases of suspected inborn errors of immunity using decision tree and random forest algorithms submission id# card Δ gene dosage: from mono-allelic protection to ibd, to bi-allelic increased fungal infection susceptibility yamanaka d , walkiewicz m , lionakis m and rosenzweig s stim mutation associated with a syndrome of immunodeficiency and autoimmunity a novel hypomorphic mutation in stim results in a late-onset immunodeficiency clinical, histological and genetic characterisation of patients with tubular aggregate myopathy caused by mutations in stim gain-of-function mutation in stim (p.r w) is associated with stormorken syndrome gain-of-function mutations in stim and orai causing tubular aggregate myopathy and stormorken syndrome stormorken syndrome caused by a p.r w stim mutation: the first italian patient and a review of the literature by studying ecs-pre and ecs-post patients we were able to describe the bona-fide effect of gcs on the immune system in general, and t lymphocytes in particular. decreased lymphocyte/thymic output, as well as increased apoptotic tcell death underlies lymphopenia in ecs/chronic gcs-exposed patients. under such conditions, il- was significantly decreased in plasma and our in-vitro studies showed that il- replenishment was able to increase bcl (anti-apoptotic molecule) and bcl expression, and efficiently counteract the apoptotic effects of gcs. recombinant il- has been explored as a co-adjuvant treatment for multiple human cancers and may offer a treatment option for lymphopenia and its genetic counselor, co-director of personalized medicine, division of hematology/oncology/bmt and the institute for genomic medicine, nationwide childrens hospital genetic counselor, division of hematology/oncology/bmt, nationwide children's hospital acknowledgments. genetic sequencing was kindly provided by drs. raif geha and janet chou at the division of immunology, allergy, rheumatology and dermatology, boston children's hospital, harvard medical school. the following grants are acknowledged: . rui . /cippt/ (usm) . bmbf eo (freiburg) the authors would like to thank the director general of health of malaysia for permission to publish this scientific presentation. while severe viral infections may also be an initial presentation of primary immunodeficiency, an immune evaluation is not always obtained in this scenario. patients with xla have an increased susceptibility to severe enterovirus infections, manifesting as chronic meningoencephalitis, which can be fatal. the following case describes a patient with newly diagnosed xla presenting as suspected coxsackievirus and confirmed hhv- meningitis, pseudomonas meningitis and bacteremia. this may be the first reported new diagnosis of xla presenting with both severe bacterial and viral coinfection. case description: a year old, partially vaccinated, hispanic male with a history of febrile seizures presented to the emergency room with fever, oliguria, watery diarrhea, lethargy, meningismus, ecthyma gangrenosum and lower abdominal pain. eight days prior to presentation, he was seen by his pediatrician for facial rash and low grade temperature, and was diagnosed with hand-foot-and mouth disease. he worsened on empiric antibiotics. he had no history of sinopulmonary infections. he did not attend daycare. his vaccines were delayed due to parental choice, and he had not received live vaccines (rotavirus, mmr or vzv). full sepsis evaluation was performed. csf demonstrated pleocytosis, and he was started on empiric antibiotics and transferred to picu. due to worsening abdominal pain, ct of the abdomen was performed, which was consistent with ruptured appendicitis and septic emboli at the lung bases. csf pcr panel was positive for hhv- and he was started on gancyclovir. csf and blood cultures subsequently grew pseudomonas aeruginosa. immune evaluation was performed. serum immunoglobulins were undetectable. in addition to iv antibiotics, he received mg/kg ivig and lymphocyte subsets revealed profound b cell lymphopenia ( . %, cells/ul). btk protein analysis revealed hemizygous btk pathogenic variant confirming the diagnosis of x-linked agammaglobulinemia. the hospital course was further complicated by brain abscesses and pyoventriculitis. he was treated with additional doses of mg/kg ivig and iv antibiotics. repeat mri of the brain nearly weeks after admission demonstrated significant improvement. there was significant clinical recovery. he was discharged home at baseline neurological status. his igg level upon discharge home was mg/dl with the plan to increase dose to mg/kg per month with close monitoring. conclusion: both severe opportunistic bacterial infections and severe viral infections as the initial presentation of xla have been well reported in the literature. this case describes the first reported severe pseudomonas aeruginosa and hhv- co-infection in a newly diagnosed xla patient. this case further highlights the necessity for an increased index of suspicion of primary immunodeficiency in a patient who presents with a severe first infection, despite lack of recurrent infections. we present two patients with dock deficiency due to compound heterozygous variants including a copy number loss at chromosome band p . spanning approximately . mb with partial deletion of the dock gene and a novel c. c>t (p.ser leu) missense variant [chr : (grch ) nm_ ] in dock . functional data is presented to support the pathogenicity of the missense change, along with a review of the literature on dock variants. the proband is a -year-old female with elevated serum ige, severe atopic dermatitis, mild persistent asthma, food allergies, and seasonal allergic rhinitis. she is currently healthy following haploidentical bone marrow transplant in june . she has a -year-old brother with dock deficiency with the same compound heterozygous variants. the brother had later onset of symptoms and a milder presentation of intermittent asthma and seasonal allergic rhinitis. each of the parents is heterozygous for one of the two variants. we evaluated the pathogenicity of the c. c>t missense variant with western blots of dock protein expression, intracellular flow cytometry, and dock stretch assays. flow cytometry showed decreased dock protein expression and stretch assays revealed t cells that were stretched in collagen gels. notably, dock is a large gene containing exons spanning kb and it is relatively common to be a carrier of a rare missense change. in fact, gnomad has approximately individuals with rare (< . frequency) missense alleles in dock . therefore, it is important to demonstrate the potential pathogenicity of any given rare missense change, since few pathogenic missense variants in dock have been reported. of the published dock variants listed in the human gene mutation database (hgmd) only are missense. the majority are gross deletions, of which were reported in hgmd. the remaining reported dock variants include nonsense, splicing, small deletions (all frameshifting), small insertions (all frameshifting), small indels, and gross insertions/duplications. this case demonstrates the relatively infrequent but important contribution of missense changes to pathogenic dock alleles. functional validation of missense alleles is critical in the complex evaluation of dock deficiency. background: hsct is the only known curative option currently for cd l deficiency, an x-linked disorder. in cd l deficiency and other x-linked immune deficiencies, there is an ongoing debate regarding the use of a carrier female sibling or mother as hsct donor. skewed lyonization despite complete donor chimerism has raised concerns for incomplete disease control post-hsct. no data exist regarding the efficacy of related female carrier as hsct donor for cd l deficiency. we herein report outcomes of three patients with cd l deficiency who underwent hsct using a related female carrier donor. method: retrospective review of patients who received hsct from carrier female related donor at three separate institutions. results: three patients with cd l deficiency underwent hsct between - . patient had recurrent episodes of pneumocystis jiroveci pneumonia (pjp) despite being on bactrim and immunoglobulin replacement. patient presented with pjp and severe neutropenia. patient presented with acute respiratory failure from severe respiratory viral infections, cmvand had severe neutropenia requiring g-csf treatment. age at the time of hsct ranged from . - yrs. all three underwent reduced toxicity hsct with busulfan and fludarabine-based preparatory regimens. two of them received matched sibling bone marrow hsct and one received tcr and cd depleted mobilized maternal pbsc haploidentical hsct. donor cd l expression varied from % - % on activated cd cells. immunoglobulin profile and lymphocyte subset were done in two of donors, they were within normal range for age, and none had significant infection history. no history of intermittent neutropenia or oral ulcers noted in donor and the absolute neutrophil count of the donor varied between /l. donor age ranged from . yrs years. cd dose ranged from . x - . x cells/kg and cd dose ranged from x . x cd + cells/kg. gvhd prophylaxis consisted of csa/mmf (n= ) and tcr-a/b depletion and no csa (n= ). neutrophil engraftment ranged from - days and platelet engraftment ranged from days. none of the patients developed acute or chronic gvhd. all three patients maintain full donor myeloid chimerism at the latest testing ( months months); t cell chimerism was % in one and mixed in two patients ( % at nine months, % at months). all three patients had excellent t cell immune reconstitution; two patients came off immunoglobulin replacement - months post hsct, whereas the rd patient is ivig dependent, though iga level was mg/dl at nine months post-transplantation. latest evaluation, months post-hsct, revealed % - % cd l expressing activated cd t cells, which correlated with donor cd l expression and t-cell chimerism. conclusion: our data suggest that hsct utilizing x-linked carrier appears to be safe and results in durable engraftment with excellent humoral and cellular immune reconstitution in patients with cd l deficiency. longer follow-up and data from a larger cohort is needed to make a definitive determination of safety and efficacy of utilizing female carrier as hsct donors in this disease. chief, immunology service, department of laboratory medicine, nih clinical center, bethesda, md, usa background: ikaros belongs to a hematopoietic-specific zinc-finger (zf) family of transcription factors. after dimerizing and dna binding to pericentric-heterochromatin (pc-hc) regions, ikaros is described as a central regulator of lymphocyte differentiation. somatic mutations/ deletions affecting ikaros n-terminal zf have been identified in b-acute lymphoblastic leukemia (all) patients, and germline n-terminal mutations were reported in cvid patients with progressive lack of b cells, hypogammaglobinemia, autoimmune diseases and b-all. methods: we performed targeted sequencing panel for known inborn errors of immunity disease-causing genes in a previously healthy male pediatric patient with burkitt lymphoma, followed by benign lymphoproliferation, thrombocytopenia and neutropenia. b-cells and immunoglobulin levels were normal. ikaros dna-binding, nuclear localization and protein binding were evaluated by emsa, fluorescence microscopy and immunoprecipitation. protein modeling was also performed. results: a novel heterozygous germline mutation in ikaros c-terminal zf dimerization domain (p.r l) was detected in this patient. this mutant showed normal pc-hc localization but dna-binding was markedly reduced in terms of ikaros dimerization and multimerization. moreover, reduced wt-mutant binding was also detected. mutant/wt cotransfection experiments suggest a haploinsufficient defect. geometry based docking of wildtype ikaros predicted that r is within the homodimer interface and may abolish cation-pi interactions and destabilize the ikaros-zf dimerization domain. conclusion: a novel germline ikaros c-terminal mutation affecting homodimerization/multimerization and resulting in reduced dna binding to its dna consensus site was detected in a patient with burkitt lymphoma, benign lymphoproliferation and cytopenias. further studies are warranted to formally establish the casual connection between this genotype and phenotype.( ) submission id# patricia pichilingue-reto, md , prithvi raj, phd , igor dozmorov, phd , quan-zhen li, md, phd , edward wakeland, phd , nancy kelly, md , maria teresa de la morena, md , nicolai s. van oers, phd methods: mice were generated by crispr/cas technology to genocopy the foxn compound heterozygous mutations identified in one of the human patients. thymopoiesis and hair follicle extrusion was analyzed in the various heterozygous and homozygous mutant mice. gene expression analyses of the hypoplastic and normal-sized thymii and the developing skin were performed. in addition, a structure-function analysis was performed with luciferase reporter assays using distinct and previously unreported foxn mutations uncovered in patients who presented with low trecs. results: mice harboring compound heterozygous mutations in foxn that match the human patient phenocopy the t-b+nk+ scid phenotype with normal hair and nails. a functional characterization of the diverse foxn mutations suggests that the severity of the block in thymopoiesis depends on whether the mutations affect the dna binding or transactivation domains of foxn . a -amino acid segment at the end of the dna binding domain appears to be essential for tec development. however, this segment is not required for normal keratinocyte functions in the skin and nail plate. gene expression comparisons are revealing key targets of foxn that suggest a dichotomy in its function in the thymus versus the skin. conclusions: novel compound heterozygous mutations in foxn are causal to a t-nk+b+ phenotype with normal hair shaft extrusion and nail plate extension. this differs from the classic nude/scid (omim # ) reported for individuals with autosomal recessive mutations in foxn . assistant professor of medicine and pediatrics, department of allergy and immunology, uva introduction: copa syndrome is a recently described monogenic immunodysregulatory syndrome. the cop protein, encoded for by the copa gene, is expressed in all cell types and is involved in trafficking from the golgi complex to the endoplasmic reticulum ( ) . the most common clinical features of copa syndrome are interstitial lung disease, pulmonary cysts or follicular bronchiolitis, pulmonary hemorrhage, arthritis, glomerular disease, and autoantibody development ( , ) . atypical features of copa syndrome identified thus far include: extrapulmonary cysts in the liver and kidney, renal and neuroendocrine malignancies, autoimmune neurological disorders such as neuromyelitis optica, and infections, such as meningitis ( ) . clinical case: we present a case of a year-old male with copa syndrome (de novo heterozygous mutation in exon , c. g>c; p.ala pro) manifesting as lymphocytic interstitial pneumonitis, peripheral blood b-cell lymphocytosis, mediastinal lymphadenopathy and persistent transaminitis (alt and ast - u/l, nl ast< u/l, alt < u/l) with normal bilirubin, alkaline phosphatase and pt/inr. the transaminitis was noted prior to diagnosis of copa syndrome, and has persisted despite seven months of therapy with pulse dose steroids, two cycles of rituximab and maintenance therapy with hydroxychloroquine and prednisone. he has had a normal ck and aldolase excluding muscle injury as a source of his transaminitis. a congenital cholestasis panel was normal. markers of autoimmune liver disease including ana, anti-liver kidney microsomal antibody and anti-smooth muscle were negative. serum ceruloplasmin and alpha- -antitrypsin level were normal and celiac serologies, were negative. liver ultrasound was normal. a liver biopsy did not demonstrate inflammatory changes, hepatocyte necrosis, mononuclear cell infiltrates or fibrosis. nonspecific biopsy findings included occasional intraparenchymal neutrophils. it is unclear if these scattered neutrophils and the transaminitis are due to an early as yet unidentified autoimmune process, perhaps in response to hepatocellular stress exacerbated by the copa mutation. discussion: liver involvement has not been reported in copa syndrome. we describe a child with copa syndrome who has had chronic transaminitis with no clear alternative cause. if the phenotypic spectrum of copa syndrome involves the liver, it may limit immunomodulatory options for the treatment of this disease. background: in humans, biallelic stat lost-of-function (lof) mutations lead to a very low or complete absence of the wild-type (wt) protein. whereas, heterozygous mutations can lead to partial loss of function. these patients are susceptible to mycobacteria and herpes virus infections. on other hand, heterozygous gain-of-function (gof) mutations in the stat gene result in a hyperphosphorylated state where patients develop recurrent or persistent chronic mucocutaneous candidiasis (cmc), other cutaneous mycosis, bacterial infections, disseminated dimorphic fungal infections, viral infections and autoimmune disease. methods: in this study, we evaluated novel stat mutations, three gof and one lof. in vitro, pbmcs from these patients were stimulated with ifn-and ifn-for , , and minutes and levels of phospho-stat were measured by flow cytometry. the stat phosphorylation and activity (firefly and renilla luciferase activities) were evaluated in u a-stat deficient cells transfected with a reporter plasmid (for luciferase), wt or mutant-stat plasmids. results: we observed higher levels of stat phosphorylation after two hours of stimulation from three gof mutations compared to wt. however, a lof mutation showed absent stat activation at baseline and in response to ifn-and ifn-. luciferase reporter assay confirmed gain of function and loss of function stat activity observed by flow cytometry. conclusions: using flow cytometry followed by a luciferase assay, we confirmed four novel stat mutations. measuring phosphorylation of stat by flow cytometry is sufficient to determine whether the stat mutation is disease causing. this assay can be translated to a clinically accessible test for stat related disease. background: variants in recombination-activating genes (rag) are common genetic causes of autosomal recessive forms combined immunodeficiencies (cid) ranging from severe combined immunodeficiency (scid), omenn syndrome (os), atypical scid (as) and cid with granulomas and/or autoimmunity (cid-g/ai). the clinical and immunological presentation is broad, ranging from severe infections secondary to near absence of t and b lymphocytes and hypogammaglobulinemia to the occurrence of autoimmunity with late manifestations with partly preserved immune subsets and near normal immunoglobulin levels and broad spectrum of autoantibodies. objective: we aim to estimate the incidence, clinical presentation, genetic variability and treatment outcome with geographic distribution of patients with the rag defects in populations inhabiting south, west and east slavic countries. due to shared ancestry, we also investigated our cohort for founder variants in rag and rag genes. methods: demographic, clinical and laboratory data were collected from rag deficient patients of slavic origin via chart review, retrospectively. results. based on the clinical and immunologic phenotype, our cohort of patients from families represented a wide spectrum of rag deficiencies, including scid (n= ), os (n= ), as (n= ) and cid-g/ai (n= ). sixty-six ( . %) patients carried rag and patients ( . %) carried rag biallelic variants. we estimate that the minimal annual incidence of rag deficiency in slavic countries varies between in , , live birth and it may vary secondary to health care disparities in these regions. in our cohort, % of the patients carried rag p.k vfs* (c. _ delaa), either in homozygous (n= , %) or compound heterozygous (n= , %) form. the majority ( %) of patients with homozygous rag p.k vfs* originated from vistula watershed area in central and eastern poland, and compound heterozygote cases distributed among all slavic countries except bulgaria. clinical and immunological presentation of homozygous rag p.k vfs* cases was highly diverse suggestive of strong influence of other genetic and/or epigenetic factors in shaping the final phenotype. survival of rag deficient patients without hematopoietic stem cell transplant (hsct) (n= , . %) is poor and dramatically improved in the last decade with access to hsct and tailored conditioning regimens. conclusion: we propose that rag p.k vfs* is a founder variant originating from the vistula watershed region in poland, which may explain a high proportion of homozygous cases from central and eastern poland and the presence of the variant in all slavs. our studies in cases with rag founder variants confirm that clinical and immunological phenotype only partially depend on the underlying genetic defect. hsct is becoming available for rag deficient patients in eastern europe with improving outcome. clinical immunologist, centre hospitalier universitaire de montréal (chum) background: acute gvhd following solid organ transplantation is a rare complication. intestinal and liver transplantation have the greatest risk of gvhd among solid organs due to high number of donor lymphocytes in these organs. prevalence of acute gvhd after liver transplantation is estimated to be around , - % and has a poor prognosis ( ) . chronic neurological gvhd is a rare form of gvhd with three subtypes described: cerebral vasculitis, demyelinating disease and immune mediated encephalitis. acute neurological gvhd has no clear definition and is still considered a controversial entity. case presentation: a year-old male underwent cadaveric liver transplantation for alcoholic cirrhosis and hepatocellular carcinoma. the donor was a year-old man who died from anoxic brain injury. the receiver was induced with basiliximab and then put on prednisone, azathioprine and tacrolimus. he was readmitted weeks later for myalgia, headache, fever and neutropenia. clinical state initially improved with empiric antibiotics. he then developed a skin eruption, colitis and dic. the latter was thought to be tacrolimus-induced. he was switched to cyclosporine. skin and rectosigmoid biopsies were compatible with acute gvhd. he received basiliximab and ivig and developed a refractory convulsive state. csf analysis showed elevated proteins and slight pleocytosis. cerebral mri showed non-specific white matter lesions and conventional angiography was normal. chimerism on peripheral blood was % but was % donor on csf. with the presence of chimerism on csf, evidence of cutaneous and digestive gvhd and no infectious cause, neurological gvhd was considered the most likely diagnosis. brain biopsy showed non specific change including neuropil spongiosis, microglial activation and reactive gliosis; but no signs of vasculitis or demyelinating disease. he was treated with atg, highdose systemic corticosteroids, cyclosporine, ivig and intrathecal methotrexate and corticosteroids. csf pleocytosis, proteins and chimerism improved with treatment ( % to % donor). no improvement was noted regarding his neurological state and he developed pancytopenia. he was then transfer to palliative care and died shortly after ( month and a half after liver transplant). discussion: to our knowledge, there is only one prior case published of neurological gvhd following liver transplantation ( ) . both patients were old, had hepatocellular carcinoma and had at least one hla match. age > year, hepatocellular carcinoma and shared hla antigen are known risk factors for gvhd following liver transplantation ( ). our patient had only one hla match with the donor. this case is intriguing as there was a great discrepancy between blood and csf chimerism. acute neurological gvhd following transplantation is a real complication. it must be taken into consideration in patients with neurological involvement after transplant, even solid organ transplantations. introduction: hyper-igm syndrome are rare. although no data are available on the frequency of activation-induced cytidine deaminase (aid) deficiency, this disorder is estimated to affect less than : , , individuals. by the year , cases worldwide ( ) with such mutation have been described. we describe a patient with hyper igm by mutation in the aicda gene. case report: mvv, -year-old boy, born to consanguineous parents, was referred with recurrent pneumonia, which started shortly after discontinuation of breastfeeding at months old. repetitive otitis evolved with bilateral tympanic and partial hearing loss. he was submitted to adenoidectomy without improvement. immunological evaluation showed normal numbers of b and t cells with cd + ( /mm , %), cd + ( /mm , %), and cd + ( /mm , %). immunoglobulin concentrations were: igg = mg/dl (p ). treatment with intravenous immunoglobulin and prophylactic antibiotic was initiated and he had no infections during the follow up except for one episode of sinusitis. at years of age, molecular evaluation was performed and a mutation in homozygosity in the aicda gene (omim * ) at position chr : . . was found, confirming the clinical suspicion. conclusion: the role of aid in the immunoglobulin class-switch recombination (csr) and somatic hypermutation (shm) have not been fully elucidated. summarizing within the shm and csr processes, aicda mutation can induce dna lesions in directed sequences in the s and v regions required for dna cleavage. recurrent infections and consanguinity raised the suspicion of inborn errors of immunity in this patient. the literature described late diagnosis as in the second or even the third decade of life. it was suggested that high levels of igm antibodies may provide effective defense, at least, against some infectious agents. it is important to emphasize that the impossibility to obtain genetic diagnosis did not prevent to introduce therapy. * aicda: activation induced cytidine deaminase gene patients with chronic granulomatous disease (cgd) are at risk for recurring infections and non-infectious inflammation, reduced quality of life and life expectancy. conventional treatment with life-long anti-bacterial and antifungal prophylaxis prolongs lifespan but does not eliminate the lifelong risk of infection and inflammation. allogenic stem cell transplantation is currently the only curative option for this disease. although sct with reduced intensity conditioning has improved treatment-related mortality and efficacy, it remains a matter of debate whether all patients with cgd benefit from sct, whether pre-existing infections and non-infectious inflammation are risk factors and at what age sct should be performed. we compared patients with cgd on conventional treatment with those after stem cell transplantation for their prognosis and evaluated potential risk factors for stem cell transplantation outcome followed up in six european centers. frequency of infections, inflammatory complications, hospitalizations, operations and immunomodulative/immunosuppressive therapy, height and weight were compared in patients on conventional treatment /before stem cell transplantation versus patients after sct. correlation between transplantation outcome and patient characteristics or medical history was tested. patients were recruited, on ct, after stem cell transplantation. before/without transplantation % of patients suffered from at least one infection, , % from inflammatory complications. patients on conventional treatment developed infection/inflammation/ hospitalization/surgery at a median of , (range [ , - , ] , iqr , ) per year, versus (range , iqr , ) in the first year after stem cell transplantation but (range [ - ], iqr , ) after the first year post stem cell transplantation. there was a significant decrease of all complications after stem cell transplantation (p < . ). growth improved significantly after stem cell transplantation (z-score weight - , versus - , (p. ), z-score height - , versus - , (p. )). nevertheless, complications post stem cell transplantation are frequent: % of patients had at least one infection, % had severe acute gvhd, % chronic gvhd, % had graft rejection, % died. preexisting active mold infection increased the risk for complications after stem cell transplantation. in summary infections and non-infectious inflammation are common in patients with cgd on conventional treatment, their growth is significantly impaired. stem cell transplantation, if successful, significantly reduces the risk for infections and non-infectious inflammation. however, treatment related mortality of stem cell transplantation in patients with cgd remains considerable. introduction: development of a diverse t cell repertoire is essential for full immune recovery following definitive treatment for severe combined immunodeficiency (scid), whether by allogeneic hematopoietic cell transplantation (hct); autologous gene therapy (gt); or, in the case of adenosine deaminase deficiency, enzyme replacement therapy (ert). however, the time course and depth of diversity of t cell receptor rearrangements have been difficult to measure directly, necessitating estimates from total and naïve t cell counts and from spectratyping, in which t cell receptor (tcr) beta chain diversity is estimated by the length distributions of cdna amplicons between a series of tcr beta chain variable (v-beta) segments that have productively recombined with the tcr beta-chain constant region. analysis of the actual sequences of rearranged tcrs could indicate more precisely the status of the t cell compartment of these patients, and might reveal oligoclonal expansion of dysregulated t cells, t cell insufficiency, or t cell exhaustion. objectives: we wished to ascertain whether deep sequencing of individual tcr v-beta rearrangements in peripheral blood could be performed sequentially following diagnosis and treatment of scid to differentiate satisfactory immune reconstitution from incomplete or skewed repertoire development that might require further cellular therapies. methods: equal amounts of total rna were obtained from peripheral blood of controls and scid patients pre-hct and at d, and mo, and yearly post-treatment(s). cdna was used as template to semi-quantitatively amplify rearrangements at the tcr-beta locus (trb). raw sequences were filtered to remove pcr errors, and resulting fastq files were converted into fasta format (seqtk software, github, inc), filtered for productive rearrangement, and analyzed for v, d, and j gene composition and length (imgt highv-quest software). the vdj statistics file (past program) was used to calculate a shannon entropy (h) index to measure repertoire diversity, taking into account both abundance and richness of the overall repertoire; and a gini-simpson index of unevenness, measuring inequality in the relative representation of species in a given sample. graphical representations of repertoire diversity were generated by hierarchical tree maps of the trb repertoires (irepertoire software): each dot represents a unique sequence and the dot size corresponds to frequency of that sequence in the total sample. results: tcr v-beta sequence analysis of scid patients (image) showed (top) baseline poor diversity due to pre-treatment ada deficiency followed by improvement to normal complexity (shannon h > . ) after receiving peg-ada and autologous lentivirus gene therapy at age m; (middle) increasing diversity in xscid after maternal t-depleted unconditioned hct, although b cells did not recover; and (bottom) failure of initial unconditioned maternal t-depleted hct in another xscid patient at m, followed by autologous lentivirus gene therapy with subsequent improvement (shannon h increasing from . to ) months later. conclusions: tcr v-beta diversity sequence analysis provided a detailed assessment of repertoire diversity in response to cellular therapies for scid. this method could become a useful predictive tool to measure successful t cell immune reconstitution, both as early as d and in the years following treatment. background: the stim (stromal interaction molecule ) protein, encoded by the stim gene, is involved in calcium regulation in the endoplasmic and sarcoplasmic reticulum. pathogenic variants in this gene are associated with three different disorders. homozygous loss-of-function (lof) pathogenic variants in stim have been reported to cause autoimmune cytopenias, lymphoproliferation, enamel defects, anhydrosis, and iris hypoplasia. the first described cases had frequent mortality in early childhood due to recurrent life-threatening infections and development of kaposi sarcoma ( ), while recently discovered cases have had more prolonged survival, though still with recurrent serious infections ( ) . heterozygous gain-of-function (gof) pathogenic variants in stim have been associated with both tubular aggregate myopathy (tam) and stormorken syndrome. tam is a clinically heterogeneous progressive muscle disorder with a variable age of onset. muscle biopsy characteristically demonstrates tubular aggregates, with type ii muscle fiber atrophy ( ) . stormorken syndrome has a phenotype that includes miosis, thrombocytopenia, intellectual disability, mild hypocalcemia, muscle fatigue, asplenia, and ichthyosis ( ) . the thrombocytopenia has not been reported to be immune-mediated; rather it is due to abnormal platelet calcium regulation ( ). we report a patient with stim pathogenic variant presenting with tam and immune-mediated thrombocytopenia, along with lymphoproliferative features, arthritis, and a mild immune deficiency. case: the patient is a -year-old with a history of congenital thrombocytopenia (platelets ranging , - , ) who presented with acute arthritis of bilateral hand joints after exposure to cold temperatures, which resolved with naproxen. he had back pain without muscle weakness, and preceding sore throat and general fatigue. labs were significant for leukocytosis and elevations in his inflammatory markers and creatine kinase. mri of his lower extremities was negative for inflammatory myositis, but did demonstrate bilateral hip and knee effusions, and significant inguinal lymphadenopathy and hyperintense linear signal changes in the mid-and distal femurs with patchy red marrow signal. abdominal ultrasound could not identify a definite spleen. bone marrow biopsy was negative for malignancy but significant for toxic granulation of neutrophils, evident of inflammation. alpha-beta double negative t cells were not elevated. interferon-gamma was mildly elevated. flow cytometry demonstrated normal t, b, and nk cell absolute counts. circulating antibodies against platelets (both igg and iga) were detected. on lymphocyte antigen and mitogen proliferation testing, he did not exhibit any proliferation when stimulated with tetanus toxoid even though he had been fully vaccinated against tetanus. muscle biopsy demonstrated large vacuoles consistent with tam on both light and electron microscopies. invitaes primary immunodeficiency panel identified a pathogenic variant in stim (c. c>t; p.arg trp), consistent with a diagnosis of autosomal dominant stim -related conditions, including stormorken syndrome ( ) . conclusion: this patient expands the phenotypic spectrum of stim related disease. based on previous evidence, gof pathogenic variants in stim are associated with tam and stormorken syndrome, while lof pathogenic variants in stim are associated with immune deficiency. however, our patient with a stim gof pathogenic variant has features of lymphoproliferation and immune dysregulation in addition to tam. stim gof pathogenic variants should be considered in the differential of patients with immune thrombocytopenia and lymphoproliferation. references: introduction / background: card is critical for protein binding upstream of nf-kb (nuclear factor kappa b) and mtorc (mammalian target of rapamycin complex ) the signaling pathway involved in t-cell activation and inflammatory response. prior testing of card mutations demonstrated variable t-cell dysfunction. in vitro studies have demonstrated reduced interferon gamma cytokine production, interference of t-cell receptor (tcr) signaling, and th phenotype skew in t-cells with card defects. while homozygous mutation causes severe combined immunodeficiency deficiency, heterozygous card defect is associated with atopy by way of inappropriate th skewing. heterozygote atopy is characterized by eosinophilia, elevated ige, and severe dermatitis. despite multiple studies demonstrating in vivo consequences of card on t-cell function, little is known of the clinical significance. moreover, few studies have demonstrated the impact of card mutations on b-cell maturation and development, despite the recognized tcr and interleukin signaling deficits. objectives: this case demonstrates a card defect that evolved from atopy to combined immunodeficiency requiring intravenous immunoglobulin therapy. it highlights the poorly understood effect of card mutation on t-cell function, and the downstream impact on b-cell quality. methods: -year-old male, with past medical history of t-cell lymphoma and no evidence of disease status post autologous stem cell transplant, was found to have card e d missense mutation by genetic testing. consistent with previous literature regarding heterozygous card defects, the patient suffered from frequent asthma exacerbations, aeroallergen sensitivity, and eczema. lab work was consistently positive for elevated ige and eosinophilia. family history was positive for a son born with congenital molluscum, and multiple other children with recurrent infections. one child was also identified with card mutation. the patient had flow cytometry demonstrating % of circulating cells with atypical immunophenotyped cd + t-cells, and positive gene rearrangement studies. his qualitative immunoglobulin levels were significant for consistently low igm, but normal quantity igg. in the patients adulthood, he had recurrent bronchitis and pneumonia requiring hospitalization and intravenous antibiotics. given his recurrent infections, the patient underwent immunodeficiency evaluation. despite previous infection with herpes zoster, the patient did not have protective titers. additionally, the patient had received the pneumococcal conjugate vaccine once, and the pneumococcal polysaccharide vaccine four times. the most recent vaccination was one year prior to evaluation. despite repeated vaccinations, titers were unprotective. consequently, the patient was diagnosed with combined immunodeficiency, and initiated on intravenous immunoglobulin therapy. results: in summary, card defect is a cause of atopy, observed to become less severe with age. studies of card heterozygote mutations have demonstrated in vitro deficiencies in t-cell activation, likely secondary to skewed or decreased inflammatory cytokine production and tcr activation. our patient demonstrates that the variable t-cell dysfunction seen in vitro can have significant clinical implications evidenced by his inadequate vaccine response, and recurrent infections. his combined immunodeficiency poses a connection between card defects and, not only t-cell, but also b-cell function. conclusions: further studies are needed to determine deficits in t-cell and b-cell function in the setting of card defect, as this case suggests the clinical implications span further than atopy. genetic variants in the scaffold gene card cause disorders of the immune system. the clinical course and treatment depends on whether the card variant causes gain-or loss-of-function. however, lymphocyte immunophenotyping and proliferation assays in cells expressing card variants don't easily distinguish between gain-and loss-of-function. to address this challenge in variant interpretation, we used multiplexed genome editing in a lymphoma b cell line (tmd ) to generate cell populations expressing all possible singlenucleotide variants in the n-terminal amino acids of card . to assess function in each variant, we tracked its relative abundance over multiple conditions using dna sequencing. since card is required for survival of tmd lymphoma b-cells, cells expressing clinically identified gain-of-function variants grew faster relative to cells expressing other variants, even in the presence of upstream pathway inhibitors. upon evaluation of the relative abundance of each variant in genomic dna and mrna, we found that clinically identified loss-of-function variants were depleted in mrna, which could be attributed to alterations in splicing or to nonsensemediated decay. to address the impact of splicing, we modeled a newly-identified splice donor mutation (c. + g>a) found in two patients from one family diagnosed with combined immune deficiency, autoimmunity and atopy that was also observed in our screen. we show that the variant causes deletion of exon four and that card missing exon four exerts a dominant-negative effect leading to decreased nf-kb signaling and cell growth. these experiments demonstrate the utility of multiplexed functional assays for determining variant effect in clinically-relevant genes, which will improve diagnosis and treatment in patients. mutations in the rag and rag genes in humans cause a wide spectrum of phenotypes, ranging from severe combined immunodeficiency (scid) with lack of t and b cells to omenn syndrome (os), atypical scid (as) and combined immunodeficiency with granulomas and/or autoimmunity (cid-g/ai). here, we sought to investigate the molecular basis for phenotypic diversity presented in patients with various rag mutations. methods: we have recently described a novel flow-cytometrybased assay in which mouse rag -/-pro-b cells containing an inverted gfp cassette flanked by recombination signal sequences (rss) are transduced with a retroviral vector expressing either wild-type or mutant human rag (hrag ). the green fluorescent protein expression directly relates to the activity of rag proteins, representing a quick and powerful tool to correlate between defective activity of hrag mutant and severity of the clinical phenotype. the genetic variants of hrag analyzed in this study were affecting the various domains of the protein: ring, zinc finger ring type domain (amino acids - ); nbr (amino acids - ); hbr (amino acids - ) and the core domain (amino acids - ). using this sensitive assay, we tested the recombination activity of human rag variants that have been reported in patients. results: we have demonstrated correlation between the recombination activity of the mutants and the in vivo clinical phenotype of patients. in particular, similarly low levels of recombination activity were observed in patients with scid and os, whereas patients with as and especially those with cid-g/ai carried mutations that retained significant residual levels of activity. conclusions: these data provide a framework to better understand the phenotypic heterogeneity of rag deficiency. here we report a case of a child with b. cepacia lymphadenitis, ultimately diagnosed with takayasu arteritis. takayasu arteritis is a large vessel vasculitis which may have a nonspecific clinical presentation in childhood possibly leading to difficulty in diagnosis. case: a -month-old female presented with two weeks of fever, respiratory distress, and lymphadenopathy, and was treated with ivig for presumed atypical kawasaki disease. imaging studies performed due to worsening respiratory distress revealed retropharyngeal abscess with bilateral cervical lymphadenopathy, culture-positive for prevotella oralis and melaninogenica, with improvement following incision and drainage and antibiotic therapy. recurrence of fever and respiratory distress prompted ct imaging of her neck significant for worsening lymphadenopathy. cultures from lymph node biopsy grew b. cepacia. following treatment, she was readmitted with respiratory distress requiring chronic steroid treatment and found to have candida albicans on bronchoalveloar lavage and necrotizing granulomatous inflammation on lung biopsy. an immunologic evaluation was notable for two normal dhr assays. cgd genetic panel was negative for pathogenic variants in cybb (p ), ncf (p ), cyba (p ), ncf (p ). testing was also notably negative for hiv pcr, bartonella pcr, cryptococcal antigen, histoplasma antigen, bal afb stain and mycobacterial cultures, cmv pcr, ebv pcr, anca, serial blood cultures, and sweat test. lymphocyte subsets were normal for age. mitogen stimulation test, myeloperoxidase antibody igg, serine protease igg, c level, lad panel, and cytokine panel were normal. autoimmune lymphoproliferative disorders (alps) panel was negative. whole exome sequencing demonstrated heterozygous mutations in cfi and jak , not considered to be clinically relevant given the patients clinical picture and laboratory evaluation. the patient was then lost to follow-up for over a year. at the age of years, the patient presented with fever and back pain. imaging revealed severe large vessel vasculitis involving the aorta and subclavian, vertebral, mesenteric, and renal arteries. she also had evidence of cardio-embolic strokes on brain mri. she had had no significant interval infections, and her immunologic evaluation remained unrevealing. in the context of her new vasculitis, evaluation for deficiency of ada (dada ) was negative. she was ultimately diagnosed with takayasu arteritis and has begun therapy with systemic corticosteroids, aspirin, and etanercept. conclusions: we describe a case of b. cepacia infection in a child without identified immunodeficiency, ultimately diagnosed with a large vessel vasculitis. the presence of b. cepacia infection warrants a thorough investigation. burkholderia has been previously associated with giant cell arteritis, another type of large vessel vasculitis, though causation has not been established. to our knowledge b. cepacia infection has not been associated with takayasu arteritis. christopher santaralas, valentine jadoul, jacqueline squire, john cannon, jessica trotter, susan aja, neil goldenberg, david graham, jennifer leiding background: chronic granulomatous disease (cgd) is a primary phagocytic immunodeficiency secondary to mutations in any of the components of nadph oxidase. in addition to infection susceptibility, patients with cgd can develop auto-inflammatory disease that is difficult to manage. metabolomics is the systematic study of small molecule biomarkers of the clinical phenotype of disease. we sought to investigate plasma metabolic profiles in cgd as we hypothesized that unique signatures may differentiate patients with cgd. methods: plasma collected from subjects with cgd ( x-linked, p phox-deficient, p phox-deficient) and x-linked cgd carriers was analyzed using a targeted multiplex assay by liquid chromatography mass spectrometry (lc-ms) and simultaneously a profiling assay by lcms. sufficient signal was present for metabolites. x-linked cgd and p phox-deficient groups were sufficiently sized for multivariate and univariate analyses in metaboanalyst. twelve patients had a single time point of plasma metabolomics analysis and three had multiple time points, including one in whom both pre-and post-hematopoetic cell transplantation time points were assessed. post-hoc comparisons were also performed for those with, versus without, clinical comorbidities of autoinflammation. results: plasma from patients with x-linked and p phox deficient cgd had a differential metabolomic signature at baseline. many metabolites as measured by ion intensity were present at high levels, particularly homocysteine, kyneurine, tryptophan, citric acid, carnitine, methionine, and adenosine. increased values of metabolites reduced to that of normal (compared to post hct). homocysteine levels were elevated among patients with (mean . x ), versus without (mean . x ), clinical comorbidities of auto-inflammation (i.e., colitis, lupus). baseline samples showed elevated kynurenine among all cgd patients, relative to historical normal controls (unmatched, separate analysis). patients with colitis had elevated citric acid levels that were higher among patients with (mean . x ), versus without (mean . x ), colitis irrespective of genotype. conclusions: preliminary data with a small patient subset suggest that patients with cgd have metabolomic signature distinguishable by phenotype. citric acid cycle metabolites are elevated in crohns disease and ulcerative colitis. based on our data, citric acid may too act as a biomarker for inflammatory bowel disease in cgd. analyzing a larger number of samples, across time points, will likely describe a metabolomics profile for cgd and identify biomarkers for auto-inflammation in cgd. no significant medical history in mother; paternal history is unknown and unavailable.no significant medical history in mother or father. rationale: ataxia telangiectasia is a disorder with variable phenotypes characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition which may correspond to the degree of atm protein expression and/or radiosensitivity. we used in vitro cytometric assessment of atm, smc and h ax phosphorylation to assess dna damage in response to radiation and found that two siblings with the same copy number gain in atm have variable clinical neurologic and immunologic phenotypes. methods: chart review and radiosensitivity assays using cytometric assessment of patm, psmc , and h ax expression after irradiation with gy. results: patient a is a month old male identified after having low trecs on newborn screening, then found to have lymphopenia and elevated igm. he has diffuse café au lait macules and no neurologic symptoms. his year old sister, patient b, was being followed by neurology for several years for ataxia. she has selective iga deficiency, normal lymphocyte counts, lymphocyte proliferative responses, gammaglobulins, and vaccine specific antibodies. both patients have a copy number gains in atm (exons - ). mother and father both have copy number gains in atm and are healthy without neurologic symptoms or recurrent infections. both patient a and b have normal atm protein expression. phosphorylated atm, smc , and h ax was assessed in lymphocyte subsets (t, b, and nk cells) after low-dose irradiation to induce dna double-stranded breaks (dsbs). these parameters were assessed at hour post-irradiation when they are expected to be maximal and at hour post-irradiation, when under conditions of normal and effective dna repair, the phosphorylation state returns to baseline. patient a had abnormal patm and psmc but normal h ax expression hour and hours after irradiation of t, b, and nk cells. patient b had normal patm, psmc , and h ax expression in t cells but abnormal patm and psmc expression in b and nk cells hour after irradiation. patient b, however, had abnormal atm phosphorylation at hours after irradiation of t, b, and nk cells.conclusions: our results indicate that a unique copy number gain in atm within a family can correspond to different clinical and immunologic phenotypes as well as variable degree of radiosensitivity. the persistence of h ax at hours post-irradiation and impaired phosphorylation of atm and smc at hour post-irradiation demonstrates defects in dna dsb repair, and this is variably altered in different lymphocyte subsets. correlation between atm phosphorylation in lymphocytes with outcomes may be an area for future studies and particularly important in counseling patients regarding outcomes. antibodies have been implicated in both protection and pathology of dengue virus infections. however, much of this data is gathered from serum/plasma responses that is a cumulative of historical and ongoing infection. to precisely understand the role of antibodies with respect to the ongoing dengue virus infection, we employed the cutting edge approach of generating of human monoclonal antibodies from individual plasmablasts from peripheral blood of dengue patients that allows us to probe for answers at a single cell level. this method involves ex vivo single cell sorting of plasmablasts from peripheral blood of well-characterized dengue infected patient followed by single cell molecular cloning of immunoglobulin heavy-and light-variable regions into expression vectors containing the defined constant region followed by transient cotransfection of hek a cells with the heavy and light chain expression vectors made from genes arising from the same cell. thus far, using this powerful technology, for the first time in india, we have made number of human monoclonals, of which are specific to dengue and neutralize dengue virus at various concentrations. all the neutralizing antibodies are dengue-envelope specific and bind the highly conserved fusion loop of the dengue virus envelope. together, with the ongoing comprehensive analysis of the b cell repertoire and somatic hypermutations, these studies provide a detailed understanding of the dengue-specific plasmablast cell response at a single cell level and create a platform for testing these antibodies for basic research, diagnostic, prophylatic and as well as therapeutic applications. surviving. six of the ( . %) surviving patients remain dependent on ig replacement despite robust donor chimerism of - % and no active gvhd. all but two received rituximab pre-hsct. of the patients who are independent of ig replacement, only one ( . %) received rituximab post-hsct, whereas / of the ig dependent patients received rituximab post-hsct. t cell immune profiling revealed that the absolute numbers of lymphocyte subsets, cd + naïve t cells, and cd + recent thymic emigrants were not statistically different between ig independent and dependent patients ( figure ). however, there was a marked decrease in the number of total b cells, the percentage of memory b cells (cd + b cells), and classswitched memory b cells (cd + igd-igm-cells) in ig dependent patients ( figure ). t follicular helper (tfh) cell populations (cd +cd ra-cxcr +pd +) were evaluated in four patients and the frequency was similar to healthy controls ( . +/- . vs. . +/- . %). the ability of the patients naïve b cells to class-switch was assessed following exposure to il- , anti-cd antibody, and anti-human igm, and revealed normal b cell class-switching and differentiation to plasmablasts ( figure ) . additionally, t cell ability to provide b cell help was assessed by coincubating naïve b cells with activated cd + t cells. this revealed comparable b cell class switching to that of healthy controls. conclusion: the high incidence of poor long-term functional b cell reconstitution following allogeneic hsct for xlp- could be related to the use of rituximab in the post-hsct setting rather than pre-hsct. normal tfh numbers and function, and ability of b-cells to class-switch in-vitro suggest that persistent hypogammaglobulinemia is these patients is unlikely from a b or t-cell intrinsic defect. the possibility of rituximab induced acquired lymph nodal stromal defect in these patients is being explored. further studies are needed to understand the biology of persistent hypogammaglobulinemia in xlp- . additionally, due to the high incidence of persistent hypogammaglobulinemia, exposure of rituximab should be limited post-hsct. background: tandem mass spectrometry (ms/ms) has emerged as a primary platform for many clinical and newborn screening laboratories. the application of ms/ms mainly focuses on the quantification of accumulated small metabolites in plasma resulting from various metabolic defects. however, many disorders do not yield such metabolic markers and would benefit from the direct quantification of intracellular target proteins. unfortunately, the extremely low (e.g., pmol/l range) protein concentrations in blood cells limit their detection via ms/ms. in recent years, peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-srm) has emerged as a promising technique for the quantification of low abundance proteins in complex matrices, including dried blood spots (dbs). our lab has demonstrated that immuno-srm methods are able to reliably distinguish affected patients from the normal controls for wilson disease (wd), wiskott-aldrich syndrome (was), severe combined immunodeficiency (scid), and x-linked agammaglobulinemia (xla) (j. proteome res., and front. immunol., in press). these results demonstrate the utilization of immuno-srm as a sensitive platform for multiplexed quantification of signature peptides in the low pmol/l range. methods: several candidate peptides for each protein were selected based on uniqueness using in silico blast tools and lc-ms/ms response. monoclonal antibodies (mabs) were then generated for peptide enrichment from dbs. blood from normal controls, wd, xla, scid, and was patients was spotted onto filter paper, dried, and stored at - °c until use. proteins were extracted from dbs, digested with trypsin, and enriched using mabs bound to magnetic beads. the enriched peptides were then eluted and analyzed using srm mode with a waters xevo tq-xs. results/conclusions: to date, immuno-srm methods have been generated for wd, was, scid, xla, and cystinosis. preliminary data shows immuno-srm methods are able to reliably quantify target proteins using signature peptides and accurately distinguish affected patients from normal controls. analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (was and btk: p = . , scid: p = . ). intra and inter-assay precision ranged from - % and - %, respectively, and the multiplexed assay showed a broad linear range ( . fmol peptide) . in a blinded sample set of pidd patients and normal controls, immuno-srm-predicted diagnoses showed excellent agreement with clinical or genetic diagnoses. every molecularly-confirmed case of was and btk was also diagnosed by immuno-srm analysis. in addition, randomly selected samples provided by the nbs laboratory of washington state were tested and peptide concentrations were found to be within normal ranges. efforts are underway to validate and incorporate peptide biomarkers for adenosine deaminase deficiency, dock deficiency, and ataxia telangiectasia, as well as general markers for nk cells and platelets into a single multiplexed assay. in addition, scid, was and xla samples continue to be run while we focus on reducing assay costs, time, and necessary sample input. our data herein provides proof of concept for the immuno-srm workflow to be extended to various other genetic diseases as potential multiplexed newborn screening methods.( ) submission id# the background: the long-term effects of glucocorticoids (gcs) on the immune system have been extensively studied in patients with different underlying conditions (e.g, malignancies or autoimmune conditions), as well as in healthy volunteers receiving short-term courses of these drugs. although these approaches provided highly relevant data, neither of them answered the unbiased/bona-fide effect of long-term gcs use on the immune system. endogenous cushing syndrome (ecs) may be caused by pituitary or ectopic acth-producing adenomas, or by tumors or hyperplasia of the adrenal cortex. patients with ecs present with different gcsdependent manifestations, including those affecting the immune system as neutrophilia and lymphopenia. when tumors are removed, most of the effects of gcs tend to progressively regress. methods: paired samples from patients with ecs due to acth-producing adenomas (age range - y, females) were studied before (ecs-pre) and - months after tumor removal (ecs-post). extended lymphocyte phenotypes and apoptosis in different cell subsets were evaluated by flow cytometry. cytokine production (elisa) and responses, as well as their effects on cell proliferation and viability, were evaluated using cell trace violet and annexin-v staining. results: among multiple immunophenotypic changes, ecs-pre patients showed significantly reduced naïve t cells and recent thymic emigrants (rte) as well as increased apoptosis in t cells when compared to themselves (ecs-post) or age matched healthy controls. moreover, significantly increased exhausted cd t cells were observed in ecs-pre patients. interestingly, ecs-post patients showed full cellularity recovery of t cells and rte with increased proliferation and reduced apoptosis, in addition to correction of most of the other changes evidenced. significantly lower il- plasma levels were also detected in ecs-pre when compared to ecspost patients. to determine the role of il- in an ecs-resembling condition, healthy control pbmcs were treated with gcs in-vitro and the effect of il- and other cytokines was tested. a significant reduction in apoptosis was observed in the il- -treated cells that almost completely countered the pro-apoptotic effects of gcs; il- was also significantly more efficient than il- , il- , ifn-alpha and ifn-gamma in rescuing cells from apoptosis. il- -specific upregulation of bcl and bcl expression was evidenced in these cells.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -wc i fc authors: frese, michael; dazert, eva title: interferon-induced effector proteins and hepatitis c virus replication date: journal: hepatitis c virus disease doi: . / - - - - _ sha: doc_id: cord_uid: wc i fc hepatitis c virus (hcv) is a small, enveloped rna virus that is often capable of establishing a persistent infection, which may lead to chronic liver disease, cirrhosis, hepatocellular carcinoma, and eventually death. for more than years, hepatitis c patients have been treated with interferon-alpha (ifn-α). current treatment usually consists of polyethylene glycol-conjugated ifn-α that is combined with ribavirin, but even the most advanced ifn-based therapies are still ineffective in eliminating the virus from a large proportion of individuals. therefore, a better understanding of the ifn-induced innate immune response is urgently needed. by using selectable self-replicating rnas (replicons) and, more recently, recombinant full-length genomes, many groups have tried to elucidate the mechanism(s) by which ifns inhibit hcv replication. this chapter attempts to summarize the current state of knowledge in this interesting field of hcv research. interferons (ifns) are a diverse class of cytokines with key functions in the innate immune response to viruses (reviewed in pestka et al., ; goodbourn et al., ; samuel, ) . three types of ifns can be distinguished that have partially overlapping biological properties. type i ifns are secreted by most virus-infected cells and by a highly specialized leukocyte population, termed natural ifn-producing cells or plasmacytoid dendritic cells (colonna et al., ) . the human genome contains many type i ifn genes encoding ifn-α subtypes, ifn-β, ifn-ε, ifn-κ, and ifn-ω. the reason why the human genome encodes so many ifn-α subtypes is not known but has been speculated that different subtypes elicit a slightly different antiviral response. furthermore, it is tempting to speculate that the most recent multiplication of ifn-α genes is a consequence of an ongoing arms race between viruses that encode soluble ifn receptors and the innate immune defense system. type i ifn genes differ from all other ifn genes by the fact that they lack introns. recently, three distantly related cytokines have been identified that share sequence similarities with type i ifns and the interleukin- (il- ) family. accordingly, these cytokines have been named ifn-λ , ifn-λ , and ifn-λ (kotenko et al., ) or il- , il- a, and il- b, respectively (sheppard et al., ) . although it seems that many biological properties of this most recently discovered group of ifn-like cytokines resemble those of type i ifns, they are referred to as type iii ifns. in contrast to types i and iii ifns, of which numerous genes have been identified, the human genome contains only one type ii ifn gene. the gene product, ifn-γ, is only expressed in specialized immune cells such as activated t lymphocytes and natural killer (nk) cells. all types of ifns bind to highly specific cell surface receptors that trigger the phosphorylation and nuclear translocation of a family of latent transcription factors, known as signal transducers and activators of transcription (stats). type i ifns bind to the ifn-α receptor (ifnar), which leads to the formation of the ifn-stimulated gene factor- (isgf- ), a heterotrimer consisting of stat , stat , and ifn-response factor- (irf- /p ). isgf- activates gene transcription via the ifn-stimulated response element (isre). type iii ifns bind to a different receptor complex consisting of the ifnlr /il- rα subunit and the il- β subunit (donnelly et al., ) but nevertheless trigger a signaling cascade that is very similar to that of type i ifns (doyle et al., ) . a slightly different signaling pathway has been described for ifn-γ. the type ii ifn binds to the ifn-γ receptor (ifngr) which leads to the phosphorytation of the gamma activation factor (gaf), a phosphorylated stat homodimer, is translocated to the nucleus, where it enhances gene expression by binding to the gamma activation site (gas). beside these wellestablished signaling pathways, alternative pathways have been described, but their contribution to the antiviral activity of ifn remains to be further elucidated (pestka et al., ) . types i and iii ifns are believed to execute their antiviral activities through the induction of proteins that accumulate inside an infected host cell. these effector proteins may interfere with distinct steps in viral replication or trigger the degradation of viral rnas. by contrast, ifn-γ predominantly induces the expression of proteins with systemic functions, such as those involved in antigen processing and presentation. in addition, ifn-γ induces the expression and release of chemokines that activate and orchestrate the adaptive immune response (e.g., . however, at least in some virus infections, ifn-γ may also contribute to the establishment of an antiviral state by the induction of proteins with direct antiviral activities (reviewed in guidotti & chisari, ) . of note, all ifns that have been tested so far inhibit hepatitis c virus (hcv) rna replication in cultured cells, although differences have been noted in respect to the ic and the kinetics of inhibition. hcv is a member of the genus hepacivirus that belongs to the family flaviviridae (van regenmortel et al., ) . hcv isolates can be grouped into at least genotypes that differ in their nucleotide sequence by % to %. furthermore, within a given genotype, subtypes can be defined that differ in their nucleotide sequence by % to %. different genotypes show a remarkable degree of heterogeneity with respect to antiviral treatment (mchutchison & fried, ) . for example, only % of patients infected with genotype mount a sustained antiviral response, whereas % to % of those infected with genotype and genotype viruses do so. this is of immediate medical significance, and numerous attempts have been made to identify the viral factor(s) that determine the outcome of current ifn therapies. the underlying molecular mechanisms are, however, still controversial. hcv has a ∼ . -kb single-stranded rna genome of positive polarity (reviewed in bartenschlager et al., ) . the genome encodes a large polyprotein that is coand post-transcriptionally cleaved by cellular and viral proteinases into proteins (core, e , e , p , ns , ns , ns a, ns b, ns a, and ns b). the production of an additional protein (f) by ribosomal frame shifting has been reported, but its function remains to be defined. the coding sequence is flanked by nontranslated regions (ntrs) that are important for rna translation ( ntr) and replication ( and ntr). both ntrs are highly structured and contain numerous stem loops, most notably in the internal ribosome entry site (ires) of the ntr (honda et al., ) but also in other regions such as the x-tail sequence of the ntr (blight & rice, ) . furthermore, ntr sequences have been shown to interact with complementary coding sequences, which further increases the amount of intramolecular base pairing (kim et al., ; friebe et al., ) . figure shows the organization of the hcv genome and depicts some of its key structural elements. the figure also summarizes major protein functions, including those that counteract the innate immune response. hcv replication takes place in the cytoplasm of persistently infected hepatocytes, the principal host cells of the virus. detailed information on the mode of rna replication is not available for hcv, but by analogy to other flaviviruses (westaway et al., ) , it has been proposed that the incoming positive-stranded rna genome is used as a template for the synthesis of a negative-stranded rna molecule that remains base-paired with its template. the resulting double-stranded rna (also called "replicative form") is then transcribed multiple times, which results in the generation of numerous full-length, positive-stranded progeny rnas that may used for replication, translation, or packaging into newly formed virus particles (for details, see bartenschlager et al., ) . double-stranded rna that is formed by intramolecular base pairing between complementary sequences of positive-stranded hcv rnas or during viral replication should alert the double-stranded rna detection system of the host cell. this would the virus genome contains a large open reading frame (orf) that encodes all major viral proteins and an alternative orf that encodes the frame shift protein (f), which has an unknown function. the structural proteins c, e , e , and p are liberated from the polyprotein by cellular signal peptidases, and all other cleavages are performed by viral proteases. rna secondary structures are drawn according to blight & rice ( ) , honda et al. ( ) , you et al. ( ), and friebe et al. ( ) . black dots indicate the position of the start and the stop codon of the large orf. the minimum regions in the and nontranslated regions (ntrs) required for replication and initiation of translation are encircled with dotted lines. rdrp, rna-dependent rna polymerase; ires, internal ribosome entry site; bsl . , stem loop . within the coding sequence of ns b normally result in the production of type i ifns and the subsequent expression of ifn-induced effector proteins, which in turn would establish an antiviral state in the ifn-producing cell itself and in neighboring cells. hcv-infected hepatocytes, however, seem not to produce much ifn, as liver biopsy samples of most chronic hepatitis c patients lack detectable amounts of type i ifn mrnas (mihm et al., ) . this finding is in line with the observation that cultured human hepatoma cells produce only small amounts of ifns in response to viral infections or other stimuli such as poly(inosine[i])-poly(cytosine[c]), suggesting that human hepatocytes are generally rather poor ifn producers (keskinen et al., ) . however, primary chimpanzee and tamarin hepatocytes were found to be highly responsive to poly(i)-poly(c), which raises the question of why the liver of chronic hepatitis c patients does not contain type i ifns (lanford et al., ) . this enigma has recently been solved by foy and co-workers, who reported that the hcv ns / a protease interferes with the ability of cells to sense double-stranded rna (foy et al., ) . ns / a-mediated cleavage of the adaptor protein trif reduces its abundance and inhibits poly(i)-poly(c)-activated signaling through the toll-like receptor pathway before its bifurcation to irf- and nuclear factor-κb (nfκb)-mediated gene activation pathways (li et al., ) . furthermore, ns / a cleaves the adapter protein mavs/ips- /visa/cardif, which interrupts the signaling between the doublestranded rna binding protein rig-i and kinases that phosphorylate the ifn regulatory factors irf- and irf- (meylan et al., ) . this act of sabotage efficiently prevents the nuclear import of the latent transcription factors irf- and irf- , a crucial step in the activation of type i ifn gene transcription (reviewed by hiscott et al., ) . taken together, these findings suggest that hcv-infected hepatocytes are prevented from producing the amount of type i ifn that is needed to assist virus clearance. nevertheless, type i ifn-induced mrnas/proteins are readily detectable in liver biopsies of hepatitis c patients, even in liver samples that do not contain detectable amounts of type i ifn mrnas (mihm et al., ) . this begs the question as to where the ifn that is not locally produced comes from. according to mihm and co-workers, natural ifn-producing cells or plasmacytoid dendritic cells may represent an important extrahepatic source of ifn in hepatitis c patients (mihm et al., ) . however, natural ifn-producing cells have the propensity to migrate to secondary lymphoid organs rather than to sites of inflammation (penna et al., ) . as a consequence, the expression of type i ifn-induced proteins may never reach levels required to eliminate the virus from already infected cells and/or to prevent the infection of new host cells. all currently licensed hcv therapies rely on the antiviral activity of type i ifn (mostly polyethylene glycol-conjugated ifn-α ) that is given alone or in combination with ribavirin. the administration of recombinant ifn bypasses the block of ifn production in hcv-infected host cells and dramatically increases the expression of type i ifn-induced proteins throughout the body. this leads in most cases to a rapid decline of hcv rna levels (first-phase response), which is believed to reflect an inhibition of virus replication. later on, hcv rna levels decline more gradually (second-phase response) as the liver is cleared of virus-infected cells (neumann et al., ; layden & layden, ) . although ifn-α initially reduces the viral load in almost all patients, a sustained response (as defined by the loss of detectable hcv rna during therapy and its continued absence for at least months after the treatment has been ended) is not experienced by all patients. especially those patients who suffer from an infection with genotype b viruses often fail to eradicate the virus (manns et al., ; fried et al., ) . the correlation between therapy success and the infecting genotype suggests the involvement of viral factors, but the underlying molecular mechanisms are not yet understood. with the development of hcv replicons (lohmann et al., ) , it became possible to analyze the role of individual cytokines in the innate immune response against hcv. because most patients respond, at least initially, to a treatment with ifn-α, it was not unexpected that this ifn and other type i ifns also block rna replication of different hcv genotypes in human hepatoma cells (blight et al., ; frese et al., ; guo et al., ; cheney et al., ; larkin et al., ; okuse et al., ; windisch et al., ; miyamoto et al., ) and in cells of nonhepatic origin, e.g., hela cells (guo et al., ) and cells (ali et al., ) . similar results were obtained by using type iii ifns (robek et al., ; marcello et al., ) and ifn-γ (cheney et al., ; frese et al., ) but not other antivirally active cytokines such as tnf-α (frese et al., ) . the idea that ifn-γ enforces the critical first line of defense in the hcv-infected liver was further elaborated by li and co-workers, who demonstrated in a co-culture experiment that nk cells block hcv replication in huh- cells through the secretion of ifn-γ (li et al., ) . clinical data are limited and it is still controversial discussed whether hepatitis c patients benefit from ifn-γ administrations. nevertheless, it is interesting to note that types i and ii ifns inhibit hcv rna replication in huh- cells in a highly synergistic manner (larkin et al., ; okuse et al., ) . given the power of combination therapies in the treatment of other persistent virus infections, it might be rewarding to elucidate the mechanism(s) responsible for the observed synergistic antiviral effects of different ifn types. for example, do ifn-α and ifn-γ enhance the expression level of one or more effector proteins in a synergistic manner as suggested by tan et al. ( ) , or do they induce the expression of different, ifn type-specific effector proteins that interfere with more than one step of the hcv life cycle as suggested by windisch et al. ( ) ? the answers to these questions may help physicians to predict the outcome of ifn therapies and lead to the improvement of ifn-based therapies. several attempts have been made to analyze systematically the ifn-induced changes in the gene expression of hcv host cells. in one approach, liver biopsy samples were taken from expetimentally infected chimpanzees and the gene expression profile was monitored by using cdna microarrays. the results revealed that the infection of the liver rapidly leads to the upregulation of numerous genes including those encoding well-known ifn-induced effector proteins such as the chimpanzee homologue of mxa (bigger et al., ; su et al., ) . in both studies, the expression of mxa and that of other type i ifn-induced proteins correlated with the magnitude and duration of the infection. however, transient and sustained viral clearances were rather associated with the production of ifn-γ and the subsequent expression of type ii ifn-induced genes, suggesting a biphasic course of the innate immune response and a crucial role for ifn-γ in virus clearance. in another approach, cdna microarrays were used to analyze ifn-induced changes in the gene expression profile of cultured human cells containing hcv replicons hayashi et al., ) . even if these and similar studies did not lead to the identification of the effector proteins that inhibit hcv replication in ifn-stimulated cells, they will guide present and future investigations by suggesting potential candidate genes. the contribution of some of the most prominent ifn-induced effector proteins (figure ) to the ifn-induced inhibition of hcv replication is discussed in the following paragraphs. antiviral pathways that may contribute to the establishment of the so-called antiviral state in ifn-stimulated human cells. from left to right: the mxa gtpase inhibits viral replication by missorting and trapping of viral components into large membrane-associated complexes (the role of gtp hydrolysis in this process is not fully understood). three different ifn-induced oligoadenylate synthetases (oas , oas , and oas ) are encoded by the human genome. binding to double-stranded rna (dsrna) leads to hetero-and/or homo-oligomerization and subsequently to the production of oligoadenylates with a , -phosphodiester bond linkage. these - a oligonucleotides activate the latent endoribo-nuclease rnase l, which leads to the degradation of viral and cellular rnas (in some cell types, the expression of rnase l is also regulated by ifns). the p isoform of the adenosine deaminase adar binds to double-stranded rna and catalyzes the conversion of adenosine to inosine (a to i). such editing may occur selectivity at one or a few positions, or more frequently, at a large number of sites. editing of viral rnas may change the coding sequence, activate an i-specific rnase, and/or destroy rna secondary structures by disrupting adenosine/uracil base pairings. the double-stranded rna-activated protein kinase pkr may block viral protein translation by the phosphorylation and thereby inactivation of the eukaryotic initiation factor eif a. furthermore, pkr may activate intracellular signaling pathways that contribute to the establishment of a robust antiviral response. the inducible nitric oxide synthetase nos produces large amounts of nitric oxide (no), which is implicated in a variety of immune functions such as the activation of macrophages mx proteins belong to the superfamily of dynamin-like large gtpases and their expression is tightly regulated by type i and type iii ifns (holzinger et al, ; reviewed in haller & kochs, ; haller et al, ) . of the two human mx proteins, mxa and mxb, only mxa has demonstrable antiviral activity. mxa is a cytoplasmic protein with a size of ∼ kda that has been shown to inhibit the replication of a broad variety of rna viruses. cell culture experiments demonstrate that mxa inhibits orthomyxoviruses, bunyaviruses, rhabdoviruses, birnaviruses, reoviruses, and togaviruses (mundt reviewed in haller et al., . in some cases, viral replication is almost completely blocked by mxa. for example, stably transfected vero cells that constitutively express mxa produce up to , , -fold lower virus titers than control cells that did not express any mx proteins . the antiviral effect of mxa has also been analyzed in vivo by using transgenic mice that constitutively express the human mxa protein but lack functional mouse mx proteins and mice that constitutively express mxa but cannot mount a proper ifn-induced antiviral response due to a disruption in the gene for the β subunit of the ifn type i receptor. in both cases, mxa-expressing animals were found to be completely resistant to thogoto virus, a tick-borne orthomyxovirus hefti et al., ) . furthermore, mxa-expressing animals exhibited an enhanced resistance against . . . influenza a virus (family orthomyxoviridae), vesicular stomatitis virus (vsv; family rhabdoviridae), lacrosse virus (family bunyaviridae), and semliki forest virus (family togaviridae) hefti et al., ) . other reports suggest that mxa has an even wider antiviral activity, but the supporting data are less convincing. the modus operandi of mxa is not completely understood, but accumulating data indicate that cytoplasmic mx proteins missort and immobilize viral components. in cells that had been infected with lacrosse virus, mxa binds and translocates the viral nucleocapsid protein into membrane-associated perinuclear complexes reichelt et al., ) . a similar phenomenon was observed in cells that had been infected with thogoto virus. in this case, however, mxa inhibited the nuclear transport of incoming viral nucleocapsids , thereby preventing primary transcription and leading to an early and very efficient block of virus replication. in healthy individuals, mxa expression is below the detection limit, but expression levels increase dramatically during many viral infections and as a consequence of ifn-α treatment (roers et al., ; chieux et al., ) . mxa mrna quantification in peripheral blood mononuclear cells has even been used to monitor the bioavailability of administered type i ifns in hepatitis c patients (gilli et al., ; jorns et al., ) . not surprisingly, elevated mxa expression levels have also been found in the liver of chronic hepatitis c patients, indicating an ongoing struggle between the innate immune system and hcv (macquillan et al., (macquillan et al., , patzwahl et al., ) . a genetic study from japan addressing a single nucleotide polymorphism at position - in the promoter sequence of the mxa gene revealed that a thymidine (t) in that position favors a sustained response of hepatitis c patients to treatment with ifn-α, whereas a guanosine (g) is more frequently found among nonresponders (hijikata et al., ) . interestingly, a t at that position increases the homology of the first isre in the mxa promoter to the isre consensus sequence (hijikata et al., ) . furthermore, experiments with reporter constructs suggest that the t allele has a higher transcriptional activity than the g allele when stimulated with ifn-α (hijikata et al., ) . a similar association between the g/t single nucleotide polymorphism at position - of the mxa gene and the response of hepatitis c patients to ifn-α therapy was found in a european study, in which the t genotype was also found to be associated with the ability to clear hcv naturally without the help of recombinant ifn (knapp et al., ) . since mxa has the ability to efficiently inhibit a variety of different rna viruses, mxa was the first ifn-induced effector protein to be analyzed for its antiviral activity in the hcv replicon system (frese et al., ) . however, no evidence was found for an involvement of mxa in the ifn-induced inhibition of hcv rna replication. the constitutive expression of mxa did not inhibit subgenomic hcv replicons, and the expression of a dominant-negative mutant of mxa did not restore hcv rna replication during ifn-α treatment (frese et al., ) . these earlier observations are in line with the more recent finding that ifn-α inhibits hcv rna replication in huh- cells and huh cells with a similar ic ( to iu/ml and to iu/ml, respectively), although the former produce nearly -fold more mxa mrnas than the latter (windisch et al., ) . taken together, the data indicate that ifn-α inhibits hcv rna replication by mxa-independent pathways. most recently, it has been noted that brefeldin a, a golgi apparatus disrupting agent, renders the replication of kunjin virus susceptible to mxa (hoemen et al., ) . since kunjin virus and hcv are both flaviviruses that use host cell-derived membranes to establish replication factories, it is tempting to speculate whether a disruption of the membranous web in hcv-infected cells would expose hcv rna-protein complexes to antivirally active proteins such as mxa. the oas/rnase l pathway (also known as ifn-inducible - a response) requires two types of enzymes, an oligoadenylate synthetase and a ribonuclease (reviewed in samuel, ) . the human genome contains four gene loci that encode ifn-induced oligoadenylate synthetases (oas , oas , and oas ) and an oas-like protein. this and alternative splicing leads to the expression of numerous isoforms with sizes ranging from to kda (rebouillat & hovanessian, ) . oas protein expression is enhanced in response to most, if not all, ifns, but the magnitude of induction can vary dramatically with different ifns and the type of the producing cell. newly produced oas proteins are believed to be inactive, but binding to double-stranded rna leads to their oligomerization and starts the production of oligoadenylates with a , -phosphodiester bond linkage ( - a oligonucleotides). these oligonucleotides bind to and activate the latent ribonuclease rnase l, a process that is associated with the formation of stable rnase l homodimers. once activated, rnase l can degrade single-stranded rnas of viral and cellular origin. cleaving of target rnas occurs preferentially on the side of uracil-adenosine (ua) and uu dinucleotides (floyd-smith et al., ; wreschner et al., ) . the cleavage of mrna and rrna may trigger a general protein shut-off in virusinfected cells, thereby limiting virus replication and spread. different oas proteins are associated with different cellular compartments, vary with respect to the amount of double-stranded rna needed for activation, and produce - a oligonucleotides of different sizes (samuel, ) . it is therefore tempting to speculate that the diversity of oas proteins and isoforms evolved to fight a rather wide spectrum of dna and rna viruses including poxviruses, reoviruses, and picornaviruses. members of the family picornaviridae seem to be especially sensitive to the oas/rnase l pathway. for example, overexpression of the -kda form of the human oas protein confers resistance to mengovirus but not vsv (chebath et al., ) , and the constitutive expression of the -kda form of the oas protein inhibited the replication of encephalomyocarditis virus but not that of vsv, sendai virus, and a reovirus (ghosh et al., ) . in this context it is interesting to note that one of the oas gene loci has recently been identified to confer increased resistance to the west nile virus (family flaviviridae) in laboratory mice (perelygin et al., ) and that the transcript of the oas b allele in susceptible mice contains a premature stop codon, which results in a truncated protein (mashimo et al., ) . experiments with congenic mice and cells derived from those mice revealed that expression of the full-length oas protein limited virus production in vivo and in cell culture. surprisingly, however, rnase l activity was highest in susceptible cells, and downregulation of rnase l activity in resistant cells did not restore virus titers to levels observed in susceptible cells (scherbik et al., ) . as with many other ifn-induced proteins, oas protein expression is slightly upregulated in hepatitis c patients (macquillan et al., ) and further enhanced in response to the administration of recombinant ifn-α (murashima et al., ) . rather indirect evidence that the oas/rnase l pathway may indeed target hcv replication/translation was recently provided by taguchi and co-workers, who reported that the n-terminal portion of ns a (amino acids to ), which lacks the so-called pkr-binding domain, binds to oas proteins and there by counteract the antiviral activity of ifn-α (taguchi et al., ) . furthermore, it has been demonstrated that purified recombinant rnase l and that from hela cell extracts efficiently cleaves hcv rna in vitro (han et al., ) . however, further investigations are needed to determine whether rnase l also cleaves hcv rnas in infected hcv host cells and to what extent an oas-induced block of hcv protein translation contributes to the ifn-induced inhibition of hcv rna replication. adar forms together with adar and the less extensively studied adar protein a small family of constitutively expressed adenosine deaminases that act on rna (reviewed in valente & nishikura, ; toth et al., ) . adar and adar bind highly structured rnas and catalyze the hydrolytic c deamination of adenosine, a reaction that converts adenosine to inosine (a to i editing). adarmediated editing may occur selectively at one or a few positions or, more frequently, at a larger number of sites (hyperediting or hypermutation). a to i exchanges may have severe consequences: ( ) editing of coding sequences may lead to amino acid exchanges because i is recognized as g by the translational machinery (of note, a to i editing does not create stop codons); ( ) editing of noncoding regions may affect rna splicing, stability, or translational efficiency (e.g., by disrupting au base pairs); ( ) editing may regulate gene silencing (e.g., by disrupting au base pairs); and ( ) hyperedited rna may be recognized and cleaved by an i-specific rnase (scadden & smith, , . a prominent example of a cellular rna that is edited by adar proteins is the mrna of the alpha-amino- -hydroxy- -methyl- -isoxazole propionate (ampa) receptor subunit glur- . adar edits a codon in exon , which results in an amino acid exchange that changes the ca + permeability of the receptor. this highly specific editing event has far-reaching consequences. adar knockout mice are prone to seizures and die young. the impaired phenotype appears to result entirely from a single underedited position in the glur- mrna, as it reverted to normal when both alleles for the underedited transcript were substituted with alleles encoding the edited version exonically (higuchi et al., ) . likewise, genetic targeting of the adar locus revealed an essential requirement for this adar protein in the embryogenesis of mice (wang et al., (wang et al., , hartner et al., ) , and it has been suggested that its expression protects against stress-induced apoptosis (wang et al., ) . a closer look at the adar gene locus revealed that protein expression is controlled by three promoters and alternative splicing (reviewed in toth et al., ) . two constitutively active promoters drive the expression of a ∼ -kda protein (p ), whereas an ifn-regulated promoter with an isre controls the expression of a larger isoform (p ) that is expressed in response to inflammation or ifn treatment (patterson & samuel, ; george & samuel, ; yang et al., a yang et al., , b . both isoforms contain multiple nuclear localization signals, but only the ifn-induced p isoform has a nuclear export signal. accordingly, p is a nuclear protein and p has been detected in both nuclear and cytoplasmic compartments. hypermutation of viral rnas has been observed for several rna viruses, including measles virus, parainfluenza virus (both family paramyxoviridae), and vsv (o'hara et al., ; cattaneo et al., ; murphy et al., ) . it has been speculated that subacute sclerosing panencephalitis (sspe), a fatal necropathic response in patients with a persistent measles virus infection of the brain, is associated with extensive editing of the matrix protein mrna. this prevents virion assembly and release because these steps in the viral life cycle require a functional matrix protein. other transcripts, however, are less frequently edited, which is thought to result in a persistent virus replication (cattaneo et al., ; baczko et al., ) . thus, an incomplete adar-mediated innate immune response might contribute to the pathology of sspe. interestingly, certain viruses abuse adar proteins to control important checkpoints in replication and particle formation. a well-known example is the hepatitis d virus (hdv), a subviral human pathogen that depends on hepatitis b virus as a helper virus (reviewed in casey, ) . hdv has a small, circular rna genome that encodes only a single protein, the hepatitis delta antigen (hdag). without editing, a -amino acid version of hdag is made that is essential for virus replication (kuo et al., ) . later on, in the viral life cycle a highly specific a to i editing event changes a uag amber stop codon to an uig tryptophan codon, and a -amino acid hdag-l is produced that mediates genome packaging (chang et al., ) . hcv rnas may also be subject to adar-mediated modifications, but in this case, editing seems to be less specific and to inhibit virus replication. it has recently been reported that the silencing of adar expression in hcv replicon cells increases the amount of hcv rna about -fold . moreover, taylor and co-workers noted that ifn-α increases the frequency of a to g mutations in subgenomic replicon rnas and that the transfection of adar-specific sirnas rescues hcv rna replication in the presence of moderate ifn-α concentrations. based on these findings, taylor et al. concluded that ifn-α inhibits hcv replication through adar-mediated hyperediting of viral rna. in our laboratory, we have used specific antibodies to determine the intracellular localization of p anddespite its nuclear export signal -, we observed that p accumulates predominantly in the nucleus of ifn-treated huh- cells. in the presence of subgenomic or full-length hcv rnas, however, we observed that p localizes to distinct cytoplasmic structures (e. dazert, r. bartenschlager, and m. frese, unpublished results) . we also analyzed the antiviral effect of constitutively expressed p on hcv replication and found that the overexpression of p in huh- cells did not block hcv rna replication. taken together, our findings support the idea of taylor et al. that p interacts with hcv rnas, but we argue that p does that only in the context off other ifn-induced proteins. further studies are under way to fully elucidate the role of p in the ifn-induced inhibition of hcv replication. another prominent protein of the innate immune defense that has long been suspected of interfering with hcv replication is the double-stranded rna-activated protein kinase pkr. this serine/threonine kinase is constitutively expressed and has multiple functions in the control of host cell transcription and translation (reviewed in garcia et al., ) . upon stimulation with ifns, most cells respond by increasing the expression of pkr. ifn-induced pkr accumulates in the cytoplasm and was found in association with ribosomes (thomis et al., ) . pkr may exert its antiviral activity through different pathways (reviewed in toth et al., ) . first, pkr is able to control the cellular translation machinery through phosphorylation of the α subunit of the eukaryotic translation initiation factor eif- α, which would affect the production of both host and virus proteins. second, pkr-mediated phosphorylation is implicated in several signaling pathways that contribute to the establishment of a robust antiviral response. for example, pkr has been shown to activate the latent transcription factor nfκb, which may lead to the enhanced expression of proinflammatory genes (gil et al., ) . in addition, pkr may activate other kinases such as the p mitogen-activated protein (map) kinase, which further intensifies and diversifies the innate immune response (goh et al., ) . the concept that pkr-mediated phosphorylation events play an important role in the innate immune response against viral infections is largely based on the fact that many rna and dna viruses try to inhibit pkr by ( ) overexpressing small rnas that bind to but do not activate pkr, ( ) producing eif- α decoys, and ( ) enhancing pkr degradation (reviewed in langland et al., ) . if pkr is a key player in ifn-induced antiviral defense, genetically targeted knockout mice that lack functional pkr proteins should be extremely sensitive to viral infections. two lines of pkr −/− mice have been generated in which the coding sequences of either the n-terminal or the c-terminal part of the protein have been disrupted (yang et al., ; abraham et al., , respectively) . pkr −/− mice are indeed more susceptible to certain virus infections than wild-type animals (balachandran et al., ; stojdl et al., ; carr et al., ; samuel et al., ) , but at least in some cases, this seems to depend on the mouse strain used, and other experimental conditions (murphy et al., ) . additional experiments have been conducted by using mefs from pkr −/− mice, but a direct antiviral activity of pkr (e.g., the inhibition of virus multiplication by blocking protein translation) is still controversial. interestingly, priming of pkr −/− mice with poly(i)-poly(c) or ifns before the virus challenge points to a rather indirect mode of pkr action, such as the enhancement of double-stranded rna-induced signaling events (yang et al., ) . however, it should be noted that most of these experiments have been performed with viruses that encode pkr inhibitors. it would be interesting to re-evaluate the phenotype of pkr −/− mice with genetically modified viruses that cannot express functional pkr inhibitors. another problem in the characterization of pkr −/− mice is the presence of related kinases that also phosphorylate eif- α (toth et al., ) . even if these kinases differ from pkr in their response to double-stranded rna and/or other physiological stress signals, they may partially substitute for the lack of pkr in pkr −/− mice, thereby making it difficult to quantify the contribution of pkr to the innate immune response (as exemplified in smith et al., ) . two hcv proteins have been described as interacting with the kinase. by analyzing hcv sequences from japanese hepatitis c patients, mutations within a discrete region of ns a, the so-called ifn sensitivity determining region (isdr), were proposed to confer resistance to ifn-α (enomoto et al., (enomoto et al., , . since the original reports by enomoto and co-workers, numerous studies have been conducted in japan as well as in other countries to determine the predictive value of ns a sequences in the outcome of ifn-based therapies, but the existence of an isdr is still controversial (reviewed in tan & katze, ; reinvestigated by pascu et al., ; brillet et al., ) . whether or not an isdr really exists, the description of such a sequence put ns a in the focus of hcv research. the subsequent finding that mutations in the isdr affect the ability of ns a to bind to and inhibit pkr (gale et al., ) led to the hypothesis that pkr blocks hcv replication and that ns a is able to counteract the antiviral activity of pkr. however, experiments with hcv replicons provided no further evidence for an involvement of ns a in ifn resistance. on the contrary, point mutations within the isdr or a deletion of amino acids encompassing the entire isdr enhanced viral replication without affecting the ifn sensitivity of hcv replicons (blight et al., ; guo et al., ) . these findings were extended by a. kaul and r. bartenschlager, who analyzed the function of ns a by using two subgenomic genotype b replicons that differ only in the ns a coding sequence. in one replicon, the isdr was identical to that of ifn-susceptible strains, whereas the isdr sequence of the other replicon contained mutations that have been suspected to confer pkr binding and ifn resistance (gale et al., ) . despite these differences, both replicons were found to be equally sensitive to ifn-α (unpublished results) . this result argues against the hypothesis that ns a counteracts an ifn-induced and pkr-mediated block of viral protein translation. however, the result does not contradict the idea that ns a inhibits other activities of pkr (e.g., a pkr-mediated priming of intracellular signaling pathways). of note, several reports suggest that ns a may sabotage the innate immune response through pkr-independent activities (discussed in macdonald & harris, ) . for example, it has been reported that ns a increases the production of interleukin (il)- , thereby attenuating the antiviral properties of ifns (polyak et al., a (polyak et al., , b . it would be interesting to study the immunomodulatory activities of ns a in an immunocompetent small animal model, which might finally put an end to the discussion about the role of pkr in hcv pathology. a second hcv protein has been reported to interact with pkr. it was found that e binds to pkr through its pkr-eif α homology domain (pephd) (taylor et al., (taylor et al., , pavio et al., ) . however, the significance of this observation has been questioned because an increasing number of clinical studies demonstrate that the pephd is a highly conserved region with no conspicuous mutations accumulating during ifn-α therapy (reviewed in tan & katze, ) . furthermore, e expression does not increase the resistance of hcv genotype b replicons toward ifns. a genomic replicon that encodes an e protein with the pephd sequence of a resistant hcv isolate had a similar degree of susceptibility as a subgenomic replicon lacking e (frese et al., ; a. kaul and r. bartenschlager, unpublished results) . the adenovirus-associated rna i (va i ), a small, highly structured rna that binds to pkr and but does not trigger its dimerization and activation, has recently been found to stimulate hcv rna replication in the replicon system . it was also reported that recombinant va i rna efficiently rescues hcv rna replication in the presence of as much as iu/ml of ifn-α . since va i rnas may also bind to other proteins of the innate immune response such as adar , more research is needed to define the role of pkr in limiting hcv protein translation. a more direct approach to the question of pkr interference with hcv rna replication/translation has recently been undertaken by using rna silencing. a. kaul and r. bartenschlager transfected cells containing subgenomic hcv replicons with sirnas that target pkr mrnas for degradation and subsequently treated the cells with different concentrations of ifn-α. in no case did they observe that a downregulation of pkr expression levels results in a restoration of hcv replication in the presence of ifn (unpublished results). with the establishment of a new generation of hcv replicons that contain the consensus sequence from a japanese genotype a isolate and replicate efficiently without the need for adaptive mutations, it became possible to study hcv rna replication in a variety of new host cells including those of nonhepatic and nonhuman origin (kato et al., ; uprichard et al., ) . most recently, genotype a replicons were employed by chang and co-workers, who set out to analyze the antiviral effect of type i ifns on hcv replication in mefs from pkr −/− mice and congenic wild-type mice. interestingly, ifn-α as well as ifn-β inhibited hcv rna replication in pkr −/− mefs as efficiently as in pkr +/+ mefs (chang et al., ) , suggesting that pkr-mediated translational control plays only a minor role in the ifn-induced inhibition of hcv rna replication. the inducible nitric oxide (no) synthetase, originally named inos but also abbreviated as nos , belongs to a small family of no-producing enzymes. in unstimulated cells, nos is virtually absent, but expression levels increase rapidly in response to pro-inflammatory cytokines, especially ifn-γ. the expression of nos results in a long-lasting production of no (karupiah et al., ) . the no free radical has been recognized for its strong antimicrobial activity against various protozoa, bacteria, and viruses. for example, the replication of a coxsackievirus is suppressed by no through inactivation of the viral cysteine protease by s-nitrosylation (saura et al., ) . furthermore, no production is essential for the t cell-mediated noncytopathic inhibition of hepatitis b virus replication in virus-transgenic mice (guidotti et al., ) . other viruses that have been reported as sensitive to no include severe acute respiratory coronavirus (akerstrom et al., ) , respiratory syncytial virus (stark et al., ) , mouse hepatitis virus (pope et al., ) , and herpes simplex virus type (adler et al., ) . however, the use of no by the infected host as an antimicrobial substance is a double-edged sword. no-induced oxidative stress may cause severe cellular and organ dysfunction. influenza a virus-infected wild-type mice, for example, suffer from an excessive production of no in the lungs, which often leads to respiratory failure and death, whereas knockout mice that cannot express functional nos survive the infection with little evidence of pneumonitis (akaike et al., ; karupiah et al., ) . in the liver of most hcv-infected individuals, nos is easily detectable (mihm et al., ; majano et al., ; schweyer et al., ) . the enhanced expression of nos in the liver of hepatitis c patients has largely been attributed to ifn-γ that is released by resident and infiltrating immune cells. in addition, it has been speculated that the hcv replication itself may stimulate the expression of nos in infected hepatocytes (machida et al., ) . based on genetic studies, it has been suggested that the production of no is involved in hcv clearance, as certain nos haplotypes were more frequently found among hcv-infected individuals who spontaneously cleared the infection and in ifn-treated hepatitis c patients who could mount a sustained antiviral response (yee et al., ) . this hypothesis, however, lacks supporting evidence from cell culture experiments. the treatment of huh- cells with the no donor (z)- -[ -(aminoethyl)-n-( -ammonioethyl)amino]diazen- -ium- , -diolate (deta nonoate) or the arginase inhibitor ng-hydroxy-l-arginine (noha) did not result in an inhibition of hcv rna replication (frese et al., ) . furthermore, the nos inhibitor l-n -( -iminoethyl)lysine (l-nil) did not even partially restore hcv replication in the presence of ifn-γ (frese et al., ) . one should, however, not overinterpret these results. the in vivo production of no may have more complex consequences than those that could be investigated in cell culture. no may act as a messenger rather than as an effector molecule, or no may induce dna damage and apoptosis (jaiswal et al., ) . thus, further studies are needed to fully elucidate the role of no in hepatitis c pathology. so far, only a few further ifn-induced effector proteins have been investigated with respect to their potential to inhibit hcv rna replication, most notably indoleamine , -dioxygenase (ido). the ido-mediated depletion of tryptophan is well known as a defense mechanism against certain intracellular parasites (carlin et al., ) . rather recently, this pathway has also been recognized as an ifn-γ-induced antiviral defense mechanism against herpesviruses (bodaghi et al., ) and poxviruses (terajima & leporati, ) . if ido inhibits hcv replication as well, inhibition of the effector protein or addition of tryptophan to the cell culture medium should restore viral protein synthesis. however, the ido inhibitor α-methyl-dl-tryptophan did not restore the replication of subgenomic hcv replicons in the presence of ifn-γ (frese et al., ) . likewise, increased concentrations of 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interferon-induced, rna-dependent p /eif- alpha protein kinase from human cells interferon action and the double-stranded rna-dependent enzymes adar adenosine deaminase and pkr protein kinase replication of a hepatitis c virus replicon clone in mouse cells adar gene family and a-to-i rna editing: diverse roles in posttranscriptional gene regulation the viith report of the international committee on taxonomy of viruses requirement of the rna editing deaminase adar gene for embryonic erythropoiesis stress-induced apoptosis associated with null mutation of adar rna editing deaminase gene replication and gene function in kunjin virus dissecting the interferon-induced inhibition of hepatitis c virus replication by using a novel host cell line interferon action -sequence specificity of the ppp(a p)na-dependent ribonuclease widespread inosine-containing mrna in lymphocytes regulated by adar in response to inflammation intracellular localization of differentially regulated rna-specific adenosine deaminase isoforms in inflammation deficient signaling in mice devoid of double-stranded rna-dependent protein kinase inducible nitric oxide synthase gene (nos a) haplotypes and the outcome of hepatitis c virus infection a cis-acting replication element in the sequence encoding the ns b rna-dependent rna polymerase is required for hepatitis c virus rna replication gene expression associated with interferon alfa antiviral activity in an hcv replicon cell line we are indebted to kerry mills, sandra thomas, ali zaid, friedemann weber, brett lidbury and ralf bartenschlager for helpful suggestions and careful reading of the manuscript; and artur kaul and r. bartenschlager for the communication of unpublished results. key: cord- - zwq do authors: guo, kejun; shen, guannan; kibbie, jon; gonzalez, tania; dillon, stephanie m.; smith, harry a.; cooper, emily h.; lavender, kerry; hasenkrug, kim j.; sutter, kathrin; dittmer, ulf; kroehl, miranda; kechris, katerina; wilson, cara c.; santiago, mario l. title: qualitative differences between the ifnα subtypes and ifnβ influence chronic mucosal hiv- pathogenesis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: zwq do the type i interferons (ifn-is) are innate antiviral cytokines that include different ifnα subtypes and ifnβ that signal through the ifn-i receptor (ifnar), inducing hundreds of ifn-stimulated genes (isgs) that comprise the ‘interferome’. quantitative differences in ifnar binding correlate with antiviral activity, but whether ifn-is exhibit qualitative differences remains controversial. moreover, the ifn-i response is protective during acute hiv- infection, but likely pathogenic during the chronic stages. to gain a deeper understanding of the ifn-i response, we compared the interferomes of ifnα subtypes dominantly-expressed in hiv- -exposed plasmacytoid dendritic cells ( , , , and ) and ifnβ in the earliest cellular targets of hiv- infection. primary gut cd t cells from donors were treated for hours ex vivo with individual ifn-is normalized for ifnar signaling strength. of , ifn-regulated genes, ‘core isgs’ were induced by all ifn-is tested. however, many ifn-regulated genes were not shared between the ifnα subtypes despite similar induction of canonical antiviral isgs such as isg , rsad and mx , formally demonstrating qualitative differences between the ifnα subtypes. notably, ifnβ induced a broader interferome than the individual ifnα subtypes. since ifnβ, and not ifnα, is upregulated during chronic hiv- infection in the gut, we compared core isgs and ifnβ-specific isgs from colon pinch biopsies of hiv- -uninfected (n = ) versus age- and gender-matched, antiretroviral-therapy naïve persons with hiv- (pwh; n = ). core isgs linked to inflammation, t cell activation and immune exhaustion were elevated in pwh, positively correlated with plasma lipopolysaccharide (lps) levels and gut ifnβ levels, and negatively correlated with gut cd t cell frequencies. in sharp contrast, ifnβ-specific isgs linked to protein translation and anti-inflammatory responses were significantly downregulated in pwh, negatively correlated with gut ifnβ and lps, and positively correlated with plasma il and gut cd t cell frequencies. our findings reveal qualitative differences in interferome induction by diverse ifn-is and suggest potential mechanisms for how ifnβ may drive hiv- pathogenesis in the gut. a a a a a the type i interferons (ifn-is) are innate antiviral cytokines that include ifnα ( different subtypes) and ifnβ [ ] . these cytokines significantly inhibited hiv- replication in vitro, but human clinical trials with ifnα showed only moderate or no inhibitory effects on hiv- infection [ , ] . all ifn-is bind to the same ifn-i receptor that is composed of two subunits, ifnar and ifnar , resulting in phosphorylation of jak and tyk . this in turn results in the phosphorylation of stat and stat that associate with irf to form the transcriptional activator, isgf [ ] . isgf translocates to the nucleus, where it binds to promoters of genes encoding ifn response elements (isres), resulting in the induction of hundreds of ifn stimulated genes (isgs), collectively referred to as the 'interferome' [ ] . recent studies highlighted ifnα as a potential adjunct to an hiv- curative strategy [ - ]. however, almost all hiv- clinical trials with ifnα were performed with only one subtype, ifnα , with mixed results (reviewed in [ , ] ). more recently, ifnα treatment of siv rhesus macaques under antiretroviral therapy did not reduce the latent hiv- reservoir [ ] . our group and others reported that ifnα only moderately inhibited hiv- in humanized mice and primary cd + t cells compared to the more potent subtypes ifnα , ifnα and ifnα [ ] [ ] [ ] [ ] . interestingly, the anti-hiv- potency of ifnα subtypes correlated with their binding affinity to ifnar , suggesting that the antiviral differences between the ifnα subtypes were due to quantitative differences in ifnar signaling strength [ , , ] . however, several human ifnα subtypes exhibit strong signals of purifying selection [ ] , suggesting that these ifnα subtypes may have evolved to have specific, nonredundant functions. some ifnα subtypes were better at inducing certain immune responses in vivo [ , ] and may trigger distinct intracellular signaling pathways [ ] . nevertheless, the notion of qualitative differences between ifnα subtypes remains controversial [ , ] . one reason is that most comparative studies normalized ifn-is using protein amounts or units/ml based on inhibition of vesicular stomatitis virus or encephalomyocarditis virus [ ] . to unravel qualitative differences between the ifn-is, it would be important to normalize ifn-is for quantitative differences in ifnar signaling strength. it is widely accepted that ifn-i signaling can prevent acute retrovirus infection. genetic ablation of ifnar resulted in higher friend retrovirus replication in mice co-infected with lactate-dehydrogenase elevating virus, a potent ifn-i inducer [ ] . moreover, ifnar blockade increased siv replication in rhesus macaques [ , ] . administration of ifnα decreased retrovirus replication in mice, monkeys and humans [ , , ] . however, in persistent virus infections, chronic ifn-i stimulation was linked to pathogenic outcomes [ ] [ ] [ ] [ ] [ ] . although clinical administration of ifnα increased the number of low-dose mucosal inoculations needed for breakthrough siv infection in rhesus macaques, once the infection was established, lower cd t cell counts were observed in ifnα -treated monkeys relative to untreated controls [ ] . these findings highlight that hiv- infection shares features with 'interferonopathies' such as aicardi-goutières syndrome [ ] , which are currently being targeted through ifn-i blockade strategies (clinicaltrial.gov nct ). in fact, ifnar blockade during chronic hiv- infection in humanized mice restored immune function, leading to better hiv- control [ , ] . neutralization of (most) ifnα subtypes in siv-infected rhesus macaques prior to infection increased viral loads as expected, but also decreased subsequent immune activation profiles [ ] . by contrast, blockade of ifn-i signaling in chronic sivtreated and untreated rhesus macaques decreased inflammation profiles associated with isgs but did not reverse t cell exhaustion or activation [ ] . the basis for the protective versus pathogenic effects of ifn-is remains unclear. one hypothesis is that the initial ifn-i response is protective due to the induction of antiviral factors, whereas chronic stimulation promotes inflammation through other isgs with sustained, elevated expression. distinct ifn-is present in the acute versus chronic stages of persistent viral infection may induce divergent cellular immune responses. tissue compartmentalization may also play a role. the gut is a critical site not only for early hiv- infection, but also in driving chronic immune activation [ ] . epithelial barrier dysfunction, partly due to the loss of th cells, leads to the translocation of enteric bacteria from the gut lumen to the lamina propria and systemic circulation, resulting in chronic immune activation [ ] [ ] [ ] . we recently reported increased ifnβ gene expression in the gut, but not the blood, in persons with hiv- (pwh) infection compared to age/gender-matched hiv- uninfected controls [ ] . by contrast, ifnα subtypes were downregulated in pwh, and ifnλ, a type iii ifn linked to mucosal immunity in mouse models [ ] , was undetectable in these samples [ ] . these findings suggest that among the diverse ifn-is, ifnβ may play a dominant role in the gut during chronic hiv- infection. here, we utilized transcriptomic approaches to evaluate whether the ifnα subtypes and ifnβ exhibit qualitatively different effects on gene expression. we then tracked how interferon-regulated genes were altered during chronic hiv- infection in the gut. our analyses highlight potential mechanisms driven by ifnβ that may influence mucosal hiv- pathogenesis. the relative anti-hiv- activity of ifnβ in primary lpmcs remains unclear, though studies using pbmcs suggest that ifnβ is relatively potent [ ] . we previously reported a spectrum of antiviral potencies of the ifnα subtypes against hiv- in primary lpmcs [ ] . these data were extended to show a -fold difference in % inhibitory concentrations (ic ) between ifnα and ifnα [ ] . using the same lpmc donors used to calculate the ic s of ifnα and ifnα , we titrated the anti-hiv- potencies of ifnα (weak), ifnα (potent) and ifnβ. the ifn-is were added into lpmc cultures at the time of infection with hiv- bal . at d post-infection, the percentage of hiv- gag p + cells were evaluated by flow cytometry. sigmoidal curves were fitted into the average inhibition data for donors, and used to calculate ic values (pg/ml protein concentration). we also calculated a metric known as 'vres', which corresponds to the level of residual virus replication at maximal doses of ifn-is [ ] . for comparison, previously reported inhibition curves for ifnα and ifnα are also shown [ ] . ifnα showed ic concentrations over -fold lower than that of ifnα (fig ) . notably, ifnβ had a similar potency as ifnα and ifnα . we also calculated ic s for individual donors (s a fig) and show significantly lower inhibition by ifnα compared to ifnα and ifnα (s b fig). ifnα and ifnα reduced hiv- infection levels to~ % at the maximal doses tested ( ng/ml), whereas ifnα , ifnα and ifnβ had higher vres between - %. however, these differences in vres were not significant (s c fig) . these findings validate prior results on the relative anti-hiv- activity of these ifnα subtypes and highlight ifnβ as a potent anti-hiv- ifn. quantitative differences were evident, as increasing the dose of weaker ifnα subtypes should enable these cytokines to inhibit hiv- just as well as the potent ifnα subtypes. the stronger anti-hiv- potencies of ifnα and ifnα compared to ifnα and ifnα were associated with higher isg induction [ ] , but it remained unknown how the other ifnα subtypes and ifnβ compare. to test the isre signaling activity of ifn-is, we used a commercially-available ilite assay (see methods). the ilite cells are human u monocytic cell lines transduced with a luciferase reporter downstream of an isre from isg , a canonical isg. serial -fold dilutions of the ifnα subtypes and ifnβ were added into the ilite cells and relative light units were measured after h. % effective concentrations (ec ) were then calculated from best-fit sigmoidal plots. fig a highlights the -fold ec difference in isre-activity between ifnα and ifnα . the ec s of the other ifnα subtypes and ifnβ fell in-between ifnα and ifnα data were normalized to mock (untreated) as % for each donor. dose-response curves were generated using a one-phase decay equation in graphpad prism . to determine ic values. vres, the percentage of cells that remain infected relative to the mock at the maximum dose tested, corresponded to plateau values from the decay equation. � note that data on ifnα and ifnα were previously published [ ] . https://doi.org/ . /journal.ppat. .g type i interferons in mucosal hiv- infection ( fig b) . notably, the isre ec of the ifnα subtypes significantly correlated with anti-hiv- potency data from our previous study (fig c) [ ] . a significant positive correlation was also observed between isre-activity and the ic values from the ifn-is tested in fig that includes ifnβ (r = . , p = . ). importantly, isre ec values correlated strongly with published ifnar binding affinity values when comparing the ifnα subtypes ( fig d) [ ] . however, the inclusion of ifnβ, which was reported to have a higher ifnar binding affinity than multiple ifnα subtypes [ , ] , abolished the correlation (fig e) . these data demonstrate that isre-activity as measured by the ilite assay can be used to evaluate quantitative differences between the ifn-is, particularly for the ifnα subtypes. our data in figs and , as well as data from other studies [ ] [ ] [ ] [ ] , provide strong evidence for quantitative differences among the ifn-is. to uncouple quantitative versus qualitative differences, we normalized our ifn-i treatments for isre-activity ( fig b) for unbiased transcriptomics. purified gut cd + t cells from different donors were treated with pg/ml ifnα , and the other ifn-is were added at higher concentrations that match the isre-activity of this ifnα dose (e.g., . ng/ml ifnα ). purified cd + t cells were evaluated instead ifnα subtypes that had potent activity against hiv- in a previous study are highlighted in black. ifnβ is highlighted in green. note that the ec s are negative log values; thus the higher the bar, the less concentration is needed to achieve an ec . (c) isre-activity versus hiv- inhibition. hiv- inhibition values were previously reported [ ] showing % inhibition of hiv- p + cells relative to mock in lpmc cultures. isre-activity versus ifnar binding affinity (d) without or (e) with ifnβ. ifnar binding affinity data were previously reported using surface plasmon resonance. for panels c to e, linear regression curves were plotted using prism . and evaluated using pearson statistics. https://doi.org/ . /journal.ppat. .g type i interferons in mucosal hiv- infection of total lpmcs to reduce potential confounders due to cell type heterogeneity in lpmcs when performing rnaseq. cd + t cells account for majority of cells in lpmcs ( %) [ ] and are the main cell types for the interaction between hiv- and antiviral isgs. given that only limited numbers of primary lpmcs can be obtained per donor, we selected ifnα , ifnα , ifnα , ifnα and ifnα , as these were highly expressed in hiv- -exposed primary pdcs in vitro and in pbmcs during chronic hiv- infection in vivo [ , ] . we also selected ifnβ because it is significantly upregulated in the gut during chronic hiv- infection on average, we obtained . million (range: . to million) sequence reads per sample (s table) . we first evaluated the transcripts per million (tpm) levels of isg , from which the isre was genetically linked to luciferase in the ilite assay. all ifnα subtypes tested and ifnβ induced isg to similar levels ( fig a, s b fig). in fact, the treatment dose used type i interferons in mucosal hiv- infection also induced the canonical isgs rsad (viperin) and mx (fig b) , as well as oasl, bst and apobec g (s table) to similar extents. these data confirmed that the treatments were indeed normalized for isre-activity and ifnar signaling strength. overall, the ifnα subtypes and ifnβ altered , genes. upregulated genes (isgs) were more prevalent ( - % of irgs) than downregulated genes across all ifn-is ( fig c) . we next compared our irg set with irgs catalogued in the interferome database [ ] . a majority ( . %) of the observed irgs were found in the interferome database ( fig d) . we then partitioned the data into individual ifn-is. strikingly, there were, on average, . -fold more ifnβ-regulated genes than those regulated by any of the individual ifnα subtypes tested (fig c) . on average, ifnβ upregulated . -fold and downregulated . -fold more genes than the individual ifnα subtypes (fig c) . there was a strong correlation between ifnar binding affinity and the number of irgs or isgs (r > . , p< . ), but these correlations were lost if ifnβ was removed (p< . ). in addition, our current analysis revealed novel irgs (s table) . many of these novel irgs may not encode protein products and/or have tentative gene designations, potentially explaining why these genes are not in the interferome database. however, some long non-coding rnas such as nrir (negative regulator of the interferon response) [ ] and bispr (bst interferon stimulated positive regulator) [ ] could have critical roles for modulating ifn-i responses. some repressed protein-coding genes such as ccr appeared specific to just one ifnα subtype (s table) . the ifnα subtypes are homologous genes that activate ifnar, raising questions on whether their differential effects were primarily quantitative. we postulated that if the differences between the ifnα subtypes are mainly quantitative, then we should observe a substantial overlap between the irgs of cells treated with different ifnα subtypes that were normalized for isre-activity. the number of irgs that overlapped between any two of the tested ifnα subtypes ranged from % (ifnα vs ifnα ) to % (ifnα vs ifnα ) ( fig a) . this included a core set of irgs altered by all ifnα subtypes tested (fig b; s table) . interestingly, many additional genes appeared to be specific to an ifnα subtype, particularly for ifnα and ifnα ( fig b and s table) . to test if the irgs unique to each ifnα subtype could have been regulated by other ifnα subtypes but excluded due to a stringent fdr cut-off of %, we investigated the median fdr of these unique genes against the other ifnα subtypes. this analysis is illustrated in s fig, where the fdr values of each of the ifnα -specific genes or ifnα -specific genes ( fig b) were plotted against the gene induction datasets for the other ifnα subtypes tested. as shown, majority of the ifnα or ifnα -specific genes had fdr values that were > % in the other ifnα subtypes. these analyses were expanded on s table, where ifnα-specific irgs had a median fdr of at least %, with most > %, when tested against genes sets from other ifnα subtypes. thus, irgs that were differently expressed by a specific ifnα subtype were unlikely to be significantly altered by the other ifnα subtypes. we next evaluated trends as to whether irgs altered by at least one ifnα subtype (a total of , genes) were similarly upregulated or downregulated. these comparisons were based on mean fold-induction values that can be visualized in the heatmap ( fig c) . surprisingly, a large number of genes (n = ) that trended to be upregulated by ifnα , ifnα , ifnα and ifnα appeared to be downregulated by ifnα (fig c, s table) . as the irgs were based on an arbitrary cut-off (� . × relative to mock, fdr � %), we next evaluated whether increasing our fc criteria changed the differential expression patterns. at ×, . × and × cut-offs, we still observed genes that were differentially regulated by ifnα to determine whether ifnα subtypes induced molecular programs distinct from each other, we subjected the irgs to ingenuity pathway analyses (ipa) [ ] . s table provides a ranked list of activated and inhibited pathways for each ifn-i. as expected, 'interferon signaling' and 'pattern recognition receptors' were the top induced pathways predicted for the . % of ifnα -regulated genes were found among ifnα -rgs, whereas . % of ifnα -rgs were found among ifnα -rgs. (b) euler diagram showing the interferome overlap between the ifnα subtypes tested. a core set of irgs altered by all ifnα subtypes were detected. ifnα-subtype specific genes were highlighted (e.g., for ifnα , for ifnα ). the total number of irgs (n = , ) include genes not shown that were shared between to ifnα subtypes. (c) heatmap of irgs from distinct ifnα subtypes. highlighted areas in red correspond to core isgs, whereas those in blue correspond to genes differentially regulated by ifnα relative to the four other ifnα subtypes tested. (d) ingenuity pathway analyses of ifnα subtypes, highlighting z-scores for shared pathways and those predicted to be specific to ifnα and ifnα . https://doi.org/ . /journal.ppat. .g ifnα subtypes tested ( fig d) . death receptor, nfκb and inflammasome signaling were also highly induced by all ifnα subtypes (s table) . interestingly, ipa also predicted distinct pathways induced by the ifnα subtypes. cd signaling, ctl and nk function, p mapk signaling and sumoylation were predicted for ifnα but not the other ifnα subtypes (s table and fig d) . only the ifnα interferome was associated with downregulated apoptosis, ppar and lxr/rxr activation, likely due to some downregulated genes such as casp (s table) . these differential predictions provide confirmatory evidence of qualitative biological differences in endpoint effector mechanisms induced by different ifnα subtypes. our data in fig c revealed~ more irgs for ifnβ than the individual ifnα subtypes. we pooled ifnα regulated genes independently of the subtype and compared them to ifnβ-regulated genes. almost half ( . %) of ifnβ interferome genes were not regulated by any of the ifnα subtypes (fig a) . these included cytokines and cytokine receptor genes such as il , ifngr , il r and tgfb ( fig a; s table) . we compared the ipa results for the ifnα subtypes versus ifnβ regulated genes. ifnβ induced more pathways (fig b) than the ifnα subtypes, such as 'th pathway', 'erk/mapk signaling' and 'regulation of actin motility'. these findings indicated that ifnβ induced a broader interferome than ifnα subtypes. moreover, compared to ifnα, ifnβ likely regulated more cellular pathways in primary gut cd + t cells. we recently reported that during chronic, untreated hiv- infection, ifn-i inducible antiretroviral genes apobec g, bst and mx , as well as ifnβ, but not ifnα, were expressed to significantly higher levels when compared to hiv- uninfected individuals [ ] . to expand on these findings, we performed rnaseq on these gut biopsies to more broadly investigate the table provides the demographic characteristics of this clinical cohort, which included pwh and hiv- uninfected individuals [ ] . on average, we processed . million sequence reads from colon biopsies per subject (table ) . filtered data were normalized using transcripts per million (tpm), trimmed mean of m values (tmm) [ , ] and deseq [ ] methods. based on relative log expression plots [ ] , the tmm method was most efficient at removing unwanted variation (s fig) . we next determined the expression levels in the colon biopsies in vivo of the 'core irgs' that were similarly regulated by the ifnα subtypes and ifnβ in gut cd + t cells in type i interferons in mucosal hiv- infection vitro. the majority ( %; n = ) of these core irgs that passed the rnaseq filter criteria were isgs (e.g., these genes were induced by the ifn-is tested, not downregulated). of these core isgs, ( %) were significantly altered in hiv- infected versus uninfected individuals at % fdr ( fig a) . the majority ( %) of these altered core isgs were upregulated during chronic hiv- infection. these included the antiretroviral genes apobec g, bst and mx , consistent with our previous report [ ] . genes linked to innate sensing, immunomodulation and cell death/proliferation, as well as negative feedback regulation of the ifn-i response, were also expressed at significantly higher levels in pwh (fig b and c , s table) . since ifnβ induced a broader interferome than all ifnα subtypes tested (fig a) , we evaluated whether isgs that were unique to ifnβ (ifnβ-specific isgs) were also induced in the gut during chronic hiv- infection. of the ifnβ-specific irgs, % (n = ) were isgs that passed the rnaseq filter criteria (fig c) . nearly a third of these ifnβ-specific isgs ( %; n = ) were significantly altered in pwh compared to hiv- uninfected controls (fig a) . only a few of the ifnβ-specific isgs were significantly upregulated in pwh (fig a) . by contrast, the vast majority (> % (n = ) of the altered ifnβ-specific isgs were downregulated during chronic hiv- infection in the gut (fig a) . these repressed ifnβ-specific isgs are involved in intracellular vesicle trafficking, transcriptional/translational regulation, protein ubiquitination and transport (fig b and c ; s table) . moreover, several known hiv- type i interferons in mucosal hiv- infection cofactors such as tsg , nup , and cul were downregulated [ ] [ ] [ ] . we again emphasize that all these genes were induced by ifnβ in gut cd t cells ex vivo (s table) . given that a great majority of these ifnβ-specific isgs were downregulated in pwh, we conclude that a significant inversion of ifnβ-specific isgs was observed during chronic hiv- infection in the gut. we next investigated potential mechanisms for how these ifnβ-specific isgs may have been downregulated in the gut in the presence of high ifnβ levels. two known negative feedback regulators, usp [ ] and unc b [ ] , were induced by all ifn-is tested ex vivo (s table) . gut ifnβ mrna levels positively correlated with usp and unc b transcripts (s table) . however, none of the ifnβ-specific isgs altered in pwh correlated with usp (s and s tables). by contrast, % of these ifnβ-specific isgs negatively correlated with unc b (s and s tables). thus, one potential mechanism for the inversion ifnβ-specific isgs in the gut during chronic hiv- infection may be the ifnβ-mediated induction of unc b . the clinical study described in table involved cohorts where paired blood (pbmcs/plasma) and colon pinch biopsies (up to per donor) were obtained. colon pinch biopsies (~ ) were type i interferons in mucosal hiv- infection pooled for same-day flow-based immunophenotyping [ , ] and the rest were frozen for later histology and transcriptomics. the study obtained comprehensive data including gut and pbmc ifnα and ifnβ transcript levels, plasma and gut viral loads, gut cd + t cell percentages (of cd + cells), myeloid activation (cd mfi in cd c+ myeloid dcs), blood cd + t cell counts, markers of microbial translocation (scd , lps, lta), inflammation (crp, il ), and epithelial barrier dysfunction (ifabp) ( table and s table) [ , , - ]. we investigated how individual altered core isgs (n = ) and ifnβ-specific isgs (n = ) correlated with these clinically relevant parameters using linear regression models, after adjusting the data for age and gender. we used a % fdr threshold for these associations. five clinically relevant parameters-gut ifnβ mrna, plasma lps, gut cd t cell frequencies, blood cd t cell counts and plasma il levels-correlated significantly with the altered core and ifnβ-specific isgs (fig and s table) . the expression of core and ifnβ-specific isgs significantly correlated with transcript levels of ifnβ (> %) rather than ifnα (< %) (s table) . interestingly, the directionality of the correlations was discordant between these gene sets: higher ifnβ levels correlated with higher expression of % of core isgs, whereas higher ifnβ levels were associated with lower expression of % of ifnβ-specific isgs ( fig a) . both core isgs and ifnβ-specific isgs were significantly associated with plasma lps levels, but again with discordant directionalities (fig b) . gut cd t cell counts (as a percentage of cd + cells) were more significantly associated with core-isgs ( % of genes negatively correlated) than ifnβ-specific isgs ( % of genes positively correlated) (fig c) . by contrast, blood cd t cell counts were more significantly associated with ifnβ-specific isgs ( % negatively correlated) than core isgs (fig d) . notably, the lower expression of a substantial fraction of ifnβ-specific isgs ( %) was associated with higher levels of plasma il . none of the core isgs correlated significantly with plasma il at the % fdr cut-off. the complete list of genes that correlated with the clinically relevant parameters (gut ifnβ mrna, plasma lps, gut cd t cell frequencies, blood cd t cell count and plasma il levels) are described in s table. we highlight several core isgs with the highest correlations in fig a, ] . nlrc and lag also positively correlated with plasma lps levels and inversely correlated with the percentage of cd + t cells in the gut (fig c and d ). since t cell exhaustion in persistent lcmv infection has been linked to the suppressive cytokine il [ ] , we evaluated if ifnβ mrna levels correlated with cytokine transcripts in the rnaseq dataset. notably, ifnβ mrna levels were associated with increased levels of il and il ra, which were in turn correlated with lag ( s a fig). ifnβ mrna levels also correlated with transcript levels of tnfα and ifnγ, but not il and tgfβ (s b fig) . these data suggest that increased ifnβ in the gut of chronic pwh may drive genes associated with sustained isg expression, antigen processing, t cell activation, inflammation and immune exhaustion. among the ifnβ-specific isgs (fig ; panels in the right half), higher ifnβ transcript levels negatively correlated with: ( ) eif h, a eukaryotic translational initiation factor (fig e) [ ]; ( ) smad , a key regulator of tgfβ [ ] , which in turn promotes mucosal barrier integrity ( fig f) ; ( ) vimp and sep , selenoproteins that regulate protein folding in the endoplasmic reticulum [ , ] that were linked to anti-inflammatory processes [ ] ; and ( ) two co-factors of hiv- vpr, nup and mus [ , ] . nup is a nuclear pore component and mus is an endonuclease; both are involved in maintaining genomic dna integrity [ , ] . reduced expression of vimp and sep were associated with lower gut cd t cell type i interferons in mucosal hiv- infection percentages (figs and g) . downregulated nup and mus were associated with higher plasma il levels (fig h and i) , lower gut cd t cell percentages (s table) and blood cd t cell counts (fig h and i) . thus, our analyses link elevated ifnβ levels in the gut of pwh to decreased protein translation and decreased protection against dna damage, protein misfolding and barrier dysfunction. fig a) and ifnβ-specific isgs (fig a) in gut biopsies from the clinical cohort were determined against (a) gut ifnβ transcripts, (b) plasma lps levels, (c) gut cd + t cell percentages, (d) blood cd t cell counts and (e) plasma il levels using linear regression models, controlling for age and gender. the clinical cohort included pwh (n = ) and matched hiv uninfected controls (n = ). correlations with fdr � % were considered significant; the proportion of those core and beta isgs are plotted as pie charts, with red, blue and gray depicting positive, negative and no significant correlations, respectively. the top genes in the relevant categories with � % representation are highlighted; a full list is available in s table. https://doi.org/ . /journal.ppat. .g two aspects of type i ifn biology that could influence its translational potential remain insufficiently addressed. first, there is an ongoing debate as to whether the biological effects of ifn-is are primarily due to quantitative differences in ifnar signaling capacity. for example, would administration of higher doses of weakly antiviral ifn-is (e.g., ifnα for hiv- infection) achieve the same in vivo effect as that of more potent ifn-is (e.g., ifnα )? second, the mechanisms governing how and why ifn-is become pathogenic during chronic hiv- infection remains unclear. which isgs are responsible for the discordant effects of ifn-is at distinct phases of infection with persistent viruses? we have undertaken an unbiased transcriptomics approach to gain deeper insights on these questions. our group and others reported diverse antiviral potencies of the ifnα subtypes and ifnβ that correlated with their ifnar binding properties. these data emphasize the contribution of quantitative differences in ifnar signaling in controlling acute viral infection [ ] [ ] [ ] [ ] ] . however, data from multiple in vivo studies also revealed that the ifnα subtypes may have expression levels were based on tmm-transformed counts. linear regression was performed in r statistical package, adjusting for age and gender. a full list of the proportion of core and ifnβ-specific genes that correlated with clinical parameters is presented in s table. https://doi.org/ . /journal.ppat. .g type i interferons in mucosal hiv- infection qualitative differences in mediating antiviral immunity (reviewed in [ ] ). in fact, ifnβ has a significantly higher binding affinity to ifnar than all ifnα subtypes [ , ] but did not exhibit higher isre activity and inhibitory activity against hiv- . recently, schaepler et al utilized saturating concentrations of various ifnα subtypes to inhibit hiv- infection in activated pbmcs in vitro. since canonical isgs were induced to similar levels at saturating ifn-i doses, the authors concluded that there were no qualitative differences between the ifnα subtypes [ ] . we argue that this conclusion is premature, as there are hundreds of isgs encompassing the interferome [ , ] . we postulate that if there were no qualitative differences, the interferomes of diverse ifn-is should have substantial overlap following stimulation of cells with ifn-is normalized for ifnar signaling strength. we utilized a luciferase reporter cell line linked to the isre of a canonical isg, isg , to normalize for ifnar signaling strength. the normalized ifn-is tested (ifnα , , , , and ifnβ) induced isg and other antiviral isgs to similar extents in gut cd + t cells as expected. interestingly, despite normalizing for quantitative differences in ifnar signaling strength, the overlap between the ifnα subtype 'interferomes' were not complete, ranging from to %. ifnα altered > genes not found in any of the other ifnα subtypes. ifnα altered > additional genes and weakly downregulated genes that were induced by other ifnα subtypes, raising the intriguing possibility that ifnα may modulate the overall ifn-i response. while it was possible that these ifnα-subtype specific genes were an artifact of the low numbers of donors used in this study, these genes were not close to statistical significance of being differentially expressed by the other ifnα subtypes, as majority had fdr values > %. the sample size we used for this work was also counterbalanced by utilizing a homogenous cell subpopulation (purified cd + t cells) treated in a controlled fashion in vitro that should decrease overall variability. in fact, > canonical isgs were captured by all ifns tested including our positive control, isg . interestingly, ifnβ altered nearly three times the number of genes compared to the individual ifnα subtypes we tested. we speculate that the higher binding affinity of ifnβ to the ifnar may contribute in part to the increased gene induction numbers, but it was notable that enhanced binding affinity did not track with isre activity. the broader interferome associated with ifnβ was also reported by other groups using pbmcs [ , ] , although it was unclear whether the doses were normalized for ifnar signaling strength. altogether, the incomplete overlap between the interferomes strongly suggests that there are qualitative differences between diverse ifn-is. the underlying molecular mechanisms for differential interferome induction remain unclear. one possibility is that subtle differences in ifnar binding may have triggered differential phosphorylation of diverse stats, jaks and mapks. this possibility is currently under investigation. we speculate that differences in interferome regulation may have consequences for the therapeutic use of ifn-is in vivo. clinical administration of ifnα and ifnβ in vivo were associated with a multitude of side-effects [ ] . one approach to potentially reduce toxicity issues is to utilize ifnα subtypes with a more 'restricted' gene regulation signature. ifnα is ten times more potent at inhibiting hiv- than ifnα , but did not induce as many genes as ifnα . thus, ifnα may be a potent anti-hiv- therapeutic with limited side-effects. however, ifnα did not seem to inhibit hiv- when administered in humanized mice as encoded plasmids via hydrodynamic injection [ ] . ifn-is with desirable immunomodulatory properties may also be useful in hiv- curative strategies [ ] , as reactivation of latent hiv- was insufficient to reduce the reservoir during antiretroviral therapy [ ] . ifn-is could be potent additive drugs for hiv- cure by activating immune cells with latent hiv- while stimulating immune responses that can kill infected cells and prevent subsequent infection through intrinsic restriction. ifnα strongly induced trail+ nk cells and apobec g-mediated hypermutation compared to ifnα in humanized mice [ ] and was predicted to induce pathways associated with immune function in gut cd + t cells compared to ifnα , , and . thus, ifnα could be a viable candidate to pursue for hiv- curative strategies. the current data could serve as a useful template to design transcriptome-wide studies that extend to the other ifnα subtypes not studied here, as well as other cell types (dcs, nk cells, cd + t cells, b cells) in various tissue compartments. such follow-up studies may aid in designing focused ifn-i biologicals for various clinical applications. our recent study suggested that ifnβ may play an important role during chronic hiv- immunopathogenesis in the gut [ ] . we observed downregulated ifnα, but elevated ifnβ levels in colon biopsies from pwh, while ifnβ was rarely detected in the pbmcs and plasma of these patients [ ] . since ifnβ induced a broader interferome than the individual ifnα subtypes, we determined how isgs induced by all ifn-is tested ('core isgs') versus isgs specifically induced by ifnβ ('ifnβ-specific isgs') were expressed in the gut biopsies following rnaseq. one limitation of our approach is that colon biopsies encompass other cell types that the interferomes based on gut cd + t cells may not capture. specifically, it is possible that the decreased expression of ifnβ-specific isgs in pwh relative to uninfected controls may be due to the significant loss cd + t cells in pwh. if this was the case, we should have also observed a decrease in core isg expression in pwh. however, over a hundred core isgs were upregulated in pwh, correlating significantly with gut ifnβ expression. alterations in ifnar expression in mucosal cd + t cells during hiv- infection may also contribute to our results, but our recent study have not detected significant changes in ifnar expression in mucosal immune cells in pwh [ ] . expanding the interferome analyses to other mucosal cell types may enable deconvolution of the bulk rnaseq data to specific cell types. consistent with our previous study [ ] , many canonical isgs that include antiviral genes remained upregulated in chronic hiv- infection in the gut. core isgs positively correlated with ifnβ rather than ifnα transcripts, suggesting that ifnβ drove these responses in the gut. interestingly, core isg expression positively correlated with plasma lps levels. previously, we showed that many isgs were upregulated in gut cd + t cells following co-incubation with prevotella stercorea, a gram-negative microbe present in colon tissue of pwh [ ] . these findings strengthen a link between microbial translocation, ifnβ, and elevated isg signatures in the gut during chronic hiv- infection. interestingly, we did not observe correlations between core isgs and the monocyte activation marker scd or myeloid dendritic cell activation. other markers such as scd [ , ] may need to be investigated in future work. irf , stat and stat (the components of isgf ) remained elevated in pwh, suggesting a mechanism for constitutive isg expression during chronic infection. notably, ifnβ expression in the gut correlated with gene markers of immune activation and inflammation (cd , psmb , nlrc , tnfa, ifng), and exhaustion (lag ). ifnβ-mediated induction of these immunomodulatory genes may account for how ifnβ contributes to pathogenesis during chronic infection. specifically, ifnβ levels in the gut were associated with il and il ra expression, which in turn correlated with lag levels, suggesting that ifnβ may drive immune exhaustion through the il pathway. it remains to be determined whether these core isgs are similarly regulated in the systemic circulation, where ifnα subtypes, and not ifnβ, are more prominent [ ] . further, the rationale for why the gut appears primed to express ifnβ remains unclear. a recent study noted that helix of ifnβ may have direct antimicrobial properties [ ] . efforts are ongoing to investigate links between ifnβ levels, ifnβspecific genes and the altered microbiome in pwh. could ifnβ have distinct biological effects that are likely not due to other ifn-is? in persistent lcmv infection of mice, neutralization of ifnβ, and not ifnα, accelerated virus clearance and improved t cell responses [ ] . in the present study, we observed that a substantial fraction of ifnβ-specific isgs, but not core isgs, correlated with plasma levels of the inflammatory cytokine il . a link between ifnβ and il has previously been reported [ ] . surprisingly, the ifnβ-specific isgs that correlated with il were downregulated during chronic hiv- infection in the gut. the magnitude of downregulation correlated with higher ifnβ levels, suggesting negative feedback control. among these negative feedback control genes, we identified unc b as a potential driver for the downregulation of ifnβ-specific isgs, possibly through the regulation of tlr signaling via endosomal trafficking pathways [ , ] . it is thought that constitutive expression of antiviral isgs may protect from virus infection [ , ] , but decreased protein translation (eif h) in pwh may minimize the contribution of antiviral isgs that are constitutively expressed. furthermore, our data suggest a potential link between ifnβ, decreased tgfβ signaling (smad ), increased unfolded protein response (vimp and sep ) and increased dna damage (nup and mus ) in chronic hiv- infection. these results raise the possibility that genes downregulated by ifnβ could have important consequences for mucosal hiv- pathogenesis. one possibility is that these altered ifnβ-specific genes (such as those that decreased protection from dna damage) may directly contribute to cd + t cell death, in line with previous studies linking ifn-is and cd + t cell depletion [ , ] . given the predicted pleiotropic effects of ifnβ, our data also raise concerns in utilizing ifnβ for treating chronic hepatitis b virus infections that became refractory to ifnα treatment [ , , ] . notably, ifnβ administered < days post-symptom onset may help resolve infection with the novel pandemic coronavirus, sars-cov- [ ] . by contrast, delayed ifnβ treatment in murine coronavirus models exacerbated immune pathology [ , ] . based on our studies as well as others, understanding the role of distinct ifns during the course of infection with diverse pathogenic viruses may yield important therapeutic insights. the mechanisms for the differential regulation of core versus ifnβ-specific isgs during chronic hiv- infection remain to be determined. studies in cell lines reveal that unphosphorylated stat , stat and irf can assemble into an unphosphorylated isgf (u-isgf ) complex that could sustain the expression of canonical isgs after a single stimulation with ifnβ [ , ] . constitutively expressed isgs regulated by u-isgf included antiviral genes such as bst , apobec g, mx that remained elevated during chronic hiv- infection. by contrast, isgs specifically regulated by phosphorylated isgf are more sensitive to negative feedback regulation. the isgf /u-isgf model would imply that core isgs are regulated by u-isgf , whereas the ifnβ-specific isgs are regulated by isgf . investigations on the phosphorylation status of stat , stat and irf in chronic hiv- infection are underway. in conclusion, we demonstrate that diverse ifn-is trigger non-overlapping interferomes in a single cell type, providing strong evidence for qualitative differences between the ifn-is. furthermore, conserved and qualitative differences in interferome induction correlated with clinically relevant markers of immunopathogenesis during chronic hiv- infection. further studies on the differences between the ifn-is and how these cytokines regulate distinct gene expression profiles in various tissue compartments could inform strategies to harness these biologicals and/or block these responses for hiv- control. the in vitro studies utilized disaggregated cells from macroscopically normal jejunum tissue that would otherwise be discarded. these tissues were obtained from patients undergoing elective surgery at the university of colorado hospital. the patients signed a release form for the unrestricted use of tissues for research following de-identification to laboratory personnel. the colorado multiple institutional review board approved the procedures and have given exempt status. colon pinch biopsies from pwh and age/gender-matched hiv-uninfected controls were obtained from archived samples from a completed, comirb-approved clinical study. this clinical study included pwh and hiv-uninfected controls, but archived colon pinch biopsies were available only for pwh and controls, respectively. study participants signed an informed consent. clinical parameters that include blood cd -t cell counts (cells/ μl), plasma viral load, tissue hiv rna (per cd t cell), tissue cd t cells (% viable cd + cells), il- (pg/ml), crp (μg/ml), ifabp (pg/ml), scd (u/ml), cd (ng/ml), lps (pg/ ml), lta (optical density), gut ifnα and ifnβ transcripts, were reported previously [ ]. ifn-is (pbl assay science, piscataway nj) were frozen into small aliquots for single-use. ilite type i ifn assay ready cells, purchased from svar life science ab (malmõ, sweden, cat# bm ) were seeded into a -well flat-bottomed cell culture plate and maintained in complete dmem (with % fbs and % penicillin-streptomycin and l-glutamine). various doses of recombinant ifn-is were added to corresponding wells. after h, the cells were lysed and luciferase activities were developed using bright-glo luciferase assay system (promega, madison, wi, cat# e ) according to manufacturer's instruction. luciferase activity was subsequently measured using a perkin-elmer victor x plate reader. % effective concentrations were computed using dose-response curve in prism . . hiv- bal (nih aids reagent program cat# ) was prepared in molt -ccr cells and titrated using an hiv- p elisa (advanced bioscience laboratories, rockville, md). ng p of hiv- bal / lpmcs (n = donors) was spinoculated in the presence or absence of titrated doses of ifnα subtypes and ifnβ (pbl assay science). the cells were harvested at d and analyzed by intracellular p flow cytometry as previously described [ , ] . % inhibitory concentrations and vres were calculated using a one-phase decay equation in prism . as previously described [ , ] . cd + t cells were purified from lpmcs (n = donors) by negative selection using easysep™ human cd + t cell isolation kit (stemcell, vancouver, bc, canada, cat# ) and followed by flow sorting to reach a purity greater than %. x of these purified cd + t cells were treated with mock, pg/ml ifnα , or an equivalent isre-activity of ifnα , , , and ifnβ. after h, rna was extracted using rneasy mini kit (qiagen, germany, cat# ). rnaseq libraries were constructed from ng rna using quantseq ' mrnaseq library prep kit (lexogen, austria, cat# . ). the rnaseq library quality was verified using bioanalyzer (agilent, santa clara, ca) before loaded into a hiseq by the genomics and microarray sequencing core facility at the university of colorado anschutz medical campus. the -well array contained primers for target genes isg , arhgef , lat and ahnak (s fig) , a reverse transcription control, a genomic dna control and a positive pcr control as well as the housekeeping gene gapdh (pph f). ninety-six-well custom rt profiler pcr arrays were performed according to the manufacturer's instructions (qiagen, valencia, type i interferons in mucosal hiv- infection california, usa) using the bio-rad cfx- real-time pcr instrument (bio-rad, hercules, california, usa) and bio-rad cfx manager software (ver. . ). cdna templates were mixed with ready-to-use rt qpcr master mixes and μl of the pcr component mix was aliquoted into each well containing predispensed gene-specific primer sets. each plate was loaded with cdna from individual samples. gene expression was normalized according to the average expression level of gapdh in the same sample. rna ( ng) from the colon pinch biopsies were used to prepare the rnaseq libraries. the rnaseq work flow of library construction, quality control, and the subsequent sequencing were similar to that of ifn-i treated gut cd + t cells as aforementioned. rnaseq data were downloaded as fastq files and their quality examined and visualized using fastqc (babraham institute, https://www.bioinformatics.babraham.ac.uk/projects/ fastqc). the removal of illumina sequencing adapters and the by-base quality screening was performed with cutadapt (https://cutadapt.readthedocs.io/en/stable). the quality screened fastq files were mapped to the current human genome assembly grch .p using hisat (johns hopkins university, https://ccb.jhu.edu/software/hisat /index.shtml), taking into account the ensembl id, gene symbol and gene length. raw gene expression counts were extracted using featurecounts from the subread package (walter+eliza hall institute of medical research, http://subread.sourceforge.net). rnaseq raw counts were obtained for gut cd t cells treated with ifn-is (ifnα , , , , and ifnβ) and mock from donors. gene counts were normalized using transcripts per million (tpm). principal component analyses necessitated removal of one donor as the transcriptome of the mock condition was extremely skewed relative to the other donors (s a fig). using edger differential expression analysis, ifn regulated genes (irgs) were defined based on a . -fold change (fc) cutoff relative to mock and a false-discovery rate (fdr) � %. the rnaseq raw counts data had gene-level read counts for hiv- -uninfected donors and pwh. lowly expressed genes which have average read counts less than per library were filtered out. as a result, out of genes were kept for further analysis and gene counts were normalized using the trimmed mean of m values (tmm) normalization method from edger (version . . ) in r (version . . ) with the "estimatedisp" function and its default settings. the tmm method calculates the scaling normalization factor for each sample by accounting for library size and observed counts, while under the assumption that the majority of genes are not de. in our case, the tmm method provided the best normalization results in terms of removing the unwanted variation, according to the relative log expression (rle) plots (s fig) . two-group differential expression (de) analysis was performed to test for differences between healthy controls and pwh, using normalized counts in edger with default settings and false discovery rate (fdr) of % to control for multiple testing. the de analysis was done in edger by the exact test using the "exacttest" function and its default settings for two-group design. the benjamini-hochberg procedure (bh step-up procedure) controlling the fdr was used as the multiple testing correction method in this study. the normalized rnaseq counts were further transformed by the variance stabilization and bias reduction method called "regularized log transformation" (rlog) from deseq (version . . ) [ ] . the transformed counts were then used as the outcome in a multivariate linear regression model. specifically, the linear regression models were fit in a gene by gene fashion to test the correlation between rna expression levels and immunopathogenic markers of chronic hiv- infection, with one marker included in the model at a time, while adjusting for age and gender. based on the results of de analysis, significantly altered genes from core-isgs and ifnβ-specific isgs were selected to test the associations with clinical parameters through the above model, separately. several immunopathogenic markers were used in this part, including gut ifnβ transcript levels, gut cd t cell percentages, blood cd t cell counts, plasma il levels and plasma lps levels. genes were considered as significantly associated with the corresponding clinical parameter with an fdr cutoff at %. in addition, the difference in proportions of significant genes in each gene list were tested with chi-squared test in r, as well as the difference in proportions of positive correlations of each gene list. all the plots were generated by ggplot (version . . ) package in r (version . . ). next generation sequencing data were deposited in the sequence read archive prjna (interferome dataset) and prjna (colon pinch biopsies). these were also deposited in the gene expression omnibus archive with accession numbers gse (interferome dataset) and gse (colon pinch biopsies). (left panels) ifnα -specific genes (n = ) were classified based on significant induction/suppression in ifnα -treated cells relative to mock at a % fdr cut-off. however, when these genes were evaluated in transcriptome datasets for ifnα , ifnα , ifnα and ifnα , most had fdr values > %, suggesting that these ifnα -specific genes were not even close to being statistically-significant in these other ifnα subtypes. (right panels) evaluation of ifnα -specific genes against gene datasets for ifnα , ifnα , ifnα and ifnα . bars correspond to the frequency of genes that fell within the fdr values noted in the x-axis. type i interferons in mucosal hiv- infection the interferons: characterization and application interferon alpha subtypes in hiv infection the significance of type-i interferons in the pathogenesis and therapy of human immunodeficiency virus infection the jak-stat pathway at twenty interferome: the database of interferon regulated genes interferon-alpha subtypes in an ex vivo model of acute hiv- infection: expression, potency and effector mechanisms interferon alpha subtype-specific suppression of hiv- infection in vivo gene therapy with plasmids encoding ifn-beta or ifn-alpha confers long-term resistance to hiv- in humanized mice dose-dependent differences in hiv inhibition by different interferon alpha subtypes while having overall similar biologic effects. msphere binding and activity of all human alpha interferon subtypes evolutionary genetic dissection of human interferons. the journal of experimental medicine interferon-alpha subtype activates nk cells and enables control of retroviral infection type i interferon differential therapy for erythroleukemia: specificity of stat activation anti-retroviral effects of type i ifn subtypes in vivo type i interferon responses in rhesus macaques prevent siv infection and slow disease progression reduced chronic lymphocyte activation following interferon alpha blockade during the acute phase of simian immunodeficiency virus infection in rhesus macaques blockade of chronic type i interferon signaling to control persistent lcmv infection persistent lcmv infection is controlled by blockade of type i interferon signaling interferons and interferon (ifn)-inducible protein during highly active anti-retroviral therapy (haart)-possible immunosuppressive role of ifn-alpha in hiv infection chronic cd + t-cell activation and depletion in human immunodeficiency virus type infection: type i interferon-mediated disruption of t-cell dynamics downregulation of robust acute type i interferon responses distinguishes nonpathogenic simian immunodeficiency virus (siv) infection of natural hosts from pathogenic siv infection of rhesus macaques an altered intestinal mucosal microbiome in hiv- infection is associated with mucosal and systemic immune activation and endotoxemia edger: a bioconductor package for differential expression analysis of digital gene expression data differential expression analysis of multifactor rna-seq experiments with respect to biological variation moderated estimation of fold change and dispersion for rna-seq data with deseq rle plots: visualizing unwanted variation in high dimensional data tsg and the vacuolar protein sorting pathway are essential for hiv- budding viral protein r regulates docking of the hiv- preintegration complex to the nuclear pore complex induction of apobec g ubiquitination and degradation by an hiv- vif-cul -scf complex usp -based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response unc b restricts systemic lethal inflammation by orchestrating toll-like receptor and trafficking gut dendritic cell activation links an altered colonic microbiome to mucosal and systemic t-cell activation in untreated hiv- infection brief report: inflammatory colonic innate lymphoid cells are increased during untreated hiv- infection and associated with markers of gut dysbiosis and mucosal immune activation low abundance of colonic butyrateproducing bacteria in hiv infection is associated with microbial translocation and immune activation enhancement of hiv- infection and intestinal cd + t cell depletion ex vivo by gut microbes altered during chronic hiv- infection elevated cd antigen expression on cd + t cells is a stronger marker for the risk of chronic hiv disease progression to aids and death in the multicenter aids cohort study than cd + cell count, soluble immune activation markers, or combinations of hla-dr and cd expression nlr family member nlrc is a transcriptional regulator of mhc class i genes roles, function and relevance of lag in hiv infection modulation of the helicase activity of eif a by eif b, eif h, and eif f tgf-beta signaling from receptors to smads selenoproteins: molecular pathways and physiological roles premature activation of the slx complex by vpr promotes g /m arrest and escape from innate immune sensing nucleoporin contributes to homologous recombination repair and post-replicative dna integrity the dna repair endonuclease mus facilitates fast dna replication in the absence of exogenous damage functional comparison of ifn-alpha subtypes reveals potent hbv suppression by a concerted action of ifn-alpha and -gamma signaling identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays ifn-beta induces greater antiproliferative and proapoptotic effects and increased p signaling compared with ifn-alpha in pbmcs of adult t-cell leukemia/lymphoma patients the interferons: years after their discovery, there is much more to learn stimulation of hiv- -specific cytolytic t lymphocytes facilitates elimination of latent viral reservoir after virus reactivation hiv infection does not alter interferon alpha/beta receptor expression on mucosal immune cells the transcriptome of hiv- infected intestinal cd + t cells exposed to enteric bacteria soluble cd made by monocyte/macrophages is a novel marker of hiv activity in early and chronic infection prior to and after anti-retroviral therapy. the journal of infectious diseases irf and unphosphorylated stat cooperate with nf-kappab to drive il expression the chaperone unc b regulates toll-like receptor stability independently of endosomal tlr transport unphosphorylated isgf drives constitutive expression of interferon-stimulated genes to protect against viral infections ifnbeta-dependent increases in stat , stat , and irf mediate resistance to viruses and dna damage cd + t-cell death induced by infectious and noninfectious hiv- : role of type interferon-dependent, trail/dr -mediated apoptosis type i interferon contributes to cd + t cell depletion induced by infection with hiv- in the human thymus development of a novel site-specific pegylated interferon beta for antiviral therapy of chronic hepatitis b virus interferon-beta and interferon-lambda signaling is not affected by interferon-induced refractoriness to interferon-alpha in vivo triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice unphosphorylated stat prolongs the expression of interferon-induced immune regulatory genes microbial exposure alters hiv- -induced mucosal cd + t cell death pathways ex vivo we thank eric lee and christine purba for expert technical assistance with the lamina propria aggregate culture model, and cheyret wood for helpful statistical advice. we also thank the patients who agreed to use discarded tissue for research purposes and the clinical study participants. key: cord- -tgt ir authors: nan title: poster session (pp. a– a) date: - - journal: hepatology doi: . /hep. sha: doc_id: cord_uid: tgt ir nan , ala- (n= ), tyr- (n= ), gly- ( n = l ) and lys- ( n = l ) . the mean time from onset of symptoms to diagnosis was . years ( - ) and patients were listed for olt an average of . months ( - ) after diagnosis. the mean time from listing to olt was . months ( - ). five patients died after olt, during a mean follow-up of . years ( months - years). one-year patient survival was % and threeyear patient survival was %. one patient required retransplantation for hepatic artery thrombosis. neurologic symptoms were the initial clinical manifestation in the majority of patients ( / ), followed by cardiopulmonary ( ) and gastrointestinal symptoms ( ). most patients experienced multiple symptoms. subjective evolution of symptoms as assessed by chart review demonstrated that symptoms referable to amyloidosis worsened after olt in seven patients, and improved or stabilized in five. following olt, neuropathy symptoms improved in four patients, worsened in seven patients and were unchanged in one patient. pre and post-olt nerve conduction velocities were available for patients and the post-olt studies showed progression of disease in , improvement in and were unchanged in patient. prior to olt, all twelve patients had an increased ivst on echocardiogram. post olt cardiac symptoms improved in six patients (five of whom also had cardiac transplantation), worsened in three patients, and were unchanged in three patients (one of whom also had cardiac transplantation backgr und :accurate delineation of the scope and magnitude of peri-operative donor risk is necessary to better allow for informed consent, to maximize the potential for donor safety, for comparative outcome analysis and ultimately to serve as a key determinant of the utility of adult-to-adult living donor liver transplantation (aaldlt). however, at present, there is lack of uniformity regarding what constitutes a complication in this setting. moreover, a system to stratify adverse events with respect to their life altering (quantity or quality) impact is lacking. aims: ) to define a graded, inclusive classification schema for both early (e) and late (l) adverse operation-related events in live liver donors a n d ) to apply this system to a retrospective review of events in individuals undergoing partial hepatectomy for live liver donation at our center. two patients ( . %) suffered cut-surface bile leaks and one ( . %) a right colon injury (grade e complications). one patient ( . %) developed a transient bilateral ulnar neuropathy (grade e). two patients ( . %) were readmitted within days of operation for nausea and dyspnea respectively (grade e). one patient underwent repair of an incisional hernia months post donation (grade l). one patient suffered positioning-related brachial plexopathy (grade l). conclusions: the definition and adoption of a graded, scale-based system of operation-related adverse events in live liver donors will allow for an inclusive, consistent and universally applicable method to collect, analyze and report donor complications. all aaldlt programs must be encouraged to fully review and report their donor related morbidity, ideally through the creation of a national donor registry initiation of antiviral therapy in the early post-transplant period, prior to overt evidence of hcv recurrence (i.e. preemptive therapy) may enhance rates of hcv eradication. aims: to determine the efficacy and tolerability of preemptive ifn versus ifnlrbv in anti-hcv positive lt patients. methods: consecutive and eligible lt recipients from a single center were enrolled. the goal was to initiate treatment within wks of lt. patients were randomized to ifn or ifn plus rbv ( mg daily x wks, then mg daily x wks, then . - . g daily based upon body weight kg). induction therapy with daily ifn was used for the first wks ( . mu daily x wks, then mu daily x wks) followed by mu tiw (n= ) or peg-ifn . uglkglwk (n= ) for wks. key inclusion criteria were stable clinical status, cr ,ooo and wbc> . . dose reductions for side effects, especially cytopenias were standardized. growth factors were given for neutropenia (anc< ) and anemia (hgb< . gldl) beginning l . virological (vr) and biochemical responses (br) were evaluated at end-of-treatment (et) and mos post-treatment (sustained virological (svr) and biochemical (sbr) responses (table below) were associated with response. histological disease was mild in the majority of patients at treatment end ( % stage fibrosis and % grade or less necroinflammatory activity). conclusions: preemptive antiviral therapy was applicable to - % of transplanted patients. biochemical responses were frequent but svrs uncommon. treatment discontinuation and lowering of rbv doses due to side effects likely reduced svrs and may have limited our ability to detect differences between treatment groups. the only predictor of svr was a negative qhcvrna pre-treatment, which implies that patients with low vl early post-lt may be the best candidates for preemptive therapy. compared to historical reports, the severity of histological disease was very mild at -year post-lt in the majority of treated patients, suggesting preemptive antiviral therapy may provide important histological benefits. the cadaveric organ shortage has led to the development of alternative strategies for organ transplantation. living donor liver transplant (ldlt) is one strategy that has offered many individuals transplantation before they die or become too sick for transplant.chronic hepatitis c (hcv) is the leading indication for liver transplantation. preliminary results from small single center studies suggest that recurrent hcv is more common after ldlt compared to cadaveric transplant with concern that graft survival may be lower after ldlt. &: to compare patient and graft survival in hcv recipients who undergo ldlt to cadaveric liver transplant recipients. methods: we analyzed the united network for organ sharing (unos) liver transplant database from january, -december, . inclusion criteria included liver transplant recipients years old transplanted from - with the transplant diagnosis of chronic hepatitis c. exclusion criteria included subjects who were hepatitis b surface antigen positive, history of prior organ transplant, concurrent kidney or heart transplant. the logrank test was used to compare survival between the two groups. results: from - . % of adult liver transplants were ldlt. ldlt recipients and , cadaveric recipients transplanted for end stage liver disease from hcv were identified. the results are shown in the table: ldlt ( a greater proportion of ldlt recipients were female and ldlt recipients were less ill at the time of transplant. and year patient and graft survival were not significantly different between the groups, (p=o. ). if only the pre-meld era is considered ( l thru / ) year graft survival for ldlt (n= ) and clt (n= ) are % and %, respectively, p=o.lo. conclusions: our analysis demonstrates that short-term patient and graft survival are equivalent in ldlt and cadaveric recipients with hcv suggesting recurrent hepatitis c does not adversely affect survival over this time period. ldlt recipients are less ill at transplantation which did not confer a short-term survival advantage. continued follow-up should determine the impact of less advanced liver disease at the time of transplant and recurrent hepatitis c after transplant on long-term graft and patient survival in ldlt recipients. disclosures: introduction: histologic injury due to recurrent hcv has been reported in up to % of hcv infected patients who undergo liver transplantation with a cadaveric graft. however, the natural history of hcv following living donor liver transplantation (ldlt) is not as well described. anecdotal evidence suggests that hcv recurrence may be more severe in ldlt recipients. we hypothesize that post-operative liver regeneration in the partial graft procured from living donors may facilitate hcv replication, or increase the vulnerability of hepatocytes to infection. methods: we performed a retrospective analysis comparing outcomes, and the incidence, timing, and severity of histologic recurrence of hcv following transplantation in patients who underwent living donor liver transplant compared to recipients of cadaveric organs. between / and / , hcv infected adult patients (age > years old) underwent liver transplantation for hcv associated cirrhosis. patients received a cadaveric graft (cad) and received a graft from a living donor (ldlt). mean time of patient follow up was months, with a range of to months. elevated serum transaminases, positive hcv rna, and liver biopsy consistent with histologic evidence of hcv defined recurrence. cholestatic hepatitis c (chc) was confirmed if all of the following criteria were met: serum total bilirubin greater than lomg/dl with no evidence of extra-hepatic biliary obstruction on ultrasound and cholangiography, and histologic features on liver biopsy consisting of portal expansion, ductular proliferation, and bile stasis with or without hepatocyte ballooning. immunosuppression, definition and treatment of rejection were standardized for both cad and ldlt. results: when comparing cad to ldlt, both the incidence of hcv recurrence and time to recurrence were not different. the overall incidence of "severe" sequelae of hcv recurrence, either cholestatic hepatitis, grade -iv inflammation, and/or hcv induced graft failure requiring re-transplantation was also not different when comparing cad to ldlt. however, when comparing cad versus ldlt, no cad patient developed cholestatic hepatitis c, compared to % of ldlt who developed this complication (p = . ). conclusions: in this patient population, the timing and overall incidence of hcv recurrence were not different when comparing cad versus ldlt, but the incidence of cholestatic hepatitis was significantly greater in patients with hcv who underwent ldlt. further multicenter studies are warranted to determine the incidence and risk factors for cholestatic hepatitis c following liver transplantation. ucsf, sun francisco, ca; fabien zoulim, inserm, lyon, france; carol l brosgart, michael wulfsohn, michael d miller, shelly xiong, gilead sciences, inc., foster city, ca background lamivudine (lam) resistance occurs in approximately % of chronic hepatitis b (chb) patients after years of lam monotherapy. in contrast, resistance to adefovir dipivoxil (adv) occurs infrequently with . % of chb patients developing the adefovir resistance mutation rtn t after years of adv therapy. pre-existing lam resistance mutations or concurrent use of immunosuppressive therapy by lt patients may increase the risk of resistance to adv. objective: to determine the incidence of adv resistance in a clinical trial of liver transplantation (pre and post) patients with lam-resistant hbv treated with adv for weeks (lam therapy was maintained in most patients). methods: the hbv reverse transcriptase domain was sequenced for lt patients with detectable hbv dna by pcr (>lo copieslml) after weeks of adv therapy (n= ). in vih-o drug susceptibility was determined following transfection of hepg cells with patient-derived hbv clones from baseline and week serum samples. results: the rtn t mutation wa!j observed in patients ( . %) at week . lam had been discontinued at weeks and after initiation of adv in these two patients respectively. the baseline lam-resistant ymdd mutation reverted to wildtype prior to week in both patients. emergence of rtn t was associated with rebound in serum hbv dna and alt elevation in both patients. in vitro phenotypic analysis showed approximately -fold reduced susceptibility to adefovir with rtn t. however, these adefovir-resistant hbv clones were fully susceptible to lam and entecavir in vitro. lam therapy was re-initiated, in addition to the ongoing adv therapy, after emergence of rtn t in these patients resulting in a > . loglo copies ml reduction in serum hbv dna in both patients. one patient also had a significant reduction (> %) in alt within months of lam treatment. no other mutations potentially associated with adefovir resistance were detected. conclusions: emergence of the adefovir resistance mutation rtn t was observed infrequently after years in liver transplantation patients ( . %, / ) infected with lam-resistant hbv, similar to observations in treatment nayve non-liver transplantation patients. the adv-resistant hbv was sensitive to lam and addition of lam resulted in clinical stabiliation in both patients. local liver immune responses are thought to play a major role in chronic autoimmune diseases directed at biliary epithelium.using the apical sodium dependent bile acid transporter (asbt) promoter to drive biliary epithelial cell -specific expression of a membrane form of ovalbumin (ova), we have previously developed ova-bil transgenic mice. because these mice are tolerant to ova, we use ova-specific t cells from ot- and ot-i transgenic mice, restricted by mhc class i and class , respectively, with well defined peptide epitopes specific for ova to induce biliary damage in a dose dependent manner. aim: ) to determine the liver mononuclear cell populations (mnc) involved in necroinflammatory disease, and ) to determine where adoptively transferred cells home and proliferate. methods: million ot-i and million ot-i nayve t cells were adoptively transferred to ova-bil mice by intraperitoneal injection. at days , , and , liver mnc were isolated by collagenase digestion, purified by discontinuous percoll gradient centrifugation, and analyzed by flow cytometry. tail bleeds were performed at days , , , and to follow serum alt. in a subset of experiments, ot- cells were labeled with carboxyfluorescein diacetate succinimidyl ester (cfse) and analyzed on day and by flow cytometry after adoptive transfer with unlabeled ot-i t cells into ova-bil mice. results: ova-bil mice develop normally without evidence of disease up to years. after adoptive transfer of ova-specific t cells, there was a marked increase in serum alt. cd + ot-i t cells were required for liver damage and ova-specific cd + t cells markedly augmented this inflammation. adoptive transfer of ot-i cd t cells alone did not induce liver injury. there was extensive portal inflammation in every portal triad, centered around the bile ducts with infiltrating lymphocytes in the bile duct epithelia, apoptotic cells, loss of biliary epithelial cells as well as interface hepatitis. liver mnc were abundant in ova-bil mice and increased after adoptive transfer of ova-specific t cells. serum alt peaked at day (mean iulml), coincident with liver mnc peak. cfse labeling studies revealed robust homing of adoptively transferred ot-i cd cells to the liver, but not to the spleen, of ova-bil mice. the ova-specific cd cells, but not cd , nk .l, or cd cells, underwent cell division. conclu-sion: recognition of biliary epithelial antigen in ova-bil mice induces a necroinflammatory response in the liver as assessed by serum alt, liver mnc numbers and immunohistochemistry. the magnitude of this response correlates with influx of nahe ovaspecific cytotoxic t cells, which are activated and divide in the liver but not in the spleen. t cell recognition of antigen expressed on bile duct epithelium occurs rapidly, causes biliary specific inflammation with interface hepatitis, which may be a model of autoimmune bile duct injury or cholangiopathy. the etiology of autoimmune hepatitis (aih) is only poorly understood although the major autoantigens, such as cytochrome p d (cyp d ), could be identified and immunodominant epitopes have been mapped. one major reason for this lack of comprehension is the fact that there are currently only few valid animal models for aih and none of them involves the autoantigen targeted in humans. thus, we generated an animal model for human aih using a natural autoantigen (cyp d ). self-tolerance in transgenic mice that express human cyp d in the liver was challenged by infecting these cyp d -mice with an adenovirus-cyp d vector (ad- d ) in order to deliver large amounts of the critical antigen to the liver and to provide a inflammatory environment that would favour autoimmunity. infection with an empty adenovirus vector resulted in a transient form of focal necrosis days after infection that subsequently disappeared after weeks. in contrast, infection with ad- d resulted in extended and persistent infiltration of cd , cd lymphocytes as well as macrophages resulting in confluentlbridging necrosis at week post-infection. in addition, the overall morphology of the liver was massively disturbed after ad- d infection. at week postinfection, the liver was approximately half the size of the control and its lobules were fused together and rounded up. first indications of fibrosis and hyperplasic nodules become apparent. the overall architecture of the liver was disrupted i disorganized and the liver parenchyma was partially collapsed. furthermore, theliver of ad- d infected mice was surrounded by multiple layers of connective tissue as seen in some stages of liver cirrhosis. additional signs of cirrhosis started to become apparent in the form of nodules that are entrapped in fibrous tissue. in addition, accumulation of infiltrating cells are visible directly under the liver capsule. these observations indicate that the cyp d -mouse displays a persistent form of hepatitis after infection with ad- d that may form the basis for a novel model system which would allow to study mechanisms involved in the initiation, propagation and precipitation of autoimmune processes involved in human autoimmune hepatitis. disclosures: urs christen -no relationships to disclose eric f johnson -no relationships to disclose michael p manns -no relationships to disclose antje rhode -no relationships to disclose matthias g von herrath -no relationships to disclose herkel, peter r galle, edgar schmitt, ansgar w lohse, johannes gutenberg university, mainz, germany background: in clinical hepatitis, hepatocytes express mhc class i molecules; we recently reported that mhc i expressing hepatocytes can function as antigen presenting cells that stimulate specific cd t cells (hepatology ; : - ) . to understand the relevance of hepatocellular antigen presentation for hepatic immunity, we now examined whether hepatocytes may induce the differentiation of primary cd t cells. because inflammatory cd t cells in the liver are most likely primed by dendritic cells of the draining lymph nodes, we also examined whether hepatocytes may also re-differentiate dendritic cell-primed cd t cells. methods: primary cd t cells from ovalbumin-specific t cell receptor-transgenic mice were stimulated by antigen presenting hepatocytes from hepatocyte-specific ciita-transgenic mice or splenic dendritic cells.the response type was determined by measuring secreted interferon-gamma (ifn) and interleukin- (il- ). results: we found that antigen presenting hepatocytes by default induced differentiation of primary cd t cells to th cells (ifn: ulml; l- : ulml). in contrast, primary cd t cells stimulated by dendritic cells differentiated to thl effector cells (ifn: ulml; l- ulml). however, these dendritic cellprimed thl type cells, when re-stimulated by hepatocytes, had a decreased capacity to produce ifn ( ulml vs. ulml after dentritic cell stimulation); and after repeated re-stimulation by hepatocytes, the dendritic-cell primed t cells even differentiated into th cells (ifn: ulml; il- : ulml). conclusions: these data show that antigen presenting hepatocytes induce th differentiation of undifferentiated cd t cells. most notably, even dendritic cell-primed cd t cells that are committed to thl differentiation could be reverted by hepatocytes to th type. thus, hepatocytes seem to have an extraordinary capacity to promote antiinflammatory hepatic immune responses. it is therefore conceivable that antigen presentation by hepatocytes associated with clinical hepatitis, in the absence of pro-inflammatory stimuli, seems to downregulate inflammatory infiltrating t cells. background nkt cells are a unique subset of regulatory lymphocytes with important immune modulatory effects. these cells recognize exogenous glycolipids anchored by a ceramide tail to the mhc-like cdld molecule, expressed by various antigen presenting cells. glycosylceramides, including the marine sponge-derived a-galactosylceramide can activate nkt cells, leading to exacerbation of hepatitis. concanavalin a induces immune mediated hepatitis in which nkt cells are key participants. glucocerebroside (gc) is a naturally occurring glycolipid. aims: to determine the immune modulatory effect of gc in a murine model of con a hepatitis. methods: five groups of balblc mice were studied group a and b mice were treated with gc ( . pg ip) two hours prior to and two hours following administration of pg con a, respectively; group c mice were treated with con a alone; group d mice were treated with gc alone; group e mice did not receive any treatment. the degree of liver damage was evaluated by serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels, and by liver histology. the immunmodulatory effect of gc was determined by facs analysis of intrahepatic and intrasplenic lymphocytes for nkt markers, and by elisa measurements of serum ifny, il , il , il , and il . the effect of gc on nkt lymphocyte proliferation was assessed in vitro. results: treatment with gc markedly reduced serum ast and alt levels in group a compared to group c ( vs. iu in group a and group c, respectively, for ast; vs. iu in group a and group c, respectively, for alt, p< . ). administration of gc alone did not change ast or alt levels compared to naive controls. histological sections of livers from group a mice revealed markedly attenuated damage compared to sections from group c livers, in which massive hepatocyte necrosis was present. the beneficial effect of gc on immune mediated hepatitis was associated with a % decrease in the intrahepatic nkt lymphocyte number, and with significant lowering of serum ifny levels ( vs. pglml in groups a and c, respectively, p< . ); administration of gc alone led to increased serum il- levels ( pglml vs. pglml in group d vs. group e, respectively, p< . ). in vitro, administration of gc decreased nkt cell pro-liferation by % in the presence of dendritic cells, but not in their absence. conclusions: administration of gc led to significant amelioration of con a hepatitis that was associated with a dendritic cell-dependent decrease of nkt cell proliferation in vitro, and with decreased intrahepatic nkt lymphocytes and serum ifny levels in vivo. these results suggest that the effect of gc may be mediated by inhibition of intrahepatic nkt cells, resulting from competitive displacement of activating elements from the cdld molecule on antigen presenting cells. glucocerebroside, a naturally occurring glycolipid, holds promise as a new immunomodulatory agent, with a possible role in treatment of autoimmune hepatitis and other immune-mediated liver disorders. disclosures: background cd + cells constitutively expressing cd have a regulatory function, their experimental removal leading to spontaneous autoimmune disease in normal rodents. cd +cd + regulatory t cells suppress both th and th responses, competing with effector cd + cells in recognizing the same peptide antigens. their ability of expansion is key to the maintenance of tolerance. autoimmune hepatitis type (aih- ) is characterized by t cell immune responses against well-defined regions on cytochrome p d , the autoantigen of aih- . aims: to investigate the ability of expansion of cd +cd + after exposure to non-antigen-specific and cypzd -specific stimuli in aih- . methods: cd +cd + t cells were analysed by triple colour flow-cytometry of freshlcryopreserved peripheral blood mononuclear cells (pbmcs) befare and after culture in the presence of a t cell expander capable of maintaining the original t cell function (cd kd dynabeads t cell expander, dynal biotech, norway) and of synthetic cyp d peptides ( pmol), spanning the antigenic regions known to induce proliferative cd + responses in aih- . patients and controls: nine patients with aih- ( female, median age . yrs, range . to yrs) were investigated, at diagnosis and while on sustained remission. nine healthy laboratory workers served as normal controls. results: before stimulation, the level of cd +cd + t cells was significantly lower in aih- patients ( . . ) than in normal controls ( . f . ; p=o.o ), a difference present both at diagnosis ( . ? . , p< . ) and during remission ( . . , p=o.ol). following exposure to the t cell expander, the level of cd +cd + t cells increased . times ( . . ) in normal controls, but only . times in am- patients ( . ? . , p=o.ol). upon exposure to cymd peptides, cd +cd + t cells remained numerically unchanged, in contrast to the pbmcs from the same patients giving a strong proliferative response (up to . times). summary & conclusion: our data show a numerical and functional impairment of regulatory t cells in am- . this defect is likely to be key to the initiation and perpetuation of the autoaggressive process in this condition. orth, mark a mcniven, nicholas f larusso, mayo medical school, clinic and foundation, rochester, mn cryptosporidium parvum (cp) opportunistically infects intestinal and biliary epithelia causing worldwide morbidity, especially in patients with aids. epithelial invasion by cp involves host cell membrane alterations resulting in a parasitophorous vacuole that envelops the parasite and a dense-band within the host cell underlying the attachment site; both processes require host cell actin remodeling. since recent studies in other systems have demonstrated that cdc , an actin-associated gtp-binding protein, plays a central role in microbial-induced actin remodeling, we tested the hypothesis that cp activates host cell cdc and its downstream effectors to induce actin rearrangement and microbial invasion. methods: we exposed cholangiocytes derived from normal human liver and immortalized by sv transformation to freshly excysted cp sporozoites and applied molecular and morphofogical approaches to test our hypothesis. results by immunofluorescent and immunoelectron microscopy, we found accumulation of cdc in cholangiocytes at the parasite-host cell interface during cp invasion. we confirmed activation of cdc in infected cholangiocytes by immunoprecipitation using an antibody to pak , a downstream effector molecule that binds exclusively to the activated form of cdc . phosphatidylinositol -kinase (pi- k), a membrane-associated kinase associated with cdc activation, was also recruited to the site of attachment, as were n-wasp, pak and p -arc, downstream effectors of cdc . inhibition of pi- k by wortmannin or ly blocked cp-induced cdc accumulation. while overexpression in cholangiocytes of a constitutively active mutant of cdc promoted cp invasion (up to %), overexpression of function-deficient mutants of pi- k, cdc or of the wa fragment of n-wasp inhibited cp invasion by - %. moreover, inhibition of host cell cdc activation by dominant negative mutation inhibited cp-induced actin accumulation, and parasitophorous vacuole and dense-band formation at the attachment site. conclusions: these combined data suggest that cp invasion of cholangiocytes (and perhaps other target epithelia) results from the organism's ability to activate a complex host cell cdc signaling pathway that induces host cell actin remodeling at the attachment site. background: hepatitis b (hbv) and hepatitis c (hcv) infections are a world-wide health concern. studies of these infections have been hampered by a lack of a convenient animal model. we previously have shown that immunocompetent rats tolerized and transplanted with human hepatocytes can support hbv infection for at least weeks. aim: to demonstrate that the immunocompetent rat which has been tolerized and transplanted with human hepatocytes can be used to sustain and study hcv infection. methods: sprague-dawley rats were injected in utero at - days gestation with one hundred thousand huh cells (human hepatocyte cell line) to tolerized them to human hepatocytes. one week after birth, the tolerized newborns were intrasplenically transplanted with million huh cells. one week after transplantation, rodents were inoculated with one hundred thousand hcv rna copies, genotype l b human serum. animals were sacrificed at and weeks. the presence of human cells was determined by immunofluorescent staining of cryosectioned liver tissue using antibodies to human albumin. pcr and rt-pcr was used to confirm the presence of human albumin in liver tissue. the presence of hcv was assayed using an antibody to ns a viral protein, and visualized using a rhodamine labeled secondary antibody. hcv infection in the liver was confirmed by the presence of negative strand rna using nested pcr. results: functional huh cells in the chimeric livers were confirmed by the presence of human albumin mrna and dna using rt-pcr and pcr. the presence of human albumin protein in the liver was further demonstrated by immunofluorescence of human albumin in frozen liver sections. eighty-one percent (n= ) of the tolerized, huh transplanted rodents were positive for human albumin in the livers. seventy-two percent (n= ) of the chimeric animals infected with hcv were positive for the presence of ns a hcv protein from immunofluorescence studies. livers of control rats that were only tolerized and control rats that were tolerized and transplanted with huh cells, but not inoculated with hcv were negative for ns a (n= ). hcv viral replication could be demonstrated in livers of tolerized, transplanted and hcv infected rats by the presence of hcv rna bands of the correct size from nested pcr using negative strand specific primers. conclusion immunocompetent rats tolerized and transplanted with human hepatocytes can be a useful laboratory model to study hcv infection. ( we have previously observed that allogeneic hepatocellular transplants are highly immunogenic and are resistant to immunosuppressive therapy with a variety of agents. donor specific transfusion in combination with anti-cd l mab is highly effective in inducing prolonged survival of skin and myoblasts and inducing indefinite and donor-specific tolerance to heart and islet allografts. the proposed mechanism of action for dst and anti-cd l mab suggests peripheral deletion of alloreactive cd + t cells and induction of a regulatory cd + t cell population. resistance to induction of indefinite acceptance has been attributed to peripheral reappearance of alloreactive cd + t cells. in preliminary studies, we observed that dst and anti-cd l mab prolonged fvbln hepatocyte (hc) survival in c e l/ mice to median survival time (mst) of days and induced indefinite acceptance (> days) in % of mice. hc rejection occurs by cd -dependent and (cd -independent) cd -dependent pathways. this model permits evaluation on these two pathways separately. the current studies address the hypothesis that dst and anti-cd l mab induces prolonged hc allograft acceptance by inducing immunoregulation of both alloreactive cd + and cd + t cells. methods: fvbln (halat-fvbin, h- ) hcs were transplanted into cd ko, cd ko, and c bl/ (all h- b) mice. hc survival was monitored by detection of reporter halat protein in serum by elisa. for comparison, donor-matched islets were transplanted into streptozotocin-induced diabetic cd ko (h- b) mice, and survival was monitored by blood glucose. recipient mice received peritransplant administration of dst ( ~ ~ fvbln splenocytes, day - ) and anti-cd ol mab ( . mg, ip, d- , - , , ) . some cd ko hosts were reconstituted cd + t cells ( x iv, d- ). results: when the cd -dependent rejection was studied in isolation (cd ko mice), dst and anti-cd ol mab prolonged hc survival to mst of days (n= ) compared to days in untreated cd ko (n= ); however, this was not significantly different from anti-cd l mab alone. when cd -dependent rejection was studied in isolation (cd ko mice), hcs were unexpectedly rejected with mst of days (n= ) despite dst and anti-cd l mab treatment. in contrast, islets of the same strain were accepted indefinitely in cd ko mice (n= , mst> days). since this siraiegy effectively controlled hc rejection in c bl mice which have both cd -and cd -dependent pathways available, the collective results suggested that perhaps host cd + t cells were necessary for the beneficial effects of the dst and anti-cd l mab. to determine whether cd + t cells were required for induction of longterm hc survival with dst and anti-cd l mab, cd ko hc recipients were reconstituted with cd + t cells prior to dst and anti-cd ol mab treatment. cd reconstituted cd kos accepted hcs for mst of days. conclusion: the effects of dst and anti-cd l mab on combined cd -and cd -initated hc rejection are more pronounced than on either pathway alone. cd + t cells appear to be required for induction of longterm hc survival under cover dst and anti-cd l mab, which offers an apparently distinct mechanism compared to the existing paradigm for the mechanism of action of dst and anti-cd l mab. engraftment of transplanted cells in the liver is affected by cellcell interactions involving hepatic endothelial cells and kupffer cells. the proximity of transplanted cells to hsc suggested that hsc could promote cell adhesion and extracellular matrix remodeling during cell engraftment. to investigate cell-cell interactions in the liver, we analyzed hsc activation in dppn-rats transplanted intrasplenically with f hepatocytes. analysis of tissues by immunostaining from animals h, h, d and d after cell transplantation showed appearance of desmin +ve hsc in periportal areas, reaching a peak on day ( - fold increase in desmin f v e hsc in cell recipients, p<o.ool). using combined immunostaining and dppiv histochemistry, desmin +ve hsc were often observed in the immediate vicinity of transplanted cells. however, a-smooth muscle actin (sma) was not expressed in hsc perturbed by cell transplantation, whereas ccl&reatment in control animals induced sma expression in hsc, as shown by tissue immunostaining, as well as western blots of tissue extracts. after cell transplantation, perturbed hsc expressed desmin extensively, especially in areas with cell transplantation-induced ischemia and consequential ggt expression. to examine whether desmin +ve hsc were perturbed following activation of kupffer cells by cell transplantation, we administered carbon particles to hepatocyte recipients intrasplenically. these studies established that the majority (up to %) of desmin +ve hsc were located directly adjacent to carbon containing activated kupffer cells. as kupffer cells are activated within hrs after cell transplantation, whereas hsc were perturbed maximally at a later time, we reasoned that these cell-cell interactions were likely mediated by soluble factors. to demonstrate whether tnf-alpha plays a role in the stellate cell activation process, we transplanted hepatocytes into animals treated with the tnf-alpha-blocker etanercept. however, etanercept did not prevent perturbation of hsc, suggesting that factors other than tnf-alpha must be involved. to eliminate the component of ischemia-mediated cell activations, we treated animals with intrasplenic nitroglycerine infusion before and during cell transplantation, which resulted in marked attenuation of hepatic ggt expression. interestingly, desmin +ve hsc were now distributed throughout the entire liver lobule, with - fold greater prevalence, compared to rats treated with cells alone without nitroglycerine, p<o.ool, suggesting that circulating soluble factors reached hsc more effectively. in contrast, treatment of animals with nitroglycerine alone did not perturb hsc. to determine effects on cell integration, bile canalicular reconstitution was analyzed with dppiv and atpase co-stainings. nitroglycerine pretreatment resulted in superior parenchymal integration of transplanted cells, in proximity with desmin +ve hsc. conclusions: transplantation of cells in liver sinusoids activates desmin, but not sma, expression in hsc. activated kupffer cells are likely mediators of hsc perturbation. improved cell engraftment following perturbation of hsc indicates that extracellular matrix remodeling could facilitate this process. these findings offer ways to further optimize liver repopulation with transplanted cells. knudsen, timothy b hummer, kim a buffers, kiyoshi ogata, cody a koch, jefiey l platt, mayo clinic, rochester, m n background hepatocyte transplantation has been of much interest as a biological liver support. how to treat hepatocytes to achieve optimum function has been uncertain. cultured, cryopreserved and hypothermically preserved hepatocytes have been employed for clinical hepatocyte transplantation, but the efficacy was rarely compared between those cell sources. using a xenogeneic hepatocyte transplantation model, the function of transplanted hepatocytes was quantified in vivo. methods: porcine hepatocytes were isolated by a two-step collagenase technique. the hepatocytes were: ( ) immediately used; ( ) kept in uw solution at °c; ( ) cultured in supplemented williams e medium; or ( ) cryopreserved in ljw solution with % dmso before use. the viability of hepatocytes was determined by trypan blue exclusion, and x lo viable hepatocytes were injected into the spleens of rag- -defficient mice. to evaluate the function of transplanted hepatocytes, the porcine albumin level in the recipient plasma was determined by a sandwich elisa. the number of surviving porcine cells in the recipients' spleen was estimated by a quantification of porcine genomic dna sequences. results: the viability of hepatocytes was as follows: freshly isolated hepatocytes, . . %; hours, hours, and hours at "c, . t . , . i . , . i . %; cryopreserved hepatocytes after thaw, . . %; cultured hepatocytes after dissociation, . t . % (means t se). after transplanting comparable numbers of viable hepatocytes, the porcine albumin concentration was found to be lower in recipients of hypothermically-stored (group ) and cultured hepatocytes (group ) than recipients of fresh hepatocytes (group ). cryopreserved hepatocytes (group ) failed to secrete any albumin. because the half-life of porcine albumin in rag- deficient mice was . hours, the porcine albumin concentration in the recipient plasma reflected real-time albumin secretion from the transplanted hepatocytes. the porcine genomic dna in the recipient spleen was quantified weeks after transplantation. fewer cells were found in the hypothermically preserved hepatocyte-recipients (group ). therefore, the lower level of the porcine albumin secretion in the group was mainly attributable to the failure of the transplanted hepatocytes to survive. the amount of porcine genomic dna was comparable between cultured hepatocyte-recipients and freshly isolated hepatocyte-recipients, although the plasma of cultured hepatocyte-recipients included less porcine albumin. it suggested that the hepatocytes had dedifferentiated during culture and secreted less albumin thereafter in vivo. the cultured hepatocytes did not seem to differentiate again in murine spleen. conclusions: both the survival and differentiation of hepatocytes were important to maintenance of good function in vivo. the hypothermically preserved and cryopreserved hepatocytes did not survive long after transplantation contrary to their good viable look. cultured hepatocytes showed relatively good survival, although their dedifferentiation was not reversible resulting in poor outcome. freshly isolated hepatocytes are recommended for cell transplantation. medicine, ube-yamaguchi, japan; hiroshi nishina, university of tokyo, tokyo, japan; kiwamu okita, yamaguchi university school of medicine, ube-yamaguchi, japan obiective; we had developed an in vivo model to monitor the trans-differentiation and proliferation of bmcs into functional hepatocytes via hepatoblast phenotype (gfpicc model). in this model, transplanted gfp-positive bmcs via tail vein efficiently migrated to the peri-portal area of liver lobules under cc induced chronic liver damage condition. transplanted bmcs gradually proliferated and spread from the peri-portal area (zone ) into the central area (zone ). under persistent liver damage condition, bmcs trans-differentiated into functional hepatocytes. previous analysis had shown that inflammatory signals had been important for the trans-differentiatin of bmcs. in this study, we focused on growth factors and analyzed which affected the transdifferentiation and proliferation of bmcs into hepatocytes in gfpl cc model. materials and methods; immunohistochemical staining and immunofluorescent double staining with antibody of gfp, several growth factors and their receptors were performed in gfpiccl model. hepatocyte growth factor (hgf), epidermal growth factor (egf), fibroblast growth factor (fgf), vascular endothelial growth factor (vegf), platelet-derived growth factor (pdgf), and transforming growth factor (tgf) were analyzed. the percentage of stained area was quantified in the sections at thirty random fields (n= in each group), and statistical analysis was performed using plsd test (anova). results; the most significant result was shown in fibroblast growth factors (fgfi, fgf ) and their receptors (flg, bek). in immunohistochemical staining, the stained region migrated and increased from zone into zone of liver lobules with the time after bmt. in immunofluorescent double staining, co-expressions both gfp and fgfs, fgf-receptors were detected at the same distribution. the percentage of stained area of fgfs and fgfreceptors gradually increased with significant difference (the data was shown in table ). although expressions of other growth factors and their receptors were detected, the significant differences were not shown. conclusions; fgfs are the most important growth factor to transdifferentiate and proliferate bmcs into hepatocytes. cholangiocytes are the target cells of chronic cholestatic liver diseases (i.e., cholangiopathies), characterized by the dysregulation of the balance between apoptosis and proliferation that leads to changes in ductal bile secretion. in rats with bile duct ligation (bdl), there is increased cholangiocyte proliferation and enhanced basal and secretin-stimulated ductal secretion. we have shown that both cholinergic (by vagotomy) and adrenergic b y administration of a single intraportal injection of -hydroxydopamine ( hda)i denervation impairs the proliferative and secretory responses of intrahepatic bile ducts to bdl through an increase in cholangiocyte apoptosis. we have shown that bile acids and nerves co-ordinately regulate the balance between cholangiocyte apoptosislproliieration in hyperplastic rat livers. in bdl rats, while ursodeoxycholate and tauroursodeoxycholate feeding prevents the loss of bile ducts caused by vagotomy in a caz+/pkc-dependent manner, taurocholate feeding prevents vagotomy-induced duct damage in a pi k dependent manner. no information exists regarding the interaction between bile acids and adrenergic innervation in the regulation of cholangiocyte function. thus, we tested the hypothesis that taurocholate feeding protects from bile duct damage induced by adrenergic denervation by -ohda. methods: immediately after bdl, rats received a single intraportal injection of -ohda (which induces degeneration of adrenergic terminal fibers, mglkglbody weight) or vehicle, and subsequently were fed % taurocholate or control diet in the presence of daily injections of wortmannin (a pi k inhibitor, . mgl kg body weight) or a control solution. we used week bdl rats as control animals. seven days later, we evaluated cholangiocyte apoptosis by : (i) tunel assay in liver sections; and (ii) measurement of caspase activity and protein expression for cleaved pro-caspase , and the number of purified cholangiocytes positive for annexin-v. cholangiocyte proliferation was evaluated by measurement of the number of pcna-and ck- -positive cholangiocytes in liver sections, and pcna immunoblots in pure isolated cholangiocytes. the changes in ductal functional activity were evaluated by measurement of (i) secretinstimulated camp levels and cl-ihcos exchanger activity in pure cholangiocytes; and (ii) secretin-stimulated bile flow and bicarbonate secretion in bile fistula rats. results: taurocholate feeding prevented the increase in the number of apoptotic cholangiocytes and the enhancement of caspase activity and protein expression for cleaved pro-caspase induced by -ohda. taurocholate feeding prevented -ohda-induced decrease in the number of pcna-and ck- positive cholangiocytes, and the loss of pcna protein expression. at the functional level, taurocholate feeding prevented the decrease in secretin-stimulated camp levels, c -l hco exchanger activity and bile and bicarbonate secretion. consistent with the concept that pi k regulates the interaction of bile acids with adrenergic nerves in cholangiocytes, we found that administration of wortmannin blocks -ohda-induced activation of cholangiocyte apoptosis, and inhibition of cholangiocyte proliferation and ductal secretion. summarylconclusion: taurocholate feeding prevents the increase in cholangiocyte apoptosis and the loss of cholangiocyte proliferation and ductal functional activity induced by adrenergic denervation by -ohda. taurocholate effects are mediated by the pi k pathway, since the simultaneous administration of wortmannin reverses such effects. these novel findings suggest that bile acids play a major role in sustaining the growth and functional activity of the intrahepatic biliary tree following liver transplantation. hepatocyte apoptosis by death receptors is emerging as a critical determinant in the initiation of hepatic inflammation and fibrosis during cholestatic liver diseases. however, the cellular sources of death ligands to activate the death receptors is unknown. we postulated that kupffer cells, which contribute to hepatic inflammation, may also contribute to hepatocyte apoptosis and liver fibrosis by generating death ligands. thus, our aim was to ascertain whether kupffer cells express death ligands and contribute to hepatocyte apoptosis and liver fibrosis in the bile duct ligated mouse, an animal model of cholestasis. methods: mice were subjected to bile duct ligation (bdl) or a sham operation and treated with gadolinium chloride, a kupffer cell intoxicant, or saline for three days. hepatocyte apoptosis in liver sections was assessed using the tunel assay. serum alt values were quantitated using commercially available kits. kupffer cells were isolated using arabinogalactan gradient centrifugation. real time pcr was used to measure expression of death ligands and fibrosis indicators. results: hepatocyte tunel positive cells, and serum alt values were . -fold, and . -fold, respectively, greater in day-(bdl) saline-treated vs. gadolinium chloride-treated mice. kupffer cell expression of the death ligands tnf-alpha and fas ligand was and -fold greater when kupffer cells were obtained from bdl vs. sham operated mice. the enhanced expression of fas ligand was verified by immunoblot analysis and tnf-alpha by an elisa. these data confirm that kupffer cells generate death ligands and contribute to hepatocyte apoptosis in cholestatic liver injury. consistent with a role for kupffer cellassociated hepatocyte apoptosis in liver inflammation, neutrophil infiltration into the bdl liver was abrogated by gadolinium chloride. hepatic mrna transcripts for a -smooth muscle actin, and collagen a (i), markers for stellate activation were all significantly attenuated gadolinium chloride vs. vehicle-treated animals. finally, the mechanisms for kupffer cell activation were explored in cell culture. isolated kupffer cells phagocytosed apoptotic bodies, which induced death ligand expression. in contrast, bile acids had no effect on kupffer cell gene expression. infection is associated with vigorous, durable cd + t cell responses against nonstructural hcv proteins. hcv persistence has been attributed to poor priming of cellular immune responses. one of the factors that contributes to poor priming of cellular immune responses may be the noncytopathic nature of hcv and to the subsequent absence or scarcity of virus-infected apoptotic or dead cells as source of exogenous antigens for crosspresentation. because crosspriming contributes to the induction of cd + t cells in vivo (nature ; - ), we hypothesized that cross-priming with dendritic cells (dcs) containing self-replicating rna might be useful to induce strong, hcv-specific cellular immune responses. methods: choosing hcv ns as a model antigen, we generated a recombinant cytopathic pestivirus self-replicating rna to amplify hcv ns in dcs. the principal characteristic of this vector is its ability to replicate in transfected cells which in turn enhances production, processing and presentation of the encoded hcv ns antigen. moreover, the availability of cytopathic and noncytopathic forms of the same replicon enabled us to study the immunologic implications of dc apoptosis and antigen reprocessing, leading to crosspriming in vivo. results: the murine dendritic cell lines dc . was transfected with cytopathic and noncytopathic recombinant replicon rna, respectively. hcv ns expression was detectable in more than % of the transfected cells by immunofluorescence. the time kinetics of apoptosis induction was monitored by flow cytometry using annexin v and propidium iodide staining. in contrast to the noncytopathic replicon, the cytopathic replicon led to dc apoptosis hours after transfection. dc . transfected with the cytopathic recombinant replicon rna were then used to immunize hla-a transgenic mice subcutaneously. ifn-positive, proliferative cd + t cells and ifn--y positive, cytotoxic cd + t cell responses were detectable after a single subcutaneous immunization with replicon-transfected dendritic cells and significantly stronger than after conventional, intramuscular dna immunization, the previous gold standard. crosspriming was demonstrated when t cells, primed by injection of h- b+ dcs into the h- b+ hla-a + mice, were tested with hla-a + antigen-presenting cells. transfer of cellular material from the vaccine dcs to the endogenous apcs, a phenomenon that underlies crosspriming, was directly visualized in vivo when fragments of csfe-labeled, injected h b+ dcs were demonstrated in hla-a + host cells in lymph nodes and spleens by flow cytometry and if microscopy. finally, the protective effect and the in vivo function of the induced hcv-specific t cells were evaluated in a surrorgate challenge model with recombinant, hcv ns encoding vaccinia virus. whereas lo to lo pfu were detected in nonvaccinated control mice, no vaccinia virus was detected in mice vaccinated with replicon-transfected dcs, indicating complete protection. summary and conclusion: these findings demonstrate and explain the immunologic potential of the proposed vaccination strategy. moreover, they support the hypothesis that experimental forcing of exogenous antigens into the mhc class i pathway and subsequent promotion of cross-priming is particularly important to generate t cell responses against noncytopathic and tissue tropic viruses such as hcv. disclosures: introduction: hepatitis c virus (hcv) infection leads to chronic liver disease in about % of infected individuals while only a minority achieves spontaneous viral elimination in the initial phase of acute hepatitis c. anti viral mechanisms clearing the infection in these patients, however, could contribute to the genera-tion of therapeutic or prophylactic vaccines. the strong association of a broad, hla restricted, vigorous and long lived anti viral cd +/ th t-cell response with spontaneously resolving acute hepatitis c has been demonstarted in the past. only few epitopes recognized by anti hcv specific cd + t cells that are associated with spontaneous viral clearance have been characterized so far. the aim of the present study was to identify and characterize hcv specific cd + t cell epitopes and their hla restriction during acute self limited hcv infection andlor viral control. methods: proliferative cd + t cell responses ( h-thymidin incorporation) against viral proteins (hcv-ns , -ns ) and against overlapping -mer pep-tides covering the entire ns and ns (aa - ) region were performed in patients with acute self limited hcv infection and in two patients during temporary viral control (hcv rna negative). ten patients with chronic hcv infection served as control group. cd + t cell clones were generated by limit-ing dilution of hcv protein specific t cell lines. hcv specific cd + t cell clones were further characterized fine specificity was assessed by proliferation assay after stimulation with overlapping peptides of the corresponding protein region, then the minimal epitope was defined with n-and c-terminal truncated peptides for each epitope. hla restriction was assessed by inhibition experi-ments (anti-dp, -dq, -dr) and cd + expression in the presence of a panel of ebv blasts by flowcytometry. results: we studied patients with acute self limited hcv infection. all pa-tients displayed significant proliferative t cell responses against hcv-ns or -ns protein, that was strongly associated with self-limited disease and absence of hcv rna. proliferative responses against -mer peptides showed multiple responses within the ns and ns region, possibly reflecting multiple epitopes in this region. in contrast no virus specific t cell responses were detectable in a control group of patients with chronic hcv. for further characterization of t cell epitopes hcv specific t cell clones were generated from patients against eight different hcv epitopes located within the ns (aa - , two epi-topes) and ns alb (aa - , six epitopes) region. the minimal epitopes consist of to amino acids. all t cell clones were hla class i restricted and could be stimulated by protein or peptide pulsed ebv blasts expressing the cor-responding hla-dr or -dq. conclusion: the hcv ns and ns proteins contain several immunodominant cd + t cell epitopes which are associated with spontaneous viral clearance. the detailed characterization of minimal epitopes and hla restriction are a pre-requisite for the synthesis of hla class i tetramers and for the development of prophylactic and therapeutic t cell vaccines. background a brief period of tissue ischemia followed by reperfusion renders tissue resistant against subsequent prolonged ischemia and reperfusion, a phenomenon called ischemic preconditioning. hepatic ischemic preconditioning protects sinusoidal endothelial cells against subsequent cold storagelwarm reperfusion injury and improves graft survival after liver transplantation in rats. aim ischemic preconditioning also reduces liver injury after warm ischemia and reperfusion, in which hepatocyte damage and kupffer cell activation are dominant features. however, it is incompletely elucidated which cell type ischemic preconditioning affects and how it works. therefore, our aim was to investigate the mechanisms of ischemic preconditioning against warm ischemia and reperfusion injury. method male sprague-dawley rats were injected intravenously with mglkg gadolinium chloride (gdcls), ulkg superoxide dismutase (sod) or mglkg n-acethyl-l-cyctein (nac) to suppress kupffer cells, superoxide formation or oxygen radical formation, respectively. livers were then preconditioned by clamping the hepatic artery and portal vein to the median and left lobes for min followed by min reperfusion. subsequently, livers were subjected to warm ischemialreperfusion injury in in vivo experiments and in liver perfusion experiments as follows. in vivo experiments: the blood flow to the median and left lobes was occluded with a vascular clamp for min. one hour after removal of the clamp, blood was collected for determination of alt activity and hyaluronic acid (ha) concentration. liver perfusion experiments: livers were perfused through the portal vein for min with physiological buffer and stored at °c in the same buffer. after min, livers were reperfused with the buffer containing ng/ml ha for min in a recirculating system for determination of alt activity in the perfusate and hepatic ha uptake as a marker of sinusoidal endothelial cell injury. livers of other non-treated rats were preconditioned by perfusion with the buffer containing h instead of ischemic preconditioning, and then subjected to warm ischemialreperfusion similarly. in other experiments, livers were subjected to ischemic preconditioning and perfused with the buffer containing . mglml nitro blue tetrazolium (nbt) for min and fixed with formalin. since nbt reacts with superoxide to form insoluble blue formazan, kupffer cell superoxide formation was evaluated in histological sections. results ischemic preconditioning decreased serum alt activity compared to control ( ? [mean semi vs ? , p < . ), but did not change serum ha concentration. pretreatment with gdc reversed the effect of ischemic preconditioning ( ), though gdc itself did not change alt activity after ischemialreperfusion ( t_ ). similar results were obtained in liver perfusion experiments, suggesting that ischemic preconditioning protected hepatocytes, but did not change sinusoidal endothelial cell injury. it is also suggested that kupffer cells mediate hepatocytes protection by ischemic preconditioning. in rats treated with sod or nac, decrease in alt activity in the perfusate by isch- background: living donor liver transplantation (ldlt) and split livers have emerged as a solution to ease the shortage of liver transplants and have achieved remarkable success in pediatric populations. in adults, ldlt is limited by the size of the graft that can be safely harvested and transplanted. similar limitations apply when extensive liver resections are needed for the treatment of hepatic tumors. we have previously demonstrated that the anti-apoptotic gene a is part of the physiologic response of hepatocytes to injury, protecting them from apoptosis and limiting inflammation by inhibiting nf-kb activation. our objective was to check whether a expression would help decrease the minimal liver mass required for mice survival. methods and results: two-thirds partial hepatectomy is well tolerated and is an accepted model of liver regeneration in rodents. we performed an extended ( %) liver resection (n= /group) in non-infected (ni) mice ( - week balb/c) and mice infected with recombinant a adenovirus (rad.a ) or a control adenovirus (rad.p-gal). surviving mice suffer a delay in regeneration as assessed by the number of proliferation cell nuclear antigen (pcna) positive nuclei h following resection ( - positive cellslhpf in mice with % liver resection vs. - positive cells/ hpf in mice with a / liver resection). interestingly, such a delay was not noted in mice expressing a ( - pcna positive cells/ hpf were detected h following % resection). a also prevented the development of coagulopathy as assessed by prothrombin time. we extended our resection to a radical hepatectomy comprising % of the liver (n= lgroup). we observed % lethality in ni and rad.p-gal infected mice. expression of a resulted in the survival of % of the animals and correlated with increased pcna positive hepatocytes as an indicator of increased cell proliferation. a mediated increase in hepatocyte proliferation was not merely dependent upon its anti-apoptotic function. expression of a in hepatocyte cultures, in a nonapoptotic milieu, increased their proliferative response to serum and hgf as compared to ni and rad.p-gal infected cells. conclusion: these results demonstrate a novel pro-proliferatif function for a in hepatocytes and argue for its critical role in accelerating liver regeneration and promoting hepatocyte survival and function, even when facing extreme metabolic demands. gene therapy with a may allow for successful transplantation with smaller ldlt, split liver allografts and even hepatocellular grafts. additionally, a based therapy to the liver may allow for extensive liver resections in the setting of cancer therapy. employee graeme currie -gilead sciences, inc.: employee samuel didier -gilead sciences, inc.: investigator stefanos hadziyannis -gilead sciences, inc.: investigator craig james -gilead sciences, inc.: employee ching-lung lai -gilead sciences, inc.: investigator nicole lama -gilead sciences, inc.: employee pietro lampertico -gilead sciences, inc.: investigator peter neuhaus -gilead sciences, inc.: investigator eugene schiff -gilead sciences, inc.: investigator norah terrault -gilead sciences, inc.: investigator hans tillmann -gilead sciences, inc.: investigator prolonged survival of porcine hepatocytes in cynomolgus monkeys. h nagata aliberti -no relationships to disclose sven erik behrens -no relationships to disclose vito racanelli -no relationships to disclose barbara rehermann -no relationships to disclose characterization of novel cd + t cell epitopes in acute selflimited hcv infection previous studies from our lab show that interleukin- (il- ) is a complete biliary epithelial cell (bec) mitogen that is normally produced by bec at low levels and upregulated by mechanical, infectious, or immunologic biliary tract injury. bile duct ligation (bdl) in il- -deficient (il- -/-) mice results in a reproducible phenotype that includes rapidly decompensating biliary cirrhosis, which is at least partially attributable to impaired biliary tree integrity. methods and results: oligonucleotide array analysis of primary mouse il- -and wild type il- +/+ bec cultures was used to search for possible molecular mechanisms that might account for impaired biliary tree integrity in il- -mice. the results showed upregulation of trefoil factor family (tff) messenger rna (mrna) in the il- +/+ bec compared to the il- -bec. tff are mucin-associated protease-resistant peptides that greatly contribute to barrier and epithelial repair in the gastrointestinal tract. various molecular analyses in vitro using bec cultures confirmed that il- -mediated gp signaling through the signal transducer and activator of transcription (stat ) importantly contributes to tff mrna and protein expression. in vivo, tff mrna and protein was expressed predominantly in the medium and large bile ducts and peribfiary glands in normal mouse liver in il- +/+ mice, but weakly present or absent in il- -mice. within several weeks after bdl, tff mrna and protein levels decreased transiently then increased to near baseline levels by twelve ( ) weeks after bdl in the il- +/+. in contrast, the il- -mice failed to show the secondary recovery of tff by weeks when mrna and protein levels were significantly lower than il- +/+ (figure) . the retarded recovery of tff mrna and protein in the il- -imice was reversed by daily treatment of recombinant il- . similar results were obtained in analysis of normal human liver and various biliary tract diseases. conclusions: tff is the predominant trefoil factor expressed in the mouse and human biliary tree; its expression appears to be upregulated by stat signaling and suppressed by the mitogen-activated protein kinases (mapk) signaling in the bec. tff expression in the biliary tree importantly contributes to biliary tree integrity and repair reactions. blackard, carolynne zander, massachusetts general hospital, harvard medical school, boston, ma; gregory beck, university of massachusetts, boston, ma; emmett v schmidt, raymond t chung, massachusetts general hospital, harvard medical school, boston, ma backgroundlaims: the innate immune response (type i ifn system) is postulated to play a critical role in control of viral infection.the role of the ifn system in controlling hcv replication has been inferred but not proven. in addition, hcv protein expression has been shown to antagonize the ifn response in vitro. until recently, it has been impossible to assess the interaction between hcv and the type i ifn system in vivo without a model that successfully supports viral replication. we have developed a cell-based binary hcv replication model that successfully recapitulates the early stages of the hcv life cycle (pnas , ) . we therefore used this system ( ) to assess the role of type i ifn in controlling hcv replication; and ( ) to assess the effect of hcv expression or replication on ifn-induced intracellular signaling. methods: full-length hcv cdna corresponding to the ph strain previously shown to be infectious in the presence of ? was transfected into huh-t cells (a t stably-transfected huh be), as well as mutant m g h human fibroblast cell lines null for the ifn signaling proteins tyk (ul), irf (u ), statl (u ), jakl (u ), and stat (us) respectively. to test the effects of known ifn antagoflists on hcv replication, the ifn signaling antagonist sendai virus y protein was cotransfected into huh-t cells. in addition, neutralizing antibodies (nab) to type i ifn were added to selected huh-ti cells. to study the effect of hcv on ifn signaling, huh-ti cells were cotransfected with the reporter plasmids pisre-luc and prl-tk. ifna ( iu/ml) was added to selected cells after transfection. reporter gene expression was monitored by the dual-luciferase reporter assay system and measured as relative luciferase activity. hcv core protein was measured using the trak-c hcv core ag elisa. results ifna significantly reduced hcv core ag production from . pg/ml (ph +/ifn-) to . pglml (ph +/ifn t) (p=o.ool). in contrast, y protein enhanced hcv core ag production to . pglml in ph y cotransfected cells (p=o.oool). control experiments confirmed that y protein inhibited ifna-induced isre-mediated signaling in huh-t cells; relative luciferase activity was reduced from (ph ifnp nab increased hcv core ag replication by % and % compared to no treatment (p=o.o ). the combination of anti- fna nab and anti-ifnp nab significantly enhanced hcv core ag production by % (p=o. ). the u cell line enhanced hcv core ag replication by over two-fold compared to the parental m g h and other mutant cell lines. hcv (ph ) transfection suppressed ifn-induced relative luciferase activity by % compared to ifn-treated pgem transfected cells (p= .w). transfection with a replication defective h mutant, subgenomic hcv core, or hcv structural protein mutant constructs had effects on suppression of isre-luc similar to h .conclusions: using a binary, full length hcv replication model, we found that (i) hcv replication is enhanced in the presence of the ifn antagonist y as well as by ifn neutralizing antibodies, confirming the criticality of the type i ifn system in innate control of hcv replication.( ) statl null cell lines enhanced hcv replication, indicating that this protein is the critical signaling component in innate antiviral immunity to hcv. ( ) hcv expression inhibits ifn signaling. hcv protein expression and specifically hcv core expression appears to be su€ficient to suppress ifn signaling. these findings demonstrate that the innate immune response exerts direct antiviral effects on hcv, and that hcv protein expression subverts type i ifn signaling. our replication model has the potential to provide new insights into the mechanisms of host control of hcv replication as well as the mechanism of hcv persistence. disclosures:gregory beck -no relationships to disclose jason blackard -no relationships to disclose raymond t chung -no relationships to disclose yoichi hiasa -no relationships to disclose yoshitaka kamegaya -no relationships to disclose wenyu lin -no relationships to disclose emmett v schmidt -no relationships to disclose carolynne zander -no relationships to disclose ifna) to . (ph / ifndy ) (p=o.o ). anti-ifna nab and anti-background cytotoxic t lymphocyte activator downregulates t cell activation by ligating b . single nucleotide polymorphisms (snps) at positions - in the promoter and + in exon of the ctla gene have been associated with an increased risk for autoimmune diseases. the + g allele has also been associated with a greater likelihood of response to interferon-alpha in patients treated for chronic hepatitis c. since some of the ctla snps either individually or linked together (haplotypes) may alter ctla expression or activity, we hypothesized that they may be associated with natural hepatitis c virus (hcv) clearance or persistence. methods: utilizing a nested case-control study design from an injection drug using cohort and a hemophiliac cohorts, we matched subjects with hcv clearance (anti-hcv positive and hcv rna negative on two occasions separated by a minimum of months) to two persistently hcv-infected individuals (hcv rna positive on two occasions) based on h n status, race, and gender. five ctla snps, which define all common haplotypes, were genotyped using either sequence specific primers or a single base extension method. conditional logistic regression was used to determine odds ratios and p-values for these snps. haplotypes were constructed using the phase program and analyzed using the snpem program. results: included in this study were and subjects who either cleared or had a persistent infection with hcv, respectively. the study cohort was % black, % white, and % other. blacks who were homozygous for the mutant allele g were more likely to experience viral clearance (or . , p= . ). this association was not found in white subjects (or . , p= . ). since, hla-cw"o is associated with viral persistence in these cohorts (thio et a j virol ), we stratified the g homozygous individuals by c w w . both blacks and whites who did not have cww were more likely to have viral clearance if they were homozygous for g (or . , p= . and or . , p=o. , respectively) . stratification by other hla alleles associated with hcv clearance or persistence did not reveal additional relationships. the g formed haplotypes with the wild type alleles at three of the four other snps examined, and those haplotypes did not have a stronger association than g alone. g was found in haplotypes with both the mutant and wild type alleles at the - promoter position, but neither of these haplotypes had a stronger association with clearance. conclusions: homozygosity for the ctla g snp is associated with clearance of hcv in blacks regardless of hla genotype, whereas in whites, homozygosity at this position trends toward an association with clearance in hla-cw*m-negative persons. since ctla g has been reported to augment t ceil activation by decreasing ctla expression, these results point to the importance of t cell activation in hcv clearance. disclosures:jacquie astemborski -no relationships to disclose james g goedert -no relationships to disclose recurrence of hcv infection post liver transplant is universal. high levels of viraemia characterize the post transplant course. in this setting, acute rejection in association with hcv re-infection of the graft (ar (hcv)) and recurrent chronic hepatitis (chi) are recognized clinical entities. we aimed to ) determine the intrahepatic gene profile in ar (hcv) and chi. ) examine whether the gene profile in ar (hcv) is different to that of acute rejection in non-hcv infected allografts (ar non-hcv). ) examine whether the gene profile of chi is different to chronic hepatitis c pre-transplant (ch). methods: genechip arrays were utilized to study pooled rna from liver tissue; patients with ar (hcv), patients with ar (non-hcv), patients with chi, patients with ch and normal livers. probe level data was analysed using the bioconductor affy library and genes with more than fold changes were considered to be upregulated. results: ar (hcv) us ar (non-hcv): compared to normal tissue, both groups were similarly characterized by upregulation of several t cell dependent immune activation genes such as the lymphocyte antigen campath- , mac- binding protein and ifn related genes. however, compared to ar (non-hcv), ar (hcv) was specifically characterized by upregulation of several ifn-alpha associated genes including - as, and p . chi vs c h compared to normal, both groups were characterized by upregulation of genes involved in antigen presentation (proteasome subunit , mdw), t cell activation (t cell receptor and ctl- ), ifn-gamma inducible genes (ifn- kd, ip- , humig and class i mhc) and ifn-alpha associated genes (p and - oas). however, the ifn-alpha and gamma genes were more upregulated in chi compared to ch. furthermore, compared to ch, the chi group displayed increased expression of genes involved in cellular proliferation (pcna, cyclin, beta integrin), apoptosis (cytochrome c, fas-l, caspase ), inflammation (macrophage mannose receptor, complement factor h) and fibrosis (angiotensin i receptor, proteoglycan core protein). conclusions: at the transcriptome level, the pathological process in hcv infected allografts with acute rejection or recurrent chronic hepatitis is characterized by upregulation of several ifn-alpha and gamma related genes compared to other livers. moreover, chronic hcv post transplant is further characterized by an increased process of cellular proliferation, apoptosis and fibrosis compared to chronic hepatitis in the pretransplant setting. collectively the data suggest that the high levels of viraemia which characterize the post transplant setting drive the immune response to act at a higher level. in addition, the upregulation of several interferon related antiviral genes supports the notion that hcv is a strong inducer of intrahepatic interferons, but is relatively resistant to their antiviral activity. (hcv) infection is thought to be mediated by a multispecific immune response. hcv-specific t cells have been described in hcv-seronegative virus-exposed individuals such as i.v.-drug addicts, family members of chronic hcv-patients and lab-personnel. however, it is not known whether these individuals had recovered from previous asymptomatic acute hepatitis c. in this study, we prospectively followed individuals who experienced an injury with an hcv-contaminated needle. methods: between january and march we collected pbmc from individuals (medical health professionals at our institution; females, males; age - years) who experienced an injury with an hcv contaminated needle. blood samples were taken on the day or the day after the event and at different time points during follow-up for up to months. low levels of hcv-rna were examined in serum by the highly sensitive transcription-mediated tma-assay (detection limit - iuiml) and in rna of pbmc. t cell-cytokine secretion (elispot-assays for ifn-gamma and il- , flow-cytometry-based ifn-g capture assays), t cell-proliferation ( -thymidin incorporation, cfse-staining), and t cell-cytotoxicity ( -chromium release assays) were investigated directly ex vivo and in t cell lines. results: none of the individuals became positive for hcv-rna in serum (tma-assay) or pbmc and all of them remained anti-hcvnegative throughout follow-up. at the time of the needle stick injury, hcv-specific cd + t cell responses were already detectable in of the individuals and became detectable thereafter in additional persons. hcv-specific cd + t cell responses could be investigated in one hla-a -positive individual in more detail at several time points. he was negative for hcv-specific interferon gamma-and il- -producing cells on the day of the injury as determined by elispot assays. however, after weeks, we detected for core- -specific ifn-gamma producing cd + t cells. out of mhc-class i-restricted hcv epitopes tested, one additional peptide (ns - ) became positive weeks later. the presence of core- and ns - -specific cd + t cells was confirmed by a second flow-cytometry-based assay. hcv-specific inf-gamma positive cells were cd -dim and hla-dr-negative. the frequency of ns - and core- -specific cd + t cells was about and pbmc, respectively, in the elispot assay and remained constant in this subject until month of followup. the increase of cd + t cell responses was accompanied by the development of an hcv-specific cd + response targeting predominantly the hcv-core and hcv-helicase antigens. conclusions: we here demonstrate in a prospective study the development of hcv-specific t cells in hcv-exposed individuals after needle stick i n j q in the absence of viremia. surprisingly, these hcvspecific t cells became detectable rather late after the potential exposure. our data are in line with previous studies demonstrating hcvspecific t cell responses in chimpanzees inoculated with subinfectious doses of hcv. t cell immunity in medical health professionals against hcv may contribute to the low prevalence of hcv among doctors and nurses since hcv prevalence in medical health personnel has been shown to be equal or even lower as compared to the general population in most studies. aims: laparoscopic cholecystectomy is the treatment of choice for symptomatic gallstone disease at present. despite its wide spread application in the past decade, the incidence of iatrogenic bile duct injuries related to this procedure has remained unchanged around . - . %. we review the investigation, management and outcomes of such injuries referred to a tertiary referral center in the united kingdom. methods: prospectively collected data on iatrogenic bile duct injuries following elective laparoscopic cholecystectomy referred to our center was analyzed. the strasberg classification was used for defining the types of injury. results: a series of patients were referred for management of iatrogenic bile duct injuries between january and june .median follow up period was months. patients had type e injuries (to%), followed by with type a ( %) with type d ( %) and each of types b ( . %) and c ( . %). an uncomplicated primary operation was described in patients ( %). despite the operation being converted to an open procedure in patients only injuries were recognized at the time of surgery.(l . %) although the median time to recognition of biliary tract injury was days ( - ) the median time to referral increased to days ( - ) for a type a injury, and days ( - ) for a type e injury. a total of patients underwent surgical intervention prior to referral of which had biliary reconstruction. nine of these patients required subsequent revision surgery. after referral ( %) patients were managed conservatively while ( %) had ercp and biliary stenting and ( %) had interventional radiology as the main procedure. surgical intervention was required in patients ( %) at our center following referral. this included t-tube insertion ( ), primary common bile duct repair (lo), biliary reconstruction ( ) and liver resection ( ) with three patients requiring subsequent revision surgery. major associated vascular injuries were present in eight patients. one patient suffers from recurrent cholangitis while five remain asymptomatic on follow up. two required liver resection due to severe ischaemic necrosis. bowel perforations were associated in three patients. both vascular and bowel injuries occurred in patients with type e bile duct injuries. nine patients ( %) had recurrent cholangitis following treatment. three patients developed end-stage liver disease due to secondary biliary cirrhosis with one undergoing orthotopic liver transplantation. there were five deaths related to bile duct injuries. two had associated vascular injuries and one had bowel perforation. a further two patients with type a injuries died following multiorgan failure due to sepsis. conclusions: iatrogenic bile duct injuries are associated with significant morbidity and mortality. delay in recognition and appropriate referral remain a continuing problem. a multi disciplinary approach in a specialized institution has a significantly better long term outcome. background radio-frequency ablation (rfa) is widely used as a treatment of inoperable tumor leasons in the liver. preliminary studies of a vx hepatoma model in rabbits showed tumor specific t lymphocytes after rfa treatment and a significantly prolonged survival of these treated animals compared to a control group. aim: the aim of the study was to assess whether the observed tumor specific t cell response after rfa treatment, found in the vx tumor model, is transferable to humans. methods: patients were included into a pilot study. patients had metastases of colonic cancer and patients were suffering from hepatocellular carcinoma (hcc) with underlying cirrhosis. all patients were treated with rfa (elektrotom him , berchtold, germany).blood samples were taken before and weeks after rfa. an interferon gamma cytokine secretion assay (miltenyi, germany) was carried out on whole blood and measured by a flowcytometer (facs calibur, bd science, germany). as test antigens served autologous liver tissue and tumor tissue received by biopsy. t cells were doublestained with cd antibody to detect cytotoxic t cells. the basic ifn gamma secretion was also measured unstimulated. the activated/non activated cd positive t cell ratio (anr) was calculated and analysed statistically with spss. only rfa naive were included. results: before rfa patients had a mean stimulation of , anr (sd= , ) against liver tissue and , anr (sd= , ) against tumor tissue (see tab. ). weeks after rfa a strong increase of anr was observed with , anr (hcc) and , anr (colorectal carcinoma) against tumor tissue. this increase was also detectable weeks after rfa ( , anr (sd= , ) (hcc) and , anr ( , ) (colorectal carcinoma)) (see fig. and tab. ) . the anr levels were allways low against liver tissue with , anr (sd= , ). discussion: rfa shows significant tumor specific t-cell stimulation in patients with primary and secondary tumors of the liver. rfa seems to overcome immune tolerance or presents alterated and/or cryptic tumor antigens, and causes a significant tumor specific cytotoxic tcell response. this effect could be exploided in multimodal antitumoral strategies. the strategy of primary hepatic resection followed by secondary liver transplantation (slt)was not evaluated in-term of operative risks and survival. between - , among cirrhotic patients transplanted for hcc with mazzafero's criteria on analysis of the specimen, had liver resection prior to slt : for recurrence (n= ), deterioration of liver function (n= ) or high risk for recurrence (n= ) with a mean time between liver resection and listing for lt was months .the comparison between slt group and patients who underwent primary liver transplantation (plt) showed similar median age ( vs. years), sex and etiology of liver disease (alcohol/viral biclother). overall time on lt waiting list of the two groups was similar ( vs. months). pathologic analysis of the specimen revealed similar number ( . vs. . ) and tumor size ( . vs. . cm). pen-and post-operative course were not different in terms of operative time ( vs. min), blood loss ( vs. ml), transfusion ( vs. units), icu ( vs. days) or hospital stay ( vs. days), morbidity ( % vs. %) or -day mortality ( . % vs. . %). during a median follow-up of months ( to months), patients recurred after plt and one after slt. after transplantation, and -year overall survivals were not different between groups ( vs. % and vs. %). results of our study showed that liver resection for hcc prior to transplantation did not increased the morbidity or impair long-term survival following lt. therefore liver resection prior to transplantation can be integrated in the treatment strategy for hcc. aims: ct- is a member of the il- family of cytokines which protects myocardiocytes against thermal and ischemic insults. in this work we analyzed the expression of ct- by normal and diseased liver and whether this cytokine might protect the liver against ischemia-reperfusion injury. methods: rat ct- cdna was cloned, recombinat rat ct- was produced and used for generation of poly and monoclonal antibodies which were employed in immunochemistry, western blot and elisa. the recombinant cytokine was assayed for therapeutic use in rat models of ischemia-reperfusion injury. wistar rats were subjected to min ischemia of % of the liver by clamping the corresponding branches of the hepatic and portal vein. after this period the clamp was released and serum and tissue samples were obtained at hours. rats were divided into three groups: group received mcg ct- minutes prior to ischemia (n= ), group received saline instead of ct- (n= ) and group were sham operated (n= ). results: immunohistochemistry of normal livers demonstrated ct- expression selectively in hepatocytes of the centrolobular area. six hours after reperfusion of ischemic livers the values of serum alt were times higher in group than in group (pco. ). severe focal hepatocellular necrosis was found in animals from group while no apparent morpholgical changes were observed in rats from group . serum ct- levels were raised after reperfusion in untreated animals as compared with sham operated rats. western blot of liver samples showed an increase of ct- expression in the non-ischemic liver lobule as compared with the damaged part of the liver. conclusions: ct- is a hepatoprotective cytokine produced by hepatocytes in centrolobular areas. ct- synthesis increases after ischemic insults. administration of recombinant ct- protects very efficiently the liver against ischemia-reperfusion injury. ct- may be a valuable therapy to attenuate hepatic damage of the donor liver during orthotopic liver transplantation. disclosures: naiara beraza -no relationships to disclose matilde bustos -no relationships to disclose pun fortes -no relationships to disclose maria ifiiguez -no relationships to disclose eduardo martinez-ans -no relationships to disclose jesus prieto -no relationships to disclose cardiotrophin- (ct- ) is a potent background: in experimental and clinical acute liver failure, both a loss of cerebrovascular autoregulation and an increase in cerebral blood flow (cbf) have been implicated in the development of brain edema. it is unclear whether the pathogenesis of these two phenomena are linked. the accumulation of glutamine within astrocytes appears to be a critical first step in the development of cerebral hyperemia, as experimental inhibition of glutamine synthetase with methionine sulfoximine (mso) attenuates brain edema and corrects cerebral hyperemia. to study cerebrovascular autoregulation without the confounding variable of brain edema, we examined animals one day after portacaval anastomosis (pca), where a decreased cerebral blood flow has been noted in the setting of a hyperdynamic systemic circulation. material and methods: a pca or sham surgery was performed in male sld rats ( - gm). at hrs, mean arterial pressure (map) was measured continuously in anesthetized rats. noradrenaline was infused at a dose of pglkglmin for minutes. mso or saline was given as an intraperitoneal injection ( mglkg) three hours prior to na infusion. relative changes in cortical blood flow (cbf) were measured with a laser doppler probe placed over the parietal cortex. the animals were mechanically ventilated and arterial blood gases were monitored throughout the experiment. three groups of rats were studied (n= ). results: the sham group had significantly higher map, in accordance with the systemic vasodilatation seen in pca rats. adding na to all groups significantly raised map and hr. the sham+na group did not exhibit an increase in cbf with a rise in map. the pca+na group had a significant increase in laser doppler flow (ldf) from baseline that coincided with an increase in map. pretreatment of the pca animals with mso prevented the rise in cbf despite a rise in map. there were no significant differences in pc among the three groups. as expected, arterial ammonia levels were higher in pca animals and increased further after inhibition of glutamine synthetase in the group that received mso. conclusions: a rise of map in rats after pca results in an increase of cbf, indicating a loss of cerebrovascular autoregulation. the restoration of autoregulation with mso, despite higher arterial ammonia levels, suggests that accumulation of glutamine within the brain is critical to the pathogenesis of altered cerebrovascular regulation in this model. as similar conclusions can be reached regarding the development of cerebral hyperemia in experimental acute liver failure, it appears that the mechanisms responsible for cerebral hyperemia and failed cerebrovascular autoregulation are linked. future studies should focus on the nature of the intracerebral signal that is responsible for the loss of cerebral autoregulation in this model. a decreased intra-hepatic production of nitric oxide (no) participates on the pathogenesis of portal hypertension in cirrhosis. although non-specific no donor substances, such as nitrates, are able to decrease the intra-hepatic vascular resistance to portal blood flow, the use of these vasodilators to treat portal hypertension in cirrhotic patients is limited by the occurrence of side effects (arterial hypotension, headaches etc). aim. to test the effect of a liver-specific no donor (ncx- ), an ursodeoxycholic acid-derived compound, on both systemic and intra-hepatic hemodynamics in portal hypertensive cirrhotic rats. method. after the development of ascites, cirrhotic rats (cc -induced) were treated with ncx- ( mglkg b.w.) or ursodeoxycholic acid ( mglkg b.w., control group) by gavage once a day for days and, then, used in of studies: ) in vivo mean arterial pressure (map), portal pressure (pp), and superior mesenteric artery (sma) blood flow measurements before and during progressive blood volume expansion (blood infusion); ) in situ liver perfusion to obtain doselresponse curves to methoxamine (alpha*-adrenergic agonist) or flowlpressure curves; and ) cgmp concentration measurement in liver tissue. results. both map and heart rate were similar in both groups, indicating that ncx- had no effect on systemic hemodynamics. pp was also similar in both groups. during blood infusion, ncx- treated rats and control rats showed similar map (p= . ) and sma blood flow (p= . ) increases; however, the pp increase observed in control rats was blunted in ncx- treated rats (p= . ). flowlpressure curves obtained during liver perfusion were similar in both groups; however, livers from ncx- -treated rats showed a decreased response to methoxamine (p= . ) than controls. the concentration of cgmp in ncx- -treated livers was significantly higher (p= . ; ) than in ursodeoxycholic acid-treated livers ( . t . vs. . . fmollmg of protein). conclusion. ncx- improves the portal system adaptability to portal blood flow increase and decreases the intra-hepatic hyper-reactivity to methoxamine observed in cirrhotic rat livers without causing systemic side effects. these beneficial effects are probably due to increase in the intra-hepatic no bioavailability. the mechanisms behind the upregulation of endothelial nitric oxide synthase (enos) in portal hypertensive animals remain unknown. the aim of this study was to investigate the mechanism that initiates enos upregulation in the superior mesenteric artery (sma) in portal hypertension. it has been shown that the vasoconstriction of the sma with the consequent increase in the sma cyclic strain is the earliest event that occurs within hours after portal vein ligation (pvl). thus, we tested the hypothesis that this early vasoconstriction of the sma may initiate enos upregulation in pvl animals. methods: portal hypertension was induced by partial ligation of the portal vein (n= ). in a separate group of rats, mesenteric vasoconstriction was achieved also by renal artery ligation (ral, n= ). corresponding sham-operated rats were used as control groups (n= ). effects of vasoconstriction of the sma in pvl and ral rats were evaluated by measuring perfusion pressure changes in the isolated sma beds in response to methoxamine, nos activity and enos protein expression. data were compared to the corresponding sham group. hemodynamic features were also determined. mean arterial pressure (map), portal pressure ( i " ) and sma blood flow (qsma) were measured by catheterization and pulsed doppler flowmetry. sma vascular resistance was calculated from map, pp and qsma. results: there was a significant increase in the sma vascular resistance in pvl ( . % . vs . . rnmhgl %flow, k . ) and ral rats ( . vs . % . mmhgf%flow, p< . ) compared to their corresponding sham-operated rats, demonstrating the presence of sma vasoconstriction in both pvl and ral groups. the mesenteric vasculature of pvl and ral animals showed hyporeactivity to methoxamine compared to their respective sham groups (p< . ). whiie pvl rats were portal hypertensive (p<o.ol), ral rats were not. the sma vascular hyporeactivity of pvl and ral were corrected by l-nmma and while there was not significant difference in enos protein expression nos enzyme activity was significantly higher in the pvl and ral rats compared to the corresponding sham-operated groups (see figure) . conclusions: mesenteric arterial vasoconstriction in itself leads to enos upregulation and therefore, plays a triggering role in the initial increase of enos catalyhc activity in sma of pvl rats. helen hendrickson, vijay shah, mayo clinic, rochester, m n background. diminished enos derived no production from the hepatic vascular endothelium contributes to hepatic vasoconstriction in cirrhotic portal hypertension. unfortunately, prior studies aimed at delivering a constitutively active form of enos (s denos) using an adenoviral vector (ads denos) were unsuccessful in correcting hepatic vasoconstriction in the bile duct ligated (bdl) rat liver. aim. to better understand the mechanism of s denos dysfunction in bdl liver by examining the influence of the enos inhibitory protein caveolin on recombinant s denos function in transduced hepatic stellate cells, which do not express enos endogenously but are a prominent cell target of systemically delivered adenoviral vectors. methods. cellular localization of caveolin was determined from fixed sham and bdl liver sections by immunogold electron microscopy. in vitro gene transfer was performed in the lx hsc line with ads denos and an adenoviral vector encoding caveolin (ad-cav). protein interactions were determined by immunoprecipita-tion and western blot analysis. nos activity was assessed by arginine to citrulline conversion assay. results. caveolin immunogold analysis in bdl liver demonstrated prominent expression not only in liver endothelial cells, but also in stellate cells. western blot analysis from lx cell lysates confirmed caveolin protein expression in vitro. in lx cells transduced with ads denos, immunoprecipitation of caveolin co-precipitated recombinant s denos and conversely, immunoprecipitation of s denos coprecipitated caveolin. to examine the influence of excess caveolin protein levels on s denos, lx cells were co-transduced with ads deno.s and adcav. co-transduction of adcav and ads d promotes the enhanced binding between s denos and caveolin as well as a decrease in the activity of recombinant s denos by % (p< . ; ads denos vs ads denos +adcav; n= ). conclusion. these studies demonstrate a functional effect of enhanced caveolin expression and binding with s denos in stellate cells and indicate that the lack of function of recombinant s denos protein delivered in vivo to cirrhotic liver may be due to inhibition of s denos by caveolin- in stellate cells. background and aims: the presence of actin-like microfilaments in the neighborhood of sinusoidal endothelial fenestrae (sef) indicates that the cytoskeleton of sinusoidal endothelial cells (sec)s plays an important role in the modulation of sef. we have reported that a potent vasoconstrictor endothelin (et) causes contractions of sef as well as hepatic stellate cells via the eta and etb receptors, leading to the elevations of sinusoidal microvascular resistance supposed to be one the essential factors in the pathogenesis of portal hypertension. rho has emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. the present study aimed to examine how a rho stimulator; lysophophatidic acid (lpa), and a rho inhibitor; y- rho-kinase inhibitor, affect the morphology of sef in vitro. methods: monolayers of cultured sec were obtained by collagenase infusion of a rat liver and then subjected to primary culture for hours. the cells were separated into three groups; control, lpa-treated (lopm), and y- -treated ( pm) groups (treatment duration, min). sef morphology was observed by scanning electron microscopy. formation of f-actin stress fibers was observed by confocal microscopy. actin cytoskeleton around sef was investigated by permealizing cells with streptolysin- ( . u) for min and examined by transmission electron microscopy. rho a and phosphorylated myosin light chain kinase were analyzed by western blotting. rho activation was measured by rhotekin binding assay. results: following lpa treatment, f-actin stress fiber and active rho increased concomitant with a decrease in diameter of sef. however, following y- treatment, f-actin stress fiber and active rho were reduced, concomitant with an increase in diameter of sef. actin cytoskeleton around sef increased in lpa-treated cell, but decreased in y- -treated cell. rho a levels were similar in control, lpa-treated, and y- treated secs. phosphorylated myosin light chain kinase levels were diminished gradually after min in y- -treated secs and increased after min in lpa-treated cells. lpa treatment increased the amount of active rho, compared to control and y- treatment. conclusion: these results indicate that rho may play an essential role in the regulation of contraction and dilation of sef through the actomyosin system. disclosures: analysis of visual-evoked response patterns suggests that gabaergic tone is increased in brain in liver failure and that this is a major cause of hepatic encephalopathy (schafer et al., troenterolow : - , ) . however, the precise mechanisms responsible have eluded researchers in this area for the last two decades. studies in brain tissue from both human and experimental animals with liver failure have consistently shown that gaba synthesis, gaba receptors and their associated benzodiazepine modulatory sites are intact and that expression of the subunit proteins of the gaba-a receptor complex are unchanged (zwingmann et al., hevatologv - , ; ahboucha et al., neurochem. int., ) . on the other hand, there is emerging evidence to suggest that neurosteroids (ns), a novel class of compounds with potent gaba agonist properties are increased in brain in endstage chronic liver failure in humans (ahboucha et al., hepatola : a, ) . in order to further assess this possibility, ns concentrations were measured using a sensitive radioassay in samples of frontal cortex obtained at autopsy from cirrhotic patients who died in hepatic coma compared to an equal number of controls matched for age, gender, and autopsy delay intervals. patient material contained up to -fold increases of ns and subsequent analysis by gas chromatography-mass spectrometry showed that the major component ns with positive allosteric properties at the gaba-a receptor was allopregnanolone. patient material did not contain significantly increased concentrations of either gaba or benzodiazepines. in a separate series of experiments, ns concentrations were found elevated up to -fold (p< . ) in brains of rats following end-to-side portacaval anastomosis (pca) and . -fold (p<o.ol) in brains of rats following hepatic devascularization (pca followed by hepatic artery ligation) compared to sham-operated controls. administration of inhibitors of the ns synthesis enzyme -hydroxysteroid dehydrogenase (trilostane or indomethacin) resulted in dose-dependent reductions in brain concentrations of allopregnanolone and a concomitant improvement in locomotor activity scores in pca rats. together, these findings offer unequivocal evidence that ns with positive allosteric modulatory properties are generated in brain in both human and experimental liver failure and that these substances contribute to the "increased gabaergic tone" characteristic of hepatic encephalopathy. pharmacological agents aimed at reduction of ns synthesis or modulation of the ns site on the gaba-a receptor complex could afford effective novel therapeutic strategies in the treatment of hepatic encephalopathy [funded by cihr canada] abstract primary hepatocellular carcinoma (hcc) is one of the most common malignancies, ranks fi&h in frequency among all cancers worldwide, and causes over million deaths annually. although a number of oncogenes and alterations in signaling pathways have been identified in hcc, current treatment for this liver tumor is largely ineffective and has poor patient prognosis. one of the potentially curative approaches in hcc therapy is based on interfering with hcc progression by blocking cell division. in regenerating liver, expression of the proliferation-specific forkhead box m l b (foxmlb) transcription factor is reactivated prior to hepatocyte dna replication and its levels are sustained throughout the period of hepatocyte proliferation. deletion of the foxmlb fllfl (loxp targeted) allele using albumin promoterlenhancer driven-cre recombinase (alb-cre) transgene resulted in significant reduction in regenerating hepatocyte proliferation, which was associated with diminished expression of cell-cycle promoting cdc a and cdc b phosphatases and increased nuclear levels of cyclin dependent kinase (cdk) inhibitor p cipl protein. in order to test the hypothesis that foxmlb is required for the development of hcc, we subjected alb-cre foxmlb -/-mice and foxmlb fllfl (control) mice to a diethylnitrosamine (den)lphenobarbital (w) liver tumor induction protocol. our data demonstrated that alb-cre foxmlb -imice are highly resistant to liver tumor formation as evidenced by their failure to develop either hepatic adenomas or hcc following weeks of denlpb tumor induction protocol. in contrast, at weeks following den/pb tumor induction protocol, foxmlb fllfl livers displayed a large number of adenomas that progressed to hcc by weeks following den/pb treatment. the alb-cre foxmlb -iliver displayed enzyme-altered foci as evidenced by protein expression of the glutathione-s-transferase placental isoform. however, alb-cre foxmlb -/hepatocytes failed to develop hyper-proliferative foci, whereas abundant brdu labeling was found in hepatic adenomas and hcc of denlpb treated foxmlb fllfl mice. this reduced dna replication of foxmlb -/-hepatocytes was associated with increased nuclear levels of the cdk inhibitor p kipl protein. furthermore, foxmlb fllfl tumors expressed high nuclear levels of m-phase promoting cdc b protein, while undetectable nuclear levels of cdc b were found in foxmlb -ihepatocytes after den/pb treatment. the cdc b phosphatase is required for mitosis because it activates the m-phase promoting cyclin b-cdkl complex, and its increased expression is a prognostic marker for a variety of aggressive tumors. consistent with this finding, foxmlb -/-hepatocytes failed to enter mitosis following denlph tumor induction protocol and this was associated with accumulation of large polyploid hepatocytes. these studies demonstrate that foxmlb is required for the proliferative expansion of hepatic tumors, suggesting that foxmlb can be used as a potential target in treatment of patients with hcc. disclosures: robert h costa -no relationships to disclose vladimir v kalinichenko -no relationships to disclose joseph kuechle -no relationships to disclose brian shin -no relationships to disclose xinhe wang -no relationships to disclose yoder yoder -no relationships to disclose key: cord- -o t srim authors: chaudhari, jayeshbhai; liew, chia-sin; workman, aspen m.; riethoven, jean-jack m.; steffen, david; sillman, sarah; vu, hiep l. x. title: host transcriptional response to persistent infection with a live-attenuated porcine reproductive and respiratory syndrome virus strain date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o t srim both virulent and live-attenuated porcine reproductive and respiratory syndrome virus (prrsv) strains can establish persistent infection in lymphoid tissues of pigs. to investigate the mechanisms of prrsv persistence, we performed a transcriptional analysis of inguinal lymphoid tissue collected from pigs experimentally infected with an attenuated prrsv strain at days post infection. a total of differentially expressed genes (degs) were detected of which degs were upregulated and degs were downregulated. specifically, genes involved in innate immune responses and chemokines and receptors associated with t-cell homing to lymphoid tissues were down regulated. as a result, homing of virus-specific t-cells to lymphoid tissues seems to be ineffective, evidenced by the lower frequencies of virus-specific t-cell in lymphoid tissue than in peripheral blood. genes associated with t-cell exhaustion were upregulated. likewise, genes involved in the anti-apoptotic pathway were upregulated. collectively, the data suggested that the live-attenuated prrsv strain establishes a pro-survival microenvironment in lymphoid tissue by suppressing innate immune responses, t-cell homing, and preventing cell apoptosis. porcine reproductive and respiratory syndrome virus (prrsv) is a positive sense, single stranded rna virus that belongs to the family arteriviridae, under the order nidovirales [ ] . based on phylogenetic analysis, prrsv was originally classified into two types: type or prrsv- , which originated in europe, and type or prrsv- , which originated in north america. the international committee on taxonomy of viruses (ictv) recently updated the arterivirus taxonomic structure in which prrsv- and prrsv- are now respectively classified as two species: betaarterivirus suid and betaarterivirus suid [ ] . prrsv infects pigs of all ages; however, clinical manifestations are more severe when the virus infects pregnant sows and young pigs, causing reproductive failure and respiratory distress, respectively (reviewed in [ ] ). prrsv is endemic in most swine producing countries worldwide, causing significant economic losses to swine producers [ ] . genes involved in innate immune response and genes encoding chemokines and receptors important for t-cell homing and trafficking were downregulated. on the other hand, genes involved in the anti-apoptotic pathway and t-cell exhaustion were upregulated. functional studies revealed that the frequencies of virus-specific ifn-γ-secreting cells are lower in lymphoid tissue than in peripheral blood mononucleated cells (pbmcs). collectively, the results shed important insight into the mechanisms of prrsv persistence in the host. the attenuated prrsv strain designated con used in this study was recovered from an infectious cdna clone constructed using viral rna extracted from the attenuated con-p [ ] . marc- cells, a monkey kidney cell line [ ] , were cultured in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) and × penicillin-streptomycin ( units/ml of penicillin, and µg/ml of streptomycin (life technologies, grand island, ny, usa). pbmcs and lymph node-derived cells were cultured in complete rpmi- medium (crpmi) supplemented with % fbs, × of glutamax- (life technologies, grand island, ny, usa), units/ml of penicillin, and µg/ml of streptomycin (sigma-aldrich, st. louis, mo, usa). mouse monoclonal antibody specific to prrsv n protein sdow was purchased from the national veterinary services laboratories (ames, ia, usa). anti-porcine cd ε (clone bb - e - c ; fitc-conjugated), anti-porcine cd (clone - - ; pecy -conjugated), anti-porcine cd (clone - - ; alexa flour -conjugated), and anti-porcine ifn-γ (clone p g ; pe-conjugated), were purchased from bd biosciences (san diego, ca, usa). alexa flour -conjugated goat anti-mouse igg was purchased from invitrogen (eugen, or, usa). the pig experiment conducted in this study was approved by the university of nebraska-lincoln (unl) institutional animal care and use committee under the protocol number . ten -week-old prrsv, porcine circovirus type (pcv ), and siv negative pigs were purchased from the midwest research swine (glencoe, mn, usa). the pigs were randomly assigned to two groups of five pigs, which were accommodated in two separate rooms in the animal biosecurity level (absl- ) research facilities at unl. after week of acclimation, pigs in group were injected with dmem to serve as negative controls, whereas pigs in groups were inoculated intramuscularly with . tcid of live-attenuated prrsv strain con . whole blood samples with anticoagulant edta were collected to obtain plasma and pbmcs. plasma samples were stored at − • c for measurement of viremia and antibody response. a portion of freshly isolated pbmcs were used for measurement of t cell responses while the rest of pbmcs were cryopreserved for future analysis. at days post-infection (dpi), sample of inguinal lymph node (iln) was aseptically collected from the pigs under anesthesia. one half of the inl was stored in crpmi for lymphocyte isolation while the other half of the inl was minced and stored in trizol reagent (life technologies, carlsbad, ca, usa) for rna extraction. viral loads in plasma and tissues were measured by a commercial rt-qpcr kit (tetracore inc., rockville, md, usa). viral loads in plasma was reported as log copies per ml, whereas viral loads in tissues were reported as log copy per µg of total rna used in the rt-qpcr reaction. for statistical purposes, samples that had no detectable levels of viral rna were assigned a value of log copies. pbmcs were isolated from edta-whole blood as previously described [ , ] . single cell suspension was isolated from iln immediately after collection as follows. the tissue was processed to viruses , , of remove connecting tissue and cut into small pieces which were placed in a -µm nylon cell strainer (corning, durham, nc, usa) in the presence of crpmi. the tissue pieces were pressed against the nylon mesh by using the plunger of a ml syringe. the resulting cell suspension was collected into a ml conical tube which was passed through a -µm cell strainer one more time to remove large tissue debris. cells were pelleted by centrifugation at × g for min at room temperature and treated with a ml red blood cell (rbc) lysis buffer (life technologies, carlsbad, ca, usa). rbc lysis reaction was ceased by adding ice cold pbs containing % fbs, followed by centrifugation at × g for min at room temperature. cell pellet was resuspended in crpmi. to determine cell concentration and viability, samples of pbmc and iln-derived cells were stained with acridine orange and propidium iodide (viastaintm aopi staining solution, nexcelom, lawrence, ma, usa) and counted using an automatic cell counter (cellometer auto , nexcelom, lawrence, ma, usa). freshly isolated cells were used for elispot and flow cytometric analysis. the remaining cells were cryopreserved in % dmso, % fbs, and % rpmi- and stored in liquid nitrogen. prrsv antibody levels in plasma were measured at the veterinary diagnostic center of the university of nebraska by using the commercial elisa idexx prrs x ab test (idexx laboratories, westbrook, me, usa) following the manufacturer's instructions. the serum-virus neutralization (svn) assay was performed as previously described [ ] using plasma rather than serum. results were expressed as the log of the reciprocal of the highest dilution that showed a ≥ % reduction in the number of fluorescent foci presenting in the control wells. the frequencies of ifn-γ-secreting cells (ifn-γ scs) in pbmcs and iln-derived cells were measured by using an ifn-γ elispot assay as previously described [ , ] . briefly two replicates of , pbmcs or iln cells freshly collected from each pig were plated into two wells of a -well plate with pvdf membrane that were coated with anti-porcine ifn-γ antibody. the cells were stimulated with µl of crpmi containing . × tcid of con . for positive control, cells were plated at cells per well, followed by stimulation with a µl crpmi containing ng/ml of phorbol myristate -acetate (pma) and µg/ml of ionomycin. for negative control, cells were simply cultured in crpmi. at h post stimulation, the plate was washed with pbs-containing . % tween (pbs-t) followed by incubation with biotin-labeled antibody against porcine ifn-γ (clone p c , bd biosciences pharmingen, san diego, ca, usa). spots were developed by using alkaline phosphatase-conjugated streptavidin (southern biotech, birmingham, al, usa) in conjunction with alkaline phosphatase substrate (vector laboratories, burlingame, ca, usa). spots were counted and analyzed using an aid elispot reader version . (aid gmbh, strassberg, germany). freshly isolated pbmcs and iln cells were ex vivo stimulated with × tcid of con as described above. a cocktail solution consisting of ng/ml of pma and µg/ml of ionomycin was included as a positive control while crpmi served as a negative control. at hrs post-stimulation, µl of crpmi containing µg/ml of golgi-plug brefeldin a (bd bioscience, san jose, ca, usa) was added and further incubated for hrs. samples were centrifuged at × g for min at room temperature. cells were resuspended in flow cytometry staining buffer (facs buffer) (pbs with % fbs and . % sodium azide), and stained with anti-porcine cd ε, cd , and cd monoclonal antibodies and incubated on ice for min in the dark. cells were washed thrice using facs buffer. the cells were fixed and permeabilized with % paraformaldehyde and . % triton x- , respectively, followed by intracellular staining with an ifn-γ antibody. cells were analyzed by using a cytek dxp cytometer (cytek biosciences, fremont, ca, usa) and acquired data were analyzed using the flowjo software (bd biosciences, san jose, ca, usa) with gating based upon fluorescence minus one (fmo) control. for each sample, , events were acquired. relevant cell population was gated on cd + prior to analyzing the cd + and cd + cells ( figure s a ). ifn-γ positive cells were counted from individual cd + , cd + , and cd + cd + double positive (dp) cells ( figure s b ). total rna was isolated from iln collected from the infected and non-infected using the trizol reagent according to the manufacturer's protocol. rna was re-purified using rneasy mini kit (qiagen, hilden, nrw, germany) according to the manufacturer instructions, followed by a dnase treatment using turbo dna-free tm kit (life technologies, v.a. graiciuno , vilnius, lithuania) to remove dna contamination. purified rna was submitted to novogene bioinformatics technology co.ltd for rna sequencing. sequencing libraries were generated using nebnext ® ultratm rna library prep kit for illumina ® (neb, ipswich, ma, usa) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. briefly, mrna was purified from mg of total rna using poly-t oligo-attached magnetic beads. fragmentation was carried out using divalent cations under elevated temperature in nebnext first strand synthesis reaction buffer ( x). first strand cdna was synthesized using random hexamer primer and m-mulv reverse transcriptase (rnase h). second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. after adenylation of ends of dna fragments, nebnext adaptor with hairpin loop structure were ligated to prepare for hybridization. in order to select cdna fragments of preferentially ~ bp in length, the library fragments were purified with ampure xp system (beckman coulter, beverly, ma, usa). then µl user enzyme (neb, ipswich, ma, usa) was used with size-selected, adaptor-ligated cdna at • c for min followed by min at • c before pcr. then pcr was performed with phusion high-fidelity dna polymerase, universal pcr primers and index (x) primer. finally, pcr products were purified using ampure xp system and library quality was assessed on the agilent bioanalyzer system. the libraries were sequenced on an illumina platform hiseq and approximately million raw bp/ bp paired-end) reads were generated. rna-seq reads were first trimmed with trim galore version . . [ , ] , which include filtering reads shorter than bp and low-quality ends from reads (phred score: < ) and the option of removing reads with ns. the quality of reads before and after trimming was checked with fastqc version . . [ ] . the filtered reads were aligned to the sus scrofa genome (sscrofa . ; gcf_ . ) using tophat version . . [ ] [ ] [ ] with a read mismatch of and a maximum multi hits of and further default parameters. alignment files generated by tophat were then used to generate gene counts using htseq version . . (htseq-count), with a minimum alignment quality of [ ] . a data matrix of gene counts for all samples was created using a custom python script and the data matrix was used to run the differential gene expression analysis in deseq version . . [ ] . genes with an adjusted p value smaller than . and a log fold change larger than were considered as differentially expressed. the differential expression result table generated by deseq was annotated with gene information obtained from ensembl biomart for sus scrofa, supplemented by annotation from the gtf file. to visualize the level of prrsv rna genome, reads mapped to the prrsv con genome were counted base by base, normalized to counts per million and subsequently used to generate a coverage track using deeptools version . . [ ] . gene ontology (go) in the database (http://www.geneontology.org/) is an international standardized classification system for gene function, and it supplies a set of controlled vocabulary to comprehensively describe the properties of genes and gene products. go enrichment analysis of degs was implemented by the goseq r package [ ] , in which gene length bias was corrected. go terms with viruses , , of corrected p-value less than . were considered significantly enriched by differentially expressed genes. kyoto encyclopedia of genes and genomes (kegg) is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). kobas version . [ ] was used to test the statistical enrichment of differential expression genes in kegg pathway. virus-specific t-cell data were analyzed by unpaired t-test analysis using graphpad prism software version . . (graphpad software, llc, san diego, ca, usa). a p < . was considered significant. sequencing data from rna-seq were deposited in the ncbi geo and are available under accession number geo: gse all pigs inoculated with con became viremic starting from dpi. the viremia level increased and peaked at dpi ( figure a ). pigs inoculated with con did not displayed any clinical signs throughout the course of this study. at dpi, iln was collected and rna was extracted. rt-pcr analysis revealed that viral rna genome was detected in all five con -infected pigs but none of the sham-inoculated pigs ( figure b) . subsequently, the rna samples were subjected to rna-seq. an average of million paired-end reads were obtained for each rna sample (table s ). to confirm the presence of viral rna during persistent infection in pigs, the reads were mapped to the con genome. viral rna reads were only detected from iln of con -infected pigs which mapped throughout the viral genome ( figure c ). the results demonstrate that all five pigs in this study were persistently infected with the attenuated prrsv strain con . to examine the host responses to con persistent infection, rna reads were mapped to the reference pig genome (sscrofa . ; gcf_ . ). principal component analysis (pca) indicated that control and con -infected pigs formed separated clusters indicating that they had distinct transcriptional profiles ( figure a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con -infected group (con_ ) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con -infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con -infected pigs. out of genes in the annotated porcine genome, there were degs (fdr < %, |log | fold change ≥ ), of which degs were upregulated and degs were downregulated ( figure b and table s ). to examine the host responses to con persistent infection, rna reads were mapped to the reference pig genome (sscrofa . ; gcf_ . ). principal component analysis (pca) indicated that control and con -infected pigs formed separated clusters indicating that they had distinct transcriptional profiles (figure a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con -infected group (con_ ) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con -infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con -infected pigs. out of genes in the annotated porcine genome, there were degs (fdr < %, |log | fold change ≥ ), of which degs were upregulated and degs were downregulated ( figure b and table s ). the control animals are indicated by red dots labeled "dmem_ animal number," while the con -infected animals are indicated by cyan dots labeled "con_animal number"; (b) the volcano plot of differentially expressed genes between con -infected and control pigs. red dots denote significantly down-regulated genes (p < . , log fold change ≤− ), green dots indicated significantly up-regulated genes (p < . , log fold change ≥ ), and grey dots represent genes that were not differentially expressed. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < . were considered statistically significant (table s ) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con -infected pigs are shown (figure a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure b and table s ). detailed analysis of selected enriched pathways is presented in the respective sections below. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < . were considered statistically significant (table s ) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con -infected pigs are shown (figure a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure b and table s ). detailed analysis of selected enriched pathways is presented in the respective sections below. the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con is capable of inducing type i ifns both in vitro and in vivo [ ] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at dpi. we observed an upregulation of rna-sensing molecules including tlr (dsrna), tlr , and tlr (ssrna) (figure a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf and irf , was observed (table s ). in addition, we observed an upregulation of tlr mrna in con -infected pigs. while the ligands and signaling pathway involving tlr remain poorly understood, it has been demonstrated that tlr can act as an anti-inflammatory receptor which can also suppress tlr -induced ifn-β production [ ] . interestingly, we observed an increased expression of cd (ox ) and its receptor cd r (figure a ). cd r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [ ] , while cd is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [ ] . cd /cd r interaction suppresses cytokine production, and inflammatory responses [ ] . the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con is capable of inducing type i ifns both in vitro and in vivo [ ] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at dpi. we observed an upregulation of rna-sensing molecules including tlr (dsrna), tlr , and tlr (ssrna) (figure a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf and irf , was observed (table s ). in addition, we observed an upregulation of tlr mrna in con -infected pigs. while the ligands and signaling pathway involving tlr remain poorly understood, it has been demonstrated that tlr can act as an antiinflammatory receptor which can also suppress tlr -induced ifn-β production [ ] . interestingly, we observed an increased expression of cd (ox ) and its receptor cd r (figure a ). cd r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [ ] , while cd is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [ ] . cd /cd r interaction suppresses cytokine production, and inflammatory responses [ ] . the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and upregulation of regulatory proteins [ ] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd ) was observed from con -infected animals (figure b) . conversely, c q, a classical complement component involved in increasing virus neutralizing ability of antibodies [ ] was down-regulated ( figure b) . collectively, the data suggest that the con virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [ ] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [ , , ] . in this study, expression of tnfrsf a (tnfα receptor ) gene that the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and up-regulation of regulatory proteins [ ] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd ) was observed from con -infected animals (figure b) . conversely, c q, a classical complement component involved in increasing virus neutralizing ability of antibodies [ ] was down-regulated ( figure b) . collectively, the data suggest that the con virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [ ] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [ , , ] . in this study, expression of tnfrsf a (tnfα receptor ) gene that contains a death domain [ ] , was downregulated in the iln of infected pigs ( figure ) . similarly, expression of pro-apoptotic genes including aifm , chac , and osr , was downregulated ( figure ). on the other hand, expression of birc and bfl- /a (bcl a ), two apoptosis inhibitors that interfere with caspase activation [ ] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl- -associated killer (bak ), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl , api, bnip , and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con . contains a death domain [ ] , was downregulated in the iln of infected pigs ( figure ) . similarly, expression of pro-apoptotic genes including aifm , chac , and osr , was downregulated ( figure ). on the other hand, expression of birc and bfl- /a (bcl a ), two apoptosis inhibitors that interfere with caspase activation [ ] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl- -associated killer (bak ), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl , api, bnip , and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con . lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [ ] . celladhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [ ] . prrsv mainly persists in lymphoid tissue of infected pigs [ , ] . in this study, expression of several important chemokine ligands (ccl , ccl , ccl , ccl , cx cl , and ccl ) and chemokine receptors (ccr and ccr ) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con -infected pigs (figure a ). on the other hand, expression of cd (pd- ), a marker of tcell exhaustion, was upregulated ( figure b) . likewise, expression of inhibitory receptors havcr (also known as tim ) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [ ] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido ) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [ ] . cell-adhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [ ] . prrsv mainly persists in lymphoid tissue of infected pigs [ , ] . in this study, expression of several important chemokine ligands (ccl , ccl , ccl , ccl , cx cl , and ccl ) and chemokine receptors (ccr and ccr ) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con -infected pigs (figure a ). on the other hand, expression of cd (pd- ), a marker of t-cell exhaustion, was upregulated ( figure b) . likewise, expression of inhibitory receptors havcr (also known as tim ) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [ ] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido ) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of regulatory t-cells (t reg ) were downregulated in iln of infected pigs (figure c) , suggesting that t reg might not be present in iln at dpi. regulatory t-cells (treg) were downregulated in iln of infected pigs (figure c ), suggesting that treg might not be present in iln at dpi. since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsvspecific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd + cd + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd + cd + dp t cells that represent memory t-cells [ ] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con , both in elispot and in flow cytometry assays (data not shown). since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsv-specific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd + cd + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd + cd + dp t cells that represent memory t-cells [ ] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con , both in elispot and in flow cytometry assays (data not shown). viruses , , x for peer review of a number of genes associated with th , or humoral response, were upregulated in the iln of con -infected pigs (figure a ). il- , rgs , and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [ ] . tnfsf b and tnfsf are potent activators of b-cell lineage and ig class switching, respectively [ ] . b-cell surface antigens ms a (cd ), activation-induced cytidine deaminase (aicda) [ ] , and rafting family member (rftn ) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con -infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure b ). however, only minimal levels of virusneutralizing antibodies (titer : ) were detected in the serum of infected pigs at dpi (figure c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con -infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). a number of genes associated with th , or humoral response, were upregulated in the iln of con -infected pigs (figure a ). il- , rgs , and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [ ] . tnfsf b and tnfsf are potent activators of b-cell lineage and ig class switching, respectively [ ] . b-cell surface antigens ms a (cd ), activation-induced cytidine deaminase (aicda) [ ] , and rafting family member (rftn ) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con -infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure b ). however, only minimal levels of virus-neutralizing antibodies (titer : ) were detected in the serum of infected pigs at dpi (figure c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con -infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). viruses , , x for peer review of prrsv persists in lymphoid tissue of infected pigs for several months [ , ] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [ ] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [ ] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [ ] . in the current study, we identified a large number of degs in persistently infected animals (figure b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv- whereas in this current prrsv persists in lymphoid tissue of infected pigs for several months [ , ] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [ ] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [ ] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [ ] . in the current study, we identified a large number of degs in persistently infected animals (figure b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv- whereas in this current study, pigs were infected with an attenuated synthetic prrsv strain [ , ] . besides, the previous study looked at a small set of selected genes while in this study we look at the genome-wide rna transcriptome. go terms and kegg analysis revealed that genes involved in the innate immune (complement and tlr) pathways, apoptosis, cytokine-chemokine signaling, t-cell exhaustion, and humoral responses are highly differentially expressed in prrsv-infected animals compared to control. the complement system is a constituent of innate immunity that serves by neutralizing cell-free viruses, lysing virus-infected cells, and boosting virus specific responses [ ] . it also links the innate and adaptive immune responses, enhances humoral immunity, regulates antibody effector mechanisms, and modulates t-cell function [ ] . many viruses have developed a strategy to evade the complement pathway by recruiting or enhancing the production of host transcription regulatory components. during acute infection ( dpi), prrsv significantly represses the expression of complement regulatory components (cd and c bpb) in lung tissue [ ] . early complement activation during acute infection facilitates the release of newly formed virions from infected cells, yet, chronic viral infection is reported to suppress the activity of complement activation proteins and increases the activity of regulatory proteins (reviewed [ ] ). in the current study, we found overexpression of cfh and cfi along with another regulatory factor cd (decay-accelerating factor). overexpression of cfh, a major soluble regulator of the alternative pathway, results in inhibition of c and c convertase enzymes. cfi suppresses the complement active proteins c b (opsonin) and c b via mediating cleavage to their inactive form [ , ] . regulatory factor cd is an inhibitor of c convertase which prevents c b deposition on the cell surface [ ] . apart from that, we also see the downregulation of c q, which increases neutralizing and hemagglutination inhibition activity of anti-influenza antibodies [ ] . available data from previous studies and the current study suggest that activation of complement pathway during acute infection helps to disseminate virus, while suppression of complement components like c q, c r, and c , and upregulation in regulatory components during persistent infection facilitates the virus to escape from the complement system to maintain persistence in lymphoid tissues. most naturally occurring prrsv strains suppress type i ifn production by inhibiting the activation and nuclear translocation of irf /irf and nf-kb [ , ] . deficiency of irf / results in severe mortality to infection with west nile virus (wnv), chikungunya virus (chikv), and ross river virus infection, and promotes viral persistence in the infected hosts [ ] [ ] [ ] . it has been reported that prrsv nsp β inhibits irf phosphorylation and nuclear translocation; thus, inhibiting ifn production [ , , ] . interestingly, the synthetic prrsv-con and its attenuated form con were able to induce type i ifns [ , ] . contrary to its nature to induce type i ifn, in the current study no upregulation of canonical type i ifn signaling pathway-associated genes was observed. however, expression of rna sensing molecules including tlr , tlr , and tlr was upregulated in con -infected animals. on the basis of available data, we hypothesize that reduced levels of viral replication and sequestration of viral rna in infected cells during persistent infection might limit further activation of type i ifn signaling pathway by preventing interaction with cytoplasmic pattern recognition receptors (prrs). apoptosis or programmed cell death is a potential host immune mechanism against virally infected cells to curtail the spread of newly formed viral progenies. apoptosis is induced by two distinct, yet inter-connected signaling pathways, the extrinsic and intrinsic pathways [ ] . in order to successfully establish persistent infection, viruses have developed mechanisms to inhibit apoptosis. for instance, adenovirus (e b- k), human cytomegalovirus (ul ), poxviruses (f l), and myxoma virus (m l) have an inhibitory effect on proapoptotic proteins bak/bax [ ] . it appears that prrsv can also modulate apoptosis. studies of pulmonary alveolar macrophages (pam) infected with prrsv in vitro reveal that the virus stimulates anti-apoptotic pathways early in infection while it induces apoptosis late in infection [ ] . on the other hand, the virus induces apoptosis in the tissues of infected animals during acute infection, but the frequencies of apoptotic cells reduced to normal levels observed in control, non-infected pigs from day post-infection [ ] . in this study, expression of multiple anti-apoptotic genes including xiap, bfl- /a (bcl a ), and birc was upregulated while expression of pro-apoptotic genes (aifm , chac , and osr ) was downregulated. xiap is an endogenous caspase and inhibitor [ ] . bfl- /a (bcl a ) is a transcriptional target of nuclear factor-kb (nf-kb) that suppresses caspase activation and apoptosis in response to death-inducing stimuli like tnfα [ ] . birc is an interaction partner to tnfrsf b and inhibits apoptosis by interfering with the caspase activation [ ] . the pro-apoptotic bcl- family member like bcl- -associated killer (bak ), which allows the release of cytochrome c via formation of homo-oligomers and stable insertion into outer mitochondrial membrane was significantly suppressed. thus, the data suggest that apoptosis was suppressed in the iln of con -infected animals during persistent infection. chemokine molecules ccl and ccl play a crucial role in migration, activation, expansion, and survival of antiviral t-cells. suppression of the ccr and ccl /ccl axis results in dysfunction of t-cells during viral infection [ , ] . ccl -ccr axis plays role in protection against multiple viruses, such as hiv [ ] , herpes simplex virus (hsv- ) [ ] , hsv- , hepatitis c virus [ ] , and pseudorabies [ ] . acute prrsv infection increases the expression of chemokines, leading to the infiltration of immune cells toward the sites of infection [ , ] . in this study, expression of both ccl and ccl was significantly downregulated in con -infected lymph node (figure a ). additionally, expression of different chemoattractant molecules (fractalkine/cx cl , ccl , ccl , ccl , ccl , and ccl ) and receptors (ccr and ccr ) was also downregulated. it is possible that downregulation of these chemokines and receptors might impair t-cells trafficking to inguinal lymph node, the site of prrsv persistence. our transcriptional data are supported by the functional data which demonstrate that the frequencies of ifn-γ sc in iln were significantly lower than in pbmcs (figure ) . chronic or persistent viral stimulation results in hierarchical loss of effector functions of t-lymphocytes, including proliferation, cytokine production (e.g., ifn-γ and il- ), and cytolytic responses [ , , ] . although the molecular signatures involved in t-cell exhaustion are not completely understood, the overexpression of cell surface inhibitory receptors (e.g., pd- , ctla- , and others) primarily mediates cd + t-cell dysfunction [ ] [ ] [ ] . t-cell exhaustion seems to be a common phenomenon caused by arteriviruses, as t-cell exhaustion was also reported in horses persistently infected with eav [ ] . recently, it was shown that prrsv infection alone or co-infection with porcine circovirus type (pcv ) can significantly upregulate surface expression of pd-l (cd ) on porcine monocytes-derived dendritic cells (modcs) [ ] . increased expression of pd-l on the surface of antigen presenting cells (apcs) possibly contributes to the ineffective t-cell responses during the prrsv infection. in the present study, expression of several markers for t-cells exhaustion such as pd- (cd ) and its ligand, cd (pd-l ) was upregulated in con -infected animals, suggesting that the t-cells in lymph nodes of persistently infected animals might be exhausted (figure b) . however, additional studies (e.g., t-cell cytotoxicity and cytokine production) are required to fully assess the functionality of t-cells during prrsv persistence. germinal centers (gcs) are the specialized locations in the secondary lymphoid tissues where b-cell maturation, differentiation, somatic hypermutation, and class switching of isotypes take place [ ] . in the current study, the gc development-associated genes, which includes il- produced by follicular helper t-cells [ ] , the regulator of g protein signaling (rgs ) [ ] , and gc-associated nuclear gtpase (nuggc) [ ] , were upregulated in the lymph node of con -infected animals. thus, prrsv infection does not seem to impair gc formation and b-cell activation. this is supported by the fact that all five con -infected pigs developed a robust antibody response, as measured by the idexx elisa ( figure b) . however, only minimal titers of virus-neutralizing antibodies were detected at dpi. perhaps, prrsv does not suppress the humoral immune response. instead, the virus evades from antibody-mediated neutralization through different mechanisms such as glycan shielding and decoy epitopes [ ] [ ] [ ] . in summary, we identified a robust host transcriptional response in the inguinal lymphoid tissue of pigs persistently infected with the attenuated prrsv strain con . genes involved in the innate immune responses are downregulated. similarly, chemokines and receptors associated with 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balancing immunity and tolerance ccr coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs ccl induces mucosal homing of hiv- -specific iga-secreting plasma cells in mice immunized with hiv- virus-like particles influence of ccr ligand dna preexposure on the magnitude and duration of immunity enhancement of immune responses by co-delivery of ccl /mip- beta chemokine plasmid with hcv core dna/protein immunization genetic co-transfer of ccr ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo molecular signature of cd + t cell exhaustion during chronic viral infection t cell exhaustion t cells and viral persistence: lessons from diverse infections pd-l expression is increased in monocyte derived dendritic cells in response to porcine circovirus type and porcine reproductive and respiratory syndrome virus infections role of nf-kappa b in cell survival and transcription of latent membrane protein -expressing or epstein-barr virus latency iii-infected cells influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank dirk anderson at flow cytometry core facility, nebraska center for biotechnology for assistance on flow cytometric analysis and the staff members of unl life sciences annex and veterinary diagnostic center for the care of animals. the use of product and company names is necessary to accurately report the methods and results; however, the united states department of agriculture (usda) neither guarantees nor warrants the standard of the products. the use of names by the usda implies no approval of the product to the exclusion of others that may also be suitable. the usda is an equal opportunity provider and employer. key: cord- -qxp gfp authors: meager, anthony title: interferons alpha, beta, and omega date: - - journal: cytokines doi: . /b - - / - sha: doc_id: cord_uid: qxp gfp interferon alpha (ifn-α) is a mixture of closely related proteins, termed “subtypes,” expressed from distinct chromosomal genes. interferon β (ifn-β) is a single protein species and is molecularly related to ifn-α subtypes, although it is antigenically distinct from them. ifn omega (ifn-ω) is antigenically distinct from ifn-α and ifn-β but is molecularly related to both. the genes of three ifn subtypes are tandemly arranged on the short arm of chromosome . they are transiently expressed following induction by various exogenous stimuli, including viruses. they are synthesized from their respective mrnas for relatively short periods following gene activation and are secreted to act, via specific cell surface receptors, on other cells. ifn-α subtypes are secreted proteins and as such are transcribed from mrnas as precursor proteins, pre-ifn-α, containing n-terminal signal polypeptides of hydrophobic amino acids (aa) mainly. pre-ifn-β contains aa, of which comprise the n-terminal signal polypeptide and comprise the mature ifn-β protein. ifn-ω contains aa—the n-terminal comprising the signal sequence and the remaining , the mature ifn-ω protein. at the c-terminus, the aa sequence of ifn-ω is six residues longer than that of ifn-α or ifn-β proteins. ifn-α, as a mixture of subtypes, and ifn-ω may be produced together following viral infection of null lymphocytes or monocytes/macrophages. the biological activities of ifns are mostly dependent upon protein synthesis with selective subsets of proteins mediating individual activities. ifns can also stimulate indirect antiviral and antitumor mechanisms, depending upon cellular differentiation and the induction of cytotoxic activity. the phenomenon of viral interference was first described nearly years ago when hoskins ( ) described the protective action of a neurotropic yellow fever virus against a viserotropic strain of the same virus in monkeys. although viral interference was further investigated in the s and s, the underlying mechanism was not discovered until when isaacs and lindemann, working at the national institute for medical research (london, uk), isolated a biologically active substance from virally-infected chicken cell cultures that, on transfer to fresh chicken cell cultures, produced a protective antiviral effect (isaacs and lindemann, ) . the word interferon (ifn) was coined for this substance. its discovery aroused considerable scientific and medical interest since by antibiotics were widely available to control bacterial infections, but, in stark contrast, viral diseases such as influenza, measles, polio, and smallpox were virtually untreatable. interest was further heightened by many subsequent studies that demonstrated that ifn could be produced by human cells and was active against a broad spectrum of viruses (see schlesinger, , for an early review). at that time, ifn was being hailed by the media as a wonder drug, but it soon became clear that ifn was being produced naturally in too small quantifies for that extravagant claim to be immediately confirmed. in fact, the low production of ifn was to bedevil attempts both to characterize it molecularly and evaluate it clinically for many years following its discovery. although the protein nature of ifn was recognized at an early stage in its development (see fantes, , for an early review), it was only following the introduction of large-scale production methods in the s (cantell and hirvonen, ) and the simultaneous development of efficient purification procedures (knight, ; rubinstein et al., ) that sufficient amounts of partially pure ifn protein became available for characterization and clinical use. gradually, it became apparent that ifn was not a single protein and that there were likely to be different types of ifn molecules. however, despite progress in the area of purification and in initial characterization by sequencing n-terminal polypeptides, ifn proteins all but defied full characterization until the advent of recombinant dna (rdna) technology in the late s. this technology, spurred on by the pharmaceutical industry's desire to produce pharmacologically active proteins cheaply, revealed that one type of human ifn, now designated ifn-cx, was a mixture of several closely related proteins, termed subtypes, expressed from distinct chromosomal genes . second and third types of ifn, designated ifn-i and ifn-y respectively, have subsequently been "cloned" (taniguchi et al., ; gray et al., ) but, unlike ifn- q are single protein species. ifn- is molecularly related to ifn-~ subtypes but is antigenically distinct from them, whereas ifn-y is both molecularly and antigenically distinct from either ifn-cx subtypes or ifn-i . (for this reason, ifn-y is considered separately elsewhere in this volume.) finally, a fourth type of ifn, antigenically distinct from ifn- ~ and ifn-i , but molecularly related to both, has more recently been cloned and characterized. rather untypically, this new ifn type has been designated ifn omega (ifn- ) (adolf, ) . the genes for ifn- ~ subtypes, ifn- and ifn- are tandemly arranged on the short arm of chromosome . they are only transiently expressed following induction by a variety of exogenous stimuli, including viruses. ifn- ~, ifn-i and ifn- proteins are synthesized from their respective mrnas for relatively short periods following gene activation and are secreted to act, via specific cell surface receptors, on other cells. early studies on the characterization of ifn receptors indicated that ifn- ~ and ifn-i were likely to share a common receptor, but it has only been comparatively recently that such receptors have been cloned (uz~ et al., ; novick et al., ) . ifn actions are initiated by activated receptors and cytoplasmic signal transduction pathways, which are now well characterized for the ifn-~/i receptor, and manifested following expression of a number of ifnspecific inducible genes. induction of the antiviral state, which is dependent on such protein synthesis, may now be viewed as just one of the many activities attributed to ifn in general; these activities include inhibition of cell proliferation and immunomodulation (see pestka et al., , for a review). in the s, two types of lfn were defined on the basis of the capacity of their antiviral activity to withstand acidification to ph . these were termed type i ifn for acid-stable ifn and type ii ifn for acid-labile ifn. type i ifn included ifn produced by virally infected leukocytes, alternatively known as leukocyte ifn, and ifn produced by virally infected human diploid fibroblasts, alternatively known as fibroblast ifn. type ii ifn, which was only produced by antigenically or mitogenically stimulated human peripheral blood mononuclear cells (pbmc), has often been referred to as immune ifn (stewart, ) . antigenic differences were described for leukocyte and fibroblast ifn and these were put on a molecular basis when knowledge of their respective n-terminal amino acid sequences became available (allen and fantes, ; knight et al., ; levy et al., ; zoon et al., ) . at that time, an international nomenclature committee (stewart et al., ) reviewed the growing evidence for the existence of distinct molecular forms of ifn and introduced the greek alphabetical system to apply to the then known antigenically distinct types of ifn. leukocyte ifn was designated ifn-~, fibroblast ifn was designated ifn- , and immune ifn became ifn-y. however, complications immediately arose when it was revealed, following the cloning of several different leukocyte ifn complementary dnas (cdnas) brack et al., ; goeddel et al., ; streuli et al., ) , that leukocyte ifn was heterogeneous and contained many different, molecularly and antigenically related species, now commonly referred to as subtypes. the research group at hoffmann-la roche labeled the subtypes produced in e. coli ra, ~b, ~c, cd, etc. (evinger et al., ; rehberg et al., ) , distinguishing them from natural components of leukocyte ifn , while the biogen group labeled these recombinant subtypes ix , ~ , c , ~ , etc. , regrettably without an appropriate alphabeticalnumerical correspondence: for example, ~k -~ , cd - ~ . however, the numerical system now prevails. when an ifn preparation is a mixture of ifn-~ subtypes, e.g., leukocyte ifn, lymphoblastoid ifn, this is often designated ifn-o~n. the later cloning of cdnas encoding ifn- ~-like proteins (capon et al., ; hauptmann and swetly, ) initially led to the naming of this new ifn as ifn-o~ subclass ii, with all of the earlier-characterized ifn- ~ subtypes being reclassified as ifn-tx subclass i. this large, unwieldy nomenclature system has been superseded by the renaming of ifn-~ ii as ifn- ; this has been generally accepted with the finding that ifn- is antigenically distinct from ifn-~ and ifn-i] proteins and thus qualifies as a separate type of ifn (adolf, ) . fortunately, the nomenclature for ifn-i] has remained straightforward since there is only one protein species, at least in humans (derynck et al., taniguchi et al., ) . from the initial cloning of ifn-o~ cdnas, there has been a plethora of reports on the cloning of new, and sometimes distinct, genomic and edna clones, and fairly disparate nomenclatures have arisen. diaz and allen ( ) therefore undertook the considerable task of compiling the ifn genes and genomic and cdna clones from the literature and introduced an arabic-alphabetical system for naming ifn genes to enable their distinction from ifn proteins. thus, ifn-oc genes became ifna genes with the addition of a numeral to denote subtype, i.e., ifna , ifna , etc. (table . ). the ifn-~ gene became ifnb and, since there is only one gene in humans, it is referred to as ifnb . for ifn- genes, w has been used; hence ifnw (table . ). besides genes that are capable of being expressed and translated into ifn proteins, there are a number of pseudogenes which are unable to give rise to ifn proteins, and which in this new nomenclature system are designated by a p, e.g., ifnap , ifnwp , or simply ifnp where pseudogenes are clearly ifn-like but cannot be definitely included in any one of the ifna, ifnb or ifnw gene families (table . ). the ifn genomic and edna clones have been designated in a variety of ways, as illustrated in table . . pseudogenic or non-translatable clones are normally prefixed with a greek ~. for the purposes of this chapter, and to reduce the complexity of naming ifn clones and proteins, the nomenclature system adopted by weissmann and colleagues will be adhered to: ifn- q, %, {x , o~, etc. . numbers, structure, and localization in humans, there are nonallelic ifna genes, one of which, ifnap , is a pseudogene (table . ). in addition, there are a further four nonallelic pseudogenes that possibly also belong to the ifna gene family. probable allelic variants of certain ifna genes, e.g., ifna , are also known to exist goeddel et al., ; dworkin-rastl et al., ; emanuel and pestka, ) . this extensive family of ifna genes are tandemly arranged on the short-arm of chromosome ( p ) and span a region of approximately kb (owerbach et al., ; shows et al., ; slate et al., ; ullrich et al., ) . the ifnb gene and the ifnw gene/pseudogene family are also located in the same region of chromosome (meager et al., a,b; owerbach et al., ; henry et al., ; capon etal., ) . there is a high degree of homology among the ifna genes, but these show much less homology to either ifnb or ifnw genes. nevertheless, all of these ifn genes share the common feature of being intron-less (taniguchi et al., ; goeddel et al., ; houghton et al., ; capon et al., ) , suggesting a very ancient origin of their common ancestral gene. it has been proposed that the primordial ifn gene arose some million years ago, with the first split occurring around million years ago to yield the first ifna and ifnb genes (wilson et al., ) . since then the ifna gene has evolved and duplicated many times to give rise to the multiple ifna genes found in present-day animals and man (mij~ita and hayashida, ; gillespie and carter, ) . around million years ago, an ifna gene appears to have diverged sufficiently from the main group to give rise to the ifnw gene family, which is present in most mammals except mice and dogs (himmler et al., ; roberts et al., ) . it is not clear what characteristic of ifna and ifnw genes enabled the numerous reduplication events to occur in comparison to the nonexistent or more limited (some mammals have more than one ifnb gene, e.g., bovines (wilson et al., ) ) reduplication of the ifnb gene (ohlsson et al., ) . it is apparent that gene conversion, as a result of mismatch repair and unequal crossover, contributed significantly to the creation of distinct, but highly homologous, nonallelic ifna (and ifnw) genes (de maeyer and de maeyer-guignard, ) . in most cases, both coding and noncoding regions have diverged, but in the case of ifna the coding region has remained identical to that of ifna , although its ' and ' flanking regions have diverged (todokoro et al., ) . the structures of ifna, ifnb, and ifnw genes are similar. each gene has a ' regulatory promoter region upstream from the transcriptional start (cap) site, a ifna ifna ifna ifnals ifna~ ifna ifnap~ ifn-oq ifn-o h, lelf-d ifn-oq (d) ;~a , ifn-a ifn adapted and modified from diaz and allen ( ) and allen and diaz ( ) , which see for specific references for genomic clones and cdnas. reproduced with permission of mary ann liebert, inc., new york, usa. coding region containing a nucleotide sequence encoding a signal polypeptide of - mainly hydrophobic amino acids, which is typical for secreted proteins, and consecutively the sequence encoding the mature ifn protein, followed by the ' flanking noncoding region, which can vary in length up to base pairs (bp) (figure . ) (derynck etal., (derynck etal., , nagata et al., ; streuli et al., ; taniguchi et al., ; degrave etal., ; goeddel etal., ; gross et al., ; lawn et al., a,b; gren et al., ; capon et al., ; henco et al., ) . the ' flanking region contains a tata or hogness box, which delineates the boundary of the upstream promoter, approximately bp from the cap site. farther upstream are found a number ofhexameric repeat sequences gaaann, where n can be any base, which in their dimeric or multimeric forms act as binding sites for nuclear transcription factors and repressor molecules (fujita et al., ; ryals et al., ) . (this area is covered in more detail in section . , inducers and transcriptional control). the ' flanking regions vary in length and contain several polyadenylation sites and thus can give rise to mrnas of different lengths (mantei and weissmann, ; henco et al., ) . they contain above-average numbers of the sequence motifs atta or ttatttat. such sequences are common, however, in many other cytokine genes and other genes, such as protooncogenes, that are inducibly and transiently expressed. it has been proposed that these sequences contribute to the relative instability and short half-lives of ifn and cytokine mrnas (caput et al., ; shaw and kamen, ). all ifn genes are normally silent and thus require some sort of stimulus to induce expression. a wide range of inducers, including viruses, bacteria, mycoplasma, endotoxins, double-stranded polynucleotides or rna (dsrna), and some cytokines, have been shown to efficiently activate transcription of ifn genes (stewart, ; de maeyer and de maeyer-guignard, ). such inducers have in general the potential to induce expression of all ifna, ifnb, and ifnw genes; however, there appears to be cell-and inducer-specific selectivity that governs the type and numbers of ifn genes expressed. for instance, virally induced human diploid fibroblasts produce mainly ifn- and only a minor amount of ifn-~ (havell et al., ) , whereas virally induced pbmc produce mainly ifn-a plus ifn-o and only a minor amount of ifn-~ (cantell and hirvonen, ; adolf et al., ) . this has to some extent been confirmed at the mrna level (shuttleworth et al., ; hiscott et al., ) . differences have also been reported in the proportions of individual ifn- ~ subtypes produced by different cell types (goren et al., ; finter, ; greenway et al., ) , suggesting that ifna genes may be differentially expressed. however, the way in which such differential expression is regulated is presently not understood. transcriptional control of ifna genes resides in their ' flanking region, upstream from the cap site. nucleotide deletions outside of position- from the cap site have little impact on transcriptional control, but deletions farther in eliminated induction, indicating that this region,- to- , contained promoter regulatory elements (ragg and weissmann, ; weidle and weissmann, ) . these have been further delineated as a purine-rich nucleotide tract between- and- , containing hexameric repeats of gaaann (gaaa g/c t/c), which appears to be necessary for inducible transcription and which has been termed the "virusregulating element" (vre) (ryals et al., ) . similar studies involving ' deletions have been carried out with the ifnb gene, and it has been found that ' sequences within- to - contain regulatory elements that are required for induction by viruses and dsrna. the minimum vre has been localized to- to- with respect to the cap site (goodbourn et al., ; goodbourn and maniatis, ) and contains two positive virus-inducible elements, termed positive regulatory domains (prdi,- to- ; prdii,- to - ), and a negative regulatory domain (nrd,- to - ) (figure . ) (fujita et al., ; fan and maniatis, ; whittemore and maniatis, ; nourbakhsh et al., ) . in addition, the hexameric repeat sequences gaaann (also present in ifna genes) spanning from - to- contain variants of the prdi sequence and two further regulatory elements, prdiii (- to- ) and prdw (- to- ) have been identified that are required for a functional vre in ifnb gene expression in mouse l cells (dinter and hauser, ; du and maniatis, ) . prdi and prdiii act as binding sites for a nuclear transcription factor, designated "interferon regulatory factor-l" (irf- ) , whose expression is transiently increased by virus infection and which appears to mediate the activation of transcription of the ifnb gene (fujita et al., ; harada et al., ; xanthoudakis et al., ) . a second virus-inducible factor, designated "interferon regulatory factor- " (irf- ), also binds to prdi but suppresses, rather than activates, transcription (harada et al., (harada et al., , . prdii is a binding site for the nuclear transcription factor nfra (clark and hay, ; fujita et al., a; hiscott et al., ; lenardo et al., ; visvanathan and goodbourn, ) , which interacts with the major groove of the dna (thanos and maniatis, ) . additionally, another protein, highmobility group y/l, also binds to prdii, interacting with the minor groove of the dna (thanos and maniatis, ) . both factors appear to be necessary for virus induction of the ifnb gene promoter. prdiv contains a binding site for a protein of the camp response element binding protein (atf/creb) family of transcription factors (du and maniatis, ) . viral induction of the ifnb gene is thought to occur following activation of pre-existing nftcb and by de novo synthesis of irf- ; these nuclear transcription factors bind to the tandemly arranged prdi and prdii and act cooperatively to initiate/activate transcription (leblanc et al., ; lenardo et al., ; visvanathan and goodbourn, ; fujita et al., b; watanabe et al., ) . reporter constructs containing prdi supported by a simian virus (sv ) enhancer, or (gaaagt) , which contains the functional equivalent of dimeric prdi (n~if et al., ) are activated not only by virus but also by overexpression of irf- macdonald et al., ) . however, in most cell lines, overexpression of irf- has led to poor induction of ifnb (and ifna) genes (harada et al., ; fujita et al., b) or none at all (macdonald et al., ; reis et al., ) . this has been attributed to the repressive effect of irf- , a homologue oflrf- , which also binds to prdi (harada et al., ) . in the undifferentiated murine embryonal carcinoma (stem) cell line p , which is refractory to viral induction oflfnb (and ifna) genes and which expresses neither irf- nor irf- , overexpression of an introduced irf- construct leads to activation of endogenous ifn genes and to activation of reporter plasmids with the ifnb promoter (harada et al., ) . in addition, cell lines permanently transformed with an antisense irf- expression plasmid exhibited strongly reduced ifnb gene inducibility that nevertheless could be restored by transient transformation with an irf-l-overproducing expression plasmid (reis et al., ) . however, the role oflrf- in virus-induced activation of the ifnb promoter remains controversial (whiteside et al., ; pine et al., ) and ruffner and colleagues ( ) have shown that in murine embryonal stem cells devoid of both irf- gene alleles (irf- ~ ) viral induction of ifnb was only slightly higher in control irf- /+ differentiated stem cells than that in irf- ~ differentiated cells. this suggests that while irf- at high levels may elicit or enhance induction of ifnb under certain circumstances, it is not essential for viral induction. in cultured mouse fibroblasts devoid of irf- , ifnb induction by the synthetic dsrna molecule poly(i):poly(c) was absent, whereas induction by newcastle disease virus (ndv) was normal (matsuyama et al., ) . however, ifn induction in vivo by either virus or dsrna has been found to be unimpaired in irf- ~ mice, indicating that irf- is not essential . it has also become clear recently that the prdi site can bind factors other than irf- and irf- , and these may be more important for regulating ifnb gene activation (whiteside et al., ; keller and maniatis, ) . in contrast, targeted disruption of the irf- gene to yield mouse fibroblasts deficient in the repressor irf- has been found to lead to upregulated induction of ifnb following ndv infection (matsuyama et al., ) . this suggests that irf- negatively regulates or represses ifnb gene induction. the induction of the ifna gene appears to be regulated differently from that of the ifnb gene. irf- is bound poorly by the equivalent prdi site in the ifna promoter and this promoter also lacks an nf~cb site ( figure . ) macdonald et al., ) . the ifna gene vre does contain a hexameric repeat nucleotide sequence (gaaatg) , designated a "tg-sequence" (macdonald et al., ), which appears to mediate virus inducibility when supported by an sv enhancer, but which does not respond to irf- (n~if et al., ) . it has, however, been reported that overexpression of irf- can induce ifna genes, at least under special circumstances (harada et al., ; au et al., ) . the ifnw gene, like ifna and ifnb genes, is virus inducible and has structural features in its ' promoter region similar to those in ifna/b promoters ( figure . ). in particular, hexameric repeat units are present, but are organized differently from those present in ifna/b genes (hansen et al., ; roberts et al., ) . however, the regulation of transcription of the ifnw gene has not been studied in detail. the ifn-(x subtypes are secreted proteins and as such are transcribed from mrnas as precursor proteins, pre-ifn-(x, containing n-terminal signal polypeptides of mainly hydrophobic amino acids (figure . ). the signal polypeptide is cleaved off before "mature" ifn-(x molecules are secreted from the cell. from their cerna sequences, mature ifn-(x subtypes have been predicted to contain amino acids (except ifn-%, amino acids) nagata et al., ; goeddel et al., ; lawn et al., a,b; gren et al., ) . the calculated molecular mass of recombinant ifn-(x subtypes is approximately . kda, although apparent molecular masses of leukocyte-derived ifn-(x subtypes in sodium dodecyl sulphate (sds)-polyacrylamide gels vary between and kda, possibly owing to variable processing of c-terminal amino acids (le w et al, ) and post-translational modifications. the amino acid sequences of ifn-(x subtypes are highly related, with complete identity at of the amino acid positions (langer and pestka, ; de maeyer and de maeyer-guignard, ) . this is illustrated in figure . , where the amino acid sequences of the subtypes are compared to an idealized consensus sequence. many of the positions where amino acids differ from subtype to subtype are conservative substitutions. interestingly, ifn-(x subtypes contain four cysteine residues whose positions ( , , , and ) are highly conserved (figure . ). these four cysteines form disulfide bridges ( - , - ) which induce folding of the ifn-(x molecule (figure . ) and whose integrity is essential for biological activity (morehead et al., ) . ifn-(x subtypes are predicted to contain a high proportion (- %) of (x-helical regions and are folded to form globular proteins (zoon and wetzel, ) . it has not yet proved possible to apply x-ray crystallographic techniques to ifn-(x subtypes, or to human ifn-~ , but the three-dimensional crystal structure of recombinant mouse ifn-[ , which is approximately % related in amino acid sequences to its human counterpart, has been solved (senda et al., ) . this has revealed that mouse ifn-[ has a structure which consists of five (x-helices folded into a compact (x-helical bundle ( figure . ). from comparative sequence analysis it is predicted that in all mammalian ifn-(x and ifn-] proteins these five (x-helical domains are conserved (korn et al, ; horisberger and di marco, ) and thus show similarities with many other cytokines, which also have (x-helical bundle structures (bazan, ) . with one exception, ifn-(x subtypes do not contain recognition sites (asn-x-ser/thr) for n-linked glycosylation (henco et al, ) ; only ifn-(x contains two of these sites (figure . ). nevertheless, o-linked glycosylation may be possible in other ifn-(x subtypes. for example, it has been found that natural ifn-%, purified from human leukocyte ifn, contains the disaccharide galactosyl-n-acetylgalactosamine in olinkage to thr- (adolf et al, a) . however, since ifn-% is the only ifn-(x subtype with a theonine at position , it may represent the only o-glycosylated ifn-(x protein (figure comprise the mature ifn-] protein (derynck et al., taniguchi et al., ; houghton et al., ) . although ifn-] is the same length as the majority of ifn-a subtypes, it shows only approximately % amino acid sequence relatedness with them ( figure . ) and is antigenically distinct. the ifn-~ protein lacks the n-terminal cys- residue present in ifn-a subtypes, but contains three other cysteines at positions , , and , the latter two corresponding to the disulfide bond pairing - in ifn-a subtypes. replacement of cys- by serine does not result in any loss of biological activity, whereas serine substitution of cys- does (mark et al., ; shepard et al., ) . as mentioned previously, on the basis of the threedimensional structure of recombinant mouse ifn-~ ( figure . ) , human ifn-[ is predicted to contain five a-helices and to fold up into an a-helical bundle structure (senda et al., ) . human ifn-[ has one potential n-glycosylation site at asn- (taniguchi et al., ) and n-linked oligosaccharides, primarily of the biantennary complextype, are known to be attached to this site in natural ifn- (hosoi et al, ) . however, these may vary considerably depending on the producer cell type (utsumi et al., ) . from cdna sequence data, it was predicted that pre-ifn-m contains amino acids, the n-terminal comprising the signal sequence and the remaining the mature ifn-m protein (capon et al., ; hauptmann and swetly, ) . the amino acid sequence of ifn-m is therefore six residues longer at the cterminus than ifn-r or ifn-[ proteins. however, it has been found that natural ifn-m is heterogeneous at the n-terminus owing to variable cleavage of pre-ifn-m; about % of mature ifn-m molecules carry two additional n-terminal amino acids (adolf, ; shirono et al., ) . it is approximately % related to ifn-cx subtype sequences, but only % related to that of ifn-[ ( figure . ), and is antigenically distinct from both ifn-~ and ifn-[ (adolf, ) . nevertheless, the four cysteines occur in the same notional positions, , , , and , as they do in ifn-~ subtypes and it is likely that ifn-m will have a similar cx-helical bundle structure to those predicted for both ifn-cx and ifn-[ proteins (senda et al., ) . ifn-m has one potential site at asn- for n-linked glycosylation and natural ifn-m has been demonstrated to be a glycoprotein with biantennary complex oligosaccharides (containing neuraminic acid) attached at this site (adolf, ; adolf et al., b) . type i ifns (ifn- t,-[ , and-m) are produced by a variety of normal cell types responding to extracellular or intracellular stimuli (stewart, ) . ifn-~, as a mixture of subtypes, and ifn-m may be produced together following viral infection of null lymphocytes or monocytes/macrophages (cantell and hirvonen, ; adolf, ) . the proportions of ifn- t subtypes may vary according to the type of virus used as inducer (hiscott, ; finter, ) . however, production of ifn-[ is usually restricted to double-stranded polynucleotide, e.g., poly-inosinic, poly-cytidylic acid, or it is well established that the biological activities of ifns are mostly dependent upon protein synthesis with selective subsets of proteins mediating individual activities. antiviral, antiproliferative and immunomodulatory activities have been ascribed to ifn-~/[ /c (reviewed in pestka et al., ; de maeyer and de maeyer-guignard, ) . the proteins and mechanisms involved in these activities are described below. virally-induced normal fibroblasts and other tissue cell types, e.g., epithelial cells (meager et al., ; stewart, ) . in all the above cases, the amount of ifn secreted is dependent on the dose of the inducer. besides normal cells, a range of transformed and tumor-derived cell lines are known ifn producers, e.g., mg human osteosarcoma cell line (meager et al., ) , and the namalwa b-lymphoblastoid cell line (phillips et al., ) . generally speaking, adherent fibroblastic cell lines produce mainly ifn-[ and only a minor quantity of ifn-~ (havell et al., ) , whereas nonadherent myeloid or lymphoid cell lines produce mainly ifn-c~ and only a small amount of ifn-i (cantell and hirvonen, ; shuttleworth et al., ; zoon et al., ) . actual production of ifns lasts only a matter of a few hours following induction. this is due to ifn mrna instability and the rapid shut-off of ifn gene transcription (caput et al., ; shaw and kamen, ) . under conditions where ifn mrna stability is increased, e.g., by blocking protein and rna synthesis following induction, ifn production has been shown to be "superinduced" once the block on protein synthesis is removed (meager et al., ; stewart, ) . despite there being vast numbers of viruses with different replication strategies, it appears that many viruses can be countered by relatively few ifn-inducible "antiviral" proteins (samuel, ) . one of the bestcharacterized of these is a family of enzymes collectively known as " - a synthetase" which, in the presence of dsrna (often an intermediate of viral rna synthesis) catalyses the formation of an unusual oligonucleotide, ppp (a 'p) na ( - a), which in turn activates an ifninduced latent endonuclease, rnase l (williams and kerr, ; wreschner et al., ; ghosh et al., ; hovanessian, ; lengyel, ; zhou et al., ) . when activated, the rnase l degrades viral (and cellular mrna) and therefore inhibits viral protein synthesis. small rna viruses (picornaviridae), e.g., mengo virus and murine encephalomyocarditis virus (emcv), whose replication is cytoplasmic are most inhibited by the induction of - a synthetase (lengyel, ; rice et al., ; chebath et al., ; kumar et al., ) . a further important ifn-induced "antiviral" protein is a dsrna-dependent protein kinase, now designated pkr, which in the active form phosphorylates the peptide initiation factor, eif , involved in polyribosomal translation of mrna (miyamoto and samuel, ; gupta et al., ; samuel, ) . phosphorylated eif is inactive and thus viral protein synthesis is inhibited. this inhibition has been associated with the loss of replicating capacity of reoviruses and rhabdoviruses such as vesicular stomatitis virus (vsv). the - a synthetase and pkr antiviral mechanisms are rather general and potentially could affect a wide range of viruses. however, ifn can induce certain proteins that inhibit specifically one class of virus. for example, the ifn-inducible mx proteins block the replication of influenza virus, probably by inhibiting the nuclear phase of viral transcription (mouse cells) or later cytoplasmic phases (human cells), without affecting the replication of many other viruses (staeheli, ; mel n et al., ; ronni et al., ) . in addition, since ifns can impair various steps of viral replication, including penetration, uncoating and assembly of progeny virions as well as transcription and translation, there are likely to be several other antiviral proteins and mechanisms (de maeyer and de maeyer-guignard, ) . for instance, some viruses, e.g., herpes virus and certain retroviruses, appear to be inhibited at the relatively late stage of virus particle maturation and budding (aboud and hassan, ) . in the course of evolution, many viruses have developed countermechanisms by which they can disrupt the antiviral mechanisms induced by ifn. such countermechanisms often point to the significance of particular "antiviral" proteins. one of the main "targets" for several different viruses is the ifn-inducible pkr. the action of this kinase is overcome in adenovirus or epstein-barr virus (a member of the herpes virus family) by the production of small viral rna molecules, vaiand eber-rnas, respectively, which bind to pkr and block its activation by dsrna (clarke et al., ; ghadge et al., ; mathews and shenk, ) . reoviruses and vaccinia virus (a member of the pox virus family) produce viral proteins (sigma and ski, respectively), that bind to dsrna and thus reduce activation of pkr (sen and lengyel, ) . interestingly, if ifn-treated vsv-infected cells are coinfected by vaccinia virus, vsv replication is rescued, presumably partly by the inhibitory effect of ski on pkr (whitaker-dowling and youngner, ) . vaccinia virus also produces a nonfunctional protein analog of elf which competes with the real eif for phosphorylation by pkr and thus dilutes out the antiviral effect of activated pkr (beattie et al., ) . other viruses, such as influenza, may activate latent cellular inhibitors of pkr activity, e.g., a kda protein (p ) (lee et al., ) . the - a synthetase-rnase l system can also be subverted. for example, emcv, a picornavirus, can inactivate rnase l in several cell lines, but this inactivation is usually blocked by ifn treatment (lengyel, ) . herpes viruses, in contrast, appear to inhibit rnase l activation by producing competing analogs of - a (cayley et al., ) . some viruses even .have the ability to block the transcription of ifn-inducible genes. the "early" ela regulatory proteins of adenoviruses prevent the activation of isgf by ifn, probably by inhibiting the transcription of the isgf subunit gutch and reich, ; kalvakolanu et at., ; nevins, ) . in the case of hepatitis b virus-infected cells, the so-called virus-specified "terminal protein" inhibits ifn-inducible gene expression . the antiviral mechanisms induced by ifn are mediated by enzymes, e.g., - a synthetase and picr, whose activities have broad implications for cell growth and proliferation. viral replication may be regarded as a form of pathological growth of a foreign, "cell-like", entity at the expense of a living cell. in the presence of ifn, enzymes are activated which curtail protein synthesis in general, but because viral protein synthesis is normally rapid, the inhibitory effect on viral replication appears more dramatic than on the slower and more complex cellular growth. possibly, the "ifn system" was evolved more as a part of a complex, interactive network of intercellular mediators of cell growth and proliferation than as one for antiviral mechanisms. recent investigations tend to support the role of ifns in regulating cell growth. for example, if a mutant form of pkr that is unable to phosphorylate eif is introduced into cells, they undergo neoplastic transformation (koromilas et al., ; lengyel, ; meurs et al., ) . this suggests that pkr normally acts as a "tumorsuppressor"' gene product. therefore, one of the mechanisms by which ifn inhibits cell proliferation could be through its capacity to induce enhanced expression/activity of pkr. ifn-~ has also been reported to inhibit cyclin-dependent cdk- kinase, which is responsible for phosphorylation of retinoblastoma (rb) protein, and this could contribute to antiproliferative activity (kumar and atlas, ; resnitzky et al., ; zhang and kumar, ) . the - a synthetase-rnase l system may also have antiproliferative and tumor suppressor activities. for instance, the levels of these two enzymes are high in growth-arrested cells: introduction of - a-like oligoadenylates into proliferating cells also causes growth impairment (sen and lengyel, ; lengyel, ; zhou et al., ) . the ifn-stimulated increases in synthesis of pkr and - a synthetase are dependent on ifn-inducible transcription factors, such as irf- (isgf ) pine et al., ; williams, ; reis et al., ) . the latter has a short half-life and thus transcription of ifn-inducible genes is rapidly repressed by the longer-lasting, inhibitory irf- (harada et al., ) . if irf- is overexpressed, cells become transformed as even low level constitutive production of pkr and - a synthetase, which can regulate normal cell growth, is abrogated. this transformation was reversed by overexpressing irf- (harada et al., ) , indicating that irf- can be viewed as a pivotal player in the growth, regulatory, and tumor suppressor machinery. a variety of other ifn-induced mechanisms, including suppression of oncogenes (contente et al., ) , depletion of essential metabolites (sekar et al., ) , and increased cell rigidity (e. wang et al., ) , could also contribute to its antiproliferative activity. the antiproliferative effects of ifns in different tumor cell lines cultured in vitro is highly variable. besides tumor cell lines, ifn- t/[ have antiproliferative activity in hematopoietic precursor cells, e.g., of the myeloid lineage (rigby et al., ; de maeyer and de maeyer-guignard, , for review). they are also potent inhibitors of angiogenesis, the process whereby blood capillaries are formed to envasculate tissues (sidky and borden, ) . besides activating intracellular processes, ifns can also activate intercellular activities, especially within the immune system, which are an essential part of host defense against infectious and invasive diseases. thus, ifns can stimulate indirect antiviral and antitumor mechanisms, which in the main rest upon cellular differentiation and the induction of cytotoxic activity. for example, in the presence of antigen-specific antibodies, macrophages can effect cell-mediated cytotoxicity. such antibody-dependent cell-mediated cytotoxicity (adcc) is enhanced by ifn, possibly through an augmentation of immunoglobulin g (igg)-fc receptor (fcr) expression (hokland and berg, ; vogel et al., ; de maeyer and de maeyer-guignard, ). in addition, another category of leukocytes comprising large granular lymphocytes and known as natural killer (nk) cells are activated, by unknown mechanisms, to kill virally-infected or tumor cell targets independently of major histocompatibility complex (mhc) antigen expression (trinchieri and perussia, ; rager-zisman and bloom, ; de maeyer and de maeyer-guignard, ) . ifns can stimulate increased expression of class i mhc antigens, i.e., hla-a, -b, -c, which are crucial for recognition of foreign antigen by cytotoxic t lymphocytes (ctl, cd +); recognition of virally infected cells by ctl depends on class i mhc antigen presentation of viral antigens at the cell membrane (heron et al., ; fellous et al., ) . ifn-(x/[ have sometimes been observed to increase class ii mhc antigen expression, which is necessary to trigger both humoral and cell-mediated immunity, but probably play a lesser role than ifn-y, which is the major class ii mhc antigen inducer (baldini et al., ; rhodes et al., ; de maeyer and de maeyer-guignard, ). all of the biological activities so far described (see above) for ifns have followed from in vitro experimentation. here it is possible to pick and choose conditions that favor particular outcomes, e.g., the antiviral response, by adjusting doses of ifn, times of incubation, levels of virus challenge, and so on. such experiments illustrate the range of biological activities of ifns but cannot define their physiological roles. that ifns have the potential for inducing antiviral and antitumor activity suggests their main role in vivo is to act as regulators of host defense mechanisms, and to prevent pathophysiological events occurring. investigations in experimental animals have supported this likelihood. for example, injection of mice with anti-ifn-~/] antibody has been shown to increase their susceptibility to a range of virus infections (virelizier and gresser, ; gresser, ) . the earliest evidence for an antitumor effect of ifn-tx/ came from inoculation of murine l cells into mice. l cells are sensitive to the antiproliferative action of ifn-~/ in vitro and in vivo, ifn~/[ prevented tumor growth by these cells. however, when a clone of l was isolated that was resistant to the antiproliferative action of lfn-~/[ , there occurred a similar retardation of tumor growth upon ifn-c~/] treatment to that observed with "sensitive" l cells, suggesting that ifn was acting indirectly in vivo by a host-mediated mechanism (gresser et al., (gresser et al., , . a similar conclusion was reached more recently using ifn-resistant b-cell lymphoma cells . in the intervening years, many studies have been conducted confirming that ifn can act directly (e.g., human ifn-a against a range of human tumor xenografts in nude mice where human ifn- ~ has no activity on the murine immune system) and indirectly (reviewed by balkwill, ) . although antitumor activity has been clearly demonstrated by the application of exogenous ifns, it is not certain that endogenously produced ifns are involved in countering tumor growth. however, some experimental evidence that endogenous ifn could play a role in host resistance to cancer or its spread has been obtained by treating mice with anti-ifn antibodies. under these conditions, the intraperitoneal transplantability of six different experimental murine tumors was observed (gresser, ) . ifn-a/~ can inhibit the growth of hematopoietic progenitor cells in vitro (rigby et al., ) and this is also likely to occur in vivo. such an occurrence is undesirable in most instances, but suppression of proliferation of bone marrow hematopoietic cell precursors has been turned to advantage in protecting tumor-bearing mice against the cytotoxicity of chemotherapeutic agents such as -fluorouracil ( -fu) (stolfi et al., ) . ifns exercise their actions in cells via ifn-specific cell surface receptors. these receptors bind ifns with high affinity (aguet, ) and transduce the signal occasioned by ligand (ifn) binding across the cell membrane into the cytoplasm. ifn-cq ifn-[ and ifn-co share the same binding sites (aguet et al., ; flores et al., ) , but ifn-y binds to different sites (branca and baglioni, ; aguet et al., ) . the binding of ifn-c~ and ifn-~ to lymphoid cells and fibroblasts has been studied extensively and has been reviewed (rubinstein and orchansky, ; branca, ; langer and pestka, ; grossberg et al., ) , and results have generally demonstrated the presence of up to a few thousand complex, high-affinity receptors per cell. chemical cross-linking studies with ~ si-labeled ifn-~ or ifn-[ to receptor-bearing cells have led to the identification of various ifn-receptor complexes, their molecular masses ranging from to kda (joshi et al., ; eid and mogensen, ; faltynek et al., ; raziuddin and gupta, ; thompson et al., ; hannigan et al., ; vanden broecke and pfeffer, ; colamonici et al., ) . such results have suggested that there are either multiple binding sites for ifn-cx, ifn-[ , and ifn-m or that there are complex multichain receptors (colamonici et al., ; hu et al., ) . although on human cells all the ifn-c~, ifn-~, and ifn-m proteins compete for common binding sites, individual ifn-c~ subtypes show different levels of activities on cells (streuli et al., ; week et al., ; rehberg et al., ) which appear to correlate with their binding behavior to the cell surface (uzd et al., ) . in particular, ifn-% shows a much lower binding for human membrane receptors than either "ifn- ~ or ifn-% (uz et al., ) . interestingly, this differential binding of human ifn-~ subtypes is not manifested in bovine cells, and all of the subtypes exhibit high specific activities (yonehara et al., ; shafferman et al., ) . ifn-i and ifn-m are also active in bovine cells (capon et al., ; adolf et al., ) , but this cross-reactivity does not extend to mouse cells, a feature that has provided experimental systems in which to characterize ifn-receptors. thus, somatic cell genetic studies with human • rodent hybrid cells containing various combinations of human chromosomes have provided evidence that the presence of human chromosome confers sensitivity of such hybrid "rodent" cells to human ifn-~, ifn-i and ifn-m (tan et al., ; slate et al., ; epstein et al., ; raziuddin et al., ) . further, it was demonstrated that antibodies raised against human chromosome encoded cell surface proteins were able to block the binding and action of human ifn-~ to human cells, indicating that this chromosome contained a gene(s) specifying the human ifn cell surface receptor . the elucidation of the full complement of components of the ifn-~/[ /m receptor has long been sought. one methodology used to isolate receptor cdnas involves transfecting mouse cells with total human dna and then selecting for cells sensitive to human ifn-cz. after several attempts, this approach led successfully to the isolation of a . kb edna from a library constructed from human lymphoblastoid (daudi) cells, which encoded an ifn-cz binding protein (uzd et al., ) containing amino acids (molecular mass da) including a signal sequence of mainly hydrophobic amino acids. this protein has a structure typical of a transmembrane glycoprotein: a large n-terminal extracellular domain, which potentially could be highly glycosylated owing to a preponderance of n-linked glycosylation sites, a short hydrophobic transmembrane domain, and an intracellular or cytoplasmic tail (figure . ) . its amino acid sequence shows little homology with any currently available sequences of proteins, including the sequence of the human ifn- receptor (aguet et al., ) ; however, the extracellular domain has been predicted to show structural similarities with the latter receptor and to a lesser extent with the so-called hematopoietin receptor supergroup (bazan, a,b) . the gene coding for this putative ifn-cz receptor has been mapped to chromosome .q , in confirmation of the earlier rodent x human hybrid cell data (tan et al., ; slate et al., ; epstein et m., ; raziuddin et al., ) . although the cloned "ifn-cz receptor" could be shown to confer sensitivity to human ifn-% in transfected mouse cells (uzd et al., ) , such cells were relatively insensitive to human ifn-% and human ifn-[ . these findings, together with those from anti-ifn-cz receptor antibody blocking studies (colamonici et al., ; revel et al., ; uz~ et al., ) and affinity cross-linking studies with ifn-% (colamonici et al., ) , have suggested that a second ifn-cz receptor exists or another component is required besides the cloned ifn-cz binding protein, to complete the receptor complex. this hypothesis is further supported by a study in which introduction of a yeast artificial chromosome (yac) containing a segment of human chromosome into chinese hamster ovary (cho) cells conferred a greatly increased response to both ifn- ~ and ifn- ~ , as well as an increased response to ifn-i and ifn-m, whereas the expression of the ifn-~ binding protein alone did not confer sensitivity (revel et al., ) . however, these increased responses can be "knocked out" by disruption of the ifn-~ binding protein gene in the yac, and then reconstituted by expression of the cdna encoding the ifn-% binding protein (cleary et al., ) , suggesting that cell surface expression of this protein is required for a fully functional receptor (see also hertzog et al., ; constantinescu et al., ) . a second human ifn-~/i receptor, which is probably the additional component of the receptor complex referred to above, has been cloned (novick et al., ) . the . kb cdna encodes a -amino-acid protein, including a signal sequence, which has the predicted structure of a transmembrane glycoprotein. the nterminal ectodomain ( amino acids) corresponds in sequence to a soluble kda ifn-(x/[ binding protein, p , isolated from urine. this domain is linked to a transmembrane segment ( amino acids) and a relatively small cytoplasmic domain of amino acids ( figure . )i overall, the primary sequence shows little ifn-a/] binding protein bind ifn-a but are insensitive to its effects, suggesting that an accessory protein, possibly the cloned ifn-c~ binding protein, is required for signaling (novick et al., ; constantinescu et al., ) . the findings that anti-p antiserum and a particular monoclonal antibody to the ifn-cx binding protein (benoit et al., ) both block the biological activity of ifn- q co indicate that the ifnhomology with that of the previously cloned ifn-a . c~/ and ifn-a binding proteins are in close proximity, binding protein (uzd et al., ) , but when the extracellular domains are compared, . % relatedness is found (novick et al., ) , suggesting that both of these ifn binding proteins belong to the same so-called class ii cytokine receptor family (uz et al., ) . two classes of cytokine receptor (class i and class ii) have been proposed by bazan ( a,b) , these being distinguished by the positions of cysteine pairs in the extracellular domain. the latter is comprised of fibronectin type iii-like units containing around amino acids and designated d (uzd et al., ) . a schematic drawing of both ifn-a/[ receptor chains is shown in figure . . subsequently, it has been found that alternative splicing of the ifn-a/ receptor gene can produce a transcript encoding a long form of the receptor protein containing a larger cytoplasmic domain of amino acids . mouse cells transfected with the cdna encoding the and thus probably interact to form a high-affinity ifna/] /co receptor complex. the most likely scenario on present evidence is for a two-chain ifn-c~/ /co receptor, comprising the cloned ifn-c~ binding protein (uz et al., ) and the long form of the ifn-c~/[ binding protein lutfalla et al., ) , each of which binds to some extent particular ifn types or ifn-(x subtypes but which together more strongly bind all ifn-~, - , and -co species and function to transmit signals across the cell membrane. however, it is not completely ruled out that other cell surface components, e.g., membrane glycosphingolipids, are required for fully functional receptors (colamonici et al., ; platanias et al., ; ghislain et al., ; uz et al., ) or, possibly, that there are alternative ifn receptors, e.g., the epstein-barr virus/complement c d receptor as an ifn-c~ receptor on b-lymphocytes (delcayre et al., ) . vaccinia virus and other orthopoxviruses contain a gene b r encoding a soluble type i ifn receptor which, unlike the class ii cytokine type receptors, belongs to the immunoglobulin superfamily (symons et al., ; colamonici et al., ) . . the intracellular domains of the two cloned ifn-binding proteins are unrelated to the tyrosine kinase class of receptors, e.g., epidermal growth factor receptor (egf-r) and platelet-derived growth factor-receptor (pdgf-r), and are not predicted to have kinase activity of any sort (uzd et al., ; novick et al., ) . however, it appears that the cytoplasmic domain of the ifn- ~ and a/ binding proteins associate with nonreceptor tyrosine kinases tyk and janus kinase (jak ), respectively, known to be involved in the signal transduction pathway of ifn-a/[ and other cytokines (novick et al., ; ghislain et al., ; ihle, ; ihle and kerr, ; velasquez et al., ) . the current understanding of this pathway is as follows. after binding of ifn-a/[ /c to their cognate receptors, the intracellular domains are phosphorylated by tyk and jak . these phosphorylated domains act as docking sites for the cytoplasmic stat (signal transducers and activators of transcrilstion ) proteins p /p (statla/b) and p (stat ) (ihle, ) . the latter undergo tyrosine phosphorylation mediated by receptor-associated tyk /jak , dimerize, translocate to the nucleus, and combine with a dna binding protein, p , to form the ifn-stimulated gene factor- (isgf ) transcription factor complex (schindler et al., ; velasquez et al., ; mtiller et al., ; platanias et al., ; shuai et al., ; gupta et al., ; yan et al., ) . both tyk and jak need to be reciprocally activated for signal transduction to occur, since cell mutants lacking either tyk or jak are unresponsive to ifn- ~ (ihle, ; ihle and kerr, ) . isgf binds to cis-acting ifn-stimulated response elements (isre), present in the promoter regions of ifn-inducible genes, to initiate their transcription (williams, ) . targeted disruption of the stat gene in mice has shown that stat has an obligatory role in ifn-cx and ifn-y signaling (durbin et al., ; meraz et al., ) . the jak /tyk -isgf pathway may not be the only "receptor-to-cell nucleus" signaling mechanism activated in ifn-stimulated cells. there has been some evidence to implicate protein kinase c (pkc) pathways as well (reich and pfeffer, ; pfeffer et al., ; c. wang et al., ) . however, this remains controversial owing to the lack of specificity of kinase inhibitors used (kessler and levy, ; james et al., ) . ifn-inducible genes have a common regulatory nucleotide sequence (g/a)ggaaan(n)gaaact in their ' flanking region and this type of sequence, which resembles the vre sequences (ryals et al., ; present in ifn genes, is designated interferonstimulated response element (isre) (williams, ) . the resemblance between isre and vre sequences probably accounts for the finding that many ifninducible genes are transcriptionally activated by virus infection or dsrna, which also activate the transcription of ifn genes (hug et al., ; wathelet et al., ) . as mentioned previously (see section . . ), ifn-receptor occupation activates cytoplasmic isgf- and this complex is translocated to the nucleus and binds to isre of ifninducible genes as a transcriptional activator. in addition, a second factor, isgf , forms complexes with isre in ifn-stimulated cells. isgf is a single, inducible phosphoprotein that has been shown to be identical to irf- pine et al., ; williams, ; reis et al., ) . the role of a third transcription factor, isgf , which is constitutively produced and requires only the central bp core of isre for binding, remains to be fully defined (kessler et al., ) . a number of other negative regulatory factors, including irf (harada et al., ) and the isgf (irf )/isgf yrelated "human interferon consensus sequence binding protein" (icsbp) (weisz et al., ; bovolenta et al., ) , which also bind to isre, are also probably involved in the regulation of transcription of ifn-inducible genes. it is clear that the regulation of expression of ifninducible genes is complex and that the mechanisms that control their selective expression are not fully understood (see taylor and grossberg, , for review) . ifninducible proteins, whose number probably exceeds , include both those proteins induced early after ifn stimulation and those proteins that may be produced at later times, often in response to the actions of "early" ifn-inducible proteins (sen and lengyel, ) . the full set of ifn-inducible proteins is probably not known, but several have been identified and characterized. table . shows an incomplete list of ifn-inducible proteins together with their likely functions. some of these proteins are not exclusively induced by ifn- ~/[ /c ; ifn-y and certain other cytokines, e.g., tumor necrosis factor-~ (tnf-~) often induce spectra of proteins that overlap with the set induced by ifn-~/ /~ (revel and chebath, ; rubin et al., ; wathelet et al., ) . it should be noted that ifn-inducible proteins tend also to be cell type-specific and thus not all proteins listed in table . will be expressed in all cell types. in some cases, ifn-inducible proteins are completely absent from a cell before ifn stimulation, but in other cases revel and chebath, ; sen and lengyel, ; samuel, ; staeheli, . revel and chebath, ; lengyel, . de maeyer and de maeyer guignard, ; heron et al, . schwemmle and staeheli, . ronni et al, . revel and chebath, . c. wang et al., . kumar and atlas, . loeb and haas, . aiidridge et al, . fellous et al, . sen and lengyel, . sen and lengyel, . bange et al., . chebath et al, . choubey and lengyel, . lawn et al, a constantoulakis et al, . porter and itzhaki, . hokland and berg, . thomas and linch, . revel and chebath, they are being constitutively produced, their synthesis augmented by ifn. the genes for mouse ifn-~ subtypes and mouse ifn-[ (no functional mouse ifn-c gene has been found) are located on mouse chromosome (dandoy et al., (dandoy et al., , de maeyer and de maeyer-guignard, , for review) . these genes are, like their human counterparts, intronless and of comparable structure. twelve mouse ifn-~ genes or pseudogenes have been identified, of which the cdnas for different genes have been cloned and expressed (langer and pestka, ; de maeyer and de maeyer-guignard, ) . mouse ifn-~ subtype proteins contain or amino acids, or exceptionally (mouse ifn-~ ) , and the four cysteines at positions (langer and pestka, ) . there is only a single-copy mouse ifn-[ gene and this encodes the i-amino-acid mature mouse ifn-[ protein (higashi et al., ; de maeyer and de maeyer-guignard, ) . mouse ifn- contains only one cysteine and thus cannot form intramolecular disulfide bonds. it has three potential n-linked glycosylation sites and is heavily glycosylated when secreted from mouse fibroblasts; the molecular mass of the native glycoprotein is approximately kda compared to the predicted kda for the nonglycosylated counterpart (de maeyer and de maeyer-guignard, ). the amino acid sequence of mouse ifn-i is about : % related to that of human ifn-i . the threedimcnsional structure of mouse ifn-i has been solved (senda et al, ) (see section . . ) and the protein has been shown to comprise five s-helices folded into a compact s-helical bundle (figure . ) . induction of transcription of mouse ifn- ~ subtype genes and the mouse ifn-i gene is probably regulated by transcription factor-binding nucleotide sequences present in the ' noncoding promoter region, in a similar way to that of human ifn-~ and ifn-[ genes (see section . ). for. example, repeated gaaa-rich sequences are present in the ' flanking regions of most mouse ifn-cz subtype genes and these are likely to be important for virus-inducible transcription (shaw et m., ; zwarthoff et al., ) . inducers of mouse ifn-~ and ifn-i synthesis, which include a number of viruses and double-stranded polynucleotides, are similar to those which induce human ifn-c~, ifn-[ , and ifn- production (stewart, ; de maeyer and de maeyer-guignard, ) . similarly, the type of ifn produced follows the pattern found among different human cell types: fibroblastic and epithelial cell lines produce mainly ifn-~, whereas leukocytes produce mainly ifn- ~ subtypes (de maeyer and de maeyer-guignard, ) . the biological properties of mouse ifn- ~ and ifn-~ are similar to those of human ifn- ~, ifn-j and ifn- (see section ). since mouse and human ifn-~ subtypes are only % homologous, there is considerable species preference in biological activity, i.e., mouse ifn-cx is weakly active in human cells and vice versa. mouse ifn- is also not active in human cells (stewart, ) . rather less is known regarding receptors for mouse ifn-~ and ifn- , than for the human counterparts, but it is probable that they comprise two or more chains, as is the case for the human ifn-~/j / receptors (uzd et al., ) . the mouse equivalent receptor chain to the ifn- ~ binding protein (uzd et al., ) has been cloned (uz~ et al., ) . the gene for this mouse ifn-~/ receptor has been located to mouse chromosome (cheng et al., ) . the mouse ifn-~/ receptor is amino acids long and is divided into a large nterminal extracellular domain ( amino acids), a short hydrophobic transmembrane segment ( amino acids), and a cytoplasmic domain ( amino acids) (uz~ et al., ) . the extracellular domain contains eight potential n-linked glycosylation sites and is predicted to exhibit the two-d domain structure of the human ifn-~ binding protein extracellular domain (figure . ) (uzd et al., ) . further mouse ifn- ~/] rceptors or components thereof await identification and characterization. signal transduction via mouse ifn-~/] receptors is expected to involve the jak /tyk -isgf (stat / ) pathway as outlined for human ifn- ~/] / receptors (see section . ). in stat genedeleted mice there are no overt developmental abnormalities, but they display a complete lack of responsiveness to mouse ifn- ~ and ifn-~, (durbin et al., ; meraz et al., ) . as a consequence, stat -/-mice are highly susceptible to infection by viruses and microbial pathogens. stat is therefore an obligatory mediator in the signal transduction pathway triggered by ifns. targeted disruption of the cloned mouse ifn- ~/] receptor gave rise to a knockout with a similar phenotype (miiller et al., ) . such mice, lacking the ifn-cx/ receptor, developed normally but were unable to respond to mouse ifn- ~/] and thus unable to cope with viral infections. the potent antiviral activity of ifn-~/] /c together with their potential antitumor actions provided the impetus for large-scale manufacture of ifns for the purpose of clinical evaluation in a variety of viral and malignant diseases. in the early s, ifn production depended on pooled, human buffy coats (leukocytes) and thus only limited quantities could be made (cantell and hirvonen, ) . later in that decade, human lymphoblastoid cells (e.g., namalwa), which could be grown to large culture volumes, became available for ifn production. by the s, following the cloning of ifn-~ and ifn-j , these ifn species were massproduced by recombinant rdna technology, leading to abundant availability of certain ifn- ~ subtypes, e.g., ifn- ~ and "stabilized" ifn-] ser . there followed production of ifn- and ifn-c by this means. clinical usage of ifn-~ preparations far exceeds that of ifn-[ and ifn-c because of early production difficulties with the latter types, though these are now solved. at the beginning of the s there was tremendous enthusiasm, both from manufacturers of ifns and from clinicians, to evaluate the therapeutic potential of ifns. however, early clinical trials had been poorly devised, were not "blinded", and often yielded only anecdotal evidence of success. it was only after many controlled, randomized studies had been conducted that it became apparent that ifns in general, administered as a single agent, were not beneficial for the treatment of the majority of malignant diseases, including the major cancers (lung, breast, colon) of the developed world. the initial optimism all but vanished and was replaced in the mid-to-late s by a more sober and realistic appreciation of the potential therapeutic value ofifns. a number of general conclusions have been drawn, as follows. (i) ifn- ~ and ifn-] , and to a lesser extent ifn- ', have antitumor activity in a small number of cancers, particularly in those that are relatively slow-growing and well-differentiated. (ii) there is no indication that heterogeneous ifn-~ preparations containing mixtures of ifn- ~ subtypes (e.g., leukocyte ifn, lymphoblastoid ifn) have different clinical effects from those of homogenous, recombinant ifn- ~ subtype or ifn-] preparations. (iii) continuous or intermittent high dosing appears to be required for antitumor efficacy. (iv) ifns probably work best in patients with a minimal tumor burden (balkwill, ) . a major concern that has emerged from clinical studies is that ifns all generate a considerable number of undesirable, clinically observable, side-effects, including fever, chills, malaise, myalgia, headache, fatigue, and weight loss, and in certain cases these have been severe enough for treatment to be halted (bottomly and toy, ; rohatiner et al., ; goldstein and laszlo, ) . in addition, a variable proportion ( - %) of patients treated with ifn- ~ or ifn-] , especially recombinant ifn-~ and recombinant ifn-] ser , develop neutralizing antibodies to the ifn species used (rinehart et al., ; antonelli et al., ) , that in some instances have been associated with clinical "resistance" to ifn (steis et al., ; oberg et al., ; freund et al., ; fossa et al., ) . a further important, but generally unrecognized, side-effect of ifn-~ treatment is the possible induction of certain types of autoimmune disease (feldmann et al., ; gutterman, ) , probably mediated via ifn-induced upregulation of mhc antigen expression and generalized immunosuppression. the most responsive cancer to ifn-a therapy is a very rare form of b-cell leukemia, known as "hairy cell" leukemia (hcl), in which a response rate up to % has been reported (gutterman, ; baron et al., ; vedantham et al., ) . in hcl patients, the "hairy cells" invade the spleen and bone marrow and the disease takes an indolent course. it has been shown convincingly that ifn-~ therapy continued over several months leads to a clearance of "hairy cells" and in some patients a long-term remission is achieved. ifn-~ ser or ifn-y were less effective against hcl (saven and piro, ) . the ifn-~-induced mechanisms whereby clearance of "hairy cells" is achieved are not fully understood, but it is believed that a direct action of ifn-~ leading to differentiation of "hairy cells" to a nonproliferating phenotype is involved (vedantham et al., ; gutterman, ) . not all patients benefit greatly from ifn-~ treatment and some develop neutralizing antibodies, particularly when ifn- ~ is used (steis et al., ) . when such neutralizing antibodies cause resistance to further ifn-~ treatment, clinical responses can be "rescued" by switching to a heterogeneous ifn- ~ preparation, e.g., leukocyte ifn-c~ (von wussow et al., ) . however, on the whole, ifn-~ therapy of hcl appears at least as effective and durable as chemotherapy with the drug pentostatin ( -deoxycoformycin) (saven and piro, ) . ifn- ~ therapy has also been shown to slow down the progression of chronic myelogenous leukemia (cml) (baron et al., ; gutterman, ) . in this malignant disease, leukemic cells grow slowly in the initial chronic, but benign, phase and persist for - years, but there follows a dramatic "blast crisis" producing rapidly proliferating myeloid leukemia cells and a fatal outcome. cml patients treated with ifn-~ in the chronic phase often achieve durable remissions, associated with the elimination of leukemic cells bearing the so-called "philadephia chromosome", sometimes lasting up to years. other malignancies in which ifn-a therapy seems to work, although with generally a lower percentage of patients responding than in hcl and cml, include lowgrade non-hodgkin lymphoma, cutaneous t cell lymphoma, carcinoid tumors, renal cell carcinoma, squamous epithelial tumors of the head and neck, multiple myeloma, and malignant melanoma. in most of these cancers, complete responses are low compared to partial responses, but ifn-~ may help with maintenance therapy of diseases in some cases, e.g., multiple myeloma (mandelli et al., ; johnson and selby, ) . the neovascularization of primary tumors is a crucial step in their development and thus the anti-angiogenic activity of ifn-~/i (sidky and borden, ) may have therapeutic value in certain early malignancies, e.g., primary melanoma (gutterman, ) . kaposi sarcoma, often found in aids patients, has been regarded as an angiogenic tumor or angioproliferative disease, which may explain why ifn-c~ treatment can lead to regression of lesions in up to % of patients with this condition (de wit et al., ; groopman and scadden, ) . in preclinical systems, the combination oflfn therapy and conventional chemotherapy has appeared to offer greater chances of producing effective treatment of many cancers, but in clinical trials this strategy has produced mostly disappointing results (see wadler and schwartz, , for review) . this may be due to (i) the inability of preclinical models accurately to predict the clinical situation; (ii) the lack of understanding of the biochemical interactions and biological consequences of combining ifns and chemotoxic agents; (iii) a failure to incorporate information on dose, scheduling, and sequence of administration of ifns and chemotoxic agents into clinical trials. despite having proven antiviral activity in vitro, ifns have not proved the hoped-for panacea for most common viral infections in man. ifn- ~/~ prevent the replication of common cold viruses (rhinoviruses and coronaviruses) in the test tube and when administered to volunteers in the form of a nasal spray, but cannot "cure" colds once they are established (scott r al., ; r.m. douglas et al., ; turner et al., ) . ifn-ix is only partially effective in preventing influenza virus infections (treanor et al., ) . topical applications of ifn-~/~ in the form of creams or ointments to herpes virus lesions, e.g., in herpes zoster (chickenpox), and genital warts (condyloma acuminatum) caused by papilloma viruses have been investigated, but have given limited beneficial effects. however, when administered parenterally, i.e., by intramuscular or intravenous injection, greater beneficial effects of ifn-(x/ on virally caused lesions and warts have been found, although not to an extent that ifn therapy has become the treatment of choice (schneider et al., ; j.m. douglas et al., ; baron et al., ; gutterman, ) . another wart-like disease, juvenile laryngeal papilloma (jlp), which can severely obstruct the airways of young children, caused by the same papilloma virus types ( and ) as cause genital warts, has also been found to respond beneficially to ifn-~ therapy. disappointingly, ifn-cz therapy appears neither curative nor of substantial value as an adjunctive agent in the long-term management ofjlp (healy et al., ) . probably the most successful application of ifn-~ therapy to viral disease is in the treatment of chronic active hepatitis, caused by either hepatitis b or c viruses (baron et al., ; gutterman, ) . up to about % of chronic active hepatitis b patients respond to ifn-~ therapy; viral infectivity markers disappear and seroconversion and cure follow. it is interesting in the case of hepatitis b virus that viral activity is responsible for inhibiting the endogenous ifn system , and thus the administration of exogenous ifn-cz constitutes a replacement therapy. in hepatitis c virus infection, some serotypes of the virus are apparently more sensitive to ifn-~ therapy than others and prolonged treatment may be necessary (> months) to prevent relapses occurring (gutterman, ) . both ifn-~ and ifn-[ have been shown to inhibit human immunodeficiency virus- (hiv- ) replication in vitro (hartshorn et al., ) . however, in vivo, there is little evidence showing that ifn-c~ therapy has any longterm beneficial effect in asymptomatic hiv-l-positive individuals or mds patients (friedland et al., ; lane et al., ) , except for limited regressions in kaposi sarcoma lesions (de wit et al., ; groopman and scadden, ) . combination therapies for hiv- infected individuals involving ifn-~ and antiviral drugs such as zidovudine (azt) have also proved to be ineffective (berglund, ) . as mentioned earlier, ifn- ~/~ inhibits hematopoiesis and therefore induces leukopenia in patients. this effect has generally been thought to be undesirable and it can lead to immunosuppression; however, it has proved useful for the treatment of diseases in which there is uncontrolled leukocytosis, e.g., thrombocytosis (markedly elevated platelet numbers), associated with various myeloproliferative diseases (gisslinger et al., ) . resistance to ifn-~ therapy has occurred in such patients when neutralizing antibodies to ifn-~ have developed, but successful retreatment with a heterogeneous ifn-~ preparation, lymphoblastoid ifn (ifn- ~n ) has been reported (brand et al., ) . the findings that production of ifn-~ and ifn- was deficient in multiple sclerosis (ms) patients (neighbor and bloom, ) stimulated clinical trials to evaluate ifns in ms. rather unexpectedly, it has repeatedly been found that ifn-] , either natural fibroblast-derived or the later recombinant ifn-j ser (ifn-j - b), injected intrathecally, subcutaneously, or intramuscularly in patients with relapsing/remitting disease leads to a reduced rate of exacerbations of the disease and thus is possibly of clinical benefit in some patients (jacobs et al., (jacobs et al., , (jacobs et al., , the ifnb multiple sclerosis study group, ; paty et al., ) . the ifn-[ -induced mechanisms that contribute to this beneficial outcome are not known, but probably immunomodulatory actions are involved, e.g., suppression of growth and activity of autoreactive t lymphocytes in the central nervous system (goodkin, ) . it is unclear whether ifn-c~ would have a similar effect. however, the results with ifn-[ treatment have been encouraging so far, although more follow-up of patients will be necessary to monitor any effects on the clinical progression of ms (ebers, ) . accumulation and breakdown of rna-deficient intracellular virus particles in interferon-treated nih t cells chronically producing moloney murine leukaemia virus inhibition of the cellular response to interferons by products of the adenovirus type eia oncogene antigenic structure of human interferon ~ (interferon alpha ii): comparison with other human interferons monoclonal antibodies and enzyme immunoassays specific for human interferon (ifn) c : evidence that ifn-c is a component of human leukocyte ifn purification and characterisation of natural interferon c : two alternative cleavage sites for the signal peptidase natural human interferon-~ is o-glycosylated human interferon c : isolation of the gene, expression in chinese hamster ovary cells and characterization of the recombinant protein high affinity binding of si-labelled mouse interferon to a specific cell surface receptor various human interferon ~ subclasses cross-react with common receptors: their binding activities correlate with their specific biological activities molecular cloning and expression of the human interferon- receptor interferon [ increases expression of vimentin at the mrna and protein levels in differentiated embryonal carcinoma (psmb) cells nomenclature of the human interferon proteins a family of structural genes for human lymphoblastoid (leukocyte-type) interferon neutralizing antibodies to interferon-cx: relative frequency in patients treated with different interferon preparations distinct activation of murine interferon-~ promoter region by irf- /isgf- and virus infection human recombinant interferon c~- c enhances the expression of class ii hla antigens on hairy cells cytokines in cancer therapy ifp is an interferon-induced leucine zipper protein that undergoes interferon-regulated cellular redistribution the interferons: mechanisms of action and clinical applications shared architecture of the hormone binding domains in type i and ii interferon receptors haemopoietic receptors and helical cytokines vaccinia virus-encoded eif- alpha homolog abrogates the antiviral effect of interferon a monoclonal antibody to recombinant human ifn-c~ receptor inhibits biologic activity of several species of human ifn-c~, ifn-[ and ifn-m combined treatment of symptomatic human immunodeficiency virus type infection with native interferon c~ and zidovudine clinical side effects and toxicities of interferon molecular interactions between interferon consensus sequence binding protein and members of the interferon regulatory factor family molecular analysis of the human interferon-cx gene family the interferon receptors evidence that types i and ii interferons have different receptors successful retreatment of an anti-interferon resistant polycythaemic vera patient with lymphoblastoid interferon-czn and in vitro studies on the specificity of the antibodies preparation of human leukocyte interferon for clinical use two distinct families of human and bovine interferon-c, genes are coordinately expressed and encode functional polypeptides identification of a common nucleotide sequence in the '-untranslated region of mrna molecules specifying inflammatory mediators activation of the ppp(a 'p)na system in interferon-treated, herpes simplex virus-infected cells and evidence for novel inhibitors of the ppp(a 'p)na-dependent rnase interferon-induced , m r protein and its mrna in human cells: molecular cloning and partial sequence of the edna constitutive expression of ( '- ') oligo a synthetase confers resistance to picornavirus infection gart, son, ifnar, and crf- genes cluster on human chromosome and mouse chromosome interferon action: nucleolar and nucleoplasmic localization of the interferoninducible -kd protein that is encoded by the if: gene from the gene cluster sequence requirement for specific interaction of an enhancer binding protein (ebp ) with dna binding of epstein-barr virus small rna eber- to the double-stranded rna-activated protein kinase dai knockout and reconstitution of a functional human type i interferon receptor complex characterization of three monoclonal antibodies that recognize the ifna receptor multichain structure of the ifn-cx receptor on hematopoietic cells vaccinia virus b r gene encodes a type i interferon-binding protein that blocks interferon transmembrane signaling role of interferon cz/[ receptor chain in the structure and transmembrane signalling of the interferon ~/[ receptor complex inhibition of revmediated hiv- expression by an rna binding protein encoded by the interferon-inducible - gene expression of gene rrg is associated with reversion of nih t transformed by ltr-c-h-ras linkage analysis of the murine interferon- ~ locus on chromosome segregation of restriction fragment length polymorphism in an interspecies cross of laboratory and wild mice indicates tight linkage of the murine ifn-[ gene to the murine ifn-cx genes nucleotide sequence of the chromosomal gene for human fibroblast ( ) interferon and of the flanking regions epstein barr virus/complement c d receptor is an interferon c~ receptor interferons and other regulatory cytokines isolation and structure of a human fibroblast interferon gene isolation and characterisation of a human fibroblast interferon gene and its expression in escherichia coli clinical 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(arg) genes in genomic dna direct evidence that the gene product of the human chromosome locus, ifrc, is the interferon-cx receptor recombinant human leukocyte interferon produced in bacteria has antiproliferative activity characterization of an interferon receptor on human lymphoblastoid cells two different virus-inducible elements are required for human ] -interferon gene regulation purification, concentration and physico-chemical properties of interferons enhanced expression of hla antigens and ~ -microglobulin on interferon-treated human lymphoid cells modulation of tubulin mrna levels by interferon in human lymphoblastoid cells why are there so many subtypes of alpha-interferons human interferon omega (co) binds to the ~/~ receptor recombinant interferon c~- a combined with prednisone in metastatic renal-cell carcinoma: treatment results, serum interferon levels and the development of antibodies expression of the terminal protein region of hepatitis b virus inhibits cellular responses to interferons c~ and ' and double-stranded rna recombinant human interferon (ifn) alpha- b in chronic myelogenous leukaemia: dose dependency of response and frequency of neutralizing anti-interferon antibodies a randomized placebo-controlled trial of recombinant human interferon alpha a in patients with aids delimitation and properties of dna sequences required for the regulated expression of human interferon evidence for a nuclear factor(s), irf- , mediating induction and silencing properties to human ifn-j] gene regulatory elements involvement of a cis-element that binds an h tf- /nf-~cb like factor(s) in virus-induced interferon-[ expression induction of the transcription factor irf- and interferon-j mrnas by cytokines and activators of second messenger pathways induction of endogenous ifn-~ and ifn-j genes by a regulatory transcription factor, irf- binding of the adenovirus vai rna to the interferon-induced -kda protein kinase correlates with function configuration of 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comparison of ~ interferon genes role of interferon in resistance to viral infection in vivo interferon and cell division. i. inhibition of the multiplication of mouse leukaemia l cells in vitro by interferon preparations mechanism of antitumour effect of interferon in mice injection of mice with antibody to interferon enhances the growth of transplantable murine tumours interferon therapy for kaposi's sarcoma associated with the acquired immunodeficiency syndrome (aids) the structure of a thirty-six kilobase region of the human chromosome including the fibroblast interferon gene ifn-~ interferon receptors and their role in interferon action interferon action against reovirus: activation of interferon-induced protein kinase in mouse l cells upon reovirus infection the sh domains of stat and stat mediate multiple interactions in the transduction of ifn~ signals regression of the interferon signal transduction pathway by the adenovirus e a oncogene cytokine therapeutics: lessons from interferon ~. proc. natl acad. sci. usa differential human interferon alpha receptor expression on proliferating and non-proliferating cells the genes for the trophoblast interferons and the related interferon ~ii possess distinct '-promoter and '-flanking sequences structurally similar, but functionally distinct factors, irf- and irf- , bind to the same regulatory elements of ifn and ifn-inducible genes absence of type i ifn system in ec cells: transcriptional activator (irf- ) and repressor (irf- ) are developmentally regulated anti-oncogenic and oncogenic potentials of interferon regulatory factors and activity of interferons alpha, beta, and gamma against human immunodeficiency virus replication in vitro a novel class of human type i interferons synthesis of two distinct interferons by human fibroblasts treatment of recurrent respiratory papillomatosis with human leukocyte interferon structural relationship of human interferon-~ genes and pseudogenes the gene for human fibroblast interferon (ifb) maps to p enhanced expression of -microglobulin and hla antigens on human lymphoid cells by interferon a gene on human chromosome located in the region q . to q . encodes a factor necessary for signal transduction and antiviral response to type i interferons structure and expression of a cloned cdna for mouse interferon-[ structure and expression in escherichia coli of canine alpha-interferon genes differential expression of human interferon genes induction of human interferon gene expression is associated with a nuclear-factor that interacts with the nf~cb site of the human immunodeficiency virus enhancer interferon enhances the antibody-dependent cellular cytotoxicity (adcc) of human polymorphonuclear leukocytes interferon-alpha hybrids a protective action of neurotropic against viserotropic yellow fever virus in macacus rhesus structural characterization of fibroblast human interferon-beta the absence of introns within a human fibroblast interferon gene interferon-induced and double-stranded rna-activated enzymes: a specific protein kinase and ', '-oligoadenylate synthetases evidence for multiple binding sites for several components of human lymphoblastoid interferon-cx interferon betal b is effective in relapsing-remitting multiple sclerosis. i. clinical results of a multicenter, randomized, double-blind, placebo-controlled trial cytokine receptor signalling stats: signal transducers and activators of transcription jaks and stats in signaling by the cytokine receptor superfamily virus interference. i. the interferon intrathecal interferon reduces exacerbations of multiple sclerosis intrathecally administered natural human fibroblast interferon reduces exacerbations of multiple sclerosis: results of a multicenter, double-blind study a phase iii trial of intramuscular recombinant beta interferon as treatment for multiple sclerosis: current status role of protein kinase c in induction of gene expression and inhibition of cell proliferation by interferon cx the treatment of multiple myelomaman important mrc trial interferon receptors. crosslinking of human leukocyte alpha- to its receptor on human cells inhibition of interferon-inducible gene expression by adenovirus e a proteins: block in transcriptional complex formation identification and characterisation of a novel repressor of interferon-~ expression protein kinase activity required for an early step in interferon-~ signalling two interferon-induced nuclear factors bind a single promoter element in interferon-stimulated genes interferon: purification and initial characterisation from diploid cells human fibroblast interferon: amino acid analysis and amino terminal amino acid sequence three-dimensional model of a human interferon alpha consensus sequence malignant transformation by a mutant of the ifn-inducible dsrna-dependent protein kinase studies on the role of the '- '-oligoadenylate synthetase-rnase l pathway in l-interferon-mediated inhibition of encephalomyocarditis virus replication interferon c~ induces the expression of retinoblastoma gene product in human burkitt lymphoma daudi cells: role in growth regulation interferon alpha in patients with asymptomatic human immunodeficiency virus (hiv) infection: a randomized, placebo-controlled trial structure of interferons interferon receptors dna sequence of two closely linked human leukocyte interferon genes dna sequence of a major human leukocyte interferon gene synergism between distinct enhanson domains in viral induction of human beta interferon gene characterization and regulation of the , -dalton cellular inhibition of the interferon-induced, dsrna-activated protein kinase the involvement of nf-~cb in -interferon gene regulation reveals its role as a widely inducible mediator of signal transducfion biochemistry of interferons and their actions tumor-suppressor genes: news about the interferon connection amino-terminal amino acid sequence of human leukocyte interferon amino acid sequence of a human leukocyte interferon molecular analysis of a human interferoninducible gene family the interferon-inducible -kda ubiquitin homolog conjugates to intracellular proteins the structure of the human interferon c~/[ receptor gene mutant u a cells are complemented by an interferon-a[ receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster different pathways mediate virus inducibility of the human ifn-~i and ifn-~ genes maintenance treatment with recombinant interferon alpha b in patients with multiple myeloma responding to conventional induction chemotherapy controlled transcription of a human m-interferon gene introduced into mouse l-cells the nucleotide sequence of a cloned human leukocyte interferon cdna site-specific mutagenesis of the human fibroblast interferon gene adenovirus virus-associated rna and translational control targeted disruption of irf- or irf- results in abnormal type i ifn gene induction and aberrant lymphocyte development involvement of a gene on chromosome in interferon production interferon production: variation in yields from human cell lines somatic cell genetics of human interferon production in human-rodent cell lines the effect of hypertonic salt on interferon and interferon mrna synthesis in human mg cells interferon-induced mx proteins form oligomers and contain a putative leucine zipper targeted disruption of the stat gene in mice reveals unexpected physiologic specificity in the jak-stat signaling pathway tumor suppressor function of the interferon-induced double-stranded rna-activated protein kinase recent divergence from a common ancestor of human ifn-~ genes mechanism of interferon action. interferon mediated inhibition of reovirus mrna translation in the absence of detectable mrna degradation but in the presence of protein phosphorylation regulated expression of a gene encoding a nuclear factor, irf- , that specifically binds to ifn-[ gene regulatory elements roles of the - disulfide bond of subtype a of human interferon in its antiviral activity and conformational stability the protein tyrosine kinase jak complements defects in the ifn-a/[ and -~, signal transduction functional role of type i and type ii interferons in antiviral defense multimerization of aagtga and gaaagt generates sequences that mediate virus inducibility by mimicking an interferon promoter element the structure of one of the eight or more distinct chromosomal genes for human interferon-c~ absence of virus-induced lymphocyte suppression and interferon production in multiple sclerosis transcriptional activation by viral regulatory proteins interferon-j promoters contain a dna element that acts as a position-independent silencer on the nf-~cb site the human interferon ~/j receptor: characterisation and molecular cloning treatment of malignant carcinoid tumors with recombinant interferon alfa- b: development of neutralizing interferon antibodies and possible loss ofantitumor activity close linkage of ~ and ~ interferons and infrequent duplication of ] interferon in humans leukocyte and fibroblast interferon genes are located on human chromosome interferon beta-lb is effective in relapsing-remitting multiple sclerosis. ii. mri analysis results of a multicenter, randomized, double-blind, placebo-controlled trial interferons and their actions transmembrane signalling by interferon ~ involves diacylglycerol production and activation of ~ isoform of protein kinase c in daudi cells large scale production of human interferon from lymphoblastoid cells purification and cloning of interferon-stimulated gene factor (isgf ): isgf (irf- ) can bind to the promoters of both beta interferon and interferon-stimulated genes, but is not a primary transcriptional activator of either tyrosine phosphorylation of the ~ and ~ subunits of the c~ and [ subunits of the type i interferon receptor gene targeting in human somatic cells: complete inactivation of an interferoninducible gene interferons and natural killer cells not more than base pairs of '-flanking sequence are required for inducible expression of a human ifn-~ gene receptors for human interferon-alpha: two forms of interferon-receptor complexes identified by chemical cross-linking receptors for human cx and [ interferon but not for interferon are specified by human chromosome specific molecular activities of recombinant and hybrid leukocyte interferons evidence for involvement of protein kinase c in the cellular response to interferon ~ a single dna response element can confer inducibility by both ~-and -interferons enhanced in vivo therapeutic response to interferon in mice with an in vitro interferon-resistant b-cell lymphoma mice devoid of interferon regulatory factor (irf- ) show normal expression of type i interferon genes critical role of a common transcription factor, irf- , in the regulation of ifn-~ and ifn-inducible genes interferons and interleukin- suppress phosphorylation of the retinoblastoma protein in growth-sensitive hematopoietic cells interferon-activated genes components of the human type antigen presentation by human monocytes: effects of modifying major histocompatibility complex class ii antigen expression and interleukin production by using recombinant interferons and corticosteroids double-stranded rna-dependent protein kinase and - a system are both activated in interferon-treated, encephalomyocarditis virus-infected hela cells the effect of recombinant-dna-derived interferons on the growth of myeloid progenitor cells phase i/ii trial of human recombinant ~-interferon serine in patients with renal cell carcinoma interferons as hormones of pregnancy central nervous system toxicity of interferons control of ifn-inducible mxa gene expression in human cells tumour necrosis factor and ifn induce a common set of proteins the interferon receptors human leukocyte interferon purified to homogeneity human leukocyte interferon: isolation and characterisation of several molecular forms induction of type i interferon genes and interferon-inducible genes in embryonal stem cells devoid of interferon regulatory factor i a -nucleotide promoter segment from an ifn-cx gene renders an unrelated promoter inducible by virus interferon induction of the antiviral state proteins induced by interferons and their possible roles in the antiviral mechanisms of action treatment of hairy cell leukaemia interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor interference between animal viruses interferon treatment of human genital papilloma virus infection: importance of viral type the interferoninduced -kda guanylate-binding protein (hgbp ) is a gtpase that converts gtp to gmp prevention of rhinovirus colds by human interferon alpha- from e. coli inhibition of ornithine decarboxylase in human fibroblast cells by type i and type ii interferons the interferon system: a bird's eye view of the biochemistry three-dimensional crystal structure of recombinant murine interferon-[ specific residues within an amino-terminal domain of residues of interferon-~ are responsible for recognition of the human interferon-a cell receptor and for triggering biological effects structure and expression of cloned murine ifn-a genes a conserved au sequence from the ' untranslated region of gm-csf mrna mediators selective mrna-degradation a single amino acid change in ifn-[ abolishes its antiviral activity existence and unique n-terminal sequence of alpha ii (omega) interferon in natural leukocyte interferon preparation clustering of leukocyte and fibroblast genes on human chromosome interferon activation of the transcription factor star involves dimerization through sh -phosphotyrosyl peptide interactions antibodies to chromosome coded cell surface components block binding of human ~ interferon but not interferon to human cells expression of interferon-~ and interferon-j genes in human lymphoblastoid (namalwa) cells inhibition ofangiogenesis by interferons: effects on tumour-and lymphocyte-induced vascular responses presence of human chromosome alone is sufficient for hybrid cell sensitivity to human interferon chromosomal location of a human cx interferon gene family expression of a functional human type i interferon receptor in hamster cells: application of functional yeast artificial chromosome (yac) screening interferon-induced proteins and the antiviral state resistance to recombinant interferon alfa- a in hairy-cell leukaemia associated with neutralizing anti-interferon antibodies the interferon system interferon nomenclature modulation of -fluorouracil-induced toxicity in mice with interferon or with the interferon inducer, polyinosinic-polycytidylic acid at least three human type a interferons: structure of a target specificity of two species of human interferon-a produced in escherichia coli and of hybrid molecules derived from them vaccinia virus encodes a soluble type i interferon receptor of novel structure and broad species specificity the linkage of genes for the human interferon-induced antiviral protein and indophenol oxidase-b traits to chromosome g- human leukocyte and fibroblast interferons are structurally related recent progress in interferon research: molecular mechanisms of regulation, action, and virus circumvention the high mobility group protein hmgi (y) is required for nf-~cb-dependent virus induction of the human ifn- gene an intracellular kda fc~,-binding protein is induced in human cells by ~-ifn characterization of human beta-interferon-binding sites on human cells two non-allelic human interferon a genes with identical coding regions intranasally administered interferon as prophylaxis against experimentally induced influenza a virus infection in humans human natural killer cells: biologic and pathologic aspects prevention of experimental coronavirus colds with intranasal alpha- b interferon nucleotide sequence of a portion of human chromosome containing a leukocyte interferon gene cluster characterization of four different mammalian-cell-derived recombinant human interferon-[ s receptor dynamics of closely related ligands: "fast" and "slow" interferons electrostatic interactions in the cellular dynamics of the interferon-receptor complex genetic transfer of a functional human interferon ~ receptor into mouse cells: cloning and expression of its cdna murine tumor cells expressing the gene for the human interferon q receptor elicit antibodies in syngeneic mice to the active form of the receptor behaviour of a cloned murine interferon alpha beta receptor expressed in homospecific or heterospecific background ~ and ] interferons and their receptor and their friends and relations characterization of interferon-alpha binding sites on human cell lines mechanism of interferon action in hairy cell leukaemia: a model of effective cancer biotherapy a protein tyrosine kinase in the interferon a/j signalling pathway distinct domains of the protein tyrosine kinase tyk required for binding of interferon-a/j and for signal transduction role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. v. protective role in mouse hepatitis virus type infection of susceptible and resistant strains of mice double-stranded rna activates binding of nf-~cb to an inducible element in human [ -interferon promoter interferon-induced enhancement of macrophage fc receptor expression: [ -interferon treatment of c h/hej macrophages results in increased numbers and density of fc receptors effective natural interferon-~ therapy in recombinant interferon-m-resistant patients with hairy cell leukaemia antineoplastic activity of the combination of interferon and cytotoxic agents against experimental and human malignancies: a review interferon c~ induces a protein kinase c-e (pkc-e) gene expression and a . kb pkc- related transcript interferon increases the abundance of submembranous microfilaments in hela-s cells in suspension culture activation of ifn- element by irf- requires a translational event in addition to irf- synthesis regulation of two interferon-inducible human genes by interferon, poly(ri):poly(rc) and viruses regulation of gene expression by cytokines and virus in human cells lacking the type-i interferon locus antiviral activities of hybrids of two major human leukocyte interferons the '-flanking region of a human ifn-c~ gene mediates viral induction of transcription human interferon consensus sequence binding protein is a negative regulator of enhancer elements common to interferon-inducible genes vaccinia rescue of vsv from interferon-induced resistance: reversal of translation block and inhibition of protein kinase activity identification of novel factors that bind to the prd region of the human [ -interferon promoter postinduction repression of the -interferon gene is mediated through two positive regulatory domains treatment of malignant carcinoid tumors with recombinant interferon alfa- b: development of neutralizing interferon antibodies and possible loss of antitumour activity transcriptional regulation of interferon-stimulated genes inhibition of protein synthesis by '- ' linked adenine oligonucleotides in intact cells a comparison of vertebrate interferon gene families detected by hybridisation with human interferon dna ribosomal rna cleavage, nuclease activation and - a (ppp(a 'p)na) in interferon treated cells multiple protein-dna interactions with the human interferon-[ regulatory element phosphorylated interferon-~ receptor subunit (ifnar ) acts as a docking site for the latent form of the kda stat protein different binding of human interferon-c~l and-c~ to common receptors on human and bovine cells. studies with recombinant interferon produced in escherichia coli interferon- ~ inhibits cyclin eand cyclin dl-dependent cdk- kinase activity associated with rb protein and e f in daudi cells expressing cloning of - a-dependent rnaase: a uniquely regulated mediator of interferon action comparative structures of mammalian interferons amino-terminal sequence of the major component of human lymphoblastoid interferon purification and characterization of multiple components of human lymphoblastoid interferon-c~ organization, structure and expression of murine interferon c~ genes key: cord- - kb qb authors: hartmann, g. title: nucleic acid immunity date: - - journal: adv immunol doi: . /bs.ai. . . sha: doc_id: cord_uid: kb qb organisms throughout biology need to maintain the integrity of their genome. from bacteria to vertebrates, life has established sophisticated mechanisms to detect and eliminate foreign genetic material or to restrict its function and replication. tremendous progress has been made in the understanding of these mechanisms which keep foreign or unwanted nucleic acids from viruses or phages in check. mechanisms reach from restriction-modification systems and crispr/cas in bacteria and archaea to rna interference and immune sensing of nucleic acids, altogether integral parts of a system which is now appreciated as nucleic acid immunity. with inherited receptors and acquired sequence information, nucleic acid immunity comprises innate and adaptive components. effector functions include diverse nuclease systems, intrinsic activities to directly restrict the function of foreign nucleic acids (e.g., pkr, adar , ifit ), and extrinsic pathways to alert the immune system and to elicit cytotoxic immune responses. these effects act in concert to restrict viral replication and to eliminate virus-infected cells. the principles of nucleic acid immunity are highly relevant for human disease. besides its essential contribution to antiviral defense and restriction of endogenous retroelements, dysregulation of nucleic acid immunity can also lead to erroneous detection and response to self nucleic acids then causing sterile inflammation and autoimmunity. even mechanisms of nucleic acid immunity which are not established in vertebrates are relevant for human disease when they are present in pathogens such as bacteria, parasites, or helminths or in pathogen-transmitting organisms such as insects. this review aims to provide an overview of the diverse mechanisms of nucleic acid immunity which mostly have been looked at separately in the past and to integrate them under the framework nucleic acid immunity as a basic principle of life, the understanding of which has great potential to advance medicine. immunology is typically categorized in innate and adaptive immunity. while the term innate is associated with conserved molecular patterns detected by germline-encoded receptors, adaptive immunity refers to t cells and b cells which use recombination and clonal selection to specifically adapt their immune receptors (t cell receptor, b cell receptors, and antibodies) in order to target foreign protein antigens. in this wellestablished concept, the innate immune system in the form of myeloid immune cells (macrophages, dendritic cells) provides information whether new protein antigens are associated with potential pathogens or damage. a limited number of germline-encoded innate immune receptors have been identified in the last two decades which are specialized to detect different classes of pathogen-or damage-associated molecules. among them are several groups of immune receptors which are specialized on the detection of foreign or damage-associated nucleic acids. one of these groups of nucleic acid-sensing immune receptors are the toll-like receptors (tlrs) tlr , tlr , tlr , and tlr (tlr not existent in humans) which are preferentially located in the endolysosomal compartment of distinct immune cell subsets and certain somatic cells. nucleic acid-detecting immune receptors located in the cytosol include the rig-i family of helicases (rig-i, mda , lgp ), cgas, and aim . although these nucleic acid-sensing immune receptors as part of the innate immune system participate in the regulation of protein antigen-directed adaptive immunity, they are now appreciated as part of a larger system of nucleic acid-directed immunity which functions to detect and eliminate foreign nucleic acids, whereas protein-directed adaptive immunity evolved to eliminate foreign proteins. although there are crossregulatory functions of both systems, nucleic acid-directed immunity has a purpose on its own. this is underlined by the fact that protein-directed adaptive immunity developed more recently in evolution, whereas nucleic acid-directed immunity dates back to the earliest forms of life represented by bacteria and archaea. with the additions of rnai and the crispr/cas system, specific nucleases including the restriction-modification (r-m) systems, and antiviral effector proteins partially discovered only recently in the context of rare hereditary inflammatory diseases, the new concept of nucleic acid immunity evolves. with crispr/cas and rnai, biology has established two mechanisms which acquire new sequence information of pathogens and memorize this information for later defense against the same type of pathogen, characteristics which functionally correspond to adaptive immunity of t cells and b cells. the various mechanisms comprising nucleic acid immunity are highly relevant for the understanding of many inflammatory and infectious diseases. this review summarizes the currently known nucleic acid recognition-based antiviral response strategies. antiviral response strategies span from ancient sequence or nucleic acid modificationdependent degradation systems (r-m, crispr, rnai) to modern innate immunity in vertebrates, in which innate nucleic acid-sensing receptors induce a broad spectrum of antiviral alarm and effector mechanisms as well as subsequent adaptive immune responses. foreign nucleic acids can be introduced by viruses or bacteriophages. however, species differ in their arsenal of defense mechanisms against such foreign nucleic acid invaders (fig. ). all species from bacteria to humans have established different types of nucleases which cleave nucleic acids that have identified themselves as foreign by their specific structure, by abundance and localization. one of the earliest forms of nucleases are the r-m systems in bacteria and archaea. in this system, modification enzymes and restriction endonucleases (reases) are directed to certain dna sequence motifs in self dna. since modified dna is not cleaved by the corresponding nucleases, dna sequence motifs without modification are identified as foreign and are degraded (for further details, see later). besides the r-m systems, a variety of rnases and dnases are established in evolution. dna outside the nucleus is degraded by dnases i, ii, and iii. fig. mechanisms of nucleic acid immunity in species relevant for human disease. biology has evolved a number of mechanisms to detect and eliminate foreign nucleic acids as introduced by viruses or bacteriophages. all species from bacteria to humans have established nucleases to directly degrade nucleic acids with structural characteristics or localizations which allow to distinguish them from regular cellular self nucleic acids. other mechanisms are predominant in certain groups of species. restrictionmodification systems in bacteria and archaea apply sequence-specific modification of self nucleic acids which allows the specific detection and degradation of foreign nucleic acids (restriction endonucleases). acquired sequence information is used by the crispr/cas system in which new sequence information about pathogenic nucleic acids is integrated into the genome and thereby memorized in order to sequencespecifically degrade foreign nucleic acids. sequence information is also used by rna interference which serves antiviral nuclease functions (sirna/dicer) as well as regulatory (microrna) functions in higher multicellular organisms. in vertebrates, innate immune-sensing receptors including dicer-related helicases rig-i and mda dominate over rnai as antiviral defense mechanism. while innate nucleic acid immune-sensing receptors elicit signaling pathways resulting in antiviral functions, a number of nucleic acid receptors (e.g., pkr, adar , ifit ) directly detect and restrict nucleic acid function and replication. since the principles of nucleic acid immunity are either established in mammals themselves or in pathogens (bacteria, parasites, helminths) or pathogentransmitting insects (e.g., mosquitoes), nucleic acid immunity as such is highly relevant for human health and disease. the rna in dna-rna hybrids is degraded by rnase h. long doublestranded rna in the cytosol is subject to dicer which cleaves rna down to short double-stranded oligoribonucleotides which enter the rnai pathway. another way to acquire sequence information for antiviral defense is used by the crispr/cas system. in this system, nucleic acid sequences derived from pathogens are integrated into the genome which allows the sequence-specific identification of the same type of pathogen during a subsequent challenge. in vertebrates, a number of highly specialized innate immune-sensing receptors such as tlr or rig-i evolved to detect pathogen-associated nucleic acids and to induce appropriate immune responses. while innate nucleic acid immune-sensing receptors elicit antiviral signaling pathways, a number of nucleic acid-detecting effector proteins (viral restriction factors, e.g., pkr, adar , ifit ) directly detect and restrict nucleic acid function and replication. various principles of nucleic acid immunity apply to different species, with only a subset applying to mammals. however, if it comes to infectious diseases, pathogens (bacteria, parasites, helminths) and pathogen-transmitting insects (e.g., mosquitoes) use additional mechanisms not present in mammals, which contribute to the interaction of the pathogen with transmitting organisms, and thus represent potential prophylactic or therapeutic targets. several fields initially developed as independent lines of research and only recently were appreciated to closely cooperate in a defense system specialized in the detection and elimination of foreign genetic material. fig. provides a rough time line of key discoveries of principles, receptors, and ligands emerging from different areas of research all in the context of nucleic acid immunity. this brief overview cannot be comprehensive or provide the exact timing of each single discovery. the idea rather is to provide a picture how different fields evolved over the years. for more detailed information on the different receptors and pathways, the reader is referred to the respective specific paragraphs of this chapter below (figs. and ). immune sensing of nucleic acids dates back to the early s with the observation that nucleic acids such as long double-stranded rna and specifically poly(i:c) can induce the antiviral factor type i interferon (isaacs, cox, & rotem, ) which was first described in (isaacs, ; lindenmann, burke, & isaacs, fig. overview of the time line of discoveries in nucleic acid immunity. this graph provides a noncomprehensive overview of the time lines when important principles, receptors, and ligands contributing to nucleic acid immunity have been described. immune sensing of nucleic acids dates back to the early s with the observation that nucleic acids such as long double-stranded rna and specifically poly(i:c) can induce type i interferon. later, it was appreciated that bacterial dna is more active than vertebrate dna. in , the activity of bacterial dna was attributed to a higher frequency of unmethylated cpg motifs in bacterial dna. in , tlr was identified as the immune receptor for the detection of unmethylated cpg motifs in dna in the endosomal compartment. sensing of cytoplasmic dna remained unclear until in aim and in cgas were identified as the cytosolic receptors responsible for dna-induced inflammasome activation and type i ifn induction, respectively. for immune sensing of rna, the story of discoveries continued in with reports on tlr -sensing long double-stranded rna and was continued in with the appreciation of tlr and tlr as receptors sensing shorter forms of unmodified single and double-stranded rna with great implications for the application of sirna. another milestone was reached with the immune sensing of cytoplasmic forms of rna, specifically the detection of -triphosphate short double-stranded forms of rna by the cytosolic receptor rig-i. the rig-i-like receptor mda added another cytosolic receptor which explained the induction of type i ifn by long double-stranded forms of rna as observed early on in the s. pkr identified in the late s was the first of the receptors restricting nucleic acid function and replication without activating immunity and cytokines. samhd that bacterial dna induces type i ifn much more vigorously than genomic dna of vertebrates (yamamoto, kuramoto, shimada, & tokunaga, ; yamamoto, yamamoto, shimada, et al., ) . it was speculated that bacterial dna in sir william coley's bacterial lysates was responsible for the antitumor activity seen using bacterial lysates for the treatment of tumor patients around years earlier (wiemann & starnes, ) . efforts were undertaken to generate synthetic oligodeoxynucleotides which mimic the type i ifn-inducing activity of bacterial dna. palindromic dna sequences were identified, and oligonucleotides containing such palindromes induced type i ifn in vitro (yamamoto, yamamoto, kataoka, et al., ) but only showed weak activity in tumor models in vivo due to rapid degradation by dnases. in , the immunological activity of bacterial dna was attributed to a higher frequency of unmethylated cpg motifs in bacterial dna (krieg et al., ) . unmethylated cpg motifs were contained in the former palindromic sequences, but a palindromic sequence was not required for the type i ifn-inducing activity. the introduction of the phosphorothioate modification in dna first described in (vosberg & eckstein, ) was used to stabilized these so-called cpg oligonucleotides which now could be successfully applied for treatment in experimental tumor models in vivo (heckelsmiller et al., ) . in , tlr was identified as the innate immune receptor required for the detection of unmethylated cpg motifs in dna (hemmi et al., ) . notably, tlr was the first innate immune receptor reported to detect a specific type of nucleic acid and to induce an immune response. despite intensive research, it took almost a decade to identify aim as the next innate immune receptor-detecting dna (hornung & latz, ) . (depletion of dntps) and adar (a-to-i conversion in dsrna) entered the field more recently in the context of genetic alterations in these genes identified in the context of inherited inflammatory syndromes (e.g., ags). ifit and ifit are two other examples of more recently described receptors which inhibit the translation of mrna. oas was identified early on soon after pkr as a factor restricting viral replication by activating rnase l. other nucleases contributing to nucleic acid immunity include rnase h structurally resolved in , which degrades the rna in dna-rna hybrids; furthermore, extracellular dnase i and endolysosomal dnase ii are known since the mid- s. knowledge around the function of the cytoplasmic dnase iii which is also called trex accumulated since and gained great impact on nucleic acid immunity like samhd and adar more recently in the context of inherited type i ifn-dependent inflammatory syndromes. antiviral rnai and the role of dicer were first described in , while the bacterial version of sequence-specific antiviral immunity, crispr/cas, was identified in . restriction-modification systems are studied since the s. however, since aim activates the inflammasome but not type i ifn, the field of dna sensing struggled until end of , when the cytosolic dnabinding enzyme cyclic gmp-amp synthase (cgas) was discovered which generates cgamp as second messenger for the downstream signaling molecule sting, resolving a big question mark in the field. at that time, sting was already known to be required for immune sensing of cytosolic dna resulting in ifn induction (barber, ) , but sting was unable to bind dna directly. although rna molecules were the first nucleic acids found to induce type ifn as described earlier, this line of research continued only in with the identification of double-stranded rna as ligand for tlr (alexopoulou, holt, medzhitov, & flavell, ) . although the activation of tlr induces some type i ifn, it could not explain the massive amounts of type i ifn induced upon cytosolic delivery of the double-stranded rna mimic poly(i:c). in parallel with the discovery of rnai and sirna, techniques of chemical synthesis of rna rapidly progressed and highly pure synthetic rna oligonucleotides at high quantities and reasonable costs became available. with easier access to highly pure synthetic rna oligonucleotides, it was found that not only long double-stranded rna but also single-stranded rna stimulated type i ifn, specifically in immune cells, and the immune receptors involved were found to be tlr and tlr (tlr nonfunctional in mouse) (diebold, kaisho, hemmi, akira, & reis e sousa, ; heil et al., ; judge et al., ) . then it was noted that even sirna induce type i ifn in tlr expressing immune cells, a surprising finding at that time since sirna was thought to be short enough to not stimulate interferon responses . this work also reported that the immune stimulatory activity of sirna can be avoided by introducing chemical modifications such as -o-methylation or by introducing pseudouridine which is used since then to avoid immunostimulation by sirna applied in cells expressing tlr in vitro or in vivo . at that time, a cheap way to generate sirna was the use of in vitro transcription. however, it was soon realized that sirna made by in vitro transcription induces high amounts of type i ifn when transfected even in cells not expressing tlr , including human myeloid immune cells. this stimulated research on the molecular mechanism responsible for type i ifn induction by in vitro-transcribed sirna in myeloid immune cells. finally, the cytosolic helicase rig-i was identified to detect -triphosphate ends in short blunt end double-stranded rna (hornung et al., ; pichlmair et al., ; schlee, roth, et al., ) . rig-i was reported earlier as immune receptor involved in antiviral responses (kato et al., ) . notably, in vitro transcription but not chemical synthesis of sirna generates such -triphosphate ends. the presence of unmodified -triphsophate ends in the cytosol indicates the presence of rna polymerase activity in the cytosol which only occurs in the course of viral replication. another member of the rig-i-like helicase family of receptors is mda which was found to be responsible for the long sought after type i ifn-inducing activity of cytosolic long double-stranded rna including poly(i:c) . as of today, most of the type i ifn-inducing activities of nucleic acids can be assigned to specific immune receptors. future may still keep some surprises for the field, for example, in the context of immune sensing in the nucleus or in the context of dna damage repair. while activation of the immune receptors described above results in the induction of immunologically active cytokines and immune responses, there is a group of nucleic acid receptors directly restricting nucleic acid function and replication largely without inducing an immune response. pkr was one of the first of these. binding of long double-stranded rna activates pkr to phosphorylate elf a leading to the inhibition of ribosomal translation of mrna to proteins (clemens & elia, ; sen, taira, & lengyel, ; thomis, doohan, & samuel, ; . soon after pkr, the - -oligoadenylate synthetase (oas) system was reported (rebouillat & hovanessian, ; yang et al., ) . in parallel to the oas system, rnase l was identified (baglioni, minks, & maroney, ; brennan-laun, ezelle, li, & hassel, ) . upon binding of oas to long double-stranded rna, oas generates - -linked oligoadenylates ( -oa). -oa activate rnase l which then cleaves cellular rnas thereby restricting viral propagation. samhd (sterile alpha motif and histidine-aspartate-domaincontaining protein ), originally described as ifn-inducible gene in (li, zhang, & cao, ) , has triphosphohydrolase activity that rapidly converts dntps to the corresponding deoxynucleoside and inorganic triphosphate, thereby depleting the supply of dntp for reverse transcriptase activity of retroviruses. in , it was learned that mutations in samhd cause inherited inflammatory syndromes with a type i ifn signature (e.g., ags) (rice et al., ), but the exact mechanism leading to type i ifn induction in this context is not well understood. originally cloned in (kim, wang, sanford, zeng, & nishikura, ) , the rna-editing enzyme adar binds to double-stranded rna and converts a to i thereby contributing to self vs nonself recognition of rna (nishikura, ) . in , it was realized that mutations in adar cause inflammatory syndromes associated with a type i ifn signature (rice et al., ) . ifit and ifit are known for many years as type i ifn-inducible rna-binding proteins which bind to single-stranded rna lacking -o-methylation at their -end and inhibiting rna translation (hyde & diamond, ) . more recent work from (pichlmair et al., ) added the information that ifit and ifit preferentially bind to viral rna containing a -triphosphate group, completing the picture how these proteins distinguish self from nonself single-stranded rna. of the proteins which function primarily as nucleases, extracellular dnase i and endolysosomal dnase ii are known since the mid- s. the cytoplasmic exonuclease trex has been identified decades later in (hoss et al., ) . only since , we know that loss of function in trex causes the type i ifn-associated inflammatory syndrome ags (crow et al., ) , suggesting that trex is critically involved in the clearance self dna within the cytoplasm of cells. rnases h are widely expressed enzymes that hydrolyze rna in rna/dna hybrids (cerritelli & crouch, ). while reports on rnase h activity date back to (stein & hausen, ) , the heterotrimeric functional complex in eukaryotes was only described in (jeong, backlund, chen, karavanov, & crouch, ) and human in (chon et al., ). in , it was reported that mutations in any of the three subunits of human rnase h cause aicardi-gouti eres syndrome (ags) (rice et al., ) . antiviral rnai and the role of dicer were first described in (wang et al., ; zambon, vakharia, & wu, ) , while the role of rna interference (rnai) in mammalian innate immunity is still poorly understood. a bacterial counterpart of acquired sequence-specific antiviral immunity is the crispr/cas system in prokaryotes first described in (barrangou et al., ; marraffini & sontheimer, ) . this prokaryotic immune system confers resistance to foreign genetic elements such as those present within plasmids and phages. information around r-m systems accumulated since the early s (luria & human, ) with reases first described in (nathans & smith, ) . the concept of nucleic acid immunity integrates different functional components which have been studied and reviewed separately in the past. only in the last few years and with the elegant genetic work on rare human inflammatory disorders associated with a type i ifn signature (of crow and others (crow & rehwinkel, )), it became evident that many of the antiviral restriction factors and various nuclease systems are tightly connected with innate immune sensing of nucleic acids inducing type i ifn. altogether, biology has established a broad spectrum of effector functions which cover most of the molecular and mechanistic possibilities to restrict the propagation of foreign genetic material. effector functions reach from direct actions on the detected nucleic acid to the elimination of cells containing foreign genetic material. fig. illustrates the functional components of nucleic acid immunity. central to all components is the detection of foreign nucleic acids by the molecular interaction of a protein (immune-sensing receptor, restriction factor, or effector protein) or a specific nucleic acid (rnai, crispr/cas) with the targeted nucleic acid. the molecular challenge on this level is the specificity of the detection and the distinction of self vs foreign. a specific molecular signature of self nucleic acid (e.g., -o-methylation at the n position in capped mrna), compartmentalization of self nucleic acids, and the rapid clearance of surplus self nucleic acids are three examples which enable specific detection of foreign nucleic acid. structural differences such as long double-stranded rna not present under physiological circumstances allow the highest confidence level of detection. upon detection, the system generates different types of responses. detection of a foreign nucleic acid can trigger an intrinsic effect which acts directly on the detected nucleic acid. examples are degradation (e.g., trex , crispr/cas), structural modification (e.g., a-to-i conversion by adar ), or disabling the function (e.g., inhibition of translation of mrna by ifit or rnai) (see fig. , middle gray layer). extrinsic effects upon detection of foreign nucleic acids require a signaling cascade finally resulting in an effect on the detected nucleic acid. extrinsic effects can occur solely inside the same cell, or they can involve functions outside the cell. extrinsic effects inside the same cell include degradation of the nucleic acid (e.g., rnase l activated by - -oa generated by oas upon binding of long double-stranded rna). extrinsic effect inside the same cell can also impact on the function of the detected nucleic acid. examples for such functional effects are inhibition of translation (e.g., phosphorylation of elf a by activated pkr) or inhibition of replication (e.g., restricting replication of retroviruses by depleting intracellular dntp pools by ifn-inducible samhd ) (see fig. , light gray layer). extrinsic effects that involve functions outside the cell in which the nucleic acid is primarily detected appear as immune responses. they range from alarming neighboring cells overview of functional components in nucleic acid immunity. the primary detection of specific forms of nucleic acids by highly specialized proteins is the central part of nucleic acid immunity. upon binding of nucleic acids, the participating specialized proteins can either exert intrinsic direct effects on the nucleic acid which they have bound, or they can have indirect extrinsic effects which require the participation of additional signaling. extrinsic effects that restrict viral replication and function can be located inside or outside cells, or both. intrinsic direct effects include degradation or structural modification of the bound nucleic acids, or direct inhibition of translation. extrinsic indirect effects via signaling pathways include mechanisms that restrict translation or replication, or that lead to degradation of nucleic acids. extrinsic effects with activities beyond the infected cell include alarming neighboring cells, activating immune effector cells, and guiding immune cells to the site of infection. together, these intrinsic and extrinsic activities represent the repertoire of nucleic acid immunity to restrict viral infection. (e.g., secretion of type i ifn or release of cgamp via gap junctions) to the guidance of immune cells to the respective cell (e.g., tlr -induced release of ip- ) and the activation of immune cells at the local site and systemically (e.g., activation of nk cells by rig-i). intrinsic and extrinsic effects act in concert to minimize the danger associated with foreign nucleic acids. in classical immunology, we distinguish innate and adaptive immunity. while innate immunity relies on receptors encoded in the germline, adaptive immunity acquires information about pathogens during the life span and memorizes such information for later use. while adaptive immunity directed against proteins relies on the mechanism of genetic recombination to adapt to novel pathogen-derived proteins, in the adaptive part of nucleic acid immunity information on pathogen-derived nucleic acid sequences is acquired and memorized (crispr/cas and rnai) (see fig. ). although the adaptive part of nucleic acid immunity requires the participation of germline-encoded proteins such as dicer, risc, innate information on structure acquired information on sequence nucleic acid receptors with effector functions crispr/cas rna interference nucleic acid receptors inducing immune functions fig. innate and adaptive components in nucleic acid immunity. in classical immunology, we distinguish innate and adaptive immunity. while innate immunity relies on receptors encoded in the germline, adaptive immunity acquires information about pathogens during the life span and memorizes such information for later usage. while adaptive immunity directed against proteins relies on the mechanism of genetic recombination to adapt to novel pathogen-derived proteins, in the adaptive part of nucleic acid immunity information about pathogen-derived nucleic acid sequences is acquired and memorized (crispr/cas and rna interference). unlike the adaptive components, the innate components of nucleic acid immunity rely on germline-encoded receptors which detect certain structures indicating viral pathogens. therefore, these receptors are identical throughout the life span, but regulation of receptor expression (e.g., by means of epigenetics) still allows adaptation to different environments (e.g., low or high burden of viral pathogens). or crispr and cas, the detection of foreign nucleic acids is mediated via acquired sequence information. in contrast, the innate components of nucleic acid immunity solely rely on germline-encoded receptors which via protein-nucleic acid interaction detect certain structures which are characteristic of foreign genetic materials. innate immune-sensing receptors are identical throughout the life span. however, it is important to note that epigenetic regulation of gene expression still allows to adapt quantitatively to different environments (e.g., low or high burden of viral pathogens). the perpetual arms race between bacteria and phages has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. such systems include r-m systems and crispr-cas. the prokaryotic dna r-m systems are based on the contrasting enzymatic activities of a sequence-specific rease and a matching sequence-specific host methyltransferase (mtase) (vasu & nagaraja, ) . by transferring a methyl group to the c- carbon or the n amino group of cytosine or to the n amino group of adenine host-specific mtases protect potential cleavage sites of host dna from reases, which on the other hand recognize and cleave foreign unmethylated or "inappropriately" methylated dna from invading phages. this r-m systems can be considered as an innate defense system. on the other hand, the crispr-cas system in prokaryotes represents a highly sophisticated adaptive immune system in which short fragments of invading dna are integrated into the crispr loci. after transcription and processing of these loci, short crispr rnas (crrnas) are generated which guide the nuclease activity of cas proteins to complementary dna or rna resulting in target cleavage (goldfarb et al., ; van der oost, westra, jackson, & wiedenheft, ). this chapter provides an overview of the well-established proteins targeting foreign nucleic acids without involving the classical immune functions such as the induction of cytokines or the activation of immune cells. such proteins can directly act on the foreign nucleic acid, or they can elicit pathways indirectly acting on the foreign nucleic acid (see fig. ). for the family of apobec proteins which detect and modify viral nucleic acids, we refer to detailed reviews by others (harris & dudley, ) . this graph provides an overview of the proteins which target foreign nucleic acids without involving the classical immune functions such as the induction of cytokines or the activation of immune cells. such proteins can directly act on the foreign nucleic acid, or they can elicit pathways indirectly acting on the foreign nucleic acid. the endonuclease dnase i is the most abundant dnase in the extracellular space which degrades dna down to tetramers. dnase ii is the predominant endonuclease in the endolysosomal compartment of cells. the cytoplasmic dnase iii (trex ) is a -to exonuclease which degrades both double-and single-stranded dna. the cytoplasmic rnase h recognizes dna-rna hybrids and cleaves the rna in such hybrids. in contrast, rnase l is indirectly activated by oligoadenylates which are formed by oas upon binding to long doublestranded rna. furthermore, adar modifies long double-stranded rna by a-to-i conversions destabilizing the double strand resulting in changes in the coding sequence of proteins. samhd depletes the pool of dntps which is the prerequisite for dna formation. samhd hydrolyzes the triphosphate in dntps resulting in deoxynucleosides. at the same time, samhd has been proposed to be a -exonuclease for single-stranded dna and rna. pkr and ifit / inhibit mrna translation by phosphorylation of the elf a and by replacing elf in the ribosomal complex, respectively. while pkr is activated by long double-stranded rna, ifit and ifit bind -triphosphate ends of singlestranded rna. adenosine to inosine (a-to-i) rna editing was originally discovered as enzymatic activity unwinding double-stranded rna in xenopus laevis oocytes and embryos (bass & weintraub, ) . soon after, it became clear that this activity is carried out by an adenosine deaminase acting on rna (adar) (bass & weintraub, ; wagner, smith, cooperman, & nishikura, ) . adenosine deaminases perform c deamination of adenosine in base-paired rna structures resulting in a-to-i conversions, a process termed a-to-i rna editing (hogg, paro, keegan, & o'connell, ) . the type i ifn-inducible isoform of adars, adar , first cloned in (kim et al., ) contains three repeats of a double-stranded rna-binding motif, and sequences conserved in the catalytic center of other deaminases. transcription from separate promoters generates two isoforms of adar , a full-length, interferon-inducible adar p and a shorter and constitutively expressed adar p . interestingly, both adar p and adar p isoforms shuttle between nucleus and cytoplasm (nishikura, ) . a-to-i editing frequently occurs in noncoding regions that contain inverted alu repeats but can also occur in proteincoding regions of mrnas resulting in the expression of altered proteins with sequences that are not encoded in the genome. recent studies indicate that adar is also found in complex with dicer to promote mirna processing and rnai efficacy (ota et al., ) , suggesting that both rnai and adar are functionally related. viral dsrna formed at different stages of replication of many viruses are substrates for rna editing by adar. it is well established that adar enzymes interfer with the virus-host interaction with adars acting as pro-or antiviral factors. the biological consequences of a-to-i changes during viral infection is not completely understood (tomaselli, galeano, locatelli, & gallo, ) . the current concept is that two effects oppose each other: on the one hand, adar -mediated a-to-i editing of viral double-stranded rna directly restricts the correct function of the edited rna and thus directly inhibits viral replication. on the other hand, a-to-i editing of double-stranded rna destabilizes long double-stranded rna thereby reducing the recognition of long double-stranded rna by double-stranded rna receptors such as mda . as a consequence, depending on the type of virus, adar has the potential to negatively interfer or to support viral replication and thus can act as proviral or antiviral factor (george, john, & samuel, ) . independently of the presence of viral infection, the lack of adar in a cell results in the activation of mda by endogenous rna species. the most likely scenario is that a-to-i editing masks endogenous rnas from detection by mda . the consequence is that if a virus actively inhibits the function of adar , endogenous rna ligands will form which activate mda and thus induce a type i ifn response. indeed, sequencing studies demonstrated that adar -deficient cells display stretches of endogenous double-stranded rna (liddicoat et al., ) . thus, a-to-i editing of endogenous dsrna is an essential function of adar preventing the activation of the cytosolic dsrna response by endogenous transcripts. samhd is composed of a sam and a hd domain. while the sam domain of samhd mediates protein-protein interactions, the hd domain possesses the triphosphohydrolase activity through which samhd hydrolyzes dntps to deoxynucleosides (goldstone et al., ; powell, holland, hollis, & perrino, ) . samhd expression has been demonstrated in monocytes, macrophages, myeloid dendritic cells, plasmacytoid dendritic cells (pdcs), and cd t cells (baldauf et al., ; gelais et al., ; kim, nguyen, daddacha, & hollenbaugh, ; laguette et al., ; pauls et al., ) . the involvement of samhd in innate immunity was initially proposed based on its mouse ortholog mg which is ifn-inducible in macrophages and dendritic cells (li et al., ) , hence the alternative name dendritic cell-derived ifn-γ-induced protein. subsequent studies showed an increased samhd expression upon stimulation of macrophages with double-stranded dna (rice et al., ) and its upregulation in the context of viral infections (hartman et al., ) . mutations in samhd have been shown to be responsible for % of patients with ags which is characterized by inappropriate and aberrant type i ifn secretion causing symptoms reminiscent of a congenital infection (rice et al., ) . a loss of function of samhd results in spontaneous type i ifn production in ags patients and samhd À/À mice (behrendt et al., ; rehwinkel et al., ) . samhd was identified as a potent restriction factor for hiv (hrecka et al., ; laguette et al., ; simon, bloch, & landau, ) , other nonhuman retroviruses (gramberg et al., ) , and herpesviruses, including hsv- (hollenbaugh et al., ; kim, white, brandariz-nunez, diaz-griffero, & weitzman, ) . the current model is that samhd through its function as dntp triphosphohydrolase decreases intracellular dntp pools in nondividing cells below the threshold level required for efficient viral reverse transcriptase or viral dna polymerase activity (lahouassa et al., ; wu, ) . the observation that functional loss of samhd leads to a spontaneous type i ifn response suggests that uncontrolled activity of endogenous retroelements may be a source of the ifn-inducing nucleic acids, but the identity of such endogenous ligands is unknown to date. besides dntp triphosphohydrolase activity, a metal-dependent -to -exonuclease activity of samhd for ssdna and ssrna was demonstrated (beloglazova et al., ) . the rnase activity was reported to directly degrade hiv- rna (ryoo et al., ) , but further work will be necessary to confirm and exactly characterize the proposed nuclease activity of samhd (rice et al., ). the interferon-inducible, double-stranded rna-activated protein kinase (protein kinase rna-activated, pkr; also known as eukaryotic translation initiation factor -alpha kinase , eif ak ) was first cloned in and represents a key mediator of antiviral activities (feng, chong, kumar, & williams, ; garcia et al., ; . pkr contains an n-terminal dsrna-binding domain (dsrbd) which consists of two tandem copies of a conserved doublestranded rna-binding motif, dsrbm and dsrbm , and a c-terminal kinase domain. binding of pkr to long double-stranded rna (longer bp) activates pkr by inducing dimerization and subsequent autophosphorylation. activated pkr inhibits -cap-dependent mrna translation by phosphorylation of the eukaryotic translation initiation factor eif a thereby preventing viral protein synthesis (farrell et al., ; levin & london, ) . besides long double-stranded rna, pkr has been shown to recognize rna with limited secondary structures (rna with a length of about nt and weak structure; short stem-loops) containing uncapped -triphosphates (nallagatla et al., ) . antiviral functions of pkr beyond the mechanism of translation inhibition are still controversial. pkr affects diverse transcriptional factors such as interferon regulatory factor , stats, p , activating transcription factor , and nf-κb. in particular, how pkr triggers a cascade of events involving ikk phosphorylation of ikappab and nf-κb nuclear translocation has been intensively studied. pkr was also reported to enhance but not being required for nf-κb-dependent type i ifn induction (chu et al., ; kumar, haque, lacoste, hiscott, & williams, ; maggi et al., ) . involvement of pkr in inflammasome activation is controversial (he, franchi, & nunez, ; lu et al., ) . pkr-mediated -cap-specific inhibition of translation is expected to perturb the proteome. since pkr-activated elf a is involved in the initiation of the translation from an aug codon, the alternative non-aug initiation takes place instead. an example of mrnas using non-aug initiation are mrnas for the heat shock proteins. another effect is the selected reduction of proteins with short half-life. reduced translation of the nf-κb inhibitor protein ikappab-alpha is one plausible explanation for the activation of the nf-κb pathway in response to pkr activation (mcallister, taghavi, & samuel, ) . other signaling pathways may be affected in the same way by the removal of an inhibitor with short half-life resulting in the activation of the pathway. interferon-induced proteins with tetratricopeptide repeats (ifits) are among the most abundantly expressed proteins of the group of interferonstimulated genes (isgs). they represent innate immune effector molecules that confer antiviral defense downstream of type i ifn through disruption of the host translation initiation machinery (daffis et al., ; pichlmair et al., ) . they are evolutionarily conserved from fish to mammals. in humans, there are four well-characterized paralogues, ifit (p /isg ), ifit (p /isg ), ifit (p /isg ), and ifit (p /isg ). like rig-i, productive binding of both ifit and ifit was shown to depend on the presence of cytosolic -triphosphate rna and is nonsequence specific. unlike rig-i, ifit and ifit preferentially bind to single-stranded rna or to double-stranded rna with a minimum three (ifit ) or five (ifit )nucleotide overhangs containing an uncapped triphosphate group at the -end of rna (abbas, pichlmair, gorna, superti-furga, & nagar, ; habjan et al., ; kumar et al., ) . replacing the triphosphate with a -cap, a -monophosphate or -oh diminishes the binding significantly (abbas et al., ) . ifit competes with eif e, the endogenous -cap binding and translation factor, in the s initiation complex formation. however, while in vitro competition experiments convincingly show that ifit can compete with eif e for binding at completely unmethylated cap rna, the out-competing of eif e from n methylated cap (cap ) structures is unclear. binding of eif e to cap structures in lysates of ifn-primed cells is rather enhanced than reduced, suggesting additional mechanisms beyond eif e competition for s disruption (habjan et al., ) . a key role for ifit in negative-strand rna viruses (vsv, influenza) and positive-strand rna viruses (wnv, mhv) except picornaviruses was reported (daffis et al., ; habjan et al., ; pichlmair et al., ) . the human - -oas family comprise four type i ifn-inducible genes: oas , oas , oas , and oasl (oas-like protein) (melchjorsen et al., ) . upon binding to long double-stranded rna, oas , oas , and oas catalyze the formation of - -oa, whereas oasl has no enzymatic activity but still has potent antiviral activity due to its coactivating role in the rig-i pathway (schoggins et al., ; zhu et al., ) . the formation of - -oligomers of adenosine ( - -oa) upon exposure to dsrna and subsequent inhibition of translation has been described early on (clemens & williams, ; farrell et al., ; hovanessian, brown, & kerr, ; zilberstein, kimchi, schmidt, & revel, ) . - -oa function as second messenger of oas binding to rnase l leading to dimer formation and subsequent degradation of cellular and viral rna (dong & silverman, ) . the structural mechanisms of rnase l activation by - -oa and its dimer formation have recently been described (han et al., ; huang et al., ) . all three human oas isoforms are activated by dsrna in vitro which is the presumed ligand in vivo as well. the full activation of the oas system in virally infected cells leads to the inhibition of protein synthesis and the induction of apoptosis, thereby interfering with viral replication (castelli et al., ) . activation of the oas-rnase l system restricts replication of a variety of viruses, in particular positive-strand viruses (e.g., picornaviruses, flaviviruses, and alphaviruses) which produce high numbers of dsrna during replication (silverman, ) . virus-encoded inhibitors of the oas-rnase l system such as the nonstructural protein (ns ) of murine coronavirus or inhibitors expressed by picornaviruses support a key role of this system in the restriction of viruses. it is interesting to note that oas and cgas (see later) share similar structural features and enzymatic function. both oas and cgas catalyze the uncommon - phosphodiester linkage upon binding to a nucleic acid ligand (hornung, hartmann, ablasser, & hopfner, ) . rnases h are a family of widely expressed nonsequence-specific endonucleases that hydrolyze solely the rna of rna/dna hybrids resulting in -hydroxyl and -phosphate terminated products and an intact dna strand (cerritelli & crouch, ) . rnases h play crucial roles in the biochemical processes associated with dna replication, gene expression, and dna repair where rna/dna hybrids can occur. furthermore, rnases h degrade rna/dna hybrids generated during viral replication. members of the rnase h family can be found in nearly all organisms, from bacteria to archaea to eukaryotes. unlike in prokaryotes and in single-cell eukaryotes, in higher eukaryotes rnases h are essential for development. the catalytic subunit of eukaryotic rnase h , rnaseh a, is well conserved and similar to the monomeric prokaryotic rnase hii. in contrast, the rnaseh b and rnaseh c subunits share very little homology between human and saccharomyces cerevisiae or bacteria. rnaseh b and rnaseh c serve as a nucleation site for the addition of rnaseh a to form an active rnase h . furthermore, they contain interaction sites with other proteins to support functions other than rnase h nuclease activity, but these functions are not well-defined yet. rnase h deficiency can cause a number of pathogenetic principles including the occurrence of ribonucleoside monophosphates accumulating in genomic dna and activating the dna damage-response pathway. rnase h deficiency leads to abundance of cytosolic rna-dna hybrids and to an increase in retroelements which both represent potential ligands for the cgas-sting signaling pathway (mankan et al., ; rigby et al., ) . in fact, mutations in any of the three subunits rnaseh a-, rnaseh b-, or rnaseh c of human rnase h cause ags, a human neurological disorder with devastating consequences (rice et al., ) . mutations that impair rnase h are also associated with systemic lupus erythematosus (sle). pathogenicity is supported by mouse models of ags-associated mutations of rnase h which show a spontaneous cgas/sting-dependent type i ifn-driven phenotype (mackenzie et al., ; pokatayev et al., ) . . dnases . . dnase i deoxyribonuclease i (dnase i) is an endonuclease which is secreted to cleave dna in the extracellular space down to an average of tetranucleotides with monophosphate and hydroxyl dna ends (baranovskii, buneva, & nevinsky, ) . both single-stranded dna and doublestranded dna are degraded by dnase i. this nuclease appears to account for the major nucleolytic activity on dna in serum and is responsible for the degradation of the majority of circulating dna derived from apoptotic and necrotic cell death and from neutrophil extracellular traps. in addition to its role in the serum, it has been proposed as one of the deoxyribonucleases responsible for dna fragmentation in the process of apoptosis (samejima & earnshaw, ) . notably, dnase l complements the activity of dnase i. although dnase l harbors nuclear localization signals, its main function appears to be in the serum, where it can degrade proteincomplexed dna (napirei, ludwig, mezrhab, klockl, & mannherz, ). in the absence of dnase i, degradation of extracellular dna is heavily reduced resulting in the activation of dna-sensing immune receptors. mice deficient in dnase i display a lupus-like phenotype with increased antinuclear antibody titers and glomerulonephritis (napirei et al., ) . mutations in the human dnase i gene and factors inactivating dnase i have been associated with sle (hakkim et al., ; yasutomo et al., ) . in a subset of sle patients, the presence of dnase i inhibitors or autoantibodies was associated with impaired dna clearance and poor prognosis, suggesting that decreased dnase i activity can also be acquired (hakkim et al., ) . notably, loss-of-function variants in dnase l have also been associated with familiar sle, supporting an important functional contribution of dnase l in clearing dna (al-mayouf et al., ) . dnase ii is a mammalian endonuclease with highest activity at low ph and in the absence of divalent cations. dnase ii cleaves dna between -phosphate and -hydroxyl resulting in the formation of nucleoside- phosphates. dnase ii is the predominant dnase located in lysosomes of cells in various tissues including macrophages (evans & aguilera, ; yasuda et al., ) . with its lysosomal localization and ubiquitous tissue distribution, this enzyme plays a pivotal role in the degradation of exogenous dna encountered by endocytosis. it has been demonstrated that digestion of large dna molecules and of cpg-a (hartmann et al., ) by dnase ii creates short dna fragments which are sensed by tlr (chan et al., ; pawaria et al., ) . dnase ii deficiency is another example of a cell-intrinsic nuclease defect driving autoimmunity. loss of dnase ii leads to a defect in the disposal of dna within lysosomal compartments (kawane et al., ; yoshida, okabe, kawane, fukuyama, & nagata, ) . accumulation of undigested dna can result in the translocation of dna into the cytoplasm which is then sensed by the cgas-sting pathway (ahn, gutman, saijo, & barber, ; gao et al., ) as well as the aim inflammasome (baum et al., ; jakobs, perner, & hornung, ) . mice lacking dnase ii display an inflammatory response that depends on both cgas and aim . besides cell-extrinsic sources of dna (e.g., nuclei expelled from erythroid precursor cells), recent work in dnase ii-deficient mice suggests that damaged nuclear dna is also subject to dnase ii degradation and might stimulate cytosolic dna immune-sensing receptors if not properly degraded (lan, londono, bouley, rooney, & hacohen, ) . in a model of cardiacspecific deletion of dnase ii, severe myocarditis and dilated cardiomyopathy developed which was attenuated if immune sensing of accumulating mitochondrial dna by tlr was inhibited (oka et al., ) . the cytoplasmic dnase iii ( -repair exonuclease , trex ) has been identified decades later than dnase i and ii (hoss et al., ) . trex is a -to -exonuclease which degrades both double-and single-stranded dna. most dna reaching the cytosol is promptly removed by trex . modifications have been reported which render dna resistant to trex . for example, oxidation of guanine bases to -hydroxydeoxyguanine ( -ohdg) protects dna from trex -dependent degradation leading to accumulation and cgas-mediated recognition of oxidized dna in the cytosol (gehrke et al., ) . only since , it is known that loss of function in trex causes the type i ifn-associated inflammatory syndrome ags (crow et al., ) , suggesting that trex is critically involved in the clearance self dna within the cytoplasm of cells which otherwise is recognized by the immune sensor cgas. defects in trex have been associated with sle and with familial chilblain lupus ; furthermore, genetic defects in trex can cause retinal vasculopathy with cerebral leukodystrophy . trex -deficient mice develop severe autoimmunity (gall et al., ; morita et al., ) . the pathology is fully rescued by additional genetic defects in cgas gao et al., ; gray, treuting, woodward, & stetson, ) or the type i ifn system demonstrating the critical pathogenic role of the ifn response triggered by endogenous dna species. the source and identity of this dna remain controversial but may derive from endogenous retroelements (beck-engeser, eilat, & wabl, ; stetson, ko, heidmann, & medzhitov, ) . alternatively, dna ligands originating during chromosomal replication have been proposed (yang, lindahl, & barnes, ) . rnai is considered one of the major mechanism for sequencespecific detection and elimination of rna genome-based viruses in plants and invertebrates (szittya & burgyan, ; zhou & rana, ) . besides its antiviral function, rnai regulates gene expression in many organisms. by suppressing transcription or translation or by targeted degradation of mrna, it controls many cellular developmental and physiological processes (burger & gullerova, ) . rnai is initiated by rnase iii family nucleases (nuclear drosha and cytosolic dicer) that cleave endogenous or exogenous double-stranded rna to finally yield short - bp exogenous sirna or endogenous mirna (bernstein, caudy, hammond, & hannon, ; elbashir, lendeckel, & tuschl, ; lee et al., ) . the mirnas/sirnas are then integrated in the rna-induced silencing complex (risc) which target complementary rna for degradation or inhibition of translation (iwakawa & tomari, ) . ago family proteins in the risc complex determine its effector function: perfectly matched mi/sirnas mediate direct target cleavage by ago , while imperfectly matched mi/sirnas inhibit translation of target mrnas by ago , , or and recruit additional effector proteins which in turn can degrade target rna (doench, petersen, & sharp, ; meister et al., ) . while an important role for rnai for the antiviral responses in helminths, insects, and plants is well established, the contribution to antiviral immunity in vertebrates is under debate. evidence accumulates for an antiviral role of rnai in mammalian cells (gantier, ) , specifically if the otherwise dominating dicer-related rig-i-like helicases are inhibited. the major obstacle is that the contribution of a sirna-mediated antiviral response cannot be studied by the knockout of dicer which is lethal at early stages of mouse embryo development (bernstein et al., ) . it was reported that type i ifn-dominated innate immune responses suppress rnai and vice versa (seo et al., ) . the absence of dicer products from small rna libraries of (+)ssrna virus (yfv, hcv) infections strengthens this assumption (pfeffer et al., ) . however, more recent studies using the more sensitive next-generation sequencing indeed provide evidence for the generation of short virus-derived small rnas (vsrnas) in complex with ago proteins and conforming to dicer cleavage fragments of - bp (sirna and pirna) in vertebrate infection systems (parameswaran et al., ) . in a seminal work, suckling mice were infected by nodamura virus (nov) or a mutant virus lacking the nov virus-encoded suppressor of rnai, b (li, lu, han, fan, & ding, ) . nov is a mosquitotransmissible (+)ssrna virus, which is highly virulent to suckling mice and suckling hamsters. loss of b leads to abundant occurrence of viral sirnas and rendered mice completely resistant to nov titers which are lethal in the presence of b (li et al., ) . since b appears not to prevent recognition by rig-i (fan, dong, li, ding, & wang, ) , an important innate immune sensor of (+)ssrna-based viruses, the data suggest a strong role of virus rna-specific rnai during nov infection. still, an impact of b on endogenous mirna networks or inhibition of other dsrnasensing receptors (mda , pkr, oas) as a major cause of lethality is not completely excluded in this work and requires further analysis. nevertheless, this study adds to the concept that virus-encoded suppressors of rnai mask the actual role of rnai in antiviral defense of vertebrates (va noncoding rna, influenza ns , vaccinia virus e l, ebola virus vp , primate foamy virus tas, hiv- tat west nile virus sfrna) (bennasser, le, benkirane, & jeang, ; haasnoot et al., ; lecellier et al., ; li et al., ; lu & cullen, ; schnettler et al., ; svoboda, ) . importantly, murine or rat oocytes or embryonic stem cells in rat and mouse express a shortened form of dicer (dicero) with enhanced cleavage activity for long dsrna which complicate the interpretation of results (flemr et al., ) . on the other hand, the finding that poly(i:c) stimulation or infection with dna or rna viruses leads to parp induced poly-adp-ribosylation of ago which induces ago degradation in all tested cells strongly indicates a competition rather than a cooperation between antiviral rnai and nucleic acid immune-sensing pathways (seo et al., ) . although some recent work propose a role of rnai in the antiviral defense of vertebrates as described earlier, the current concept is that immune-sensing receptors represent the major antiviral defense strategy in vertebrates. immune-sensing receptors recognize characteristic features of foreign and invading nucleic acids such as unusual localization, specific structural elements, and modifications. stimulation of nucleic acid-sensing receptors results in the induction of cytokines (e.g., type i interferons) and chemokines to alarm neighboring cells, in the recruitment of immune cells, in the activation of cell autonomous mechanisms interfering with virus assembly and protein translation, or in the induction of several types of cell death including apoptosis, necroptosis, and pyroptosis. this chapter provides an overview of the most important immune-sensing receptors of nucleic acids for which robust evidence exists regarding the molecular mechanism of detection, the structural aspects of receptor ligand interaction, and the downstream signaling pathways (fig. ). tlr detects long double-stranded rna (alexopoulou et al., ) , and unlike other nucleic acid-sensing tlrs, besides its endolysosomal localization, it is also expressed on the surface of certain cell types (matsumoto, kikkawa, kohase, miyake, & seya, ; pohar, pirher, bencina, mancek-keber, & jerala, ) . tlr detects dsrna longer than bp. the ectodomains of two tlr molecules bind one dsrna molecule in a way that the cytoplasmic c-terminal signaling domains are juxtaposed to each other resulting in downstream signaling (liu et al., ) . tlr interacts with the ribose-phosphate backbone of dsrna and has no specific sequence requirements. given the absence of long dsrna under physiological conditions, tlr should be inactive in the absence of an infection. still, a number of studies proposed recognition of endogenous dsrna by tlr in situations of sterile tissue damage, but the specific ligand is not well defined. the identification of ligand specificities of tlr and tlr has been hampered by their mutually exclusive expression in different cell types and by considerable differences between mouse and human. tlr and tlr are examples where distinct function of nucleic acid-sensing tlrs is determined by their differential expression in immune cell subsets. while the expression of tlr in the human immune system is almost restricted to b cells and pdc, tlr is preferentially expressed in myeloid immune cells. consequently, tlr ligands drive b cell activation and the production of large amounts of ifn-alpha in pdc, while tlr induces the secretion of high amounts of il- p in myeloid immune cells. fig. immune-sensing receptors-detecting foreign nucleic acids and inducing indirect effector responses. this graph provides an overview of immune-sensing receptors of nucleic acids. tlr is the only one which besides its endosomal localization is also reported to be expressed on the cell membrane. tlr binds long double-stranded rna which is not present in the cytosol of normal cells and is an indicator of foreign. tlr is expressed in myeloid immune cells and in a number of somatic cells including fibroblasts and endothelial cells. the other three tlrs expressed in the endolysosomal compartment of distinct immune cell subsets are tlr , tlr , and tlr . tlr detects even short rna, preferentially double-stranded and containing g and u. tlr detects single-stranded rna. while tlr is expressed in human myeloid immune cells, tlr and tlr are predominantly expressed in human b cells and plasmacytoid dendritic cells. tlr detects single-stranded dna containing unmethylated cpg dinucleotides. in the cytoplasm, rig-i specifically detects rna if it contains at least a short double strand with a blunt end and a -triphosphate. the rig-i-like receptor mda detects long irregular forms of double-stranded rna, but the exact definition of the ligand structure is unclear. both rig-i and mda are widely expressed in immune cells and nonimmune cells, and induce a broad array of cell autonomous and extracellular antiviral responses including the production of type i interferon. mda ligands also activate multiple other receptor pathways that depend on the detection of long double-stranded rna, including pkr, adar , and tlr . the cytosolic receptor aim detects long double-stranded dna and activates the inflammasome. the other key receptor for the detection of dna in the cytoplasm is cgas. cgas is activated by long double-stranded dna and short forms of double-stranded dna with single-stranded overhangs containing gs, a structure which was termed y-form dna and which is presented during retroviral infection or by endogenous retroelements. upon activation, cgas catalyzes the formation of - -cgamp from gtp and atp. - -cgamp acts as a second messenger which binds to the downstream signaling protein sting which induces type i interferon via tbk and irf . - -cgamp can travel to and alarm neighboring cells via gap junctions. sting also activates nf-κb activation and inflammatory cytokines. tlr and tlr are preferentially activated by polyu or by g-and u-rich sequences (diebold et al., ; heil et al., ; hornung et al., ; judge et al., ) . however, confounding factors need to be considered while interpreting these results (forsbach et al., ) . furthermore, it has been demonstrated that tlr selectively detects ssrna, while tlr primarily detects short stretches of dsrna but can also accommodate certain ssrna oligonucleotides sioud, ) . however, since neither polyu or g-and u-rich sequences nor ssrna or short dsrna structures are overrepresented in microbial or viral rna, the distinction of self vs nonself by tlr and tlr requires additional information. in fact, endogenous rnas are posttranscriptionally modified at their nucleobases and backbone. the current concept is that the addition of certain modifications to self rna inside the nucleus provides a signature for self. one example is -o-methylation which is a common nuclear modification of rna performed by a specific mtase located in the nucleolus. the mtase adds a methyl group to the -hydroxyl group of the ribose. this modification represents a marker of self in higher eukaryotic cells and prevents the recognition of endogenous rna by tlr and tlr judge et al., ; kariko, buckstein, ni, & weissman, ) . other modifications of rna molecules which potently inhibit the detection of transfer rna and ribosomal rna by tlr and tlr are the incorporation of pseudouridine (Ψ), -methylcytidine (m c), -thio-uridine (s u), or n -methyladenosine (m a) (kariko et al., ) . the presence of such modifications in part explains the lack of immunostimulation of host-derived rna vs microbial rna (gehrig et al., ; jockel et al., ) . however, endogenous rna from apoptotic or dying cells still activates tlr and tlr once entering the endolysosomal compartment (busconi et al., ; ganguly et al., ; vollmer et al., ) . thus, additional factors such as intracellular localization and degradation by nucleases likely support a faithful discrimination of self from nonself by tlr and tlr . it is interesting to note that there is an obvious need to sense and eliminate certain endogenous rnas as well. in this context, it has been reported that the loss of tlr function causes retroviral viremia (yu et al., ) indicating that endogenous rnas transcribed from rna polymerase ii promoters are not generally excluded from tlr-mediated recognition. tlr senses dna in the endolysosomal compartment of certain immune cells (hemmi et al., ) . like tlr and tlr , tlr travels to the endolysosomal compartment via the chaperone protein unc b (protein unc- homolog b ) (latz et al., ; pelka, shibata, miyake, & latz, ) . there, tlr is proteolytically processed at a defined protruding loop structure without disrupting the horseshoe shape of the protomer (bauer, ; onji et al., ; peter, kubarenko, weber, & dalpke, ) . cleavage is necessary for the activation of tlr signaling (ewald et al., ; park et al., ) . tlr preferentially detects dna with unmethylated (no methylation at the c carbon of cytosine) cpg dinucleotides (cpg dna) with a preference for certain sequence contexts (hexamer cpg motifs, in humans -gtcgtt- ) which vary between species and which altogether are less frequent in eukaryotic self dna hartmann, weiner, & krieg, ; hemmi et al., ; krieg et al., ) . activation of tlr signaling is preceded by dimer formation where two cpg dna molecules symmetrically bind two tlr molecules (ohto et al., ) . both cpg dna molecules bind to the c-terminal fragment of one protomer and the cpg-binding groove in the n-terminal fragment of the other. inhibitory dna oligonucleotides only bind to the n-terminal fragment. methylated single-stranded cpg dna and double-stranded dna exhibit lower binding to tlr and are less potent to induce tlr dimer formation. of note, digestion of dna molecules by the lysosomal endonuclease dnase ii creates short tlr stimulatory dna fragments (chan et al., ; pawaria et al., ) . notably, specificity for unmethylated cpg motifs is reduced if the cpg motif is within a phosphorothioate-modified dna often used to stabilize oligodeoxynucleotides against dnases. nevertheless, a high degree of specificity is well established for unmethylated cpg motif containing dna within a natural phosphodiester backbone including microbial dna hartmann & krieg, ) . it is specifically noteworthy that genomic microbial dna displays a much stronger activity to stimulate tlr as compared to genomic dna of vertebrates. although eukaryotic dna presents with a lower frequency of nonmethylated cpg motifs than microbial dna, this difference in frequency of unmethylated cpg motifs does not allow a clear cut distinction of self from nonself on a structural basis. endogenous dna at high concentrations can activate tlr once delivered into the endolysosome (marshak-rothstein, ) . it is also of great importance to be aware of the differences of tlr mediated dna recognition between species. in humans, tlr is almost exclusively expressed in b cells and pdc (hornung et al., ) , while in mice, tlr is expressed more widely including myeloid immune cells. in humans, tlr predominantly induces type ifn production in pdcs and polyclonal activation in b cells via myd /irf -dependent signaling. species-specific expression patterns of tlr are responsible for the fundamental functional differences of tlr in mouse and man. another important issue is that in preparations of human immune cell subsets, minute amounts of pdc indirectly activate other immune cell subsets such as monocytes, myeloid dendritic cells, or nk cells. this needs to be carefully considered when direct effects of tlr activation in immune cells other than b cells and pdc are claimed, such as direct tlr effects in human myeloid immune cells and nk cells. three different classes of cpg oligonucleotides have been described, cpg-a, cpg-b, and cpg-c (avalos et al., ; hartmann et al., ; kerkmann et al., ; krug et al., ; rothenfusser et al., ) . based on the palindromic structure, cpg-a spontaneously forms nanoparticle-like complexes (kerkmann et al., ) that explain much higher type i ifn-inducing capacity in pdc as compared to cpg-b which are monomeric. monomeric cpg-b potently activates b cells which do not internalize larger particles of dna as with cpg-a complexes. cpg-c potently stimulates both b cells and pdcs. in cell culture, delayed tlr activation due to slower uptake of cpg-a nanoparticles allows a longer self-priming of pdc by minute amounts of spontaneously released type i ifn. priming of pdc results in higher ifn-inducing activity of cpg-a seen in cell culture . rig-i belongs to the cytosolic dexd/h box rna helicases and is one of three members of the so-called family of rig-i-like helicases (others: mda and lgp ). rig-i is closely related to the dicer family of helicases of the rnai pathway. rig-i contains a rna helicase domain and a two n-terminal card domains which relay the signal to the downstream signaling adaptor mavs (mitochondrial antiviral-signaling protein). rig-i signaling via mavs not only leads to the induction of type i ifn responses via tbk and irf / , it also activates caspase- -dependent apoptosis, preferentially in tumor cells (besch et al., ; el maadidi et al., ; glas et al., ; kumar et al., ) . furthermore, rig-i was also found to mediate mavs-independent inflammasome activation (poeck et al., ) , specifically in the context of viral infection (poeck et al., ; pothlichet et al., ) . rig-i detects blunt ends of double-stranded rna containing a -triphosphate or a -diphosphate (goubau et al., ; hornung et al., ; marq, hausmann, veillard, kolakofsky, & garcin, ; pichlmair et al., ; schlee, roth, et al., ) . such rna ligands are presented, for example, by negative-strand rna viruses which form panhandle structures with their matching -and -ends of the single-stranded genomic rna (rehwinkel et al., ; schlee, roth, et al., ) . while crystal structures confirmed the structural requirements as determined in functional studies (civril et al., ; jiang et al., ; kowalinski et al., ; luo et al., ; wang et al., ) , the minimum length of the double-strand required for rig-i activation is still controversial. while approaches with synthetic or highly purified enzymatic double-stranded -triphosphate rna revealed a minimum length of - bp (marq, hausmann, et al., ; schlee, hartmann, et al., ) , bp were demonstrated to be sufficient for a hairpin forming oligonucleotide (kohlway, luo, rawling, ding, & pyle, ) . however, alternatively to the predicted hairpin, these oligonucleotides may form mer duplexes when entering the cell. although oligomerization of card modules of each rig-i protein along the rig-i filament bound to a longer double-stranded rna molecule induces robust rig-i signaling, the minimal signaling unit is sufficient for rig-i to trigger signal transduction. in the latter case, a card tetramer is stabilized by ubiquitin chains . furthermore, it is interesting to note that rig-i mutants deficient in atp hydrolysis of their helicase domain cannot detach from suboptimal endogenous rna ligands leading to erroneous signaling which can cause autoimmunity (lassig et al., ) . importantly, n -methylation ( -o-methylation at the first nucleotide of capped rna) serves as a signature of self rna and completely abrogates rig-i sensing of rna, while in the absence of n methylation, rig-i binding is hardly impaired by the ppp -linked m g cap structure itself (schuberth-wagner et al., ) . rig-i is ubiquitously expressed in all cell types including tumor cells. however, the type of rig-i induced responses differs between cells. while normal healthy cells such as melanocytes and fibroblasts are quite resistant to rig-i-induced apoptosis, tumor cells are highly susceptible to rig-iinduced cell death (besch et al., ; kubler et al., ) . based on this tumor selective activity and a favorable toxicity profile, rig-i-specific ligands are currently being developed for immunotherapy of cancer (duewell et al., , ellermeier et al., ; schnurr & duewell, . part of the potent antitumor activity of rig-i ligands is its ability to promote crosspresentation of antigens to cd t cells and to induce cytotoxic activity (hochheiser et al., ) . rig-i ligands show strong therapeutic activity in viral infection models such as influenza (weber-gerlach & weber, ) . notably, rig-i has also been shown to be involved in the detection of intracellular bacteria (abdullah et al., ) . rare genetic gain-of-function variants of rig-i have been associated with an atypical form of singleton merten syndrome (jang et al., ) . like rig-i, melanoma differentiation associated gene (mda ) is a cytosolic dexd/h box rna helicase which signals through mavs and irf / irf . despite its similar structure, mda senses a different type of ligand which has been described as higher order rna structures (pichlmair et al., ) . so far, a mda -specific ligand has not been described. double-stranded rna ligands activating mda are typically promiscuous ligands, such as poly(i:c) which also activates tlr and antiviral effector proteins which inhibit translation upon binding to doublestranded rna, such as pkr and oas. multiple effects of mda ligands cause a high degree of toxicity in vivo strictly limiting the clinical application of mda ligands. unlike rig-i which primarily binds to the ends of rna, mda proteins bind double-stranded rna internally, independently of its terminal structures. additional mda molecules then closely stack in a helical headto-tail arrangement around dsrna resulting in the formation of long mda filaments which initiate signaling toward activation of mavs (del toro duany, . lgp (laboratory of genetics and physiology ) is a third cytosolic rig-i-like helicase lacking card domains for signaling. lgp appears to contribute to the fine tuning of immune responses by inhibition of rig-i and supporting mda signaling venkataraman et al., ) . while mda contains the signaling card domains but has relatively weak binding to double-stranded rna, lgp readily detects diverse double-stranded rna species but lacks a signaling domain. the current concept is that lgp assists the interaction of mda with double-stranded rna and filament formation, thereby enhancing mda -mediated antiviral signaling (bruns & horvath, ; bruns, leser, lamb, & horvath, ) . notably, genetic gain-of-function variants of mda have been associated with autoimmune disorders (junt & barchet, ; kato & fujita, ) . the hin- (hematopoietic interferon-inducible nuclear proteins with a -amino acid repeat) family member aim (absent in melanoma ) binds and oligomerizes on cytoplasmic double-stranded dna through its c-terminal hin domain in a sequence-independent manner (fernandes-alnemri, yu, datta, wu, & alnemri, ; hornung et al., ; roberts et al., ) . dna binding of the hin domain relieves the autoinhibitory conformation of aim and allows the n-terminal pyrin domain of multiple aim proteins to form a helical structure which nucleates the helical assembly of asc (apoptosis-associated speck-like protein containing a card) filaments (lu, kabaleeswaran, fu, magupalli, & wu, ; lu et al., ) thereby forming an inflammasome that results in the release of il- beta. the formation of an aim inflammasome requires a minimal length of double-stranded dna of - bp (jin et al., ) . the cytosolic immune-sensing receptor cgas (cai, chiu, & chen, ; wu et al., ) detects long double-stranded dna (dsdna) or short dsdna with unpaired open ends containing guanosines (y-form dna) as, for example, presented in highly structured single-stranded dna of retroviruses or certain endogenous retroelements . it is important to note that trex has a gate keeper function for cgas. usually, cytosolic dna is efficiently degraded by trex . only in the case of excess cytosolic dna, or dna modifications rendering dna resistant to trex mediated degradation, dna gains access to cgas resulting in downstream signaling. along these lines, it has been reported that oxidized dna (e.g., -hydroxyguanosine, -ohg) as occurring in the context of uv radiation or upon exposure to reactive oxygen species resists trex -mediated degradation (gehrke et al., ) . this results in an accumulation of dna in the cytosol. oxidized dna has the same affinity to cgas than nonoxidized dna. cgas is monomeric in its unligated state. however, two cgas molecules bind to two dsdna molecules in a way that each cgas protomer presents an additional interaction site with the dna bound to the other cgas protein. upon activation by cytosolic dna, cgas catalyzes the formation of - -cgamp from gtp and atp (ablasser, goldeck, et al., ; gao, ascano, wu, et al., ) . - -cgamp acts as a second messenger which binds to the downstream signaling protein sting (gao, ascano, zillinger, et al., ) which induces type i interferon via tbk and irf . - -cgamp can travel to and alarm neighboring cells via gap junctions (ablasser, schmid-burgk, et al., ) . sting activation is also associated with nf-κb activation and prominent induction of inflammatory cytokines such as il- and tnf-a. the general view is that most of the relevant immune receptors, effector proteins, and pathways participating in nucleic acid immunity have now been identified. these different players have in common that they all serve the function to detect and to disable foreign nucleic acids. altogether they constitute the system of nucleic acid immunity. all of these pathways are relevant for human disease, either as part of the human antiviral defense system, or indirectly by being active in pathogens or pathogen-transmitting organisms. examples are arboviruses (zika, dengue, yellow-fever, west nile) which are transmitted by arthropod vectors. successful arboviruses need to escape both rnai in insects and immunoreceptors such as rig-i in humans. only if they manage to inhibit both pathways, they can establish as pathogens. therefore, it will be interesting to understand the molecular evolution of escape strategies in emerging viruses such as zika, which may lead to the identification of the molecular step that allowed the virus to spread more efficiently (rasmussen & katze, ) . another example is the important role of rnai in pathogens such as filariae and other human pathogenic helminths. there, rnai may be useful as a therapeutic target. another example is the presence of endosymbionts such as wolbachia, a genus of bacteria, in filarial nematodes (taylor, bandi, & hoerauf, ) . the release of wolbachia nucleic acids may contribute to the pathogenesis of filarial infection. furthermore, crispr/cas is involved in the evolution of pathogenic bacteria. nucleic acid sensing in vertebrates is required for antimicrobial immunity and is involved in the pathogenesis of many inflammatory diseases. although much is known about the structure and the function of the single pathways, the functional interaction of the different pathways is far from being understood. distinct expression patterns of the receptors in different cell types and cell-type-dependent differences in the expression of downstream signaling components and transcription factors contribute to the complexity of nucleic acid immunity. as a result, the same type of ligand can have different functional outcomes in different cell types. furthermore, since different receptor pathways are activated by the same type of ligand, it is currently unclear whether competition of receptor binding or distinct molecular trafficking to the corresponding receptors or both impact on the functional outcome of ligand exposure. for example, long doublestranded dna in principle binds to both aim and cgas, and functional cooperation or inhibition of the respective pathways are unclear. now since most of the individual molecular pathways of nucleic acid immunity are on the table, we see ourselves just at the dawn of an exciting new research field which is expected to advance medicine specifically in the areas of infection and inflammation and with broad implication for human diseases. this work was supported by the dfg-funded excellence cluster immunosensation, and the helmholtz-funded german center for infection research (deutsches zentrum f€ ur infektionsforschung, dzif). structural basis for viral '-ppp-rna recognition by human ifit proteins rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate cgas produces a '- '-linked cyclic dinucleotide second messenger that activates sting trex deficiency triggers cell-autonomous immunity in a cgas-dependent manner cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp sting manifests self dnadependent inflammatory disease recognition of double-stranded rna and activation of nf-kappab by toll-like receptor loss-of-function variant in dnase l causes a familial form of systemic lupus erythematosus differential cytokine production and bystander activation of autoreactive b cells in response to cpg-a and cpg-b oligonucleotides interferon action may be mediated by activation of a nuclease by pppa 'p 'a 'p 'a samhd restricts hiv- infection in resting cd (+) t cells human deoxyribonucleases sting-dependent cytosolic dna sensing pathways crispr provides acquired resistance against viruses in prokaryotes a developmentally regulated activity that unwinds rna duplexes an unwinding activity that covalently modifies its double-stranded rna substrate toll-like receptor processing: the key event in toll-like receptor activation? cutting edge: aim and endosomal tlrs differentially regulate arthritis and autoantibody production in dnase ii-deficient mice an autoimmune disease prevented by anti-retroviral drugs mouse samhd has antiretroviral activity and suppresses a spontaneous cell-intrinsic antiviral response nuclease activity of the human samhd protein implicated in the aicardi-goutieres syndrome and hiv- restriction evidence that hiv- encodes an sirna and a suppressor of rna silencing role for a bidentate ribonuclease in the initiation step of rna interference dicer is essential for mouse development proapoptotic signaling induced by rig-i and mda- results in type i interferonindependent apoptosis in human melanoma cells rnase-l control of cellular mrnas: roles in biologic functions and mechanisms of substrate targeting lgp synergy with mda in rlr-mediated rna recognition and antiviral signaling the innate immune sensor lgp activates antiviral signaling by regulating mda -rna interaction and filament assembly swiss army knives: non-canonical functions of nuclear drosha and dicer dna and rna autoantigens as autoadjuvants the cgas-cgamp-sting pathway of cytosolic dna sensing and signaling the role of '- ' oligoadenylate-activated ribonuclease l in apoptosis ribonuclease h: the enzymes in eukaryotes dnase ii-dependent dna digestion is required for dna sensing by tlr contributions of the two accessory subunits, rnaseh b and rnaseh c, to the activity and properties of the human rnase h complex jnk and ikkbeta are required for activating the innate response to viral infection the rig-i atpase domain structure reveals insights into atp-dependent antiviral signalling the double-stranded rna-dependent protein kinase pkr: structure and function inhibition of cell-free protein synthesis by pppa 'p 'a 'p 'a: a novel oligonucleotide synthesized by interferon-treated l cell extracts higher activation of tlr in plasmacytoid dendritic cells by microbial dna compared with self-dna based on cpg-specific recognition of phosphodiester dna mutations in the gene encoding the '- ' dna exonuclease trex cause aicardi-goutieres syndrome at the ags locus aicardi-goutieres syndrome and related phenotypes: linking nucleic acid metabolism with autoimmunity '-o methylation of the viral mrna cap evades host restriction by ifit family members mda -filament, dynamics and disease. current opinion in virology innate antiviral responses by means of tlr -mediated recognition of single-stranded rna sirnas can function as mirnas a bipartite model of - a-dependent rnase l targeted activation of melanoma differentiation-associated protein (mda ) for immunotherapy of pancreatic carcinoma rig-i-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by cd (+) t cells a novel mitochondrial mavs/caspase- platform links rna virus-induced innate antiviral signaling to bax/bak-independent apoptosis rna interference is mediated by -and -nucleotide rnas therapeutic efficacy of bifunctional sirna combining tgf-beta silencing with rig-i activation in pancreatic cancer dnase ii: genes, enzymes and function the ectodomain of toll-like receptor is cleaved to generate a functional receptor rig-i-dependent antiviral immunity is effective against an rna virus encoding a potent suppressor of rnai interferon action: two distinct pathways for inhibition of protein synthesis by doublestranded rna identification of doublestranded rna-binding domains in the interferon-induced double-stranded rnaactivated p kinase aim activates the inflammasome and cell death in response to cytoplasmic dna a retrotransposon-driven dicer isoform directs endogenous small interfering rna production in mouse oocytes identification of rna sequence motifs stimulating sequence-specific tlr -dependent immune responses autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease self-rna-antimicrobial peptide complexes activate human dendritic cells through tlr and tlr processing of double-stranded rna in mammalian cells: a direct antiviral role cyclic [g( ', ')pa( ', ')p] is the metazoan second messenger produced by dna-activated cyclic gmp-amp synthase structure-function analysis of sting activation by c[g( ', ')pa( ', ')p] and targeting by antiviral dmxaa activation of cyclic gmp-amp synthase by self-dna causes autoimmune diseases impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action identification of modifications in microbial, native trna that suppress immunostimulatory activity oxidative damage of dna confers resistance to cytosolic nuclease trex degradation and potentiates sting-dependent immune sensing samhd restricts hiv- infection in dendritic cells (dcs) by dntp depletion, but its expression in dcs and primary cd + t-lymphocytes cannot be upregulated by interferons an rna editor, adenosine deaminase acting on double-stranded rna (adar ) targeting the cytosolic innate immune receptors rig-i and mda effectively counteracts cancer cell heterogeneity in glioblastoma brex is a novel phage resistance system widespread in microbial genomes hiv- restriction factor samhd is a deoxynucleoside triphosphate triphosphohydrolase antiviral immunity via rig-i-mediated recognition of rna bearing '-diphosphates restriction of diverse retroviruses by samhd cutting edge: cgas is required for lethal autoimmune disease in the trex -deficient mouse model of aicardigoutieres syndrome the ebola virus vp protein is a suppressor of rna silencing sequestration by ifit impairs translation of 'o-unmethylated capped rna impairment of neutrophil extracellular trap degradation is associated with lupus nephritis structure of human rnase l reveals the basis for regulated rna decay in the ifn response apobecs and virus restriction adenovirus infection triggers a rapid, myd -regulated transcriptome response critical to acute-phase and adaptive immune responses in vivo rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells cpg dna and lps induce distinct patterns of activation in human monocytes mechanism and function of a newly identified cpg dna motif in human primary b cells delineation of a cpg phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo cpg dna: a potent signal for growth, activation, and maturation of human dendritic cells the protein kinase pkr is critical for lps-induced inos production but dispensable for inflammasome activation in macrophages peritumoral cpg dna elicits a coordinated response of cd t cells and innate effectors to cure established tumors in a murine colon carcinoma model species-specific recognition of single-stranded rna via toll-like receptor and a tolllike receptor recognizes bacterial dna sequence-specific activation of the dna sensor cgas by y-form dna structures as found in primary hiv- cdna cutting edge: the rig-i ligand prna potently improves ctl cross-priming and facilitates antiviral vaccination rna editing by mammalian adars host factor samhd restricts dna viruses in non-dividing myeloid cells aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc '-triphosphate rna is the ligand for rig-i sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr oas proteins and cgas: unifying concepts in sensing and responding to cytosolic nucleic acids intracellular dna recognition quantitative expression of toll-like receptor - mrna in cellular subsets of human peripheral blood mononuclear cells and sensitivity to cpg oligodeoxynucleotides a human dna editing enzyme homologous to the escherichia coli dnaq/mutd protein synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells vpx relieves inhibition of hiv- infection of macrophages mediated by the samhd protein dimeric structure of pseudokinase rnase l bound to - a reveals a basis for interferon-induced antiviral activity innate immune restriction and antagonism of viral rna lacking -o methylation particle counts and infectivity titrations for animal viruses foreign nucleic acids as the stimulus to make interferon the functions of micrornas: mrna decay and translational repression aim drives joint inflammation in a self-dna triggered model of chronic polyarthritis mutations in ddx , which encodes rig-i, cause atypical singleton-merten syndrome rnase h of saccharomyces cerevisiae is a complex of three proteins structural basis of rna recognition and activation by innate immune receptor rig-i structures of the hin domain:dna complexes reveal ligand binding and activation mechanisms of the aim inflammasome and ifi receptor the '-o-methylation status of a single guanosine controls transfer rna-mediated tolllike receptor activation or inhibition sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna translating nucleic acid-sensing pathways into therapies suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna rig-i-like receptors and autoimmune diseases cell type-specific involvement of rig-i in antiviral response chronic polyarthritis caused by mammalian dna that escapes from degradation in macrophages spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by cpg-a in plasmacytoid dendritic cells activation with cpg-a and cpg-b oligonucleotides reveals two distinct regulatory pathways of type i ifn synthesis in human plasmacytoid dendritic cells self-priming determines high type i ifn production by plasmacytoid dendritic cells tight interplay among samhd protein level, cellular dntp levels, and hiv- proviral dna synthesis kinetics in human primary monocyte-derived macrophages molecular cloning of cdna for double-stranded rna adenosine deaminase, a candidate enzyme for nuclear rna editing samhd restricts herpes simplex virus in macrophages by limiting dna replication defining the functional determinants for rna surveillance by rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna cpg motifs in bacterial dna trigger direct b-cell activation cpg-a oligonucleotides induce a monocyte-derived dendritic cell-like phenotype that preferentially activates cd t cells targeted activation of rna helicase retinoic acid-inducible gene-i induces proimmunogenic apoptosis of human ovarian cancer cells double-stranded rna-dependent protein kinase activates transcription factor nf-kappa b by phosphorylating i kappa b ips- differentially induces trail, bcl , birc and prkce in type i interferonsdependent and -independent anticancer activity samhd is the dendritic-and myeloid-cell-specific hiv- restriction factor counteracted by vpx samhd restricts the replication of human immunodeficiency virus type by depleting the intracellular pool of deoxynucleoside triphosphates dnase a deficiency uncovers lysosomal clearance of damaged nuclear dna via autophagy atp hydrolysis by the viral rna sensor rig-i prevents unintentional recognition of self-rna tlr signals after translocating from the er to cpg dna in the lysosome a cellular microrna mediates antiviral defense in human cells the nuclear rnase iii drosha initiates microrna processing a mutation in trex that impairs susceptibility to granzyme a-mediated cell death underlies familial chilblain lupus mutations in the gene encoding the '- ' dna exonuclease trex are associated with systemic lupus erythematosus regulation of protein synthesis: activation by doublestranded rna of a protein kinase that phosphorylates eukaryotic initiation factor . proceedings of the national academy of sciences of the united states of america interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing rna interference functions as an antiviral immunity mechanism in mammals identification of human homologue of mouse ifngamma induced protein from human dendritic cells rna editing by adar prevents mda sensing of endogenous dsrna as nonself studies on the production, mode of action and properties of interferon structural basis of toll-like receptor signaling with double-stranded rna adenovirus va noncoding rna can inhibit small interfering rna and microrna biogenesis crystal structure of the f g aim pyd mutant and similarities of its self-association to ded/ded interactions plasticity in pyd assembly revealed by cryo-em structure of the pyd filament of aim unified polymerization mechanism for the assembly of asc-dependent inflammasomes novel role of pkr in inflammasome activation and hmgb release structural insights into rna recognition by rig-i a nonhereditary, host-induced variation of bacterial viruses ribonuclease h mutations induce a cgas/sting-dependent innate immune response potential role of pkr in double-stranded rna-induced macrophage activation cytosolic rna:dna hybrids activate the cgas-sting axis short doublestranded rnas with an overhanging ' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys unpaired ' ppp-nucleotides, as found in arenavirus double-stranded rna panhandles, are not recognized by rig-i crispr interference: rna-directed adaptive immunity in bacteria and archaea tolling for autoimmunity-prime time for establishment of a monoclonal antibody against human toll-like receptor that blocks double-stranded rna-mediated signaling protein kinase pkr amplification of interferon beta induction occurs through initiation factor eif- alpha-mediated translational control human argonaute mediates rna cleavage targeted by mirnas and sirnas differential regulation of the oasl and oas genes in response to viral infections genetargeted mice lacking the trex (dnase iii) '-> ' dna exonuclease develop inflammatory myocarditis '-triphosphate-dependent activation of pkr by rnas with short stem-loops features of systemic lupus erythematosus in dnase -deficient mice murine serum nucleases-contrasting effects of plasmin and heparin on the activities of dnase and dnase -like (dnase l ) restriction endonucleases in the analysis and restructuring of dna molecules a-to-i editing of coding and non-coding rnas by adars structural basis of cpg and inhibitory dna recognition by toll-like receptor mitochondrial dna that escapes from autophagy causes inflammation and heart failure an essential role for the n-terminal fragment of toll-like receptor in dna sensing adar forms a complex with dicer to promote microrna processing and rnainduced gene silencing six rna viruses and forty-one hosts: viral small rnas and modulation of small rna repertoires in vertebrate and invertebrate systems proteolytic cleavage in an endolysosomal compartment is required for activation of tolllike receptor p regulates the hiv- restriction factor samhd . proceedings of the national academy of sciences of the united states of america cutting edge: dnase ii deficiency prevents activation of autoreactive b cells by doublestranded dna endogenous ligands nucleic acid-sensing tlrs and autoimmunity: novel insights from structural and cell biology identification of an n-terminal recognition site in tlr that contributes to cpg-dna-mediated receptor activation identification of micrornas of the herpesvirus family ifit is an antiviral protein that recognizes '-triphosphate rna rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates activation of mda requires higher-order rna structures generated during virus infection recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production the role of unc b protein in surface localization of tlr receptor and in cell priming to nucleic acid agonists rnase h catalytic core aicardi-goutieres syndrome-related mutant invokes cgas-sting innate immune-sensing pathway in mice type i ifn triggers rig-i/tlr /nlrp -dependent inflammasome activation in influenza a virus infected cells aicardi-goutieres syndrome gene and hiv- restriction factor samhd is a dgtp-regulated deoxynucleotide triphosphohydrolase genomic signatures of emerging viruses: a new era of systems epidemiology the human ', '-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties samhd -dependent retroviral control and escape in mice rig-i detects viral genomic rna during negative-strand rna virus infection mutations involved in aicardi-goutieres syndrome implicate samhd as regulator of the innate immune response mutations in adar cause aicardi-goutieres syndrome associated with a type i interferon signature clinical and molecular phenotype of aicardi-goutieres syndrome c-terminal truncations in human '- ' dna exonuclease trex cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy rna:dna hybrids are a novel molecular pattern sensed by tlr hin- proteins regulate caspase activation in response to foreign cytoplasmic dna the rna helicase lgp inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i cpg-a and cpg-b oligonucleotides differentially enhance human peptidespecific primary and memory cd + t-cell responses in vitro the ribonuclease activity of samhd is required for hiv- restriction trashing the genome: the role of nucleases during apoptosis approaching the rna ligand for rig-i? recognition of ' triphosphate by rig-i helicase requires short blunt doublestranded rna as contained in panhandle of negative-strand virus noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells breaking tumor-induced immunosuppression with '-triphosphate sirna silencing tgfbeta and activating rig-i. oncoimmunology, . e induction of immunogenic cell death by targeting rig-i-like helicases in pancreatic cancer a diverse range of gene products are effectors of the type i interferon antiviral response a conserved histidine in the rna sensor rig-i controls immune tolerance to n - 'o-methylated self rna characteristics of a double-stranded rna-activated protein kinase system partially purified from interferon treated ehrlich ascites tumor cells reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells viral encounters with ', '-oligoadenylate synthetase and rnase l during the interferon antiviral response intrinsic host restrictions to hiv- and mechanisms of viral escape single-stranded small interfering rna are more immunostimulatory than their double-stranded counterparts: a central role for '-hydroxyl uridines in immune responses enzyme from calf thymus degrading the rna moiety of dna-rna hybrids: effect on dna-dependent rna polymerase trex prevents cellintrinsic initiation of autoimmunity renaissance of mammalian endogenous rnai rna interference-mediated intrinsic antiviral immunity in plants wolbachia bacterial endosymbionts of filarial nematodes mechanism of interferon action: cdna structure, expression, and regulation of the interferon-induced, rna-dependent p /eif- alpha protein kinase from human cells mechanism of interferon action: autoregulation of rna-dependent p /eif- alpha protein kinase (pkr) expression in transfected mammalian cells adars and the balance game between virus infection and innate immune cell response unravelling the structural and mechanistic basis of crispr-cas systems diverse functions of restriction-modification systems in addition to cellular defense loss of dexd/h box rna helicase lgp manifests disparate antiviral responses immune stimulation mediated by autoantigen binding sites within small nuclear rnas involves toll-like receptors and incorporation of phosphorothioate groups into fd and phi x dna a double-stranded rna unwinding activity introduces structural alterations by means of adenosine to inosine conversions in mammalian cells and xenopus eggs rna interference directs innate immunity against viruses in adult drosophila structural and functional insights into '-ppp rna pattern recognition by the innate immune receptor rig-i standing on three legs: antiviral activities of rig-i against influenza viruses coley's toxins, tumor necrosis factor and cancer research: a historical perspective cellular and biochemical mechanisms of the retroviral restriction factor samhd how rig-i like receptors activate mavs cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna in vitro augmentation of natural killer cell activity and production of interferon-alpha/beta and -gamma with deoxyribonucleic acid fraction from mycobacterium bovis bcg unique palindromic sequences in synthetic oligonucleotides are required to induce ifn [correction of inf] and augment ifn-mediated [correction of inf] natural killer activity dna from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth trex exonuclease degrades ssdna to prevent chronic checkpoint activation and autoimmune disease interferons, double-stranded rna, and rna degradation. isolation and characterization of homogeneous human ( '- ')(a)n synthetase molecular cloning of the cdna encoding human deoxyribonuclease ii mutation of dnase in people with systemic lupus erythematosus shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested dna nucleic acidsensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors rnai is an antiviral immune response against a dsrna virus in drosophila melanogaster rna-based mechanisms regulating host-virus interactions antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor isolation of two interferoninduced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase key: cord- -btbf wkg authors: hoffmann, h. j.; nielsen, l. p.; blumberga, g.; dahl, r. title: decrease in fine t‐cell subset ratio mt /mt during steroid reduction of asthmatic patients date: - - journal: scand j immunol doi: . /j. - . . ah.x sha: doc_id: cord_uid: btbf wkg combining inhaled long‐acting β‐ agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t‐cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd (+)cd ra(–)cd l(+)cd adim) and mt (cd (+)cd ra(–)cd l(–)cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) – µg daily or equivalent, were randomized to receive, double‐blinded, either seretide(®)[salmeterol and fluticasone propionate (sfc, n = )] µg/ µg bd or fp µg bd (n = ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or µg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p = from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p = from first to final visit). in patients receiving laba + ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -rhji io authors: popko, brian; corbin, joshua g.; baerwald, kristine d.; dupree, jeffrey; garcia, annie m. title: the effects of interferon-γ on the central nervous system date: journal: mol neurobiol doi: . /bf sha: doc_id: cord_uid: rhji io interferon-gamma (ifn-γ) is a pleotropic cytokine released by t-lymphocytes and natural killer cells. normally, these cells do not traverse the blood-brain barrier at appreciable levels and, as such, ifn-γ is generally undetectable within the central nervous system (cns). nevertheless, in response to cns infections, as well as during certain disorders in which the cns is affected, t-cell traffic across the blood-brain barrier increases considerably, thereby exposing neuronal and glial cells to the potent effects of ifn-γ. a large portion of this article is devoted to the substantial circumstantial and experimental evidence that suggests that ifn-γ plays an important role in the pathogenesis of the demyelinating disorder multiple sclerosis (ms) and its animal model experimental allergic encephalomyelitis (eae). moreover, the biochemical and physiological effects of ifn-γ are discussed in the context of the potential consequences of such activities on the developing and mature nervous systems. interferon-gamma (ifn-?) was originally discovered by wheelock ( ) as an activity that interfered with viral replication. tlymphocytes and natural killer cells are the only cells known to be capable of expressing this cytokine (reviewed in trinchieri and perussia, ) . because under normal condi-under these conditions, neuronal and glial cells that normally do not encounter ifn-',v are exposed to the potent, pleotropic effects of this cytokine. there is considerable evidence that suggests that the effects of ifn-?~ are important in a variety of cns disorders (reviewed in ransohoff and benveniste, ) . in this article, we discuss various molecular, biochemical, cellular, and physiological properties of ifn-~, and its evoked response. moreover, the potential role this cytokine plays in cns disorders is discussed. a considerable amount of information has been learned concerning the molecular biology and biochemistry of ifn-~, since the human and mouse cdnas were cloned over a decade ago (gray et al., ; goeddel, , ) . the human and mouse proteins are and amino acids in length, respectively, and are encoded for by four exons that reside on kb of dna. interestingly; the human and mouse proteins share only limited homology, approx % at the amino acid level, and do not bind to the other's receptor to an appreciable extent (reviewed by farrar and schreiber, ) . both proteins contain two glycosylation sites (gray and goeddel, ; ealick et al., ) , although glycosylation is not essential for ifn-'i activity (kelker et al., ) . one factor that may play a role in regulating the biological activity of ifn-y is message stability, since the ifn-y mrna contains an au rich sequence in the '-untranslated region that has been shown to reduce [he mr_na halfqife of other cytokines dramatically (shaw and kamen, ) . biologically active ifn-~/from both human and mouse is a noncovalently bound homodimer that contains two receptor binding sites . the ifn- receptor is a multimeric receptor complex composed of a ligand binding subunit (a-chain) (aguet et al., ) and a transmembrane, accessory factor (~-chain) (soh et al., ; hemmi et al., ) . the c~-chain is a ubiq-uitously expressed, glycosylated, cell-surface protein that binds ifn-~ , with high affinity (reviewed by farrar and schreiber, ) . the extent of glycosylation is variable depending on cell type and accounts for the range of sizes of the a-chain, between and kda. on binding, ifn- induces (r-chain dimerization, which is believed to be critical to the initiation of the signal transduction cascade (greenlund et al., ; fountoulakis et al., ) . this feature of the ifn- receptor has been exploited to generate mutant forms that confer ifn- , unresponsiveness to cells in a dominant manner (dighe et al., (dighe et al., , . the crystal structure of the complex between ifn-y and the a-chain of its receptor has recently been reported (walter et al., ) . the ~-chain of the receptor, which associates with the or-chain in an ifn-,f-dependent manner, is essential for the induction of the ifn- signaling cascade (bach et al., ) . recently, considerable progress has been made in the elucidation of the ifn-,~-induced signal transduction pathway (sadowski et al., ; darnell et al., ; shuai, ; vilcek and oliveira, ; david, ; schindler, ) . the janus kinases jak and jak become phosphorylated following binding of ifn- to its receptor, with which the jak kinases associate (muller et al., ; wafting et al., ) . the jak kinase appears to associate with the a-chain, and the jak kinase associates with the [ -chain (sakatsume et al., ) . following activation, the jak kinases phosphorytate the transcriptional factor known as signal transducer and activator of transcription (stat-i~z), w~hich binds to a specific dna element referred to as ~,-activation site (gas) . stat-la binding results in the rapid transcriptional induction of genes containing the gas element. for example, the genes encoding the gaunylate binding protein and the igg fc receptor are induced in this way: other genes that are stimulated by ifn-~/, such as the class i and class ii molecules of the major histocompatibility complex (mhc), require a longer period (several hours) before transcriptional induction is detected. these genes lack the gas element and are likely activated through ifn-?-induced intermediates (benveniste and benos, ) . (summarized in table ) ifn-? plays a critical role in the regulation of the immune response (young and hardy, ) . for this reason, it is often referred to as immune interferon. ifn-y facilitates the stimulation of the ~ subpopulation of t-lymphocytes, which control cell-mediated immunity and which, in fact, express ifn-y; whereas it impedes the stimulation and activity of the th subpopulation of t-cells, :which express il- and are involved in regulating humoral immu-ni~ (gajewski et al., ; swain et al., ; paul and seder, ; seder and paul, ; reiner and seder, ) . cytotoxic t-cells and natural killer cells are also activated by ifn- (trinchieri and perussia, ) . ifn-? also plays a role in regulating antibody production, promoting an igg a response through a direct effect on b-cells (snapper and paul, ) . importantly, ifn-? is a potent stimulator of expression of the antigen-presenting components of the immune system. mhc class i and ii molecules are dramatically upregulated in the presence of ifn-'y on a variety of cell types (reviewed in vilcek et al., ; benveniste and benos, ) . ifn-? is also a powerful effector molecule of the inflammatory response (reviewed in schreiber and celada, ) . ifnq,-stimulated macrophages release a number of inflammatory cytokines, including tumor necrosis factor-alpha (tnf-a), il- , il- , and il- . in addition, ifn-? increases the microbicidal activities of macrophages through the induction of the synthesis of hydrogen peroxide and nitric oxide . ifn-? is also a potent stimulator of the expression of the fc receptors for igg immunoglobins on macrophages (erbe et al., ) . all of these actions of ifn-? on macrophages serve to stimulate the cytocidal activity of these cells. multiple sclerosis (ms) is the most common human demyelinating disease that affects the cns. although the etiology of ms has not been established, it is widely believed that immunological mechanisms are involved (reviewed in martin et al., ; raine, a,b) . re cns is immunologically distinct owing to the presence of the blood~brain barrier (crone, ) , which inhibits entry of both immune cells and humoral factors. in ms patients, this barrier is disrupted (martin et al., ) allowing increased access of blood components (cellular and noncellular) to the cns. the enhanced permeability of the blood-brain barrier is thought to be a contributing factor in immunemediated demyelinating disorders (reviewed in poser, poser, , . importantly, there is an increased infiltration of t-lymphocytes (cd + and cd +) into the cns of ms patients (martin et al., ; utz and mcfarland, ) , with cd + (t-helper) t-cells being predominant at the sites of active demyellnation (booss et al., ; traugott et al., ) . t-cell responses to a variety of myelin antigens, including myelin basic protein (mbp) and proteolipid protein (plp), have been identified in ms patients (reviewed in miller et al., ) . experimental allergic encephalomyelitis (eae) is the primary animal model used in the study of ms (reviewed in alvord et al., ; zamvil and steinman, ; martin and mcfarland, ) . eae can be induced in a variety of laboratory animal species by immunization with either cns tissue, myelin, or volume i , myelin components. as demonstrated through passive transfer studies, eae is a t-cell (cd +, thl) mediated disease model (zamvil and steinman, ; voskuhi et al., ) . a relapsing/remitting form of eae is induced at high incidence in sjl mice immunized with an encephalitogenic epitope of plp (tuohy et al., a (tuohy et al., ,b, (tuohy et al., , mcrae et al., ) . this model of eae exhibits many of the clinical, pathological, and immunological feat~ares of ms. the t-cells that infiltrate into the cns in ms and eae are activated and produce ifn-i, which is believed to play a role in the pathogenesis of irnmune-rnediated demyelinating disorders (reviewed in hartung et al., ; olsson, ; panitch, ). there is considerable circumstantial evidence that suggests that ifn-~, is a key mediator of inflammation in ms and eae. many of the pathological events observed in ms and eae, such as increased mhc expression, macrophage activation, increased expression of leukocyte adhesion molecules on blood-brain barrier endothelial cells (olsson, ) , and reactive gliosis (balasingam et al., ) , are consistent with the known effects of ifn-~,. ifn- is detected in active ms lesions (traugott and lebon, ) , and increased numbers of ifn-~,-secreting tlymphocytes are present in the cerebrospinal fluid of ms patients (olsson et al., ) . beck et al. ( ff) reported a cor~iation between the onset of clinical symptoms of ms and mitogenstimulated ifn-y production. moreover, eae can be transferred to naive animals using ifn-~,-secreting tqymphocytes isolated from animals experiencing eae (zamvil and stelnman, ) , and a significant increase in the level of ifn-?, mrna is observed in the cns of animals with severe eae compared to animals with mild eae (renno et al., ) . perhaps the best direct evidence for the deleterious effects of ifn-y in ms came inadvertently from a small clinical trial in the early s. of ms patients receiving ifn-~/, seven experienced exacerbations during the -wk treatment period, which was significantly higher than the pretreatment exacerbation rate (panitch et at., ; panitch, ) . not only did this study clearly eliminate ifn-~/as a potential therapeutic agent for ms, but it also demonstrated that ifn-~, activity can lead to a wo~,ens of tlne disease course, suggesting a role for this cytokine in normal disease progression. there are, however, contradictory experimental data concerning the role of ifn-~, in eae. the administration of antibodies to ifn-~, e~anced the severity of eae in mildly susceptible mice (billiau et al., ; voorthuis et al., ; duong et al., ) , and resulted in eae susceptibility in resistant strains (duong et al., ) . moreover, treatment of sjl/j mice, which are very sensitive to eae, with ifn-~, results in enhanced survival (billiau et al., ) . nevertheless, the protective effect of ifn-~, is not observed when eae is passively transferred. voorthuis et al. ( ) reported that intraventricular administration of ifn-~/ into eae-induced rats resulted in complete suppression of clinical signs, and willenborg et al. ( ) , using a viral delivery system, suggested that ifn-y had no effect on disease. recently, ferber et al. ( ) examined the eae susceptibility of b .pl mice that contained an experimentally inactivated ifn-~, gene (dalton et al., ) . b .pl mice are normally very susceptible to mbp-induced eae, and the ifn-?, ka~ockout mice appear equally sensitive to disease induction. although morphological data were not presented, this work dearly demonstrates that ifn-~, is not essential for eae induction, at least not in b .pl mice. to understand better the effects of ifn-u on cns cells in vivo, in the absence of other t-cellderived factors, a variety of approaches have been used to deliver this cytokine to the cns. the direct injection of ifn-~, into various animal models has been shown to upregulate the volume , expression of both mhc class i and class ii glycoproteins on various neuronal celt types that do not normally express these proteins at detectable levels. wong et al. ( ) demonstrated a dramatic increase in the expression of the mhc class i glycoprotein in astrocytes, neurons, oligodendrocytes and microglia following the delivery of ifn-y to the cns of -dold mice. direct injection of ifn-y into the cns has also been demonstrated to increase the expression of mhc class i on rat endothelial and ependymal cells and class ii on microglia, ependymal, and perivascular cells (sethna and lampson, ) . continuous iv infusion of ifn-~f into lewis rats for a period of d induced mhc class ii expression on microglia, ependymal and endothelial cells of large blood vessels (steiniger and van der meide, ) . these findings support the earlier work of momburg et at. ( ) who reported that the iv infusion of ifn-y into mice resulted in class ii expression in many organs, including the brain, where "round cells in the vicinity of meningeal blood vessel," presumably microglia, were class ii immunoreactive. ifn-y has also been suggested to play a role in the breakdown of the blood-brain barrier and the recruitment of inflammatory cells into the cns. seth_ha and lampson ( ) reported that a single injection of ifn-y into the rat basal ganglia induced the infiltration of cd + t-cells into the perivascular space and monocytes/ macrophages, and presumptive natural killer cells into the brain parenchyma. these authors suggested that such cellular recruitment may be facilitated by ifn-y-induced changes in the endothelial cells, resulting in an alteration in the permeability of the blood-brain barrier. moreover, simmons and willenborg ( ) , using direct injection of ifn-~. into the spinal cord of rats, reported an inflammatory response similar in pattern to that observed in early eae with the accumulation of inflammatory cells in the meninges and around spinal cord veins also suggestive of a compromised blood-brain barrier. tjuvajev et ai. ( ) reported an ifn-y-induced breakdown of the blood-brain barrier in an in vivo brain tumor model, and huynh and dorovini-zis ( ) demonstrated that brain endothelial cells undergo dramatic morphological, functional, and permeability changes in response to ifn-y, suggesting that this cytokine alters bloodbrain barrier function. steffen et al. ( ) demonstrated increased expression of vascular cell adhesion molecules (vcam) and intercellular adhesion molecule- (icam- ) on the brain endothelium of sjl mice experiencing eae, and antibodies to these molecules inhibited binding of peripheral lymphocytes to inflamed brains. furthermore, mccarron et al. ( ) showed that ifn-y significantly increased the adhesion of mgp-specific encephatitogenic t-ceils to mouse cerebrovascular endothelial cells, and that this effect correlated with the induction of icam- expression by the endothelial cells. the studies discussed above consistently indicate a role for ifn-y in the inducement of expression of mhc and cell adhesion molecules, and the recruitment of inflammatory cells; however, the infusion of ifn-y into the cns has also been implicated in a variety of other processes. for example, yong et al. ( ) reported that the direct administration of ifn-~/ into the adult mouse brain following corticectomy resulted in an increase in trauma-initiated gliosis. brosnan et al. ( ) , using the visual pathway of the rabbit, demonstrated that the injection of ifn-y significantly reduced neural conduction and induced subtle axonal pathology. in addition, the simultaneous injection of ifn- and a myelin/oligodendrocyte glycoprotein antibody significantly delayed cervical somatosensory evoked potentials, and caused massive demyelination in the spinal cord (vass et al., ) . vass et al. ( ) also reported an ifn-y-induced increase in mhc expression, which accompanied spinal cord demyelination. in complement with various delivery systems previously discussed, we have used a transgenic approach to examine the effects of ifn-y on the developing nervous system, (corbin et al., ) . transgenic mice were generated in which the expression of ifnq was volume , i placed under the transcriptional control of the mbp gene (mbp/ifn-y transgenic animals). transgenic mice generated with tl-ds construct have a shaking/shivering phenotype that is similar to that observed in naturally occurring mouse models of hypomyelination (e.g., sb_iverer, jimpy, quaking), and these transgenic animals have dramatically less cns myelin than control animals. reactive gliosis and increased macrophage/microglia f / immunostaining were also observed. additionally, mhc class i and class ii mrna levels were increased in the cns of mbp/ifn-y transgenic mice, and the increase in mhc class i mrna expression was detected in both white and gray matter regions. surprisingly, cerebelfar granule cell migration was also disrupted in these animals. these results strongly support the hypothesis that ifn-~, is a key effector molecule in irnmtme-mediated demyelinating disorders, and suggest that the presence of this cytokine may also disrupt the cytoarchitecture of the developing nervous system. the mechanism by which ifn- exerts its action within the cns remains unresolved. one idea concerning the potential cellular site of action of ifn-y in cns demyelinating disorders is that ifn- activates macrophages/ microglial cells, which in turn mediate cytotoxic effects on oligodendr~ytes (fig. ) . tnf-(z, which has previously been shown to be toxic to oligodendrocytes in vitro, is expressed by ifn-y-activated macrophages/microglia (selmaj et al., ) . more recent in vitro experiments suggest that the microglial cell toxicity to otigodendrocytes is mediated through cellsurface expression of tnf-cz and the production of nitric oxide (merrill et al., ) . nitric oxide is very labile, such that to elicit their cytotoxic effects, the activated microglia need to be in intimate contact with the target oligodendrocytes. oligodendrocytes undergo necrotic cell death following exposure to nitric oxide (mitrovic et al., ) . in support of the hypoth~ esis that nitric oxide is important in the pathogenesis of immune-mediated demyelinating diseases, it has been shown that nitric oxide synthase mrna levels are increased in mice experiencing eae (koprowski et al., ) , and nadph-diaphorase histochemical staining, a marker for nitric oxide production, as well as nitric oxide synthase rnrna levels were found to be elevated in the brains of ms patients (b et al., ) . ifn-y may also have a direct deleterious effect on oligodendrocytes (fig. i) , which have been shown to express ifn- receptors (torres et al., ) . we have examined the effects of ifn- on oligodendrocytes using the moch- cell line, which was derived from an oligodendroglioma that developed in a transgenic line of mice and expresses a variety of features of oligodendrocytes (hayes et al., ; li et al, ) . when moch- cells are cultured in medium containing low concentrations of volume , serum ( % fetal bovine serum [fbs]) they extend multiple, thin, branched processes and have phase-bright cell bodies, which are typi ~ cal features of cultured primary oligodendrocytes (reviewed in popko et al., ) . furthermore, these cells are immunoreactive against antibodies to galactocerebroside, a glycolipid marker of mature oligodendrocytes, and abundantly express myelin protein genes (hayes et al., ) . mochq cells cultured in % fbs or chemically defined medium do not express detectable levels of the astrocyte marker glial fibrillary acidic protein (gfap). when moch- cells are cultured in % fbs medium containing ifn- . however, they transform to flat, astrocyte-like cells with enlarged cell bodies and appear more adhesive ~ in addition to these morphological changes, ifn- stimulates the expression of abundant levels of the astrocyte marker gfap, as well as slightly elevated levels of mbp and plp mrna. ifn- also induces an enormous increase of mhc class i expression, as well as a comparatively modest increase of mhc class ii mrna expression in mochq cells. the morphological and molecular changes mc)ch- cells undergo in response to ifn- suggest that ifn- may induce a direct effect on oligodendrocytes, or a subpopulafion of these cells, in vivo . recent studies further support the hypothesis that ifn-y has a direct, deleterious effect on oligodendrocytes. using a morphological assay, oligodendrocytes in vitro were shown to undergo apoptotic cell death at very high frequency following exposure to relatively low concentrations of ifn- (vartanian et al., ) . the addition of rnicroglia to the culture did not significantly increase the frequency of otigodendrocyte death, further supporting a direct effect of the cytokine. interestingly, vartanian et ai. ( ) also detected apoptotic oligodendrocytes, along with ifn- , in the advancing margin of active ms plaques, but not in the chronic portion of the plaque or in adjacent unaffected white matter. agresti et al. ( ) have also observed deleterious effects of ifn-y on cultured oligodendrocytes. in the absence of cell death, they observed a decrease in the ability of oligodendrocyte progenitors to divide and differentiate, as well as changes in myelin protein gene mrnas and a general metabolic depression. these effects were shown to be fully reversible following cytokine withdrawal (agresti et al., ) . the harmful effects of ifn-y on myelination might be mediated through the induction of mhc expression in oligodendrocytes, which normally do not express these molecules at an appreciable level. in support of this possibility, transgenic mice in which mhc class i expression has been targeted to oligodendrocytes are severely hypomyelinated (turnley et al., b; yoshioka et al, ; turnley and morahan, ) . this hypomyelh~ation occurs in the absence of immune celt infiltration, suggesting that mhc class i expression in oligodendrocytes is sufficient to disrupt myelination. recently; power et al. ( ) examined these transgenic animals in more detail and demonstrated that cell-surface expression of mhc class i in oligodendrocytes of these mice was minimal moreover, immunocytochemistry for f~ -microglobulin, which is required for cellsurface expression of mhc class i, revealed that expression of ~ -microglobulin was undetectable in oligodendrocytes and limited to microglia in lhe cns. power et al. ( ) suggest that the accumulation of mhc class i protein m the cytoplasm of the oligodendrocytes, owing to the absence of f~ -microglobulin, may interfere with normal myelin protein trafticking, leading to the observed hypomyelination in these mice. in vitro, oligodendrocytes at all stages of differentiation can be induced by ifn- to express cell-surface mhc class i (suzumura et al., ; turnley et al., a; massa et al., ) . nevertheless, it is unclear what the consequences of oligodendroglial expression of mhc class i are. as mentioned previously, cd + t-cells are present in the cns of ms patients (martin et al., ; utz and mcfarland, ) . it is, however, unknown whether oligodendrocytes in vivo can present antigen to these cells. in contrast, many investigators have searched for, but failed to demonstrate, ifn- mediated induction of mhc class ii on oligodendrocytes in culture (wong et al., ; suzumura et al., ; turnley et al., a; satoh et al., b) , suggesting that oligodendrocytes are refractory to mhc class ii induction. calder et al. ( ) demonstrated that oligodendrocyte precursors (o- a progenitor cells) can be induced to express mhc class ii by ifn-~/, but that maturation into oligodendrocytes leads to loss of inducibility. nevertheless, bergsteinsdottir et al. ( ) were able to induce mhc class ii expression in a subset of mature oligodendrocytes in culture with ifn- in the presence of the synthetic glucocorticoid dexamethasone. although gtucocorticoids are normal cns constituents (birmingham et al., ; kumar et al., ) , the in vivo significance of these in vitro observations is unclear. another activity of ifn-t that might be relevant to the pathogenesis of immune-mediated demyelinating disorders concerns the ability of this cytokine to stimulate the expression of mat-shock (stress) proteins. these are ubiquitously expressed proteins that are believed to play a role as chaperones in normal protein synthesis and degradation (reviewed in jindal, ) . the expression of these proteins increases following cell stress, and this response is thought to be protective. heat-shock proteins are highly conse~rved across divergent species and are very irnmunogenic. it has been demonstrated that there is increased heat shock protein expression in ms and eae lesions, and importantly, that there is an immune response to [he stress proteins in these disorders (selmaj et al., ; prabhaker et al., ; gao et al, ; van noort et al., ) . it has also been demonstrafed that fn-'f is capable of potentiating the induction of stress protein synthesis (morange et al., ; ferm et al., ) , such that this effect might facilitate the induction of the immune response to the heat-shock proteins in immune-mediated disorders. the characterlzation of the heat-shock response in these disorders should provide further insight into the role these proteins play in the pathogenesis of disease. a potential direct effect of ifn-? on oligodendrocytes in immune-mediated demyelinating disorders is consistent with recent studies that examined biopsy specimens from patients with acute ms (rodriguez and scheithauer, ) . they observed degeneration of inner myelin loops with preservation of outer loops and axons in early plaques, as well as areas in which well-myelinated and demyelinated axons were in close proximity. these observations suggest that the early pathological lesions that occur in ms are compatible with a primary disturbance in the myelinating function of oligodendrocytes, not a nonspecific inflammatory reaction that affects the myelin sheath, as might occur if microglia were activated subsequently to phagocytose the myelin sheath. astrocytes, which have been shown to express the receptor for ifn- (rubio and de felipe, ) , respond to the presence of this cytokine in a variety of ways. as mentioned previously, mhc class i expression on astrocytes increases substantially in the presence of ifn- (wong et al., ; mauerhoff et al., ; massa et al., ) . this expression appears functional in that astrocytes are susceptible to lysis by cytotoxic t-cells in an antigen-specific manner (skias et al., ) . ifnq,-induced mhc class ii expression by astrocytes has also been demonstrated in vitro and in vivo (fontana et al, ; fierz et al., ; pulver et al., ) , although fine in vivo levels are low relative to that detected on microglial cells (reviewed in benveniste, ) . the biological significance of the antigen-presenting potential of astrocytes remains to be resolved in vivo. there is also evidence that suggests that ifn- plays a role in the reactive astrogliotic response, particularly in immune-mediated disorders. reactive astrogliosis is the response in which astrocytes express increased levels of gfap, undergo hypertrophy, and proliferate (eng, ) . there are dramatically elevated levels of geap expression in the cns of the volume i , mbp/ifn- transgenic animals, although it is unclear whether this represents a direct response of astrocytes to i~- or an indirect response elicited by the myelin abnormalities that occur in these mice (corbin et al., ) . moreover, as mentioned, yong et al. ( ) showed that if n< , as well as a number of other cytokines (balasingam et al., ) , increased trauma-induced gliosis in mice. these investigators also demonstrate that ifn-? promotes the proliferation of adult human astrocytes in vitro (yong et al., ) . nevertheless, in a follow-up study, yong et al. ( ) were unable to reproduce these results using mouse astrocytes, suggesting that there are species differences in the astrocytic response to ifn- . ifn- has also been shown to increase the expression of intercellular adhesion molecules on astrocytes. human fetal (frohman et al., ) and adult (satoh et al., a) astrocytes, as well as mouse astrocytes (satoh et al., b) , have been shown to express icam- in the presence of ifn- . such expression might facilitate the interaction of these cells with lymphocytes. ifn-~,-stimulated human and rat astrocytes have also been shown to express vcam- , possibly suggesting a role in lymphocytic infiltration across the blood-brain barrier into the cns (rosenman et al., i ) . although ifn- readily induces the expression of mhc molecules in astrocytes, oligodendrocytes, and resident cns microglia, !fn- -induced expression of mhc molecules in neurons is under tighter control. early evidence demonstrating the ability of brain cells, including neurons, to express mhc molecules includes experiments in which ifn-y was either added to mixed brain cultures or i~ected directly into the cns (wong et al., (wong et al., , . as detected by immunohistochemical analysis, the addition of ifn- to brain cells in culture results in % of neurons expressing mhc class i, whereas direct injection into the cns results in approx % of neurons expressing mhc class i. moreover, the response of the olb neuronal cell line to ifn-? has been investigated (joly and oldstone, ) . the olb cell line was developed by retroviral-mediated oncogene delivery to cells from the olfactory bulb (ryder et al., ) . in these cells, ifn- stimulates the expression of mhc class i both at the mrna level and on the cell surface. furthermore, in the presence of ifn- , the peptide transporters ham and ham , which are involved in the processing of class i antigens, are induced in these cells, whereas an increase in the expression of ~ -microglobulin also occurs (joly and oldstone, ) . in other studies, cell lines derived from two medulloblastomas, which are immunoreactive against neurofilament antibodies, but unreactive against gfap and s- antibodies, have also been shown to empress mhc class i and class ii antigens following exposure to ifn-y (tamura et al., ) . although these studies further demonstrated that cells of neuronal origin are intrinsically capable of expressing mhc molecules and suggest that this expression may serve a functional role, the physiological conditions under which functional neuronal mhc expression can occur have not yet been fully elucidated. toward this goal, neumann et al. ( ) have combined whole-cell patch-clamp recording techniques with single-cell rt-pcr analysis to demonstrate that in the presence of ifn- , both mhc class i and -microglobulin are significantly more inducible on electrically silent neurons compared to neurons spontaneously firing action potentials. this result suggests that functional mhc class i expression may occur only in neurons that are no longer biologically active. this would then allow these cells to be recognized and killed by cytotoxic t-lymphocytes, thus permitting active virus to be cleared from inactive neurons. this result also suggests that active neurons may not possess the potential to express functional mhc, thus allowing neurons to survive at the cost of continued viral infection (joly et al., ) . future investigation in this area will likely yield additional insight into our understanding of the functional interactions between neurons and cells of the immune system. although the majority of the studies addressing the effect of ifn-~, on neurons have been directed at examining the expression of mhc glycoproteins, several studies have reported other ifn-~,-induced neuronal consequences. as previously discussed, brosnan et al. ( ) reported a significant reduction in neural conduction and modest axonal pathology in the visual system of the rabbit following injection of ifn-,f. recently, mcmillian et al. ( ) reported that the treatment of rat primary basal forebrain mixed cultures with ifn- decreased the number of choline acetyl transferase (chat) immunopositive neurons, whereas the number of chat immunonegative neurons was unaffected. this selective neuronal killing was shown to be mediated through ifn-~,-induced microglia activation. moreover, birdsall ( ) demonstrated that following exposure to ifn-~[, two human neuroblastoma cell lines, but not cultured human cortical neurons, expressed icam. ifn-y also enhanced tnf-el-induced binding of these cells to neutrophils. finally, collins ( ) demonstrated that susceptibility to viral infection of a human cerebral cortical neuronal cell line was dramatically increased following treatment with human ifn-~, and that this increased susceptibility was owing to membrane expression of hla class i molecules. ifn--f has also been shown to affect neuronal differentiation in vitro. chang et al. ( ) demonstrated that ifn-~, retarded the death of cultured rat sympathetic neurons following nerve growth factor (ngf) deprivation. moreover, ifn-y has been shown to potentiate ngfstimulated differentiation of pc cells (improta et al., ) . barish et al. ( ) also demonstrated that ifn-y increased the differentiation of cultured cortical and hippocampal neurons. nevertheless, it is unlikely that the developing nervous system is exposed to appreciable levels of ifn- under normal conditions, such that the in vivo relevance of these observations is unclear. in contrast, there is in vivo evidence that suggests that ifn-~, might have a detrimental effect on the developing nervous system. as mentioned earlier, transgenic mice in which ifn-y was targeted to the cns demonstrate a disruption in cerebellar granule cell migration (corbin et al., ) . during development, cerebellar granule cells migrate from the external granule cell layer (egl), through the molecular layer, to the internal granule cell layer (igl) along processes extended by radial gliai cells (chuong, ) . in transgenic mice expressing ifn-? within the cns, many granule cells are still observed in the egl and in the molecular layer. this observation is reminiscent of other mouse mutants (e.g., weaver, staggerer) in which the interaction between granule cells and radial glia is disrupted (chuong, ) . perhaps the ectopic expression of ifn-~, in the cerebellum of transgenic mice has a deleterious effect on lhe interaction between granule cells and radial glia, thus disrupting the migration of these cells from the egl to the igl. further investigation is warranted to characterize the potentially deleterious developmental effects of ifn-g on the cns in more detail. recently, meda et al. ( ) suggested a role for ifn-y in alzheimer's disease. the senile plaques that develop in alzheimer's patients are characterized by the extracellular deposits of [ -amyloid protein. meda et al. ( ) demonstrated that ifn-y acted synergistically with fj-amyloid in the activation of microglia, which produce tnf-o~ and nitric oxide. in coculture experiments, it was shown that microglia activated by ifnq, and ~-amyloid caused neuronal injury. clearly, these are exciting findings that lead to the speculation that ifn-~/might also be involved in other neurodegenerative disorders. ifn-? is a multifunctional cytokine involved in regulating a variety of immune responses. this cytokine also has a multitude of effects in the cns (table ) . although normally excluded from the cns, this cytokine is present during many disorders in which the cns is affected, including the most common human demyelinating disease, ms. the presence of table effects of ifn-? on the cns disruption of myelination exacerbation of reactive gliosis disruption of the blood-brain barrier recruitment of inflammatory cells from the periphery induction of mhc expression stimulation of macrophage/microglial cells disruption of cerebellar granule cell migration this cytokine in the cns leads to an abundance of pathological effects, including decreased myelination, increased gliosis, disruption of blood-brain barrier func~on, increased mhc expression, stimulation of macrophage/microglial activation, and disruption of cerebellar granule cell migration. taken as a whole, these observations demonstrate that ifn-? can either directly or indirectly mediate many of the cns pathological effects that are observed in disorders in which immune cell traffic in the cns increases. understanding the mechanism(s) by which ifn-~,r exerts its actions on cns function will undoubtedly prove useful toward generating rational therapeutic approaches for the treatment of cns disorders in which this cytokine plays a harmful role. we thank numerous colleagues for many helpful discussions that guided the formulation of the ideas presented here. we also thank kathy toews for assistance in preparing the manuscript. the ifn-? work in brian popko's laboratory has been supported by the national institutes of health (nih) research grant roi ns , and the national multiple sclerosis society research grant rg . j. d. is supported by a training grant hd from the nih. b. p. is a recipient of an nih research career development award (ns ) from ninds. reversible inhibitory effects of interferon-y and tumouar necrosis factor-o: on oligodendroglial lineage cell proliferation and differentiation in vitro molecular cloning and expression of the human interferon-? receptor experimental allergic encephalomyelitis: a usefid model for multiple sclerosis ligand-induced assembly and activation of the gamma interferon receptor in intact cells reactive astrogliosis in the neonatal mouse brain and its modulation by cytokines, f neurosci gamma-interferon promotes differentia* tion of cultured cortical and hippocampal neurons increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in 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disease: role of myelin autoreactive t cell production of interferon-gamma autoreactive t lymphocytes in multiple sclerosis determined by antigen induced secretion of interferon-gamma interferons in multiple sclerosis treatment of multiple sclerosis with game, ha interferon: exacerbations associated with activation of the immune system lymphocyte responses and cytokines mochq cells: an oligodendrocyte cell line generated using a transgenic approach trauma and multiple sclerosis the pathogenesis of multiple sclerosis. additional considerations major histocompatibility complex class i expression in oligodendrocytes induces hypomyelination in transgenic mice heat shock protein immunoreactivity in csf: correlation with oligoclonal banding and demyelinating disease~ neurology cultured human fetal astrocytes can be induced by interferon-gamma to express hla-dr the dale e. mcfarlin memorial lecture: the immunology of the multiple sclerosis lesion presidential address: multiple sclerosis: immune system molecule expression in the central nervous system t helper cell differentiation in immune response cytokine production by cells in cerebrospinal fluid during experimental allergic encephalomyelitis in sjl/j mice ultrastructure of multiple sclerosis cytokineinduced expression of vascular cell adhesion molecule- (vcam-t) by astrocytes and astrocytoma cell lines demonstration of the presence of a specific interferon-~, receptor on murine astrocyte cell surface establishment and characterization of multipotent neural cell lines using retrovirus vectormediated oncogene transfer a common nuclear signal transduction pathway activated by growth factor and cytokine receptors the jak kinases differentially associate with the ot and [ (accessory factor) chains of the interferon i receptor to form a functional receptor unit capable of activating stat transcription factors cytokine-induced expression of intercellular adhesion molecule- (icam- ) in cultured human oligodendrocytes and astrocytes expression and induction of intercellular adhesion molecules (icams) and major histocompatibility complex (mhc) antigens on cultured murine oligodendrocytes and astrocytes molecular characterization of interferon gamma as a macrophage activating factor acquisition of lymphokine-producing phenotype by cd + t cells cytokine cytotoxicity against oligodendrocytes. apoptosis induced by lymphotoxin expression of heat shock protein- by oligodendrocytes in vivo and in vitro: implications for multiple sclerosis immune modulation within the brain: recruitment of inflammatory cells and increased major histocompatibility antigen expression following intracerebral injection of interferon-gamma a conserved au sequence from the ~ untranslated region of gm-csf ml-hna mediates selective mrna degradation i ) interferon-activated signal transduction to the nucleus a single phosphotyrosine residue of stat required for gene activation by interferongamma direct injection of cytokines into the spinal cord causes autoimmune encephalomyelitis-like inflammation susceptibility of astrocytes to class i mhc antigen-specific cytotoxicity interferongamma and b cell stimulatory factor- reciprocally regulate ig isotype production identification and sequence of an accessory factor required for activation of the human interferon %, receptor evidence for involvement of icam- and vcam- in lymphocyte interaction with endothelium in experimental autoimmune encephalomyelitis in the cen~al nervous system in the sjl/j mouse rat ependyma and microglia cells express class ii mhc antigens after intravenous infusion of recombinant gamma interferon the expression of mhc antigens on oligodendrocytes: induction of polymorphic h- expression by lymphokines helper t-cell subsets: phenotype, function and the role of lymphokines in regulating their development expression of major histocompatibility complex on human medulloblastoma cells with neuronal differentiation rg- glioma growth attenuation and 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molecules in oligodendrocytes the role oft cells in multiple sclerosis: implications for therapies targeting the t cell receptor the small heat-shock protein alpha b-crystalline as candidate autoantigen in multiple sclerosis interferon-y-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis interferon- potentiates anti~ body-mediated demyelination in vivo recent progress in the elucidation of interferon-gamma actions: molecular biology and biological functions interferon gamma: a }nnphokine for all seasons suppression of experimental allergic encephalomyelitis by intraventricular administration of interferon-gamma in lewis rats t helper (tftl) functional phenotype of human myelin basic protein-specific t lyrnphocytes crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor complementation by the protein tyrosine kinase jak of a mutant cell line defective in the interferon-gamma signal transduction pathway interferon-like virus-inhibitor induced in human leukocytes by phytohernagglutinin cytokines and murine autoimmune encephalomyelitis: inhibition or enhancement of disease with antibodies to select cytokines, or by delivery of exogenous cytokines using a recombinant vaccinia virus system inducible expression of h- and ia antigens on brain cells interferon-gamma induces the expression of h- and la antigens on brain ceils r ( ) y-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo differential proliferafive response of human and mouse astrocytes to gala-interferon ~ ) try_ ~nsgewic mouse model for central nervo~is system demyelknation role of interferon-y in immune cell regulation the t lymphocyte in experimental allergic encephalomyelitis key: cord- -niiib f authors: kristensson, krister title: neuronal targeting and functional effects of infectious agents transmitted from animals to man date: journal: nan doi: . /bf sha: doc_id: cord_uid: niiib f the nervous system is an «immune-privileged» site and can provide a reservoir to harbor as persistent or latent infections certain microbes that find their way to the brain. from an evolutionary standpoint, such infections are characterized at most times by low levels of the infectious agent in the systemic domain, except when multiplication has just taken place. hence the ability for transmission of the pathogens from animals to man will be determined by the availability of microbes to be transferred by a vector (e.g. in trypanosomiasis), or the amount of infective forms of the microbes shed into an environment (e.g. in toxoplasmosis). using african trypanosomes, toxoplasma,listeria and influenza a virus as examples, mechanisms by which microbes can spread and be targeted to and within the brain to cause various types of nervous system dysfunctions is reviewed. newly revealed potentials of certain cytokines to stimulate neurons to control the growth, and even kill, microbes in their cell bodies is also described. za, west nile encephalitis, borna disease, rabies, japanese encephalitis, sars), and prions (e.g. bovine spongiform encephalopathy, variant creutzfeldt-jakob disease). while the detection of the negri bodies in rabies marks the beginning of the th century, the threat of a prion epidemic spreading from cows to humans has created a wave of anxiety at the end of the century. prion infections are covered in the lecture by a. aguzzi in this symposium, and this presentation will focus on transmissible diseases caused by the other categories of pathogens. these pathogens will be exemplified by african trypanosomes, listeria monocytogenes and influenza a virus, which have been the subject of experimental studies in our laboratory, and by toxoplasma gondii, which in spite of its high prevalence in the human population ( - % of the world's population), seems to have been largely neglected in neuroscience research. the choose of these infections is also given by the fact that they illustrate how microbes can use different pathways for spread and targeting to and within the brain as well as how they can use different strategies to interact with neurons to cause degeneration or functional disturbances of the host brain. by excluding most macromolecules from passing into the brain, the blood-brain barrier (bbb) also prevents most microbes from entering the nerve parenchyma. in spite of this, a number of pathogens have adopted mechanisms for a hematogenous spread to the brain. for instance, toxoplasma gondii are carried by macrophages in the blood. from these macrophages they can be released to infect and multiply in brain endothelial cells for further passage into the nerve parenchyma. other pathogens, e.g. rabies virus, spread instead to the central nervous system along peripheral nerves and some, e.g. influenza a virus and listeria monocytogenes, may spread both via the blood and along peripheral nerves as will be discussed in a following section. we have focused our interest on mechanisms by which subspecies of the extracellular parasite trypanosoma brucei (tb) cross the bbb. tb causes african trypanosomiasis (sleeping sickness), which is an example of a re-emerging zoonosis. although the disease in humans, human african trypanosomiasis, had almost disappeared in the early ths , the incidence of the disease has now almost returned to the same levels as in the ths . the african trypanosomes spread by the tsetse fly in sub-saharan africa and use both wild game and domestic animals, e.g. cattle and pigs, as reservoirs. the subspecies tb rhodesiense in east and southern africa causes a relatively rapid disease, while tb gambiense in west and central africa causes a more protracted disease hallmarked by disturbances in sleep patterns. infections with both subspecies lead invariable to death when left untreated. for therapeutic purposes, the question of how and when the parasites pass the bbb during the course of disease has important consequences. at an early stage of the disease relatively non-toxic drugs, which do not pass the bbb, are available, but for the late stage with involvement of the nervous system arsenic preparations are the drug of choice. these arsenic compounds are trypanocidal and pass the bbb, but they are also neurotoxic and cause lethality in a high proportion, up to %, of the patients. the diagnosis of the late stage relies on the finding of parasites and/or white blood cells in the cerebrospinal fluid. the mechanisms of trypanosome entry into the brain are still not known, and the proper staging of the disease, i.e. determination of the stage when the parasites has passed beyond the bbb, remains to be clarified. with the aim of determining how and when trypanosomes pass the bbb, we are undertaking a series of studies on invasion of the rodent-pathogenic tb brucei strain into the brains of mice and rats. in both animals, the trypanosomes localize early during infection to regions in the brain that lack a bbb, i.e. the choroid plexus and the circumventricular organs. at later stages, however, we have recently found that the parasites can penetrate into the brain parenchyma through the cerebral vessels ( fig. ), although the tight junction proteins of the endothelial cells are preserved (mulenga et al., ) . this indicates that the penetration of trypanosomes into the brain parenchyma is an active process and not a consequence of a damaged bbb. we have obtained preliminary evidence that the pro-inflammatory cytokine, interferon (ifn)-g may be involved in regulating the neuroinvasion. in ifn-g receptor knock out mice, the parasites stay in or around the cerebral vessels and do not spread into the neuropil (kristensson et al., unpublished observation) . the mechanisms by which ifn-g may facilitate invasion of the trypanosomes may involve effects on the brain tissue or on the trypanomes. for instance, this cytokine is a most potent inducer of expression of chemokines in astrocytes and microglial cells in the brain and of proteases, and may regulate cell junctions as well. such factors are involved in attraction and penetration of different populations of white blood cells across the bbb, and may -invasion of african trypanosomes (tb brucei) through the blood-brain barrier. brains from rats sampled at (left) and (right) after intraperitoneal injection of the parasites. double-immunolabelling of the variable surface glycoprotein of the parasites (red) and glucose transporter- of the cerebral endothelial cells (green). at days after infection, the parasites are confined to the vessel lumens, while at days after infection they have invaded the brain parenchyma. hypothetically therefore facilitate the invasion of trypanosomes. the trypanosomes may have an advantage to pass through an intact bbb into the brain, because this would protect them from neutralization by circulating antibodies. in fact, treatment of mice with drugs that do not pass the bbb can clear the infection from the organism except from the brain, where the parasites have been suggested to persist for extended periods to cause late relapses (jennings et al., ) . the most characteristic clinical feature of nervous system involvement by the parasites is the sleep disturbances manifested as a disruption of the sleep pattern and circadian rhythms. in collaboration with prof. m. bentivoglio we have described marked dysfunctions of the suprachiasmatic nucleus, which is the main pacemaker for circadian rhythms, in an experimental model of the disease. these findings have been reviewed elsewhere (bentivoglio et al., ; kristensson et al., ) . trypanosome infections evoke a marked inflammatory response in the brain, which in humans is most intense in the white matter; a leukoencephalitis. during this process, inflammatory cytokines are induced in astrocytes and microglial cells (hunter et al., ; quan et al., ) . in spite of the long-standing induction of proinflammatory cytokines, which potentially may be neurotoxic, there are only minor signs of neurodegeneration in trypanosome-infected brains, both in human clinical materials and in experimental rodents (kristensson and bentivoglio, ) . this is interesting in view of the fact that over-expression of inflammatory cytokines have been implicated in the pathogenesis hiv dementia and in neurodegenerative diseases, such as alzheimer's disease. many of these cytokines are activated by nuclear factor-kb (nf-kb). aspirin and its metabolite sodium salicylate inhibit nf-kb-initiated transcription of immune response genes and treatment regimes with this drug have been considered in neurodegenerative diseases. since a chronic rodent infection with trypanosomes would provide an in vivo model of long-term inflammatory neurodegeneration, we have examined the effects of chronic treatment with sodium salicylate on neurodegeneration and cytokine rna expression in trypanosome-infected rats. in brains from untreated trypanosome-infected rats, degeneration, as detected by a modified cupric silver stain, was limited to certain nerve fibers in the vagus, lateral olfactory tract and some other white matter tracts, while sodium salicylate treatment resulted in extensive terminal and neuronal cell body degeneration in the cortex, hippocampus, striatum, thalamus and anterior olfactory nucleus. this exaggerated neurodegeneration was temporally and spatially associated with enhanced mrna expression of interleukin- b, interleukin- b converting enzyme, tumor necrosis factor-a, and inhibitory factor kba in the brain. restricted areas showed elevations in mrna expression of interleukin- receptor antagonist, interleukin- , inducible nitric oxide synthase, ifn-g, and inducible cyclooxygenase (quan et al., ) . our results reveal an unexpected, serious complication in using aspirin drugs for treatment of chronic inflammatory brain conditions. in contrast to the effect of ifn-g in facilitating invasion of the extracellular african trypanosomes into the brain parenchyma, we have found that the same cytokine plays a crucial role in preventing spread of the intracellular bacterium, listeria monocytogenes, to the brainstem along the trigeminal nerves. listeria is widespread in nature and can infect several animal species. the bacteria pose a particular problem in sheep farming. they cause the so-called «circling disease» in sheep, which is a lethal disease with signs of brainstem encephalitis. by electron microscope, listeria has been found within axons of the trigeminal nerve in infected sheep (otter and blakemore, ). an encephalitis similarly targeted to the brainstem has, since its first description by eck ( ) , been reported in hundreds of human patients and the disease has commonly followed the eating of listeria-contaminated, unpasteurized cheese (armstrong and fung, ) . for study of factors that control spread of listeria monocytogenes along the trigeminal nerve to the brainstem, we have developed a mouse model in which the bacteria are injected into the snout of different immuno-deficient mice and the appearance of the bacteria in neurons of the trigeminal ganglia and brainstem recorded. in recombination activating gene (rag- )-deficient mice a neural route of infection was suggested after snout injection of the bacteria (jin et al., ) . this suggestion was based on immunostaining of listeria monocytogenes in the trigeminal ganglia and brainstem, but not in other areas of the brain; the kinetics of bacterial loads in snout, trigeminal ganglia and brain; and the increased resistance of mice infected with the plcb bacterial mutant (unable to spread from cell to cell). by using mice genomically lacking the mononuclear phagocytic growth factor colony-stimulating factor , and thereby deficient in macrophage and dendritic cell populations, we found that these cells play a dual role in listeria infections (jin et al., ) . on the one hand, they constitute a major defense against systemic infections, but on the other hand, they facilitate the invasion of the bacteria along the trigeminal nerve after snout injection. since dendritic cells of the cd c phenotype are in direct contact with peripheral nerve fibers, it may be suggested that these cells serve as an amplifier of listeria replication and thereby increasing the chances of bacterial spread to nerve fibers, which is dependent on direct cell contacts ( fig. ) . ifn-g plays a protective role against listeria neuroinvasion along the trigeminal nerve and inducible nitric oxide synthase accounts partially for this protection. this was shown by comparison of the susceptibility to infection by listeria monocytogenes in the snout of mice deleted for the genes of both the ifn-g receptor and the inducible nitric oxide synthase with wild-type mice. the source of ifn-g appeared to be natural killer cells as shown by the use of combined rag- -deficient, g-chain receptor gene-deficient mice. in vitro experiments showed that ifn-g can directly inhibit growth of listeria in dorsal root ganglia cells (jin et al., ) , and recent experiments ha- . taken together these observations show that listeria monocytogenes after initial replication in macrophages/dendritic cells have the propensity to infect peripheral sensory neurons and spread to the central nervous system. at the same time, however, sensory neurons can control the replication of listeria and ifn-g may induce neurons to kill bacteria. this observed new function of a neuron, i.e. a bacterocidal activity, may be of relevance for an understanding of how spread and replication of infectious agents can be controlled in the immuno-priveledged nervous system. during infections with the parasite toxoplasma gondii, direct effects of ifn-g on replication of a pathogen in somatic cells have also been observed. this parasite replicates rapidly in macrophages, and ifn-g activation of these cells was previously considered as the major effectors for resistance to this intracellular pathogen. however, recent studies employing experiments on chimeric mice have shown that also nonhemopoietic cells can directly mediate ifn-g-dependent resistance of the host during toxoplasma gondii infections. in fact, it was speculated that control of intracellular parasitic replication is a reason why the ifn-g receptor is retained in nucleated somatic cells (yap and sher, ) . after infection of neurons (and skeletal muscle cells), the growth of toxoplasma gondii in a host is much retarded and the parasites persist in the brain as slowly replicating bradyzoites (fagard et al., ; lüder et al., ) . also in the nervous system, growth of toxoplasma may be under the control of ifn-g (fagard et al., ) , although the precise role of the cytokine remains to be determined (schlüter et al., ) . in spite of the long-standing infections with release of cytokines that control the intracellular growth of the parasites there are, like in african trypanosome-infected brains, no or only minor signs of neurodegeneration. the question how potentially neurotoxic cytokines can control growth of the parasite in neurons without causing their damage has been addressed in cell culture systems. both astrocytes and neurons are infected by the toxoplasma parasites, but supernatant from infected astrocytic cultures could prevent infected neuronal cultures from degeneration (rozenfeld et al., ) . the suggestion of a neuroprotective effect of astrocytes during infections is accordance with our studies showing such an effect in mumps virus-infected hippocampal neurons (owe-larsson et al., ) . these studies emphasis that infections in the brain are controlled not only by molecules that arrest intraneuronal microbe growth, but also by molecules that may protect neurons from damage. since microbes, like toxoplasma, may persist in the brain for the life span of the host, the questions arise whether the balance in release of microbe growth control and neuroprotective molecules can be altered to cause neurodegeneration later in life, and whether the infected neurons or the release of the molecules have any effect on neuronal function and behavior of the host. the latter question, i.e. potential behavior consequences of latent toxoplasma infections in the brain, was recently addressed by berdoy et al. ( ) . the definite host animal of toxoplasma gondii is the cat, while rodents, birds and by accident humans are intermediate hosts ( fig. ) . although the brain provides a protected site for hiding of the parasite, it may also be a cul-de-sac: if the intermediate host dies, the parasite will die, unless the predator eats the intermediate host. according to the hypothesis of parasite manipulation of host behavior, a parasite may alter the behavior of a host to favor its transmission to another animal (poulin, ) . rats fear the smell of urine of their predator, the cat, but not that of a rabbit. however, this fear was lost in toxoplasma-infected rats; the infected rats were even attracted to the cat urine, and this «fatal attraction» will increase the chances of predation (berdoy et al., ) . in a review of human latent toxoplasma infections, it was noted that infected children may have a somewhat reduced iq, and that both men and women may have certain traits in their personality profile (webster, ) . infected men had a higher tendency to disregard rules of the society, they were more suspecting, jealous and dogmatic, while infected women were more warm-hearted, out-and easy-going, but also more conscientious, persistent, moralistic and staid (flegr and hrdá, ; flegr et al., ) . whether these findings are reproducible in other studies and societies remains to be evaluated as well as whether they represent causes or consequences of the infection. however that may be, these very common infections of the brain deserve studies in more detail. another common infectious agent transmissible from animals to man, which has been associated with human behavior disturbances is influenza a virus. there are well-documented cases of transmission from pigs to humans, and a few years ago direct spread of influenza a virus from hens to humans caused an epidemic in hong kong. pigs, ducks and humans are considered as a melting pot for this virus, and since the virus is segmented, reassorted variants can appear. thus, there is always the threat that new variants with neurotropic properties may araise. even in current epidemics, encephalitis due to influenza is not uncommon; in a recent study of , patients in finland (population million) with acute central nervous system disease of suspected viral origin collected during - , varizella-zoster virus was the main cause of disease followed by herpes simplex virus, enteroviruses and influenza a virus (koskiniemi et al., ) . there are anecdotal reports that psychiatric disturbances followed influenza in past epidemics; for instance, it was said that the world was never the same again, mentally, after the epidemic, and a reversible schizophrenia-like syndrome was associated with the / epidemic (menninger, ) . there are also a number of studies, although the data are equivocal, suggesting an association between influenza a virus infections during pregnancy (the second trimester) and schizophrenia. although, taken together the epidemiological studies seem to have shown such an association, these type of studies may not contribute more than to indicate a relationship and the «the initiative now rests on cellular anatomy and neurophysiology» (munk-jørgensen and ewald, ) to forward our knowledge. in order to study the potentials of influenza a virus to cause developmental and/or functional disturbances of the nervous system, we have used a mouse-neuroadapted strain, wsn/ , of influenza a virus, which is derived from the first influenza a virus isolate , in a series of studies in mice. this virus causes a lethal disease in mice with virus targeting to neurons in the hippocampus and substantia nigra following intracerebral or systemic injections (takahashi et al., ) . following injection into the olfactory bulbs, the virus spread to the anterior olfactory, the midline thalamic and medial habenular nuclei as well as to the ventral tegmentum areas. following this way of injection, the mice survive and the virus is cleared from the brain (mori et al., ) . our preliminary studies have shown that these surviving mice show certain disturbances consisting of learning disabilities and altered behavior in an elevated plus maze test (aronsson et al., unpublished data) . in immunodefective transporter associated with antigen processing (tap ) mutant mice similarly infected with the virus, all segments of the genome of the virus could persist in the brain for more than months after infection. viral rna encoding the nonstructural ns protein was detected in sections from the brain at midbrain levels by rt-pcr in almost all animals. both negative-strand genomic rna (vrna) and positive-strand rna, including mrna, were found (aronsson et al., ) . this observation shows that certain regions of the brain in immunodefective mice may harbor the genome of influenza a virus, including the ns gene the products of which may play a regulatory role in a host cell metabolism. in order to investigate if maternal influenza a virus has the potential to cause brain dysfunctions in the offspring, the wsn/ strain was instilled intranasally into mice at day of pregnancy (aronsson et al., ) . when the fetuses were examined days later, viral nucleoprotein and rna were detected in their brains. after showing that influenza a virus could pass the placenta and be targeted to the fetal brains we studied in another series of experiments the effect of the maternal infection on the offspring. at an infectious dose of , plaque forming units (pfu) intranasally instilled during pregnancy, mice were born but all died within - days after birth. when the viral dose was reduced to pfu, all offspring survived. about % of brains sampled from these offspring at , , , and days after birth showed presence of viral rna encoding the matrix and nucleocapsid proteins by rt-pcr ( fig. ) . these studies show that influenza a virus can persist in the brain even of non-immunocompromised individuals after an infection at early life. in our preliminary studies, we have observed that there are certain changes in the gene expression profile, verified by real time pcr, in brains from the offspring. interestingly some of these changes did not appear until days of age, which may indicate that a maternal influenza a virus infection can cause gene expression changes that progress over time to become manifested later in life. new observations on the neuropathogenesis of infectious agents that can be transmitted from animals to man have been highlighted and these include: i) the role of cytokines for spread and targeting to the brain. while the host derived inflammatory molecule ifn-g seems to inhibit the invasion of the intracellular listeria monocytogenes along trigeminal nerves, it had the paradoxical effect of facilitating entry of the extracellular african trypanosomes into the brain through the bbb. ii) populations of neurons that are attacked by pathogens have the capacity to control growth, and even to kill, the microbes. this occurs by mechanisms that at the same time protect neurons from being damaged. pharmacological treatment may tilt a balance between potentially neurotoxic and neuroprotective molecules in an unpredictable direction to cause severe neurodegeneration in the brain. iii) microbes may induce imbalances in neuronal circuits and gene expression changes that may progress in later life. questions may therefore be addressed whether microbes transmitted from animals could be included brainstem encephalitis (rhombencephalitis) due to listeria monocytogenes: case report and review persistence of the influenza a/wsn/ virus rna at midbrain levels of immunodefective mice persistence of viral rna in the brain of offspring to mice infected with influenza a/wsn/ virus during pregnancy trypanosoma brucei and the nervous system fatal attraction in rats infected with toxoplasma gondii encephalomyelitis listeria apostematosa differential development of toxoplasma gondii in neural cells influence of chronic toxoplasmosis on some human personality factors. folia parasit correlation of duration of latent toxoplasma gondii infection with personality changes in women astrocyte activation correlates with cytokine production in central nervous system of trypanosoma brucei brucei-infected mice the brain as a source of relapsing trypanosoma brucei infection in mice after chemotherapy a neural route of cerebral listeria monocytogenes murine infection -role of immune mechanisms in the control of bacterial neuroinvasion colony-stimulating factor- dependent cells protect against systemic infection with listeria monocytogenes, but facilitate neuroinvasion infections of the central nervous system of suspected viral origin: a collaborative study from finland pathology of african trypanosomiasis parasites and the brain: neuroinvasion, immunopathogenesis and neuronal dysfunctions toxoplasma gondii in primary rat cns cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation influenza and schizophrenia. an analysis of post-natal 'dementia precox', as of , and five years later selective targeting of habenular, thalamic midline and monoaminergic brainstem neurons by neurotropic influenza a virus in mice trypanosoma brucei brucei crosses the blood-brain barrier while tight junction proteins are preserved in a rat chronic disease model epidemiology in neurobiogical research: exemplified by the influenza-schizophrenia theory observation on the presence of listeria monocytogenes in axons reduction of voltage-dependent calcium currents in mumps virus-infected cultures of rat hippocampal neurons the evolution of parasite manipulation of host behaviour: a theoretical analysis chronic sodium salicylate treatment exacerbates brain neurodegeneration in rats infected with trypanosoma brucei soluble factors released by toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration toxoplasma gondii infection of neurons induces neuronal cytokine and chemokine production, but gamma interferon-and tumor necrosis factor-stimulated neurons fail to inhibit the invasion and growth of t. gondii the substantia nigra is a major target for neurovirulent influenza a virus rats, cats, people and parasites: the impact of latent toxoplasmosis on behaviour effector cells of both nonhemopoietic and hemopoietic origin are required for interferon (ifn)-g and tumor necrosis factor (tnf)-a-dependent host resistance to the intracellular pathogen, toxoplasma gondii in the list of potential causes of behavior disturbances or even neuropsychiatric diseases in man. key: cord- -bq w jk authors: utermöhlen, o.; karow, u.; baschuk, n.; herz, j.; loegters, t. t.; krönke, m. title: delayed elimination of the lcm virus from acid sphingomyelinase‐deficient mice due to reduced expansion of virus‐specific cd (+) t lymphocytes date: - - journal: scand j immunol doi: . /j. - . . l.x sha: doc_id: cord_uid: bq w jk the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn‐γ generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase(–/–) mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd (+) t cells. the secretion of ifn‐γ in response to contact with target cells as well as the cytolytic activity of virus‐specific cd (+) t cells was severely impaired. additionally, both phases of the lcm virus‐specific dth response, mediated by cd (+) and cd (+) t cells consecutively, were diminished in asmase(–/–) mice. however, the secondary memory response of virus‐specific ctl was not altered, and the virus was effectively controlled for at least months by asmase(–/–) mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus‐specific cd (+) t cells during the acute infection of mice with the lcm virus. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -tyeh wz authors: hvas, c. l.; kelsen, j.; agnholt, j.; höllsberg, p.; dahlerup, j. f. title: probiotic bacteria induce regulatory cytokine production via dendritic cells date: - - journal: scand j immunol doi: . /j. - . . au.x sha: doc_id: cord_uid: tyeh wz probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : – ), and dendritic cells were matured from their peripheral blood mononuclear cells. t‐cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf‐α, ifn‐γ, il‐ and gm‐csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf‐α and il‐ than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn‐γ and tnf‐α were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd (+)), in part mediated by dendritic cells. future studies will address whether this shift to a cd (+) phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - iqpl p authors: mackay, ian m.; arden, katherine e. title: rhinoviruses date: - - journal: viral infections of humans doi: . / - - - - _ sha: doc_id: cord_uid: iqpl p picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide. hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under years and occur in all seasons. aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing and with significant direct and indirect healthcare expenditure. ari incidence is highest in the first years of life, with up to thirteen episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month. picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide [ ] . hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under years and occur in all seasons [ , ] . aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing [ ] and with signifi cant direct and indirect healthcare expenditure [ , ] . ari incidence is highest in the fi rst years of life, with up to episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month [ , - ] . in preschool-aged children, nearly % of general practitioner visits are for ari [ ] , many of which are self-limiting. aris can often be managed in the community with supportive care from parents, but complications can arise that require a medical visit for management of asthma, otitis media, or sinusitis [ ] . hrvs replicate in nasal cells, sinus cells, bronchial epithelial cells (becs) [ , ] , and smooth muscle cells [ ] but not in monocytes [ ] or dendritic cells (dcs) [ ] . the infl ammatory immune response they trigger very soon after infection has its greatest impact in the young, the elderly, those with asthma or chronic obstructive pulmonary disease (copd), and in the immunocompromised. first infections usually elicit a stronger response. antiviral interventions have been under development for decades; to date most have met with varying degrees of failure or unacceptability. vaccines have been considered unachievable because of the large number of diverse and distinct viral types. there are classically defi ned and recognized hrv serotypes grouped into two species, hrv-a and hrv-b, and a recently defi ned third species, hrv-c, containing more than genotypes identifi ed and characterized entirely by molecular means. their cousins, the four enterovirus species (ev-a, ev-b, ev-c, and ev-d), are also found in the airways at times. most systematic and mechanistic studies of hrv etiology and pathogenesis have been informed by studies in adults, mostly prior to the discovery of hrv-cs. adults exhibit reduced symptoms from hrv infections because of prior exposure and the resultant protective immune memory which that imparts (see sect. . ). furthermore, many modern studies ( ) draw conclusions about lower respiratory tract (lrt) disease using urt specimens and ( ) infrequently sample, doing so across small cross sections of time. these limitations have hampered attempts to associate virus detection and disease. current thinking is that hrv-cs may be key players in asthma exacerbations although our inability to culture them routinely has hindered our progress in understanding their role. the impact of the hrvs has been underestimated for decades, and the concept of the hrvs as a very large assemblage of genetically, immunogenically, antigenically, and temporally distinct and stable viral entities remains rare; they are more commonly considered a single variable virus, a view that science does not support. the disease most commonly associated with the airways and resulting from hrv infection is the common cold, a selflimiting coryzal illness [ - ] . the term dates back to ancient greece, but evidence that the syndrome and asthma, another disease most frequently due to hrv infection, has been with us since ancient times can be viewed in writings on the ebers papyrus, a medical document written in the sixteenth century bc [ , ] . in the common cold was considered either to be due to exposure to the elements or to infection by bacteria [ ] . it was later understood to be largely due to something in bacteria-free fi ltrates, and so the search for viral causes began [ , ] . the common cold unit (ccu) was established in salisbury, uk, to seek solutions to the mysteries of the common cold, mostly through adult volunteer infection studies and careful systematic science [ ] . the ccu functioned for years ( - ) , and it was here in that the fi rst in vitro culture of an hrv was achieved using lung tissue from a particular embryo ( fig. . ) [ , ] . propagation failed once this tissue was expended [ , ] . once hrv isolation was possible, viral serotyping developed and culture techniques were further refi ned. this leads to an international effort to characterize and name the hrvs [ - ] . in renewed interest in hrv research was triggered by the description of a distinct clade of hrv types [ ] found using molecular typing. the resultant fl urry of hrv research raised questions about many earlier paradigms of rhinovirology and of the role of established respiratory viruses in aris. the novel clade was proposed as a new species, hrv-c, which was taxonomically confi rmed in [ - ] . prior to the discovery of the hrv-cs, the genus rhinovirus had been abolished and the hrv-a and hrv-b species assigned to the genus enterovirus within the family picornaviridae [ ] . the hrv-cs have been assigned a new naming scheme based on genetic sequence in the absence of antigenic or serological data. while the sequencing of all serotyped hrv genomes was completed in , few of the hrv-cs or apparently novel hrv-as or hrv-bs have been similarly characterized, so the full spectrum of hrv genomes, the rhinovirome, remains incomplete. in this chapter we have described individual serotyped hrvs as the "classical" types, a type being the description for a single, genetically stable, stand-alone hrv. methods for epidemiologic analysis the original clinical defi nition of an hrv infection was written using data from cell and tissue culture and adult human infection studies. after in vitro isolation methods employed a virus interference test to more easily determine successful isolation; cultures suspected of infection with an uncharacterized hrv prevented infection by another, readily titratable virus [ ] . later, price ( ; the jh strain) and then pelon and co-workers ( ; , strain) developed culture systems that permitted hrv replication to be more easily identifi ed [ , ] . the early hrvs were initially classifi ed as echoviruses (echo ; later hrv- ) [ ] . at the same time, propagation of the hgp (hrv- ) strain resulted from using increased acidity, lowered cultivation temperatures, and constant motion (rotation) [ , ] . despite the challenges [ ] , virus isolation was a more sensitive indicator of infection than an antibody rise in paired sera [ ] . it was found that several cell lines and methods were required to encompass virus concentrations ranging from to tcid /ml [ - ] and growth differences among the different virus types. additionally, cell age after plating (< h), inoculum volume (relevant to the culture vessel), medium ph ( . - . ), and cell density were important factors for the reproducible appearance of hrv-induced plaques and for higher virus yields [ - ] . the hrvs can grow at temperatures above °c (some prefer that under certain conditions) [ ] , but rolling at °c, preceded by a - -h stationary incubation period [ ] , has historically provided the highest yield and fastest in vitro hrv growth [ , , , ] . serodiagnosis grew increasingly impractical as the number of serotypes increased [ , ] . however, antibody-based methods were essential for type-specifi c neutralization of infection [ ] from which early epidemiology data were derived and around which the hrv nomenclature system evolved in [ ] . the fi rst classical strains were officially named in [ ] , the last in [ ] . today we know that cell culture-based methods are unreliable for accurately representing respiratory virus epidemiology; although enhanced by immunofl uorescence, they are still used [ ] . the hrv-cs have not been successfully cultured in any cell lines or primary cell culture, although many attempts have been described [ , - ] . in hrv-c and w (another hrv-c) were shown to grow using organ culture [ ] . sinus tissue hosted increasing levels of viral rna, as did adenoid, tonsil, and nasal polyp tissue, but much less effectively, as measured by in situ hybridization [ ] . the sinus organ culture system also allowed testing of the fi rst reverse engineered hrv-c (pc ) [ ] . isolation identifi ed hrvs in ~ % of adults with aris, associated with . illnesses per year [ ] . because culture is ineffi cient and subjective and requires expertise, even for the culturable hrv types, it is becoming an art lost to clinical laboratories the world over. it is unsurprising that pcr-based methods now prevail, providing a much improved understanding of the nature and scope of hrv infections. the virological and immunobiological cost of this improvement is a paucity of low passage "wild" hrv isolates to work with; thus, many research fi ndings from recent years have employed easy to grow highly passaged and adapted hrv isolates. the impact of virus adaptation on the reliability of data from use of such viruses is unknown. pcr-based assays have dramatically increased the frequency of hrv detection [ - ] . the improved sensitivity and reduced turnaround time have shown that hrvs, as a group, are usually the predominant viruses in ari cases [ - ] . with reliable detection levels that extend from as few as tcid /sample to well above clinically relevant loads, pcr can detect virus levels which are commonly shed during all stages of experimental infection studies [ , ] . the common understanding of the systemic [ - ] or symptomatic [ , ] context of hrv detections was established during the era of culture detection, and pcr has challenged these paradigms by detecting virus more often than culture. hrvs are sometimes found in "healthy controls"; however, it is likely that with more thoughtful defi nitions of "healthy," these detections would reduce. it is not uncommon to experience a feeling that one is "coming down" with something that never develops further. this is likely due to a transient infection or reinfection by an hrv or other respiratory virus that is eliminated quickly by the host response. it is possible to correlate viral nucleic acid load at the sampling site with disease severity; however, this is made diffi cult by the highly variable sampling effi ciency of respiratory tract specimens which only permit the generation of reliable quantitative pcr (qpcr) data if serial specimens are available [ ] . the ′ untranslated region (utr; figs. . and . ) is the most common target for diagnostic oligonucleotides since the fi rst hrv rt-pcr in [ ] , and the region has retained relevance for virus detection by its adaptation to reverse transcriptase real-time methods (rt-rtpcr) [ , , , , - , , - ] . the ′utr is comprised of a number of conserved sequence "islands" (fig. . ) that permit the robust detection of the majority of hrvs and those "respiratory evs" which can be regularly detected in the respiratory tract [ , ] . the detection of respiratory evs in no way detracts from the importance of supporting clinical decision making using these assays. however, repositioning [ ] . the pcr primers of broadly reactive conventional rt-pcr [ , ] and rt-rtpcr [ , ] assays are shown these primers or changing the method of employing them [ - ] may undermine assay performance, as evidenced by predicted hybridization mismatches, uncommonly low detection frequencies [ ] , and by comparison of multiple primer sets using the same specimens [ ] . the addition of an oligoprobe rtpcr method increases amplicon detection sensitivity and specifi city, identifying -fold fewer tcid /ml or fold fewer genome copies than agarose gel detection of amplicon [ , , ] . other molecular tools, capable of detecting multiple targets, have evolved in recent years [ , , - ] , and some have gone on to be approved for clinical laboratory use [ ] . microarrays can detect thousands of viral targets, but are expensive for routine use (usd - per sample) and not sensitive enough to avoid a pre-hybridization pcr amplifi cation when using clinical specimens. at their most robust, microarrays, like pcr, rely on the existence of conserved regions of sequence to detect unknown viruses allowing them to detect previously unknown hrv types [ ] . highthroughput or "deep" sequencing platforms have become less expensive and more readily available, and they have succeeded in fi nding new diversity within the hrv species [ ] . the experiments remain costly so have not yet found a place for regular screening tasks and remain coupled to a need for pre-pcr steps. rapid protein-or virion-based assays are not (yet) adequately sensitive [ , ] . because of the high number of hrvs and the high frequency of infections, genotyping methods have become an essential accompaniment for understanding hrv epidemiology. nucleotide sequencing of the vp , ′utr+vp +vp (called hereafter vp /vp ), or ′utr region has replaced traditional serological methods, because of its speed and need for fewer specialized reagents compared to serotyping. vp yields the most comprehensive subgenomic genotyping information and is essential for the minimal defi nition of a new hrv type [ ] . the vp /vp region (fig. . ) is considered easier to use because it encompasses suffi cient genetic diversity to confi rm the identity of a clinical hrv type while also providing broad enough sensitivity to amplify the ~ hrvs from a challenging biological substrate, clinical specimens [ ] . screening of airway specimens for hrvs is not routine [ ] due to factors including cost and the perceived low clinical relevance of detection. genotyping is mostly relegated to research facilities. because of this, hrv molecular epidemiology studies tend to be smaller and focused on a specifi c disease or research question. most in-depth molecular studies of hrv replication have focused on a single hrv type. generally, it is presumed that results can be extrapolated to the other hrv types and to the in vivo situation. hrvs replicate in the cytoplasm (fig. . ) [ ] with membrane-associated replication structures containing double-stranded rna (dsrna) replicative intermediates (ri) which are formed in cells h after infection [ , ] . single-stranded infectious rna forms after ris start to accumulate [ ] . genomic rna (plus strand) is the template for complementary minus strand synthesis which in turn is the template for new genomic plus strands that become incorporated into virions [ ] . virions are synthesized from to h after infection and reach maximum release levels at - h [ ] . hrv replication in epithelial cells may shut off host cell transcriptional activity via direct cleavage of transcription factors and nuclear pore complex components. protease a ( a pro ) of hrv-b may directly cleave eukaryotic initiation factor g (eif g) when bound to eif e [ , ] . the eifs have key roles in initiation and rate control of host cell translation [ ] . host cellular protein production is virtually replaced by hrv-b proteins after only h of infection [ ] . hrv-b -infected cells also display reduced nuclear importing and degraded nuclear pore complex (npc) components [ ] . this may represent another hrv strategy for limiting the host response by preventing or reducing key signaling pathway molecules (e.g., irf- , stat , nf-κb) and shutting down host cell protein synthesis. protease c ( c pro ) from hrv-a targets the nucleus and can disrupt active and passive nucleocytoplasmic transport [ , ] . recombinant a pro protein from hrv-a ,hrv-a , hrv-b , hrv-b , hrv-c , and hrv-c exhibited differing specifi cities and kinetics against eif g as well as npc components demonstrating functional diversity between hrv types [ ] . this fi nding underscores the functional diversity within the hrv species and the risk of extrapolating too greatly from the study of single hrv types. it is apparent from a wealth of immunobiological data that hrvs still effi ciently trigger a proinfl ammatory immune response that has considerable clinical impact among at-risk groups, and that their putative interruption of host cell machinery does little to hinder this. the virion encapsulates an approximately kb positive sense rna genome (fig. . ), which tends to be more adenine and uracil (a+u) rich than the ev genome [ ] . in particular, a+u more frequently occupies the third or "wobble" genome replication in association with membranes produces the viral polyprotein which is co-and posttranslationally processed by a pro and c pro into the proteins ( p -p ) and structural peptides ( vp -vp ; vp and vp derive from the vp precursor protein) that assemble into protomers, pentamers, and fi nally capsids. nonstructural proteins are also released in these cleavages as well as through autoproteolytic cleavage. mature hrv virions packaged with an ssrna genome escape by cell lysis (adapted with permission from arden et al. [ ] ) codon position. the single rna "gene" acts as messenger rna to encode the single multi-domain, proteolytically processed "polyprotein." the coding region is bracketed by utrs which perform regulatory functions necessary for genome duplication [ ] . these are very similar genomic, transcriptional, and translational features to those of their close cousins, the evs. most of the information currently required for virus identifi cation by the international committee on taxonomy of viruses (ictv) can be found through analysis of the genetic features of hrvs ( fig. . ). there are complete hrv polyproteins on the genbank database ( fig. . ). the fi rst complete hrv genome sequence (hrv-b ) was described in [ ] followed by hrv-a in [ ] and hrv-a b in [ ] (fig. [ ] . sequencing of the vp /vp region was completed for all classical strains in [ ] , and the complete set of d regions were available in [ ] . currently there are at least named hrv-c vp regions the current spectrum of complete hrv complete polyprotein amino acid sequences available on the genbank database. the alignment was conducted using mafft within geneious pro v . [ ] . the phylogenetic and molecular evolutionary analyses were con-ducted using mega version (poisson model, bootstraps with consensus support shown at the nodes where space permitted [ ] ) (reprinted with permission from miller and mackay [ ]) available and complete hrv-c genomes. many more genomes are appearing as part of the rhinovirus consortium's efforts to complete and study the rhinovirome using highthroughput sequencing technologies to genetically characterize hrvs from their combined clinical specimen stores ( http://www.international-rhinovirus-consortium.org/ ). many ′utr and vp /vp sequences reside on the genbank database, most of which are labeled using in-house laboratory schemes rather than an approved nomenclature. analysis of the full-length genomes supports the use of ′utr, vp , and vp /vp subgenomic regions for useful representation of hrv species and types [ , ] . recombination, the process of genetic exchange which results in a chimeric genome [ ] , can only be detected in mature viruses after the fact, and it must therefore be inferred indirectly through genomic analysis and comparison. predictions of infrequent recombination among the hrvs [ ] have been made based on examination of the available set of hrv coding and noncoding regions [ ] . intensive analyses reported that recombination is not a driving force for the evolution of hrv types [ , , ] . some discrepancies are likely because of the different number of sequences used, the different origins of the viruses used for sequencing, and the analysis methods employed. hrv-c evolution seems to have been more affected by prior recombination, than is apparent for members of hrv-a or hrv-b. this is similar to the ev species but with far fewer predicted recombination events than for ev evolution [ , , , ] . most of the recombination proposed to have affected the hrvs occurred between hrv-c and hrv-a and is often found within the ′utr or at the ′utr/vp junction [ , , ] but rarely in coding sequence ( a [ ] or c [ ] ). the high sequence diversity among the individual hrv polyprotein coding sequences may keep recombination events to a minimum in order to retain viral fi tness [ ] . the ability of hrvs to recombine in practice awaits empirical evidence; the extent of recombination among all hrv or ev types and the frequency with which viable recombinants arise are entirely unquantifi ed. the - nm hrv virion has been visualized for only a handful of hrv-a and hrv-b types (including hrv-a a, hrv-a , hrv-b , hrv-b , and hrv-a ), but no hrv-c structures have been empirically determined to date. the fi rst, hrv-b , was described in [ ] followed by hrv-a a in [ ] , hrv-a in [ ] , hrv-a in [ ] , and hrv-a in [ ] . hrv-c structure has only been predicted using computer modeling, but their basic structure seems to be that expected of an hrv ( fig. . ) [ ] . the hrv capsid shell is composed of protomers, each comprising one copy of the viral proteins vp , vp , vp , and vp . vp , vp , and vp (each ~ kda) are to some extent exposed on the capsid surface, whereas vp (~ kda) is internalized and associated with viral rna. five protomers come together at a point around a fi vefold axis, and this cluster is called the pentamer. the fi vefold axis is circumscribed by a cleft referred to as the "canyon." vp , vp , and vp are each formed by a convoluted set of protein sheets and loops [ ] . the loops protrude beyond the external capsid surface and contain discontinuous antigenic sites. of the hrv types studied, four neutralizing antibody immunogenic (nim) regions have been identifi ed on hrv-b and hrv-a : nim- a (located in vp ), nim- b (vp ), nim-ii (vp and vp ), and nim-iii (vp and vp ) [ ] . antigenic sites identifi ed on hrv-a are called a, b, and c [ ] . the scope and location of antigenic and immunogenic moieties among the hrv-cs is unknown. using known receptor binding sequence as a guide for computer modeling ( fig. . ), it has been predicted that when discovered, the receptor for the hrv-cs will differ from the major and minor receptors defi ned for the hrv-as and hrv-bs [ ] . the three hrv species within the genus enterovirus are a genetically, immunogenically, and antigenically diverse assemblage of > viral types (table . ). this accounts for the combination of hrv-a a and -a b, exclusion of hrv- , which is actually ev-d despite confusion over acid liability [ - ] and combination of hrv-hanks which is actually hrv-a [ ] . serological studies indicate that some hrv-a and hrv-b types may not be distinct enough to deserve a unique identity [ ] . species within the genus share > % amino acid (aa) identity in the polyprotein and in c+ cd and > % aa identity in p ( fig. . ) as well as their host cell receptors, a limited natural host range, a genome base composition (g+c) that varies by no more than . %, and a similar compatibility of proteolytic processing, replication, encapsidation, and genetic recombination [ ] . a variant of the same hrv type shares - % aa identity or more in vp [ ] . much of the nongenetic criteria remain undefi ned for the hrv-cs. in the genera enterovirus and rhinovirus were offi cially combined, retaining the former genus name enterovirus with the human enterovirus c as the prototype species. a genus in the order picornavirales , family picornaviridae , is at least % different in its amino acid identity from any other genus. in a proposal establishing the species human rhinovirus c was ratifi ed by the ictv. formal hrv-c numbering commenced in , and type numbers were initially assigned based on the date of submission of relevant sequences to genbank (hrv-c , formerly nat ; hrv-c , f. nat ; hrv-c , f. qpm; hrv-c , f. c , etc.; table . ) [ ] . a clinical detection of an hrv-c can be considered a novel type principally based on its vp sequence or provisionally ("c_pat," table . ) based on vp /vp [ ] and could be confi rmed as a variant of a previously characterized hrv-c by identity thresholds to either region. the ′utr can be and still is used [ , ] for hrv genotyping, but it is a more problematic region than vp or vp /vp because of the recombination activity that affects this region, especially among the hrv-cs [ ] . this is presented as phylogenetic intermingling of some hrv-a and hrv-c types [ ] . nonetheless, careful application of sequence identity thresholds when comparing clinical sequences to the genbank database (≥ % identity required before assigning a clinical detection to a particular type) succeeds in characterizing hrv species and types [ ] . there are currently types within hrv-c (which includes the types once grouped together under hrv-"a ," hrv-x, and hrv-ny clades), hrv-a types, and hrv-bs. the most up-to-date information on current taxonomic trends can be found at the ictv picornaviridae study group website ( http://www.picornastudygroup.com/ ). ( c ) hrv-c versus hrv-a simplot data projected onto the hrv-c pentamer. the domains of interest are mostly shown within a single asymmetric unit. ( d ) a minor group pentamer (hrv-a , gray ) including antigenic sites (sites a -c , green ) and very-low-density-lipoprotein receptor (vldlr) footprint ( red ) [ ] . attachment of the vldl-r involves adjacent vp molecules. magnifi ed vp area represents one half of a vldl-r footprint [ ] . amino acid substitutions ( arrowed ) contributed to the differences between minor group sites b and c (adapted with permission from mcerlean et al. [ ] ) historically a key feature distinguishing the hrvs from the evs was the instability of the hrv capsid in the presence of acid and their lower preferred laboratory propagation temperature ( - °c versus °c for evs). over time hrvs have been subclassifi ed in different ways. the fi rst was based on tissue tropism and host range. hrvs that preferred growth using monkey cells were called "m" strains and those (the majority) that grew only in human cell cultures, "h" strains [ , - ] . these two groups correlate with receptor usage [ ] (table . ) and possibly with the titer of the inoculum employed [ ] . in it was proposed to abandon this terminology in favor of a sequential numbering system [ ] . picornaviruses recognize a variety of cellular receptors [ , , ] . hrv types are also subdivided into major and minor groups defi ned by use of one of the two main receptor molecules [ , ] . the capsid of the majority of classical hrvs ( n = ) [ ] interacts with the amino-terminal domain of the kda intercellular adhesion molecule (icam- ; cd ) [ - ] . receptor binding destabilizes the hrv capsid, probably by dislodging the "pocket factor," and initiates uncoating [ , , ] . icam- interacts with its receptor, leukocyte function antigen- (lfa- ), and plays a role in recruitment and migration of immune effector cells [ ] . the minor group [ ] of classical viruses employ members of the low-density lipoprotein receptor (ldlr) family to attach to cells [ ] . binding of vldl-r occurs outside of the canyon employing a different destabilizing and uncoating mechanism. heparan sulfate may act as a receptor under specifi c conditions [ , , ] . in andries et al. defi ned, and laine et al. refi ned, two "antiviral groups" (a and b) based on their susceptibility to a panel of antiviral molecules [ , ] . these groupings refl ected the nature of the amino acid (and hence nucleotide) sequence of the region interacting with the antiviral molecules. these antiviral groups can also be visualized using phylogeny [ ] . when sequences from other subgenomic regions, including p , c, and cd, were examined by phylogeny, the species were found, in most cases, to inversely correlate with antiviral grouping labels (table . ). m and h indicate early cell tropism-based classifi cation (monkey, human) abandoned in favor of a sequential numbering system [ ] . hrv types were later divided into the major and minor groups defi ned by receptor tropism [ , ] . receptor-designated minor group hrv types are underlined, and major group types are shown in bold. antiviral groups (a and b) are labeled [ , ] . hrv-a and hrv-a are also likely the same serotype [ ] . a full list of genetically close serotype pairings was presented by ledford et al. [ ] hrv-c nomenclature was defi ned in and currently includes a number of p rovisionally a ssigned t ypes (pat) which are confi rmed once preliminary vp /vp data can be confi rmed with vp sequence and the provisional number removed (e.g., c_pat to c_pat have already been reassigned) today, sequencing and phylogeny play a central role in species classifi cation within the genus, and together, they are surrogates for the important biological classifi cation criteria [ , , , - ] . for the hrv-cs, fi rst described as the "hrv-a " clade (not to be confused with the single virus, hrv-a , this naming scheme appeared after the hrv-c clade's name was proposed) of viruses in [ ] , sequencing of ′utr and vp /vp has provided the bulk of hrv information from clinical studies. while culture in primary sinus tissue has been reported [ ] , no receptor is yet defi ned. hrvs are the most numerous and frequently detected of all the "respiratory viruses," so-called because of their predominant detection in and tropism for the human urt or lrt ( fig. . ). the circulation of hrvs varies with population age, underlying disease, immunocompromise, over time, and across distance. circulation is infl uenced by the nature, strength, distinctiveness, and memory of the immune response hrvs trigger and by the nature and prevalence of other concurrently circulating respiratory, and perhaps nonrespiratory, viruses. with the recent discovery of the unculturable hrv-cs came the realization that previous hrv epidemiology was only reliable if conducted by one or more suitably broad-spectrum hrv pcr assays [ ] ; hence, prior to , detection of the full spectrum of ≥ hrvs did not occur. after , the ability to detect all types very much depended on the nature of the pcr primers and detection methods used. the great number of distinct hrv types has burdened the search for answers to epidemiology-related questions. however, as for other important respiratory viruses including human respiratory syncytial virus (hrsv) and the infl uenza viruses (ifvs), the virus types within a species show evidence of being both distinct and discrete viruses that are independently recognized by their host and consequently independently infect their hosts. each hrv type is also genetically stable [ ] . the hrv species circulate variably from year to year with evidence of epidemics of distinct types. a prospective longitudinal cohort study over months examined hrv frequency and diversity in specimens from healthy children ( - years of age) [ ] . a median of three hrvs and a maximum of six were detected per child. a similar outcome resulted from an australian cohort study [ ] . genotyping reveals more of the hrv diversity at a single site than culture ever could with molecular studies fi nding between and distinct hrvs at a single location [ , , ] . the number of additional hrv cases that occur in children outside of specifi cally defi ned symptomatic periods remain to be defi ned, with current studies indicating that a much higher number of hrv infections may occur. more comprehensive investigation of hrv type and illness will be undertaken during analysis of data from the australianbased observational research in childhood infectious diseases (orchid) study ( http://clinicaltrials.gov/show/ nct ). interestingly, the hrv-bs are often underrepresented, even when accounting for the smaller number of known hrv-b types [ ] . a number of studies have not found any robust patterns between the circulating hrv types or species and clinical outcome, but the majority of studies seeking this information are short and sample infrequently, limiting their ability to fi nd the patterns they seek [ ] . studies into the relative sensitivities of nasopharyngeal aspirates (npa) and swab sampling methods produce differing results, but generally, if seeking the best diagnostic yield for as many respiratory viruses as possible (i.e., seeking a laboratory diagnosis to support clinical decision making), npas are the sample of optimal choice. one study reported similar clinical sensitivities between swabs and npas for human coronaviruses (hcovs), ifvs, and hrsv, but reduced sensitivities using swabs for hrvs, human adenoviruses (hadvs), human metapneumovirus (hmpv), or parainfl uenza viruses (hpivs) [ ] . a second study reported no difference in sensitivities for hrvs, hadvs, and hpivs but a reduced sensitivity for hrsv and ifvs when using swabs [ ] . nasopharyngeal washes also yield more viral culture success than either nasal or pharyngeal swabs. nonetheless, many studies use nasal swabs as the sample of choice because they allow self-collection and involve much less discomfort than npas, and pcr has meant that infectious virus is not required, only viral nucleic acid which relaxes some limitations imposed by the need for rapid, careful, temperaturecontrolled, and expensive transport requirements [ , , ] . bronchoalveolar lavage samples are best for seeking lrt etiologies, especially in adults where nasal wash viral loads can be low compared to those in children, but this is an invasive method with some risk attached [ ] . hrvs infect all people, all around the globe. spread of hrvs is most obvious and frequent from child to child and from child to parent [ ] . in populations of mixed age, the majority of hrv detections occur in children [ ] . among specimens from healthy children, over a third ( %) were hrv positive. children less than years of age ( % of whom were hrv positive) were shown to have more hrv infections and a wider diversity of hrv types than children more than years old ( % hrv positive) [ ] . healthy adults in the military [ , ] , at university [ ] , at home [ - ] , and in the workplace [ ] have also featured prominently in historical, culturebased, and volunteer infection studies and heavily infl uenced our view of hrv infection outcomes [ , ] . although studies of children in hospital-based populations usually report more signifi cant clinical outcomes (relating to the lrt) [ ] than community-based studies, these data are still broadly applicable. hospital populations originate from the community and refl ect the more serious and perhaps fi rst exposures to the virus. hospital-based populations defi ne the potential of a virus to cause severe clinical outcomes. disease at this end of the spectrum has the strongest infl uence on future prioritization of therapeutic research and developments [ ] . modern air travel contributes to the rapid spread of respiratory viruses as seen in their often frequent detection among travelers [ ] including those with febrile illnesses [ ] . apart from children, hrvs are found with the great clinical impact in the elderly (described as - years of age) with % of aris positive for an hrv, sometimes with a greater burden of disease than ifvs [ ] . those with asthma or copd are also affected by the ari triggering exacerbations of wheezing illness (see sect. . ). it is thought that this is not a different type of infection but rather a different response to infection by the host. wheezing can also result from infection in atopic people who do not have underlying asthma or copd. hrvs cause signifi cant impact in the immunocompromised, and this group is the only population to date that has been found to host truly persistent hrv infections (see sect. . ). because the hrvs are the largest group of viruses to infect humans, it is not surprising that they confuse differential diagnoses during pandemics and have key roles in co-detections and asymptomatic disease. the study of hrvs is the study of all respiratory viruses; while each can be considered in isolation, this will likely be detrimental to a greater understanding of respiratory virus pathogenesis. hrvs circulate throughout the year but usually with a bimodal peak in temperate locations in both hemispheres. the highest peaks, mostly defi ned using adult populations, are in the autumn (fall) and spring [ , , ] (and, peculiarly, on a monday [ ] ). the major winter dip in hrv prevalence closely coincides with the peaks of other respiratory viruses, particularly ifvs [ ] and hrsv [ ] . one hypothesis states that a miasma exists in the school classroom, of particular relevance to those who suffer asthma exacerbations, and this miasma maintains immune stimulation, which subsequently wanes among school children during holidays, to be challenged anew upon return to school [ ] . it is clear that an interplay or interference takes place between viruses at the population level, particularly evident among rna viruses. there is a correlation between spiking spring and autumnal hrv case numbers and an asthma exacerbation "season" - days after return to school from holidays, in a range of climates [ - ] . this was particularly obvious among asthma hospitalizations of children ( - years of age) in ontario, canada, which peaked at weeks - across a decade [ ] . upon investigation, hrvs were the most prevalent of the viruses found in a -year analysis of emergency room presentations in ontario [ ] . hrvs also predominate during "hay fever season" [ ] . although a defi ned seasonality is not always found in the tropics [ ] , this may sometimes be due to testing that does not include hrvs [ , ] or only some hrvs [ ] . all the hrv types continue to circulate today, including those named in the earliest of the nomenclature assignments. at a single site during - months, or more types can co-circulate [ , ] [ ], dropping [ , ] if the study time frame at the site is shortened. a recurring hrv type, defi ned using molecular tools, accounted for . % of any virus detected in a birth cohort followed for months [ ] and, in another cohort, occurred twice in two children, within a -month period [ ] . within a given year and across different years, it is apparent that hrv species exchange predominance [ , , , - ] . no evidence exists to satisfactorily explain this; however, herd immunity may be a factor. the use of cell and tissue culture underestimated the frequency of multiple infections in patients, most likely because the dominant virus out-replicated any others, or due to viral load differences, specimen quality issues, differing cell tropisms, or the triggering of an antiviral state by the fi rst virus. when the majority of respiratory viruses are sought using pcr techniques, multiple virus-positive specimens can comprise a third of those tested [ ] , dropping to around a fi fth of ari episodes when fewer viruses are sought [ ] . there is sometimes an emphasis on the high number of hrv cases that are identifi ed in the presence of another virus, and including hrv testing does raise the frequency of pathogen detection above one per sample [ ] . coinfections, or, more correctly for pcr-based studies, co-detections (since pcr cannot determine infectivity), have been found to either increase [ , - ] or have no impact on the clinical outcome in their host [ - ] , and so the issue of clinical relevance of co-detections is still uncertain. in extreme cases, half of all hrv detections can be found concurrently with another virus. on the surface, this is a signifi cant fraction, and yet % or more of hrsv, hmpv, ev, and ifv detections and % of hcov-nl detections can be found in the company of another virus [ ] . other studies fi nd different, but still higher proportions of co-detections involving non-hrvs [ ] . whether co-detections represent a particular synergism between the involved viruses, a differential capability to manipulate the host immune response, a sign of innocuousness for the most frequently involved virus [ ] , or a chance due to overlapping seasons remains unclear. it is clear, however, that co-detections are not an anomaly or an error due to "overly sensitive" pcr tests; they are evidence of further biological complexity that, until recently, remained hidden from us. recent studies have shown that the initial impression of hrvs being overrepresented in these cases was incorrect. closer analysis of viral co-detections has revealed patterns [ , ] . these became clear when codetections were examined bidirectionally, not just how many hrvs were positive for virus x but also how many of virus x cases were positive for an hrv. whether in a hospital or a community setting, hrvs more often occur as the sole virus detected in aris [ , ] . considering their ubiquity, it is interesting that relatively low numbers of concurrent detections occur [ , ] , supporting the concept that hrvs have a direct role in the clinical outcome of their infection [ ] . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens, while the host provides a vessel for hrv replication and transmission. studies of single respiratory viruses without being in the context of the respiratory virome are of limited value in drawing conclusions about clinical impact. much of the longitudinal epidemiology data previously relied upon to form assessments of hrv signifi cance was acquired using culture-based techniques. with improved and more comprehensive testing, patterns can be seen among the interactions of hrvs and other respiratory viruses. virus interference is a type of virus-virus interaction (vvi) that has been known for decades. vvi has recently been categorized into types [ ] . at the population level, it has been noted that during trials of live attenuated ifv (laiv) vaccines, an interferon (ifn) response was triggered that protected vaccinees against off-target viruses for days postvaccination [ ] . this study went so far as to suggest such effects could be maintained for a prolonged period using a regime of consecutive schedule vaccinations, each separated by days or more, during times of a prolonged epidemic [ ] . a similar effect was produced using live ev vaccines (lev) to replace pathogenic ev types and interrupt outbreaks [ ] . orally administered levs succeeded in their principal task but also reduced the incidence of aris during epidemics by % overall [ ] . this shows that immune activation in the gastrointestinal system generates an anatomically distinct protective effect and there may be a similar effect on the gut's infl ammatory status after respiratory virus infection. in contrast to the laiv results, the offtarget protective effect was reversed in a study using a trivalent inactivated ifv vaccine [ ] . the mechanism underneath these opposing outcomes is unclear. during the heyday ( s) of tissue culture for virus studies, a common biological assay for infection with hrv involved attempted infection of the culture with an enterovirus (ev) or hpiv- [ , ] . failure of the superinfecting virus to grow heralded the likely presence of a noncytopathogenic hrv. virus interference has been used to measure ifn in specimens through its inhibition of hrv growth [ ] . more recently hrv-hadv dual pcr-positive cases were found less often than expected and harbored lower viral loads of hrv than did specimens from cases of sole hrv infections [ ] . signifi cantly, the majority of these instances of vvi involve rna viruses [ ] . it has been shown that dual infections of peripheral blood mononuclear cells (pbmcs) with viruses other than hrsv (including hrvs) induced immune responses similar to those of single infections, but coinfections including an hrsv resulted in reduced ifn-γ responses [ ] . vvis are affected by the ability of each to moderate the host response against them. virus interference has also been identifi ed in virus positives as a series of patterns among respiratory specimens tested for up to respiratory viruses (fig. . ) [ , ] . statistical analyses supported that many of the co-detections occurred in patterns, in particular that fewer co-detections involved an hrv than would have been expected by chance alone ( p ≤ . ). for some period, rna virus infection, especially the hrv group, may render the host less likely to be infected by other viruses and, by extrapolating to the community level, help constrict the epidemic periods of other viruses by reducing the number of fully susceptible hosts. virus interference as a feature of respiratory virus epidemiology can also be seen in results of other studies [ ] . during an -week period that spanned peak h n pandemic infl uenza season in wisconsin, it was infl uenza a virus (ifav) that seemed to dominate hrv in children with asthma who were sampled weekly [ ] . whether this refl ects all ifv-hrv interactions or just those involving a novel ifv such as h n is unclear. it was found that pbmcs from these children exhibited normal immune responses [ ] . reports of subjects with continuous and extended (greater than - weeks) periods of hrv positivity [ , ] increased as pcr methods replaced cell culture for hrv detection. this had only rarely been recorded using culture [ ] . hrv rna has been detected days prior to symptoms commencing and for as long as or more weeks after they cease [ , - ] . studies that only defi ne the period between aris in children as that time when specimens are rt-pcr negative [ ] will not detect overlapping serial infections (fig. . ). epidemiology that incorporates hrv typing generally does not fi nd chronic shedding [ ] . hrv shedding normally ceases within - days, after signs and symptoms have stopped [ , , , , , ] . thus, the perception of persistence is probably due to serial or overlapping infections by multiple untyped strains [ , , , ] . few studies [ ] have suitably addressed persistence in hrv infections involving healthy subjects since pre-and post-sampling clinical data are rarely described [ , ] . to date, true persistence-an ongoing detection of a single confi rmed hrv type-has been limited to individuals with underlying immunosuppression or immune dysfunction [ ] . hrv-cs were detected more than three times longer in immunocompromised young patients than in immunocompetent children, with a mean of versus days [ ] . multiple detection of the same hrv type ( % identical hrv- a sequence in each patient over time) extended to months in hematopoietic stem cell transplant recipients. the proof of causality is as diffi cult to achieve as the proof of innocuousness when it comes to respiratory viruses and aris. the defi nition of "well" subjects prior to or at the time of sampling or inoculation is sometimes not clear, especially for young children who cannot reliably report symptoms [ , , ] . often parents notice a symptomatic illness before an infection is detected in the laboratory [ ] , supporting the importance of diaries in longitudinal home-based community studies. nonetheless, even with the support of telephone interviews and home visits, milder cold symptoms may be missed. it is not uncommon for an asymptomatic control to subsequently become symptomatic or have been symptomatic before sampling [ , ] . some studies employ sensitive symptom scoring systems [ ] , but the criteria for being symptomatic are usually designed to describe and clearly discriminate overt or more "severe" illnesses, those with obvious and measurable signs. strict defi nitions help improve patient management and the commencement or better direction of treatment or cohorting. however, in research studies the arbitrary degree of severity required for reporting a symptomatic event often overlooks very simple changes in host biology due to a virus's replication. these changes to the norm are mild but nonetheless represent disease (a disorder of structure or function that produces specifi c symptoms or that affects a specifi c location and is not simply a direct result of physical injury) in the literal sense. such minor or short-lived, often unrecorded [ ] , indications of infection include sinus pain, headache, sore throat, earache, watery eyes, fatigue, muscle aches and pains, and mood changes. within families, hrvs are frequently transmitted from vsig activated "shields up" a b children who are usually symptomatic [ ] . infants frequently exposed to other children have more asymptomatic viral infections [ ] . among infected adult family members, asymptomatic infections are more likely [ ] . among older parents, whether their children live at home or not, asymptomatic infections are more frequent following hrv challenge than among adults without children or in younger parents [ ] . in a study of viral species in age-stratifi ed cases and controls, signifi cantly lower viral loads were found in those without the required symptoms [ ] . qpcr may prove useful to determine viral load cutoffs to address this issue in the future, although the respiratory tract is a diffi cult tissue for qpcr [ ] . the high sensitivity of pcr-based methods has raised concerns over the clinical relevance of a virus-positive result [ ] . it is clear that a proportion, around fi ve to % of study-defi ned asymptomatic control populations [ , , ] , are virus positive using sensitive pcr-based methods. this may vary up to nearly % of cases when stratifi ed by age, virus, and season or when including highrisk populations [ , ] . every respiratory virus, even ifvs and hrsv, can be found in cases without symptoms at the time of specimen collection even after specifi c inoculation of adults [ , , ] . this is a complex and incomplete story in need of more research, and so it is frustrating that positivity in asymptomatic people is often used to rank viral importance. better data are required from asymptomatic controls for any conclusion to be drawn about causality [ ] , but this requirement often disregards the memory of a normal functioning protective host immunity. it is the host response that defi nes the degree of clinical severity for the infl ammatory disease that is the hallmark of an ari [ ] . it is well known that previous exposure to a virus affords protection from the full clinical spectrum of disease upon repeat exposure to that virus. it should come as no surprise then that hrvs, which usually cause brief infection anyway, could well produce only minor signs and symptoms upon reinfection. the unique and extremely personal infection history of each member of a control group cannot be determined unless they are part of a longitudinal cohort. so, what do cohort studies, supported by comprehensive pcr-based testing, tell us about asymptomatic virus infections? some cohort studies do not look in asymptomatic children, seeking samples only at times of symptomatic illness [ , , ] . a birth cohort of children enrolled and sampled when ill and every months for months identifi ed hrvs - % of infants and toddlers who had no nasal symptoms (defi ned solely by the presence of rhinorrhea) [ ] . the childhood origins of asthma (coast) birth cohort followed infants at high risk for allergies and asthma for months and identifi ed hrv infections as preceding (mean age of fi rst detection, months) those of hrsv (mean age at least months), and hrvs were found in % of asymptomatic versus % of moderately to severely ill patients; the most frequently symptomatic children also had the greatest proportion of asymptomatic infections [ ] . in a study of children with asthma sampled weekly for weeks during each of two peak hrv seasons, nearly two-thirds who were virus positive but not sensitized to at least one allergen showed no asthma symptoms, and nearly half showed no ari symptoms; in the children who were sensitized, less than one-third showed no asthma symptoms, and only a fi fth had no ari symptoms [ ] . a convenience population of healthy children ( - years old) without asthma were followed during at least three seasons, and picornaviruses were detected in % of specimens ( % of infections) not associated with symptoms, the impact of hrv typing and of sampling based only on symptoms. the example provided here diagrammatically represents a single, hypothetical monitoring period, starting at time = , for a single individual. the period of potentially detectable hrv is indicated by an open box. if sampling occurred at each time point ( - ) and hrv positives were genotyped, it would be apparent that three different strains infected the individual, although discerning hrv-x from hrv-z at time point would require a molecular cloning approach. illness, in different forms, may have continued over the entire period depending on the symptoms required/recorded and the period of time represented by the monitoring period. in this case a clinical diagnosis may record only a single symptomatic episode. genotyping may not be performed, and sampling may be intermittent, and so association between viral type or species and disease is impossible. in the study examples indicated by ( a ) start and fi nish sampling or ( b ) symptomatic sampling, ( asterisks mark sampling times in fi lled bars), the laboratory data would have made only one or two identifi cations, respectively. in the third example, ( c ) frequent sampling of this type has previously led to conclusions of hrv persistence or chronic shedding; when combined with genotyping, it becomes apparent that different hrv types are present although of the infections came from households with an infected sibling [ ] . in summary, there is clear evidence for the presence of hrvs in asymptomatic controls. a precise proportion cannot yet be defi ned. some study controls show signs of a "lead-in" period where rna positivity precedes an ari defi ned on follow-up, while others may have been defi ned as symptomatic if more symptoms had been accounted for. mechanisms and routes of transmission hrvs have been found at extra-respiratory sites. viremia was determined in the blood of children with lrt infection or pericarditis [ , ] , and hrv-c was more commonly associated with viremia than was hrv-a, supporting possible increased pathogenicity [ ] . blood was also positive for hrv rna and infectious virus from infants at necropsy [ , ] , and hrv rna was detected in the plasma of children with asthma, bronchiolitis, or common cold [ ] . an hrv was once isolated from feces [ ] , and more recently higher than expected loads of hrvs were detected in fecal specimens from children with suspected meningitis and fever of unknown origin [ ] , with gastroenteritis [ ] , and in a child with pericarditis [ ] . nonetheless, the nasopharynx is still considered the main site of focal virus production [ ] , regardless of inoculation route [ ] , and most studies of transmission routes have centered on the urt. in contrast to ifv and hrsv, hrv infection involves less destruction of tissue. ciliated epithelial cells are sloughed off in proportion to the severity of an hrv ari, but this damage is minimal and does not occur during the viral incubation period or with subclinical infections [ , ] . the incubation period between infection and onset of virus shedding into nasal secretions is - days with shed viral titers peaking in adults between days and [ , ] . the time until successful hrv transmission among adults in a childless family setting is usually - days and requires the donor to be shedding at least tcid at some stage, to have recoverable virus on the hands and in the nares, enough shared time, and a moderate to severe ari [ ] . the lungs have been shown to host replicating hrv [ ] , and the reader of such reports may be left with the perception that detection of hrv replication in the lrt explains all lrt symptoms. however, relatively few studies seek or identify true hrv replication in the lrt. while the overwhelming majority of lrt cases detect hrv from the urt, a correlation between urt positivity and lrt disease does exist [ ] . it is well known from experimental inoculation studies that hrv infection can result from inoculation of the conjunctival sac after virus is moved through the nasolacrimal duct [ ] . in these studies virus was commonly delivered by aerosol or intranasal instillation of . ml to ml of suspension [ - , , - ] . in the laboratory, hrvs can retain infectivity for hours to days on suitable, nonporous solid surfaces, especially if the inoculum remains damp [ , ] , which supports direct self-inoculation especially in the family setting and indirect inoculation via fomites [ ] . in a trial to defi ne the movement of virus from a contaminated donor to a recipient via multiple surfaces or by hand-to-hand contact, % (donor to objects to recipient) and % (donor to recipient fi ngers) of the virus recoverable from the donor's fi ngertips were recoverable from the recipients' [ ] . even under observation, eye rubbing ( . h − - . h − ) and nosepicking ( . h − - . h − ) occur frequently [ , ] , suggesting self-inoculation could outpace personal hygiene, particularly in the young. it was once thought strange that aris were so common, but isolation rates for the expected viruses were so low [ , ] . with a better understanding of the importance of preexisting antibody (something common among the predominantly adult volunteers used by many studies), the discovery of a third, unculturable species of hrv (still causing aris but impossible to isolate or detect using antibody-based systems for which no reagents existed), and a vastly improved diagnostic sensitivity, this is much less confounding. in the past, household cross infection, determined by ari, was low, about fi ve exposures to infected members required for infection [ ] despite viral loads in nasal washings peaking at . × tcid /ml [ ] . experimental transmission was also reportedly ineffi cient [ ] . in contrast, "naturally" close-quartered military populations, interacting over - weeks, experienced rapid spread of hrvs to > % of the group [ ] . the use of pcr recently clarifi ed this discrepancy, confi rming that frequent transmission in families is more common than culture-based studies had identifi ed, often resulting in asymptomatic infection among older siblings and parents [ ] . pcr has helped defi ne the scope of viral rna, if not actual infectious virus, survival, and spread. transmission studies require infectious hrv, and so the hrv-cs do not contribute to the historical data. under crowded or intimate conditions and with more severe colds, transmission reaches - % [ , ] . in some studies, both large-and small-particle aerosols proved ineffi cient, supported by a low isolation rate from saliva ( % compared to % of hand washes and % of nasal swabs) [ , , ] and from only . % of participants exposed to large-particle aerosols [ ] . in other human donor-recipient model studies however, aerosol proved to be the main transmission route among antibody-free adults [ , ] . the discrepancy may have been due to insuffi ciently long or intense exposure in the earlier aerosol experiments [ , ] . apart from particle size, spread of virus by aerosol is affected by existing nasal obstruction which can divert secretions from the nares to contaminate saliva, the presumptive source of virus in coughs and sneezes [ ] . when exposed to liters of a small-particle aerosol, tcid of hrv- was associated with fever and prominent tracheobronchitis in antibody-free (< : ) adult volunteers but not when delivered via nasal drops or a coarse aerosol [ ] . it has also been found that simple breathing releases hrv rna (the same type was also identifi ed from nasal mucous) from at least a third of adults and children with symptomatic aris and infectious hrv could be isolated from a fi fth [ , ] . it is apparent that hrvs accumulate at sites with heavy human traffi c, potentially forming a secondary source of infection. hrv rna can be detected from % of ~ -hourold fi lters placed to sample air in offi ce buildings [ ] . in aircraft, high effi ciency particulate air (hepa) fi lters have been found to harbor hrv rna more than days after they were removed for servicing [ ] . hrv infections trigger a vigorous proinfl ammatory immune response that is thought to drive the symptoms experienced as illness [ , , ] , but they do not seem to actively prevent or interfere with the host's immune response the way most other viruses have evolved to do. there may be a role for repeated challenge by hrvs and other respiratory viruses leading to infl ammation and tissue remodeling. the host response to hrv infection can be broadly broken into the innate (very fast, encoded in the germ line, nonadaptive) and adaptive (slower to develop, reliant on t cells, b cells, and the generation of antibody) responses. while the innate system is "always watching," it is signifi cantly amplifi ed by virus infection. the adaptive response is initiated by the host's fi rst infection with a particular virus and then functions to limit subsequent infections through the production of neutralizing antibodies and amplifi cation of existing cell-mediated immunity. after virus-receptor binding and internalization, the earliest host cell immune response to an hrv infection is elicited by the innate immune system (fig. . ). epithelial cells represent the front line against hrv invasion although alveolar macrophages and dcs are better equipped to respond [ ] and do so despite not hosting hrv replication directly [ ] . virus detection is mediated by pattern recognition receptors (prrs) that have evolved to recognize conserved molecular structures shared among diverse pathogens. internal-or surface-mounted prrs include sentinels that specifi cally recognize picornavirus rna and protein and, in doing so, trigger an immune circuit that results in the production of ifns and subsequently hundreds of ifn-stimulated gene products. the innate response to viral infection hinges on inducing two type i ifns (initially ifn-ß then ifn-α), secreted cytokines that produce antiviral, antiproliferative, and immunomodulatory outcomes [ ] . the type iii ifns (ifn-λ or il- , ifn-λ or il- a, and ifn-λ or il- b) are also produced in response to viral infection in a range of cells, although their receptor is not as widespread [ ] . the type ii ifn, ifn-γ, is produced by activated t cells and natural killer cells rather than in direct response to virus [ ] . detection of viral components triggers protein signaling cascades that regulate ifn synthesis through the activation of viral stress-inducible genes (vsigs) [ , ] . these are sometimes expressed constitutively but upregulated after ifn induction following hrv infection [ ] . released ifn-ß binds to the ifn-α/ifn-ß receptor in an autocrine (the same cell) and paracrine (neighboring cells) manner, starting a positive feedback loop for type i ifn production, the "second wave." vsigs include the antiviral proteins protein kinase r (pkr), ′ ′oas/rnasel, and the mx proteins [ ] . ifn-α upregulates expression of mxa, ′ ′-oas, and pkr [ ] . the mx pathway is also induced after virus infection but is not constitutively expressed [ ] . depending on the sentinel system stimulated, there are different pathways to vsig activation. those vsigs with antiviral properties (e.g., mxa, pkr, ′ ′oas/rnasel) inhibit different stages of virus replication and strengthen an antiviral state in the host. while this state is well known, the nature of its induction by different respiratory viruses and the impact of induction upon the replication of other respiratory viruses are topics for considerable ongoing research. one pathway to ifn induction relies on the ifn-upregulated cytosolic sentinels retinoic acid inducible gene rig-i-like receptors (rlrs) rig-i (specifi c for ifav and others) and melanoma differentiation-associated gene (mda , specifi c for picornaviruses and others) [ , ] . these rna helicases recognize either rna with a ′-triphosphate or distinct dsrnas, which results in activation of nf-κb leading to "classical" type i ifn induction [ , ] . studies into the innate response to hrv infection have been limited to the use of a very few easily cultured types. it is presumed that the result can be extrapolated to most if not all types. this is yet to be tested. rig-i is degraded by hrv-a [ ] , ifn regulatory factor (irf)- homodimerization is interfered with hrv-b which limits ifn-β induction [ , ] , and mda is degraded by hrv-a a but not hrv-a [ ] . another pathway for recognizing hrv infection involves the toll-like receptors (tlrs), transmembrane prrs that terminate in an intracellular signaling region. the endosomally localized tlr , tlr , tlr , and tlr recognize nucleic acids and are also involved in innate antiviral responses. tlr and tlr identify g/u-rich ssrna from endocytosed viruses, while tlr recognizes unmethylated cpg dna present in dna viruses [ , ] . tlr and tlr are found on the cell surface and recognize hrv or hrsv proteins, respectively [ , ] , and tlr recognizes dsrna. tlrs operate mainly, but not exclusively, in plasmacytoid dc [ ] . the particular tlr that notifi es of an hrv incursion may depend on the method of virus approach [ ] . tlr activation can reduce ′ ′oas and mxa mrna expression and ip protein in adolescents with asthma compared to healthy controls [ ] . tlr activation did not result in a similar disparity [ ] . it has been suggested that hrvs may have evolved with humans to such an extent that their symbiotic relationship serves to help train the human immune system [ ] . intriguingly, within the hrv species, there are differences in the type and level of host response induced [ ] which may refl ect receptor usage, route of entry and cell type infected, hrv species, or the degree of laboratory-adapted virus used during in vitro studies. after initial hrv infection, the innate response results in production of proinfl ammatory cytokines, vasoactive peptides, and chemokines that attract leukocytes, granulocytes, dcs, and monocytes (table . ) [ , , ] . the t-lymphocyte response to viral intrusion can be broadly categorized as t h - -like and t h- -like. other t-cell subsets exist, but most work in relation to hrv has been conducted on the earliest defi ned subsets. the t h - cellular response is important in managing cellular immunity and producing interleukin (il)- and ifn-γ. the t h - cellular response manages humoral immunity and stimulates b cells via il (initiating production of ige), il (infl uencing eosinophils), and il (crucial component of allergen-induced asthma). these two t-cell responses act in concert with epithelialderived chemokines (e.g., eotaxin) to promote the recruitment and activation of eosinophils and mast cells, contributing to chronic airway infl ammation and the hyperresponsiveness of airways to a variety of nonspecifi c stimuli [ ] . t h- lymphocytes, opposing t h- lymphocytes, contribute to an allergic infl ammatory cascade, akin to what occurs to rid humans of parasites [ ] . the t h- response can also be repressed by binding of microrna, which leads to an altered balance favoring a t h- state in mice and probably in humans [ ] . regulatory t cells (t reg ) suppress allergic infl ammatory pathways and are therefore fundamental in protecting the airway from allergen sensitization [ ] . considerable immunobiological research has focused on asthma exacerbation, with which hrvs are intimately involved. although upregulated by hrv infection, the t h - response is comparatively defi cient in people with asthma [ , ] . this is problematic as an increased t h - -like cytokine response, deduced from higher sputum mrna ifn-γ/il values, speeds clearance of hrv and symptom amelioration [ ] . one possible cause of the t h - defi ciency in people with asthma is inadequate maturation of type i and iii ifn responses due to reduced exposure to infections early in life [ ] . the "hygiene hypothesis" [ , ] posits a pathway for an asthma etiology described [ ] in terms of the young, unchallenged immune system, dependent on infections to stimulate the development of its t h- -like functions. one theory suggests that hrvs play a central role in developing that effi cacious antiviral immunity, particularly in infancy, via their frequent, usually mild self-limiting infections [ ] . genome-wide expression analysis of becs from healthy and asthmatic adult subjects after hrv-a a infection revealed some signifi cant differences that were found between cell types and response to infection [ ] . these included immune response genes (il b, il , il , il f , il ) and airway remodeling genes (loxl , mmp , fn ) and an overall proinfl ammatory response and metabolic slowdown consistent with proteolytic cleavage of transcription factors by some hrvs [ , - ] in the infected cells. this study further noted some similarities to gene expression changes observed in brushings from people with mild asthma after allergen exposure and in bal cells from subjects with corticosteroidresistant asthma [ ] . overall, hrv replication and the host transcriptional response to it were similar in normal or asthmatic bec cells [ ] . this indicated, at least in adults, that something beyond the epithelial cell is an important contributor to more severe clinical outcomes in asthma. the application of inactivated hrv-b was found to promote release of il from monocytes (an immunosuppressive cytokine) and to inhibit the stimulation of il (drives t h - development) [ ] . however, neither il nor il was signifi cantly induced in asthmatic adult volunteers in response to hrv-a compared to healthy subjects [ ] . while ifn-α was detected after transfection of dcs with hrv-b ssrna, low tnf-α and il levels were also noted [ ] . it was posited that the reduced il could indicate negatively affected local immunity possibly predisposing to secondary infections [ ] . infection of stromal lung cells by hrv-b triggered exaggerated levels of the pleiotropic il (an il type cytokine), akin to those triggered by hrsv, which were also detected in nasal secretions from children with wheezing [ ] . other cytokine changes have been identifi ed in atopic adult volunteers challenged with hrv-a . g-csf and il (chemo-attractant for neutrophils) levels rose in the urt (as examined by protein detection in nasal lavage) and lrt (mrna detection in sputum) with concomitant rises in blood and nasal neutrophil numbers [ , , ] . the nasal epithelial cells of atopic individuals, especially in season, express more icam- than those of nonatopic adults [ ] as do normal subjects infected by the major group hrv-b [ ] . by contrast, ifn-γ and il , which appear later postinfection, downregulate icam- expression in infected cells [ ] and encourage infi ltration of neutrophils [ ] , respectively. changes in icam- levels may modify participates in creation of an antiviral state; produced by and infl uences the maturation of dcs il- β proinfl ammatory properties; enhances adhesion molecule expression including icam- ; induces il- receptor gm-csf a granulocyte and monocyte growth factor il- stimulates growth and differentiation of t and b lymphocytes and cytotoxic activity of nk cells and monocytes il- t h differentiation, promotes ige synthesis il- activation, differentiation, and proliferation effects on t and b lymphocytes; induces c-reactive protein stimulating pyrexia il- /cxcl- neutrophil chemoattractant resulting in neutrophilic, monocytic, and lymphocytic recruitment and degranulation activity il- anti-infl ammatory factor produced by monocytes that acts by inhibiting proinfl ammatory cytokines il- , il- , and tnf-α irf a master hub, regulating antiviral immunity ip /cxcl chemoattractant for activated t h and nk cells tnf-α proinfl ammatory activity similar to il- β; activates neutrophils; induces vascular permeability mpc- a monocyte attractant bradykinin potent infl ammatory mediator, increases vascular permeability tslp an il- -like cytokine that activates myeloid dcs to induce naive t cells into t h cells producing il- , il- , and tnf-α; induced by hrv in the presence of il- bec bronchial epithelial cells, dc dendritic cell, irf interferon regulatory factor, ifn-γ inducible cytokine protein, nk natural killer, pbmc peripheral blood mononuclear cells, il interleukin, tnf tissue necrosis factor, tslp thymic stromal lymphopoietin t-lymphocyte-mediated cytotoxic or t h interactions with hrv-infected cells, upregulating receptor expression and encouraging eosinophil and t-cell infi ltration into the lower airways of asthmatic individuals [ , ] . before an hrv can enter a cell, it must pass through a defensive barrier of secreted anti-hrv antibody, mostly iga. the ease with which this passage occurs is proportional to the progression of clinical disease. healthy adult volunteers were found to develop iga by at least days to weeks after inoculation-about the same time as serum antibody-and retain peak levels for at least weeks [ , - ] , falling faster than serum levels [ , ] . there is also some evidence for a degree of nasal immune memory [ ] . volunteers with pre-study serum antibody could still be infected in some studies [ , , ] , but not in others [ ] . infection is more clear in volunteers without preexisting nasal antibody to experimental challenge virus; they become infected, exhibit more severe ari, and shed more virus for longer [ , ] . iga does not seem to modify illness severity or virus shedding, but high levels prevent reinfection by the initiating virus type. low levels or absence of iga does not prevent reinfection by the same hrv type, which may manifest as symptomatic or asymptomatic disease [ ] . older children, adolescents, and adults have greater amounts of hrv-neutralizing antibody than young children [ ] , accompanying a trend toward decreasing numbers of symptomatic aris with increasing age [ , ] . this feature raises an issue: did the use of older subjects in many common cold studies underplay the pathogenic potential of the hrvs because protective or partially cross-protective antibodies moderated the impact of infection? consequently, quantifying levels of type-specifi c serum antibody became routine practice prior to some studies. adult volunteer studies determined that no infections resulted if preexisting neutralizing antibody titers ≥ : existed; as levels grew from , so did levels of resistance to infection [ , ] . adults were protected by serum titers of : - : [ , ] . the trend was interrupted by adults in the - year age group, presumably because they had begun families and their young children acquired and amplifi ed currently circulating types from the community and transmitted them into a household that was either immune naïve or lacking suffi cient antibody or cell-mediated memory for protection [ ] . traditional vaccine strategies were quickly ruled out as a prophylactic intervention for hrv illness because of the extensive antigenic variability that is a hallmark of the genus enterovirus [ , ] . however, if it were possible to identify "master" strains [ ] that exhibit suffi cient antigenic cross-reactivity to induce broad heterotypic responses against many other hrv strains, then an effective vaccine could still be possible. in fact, boosting host immunity to an hrv type by repeat infection does heighten immunity to one or more other types [ , ] . the highest of these heterotypic antibody titers develop against those types with the highest preexisting antibody levels [ ] . the fi rst description of a unifying hrv numbering system recounted the appearance of minor serological cross-reactions, which were removed by modifi cation of the technique [ ] . subsequently, cross-reactions were better defi ned during experimental inoculation when multiple hrv immunogens and antigens were used to deduce the extent of heterotypic responses [ , , , ] . less promising for hrv vaccinology was the description of antigenic variation within hrv types which suggested that immunity to one variant of the type might not protect against infection by other variants [ , ] . the "prime strain" is a specifi c antigenic variant of a prototype hrv type that is neutralized to a lesser extent by antisera from the prototype, while yielding antisera that effectively neutralize both itself and the prototype [ ] . another form of this cross-neutralization is ascribed to the "intertypes," which are hrv isolates that share a lower-level serological relationship with a pair of hrv strains, which themselves share neutralizing reactivity, e.g., hrv-a and hrv-a [ ] . the low-level reciprocal neutralizing activity was not equivalent in both directions; anti-hrv-a sera had a higher titer for hrv-a than anti-hrv-a sera did for hrv-a [ ] . over strains were linked directly by such one-or two-way cross-reactions or indirectly through two or more strains. hrv-a and hrv-a are linked via hrv-a , hrv-a , and hrv-a (anti-a serum neutralizes hrv-a , anti-a neutralizes hrv-a , and anti-a neutralizes both hrv-a and hrva-a [ , ] ). a surrogate molecular method which provided insight into these interrelationships, perhaps expanding upon them to identify useful patterns for vaccine immunology purposes, would be most welcome. in summary, heterotypic immunity and hrv intertypes might be exploitable features of hrv immunobiology that could confer maximum protection upon the host from the minimum number of hrv types [ ] . hrvs circulate in great numbers, and any specifi c roles for distinct hrv types in initiating disease remain to be defi ned. the relatively inconsequential common cold is the most frequent manifestation of viral infection in humans, with to > % of colds positive for an hrv [ , , ] . furthermore, aris due to hrv infection can exacerbate or result in a much greater burden of disease in those with asthma, copd, or cystic fi brosis. other complications include otitis media, pharyngitis, and wheeze in atopic people without asthma. the role of viruses in the origin of some of these diseases or their exacerbation is still unresolved. the lrt disease may mask the urt nature of the infection, favoring clinical diagnosis of an lrt illness. interestingly, during the h n pandemic, much of the parentinitiated healthcare visits from a birth cohort in the united states were not due to pandemic virus but hrv and hrsv [ ] . there is no known natural murine rhinovirus on which to base a small animal model of hrv infection, and mice are not natural hosts for hrvs. a recently developed model of airway disease using mengovirus (a picornavirus infecting rodents) may yield valuable in vivo airway infection and infl ammation data [ ] . hrvs are often detected in neonates and infants with lrt signs and symptoms because the very young have narrow, immature airways and are more signifi cantly affected by airway swelling, excessive secretions, and smooth muscle contraction [ ] . this may also be due to the relatively naive immunity of very young children. much of the more severe disease in hrv-positive children occurs in the youngest of them. some key examples are addressed below. for the common cold, as for any illness, accurate epidemiology and burden of disease data underpin the prioritization of preventing, treating, and further researching the etiological agent. to assign funds for researching the agent, health policy makers also need to understand how effi cacious and costeffective the development of an intervention will be [ ] . the host immune response to hrv replication is the main cause of the signs (quantifi able fever, rhinorrhea) and symptoms (feeling of fever, myalgia, headache, fatigue, and mood change) of a cold that the host experiences [ , , ] . a feature of common colds is increased vascular permeability which, enhanced by kinins, results in increased plasma protein (albumin and immunoglobulin [ig] g) levels in mucus, approaching the levels in serum [ ] . histamine levels do not rise in nasal secretions of otherwise healthy cold sufferers [ ] . during the resolving phase of the ari, glandular proteins (lysozyme, siga) predominate [ ] . the common cold syndrome is also described as rhinosinusitis (the agglomeration of rhinitis and sinusitis since they frequently clinically coexist) [ , ] . this consists of nasal discharge or rhinorrhea, nasal obstruction, sore throat, sinus pain, headache, sneezing, watery eyes, cough, fever, fatigue, muscle aches and pains, and mood changes [ , ] . these are caused directly or indirectly by viral infection; cough is the result of vagus nerve irritation by mucus; sneeze results from trigeminal nerve irritation; sore throat is likely due to the action of prostaglandins and bradykinins; and fever, psychological effects, fatigue, and myalgia are mediated by cytokines [ ] . hypertrophic adenoids have also been found to have a high proportion of viral, especially hrv, occupation regardless of host symptomatic state [ ] . observation of natural culture-confi rmed hrv colds in adults noted that cough usually started by day and was more persistent up to days later [ , ] . rhinorrhea, sneezing, and sore throat were reported by half or more of patients and headache by at least a quarter of cases [ , ] . as neutrophils accumulate at the site of primary urt infection, the myeloperoxidase in their azurophilic granules creates the yellow-green coloration of nasal mucus that was once considered a sign of bacterial superinfection [ , ] . a common cold caused by an hrv cannot be clinically distinguished from one that caused by any of the other respiratory viruses [ , ] . as is likely for a single hrv type, once the host has been infected by an hmpv, hpiv, ifv, etc., a secondary exposure to that same virus type will produce less severe clinical outcomes due to pre-primed host immunity. asthma is a clinical diagnosis made on the basis of patient history, physical examination, assessment of airway obstruction or reversibility, and response to bronchodilators [ ] . it is a complex chronic respiratory disease involving airway infl ammation, airfl ow obstruction, and airway hyperresponsiveness, which manifests as recurrent reversible attacks with deteriorating asthma control that are generated by interactions between infectious agents and other environmental and genetic factors that remain incompletely characterized [ ] . the mechanistic role for hrvs in asthma inception and exacerbation is not yet defi ned [ , ] but is being revealed as the extremely complex interplay between infl ammation due to virus versus that due to atopy is explored [ ] . possible virus-host interactions include (i) severe hrv infection of healthy infants which may result in subsequent development of asthma; (ii) hrvs may trigger asthma in children with a genetic predisposition toward atopy; (iii) repeated mild infections may protect against more asthmogenic/cytopathic viruses or the overdevelopment of the t h type response; and (iv) hrvs may simply exacerbate that which already exists [ ] . it is unclear if the risk of atopic asthma during infancy is increased by aris which affect the development of the immune system, or whether aris lead to asthma development in children with a genetic predisposition to more severe responses to infection [ , , ] , or a mix of both. in children with asthma, viruses have been detected in at least % of exacerbations ( % picornaviruses, probably hrvs [ ] ) and in % of adults [ ] . acute wheezing episodes (including bronchiolitis and acute asthma) are a frequent, epidemic, and seasonal lrt manifestation of urt respiratory virus infection of children from all ages, especially during the fi rst year of life [ , - ] . bacteria are not major factors in wheezing exacerbations [ ] . wheezing is blamed for high socioeconomic and healthcare costs, overuse of antibiotics, being the primary cause of hospitalization among children, and, rarely, for death [ , , ] . traditionally hrsv infection has most often been the virus causally associated with expiratory wheezing, wheezy bronchitis, or asthma exacerbations because of the virus's well-known ability to infect the lrt, its more frequent detection in some studies [ ] , and the low perceived likelihood of urt viruses such as hrvs replicating in the warmer lrt. nonetheless, periods of epidemic wheezing in the absence of high rates of hrsv detection are common [ , ] . hrvs even predominated in some culture-based studies of wheeze [ , ] . the coast study used sampling criteria that were intentionally designed to investigate the role of hrsv in illness, but instead indicated that hrvs were the most important predictor of subsequent wheezing in early childhood, and this is supported worldwide [ , , ] . the asthmatic airway is characterized by an infi ltration of eosinophils and th -type t cells (th cells) [ ] . in those with an atopic background, eosinophilia was more common, and the virus isolation rate was higher than in the nonatopic group [ ] . the cytokine and eosinophil activation profi les for hrsv-induced wheezing differ from those induced by hrv in which il is signifi cantly higher in serum and nasal aspirates than for hrsv [ ] . ip was the only cytokine signifi cantly elevated in all symptomatic wheezing groups [ ] . signifi cantly higher rates of hrv detection with more obvious lrt symptoms are more common in children with asthma than in non-asthmatic populations [ , , , ] . exacerbations of asthma are often preceded by a symptomatic rather than asymptomatic hrv infection [ , , , - ] although, in some instances, an exacerbation is the only sign of infection [ ] . reduced peak expiratory volume in children is especially associated with detection of respiratory picornaviruses [ ] . severe "wheezy bronchitis," a historical term describing an acute illness with preceding ari and characterized by cough, wheezing, breathlessness, and mucous production, was more often positive for a virus than mild disease [ ] . even the use of culture found that hrvs predominated in both urt and lrt (sputum containing becs) or combined respiratory tract samples [ ] . bacteria were often present with ifv, but not with hrvs [ ] . the airway epithelial cells form a physical barrier in addition to their roles in immune surveillance and regulatory control [ ] . however, the asthmatic bronchial epithelium is compromised by incomplete tight junctions that are more sensitive to airborne pollutants [ ] and most likely to allergens and respiratory viral infections. this is further specifically disturbed by hrv infection which reduces expression levels of tight and adherens junction proteins [ ] . in those with asthma, the presence of an hrv can induce illness that, while often more severe than in non-asthmatics, has been associated with signifi cantly different hrv load or duration of hrv rna detection in people with asthma compared to those without [ ] . hrv-c types are often detected in more serious clinical outcomes than hrv-a or -b [ ] although hospitalizations may be fewer for hrv-cs than the other species [ ] . aom is diagnosed by middle ear effusion (otorrhea) with simultaneous signs and symptoms of ari including fever, earache, rhinitis, cough, sore throat, chest wheeze, nocturnal restlessness, irritability, poor appetite, diarrhea, and vomiting. transient abnormal (negative) ear pressure upon tympanometry occurs in two-thirds to three quarters of uncomplicated colds among healthy children [ , ] . aom is a frequent reason for outpatient antibiotic therapy which can reduce the time to resolution of symptoms in infants and has been attributed to reducing the overall hospital burden of aom [ - ] . since a longitudinal day-care study in , the association between aom and viral urt infection has been coalescing, and it is now clear that aom often occurs with or shortly after a viral ari, most frequently in the young and occurring more often during winter than summer [ , ] . the use of infl uenza vaccines reduced aom occurrence by a third during an epidemic period [ ] , but the use of pneumococcal vaccine did not reduce the occurrence of aom overall, just that relatively small fraction ( %) due to the target bacteria [ ] . the isolation by culture and pcr detection of viruses from middle ear fl uids and the refractory nature of some aom cases to antibiotic therapies confi rmed that viruses play an important role in this illness [ , , ] . studies relying on underperforming culture-based techniques underestimated the role for viral aris [ , ] , but other studies using pcr techniques and including hrvs found them to be the most frequently detected virus in middle ear fl uids and nasopharyngeal secretions [ , ] . the use of pcr has identifi ed respiratory viruses, most often hrvs, in nasal secretions of - % of children with aom [ , ] . because virus is often detected in the nasopharynx at the same time as the middle ear fl uid, the question of the relevance of a pcr positive is a valid one [ ] . picornaviruses have been detected in % of nasopharyngeal swabs taken during cold season from aom-prone infants and young children, and large quantities of hrv rna have been detected by in situ hybridization of adenoid tissues from % of children with recurrent aom and/or adenoid hyperplasia [ , ] . in a cohort of children followed from to months and using culture-rt-pcr, hrvs in the urt were the second most frequent pathogens associated with aom, after hrsv [ ] . viruses, most often hrvs ( . % of aom with ari), were also detected concurrently with non-ari periods associated with aom episodes ( % of aom without ari) [ ] suggesting that aom may be the only manifestation of some hrv aris, just as wheezing sometimes is. in the united states, subjects were enrolled and followed in a birth cohort until the fi rst aom episode or between and months of age ]. hrvs accounted for % of viruses detected and % of specimens with a single virus detected. this dominance was maintained even through the h n infl uenza pandemic [ ] . in the day-care aom study mentioned above, primary acquisition of streptococcus pneumoniae or haemophilus infl uenzae had minimal importance as an initiation factor for aom with effusion, but nasopharyngeal colonization was important [ ] . animal studies have shown that virusbacteria interactions have a role in nasopharyngeal colonization and aom development [ ] . positive correlation has been made between hrv detection in aom-prone children and moraxella catarrhalis infection as well as a tendency toward the copresence of streptococcus pneumoniae [ ] . the presence of hrv-b was shown to increase adherence of s. pneumoniae in human tracheal epithelial cell cultures [ ] . it is believed that these three bacterial pathogens can colonize without symptoms until a viral ari shifts the balance toward a cytokine-mediated infl ammatory state [ ] . other diseases in which hrvs are often detected this disorder of older patients encompasses emphysema (alveolar destruction) and chronic bronchitis (large airway infl ammation with chronic mucous production) and describes a long-term obstruction to airfl ow in the lung (compared to asthma which is a reversible obstruction with normal fl ow between exacerbations). while bacteria are found in half of all exacerbations, antibiotic therapies have often yielded poor outcomes [ ] . hrv infections result in more copd exacerbations (~ % of cases [ ] ) than any other virus identifi ed to date [ , ] . an experimental human model of hrv infection in copd provided preliminary evidence that hrvs cause exacerbations [ ] . viral culture associated symptomatic hrv infections with exacerbations among chronic bronchitics, including cases of isolation from sputum (lrt sample) in the absence of hrv in the urt [ ] . adding the measurement of an infl ammatory marker in the serum, like il- , further improves the speed of predicting an infectious etiology for exacerbations of copd [ ] . pneumonia is a disease that often occurs early in life, is responsible for millions of deaths each year [ ] , and is caused by viral and/or bacterial infections. a diagnosis of pneumonia requires a radiologically confi rmed infl ammatory infi ltration of the lung tissue. childhood communityacquired pneumonia (cap) is common in developing countries [ ] . cap also complicates existing chronic medical conditions and takes advantage of immunosenesence [ ] . the role of hrvs in contributing to the development of bacterial pneumonia is likely underestimated [ , ] . determining an etiology is confounded by the rarity of obtaining lrt specimens, by short-term studies, and by the complex milieu of viruses and bacteria involved. less invasive sampling of the urt permits more routine sampling and screening, and so convenience and reduced risk have led to the detection of putative pathogens in the urt with the general assumption that they account for lrt disease, especially in children under the age of years [ ] . pneumonia studies are complicated by the lack of a suitable control group; sputum is not produced from the healthy lower airway and needle aspiration, while a gold standard is also a hospital procedure with some risk [ ] . studies that are comprehensive and use sensitive molecular testing are also rare for the study of cap etiology. when used for cap investigations, pcr methods almost double the microbiological diagnoses over conventional culture and serology techniques, especially improving the identifi cation of mixed infections and fastidious viruses [ ] . rapid diagnosis aids management and helps make decisions about treatment, while prolonged searching for an etiological agent leads to further invasive testing [ , ] . at least a quarter of clinical cap cases remain unsupported by microbiological fi ndings [ , ] . infections causing pneumonia vary with age and vaccination status [ ] . viruses can be detected in up to % of infants ( - months of age) with pneumonia, and these cases follow a seasonal pattern [ , ] . bacteria can also be detected in over % of infants and older children, the elderly, and those with severe cap [ , ] . studies that predated the use of pcr pronounced hrsv, followed by hrvs, the major viral contributors to cap, with viruses comprising - % of childhood pneumonia cases [ , ] . in the pcr age, the role of hrvs has received increasing attention, and they are increasingly the major viral group detected from both urt and lrt (sputum) specimens of children with cap. this holds true even when studies extend across or more years, which presumably would account for seasonal variation in virus prevalence [ , , , ] . it is suspected that viruses such as hrv prepare the way for subsequent bacterial infection in some direct or indirect fashion [ , , , ] . there are laboratory data which support this [ ] as well as observational data showing a high proportion of hrvbacterial co-detections [ , ] . mixed infections including viruses are a possible cause of antibacterial treatment failure and sometimes a puzzle for physicians. mixed infections occur frequently in lrt diseases such as pneumonia, which is not surprising since new techniques make it clear that the lungs are not the sterile environments we once thought [ , , , ] . viral-bacterial coinfections can comprise % of patients, while viral-viral ( - %) and bacterial-bacterial ( - %) are much less common [ , , , , ] . hrsv or hrv is often co-detected with s. pneumoniae in urt samples [ , , ] . hrv detections dominate in younger children with pneumonia during peak hrv seasons, although frequently in co-detections with other viruses [ ] . acute bronchitis (less than -week duration in children) is defi ned as a sudden cough that often results from large airway infection and frequently involves viruses. croup or laryngotracheobronchitis (viral or recurrent [ ] ) is a common lrt illness in children that includes the trachea and larynx as well as the larger airways, resulting in a barking cough. patients with croup most often have a viral infection with some role for hrvs, although the extent of this is unclear [ , ] . despite testing, a third of cases remain without a viral etiology [ ] . tracheobronchitis resulted from some hrv-a infection of volunteers [ ] . chest pain and cough have been reported in half or more of adults with hrv infection [ ] as well as in children and adults with hrvs detected during exacerbations of bronchitis, with or without an associated ari [ , ] . bronchiolitis occurs seasonally, especially in winter, in infants ( - months of age), affecting the small peripheral bronchioles. winter is the peak season for hrsv circulation, but not usually for hrv. bronchiolitis is a clinical diagnosis encompassing various disease entities and is most often reported in association with detection of hrsv, a winter virus [ , ] . however, hrvs make up the majority of hrsv-negative bronchiolitis cases [ ] , and hrvs are co-detected with hrsv for which hospitalization is prolonged compared to cases positive for either virus alone [ ] . those children positive for an hrv during a clinically diagnosed bout of bronchiolitis have a signifi cantly higher risk of recurrent wheezing in the subsequent year than those in whom another virus is detected [ ] . hrvs were reported in over fi vefold more cases of bronchiolitis than hrsv among patients in a -year prospective cohort of very low birth weight infants in buenos aires, argentina [ ] . after a viral ari, some proportion of infections may be complicated by sinusitis (infl ammation of the sinus mucosa), the extent of which may be underestimated in children if the ari is mild and unattended by parents [ ] . symptoms may include sinus pain, headache, facial pain, discolored nasal discharge, postnasal drip, cough, sore throat, malaise, and sometimes fever (more so in children) [ , ] . the precise role for viruses and bacteria in sinusitis is still unclear [ ] . sinusitis is a common comorbidity in those with asthma [ ] . the in situ presence of hrv-b rna in maxillary sinus epithelium was reported in seven of adults with acute sinusitis [ ] . hrvs were also detected by pcr in half of adults with acute maxillary sinusitis; half of the hrv positives were negative for any bacteria [ ] . the common cold is often associated with computed tomographically confi rmed sinus cavity occlusion or abnormality in adults with self-diagnosed aris [ , ] . magnetic resonance imaging identifi ed reversible abnormalities of the paranasal sinuses in a third of healthy adult volunteers following challenge with hrv-a [ ] . further evidence of the tropism of hrvs for sinus tissue comes from it being, so far, the only successful host for in vitro hrv-c replication [ ] . culture-and serology-based testing has shown that virus infections in cystic fi brosis (cf) patients occur with the same prevalence as the general community, but the consequences of infection are more obvious or severe. these include deterioration of lung function, cough, increased expectoration and weight loss, and a synergistic increase in bacterial growth or acquisition of new bacterial infections [ , - ] . the mechanism behind the acquisition of new bacteria is still unknown and not always observed [ ] , but may involve a reduction in the host's immune response or viral damage to the respiratory epithelium. there is circumstantial evidence that hrv infections have been associated with respiratory exacerbations in cystic fi brosis patients [ , ] , albeit in very low numbers by nonmolecular studies [ ] and without a significantly different clinical outcome from non-hrv aris in these patients [ ] . molecular methods have not yet been applied regularly, thoroughly, and systematically, but they generally fi nd hrvs to be prominent among cf children with ari-associated respiratory exacerbation and involved in mixed viral-bacterial infections [ ] . hand washing and disinfectant wipes have been shown to be effective methods of interrupting transfer from fomites to the nose or to conjunctivae [ , , ] . however, with eye rubbing, face touching, and nose-picking occurring frequently [ , ] , self-inoculation often outpaces personal hygiene, particularly in the young. hand disinfection is frequently recommended for prevention of hrv infection but has not been supported by controlled clinical trials in a natural setting [ ] despite good results in experimental tests [ ] . ethanol-containing disinfectants were more effective than simple hand washing with soap and water for removal of hrv-a inoculum, as assessed by culture, and the inclusion of organic acids afforded a residual antiviral effect [ - ] . however, continual hand washing with extra ingredients resulted in skin irritation [ ] . the experimental testing [ , ] may have been biased by short study periods, the absence of a mucus carrier to mimic natural surface deposition and overly stringent control over virus application/hand disinfection compared with the natural study. additionally, the natural setting study used pcr [ ] which detects hrvs more often than culture. the disparity between outcomes may also refl ect the contribution of airborne hrv transmission. because of the absence of a vaccine or specifi c antiviral, the most popular method of intervention in uncomplicated hrv aris is treatment of the symptoms. this is achieved using analgesics, decongestants, antihistamines, and antitussives. due to a lack of studies, data are limited on the effectiveness of over-the-counter common cold medications for children [ ] . anticholinergic agents have proven useful to reduce rhinorrhea [ ] . for controlling symptoms in those with exacerbated asthma, most of which do not require hospitalization, bronchodilators and oral corticosteroids are the main treatments [ ] . the interruption of proinfl ammatory immune responses or specifi c signaling pathways using steroids, or other novel therapeutics, may prove to be a more robust approach for treating hrv infections; they have not been successful for hrsv [ ] . when initiated early in the illness, a combination of antiviral (ifn-α b) and anti-infl ammatory (chlorpheniramine) components showed promise for interrupting nasal viral replication and symptoms [ ] . antiviral agents (table . ) require early application to effectively precede the pathogenic immune response to hrv infection [ ] , but they often fail to reproduce their in vitro successes in vivo. most antirhinoviral drugs are based on capsid-binding agents (fig. . ) . additionally, oral delivery can complicate drug safety because this route increases the risk of systemic side effects compared to a nasal or topical route, but these risks must be considered alongside the disease to be treated; drug side effects are disproportionately severe compared to a common cold than to a severe asthma exacerbation. a systemic route is benefi cial if an effect is sought on hrv replication sites that are otherwise inaccessible, such as those not associated with respiratory tract illness [ ] . the recent discovery of the new species, hrv-c, has shone a bright light on how little was known about the hrvs. the hrv-cs and also the newly discovered hrv-as and hrv-bs are fastidious in culture, with a single report of hrv-c growth in primary sinus tissue, and the identity of a cellular receptor still unknown. thus, it is diffi cult to proceed in many areas, including basic virology, seroepidemiology, immunobiology, and antiviral testing. determination of the receptors for these new hrvs would aid the search for a more accessible culture system. there would be great interest in a vaccine for some or all of the hrvs, but with increasing evidence of the interactions between hrvs, their hosts, and other respiratory viruses, it may not be wise to interfere before we fully understand what the impact of losing a constantly circulating hrv challenge would be. antivirals specifi cally targeting the hrvs may be a better bet, but routine hrv testing and genotyping will fi rst need to be more widespread as surveillance for antiviral resistance will be an important component of monitoring the success of any intervention. studies to determine whether there are differences in clinical and immunobiological impact between the many different types are lacking but would greatly improve our ability to plan future routine testing, understand all the clinical responses to the diverse hrvs and to outbreaks of ari, and improve hrv epidemiology. it is interesting to note that the hrv-bs are signifi cantly underrepresented in hrv detections. we do not yet know their niche or clinical impact. it may be possible that hrv-bs are the most well adapted of the hrvs, causing little to no detectable clinical impact, or they may create a different impact than that which we expect, or they may be a species in decline. the jury remains out on whether hrvs cause or are involved in the development of asthma or merely trigger exacerbations once asthma is established. with a very high healthcare impact from asthma around the world and atopic conditions that may be exacerbated by hrvs on the rise, this is an important area for 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and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures viral stress-inducible genes rhinovirus induces airway epithelial gene expression through double-stranded rna and ifndependent pathways the interferon response circuit: induction and suppression by pathogenic viruses il- and ifn-alpha regulate the expression of mxa, ′, ′-oas and pkr genes in association with the activation of raf-mek-erk and pi k-akt signal pathways in hepg . . cells the interferon-inducible rna helicase, mda- , is involved in measles virus-induced expression of antiviral cytokines rig-i is cleaved during picornavirus infection human rhinovirus attenuates the type i interferon response by disrupting activation of interferon regulatory factor attenuation of the type i interferon response in cells infected with human rhinovirus mda- is cleaved in poliovirus-infected cells interferons: signaling, antiviral and viral evasion sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion viral infections and atopy in asthma pathogenesis: new rationales for asthma prevention and treatment innate and adaptive immune responses to viral infection and vaccination principles of intracellular viral recognition viruses and toll-like receptors human rhinovirus recognition in non-immune cells is mediated by toll-like receptors and mda- , which trigger a synergetic pro-infl ammatory immune response toll-like receptor function is reduced in adolescents with asthma modulation of the immune system by human rhinoviruses diversity in the bronchial epithelial cell response to infection with different rhinovirus strains host defense function of the airway epithelium in health and disease: clinical background the common cold at the turn of the millennium relationship of viral infections to wheezing illnesses and asthma innate and adaptive immune responses in asthma microrna- limits in vivo immune response-mediated activation of the il- /ifn-gamma pathway, th polarization, and the severity of delayed-type hypersensitivity a defective type response to rhinovirus in atopic asthma acute severe asthma innate immunity in the pathogenesis of virusinduced asthma exacerbations hay fever, hygiene, and household size family size, infection and atopy: the fi rst decade of the "hygiene hypothesis microbial manipulation of immune function for asthma prevention rhinovirus c protease precursors cd and cd' localize to the nuclei of infected cells human rhinovirus a proteinase cleavage sites in eukaryotic initiation factors (eif) gi and eif gii are different rhinovirus a proteinase mediated stimulation of rhinovirus rna translation is additive to the stimulation effected by cellular rna binding proteins human major group rhinoviruses downmodulate the accessory function of monocytes by inducing il- rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production interleukin- : stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness nasal-secretion leukocyte populations determined by fl ow cytometry during acute rhinovirus infection expression of intercellular adhesion molecule- (icam- ) in nasal epithelial cells of atopic subjects: a mechanism for increased rhinovirus infection? rhinovirus infection of human embryonic lung fi broblasts induces the production of a chemoattractant for polymorphonuclear leukocytes immune responses to viral infections: relevance for asthma the role of nasal secretion and serum antibody in the rhinovirus common cold an elisa for the detection of rhinovirus specifi c antibody in serum and nasal secretion analysis of nasal secretions during experimental rhinovirus upper respiratory infections further characterization of the local respiratory tract antibody response induced by intranasal instillation of inactivated rhinovirus vaccine the time course of the humoral immune response to rhinovirus infection evaluation of an enzyme-linked immunosorbent assay that measures rhinovirusspecifi c antibodies in human sera and nasal secretions effect of rhinovirus infection on cellular immune parameters in allergic and nonallergic subjects rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes antibody to rhinovirus in human sera. ii. heterotypic responses homologous and heterologous resistance to rhinovirus common cold antigenic groupings of rhinovirus serotypes antigenic variation among strains of rhinovirus type hyper-antigenic variation occurs with human rhinovirus type antigenic variation of rhinovirus type isolation of rhinovirus intertypes related to either rhinoviruses and or and is a rhinovirus vaccine possible? allergens, viruses, and asthma exacerbations viruses and bacteria in the etiology of the common cold effect of the infl uenza a/h n pandemic on viral respiratory infections in the fi rst year of life lower respiratory tract infection induced by a genetically modifi ed picornavirus in its natural murine host viruses as precipitants of asthma symptoms ii. physiology and mechanisms infl uenza vaccination: policy versus evidence: no gap between policy and evidence mechanisms of symptoms of the common cold and infl uenza mechanisms of the symptoms of rhinosinusitis kinins are generated in nasal secretions during natural rhinovirus colds european position paper on rhinosinusitis and nasal polyps european position paper on rhinosinusitis and nasal polyps . a summary for otorhinolaryngologists development of a predictive index for picornavirus infections frequent detection of respiratory viruses by real-time pcr in adenoid samples from asymptomatic children rhinovirus infections in an industrial population. ii. characteristics of illness and antibody response prevalence of asthma symptoms and atopic disorders in preschool children and the trend over a decade acute exacerbations of asthma: epidemiology, biology and the exacerbation-prone phenotype gene-environment interactions in asthma viral infections and asthma inception rhinovirus infections more than a common cold community study of role of viral infections in exacerbations of asthma in - year old children respiratory viruses and exacerbations of asthma in adults rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care virus-induced eosinophil mediator release requires antigen-presenting and cd + t cells the etiologic and epidemiologic spectrum of bronchiolitis in pediatric practice role of viral infection and host factors in acute episodes of asthma and chronic bronchitis the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children how viral infections cause exacerbation of airway diseases viruses as precipitants of asthma symptoms. i. epidemiology the childhood origins of asthma (coast) study viral infection in wheezy bronchitis and asthma in children viral specimen collection by parents increases response rate in populationbased virus studies rhinovirus illnesses during infancy predict subsequent childhood wheezing tlr -and th cytokinedependent production of thymic stromal lymphopoietin in human airway epithelial cells role of viruses and bacteria in acute wheezy bronchitis in childhood: a study of sputum greater frequency of viral respiratory infections in asthmatic children as compared with their nonasthmatic siblings asthma exacerbations in children are associated with rhinovirus but not human metapneumovirus infection role of viral infections, atopy and antiviral immunity in the etiology of wheezing exacerbations among children and young adults synergism between allergens and viruses and risk of hospital admission with asthma: case-control study viruses as precipitants of asthmatic attacks in children viral respiratory infections in asthmatic children staying in a mountain resort defective epithelial barrier function in asthma rhinovirus infection-induced alteration of tight junction and adherens junction components in human nasal epithelial cells enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increased viral load in the upper respiratory tract prevalence and clinical characterization of a newly identifi ed human rhinovirus c species in children with acute respiratory tract infection effect of upper respiratory tract infection on eustachian tube ventilatory function in the preschool child the antiviral compound enviroxime targets the a coding region of rhinovirus and poliovirus importance of respiratory viruses in acute otitis media prevalence of various respiratory viruses in the middle ear during acute otitis media respiratory virus infection as a cause of prolonged symptoms in acute otitis media is acute otitis media a treatable disease? antimicrobials for acute otitis media? a review from the international primary care network a placebo-controlled trial of antimicrobial treatment for acute otitis media a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion role of viruses in middle-ear disease infl uenza vaccination in the prevention of acute otitis media in children effi cacy of a pneumococcal conjugate vaccine against acute otitis media rhinovirus in acute otitis media temporal relationships between colds, upper respiratory viruses detected by polymerase chain reaction, and otitis media in young children followed through a typical cold season presence of viral nucleic acids in the middle ear: acute otitis media pathogen or bystander? rhinovirus in adenoid tissue effects of rhinovirus infection on the adherence of streptococcus pneumoniae to cultured human airway epithelial cells diagnosis of pathogens in exacerbations of chronic obstructive pulmonary disease overview of virus-induced airway disease virological studies in chronic bronchitis identifying viral infections in vaccinated chronic obstructive pulmonary disease (copd) patients using clinical features and infl ammatory markers. infl uenza other respi viruses viral pneumonia viral pneumoniae in children: incidence and aetiology community-acquired viral pneumonia childhood community-acquired pneumonia community-acquired pneumonia in children etiological diagnosis of childhood pneumonia by use of transthoracic needle aspiration and modern microbiological methods improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction diagnosis and management of pneumonia in children viruses and bacteria in sputum samples of children with community-acquired pneumonia a study of pneumonia in a rural area in southern alabama respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell type-dependent manner etiology of communityacquired pneumonia in hospitalized children atypical bacterial infections explained by a concomitant virus infection metagenomic analysis of respiratory tract dna viral communities in cystic fi brosis and non-cystic fi brosis individuals evolution of the df cftr mutation the viral aetiology of croup and recurrent croup croup: an -year study in a pediatric practice rhinovirus neutralizing antibody responses and their measurement rhinovirus infection in acute exacerbations of chronic bronchitis: a controlled prospective study respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants rhinovirus bronchiolitis and recurrent wheezing: -year follow-up prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis human rhinoviruses in severe respiratory disease in very low birth weight infants intranasal fl unisolide spray as an adjunct to oral antibiotic therapy for sinusitis respiratory illness caused by picornavirus infection: a review of clinical outcomes rhinitis, sinusitis, and asthma rhinovirus rna in the maxillary sinus epithelium of adult patients with acute sinusitis computed tomographic study of the common cold physiologic abnormalities in the paranasal sinuses during experimental rhinovirus colds viral infections of the respiratory tract in patients with cystic fi brosis respiratory infections in cystic fi brosis patients caused by virus, chlamydia and mycoplasma -possible synergism with pseudomonas aeruginosa clinical manifestations of exacerbations of cystic fi brosis associated with nonbacterial infections association of respiratory viral infections with pulmonary deterioration in patients with cystic fi brosis the role of respiratory viruses in cystic fi brosis rhinovirus c and respiratory exacerbations in children with cystic fi brosis infective respiratory exacerbations in young adults with cystic fi brosis: role of viruses and atypical microorganisms a randomized trial of the effi cacy of hand disinfection for prevention of rhinovirus infection effectiveness of hand sanitizers with and without organic acids for removal of rhinovirus from hands effi cacy of organic acids in hand cleansers for prevention of rhinovirus infections virucidal hand treatments for prevention of rhinovirus infection over-the-counter cold medications: a critical review of clinical trials between and ipratropium nasal spray: a new treatment for rhinorrhea in the common cold a -versus -day course of oral corticosteroids for children with asthma exacerbations who are not hospitalised: a randomised controlled trial combined antiviralantimediator treatment for the common cold in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus c protease treatment of picornavirus infections the common cold: control? uncommon(ly considered) manifestations of infection with rhinovirus, agent of the common cold intranasal interferon-a treatment of experimental rhinoviral colds human tolerance and histopathologic effects of long-term administration of intranasal interferon-a safety and effi cacy of intranasal pirodavir (r ) in experimental rhinovirus infection combating enterovirus replication: state-of-the-art on antiviral research rhinovirus chemotherapy a comparison of the anti-rhinoviral drug binding pocket in hrv and hrv a a new oral rhinovirus inhibitor bta human rhinovirus c protease as a potential target for the development of antiviral agents in vitro resistance studies of rupintrivir, a novel inhibitor of human rhinovirus c protease effi cacy of tremacamra, a soluble intercellular adhesion molecule , for experimental rhinovirus infection inhibitory effects of tiotropium on rhinovirus infection in human airway epithelial cells levofl oxacin inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells pellino- selectively regulates epithelial cell responses to rhinovirus azithromycin induces antiviral responses in bronchial epithelial cells suggested reading newly identifi ed human rhinoviruses: molecular methods heat up the cold viruses do rhinoviruses reduce the probability of viral codetection during acute respiratory tract infections? human rhinoviruses: the cold wars resume proposals for the classifi cation of human rhinovirus species c into genotypically-assigned types cold wars: the fi ght against the common cold acknowledgments we wish to sincerely thank the following for valuable discussions: john upham, anne chang, danielle wurzel, michael nissen, ron turner james gern, and stephen b. liggett. we are grateful for the extreme patience of corin and ronan mackay, slightly less so for their frequent provision of our fi rsthand experience in hrv clinical symptoms. key: cord- - hcgdlmt authors: bhuvanath, s.; nilkaeo, a. title: inflammatory cytokine modulation of cancer cell proliferation date: - - journal: scand j immunol doi: . /j. - . . bi.x sha: doc_id: cord_uid: hcgdlmt inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell‐mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il‐ ( and il‐ can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf‐ ), oral carcinoma cell line (kb), colon cancer cell line (caco‐ ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - ewi dka authors: nan title: primary immunodeficiencies date: journal: pediatric allergy, asthma and immunology doi: . / - - - - _ sha: doc_id: cord_uid: ewi dka primary immunodeficiencies (pids), once considered to be very rare, are now increasingly recognized because of growing knowledge in the immunological field and the availability of more sophisticated diagnostic techniques and therapeutic modalities [ ]. however in a database of > , inpatients of a general hospital for conditions suggestive of id patients were tested, and an undiagnosed pid was found in ( %) of the subjects tested [ ]. the publication of the first case of agammaglobulinemia by bruton in [ ] demonstrated that the pid diagnosis is first done in the laboratory. however, pids require specialized immunological centers for diagnosis and management [ ]. a large body of epidemiological evidence supports the hypothesis of the existence of a close etiopathogenetic relation between pid and atopy [ ]. in particular, an elevated frequency of asthma, food allergy (fa), atopic dermatitis and enteric pathologies can be found in various pids. in addition we will discuss another subject that is certainly of interest: the pseudo-immunodepressed child with recurrent respiratory infections (rris), an event that often requires medical intervention and that very often leads to the suspicion that it involves antibody deficiencies [ ]. structural genes, and also perhaps on the lack of / c genes [ ] . mbl deficiency is due to one of three point mutations in the gene for mbl, each of which reduces levels of the lectin by interfering with the protein oligomerization [ ] . in children with this kind of deficiency, the level of mbl is . mg/l compared to the mg/l in controls [ ] . regardless of whether the children are homozygote (hz) or heterozygote (het) in relation to a given mutation, the defect appears to be more consistent in small babies aged - months [ ] , who show an immaturity in providing immune response to capsular bacteria and in whom low levels of opsonin are incapable of compensating for this [ ] . the risk of contracting infections is similar in hzs [ ] and hets [ ] , though it persists throughout life in hzs because of an abnormal allele, while it exhausts itself in the hets, where the frequency of abnormality is similar to that of the general population [ ] . anomalies in immunoglobulins (ig) and in opsonization have been observed, respectively in %- % of children suffering from frequent asthma and igg subclass deficiencies. children suffering from cystic fibrosis also present an elevated prevalence of immediate cutaneous reactions to aeroallergens, and although without primary defects of adoptive immunity, they are susceptible to severe rris; therefore it may be possible that they suffer from the mucosal antigenic exclusion [ ] . unlike asthmatic children, in whom a relatively high concentration of ige for respiratory viruses was observed [ , , ] , positive skin prick tests (spts) are more common for aspergillus fumigatus [ ] . the hypothesis suggested by these observations is that atopy derives from an unbalanced immune response to foreign antigens, with a consequent lack of their early identification or the capacity to neutralize or eliminate them. this hypothesis is based on the evidence that id precedes the development of atopy: in the taylor et al studies, newborn babies, the children and/or siblings of atopic patients, presented a significant reduction in serum concentrations of iga when aged months: this was transient hypogammaglobulinemia (hgg) of infancy (thi). the association of very low iga levels with atopy has been proposed again in the classic prospective study on the association of viral respiratory infections (vri) and the onset of allergic manifestations, which proved serum iga levels at the lowest normal levels in the children studied [ ] . this data has been confirmed within primary immunodeficiencies (pids), once considered to be very rare, are now increasingly recognized because of growing knowledge in the immunological field and the availability of more sophisticated diagnostic techniques and therapeutic modalities [ ] . however in a database of > , inpatients of a general hospital for conditions suggestive of id patients were tested, and an undiagnosed pid was found in ( %) of the subjects tested [ ] . the publication of the first case of agammaglobulinemia by bruton in [ ] demonstrated that the pid diagnosis is first done in the laboratory. however, pids require specialized immunological centers for diagnosis and management [ ] . a large body of epidemiological evidence supports the hypothesis of the existence of a close etiopathogenetic relation between pid and atopy [ ] . in particular, an elevated frequency of asthma, food allergy (fa), atopic dermatitis and enteric pathologies can be found in various pids. in addition we will discuss another subject that is certainly of interest: the pseudo-immunodepressed child with recurrent respiratory infections (rris), an event that often requires medical intervention and that very often leads to the suspicion that it involves antibody deficiencies [ ] . in several pids (table . ) [ , , , , , , , , , ] , atopic symptoms are present: gastroenteric and rhinitis in selective iga deficiency (sigad), severe ad in wiskott-aldrich syndrome (was) and hyper-ige syndrome (higes), in which it spreads over the entire body, and to which other allergic manifestations can also be associated such as asthma, rhinitis and angioedema. various acquisitions indicate that pid is also an opsonization deficiency, observed in % of the normal population [ ] . in this disease, microorganism phagocytosis by polymorphonuclear (pmn) leukocytes appears annulled, and the patient is subject to severe infections supported by capsular bacteria: the deficiency, described in association with severe and recurrent infantile infections [ , , ] , depends on the lack of mannose-binding lectin (mbl) [ ] , its primary immunodeficiencies a possible atopy dependence on iga underproduction rather than on ige hyperproduction ( fig. . ): in children with levels of iga at the minimum normal level, and followed from birth until the age of - months, a greater severity of atopic manifestations and an increased cumulative incidence of asthma, ad and otitis media with effusion (ome) were observed compared to controls. the close links between id and atopy are confirmed by symptoms similar to ad present in some forms of was ( %), higes ( %), xla (x-linked agammaglobulinemia) or autosomal recessive (ar), ataxia-telang- chapter primary immunodeficiencies proposed that in these patients cd -th levels are sufficient for modulating ige synthesis, but cd t-cell levels are inadequate for inhibiting ige synthesis, which results in increased ige synthesis. this hypothesis is supported by the observation that omenn syndrome, was and especially higes, with an immunological phenotype characterized by a quantitative and qualitative reduction of cd t cells, are accompanied by extremely high levels of serum ige [ , , ] . lymphocytes in subjects with normal levels of ige are incapable of producing them, not even after stimulation with polyclonal activators such as pwm (pokeweed mitogen) or ebv (epstein-barr virus), while patients with high antibody levels spontaneously synthesize in culture sige (specific) levels between and , pg/ml, also releasing factors capable of increasing ige secretion (ige-pf) [ ] . supernatant derivatives from the t cells of patients with higes are in fact capable of inducing in vitro the pre-b cells to increase ige production; furthermore, when the t lymphocytes in these patients are isolated on the basis of receptors for the ige fc fragment, the remaining cells release ige-pf [ ] . considering the suppressive activity of human lymphocytes with cd phenotype on sige, it has been observed that these lymphocytes are able to suppress sige synthesis in patients with high antibody levels; similarly cd + cells from a bone marrow transplant (bmt) can suppress ige production in the hla-compatible recipient [ ] . the study of patients with id associated with hyper-ige has supplied useful information concerning ige system biology, although the immune defect essentially responsible for ige increased production and for severe atopic iectasia (ata), thymic alymphoplasia, scid (severe combined id) ( %) [ ] and, occasionally, by digeorge syndrome (dgs), id with hyper-igm (higms) now cd /cd l deficiency, selective igm deficiency, biotin-dependent carboxylase deficiency, cgd (chronic granulomatous disease), primary neutropenia, and in netherton, nezelof, omenn and shwachman syndromes [ ] . other forms, in addition to those discussed, are associated with gastrointestinal symptoms: diarrhea and malabsorption of xla and thi, diarrhea in was and dgs, food-related allergies ( %) in sigad and also an elevated frequency of asthma [ ] .among secondary id, only aids is associated with ad (chap. ). the association between a deficiency of t cells and high levels of ige, observed in patients with higes, nezelof syndrome, ata, was and other diseases, has been known for some time (table . ) [ ] . experimental studies on animals indicate that there may be an inverse correlation between serum ige levels and t-cell functions: this could be attributed to a t-lymphocyte deficiency in atopics, genetically determined, which makes them more vulnerable to the camp inhibiting activity, and consequently causing an imbalance between the two subclasses of t cells, which could lead to ige hyperproduction and atopy development; however, in no case is there evidence of a relationship between cd deficiency, ige levels and allergic symptoms. it has been serum ige concentrations chronic granulomatous disease m- y < - , hyper -ige syndrome - y - , , nezelof syndrome m- y - , non-x-linked agammaglobulinemia - y - other variable immunodeficiency normal infants and adults - y - data from reference [ ] . m months, y years, gm geometric mean. manifestations has not yet been identified. the most interesting syndromes from this point of view are the three syndromes analyzed above, characterized by common clinical indications such as early ad onset, increased susceptibility to all varieties of pathogens, as well as an exceptionally high ige serum level [ , ] ( table . ). several pids have autoimmune features, including chédiak-higashi syndrome, cgd, complement deficiencies c q, c r, c s, c , c , griscelli syndrome, higms (cd deficiency), lad (leukocyte adhesion deficiency), hla class i deficiency, hla class ii deficiency, omenn syndrome, was, and xlp ( ) , which will be dealt with subsequently. in children with a mean age of months, autoimmunity was chronic and severe requiring prolonged immunosuppression, however with no spontaneous remission of such manifestations [ ] . definition pids ( fig. . ) [ ] consist of a heterogeneous spectrum of congenital, individual and combined anomalies of the immune system (humoral deficiencies, combined deficiency of b and t cells, the complement, phagocytes, neutrophils, etc.), as well as syndromes and diseases associated with id that are traditionally classified as pids. the updated classification (table . ) has divided ª pids into six main groups, also including secondary id with infections (first among them all aids) that cause deficiency and immunosuppression [ ] . the classifications of pids is based on characteristic clinical features and specific alterations in immune status. advances in molecular genetics now make it possible to complete the table according to the types of genetically altered molecules involved [ ] . to complete this data, see table . and table . [ , , , ] showing the behavior of antibodies and circulating b and t cells. pids occur infrequently and are highly heterogeneous in nature, relatively few centers gain extensive experience in the diagnosis, so it is difficult to estimate the prevalence of these disorders from routinely collected health statistics [ ] . studies in countries on all continents have included , patients: tables . and data concerning incidence has increased considerably thanks to a greater availability of specific tests and more widespread knowledge in the medical profession related to these pids, including ata [ ] . however, because primary immunodeficiencies [ , ] ; other data from [ ] (omenn syndrome) and [ ] (jak ). ada adenosine deaminase, id immunodeficiencies, jak janus-family kinase, pnp purine nucleoside phosphorylase, Ø decreased, ØØ markedly decreased, ≠ increased, -absent, + present, n normal. a progressive. b not functional. predominantly t-cell defects the total may not correspond to the sum of the cases because it may include some pid with very low incidences. the figures should be divided into the years that were considered. a incidence × live births; the thi figure includes probable cases. thi selective igg subclass deficiency autosomal hyper-igm syndrome selective antibody deficiency with normal igs cellular and antibody id syndromes associated with other major defects ata wasp syndrome digeorge anomaly hie nijmegen anomaly immunodeficiency associated with or secondary granulocyte dysfunctions defects of phagocyte number and function cgd cyclic neutropenia kostmann's syndrome schwachman syndrome complement deficiency -undefined total pid , time period years years / - / latin america includes eight countries. xla x-linked agammaglobulinemia, cvid common variable immune deficiency, thi transient hypogammaglobulinemia of infancy, scid severe combined id, was wiskott-aldrich syndrome, ata ataxia-telangiectasia, cgd chronic granulomatous disease. to be reliable and can also be used for other recessive x-linked pids to identify cell lines with genetic defects, as is the case of was [ ] . one must, however, properly consider the phenomenon of mutations, that can render useless the inactivation method, as has been proved in was, in xla and also in scid, in which the mutation is not in the maternal t cells but in the germlines [ ] . the main clinical aspects of humoral and cellular pid are schematized in table . [ ] ; further in numerous pids there is a deficiency of chemotaxis (table . ) as in cgd [ ] . in antibody deficiencies, current treatment, while waiting for genetic treatment to become available, complicated in xla by several btk mutations, is based on the prophylactic administration of ivig, combined with quick antibiotic treatment during infectious episodes. the genes responsible for id linked to chromosome x have been recently mapped on the respective chromosome bands (fig. . ): the bands on the short limb are designated "p" and those on the long limb "q" (table . ). this was possible thanks to the refinement of dna recombinant technology (rdna), including dna probes (sequences of radio-marked dna) and restriction fragment length polymorphism (rflp). the closer the gene segregates to rflp, the lower the chance that they might be separated by recombination phenomena when meiosis occurs: the identification of deficient genes allows early diagnosis, even prenatal, and if necessary gene therapy or bmts [ ] . furthermore, the observation that numerous pids are transmitted with an x-linked modality allows a relatively simple diagnosis of males with a positive family history (fh); if fh is negative ( %- % of xla cases) or there are females presenting a clinical pattern of pid, or when sporadic cases are caused by a new mutation, carrier identification is based on the study of immunologically normal female carriers, with two populations of b precursors, using x-chromosome inactivation analysis. this test does not take into account the existence of possible gene mutations and the availability of already affected relatives, and it is also relatively simple and fast [ ] . molecular studies follow the hypothesis that, at an early stage during embryogenesis, one of the two x chromosomes is randomly inactivated in the cells of all tissues of female embryos (persisting as barr's chromatin) [ ] . therefore in normal conditions, one has a cell mosaic that actively expresses for % the paternal x chromosome and for the remaining % the maternal x chromosome (lionization) [ ] (fig. . a) [ ] . in female carriers of xla, the cell mosaic expresses % for an x chromosome with btk in an active form and the remaining % for an x with a mutated btk (bruton tyrosine kinase). this means that in the carrier mother it is inactivated in preference to the x chromosome carrier of the defective gene in b, which matures therefore in an unbalanced manner (not randomly), while in all other cells activation occurs randomly. it follows that in fixed carriers only the b lymphocytes that have the x carrier of a normal gene complete the differentiating route, while the precursors that express the x chromosome with a mutated btk do not mature into b cells, but remain blocked [ , ] (fig. . b) . in x-scid, the study of fixed carriers follows the corrected lyon hypothesis, because the cells with a normal active x develop into normal t lymphocytes; however, when t precursors with a mutant x reach the stage where the x is needed, they do not find it and consequently do not develop: thus female carriers have only one normal and active x, instead of the random mixture of cells with one of the two active x [ ] . the inactivation test appears predominantly b-cell immunodeficiency inherited in an x-linked trait, only % of males have a fh positive for pid; female cases are also known, supporting an ar trait [ ] . classically affected subjects present levels of igg at < mg/dl, with very low circulating iga, igm and b cells (table . ), in which are found, in addition to bm, pre-b lymphocytes in an almost normal quantity [ ] . xla is characterized by a blocking of b-cell differentiation that results in an arrest of the evolution of pre-b a cells: low levels of cytoplasmic igm and high levels of surrogate light (l) chains (cd b) into later-stage b cells [ ] .the b-cell differentiation arrest in the majority of xla patients appears to be homogeneous, with approximately % of the pro b-cell compartment being negative for cytoplasmic igm expression [ ] . the size and nature of the residual more mature b-cell population (leakiness) varied among patients, independent of the type of btk mutation. further, it appears that the pro b-cell compartment composition in bone marrow (bm) of some xla patients can be influenced by low levels of wild-type btk mrna [ ] . on the contrary, t cells are normal both in function and in number, as is thymus architecture, including hassall's corpuscles and thymus-dependent areas of spleen and lymph nodes. b lymphocyte zones are typically depleted, with an absence of gcs, plasma cells, and cortical and medullar differentiation compared to normal (figs. . and . ) and an absence of adenoidal tissue (fig. . ). the intestinal lamina shows a similar deficiency [ ] , even if both b and t cells use the same recombination (chap. ). in the bm, increased pre-b lymphocytes without cd and cd can be observed. the pre-b cells are capable of transcribing and translating microgram intracytoplasmic (ic) h chains, but not the l chains [ ] , thus pre-b only form microgram chains not associated with v h , while only % of normal cells produce incomplete chains [ ] . experimental data currently indicate that the defect lies in the xla gene mutations that codify for btk [ , ] . the xla gene is expressed by b cells during differentiation, but is not transcribed in the t cells, thereby explaining the b lymphocyte maturative block at the pre-b level [ ] (fig. . ), immediately after their appearance in the bm [ ] . xla, however, presents a genetic heterogeneity, explained by mutations in the btk domain (ph, th, sh , sh , kinase), with a frequency proportional to the pertinent domain dimensions [ ] . the mutation size was ascertained by finding mutations in patients ( fig. . ) . equally, genes codifying for marker proteins and receptors that are essential for b-cell maturation and development are also involved: in fact many of these proteins, including btk, hm chains and surface proteins are crucial for b-cell differentiation [ ] . studying chil-dren of both sexes with xla, various mutations of hm germline have been identified, in addition to deletions affecting the d, j h and cm genes and other gene alterations capable of blocking h chain synthesis on b lymphocytes [ ] . an equivalent molecular defect was observed in an infant girl with xla, with differentiation block preceding the ig gene rearrangements by early pre-b cells [ ] . there are also the so-called leaky forms, with absent or few b cells and various antibody deficiencies [ ] , which can be attributed to individual mutations of btk [ ] , for example in the non-kinase domain, which permits the expression of normal btk levels [ ] . however, btk mutations can be even more detrimental for b lymphocyte proliferation compared to the total kinase absence [ ] . xla is clinically characterized from its onset in male babies, at - months of life (but also at the end of the st year), when the maternal igg passive protection ceases. it usually attracts attention due to delayed growth and mostly recurrent and severe bacterial infections dominate (sinusitis, otitis, bronchitis, [ ] . although representing the phenotypic picture of humoral id, confirmed as a separate deficiency [ ] , xla associated with ghd is mapped in the same region as the x chromosome of the isolated xla. the observation of hz deletion of one or more c h genes for the h chains of ig in %- % of normal patients has led to the identification of various polygenic deletions concerning the genes of one or more isotypes and subclasses [ , ] . some subjects are lacking in genes of all or some igg subclasses, associated with iga and ige deficiency, with no clinical symptoms in % of cases [ , ] . in italy these deletions have a frequency of . % and the expected frequency in hzs is of : , [ ] . only some of the families that produce l chains and not the k chains are known. in one family the molecular bases of the deficiency were ascribable to two different punctiform mutations, one in each cκ allele that prevented the formation of -s-s bridges between the k and h chains. the k:l ratio in human ig is : , and the relative alterations can be observed in numerous primary or secondary ids [ ] . only one patient is known with an l chain deficiency, hgg and rri (upper and lower respiratory tract) [ ] . [ , , ] . rotavirus and echo viruses also cause severe meningoencephalitis in %- % of patients [ ] . phenotypic variability may occasionally be present, as in a family spanning three generations [ ] . in patients with a median age of . years the median age at the xla onset was months and the median age of diagnosis was years, with a median diagnosis delay of months. the common infectious diseases were pneumonia, otitis, diarrhea, sinusitis, and arthritis. the most common chronic infections were seen in . % of the patients: in the respiratory tract in . %, in the gastrointestinal tract in . %, in the central nervous system (cns) in . %, and in the musculoskeletal system in . % of patients [ ] . bronchiectasis, malabsorption, arthritis, autoimmune and tumor-related diseases are the most common complications, as well as edema, contractures, etc. (fig. . ). one must predict the onset of bronchiectasis and intervene quickly with specific physiotherapy, because forms that are initially localized later spread, causing respiratory failure in older children and adolescents. one-third of all cases start with mono-or rheumatoid arthritis (ra) caused by ureaplasma urealyticum with a sterile exudate, which usually regresses following treatment with ivig [ ] .anti-polio vaccinations with live attenuated viruses should be forbidden, because they can cause very severe pneumonia [ ] . about ten cases of xla associated with ghd are known. in addition to reduced growth, clinical symptoms are typical of xla, though it is not a variant, sporadic cases have been described, occasionally associated with sigad ( %- %) and more often with ata ( %) and susceptibility to infections [ ] or without rris [ ] , differentiating patients with probable pid from those with low levels of igg (table . ) [ ] , in whom it may represent delayed maturation [ ] . selective deficiency of igg subclasses presents three different aspects: ∑ total lack of a subclass ∑ two sd levels below average ∑ inability to produce antibodies relative to the subclass in question, even when hematic concentrations are normal [ ] the following selective deficiencies are present [ , ] : ∑ isolated igg : deficiency in only a few cases has been described, also because this subclass represents %- % of all iggs (the others account for % [igg ], % [igg ] and % [igg ]), its absence is very probably an indication of an evident hgg; these patients usually have a reduced level of total iggs and react normally to antigens with a polysaccharide capsule. ∑ other combined id (cid): for example igg +igg , igg +igg , igg +igg +igg , igg +igg , igg +igg + igg , some of which are associated with a deficiency of iga or its subclasses [ , ] . the association of sigad and defects of igg subclasses is explainable in view of ig production ontogenesis by b cells: one starts with igm, moving on to igd, and then to igg ending up with iga passing by ige [ ] ; therefore the deficiency could originate with an immunological defect involving the t lymphocyte regulating work or b cells secreting ig, with an effect on the final stages of their production. in rare cases, more or less extended deletions in chromosome have been observed, in the region that codifies the h chains; in most cases the genome is instead intact, confirming a possible defect in b lymphocyte switching [ ] . very rarely this deficiency depends on gene hz deletions [ ] . this pid is probably the most common of all (tables . , . ), especially in nonselected populations of caucasian origin [ ] . it is defined by the presence of a serum level of iga < mg/dl and the absence of siga in the total deficiency; in the partial sigad levels are < mg/dl but < sd compared to normal levels for age, with measurable siga [ ] . in total deficiencies, igm and igg levels can be normal [ ] (table . ), but igg and igg levels are low [ ] . in several cases, the partial deficiency is transient [ ] , returning to normal levels of iga in % of cases by the age of and in % by ( fig. . ) [ ] . the sigad is transmitted sporadically; however, cases of multifactorial and dominant ar, with a variable or incomplete expression [ , ] transmitted within the same family have been reported. functional alterations reflect on the final maturing process of b lymphocytes, given that about % of b iga + lymphocytes show an igm+igd+iga+ membrane phenotype, a normal aspect only in newborn babies [ ] . studies on chromosome have not led to conclusive results, because deletions in children with sigad are associated with mental retardation, facial dysmorphisms, failure to thrive, etc. an association with hla haplotypes situated on chromosome is instead more consistent, and common in patients with cvid. interesting indications for understanding the pathogenesis come from molecular genetic studies that have allowed the formulation of a hypothesis of multifactorial origin, given that the combinations of more widely involved hla haplotypes and extended haplotypes involve the class i-iii genes, to the extent that they are more often encountered in the general population [ ] .among the class iii alleles the most studied is the gene that dictates the c a, among class i and ii the most common are a , a , b , b , b , cw , dr , dr , dr , dqw , associated with haplotypes such as a , b , dr ; b , dr ; a , b or a , b . other haplotypes are extended, such as b /sco /dr , bw /sc [ , ] /dr , bw /sc /dr and b f/fc /dr , the first of which is increased in patients with a combined iga, igg and ige deficiency [ , , ] . the association with dr gives sigad a risk factor of (table . ). one hla supertype is also found in deficiencies of -hydroxylase with a late onset, suggesting an important locus for iga differentiation close to class iii hla genes [ ] . it has been thought that to induce sigad a non-hla gene or an environmental penetration factor might be necessary, due to the possible sigad discordant expression in hla-identical twins [ ] . however, the analyzed sequence of involved alleles showed a significant sigad correlation with some alleles belonging to hla-dq locus, composed of a protective allele with aspartic acid and a susceptible one with valine or alanine in the b chain in position [ ] . we have observed that the presence of aspartic acid ensures protection in dm. furthermore, the immunological deregulation extends to the typical formation of auto-antibodies and characterized by the presence of igg anti-iga [ , ] . in these subjects, there is a wide symptom range, mostly represented by rris, allergic and autoimmune diseases (aids), among which is diabetes. allergic diseases are twice as common in partial deficiencies, unlike aids [ ] . from a clinical point of view, the frequency of chronic diarrhea and malabsorption, associated with celiac disorders and giardia lamblia infestation are not surprising, considering siga's prominent iga , igg , igg and ige due to deletion of ig h chain constant region genes were associated with undue susceptibility to infection [ ] (see "rri"). a few cases of selective igm deficiency are known, associated with rris and various other symptoms [ ] . igm deficiency was detected in four children with rris. isolated igm defect was present in two children, and two more children had an associated igg subclass deficiency [ ] . cvid includes a heterogeneous group of unhealthy conditions that have in common hgg and rris; it has an incidence of between : , and : , [ ] . the inheritance of two susceptibility genes within the hla on the short arm of chromosome : one located near the class ii region and the other near the junction between the class iii and class i regions is a serious risk for the development of cvid [ ] . there are autosomal dominant or ar forms also linked to sex; sporadic cases are the most common [ ] . the molecular bases are not totally clarified as yet: the pathogenetic mechanisms may depend on b lymphocyte ( % of patients) and t lymphocyte ( %) defects [ ] . the b-cell intrinsic defect is attributable to an alteration of the differentiating line at different stages of maturation, resulting in a poor formation of antibodies, with hgg of variable degrees, while in patients with xla the circulating b lymphocytes are virtually absent. the iggs are < mg/dl (with a reduction in all the subclasses: a normal phenotype is observed in only % of patients) [ ] . more often there is a hierarchical order in the shortage: igg < igg < igg < igg [ ] . iga and igm antibodies are < - mg/dl [ ] , reflecting the potential cd underexpression, implying an activation deficiency [ ] or a t-b cooperation defect [ ] . a study of t lymphocyte subpopulations indicates various subgroups of patients: % have t cells with scarce il , il and il levels, while % have a reduced cd :cd ratio, with an increase in cd bearing the cd marker, which suppresses igg production, elaborates normal il levels and increases ifn-g production [ ] . it can also accompany a deficiency of interleukins (ils: il , ifn-g), suggesting a defect in the signaling mechanisms based on the tcr/cd [ ] . a t-lymphocyte deficiency is therefore difficult to evaluate [ ] , also because this could be a vri effect [ ] . cvid can also be observed following congenital rubella or ebv infections; it can also be induced by some drugs such as phenytoin [ ] . in / cvid families, subjects were identified with identical large mutations in the icos (inducible costimulator) gene, expressed on the surface role in the formation of a barrier against the penetration of polypeptidic macromolecules through the intestinal mucosa. sigad therefore facilitates the penetration of food antigens through the mucosa followed by the formation of specific antibodies. for example, % of patients present cics and precipitins to cm, % to bovine anti-serum and % to anti-serum of calf fetus [ ] . symptoms affecting the respiratory tract are also caused by the absence of siga, as in / children aged - with increased susceptibility to rris [ ] . patients balance the siga deficiency with the sigm, but in some cases the compensation is insufficient for exempting them from rris and asthma [ ] . sigad should be diagnosed on the basis of both serum and secretory iga, because normal levels in adults are achieved at different times (table . ) . some drugs such as phenytoin (an anticonvulsant) can determine sigad, sometimes persisting in time after the drug has been discontinued. the clinical symptoms in these cases are not different from those of patients with sigad [ ] . there is no random treatment; these patients do not benefit from therapy with ivig, even when enriched in iga. there are no counter-indications for obligatory and optional vaccinations [ ] . whole blood or plasma transfusions containing iga can sensitize patients or cause anaphylactic shock in those already sensitized [ ] . life expectancy is excellent; however, the random discovery of sigad in asymptomatic children should not be underestimated. they should in fact undergo periodic clinical and laboratory controls so as to identify as early as possible any possible pertinent symptoms. at the same time, there is the need to ensure a good life quality with adequate prevention of rris in those patients whose respiratory tract is affected [ ] . sadni translates into the inability to respond to certain antigens, especially if polysaccharide. while some individuals are normal, others contract sinopulmonary infections. the reduction of igg levels is more of an associative relationship than a random one; igg levels, on the other hand, do not predict antibody responses. subjects who do not respond to anti-hepatitis vaccination may fall into this category [ ] . in one retrospective survey at a pediatric tertiary care center, sadni was the most frequent diagnosis, accounting for % of id diagnoses [ ] . there are cases of patients in good health, without ige due to gene deletion [ ] . in two siblings, deficiency of of activated t cells, which interacts with the icos ligand gene expressed on b cells. an additional patients with sporadic cvid were examined, and no mutations were found. only in patients with cvid screened thus far (< %) have been found to have icos mutations. one unexplained feature of cvid is that the onset of clinical symptoms does not occur until late childhood or adulthood [ ] . pid is variable either in the clinical and immunological pattern, or in the onset period, more common during the school years or in adults, but also between the ages of and [ ] . the acute bacterial recurrent and/or severe lower respiratory tract infections (lrti) are characteristic: sinusitis ( % of pediatric cases), otitis media ( %), bronchitis, pneumonia ( %) and/or digestive tract infections (diarrhea %) [ ] . the prevalence of infections caused by mycetes has increased as well as cases of pneumonia caused by pneumocystis carinii, a signal for cell-mediated immunity (cmi) [ ] . the gastroenteric tract is dominated by symptoms similar to those seen in celiac disease, with generalized malabsorption, steatorrhea, lactose intolerance, protein-losing enteropathy, inflammatory bowel disease (ibd), saccharidase deficiency and malabsorption of vitamin b and folic acid, supported also in this case by intestinal infestation caused by giardia lamblia [ ] . the tumor necrosis factor receptor family (tnfr) member taci (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) mediates isotype switching in b cells. in / unrelated subjects with cvid and / subjects with sigad there was a missense mutation in one allele of tnfrsf b (encoding taci). none of these mutations were present in healthy subjects. tnfrsf b mutations cosegregated with the phenotype of cvid or sigad in family members of the index subjects. b cells from subjects with taci mutations expressed taci but did not produce igg and iga in response to the taci ligand april (a proliferation-inducing ligand), probably reflecting impaired isotype switching [ ] . other characteristics are hemopathies, hepatosplenomegaly, autoimmune hemolytic anemia (aiha) and x-linked lymphoproliferative disease (xlp), and cutaneous and internal organ granulomas (which differentiate it from xla), in particular ra, thrombocytopenia, and neutropenia [ ] . offspring of cvid patients are at risk throughout their lives for cvid development and should be monitored with a high index of suspicion [ ] . based on experimental evidence, it has been hypothesized that iga-and cvid-associated deficiencies may be the extreme opposites of one clinical spectrum: there is a block of b-cell differentiation, different only in the isotype involved. both defects often appear in different members of the same family groups and more or less the same alleles are present [ ] . the most accredited hypothesis is that a number of extended haplotypes of the hla system are shared, to which gene duplications, deletions and polymorphisms codifying for some class ii and iii alleles correspond [ ] . in fact, a number of common hla haplotypes, especially belonging to class iii, are observed in patients, and at least two haplotypes in % of cases [ ] , such as hla-dqb * , hla-dr , c b-sf, c a, g - , bf- . , c a, hsp- - . , tnfa- , hla-b and hla-a , postulating therefore the existence of a common genetic basis [ ] , with a susceptible gene ( p . ) possibly the association marker [ ] . for example in five members of a large family with one of the two pids, duplications of the c genes were associated with a selected group of hla class ii and iii genes [ ] . the fact that four members without pid also had these haplotypes indicates that their presence alone is not sufficient for expressing pid, leaving room therefore for other factors [ ] such as overlapping relations with celiac disease. the analysis of linked genes has confirmed a strong association with locus a, suggesting that an important role in both pid is played by the gene codifying c a or an adjacent one [ ] . see "x-linked hyper-igm (or hyper-igd) or cd deficiency (xhigms)" for further discussion. unlike transient hgg that occurs when the maternal iggs gradually disappear from circulation (table . ), in the original study by taylor et al iga levels had fallen, becoming regular in newborn babies with thi after year, corresponding to the nonatopic levels [ ] . it consists therefore of a pathological delay in the normal antibody production maturing process. walker et al have calculated that the prevalence of thi is × in children, equal to the prevalence of symptomatic sigad ( × ) [ ] . in all children the igg and in / ( %) the iga were < th percentile, / ( %) had igm levels < th percentile resolved around the nd month; further confirmation consisted in the fact that the children had symptoms either of ad or of fa or food intolerance [ ] . from table . the mean incidence is from to × . during years children aged - months were diagnosed with thi and an incidence of . /year [ ] . in other studies the main defect was in the igg: in one it had normalized between and months [ ] , in another trial / babies ( . %) exhibited at the age of months an absence of serum igg levels and of specific antibodies to viral agents, which in eight children were detected before the serum igg levels returned to normal, whereas in basis is an il r deficiency [ ] , more precisely of the g receptor mapped on chromosome xq [ ] . the sole deficiency of il r is not sufficient for producing an immunological phenotype as devastating as scid [ ] . because the g chain of il r (il rg), a shared component of il r, il r, il r, il r, il r, il r and il r [ ] , gc mutations interfering with its link to the ils deprive the lymphoid progenitor cells of the crucial signals for normal lymphocyte intrathymic development [ ] . mutations in any of the genes: il rg, il ra, jak , artemis, rag , rag , cd , ada, cd cause scid [ , , , , , , , , , , ] . a total of il rγ gene mutations have been sequenced, of which are unique [ ] . each of these mutations has resulted in γc deficiency with varying degrees of id. the mutations are distributed throughout the eight exons of the gene, as well as in the regions necessary for proper transcription and translation. the penetrance of each of the above il rg mutations is unknown. exons and have mutation hot spots. the types of mutations identified include missense, nonsense, insertions, deletions, splice mutations, and mutations that affect rna processing and translation [ ]. among mutations in patients, the most numerous ( %) are punctiform mutations (fig. . ) plus one missense related to amino acid residues [ ] , with a lack of jak and gc interactions [ ] . in an atypical form, the substitution with residual cysteine of the arginine at position appears to be decisive for gc chain expression, probably a mutation reversion at the basis of the molecular defect, with a numeric and functional t-cell normalization [ ] . the mutations, by inactivating the common g chain, render the t cells of boys with scid-x unresponsive to several ils. the result is a block in t-cell development and a severe deficiency of mature t cells. b cells, although pre-two children normal igg levels were detected even before the appearance of specific viral antibodies. igg levels usually normalize at between and months [ ] , at the age of years (fig. . ) or before months of age in / children; however, / still had low ig levels at - months of age [ ] . at was in / children ig levels were still < sd for age and in / various igg subclass deficiencies were detected [ ] . a prospective study with an -year follow-up found that igg and iga deficiency is normalized by the age of , but in a minority of cases this may be a prodrome of sigad or another humoral deficiency [ ] . a study with a -year follow-up of children with igg deficiency as well as iga deficiency in % of the cases, observed multiform clinical symptoms. since thi can gradually normalize, some children have low antibody titers, and others low igg levels. however, both groups experienced significant infections [ ] . in some cases, thi is asymptomatic; in others infections, especially of the respiratory tract, are present. the designation of thi may be a misnomer, and an alternative designation could be added to thi such as "with recovery" or "with development of other dysgammaglobulinemia" [ ] . general characteristics of combined t-cell/b-cell immunodeficiency are summarized in table . [ ] . t -b + scid is a heterogeneous group with an incidence of between : , and : , livebirths [ ] . xlinked fh is positive in % of cases [ ] . the genetic sent in normal or even increased numbers than in other forms of scid, are dysfunctional [ ] . b cells do not mature or produce antibodies due to a complete b-cell differentiation arrest at the pre-bcr checkpoint, showing the absence of complete vdj recombination [ ] . other forms are also known with an attenuated phenotype and a partial t-cell function [ ] . typical scid-x represents the most common form, with . % of cases [ ] ( . % in tables . , . ) . in the thymus, there is a severe hypocellularity, without lymphocytes and hassall's bodies where thymic epithelial cells predominate without grossly evident corticomedullary differentiation (fig. . ). severe lymphopenia is often associated with eosinophilia; nk cells are within the norm or rare. the majority of in-fants with scid-x lack both t and nk cells (t -b + nkphenotype) [ , ] . cd t cells, if present, are of maternal origin, because the block, as also in scid ar, occurs at the level of cd -, cd -, cd -, cd + and cd a + ; developing t cells and cd + thymic dc are reduced > -fold when compared to age-and gendermatched control thymus [ ] (fig. . , pre-t, tn), thus scid t -b + . the study of other subpopulations distinguishes the scid subtypes: t cells are reduced in all variants, the absolute cord blood (cb) number is - , lymphocytes/mm (tables . , . , so that any count below , /mm is lymphopenic). moreover, in ada deficiency (adenosine-deaminase) there is a maximum reduction in total lymphocytes, in scid-x and in jak defi- chapter primary immunodeficiencies alies) and against a common t and nk cell drop [ ] . furthermore, in two cohorts [ , ] the affected females had the same phenotype, indicating a possible complex molecular defect [ ] . there is no response to delayed spts, and there is an absence of lymphocyte proliferative responses to mitogens and to a specific antigen such as tetanic toxoid [ , , ] . the average age at diagnosis was . months in children [ ] , similar to the ages reported in > children with scid from all causes [ , ] . the male:female ratio is : and diagnosis is often missed or occurs too late to save the lives of those infected infants who may manifest gvhd early on with morbilliform eruption in the first few days of life due to the transplacentally acquired maternal t cells, intractable diarrhea resulting in a severe malabsorption ( fig. . ), severe interstitial pneumonia (fig. . ), or giant cells caused by anti-measles vaccination or bcg (bacillus calmette guérin), with death caused by chickenpox or infections caused by pneumocystis carinii, herpes, adenovirus, cmv (cytomegalovirus), etc. [ ] . the absence of tonsils is observed and also lymphoid tissue [ ] and thymus [ ] hypoplasia. these children must be transferred urgently to a specialized center and be placed in a sterile room to receive a bmt [ ] . on rare occasions, il rγ mutations have caused an atypical mild scid that presented beyond infancy [ ] . up-regulation of bcl- by an il r lacking il rb tyrosine residues leads to increased cell survival after il deprivation; astonishingly, this survival signal does not occur when gc tyrosine residues are absent. thus, if ciency, the number of b cells is the highest and that of nk cells is the lowest (b + >t ->nk -); nk cells on the contrary reach their highest levels in the ar form [ ] . in scid there can also be b alymphocytosis [ ] . this divergent data is, however, characteristic of t -b + nkmolecular defects (gc, jak defects), b + t -nk -(ada deficiency), or t and b (possible recombination anom- gc-dependent signals are revealed only in the absence of il rb tyrosine, il r engages at least two distinct signaling pathways to regulate apoptosis and ccl- expression [ ] . in two clinical series, patients with mutations in il rg represent %- % of all scid cases [ , ] . children with il rg mutations have lymphopenia in % of cases, with total lymphocyte counts < , /mm (normal levels, , - , /mm ), based on clinical case series [ , ] . all patients have very low or absent t cells, and approximately % have low or absent nk cells [ ] . ar mutated genes on autosomal chromosomes have been identified in ada deficiency, jak deficiency, and rag or rag deficiency [ ] . the existence of b-and t-lymphocyte lymphoid precursor differentiated defect is particular, in some cases a rag and rag mutation, the two genes that activate vdj recombination [ ] was observed; however, this rag gene function has been questioned [ ] since a rag defect is more present in t -b -scid and the omenn syndrome [ ] . in babies suffering from scid, there is a marked reduction of t and b lymphocytes (table . ) and all in vivo and in vitro responses are absent. onset and clinical and histological pattern is similar to that of x-scid. the jak gene mutation (tables . - . ) variant has a frequency of . %- . % [ , ] among babies affected by scid and in the absence of t ( ± %) and nk cells ( ± %) [ ] . the molecular base is the mutation affecting the jak , which prevents it from associating with the gc chain and from sending signals to the abovementioned ils [ ] and to other marker proteins belonging to the jak-stat complex [ ] . at the origin is a lack of t lymphocytes that transform into the scid phenotype [ ] . these patients present b + t -nk -: the b ( ± %) with iga equal to ± % [ ] , and those with x-scid present a defective differentiation, but are capable of producing elevated levels of ige in the absence of other isotypes [ ] . this data indicates that gc and jak are essential for t-and nk-cell development [ ] . the clinical characteristics are identical to those in x-scid, with the difference that the scid-jak phenotype is also observed in females ( %) [ ] . furthermore, a jak deficiency could be an important cause of scid ar and should be considered in all patients with the b + t -nkphenotype, without an x-recessive heritage [ ] . in a -month scid-x infant presenting with a history of recurrent infection and failure to thrive, a novel splice mutation, gc-dependent, was described, characterized by near-normal count of functionally deficient nk cells (b + t -nkcell phenotype). cell surface gc expression was undetectable on nk cells and in trace amounts in the minority of b cells. t cells were absent, igg and iga undetectable, and igm were within the normal range [ ] . bmt is not a perfect therapy, because b-cell function developed in / children, and nk functions normalized in / children after bmt [ ] . the family pedigree shows an inbred family with consanguinity across five generations. two brothers were diagnosed with scid. one, at the age of months, presented with persistent oral thrush, oral ulcers, and failure to thrive. he had no palpable lymph nodes and no thymus shadow on a chest x-ray film. the second was diagnosed soon after birth and the third brother has always been healthy. three other male cousins died in infancy from severe infections consistent with scid; a th cousin presented with oral candidiasis at the age of weeks and failure to thrive. no thymic shadow was detected on chest x-ray film and peripheral blood lymphocytes showed persistent lymphopenia. he had no lymph nodes, failed to reject a skin allograft and did not show an increase in the blood igg and igm antibodies for dtp after three vaccinations. the three affected patients were hz for a caet transition at nucleotide in exon , leading to a proline to serine substitution (p s) in the extracellular domain of il r. the cousins and their parents harbored both wild and mutant alleles. this partial deficiency is sufficient to block t-cell development and lead to a scid phenotype. the fh of severe pid with multiple affected male infants strongly suggested an x-linked inheritance. nevertheless, this family consanguinity is in favor of an ar inheritance [ ] . defective il r expression caused in three patients a t -b + nk + scid, indicating that the t-cell defect in scid-x resulted from inactivation of il ra signaling. thus il r-mediated signaling is required for t cells but not for nk ontogenes. mutations in the gene for the il r chain on chromosome p were found in all three patients [ ] . these infants resemble those with other types of scid with respect to their susceptibility to infection and the absence of functional t cells and b cells. however, they differ in that their circulating lymphocytes are primarily nk cells. rag and rag are required for the rearrangement of tcr and bcr genes [ ] . half of the patients with t -b -scid had mutations in their rag or rag genes, thus highlighting the crucial role of these genes in normal v(d)j recombination machinery [ ] . rag-thymocytes lack a functional pre-tcr and hence arrest at the cd -/cd + stage of differentation [ ] : without rag and rag , mature ig and tcr genes cannot be assembled, and lymphocyte development is arrested at very early stages [ ] . abnormalities of the chest, scapula and iliac bones and short and stumpy limbs [ , ] . x-ray abnormalities are documented in fig. . : the absent thymic shadow and a notable cupping and flaring of the ribs' ends (arrows) can be observed, while histological studies of the chondrocostal junctions document their total cellular disorganization (fig. . ). this deficiency is the object of a great deal of attention because it was the first to be treated using gene therapy [ ] . the lack of ada is observed in . % of patients with scid [ ] . the ada enzyme catalyzes the conversion of adenosine and deoxyadenosine into inosine and deoxynosine; although ada is found in all cells (cd anchors ada to the lymphocyte cell surface (table . ), the deficiency damages above all the immune system [ ] . table . [ ] summarizes the biochemical foundations of this pid. more than ada mutations are known, including > amino acid substitutions, deletions and punctiform mutations or anomalies of the gene itself, such as exon deletion and exons , , and - mutations with a total of , nine of these in patients with ada-scid and six in those with a partial deficiency [ ] . an additional mutant alleles have been found ( missense and single-codon deletion) [ ] . adenosine and deoxyadenosine are also apparent suicide inactivators of the enzyme s-adenosylhomocysteine (sah) hydrolase, with consequent accumulation of sah, a powerful inhibitor of virtually all cellular methylation reactions [ ] . the accumulation of metabolites, including camp, deoxy-atp and '-o-methyladenosine, has a toxic effect on the cells by blocking dna synthesis and dividing and resting t lymphocyte proliferation [ ] (fig. . ). four different clinical phenotypes have been described for ada-deficient subjects (table . ) [ ] , which cover a broad spectrum of immunological aberrations, from the complete absence of b and t immunity, indicating scid ( %- % of patients) (the thymus in fig. . ) to forms with a delayed onset or partial deficiency ( %- %) [ ] . in children, the delay between onset of symptoms and diagnosis has been estimated to average months [ ] . if clinical symptoms indicate an early onset, in addition to typical scid symptoms, there are also x-ray pathognomonic skeletal abnormalities of the chondrodysplasia type, especially this very rare ar type of scid was observed for the first time in in identical twin male infants who exhibited a total lack of both lymphocytes and granulocytes in their peripheral blood and bone marrow. it has a frequency of % in cases of scid [ ] . the children are symptomatic in % of cases within the first days after birth [ ] and is usually fatal within the rd month of life without a bmt [ ] . due to the common stem cell (sc) non-maturation [ ] , it is characterized by total block in lymphoid and myeloid precursor differentiation, therefore not only by an extraordinary lymphopenia, but also by a marked cytopenia in all sections (table . ) [ ] , in the spleen, in the lymph nodes and in the gastroenteric tract, and a high frequency of severe successive infections [ ] . the thymus is always much reduced in volume, no hassall's bodies are seen [ ] . seven of the eight infants reported by who with this defect died between and days of age from overwhelming infections; the eighth underwent complete immunological reconstitution from a bmt [ ] . an additional three of five children who required two hscts (hematopoietic sct stem-cell transplantation) and received intensive conditioning therapy before haploidentical hsct (matched for of the hla loci) are alive and well an activated phenotype and poor functional capacity [ ] . studies involving hla typification and dna polymorphism show that t cells belong to the host, ruling out, therefore, the etiology of maternal cell engraftment [ ] , unlike other types of scid [ ] . the absence of circulating b is also characteristic [ ] , equal to . %- . % of normal levels [ ] , reaching % [ ] , high ige levels ( ui/l), hypereosinophilia reaching , ¥ cells/l (normal, - . cells/l), and low ig levels at the beginning [ ] then declining to the point of agammaglobulinemia [ ] , comparable to that in reticular digenesis (table . ) [ ] . the marked b-cell depletion can also bear rag and rag gene missense mutations that decrease the efficiency of vdj recombination, which results in impaired but not absent rearrangement of both bcr and tcr. four missense mutations were detected in the rag- in / patients [ ] . in / patients ( %) the mutations affected the rag gene, and in / ( %) the rag gene [ ] . increased ige is linked to th primary infiltration, with spontaneous production of il ifn-g, il , il and il , which is down-regulated by ifn-g therapy [ ] . clonal expansion of vb + cd + , cd -cd secreting high il levels and low il and ifn-g levels [ ] could indicate an analogy with the fas (cd ) defect. t lymphocytes show an activated phenotype and a spontaneous apoptosis associated with reduced expression of bcl- gene product, and a higher cell death of cd + cd r + cells [ ] . given that high cd levels in the lymph nodes, skin and serum of three children generated th lymphocytes [ ] , a th -mediated pathogenesis is possible: the cd are th markers (chap. ). as in human scid, b and t cells are found in mice with scid, but with a final repertoire that is decidedly oligoclonal and lacks the heterogeneity characteristic of a normal immune system, so lymphopenic scid and omenn syndrome could be two aspects of the same disease with different clinical expressions, especially of time [ ] . clinically, young babies soon after birth show a generalized exudative erythroderma and desquamation, often mistaken as ad, alopecia, widespread lymphadenopathy, hepatosplenomegaly, persistent and profuse diarrhea, failure to thrive with malnutrition ( fig. . ) , aiha, recurrent infections caused by common and opportunistic germs ( fig. . ), and markedly elevated serum ige levels [ , , , ] . this outline included four babies from the same family with the same symptoms until death occurred at - months, but who did not present hypereosinophilia and were diagnosed as dm [ ] . differential diagnosis may be challenging since omenn syndrome and gvhd show dyskeratosis and basal vacuolation, but the first always shows acanthosis and usually parakeratosis. gvhd shows a flat epidermis and rarely parakeratosis. both can be distinguished after immunohistochemical staining for cd and cd , which shows predominantly lymphocytes in the dermal infiltrate in omenn syndrome, and relatively more macrophages in gvhd [ ] . with myeloid and t-and b-cell lymphoid reconstitution [ ] ; another child is alive and well after months [ ] . b-scid, characterized by increased cell sensitivity to radiation secondary to mutations of the artemis gene, could carry a poorer prognosis because of defective repair of dna breaks [ ] , occurring around the time of bmt, from the effects of chemotherapy, infections, and gvhd [ , , ] . one group of patients with scid with an additional sensitivity to radiation was found to harbor large deletions or truncation mutations in the artemis gene mapped on chromosome p [ ] , implying a role for artemis in dna double-strand break repair, which is mutated in human scid [ ] . omenn syndrome is classified as a scid because newborn babies exhibit symptoms similar to a gvhd, due to a antigen expression and cd a absence [ ] , and because it can coexist in families with alymphocytosis [ ] . this is an ar syndrome with an unknown pathogenesis, sharing characteristic clinical and immunological abnormalities with t + b -scid [ ] . severe cutaneous lesions with hyperkeratosis, apoptotic malpighian necrosis and basal membrane destruction can be associated [ ] . no lymphoid cells or hassall bodies are found in the thymus [ ] . the immunological structure reveals histiocyte infiltration of the skin, bm and lymph nodes, with proliferation of t infiltrating the epidermis and the enteric mucosa, increased t cells with combined t-cell and b-cell deficiency igg (g/l) . - . igm (g/l) neutrophils (cells/ml) , - , data from [ ] . in a child with scid and circulating t cells within the norm, a gene transcription deficiency was ascertained [ ] . a male infant of first cousin parentage presented at the age of months with cmv pneumonia, persistent oral and esophageal candidiasis, adenovirus gastro-enteritis, and failure to thrive. he developed lymphadenopathy, hepatosplenomegaly, iron deficiency anemia with no evidence of hemolytic anemia, and chronic inflammation of his lungs and mandible. biopsies showed extensive lymphocytic infiltration of his lung, liver, gut, and bone. serum igg and igm were elevated, but iga was low. he had t-cell lymphocytopenia, with an abnormal cd :cd ratio of : . the t cells responded poorly to anti-cd , phytohemagglutinin and other mitogens, and to il . he was found to have a truncated mutation of the il ra chain (cd ). he was given a successful allogeneic bmt after cytoreduction [ ] . about cases have been reported, [ , , ] , % [ ] or % [ ] of which have the x-linked form of cd deficiency [ ] . patients are generally male, but there can be a non-x-linked form [ ] , in which % of patients are females [ ] . an estimated minimal incidence was calculated of in , , live births. over half of patients developed id symptoms and were diagnosed by year of age, and over % by years of age [ ] .although carriers of xhigms are considered to be asymptomatic, an extreme lyonization of the normal x can lead to a mild expression of the xhigms which is similar to cvid [ ] . it can be secondarily caused by environmental factors and also stem from congenital rubella [ ] : this indicates its heterogeneity. mutations in the tnfrsf encoding cd in xhigm patients result in a lack of b-cell signaling by activated t cells [ ] . however, boys out of failed to express cd , and tnfrsf mutations were found in of these boys, whereas no tnfrsf mutations were found in boys with weak expression of cd [ ] . as a result, xmigm b cells fail to undergo isotype switching and produce only igm due to a defect in the rna editing enzyme, activation-induced cytidine deaminase (aicda), an enzyme expressed only in b cells and required for the processes of class-switching and somatic hypermutation of ig genes [ ] . the marked reduction of igg (< mg/dl), ige and iga is accompanied by a sharp increase in mature igm and circulating igd, but b cells do not express other ig [ ] . interestingly, % of patients with confirmed xhigms who had tnfrsf mutations had low concentrations of igg, iga, and igm. most of the remaining patients with xhigms had the classic pattern of normal or raised igm with low concentrations of iga and igg [ ] . the cd gene defect is usually expressed on the membrane by activated t lymphocytes, which therefore cannot bind b-cell cd [ , ] . figure . shows cd localizations and mutation frequencies, in . % of cases mistakenly. for example, a sense codon substitutes for a missense one, creates a premature stop signal: therefore specific pertinent mutations, such as g e, tunistic infections. this disorder is related to mutations in the gene that encodes the nuclear factor kb (nf-kb), which is required for activation of the transcription factor nf-kb, or nemo (nf-kb essential modifier), also known as ikk (inhibitor of b kinase). the phenotype observed in x-higms-eda patients shows that the putative zinc-finger domain of nemo has a regulatory function and demonstrates the definite requirement of cd -mediated nf-kb activation for b cell ig classswitching [ ] . three other genes, expressed by b cells, have been associated with the higm phenotype giving place to higm - . mutations of activation-induced cytidine deaminase (aicda) (higm ) and uracil glycosylase (ung) (higm ), both expressed by follicular b lymphocytes, lead to defective class switch recombination and somatic hypermutation. mutations of cd , the cd receptor, cause a rare autosomal form with a clinical phenotype similar to cd deficiency (higm ). these rare pids may shed light on the complex events leading to the production of high-affinity, antigen-specific antibodies of different isotypes [ , ] . early treatment with ivig associated with antibiotic prophylaxis have reduced the incidence of life-threatening infections and improved the growth of children with higms [ ] . cycles of g-csf (granulocytecolony stimulating factor) in the presence of severe neutropenia are advised [ ] . substitute therapies with soluble forms of recombinant or gene type cd [ ] are being studied. a recent review of cd -deficient patients showed that % develop liver disease and only % survive into the third decade of life [ ] . bmt has a successful outcome in young children ( %); older patients with more ad-can interfere directly with the link site for cd ( fig. . ). consequently the signal which indicates that b cells should begin isotype switching, limited to the production of low-affinity igm, is missing [ ] . without isotype switching, gc formation is minimal [ ] (fig. . ) and follicular dendritic cells (fdcs) are reduced in number, also having an abnormal phenotype [ ] . as shown by figs. . - . , the lack of cross-linking of cd by cd results in b-cell failure to up-regulate cd and cd , important costimulatory molecules that interact with immunoregulatory molecules on t cells such as cd and ctla- . two patients with normal levels of cd have also been described [ ] . as in males with xla, infections start during the th month, those most often observed are otitis, pneumonia or sepsis cased by pyogenic bacteria, opportunistic infections, in particular caused by pneumocystis carinii [ ] , and also ulcerative stomatitis, ra, neutropenia, aiha, lymphoproliferating complications and type b gastroenteric lymphomas with igm [ , ] . the most prominent clinical infections were pneumonia ( % of patients), upper respiratory infections (urti) ( %- %) including sinusitis ( %) and recurrent otitis ( %), lrti ( . %) recurrent/protracted diarrhea ( %- . %), cns infections ( . %- %), sepsis ( %- . %), cellulitis ( %), hepatitis ( %- . %), and osteomyelitis ( %) [ , ] . lymphoid tissues are normal or hyperplastic [ ] . recently, a rare form of higms associated with hypohydrotic ectodermal dysplasia (eda) characterized by the absence or hypoplasia of hair, teeth, and sweat glands has been described. unlike patients with higms, these patients failed to have a history of oppor- purine-nucleoside phosphorylase (pnp) deficiency, ar, for which patients have been reported [ ] , is characterized by the absence of an enzyme necessary for the catabolism of purines, which converts inosine, deoxynosine, guanosine and deoxyguanosine into hypoxanthine and guanine ( fig. . ); the responsible gene has been mapped to chromosome q at position . [ ] . this has also been observed in patients with nezelof syndrome [ ] . a variety of mutations have been found in the pnp gene in patients with pnp deficiency [ ] . although ada and pnp are both purine salvage pathway enzymes, pnp deficiency does not lead to as severe an id as ada deficiency. patients have considerably reduced concentrations of serum and urinary uric acid. numbers of t cells fall progressively, more than that of b cells (table . ), just like the proliferating responses to mitogens and antigens, especially because pnp deficiency causes an intracellular accumulation of deoxy-gtp (guanosine triphosphate) inhibiting ribonucleotide-reductase and t-and b-lymphocyte proliferation, so combined t and b defects are critical. pnpdeficient patients are as profoundly lymphopenic as those with ada deficiency, with absolute lymphocyte counts usually < /mm . ig levels and production of specific antibodies are all normal [ ] . onset may be early, as for scid, but also delayed until the age of - years. the clinical pattern is dominated by recurrent bacterial, viral and fungal infections, with an abnormal susceptibility to opportunisic germs. two-thirds vanced liver disease may die because cryptosporidia infection that has progressed rapidly following pretransplantation cytotoxic conditioning therapy [ ] . therefore, a patient with end-stage liver disease related to cd deficiency first received a liver graft, and as soon as liver-graft function was satisfactory, bmt was performed with a nonmyeloablative conditioning protocol of fludarabine and melphalan [ ] . the screening for cd deficiency should include children with severe rri, and with dysgammaglobulinemia with a normal or increased igm level [ ] . conventional allogeneic hsct from an hla-matched or a matched unrelated donor (mud) is curative and feasible, if performed before significant infections and organ damage occur [ ] . an approach for high-risk patients including nonmyeloablative hsct was workable in a retrospective analysis of european patients undergoing hsct for cd deficiency in eight european countries between and . the donor sc source included hla-identical siblings, muds, and two phenotypically matched parental stem cells (scs) ( tcd [t-cell depleted]). of these patients, ( %) died from infection-related complications, with a positive result in . % of patients [ ] . carriers can be detected, and this is useful for making a prenatal diagnosis [ ] . of patients suffer from neurological alterations, ranging from spastic symptoms and alterations, etc., to mental retardation and one-third from aids, the most common of which is aiha. the consequence of severe infections, generalized vaccination, severe chickenpox, lymphosarcoma and gvhd caused by blood transfusions in the first decade of life is death [ , ] unless bmt is successful [ , , , , ] . however, poor neurodevelopmental progression may result [ ] or may not [ ] . since the biochemical bases of pnp and ada deficiencies are similar, it is hoped that genetic treatment will also be effective in children with this pid [ ] . this deficiency of hla molecule expression occurs in the more severe forms of pid if they are class ii: about cases [ , ] are known of this ar syndrome [ ] , heterogeneous for the numerous complementation groups the patients are divided into [ ] . hla class ii molecules are absent in all tissues [ ] , to the extent that the cells of patients maintained in cultures for years preserve the negative phenotype [ ] . there is a deficiency of class ii gene transactivator (ciita) codified by chromosome , the expression of which plays an important role in t-cell activation: its absence makes class ii gene expression impossible [ ] . this function is shared with another protein mapped on chromosome , rfx , with a binding site in the promoter region of genes codifying class ii chains [ ] . two additional class ii-specific transcription factors are rfxap and rfxank [ ] . these act on the class ii promoter region and are essential and also nonreplaceable, to the extent that alternative routes cannot compensate for their absence [ ] . furthermore inactivation, or the deficiency of these factors, has a specific effect on the genes dictating hla class ii, the li chain and hla-dm, because there is no indication that other regulating systems may be involved [ ] . the absence of hla class ii is associated with a cd lymphopenia. hla class i expression is normal in the patients tested and cd lymphocyte numbers are not reduced [ ] . interestingly, in a twin study, despite the deficiency, there were antibody responses and class ii-dependent t cells; hence the authors envisage that this represented a hla class ii residual expression below the test sensitivity [ ] . the clinical outline is dominated very early on, before the age of months (range, weeks to months) [ ] , by severe and recurrent gastroenteric and pulmonary infections, with a severe and prolonged course, associated with malabsorption and failure to thrive [ ] . bacterial and viral infections, bronchopneumonia, hepatitis, cholangitis, viral meningoencephalitis and various autoimmune manifestations are common complications [ ] . even though an hla class ii deficiency is clinical-ly less severe than scid, the result is uniformly fatal during the first or second decade of life [ ] . the most evident immune defect consists in the complete lack of reactivity to exogenous antigens, which in vivo reflects an anergy to spts, as well as the complete lack of hla class ii expression and absence of cellular and antibody responses to antigen stimulation [ ] , which are instead positive to mitogens (table . ; fig. . ) [ ] . laboratory investigations show a normal b lymphocyte number, but children may be agammaglobulinemic [ ] . the thymus and other lymphoid organs are remarkably hypoplastic, with a severe cd lymphocyte depletion, while cd and b-cell levels are normal. the syndrome involving a deficiency of hla antigens confirms an important hla biological role in the complex system of t-b cooperation [ , ] . some studies suggest that there are more types of deficiency. when placed together in a culture, the b lymphocytes of these patients, previously transformed by ebv, the lymphocytes correct each other so as to allow hla class ii molecule expression. this has led to the identification of so-called complementary groups [ ] . the specification that the gene is mapped on chromosome p . can lead to an earlier prenatal diagnosis [ ] . longterm survival seems to depend primarily on hla-identical and hla-haploidentical bmt performed in the first years of life, before the acquisition of chronic virus carriage and sequelae of infections [ , ] . a child recently received a transplant [ ] with a novel protocol [ ] : the cd count increased up to cells/ml [ ] . a direct correction of the genetic defect is based on the transduction of cells from patients with lentiviral vectors encoding ciita, rfxank, rfx , or rfxap. the rfxank vector restored class ii expression in a t-cell line from one patient. the rfxap vector corrected primary cells from a second patient [ ] . the study of the common association of hla class i molecule deficiency, already known as bare lymphocyte syndrome, has led to the identification of various patients with an isolated deficiency and of one patient with a deficiency associated with class ii, the most severe [ ] . the deficiency is caused by tap- -tap- mutation, accompanied by severe and chronic bacterial rris [ ] . in two brothers with the ar hla class i defect, the onset of rris took place between the ages of and ; the poor expression of nk cells was so severe that it led to the development of bronchiectasis [ ] . the immunological structure is characterized by few cd : the deficient expression of hla class i molecules is diagnostic [ ] (tables . , . ). mutations with no symptoms referable to an id, therefore integrating a genetic heterogeneity. two other brothers and both parents were healthy [ ] . while the ε chain deficiency produces modest clinical symptoms, the other two are severe also from an immunological point of view: in the γ chain deficiency resulting from the profound cd and cd ra decrease caused by altered thymic activity that leaves the cd ro unaffected [ ] , and in those of the ζ chains due to the severe thymic atrophy [ ] and thymocytes falling to % of normal levels, with limits at between % and % [ ] . the cd δ deficiency due to a heritable mutation of the cd gene that prevents the synthesis of the cd protein has been reported in cases hz for the cd mutation. two cousins died at - months of age because of overwhelming infection. the thymus shadow is clearly visible on chest x-rays. the thymus becomes populated with developing thymocytes, with an arrest of differentiation at the cd -cd stage of t-cell development. a girl ( rd patient) survives after a bmt [ ] . this rare deficiency transmitted as an ar trait is caused by mutations of the zap- gene, a non-src family protein tyrosine kinase (ptc) important in t-cell signaling (tables . - . ). zap- , known to be crucial for t cell activation, is a key player in tcr down-modulation and z degradation [ ] . zap- has an essential role in the cd g chain deficiency due to g or e gene mutations [ ] determines a lack of cd and the absence of cd ra [ ] . in the first two cases described, one brother died at months because of viral pneumonia after a clinical history indicating scid with severe aiha, while the other was asymptomatic at the age of years, although with the same molecular defect [ ] . the study of these brothers proved that, in spite of the absence of functioning g chains and % of the expressive levels of the cd /tcr complex, the lymphocytes were normal. according to the authors, other chains may act in the place of missing ones; however, the correlated scarcity of cd may have negatively interfered with the mechanisms discriminating between self and non-self, while g chain deficiency could have modulated the onset of the deceased brother's severe autoimmune disease (aid) [ ] . a cd e deficiency was found in a -year-old child with mild rri symptoms and otitis media; the expression of the cd /tcr complex was only %, but the stimulation with anti-cd induced a normal proliferating response. in fact, despite the ongoing mutation, a northern blot analysis showed production of a low amount of transcribed rna, corresponding to a small quantity of e normal chains, even though their dimensions were smaller than normal ones [ , ] . the z chain deficiency found in the two brothers is similar to the deficient expression of cd /tcr [ ] . in the younger brother, the thymus, markedly reduced, showed no hassall bodies; the elder brother had similar chain [ ] . in several babies (most of mennonite origin) with scid [ , , , ] , the nonfunctional cd t cells (table . ) were either normal or increased (cd + cd + , %). cd absence in the thymus and in circulation (cd + cd + , %- %) [ , ] suggests that the selective process is arrested during the transition from double-positive (dp) to mono-positive (mp) t cells [ , , ] .arrested thymocytes had terminated rag gene expression and up-regulated tcr and bcl- expression, but failed to differentiate into mature cd or cd mp thymocytes, to be rescued from death by neglect or to sustain il ra expression [ ] . zap- deficiency results in an impairment of transendothelial migration that can be rescued by the transfection of zap- because cross-talk between the zap- signaling pathway and the chemokine receptor cxcr is required for t-cell migration [ ] . although the thymic architecture is normal with presence of hassall bodies [ ] and cd seem normal in the cortex, very few migrate to the medulla [ ] . the near absence of cd + cells and an increased cd :cd ratio dominate [ ] . the few cd coexpress cd + , the nk-cell marker; b cells appear normal and functional, cd -cd + is at a level of %- % [ , ] , and serum ig values are normal [ ] . the same phenotype was found in the brothers [ ] ; other relatives were het [ , ] . the absence of cd expression was shown to correlate with a missense mutation in both ig alleles of the cd a gene domain in a -year-old man and his sister, whereas high percentages of cd -cd -tcrab + t cells were found in the three siblings [ ] . the proliferative responses in vitro to phorbol myristate acetate (pma) and ionomycin, pkc activators (protein kinase c), were normal, unlike pha (phytohemagglutinin), pwm, tetanic toxoid, anti-cd , etc. [ , ] . the positives operate below the tcr, while the negatives react directly with the cd /tcr complex [ , , ] , confirming the zap- deficiency [ ] . the cd are present despite the deficiency because syk, the other member of the family, ensures a compensatory role in the infrathymic cd selection, although with a limited efficacy [ ] . seven months after bmt, a child was clinically well and immunologically recovered [ ] . studies in two siblings hz for a stop mutation in the tap- gene suggest that nk cells express still unknown inhibitory receptor(s) (the missing receptor, discussed in chap. ) capable of down-regulating the nk cell cytotoxicity on binding to surface ligand(s) expressed by t cell blasts. functional analyses were consistent with the concept that this putative inhibitory receptor is expressed by virtually all tap- /nk cells, whereas it is present only in rare nk cells from healthy persons. another prospect would be that tap- /nk cells are actually missing this still unidentified triggering receptor involved in nk cell-mediated killing of pha blasts. since cells derived from patients displaying defective expression of either of the tap subunits are characterized by a strong reduction of mature hla class i molecules at the cell surface, a tap deficiency is connected with hla class i deficiency [ ] . as discussed in chap. , nfat (nuclear factor of the activated t cells) is a transcription factor that forms a powerful transcriptional activating complex and, by linking with specific dna-regulating sites, plays a critical role in the synthesis of various t-cell ils which, due to the deficiency or excessive migratory mobility of nfat, although normal in number and in distribution, are incapable of activating and/or secreting the genes of il , il and ifn-g [ ] . a -year-old girl with scid presented during infancy with severe recurrent infections and failure to thrive; her mrna was not produced for il - and ifn-g due to poor t-cell proliferation, although these were normal in number and in distribution, to initiate the transcription of the relative genes, regulated by nfat, with a binding site in the proximity in the ¢ region. this severe clinical picture is accompanied by evident hgg [ , ] . nk-cell deficiency is found in scid, cvid, reticular dysgenesis, chédiak-higashi syndrome, xlp, lad in tap- deficiency and in cfs (chronic fatigue syndrome), in particular cid such as scid, suggesting an association between nk-and t-cell deficiencies [ ] . there is one known case of an adolescent with an isolated numerical and functional deficiency of nk cells and of precursors, recurrent neutropenia, severe and recurrent ebv, cmv, herpes simplex virus (hsv) infections and life-threatening chickenpox. another child, diagnosed at the age of . years with a cd deficiency, suffers from severe viral and bacterial infections although he has antibodies to various viruses [ ] . the growing list of human genetic defects that impair nk-cell function has been recently joined by nemo-id [ ] which occurs in a group of patients with antibody deficiency combined with exquisite susceptibility to infection with nontuberculous mycobacteria. infectious susceptibilities common to these disorders stress the important role for nk cells in host defense [ ] . the natural history of three boys with nemo mutations outside of the th exon has been described. including these boys, there have been families described as having nemo-id. the resulting estimated incidence of nemo-id is : , live male births, making this disorder significantly less common [ ] . cd ra cells and a marked clinical improvement. these data indicate that the thymus is differentially required in the maintenance of the tcr repertoire complexity [ ] . known also as idiopathic lymphocytopenia, primary cd t-cell deficiency is revealed by a profound and persistent reduction in circulating cd and with a cmi deficiency. it is documented in patients suffering from infections caused by opportunistic germs such as cryptococcus-induced meningitis and oral candidosis, also including ten children and a number of adolescents, for whom the following minimum levels of cd per age have been established: < , cells/mm from to months and < /mm from to years, or a total lymphocyte count of < % on two separate occasions without being hiv-infected [ ] . a family has been reported involving two brothers aged and with t counts between and /mm , recurrent respiratory, intestinal and cutaneous infections, and failure to thrive. the mother showed a low cd :cd ratio [ ] , while the entire family showed normal levels of ig and subclasses and hla molecules [ ] . other symptoms included mental retardation, pansinusitis, bronchiectasis [ ] , but no infections caused by opportunistic germs such as those reported by the who scientific group [ ] . one case of primary cd deficiency is known of a child with scid without genetic transmission of the deficiency. t-cell proliferative responses to mitogens were defective and il r expression was deficient on his t lymphocytes, and b cells did not differentiate into antibodysecreting cells when provided with the help of normal t cells [ ] . the index patient for primary cd deficiency was the first child of consanguineous kurdish parents. she presented aged months with a rash, pyrexia, hepatosplenomegaly, lymphadenopathy, pneumonitis, pancytopenia, and disseminated cmv infection. laboratory analysis showed absolute lymphopenia, low t cell numbers, with markedly low cd + and low cd + and normal b cell numbers. she responded well to anti-cmv treatment and at months underwent a mud bmt. t-cell engraftment was demonstrated weeks after bmt. despite continuous anti-cmv treatment, her undifferentiated scid human p lck deficiency p lck deficiency is an ar scid due to a defect of an src kinase critical for the generation of mature thymocytes in adult mice. p lck is important in tcr signaling and phosphorylation of the itams of the cd /tcr complex proteins. mutant mice lacking p lck have pronounced thymic atrophy, a critical reduction in dp (cd + cd + ) thymocytes, no detectable mp thymocytes, and only a few peripheral t cells. both proliferation and development of a given defined cell subpopulation depend on meuse age. the absolute numbers and proliferation of dn and isp (immature single positive) thymocytes only proliferate during fetal and early postnatal life up to days after birth, whereas the proliferation is significantly decreased beyond that age, thus lck may have differential roles in the proliferation and maintenance of dn, isp, and mp/dp thymocyte populations [ ] . the first demonstration of a human scid patient with an abnormal expression of p lck is an scid infant hospitalized at months for dehydration, failure to thrive, and sepsis. the immune phenotype included hgg, selective cd lymphopenia, lack of cd expression on cd + t cells and poor t cell blastogenic responses to various mitogens and il . p lck protein expression was only minimal with an unusual mrna splicing pattern of the lck gene. the levels of p fyn were normal and it is therefore possible that p fyn played a role, albeit incomplete, in the development of his mature t cells. the child has since undergone an allogeneic bmt (at months) from a matched unrelated donor (mud) [ ] . unfortunately the boy died months later due to cmv infection and gvhd (fd goldman, pers. comm., nov. ). whn (winged-helix-nude) encodes for a transcription factor that is crucial for maturation of the thymus microenvironment [ ] . nu/nu mice fail to develop a thymus and mature t cells due to a defect in the whn gene encoding a transcription factor necessary for terminal epithelial cell differentiation. a defective whn gene could lead to the disrupted early t cell development in the bm. t cell progenitors were associated with a lack of pta gene expression and a failure to give rise to mature t cells in adoptive euthymic hosts. wild-type hscs rapidly matured into functional t cell progenitors in the marrow of euthymic or thymectomized but not nu/nu hosts. therefore defects in bm prethymic t cell development can contribute to t cell deficiency in nu/nu mice [ ] . in two sisters a severe scid caused by mutation of the whn gene was associated with complete alopecia. hla-identical bmt in one of the two girls resulted in a clear reconstitution of cd + and cd + cmv reactivated, and she died days after bmt [ ] . a -bp deletion in the gene encoding cd resulted in the loss of glutamic acid and tyrosine in the first fibronectin type iii module of the extracellular domain of cd , identifying a region important for cd structural integrity and lack of surface cd expression. this was almost certainly responsible for the id in this girl [ ] . a second child presented at months of age with severe cid, showing similar t-cell defects. despite normal b-lymphocyte numbers, serum ig levels decreased with age [ ] . introduction of a functional cd minigene was sufficient to overcome the main scid-associated defects and represents a potential route to a gene therapy for human cd -deficient scid [ ] . two male infants born to consanguineous parents had scid despite phenotypically normal blood lymphocytes. their t cells were unable to produce il , ifn-g, il and tnf-a [ ] . another child with scid had defective transcription of il genes encoding il -il [ ] . dna binding of activation protein (ap- ), oct, creb, sp , and nf-k b was normal, but the binding of nfat to its il promoter response element [ ] , or the ability of nuclear factors from the child's t lymphocytes to bind response elements present in the il regulatory region [ ] was barely detectable [ , ] both before and after t-cell stimulation [ ] . these results indicate that the nfat abnormality may underlie the multiple il deficiency in these boys. nezelof syndrome, also known as cellular id with ig, or combined with a predominant t-cell defect, or as a scid variant, clinically less severe compared to the previous ones, is characterized by a form of ad, concentrations of ige that may also be extremely elevated (table . ), and normal or increased serum levels of other ig classes [ ] . the cmi study emphasized the mature t-cell reduction or absence, various expressions of immature cells, with cutaneous anergy to spts and a reduced or absent in vitro lymphocyte response to mitogens. from infancy, patients present recurrent or chronic pulmonary infections, pondostatural retardation, oral and/or cutaneous candidosis, chronic diarrhea, recurrent cutaneous and urinary tract infections, gram-negative bacterial sepsis and a particularly severe form of chickenpox [ ] . differential diagnosis must include pediatric aids, also marked by proportionably increased ig and a lack of antibody and t-cell function [ ] . inherited through ar modalities, cd deficiency has been observed in children, two of whom were brothers, with mutations of the fas gene, one hz and het [ , ] , as well as in unrelated children [ ] . these mutations most often arise as a result of mutations in the gene encoding the lymphocyte apoptosis receptor fas/apo-l/cd . a novel mutation has been identified in the intracellular apoptosis signaling domain of fas in members of a family, with several members monitored for up to years [ ] . thus, the deficiency is inherited in an autosomal dominant fashion but with a high degree of variability in clinical expression [ ] , but also in an ar fashion [ ] . the clinical picture is dominated by imposing hepatosplenomegaly with an early onset, even neonatal, accompanied by t-cell hyperproliferation, chronic and persistent lymphadenopathy, and failure to thrive [ ] . an extensive lymphocyte infiltration of lymph nodes, spleen and liver is observed, with t cells reaching , /ml [cd + , cd -cd -(dn) equal to - cells/ml compared to - in controls], as in omenn syndrome, also in the bloodstream, with possible oligoclonality of t cellularity. dn t cells expressed the a/b tcr [ ] . immune dysregulation is associated with g and a hgg (hypergammaglobulinemia, auto-antibodies and aids, especially of the hematological type, such as aiha, and with a severe and recurrent thrombocytopenia [ , ] . autoimmune features are discussed in chap. . an overlapping mechanism could belong to the etiopathogenesis of xlp and omenn syndrome. was has a prevalence of approximately × live births [ ] . it is transmitted as a recessive hereditary trait linked to the chromosome x, localized in a pericentrometric position on the short limb of chromosome x (xp . -p . ) [ ] . it is therefore possible to identify the female carriers and to provide prenatal diagnosis [ ] . the gene that codifies the was defective protein (wasp) has been isolated [ ] and has mutations distributed among all exons of the entire gene, of which are unique and familiar, with two large deletions, one embracing exons - and one intron [ , ] (fig. . ). six novel mutations have been identified that involve nonsense mutations, or small deletions, all of which result in predicted truncation of wasp synthesis [ ] . a new, recurrent mutation is v m, due to a cpg island was found in a hz girl, who showed microthrombocytopenia and infections to the same degree as her hemizygous father and brother. the amount of was protein was about % in platelets and % in mononucleated white cells [ ] . involved in ensuring the t lymphocyte functional polyvalence, also explaining why microvilli and platelet defects are absent [ ] .wasp and several related proteins (the wasp family) are all involved in the organization of the actin cytoskeleton. to carry out vital functions, cells have to rearrange their actin cytoskeletons [ ] . the characteristics peculiar to wasp as a meeting point for the marking pathways is illustrated in fig. . [ ] . the wasp function is absent in cases of was, in ten with attenuated was and in with xlt [ ] . in normal subjects, it is found in the cyto-molecular biology has proven that wasp found only in blood cells binds the small gtpase cdc h in the gtp but not in the gdp [ , , ] . cdc h plays a critical role in the assembly of actin filaments [ ] and in t-cell polarization when they encounter a b-lymphocyte apc [ ] . wasp activity is regulated by several proteins acting in concert to control wasp configuration. the wasp-interacting protein, when phosphorylated, releases wasp from its grip, allowing wasp to be activated by rho-family gtpases [ ] . experimental data also indicate that cdc , wasp and actin might be plasm but not in the nucleus of various cells such as platelets, t and b lymphocytes and monocytes [ ] . the xlt gene is located on the same locus as the was and could therefore be a variant [ ] ; the main immunological anomalies are summarized in table . [ , ] . children with was have significantly elevated levels of il and ige (table . ) and decreased levels of ifn-γ [ ] . the pathogenetic mechanism unifying the symptom triad is not clear; the glycosylation defect has been proved, primarily concerning sialidation, therefore resulting in an instability on the membranes of platelets, neutrophils and lymphocytes expressing a glycoprotein sialopherin (cd ) [ ] , localized on chromosome , which makes it an improbable candidate, even though cd is indeed the binding agent of cd and could therefore play a role in t-cell maturation, differentiation and activation, thereby acquiring marking capacities that are independent of tcr/cd [ ] . however, the tcr-mediated signaling defect is characteristic of was [ ] , in addition to the reduced expression of cd [ ] , which can explain immune and hematological defects. in the lymph nodes, there is a shortage of lymphatic follicles and the thymus-dependent and -independent areas are depleted, moderately at the age of years ( fig. . ) and to a greater extent at years ( fig. . ). the predominant immunological outline is constituted by elevated iga and ige levels, low igm and all igg levels, as well as the absence of a response to polysaccharide antigens, which is why the children's serum lacks isohemagglutinin [ , ] . in unweaned babies, the most striking finding is the cd :cd ratio = [ ] , compared to . in normal children aged . - . (tables . - . ). was usually starts at . months (range, - ) [ ] with hemorrhagic manifestations, petechiae and prolonged bleeding from the umbilical scar or the circumcision site, observed in newborn babies [ ] . the clinical triad is characterized by cutaneous lesions that are practically indistinguishable from rather severe ad ( %), congenital thrombocytopenia ( %), a marked susceptibility to rris ( %) [ ] (figs. . , . ) and gastroenteric symptoms such as hematemesis, melena and chronic diarrhea [ ] . other complications may include neutropenia ( %), arthritis ( %), skin vasculitis ( %), cerebral vasculitis ( %), inflammatory bowel disease ( %), and renal disease ( %) [ ] . a reduced thrombopoiesis (level < , /ml), with microthrombocytes and an accelerated turnover in boys must allow for a suspected diagnosis [ ] . it has recently been proven that the classic presentation is more common in children aged . months than in those aged . months ( % compared to %), unlike platelet counts [ ] . however, only % of unselected children with persistent thrombocytopenia, positive fh, small platelets and defects associated with t and/or b lines had the classic triad and % only thrombocytopenia before diagnosis [ ] . primary immunodeficiencies infections, appearing during the first months of life, are often marked by otitis media, pneumonia, meningitis and sepsis, caused by viruses (cmv and herpesvirus) and by bacteria (pneumococci or other capsular polysaccharide). these are followed by more common infections caused by opportunistic germs, pneumocystis carinii and mycetes such as candida albicans. differential diagnosis should also include a rare ar syndrome similar to was, also reported in female patients, characterized by ad, rris and thrombocytopenia with microthrombocytes [ ] . when caring for these children one must monitor the platelet count, the immunological structure (ig, lymphocyte and subpopulation counts) and the potential onset of autoimmunity and tumors [ ] .aiha may be found in % of children [ ] . prophylactic treatment for infections is done with ivig; mg/kg every weeks) and sulfamethoxazole ( mg/ kg/ days) after diagnosis [ ] . splenectomy may decrease the bleeding tendency [ ] , but early relapse of thrombocytopenia after splenectomy is predictive of a poor prognosis [ ] . on average death occurs around the age of ( in untreated children), but can occur between . and . [ ] , with survival also > . death is caused by massive hemorrhages ( %), tumors ( %) and severe infections ( %) [ ] . the second largest group of patients with id given bmts since are those with was, with . % of children aged < years [ ] . fourteen out of patients underwent phenoidentical (n= ) or haploidentical (n= ) hscts; the other four died before hsct could be undertaken [ ] . boys who had received a mud hsct transplant < years had survival rates similar to those receiving hla-identical sibling transplants, but the success rate decreases dramatically at the age of - [ ].wasassociated t-cell signaling defects can be improved upon retrovirally transduced hscts [ ] . recently, correcting the t-cell defects has been proposed. the potential for correction of the t-cell defects has recently been demonstrated by transduction with an oncoretroviral vector encoding the wasp, which resulted in correction of the deficient proliferative response to tcr stimulation characteristic of was [ ] . ata is a complex ar inherited syndrome, associated with neurological, immunological, endocrinological, hepatic and cutaneous abnormalities, characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, and increased susceptibility to rris [ ] with an incidence estimated at : , - , , live births [ ] . in italy the frequency on the general population, is of . × , with an increase in hets from . % to . % [ ] . it is characterized by a genetic heterogeneity, which is reflected in the division into four main groups of complementation, to which one must add the nijmegen and at-fresno variants, perhaps caused by other well-defined id syndromes the same gene, also localized on the long arm of chromosome q . [ ] . the -kb gene, called atm (at mutated) because of its mutations by defective splicing in all patients with ata, permits het identification [ ] . a dna clone complementary to atm shows considerable affinity to factors responsible for signals involved in regulating the cell cycle and codifying a protein similar to phosphatidylinositol- -kinase (pi k) [ ] , involved in mitotic signal transduction, meiotic recombination, and cell cycle control. a result could be a recombination defect which interferes with b and t lymphocyte gene rearrangement, involving tcr and isotype switching, consequent to a damaged dna triplication and therefore accounting for ig deficiencies [ ] . cells from these patients progress too rapidly from the g phase, in which they receive ionizing radiations, to the s phase, then continuing irradiation, to the g /m phase with further delay, evolving in apoptosis [ ] . this hypothesis has received further credit after observing that the p gene expression does not increase in human cells exposed to radiations [ ] . the p gene is part of the normal cell cycle and during the s phase provides time for the dna physiological repair after exposure to radiation that may also be cosmic [ ] . the thymic tissue is either absent or degenerated with a fetal appearance (fig. . ) : some follicles, also with b cells, are visible at the age of (fig. . ) , at there is complete cellular depletion (fig. . ). immune deficiencies are humoral and cellular (cutaneous anergy and depressed proliferative responses) [ ] . the karyogram shows that the lymphocytes have common rupture points at the chromosomal level with inversions and translocations involving precisely the tcr and ig genes [ ] . most chromosomal translocations involve the genes encoding tcr on chromosome and the ig h chains on chromosome : most breakpoints occur at the loci that encode ig and tcr for antigen (regions q , p , q , q ) [ ] , in areas typical for cod-ification of molecules of immunological importance (chap. ). an important role is played by genes belonging to ig gene superfamily (igsf) (table . ) . possibly the progressive id of ata, like its apparently unlinked manifestations, is at least in part linked to the accumulation of clonal anomalies affecting the tcr and the igsf: this suggests the intervention of "illegitimate" recombinations damaging above all the t cells [ ] . t-cell immunological deficiency is completed with lymphopenia, a decreased cd :cd ratio due to the drop in cytotoxic cd , and a rise of immature forms with tcrgd [ ] . another consequence is the isotype deficiency: about % of patients present sigad; > % are also affected by an igg -igg deficiency with igm becoming monoclonal, and % by serum igg deficiency [ , , ] . fig. . ,the degree of depletion of lymphocytes is extensive in both thymus-dependent and thymus-independent areas chromosome into cells, the chromosomal aberrations induced by x-rays were suppressed [ ] . the rare ar nijmegen breakage syndrome, so called because it was initially seen in two brothers of secondcousin parents living in that city, and at the moment observed in approximately patients, has various characteristics of ata but without ataxia, telangiectasia, or high concentrations of afp. clinical characteristics are singular: short stature and microcephaly with prenatal onset, bird-like profile, prominent midface, a long nose, low-set ears, cutaneous depigmentation with caféau-lait spots, an almost normal intelligence, and also rris and bronchiectasis. humoral and cellular id includes reduction of antibodies and lymphoproliferative responses [ , ] . during an -year period of observation, the id was found to be profound, highly variable, and with a tendency to progress over time in / children [ ] . there is a high proclivity to expressing rearrangements of chromosomes and as in ata [ , ] . dgs is usually sporadic, with known cases of positive fh [ ] . it is caused by a defective development of the rd and th branchial pouches which takes place before the th week of gestation, with consequent thymic hypoplasia or aplasia and parathyroid hypoplasia; the th and th pouches and branchial arches can also be affected [ ] . the cause can be found in the neural crest cell incapacity to migrate and interact appropriately with endothermic cells of the brachial pouches and arches [ ] . deletions (often microdeletions) at the pericentrometric region of chromosome q -pter have been described in %- % of cases [ ] . a microdeletion q . was recorded in children aged - months, % of whom had developmental delays, mild hypotonia, as well as language and speech delays [ ] . another children had deficits in the areas of attention, story and visuospatial memory, arithmetic performance relative to other areas of achievement, psychosocial functioning [ ] , and mental retardation in % of children [ ] , thus indicating the need for early intervention beginning in infancy [ ] . overlapping alterations are present in the syndrome complex known as catch , which in turn includes the charge association. other cases of dgs can derive from microdeleted chromosome p (fetal-alcoholic syndrome, retinoic embryopathy, maternal diabetes) [ ] . this variable phenotype is reliably referred to microdeletion q . ; the greater it is the more complex is the associated phenotype [ ] . another difference depends on the variable spectrum of t-cell abnormalities in individuals with dgs who might have normal t-cell numbers and function, low t-cell numbers but fairly normal t-cell proliferative function [ ] or no t cells purkinje cells (pcs) and degenerated granular cells. that the number of basket cells, so called because they form with the axons bunches of fibrils distributed so as to form a nest in which the nucleus of each pc settles, is almost normal, proving that pcs are probably normal at birth and degenerate only later [ ] . typical clinical manifestations are ataxia, telangiectasia of both auricular lobes and sclera, rris and an elevated incidence of neoplasia [ ] . from a review of patients [ ] , the percentages of symptoms are as follows: progressive ataxia ( %), typically cerebellar, becomes evident when children start walking or a little later, affecting intentional movement and becoming complicated by dysarthria ( %) and involuntary choreic movements ( %), causing the majority to be unable to walk by about the age of - [ ] . at a later stage it is possible to observe nystagmus ( %), strabismus, oculomotor apraxia ( %), reduction or absence of reflexes ( %), and dyslalia, increasingly amplified and, in some patients, also mental retardation [ ] . telangiectasias develop between the ages of and on the bulbar conjunctiva ( %) (fig. . ) , on the flexor surfaces of the limbs and areas exposed to sun rays ( %). height and weight are < th percentile ( %) and the appearance is progeric ( %). severe rris are common ( %), encouraged by antibody deficiencies. the pathogens involved can be bacterial or viral, often resulting in lung bronchiectasis, all starting after the onset of neurological manifestations [ , ] . associated neoplasia ( %) is usually lymphoreticular, less common than adenocarcinoma, with an eightfold increased trend for all kinds of tumors [ ] . in cultures the fibroblasts of these patients are three times as sensitive, compared to controls, to ionizing radiations and to radiomimetic chemical substances, but not to uv rays, unlike what is observed in the cells of subjects affected by xeroderma pigmentosum [ ] . in addition, the persistence of elevated serum a- -fetoprotein (afp) levels was observed in all patients. an interesting in vitro study has reported that by introducing a normal human other well-defined id syndromes fig. . . conjunctival telangiectasia in a girl with ata [ ] . a second group is referred to as having partial dgs (dgsp) or transient forms (dgst), with mild symptoms. the designation "complete digeorge syndrome" (dgsc) is reserved for the third group of infants who have absence of thymic function in addition to other defects of the rd and th pharyngeal pouches, < % of patients with dgs, although they can have high t-cell numbers that respond to mitogens [ , ] . these patients have profound id, with its associated clinical findings [ ] . dgst includes cases with a spontaneous quantitative and qualitative t lymphocyte recovery [ ] . the thymus can also be ectopic: in dgsc the t zones are depleted, the cd /cd markedly reduced both in number and in function with spt anergy, and b cells appear unaffected or increased [ ] . in dgsp, the most common type, t-cell number and function are instead usually normal, as are the cd /cd cells with a nk phenotype, or they may be moderately reduced [ ] . the proliferative response to mitogens can be pathologically reduced [ ] and the response to polysaccharide antigens may be absent [ ] . from neonatal age, there are malformations of other structures that form during the first weeks of embryogenesis, presenting a suggestive but not pathognomonic picture (table . ) [ ] . diagnosis is usually suspected within the first days after birth, due to the presence of hypocalcemic tetany caused by hypoparathyroidism and cardiac malformation. the facial dysmorphism is also characterized by a small mouth with thin lips described as fish-like [ , ] (fig. . ) . the two rare cardiopathies indicated in table . depend on neural crest nonintegration, as mentioned, which accounts for > % of the alterations alone [ ] . others can be observed affecting the right heart, such as fallot tetralogy, pulmonary athresia with an interventricular septum defect, and pulmonary infundibular stenosis [ ] . babies surviving the neonatal period manifest from the very first months an increased susceptibility to infections, particularly those of the respiratory and digestive tract, viral and/or fungal, but also caused by pneumocystis carinii, which can be fatal in dgsc [ ] . other findings include gastroesophageal reflux, speech delay, laryngomalacia, absent kidney, conductive or sensorineural deafness, th cranial nerve palsy, and hypothyroidism [ ] . treatment with high doses of vitamin d and diets enriched with ca gluconate are needed immediately, also ensuring that calcemia remains at the lower limit of normal values so as to avoid snc and renal damage. subsequently the possible correction of cardiac malformations should be evaluated. id may be severe, but can regress spontaneously with reconstitution of cmi and t functions; compensating hyperplasia of the residual parathyroid tissue can make it possible to discontinue ca and vitamin d treatment [ ] . dgs natural history is, however, complicated by mental retardation and the difficulties encountered in correcting cardiac malformations and in controlling hypoparathyroidism [ ] . because of variability in the id severity, it is difficult to evaluate claimed benefits of bmt: in two cohorts of [ ] and transplanted infants [ ] , the survivors were out of ( . %). recently, / and / infants underwent postnatal transplantation with cultured unrelated thymic tissue, with immunosuppression, with positive results [ ] . del q . syndrome, characterized by a -mb deletion on chromosome q . is the most frequent known chromosomal microdeletion syndrome, with an incidence of in , - , livebirths. patients show chapter primary immunodeficiencies clinical outlines not unlike gvhd [ ] (fig. . ) [ ] , suggesting a possible connection to a fas deficiency (cd ) or apoptosis syndrome. the sh d a gene was found altered in two families, thus indicating that xlp must be considered when more than one male patient with cvid is encountered in the same family, and sh d a must be analyzed in all male patients with cvid [ ] . recently a critical revisitation of this experiment in nature has allowed the identification of links between id and allergy [ , , , ] . the rare higes is associated with bacterial rris, chronic ad, coarse facial features and very elevated ige levels [ , ] ( table . ), up to , iu/ml [ ] . linkage to a region on chromosome q has been demonstrated in several affected families; however, neither the fundamental host defect nor the defective gene has yet been identified [ ] . fh is frequently positive for atopic disease, at times higes is combined with an unusual predisposition to staphylococcus aureus infections [ , , , ] . in buckley's study, it was present in . % of cases, of both sexes, indicating an autosomal dominant transmission with incomplete penetrance [ ] . onset occurs in the pediatric age in % of cases [ ] . clinical presentation is unusual: there are no complaints during the first months of life, toward the rd- th month a severe form of chronic ad appears all over the body, which can be associated with other allergic manifestations, including asthma in . % of cases [ ] . skin biopsy specimens reveal spongiosis and perivascular dermatitis and/or folliculitis with a predominance of eosinophils [ ] . there is an excessive predisposition to cutaneous and respiratory tract infections (deep and superficial abscesses, otitis, pneumonia, sepsis) ( fig. . ), also encouraged by neutrophil chemotactic deficiency caused by defective cellular functions (table . ), which, if present, is so pronounced that it becomes a characteristic, unlike ad where it is secondary [ ] . the subcutaneous abscesses, described as cold, not covered by warm and reddened skin, are pathognomonic to higes but not essential to the diagnosis [ ] . the abscess is filled with pus that always grows staphylococcus aureus; in some cases mucocutaneous candidosis and chronic herpetic keratitis are associated [ ] . infections appear within the first months [ ] . the face shows coarse and dysmorphic features, midline facial defects such as a prominent nose and a high, arched palate, and disproportionate cheekbones and mandible; pondostatural growth notably retarded [ ] , pneumatocele (fig. . ) and osteoporosis caused by reduced bone density with a tendency to fracture [ ] complete the picture. six consanguineous families have been reported with an ar form of higes, including cardiac abnormalities, t-cell deficits, cleft palate facial anomalies, and hypocalcaemia. at least genes have been mapped to the deleted region. recently, in / patients with del q . syndrome without q deletion mutations were found in t-box that is a major genetic determinant of the del q . syndrome [ ] . xlp is caused by a defect in the sh d a gene (table . ), which binds to the cytoplasmic domains of cd slam (signaling lymphocyte activation molecule) and b , and may regulate signals transmitted by these receptors in t and nk cells, respectively [ ] . xlp has been reported in > males from > families [ , ] , and in an other males [ ] , it is inherited with the x-linked model. it is set off in males aged - by an ebv infection that became manifest with a very polymorphous pattern, often with unusually severe or fatal infections mononucleosis caused by the immune system incapacity to respond to ebv, or evolving into a hgg with iga and igg deficiency and higms, or medullar aplasia and/or a burkitt type lymphoma [ ] . the disease has been reported in female subjects [ ] . xlp polymorphism could be explained by the fact that the ebv receptor is expressed on differentiating b lymphocytes starting with the preceding isotypic conversion stage [ ] . it has recently been verified that before ebv infection, males already suffer from dys-or pan-hgg, incapable of regulating the expression of ig and/or containing b or t lymphoproliferation. even after ebv infection, the immune system is unable to provide adequate th responses, and therefore releases cytotoxic alloreactive cd and th -like t cell ils, causing extensive damage to the entire parenchyma, exemplified by fulminating hepatitis, cellular infiltrations and tissular necrosis. the lymphoid tissues with an altered structure are also affected by necrosis, with a high incidence of mostly nonlocalized lymphomas [ ] . the thymus is also affected by thymocyte rarification, with other well-defined id syndromes fig. . . bone marrow biopsy specimen in a -year-old boy: numerous histiocytes in erythrophagocytosis affected children aged months to . years, with ar-higes presenting with the classic immunological findings, including rri, eczema, elevated serum ige, hypereosinophilia, and severe recurrent fungal and viral infections [ ] . notably, patients with ar-higes did not have skeletal or dental abnormalities and did not develop pneumatoceles, as seen in autosomal dominant-higes [ ] . among the immunological characteristics (table . ) [ , ] , cutaneous anergy to several antigens such as candida and tetanic toxoid is characteristic, which is associated with the anomaly of proliferative responses by the t cells to antigens and mitogens, in contrast with the integrity of other functions tested in vitro [ ] . t subpopulations appear to be normal [ ] . however, the lymphocyte proliferation to anti-cd /cd monoclonal antibodies can be impaired [ ] . as noted, ifn-γ deficiency associated with a pathological th prevalence has a fundamental impact on ige hyper-production [ , ] . in higes, some studies have confirmed ifn-g deficiency compared to controls [ , ] , also due to an impaired response to il [ ] , while others have not [ , ] ; however, compared to ad, normal levels of t producers of il are characteristic [ ] . considering the ifn-g/il + correlation of ad, in higes no specific t-cell anomalies are noted, nor does the hyper-ige explain this pediatric abnormal susceptibility to infections: high ige levels are also seen in children with ad, who do not, however, have an unusual predisposition to abscess formation [ ] . one typical characteristic is sige directed against microbial antigens: the anti-staphylococcal sige rise to . % compared to normal levels of . %- . %. another constant finding is the increase in % of cases of eosinophil concentrations, which make up %- % of leukocytes [ ] , reaching %- % [ ] . by expressing the cd -cd duo, they stimulate the isotype b-cell switching to ige.we studied children affected by severe ad, chronic fa-induced diarrhea and asthma. the allergens responsible were cm and der p [ ] . in case of higes caused by fa, atopic manifestations can clearly improve following an exclusion diet, reducing the frequency of infections and partially correcting the immune defect [ ] , revealing how fa can induce several immunological anomalies. diagnosis is made on the basis of the data in table . ; differential diagnosis with ad is schematized in table . [ ] . treatment with cromolyn is extremely effective, anti-staphylococcal antibiotic treatment [ , ] and if necessary antifungal therapy provide good results [ ] . griscelli disease, mapping to chromosome q [ ] , is an ar syndrome caused by mutations in the myo a (gs ), rab a (gs ), or mlph (gs ) genes, all of which lead to a similar pigmentary dilution [ , ] . the disease is also characterized by partial oculocutaneous albinism, predisposition to pyogenic infections and in most patients by abnormal regulation of the immune system, which results in a syndrome of macrophage hyperactivation, known as hemophagocytic lymophohistiocytosis [ ] . mutations in the gtp-binding protein rab a (gs ), which appears to be involved in an uncontrolled t lymphocyte and macrophage activation syndrome, leading to death in absence of bmt, occur in this syndrome [ ] . a mutation was found in the myo a gene (gs ) associated primarily with neurological impairment [ ] . two identical twin boys aged months were reported with persisting fever, mouth ulcers, hepatosplenomegaly, pancytopenia and failure to thrive [ ] , as was an -month-old infant [ ] . both infants had silvery-gray hair and pigment clumps on the hair shafts, and skin biopsy showed accumulation of melanocytes on melanosomes. their parents were first cousins and a sibling with similar manifestations had already died, as did the twins. a genetic study revealed a -bp deletion in the rab a gene ( del aagcc in exon ) [ ] . in a -year-old child with hemophagocytic syndrome, id, and secondary neurological disorders, typical melanosome accumulation was found in skin melanocytes and pigment clumps were observed in hair shafts. two heterozygous mutant alleles of the rab a gene, a c-t transition (c t) leading to q stop and a g-c transversion on the exon splicing donor site (g + c) were found [ ] . the finding of gray strands of hair, gray eyebrows, and eyelids in childhood should alert pediatricians to considering griscelli syndrome since an early diagnosis is life-and health-saving [ ] . the phagocyte system with the biochemical basis of cgd is analyzed within the framework of innate immunity. chronic granulomatous disease (cgd) has an overall prevalence of : , to : , although this could be underrated (table . ), considering that some subjects may have a very mild clinical phenotype that escapes diagnosis [ ] . a us registry of birth rates found a prevalence of : , to : , live births for the period - [ ] . the youngest patient was days old [ ] and in children with cgd the mean age at the onset of infections was months, with a median delay in diagnosis of . years [ ] . otherwise the the clinical features of this rare ar disease include oculocutaneous albinism and susceptibility to especially s. aureus and b-hemolytic streptococcus [ ] . approximately % of patients develop an accelerated phase of the disease, with deposition of lymphohistiocytes in the liver, spleen, lymph nodes and bm, resulting in hepatosplenomegaly, lymphadenopathy, bm infiltration hemophagocytosis, pancytopenia as well as fever, jaundice, prolonged bleeding, easy bruisability, neurological changes (nystagmus and neuropathy), mild mental retardation, and partial ocular and cutaneous albinism [ , , ] . the cellular hallmarks of the disease include large lysosomal granules in leukocytes, giant melanosomes in melanocytes and affecting other cells of the body such as neural schwann cells, renal tubular cells, gastric mucosa, pneumocytes, hepatocytes, langerhans cells of the skin, and adrenal cells [ , ] . the fundamental defect in this disorder was found to be caused by mutations in a gene mapped to chromosome q -q [ ] encoding a cytosolic protein on chromosome named lysosomal-trafficking (lyst) regulator, encoding a -kd protein whose function remains unknown [ ] . bmt is resolutive in these children [ ] . normal protein expression (x + form), but with a total absence of oxidase due to incorrect binding [ ] . ar-cgd is caused by a mutation in the genes encoding the remaining oxidases of kd (p phox ) (phox, phagocytic oxidase) [ ] (ncf- ), p phox of kd (cyba), and p phox of kd (ncf- ) [ , ] . the ar-cgd forms ( . %- % of cases) [ , ] are identified using the immunoblotting technique, depending on whether they affect the p phox , the p phox or the p phox [ , , , ] , with greater prevalence in an american study [ ] . patients with the x-cgd appear to have a more serious clinical phenotype than patients with the ar-cgd, based on the fact that they are diagnosed significantly earlier (mean, . years of age vs . years of age, respectively), have a significantly higher prevalence of infections and a higher mortality ( . % vs . %) [ ] . mutations in any of the structural molecules (table . ) lead to cgd. mutation of rac (see lad), the predominant g protein in neutrophils, leads to defects in so production, as well as in chemotaxis [ ] . activation of the nadph oxidase requires complex rearrangements between the protein median age at onset was . months, and the median age at diagnosis was . years [ ] . the deficiency appears in two forms ( [ , ] , with an h-chain deficiency, is divided into four x subtypes (table . [ , , , , , ] ), also identified on the basis of nbt results, depending on whether the x is absent (the most common form), reduced or present but inactive; subtype x is divided into two variants: in one of them the nbt is slightly positive in %- % of cells ( % of patients), in the other in %- % ( % of patients) [ , , , ] . more precisely, the four subtypes are caused by mutations in the four gp phox regions, many of which depend on cybb gene mutations, causing the x form, while mutations depend on the nadphoxidase activity (x form) and eight others lead to a phagocyte deficiency subunits, which are in part mediated by noncovalent binding between src-homology domains (sh domains) and proline-rich motifs [ ] . cgd is a hereditary disease (table . ) characterized by severe recurrent pyogenic infections. this marked susceptibility is caused by the phagocytes' incapacity to kill in particular the catalase-positive bacteria, because of a genetic defect of the nadph-oxidase enzymatic system situated in the wall of the phagocytic vacuole. in cgd, phagocytosis occurs normally, but the nadph-oxidase is unable to markedly produce anion superoxide (o •-), h o and other o free radicals, thereby permitting the survival of microorganisms within the cells, where they are protected from the antibodies and from most antibiotics [ ] . another consequence of the lack of o radicals is the development with countless inflammatory episodes, which then result in typical granulomas [ ] . the nitroblue tetrazolium (nbt) reduction test is based on the chemical characteristics: in fact, the phagocytes without o •are unable to reduce the yellow nbt of products activated by pha aspecifically stimulated phagocyte o , or specifically with corpuscle particles such as preopsonized yeasts (fig. . ) . the result was % in children [ ] . at a molecular level, the genes that codify the two subunits of flavocytochrome b , gp phox and p phox have been cloned: respectively the cytochrome, h (b) and l (a) chains situated on the phagosome vacuole membrane, and also the cytosolic factors p phox , p phox and p phox , deriving from the nadph-oxidase activation, all proteins placed inside the cytoplasm and that belong to innate immunity. it has therefore been possible to identify molecular lesions at the cgd origin, with the exception of the p rac [ , , ] . the clinical pattern is severe in the x form and variable in the other two x forms; onset occurs within the st year of life in / of cases, and in others within the nd year [ ] , although it can appear also at the age of [ ] . purulent recurrent infections, with a granulomatous evolution, predominantly affect the epithelial surfaces normally colonized by bacteria, such as cutaneous, subcutaneous, mucous membranes, the respiratory tract and the intestine: cutaneous and mucosal infections, and lymphadenitis lead to suppuration and fistulation (fig. . ) , pneumonia or lung abscesses (fig. . ) are more frequently characterized by persistent fever and diarrhea [ ] (table . ) [ , ] . pneumonia was the most prevalent infection in patients ( %) (mostly by aspergillus), followed by suppurative adenitis ( %), subcutaneous abscess ( %) and liver abscess ( %); mostly by staphylococcus, osteomyelitis ( %) mostly by serratia, and sepsis ( %), and by salmonella [ ] . in a long-term trial, pneumonitis was the most prevalent infection ( %) followed by lymphadenitis ( %), aphthous stomatitis ( %), liver abscesses ( %) and chronic lung disease the rates (%) consist of a first [ ] and of a second number related to the european study [ ] ; us data regarding ar cgd are in parentheses [ ] . data from [ , , , , ] . x x-linked, ar autosomal recessive, ad autosomal dominant inheritance, nd not done. a the superscript symbols indicate the level of immunoreactive proteins: undetected,diminished, + normal protein levels. granulomas in the entire lung parenchyma, which are formed by mononucleates (fig. . a) with giant cells (fig. . b) . the chronology of infection onset is summarized in table . [ ] : lymphadenitis is the earliest. osteomyelitis is usually a worrying complication: ( %) [ ] . lymphadenitis, lung infections, enteral infections, and hepatic abscesses were the most frequent infections in a cohort of children [ ] . staphylococcal liver abscesses are almost pathognomonic of cgd [ , ] . because the infections develop in areas drained by lymphatics, they tend to diffuse via the lymphohematogen route, thus causing arthritis and osteomyelitis and abscess formation, especially affecting the bones, which are the most severe manifestation, and hepatitis with common upsurge of hepatosplenomegaly. lung infections are almost the rule: those initially segmented and parallel tend to gradually spread over the entire lobe [ , ] . histological examination shows widespread gastric outlet obstruction urinary obstruction extensive bone destruction involves various segments, for example the vertebra, the metacarpus and the metatarsus, causing widespread damage, which is difficult to treat and is also irreversible [ , ] . aspergillus, pulmonary, bone (fig. . c) or encephalic infections constitute a severe therapeutic problem and are threatening events, with a mortality rate of %, but with specific treatment the prognosis is good as far as recovery is concerned [ ] . the treatment includes prophylaxis with trimethoprim-sulfamethoxazole (tmp/smx) ( mg/day given in two divided doses), and ifn-g ( mg/m subcutaneously thrice weekly) in all patients with cgd, regardless of genotype [ , ] . itraconazole therapy ( and then mg/kg/day) has an excellent tolerance in all cases and was effective in of children ( . %) [ ] . survival until the age of and beyond is achieved by % of patients with cgd xl and % of those with cgd ar [ ] . because the prognosis is uncertain, as observed, the only possibility for a definite resolution is with a bmt, from family donors who are x-cgd or x-cgd-identical [ ] . bmt was successful in children out of ( . %) (see table . ), including a -year-old boy with x-cgd who underwent successful hla-identical peripheral blood sc transplantation during invasive pulmonary aspergillosis and osteomyelitis, which was unresponsive to antifungal treatment [ ] . lad is due to mutations in the gene on chromosome at position q . encoding cd (table . ). it is divided into five types: lad type i to lad type v [ , , , ] . the three subunits of the cd /cd complex are involved in pid (lad type i syndrome), ar, linked to the lack of a m b equal to cd b/cd (table . ) surface expression on all leukocyte populations caused by different mutations in the cd encoding gene, often severe in infancy [ , ] . children with a deficiency of these integrins have a defect above all in phagocyte action, suffer from severe infections from the neonatal period [ ] due to absent b -integrin activity, which impairs neutrophil ability to exit the circulation and travel to sites of infection. on the contrary, leukocyte movements are not prevented, indicating the normal involvement of cd and cd (table . ) . the clinical basis for defining this disease, described in over cases [ ] , dates back to a study at the soothill school in [ ] . there are two forms of lad type i [ ] : if the deficiency is full blown (no detectable cd ), the clinical symptoms (table . ) [ , ] are dominated by severe and recurrent infections with a negative prognosis in the first years of life unless corrected by an allogenic bmt, the only resolutive treatment [ ] . if instead it is a partial deficiency with residual cd expression, the clinical outline is less severe and some patients, with appropriate treatment, can live to adult age [ ] . lad type ii, ar (cd levels are %- % of the normal levels), with a molecular base represented by an slex ligand (cd s) is a defect common to cd e and p, which mediate neutrophil rolling. in the absence of a gdp-fucose transporter, the slex is not made. lad type results from mutations in this transporter that takes fucose into the golgi apparatus for posttranslational fucosylation of newly synthesized proteins. this is the ligand for cd e; without it, leukocytes cannot make initial attachment to vascular endothelium [ ] . lad has been described in two children aged and with mental retardation, from different families, but both with parents who were blood relatives [ ] . it has a lower mortality rate [ ] . mice with a deficiency of both selectins show a lad-like syndrome, providing a useful model for studying these syndromes [ ] . lad type iii shows defective tethering and adhesion and bleeding diathesis. this is a new syndrome where in vitro leukocytes showed normal rolling along endothelial cell cultures but defective tethering and tight adhesion. thus this is a defect in the capability of vascular integrins on circulating leukocytes to rearrange with their endothelial ligands at adhesive contacts and rapidly arrest on target vascular endothelium in response to endothelial-displayed chemoattractants. however, the expression levels of the major integrins on lymphocytes and neutrophils were largely conserved in the patient cells, ruling out a lad-i syndrome. patient leukocytes showed no lad-ii like fucosylation defect, since they expressed normal levels of the fucosylated marker cd s, comprising the slex carbohydrate selectin ligand [ ] . defects in both leukocyte and platelet functions that are biochemically and molecularly distinct from the adhesion disorders previously described suggest a mutation in an early myeloid pathway. the defect is associated with regulation of the gtpase activating protein rap , as demonstrated by the intact rap expression and activation by phorbol esters, thus ruling out an lad defect in rap gtp loading [ ] . lad type iv manifests defective cd e expression or tethering. a girl developed pseudomonas omphalitis at weeks of age, recurrent ear and urinary tract infections, and had clinical evidence of impaired pus formation reminiscent of a lad syndrome, but her neutrophils were functionally normal and expressed normal levels of cd , cd e, and slex. however, the patient showed an absence of cd e from the endothelium, although e-selectin mrna was present. in contrast to patients with lad , she had mild chronic neutropenia but appropriate leukocyte increases in response to infections or gm-csf. a bm biopsy performed during a period of health showed normal cellularity for her age. her fh is remarkable only for a previous sibling who had died at weeks of gestation of a staphylococcal infection of the fetus, amniotic fluid, and phagocyte deficiency placenta. she also has two half-sisters who are completely well. the fh is negative for recurrent infections in either parent or more distant relatives [ ] . lad type v caused by rac deficiency. a -week-old boy born to unrelated parents had delayed uc separation, perirectal abscesses, poor wound healing, and absent pus at sites of infection in the setting of neutrophilia, suggesting a neutrophil defect. his neutrophils exhibited decreased chemotaxis, polarization, azurophilic granule secretion, as well as significantly reduced stimulated superoxide production but had normal expression and up-regulation of cd b. rac constitutes more than % of the rac in neutrophils [ ] . a -yearold boy who had multiple recurrent, life-threatening infections characterized by leukocytosis and notable for the absence of pus in the inflamed tissues was reported. the presence and density of cd b, cd c, and cd were normal. the expression of cd p and cd l were also normal. a bmt was curative. the boy shared a phenotype that closely mimicked that of a mouse mutant deficient in the rho gtpase, rac [ ] . the disease was shown to be attributable to an ad mutation in the rho gtpase rac at an amino acid needed for proper interaction with other intracellular proteins. rac comprises > % of the critically important g protein rac in neutrophils. each member of the family appears to control a distinct function of the actin cytoskeleton (chemotaxis and degranulation) and nadph oxidase (superoxide production) function [ ] . a male child from the mother's first pregnancy was born at term from parents of arab ethnic origin who were first cousins. he had a severe genetic disorder associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis and a bleeding diathesis. platelet transfusions and antibiotic courses reduced the symptoms, which remained a significant clinical problem. at age years, he died from disseminated fungal infection after a mismatched bmt. a younger brother presented with the same clinical and hematological phenotypes at birth and died at age week from sepsis. g pd converts g p to -phosphogluconolactone, generating nadph and a h + ion from nadp + . nadph oxidase catalyzes the monovalent reduction of o to o •-, with the subsequent conversion to h o by superoxide dismutase [ ] . in the form of a partial deficiency, known as the cause of hemolytic anemia or favism, the enzyme's residual activity ( %- %) permits nor-mal bactericidal activity. the g pd variants have been classified by the level of residual enzyme activity and propensity for hemolysis and grouped into five classes: class i, severely deficient with chronic hemolytic anemia; class ii, severely deficient with occasional hemolytic anemia (< % residual activity); class iii, moderately deficient ( %- % residual activity); class iv, normal activity ( %- %); class v, increased activity [ ] . in a trial on g pd-deficient subjects originating from different parts of italy, a greater molecular heterogeneity than described by others was observed, especially in sardinia [ ] . in a complete deficiency, sexually transmitted, whose gene is localized on the chromosome x at position p and characterized by several mutations and their variants, with a consequent deficiency of bactericidal activity, the neutrophils are unable to kill s. aureus, e. coli and serratia, and therefore there is an increased susceptibility to infections, rather like cgd [ ] . the diagnostic work-up of children may reveal a child with recurrent infections who initially received the diagnosis of g pd deficiency, subsequently shown to have the phenotype of x-linked cgd [ ] . the disorder has a higher incidence in mediterranean countries and asia, in japan ( . %) than in indonesia ( . %), as ascertained with a novel screening kit [ ] , and is low in newborns in tehran, iran ( . %) [ ] . within the framework of oxygen-dependent killing defects, hereditary myeloperoxidase deficiency (mpo) is the most common neutrophil biochemical defect and plays an important role in the host defense mechanism against microbial diseases. the neutrophil disorder characterized by the lack of mpo activity is speculated to be associated with a decreased level of immunity. mpo is unusually accompanied by a specific pathology. ar transmitted, it appears far more common than previously suspected ( : , for the partial deficiency to : , for the total deficiency). it is a disorder that is prevalently recorded in entirely healthy patients and therefore, in most cases, a random laboratory finding. in addition to three already-known mutations, the genetic characterization of an italian population showed the presence of six novel mutations: four missense mutations, a deletion of an adenine within exon (c. dela) and a mutation within the ¢ splice site of intron (c. - a>c). the c. dela deletion causes a shift in the reading frame with the occurrence of a premature stop codon within the pro-peptide. the activation of a cryptic ¢ splice site located nt upstream of the authentic ¢ splice site causes a shift in the reading frame that may lead to the generation of an abnormal mpo precursor lacking the enzymatic activity [ ] . in a japanese patient with complete mpo deficiency, neutrophil function analysis revealed that mpo activity was alkaline phosphatase [ ] . monocyte functional alterations in the second individual suggest that c/ebph plays a critical role in monocyte/macrophage development of humans and implicates abnormalities in monocytes/macrophages and neutrophils in the onset and development of the disorder [ ] . severe congenital neutropenia (scn) and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. recently, mutations of the gene encoding neutrophil elastase (ela ) have been indicated as the most common cause for scn as well as the cause for autosomal dominant cyclic neutropenia [ ] . deficiency of ela leads to regularly fluctuating levels of neutrophils [ ] . linkage analysis on affected pedigrees have shown that cyclic neutropenia and sporadic cases of this disease are due to a mutation in the gene for ela , located at p . [ ] . this enzyme is synthesized in neutrophil precursors early in the process of primary granule formation [ ] . a mutation in the ela gene was detected in one of three apparently autosomal dominant kindreds with familial scn. no mutations were identified in the apparently ar families [ ] . these results fit those showing that mutations were found in all five scn families [ ] , but they suggest that not all cases of autosomal dominant scn caused by mutations in ela [ ] . however, the high frequency of het mutations in the neutrophil elastase gene in sporadic scn confirms a previous report [ ] . considering that four novel mutations and a low-frequency polymorphism were detected, nearly all cases of sporadic scn may result from de novo het mutations in ela [ ] . in recurrent scn, an absolute neutrophil count of < cells/mm (or < . ¥ /l) [ ] oscillates with an approximate -day periodicity. circulating neutrophils vary between almost normal numbers and zero [ ] . in about % of patients with cyclic neutropenia, however, the cycles range from to days [ ] . in children referred during a -year period pids were as follows: cyclic neutropenia ( . %), shwachman-diamond syndrome ( . %), kostmann syndrome ( %), and chédiak-higashi syndrome ( . %). the mean absolute neutrophil count of children was . ± . cells/mm (range, - , /mm) at the first visit. the children first experienced symptoms of infection suggesting neutropenia at a median age of . months (range month to years), also suffering from oral ulcer, otitis, pneumonia, diarrhea, cutaneous abscess, and oral candidiasis [ ] . fever, stomatitis, and periodontitis and skin infections occur during periods when the neutrophil count is low. significantly diminished with slightly elevated superoxide production. mutational analysis of the patient revealed a glycine to serine substitution (g s) in the exon region [ ] . because the granulocytes without mpo cannot kill candida, some subjects, presumably carriers of a more extensive mutation and in association with other diseases, present severe and recurrent candida infections. the mpo defect can be diagnosed via a cytochemical investigation or a quantitative count of enzyme levels [ ] . neutrophil-specific granule deficiency is a rare autosomal dominant disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins [ ] . the markedly decreased level of mrna expression for the bactericidal/permeability-increasing (bpi) protein, the activation factor pu- and defensins in these patients suggests a role for ccaat/enhancer binding protein (c/ebpη) gene in earlier phases of the myeloid differentiation program [ ] . c/ebpη is a member of the leucine zipper family of transcription factors, expressed primarily in myeloid cells [ ] . recessive mutations in the c/ebpη gene were described in one patient; analyses of the c/ebpη locus indicated that the disorder could have resulted from hz recessive inheritance of the mutant allele from an ancestor shared by both parents [ ] . loss of c/ebph function is the primary genetic defect in this disease [ ] . in a second individual lacking functional c/ebph, analysis of peripheral blood leukocytes revealed aberrant expression of cd , cd b, cd , cd , and cd on the proband cells [ ] . a male patient lacking neutrophilspecific granules died from complications of pneumonia at age [ ] . neutrophil-specific granules contain important microbicidal components (table . ) . among other deficiencies of oxygen-independent killing, this ar defect is characterized by severe recurrent bacterial deep-tissue skin infections without patients showing an increased susceptibility to a particular pathogen. they have defects in chemotaxis, disaggregation, and receptor up-regulation. deficiencies of the oxidoreduction and microorganism-killing mechanisms have also been described. the markedly decreased level of mrna expression for the bactericidal/ permeability-increasing (bpi) protein, the activation factor pu- and defensins in these patients suggests a role for c/ebph in earlier phases of the myeloid differentiation program [ ] . the defect is identified through a blood test colored with a wright reactive in which polymorphonucleates do not present the specific granules that normally contain lactoferrin. from a morphological point of view, the nuclei appear bilobated and the nuclear membrane may show intro-and extroversions. it is also possible to identify the membrane's lack of cyclic neutropenia is an autosomal dominant disorder in which cyclic hematopoiesis causes intervals of neutropenia and susceptibility to opportunistic infection. in nine families whose children displayed typical blood patterns, pedigrees confirmed dominant inheritance without evidence of heterogeneity or decreased penetrance; three pedigrees suggested new mutations [ ] . a wide spectrum of symptom severity, ranging from asymptomatic to life-threatening illness, was observed within the nine families. the phenotype changed with age. children displayed typical neutrophil cycles with symptoms of mucosal ulceration, lymphadenopathy, and infections [ ] . patients are usually asymptomatic, but during the period of severe neutropenia, recurrent overwhelming infections, inflammation, and ulcers occur in about % of patients and can lead to significant chronic morbidity [ ] . severe neutropenia was shown by children, moderate by , and mild by : of these children had leukopenia, anemia, thrombocytopenia, and monocytosis. during follow-up, respiratory infections developed in , oral manifestations in children. the most common infections, in descending order of frequency, were otitis media, abscesses, pneumonia, oral ulcers, acute diarrhea, cutaneous infections, oral candidiasis, and periodontits. sinusitis, cystitis, conjunctivitis, meningitis, and osteomyelitis were less frequently observed. hepatomegaly was also detected in children and splenomegaly in one; children died of recurrent infections. therefore, recurrent infections always deserve further evaluation for detecting such disorders [ ] . abdominal pain must be assessed aggressively because of the high frequency of clostridium infections during the period of severe neutropenia [ ] . during the course of scn, bm shows lack of maturation of granulocyte precursors beyond myelocytes, and there is myeloid hyperplasia during the remainder of the cycle. occasionally, there is a reduction in the severity of neutropenia and the accompanying infections over time [ ] . a complete clearing of symptoms and a significant increase in quality of life is noteworthy in children [ ] . however, while the disease is commonly described as benign, four children in three of the nine families died of clostridium or e. coli colitis, documenting the need for urgent evaluation of abdominal pain [ ] . pediatric cyclic neutropenia is effectively treated with rhug-csf (recombinant human g-csf), usually at doses of - mg/kg/day (median dose, . mg/kg/day) [ ] or twice weekly, or once a month. typically, children are noted in early infancy to have persistent scn with absolute neutrophil counts < . ¥ /l lasting for months or years [ ] . in children aged days to months, the initial and lowest median absolute neutrophil counts were . ¥ /l and . ¥ /l, respectively [ ] . usually, children suffer from long-term recurrent bacterial infections, and maturation arrest of myelopoiesis at the promyelocytemyelocyte stage of bm development [ ] . the disease begins during the st year of life, and its infectious complications include cellulitis, perirectal abscess, peritonitis, stomatitis, and meningitis, commonly as a result of infections with s. aureus, e. coli and pseudomonas aeruginosa [ ] . the numbers of circulating monocytes and eosinophils are often increased [ ] . missing the most important cells in the defense against bacterial infections, the neutrophil granulocytes, children suffer from episodes of severe, often life-threatening bacterial infections [ ] . they spend many days in hospital, requiring iv antibiotic treatment. recurrence of bacterial infections leads to irreversible tissue damage, for example in the lungs, requiring often disabling surgical interventions. a high incidence of significant bone mineral loss was seen in children with scn [ ] . the presence of qualitative and quantitative abnormalities of primitive myeloid progenitor cells expressing g-csfr may play an important role in the impairment of granulopoiesis in these patients, thus nearly all patients have a response to pharmacological doses of rhug-csf: neutrophil counts rise, infection rates fall, and mortality is reduced [ ] . since the introduction of rhug-csf, most children enjoy a normal life span and a greatly improved quality of life, although they still have problems with infections, especially chronic gingivitis and periodontitis [ ] . it is more likely that the bone loss was caused by the pathophysiological features of the underlying disease, but it is possible that rhug-csf accelerates bone mineral loss [ ] . prolonged administration of rhug-csf at a dose of u/kg bw twice daily may be associated with increased bone resorption, mediated by osteoclast activation and leading to bone loss. in children, the resulting osteopenia can be successfully managed with antiresorptive bisphosphonate therapy with significant improvement in bone density [ ] . a child maintained on long-term rhug-csf therapy developed acute myelogenous leukemia associated with a g-csfr mutation.after having undergone successful allogeneic bmt, both ela- mutation and g-csfr mutation became undetectable by pcr [ ] . shwachman syndrome, a rare ar condition, characterized by pancreatic insufficiency, reduced mobility and neutrophil chemotaxis, cyclic neutropenia, thrombocytopenia, metaphyseal dysostosis, delayed growth and recurrent pyogenic infections, in two cases was associated with isolated gh deficiency [ , ] . in addition to metaphyseal chondrodysplasia, neutropenia, and pan-poor to absent granuloma formation [ ] . salmonella and certain viral infections [hsv, cmv, parainfluenza, and respiratory syncytial virus (rsv)] are also seen [ ] . most patients bearing an ifn-gr deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome [ ] . mortality in these children is high, and infections are severe and recurrent [ ] , as in an -year-old girl before receiving a bmt [ ] . a point mutation may be fatal: an individual, probably hz for the mutation, died from meningitis due to mycobacterium bovis [ ] .a hz missense ifn-gr mutation was identified in two siblings who did not respond to low or intermediate concentrations, yet responded to high ifn-g concentrations, probably for a reduced affinity of ifn-gr for its ligand [ ] . otherwise the mutation results in normal surface expression of ifn-gr that do not bind ifn-g [ ] . a dominant deletion in the ifn-gr gene has been reported in a female patient hz for a -bp deletion in exon of ifn-gr who developed postvaccinal disseminated bcg infection [ ] . the ar form of partial ifn-gr deficiency was reported in patients of unrelated kindred with susceptibility to mycobacterial infection [ ] . an -year-old girl with ifn-gr deficiency, also with recurrent mycobacterial infections and liver cirrhosis with portal hypertension, received red cell-depleted bmt from her hla-identical sister. the transplantation course was uneventful and years later the child remains in excellent clinical condition and free of mycobacterial infections [ ] . a complete ifn-gr deficiency was found in a child due to a hz dinucleotide deletion resulting in a premature stop codon in the protein extracellular domain. this gene defect emphasizes the critical role that ifn-g plays in host defense against mycobacteria [ ] . a girl with bcg and salmonella enteritidis infection and a hz recessive deletion in the p subunit of il leading to a complete il p deficiency has been reported. a large hz deletion within the il p subunit gene was found, precluding il p (composed of p and p subunits) functional expression by activated dcs and phagocytes. the net result was a markedly impaired ifn-g production by lymphocytes. however, addition of recombinant exogenous il p in the assay was able to restore normal ifn-g production in vitro [ ] . the girl suffered from well-organized granulomas, possibly due to residual il -independent ifn-g production [ ] . another kindred [ ] and two siblings and one unrelated patient [ ] carried the same large deletion, also accompanied by disseminated infections. a -year-old female was repeatedly hospitalized since the age of weeks for recurrent episodes of pneumococcal pneumonia with sepsis and other infections in the absence of creatic exocrine insufficiency, the findings in children are noted as variable extremity shortening, cup deformation of the ribs, metaphyseal widening and hypoplasia of the iliac bones, and increased echogenicity of the pancreas with no change in size [ ] . recurrent infections begin during the st year of life and commonly involve the sinuses, lungs, bones, skin, and urinary tract [ ] . neutropenia, either cyclic or intermittent, occurs in all patients, and %- % of patients also have pancytopenia [ ] . immune functions may be involved in this syndrome, including marked pan-hgg, especially of the iga, normal/increased cellular immunity, but depressed humoral and nk cell immunity [ ] . in patients diagnosed in infancy, a significant growth improvement and a decreasing frequency of infections were observed over time, in addition to improvement or normalization of exocrine pancreatic function [ ] . a continuous spectrum from systemic bcg infection to local recurrent nontuberculous mycobacterial infection covered by the clinical features of affected children has recently helped to identify several genetic defects in the monocyte-macrophage-th t-cell pathway [ ] . different types of mutations in four genes (ifn-gr , ifn-gr , il p , il rb ) forming the ifn-γ/il axis [ ] have revealed both allelic and nonallelic heterogeneity and result in different disorders whose common pathogenic pathway is impaired ifn-g-mediated immunity [ , ] . several children have been reported who presented a new kind of hereditary id with severe and/or recurrent infections caused by only one microorganism family, in opposition to other patients with classic pid. five new syndromes may encompass these children with a genetic predisposition to infectious diseases. if the ifn-g/il axis is impaired, the host becomes highly susceptible to infection with organisms that replicate intracellularly (susceptibility to mycobacterial disease). stat- (signal transducer and activator of transcription- ) deficiency predisposes to viral disease, nemo and irak- (il r-activating kinase- ) deficiencies predispose to infections caused by pyogenic bacteria [ ] . this pid encompasses several defects: complete, partial, and ar ifn-gr deficiency, and complete, partial, and ad ifn-gr deficiency [ ] . ifn-g and the cellular responses induced by it are essential for controlling mycobacterial infections. patients with ar mutations leading to complete loss of ifn-gr or ifn-gr expression have the most severe phenotypes, and they present early in life with disseminated severe infections, especially if they have received bcg vaccination, and have fever. she exhibited il deficiency that was associated with an abnormality of the il p gene. although present, ifn-g was reduced [ ] . a genetic lack of il rb surface expression predisposes to severe infections by pathogenic mycobacteria or salmonella and causes strongly decreased, but not completely abrogated ifn-g production [ ] . the deficiency may be complete as well as partial [ ] . several patients with these features have been reported [ ] . three unrelated individuals with severe, idiopathic mycobacterial and salmonella infections were found to lack il rβ chain expression. il rβ sequence analysis revealed genetic mutations that resulted in premature stop codons in the extracellular domain [ ] . a patient with severe infections as above and multiple adverse drug reactions had t cells unable to produce ifn-g or proliferate in response to il , despite the expression of wild-type il rβ and il rβ [ ] . defective il r signaling leads to low t-cell and nk-cell ifn-g production [ ] . il rb and il rb chains are associated in an ar deficiency with susceptibility to mycobacteria and salmonella infections [ ] . the stat ( forms, ad and ar [ ] ) s mutant failed to restore ifn-g production in stat -deficient il rb transgenic cells [ ] . stat , - , and - activation by il was lost, an impairment specific for il ; nor is activation of stat alone sufficient for il -induced ifn-g production and proliferation [ ] . two unrelated infants hz with respect to mutated stat suffered from mycobacterial disease, but unlike patients with ifn-gr deficiency both died of viral disease [ ] the complement is an integral part of the humoral defense system against infections and also for promoting inflammatory process (figs. . , . ). complement deficiency was found in / dutch patients ( . %) over a -year period ( . % × year) [ ] . from the study of blood donors the prevalence may be of . % in the general population [ ] . congenital deficiencies have been described for most of the proteins it is composed of (tables . and . f [ , , , ] , usually following the ar model. properdin deficiency is the only complement deficiency that is x-linked [ ] . hets can be easily identified because their relevant component is present in the serum with a % concentration. the lack of one component at the hz level serologically involves the blockage of enzyme release below and the absence of hemolytic activity, while that of controlling proteins causes its uncontrolled activation, consuming the factor that is the object of control and, in various ways, also of successive components [ ] . nonfunctional c q variants have been observed, c r and c s deficiencies are often associated, probably because they are mapped on contiguous genes of chromosome (c q on ) [ ] . the b, c and c genes, situ-ated on the short limb of chromosome , constitute along with others the hla class iii (chap. ). c and c are codified on chromosome p and have a similar structure; c shows a different structure, because the molecule consists in three a, b and g chains, united to form two subunits, a-g and b dictated by different genes [ ] . alternative pathway deficiencies are extremely rare [ ] . complement deficiencies are accompanied by an increased frequency of infectious pathologies [ ] , although it is not rare to come across them in individuals who are apparently in good health, as in the case of c hereditary deficiency [ ] . also frequent are teens and young adults with autoimmune manifestations (chap. ). classic pathway deficiencies are often associated with sle-like diseases (systemic lupus erythematosus), id of the early components of complement (c -c ) are associated with risks of infections caused by encapsulated bacteria such as streptococcus pneumoniae, haemophilus influenzae type b, as well as by meningococci [ ] . the incidence of sle in patients with c q, c , or c deficiency is %, %, and %, respectively [ ] . partial c deficiency is also associated with sle; % of patients with sle exhibit c a deficiency [ ] . several components are associated with development of membranoproliferative glomerulonephritis (table . [ , , , ] ). in alternative pathway id, the infections recognize pyogens as the most common etiological agents, while final common pathway id (c -c ) or properdin (p) have been associated with recurrent or invasive infections by neisseria (n) gonorrhoeae or n. meningitidis, gramnegative bacteria, and asplenia, agammaglobulinemia [ , , , ] . it is estimated that the frequency of meningitis in subjects with hz deficiency of the final c -c pathway is %, , -fold higher than in non-id individuals [ , ] . some characteristics appear to associate the patients with complement deficiency and meningococcal disease: frequent recurrent episodes, an older age at the first onset, lower mortality compared to patients with a normal complement, and a prevalence of males [ ] . over patients with c q, c r and c s deficiencies have been described, c s deficiency only in two cases [ ] . a selective and complete c s deficiency in a -year-old girl with complex aids including sle-like syndrome, hashimoto's thyroiditis, and autoimmune hepatitis has been reported. exon-specific amplification of genomic dna by pcr followed by direct sequence analysis revealed a mz nonsense mutation in the c s gene exon xii at codon . both parents were het for this mutation [ ] . a deficiency in one of these proteins is sufficient to block the classic pathway activation; deficiency results as a consequence of non-synthesis, which in the unlike c , hz c deficiency is very rare and is caused by the non-expression of all alleles ( of c a and of c b, maternal and paternal alleles), which can occur due to punctiform mutations, gene deletions, or other gene alterations that prevent gene transcription [ ] . the two a and b genes are polymorphous, just like c , c , c , c a and c b and the b factor (bf); polymorphic variants of the other proteins are rare, with different alleles for c a and for c b identified at the moment. moreover, two loci c a and c b null alleles q (quantity ), do not codify for any phenotype, although often present in the general population [ ] . in a -year-old caucasian child who suffered from several bouts of pneumonia caused by respiratory viruses, eight episodes of acute otitis media, prolonged respiratory and urinary tract infections, molecular studies of the c gene region revealed hz deletion of hla class iii cyp a-tnxa-rp -c b, generating total deficiency of case of c q amounts to % of cases, while in the remaining % the molecules are malfunctioning but cross-reacting with the native molecule [ ] . c r and c s deficiencies are usually combined, due to the contiguity of the two genes; typically in these patients c r is absent and c s levels are reduced ( %- %) [ ] . affected patients suffered from a sle-like syndrome and sporadically from an extended predisposition to infections [ ] . associated symptoms in c q deficiency are sle-like syndrome, rheumatic disease, and infection. several children suffered from meningitis, recurrent septicemia, recurrent otitis media, pneumonia, and stomatitis; two died from meningitis septicemia [ ] . the incidence is based on the data from [ ] ; the inheritance is always ar, with the exception of c -inh deficiency (autosomal dominant) and factor p deficiency (autosomal recessive or x-linked). data from [ , , , ] . gn glomerulonephritis, jra juvenile rheumatoid arthritis. c b and the flanking ¢ region up to c a [ ] . moreover, in / cases the c a*q alleles were related to a c a/cyp p gene deletion within the hla-b c c bfs c aq b dr haplotype. in / cases, the c b*q allele was related to a c b/cyp p gene deletion within the hla-b c c bff c a bq dr haplotype [ ] . the c null allele incidence is so elevated that % of the population expresses all c genes, while % lacks - alleles [ ] . the elevated number of q probably derives from the marked similitude of the two genes, which facilitates the unequal crossover, but this crossover in the hla can modulate the expression of three c a alleles and one c b or vice versa; the c aq allele spreading amplifies the risk of contracting sle and juvenile ra (jra). hz c deficiency, the most common in the caucasian population, has an incidence that varies between : , and : , , whereas the het carrier rate is . % [ ] . it is usually found in the a , b , dr , bfs, c q , c a, c b haplotype context which, due to its considerable rarity, could assume a predictive value [ ] . two different types of c deficiency are known: in type i the synthesis is deficient due to the protein nontranslation, in type ii there is a selective absence of secretion but not of synthesis, therefore c levels are . %- % of normal values [ ] . hets have a nonfunctioning gene, the complement profile is characterized by serum c concentrations equal to % of normal values; about % are asymptomatic, while the other half exhibit frequent infections and quite a few suffer from sle and correlated syndromes [ ] . het c deficiency was associated with a -bp deletion in the c gene (type i), mainly within the hla-a b c q bfs c a b dr haplotype [ ] . in a certain number of cases, a c deficiency is accompanied by a partial bf malfunctioning, genetically close to it. hzs can also have a deficient function of the alternative pathway [ ] . possibly this deficiency, like other quite common ones, may not always be reported in the literature: the total number of cases therefore underestimates the real prevalence, as is also found in children with c deficiency [ ] . c deficiency must be suspected in all patients presenting pneumococcal infections after the age of years [ ] . both c and c predispose to sle, but this is not the expression of a particular genetic association caused by the same gene localization, because c q, r, s, deficiencies, which also cause sle, are situated as mentioned outside the hla system [ ] . the molecular bases of this pid appear heterogeneous. the c gene exists in different allelic forms, some of which have reduced functionality. one must remember the c important role in immune responses, also as far as apc and b cells are concerned, as well as the defensive role played in innate immunity along with c . since both pathways converge in the cleavage and activation of c , there is no way that this defect can be corrected, and furthermore the opsonic power is greatly deficient, as is the c chemotaxis; therefore patients affected by hz deficiency mostly present clinical symptoms totally similar to a congenital hgg with severe recurrent infections and at times the symptoms of cic disease [ ] . although id is severe, some patients apparently remain in good health and the syndrome may also in time become less severe, probably due to the higher number of immune experiences that allow a better effector function to antibody reactions mediated by the fc receptor [ ] . c protein was defective in noninfected nigerian children with protein-energy malnutrition (pem), but rose significantly in the presence of bacterial infection, thus sharing the values found in healthy controls [ ] . in its clinical expressions, c deficiency does not differ from the other deficiencies discussed here; the clinical consequences of absent c a anaphylotoxin are unclear [ ] . one-fourth of all patients are asymptomatic. in caucasians incidence is : , [ ] . deficiencies associated with c -c are rare but reflect the close genetic proximity of their pertinent genes; in c -c deficiencies, % of patients lacking one component experience at least one severe episode of neisseria infection and . % one aid [ ] . rare in europe, c deficiency is the second most common complement deficiency in the japanese ( . %) [ ] . in italian children it has a prevalence of % and has been identified also in healthy siblings [ ] . in japan c deficiency is more associated with meningococcal meningitis than with neisseria infections [ ] . in a highly inbred arab population, a c deficiency was associated with a mutation (g c) that is also prevalent among israeli jews of moroccan ancestry [ ] . a total deficiency of this -kd protein, inherited as an ar trait, has been described in a young patient with a hemolytic-uremic syndrome and in one case in italy whose parents were first cousins [ ] . among relatives of the proband studied, encompassing generations, ten had low factor h levels, including her two children, indicating a het factor h deficiency [ ] . h deficiency results in uncontrolled breakdown of c , and in depletion of bf, p and c [ ] . c and c components are decreased in varying degrees, while c and c are found in plasma in traces and only as activated molecules [ ] . inheritance of factor d deficiency is for the moment uncertain [ ] (table . ): a partial deficiency ( %- % of normal concentrations) has been described in two mz twins, and a total deficiency in one male. this deficiency, serologically characterized by the non-functionality of the alternative pathway, is clinically accompanied by an increased susceptibility to neisseria infections [ ] , leading us once more to emphasize the alternative pathway significance as a substantial means of defense for a broad spectrum of damaging actions caused by bacterial infections. properdin deficiency is the only one inherited as a characteristic linked to the chromosome x and only affects males [ ] . at the moment, > cases have been described. the deficiency can be materialized by a total p absence, with levels reduced to %, with normal levels, however, showing an altered functional activity [ ] . specific research has shown a remarkable reduction of c a and b titers, which represent the c -convertase proteins, with a consequent heightened consumption due to the alternative pathway spontaneous activation. males are affected by septic episodes caused by neisseria, sometime fulminating, with onset even occurring during the st year of life [ ] . there is no evidence of increased susceptibility to cic diseases or infections caused by other organisms [ ] , thus implying that a functioning alternative pathway is particularly important for a defense against infections [ ] . correctly identifying children with complement abnormalities is important and worthwhile if any of the following factors are present: id (such as repeated or unusual infections with other organisms, fh, unusual course of the illness, etc.), repeated neisserial infections, infection with an unusual serogroup, fulminant disease in males (p deficiency), coexisting angioedema, autoimmune, or connective tissue disorders [ ] . in the two forms of c deficiency (c a + c g and c b mapped on chromosome ), the subunit not involved is present in the serum, also with reduced levels, and accompanied by altered functionality of the one involved. c deficiency has different characteristics due to the diverse associations of the b and a-g chains; therefore caucasians with this deficiency lack the b chain ( %), while colored patients lack the a and g chains ( %) [ ] . c deficiencies are poorly considered because they are often asymptomatic [ ] ; however, in japan there is an incidence of . % [ ] and between % [ ] and % of cases [ ] present meningococcal meningitis. congenital c deficiencies are very common among the japanese ( . %- . %) and represent . % of all deficiencies (table . ). there have been > cases of congenital c -c deficiencies reported, distributed unevenly between the various ethnic groups: c and c deficiencies are prevalent in colored patients and the c deficiencies appear to be more common in caucasians [ ] . an analysis of published studies shows that % of patients with sporadic meningococcal infections may have a c -c deficiency [ ] . clinical patterns are overlapping. the most common complement deficiency is c -inh deficiency (c inhibitor), responsible for hereditary angioedema that causes symptoms in hets (chap. ). at least cases of factor i deficiency are known [ ] , with autosomal co-dominant transmission because parents show normal complement levels at % of factor i [ ] . as in an h deficiency, serologically one has the alternative pathway activation, due to c b non-catabolization that continuously forms c conversion with severe c deficiency, the levels of which do not exceed % of normal values [ ] . in hzs, in addition to subsequent pyogenic infections, as in c deficiency [ ] , cutaneous rashes and urticaria caused by massive release of histamine and pro-inflammatory cellular products by anaphylotoxic fragments are reported [ ] . the fact that a child during the initial period of life should experience a certain number of urtis or lrtis is within the norm: rris are mainly caused by immunological immaturity or inexperience, both transient. a typical symptom outline is difficult to define, and prevalence is also little known. in id children, a basic pathology is present instead, which encourages the recurrence of infections. although a distinction between infection and associated disease is important, childhood infections might have a key role in stimulating the maturation of the immune system, and the microbial burden in early life has been invoked as a protective factor against wheezing and asthma (chap. ). preschool-age children have an especially high frequency of vris, with most having three to eight infections per year and %- % have ≥ vris per year. the rate in children aged - has been about in children compared with about in for children aged - , and about in , for those aged - [ ] . rris may be defined as >six episodes of urti and/or > lrtis in the previous year, or based on age and ≥ episodes per year if aged < or ≥ episodes per year if aged ≥ . several risk factors can influence the onset and recurrence of infections [ ] . it is clear that the younger the child is, the more he/she may fall ill: this is also related to serum ig levels (table . ). the dogma of primary and secondary responses also may not apply to infections, at least those caused by rotavirus, which confers considerable protection only after various infectious events and in children aged [ ] . environmental factors in the absence of a basic pathology are important. it is also obvious that the more crowded the environment the child lives in, the more probable that infection becomes. in addition to the number of siblings, other factors such as socioeconomic status, age (preschool children), contact with outside persons, especially babysitter, early social contacts, exposure to passive smoke, indoor and outdoor pollution may be found to be related to the hygiene hypothesis (chap. ). however, daycare attendance, which was considered to be an indicator of exposure to respiratory pathogens, and the presence of siblings, increased the risk of urti in preschool children aged - [ ] , and in the st year of life for children with fha [ ] . among children with fha, the protective effect of day care attendance in early life against the development of atopy only begins by years, and against wheezing this may not be observed until after years [ ] . the particular ease of smoking parents and/or relatives who fall ill with influenza has been known for some time, to the same extent that the children who live with them are affected by rris (table . ). the impact on rri incidence as it is related to children in kindergartens, has been reflected by a significantly increased morbidity observed in babies aged months to years who go to daycare, who show a number of more severe and longer lasting infections per year [ ] , with an incidence of . % in children who stay at home compared to . % of those in kindergartens [ ] and an increase of % of ome persistence (chap. ). in children exposed to cigarette smoke, the risk increased -fold for lrti [ ] or by . -fold, equal to ≤ episodes of respiratory infections each year [ ] . studies on environmental pollution have identified the most damaging agents: conclusive data on fine particles in suspension and polluting derivatives is available, proving a significantly increased risk of infantile rris: table . indicates that no reduces immune defenses against rtis, provoking alterations of the epithelium and of the lymph node cells, with negative effects on mucociliary clearance and macrophages. the biological role played by no in the domestic pollution derived from the home has been ascertained to be related to cooking and the smoke released by combustion [ ] . using wood for heating leads to so development, while radiators cause the air to dry, which in turn causes potentially infected particles to remain in suspension. pollutants are increasingly responsible for indoor pollution (chap. ). although levels of micro-pollution are not easily ascertained, significant associations with acute rris and conditions such as polypnea and dyspnea have been reported, especially in < year-old children [ ] . these children's capacity to evoke adequate responses is genetically controlled; however, it is commonly known that their parents or siblings have suffered similar illness as children. in subjects with physiological immaturity of the immune system, vris more easily cause infectious episodes, which are important factors to be considered only in the presence of recurrent or incompletely cleared conditions [ ] . predisposing factors related to a basic pathology derive from perinatal factors, more common in premature babies, which can lead to respiratory tract alterations and consequently to bronchopulmonary dysplasia; anatomical anomalies; cystic fibrosis, which can become recurrent pneumonia; adenoiditis causing otitis and ome; congenital ciliary dyskinesia; humoral deficiency and pid characterized by recurrent sinopulmonary infections [ ] (table . ) [ ] . viruses are the principal etiological agents, and over a novel member of the coronavirus family has been characterized, which is associated with cases of sars (severe acute respiratory syndrome). phylogenetic analyses and sequence comparisons showed that sarscoronavirus is not closely related to any of the previously characterized coronaviruses [ ] . as investigated by a -year study the overall prevalence is age-related, and different between children aged - ( fig. . ) [ ] and those aged - ( fig. . ) [ ] : rsv and rhinovirus have a different impact on the first group ( % vs %) and the influenza viruses on the second group ( %- %). incidence in young children was . -fold higher than in those aged - [ ] . rsv causes bronchiolitis in breast-fed babies, with a higher frequency the younger the child is (tables . , . ) , and rhinitis in older siblings. even an infectious agent neglected for some time, such as ureaplasma urealyticum, causes a lung pathology in younger children while sparing those > years old [ ] . the bacteria most commonly involved are: streptococcus pyogenes (pharynx and larynx); haemophilus in- complement deficiency fluenzae (middle ear and larynx reaching the epiglottis); and streptococcus pneumoniae (middle ear). there are often secondary bacterial infections such as complications caused by vris, which certainly contribute to recurrent infections and/or the onset of chronicity [ ] . in chap. we reported several studies which concluded that early infections may protect from atopy development (hygiene hypothesis). we must distinguish pid outlines and pseudo-id in children with rris. summarizing the aforementioned, severe and recurrent lrti and sinusitis are the principal clinical manifestations in children affected by deficiencies prevalently involving humoral immunity (table . ). these children fall ill during the first weeks of their lives and often contract infections caused by opportunistic agents, fungi, protozoa and viruses, and as months go by are also affected by malnutrition and failure to thrive. episodes affecting the airways, particularly common in cellular and combined id, tend to become longer or severe, especially if complicated by pneumonia [ ] . chronic disorders such as sinusitis and bronchiectasis (sinobronchial syndrome) are not rare; interstitial pneumonia is in most cases caused by pneumocystis carinii and results in tachypnea and lung hyperinflation [ ] . infections supported by the herpes simplex, ebv and cmv are also common: males with xlp exhibit a deficient response to ebv [ ] . severe and recurrent sinopulmonary, was, higes and ata infections as well as severe rris in cgd, complement deficiencies and lad to v must also be borne in mind. cases of recurrent pneumonia should be warning signals to rule in . % pediatric cases of pids [ ] . the second most important manifestation is chronic diarrhea: in some cases the infections are caused by rotavirus and enterovirus among which the echo: giardia lamblia, salmonella and campylobacter can also cause chronic enteric infection; malabsorption resistant to treatment can be ascribed to cryptosporidium [ ] . rris are common in children. they reflect the immaturity of the immune system in its encounter with environmental antigens; this developmental delay during the first years of life fosters the development of rris. thus, rris are part of the growing-up process of any child [ ] . the consequences of rris can be of a profound and sometimes protracted alteration of the different immune defense mechanisms, which place the child in an undefended position, similar to the condition observed in children with pids, compromising the phagocytes, lymphocytes, nk cells, antibody production, and ils at every occasion [ ] . the responsible viruses for these infections have a development limited to surface mucous cells, spreading from cell to cell due to contiguity, while the viremic stage is absent or remains marginal. the incubation period is therefore brief, normally < days; consequently the immune response may not be capable of ensuring a protective function, or it only intervenes partially, clarifying the potentially unlimited number of infectious episodes [ ] . from a pathogenetic point of view, the virus works by triggering the development of ige and allergic sensitization and/or damaging the immune structures [ , ] . in the first case, it is known that experimental infection in mice with rsv is capable of significantly increasing the absorption of ovalbumin (ova) administered by aerosol, of igg, ige and anti-ova siga (fig. . ) and of increasing the synthesis of ige and specific igg to ragweed also administered by aerosol. the result is that the majority of infants become infected with rsv, although lrtis develop in only about % [ ] . approximately %- % of those subsequently experience recurrent acute asthma from vri [ ] . the mechanism by which vris induce atopic sensitization in experimental models is identified with antigen penetration and sige synthesis [ ] . studies show that viruses increase mucosal permeability, by modulating antigen uptake and altering antigen processing by the mucosa, which results in the ige-suppressor t-cell depression, while ifn-g-modulated histamine release further increases mucosal permeability [ ] . it is probable that the immune deficiency is secondary to vris, because many viruses are capable of inducing transient [ ] . the lack of nk cells, which are in the front line of defense against viral infections, and with which the altered production of il and factors activating the phagocytes are associated, appears significant, but it is unclear whether the deficiency is primary or secondary to viral infections [ ] . the basic question with potential therapeutic consequences therefore remains unanswered, and is probably destined to remain so until more sophisticated tests are available to clarify this issue, although the nk-cell reduction in these children corroborates the first hypothesis. recent data emphasizes the important defensive activity of cytotoxic cd t cells and by different ils, including il , il and il active in antiviral defense: if the infected cells express on the surface the viral antigen processed in association with hla class i molecules, cytotoxic cd take care of killing the cell. normally the cd are activated by the virus itself or by soluble factors released by the infected cells and mediate the cellular lyses after recognizing hla class i antigens on the same cell. however, if the apc is a macrophage, it can be parasitized by the virus, with consequently reduced chemotaxis and microbicidal activity and in particular the capacity to cooperate with t cells [ ] . considering that macrophages may have evolved specific mechanisms for directing t-cell development toward the th , since cmi can solve some infections, it is clear that their dysfunctions appear in the insufficient nk cell activation and inadequate th development in response to infections. ifn-g, produced not as a direct consequence of infection, but probably by il and/or il , stimulates the nearby cells to block the nucleic acid transcription and therefore the viral replication, preventing the infection of those cells to which virus spreads due to contiguity. therefore the ifn-g species-specific antiviral activity takes place at the very first stages of the infection, preceding the antibodies [ ] . the response to germs' capsular polysaccharides, particularly deficient until the age of , although encounters with these germs are abundant during this period of time, still remains to be evaluated [ ] . however, selected children, although normal from an immunological point of view, may have a deficient antibody response even aged - [ ] with a percentage of modifications of both humoral and cmi, therefore not only of antibody synthesis or phagocyte and neutrophil functions, but also of t lymphocytes and related ils [ ] . it is also possible to hypothesize that persistent vris are capable of inducing th activation by antigens or super-antigens: th t cells with il help induce virus-specific cd + to produce il , which recruits eosinophils in the respiratory tract, thus reducing ifn-g secretion. thus there may be an increased interaction of ige mast cells in these subjects with immunoregulatory alterations, due to a marked lymphoproliferative response and the elaboration of other ils, which consequently amplifies ige production in the respiratory tract. the ige response is thought to lead to a greater production of bronchoconstrictor mediators by effector cells; viral infections themselves may induce these cells to release histamine. it is also known that humoral deficiency, especially of iga, very often opens the way to gram-positive germs causing viral and bacterial infections, thereby completing the circle [ ] . interestingly, only / children have at least a significant production of antigen-specific salivary iga against klebsiella pneumoniae [ ] . even if the alterations are transient and aspecific in children with rris, their occurrence and persistence for a number of months, for a year and even longer, leads to believe that the immunosuppressive mechanisms set off by the first episode occasion more severe, profound and lasting consequences for the immune functions than those occurring in their normal peers [ , ] . having at least one physician-diagnosed lrti in the st year of life was significantly associated with recurrent wheezing (or, . ) and asthma (or, . ) [ ] . at the base of this exclusive predisposition in children for contracting rris, there is a nk cell reduction [ ] and immune deficiencies related to the global lymphocyte population, the cd and the cd :cd ratio, unbalanced toward cd t cells, prevalent in children expressing coughing compared to those with bronchial hyperreactivity (bhr) [ ] . other t-cell deficiencies in children with humoral anomalies include a considerable spontaneous production of il and il , both generated by th phenotypes, or alternatively by the th , which express both ils [ ] . it is similarly feasible that virus-specific cd deprived of cytolytic activity are converted into th -like t cells when il is present [ ] . cmi in these children consists above all in the transient t-cell numeric and functional depression, coinciding with a deficiency of ils necessary for their activation, proliferation and differentiation. the virus toxic effect also acts directly on the t cells, inciting rough structural modifications including giant polynucleate cells, which jeopardize homing and recirculation capacities, to the point of immunosuppression, affecting the specific lymphocytes for that particular virus, thus favoring the attacking organism [ ] . a condition of immunosuppression occurs also as a result of superantigen (sa) orchestration, which, stimulating a large num-nonreactive children of %- % [ , ] , rising to %- % if with an iga and/or igg subclass deficiency [ , , ] . while % of children have low antipneumococcus igg , antibody responses are totally absent in % of dysimmunoglobulinemics with a virtual absence of iga and igg [ ] , confirming previous data [ ] . iga and igg -deficient children show a clinical pattern with elevated susceptibility to s. pneumoniae infections [ ] . von waldeyer ring (nalt) is a significant constituent of immune response, and of resistance to nalt-dependent infections. among the consequences of chronic adenotonsillitis (table . ) the statistically significant decrease in ig levels, including siga, must be evaluated eventually in relation to rri complications (chap. ). igg subclass deficiencies can be present in children with chronic and/or severe asthma, associated or not with sigad, in children affected or not by asthma with severe rris or with chronic nonallergic respiratory clinical symptoms, in children with sigad, ata, was, cvid, scid and in healthy subjects [ ] . the anti-virus antibodies generally belong to the igg and igg isotypes while the igg protect from microbes with polysaccharide antigens, such as s. pneumoniae and group a and type b h. influenzae [ ] . table . shows normal values for igg subclasses in subjects aged - . unlike total igg concentrations, igg and igg reach normal levels within the st year, igg mature more slowly, ensuring an effective antibody response only after the nd year, and igg develop even more slowly. various authors believe that the role played by igg subclasses is unique and vital in defending from infections: igg deficiency is associated with an increased susceptibility to infections by bacteria expressing capsular polysaccharides, such as pneumococci, meningococci, h. influenzae, bordetella pertussis, etc., as well as other factors capable of setting off an inflammation [ , ] . igg deficiency instead seems associated with a marked predisposition for rris [ ] . however, undetectable igg subclass levels are a common finding in normal individuals and an accurate detection of very low levels of igg is technically difficult to achieve [ , ] . babies aged ≥ month have levels of circulating lymphocytes secreting all four subclasses in a higher number than adults; therefore the capacity to produce antibodies exists well before a full humoral response is developed. subnormal igg subclass concentrations, especially of igg , are also observed in healthy children who do not present an increased susceptibility to infections. low subclass levels do not necessarily indicate that the subject will experience immune disorders exclusively linked to these, nor do normal concentrations guarantee that the child will be spared complications. sub-jects have been observed both with normal igg levels and with recurrent infections and with a subclass deficiency, which often do not form other types of antibodies [ ] . igg subclass determination does not indicate what the humoral level restricted to that molecule is: this is a characteristic in children, unlike adults, even though research was carried out using the same methods in the two studies [ ] . interesting hints come from studies involving children with rris: of children aged - , with a confirmed diagnosis of susceptibility to infections, / ( . %) had a igg subclass deficiency, almost all concerning igg , with several associations [ ] . in other children, the selective igg deficiency was statistically significant compared to atopic controls without rri, associated not so much with a well-defined state of id as to a respiratory tract defense mechanism deficiency, in view of the fact that the relative prevalence of this subclass in secretions may indicate a role in the mucosal defense [ ] . other children with typical symptoms of recurrent infections, lymphadenopathies, failure to thrive and hgg exhibited low igg levels, confirming that normal levels of total igg do not exclude a subclass deficiency [ ] . in a cohort of young babies, igg deficiency was only present in / subjects ( %) [ ] ; however, the absence of a control group makes the results incomparable. an igg subclass deficiency is therefore able to induce or worsen chronic respiratory symptoms in allergic and nonallergic children, especially if predisposed to developing these affections [ ] , or in subjects with sigad [ ] . with time these deficiencies, and eventually also those associated with iga, can normalize [ ] . in conclusion, transient and persistent iga and/or igg deficiencies have been reported in a small percentage of asymptomatic children [ ] , but even if iga and igg subclasses are not always required as such for a normal immune response, their deficiency may predispose to rris [ ] . as previously illustrated in chap. , the close links between atopy and rris are known, and this is confirmed by the observation that asthmatic children have a higher incidence of rris than their nonasthmatic siblings. it has been known that rris during the early periods of life can play a role in the development of bhr and atopy: in the classic study by frick et al [ ] , in out of allergic children sensitization was propitiated by rris. with continued observation, the authors noted the presence of high ige levels, positive rast and histamine released by leukocytes after infections [ ] . in a cohort of asthmatic children aged . - . , the affected by th week of pregnancy is possible, so as to evaluate the immune phenotype in the fetal blood [ ] . wccs (white-cell counts) in cb and differential counts can be used to detect the lymphopenia that is commonly present in infants with scid. however, subset analysis by flow cytometry is necessary to enumerate t, b and nk cells. subsequently, scid diagnosis will be suspected when overwhelming opportunistic infections occur [ ] . depending on whether one suspects a humoral, cellular or innate immune deficiency, we begin with the algorithm in table . [ ] , positive if infants or children have ≥ of these signs, then , ] on laboratory tests can be consulted. however, children with variable levels of antibody id may end up with different diagnosis [ ] . children with higms presented initially with a history of an increased susceptibility to infection including pneumocystis carinii pneumonia [ ] in % of children [ ] . in pids affecting phagocytes, because of the relatively narrow spectrum of disease-specific infections (such as aspergillosis in cgd), careful attention to the microbiology laboratory early in the course of evaluation of a patient suspected of having a pid is crucial to orient the work-up in the appropriate direction [ ] . in a male newborn referred to hospital at days of age for fever, hemodynamic failure and an inflammation syndrome caused by pulmonary infection, culture of tracheal, bronchoalveolar lavage samples and lung biopsy grew positive for a. fumigatus, enabling the diagnosis of cgd [ ] . to investigate whether patients with undiagnosed id could be identified in diverse inpatient hospital populations, a scoring algorithm and computer screening method was updated [ ] on the basis of icd- codes to survey the discharge diagnoses of all hospitalized patients over periods of time. thus id patients were identified, eight of whom were children aged - ( %), two with neutropenia, two with igg deficiency, one with lad, one with dgs, etc. [ ] . we also suggest including congenital phagocytic defects in the differential diagnosis of recurrent bacterial or fungal infections in a child [ ] . a congenital complement deficiency should be suspected if levels of even one component are reduced [ ] . early diagnosis is essential for choosing the necessary treatment [ ] . the differential diagnosis of pid will emphasize the different characteristics schematized in table . , to which one must add objective rarity, while fh and child gender become important [ ] . in subjects suffering from omenn syndrome, was, severe combined and cellular ids, the screening of clinical symptoms may be useful at birth and during the first few months after birth (table . ) [ , , ] . the localization of infections is multiple, the id child usually appears to be ill, and the peripheral lymph nodes and lymphatic rris had a higher incidence of fh positivity (p= . ), increased ige (p= . ), as well as a combined iga (p= . ) and igg (p= . ) deficiency [ ] . ige hyperproduction could be the result, not only of the wellknown association between igg subclasses and ige and their coregulation of il expression [ ] , but also of a virus-caused unbalanced cd :cd ratio [ ] . these results link atopy to rris, confirming that the state of chronic inflammation and bhr induced by allergic sensitization is an ideal substratum for the adhesion and chronic evolution of bacterial and/or viral infections. there are no specific clinical outlines for rris. on the contrary, symptoms are extremely varied, with, as previously mentioned, infections caused by bacteria and viruses. urtis are common at age . during the last months, . % of the children experienced more than one bout of acute otitis media, . % had more than one pharyngotonsillitis episode, . % contracted > common colds, and . % had rhinitis weekly or monthly [ ] . there are children who, during the period of maximum exposure due to biological immaturity and immunological inexperience, suffer from one episode each month affecting different organ systems, as well as lymphadenopathies and failure to thrive. the capacity for inducing bhr in normal subjects and worsening the symptoms in those already ill are precisely caused by vri, also facilitating greater penetration of inhaled viral allergens [ ] (table . ); rris in turn predispose to sinusitis. lower ifn-g levels produced by of children at months of age were even greater if the comparison was made between children with rris and those with no or maximally one rri during the follow-up period [ ] . rhinovirus-induced infections (table . ) take the appearance of common rhinitis, but stimulate mastocytes to release histamine, contributing to bhr development and the perspective of delayed reactions. a differentiating feature is the respiratory infections in the id child that may also result from opportunistic pathogens [ ] (table . ). respiratory infections should be under control. a screening of humoral immunity revealed low ig levels in . %, low iga levels in . %, and sigad in . % of children [ ] . during the last few years, increasingly sophisticated diagnostic techniques have permitted prenatal diagnosis in many cases (table . ) [ , , , , , ] : in forms supported by rag- and/or rag- mutations a diagnosis even at the th- th or at the pharyngeal tissue are almost imperceptible [ , ] . the seriously undernourished appearance should be noted, more often observed in children with scid [ ] . rris can be observed in other cmi forms [ ] : deficiencies of cd g and e chains [ , , ] , zap- [ , ] and hla class ii [ , ] . finally, children with aids will seem to be in severe general condition and this disease is a paradigmatic example of how hiv can overturn the t lymphocyte immune defense with regards to opportunistic infections [ ] . in some cases of pediatric aids there is hgg that is indistinguishable from pid and that belongs to the differential diagnosis of severe recurrent infections during the first few months after birth [ ] . as far as tih is concerned, the confirmation of a normal presence of the b lymphocytes and low levels of intrinsically produced ig is resolutive, compared to agammaglobulinemia [ ] . an articulate case history often identifies the familiarity of rris, usually with an absent basic pathology and the frequent predisposing environmental factors (table . ), among which passive cigarette smoking stands out. maximum prevalence occurs during the first years of life or during first contacts with school, the disease is limited in time, and there is usually a single location. in most cases the pseudo-immunodepressed child is clinically normal in all other respects [ ] . in ten reported sars-infected children from hong kong, fever, cough, and runny nose were complement deficiency lateral pharyngeal x-ray to visualize adenoidal tissue cytokine production (il , il , il , il , ifn-g, il ) chromosome fragility (ataxia-telangiectasia, bloom's syndrome, etc.) data from [ , , ] . ada adenosine-deaminase, adcc antibody dependent cellmediated cytotoxicity, ctl cytotoxic t lymphocytes, ltt lymphocyte transformation test, pha phytohemagglutinin, pma phorbol myristate acetate, pnp purine nucleoside phosphorylase, ppd purified protein derivative. sars seems to have a less aggressive clinical course in younger children [ ] . when in doubt, a broad spectrum of laboratory tests are available: cbc, proteinemia and protidogram, serum ig levels, or in secretions and igg subclasses (table . ), immunoelectrophoresis (homogeneous components, k/l), dosage of isohemagglutinin, five other natural antibodies, the sweat test [ ] , in strictly selected cases also a lymphocyte population and subpopulation count (tables . - . ), and x-ray of paranasal sinuses. analysis of the lymphocyte profile sometimes shows a number of deficiencies, statistically differentiated from those found in other children affected by an asthmatic pathology; however, none of the immunological deficiencies indicated (table . ) are characteristic in pseudo-immunodepressed children. common diagnostic methods may not be capable of revealing a deficiency of igg subclasses or of selective igg : the chance that there may be abnormal igg or igg levels is not excluded by the normality of igg serum concentrations [ ] . furthermore, the distribu-tion of igg into four subclasses makes it difficult to identify these deficiencies simply by measuring total serum igg levels [ ] ; only for the past few years have there been highly specific reagents for measuring individual subclass levels and methods such as radial immunodiffusion (rid) [ , ] . the aaaai has recommended not relying on subclass levels [ ] , especially igg levels, which seem to be unmeasurable in % of the population [ ] . rid, which has proven to be more sensitive than the elisa used by the cdc in atlanta, has shown that % of normal children have values below normal for at least one subclass [ ] ; a similar deficiency was present in % of children with rris [ ] . a recent study measuring the igg with both methods, has proved that the rid can show higher values of igg and igg in low serum levels of both ig [ ] , data with an unquestionable negative effect in pediatrics. in conclusion, at the moment our knowledge suggests that we should also carefully interpret low levels of one or more subclasses, because on the one hand this might indicate a transient or paraphysiological condition, on [ ] . igg levels in all patients also rose considerably compared to previous treatment: the average levels in different determinations was > mg [ ] . finally, all children grew normally; the height achieved by each child is between the rd and th percentile, within the limits of theoretical values calculated on the height of their parents. substituting therapy with ivig has allowed patients to return to their normal activities, with a considerable improvement in quality of life. in all these years, we have never come across substantial unwelcome reactions or infectious complications [ ] , as also found by other authors [ , ] . ivig could also be effective for reducing the allergic symptoms discussed thus far: presuppositions are not lacking, such as the blocking of allergens and mastocyte fcr thanks to the modest quantities of igg present in the preparations [ ] . in addition, the increased understanding of the igg transplacental passage (chap. ) can absolve the function of timing their transfusion in the case of mothers with antibody id, so that the fetal defenses can be complete and quantitatively adequate. in sigad common ivig preparations cannot be used, even if with a low content of iga, nor enriched, both because of the extremely short iga life-span, which would therefore suggest iga administration every - days, and because the infused iga do not reach the secretions [ ] . should ivig be indicated for a deficiency associated with igg , or should transfusions of blood derivatives become necessary, one must first investigate serum antibody anti-iga levels (igg and ige) and, should these be positive, avoid infusions or administer them in a hospital under strict medical supervision, or use washed red blood cells [ ] . the same precaution must be taken for subjects with ata for whom ivig, if is appropriately administered, also ensure beneficial effects on quality of life, while there are no known therapies for contrasting neurological symptoms. in patients with humoral deficiency, alongside ivig, if appropriate, an antibiotic prophylaxis is suitable with monthly cycles, alternating amoxicillin, cephalosporin, co-trimoxazole, etc., bearing in mind family compliance. in cvid recurrent infections caused by giardia lamblia should be treated using furazolidone ( mg/kg/day) or methronidazole ( mg/kg/day) for days, if necessary to be repeated. cvid treatment in specialized centers involves recombinant il , il and cimetidine [ ] . some t-cell pids represent a severe clinical emergency, such as omenn syndrome, in which hypovolemic shock and reticular dysgenesis are immanent in the battle for survival. although precise figures are unavailable, thousands of patients worldwide with different forms of the other a modest deficiency can result in clear hgg [ ] . subnormal igg levels can indeed be associated with various manifestations of immune dysfunctions; it is therefore advisable in this case to proceed with specific investigations, measuring the response to polysaccharide antigens and studying the lymphocyte activity in vitro [ ] . in children with normal serum levels, who are instead lacking in antibody responses to polysaccharide antigens [ , ] , this is a conclusive investigation [ ] (fig. . ) . a study of children aged > , half atopic and half not, has confirmed this thesis, concluding that the answers were similar in both groups, therefore excluding a greater rri predisposition in the atopic children [ ] . many patients with high ige levels do not present atopic manifestations: it is thought that an increased concentration is related to a reduced inhibiting activity of the thymus in ige synthesis [ ] . recurrent sinopulmonary infections must make one also consider cystic fibrosis and immotile cilia syndrome [ ] . children with malnutrition (chap. ) suffer from numerous ids, prevalently concerning cmi; their vulnerability makes them succumb to severe bacterial me infections and urtis, often also risking death. obese subjects may also be affected by rris due to a possible adipose tissue hypovascularization or to a defect in the granulocyte microbicidal activity [ ] . in the presence of antibody deficiency, antibiotic treatment is chosen as a preventive therapy in less severe cases, otherwise the preferable therapy consists in ivig (fig. . ). this treatment is restricted to a limited number of diseases, including some forms of id, secondary or cytopenic id, in which effectiveness has been proved in dbpc studies [ , , ] , like other positive forms of intervention described, while it appears to be of no use in uncomplicated thi [ , ] . two children aged . and with higes and kawasaki disease were administered mg/die of ivig for days and one . -year-old with higes received only one dose, with ige levels falling from , - , to - , ui/ml on the th day. hence there was almost a normalization of ige production with symptom relapse after months; similar results using a single dose were also obtained in two children with higes and severe ad [ ] . the following data represent a number of clinical and immunological parameters in children suffering from humoral pid and ata, with igg levels < mg/dl. treatment with ivig, also at a higher dosage, was very well tolerated by patients: all children presented a clear- verely affected by several factors [ ] . either hla-identical marrow or t-cell-depleted (tcd) haploidentical parental marrow is the standard of care for scid ( fig. . ) . when histocompatible related donor bmt is unavailable, a bmt either with hla-identical unfractionated or tcd haploidentical parental marrow is the standard of care for scid [ ] . all but one ( %) of scid infants who received tcd identical or haploiden-genetically determined id have been given bmt in attempts to correct their underlying id [ ] , including a recent series [ ] . specific treatment for cellular pid consists in a bmt from a hla-compatible donor [ ] . the ideal sc donor is normally a sibling who shares identical hla class i and class ii loci. without such a donor, these transplantations usually resulted in fatal gvhd. if death did not occur, event-free survival was se- treatment tical bmt in the first days of life are currently alive, with the period of survival ranging from months to > . years after transplantation. this compares favorably with a % survival rate of infants receiving transplants at a median age of days (range, - days) [ ] .a girl with t -b -scid received a full matched bmt from her sister at age weeks [ ] . a worldwide survey conducted by buckley from through , with subsequent additions of published cases from the literature, revealed that of ( %) patients with pid transplanted with hla-identical marrow during a period of years were alive [ ] . there are > patients worldwide who have survived scid as a result of successful transplantation of hla-identical or haploidentical bm [ ] . most importantly, of infants ( %) undergoing transplantation in the first . months of life are currently alive [ , ] , compared with a cut-off at months ( % vs %, children younger vs those aged > months) receiving bmt (or . [ ] ). we stress that neonates developed higher lymphocyte responses to phytohemagglutinin and higher numbers of cd + and cd ra + t cells in the first years of life than those receiving bmts late. t-cell antigens peaked earlier and with higher values in the neonatal bmts ( days to year) than in the late bmts ( - years) [ ] . over the past years % of all scid patients ( / ) receiving bmt at duke university medical center survive to varying ages up to ≥ years after bmt. only had an hla-identical donor. all others received rigorously tcd haploidentical bm from a parent, most often the mother. the soy lectin, srbc rosetting technique was used (r. buckley pers. comm. november th, and april th, ). an uncommon bmt to treat ar scid was undertaken in a -month-old girl. the donor was her hla-mismatched -year-old sister, who had previously received a bmt from her father [ ] : presently, they are aged and and are affected by molloscum contagiosum infection [ ] . bmt, both hla identical unfractionated and tcd hla aploidentical [ ] .a recent trial found that because only %- % of affected children have a familial hlaidentical donor (rid), the alternative therapeutic options are bmt from a mud or a haploidentical bmt or from hla-mismatched related donors (mmrds). only % of these children may find a matched donor; therefore, the remaining pid-affected children are candidates for a tcd haploidentical bmt [ ] . mud hsct is successful in young children [ ] , but the success rate decreases dramatically above the age of - years [ ] (table . ) [ , , , , , , , , , , , , , , , , , , , , , , , , , - , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ] . bmt survived. compared with mmrd bmt, survival was significantly higher with rid or with mud ( / = . %) [ ] . when an hla-identical sibling as the donor is unavailable, a phenotypic hla-matched unrelated bmt is needed, also used in cd deficiency [ , ] , with a clinical and immune outline normalization [ ] and a variable effect on igg subclasses treatment [ ] . c median ( . - years after bmt). p, at the time of publication. [ ] . possibly because of earlier diagnosis before untreatable opportunistic infections develop, the results have improved considerably during the last two decades [ ] . bmts have been successful when applied within the first - days of life in infants with scid, ( %) of those still alive range from months to years, not justifying in utero transplants. a completely normal t-cell function was obtained within - days [ ] and in an other patients was still present after - years [ ] . of the children who received a mismatched parental bmt from to , ( %) remain alive with t-cell immune reconstitution, a median of . (range, - . ) months after bmt [ ] . of children out of who received allogeneic bmt after the first days of life, ( %) are alive [ ] . three out of five children who received a hsct are alive and well after - months [ ] . breast feeding appeared correlated to an earlier reconstitution when the donor was the mother [ ] . intra-amniotic gene transfer has been successfully carried out on a laboratory animal, registering in a dose-dependent manner the fetal gastroenteric and respiratory effects [ ] . if confirmed in human beings, this method of treatment will certainly prove useful for prenatal correction of pid. bmt/hsct should be completed by conditioning regimens with busulfan and cyclophosphamide, less toxic than total lymphoid irradiation or a combination of nucleoside analogs and anti-lymphocyte antibody preparations [ ] . for example, busulfan ( mg/kg), melphalan ( mg/m ) and anti-thymocyte globulin ( mg/kg) [ ] . to enhance the engraftment rate in haploidentical bmt in pid, it was recently suggested to add donor peripheral scs after mobilization with g-csf ( mg/kg for days) and bm cells. with this procedure the cell load is increased, which allows intensification of the conditioning regimen for induction of faster engraftment [ ] . in utero bmt suggested advantages include the sterile environment in utero, and immaturity of the fetal immune system enabling the prevention of clinical manifestations of the disease in the neonate, and the engraftment without the use of cytotoxic conditioning regimens: a child thus treated was well at age months [ ] . two series of six and four patients [ , ] and two additional case reports [ , ] of in utero transplants have been published, yet failure of b-cell engraftment and function may result in long-term dependence on ivig replacement [ ] . better results than those published for in utero bmt for scid were implicit in infants admitted and diagnosed at a median age of days because of a fh of a previously affected infant. bmt was successful and all children are alive and well with follow-up to . years [ ] or < days vs approximately months [ ] . however, it is suggested that in utero transplants may carry the risks associated with injecting the fetus and the inability to detect gvhd during gestation [ ] . umbilical cb transplantation (ucbt) was done in two children affected by a zap- deficiency and an omenn-like syndrome. both are alive and well at . and . years after ucbt [ ] . unrelated ucbt in eight children with severe t-cell id [ ] and in three with was [ ] resulted in consistent and stable t -, b -, and nk cell development [ , ] . faster availability of ucbts is a meaningful advantage for patients requiring urgent transplantation: a median of days more rapidly than did those receiving bone marrow [ ] . in a large report [ ] , patients with scid, seven with was, and other unspecified pid received an unrelated ucbt. ucb was evaluated as a sc source for immune reconstitution in children with severe primary t-cell ids such as scid, reticular dysgenesis, thymic dysplasia, cid, dgs, and was when a matched sibling donor was unavailable, and has been used to date in more than , patients [ ] . three infants who rejected a tcd-mismatched parental bmt without prior cytoreduction engrafted after infusion of ucbt [ ] . a . -year-old girl with hla class ii deficiency had a successful related ucbt for graft failure following tcd nonidentical bmt [ ] . a girl with reticular dysgenesis failed to engraft following her first transplant, but fully engrafted after a second unrelated ucbt. five of six patients showed grade i gvhd, although one child experienced grade iv skin and gut gvhd. immunological ucbt resulted in consistent and stable t-cell, b-cell, and nk-cell development [ ] . long-term event-free survival (≥ months) with recovery of antigen-specific responses was reported following an unrelated ucbt in a child with omenn's syndrome [ ] . gene therapy, which has revolutionized and could revolutionize even more pid treatment in the near future, is analyzed in table . [ , ] . the requisite for applying this form of genetic engineering treatment is that the responsible gene must be cloned; the most common techniques involve knock-out mice and inactivating a particle [ ] , or employing retroviral vectors (table . ) [ ] , totally deprived of their genomic factors except for the normal copy of the gene to be inserted. this is indispensable for allowing the vector to reach the human nucleus, where it will integrate with the cellular genome [ ] . in this case, pbls are collected through leukapheresis and cultivated in vitro with retroviral particles containing a normal gene rna copy: thus the healthy gene is introduced into the cell genome using a vector and the manipulated cells are reinfused, so as to restore normal immune functions. this system has been used to treat two children aged months suffering from scid [ ] and from ada deficiency by employing autologous pbls [ ] , bm cells [ ] , and cb cells [ ] . three reports [ , , ] of successful gene therapy in infants with x-linked scid [ , ] and in t -b -scid [ ] are a major step forward among repeated efforts to achieve better immune reconstitution in ada-scid with gene therapy than with bmt/sct [ ] . immediately after the diagnosis had been made in two capacity of wild-type, replication-competent retroviruses to cause leukemia in immunologically immature neonatal mice [ ] , and in humans (two children out of ) [ , , ] . retroviruses can cause insertional oncogenesis, a long-known potential complication of retroviral gene transfer attempts, because gene integration occurs at random in the genome, thus deregulating the expression of cellular oncogenes [ ] . this complication has been thought to be unlikely with such vectors, because they are capable of inserting only once into the cell's chromosomes and cannot repeatedly reproduce and integrate. lentiviruses may be more effective than murine retroviruses for gene transfer into human hematopoietic scs and t lymphocytes [ ] . in ada-scid, the safety and efficacy of hsc gene therapy combined with nonmyeloablative conditioning for the treatment of scid has allowed two children to live at home and clinically well, with normal growth and development [ ] . on january , , fda placed on "clinical hold" all active gene therapy trials using retroviral vectors to insert genes into blood scs after having learned that a second child treated in the french gene therapy trial developed a leukemia-like condition. gene therapy is on hold despite enormous promise for certain scid/ cid variants. survival. in scid, the european experience with unfractionated hla-identical and tcd or non-tcd haploidentical or mud bmts in patients with scid reported that between and , a -year survival with sustained engraftment was significantly better after hla-identical than after mismatched transplantation ( % vs %).within the hla-identical group, survival after bmt from genotypically or phenotypically identical related or muds was %, %, and %, respectively [ ] . in non-scid, -year survival after genotypically hla-matched, phenotypically hla-matched, mmrd, and mud bmt was %, %, %, and %, respectively [ ] . in a retrospective analysis of bmts performed between and at european centers in children as young as month with different pids (excluding scid), the overall survival among recipients of hla genetically identical bmt was %, . % in patients who received closely matched bmt, and % in recipients of bmt with two or three mismatched hla antigens. a significant improvement in survival has been achieved in most pids (overall survival, . % vs . %, primarily because of a decrease in the frequency of infectious complications [ ] . in the similar analysis performed in children with scid at european centers between and , out of ( . %) patients were alive with evidence of engraftment months after bmt. however, patients died > months post-bmt, mainly due to cgvhd and/ or viral infection. thus gvhd months after bmt and b -scid vs b + scid were the main factors associated with a poor outcome [ ] . the disease-free survival was significantly better for patients with b + scid ( . %) than for those with b -scid ( . %) [ ] . in a children aged and months including a novel splice imitation in the common gc chain [ ] , haploidentical cd + peripheral progenitor cells mobilized with gm-csf were isolated to a purity of more than %. these cells were infused with no prior chemoablation and no prophylaxis against gvhd. both children showed signs of t-cell reconstitution beginning weeks after the cd + infusion and were weaned from continuous cures. they are in excellent health, without gvhd, and months after transplantation. one child does not need replacement ig. the other received a booster infusion of cd + scs from the original donor year later to improve b-cell function and now receives ig every months. both were followed for months after gene transfer [ ] . however, retroviral vectors have the treatment data from [ , , ] . ciita class ii transactivator, cgd chronic granulomatous disease, xla x linked agammaglobulinemia; for other abbreviations see table . . a done with success, see text for details. b in utero transplant of maternal stem cells. trial on children with pid receiving bm from hla-nonidentical related donors or from hla-identical unrelated donors at european centers between august and june , out of children ( . %) survived - months. bm was tcd by use of either erythrocyte rosetting or monoclonal antibodies to prevent gvhd [ ] . additional survival rates were reported previously (table . ). in a series of consecutive ud bmts / children with scid and non-scid pids who received a bmt with reduced-intensity conditioning (ric) regimen between and survived after a . -year follow-up, as well as / children who received a bmt with myeloablative conditioning (mat) between and and survived after an . -year follow-up. therefore a ric regimen results in improved survival and reduced bmt-related mortality compared with mat in hr children undergoing an ud bmt [ ] . in transplanted patients with was, the -year probability of survival differed according to donor type: % with hla-identical sibling donors, % with other related donors, and % with mud. significantly, boys who had received a mud transplant before years of age had survival rates similar to those receiving hlaidentical sibling transplants [ ] . however, the time required to develop immune function after haploidentical scts is quite different from that after unfractionated hla-identical bm. lymphocytes with mature t-cell phenotypes and functions fail to rise significantly until - months after bmt; normal t-cell function is reached between and months [ ] . b-cell function develops much more slowly, averaging - . years for normalization; many do not have b-cell function, despite normal t-cell function. [ ] . ex vivo rigorous depletion of post-thymic t cells from donor marrow that cause gvhd is efficient and feasible, even in haploidentical settings [ ] , presumably because of more effective infection-control measures and better transplantation strategy [ ] . for non-scid, sct can provide a cure, and grafts from unrelated donors are almost as beneficial as those from genetically hla-identical relatives [ ] . in most patients, deficient b-cell function persists after transplantation and requires lifelong ivig therapy [ , ] , which is necessary to prevent bacterial and common viral infections [ , ] . some patients also have persistent deficiencies of t-cell function after sct [ , ] . in children with rris, depending on the nature of the infection, the pediatrician will prescribe the most appropriate symptomatic and/or antibiotic therapy. in the presence of persistent inflammation, or during the winter, when the risk of close acute recurrent episodes is higher, anti-inflammatory preparations will be prescribed via aerosol, chromones, ketotifen, b -adrenergic and if necessary steroids for topical use, strictly depend-ing on the need. we suggest monitoring measures, such as keeping a clinical diary, in which each acute episode should be briefly noted, continuing registration until clinical symptoms have not regressed for at least days and returning to keep notes in the diary each time there is a cough and/or nasal and/or bronchial inflammation, completing this with pef as well as some respiratory parameters right at the beginning and then every months. it is obvious that if medical intervention is not resolutive a center specialized in infantile respiratory physiopathology should be contacted [ ] . children with sars were treated with high-dose ribavirin, oral prednisolone, or iv methylprednisolone, with no shortterm adverse effects [ ] . antibiotics must be used very carefully in these children because they can influence positively or negatively the innate, cellular or humoral immunity (chap. ), interaction with ils and growth factors are not known, repeated use often causes phenomena involving allergy/ intolerance [ ] , and most infection-prone children suffering from vris are given antibiotics unnecessarily. in italian children ( . % of males) aged months to years (median, years) with a history of rris, macrolide therapy of acute respiratory infections influenced the natural history of rris, probably because of their elective activity on atypical bacteria [ ] . considering the emergence of antibiotic-resistant bacterial stock, as for example s. pneumoniae, immunotherapy has been proposed as a means of preventing rris by providing children with small doses of inactive bacterial antigens liable to trigger specific and protective immune responses (table . ) [ ] . for example, om- bv significantly reduces the urti rate, particularly in a dbpc study in children aged - with a history of acute urtis [ ] , is active in preventing rri episodes [ ] with a meaningful reduction in the number of days of suffering acute urtis [ ] . bacterial ribosomal and membrane proteoglycans of s. pneumoniae, which stimulate b cells with secretory responses, as well as memory cells, may be used for responding to future infections [ ] . ribosomal immunotherapy appears to be not only well tolerated, but also ideally targeted to induce mucosal responses [ ] . among the preparations reserved for specific use, a study of pidotimod in dbpc trials proved its effectiveness in a sample of children with rris, also showing increased cd , absent in placebo-treated children [ ] . the use of immunostimulants should be limited to children with proven high susceptibility to acute urti, or overexposed children attending daycare facilities, or attending kindergarten or elementary school [ ] . however, according to a meta-analysis, immunostimulants are an effective treatment for the prevention of acute urti in children [ ] . furthermore the indiscriminate and purely empiric use of ivig must be discouraged in every child with rri, whereas in a prospective, dbpc study of ivig and co-trimoxazole, of children < years referred for recurrent bacterial rris became infection-free over a -month obser-if caused by pneumococci. other kinds of vaccines have provided disappointing results: fig. . [ ] indicates the immune bases of a specific immunization and the possibility of specific interventions. in children with pid, one should bear in mind all the aforementioned facts. until the past few years, there was a busy motion into the fundamental problems underlying a majority of these conditions. many have now been mapped to specific chromosomal locations, and an impressive number vation period [ ] , in addition to having an extremely unfavorable cost-benefit ratio [ ] immunization children with rris and deficient antibody responses to germs expressing a capsular polysaccharide can be successfully vaccinated, but avoiding the administration of live virus vaccines and integrating this if necessary with an igg replacement therapy [ ] . furthermore, in view of the availability of conjugated vaccines, it will be possible to induce antipneumococcus-igg , providing an effective treatment for children with rris, especially treatment of the fundamental biological errors have been identified. the pediatrician is entrusted with a more difficult job, that of identifying as early as possible the possible existence of pid, remembering the suggestions for case history in chap. , with the exception of clinical emergencies such as omenn syndrome and reticular dysgenesis. this specific research becomes a necessity thanks to the new diagnostic and therapeutic advances that have been conceived over the past few years: the earlier one acts, on the one hand with a prenatal diagnosis and on the other with a bmt or sct therapy, the greater the chance to increase life expectancy for these children, in addition to ensuring better quality of life. the discovery and cloning of the genes for these diseases have obvious implications for the potential of gene therapy. the rapidity of these advances suggests that there will soon be many more to come. one of the most common differential diagnoses will occur with a child affected by rris, for whom we believe the number of infections 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bone marrow transplantation in patients with wiskott-aldrich syndrome from a single center nuovi aspetti diagnostici e patogenetici dei difetti primitivi dell'immunità umorale selective deficiency of interferon-gamma production in the hyper-ige syndrome: relationship to in vitro ige synthesis genetics, phenotype, and natural history of autosomal dominant cyclic hematopoiesis atypical x-linked agammaglobulinemia (letter) long-term followup and prognosis of chronic granulomatous disease in yugoslavia: is there a role for early bone marrow transplantation? griscelli disease maps to chromosome q and is associated with mutations in the myosin-va gene thymic function after hemapoietic stem cell transplantation for the treatment of severe combined immunodeficiency il bambino che si ammala spesso: profilo immunologico-clinico xlinked immune dysregulation, neonatal insulin dependent diabetes, and intractable diarrhoea new hereditary immunodeficiencies and genetic predisposition to infective diseases in children inherited interleukin- deficiency: il b genotype and clinical phenotype of patients from six kindreds links between complement abnormalities and systemic lupus erythematosus ataxia telangiectasia syndrome: clinical picture and immunological abnormalities human equivalent of the mouse nude/scid phenotype: longterm evaluation of immunologic reconstitution after bone marrow transplantation immunodeficiency-related lymphoproliferative disorders: prospective data from the united kingdom children's cancer study group registry lowered yields of virus-induced interferon production in leukocyte cultures and risk of recurrent respiratory infections in children clinical heterogeneity and reversibility of selective immunoglobulin a deficiency in children two siblings with deficiency of iga , igg , igg and ige due to deletion of immunoglobulin heavy chain constant region genes extensive deletion of immunoglobulin heavy chain constant region genes in the absence of recurrent infections: when is igg subclass deficiency clinically relevant? il trapianto prenatale e postnatale di cellule staminali emopoietiche in bambini affetti da immunodeficienza primitiva methodologic problems in establishing normal values for igg subclass concentrations in a pediatric population: comparison of radial immunodiffusion and elisa methods recurrent v m mutation within the wiskott-aldrich syndrome protein: description of a homozygous female patient prenatal diagnosis and genetic analysis of x-linked immunodeficiency disorders the interleukin- receptor gamma chain maps to xq . and is mutated in x-linked severe combined immunodeficiency, scidx defective il r expression in t(-)b(+)nk(+) severe combined immunodeficiency a partial deficiency of interleukin- r is sufficient to abrogate t-cell development and cause severe combined immunodeficiency the scid but not the rag- gene product is required for sm-se heavy chain class switching chronic granulomatous disease the primary immunodeficiencies primary immunodeficiency diseases: report of an iuis committee the common cold -principles of judicious use of antimicrobial agents phagocyte immunodeficiencies and their infections del defines a novel small deletion hotspot in the interferon-gamma receptor chain naturally occurring immune response against bacteria commonly involved in upper respiratory tract infections: analysis of the antigen-specific salivary iga levels ridotta funzionalità natural killer in bambini con infezioni respiratorie ricorrenti a case of hyperimmunoglobulinemia e treated with cow's milk-and egg-free diet characterization of a novel coronavirus associated with severe acute respiratory syndrome the complement system outcomes among recipients of placental-blood transplants from unrelated donors interaction of il- rb and gc chains with jak and jak : implications for xscid and scid primary immunodeficiencies in switzerland: first report of the national registry in adults and children intravenous immunoglobulin consensus statement brief report: a point mutation in the sh domain of bruton's tyrosine kinase in atypical x-linked agammaglobulinemia clinical course of patients with major histocompatibility complex class ii deficiency recurrent pneumonia as warning manifestation for suspecting primary immunodeficiencies in children disorders of the polymorphonuclear phagocytic system. in: stiehm er (ed) immunologic disorders in infants and children difetti dell'immunità cellulare e immunodeficienze combinate clonal selection and learning in the antibody system improved survival after unrelated donor bone marrow transplantation in children with primary immunodeficiency using a reduced-intensity conditioning regimen griscelli syndrome congenital neutropenia and primary immunodeficiency disorders: a survey of iranian patients recurrent sinusitis and immunodeficiency congenital immunodeficiency with a regulatory defect in mhc class ii gene expression lacks a specific hla-dr promoter binding protein molecular defects in the bare lymphocyte syndrome and regulation of mhc class ii genes sialophorin (cd ) and the wiskott-aldrich syndrome impaired interferon gamma-mediated immunity and susceptibility to mycobacterial infection in childhood autosomal recessive hyperimmunoglobulin e syndrome: a distinct disease entity correction of complete interferon-gamma receptor deficiency by bone marrow transplantation the repair of dna damages/modifications during the maturation of the immune system: lessons from human primary immunodeficiency disorders and animal models activation-induced cytidine deaminase (aid) deficiency causes the autosomal recessive form of the hyper-igm syndrome jak ) deficiency: clinical, immunologic, and molecular analyses of patients and outcomes of stem cell transplantation host defense mechanisms in respiratory infection icos deficiency in patients with common variable immunodeficiency immunoglobulin isotype-specific antibody responses to pneumococcal polysaccharide vaccine in patients with recurrent bacterial respiratory tract infections a rare syndrome in the differential diagnosis of hepatosplenomegaly and pancytopenia: report of identical twins with griscelli disease direct evidence of autosomal recessive inheritance of arg to termination codon in purine nucleoside phosphorylase gene in a family with a severe combined immunodeficiency patient mechanism of recruitment of wasp to the immunological synapse and of its activation following tcr ligation cutaneous symptoms in primary immunodeficiencies a single ataxia-telangiectasia gene with a product similar to pi- kinase immunostimulation with om- in children with recurrent infections of the upper respiratory tract: a double-blind, placebo-controlled multicenter study t helper type -like cells and therapeutic effect of interferon-gamma in combined immunodeficiency with hypereosinophilia (omenn's syndrome) omenn's syndrome: differential diagnosis in infants with erythroderma and immunodeficiency indications for the use of intravenous gammaglobulin bone marrow transplantation (bmt) for the syndrome of pigmentary dilution and lymphohistiocytosis (griscelli's syndrome) the complex genetics of common variable immunodeficiency selective polysaccharide antibody deficiency in familial digeorge syndrome rag mutations in human b cell-negative scid waspbase: a database of was-and xlt-causing mutations defective activation of the alternative pathway of complement in patients with homozygous c deficiency: studies in two unrelated families x-linked lymphoproliferative disease: twenty-five years after the discovery genetic, biochemical, and clinical features of chronic granulomatous disease treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the european experience severe osteopenia in a young boy with kostmann's congenital neutropenia treated with granulocyte colony-stimulating factor: suggested therapeutic approach clinical and immunologic characteristic of healthy children with subnormal serum concentrations of igg subnormal serum concentrations of igg in children with frequent infections associated with varied patterns of immunologic dysfunction human immune disorder arising from mutation of the a chain of the interleukin- receptor laboratory assessment of immune deficiency disorders immunodeficiency presenting as hypergammaglobulinemia with igg subclass deficiency phenotypic and functional alterations of peripheral blood monocytes in neutrophil-specific granule deficiency recent advances in the genetics of primary immunodeficiency syndromes recurrent and chronic upper respiratory infections and chronic otitis media defective expression of cd and autocrine growth-stimulation in epstein-barr virus-transformed b cells from patients with wiskott-aldrich syndrome confirmation of x-linked hypogammaglobulinemia with isolated growth hormone deficiency as a disease entity a new kindred with x-linked lymphoproliferative disease treatment of hypogammaglobulinaemia with intravenous immunoglobulin regulation of the polarization of t cells toward antigen-presenting cells by ras-related gtpase cdc primary immunodeficiency diseases in norway functional correction of t cells derived from patients with the wiskott-aldrich syndrome (was) by transduction with an oncoretroviral vector encoding the was protein a multi-institutional survey of the wiskott-aldrich syndrome molecular basis of opsonic defect in immunodeficient children associations of mutations in mannose binding protein gene with childhood infection in consecutive hospital series association of low levels of mannan-binding protein with a common defect of opsonisation complement deficiencies in infections with neisseria meningitidis genetic aspects of ataxia-telangiectasia wiskott-aldrich syndrome protein, a novel effector for the gtpase cdc , is implicated in actin polymerization detection of rag mutations and prenatal diagnosis in families presenting with either t-b-severe combined immunodeficiency or omenn's syndrome two siblings with recurrent infections transient iga deficiency and pathogenesis of infantile atopy a deletion in the gene encoding the cd antigen in a patient with scid inherited deficiencies of the terminal complement components structural analysis of low tcr-cd complex expression in t cells of an immunodeficient patient embryologic and other developmental considerations of thirty-eight possible variants of the digeorge anomaly results of allogeneic bone marrow transplantation in patients with leukocyte adhesion deficiency brief report: correction of x-linked hyper-igm syndrome by allogeneic bone marrow transplantation genetically determined immunodeficiency diseases: a perspective unexplained opportunistic infections and cd + t-lymphocytopenia without hiv infection. an investigation of cases in the united states haematological abnormalities in shwachman-diamond syndrome igg subclasses t cell depleted haploidentical bone marrow transplantation for the treatment of children with severe combined immunodeficiency a family of wasps clinical, immunologic, and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis defective opsonization. a common immunity deficiency immunology in the pediatrician's office independent mutations of the human cd e gene resulting in a t cell receptor/cd complex immunodeficiency genetically determined immunodeficiency disease and malignancy report from immunodeficiency cancer registry complementation cloning of an mhc class ii transactivator mutated in hereditary mhc class ii deficiency (or bare lymphocyte syndrome) severe combined immunodeficiency: a retrospective single-center study of clinical presentation and outcome in patients atypical x-linked severe combined immunodeficiency due to a possible spontaneous reversion of the genetic defect in t cells molecular genetic analysis of x-linked hypogammaglobulinemia and isolated growth hormone deficiency studies of the expression of the wiskott-aldrich syndrome protein new and old immunodeficiencies bone marrow transplantation in severe combined immunodeficiency from a sibling who had received a paternal bone marrow transplant immunodeficiency disorders: general considerations severe combined immunodeficiency in man with an absence of immunoglobulin gene rearrangement but normal t-cell receptor assembly chronic granulomatous disease signaling through zap- is required for cxcl -mediated t-cell transendothelial migration allogeneic hematopoietic stem cell transplantation for seven children with x-linked hyper-igm syndrome: a single center experience stem cell transplants in utero for genetic diseases: treatment and a model for induction of immunological tolerance deficient expression of a b cell cytoplasmic tyrosine kinase in human x-linked agammaglobulinemia mannose-binding lectin: the pluripotent molecule of the innate immune system consensus conference: il bambino con deficit di iga: le caratteristiche cliniche, immunologiche e genetiche per una corretta gestione il bambino immunodepresso th t-cell and monocyte defects autoimmune lymphoproliferative syndrome (alps) in a child from consanguineous parents: a dominant or recessive disease? rotavirus infection in infants as protection against subsequent infections regulation of immunoglobulin (ig)e synthesis in the hyper ige syndrome the gene involved in x-linked agammaglobulinemia is a member of the src family of protein-kinases primary immunodeficiency mutation databases v(d)j recombination defects in lymphocytes due to rag mutations: severe immunodeficiency with a spectrum of clinical presentations a cd minigene restores regulated isoform expression and immune function in cd -deficient mice: therapeutic implications for human cd -null severe combined immunodeficiency analysis of natural killer cells in tap -deficient patients: expression of functional triggering receptors and evidence for the existence of inhibitory receptor(s) that prevent lysis of normal autologous cells major histocompatibility complex class iii genes and susceptibility to immunoglobulin deficiency and common variable immunodeficiency release of leukotriene c in respiratory tract during acute viral infection affinity maturation and class switching evaluation of the child with suspected primary immunodeficiency frequency and severity of infections in day care upper respiratory tract infections in young children: duration and frequency of complications features of transient hypogammaglobulinaemia in infants screened for immunological abnormalities chediak-higashi syndrome: a clinical and molecular view of a rare lysosomal storage disorder clinical and cellular features of "common variable" hypogammaglobulinemia igg subclass levels in the serum of patients with primary immunodeficiency severe combined immunodeficiency due to a specific defect in the production of interleukin- clinical patterns and natural history of asthma clinical and laboratory evaluation of complement deficiency in-utero transplantation of parental cd haematopoietic progenitor cells in a patient with x-linked severe combined immunodeficiency phox , a third cytosolic component of the activation complex of the nanph-oxidase to contain sh domain dominant negative mutation of the hematopoietic-specific rho gtpase, rac , is associated with a human phagocyte immunodeficiency a prospective cytogenetic study of cases of digeorge syndrome family studies of iga deficiency carrier detection of the x-linked immunodeficiency diseases using x-chromosome inactivation analysis report on a national registry of patients topical imiquimod for molluscum contagiosum in t-cell immunodeficiency mutations in the mu heavy-chain gene in patients with agammaglobulinemia immunological findings in infants with wiskott-aldrich syndrome chromoglycate treatment of patient with hyperimmunoglobulinaemia e syndrome (letter) primary immunodeficiency diseases in latin america: first report from eight countries participating in the lagid. latin american group for primary immunodeficiency diseases bcl activates the nf-kappab pathway through ubiquitination of nemo virally induced immunosuppression the xlinked hyper-igm syndrome: clinical and immunologic features of patients brief report: twin boys with major histocompatibility complex class ii deficiency but inducible immune responses neuropsychological profile of children and adolescents with the q . microdeletion case definition for surveillance of severe acute respiratory syndrome sars primary immunodeficiency diseases role of tbx in human del q . syndrome high incidence of significant bone loss in patients with severe congenital neutropenia (kostmann's syndrome) key: cord- -ft cp op authors: rahman, q. k.; wikman, m.; vasconcelos, n.‐m.; berzins, k.; ståhl, s.; fernández, c. title: the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th‐ type of response date: - - journal: scand j immunol doi: . /j. - . . aw.x sha: doc_id: cord_uid: ft cp op finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less‐conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb ‐specific antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th‐ antibody response. in contrast, no priming effect was observed for ex vivo ifn‐γ production but stimulation with the hsp‐chimeric fusion protein induced a stronger secretion of ifn‐γin vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - wh kb p authors: melchjorsen, j.; bowie, a. g.; matikainen, s.; paludan, s. r. title: differential requirements for toll‐like receptor signalling for induction of chemokine expression by herpes simplex virus and sendai virus date: - - journal: scand j immunol doi: . /j. - . . r.x sha: doc_id: cord_uid: wh kb p toll‐like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress‐associated molecules. tlr–ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus‐induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus‐encoded inhibitor of tlr‐signalling a r or dominant‐negative myd totally inhibited hsv‐induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv‐induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr‐dependent and ‐independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - gdfo vd authors: nan title: tsanz oral abstracts date: - - journal: respirology doi: . /j. - . . .x sha: doc_id: cord_uid: gdfo vd nan introduction very little is known about adult health of survivors of extreme preterm birth. the aim of this study was to assess the burden of respiratory symptoms in a cohort of young adults born prematurely compared to sibling controls. method one hundred and fifty six children born prematurely ( - weeks gestation) at the mater hospital brisbane between [ ] [ ] were mailed questionnaires to assess their respiratory symptoms using a modified version of the european community health survey. term-born siblings were invited to act as controls. results thirty six responses were received ( %). the studied cohort consisted of cases ( % female) and controls ( % female). the median age was years ( - ) in the cases and years ( - ) in the control group (p = ns). shortness of breath (sob) was reported in % of the preterm cases, but nil in the control group (p = . ). there was a higher incidence of day/night cough ( % vs. %, p = . ) and morning cough ( % vs. %, p = . ) in the preterm cases compared to controls. the preterm cases were also more likely to experience a chest infection before the age of five ( % vs. % of controls, p = . ). sob was unrelated to a history of asthma, atopy, exposure to smoke or domestic animals as there was no difference between controls and cases. conclusion a higher incidence of sob was reported in young adults who were born prematurely compared to sibling matched controls, and this appears to be unrelated to asthma and atopy. further subjects will be enrolled interstate to assess predictors of respiratory symptoms in early adulthood. background currently all protocols for checking patient readiness for oral intake post fibre optic bronchoscopy (fob) are non-evidence based. there is a need to establish the shortest safe time to implement resumption of oral intake following administration of local anaesthesia for various reasons. we examined return times for the gag reflex and swallow response. method a prospective study of consecutive patients presenting for a fob, age - , were assessed for optimum time to check the gag and swallow reflex. the gag was checked pre and post fob by the touch method (tickle back of throat), swallow was checked post fob with a sip of water at various times. results after hour % and % of patients had a gag reflex (n = ) and swallow response (n = ) respectively. this increased to % and % at ½ hours, % and % at hours, % and % at ½ hours. the amount of sedation or length of procedure did not correlate with the return of the gag reflex or swallow response. none of the patients who swallowed with the gag still absent coughed or aspirated. eighteen patients did not have a gag reflex pre fob. conclusion the gag reflex and swallow response are separate. data shows it is possible to swallow safely without a gag reflex. the time to safe swallow is much shorter than recommended in current protocols. however, we consider it safer to allow oral intake when both reflexes are present, if the gag is present pre fob. otherwise the patient should wait for ½ hours post fob as % have a returned gag reflex, if swallow response is also back. nomination none. introduction home monitoring in copd may identify acute exacerbations earlier, enabling prompt treatment, thus improving morbidity and mortality. reactance (x rs ), measured by forced oscillation technique has a potential role in home monitoring. the aim of this study was to determine within-and between-day repeatability of resistance (r rs ), x rs , and spirometry in stable copd subjects. methods ten copd subjects underwent seven consecutive home visits consisting of three measures of fot ( minute recording) and spirometry before and after mcg ventolin via spacer. results subject characteristics mean (sd) -age . years ( . ), smoking history . pack years ( . ), post-bd (post-bronchodilator) fev . % predicted ( . ), fev /fvc ratio . ( . ). repeatability measures are reported as intra-class correlation coefficient (icc) and sd of within subject variance (sw). introduction chronic obstructive pulmonary disease (copd) is the single most important risk factor for lung cancer (affecting - % of those diagnosed). considerable overlap exists between smokers who develop copd and/or lung cancer, suggesting involvement of shared pathogenic pathways (inflammation, matrix remodeling and cell death). in a prospective study of high and low risk smokers we have combined snps from gwas and candidate gene studies to develop a susceptibility score for lung cancer (lcss). methods seven hundred and twenty eight high risk individuals (chronic smokers, > years old, > pack years, spirometric confirmed copd), and smokers without copd were recruited and followed for a mean of years. cohorts were matched for smoking history, ethnicity, gender and age, thereby excluding confounding from these variables. all volunteers completed spirometry, modified ats respiratory questionnaire and gave blood for dna. iplex and taqman systems were used to genotype the snp panel. cases are in the high risk (copd) cohort ( % over years, mean score = . ) and ( %) from the low risk cohort, normal lung function ( % over years, mean score . ). the healthy unaffected smokers' mean score was . . this prospective study confirms the risk status assigned by the lcss with ( %) vs. ( %) lung cancers over the year follow up (or = . (%% ci . - . , p = . ).the performance characteristics of the lcss reported here, confirm its utility, in correctly identifying smokers at greatest lung cancer risk. conclusion in this prospective study, we show that the lcss identifies those at greatest risk of lung cancer who might benefit from aggressive preventive strategies such as cessation and chemoprevention. the author is not aware of any conflict of interest. malignant mesothelioma (mm) remains an incurable cancer and its global incidence is rising rapidly. alternative therapeutic strategies are therefore required. bacterial products have been trialled in an effort to enhance local immunity and have demonstrated tumouricidal activity. staphylococcal enterotoxins (se) are classic models of superantigens that have potent mitogenic activity on t cells and demonstrated anti-tumour effects in several cancer models. intrapleural delivery of staphylococcal enterotoxin c (sec) has been used in china for many years as a pleurodesing agent. however, it is unknown whether sec actually kills cancer cells. in this study, we examined the efficacy of sec in the treatment of mm. sec was added at various concentrations ( - ng/ml) to several human and murine mm cell lines and a human benign mesothelial cell line in vitro. dose dependant cytotoxicity was observed in all cell lines resulting in a significant reduction in viability at higher doses ( ng/ml) when using trypan blue exclusion and wst- assays ( . < p < . ). in an effort to elucidate the mechanism of action of sec, annexin v staining and flow cytometry were used to measure apoptosis. results demonstrated a significant increase in apoptosis in mm cells when treated with sec compared to untreated controls ( . < p < . ). on the contrary, benign mesothelial cells appeared to be resistant to the apoptotic effects of sec at equivalent concentrations (p > . ). elisa based assays were used to examine cytokine profiles in culture supernatants of sec treated mm cells and benign mesothelial cells. levels of the pro-inflammatory cytokine il- decreased in sec treated mm cells ( . < p < . ) compared to a significant increase in levels observed in benign mesothelial cells ( . < p < . ). these results suggest that sec kills mm cells in vitro with some specificity and its activity against mm in vivo warrants investigation. conflict of interest no. purpose we examined age trends in the distribution of stage at diagnosis in patients presenting with non-small cell lung cancer (nsclc) at tertiary hospitals. methods we used the queensland integrated lung cancer outcomes project (qilcop), a clinical registry which collects information on about % of all lung cancer patients in queensland, to analyse the distribution of clinical (tnm) stage among , patients diagnosed with nsclc between and . differences in stage distribution across age were analysed using tests of proportions and multivariable logistic regression with stage as the dependent variable and other demographic characteristics as covariates. results the median age at diagnosis of patients in the study was years (range - ) and % were males. the overall proportions of stages i, ii, iii, and iv were, respectively, %, %, %, and %. the percentage of stage i disease increased with age (p < . ), from % in those younger than years to %, %, and % in those aged - , - , and years or older, respectively. age differences in stage distribution remained significant in multivariable analysis controlling for gender, rural residence, and socioeconomic status. among the other characteristics, only gender differences in stage was significant, with stage i cancer being more common in women compared to men ( % vs. %, p = . ). australia has the world's highest incidence of mesothelioma, a disease which has no proven effective therapy. the median survival of less than months and five year survival of % have not changed in two decades. radical resection has a high attrition rate with most cases recurring locally, and few patients have durable responses to chemotherapy. symptoms are related to local disease which compresses the lung and causes severe chest pain from enlarging tumour masses. aim to improve local control of mesothelioma by high dose radiotherapy using advanced technologies that precisely define the active tumour and reduce toxicity to normal tissues. methods all patients had fdg pet scans co-registered with a simulation ct scan to define the target volume, and follow-up pet scans were analysed to assess the residual total glycolytic volumes (tgv) after radiotherapy. acute and long term toxicities were assessed. results between and thirty patients with incompletely resected pleural mesothelioma were treated with radiation doses of to gy to part or all of one hemithorax. all patients who received chemotherapy had progressed prior to radiotherapy. in we introduced a program using a new technique called intensity-modulated radiotherapy (imrt). tgvs reduced by % after radiotherapy, median survival was extended to months and there were no major radiation toxicities, the most common being grade two pneumonitis. relapses were frequent on extended follow-up, the majority in areas outside the radiotherapy field. conclusions imrt is effective in maximising local control in mesothelioma patients who have had extrapleural pneumonectomies, and for selected patients with an intact lung. toxicities are manageable and locoregional control is very good. high dose radiotherapy is recommended for most mesothelioma patients for long term palliation and control of locoregional progression. introduction mortality benefits for ldct screening are not yet known. the queensland lung cancer screening study is screening up to high risk volunteers based on the nlst/acrin protocol. aims observational cohort study to assess: disease detection rate; lung nodule work-up; cost; quality of life issues; smoking cessation; biomarker collection feasibility. methods recruitment via local advertisement and press release. major inclusion criteria: age - years; smoking history ‡ pack years; fit for surgery. volunteers have one prevalence and two incidence scans and follow-up for three more years. ldct parameters: phillips brilliance slice multidetector scanner; low-dose protocol; . mm slice width. scan reporting: two radiologists independently; independent cad reading (phillips brilliance software); final report is agreed by consensus. results see table -to be updated. magnetic resonance imaging (mri) is a useful modality for assessing chronic thromboembolic pulmonary hypertension (cteph) before pulmonary endarterectomy (pea). cardiac mri provides more accurate right ventricular (rv) data than echocardiography. mr angiography demonstrates vascular changes reliably to a segmental level and mr perfusion shows disease distribution. aim to examine the relationship between changes in mri parameters with clinical and haemodynamic outcomes post-pea. methods rv end-diastolic volume (rvedv), rv ejection fraction (rvef), vessel abnormalities and lobar perfusion defects were determined with mri before and after peas performed during [ ] [ ] [ ] [ ] . changes in new york heart association (nyha) functional class, six minute walk distance ( mwd), mean pulmonary artery pressure (mpap) and cardiac output (co) were collected retrospectively from patient charts. results nineteen patients assessed pre-pea were of mean ± sd age ± years, nyha class . ± . , mwd ± metre and mpap ± mmhg. immediately post-pea, mpap fell by ± mmhg which was related to changes in rvedv of ± % (r = . , p = . ). at - months post-pea, mwd improvement ( ± metre) was related to changes in rvef of ± % (r = . , p = . ) but angiographic changes had a weak relationship with nyha class shift (r = . , p = . ). perfusion generally improved after pea relating weakly with rvedv (r = . , p = . ) but not clinical outcomes. conclusions this study shows that improvements in mri parameters (rv data more than angiographic findings) after pea correspond to clinical and haemodynamic outcomes. we have demonstrated a useful imaging test for monitoring patients post-pea with no radiation exposure. purpose allergic reactions to antibiotics are very common in cystic fibrosis (cf) patients and can complicate treatment in patients with multi-or pan-resistant bacterial species. we hypothesised that post-transplant immunosuppression may reduce the requirement for desensitisation. methods and materials a retrospective review was performed in june to detect prescribing practices and any changes in allergy patterns before and following lung transplantation in cf. since antibiotic desensitisation has not been used post-transplant at our institution, our aim was to review our experience with antibiotic rechallenge, without desensitisation, in the post-transplant setting. results fifty eight cf patients, ( %) female, aged years, range - years, heart-lung, heart-lung liver have undergone transplantation at our institution. ( %) had a pre-transplant history of ige (angioedema - %) or non-ige (e.g. rash, nausea, arthralgia or liver dysfunction - %) mediated reactions to at least one antibiotic ( % penicillin, % cephalosporin, % carbapenem, % aztreonam and % others). desensitization to antibiotics with non ige mediated reaction was attempted in out of the patients pre-transplant and successful on all occasions. in other patients, alternative antibiotics were selected and desensitisation was not required. after transplantation, ( %) patients with non ige mediated reactions were rechallenged without desensitisation on occasions. no life-threatening reactions were observed. only two episodes required antibiotic cessation and both recovered without incident. new onset of adverse reaction to iv colistin and voriconazole occurred in two patients following transplantation. conclusions cautious antibiotic rechallenge can be successfully achieved without desensitisation in the majority of patients who have had non ige mediated allergic phenomena to the same compound prior to transplantation. idiopathic pulmonary fibrosis (ipf) is characterised by marked collagen deposition. the receptor subunit gp has been associated with the progression of fibrosis. the interleukin (il)- family of cytokines all require gp to initiate signal transduction to activate either the extracellular regulated kinase (erk) or signal transducer and activator of transcription (stat) pathways. the il- family of cytokines consist of il- , il- , oncostatin m (osm), leukaemia inhibitory factor (lif), cardiotrophin- (ct- ), ciliary neurotrophic factor (cntf), il- and il- . previous studies performed in our laboratory have demonstrated that exaggerated gp -stat signalling is fundamental to the development of bleomycin-induced lung fibrosis in a murine model. we hypothesise that pulmonary fibrosis is mediated by il- family cytokine/gp -stat / signalling. the aim of the current study was to identify which of the il- family cytokines are important in the development of bleomycin-induced pulmonary fibrosis. bleomycin or control saline was administered intranasally to individual il- knockout (il- -/-) mice and dual il- and il- a-receptor knockout (il- -/-;il- ar -/-) mice. collagen production was examined by histology and hplc in lung tissue days post treatment. no significant increase in collagen was observed in bleomycin treated il- -/or il- -/-;il- ar -/mice implicating a role for il- in the development of pulmonary fibrosis. interestingly, the histology of il- -/-;il- ar -/mice displayed marked emphysema which was not observed in individual il- -/mice suggesting this is an il- -mediated response. the role of il- family cytokines in proliferation, myofibroblast differentiation and collagen expression were examined using fibroblasts isolated from wildtype (wt) and genetically engineered mice containing point mutations to prevent gp -erk / signalling (gp f ) or gp -stat / signalling (gp stat ). overall, there was no significant increase in proliferation hours post cytokine stimulation as assessed by wst- reagent. il- and il- did not stimulate a-sma or collagen expression above control, measured by real time pcr. in conclusion, increasing evidence suggests that il- plays an important role in the development of bleomycininduced pulmonary fibrosis but this does not appear to be induced by direct effects of il- on fibroblast proliferation, differentiation or collagen production. mannose binding lectin (mbl) is a key mediator of innate immunity and efferocytosis (clearance of apoptotic cells) and is thus important in protecting against tissue damage. reduced mbl is implicated in airways disease including infection, copd and bos, however, 'normal' plasma levels of mbl are highly variable due genetic polymorphisms complicating correlation with disease processes. we have previously shown reduced mbl and defective efferocytosis in bal from patients with post-transplant bos, but there are conflicting reports of the link between low plasma mbl levels, increased complement activation and graft rejection. to compare mbl levels in the peripheral blood and bal compartments, we investigated mbl in paired plasma and bal from lung transplant recipients ( stable; stable with infection, with lymphocytic bronchiolitis and with bos) and in plasma from and bal from controls. in plasma, mbl levels were highly variable. no significant differences were noted among the transplant groups although levels were significantly reduced in all transplant patients vs. controls. there was no correlation between mbl and time post-transplant or pre-transplant diagnosis. in bal, mbl levels were less variable and significantly reduced in patients with bos (mbl ng/ml: controls: . ± ; stable . ± . ; stable infected . ± . ; bos . ± . ). interestingly, in all patients with lb, mbl levels were very low in both plasma (mean . ng/ml) and bal ( ng/ml). low levels of mbl in the airway may play a role in reduced efferocytosis, leading to tissue damage and airways disease post-transplant. in normal subjects, external dead space with exercise is associated with a slower deeper breathing pattern compared at the same ventilation. with simulated lung restriction and constant intensity exercise, we tested the hypothesis that dead space combined with reduced exercise intensity to match ventilation, would alter pattern of breathing, reduce inspiratory reserve volume (irv) and thus increase dyspnea. methods eleven healthy male subjects, aged ( ) (sd) years completed separate visits with (a) no restriction and (b) chest wall strapping to reduce fvc by ( ) introduction glossopharyngeal breathing (gpb) is used by competitive breath-hold divers to increase lung gas content above tlc to improve performance. this occurs by both lung expansion and gas compression. whilst gpb is known to induce hypotension and tachycardia, little is known about the changes that occur to both the pulmonary circulation and the structural integrity of the thorax. the aim of this study was to investigate these changes within an elite cohort. methods six male breath-hold divers were studied. exhaled vc was measured before and after gpb. subjects were studied in the supine position at baseline tlc and after maximal gpb above tlc at least hours apart. tc m labelled macro aggregated albumin was injected and a computed tomography (ct) of the thorax was performed during breath-hold. dynamic and single photon emission ct (spect) images were generated and analysed by two blinded nuclear medicine physicians for perfusion intensity (wilcoxon signed rank test) and dynamic regional blood flow. a paired t-test was used to assess physiological parameters. registered ct images were used to determine structural change in the thorax. results five subjects increased exhaled vc with gpb [mean (sd)] by . ( . ) l (p < . ). there was a reduction in perfusion intensity following gpb in the anterior (p < . ) and inferior (p < . ) lung segments. there was no change in the timing of blood flow. % of the increase in expired lung volume above baseline tlc was via thoracic expansion ( . ( . ) l (p < . )) with a caudal displacement of the diaphragm. one subject who was not proficient at gpb had no change in exhaled volume, ct appearance or lung perfusion. background we have developed a sensitive method to determine conductance-lung volume (conductance profile) and distensibility-lung volume (distensibility profile) relationships using the forced oscillation technique (fot). using this method, we aimed to assess the effect of a short-acting bronchodilator (bd) on these profiles in asthma. methods twenty two asthmatics and healthy controls completed distensibility measurements (fot) and lung function tests before and after bd. the conductance and distensibility profiles were described continuously and determined at specific lung volumes, residual volume (rv), frc, tlc and midway between frc and tlc (mid). results following administration of bd: the conductance profile in the asthma group was shifted upwards, and the distensibility profile was altered such that significant increases in distensibility were observed at rv (p < . ) and frc (p < . ), but not at mid or tlc. in contrast, no changes were seen in the conductance or distensibility profiles in the control group. post-bd distensibility in asthma remained reduced compared to controls. conclusion using a sensitive method for determining conductance and distensibility profiles, we found that both conductance and distensibility are reduced in asthma across a range from low to high lung volumes. both these profiles are altered in asthma after bd but not in controls. we propose that in asthma the remaining deficit in distensibility after bd will provide unique insight into to altered airway mechanical function due to airway remodelling. cigarette smoke exposure is a major risk factor in susceptibility to serious respiratory infections, particularly in children. although smoke exposure is known to alter immunity to infection, the underlying molecular mechanisms are not well understood. aim identify regulatory mechanisms that drive impaired macrophage function. methods the mh-s alveolar macrophage cell line was exposed to a short minute pulse of cigarette smoke extract (cse) prior to challenge with lps or fitc-e.coli. results cse blocked phagocytosis of e.coli and inhibited lps activation of canonical and alternative tlr pathways. both nfjb translocation and transactivation pathways were compromised as cse inhibited ijba degradation and p phosphorylation. cse also blocked ap- activity by inhibiting p , but not jnk or erk / . we next excluded lps tolerance mechanisms involving receptor internalisation or induction of negative regulators. as free radical species are abundant in cse we investigated their role using the potent scavenger, reduced glutathione (gsh). since gsh restored all responses, we screened a panel of oxidative/nitrosative stress markers and identified carbonylation as the only cse inducible marker. oxyblot analysis confirmed that cse potently introduced carbonyl groups to many proteins ( - kda range) in a dose and time dependent manner that inversely correlated with tnf-a expression. cse treated macrophages also displayed heavily carbonylated pseudopodia that was reversed by gsh as determined by immunocytochemistry (icc). conclusion macrophage sensing and ingestion of pathogen is compromised by protein carbonylation of the outer membrane where phagocytic receptors cluster and also penetrates cytoplasmic regions where signalling moieties reside. therefore, targeting single pathways will not restore macrophage function due to the global nature of cse mediated carbonylation. support nhmrc. background asthma shows varying levels of resistance to effective treatment by glucocorticoids. in studies on tgfb-induced epithelial mesenchymal cell transition (emt) using the a type ii human epithelial cell line, the regulatory effect of the glucocorticoid, dexamethasone (dex, . - nm) on interleukin- a (il- a)-induced interleukin- (il- ) generation was markedly reduced in the presence of tgf-b pm (n = ; p < . ). aim our studies were designed to characterise the mechanism of the glucocorticoid resistance. results the resistance induced by tgf-b was: concentration dependent ( - pm); a glucocorticoid class effect as it also occurred with budesonide; independent of emt: it was observed within hours, whereas emt requires days; also observed in the central airway epithelial cell line, beas- b and passaged, primary bronchial epithelial (nhbe) cells. treatment of a cells with sb , a tgfb receptor type i kinase inhibitor, restored the dex inhibitory effect on il- release (from control ± % to ± %, inhibition in sb lm, n = ; p < . ). in a cells transfected with a glucocorticoid response element (gre)-driven reporter gene, tgfb ( pm) inhibited the gre response to dex ( . - nm) by more than %. in addition, tgf-b impaired dex regulation of the gre-dependent gene, ijb. pge plays a protective role in asthma by inhibiting airway inflammation. it is predominantly produced by epithelial cells in response to pro-inflammatory stimuli and acts as an autocrine and paracrine mediator. prostanoids have been shown to regulate expression of enzymes involved in their metabolism, as well as expression of their receptors and that regulation is tissue-and cell-specific. despite its importance, however, mechanisms underlying the regulation of expression of enzymes involved in pge metabolism and its receptors in human lung epithelial cells have remained elusive. therefore, we hypothesised that pge regulates expression of pge synthase (pges ) and its receptors (e prostanoid (ep) - ) in human airway epithelial cells. methods real time rt pcr and facs analysis were used to assess mrna and protein expression, respectively in human airway epithelial cells hbe before and after pge stimulation. results pge up-regulates pges in time and concentration dependant manner. in addition, ep receptors (ep , ep and ep ) were up-regulated following pge stimulation at mrna level. however, these receptors show different dynamics in expression. while ep reaches peak in mrna expression at hour, peak expression for ep and ep is at hour post stimulation. monocyte derived dendritic cells (dcs) have been recognised for their potential role in immune responses and their functional relevance with regard to adaptive immune responses although more detailed knowledge of dc biology in human airways is required. the objective of this study was to modify the monocyte derived dcs from peripheral blood and direct the cells via exposure to pro-inflammatory conditions as seen in copd, with a view to identifying novel targets for cellular therapy. we characterised monocyte-derived dcs in culture and evaluated the effects of human rhinovirus infected bronchial epithelial cells, pi:c (polyinosine-polycytidytic acid) and bc (bacterial extract) on directing dc differentiation and maturation in culture. we found an impaired adaptive immune response, and in particular, an impaired cd effector cell function in copd patients. our dc culture results showed that both mhc-i and mhc-ii expression on dcs from copd were significantly down regulated compare to healthy controls, which could affect mhc restricted ag presentation, and lead to a failure to activate responder t cells. furthermore, we tested the capability of monocyte-precursors to differentiate into functional dcs. only a very small percentage of cultured monocytes from patients with copd was capable of differentiating into mature dcs, compared with healthy controls. during dc activation, there was up-regulation of co-stimulatory (cd / ) and maturation markers (mhcs), enabling dc to activate naïve t cells in mixed lymphocyte culture both in copd patients and healthy controls. our preliminary data indicated that this activation leads to the generation of effector t cells, further study is needed. defective dc activation of t cells may underlie poor t cell responsiveness in copd in response to inflammation and may, in part, determine the response to therapy. our data suggest a promising role in vitro for pharmacologic treatment as a means of generating functional dcs and will further stimulate speculation regarding their potential clinical application. support nhmrc australia. anxiety and depression are the two most common and the least treated comorbidities associated with chronic obstructive pulmonary disease (copd). they have been found to have a statistically significant and independent adverse association with mortality, longer length of hospital stay, persistent smoking and worse physical and social function. evidence for various interventions to overcome these symptoms in copd is limited or inconclusive. methods this is a cochrane review. all randomised controlled trials (rcts) and cross over trials (cots) dealing with pharmacological and/or psychological interventions for anxiety and/or depression in adults with copd were considered. search was performed via various medical search engines and cochrane database register. duration of follow-up in the studies was generalised as short-term ( - months), medium-term ( - months) and long-term ( - months). the data was analysed using the fixed effect model and difference in symptom control by interventions as mean difference (md) or the standardised mean difference (smd) depending upon the heterogeneity of various scales used. results eight rcts/cots were included. pharmacotherapy showed nil significant effect to control anxiety (smd of - . , p value . , n = ), however, showed significant benefit in controlling depression symptoms (smd - . , p value . , n = ) in copd over short-term. on the other hand, cbt was found to be beneficial in controlling both anxiety (md - . , p value . , n = ) as well as depression (md - . , p value . , n = ) in copd over short term. conclusion cbt is superior as compared to pharmacotherapy over short-term to control anxiety and depression in copd. background the emergence of clonal pa and associated risk of cross-infection is a major cause for concern in cf centres in several countries, including australia. two clonal pa strains (aes and aes ) have been detected in several eastern australian cf centres, but the overall prevalence of these and other strains throughout australia remains unknown. methods a cross-sectional study involving cf centres ( paediatric, adult, combined) was performed. in total, sputum-producing children and adults with documented pa infection provided two sputum samples, months apart, and pa isolates from each sample were genotyped by reppcr-based cluster analysis. results collectively, aes and/or aes strains were identified in % of pa infected cf patients and found in all participating cf centres. several other minor clonal strains were also identified in many centres. only % of patients were infected with unique (non-clonal) pa strains and % of patients were infected with more than one clonal strain. conclusion clonal pa strains are common in australian patients with cf. there is marked variation in prevalence of both major and minor clonal strains between cf centres and within states. longitudinal analysis of the clinical impact of clonal pa infection is urgently required so as to allow an evidence-based approach to patient management and infection control. reduced glutathione (gsh), a major component of anti-oxidant defence, is transported into the lung via cftr. gsh protects against myeloperoxidase (mpo)-induced oxidative stress by undergoing oxidation to gssg and gsa. excess mpo induces chlorination of tyrosine ( -cl-tyr) via the production of hypochlorous acid. we undertook a cross-sectional survey of young children with cf participating in our early surveillance program that includes annual bronchoalveolar lavage (bal) and chest ct scan. markers of neutrophilic inflammation and of the gsh system were determined in bal and the presence of bronchiectasis (b) and air trapping (at) determined on chest ct. samples from children with cf (mean age . years) and nine samples from children without cf (mean age . years) undergoing investigation for chronic respiratory symptoms (ncf) were studied. cf samples had more neutrophils that ncf samples (geometric mean . vs. . · /ml fluid retrieved), more mpo ( . vs. . ng/ml, p < . ), lower levels of gsh ( . vs. . nm, p < . ) and a trend for a lower gsh:gssg ( . vs. . , p = . ). within the cf samples levels of mpo correlated with gssg (p = . ), and gsa (p < . ) and -cl-try correlated with gssg (p = . ) and gsa (p < . ) indicating neutrophilic inflammation exceeding anti-oxidant defence capability. however, after controlling for age and the presence of free neutrophil elastase, there were no relationships between gsh, gssg or gsa and either b or at. while our data demonstrate gsh deficiency and defective anti-oxidant defence, these are not related to structural lung disease. longitudinal studies will be required to determine the impact of gsh deficiency in the initiation and progression of lung disease in cf. support cfft, inc (usa); nhmrc, acfrt, mcri and pmh foundation (aust). introduction there are no australasian guidelines for the detection and eradication of pseudomonas aeruginosa in preschool children with cf. the optimal eradication regimen for preschool children remains uncertain. aims to develop australasian guidelines for p. aeruginosa detection and eradication in preschool children with cf based on a national multi-centre randomized control trial. methods an electronic web-based questionnaire was sent to every tertiary paediatric cf centre in australasia to determine current detection and eradication practices. results all eleven centres completed the survey. a combination of positive oropharyngeal culture (opc) and confirmatory bronchoalveolar lavage (bal) culture was the most common method of p. aeruginosa detection ( %), with surveillance frequencies varying; annually (n = , %), every clinic visit ( , %) and clinically indicated ( , . %). eradication treatment was instigated on one positive culture (opc n = , bal culture n = centres) for p. aeruginosa at any bacterial density ( %) or bacterial density ‡ cfu/ml ( % of centres). eradication regimens varied between centres with most ( %) using intravenous antibiotics either alone (n = - ); in combination with - months nebulised antibiotics (n = ); or with - months nebulised and oral antibiotics (n = ). choice of regimen was influenced by clinical status in three centres. two centres used combinations of inhaled and oral antibiotics alone. failure to eradicate resulted in a change in treatment in % of centres. inhaled tobramycin mg ( days and months) was considered the least acceptable regimen for preschool children in terms of efficacy and burden of care with most favouring more aggressive iv treatments. conclusion this survey supports a call for evidenced based australasian guidelines for the detection and eradication of p. aeruginosa in preschool children. comparative trials of the most favoured eradication regimens would enhance this process. pseudomonas aeruginosa is the most important respiratory pathogen in cystic fibrosis (cf). although unproven, it is generally thought to be acquired from the environment. however, molecular typing studies indicate person-to-person transmission by some clonal strains may also occur. clonal p.aeruginosa strains have not been compared previously with isolates collected from non-cf patients, animals or the environment. aim to determine sequence-based clonality of p.aeruginosa isolates collected from several different ecological niches. methods mlst and sequence type (st) analysis was performed on isolates collected from cf patients (n = ), non-cf patients (n = ), animals (n = ), and the natural environment (n = ). cf isolates included each of the major and minor clonal australian strains and a range of unique strains isolated from patients residing in se qld. non-cf, animal and environmental isolates were collected from the same region. results of the individual strains detected, ( . %) were found in more than one niche. overall, unique and minor clonal cf strains were detected in at least one other niche; including cf strains found in the environment. to date, none of the three major qld cf clonal strains have been detected in another niche. conclusions in cf, environmental exposure to p aeruginosa seems important for acquiring unique and minor clonal strains. finding that the three major clonal strains were confined to cf patients further suggests person-to-person transmission is occurring and/or strain associated adaptation to the cf lung. support nhmrc, acfrt, tpch foundation. background clostridium difficile colitis remains a rare but potentially life threatening complication in cf patients particularly in the post lung transplant setting. aim ( ) to review the incidence of c. difficile colitis among non-transplant and post-lung transplant cf patients attending our centre. ( ) to identify the clinical features in cf patients presenting with cdad. method retrospective study on c. difficile toxin status in all fecal samples collected in cf patients between to was reviewed. patients with positive c. difficile toxin were identified as index cases, those with negative samples were selected as control cases. results two hundred and twenty-three fecal samples were collected from cf patients. nineteen cdad cases were identified in patients including non-transplant and two post-transplant patients. thirteen had mild colitis and two had fulminant colitis. incidence density of cdad in non-transplanted cf patients ( . / patient-days) is comparable to the transplanted group (two episodes/ patient-days). time to diagnose cdad among post-transplant patients is shorter than non-transplant patients ( vs. days). a significantly proportion in the cdad group had recent ciprofloxacin when comparied to controls ( . % vs. %, p-value: . .). a significantly higher proportion of patients in the cdad group are currently on gastric suppression therapy than control ( % vs. %, p-value: . ). conclusion the incidence density of cdad is comparable between pre and post transplant cf patients. cdad is more common among patients with prolonged ciprofloxacin treatment and concurrent gastric acid suppression. improving patient awareness by optimising patient education particularly during prescription of ciprofloxacin or gastric acid suppression treatment can prompt early presentation and management of cdad. conflict of interest no. g karmakar , d milne , m wilsher green lane respiratory services, and department of radiology, auckland city hospital, auckland, new zealand introduction major (massive or submissive) pulmonary embolism (pe) is a potentially lethal condition particularly if associated with cardiogenic shock. thrombolysis is accepted as standard treatment for massive pe with hypotension, but it carries substantial risk of bleeding and risk may outweigh the benefit in sub-massive pe. the objective of this study was to examine risk factors for and the outcome of major pe in this institution. methods we collected data retrospectively from all patients with mpe requiring icu admission in auckland city hospital (ach) over a year period. the primary outcome variable was mortality with secondary variables including precipitating factors and morbidity. results twenty-eight subjects ( massive pe) were identified. eight of with massive pe were thrombolysed and four were treated conservatively with day mortality of % and % respectively. twelve of patients with sub-massive pe received thrombolysis with no mortality. significant bleeding complications were reported in four of thrombolysed patients. prior surgery without dvt prophylaxis was identified as a precipitant in patients. conclusions massive pe carries significant mortality irrespective of thrombolysis but such treatment appears safe in sub-massive pe. in spite of evidence of efficacy, failure to offer prophylaxis of dvt in the perioperative setting appears an ongoing risk factor for major pe. introduction there is a need to improve general understanding of the epidemiology, pathophysiology, outcome and therapies for rare (orphan) lung diseases. aims ) to establish an electronic reporting registry of orphan lung diseases in australasia. ) to provide a useful resource for physicians and patients. methods a website (www.arnold.org.au) was developed containing information on orphan lung diseases, useful links and an on-line patient discussion forum. tsanz members were invited to participate in reporting cases electronically by a quarterly email reminder. the following data were entered: first two letters of given and family name, date of birth, postcode and whether the patient was new or old to the physician. results ethical approval in new zealand is still awaited. results twenty-three h n cases of mean ± sd age . ± years were confirmed from known lt recipients (incidence . %). cases peaked in new south wales (n = ) and queensland (n = ) consistent with epidemiology in the general community. clinical manifestations included allograft dysfunction in of ( . %), upper respiratory tract symptoms only . %, fever . %, myalgias . %, hypoxia . % and radiological infiltrates . %. mean hospitalisation was ± . days. cases were diagnosed in the hospital setting (n = ) and in the community (n = ). other risk factors included obesity n = , diabetes n = and malignancy n = . treatment consisted of oseltamivir mg bd initially for days in all cases, extension beyond days n = (mean ± . days) due to ongoing symptoms, steroid therapy n = , mechanical ventilation n = and noninvasive support n = . patients ( . %) have not returned to baseline lung function and there were four deaths (bos grade three n = and grade n = prior to diagnosis). conclusions the incidence of h n in the australian lt population was less than that estimated for the broader community but mirrored the geographical distribution. the majority experienced allograft dysfunction with most deaths recorded in those with preexisting bos grade . we recommend h n vaccine (panvax, csl) for all lt recipients. as the only commonly transplanted organ exposed to the atmosphere, the lung allograft may be uniquely susceptible to iga deficiency. since igg deficiency is common after transplantation, we hypothesised that iga production may be similarly affected and may contribute to bos pathogenesis. methods we conducted a cross-sectional evaluation of our transplant cohort. total iga, igm, igg, and igg subclasses were measured in serum using elisa. demographic data, immunosuppression and bos status were recorded. results one hundred and twenty three patients ( % of the total cohort, aged . years (range . - . ), % female, % cf, % copd) were evaluated. the median iga level was . g/l (normal range - g/l). patients ( %) were iga deficient, and ( %) of these were pan-hypogammaglobulinaemic. iga levels were lower in patients with bos (median, iq range; . , . - . vs. . , . - . , p = . ). iga deficiency was not associated with age, sex, diagnosis, transplant type or time post-transplant. the median igg level was . g/l (normal range - g/l) and patients ( %) were deficient. igg levels were lower in bos (median, iq range; . , . - . vs. . , . - . , p = . ) and igg deficiency was more common in copd ( %, p = . ). igg levels were negatively correlated with age (r = . , p = . ). multivariate logistic regression models identified iga deficiency as independently associated with bos (p = . ), while the association of igg deficiency with bos was explained by interaction between bos, age and copd. igm deficiency was present in patients ( %) and was more common in males ( %, p < . ), but was not significantly different in bos. igg, iga and igm levels were not associated with immunosuppression or the use of basiliximab induction conclusions serum iga deficiency is common after transplantation and is independently associated with bos. impaired defence of mucosal surfaces because of iga deficiency may contribute to bos pathogenesis. results rv infection alone led to induction of, pstat- , cxcl and release of ifn-k sirna, knockdown of mda- reduced cxcl , ifn-b and pstat- and also reduced ifn-k. silencing of tlr- and rig-i alone had no effect. when combined both sirna for tlr- and mda- had no additional effect. treatment of becs with bx inhibited the production of type i ifn but only partially inhibited ifn-k release. blockade of p mapk however led to substantial blocking of ifn-k release and also led to a marked reduction in the typeiifn response. conclusion we found that following infection with rv, becs type i ifn responses, pstat- induction as well as release of ifn-k was dependent on mda- . blockade of tbk- /ikk by bx reduced type i ifn response, but ifn-k release was only partially inhibited blockade of p mapk resulted in suppression of both ifn-k and type ifn, suggesting this pathway is crucial in the antiviral response to rv infection. conflict of interest none. introduction pregnant women have increased susceptibility to respiratory virus infection. human rhinovirus (hrv) and influenza are the most common respiratory viruses isolated during severe exacerbations in pregnant asthmatics and are high risk factor for respiratory-related maternal and neonatal morbidity and mortality. understanding the innate and adaptive immunological processes underlying respiratory virus infection in pregnancy will lead to improved treatments, resulting in better health outcomes for both mother and baby. methods cross-sectional study of pregnant asthmatics, pregnant non-asthmatics, non-pregnant asthmatics and healthy non-pregnant women. blood mononuclear cells were cultured with hrv (rv or rv b), influenza a (h n ), phytohaemagglutinin or tlr and tlr agonists, polyinosinic:polycytidylic acid and imiquimod, respectively. protein concentrations of ifn-a, ifn-k ifn-c, il- and il- were quantified from culture supernatant by elisa and cba. results pregnant asthmatics had significantly increased il- a and il- (median . pg/ml and . pg/ml, respectively) compared to healthy women (median pg/ml and . pg/ml, respectively, p < . ). ifn-c was significantly reduced in pregnant asthmatics (median . pg/ml) compared to healthy women ( . pg/ml, p = . ). ifn-a and ifn-k were induced by hrv and influenza in response to rv , pregnant women had decreased ifn-a production ( . pg/ml) compared to healthy controls ( . pg/ml, p = . ) whilst ifn-k production was significantly reduced in both pregnant asthmatics ( . pg/ml, p = . ) and pregnant non-asthmatics ( pg/ml p < . ). conclusion increased il- and il- production and reduced ifn-c, ifn-a and ifn-k are important inflammatory and anti-viral alterations during pregnancy and asthma; providing important insight into why pregnant women are more susceptible to respiratory virus infections. support nhmrc. conflict of interest no. nomination nil. the incidence of ntmld is increasing markedly in australia. it is not known why patients with ntmld are susceptible to these organisms, as most patients do not have identifiable risk factors or a documented immune defect. as cell-mediated immunity is crucial for control of mycobacterial disease, we assessed whether ntmld is associated with diminished th immune responses. methods our study cohort consisted of patients with ntmld at different stages of treatment, offspring of patients and unrelated healthy controls. plasma levels of cxcl and il- were assayed on all subjects by cytometric bead array or elisa. in a subset of subjects, pbmc were assessed for production of ifng, il- , il- and il- in response to stimulation with mitogen (seb) and purified protein derivative (ppd). all data was analysed using non-parametric statistical tests. results plasma levels of both cxcl and il- were higher in ntm patients compared with unrelated controls and/or offspring (p < . ). cxcl levels were lower in patients who responded well to treatment compared to those who responded poorly (p = . ). compared with healthy controls, pbmc from ntm patients produced similar levels of ifng, il- and il- , less il- (p < . ) in response to seb, but more il- in response to ppd (p < . ). conclusions ntmld is not associated with diminished th responses. elevated levels of cxcl indicate ongoing ifng release in vivo. ntm patients may have a bias towards il- production in response to mycobacterial antigens and/or harbour an intrinsic defect in th immunity. paracetamol is commonly used in infants as an analgesic and antipyretic. cross sectional studies have reported an association between frequent paracetamol consumption in early life and risk of childhood asthma. to date, no study has controlled for indication for use of paracetamol, or confounders such as history of respiratory infections, which is independently associated with asthma. aim to examine, in a prospective cohort study, whether frequent paracetamol exposure during early life increases the risk of childhood asthma. method six hundred and twenty infants with an atopic family history were recruited. paracetamol exposure was prospectively documented on occasions to years of age, including the number of days and the indication for use. an interviewer-administered questionnaire was used at and years to ascertain asthma in the previous months. results exposure to paracetamol occurred in % of participants by two years of age. increasing frequency of use of paracetamol was associated with increased risk of childhood asthma (or = . , %ci . - . per doubling of days of use). adjustment for frequency of respiratory infections substantially reduced the strength of these associations (aor = . , . - . ). paracetamol use for non-respiratory tract infections and injury was not associated with asthma (or = . , . - . ). conclusions in children with a family history of allergic disease, we found no association between early paracetamol use and risk of subsequent allergic disease when adjusted for respiratory infections, or when only paracetamol use for non-respiratory tract infections was examined. these findings do not support suggestions that paracetamol use up to age two years increases the risk of asthma. support nhmrc. aim to determine whether a water-based exercise program was effective in improving exercise capacity and quality of life in people with copd with physical co-morbidities compared to a land-based exercise program or no exercise. methods participants with copd referred to pulmonary rehabilitation and who had a physical co-morbidity were randomly allocated to one of three groups: land-based exercise, water-based exercise or a control group of no exercise. the two exercise groups trained for eight weeks, three exercise sessions per week. participants underwent measurements of respiratory function, exercise capacity and quality of life by a blinded investigator at baseline and following intervention. results of participants (mean (sd) age ( ) knowledge of airway dimensions is critical for bronchoscopists assessing airway stenoses requiring interventions. it is also important for evaluating phenotypic features of obstructive lung diseases (old). however, real-time quantification of airway dimensions during bronchoscopy is lacking. inserted into the airways via a bronchoscope, anatomical optical coherence tomography (aoct) is a light-based imaging technique with the unique capacity to obtain such measurements. we describe the validation, research and clinical applications of bronchoscopic aoct. methods (study ) aoct was validated in a phantom model, excised porcine airways and in human subjects. (study ) airway compliance curves were constructed and compared from aoct-based measurements in volunteers with and without old during bronchoscopy. (study ) stenosis dimensions (length, calibre) were measured compared in patients with symptomatic airway stenosis using pre-procedure computed tomography (ct) and intra-procedure aoct (bland altman analysis) to determine interventional strategy. results in phantom and porcine airways, aoct measurements were accurate and reliable. mean ct-aoct diameter measurements differed by . ± . mm. ( ) airway compliance was increased in copd (n = ) relative to control (n = ) and asthma ( ) subjects, which were similar. ( ) in patients, the mean difference between ct and aoct-based stenosis measurements was . ± . mm. aoct proved more reliable when ct image quality was poor or where a delay occurred between ct and bronchoscopy. conclusions aoct provides real-time measurements of airway dimensions which are accurate and reliable and can be used for research and clinical applications. nomination ann woolcock young investigator award. support nhmrc. disclosure to declare yes. a notable feature of allergic asthma is the infiltration of mast cells into human airway smooth muscle (hasm) bundles. thus, mast cells and hasm can likely exhibit mutual functional modulation via direct cell-cell contact or through released factors. to examine this possibility, we have used a human mast cell line (hmc- ) to evaluate mast cell modulation of hasm cell function. methods hmc- cells were transfected with human fcria, and fcri expressing cells were flow sorted. the functional activity of these cells (hmc- a) was examined by measurement of released cytokines via ige/antigen stimulation. hasm cells were co-cultured with hmc- a cells, or with conditioned media derived from hmc- a cells, to examine the impact on cytokine release. methods hmc- a cells were activated by ige/antigen to release il- . the co-culture of hasm cells with hmc- a cells, induced both il- (n = , p < . ) and eotaxin (n = , p < . ) production from hasm cells and this effect was greatly amplified when hmc- a cells were antigen-activated. the effect of hmc- a co-culture could be reproduced by addition of conditioned media derived from activated hmc- a cells. a bio-plexÔ cytokine array showed release of mcp- and mip- b was strongly induced from hmc- a cells upon ige/antigen stimulation. however, treatment of hasm cells with these cytokines did not elicit il- release. conclusions our study provides further evidence that the release of soluble mediators from activated mast cells can induce cytokine production from hasm cells. further work is currently ongoing to identify the factor/s responsible for this effect. we have previously demonstrated that gp -mediated stat signaling is required for bleomycin-induced lung fibrosis in mice. to determine the role of phosphorylated stat (pstat ) in the development of human lung fibrosis we examined stat and the regulation of stat expression in lung tissue from idiopathic pulmonary fibrosis (ipf) patients. immunohistochemistry revealed nuclear localization of pstat in fibroblastic cells within fibrotic foci. suppressor of cytokine signaling- (socs ) is a known negative regulator of gp -induced stat activation. we tested the hypothesis that reduced socs expression may account for elevated pstat in cells within the fibroblastic foci of ipf lungs. rt pcr profiler analysis of the jak/stat pathway demonstrated that il- up-regulated socs mrna in both ipf and hlf. western blot analysis confirmed that socs expression was not aberrant in these cells, although a trend towards reduced socs expression was evident. our analysis identified six jak-stat pathway associated genes that were significantly altered in ipf cells following il- exposure for minutes but of those, only il- was up-regulated. examination of other known regulators of stat signaling including socs , socs , socs , socs , pias , pias and protein tyrosine phosphatase non-receptor type (ptpn ) was performed but only socs expression was reduced in ipf. in summary, dysregulation of socs does not appear to cause high stat levels in ipf tissue but the potential regulation by socs is the subject of ongoing investigations. the phosphoinositide kinase (pi k) signal transduction pathway contributes to the airway remodelling associated with asthma; however, the precise roles of the specific pi k isoforms are currently unknown. in this study, we investigated the roles of the class ia pi k isoforms p a, p b and p d in airway smooth muscle (asm) cells derived from asthmatic subjects and asm cells and lung fibroblasts from non-asthmatic subjects. methods cells were stimulated with transforming growth factorb (tgfb; ng/ml) and/or % fbs in the presence or absence of specific pi k inhibitors pik (p a), tgx (p b) or ic (p d) (all . - lm) or vehicle control (dmso). fibronectin deposition, vegf and il- secretion were measured using elisa, mitochondrial activity was assessed by mtt assay and proliferation by bromodeoxyuridine (brdu) incorporation assay. results in non-asthmatic asm cells inhibition of p a and p b decreased vegf and il- secretion and cell proliferation (n = ; p < . ), whereas in asthmatic asm cells, only inhibition of p a (n = - , p < . ) but not p b (n = - , p > . ) had an effect. furthermore, we demonstrated isoform specific roles with p a (n = , p < . ) but not p b (n = - ) or p d (n = - ) modulating fibronectin deposition in asm cells and lung fibroblasts. conclusion specific pi k isoforms have distinct roles in the regulation of inflammatory cytokines (il- ), growth factors (vegf) and extracellular matrix proteins (fibronectin) associated with airway remodelling. intrinsic differences exist in the roles of the pi k isoforms in asthmatic asm. acute respiratory distress syndrome is characterized by inflammation and fibrosis. cellular therapies potentially restore pneumocytes and reduce inflammation. aims we evaluated the role of term human umbilical cord mesenchymal stem cells derived from wharton's jelly (umscs) and human amnion epithelial cells (haecs) in treating a bleomycin-induced model of lung injury. methods cells were administered systemically into a mouse model of acute lung injury hours following intra-nasal administered bleomycin. results both haecs and umscs reduced inflammation with decreased tnf-a, il- , il- and tgf-b. collagen in the lung was significantly reduced by both umscs and haecs as a possible consequence of increased degradation by matrix metalloproteinase- (mmp- ) and down-regulation of their endogenous inhibitors the tissue inhibitors of matrix metalloproteinases (timps) - and - . umscs were detected in the lung at weeks postinjection, vs. weeks for haecs. in addition, umscs did not demonstrate lung differentiation while haecs developed an alveolar phenotype. conclusions both umscs and haecs, have anti-inflammatory properties and reduce fibrosis in lung injury but haecs adopt a lung phenotype support small grant monash university. nomination nil. background with improvement in clinical care and longer survival of patients with cystic fibrosis, pregnancy has become commonplace. however the impact of cystic fibrosis on maternal health and foetal outcomes requires ongoing review. methods a retrospective study of pregnancies from women with cystic fibrosis during the period - was performed. changes in lung function, body mass index, and development of gestational diabetes were recorded. foetal outcomes and maternal survival were examined and the influence of pre-pregnancy parameters on outcomes were evaluated. results mean age of pregnancy was . years with a mean pre-pregnancy fev of . % predicted. eleven out of twenty pregnancies had a pre-pregnancy fev < % predicted. during pregnancy, fev fell by . % (ci . - . ), but recovered to baseline within months post-partum. mothers gained a mean weight of . kg and gestational diabetes developed in . % of women. all women delivered live births apart from one therapeutic abortion. five infants were preterm and three had low birthweight for age. four mothers either died or required lung transplantation after pregnancy on follow up. fev < % predicted and body mass index < kg/m were significant predictors of foetal complications. background carrier screening for cf has been available for many years but there is no national program for population-based screening in australia. knowledge of australian cf healthcare professionals' attitudes towards carrier screening would provide useful information about how a program could be implemented. the aim of this study was to investigate the attitudes of cf respiratory physicians and cf clinic coordinators in australia towards population-based carrier screening for cf. method a purposed designed questionnaire assessing knowledge and attitudes towards cf carrier screening was distributed to respiratory physicians and cf clinic coordinators throughout australia. results there were respiratory physicians registered with the cf special interest group of tsanz and cf clinic nurses identified through the cf coordinators network. seventy-five responded, respiratory physicians ( . %) and coordinators ( %). forty-two ( %) respondents were in favour of population-based carrier screening for cf. sixty-four ( %) rated raising a child with cf as difficult/very difficult, ( %) rated the shortened life span as a significant concern and ( %) the daily treatment regimen as a significant concern. disadvantages of screening were perceived anxiety amongst carriers (n = , %) and discrimination of carriers (n = , %). respondents rated the following barriers as most important: limitations of predicting clinical outcomes (n = , %) and insufficient time and resources for providers (n = , %). fifty-four ( %) of respondents believed they had a role in the development of a cf carrier screening program. adherence to medication regimens in patients with cystic fibrosis (cf) vary substantially. direct measures of adherence using electronic monitors attached to medication bottles enable the precise recording of usage. the macrolide antibiotic, azithromycin has demonstrated clinical benefit when used in cf patients with moderate to severe impairment of lung function. aim this study compares the adherence levels of cf patients randomised to two different treatment regimens of azithromycin, measured by electronic monitoring. methods patients were prescribed the medication once ( mg) or three times ( mg) a week. data were collected over weeks using electronic monitoring devices. adherence measures were defined as: total adherence (total amount of medication taken divided by total medication prescribed) and total number of days adhered (number of days where prescribed doses were taken divided by number of days monitored). results the study recruited participants ( % male; mean age = . sd = years, mean fev % = . , sd = . ). total adherence in patients prescribed the weekly regimen (m = . , sd = . %) were significantly different compared to the three times a week regimen (m = . , sd = . %, p < . ). no significant difference was observed in total number of days adhered. fev % predicted negatively correlated with adherence to medication (total adherence r = - . , p< . , days adhered r = - . , p < . ) and to bmi (r = . ,p < . ). positive correlation was observed between age of the participant and adherence to medication (total adherence r = . , p < . , days adhered r = . , p < . ). conclusion participants adhered better to a once weekly regimen than a three times a week regimen. preclinical studies in non-human primates (nhp) are essential to estimate effectiveness and safety in developing gene transfer protocols to treat cystic fibrosis (cf) airway disease prior to clinical trials. lentiviral (lv) vectors can provide in vivo gene expression and persistence suited to long lasting cf airway correction in mice. we have begun examination of lv gene transfer in lungs of marmosets (callithrix jacchus), a small non-human primate with lung anatomy and physiology similar to humans. methods lysophosphatidylcholine (lpc, . %) pre-treatment was followed by a lv vector encoding the lacz (lv-lacz) reporter gene, pseudotyped with the vsv-g surface protein. doses were delivered into the trachea of four intubated marmosets. trachea and lungs in two animals were examined after week; blood taken daily was tested for presence of vector particles. results epithelial cell lacz gene expression was present primarily in conducting airways in a patchy distribution. a transient o desaturation was noted in some animals after lpc administration; behavioural and physiological indices were normal postoperatively. limited patches of haemorrhage and neutrophil / mononuclear cell infiltration were deemed unremarkable by a veterinary pathologist. serum p lv capsid protein levels that appeared after dosing were absent after day two. conclusions these first studies indicate lpc/lv dosing procedures are well tolerated and can induce target-cell gene expression. further histological and immunological analyses are in progress. the remaining two animals will undergo longer-term assessment of the success of lentiviral lung gene transfer. background bronchiectasis and air trapping are important features of cystic fibrosis (cf) structural lung damage, however data on disease progression in young children are lacking. aim to assess longitudinal changes in ct-detected early structural lung damage in. methods subjects included children (age range . to . years) who underwent annual ct scans, with paired scans. each ct scan consisted of three slices at endinspiration and three slices at end-expiration. the left and right upper, middle and lower zones were assessed for the presence and extent (none, less than %, more than %) of bronchiectasis and air trapping using previously described methods. infection and inflammation were assessed using bronchoalveolar lavage at the time of ct scan. results bronchiectasis was present in % of initial scans, persisting in % of subsequent scans. median extent increased from initial to subsequent scan (p = . ). previous psa infection and neutrophilic inflammation was associated with increased prevalence of bronchiectasis at subsequent scan (or = . ). air trapping was present in % of initial scans, persisting in % of subsequent scans. median extent increased from initial to subsequent scan (p = . ). infection and inflammation did not increase risk of air trapping, but air trapping was more common in girls (or = . ). bronchiectasis and air trapping commonly occurred together. the minute walk test ( mwt) and incremental shuttle walk test (iswt) are commonly used to assess functional exercise capacity, prescribe the training intensity and measure the efficacy of pulmonary rehabilitation. no studies have compared these tests in patients with non-cf bronchiectasis. aims to compare peak dyspnea and heart rate (hr), and nadir oxygen saturation (spo ) during the mwt and iswt in subjects with non-cf bronchiectasis. methods twenty-seven participants (aged ± year, fev ± %pred, fvc ± %pred) with non-cf bronchiectasis enrolled in a trial of pulmonary rehabilitation, completed two mwts and two iswts in random order. results the minute walk distance ( mwd) and the incremental shuttle walk distance (iswd) were significantly greater on the nd test (both p < . ). the mean ( % ci) increase in the mwd was m ( to m); % ( to %) and in the iswd was m ( to m); % ( to %). the greatest mwd and iswd was ± m and ± m respectively. there was a strong relationship between the mwd and iswd (r = . , p < . ). peak dyspnoea was higher for the iswt ( . ± . vs. . ± . , p = . ) but there was no difference in peak hr ( ± vs. ± % age pred maximal hr, p = . ) or nadir spo ( . vs. . %, p = . ). conclusion although peak hr was similar, the externally paced, incremental nature of the iswt may account for the higher dyspnea scores in these subjects with non-cf bronchiectasis. future research will determine the responsiveness of the iswt and mwt following pulmonary rehabilitation in this population. the six minute walk test ( mwt) is extensively used in clinical practice. however, the role of this test in people with dust-related lung disease remains unclear. aim the aims of the study were to investigate the relationships between exercise capacity measured by the mwt and the incremental peak and endurance cycle tests, the mwt and health-related quality of life, and the mwt and activity levels in people with dust-related lung disease. methods thirty male participants with asbestos related pleural disease, asbestosis or silicosis performed two mwts separated by minutes with the better of the tests used for analysis. during the rest period, participants completed the st george's respiratory questionnaire (sgrq). on a separate day, participants performed spirometry, lung volumes, dlco and a peak and endurance cycle test. participants wore an activity monitor (sense-wear pro ) for a period of seven days. results mean (sd) age of participants was ( ) there was a significant correlation between mwt distance and peak watts (r = . , p < . ) but not with endurance cycle time. there were significant correlations between the mwt and all components of the sgrq, the strongest correlation being with the 'activity' domain (r = - . , p < . ) and significant correlations between mwt and average daily steps (r = . , p = . ) and average mets (r = . , p < . ). conclusion findings suggest the mwt may be a useful measure of exercise capacity and may reflect daily activity in people with dust-related lung disease. the minute walk test ( mwt) is used to assess prognosis and evaluate exercise capacity in interstitial lung disease (ild), however the physiological load imposed by the mwt is unknown. this study compared cardiorespiratory responses to mwt and cardiopulmonary exercise testing (cpet) in ild. methods fifteen participants with ild (nine ipf), mean age (standard deviation ) years and tlco ( ) %predicted undertook cpet and mwt on the same day in random order. pulmonary oxygen uptake (vo ), ventilation (ve), carbon dioxide production (vco ), oxyhaemoglobin saturation (spo ) and heart rate were compared between the tests using a portable metabolic cart. relationships between minute walk distance ( mwd) and peak cardiorespiratory responses on cpet were evaluated using correlations. results peak vo measured during the mwt was lower than during cpet ( . ( . ) vs . ( . ) ml.kg/min, p = . ). oxygen consumption during mwt reached a mean of % of vo peak achieved on cpet ( % confidence interval - %vo peak). peak ventilation, carbon dioxide production and peak heart rate were significantly lower during mwt, but there was no difference in nadir spo ( ( )% vs. ( )% on mwt and cpet respectively, p = . ). a higher mwd was associated with a higher peak work rate (r = . , p < . ) but there were no relationships between mwd and peak cardiorespiratory responses on cpet. conclusions the mwt elicits a high but submaximal oxygen uptake in people with ild. given the poor relationship between mwd and peak cardiorespiratory responses elicited by cpet, the prognostic value of the mwt may be related to the degree of oxygen desaturation elicited by this test. results there were no differences between groups at baseline for age, gender, body mass index or respiratory function. after twelve months both groups had similar improvement in fef - %(mean . l/sec, % confidence interval . - . l/sec). there was no significant effect on fev or fvc due to either treatment allocation or time. both groups demonstrated significant improvements in all domains of both the sgrq and lcq but there was no difference between groups. conclusion the inhalation of both isotonic ( . %) and hypertonic ( %) saline improved small airways function and improved quality of life over months, however both treatments were equally effective. introduction this project aimed to develop, implement and evaluate an innovative primary care model in community pharmacy for screening, monitoring and education of people with or at risk of sleep disorders (sd). methods a randomised control trial comparing two approaches was conducted: ) risk assessment only (rao) or ) risk assessment plus overnight nasal flow monitoring using the flowwizardÒ device (ra+). the risk assessment tool collected data on lifestyle, medical conditions, medications and included validated instruments for detecting sd. twentythree pharmacies ( rao/ ra+) recruited patients during a month period. patients at significant risk for a sd were provided with information and referred to a gp. results patients were recruited (rao n = , ra+ n = ). of these, . % (rao %, ra+ %) had an increased risk of daytime sleepiness, . % (rao %, ra+ %) were at risk of significant insomnia, . % (rao %, ra+ %) at risk of obstructive sleep apnea, and . % (rao %, ra+ %) at risk of restless legs syndrome (rls). pharmacists recorded a total of interventions and patients were referred to their gp ( of screened). preliminary results showed that patients (rao, n = , ra+, n = ) have completed the follow-up questionnaire with gp referrals being taken up and seven sd diagnosed ( % of those who took up a referral, rao, n = , ra+, n = ). nine patients ( %) received a diagnosis other than a sd and patients reported still awaiting sleep specialist assessment or testing. conclusion the results of this study indicate that community pharmacy is a potential site for sd screening. support the pharmacy guild of australia, investigator initiated grants. nomination nil. conflict of interest nil. jm foster , l smith , sz bosnic-anticevich , t usherwood , sm sawyer , cs rand , hk reddel university of sydney, australia, royal childrens hospital melbourne, australia, and johns hopkins university, baltimore, usa aim to identify beliefs and behaviours associated with poor adherence which could be used to guide tailored interventions in primary care. methods patients aged ‡ years with doctor-diagnosed asthma and a current ics/laba prescription completed questionnaires on beliefs and behaviours, side-effects, asthma control (acq), and underwent spirometry. adherence with ics/laba was measured over weeks by smartinhalers which electronically recorded the time and date of each actuation. univariate and multivariate analyses of questionnaire items identified predictors of adherence. results ninety-nine of patients completed the study ( female; mean ± sd fev % predicted ± ; acq . ± . ). mean adherence was % ± (n = ). thirty one beliefs or behaviours were significantly associated with poor adherence (p < . ). factor analysis of these items identified themes: f . perceived necessity; f . safety concerns; f . acceptance of asthma chronicity and ics/laba effectiveness; f . advice from friends/family; f . motivation/routine; f . ease of use; and f . satisfaction with asthma management. regression analysis demonstrated that items in themes independently predicted poor adherence (model adj. r sq. = . ; p < . ) including 'my preventer is necessary to keep my asthma under control'(f ), 'i get side effects from my steroid inhaler'(f ), 'i think i will have asthma for a long time'(f ), 'my family/ friends tell me i should use my preventer inhaler more often'(f ), 'i have a fixed daily routine for taking my asthma medications'(f ). adherence was lower for patients who attributed dental deterioration or dry eyes to their ics, but not for hoarseness. conclusions this study identified key beliefs or behaviours associated with poor adherence which may be amenable to change in patient-specific primary care interventions. support asthma foundation nsw, glaxosmithkline (medications investigation of pulmonary embolism with ctpa is often performed despite low clinical risk and results in unnecessary exposure to radiation and radiocontrast as well as inefficient use of medical resources. risk stratification with a validated prediction tool (wells score) complements clinical decision making and rationalises the use of ctpa to appropriate patient groups. methods prospective assignment of wells score by requesting clinicians on a formal algorithm form was instituted in . all patients being investigated for pulmonary embolism were required to have the form filled prior to performance of ctpa. patients stratified low clinical risk (wells £ ) did not proceed to ctpa unless a senior physician override was applied. intermediate risk patients (wells - ) proceeded to d-dimer measurement and if above the laboratory cutoff ( . ) proceeded to imaging. all high risk patients (wells > ) proceeded to ctpa directly. ctpa outcomes, d-dimer levels, request locations and dates were recorded. data were collected from february to august . results a total of patients were investigated with ctpa in this period. patients ( %) did not have the wells score assigned but patients ( %) had complete data. ( %) request originated from the emergency department, ( %) from inpatient wards and ( %) and ( %) from icu and outpatients respectively. the prevalence of pulmonary embolism in our study population was % similar to data from wells and others. ( %) patients were stratified to low risk, ( %) to intermediate risk and ( %) to high risk. the prevalences of pulmonary embolism were %, % and % respectively in these risk groups, comparable with published data. when evaluated against the same period in , there was an absolute reduction of ( %) ctpas performed. conclusion institutional implementation of a formal clinical prediction tool into the decision making process is feasible and yields significant reduction in ctpas performed, with substantial cost savings and patient benefits. aim to conduct an rct to measure the impact of the practitioner asthma communication and education program (pace) on general practitioner (gp) management of paediatric asthma. methods gps recruited through local practice networks identified patients aged - with diagnosed asthma. gps received two hour interactive workshops. results outcome data were collected from intervention and control gps, and intervention and control families. a significantly higher percent of intervention gps than control gps reported frequently providing a written asthma action plan ( . %, p = . , nnt = . ).intervention gps reported higher rates of giving written instructions to adjust medication ( . %, p = . , nnt = . ) and of providing spacers ( . %, p = . , nnt = . ). a significantly higher percent of intervention group children had received a written asthma action plan in the last year ( . %, p = . , nnt = . ). fewer intervention group children with infrequent intermittent symptoms were using regular ics ( . %, p = . , nnt = . ). intervention gps had higher improvements in confidence ( . %, p = . ), helpfulness ( . %, p = . ) and frequency of using the taught communication strategies ( . %, p = . ). conclusion the pace program is the most robustly evaluated program of gp asthma education in australia. our results provide high level evidence that paediatric asthma management is improved by pace. pace may be useful for educating other health professionals involved in chronic disease management. support australian government department of health and ageing. nomination nil. conflict of interest nil. vincent siaw, karmen yai, anand rose department of respiratory medicine, flinders medical centre, bedford park, south australia, australia pulmonary embolism is a common cause for presentation to the emergency departments of tertiary hospitals. if undiagnosed it has a % mortality. the diagnostic algorithm includes a clinical assessment, d'dimer assays and imaging in the appropriate patient. the preferred tool in our institution for confirming a diagnosis of a pulmonary embolism is the ct pulmonary angiogram (ctpa). we decided to audit our practise of doing a clinical assessment using a standardised risk score (eg.wells score) prior to requesting a ctpa. aim to audit the use of standardised clinical risk assessments prior to requesting a ctpa when a pulmonary embolism is suspected. methods ctpa requests from the emergency department from february and march were retrieved. cases notes were screened for mention of the wells or geneva scores. individual symptoms were also studied in attempt to reconstruct the score from the notes. results of the ct pulmonary angiograms performed - requests were from the emergency department. of these requests . % ( patients) were positive studies. systematic clinical risk assessment had been used in . % ( cases). when a systematic clinical score was performed . % of the ctpa studies were positive. this was greater than when no risk score was performed ( % of ctpa studies returned positive). we aimed to compare gp and parent reports of asthma management styles from an rct of practitioner asthma communication and education (pace). methods gps recruited through local networks identified patients aged - with diagnosed asthma. intervention gps participated in two hour workshops of patient education and communication techniques. results at months, gps ( intervention, control) and parents ( intervention, control) provided data. more intervention gps ( . %) reported checking device use (vs. . %; diff = . %, p = . ). intervention parents ( . %) reported that gps checked their device use more frequently (vs. . %; diff = . %, p = . ). more intervention gps ( . %) reported providing educational messages (vs. . %; diff = . %, p = . ). however, more control parents ( . %) (vs. . %; diff = - . %, p = . ) reported receiving messages. more control gps ( . %) said they asked patients about new medication fears (vs. . %; diff = - . %, p = . ), but more intervention parents ( . %) reported being asked about this (vs. . %; diff = . %, p = . ). conclusions gp and parental reports of device use checking were consistent. reports of educational messages and communication were less consistent, though these may have been provided or used but not recognised by parents. these findings highlight that parents and their gps can have very different perceptions of some aspects of a child's asthma management. care should be taken when selecting outcome measures for clinical trials. support australian government department of health and ageing. nomination nil. conflict of interest nil. to background smoking cessation interventions in outpatient settings has been clearly demonstrated to be one of the most cost effective strategies available in reducing disease burden. given the evidence of superior benefits with over nicotine replacement therapy, we aimed to evaluate its benefit in the inpatient setting for smokers admitted with acute smoking related events. methods adult patients (n = , - years) recruited from the respiratory, cardiology, neurology, vascular and general medical wards of the queen elizabeth hospital, lyell mcewin health service and the royal adelaide hospital were randomised to receive either vt (varenicline tartrate) plus quit sa counselling (n = ) or quit sa counselling alone, (n = ). results preliminary analysis shows that after three months of follow-up, smoking abstinence was achieved by . % in the control and . % in the intervention group, (p = . ). preliminary subgroup analysis indicates that cardiac patients, (n = ) have gained the most benefit with . % obtaining continuous abstinence. conclusion whilst a beneficial smoking cessation trend is evident at three months, these are only preliminary results. our recruitment target sample size is likely to provide sufficient power to identify significant differences in abstinence rates between treatment and control groups, and permit sub-group analyses of treatment effect based upon inpatient characteristics. support nil. nomination nil. introduction cigarette smoking prevalence has been in decline in australia over many decades but prevalence remains high in lower socioeconomic groups. hospital employees span the socioeconomic spectrum but there are few data on smoking prevalence from these sites. the visibility of smoking on campus conflicts with the health message that hospitals should promote but cessation services are often not provided. the queen elizabeth hospital (tqeh) has had an ongoing stop smoking service (using cost-price nrt and counselling) provided by tej since and -yearly surveys are conducted to assess benefits and ongoing need. methods employees of three metropolitan teaching hospitals (royal adelaide -rah, flinders medical centre -fmc and tqeh) and the alice springs hospital (ash) were sent a single page questionnaire asking about smoking status and views about smoking on campus. returns were voluntary but encouraged via a small monetary prize. tqeh was surveyed thrice ( , and ) , the other hospitals were surveyed once (late / early ) . results almost all employees reported knowing smoking is a health hazard. most employees (smokers & non-smokers) at all hospitals, thought smoking in public view was unacceptable but support for a total ban was less than for suitable areas where smoking was allowed. tqeh smoking prevalence is much lower than the comparator hospitals where prevalence is similar to national prevalence ( introduction interleukin- a is a cytokine released from t helper (th ) cells which induces and mediates various pro-inflammatory responses. as a result, il- a has been linked to many immune/autoimmune related diseases but its role in copd has not been explored. in the present study we investigated whether il- a regulates cigarette smoke (cs)-induced lung inflammation. methods wild-type (wt) or mice deficient in il- a (il- a -/-) were placed in a perspex chamber and exposed to cs generated from nine cigs per day for days. in separate experiments, cs-exposed wt mice were treated with anti-il- a antibody. on the fifth day, mice were killed, the lungs lavaged with pbs and then harvested for genomic analysis. results wt mice exposed to cs for days had significantly more balf macrophages ( . ± . (sem) · ) and neutrophils ( . ± . · ) than sham-exposed mice ( . ± . · and , respectively) (n = - , p < . ). however, cs-exposed il- a -/mice had significantly fewer macrophages ( . ± . · ) and neutrophils ( . ± . · ) than cs-exposed wt mice (n = - , p < . ). macrophage and neutrophil numbers in sham-exposed il- a -/mice ( . ± . · and . ± . · ) were similar to those of sham-exposed wt mice. gene expression analysis by qpcr showed that cs-exposed il- a -/mice had markedly reduced mcp- , tnfa, il- a, il- and mmp- expression compared to cs-exposed wt mice. treatment of cs-exposed mice with anti-il- a antibody significantly reduced cs-exposed balf macrophages and neutrophils (n = , p < . ). in addition, we found that lungs of nod-scid mice deficient in t & b lymphocytes expressed il- a in response to cs. conclusions these data show that il- a regulates cs-induced lung inflammation and that targeting il- a may have therapeutic utility in inflammatory lung diseases where cs plays a role. introduction in utero exposure to tobacco constituents may contribute to respiratory health problems later in childhood. glutathione s-transferases (gsts) are important in detoxification of xenobiotics. a reduction in the mother and fetus's detoxification ability due to genetic variation in gsts could expose the fetus to higher levels of toxins. objective to investigate the interactive effects of maternal smoking during pregnancy with maternal and infant gst genotypes on airway responsiveness (ar) and lung function in infancy at , and months and longitudinally throughout the first year. methods gstt , gstp and gstm were genotyped in infants and mothers using pcr. in utero exposure to maternal smoke was evaluated by questionnaire, ar was assessed by histamine challenge and v'maxfrc was measured using the rapid thoracoabdominal compression technique. results gstt non-null in infants, mothers or both was associated with reduced ar at months and throughout the first year and increased v'maxfrc at months. maternal gstp val/val or ile/val was associated with increased v'maxfrc at months. in infants exposed to in utero smoke, gstt non-null infants, mothers or both was associated with reduced ar at month and throughout the first year and increased v'maxfrc throughout the first year. there were no significant associations with gstm . conclusion gst genes may be especially important during fetal development as they may modify, through proficient detoxification, the effects of in utero maternal smoke exposure on ar and lung function in infants. funding nhmrc. conflict of interest no. introduction there are few birth cohort studies in which frequent, contemporary measures of tobacco smoke exposure have been related to lung function and airway responsiveness in later childhood. aim to examine the effects of in utero and post natal exposure to ets on lung function and airway responsiveness at age years. methods children with a family history of asthma were recruited antenatally into a randomized trial of house dust mite avoidance and dietary modification results a total of subjects were enrolled ( indigenous australians, indonesians). in the indigenous australian setting the sgrq total score was independently associated with exacerbation frequency and lung function (% predicted fev ) whilst the symptom score was associated more strongly with ae frequency and activity score with lung function. in indonesians with ptb the total sgrq score correlated with treatment response over time as well as lung function (% predicted fvc), exercise tolerance ( mwt) and the extent of involvement on cxr. conclusions in an indigenous australian and indonesia, setting respiratory-related qol using a modified sgrq correlates with lung function, exercise performance, disease activity and treatment. these tools should be a useful addition to evaluating interventions in this setting. background epithelial mesenchymal transition is a process in which airway epithelial cells disaggregate and then migrate through the reticular basement membrane (rbm) into the lamina propria to become myofibroblasts. the aim of this study was to identify if emt is active in the airways in smokers, and whether relevant to copd. methods endobronchial biopsies (ebb) from current smokers with copd (cs; n = ) and ex-smokers with copd (es; n = ), smokers with normal lung function (ns; n = ) and never-smoking controls (nc; n = ) were stained for emt markers, s a a fibroblast protein, epidermal growth factor receptor (egfr) and matrix metalloproteinase- (mmp- ). computer-assisted image analysis was used to quantify the expression of markers in biopsies and slides were counted by an observer blinded to subject and diagnosis. we used non-parametric statistics. results compared to nc, there was significant fragmentation of the rbm in cs, es and ns groups (p < . ), which was especially marked in cs and was positively related to pack years in copd subjects (r = . , p = . ). cs, ns and es demonstrated increased staining for: basal epithelial s a (p < . ), epithelial egfr (p < . ) and mmp- (p < . ) for cells in rbm 'clefts', and rbm cell s a (p < . ) compared to nc. there was increased rbm cell s a staining in cs vs. es and ns (p < . ). basal epithelial cells staining for s a correlated negatively with airflow limitation (r = - . , p = . ) in cs, and dual staining revealed that basal s a positive cells co-stained with vimentin (an additional mesenchymal marker). conclusions our findings suggest that emt is active in smokers, and is most evident in current smokers with copd, suggesting a role in copd pathogenesis. pulmonary emphysema is a major component of the chronic obstructive pulmonary disease (copd), and also predisposes affected individuals to lung cancer. emphysema can be a familial or acquired disease, with the great variation in development of disease in atrisk populations reflecting the influence of other susceptibility determinants. in this regard, the il- cytokine family has been linked with emphysema pathogenesis. however, studies into the definitive mechanisms by which these cytokines cause emphysema have been hampered by the absence of informative animal disease models. to address this issue, we have utilized a sophisticated animal model (gp f/f mice) with a subtle mutation in the il- cytokine family receptor gp which, as a consequence of abolishing binding of both shp and socs , simultaneously mediates stat / hyper-activation and impaired shp -mapk and -pi k activation. the gp f/f mice spontaneously develop emphysema by months of age characterized by increased static compliance. lung stereology has further confirmed emphysematous changes, revealing increases in volumes of airspace and lung. among the il- cytokine family, il- expression is significantly up-regulated in the lungs of gp f/f mice, and genetic ablation of il- in gp f/f mice prevents the development of emphysema. notably, an increased apoptosis of alveolar cells has been identified as the underlying cellular mechanism associated with the emphysema in gp f/f mice. collectively, our observations identify for the first time that deregulated gp signalling by il- cause's alveolar cells to undergo apoptosis, which coincide with the pathogenesis of emphysema. furthermore, this mouse model has the enormous potential to allow us to explore common mechanistic links between copd and lung cancer. supported by the nhmrc, australia. conflict of interest no. introduction despite smoking cessation, susceptible copd patients continue to decline in lung function. understanding biological pathways and their gene ontologies would help to develop better treatments and diagnostic methods for copd. the aims of this study were to identify gene ontologies associated with mild and moderate copd by (i) profiling mrna and (ii) mirnas and their predicted targets. methods profiling was performed on total rna extracted from lung tissue of copd patients undergoing resection for lung cancer. microarray platforms (operon v and agilent g v ) were used to characterise mrnas and mirnas respectively. analysis was performed using brb array tools v . and gsea. results the patients were caucasian former smokers with mean (sd) age ( ), fev ( ) % predicted, kco ( )% predicted and pack years ( ). we identified authentic candidate genes (p < . ) that predicted copd progression with % accuracy in in-house and public datasets. genes involved in cell cycle, proliferation, development and growth were identified. increasing expression of mir- c, a candidate mirna for emphysema progression, on lung fibroblast and epithelial cells downregulated predicted mrna targets with potential biological role in copd. conclusions we have identified multiple gene ontologies associated with copd severity. these targets have promising biological roles in copd and can be further developed as biomarkers or therapeutic targets. cigarette smoke (cs)-induced oxidative stress is known to drive the pathogenesis of copd. the antioxidant glutathione (gsh) is essential for efficient macrophage functions including phagocytosis of apoptotic cells (efferocytosis) which we have shown to be defective in copd. gsh synthesis is controlled by a cd /xct cysteine transporter pathway. cd is also a ligand for galectin- (gal- ), a lectin important for macrophage phagocytosis and gsh synthesis. we hypothesised that targeting oxidative stress in copd by increasing gsh would increase gal- levels and improve efferocytosis. we investigated (a) ex vivo: oxidative stress markers ( -isoprostane; mmp ), gsh and gal- in bal from controls and current-and ex-smoker copd subjects (b) in vitro: the effects of cs on alveolar macrophage production of gal- and gsh (c) in vivo: the effects of treatment with a gsh precursor, procysteine, on efferocytosis, gal- and gsh in smokeexposed mice. procysteine was administered in semi-solid mouse feed. efferocytosis was investigated in lung tissue and bal macrophages. -isoprostane and mmp were significantly increased in bal in current-and ex-smokers with copd. gsh and gal- were decreased in copd (gal- , ng/ml: current . ± . , ex-smoker . ± . vs. controls . ± . ). in vitro cs treatment decreased gal- expression. in vivo, cs caused decreased efferocytosis that was significantly improved by procysteine (control; smokeexposed; procysteine + smoke-exposed: bal . %; . %; . %; tissue . %; . %; . %). gsh and gal- were also significantly increased by procysteine (gal- , ng/ml: control . ± . ; smoke-exposed . ± . ; procysteine + smoke-exposed . ± . ng/ml). targeting oxidative stress is a viable approach to improve macrophage dysfunction in copd. support nhmrc, arc. introduction our knowledge about the effects of inhaled corticosteroids (ics) on airway remodelling in chronic obstructive pulmonary disease (copd) is limited. we have previously reported that in bronchial biopsies (bb) from copd subjects the reticular basement membrane (rbm) is fragmented and hypervascular. in this study we have examined the effects of ics on these airway remodelling changes in copd. methods in a double blind and randomised study we compared the effects of months of fluticasone propionate (fp, . mg/twice daily) with placebo. bb were stained with collagen iv antibody to mark vessel endothelial basement membrane. the length of rbm splits and the number and area of vessels in the rbm were compared before and after treatment. results copd subjects were randomized : to receive either fp (n = ) or placebo (n = ). there were no differences between the groups before treatment. introduction copd is a complex disease characterised by fixed airflow obstruction and neutrophilic airway inflammation. markers of systemic inflammation such as serum amyloid a (saa) are elevated in copd. however, little is known about the relationship between airway and systemic inflammation. this study tested the hypothesis that systemic inflammation is associated with airway neutrophils in copd. methods participants with copd (n = , > years, with fev /fvc < and fev % predicted < ) and healthy controls (hc; with normal lung function n = > years) underwent clinical assessment, spirometry, blood collection for saa, il- and crp and sputum induction. sputum was processed for differential cell count and mediators. results airway proportions of neutrophils and eosinophils, levels of il- , total mmp- and gene expression of il- were increased in participants with copd. serum il- (median q -q ; ( . ( . - . )) vs. asbestos-related lung cancers (arlc) account for - % of all lung cancer, and are difficult to distinguish from non-asbestos related tumours (narlc) by clinical and histological criteria. we hypothesised that whole genome array comparative genomic hybridization (acgh) profiling could identify regions of gain and loss common and specific to asbestos-related lung cancer. methods the acgh profiling by agilent cgh b arrays was performed on primary non-small cell lung cancers obtained from the prince charles hospital (tpch) lung tumour bank. lung cancers occurring in individuals with >= asbestos bodies/gram wet weight (ab/gww) of lung tissue were defined arlc and individuals with ab/gww were defined narlc. genome breakpoints were called using the circular binary segmentation algorithm implemented in dnacopy. recurrent regions of amplification and deletion were identified using the genomic identification of significant targets in cancer (gistic) algorithm developed by the broad institute, controlling for false discovery rates (q < . ). results gistic identified recurrent copy number gains in q and narlc at q < . but none for arlc at the same threshold. to introduction the relationship between asbestos exposure and asbestos related diseases (ard) such as asbestosis, lung cancer and mesothelioma are well established. less is known about asbestos exposure and non-ard respiratory diseases. aim to investigate respiratory symptoms and lung function in former workers and residents from wittenoom who have not developed an ard. methods an annual review, which includes lung function, plain chest x-ray and respiratory questionnaire, is conducted on a cohort of ex-workers and ex-residents from wittenoom. only those who had been reviewed within the previous years and had not developed an ard, nor had plain chest radiographic evidence of asbestosis, were included in the analyses. the prevalence of respiratory symptoms was determined and standardised lung function z-scores calculated. predictors of symptoms and lung function were assessed using both multiple logistic and linear regression. results questionnaire data was available for subjects ( women, ex-workers; mean age . ± . years), while acceptable lung function data was available for subjects ( women, ex-workers). the prevalence of reported symptoms ranged between and % for wheeze, cough, sputum, shortness of breath and bronchitis. pack years of smoking and/or being an ex-worker were the main risk factors for symptoms. standardised lung function scores ( %ci) for the total group were - . (- . -- . ), - . (- . -- . ) and . ( . - . ) for fev, fvc and fev/fvc respectively. both pack-years and cumulative asbestos exposure were independently associated with reduced fev and fvc. conclusions people previously exposed to asbestos, particularly ex-workers, have high rates of respiratory symptoms which are mostly related to smoking. reduced lung function in the cohort was associated with both smoking and cumulative asbestos exposure. conflict of interest none. introduction malignant mesothelioma (mm) is an aggressive cancer with a very poor prognosis. interactions of the components of the extracellular matrix (ecm) are now known to be important for the growth and regulation of cancer cells. tgfb is an important regulator of the ecm and in particular collagen. previous data in our laboratory has shown that blocking tgfb signaling by using tgfb antibodies inhibits collagen production and mm growth. aim to determine the signaling pathways downstream of tgfb that are important in the regulation of collagen expression in mm. methods components of the tgfb pathway were inhibited by use of chemical inhibitors and overexpression of the endogenous inhibitor smad in control and mm cell lines. collagen levels were measured by realtime pcr. results collagen regulation is thought to occur through the classic smad / signaling pathway. our data show that smad overexpression inhibits tgfb-induced collagen production in normal mesothelial cells and the mesothelial cell line met- a but not in the mm cell lines investigated. therefore, the smad / pathway for collagen regulation appears to be altered in malignant mesothelioma. it was shown that smad / are expressed, phosphorylated and activated by tgfb in the mm cell lines. our results indicate that nuclear import of smad , which is responsible for the nuclear import of smad / , is altered in mm. aim to characterise impedance variability at hz in asthma and its relationship to asthma severity. methods a school-based cohort of non-asthmatic children, aged (mean (sd)) . ( . ) years (uptech feasibility study) were tested on two occasions weeks apart. an asthma camp cohort of asthmatics, aged . ( . ) years, were tested daily for days. mean resistance (rrs ) and reactance (xrs ) of at least three technically acceptable one minute recordings were reported. medications were not withheld. variability was assessed by intraclass correlation coefficient (icc) and within-subject standard deviation (sd w ) using first and last testing day data, and all days of data for sd w severity comparison amongst asthmatics. results repeat fot measures at hz were obtained in / non-asthmatic children. mean (sd) rrs and xrs was . ( . ) and - . ( . ) for the uptech cohort, and . ( . ) and - . ( . ) cmh o/l/s in the asthma camp cohort respectively. rrs variability was increased in asthmatics. rrs variability tended to be higher in persistent vs. intermittent asthmatics but did not reach statistical significance (p = . ). non asthmatic (n = ) introduction our cochrane review examining the efficacy of using feno to tailor the dose of inhaled corticosteroid showed that feno cannot be routinely recommended for clinical practice at this stage and remains uncertain. however all the studies used a single feno cut-off. in this rct we determined if asthma monitoring using feno (using two different cut-offs dependent on atopy) is better than control (symptoms and fev ) in preventing asthma exacerbations in children on inhaled corticosteroids. methods over -months, children underwent spirometry, feno, qol and asthma/ cough diary during every visit. treatment for asthma was adjusted according to pre-determined criteria taking into account atopy status and dependent on allocation group (feno or control). results about children were randomised-feno group (n = , median age . , iqr . ), or control group (n = , median age . , iqr . ). significantly fewer children in the feno group had asthma exacerbations compared to the control group ( vs. ; p = . ) over -months. number needed to treat (nnt) to prevent one child from having any exacerbation in months = ( %ci , ). parental qol improved in feno group at final visit in comparison to the qol in control group (p = . ). fev increased in both groups over the duration of the study but there was no difference between the groups when measured at baseline (p = . ) and at final (p = . ). conclusion tailoring of asthma medications in accordance to feno levels (compared to usual management), taking into account atopy status, reduces asthma exacerbations and improves asthma qol. however both strategies equally improved fev . background inhaled corticosteroids have a modest effect on improving symptom control in preschool asthmatic children. delivery of inhaled steroids with pmdi-spacers are influenced by children's proficiency in spacer technique, and adherence to prescribed medication. aim to investigate the influence of an incentive spacer (funhaler), on spacer technique, adherence to treatment, and asthma control in preschool asthmatic children. methods about children aged - years, and being prescribed regular inhaled steroids in the community were randomised to receive regular inhaled fluticasone through either an aerochamber plus Ò , of a funhaler Ò . subjects were followed up three monthly for a year. proficiency in spacer technique was measured at each visit by measuring the amount of salbutamol inhaled from spacer onto a filter interposed between subject and spacer. adherence was monitored by smartinhaler Ò electronic devices. symptoms were recorded on diary cards for a week before each study visit. results there was no difference between the funhaler group and the aerochamber group in terms of adherence to medication or measures of asthma control (p > . ). spacer technique was significantly better in the funhaler group in subjects younger than years of age at time of randomisation (p < . ). there was large inter subject variation in drug dose inhaled on filter, ranging from - % (drug dose recovered from filter, as percentage of total dose recovered), and mean adherence over each month period ranging from - %. discussion the funhaler Ò does not improve clinical outcome, but improves spacer technique in children younger than years of age. the large variability in adherence and drug delivery should encourage both efforts to improve adherence, and efforts to standardise inhaled drug delivery in preschool children. - and - . about % of ob cases were notified within year of arrival. of the australianborn cases were close household contacts of an adult tb case. about cases had culture confirmed disease ( fully sensitive to first line drugs, one multidrug resistant). % had pulmonary and % had lymph node tb. about cases completed the treatment, two were lost to follow-up and one died. compared to adult tb cases, children were more likely to be refugees (or . (ci . - . )), diagnosed on contact screening (or . ( . - . )), have lymphatic tb (or . (ci . - . )), and less likely to be culture-confirmed (or . (ci . -. )). the png child visitors' cases diagnosed in queensland had a higher level of severe and culture-confirmed disease. conclusion queensland has a very low burden of childhood tb, indicating low levels of tb transmission in the community. hrgm children, especially refugees, will remain at risk due to infection acquired overseas. contact screening is an important method of diagnosing early tb, and refugee screening and preventive treatment may play a role in protecting this group. funding support nil. conflicts of interest nil. introduction in response to injury, normal and efficient epithelial repair is essential in order to maintain barrier integrity and immune function. however, aberrant repair has been suggested as a contributor to disease progression in asthma. many studies have only included subjects with atopic asthma and thus any intrinsic epithelial abnormality common to all asthmatic phenotypes is difficult to isolate. this study aimed to assess whether epithelial repair is dysregulated in asthmatic subjects and if this is common to the disease or is phenotype specific. the regulatory mechanisms promoting the cellular proliferation and migratory aspects of the repair process were also assessed. methods paediatric airway epithelial cells (paec) of atopic and non-atopic healthy and asthmatic subjects were isolated by non-bronchoscopic bronchial brushings. culture monolayers were wounded using an in-house wounding device, and the percentage of wound closure determined daily. proliferation and migration were also assessed over the course of repair using western blot. results paecs from healthy non-atopic (paec hna ) and healthy atopic (paec ha ) subjects successfully achieved full wound closure between - days. in contrast, atopic asthmatic (paec aa ) and non atopic asthmatic (paec naa ) subjects failed to fully repair and only achieved % wound closure by days. protein analysis showed a -fold increase in proliferation and -fold increase in migratory markers during repair in paec hna . however, reduced proliferation and no migration activity were seen in paec aa. conclusion atopic and non-atopic asthmatic epithelial cells possess dysfunctional repair profiles in response to mechanical wounding. results suggest dysregulated repair is an intrinsic epithelial abnormality in asthma and this appears to be independent of phenotypic criteria or atopy. introduction refractory chronic cough is associated with increased cough sensitivity. speech pathology intervention has been shown to be an effective intervention for refractory cough but the mechanism behind the improvement is not known. this study provides objective measures of the mechanism and the number of treatments required to effect a response. methods adults with chronic cough (n = ) were assessed before, during and after speech pathology intervention. the primary outcome measures were capsaicin cough reflex sensitivity, automated cough frequency detection and cough-related quality of life. results participants responded to the treatment with a significant improvement in coughrelated quality of life, p = . , cough reflex sensitivity, c : mean ± sd . ± . vs. c : . ± . lmol/l, p = . , cough frequency cf: . ± . vs. ± . coughs/hr, p = . , cough threshold ct: . ± . vs. . ± . lmol/l, p = . , and urge-to-cough utc: median (iqr), ( ) vs. ( ), p = . . conclusion speech pathology management is an effective treatment for refractory chronic cough. the mechanism behind the improvement is due to reduced laryngeal irritation which results in decreased cough sensitivity, improvement in cough symptoms, laryngeal symptoms, and cough quality of life. introduction airway hyperresponsiveness (ahr) is a characteristic feature of asthma. in young asthmatics, severity of ahr is related to exhaled nitric oxide (eno), a marker of eosinophilic airway inflammation, and ventilation heterogeneity in the conducting airways (scond). with increasing age, eosinophilic inflammation decreases and ventilation heterogeneity in the very peripheral, acinar, airways worsens. aim to determine if the predictors of ahr differ in young and older asthmatics. methods about young ( - ) and older ( - ) asthmatic subjects underwent baseline spirometry, body plethysmography, eno, multiple breath nitrogen washout (mbnw), and methacholine (mch) challenge. ahr was expressed as dose response slope (drs = %fall fev /lmol mch). ventilation heterogeneity of the conducting (scond) and acinar (sacin) airways were calculated from the mbnw. predictors of ahr in each group were determined by multiple linear regression. results compared to younger asthmatics, older asthmatics had lower values of eno, less severe ahr, worse acinar heterogeneity; however there were no differences in scond values. in younger asthmatics, ahr was predicted by fev /fvc (partial r = . ), eno (partial r = . ) and scond (partial r = . ) (overall r = . , p < . ). in older asthmatics, ahr was predicted by rv % predicted (partial r = . ), sacin (partial r = . ) and fev % predicted (partial r = . ) (overall r = . , p < . ). conclusions the predictors of ahr are different in young and old asthmatics. in older asthmatics, eno is not a significant predictor of ahr, which may reflect the changing inflammatory profile associated with aging. the association between ahr and both rv and sacin suggests that ahr in older asthmatics is determined by abnormalities in very peripheral airways. introduction in this systematic review and meta-analysis, we sought to establish if maternal asthma is associated with an increased risk of adverse perinatal outcomes associated with size at birth and timing of birth. methods electronic databases were searched for the following terms: (asthma or wheeze) and (pregnan* or perinat* or obstet*). cohort studies published between and march were considered for inclusion. articles were identified, and publications involving , , subjects met the inclusion criteria, by reporting at least one perinatal outcome in pregnant women with and without asthma. meta-analysis was conducted with subgroup analyses by study design and active asthma management. results maternal asthma was associated with an increased risk of low birth weight (relative risk [rr] . , % confidence interval [ci] . , . ), small for gestational age (sga, rr . , ci . , . ), very sga (rr . , ci . , . ), significantly reduced mean birth weight (weighted mean difference - g, ci - , - g), and reduced risk of high birth weight (rr . , ci . , . ). maternal asthma was associated with an increased risk of preterm labor (rr . , ci . , . ), early preterm labor (rr . , ci . , . ) and preterm delivery (rr . , ci . , . ). the risk for preterm labor and delivery was reduced to a non-significant level in those studies reporting active management of asthma during pregnancy (rr . , ci . , . ; rr . , ci . , . ). conclusion pregnant women with asthma are at increased risk of perinatal complications which affect the baby's size and timing at birth. active asthma management may reduce the risk of preterm labor and delivery. with threats of new pandemic strains of influenza a virus and resistance to anti-virals there is a need for novel therapeutics that reduce viral replication and lung pathology. the pathology arising from pandemic influenza is due to an excessive host response characterised by a rapid, massive infiltration of inflammatory cells of the innate immune system into the airways leading to excessive reactive oxygen species (ros) production. thus, we investigated the primary enzymatic source of inflammatory cell ros, nox -containing nadph oxidase, as a novel target against lung inflammation and pathology caused by influenza a viruses of varying virulence. wt and nox -/--mice were pfu/mouse) or high virulence following infection with x- , lungs of nox -/-mice displayed a significant reduction in viral titre (~ - %), macrophages, peribronchial inflammation and mcp- compared to virusinfected wt mice. lung levels of il- b were approximately -fold higher in nox -/-mice. balf macrophages, neutrophils, and t lymphocytes of nox -/-mice produced minimal superoxide compared to controls. the magnitude of balf and spleen influenza-specific dbnp + and dbpa + cd + t cells were similar in wt and nox -/-mice indicating that the major mechanisms of the adaptive immune response that effectively clear influenza a virus are preserved in nox -/-mice. in vivo administration of the nox inhibitor apocynin ( mg/kg/day) significantly suppressed viral titre, airways inflammation and inflammatory cell superoxide following infection with x- or pr/ strains. in conclusion, nox inhibition should be considered for seasonal and pandemic control of the mortality/ morbidity induced by influenza a virus, irrespective of the strain department of respiratory medicine australia, cooperative research centre for asthma and airways, new south wales, australia, and department of respiratory medicine therefore the aim, of this study was to examine the impact of a standard, -week exercise-based pr program on pal. methods about subjects ( ( ) years) with copd (fev % predicted = ( )) completed pr where they undertook twice weekly exercise classes consisting of one hour of upper and lower limb strengthening exercise and aerobic exercise. pal was estimated using a multi-sensor device (sensewear, healthware bodymedia) worn for a day period. an index of pal was derived by dividing total daily energy expenditure in metabolic equivalents (mets) by whole night sleeping energy expenditure (average of nights sleeping). pal was measured in the week immediately prior and in the immediately following pr. results despite a significant increase in six minute walk distance ( mwd), pr resulted in no change in pal copd patients failed to increase their pal. while changes in pal may take longer to elicit i.e. the change in pal may be delayed following pr, it is possible that the current focus of pr on increasing outcomes such as mwd may be too narrow to elicit changes in pal little is known regarding the use of acts in patients admitted with aecopd in australia. this survey aimed to identify current practice and opinion of australian hospital physiotherapists concerning acts. methods paper-based surveys were distributed to physiotherapists of 'large' and 'principal referral' australian public hospitals (identified via a government health report). a response rate of % (n = hospitals) yielded surveys for analysis. results most physiotherapists ( %) prescribe acts for - % of patients with ae-copd, with % of act treatments lasting - minutes. the techniques most frequently used for airway clearance were physical exercise ( %) and the active cycle of breathing technique ( %). the main influences on choice of act were precautions or contraindications to individual techniques ( %) and the degree of patient dyspnoea ( %). many physiotherapists ( %) prescribe acts with the aim of enhancing a patient's recovery from aecopd and % perceive airway clearance to be fairly or very important to the overall management of aecopd. there was mixed awareness of the evidence for acts in aecopd, with % of physiotherapists citing it as supportive conclusion australian physiotherapists frequently use acts for patients with aecopd and perceive their role to be important. physical exercise is the present modality of choice to achieve airway clearance acknowledgements nh&mrc, cure cf foundation sa. conflict of interest no. aim to examine the health outcomes of , children first exposed to blue asbestos at wittenoom when they were less than years of age. methods standardised mortality ratios (smr's) calculated to compare wittenoom children's mortality with the western australian population. results about , females and , males were children at wittenoom, mean age of arrival years (sd years); ( %) were born there or moved there soon after birth. median duration of residence was months (iqr - months). there were deaths ( females and males) between and end of . deaths were from malignant mesothelioma ( % of all deaths - females, males, - pleural, peritoneal). among males, there was excess mortality from all causes (smr = . ), all cancers (smr = . ), mm (smr = ), accidents, injuries and poisonings (smr = . ) and circulatory disease (smr = . ). mortality from suicide and transport accidents were also in excess but not statistically significantly increased. among females there was excess mortality from all causes (smr = . ), and all cancers (smr = . ) and mm (smr = ). conclusion former children of wittenoom experience high cancer mortality. support nhmrc. nomination nil. conflict of interest nil. introduction blue asbestos (crocidolite) was mined and milled at wittenoom between and . tailings from the mine were distributed and used extensively throughout the town. exposure to children also occurred from the laundering of workers clothes at home. earlier work has shown a lower risk of malignant mesothelioma (mm) in children from wittenoom than in those exposed to blue asbestos as adults.a case report of a year old ex-forestry worker with an pack year smoking history is presented. he was referred with two distinct periods of hemoptysis, one months prior to referral for which he declined investigation or follow up, and another three weeks prior to referral. on each occasion, he described two tablespoons of hemoptysis daily lasting approximately month. he lives on an acreage at mt kilcoy, km north of brisbane, with his wife, one goat, three dogs, one cat, deer and wild birds. emphysema manifesting as gradually worsening exertional dyspnoea with wheeze, had been diagnosed years ago by his local doctor. he described symptoms of obstructive sleep apnoea including witnessed apnoea, choking arousals and loud snoring. he also reported intermittent diarrhoea for years but denied weight loss or rectal blood or mucous. a ct chest showed multiple bilateral nodules of varying size the largest being . cm, and multiple low density liver lesions. the provisional diagnosis was metastatic colorectal cancer. bronchoscopy with ebus guidance did not yield the diagnosis which was eventually made by trans-thoracic needle aspiration without complication. echinococcus serology performed post procedure was > , consistent with echinococcus infection. this case of echinococcus disease is presented and the vectors discussed. echinococcus disease was previously prevalent in australia and new zealand, with a reduction in incidence from improved animal husbandry. with an increasing deer population in south east queensland and subsequent rising human contact, clinical awareness is necessary to avoid potential complications from biopsy and ensure cases are promptly treated rather than mistakenly diagnosed as incurable disease. introduction community-acquired pneumonia (cap) is a leading cause of mortality, morbidity and hospital admission places strain on our healthcare system. procalcitonin (pct) is a biomarker of bacterial infection which may help gauge the severity and prognosis of patients with cap. aim to examine the role of pct measurement in reducing hospital admissions, length of stay (los), and antibiotic (ab) usage in patients with cap. methods prospective, single-blinded, externally controlled study of consenting adult patients admitted with cap. pct levels were obtained on day and day (if indicated). the investigator evaluated clinical parameters and the pct values to determine the timing of oral ab switch and discharge. this process was used to compare with standard practice but was not actually implemented for the purpose of this study. results sixty patients were included in the study. the mean age was . ± . y and . % were male. the average psi was ± (class iv) and the median curb- was . the mean los for this cohort was . ± . d and the calculated los using pct guidance pathway was . ± . d. (p = . ) a multivariate analysis will be presented. conclusions our study supports the hypothesis that the incorporation of pct levels can reduce the requirement for hospital admission and los in patients with cap. a randomised prospective clinical trial is planned to help clarify these findings. support nil. introduction children in the highlands of papua new guinea (png) suffer on average . acute lower respiratory infections (alris) before age months, / of which are moderate or severe. while streptococcus pneumoniae and haemophilus influenzae are the primary bacterial cause of alri in png, the role of viruses in the aetiology of alri is uncertain. aim determine identification rates of respiratory viruses in nasal samples collected from children with moderate/severe alri and healthy children aged < months in png. methods as part of a neonatal pneumococcal conjugate vaccine trial in the png highlands, we collected pernasal swabs from children with moderate/severe alri (n = ) and at routine follow-up (n = ). rt-pcr methods were used to identify a broad range of respiratory viruses. the frequency of viral detection was compared between groups of samples collected during an alri and routinely using chi-square analysis. results several viruses were detected more frequently in alri than routine samples: adenoviruses . / . (% of alri samples positive/% of routine samples positive) p = . , influenza viruses . / . p = . and respiratory syncytial virus (rsv) . / . p = . . human metapneumovirus and parainfluenza viruses were detected in four and three samples, with no difference between groups. human coronaviruses and human rhinoviruses (hrv) were less commonly detected in alri than in routine samples ( . / . p = . and . / . p = . , respectively). a total of different hrv strains were identified. conclusion in young children in png, viral identification rates are high, with rsv, adenoviruses and influenza viruses associated with moderate/severe alri and a large amount of genetic diversity of rhinoviruses in both sick and healthy children. introduction to increase the documentation and documented provision of an electronic asthma action plan (e-aap) to children discharged with asthma from the emergency department (ed) at chw. methods an electronic aap (e-aap) was introduced in april by a multidisciplinary team comprising representatives. at chw, aaps were available to be printed off by the intranet. to be entered into the electronic medical record (emr), the medical officer had to photocopy the completed plan which would then be scanned into the emr. evidence suggested that busy doctors, particularly in the ed, were either not providing patients with aaps on discharge or not photocopying them for the medical record. the evaluation of the e-aap consisted of a review of the documented provision of asthma action plans in the hospital wide emr (powerchart) for a year pre & post the introduction of the e-aap, a review of patients discharged from the ed with a diagnosis of asthma for similar six month periods pre and post intervention and a medical staff satisfaction survey. results the total number of plans recorded in emr increased by %, from - to - . the number of plans recorded for ed discharges increased significantly from % to % [p < . ]. the number of patients recorded as leaving the ed with a plan increased significantly from % to % (p < . ). the use of the e-aap in the ed is now standard of care and this is also being adopted hospital wide as more staff became familiar with its usefulness.conclusion the e-aap significantly increased the number of recorded aaps and patients discharged with a recorded aap. support nil. nomination asthma/allergy. conflict of interest no. lisa wood , , manohar garg , amber wood , , peter gibson , centre for asthma and respiratory diseases, university of newcastle, new south wales, australia, and nutraceuticals research group, university of newcastle, new south wales, australiaintroduction dietary fat activates innate immune responses, leading to an increase in systemic inflammation. however, the effect of dietary fat on airway inflammation has not been investigated. we hypothesised that a high fat intake may lead to increased airway neutrophilia in asthma. the aim of this study was to examine the effect of a high fat versus low fat food challenge on airway inflammation in asthma. methods non-obese subjects with asthma were randomized to receive a high fat/ high energy (hf) (n = ) or low fat/ low energy (lf) (n = ) food challenge. obese subjects also received a hf challenge. subjects on the hf challenge consumed a meal containing kj, including % of energy ( g) from fat. subjects on the lf challenge consumed a meal containing kj, including % of energy ( g) from fat. at baseline, hypertonic saline challenge and clinical assessment were performed. induced sputum samples were collected at baseline and at hours. airway inflammatory markers included induced sputum total and differential cell counts, il- and neutrophil elastase, measured by commercial assay. tlr mrna expression from sputum cells was measured using rt-pcr. results at hours after the food challenge, subjects on the hf challenge, had a significantly higher increase in %sputum neutrophils ( . ( . (sem)) % vs. . ( . ) %, p = . ) and higher fold increase in tlr mrna expression ( . ( . - . (iqr)) vs. . ( . - . ), p = . ), compared to the lf challenge. subjects on the hf challenge also had an impaired bronchodilator response, with a lower increase in fev /fvc% at hours compared to the lf challenge ( . (- . - . (iqr)) % vs. . ( . - . ) %, p = . ). there were no differences in the responses of obese vs. non-obese asthmatics to the hf challenge. conclusions a high fat/ high energy challenge causes an increase in airway inflammation and suppresses bronchodilator response in asthma. strategies aimed at reducing dietary fat intake may be useful in reducing inflammation in asthma. support nhmrc project grant. introduction longitudinal fev data in children with non-cystic fibrosis (non-cf) bronchiectasis is contradictory and there is no multi-factor data on the evolution of lung function and growth in this group. we longitudinally reviewed lung function and growth in children with non-cf bronchiectasis and explored biologically plausible factors associated with changes in these parameters over time.methods fifty-two children with ‡ years of lung function data were retrospectively reviewed. changes in annual anthropometry and spirometry at year- and year- from baseline were analysed. the impact of gender, age, aetiology, baseline fev , exacerbation frequency, radiological extent and period of diagnosis was evaluated. results over years, the group mean fef - %predicted and bmi z-score improved by . (p = . , %ci . - . ) and . (p = . , %ci . - . ) per annum, respectively. fev %predicted, fvc %predicted and height z-score all showed non-significant improvement. over years, there was improvement in fvc %predicted (slope . , p = . ) annually but only minor improvement in other parameters. children with immunodeficiency and those with low baseline fev had significantly lower bmi at diagnosis. frequency of hospitalized exacerbation and low baseline fev were the only significant predictors of change in fev over years. decline in fev %predicted was large (but nonsignificant) for each additional year in age of diagnosis. conclusions spirometric and anthropometric parameters in children with non-cf bronchiectasis remain stable over - year follow-up period once appropriate therapy is instituted. severe exacerbations result in accelerated lung function decline. increased medical cognizance of children with chronic moist cough is needed for early diagnosis, better management and improving overall outcome in bronchiectasis. introduction it is well established that many survivors of very low birth weight (vlbw; < g at birth) have impaired lung function. the aim of this study was to determine whether abnormal lung function at years of age is established in childhood. a second aim was to see if abnormal lung function at years of age tracks through the period of normal lung development to predict impaired maximal lung function and may be a precursor to copd in adult years. methods a cohort of vlbw (n = ) and normal birth weight (nbw; > g at birth; n = ) has been followed for years. very low birthweight participants completed spirometry and lung volumes at , , , and years of age and nbw at , and years of age. restricted maximum likelihood modeling was used for longitudinal fev z-score as it allows for analysis of data from different time points that are not necessarily evenly spaced, without being affected by missing data. results about vlbw children completed lung function testing at years of age, ( . %) had abnormal fev z-scores (defined as > sd's below the mean). vlbw survivors showed minimal 'catch-up' in fev z-score over the years of the study; those without (bronchopulmonary dysplasia) fev improved . (p = . ) z-scores, those with bpd fev improved . (p = . ) z-scores. vlbw with bpd survivors did not return to within normal limits. conclusions the reduced lung function in adult survivors of low birth weight is established in early childhood. while there is some improvement in growth of those with abnormal fev z-scores in early childhood, those with bpd remain below two sd's from the mean, and at the age of have a reduced peak lung function. introduction bronchopulmonary dysplasia (bpd) is a common complication of preterm birth. although there is evidence that individuals with a history of bpd have respiratory abnormalities in childhood, there remains a paucity of evidence regarding the outcome of the disease in adulthood. in a pilot study we recently described high resolution computed tomography (hrct) appearances of emphysema in young adults with a history of bpd. aims to describe the structural pulmonary sequelae of bronchopulmonary dysplasia in adulthood and to evaluate a scoring system originally designed for paediatric subjects. methods about adult survivors of bpd underwent hrct of the chest, along with lung function testing (spirometry, lung volumes and diffusing capacity) and a respiratory health survey. the ct studies were scored by two thoracic radiologists blinded to the patient's clinical details, using a previously described system developed for children and adolescents who were born prematurely using parameters. results abnormal findings were seen in all scans, the most common findings were subpleural triangular opacities ( %), linear opacities ( %), air trapping ( %) and emphysema ( %). agreement between the two observers for total score and common abnormalities varied with a linear weighted kappa value of . for linear opacities, . for triangular opacities, . for air trapping, and . for emphysema. conclusions linear and sub-triangular opacities on hrct chest are almost universal in young adults with a history of bpd. findings of emphysema and gas trapping are common and there is good interobservor agreement for these abnormalities. introduction pulmonary surfactant (ps) is synthesised by alveolar type ii epithelial cells to regulate the surface tension at the air-liquid interface of the air breathing lung. developmental maturation of ps is controlled by many factors including oxygen, glucose, catecholamines and cortisol. the intrauterine growth restricted (iugr) fetus is hypoxemic and hypoglycaemic, with elevated plasma catecholamines and cortisol. aim to determine the impact of iugr induced by chronic placental restriction via the carunclectomy model on ps maturation. methods we investigated the expression of surfactant protein (sp) -a, -b and -c and their genes in lung tissue of fetal sheep at days and days gestation (term, ± days) from control and carunclectomised merino ewes. results placentally restricted (pr) fetuses had a body weight < sd from the mean of control fetuses and a mean gestational pao < mmhg. pr fetuses had a reduced absolute, but not relative lung weight, decreased plasma glucose and increased plasma cortisol concentration. lung sp-a, -b and -c protein and mrna expression were reduced in pr compared with control fetuses at both ages. sp-b and -c, but not sp-a mrna expression and sp-a, but not sp-b or -c protein expression increased with gestational age. mean gestational pao was positively correlated with sp-a, -b and -c protein and sp-a and -c mrna expression. sp-a and -b gene expression were inversely related to plasma cortisol concentration. conclusion chronic placental restriction and hypoxemia results in an inhibition of ps maturation and thus iugr fetuses are at risk of lung complications, especially if born prematurely. support by the arc & nhmrc. nomination nil. conflict of interest nil. to sue jenkins , , , nola cecins , , sir charles gairdner hospital, perth, western australia, australia, curtin university of technology, perth, western australia, australia, and lung institute of western australia, perth, western australia, australiaintroduction the mwt is widely used to assess patients with chronic lung disease (cld). anecdotal reports and studies in small numbers of patients suggest that adverse events associated with the mwt are rare in patients with cld. this study reports observed adverse events and predictors of oxygen desaturation during the mwt in patients with stable cld referred to an out-patient pulmonary rehabilitation service. methods about consecutive patients completed the mwt in accordance with a standardised protocol that included continuous monitoring of oxygen saturation (spo ) and heart rate (hr, polar). the respiratory diagnoses of the patients were chronic obstructive pulmonary disease (copd), n = ( %); interstitial lung disease (ild), n = ( %); bronchiectasis, n = ( %) and asthma n = ( %). results observed adverse events occurred in tests ( %). one test was terminated when the patient reported chest pain and one patient developed persistent tachycardia (hr > bpm) immediately following the test. in tests ( %), the tester instructed the patient to stop walking due to profound oxygen desaturation (spo < %). six patients prematurely terminated the mwt due to intolerable symptoms. forty-seven per cent (n = ) of patients demonstrated oxygen desaturation, defined as a decrease in spo ‡ % to < % during the test. pre-exercise spo was a significant predictor of desaturation in the copd ( . , . to . , adjusted odds ratio [or], % confidence intervals) and ild (or . , . to . ) cohorts with fev also a predictor in patients with copd (or . , . to . ). conclusions profound oxygen desaturation is the commonest adverse event observed during the mwt in patients with stable cld. this finding questions the american thoracic society guidelines for the mwt which state that oximetry is optional. introduction there is weak support for use of opiates in palliation of refractory dyspnea; respiratory depression is perceived as a major risk. we evaluated the effect of i.v. morphine on dyspnea in controlled conditions and related this to concomitant respiratory depression. methods with ethical approval, healthy subjects received . mg/kg morphine sulphate or saline on separate days in a randomised controlled design. before and for hours after administration, subjects performed (i) dyspnea responses (measured with a visual analog scale) to increasing p et co with ventilation () constrained at resting levels (ii) unconstrainedresponses to increasing p et co to assess respiratory drive. results pre morphine with constrained , all subjects tolerated an elevated p et co of - mmhg; mean dyspnea rating was % ( (sem)). post morphine, at the same p et co , mean dyspnea rating fell to % ( , p < . , paired t). all subjects reported reduced dyspnea at minutes and this was sustained for hours. no changes in dyspnea scores were seen following saline. with unconstrained, at equivalent levels of p et co , morphine, but not saline, was associated with a lower in each subject for up to hours; mean fell from ( ) to ( ) l/min (p < . ). to assess if respiratory depression could account for reduced dyspnea, scores were compared at the different p et co levels that induced equivalent unconstrainedlevels with and without morphine; mean dyspnea scores were not different ( ( ) vs. ( ) %). conclusion a clinically moderate dose of morphine results in substantial and sustained relief of laboratory dyspnea in a small group of healthy subjects consistent with the associated degree of respiratory depression. support breathlessness research charitable trust uk; nih, usa. nomination nil. introduction pulmonary rehabilitation (pr) is a cornerstone of management for patients with chronic obstructive pulmonary disease (copd) and its efficacy is supported with level evidence. despite the known benefits of pr, up to one third of those people with copd who are referred to pr choose not to participate. there is little information regarding perceived barriers to attendance in an australian health care context. methods nineteen people with copd ( women and nine men, gold stage i-iv, age range - years) who had declined to take part in an outpatient pr program at a metropolitan teaching hospital participated in a qualitative study. semi-structured interviews were used to establish reasons for failing to attend the pr program. these interviews were transcribed verbatim and analysed using the principles of grounded theory. results three major themes were identified regarding barriers to attendance at pr. the first related to difficulties with getting there, including a lack of available transport and poor mobility. the second theme related to a lack of perceived benefit, including perceptions that pr would not improve their health or that they were currently doing enough exercise. the third major theme involved restrictions imposed by underlying medical conditions and included the influence of comorbidities and pain. minor themes that arose included competing demands, age, fatigue and program timing. conclusions in australia many patients with copd who are invited to attend pr do not perceive the program would be beneficial, feel they are too unwell to attend or have difficulty with access. further support should be offered to pr candidates and alternative methods of delivering pr to enhance uptake should be considered. introduction pulmonary rehabilitation has emerged as recommended standard care for people with chronic lung disease. however potential demand to access these services far exceeds the available resources. this study's aim was to determine if baseline measures of the bode index, dyspnea (modified medical research council questionnaire), minute walk distance ( mwd), physical activity, taunton respiratory quality of life questionnaire (trq), smoking status, and frequency of hospitalisations can predict responders to pulmonary rehabilitation. methods we retrospectively evaluated all participants with a diagnosis of copd, who attended the pulmonary rehabilitation program at the prince charles hospital between and . a participant was considered a responder to pulmonary rehabilitation if benefit was achieved in exercise capacity ( ‡ % increase in mwd) and/or quality of life ( ‡ . sd decrease in trq as described by cohen's effect size). prediction of responders was assessed using chi square cross tabulations and t-tests with significant measures analysed using a binary logistic regression model. results one hundred and forty-two participants ( males, mean age ( sd) years, mean fev . ( . ) %) who completed pulmonary rehabilitation were analysed. sixtyfive ( . %) people were categorised as responders using the above criteria. significant mean differences were: trq . ( . ) for responders vs. . ( . ) for non-responders p < . ; bode index . ( . ) vs. . ( . ) p = . ; mwd ( ) m vs. ( ) m p = . . the binary logistic regression model showed a higher trq score was the only factor that predicted a responder to pulmonary rehabilitation. no other measure added to the predictive power of the model. conclusion higher trq scores may be useful in predicting which participants are most likely to benefit from referral to pulmonary rehabilitation. further study is underway investigating other factors that may improve these findings. support nil. nomination nil. conflict of interest no. key: cord- -axd z authors: hansen, t. k.; tarnow, l.; thiel, s.; steffensen, r.; parving, h.‐h.; flyvbjerg, a. title: association between mannose‐binding lectin and vascular complications in type diabetes date: - - journal: scand j immunol doi: . /j. - . . i.x sha: doc_id: cord_uid: axd z complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose‐binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . – . ), p = . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ µg/l (iqr – µg/l) versus µg/l (iqr – ), p = . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ µg/l (iqr – µg/l) versus µg/l (iqr – µg/l), p = . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -ptfjxpo authors: isa, a.; norbeck, o.; pöhlmann, c.; tolfvenstam, t. title: mapping of the ex vivo cellular immune response against the complete human parvovirus b genome during acute infection date: - - journal: scand j immunol doi: . /j. - . . n.x sha: doc_id: cord_uid: ptfjxpo background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self‐limiting disease, but also capable of causing both significant pathology and long‐term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifnγ enzyme‐linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of – sfc/million pbmcs, roughly corresponding to . – . % b ‐specific cd (+) cells circulating in peripheral blood at – weeks post‐infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow‐up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post‐infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -jzdrvfvr authors: ahlfors, e.; sveinhaug, m. m.; nango, g.; johansen, c.; lyberg, t. title: proliferation of cells in the oral mucosa, the ear skin and the regional lymph nodes in mice sensitized and elicited with a hapten date: - - journal: scand j immunol doi: . /j. - . . ac.x sha: doc_id: cord_uid: jzdrvfvr during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti‐brdu antibody and developed using abc‐kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, – h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten‐exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - ob exq authors: sukhija, s.; gupta, v. k.; shah, a.; thiel, s.; sarma, p. u.; madan, t. title: levels, complement activity and polymorphisms of mannan‐binding lectin in patients of bronchial asthma with allergic rhinitis date: - - journal: scand j immunol doi: . /j. - . . ai.x sha: doc_id: cord_uid: ob exq activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan‐binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl‐induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age‐matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl‐induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p = . , or = . , % ci: . < or < . ). individuals with ‘a’ allele at position showed increased mbl levels, activity and disease severity. our results suggest that ‘a’ allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - qfmo rq authors: reinholdt, j.; baxendale, h.; ekström, n.; kayhty, h.; poulsen, k.; kilian, m. title: pneumococcal iga protease activity interferes with opsonophagocytosis of streptococcus pneumoniae mediated by serotype‐specific human monoclonal iga antibodies date: - - journal: scand j immunol doi: . /j. - . . t.x sha: doc_id: cord_uid: qfmo rq bacteria‐specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody‐treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as fabα (monovalent) deprived of fcα which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody‐coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero‐steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type‐ polysaccharide‐induced phagocytosis of iga protease‐deficient type‐ pneumococci equally well in the absence as in the presence of complement. iga antibody to type‐ polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type‐ polysaccharide served as opsonin. iga antibody extracted from iga protease‐producing target bacteria was almost exclusively in the form of fabα. conversely, iga from protease‐deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody‐mediated opsonophagocytosis. besides, in these experiments, iga‐mediated opsonophagocytosis was independent of complement. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -n lr z u authors: phillpotts, robert title: interferon prophylaxis of the common cold date: - - journal: trends pharmacol sci doi: . / - ( ) - sha: doc_id: cord_uid: n lr z u interferon is a potential prophylactic agent for the common cold. but there are problems. the present levels of side-effects that have been observed don't justify its use in the long term. robert phillpotts describes the mechanism of interferon action and the future hopes and developments for its use in preventing colds. mrc common cold unit, coombe road, solisbuty, wil~chire sp bw, uk, interferon is a potential prophylactic agent for the common cold. but there are problems. the present levels of side-effects that have been observed don't justify its use in the long term. l~'obert phillpotts describe~ the mechanism of interferon action and the future hopes and developments for its use in preventing colds. interferon appears to be the ideal antiviral drug for use in preventing colds; it is extremely potent, and active against a wide range of viruses. however, it is too toxic for systemic use in minor respiratory illnesses, and when taken in adequate doses intranasahy it causes a mild inflammation of the mucosa. current research is directed towards overcoming this problem. for most people the common cold is a mild, self-limiting illness. however, the high incidence of colds, and their ability to cause an exacerbation of chronic underlying respiratory disease leads to considerable morbidity. research has established that over serologicaily distinct viruses can cause a cold, and there is little hope that infectica can be controlled by vaccination. therefore control by means of a broad-spectrmn antivizal agent is the most rational approach, and interferon is without doubt the most pote=t, and broadly active antiviral agent yet discovered. c~ course there are limitations upon the k~nd of medication which could be used to prevent a cold. for example it should be cheap and easy to manufacture, as well as being highly effective, and virtually free from side-effects. i what is interferen? i human interferons (hulfn) are pro-/ teins or glycoproteins of which there are: principal types, a, , and "y. hulfn a and p: ~re both produced by cel~s after exposure to a virus: wn a by" leucocytes and lfn by fibroblast,,,. hulfn ~ is also produced by fibroblasts exposed to double-stranded rna, while ifn ~/is produced by lymphocytes only in response to an antigenic or mitogenic stimulus. interferons do not directly inhibit virus growth, but exposure of :mittfected cells to interferon makes them resistant to attack by viruses, interferon is not internalized by cells but binds to cell surface receptors (there is one receptor for ifn a and [ , and another receptor for ifn y) triggering off a series of events within the cell. multiple effects have been observed in cells, including the inhibition of penetration and maturation of certain viruses. however, the principal effect of interferon treatrnent seems to be to inhibit viral protein synthesis t. a system of interferoninduced enzymes has been described, some of which are activated by doublestranded rna molecules such as those produced during the replication of rna 'druses. the effect of these enzymes is to inhibit the initiation of viral protein synthesis, and to increase the rate at which viral mrna is degraded within the cell (see fig. ). future research will mldoubtedly disclose other mechanisms by which virus growth is prevented. "[here is already evidence to suggest that i[fn ~ has a different mechanism of ~k~tion from ifns a and , as combinations of ifn ~ with ifn ct or ifn (but not ifn a with ifn ) exhibit synergy. the potency of various interferon preparations may therefore be compared in terms of units of antiviral activity. there is species specificity in this process v ,' , while not absolute, means in practice that human interferon must be used to treat human cells. human interferon may be produced from cells cultured in vitro. a partially purified human leucocyte (a) interferon is produced by the finnish red cross from huffy coat cells (derived from blood donations) which have been stimulated with sendal virus. however, this procedure is expensive, and the amount of interferon which can be produced is limited by the availability of buffy coat cells. more recently, dna copies of the mrnas coding of all types of hulfn have been synthesized, and inserted into plasmid vectors downstream from a suitable prokaryotic promoter. such plasmis are introduced into bacteria by transfection, where they multiply, and high levels of expression of the interferon gene sequence can be cheap, costs will undoubtedly be further reduced. intranasal interferon prevents colds in an initial experiment at the common cold unit in wiltshire, uk, intranasal administration of partially purified human leucocyte interferon to volunteers was shown to prevent colds caused by rhinovirus type (ref. ). rhinoviruses cause approximately % of colds. however, leucocytes from - donations of blood were required to produce the mu of interferon used to treat each volunteer. furthermore, the use of partially purified material in which only about % of the protein was interferon also raised the question of whether interferon itself was responsible for preventing rhinovirus infection. could the activity have been due to contaminating, biologically active molecules also derived from human leucocytes? this question was answered in a further experiment carried out in , in which hulfn a, purified to virtual homogeneity on a monoclonal antibody affinity column was shown to prevent colds caused by another rhinovirus, type (ref. ). a total dose of mu was given intranasally to each volunteer over a period of days ( doses, doses daily). four doses were given before, and after vixus challenge. there was a dramatic reduction in the frequency of colds in the interferon treated group (table ) . this was accompanied by lesser reductions in the number of volunteers who shed virus, or who had an increase in serum neutralizing antibody to the challenge virus. in a further experiment conducted under an identical protocol, closely similar results were achieved using highly purified, hulfn a- , pro-duced in escherichia coil (manufactured by schering plough). this experiment clearly demonstrated that interferons produced in bacteria by recombinant dna techniques could be as active as those produced by human celk. how can interferon be used in prm'tke? in these experiments, interferon was given intranasally in large doses by a physidan. therefore ffinterferon is to find application as a prophylactic against the common cold. two questions have to be answered. firstly, could people treat themselves with interferon using a simple design of nasal spray? sc-condly, what is the minimum quantity of interferon necessary to protect against infection with cold viruses? answers to these questions were sought in a dose-ranging study, in which volunteers gave themselves various doses of hulfn a- using a finger-actuated nasal spray s . interferon was given for one day before, and days after, challenge with virulent human rhinoviruses. not only was the dose of interferon varied, but also the time of virus challenge in relation to a dose, so that the period of maximum vulnerability to virus infection could be identified. the results of this study suggest that at least - mu of ifn a- self-administered intranasally times daily are necessary to protect against experimental rhino~rus infection. subsequent experiments have shown that - mu of huifn a-a (a similar molecule to hulfn a-, produced by roche) can protect against experimental infect/on with a human respnratory coronavtrus (see fig. )". coronaviruses are the ~econd most frequently encouatered cause of a l.oid. and are responsible for - e/r of cases. however. one more requirement must be. met by a prophylactic against the common cold -almost complete freedom from unpleasant snde-effects, it is here that research has run into difficulties. further studies have shown that while regimens of ifn a similar to that proposed seem to he necessa~" for protection against natural colds, such doses are also toxi=, and produce a mild inflammation of the nasal mucosa. in a recent stuly from the university of virginia, prclorge.d intranasal administration of the hulf?,~ or- was associated with muxr~d irritation in % ot recipients"l histologically, marked epithelial acute t qfiammation with ulceration occurr,'d in %, and % had pronounced submucosal l.~anphoq,/tic and mononuclear cell ~filtrates. although these abnormalities re'solved within weeks after stopping treatment, long-term administration of ifn a would not be acceptable. however. there has been little sign of irritation of volumeers wen interferon f~r days. therefore interferon prophylaxis could be used when the time of exposure to virus (or fear of exposure to virus) can be predicted. examples of this situation would be contact with a cold sufferer, or before an examination or some other important event. there is ever' reason to believe that the problem of poor tolerance to intranasal interferon will be overcome. only a very. small number of interferons have been tested for antiviral activity and toxicity in the nose, and it is conceivable that a molecular subspecies of hulfn c~. huifn t~ or hulfn -¢, or a hybrid interferon molecule prepared from these, may have an improved therapeutic ratio. interferon could then be taken for prolonged periods as a prophylactic against the common cold by patients with underlying chronic respiratory disease, such as bronchitis or asthma. not surprisingly therefore, the discovery of inteffemns with this desirable property is one of the major goals in common cold research today. the question of whether interferon can be used to treat a cold also remains open. the relatively short course of the illness suggests that virus replication in the nose oomns rapidly, and may even be essentially complete by the time symptoms have begun. however, this pessimistic view has yet to be confirmed, and there is some evidence to suggest that even a relatively we~k antiviral agent, such as enviroxime (registered trade name, produced by eli lilly and company), administered locally after virus infection can affect the course of a cold tt. perhaps a suitable preparation of interferon, which could be over times more potent than enviroxime in vitro, could prove clinically useful. as modulators of appetite. the intiuen~es of other neurotransmitters and the physicochemical properties of food will, therefore, receive less prominence. at the outset it should be stres~d that the complexities of appetite regulation make it extremely unlikely that any single agent will prove to be the holy grail of appetite suppressants. future pharmacological approaches will need to take cognizance of this fact and attempt to tailor the treatment to the individual rather than the individual to the treatment. this is particularly important in view of the fact that the majority of peptides appear to have multiple effects, making it likely that the indiscriminate use of these agents, in subjects in whom a deficit or an excess has not been demonstrated, will lead to an unacceptably high incidence of side effects. opuad reading m a large body of evidence has now accumulated supporting a role for opioid peptides in the modulation of feeding behavior t. in particular, the relatively specific opioid antagonist, naloxone, has been demonstrated to decrease fe~ding under a variety of conditions in many species including humans. in humans, however, the major effect o! opioid blockade with naloxone appears to be to reduce carbohydrate intake rather than to produce an absolute reduction in calories. this reduction is offset by an increase in fat intake. in addition, the first ion term study in humans by atkinson using the long-acting opiate antagonist, naltrexone, produced disap- s ) erin. ciples of gme nmnipulatwn key: cord- -fcno z authors: nan title: molecular aspects of viral immunity date: - - journal: j cell biochem doi: . /jcb. sha: doc_id: cord_uid: fcno z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv ). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between and days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd + and cd + t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd + and cd + cells were detected by immunohistochemistry at days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il- , il- , il- , il- , il- , tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il- , tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd + cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp was both necessary and s f i c i e n t to cause retention of class i and icp expression in fibroblasts caused the cells to resist lysis by cd + t lymphocytes. icp is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp are being assessed in proteosome and tap transporter assays. gst-icp fusion proteins tightly bind a . kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp binds to a single cellular protein of = kda on ligand blots. these proteins are good candidates as cellular targets of icp and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp is very effective i n blocking cd + t lymphocyte responses in vivo, perhaps explaining the predominance of cd + vs. cd + anti-hsv ctl i n vivo. we expect that icp may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma , john p. carroll , sara r. abromson-leeman , joseph c. laning , martin e. dorf , and thomas l. benjamin . idepartment of pathology, emory university school of medicine, atlanta, ga , and department of pathology, harvard medical school, boston, ma . susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h- haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c wbida mouse and the highly resistant but mhc-identical (h- k) c bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c wbida x c bwcd.i) x c bwcdj backcross mice cosegregated with mtv- . inheritance of mtv- showed perfect concordance with absence of peripheral vp + t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv- showed no evidence of recombination between pyvs and mtv- . strongly biased usage of vp by (a) polyoma-specific cd + ctl from virus-infected c bwcdj mice and by @) cd + t cells infiltrating a polyoma tumor in a virus-immune c bwcd.i host provide further evidence that t cells bearing this mtv- sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv- sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h- , non h- , level of cd + t cells, of cd + t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd + t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd + t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd + t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd + t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type (ad ) and human papillomavirus type (hpv )) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad e a protein to which ctl clones were directed that could eradicate ad e induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad e b protein. these ad eib specific ctl clones, however, were as effective as the ad e a specific ctl clones in the eradication of ad e induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad e induced tumor cells with the activated ras oncogen the possibility of ad e b specific ctl to recognize the ad e induced tumors was eliminated whereas the ad e a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv induced mouse tumor model an immunosubdominant ctl epitope was identified in the e protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv , that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a * , a * , a" , a* and a * ). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of . class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment . , biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual ' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are - 'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the -month old infant, an rsv vaccine will need to be effective when given to -month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: ) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: ) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd + cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd ' and cd + t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd ' t cells were more pathogenic than cd + t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m ( k) protein were predominantly cd ' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd + t cells, and ctl. mice primed to g protein gave rise to predominantly cd ' t cells producing th cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type cytokines, notably il- , appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd t cells and protective effects by cd ' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d ' t cells and b cells (immune complexes) in maintaining cd + t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn . the analysis of the development and memory phases of the cd + "helper" n h ) and cd + cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type ) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose ( mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd + th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd -high, l-selectin-low, cd d (vla- ) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il- production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b . and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il- vs il-i /ifny are present at the initiation of the response of either cd or cd cells they dictate that the effectors generated will be polarized either towards il- and il- secretion or il- and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd effector cells not re-exposed to ag, produce no cytokines and they die within - days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il- produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il- and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to . , the s ikes re ain their neutral configuration ( ). probably as a consequence, the viral infectivity is totally preserved after an exposure of hours at p . an cf t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein ( ). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position , which is part of antigenic site , is important, although not essential, for the viral virulence. similarly, the arginine , which belongs to antigenic site , is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in ). viral strains mutated at arginine have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody ( ). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus ( ). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg a antibody; (c) activated (le., ll- rc) t cells infiltrating the cns are exclusively of the cd + phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids - of the vp capsid protein. a thl line specific for vp - exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein . prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by amino acids between residues and . au of the tg(mhu m) mice developed neurologic drsease - days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu m) mice with cjd prims produced mhu mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu m) mice wen differenl about % of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane > days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene - -fold was not deleterious to themiw but it did shorten scrapie incubation times from - d to - d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon (corresponding to codon in hurp) pmdnced spontaneous, fatal neurcdegeneration between and d of age in two lines of tg(mohp-pio l) mice designated and . genetic crosses of tg(moprp-p l) mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between and days of age. the t g~o p r p -p l o l l ) ~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast the tg and mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg overexpress the mutant transgene - -fold and develop fatal neurodegeneration behveen and d of age. tg mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md - . cd ' lymphocytes (tcd +) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of to residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors ( residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh -terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the residue precursor than from the nh -terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv- envelope protein that contain overlapping determinants each presented by different class i mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv- envelope to provide help for neutralizing antibody (ab) and cd + cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p peptide from the v loop of hiv- iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p segment of both iiib and mn strains of hiv- in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all responses, neutralizing ab, ctl, and th helper cells. the ctl specific for the mn strain p peptide crossreacted with strains sc, sf , , and cdc . the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it to -fold more potent in binding to the class i mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv- sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a , with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/ . cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m ) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/ and a/hawaii/ . this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/ virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il- , months after immunization were also observed . immunization of rabbits with dna encoding l , the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type (hsv- ), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv- . dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, - , dna cell biol, the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e ) codes for more than polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e gpl k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by distinct viral polypeptides, of which (the ad e . k or the complex of the . k and . k proteins) are coded in the e region. the e polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e . k or ad e gpl k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e genes in viral constructs, several transgenic mouse lines containing the ad e genes have been constructed for these experiments. the e genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e transgenic animals (h- b'd) have been transplanted allogeneically to h- d recipients and remained viable, secreting insulin until the end of the experiment at days; in contrast, control nontransgenic islets of the same genotype were rejected by - days. the e genes behind the native e promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e promoter of the transgene is responsive to stimulation by the ad e a following infection with an e minus ad and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr .) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than , cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in , a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus . k promoter and the s segment under control of the k promoter. the m segment, which encodes the g and g envelope proteins, was included because of our findings that: ( ) immunization with vaccinia or baculovirus-expressed g and g induced a neutralizing and protective immune response in hamsters; and, ( ) neutralizing antibodies to g or g could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd t cells and in the induction of virus-specific ctl activity. thereafter, the cd t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl- in the t and b cells and in c bl/ ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd , thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma thfectious disease unit, massachusetts general hospital, boston, ma §tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo for gag and / for envelope. cd + lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd + cells. suppression mediated by unstimulated cd autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd + cells activated by an anti-cd -specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv- molecular clone (nl ) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p and gp were observed in chimpanzees infected with n u and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv- , and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to years with cd cells > /mm . methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn- infected autologous cd cells, using target cells infected with recombinant vaccinia viruses expressing hn- proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for years, who by qc-pcr had <i viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv- variants, and both the p and p epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr region of the tcr in an individual with a vigorous response to gp suggested that approximately % of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv- infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf- ) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il- and il- receptor genes. to investigate the role of irf- related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o pfu of coxsackie virus (cvb ). homozygous irf- (+) and heterozygous irf- (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf- -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf- regulated gene products such as inos and ifn are essential for the early defense against cvb -induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd and b-cell populations and a higher proportion of macrophages and cd cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il , il and il , but higher levels of il and ifng in the elderly as compared to the young mice. the addition of mf to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il , il and il levels for both young and old, while il and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus (cvb ), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb is able to infect normal c bl/ mice and immunocompromised (cd ko and m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers - days pi.. large pathological changes were detectable only in heart tissue of cd ko mice, and were maximal at - weeks after infection. these results suggest the possibility that cd ' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd + and cd ' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p lck are resistant to coxsackievirus b induced heart disease. tamara martino, karen aitken, joseph penninger, jan nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus (cvb ) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p kk are resistant to cvb induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb induced heart disease, cytokine expression in the hearts of cvb infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd + t cells from m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd + t cells seen in normal mice days after lcmv infection, previously presumed to be due to the activity of cd + ctl, still occurs in m-/-mice. however, mice show an earlier rise in percentage and absolute numbers of cd + t cells by days after infection. cd + t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd + t cells in normal mice after lcmv infection. cd + ciz activity is lower in m-/mice and have a later peak than cd + ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in -mice. mice can assume some of the cytotoxic functions of the cd + cell population from normal mice, including immune-mediated pathology. further, cd + t cells from m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd + t cells from normal mice. mice genetically engineered to be deficient in -microglobulin in conclusion, these data suggest that cd + t cells from am-/- australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd and its ligand, in the presence of b cell acting cytokines, such as interleukin (il- ), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd l and il- , did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w lpg, u. k. and *nationallnstirute formedica research, london, nw aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il- has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il- on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il- gene was disrupted in /sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il- . mice were infected intranasally with a strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il- deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd + cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd +t helper (th) cells of the - subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within days pi. c t l s were detected by h- specific lysis of ldv-specific target cells. these target cells were generated by transfection of t -nm cells with a retrovirus vector carrying the gene (ow ) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected t -nih cells and cultured for days in the presence of il- . the ctls had largely disappeared in mice by days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v ) or an isogenic single-site attenuated vee mutant (v ) (single amino acid change at e glycoprotein position from glu-lys) and at , , , , , and hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il- , ifn-y, il- , il- , il- , and il- gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il- , ifn-y, il- , and il- were detected in the lymph node, with no changes in either il- or il- ; however, whereas cytokine elevations were detected by hours after injection of v , elevations were delayed until hours following infection with v . cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il- , and il- were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il- and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il- andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il- . to confirm the importance of the immune system in clearing rotavirus infection we infected rag knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. , : - ) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement b binding protein (bc -bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature , : - ) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc bbp ( kotwal, am. biotech. lab., ) and has also been shown to bind to two important complement components, c and c , and block the complement cascade at multiple sites in vitro (kotwal et al., science , - ; mckenzie, kotwal et al., j. inf. dis. , - . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas , : - ) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature , : - ) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, mol. immunol. : ) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h- haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, ) . various immune regulatory molecules were investigated in inbred susceptible /j ( ) mice and resistant c bl/ j (b ) mice which share the same mhc haplotype. when they were given eid, of sendai virus intranasally, mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in mice. virus was eliminated from the infected lung days earlier in b mice than in mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il- , ifn-y, il- , and il- producing cells were comparable for each strains of mice, while more il- producing cells were present in the lung of mice. however, the numbers of these cytokine producing cells peaked in mice days later than in b mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il- , and il- were present in mice than in mice. recall cytokine production in the draining mln showed only the presence of il- , ifn-y, il- , and il- , while no il- was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in mice. therefore, the susceptibility of mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il- , ifn-y, tnf-a or il- in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il- by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il- by rvv was shown to inhibit the host il- response required for the development of ctl's. no production was also significantly suppressed by il- . rvvs expressing il- or il- , although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i (il- ) doses of >i ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd + t cell expansion and ctl activation, and up to log increases in viral replication [orange, wolf, and biron, j. immunol. : , . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il- induced tnf-cx expression and that lcmv infection synergized with il- for this induction. administration of antibodies neutralizing tnf reversed the il- -induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd + t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il- -treated uninfected mice and were dramatically elevated in il- -treated virus-infected mice, including: ) decreases in body weights; ) elevation of circulating glucocorticoid levels: and ) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il- for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a % decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c bu mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl- , , ', '-tetramethyl indocarbocyanine perchlorate-or pkh -gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately % of the transferred bone marrow cells accumulated in spleens after hrs and % of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn- mrna by in sifu hybridization. treatment with anti-agmi or anti-nk .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box , canberra, act, australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c bu (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c bu mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn- and il- . in contrast spleen cells from infected c bu mice produced ifn-y and l- to levels that were only slightly less than uninfected controls. a- production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd stimulated lymph node cells from infected mice produced elevated ifn- suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il- or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl- proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl- does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il- produced a fully productive vsv infection at all times analyzed, up to weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells ( - fold by week and , fold by week ). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il- and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or -galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed or days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th type cytokines when stimulated with rsv infected spleen cells . however, the levels of - and fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il- were higher in the bulk culture from vac-f primed animals. the il- and il- production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed days after intranasal inoculation produced much lower levels of il- and - while the levels of il- and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day and subside as cd + t cell responses are activated at day post-infection. in contrast, m-/-mice, lacking cd + t cells, have dramatically elevated nk cell responses on day postinfection. the m-/-response is evidenced by increased nk cell activity, as well as up to -fold increases in blast and total nki.i+cd -cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)- independent, whereas day nk cell responses in m-/-mice are csa-sensitive and il- -dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il- and transforming growth factor (tgf- ) were examined. induction of il- mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days , , , , , and post-infection demonstrated delayed induction of il- protein as compared to ctl activation. tgf- , evaluated in biological and elisa assays, was induced maximally at days to post-infection. the kinetics of tgf- production by cells from infected m-/mice was similar to that of normal mice. however, cells from m-/-mice produced il- at early but not at late times postinfection. together, these results suggest that either il- is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il- p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days - after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with pfu of either a wild type strain of mv(chicago- ,chi- ) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi- on d ( . pfu/ third of implant), and moron d ( . pfu/ third of implant). hematoxylideosin stained sections of chi- -infected thymuses showed marked distortion of the cortex and medulla by d with thymocyte poilolosis and decreased cellularity. by d , these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd ,cd and cd were analyzed by flow cytomehy. there were significant decreases in the cd +cd + cell pop-ulation by d with complete loss of all such cells by d with chi- , and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae ) or cd co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d . only rare cells stained both with mv ha and cd or cd . mv ha was not expressed over background on any cd + cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl- ) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd were found to drop dramatically. other cell surface antigens such as cd , cd , and cd were unaffected during infection with myxoma virus. further more, the downregulation of cd by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p lck associates with the cytoplasmic domain of cd we have also examined the association of p lck with cd as well as steady state levels of p lck during viral infection. the modulation of surface cd has also been described in hiv infected t cells suggesting that the loss of cell-surface cd may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s o r us - gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd +. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p a(paucisymptomatic) group than in the pl(asymptomatic) and the p b-f groups (symptomatic group). the cohort of children has now been followed during years, and children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c weeks of age) showed no deletion of their cognate vp * t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp + t cells characteristic of c h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv- endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i (pol ) template. the promoter contains a tata box bp upstream of the transcription initiation site, binding sites for several pol i transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol . in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt- , but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i promoter ( pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used ( pglml), in the range of a typical polymerase in (pol ) promoter (va-i). purified tax, transactivates this promoter -to -fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the . m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i promoter and a unique pol i promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i template containing the base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within h of virus infection, while an indirect mechanism, regulated by type interferon (ifn), operates within h of virus infection. cd expression increased by - fold within h of wnv infection on hef, and by - -fold within h. wnv-inactivated, conditioned supematants removed from infected hef cultures after h incubation did not alter cd expression on unqimulated hef. whereas conditioned supernatants from h-infected cultures increased cd expression by about . - -fold after incubation for h, but not after h, similar to cd induction by ulml of ifn-p. increased cd expression on hef by wnv was also cell-cycle dependent. cd increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd by types and ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus and , and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd expression on hef after h. another alphavirus, ross river, was able to induce cd but only by the indirect mechanism of type ifn-dependent release. poly i.c, also, increased cd expression to the same extent as ifn-p after h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd expression in a manner similar to wnv. the ability of flavivhses to induce increased cd expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position of the pi gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position , not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p microglobulin in the er results in stable cell surface expression of the trimeric mhc- molecule. in previous studies we showed that in hpv- positive cervical carcinomas there was a loss of mhc- protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc- is mediated by an hpv- encoded protein. human keratinocytes were transfected withvarious hpv- constructs including pat , the full length genome, pat esx the full length genome with a premature stop codon in e , puc.et , the e and e oncogenes only, and pkve , expressing e from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap- and mhc- heavy chains in the cells with pat as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat esx and puc.et lines showed comparable tap- and mhc- protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap- and mhc- mrna between the different cell lines. the data indicate that expression ofhf'v- e leads to post-transcriptional loss of mhc- , presumably by interfering with tap. to map and characterize functional differences between e a of ad and adl , we previously constructed a series of hybrid ad / e a genes and used them with ad e b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad e a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd + as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin- from a variety of organisms. many of the numerous immunological properties ascribed to interleukin- are shared by the product of bcrfl and this has led to it being termed viral interleukin- . in order to investigate the activity of viral interleukin-i (vil- ) and its interactions with the human interleukin- receptor we have expressed the protein in a bacterial and the eukaryotic cos- expression systems. the bacterially expressed vil-i was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt .l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp- . a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil- protein that interact with the hil- receptor and confer its biological activity. a number of these mutants have been expressed in the cos- expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil- that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the . k or . k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k l protein shows sequence similarity to the eukaryotic initiation factor a (eif a) that is phosphorylated and inactivated by pkr. the k l protein competitively binds the kinase and blocks host eif a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin- , tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of i-ifna to u cells and binding of i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for fnc fp. direct binding of -ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd + t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase , wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa . antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p -microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids - (h -kk), - (h -kd) and - (h -db). the frameshift was introduced amino acids upstream of the h -kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h -i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h -kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h -dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np - srywairtr was studied using hla b -restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b' in vitro, and for presentation to ctl clones by hla positive targets. even conservative amino acid substitutions of the peptide residues p , , and profoundly influenced ctl recognition, without affecting binding to hla ' . these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l gene segment (but not those using tcr va ) were also sensitive to p substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class . these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla , p i , p and p are "flag" residues with tcr accessible side chains. the e / k protein of human adenovirus type (ad ) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e / k protein mediates binding to mhc molecules. apart from a stretch of approximately conserved amino acids in front of the transmembrane segment, e / k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad e / k protein. we show that e / k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in cells. based on the differential binding of monoclonal antibody tw . and cyanogen bromide cleavage experiments, a structural model of e / k is proposed, in which cys and cys as well as cys and cys are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e / k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e / k molecules. mutation of the three other cysteines at position , and had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e / k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd ' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent - % of the u.s. population are being tested to determine which of abundant cmv proteins they recognize by a cd ' ctl response: p , p , p , ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd ' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a . developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p and gb together with either of two lipid-based adjuvants, commercially available d tapm (bcehringer-mannheim) or mf gth (chiron, emeryville, ca). our preliminary studies with hsv- gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa as a restriction element in the transgenic model. the ctl response to sendai virus in c by mice is directed almost exclusively to a single h- kb-restricted epitope derived from the virus nucleoprotein, npj - (sev- ). analysis of independent t cell hybridomas generated from c by mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev- bound to kb has shown that the side chaiis of peptide residues phpi, gl , a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi and a d protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev- . to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev- analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev- . these data are consistent with a critical role for the gi and a d in governing tcr-sev- eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla . restricted ctl from hla-a . uansgenic mice was the m peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/ / -specific murine ctl that recognized an hla-a . restricted epitope distinct from m . using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/ nsl protein. because these ctl did not recognize targets infected with the a/alaska/ / saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a . specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common amino acid sequence and two distinct sets of bmding mtif residues. however, the n name.r peptide was able to sensitize ctl for half maximal lysis at - fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions and and was not recognized at any tested concentration, although it bound with higher &ity to hla-a . than the peptide from a/pw . the a/pr/ nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c bv mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd + immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd ' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h- b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd + ctl response directed against three wellcharacterized epitopes presented by h- db molecules: " - (fqpq-ngqfi), gp - (kavynfatcgi) and gp - (sgven-pggycl). the h- db motif is characterized by a sequence of to a.a. with two anchor residues: asn at position and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the lcmv peptides and known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides ( / ) did not bind to db. the other (including the epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt .aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt .aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently ~ . rt .a alleles are naturally linked with the tapa or the tapb allelic group . we have set out to characterise various alleles for the rt .a molecule, and find that, for the majority of tapaassociated rt .a molecules, acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt .a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b . seventeen variant peptides were examined for their binding to hla b ; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b positive hiv- infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd + human t cell lines and clones specific for different hiv antigens (gp , p , p ), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p is processed by the host cell into the nonamer peptide p - and is presented to cytotoxic t lymphocytes by the h- kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p to show that the rate of p - production is proportional to the amount of antigen secreted into the host cell cytosol. p is degraded in the host cell cytosol with a half life of minutes. the appearance of p - is coupled to the degradation of newly synthesized p . we have determined the rate of intracellular p secretion and by accounting for the rate of p degradation we estimate that approximately p molecules are degraded to produce one p - epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd + and class i hlarestricted by the b molecule. all three clones recognized lllb and rf but not mn strains of hiv- . using vaccinia vectors expressing truncated versions of the hiv- envelope, the clones were found to recognize an epitope within amino acids - , but not including - of gp . further mapping of the epitope with synthetic -mer peptides overlapping by , or -mers overlapping by , was unsuccessful. the sequence of the region of gp recognized by these clones was compared to the predicted hla- peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of allelic variants of ebv-lcl. by restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described ' peptide epitope from pp was not able to prime the autologous ebv-lcl for killing by the pp -specific ctl, whereas a recombinant vaccinia virus expressing whole pp could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp -specific cd ' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b' allele are recognized and killed by the pp -specific t cell clone, and cell lines carrying ' alleles are recognized by the hiv gp -t cell clone. we conclude that the reported cmv pp b" restricted epitope is not correct, since the ctl in question will only recognize ' alleles in combination with the correct pp epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus " - , ha - and ha - was found to be similar. an amino acid peptide corresponding to ha - which contains the - epitope was transported at a similar efficiency as the amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue , this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at . fmolelminute (+/- . ). the km for this peptide was found to be . nm(+/- . ). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., . previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids - ) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct ( - /r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus ( < n < amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs ( - /r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the r construct was presented at a level equivalent to that seen with the wild-type - /rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den- was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den- will interact with mim and lymphocytes in different manners, we used two isolates --plo strain (obtained from a df patient during taiwan outbreaks) and strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd icdi mab and complement prior to adherence procedure and the purity of mim culture was checked by cd surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den- strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den strain. in addition, this dhf-den strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv- ) and lymphocytic (h ) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that ( ) different strains of dengue virus could orchestrate quite differently with immune cells, ( ) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and ( ) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. ( ) when viral yields were enhanced early than day post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv- using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny . primary isolates of hiv- have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd -based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd binding site (cd -bs) on gp . cd -lgg is a recombinant molecule in which the variable domains of both heavy and light chains of lgg were replaced with the first and second immunoglobulin-like domains of human cd . both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg and cd -lgg were added at pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p production was measured over days of culture and an end-point titer of hiv- in the presence and absence of added antibody was determined. both igg and cd -lgg were found to reduce the original hiv titer from seven plasma samples with high virus titer (> tcid /ml) by up to -fold. this is in comparison to soluble cd which only reduced viral infectivity by -fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd -bs of hiv- gp are able to effectively neutralize primary isolates of hiv- and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of overlapping synthetic peptides corresponding to the entire hpv capsid proteins was used to generate hyperimmune sera. several antisera against different peptides were reactive with intact hpv capsids at titers up to : . . hiv- serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md . previously, we showed that hiv- proteins conjugated to . abortus (ba) could generate anti-hiv- neutralizing antibodies in mice even after depletion of cd * t cells. in this study a -mer peptide from the v loop of hiv- (mn) was synthesized ) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v -klh induced mainly igg , whereas v -ba induced all igg isotypes but lgg a predominated. fecal extracts from mice immunized with v -ba were shown by elsa to contain iga antibodies. sera from these mice bound gp , expressed on the surface of infected cells. sera from mice immunized with v -ba inhibited syncytia formed between cd ' t cells and chronically infected [hiv-i (mn)] h cells. inhibition of syncytia, formed by other hiv- lab. strains correlated with the degree of their homology with the v region of hiv-i (mn). to mimic the efffect of hiv- , mice were depleted of cd ' cells using anti-l t at the time of primary or secondary immunization. following primary immunization, cd + t cell depletion abrogated v -klh antibody responses, whereas responses to v -ba were retained and sera from these mice were able to inhibit gp- mediated syncytia. in secondary responses, cd ' t cell-depletion prevented boosting to v -klh, but v -ba increased anti and syncytia-inhibiting antibodies. these results suggest that: . . abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and . that infection with hiv- with subsequent impairment of cd ' t cell function would not abrogate anti-hiv- antibody responses if . abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh - p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn i, departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c bl/ j(b ) mice treated with the mab mel- to l-selectin have been analysed following i.n. infection with sendai virus. mel- treatment caused a - % decrease in the lymphocyte recruitment to the mediastinal (h ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd + ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel- antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel- -treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd + th precursor cells into lymph nodes. facs sorting for l-selectin+, +, and l-selectin-, b + cell populations from the mln and the cln of normal b mice days post sendai virus infection, showed that the afcs were from the l-selectin-, b + cell population, a population which comprised - % ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv- infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position in gp , is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position , upstream from the v loop, does not involve the same site and does not involve v . substitution of thr or ile also confers resistance. replacement of the v loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v peptide of mn but is affected by regions outside v . the other appears to be conformational and outside v , but its functional recognition is influenced by the v loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids - of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus to fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids - of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p< . ). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e a n d e genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e -specific and cd + t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e protein of hpv was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in groups of mice ( ) those not challenged with e ( ) mice not grafted but challenged with e protein in the ear ( ) mice primed by grafting with hpv e expressing cells and challenged with e protein ( ) mice primed by grafting with x hpv e cells on day , grafted again with lo hpv e cells on day and challenged with e protein in the ear. mice optimally grafted and challenged (group ) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group ) exhibited igg antibody levels comparable to the control group ( ). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp ) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr antibodies was also investigated in hepatitis c viremic individuals and non-viremic patients. a high frequency of positive reaction ( %) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp v loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., ) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = , ) and with serum rotavirus iga titers stimulated by the different reassortants (p < , ). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v profiles were determined using synthetic proteins derived from the amino acid sequences of the v region of laboratory strains of hiv- in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v regions of mn, sf , ny and han/sc. less than % of individual in each group recognized the v peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< . ). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p ag inhibition assays. although most patient serum was capable of inhibiting p ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at days post-infection, timepoints from days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about -fold by days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd + lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd were evaluated in uninfected controls (group l), hiv- positive patients with % cd + t cells (group ), and hiv-i-infected patients with ~ % cd + t cells (group ). most of these subjects also had -color staining for cd \cd ro\cd ra. the appearance of positive cd and cd ro on hivinfected and uninfected cells correlated well (r=. p<.ool). the percentage of cells staining cd +\cd +.(bright plus dim) was . ( %cl . - . ) in group , . ( . - . ) in group , and . ( . - . ) in group . the respective values for these groups that were cd +\cd gbwm was . ( . - . ), . ( . - . ), and . ( . - . ). values for cd +\cd ro+ were . ( . - . ), . ( . - . ), and . ( . - . ), respectively. in single factor discriminate function tests, the %cd +\cd + cells best predicted subject group ( % correct), proving to be a better discriminator than %cd +\cd b'h' ( % ~orrect),cd +\cd ~"" ( l%), cd +\cd ro+ ( %) and cd +\cd ro+\cd ra-( %). overall, no advantage was seen to splitting the cd +\cd + cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd +\cd ro+ compartment into cd ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv- infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp were performed at month and and two boosts of recombinant gpl mn/lai at month and in alum or incomplete freund adjuvant( fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from out of donors : seven out of eighteen ( %) were found to present envelope specific cytotoxic activity at months , , or post immunization ; this activity was characterized as a cd +,cd +, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd + cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al . mhc class i restricted cd ' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il- and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd ' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd + and cd ' t cells by their expression on the surface of distinct d r isoforms. cd ra is expressed on naive or virgin t cells, while cd ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd + t cells. the cd ' cells were stained for cd ro' (pe) and cd ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd +/cd ro+ t cells. similarly, cd +/cd rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd +/cd rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd + t cell response in lcmv infection we have observed a profound activation and proliferation of cd + t cells with a - fold increase in total number peaking at day - post infection. in c bw mice, most of the viral antigen is cleared by day seven, and after day the total cd + number per spleen drops about -fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd + cell remains remarkably stable between day - of the acute infection and for many months thereafter. thus, the decline in the cd ' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd + cells from c bl female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd + cells either by magnetic bead depletion of non-cd + cells, or by facs after staining with anti-cd monoclonal antibody these enriched (> % cd +) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at cd + cells, compared to a frequency greater than / ooooo in naive controls. the memory cd + cells were further characterized as cd rb-low ( / ) , cd -high ( / ), lselectin-low ( / ), and cd d-high (vla- -high) (v ). this is close agreement with other phenotyping studies on cd + memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn . the immune response of influenza a and sendai-virusspecific, memory cd ' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c bu mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated or days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd ' t cells from the ln and spleen resulting in dna fragmentation of - % ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb a cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb a cells were treated with pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by pm of nmda was reversed by pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a to carol s. reiss. pediatrics, university of iowa, iowa city, ia. mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. . % of suckling c bu (kbdb) mice inoculated intranasally with mhv-jhm at days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at - weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c bu mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c bv mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids - (cslwngphl, db), - (rcqifani, kb), and - (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c bu mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of - week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of - week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd + t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases research units, national animal disease center, usoa, agricultural research service, ames, ia a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a . kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in -week-old or -day-old pigs, lltba replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba -infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba , -day-old pigs were infected intranasally with lltpa or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about -fold for lltpa . abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba -infected pigs. while replication of lltbres in the brain progressed until death at days post-infection, replication of lltpa in the brain ceased by days post-infection and the pigs exhibited only mild clinical signs. since lltba is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type (hsv- ) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv- in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of - week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day post-infection. in contrast, in a large group of cba mice studied up until days post-infwtion, viral rna could be detected by both techniques in % of mice until as late as days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v region of the siv env gene. the majority (> %) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd + t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd + t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd + t cells with monoclonal antibody gk . . both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd + t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md ; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn- epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn- subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv- infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv- of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp . because of its early expression during the hsv replication cycle, lcp represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein (nsi) and the lcp sequence of hsv- . the nsi-icp protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing pg of nsi-icp . responder cells obtained from draining lymphnodes were re-stimulated in vitro with p cells lransfected with icp and then lesled for cytolytic activity on icp -p and control p . the induction of icp specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h- d haplotype: a dd restricted epitope from the gp protein of hiv- and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately ?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd- mice were inoculated subcutaneously with x ' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo ed, of influenza h s . au corn mice suffered severe disease and % died. only one of ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was , while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, . in a parallel experiment, no influenza infectivity was detected in the lungs of ha vector immunized mice at days postchallenge. in contrast, / pbs-inoculated mice and / inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of . and . x lo pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from to . ug of dna was inoculated by intramuscular and intradermal routes. from . ug to . ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required to , times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations ( ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl e hpv associated cervical cancer cells express hpv e protein and antibody to hpv e can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e protein of hpv , and expressing e protein in the skin, has recently been described ( ) and these mice develop spontaneous humoral immunity to e protein similar to patients with cervical cancer( ). to determine whether immunisation could induce immunity to e sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv e protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv e expressing tumour cells in nrro. we then used similar immunisation with e iquil a to induce e specific immunity in fvb (h- ) mice. h- qskin @s expressing e were not rejected by e immunised h-zq mice, though immunisation induced antibody to e , and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl e lack a tc epitope in the context of h-zq, or that expression ofe in the skin from the e transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types or , and continued expression of the e and e gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e and e from hpv and with the aim of inducing e and e specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e /e reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv € specific ctl from c bu mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi e alone, with similar recognition of the defined immunodominant h- db restricted epitope, e residues - . ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa mice infected with the laboratory strains of a/pr/ / (hln ) or the mouse adapted a/hk/ (h n ) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h and h strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/ were susceptible to subsequent infection with the a/hk/ strain although they were resistant to weight loss and death. other strains such as a/beijing/ or a/georgia/ provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/ challenge than mice previously infected with a/bei/ and a/ga/ , and were similar to mice that had been previously infected with a/bei/ . thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h- -ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd + ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day after infection. there was little effect on virus clearance. as found previously with cd -depleted h- b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd + t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd + cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd + ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd +, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd + responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type (hsv- ) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv- to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day and day resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv- vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv- . viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl - and plasmodium berghei cs protein - were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam cyj) and dissolved in a % dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and % soya oil in water. wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of mice intra-peritoneally or subcutaneously at . and days. days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of w:l the plasmodium berghei peptide encapsulated in micmpartides gave % iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p ccs - gave a level of lysis of % at an e:t ralioof w . these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type poliovirus (shah sauketf) together with the regular type and type canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp . as previously shown for hypsin-mated type poliovirus @vm alone. trypsin used to modify the type component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with -fold concentrated type component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type poliovirus. no unexpected sideeffects were recorded phase i trials comprised adult volunteers with at least years since the last dose of poliovirus vaccine and children who were due to receive the third dose of the regular immunization schedule at about years. in both groups. individuals received tryipv and were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with . ug of an h expressing plasmid dna (pcmvri ). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at days post boost protection against the lethal challenge was complete at days, month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv- t-cell epitopes: generation of hla-b* -restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv- patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys) and synthetic htlv- peptides which seem suitable for vaccine design. htlv- amino acid sequences were searched for eight to mer patterns carrying the anchor residues of the hla-b* peptide motif at positions two and eight to fourteen. candidate peptides were synthesized according to the matched sequence patterns. their hla-b* affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b* cells. the fourth group (controls) were inoculated with h n (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h n group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp la sequence. seventeen patients participating in a phase i gp protocol and patients participating in a phase i gp protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into well plates containing peptides overlapping the la sequence of gp , pulsed on day with tritiated thymidine and harvested and counted on day . results: the percentage of patient's pbmcs from each trial with an lsi to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp or gp proliferate to multiple peptides within the gp molecule. reactivity from the end of c through early c (lai #i - ) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v loop peptide (la # ). although the percent reactivity to the entire gp molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c (lai # - ). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il- production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] and a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna proteins ( a, b, c). and ebna c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna , , lp or lmp . the ebv-specific ctl lines expressed cd /cd and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class- . these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna family of proteins and lmp a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology, genetic therapy, inc., gaithersburg, maryland replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. : - ; ) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of , and . in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: . f . % vs . * . %. . * . % v s . * . %. and . * . % vs . * . % (meanrts.e.m., n= ; p<o.ol, all comparisons) a t e:t ratios of , and , respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping -mer peptides derived from p . two individuals ( and ) had ctl against p . (a(mino) a(cids) - , krwiilglnknrmysftsi), two others ( and ) recognized p . (aa - , frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p . ( ) and p . (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of aa overlapping -mers and to test for hiv- isiv crossreactivity. both two p . epitopes (possibly restricted by hla-a ) and the p . epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p . epitopes is in progress. the sn p . epitope was also non-crossreactive, despite only one aa difference (position , s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p in humans (johnson ji , , , buseyne jv , , and to p . in cynomolgus macaques (gotch jmprim . , ) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd- and cd- -cells, b-cells and adherent cells, while an average of % of virus was associated with lps-stimulated -cells and adherent cells. in the second experiment the combined function of antigen presenting cells, -helper cells and -cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day , and decreased at days , and post-cvb , infection relative to unlnlected animals. conclusions: these data indicate that cvb , associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb , modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb ,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih t fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of . kb in hind i j and of . kb in hind i j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the . kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv - progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v - cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv - was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a kb transcript in northern blot analysis. a kb cdna was isolated and codes f o r a novel polypeptide o f amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/ / in in uitro assays of viral infectivity of the mouse b-lymphoma a - . . we have found that although infection sensitizes the a cells for recognition by both class i and class i restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/ / from a different passage history, we find that n o t only are the infected a cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a - the therapeutic and prophylactic antiviral efficacy of interleukin- (il- ) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il- commenced hours after mice were infected w i t h hsv, and was continued daily for a total of days. il- therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. . x we~ells/well of both adherent and nonadherent cell types were infected with two different den- virus strains : ( ) thiland strain isolated from the dengue hemorrhagic fever(dhf patient and ( ) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den and dhf virus strain replicated much more efficiently ( . x pfu/ml on day p.i. than df strain; (b) nonadherent cells were times less efficient to produce viral yields than adherent cells (dhf and df strains peaked at . ~ ' pfu/ml and . ~ pfu/ml on day p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi- /crmn k gene trigger an inflammatory response by - hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at - hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice - months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for days. by this method, pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted :lo. scid mice were used as a second detection assay for mcmv. using scid mice and different doses of virus, the lds, of mcmv in scid mice was - pfu. mcmv infections were detected when - pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of spleens, kidneys, and salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after - days. in addition, lytic mcmv was detected in scid mice days after injection with ( )' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp , but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp -negative cell line showed no difference from control viruses, and the presence of hsp did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp proteins act as molecular chaperones and that hsp can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs /bl and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days and postinfection, mln or spleen cells from both strains of mice produced il- , il- , il- and ifn-y, but little or no il- or il- . however, following in vivo treatment with anti-ifn-y antibody, production of l- and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs /bl mice showed little change in cytokine profile. an increase in il- and l- production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of l- and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c /bl mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a amino acid type i membrane glycoprotein with potential n-glycosylation sites at asn , asn , and asn . due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble amino acid form of rgp lacking a transmembrane domain (rgpt ). rgpt was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt was secreted only when asn l was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt . although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of his residues was added to the cooh-terminus of rgpt . this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+ -agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev- locus is reduced to % compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci ( , , and ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad . one of the major isletcell antigens, shows a high degree of homology with the c protein of cbv- . studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp and gad , are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p expression in of ( %). p detection by ipox is usually associated with mutated p . sscp for exons - revealed p alterations in of specimens, and loh at exon was seen in only of evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p . we recently reported that sv induces mesotheliomas in rodents, and that sv -like sequences are present and expressed in mpm. of note, sv large t antigen (tag) binds and inactivates wt p i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p may result from its physicdl binding with tag. tag was detected by ipox in ( %) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p (p = . , fisher's exact text), and preliminary experiments suggest co-precipitation of p and tag. finally, we demonstrate that waf- is not detectable in mpm expressing wt p suggesting that p is inactive. we speculate that tag expression in mpm binds to and inactivates p contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in % of cervical carcinomas and possibly % of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type ' and hla-a '. culture media consisted of rpmi- % human serum supplemented in three cases with u/ml il- , in another three cases with u/ml il- and u/ml il- , and in the final three cases with u/ml il- and u/ml il- . the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c a (hpv, hla-a ') transfected with either the hpv type e or e genes. one patient's cells maintained with uiml il- and ulml il- showed hpv e protein specific cytotoxicity in a hla-a restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k- . the ctl efficiently lysed a c a-e clone but in contrast a vector only transfectant was not lysed. the lysis of c a-e was blocked with a-cd and a-cd antibodies at % and % respectively. facs analysis determined that this ctl population was . % cd 'cds' and . % cd 'cd '. limited e recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv- infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv- infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b restricted epitopes: p - gag and amino acids - of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv- infection may be influenced by an effective host immune response against hiv- and/or by viral properties. to investigate the cellular immune response to hn- we analyzed ctl activity against different hiv- proteins in long-term asymptomatic hiv- infection as well as during rapid progression to aids. methods: longterm asymptomatic individuals with cd counts > celllpl after more than years of infection were selected from the amsterdam cohort study on aids versus subjects who progressed to aids < years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv- ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus isolates from an outbreak of disease in southem india. sequence analysis within the ' untranslated region reveals that these isolates fall into two groups that differ by - % (equivalent diversity to that seen between between published sequences of poliovirus and coxsackie a virus). these two groups of viruses also differ in their cell tropism. isolates defined as group by their 'utr sequence grow equally well on ht cells (a human colon carcinoma cell line) and vero cells. isolates of group , with one exception, grow only on ht cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the ' untranslated region sequence of a group virus but the protein profile and cellular tropism of a group virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c -i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py is the mouse mammary tumor virus superantigen (mtv sad gene, which deletes t cells required for py tumor immunosurveillance in h- ' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i and pedatteck (peru). both strains are susceptible to py as % of infected animals develop a full profile of tumors. crosses between cs br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech , yield f progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs br) x c br) and ((czech i x cs br) x c br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i and peru mice by pcr using primers specific for mtv sag indicates that both strains are negative for proviral mtv sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i genomic dna. these results indicate that, like the c hibi, the pedatteck and czech i contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis- is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis- suppresses primary antibody response more efficiently in infected mice. some of the fts- infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis- show a high degee of homology between this variant and the prototype f-mulv clone . in this study we have attempted to localize the genomic determinant of fis- which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis- and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd ) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th cd t cell subset. interestingly, when cd responses were observed, cd tcell responses were also detectable. . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex- xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp was strain specific, and the polymerase ( d) was the most immunogenic and cross reactive. d) a construct comprising d and the immunodominant vp epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus (ehv- ; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv- . to generate useful amounts of individual ehv- glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv- infection. au three glycoproteins induced serum (elisa) antihodies to ehv- , and ehv- gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv- , protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv- antigen were observed for each of the ehv- glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv- . molecular pathogenesis of ural infeetiom - enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb , infection in adolescent c h/hesnj mice. antigen presenting cell, -helper cell and -cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo plaque forming units of cvb , at day and with lo srbc's at days , , and post-cvbb, infection. splenocytes were harvested days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb , was associated with . % to . % of cd- positive t-cells and w a % to % of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a % and % under non-stimulated conditions. an average of % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. , in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by % and % of residues, and the nt s genome is shorter by nt. we surveyed reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; ( . %) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california ( ), arizona ( ), new mexico (l), and mexico ( ). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to . %. but amino acid distances were less than %. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd + ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a + patients, htlv i-specific cd + ctl restricted by hla-a recognize a peptide derived from the htlv i tax protein (tax - llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd ' tax - specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag - vl&sdfri) presented by hla-a . these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd + t helper cells in this autoimmune response by treating mice with the cw-specific gk . monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c h mice by showing that depletion of cd + cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b o.br mice than in c h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl (h n ) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues - and . . when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl -immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha - subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha - epitope of influenza strain a i m in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha - specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv- gp and in the n-terminus of hla class i beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = . by fisher two tail analysis)in a second, year-longitudinal study of progre.ssors and stable patients we found: ( ) the production of crab was seen in - % of rapid progresson, while the true stables produce only infrequent low-titers crab. ( ) in rapid pmgressors, production of crab preceded by - years the marked drop in cd counts. ( ) crab production did not correlate with the degree of hyperglobulinemia in these patients. ( ) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn- receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn- and - production, without detectable - , and with decreased - secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i - producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th- -type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord- -by aczt authors: vilhelmsson, m.; ekman, g. j.; zargari, a.; scheynius, a. title: the malassezia sympodialis allergen mala s with sequence similarity to manganese superoxide dismutase induces maturation and production of inflammatory cytokines in human dendritic cells date: - - journal: scand j immunol doi: . /j. - . . ae.x sha: doc_id: cord_uid: by aczt the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t‐cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen‐presenting dendritic cells. monocyte‐derived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il‐ ( ‐fold), tnf‐α ( ‐fold) and il‐ (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross‐reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -ilrp vb authors: wefer, j.; harris, r. a.; lobell, a. title: protective dna vaccination against mog( ‐ )‐induced experimental autoimmune encephalomyelitis involves induction of ifnβ date: - - journal: scand j immunol doi: . /j. - . . j.x sha: doc_id: cord_uid: ilrp vb dna vaccine coding for the encephalitogenic peptide mog( ‐ ) protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t‐cell immunoglobulin‐ and mucin‐domain‐containing molecule (tim‐ ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen‐specific lymphocyte population upregulating ifnγ upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigen‐specific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen‐specific upregulation of ifnβ upon recall stimulation and ( ) reduced il‐ rβ on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnβ. we are currently investigating the cellular mechanisms behind this ifnβ‐mediated protection. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -xxw kptr authors: chistensen, h. r.; frøkiær, h. title: characterization of a large panel of lactic acid bacteria derived from the human gut for their capacity to polarize dendritic cell date: - - journal: scand j immunol doi: . /j. - . . ap.x sha: doc_id: cord_uid: xxw kptr dendritic cells (dcs) are the principal stimulators of naïve t helper (th) cells and play a pivotal regulatory role in the th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut‐derived lactobacillus and bifidobacterium spp. was screened for dc‐polarizing capacity by exposing bone marrow‐derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc‐surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin‐ (il‐ ) and tumour necrosis factor (tnf)‐α, while the differences for il‐ and il‐ were less pronounced. bifidobacteria tended to be weak il‐ and tnf‐α inducers, while both strong and weak il‐ inducers were found among the strains of lactobacillus. remarkably, strains weak in il‐ induction inhibited il‐ and tnf‐α production induced by an otherwise strong cytokine‐inducing strain of lactobacillus casei, while il‐ production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il‐ were most effective in upregulating surface mhc class ii and cd . moreover, l. casei‐induced upregulation of cd was reduced in the presence of a weak il‐ ‐inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg‐driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - nye nc authors: krarup, a.; sørensen, u.; matsushita, m.; jensenius, j. c.; thiel, s. title: mannan‐binding lectin, l‐ficolin and h‐ficolin selectively binds to different bacteria date: - - journal: scand j immunol doi: . /j. - . . al.x sha: doc_id: cord_uid: nye nc mannan‐binding lectin (mbl), l‐ficolin and h‐ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l‐ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type‐ , ‐ , ‐ , ‐ and ‐ ). h‐ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h‐ficolin initiated activation of complement factor c , whereas l‐ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . μg of mbl/ml, . μg of l‐ficolin/ml and . μg of h‐ficolin/ml, respectively. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -hhvmsn b authors: karlsson, h.; larsson, p.; wold, a. e.; rudin, a. title: pattern of cytokine responses to gram‐positive and gram‐negative commensal bacteria is profoundly changed when monocytes differentiate into dendritic cells date: - - journal: scand j immunol doi: . /j. - . . at.x sha: doc_id: cord_uid: hhvmsn b the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigen‐presenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte‐derived dcs were stimulated with uv‐inactivated gram‐positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram‐negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il‐ p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il‐ p , tnf, il‐ and il‐ in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram‐positive strains correlated with a lower surface expression of toll‐like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram‐negative bacteria. ifn‐γ increased the expression of tlr on dcs and also potentiated the cytokine response to gram‐negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram‐positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -eblcf authors: kollgaard, t. m.; reker, s.; petersen, s. l.; masmas, t. n.; vindelov, l. l.; straten, p. t. title: clonally expanded cd (+) t cells in allogeneic bone marrow transplantation date: - - journal: scand j immunol doi: . /j. - . . bm.x sha: doc_id: cord_uid: eblcf allogeneic bone marrow transplantation (bmt) is a potentially curative therapy for patients with haematologic malignancies. several lines of evidence demonstrate that donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t‐cell‐depleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft‐versus‐host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft‐versus‐tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t‐cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd (+) t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t‐cell receptor clonotype mapping based on rt‐pcr and denaturing gradient gel electrophoresis (dgge). using this gel‐based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd (+) t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - zuewjv authors: nilkaeo, a.; bhuvanath, s. title: interleukin‐ inhibition of oral carcinoma cell proliferation date: - - journal: scand j immunol doi: . /j. - . . bg.x sha: doc_id: cord_uid: zuewjv interleukin‐ (il‐ ), a pro‐inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn‐γ production and stimulation of nk‐cell‐cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il‐ effect on an oral carcinoma (kb) cell line. with rt‐pcr technique, kb‐cell line was found to express il‐ receptors (il‐ rα and il‐ rβ), indicating that this oral carcinoma line is a target for il‐ study. we showed that recombinant human il‐ inhibited kb‐cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il‐ suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il‐ treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death‐controlling genes (bcl‐ and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb‐cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il‐ , as this cytokine is an important regulator of anticancer mechanisms. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -ajafw authors: bozza, fernando a; cruz, oswaldo g; zagne, sonia mo; azeredo, elzinandes l; nogueira, rita mr; assis, edson f; bozza, patricia t; kubelka, claire f title: multiplex cytokine profile from dengue patients: mip- beta and ifn-gamma as predictive factors for severity date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ajafw background: dengue virus pathogenesis is not yet fully understood and the identification of patients at high risk for developing severe disease forms is still a great challenge in dengue patient care. during the present study, we evaluated prospectively the potential of cytokines present in plasma from patients with dengue in stratifying disease severity. methods: seventeen-cytokine multiplex fluorescent microbead immunoassay was used for the simultaneous detection in dengue patients. glm models using bimodal or gaussian family were determined in order to associate cytokines with clinical manifestations and laboratory diagnosis. results: il- β, ifn-γ, il- , il- , il- , il- and gm-csf were significantly increased in patients with severe clinical manifestations (severe dengue) when compared to mild disease forms (mild dengue). in contrast, increased mip- β levels were observed in patients with mild dengue. mip- β was also associated with cd +nk cell circulating rates. il- β, il- , tnf-α and mcp- were associated with marked thrombocytopenia. increased mcp- and gm-csf levels correlated with hypotension. moreover, mip- β and ifn-γ were independently associated with both dengue severity and disease outcome. conclusion: our data demonstrated that the use of a multiple cytokine assay platform was suitable for identifying distinct cytokine profiles associated with the dengue clinical manifestations and severity. mip-β is indicated for the first time as a good prognostic marker in contrast to ifn-γ that was associated with disease severity. during the last decades dengue became the most important arthropod-borne emerging viral disease in tropical countries [ ] . it is estimated that about . % notified cases are classified as dengue haemorrhagic fever (dhf) and about . - % of dhf cases are lethal [ ] [ ] [ ] . in the last two decades, latin america saw a dramatic increase in frequency and in geographic extension of dengue fever. specifically, the situation in brazil has worsened during the last decade since the introduction of the dengue- serotype. in the past years brazil had dengue outbreaks with at least million cases ( ) ( ) and within the last months thousand cases were reported [ ] . in addition, severe disease forms are occurring with increased frequency and mortality rates. dengue pathogenesis is not completely understood, and the main determinants of the development of severe forms are not yet well established. increase in capillary permeability associated with endothelial activation and haemorrhagic phenomena are landmarks of severe clinical manifestations, strongly suggesting an alteration in immunoregulation [ ] . cytokines are proteins secreted during innate and adaptive immunological responses, acting as inflammatory mediators or modulatory molecules during several haemorrhagic fevers [ ] . clinical studies support a key role for cytokines in the dhf pathogenesis [ ] [ ] [ ] [ ] [ ] [ ] . during dengue virus infections, cytokines are involved in the disease onset and homeostatic regulation. specifically, tnf-α, il- β and il- have been associated with both coagulation (f + and tatc) and fibrinolysis (t-pa, papc, and d-dimmer) activation markers [ ] . this activation is more striking in patients with severe clinical manifestations, although it can be found at lower degrees in patients with mild disease [ , ] . despite the fact that cytokine network and their multiple regulatory pathways are highly complex and not fully elucidated during dengue fever, these molecules seem to represent interesting markers for patient stratification or prognosis. an emerging interest has appeared in order to define biomarkers that may have pathophysiological roles during disease and that may be used as future therapeutic targets. new technologies have been developed in order to detect multiple biomarkers within a single and small blood sample. such approaches may lead to the development of specific marker panels for dengue fever. accordingly, cytokine patterns have been indicated as serum biomarkers during infectious diseases such as hepatitis c [ ] , ards [ ] and sepsis [ ] . in this study, we prospectively evaluated the potential use of plasma cytokine concentrations for severity stratifica-tion of patients with dengue, using a cytokine-multiplex assay. among tested cytokines, we were able to recognize ten significantly altered circulating factors and to characterise cytokine patterns related to determined clinical manifestations and disease severity. the ethics committee of the oswaldo cruz foundation approved this study protocol and written informed consent was obtained from all patients or their guardians prior to blood collection. we included prospectively dengue-infected patients ( a detailed history and physical examination was performed to detect hemorrhagic manifestations (positive tourniquet test for capillary fragility, skin haemorrhages, epistaxis, gingival, gastrointestinal, or urinary tract haemorrhage), signs of plasma leakage (pleural or pericardial effusion, ascites), signs of circulatory failure (cold extremities, cyanosis, hypotension, tachycardia, shock), and hepatomegaly. in addition to the suggestive clinical diagnosis, all patients had the dengue virus infection confirmed either by antidengue enzyme-linked immunoabsorbent assay (elisa)-igm, serotype specific reverse transcription-polymerase chain reaction (rt-pcr) or by virus isolation [ ] [ ] [ ] . dengue immune response was considered as primary or secondary by igg elisa according to previously established criteria [ ] . as previously reported [ ] [ ] [ ] , we also were often unable to characterize the severe disease forms based on who criteria [ ]. in nicaragua, harris et al. [ , ] described four key severe clinical manifestations associated with dengue -shock, plasma leakage, marked thrombocytopenia or internal haemorrhage -that do not fit dhf/dss classification as single parameters. according to these criteria, we considered: • severe dengue -dengue confirmed cases plus severe thrombocytopenia (< , platelets/mm ) and/or hypotension (postural hypotension with decrease in systolic arterial pressure in mm hg in supine position or systolic arterial pressure < mm hg) and/or plasma leakage (either haemoconcentration fluctuation of packed cell volume ≥ % during illness course and recovery or clinical signs of plasma leakage, such as pleural effusion) and/or severe haemorrhagic manifestations. • mild dengue -dengue confirmed cases in absence of severe thrombocytopenia, hypotension, plasma leakage signs or haemorrhagic manifestations. blood samples were collected from a peripheral vein and kept on ice. plasma was collected by centrifugation at g for min at °c, aliquoted, and stored at - °c until the analysis day. a multiplex biometric immunoassay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokine measurement according to the manufacturer's instructions (bio-plex human cytokine assay; bio-rad inc., hercules, ca, usa). cytokines measured were: il- β, il- , il- , il- , il- , il- , cxcl (il- ), il- , il- (p ), il- , il- , granulocyte colony stimulating factor (g-csf), granulocyte-monocyte colony stimulating factor (gm-csf), monocyte chemoattractive protein (mcp- /ccl ), macrophage inflammatory protein (mip- β/ccl ), and tnf-α. briefly, μl plasma samples were diluted : and incubated with antibodycoupled beads. complexes were washed, then incubated with biotinylated detection antibody and, finally, with streptavidin-phycoerythrin prior to assessing cytokine concentration titres. concentrated human recombinant cytokine was provided by the vendor (bio-rad laboratories). a range of . - , pg/ml recombinant cytokines was used to establish standard curves and to maximize the sensitivity and the assay dynamic range. cytokine levels were determined using a multiplex array reader from luminex™ instrumentation system (bio-plex workstation from bio-rad laboratories). the analyte concentration was calculated using software provided by the manufacturer (bio-plex manager software). liquid nitrogen cryopreserved peripheral blood mononuclear leukocytes were isolated by histopaque- (sigma chemical co., saint louis, mo, usa) from out of dengue patients. cells were stained for cd surface marker using anti-cd -cy (igg , clone b ) from pharmingen (san diego, ca, usa) and positive cells were detected by flow cytometry as described before [ ] using facscalibur (becton-dickinson). events ( , - , ) were acquired and analyses were carried out with flowjo (treestar, version . ) software. the nonparametric mann-whitney u test was used to evaluate differences between cytokine ratios from severe and mild dengue patients. glm models were used to evaluate factors independently associated with quantitative variables. analysis of factors independently associated with dengue severity and other clinical manifestations was performed with glm with logistic regression or gaussian family. results from the logistic regressions are given as odds ratio (or). the confidence interval (ci) was established at %. alternatively, for a glm gaussian family t values were recorded. a probability value of p< . was considered to be significant. the statistical programs r [ ] and prism (graph-pad software, san diego, ca, usa) were employed. the fisher's exact test was applied to determine the significance of positive samples from patients when comparing different virus serotypes or sequential infections. correlation between platelet counts and cytokine production in blood samples was estimated by spearman's correlation. from the patients included, were classified as severe dengue and as mild dengue. detailed demographic, clinical, and laboratorial data from dengue patients are summarized in table . blood collection was performed between and days after disease onset. in order to avoid effects due to differences in the blood collection time, we compared mild and severe dengue patient groups using mann-whitney u test, which showed no differences in the disease onset time at the moment of sample collection [see additional file ]. the original data used to perform this analysis is shown at figure . patients with mild and severe dengue were investigated for prior incidence of infection, detected by serologic immune response (igg antibodies for denv). patients with severe dengue ( %; out ) were more likely to be experiencing a secondary dengue virus infection than patients with mild dengue ( %; out ), although no statistical significance was found in fisher's exact test (p = . ). among patients with denv- , were classified as secondary infection, whereas among patients with denv- , were classified as secondary infection (p = . ). dengue fever is characterised by a high fever phase and an abrupt drop in body temperature that has been called defervescence phase. characteristically the disease outcome is defined during this phase, when patients can either recover rapidly or progress to a severe life-threatening stage. cytokines and immunoactivation markers such as ifn-γ, il- , soluble cd and receptors for tnf-α [ , ] are associated with the defervescence phase and with disease severity. ifn-α levels are higher in dhf than in df during defervescence [ ] . during the febrile phase significant increase in cytokine circulating levels was detected including il- , il- , il- , mcp- and mip- β levels, which were maintained also elevated in defervescence (data not shown, analysed by non parametric kruskal-wallis test and dunn's multiple comparison test when compared with controls, p < . ); no significantly altered febrile levels were found when compared to defervescence. during the febrile cytokine levels in plasma from patients with mild and severe dengue figure cytokine levels in plasma from patients with mild and severe dengue. box-and-whiskers graph. the box extends from the th to the th percentile and the line at the middle is the median. the error bars, or whiskers extend down to the lowest value and up to the highest. factors were sorted according to their functional groups. mann-whitney u test was used to evaluate differences between cytokine concentration from severe and mild dengue patients. * p < . , ** p < . and ** p < . . phase il- was significantly higher than in defervescence. il- β, il- , ifn-γ were significantly increased during defervescence as compared to control samples. significant levels of il- , il- , and il- were not detected during dengue disease in our patients. il- was detected both in healthy individuals and in dengue patients but no difference between these two groups was detected [see additional file ]. we studied the cytokine profile from brazilian patients in order to compare severe and mild dengue cases during the acute phase of the disease. figure shows data from patients with regard to their plasma cytokine contents, which were sorted in four groups according to their reported function. we observed that cytokine concentrations of il- β, ifn-γ, il- , il- , il- , il- and gm-csf were significantly increased during severe dengue as compared to mild dengue, while mip- β levels are higher in mild dengue. mip- β and ifn-γ were independent variables associated disease outcome as determined by a logistic regression model (table and figure ). while mip- β was increased during mild dengue with odds ratio (or) of . and confidence interval (ci) . - . , ifn-γ was associated with severity with or of . (ci, . - . ). to assess relationships between cytokine levels and several clinical manifestations, the patient cohort with severe dengue was divided into distinct subgroups: those with hypotension, thrombocytopenia (≤ . counts/mm ) and/or haemorrhagic manifestations. a logistic regression model was used for binomial response subgroups and glm models using gaussian family were employed for subgroups with continuous response in order to determine cytokine profiles. il- β was associated with marked thrombocytopenia with or = . (ci, . - . ) in dengue patients. tnf-α was inversely related to thrombocytopenia with or = . (ci, . - . ) (table , figure ). considering platelet counts as a continuous variable for statistical analysis with a gaussian family, it was possible to determine that il- (p = . ) and mcp- (p = . ) levels are inversely related to their counts, displaying therefore an association with thrombocytopenia, while mip- β (p = . ) confirms its association with higher counts -normal or tending to normal (table ) . gm-csf (or = . ; ci, . - . ) was related with hypotension, whereas il- β had a negative predictive table and figure . natural killer (nk) cells have been earlier related to mild cases of dengue [ ] . forty-eight pbmc samples from thirty-five patients had their cd + rates determined by flow cytometry and a good correlation was observed with cytokines detected in plasma as independent factors in predicting severity table . their respective mip- β plasmatic levels (r = . ; p = . ). considering that different cytokines act in the immunological network as stimulating/up regulating factors and also in a feedback loop as down regulators, the cytokine balance might play a role in the immune response outcome. therefore we calculated mip- β/ifn-γ ratios for every patient and compared those from mild dengue with those from severe dengue. ratio averages ± sem were respectively ± and ± (p = . ; mann whitney u test), confirming our earlier data that these cytokines are acting as opposing factors. the different models built here using clinical manifestations as independent variables each exhibit specific cytokine profiles. the cytokine profile identified in patients with dengue may represent a valuable tool for the characterisation of immunological response patterns and may assist the identification of patient groups at risk for developing severe disease. in the present study, the use of a multiplex analysis for cytokine plasma detection in patients with dengue could identify cytokine profiles associated with the disease severity. early identification and management of severe dengue disease are essential to prevent death. it has been increasingly recognized that the inflammatory response and deregulated cytokine production play key roles in the development of severe clinical manifestations [ ] . however, cytokine profiles associated with dengue evolution and prognosis are not well established. new technologies for cytokine quantification were developed including the multiplex immunofluorescent bead array analysis system, allowing multiple biomarkers to be tested simultaneously in a small volume from one single plasma aliquot. recently, this methodology has been used for cytokine profile evaluation during several infectious diseases including viral infections [ , , ] and sepsis [ ] , among others. we were able to identify models for cytokine circulation during dengue acute phase that may vary with clinical manifestations. mip- β was for the first time associated with a good prognostic and was identified in the different disease models presented here. mip- β has been earlier detected after dengue virus cell stimuli in vitro [ , ] but preliminary studies in vivo did not report their role in severity. in accordance with a protective role for mip- β, changes in mip- β levels were significantly correlated with decreases in viral titre after hepatitis c treatment [ ] . in addition, mip- β was up regulated in acute infection in chimpanzees only when viral clearance took place, but not in those animals which failed to eradicate the virus [ ] . mip- β is produced by human monocytes and dendritic cells upon different stimuli [ ] as well as by activated nk cells [ ] and lymphocytes [ , ] . activated nk cells release granzyme a, which displays cytolytic functions and mip- β is chemoattractant for nk cells, recruiting them to inflammatory sites. nk cells have been associated with mild dengue [ ] . here a good correlation between mip- β plasma levels and nk cells was observed, reinforcing the relevance of these pathways and strongly suggest- ing their role in dengue protective mechanisms. an early and efficient virus clearance by direct or indirect nk functions is likely controlling virus replication, restricting intense immunological activation and the dengue immunopathology and therefore favouring a mild dengue disease. in previous studies, tnf-α has been reported to be associated with severity, mainly during dhf in brazilian patients [ , , ] . in the present study, however, this cytokine was not strongly associated with severity. indeed, other authors also found inconsistency or no difference in tnf-α levels in severe vs. mild disease forms [ , ] . we may hypothesize that differences in dengue virus serotypes or in host immune response such as different tnf-α genetic polymorphisms may explain the disease outcome. in our study from (braga et al., ) , patients were dengue- infected, while in the present study, patients were dengue- and - infected. a recent report [ ] describes non-significant tnf-α serum levels in adult patients and suggests that the discrepancy may have been caused by a transient tnf-α peak which was not detected at a later time point. in the present study we observed an association of ifn-γ with disease severity. indeed, increased ifn-γ concentrations have been detected in dengue patients in a variety of studies [ , [ ] [ ] [ ] [ ] . dhf induced by dengue- was associated with higher viremia early in illness and earlier peak plasma ifn-γ levels; maximum plasma viremia levels correlated with the degree of plasma leakage and thrombocytopenia [ ] . however, in a previous study from our group we failed to observe association of ifn-γ with disease severity [ ] , probably due to the small number of severe patients analyzed or to the dengue- incidence. ifn-γ is produced during a t-lymphocyte helper response type and may reflect cd + t cell activation with production of inflammatory cytokines. high levels of ifn-γ were observed in patients with dengue from asian and latin america and were associated with severity [ ] . ifn-γ produced by t cells may also activate mononuclear phagocytes (monocytes and dendritic cells), which would produce factors such as tnf-α, tissue factor, and plateletactivating factor, among other mediators. these factors may all participate in platelet and endothelial cell activation, leading to platelet consumption, increase in endothelial permeability, hypotension and ultimately to shock. ifn-γ has also been associated with secondary heterologous dengue virus infections [ , ] inducing a strong antigenically cross-reactive inflammatory response, probably inefficient in terms of antibody and t-cell specific response. indeed, we observed earlier in several patients a cd +t cell activation with hla-dr+ subset increase that was associated with severity [ ] . gm-csf acts at early differentiation processes at myeloid progenitors or resting monocytes [ ] . an additional stimulus may be required to activate monocytes or dendritic cells in order to produce proinflammatory cytokines [ ] . gm-csf was associated with hypotension as well as mcp- , likely acting both in concert, contrasting with mip- β, once more associated with good prognostics. mcp- was earlier detected in dhf patients [ ] but for the first time we reported clinical and laboratory findings associated with severity. il- and mcp- , here associated with thrombocytopenia, are chemokines and may contribute to platelet activation, either by their chemoattractant properties or by their effect on endothelial permeability. both factors were detected in patients with dhf [ , ] . these cytokines are produced by monocytes after various activation stimuli, such as virus infection, and increase the endothelial permeability by disrupting tight junctions among cells [ ] . despite the fact that our study could identify cytokines with good accuracy for the stratification and/or prognosis of dengue, it has potential limitations. here we identified cytokines related to dengue severity, but the small sample size represents a shortcoming regarding the generalization of our results. in addition, only one time point was used for the measurement of cytokines, not allowing further insights provided by sequential measurements. moreover, classification of disease severity has been a matter of debate, especially for adult patients' management and classification. indeed, the who criteria for dhf has failed to identify severe disease, including fatal cases, in adult latin america population [ , ] (s.m.o. zagne, r.m.o. nogueira, unpublished) and clearly do not satisfy the stratification of our studied population. accordingly, in the present study severe disease forms were classified following other proposed criteria [ ] . while a direct correlation of cytokine concentrations and the pathophysiology of severe dengue is tempting, we believe that the full burden of disease severity cannot be attributed to a single cytokine. cytokines may be increased simply as one of the several steps in the network loops without necessarily playing a direct harmful role and most likely more than one factor may be involved, including others not tested here such as il- , tgf-β, and mif among others [ ] . we can suggest a mechanism explaining our cytokine models for dengue fever (figure ) . mip- β would be associated with a protective pathway for its chemoattractive and activating effect on nk cells, which in turn are efficient cells in early virus clearance, by their antiviral cytokine production and cytotoxic activity against infected target cells. ifn-γ has a deleterious effect for the host regarding its action in activating t cells for virus anti-genic cross-reactive response and monocyte/dendritic cell activation. mononuclear monocytes are activated by ifnγ and gm-csf among other cytokines and in turn produce several factors such as il- β and mcp- that may act on vascular permeability leading to plasma leakage and haemoconcentration. as suggested by other authors, it is likely that viral replication in antigen presenting cells, cytokine liberation and circulation, and t cell activation may not be a linear process [ ] , but in fact a complex interaction network, with positive and negative feedbacks, where viral clearance and pathologic events take place, such as increased vascular permeability and circulatory collapse, and their balance may determine the disease outcome. our study demonstrated the plasma cytokine profile in dengue fever from a brazilian population detected by a multiplex bead immunoassay. mip-β is indicated for the first time as a good prognostic marker and in contrast to ifn-γ that was associated with the disease severity. both cytokines can discriminate mild from severe cases. moreover, we show here for the first time that during the dengue course different cytokine profiles may be present and vary according to determined clinical manifestations. the cytokine profiles identified herein by bead array multiplex system may favour an early 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cytokine responses of dengue-specific cd + t cells to heterologous serotypes dengue-specific t cell responses in peripheral blood mononuclear cells obtained prior to secondary dengue virus infections in thai schoolchildren alternative activation of macrophages effects of granulocyte-monocyte colony-stimulating factor (gm-csf) on expression of adhesion molecules and production of cytokines in blood monocytes and ovarian cancer-associated macrophages mcp- , a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells il release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers predictors of spontaneous bleeding in dengue dengue and dengue hemorrhagic fever, brazil of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (dhf/dss) this work was supported by fundação oswaldo cruz (pdtsp-dengue), decict/conselho de desenvolvimento científico e tecnológico (cnpq), fundação de amparo à pesquisa do estado do rio de janeiro (faperj). the authors thank the program for technological development in tools for health-pdtis-fiocruz for use of its luminex facilities. we acknowledge in memoriam dr. jussara p. nascimento for her constant encouragement, dr. marcelo a. pinto for suggestions and dr. andrea schwager for the manuscript revision. the authors declare that they have no competing interests. fab and ogc contributed equally to the study. fab contributed to the study conception and design, carried out clinical studies, helped in data analysis and in drafting the manuscript. ogc performed data and statistical analysis. smoz carried out the clinical studies. efa and carried out the luminex immunoassays. ela collected and stored samples and patient data and helped in the luminex immunoassays. rmrn was responsible for the confirmatory diagnostics. ptb conceived the study and design and helped to draft the manuscript. cfk conceived the study and design, participated in data and statistical analysis and drafted the manuscript. all authors read and approved the final manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -g k vvdc authors: krog, j.; jepsen, c. f.; tønnesen, e.; parner, e.; hokland, m. title: the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets date: - - journal: scand j immunol doi: . /j. - . . aa.x sha: doc_id: cord_uid: g k vvdc materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr‐release assay using k as nk‐sensitive target cells. the pbmcs were characterized, using ‐colour flow cytometry, with special emphasis on the nk‐cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -q y sk authors: draborg, h.; roggen, e. l.; soni, n. k.; patkar, s.; friis, e. p.; lyngstrand, s. t.; christensen, l. l. h.; batori, v.; danielsen, s.; ernst, s. title: recominant expression and immunological characterization of house dust mite allergen der p date: - - journal: scand j immunol doi: . /j. - . . ag.x sha: doc_id: cord_uid: q y sk the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige‐mediated allergic reactions, while maintaining its ability to trigger proper th‐cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro‐der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro‐enzyme to a fungal signal peptide. the n‐glycosylation site of der p was mutated resulting in a deglycosylated pro‐enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active‐site‐titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro‐der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige‐binding assays and t‐cell proliferation assays. by in silico epitope mapping of a modelled ‐dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -zjyrhlxn authors: sigmundsdóttir, h.; johnston, a.; gudjónsson, j. e.; valdimarsson, h. title: differential effects of interleukin‐ and interleukin‐ on superantigen‐induced expression of cutaneous lymphocyte‐associated antigen and αeβ integrin (cd ) by cd (+) t cells date: - - journal: scand j immunol doi: . /j. - . . ab.x sha: doc_id: cord_uid: zjyrhlxn the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin‐ (il‐ ), il‐ and transforming growth factor (tgf)‐β are important regulators of chronic inflammatory disease, where cutaneous lymphocyte‐associated antigen (cla) and αe integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue‐specific homing but may help to retain t cells within epithelial layers. we have previously shown that il‐ alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd (+) but not cd (+) t cells. while il‐ increased superantigen‐stimulated expression of cla, this cytokine strongly inhibited the cd expression, and a combination of il‐ and tgf‐β completely abrogated the induced cd expression. conversely, il‐ suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th ‐mediated inflammatory responses, il‐ may also inhibit the migration of cd (+) t cells into the skin while il‐ promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il‐ and il‐ , and the balance between these cytokines could influence the t‐cell migration of inflammatory cells into epithelial tissues. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -pvz dll authors: nan title: abstracts for the scandinavian society for immunology th annual meeting and th summer school date: - - journal: scand j immunol doi: . /j. - . . .x sha: doc_id: cord_uid: pvz dll nan the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - sjsogvq authors: røntved, c. m.; dernfalk, j.; ingvartsen, k. l. title: do high and low tumour necrosis factor‐α responders exist in dairy cows? date: - - journal: scand j immunol doi: . /j. - . . v.x sha: doc_id: cord_uid: sjsogvq a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor‐α (tnf‐α) ex vivo. initially, a time‐ and dose‐dependent study was carried out to find the optimal stimulation conditions for the tnf‐α response. the tnf‐α response peaked between and h at . °c. a dose in the range of – g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf‐α response. thirty‐eight danish–holstein dairy cows were investigated for their tnf‐α responsiveness ex vivo in the periparturient period. heparin‐stabilized blood samples were collected seven times over a period of months (weeks − , − , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf‐α responsiveness occurred over time. moreover, the mean tnf‐α responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf‐α response, whereas others had high a tnf‐α response. we are currently investigating whether high and low tnf‐α responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf‐α responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -p hhd ed authors: Şahar, esra atalay; can, hüseyin; İz, sultan gülçe; döşkaya, aysu değirmenci; kalantari-dehaghi, mina; deveci, remziye; gürüz, adnan yüksel; döşkaya, mert title: development of a hexavalent recombinant protein vaccine adjuvanted with montanide isa v and determination of its protective efficacy against acute toxoplasmosis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: p hhd ed background: toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. a vaccine against t. gondii is required to prevent consequences of the infection. the aim of this study is to generate a multivalent recombinant protein vaccine against t. gondii. methods: previously discovered antigenic proteins of t gondii were evaluated by their expression level in e. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with montanide isa v. humoral and cellular immune responses were determined by flow cytometry and elisa. vaccinated mice were challenged with t. gondii ankara strain tachyzoites. results: in mice vaccinated with hexavalent vaccine, strong total igg (p < . ) and igg a (p < . ) responses were induced compared to controls, the ratio of cd (+) and cd (+) t lymphocytes secreting ifn-γ increased, and significantly higher extracellular ifn-γ secretion was achieved compared to the controls (p < . ). the survival time of the vaccinated mice increased to . ± . days which was significantly higher than controls (p < . ). conclusions: altogether, these results show that the hexavalent vaccine which is developed for the first time against t. gondii induced strong and balanced th and th immune responses as well as conferred significant protection against challenge with lethal toxoplasmosis in murine model. toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis in all warm blooded animals and humans. it is reported that one third of the world's population is estimated to be infected with t. gondii. t. gondii usually causes an asymptomatic infection in healthy people, but can be life-threating in immunecompromised patients (organ transplant recipients, acquired immunodeficiency syndrome patients, and cancer patients). congenital toxoplasmosis may cause abortion, neonatal death or foetal abnormalities in the foetus [ ] . farm animals such as sheep, goats, and pigs have also been shown to be susceptible to t. gondii infection. toxoplasmosis infection in farm animals causes significant economic losses as a result of prenatal death, abortion, and neonatal death. moreover, t. gondii is linked to mental disorders and may affect human behaviour, personality, and other phenotypic traits [ ] . cats and other felines are the definitive hosts of t. gondii [ ] . in the life cycle of t. gondii the main infective forms are tissue cysts (containing bradyzoites) and oocysts (containing sporozoites). the infection sources for humans are consumption of vegetables, fruits or water contaminated with faeces of infected cats containing oocysts and raw or undercooked meat contaminated with tissue cysts [ ] . for these reasons, development of a safe and protective vaccine against t. gondii infection that can be used in animals and humans has utmost importance. recombinant protein vaccines are safe and efficient, and have a great potential for prevention or eradication of diseases. one of the most important issues during a recombinant protein vaccine development is the selection of the vaccine candidate antigen(s) [ ] . antigens to be used in vaccine formulations against toxoplasmosis should actively induced strong immune response, produce long-lasting immunity, and be antigenic in each stage of the parasite [ ] . in our study group's previous studies, we used protein microarray containing candidate exon products of t. gondii to screen well-characterized sera from acute and chronic toxoplasmosis human cases and murine model infected orally with oocysts or tissue cysts. during these studies, screening human sera prioritized antigenic proteins and screening these antigens with murine sera prioritized proteins based on their immunogenicity [ ] [ ] [ ] . in this study, we selected six proteins based on their antigenic epitopes using bioinformatics and protein expression level in e. coli. thereafter, we developed a hexavalent recombinant protein vaccine adjuvanted with montanide isa v and administered to a murine model to determine its immunogenicity and protection efficiency against lethal toxoplasmosis. animals - week old female healthy swiss outbred mice were obtained from the bornova veterinary control institute animal production facility and used during the experiments. animals were housed under standard and suitable conditions. specifically rooms had ambient temperature and humidity, adequate light cycle, and diet was specific for each animal type. all animals were checked for humane endpoints every day such as rapid weight loss more than~ % of gross body weight, inability to assess water or food, or loss of skin elasticity indicative of dehydration. in any of these circumstances, we pre-euthanized the animals with ketamine hydrochloride ( mg/kg) and % xylazine ( mg/kg) and then euthanized with cervical dislocation. based on the data obtained from previous studies [ ] [ ] [ ] , proteins were selected based on their immunogenicity in humans and murine sera (table ). in order to determine the vaccine candidate antigens, expression levels of these proteins were analysed using western blot and moreover, a comprehensive bioinformatics analyses was performed. the gene sequences of the different genes were accessible from the toxoplasma genomic resource (http:// www.toxodb.org/toxo/). the plasmids expressing these recombinant proteins were constructed as previously described [ ] [ ] [ ] . thereafter, the plasmids were cloned into chemically competent escherichia coli (e. coli) bl star (de ) plyss cells according to the manufacturer's protocol (invitrogen, usa). e. coli bl star (de ) plyss cells containing different plasmids were incubated at °c with shaking at rpm for - h until the optical density (od ) reached an absorbance of , ng/μl. then, recombinant protein expression was induced with . mm iptg (isopropyl-d-thiogalactopyranoside) and the bacterial cultures were incubated for h, °c with shaking at rpm. next, cell cultures were harvested by centrifugation at ×g for min. the pellets were homogenized with lysis buffer [ . % triton x- , mm tris-cl and . m nacl (ph: . )] and were freeze-thawed times. the homogenates were incubated on a rotator at room temperature for min and then were centrifuged at . ×g for min. the supernatants were incubated with ml ni-nta superflow beads (qiagen, usa) for min with shaking at rpm. at the end of incubation, the suspension was centrifuged at rpm for min and the supernatant was discarded. then, the ni-nta beads were washed with mm tris-cl and . m nacl and mm imidazole (ph: . ) for min with shaking at rpm. thereafter, the suspension was centrifuged at rpm for min and the supernatant was discarded. next, ni-nta beads were incubated with mm tris-cl and . m nacl and . m imidazole (ph: . ) for min with shaking at rpm. finally, the suspension was centrifuged at rpm for min and supernatants were analysed with western blot as described below to determine the expression level of recombinant proteins. the most abundantly expressed recombinant proteins were selected for vaccine development. the protective immune response against toxoplasmosis is conferred by mainly by cellular immune response and through humoral immune response [ , ] . in addition, the expression of recombinant proteins of t. gondii will be performed in e. coli. t. gondii is a eukaryotic obligate intracellular parasite. protein post-translational modifications are common events in most eukaryotes, such as glycosylation. glycosylation of peptide effects protein immunogenicity and major histocompatibility complex (mhc) binding [ ] . thus, apart from the determination of the expression levels, proteins will analysed by bioinformatics tools to determine the presence of antigenic epitopes as well as glycosylation sites. at the end of the bioinformatics analyses, we aimed to select the proteins that have cellular and humoral immune response inducing epitopes with the least glycosylation sites. since t. gondii is an intracellular parasite, immunemediated protection against toxoplasmosis is mainly conferred by interaction between cd + t cells and mhc class i and cd + t cells and mhc class ii [ ] [ ] [ ] [ ] . antigenic sites recognized by cd + t cells and cd + are peptides containing to and amino acids associated with the mhc class i (mhc-i) and mhc-ii molecules, respectively [ ] [ ] [ ] . predicted epitopes of cd + t cells table the plasmids encoding selected t. gondii antigenic proteins toxodb a name plasmid name toxodb a name plasmid name tgme _ _ pa tgme _ _ pb tgme _ _ pb tgme _ _ pc tgme _ _ pc tgme _ _ pd tgme _ _ pd tgme _ _ pe tgme _ _ pe tgme _ _ pf tgme _ _ pf tgme _ _ pg tgme _ _ pg tgme _ _ ph tgme _ _ ph tgme _ _ pa tgme _ _ pa tgme _ _ pb tgme _ _ pb tgme _ _ pc tgme _ _ pc tgme _ _ pd tgme _ _ pd tgme _ _ pe tgme _ _ pe tgme _ _ pf tgme _ _ pf tgme _ _ pg tgme _ _ pg tgme _ _ ph [ ] [ ] [ ] [ ] [ ] [ ] . the potential linear b-cell epitopes of antigenic proteins were also analysed by support vector machine (svm) which has been utilized by combining the tri-peptide similarity and propensity scores (svmtrip; http://sysbio.unl.edu/ svmtrip/prediction.php) [ ] . in many eukaryotic pathogens, proteins may have nand o-linked glycosylation sites through post translational modification which may affect their interaction with their host organisms. protein glycosylation is common in t. gondii as it is in other eukaryotes [ ] . in this study, e. coli was used as the expression system and we aimed to select the vaccine candidate proteins among the recombinant proteins with least glycosylation sites in predicted antigenic epitopes. the potential n-glycosylation and oglycosylation sites of antigenic proteins were analysed using netnglyc . server (http://www.cbs.dtu.dk/services/netnglyc/) netoglyc . server (http://www.cbs. dtu.dk/services/netoglyc/) [ ] . according to protein expression levels and bioinformatics results, proteins were [ph (tgme _ _ ), pa (tgme _ _ ), pe (tgme _ _ ), pd (tgme _ _ ), pe (tgme _ _ ), ph (tgme _ _ )] were selected as vaccine candidate. thereafter, e. coli bl star (de ) plyss cells containing the ph , pa , pe , pd , pe , and ph stocks were inoculated into individual ml lb broth medium supplemented with ampicillin ( μg/ml) and incubated overnight at °c with rpm shaking. next day, the overnight cultures were inoculated into the bioreactor (bioflo , new brunswick, usa) containing . l enrichment medium supplemented with ampicillin ( μg/ml). the dissolved oxygen and ph levels were maintained at - and . ± . with vigorous mixing ( rpm) at °c until od reached . . then, the cell cultures were induced at a final concentration of . mm iptg and incubated for h at °c. the cells were centrifuged at ×g for min and the pellet was resuspended with ml pre-chilled loading buffer ( mm tris-cl, . m nacl, ph . ) and homogenized with a blender for s (waring, usa). then, the homogenized cells were disrupted twice using a microfluidizer processor (microfluidics m- l pneumatic, usa) at a low temperature under internal pressure of , psi and centrifuged at , g for ½ h at °c. the homogenates were centrifuged at , ×g for min at °c. the clarified supernatants were filtered using . μm pore filter (corning, usa). the filtered samples were purified with akta fast protein liquid chromatography (fplc) system, controlled by uni-corn™ software (ge health, usa), using ml hitrap ni + chelating hp column (ge health, usa). approximately ml filtered supernatant was loaded to the hitrap ni + chelating hp column. after binding, the column was washed with buffers containing increasing concentrations of imidazole ( mm, mm, mm). the recombinant proteins (rh , ra , re , rd , re , and rh ) were eluted by raising the imidazole concentration to . m. purity and identity of the purified proteins were analysed by % (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) sds-page and western blot analysis and concentrated with vivaspin (sartorius, germany). the proteins were further purified by fplc on a superdex / - gl [ - , molecular weight cut-off (mwco)] column (ge health, usa) to remove excess endotoxin. the subsequent protein fractions were pooled, concentrated and quantitated by bradford method (pierce, usa). to observe the expression levels, purity and immunoreactivity, proteins were separated by % sodium dodecyl sulfate-polyacrylamide gel (sds-page). the separated proteins were transferred to polyvinylidene difluoride (pvdf) transfer membrane (immobilon-p, millipore, ma), blocked by . % non-fat dry milk for h at room temperature. the membranes were probed with a / dilution of monoclonal anti-polyhistidine antibody (sigma-aldrich, usa) for . h at room temperature. then the membranes were probed with a / dilution of alkaline phosphatase-conjugated goat anti-mouse igg (h + l) antibody (sigma-aldrich, usa) for h at room temperature. the blot was developed with diethanolamine buffer ( % diethanolamine, m hcl ph: . , the amount of endotoxin in the purified vaccine candidate recombinant proteins were determined with limulus ameobocyte lysate (lal) gel-clot test using pyrotell single test vials according to the manufacturer's protocol (cape cod inc., usa). the test sensitivity was . endotoxin units (eu)/ml. e. coli o :h control standard endotoxin (cape cod inc., usa) was used for positive control and lal reagent water was used for negative control. the concentration of endotoxin-depleted, purified recombinant protein samples were calculated with bradford method (pierce, usa) and stored at − °c until use. vaccination and t. gondii challenge infection - weeks old female swiss webster outbred mice were randomly divided into four groups, each group consisting of eleven mice (n: ). mice were vaccinated intraperitoneally (i.p.) twice at weeks intervals. vaccines and control groups as shown in table . the first group was immunized with hexavalent ( antigens) recombinant proteins adjuvanted with montanide isa v (seppic, france) prepared according to the manufacturer's protocols. montanide isa v was used as adjuvant due to its efficiency in inducing both humoral and cellular immune response. three groups were considered as control; one control group was administered only with hexavalent recombinant proteins without montanide isa v adjuvant. the adjuvant control group was inoculated with only montanide isa v [ μl montanide isa v adjuvant + μl phosphate buffered saline (pbs)] and the last control group was inoculated only pbs ( μl pbs). tail bleeds were performed weeks after each vaccination. nine weeks after first vaccination, eight mice from all four groups were challenged intraperitoneally with × tachyzoites of t. gondii local strain called ankara [ ] . thereafter, the infected mice were observed for the symptoms of toxoplasmosis such as loss of fur brightness and appetite and survival times were recorded on a daily basis. detection of total igg and igg subclass antibody response using rec-elisa determination of t. gondii specific total igg, igg and igg a antibodies in vaccinated mice were performed by recombinant enzyme-linked immunosorbent assay (rec-elisa) as described [ , ] . in brief, each well of microplates (nunc, usa) were coated with μl of recombinant proteins solution (containing μg/ml of each rh , ra , re , rd , re and rh in × pbs) and incubated overnight at °c. next day, plates were washed times with μl pbs-t ( . % tween in pbs) and then blocked with % nonfat dry milk containing . % pbs-t for h at room temperature. mice sera were diluted to / with blocking buffer supplemented with e. coli lysate at a final concentration of mg/ml protein to block anti-e. coli antibodies and incubated for min at °c. then, the mouse sera were added to the wells in duplicate and incubated for h at °c with gentle shaking. after three washes with μl pbs-t, the plates were incubated with μl of anti-mouse igg to evaluated cytokine production, three mice from per group were euthanized weeks after the prime vaccination and their spleens were removed. single-cell suspensions of splenocytes were prepared as previously described [ , ] . aliquots of × viable splenocytes in growth medium [ × rpmi supplemented with % fcs (nbcs, hyclone, thermo fisher scientific, usa), mm l-glutamine (gibco, invitrogen,usa) penicillin ( u/ml) and streptomycin ( μg/ml) (sigma-aldrich, usa), . mm non-essential amino acid and thereafter, the cells were permeabilized and labelled with pe conjugated rat anti-mouse ifn-γ (bd biosciences, usa) or pe conjugated rat anti-mouse il- antibodies (bd biosciences, usa) according to the manufacturer's recommendations. antibodies were diluted in perm/wash solution (bd biosciences, usa) for intracellular staining. all antibodies were used at a final concentration of . μg/ cells. t cell populations of cells, gated with cd + positive expression, were analysed to quantify: the percentage of rh , ra , re , rd , re and rh proteins specific cd + t lymphocytes secreting ifn-γ and cd + t lymphocytes that secreted il- and ifn-γ using facs diva software (bd biosciences, usa). all data were obtained on a bd facsaria flow cytometer (facsaria; bd biosciences, usa). data obtained during the experiments were processed using microsoft excel software and prism . program (graphpad, san diego, ca). a two-tailed unpaired t-test with % confidence interval was used to determine the significance between the vaccination groups. kaplan-meier survival curves were constructed to illustrate protection from lethal toxoplasmosis. humoral and cellular immune responses and survival time were expressed as mean ± standard deviation (s.d.). the expression of recombinant proteins were induced with . mm iptg when growing cells reached an absorbance of . ng/μl at od nm. the cell cultures were harvested h after induction, homogenized with lysis buffer and purified by ni-nta beads. then, the recombinant protein expression levels were assessed by western blotting and rh (tgme _ _ ), ra (tgme _ _ ), re (tgme _ _ ), rd (tgme _ _ ), re (tgme _ _ ), and rh (tgme _ _ ) had detectable bands at . kda, . kda, . kda, . kda, . kda, and . kda, respectively. bioinformatic analyses to predict mhc-i, mhc-ii, and b cell epitopes of the recombinant proteins using immune epitope database and analysis resource (iedb) and svmtrip showed that there were , , , , , and predicted mhc-i epitopes (fig. a) ( table ) , , , , , , and predicted mhc-ii epitopes (fig. b) (table ) and , , , , , and predicted b-cell epitopes (fig. c) (table ) in re , rd , rh , re , ra and rh , respectively. the n-glycosylation and o-glycosylation sites of antigenic proteins were predicted using netnglyc . server and netoglyc . server. moreover, n and oglycosylation sites on the predicted mhc-i, mhc-ii, and b-cell epitopes were also analysed ( table ) . the results showed that rh , re , rd , re , ra , and rh have ( . %), ( . %), ( . %), ( . %), ( . %), and ( . %) o-glycosylation sites, respectively (fig. a) ( table ) . regarding n-glycosylation, rd , ra , re and rh proteins have ( . %), ( . %), ( . %), ( . %) sites respectively, while rh and re proteins didn't have n-glycosylation site (fig. b) (table ) . next, we next focused on the potential glycosylation sites inside predicted mhc-i, mhc-ii, and b cell epitopes of re , rd , rh , re , ra , and rh . the results showed that in the mhc-i epitopes, re and rh have only one n-glycosylation site, re , re , rh , and rd have , , and o-glycosylation sites, respectively (table ). in mhc-ii epitopes, re and rh have and one n-glycosylation site, re , rd , rh , re , and rh have , , , , and o-glycosylation sites, respectively (table ) . on the other hand, b-cell epitopes of re , rd , rh , re , and ra have , , , and oglycosylation sites but did not contain any n-linked glycosylation site (table ) . based on the protein expression levels and mhc-i, mhc-ii, and b cell epitope prediction results, rh , ra , re , rd , re , and rh were grown in big batches of lb using a bioreactor, purified in on a hitrap ni + chelating column and then polished on a gel filtration column to remove excess endotoxin. the purity of rh , ra , re , rd , re and rh were assessed by sds-page and western blotting as shown in fig. a and b. the purified rh , ra , re , rd , re and rh had apparent molecular weight of approximately . kda, . kda, . kda, . kda, . kda, and . kda, respectively. some of the proteins gave several bands possibly due to multimerization of recombinant protein, degradation or stalling of protein synthesis in e. coli. to determine total igg, igg and igg a antibodies against rh , ra , re , rd , re , and rh proteins in adjuvanted and control group mice serum samples, rec-elisa was performed. according to the results, total igg response detected at day was significantly higher in sera of mice administered with hexavalent recombinant protein mixture (+) montanide isa v vaccine and the control group administered with only hexavalent recombinant protein mixture compared to the pre-vaccination sera (p < . , ***). in control groups administered with pbs or montanide isa v didn't induce a significant total igg response. significantly high levels of total igg immune response was detected in the hexavalent recombinant protein mixture (+) montanide isa v when compared with the mice administered with hexavalent recombinant protein mixture (p = . , **) (fig. ) . to assess the polarization of igg /igg a which is a preliminary marker of whether vaccine induced the th or th immune response, the igg and igg a response was analysed by rec-elisa. the polarization of igg and igg a response is shown in fig. . hexavalent recombinant protein mixture (+) montanide isa v vaccine induced significantly high levels of igg and igg a at day compared to the controls groups (p < . , **). overall, in mice administered with hexavalent recombinant protein mixture (+) montanide isa v, igg a and igg levels showed a strong and balanced immune response. in control groups, pbs or montanide isa v didn't induce a significant igg or igg a response. (fig. ) . single-cell suspensions of splenocytes obtained from mice administered with hexavalent recombinant protein mixture (+) montanide isa v and controls were stimulated with purified rh , ra , re , rd , re , and rh proteins. concentration of extracellular cytokine il- and ifn-γ were determined using elisa. according to the results, ifn-γ level was significantly higher in mice vaccinated with the hexavalent recombinant protein mixture (+) montanide isa v compared to mice vaccinated with only montanide isa v (p = . ,***) or pbs (p = . ,***) (fig. a) . on the other side, il- level was also significantly high in mice vaccinated with the hexavalent recombinant protein mixture (+) montanide isa v compared to control groups administered with pbs (p = . , *) or hexavalent recombinant protein mixture (p = . , *) (fig. b) . flow cytometry analysis was used to determine the ratio of cd + t lymphocytes secreting ifn-γ and cd + secreting ifn-γ and il- in vaccinated and control groups. protection against intracellular parasite like t. gondii is primarily achieved by cd + t lymphocytes secreting ifn-γ. for this reason, cd + t cell response is important for an ideal vaccine against toxoplasmosis [ , , ] . t cell population has been gated with cd + staining. in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v vaccine, the ratio of cd + t lymphocytes secreting ifn-γ increased , . and . times compared to control groups vaccinated with pbs, montanide isa v, and only hexavalent recombinant protein mixture (fig. a) . besides, the ratio of cd + t lymphocytes secreting ifn-γ increased . and . times in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v compared to control groups vaccinated with pbs, montanide isa v, and only hexavalent recombinant protein mixture (fig. b) . on the other hand, the ratio of cd + t lymphocytes secreting il- in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v increased . , . , and . times compared to control groups vaccinated with pbs, montanide isa v, and only hexavalent recombinant protein mixture (fig. c) . cd + t cells from all experimental groups proliferated to comparable ratios in response to cona (data not shown). protection against lethal toxoplasmosis in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v was determined using t. gondii ankara strain tachyzoites. eight from each the results are summarized in fig. . recently, our study group has discovered some t. gondii proteins that can be used as vaccine candidate against toxoplasmosis using in silico and immunoscreening approaches based on protein microarrays [ , ] . we selected the antigens from this screening approach because antigens selected without a screening approach ended with disappointing clinical results such as the malaria vaccine rts,s which is a hybrid protein particle designed in s. the major surface glycoprotein (msg) containing hybrid protein was formulated in a multi-component adjuvant (as ) and showed % protection in east african children in [ ] . for these reasons, development of a multivalant recombinant protein vaccine using some of these discovered proteins was the aim of this study. for this purpose, we used the data from protein microarray screening and prioritized proteins based on their immunogenicity. to further analyse these proteins for their availability for vaccine development against toxoplasmosis, we conducted small scale protein expression experiments in conjunction with bioinformatics analyses. according to protein expression levels proteins were suitable to be used in the vaccine development. among them, rh (tgme _ _ ) is a dnak family protein, ra (tgme _ _ ) is rop , re (tgme _ _ ) is a sag-related sequence srs a, rd (tgme _ _ ) is an fig. extracellular a ifn-γ and b il- levels elicited by hexavalent recombinant protein mixture (+) montanide isa v and control groups. the red bars represent ifn-γ response and the green bars, il- response. each bar represents the mean ± sd value of ifn-γ and il- responses of mice from each group. each bar represents the mean ± sd value of ifn-γ and il- responses of mice from each group. single cell suspensions were stimulated with rh , ra , re , rd , re , and rh recombinant proteins with a final concentration of μg/ml. in figure, *** represent p ≤ . and * represent p ≤ . ubiquitin carboxyl-terminal hydrolase, re (tgme _ _ ) is a hypothetical protein, and rh (tgme _ _ ) is a plectin. the protection against t. gondii infection is dependent on cd + t-cytotoxic lymphocytes which play a significant role in cell mediated protection as well as b cells which is important in humoral immune response [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for this reason, mhc-i, mhc-ii, and b-cell epitopes of the vaccine candidate proteins were predicted by bioinformatics. svmtrip online service was used to analyse the b-cell epitopes of re , rd , rh , re , ra , and rh proteins. as shown in fig. c , the presence of b-cell epitopes on the vaccine candidate proteins suggests that they have a strong potential to act as a bcell antigen. we also used the online service iedb to analyse mhc-i and mhc-ii epitopes of re , rd , rh , re , ra , and rh proteins and found mhc-i and mhc-ii epitopes on the vaccine candidate proteins ( fig. a and b) . protein glycosylation has great importance in terms of protein stability, three-dimensional structure, surface expression, activity, and antigenicity [ ] . previously, it was shown that t. gondii contains n-linked glycosylated proteins and o-linked glycosylated proteins by lectin-probed western blot analysis. moreover, it is reported that n and o-linked glycosylated proteins are found throughout the secretory pathway of the t. gondii and n-linked glycosylation of proteins is essential for the survival of parasite [ ] . for this reason, nand o-linked glycosylation sites of antigenic proteins, which may be candidates for vaccination, was predicted by bioinformatics. the results suggest that the vaccine candidate proteins contain n-linked glycosylation sites except for the rh and re proteins (fig. b) . o-linked glycosylation sites are found on re , rd , rh , re , ra , and rh proteins (fig. a) . at this stage, we further examined the n-and olinked glycosylation sites in b cell, mhc-i, and mhc-ii epitopes of the vaccine candidate antigens by bioinformatics. the results demonstrate that b-cell epitopes of vaccine candidate proteins were not containing n-linked glycosylation sites. b-cell epitopes of re , rd , rh , re , ra were o-glycosylated ( table ). the n-linked glycosylation site in the mhc-i epitopes were only found in the re and rh proteins and the o-linked glycosylation is detected in the re , re , rh , and rd proteins ( table ). the n-linked glycosylation site in the mhc-ii epitopes were similarly found in the re and rh proteins and the o-linked glycosylation is detected in the re , rd , rh , re , and rh proteins (table ). these results show that the vaccine candidate proteins and their epitopes are glycosylated at various ratios and their antigenicity is high. thereafter, we developed a hexavalent recombinant protein protein vaccine adjuvanted with montanide isa v which has shown to induce strong cellular and humoral immune response. after vaccination of swiss webster outbred mice, strong total igg, igg and igg a responses were detected in mice administered with hexavalent recombinant protein mixture (+) montanide isa v compared to control groups vaccinated with only montanide isa v or pbs (p < . ) indicating strong and balanced th and th responses (figs. and ) . the production of extracellular ifn-γ was significantly higher in mice vaccinated with the hexavalent recombinant protein mixture (+) montanide isa v compared to mice vaccinated with only montanide isa v or pbs (p < . ) (fig. a) . flow cytometry results were also in compatible with extracellular elisa in which hexavalent recombinant protein mixture (+) montanide isa v showed increment in ratio of cd + and cd + t lymphocytes secreting ifn-γ ( fig. a and b) . cd + t lymphocytes secreting il- were also increased according to flow cytometry results as well as there was an increase in extracellular il- secretion according to elisa (figs. b and c) . flow cytometry results, specifically cd + t lymphocytes secreting il- cell ratio is bigger than cd + t lymphocytes secreting ifn-γ which contradict with elisa results in which ifn-γ was higher than il- secretion. this discrepancy can interpreted as ifn-γ levels detected by elisa can be related to cd + t lymphocytes secreting and macrophages other than cd + t lymphocytes. on the other side, the main protective cells against toxoplasmosis are conferred by cd + t lymphocytes secreting ifn-γ which has increased with recombinant protein mixture (+) montanide isa v compared to controls. the protective efficacy of hexavalent recombinant protein mixture (+) montanide isa v against lethal toxoplasmosis was evaluated by infecting mice intraperitoneally with t. gondii ankara strain tachyzoites. t. gondii ankara strain is africa genotype and causes death in mice in approximately - days [ ] . challenging study showed that survival was prolonged from to days which was observed in control group mice administered with only montanide isa vand pbs to more than days in two mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v (fig. ) . in this study, montanide isa v was selected as an adjuvant due to its efficiency in inducing both humoral and cellular immune responses. montanide isa v was used as adjuvant in previous studies against bovine herpesvirus , boophilus microplus, foot and mouth disease virus (fmdv), and leishmania major [ ] [ ] [ ] [ ] . in the vaccine trial against fmdv, boophilus microplus, and leishmania major, montanide isa v induced significant levels of protective cytokine production and/ or antibody response. during the vaccine trial with recombinant glycoprotein d of bovine herpesvirus , a mixed th /th response was elicited [ ] . in this study, hexavalent recombinant protein mixture (+) montanide isa v showed strong and balanced th and th responses. overall, the th part of the immune response elicited by hexavalent recombinant protein mixture (+) montanide isa v induced significant levels of cd + and cd + t lymphocytes secreting ifn-γ and conferred significant protection in swiss outbred mice challenged with lethal dose of t. gondii ankara strain tachyzoites. in literature, multivalent recombinant protein vaccines have been developed against toxoplasmosis. in these studies, surface related antigens rsag , rsag , rsag , rsrs , rp , rsrs , and rsrs ; dense granule proteins rgra , rgra , rgra , rgra , rgra , and rgra ; rhoptry proteins rrop , rrop , and rrop as well as rtgpi- , rmag and rbag have been used [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in all of the recombinant protein or dna vaccine studies against toxoplasmosis, murine models are being used to determine the immunity and protection conferred by vaccinations. michima et al., vaccinated mice with a mixture of rsag , rsag , rsag , rsrs , and rp proteins adjuvanted with freund and achieved % survival up to days after i.p. challenging with t. gondii beverley strain bradyzoites [ ] . golkar et al., vaccinated mice with a mixture of rgra and rgra proteins adjuvanted with monophosphoryl lipid a and achieved . % decrease in brain cyst formation after i.p. challenging with t. gondii pru strain cysts [ ] . in one study, rrop , rgra , rgra proteins and cholera toxin were administered through intranasal route to mice and oral challenge with veg cysts resulted in . % decrease in brain cysts compared to controls [ ] . in a study that used antigenic epitopes of sag , gra , and mag proteins adjuvanted with freund decreased the brain cysts formation by % [ ] . dziadek et al., , evaluated rrop , rrop , rgra , and rsag in three vaccine formulations adjuvanted with incomplete freund administered subcutaneously to mice and challenge with t. gondii dx cysts results with to % decrease in parasite burden [ ] . in another study, a synthetic peptide, generated using b-cell and two t-cell epitopes derived from sag , gra , and gra antigens was adjuvanted with freund and challenge with gjs tachyzoites increased survival time [ ] . in other study, mice were vaccinated with the mixture of rrop and rsag adjuvanted with freund and challenged with the t. gondii rh strain. the results showed that t. gondiispecific igg antibodies levels and lymphocyte proliferative responses are increased in vaccine group and conferred more efficient protection compared to the control groups [ ] . sun et al., vaccinated mice with a mixture of rbag , rsrs , and rsrs proteins adjuvanted with freund or recombinant mindin. the results showed that vaccine using mindin as an adjuvant efficiently stimulated humoral and cellular responses, including antigen-specific igg and igg a, as well as lymphocyte proliferation. also the improved protection against t. gondii infection was observed in the mindin adjuvanted vaccine group compared with the other controls [ ] . in another study, t-and b-cells epitopes of ama , ron , and ron proteins was used to develop multivalent peptide vaccine formulations. the iga levels were increased in the mice immunized with single rron , while the igg levels were higher in the mice immunized with rama (+) rron. significant level of ifn-γ was detected in mice immunized with rama (+) rron . infection with t. gondii is naturally occurs through the oral route by water or food contaminated with tissue cysts (containing bradyzoites) or oocysts (containing sporozoites). interestingly, in this study, the mice were challenged orally with × tachyzoites of t. gondii rh strain. it was reported that the control mice died within days. the mice immunized with a + r achieved % survival rates. the mice immunized with ama , a + r + r , and ron achieved , , and % survival rates, respectively [ ] . another vaccine trial using t-and b-cells epitopes of sag , gra , gra , and rop proteins resulted with higher levels of igg and igg a subclass titters, significant production of ifn-γ, percentage of t lymphocyte subsets and longer survival times against intraperitoneal challenge with t. gondii rh strain tachyzoites [ ] . rgra and rbag were used in a multivalent recombinant protein vaccine adjuvanted with alum. significant increase in igg, igg a subclass titters as well as significant increment in ratio of ifn-γ secreting cd + and cd + t lymphocyte subsets were achieved. mice were challenged orally with - t. gondii pru strain tissue cysts and the amount of tissue cysts in vaccinated group decreased . % compared to control groups. in sum, multistage and multivalent rbag and rgra vaccine increased immune response but induced partial protection against toxoplasmosis [ ] . picchio et al., vaccinated mice with rtgpi- , rrop , and rgra proteins ajuvanted with alum intradermally or with cpg-odn intranasally and mice were orally challenged with the t. gondii me tissue cyst. p + r + g vaccine formulations induced significant decreases in the number of cysts per brain compared to the control group. according to the levels of igg and igg a subclasses p + r + g vaccine groups showed a mixed th /th immunity [ ] . overall, in the present study a hexavalent recombinant protein vaccine adjuvanted with montanide isa was first time developed and administered to mice to protect against lethal toxoplamosis. moreover, the immunogenic and protective efficiency of rrop , dnak family protein, srs a, ubiquitin carboxyl-terminal hydrolase, and plectin was first time tested in an animal model. in addition, montanide isa v was first time used as an adjuvant in mice model against toxoplasmosis. apart from these, multiplexing recombinant proteins induced strong and balanced th and th immune 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toxoplasma gondii in balb/c mice protective immunity induced by peptides of ama , ron and ron containing t-and b-cell epitopes via an intranasal route against toxoplasmosis in mice toxoplasma gondii: vaccination with a dna vaccine encoding t-and b-cell epitopes of sag , gra , gra and rop elicits protection against acute toxoplasmosis in mice vaccine potential of antigen cocktails composed of recombinant toxoplasma gondii tgpi- , rop and gra proteins against chronic toxoplasmosis in c h mice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions eaŞ, hc, ayg, md conceived the study and participated in its design. eaŞ, hc, sgİ, add, mkd, md participated in in vitro and in vivo studies. eaŞ, hc, rd, ayg, md helped in discussion of results. eaŞ and md performed the statistical analyses. eaŞ, hc, ayg, md interpreted the results and drafted the manuscript. all authors read and approved the final manuscript. this study was supported by the scientific and technological research council of turkey (tubitak) grant s to ayg. the funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. the dataset analyzed during the current study is available from the corresponding author on reasonable request. the animal study was performed under the instructions and approval of the institutional animal care and use committee (iacuc) of ege university for animal ethical norms (permit number: - ). not applicable. the authors declare that they have no competing interests. key: cord- -eg wcd authors: zheng, zhihang; li, min; liu, zhihua; jin, xia; sun, jin title: establishment of murine infection models with biological clones of dengue viruses derived from a single clinical viral isolate date: - - journal: virol sin doi: . /s - - -y sha: doc_id: cord_uid: eg wcd dengue virus (denv) is a single-stranded rna virus transmitted by mosquitoes in tropical and subtropical regions. it causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome in patients. each year, million people are estimated to be infected by four serotypes of dengue virus, creating a great burden on global public health and local economy. so far, no antiviral drug is available for dengue disease, and the newly licensed vaccine is far from satisfactory. one large obstacle for dengue vaccine and drug development is the lack of suitable small animal models. although some denv infection models have been developed, only a small number of viral strains can infect immunodeficient mice. in this study, with biologically cloned viruses from a single clinical isolate, we have established two mouse models of denv infection, one is severe lethal infection in immunocompromised mice, and the other resembles self-limited disease manifestations in balb/c mice with transient blockage of type i ifn responses. this study not only offers new small animal models of dengue viral infection, but also provides new viral variants for further investigations on dengue viral pathogenesis. dengue virus (denv) is an arbovirus transmitted by aedes aegypti and aedes albopictus mosquitoes. it belongs to flavivirus genus of flaviviridae family and contains a positive single-stranded rna genome. denv can be antigenically classified into four serotypes (denv- - ), which are often co-circulating in the endemic regions with similar clinical manifestations. though the majority of denv infections are asymptomatic, a minority of which can result in self-limited disease including dengue fever (df), and less than % may develop into dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). because of the global warming, increased air travel, and the lack of an efficacious vaccine, denv has become the most prevalent mosquito-borne viral pathogen in recent decades (guzman et al. ) . each year, denv is estimated to infect million people globally, affecting nearly half of the world population. asia accounts for % of the dengue disease burden, followed by latin america and africa (bhatt et al. ) . as an imported pathogen in china, dengue virus had caused sporadic outbreaks in southern china before . however, an unexpected large dengue outbreak attacked guangzhou in , resulting in , national reported cases that year (jin et al. ) , in which the majority was caused by dengue virus serotype (denv- ), and the others by serotype (denv- ) (zhao et al. ; li et al. ) . since then, - confirmed cases were reported to china cdc (chinese centers for disease control and prevention) each year. in nature, non-human primates and human are the only mammalian hosts for denv infection. naturally acquired dengue virus does not infect any outbred or inbred mice, unless viruses are inoculated with extremely high dose (e.g. - pfu of denv- in -week old c /bl ) (chen et al. ) or through intracerebral injection. in some other models, mouse-brain adapted strains are used. but such infection of adapted strain or intracerebral infection usually results in neurological manifestations, which are not routine clinical signs of human diseases. and, after passaging in mice, characteristics of these mouse brain-adapted viral strains may differ from strains naturally acquired, attenuation in their ability to cause human disease was documented (sabin and schlesinger ; hotta ; sarathy et al. ) . thus, the inability of dengue virus to replicate in murine model had hampered the development of dengue vaccine and antiviral therapy. monkey models, while more useful, are not suitable for routine preclinical testing of vaccine candidates or drug prototypes because of their high cost, limited animal availability and ethical concerns. the species specificity of denv infection may be partially attributed to the differences of innate immunity between primates and mice. interferon (ifn) signaling plays an essential role in the protection against dengue disease in humans, nevertheless denv has also evolved many mechanisms to antagonize ifn signaling and ifn production in vivo (green et al. ) . one of them is the binding and degradation of human stat by dengue viral protein ns (ashour et al. ) . another is the cleavage of human sting molecules by dengue viral protease ns b (aguirre et al. ; yu et al. ) . interestingly, both of these two antagonistic mechanisms only exist in human. denv ns does not bind to murine stat , neither does ns b cleave murine sting proteins, because the target sequences on these two molecules are highly species specific (ashour et al. ; aguirre et al. ; stabell et al. ) . but deletion of mstat or msting could enhance the replication of denv in mouse or mouse derived primary cells (ashour et al. ; aguirre et al. ) . these observations also imply the necessity of ifn signaling and ifn production in restricting denv infection in mice. therefore, immunocompromised mice without complete ifn responses or humanized mice are presumed more susceptible to denv infection. as humanized mice are laborious to prepare, and often introduce mouse-to-mouse variation, they are less suitable for application in vaccine development (akkina et al. ) . in contrast, knockout mice are easier to manipulate and a series of ifn deficient mice have been tested in the development of dengue infection model, including those of ifna/br -/-, ifncr -/-, ifna/b/cr -/-, mavs -/-, stat -/-, stat -/-, irf -/and irf -/- (shresta et al. ; perry et al. ; prestwood et al. ). among them, type i/ii ifn receptors double knock out mice usually develop lethal diseases when challenged with suitable denv strains, thus are used more frequently. however, only a limited number of dengue virus strains are able to replicate in mice and reproduce the severe symptoms (e.g., vascular leakage) of human disease. these viruses are usually based on clinical isolates (denv- c / , denv- - , denv- tvp- ), obtained through alternate passage between mosquito c / cells and ag mice (denv- d s ), or isolated after plaquepurification (denv- d y p-pp , denv- s ) (sarathy et al. ) . such mouse models are valuable in testing vaccine efficacy and studying viral pathology or transmission. notably, there are an increasing number of investigations that suggest t cell immunity is an essential component for flavivirus vaccine (st john and rathore ). but, type i interferon are vital for cd ? and cd ? t cell generation and maturation (crouse et al. ) . and ifn-c is also one of the major cytokines mediating th /ctl function and t cell development. therefore, ifn deficient transgenic mice are not ideal for studies of t cell immunity or t cell vaccines in an active immunization model, for the lacking of cross-talk between ifn pathway and adaptive immunity. in this study, we have purified two dengue virus strains of serotype exhibiting different plaque sizes from one clinical isolate denv- gz. through subcutaneous injection of low doses of these viruses to type i/ii ifn receptor knock-out mice, we successfully established mouse models of lethal infection. the pathogenicity of the two variants in ag was demonstrated to be different. further, we compared the infectivity of these two viral variants in a self-limited infection model, in which type i ifn receptor of wild-type balb/c mice had been transiently blocked before infection, and found only the virus strain exhibiting larger plaque size caused infectious viral particles in sera. our research here provides two kinds of dengue virus infection mouse models, which reproduce both severe and self-limited manifestations of human diseases, expanding the current choice of dengue viral infection small animal models. dengue virus serotype clinical strain, denv- gz, was originally isolated from a patient in guangzhou in , and was kindly provided by dr. xiaoyan che of zhujiang hospital in guangzhou, china. the virus was propagated in mosquito c / cells for no more than three passages in our lab. virus titer was determined in vero cells by foci forming assay. vero cells were passaged in dulbecco's modified eagle's medium (dmem, gibco, ny, usa) supplemented with % fetal bovine serum (fbs, gibco, auckland, new zealand) and % penicillin/streptomycin (p/s, gibco, ny, usa), and cultured at °c in a % co incubator. mosquito c / cells were cultured in minimum essential medium (mem, gibco) supplemented with % fbs, % penicillin/streptomycin and % non-essential amino acids (gibco, ny, usa) in a °c incubator under % co atmosphere. to purify viruses of different plaque morphology from the original clinical isolate, virus stock of denv- gz was serially diluted, and used to infect c / cell at , , , , and / pfu per well in a -well plate. seven days post infection, supernatant of each well was harvested. the foci size of virus that produced in each well was determined subsequently in vero cells by foci forming assay in -well plate. only supernatant containing virus with uniform foci size was kept for further propagation. after three passages, two viral clones maintained different plaque sizes, herein named as denv- d - -sp (variant with small plaque size) and denv- h - -lp (variant with large plaque size), were stored in a - °c freezer. titer of virus stock and infectious viral particle in serum of mice were determined using foci forming assay in vero cells. specifically, vero cells were seeded at cells/ well in a -well tissue culture plate and incubated overnight at °c, % co . on the second day, sera of infected mice or viral stocks were serially diluted in dmem and added into wells containing vero cells for a h incubation period. subsequently, the viral inoculum was removed and dmem supplemented with . % fbs, % p/s, . % carboxymethylcellulose (cmc, sigma, mo, usa) was added. after incubation for h at °c, cells were fixed with % paraformaldehyde (pfa). following the removal of pfa, infected cells were stained with antidengue monoclonal antibody d - ( : , santa cruz biotechnology, ca, usa) for h, followed by addition of biotin labeled anti-mouse igg (santa cruz biotechnology, ca, usa), and then streptavidin-alkaline phosphatase (sigma, mo, usa). finally, the foci of dengue virus were visualized by adding bcip/nbt substrate (beyotime biotechnology, jiangsu, china). neutralizing activity of mouse sera was evaluated using foci reduction neutralization test (frnt) as previous described (sun et al. ). genome information of two viruses was acquired through sanger sequencing. briefly, viral genomic rna was extracted from the virus stocks. after synthesis of viral cdna with specific primers, fragments covering the whole coding region of genome were amplified using phusion high-fidelity pcr polymerase (thermofisher, lithuania). when the pcr product concentration was high enough, they were sent directly for sequencing. otherwise, fragments were inserted into the peasy-blunt plasmid (transgene, beijing, china), which was then used to transform e. coli bacteria. three colonies containing each of the fragments were sequenced. full-length sequence of the single open reading frame was achieved based on the consensus sequence of each fragment, and then stringed together by overlapping them with each other. after that, sequence information of these two viruses was submitted to genbank, and the access numbers are mn (denv- h - -lp) and mn (denv- d - -sp) respectively. ifn-c elispot assay was performed according to manufacturer's protocol (mabtech, nacka strand, sweden). splenocytes of infected or control mice were isolated by homogenizing and filtering through lm cell strainers. after depleting erythrocytes, cells were re-suspended in % fbs/prmi- and added to elispot plates (millipore, co. cork, ireland) pre-coated with ifn-c antibodies. then pbs, lg/ml peptide, ns or e protein or lg/ ml cona was added to each well to stimulate splenocytes, and the plates were incubated at °c for - h. after being washed with pbs for times, plates were stained with biotin conjugated antibody, and subsequently streptavidin-horse radish peroxidase (hrp). finally, immune spots were visualized with the addition of tmb substrate. the image of spots was captured and analyzed by immu-nospot analyzer (cellular technology ltd. usa). balb/c mice were purchased from beijing vital river laboratory animal technology. type i and type ii ifn receptor double knock-out c bl/ mice (ag ) were originally provided by dr. guangxun meng of institut pasteur of shanghai, and raised in our laboratory. all mice were bred and maintained in specific pathogen-free barrier facilities and used at - weeks of age. for virus infection experiments, all procedures were completed under bsl- and absl- laboratory conditions as approved by the biosafety committee of institut pasteur of shanghai. female balb/c mice of - weeks old were injected with mg anti-type i ifn receptor monoclonal antibody (mar - a , bioxcell, usa) intraperitoneally (i.p.) at one day prior to challenge with . pfu parental strain, h - -lp or d - -sp through subcutaneous (s.c.) injection. blood samples were collected though retro-orbital bleeding daily from the st day post infection ( dpi) to th day post infection ( dpi). whole blood was lysed and homogenized within trizol-ls reagent (invitrogen, ca, usa) for measuring viral rna copy. meanwhile, for further detection of infectious viral particles in serum, sera were isolated from the blood through centrifugation. female ag mice of - weeks old were infected with pfu, pfu or pfu denv viruses through subcutaneous (s.c.) injection on day . body weight and survival rate were monitored from dpi to dpi, during which moribund mice with weight loss over % were euthanatized and recorded as death. vascular leakage was determined following intravascular administration of evans blue dye (sigma, louisiana, usa). briefly, ll evans blue dye ( % in pbs) was intravenously injected to mice on the th days post-infection of the two viral variants. after h dissemination, the mice were anaesthetized and perfused with ml pbs. digestive tracts and livers were collected and photographed. to quantify viral genomic copy in blood, rna was extracted following the user's guide of trizol ls reagent. a two-step quantitative rt-pcr (qrt-pcr) was then performed. first, cdna was synthesized using the fas-tquant rt kit (with gdnase) (tiangen biotech, beijing, china). second, amplifications were carried out using the fastfire qpcr premix (probe) kit (tiangen biotech, beijing, china) with the primer pairs designed according to the conserved sequence in denv utr: (forward, -tgayaagcartcagacac- ; reverse, -tcac-carrctccctttgc- ; fluorescence probe -cca-gagatcctgctgtctc- ). amplification cycles consisted of an initial incubation step of °c for min, cycles at °c for s, °c for s, and °c for s. standard rna were in vitro transcribed from a dna template that contains a t promoter and the target polynucleotide fragment for the primers indicated above. to obtain the standard curve, serially diluted standard rna ( - copy) were used as templates in parallel with sample rnas. copy of viral rna was calculated according to standard curves. all data were analyzed using graphpad prism software. t test or two-way anova were used to analyze the significance as indicated in legends. all results were expressed as means plus and minor standard errors of mean (sem). p values of . or less were considered statistically significant (*p \ . ; **p \ . , ***p \ . ; ****p \ . ). during in vitro propagation of a clinical strain of denv- obtained from guangzhou, china, heterogenous foci of different plaque sizes were observed in vero cell cultures (fig. a) . through limiting dilution and culture in mosquito c / cells, two viral variants were separated. after subsequent passage for three rounds in c / cells, two virus strains with stable foci morphology were obtained. one of them forms larger foci of about . mm diameter after h culture, and it was termed as denv- h - -lp (fig. a) ; the other forms smaller foci of about . mm diameter and was termed as denv- d - -sp (fig. a) . we next compared the replication abilities of these two viruses in both vero cells and c / cells using the onestep growth curve. denv- h - -lp had a higher replicating efficiency in vero cells than denv- d - -sp during the first h after infection, followed by a decline afterwards, due to the probability of running out target cells for infection; in comparison, denv- d - -sp rose slowly to a peak at h post infection (hpi), and kept its plateau until hpi (fig. b) . distinct from what was observed in vero cells, there was no obvious growth difference between the two viruses in mosquito cells (fig. c) . taken together, these data demonstrated that two viral variants with different foci sizes are isolated from one clinical virus isolate. and these two variants have different replication abilities in vero but not mosquito c / cells. to determine whether the different replication phenotypes reflect underlying genetic divergence between the lp and sp viruses, we sequenced the whole coding sequence of denv- h - -lp and denv- d - -sp, and aligned their envelope sequence with those of representative viruses belonging to different denv- genotypes, including asian genotype, american asian genotype, cosmopolitan genotype and sylvatic genotype. additionally, denv- strains isolated in guangdong, china, from to were also included for comparison. the phylogenetic analysis showed that denv- h - -lp and denv- d - -sp belonged to the cosmopolitan genotype and were clustered together with many strains isolated in west pacific regions, with percentages of homology ranging from . % to % (fig. ) . further comparison of genomic information between these two viruses also indicated that they had sequence variations in coding regions as illustrated in table . collectively, we found sequence variations in e, ns , ns b and ns regions, including synonymous mutations and amino acid changes. among the nonsynonymous mutations, two are in domain i/ii regions of e protein, two are in peptidase domain and helicase domain of ns respectively, another is located in a transmembrane helix of ns b, and the rest two are in ns rdrp domain. all of these domains are components of viral binding, replication or processing machinery, and highly related to viral antagonisms to host immunity. the conservation of mutations was also analyzed in comparison with representative strains from different genotypes, interestingly, though most synonymous mutations were also observed in other dengue- viruses, the seven nonsynonymous mutations were unique, and these sites seem more conserved in dengue- viruses (table ) . these genomic characteristics may contribute to the different infectivity both in vitro and in vivo. as type i/ii interferon deficient mice are more susceptible to dengue virus infection, we determined the in vivo infectivity of these two viruses in type i and type ii ifn receptors double knock-out ag mice. ag mice between and weeks old were infected through s.c. injection with pfu, pfu and pfu denv- h - -lp or denv- d - -sp (fig. ) . it is observed that infection of either virus led to % mortality within - days, even at a dose as low as pfu, though denv- h - -lp caused more weight loss and faster death than denv- d - -sp at the same dosage. specifically, weight loss began on dpi, dpi and dpi in mice infected with , , and pfu denv- viruses were used to infect cells at moi . , supernatant of infected cells were collected at the indicated timepoints. titers of infectious virus in supernatant were measured using foci forming assay in vero cells. detection limits of infectious virus titer were indicated with dashed lines. significance of the differences was calculated with two-way anova test, ***p \ . , n.s., not significant. h - -lp, respectively (fig. a , b, c), and death began on dpi, dpi and dpi correspondingly (fig. d, e, f ). in contrast, weight loss was first observed - days later in mice infected with denv- d - -sp, on dpi or dpi with inoculation of - pfu or pfu, respectively. the death was also delayed, began on dpi or dpi correspondingly. as a mixture of two types of viral variants, the parental strain was also able to cause weight loss and lethal diseases in ag mice. comparing to the two purified variants, the parental quasispecies presented intermediate level of pathology, which was most obvious in the dose groups of pfu. through evans blue staining, we also detected plasma leakage in sick mice on day post infection by either denv- h - -lp or denv- d - -sp viruses (fig. ) . consistent with the progress of weight loss and survival curves, leakage of evans blue in liver or digestive tract was more severe in mice infected with h - -lp on dpi. the evidence above demonstrated that the two viral isolates show differences on the pathology and disease kinetics, though they both have capabilities for causing lethal infection in ifn deficient mice. although both viruses can cause lethal infection in type i and type ii double knock-out mice, whether the lp and sp viruses behave differently in type ii ifn competent host is unknown. we have recently developed a zikv infection model in balb/c mice with transient blockage of type i ifn fig. phylogenetic analysis of denv- d - -sp and denv- h - -lp with representative serotype- dengue viruses of different genotypes isolated from different geographical regions. the phylogenetic tree was obtained after analyzing envelope sequences of denv- with mega x software, using the maximum likelihood method and kimura -parameter model. members of six reported genotypes of denv- were included. denv- d - -sp (mn ) and denv- h - -lp (mn ) were denoted by red dots in the tree. the scale bar denotes an evolutionary distance of . nucleotides per position in the sequence. virologica sinica receptor (ifnar) (liang et al. ) , we thus used the same strategy to examine these two dengue viruses for further comparison. wt balb/c mice were injected with antibody specific for ifnar at one day prior to s.c. infection with pfu of either viral strain, and monitored for seven days. the viral rna of denv- h - -lp could be detected throughout the week after infection, and the peak rna level of about genomic copy per microgram total rna was reached on day post infection, followed by gradually dropping to copy on the th day (fig. a) . in contrast, viral rna of denv- d - -sp was not detected until day post infection, when merely copy per microgram of total rna was transiently detected for days. consistently, infectious virus was detected in sera of denv- h - -lp infected mice from to dpi, during which peak viremia of pfu/ml appeared on day post infection. meanwhile, infectious virus was not detectable in mice infected by denv- d - -sp (lower than detection limit of pfu/ml) (fig. b) . moreover, h - -lp viral rna was also detected in liver, spleen and eyes on dpi (fig. c ), further confirming replication of h - -lp in susceptible organs. the parental strain also replicated in balb/c mice ( fig. a and b) , and the kinetics of viremia were closer to that of h - -lp, except for the delay of peak viremia to the th day post infection in both assays. to examine whether the above observed different virological properties of the two denv variants affect adaptive immunity in host, we evaluated adaptive immune responses induced by their infection in balb/c mice. denv- h - -lp infection elicited antibodies crossneutralizing the reference strain denv- (fig. a ) and it also stimulated t cell responses specific to peptides of denv- ns and zikv ns (fig. b) . similarly, neutralizing antibody and t cell responses were also detected in mice which were previously infected by denv- d - -sp, though both responses were weaker than those elicited by denv- h - -lp. considering genome similarity between the two variants, such different magnitudes of adaptive immune responses are mainly related to the different viral replication of two variants and different expression level of immunogens in vivo. in summary, we isolated from the same viral quasispecies two denv variants that showed distinct in vitro replication phenotype in vero cells, and different in vivo infectivity and pathogenicity in mice. using either of these two viral variants, we have established a lethal infection model in immune deficient ag mice. with the variant denv- h - -lp, we have also developed a self-limited infection model in wt mice pretreated with type-i ifn receptor antibodies. these two closely-related viruses not only offer new tools for in vivo studies, but also provide table sequence variation between h - -lp and d - -sp. in this study, we have successfully separated two viral variants that show different foci sizes in vero cells from a common serotype- clinical isolate of dengue virus. using these two viral variants, we established mouse infection models that reproduce severe manifestations of dengue diseases in ag mice. such lethal model with h - -lp or d - -sp viruses in ag mice can be used in pathology study, for example, to explore the viral and host factors that mediate hemorrhagic manifestation of dengue viruses. on the other hand, denv- h - -lp was used in another infection model which resembles self-limited infection of dengue virus in balb/c mice. and this transient infection model is more suitable for study of t cell responses, since the immune cell lineages are intact and the ifn-c responses are normal during the development of adaptive immunity. additionally, this self-limited infection model is also suitable for investigating how murine host resolves dengue infection. in previous investigations of dengue infection, only a limited number of viral strains were found to infect immune deficient mice, in which human illness without neurological disease can be studied, and lethal doses of these viruses ranged from to pfu (sarathy et al. ) . one often used virus is dengue serotype- strain d , which was obtained from a neuropathic strain pl through alternative passage between c / mosquito cells and ag mice. the ld of this virus in ag is - pfu (orozco et al. ) . another is d y p-pp , a double-plaque purified clone from a laboratory strain d y p, which had been passaged in c / cell for times. and the lethal dose of d y p-pp in ag mice fig. weight loss and survival curve of ag mice after infection with denv- h - -lp and denv- d - -sp. ag mice between - weeks old were infected with two viral variants and the parental isolate through s.c. injection. three different dose of viruses, pfu (a, d; n = ), pfu (b, e; n = ), and pfu (c, f; lp and sp n = , parental n = ), were inoculated into mice. mice injected with pbs were included within each panel for control (ct, n = ). weight change was recorded and presented as a ratio to the initial weight on day . moribund mice with weight loss over % were euthanatized and recorded as death. two-way anova was used in statistical analysis of differences among groups, *p \ . , **p \ . , ***p \ . ,**** p \ . , n.s., not significant. was documented higher than pfu (tan et al. ) . in contrast, the virus strains in this study had % mortality within days after inoculation in ag mice of - weeks old even when the dose was reduced to pfu, i.e., approximately times more lethal than the d and d y p-pp viral strains, and thus making denv- h - -lp virus a unique tool for studying the pathogenesis of dengue virus. through evans blue staining, we also confirmed the manifestation of plasma leakage in infected mice, which is a characteristic of severe dengue diseases in human. collectively, we show the highly virulent dengue strain h - -lp is ideal for establishing lethal infection models at low inoculum. to increase the utility of the new dengue viral variants in the context of relatively normal immune system, we have also adapted a recently developed model that transiently blocks ifnar with antibodies in balb/c mice. high level of viral rna was maintained within the first week after infection by denv- h - -lp, but only low level of transient viral rna was detected at - dpi in mice administered with denv- d - -sp. though no clinical symptom was observed, in mice infected with denv- h - -lp, we successfully detected infectious virus in sera, and viral rna in multiple organs including liver, spleen and eyes. the viruses might have been eliminated from mice after days of infection, accompanied by the maturation of denv adaptive immunity. thus, such characteristic and progress of infection is consistent with what was observed in patients with mild disease (who ). in previous animal models, denv infection of adult wt mice required extremely high inoculum, intracranially injection, or mouse adapted viruses. a portion of these models led to neurological symptoms or additional clinically irrelevant manifestations, limiting their use for pathology and therapeutic study (plummer and shresta ; sarathy et al. ) . to our knowledge, this is the first study which developed a denv infection model in wt mice, using a moderate dose of a non-adapted virus strain through physiologically more relevant s.c. injection route. it is often observed that many viruses form heterogenous plaques of different sizes in vitro, indicating that viruses exist as quasispecies during transmission or passaging (kato et al. ; moser et al. ), but the virological differences among various phenotypic variants have not been carefully examined. clinically, such viral diversity may assist arbovirus to survive under different selection pressure from two fig. vascular leakage in ag mice induced by infection of denv- h - -lp and denv- d - -sp. ag mice of weeks old were infected with pfu denv- h - -lp or d - -sp through s.c. injection. seven days post infection, mice were injected intravenously with evans blue and denv-induced vascular leakage was visualized in different tissues. pbs infected mice were used as negative controls. female balb/c mice of - weeks old were injected through i.p. with mg antibody to type i ifn receptor (mar - a ) at one day prior to infection. on the next day, pfu of viruses were inoculated into mice through s.c. route. then, blood samples were collected daily after infection. a viral rna copies in blood were measured using qrt-pcr, two-way anova was used in statistical analysis of the differences among groups, **p \ . , ****p \ . , and b titers of infectious virus in serum samples were determined through foci forming assay in vero cells; n = . t test was used in statistical analysis of the differences among groups, *p \ . , **p \ . , *** p \ . , ****p \ . . c eye, liver and spleen were collected from h - -lp virus-infected mice, and viral rna loads were determined, n = . detection limits of rna copy and infectious virus titer were indicated with dotted lines. n.d. not detected. distinct hosts, invertebrate and vertebrate. denv- h - -lp exhibits better replication capability than denv- d - -sp in vero cells, whereas their growth in mosquito cells is similar, reflecting possibility that the large-plaque variants in the original isolate contribute more to viral fitness in mammalian host. consistently, in mouse model, denv- h - -lp presents higher pathogenicity in ag mice and better replication in balb/c mice. comparing the coding sequence of denv- h - -lp and denv- d - -sp, we found amino acid variations on e, ns , ns b and ns proteins. e protein is responsible for virus attachment and entry; ns , ns b and ns are all the components of replication complex and essential antagonists for type i ifn responses (green et al. ). thus, further investigation of the underlying mechanism for the different phenotypes of these two viruses is valuable for understanding the molecular basis of dengue virulence and denv-host interaction. denv inhibits type i ifn production in infected cells by cleaving human sting humanized rag -/-gammac-/-mice support multilineage hematopoiesis and are susceptible to hiv- infection via systemic and vaginal routes mouse stat restricts early dengue virus replication the global distribution and burden of dengue both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage regulation of antiviral t cell responses by type i interferons innate immunity to dengue virus infection and subversion of antiviral responses dengue: a continuing global threat experimental studies on dengue. i. isolation, identification and modification of the virus at days post infection, frnt (foci reduction neutralization test) a and ifn-c elispot assay (b) were performed with mouse sera and splenocytes, respectively. reference strain denv- was used in frnt assay, peptides and proteins derived from denv- (d -ns , d -ns , d -e ) and zika virus (z-ns , z-ns ) were included in elispot assay to measure specific and crossreactive responses dengue fever in china: an emerging problem demands attention characterization of large and small-plaque variants in the zika virus clinical isolate zikv/hu/s /chiba/ molecular epidemiology demonstrates that imported and local strains circulated during the dengue outbreak in guangzhou recombinant zika virus envelope protein elicited protective immunity against zika virus in immunocompetent mice growth and adaptation of zika virus in mammalian and mosquito cells characterization of a model of lethal dengue virus infection in c bl/ mice deficient in the alpha/beta interferon receptor stat mediates innate immunity to dengue virus in the absence of stat via the type i interferon receptor mouse models for dengue vaccines and antivirals gamma interferon (ifngamma) receptor restricts systemic dengue virus replication and prevents paralysis in ifn-alpha/beta receptor-deficient mice production of immunity to dengue with virus modified by propagation in mice mouse models of dengue virus infection for vaccine testing murine model for dengue virus-induced lethal disease with increased vascular permeability adaptive immune responses to primary and secondary dengue virus infections dengue viruses cleave sting in humans but not in nonhuman primates, their presumed natural reservoir elaboration of tetravalent antibody responses against dengue viruses using a subunit vaccine comprised of a single consensus dengue envelope sequence subcutaneous infection with non-mouse adapted dengue virus d y p strain induces systemic vascular leakage in ag mice in: dengue: guidelines for diagnosis, treatment, prevention and control, new edn. who guidelines approved by the guidelines review committee dengue virus targets the adaptor protein mita to subvert host innate immunity epidemiological and virological characterizations of the author contributions js and xj designed and supervised the study. zhz and js carried out the experiments. zhz, js and xj analyzed the data and wrote the paper. zhl and ml assisted with animal experiments on ag mice. all authors read and approved the final manuscript. conflict of interest all authors declare that they have no conflict of interest. key: cord- -lnmc vv authors: steinhauer, c.; wingren, c.; borrebaeck, c. a. k. title: high‐throughput proteomics on antibody‐based microarrays: the importance of probe and surface design date: - - journal: scand j immunol doi: . /j. - . . ax.x sha: doc_id: cord_uid: lnmc vv in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive high‐throughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single‐chain fv antibody library, genetically constructed around one framework, the ncoder‐library, containing × clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on‐chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in‐house‐designed substrate, macroporous silicon coated with nitrocellulose (map ‐nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double‐(his) ‐tag, we increased the binding efficiency of sinfab‐molecules to ni (+)‐coated solid supports, thereby allowing nonpurified probes to be directly applied. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -s g g x authors: huang, y.‐m.; liu, x.; steffensen, k.; sanna, a.; arru, g.; sominanda, a.; sotgiu, s.; rosati, g.; gustafsson, j.‐Å.; link, h. title: anti‐inflammatory liver x receptors and related molecules in multiple sclerosis patients from sardinia and sweden date: - - journal: scand j immunol doi: . /j. - . . d.x sha: doc_id: cord_uid: s g g x the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing‐remitting ms from sassari, sardinia and stockholm, sweden. sex‐ and age‐matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real‐time pcr. lxr‐α was lower (p < . ) in ms (mean ± sem: . ± . ; n = ) compared to hc ( . ± . ; n = ). lxr‐α was lower in ms from stockholm ( . ± . ; n = ) compared to corresponding hc ( . ± . ; n = ; p < . ) and compared to ms ( . ± . ; n = ; p < . ) and hc ( ± . ; n = ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr‐β (− . ± . ) compared to corresponding hc (− . ± . ). ms from stockholm was associated with higher abca‐ ( . ± . versus . ± . ; p < . ) and higher estrogen receptor‐β‐cx ( . ± . versus . ± . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ± . ) compared to ms from sassari ( . ± . ; p < . ), ms ( . ± . ; p < . ) and hc from stockholm ( . ± . ; p < . ). ms from sassari had lower cyclooxygenase‐ compared to corresponding hc ( . ± . versus . ± . ; p < . ) and lower prostaglandin‐e (− . ± . ) compared to the hc ( . ± . ; p < . ) and ms ( . ± . ; p < . ) and hc from stockholm ( . ± . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -c tqioz authors: lauridsen, c.; jensen, s. k. title: supplementation of vitamin c to weaner diets increases igm concentration and improves the biological activity of vitamin e in alveolar macrophages date: - - journal: scand j immunol doi: . /j. - . . u.x sha: doc_id: cord_uid: c tqioz vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay‐c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr‐(α‐tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - njndob authors: broggi, achille; ghosh, sreya; sposito, benedetta; spreafico, roberto; balzarini, fabio; lo cascio, antonino; clementi, nicola; de santis, maria; mancini, nicasio; granucci, francesca; zanoni, ivan title: type iii interferons disrupt the lung epithelial barrier upon viral recognition date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: njndob lower respiratory tract infections are a leading cause of mortality driven by infectious agents. rna viruses such as influenza virus, respiratory syncytial virus and the new pandemic coronavirus sars-cov- can be highly pathogenic. clinical and experimental evidence indicate that most severe and lethal cases do not depend on the viral burden and are, instead, characterized by an aberrant immune response. in this work we assessed how the innate immune response contributes to the pathogenesis of rna virus infections. we demonstrate that type iii interferons produced by dendritic cells in the lung in response to viral recognition cause barrier damage and compromise the host tissue tolerance. in particular, type iii interferons inhibit tissue repair and lung epithelial cell proliferation, causing susceptibility to lethal bacterial superinfections. overall, our data give a strong mandate to rethink the pathophysiological roles of this group of interferons and their possible use in the clinical practice against endemic as well as emerging viral infections. efficiently than mice that bear wt stromal cells, and phenocopy ifnlr -/à ifnlr -/chimeras (fig. g) . these data further support the direct activity of ifn-λ on epithelial cells. interestingly, the gene that was most downregulated in ifnlr -/epithelial cells compared to wt cells is the e ubiquitin-protein ligase makorin- (mrkn ) (fig. a) , which controls p and p stability by favoring their proteasomal degradation ( ) . under oxidative stress condition and dna damage, a hallmark of severe viral infections ( ), p is stabilized by phosphorylation and p degradation, mediated by mkrn , favors apoptosis over dna repair ( ). indeed, ifnlr -/epithelial cells, that express lower levels of mkrn , have elevated levels of p as measured by flow cytometry (fig. h , i). these data indicate that the capacity of ifn-λ to reduce tissue tolerance stems from its capacity to inhibit tissue repair by directly influencing epithelial cell proliferation and viability. also, that p degradation via mrkn upregulation is potently influenced by ifn-λ signaling. rna viruses can use several strategies to modulate the immune response to their advantage( , ), therefore it is crucial to understand the molecular pathways involved in the maintenance of sustained ifn-λ production. moreover, the difference between mrna expression and protein levels of interferons suggest that a low abundance cell type with high secretory capacity may be responsible for long term ifn-λ production. we thus investigated the cellular source and molecular pathways that drive ifn-λ production in our model. early after initial influenza virus infection, ifn-λ is expressed by infected epithelial cells, however, at later time points, dcs from the parenchyma of the lung start to express high levels of the ifn-λ transcript( ). we thus hypothesized that lung dcs are the main producers of ifn-λ and are responsible for the secretion of ifn-λ during viral infections. accordingly, sorted lung resident dendritic cells express high levels of ifn-λ transcript after days of poly (i:c) treatment, in contrast to epithelial cells, alveolar macrophages and monocytes (fig. a) , which, instead, express inflammatory cytokines (fig. s a, b) . moreover, diphtheria toxin (dt)-mediated depletion of cd c + cells in cd c-dt receptor (dtr) mice was sufficient to completely abolish ifn-λ transcript and protein upregulation upon days of poly (i:c) treatment (fig. b, c) , while production remained unaltered (fig. s c with the response measured in vivo, tlr stimulation did not induce ifn production while it induced upregulation of pro-inflammatory cytokines, and intracellular delivery of poly (i:c) induced high levels of ifn-i but not ifn-λ (fig. d, fig. s a , b). in agreement with the key role of tlr , ifn-λ production upon extracellular poly (i:c) encounter was abolished by genetic deletion of the signaling adaptor trif (encoded by the gene ticam ) but not by deletion of the rig-i/mda adaptor mavs (mavs) (fig. d) . conversely, ifn-i production in response to intracellular delivery of poly (i:c) was largely dependent on the signaling adaptor mavs (fig. s a ). consistent with our previous data, when the rig-i/mavs pathway was activated by transfection of the influenza a virus derived pathogen-associated molecular pattern (pamp) -phosphate-hairpin-rna ( p- hprna), ifn-i but not ifn-λ, was efficiently induced in a mavs-dependent manner ( fig. s a - e, poly (i:c) was used as a control). finally, inhibition of endosomal acidification by treatment with the pharmacological agent chloroquine abolished ifn-λ induction in response to extracellular poly (i:c), while it preserved ifn-i production upon intracellular poly (i:c) delivery (fig. s a, b) . these evidences clearly indicate that tlr stimulation potently induces ifn-λ production by dcs in vitro. we, thus, explored the importance of the tlr -trif pathway in vivo under our experimental conditions. dendritic cells sorted from ticam -/mice treated with poly (i:c) for six days did not express appreciable levels of ifn-λ transcripts while still produced type i interferons ( fig. e, f) . moreover, poly (i:c) treated ticam -/mice were protected from s. aureus superinfections (fig. g) , and the decrease in bacterial burden correlated with lower ifn-λ transcript levels in the lung, although ifn-i levels remained similar to those of wt mice (fig. h , i). confirming the crucial role of tlr signaling in dcs for ifn-λ production, chimeric mice in which ticam -/bone marrow (bm) cells are transferred in a wt irradiated host (ticam -/ àwt) phenocopied ticam -/animals ( fig. j-l) . the immune system evolved to prevent and resist to pathogen invasion but doing so often threatens host fitness and causes disease in the form of immunopathology ( ). rna viruses are the major cause of most severe lower respiratory tract viral infections ( , ). while most virus infections manifest as self-limiting upper respiratory tract infections, influenza viruses, sars- cov, sars-cov- and mers-cov can progress to severe lung disease with potentially lethal outcomes ( , , ) . although different viruses vary in their virulence and pathogenic potential, the most severe cases of lung rna viral infections share similar features that suggest an immune pathological etiology. in covid- , sars, mers and flu, severe symptoms and death occur late after the initial symptoms onset, and after the peak in viral load ( - ) further indicating a central role for an immune etiology of the most severe forms. while ifn-λ is uniquely equipped to induce a gentler immune response that favors viral clearance in the lungs without inducing overt immune activation ( , , ), its impact on epithelial cell biology and its effect on the maintenance of tissue integrity and tolerance to pathogen invasion is incompletely understood. in a system that allowed us to isolate the effect of immune activation from resistance to viral infection, we demonstrate that sustained ifn-λ production in the lung in response to viral pamps compromises epithelial barrier function, induces lung pathology and morbidity and predisposes to lethal secondary infections by impairing the capacity of the lungs to tolerate bacterial invasion. loss of lung barrier tolerance is sufficient to induce lethality upon bacterial challenge independently of bacterial growth ( ), and alteration of the repair response in the lung can favor bacterial invasion independently from immune cell control ( ). in our model immune cell recruitment is not affected by ifn-λ and neutrophils are dispensable for the impaired control of bacterial infections, while ifn-λ signaling on epithelial cells is necessary and sufficient to cause heightened bacterial invasion. under our experimental conditions, tlr -trif signaling in conventional lung dcs is responsible for the induction of ifn-λ. this is consistent with reports indicating that tlr -deficient mice are protected from influenza-induced immune pathology( ). moreover, tlr detects replication intermediates from necrotic cells ( ) and is, thus, insensitive to viral immune evasion. this is of particular interest during highly pathogenic human coronavirus infections, whose success in establishing the initial infection is partly due to their ability to dampen tlr and mavs dependent early ifn responses ( ) ( ) (available at https://clinicaltrials.gov/ct /show/nct ). clin. microbiol. rev. , - ( ) . intratracheal instillations (i.t.) were performed as previously described in ( ) rectal temperature and body weights were monitored daily. mice were deemed to have reached endpoint at % of starting weight or after reaching body temperature of °c or lower. to generate mice with hematopoietic-specific deletion of ifnlr or ticam , -week-old cd . + mice were exposed to lethal whole-body irradiation ( rads per mouse) and were reconstituted with × donor bone marrow cells from -week-old wild-type, ifnlr -/or ticam -/mice. mice were treated with sulfatrim in the drinking water and kept in autoclaved cages for weeks after reconstitution. after weeks, mice were placed in cages with mixed bedding from wild-type, and ifnlr -/or ticam -/mice to replenish the microbiome and were allowed to reconstitute for more weeks. a similar procedure was used to generate bone-marrow chimeras with stromal cellsspecific deletion in ifnlr . here, recipient wt or ifnlr -/mice underwent irradiation and were reconstituted with bm cells derived from cd . + mice similarly as described above. to evaluate the percentage of chimerism, a sample of peripheral blood was taken from chimeric mice after weeks of reconstitution and stained for cd . and cd . (antibodies as identified under 'reagents and antibodies') and were analyzed by flow cytometry. in order to deplete cd c + cells, cd c-dtr mice received μg/kg diphtheria toxin (dtx) intravenously starting one day before tlr ligand or saline administration and continuing every other day until day post-treatment to maintain depletion. in vivo depletion of neutrophils was carried out by injecting anti-ly g antibody ( μg/mouse) intraperitoneally, starting one day before treatments and then continuing every other day through the duration of the treatment. as controls for no depletion, mice were injected with rat igg isotype control. to assess lung permeability, treated mice were administered fitc-dextran ( μg/mouse) i.t. before or after s. aureus infection. after hr of dextran instillation, blood was collected from the retro-orbital sinus, and the plasma was separated by centrifugation. leakage of dextran in the bloodstream was measured as fitc fluorescence in the plasma compared to plasma from mocktreated mice. bal was collected as described in ( ) briefly, the lungs of euthanized mice were lavaged through the trachea with ml pbs to collect the bal. samples were centrifuged and the supernatants were used for total protein measurement (pierce bca protein assay, thermo fisher scientific # ) and ldh quantification (pierce ldh cytotoxicity assay, thermo fisher scientific #c ). lungs were excised and used for rna extraction using tri reagent (zymo research #r - - ). the left lobe of the lung was weighed and homogenized in ml of sterile d.i. water in a fisherbrand™ bead mill homogenizer. to calculate bacterial load, homogenate was serially diluted and plated on tsb-agar plates in duplicate. colonies were counted after h incubation, and bacterial burden in the lungs was calculated as cfu normalized to individual lung weight. cytokines production in the lungs was measured in the supernatants collected after centrifuging the lung homogenates. lung cells were isolated as described in ( ) briefly, mice were euthanized and perfused. ml of warm dispase solution ( mg/ml) were instilled into the lungs followed by . ml of % low-melt agarose (sigma #a ) at °c, and allowed to solidify on ice. inflated lungs were incubated in dispase solution, for ' at rt. the lungs were then physically dissociated, incubated ' with dnase i μg/ml and filtered through μm and μm strainers. red blood cells were lysed using ack buffer. single cell suspensions were stained for live/dead using zombie red or zombie violet, and then with antibodies against surface antigens diluted in pbs + bsa . % for minutes at °c. cells were then washed, fixed with . % paraformaldehyde for minutes at room temperature, washed again and resuspended in pbs + bsa . %. samples were acquired on a bd lsrfortessa flow cytometer and data were analyzed using flowjo v. software (bd biosciences). countbright absolute counting beads (invitrogen #c ) were used to quantify absolute cell numbers. purified rna was analyzed for gene expression on a cfx real-time cycler (bio-rad) using a taqman rna-to-ct -step kit (thermo fisher scientific) or sybr green (bio-rad). probes specific for ifnl / , ifnb , il b, rsad , gapdh were purchased from thermo fisher scientific, and sybr-green primers for rsad , cxcl , gapdh were purchased from sigma. cytokine analyses were carried out using homogenized lung supernatants, and cell supernatants from stimulated flt l-dcs. ifnλ / elisa (r&d systems dy b) and mouse ifnβ, il β, il- , tnfα elisa (invitrogen) were performed according to manufacturer's instructions. bronchoalveolar lavages (bal) were obtained from five intensive care unit (icu)-hospitalized sars-cov- -positive patients. in parallel, five naso-oropharyngeal swabs were collected from both sars-cov- -positive and -negative subjects. among positive patients, two were hospitalized but without the need of icu support, whereas three out of five did not require any hospitalization. the negative swabs were obtained from subjects undergoing screening for suspected social contacts with covid- subjects. swabs were performed by using floqswabs® (copan) in utm® universal transport medium (copan). all samples were stored at - °c until processing. the study involving human participants was reviewed and approved by san raffaele hospital irb in the covid- biobanking project. the patients provided written informed consent. rna extraction was performed by using purelink™ rna thermo fisher scientific according to manufacturers' instruction. in particular, µl for each bal and swab analyzed sample were lysed and homogenized in the presence of rnase inhibitors. then ethanol was added to homogenized samples which were further processed through a purelink™ micro kit column for rna binding. after washing, purified total rna was eluted in µl of rnase-free water. system (invitrogen™) protocol by using µl of rna extracted from each bal and swab sample. qrt-pcr analysis for was then carried out for evaluating il , il b, ifnb , ifna , ifnl and ifnl expression. all transcripts were tested in triplicate for each sample by using specific primers. gapdh was also included. real-time analysis was then performed according to manufacturer instructions by using taqman® fast advanced master mix (applied biosystems™ by thermo fisher scientific). real-time pcr analysis was performed on abi by applied biosystems. statistical significance for experiments with more than two groups was tested with one-way anova, and dunnett's multiple-comparison tests were performed. two-way anova with tukey's multiple-comparison test was used to analyze kinetic experiments. two-way anova with sidak's multiple-comparison test was used to analyze experiments with grouped variables (i.e. treatment, genotype). statistical significance for survival curves were evaluated with the log-rank (mantel-cox) test and corrected for multiple comparisons with bonferroni's correction. to establish the appropriate test, normal distribution and variance similarity were assessed with the d'agostino-pearson omnibus normality test using prism (graphpad) software. when comparisons between only two groups were made, an unpaired two-tailed t-test was used to assess statistical significance. to determine the sample size, calculations were conducted in nquery advisor version . . primary outcomes for each proposed experiment were selected for the sample size calculation and sample sizes adequate to detect differences with an % power were selected. for animal experiments, four to ten mice per group were used, as indicated in the cells were gated on fsc and ssc to eliminate debris, on fsc-a -fsc-h to select single cells and cells negative for live/dead dye and lineage markers (cd , cd , ter ). epithelial cells were gated as cd -epcam + cd -. the epcamcells were sorted for immune cells as follows: amac were gated as cd + ly g -cd c hi siglec-f + , monocytes and monocyte-derived cells (mo) were gated as cd + ly g -siglec-f -ly c + , cdcs were gated as cd + ly g -siglec-f -ly c -cd c + mhc-ii hi . estimates of the severity of coronavirus disease : a model-based analysis clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study of the n. t. u. (ntu) c. of m. hospital progression in patients with severe acute respiratory syndrome virological assessment of hospitalized patients with covid- influenza and rhinovirus viral load and disease severity in upper respiratory tract infections innate and adaptive immune responses in patients with severe acute respiratory materials and methods mice c bl/ j jax ) mice were purchased from jackson labs. c bl/ il- r -/-(ifnlr -/-) mice were provided by bristol-myers squibb. mice were housed under specific pathogen-free conditions at boston children's hospital. staphylococcus aureus infections were conducted in the biosafety level- facility at boston children's hospital. all procedures were approved under the institutional animal care and use committee (iacuc) and conducted under the supervision of the department of animal cd (m / ), mhc-ii i-a/i-e (m / r (tlr-r ) and p-hprna (tlrlhprna) were purchased from invivogen. for in vivo administration of type iii ifn, we used polyethylene glycol-conjugated ifn-λ (peg-ifn-λ) (gift from bristol-myers squibb). diphtheria toxin (unnicked) from corynebacterium diphtheriae was purchased from cayman chemical. anti-ly g antibody, clone a (be - ) and rat igg a isotype control (be ) for in vivo administration was purchased from bioxcell. '-deoxy- -ethynyl uridine (edu) was purchased from carbosynth (ne ) epithelial cell proliferation') before being stained with antibodies against cell-surface antigens. intracellular staining of ki and p were carried out using foxp fix/perm buffer set (biolegend # ) following the manufacturer's instructions. epithelial cell proliferation proliferation of lung after permeabilization cells were washed and incubated with mm copper sulphate (millipore-sigma), mm sodium ascorbate (millipore-sigma) and μm sulfo-cyanine -azide (lumiprobe #a ) in tris buffered saline (tbs) mm, ph . , for min at room temperature. ion torrent for targeted transcriptome sequencing, ng of rna isolated from sorted cells was retro barcoded libraries were prepared using the ion ampliseq transcriptome mouse gene expression kit as per the manufacturer's protocol and sequenced using an ion s system genes were called expressed (n= , ) if they had average log expression of or greater in either wt or ifnlr -/-. differentially expressed genes (degs) between wt and ifnlr -/-were selected by thresholding on fold change (+/- . ) and p value ( . ). in heatmaps, degs were z-scaled and clustered (euclidean distance, ward linkage). pathway analysis was performed with the r package hyper cell culture flt l-dcs were differentiated from bone marrow cells in iscove's modified dulbecco's media (imdm; thermo fisher scientific), supplemented with % b -flt l derived supernatant and % fetal bovine serum (fbs) for where indicated poly (i:c) stimulated cells were pre-treated with μg/ml chloroquine for minutes prior to stimulations. qrt-pcr and elisa rna was isolated from cell cultures using a genejet rna purification kit (thermo fisher scientific #k ) according to manufacturer's instructions. rna was extracted from excised lungs by homogenizing them in ml of tri reagent. rna was isolated from tri reagent samples using phenol-chloroform extraction or column-based extraction systems (direct-zol rna microprep and miniprep, zymo research #r and #r ) according to the manufacturer's protocol flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies wt mice were treated daily with i.t. . mg/kg poly (i:c), . mg/kg r or saline for days and infected i.t. with . x cfu of s. aureus at day . (a) body temperature, (b) total protein in the bal and **p < . and ***p < . (one-way anova). each mouse represents one point a-h) wt mice were treated daily for , , or days with i.t. . mg/kg poly (i:c) or days of saline, and infected with i.t. . x cfu of s. aureus for h. total lung homogenates were analyzed by qpcr for bacterial burden was evaluated in total lung homogenate **p < . and ***p < . (one-way anova compared to saline treatment). each mouse represents one point wt and ifnlr -/-mice were treated daily with i.t. . mg/kg poly (i:c) for days and infected with i.t. . x cfu of s. aureus for h. (a) weight change, (b) total protein in the bal **p < . and ***p < . (two-way anova). each mouse represents one point lung resident dcs are the primary producers of ifn-β upon poly (i:c) treatment mg/kg poly (i:c) or saline for days were sorted for epithelial cells (ec), resident dc (resdc), monocyte-derived dc (modc), and alveolar macrophages (amac) and assessed for (a) il b and (b) ifnb relative mrna expressions. cd c-dtr mice depleted for cd c + cells in vivo by dtx injections were treated daily with i.t. . mg/kg poly (i:c) or saline for days. total lung lysates of the treated mice were analyzed for (c) ifnb relative mrna expression, and (d) ifn-β protein expression by elisa , **p < . and ***p < . (two-way anova) rig-i or tlr ligands. flt l-dcs from wt, ticam -/-or mavs -/-mice were treated with μg/ml poly (i:c), μg/ml transfected poly (i:c) or μg/ml r for h. ifnb (a), and il b (b) relative mrna expressions were evaluated by qpcr , **p < . and ***p < . (two-way anova) mean and sem of independent experiments is depicted flt l-dcs upregulate ifn-λ uniquely upon activation of tlr signaling and not in response to the rig-i specific ligand p-hprna. flt l-dcs from wt mavs -/-mice were treated with μg/ml poly (i:c), or μg/ml transfected p-hprna for h or h ifnb (b), and il b (c) relative mrna expressions were evaluated by qpcr after h and ifn-β (e) levels in the supernatants were evaluated by elisa after h the endosomal tlr inhibitor chloroquine inhibits poly (i:c) dependent ifn-λ expression in flt l-dcs. flt l-dcs from wt mice were treated with μg/ml poly (i:c), or μg/ml transfected poly (i:c) for h in the presence or absence of μg/ml chloroquine we thank dr. jc kagan for discussion, help and support. funding: iz is supported by nih grant r ai , r dk , and niaid-dait-nihai . ab is supported by ccfa key: cord- -d b qvhs authors: siassi, m.; hohenberger, w.; croner, r. title: expression of human collectins in colorectal carcinoma date: - - journal: scand j immunol doi: . /j. - . . bo.x sha: doc_id: cord_uid: d b qvhs introduction: the human collectins, mannan‐binding lectin (mbl), surfactant protein‐a (sp‐a) and surfactant‐protein‐d (sp‐d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy‐kit). gene expression profiles were analysed using gene‐chips (affymetrix, hg‐u ). we analysed the data for the expression of mbl, its associated serine proteases mannan‐binding lectin‐associated serine protease / (masp / ), sp‐a and sp‐d. the signal intensity of the genes of interest was compared using the mann–whitney u‐test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp‐a and masp‐ reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this study. only sp‐a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -iszb qlk authors: astrinidou‐vakaloudi, a.; xytsas, s.; diamanti, i.; . ioannidis, h; pangidis, p. title: presence of helicobacter pylori antibodies in haemodialysis patients date: - - journal: scand j immunol doi: . /j. - . . p.x sha: doc_id: cord_uid: iszb qlk aim: renal dysfunction may influence the colonization of gastric mucosa by urea‐splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end‐stage renal disease (esrd), receiving long‐term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(+) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -h o yqv authors: nan title: oral communications and posters date: - - journal: inflamm res doi: . /bf sha: doc_id: cord_uid: h o yqv nan the drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : ( ) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? ( ),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; ( ) how does drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. there is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. several myelin-derived autoantigenic targets have been described and include myelin basic protein (mbp), proteolipid protein and myelin oligodendrocyte glycoprotein. there has been a particular focus on mbp for at least two reasons: mbp-specific cd + tcell receptors (tcrs) have been found in multiple sclerosis brains, and cells presenting an immunodominant mbp( - ) peptide in complex with hla-dr b have been shown to be present in multiple sclerosis lesions. also, humanized mice expressing the hla-dr b gene and a human t-cell receptor (tcr) that recognises the mbp - peptide in the context of hla-dr b either spontaneously or after immunization with mbp - develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. this talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. to resolve the pathogenic mechanisms of rheumatoid arthritis (ra) we need to identify the causative genetic and environmental factors. this has however proven to be complex, with many factors and genes interacting. inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as ra. animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. there are several arthritis models, which each may reflect various variants of the heterogeneity of ra in humans. examples are collagen induced arthritis (cia) and pristane induced arthritis (pia), which both fulfills the clinical diagnostic criteria for ra. both diseases are genetically complex and the susceptibility is, as ra, dependent on many polymorphic genes operating in concert. so far genes in this concert have been identified; the mhc class ii ab gene in the mouse ( ) and the ncf gene in the rat ( ) and in the mouse ( ) . the ncf protein is a part of the nadph oxidase complex mediating oxidative burst. the discovery of the ncf polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive t cells. mice with the deficient ncf allele are more susceptible to cia, and also developed a chronic form of arthritis. interestingly, the immune response to cii was enhanced by the ncf deficiency linking the ncf pathway to the adaptive immune response. oxidation of t cell membranes seem to be a key event in the pathogenic mechanism as reduction of t cell membranes induces arthritis in rats ( ). on the basis of these findings a new type of therapy myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (achr) at the neuromuscular junction. the antibodies reduce the number of achr leading to failure of neuromuscular transmission and muscle weakness. the achr antibodies as measured in conventional immunoprecipitation assays are igg, high affinity, polyclonal and specific for human achr. they reduce the numbers of achr by antigenic modulation and by complement-mediated damage to the neuromuscular junction. myasthenia gravis has a very intriguing relationship with the thymus gland. in many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express achrs. there are many b cells and plasma cells and thymic lymphocyte preparations synthesise achr antibodies. the possibility that the thymic achr induces the germinal centre formation and achr antibody synthesis is supported by much evidence. some patients, however, have thymic tumours and in these the role of the thymus is less clear. moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age. there are up to % of myasthenia patients that do not have the typical achr antibodies. some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. the pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed. finally, among the patients who have neither achr nor musk antibodies by conventional testing, we have evidence for lower affinity antibodies to achr which can now be detected using molecular approaches which will be described. arry- (azd ) is an inhibitor of mek / currently in development for cancer. phase determined the msd ( mg) and the safety of the compound given continuously. in decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. paired pre-and postdose tumor biopsies showed a reduction in perk (- %) and proliferative index (- %). the trough plasma concentration ( ng/ml) corresponded to~ % inhibition of perk. about % of pts had stable disease after months. these results demonstrate that arry- (azd ) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. there are several on-going phase studies, in melanoma, colorectal, lung and pancreatic cancer. arry- is another potent, selective mek / inhibitor, currently in development for inflammatory diseases. when human whole blood was stimulated with tpa, arry- inhibited tnfa, il- b and il- (ic s of , and nm, respectively). % inhibition of perk required nm. arry- was highly efficacious in cia and aia rat models, with ed s of and~ mg/ kg, respectively. in normal volunteers arry- was well-tolerated and there was a dose-proportional increase in drug exposure. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpa-induced tnfa and il b. an on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of arry- monotherapy. in addition, we have initiated a clinical trial designed to evaluate arry- in combination with methotrexate in patients with rheumatoid arthritis. rho kinases (rock) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.rocks prominent role in cytoskeletal architecture suggests that rock inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.we have synthesized small molecule inhibitors of rock which are specific for the rock- isoform.these rock- inhibitors, typified by slx- and slx- , are potent (ic < nm), selective for rock- (> fold selectivity for rock- over rock- ) and exhibit good oral bioavailability.this talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.slx- inhibits cell proliferation and migration in several tumor cell lines including ht- , panc- and mda-mb- . moreover in xenograft studies using nude mice, slx- significantly inhibited tumor growth with these same cell lines. in liver fibrosis, slx- prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.slx- is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.these data suggest that rock- selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as slx- and slx- will provide effective treatment for oncology and fibrotic disease. cyclooxygenases (cox) catalyze the first step in the synthesis of prostaglandins (pg) from arachidonic acid.cox- is constitutively expressed.the cox- gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.levels of cox- are increased in both inflamed and malignant tissues.in inflamed tissues, there is both pharmacological and genetic evidence that targeting cox- can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).multiple lines of evidence suggest that cox- plays a significant role in carcinogenesis.the most specific data that support a cause-and effect relationship between cox- and tumorigenesis come from genetic studies.overexpression of cox- has been observed to drive tumor formation whereas cox- deficiency protects against several tumor types.selective cox- inhibitors protect against the formation and growth of experimental tumors.moreover, selective cox- inhibitors are active in preventing colorectal adenomas in humans.increased amounts of cox- -derived pge are found in both inflamed and neoplastic tissues.the fact that pge can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of cox- contributes to both wound healing and tumor growth.taken together, it seems likely that cox- induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. ( ), k hagihara( ), t nishikawa( ), j song( ), a matsumura ( ) ( ) health care center, osaka university, japan ( ) osaka university graduate school of medicine, japan it is still less known about the actual pathogenic role of il- in the inflammatory status. to know the pathogenic role of il- and the efficacy of il- blockade in inflammation, a humanized anti-il- receptor antibody, tocilizumab, was used for the treatment in chronic inflammatory diseases, such as castlemans disease, rheumatoid arthritis and crohns disease. since il- blocking therapy improved the clinical symptoms and the laboratory findings, the il- function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid a (saa). saamrna induction, saa promoter activity and assembling of transcriptional factors on saa promoter were analyzed by the real time rt-pcr, gel shift assay and dna affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, il- , il- and tnf-alpha. in result, il- was an essential cytokine in induction of saamrna through the activation of stat which constructed the complex with nf-kappab p and a cofactor p . although there was no stat consensus region on saa promoter, stat bound at the site of nf-kappab re. the above research proved that il- signal is essential on the synergistic induction of saa via newly discovered stat transcriptional mechanism, suggesting the presence of this stat mechanism in inflammation, and confirming the normalization of serum saa level by il- blocking therapy in inflammatory diseases. this research method develops a subsequent therapy for serious aa amyloidosis by inhibition of saa production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases. takashi wada( ), k matsushima( ), s kaneko ( ) ( ) kanazawa university, japan ( ) university of tokyo, japan accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.the possible positive amplification loop from cxc chemokines to cc chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.it is of note that monocyte chemoattractant protein (mcp)- / monocyte chemotactic and activating factor (mcaf)/ ccl , a prototype of cc chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. new insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. in addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.the selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. we focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this symposium. ( ), p lacamera ( ), b shea ( ), g campanella ( ), b karimi-shah ( ) , n kim ( ), z zhao( ), v polosukhin ( ), y xu( ), t blackwell ( ) aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.here we demonstrate that lysophosphatidic acid (lpa) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, lpa , are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. the absence of lpa markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.the increase in vascular permeability caused by lung injury was also markedly reduced in lpa -deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.these results demonstrate that lpa links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.lpa therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. we have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. we have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. we have started to explore the role of tnf-alpha for adult neurogenesis. infusion of an antibody to tnf-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the tnf-r receptor. we have shown that tnf-r is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. we have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. we found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. in contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. the role of inflammation for these differences is currently being explored. proteinase-activated receptor- (par- ) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with sligkv that binds intramolecularly and activates the receptor. par- is implicated in innate defense responses associated with lung inflammation. we showed that par- is expressed by human alveolar (a ) and bronchial ( hbe) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide sligkv-nh . in cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin g, two serine proteinases, and by pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.we demonstrated that these three proteinases do not activate par- , but rather disarm this receptor, preventing its further activation by trypsin but not by sligkv-nh . preincubation of a par- transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of par- , generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of par- in the respiratory tract, thereby altering the host innate defense mechanisms. caspase- -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. ongoing research is investigating whether par agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways. the intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.given that protease-activated receptor (par ) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.scbn, caco-ii and t epithelial cells were grown to confluence on filter supports and mounted in ussing chambers to study short circuit current (isc) and transepithelial resistance (rte).cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to rte.apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in rte and decreased the transepithelial flux of a mw dextran.interestingly, the effect of trypsin could not be replicated by activators of pars , and , suggesting that the effect on rte was not due to activation of pars.subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase c.a trypsin-induced increase in intracellular calcium was not involved.inhibition of pkc-zeta, but not of typical pkcs, also blocked the response.our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation. clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. the high concordance in monozygotic twins (> %), which is not seen in dizygotic twins (< %) points to strong contribution of genetic susceptibility to the overall risk for disease. ibd represents a "complex disease" and may involve a large number of interacting disease genes. crohns disease has become an example for the successful molecular exploration of a polygenic etiology. crohns disease was not known before . incidence has increased since now leading to a lifetime prevalence of up to . % in western industrialized countries. the current hypotheses propose unknown trigger factors in the life style of western industrialized nations that interact with a polygenic susceptibility. it appears that increased expression and production of tnf and an enhanced state of activation of the nfkb system are main drivers of the mucosal inflammatory reaction. the exploration of inflammatory pathophysiology of crohns disease using full genome, cdna and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. while this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome being most consistent between different populations. in three coding variations in the card gene were identified that are highly associated with development of the disease. all variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of nfkb in macrophages and epithelial cells. interestingly, the three disease associated snps are never found on the same haplotype. in compound heterozygotes or homozygotes they result in a rr of > to develop crohns disease as an adult. a particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the card gene. variations in the card gene do not fully explain the linkage finding in the pericentromeric region of chromosome . after stratification for card variants, the broad linkage peak is reduced to two more defined peaks on p and q, respectively. while the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes and , respectively). dlg- is the example of a low-risk susceptibility gene with a modest associated odds ratio ( . - . ) . interestingly, the associa-tion signal appears to be confined to young males. slc a / which encode the kation-transporters octn and have been suggested to represent the disease gene in the + kb haplotype block on chromosome q . mdr has also been implicated as a disease gene in ibd. although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes mdr likely as a low risk susceptibility gene. with the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. recently a k illumina scan has been published identifying il- r as a further disease gene. we used a genome wide candidate gene approach (with appr. . csnps) to identify atg l as a further disease genes. both genes were confirmed and a further regulatory snp involving ptger was annotated by a belgian genome wide scan. by the time of presentation three further genome wide snp scans in crohn disease will most likely have entered the public domain. the further exploration of crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. the creation of a medical systems biology of disease will lead to new models and eventually new therapies. the chemokine receptor ccr plays a pivotal role in mediating the migration of t cells to the gastrointestinal mucosa. the ligand for ccr , teck, s highly expressed in the gi tract. the pathogenicity of intestinal ccr +/ cd + t cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of crohns and celiac disease patients. ccx -b is a highly selective and potent, orally bioavailable, small molecule antagonist of ccr .the compound proved to be highly efficacious in the tnf-aare and mdr a-/-murine models of inflammatory bowel disease (ibd). in phase i trials, ccx -b was well-tolerated, and no drug-related saes were reported.a -day placebo-controlled phase ii study was recently completed in patients with moderate to severe crohns disease. ccx -b was shown to be both safe and to have encouraging clinical results: % of patients on ccx -b (cdai ! , crp > . mg/l) exhibited a -point drop in cdai compared to % on placebo. furthermore, a crp decrease of mg/l was seen in the ccx -b group compared to placebo. colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. a mean decrease from baseline in the concentrations of tnf-alpha, il- p , ifn-gamma, and the chemokine rantes was shown in the ccx -b group while levels remained stable in the placebo group. ccx -b is the first chemokine-based inhibitor of leukocyte trafficking to be tested in ibd. the compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of crohns disease. the activating receptor nkg d seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for type diabetes and multiple sclerosis. the aim of this study was to asses the role of nkg d in a model of inflammatory bowel disease, where cd +cd -t cells from balb/c mice are adoptively transferred to scid mice, and to evaluate the therapeutic effect of an anti-nkg d antibody therapy. the expression of nkg d was evaluated by flow cytometry, immunohistochemistry and by pcr. we found a marked up-regulation of nkg d on the cell surface as well as increased levels of nkg d mrna in cd + t cells from colitic scid mice as compared to normal balb/c mice. we next studied the effect of anti-nkg d antibody (cx ) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. cx treatment decreased the cellsurface expression of nkg d and prophylactic administration of cx attenuated the development of colitis significantly. a moderate reduction in the severity of disease was observed after cx administration to mildly colitic animals, whereas cx did not attenuate severe colitis. thus, nkg d may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of nkg d in cd + t cells increased markedly during the development of disease and since administration of cx early but not late in the course attenuated the disease severity. proteins are used already for more than a century in the treatment of disease. the first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. the second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. the development of the recombinant dna and cell fusion technology in the seventies of the th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. in human insulin was introduced as the first recombinant dna derived biopharmaceutical and since than more than have gained approval. the pipeline contains many more potential biopharmaceuticals and at present in new drug applications concerns a biotechnology derived product. a major problem of therapeutic proteins is the induction of antibodies. for foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. however the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. and also the proteins which are homologues of endogenousfactors such as gm-csf, interferons etc. induce antibodies, sometimes even in the majority of patients. by definition we are immune tolerant to products which are copies of endogenous proteins. the products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. when human therapeutic proteins induce antibodies, they are breaking b cell tolerance, which starts with the activation of autoreactive b-cells. presenting the self-epitopes in an array form is very potent activator of these b-cells. this explains why aggregates of human proteins are the most important factor in induction of antibodies. these aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. there are only a few studies in experimental model systems on the properties of the aggregates which break b-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved. we study the capacity of a protein product to break b-cell tolerance in mice made transgenic for the specific protein. these mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. although these models have helped to identify the factors important for breaking b-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. they also allow to study the involvement of tcells in breaking b cell tolerance. all data obtained untilnow indicate this process to be t-cell independent. contact information: dr huub schellekens, central laboratory animal institute, utrecht university, utrecht, the netherlands e-mail: h.schellekens@gdl.uu.nl biomonitor aps, and institute for inflammation research (iir), rigshospitalet, copenhagen, denmark using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (abs). many have assumed that these drugs may be administered with little or no risk of triggering specific t-and/or b-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. unfortunately, this is not the case, and even socalled % human biologicals are potentially immunogenic ( ) ( ) . i shall discuss two examples: ) recombinant human cytokines (ifn-beta- a and - b), and ) anticytokine ab constructs (anti-tumor necrosis factor (tnf)-alpha). ifn-beta has been used for treatment of patients with multiple sclerosis since the early nineties. though initially neglected as a clinical problem, ifn-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. the reported frequencies and titers of anti-ifn-beta ab vary considerably depending upon ifn-beta preparations and administration, and the types of assays being used ( - ). it took more than years of clinical use before abmediated decrease in bioactivity of ifn-beta, a condition in which the clinical effect of continued injection of rec. ifn-beta is minimized or abrogated, was universally recognized ( , ). ) anti-tnf-alpha human ab constructs tnf-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of tnf-alpha have long been a goal in the treatment of these conditions. currently, there are three approved and two other anti-tnf-alpha biopharmaceuticals in clinical use. unfortunately, response failure is frequently encountered. thus, - % of patients are primary non-or lowresponders to the anti-tnf constructs, and secondary response failure is commonplace, mostly due to induction of anti-abs. several different methods have been used to assess circulating levels of anti-tnf drugs as well as anti-abs. most of these have been based on elisa technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc. interferon beta (ifnbeta) has been an important step forward in the treatment of multiple sclerosis(ms), an inflammatory disease of the human central nervous system. however, one of the problems of ifnbeta is its immunogenicity; a substantial percentage of ms patients treated this recombinant protein develop anti-ifnß antibodies, primarily of the igg class. the level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. most studies of these antibodies have measured their ability to neutralize ifnbetas effect in vitro, using assays in which sera from ms patients inhibit the protective effect of ifnbeta on viral killing of target cells. this antibody population is called neutralizing antibodies (nabs). tests measuring binding of antibodies to ifnß in vitro are called binding antibody (bab) assay. anti-ifnbeta antibodies detected by bab assays are present in a high percentage of ms patients, and can occur at low levels without any apparent adverse effect on ifnbeta bioactivity. the distinction between babs and nabs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of babs and nabs is a continuum with the assay systems simply measuring the strength of the antibody response. in many treated patients, the anti-ifnbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. high levels of anti-ifnbeta antibodies with high affinity results in loss of ifnbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or adb. adb can be considered the in vivo correlate of the neutralizing effect of the anti-ifnß antibody population, while the nab assay measures the in vitro neutralization of this population of immunoglobulins in the serum. the three ifnbeta preparations have different incidences of nabs and different patterns of appearance and disappearance over time of nabs. because there is no direct correlation between nab levels and bioactivity at moderate levels of nab, in vivo bioactivity assays for ifnbeta have become increasingly utilized. in a large multicenter study in the us, called the insight study, bioactivity as measured by ifnbeta induced upregulation of the ifn-response genes mxa, viperin, and ifit , was shown to be highly correlated with nab levels, confirming a single center study (pachner, a.r., pak,e., narayan, k., multiplex analysis of expression of three ifnbeta-inducible genes in antibody-positive ms patients, neurology, : - , ) . multiple studies, including a large multicenter study in denmark and a recent study from our center using high resolution mri of the brain once a month, have demonstrated that nabs abolish the salutary effects of ifnbeta on clinical aspects of ms, especially inflammation. recent guidelines for european neurologists recommend stopping ifnbeta in nab-positive patients. in order to maintain bioactivity of this important medication for ms, some neurologists have attempted to use immunosuppressives either to prevent the development of nabs, or to treat them once they have developed. however, at this point in time, there is no clearly optimal way to treat nabs. major efforts have been underway to decrease the immunogenicity of ifnbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested. proteinase-activated receptors (pars). endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin g, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. thus, the expression of pars on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of pars as a part of the communication system in the skin during inflammation and the immune response. for example, par activates nf-kb in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. on sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. par and par also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. they also stimulate signal transduction pathways involved in cutaneous inflammation. in sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. novel compounds regulating protease and par function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. in addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the gprotein coupled receptor family called the proteinase activated receptors (pars). these receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. our study examined the effect of par activation in joint inflammation and pain. male c bl/ mice received an intra-articular injection of either the par activating peptide aypgkf-nh or the inactive peptide yapgkf-nh ( mg) into the right knee. knee joint blood flow was then measured in these mice by laser doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. mechanical allodynia was also assessed in these animals by application of von frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. intra-articular injection of the par activating peptide caused knee joint blood flow to gradually increase by up to % over the succeeding hrs. knee joint swelling was also observed as well as the development of a mechanical allodynia. all responses could be blocked by pre-treatment with the selective par antagonist pepducin p pal ( mg i.p.). the control peptide yapgkf-nh had no discernible effect on joint inflammation or pain. these experiments show that peripheral activation of par receptors in mice knees causes joint inflammation and pain. vincent lagente ( ), e boichot ( ) ( ) air liquide, centre de recherche claude-delorme, jouy en josas, france ( ) inserm u , universitØ de rennes matrix metalloproteinases (mmps) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. these led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. among metalloproteinases (mmps) family, macrophage elastase (mmp- ) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (copd). pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. pulmonary fibrosis is associated with deposition of extracellular matrix (ecm) components in the lung interstitium. the excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-mmp- /timp- ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day after bleomycin administration. finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. in addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. serine proteases cleaving specifically at an arginin site are able to activate protease-activated receptors (pars), which then send specific messages to cells. we have demonstrated that members of the par family (par , par and par ) are present on peripheral sensory neurons, where they can be activated by different proteases.the activation of par and par in isolated sensory neurons provokes calcium mobilization and the release of substance p and cgrp, while the activation of par inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.thrombin and pancreatic trypsin caused inflammation respectively through a par and par -dependent mechanism involving the release of neuropeptide.the extrapancreatic form of trypsin (mesotrypsin or trypsin iv) also caused neurogenic inflammation through a par and par dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a par -dependent mechanism.in contrast, activation of par on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of pars on peripheral sensory neurons.determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs. introduction: a scoring system for disseminated intravascular coagulation (dic) in humans has been proposed by the international society on thrombosis and haemostasis (isth). it was the objective of this study to develop and validate a similar scoring system for dic in dogs in order to establish the dog as a spontaneous animal model. methods: for the developmental study, consecutive dogs admitted to the intensive care unit (icu) were enrolled prospectively (group a). blood samples were collected daily and a broad panel of coagulation assays performed. diagnosis of dic was based on the expert opinion of one human physician and two veterinarians. a multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of dic. integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the stard criteria. the validation study prospectively enrolled consecutive dogs (group b). results: dogs were excluded from group a where / dogs ( %) had dic. final multiple logistic regression model was based on aptt, pt, d-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. diagnostic accuracy of the model was sustained by prospective evaluation in group b. conclusion: based on generally available assays, it was possible to design an objective diagnostic model for canine dic, which has both a high sensitivity and specificity. such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients. in , a coagulation independent change in light transmittance (biphasic waveform [bpw] ) was reported in automated activated partial thromboplastin time assays (aptt) in patients with disseminated intravascular coagulation (dic). a calcium-dependant precipitate of creactive protein and very-low-density-lipoprotein was causing the bpw. our group recently identified this phenomenon in dogs also. initially, bpw was introduced as a complementary tool to assist diagnosing dic. however, recent studies reported that bpw may have a stronger potential as a prognostic marker for survival. the aims of the study were to prospectively investigate (a) the diagnostic significance of bpw regarding dic and (b) the significance of bpw to outcome, in dogs with diseases known to predispose for dic. the study was performed as a prospective, observational study including consecutive dogs with a final diagnosis known to predispose for dic ( % were finally diagnosed with dic). outcome was -day survival. bpw was assessed by means of a hirudin-modified aptt assay (kjelgaard-hansen et al., jvim : ; - ) . relative risk according to bpw (rr [ % confidence interval]) for (a) a dic diagnosis and (b) -day mortality, were assessed. -day mortality in the study population was %. % were bpw positive. bpw was not a significant diagnostic factor for dic (rr= . [ . ; . ] ), but strongly so for outcome (rr= . [ . ; . ] ) with a % ( / ) mortality amongst bpw positive dogs. in conclusion, bpw was observed in dogs predisposed to dic, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. ( ), b hideo ( ) ( ) department of molecular pathology, kumamoto university, japan ( ) department of gastroenterological surgery, kumamoto university, japan aeromonas species are facultative anaerobic gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (dic) is a life-threatening complication of sepsis patients, causing multiple organ failure.however, a mechanism leading to coagulation induction in the bacterial infection has not been known. to study the dic induction by aeromonas species infection, we investigated coagulation activity of a serine protease (asp) from aeromonas sobria, predominantly isolated in patients blood. proteolytically active asp shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of nm. asp activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate boc-val-pro-arg-mca, but no enzymatic activity was produced from coagulation factors ix and x. analysis by sds-page revealed that asp released a prothrombin fragment with a molecular weight identical with that of f¿-thrombin in an incubation timedependent manner. western blotting using biotinylated phe-pro-arg-chloromethylketone, a thrombin inhibitor, showed that asp produced an enzymatically active fragment whose molecular weight was same as that of f¿-thrombin. prothrombin incubated with asp but not the protease itself caused platelet aggregation. these results indicate that asp activates prothrombin, producing f¿-thrombin that converts fibrinogen to fibrin clot, and suggest that asp coagulation-inducing activity contributes to dic development in sepsis caused by aeromonas sobria infection. the present study shows a link between inflammation and coagulation mediated by a bacterial protease. hemolytic episodes are often associated to high amounts of free heme in circulation (up to um) and the development of an inflammatory response that may develop to a chronic inflammation. our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and nadph oxidasederived reactive oxygen species (ros) generation, as well as pkc activation and interlukin- expression (graÅa-souza et al., ) . moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. heme protective effect requires nadph oxidase-derived ros and involves the activation of mapk, pi k and nf-kb signaling cascades as well as heme oxygenase (ho) activity (arruda et al., ) . more recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of bax insertion into mitochondria and a dramatic increase on bcl-xl/bad protein ratio in a ros-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. these findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. the recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.financial support: faperj, cnpq, capes. ( ) ( ) university of melbourne, victoria, australia ( ) monash university, victoria, australia we have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (gpx ), show significantly larger infarcts after stroke. recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. to explore this, gpx -/-mice were subjected to transient mid-cerebral artery occlusion (mcao) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. after hr mcao, leukocyte-endothelium interaction was significantly reduced in gpx -/-mice compared to wt counterparts during the second hour of reperfusion. laser doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in gpx -/-mice following transient mcao. this suggests that the reduction in nutritive blood flow following stroke in gpx -/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. furthermore, matrix metalloproteinase- (mmp ) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in gpx -/-mice to a greater extent than in wt mice after mcao, suggesting a role for oxidative stress in cerebral microvascular injury. the data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of mmp . chris bolton( ), c paul( ), s barker ( ), r mongru ( ) ( ) william harvey research institute, london, uk ( ) university west of england, bristol ( ) queen mary university of london adrenomedullin (am) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.in vitro work has shown that am beneficially regulates blood-brain barrier (bbb) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.our preliminary studies in acute experimental autoimmune encephalomyelitis (eae), a model of the human disease multiple sclerosis (ms), have demonstrated significant elevations in am peptide levels corresponding with am mrna changes during late, neurological disease where am production may be linked to the restoration of bbb function. however, am is not exclusively produced as result of am gene upregulation. furthermore, am peptide levels do not always match am mrna changes during other disease phases of eae.the current study has investigated, more closely, the relationship between am gene expression and subsequent levels of associated peptides. am mrna levels were determined, by rt-pcr, in the cerebellum, medulla-pons and spinal cord of normal and eae-inoculated lewis rats at the height of disease. am and proadrenomedullin peptide (pamp) levels were measured in the tissues by radioimmunoassay.all tissues examined showed an increase in am gene mrna compared to control levels.am and pamp changes were observed in the samples and differences between the peptide profiles were recorded.an understanding of alterations in the generation of am and related peptides during neuroinflammation may provide insight into mechanisms affecting bbb permeability and be of relevance to the changes in neurovascular function seen during ms. platelet-activating factor (paf) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. although, paf is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that paf may be a mediator of noxious signaling in spinal cord in case of neuronal injury. paf-induced tactile allodynia may be mediated by atp, glutamate and the generation of nitric oxide (no). the present study elucidated down-stream signaling pathway for paf-induced tactile allodynia. paf-and glutamate-induced tactile allodynia was blocked by the pretreatment with no scavengers and inhibitors of no synthase, soluble guanylate cyclase or cgmp-dependent protein kinase (pkg). recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. to explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a subunit (glyr a ) by sirna spinally transfected with hvj-e vector was examined. in mice spinally transferred with sirna for glyr a , the reduction of glyr a was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pcpt-cgmp, paf, glutamate failed to induce tactile allodynia in mice spinally transferred with sirna of glyr a , while these compounds produced tactile allodynia in mice transferred with mutant sirna of glyr a as a control. glycine tranporter inhibitors ameriolated paf-and pcpt-cgmp-induced allodynia. these results suggest that glutamate-no-cgmp-pkg pathway plays a key role for paf-induced tactile allodynia in spinal cord and glyr a may be a target molecule for pkg to induce allodynia. ( ), r leite( ), ys bakhle ( ) ( ) federal university of minas gerais, belo horizonte, brazil ( ) medical college of georgia, augusta, usa ( ) imperial college, london, uk selective cyclooxygenase inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.we have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (cx). male holtzman rats ( - g; - animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin b, cytochalasin b) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and min later given cx ( mg/kg, s.c.). after a further min, rats were injected (ipl) with the inflammatory stimulus, carrageenan ( mg/paw). mechanical pain threshold was hourly measured over the next h, using the randall-sellitto method. the cxinduced hypoalgesia was reversed by low doses of latrunculin b or cytochalasin (latrunculin % reversal = . nanomoles), higher doses of microtubule inhibitors (taxol % reversal = . nanomoles) with no effect of acrylamide ( up to nanomoles).we conclude that ) local changes in (paw) cytoskeleton occurred during cxinduced hypoalgesia and ) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.financial support: cnpq, fapemig and capes there are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. cox- in brain during neurodegenerative or neuropsychiatric disorders.in the present study, we examined the effect of nimesulide (a preferential cox- inhibitor) in subchronic immobilization stress. mice were subjected to immobilization stress for hrs daily for a period of seven days. nimesulide ( . mg/kg, i.p.) was administered daily for days before challenging them to immobilization stress. behavioral analysis revealed the hyperlocomotor activity and increased anxious response. subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. these results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. there is accumulated evidence for ngf role as a peripheral pain mediator. ngf is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. ngf increase was also observed in temporomandibular join (tmj) after cfa injection, indicating its possible involvement in local hyperalgesic states. therefore, the objective here was to evaluate if ngf participate in the tmj nociception. to test this hypothesis, the ngf was injected into the tmj alone or after carrageenan (cg) and the spontaneous nociceptive behavior of head flinches was counted for up min. further evidence for the ngf nociceptive activity was obtained quantifying the local production of ngf after cg injection, by elisa, and the fos-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after ngf injection. injections were performed in . ul. ngf ( . , and ug) injected in the tmj challenged h prior by cg ( ug) induces a dose-dependent increase in the number of head flinches. this increase was reduced by k a ( and ug), indicating a trka receptor-mediated effect. we detected a significant increase in the ngf production and h after the tmj cg ( ug) injection. the tmj injection of ngf ( ug) alone did not induce detectable spontaneous nociceptive behavior. however, the ngf ( ug) injection induces a significant increase in the fos like immunolabeling (fli) in the sensory trigeminal nucleus compared to the saline injection. these results indicate that the ngf participates in the nociceptive activity in the tmj, specially in inflammatory conditions. mif was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (ra) several years ago, but it now clear that mif is also involved in the pathogenesis of systemic lupus erythematosus (sle) and atherosclerosis. mif-deficient lupus-prone mrl/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to mif-expressing mice. similarly, mif-deficient atheroma-pone ldlr-deficient or apoe-deficient mice are significantly protected from disease and antimif mab therapy is beneficial. ra and sle are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of mif. given the effects of mif on atherosclerosis it can be hypothesised that mif overexpression participates in the risk of atherosclerotic vascular disease in ra and sle. recent data have provided insights into mechanisms of action for mif relevant to all these concepts. firstly, the newly described role of mif in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to ra, sle, and atherosclerosis, with evidence that mif mediates macrophage recruitment in sle and atherosclerosis. secondly, glucocorticoid (gc) therapy is possible risk factor for atherosclerosis in patients with ra and sle, and it is now clear that gc increase the expression and release of mif, potentially implicating mif in gc-related increases in atherosclerosis in ra and sle. specific therapeutic targeting of mif in ra and sle may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases. to enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.cytokines such as tumour necrosis factor (tnf) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.however, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (mif) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.therefore we explored the ability of mif to regulate leukocyte recruitment.this was achieved using intravital microscopy to examine the intact microvasculature in mice following local mif treatment. these experiments showed that mif induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly cd +/f / -ve/cd c-ve monocyte/ macrophage lineage cells.mif did not induce upregulation of adhesion molecules p-selectin and vcam- , although their constitutive expression contributed to recruitment.in contrast, mif-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, ccl /mcp- , and its receptor ccr , and in response to anti-cxcr .this was supported by in vitro experiments showing that mif induced ccl /mcp- release from cultured murine endothelial cells.finally, mice lacking cd , the putative mif binding molecule, did not respond to mif.these data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from cd .this function may be critical to the ability of mif to promote diseases in which macrophages are key participants. gm-csf and m-csf (csf- ) can enhance macrophage lineage numbers as well as modulate their differentiation and function. of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. what the critical actions are of these csfs on macrophages during inflammatory reactions are unknown. to address this issue, adherent macrophages (gm-bmm and bmm) were first derived from bone marrow precursors by gm-csf and m-csf, respectively, and stimulated in vitro with lps to measure secreted cytokine production, as well as nf-kb and ap- activities. gm-bmm preferentially produced tnfa, il- , il- and il- while, conversely, bmm generated more il- and ccl ; strikingly the latter population could not produce detectable il- and il- . following lps stimulation, gm-bmm displayed rapid ikba degradation, rela nuclear translocation and nf-kb dna binding relative to bmm, as well as a faster and enhanced ap- activation. each macrophage population was also pre-treated with the other csf prior to lps stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and nf-kb activity. thus gm-csf and m-csf demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for m-csf. granulocyte macrophage-colony stimulating factor (gm-csf), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. gm-csf is able to prime macrophages for increased pro-inflammatory responses, including the increased release of tnfa and il- following stimulation with, for example, lps. in addition, gm-csf has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. gm-csf-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of gm-csf with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. t cells appear to be the major cell type responsible for gm-csf production required for arthritis, and gm-csf appears important in the effector phase of disease, subsequent to t cell activation. blockade of gm-csf results in fewer inflammatory cells, particularly macrophages, and cytokines such as tnfa, at the site of inflammation. these findings suggest that blockade of gm-csf may be an effective treatment in a range of inflammatory diseases. the autoimmune disease type diabetes mellitus (t dm) is thought to be mediated by autoreactive t cells recognizing islet autoantigens, including gad , ia- and proinsulin. this disease arises on a distinctive genetic background, mapping most notably to the mhc, and is also open to strong environmental influence. to investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive t cells are important, we have developed an approach to epitope identification that is mhc allele and autoantigen specific, and operates for both cd and cd t cells. utilizing this, we have uncovered populations of islet antigen-specific t cells that have the immunological credentials to be both pathogenic (eg th , tc ) and protective (treg) in the disease. we have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions. tcr transgenic targeting b: - cause diabetes . knockouts of the insulin gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin gene (islet expression) prevents % of diabetes . dual insulin knockout with transgenic insulin with altered peptide (b :a) prevents all diabetes . islets with native b: - sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by t cells .anti-b - t cells have conserved valpha and jalpha chain usage but no conservation n region or beta chain . alpha chain as transgene sufficient to engender anti-insulin autoantibodies . kay and coworkers demonstrate insulin reactivity "upstream" of igrp and igrp reactivity nonessential.future studies in nod directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.in man we can now identify at birth genetic risk as high as % of activating anti-islet autoimmunity with mhc analysis and restricted heterogeneity suggesting dominant target.insulin autoantibodies in prospective studies such as daisy usually appear initially and levels are related to progression to diabetes.analysis of cadaveric donors is underway to elucidate primary targets. (t d) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. the holy grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. this has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of t-cell co-stimulation molecules) or form (as an altered peptide ligand). compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (nod) mouse model of t d, and possibly in humans. proinsulin/ insulin dna, protein or t-cell epitope peptides administered in a tolerogenic manner to the nod mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector t cells, induction of regulatory t cells). administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory t cells in the rodent, is the most immediately translatable approach to humans. initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for t d. combination autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established t d. leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). the initial paradigm of a -step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. additionally, organ-specific differences regarding the role of distinct molecules were established. finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. in-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-lewisx, or icam- and lfa- , have been proven sufficiently relevant to make them candidates for potential therapies. with the anti lfa- antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuíon molecules which obviously mediate more than just diapedesis. finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa. department of dermatology, heinrich-heine-university, düsseldorf, germany atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. the recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. during recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. with regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory t and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. the skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. a network of pro-inflammatory cytokines including il- and tnf-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. mitogen-activated protein kinases (mapks) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. increased activity of mapks, in particular p mapk, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. a careful examination of different signalling pathways in various inflammatory conditions is therefore needed. this presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin- , tnf-µ, p mapk, msk / , mk , nf-kappab and ap- . histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. in contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. the discovery of the histamine h receptor (h r) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. h r deficient mice and mice treated with h r antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in th responses. ex vivo restimulation of primed t cells showed decreases in th cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the h r was responsible for the the t cell effects. the influence of h r on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and h r deficient mice. the h r was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the h r was minor. surprisingly the h r effect was independent of mast cells or other hematopoetic cells. this work suggests that the h r can modulate both allergic responses via its influence on t cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. the studies show that the h r mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of h r antagonists. ( ), bsp reddy ( ) ( ) nizams institute of medical sciences, hyderabad; india ( ) genix pharma, india rupatidine, carries the majority of the histamine h receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria. objectives: the aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response. methodology: male volunteers were enrolled after written informed consent before to ethic committee approved protocol. in this randomised, double-blind, single oral dose, cross overstudy, they were randomized to receive either mg rupatidineformulation after overnight fast. washout was days. wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. ten minutes later, wheal and flare were visualized under a bright lamp. histamine induced wheal and flare skin test was performed before and regularly to hours after drug administration. results: administration of reference and test formulations of rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. the least square mean ratio (%) t vs r for peak activity imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (auc - mmsq/hr and auc - %/hr) both for untransformed and log transformed data were found to be within - % of % ci limits both formulations well tolerated. conclusions: it can thus be concluded that be concluded that test formulation of rupatidine tablet is bioequivalent to reference rupatidine tablet ( ), h yoshimura( ), k ohara ( ), y mastui ( ), h hara ( ), h inoue ( ), h kitasato ( ), c yokoyama( ), s narumiya ( ), m majima ( ) ( ) kitasato university school of medicine, kanagawa, japan ( ) tokyo dental medical colledge, tokyo, japan ( ) kyoto university school of medicine, kyoto, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported the magnitude of cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitorsconstitute revascularization. we hypothesized that txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggrega- inflamm. res., supplement ( ) tion.to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb model. recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out(ipko) and thromboxane (tx) a receptor (tp) knock out(tpko). blood recovery in tp-/-significantly delayed compared to wt and ipko. immunohistochemical studiesrevealed that tp-/-mice were less stained against pecam positive cells compared to wt and ipkoplasma sdf- and vegf concentration were significantly reduced in tp-/-mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. purpose: the s calcium-binding proteins, a , a and a are constitutively expressed in neutrophils and induced in activated macrophages. high levels are found in sera from patients with infection and several chronic inflammatory diseases. the calgranulin complex, a /a is anti-microbial; a has oxidant-scavenging functions. a is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (mc). effects of these mediators on mc and monocyte function were compared. methods: human pbmc or murine mc were activated in vitro with s and mediator release and cytokine induction (assessed by quantitative rt-pcr/elisa), determined. a cys to ala a mutant was used to determine whether effects on mc are mediated by redox. immunohistochemistry was used to demonstrate s s in asthmatic lung. the s s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of a . a , a or the a /a complex had relatively low ability to induce il , tnf, il , and chemokines mrna from pbmc compared to a . only a induced significant levels of il ; none induced il or gm-csf mrna compared to lps. in contrast to a which is activating, a significantly inhibited mc degranulation provoked by ige cross-linking; suppression was dependent on cys . conclusions: the cytokine profile generated by a in mc and monocytes strongly supports a role the pathogenesis of asthma. in contrast, results strongly support a role from a in oxidant defence, particularly to hypobromite generated by activated eosinophils. ( ), d mankuta( ), g gleich ( ), f levi-schaffer ( ) ( ) hebrew university of jerusalem, israel ( ) hadassah university hospital, israel ( ) university of utah, usa the onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. eosinophil major basic protein (mbp), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. characterized more than two decades ago, the exact nature of this activation has not been clarified as yet. here we demonstrate that mbp exerts its activating effect on human mast cells and basophils through cd and hematopoietic cell kinase (hck). a genome-wide analysis showed that hck displays shifts in mrna levels specifically upon mbp-induced mast cell activation. hck also shows a unique priming pattern prior to this activation. cd is phosphorylated specifically upon activation with mbp and deploys a signaling complex that critically depends on hck. extracellular neutralization of cd interferes with mbp entry into the cell, and this as well as rna silencing of hck results in defective mbp-induced activation. finally, cd neutralization abrogates mbp-induced anaphylaxis in-vivo. these findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. alexander robinson ( ), d kashanin ( ), f odowd( ), v williams( ), g walsh ( ) ( ) cellix ltd, institute of molecular medicine, dublin, ireland ( ) university of aberdeen, scotland, uk leukocyte adhesion to endothelial cell bound proteins, such as icam- and vcam- , is an initial step of the inflammatory response. we have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. - dynes/cm ). the adhesion profiles of resting and pmastimulated peripheral blood lymphocytes (pbls) were recorded, with respect to vcam- , icam- , and bsa. images at each shear stress level were captured using a digital camera, and analysed using our in-house ducocell software package. distinct morphological changes in pma-stimulated pbls, compared to non-stimulated cells, can be observed. these include a less rounded appearance of the pma-stimulated pbls, and evidence of "uropod" formation, which anchor the t cell to the endothelium as part of the migration process. levels of adhesion to vcam- are high ( - %, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and pma-stimulated pbls.however, there is a distinct difference between the adhesion levels of non-stimulated and pma-stimulated pbls to icam- , with pma-stimulated cells showing a higher affinity for icam- than nonstimulated cells (approx. % and %, respectively).pbl adhesion to bsa is negligible. we present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. this platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. introduction: pulmonary aspiration of gastric contents is a common complication observed in icu patients and a potential trigger of ards. in this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (gj). methods: gj was obtained from donor rats (ph . ). male c bl/ were instilled with ml/kg of gj. after or h, the animals were sacrificed and lung and balf were collected. control group consisted of non-manipulated mice. . ae . , sg h: . ae . ; pg/ml). discussion: gj aspiration induced an initial adherence of pmn to lung tissue that is correlated with increased tnf-a/il- ratio in balf at the nd h. the reduction of mpo activity is correlated with the decrease in tnf-a/il- ratio. the late increase of pmn in balf might be a consequence of the early production of tnf-a. the results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of tnf-a. objectives: intestinal i/r is implicated as a prime initiating event in the development of acute respiratory distress syndrome (ards) after trauma and hemorrhagic shock. we investigated the effects of lps challenge to mice previously submitted to i-i/r, a two-hit model of acute lung injury. methods: male c bl/ mice were subjected to min of intestinal ischemia and challenged with . mg/kg of intranasal lps at the th hour of reperfusion (two-hit). balf and culture of lung explants were performed h after lps challenge. mice subjected to i-i/r or lps alone were used as controls. results: two-hit mice showed marked increase in lung evans blue dye leakage compared to i-i/r ( . ae . vs . ae . , mg/mg). lung mpo was increased ( . ae . vs . ae . ; od nm) whereas the neutrophil recruitment to balf was inhibited in the two-hit group compared to lps group ( . ae . vs . ae . ; x e cells/mouse). the levels of nox-in the two-hit group were significantly increased when compared to i-i/ r controls in balf ( . ae . vs . ae . ; mm) and in lung explants ( . ae . vs . ae . ; mm/mg of tissue). conclusions: intestinal i/r predisposes the animal to an exacerbated response to a low dose lps insult. the exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. the results are suggestive that patients exposed to systemic inflammatory response might develop ards when in contact with secondary inflammatory stimuli. nitric oxide may play an important role in this process. ( ) ( ) novartis institutes for biomedical research, horsham, uk ( ) university of michigan, usa obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like copd. to test this hypothesis, we exposed high capacity runner (hcr) and low capacity runner (lcr) rats to months of whole-body smoke exposures.the animals, bred over successive generations on the same background strain for high or low running capacity, differ by over % (p< x - ) for exercise capacity, measured by running on a treadmill.after months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the hcr-and lcr-smokeexposed(se) animals compared to air-exposed controls (p< . ); however there was a - -fold increase in the number of neutrophils and lymphocytes in the lcr-over the hcr-se group (p< . ).histopathology revealed there was greater inflammation and lung damage present in the lcr-versus hcr-se group (p< . ). metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both se groups, oxidative damage to the lung surfactant layer was significantly more extensive for the lcr-se. systemic oxidative damage was also more apparent in the lcr-se group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. the metabolic data suggest that repair processes maybe more effective in the hcrs. in summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. ( ), ap ligeiro-oliveira( ), jm ferreira-jr( ), sr almeida( ), w tavares de lima( ), shp farsky ( ) ( ) university of s¼o paulo, brazil ( ) regional integrated university of alto uruguai and missðes, brazil methods: male wistar rats were exposed to vehicle or hq ( mg/kg; ip.;daily, days, two-day interval every five days). on day , animals were ip sensitized with ovalbumin (oa). assays were performed on day . results: hq-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro oa-induced tracheal contraction. the latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of oa. the oa-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound / . in fact, lower levels of circulating oa-anaphylactic ige antibodies were found in hq-exposed rats. this latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules cd and cd on oa-activated lymphocytes indicated the interference of hq exposure on signaling of the humoral response during an allergic inflammation. contact information: ms sandra manoela dias macedo, regional integrated university of alto uruguai and missðes / university of s¼o p, department of clinical and toxicological analyses, s¼o paulo, brazil e-mail: smdmacedo@yahoo.com.br ( ) ( ) radboud university nijmegen, medical centre, nijmegen, the netherlands ( ) university hospital, zürich, switzerland toll-like receptors (tlr) are essential in the recognition of invading microorganisms. however, increasing evidence shows involvement of tlr in autoimmunity, such as rheumatoid arthritis (ra), as well. here we investigated whether synovial expression of tlr and tlr was associated with the expression of ifna, tnfa, il- b, il- , il- , and il- and studied in what way these receptors and cytokines were associated in vitro. using immunohistochemistry, we found that tlr / tlr expression in synovial tissue was associated with the presence of ifna, il- b and il- , but not tnfa, il- and il- . to investigate whether ifna, il- b and il- could induce tlr / tlr upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined tlr / mrna expression. ifna incubation resulted in significant tlr /tlr upregulation, whereas il- b and il- did not. pre-incubation with ifna and subsequent stimulation of tlr /tlr significantly enhanced il- , tnfa and ifna/b production, indicating that the ifn-induced tlr upregulation was functional. low amounts of biologically active il- b were produced upon stimulation with atp, but not upon tlr / tlr stimulation, although mrna levels were high. interestingly, ifna-priming significantly increased the atp-induced il- b production. here, we demonstrated a dual role for ifna in vitro, which could explain the association between tlr and il- b / il- in synovial tissue. first, involvement in tlr /tlr regulation and second, involvement in atp-induced production of biologically active il- b. these results suggest involvement of anti-viral immune responses in ra and ifna as a key player in chronic inflammation. the pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (ppr) has been suggested recently. here, we described the role of two members of the nacht-lrr (nlr) family, nod (nucleotide/ binding oligomerization domain) and nod in model of acute joint inflammation induced by intraarticular injection of tlr (toll-like receptor) agonist streptococcus pyogenes cell wall fragments. we found that nod deficiency resulted in reduced joint inflammation and protection against early cartilage damage. in contrast, nod gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. to explore whether the different function of nod and nod occurs also in humans, we exposed pbmcs carrying either nod frameshift or nod frameshift mutation with scw fragments in vitro. both tnfa and il- b production was clearly impaired in pbmcs carrying the nod fs compared to pbmcs isolated from healthy controls. in line with the nod gene deficient mice, human pbmcs bearing the nod mutation produced enhanced levels of proinflammatory cytokines after h stimulation with scw fragments. these data indicated that the nlr family members nod and nod have a different function in controlling tlr -mediated pathways. we hypothesize that intracellular nod -nod interactions determine the cellular response to tlr triggers. whether lack of controlling tlr -driven pathways by nod signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (ra), remains to be elucidated. leukocyte immunoglobulin-like receptors (lilrs) are a family of receptors with potential immune-regulatory function. activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (ra). ra is a chronic inflammatory disease of joints caused by mediators (i.e. tnf-a) produced by activated leukocytes. we recently demonstrated expression of activating lilra in synovial tissue macrophages from ra patients. the aim of this study was to determine expression and function of lilra in monocytes and macrophages. peripheral blood mononuclear cells (pbmc) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating thp- cells with vitamin-d . lilra expression was measured by flow cytometry before and after modulation with cytokines. differentiation to macrophages significantly up-regulated lilra expression (p= . ). treatment of macrophages with lps, tnf-a, il- b and ifn-g but not il- caused significant down-regulation of lilra (p< . ). function of lilra was assessed by cross-linking with plate-immobilised lilra -specific mab. soluble tnf-a was measured by elisa. activation of cells elicited tnf-a production in a dose-dependent manner while time-course analysis shows maximal production at h. correlation between lilra expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. in ra, abnormal regulation of lilra could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. pharmacological blocking of lilra could potentially provide therapeutic benefit. ( ) ( ) university of valencia, spain ( ) northwick park institute for medical research, uk co-releasing molecules (co-rms) mimic the biological actions of co derived from heme oxygenase activity. in the present work we studied the effects of a water-soluble co-releasing molecule (corm- ) on an animal model of human rheumatoid arthritis. dba- /j mice were treated with corm- ( , or mg/kg/day, i.p.) from day to after collagen-induced arthritis (cia) and sacrificed on day . administration of corm- resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. all these parameters were significantly reduced by corm- treatment with the most pronounced protective effect observed at mg/kg. the levels of pro-inflammatory mediators (pge , il- beta, tnfalpha, il- and il- ) in the hind paw homogenates were significantly inhibited by corm- . in addition, comp levels in serum, a marker of cartilage degradation, was reduced by the co-releasing agent. our studies show that therapeutic administration of corm- alleviates the clinical features of murine cia at the late phase of this response. the beneficial action of co liberated from corm- appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. aim: to setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis. methods: full thickness bovine articular cartilage punches were cultured with or without ng/ml il- a, tnf-a and oncostatin m. after three weeks the cartilage and culture medium were analyzed for weight changes, water content, dna content, glycosaminoglycans (gag), hydroxyproline (hyp), damaged collagen molecules, mmp activity, ctx-ii and comp. diclofenac, dexamethasone, pioglitazone, remicade, risedronate, galardin and a - were tested for their effect on cartilage degradation. results: exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active mmps (all p < . ). comp release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment. discussion: stimulation of bovine articular cartilage explants with a cocktail of il- a, tnf-a and osm results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. toll-like receptors (tlrs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. involvement of tlr and tlr activation in the expression of arthritis was studied using interleukin- receptor antagonist deficient (il- ra-/-) mice, which spontaneously develop an autoimmune t-cell mediated arthritis. spontaneous onset of arthritis was dependent on tlr activation by microbial flora, as germ-free mice did not develop arthritis. after crossing with tlr knockouts, il- ra-/-tlr -/-mice developed more severe arthritis compared to il-ra-/-tlr +/+ littermates; whereas, tlr -/-il- ra-/-mice were protected against arthritis. to clarify the mechanism by which tlr and tlr differentially regulated the disease expression, we studied the role of these tlrs in il- production and th development, both important in il- ra-/-arthritis. wild type bone-marrow-derived dendritic cells (bmdcs) produced similar levels of il- upon stimulation with tlr and tlr ligands; however, il- ra-/-bmdcs produced less il- than wild type dcs upon tlr stimulation and more il- than wild type dcs upon tlr stimulation. furthermore, il- ra-/-t cells produced lower amounts of il- when cultured with tlr -activated apcs and higher amounts of il- when cultured with tlr -stimulated apcs, both in combination with cd stimulation. facs analysis of th (cd +/il- +) cells from both spleen and draining lymph nodes revealed % reduction in il- ra-/-tlr -/-mice compared to il- ra-/-tlr +/+ littermates. specific cd /cd stimulation of non-adherent splenocytes confirmed lower il- production in il- ra-/-tlr -/-. these findings suggest important roles for tlr and tlr in regulation of th development and expression of arthritis. prostaglandine (pge ) stimulates the transactivational activity of p through p map kinase-dependent ser phosphorylation (jbc ) .p controls cell-cycle progression, in part, by differential regulation of ap- proto-oncogenes (jun/fos).currently we studied pge control of cyclin d promoter activity with particular attention to the role of ap- oncogenes.pge induced a . fold increase in junb mrna expression (northern blot), a . -fold increase in junb promoter activity (luciferase assay), and increased ser junb phosphorylation in human synovial fibroblasts (hsf) (western blot).c-jun was strongly inhibited while jund, c-fos, fra / , and fosb expression were upregulated by pge .in cell-cycle experiments, transformation with a constitutively active ha-ras construct (ras g v) resulted in a . fold increase in cyclin-d promoter activity, cyclin-d synthesis, thr /tyr phosphorylation of erk / ( . fold) and ap- (c-jun)-dependent transactivity ( . fold); cyclin d /cdk - inhibitor p ink a synthesis was suppressed. addition of excess rass n dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.ectopic expression of c-jun, c-fos and especially jund expression constructs stimulated cyclin d promoter activity/protein synthesis, blocked p ink a synthesis; the latter effects were reversed by the addition of excess junb.pge exerted temporal and bi-phasic dose-dependent control of the cyclin d promoter activity, largely through differential ap- activation and promoted cell cycle arrest and apoptosis in hsf at high physiological concentrations.the results provide further insight into the biology of the cpla / cox/pges biosynthetic axis and highlight the complexity of pge action in terms of cell-cycle progression. di-glucopyranosylamine (diga) is an antikeratitic (roberts et al., , acvo conference, scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (bolton et al., , inflammation res. (s ) s ) and unknown mechanism of action.interestingly, anti-tnf therapy is anti-keratitic.-diga hydrolyses to monoglucosylamine (mga) and glucose, which is prevented by n-acetylation (nacdi-ga).lider ( , pnas. : - ) showed that sulphated disaccharides are orally active, inhibit tnf synthesis and the dth reaction.we have investigated the anti-tnf and anti-rheumatic activity of the sulphated and free digas.human whole blood (hwb) was stimulated with pha ( mg/ml) to synthesise tnf.antigen induced arthritis (aia) was induced in methylated bovine serum albumin (mbsa) sensitised c bl/ mice challenged i.a. into the stifle joint.collagen arthritis (cia) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day . hwb tnf synthesis was inhibited by diga, mga and nacdiga(ic < . mm). diga ( ml, mm) i.a. prevented hour aia (- . +/- . mm).diga at mg/kg reduced aia when administered i.v. (- . +/- . mm, p< . ) and i.p. (- . +/- . mm, p< . ), but is hydrolysed p.o. ( . +/- . mm ns). polysulphated diga (diga s) was unstable, but stabilised by n-acetylation (nacdi-ga s).tnf synthesis was potently inhibited by both nacdiga and nacdiga s (ic < . mm).nacdiga s ( mg/kg p.o.) inhibited aia (- . +/- . mm), and nacdiga s with lower degrees of sulphation (mw and kda) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of tnf synthesis and possess oral anti-rheumatic activity. excessive no appears to play a key role in the pathogenesis of chronic inflammatory diseases. in this study we aimed to evaluate no synthesis in rheumatoid arthritis (ra) before and after therapy. it was performed on persons, divided into groups: a negative control group of healthy volunteers, a positive control group with ra, a group with ra and physiotherapy (phys), a group with ra and low doses of cimetidine (cim) + doxycycline (dox), a group with ra and combined treatment phys + cim + dox, and a group treated with usual doses of ibuprofen (ibu). serum nitrite/nitrate (griess) was measured in order to evaluate no synthesis. results: compared to the positive control group, in all the treated groups no synthesis decreased significantly. there was no significant difference between phys and cim+dox effect alone. the combined treatment, phys + cim + dox had a much better inhibitory effect on no synthesis. between the phys, cim + dox and phys + cim + dox groups and that treated with ibuprofen, there was no significant difference in reducing no synthesis. conclusions: ) in ra phys + cim + dox treatment was as efficient as ibuprofen in reducing no synthesis. ) the low doses of cim and dox may allow a longer treatment due to the lower side effects risk enhanced socs expression following exposure of murine macrophages to lps implicated socs in the control of lps-mediated signaling. socs regulates nfkb signaling in murine macrophages, blocking at the level of mal or ikba phosphorylation. we investigated the role of socs in regulating the production tnf by lps and pam csk -activated primary human monocytes. blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing socs (adv-socs ), control vector (adv-gfp) or left untreated. adv-socs monocytes were exposed to tlr and tlr ligands, lps ( ng/ml) or pam csk ( ng/ml). facs analysis demonstrated infection efficiencies of ae % and ae % (n= , mean ae sem) of monocytes expressing adv-gfp or adv-socs at moi . adv-socs blocked lps and pam csk induced tnf mrna and protein production in a dose-dependent manner. in contrast, il- and il- production by adv-socs -infected monocytes was not blocked. adv-socs also blocked lps and pam csk induced tnf production by macrophages isolated from synovial fluid. infection efficiencies of ae % or ae % were obtained. quantitative western blot analysis revealed that the classically defined nfkb pathway was not altered at the level of ikba or p activation. furthermore, the kinetics of lps and pam csk induced ikba phosphorylation and degradation in adv-socs monocytes remained unaffected (n= and donors, respectively). further, analysis of parallel mapk pathways demonstrated no block in p or erk mapk pathways. these data suggest that socs regulation of lps and pam csk -induced tnf production by human monocytes occurs downstream of tlrs, possibly at the level of transcription. recently, beta-nad+ has emerged as a novel extracellular player in the human urinary bladder. beta-nad+ is the natural substrate of cd which catalyzes the conversion of beta-nad+ to cadpr. under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-nad+ compared to intracellular levels ( - mm). during inflammation cell lysis may cause bursts of high local beta-nad+ levels. however, the effect of beta-nad+ on the human detrusor smooth muscle cells (hdsmc) was unknown. the effect of beta-nad+ on cultured (explant technique) hdsmc was determined by: ) measuring cytosolic free calcium ([ca +] i) in fura- loaded hdsmc using spectrofluorometry and ) force measurements in - mg detrusor strips. hdsmc responded to beta-nad+ ( - mm) with an immediate and transient increase in [ca +] i. the ca + transient was followed by one or two much slower and transient increases in [ca +] i, indicative of beta-nad+ enzymatic conversion into cadpr. the ca + responses persisted in the absence of extracellular calcium. the ca + responses to beta-nad+ were not affected by exposure of hdsmc to atp supporting the notion that the effects of beta-nad+ were not mediated via p x purinoceptors. furthermore, beta-nad+ caused a concentration-dependent detrusor muscle relaxation. this is the first study to report that extracellular beta-nad+ affect intracellular calcium homeostasis and force in hdsmc. these powerful actions of beta-nad+ suggest a role for beta-nad+/cadpr system as a novel extracellular player in the human detrusor during inflammation. aids remains a worldwide threat more than two decades after identification of hiv as the etiological agent. its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. hiv trans-activator (tat) is one of the regulatory proteins that mediates hiv replication and dysregulates cellular functions such as apoptosis and cytokine expression. for example, tat induces tumor necrosis factor (tnf) and enhances gp -induced neurotoxicity. we recently showed that tat induces the overexpression of il- via cellular kinase pkr and activation of transcription factor ets- . in this study, we examined whether tat plays a role in perturbing interferon-& (ifn&) signal transduction. we showed that tat impaired ifn&-induced stat tyrosine phosphorylation, but had no effects on the serine residue of stat and jak kinases in primary human blood monocytes. furthermore, we found that the nuclear translocation of phospho-stat was abrogated by tat. the inhibition of phospho-stat led to the deformation of stat homodimers and subsequent stat-dna complex. to investigate the cellular consequences, we measured the expression of ifn&-stimulated genes including human leukocyte antigen (hla) and , oligoadenylate synthetase ( , oas), a key enzyme in the activation of latent ribonuclease l. the results showed that tat inhibited transcriptional activation of , oas and hla. taken together, we identified a new role for tat in which it impairs ifn& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to hiv persistence, opportunistic infections and disease progression. caspase- belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin- (il- ), a cytokine known to play an important role in the pathogenesis of psoriasis. the purpose of this study was to determine the expression of caspase- in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase- and il- . interestingly, increased caspase- activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. in vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p mapk dependent increased secretion of procaspase- and active caspase- . furthermore, anisomycin increased the mrna expression of il- through a p mapk dependent but caspase- independent mechanism, reaching a maximum level after hours of stimulation. finally, anisomycin caused a rapid ( hours) increase in the secretion of proil- and active il- . secretion of active il- was mediated through a p mapk/caspase- dependent mechanism, whereas secretion of proil- was mediated by a p mapk dependent but capsase- independent mechanism. these data demonstrate that the activity of caspase- is increased in psoriatic skin and that il- secretion is regulated by a p mapk/caspase- dependent mechanism, making caspase- a potential target in the treatment of psoriasis. prostaglandin e (pge ) regulates the stability of cyclooxygenase- (cox- ) mrna through adenylate/uridylate-rich elements (ares) in the untranslated region ( utr) by a positive autocrine/paracrine feed-forward loop. the principal objective of this study was to elucidate the molecular mechanisms involved in the pge dependent stabilization of cox- in human synovial fibroblasts (hsfs). transfection of well-known are binding proteins (aubps) demonstrated that tristetraprolin (ttp) potently destabilized a [luciferase-cox- utr] reporter fusion mrna ( ae . % decrease in luciferase activity vs. control). ttp protein levels in hsfs remained constant despite il- b-induced changes in ttp mrna levels, thus suggesting translational regulation of its expression. pge did not affect the transcription or translation of this gene in hsfs. western blot analysis of hsf ttp demonstrated the existence of a specific, covalent~ kda heterocomplex containing ttp (ttphcx). although ttphcxs exact composition and stoichiometry is yet to be defined, pge selectively regulated the amount of this heterocomplex in a time-dependent manner. furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that pge can induce export of a small nuclear pool of ttp-gfp. finally, transfection of ttp into hsfs also influenced cox- gene transcription, thus enabling ttp to regulate cox- gene expression at both the transcriptional and post-transcriptional level. in conclusion, we have demonstrated that ttp is an rna binding protein capable of influencing cox- mrna stability and transcription and whose localization and interaction with other factors is regulated by pge . these data can provide important insight into deciphering the role of pge in fine-tuning physiological and pathophysiological gene regulation. ( ) ( ) chinese academy of sciences, shanghai, pr china ( ) ohio state university, usa mitogen-activated protein (map) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. map phosphatases (mkp)- is an archetypical member of the dual-specificity phosphatase family that deactivates map kinases. induction of mkp- has been implicated in attenuating the lipopolysaccharide (lps) and peptidoglycan (pgn) responses, but how the expression of the mkp- is regulated is still not fully understood. here, we show that inhibition of p map kinase by specific inhibitor sb or rna interference (rnai) markedly reduced the expression of mkp- in lps or pgn-treated macrophages, which is correlated with prolonged activation of p and jnk. depletion of mapkap kinase (mk ), a downstream substrate of p , by rnai also inhibited the expression of mkp- . the mrna level of mkp- is not affected by inhibition of p , but the expression of mkp- is inhibited by treatment of cycloheximide. thus, p mapk plays a critical role in mediating expression of mkp- at a posttranscriptional level. furthermore, inhibition of p by sb prevented the expression of mkp- in lpstolerized macrophages, restored the activation of map kinases after lps restimulation. these results indicate a critical role of p -mk -dependent induction of mkp- in innate immune responses. the i-kb kinase (ikk) complex regulates the activation of nf-kb a key transcription factor in inflammation and immunity. whilst ikka activity is necessary for proinflammatory and anti-apoptotic gene expression, ikka has distinct roles in lymphorganogenesis and b cell maturation. here we describe a role for ikka in cell mediated immunity (cmi). paw inflammation in methylated bsa-induced cmi was significantly reduced in transgenic mice expressing a mutant ikka protein that cannot be activated (ikka aa/aa ) compared to wild-type (wt). antigen-induced il- and ifng production by ikka aa/aa splenocytes and ikka aa/aa t cell:dc cocultures were also significantly reduced ex vivo. this could be normalised by using wt t cell: ikka aa/aa dendritic cell (dc) but not ikka aa/aa t cell:wt dc combinations. this suggests that reduced cmi in ikka aa/ aa mice is due to a defect in ikka aa/aa t cells not dcs. this is not due to a requirement of ikka in tcrmediated activation of t cells, since anti-cd /cd mediated activation of ikka aa/aa t cells was unaltered. however, lps-induced production of the important th cytokine il- is impaired in ikka aa/aa dcs. we are currently addressing the hypothesis that ikka activity may be required for the generation and maintenance of antigen-specific t cells in vivo. recently we described a role for ikkµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. ikka may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. as a latent transcription factor, nf-kb translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. although extensive studies have been done regarding how nf-kb is triggered into nucleus, little is known about how it is regulated inside nucleus. by twohybrid approach, we identify a prefolding-like protein snip that is expressed predominantly and interacts specifically with nf-kb inside nucleus. we show that rnai knockdown of snip leads to impaired nucleus activity of nf-kb and dramatically attenuates expression of nf-kb dependent genes. this interference also sensitizes cells to apoptosis by tnf-a. furthermore, snip forms a dynamic complex with nf-kb and is recruited to the nf-kb enhancesome upon stimulation. interestingly, snip protein level correlates with constitutive nf-kb activity in human prostate cancer cell lines. the presence of nf-kb within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous snip. our results reveal that snip is an integral component of nf-kb enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of nf-kb regulation. bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. an imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. in vitro, osteoclast formation from bone marrow macrophages is induced by rankl (receptor activator of nf-kappa b ligand) in the presence of m-csf (macrophage colony stimulating factor). osteoclastogenesis is markedly enhanced in bone marrow macrophages from ifnar -/-and ifnar -/-mice and results in increased number of multinucleated cells positive for osteoclast marker, trap (tartrate-resistant acid phosphatase). consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density. these findings suggest that the ifn alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that rankl signaling is essential for the induction of osteoclast differentiation. atp acting on p x receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin- beta (il- b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. here we identify pannexin- , a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin- is required for processing of caspase- and release of mature il- b induced by p x receptor activation. furthermore, maitotoxin and nigericin, two agents considered to evoke il- b release via the same mechanism were studied. maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin- -independent phase induced by p x receptor activation, and this was unaltered by pannexin- inhibition.nigericin did not induce dye uptake.inhibition of pannexin- blocked caspase- and il- b processing and release in response to this two stimuli.thus, while pannexin- is required for il- b release in response to maitotoxin, nigericin and atp, a mechanism distinct from pannexin- hemichannel activation must underlie the former two processes. introduction: saa is a classic acute-phase protein upregulated during inflammatory response. saa is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine il- . here we verify the effect of saa on the mrna expression and release of mip- alpha, a chemokyne involved in the recruitment of dendritic cells. methods: peripheral blood mononuclear cells (pbmc) isolated from peripheral blood by density gradient were cultured in rpmi medium in the presence of saa. mip- alphaconcentration was determinated in the supernatant of cell cultures by elisa. mrnawas analyzed bythe ribonuclease protection assay (rpa). results: pbmc stimulated with saa ( ug/ml) induced the expression of mip- alpha mrna at , and hours. mip- alpha protein was found in the suppernatant of and hours cultures (p< , ) and the addition of sb (p inhibitor) and pd (erk / inhibitor) completely abolished the release of mip- alpha. conclusions: saa is an inducer of mip- alpha expression in pbmc and p and erk / are important pathway signaling to this effect. saa is one of the factors responsible by the recruitment of dendritic cells. the p pathway is activated in numerous inflammatory conditions, including ra, ibd asthma, acute coronary syndrome, and copd, and its activation helps drive the production of inflammatory mediators. inhibitors of p decrease mediator production and therefore can produce profound anti-inflammatory effects. arry- is a potent inhibitor of p enzyme (ic < nm) with a novel pharmacophore and physiochemical properties distinct from those of other p inhibitors, being very water soluble. it is extremely potent in human whole blood, blocking lps-stimulated tnf production with an ic < nm.in animal models of rheumatoid arthritis (cia and aia) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ed ~ mg/kg). a phase i single ascending dose clinical study was run in healthy volunteers. after an oral dose of , , , or mg), blood was drawn at various times, stimulated ex vivo with lps, and analyzed for cytokines and inflammatory mediatorsfj arry- was well-tolerated and drug exposure was proportional to dose. in ex vivo samples, there was both a time-and concentrationdependent inhibition of il , pge and tnffz with > % inhibition observed at the mg dose level. the plasma concentrations of drug peaked at~ ng/ml at the mg dose and cytokine inhibition was sustained for > hours, showing that low doses of arry- produced profound effects on clinical biomarkers. further evaluation of arry- in patients with inflammatory diseases is planned. introduction: we demonstrated that in vivo chronicle blockage of nos (l-name, mg/kg; oral route; days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. these effects may be depending, at least in part, on decreased expression of l-selectin on leukocytes and pecam- on endothelium. aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of l-name treatment on secretion of tnf and il- b; by circulating leukocytes and migrated peritoneal neutrophils. methods: male wistar rats were treated with l-name ( mg/kg; oral route; days) or sterile saline (control). circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained hours after i.p. injection of oyster glycogen ( %; ml). no (griess reaction) and cytokines (elisa) were quantified in supernatants of x cultured cells before and hours after lps stimulation ( m;g/ml). results: levels of no, tnf and il- b; were reduced in circulating leukocytes from l-name-treated rats in both basal and lps stimulated conditions. on the other hand, only secretion of il- b; was impaired by migrated neutrophils. conclusions: results show that in vivo l-name treatment, which partially reduces no production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. however, the same pattern of inhibition is not detected if neutrophils are in vivo primed. objectives: to investigate the ability and mechanism of ifn-g to suppress interleukin- (il- )-induced mmp- expression in articular chondrocytes. methods: human chondrocytes were treated with ifn-g or il- beta alone or in combination. mmp- mrna was analyzed by rt-pcr. mmp- protein, phospho-stat and p / mapk levels were measured by western blotting. mmp- promoter-luciferase, cmv-cbp/p plasmids and stat sirna were transfected by calcium phosphate method. ap- activity was monitored by elisa. stat -cbp/p interaction was studied by immunoprecipitation. results: ifn-gpotently suppressed il- -induced expression of mmp- and promoter activity. blockade with neutralizing ifn-gr antibody revealed that mmp- inhibition by ifn-¼ was mediated by the ifn-¼ receptor. ifn-beta-stimulated activation of stat was directly correlated with mmp- suppression. knockdown of stat gene by specific sirna or its inhibition with fludarabine partially restored the il- induction of mmp- expression and promoter activity. ifn-g did not alter activator protein (ap- ) binding ability but promoted physical interaction of stat and cbp/p co-activator. p overexpression reversed ifn-g inhibition of endogenous mmp- mrna expression and exogenous mmp- promoter activity. conclusions: ifn-g through its receptor activates stat , which binds with cbp/p co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of mmp- gene in chondrocytes. ifn-g and its signaling pathways could be targeted therapeutically for ( ), p asmawidjaja ( ), r hendriks( ), erik lubberts ( ) ( ) erasmus medical center, department of rheumatology, rotterdam, the netherlands ( ) erasmus medical center, department of immunology, rotterdam, the netherlands the objective of this study was to identify the role of il- in th polarization in the prone autoimmune dba- mice with and without collagen-induced arthritis and to evaluate th specific cytokine and transcription factor expression. il- induced th cells in vitro from spleen cells of naïve and collagen-type ii (cii) immunized dba- mice. the percentage of th cells is markedly higher in cii-immunized versus naïve dba- mice. adding il- to tgf-beta/il- stimulated cd + t cells did not significantly increase the percentage of th cells. tgfbeta/il- in contrast to il- induced a relatively high percentage of il- +/ifn-gamma-cells and low il- -ifn-gamma+ cells. tgf-beta/il- did not increase il- receptor expression, which may explain why adding il- directly or two days after tgf-beta/il- did not result in an increase in the percentage of th cells. elevated expression of il- a and il- f as well as the th specific transcription factor rorgammat was found under il- as well as tgfbeta/il- conditions. interestingly, il- but not tgf-beta/il- is critical in the th cytokine il- expression in t cells from ciiimmunized dba- mice. these data show that il- was more pronounced in inducing il- +/ifn-gamma-(th ) cells under cii-immunized conditions. furthermore, il- did not markedly increase the percentage of th cells induced by tgf-beta/il- . however, il- is critical for the induction of il- expression, suggesting a unique role for il- in the induction of specific th cytokines ebi was initially discovered as a transcriptionally activated gene in epstein-barr virus-infected human b lymphocytes, and similar to p of il- . ebi protein has been shown to form heterodimers with p . p /ebi termed il- , can influence the function of multiple t cell subsets, including naive, effector, regulatory and memory t cells. however, previous studies showed that the overlapped expression of ebi and p is very limited. these data lead to the hypothesis that ebi may play a role independently from its association with p . thus, to define the function of ebi , we generated ebi transgenic (tg) mice expressing in multiple tissues. ebi tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory cd +, cd + t cells, b cells, nk cells and nkt cells. cd +t cells isolated from spleens of ebi tg mice, however, produced less ifn-g than cells from wt (wild type) control mice after in vitro stimulation with anti-cd and anti-cd antibodies. in vivo studies, delayed-type hypersensitivity (dth) and contact hypersensitivity (chs) responses were significantly reduced in ebi tg compared with wt mice. moreover, the chs responses in ebi tg mice were recovered with anti-ebi polyclonal antibody. notably, chs reaction in wt mice was increased by anti-ebi antibody. in contrast, anti-p antibody suppressed chs responses in wt mice. these data suggest that ebi acts in different from il- , and reduces th responses. ( ), o thaunat( ), x houard ( ), o meilhac ( ), g caligiuri( ), a nicoletti ( ) ( ) inserm u and university denis diderot-paris , chu xavier bichat, paris, france ( ) inserm umr s , universitØ pierre et marie curie-paris , centre de recherche des cordeliers, paris, france arteries are composed of three concentric tissue layers which exhibit different structures and properties. because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). in contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. in the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. these mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. we propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. hence, an adventitial adaptive immune response predominates in chronic rejection. inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. these adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial cox-derived prostaglandins such as pge and prostacyclin acting on ep and ip receptors, respectively.activation of vascular ip receptors is especially important in limiting the atherogenic properties of thromboxane a acting on tp receptors.more recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.as well as the complex crosstalk between g-protein-coupled receptors (gpcrs), recent evidence indicates that many gpcrs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.we have found that ep -i, tp and ghrelin receptors readily form homodimers, but that co-transfection of hek cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.since inflammatory conditions are thought to change the relative expression levels of gpcrs in the vasculature, and since varying the expression levels of gpcrs will affect their ability to form heterodimers, then one might predict that gpcr heterodimerization would indeed influence the reactivity of vascular tissue during inflammation. [this work was fully supported by grants from the research grants council of the hong kong special administrative region (cuhk / m and vascular inflammation leads to formation of leukotrienes through the -lipoxygenase pathway of arachidonic acid metabolism. leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the blt and cyslt receptor subtypes. recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. leukotriene b (ltb ) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. initially identified on neutrophils, blt receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in ltb -induced atherogenesis. targeting ltb -induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. furthermore, blt receptor expression has been demonstrated on t-cells, suggesting ltb as a potential link between innate and adaptive immunological reactions. taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. in normal physiological conditions, the prostanoid (prostaglandin (pg) and thromboxane (tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (cox- ). this synthesis and release happen few minutes after cell or tissue stimulation. in vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (cox- ) and other prostanoid synthases can be observed. as an illustration of the previous experimental results, there is an increased presence of cox- and the inducible enzyme responsible for pge synthesis (mpges- ) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. in vascular cells in culture, pgi is the major biological active prostanoid produced in the normal physiological conditions. however, when cox- is induced, pgi and pge are equally produced. the role of cox- , cox- and mpges- activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. there is increasing evidence for the presence and a role of the ep receptor subtypes (ep , ep , ep or ep ) preferentially stimulated by pge in the vascular wall. for these reasons, we have characterized the receptors activated by pge in human mammary arteries. in these vessels incubated with a pro-inflammatory cytokine (interleukin- â) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. this effect is abolished by treatment of the vascular preparations with a selective cox- inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. rheumatoid arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for ra).we show that the different hla class ii alleles contribute to the development of anti-ccp-positive and anti-ccp negative ra.the se alleles do not independently contribute to the progression to ra, but rather contributed to the development of anti-ccp antibodies. next we determined the effect of smoking and observed that smoking only conferred risk to contract ra in the ccp-positive group and not in the anti-ccp negative group. for the risk factor ptpn (a gene that regulates treshold of lumphocyte activation) the allele c t only contributed to ccp-positive ra. in contrast to hla two other risk factors were found to be associated with both ccp-positive and ccp-negative ra. the risk factor in the fcrl-gene as has been identified in the japanese population was also tested in dutch ra cases and unrelated dutch controls. carrier analysis of the snp (rs ) revealed association of cc genotype with higher risk of developing ra as compared to tt & tc carriers (p = . and or = . ). in a meta-analysis of all studies comparing individuals, the or for the cc genotype to develop ra was . and the p-value < . . in conclusion, different steps in pathogenesis of the syndrome ra can be delineated this talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (crohns disease and ulcerative colitis). proven genes containing genetic variants predisposing to crohns disease include ibd / q , card /nod and il r. data is suggestive but not yet as convincing for many other genes. a common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. only for card /nod is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. some mouse models (gene targeted) of card appear to show opposite effects to other models and human systems. in humans, card mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. it is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. pathogen-recognition receptors (prrs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. since their discovery in vertebrates, toll-like receptors (tlrs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. recently a new family of intracellular prrs, the nod-like receptors (nlrs), which include both nods and nalps have been described. mutations within the nalp /cryopyrin/ cias gene are responsible for three autoinflammatory disorders: muckle-wells syndrome, familial cold autoinflammatory syndrome, and cinca/nomid. the nalp protein associates with asc and caspase- (thereby forming a molecular machine termed inflammasome that displays high proil- beta-processing activity. macrophages from muckle-wells patients spontaneously secrete active il- beta. increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with nalp -dependent autoinflammatory disorders. here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. allergic inflammation (ai) is a complex phenomenon initiated by allergen binding to ige sensitized mast cells and consequent mast cell activation. this causes the symptoms of the early phase of ai and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the ai becomes a chronic process. we defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. in the synapse these two old cellular players of ai have a cross talk via soluble mediators and receptor-ligand interactions. this results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. in addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. we propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. ( ) ( ) erasmus mc, rotterdam, the netherlands ( ) department of immunology weizmann institute of science, rehovot, israel allergic asthma is one of the most common chronic diseases in western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with th cells, eosinophils, and mast cells. if we are to devise new therapies for this disease, it is important to elucidate how th cells are activated and respond to intrinsically harmless allergens. dendritic cells (dcs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. we have shown that intratracheal injection of ovalbumin (ova) pulsed dcs induces sensitization leading to eosinophilic airway inflammation upon ova aerosol challenge. we investigated the role of dcs in the secondary immune response in a murine asthma model. ova aerosol challenge in ova-dc sensitized mice, induced an almost fold increase in the number of airway dcs as well as an increase in eosinophils and t cells. to investigate the functional importance of dcs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous dcs in sensitised cd c-diphtheria toxin (dt) receptor (cd cdtr) transgenic mice by airway administration of dt h before ova aerosol ( x) challenge or during an ongoing inflammation (depletion after x ova aerosols continued with additional ova aerosols). numbers of balf eosinophils, th cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway dcs during secondary challenge. karolinska institute, stockholm, sweden nk cells are innate lymphocytes with potent immunoregulatory functions. they are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. for example, they are involved in, and affect, acute as well as chronic inflammatory responses. in the present talk, a general overview on our current knowledge of nk cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity ige receptors (fc<epsilon>ri) with allergens. although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate th -type cytokines (e.g. il- and il- ), subsequently supporting ige synthesis and underlying atopy. importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. s. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing th -type adaptive immune responses. while we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. recent advances, however, have shed light upon the major intracellular signal transduction processes involved in fc<epsilon>ri activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. an important discovery in this regard is the phosphatase ship, which downregulates pi -kinase signalling in both basophils and mast cells. recent data shows that ship expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with ship recruitment (cd r, cd r). identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. mitogenesis and proliferation of vsmc play an important role in atherogenesis. pro-inflammatory secretory phospholipases a (spla ) hydrolyse glycerophospholipids of hdl and ldl and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.spla s lipolysis products localize in vascular wall in vicinity of vsmc.we have tested the impact of spla , hdl and ldl and of their hydrolysis products on mitogenesis and pge and ltb release from vsmc.mitogenesis was significantly enhanced by native hdl, and ldl, and by group v spla . spla hydrolysis of hdl and ldl enhanced mitogenic activity in order v>x>iia.the release of pge from vsmc was enhanced by group x spla s but not iia or v.the greatest effect was seen for hdl hydrolysed by group v and x spla .native ldl and its spla hydrolysis products enhanced the release of pge in order x>v>iia.the release of ltb from vsmc was markedly increased by native ldl and hdl, and hydrolysis products of group v and x, but not iia spla .migration of vsmc was significantly enhanced by spla iia and inhibited by hdl.this study demonstrates a complex interaction of hdl and ldl with pro-inflammatory spla s, which affects mitogenesis, eicosanoid release and migration of vsmc.study of biocompatible spla blockers in the therapy of atherosclerosis is indicated. contact information: professor waldemar pruzanski, university of toronto, department of medicine, toronto, ontario, canada e-mail: drwpruzanski@bellnet.ca ( ) ( ) ipmc-cnrs umr , valbonne, france ( ) university of washington, seattle, usa ( ) inserm umrs , paris, france ( ) university of naples, italy the superfamily of phospholipase a comprises at least intracellular enzymes and up to secreted pla s (spla s). elucidating the biological roles of each pla member is currently the most challenging issue in the pla field. the different spla s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). increasing evidence suggests that spla s iia, iii, v, and x are involved in inflammatory diseases including atherosclerosis. among spla s, the human group x (hgx) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (ldl). on human alveolar macrophages, hgx spla can trigger secretion of tnf alpha, il and il in a non-enzymatic manner. on colorectal cancer cells, hgx spla stimulates cell proliferation, produces potent eicosanoids including pge , and activates the transcription of key genes involved in inflammation and cancer. the enzyme can also hydrolyze pc and platelet-activating factor (paf) of ldl particles very efficiently. finally, hgx spla is present in human atherosclerotic lesions and converts ldl into a proinflammatory particle that induces macrophage foam cell formation, as well as map kinase activation, arachidonic acid release, and expression of adhesion molecules in huvec cells. some other key molecular features of spla s including hgx will be presented. we have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (ptdcho) by spla s, but the mechanism of this selectivity is not known. since both sphingomyelin (sm) and lysoptdcho inhibit the activity while increasing fatty acid specificity of other pla s, we have examined fatty acid release by spla siia, v and x in relation to relative increases in proportion of endogenous sm and lysoptdcho during lipoprotein digestion. the analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (lc/esi-ms) and lc/collision induced dissociation (cid)/esi-ms using conventional preparations ofldl and hdl. the highest preference for arachidonate release from ldl by group x spla was observed when the residual sm/ pdcho molar ratio had reached . compared to a starting ratio of . .group v spla showed preferential release of linoleate at residual sm/ptdcho molar ratio . , while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. the relative increases in lysoptdcho and sm during the digestion with spla iia were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. these results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of ptdcho. the residual sm/ptdcho ratios reached during group v and x spla digestion are similar to those observed for lesional ldl, which promote release of ceramides by smase leading to ldl aggregation. the above findings support a potential role of sphingomyelins in atherogenesis. although sphingomyelin (sm) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (pc),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (pla). we showed that sm inhibits several lipolytic enzymes including secretory pla iia, v, and x, and hepatic and endothelial lipases, all of which hydrolyze pc. treatment of sm in the substrate with smase c not only relieved the inhibition but also activated the pla reaction further, suggesting that ceramide, the product of smase c, independently stimulates pla , possibly by disrupting the bilayer structure. smase d treatment, which produces ceramide phosphate, did not stimulate the spla . the fatty acid specificity of pla is significantly affected by sm. thus spla x exhibited enhanced specificity for the release of arachidonic acid ( : ) in presence of sm, due to a preferential inhibition of hydrolysis of other pc species. in contrast, sm inhibited the release of : by spla v. ceramide selectively stimulated the release of : by both enzymes. only the long chain ceramides (> carbons) were effective, while ceramide phosphate did not stimulate spla activity. sm-deficient cells released more : in response to spla -treatment than normal cells, and pretreatment of normal cells with smase c increased their susceptibility to spla attack. these studies show that sm and ceramide regulate the activity and specificity of pla, and consequently the inflammatory response. secretory phospholipase a (spla ) types iia, v, or x, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.given the link between spla and atherogenesis, a mouse model of atherosclerosis (apoe-/-) was used to study the effects of a- , an inhibitor of spla enzymes, on atherosclerosis and cholesterol levels over weeks of treatment. mice were fed with a high-fat, high cholesterol diet alone during the study ( % fat; . % cholesterol, . % casein) and were treated with vehicle or a- bid at mg/kg or mg/kg by oral gavage. total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.treatment with a- significantly reduced aortic atherosclerotic plaque formation in apoe-/-mice fed a high fat diet when compared to the untreated control by approximately %. in a different model that used angiotensin ii in conjunction with a high fat diet in a background of apoe-/-deficient mice for weeks, oral dosing of a- ( mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. these data suggest that a- is a potential novel therapeutic agent for the treatment of atherosclerosis. ( ), s doty( ), c antonescu ( ), c staniloae ( ) ( ) saint vincents hospital manhattan, new york, usa ( ) hospital for special surgery, new york, usa ( ) sloan-kettering institute for cancer research, new york, usa tnf-stimulated gene (tsg- ) is induced by tnf-a during inflammation and its secreted product tsg- glycoprotein is involved in immune-mediated inflammatory diseases and fertility. it regulates cox- and prostaglandin synthesis, and participates in extracellular matrix remodeling. considering the chronic inflammatory nature of atherosclerosis we hypothesized that tsg- is expressed in atherosclerotic plaques and investigated tsg- protein expression and cellular distribution on superficial femoral artery endarterectomy specimens from diabetic and non-diabetic patients with peripheral vascular disease. six histologically normal radial artery specimens were analyzed as control. paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-tsg- antibody. tsg- expression was consistently present in all atherectomy specimens but not in control specimens. a distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. patchy tsg- expression was noted in the extracellular matrix. within the inflammatory plaques from diabetic patients, tsg- stained the foamy macrophages. tsg- expression was also confirmed and quantified by qrt-pcr that showed a significant up-regulation of tsg- gene (more that fold induction compared to housekeeping genes). our study identifies for the first time the preferential expression of tsg- in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. tsg- is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. ( ), r krohn ( ), h lue ( ), jl gregory( ), a zernecke ( ), rr koenen ( ), t kooistra ( ), p ghezzi( ), r kleemann ( ), r bucala( ), mj hickey ( ), c weber ( ) ( ) university hospital of the rwth aachen, germany ( ) centre for inflammatory diseases, monash university, melbourne, australia mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (clf)-chemokines. the pleiotropic cytokine macrophage migration inhibitory factor (mif) plays a critical role in inflammatory diseases and atherogenesis. the underlying molecular mechanisms are poorly understood, but, interestingly, mif displays structural features resembling chemokines. we have identified the chemokine receptor cxcr as a functional receptor for mif. mif triggered galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through cxcr , inducing rapid integrin activation. mif directly bound to cxcr with high affinity (kd of . nm). monocyte arrest mediated by mif in inflamed or atherosclerotic arteries involved cxcr as well as cd , a recently identified membrane receptor moiety for mif. accordingly, cxcr and cd were found to occur in a receptor complex. in vivo, mif deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. thus, mif displays chemokine-like functions by acting as a non-cognate ligand of cxcr , serving as a regulator of inflammatory and atherogenic recruitment. these data harbor an intriguing novel therapeutic prospect by targeting mif in atherosclerosis and add a new dimension to mif and chemokine receptor biology. ( ), r toes ( ), h van bockel( ), paul quax ( , ) ( ) tno bioscienses, leiden, the netherlands ( ) department of vascular surgery, leiden university medical center, the netherlands ( ) department of rheumatoly, leiden university medical center, the netherlands the immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. arteriogenesis was impaired in c bl/ mice depleted for natural killer (nk)-cells by anti-nk . antibodies and in nk-cell-deficient transgenic mice. arteriogenesis was, however, unaffected in jµ knockout mice that lack nk . + natural killer t (nkt)cells, indicating that nk-cells, rather than nkt-cells are involved in arteriogenesis. furthermore, arteriogenesis was impaired in c bl/ mice depleted for cd + tlymphocytes by anti-cd antibodies, and in major histocompatibility complex (mhc)-class-ii-deficient mice that lack mature peripheral cd + t-lymphocytes. this impairment was even more profound in anti-nk . treated mhc-class-ii-deficient mice that lack both nkand cd + t-lymphocytes. finally, collateral growth was severely reduced in balb/c as compared with c bl/ mice, two strains with different bias in immune responsiveness. correspondingly, fewer cd -positive lymphocytes accumulated around collaterals in balb/c mice. these data show that both nk-cells and cd + t-cells play an important role in arteriogenesis. moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (ih) and accelerated atherosclerosis, often causing graft failure. inflammation is an important trigger for these processe. complement is an important part of the immune system and participates in regulating inflammation. although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. the involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic apoe leiden mice. in these veins ih and accelerated atherosclerotic lesions develop within days, consisting mainly of foamcells and smc. to study the functional role of complement in vein graft remodeling, cobra venom factor (cvf: u daily) was used to deplete complement starting one day prior to vein graft surgery. cvf-treatment reduced vein graft thickening by % (p= . ), when compared to saline treated controls (n= ).to confirm that the reduction by cvf was due to hampered complement function and not a direct effect of cvf, complement activation was blocked using crry-ig (inhibiting c convertases). crry-ig ( mg every other day) led to % decrease in vein graft thickening (p= . ) compared to controls receiving non-relevant control igg. these data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. ( ), m-c koutsing tine( ), p borgeat( ), h ong ( ), sylvie marleau ( ) ( ) universite de montreal, quebec, canada ( ) centre de recherche en rhumatologie et immunologie, canada we have previously shown that ep , a growth hormone-releasing peptide (ghrp) analogue binding selectively to the scavenger receptor cd , elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoe-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. we investigated the effect of ghrp analogues on i/r-elicited remote lung injury in week-old apoe-/-mice fed a high fat high cholesterol (hfhc) diet from weeks of age. at weeks old, mice were treated daily with a s.c. injection of ep ( mg/kg) for days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for minutes, followed by minutes reperfusion. our results show that ep significantly reduced leukocyte accumulation by % in the lungs, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice (n = - per group), as assessed by myeloperoxidase assay. this was associated with a % reduction of opsonized zymosan-elicited blood chemiluminescence. in contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoe-/-/cd -/-deficient mice, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice. we conclude that ep protects i/r-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a cd -mediated pathway. glycogen synthase kinase beta (gsk- beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. dysregulation of gsk- beta has been implicated in the pathogenesis of several diseases including sepsis. here we investigate the effects of two chemically distinct inhibitors of gsk- beta, tdzd- and sb , on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. male wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to mmhg for min) and subsequently resuscitated with shed blood for h. hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. treatment of rats with either tdzd- ( mg/kg, i.v.) or sb ( . mg/kg, i.v.) min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. the protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. in addition, tdzd- , but not sb , attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin . neither of the gsk- beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. thus, we propose that inhibition of gsk- beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. ( ), y ito ( ), h yoshimura ( ), h inoue ( ), n kurouzu ( ), h hara ( ), y mastui ( ), h kitasato ( ), s narumiya( ), c yokoyama ( ), m majima ( ) ( ) kitasato university school of medicine, japan ( ) kyoto university school of medicine, japan ( ) tokyo medical and dental university, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitors constitute the major determinant of revascularization. we hypothesized txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggregation. to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb ischemia. recovery from acute hind limb ischemia, as assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out mice (ipko) and thromboxane (tx) a receptor (tp) knock out mice (tpko) by using lase doppler. blood recovery in tpko significantly delayed compared to wt and ipko. immunohistochemical studies revealed that the number of cd positive cells in the ischemic quadriceps were less stained in tpko compared to wt and ipko.plasma sdf- and vegf concentration were significantly reduced in tpko mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. administration of selective tp agonist may open new therapeutic strategy in regenerative cardiovascular medicine. during renal ischemia/reperfusion (i/r) injury, apoptosis has been reported as a very important contributor to the final kidney damage. the determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. the aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. based in a rat kidney i/r model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the i/r derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. thus, the control in the peroxynitrite generation during the i/r could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal i/r injury. metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. using nmr spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. serum from patients with synovitis of "t months duration whose outcome was determined at clinical follow-up was used. vitreous samples were from patients undergoing vitrectomy for vitreoretinal disorders. one dimensional h nmr spectra were acquired. principal components analysis (pca) of the processed data was conducted along with a supervised classification. with the arthritis serum there was a clear relationship between each samples score in the pca analysis and the level of crp. supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. a similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. in this case differences were not due to a straightforward relationship with inflammatory markers (il- , ccl ), which did not correlate with pca in these samples. similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. these results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. h-nmr-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. ( ), am artoli( ), a sequeira( ), c saldanha ( ) ( ) instituto de medicina molecular,faculdade de medicina de lisboa, portugal ( ) cemat, instituto superior tØcnico, universidade de tØcnica de lisboa, portugal the recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. the precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. in this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. leukocyte-endothelial cell interactions in post-capillary venules of wistar rats cremaster muscle were monitorized by intravital microscopy. from these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice boltzmann solver for shear thinning fluids. the dynamics of leukocyte clusters under non-newtonian blood flow with shear thinning viscosity was computed and discussed. in this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. we have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. we verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. it was found that the lattice boltzmann solver used here is fully adaptive to the measured experimental parameters. this study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. one family of transcripts that are highly expressed in activated macrophages are members of the schlafen (slfn) gene family; a recently identified family whose function is still unknown. this study examined the mrna expression of slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (cia) in vivo. real-time pcr expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (lps) over a time course, revealed differential expression of individual slfn family genes. in particular, slfn- , slfn- , and slfn- were maximally induced after hours. the maximal induction of slfn- and slfn- was observed after hours of lps treatment. individual members of the slfn family were also differentially expressed in cia, a model of rheumatoid arthritis. mrna levels of slfn- , slfn- , slfn- and slfn- were elevated in joints affected by cia. to investigate the role of slfn- , we have generated a transgenic mouse line, which over expresses slfn- specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and gal . further characterisation of the slfn- over expressing mouse line will be used to assess the function of slfn- in macrophage biology and inflammation, and its potential as a therapeutic target. macrophages play an important role in resolving inflammation. it is known that the resolution of inflammation requires alternative activation of macrophages. but the precise events of phenotype switching in macrophages remain poorly understood. we show that lipocalin , lcn- , is able to provoke a switch in macrophage activation. in an in vitro co-culture model for renal epithelial cells and macrophages, we detected by sirna technique that the presence or absence of lcn- determines proliferation processes in damaged renal epithelial cells. the proliferative response was dependent on proinflammatory or anti-inflammatory environment. as lcn- is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. we anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by lcn- . ( ), y cao ( ), s adhikari ( ), m wallig ( ) ( ) national university of singapore, department of pharmacology, singapore ( ) university of illinois at urbana champaign, usa it has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. the current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. acute pancreatitis was induced in the mouse by administering hourly injections of caerulein ( mg/kg) for , and hours respectively. neutralizing monoclonal anti-il- antibody ( . mg/kg) was administered either with or without crambene ( mg/kg) hours before the first caerulein injection. rt-pcr, western blotting and immunostaining were performed to detect cd expression. apoptosis in pancreatic sections was visualized by tunel. severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (mpo), water content, and morphological examination. pancreatic levels of inflammatory mediators were examined by elisa. the protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as mcp- , tnf-a and il- â and up-regulating anti-inflammatory mediators like il- . phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor cd .the anti-inflammatory mediator il- plays an important role in crambene-induced protective action against acute pancreatitis. the release of anti-inflammatory mediator il- is downstream of phagocytosis. these results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. ( , ) ( ) northern ontario school of medicine, thunder bay, ontario, canada ( ) lakehead university, canada integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including pseudomonas aeruginosa. we have recently established that beta integrins in lung epithelial cells (lec) provide co-stimulatory signals regulating inflammatory responses (ulanova et al, am j physiol, , : l -l ). we hypothesized that lec integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to p. aeruginosa. to determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of p. aeruginosa infection of a cells. to investigate interactions of bacteria with lec, p. aeruginosa strain pak was chromosomally labeled with a green fluorescent protein gene using a mini-tn delivery system.using several fluorescence-based detection systems, we established that the natural beta integrin ligand, fibronectin, mediates bacterial adhesion to lec.p. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta integrin expression followed by the increased cell surface protein expression. the surface expression of integrin beta increased shortly following bacterial exposure without alterations of mrna expression, suggesting rapid protein redistribution within the cells. the data indicate that p. aeruginosa are capable to modulate integrin gene/protein expression in lec, potentially using fibronectin to alleviate bacterial binding to beta integrins. upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. integrin receptors in lec may represent significant therapeutic targets in pulmonary infection caused by p. aeruginosa. the purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. through activation of the a a receptor (a ar) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene b formation while potentiating that of prostaglandin e through the up-regulation of the cyclooxygenase (cox)- pathway. moreover, our laboratory determined that a ar engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including tnf-and mips. in mice lacking the a ar, migrated neutrophils expressed less cox- than their wild type counterpart while displaying higher mrna levels of tnf-and mip- . mononuclear cells from a ar knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following a ar engagement, implicate pivotal metabolic pathways such as intracellular cyclic amp, p and pi- k. together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. the newest developments regarding adenosines effects on neutrophil functions will be presented.this work is supported by the canadian institutes of health research (cihr). human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (amps). to identify human skin amps, we analysed extracts of healthy persons stratum corneum by reversed phase-hplc and purified a novel kda amp that showed sequence similarity to mouse hornerin. suggesting that it originates from the human ortholog, we cloned it. human hornerin encodes a amino acid protein that contains a s domain, an ef-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused s protein family. strongest constitutive hornerin mrna expression was seen in differentiated keratinocyte cultures. to follow the hypothesis, that hornerin fragments represent amps, we recombinantly expressed three hornerin peptides, rhrnr (tandem repeat unit b), rhrnr (tandem repeat unit a) and rhrnr (c-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. the rhrnr peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against escherichia coli, pseudomonas aeruginosa and candida albicans. the other peptides were found to be not or nearly not antimicrobially active. our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed amps, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. ( ), n lu( ), r jonsson( ), d gullberg ( ) ( ) department of biomedicine, university of bergen, norway ( ) the gade institute, university of bergen, norway a ß is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. in this study we investigated the factors that regulate mouse integrin a ß expression. specifically, we have analyzed the influence of cell passage, growth factors and the -d microenvironment. using sv immortalized as well as primary fibroblasts, we show that a ß integrin is up-regulated when these cells are cultured within stressed collagen type i lattices. however, a ß is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. we also show here that a is upregulated by tgf-a on planar substrates. these findings suggest that mechanical tension and tgf-a are important factors in the regulation of a ß that need to be to taken into consideration when evaluating the role of a ß in wound healing and fibrotic disorders. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada the objective of this study was to investigate the effects of par deficiency in various models of colonic inflammation in order to elucidate the role of endogenous par in the process of inflammation in the gut.colonic inflammation in c bl wildtype and par -/-mice was induced by treatment with . % dss (in drinking water) or tnbs ( mg or mg in ul of % ethanol, single intracolonic injection) or pre-sensitizing mice with % oxazolone (in olive oil) applied to the skin of the abdomen, and days later, a single intracolonic injection of % oxazolone (dissolved in % ethanol).intravital microscopy was performed, days (tnbs/dss) or days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.lastly, various parameters of inflammation were assessed following the intravital microscopy.par -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in dss, and tnbs challenge. in all three challenges, mpo activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to par -/-.our evidences indicate that deficiency in par attenuates inflammatory responses in the experimental models of colitis associated with either th (tnbs/dss) or th (oxazolone) cytokine profile.therefore, par deficiency in the gut exerts antiinflammatory properties that are independent of th or th cytokine profile.the present study further highlights par as a potential target for inflammatory bowel diseases. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada in a previous study, inflammatory responses induced by three different models of colitis (tnbs/dss/oxazolone) were significantly attenuated in mice deficient for par (par -/-). among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by par deficiency. aim of this study was to assess the effects of par deficiency (via par -/-mice) on the recruitment of leukocyte in colonic venules. in anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fmlp ( mm) or paf ( nm) or by intraperitoneal injection of tnf-a; ( . mg -given hours before the intravital microscopy). using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of par -/-versus par +/+ mice. fmlp and paf as well as tnf-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. par -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fmlp topical administration. leukocyte adherence induced by fmlp and tnf-a; was significantly lower in par -/-mice compared to wild types as well. no difference was observed between par -/-and wildtype for leukocyte rolling/adherence-induced by paf. the lack of functional par attenuated leukocyte trafficking in response to fmlp and tnf-a; but not to paf. the involvement of par activation in mouse colon leukocyte trafficking highlights par as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. ( ), kk hansen( ), k chapman( ), n vergnolle ( ), ep diamandis ( ), md hollenberg ( ) ( ) advanced center for detection of cancer, mount sinai hospital, university of toronto, toronto, on, canada ( ) proteinases and inflammation network, university of calgary, calgary, ab, canada kallikreins (klks) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. we have recently shown that klks can activate proteinaseactivated receptors (pars), a family of g-protein coupled receptors associated with inflammation. we hypothesized that like trypsin, kallikreins can trigger inflammation via the pars. we studied the ability of klks and to activate pars , and in vitro and to cause oedema in a mouse model of paw inflammation in vivo. we found that klk is able to activate both of pars and and to prevent thrombin from activating par . on the other hand, klk was a specific activator of par . kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. the oedema was accompanied by a decreased threshold of mechanical and thermal nociception. our data demonstrate that by activating pars and and by inactivating par , kallikreins, like klks and , may play a role in regulating the inflammatory response and perception of pain. ( ), d park ( ), b short( ), n brouard( ), p simmons( ), s graves ( ), j hamilton ( ) ( ) melbourne university, melbourne, victoria, ( ) peter maccallum cancer institute. melbourne, australia mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cyto-kines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. using are-driven and nf-kb-targeted reporter genes, transfection of the nf-kb p subunit and nrf into hepg or other cells, as well as sirna technique to knockdown endogenous p in cells, we found that nf-kb p subunit repressed the anti-inflammatory and anticarcinogenetic nrf -are pathway at transcriptional level. in p -overexpressed cells, the are-dependent expression of heme oxygenase- was strongly repressed. in the cells where nf-kb and nrf were simultaneously activated, p unidirectionally antagonized nrf transcriptional activity. the p -mediated are inhibition was independent of the transcriptional and dna-binding activities of p . co-transfection and rna interference experiments revealed two mechanisms which coordinate the p -mediated repression of are: ( ) p selectively deprives creb binding protein (cbp) from nrf , but not mafk, by competitive interaction with the ch -kix domain of cbp, resulting in inactivation of nrf transactivation domain and concomitant abrogation of the nrf -stimulated coactivator activity of cbp; ( ) p promotes recruitment of histone deacetylases (hdac ) to are by enhancing the interaction of hdac with either cbp or mafk, leading to inactivation of cbp and deacetylation of mafk. this study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the are-dependent gene expression by p subunit. since various inflammatory and tumor tissues constitutively overexpresses p in their nuclei, the finding in this study implies a strong repression of are-dependent gene expression must take place in those tissues. in this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. dendritic cells (dc) play a pivotal role in the induction of immune response and tolerance. it is less known that dc accumulate in atherosclerotic arteries, where they might activate t-cells and contribute to the progression of disease. the serine protease thrombin is the main effector protease of the coagulation cascade. thrombin is also generated at sites of vascular injury and during inflammation. hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. thrombin activates various cells via protease-activated receptors (pars). immature dc do not express pars. upon maturation with lps, tnfalpha, or cd l, only lps-matured dc expressed par and par on their surface. stimulation of dc with thrombin, par -or par -activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in lps-, but not in tnf-alphamatured dc. the thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. stimulation of pars with thrombin or respective receptoractivating peptides led to activation of erk / and rho kinase i (rock-i) as well as subsequent phosphorylation of the regulatory myosin light chain (mlc ). the erk / -and rock-i-mediated phosphorylation of mlc was indispensable for the par-mediated chemotaxis as shown by use of pharmacological inhibitors of rock, erk and mlc kinases. in addition, thrombin significantly increased the ability of mature dc to activate proliferation of naive t-lymphocytes in mixed leukocyte reactions. in conclusion, our work demonstrates expression of functionally active thrombin receptors on lps-matured dc. we identified thrombin as a potent chemoattractant for mature dc, acting via rho/ erk-signaling pathways. data concerning the role of circulating modified low density lipoproteins (modldl) in atherogenesis and other pathologies are scarce. one reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modldl in vivo. we report a novel approach for specific labeling of human native ldl (nldl) and modldl (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated ldl) with the positron emitter fluorine- ( f) by either nh -reactive n-succinimidyl- -[ f]fluorobenzoate or sh-reactive n-[ -( -[ f]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, - %; specific radioactivity, - gbq/ mmol). radiolabeling itself caused neither additional oxidative structural modifications of ldl lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. the approach was evaluated with respect to binding and uptake of f-nldl and f-modldl in cells overexpressing various lipoproteinrecognizing receptors. the metabolic fate of f-nldl and f-modldl in vivo was delineated by dynamic small animal pet studies in rats and mice. the in vivo distribution and kinetics of nldl and modldl correlated well with the anatomical localization and functional expression of ldl receptors, scavenger receptors, and receptors for advanced glycation end products. the study shows that ldl modification, depending on type and extent of modification, in part or fully blocks binding to the ldl receptor, and reroutes the modldl to tissuespecific disease associated pathways. in this line, flabeling of modldl and the use of small animal pet provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. the p mapk signaling pathway, which regulates the activity of different transcriptions factors including nuclear factor-Þb (nf-Þb), is activated in lesional psoriatic skin. the purpose of the present study was to investigate the effect of fumaric acid esters on the p mapk and the down stream kinases msk and in cultured human keratinocytes. cell cultures were incubated with dimethylfumarate (dmf), methylhydrogenfumarate (mhf) or fumaric acid (fa) and then stimulated with il- b before kinase activation was determined by western blotting. a significant inhibition of both msk and activations was seen after pre-incubation with dmf and stimulation with il- b whereas mhf and fa had no effect. also, dmf decreased phosphorylation of nf-kb / p (ser ), which is known to be transactivated by msk . furthermore, incubation with dmf before stimulation with il- b resulted in a significant decrease in nf-kb binding to the il- kb and the il- kb binding sites as well as a subsequent decrease in il- and il- mrna expression. our results suggest that dmf specifically inhibits msk and activations and subsequently inhibits nf-kb induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. these effects of dmf explain the anti-psoriatic effect of fumaric acid esters. a humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking b, t and nk cells. in this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. inflammation is associated with the expression of activation markers and inflammatory medi-ators such as tnf-alpha, hla-dr and cd a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of ki- and ck- . epidermal hyperplasia is a typical readout in this model. in a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of tnf-alpha, antibodies directed against growth factors, mmp-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine a.in addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (caspary et al. brit j dermatol ; , - ) . employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. kristian otkjaer ( ), e hasselager( ), j clausen( ), l iversen ( ), k kragballe ( ) ( ) aarhus university hospital, denmark ( ) novo nordisk a/s, denmark interleukin- (il- ) is assumed to be a key cytokine in the pathogenesis of psoriasis. increased levels of il- are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. whether il- is derived from antigen-presenting cells or keratinocytes remains unsolved. the aim of the present study was, therefore, to characterize il- expression in non-lesional psoriatic skin ex vivo. mm punch biopsies from nonlesional psoriatic skin were collected. biopsies were transferred to cacl enriched keratinocyte basal media and cultured with vehicle or il- beta ( ng/ml) for , , , , , and hours, respectively. the samples were analyzed by in situ hybridisation, qrt-pcr, immunofluorescent staining and elisa. incubation with il- beta rapidly induced il- mrna expression in the biopsies. the highest level of il- mrna was detected after hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of il- mrna. increased levels of il- protein were detected in the supernatant of the il- beta stimulated biopsies. immunofluorescent staining of the biopsies showed no il- protein in the keratinocytes, whereas the il- protein was present in epidermal cd a positive dendritic cells. our data emphasize the keratinocyte as the cellular source of il- expression in human skin. interestingly, immunofluorescent staining of our cultured biopsies showed il- protein in epidermal dendritic cells whereas no il- was detected in the keratinocytes. this indicates that epidermal dendritic cells are the target for keratinocytederived il- . one response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (mmps) that participate in tissue remodeling. excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. calcitriol, the hormonally active form of vitamin d, is known to have beneficial effects during cutaneous inflammation. we hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive mmp proteolytic activity. our experimental model consists of hacat keratinocytes cultured with tnf to simulate an inflammatory state. pro-mmp- was quantified by gelatin zymography and mrna by real-time pcr. the levels and activation of signaling proteins were determined by immunoblotting. the increase in pro-mmp- activity and mrna levels induced by tnf was inhibited by~ % following h treatment with calcitriol. using specific inhibitors we established that the induction of mmp- was dependent upon the erk pathway, while p -mapk and pkc inhibited, and jun-kinase, pi- -kinase and src did not affect it. levels of c-fos, a component of ap- transcription complex known to mediate mmp- induction, were elevated by tnf and further increased by calcitriol. the induction of mmp- by tnf was abolished by inhibition of the egfr tyrosine kinase attesting to the requirement for egfr trans-activation. calcitriol also inhibited the induction of mmp- by egf. we conclude that calcitriol inhibits the induction of mmp- gene expression by tnf in keratinocytes by affecting an event downstream to the convergence of the egfr and the tnf signaling pathways. ( ), p verzaal ( ), t lagerweij ( ), c persoon-deen ( ), l havekes ( ), a oranje ( ) ( ) tno pharma, department of inflammatory and degenerative diseases, leiden, the netherlands ( ) erasmus medical center, department dermatology and venereology, rotterdam, the netherlands mice with transgenic overexpression of human apolipoprotein c in liver and skin display a strongly disturbed lipid metabolism. moreover, these mice show a loss of skin-barrier function evident from increased trans epidermal water loss. these mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. both hyperplasia of epidermis and dermis are observed. histological analysis shows increased numbers of cd + t cells, eosinophils, mast cells and ige-positive cells in the dermis. serum levels of ige are increased as well. cytokine profiling of draining lymph nodes is in favor of a th -mediated disease. development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.this model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. topical immunosupppressants such as elidel and protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-when delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. we speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.accordingly, we have designed and discovered a series of "soft" cyclosporin a (csa) derivatives as potentially safer alternatives.in general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.in this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.the results or our drug discovery efforts around soft csa derivatives will be presented. ( ), y sawanobori ( ), u bang-olsen ( ), c vestergaard( ), c grønhøj-larsen ( ) background: a strain of japanese fancy-mice, nc/nga, serves as a model for atopic dermatitis. under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of il- mrna has been shown. also, transgenic mice over-expressing il- exhibit increased scratching behaviour and develop severe dermatitis. consequently we decided to explore the therapeutic effect of an anti il- antibody on scratching behaviour and dermatitis in nc/nga mice. methods: prior to clinical manifestation of dermatitis, we commenced treatment of nc/nga mice with il- ratanti-mouse mg/kg intraperitoneally every fifth day for seven weeks. clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. serum analysis for ige and il- as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point. results: taken over the entire intervention period, treatment with anti il- antibody in nc/nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. however, post hoc analysis revealed a significant reduc-tion of scratch by the anti il- antibody treatment in the time interval day - . our results suggest an anti pruritic role for il- antibody in an atopic dermatitis-like animal model. anti il- antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. ( ), p ferro ( ), hm asnagli ( ), v ardissone ( ), t ruckle ( ), f altruda ( ), ch ladel ( ) ( ) rbm merck serono/university of torino, italy ( ) merck serono pharmaceutical research institute, geneva, switzerland ( ) university of torino, dipartimento di genetica, biologia, biochimica, italy class-i phosphoinositide -kinases (pi ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. since pi k-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of pi k-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. in our study the inhibitory properties of a selective pi k-g inhibitor as on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. two mouse models were used: the first, irritant contact dermatitis (icd), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. the second, contact hypersensitivity (chs) is a t-cell dependent model, modeling in part t-cell-mediated skin diseases such as psoriasis. we demonstrated the therapeutic effect of pi kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective pi k-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (icd) -effective dose , mg/kg p.o. once -as well as in t-cell mediated skin pathology (chs)effective dose mg/kg p.o. bid. we conclude that the mechanism of action related to inhibition of pi k-g are demonstrable after oral administration of selective inhibitors like as in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. introduction: high mobility group box (hmgb ) has recently been identified as a late mediator of endotoxin lethality. we newly developed an extra corporeal hmgb absorber. the purpose of this study was to test the hypothesis that hmgb removal could prevent or reduce endotoxin induced lethality or tissue injury of rats. methods: all experiments were conducted in accordance with the institutional care and use committee.male wistar rats were randomly allocated into three groups; hmgb absorber group (group i),hmgb nonabsorber group (group ii), and vacant column group (group iii).we applied these columns for each groups at hours after lps injection. the rats were sacrificed hours after lps injection for pulmonary histology. we statistically analyzed survival rate with kaplan-meier and the levels of hmgb with anova. results: survival rate was % in the group i at hours after lps injection, as compared with % in the group ii and % in the group iii. the pulmonary histology in both group ii and group iii showed acute inflammatory injuries, whereas group i showed less inflammatory changes.the level of hmgb in the group i was significantly lower than those of group ii and iii. discussions:these results demonstratethat specific absorption of endogenous hmgb therapeutically reverses lethality of established sepsis indicating that hmgb inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines. contact information: dr hideo iwasaka, oita university, anesthesiology and intensive care unit, yufu city, japan e-mail: hiwasaka@med.oita-u.ac.jp ( ) ( ) department of pharmacology, national university of singapore, singapore ( ) dso national laboratories, singapore hydrogen sulfide (h s) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. in this study, we have investigated the role of h s in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. male swiss mice were subjected to cecal ligation and puncture (clp) induced sepsis and treated with saline, dl-propargylglycine (pag, mg/kg i.p.), an inhibitor of h s formation or sodium hydrogen sulfide (nahs) ( mg/kg, i.p.), a h s donor. pag was administered either hour before or hour after induction of sepsis while nahs was given at the time of clp. using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of pag significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mrna and protein levels of adhesion molecules (icam- , p-selectin and e-selectin) in lung and liver. in contrast, injection of nahs significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. on the other hand, normal mice were given nahs ( mg/kg i.p.) to induce lung inflammation with or without pretreatment of nf-x×b inhibitor, bay - . h s treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. these alterations were reversed by pretreatment with bay - . moreover, expression of cxcr in neutrophils obtained from h s treated mice was significantly upregulated leading to an obvious elevation in mip- directed migration of neutrophils. therefore, h s act as an important endogenous regulator of leukocyte trafficking during inflammatory response. transient receptor potential vanilloid (trpv ) is primarily found on sensory nerves. we have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. selective trpv antagonists are not available for use in the mouse in vivo, thus established trpv knockout (-/-) mice were used. c bl wt and trpv -/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (lps). the response was monitored for h. blood pressure, measured before and at intervals after lps in conscious mice via a tail cuff was reduced in both wt and trpv -/-mice, with trpv -/-mice showing an enhanced drop at h. in a separate group temperature, a proposed pre-mortality marker was also reduced by h, again with a significantly increased drop in trpv ko. furthermore higher levels of two inflammatory markers tnfa and nitrite (as an indicator of no) were measured in peritoneal lavage and higher levels measured intrpv -/-as compared with wt samples. finally aspartate aminotransferase (ast) levels also enhanced in trpv -/-versus wt mice, although markers for kidney and pancreatic damage were similar in both genotypes. we conclude that trpv plays a protective role in sepsis. trpv is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. ( ), da souza-junior( ), l de paula ( ), mc jamur ( ), c oliver ( ), sg ramos ( ), cl silva ( ), lucia helena faccioli ( ) ( h rv) . infected balb/c mice developed an acute pulmonary inflammation and higher levels of tnf-a, il- , kc, mcp- and mip- were detected in the lungs by day . in vivo degranulation of mast cells by c / led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. the magnitude of cellular immune response was also partially impaired in infected mice and treated with c / . histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with c / . of interest, the number of mycobacterial bacilli recovered from the lungs was log higher after treatment of infected mice with c / . these findings suggest that mcs participate in host defense against m. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. ( ), ms chadfield ( ), db sørensen ( ), h offenberg ( ), m bisgaard ( ), he jensen ( ) ( ) department of veterinary pathobiology, faculty of life sciences, university of copenhagen, denmark ( ) novo nordisk a/s, cell biology, gentofte, denmark introduction: pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. the aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of p. multocida of different origin in an aerogenous murine model. materials and methods: female balb/ c-j mice (app. g, taconic, denmark) were infected intranasally with two clinical isolates of p. multocida of avian (vp ) and porcine (p ) origin, at three different levels of inocula concentration. after euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase mmp- and metalloproteinase inhibitor timp- . results: all mice infected with the avian strain were euthanized after hours. viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. in the liver, disseminated necrosis with formation of microabscess was also seen. on the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after , and h. furthermore, differences were seen in the nature of the lesions caused by the two strains. there was a difference in expression of mmp- and timp- between infected and noninfected mice. the model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of mmp- and timp- . ( ), r molinaro ( ), a franÅa ( ), m bozza ( ), f cunha( ), s kunkel ( ) ( ) universidade do rio de janeiro, brazil ( ) universidade de s¼o paulo, brazil ( ) university of michigan, usa introduction and objectives: studies reveal that regulatory t (treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. ccr knockout mice (ccr -/-) are more resistant to lps shock. so, our aim is to study the mechanisms involved in the resistance of ccr -/-subjected to severe sepsis by cecal ligation and puncture (clp) and how tregs modulate this effect. results: c /bl mice were subjected to clp model, whereby the cecum was partially ligated and puncture nine times with a g needle. sham-operated mice were used as control. mice subjected to clp and sham surgery were treated with antibiotic since h after and until days. ccr -/-mice subjected to clp presented an increase in survival rate ( %) compared with wildtype mice ( %), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. besides, tregs from ccr -/-clp mice did not inhibit proliferation of t effector cells as observed for treg from wild clp mice, at a proportional ratio of teffector:treg. interesting, treg from ccr -/-clp mice did not inhibit neutrophil migration to bal when co-injected with fungal challenge as secondary infection, while the treg from wild clp mice did, as expected. conclusions: these results suggest that treg cells from ccr -/-mice did not present suppressive response and it could be an important factor in their survival. inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. inflammation has been associated with diseases like cancer, diabetes and many other. proinflammatory cytokine (tnf -µ and il- â) and no are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. thus inhibition of pro-inflammatory cytokines and no production are important targets for treatment of inflammatory disorders. nowadays due to the emerging side effects of cox inhibitors, these targets have been paid more attention for the treatment of these conditions. some medicinal plants such as curcuma longa ( ), commiphora mukul ( ) in humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated b lymphocytes, eliminated after a few days or weeks by apoptosis. however, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without dna-synthesis and refractory to signals from antigen or antigen-antibody complexes. the lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. the niche provides survival signals like il- , cxcl and tnf. within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. the number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. this competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like cxcl , which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. plasmablasts and plasma cells express cxcr , the receptor for cxcl . while plasmablasts migrate in response to cxcl , plasma cells depend on it for survival in the niche, but are no longer migratory. thus once disloged from their niche, they will die. plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express cxcr , the receptor for the interferon-gamma-induced chemokines cxcl , and , which may lure the plasmablasts into inflamed tissue. the switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. inflamed tissue contains survival niches for plasma cells. in the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. upon resolution of the inflammation the plasma cells will be dislodged and die. longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory b cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. in chronic inflammation, this mechanism can contribute to pathogenesis. thus in the nzb/w model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. later, in established disease, autoreactive plasma cells are shortlived and continuously generated. they do not compete with the longlived plasma cells, and both populations coexist as prominent populations. interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. this may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, ( ), h lee ( ) c a is a potent inflammatory mediator produced during complement activation. unregulated c a signalling through its receptor (c ar) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. considerable effort has gone into development of c ar antagonists for human therapy. we took neutrophils from genetically modified human c ar knock-in mice in which the mouse c ar coding region was replaced with human c ar sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mabs) to human c ar. these mabs inhibit c a-induced neutrophil migration and calcium-flux, and bind to a region of the nd extracellular loop of c ar loop that seems to be critical for receptor activity. this study investigated the effectiveness of these mabs in the k/bxn serum-transfer model of inflammatory arthritis. human c ar knock-in mice were given - mg/kg mab intraperitoneally, before or after inflammatory arthritis developed. mice treated with anti-c ar mab one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. histopathology of the joints in anti-c ar mab-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. furthermore, and most significantly, a single mg/kg dose of anti-c ar mab given days after initiation of disease completely reversed inflammation. in the collagen-induced arthritis (cia) model, injection of anti-c ar mab after development of inflammation also reversed inflammation to baseline. these potent new antibodies to human c ar are in preclinical development. the cytokine macrophage migration inhibitory factor (mif) participates in fundamental events in innate and adaptive immunity. the profile of activities of mif in vivo and in vitro is strongly suggestive of a role for mif in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (ra), asthma, and sepsis. mif also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of mif is in fact induced by glucocorticoids. thus, mif functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. therapeutic mif antagonist may therefore provide a specific means of steroid sparing. since mif are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.we developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against mif.the antibody can neutralize mif activity in cell based assays, and is very effective in a lps induced mouse sepsis model. using this antibody as a tool, we are studying the function of mif in comparison with the function of lps.we found that lps induced inos expression and no secretion are dependent on the secretion of mif.we also found that although both lps and mif induce g arrest in macrophage cell line raw . , their functions are independent to each other. structure-based small molecule drug design. an effective agent would be the first orally active cytokine antagonist. methods: collagen-induced arthritis (cia) was induced in dba- mice by immunisation with bovine type ii collagen/adjuvant on day and . cor , synthesized on the basis of computer modeling of mif protein x-ray crystallographic data, was administered by daily oral gavage from day . etanercept ( mg/kg ip q d) was used as a positive control. the mek-erk pathway is activated in numerous inflammatory conditions, including ra, ibd and copd. arry- is a potent (ic = nm), selective, atp-uncompetitive mek / inhibitor.arry- is highly efficacious in cia and aia rat models, with ed s of and mg/kg, respectively, equal to or better than standard agents. addition of arry- to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. in tpa-stimulated human whole blood, this compound inhibited tnf, il- and il- production (ic s of , and nm, respectively). in contrast, inhibition of perk required nm to achieve a % reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to perk inhibition.in clinical studies, healthy volunteers were administered a single oral dose of , , , or mg); blood was drawn at various times after dosing and stimulated ex vivo with tpa. arry- was well-tolerated and drug exposure was dose-proportional. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpainduced il and tnffz with > % inhibition observed at plasma concentrations of and ng/ml, respectively. similar inhibition of perk required ng/ml. a multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of arry- and helped define tolerability. clinical evaluation of arry- in combination with methotrexate in patients with rheumatoid arthritis is on-going. p (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as tnfand il- a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. inhibition of p activity is expected to regulate the levels of tnf-a and il- b thereby alleviating the effects of inflammation in ra. a new class of p inhibitors based on the naphthyridininone scaffold have been discovered. x-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template. this presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. ( ), h aaes ( ), w-h boehncke( ), j pfeffer( ), t skak-nielsen( ), i teige ( ), k abell ( ), ph kvist ( ), e ottosen ( ), tk petersen ( ), lars svensson ( ) ( ) discovery, leo pharma, industriparken, ballerup, denmark ( ) department of dermatology, johann wolfgang goethe-university, frankfurt am main, germany p map kinase plays an important role in mediating an inflammatory response in mammalian cells. as a consequence of activation, several inflammatory mediators are released including il- b] and tnfa. both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. approximately % of psoriasis patients develop psoriatic arthritis. leo is a member of newly developed class of selective p map kinase inhibitors. the compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. the in vivo models selected include the cia arthritis model, the human psoriasis xenograft scid mouse model, the uvb-induced dermatitis model, the lps induced tnfa model and a local gvh model. treatment with leo led to an amelioration of the ongoing inflammation in all investigated in vivo models. in the cia model, a clear dose response effect was observed on the developing arthritis in both rats and mice ( % reduction in mice and % in rats at mg/kg p.o.). in the humanised psoriasis model, leo at a dose of mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by %) and on the infiltrating inflammatory cells. the anti-inflammatory effect of leo was even close to the effect of systemically delivered steroids in both models. we believe that the new highly selective class of p map kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. slx- is a potent, selective, orally bioavailable inhibitor of the rho-kinase rock- . its ic for rock- and rock- inhibition is nm and > mm respectively. the ability of slx- to inhibit septic liver injury was investigated in c bl/ j mice challenged with lipopolysaccharide (lps) and d-galactosamine (d-gal). mice were given lps ( mg/mouse) and d-gal ( mg/ mouse) i.p and . hours later were sacrificed for analysis of liver injury. mice challenged with lps/d-gal had a > fold increase in serum alt and ast levels. this increase was reduced by > % in mice pretreated with slx- either orally ( mg/kg, and hrs prechallenge) or i.p. ( - mg/kg, min pre-challenge). slx- inhibited the increase in hepatic levels of tnffalpha produced by lps/d-gal by > %. to assess the kinetics of slx- s benefit, slx- ( mg/kg i.p.) was given min prior to, or or min after the lps/ d-gal. slx- was effective at inhibiting the rise in alt and ast levels at all time points suggesting that inhibiting rock- even after the initiation of the lps/d-gal driven cascade protects against septic liver injury. in a survival study, out of mice given lps/d-gal were dead by hrs whereas in mice given slx- ( mg/kg orally) animal died at hours and the remaining mice were alive hrs later. these results show that specific inhibitors of rock- may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.they potently inhibit protein kinase c activity in vitro as demonstrated by inhibition of il- secretion in human purified t-cells stimulated with anti-cd and anti-cd or whole blood seb challenge.the pkc-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. ( ), j zhang ( ), k henley ( ), m white ( ), d hilton ( ), b kile ( ) ( to investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human fcgam-mariia in inducible and passively transferred models of arthritis. transgenic mice developed severe ra-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. disease could be reduced by the administration of either specific monoclonal antibodies to fcgammariia or small chemical entities (sce) designed to bind to the fcgam-mariia dimer. to investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to c bl/ control mice was examined using phagocytosis of fluorescent beads coated with ova or ova/anti-ova immune complex or opsonised sheep red blood cells. in both assays macrophages from fcgammariia transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at hours.this difference diminished over time andwas only seen where particles were opsonised. treatment of macrophages with specific fcgammariia blocking monoclonal antibody fragments . f(ab) or iv. f(ab) reduced phagocytosis to background levels . macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines tnf-alpha and il- beta when stimulated in vitro with heat aggregated immunoglobulin (hagg). moreover, this response was also blocked by specific fcgammariia monoclonal antibody fragments or sce. thus, expression of the fcgammariia transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased th cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. harald burkhardt ( ), u hüffmeier ( ), i kçnig ( ), j lacorz ( ), a reis ( ), k reich ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander-unisversity of erlangen-nuremberg, germany results: whereas the earlier described strong association of allele tnf*- a with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the psors risk allele. for psa, but not psoriasis vulgaris without joint involvement strong association with the allele tnf*- t was detected (or= . , % ci . - . ; pcorr= . ) also in patients negative for the psors risk allele. our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. while the previously reported association between tnf*- a and psoriasis seems to primarily reflect ld with psors , tnf*- t may represent a risk factor for psa independent of psors . ( ), n modi ( ), m stanford ( ), e kondeatis ( ), r vaughan ( ), f fortune ( ), w madanat ( ), c kanawati( ), p murray ( ) methods: dna was obtained from patients with bd, from the uk and from the middle east (me), and controls individuals, from the uk and from me. dna was prepared by proteinase k digestion, and salt extraction and - and snp were detected by a pcr-ssp. results: there was no significant difference in expression of - c/t or a/g when all bd patients were compared to all control individuals, (p= . and . , respectively). as we have previously shown differences in snp expression in different patient groups we tested the uk and me patients separately. however, there was no significant difference in either snp in uk bd patients, (p= . , p= . ) or me bd patients (p= . , p= . ) when compared to the appropriate controls. ( ), da brown ( ), h johnen ( ), mr qiu ( ), t kuffner ( ), pgm curmi ( ), l brown ( ), m mazzanti ( ), sn breit ( ) ( introduction: cerebral palsy (cp) is a nonprogressive motor disorder caused by white matter damage in the developing brain. it is often accompanied with neurocognitive and sensory disabilities. the cause and pathogenesis of cp is multifactorial and continues to be poorly understood. chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for cp both in term and preterm infants. il- gene is single copy gene located on chromosome q . - in humans. interleukin- is synthesized by a variety of immune (t cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. it is also detected in organ-specific secretions in a number of inflammatory processes. amniotic fluid interleukin- concentrations decreased with advancing gestational age. but, women with preterm labor and women with chorioamnionitis have higher interleukin amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. the aim of our study was to estimate allelic frequency for regulatory il - snp in the children with the cp. methods: dnas obtained from peripheral blood of cp patients and unrelated healthy volunteers were genotyped for the il - snp pcr-rflp method. results and conclusions: il - genotype fncc was more common in the population with cerebral palsy in the comparison to healthy volunteers. the significance of the association between il fngene polymorphisms and cerebral palsy has to be investigated in the future studies. ( ), d delbro ( ), e hansson ( ) ( ) kalmar university, sweden ( ) gçteborg university, sweden background: acetylcholine (ach) is a major signalling molecule, binding partly at nicotinic receptors (nachrs; a family of ions channels with nicotine as a selective ligand). one subtype, the alpha nachrs, has antiinflammatory effects by way of down-regulation of tnfalpha release from macrophages. the alpha nachrs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. aim. in rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: . the expression of alpha nachrs and alpha nachrs by immunofluorescence and western blot. . intracellular ca +-transients spread within the astroglial networks after stimulation with nicotine. . pro-inflammatory cytokines, il- b, il- , and tnfalpha, released from microglial cells after stimulation with nicotine. results: alpha nachrs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. the ca + transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. these ca + responses were blocked by the alpha nachr antagonist alpha-bungarotoxin. the release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine. conclusion: alpha nachrs appear to be involved in some of the effects of nicotine administration to rat astrocytes. an anti-inflammatory action of cholinergic nerves on the astrocytes via nachrs seems probable, which may have therapeutic implications. university, department of natural sciences, kalmar, sweden e-mail: ann.pettersson@hik.se gedeon richter plc., budapest, hungary tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of nsaids. review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (cohran database ). efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. our aim was to assess oral efficacy of tramadol at side effect free doses in rats. anti-hyperalgesic effect was determined in complete freunds adjuvant (cfa) induced thermal hyperalgesia test (th) and in model of cfa-induced knee joint arthritis. effect on inflammation was characterized in carrageenan induced paw edema test (et). for the characterization of side effect accelerating rotarod assay (arr) was used. significant impairment in arr was noted at mg/kg dose, and above. in the th test weak % effect was seen at side effect free dose ( mg/kg). in the arthritis model b.i.d. mg/kg dose of tramadol given on days - after cfa caused a maximum % effect on weight bearing incapacitance (day ). however, its effect seemed to diminish ( % on day ) upon repeated treatment. in et tramadol had significant anti-inflammatory effect at but not at mg/kg dose. these results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. chronic relapsing experimental allergic encephalomyelitis (cr eae) is a disease that bears striking similarities to the human condition multiple sclerosis (ms). in particular, cr eae and ms have major inflammatory events in the central nervous system (cns) that culminate in demyelination and disruption of axonal function.one group of mediators involved in the progressive cns inflammation are the prostaglandins (pgs).pg generation is regulated in experimental non-immune conditions of the cns by n-methyl-d-aspartate (nmda) receptor activation.nmda receptor-mediated events are also evident in eae and may therefore have the potential to influence pg production.the study was designed to profile pge and pgd in cns tissues during the development of cr eae and to examine the role of the nmda receptor in pg production through the use of the specific antagonist mk- .biozzi mice were inoculated for cr eae and cns tissues were sampled during the course of disease.enzyme immunoassay of processed samples revealed pge and pgd levels within normal limits during the acute and subsequent remission phases of cr eae.in contrast, dramatic changes in pg concentrations were observed with a relapse of symptoms and a remission of disease.mk- was therapeutically administered to cr eae-diseased mice at the onset of relapse and changes in cns pg levels were recorded.the relapse phase of cr eae, but not the acute stage of disease, is characterised by an increase in cns pg production that may be influenced by nmda receptor activation. livia l camargo( ), lm yshii ( ) ( ), sk costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college, london, uk ( ) butantan institute, s¼o paulo, brazil objectives: the neuropeptide substance p (sp) released by capsaicin-sensitive nerves (csn) plays a pivotal role in neurogenic inflammation. despite the prevalence of arthritis, the contribution of sp to the progression of arthritis has not been established. this study investigated the effect of csn ablation and sr , a sp antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin ( %, h time course) in female wistar rats. the kaolin-injected knee (ipsilateral -ipsi) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. in addition, increased pain score and high levels of myeloperoxidase (mpo, marker of neutrophil accumulation) and both pro-inflammatory (il- b and il- , but not tnfa) and anti-inflammatory (il- ) cytokines was detected in the ipsi synovial fluid of these animals. both destruction of knee joint csn fibres by neonatal capsaicin treatment and i.a. injection of rats with sr ( nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. in contrast, the same treatment caused increased mpo activity and cytokine concentrations measured h post kaolin injection. conclusions: peripheral release of sp after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. however, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. ( ) ( ) university of s¼o paulo, s¼o paulo, brazil ( ) butantan institute, s¼o paulo, brazil objectives: previously, intra-tracheal (i.tr.) injection of dep or , -naphthoquinone ( , -nq) evoked plasma extravasation and cell influx in rat airways. we now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. we also determined the ability of dep and , -nq-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (rta) and corpus cavernosum (rcc) reactivity using organ bath assays. results: capsaicin-or vehicle-treated male wistar rats received i.tr. injection of dep ( mg/kg) and , -nq ( nmol/kg). after min, dep and , -nq produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. in capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for c-fibres, primarily tachykinins. increased mpo and cytokine levels were detected in bronchi of capsaicin-treated rats following h treatment with pollutants. this treatment contributes to augment the ach ( - - - m)-induced relaxation in the rta but not in the rcc. in capsaicin-treated rats, the rta response to the pollutants was not affected but it was capable of evoking a marked relaxation in rcc in animals challenged with pollutants. conclusions: , -nq exacerbates dep-induced plasma extravasation and mpo activity in the airways, indicating a neurogenic mechanism through tachykinins. the exposure to dep and , -nq affects the endotheliumdependent response in rta without interfering with rcc. neuropeptides are unlikely to affect the pollutantsinduced changes in the rta. acknowledgements: capes, cnpq, fapesp. we thank ma alves for technical assistance. trans-resveratrol (rv) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. the purpose of this study was to determine the effect of rv on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (lps) in glial cells. rt-pcr showed that rv ( , , mm) dose-dependently inhibited ng/ml lps induced tnfa, il- b, il- , mcp- and inducible nitric oxide synthase mrna expression. rv also inhibited lps-induced production of these cytokines (elisa), nitric oxide and reactive oxygen species in a dose-dependent manner. western blot analyses showed that resveratrol could inhibit lps-stimulated phosphorylation of erk / and jnk but not p . nf-kb reporter assay showed rv could inhibit nf-kb activation by lps in microglia and astrocytes. these results suggest that rv may inhibit lps-induced microglial and astrocyte activation through erk / , jnk and nf-kb signaling pathways. therefore, rv is a natural product with therapeutical potential against disease conditions in cns that involve an overproduction of proinflammatory cytokines and reactive oxygen species. ( ), cs patil( ), sv padi ( ), vp singh ( ) ( ) university institute of pharmaceutical sciences, panjab university, chandigarh, india ( ) pharmacology r & d, panacea biotec ltd. lalru, punjab, india persistent stimulation of nociceptors and c-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. the important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (no). the aim of the present study was to test the sensitivity of pde inhibitor sildenafil in chronic constriction injury (cci) model, a rat model of neuropathic pain. sciatic nerve injury is associated with development of hyperalgesia days after the nerve ligation. sildenafil ( and fÝ g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. the hyperalgesic effect of sildenafil was blocked by l-name and methylene blue (mb), which on per se treatment showed antinociceptive effect in nerve ligated rats. the results from the present study indicated that the major activation of no cgmp pathway in the chronic constriction injury model of neuropathic pain. the aggravation of hyperalgesic response might be due to the increased cgmp levels resulting in pkg-i activation and its upregulation. glycine transporter (glyt) and glyt are expressed in glia and neurons, respectively. glyt make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to nmda receptors, while glyt supplies glycine into synaptic vesicles in glycinergic neurons. therefore, glyt inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. the present study examined the effects of glyt inhibitors on pain in animal models. inhibitors of glyt (sarcosine and org ) and glyt (alx and org ) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. glyt inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in complete freund adjuvant (cfa)-induced inflammation mice but the antiallodynia effects appeared after latent period. on the other hand, glyt inhibitors produced antiallodynia effects immediately after the i.t. injection in cfa-treated mice. these inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.pretreatment of specific antagonists of glycine site of the nmda receptors disappeared the latent periods of glyt inhibitors and potentiated the antiallodynia effect. glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected glyt inhibitors. these results suggest that both glyt and glyt inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. the tumour microenvironment in particular tumour associated macrophages (tams) play a role in determining tumour outcome. despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of tams in this disease. we have utilized our mouse model of gastric tumourigenesis, the gp ff mouse to assess the effect of the gp ff mutation on macrophage function and ascertain the role of macrophages in tumor formation. this mouse has a knockin mutation in the il- family cytokine receptor gp preventing shp /ras/erk signaling and leading to constitutive stat transcriptional activation. tumour development is inflammation dependent, there is a requirement for il- , and development is inhibited by nsaid treatment or microbial eradication, however an adaptive immune response is s inflamm. res., supplement ( ) posters dispensable for tumorigenesis. in gastric antrum the gp ff mutation results, decreased erk / activation and constitutive phosphorylation of stat , abnormal signaling is replicated in macrophages. antral stat activation is unaffected by depletion of il- . however in macrophages an absence of il- results in higher stat activation demonstrating the anti-stimulatory role of il- on macrophages. the gp ff mutation results in macrophages with decreased il- and increased inos mrna expression reflecting a more basally activated phenotype. manipulations of the gp ff mouse that reduce tumor size (eg. antibiotic or nsaid treatment or stat hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. macrophages of gp ff mice display aberrant gp signaling potentially resulting in exaggerated response to stimuli. the key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. ( ), y riffo-vasquez( ), s brain( ), s costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college london, uk objectives: we have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (dep) and , -naphthoquinone ( , -nq) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. this study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (wt) and trpv knockout (ko) mice exposed to dep and , -nq. dep ( mg/kg) and , -nq ( nmol/kg)-induced airway inflammation was assessed via i-labelled albumin h post pollutants injection. mpo assay and histopathology were performed h after treatment. staining of lung and trachea specimens with h&e provided a profile of cell infiltration in both wt and ko animals. results: injection of dep and , -nq evoked a potent plasma extravasation into the trachea and lung of wt, but not trpv ko, suggesting these pollutants act via a trpv -mediated mechanism. in contrast, mpo activity in the airways of trpv ko mice was exacerbated compared to wt mice data. likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of trpv ko mice compared to wt. conclusions: inhibition of increased microvascular permeability in the airways of trpv ko mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. however, neutrophils/macrophages accumulate more in trpv ko, indicating that the lack of trpv receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for trpv receptors. acknowledgements: capes, cnpq, fapesp. ( ), n sato( , ), y endo ( ), s sugawara ( ) ( ) division of oral immunology, department of oral biology, tohoku university graduate school of dentistry, sendai, japan ( ) division of fixed prosthodontics, department of restorative dentistry, tohoku university graduate school of dentistry, sendai, japan biotin is a water-soluble vitamin of the b complex and functions as a cofactor of carboxylases.biotin deficiency causes alopecia and scaly erythematous dermatitis.moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.however, immunological effects of biotin on allergic inflammation remain unclear.in this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (ni)-allergy model mouse and a murine macrophage cell line j . . female balb/c mice ( weeks old) received a basal diet or a biotin-deficient diet for weeks.ten days after sensitization with intraperitoneal injection of lps and nicl , the mice were challenged with intradermal injection of nicl into the pinnas.allergic inflammation was measured by ear swelling.the ethical board for nonhuman species of the tohoku university graduate school of medicine approved the experimental procedure followed in this study.j . cells were cultured in biotin-sufficient or deficient medium for weeks. ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.il- beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.moreover, biotin-deficient j . cells produced il- beta significantly higher than biotinsufficient j . cells.to investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for weeks.ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.these results indicated that biotindeficiency deteriorates allergic inflammation.the augmentation of il- beta production is probably involved in those deteriorations. one of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. applying the elisa technique, levels of il- , icam- and e-selectin were studied in patients with acute pancreatitis. mediators levels were studied in arterial, venous and pancreatic ascites samples. according the atlanta criterion the mild pancreatitis was established in patients and severe -in patients. the highest levels of il- were noted in ascites and lowest in arterial samples. the highest concentration of adhesion molecules was in venous samples and lowest in ascites. it was a clear correlation between levels of il- and adhesion molecules and severity of pancreatitis. during first week the levels of il- gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. icam- levels gradually increased during first three day with the following decrease after this term. the highest levels of e-selectin were noted at the time of admission. the clear correlation between il- and adhesion molecules was noted in both groups of patients. besides that, the clear strong correlation was observed between il- and quantity of circulating granulocytes and between e-selectin and hematocrite in patients with necrotizing pancreatitis. our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. subsequently eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. this is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. mc/eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. we have previously described an mc/eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. still, pathways that mediate mc/eos cross-talk in allergy are not fully characterized. methods: human cord-blood derived mc were cocultured with peripheral blood eos, and activated with anti-ige. mc/eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. expression of surface molecules was analyzed by facs. mc activation was measured by chromogenic assays for â-hexosaminidase. relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation. results: mc and eos physically interact, forming welldefined couples in-vitro and in-vivo. in the presence of eos, mc are more releasable under baseline or igeactivating conditions. this effect is partially mediated by cd and dnam- on mc, and b and nectin- on eos. we describe a novel physical interaction between mc and eos that we name "the allergic synapse". this synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. methods: mc were obtained from cord blood mononuclear cells ( - weeks with scf, interleukin- and prostaglandin e , cbmc). cbmc were activated, after their culture for days with myeloma ige ( mg/ml), by rabbit anti-human ige antibodies ( mg/ml), for , and h at c. activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. the expression of flip, mcl- , bcl- , bcl-xl, bak and bax was assessed by immunoblot analysis. results: two anti-apoptotic proteins were found to be upregulated: flip, which is involved in the extrinsic apoptotic pathway and mcl- that is mainly implicated in the intrinsic apoptotic pathway. in contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. bcl- and bcl-xl) was not altered. likewise, the expression of pro-apoptotic proteins from the bcl- family (i.e. bak and bax) was either undetectable or unchanged. conclusions: our findings reveal that ige-dependent activation of human mc mainly induces the selective increase in the expression of two pro-survival molecules. this may be one of the mechanisms that underlay mc hyperplasia in the chronic allergic inflammation. ( ), c armishaw( ), z yang ( ), h cai ( ), p alewood( ), c geczy ( ) ( ) university of new south wales, faculty of medicne, sydney, australia ( ) university of queensland, institute for molecular biosciences, st lucia, australia purpose: human s a , s a and s a are closely related proteins associated with inflammation. s a is expressed in the human genome, but not in the mouse. s a is a potent monocyte chemoattractant and mast cell (mc) activator. mouse (m) s a and human (h) s a share % structural identity, but the hinge domains are more divergent. the ms a hinge (ms a - ) is a potent chemoattractant for leukocytes whereas this sequence in hs a (hs a - ) is inactive. methods: s a hinge domain and its alamine scan mutants were synthesized and their activities were tested by using thp cells or murine mc in vitro and mouse footpad injection. results: s a hinge domain (s a - ) was chemotactic for monocytes and mc and provoked mc degranulation in vitro and oedema, and leukocyte recruitment in vivo. in contrast to s a , the hinge domain only weakly induced cytokine production (il , il ) by macrophages. residues essential for oedema were hydrophobic in nature (leu , isoleu , isoleu and isoleu ). n and i were essential for responses provoked by - m s a - whereas mutation of k , l , n , i , d , k and i significantly reduced migration with - m s a - . conclusions: s a and ms a may be functional chemattractant equivalents; s a may have arisen by duplication of human s a . isoleucine residues in s a hinge domain are essential for its proinflammatory properties. ( ), g kiriakopoulou ( ), e tsimara( ), a voultsou( ), k zarkadis ( ) ( ) general hospital of zakynthos, greece ( ) medical centre of katastari, zakynthos, greece schistosome is a disease which pests in tropical countries. the endemic areas are south america, far and middle east, and africa. our aim is to present an incident which concerning a parasitic infection, not endemic in greece. patient: man years old who immigrated from pakistan (at the side of the aparkenar river) in greece, a year ago. he turned out with anlage, headache and weight loss. he is a swimmer. he mentioned also a fever before migrating to greece. clinically: largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. laboratory examination: leucocytes . eo . %, hb . g/dl, ht . %, mcv , fl, mch . pg, thrombocytes . , blood sedimentation mm, fe mg/ml, ferritin . ng/ml. normal biochemical examinations. u/s: livers size lightly increased . mm, hepatoportal vein with normal amplitude. parasitology of feces: in the immediate confection with lugol of the second sample, there were observed: big scattering oval ovules ( - mm) with a thin wall and quills on the side, as well as three imago worms (> mm): schistosoma mansoni. treatment: praziquantel was given. recheck in a months time: blood examinationleucosites . , eo . %, hb , g/dl, ht . %. parasitology of feces is negative. in the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis posters inflamm. res., supplement ( ) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as t lymphocytes and eosinophils. recently, high-sensitivity crp (hs-crp) has become available for detecting small changes in crp levels within the normal range, allowing for assessment of subclinical inflammation. this study was undertaken to investigate the relationship between hs-crp and bronchial asthma. we collected blood samples from patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ecp) and pulmonary function as well as serum hs-crp. serum crp levels in patients without attacks (average . mg/ l; p < . ) and with attacks (average . mg/l; p < . ) were significantly higher than those of normal controls (average . mg/l). serum hs-crp levels were inversely correlated with fev . % in asthmatic patients (r = - . , p < . ). in conclusion, these results indicate that serum hs-crp as well as ecp may be related to the state of asthma exacerbation and allergic inflammation. objectives: the pshr assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type i hypersensitivity reaction. in this study we tested methods for removing ige-antibodies (stripping) from donor basophils. moreover a method was developed for improving the antigen specificity profile in pshr. proof-of-concept was provided using absorption with complete allergen extracts. methods: buffy coats were screened to exclude reactivity against relevant allergens. subsequently pbmcs were purified and basophils were stripped with either lactate or a phosphate buffer. cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. released histamine was measured spectrofluorometrically (hr-test, reflab). cutoff was % hr. for absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and bsa as control). after centrifugation the supernatant was used to passively sensitize stripped basophils. hr was measured as described above. results: stripping experiments using the two buffers only partially removed surface ige but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. absorption experiments showed that it was possible to specifically remove peanut specific ige from patient serum. removal of specific ige reactivity to peanut extract was verified by western blotting. conclusions: using peanut extract as a model it was demonstrated that in pshr antigen specificity can be modified. ( ), sk bk( ), v sharma( ), rp bhandari ( ) ( ) nepal medical college teaching hospital, nepal ( ) all india institute of medical sciences, new delhi, background: nepal has one of the highest maternalmortality rates in the world. this study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women. methods: women with thromboembolic diseases were identified and their case records retrieved and reviewed s inflamm. res., supplement ( ) posters from january to december . demographiccharacteristics were compared between women with and without thromboembolism. the total number of deliveries over the study period was , , giving an incidence of . per deliveries. there were two cases of pulmonary-embolism and one resulted in a maternal-death. the others had deep-vein-thrombosis of which over % were limited to calf veins only. the ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were . and . per deliveries (p <. );the corresponding cases of deepvenous-thrombosis diagnosed were . and . per deliveries, respectively (p <. ). the majority ( %) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. women with venous-thromboembolism were older, had higher bmi, and a higherincidence of preeclampsia. there were approximately twice as many postpartum as antepartum events. bloodgroup a, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of < weeks, a bmi of , or more and maternal-age of or over were all found to increase incidence of venous-thromboembolism. the long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like nepal. ( ), ch ladel ( ), t ruckle( ), c rommel( ), r cirillo ( ) ( ) rbm-merck serono, colleretto giacosa, italy ( ) serono pharmacological research institute, geneva, switzerland rheumatoid arthritis (ra) is a severe articular disease. massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. blockade of pi k signalling pathway has been demonstrated to be curative in a murine model of ra, collagen-induced arthritis (cia). in this study we explored the molecular mechanisms by which pi k signalling inhibition resulted in clinical amelioration of disease symptoms. as , a novel isoform non-selective, yet specific class-i-pi k inhibitor, administered at mg/kg twice-a-day for days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. at the end of treatment, post-arthritic paws were removed and phosphrylation levels of akt (p-akt), downstream target in pi k-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. akt phosporylation resulted to be significantly enhanced by disease induction and as was able to decrease its levels down to values comparable with naïve animals. controls and as treated mice were bled before treatment, after two days of treatment and at treatments end. no changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. t cell number was not affected, however a significant decrease of natural-killer, memory and regulatory t cells was observed after as administration. finally, a non-significant, moderate reduction of bcell number was also observed. these data demonstrate that efficacy of as in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via pi kg) and immunosuppressive (via pi kd/a/b) activity. ( ) ( ) institute of biomedical science, university of sao paulo, brazil ( ) ibilce -sao paulo state university, brazil epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. we investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. seven days after being ovariectomized (ovx), groups of rats were sensitized with ovalbumin (oa). fourteen days after sensitization, the animals were oachallenged and used day thereafter. allergic,shamoperated animals were used as controls. some ovx animals were treated with estradiol ( ìg/kg) or progesterone ( ìg/kg) h before being challenged. mast cell degranulation was quantified in samples of isolated, oa-challenged bronchi. the airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. ovx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. cultured bal and bone marrow cells from allergic rats increased posters inflamm. res., supplement ( ) s the release of il- and reduced that of il- and tnf. the release of il- by bone marrow cells was significantly reduced. these effects were reverted by estradiol, and progesterone reduced il- and increased il- production in bal and increased that of il- and tnf in bone marrow cells. bronchial mast cell degranulation upon direct contact with oa in ovx rats was less than in controls. it is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. objectives: to study the participation of fsh on modulation of e-and l-selectin, icam- and mac- expression in ovariectomized (ovx) rats made allergic. methods: female rats were sensitized (oa/alumen) after (ovx- ) or days (ovx- ) of ovx or sham-operated (sh). fourteen days thereafter animals were challenged (oa, %; aerosol) and sacrificed h after. bronchoalveolar lavage (bal) was collected and flow citometry analyses oficam- , mac- and l-selectin expression was studied. in parallel, lungs were frozen and sections were analysedfor e-selectin expression by immunohistochemistry. results: at day , e-selectin expression increased(ovx- = , ae , ) and at day , decreased (ovx- = , ae , ) as compared to respective controls (sh- = , ae , ; sh- = , ae , ). estrogen treatment reverted this profile in both groups (ovx- +e= , ae , ; ovx- +e= , ae , ). mean fluorescence intensity of bal cells showed increase of mac- expression (ovx- = ae , vs sh- = , ae , ), icam- (ovx- = , ae , vs sh- = , ae , ) and l-selectin (ovx- = , ae , vs sh- = , ae , ) at day i.e., ovx- group. on the other hand, we observed a decrease in icam- (ovx- = , ae , vs sh- = ae , ) and mac- expression (ovx- = , ae , vs sh- = , ae , ) was seen in ovx- group. conclusions: oscillation of hormone levels during immunization with oa increased (ovx- ) and decreased (ovx- ) expression of adhesion molecules. estradiol treatment reverted this effect. this results suggest that fsh modulates the ali in rats by acting on cell ( ), s lim ( ), y lin ( ), bp leung ( ), c thiemermann ( ), wsf wong ( ) ( ) national university of singapore ( ) the william harvey research institute, london, uk glycogen synthase kinase b (gsk- b) is known to regulate various cellular functions including inflammatory responses. we hypothesized that inhibition of gsk- b may have anti-inflammatory effects in a mouse asthma model. balb/c mice were sensitized with ovalbumin (ova) and challenged with aerosolized ova. tdzd- , a non-atp competitive gsk- b inhibitor, was administrated by i.v. injection one hour before ova challenge. tdzd- significantly reduced the ova-induced eosinophilia in a dose-dependent manner and inhibited the levels of il- , il- and eotaxin in bronchoalveolar lavage (bal) fluid. tdzd- also suppressed the mrna levels of icam- , vcam- and chitinase proteins in the lung. histological studies revealed that tdzd- substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. tdzd- also decreased ova-specific ige level in the serum. in addition, ova-induced increase in airway resistance and reduction in dynamic compliance were attenuated by tdzd- . our findings suggest that inhibition of gsk- b may have therapeutic potential for the treatment of allergic airway inflammation. ( ), a yildirim ( ), f ercan ( ), n gedik ( ), m yuksel( ), inci alican ( ) ( materials and methods: sprague-dawley rats ( - g) were exposed to oc (burn group) or oc water bath (control group) for s under ether anesthesia. adm ( ng/kg; s.c) was administered min before burn and all rats were decapitated h after the burn insult. trunk blood was collected for the measurement of tnf-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (lp), myeloperoxidase (mpo) activity and formation of reactive oxygen metabolites (roms) using chemiluminescence assay. results: burn resulted in severe morphologic damage in tested tissues. lp increased in lung and kidney (p< . - . ) and mpo activity showed a marked increase in all tested tissues (p< . ) of the burn group. adm reversed these parameters effectively (p< . - . ). luminol chemiluminescence levels showed increases in both ileum and lung (p< . - . ) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p< . ) of the burn group. adm treatment was also beneficial in reducing chemiluminescence levels near to controls (p< . ). adm reduced plasma tnf-alpha level (p< . ) which showed a significant increase in burned animals compared to controls (p< . ). conclusions: adm is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, rom generation, lp, and the release of proinflammatory cytokine tnf-alpha. kalpana panday ( ), sd joshi ( ), kr reddy ( ) ( we determined the crystal structure of human hematopoietic prostaglandin (pg) d synthase (h-pgds) as the quaternary complex with glutathione (gsh), mg +, and an inhibitor, hql- , with anti-inflammatory activities in vivo, at . resolution. hql- was found to reside within the catalytic cleft between trp and gsh in the quaternary complex. hql- inhibited h-pgds competitively against the substrate pgh as well as noncompetitively with respect to gsh. surface plasmon resonance analysis revealed that hql- bound to h-pgds with an affinity that was -fold higher in the presence of gsh and mg + than in their absence. hql- inhibited selectively pgd production by human h-pgds-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between h-pgds and cyclooxygenase. orally administered hql- inhibited antigen-induced production of pgd , and airway inflammation in mice without affecting the production of pge and pgf f¿. knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit pgd production specifically. introduction: it has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. this study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung. methods: male wistar rats ( - g) were anesthetized and paralyzed, and the two lungs were independently intubated. differential ventilation was applied for h ( breath/min). one lung was subjected to hyper-ventilation ( ml/kg/lung) and the other was ventilated with a normal volume ( ml/kg/lung). in a control group, both lungs were ventilated with a normal volume. after sacrifice, samples of lung, plasma and liver were collected. the expression of the pro-inflammatory chemokine mip- was evaluated by rt-pcr and the edema was assessed by the ratio between the wet and dry weight of the lung. systemic inflammation was estimated in liver by measuring the expression of tnfa by rt-pcr as well as its levels in plasma. the hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in mip- expression compared to the normal ventilated lung. no differences were found in edema, neither expression of mip- between the normally ventilated lung and control lungs. no significant changes were observed in liver expression and plasma levels of tnfa as a consequence of unilateral lung hyper-ventilation. the over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. the normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. ( ), j-y gillon( ), v lagente ( ), e boichot ( ) ( ) university of rennes , rennes, france ( ) serono international s.a., geneva, switzerland macrophage elastase (mmp- ) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with copd (chronic obstructive pulmonary disease). the mechanism of action of mmp- in the development of pulmonary inflammatory process is still unknown. in the present study, we investigated the effect of recombinant human mmp- (rhmmp ) on il- /cxcl release from alveolar epithelial cell line a and we explored the underlying mechanisms. a cells were stimulated with rhmmp- ( . - - . - u/ml) during hours and il- /cxcl level in supernatant was determined by elisa. involvement of map (mitogen activated protein)-kinases was studied by western-blotting and also using chemical inhibitors. nfkb activation was examined with the transam nfkb p kit. we observed that mmp- elicited il- /cxcl release in a dose-dependent manner. this production could be prevented by a pretreatment for hour with a selective mmp- inhibitor (as - mm) or with the nonselective inhibitor batimastat ( - mm) . the il- /cxcl production was also inhibited by actinomycin d ( mg/ml), erk / inhibitors (u mm and pd mm) and nfkb inhibitors including bay - ( mm) and nfkb activation inhibitor ( nm), whereas p kinase inhibitor (sb ) had no effect. stimulation with mmp- was rapidly followed by a phosphorylation of erk / ( min) and an nfkb nuclear translocation and activation ( h). the nfkb activation was not inhibited by a treatment with u . these data suggest that alveolar epithelium is a target of mmp- since it upregulates gene expres-s inflamm. res., supplement ( ) posters sion and release of il- /cxcl , via erk / and nfkb activation, but these two pathways appears to be distinct. agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (lps), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (il)- , in particular, is often used a measure of relative toxicity.in this study we have compared mrna expression and mediator release in nci-h human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (tpm), lps, bleomycin, diesel exhaust particles, residual oil fly ash (rofa), carbon black and vanadyl sulphate.polystyrene, poly(methyl-methacrylate) and the tpm vehicle, dimethyl-sulphoxide were used as negative controls.confluent monolayers of h cells were exposed to serial dilutions of test agents in serum-free medium for hours.the conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by luminex technology.the levels of gene expression of il- , matrix-metalloprotease- (mmp- ), the gel-forming mucin muc ac, heparinbinding epidermal growth factor-like growth factor (hb-egf) and the cytochrome p s cyp a and cyp b were determined by quantitative-polymerase chain reaction.all of the toxicants induced similar responses whereas the negative controls were largely ineffective.-such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of il- alone, particularly in the case of agents such as tpm, where the conventional vehicle is found to have some biological activity. respiratory tract infections are a major public health issue. prevention in high risk populations relies mainly on vaccination. vaccination is highly recommended in decrease absenteeism. immunomodulating drugs are important tools in the treatment of infectious diseases. immunomodulatory agents are probably contributive in decreasing exacerbation rates. the authors present different classes of immunomodulators that are currently in use. the vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. by preventing infections, vaccines prevent the development of a strong t helper (th ) response. the challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. deoxypodophyllotoxin (dpt) is a medicinal herbal product that is isolated from anthriscus sylvestri. that inhibits cyclooxygenase- (cox- ) and cox- dependent phases of prostaglandin d (pgd ) generation in bone marrow-derived mast cells (bmmc) in a concentration-dependent manner with ic values of . mm and . mm, respectively. this compound inhibited cox- and -dependent conversion of the exogenous arachidonic acid to pgd in a dose-dependent manner with an ic values of . mm and . mm, respectively. however, dpt did not inhibit cox- protein expression up to a concentration of mm in the bmmc, indicating that dpt directly inhibits cox- activity. furthermore, this compound consistently inhibited the production of leukotriene c (ltc ) in a dose dependent manner, with an ic value of . mm. these posters inflamm. res., supplement ( ) s results clearly demonstrate that dpt has a dual cox- / -lox inhibitory activity in vitro. therefore, this compound might provide the basis for novel anti-inflammatory drugs. in order to determine anti-allergic and antiasthmatic activity of dpt in vivo, we used rat pca model which was activated by anti-dinirophenyl ige and ovalbumin/alum induced mouse asthmatic animal model, respectively. as a result, dpt strongly inhibited pca reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. furthermore, dpt decreased the mrna levels of the th cytokines in a murine asthmatic model. in addition, northern blot analysis showed that dpt also reduced both the eotaxin and arginase i mrna levels in a dose dependent manner. these results suggest that dpt may be beneficial in regulating various inflammatory diseases. an imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (copd), a smokingrelated disorder associated with accelerated lung function decline.in particular, the activity of matrix metalloproteases (mmps) have been implicated in driving both the inflammation and parenchymal destruction observed in copd patients.here, we tested whether a broad spectrum mmp inhibitor, pkf- , could block the inflammation induced by an acute exposure to cigarette smoke in strains of mice, balb/c and c /bl .animals were administered the compound ( - mg/kg) either per os (p.o.) or intranasally (i.n.) hour before and hours after exposure to smoke on three consecutive days.bronchoalveolar lavage (bal) was performed and lungs were flash frozen for inflammatory marker analysis.pkf- dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~ % at mg/kg; p < . )or i.n. (~ % at mg/kg; p < . ).however, the compound had no clear effect on bal neutrophil infiltration in c /bl mice by either route of administration.interestingly, in both strains bal macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < . ). examination of lung tissue cytokine levels revealed that while smoke-exposure increases il- beta, kc, and mip- , pkf- had little effect on these cytokines.these data suggests the ability of broad spectrum mmp inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. to investigate the role of seh in the regulation of the pulmonary inflammatory response, we have used seh deficient mice in a locally administered lps model. male seh deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled lps.four hours later they were sacrificed and bal was performed. differential counts and cytokine levels in bal were evaluated. results: lps induced a significant increase in total cell number and neutrophil number in bal in the seh deficient mice and in wt mice;no significant differences between the groups were seen (table ) cytokine analysis showed significant increases in tnfalpha, il- , kc, gm-csf, mcp- , il- beta and rantes in the lps-exposed wt mice. no significant differences were seen between lps exposed wt and ko mice except for a significant increase in tnfa in ko mice. our results show that seh has no pivotal role for the regulation of the acute inflammatory response to lung administered lps. fam.liliaceae. based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. the antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by freunds adjuvant in rats. in the first test, the antiinflammatory effect of steroidic saponins mg/kg is weak, statistically insignificant. in the arthritis test, steroidic saponins mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after days). in both tests, the suprarenal glands weight was modified. objectives: dendritic cells (dcs) are professional antigen presenting cells. many types of dc with subtle difference in phenotype have been reported in several organs. existence of dc was reported in synovial tissue of rheumatoid arthritis (ra), however, the details in ra dc still remains unclear. in this study, we generated a new lineage of dc with gmcsf (+tnfa) and investigated their functions. furthermore the ability of osteoclastgenesis was examined. methods: monocyte-derived dcs or macrophage were generated in ( ) tnfa + gm-csf ( ) gm-csf ( ) il- + gm-csf ( ) mcsf. the phenotypes of these cells were analyzed by morphological examination and flow cytometry (facs calibur). cell proliferation was examined by wst- assay. dc functions were assessed in antigen presenting ability (mlr assay), cytokine production (elisa), and endocytosis (fitc-dextran uptake). concerning osteoclastgenesis, monocyte-derived dcs were incubated with rankl and mcsf. trap stain was performed and the resorption ability was assessed on osteologic cell culture system and dentine slices. results: these cells were dendritic-like and their surface markers were cd a low cd b + cd c + cd + cd + cd low hla-dr + dc-sign low and different from conventional dc or macrophage. they had an antigen presenting ability to induce na*ve cd + t cell proliferation, il- production and endocytosis. in the presence of rankl and mcsf, they differentiated multinuclear trap-positive cells with bone resorption ability, which was strengthened by tnfa. we generated a new lineage of dc with gm-csf (+ tnfa). the dc seemed to play a pivotal role in inflammatory arthritis under tnf immunity. the clinical effectiveness of rituximab and other b cellattenuating rheumatoid arthritis (ra) therapies has increased interest in understanding the role of b cells in ra pathogenesis.the possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the entelos ra physiolab platform, a mathematical model of the joint of an ra patient.the platform dynamically integrates the contributions of immune cells (t cells, b cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in ra.the b cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.the dynamics of these b cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from ra patients (e.g., acr score and radiographic progression rates).an assessment of the contribution of individual b cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in ra patients. in contrast, proinflammatory cytokine production by b cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to t cells.biosimulation research in the ra physiolab platform is advancing our understanding of the mechanisms underlying effective b cell-targeting ra therapies, and may guide the development of improved second-generation therapeutic approaches. methods: the hmscs were obtained at the operation.the mononuclear cells were extracted and the colony forming assay were performed after weeks. the hmscs were cultured and in passage ,the cell surface antigens of both groups were analyzed by flow cytometory.in passage , in control group, the cells were cultured with beta glycerophosphate(bgp) and in osteogenic group, the cells were cultured with bgp and ascorbic acid(aa) and dexamethasone(dex).after weeks, alp staining and activity were measured in each group.after weeks, alizarin red s assays were performed.rna was extracted from the cells cultured with bgp and aa and dex for weeks and weeks and the gene expressions of bone formation markers were examined by real time pcr. the mann-whitney test was used for the statistical analyses. the colony forming assays showed no significant differences in oa and ra. in flow cytometory, the cell surface antigens in oa and ra were almost same.in alp activity,there were no significant differences in oa and ra.in alizarin red s, there were significant differences.in real time pcr,the gene expressions showed no significant differences in oa and ra. conclusions: the hmscs of ra will be able to use for regenerative therapy. silje vermedal høgh ( ), hm lindegaard ( ), gl sorensen ( ), a høj ( ), c bendixen ( ), p junker ( ), u holmskov ( ) ( cytosolic phospholipase a (cpla ) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. in addition, cpla regulates the phagocyte nadph oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. collageninduced arthritis (cia) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling rheumatoid arthritis in humans. previous studies show that cpla -deficient mice are resistant to cia. thus we aimed to study whether cpla is up-regulated during the development of cia and to detect its exact location in the inflamed joints. immunoblot analysis revealed an increase in the level of joint cpla protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. immunohistochemistry with specific anti-cpla antibodies revealed low positive cpla protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. in the joints of the cia mice, large amounts of inflammatory infiltrate containing cpla were detected. in addition, robust cpla protein expression in the skeletal muscles surrounding the joints and strong cpla positive staining in sebaceous glands were detected. the high correlation between the severity of inflammation and the elevated cpla protein, due to an inflammatory infiltrate and increased cpla expression, suggests an important role of cpla in the development of arthritis. rheumatoid arthritis (ra) is the complex disease depending on environmental as well as genetic factors. in spite of the large research efforts we know in fact only small number of genes involved in this disease. animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. the aim of the current project is to identify the genes and their functional role importance for arthritis. two loci associated with arthritis were identified using a cross between the b .q (intermediate susceptible) and nod.q strains (resistant to arthritis). one on chromosome a disease protective locus (cia ) and another on chromosome a disease-promoting locus (cia ). nod.q allele at cia promotes arthritis whereas cia , has a protective effect in contrast to the b .q allele on chromosome . a promising candidate gene in cia locus is complement factor (c ) as the nod.q allele produced defective c protein. cia locus contains several genes of potential importance for disease such as fc riib, fc riii, fc riv ncf , fh. the results of cia (collagen induced arthritis) and caia (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains fc r region showed a significant difference in incidence and severity of arthritis. the disease is controlled in a recessive pattern. the fragment devoid of fc r region seems to be protective. the subcongenic for the cia locus has been generated recently which will be tested for cia and caia susceptibility. the results from these experiments will be discussed in detail. angela pizzolla, ka gelderman, r holmdahl ncf is a component of the nadph oxidase complex, which produces reactive oxygen species (ros) upon activation into phagosomes and extracellularly. polymorphisms in the ncf gene that impair the capacity to produce ros, enhance susceptibility of both mice and rats to arthritis. activation of autoreactive t cells drives arthritis development but neither ncf expression nor oxidative burst have been detected in t cells. we hypothesize that antigen presenting cells influence t cell activity by producing ros during antigen presentation. we aimed to clarify the role of ros produced by dendritic cells (dc) on t cell activation. dc were grown from bone marrow from ncf mutated and wildtype mice. we could show that ncf mutated dc proliferated and differentiated better, had higher expression levels of costimulatory molecules and mhc ii upon stimulation as compared to ncf wildtype dc. in addition, ncf mutated dc induced higher levels of il- production by hybridoma t cells. to analyze the role of ncf in dc on arthritis, mice were developed expressing functional ncf restricted to dc (b .qdcn). these mice are characterized for burst, ncf expression and ability to present antigen. we published that immunization with myelin oligodendrocyte glycoprotein (mog) protein resulted in higher disease severity than immunization with mog peptide in ncf deficient mice. this suggests that ncf plays a role in the uptake and processing of posters inflamm. res., supplement ( ) s antigens, probably by dc. this will be further investigated with the b .qdcn mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. purpose: macrophage migration inhibitory factor (mif) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. it is expressed in human ra synovial tissue and its suppression inhibits t or b cell dependent animal models of ra. we investigated the role of mif in k/bxn serum transfer arthritis. methods: arthritis was induced by injection of k/bxn serum on days and in littermate wt and mif-/-mice. arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage. results: wt mice exhibited arthritis as early as day and % incidence was observed on day . mif -/-mice exhibited delayed arthritis, with onset on day and % incidence on day . mif -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( methods: osteoarthritis was induced by bilateral transection of the medial meniscus in dunkin-hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding. results: the first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. cartilage destruction due to meniscal transection was also histologically detected. however, biomarkers for cartilage destruction (ctx-ii, hp/lp ratio, comp) were not increased. treatments aiming at different processes in osteoarthritis, such as bone destruction (risedronate), inflammation (pioglitazone and anakinra), and cartilage destruction (galardin) were not effective in this model. the early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. the ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. the meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments. livia l camargo ( ), a denadai-souza ( ), lm yshii ( ), a schenka ( ), ma barreto ( ), d boletini-santos ( ), c lima ( ), v rioli ( ), mn muscar ( ), e ferro ( ), sk costa ( ) ( methods: aia was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. knee oedema and pain score were assessed daily in controls and animals treated with hemopressin ( or ìg/day; i.a.). histopathological changes, cell number and cytokines in the synovial fluid of aia rats were determined at day . results: aia rats developed a severe mono-arthritis characterized by a joint oedema and pain. at day , there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by elisa. both doses of hemopressin significantly reduced the knee oedema, but only mg of hemopressin attenuated the pain score. acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.). conclusions: hemopressin has potential for treating acute signs of aia by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis. calcitriol, the hormonal metabolite of vitamin d and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. the map kinase erk plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.we hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on erk activation in the presence or absence of inflammatory mediators. our experimental model was immortalized non-tumorigenic human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators. inflammation was mimicked by exposure to tnf. level and activation of signaling molecules were determined by immunoblotting. by using the specific egf receptor (egfr) tyrosine kinase inhibitor, ag , we established that tnf activates erk in an egfr-dependent and egfrindependent modes. the egfr-dependent activation resulted in the induction of the transcription factor, c-fos, while the egfr-independent activation was of a shorter duration and did not affect c-fos expression. treatment with calcitriol alone increased erk activation and c-fos induction. pretreatment with calcitriol enhanced egfr-dependent erk activation and tyrosine phosphorylation of the egfr, but completely abolished egfr-independent tnf-induced erk activation. pretreatment with calcitriol increased the rate of de-phosphorylation of activated erk, accounting for the inhibition of egfr-independent erk activation by tnf. it is possible that effects on the erk cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation. christina barja-fidalgo ( ), r saldanha-gama ( ), ja moraes ( ), r zingali ( ), c marcienkewicz ( ) ( ) universidade do estado do rio de janeiro, rio de janeiro, brazil ( ) universidade federal do rio de janeiro, rio de janeiro, brazil ( ) temple university, philadelphia, usa neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an rgd sequence, a cell attachment site of ecm and cell surface proteins recognized by integrins. they are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ecm interactions. we reported that rgd-disintegrins, selectively interact with integrin amb , a b and/or avb ) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways. recently showed that, a selective ligand of a /a b integrins, vlo , induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. these effects are mediated vlo interaction with a b integrin, activating the focal adhesion cascade. vlo effects on the delay of neutrophil is modulated by pi k, erk- mapk and nf-kb pathways that seems to interfere with the balance between anti-and pro-apoptotic bcl- family members and with mitochondrial membrane potential. data emphasize mechanistic details of the role of a b integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (faperj, cnpq, capes, ifs-sweeden) ( ), h serezani ( ), m peters-golden( ), sonia jancar ( ) '( ) university of s¼o paulo, brazil ( ) university of michigan, usa it is been shown that leukotriene (lt) b and cysteinyl lt (ltc , ltd and lte ) enhances fcr-mediated phagocytosis in alveolar macrophages (am), dependent on protein kinase c (pkc). in contrast, ltb but not cyslt effects are exclusive on syk activation. in the present study we investigated the role of specific pkc isoforms and its upstream and downstream targets involved in lt-enhanced fcr-mediated phagocytosis. to this purpose, ams were pretreated or not with inhibitors of pkc-d (rottlerin - um), pkc-a (ro- - - nm), pi k (ly - um and wortmannin- nm), erk / (pd - um), cpla (aacocf - um), p mapk (sb - um) and ca++ (bapta/am- um), before stimulation with ltb or ltd and addition of igg-opsonized red blood cells for min. activation (phosphorylation) of signaling molecules by lts were analyzed by western blot. our results demonstrate that ltb -enhanced phagocytosis is dependent on pkc-a, while ltd effects are mediated by pkc-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. while galectin- mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin- is known as a strong pro-inflammatory and anti-apoptotic signal. we have recently recognized galectin- as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. in this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin- at gene and protein level in monocytic thp- cells. we have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from e. coli (lps). the targeted mrna level was evaluated using quantitative rt-pcr technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. the results showed that immunomodulatory drugs affected the expression of galectin- on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. the modulatory effects of the applied drugs on galectin- and - expressions were also observed in the cells activated by lps. these findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin- and- expressions, as well as the role of these lectins in the physiology of monocytes. introduction: pancreatitis-associated protein (pap) has been recently described as an endogenous mechanism involved in the regulation of inflammation. in the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by pap. the pancreatic human cell line panc was incubated with pap ( ng/ml) and/or tnfa ( ng/ml). total rna was obtained and the expression of tnfa was examined by rt-pcr. in addition, the effect of pap on nfkb activation was measured by inmunofluorescence in cells. western blot analysis was used to determine the expression of nfkb mediators: phosphorylated ikk, ikba and p . results: we observed that pap administration to cells prevented nfkb translocation to the nucleus as well as the tnfa-induced tnfa gene expression. when tnfastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of tnfa gene expression was completely restored. on the other hand, pap had no effect on ikk phosphorylation or ikba degradation. conclusions:in this study we have provided evidence that pap modulates the inflammatory response by blocking nfkb translocation to the nucleus. this pap-induced nfkb inhibition requires a jak/stat-dependent de novo protein synthesis. objectives: this study investigated the inhibitory mechanism of hyaluronan (ha) on lipopolysaccharide (lps)stimulated production of proinflammatory cytokines in u macrophages. methods: ha was added to u macrophage cultures in the presence of lps, with or without pretreatment with anti-intercellular adhesion molecule- (icam- ) antibody. secreted levels of tumor necrosis factor a (tnfa), interleukin (il)- b, and il- were determined by enzyme-linked immunosorbent assay. the phosphorylation of nuclear factor (nf)-kb, ikba, and mitogenactivated protein kinases (mapks) was analyzed by immunoblotting. results: lps stimulated production of tnfa, il- b, and il- . in contrast to kda ha, kda ha at mg/ ml inhibited lps-induced cytokine production. anti-icam- antibody blocked the effects of ha on the lps actions on u cells. lps activated nf-kb and mapk pathways, whereas ha down-regulated p nf-kb and ikba phosphorylation by lps without affecting mapks. inhibition studies revealed the requirement of nf-kb for lps-stimulated cytokine production. anti-icam- antibody reversed the inhibitory effects of ha on phosphorylation of p nf-kb and ikba. conclusions: ha of intrinsic molecular weight suppresses lps-stimulated production of proinflammatory cytokines via icam- through down-regulation of nf-kb and ikb. exogenous ha into arthritic joints could act as an anti-nf-kb agent by the mechanism demonstrated in the present study. the principal eicosanoid product of endothelial cox- is prostacyclin (pgi ), which is a potent vascular patency factor. induction of endothelial cox- under hypoxic conditions is well documented. this response, along with associated pgi release, is likely an important protective homeostatic response. in order to explore the role of candidate signalling agents in cox- expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the cox- promoter region in huvec. cox- induction under hypoxic conditions was confirmed with the wild type construct. strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (hifs) were more important for cox- expression than those for nfkb. furthermore, expression of cox- was increased under normoxic conditions by transfection of huvec with normoxia-stable hif mutants. emsa showed hif binding in nuclear extracts from untransfected hypoxic huvec. under these hypoxic conditions, increased release of pgi but not vaso-occlusive thromboxane a was seen. thus the putative protective induction of cox- in endothelial cells in response to hypoxia involves signalling by hifs. crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of th immune response plays a crucial role. however, the therapeutic efficacy of the regulation of th -predominant immune responses remains to be investigated. therefore, the effects of a th selective inhibitor tak- were investigated in a model of crescentic glomerulonephritis in wky rats. methods: tak- was administered orally, starting at the time of induction of glomerulonephritis. in group , the drug was administered daily for the initial days. tak- was administered on day only in group , and from day to in group . in each group, nephritic rats were killed on days and . results: in group , glomerular damage, including crescent formation, was improved on day , with reductions in the numbers of cd , cd and ed- positive cells, as well as in urinary protein excretion. protein and transcript levels of th cytokines in the diseased kidneys were markedly decreased by tak- treatment. renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. elevated levels of serum creatinine showed concomitant improvement. in group , in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. treatment only on the first day in group , partially rescued renal dysfunction. conclusions: these results suggest that the initial inhibition of th immune response has an appealing therapeutic potential for crescentic glomerulonephritis. parasitic nematodes and their hosts are now known to produce a wide range of galectins. whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. studies at moredun have demonstrated that both the endoparasitic helminth, haemonchus contortus, and the ectoparasitic mite, psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. in the case of h. contortus, there was homolgy with known est sequences, which allowed subsequent cloning and expression studies to be undertaken. a functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. this may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear s inflamm. res., supplement ( ) posters to provide the primary nutrient source for their survival. moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. the aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (lps)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mrna levels in these tissues. male cd- mice were injected intraperitoneally with mg/kg lps alone or concomitantly with an intravenous dose of pentoxifylline ( mg/kg), lisofylline ( mg/kg) or prednisolone ( mg/kg). the tissues were harvested , , , , and h following lps administration and stored at - c. relative quantification of tumor necrosis factoralpha (tnf-alpha), interleukin- beta (il- beta), interleukin- (il- ), and interferon-gamma (ifn-gamma) mrna levels was performed by real-time rt-pcr. the highest levels of cytokine mrna were observed at or h after lps administration, whereas the expression of tnf-alpha and il- beta in lungs and il- in liver reached the peak values at h and then decreased gradually. in addition, the lps effects on cytokine mrna were more pronounced in liver when compared to lungs. all administered compounds inhibited the lpsinduced tnf-alpha mrna expression (by up to approximately %), whereas lisofylline significantly increased ifn-gamma mrna levels in both tissues at most investigated time points. for other cytokines, the observed differences did not reached statistical significance. in conclusion, with the exception of ifn-gamma, the time course of cytokine mrnas differed considerably depending on the type of tissue. in the murine model of lps-induced septic shock, only tnf-alpha gene expression was suppressed by all compounds under investigation. maria sanz( ), m losada ( ), c company ( ), c lope-gines( ), l piqueras( ), j cortijo ( ) the migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. fractalkine/ cx cl (fr) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. clinical studies and animal disease models have shown that fr is also involved in the pathogenesis atherosclerosis. we have demonstrated that angiotensin-ii (aii) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. in the present study we have investigated whether aii can cause the synthesis and expression of fr on human umbilical arterial endothelial cells (huaecs). huaecs were stimulated with ang-ii microm or with tnfalpha ( ng/ml) for , and h. fr was determined in the culture supernatants by conventional sandwich elisa. fr was only detected after h and h stimulation with tnfalphafnwhereas aii was unable to provoke the cleavage of the chemokine. semiquantitative rt-pcr analysis of huaecs showed increased fr mrna expression in aii-stimulated cells for and h. these effects were caused by the interaction of aii with its at receptor since they were abolished by losartan (at receptor antagonist). tnfalpha also increased fr mrna. immunohistochemical analysis of the cultured endothelial cells showed a clear expression of fr in huaecs stimulated with aii or tnfalpha for and h. these results suggest that fr could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by aii. the lipophilic yeast malassezia is an exacerbating factor in atopic dermatitis (ad).among organisms of the malassezia species, m. globosa and m. restricta are particularly dominant on the skin of ad patients. our previous study has demonstrated that human keratinocytes responded to the two malassezia species with different th -type cytokine profiles, i.e. m. globosa induced il- , il- , and il- secretion from the keratinocytes, whereas m. restricta induced il- secretion.these findings suggest that m. globosa and m. restricta play a synergistic role in triggering or exacerbating ad by stimulating the th immune response. pattern recognition receptors (prrs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. in this study, we assessed the role of prrs for cytokine production by human keratinocytes in response to malassezia species. human keratinocytes were pretreated with various anti-prr monoclonal antibodies (mabs) and stimulated with m. globosa or m. restricta. cytokine secretion from keratinocytes was measured by using fast quant elisa kit. exposure of human keratinocytes to m. globosa and m. restricta resulted in enhanced secretion of il- and il- , respectively. the m. globosainduced increase in il- secretion was inhibited by mabs against cd and cd . in case of m. restricta, mabs against toll-like receptor (tlr ) and cd suppressed significantly il- secretion from keratinocytes. these findings suggest that the distinct prrs interactions with fungal pathogen-associated molecular patterns (pamps) are key factors in differential cytokine secretion from keratinocytes stimulated with malassezia species. atopic dermatitis (ad) is a chronic, relapsing inflammatory skin disease associated with allergy. mdc (macrophage-derived chemokine/ccl ) and tarc (thymus and activation-regulated chemokine/ccl ) are th type cytokines, and it has been reported that serum mdc and tarc levels are associated with ad disease. in present study, we investigated the effect of prunus yedoensis matsum barks on the inflammatory chemokines (mdc and tarc) and jak-stat pathway in hacat keratinocytes. as a result, etoac fraction and e sub-fraction inhibited the mrna expression and protein level of mdc and tarc in a dose-dependent manner. also, e sub-fraction showed inhibitory activity on the stat protein level. these results suggest that p. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (mdc & tarc). the il- family now consists of members, most of which have assigned functions.there are members of the il- receptor family (including the decoy receptor type ii il- r).many of the il- family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.the il- family members il- f , f and f signal through a complex of the il- r family member rp in association with il- r acp to activate common inflammatory pathways.the specific activity is is low, on the order of - ug/ml ec .we have found that removal of a few n-terminal amino acids from il- f , f , f and f can increase their bioactivity approximately -fold. the location of the n-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.in addition, n-terminally truncated il- f is capable of antagonizing signaling via il- rrp , but full-length f is inactive. ( ) university of ulsan, japan kyoto university, japan inflammation plays a pathogenic role in the development of obesity-related complications such as type ii diabetes and atherosclerosis. tumor necrosis factor alpha (tnfa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. tr (hvem/tnfrsf ), which is a member of the tnf receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells (light/tnfsf ), is a potent mediator of inflammatory responses. the purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting tr pathway. c bl/ tr knockout mice and their wild-type control were fed a high-fat diet for weeks and the obesity phenotypes were determined in the obese tr knockout mice and the control. the obese tr mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese tr knockout mice compared with those of the control. our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese tr knockout mice fed a high-fat diet. objectives: the present study was undertaken to investigate the role of insulin on allergic airway inflammation. methods: diabetic male wistar rats (alloxan, mg/kg, i.v.) and controls were sensitized with ova ( ìg) and al(oh) ( mg, s.c.) days after alloxan or saline injection. the animals were challenged days later by the intratracheal instillation of ova ( mg/ . ml). the following analyses were performed h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (bal) fluid; (b) quantification of tnf-alpha and il- beta in the bal by elisa; and (c) immunohistochemistry for p-and e-selectins on lung vessels. results: compared to the control animals, diabetic rats exhibited reduced number of neutrophils ( %) and mononuclear cells ( %); reduced levels of tnf-alpha ( %) and il- beta ( %), and reduced p-selectin expression ( %) in response to ova challenge. these abnormalities were corrected after treatment of diabetic rats with a single dose of nph insulin ( iu, s.c.), h before ova challenge. although we did not find differences in e-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin. conclusions: data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of tnf-alpha and il- beta production and selectin expression. supported by fapesp and pronex. hormonally active vitamin d derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.we examined whether vitamin d also interferes with the pro-inflammatory action of the keratinocytes themselves. human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to tnf to simulate an inflammatory challenge and their response was monitored by assessing mrna levels of the cytokine tnf, the chemokine il- and the adhesion molecule icam- by real-time pcr. icam and il- were induced rapidly peaking after h, their mrna levels increased again from h to reach a plateau between h to h after exposure to tnf, whereas tnf mrna levels increased steadily between h and h. h pretreatment with calcitriol, the hormonal form of vitamin d, inhibited induction of il- but did not affect that of icam- or tnf h following exposure while calcitriol markedly inhibited the induction of all pro-inflammatory genes h after the tnf challenge.calcitriol inhibits the activation of jun kinase (jnk) and p by tnf. this action was mimicked by the posters inflamm. res., supplement ( ) s jnk inhibitor sp and the p inhibitor sb .the combination of the two inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. we conclude that vitamin d attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the jnk and p cascades. ( ), cm lotufo ( ), p borelli ( ), zs ferreira ( ), rp markus ( ), shp farsky ( ) ( ) department of clinical and toxicological analyses, school of pharmaceutical sciences, university of s¼o paulo, brazil ( ) department of physiology, bioscience institute,university of s¼o paulo, braziil introduction: we showed that endogenous glucocorticoids (eg) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of l-selectin on segmented cells. aims: we evaluated the role of eg on endothelial cells (ec) and the molecular mechanisms responsible for hormonal actions in neutrophils and ec. methods: neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and ec were cultured from testis vessels. cells were obtained from adrenalectomized (adx), ru -treated, shamoperated, vehicle-treated and non-manipulated (nm) wistar rats. results: circulating neutrophils and segmented cells from ru -treated rats presented elevated and decreased expressions of l-selectin vs cells from control animals, respectively. the effects were not dependent on alterations of l-selectin mrna levels. ec from adx animals presented more ability to adhere neutrophils from nm rats and enhanced mrna levels and membrane expressions of icam- , vcam- and pecam- . participation of the glucocorticoid cytosolic receptor(gcr) on these effects was shown by similar results in cells from ru treated rats. nfkappab translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in ec from adx or ru -treated rats. conclusions: data show the participation of the gcr on events involved in neutrophil mobilizations, but nfkappab transcription is only involved on ec. ( ), y naito( ), t okuda ( ), k mizushima ( ), t okayama ( ), i hirata ( ), h tsuboi ( ), t suzuki ( ), o handa ( ) background: despite the inhalation of co at high concentrations had been considered as a toxic gas, the inhalation of co at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. however, it is unclear whether the direct exposure of co to the intestinal inflamed mucosa is effective or not. in this study, we investigated the therapeutic efficacy of the rectal co administration for rat colitis model. acute colitis was induced with trinitrobenzene sulfonic acid (tnbs) in male wistar rats. co( ppm- ml) was intrarectally administrated twice a day after the induction of colitis. rats were sacrificed at days after the administration of tnbs. the distal colon was removed, and the ulcer lesions were measured. thiobarbituric acid (tba)-reactive substances and tissueassociated myeloperoxidase (mpo) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. moreover, we evaluated the expressions of cinc- mrna/protein and p-p mapk protein. the intracolonic administration of co ameliorated tnbs-induced colonic ulceration. the increases in tba-reactive substances and mpo activity after tnbs administration were both significantly inhibited by treatment with co. moreover, the rectal administration of co significantly inhibited the increased expression of cinc- mrna/protein and p-p mapk protein after the induction of tnbs-induced colitis. the rectal administration of co protected from the intestinal inflammation in rats. based on these data, the beneficial effects of co on the intestinal mucosal injury may be attributed to its anti-inflammatory properties. alessandra gambero ( ), m maróstica ( ), m saad( ), j pedrazzoli jr ( ) ( ) s¼o francisco university medical school, brazil ( ) state university of campinas, brazil recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines. the adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. in this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic tnbs instillations were evaluated. the adipocyte size was measured after collagenase digestion. the basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. the colitis animals showed higher mesenteric fat mass ( . +- . and . +- . % of body weight for colitis and control, respectively; p< . ) in despite of the lower body weight. the mesenteric adipocytes from colitis animals presented reduced diameter ( . +- . and . +- . um for colitis and control, respectively; p< . ), higher basal lipolysis ( . +- . and . +- . ug.mg- for colitis and control, respectively; p< . ) and tnf-alpha production ( . +- . and . +- . ng.mg- for colitis and control, respectively; p< . ). perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis ( . +- . and . . +- . ug.mg- for colitis and control, respectively; p< . ), increased tnfalpha ( . +- . and . +- . ng.ml- for colitis and control, respectively; p< . ), leptin ( . +- . and . +- . pg.ml- for colitis and control, respectively; p< . ) and adiponectin production ( +- and +- ng.ml- for colitis and control, respectively; p< . ). the mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one. this work was financially supported by fapesp. inflammatory bowel disease (ibd) is a group of chronic inflammatory disorders of the intestine. the role of the pro-inflammatory p mapk signalling cascade in the pathogenesis of ibd is highly controversial. we therefore aimed to investigate the role of p mapk in chronic dextran sodium sulfate (dss) induced colitis, an experimental model of ibd. chronic intestinal inflammation was induced by oral cyclic administration of % dss in sjl mice. clinically, the dss treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. at the molecular level, this was accompanied by an up-regulation of tnfa, il- â, il- , il- , kc, cox- , igg heavy chain, and phospho-stat in the dss treated mice.the clinical and molecular features described above recapitulate findings reported in human ibd. in order to assess the role of p mapk, the activation of the p mapk signalling cascade was analysed by western blot analysis. the expression and phosphorylation levels of both p mapk and of mk and hsp , two down-stream targets, were not increased in dss treated animals compared to controls. leo , a potent inhibitor of p activity in vivo, was dosed as pretreatment and after completion of dss treatment. pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. collectively, these results strongly suggest that the p kinase pathway only plays a minor role, if any, in the dss model. (sp) were gmcsf differentiated, dcs purified through gr + cell depletion, and spleen tcells isolated by pan tcell negative selection. spdcs or bmdcs were stimulated +/- ng/ml lps. for mlr, balb/c tcells were added for days. cells were incubated with sb ( -( -fluorophenyl)- -( -ethylsulfinylphenyl)- -( -pyridyl) h-imidazole, sb) or ml ((rs)-{ -[ -( -fluorophenyl)- -methylsulfanyl- h-imidazol- -yl]pyridine- -yl}-( -phenylethyl)amine, ml) and washed prior to lps stimulation (bmdcs) or cell mixing (t cells). hthymidine incorporation was measured, cell viability by mtt assay, tnfa and il- production by elisa. mlr tcell proliferation inspdcs or bmdcs was inhibited by sb (ic spdc . mm, bmdc . mm) and ml (ic spdc . mm, bmdc . mm). preincubation with dcs had no effect, despite reduced lps stimulated il- and tnfa synthesis by sb (ic il- . mm, tnf . mm) and ml (ic il- . mm, tnf . mm). preliminary data shows that preincubation of t cells with sb and ml modifies the mlr response. p plays a role in the interaction of dcs and t cells in antigen recognition. however, pre-incubation of drugs with dcs was ineffective. the role of t cell p mapk in the mlr is under investigation. p inhibitors may possess disease modifying properties because of reduced tcell antigen reactivity to dc antigen presentation. ( ), s luik( ), s laufer( ), m seed ( ), v holan( ), s fiorucci ( ) ( ) synovo gmbh, tübingen, germany ( ) university of tübingen, germany in vivo anti-inflammatory activity of certain p kinase inhibitors is limited by bioavailability. however, it is possible that they may be useful in the therapy of ibd should it be possible to mediate there uptake in and around bowel lesions. we reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (wbc) associated with lesions. we prepared prodrugs of p inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically ( d post boost) or in a dss or tnbs model of ibd in the mouse. in both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at mmolkg- d- . we propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. the plasma levels of interleukins (il- , il- ) and adhesion molecules (e-selectin and icam- ) were measured in patients with necrotizing pancreatitis. the measurement was performed immediately after admission, at the , , and day. all patients were divided on two groups: first group compiled patients, in which dexamethasone ( mg/day during - days) was applied in the complex management of acute pancreatitis, and control group - patients that did not receive corticosteroids. all patients received the initial therapy. the increased levels of il- , il- , il- , icam- , and eselectin were noted in both groups of patients at the time of admission. the gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. its levels clear correlated with the severity of mods and spreading of necrotic processes confirmed by ct. starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. the incidence of contamination of necrotic foci had no difference in both groups of patients. the ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of nf-kappa b pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. the objective of this study was to examine whether t cell specific overexpression of the th transcription factor gata- can inhibit th /th cell mediated experimental mbsa arthritis. mbsa-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day . at day , t cell specific gata- tg mice did not show any difference in arthritis score compared to wild type mice. however, at day , wild type mice had developed severe joint inflammation having the maximum arthritis score. in contrast, gata- tg mice showed only mild joint inflammation, suggesting that t cell specific overexpression of gata- protects against development of severe joint inflammation. facs analysis revealed low levels of il- +/ifn-gammacells in wild type as well as in gata- tg mice at day . as expected, il- positive cells were higher in gata- tg mice compared with wild type mice. interestingly, at day , the percentage of il- +/ ifn-gamma-cells were markedly increased in wild type mice but not in gata- tg mice, suggesting prevention of th expansion under gata- overexpression in vivo. these data revealed that t cell specific overexpression of the th transcription factor gata- protects against progression of severe joint inflammation during mbsainduced arthritis. furthermore, il- +/ifn-gammacells play a critical role in the progression of joint inflammation in this model and gata- overexpression in t cells prevents expansion of the il- +/ifn-gamma-t cell subset. pingping jiang ( ), pt sangild( ), t thymann ( ), hh-y ngai ( ), w-h sit ( ), k-l chan ( ), jm-f wan ( ) ( necrotizing enterocolitis (nec) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. preterm delivery and enteral milk formula feeding are factors predisposing to nec. to understand the pathophysiology of nec, two-dimensional gel electrophoresis ( d page) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous nec occurring in response to days of parenteral feeding followed by day of enteral formula feeding. the intestinal proteomes of pigs with clinical symptoms of nec (n = ) were compared with corresponding pigs remained healthy (n = ). syproruby staining was used and differently expressed proteins were identified by maldi-tof ms or maldi-tof/tof ms. the proteins with significantly different expression between nec and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase and chain a, medium-chain acyl-coa dehydrogenase with -thiaoctanoyl-coa etc.), inflammation (peptide-binding protein (pbp ) and snail homolog ), signal transduction proteins (thyroid hormone binding protein precursor, park protein and chain b, structure of the rho family gtp-binding protein cdc in complex with the multifunctional regulator rhogdi etc.) and anti-oxidation (manganese-containing superoxide dismutase(sod)). these data underscore the significant impact of intestinal proteomics in unraveling nec pathophysiology and biomarker discovery. blood are used to monitor the progression of inflammation. the aim of our study were to investigate systemic markers of disease in a rat model of lps induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. animals were exposed to bacterial lipopolysacharide (e.coli :b ) by inhalation. the lungs were lavaged hours post provocation and the level of cell influx was determined. relevant mediators of acute pulmonary inflammation were analysed with standard elisa technology in bronchoalveolar lavage fluid and in blood. in addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. results showing the effects of lps challenge on local and systemic parameters will be presented and the possible link to lps responses in man discussed. pulmonary inflammation models are widely used in pharmacological research. however, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. also, results derived from bronchioalvelar (bal) fluid are subject to variability if the retrieval techniques are non standardized. here we describe a non-invasive standardized method for retrieval of bal fluid to be used in mice. we present the characterisation of these techniques using the inflammatory response to lps and propose that this non invasive method can be used to refine lps and other challenge models. the objectives were to evaluate the dynamic response after a single intra-tracheal administration of of mg ( ml/animal) of lps (p.aeruginosa) to c bl/ j mice. control animals received a single dose of sterile ml . % saline/animal. the mice were terminated , , , and h after instillation using a non invasive and operator independent lavage technique. results showing the effects of single lps challenge on bal parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. these techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from bal fluid retrieval. the human psoriasis xenograft scid mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. the model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. in order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient scid mice. transplanted mice were treated with daivonex /dovobet (calcipotriol) and bms (betamethasone). the results show a strong antipsoriatic efficacy after treatment with bms (epidermal thickness reduced by %). treatment with daivonex / dovobet also showed an anti psoriatic effect with a % reduction in epidermal thickness. serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. the observed effects are in concordance with clinical results of treatment of psoriasis. it is concluded that the model is useful for testing topical treatments. we have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (barja-fidalgo; inflamm res ( ): ) . here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (un-group) or % protein diet (c-group) during the first days of lactation. un rats showed lower number of blood pmn, though no difference in bone-marrow neutrophils number was observed. blood neutrophils from un-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed inos and spontaneously produced o -, no and tnf-alpha. in vivo treatment with lps, at non-lethal doses, significantly increases tnf-alpha and superoxide production by neutrophils, compared with controls. lps increased no production by neutrophils from both groups, inducing inos expression in control cells, but no further increase in inos expression in un rats. nucleare nf-kb is constitutively augmented in un rats. un animals presented a higher survival rate in a model of clp-induced severe sepsis. these results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. transgenic mice over-expressing vascular endothelial growth factor (vegf) in the epidermal basal layer under the human keratin (k ) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of t-cells in the dermis and epidermis and also increased dermal angiogenesis. the aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with t-cells in transgenic k /vegf mice. we induced a cutaneous inflammation in the ear skin by repeated topical treatments with -o-tetradecanoylphorbol- -acetate (tpa), in order to investigate the inflammatory response. the in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone- -valerate, was also investigated. the ear thickness was significantly increased in transgenic animals compared to wild type animals following tpa-induction. the epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. a marked infiltration with cd -positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. finally, topical treatment with betamethasone- -valerate significantly reduced the ear swelling and epidermal thickness. we conclude that over-expression of vegf in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. the model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. background and aims: the diabetes-prone bb (bbdp) rat spontaneously develops insulin-dependent diabetes resembling type diabetes (t d) in man. the bbdp rat is t-cell lymphopenic with a profound lack of regulatory t cells. the recent thymic emigrants in bbdp rapidly undergo apoptosis unless rescued from apoptosis by tcr stimulation. the increase in apoptosis is due to a frameshift mutation in gimap which causes a severe truncation of the protein. the mutation is the strongest genetic factor for rat t d. we aim to detect how gimap affects the lifespan of t cells. results: overexpression in c cells of both wt gimap and gimap with the bbdp mutation causes an increase in apoptosis -the latter with a very rapid onset. reduction of human gimap by rna-interference in jurkat cells did not affect the number of apoptotic cells. overexpression of human gimap causes apoptosis in jurkat cells and primary naïve t cells but not in activated t cells. finally, gimap -mrna is upregulated in in vitro activated human primary t-cells (detected by rt-pcr). conclusions: based on the phenotype of the bbdp, rat gimap was expected to be anti-apoptotic. however, we report here that overexpression of both mutated and wt gimap causes rapid death of the cells. this suggests that gimap is pro-apoptotic. the results with human wt gimap support this conclusion: recently, much focus has been on the cellular cd / cadpr signaling system during inflammatory processes. the cd /cadpr system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine il- . to our knowledge, the expression and function of the cd /cadpr signaling system in the human detrusor muscle have not been described. cd protein expression in cultured (explant technique) human detrusor smooth muscle cells (hdsmc) was demonstrated by western blot (wb) and confocal microscopy (cm). cytosolic free ca + concentration ([ca +] i) in hdsmc and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. wb and cm showed that hdsmc expressed cell surface cd which could be upregulated by il- ( ng/ml). in hdsmc briefly activated with il- ( ng/ml) cadpr induced a rapid, transient dose-dependent increase in [ca +]i. cyclic adpr-mediated ca + increase was greatly reduced in ca + free medium suggesting ca + entry as well as ca + release. cyclic adpr -elicited ca + increase was mimicked by -deaza-cadpr, and blocked by -bromo-cadpr, a cadpr antagonist, but not by nifedipine or verapamil. in the presence of il- , cadpr caused concentration-dependent relaxations of detrusor muscle. we report for the first time that ) hdsmc express cell surface cd , ) the expression and function of cd are augmented by il- , ) externally added cadpr elicited a rapid, il- -dependent, and -bromo-cadpr-inhibitable ca + mobilization, ) cadpr induces relaxation of human detrusor muscle. the study indicates a role of cd /cadpr in human urinary bladder inflammation. miao lin is a formulation of sen miao san and lingzhi that consists of cortex phellodendri, atractylodisa rhizoma, radix achyranthis bidentatae, and ganoderma lucidum. these ingredients are reported to have anti-inflammatory and analgesic effects. in this study, we have investigated the anti-arthritic property of miao lin in an animal model of arthritis induced by unilateral injection of freunds complete adjuvant (fca) into rat knees. contents of the miao lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for days before induction of arthritis and days after. extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. single unilateral injection of fca into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. intra-peritoneal injection of miao lin ( mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration ( mg/kg/day) suppressed oedema and hyperaemia. histological examination showed that both routes of administrations of miao lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered miao lin also attenuated synovial tissue proliferation. these findings suggest treatment with intra-peritoneal or oral miao lin could provide significant anti-arthritic effects. an extract of the anti-arthritic thermalife cream contains trace elements. diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. non-penetrating trace elements were discarded from the test formula (t ), and compared with the original formula (t ) for in vitro anti-inflammatory efficacy (tnf-a secretion in lps-challenged human monocytes). methods: human epidermis was mounted in vertical franz type diffusion cells (stratum corneum facing up). t cream (n= ) or no cream (n= ) was applied to the donor compartment of diffusion cells, with pbs in the receptor compartment ( . ml ; stirred continuously at c). min after administration the receptor fluid was analysed for presence of metal ions by icp-ms. a replication study used a different skin donor. subsequently, human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ,) or not in the presence of % t , % t , or no treatment. hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). statistical analyses used a treat by lps anova (p < . ). results: zinc was the only trace element to penetrate the human epidermis significantly. both formulations strongly suppressed lps-induced tnf-a secretion. t with zinc only was more effective than t (treat:f , = . , p < . ; lps:f , = . , p < . ; treat by lps:f , = . , p < . ). conclusions: anti-tnf efficacy from thermalife extracts was retained with zinc chloride as the only trace element. ( ) ( ) osprey pharmaceuticals limited, canada ( ) probetex, inc., texas, usa the ccl /ccr chemokine/receptor axis, infiltrating monocytes/macrophages (m/m), th cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. the eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. a recombinant fusion protein comprised of the human ccl chemokine fused to a truncated form of the enzymatically active a domain of shiga toxin has been developed. the ccl portion binds specifically to ccr -bearing leukocytes and enters the cells, where the sa portion inhibits protein synthesis. the compound was tested in a model of anti-thymocyte serum (ats)-induced mesangioproliferative glomerulonephritis. male rats were injected with ats on day and treated intravenously with vehicle, or mg/kg of the recombinant protein q d from day until day . urine and blood collections were made prior to ats injection and on days and . animals were sacrificed on day . no treatment related effects on body weight or signs of clinical toxicity were observed. urine protein levels were decreased in treated animals. histopathological analyses of kidney sections revealed maximum reductions of , , , and % for glomerular lesions, m/m count, fibronectin and µ-smooth muscle actin, respectively. the latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. these results indicate a significant renal-protective effect in this model of nephritis. further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. inflamm. res., supplement ( ) posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cytokines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. coronary artery disease (cad) is characterized by enrichment of inflammatory cells in the vessel wall. we hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. in vivo transmigration was studied by use of a skin blister model. blisters are raised by suction and cells are analysed the following morning ( h blister) and after additional ten hours of incubation with pbs or autologous serum, corresponding to intermediate and intense blister. monocytes were analysed by flow cytometry for the expression of cd b, before and after in vitro fmlp stimulation. chemokines in serum and blister fluid was analysed in parallel. cd b expression on resting monocytes harvested from h blister was lower in patients as compared to controls (p= . ). lower expression of cd b in patients was also observed in the intermediate and intense blisters after stimulation with fmlp (p= . and p= . , respectively). the number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. serum concentration of mip- µ was higher among patients (p= . ) and similar levels were seen in blister fluids. concentration of mcp- was similar in both serum and blister fluid. we demonstrate that monocytes from patients with cad have a reduced expression and ability to up-regulate the adhesion molecule cd b at sites of inflammation. these differences may modulate events related to the transmigration process and indicate a changed activation pattern. to which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. in inflammation, nitric oxide (no) is produced by inducible nitric oxide synthase (inos) induced by bacterial products and cytokines, and no acts as a regulatory and proinflammatory mediator. one of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of no production. the aim of the present study was to investigate the mechanisms how glucocorticoids inhibit inos expression and no production. dexamethasone and a dissociated glucocorticoid ru inhibited no production, and inos protein and mrna expression in murine j macrophages exposed to bacterial lipopolysaccharide (lps). in the presence of a glucocorticoid receptor (gr) antagonist mifepristone, the effects of dexamethasone and ru on no production were reduced. the role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (hdacs); non-selective trichostatin a and apicidin, and hdac selective mc . hdac inhibitors reversed the effects of dexamethasone and ru on inos expression or no production. stably transfected a / cells containing human inos promoter were used in promoter-activity studies. cytokine-induced inos promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin a. these results suggest that glucocorticoids inhibit inos expression and no production by a gr-mediated and gre-independent manner possibly through histone deacetylation and transcriptional silencing. we are investigating mechanisms involved in tnfainduced hyperalgesia in the mouse paw. previously, we have seen that tnfa causes a trpv -dependent bilateral hyperalgesia. here we investigate the role of cox in this process. female cd mice ( - g) were given intraplantar injections (i.pl.) of tnfa ( pmol/ microl) and tyrode (as vehicle, contralateral paw; microl). thermal hyperalgesic thresholds were measured using the hargreaves technique before and h after injection. indomethacin ( mg/kg) was co-injected with tnfa whilst contralateral paw was injected with tyrode and corresponding amounts of indomethacin vehicle ( % nahco ). another group of mice were injected with tnfa i.pl. plus % nahco with the contralateral paw injected with tyrode plus indomethacin ( mg/kg). results are expressed as mean ae s.e.m and statistical analysis performed using students t-test. tnfa ( pmol) leads to significantly reduced (p< . compared to baseline values) paw withdrawal latency in both paws h after injection i.e bilateral hyperalgesia. however, local injection of indomethacin ( mg/kg) with tnfa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. interestingly, indomethacin co-injected with tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. the same results were seen using the selective cox- inhibitor, nimesulide. in conclusion, cox- derived prostaglandins are important in the development of hyperalgesia. local cox- inhibition at the site of tnfa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. hydrogen sulfide (h s) is synthesized naturally in the body from cysteine by cystathionine g lyase (cse). h s has been variously reported to exhibit both pro-and antiinflammatory activity. in an attempt to obtain further information about the role of h s in inflammation we examined the effect of dexamethasone on lipopolysaccharide (lps)-mediated endotoxic shock. male sprague dawley rats ( - g) were administered dexamathasone ( mg/kg, i.p.) either h before or h after lps ( mg/kg, i.p.) injection. animals were killed h after lps administration and plasma and tissues harvested. as expected, lps injection significantly increased plasma tnfa and il- b as well as liver and lung myeloperoxidase (mpo) activity. lps also increased plasma nitrate/ nitrite (nox), h s concentration and liver and kidney h s synthesis from exogenous cysteine indicative of upregulation of cse in these tissues. either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma h s and tissue h s synthesizing activity. in separate in vitro experiments, exposure of rat peripheral leucocytes to lps ( ng/ml, h, oc) resulted in upregulation of both cse and inos (measured by western blot). dexamethasone ( nm) significantly (p< . ) reduced expression of both cse and inos. these data provide further evidence that h s is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of h s production. ( ), u jalonen ( ), h kankaanranta ( ), r tuominen( ), e moilanen ( ) ( ) the immunopharmacology research group, medical school, university of tampere and tampere university hospital, tampere, finland ( ) the division of pharmacology and toxicology, faculty of pharmacy, university of helsinki, helsinki, finland tristetraprolin (ttp), also known as nup , tis , g s and zfp , is a factor that binds to utr of mrna of some transiently expressed inflammatory genes and regulates mrna stability. ttp has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. however, the regulation of the expression of ttp itself is largely unknown. in the present study, we investigated the role of classical protein kinase c (cpkc) isoenzymes in the regulation of ttp expression. in j macrophages ttp expression is induced by lipopolysaccharide (lps) and this can be further enhanced by addition of nm phorbol myristate acetate (pma). this additive effect of pma on ttp was abolished by a prolonged preincubation with a higher s inflamm. res., supplement ( ) posters concentration of pma for h, which also downregulated the expression of pkca, pkcbi and pkcbii isoenzymes. pkc inhibitors ro (inhibits pkcb, & and e), gÖ (inhibits pkca, b and &) and cgp (inhibits pkcbii) reduced lps + pma -induced ttp protein and ttp mrna expression. pkcbii inhibitor cgp did not affect ttp mrna half-life and therefore we measured the effects of cgp on the activation of transcription factors involved in ttp expression. cgp had no effect on the activation of nf-kb, egr or sp . in contrast, cgp reduced the activation of transcription factor ap- , which may explain its inhibitory action on ttp expression. the results suggest that pkcbii is involved in the regulation of ttp expression in activated macrophages, possibly through the activation of transcription factor ap- . the most widespread gracilaria verrucosa in the sea of korea is the attached form of red algae growing on a rockly substrate. in this study, we isolated fourteen compounds from g. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (tnf-a il- , il- and no) in raw . cells. among them, -oxooctadec- -enoic acid and -oxooctadec- -enoic acid inhibited the production of tnf-a, il- , il- and no at the concentration of mg. also, these two compounds showed inhibitory activity on the mrna expression and protein level of inflammatory markers (tnf-a il- , il- and inos) in a dose-dependent manner. these results suggest that g. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and inos. lene jensen( ), p hjarnaa ( ), j fensholdt ( ), p-p elena( ), k abell ( ), tk petersen ( ) ( ) discovery, leo pharma, ballerup, denmark ( ) iris pharma, la gaude, france angiogenesis is known to play an important role in many inflammatory diseases including arthritis. additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (amd). we have synthesized a potent angiogenesis inhibitor, leo-a, targeting kinases related to angiogenesis, e.g. vegfr- . additionally, leo-a has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. the compound was tested systemically in inflammatory in vivo models in mice and rats. the in vivo models selected include the cia arthritis model (mice and rats), the local gvh rat model, the lps induced tnfa model (mice and rats), the anti-cd induced il- response mouse model and the rat argon laser-induced choroidal neovascularisation (chnv) model, a model for amd. the following results were obtained after systemic treatment with doses of up to mg/kg i.p. or mg/kg p.o. once daily: in the local gvh model, leo-a significantly inhibited the growth of the local lymph node by %. in the cia model, leo-a had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). in the lps induced tnfa model in mice, high doses of leo-a were found to inhibit the tnfa release. in the chnv model, a significant effect was obtained following systemic treatment. in conclusion, leo-a has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved. mice lacking pi kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. we show here that mice harbor pi kg gene depletion and pi kd kinase-inactive mutation, pik cgd koi, exhibited thymus atrophy, similar to previously reported pi kg and d double knockout (p g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory t cell activity. in particular, serum igg / igg a ratio and ige level were elevated in pik cgd koi mice corresponding to a skewed th profile in vitro. histological analysis revealed eosionophil-and t celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik cgd koi mice, but organ-specific auto-antibody was not detected in circulation. on the contrary, when mature wt t cells were treated with pi k d or together with pi k g selective inhibitors, while th cytokines were suppressed th cytokines were not augmented in vitro. thus, t cell development, but not peripheral t cell proliferation or cytokine production, requires cooperativity of pi kg and d. genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type ig and t cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote th reaction but attenuate th medicated disorders. platelet activating factor (paf) is an important mediator in several pathophysiological processes. paf receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. paf can up-regulate kinin b receptor expression by various mechanisms. our aim was to investigate the role for kinases in paf-induced kinin b receptor up-regulation. wistar rats were treated with paf, or left untreated as controls, h before i.d. injection of . ml pbs containing des-arg -bradykinin (dapk, nmol right hind paw) and . ml pbs (for control, left paw). various kinase inhibitors were administered to the rats after paf treatment and oedema was measured by the use of a plethysmometer (ugo basile) - minutes after dapk-injection. oedema was expressed in ml as difference between right and left paws.additionally paw samples were taken for western blot analysis for total and phosphorylated forms of jnk and erk / . dabk-induced paw oedema after pafinjection is significantly inhibited by the selective jnk sp and erk / pd inhibitors. western blot analysis shows that phosphorylation of jnk and erk / is important in the up-regulation of b receptors. our results clearly show that the phosphorylation of both erk / and jnk mapkinases is an important step for the in vivo up-regulation of b receptors by paf. however, the exact mechanisms (transcriptional and post-transcriptional) by which paf can trigger kinase phosphorylation and then up-regulate the b receptor require further investigation. continued interest in development of small molecule inhibitors of p mitogen-activated protein (map) kinase is based on the central role this enzyme plays in inflammatory cell signaling. activation of p leads to increase production of pro-inflammatory cytokines such as tnf-a and il- b making it an prominent target for antiinflammatory drug discovery. a virtuell screening approach identified -( -chlorophenyl)- -(( -methoxyphenoxy)methyl)- [ , , ] triazolo [ , -b] [ , , ] thi adiazole as a potential hit. this was confirmed by synthesis and testing. to explore further sar, a first set of derivates was prepared by cyclization of the -substituted- -amino- -mercapto- h- , , -triazoles with carboxylic acids in presence of phosphorus oxychloride. the synthetic strategy used allows both variation at position and . synthesis and sar will be presented. cytokines like il- b and tnfa play central roles in inflammatory diseases like rheumatoid arthritis. production of cytokines in monocytes, macrophages and other cells is triggered by factors such as lps, uv-light, osmotic and cellular stress or physical and chemical attraction. in particular il- b and tnfa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. involved in this signal pathway, p mapk as a pivotal enzyme is considered to be a validated drug target and therefore, p mapkinhibitors are of therapeutical interest. in this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p mapk-inhibitors with promising in vitro activity. the assay procedure involves defined blood cell stimulation by lps and isolation of tnfa or il- b, which are subsequently quantified by tmb-elisa technique via photometric measurement. the validation of the assay conditions involved well characterized p mapk inhibitor sb and a highly active compound developed in our lab. a data set was generated by determining whole blood samples consisting of in each case three male and female individuals on three different days. statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. p mitogen-activated protein (map) kinase is required for the biosynthesis and release of pro-inflammatory cytokines il- and tnf a. inhibition of p map kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. trisubstituted pyridinylimidazoles are potent inhibitors of the p map kinase. scope of this work was to investigate -thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. we synthesised -alkylsulfanyl, -( -fluorophenyl), -( -aminopyridin- -yl) substituted imidazoles as p map kinase inhibitors. as substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. the synthesis and biological testing of effective the -aminopyridin- -yl imidazoles with low inhibitory concentrations are described. biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.variation at the -thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone nh group of met , for inhibitory activity. less clear is the importance of the hydrogen bond between n of the imidazole ring and lys of p map kinase.to investigate the role of lys in interacting with the scaffold we prepared two sets of , diaryl-substituted isoxazoles. these data suggest a dynamic interaction of the core heterocycle with lys , contrary to the observation on the compound vk- and p map kinase, that a nitrogen atom bearing a lone pair in position of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of lys rather than to form an attractive interaction with p map kinase. to complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of -substituted and unsubstituted , -diarylisoxazoles investigating the interaction with the hydrophobic pocket ii of p . these data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. it is a consequence of acute inflammatory response to lipopolysaccharide (lps), a major component of the outer membrane of gram-negative bacteria. natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. in this study, we investigated the effect of ferulaldehyde (fa), a natural compound of red wine, on lps-induced endotoxic shock in mice and on lps-stimulated murine macrophage-like raw . cells. treatment of c bl/ mice with fa significantly attenuated the lps-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines tnf-a and il- b in the serum. the serum level of the anti-inflammatory cytokine il- was higher in mice treated with fa and lps compared to lps treatment alone. lps-induced phosphorylation and thereby activation of akt, and jnk was also strongly inhibited by fa treatment whereas the phosphorylation level of erk / and p mapks remained unaltered. activation of nuclear factor-kappab (nf-kb) in liver of fa-treated mice were significantly suppressed. although fa had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in raw . cells either, it decreased the lps-induced ros and nitrite production in a dose-dependent manner. our results suggest that fa has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress nf-kb activity via the down-regulation of akt and jnk. myeloperoxidase (mpo), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. on the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. the formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. we investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (maldi tof ms). specific reaction products play an important role in the modulation of the immune response. comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. objectives: to study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (ra). methods: twenty patients with active ra received leflunomide mg for days followed by mg daily for weeks. at week all patients started infliximab mg/kg, and received a further four infusions at weeks , , and . results: the commonest adverse event was pruritis associated with an eczematous rash. there was no relationship between the serum concentration of a , the active metabolite of leflunomide, and adverse events. the mean disease activity score (das ) fell from . at week to . (p< . ) at week and remained between . and . up to week . in those patients remaining on treatment, more than % achieved an acr response from week to week , and up to % achieved an acr response. conclusions: infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult ra. however, widespread use may be limited by adverse events, which were common and in some cases severe. objective: the transcription factor nuclear factor-kb (nfkb) regulates the expression of proinflammatory cytokines such as tnfa and il- those play pivotal roles in pathogenesis in rheumatoid arthritis. parthenolide, a sesquiterpene lactone, was reported to inhibit the dna-binding of nfkb. the objective of this study is to investigate the potential of parathenolid to inhibit the pathogenesis of collagen-induced arthritis. methods: mice were injected i.p. with a cocktail of anticollagentype ii mabs on day , followed by i.p. injection of lps on day to induce anti-collagen mab-induced arthritis. the mice were orally administrated with parathenolide ( mg/kg/day) starting on the day of first immunization (day ) in prophylactic treatment group and after the onset of arthritis (day ) in the therapeutic treatment group. clinical disease score, radiographic and histological scores were evaluated. mrna expression of il- b and tnfa in the affected joints were measured by real-time pcr. results: clinical disease scores were significantly reduced both in prophylactic treatment group ( . ae . ) and therapeutic treatment group ( . ae . ) compared to untreated group ( . ae . , p = . and p = . respectively). histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p< . ). steady state mrna levels of il- b and tnfa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p< . ). the results in this study suggest that nfkb is an important therapeutic molecular target for treatment of inflammatory arthritis. fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. the concentration of this glycoprotein increase in inflammatory conditions. a fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. the objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine ( ìm) to detect oxygen free radical production. the cells ( , x cell/ml) were then incubated with a range of concentrations of fibrinogen ( - mg/dl) for minutes. our results show that posters inflamm. res., supplement ( ) s fibrinogen leads to an increase in neutrophil activation as measured by free radical production. this effect becomes evident at borderline-high concentrations ( - mg/ dl), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. we hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. cyclooxygenases (cox- and cox- ) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. the constitutively expressed isoform cox- is mainly involved in homeostatic processes, while the inducible isoform (cox- ) is associated with inflammatory reactions. various in vitro assays have been developed in order to define the selectivity against cox- and cox- of nonsteroidal anti-inflammatory drugs (nsaids). however, these in vitro assays can give discordant results related to several parameters. the aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new nsaid candidates. first, in an enzymatic cox assay, the arachidonic acid concentration (aa; cox substrate), and the species of cox enzymes tested (ovine vs. human), two factors able to conceal the anti-cox activity of nsaids, have been evaluated and optimized. next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. this cell-based assay allows concomitant measurement of anti-cox- and anti-cox- effects by prostaglandin e (pge ) measurement after a (calcimycin) and bacterial lipopolysaccharide (lps) stimulations, respectively. both assays have been calibrated and compared by testing reference nsaids, selective or not for cox- or cox- . fifty % inhibitory concentration (ic ) values against cox- and cox- and cox- :cox- ratios obtained were in accordance with the previously described nsaid specificity and coherent between both assays (r= . ). in conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new nsaid candidates against human cox- and cox- . for increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. the human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. we aimed to study the translational aspects of these models. material and methods: in humans, epidermal skin chambers were stimulated with autologous serum for hours. in rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day . the inflammatory response was measured after , and hours. the cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated. results: at / hours the cellular distribution was similar in air pouch and skin chambers. the major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. both in human and rats the concentrations of mpo and mcp- were increased. furthermore, transmigrated cells displayed a different chemokine receptor pattern. in rats transmigrated cells expressed cd b, were cd lo, ssclo and rp- + (granulocyte marker). in humans, transmigrated granulocytes expressed cd and cd b. these cells had a significantly higher cd b expression compared to corresponding cells in peripheral circulation. our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. these models may be useful for bridgingpreclinical and clinical drug discovery. furthermore, they may work as translatable proof of mechanism (pom) models for drug candidates targeting different inflammatory components. objectives: to analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke. material and methods: we investigated consecutive patients with mean age ae . years who were admitted within h after ischemic stroke. a control group of patients with mean age ae . years without ischemic stroke was also tested. measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay. results: patients with acute ischemic stroke had significantly higher serum levels (mean value+sd) of neopterin than those without acute ischemic stroke: . ae . and ae . nmol/l. correlation analysis revealed p< . . discussion: immune mechanisms contribute to cerebral ischemic injury. the finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin. our results indicate increased macrophage activation after ischemic stroke. in patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. however, these preliminary results need be confirmed by controlled studies. produced a marked (p< . ) reduction in the number and duration of ventricular tachycardia (vt) during both ischemic and reperfusion phases. the total number of ischemic ventricular ectopic beats (vebs) reduced from ffae in the control to ffae at the concentration of ffmg/ml (p< . ). in the ischemic phase, cynodon dactylon ( ffmg/ml) also decreased the incidence of vt from % (control) to %. in addition, incidences of reperfusion-induced vt, total vf and reversible vf duration were significantly lowered by the same concentration (p< . for all). the results show that cynodon dactylon has a protective effect against i/r-induced cardiac arrhythmias in isolated rat hearts. regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of cynodon dactylon probably is due to its anti-inflammatory properties.key words: cynodon dactylon; arrhythmias; anti-inflammatory; isolated heart; rat objectives: intestinal ischemia-reperfusion (iri) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. we thus decided to investigate the effects of iri on nos and cox isoforms, neutrophil infiltration (mpo), lipoperoxidation (tbars) and protein tyrosine nitration (nt) in different brain areas and diaphragm muscle of wistar rats. methods: iri comprised the occlusion of superior mesenteric artery during min followed by h of reperfusion. sham animals were submitted to the surgical procedure with no interference on the blood flow. results: iri resulted in increased expression of mrna for nnos (cortex) and cox- (hypothalamus) associated to a marked reduction of ca +-dependent nos activity in cortex, hypothalamus and hippocampus (but not in cerebellum). tbars contents were also reduced in cortex and hypothalamus. neither mpo activity nor nt was altered by iri in the brain. diaphragms from animals with iri exhibited increased mpo and ca +-dependent nos activities, as well as tbars content and nt. in contrast, enos protein expression and both gene and protein nnos expression were reduced. no effects were observed on cox isoforms or enos gene expression. conclusions: these findings suggest that, within the first h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. in the cns, distinctive susceptibility to the iri seems to occur in the different areas, probably as a defensive strategy aimed to counteract the iri-mediated systemic injury. anne-sofie johansson ( ), h qui ( ), m wang ( ), i vedin ( ), jz haeggstrçm ( ), j palmblad ( ) ( ( ), r carnuccio( ), p romagnoli ( ), f rossi ( ) ( ) second university of naples, italy ( ) university of naples, italy ( ) university of florence, italy we previously found that several inflammatory markers, e.g., nuclear factor-kb (nf-kb), were increased and a neointima was formed in a model of carotid surgical injury ( ) . the purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. to this aim we measured cox- , nf-kb, platelet aggregation and neointima formation in rats administered rosiglitazone ( mg/kg/ die, by gavage) for days before carotid injury and days after injury. control rats received physiological solution. days after injury cox- expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p< . ). rosiglitazone also caused a significant decrease of nf-kb/dna binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. platelet aggregation was reduced by % in treated versus control carotids (p< . ). the influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting days after rosiglitazone treatment. the results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. the aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (eae). rats of eae-susceptible dark agouti and eae-resistant albino oxford strain were immunized with guinea pig spinal cord homogenate (dagpsc and aogpsc), while non-immunized rats served as controls (danim, aonim). on day after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. macrophages from aonim rats exhibited lower adherence capacity and higher phagocytosis and h o production then macrophages from danim rats. immunization decreased adherence and phagocytosis and increased h o production in macrophages from ao rats, but did not influence these activities in macrophages from da rats. our results suggest that inflammatory activities of macrophages from ao rats could be considered as regulatory mechanisms connected with the resistance to eae induction ( ( ), b sehnert ( ), h lanig( ), s päßler( ), r holmdahl ( ), h burkhardt ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander university of erlangen-nuremberg, germany ( ) lund university, sweden objectives: the aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine igg mab ciic that is highly somatically mutated and its epitope on type ii collagen (cii, aa - ). methods: the establishment of a dynamic simulation modelling of a ciic single-chain fragment (scfv) in complex with the triple helical ciic epitope permitted structural insights into immune complex formation. the computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the c scfvs and the respective ciic epitope that were produced as recombinant constructs. the binding affinities of the mutated scfvs were determined by elisa and surface plasmon resonance measurements. the mutation experiments confirmed the predicted interaction sites of cii in the cdr and cdr regions of both heavy andlight chain. surprinsingly also the model prediction, that the conversion of the c scfv sequence into the respective germline does not affect cii binding affinity (kd x - ) could be confirmed experimentally by the mutagenesis of (!) positions. our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. the molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding cdrregions of the antibody considerably compared to globular antigens. these sterical constraints might provide an explanation why somatic mutations have no obvious impact on cii recognition by the arthritogenic autoantibody. moreover, the structural insights into cii-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. we observed a significant association between the mbp-elicited cd + t-cell proliferation and active brain lesions, on the one hand, and il- , il- and ifn-gamma, on the other. when grown in the presence of standard serum from a healthy donor, pbmc from healthy individuals responded to mbp with a higher il- production than pbmc from ms patients. thus, normal pbmc respond to mbp with production of tnf-alpha, ifn-gamma and il- , but ms is associated with enhanced tnf-alpha-, ifn-gammaand decreased il- responses, and disease activity is associated with mbp-induced proliferation of cd + t cells. ( ), k goula ( ), p georgakopoulos ( ) ( ) renal unit, st. anrdew hospital, patras, greece ( ) intensive care unit, st. anrdew hospital, patras, greece background: urethritis is an infection of the urethra. most cases are sexually transmitted. haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. patients with underlying diabetes are also a specific population at risk. urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (e. coli or klebsiella). viral causes of urethritis include herpes simplex virus and cytomegalovirus. neisseria gonorrhoeae and c trachomatis account for most cases of urethritis in men ( %). the aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. we also determined diabetes as a coexisting factor in the infected patients. we retrospectively reviewed all cases of urethritis of maintenance haemodialysis patients at our center over the past years. the diagnosis was made according to patients symptoms and signs but also using urine specimens for culture. patients ( . %) from the study group were diabetic. results: cases of urethritis were determined. all infected patients were diabetic. isolated microorganisms were e. coli ( cases), enterobacter aerogenes ( case objectives: to explore the ability to use paquinimod as a steroid sparing drug in an animal model for sle. methods: mice were initially treated with a high dose of prednisolone ( mg/kg/day). thereafter the amount of steroid was reduced to . mg/kg/day and a low dose of paquinimod ( . mg/kg/day) was added. the development of glomerulonephritis was measured as hematuria during the experimental period. serum was collected for analysis of anti-dsdna antibodies. kidneys were collected and histopathological observations were performed. organ weight and lymphocyte sub-populations were assessed in the spleen. results: when treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. a significant reduction in the level of hematuria, in spleen enlargement and in the total number of cd +, cd + and on cd -cd -t cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. the development of glomerulonephritis was also significantly reduced in these mice. an almost complete inhibition of anti-dsdna in serum was seen in all treated groups. conclusions: when high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, t-cell sub-populations and development of glomerulonephritis were examined. this setting could be of great importance in future treatment of human sle in order to reduce the steroid dose needed in the treatment of this disease. and la(ssb). the purpose of this study was to screen for novel antibodies against cell surface antigens in primary sjs. proteins (mp) were isolated from cell membranes (hela cells), and were tested with sera from sjs patients or healthy blood donors individually in western blot (wb) at : . mp were separated on -d gels and tested in wb ( : ) to locate the appropriate spots for mass spectrometry (ms) analysis. paraformaldehyde fixed hela cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. antigens were isolated at around , , , kda ( total positive/ tested patients). the dominant antigen was at kda. large quantities of endogenous proteins were obtained and the membrane fraction was enriched. one of the main obstacles to further study possible surface antigens as m muscarinic receptor was overcome. proteins were separated on d-gels and tested in wb to locate the relative spots for ms. the correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. in conclusion, membrane or membrane-associated antigens were recognised by sera from sjs patients. one of them might correspond to m muscarinic receptor. this identification might help in developing a diagnostic assay for sjs. osamu handa, s kokura, k mizuahima, s akagiri, t takagi, y naito, n yoshida, t yoshikawa aim: various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. however the precise mechanism of cytotoxicity of these additives are not known. in this study, we investigated the effects of ultraviolet-b (uvb) exposure on additives-treated human normal skin keratinocytes (hacat). most popularly used additives in cosmetics such as methylparaben (mp), octandiol (od) and phenoxyethanol (pe) were used. hacat keratinocyte was cultured in mp-containing medium for h, exposed to uvb and further cultured for another h. subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with hoechst and propidium iodide or annexin-v. same experiments were done using od and pe respectively instead of mp under same condition. in addition, gene chip analysis was performed in each group. results: uvb exposure enhances cytotoxicity of these additives even at low concentration. gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group. these results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight. these kinases in the pathogenesis of psoriasis. recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase (msk ) was demonstrated in psoriatic epidermis. the purpose of this study was to investigate msk and the transcription factor camp-response-element-binding protein (creb) in psoriatic skin and in cultured normal human keratinocytes. keratome and punch biopsies were taken from patients with plaque-type psoriasis. normal human keratinocytes were cultured and stimulated by interleukin- â (il- ß) or anisomycin. some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. on human skin inflammation, matrix metalloproteinase- (mmp- ) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. in the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on mmp- activity and mmp- expression were examined. from the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant mmp- with the ic s of . - . ìm, while the flavones such as apigenin and wogonin showed weak inhibition. when the effects of flavonoids on mmp- induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin ( . - . ìm) strongly inhibited mmp- induction from tpa-treated human dermal fibroblasts, but naringenin (flavanone) did not. by gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, ap- , whereas naringenin did not. among mapks, quercetin inhibited the extracellular signal-regulated protein kinase (erk) and p mapk activation, and kaempferol inhibited the p mapk and c-jun n-terminal kinase (jnk) activation. on the contrary, the flavones and naringenin did not inhibit the activation of these three mapks. all these results indicate that the capacity of mmp- inhibition and mmp- down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. ( ) ( ) university of valencia, spain ( ) istituto di chimica biomolecolare cnr, napoli, italy avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. recently, its derivative avarol- -thiosalicylate (ta) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.it is interesting to note that avarol and ta inhibited nf-Þb activation in hacat keratinocytes. now, the effect of avarol and ta was investigated in the tpa-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. topical treatment with ta ( mg/ml) produced a % inhibition of oedema and a strong reduction of pge ( %), ltb ( %) and mpo activity ( %) in skin homogenates. the inhibitory effect of avarol at the same dose was % for oedema, % for pge , and % for ltb and mpo activity. histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to tpa treatment. besides, the reduction of cutaneous tnf-a by avarol and ta was also detected by immunohistochemical analysis. these compounds were also capable of suppressing nf-Þb nuclear translocation in mouse skin. in summary, our results suggest that inhibition of proinflammatory metabolites by ta and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. its mechanism of action is related to the inhibition of nf-Þb activation and can be mediated by the downregulation of intracellular signal-transduction ( ), ams silva( ), cmm santos( ), dcga pinto( ), jas cavaleiro( ), jlfc lima ( ) ( ) requimte, departamento de química-física, faculdade de farmµcia da universidade do porto, porto, portugal ( ) departamento de química, universidade de aveiro, aveiro, portugal -styrylchromones are a novel class of chromones, vinylogues of flavones ( -phenylchromones), which have recently been found in nature. several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. however, the anti-inflammatory potential of -styrylchromones has not been explored so far. thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic -styrylchromones by studding their influence on different systems that are related to the inflammatory process. the putative inhibitory effects of several -styrylchromones on the proinflammatory enzymes cyclooxygenase (cox- ), cyclooxygenase (cox- ) and -lipoxygenase ( -lox) was evaluated in vitro and compared with structurally related flavonoids. the capacity of the studied -styrylchromones to scavenge reactive oxygen (ros) and nitrogen species (rns) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. from the tested -styrylchromones, those having a catecholic bring where shown to be the most effective scavengers of ros and rns, being, in some cases, more active than flavonoids. no considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. the results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. the usefulness of -styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. the importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. we therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (pet) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. uptake of radiotracers f-fluorodeoxyglucose (fdg), -o-methyl- - f-fluoro-l-dopa (omfd), and f-labeled native/oxidized low density lipoproteins (nldl, oxldl) in single-or cocultivated human myeloid (monocytic) leukemia cell line thp- was compared with that by squamous cell carcinoma (fadu), mamma (mcf- ) and colorectal adenocarcinoma (ht ) cell lines (without or in the presence of specific inhibitiors). several thp- phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. differentiated thp- cells show, when compared with tumor cells, a comparable fdg accumulation, a considerably lower omfd uptake, and a significantly higher oxldl uptake. on the other hand, during differentiation and retrodifferentiation thp- cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. the observed differences in uptake of several radiotracers in vitro in-between thp- phenotypes and between thp- phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. genomic and full-length cdna sequences provide opportunities for understanding human gene expression. determination of the mrna start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. although the mrna start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. recently, we have developed a -end sage ( sage) that can be used to globally identify the transcriptional start sites and frequency of individual mrnas. a strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (hdac) inhibitor is currently in clinical trials. however, how epigenetic drugs mediate its effects is poorly understood. to assess the effects of epigenetic drugs, the gene expression by sage in colon cancer cell lines treated with epigenetically affecting agents, -aza- deoxycytidine, a potent inhibitor of genomic and promoter-specific dna methylation and trichostatin a, a hdac inhibitor was investigated. epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. colon cancer is one of the most frequently diagnosed cancers in western societies. interleukin- (il- ) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. il- is produced by many different cell types. the main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. a variety of studies have demonstrated that over expression of il- contributes to the pathogenesis of various inflammatory diseases as well as cancer. it has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce il- . serum levels of il- are elevated in patients with colorectal cancer, however serum levels of il- were found to be independent of il- mrna expression in tumor tissue. in this study we analyzed il- mrna expression by real-time pcr in sporadic colon cancer tissue as well as corresponding normal mucous tissue. il- mrna expression in tumor tissue was lower than in the corresponding normal mucous tissue (p= , ). there was no correlation betweenil- mrna expression and tumor grade or stage. thus we can conclude that il- produced at the tumor site is not involved in sporadic colon cancer progression. ( ), t aiamsa-ard ( ), v chinswangwatanakul ( ), ki techatraisak ( ), s chotewuttakorn ( ), a thaworn ( ) ( material and methods: huvec were cultured as standard techniques and grown to confluence until use. serum was obtained from cholangiocarcinoma patients and normal healthy subjects. huvec were treated with % of serum and incubated for hours. cells were analyzed by using [ h] thymidine and immunoblotting assay for cell proliferation and cox- /nos- protein expression, respectively. results: serum of cca patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. on the protein expression, cca serum significantly increased the expression of cox- but not nos- in hevec. however, the proliferate effect on endothelial cells by cca sera did not correlate with the expression of cox- . conclusions: this result suggested that some factors in serum of cancer patients could induce cox- protein expression in huvec. the increasing of cox- might be one of various factors involve in the proliferation process. aim: superoxide is responsible for the neutrophil-mediated tumoricidal activity. the aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of ok- @on neutrophil-derived superoxide production and tumor growth. methods: ah a rat hepatocellular carcinoma cells were implanted into the hind leg of male donryu rats. pmns were harvested from rat peritoneal cavity h after intraperitoneal injection of oyster glycogen. superoxide production were measured by the method of cladependent chemiluminescence, which has high sensitivity and specificity to superoxide. the counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after days of tumor inoculation. both pma and oz-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. the suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. the subcutaneous administration of ok- , a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression. our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. furthermore, the effect of ok- on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of ok- . ( ), m jokic ( ), v zjacic-rotkvic( ), s kapitanovic ( ) ( ) university hospital sestre milosrdnice, bucharest, romania ( ) division of molecular medicine rudjer boskovic institute, bucharest, romania introduction: il- is a pleiotropic cytokine mapped to chromosome p - . its promoter snp - g/c is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. we tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (gep-nets). patients and methods: dnas from patients diagnosed with gep-net and age and sex-matched volunteers were analyzed for - g/c snp of the il- gene. to analyze il - c/g polymorphism we used pcr -nlaiii rflp method. for statistical analysis Ä test and fishers exact test were used. the level of significance was . . results: there were no differences observed in the frequencies of the - high expression (gc and gg) genotypes between the patients and healthy volunteers (p= . ), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p= . ). - g/c genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (pets) than in those with functional pets (p= . ). conclusions: high expression genotypes of il- - snp are more frequent in non-functional pets and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. how mast cells influence tumor growth is not well understood. the neuroendocrine peptide, neurotensin (nt) is a potent secretagogue of mc that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its gpcr nt-type receptor (nts ). here we show that hmc- human mc express nt-precursor (pront) mrna and protein, and secrete immunoreactive nt when stimulated. rt-pcr on hmc- cell rna yielded a band with % sequence identity to pront and a band corresponding to the pront processing enzyme, pc a.immunocytochemistry on hmc- cells showed specific staining for pront. stimulation of hmc- cells with a + pma, pge , c / or mastoparan released immunoreactive nt.rt-pcr on hmc- cell rna yielded a band with % sequence identity to human nts . western blotting gave bands corresponding to unglycosylated ( kda) and glycosylated ( kda) nts .immunocytotochemistry on hmc- cells showed specific staining for nts . these finding have significance for the role of mast cells in tumor growth. ( ), j buddenkotte ( ), mp schçn( ), m steinhoff ( ) ( ) university hospital münster, germany ( ) university hospital würzburg, germany the proteinase-activated receptor par- has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. however, the role of par- in cutaneous cancerogenesis is still unknown. here we could show a protective role of par- in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. to this end, par- -deficient and wild-type mice were painted once with dmba ( , -dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester pma (phorbol myristate acetate ( -o-tetradecanoylphorbol- -acetate)) twice per week at the same sites. tumors started to appear after eight weeks. after weeks, par- -deficient mice showed a significantly increased number of skin tumors ( per animal on the average) in contrast to the wild-type (eight tumors per mouse). analysis of possible signal transduction pathways activated upon par- stimulation in hacat keratinocytes showed an involvement of extracellular signal regulated kinase / (erk / ) and profound epidermal growth factor receptor (egfr) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta (tgf-â ). thus, our results provide the first experimental evidence for a tumor-protective role of par- . ( ), ma arbós ( ), a fraga ( ), i de torres( ), j reventós ( ), j morote ( ) ( pathogenesis of benign prostatic hyperplasia (bph) and prostate cancer (pca) is still unresolved, although chronic inflammation may play a significant role in disease progression. prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. to better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. we analysed primary human fibroblast cultures from normal transition zone (tz), normal peripheral zone (pz), benign prostatic hyperplasia (bph), and pathologically confirmed prostate cancer (ca). cells of different origin displayed distinct mrna expression profiles for the core proteins of proteoglycans and both sdf /cxcr and mcp /ccr chemokine axis. when incubated with monocyte-conditioned medium all four cell types significantly changed sdf /cxcr and mcp /ccr expression in a fibroblast population dependent manner. monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. monocytes adhered rapidly to fibroblasts and preferentially to bph and pz cells. in addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. supported by the spanish urology society (madrid, spain). we have recently shown that paf-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with paf-like molecules present on the surface of these cells. removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. in the present study we investigated the impact of apoptotic cells or treatment with paf-r antagonist on ehrlich ascitic tumor (eat, ip) and melanoma b f (sc). paf-r antagonist, web ( mg/kg, ip) was given daily for days. we found that eat growth was significantly reduced by pretreatment with web , and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by web pretreatment. eat growth was accompanied by increased production of prostaglandin e , vegf and no which was reduced significantly by web treatment. in b f melanoma, web , alone or in association with an apoptosis inducer chemotherapeutic agent, dacarbazin (dcb, ug/kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. in association with dcb, web- reducedactive caspase- expression in the tumor andmarkedly increased the survival of tumor-bearing mice. the data obtained here show that during tumor growth, activation of paf-r by molecules present in the surface of apoptotic/necrotic cells, or by paf produced in the milieu, favors tumor growth and suggests that pafantagonists could be useful in tumor treatment, particularly when in association with chemotherapy. financial suport by fapesp and cnpq. ( ), mt quiles ( ), a figueras( ), r mangues( ), f vinals( ), jr germa( ), g capella ( ) ( ) institut de recerca vall de hebron, barcelona, spain ( ) translational research laboratory, idibell -institut cataladoncologia, spain the malignant potential of tumor cells may be influenced by the molecular nature of k-ras mutations. we have previously shown that codon mutations associate with an increased resistance to apoptosis. we hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the cxcl (sdf- )/ cxcr chemokine axis. to do so we have combined in vitro and in vivo studies using stable cys and asp nih t transfectants. cys tumors showed a higher microvessel density associated with shorter latency period. prominent vessels with µ-smooth muscle actin positives cells surrounded by f / macrophages were only observed in asp tumors associated with a shorter growth period. asp tumors displayed increased vegf expression both at the rna and protein levels, mainly produced by tumor cells. tsp- protein levels were similarly diminished in both transfectants. higher mmp and mmp activities and expression were observed in asp tumors probably produced by macrophages or stromal cells. total and active mmp levels were higher in cys tumors. the expression of sdf- and cxcr remained unchanged while sdf- g isoform was selectively induced in cys tumors, suggesting sdf- a or b are induced in asp tumors. these results show distinct k-ras mutations induce specific angiogenic phenotypes. the differential stimulation of vegf expression, metalloprotease activities and sdf- expression observed is the result of the joint action of tumor cells and the local microenvironment. contact information: dr maria a arbos via, institut de recerca vall de hebron, unitat de recerca biomedica, barcelona, spain e-mail: maarbos@ir.vhebron.net incisional hernias (ihs) represent a common complication of laparotomies, involving remarkable healthcare costs. representative ih animal models are lacking and characterization of human tissue resources is scant. this limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of ihs. here, we compared tissue specimens (carefully obtained > cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without ih hernia. the most prominent morphologic characteristics of ih tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ecm), and a paucity of fibroblasts. in ih muscles, inflammatory infiltrates were observed. other significant changes were: decreased collagen type i/iii ratio; differential proteoglycans mrna expression; enhanced metalloproteinases/ endogenous inhibitors ratio (mmps/timps); and upregulation of apoptosis effectors (caspase- and substrates; tnf-alpha; il- ). in vitro, hernia fibroblasts (ihfs) exhibited significantly higher ( -fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. moreover, ihfs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. based on these descriptive results in human tissues, a novel hypothesis emerges regarding ih formation. specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. overall, this may be causally involved in the mechanisms of ecm destruction yielding ih (supported by fis pi_ and gencat_agaur_ xt_ ). ( ), m spinola( ), c pignatiello( ), w cabrera ( ), og ribeiro ( ), n starobinas( ), t dragani ( ) ( ) butantan institute, sao paulo, brazil ( ) istituto nazionale tumori, milan, italy airmax and airmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. in a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, airmin mice developed a persistent subacute lung inflammation and a fold higher lung tumor multiplicity than airmax mice, which showed a transient lung inflammatory response. we have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant c bl/ and lung cancer susceptible a/j mouse strains. gene expression profile analysis of urethane-treated and untreated animals was performed using the applied biosystems mouse genome survey microarray containing , mouse transcripts. mrna expression of candidate differentially expressed genes was validated by quantitative real-time pcr and the over-represented biological themes were analyzed with the ease software. urethane treatment modulated the gene expression profile in all four lines. among the confirmed genes, vanin (vnn ) and major histocompatibility antigen e alpha (h -ea) resulted common to both mouse models. the most represented gene categories in air model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. mhc/antigen process and presentation and immunoresponse were the major themes in the inbred model. moreover, a gene cluster in chromosome ( . cm) was observed. the study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.it is well established that the vegf signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. however, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of vegf signalling only. we have developed a nanomolar inhibitor (compound a) of the receptor tyrosine kinase vegfr-r (kdr), which was subsequently shown to be a potent inhibitor of closely related kinases (vegfr- and - , pdgfr, kit, csf- r) but also unrelated soluble tyrosine kinases (src-familily of kinases and raf). compound a potently inhibits vegf stimulated endothelial cell proliferation but has no effect on non-ec proliferation, which is suggestive of a selective antiangiogenic potential. the unique kinase inhibitory profile of compound a combined with excellent oral bioavailability ( %) has translated into superior in vivo anti- inflamm. res., supplement ( ) posters tumour efficacy when compared to the relatively selective kdr inhibitor ptk . thus, treatment of nude mice implanted with either commercial atcc derived tumour cells (a and du- ) or low passage patient derived tumors (cxf ; colon cancer, rxf ; renal cancer) with compound a resulted in inhibition of tumour growth which was significantly better than for ptk treated mice. compound a is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. ( ), p bobrowski( ), m shukla ( ), t haqqi ( ) ( ) albany medical college, usa ( ) rainforest nutritionals, inc, usa ( ) case western reserve university school of medicine, usa background: the amazonian medicinal plant sangre de grado (croton palanostigma) has traditional applications for wound healing and inflammation. we sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants. methods: acute oral safety and toxicity was tested in rats according under oecd protocol # . proanthocyanidin oligomers were quantified by hplc and progrados antioxidant activity assessed by the orac, norac and horac assays. human cartilage explants, obtained from surgical specimens, were treated with il- b ( ng/ ml) to induce matrix degradation and glycosaminoglycans (gag) release. progrado ( - mg/ml) was tested for its ability to maintain optimal igf- transcription and translation in cartilage explants and cultured chondrocytes. results: progrado displayed no evidence of toxicity ( mg/kg po) leading to gsh safety rating of /unclassifiable. oligomeric proanthocyanidin content was high ( mg/kg) with the majority of oligomers > mers.progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and il- b induced glycosaminoglycan release from human cartilage explants. progrado prevented il- b induced suppression of igf- production from human cartilage explants as well as stimulating basal igf- production (p< . ). comparable changes in igf- gene expression were noted in cultured human chondrocytes. conclusions: progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, igf- . this suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. the solvent extracts from korean fermented soybean (chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. the activities of chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. in addition, the protective effects against h o -induced cytotoxicity and oxidative dna damage in the nih/ t fibroblasts line were examined. the extracts from chungkukjang and soy isoflavones inhibited the generation of , -diphenyl- picryl hydrazine (dpph) radicals, and had an inhibitory effect on ldl oxidation. the extracts from chungkukjang and soy isoflavones strongly inhibited h o -induced dna damage in the presence or absence of endonuclease iii and fpg. furthermore, they showed cytoprotective effects against h o , without cytotoxicity except for the hexane extract at high concentrations (> mg/ml). the ethanol and n-butanol extracts appeared to have most potent antioxidant activities. these in vitro results show that the extracts of chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative dna damage without having cytotoxic effect. moreover, the extracts of chungkukjang inhibited mda formation in the liver, dna damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in kbro -treated mice. these in vivo results were similar to those of in vitro experiments. therefore, chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress. (supported by bk project from korea research foundation). sirt is a histone deacetylase, involved in oxidative stress and aging. because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the sirt activity, comparing heart (h) and adipose (a) tissue of sedentary young (n ), sedentary old (n ) and trained old (n ) rats. the trained old rats performed a -weeks moderate training on treadmill. on h and a tissue of all rats sirt activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (mda) and protein-aldehyde adducts -hydroxynonenal ( -hne), mnsod, catalase and foxo a by western blot, and gadd a, cyclin d and foxo a mrna by rt-pcr. aging reduced sirt activity in h (p< . ) without effects in a, producing an increase of mda (h, p< . ; a, p< . ) and -hne (h, p< . ; a, p< . ), and a decrease of mn-sod (p< . ) and catalase (p< . ) expression in both h and a. aging did not affect foxo a protein expression in h, and foxo a mrna in a. exercise produced an increase in h foxo a protein expression (p< . ) and in a foxo a mrna, associated to higher mn-sod (h, p< . ; a, p< . ) and catalase (h, p< . ; a, p= . ) levels in both h and a of aged rats. in heart exercise-induced higher sirt activity bring on decrease in cyclin d and increase in gadd a mrna expression. in a we found a similar decrease in cyclin d , without changes in gadd a mrna expression. these findings suggest that exercise is able to increase sirt activity in aged rats. ( ), t horiguchi( ), k abe( ), h inoue( ), t noma ( ) ( ) institute of health biosciences, the university of tokushima graduate school, tokushima, japan ( ) minophagen pharmaceutical co. ltd, japan objectives: glycyrrhizin (gl) is a major component of glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. gl has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. however, the molecular mechanisms of the effects remain unclear. to analyze the molecular basis of gl signaling, we performed the microarray analysis using ccl -induced mouse hepatitis models. methods: eight-week-old icr male mice were treated intraperitoneally with f×l/ kg bw of ccl w/wo mg/ kg bw of gl. after hours and hours, livers and serum were collected and analyzed. for microarray analysis, the expression patterns of genes between hour-treated-livers (ccl or ccl and gl) and no treated-livers were compared. results: gl-treatment dramatically decreased the gpt activity in plasma at hours compared to that in ccl treated plasma. however, the levels of mrna expression of inflammatory genes such as phospholipase a , hsp , and procollagen were still very high in gl-treated liver. after hours, the mrna levels of them were significantly reduced in gl-treated mice compared to those of ccl -treated liver. then, we screened , genes by microarray and found that genes were up-regulated and genes were down regulated in ccl +gl compared to ccl treatment. interestingly, ros scavenger-related genes were significantly up-regulated in ccl + gl. detail analysis is currently ongoing. we found the unique relationship between gl activity and ros regulation. this finding suggests a novel way to treat inflammatory diseases including hepatitis. objectives: experimental autoimmune encephalomyelitis (eae) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the cns. it is widely employed as an animal model of human multiple sclerosis. interestingly, the number of studies relating these diseases with peripheral organs is limited. we thus investigated the consequences of eae on the degree of lipoperoxidation (tbars) and mpo activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder). university of waikato, hamilton, new zealand mitochondria play a fundamental role in the life and death of all eukaryotic cells. cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn ). this protein is increasingly being implicated to play a role in modulating cellular inflammation. we have developed an in vitro model cell system using thp- monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn expression and modulation of proinflammatory cytokine responses. we have found that the ability of cpn to modulate tnf-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our thp- cells. we also demonstrate that such modulation involves both erk / and p mapk pathways. the significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. ( ), b arnold( ), g opdenakker ( ) ( ) jagiellonian university, department of evolutionary immunobiology, krakow, poland ( ) german cancer research center, department of molecular immunology, heidelberg, germany ( ) rega institute for medical research, university of leuven, laboratory of immunobiology, leuven, belgium we showed that in mice genetically deprived of metalloproteinase (mmp- -/-) at least one compensatory mechanism operates as there are elevated levels of pge of cox- origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. also infiltration of peritoneal cavity by inflammatory neutrophils is changed in mmp- -/-mice as at hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. in contrary, at hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in mmp- -/-mice. for this numbers of apoptotic (annexin v) and necrotic ( -aad) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. the results revealed that both, numbers of apoptotic cells and levels of active caspase were significantly lowered in mmp- -/-mice while levels of caspase , and were significantly elevated in comparison to control animals. we conclude that an impairment of apoptosis is observed in mmp- -/mice during zymosan peritonitis and it is due to the decreased levels of active caspase . the increased activity of other examined caspases is most probably independent of apoptosis. ( ), h james ( ) the selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(nos) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. the aim of this study was to evaluate the activity of nos inhibitors in experimentally induced inflammation, pain and hyperalgesia. the effect on acute inflammation was studied in carrageenan-induced paw edema in rats. the effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. nos inhibitor ng-nitro-l-arginine methylester (l-name), and mg/kg produced a dose-dependent inhibition of paw edema ( % and % at h; % and % at h). a marked reduction in paw edema was observed with ng-monomethyl-l-arginine acetate (l-nmma), mg/kg( % at h; % at h). selective inducible nos(inos) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of mg/ kg( % at h; % at h) but not with a dose of mg/kg . the effects were comparable to nonselective cox inhibitor indomethacin mg and mg/kg ( % and % at h; % and % at h respectively) and selective cox- inhibitor rofecoxib, mg/kg ( % and % respectively). nos and inos inhibitors significantly increased the pain threshold latency in the tail-flick test. these inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. however, carrageenan-induced paw hyperalgesia was not inhibited. the results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both nosand inos inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. ( ), p hart( ), j edwards ( ), c quirk ( ) ( ) molecular pharmacology limited (usa), australian division, perth, western australia ( ) telethon institute for child health research, perth, western australia thermalife cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. a novel product, derived from thermalife, was assessed on its therapeutic potential in oxsoralen-uvb burns. as a possible mechanism for the sunburn efficacy, suppression of tnf-a and il- â production by human monocytes was assessed in vitro. methods: sunburn: four sites were marked on the arm of the subject. three sites were exposed to oxsoralen ( %) plus uva/uvb light, one site was exposed to oxsoralen only. cream was applied at min, or at hrs after injury. a third injury site was not treated. photographs were taken before, hrs, and weeks after injury. cytokines: human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of % or % active ingredient. hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine elisa). results: at hrs after oxsoralen-uv, the min treatment site showed slight erythema, the hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. weeks after injury, the min site was normal, the hr site was a dark colour, whereas the untreated site had a significant scar. oxsoralen alone had no effect on the skin. the novel product suppressed lps-induced tnf-a and il â secretion by . % and . %, respectively. conclusions: a novel thermalife-derived product reduced total injury after oxsoralen enhanced uva/ uvb burns, which is possibly related to cytokine suppression. ( ), p hart( ), j snowden ( ), maud eijkenboom ( ) ( ) molecular pharmacology limited (usa), australian division,perth, western australia ( ) telethon institute for child health research, perth, western australia a mixture of bovine plasma protein fractions and zinc chloride (bov-zn) was assessed for its ability to regulate cytokine production by lps-stimulated monocytes. dosereponse curves for tnf-a suppression were generated. further, competition with fcs in the culture medium and the metabolism of monocytes under influence of bov-zn were assessed. in all experiments the culture medium environment was similar. human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of %, . %, %, . %, %, % or % bov-zn (two pooled experiments). hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). a competitive inhibition design for the standard tnf-a assay was set up for %, %, %, % fcs against %, . %, %, % bov-zn. the culture media were treated as above. metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (promega celltiter ) in treated and untreated cell cultures over - hrs at intervals. the ic for tnf-a suppression was reached at . % bov-zn in each of two experiments. fcs did not compete with bov-zn in suppressing tnf-a in lpsstimulated monocytes. at low fcs concentrations bov-zn stimulated tnf-a production in the absence of lps. this tnf-a increase was countered with increasing concentrations of fcs. metabolism of cells was not affected by % bov-zn. conclusions: bov-zn could reliably and effectively reduce tnf-a secretion in vitro, without competing with the fcs in the culture medium, and without disturbing the metabolism of monocytes. inflammatory diseases such as rheumatoid arthritis (ra) result from overproduction of cytokines including tnf-£\ and il- fÒ. these cytokines are known to be regulated by the stress-activated p fnfnmap kinase pathway. because of this, inhibition of p map kinase has been one of the most compelling targets for the treatment of inflammatory disease. over the last years, numerous groups have reported on the development of p map kinase inhibitors. x-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. regions of the binding site are known to be unique to p vs other kinases, enabling the development of p selective molecules. inflamm. res., supplement ( ) posters anti-inflammatory activities. a series of labdane-type diterpenoids ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) with various patterns of substitution were tested for potential anti-inflammatory activity.of these compounds, and were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. these derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of and % were noted in the -o-tetradecanoyl-phorbol- -acetate (tpa)-induced ear oedema in mice. in addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. the diterpenoids also reduced the production of nitric oxide, prostaglandin e , and tumour necrosis factor-alpha in bacterial endotoxin-activated raw . macrophage cells with ic in the range - mm. inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase- , as determined by western-blot analysis and rt-pcr. since nuclear factor-kappab (nf-kb) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. our results indicate that both compounds interfere with the phosphorilation of ikbalpha and ikbß, resulting in inhibition of their degradation. in summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking nf-kb activation and provide potentially therapeutic perspectives in inflammatory conditions. the aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. to fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. a comparison with diclofenacum at a dosage of mg/kg and substance ffw- Ñ (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect ( %) at a dosage of mg/kg) was made. the results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. fepolen revealed the highest effect during first min and hour of inflammation that was higher then action of diclofenacum. these data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. during next hours therapeutic effects of fepolen and diclofenacum were at the same level. fepolen showed the same dynamics of anti-inflammatory action as ffw- Ñ that demonstrates a property to influence both routs of arachidonic acid metabolism. in summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. the aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. the gastrointestinal inflammatory condition crohns disease involves a th -response with increased levels of proinflammatory cytokines like tnfa and il , and mouse models of crohns disease show that the balance of il / is crucial for disease progression. we have used mouse bone marrow derived dendritic cells (bmdc) to model a proinflammatory crohns disease like condition in vitro with cocktail-induced bmdc secretion of il , il and tnfaf jthe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin d , which were able to suppress the cocktail induced il secretion. further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress tnbs induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. in summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development. p - inflammation and human incisional hernia pathophysiology maria antonia arbos via( ) eae was induced by immunization of female lewis rats with guinea-pig myelin basic protein (mbp) in complete freunds adjuvant (cfa) and the animals were studied at the stage iii of the disease (characterized by complete paralysis of the hind-limbs) compared to cfa rats, eae resulted in increased mpo activity (u/mg tissue) in kidney ( ae vs. ae ae ; p< . ), and higher tbars contents (nmol mda/mg tissue) in liver acknowledgements: capes, cnpq, fapesp. contact information: ms simone teixeira, university of s¼o paulo, department of pharmacology, campinas, brazil e-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption supported by bk project from korea research foundation) contact information: mr young hoon kim here we report on the development of the pge mimetic combination therapy (dp- ) that inhibits basal and lps/tlr induced tnf-?, il- ß, and mmp- , , , in human and murine synovial membranes and peripheral macrophages.in a murine model of chronic synovitis (dorsal skin air pouch), dp dramatically inhibited il- ß, tnf-?, mip , mcp- and il- expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.in a model of inflammatory arthritis (collagen induced arthritis, cia), dp markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.in addition, tnf-?, il- ß, mmp- and to a lesser extent mmp- expression/synthesis levels were strongly suppressed as judged by rt-pcr and elisa measurements we have developed two rias, one for functional blood levels of the above mentioned anti-tnf-alpha constructs, and one for anti-abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/remicade and etanercept/enbrel ; i shall present some of these data ( , anatomy-physiology, faculty of medicine, laval university, quebec, canada neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate ltb from the -lipoxygenase pathway, pge through the inducible cyclooxygenase (cox- ) pathway and cytokines/chemokines as tnf-alpha, il- beta, il- , mip- alpha, mip- beta, mip- alpha and mip- alpha. engagement of the adenosine a a receptor (a ar) blocks the in vitro synthesis of ltb while it potentiates the cox- pathway in fmlp-treated neutrophils. in addition, it selectively prevents the expression and release of tnfalpha, mip- alpha, mip- beta, mip- alpha and mip- alpha in toll-like receptor- -stimulated human neutrophils. little effect was observed on il- beta and il- . using the murine air pouch model of inflammation with a ar-knockout mice, we observed that the activation of a ar positively impacts the expression of cox- in vivo, with particular magnitude in inflammatory leukocytes. in mice lacking the a a receptor, neutrophils that migrated into the air pouch h following lps injection expressed higher mrna levels of tnf-alpha, mip- alpha and mip- beta than neutrophils from wild type mice. together, these results indicate that neutrophils are important mediators of adenosines protective effects. given the uncontrolled inflammatory phenotype observed in a ar-knockout mice and in view of the potent inhibitory actions of pge on inflammatory cells, an increased cox- expression and a prevented release of tnf-alpha, mip- alpha and mip- beta caused by a ar activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. sepsis induced by endotoxins including lipopolysaccharide (lps) is a big problem in clinical medicine. for a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis ( d-page) and maldi-tof to follow the changes of significant proteins in a murine macrophage cell line -raw . after challenged with lps (escherichia coli :b ) for , and hours. we identified proteins from approximately detected protein spots with either increased or decreased in relative abundance as a result of lps treatment. the proteins identified with increased expression are the retinoblastoma binding protein , capg protein, poly(rc) binding protein , isocitrate dehydrogenase (nad+) alpha, lactate dehydrogenase , a chain, guanine nucleotide binding protein (g protein), beta polypeptide like , triosephosphate isomerase and proteasome alpha subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein p , malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type and rho, gdp dissociation inhibitor (gdi) beta). many of these altered proteins have interesting functions in inflammation. with the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. the ubiquitous mitogen-activated protein (map) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. they are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. based on a virtual screening approach we identified -amino- -benzyl- -( -bromophenyl)- -methyl- , -dihydropyrano[ , c] pyrazole- -c arbonitrile as a potential novel lead structure as p map kinase inhibitors. a set of compounds were prepared starting from different substituted pyrazol- ( h)-ones via a base-catalyzed condensation with aldehydes and ch acids, such as malononitrile, to provide them for biological tests in a p enzyme assay. first structure-activity relationship confirm the value of this novel lead. this study was conducted to determine the physiological c-reactive protein (crp) and alpha -acid glycoprotein (aag) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. serum crp levels were measured by elisa and aag levels were measured in healthy beagles of various ages by tia, and then separately -in pregnant beagles -by srid. serum crp levels ranged from . to . ìg/ml in male, and from . to . ìg/ml in female dogs. no significant sex-related differences were observed in the values. further, there were no significant age-related differences either. serum crp levels increased during pregnancy and peaked at . - . ìg/ml or days after ovulation, demonstrating two characteristic features of crp levels change in pregnant dogs. serum aag levels ranged from to ìg/ml in male, and from to ìg/ml in female dogs, without any significant sex-or age-related variation. serum aag levels increased in all pregnant beagles and peaked in the middle of gestation at - , ìg/ml. despite a high value of , - , ìg/ml being observed for serum aag levels in pregnant beagles inoculated with staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of srid ( ìg/ml). no significant sex-/-age related differences were observed in serum both crp and aag levels and these levels increased during pregnancy. the results of aag levels in umbilical cords were below the detection levels suggest aag is not transported to the placenta. polymorphonuclear neutrophils (pmns) play a key role in the inflammatory response against infectious agents.however, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.in this study, we examined the effect of pbi- , a low molecular weight immunomodulatory molecule, on pmn activation by lps both in vitro and in vivo. we measured by elisa the production of tnf-a by human lps-activated pmn in the presence or absence of pbi- .the ability of pbi to modulate pmn activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.exudates from different groups of animals (controls and pbi- treated animals, n= ) were used to assess leukocyte infiltration and to measure by elisa tnf-µ, mcp- and pge production.in vitro, pbi- is able to significantly decrease by . % ae . % (p< . ), tnf-a production by human lpsactivated pmn.in vivo, pbi- significantly decreased the production of tnf-a ( . % ae . %; p< . ), mcp- ( . % ae . %; p< . ) and pge ( . % ae . %; p< . ) induced by lps injection.however, pbi- did not significantly inhibit leukocyte infiltration.these results show that pbi- is able to modulate pmn activation and inflammatory response and suggest potential use as anti-inflammatory agent. ( ), lj lowenstine,( ), aj norris( ), t spangler( ), lm woods ( ) ( ) zoological society of san diego, usa ( ) department of pathology, microbiology, and immunology, university of california, davis, usa this study investigated the role of a novel reovirus in a outbreak of necrotizing typhlocolitis in american crows in california. included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. two control groups (n= each) were selected for parasitology and em (group ), and gross and histopathology (group ). all outbreak cases and group controls tested negative for west nile virus by pcr.all outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.two cases had multifocal hepatic necrosis. negative contrast em revealed reovirus particles in % ( / ) of outbreak cases and in % ( / ) of controls. supplemental tests failed to suggest other concurrent or confounding etiologic agents.overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.there was a noteable absence of similar typhlocolitis in archived crows submitted to the vmth from - , suggesting an emerging corvid disease in california, which bears further investigation. mitogen-activated protein kinase (mapks) pathways play an important role in the signalling system activated by proinflammatory cytokines. among the most important cascades the activation of erk / by mek / is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. as , a selective mek inhibitor, demonstrated anti-inflammatory properties in reducing tnf-alpha production induced by lps injection (ic mg/kg). therefore, primary aim of the present study was to assess the therapeutic strength of the as in a mouse model of collagen-induced rheumatoid arthritis (cia) assessing the effect of the compound on structural changes related to the cartilage. cia is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. as treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for days (twice daily), by oral route at the doses of , and mg/kg. as at and mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. at histology, vehicle-treated animals showed severe inflammation and joint surface erosion. administration of as significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. in conclusion, the results obtained in this study clearly demonstrate that the selective blockade of mek could be considered as an innovative therapeutic approach to treat rheumatoid arthritis. experimental evidences have shown that the toxicity of ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ros) and carcinogenicity. neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. however, relatively little is known about the potential of nickel salts in activating human neutrophils.thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. the measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ros and reactive nitrogen species (rns). enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. the obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. in the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (o -.), hydrogen peroxide (h o ), hydroxyl radical (ho.) and perchloric acic (hocl). the observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. ( ), h spalteholz ( ), u reibetanz ( ), p salavei ( ), m fischlechner ( ), h-j glander( ), j arnhold ( ) ( ) university of leipzig, medical faculty, institute for medical physics and biophysics, germany ( ) university of leipzig, derpartment of dermatology, andrology training centre of the european academy of andrology, germany unintentional childlessness often caused by common reasons as inflammation affects - % of german couples. inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (pmn), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (ar) and apoptosis as well as changes in the lipid structure and reduced mobility. stimulated pmn release the strongly cationic heme protein myeloperoxidase (mpo), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (ps). a population of freshly prepared spermatozoa shows only a very small amount of cells with mpo binding ability as well as externalization of ps. the number of spermatozoa able to bind mpo raises considerably in samples containing predamaged cells or introducing the ar as could be observed with rhodamine b isothiocyanate (ritc)labelled mpo and antibody techniques by fluorescence microscopy as well as flowcytometry. the activation ofmpo with its substrate hydrogen peroxide (h o ) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (hocl) and enhanced markedly the number of annexin v positive and non-vital cells. components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of mpo. the coincidence of ps externalization and mpo binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. recent findings suggest a crucial role of proteinaseactivated receptor- (par ) in inflammation and innate immunity. par is the second member of a novel g protein-coupled receptor subfamily with seven putative trans-membrane domains. this subfamily is characterized by a unique mechanism of receptor activation. accessible serine proteases cleave the receptor to expose a new, previously cryptic, n-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. tryptase, trypsin, and bacterial serine proteases are capable of directly activating par . par is expressed by human neutrophils, however its functions on these cells remained unclear. the data of our present study indicate that par agonists enhance interferon gamma (ifna)-induced up-regulation of cell surface fca;ri, one of the key receptors involved in neutrophil phagocytic activity. moreover, par agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. additionally, there is a significant increase of par expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. together, our results indicate that par may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and fca-receptor expression. aim: to ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process. we determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal i/r could restore reparation and assessed the influence of inflammation in the process.results: i/r provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and pcr mrna of stathmin and pcna. the cytokine profile revealed the influence of the inflammatory environment on kidney repair. regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation ( hours). pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.conclusions: macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. ( ), k bendtzen ( ), f sellebjerg ( ), ch nielsen ( ) ( antibodies against myelin basic protein (mbp) are present in sera from patients with multiple sclerosis (ms), but the role of these antibodies is controversial. we collected sera from ms patients and healthy individuals and found that both groups contained igm anti-mbp antibodies, while ms sera contained small amounts of igg anti-mbp. however, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. addition of mbp to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (pbmc) resulted in a significant deposition of igm on cd + monocytes, indicating that formation of mbp/igm complexes had occurred. this deposition was strongly inhibited by addition of mm edta to the sera, indicating that it was complement dependent. the pbmc produced significant amounts of il- , tnf-alpha and ifn-gamma upon stimulation with mbp, and the extent of the cytokine production did not depend upon whether sera from ms patients or from healthy controls were present. however, disruption of the tertiary structure of mbp by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to mbp. we propose that natural igm autoantibodies may form complexes with mbp, facilitating the uptake of mbp by antigenpresenting cells (apc). since sera from ms patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. we currently investigate this possibility. loredana postiglione( ), g tarantino( ), a spanò( ), p ladogana ( ), fl perrone( ), s padula( ), a riccio ( ) ( ) federico ii university medical school of naples, department of molecular and cellular biology and pathology l.califano, naples, italy ( ) federico ii university medical school of naples, departmentof clinical and experimental medicine, naples, italybackground: hepatitis c virus (hcv) infection can induce immunological disorders with different clinical expression such asarthritis, sjogren sindrome and various form of vasculitis.aim: to study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-ccp) in a group of patients affected by hcv-related arthritis and the eventual correlations with rheumatoid factor (rf) and/orantinuclear antibodies (ana), and articular involvement. study design: patients with arthritis were selected in a population of subjects affected by hcv infection. each patients was evaluated by clinical examination ( denoted poliarticular and mono-oligoarticualr involvement), by x-graphic aspects of joint involvement ( patients presented join erosions), by ana, rf and anti-ccp positiveness.results: , % of patients presented positivenessfor anti-ccp, without significant correlation between suchparameter and ana, rf and articular involvement. anti ccp resulted positive in out of the patients with joint erosions, and only in out of the patients without joint erosions. such frequency analyzed by chi square ended up in no significant differences. our patients presented an interesting prevalence of the positiveness for anti-ccp. these data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. expression of nkg d on cd + t cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, crohns disease and an animal model of type diabetes. the monoclonal antibody cx recognizes murine nkg d and has been shown to block ligandbinding and mediate internalization of nkg d. furthermore, cx can inhibit and/or ameliorate disease in animal models of type diabetes and inflammatory bowel disease. thus, it is very likely that nkg d plays an important role in the development of inflammatory and autoimmune diseases. since little is known about the pharmacokinetics and pharmacodynamics of the cx antibody, we decided to study this in both regular balb/ c mice and immunodeficient cb .scid mice. different doses of cx antibody was injected intraperitoneally and pk and pd was measured by elisa (anti-cx elisa in serum) and flow cytometry (down-regulation of nkg d on cd b+ nk cells) for up to two weeks after administration. we found that cx very efficiently down-regulate nkg d on cd b+ nk cells and that the effects of the antibody can be seen for more than two weeks after one single injection. finally, we propose a model which may be helpful in predicting the effects of different doses of cx antibody in vivo. ( ), k mehta( ), n deo( ), j chaudhary( ), p bobrowski ( ) ( ) albany medical college, usa ( ) vedic lifesciences, usa ( ) rainforest nutritionals, inc, us background: the efficacy and safety of reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a mumbai-based multi-center, randomized, double-blind study.methods: subjects (n= ) were screened and randomized to receive glucosamine sulfate (n= , mg/day) or reparagen (n= , mg/day), a polyherbal consisting of vincaria (uncaria guianensis) and rni (lepidium meyenii) administered orally, twice daily. primary efficacy variables were womac scores, visual analog score (vas) for pain, and response to treatment defined as a % improvement in womac pain, with assessments at , , , and weeks. secondary variables were results: subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (p< . ), with a similar, progressive improvement over the course of the week treatment protocol ( - % reduction in total womac or vas scores). the response rate was substantial for both glucosamine ( %) and reparagen ( %), which exceeded placebo responses ( %, p < . ) and supported by investigator and subject assessments. tolerability was excellent and safety parameters were unchanged. rescue medication use was significantly lower in the reparagen group (p < . ), and serum igf- levels were unaltered.conclusions: both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. we speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. we investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. tested nutrient mixture (nm) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. systemic inflammation in mice challenged with bacterial lipopolysaccharide (lps) was monitored by blood plasma levels of fourteen key inflammatory cytokines. two week supplementation with mg nm/kg body weight prior to lps challenge provided significantly greater protection than did supplementation with ibuprofen. induction of interleukin- (il- ) and monocyte chemoattracting protein- , two cytokines especially responsive to lps challenge, was reduced in nmsupplemented animals by % and %, respectively. corresponding reduction in ibuprofen group was % and %. protective mechanisms involved were assessed in human cultured u macrophages stimulated with lps.the cytokines most responsive were tumor necrosis factor alpha ( % and % reduction by supplementation with nm and ibuprofen, respectively) and il- ( % and % in corresponding reduction). nm supplementation dramatically reduced prostaglandin e secretion by stimulated macrophages along with cyclooxigenase- (cox ) cellular protein expression. mrna levels forcox and inflammatory cytokines were also dramatically reduced. quercetin was the most effective nutrient when tested individually. however, nm appeared to surplus the combined effect of individual components. we conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. ( ), hp kim ( ), kh son ( ) ( ) college of pharmacy, kangwon national university, south korea ( ) department of food and nutrition, andong university, south korea chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (inos)-catalyzed no production in cell cultures. in the present study, to find the optimal chemical structures and to elucidate their action mechanisms, synthetic chalcones having the substituent(s) on a-and b-rings were prepared and their effects on inos-catalyzed no production were evaluated using lps-treated raw . cells. among the tested compounds, -methoxy- , -dichlorochalcone (ch ), -hydroxy- -methoxychalcone (ch ), -hydroxy- -bromo- -methoxychalcone (ch ) and -hydroxy- , -dimethoxychalcone (ch ) potently inhibited no production (ic s, . - . mm). the favorable chemical structures were found to be a methoxyl substitution in a-ring at adjacent position ( or ) to carbonyl moiety with/without -(or -)hydroxyl group and -halogen substitution in b-ring. when the cellular action mechanisms of ch , ch and ch were further examined, it was revealed that ch and ch clearly down-regulated inos expression while ch did not. moreover, ch and ch were proved to suppress the nuclear transcription factor-kb activation. from the results, it is suggested that certain chalcone derivatives potently inhibit inos-catalyzed no production by the different cellular mechanisms, inos down-regulation or inos inhibition, depending on their chemical structures. these chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. an oligomeric stilbene alpha-viniferin (avf) was isolated from root of carex humilis (cyperaceae) as an inhibitor of cyclooxygenase (cox)- activity by bioassayguided fractionation. the avf was later found to downregulate lipopolysaccharide (lps)-induced cox- expression as well as to inhibit nuclear factor (nf)-kb activation, in addition to its inhibitory effect on cox- activity. furthermore, the compound exhibited antiarthritic effect in vivo. avf is a trimer of resveratrol and contains benzofuran moieties in its central part. starting from benzofuran and its related chemicals, cyclohexylimino- -methyl- , -dihydro- h-benzo [ , ] oxathiol- -one (lyr- ) was discovered to inhibit lpsinduced nf-kb transcriptional activity in macrophages raw . . the lyr- reduced lps-induced dna binding activity and nuclear translocation of nf-kb as well as inhibited lps-induced degradation and phosphorylation of inhibitory kb (ikb) protein. these results suggest that lyr- could suppress lps signaling molecule, putatively ikb kinase (ikk) complex, upstream ikb degradation in nf-kb activating pathway. lyr- inhibited in vitro kinase activity, gst-ikb phosphorylation, of wild type ikkbeta or a constitutively active ikkbeta mutant (c/a, cys- to ala) but did not affect that of another constitutively active ikkbeta mutant (ss/ee, ser- and to glu). therefore, lyr- could inhibit lps-induced nf-kb activating pathway by targeting ser- and/or residues on the activation domain of ikkbeta. as pharmacological actions, lyr- prevented nf-kb-dependent expression of inducible nitric oxide synthase, cox- , or inflammatory cytokines at the transcription level in lps-stimulated macrophages raw . . furthermore, lyr- protected lpsinduced septic shock in vivo. faculty of medicine, institute of pharmacology, ljubljana, slovenia a part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. in mammals histamine is mainly degraded by two enzymes: histamine-n-methyltransferase (hnmt) and diamine oxidase (dao). the aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. their effects on enzyme activity and mrna expression were studied in guinea pig tissues. plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. specific enzymatic activities of dao and hnmt were determined by radiometric assay. in addition, guinea pigs were treated with saline or amitriptyline ( mg/kg, ip), afterwards dao and hnmt mrnas were detected by pcr in different tissues. results showed thatamitriptyline, nm, , and mm, increased guinea pig plasma dao activity by , , and %, respectively, while sertraline increased it at mm (by %). at higher concentrations ( and mm) sertraline decreased dao activity. in the guinea pig tissues hnmt activity changes were found only when incubated with amitriptyline; sertraline had no effect. at and nm amitriptyline the activity of hnmt increased by and %, respectively. in animals treated with amitriptyline an induction of dao and hnmt mrna expression was noticed in several tissues. our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. the effect might be the opposite with higher amitriptyline concentrations. steven hefeneider( , ), c macarthur ( ), d trune ( ), s mccoy ( ) ( ) oregon health and science university, portland, oregon, usa ( ) targeted gene delivery, inc., portland, oregon, usa engagement of toll-like receptors (tlrs) by bacterial components such as lps and dna initiates inflammation.the current study examines a novel anti-inflammatory peptide, termed p , for treatment of inflammation induced by either lps or bacteria.peptide p was derived from an immunoregulatory protein of vaccinia virus, and interferes with tlr signaling.in this study we examined the efficacy of p to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (aom).we demonstrate in the sepsis model, that in vivo treatment of mice with p inhibited lps-induced production of serum inflammatory mediators.moreover, p treatment, administered after initiation of inflammation, significantly increased survival of mice injected with lps.in the aom model, peptide p significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by h. influenza.assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide p .simultaneous injection of bacteria and peptide p resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.fluid accumulation within the eustachian tubes was also significantly reduced following p treatment.-subcutaneous and oral administrations of p , but not intravenous administration, were also efficacious in reducing inflammation. administration of p after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.taken together, these results demonstrate the therapeutic potential of peptide p to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. ( ), c zhou( ), y zhang( ), m sun( ), x wan ( ), h yu( ), x yang( ), rd ye ( ), j-k shen ( ) formyl peptide receptor-like (fprl ) is a structural homologue of fpr, which binds chemotactic peptides of as small as amino acids (e.g., fmet-leu-phe, fmlf) and activates potent bactericidal functions in neutrophils. in comparison, fprl ligands include peptides of - amino acids, such as trp-lys-tyr-met-val-[d]met (wkymvm) and other synthetic peptides. to determine the core peptide sequence required for fprl activation, we prepared various analogues based on wkymvm and evaluated their bioactivities in an fprl -transfected cell line. although substitution of d-met resulted in loss of activity, removal of val together with d-met produced a peptide that retained most of the bioactivities of the parent peptide. the resulting peptide, wkym, represents a core structure for an fprl ligand. further substitution of lys with nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both fprl and fpr. based on these structure-activity studies, we propose a model in which the modified tetrapeptide trp-nle-tyr-met (wnleym) binds to fprl through aromatic interactions involving the side chains of trp and tyr , hydrophobic interaction of nle , and the thio-based hydrogen bonding of met , with the respective residues in fprl which have not been identified. the identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for fprl -related disorders.there is a growing awareness of the interaction of food constituents with the immune system. the present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. ear swelling, as a measure for inflammation, as well as il- , tnf-a and il- levels in the ear and the number of tnf-a producing spleen cells were analyzed.-glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of tnf-a producing spleen cells).glycine effects ( , and mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses ( . and mg/mouse/ day) inhibited the inflammatory response significantly. surprisingly higher doses of lactoferrin ( and mg/ mouse/day) failed to influence the inflammatory reaction. a combination of both nutrients (lactoferrin . mg/ mouse/day in combination with glycine or mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. additionally an additive effect of both components was seen on the number of tnf-a producing spleen cells. the present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of tnf-a producing spleen cells. ( ), p sambrook( ), k fukudome( ), m xue ( ) ( ) university of sydney, st leonards, nsw, australia ( ) saga medical school, saga, japan objectives: to investigate the i) expression of endothelial protein c receptor (epcr) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (ra) and osteoarthritis (oa) and ii) role of epcr and its ligand, activated protein c (apc), on the function of monocytes from ra patients.methods: epcr, cd and pc/apc in synovial tissues were detected by immunostaining and in situ pcr. monocytes were isolated from peripheral blood of patients with ra and treated with apc, lipopolysaccharide (lps), and/or epcr blocking antibody, rcr . cells and supernatants were collected to analyze the expression/activation of epcr, nuclear factor nf-kb and tumour necrosis factor tnf-a.results: epcr was expressed by both oa and ra synovial tissues but was markedly increased in ra synovium. epcr was colocalized with pc/apc mostly on cd positive cells in synovium. in ra monocytes, apc upregulated epcr expression reduced monocyte chemoattractant protein- -induced chemotaxis of monocytes by approximately %. apc also completely suppressed lps-stimulated nf-kb activation and attenuated tnf-a protein by more than % in ra monocytes. the inhibitory effects of apc were reversed by rcr , indicating that epcr modulates the inhibitory effects of apc.conclusions: our results demonstrate for the first time that epcr is expressed by synovial tissues, particularly in ra, where it co-localizes with pc/apc on monocytes/ macrophages. in addition, apc inhibits the migration and activation of ra monocytes via epcr. these inhibitory effects on ra monocytes suggest that pc pathway may have a beneficial therapeutic effect in ra. key: cord- -r y j authors: hedegaard, c. j.; bendtzen, k.; nielsen, c. h. title: the role of immune complexes consisting of myelin basic protein (mbp), anti‐mbp antibodies and complement in promoting cd (+) t‐cell responses to mbp in health and multiple sclerosis date: - - journal: scand j immunol doi: . /j. - . . k.x sha: doc_id: cord_uid: r y j multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti‐mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen‐presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp‐derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease‐associated anti‐mbp antibodies in ms patients, respectively. we have investigated the formation of mbp‐containing ic, the binding of mbp to b cells, the mbp‐elicited induction of th‐cell and b‐cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c‐inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd (+) t cells, we observed the production of tnf‐α, ifn‐γ and il‐ by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il‐ , il‐ and il‐ was detected. we are currently investigating the capability of ms sera to promote the formation of mbp‐containing ic and thereby enhance the cytokine responses, by virtue of elevated anti‐mbp contents. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - d gf az authors: sønder, s. u. s.; hedegaard, c. j.; bendtzen, k. title: monitoring patients treated with type interferons: antiviral versus mxa induction assays date: - - journal: scand j immunol doi: . /j. - . . bb.x sha: doc_id: cord_uid: d gf az interferon‐α/β (ifn‐α/β) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large‐scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large‐scale screening in specialized laboratories. the read‐out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn‐α or ‐β, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- - at rhqk authors: cann, alan j. title: infection date: - - journal: principles of molecular virology doi: . /b - - - - . - sha: doc_id: cord_uid: at rhqk virus infection of higher organisms is the cumulative result of all the processes of replication and gene expression described in the previous chapters. together, these determine the overall course of each infection. infections range in complexity and duration from a very brief, superficial interaction between the virus and its host to infections that may span the entire life of the host organism, from before birth to its eventual death. a common misconception is that virus infection inevitably results in disease. in reality, the reverse is true—only a small minority of virus infections gives rise to any disease symptoms. this chapter provides an overview of the numerous patterns of virus infection and forms an introduction to the discussion of virus pathogenesis in chapter . unlike previous and subsequent chapters, this chapter deals primarily with the interaction of viruses with intact organisms rather than with the molecular biologist’s usual concern about the interaction between a virus and the cell. i explain how the immune responses to viruses enables the body to resist infection, and how viruses respond to this pressure. i describe and understand how virus infections are prevented and treated. life on earth depends on the primary productivity of plants-the production of organic molecules from inorganic molecules such as co -(with some an additional contribution from some bacteria). from the smallest single-celled alga in the ocean to the largest forest giant tree (and everything in between, such as broccoli), they are vitally important. photosynthetic algae in the oceans play a major role in controlling the atmosphere and the climate, and interaction with viruses is one of the major mechanisms which in turn control the algae. all higher animals depend on the primary productivity of plants for their food. so plants are a big deal, and anything which affects plant growth is of great importance. in purely economic terms, viruses are only of importance if it is likely that they will affect crops during their commercial lifetime, a likelihood that varies greatly between very short extremes in horticultural production and very long extremes in forestry. some estimates have put total worldwide cost of plant virus infections as high as us$ per year. by which plant viruses are transmitted between hosts is therefore of great importance. there are a number of routes by which plant viruses may be transmitted: i seeds: these may transmit virus infection either by external contamination of the seed with virus particles or by infection of the living tissues of the embryo. transmission by this route leads to early outbreaks of disease in new crops which are usually initially focal in distribution but may subsequently be transmitted to the remainder of the crop by other mechanisms. i vegetative propagation/grafting: these techniques are inexpensive and easy methods of plant propagation but provide the ideal opportunity for viruses to spread to new plants. i vectors: many different groups of living organisms can act as vectors and spread viruses from one plant to another: i bacteria (e.g., agrobacterium tumefaciens-the ti plasmid of this organism has been used experimentally to transmit virus genomes between plants) i fungi i nematodes i arthropods: insects (e.g., aphids, leafhoppers, planthoppers, beetles, thrips) i arachnids (e.g., mites) i mechanical: mechanical transmission of viruses is the most widely used method for experimental infection of plants and is usually achieved by rubbing virus-containing preparations into the leaves, which in most plant species are particularly susceptible to infection. however, this is also an important natural method of transmission. virus particles may contaminate soil for long periods and be transmitted to the leaves of new host plants as wind-blown dust or as rain-splashed mud. some of the most original experimental biology currently being done involves plant science. biologists can do experiments with plants that they can only dream of being able to perform with animals. and yet the idea persists among many that botany is boring. much of the most exciting plant science involves plant viruses, either as experimental tools or in terms of finding ways to prevent infection. and as this section describes, the biology of plant viruses has some striking differences from that of animal viruses. so if you think botany is boring, you probably need to find out more about plant viruses. the problems plant viruses face in initiating infections of host cells have already been described (chapter ), as has the fact that no known plant virus employs a specific cellular receptor of the types that animal and bacterial viruses use to attach to cells. transmission of plant viruses by insects is of particular agricultural importance. huge areas of monoculture and the inappropriate use of pesticides that kill natural predators can result in massive population booms of pest insects such as aphids. plant viruses rely on a mechanical breach of the integrity of a cell wall to directly introduce a virus particle into a cell. this is achieved either by the vector associated with transmission of the virus or simply by mechanical damage to cells. transfer by insect vectors is a particularly efficient means of virus transmission. in some instances, viruses are transmitted mechanically from one plant to the next by the vector and the insect is only a means of distribution, through flying or being carried on the wind for long distances (sometimes hundreds of miles). insects that bite or suck plant tissues are the ideal means of transmitting viruses to new hosts-a process known as nonpropagative transmission. however, in other cases (e.g., many plant rhabdoviruses), the virus may also infect and multiply in the tissues of the insect (propagative transmission) as well as those of host plants. in these cases, the vector serves as a means not only of distributing the virus but also of amplifying the infection. initially, most plant viruses multiply at the site of infection, giving rise to localized symptoms such as necrotic spots on the leaves. the virus may subsequently be distributed to all parts of the plant either by direct cell-to-cell spread or by the vascular system, resulting in a systemic infection involving the whole plant. however, the problem these viruses face in reinfection and recruitment of new cells is the same as the one they faced initially-how to cross the barrier of the plant cell wall. plant cell walls necessarily contain channels called "plasmodesmata" which allow plant cells to communicate with each other and to pass metabolites between them. however, these channels are too small to allow the passage of virus particles or genomic nucleic acids. many (if not most) plant viruses have evolved specialized movement proteins that modify the plasmodesmata. one of the best known examples of this is the -k protein of tobacco mosaic virus (tmv). this protein is expressed from a subgenomic mrna (figure . ), and its function is to modify plasmodesmata causing genomic rna coated with -k protein to be transported from the infected cell to neighboring cells (figure . ). other viruses, such as cowpea mosaic virus (cpmv; comoviridae) have a similar strategy but employ a different molecular mechanism. in cpmv, the -/ -k proteins form tubular structures allowing the passage of intact virus particles to pass from one cell to another (figure . ). typically, virus infections of plants might result in effects such as growth retardation, distortion, mosaic patterning on the leaves, yellowing, wilting, etc. these macroscopic symptoms result from: i necrosis of cells, caused by direct damage due to virus replication, i hypoplasia-localized retarded growth frequently leading to mosaicism (the appearance of thinner, yellow areas on the leaves), i hyperplasia-excessive cell division or the growth of abnormally large cells, resulting in the production of swollen or distorted areas of the plant. plants might be seen as sitting targets for virus infection-unlike animals, they cannot run away. however, plants exhibit a sophisticated range of responses to virus infections designed to minimize harmful effects. plants fight virus infections in a number of ways. first, they need to detect the infection, which they do by means of sensing virus signature molecules (so-called pathogen-associated molecular patterns or pamps, e.g., particular proteins) via dedicated receptors. when this happens, the production of resistance proteins that activate highly specific resistance mechanisms is triggered. in response, plant viruses attempt to evade these defense mechanisms by altering protein structures where possible and by producing proteins which bind to and hide small rnas which would trigger rna silencing. infection results in a "hypersensitive response," manifested as: i the synthesis of a range of new proteins, the pathogenesis-related ("pr") proteins, i an increase in the production of cell wall phenolic substances, i the release of active oxygen species, i the production of phytoalexins, i the accumulation of salicylic acid-amazingly, plants can even warn each other that viruses are coming by airborne signaling with volatile compounds such as methyl salicylate. the hypersensitive response involves synthesis of a wide range of different molecules. some of these pr proteins are proteases, which presumably destroy virus proteins, limiting the spread of the infection. there is some similarity here between the design of this response and the production of interferons (ifns) by animals. systemic resistance to virus infection is a naturally occurring phenomenon in some strains of plant. this is clearly a highly desirable characteristic that is prized by plant breeders, who try to spread this attribute to economically valuable crop strains. there are probably many different mechanisms involved in systemic resistance, but in general terms there is a tendency of these processes to increase local necrosis when substances such as proteases and peroxidases are produced by the plant to destroy the virus and to prevent its spread and subsequent systemic infection. an example of this is the tobacco n gene, which encodes a cytoplasmic protein with a nucleotide-binding site which interferes with the tmv replicase. when present in plants, this gene causes tmv to produce a localized, necrotic infection rather than the systemic mosaic symptoms normally seen. there are many different mechanisms involved in systemic resistance, but in general terms there is a tendency toward increased local necrosis as substances such as proteases and peroxidases are produced by the plant to destroy the virus and to prevent its spread and subsequent systemic disease. virus-resistant plants have been created by the production of transgenic plants expressing recombinant virus proteins or nucleic acids which interfere with virus replication without producing the pathogenic consequences of infection, for example: i virus coat proteins, which have a variety of complex effects, including inhibition of virus uncoating and interference of expression of the virus at the level of rna ("gene silencing" by "untranslatable" rnas), i intact or partial virus replicases which interfere with genome replication, i antisense rnas, i defective virus genomes, i satellite sequences (see chapter ), i catalytic rna sequences (ribozymes), i modified movement proteins. this is a very promising technology that offers the possibility of substantial increases in agricultural production without the use of expensive, toxic, and ecologically damaging chemicals (fertilizers, herbicides, or pesticides). in some countries, notably in europe, public resistance to genetically engineered plants has so far prevented the widespread adoption of new varieties produced by genetic manipulation without considering the environmental cost of not utilizing these new approaches to plant breeding. the most significant response to virus infection in vertebrates is activation of both the cellular and humoral parts of the immune system. a complete description of all the events involved in the immune response to the presence of foreign antigens is beyond the scope of this book, so you should refer to the books mentioned in the further reading at the end of this chapter to ensure that you are familiar with all the immune mechanisms (and jargon!) described below. a brief summary of some of the more important aspects is worth considering however, beginning with the humoral immune response, which results in the production of antibodies. the major impact of the humoral immune response is the eventual clearance of virus from the body. serum neutralization stops the spread of virus to uninfected cells and allows other defense mechanisms to mop up the infection. figure . shows a very simplified version of the mammalian humoral response to infection. virus infection induces at least three classes of antibody: immunoglobulin g (igg), igm, and iga. igm is a large, multivalent molecule that is most effective at cross-linking large targets (e.g., bacterial cell walls or flagella) but is probably less important in combating virus infections. in contrast, the production of iga is very important for initial protection from virus infection. secretory iga is produced at mucosal surfaces and results in "mucosal immunity," an important factor in preventing infection from occurring. induction of mucosal immunity depends to a large extent on the way in which antigens are presented to and recognized by the immune system. similar antigens incorporated into different vaccine delivery systems (see "prevention and therapy of virus infection") can lead to very different results in this respect, and mucosal immunity is such an important factor that similar vaccines may vary considerably in their efficacy. igg is probably the most important class of antibody for direct neutralization of virus particles in serum and other body fluids (into which it diffuses). direct virus neutralization by antibodies results from a number of mechanisms, including conformational changes in the virus capsid caused by antibody binding, or blocking of the function of the virus target molecule (e.g., receptor binding) by steric hindrance. a secondary consequence of antibody binding is phagocytosis of antibody-coated ("opsonized") target molecules by mononuclear cells or polymorphonuclear leukocytes. this results from the presence of the fc receptor on the surface of these cells, but as has already been noted in chapter , in some cases opsonization of virus by the binding of nonneutralizing antibodies can result in enhanced virus uptake. this has been shown to occur with rabies virus, and in the case of human immunodeficiency virus (hiv) may promote uptake of the virus by macrophages. nonphagocytic cells can also destroy antibody-coated viruses via an intracellular pathway involving the trim protein. antibody binding also leads to the activation of the complement cascade, which assists in the neutralization of virus particles. structural alteration of virus particles by complement binding can sometimes be visualized directly by electron microscopy. complement is particularly important early in virus infection when limited amounts of low-affinity antibody are made-complement enhances the action of these early responses to infection. despite all the above mechanisms, in overall terms cell-mediated immunity is probably more important than humoral immunity in the control of virus infections. this is demonstrated by the following observations: i congenital defects in cell-mediated immunity tend to result in predisposition to virus (and parasitic) infections, rather than to bacterial infections. i the functional defect in acquired immune deficiency syndrome (aids) is a reduction in the ratio of t-helper (cd ), which may have been present before the onset of aids but were previously suppressed by the intact immune system. cell-mediated immunity depends on three main effects ( figure . ). these all act via molecular mechanisms that will be explained later in this chapter (see "viruses and apoptosis," below): i nonspecific cell killing (mediated by "natural killer" [nk] cells), i specific cell killing (mediated by cytotoxic t-lymphocytes [ctls]), i antibody-dependent cellular cytotoxicity (adcc). nk cells carry out cell lysis independently of conventional immunological specificity, that is, they do not depend on clonal antigen recognition for their action. they are not major histocompatibility complex (mhc) restricted. in other works, nk cells are able to recognize virus-infected cells without being presented with a specific antigen by a macromolecular complex consisting of mhc antigens plus the t-cell receptor/cd complex. the advantage of this is that nk cells have broad specificity (many antigens rather than a single epitope) and are also active without the requirement for sensitizing antibodies. they are therefore the first line of defense against virus infection. nk cells are most active in the early stages of infection (i.e., in the first few days), and their activity is stimulated by ifn-α/β. nk cells are not directly induced by virus infection-they exist even in immunologically naive individuals and are "revealed" in the presence of ifn-α/β. they are thus part of the "innate" rather than the "adaptive" immune response. their function is complementary to and is later taken over by ctls which are part of the "adaptive" immune response. not all of the targets for nk cells on the surface of infected cells are known, but they are inhibited by mhc class i antigens (which are present on all nucleated cells), allowing recognition of "self" (i.e., uninfected cells) and preventing total destruction of the body. it is well known that some virus infections disturb normal cellular mhc-i expression and this is one mechanism by which nk cells recognize virus-infected cells. nk cell cytotoxicity is activated by ifn-α/β, directly linking nk cell activity to virus infection. unlike nk cells which may be either cd or cd , ctls are usually of cd (suppressor) phenotype, that is, they express cd molecules on their surface. ctls are the major cell-mediated immune response to virus infections and are mhc restricted-clones of cells recognize a specific antigen only when presented by mhc-i antigen on the target cell to the t-cell receptor/ cd complex on the surface of the ctl. (mhc-i antigens are expressed on all nucleated cells in the body; mhc class ii antigens are expressed only on the surface of the antigen-presenting cells of the immune system-t-cells, b-cells, and macrophages.) ctl activity requires "help" (i.e., cytokine production) from t-helper cells. the ctls themselves recognize foreign antigens through the t-cell receptor/cd complex, which "docks" with antigen presented by mhc-i on the surface of the target cell ( figure . ). the mechanism of cell killing by ctl is similar to that of nk cells (explained below). the induction of a ctl response also results in the release of many different cytokines from t-helper cells, some of which result in clonal proliferation of antigen-specific ctl and others that have direct antiviral effects-for example, ifns. the kinetics of the ctl response (peaking at about days after infection) is somewhat slower than the nk response (e.g., À days cf. . À days)-so nk cells and ctls are complementary systems. the induction of a ctl response is dependent on recognition of specific t-cell epitopes by the immune system. these are distinct from the b-cell epitopes recognized by the humoral arm of the immune system. t-cell epitopes are more highly conserved (less variable) than b-cell epitopes, which are more able to mutate quickly to escape immune pressure. these are important considerations in the design of antiviral vaccines. the specificity of cell killing by ctls is not absolute. although they are better "behaved" than nk cells, diffusion of perforin and local cytokine production frequently results in inflammation and bystander cell damage. this is a contributory cause of the pathology of many virus diseases (see chapter ), but the less attractive alternative is to allow virus replication to proceed unchecked. adcc is less well understood than either of the two mechanisms mentioned above. adcc can be carried out by nk cells or by ctls. the mechanism of cell killing is the same as that described in the next section, although complement may also be involved in adcc. the distinguishing feature of adcc is that this mechanism is dependent on the recognition of antigen on the surface of the target cell by means of antibody on the surface of the effector cell. the antibody involved is usually igg, which is bound to fc receptors on the surface of the t-cell. adcc therefore requires a preexisting antibody response and hence does not occur early during primary virus infections-it is part of the adaptive immune response. the overall contribution of adcc to the control of virus infections is not clear, although it is now believed that it plays a significant part in their control. apoptosis, or "programmed cell death," is a critical mechanism in tissue remodeling during development and in cell killing by the immune system. there are two ways in which a cell can die: necrosis or apoptosis. i necrosis is the normal response of cells to injury caused by toxins or environmental stress. necrosis is marked by nonspecific changes such as disruption of the plasma membrane and nuclear envelope, rupture of membrane-bounded organelles such as mitochondria and lysosomes, cell swelling, random fragmentation of dna/rna, influx of calcium ions into the cell, and loss of membrane electrical potential. the release of cellular components from the dying cell causes a localized inflammatory response by the cells of the immune system. this frequently leads to damage to adjacent cells/tissue-"bystander" cell damage. i apoptosis is, in contrast, a tightly regulated process that relies on complex molecular cascades for its control. it is marked by cell shrinkage, condensation, and clumping of chromatin, a regular pattern of dna fragmentation, and "bubbling off" of cellular contents into small membrane-bounded vesicles ("blebbing") which are subsequently phagocytosed by macrophages, preventing inflammation. when triggered by the appropriate signals, immune effector cells such as ctls and nk cells release previously manufactured lytic granules stored in their cytoplasm. these act on the target cell and induce apoptosis by two mechanisms: i release of cytotoxins such as: ( ) perforin (aka cytolysin), a peptide related to complement component c which, on release, polymerizes to form polyperforin, which forms transmembrane channels, resulting in permeability of the target cell membrane; and ( ) serine proteases related to trypsin. these two effectors act collaboratively, the membrane pores allowing the entry of granzymes into the target cell. the membrane channels also allow the release of intracellular calcium from the target cell, which also acts to trigger apoptotic pathways. i in addition, ctls (but not nk cells) express fas ligand on their surface which binds to fas on the surface of the target cell, triggering apoptosis. binding of fas ligand on the effector cell to fas (cd ) on the target cell results in activation of cellular proteases known as "caspases," which in turn trigger a cascade of events leading to apoptosis. the process of induction and repression of apoptosis during virus infection has received much attention during the last few years. it is now recognized that this is an important innate response to virus infection. the regulation of apoptosis is a complex issue that cannot be described fully here (see further reading and figure . for a summary), but virus infections disturb normal cellular biochemistry and frequently trigger an apoptotic response, for example: the pathways controlling apoptosis are very complex. this diagram represents only a simple summary of some of the mechanisms of major significance in virus infections. i pkr activation: the ifn effector pkr (rna-activated protein kinase may be activated by some viruses (e.g., hiv, reovirus). i p activation: viruses that interact with p (chapter ) may cause either growth arrest or apoptosis (e.g., adenoviruses, sv , papillomaviruses). i transcriptional disregulation: viruses that encode transcriptional regulatory proteins may trigger an apoptotic response (e.g., htlv tax). i foreign protein expression: overexpression of virus proteins at late stages of the replication cycle can also cause apoptosis by a variety of mechanisms. in response to this cellular alarm system, many if not most viruses have evolved mechanisms to counteract this effect and repress apoptosis: i bcl- homologues: a number of viruses encode bcl- (a negative regulator of apoptosis) homologues (e.g., adenovirus e b- k, human herpesvirus [hhv- ] ksbcl- ). i caspase inhibition: caspases are a family of cysteine proteases that are important inducers of apoptosis. inhibiting these enzymes is an effective way of preventing apoptosis (e.g., baculovirus p , serpins, viaps-"inhibitors of apoptosis"). i fas/tnf inhibition: viruses have evolved several mechanisms to block the effects of fas/tnf, including blocking signaling through the plasma membrane (e.g., adenovirus e ), tumor necrosis factor receptor (tnfr) mimics (e.g., poxvirus crma), mimics of death signaling factors (vflips), and interactions with signaling factors such as fas-associated death domain (fadd) and tnfr-associated death domain (tradd) (e.g., hhv- [ebv] lmp- ). i p inhibition: a number of viruses that interact with p have evolved proteins to counteract possible triggering of apoptosis (e.g., adenovirus e b- k and e , sv t-antigen, papillomavirus e ). i miscellaneous: many other apoptosis-avoidance mechanisms have been described in a wide variety of viruses. without such inhibitory mechanisms, most viruses would simply not be able to replicate due to the death of the host cell before the replication cycle was complete. however, there is evidence that at least some viruses use apoptosis to their benefit. positive-sense rna viruses such as poliovirus, hepatitis a virus, and sindbis virus with lytic replication cycles appear to be able to regulate apoptosis, initially repressing it to allow replication to take place, then inducing it to allow the release of virus particles from the cell. by the s, interference (i.e., the blocking of a virus infection by a competing virus) was a well-known phenomenon in virology. in some cases, the mechanism responsible is quite simple. for example, avian retroviruses are grouped into nine interference groups (a through i), based on their ability to infect various strains of chickens, pheasants, partridges, quail, etc., or cell lines derived from these species. in this case, the inability of particular viruses to infect the cells of some strains is due to the expression of the envelope glycoprotein of an endogenous provirus present in the cells which sequesters the cellular receptor needed by the exogenous virus for infection. in other cases, the mechanism of virus interference is less clear. in , alick issacs and jean lindenmann were studying this phenomenon and performed the following experiment. pieces of chick chorioallantoic membrane were exposed to ultraviolet (uv)-inactivated (noninfectious) influenza virus in tissue culture. the "conditioned" medium from these experiments (which did not contain infectious virus) was found to inhibit the infection of fresh pieces of chick chorioallantoic membrane by (infectious) influenza virus in separate cultures ( figure . ). their conclusion was that a soluble factor, which they called "interferon," was produced by cells as a result of virus infection and that this factor could prevent the infection of other cells. as a result of this provocative observation, ifn became the great hope for virology and was thought to be directly equivalent to the use of antibiotics to treat bacterial infections. the true situation has turned out to be far more complex than was first thought. ifns do have antiviral properties, but by and large their effects are exerted indirectly via their major function as cellular regulatory proteins. ifns are immensely potent; less than molecules per cell show evidence of antiviral activity. hence, following isaacs and lindenmann's initial discovery, many fairly fruitless years were spent trying to purify minute amounts of naturally produced ifn. a b figure . discovery of ifns. in , alick issacs and jean lindenmann discovered ifns by performing the following experiment. (a) pieces of chick chorioallantoic membrane were exposed to uv-inactivated (noninfectious) influenza virus in tissue culture. (b) the "conditioned" medium from these experiments (which did not contain infectious virus) was found to inhibit the infection of fresh pieces of chick chorioallantoic membrane by (infectious) influenza virus in separate cultures. they called inhibitory substance in the condition medium "interferon." this situation changed with the development of molecular biology and the cloning and expression of ifn genes, which has led to rapid advances in our understanding over the last years. there are a number of different types of ifns: i ifn-α: there are at least molecular species of ifn-α, all of which are closely related; some species differ by only one amino acid. they are synthesized predominantly by lymphocytes. the mature proteins contain amino acids, with a minimum homology of % between the different types. all the genes encoding ifn-α are located on human chromosome , and gene duplication is thought to be responsible for this proliferation of genes. i ifn-β: the single gene for ifn-β is also located on human chromosome . the mature protein contains amino acids and, unlike ifn-α, is glycosylated, with approximately % homology to other ifns. it is synthesized predominantly by fibroblasts. i other ifns: the single gene for ifn-γ is located on human chromosome . the mature protein contains amino acids, is glycosylated, and has very low sequence homology to other ifns. it is synthesized predominantly by lymphocytes. other ifns, such as ifn-γ, -δ, -k, -τ, etc., play a variety of roles in cellular regulation but are not directly involved in controlling virus infection. because there are clear biological differences between the two main types of ifn, ifn-α and -β are known as type i ifn, and ifn-γ as type ii ifn. induction of ifn synthesis results from upregulation of transcription from the ifn gene promoters. there are three main mechanisms involved: i virus infection: this mechanism is thought to act by the inhibition of cellular protein synthesis that occurs during many virus infections, resulting in a reduction in the concentration of intracellular repressor proteins and hence in increased ifn gene transcription. in general, rna viruses are potent inducers of ifn while dna viruses are relatively poor inducers; however, there are exceptions to this rule (e.g., poxviruses are very potent inducers). the molecular events in the induction of ifn synthesis by virus infection are not clear. in some cases (e.g., influenza virus), uv-inactivated virus is a potent inducer; therefore, virus replication is not necessarily required. induction by viruses might involve perturbation of the normal cellular environment and/or production of small amounts of double-stranded rna. i double-stranded (ds) rna: all naturally occurring double-stranded rnas (e.g., reovirus genomes) are potent inducers of ifn, as are synthetic molecules (e.g., poly i:c); therefore, this process is independent of nucleotide sequence. single-stranded rna and doublestranded dna are not inducers. this mechanism of induction is thought to depend on the secondary structure of the rna rather than any particular nucleotide sequence. i metabolic inhibitors: compounds that inhibit transcription (e.g., actinomycin d) or translation (e.g., cycloheximide) result in induction of ifn. tumor promoters such as tetradecanoyl phorbol acetate or dimethyl sulfoxide are also inducers. their mechanism of action remains unknown but they almost certainly act at the level of transcription. the effects of ifns are exerted via specific receptors that are ubiquitous on nearly all cell types (therefore, nearly all cells are potentially ifn responsive). there are distinct receptors for type i and type ii ifn, each of which consists of two polypeptide chains. binding of ifn to the type i receptor activates a specific cytoplasmic tyrosine kinase (janus kinase, or jak ), which phosphorylates another cellular protein, signal transducer and activator of transcription (stat ). this is transported to the nucleus and turns on transcriptional activation of ifn-responsive genes (including ifn, resulting in amplification of the original signal). binding of ifn to the type ii receptor activates a different cytoplasmic tyrosine kinase (jak ), which phosphorylates the cellular protein stat , leading to transcriptional activation of a different set of genes. the main action of ifns is on cellular regulatory activities and is rather complex. ifn affects both cellular proliferation and immunomodulation. these effects result from the induction of transcription of a wide variety of cellular genes, including other cytokines. the net result is complex regulation of the ability of a cell to proliferate, differentiate, and communicate. this cellregulatory activity itself has indirect effects on virus replication. type i ifn is the major antiviral mechanism-other ifns act as potent cellular regulators, which may have indirect antiviral effects in some circumstances. the effect of ifns on virus infections in vivo is extremely important. animals experimentally infected with viruses and injected with anti-ifn antibodies experience much more severe infections than control animals infected with the same virus. this is because ifns protect cells from damage and death. however, they do not appear to play a major role in the clearance of virus infections-the other parts of the immune response are necessary for this. ifn is a "firebreak" that inhibits virus replication in its earliest stages by several mechanisms. two of these are understood in some detail, but a number of others (in some cases specific to certain viruses) are less well understood. ifns induce transcription of a cellular gene for the enzyme , -oligo a synthetase ( figure . ). there are at least four molecular species of , -oligo a, induced by different forms of ifn. this compound activates an rna-digesting enzyme, rnase l, which digests virus genomic rnas, virus and cellular mrnas, and cellular ribosomal rnas. the end result of this mechanism is a reduction in protein synthesis (due to the degradation of mrnas and rrnas)-therefore the cell is protected from virus damage. the second method relies on the activation of a -kda protein called pkr (figure . ). pkr phosphorylates a cellular factor, eif α, which is required by ribosomes for the initiation of translation. the net result of this mechanism is also the inhibition of protein synthesis and this reinforces the , -oligo a mechanism. a third, well-established mechanism depends on the m x gene, a single-copy gene located on human chromosome , the transcription of which is induced by type i ifn. the product of this gene inhibits the primary transcription of influenza virus but not of other viruses. its method of action is unknown. in addition to these three mechanisms, there met-trna met .elf .gtp (initiation of translation) gdp + "gef" the modified nucleic acid , -oligo a is involved in one of the major mechanism by which ifns counteract virus infections. are many additional recorded effects of ifns. they inhibit the penetration and uncoating of sv and some other viruses, possibly by altering the composition or structure of the cell membrane; they inhibit the primary transcription of many virus genomes (e.g., sv , hsv) and also cell transformation by retroviruses. none of the molecular mechanisms by which these effects are mediated has been fully explained. ifns are a powerful weapon against virus infection, but they act as a blunderbuss rather than a "magic bullet." the severe side effects (fever, nausea, malaise) that result from the powerful cell-regulatory action of ifns means that they will never be widely used for the treatment of trivial virus infections-they are not the cure for the common cold. however, as the cell-regulatory potential of ifns is becoming better understood, they are finding increasing use as a treatment for certain cancers (e.g., the use of ifn-α in the treatment of hairy cell leukemia). current therapeutic uses of ifns are summarized in table . . the longterm prospects for their use as antiviral compounds are less certain, except for possibly in life-threatening infections where there is no alternative therapy (e.g., chronic viral hepatitis). in total, the many innate and adaptive components of the immune system present a powerful barrier to virus replication. simply by virtue of their continued existence, it is obvious that viruses have, over millennia, evolved effective "counter-surveillance" mechanisms in this molecular arms race. as described above, ctls can only respond to foreign antigens presented by mhc-i complexes on the target cell. a number of viruses interfere with mhc-i expression or function to disrupt this process and evade the ctl response. such mechanisms include downregulation of mhc-i expression by adenoviruses and interference with the antigen processing required to form an mhc-iÀantigen complex by herpesviruses. the mhc-ii antigens are essential in the adaptive immune response in order to stimulate the development of antigen-responsive clones of effector cells. again, herpesviruses and papillomaviruses interfere with the processing and surface expression of mhc-iiÀantigen complexes, inhibiting the ctl response. the poxvirus molluscum contagiosum encodes a homologue of mhc-i that is expressed on the surface of infected cells but is unable to bind an antigenic peptide, thus avoiding killing by nk cells that would be triggered by the absence of mhc-i on the cell surface. similar proteins are made by other viruses, such as hhv- (cmv), and herpesviruses in general appear to have a number of sophisticated mechanisms to avoid nk cell killing. see viruses and apoptosis earlier in this chapter. cytokines are secreted polypeptides that coordinate important aspects of the immune response, including inflammation, cellular activation, proliferation, differentiation, and chemotaxis. some viruses are able to inhibit the expression of certain chemokines directly. alternatively, herpesviruses and poxviruses encode "viroceptors"-virus homologues of host cytokine receptors that compete with cellular receptors for cytokine binding but fail to give transmembrane signals. high-affinity binding molecules may also neutralize cytokines directly, and molecules known as "virokines" block cytokine receptors again without activating the intracellular signaling cascade. ifns are cytokines which act as an effective means of curbing the worst effects of virus infections. part of their wide-ranging efficacy results from their generalized, nonspecific effects (e.g., the inhibition of protein synthesis in although direct humoral immunity is less significant than cell-mediated immunity, the antiviral action of adcc and complement make this a worthwhile target to inhibit. the most frequent means of subverting the humoral response is by high-frequency genetic variation of the b-cell epitopes on antigens to which antibodies bind. this is only possible for viruses that are genetically variable (e.g., influenza virus and hiv). herpesviruses use alternative strategies such as encoding viral fc receptors to prevent fc-dependent immune activation. poxviruses, herpesviruses, and some retroviruses encode mimics of normal regulators of complement activation proteins (e.g., secreted proteins that block c convertase assembly and accelerate its decay). poxviruses can also inhibit c polymerization, preventing membrane permeabilization. viruses do not set out to kill their hosts. virus pathogenesis is an abnormal situation of no value to the virus-the vast majority of virus infections are asymptomatic. however, for pathogenic viruses, a number of critical stages in replication determine the nature of the disease they produce. for all viruses, pathogenic or nonpathogenic, the first factor that influences the course of infection is the mechanism and site of entry into the body (figure . ): i the skin: mammalian skin is a highly effective barrier against viruses. the outer layer (epidermis) consists of dead cells and therefore does not support virus replication. very few viruses infect directly by this route unless there is prior injury such as minor trauma or puncture of the barrier, such as insect or animal bites or subcutaneous injections. some viruses that do use this route include hsv and papillomaviruses, although these viruses probably still require some form of disruption of the skin such as small abrasions or eczema. i mucosal membranes: the mucosal membranes of the eye and genitourinary (gu) tract are much more favorable routes of access for viruses to the tissues of the body. this is reflected by the number of viruses that can be sexually transmitted; virus infections of the eye are also quite common (table . ). i alimentary canal: viruses may infect the alimentary canal via the mouth, oropharynx, gut, or rectum, although viruses that infect the gut via the oral route must survive passage through the stomach, an extremely hostile environment with a very low ph and high concentrations of digestive enzymes. nevertheless, the gut is a highly valued prize for viruses-the intestinal epithelium is constantly replicating and a good deal of lymphoid tissue is associated with the gut which provides many opportunities for virus replication. moreover, the constant intake of food and fluids provides ample opportunity for viruses to infect these tissues (table . ). to counteract this problem, the gut has many specific (e.g., secretory antibodies) and nonspecific (e.g., stomach acids and bile salts) defense mechanisms. i respiratory tract: the respiratory tract is probably the most frequent site of virus infection. as with the gut, it is constantly in contact with external virus particles which are taken in during respiration. as a result, the respiratory tract also has defenses aimed at virus infection-filtering of particulate matter in the sinuses and the presence of cells and antibodies of the immune system in the lower regions. viruses that infect the respiratory tract usually come directly from the respiratory tract of others, as aerosol spread is very efficient: "coughs and sneezes spread diseases" (table . ). the natural environment is a considerable barrier to virus infection. most viruses are relatively sensitive to heat, drying, uv light (sunlight), etc., although a few types are quite resistant to these factors. this is particularly important for viruses that are spread via contaminated water or foodstuffsnot only must they be able to survive in the environment until they are ingested by another host, but, as most are spread by the fecalÀoral route, they must also be able to pass through the stomach to infect the gut before being shed in the feces. one way of overcoming environmental stress is to take advantage of a secondary vector for transmission between the primary hosts ( figure . ). as with plant viruses, the virus may or may not replicate while in the vector. viruses without a secondary vector must rely on continued host-to-host transmission and have evolved various strategies to do this (table . ): i horizontal transmission: the direct host-to-host transmission of viruses. this strategy relies on a high rate of infection to maintain the virus population. i vertical transmission: the transmission of the virus from one generation of hosts to the next. this may occur by infection of the fetus before, during, or shortly after birth (e.g., during breastfeeding). more rarely, it may involve direct transfer of the virus via the germ line itself (e.g., retroviruses). in contrast to horizontal transmission, this strategy relies on long-term persistence of the virus in the host rather than rapid propagation and dissemination of the virus. having gained entry to a potential host, the virus must initiate an infection by entering a susceptible cell (primary replication). this initial interaction frequently determines whether the infection will remain localized at the site of entry or spread to become a systemic infection (table . ). in some cases, virus spread is controlled by infection of polarized epithelial cells and the preferential release of virus from either the apical (e.g., influenza virus-a localized infection in the upper respiratory tract) or basolateral (e.g., rhabdoviruses-a systemic infection) surface of the cells (figure . ). following primary replication at the site of infection, the next stage may be spread throughout the host. in addition to direct cellÀcell contact, there are two main mechanisms for spread throughout the host: i via the bloodstream: viruses may get into the bloodstream by direct inoculation-for example, by arthropod vectors, blood transfusion, or intravenous drug abuse (sharing of nonsterilized needles). the virus may travel free in the plasma (e.g., togaviruses, enteroviruses) or in association with red cells (orbiviruses), platelets (hsv), lymphocytes (ebv, cmv), or monocytes (lentiviruses). primary viremia usually precedes and is necessary for the spread of virus to other parts of the body via the bloodstream and is followed by a more generalized, higher titer secondary viremia as the virus reaches the other target tissues or replicates directly in blood cells. i via the nervous system: as above, spread of virus to the nervous system is usually preceded by primary viremia. in some cases, spread occurs directly by contact with neurones at the primary site of infection; in other cases, it occurs via the bloodstream. once in peripheral nerves, the virus can spread to the central nervous system (cns) by axonal transport along neurones. the classic example of this is hsv (see "latent infection," below). viruses can cross synaptic junctions as these frequently contain virus receptors, allowing the virus to jump from one cell to another. the spread of the virus to various parts of the body is controlled to a large extent by its cell or tissue tropism. tissue tropism is controlled partly by the route of infection but largely by the interaction of a virus-attachment protein (vap) with a specific receptor molecule on the surface of a cell (as discussed in chapter ) and has considerable effect on pathogenesis. at this stage, following significant virus replication and the production of virus antigens, the host immune response comes into play. this has already been discussed earlier and obviously has a major impact on the outcome of an infection. to a large extent, the efficiency of the immune response determines the amount of secondary replication that occurs and, hence, the spread to other parts of the body. if a virus can be prevented from reaching tissues where secondary replication can occur, generally no disease results, although there are some exceptions to this. the immune response also plays a large part in determining the amount of cell and tissue damage that occurs as a result of virus replication. as described above, the production of ifns is a major factor in preventing virus-induced tissue damage. the immune system is not the only factor that controls cell death, the amount of which varies considerably for different viruses. viruses may replicate widely throughout the body without any disease symptoms if they do not cause significant cell damage or death. retroviruses do not generally cause cell death, being released from the cell by budding rather than by cell lysis, and cause persistent infections, even being passed vertically to the offspring if they infect the germ line. all vertebrate genomes, including humans, are littered with retrovirus genomes that have been with us for millions of years (chapter ). at present, these ancient virus genomes are not known to cause any disease in humans, although there are examples of tumors caused by them in rodents. conversely, picornaviruses cause lysis and death of the cells in which they replicate, leading to fever and increased mucus secretion, in the case of rhinoviruses, and paralysis or death (usually due to respiratory failure due to damage to the cns resulting, in part, from virus replication in these cells) in the case of poliovirus. the eventual outcome of any virus infection depends on a balance between two processes. clearance is mediated by the immune system (as discussed previously); however, the virus is a moving target that responds rapidly to pressure from the immune system by altering its antigenic composition (whenever possible). the classic example of this phenomenon is influenza virus, which displays two genetic mechanisms that allow the virus to alter its antigenic constitution: i antigenic drift: this involves the gradual accumulation of minor mutations (e.g., nucleotide substitutions) in the virus genome which result in subtly altered coding potential and therefore altered antigenicity, leading to decreased recognition by the immune system. this process occurs in all viruses all the time but at greatly different rates; for example, it is much more frequent in rna viruses than in dna viruses. in response, the immune system constantly adapts by recognition of and response to novel antigenic structures-but it is always one step behind. in most cases, however, the immune system is eventually able to overwhelm the virus, resulting in clearance. i antigenic shift: in this process, a sudden and dramatic change in the antigenicity of a virus occurs owing to reassortment of the segmented virus genome with another genome of a different antigenic type (see chapter ). this results initially in the failure of the immune system to recognize a new antigenic type, giving the virus the upper hand ( figure . ). the occurrence of past antigenic shifts in influenza virus populations is recorded by pandemics (worldwide epidemics; figure . ). these events are marked by the sudden introduction of a new antigenic type of hemagglutinin and/or neuraminidase into the circulating virus, overcoming previous immunity in the human population. previous hemagglutinin/neuraminidase types become resurgent when a sufficiently high proportion of the people who have "immunological memory" of that type have died, thus overcoming the effect of "herd immunity." the other side of the relationship that determines the eventual outcome of a virus infection is the ability of the virus to persist in the host. long-term persistence of viruses results from two main mechanisms. the first is the regulation of lytic potential. the strategy followed here is to achieve the continued survival of a critical number of virus-infected cells (i.e., sufficient to continue the infection without killing the host organism). for viruses that do not usually kill the cells in which they replicate, this is not usually a problem; hence, these viruses tend naturally to cause persistent infections (e.g., retroviruses). for viruses that undergo lytic infection (e.g., herpesviruses), it this chart shows the history of influenza pandemics throughout the twentieth century. the first pandemic of the twenty-first century occurred in and was caused by an h n type virus, although this was not as damaging as earlier pandemics. is necessary to develop mechanisms that restrict virus gene expression and, consequently, cell damage. the second aspect of persistence is the evasion of immune surveillance, discussed above. patterns of virus infection can be divided into a number of different types. abortive infection occurs when a virus infects a cell (or host) but cannot complete the full replication cycle, so this is a nonproductive infection. the outcome of such infections is not necessarily insignificant, for example, sv infection of nonpermissive rodent cells sometimes results in transformation of the cells (see chapter ). this pattern is familiar for many common virus infections (e.g., "colds"). in these relatively brief infections, the virus is usually eliminated completely by the immune system. typically, in acute infections, much virus replication occurs before the onset of any symptoms (e.g., fever), which are the result not only of virus replication but also of the activation of the immune system; therefore, acute infections present a serious problem for the epidemiologist and are the pattern most frequently associated with epidemics (e.g., influenza, measles). these are the converse of acute infections (i.e., prolonged and stubborn). to cause this type of infection, the virus must persist in the host for a significant period. to the clinician, there is no clear distinction among chronic, persistent, and latent infections, and the terms are often used interchangeably. they are listed separately here because, to virologists, there are significant differences in the events that occur during these infections. these infections result from a delicate balance between the virus and the host organism, in which ongoing virus replication occurs but the virus adjusts its replication and pathogenicity to avoid killing the host. in chronic infections, the virus is usually eventually cleared by the host (unless the infection proves fatal), but in persistent infections the virus may continue to be present and to replicate in the host for its entire lifetime. the best-studied example of such a system is lymphocytic choriomeningitis virus (lcmv; an arenavirus) infection in mice (figure . ). mice can be experimentally infected with this virus either at a peripheral site (e.g., a footpad or the tail) or by direct inoculation into the brain. adult mice infected in the latter way are killed by the virus, but among those infected by a peripheral route there are two possible outcomes to the infection: some mice die but others survive, having cleared the virus from the body completely. it is not clear what factors determine the survival or death of lcmv-infected mice, but other evidence shows that the outcome is related to the immune response to the virus. in immunosuppressed adult mice infected via the cns route, a persistent infection is established in which the virus is not cleared (due to the nonfunctional immune system), but, remarkably, these mice are not killed by the virus. if, however, syngeneic lcmv-specific t-lymphocytes (i.e., of the same mhc type) are injected into these persistently infected mice, the animals develop the full pathogenic symptoms of lcmv infection and die. when newborn mice, whose immune systems are immature, are infected via the cns route, they also develop a persistent infection, but, in this case, if they are subsequently injected with syngeneic lcmv-specific t-lymphocytes, they clear the virus and survive the infection. the mechanisms that control these events are not completely understood, but evidently there is a delicate balance between the virus and the host animal and the immune response to the virus is partly responsible for the pathology of the disease and the death of the animals. not infrequently, persistent infections may result from the production of defective-interfering (d.i.) particles (see chapter ). such particles contain a partial deletion of the virus genome and are replication defective, but they are maintained and may even tend to accumulate during infections because they can replicate in the presence of replication-competent helper virus. the production of d.i. particles is a common consequence of virus infection of animals, particularly by rna viruses, but also occurs with dna viruses and plant viruses and can be mimicked in vitro by continuous high-titer passage of virus. although not able to replicate themselves independently, d.i. particles are not necessarily genetically inert and may alter the course of an infection by recombination with the genome of a replication-competent virus. the presence of d.i. particles can profoundly influence the course and the outcome of a virus infection. in some cases, they appear to moderate pathogenesis, whereas in others they potentiate it, making the symptoms of the disease much more severe. moreover, as d.i. particles effectively cause restricted gene expression (because they are genetically deleted), they may also result in a persistent infection by a virus that normally causes an acute infection and is rapidly cleared from the body. in a latent state, the virus is able to downregulate its gene expression and enter an inactive state with strictly limited gene expression and without ongoing virus replication. latent virus infections typically persist for the entire life of the host. an example of such an infection in humans is hsv. infection of sensory nerves serving the mucosa results in localized primary replication. subsequently, the virus travels via axon transport mechanisms further into the nervous system. there, it hides in dorsal root ganglia, such as the trigeminal ganglion, establishing a truly latent infection. the nervous system is an immunologically privileged site and is not patrolled by the immune system in the same way as the rest of the body, but the major factor in latency is the ability of the virus to restrict its gene expression. this eliminates the possibility of recognition of infected cells by the immune system. restricted gene expression is achieved by tight regulation of α-gene expression, which is an essential control point in herpesvirus replication (chapter ). in the latent state, hsv makes an . -kb rna transcript called the latent rna or latency-associated transcript (lat). the lat is broken down into even smaller strands called micrornas (mirnas), and these block the production of proteins which reactivate the virus. drugs which block production of these mirnas could in theory "wake up" all the dormant viruses, making them vulnerable to the immune system and to antiviral therapy, and this raises the eventual possibility of a cure for herpes infections. expression of the lat promotes neuronal survival after hsv infection by inhibiting apoptosis. this anti-apoptosis function could promote reactivation by: i providing more latently infected neurons for future reactivations, i protecting neurons in which reactivation occurs, i protecting previously uninfected neurons during a reactivation. when reactivated by some provocative stimulus, hsv travels down the sensory nerves to cause peripheral manifestations such as cold sores or genital ulcers. it is not altogether clear what constitutes a provocative stimulus, but there are many possible alternatives, including psychological and physical factors. periodic reactivation establishes the pattern of infection, with sporadic, sometimes very painful reappearance of disease symptoms for the rest of the host's life. even worse than this, immunosuppression later in life can cause the latent infection to flare up (which indicates that the immune system normally has a role in helping to suppress these latent infections), resulting in a very severe, systemic, and sometimes life-threatening infection. in a manner somewhat similar to herpesviruses, infection by retroviruses may result in a latent infection. integration of the provirus into the host genome certainly results in the persistence of the virus for the lifetime of the host organism and may lead to an episodic pattern of disease. in some ways, acquired immunodeficiency syndrome (aids), which results from hiv infection, shows aspects of this pattern of infection. the pathogenesis of aids is discussed in detail in chapter . there are two aspects of the response to the threat of virus diseases: first, prevention of infection, and second, treatment of the disease. the former strategy relies on two approaches: public and personal hygiene, which perhaps plays the major role in preventing virus infection (e.g., provision of clean drinking water and disposal of sewage; good medical practice such as the sterilization of surgical instruments) and vaccination, which makes use of the immune system to combat virus infections. most of the damage to cells during virus infections occurs very early, often before the clinical symptoms of disease appear. this makes the treatment of virus infection very difficult; therefore, in addition to being less expensive, prevention of virus infection is undoubtedly better than cure. to design effective vaccines, it is important to understand both the immune response to virus infection and the stages of virus replication that are appropriate targets for immune intervention. to be effective, vaccines must stimulate as many of the body's defense mechanisms as possible. in practice, this usually means trying to mimic the disease without causing pathogenesis-for example, the use of live attenuated viruses as vaccines such as nasally administered influenza vaccines and orally administered poliovirus vaccines. to be effective, it is not necessary to get % uptake of vaccine. "herd immunity" results from the break in transmission of a virus that occurs when a sufficiently high proportion of a population has been vaccinated. this strategy is most effective where there is no alternative host for the virus (e.g., measles) and in practice is the situation that usually occurs as it is impossible to achieve % coverage with any vaccine. however, this is a risky business; if protection of the population falls below a critical level, epidemics can easily occur. synthetic vaccines are short, chemically synthesized peptides. the major disadvantage with these molecules is that they are not usually very effective immunogens and are very costly to produce. however, because they can be made to order for any desired sequence, they have great theoretical potential, but none are currently in clinical use. recombinant vaccines are produced by genetic engineering. such vaccines have been already produced and are better than synthetic vaccines because they tend to give rise to a more effective immune response. some practical success has already been achieved with this type of vaccine. for example, vaccination against hepatitis b virus (hbv) used to rely on the use of australian antigen (hbsag) obtained from the serum of chronic hbv carriers. this was a very risky practice indeed (because hbv carriers are often also infected with hiv). a completely safe recombinant hbv vaccine produced in yeast is now used. dna vaccines are the newest type of vaccine and consist of only a dna molecule encoding the antigen(s) of interest and, possibly, costimulatory molecules such as cytokines. the concept behind these vaccines is that the dna component will be expressed in vivo, creating small amounts of antigenic protein that serve to prime the immune response so that a protective response can be rapidly generated when the real antigen is encountered. in theory, these vaccines could be manufactured quickly and should efficiently induce both humoral and cellmediated immunity. initial clinical studies have indicated that there is still some way to go until this experimental technology becomes a practical proposition. subunit vaccines consist of only some components of the virus, sufficient to induce a protective immune response but not enough to allow any danger of infection. in general terms, they are completely safe, except for very rare cases in which adverse immune reactions may occur. unfortunately, they also tend to be the least effective and most expensive type of vaccine. the major technical problems associated with subunit vaccines are their relatively poor antigenicity and the need for new delivery systems, such as improved carriers and adjuvants. virus vectors are recombinant virus genomes genetically manipulated to express protective antigens from (unrelated) pathogenic viruses. the idea here is to utilize the genome of a well-understood, attenuated virus to express and present antigens to the immune system. many different viruses offer possibilities for this type of approach. one of the most highly developed systems so far is based on the vv genome. this virus has been used to vaccinate millions of people worldwide in the campaign to eradicate smallpox and is generally a safe and effective vehicle for antigen delivery. such vaccines are difficult to produce. no human example is clearly successful yet, although many different trials are currently underway, but vvÀrabies recombinants have been used to eradicate rabies in european fox populations. vv-based vaccines have advantages and disadvantages for use in humans-a high percentage of the human population has already been vaccinated during the smallpox eradication campaign, and this lifelong protection may result in poor response to recombinant vaccines. although generally safe, vv is dangerous in immunocompromised hosts, thus it cannot be used in hiv-infected individuals. a possible solution to these problems may be to use avipoxvirus vectors (e.g., fowlpox or canarypox) as "suicide vectors" that can only establish abortive infections of mammalian cells and offer the following advantages: i expression of high levels of foreign proteins, i no danger of pathogenesis (abortive infection), i no natural immunity in humans (avian virus). inactivated vaccines are produced by exposing the virus to a denaturing agent under precisely controlled conditions. the objective is to cause loss of virus infectivity without loss of antigenicity. obviously, this involves a delicate balance. however, inactivated vaccines have certain advantages, such as generally being effective immunogens (if properly inactivated), being relatively stable, and carrying little or no risk of vaccine-associated virus infection (if properly inactivated, but accidents can and do occur). the disadvantage of these vaccines is that it is not possible to produce inactivated vaccines for all viruses, as denaturation of virus proteins may lead to loss of antigenicity (e.g., measles virus). although relatively effective, "killed" vaccines are sometimes not as effective at preventing infection as "live" virus vaccines, often because they fail to stimulate protective mucosal and cell-mediated immunity to the same extent. a more recent concern is that these vaccines contain virus nucleic acids, which may themselves be a source of infection, either of their own accord (e.g., ( )sense rna virus genomes) or after recombination with other viruses. virus vaccines do not have to be based on virion structural proteins. the effectiveness of attenuated vaccines relies on the fact that a complete spectrum of virus proteins, including nonstructural proteins, is expressed and gives rise to cell-mediated immune responses. live attenuated virus vaccines are viruses with reduced pathogenicity used to stimulate an immune response without causing disease. the vaccine strain may be a naturally occurring virus (e.g., the use of cowpox virus by edward jenner to vaccinate against smallpox) or artificially attenuated in vitro (e.g., the oral poliomyelitis vaccines produced by albert sabin). the advantage of attenuated vaccines is that they are good immunogens and induce long-lived, appropriate immunity. set against this advantage are their many disadvantages. they are often biochemically and genetically unstable and may either lose infectivity (becoming worthless) or revert to virulence unexpectedly. despite intensive study, it is not possible to produce an attenuated vaccine to order, and there appears to be no general mechanism by which different viruses can be reliably and safely attenuated. contamination of the vaccine stock with other, possibly pathogenic viruses is also possible-this was the way in which sv was first discovered in oral poliovirus vaccine in . inappropriate use of live virus vaccines, for example, in immunocompromised hosts or during pregnancy may lead to vaccineassociated disease, whereas the same vaccine given to a healthy individual may be perfectly safe. despite these difficulties, vaccination against virus infection has been one of the great triumphs of medicine during the twentieth century. most of the success stories result from the use of live attenuated vaccines-for example, the use of vv against smallpox. on may , , the world health organization (who) officially declared smallpox to be completely eradicated, the first virus disease to be eliminated from the world. the who aims to eradicate a number of other virus diseases such as poliomyelitis and measles, but targets for completion of these programs have undergone much slippage due to the formidable difficulties involved in a worldwide undertaking of this nature. although prevention of infection by prophylactic vaccination is much the preferred option, postexposure therapeutic vaccines can be of great value in modifying the course of some virus infections. examples of this include rabies virus, where the course of infection may be very long and there is time for postexposure vaccination to generate an effective immune response and prevent the virus from carrying out the secondary replication in the cns that is responsible for the pathogenesis of rabies. other potential examples can be found in virus-associated tumors, such as hpv-induced cervical carcinoma. most existing virus vaccines are directed against viruses which are relatively antigenically invariant, for example, measles, mumps, and rubella viruses, where this is only one unchanging serotype of the virus. viruses whose antigenicity alters continuously are a major problem in terms of vaccine production, and the classic example of this is influenza virus (see earlier). in response to this problem, new technologies such as reverse genetics could be used to improve and to shorten the lengthy process of preparing vaccines. rna virus genomes can be easily manipulated as dna clones to contain nucleotide sequences which match currently circulating strains of the virus. infectious virus particles are rescued from the dna clones by introducing these into cells. seed viruses for distribution to vaccine manufacturers can be produced in as little as À weeks, a much shorter time than the months this process takes in conventional vaccine manufacture. using the same technology, universal influenza vaccines containing crucial virus antigens expressed as fusion proteins with other antigenic molecules could feasibly be produced, making the requirement for constant production of new influenza vaccines obsolete. although this has not yet been achieved, advances toward these goals are being made. the explosion of molecular techniques described in earlier chapters is now being used to inform vaccine design (as well as the design of antiviral drugs) rather than simply relying on trial-and-error approaches. however, developing safe and effective vaccines remains one of the greatest challenges facing virology. rna interference (rnai) is a posttranscriptional gene silencing process that occurs in organisms from yeast to humans. in mammals, small rnas include small interfering rnas (sirnas) and mirnas. sirnas, with perfect base complementarity to their targets, activate rnai-mediated cleavage of the target mrnas, while mirnas generally induce rna decay and/or translation inhibition of target genes (figure . ). mammals, including humans, encode hundreds or thousands of mirnas. some viruses with eukaryotic hosts also encode mirnas. herpesviruses in particular encode multiple mirnas; most other nuclear dna viruses encode one or two mirnas. rna viruses and cytoplasmic dna viruses appear to lack any mirnas. virus mirnas may serve two major functions. several have been shown to inhibit the expression of cellular factors that play a role in cellular innate or adaptive antiviral immune responses, so reducing the effectiveness of the immune response. alternatively, virus mirnas may downregulate the expression of virus proteins, including key immediate-early or early regulatory proteins. in hsv, mirnas are expressed at high levels during latency, but not during productive replication, so their action is thought to stabilize latency. recently there have been controversial claims that mirna can exert antiviral activity in mice, at least in some circumstances. antiviral sirna activity is only seen in stem cells and in newborn mice and many scientists think sirna is not a major part of the innate immune system in adult animals. there is evidence that sirna may be turned on in responses to virus infection, but rather than acting directly against the virus, it may be used to regulate the ifn response. that still leaves the fact that in mammals mirna is a powerful regulator of gene expression, including virus genes. many viruses use mirna to control their own gene expression and that of their host cells. on infection of a host cell, viruses encounter a range of mirna species, many of which have been shown to restrict virus gene expression. thus they have had to evolve a range of mechanisms to evade mirna restriction is the same way that they have evolved other mechanisms to mitigate the impact of innate immunity. these include: i blocking mirna function i avoiding utr targets complementary to cellular mirnas i evolving very short utrs i evolving structured utrs rnai expression can be induced by dsrna, and this approach has been used to investigate gene function in a variety of organisms including plants and insects. however, this method cannot be applied to mammalian cells as dsrnas longer than nucleotides induces the ifn response (see earlier), which results in the degradation of mrnas and causes a global inhibition of translation. to circumvent this problem, chemically synthesized sirnas or plasmid-vectors manipulated to produce short hairpin rna molecules can be used to investigate gene function in mammals. in the future it may be feasible to treat virus diseases by shutting off gene expression by directing the degradation of specific mrnas, and many clinical trials are currently underway. although rna interference has been used widely in cultured cells to inhibit virus replication and to probe biological pathways, considerable problems must be overcome before it becomes a useful therapy, including the development of suitable delivery and targeting systems and solving the issue of stability in vivo. the natural world is a soup of bacteriophages. so how do bacteria survive against this constant onslaught? with their own form of adaptive immunity. crisprs (clustered regularly interspaced short palindromic repeats) are short, direct repeats of dna base sequences. each crispr contains a series of bases followed by the same series in reverse (a palindrome) and then by or so base pairs known as "spacer" dna. the spacers are short segments of virus or plasmid dna. crisprs are found in the genomes of approximately % of bacteria and % of archaea. crispr loci are typically located on the bacterial chromosome, although some are found on plasmids. bacteria may contain more than one crispr locus-up to in some cases. crisprs function as a sort of prokaryotic immune system, conferring resistance to exogenous genetic elements such as plasmids and bacteriophages. intriguingly, the crispr system provides a form of acquired immunity, allowing the cell to remember and respond to sequences it has encountered before. how do crisprs work? crisprs are often adjacent to cas (crispr-associated) genes. the cas genes encode a large and heterogeneous family of proteins including nucleases, helicases, polymerases, and polynucleotide-binding proteins, forming the crispr/cas system. (note: cas genes, cas the proteins encoded by these genes.) the interesting bits are the unique spacer elements (derived from exogenous sequences such as viruses and plasmids) rather than the repeats themselves. the spacer elements originate from exogenous dna the bacterium (or its ancestors) has previous encountered-they are typically pieces of phage or plasmid dna. this allows the cell to recognize these sequences via base homology if they enter the cell again, for example, if the bacterium is infected with a bacteriophage whose genome contains this sequence: . cas-encoded nucleases cleave invading dna into short pieces. . other cas proteins allow a fragment of the foreign dna to be incorporated as a novel repeat-spacer unit at the leader end of the crispr site. . the crispr array is then transcribed to form a pre-crispr rna (crrna) transcript. . the pre-crrna is cleaved within the repeat sequence by cas proteins to generate small crispr rnas, crrnas. . the crrnas to work in a similar way to rnai in eukaryotic cells, although there are important differences in the machinery by which this happens. crisprs are an important way in which bacteria are able to survive constant attack by bacteriophages in the environment, but phages have been around for a very long time too, so they must have found ways of counteracting the crispr system. eukaryotic viruses may express inhibitors such as dsrnabinding proteins that interfere with the rna silencing machinery, whereas bacteriophages acquire mutations or recombine the sequence corresponding to the crispr spacer to avoid recognition in an analogous way to how viruses of eukaryotes acquire mutations in b-cell and t-cell epitopes in proteins to evade the mammalian immune system. so who (apart from bacteria) cares about crisprs? altering the spacer via genetic manipulation can provide novel phage resistance, whereas spacer deletion results in loss of phage resistance. although crisprs originate in bacteria, they also work in eukaryotic cells if introduced by genetic engineering. this provides a convenient way of targeting genes in cells, including human cells. recent work suggests that crisprs might also be involved in control of bacterial gene expression as well as in immunity. we will undoubtedly see much more widespread use of crisprs in biotechnology over the next few years. phage therapy, the use of bacteriophages to treat or prevent disease, stretches back a century to the earliest days of the discovery of phages. long before the discovery of antibiotics, the thought that viruses which lyse bacteria could be used to treat diseases was highly attractive. yet this idea has never become a widespread practical reality. devotees of phage therapy defend their cherished belief with almost religious fervor, but there are serious obstacles to be overcome, such as the narrow host range of most phages (a few strains of bacteria, not even an entire species) and the speed at which bacteria develop resistance to infection. as the spectrum of clinically useful antibiotics dwindles, phage therapy increases in attractiveness, but is unlikely ever to replace the antibiotic golden era of disease treatment we are now leaving behind. another aspect of "virotherapy" is the growing interest in oncolytic virusesviruses engineered to kill only cancer cells. the usefulness of many different types of virus has been investigated, including adenoviruses, herpesviruses, reoviruses, and poxviruses. although safety is a concern even in patients with terminal illnesses, this is one area of medical research where optimism is considerable. many clinical trials are underway at it seems certain that this approach to cancer treatment will eventually become more common, possibly as an adjunct to other forms of therapy such as surgery, drugs, and radiotherapy. viruses have also developed as gene delivery systems for the treatment of inherited and acquired diseases. gene therapy offers: i delivery of large biomolecules to cells, i the possibility of targeting delivery to a specific cell type, i high potency of action due to replication of the vector, i potential to treat certain diseases (such as head and neck cancers and brain tumors) that respond poorly to other therapies or may be inoperable. the very first retroviral and adenoviral vectors were characterized in the early s. the first human trial to treat children with immunodeficiency resulting from a lack of the enzyme adenosine deaminase began in and showed encouraging although not completely successful results. like most of the initial attempts, this trial used recombinant retrovirus genomes as vectors. in , the first successful gene therapy for motor neurons and skin cells was reported, while the first phase three (widespread) gene therapy trial was begun in . in , the first successful treatment of a patient with severe combined immunodeficiency disease (scid) was reported, but, sadly, the first death due to a virus vector also occurred, and in the occurrence of leukemias due to oncogenic insertion of a retroviral vector was seen in some scid patients undergoing treatment. several different viruses are being tested as potential vectors ( integrate into cellular dna at high frequency to establish a stable latent state; not associated with any known disease; vectors can be constructed that will not express any viral gene products. only b kb of dna can be packaged into the parvovirus capsid, and some virus sequences must be retained for packaging; integration into host-cell dna may potentially have damaging consequences. herpesviruses relatively easy to manipulate in vitro; grows to high titers; long-term persistence in neuronal cells without integration. (long-term) pathogenic consequences? integrate into cell genome, giving long-lasting (lifelong?) expression of recombinant gene. difficult to grow to high titer and purify for direct administration (patient cells must be cultured in vitro); cannot infect nondividing cells-most somatic cells (except lentiviruses?); insertional mutagenesis/activation of cellular oncogenes. can express high levels of foreign proteins. avipoxvirus vectors (e.g., fowlpox or canarypox) are "suicide vectors" that undergo abortive replication in mammalian cells so there is no danger of pathogenesis and no natural immunity in humans. a high proportion of the human population has already been vaccinated-lifelong protection may result in poor response to recombinant vaccines (?). dangerous in immunocompromised hosts. the alternative to vaccination is to attempt to treat virus infections using drugs that block virus replication (table . ). historically, the discovery of antiviral drugs was largely down to luck. spurred on by successes in the treatment of bacterial infections with antibiotics, drug companies launched huge blind-screening programs to identify chemical compounds with antiviral activity, with relatively little success. the key to the success of any antiviral drug lies in its specificity. almost any stage of virus replication can be a target for a drug, but the drug must be more toxic to the virus than the host. this is measured by the chemotherapeutic index, given by: dose of drug that inhibits virus replication dose of drug that is toxic to host the smaller the value of the chemotherapeutic index, the better. in practice, a difference of several orders of magnitude between the two toxicity values is usually required to produce a safe and clinically useful drug. modern technology, including molecular biology and computer-aided design of chemical compounds, allows the deliberate design of drugs, but it is necessary to "know your enemy"-to understand the key steps in virus replication that might be inhibited. any of the stages of virus replication can be a target for antiviral intervention. the only requirements are: i the process targeted must be essential for replication. i the drug is active against the virus but has "acceptable toxicity" to the host organism. what degree of toxicity is "acceptable" clearly varies considerably-for example, between a cure for the common cold, which might be sold over the counter and taken by millions of people, and a drug used to treat fatal virus infections such as aids. the attachment phase of replication can be inhibited in two ways, by agents that mimic the vap and bind to the cellular receptor or by agents that mimic the receptor and bind to the vap. synthetic peptides are the most logical class of compound to use for this purpose. while this is a promising line of research, there are considerable problems with the clinical use of these substances, primarily the high cost of synthetic peptides and the poor pharmacokinetic properties of many of these synthetic molecules. it is difficult to target specifically the penetration/uncoating stages of virus replication as relatively little is known about them. uncoating in particular is largely mediated by cellular enzymes and is therefore a poor target for intervention, although, like penetration, it is often influenced by one or more virus proteins. amantadine and rimantadine are two drugs that are active against influenza a viruses. the action of these closely related agents is to block cellular membrane ion channels. the target for both drugs is the influenza matrix protein (m ), but resistance to the drug may also map to the hemagglutinin gene. this biphasic action results from the inability of drug-treated cells to lower the ph of the endosomal compartment (a function normally controlled by the m gene product), which is essential to induce conformational changes in the ha protein to permit membrane fusion (see chapter ). many viruses have evolved their own specific enzymes to replicate virus nucleic acids preferentially at the expense of cellular molecules. there is often sufficient specificity in virus polymerases to provide a target for an antiviral agent, and this method has produced the majority of the specific antiviral drugs currently in use. the majority of these drugs function as polymerase substrates (i.e., nucleoside/nucleotide) analogues, and their toxicity varies considerably, from some that are well tolerated (e.g., acyclovir) to others that are quite toxic (e.g., azidothymidine or azt). there is a problem with the pharmacokinetics of these nucleoside analogues in that their typical serum half-life is to hours. nucleoside analogues are in fact pro-drugs, as they must be phosphorylated before becoming effective-which is key to their selectivity: i acyclovir is phosphorylated by hsv thymidine kinase times more efficiently than by cellular enzymes. i ganciclovir is times more effective against cmv than acyclovir but must be phosphorylated by a kinase encoded by cmv gene ul before it becomes pharmaceutically active. i other nucleoside analogues derived from these drugs and active against herpesviruses have been developed (e.g., valciclovir and famciclovir). these compounds have improved pharmacokinetic properties, such as better oral bioavailability and longer half-lives. in addition to these there are a number of nonnucleoside analogues that inhibit virus polymerases; for example, foscarnet is an analogue of pyrophosphate that interferes with the binding of incoming nucleotide triphosphates by virus dna polymerases. ribavirin is a compound with a very wide spectrum of activity against many different viruses, especially against many (À)sense rna viruses. this drug acts as an rna mutagen, causing a -fold increase in mutagenesis of rna virus genomes and a % loss in virus infectivity after a single round of virus infection in the presence of ribavirin. ribavirin is thus quite unlike the other nucleoside analogues described above, and its use might become much more widespread in the future if it were not for the frequency of adverse effects associated with this drug. virus gene expression is less amenable to chemical intervention than genome replication, because viruses are much more dependent on the cellular machinery for transcription, mrna splicing, cytoplasmic export, and translation than for replication. to date, no clinically useful drugs that discriminate between virus and cellular gene expression have been developed. as with penetration and uncoating, for the majority of viruses the processes of assembly, maturation, and release are poorly understood and therefore have not yet become targets for antiviral intervention, with the exception of the antiinfluenza drugs oseltamivir and zanamivir, which are inhibitors of influenza virus neuraminidase. neuraminidase is involved in the release of virus particles budding from infected cells, and these drugs are believed to reduce the spread of virus to other cells. the most striking aspect of antiviral chemotherapy is how few clinically useful drugs are available. as if this were not bad enough, there is also the problem of drug resistance to consider. in practice, the speed and frequency with which resistance arises when drugs are used to treat virus infections varies considerably and depends largely on the biology of the virus involved rather than on the chemistry of the compound. to illustrate this, two extreme cases are described here. acyclovir, used to treat hsv infections, is easily the most widely used antiviral drug. this is particularly true in the case of genital herpes, which causes painful recurrent ulcers on the genitals. it is estimated that to million people suffer from this condition in the united states. fortunately, resistance to acyclovir arises infrequently. this is partly due to the high fidelity with which the dna genome of hsv is copied (chapter ). mechanisms that give rise to acyclovir resistance include: i hsv pol gene mutants that do not incorporate acyclovir i hsv thymidine kinase (tk) mutants in which tk activity is absent (tk ) or reduced or shows altered substrate specificity strangely, it is possible to find mutations that give rise to each of these phenotypes with a frequency of to in clinical hsv isolates. the discrepancy between this and the very low frequency with which resistance is recorded clinically is probably explained by the observation that most pol/tk mutants appear to be attenuated (e.g., tk mutants of hsv do not reactivate from the latent state). conversely, azt treatment of hiv infection is much less effective. in untreated hiv-infected individuals, azt produces a rise in the numbers of cd cells within À weeks. however, this beneficial effect is transient; after weeks, cd t-cell counts generally revert to baseline. this is due partly to the development of azt resistance in treated hiv populations and to the toxicity of azt on hematopoiesis, as the chemotherapeutic index of azt is much worse than that of acyclovir. azt resistance is initiated by the acquisition of a mutation in the hiv reverse transcriptase (rt) gene at codon . in conjunction with two to three additional mutations in the rt gene, a fully azt-resistant phenotype develops. after weeks of treatment, À % of azt-treated patients develop at least one of these mutations. this high frequency is due to the error-prone nature of reverse transcription (chapter ). because of the large number of replicating hiv genomes in infected patients (chapter ), many mistakes occur continuously. it has been shown that the mutations that confer resistance already exist in untreated virus populations. thus, treatment with azt does not cause but merely selects these resistant viruses from the total pool. with other anti-rt drugs, such as didanosine (ddi), a resistant phenotype can result from a single base pair change, but ddi has an even lower therapeutic index than azt, and relatively low levels of resistance can potentially render this drug useless. however, some combinations of resistant mutations may make it difficult for hiv to replicate, and resistance to one rt inhibitor may counteract resistance to another. the current strategy for therapy of hiv infection is known as haart (highly active antiretroviral therapy) and employs combinations of different drugs such as a protease inhibitor plus two nucleoside rt inhibitors. molecular mechanisms of resistance and drug interactions are both important to consider when designing combination regimes: i combinations such as azt ddi or azt tc have antagonistic patterns of resistance and are effective. i combinations such as ddc tc that show cross-reactive resistance should be avoided. certain protease inhibitors affect liver function and can favorably affect the pharmacokinetics of rt inhibitors taken in combination. other potential benefits of combination antiviral therapy include lower toxicity profiles and the use of drugs that may have different tissue distributions or cell tropisms. combination therapy may also prevent or delay the development of drug resistance. combinations of drugs that can be employed include not only small synthetic molecules but also "biological response modifiers" such as interleukins and ifns. virus infection is a complex, multistage interaction between the virus and the host organism. the course and eventual outcome of any infection are the result of a balance between host and virus processes. host factors involved include exposure to different routes of virus transmission and the control of virus replication by the immune response. virus processes include the initial infection of the host, spread throughout the host, and regulation of gene expression to evade the immune response. medical intervention against virus infections includes the use of vaccines to stimulate the immune response and drugs to inhibit virus replication. molecular biology is stimulating the production of a new generation of antiviral drugs and vaccines. further reading rna-based viral immunity initiated by the dicer family of host immune receptors viral subversion of apoptotic enzymes: escape from death row understanding hiv- latency provides clues for the eradication of long-term reservoirs implications of high rna virus mutation rates: lethal mutagenesis and the antiviral drug ribavirin five questions about viruses and micrornas how do viruses avoid inhibition by endogenous cellular micrornas? clinical applications of dna vaccines: current progress modulation of natural killer cell activity by viruses pros and cons of phage therapy microrna in the immune system, microrna as an immune system crispr interference: rna-directed adaptive immunity in bacteria and archaea new viruses for cancer therapy: meeting clinical needs how do plant viruses induce disease? interactions and interference with host components interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures antiviral drugs for viruses other than human immunodeficiency virus plant virus ecology the prospects and challenges of universal vaccines for influenza viral subversion of the immune system viral tricks to grid-lock the type i interferon system oncolytic viruses as anticancer vaccines. front. oncol. , key: cord- - qu oh authors: dandekar, ajai a.; anghelina, daniela; perlman, stanley title: bystander cd t-cell-mediated demyelination is interferon-γ-dependent in a coronavirus model of multiple sclerosis date: - - journal: the american journal of pathology doi: . /s - ( ) - sha: doc_id: cord_uid: qu oh mice infected with the coronavirus mouse hepatitis virus, strain jhm (jhm) develop a disease that shares many histological characteristics with multiple sclerosis. we previously demonstrated that jhm-infected mice that only have cd t cells specific for an epitope not in the virus develop demyelination on specific activation of these cells. herein we show that this process of bystander t-cell-mediated demyelination is interferon-γ (ifn-γ)-dependent. the absence of ifn-γ abrogated demyelination but did not change t-cell infiltration or expression levels of inflammatory cytokines or chemokines in the spinal cord. these results are consistent with models in which ifn-γ contributes to cd t-cell-mediated demyelination by activation of macrophages/microglia, the final effector cells in the disease process. the human disease multiple sclerosis (ms) is an immunemediated, chronic inflammatory disease of the central nervous system (cns) characterized histologically by multiple focal demyelinating lesions. , although t cells are found in the demyelinating lesions, the specificity of the majority of these t cells is largely unknown. these cells do not generally appear to be autoreactive. intercurrent illnesses in patients with autoimmune disease, including keratitis and ms, often result in temporary worsening of disease. , in some cases, intercurrent infection is the triggering event for the development of disease. several mechanisms have been proposed to account for this phenomenon. [ ] [ ] [ ] the first of these is molecular mimicry, a phenomenon in which t-cell epitopes from a pathogen are sufficiently similar to a host peptide to induce a misdirected t-cell response. another possibility is the release of previously hidden selfepitopes to which t cells have not been tolerized, called epitope spreading. alternatively, the cytokine milieu in the setting of infection might activate and induce the proliferation of nearby autoreactive t cells. finally, t cells activated specifically against an unrelated infectious agent may migrate into areas of chronic inflammation. these cells could then interact with resident cells and soluble mediators at the site of inflammation to induce bystander pathology. mice infected with the neurotropic coronavirus mouse hepatitis virus, strain jhm (jhm) develop acute and chronic demyelinating disease with histopathological similarities to ms. , the process of demyelination is immune-mediated, as rag Ϫ/Ϫ or rag Ϫ/Ϫ (mice deficient in recombination activation enzyme) or scid (mice with severe combined immunodeficiency) mice, which lack b and t lymphocytes, did not develop demyelination on infection with jhm , ; however, if rag Ϫ/Ϫ mice were reconstituted with either jhm-immune cd or cd t cells, demyelination proceeded as in the immunocompetent mouse. in either reconstituted rag Ϫ/Ϫ or wildtype mice, jhm-specific t cells were detected in the infected cns and constituted the majority of the infiltrating cells. [ ] [ ] [ ] although t cells reactive to myelin or other cns epitopes were not identified in jhm-infected mice, the specificity of a large fraction of these cells was not determined in those studies. while some of these cells may target jhm-specific epitopes that are, as yet, unidentified, others are likely to be specific for neither jhm nor cns antigens. previously, we showed that cd t cells specific for either an epitope in lymphocytic choriomeningitis virus (lcmv) or vesicular stomatitis virus were able to mediate demyelination in infected rag Ϫ/Ϫ mice. demyelination was demonstrated most effectively when mice transgenic for t-cell receptors recognizing one of these epitopes were directly infected with jhm and t cells were activated by exposure to cognate antigen in adjuvant. this effect was limited to cd t cells because we showed that specific activation of non-jhm specific cd t cells did not result in bystander demyelination. furthermore, mere activation of transgenic cd t cells with peptide in adjuvant, in the absence of jhm infection, did not result in demyelination. interferon-␥ (ifn-␥) is a critical mediator of homeostasis and inflammation in ms and several of its rodent models, including experimental autoimmune encephalomyelitis (eae) and demyelination mediated by theiler's encephalomyelitis virus. - similarly, in jhm-infected mice, transfer of virus-specific, ifn-␥ Ϫ/Ϫ cd lymphocytes to infected rag Ϫ/Ϫ mice resulted in an % decrease in demyelination as compared to those mice receiving ifn-␥ ϩ/ϩ cd t cells ( . % vs. . %). this decrease was specific for the defect in ifn-␥ as transfer of cd t cells deficient in tumor necrosis factor (tnf)-␣ did not result in a reduction in demyelination. because of the central role of ifn-␥ in demyelination induced by jhm-specific cd t cells, we reasoned that this mechanism might be shared by non-jhm specific cd t cells. to test this hypothesis, we reconstituted rag Ϫ/Ϫ mice with bone marrow cells derived from p transgenic ifn-␥ Ϫ/Ϫ rag Ϫ/Ϫ mice. these mice exhibited little demyelination after jhm infection when compared to mice reconstituted with bone marrow from p transgenic ifn-␥ ϩ/ϩ rag Ϫ/Ϫ mice, indicating that ifn-␥ production is critical for bystander demyelination. pathogen-free p transgenic (p ) rag Ϫ/Ϫ mice were purchased from taconic labs (germantown, ny). these mice are transgenic for a tcr specific for residues - of the lcmv glycoprotein (epitope gp ). ifn-␥ Ϫ/Ϫ mice were a gift from dr. john harty (university of iowa). we bred p ifn-␥ Ϫ/Ϫ rag Ϫ/Ϫ mice from these strains at the university of iowa animal care facility. rag Ϫ/Ϫ mice were purchased from the jackson laboratory (bar harbor, me). all mice were on the c bl/ background. all animal studies were approved by the university of iowa animal care and use committee. the . -v- strain of jhm was kindly provided by dr. john fleming (university of wisconsin, madison, wi). mice were infected with pfu of jhm intracranially and harvested at days post-inoculation (p.i.). to activate t cells, g gp peptide (kavynfatm) (bio-synthesis, lewisville, tx) was emulsified in complete freund's adjuvant (cfa) and administered intraperitoneally. virus was titered by a plaque-reduction assay as previously described. bone marrow chimeras rag Ϫ/Ϫ mice were irradiated with . gy and allowed to rest for hours. bone marrow was harvested from either p rag Ϫ/Ϫ mice or from p ifn-␥ Ϫ/Ϫ rag Ϫ/Ϫ mice, and ϫ cells were injected retro-orbitally into the recipient rag Ϫ/Ϫ mice. six weeks after transfer, peripheral blood mononuclear cells were stained by flow cytometry to ensure that p transgenic t cells were present. staining for peptide-stimulated production of ifn-␥ and tnf-␣ was done as previously described. in brief, lymphocytes were harvested from the cns using a % percoll (pharmacia, uppsala, sweden) gradient and incubated with peptide coated-el- cells at a ratio of : . p peptide or s peptide (the immunodominant cd t-cell epitope recognized in jhm-infected b mice; cslwngphl , ) was used at a final concentration of mol/l. cns-derived lymphocytes were incubated with apcs and peptide for hours in the presence of brefeldin a (bd pharmingen, san diego, ca). cells were stained with fluorescein isothiocyanate (fitc)-conjugated anti-cd mab and pe-conjugated anti-ifn-␥ or tnf-␣ mabs (bd pharmingen). spinal cords were removed from jhm-infected mice, fixed in zinc formalin (labsco, solon, oh) and embedded in paraffin. to determine areas of myelin damage, -m sections were stained with luxol fast blue (lfb) and counterstained with hematoxylin and eosin. areas of total myelin and demyelination were quantified using nih imagej software and the percentage of demyelination was determined. to stain for macrophages/microglia or virus, sections were incubated with antibodies directed against f / (macrophages/microglia, serotec, oxford, uk), diluted : in normal goat serum or the nucleocapsid (n) protein of jhm (mab b . ; : ; a gift from dr. michael buchmeier, the scripps research institute, la jolla, ca). secondary antibodies were biotinylated goat anti-rat ( : ; vector laboratories, burlingame, ca) or anti-mouse igg ( : ; jackson immunoresearch, west grove, pa), respectively. the staining was visualized using streptavidin-peroxidase (jackson immunoresearch) and diaminobenzidine (-aldrich, st. louis, mo). sections were counterstained with hematoxylin. axons were detected with monoclonal antibody cocktail smi- ( : ; sternberger monoclonals, lutherville, md), followed by fitc-conjugated goat anti-mouse secondary antibody ( : ; icn biomedicals, aurora, oh). all mi-croscopy was done on a leica dmb microscope and photographed using an optronics digital camera. rna was extracted from the spinal cords of mice using tri-reagent (molecular research center, cincinnati, oh). we performed rnase protection assays (rpa) as previously described, using probes for the cytokines lymphotoxin-␣, tnf-␣, interleukin (il)- , il- p , il- , and il- ␤; and the chemokines ccl (mcp- ), ccl (mip- ␣), ccl (mip- ␤), ccl (rantes), ccl (mcp- ), cxcl (ip- ), and mip- . these probes were generously provided by dr. iain campbell (the scripps research institute). templates for the probes were linearized with ecori and p-labeled antisense probes were synthesized using t rna polymerase. ten micrograms of spinal cord rna was used for each hybridization reaction. samples were hybridized to probe, treated with rnase and run on a denaturing % polyacrylamide mol/l urea gel. radioactivity was measured using phosphorimagery and quantified using nih image software. statistical significance was determined by unpaired ttests. all results are expressed as means Ϯ sem. values of p Ͻ . were considered statistically significant. given the key role that ifn-␥ has in demyelination mediated by jhm-specific cd t cells, we investigated whether ifn-␥ produced by bystander cells was also important for their ability to cause myelin destruction. for this purpose, we created bone marrow chimeras in which ifn-␥ was only deficient in the t-lymphocyte compartment. the resulting chimeras had only transgenic cd t cells deficient in the ability to produce ifn-␥, but other cells of hematopoetic lineages cells were derived, in part, from the recipient bone marrow, ensuring that such sources of ifn-␥ as nk cells were present. in all experiments, we used chimeras created with bone marrow from p ifn-␥ ϩ/ϩ rag Ϫ/Ϫ mice as controls. chimeras with p transgenic t cells deficient or sufficient for ifn-␥ were infected with jhm. to activate the transgenic t cells, we injected these mice on the day of infection with g lcmv gp peptide in cfa (cfa: gp ). this amount of peptide was previously shown to provide optimal stimulation of the gp -specific tcr transgenic cells. these mice were monitored for the development of signs of demyelination, including hindlimb paralysis, in the weeks following infection. cns tissue was harvested from mice on day p.i. we stained the spinal cords from jhm-infected, cfa: gp -treated, ifn-␥-sufficient or -deficient chimeras with lfb to determine the amount of bystander demyelination in these mice (figure , a and e) . chimeras with p transgenic t cells deficient for ifn-␥ did not develop demyelination substantially above the background level (table ) seen in rag Ϫ/Ϫ or rag Ϫ/Ϫ mice infected with jhm. as such, this % reduction represented a nearly complete abrogation in demyelination in chimeras reconstituted with ifn-␥ Ϫ/Ϫ t cells. wild-type mice infected with jhm develop demyelination that is largely primary demyelination with relative sparing of axons. to test whether jhm infection in our p chimeras similarly resulted in primary demyelination, we stained spinal cord sections with an antibody cocktail that recognizes all axons, as described in materials and methods. areas of demyelination in chimeras containing ifn-␥ ϩ/ϩ t cells all demonstrated positive staining for axons, indicating that the demyelinating process in these mice, as in wild-type mice, is primary ( figure d ). there was no evidence for axonal loss in ifn-␥ Ϫ/Ϫ chimeras ( figure h ), reflecting the minimal amount of demyelination observed in these animals. jhm infection resulted in similar clinical disease in the two groups of mice with cfa:gp -stimulated t cells. signs were largely those of encephalitis (hunching, ruffled fur, lethargy) rather than demyelination (hindlimb paralysis, righting inability), probably as a result of the low amount of demyelination observed. additionally, to determine whether differences in virus clearance contributed to the differences in demyelination, we measured viral titers in the brain at day p.i. (table ). there was not an appreciable difference in viral titers between the two groups. macrophages/microglia are likely the terminal effectors of demyelination in jhm-infected mice. as such, we reasoned that the lack of ifn-␥ produced by activated, non-jhm-specific t cells would result in a decrease in macrophage/microglia infiltration into the white matter of the spinal cord. we used the macrophage/microglia marker f / to identify the location and number of these cells in the white matter of chimeras with ifn-␥-sufficient or -deficient t cells. consistent with previous studies of jhm-induced demyelination, , , ifn-␥-sufficient chimeras had infiltration of macrophages/microglia into the white matter in areas of demyelination (figure , a and b) . ifn-␥-deficient chimeras, however, had limited macrophage/microglia infiltration into the spinal cord. these areas may represent sites of incipient myelin destruction, although little frank demyelination was detected ( figure , e and f). demyelination occurred in the vicinity of virus-infected cells. however, virus antigen was not found in areas of demyelination, suggesting that myelin destruction occurred during the process of virus clearance. (figure , c and g) . an explanation for the lack of demyelination and macrophage/microglia infiltration in chimeras containing ifn-␥ Ϫ/Ϫ lymphocytes is a reduction in t-cell activation or infiltration into the cns, or both. to address this possibility, we used flow cytometric analysis to determine the number of lymphocytes in the cns and their activation status. there were similar influxes of cd transgenic t cells in the cns (table ) , with approximately . ϫ cells in the cns of ifn-␥ ϩ/ϩ chimeras, and . ϫ cells in the cns of ifn-␥ Ϫ/Ϫ chimeras. to determine whether these cns-derived, transgenic t cells were functionally activated, we stained for the intracellular production of cytokines ifn-␥ or tnf-␣ (fig-ure ,a to h) . these cytokines are produced by t cells in response to specific activation through recognition of cognate peptide by the tcr. as expected, ifn-␥ was expressed only by transgenic cd t cells harvested from recipients of ifn-␥ ϩ/ϩ bone marrow (figure , a and e) . there was a similar frequency and number of activated cells in both ifn-␥-sufficient and -deficient chimeras as measured by tnf-␣ staining (figure , b and f; table ). therefore, we conclude that the production of ifn-␥ by specifically activated cells, and not merely activation of t cells, is responsible for bystander demyelination in this system. additionally, there were no cd t cells specific for the immunodominant epitope of jhm (residues - of the spike glycoprotein) in any mice tested ( figure , c, d, g, h) . a recent study demonstrated that, even in the absence of cognate antigen in the cns, activated cd t cells could infiltrate the cns, with subsequent activation of microglia. we were able to detect activated p t cells in the cns of cfa:gp -treated mice, in the absence of jhm infection, although there were roughly six-fold fewer t cells in the cns of uninfected mice than in their jhm-infected counterparts (data not shown). however, in this model system, we were unable to find histological evidence of macrophage/microglia activation or demyelination in the absence of jhm infection ( figure , i and j). we next asked whether the difference in ifn-␥ production by activated lymphocytes in the cns would result in differential expression of other cytokines or chemokines within the cns. to answer this question, we extracted rna from whole spinal cords of chimeras with ifn-␥sufficient and -deficient lymphocytes, and quantified the mrna levels of eight chemokines and six cytokines by rpa, as described in materials and methods. tnf-␣ was the only cytokine detected of the six assayed and it was present at similar levels in both groups of mice (data not shown). message levels of the chemokines tested were similar in the two experimental groups (figure ). there was a trend toward higher levels of cxcl and ccl in those mice containing ifn-␥ ϩ/ϩ lymphocytes, although this did not reach statistical significance. in mice treated with cfa:gp but not infected with jhm, we could not detect significant amounts of any chemokine by rpa, consistent with our finding that macrophages/microglia were not activated in this setting (figure and figure j ). a key element of the present study was the use of bone marrow chimeras to address the role of ifn-␥ in by-stander demyelination mediated by cd t cells. the production of these chimeras allowed us to address the hypothesis that ifn-␥ produced by cd t cells, and not from other sources, was the critical element in mediating bystander demyelination. furthermore, this approach did not compromise ifn-␥ production by cells such as nk cells and dendritic cells, thereby preserving the innate immune response to the virus. , the results showed that ifn-␥ produced by these innate cells was unable to initiate the demyelinating process, even in the context of activated cd t cells lacking only the ability to produce ifn-␥. our results show that bystander demyelination mediated by cd t cells is ifn-␥-dependent and thereby is similar to that mediated by jhm-specific cd t cells. these results highlight the important role that cd t cells have in demyelination in jhm-infected mice. ifn-␥ has also been shown to be critical in other models of demyelination and in ms. in rodents with eae induced by cd t cells specific for a cns antigen, demyelination is greatly diminished in the absence of ifn-␥. in all cases, the presence of cd t-cell-produced ifn-␥ results in increased macrophage/microglia infiltration into the white matter, with subsequent demyelination. similarly, cd t cells are abundant in ms lesions and treatment with anti-ifn-␥ antibody appeared to reduce disability in a small (n ϭ patients receiving treatment) clinical trial. consistent with these data, administration of ifn-␥ to patients with ms resulted in disease exacerbation. in patients with ms, the source of the ifn-␥ that leads to disease exacerbation is unknown, but based on our results, it may be from cd t cells responding to an intercurrent infection. from our results, it appears that ifn-␥ and not any other cytokine or chemokine was the key mediator of bystander demyelination (figure ). however, because our screen for chemokines and cytokines was not exhaustive, it is possible that other soluble factors were affected by the presence or absence of cd t-lymphocyte-produced ifn-␥. alternatively, differences in local concentration of chemokines or cytokines may be critical in the demyelinating process and might not be evident in this assay. it is also possible that ifn-␥ affects we also measured cytokine and chemokine levels for uninfected, but cfa: gp -treated, ifn-␥ ϩ/ϩ (hatched bars) and ifn-␥ Ϫ/Ϫ (shaded bars) chimeras. *, no mrna for this chemokine was detectable in these mice. values are normalized to expression of the housekeeping gene l . there were no statistically significant differences in measured chemokine levels between jhm-infected ifn-␥ Ϫ/Ϫ and ifn-␥ ϩ/ϩ chimeras or between uninfected ifn-␥ Ϫ/Ϫ and ifn-␥ ϩ/ϩ chimeras. we analyzed ifn-␥ ϩ/ϩ and ifn-␥ Ϫ/Ϫ jhm-infected, cfa:gp -treated chimeras, and mice in each group of the uninfected limb. the functions of non-soluble molecules that may be involved in macrophage/microglia activation. the amount of demyelination observed in these jhminfected bone marrow chimeras was substantially less than that seen in infected, rag Ϫ/Ϫ mice reconstituted with jhm-immune splenocytes. this relatively low level of demyelination was also seen in our previous study using nonchimeric mice. because the p t cells are not specific for jhm, this suggests that nonspecific t cells are substantially less efficient at mediating demyelination than are virus-specific cells, probably because cognate antigen-tcr interactions do not take place within the cns in this model. analogously, such a low but significant level of demyelination would be consistent with a role for activated, bystander t cells in clinical exacerbations in patients with ms. in contrast to the results obtained with cd t cells, ifn-␥ is a negative regulator of demyelination in eae mediated by cd t cells. , , in ifn-␥ Ϫ/Ϫ or ifn-␥r Ϫ/Ϫ mice with eae, disease was more severe and greater numbers of cd t cells were detected in the cns than in wild-type mice with eae. most strikingly, a neutrophilic infiltration into the cns occurred in the absence of ifn-␥ and chemokine production was greatly altered. similarly, in jhm-infected rag Ϫ/Ϫ mice, adoptive transfer of jhm-immune cd t cells from ifn-␥ Ϫ/Ϫ donors resulted in more severe disease and enhanced demyelination when compared to recipients of wild-type cd t cells. thus, in the context of cd t cells, ifn-␥ acts, at least in part, to down-modulate the immune response, resulting in less myelin destruction. collectively, these results highlight the importance of cd t cells in demyelinating diseases, including ms. the results of the study presented here also emphasize the central role that ifn-␥ has in this process, both in the context of cd 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the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity the jhm strain of mouse hepatitis virus induces a spike protein-specific d b -restricted ctl response cd ϩtcrϩ and cd ϩtcr-cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus a central role for cd ϩ t-cells and rantes in virus-induced central nervous system inflammation and demyelination intracellular staining for tnf and ifngamma detects different frequencies of antigen-specific cd (ϩ) t cells effective and selective immune surveillance of the brain by mhc class i-restricted cytotoxic t lymphocytes improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood a pathogenic role for myelin-specific cd (ϩ) t cells in a model for multiple sclerosis clonal expansions of cd (ϩ) t cells dominate the t cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction randomized study of antibodies to ifngamma and tnf-␣ in secondary progressive multiple sclerosis exacerbations of multiple sclerosis in patients treated with gamma interferon cd t-cell-mediated demyelination is increased in the absence of gamma interferon in mice infected with mouse hepatitis virus we thank dr. jodie haring and taeg kim for critical review of the manuscript. key: cord- -b xb f authors: hulst, marcel; kerstens, hinri; de wit, agnes; smits, mari; van der meulen, jan; niewold, theo title: early transcriptional response in the jejunum of germ-free piglets after oral infection with virulent rotavirus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: b xb f germ-free piglets were orally infected with virulent rotavirus to collect jejunal mucosal scrapings at and hours post infection (two piglets per time point). ifn-gamma mrna expression was stimulated in the mucosa of all four infected piglets, indicating that they all responded to the rotavirus infection. rna pools prepared from two infected piglets were used to compare whole mucosal gene expression at and hpi to expression in uninfected germ-free piglets (n = ) using a porcine intestinal cdna microarray. microarray analysis identified down-regulated and up-regulated genes. northern blot analysis of a selected group of genes confirmed the data of the microarray. genes were functionally clustered in interferon-regulated genes, proliferation/differentiation genes, apoptosis genes, cytoskeleton genes, signal transduction genes, and enterocyte digestive, absorptive, and transport genes. down-regulation of the transport gene cluster reflected in part the loss of rotavirus-infected enterocytes from the villous tips. data mining suggested that several genes were regulated in lower- or mid-villus immature enterocytes and goblet cells, probably to support repair of the damaged epithelial cell layer at the villous tips. furthermore, up-regulation was observed for ifn-γ induced guanylate binding protein , a protein that effectively inhibited vsv and emcv replication in vitro (arch virol : – , ). this protein may play a role in the small intestine’s innate defense against enteric viruses like rotavirus. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. with an estimated death rate of more than , per year, mainly affecting children less than years of age in developing countries, rotavirus is recognized as one of the major infectious diseases of the gastrointestinal tract [ ] . rotaviruses are members of the family reoviridae, viruses with segmented double-stranded rna genomes [ ] . in the small intestine, mature enterocytes near the top of the villi are the primary target cells for virus replication [ ] . replication in these cells provokes numerous intraand extracellular pathological changes that inevitably lead to disruption of the absorptive and digestive functions of the small intestine, and consequently, to malabsorption and diarrhea. these changes include destruction of enterocyte brush borders, enterocyte vacuolization, loss and destruction of enterocytes, villus blunting and atrophy, thinning of the intestinal wall, and crypt hyperplasia (for comprehensive reviews, see [ , ] ). however, the nature and severity of histopathological alterations in vivo can be quite different depending on the species and virulence of the rotavirus strain. there is no clear correlation between these alterations and manifestation of clinical symptoms. a systemic inflammatory response can be absent, and rotavirus infections can be asymptomatic [ , ] . this suggests that the interplay between host and viral factors is important for determining the course of this disease. for electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. instance, rotavirus nsp acts as an enterotoxin that induces diarrhea in mice in the absence of rotavirus replication [ ] . nsp affects ca + and electrolyte homeostasis in an auto-and paracrine fashion in both rotavirus-infected and uninfected intestinal cells [ ] . nsp increases ca + permeability of the er and plasma membrane, resulting in an increased ca + concentration in the cytosol ([ca + ] cyt ), causing derailment of numerous ca + -dependent cellular processes [ ] . in uninfected enterocytes and crypt cells this rise in [ca + ] cyt is induced by binding of exogenous nsp to an apical receptor that modulates the plc-ip pathway [ ] . the higher [ca + ] cyt triggers laminal secretion of peptides and amines by uninfected enterocytes, and luminal cland h o secretion by crypt cells [ , ] . in infected enterocytes, the rise in [ca + ] cyt is believed to be independent of plc modulation [ ] , and this rise perturbs cytoskeleton and tight junction integrity, which ultimately leads to cell lysis [ , , , ] . in vitro studies with cell lines, mainly derived from colon, have contributed significantly toward understanding the pathogenesis of rotavirus on a molecular level. however, the intestinal mucosa consists of a diversity of specialized cell types in different states of differentiation. presumably, all these different types of cells respond differently to environmental changes, and accordingly to changes in their neighboring cells. therefore, the regulation of genes responsible for these complex phenotypic responses in vivo may not be detected by challenging single types of cultured cells with rotavirus. to address this issue, we studied the early transcriptional response in jejunal mucosa of -week-old, just-weaned piglets after oral infection with virulent group a rotavirus. to assign measured responses exclusively to rotavirus, we performed these experiments in germ-free piglets. differential expression patterns of uninfected versus infected jejunum were recorded and h before severe diarrhea was expected, using a homemade pig intestinal cdna microarray [ ] . the biological significance of elevated or reduced expression of these genes for rotavirus pathogenesis is discussed. seven germ-free piglets (groot yorkshire [cofok large white]) were obtained by caesarean section and housed in isolators, fed with sterilized condensed milk till the age of days and thereafter with pelleted feed (sterilized by x-ray radiation) and water ad lib. on day , three of the seven piglets were transported to the necropsy room and served as uninfected control piglets. the four remaining pigs were orally infected with virus suspension diluted in a total volume of ml pbs and containing rotavirus particles (as determined by negative-stain semi-quantitative electron microscopy) of strain rv [ ] . the virus suspension was prepared from the contents of the small and large intestine of a rotavirus-infected gnotobiotic piglet [ ] . the above applied oral dose caused severe diarrhea from hpi (hours post infection) in -week-old gnotobiotic piglets [ ] . infected piglets were housed in their isolators under the same conditions as described above for another period of (two piglets) or h (two piglets) before they were transported to the necropsy room. immediately after arrival in the necropsy room, ml of edta blood for hematological analysis was collected from the jugular vein. subsequently, animals were killed by barbiturate overdose and their intestines were taken out. the jejunum was opened and rinsed with cold saline, and cm of mucosa in the middle of the jejunum was scraped off with a glass slide, frozen in liquid nitrogen, and kept at - °c until rna and dna extraction. an adjacent part of the collected jejunum was fixed in % formaldehyde and used to determine the villus height and crypt depth. villus and crypt dimensions were determined on hematoxylin-eosinstained -lm tissue sections [ ] . during the experiment, fecal samples were collected at , and hpi from the rectum for determination of the percent dry matter [ ] . fecal samples were tested for the presence or absence of rotavirus by elisa [ ] . the germ-free status of each piglet was confirmed by analyzing throat saliva and feces samples, collected on days , and , and on the day of slaughter, for the presence of microorganisms. isolation of rna and dna from g of frozen mucosal scrapings, total rna (dnasefree) was isolated using trizol Ò reagent (invitrogen) as described recently [ ] . the yield per gram of tissue and the purity of the rna were calculated from measurement of the extinction at and nm. the integrity of all rna samples was checked by analyzing lg of rna on a denaturizing % (w/v) agarose gel. after ethidium bromide staining, the gel was scanned to calculate the s/ s peak ratio (volume s over volume s) for each preparation. rna with a ratio [ was considered of adequate quality to be used for real-time pcr and microarray analysis. a part of the isolated rna was used to prepare rna pools for microarray analysis. a control pool was prepared by mixing equal amounts of rna isolated from the jejunum of the three uninfected piglets (n = ). the same was done for the two infected piglets slaughtered at h and for the two piglets slaughtered at h. after gentle homogenization in lysis buffer, dna was extracted from . g of frozen mucosal scrapings, and lg of purified dna was analyzed on a . % agarose gel [ ] . the relative concentrations of interferon-gamma (ifn-c) and ornithine decarboxylase antizyme (oaz ) mrna in all rna samples was determined by real-time pcr. two hundred ng of total rna was reverse transcribed in a standard rt reaction using superscript ii reverse transcriptase (invitrogen) and pd(n) primers. ifn-c cdna in these rt reactions was quantified using labeled light-cycler probes (roche diagnostics) as described [ ] and expressed as pg/ll control plasmid. a -mer forward primer ( -gacccgacgcttgcttcatg- ) and a mer reverse primer ( -gagtgagcgtttatttgcac- ), generating a cdna fragment homolog to nucleotide - of the human oaz mrna reference sequence (gi: ), were used to quantify oaz cdna using cybergreen as label in a standard lightcycler reaction. the relative concentration of oaz mrna was calculated by extrapolation on a standard curve prepared from dilutions of an rt reaction prepared from a reference rna sample [ ] . the quantity of s rrna in each rna sample was determined using the above described rt reactions by real-time pcr [ ] and used to normalize the ifn-c and oaz concentrations. the quantity of s rrna showed no essential differences among all individual rna samples (average concentration ± sd; . ± . lg/ll of control plasmid). the same collection of pig probes (ests) used in earlier studies [ , ] were spotted in triplicate on corning ul-tragaps slides. briefly, this collection consisted of , probes prepared from jejunal mucosal scrapings collected from -week-( ) and -week-( , ) old pigs, probes coding for porcine cytokines (ifn-c, tnf-a, gmcsf, il- , , , , and ) and lung surfactant proteins sftpa and sftpd, and marc and marc probes (porcine ests) homolog to trefoils, collectins, defensins, and glycosyltransferases [ ] . a list of the probes already sequenced/ annotated is accessible on the website of arch virol (gene list hulst et al. pdf). dual-color (cy -cy ) hybridization of slides was performed using the rna micromax tsa labeling and detection kit (perkinelmer) as described earlier [ ] . messenger rna levels in both infected pools ( and hpi) were independently compared to the expression levels in the control (uninfected) pool. for each comparison, a dye swap was performed. in addition, a control hybridization experiment was performed in which a microarray slide was simultaneously hybridized with cy labeled control rna and cy -labeled control rna. scanning of slides, processing of raw images, creation of data reports, data-normalization and statistical analysis were performed as described by niewold et al. [ ] with minor modifications. briefly, probes were considered to be differentially expressed when at least four of the six data points (spots) on both dye swap slides hybridized with a ratio of . -fold (m = [log (cy /cy )] \ - . or [ . ) or more ( . is considered significant according to the manufacturer of the tsa kit) and were identified by significant analysis of microarrays (sam) [ ] with a median false discovery rate (fdr or q value) of \ %. equal amounts of total rna ( lg) were separated on a denaturizing % (w/v) agarose gel. after several washes with rnase-free water, the gel was blotted on hybond-n membranes (amersham), and blots were hybridized with p-labeled dna fragments homolog to the mrna in question, in the same manner as was described in an earlier study [ ] . after post-hybridization washes, the blots were scanned using a storm phosphor-imager (molecular dynamics, sunnyvale, california, usa). four -week-old germ-free piglets were orally infected with a dose of rotavirus that caused severe diarrhea from hpi in -week-old gnotobiotic piglets [ ] . for practical reasons, three uninfected germ-free piglets were slaughtered at the zero time point (mock, see table ). in order to isolate highquality rna from jejunal mucosal scrapings, infected piglets were slaughtered and hpi. thus, and h before severe diarrhea would have been induced. in three of the four infected animals, rotavirus was detected in their feces. determination of the percent dry matter showed that only the fecal samples collected hpi (piglets and ) had a significantly lower (pasty) consistency [ ] . this indicated that not all of the piglets developed diarrhea before hpi, and that the two piglets slaughtered hpi did not develop the severe form of diarrhea normally observed at hpi [ ] . in jejunal tissue sections prepared from these two -h piglets, villus length was decreased to two-thirds of the average length measured in corresponding sections prepared from the three control piglets. no significant differences in crypt depths were observed between infected and control animals. for both piglets that were slaughtered at h, these results indicated that the orally applied rotavirus reached the transcriptional response to rotavirus jejunum and induced the desired limited (not severe) pathological symptoms. in addition, the lower concentration of lymphocytes in the blood indicated that the animals were effectively infected with rotavirus [ ] . in the feces of piglet , slaughtered at h, no rotavirus could be detected. moreover, only a small decrease in villus length ( %) was observed for this piglet and its -h replicate. to find additional evidence whether the jejunum of piglet was effectively challenged with rotavirus, the level of ifn-c mrna in jejunal mucosal scrapings was measured by real-time pcr (fig. ). in rna samples isolated from the three uninfected pigs, hardly any ifn-c mrna could be detected. in contrast, the ifn-c mrna levels in scrapings of all infected piglets were up to -fold higher than in uninfected piglets, whereas oaz mrna levels were nearly equal for all rna samples. this indicated that the jejunum of all piglets, including piglet , responded to the orally applied rotavirus challenge. despite the loss of cells from the tip of the villi, the amount of rna (table ) isolated from all infected piglets was comparable to that of uninfected piglets. on an ethidium-bromide-stained agarose gel, no degradation of rna was visible for any of the extracted rna samples (see also fig. a ). in addition, s/ s peak ratios were [ for all these samples (table ). these results showed that scrapings collected from all piglets yielded high-quality rna suitable for real-time pcr and microarray analysis. no random (necrotic) and/or fragmentized (apoptotic) dna was visible after gel electrophoreses of dna samples extracted from any of the piglets, indicating that the majority of cells imbedded in the epithelial layers of all the piglets were not apoptotic or necrotic (results not shown). mid-jejunal mucosal gene expression analysis was performed using a homemade pig cdna small intestinal microarray [ ] . in two separate hybridization experiments, mrna expression levels in an uninfected rna pool (n = ) were compared to expression levels in rna pools prepared from -and -h infected piglets (both n = ). for both comparisons, dye swaps were performed. probes that hybridized differentially with a ratio (fc; infected over uninfected) of . or [ . in both slides of the dye-swap and that were identified with the lowest possible false-discovery rate (fdr; based on sam, [ ] ), i.e., . % for the -h comparison and . % for the -h comparison, were selected for further analysis. raising of the fdr to a maximum of % did not identify additional probes with a fc \ . or [ . in either comparison. selected probes were sequenced and annotated after blastn or blastx analysis (when not yet annotated). for each differential expressed probe, the mean fc calculated from the two dye-swap slides is presented in table . when cy -and cy -labeled cdna was prepared from the same uninfected rna pool and simultaneously hybridized on the array, none of the probes that hybridized differentially in the -and -h comparisons hybridized differentially (results not shown). six out of the nine probes that hybridized with a fc of . -fold or more in the -h comparison (panel ''higher in infected'') also hybridized significantly more strongly in the -h comparison. for three of these probes (r -r ), the ratio of differential expression further increased with time. in contrast, only one probe (r ) hybridized significantly less strongly at both time points. based on literature search and data mining, a tentative function was assigned for the genes identified by blast analysis (see table ). in addition, the fc (infected over uninfected) of genes which were also found to be regulated in a previous study by cuadras et al. [ ] , i.e., h after infection of human intestinal epithelial caco- cells with rotavirus live virus vaccine rvv, is provided in parentheses in table after the annotations. probes coding for ifn-c, tnf-a, gm-csf, and il- , , , , and were spotted on the array. however, the fluorescence intensity was not higher than the background threshold for any of these probes, indicating that mrna concentrations of these cytokines (including ifn-c) in infected and uninfected mucosal scrapings were too low to detect by microarray analysis, probably due to the relatively low percentage of cytokine-producing (immune) cells present in intestinal mucosa [ ] . northern blots (nb) loaded with equal amounts of rna from each of the piglets were hybridized with p -labeled cdna probes homologous to six differentially expressed mrnas and to three mrnas that were not identified as differentially expressed. for all nine mrnas, the length of the transcript(s) detected on blots were comparable to the length of porcine or human mrna reference sequences posted in the ncbi databank or reported in the literature. in accordance with array data, nb analysis showed that expression levels of gbp- , krt , sat, mgam, and casp mrnas were significantly higher in both -hinfected piglets than in uninfected piglets. for all of these mrnas, hybridization intensities for piglet were nearly equal to that of piglet . gbp- , krt , sat, and mgam mrna expression was also up-regulated in infected piglet , slaughtered at hpi. this indicated that the response to rotavirus infection in this -h piglet was comparable to the response observed in both -h piglets. however, no significant up-regulation of these mrnas was observed in the other piglet slaughtered at h ( ), indicating that this piglet responded differently to the rotavirus infection than its -h replicate and the two piglets slaughtered at h. in accordance with array data, nb analysis showed that the expression level of ifabp mrna was significantly lower in -h-infected piglets than in uninfected piglets. for mrnas that showed no significant differential expression on the arrays (glutathione-s-transferase, calbindin-d, and aldolase-b), no large differences in hybridization intensities were observed between uninfected piglets and the two piglets slaughtered hpi and one of the piglets slaughtered hpi ( ). however, significantly lower hybridization intensities were observed for calbindin-d and aldolase-b mrnas for piglet than for its -h replicate. using microarray analysis, we detected a set of genes that are differently expressed in rotavirus-infected jejunal mucosa compared to uninfected mucosa. for nine mrnas, expression levels in individual piglets were analyzed by nb. these analysis confirmed the array data. in addition, nb analysis showed that one piglet slaughtered at hpi responded quite similarly to rotavirus infection as both h piglets did, whereas its -h replicate did not, despite the fact that this latter piglet also showed an ifn-c mrna response. in addition, out of the genes differentially expressed at hpi also reacted at hpi. these results indicated that three out of four infected piglets responded quite analogously. because we used a limited number of germ-free piglets per time point and measured responses in a mixed population of cells, we imposed stringent criteria for selection of genes ([ . -fold up-or down-regulation and a false-discovery ratio of less than %). using this approach, we minimized the chance of selecting genes hybridizing differentially solely due to inter-animal variation in gene expression and/or cell composition. however, such stringent selection criteria could have excluded the detection of more rotavirus-regulated genes, especially of genes regulated exclusively in specific types of cells that are present in low quantities in the jejunal mucosa. the different responsiveness of one of the -h piglets, however, obliged us to interpret our overall results carefully, especially, concerning the five genes that reacted solely at hpi (txn, frk, dppc- , if , and actb; see table ). nevertheless, data mining revealed relevant relationships between these five genes, h response genes, and processes known to be important for rotavirus pathogenesis. cuardras et al. [ ] measured the transcriptional response in the human enterocyte cell line caco- , , , and hpi with rhesus rotavirus live vaccine. four genes up-regulated in our experiments (gbp- , sat, mgam, ppa ) were also up-regulated hpi in caco- cells. recently, aich et al. [ ] profiled the transcriptional response in surgically prepared jejunal loops from -dayold colostrum-deprived calves after h of perfusion with bovine rotavirus (brv). several genes for which we detected more than . -fold up-or down-regulation (txn, nadh , sglt , actb, sat, casp , and ppa ) were also present on the cdna array they used (ncbi geo acc. number gpl ). none of these genes showed a differential expression of twofold or more in their study. the different route of administration and virulence of the strain used, the digestive differences between the jejunum of omnivores and herbivores, and, in the case of the study of cuardras et al., the various specialized cell-types present in the jejunum of living animals versus cultured colon-derived caco- cells are probably responsible for the poor correspondence between these three studies. based on relevant literature and functional information in databanks, we assigned a function and a possible type(s) of cell(s) responsible for expression for most of the genes on our list (fig. ) . in this hypothetical model, information from existing models dealing with the pathogenesis of rotavirus infection [ , , ] and the development and maintenance of the small intestinal epithelium [ ] were used to fit in our data. possible functions of these genes in relation to processes and pathways known to be important for rotavirus pathogenesis are discussed below. measurements of villus length indicated that considerable numbers of epithelial cells were lost from the tip of the villi, including (infected) mature enterocytes. in part, down-regulation of genes involved in transport of ions and nutrients over the membranes of mature enterocytes, like meprin a, sglt , and ifabp , may be a direct result of this loss. in another part, replication of rotavirus in enterocytes imbedded in the epithelial layer could have down-regulated transcription of these genes. this may also be the case for other down-regulated genes from our list, especially for genes detected only at hpi (r -r , table ). in addition to down-regulation of sglt , ifabp , and meprin a, we observed up-regulation of two other genes that may affect the absorptive and digestive function of the intestine: a gene coding for a protein carrying a ca + -permeable cation channel cd /ige fc receptor subunit b domain (ms a ) and a gene homolog to a bacterial oligo/dipeptide permease (dppc- ). it is tempting to link up-regulation of ms a directly to nsp induced enhancement of ca + permeability of the plasma and er membranes in intestinal epithelial cells [ , ] . likewise, up-regulation of the dppc- homolog may be related to enhanced laminal secretion of peptides and amines by uninfected epithelial cells, a process believed to be triggered by raised [ca + ] cyt [ ] . characterization of these porcine transcripts/proteins is needed to provide further insight in the role of these genes in rotavirus pathogenesis. the same applies for the tmem b gene. this gene showed the highest level of up-regulation. so far, only a duf protein domain with unknown function has been predicted in tmem b. recently, it was reported that rotavirus infection in infant mice induced apoptosis in vivo [ ] . although dna analysis showed that the majority of cells present in infected mucosal scrapings were not apoptotic, we observed upregulation of the apoptosis effector protein casp . this suggests that programmed cell death in the epithelial layer was stimulated by rotavirus infection. nb analysis detected a considerable level of casp mrna expression in uninfected mucosal scrapings. this constitutive expression of casp is, most likely, related to the process of maintenance of the absorptive status of the intestinal epithelial layer. a process in which mature enterocytes continually die due to apoptosis and are replaced by differentiating cells migrating from surrounding crypts to the tip of the villi [ ] . in contrast to mature enterocytes, an in vivo study in mice showed that goblet cells are largely spared from apoptosis in rotavirus-infected mice [ ] . moreover, migration of goblet cells from the crypt to the tips of the villi was stimulated in these mice. we found up-regulation of the goblet cell marker gene krt [ ] and the lower and mid-villus immature enterocyte marker gene mgam [ ] . this could indicate that transcriptional activity in both of these cell types was promoted. stimulation of apoptosis in rotavirus-infected enterocytes and higher proliferation/differentiating activity in goblet cells and immature enterocytes could be a coordinated response of the jejunum to remove infected enterocytes and overlay villus tips with fresh enterocytes, goblet cells, and mucus layer. we did not detect genes on our array that were directly associated with cell-cycle progression/arrest. however, down-regulation of the nuclear kinase frk (an antagonist of cell proliferation) and tms f may be associated indirectly with this process. in humans, tms f is strongly homologous to tm sf , a protein that reduced the ability of the crypt cell line ht to proliferate [ ] . several other genes that may play a role in cell death and repair were regulated. sat was up-regulated, and txn and prr were down-regulated. for this later protein, reduced expression in cells was correlated with increased sensitivity to taxane-induced cell death, a caspase-independent process characterized by the polymerization of tubulins to extraordinarily stable microtubules [ , ] . txn is the major carrier of redox potential in cells, and it is crucial for the defence of cells against oxidative-stressmediated apoptosis. txn also regulates gene expression by increasing binding of redox-sensitive transcription factors like p [ ] , nf-jb [ ] , and the nrf- /polyamine- table . information about the paneth cell marker thoc is provided in reference [ ] transcriptional response to rotavirus inhibited sat expression [ ] . therefore, up-regulation of sat gene expression at hpi may be directly related to down-regulation of txn at hpi. sat is a rate-limiting enzyme in spermine/spermidine metabolism. acetylation of these polyamines by sat promoted their degradation and excretion [ ] . recently, it was reported that depletion of polyamines suppresses apoptosis in normal intestinal epithelial cells by akt-kinase-mediated inhibition of casp activity [ ] . nb analysis showed that the increase in sat mrna expression coincided with the goblet cell marker krt . therefore, it would be interesting to determine whether sat expression in goblet cells can be stimulated by rotavirus infection and whether it plays a role in protecting these cells from apoptosis [ ] . interestingly, it was recently demonstrated that rna viruses can directly modulate polyamine metabolism by regulation of sat transcription and splicing [ ] . a most interesting gene that we found more than tenfold up-regulated codes for an as yet not-well-characterized hypothetical human protein (loc ) carrying a phospholipase a inhibitor domain (pla -inh). the magnitude and kinetics of up-regulation of this gene corresponded exactly with gcnt , suggesting that this gene was expressed in the same types of cells as gcnt , most likely mucus-producing goblet cells and/or differentiating (immature) enterocytes. the amino acid sequence translated from our pla -inh est showed an overall amino acid identity of %, and all cysteines aligned perfectly with cysteines of the human loc protein and of other proteins that bear a typical pla -inh domain. pla s comprise a diverse family of cytosolic and secreted enzymes that hydrolyze membrane phospholipids to free fatty acids. they play an important role in many exogenous and intracellular processes, ranging from fatty acid metabolism and lysis of membranes to the synthesis of arachidonic acid (a-acid), an essential precursor for the production of inflammatory mediators such as eicosanoids. secreted pla s are calcium-dependent enzymes. cytosolic pla s (cpla ) can also be calcium-independent. a moderate increase in [ca + ] cyt mediated translocation of calcium-dependent cpla to intracellular membranes where it hydrolyses phospholipids to a-acid [ ] . a similar effect was observed after activation of the ms a calcium-permeable cation channel (up-regulated here at hpi, see above) on the surface of mast cells [ ] . perhaps, enhanced expression of a pla -inh in our study may be a countermeasure of specific intestinal epithelial cells to normalize and/or inhibit pla enzyme activity in response to extra-and intracellular changes in [ca + ] evoked by rotavirus replication, either to protect specific cells from extra-and intracellular membrane-damage or to negatively regulate a-acid production. with respect to the latter process, we observed down-regulation of the enzymes cyp c and acsl , which both utilize a-acid acid as substrate. interestingly, the capsid protein of parvovirus possesses pla activity [ ] , and hmcv particles carry a cell-derived pla activity [ ] . for both viruses, pla activity appeared to be essential for infectivity. our results showed that ifn-c mrna expression in the jejunum of infected piglets peaked around hpi and tended to decline beyond this time point (see fig. ). this suggests that ifn-c was produced for a short period. recently, aich et al. [ ] measured the mrna expression levels of several cytokines in jejunal loops perfused for h with brv. however, they observed no ifn-c mrna response. because our orally administered rotavirus needed time to reach the jejunum, our h infection period represents a shorter period than h of perfusion. after h of perfusion, expression of ifn-c mrna may have dropped to normal levels. interestingly, they did detect a rotavirus-induced il- (alias ifn-b ) mrna response at hpi [ ] . recent studies showed that the interplay between ifn-gamma and il- controls the influx and clearance of neutrophils and, subsequently, the transition to a more sustainable influx of mononuclear cells during acute inflammation [ , ] . therefore, it would be interesting to study which immune cells produce ifn-c and il- and whether an orchestrated action of these cells regulates an influx of vital immune cells in the jejunum after rotavirus infection. the ifn-c-inducible gbp- gene was up-regulated hpi. overexpression of gbp- and gbp- in hela cells and nih t cells abrogated the cytopathogenic effect mediated by vsv and emcv, respectively, by an unknown mechanism [ , ] . furthermore, it was shown that expression of murine gbp- in nih t cells neutralized the cytotoxic effect of the taxane drug paclitaxel [ ] . this drug specifically stimulates polymerization of tubulins to extraordinarily stable microtubules. these stable microtubules interfere with the function of normal microtubule filaments and inevitably induce cell death [ ] . the reduced expression of prr we observed (discussed above) may be an indication that the formation of extraordinarily stable microtubules in intestinal epithelial cells actually takes place in response to rotavirus infection. in fact, several studies have shown that rotavirus infection induces disorganization of the cytoskeleton network and microtubule filaments in enterocytes [ , , ] . moreover, we also observed the up-regulation of the ifn-a/b-inducible gene ifi , a 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regulate cell density-dependent proliferation a viral phospholipase a is required for parvovirus infectivity akt kinase activation blocks apoptosis in intestinal epithelial cells by inhibiting caspase- after polyamine depletion keratin serine phosphorylation is a stress and intestinal goblet cell marker acknowledgments the authors would like to thank arie hoogendoorn for his assistance in the animal experiment.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord- -p ew w authors: ramana, chilakamarti v. title: regulation of early growth response- (egr- ) gene expression by stat -independent type i interferon signaling and respiratory viruses date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: p ew w respiratory virus infection is one of the leading causes of death in the world. activation of the jak-stat pathway by interferon-alpha/beta (ifn-α/β) in lung epithelial cells is critical for innate immunity to respiratory viruses. genetic and biochemical studies have shown that transcriptional regulation by ifn-α/β required the formation of interferon-stimulated gene factor- (isgf- ) complex consisting of stat , stat , and irf transcription factors. furthermore, ifn α/β receptor activates multiple signal transduction pathways in parallel to the jak-stat pathway and induces several transcription factors at mrna levels resulting in the secondary and tertiary rounds of transcription. transcriptional factor profiling in the transcriptome and rna analysis revealed that early growth response- (egr- ) was rapidly induced by ifn-α/β and toll-like receptor (tlr) ligands in multiple cell types. studies in mutant cell lines lacking components of the isgf- complex revealed that ifn-β induction of egr- was independent of stat , stat , or irf . activation of the mek/erk- / pathway was implicated in the rapid induction of egr- by ifn-β in serum-starved mouse lung epithelial cells. interrogation of multiple microarray datasets revealed that respiratory viruses including coronaviruses regulated egr- expression in human lung cell lines. furthermore, egr- inducible genes including transcription factors, mediators of cell growth, and chemokines were differentially regulated in the human lung cell lines after coronavirus infection, and in the lung biopsies of covid- patients. rapid induction by interferons, tlr ligands, and respiratory viruses suggests a critical role for egr- in antiviral response and inflammation with potential implications for human health and disease. interferons (ifns) are pleiotropic cytokines that play a central role in innate and adaptive immunity ( , ) . there are major types of interferons. the type i interferons consists of ifn-alpha/beta (ifn-α/β ). type ii interferon represented by interferon-gamma (ifn-γ), and type iii interferons represented by interferon lambda (ifn-λ ) . the biological effects of ifn are mediated mainly by the rapid and dramatic changes in gene expression ( )). type i ifn signaling involves the binding of ifn-α/β to its receptor (ifnar) and activation of receptor-associated janus protein tyrosine kinases jak and tyk and the phosphorylation of stat and stat to form a heterodimer. this heterodimer associate with the interferon responsive factor (irf ) to form the interferon-stimulated gene factor- (isgf- ) complex on interferon-stimulated response elements (isre) in the promoters of type i ifn responsive genes to regulate transcription ( , , ) . stat homodimer or heterodimer of stat /stat can also bind to gamma activated sequence (gas) in the promoter and regulate transcription of some type i ifn responsive genes ( , ) . in addition to this classical canonical jak-stat pathway, non-canonical pathways involving stat , irf , and unphosphorylated isgf- components in the regulation of gene expression have been described ( ) . furthermore, several signal transduction pathways are activated by ifn receptors in parallel to jak-stat including extracellular signal-regulated kinases (erk- / ) and phosphoinositide '-kinase/akt pathways ( , ) . the first wave of interferon signaling is followed by induction of transcription factors such as interferon regulatory factors (irfs) that sustain the secondary and tertiary transcriptional responses. tlr recognition of pathogens by immune cells results in the production of multiple cytokines such as ifn-α/β , tumor necrosis factor-α (tnf-α ), and interleukin-β (il- β ) in innate immunity ( ) . activation of multiple signal transduction pathways and cross-talk between the pathways enables fine-tuning of gene expression in innate immunity ( ) . cross-talk between tnf-α and ifn signaling pathways regulate inflammatory gene expression to influence the immune responses ( , ) . clinical significance of the balance between immune modulation and inflammation in signal transduction pathways has been demonstrated in a variety of autoimmune diseases ( , ) . early growth response (human egr / mouse egr ; referred in this manuscript as egr- ) belongs to a family of immediate-early response genes that contain a conserved zinc finger dna-binding domain and binds to a gc-rich sequence in the promoters of target genes ( ) . a variety of signals, including serum, growth factors, cytokines, and hormones stimulate egr- expression ( , ) . egr- has been shown to play an important role by regulating inflammatory gene expression in a variety of lung diseases and in mouse lung injury models including asthma, emphysema, airway inflammation, and pulmonary fibrosis ( ) ( ) ( ) . ischemia-mediated activation of egr- triggers the expression of pivotal regulators of inflammation including the chemokine, adhesion receptor, and pro-coagulant gene expression ( ) . egr- stimulates chemokine production in interleukin- mediated airway inflammation, and remodeling in the lung ( ) . high levels of expression of egr- and egr- inducible genes were reported in atherosclerosis, an inflammatory disease ( ) . egr- may have a potential role in liver injury and in acute pancreatitis ( ) ( ) ( ) . lipopolysaccharide (lps) induction of egr- was mediated by the activation of erk- / pathway and serum response elements ( ) . in this study, transcription factor profiling in interferon-mediated gene expression data sets and rt-pcr revealed that egr- was rapidly induced by ifn-α/β and tlr ligands in multiple cell types. studies in mouse and human fibroblast mutant cell lines revealed that egr- induction by type i interferons was independent of transcription factors stat , stat or irf . furthermore, the regulation of egr- by ifn-β was mediated by the activation of the erk- / pathway in serum-starved mouse lung epithelial cells. respiratory pathogens including coronaviruses (sars-cov- and ) and influenza viruses regulated the expression of egr- in human lung cell lines and in lung biopsies and peripheral blood cells of covid- patients, these studies suggest that the regulation of egr- may play an important role in the antiviral response and inflammatory disease. gene expression in response to interferon, tnf-α. and tlr agonist treatment in human peripheral blood mononuclear cells (pbmc), human hepatoma cells (huh- ), mouse bone marrow-derived macrophages (bmdm) were reported previously ( ) ( ) ( ) . supplementary data was downloaded from the journal publisher websites and geo datasets were analyzed with the geor r method (ncbi). cluster analysis was performed using gene expression software tools at www.heatmapper.ca. gene expression datasets representing human lung cell lines infected with respiratory viruses and from covid- patients were reported previously ( , ) . gene expression resources from immgen rna seq skyline were used ( http://rstats.immgen.org/skyline_covid- /skyline.html ). outliers of expression were not included in the analysis. mouse lung epithelial (mle-kd) and macrophage (raw . ) cell lines were used ( , ) . human fibrosarcoma cell line ( ftgh) and mutant cell lines lacking stat (u a), stat (u a), and irf (u a) were described previously ( ) . wild -type and stat -knockout mouse embryo fibroblast (mef) cell lines were used ( ) . cells were maintained in dmem supplemented with % fbs and % penicillin and streptomycin. cells were plated at - % density and maintained in full medium for a day. cells were incubated in serum-free medium for another hours. cells were treated with tnf-α ( ng/ml) or ifn-β were performed using ambion retroscript (austin, tx), according to the manufacturer's protocol. primer sequences for egr- , irf , and gapdh were obtained from the molecular reagents section of mouse genome informatics (mgi). human egr- and β− actin primer sequences were previously described ( ) . pcr products were resolved on a % agarose gel containing ethidium bromide and visualized with u.v. light and images were captured on a digital system. image files were processed with imagej (nih) software. specific gene expression was normalized to gapdh or β -actin and fold changes in the treated samples were calculated with respect to controls. mle-kd cell extracts were prepared and proteins were separated by electrophoresis using %- % sds-page gels. proteins in the gel were electrophoretically transferred to polyvinylidene difluoride membranes (bio-rad, ca) and subjected to immunoblotting with the antibodies for phosphorylated erk- / (thr /tyr ) or total erk- / from cell signaling technology (beverly, ma). blots were visualized by enhanced chemiluminescence western detection system (pierce, il). (figure a and b). these results were consistent with previous studies in human fibroblasts ( ) . tnf-α induction of egr- was much higher than ifn α/β in ftgh cells ( figure b ). interestingly, tnf-α but not ifn-α induced egr- mrna in hela cells suggesting differential pathway regulation ( ) . furthermore, ifn-λ induction of egr- was higher than with ifn-α/β in huh cells ( figure c ) irf (u a) demonstrated that ifn-β induction of egr- was independent of isgf- components ( figure b ). tnf-α and tlr ligands activate several mitogen-activated protein kinase (mapk) pathways such as extracellular signal-regulated kinase (erk), jun n-terminal kinase (jnk) and p ( ) . activation of the erk- / pathway leading to the phosphorylation of transcription factors such as elk and srf was implicated in the rapid induction of egr- in response to multiple stimuli ( , ) . ( ) . detailed promoter analysis revealed that multiple distal and proximal cis-elements were involved in egr- induction ( ). the generation of an inflammatory response is a complex process involving multiple cytokines acting in parallel and in concert in innate and adaptive immunity ( , ) . influenza virus-infected dendritic cells and macrophages, which reside in close proximity to lung epithelium, can produce significant amounts of tnf-α and type i ifn in response to virus infection ( ) . ifn-α / β is involved in signaling cross-talk with tnf −α that enhance or dampen the severity of inflammatory response ( , ) . interaction between interferon-α/β and tnf-α signaling was reported in autoimmune diseases. ( , ) . a select list of genes was induced by both tnf-α and interferon-a /β in pbmc and bmdm gene expression datasets ( figure ). tnf-α induction of these genes was much higher than ifn α/β in the mouse bmdm cells. interestingly, egr- was induced by both cytokines in pbmc and bmdm. these genes are involved in transcriptional regulation and integrating signal transduction pathways that are likely to play an important role in the cytokine storm and shift to hyperinflammatory gene expression in response to coronavirus infections ( , ). egr- is a zinc finger transcription factor that binds to a gc-rich sequence in the promoter regions of target genes. a shortlist of egr- regulated genes was generated from the truust transcription factor database and published studies in different cell types and under different stimuli ( ) ( ) ( ) ) . egr- regulates genes involved in cell growth (egr , atf , pdgfrb, cdkna , ccnd , tnfsf , and chemokines involved in inflammation such as (cxcl , cxcl , ccl , ccl ). it is important to note that many of the egr- target genes were also known targets of nf-kb, ap , and stat in cytokine signaling ( ) . furthermore, egr- interacts with several other transcription factors such as ets , ets , elk that are common in cytokine responsive genes ( ) . previous studies have shown that transcription factors of innate and adaptive immunity show functional connectivity involving shared interacting protein partners and target genes. ( ) . cluster analysis revealed that egr- induction was correlated with the expression of egr- target genes by ifn-α/β in pbmc and in bmdm cells ( figure ). severe acute respiratory syndrome coronaviruses (sars-cov) and influenza viruses are major respiratory pathogens in humans with seasonal epidemics and potential pandemic threats ( , ) . gene expression profiling studies of human lung epithelial cell lines such as calu- , a , and nhbe in response to respiratory viruses were reported recently ( , ) . interrogation of infection also induced egr- mrna in a cells, hours post-infection ( figure c ). these studies suggest that egr- expression is a common host response to many respiratory viruses gene expression profiling studies of a limited number of human lung biopsies and pbmc of healthy controls and covid- patients were reported recently ( , ) . interrogation of the gene expression data in lung biopsies revealed that egr- expression levels were significantly lower in covid- patients compared with healthy controls. expression of egr- target genes related to cell growth such as ccnd , egr , and pdgfrb were also down-regulated ( figure and data not shown). in contrast, expression of egr- target genes involved in inflammatory responses such as cxcl , ccl , ccl , ccl , and tnfsf were dramatically enhanced in covid- patients compared with healthy controls (figure and data not shown). interestingly, stat expression levels were significantly increased in covid- patients compared with healthy controls (figure ). there are several limiting factors to interpreting the data. the lung is a complex tissue consisting of more than thirty cell types with differential contributions with respect to cell mass and gene expression. for example, type i and type ii cells in mouse lungs constitute the major and minor cell types and express distinct cell markers ( ) . without information on the expression levels in distinct cell types, it is difficult to correlate egr- expression with target genes in the whole lung tissue. in support of this view, in a mouse model of cd + t cell -mediated lung injury, chemokine expression was dependent on egr- activation in alveolar type ii cells ( ) . another intriguing possibility is that enhanced stat expression compensates for decreased egr- expression in some cell types in the lung. it is important to consider that in addition to changes in mrna levels, the phosphorylation status of stat and egr- may play an important role in the transcriptional regulation of chemokine target genes. it is likely that differential expression in distinct cell types may account for the disparity of egr- has emerged as a key regulator of cell growth, reproduction, and response to tissue injury ( , ( ) ( ) ( ) . egr- was rapidly induced by interferons and pro-inflammatory cytokines such as tnf-α, and il- β ( , , ) . recent studies have demonstrated dramatic changes in type i interferon, tnf-α, and il- β production by immune cells and cytokine-mediated lung inflammation in covid- patients ( , , , ) . however, the role of egr- in innate immunity and antiviral response to respiratory viruses in general and sars-cov- , in particular, remains to be investigated. transcriptional factor profiling in the transcriptome revealed that egr- was induced by ifn-α/β , tlr ligands, and tnf-α in human and mouse cells. studies in mutant cell lines lacking stat , stat , and irf revealed that ifn-α/β induction of egr- was independent of isgf- components and mediated by the activation of erk- / pathway. furthermore, respiratory viruses such as sars-cov- induced egr- and its target genes in several lung epithelial cell lines and in covid- patients. activation of egr- by ifn-α/β and tnf-α and cross-talk between the pathways modulates signal transduction and inflammatory response in innate immunity. how cells respond to interferons the jak-stat pathway at twenty induction of human mrnas by interferon complex roles of stat in regulating 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inflammatory misfiring in severe covid patients identification and characterization of a new member of the tnf family that induces apoptosis multiple redundant effector mechanisms of cd + t cells protect against influenza infection angiotensin -converting enzyme is a functional receptor for the sars coronavirus early growth response- (egr- ) -a key player in myocardial cell injury protein kinase c beta/early growth response- pathway. a key player in ischemia, atherosclerosis, and restenosis role of alveolar epithelial early growth response (egr- ) in cd + t cell-mediated lung injury transcriptional regulation of egr- by the interleukin- -jnk-mkk -c-jun pathway impaired type i interferon activity and inflammatory responses in severe covid- patients immunophenotyping of covid- and influenza highlights the role of type i interferons in development of severe covid- key: cord- -x gbklw authors: ruseva, m.; gajdeva, m.; takahashi, k.; ezekowitz, a.; thiel, s.; jensenius, j. c. title: mannan‐binding lectin inhibits humoural responses date: - - journal: scand j immunol doi: . /j. - . . an.x sha: doc_id: cord_uid: x gbklw chronic hepatitis b virus (hbv) infection affects about – million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n‐linked glycosylation side and is recognized by both mbl‐a and mbl‐c in a ca‐dependent manner. hbsag–mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl‐a and mbl‐c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag‐specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was ‐fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein‐ag‐mbl‐rich complexes inhibit b‐cell responsiveness via putative mbl receptors. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < . ) in ms (mean ae sem: . ae . ; n ¼ ) compared to hc ( . ae . ; n ¼ ). lxr-a was lower in ms from stockholm ( . ae . ; n ¼ ) compared to corresponding hc ( . ae . ; n ¼ ; p < . ) and compared to ms ( . ae . ; n ¼ ; p < . ) and hc ( ae . ; n ¼ ; p < . ) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < . ) lxr-b (À . ae . ) compared to corresponding hc (À . ae . ). ms from stockholm was associated with higher abca- ( . ae . versus . ae . ; p < . ) and higher estrogen receptor-b-cx ( . ae . versus . ae . ; p < . ) compared to corresponding hc. the hc from sassari had higher androgen receptor ( . ae . ) compared to ms from sassari ( . ae . ; p < . ), ms ( . ae . ; p < . ) and hc from stockholm ( . ae . ; p < . ). ms from sassari had lower cyclooxygenase- compared to corresponding hc ( . ae . versus . ae . ; p < . ) and lower prostaglandin-e (À . ae . ) compared to the hc ( . ae . ; p < . ) and ms ( . ae . ; p < . ) and hc from stockholm ( . ae . , p < . ). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd + bdca -dendritic cells that produce il- and il- and have no enhanced type i interferon production y. m. huang, s. adikari, u. båve, a. sanna , & g. alm dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd þ dc from human blood monocytes, which coexpress bdca þ but are negative for bdca -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il- and il- , but no il- p and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd c, bdca- and cd a expression, having potent th -promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins (k ) and (k ) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k were selected on the basis of predicted binding to hla-cw and sequence similarities with m protein. matched peptides from the sequence of m protein and a set of peptides with poor predicted binding were also selected. cw þ individuals with psoriasis and cw þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd þ , cd þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd þ t cells was evaluated by cd expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw þ psoriasis patients had significant cd þ t-cell ifn-g responses to peptides from k and m protein selected on the basis of sequence homology and predicted hla-cw* binding. these responses were about times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd þ t cells. cd þ t cells showed only borderline responses. cd þ t cells from cw þ nonpsoriatic individuals responded to some m peptides but very rarely to k peptides, and this also applied to the cla þ cd þ subset. these findings indicate that psoriatic individuals have cd þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly % in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately / of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in / ( %) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ . ). in the gc þ group, . % of the patients presented with anti-ro/ssa compared to . % in the gcgroup. anti-la/ssb was detected in . % of the gc þ patients compared to . % of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to . % in the gcpatients (p ¼ . ). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type diabetic (t dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in t dm patients with overt nephropathy and t dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was . ( . - . ), p ¼ . . median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [ mg/l (iqr - mg/l) versus mg/l (iqr - ), p ¼ . ], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [ mg/l (iqr - mg/l) versus mg/l (iqr - mg/l), p ¼ . ]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < . ). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog - protects lew. av from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim- ), reported to be exclusively expressed on differentiated t cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation weeks after protective dna vaccination. in protected rats, we observed ( ) no alterations in antigenspecific th or th responses, ( ) reduced mhc ii expression on splenocytes early after eae induction, ( ) antigen-specific upregulation of ifnb upon recall stimulation and ( ) reduced il- rb on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd þ t cells, we observed the production of tnf-a, ifn-g and il- by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il- , il- and il- was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd þ and cd þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins and in parallel with the th /th dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase , we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl and mmgl , is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin- and interleukin- upregulate mmgl and mmgl expression and that, in vivo, induction of mmgl and mmgl is dependent on interleukin- receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th conditions. background: human parvovirus b (b ) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b infection were included in the study and followed consecutively for up to weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of - sfc/ million pbmcs, roughly corresponding to . - . % b specific cd þ cells circulating in peripheral blood at - weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b infection are surprisingly narrow in distribution and remain at high levels for up to weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !À ), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<À ) during the period prior to assessment were . ( % ci . - . ; ¼ . ) and . ( . - . ; . ), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children - years old who were subsequently characterized as wasted was . ( . - . ; p ¼ . ), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr . ; . - . ; p < . ). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first years of life. a. astrinidou-vakaloudi, s. xytsas, i. diamanti, h. ioannidis & p. pangidis microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and nefrology, nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included sera from patients with esrd ( male and female) undergoing periodic haemodialysis; mean time of treatment was . months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in out of ( %) patients in the dialysis group, while / ( . %) tested positive for iga. igg caga antibodies were present in out of ( . %). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was , and %, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd þ and cd þ t cells and the ability to respond with th -type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor / agonist that, when given subcutaneously, induces type- immune responses against heterologous antigens. here, a fusion of opri to ag a of mtb (opri-ag a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag a was combined with an ag a-encoding dna vaccine (ag a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag a dna induced significant systemic th immune responses. intranasal boosting with opri-ag a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a r or dominant-negative myd totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. - -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse -specific nucleotidyl transfer. this first crystal structure of a - oligoadenylate synthetase reveals a structural conservation with the -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the -and -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the - oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing - -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd ). expression of cd can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga protease that cleaves human iga , but not iga , molecules in the hinge region. this leaves iga as faba (monovalent) deprived of fca which contains the docking site for cd . iga is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl ). materials and methods: iga and iga monoclonal antibodies to serotype pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype pneumococci with and without iga protease activity, respectively, were obtained after inactivation of the iga gene of the tigr strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga and iga antibody to type- polysaccharide-induced phagocytosis of iga protease-deficient type- pneumococci equally well in the absence as in the presence of complement. iga antibody to type- polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga protease deficient compared to homologous wildtype target bacteria. a similar effect of iga protease activity of the target bacteria was not observed in a parallel experiment where iga antibody to type- polysaccharide served as opsonin. iga antibody extracted from iga protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga from protease-deficient bacteria and iga from both types of bacteria were intact. conclusions: these results indicate that the iga protease activity of s. neumoniae may help the bacteria escape iga antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following weeks fed either a control diet or this diet supplemented with mg stay-c per kg. blood sampling was obtained weekly from day and until day of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b was decreased weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o :b ) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between and h at . c. a dose in the range of - g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of months (weeks À , À , , , , and around calving) and stimulated with g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of cows was found to be significantly increased (p < . ) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk / pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor (svegfr ) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < . ) and a less pronounced but still significant increase in svegfr . release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < . ). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr , which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il- and il- , with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, , t. rix, , j. krog, , e. tønnesen & m. hokland department of anaesthesiology and intensive care, aarhus university hospital, and institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il- and il- . this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k cells. this method was compared to the current standard cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il- and il- is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd -cd dim and the cd -cd bright subsets, with a much greater proportion of ifn-g-positive cells in the cd -cd bright subset. the effects of stimulation with il- and il- on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th and th effector cells. now the importance of regulatory cd þ cd þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd þ t cells of th phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin (il- ) and il- and demonstrate spontaneously arising cd þ cd þ populations and high concentrations of il- in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il- and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a h cr-release assay using k as nk-sensitive target cells. the pbmcs were characterized, using -colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata . ). p values < . was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < . and pcontrols < . ). although the cytotoxicity increased relatively more (p < . ) in the group of divers compared to the group of control donors between day and . discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin- (il- ), il- and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd ) may be expressed. unlike cla, cd is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il- alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd by cd þ but not cd þ t cells. while il- increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd expres-sion, and a combination of il- and tgf-b completely abrogated the induced cd expression. conversely, il- suppressed cla but increased cd expression. these findings indicate that, in addition to suppressing the development of th -mediated inflammatory responses, il- may also inhibit the migration of cd þ t cells into the skin while il- promotes such migration. thus, the expression of cla and cd may be antagonistically regulated by il- and il- , and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu ( mg/ kg animal) was injected i.p. h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in % paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, - h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells h after elicitation. after h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a a receptor expression in peripheral blood cd þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd þ cells of asthmatic patients expressed significantly lower levels (p < . ) of a a receptor in protein level (mean percentage of cells positive ae sem: . ae . , n ¼ ) compared to healthy individuals ( . % ae . , n ¼ ). double staining for cd expression showed that stimulation of cd þ cells decreased a a expression in both groups but indicated that the detected lower levels of a a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a a mrna expression in unstimulated cd þ cells was significantly higher (p < . ) in asthmatics (n ¼ ) compared to healthy controls (n ¼ ). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a a adenosine receptor on their peripheral cd þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost % of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s ( . kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s for different time periods. it was found that the maturation marker cd and the costimulatory molecules cd and cd were upregulated on the mddcs exposed to mala s for h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s for h induced an increased production of the inflammatory cytokines il- ( -fold), tnf-a ( -fold) and il- (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th -like activity (grönlund et al. immunology, ) . we here coupled the recombinant major cat allergen fel d to cbps (cbp-fel d ) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd a þ mddcs were capable of taking up fitc-labelled cbp-fel d , as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d resulted in an upregulation of the costimulatory molecule cd on the mddcs, which was not observed with fel d or cbps alone. finally, cbp-fel d induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin- from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about % of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of kda. protein purification procedure was developed to obtain nearly pure der p protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e . the deglycosylated recombinant pro-der p revealed immunologic similarity to the native der p molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled -dimensional structure of der p , five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p and screening for hypoallergenic variants was performed. combining inhaled long-acting b- agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt (cd þ cd ra -cd l þ cd adim) and mt (cd þ cd ra -cd l -cd abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt correlates with a th phenotype and mt with a th phenotype. stable asthmatics, requiring fluticasone propionate (fp) - mg daily or equivalent, were randomized to receive, double-blinded, either seretide [salmeterol and fluticasone propionate (sfc, n ¼ )] mg/ mg bd or fp mg bd (n ¼ ). if asthma was controlled based on lung function and symptoms at clinic visits every weeks, ics dose was tapered until asthma exacerbated or mg was reached. the frequency and ratio of mt and mt t cells of the patients was monitored at week intervals. as treatment tapered, the frequency of mt cells decreased (p ¼ from first to final visit), whereas that of mt cells increased. the ratio of mt /mt decreased (p ¼ from first to final visit). in patients receiving laba þ ics, the fall in mt /mt ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c a and c a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in patients of bronchial asthma with allergic rhinitis and unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev values of the patients. association of single nucleotide polymorphisms (snps) in exon and intron of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a g in exon and g a in intron of the mbl, were novel. snp g a was significantly associated with the disease (p ¼ . , or ¼ . , % ci: . < or < . ). individuals with 'a' allele at position showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p ). in addition, a second class of factor h-binding proteins of approximately - kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs - ) specifically bind to ospe-related lipoproteins. we also found fhr- , whose c-terminal scrs - are homologous to scrs - of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal amino acid residues from region v of p abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o: mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes ( a, d and f) as well as some capsulated s. aureus serotypes (type- , - , - , - and - ). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c , whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in plasma samples were determined and the median values were estimated at . mg of mbl/ml, . mg of l-ficolin/ml and . mg of h-ficolin/ ml, respectively. the absence of early complement components (c , c and c but not c ) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c -deficient (c def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c def. mice. we hypothesized that splenic cd b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin- (il- ) and ifn-a (ifn-a). we observe a moderate increase of il- and ifn-g mrna in cd b þ cells of c def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c def. mice. preliminary results suggest that mrna in cd b þ cells of c def. mice is even lower than that in wt. six hours following i.v. application of mg of a abstracts .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il- , ifn-g and ifn-a mrna is increased in cd b þ cells of both c def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c -deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c is not a predisposing factor for sle and our observation that c def. animals display low levels of ifn-a mrna. - million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c q, c and c result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was -fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c -activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)- , - and - and a small, nonenzymatic component, map . masp- has been shown to elicit complement activation through the sequential proteolytic cleavage of c and c upon binding of mbl/masp- complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp- /map gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp- protein. recombinant wildtype masp- and masp- containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp- function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp- to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th , th and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin- (il- ) and tumour necrosis factor (tnf)-a, while the differences for il- and il- were less pronounced. bifidobacteria tended to be weak il- and tnf-a inducers, while both strong and weak il- inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il- production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il- were most effective in upregulating surface mhc class ii and cd . moreover, l. casei-induced upregulation of cd was reduced in the presence of a weak il- inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco- cells), which should mimic the intestinal environment, was studied. cytokine secretion (il- , il- and tnf-a) and upregulation of maturation surface markers on dcs (cd and cd ) was measured. different lab induced diverse cytokine responses. some strains were strong il- and tnf-a inducers and others weak. all strains induced il- . different lab also differentially modulated expression of cd and cd on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco- coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd -cd þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with mg/ml uv-inactivated bacteria or mg/ml phytohemagglutinin (pha) for days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab ) and anti-idiotypic antibody (ab ). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab and ab were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with mg of igm dj) than in sera of mice immunized with times lower doses of immunogen ( . mg per doses). moreover, ab and ab antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il- p and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il- p , tnf, il- and il- in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor (tlr ) on dcs compared to monocytes. the surface expression of tlr on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet ; : - ) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il- and gm-csf) and phenotype (cd , cd , cd and cd ) were measured on day . results: lactobacillus spp. induced higher productions of tnf-a and il- than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd expression without simultaneous upregulation of cd . the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein : immunization with a dna construct based on the malarial antigen fused with a fragment of hsp primes for a th- type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp (pf c) and compared it to the whole hsp molecule from trypanosoma cruzi (tchsp ). we found that pf c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf c against the malarial antigen eb in a chimeric dna construct. no appreciable levels of eb -specific abstracts .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th- antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing  clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the attomol (mass spectrometry) and the zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map -nc ), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his) -tag, we increased the binding efficiency of sinfab-molecules to ni þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp- , - and - ). after recognition of a microorganism by mbl, activation of the complement system occurs. masp- and masp- share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp- , an assay for masp was presented, based on antibodies against the a-chain of masp- . with the new knowledge of the three masps, and the sharing of domains by masp- and masp- , assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp- in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp- / and a developing secondary antibody against the c-terminal part of the protease domain of masp- . we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp- protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b . (cd ) and b . (cd ). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon , were sequenced with flanking introns in healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a . we studied the simultaneous expression of , transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a cell line is measured. in an attempt to find a new and better reporter gene for type ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u a chip from affymetrix. the expression of , genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering > % of all major human populations. for each hla supertype, we have selected the top candidates for test in biochemical-binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b* and hla-b* . methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b* and hla-b* are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a* was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in of the tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin- (il- ), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il- effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il- receptors (il- ra and il- rb), indicating that this oral carcinoma line is a target for il- study. we showed that recombinant human il- inhibited kb-cell proliferation by % at concentration of ng/ml (p < . ), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il- suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il- treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl- and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il- , as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c cells, after h of cultivation. accordingly, the viability of c cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a , a , a , a , b and b . furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th , but not th , cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase , a well-established marker for alternatively activated myeloid cells or m , a strong upregulation of fizz and ym, two additional recently identified markers for m . further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l ) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type cytokines (il- , il- and il- ) and are therefore rather m associated. overall, our data provide new markers for msc in cancer and further establish their m activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b* and hla-b* , and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl- is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and healthy controls were used to investigate whether bcl- protein in involved in breast cancer (grade iii). full length bcl- cdna was retrovirally transduced into eph- cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl- in breast cancer (iii). results: restoration of bcl- into eph- cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl- -transduced eph- cells is prolonged by about h, presumably as a result of the action of bcl- at the bcl- at the g /s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph- cells, control-transduced eph- cells and bcl- -transduced eph- cells. consistently, we demonstrated that bcl- is expressed in % of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl- is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il- ( and il- can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf- ), oral carcinoma cell line (kb), colon cancer cell line (caco- ) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate ( ) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and ( ) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from patients and healthy controls. the patients were observed for years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < . ) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ . ), and pneumonia (n ¼ ) was associated with poor survival (p ¼ . , hr ¼ . , % ci . - . ). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ . , % ci À . - . ) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from crc patients and normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u ). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease / (masp / ), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp- reached the noise threshold of signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord- -hj mq lq authors: matthew, j.; pintopereira, l. m.; pappas, t. e.; swenson, c.; grindle, k.; roberg, k.; lemaske, r. f.; lee, w.; gern, j. e. title: seasonal distribution of respiratory viruses in pediatric asthma in trinidad west indies date: - - journal: journal of allergy and clinical immunology doi: . /j.jaci. . . sha: doc_id: cord_uid: hj mq lq nan p . ) prevalence in children who needed to be nebulized (n , . %) compared with stable (n , . %) asthmatics. conclusions: rhinovirus is the major viral trigger for pediatric asthma in trinidad and is not associated with seasonality in this tropical climate. we hypothesize that children identified as wheezers will present with increased levels of inflammatory cytokines and decreased levels of anti-viral cytokines in nasal lavage samples during symptomatic rv infection compared to non-wheezers during symptomatic rv infection. methods: to test this hypothesis, children enrolled in the childhood origins of asthma (coast) project provided nasal lavage samples during symptomatic respiratory infections and at scheduled well visits at ages and years. children with a history of wheezing were sampled when well, during an uncomplicated cold and during a wheezing illness. children with no history of wheezing were sampled when well and ruing an uncomplicated cold. innate cytokine profiles were assayed using a beadlyteÒ human multi-cytokine flex kit, and detection of the cytokines were evaluated using the luminex ä is for the following cytokines: il- , il- , il- (p ), ifn-g, ifn-a, and tnf-a. interleukin- levels were evaluated by elisa. results: interleukin- , il- , ifn-g, and il- were present in measurable concentrations. concentrations of il- were significantly higher during symptomatic rv infection than when well ( . vs. . , p< . ). the % detectability of il- and ifn-g were significantly higher during symptomatic rv infection than when well ( . vs. . , p . ; . vs. , p . respectively). concentrations of ifn-g tended to be higher in non-wheezers than in wheezers during symptomatic rv infection ( . vs. . , p . ). conclusions: during symptomatic rv illness, non-wheezers presented with higher levels of ifn-g than wheezers, suggesting the possibility that wheezers may have a depressed anti-viral response, which may not be as effective at fighting viral infections. funding: nih myosin light chain kinase (mylk) variants that confer rationale: asthma is a heritable trait characterized by lower airway smooth muscle contraction and inflammation. myosin light chain kinase is a multifunctional protein involved in regulation of bronchial contractility and other activities relevant to asthma key: cord- -eiphxwmn authors: trouillet-assant, sophie; viel, sebastien; gaymard, alexandre; pons, sylvie; richard, jean-christophe; perret, magali; villard, marine; brengel-pesce, karen; lina, bruno; mezidi, mehdi; bitker, laurent; belot, alexandre title: type i ifn immunoprofiling in covid- patients date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: eiphxwmn covid patients in icu present a high mortality rate and immunoprofiling reveals heterogeneous ifn-α production with about % of critically-ill patients unable to produce ifn-α , highlighting the immune response heterogeneity and opening avenues for targeted therapies. sophie trouillet-assant , * , phd, sebastien viel , , , * , pharmd, phd, alexandre gaymard merazga for their excellent work. we thank fabien subtil for his helpful advice for statistical analysis. we also thank the life (lyon immunopathology federation) community for fruitful discussion. capsule summary: covid patients in icu present a high mortality rate and immunoprofiling reveals heterogeneous α production with about % of critically-ill patients unable to produce ifn-α , highlighting the immune response heterogeneity and opening avenues for targeted therapies. to the editor, severe acute respiratory syndrome coronavirus (sars-cov- ) infection (covid- ) is characterized by a wide spectrum of disease encompassing asymptomatic carriage, mild to severe upper respiratory tract illness that can evolve into respiratory failure or rapidly progressing severe viral pneumonia with acute respiratory distress syndrome (ards). disease severity depends on viral strain and host risk factors have been identified such as age and male gender. in addition, an excessive immune response has been identified in patients showing a cytokine storm associated with ards . various immunosuppressive drugs, including il- blockers or jak-stat signaling inhibitors have been suggested for the treatment of sars-cov- infection whereas additional clinical trials are evaluating the use of recombinant interferon to foster host antiviral response. (clinicaltrials nct , nct ). type i interferons (ifn-i) are major components of the innate immune system and represent critical antiviral molecules . to date, ifn-i response has not been evaluated in covid- patients and its contribution to the viral control and inflammation is unknown. in this study, we assessed the kinetics of plasma ifn-i in covid- patients with a spectrum of severity degree. this study was approved by an ethical committee for biomedical research (comité de protection des personnes hcl). (supplemental material and method of this article online repository). firstly, we explored three patients issued from the first covid cluster diagnosed in france (les contamines, haute savoie, france) in february . we took advantage of the new digital elisa technology single-molecule arrays (simoa) and analyzed the kinetics of plasma inflammatory cytokines. interleukin (il)- , c-reactive protein (crp) and interferon γ-induced protein (ip- ) were elevated in the two symptomatic patients (pt , ) (supplementary figure in the online repository). strikingly, no ifn-α was detectable in these two patients. in contrast, il- , crp and ip- elevation of plasmatic ifn-α was observed. viral loads were low with no obvious quantitative difference between all three patients. we further explored a larger cohort of critically ill covid patients from one of the intensive care unit (icu) at hospices civils de lyon (lyon, france). of note, all the patients were treated with standard of care and none received antiviral or immunotherapies. considering the first days of infection, more than half of critically ill patients required invasive mechanical ventilation ( / ). we observed that patients demonstrated a peak in ifn-α at day - of symptoms onset corresponding to the viral replication phase, that decreased overtime to low but still detectable ifn-α the timing of interferon exposition may be critical to control the virus and avoid immunopathogenesis. channappavanar et al. have shown that delayed ifn-i expression can be detrimental in mice in the context of sars-cov- infection . our data suggests that screening patients for ifn production is instrumental to select those who could benefit from early intervention with ifn. following day , il- remains increased while ifn-α tapered. this kinetics highlight that cytokine inhibitors could be helpful at the second phase of the disease following ifn-i decrease. viral characteristic or individual genetic susceptibility should be explored to understand the defect of ifn- α production in some covid patients. some ifn-α positive patients also experienced fatal outcome highlighting the multifactorial causes of disease severity. we acknowledge limitations of this study, related to the small number of included patients and the technical limitation for the measurement of ifn-β and ifn-λ, in this proof of concept study. here, we provide new argues for an early intervention with recombinant ifn-α and we also highlight the window of opportunity for immunosuppressors at the second phase of the disease, delay between symptom onset and icu admission (days) [ - ] [ - ] . bacterial co-infection during icu stay (n (%)) ( %) ( %) diabetes (n (%)) ( %) ( %) chronic obstructive pulmonary disease (n (%)) ( %) ( %) cardiovascular disease (n (%)) ( %) ( %) hypertension (n (%)) ( %) ( %) cancer (n (%)) ( %) ( %) active smokers (n (%)) ( %) ( %) mortality at d after symptom onset(n(%)) ( %) ( %) . crp -c-reactive protein, icu -intensive care unit, bmi -body mass index table -clinical characteristics of covid- patients in intensive care unit p-value are calculated using mann-whitney test for quantitative values and using fisher-exact test for qualitative ones. a. plasma ifn-α concentrations (fg/ml) were determined by single molecule array (simoa) b.c.d. il- , crp and ip- concentrations were measured using a multiplexed assay with the ella platform. e. viral load is represented as cycle threshold of ip rt-qpcr using assay designed by pasteur institut in paris. ifn-interferon ; il- -interleukin ; crp -c-reactive protein ; ip- -interferon γ-induced protein a. ifn score is a transcriptionnal signature defined by interferon-stimulated gene (isg) quantified using nanostring technology and obtained from paxgene tubes in covid- patients. b-d. normal values for healthy volunteers was indicated by grey area. vertical bar indicates median delay between symptom onset and icu admission. concentrations of ifn-γ were quantified in only / patients because of lack of material. clinical features of patients infected with novel coronavirus in wuhan, china covid- : consider cytokine storm syndromes and immunosuppression type i interferons (α/β) in immunity and autoimmunity / and directive / /ec) and the french data protection law (law n° - on / / and décret n° - on / / ), we obtained consent from each patient or his next of kin usa) on plasma samples of covid- patients. the assay was based on a -step protocol using an hd- analyzer (quanterix). il- , crp and interferon γ-induced protein (ip- ) concentrations were measured using a multiplexed assay with the ella platform (protein simple© ca, usa), according to manufacturer's instructions. plasma il a/b and il- (type iii interferon) have been quantified by elisa (pbl laboratories rna integrity was then evaluated by agilent rna microarray (agilent technologies© data standardization was obtained using the geometric mean of internal control and housekeeping genes count number. interferon score was calculated as previously described . virus quantification load viral load was quantified from nasopharyngeal swabs or endotracheal aspirates. rna extraction was performed by the automated nuclisens® easymag® (biomérieux, marcy l'etoile, france) using manufacturer's instructions. a μl reaction contained μl of rna p-value were calculated using mann-whitney test for quantitative values and using fisher-exact test for qualitative ones comparison of rt-qpcr and nanostring in the measurement of blood interferon response for the diagnosis of type i interferonopathies walzer international center of research in infectiology, lyon university, inserm u , cnrs umr , ens, ucbl, lyon, france we explored the first three sars-cov- positive patients diagnosed in france (les contamines, france) in february . patient : a high risk contact (a -year-old man) initially negative for sars-cov- developed fever and cough with respiratory crackles at auscultation on the fifth day of hospital isolation. a bilateral interstitial syndrome at the ct-scan with bilateral ground-glass opacification predominant on the left. sars-cov was detected from endotracheal aspirates (eta), all nasopharyngeal swabs were always negative. the daily follow-up revealed a short-lasting excretion with only two successive eta for these three patients, no other respiratory pathogens were detected. these patients did not need oxygenation, nor antibiotics, steroids or antiviral agents. plasma samples and paxgene® tubes were collected from covid- patients hospitalized in the university hospital of lyon (hospices civils de lyon), france. diagnosis of covid- was confirmed in all patients by rt-pcr.all critically ill patients, admitted to icu, were included in the mir-covid study. this study was registered to the french national data protection agency under the number - and was approved by an ethical committee for biomedical research (comité de protection des personnes hcl) under the number n° - . in agreement with the general data protection regulation (regulation key: cord- -jh msz c authors: olagoke, olusola; quigley, bonnie l.; timms, peter title: koalas vaccinated against koala retrovirus respond by producing increased levels of interferon-gamma date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: jh msz c koala retrovirus (korv) is believed to be in an active state of endogenization into the koala genome. korv is present as both an endogenous and exogenous infection in all koalas in northern australia. korv has been linked to koala pathologies including neoplasia and increased susceptibility to chlamydia. a korv vaccine recently trialled in northern koalas improved antibody response and reduced viral load. this communication reports the expression of key immune genes underlining the innate and adaptive immune response to vaccination in these northern koalas. the results showed that prior to vaccination, il- was expressed at the highest levels, with at least -fold greater expression compared to other cytokines, while cd mrna expression was significantly higher than cd mrna expression level. interferon-γ was up-regulated at both - and -weeks post-vaccination while il- was down-regulated at -weeks post-vaccination. korv is currently undergoing endogenization into the koala genome, thus offering an exciting opportunity to study retroviral endogenization in real time [ , ] . nine korv subtypes (korv-a to -i) have been identified with the subtypes a and b being the most studied [ ] . all northern koalas (queensland and new south wales) are thought to be harbour at least korv-a, a korv subtype which is both endogenous and infectious in these koala populations. korv infection has been linked to immune alterations, with infected southern koalas shown to have altered immune profiles [ ] and korv-b infected northern koalas shown to possess levels of immune dysregulation [ ] . korv has also been linked to increased susceptibility to diseases in koalas [ ] . we recently described antibody production and reduction in viral load following vaccination in koalas harbouring endogenous korv [ ] . briefly, koalas were vaccinated with a recombinant korv env protein along with a triadjuvant of poly i:c, polyphosphazine and host defence peptide . each animal received the vaccine subcutaneously on day zero with a booster dose delivered four weeks after. the vaccine was shown to lead to a significant increase in serum anti-korv igg levels and neutralising antibody titres. the antibodies were shown to be cross-reactive against exogenous korv subtypes. in the present study, we examined the expression of important koala cytokines, immune markers and host restrictions factors to determine their pre-and post-vaccination levels in northern koalas harbouring endogenous korv. to understand the expression profiles of key immune genes associated with vaccine response, we compared the pre-vaccination levels to -and -weeks post-vaccination levels. we chose to analyse the gene expression changes in a non-antigen stimulated model. genes targeted included nine cytokines (ccl l, interleukin open access *correspondence: oolagoke@usc.edu.au genecology research centre, university of the sunshine coast, sunshine coast, qld, australia (il)- β, il- , il- , il- , il- , il- a, il- and interferon gamma (ifn-γ), four host restriction factors (bst , isg , rsad and trim ) and two t-cell markers (cd and cd β). total rna was purified from µl whole koala blood using trizol ® ls (thermofisher), as per manufacturer's instructions. rna was treated with the turbo dna-free kit (thermofisher) before target gene transcripts were quantified using a custom nanostring probe panel (systems biology and data science, griffith university, australia). five koala housekeeping genes (actb, gapdh, hmg a, nckap l and stx ) were included in the panel for normalization and quality control. normalized transcripts were quantified pre-vaccination and converted to fold-change for post-vaccination analysis. expression levels of the target genes prior to vaccination were examined to gain an insight into the baseline immune genes expression of koalas harbouring endogenous korv. il- was by far the most abundant cytokine mrna expressed in these korv infected koalas (fig. a) , with over -fold higher expression compared to other cytokines. both il- and il- β were the next most abundantly expressed cytokines and they had comparable expression levels (fig. a ). the remaining cytokines tested (ccl l, il- , il- a, il- , il- and ifn-γ) had low, but detectable levels in all koalas investigated (fig. a) . additionally, all four host restriction factor genes (bst , isg , rsad and trim ) were expressed at quantifiable levels in all koalas (fig. b) . finally, cd gene expression levels were significantly lower than cd β gene expression levels (~ folds) (fig. c) . after vaccination, ifn-γ had the most pronounced expression change, with significant up-regulation at -weeks (log fold change = . ; p = . ) and continued up-regulation at -weeks (log fold change = . ; p = . ) post-vaccination (fig. a) . there were also a small but statistically significant down-regulation of il- a at -weeks post-vaccination (log fold change = − . ; p = . ) and il- by -weeks post-vaccination (log fold change = − . ; p = . ) (fig. a) . there were no significant changes in the mrna expression of the other cytokines tested (ccl l, il- β, il- , il- , il- , and il- ) at either -or -weeks post-vaccination, data not shown. in addition, none of the host restriction factors tested (bst , isg , rsad and trim ) had significant changes in their expression either at -or -weeks post-vaccination when compared to prevaccination levels. interestingly, the post-vaccination expression of cd mrna remained largely unchanged relative to pre-vaccination levels, while cd β mrna was slightly upregulated at -weeks post-vaccination (log fold change = . ; p = . ) (fig. b) . in this cohort of korv vaccinated and endogenously infected koalas, a small but significant increase in the expression of ifn-γ at both -and -weeks post-vaccination was observed, compared to pre-vaccination levels. while the biological impact of such sustained up-regulation is not fully known yet, a recent study showed that korv-positive southern (victorian) koalas had significantly lowered ifn-γ levels compared to korv-negative koalas [ ] . the authors of that study argued that such decreased ifn-γ levels may increase susceptibility to chlamydia and other opportunistic infections in korvpositive koalas. this work is the first to report an increase in ifn-γ in response to korv vaccination and, as such, suggests a means to counteract korv-associated reduction in ifn-γ levels in koalas. the antiviral properties of ifn-γ have been well documented. for instance, longlasting porcine circovirus vaccine efficacy was shown to be sustained, in part, by ifn-γ producing cells [ ] . ifn-γ has also been linked to b-cell differentiation into igg production [ ] . the increase in ifn-γ detected in this work may offer insight into the sustained anti-korv igg production and reduction in viral load reported in koalas following vaccination [ ] . a possible mechanism for the observed increase in ifn-γ levels post-vaccination may be the inclusion of poly[di(sodium carboxylatoethylphenoxy)phosphazene] (pcep) as an adjuvant component in the korv vaccine. pcep has been previously shown to induce a strong ifn-γ response and enhance antigenspecific immune response in mice following vaccination [ ] . several host restriction factors able to interfere with multiple stages of the viral life cycle have been identified in humans and animals [ ] . our study investigated the expression of four host restriction factors in koalas harbouring endogenous korv: bst , isg , rsad and trim . all four genes were found to be expressed at quantifiable levels. surprisingly, bst , isg and rsad were expressed at relatively high levels, suggesting that these restriction factors may have biological importance in koalas harbouring endogenous korv. although there was not a vaccine-induced up-regulation in these host restriction genes, the detection and investigation of these genes nonetheless present a new line of research into innate antiviral mechanisms for koalas. the role of korv in modulating the cd :cd ratio in koalas has been previously investigated. a study of captive koalas harbouring endogenous korv reported a cd :cd expression median ratio of . (range: . - . ) [ ] . surprisingly, all koalas in the current study had very low levels of cd mrna expression when compared to cd β mrna expression. this observation was not improved by vaccination. when compared to the normal range in humans ( . - . ) [ ] , the levels observed in this work suggest an abnormally low cd expression in koalas. unfortunately, the lack of koala-specific reagents prevented further investigation into whether the reduced cd mrna expression directly translated into fewer cd t cells. however, a positive association between cd molecule and cd mrna expression has been previously shown in human studies [ ] . as such, these results could suggest an ongoing korv-associated immunosuppression, possibly through the preferential loss of cd t cells in korv-infected koalas, similar to what is seen in cats infected with feline leukemia virus and feline immunodeficiency virus [ ] . finally, il- was by far the most expressed cytokine detected in tested koalas. increased il- expression has been implicated in several pathologies including leukemia [ ] . high levels of il- is also associated with progression to disease in people infected with hiv- , when compared to those who were able to maintain natural control of the infection [ , ] . the extremely high level of il- seen in these korv infected koalas is highly interesting, as it may be an important biomarker for underlying conditions that may be korv-associated. in these endogenously infected koalas, korv vaccination led to a small but significant decrease in il- mrna expression. a recent study showed that reduction in il- levels was required for chlamydia clearance in korvinfected koalas [ ] . if further studies can conclusively link il- to koala pathologies, then the role of il- inhibitors, such as through vaccination, should be further explored. this work described increased ifn-γ mrna expression following korv vaccination in koalas harbouring endogenous korv. a very high expression level of il- mrna prior to vaccination, followed by a slight but significant decrease post-vaccination, was also observed. finally, this work provides new insight into possible mechanisms for korv-associated immunosuppression in korv infected koalas. korv: koala retrovirus; il: interleukin; ifn: interferon. retroviral invasion of the koala genome long-read genome sequence assembly provides insight into ongoing retroviral invasion of the koala germline helping koalas battle disease-recent advances in chlamydia and koala retrovirus (korv) disease understanding and treatment in koalas altered immune parameters associated with koala retrovirus (korv) and chlamydial infection in free ranging victorian koalas (phascolarctos cinereus) altered immune cytokine expression associated with korv b infection and season in captive koalas an exogenous retrovirus isolated from koalas with malignant neoplasias in a us zoo therapeutic vaccination of koalas harbouring endogenous koala retrovirus (korv) improves antibody responses and reduces circulating viral load memory t cell proliferative responses and ifn-γ productivity sustain long-lasting efficacy of a cap-based pcv vaccine upon pcv natural infection and associated disease b cells responses and cytokine production are regulated by their immune microenvironment poly[di(sodium carboxylatoethylphenoxy)phosphazene] (pcep) is a potent enhancer of mixed th /th immune responses in mice immunized with influenza virus antigens restriction factors: from intrinsic viral restriction to shaping cellular immunity against hiv- expression profiles of the immune genes cd , cd β, ifnγ, il- , il- and il- in mitogen-stimulated koala lymphocytes (phascolarctos cinereus) by qrt-pcr imbalance in the game of t cells: what can the cd /cd t-cell ratio tell us about hiv and health? plospathog correlation between the expression of cd and the level of cd mrna in human b-cell lines clinical aspects of feline retroviruses: a review plasma interleukin level predicts for survival in chronic lymphocytic leukaemia host factor predictors in long-term nonprogressors hiv- infected with distinct viral clades il- alterations in hiv- infected children with disease progression antibiotic treatment of chlamydia-induced cystitis in the koala is linked to expression of key inflammatory genes in reactive oxygen pathways publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the many groups that have significantly supported the overall koala vaccine development work, including the departments of transport and main roads, and environment and heritage protection of the queensland government, the moreton bay rail project team, moreton bay regional council, city of gold coast, redland city council, lone pine koala sanctuary, endeavour veterinary ecology, koala action inc., friends of koala (lismore), australia zoo wildlife hospital, staff and volunteers at the adelaide koala and wildlife hospital, as well as all koala rescue groups, zoos south australia, and vaccine and infectious diseases organization (vido), canada. we would also like to thank dr nic west and dr jelena vider (systems biology and data science, griffith university, australia) for help with nanostring experimentation. this work was financially supported by the australian research council linkage grant lp received by pt. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the data that support the findings of this study are available from the authors on request. all animal work reported in this study was approved by university of the sunshine coast animal ethics committee (ana ). this study was conducted in compliance with approved institutional guidelines. not applicable. the authors declare no competing interests.received: july accepted: october key: cord- -rgnhfcbx authors: du, fenghe; liu, bao; zhang, shuyang title: covid- : the role of excessive cytokine release and potential ace down-regulation in promoting hypercoagulable state associated with severe illness date: - - journal: j thromb thrombolysis doi: . /s - - - sha: doc_id: cord_uid: rgnhfcbx [image: see text] keywords covid- · excessive cytokine release · ace · hypercoagulable state · vascular thrombotic events highlights • dysregulated immune responses in covid- could lead to inefficient virus clearance, which further brings about over-activated inflammatory response and excessive cytokine release. • excessive cytokine release in severe cases of covid- is characterized by enhanced serum levels of il- , il- , il- , and ifn-γ. increased serum level of the anti-inflammatory cytokine il- might be a compensatory mechanism to reduce the over-activated inflammatory response. • ace expression levels could be down-regulated, according to the recent bio-informatic analysis with the interactome model based on characteristics of human protein/sars-cov interactions, but a direct experimental evidence is still required to confirm this prediction. • in severe covid- , the excessive cytokine release and potential down-regulation in ace expressions can not only promote a hypercoagulable state individually, but also reciprocally enhance the pro-thrombotic functions of each other. • anti-inflammatory therapies, including tocilizumab, chloroquine, and hydroxychloroquine, which can be promising treatment to control excessive cytokine release in severe covid- , have the potential to reduce the risk of vascular thrombotic events, but more clinical data are needed for optimum instruction of drug use and drug selection. the novel coronavirus disease , caused by severe acute respiratory syndrome coronavirus type (sars-cov- ) infection, is currently leading to a global pandemic. clinical studies so far have demonstrated an association between covid- severity and vascular thrombotic or thromboembolic events [ ] . moreover, covid- severity is also frequently associated with over-activated inflammatory response characterized by excessive cytokine release [ ] [ ] [ ] [ ] [ ] . to prevent the vascular thrombotic events in severe covid- , it is urgently necessary to understand the underlying pathogenic mechanisms. based on currently available evidence, we discuss the mechanisms of initiation, the characteristics, and the immuno-pathological basis of the over-activated inflammatory response associated with severe covid- . furthermore, we summarize cytokines that have consistently displayed enhanced serum levels in severe patients. mechanisms through which these cytokines influence the coagulable state are reviewed in detail after an overview of the role of excessive pro-inflammatory cytokine release in promoting hypercoagulable state in covid- . additionally, we discuss the mechanisms and potential of anti-inflammatory medications investigated in recent clinical trials to prevent the hypercoagulable state in severe covid- . despite a lack of direct evidence showing a downregulation of angiotensin-converting enzyme (ace ) expression levels in covid- , a recent bio-informatic analysis predicts a downregulated ace expression in sars-cov- infection [ ] . by interpreting the antithrombotic and anti-inflammatory functions of ace , we propose that the potential ace down-regulation in covid- can contribute to the pathogenesis of hypercoagulable state and excessive inflammation in severe cases. moreover, we further discuss the mutually promoting effects of excessive cytokine release and potential ace down-regulation on the pro-thrombotic activities of each other. ultimately, based on previous preclinical studies and recent clinical studies, we analyze the effects of angiotensin-converting enzyme inhibitors (aceis) and angiotensin ii receptor blockers (arbs) treatment on the risk of vascular thrombosis. by interpreting the pathological mechanisms, we aim to illustrate that excessive pro-inflammatory cytokine release and potential ace down-regulation can promote the hypercoagulable state in severe covid- , and propose that the anti-inflammatory medications, as well as acei/arb, can benefit severe covid- patients by reducing the risk of vascular thrombotic events. although pneumonia is the most common manifestation of severe covid- , recent clinical studies have suggested a significant association between vascular thrombotic or thromboembolic events and covid- severity [ , , ] . in a retrospective analysis of covid- patients, a marked increase in serum d-dimer level, indicating a hypercoagulable state, is frequently observed in non-survivors. disseminated intravascular coagulation (dic) has occurred in . % of non-survivors, compared to an only . % chance of dic in patients who survived [ ] . additionally, a pooled analysis has further confirmed the association between increased serum d-dimer level and poor clinical prognosis [ ] . in a recent covid- clinical case report, anti-phospholipid antibodies (apl) have been detected in icu patients with multiple infarcts, and anti-phospholipid syndrome (aps) is suspected to be a potential cause of the vascular thrombotic events in these severe cases [ ] . as evidenced by previous clinical studies, persistent positive for serum apl is not necessarily related to the clinical characteristics of aps, including thrombotic events, in patients with virus infection [ ] . in addition to the increased apl, a second thrombophilic factor is necessary for the thrombotic events associated with aps to occur [ ] . therefore, a hypercoagulable state might be an essential factor that synergistically contributes to the observed multiple infarctions along with the enhanced serum apl in the icu patients with covid- . the hypercoagulable state, manifested by the multiinfarctions and the increased serum level of d-dimer, is related to covid- severity and indicate a poor prognosis. understanding the pathophysiology behind the vascular thrombotic events in severe covid- is beneficial for the prevention and treatment of these events. a retrospective clinical study indicates that, compared to mild covid- patients, severe patients display lower levels of cd + t cells and cd + t cells, as well as higher levels of interleukin- (il- ) and interleukin- (il- ) in the peripheral blood before receiving any treatment for the disease. no significant difference is found in the t cellpolarizing cytokines, including interleukin- (il- ), interferon γ (ifn-γ), and interleukin- (il- ), responsible for the polarization of t helper cell (th) , th , and th , respectively [ ] . however, compared to healthy individuals, covid- patients have displayed elevation of serum levels of cytokines that are suggestive of pro-inflammatory th activation, including ifn-γ and ifn-γ inducible protein (ip- ). besides the enhanced levels of pro-inflammatory cytokines, increased plasma levels of anti-inflammatory cytokines, including il- , have been simultaneously detected in covid- patients, suggesting a dysregulation of the adaptive immune system following the virus infection [ ] . although the mechanism underlying the dysregulated immune response in covid- is by far beyond comprehension, clinical studies have shown that, both declined levels of innate and adaptive lymphoid cell, and an increase in the percentage of naïve cd + t cells accompanied by a reduction in the percentage of memory cd + cells, are associated with sars-cov- infection, indicating a reduced efficacy for virus clearance in infected patients [ , ] . thus, dysregulated t cell immune responses are observed in covid- , potentially contributing to the commonly seen lymphocytopenia in infected patients, leading to inefficient clearance of the virus [ , ] . previous animal experiments using mouse models with mers (middle east respiratory syndrome) and sars-cov (severe acute respiratory syndrome coronavirus) infections have shown that the timing of immune response in the virus infections is critical for optimal virus clearance: an early anti-viral response before the peak of virus replication after an infection is important for the inhibition of virus replication and the activation of anti-viral adaptive immune responses; however, a delayed immune response to the virus infection not only inhibits the virus specific adaptive immune responses, but also enhances the excessive inflammatory cytokine release and the leukocyte infiltrations into tissues or organs, leading to a poor disease outcome [ , ] . therefore, ineffective clearance of sars-cov following its infection can lead to a delayed immune response, which can bring about continuous inflammations and excessive cytokine release. according to the result of a retrospective clinical study, excessive cytokine release has occurred in severe patients with covid- . among covid- patients, intensive care unit (icu) patients (n = ) have higher serum levels of interleukin- (il- ), interleukin- (il- ), il- , granulocytecolony stimulating factor (g-csf), ip- , monocyte chemoattractant protein- (mcp- ), macrophage inflammatory protein -α (mip- α), and tumor necrosis factor-α (tnfα) than non-icu patients (n = ). compared to healthy adults, both icu and non-icu patients have shown enhanced serum levels of interleukin- β (il- β), interleukin- receptor antagonist (il- ra), il- , interleukin- (il- ), interleukin- (il- ), interleukin- (il- ), basic fibroblast growth factor (fgf ), g-csf, granulocyte-macrophage colony-stimulating factor (gm-csf), ifn-γ, ip- , mcp- , mip- α, macrophage inflammatory protein -β (mip- β), platelet-derived growth factor (pdgf), tnf-α, and vascular endothelial growth factor (vegf) [ ] . besides, yang et al. have revealed a positive correlation between the levels of ip- , monocyte chemoattractant protein- (mcp- ), il- ra in the peripheral blood, and the severity of the coronavirus infection based on the murray score [ ] . serum cytokine profiles, including those of il- , il- , il- , il- , ifn-γ, and tnf-α, change dynamically after the disease onset in severe patients, reaching their peak levels in to days after the disease onset [ ] . after the disease onset, while mild patients have not had remarkable fluctuations in the serum levels of any of these cytokines, significant fluctuations in il- and ifn-γ serum levels have been discovered in severe patients. in severe patients, il- and ifn-γ serum levels have declined to the initial level in - days after the disease onset. the serum level of il- has remained continuously higher in severe patients than in mild patients, and the serum level of il- has remained higher in severe patients than in mild patients by the end of - days after the disease onset. however, no significant difference in serum levels of il- and tnf-α between mild and severe patients has been detected throughout the whole course of the disease [ ] . a report by zhou et al. reveals the immuno-pathological basis behind the hyper-inflammatory immune response associated with severe covid- . by intracellular staining of potential cytokines responsible for promoting cytokine storm in sars and mers, zhou et al. have demonstrated that compared to healthy individuals, icu and non-icu patients have increased expressions of il- and gm-csf in the cd + t cells. moreover, the extent of the increment in il- and gm-csf expressions in cd + t cells is correlated with the severity of disease manifestations (fig. ). th cells, positively stained for both gm-csf and ifnγ, are only present in icu patients, but not in non-icu patients. this phenomenon, in part, explains the over-activated inflammation in severe patients with covid- [ ] . enhanced plasma levels of activated cd + cd + monocytes capable of producing pro-inflammatory cytokines il- and gm-csf are found only in covid- patients, but not in healthy individuals [ ] . acting on the gm-csf receptors on myeloid cells, gm-csf leads to activation of the janus kinase -signal transducer and activator of transcription (jak -stat ) pathway, influencing both the differentiation of myeloid precursor cells and the polarization of macrophages [ ] . although the activity of gm-csf is redundant in physiological myeloid hematopoiesis because of the presence of g-csf and macrophage-colony stimulating factor (m-csf), gm-csf is responsible for emergent hematopoiesis in acute pathological conditions such as acute infections, in accordance with the sudden increment of its serum level following an acute infection [ ] . in covid- , enhanced gm-csf level can increase the gm-csf receptor signal strength in myeloid cells, promoting their differentiation into proinflammatory macrophage subtype [ ] . an in vivo study suggests that gm-csf dependent pu. expression mediates the terminal differentiation of macrophages and the enhancement in phagocytic activities of macrophages [ ] . besides the role of gm-csf in regulating macrophage differentiation and function, gm-csf can also up-regulate the immunological activities of neutrophils, including tissue entries, phagocytic activities, and reactive oxygen species (ros) productions ( fig. ) [ ] . during virus infections, activated t cells can enter the bone marrow and influence hematopoiesis by secreting ifn-γ [ , ] . both in vitro and in vivo evidence has shown that t-cell-derived ifn-γ favors monocyte differentiation over neutrophil differentiation during myelopoiesis in the bone marrow. this tilted balance of myeloid progenitor development away from neutrophil towards monocyte is mediated by both ifn-γ-stimulated expressions of pu. and interferon responsive factor (irf- ), which are inducers of monocyte differentiation, and ifn-γ-induced activation of suppressor of cytokine signaling (socs ), which inhibits the phosphorylation of stat in response to g-csf, in granulocyte-macrophage progenitors ( fig. ) [ ] . ifn-γ can also skew the development of multipotent hematopoietic progenitors to the monocytic lineage in an indirect manner by stimulating il- production in the bone marrow mesenchymal stem cells [ ] . moreover, in response to ifn-γ, the production of il- in macrophages is also positively regulated through direct il- promoter binding by notch [ ] . when encountering the novel coronavirus, the immune system displays low efficacy for virus clearance [ , ] . constantly exposed to the viral pathogen, the immune system may initiate a continuous systemic inflammation through gm-csf and ifn-γ production by t cells. gm-csf and ifn-γ could promote both the function and the production of macrophages. activated macrophages could further continue the vicious cycle of excessive systemic inflammation by promoting the production and immunological activities of itself through gm-csf secretion ( fig. ) [ ] . the excessive inflammatory response in severe patients, potentially attributed to unresolved virus infection, could eventually result in t cell depletion. in accordance, a high neutrophil to cd + t cell ratio (n r) in the blood has been suggested to predict a worse prognosis in covid- patients [ ] . this observation might be explained by the t cell depletion in severe patients, in conjunction with the role of ifn-γ, secreted by t fig. schematic diagram showing the immuno-pathological basis of excessive cytokine release in severe covid- patients. sars-cov- severe acute respiratory syndrome coronavirus type , il- interleukin- , gm-csf granulocytemacrophage colony-stimulating factor, ifn-γ interferon γ, gmp granulocyte-macrophage progenitor, socs suppressor of cytokine signaling , g-csf granulocyte-colony stimulating factor, irf- interferon responsive factor cells, in favoring monocytes differentiation versus neutrophil differentiation in the bone marrow during virus infections. age and human leucocyte antigen (hla) polymorphisms can be factors that predispose covid- patients to prolonged and excessive inflammations. an aged immune system is not only associated with a decreased efficiency for virus clearance due to declined b and t cell productions and functions, leading to constant viral exposure and excessive inflammations, but also associated with an increased propensity for excessive pro-inflammatory cytokine release because of reduced productions of the immunosuppressive thymusderived regulatory t cells (tregs) [ , ] . additionally, hla displays extensive polymorphisms, and is crucial for the immune response to viral antigens. sars-cov- infection may be similar to other types of virus infection, where associations can exist between certain hla haplotypes and disease severity [ ] . certain hla haplotypes might predispose covid- patients to a more excessive inflammatory response. future genetic sequencing studies are required to understand this association, which can guide the treatment and management of patients in the clinic, as well as vaccine productions. early anti-viral immune responses can facilitate the clearance of pathogens; however, the prolonged and excessive inflammatory activities, observed in severe covid- patients, can result in tissue damage and hypercoagulable state. differences in standards for severe illness and variations in health conditions among patients may lead to discrepancies of the results from different retrospective clinical studies that compare serum levels of cytokines between mild and severe covid- patients. however, most retrospective clinical studies so far have consistently displayed enhanced serum levels of pro-inflammatory cytokines, including il- , th related ifn-γ, il- along with increased serum levels of the anti-inflammatory cytokine il- in severe patients, in comparison with the serum levels of these cytokines in mild patients [ ] [ ] [ ] [ ] [ ] . based on previous experimental and clinical evidence, generally, the pro-inflammatory cytokines, including il- , ifn-γ, il- , can promote a hypercoagulable state in favor of thrombus formation, whereas the anti-inflammatory cytokine, il- , conducts anti-coagulative activities which prevent vascular thrombotic events (fig. ) . in severe covid- , the excessive pro-inflammatory cytokine release can contribute to the activation of coagulation cascade. thrombin, an essential candidate in the coagulation cascade, not only mediates the blood clot formation by converting fibrinogen to fibrin, but also can act on the proteinase-activated receptors to amplify the inflammation in a positive feedback manner [ ] . the presence of positive regulations between inflammation and coagulation may explain the concurrent existence of excessive pro-inflammatory cytokine release and serum markers of hypercoagulable state in severe covid- patients. although the excessive pro-inflammatory cytokine release in severe covid- patients can promote vascular thrombosis through complicated interplays with factors that participate in the blood coagulation, it might not be the only etiology of hypercoagulable state and vascular thrombus formation in severe covid- . a recent autopsy study has demonstrated that sars-cov- can directly invade and damage vascular endothelial cell (ec), as evidenced by the presence of viral inclusions inside the vascular ecs in the histological analyses [ ] . besides, aggregation of inflammatory leukocytes around vascular ecs has also been detected in the histological analyses, indicating that sars-cov- can possibly induce vascular endothelitis by directly invading ecs, facilitating vascular thrombosis [ ] . therefore, the significance of excessive pro-inflammatory cytokine release as an etiology of hypercoagulable state and thrombus formation in severe covid- should be further assessed in future experimental studies. according to the previously described characteristics of the dynamically changing cytokine profiles, in severe covid- patients, enhanced il- and ifn-γ serum levels persist only for the first - days following disease onset, whereas the il- serum level is continuously elevated throughout the whole course of the disease [ ] . hence, in severe covid- , enhanced serum levels of il- and ifn-γ could be a trigger for the elevation in serum il- level by increasing macrophage differentiation and activation (fig. ) . the mechanisms by which the pro-inflammatory cytokines, il- , ifn-γ, il- , and the anti-inflammatory cytokine, il- , affect the coagulable state are interpreted in detail subsequently. the order of discussion on the proinflammatory cytokines is arranged based on the potential course of sequence by which their release is triggered during the over-activated inflammatory response associated with severe covid- . the excessive release of il- , ifn-γ, il- , and il- in severe covid- could result from the sars-cov- infection itself, the bacterial infection secondary to the sars-cov- infection in immune-compromised individuals, or the excessive innate immune response to damage-associated molecular patterns (damp) produced by tissue damages due to sars-cov- invasion [ , ] . hence, blocking the host cell entry and the replication of sars-cov- during early infection could prevent the excessive release of the pro-thrombotic cytokines in covid- . however, for severe patients who have already developed excessive inflammatory response, antagonizing the productions and activities of the pro-thrombotic cytokines could be a better approach to attenuate the over-activated inflammatory response and to reduce the risk of vascular thrombosis. cytokines with pro-thrombotic activities il- il- is pleiotropic regarding its influence on the differentiation of various cd + t cell subsets. il- induces th and th differentiation by enhancing interleukin- (il- ) receptor expression and il- receptor expression, respectively, through stat [ , ] . however, il- plays an inhibitory role in the differentiation of th [ ] . additionally, il- has been shown to increase the thymic or peripheral production of tregs and the immuno-suppressive activity of tregs [ , ] . il- can stimulate endothelial plasminogen activator inhibitor (pai- ) release in vitro, indicating a potential for il- to inhibit fibrinolysis [ ] . results of clinical study have shown that il- can activate both coagulation and fibrinolysis in vivo, possibly mediated by il- induced cytokines that can impair the anti-coagulant function of the vascular endothelium [ ] . by initiating the differentiation of th , il- can induce the production of ifn-γ in t cell subsets [ , ] . ifn-γ is associated with pro-coagulant effects by several mechanisms introduced below. ifn-γ ifn-γ plays a dual role in vascular thrombosis by promoting thrombus formation, while delaying thrombus resolution. according to previous experimental evidence, ifn-γ promotes thrombus formation mainly through increasing the platelet activities and impairing the vascular endothelium. in vitro experiment on monocytic u cell in the condition of a low thrombin concentration has shown that ifn-γ increases platelet adhesion to the monocytic cell, thereby promoting platelet activation, which is indicated by enhanced platelet -hydroxytryptamine ( -ht) release [ ] . furthermore, in vitro evidence has also indicated that th related activities, especially ifn-γ secretion, increase the expression of tissue factor (tf) in monocytes, facilitating the pro-coagulant activity of monocytes [ ] . ifn-γ plays a crucial role in inflammation by mediating macrophage activation and leukocyte recruitment. results of animal studies suggest that increased ifn-γ production mediates monocyte activation and monocyte recruitment to the vessel, promoting vascular inflammation, ros production, together with vascular ec and vascular smooth muscle cell (smc) dysfunction [ ] . in physiologic conditions, vascular ecs maintain their anti-thrombotic, anti-inflammatory, and vasodilative properties by producing substances, including anti-thrombin, thrombomodulin, prostaglandins, and nitric oxide (no) [ ] . ifn-γ induces vascular dysfunction by promoting the expression of chemoattractant molecule mcp- , changing the physiologic anti-inflammatory phenotype of the vascular ecs into the pathological pro-inflammatory phenotype, thereby leading to enhanced ros production and decreased vasodilative function of the vascular endothelium, in favor of thrombus formation [ ] . damage of vascular ecs exposes the sub-endothelial pro-thrombotic matrix to the circulating platelets. interactions between subendothelial pro-thrombotic proteins and platelets result in platelet adhesion, activation, and aggregation through complex receptor-mediated signalings [ , ] . additionally, a recent animal experiment shows that ifn-γ can promote deep venous thrombosis by mediating neutrophil extracellular traps (nets) formation [ ] . after thrombus formation, ifn-γ could impede the thrombus resolution through decreased vegf and matrix metalloproteinase (mmp ) expressions in macrophages mediated by ifn-γ /stat signal pathway activation [ ] . overall, il- contributes to a hypercoagulable state by enhancing the activity and production of platelets, promoting an imbalance between plasma levels of coagulative factors and anti-coagulative factors, and inducing endothelial dysfunction. il- induces thrombus formation by increasing platelet production and activity. il- has been shown to promote a hypercoagulable state and accelerate blood clot formation via whole blood thromboelastography, as evidenced by the markedly reduced time before maximum velocity of clot growth (tmrtg). the il- -induced hypercoagulable state in the whole blood has been further confirmed by the hyper-activation of platelets under scanning electron microscopy [ ] . il- contributes to inflammatory thrombocytosis by up-regulating thrombopoietin (tpo) production in hepatocytes, indicated by in vivo experiments involving il- administration and tpo neutralization in c bl/ mice [ ] . increased platelet production plays a role in promoting primary hemostasis during inflammation. furthermore, il- can promote hypercoagulability by modulating plasma levels of coagulant factors. in vitro evidence has suggested that, in ecs and monocytes, il- increases the expression of tf, which initiates the activation of extrinsic coagulation pathway that is responsible for thrombus formation [ , ] . results of animal experiments also confirm the capability of il- to stimulate the initiation of coagulation. in response to an escherichia coli (e coli.) endotoxin injection, an increase in plasma levels of thrombin fragments f f and thrombin-antithrombin iii complexes, indicating coagulation activation, is abrogated by a simultaneous il- antibody injection in chimpanzees [ ] . in canine, following il- injection, plasma levels of fibrinogen and von willebrand factor (vwf) are increased, while the plasma concentration of free protein s is reduced, suggesting that increasing the plasma level of il- can induce a hypercoagulable state [ ] . last but not least, il- signaling in ecs results in endothelial dysfunction, increasing the risk of vascular thrombotic events. although ecs do not have il- receptor expression, they do have the expression of glycoprotein (gp ), which can bind to the complex of il- and its soluble receptor sil- r in the plasma, allowing ecs to respond to il- stimulation [ ] . il- trans-signaling can activate vascular ecs by increasing the expressions of chemokines and cell adhesion molecules, as well as the secretion of il- by the vascular ecs, turning the physiologic phenotype of the vascular ecs into the pathologic phenotype [ ] . additionally, in a cell culture experiment, il- trans-signaling in microvascular ecs leads to endothelial damages by promoting apoptosis [ ] . despite plenty of evidence showing the contribution of il- to increased blood coagulability and thrombus formation, il- could also be necessary for venous thrombus resolution by recruiting macrophages and enhancing expressions of mmp , mmp , and urokinase-type plasminogen activator (plau) through il- /stat signaling in macrophages [ ] . however, the exact role il- plays in thrombus resolution can be controversial because of the in vitro evidence showing up-regulated pai- expression in adipose tissue by il- [ ] . the role and mechanism of il- production during acute virus infection were elegantly reviewed by josé m. rojas et al. [ ] . by mediating negative regulation of mhc ii expression on antigen-presenting cells, il- can impair the priming of th , which plays a vital role in controlling virus infections by facilitating the effector functions of cytotoxic leukocytes and lymphocytes [ , ] . although it is unclear whether the elevation of serum il- in covid- patients is a mechanism by which sars-cov- circumvent the surveillance of adaptive immunity, il- enhancement in severe cases is likely a compensatory mechanism to quench the over-activated inflammation, having an inhibitory effect on coagulation and thrombus formation. because of the anti-inflammatory function of il- , it could prevent vascular thrombosis by alleviating the pro-thrombotic effects associated with pro-inflammatory cytokines [ , ] . by inhibiting the production of proinflammatory cytokines, il- can indirectly decrease mitochondrial ros production in ecs, reducing the acute vascular endothelial cell activation [ ] . il- is elevated in response to venous thrombosis in the vascular wall, playing a role in suppressing vascular inflammation and reducing venous thrombosis, as evidenced by animal experiments in rats [ ] . in a double-blinded cohort study involving healthy volunteers, il- injection has resulted in decreased bone marrow platelet production compared to placebo, therefore reducing the risk of thrombus formation [ ] . a clinical study has further revealed the association between decreased serum il- and endothelial dysfunction in patients with venous thrombosis [ ] . by targeting the excessively released pro-thrombotic cytokines in severe covid- , anti-inflammatory therapy can theoretically alleviate the hypercoagulable state. currently, it is believed that the timing of anti-inflammatory therapy is an essential factor to be considered when weighing the risks and benefits for covid- patients. during the incubation period and the non-severe symptomatic stage of covid- , anti-inflammatory therapy can impair the antiviral immune response, increasing the risk of aggravating disease severity; however, anti-inflammatory therapy in the severe stage of covid- , when the viral load is high and the optimal time window for virus clearance is missed, can benefit covid- patients by alleviating tissue damages due to the excessively activated inflammatory response [ ] . the use of anti-inflammatory medications, including tocilizumab, as well as chloroquine and hydroxycloroquine, to suppress the over-activated inflammatory response in severe covid- has been undergoing clinical investigations. by antagonizing the cytokines with pro-thrombotic effects, these anti-inflammatory medications also have the potential to lower the risk of vascular thrombosis. conventionally, corticosteroids are used as a treatment for excessive inflammation, but high doses and long durations of drug intake are often needed, usually leading to serious side effects [ , ] . il- is considered as a key mediator of excessive pro-inflammatory cytokine release in covid- , and tocilizumab, an anti-il- receptor monoclonal antibody, is used to suppress the over-activated inflammatory response in severe covid- cases in several recent observational clinical studies. these studies indicate that tocilizumab, without significant associated adverse effect, is effective for controlling excessive inflammations and reducing disease mortality in severe covid- patients, especially for those with significant elevation in serum il- level [ , ] . besides, a systemic review also indicates that tocilizumab could be a promising treatment for the excessive inflammation in severe covid- [ ] . however, considering the number of patients participated in these studies are relatively small, and these studies are merely observational, the conclusions of these studies need to be confirmed by randomized controlled clinical trials with larger numbers of participants. by suppressing the over-activated inflammation, tocilizumab can theoretically mitigate vascular thrombosis by reducing the pro-thrombotic effects of the pro-inflammatory cytokines during excessive inflammations in severe covid- (fig. ) . hence, future clinical studies of tocilizumab could evaluate the potential of tocilizumab to reduce vascular thrombotic events by adding vascular thrombotic complications, including dic, as a secondary endpoint. according to recent clinical studies, low dosage of adjunctive chloroquine and hydroxychloroquine treatment could be an effective solution for excessive pro-inflammatory cytokine release in severe covid- patients. in a retrospective observational study with patients, in addition to the basic treatments for covid- , low doses of hydroxychloroquine significantly reduce the fatality and the serum level of pro-inflammatory il- in severe covid- patients [ ] . a randomized controlled clinical trial suggests that compared to a low dosage of chloroquine treatment, a high dosage of chloroquine treatment for severe covid- patients is associated with increased incidences of adverse effects, especially when used adjunctively with other antibiotics or anti-viral medications [ ] . however, severe covid- patients in the group receiving high dosage of chloroquine treatment are of older ages, which could be a confounding factor in this randomized clinical trial. chloroquine and hydroxychloroquine, which can be promising in the treatment of excessive inflammatory response in severe covid- , have both immuno-modulatory and anti-thrombotic functions. chloroquine and hydroxychloroquine can inhibit the production of ifn-γ, il- , and tnf-α by peripheral blood mononuclear cells (pbmcs) [ ] . this modulation in immunological activities of pbmcs is critical in preventing the viscious cycle of excessive inflammations in covid- (fig. ) , hence, reducing the risk of excessive pro-thrombotic cytokine release and hypercoagulable state (fig. ) . despite the negative regulating effects on pro-inflammatory cytokine productions, chloroquine and hydroxychloroquine are less associated with secondary infections following their treatment than immuno-suppressive medications are, as their effects on the immune system is immuno-modulatory [ ] . in addition, hydroxychloroquine reduces the risk of vascular thrombosis by inhibiting platelet activation in response to pro-coagulant factors, as evidenced by the in vitro study showing that hydroxychloroquine, at the concentration of mm, abrogates expressions of platelet cd a and cd , which indicate platelet activation, following apl and thrombin agonist receptor peptide treatment. this inhibitory effect of hydroxychloroquine on platelet activation is also present even when the concentration of hydroxychloroquine is as low as . mm [ ] . similar function in the inhibition of platelet activation has also been demonstrated in vitro with chloroquine [ ] . in agreement with its function of platelet inhibition, hydroxychloroquine can reduce the incidence of vascular thrombotic events after operations or in autoimmune diseases including systemic lupus erythematosus (sle) and aps [ , ] . furthermore, in vitro evidence has shown that chloroquine prevents sars-cov- infection in vero e cells by preventing cell entry and interfering with the process of virus-cell fusion [ ] . chloroquine can interfere with host cell-entry of sars-cov- by preventing the terminal glycosylation of its receptor ace [ ] . therefore, chloroquine and hydroxychloroquine can theoretically lower the risk of hypercoagulable state and vascular thrombosis by alleviating the endothelitis caused by direct sars-cov- invasion into the host vascular ecs in covid- [ ] . sars-cov- and sars-cov bind to ace for cell entry, and share a similar amino acid sequence in their receptor-binding spike proteins, with a % amino acid sequence similarity between the spike protein of sars-cov- and that of sars-cov s urbani [ , ] . these two viruses also bind to the same receptor, ace , for cell entry [ ] . as in the case of sars-cov infection, the sars-cov- spike protein is primed by the transmembrane serine protease (tmprss ), which facilitates viral entry into host cells [ ] . a recent study using single-cell rna-sequencing (scrna-seq) has identified cells with co-expressions of ace and tmprss , including type ii pneumocytes, ileal enterocytes, and nasal goblet cells [ ] . these cells are, therefore, targets for sars-cov- invasion in the host. interactome model for interactions between sars-cov- and human cell, based on characteristics of human protein/ sars-cov and human protein/mers-cov interactions, predicts a down-regulating effect of sars-cov- infection on ace expression in human cells [ ] . ace protein expression is found in multiple organs and tissues, including vascular ecs and smcs [ ] . both in vitro and in vivo experiments have pointed out the role of sars-cov spike protein in down-regulating ace expression through ace binding [ ] . sars-cov infection can result in a reduction in myocardial ace expression at mrna and protein levels in mice [ ] . in vitro experiments have proved the ability of sars-cov spike protein to induce the activity of a disintegrin and metalloprotease (adam ), which sheds ace from the membrane, down-regulating the expression of ace in vero e cells [ ] . although tmprss may interfere with the ace shedding activity of adam , coexpressions of tmprss and ace can only be found in restricted types of cells, as indicated by the result of the recent scrna-seq experiment [ , ] . due to the similarity between amino acid sequences of sars-cov and sars-cov- spike proteins, sars-cov- could lead to a downregulation in ace expression in covid- through ace binding. however, further experiments using animal models with sars-cov- infection are still required to better clarify the effect of sars-cov- infection on ace expression in different tissues or cell types. ang ii (angiotensin ii) is the effector molecule in the ace/ ang ii/at receptor (angiotensin ii receptor type ) axis, associated with pro-thrombotic activities including increased tf and pai- production by aortic ecs and smcs [ , ] . upon binding to at r, ang ii can induce ros production by stimulating the activity of nadh and nadph oxidases in vascular smcs, promoting oxidative stress and vascular inflammation, which can lead to vascular injury and thrombus formation [ ] . many pieces of experimental evidence have suggested the anti-thrombotic activity of the ace /ang - (angiotensin - )/mas receptor axis. in rats, low ace activity is related to thrombus formation, whereas enhancing ace activity decreases both platelet accumulation and size of thrombi, delaying the thrombosis [ ] . ace catalyzes ang ii to ang - , which does not have the pro-thrombotic functions associated with ang ii [ ] , preventing the adverse effect of ang ii on vascular injury and thrombosis [ ] . furthermore, ang - can promote platelet no release by binding to the mas receptor on the platelet [ ] . therefore, ace down-regulation, which possibly occurs in covid- , enhances the activation of the ace/ ang ii/at receptor axis. in addition to the pro-thrombotic activities of the ace/ang ii/at receptor axis, enhanced ang ii level can further inhibit the expression of ace by activating mitogen-activated protein kinase (mapk ) and mapk , worsening the vascular injury and thrombosis [ ] . excessive cytokine release in severe covid- can be a crucial factor contributing to the pro-thrombotic activities related to ace down-regulation (fig. ) . emerging amounts of evidence have shown the critical role of pro-inflammatory cytokines in ang ii-mediated vascular fig. schematic diagram showing the mechanisms by which potential ace expression down-regulation in covid- contributes to vascular thrombosis and the reciprocal promoting relationship between the effect of ace expression down-regulation and that of excessive cytokine release on vascular thrombosis. ace angiotensin-converting enzyme , ifn-γ interferon γ, il- interleukin- , pai- plasminogen activator inhibitor- , tf tissue factor dysfunctions. in ifn-γ knockout mice, characteristics of ang ii-mediated vascular injury are attenuated. this attenuation of vascular injury is due to decreased ifn-γ mediated nature killer (nk) cell and monocyte recruitment, and reduced mutual activation of nk cells and monocytes at the vessel [ ] . in addition, a recent animal study suggests that ang ii promotes thrombosis through increased platelet activation induced by t cell-dependent il- signaling [ ] . hence, the elevated serum levels of pro-inflammatory cytokines in severe covid- tend to favor the ang iimediated vascular injury and thrombosis. ace down-regulation can, in turn, lead to enhanced levels of cytokines with pro-thrombotic activities (fig. ) . a randomized double-blind clinical study has shown that in patients with coronary artery disease, acei/arb treatment elevates the serum level of il- , but reduce the serum level of il- , thereby decreasing the thrombin stimulated platelet aggregation in the plasma [ ] . thus, in covid- , the possible down-regulated ace activity can leave the activity of ace/ang ii/at axis uncontrolled, resulting in excessive inflammation characterized by increased serum levels of pro-thrombotic cytokines but decreased serum levels of antithrombotic cytokines, contributing to vascular thrombosis. acei/arb has been essential for treating hypertension and heart failure, but the use of acei/arb during the covid- pandemic was concerned because of the potential risk of acei/arb in enhancing ace expression, which may increase the chance or severity of infection. however, most of recent clinical studies do not indicate an association between acei/arb treatment and a higher risk of sars-cov- infection. a meta-analysis on , cases of covid- suggests that acei/arb does not increase the risk of sars-cov- infection and the disease transition from mild to severe, and that acei/arb treatment reduces the incidence of mortality compared to other anti-hypertensive agents in covid- patients with hypertension [ ] . moreover, in a retrospective analysis of adult hypertensive covid- patients, compared to patients not taking acei/arb or taking other anti-hypertensive drugs, those taking acei/arb are associated with a lower risk of dic and mortality [ ] . as described at the end of the previous section, acei/ arb can lower the risk of hypercoagulability by controlling systemic inflammations. besides, acei/arb can also reduce vascular inflammations, preventing vascular injuries and platelet activations (fig. ) [ ] . acei can prevent vascular inflammation by reducing the activity of nuclear factor kappa-b (nf-κb) in vascular cells and macrophages [ , ] . furthermore, acei reduces levels of pro-inflammatory cytokine expressions in the aorta in patients with abdominal aortic aneurysm, indicating the ability of acei to reduce vascular inflammation [ ] . additionally, arb can normalize the vascular inflammation and vascular dysfunction caused by ang ii [ ] . antagonization of at by arb can result in increased ang ii-induced ec at activation, which can promote no production, preventing vascular thrombosis [ , ] . hence, hypertensive covid- patients can benefit from the vasoprotective mechanisms of acei/arb. better clinical outcomes of hypertensive covid- patients who received acei/arb in recent clinical studies can be in part attributed to the capability of acei/arb to prevent a hypercoagulable state. the severity of covid- is associated with both excessive cytokine release and hypercoagulable state. age and hla polymorphisms could be factors that determine the propensity to develop the over-activated inflammations in covid- . in severe covid- patients, the excessive release of pro-inflammatory cytokines, including il- , ifnγ, and il- , has been consistently shown in recent retrospective clinical studies. abundant pieces of evidence have indicated the role of these pro-inflammatory cytokines in promoting a hypercoagulable state and, therefore, vascular thrombosis. however, excessive pro-inflammatory cytokine release might not be the only etiology of hypercoagulable state in covid- , and its significance in the pathogenesis of hypercoagulable state in severe covid- should be investigated in future studies. because of structural similarities between receptor binding proteins of sars-cov and sars-cov- , sars-cov- infection is likely to result in ace down-regulation, which has been confirmed in sars-cov infection previously. this hypothesis is currently supported by the prediction using an interactome model, but further experimental studies are needed to confirm the hypothesis. ace down-regulation can lead to ang ii-mediated vascular dysfunction and thrombosis. furthermore, ace down-regulation and excessive pro-inflammatory cytokine release can mutually promote the pro-thrombotic activities of each other. understanding the mechanisms behind the vascular thrombotic events in severe patients with covid- is essential for the prevention and treatment of vascular thrombosis in covid- . acei/arb can prevent hypercoagulable state by activating ace /ang - /mas receptor axis while inhibiting ace/ang ii/at receptor axis, which possibly explains the reduced incidence of dic in hypertensive covid- patients receiving acei/arb treatment. anti-inflammatory medications, including tocilizumab, chloroquine, and hydroxychloroquine, could be an effective treatment to control the over-activated inflammatory response and have the potential to reduce the risk of vascular thrombosis in severe covid- . the potential of these anti-inflammatory medications to reduce vascular thrombotic events, along with the optimum choice of an anti-inflammatory drug to control the excessive cytokine release, detailed standards for patient selection, as well as the optimal time of initiation and the optimal duration of the anti-inflammatory therapy, are remained to be 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critically ill patients with covid- team ftc-( ) effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial chloroquine and hydroxychloroquine equally affect tumor necrosis factor-alpha, interleukin , and interferongamma production by peripheral blood mononuclear cells predictors of major infections in systemic lupus erythematosus hydroxychloroquine reverses platelet activation induced by human igg antiphospholipid antibodies on the inhibitory effect of chloroquine on blood platelet aggregation prevention of postoperative deep venous thrombosis in legs by orally administered hydroxychloroquine sulphate what is the role of hydroxychloroquine in reducing thrombotic risk in patients with antiphospholipid antibodies? remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro a pneumonia outbreak associated with a new coronavirus of probable bat origin veesler d ( ) structure, function, and antigenicity of the sars-cov- spike glycoprotein sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury sars-coronavirus modulation of myocardial ace expression and inflammation in patients with sars modulation of tnf-alpha-converting enzyme by the spike protein of sars-cov and ace induces tnf-alpha production and facilitates viral entry tmprss and adam cleave ace differentially and only proteolysis by tmprss augments entry driven by the severe acute respiratory syndrome coronavirus spike protein angiotensin ii increases plasminogen activator inhibitor type and tissue-type plasminogen activator messenger rna in cultured rat aortic smooth muscle cells angiotensin ii increases plasminogen activator inhibitor- and tissue factor mrna expression without changing that of tissue type plasminogen activator or tissue factor pathway inhibitor in cultured rat aortic endothelial cells angiotensin ii stimulates nadh and nadph oxidase activity in cultured vascular smooth muscle cells ace activation promotes antithrombotic activity the effects of angiotensin metabolites on the regulation of coagulation and fibrinolysis in cultured rat aortic endothelial cells ace internalization and degradation is controlled by ubiquitin ligase nedd the antithrombotic effect of angiotensin-( - ) involves mas-mediated no release from platelets map kinase/ phosphatase pathway mediates the regulation of ace by angiotensin peptides angiotensin ii-induced vascular dysfunction depends on interferon-gamma-driven immune cell recruitment and mutual activation of monocytes and nk-cells novel role of t cells and il- (interleukin- ) in angiotensin ii-induced microvascular dysfunction comparative effects of at -antagonism and angiotensin-converting enzyme inhibition on markers of inflammation and platelet aggregation in patients with coronary artery disease acei/arb use and risk of infection or severity or mortality of covid- : a systematic review and meta-analysis association of inpatient use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid- ace inhibitor quinapril reduces the arterial expression of nf-kappab-dependent proinflammatory factors but not of collagen i in a rabbit model of atherosclerosis angiotensin-converting enzyme inhibition prevents arterial nuclear factor-kappa b activation, monocyte chemoattractant protein- expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis ace inhibitors potently reduce vascular inflammation, results of an open proof-of-concept study in the abdominal aortic aneurysm angiotensin ii-mediated hypertension in the rat increases vascular superoxide production via membrane nadh/nadph oxidase activation. contribution to alterations of vasomotor tone losartan reduces phenylephrine constrictor response in aortic rings from spontaneously hypertensive rats role of nitric oxide and angiotensin ii type receptors the possible role of angiotensin ii subtype at receptors in endothelial cells and isolated ischemic rat hearts publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -cxi cr authors: yoshida, asuka; kawabata, ryoko; honda, tomoyuki; tomonaga, keizo; sakaguchi, takemasa; irie, takashi title: ifn-β-inducing, unusual viral rna species produced by paramyxovirus infection accumulated into distinct cytoplasmic structures in an rna-type-dependent manner date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: cxi cr the interferon (ifn) system is one of the most important defensive responses of mammals against viruses, and is rapidly evoked when the pathogen-associated molecular patterns (pamps) of viruses are sensed. non-self, virus-derived rna species have been identified as the pamps of rna viruses. in the present study, we compared different types of ifn-β-inducing and -non-inducing viruses in the context of sendai virus infection. we found that some types of unusual viral rna species were produced by infections with ifn-β-inducing viruses and accumulated into distinct cytoplasmic structures in an rna-type-dependent manner. one of these structures was similar to the so-called antiviral stress granules (avsgs) formed by an infection with ifn-inducing viruses whose c proteins were knocked-out or mutated. non-encapsidated, unusual viral rna harboring the ′-terminal region of the viral genome as well as rig-i and typical sg markers accumulated in these granules. another was a non-sg-like inclusion formed by an infection with the cantell strain; a copyback-type di genome, but not an authentic viral genome, specifically accumulated in the inclusion, whereas rig-i and sg markers did not. the induction of ifn-β was closely associated with the production of these unusual rnas as well as the formation of the cytoplasmic structures. eukaryotic cells are equipped with various defense mechanisms to detect and respond to viral infections rapidly. the interferon (ifn) system is one of the most important natural defenses of mammalian cells in the early phase of viral infection. host cells sense the invasion of viruses by recognizing their pathogen-associated molecular patterns (pamps), including the structural characteristics of viral rnas that differentiate them from cellular rnas . viral rnas are detected by non-self rna sensors such as toll-like receptors (tlr), and a family of cytosolic rna helicases termed rig-i-like receptors (rlrs), including retinoic-acid inducible gene-i (rig-i), melanoma differentiation-associated gene (mda ), and laboratory of physiology and genetics gene (lgp ). this is followed by the subsequent induction of ifn-β (gitlin et al., ; kaisho and akira, ; saito et al., ) . autocrine or paracrine ifns bind to ifn receptors on the cell surface, leading to the expression of s of ifn-stimulated genes (isgs) through the jak/stat signaling pathway, which ultimately exerts various antiviral effects . translational arrest is one of the ifn responses of host cells triggered by viral infections. various eukaryotic translation initiation factor (eif ) kinases such as protein kinase r (pkr) are activated in response to ifns, and the accumulation of phosphorylated eif α inhibits the translation of both cellular and viral mrnas kedersha and anderson, ; holcik and sonenberg, ) . cytoplasmic stress granules (sgs), which are the foci of concentrated s translation preinitiation complexes and defined by certain marker rna binding proteins such as t-cell intracellular antigen- (tia- ), tia- -related protein (tiar), and ras-gap-sh domain-binding protein (g bp ), are formed under these conditions kedersha and anderson, ) . because they contain stable inert mrna, sgs are believed to serve as temporary sites at which mrnaprotein complexes are stored to pause active translation or be decayed in adjacent processing bodies (anderson and kedersha, ; balagopal and parker, ; buchan and parker, ) . a number of viruses have been shown to induce the formation of sgs in infected cells, and this may be related to the virus-induced shut-off of cellular protein translation. for example, hepatitis c virus, poliovirus, semliki forest virus, and mammalian orthoreovirus promote the shut-off of cellular proteins and the assembly of sgs at the early phase of infection, and this is inhibited as the infection progresses (mcinerney et al., ; white et al., ; qin et al., qin et al., , ariumi et al., ; white and lloyd, ; garaigorta et al., ; panas et al., ; ruggieri et al., ; fitzgerald and semler, ; pager et al., ; carroll et al., ) . two rna viruses, respiratory syncytial virus and coronavirus, are also known to utilize sgs as part of the machinery to inhibit host cellular protein translation (raaben et al., ; lindquist et al., lindquist et al., , . sgs were not detected during infection by influenza a virus (iav); however, a recombinant iav lacking non-structural protein (ns ), an inhibitor of pkr, efficiently induced the formation of sgs and the production of ifn-β in a pkr-dependent manner (khaperskyy et al., ; mok et al., ; onomoto et al., ) . in this case, sgs were suggested to play an important role as the sites of viral rna sensing and subsequent anti-viral responses, because rlrs localized together with viral nucleoproteins and rna as well as anti-viral proteins in sgs (onomoto et al., ; ng et al., ) . the rna species produced during the course of rna viral replication, such as mrna, dsrna, and -triphosphate ( -ppp) rna including leader, trailer, genome, and antigenome rnas as well as defective interfering (di) genomes, have been shown to trigger the production of type i ifns (yoneyama et al., ; hornung et al., ; pichlmair et al., ; strahle et al., ; hausmann et al., ; baum et al., ; baum and garcia-sastre, ; kato et al., ; marq et al., ; davis et al., ; bowzard et al., ; weber et al., ; runge et al., ; schmolke et al., ) . we and other groups have recently reported that recombinant viruses of sendai virus (sev), a prototype of the family paramyxoviridae, in which the c proteins are knocked-out or mutated, generate dsrna in infected cells at levels similar to the production of ifn-β (takeuchi et al., ; irie et al., ) . previous studies also reported that, in the cases of sev and iav, copyback (cb)-and internal deletion (id)-type di genomes, respectively, rather than full-length viral genomes, preferentially associated with rig-i and strongly induced the production of ifn-β (baum et al., ; baum and garcia-sastre, ) . in spite of the large number of studies conducted in this field, it remains unknown what kinds of viral rna species are recognized by rlrs and where the sites of recognition are in real infections by rna viruses. in the present study, we compared some types of ifn-β-non-inducing and ifn-β-highly inducing viruses in the context of sev infection. one of the biggest advantages of this study is that the comparison can be performed within the context of the same viral species. we found that some types of unusual rna species that were distinguishable according to specific detectability by the anti-dsrna antibody or fish analysis were produced during the viral replication of ifn-inducing sevs, but not ifn-non-inducing sev strains, and accumulated into distinct cytoplasmic structures in an rna-type-dependent manner. these unusual rnas exhibited distinct properties in infected cells in terms of encapsidation with the viral n protein and subcellular distribution with sg marker proteins and rlrs. our results suggest that rna-typedependent mechanisms recognize and accumulate virus-derived, ifn-β-inducible, unusual rnas into specific compartment to trigger the production of ifn-β, and that sev may evade detection by the host innate immune system by preventing the production of these rna species. llc-mk cells (macaque monkey kidney-derived cells, described in kiyotani et al., ) and hela cells (ccl- ; purchased from atcc) were maintained as described previously (irie et al., ) . all of the sevs, even the wt of strain z and hamamatsu (hmt), used in this study were recovered from cdna using a reverse genetics technique as described previously (kato et al., ; fujii et al., ) , except for the cantell (cnt; vr- ; atcc), fushimi (fsm), and nagoya (ngy) strains. the sev recombinants, c /c(-), c(-), and v(-), were kindly provided by kato (national institute of infectious diseases, japan; kato et al., ; kurotani et al., ) . all of the sevs as well as virulent and avirulent newcastle disease virus (ndv) miyadera and d strains (toyoda et al., ) , respectively, were propagated in embryonated chicken eggs. sev and ndv titers were determined by an immunofluorescent infectious focus assay in llc-mk cells and expressed as cell infectious units (cius)/ml, as described previously (kiyotani et al., ) . one-step growth kinetics of the viruses was determined as described previously (irie et al., ) . plasmids encoding the p, c, and v proteins of sev strain z in the pcaggs. mcs vector have been described previously (sakaguchi et al., (sakaguchi et al., , irie et al., b irie et al., , . the polyclonal antibodies (pabs) against whole virions of sev and ndv were described previously (kiyotani et al., ) . the pabs against the sev p and c proteins were kindly provided by kato. the monoclonal antibody (mab) against the sev n protein was kindly provided by suzuki (national institute of infectious disease, japan). the mab and pab against g bp (sc- , santa cruz biotechnology; and ab , abcam, respectively), pab against rig-i ( ; immuno-biological laboratories, japan), mab against tiar, and dsrna (# , cell signaling technology; and j , scicons, hungary, respectively) were used according to the protocols of the suppliers. hela cells cultured on glass coverslips were infected with the indicated viruses or transfected with the indicated plasmids. the culture medium was replaced by serum-free dmem h postinfection (p.i.) or post-transfection (p.t.), and cells were treated with sodium arsenite (naaso ; sigma) at a final concentration of . mm for min or with ifn-α ( , iu/ml; r&d systems) for h. hela cells cultured on glass coverslips were transfected with the indicated plasmids using fugene hd transfection reagent (promega) or infected with the indicated viruses at an moi of . at h p.t. or p.i., cells were fixed and immunostained as described previously using appropriate combinations of primary and secondary antibodies (irie et al., ) . to detect rig-i, and dsrna, the tyramide signal amplification (tsa) kit with hrp-goat anti-mouse igg and alexa fluor tyramide (molecular probes) were used to increase the detection sensitivity. coverslips were mounted on glass slides with the slowfade gold antifade reagent with or without dapi (molecular probes) and observed using an lsm confocal microscope (carl zeiss). hela cells were infected with the indicated viruses at an moi of . at h p.i., total rna was prepared using the high pure rna isolation kit (roche diagnostics). viral rna in the working viral stocks was prepared using the high pure viral rna kit (roche diagnostics). quantitative (q) rt-pcr was performed as described previously (irie et al., (irie et al., , . qrt-pcr samples were analyzed using the eco real-time pcr system (illumina). for direct comparison, all of the indicated samples were analyzed in the same experiments. to detect the genome-length viral rnas and cbdi genomes of sev stocks, qrt-pcr was performed using the primer sets of sevz + sevz , as described previously (irie et al., a (irie et al., , , and cbdidetect , - , + cbdidetect , - , , which were complementary to the regions of the indicated positions of sev genome rna, as described recently by baum et al. ( ) . hela cells were lysed in ripa buffer ( . % np- , mm tris-hcl [ph . ], mm nacl) after h of infection by the indicated viruses or min of the arsenite treatment, and the insoluble fraction was removed by high-speed centrifugation. the viral proteins in the lysates were removed by three consecutive immunoprecipitation steps using anti-sev or anti-ndv pabs and protein g sepharose beads (ge healthcare life sciences). supernatants were harvested from the final immunoprecipitation samples, and were then subjected to rna preparation, as described above. hela cells cultured on glass coverslips were transfected with μg of the indicated rna samples prepared above, together with . μg of an empty puc vector, using the fugene hd transfection reagent. cantell samples ( . × ciu/ml) that were ∼ -fold serially diluted were inoculated into the allantoic cavity of -days-old embryonated chicken eggs, and incubated for h at • c. allantoic fluid was harvested from the eggs, and the titers of each fluid stock were determined as described above. rna samples were prepared from μl of each fluid stock, and the ratios of the cbdi genomes to viral genomes were determined by qrt-pcr as described above. hela cells cultured on glass coverslips were infected with the indicated viruses. at h p.i., cells were fixed with % paraformaldehyde solution in pbs, and then subjected to fish analysis using the fish tag rna green kit with alexa fluor dye (molecular probes) according to the protocol of the supplier. the rna probe was designed to be complementary to the region of , - , in sev genome rna. after fish, some samples were further subjected to fluorescent immunodetection as described above using the indicated antibodies. final samples were observed using an lsm confocal microscope. we first examined whether g bp -positive granular structures were formed during infections by a series of c knockedout and mutated sev recombinants and the parental z strain by immunofluorescence microscopy (figure ; supplementary figure s ). treating hela cells with sodium arsenite (naaso ) caused the formation of granular structures in the cytosol, which were figure | subcellular distribution of g bp and tiar in hela cells treated with or without sodium arsenite (a), and infected with a series of c-deficient rsevs, dy , dy , d y, c /c(-), and c(-), and a v-deficient rsev, v(-) (b). hela cells treated with ethanol (etoh) or sodium arsenite (naaso ) or infected with the indicated virus were immunostained with anti-g bp mab, anti-tiar mab, and anti-sev pab. (c) the g bp foci in the samples of (b) were observed under a fluorescent microscope, and the number of g bp granule-positive cells was counted and is presented as percentages against the number of sev antigen-positive cells (closed bars). the relative amounts of ifn-β mrna in cells infected with the indicated viruses were determined by qrt-pcr, and normalized to those of β-actin mrna (open bars). (d) similar experiments were performed for a series of c-mutated sev recombinants, and the results are presented as bar graphs, similarly to (c). defined as sgs based on the expression of related proteins such as g bp and tiar. g bp and tiar are well-established sg-associated proteins that are typically and diffusely present throughout the cytoplasm and dominantly present in the nucleus, respectively. however, treating cells with arsenite markedly changed the localization to form sgs containing these proteins in nearly all cells ( figure a) . when cells were infected with c(-), g bp -positive granular structures were observed in almost % of sev antigen-positive cells, and were considered to be sg-like structures since tiar was also detected in the majority of the granules (figures b,c) . in this situation, tiar was mostly in the cytoplasmic structures, whereas a larger part of tiar was still observed in the nucleus in the arsenite-treated cells. this difference is probably due to the different exposure time to the stimuli: min. for the treatment with arsenite and h for the infection. in contrast, the percentages were only % or less in cells infected with the parental z-wt as well as the dy , dy , and d y recombinants, which lacked the smaller c proteins, y , y , and both y and y , respectively ( figure c; supplementary figure s a ). an infection by c /c(-) and v(-), lacking the larger c proteins, c and c, and the v protein, respectively, resulted in a slight increase in the number of granules ( figure c ; supplementary figure s a ). of note, unlike the viruses reported previously, such as ns -deficient iav and vesicular stomatitis virus (mok et al., ; onomoto et al., ; dinh et al., ) , the fluorescence of the sev antigen was not colocalized with that of the representative sg marker g bp in the granules (figure b ; supplementary figure s ). ifn-β mrna levels in the infected cells were also compared between the viruses ( figure c) . strong correlations were observed between ifn-β mrna levels and the percentages of granular structure-forming cells against infected cells. similar strong correlations were observed for a series of c mutant viruses that possessed single-to triple-amino-acid substitutions of highly conserved, charged amino acids within the c proteins, which diminished their ability to antagonize the host ifn system to various degrees (figure d ; supplementary figure s b ; irie et al., ) . the marked difference observed in granular formation and ifn-β mrna levels among the viruses was not due to their different growing abilities. the one-step growth kinetics of all the recombinants used above was previously reported to be similar to that of the parental z-wt, except for c(-) and c /c(-), the titers of which were - logs lower than that of z-wt throughout the time course, and viral protein synthesis in the infected cells did not significantly differ among the viruses examined (kurotani et al., ; irie et al., a irie et al., , . treatment of hela cells with ifn-α did not induce g bp -positive granules, indicating that the formation of sglike structures observed by sev infection was triggered by sev infection, but not by sev-induced type i ifns (supplementary figure s c) . taken together, these results strongly suggested that a relationship may exist between the formation of g bp -positive granules and the induction of ifn-β in the c recombinants, and also that c proteins may suppress the formation of granules. we examined whether expression of the c protein alone and infection of the non-granule-forming sev could inhibit the formation of granules in cells treated with arsenite or infected with ndv (figure ; supplementary figure s ). ndv, another prototypic paramyxovirus, which was considered to be an ifn-β-inducing virus, could induce g bp -positive granules in nearly all cells infected with virulent as well as avirulent strains (miyadera and d strains, respectively; figure b ). the c protein alone failed to inhibit the formation of granules in cells treated with arsenite (figure a) as well as infected with ndv ( figure b) , although the number of granules was slightly reduced in the c-expressing cells treated with arsenite compared to that observed in the neighboring non-c-expressing cells (figure a) . in addition to c, the other p gene products, p and v proteins, also failed to inhibit the formation of both types of granule (supplementary figure s ) . infection by sev-z-wt and c-deficient recombinant c(-) also failed to inhibit the arsenite-induced formation of granules (figures c,d) . small granules dispersed in the cytoplasm were induced by arsenite in both cases of infection by wt and c(-). of note, the g bp -positive granules induced by arsenite and viruses, such as c(-) and ndv, differed in size; the granules induced by the infection were apparently larger than those induced by arsenite (figures and ) , implying a possible difference in cellular pathways leading to these two types of granular structure. these results demonstrated that sev did not have the ability to inhibit the formation of both types of granules. a marked difference was noted in the abilities of the sev strains to induce ifn-β. although most of the sev strains including z have been characterized by their strong ability to counteract the innate immune system, the cnt strain has been widely used as a virus that induces high levels of ifn-β (baum et al., ; tapia et al., ) . therefore, we compared the abilities of some sev strains to induce ifn-β and sg-like structures (figure ; supplementary figure s ). in all cases, g bp -positive granular structures were only detected in less than % of sev antigen-positive cells (figure ; supplementary figure s a ). ifn-β mrna was not highly induced in cells infected with the sev strains, except for cnt, whereas cnt induced ifn-β mrna at a level that was -fold higher than that by z (figure ) . regarding the sev strains, unlike that observed in the c recombinants, a correlation was not observed between ifn-β mrna levels and the percentage of granular structure-forming cells against infected cells (figure ) . the different levels of ifn-β mrna induced by the strains could not be attributed to differences in viral growth (supplementary figure s b) . these results suggested that the formation of g bp -positive granules was not necessarily required to sense the sev cnt infection, followed by the production of ifn-β, unlike the c recombinants. we attempted to identify the reason for the difference in granular formation between these two types of ifn-β-inducing virus, c(-) and cnt. to address this issue, we first examined the ability of total rna prepared from virus-infected as well as arsenite-treated cells to induce g bp -positive granules ( figure a; supplementary figure s a ). total rna samples prepared from cells infected with any of the ifn-β-inducing viruses, sev-cnt, c(-), and ndv, were able to induce the granules as well as ifn-β in hela cells despite no formation of the granules by the cnt infection, whereas those from cells infected with the ifn-β-non-inducing sev-z-wt or treated with arsenite were not (figure a) . we then examined the content rates of cbdi genomes, a potent ligand for rig-i, against viral genome-length rnas in the rna samples used above ( figure b ). the cnt sample contained cbdi genomes at a level that was ∼ , -fold higher than that in the z-wt sample, although the sample of c(-) contained similar levels to the z-wt sample ( figure b) . we further examined the effect of removal of encapsidated viral rna species, such as viral full-length and di genomes, by three consecutive rounds of immunoprecipitation using antisera against the whole virions of sev and ndv prior to the preparation of rna samples (supplementary figure s b) . total rna prepared from the postimmunoprecipitation samples of c(-) and ndv was still able to induce the granules as well as ifn-β, but that of cnt lost these abilities (figure c ). since the naked cbdi genomes have been reported readily to form an ideal structure as the rig-i ligands of -triphosphated, blunt-ended dsrna (kolakofsky, ) , these results indicated that the major ifn-β-inducing viral rna species produced in the cells infected with cnt was encapsidated cbdi genomes, whereas those for sev- c(-) and ndv were not. the results above suggested that there may be at least two types of ifn-β-inducing viral rna species with a marked difference in the formation of sg-like granules. to elucidate this difference, we examined the subcellular distribution of unusual viral rna species produced by c(-) and cnt (figures and ) . we and other groups previously reported a strong correlation between the production of dsrna detected by the anti-dsrna antibody j (j -dsrna) and the induction of ifn-β in infections of the c-mutated sev recombinants and ndv (takeuchi et al., ; irie et al., ) . therefore, we first examined the production and subcellular distribution of j -dsrna in the cells infected with the ifn-β-inducing viruses by immunofluorescence microscopy (figure ) . dsrna fluorescent signals were absent in cells infected with ifn-non-inducing z-wt as well as in those with the ifn-β-inducing strain cnt ( figure a ). in contrast, dsrna fluorescent signals were clearly observed in the cytoplasm of cells infected with ifn-β-inducing sev- c(-) and ndv with dispersed and granular distributions, respectively ( figure b) . a previous study reported that ns -deficient iav infections caused rig-i to form granular aggregates that contained sg markers as well as viral rna (onomoto et al., ) . therefore, the cells infected with c(-) and ndv were co-stained with anti-rig-i and anti-g bp antibodies ( figure c ). similar to iav, rig-i almost perfectly colocalized with the virus-induced g bp -positive granules in both cases of infection ( figure c) ; however, unlike iav, these granules did not colocalize with the viral antigen or viral j -dsrna ( figure b, arrowheads) , as shown in figures and . together with the results of figure , j -dsrna appeared to be not or less encapsidated by viral nucleoproteins because the access of the antibody to and the formation of dsrna by tightly encapsidated viral rna, such as viral genomes, was unlikely. we further attempted to visualize the subcellular distribution of the unusual viral rna species that were not fully encapsidated, unlike the genome-length viral rnas, by fish analysis (figure ) . infected cell samples were prepared without a protease treatment to exclude fully encapsidated viral rna species, and were then stained with an rna probe complementary to the , - , region of the (-)-sense viral genome rna, designed by referring to two wellcharacterized cbdi genomes of sev (calain et al., ; -gil et al., ) . fluorescence-positive cytoplasmic inclusions were observed in the c(-)-as well as in the cntinfected samples, while an apparent signal was not detected in z-wt-infected or uninfected samples ( figure a ). when the z-wt-infected sample was treated with proteinase k, apparent signals were observed throughout the cytoplasm ( figure a) . these results strongly suggested that fully encapsidated viral genomes were not detected, whereas nonor partially encapsidated viral rna species were detectable in this system. the fish-positive inclusions observed in cntinfected cells were no longer detected in the cells infected with the cnt-lowdi (figure a) , which contained ∼ -fold fewer cbdi genomes than the original cnt sample ( figure b) . together with the results of figure , the fish signals observed in the cnt-infected cells were considered as the cbdi genomes. the fish samples of c(-) and cnt-infected cells were further immunostained to examine the subcellular colocalization of fish signals, the sev n protein, rig-i, and g bp (figures b-d, respectively) . the sev n protein was detected in the fish-positive inclusions of cnt-infected cells, suggesting that the rna species detected in the cnt samples were only partially, not fully, encapsidated, unlike the fully encapsidated, full-length, intact viral genomes ( figure b ). in contrast, the n protein was not detected in the inclusions of c(-)-infected cells, which suggested that the rna species detected in the c(-) samples were not encapsidated ( figure b) . the fish-positive inclusions of cnt-infected cells were not apparently colocalized with rig-i or g bp , whereas most of those in the c(-)-infected cells colocalized obviously with rig-i and g bp , unlike the j -dsrna (figures c,d) . these results indicated that unusual viral rna species harboring the -region of (-)-sense sev genome rna, which was not produced during infection by ifn-β-non-inducing z-wt, was produced in infections by , and were selectively formed into distinct cytoplasmic inclusions in an rna-type-dependent manner. the inclusions in the c(-)-infected cells could be identified as avsgs, and may be the site to detect viral rna by rig-i, whereas those in cnt-infected cells were not avsgs, but as-yet-unidentified structures. although a number of studies have examined host innate immune responses against pathogenic microbes including rna viruses, the virus-derived rna species that serve as pamps in real viral infections and the sites at which pamps are recognized by rlrs have yet to be clarified in detail. two important insights were reported recently. regarding the real pamps in infections by rna viruses, the cb and id types of di genomes were identified as the ligands of rig-i in sev-and iav-infected cells, respectively, both of which could form ideal structures as the rig-i ligands (baum et al., ; baum and garcia-sastre, ; martinez-gil et al., ) . the sg-like structures have been suggested to serve as the sites at which the rlrs encounter viral rna and subsequently activate the ifn signaling pathways in infections by rna viruses (onomoto et al., ; yoo et al., ) . in order to establish what and where viral rna species were detected by rlrs, in the present study, we compared two types of ifnβ-inducing sev, a recombinant c(-) and a strain cnt, with a non-ifn-β-inducing z strain, in terms of the formation of sglike granules and the production of unusual viral rna species. a major advantage of our study is that the comparison can be performed within the context of the same viral species. several types of unusual viral rna species were found to be generated in cells infected with the ifn-inducing sevs but not those with the ifn-non-inducing sevs (summarized in table ). one was a dsrna (j -dsrna) that was detected by the anti-dsrna antibody, j (type i in table ) . as for sev, the generation of j -dsrna may have been restricted by the c proteins because it was only detected in cells infected with c-deficient or mutated recombinants, but not in those with intact sevs (figure ; takeuchi et al., ; irie et al., ) . we and other groups demonstrated that j -dsrna activated pkr, and this was followed by the phosphorylation of eif and the production of ifn-β, both of which resulted in antiviral effects in the host cells. the activation of pkr has been reported to induce the formation of sg-like structures during infections by some rna viruses, such as iav and measles virus (mev; mok et al., ; onomoto et al., ; okonski and samuel, ) , and this also appears to be the case for the sev c recombinants. unlike these viruses, the sg-like structures formed during c(-) infections did not include the j -dsrna, which was dispersed in the cytoplasm, although they contained rig-i (figure ) . this may lead to the assertion that the sg-like structures induced during infections by c recombinants are not the sites at which to detect sev infections. however, the sg-like structures formed by c(-) were revealed to contain another type of unusual viral rna species by fish analysis, in which an rna probe targeting the nt region of the -end of the (-)-sense sev genome was used (type ii in table ; figure ). this now strongly suggests that the sg-like structures found in the c(-) infection are defined as avsgs. unlike the iav infection, the type i and ii rna species seemed to be not or less encapsidated, given that they were not colocalized with viral antigens and were not removed from the infected cell lysates by immunoprecipitation using anti-sev antibody (figures and - ) . sev trailer rna, which is transcribed from the -ends of (+)-sense antigenome rna, was previously reported to interact with tiar to inhibit apoptosis and the formation of sgs induced by infection (iseni et al., ) . the c(-) virus was shown to induce apoptosis more quickly and severely in infected cells than the wt virus (irie et al., ) . although appearing to contain the nonencapsidated -end of a (-)-sense genome rna, at least in the part that is concordant with the trailer rna, it is unlikely that the type ii rna observed in the c(-)-infected cells has the ability of the trailer rna to inhibit apoptosis and sg formation. although details of the type i and ii rnas remain to be solved, given the cytoplasmic replication of sev rna without forming inclusion bodies and the differences of the rna species in subcellular distribution and reactivity with the j and fish probe, the type i dsrna somewhat unwound actively or incidentally into the type ii rna might be accumulated into the avsgs. although the sg-like structures and j -dsrna were not detected during cnt infections, fish-positive non-granularshaped inclusions were observed (type iii in table ; figure ). these cnt inclusions were markedly different from those of c(-). the inclusions did not include rig-i or g bp , but contained the n protein (figure ) , suggesting that the inclusions were not avsgs, and that the type iii rna was at least partially encapsidated. the cnt stock used in the present study contained a larger amount of cbdi genomes than the other viral stocks ( figure b) . the sev cbdi genomes have been shown to be partially and/or more loosely encapsidated than intact genomes (kolakofsky, ; strahle et al., ) . the sev cbdi genomes were recently identified as strong ligands for rig-i (baum et al., ; tapia et al., ) . indeed, the cnt-lowdi had lost the ability to induce ifn-β and the inclusions had not been found in the infected cells (figures b and ) . taken together, the type iii rna was identified as the cbdi genome. these results indicated that the process of detecting infections and the subsequent induction of ifn-β differed largely between c(-) and cnt: avsg-dependent and -independent mechanisms for c(-) and cnt, respectively. most viruses have been shown to possess the ability to antagonize host ifn pathways in order to avoid activating host antiviral actions, and this strategy is mostly based on a counteraction against the molecules involved in these pathways (versteeg and garcia-sastre, ) . however, a recent study reported that encephalomyocarditis virus (emcv) has the ability to disrupt sgs by cleaving g bp in order to avoid the innate immune detection of its infection and subsequent induction of ifn-β (ng et al., ) , suggesting that another effective strategy for viral evasion from the ifn system by preventing avsg formation exists. generation of the unusual viral rna species triggering the production of ifn-β seems to be suppressed during intact rna viral replication. similarly to sev, another paramyxovirus mev c protein was recently shown to impair the production of j -dsrna and the activation of pkr coupled with the formation of sg-like structures (okonski and samuel, ; pfaller et al., ) . unlike the case of sev, in which the knockout of c resulted in the production of j -dsrna (figures and ), but not cbdi genomes, the j -dsrna produced during the infection by a c-deficient mev recombinant was reported to be a cbdi genome (pfaller et al., ) . although the cbdi genomes were more dominantly produced by sev-cnt than by the other strains and c-recombinants tested ( figure b and data not shown), this unique property of cnt might be attributed to its c protein that possibly have a functional difference with those of the other sevs. the c proteins of both sev and mev have been shown to play critical roles in modulating viral rna synthesis and maintaining its integrity by potentially stabilizing the ribonucleoprotein (rnp)-polymerase complex (tapparel et al., ; reutter et al., ; bankamp et al., ; irie et al., a irie et al., , ito et al., ) . dysfunctions in the c proteins may result in a disturbance in integrity, leading to the production of the unusual, ifn-β-inducing rna species. the results of the present study indicate that several types of ifn-β-inducible, unusual viral rna species may be produced during sev infections and included in avsg-like and non-avsg-like cytoplasmic inclusions, which suggests that rna-typedependent mechanisms recognize and accumulate such unusual viral rnas in specific compartments. in addition, the production of these unusual rna species may be restricted during intact viral replication in order to avoid detection by host innate immunity. pathogen recognition and innate immunity visibly stressed: the role of eif , tia- , and stress granules in protein translation rna granules hepatitis c virus hijacks p-body and stress granule components around lipid droplets polysomes, p bodies and stress granules: states and fates of eukaryotic mrnas identification of naturally occurring amino acid variations that affect the ability of the measles virus c protein to regulate genome replication and transcription differential recognition of viral rna by rig-i preference of rig-i for short viral rna molecules in infected cells revealed by nextgeneration sequencing rig-i goes beyond naked recognition eukaryotic stress granules: the ins and outs of translation molecular cloning of natural paramyxovirus copy-back defective interfering rnas and their expression from dna amino acids and of mammalian orthoreovirus protein microns are necessary for stress granule localization, core protein lambda interaction, and de novo virus replication the ' untranslated regions of influenza genomic sequences are 'ppp-independent ligands for rig-i induction of stress granule-like structures in vesicular stomatitis virus-infected cells poliovirus infection induces the colocalization of cellular protein srp with tia- , a cytoplasmic stress granule protein involvement of the leader sequence in sendai virus pathogenesis revealed by recovery of a pathogenic field isolate from cdna hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus rig-i and dsrna-induced ifnbeta activation translational control in stress and apoptosis '-triphosphate rna is the ligand for rig-i inhibition of interferon regulatory factor activation by paramyxovirus v 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sendai virus-derived rna agonist of rig-i as a virus vaccine adjuvant importance of eif alpha phosphorylation and stress granule assembly in alphavirus translation regulation the ns protein of influenza a virus interacts with cellular processing bodies and stress granules through rna-associated protein (rap ) during virus infection encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses stress granule formation induced by measles virus is protein kinase pkr dependent and impaired by rna adenosine deaminase adar critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity modulation of hepatitis c virus rna abundance and virus release by dispersion of processing bodies and enrichment of stress granules sequestration of g bp coupled with efficient translation inhibits stress granules in semliki forest virus infection measles virus c protein impairs production of defective copyback double-stranded viral rna and activation of protein kinase r rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eif alpha phosphorylation and pkr mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies mutations in the measles virus c protein that up regulate viral rna synthesis dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection in vivo ligands of mda and rig-i in measles virus-infected cells regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp analysis of interaction of sendai virus v protein and melanoma differentiation-associated gene aip /alix is a binding partner of sendai virus c protein and facilitates virus budding rig-i detects mrna of intracellular salmonella enterica serovar typhimurium during bacterial infection sendai virus defective-interfering genomes and the activation of interferon-beta activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity inhibition of sendai virus genome replication due to promoterincreased selectivity: a possible role for the accessory c proteins structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of newcastle disease virus viral tricks to grid-lock the type i interferon system incoming rna virus nucleocapsids containing a '-triphosphorylated genome activate rig-i and antiviral signaling inhibition of cytoplasmic mrna stress granule formation by a viral proteinase poliovirus unlinks tia aggregation and mrna stress granule formation the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses dhx enhances rig-i signaling by facilitating pkrmediated antiviral stress granule formation we thank the staff of the analysis center of life science, hiroshima university, for the use of their facilities. we also thank dr. k. takeuchi (university of tsukuba) for fruitful discussions. this work was supported by jsps kakenhi (grant numbers and ). the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. . key: cord- -wgqierp authors: brook, cara e; boots, mike; chandran, kartik; dobson, andrew p; drosten, christian; graham, andrea l; grenfell, bryan t; müller, marcel a; ng, melinda; wang, lin-fa; van leeuwen, anieke title: accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: wgqierp bats host virulent zoonotic viruses without experiencing disease. a mechanistic understanding of the impact of bats’ virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. we carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. in general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats. bats have received much attention in recent years for their role as reservoir hosts for emerging viral zoonoses, including rabies and related lyssaviruses, hendra and nipah henipaviruses, ebola and marburg filoviruses, and sars coronavirus (calisher et al., ; wang and anderson, ) . in most non-chiropteran mammals, henipaviruses, filoviruses, and coronaviruses induce substantial morbidity and mortality, display short durations of infection, and elicit robust, long-term immunity in hosts surviving infection (nicholls et al., ; hooper et al., ; mahanty and bray, ) . bats, by contrast, demonstrate no obvious disease symptoms upon infection with pathogens that are highly virulent in non-volant mammals (schountz et al., ) but may, instead, support viruses as longterm persistent infections, rather than transient, immunizing pathologies (plowright et al., ) . recent research advances are beginning to shed light on the molecular mechanisms by which bats avoid pathology from these otherwise virulent pathogens (brook and dobson, ) . bats leverage a suite of species-specific mechanisms to limit viral load, which include host receptor sequence incompatibilities for some bat-virus combinations (ng et al., ; takadate et al., ) and constitutive expression of the antiviral cytokine, ifn-a, for others (zhou et al., ) . typically, the presence of viral rna or dna in the cytoplasm of mammalian cells will induce secretion of type i interferon proteins (ifn-a and ifn-b), which promote expression and translation of interferon-stimulated genes (isgs) in neighboring cells and render them effectively antiviral (stetson and medzhitov, ) . in some bat cells, the transcriptomic blueprints for this ifn response are expressed constitutively, even in the absence of stimulation by viral rna or dna (zhou et al., ) . in non-flying mammals, constitutive ifn expression would likely elicit widespread inflammation and concomitant immunopathology upon viral infection, but bats support unique adaptations to combat inflammation (zhang et al., ; ahn et al., ; xie et al., ; pavlovich et al., ) that may have evolved to mitigate metabolic damage induced during flight (kacprzyk et al., ) . the extent to which constitutive ifn-a expression signifies constitutive antiviral defense in the form of functional ifn-a protein remains unresolved. in bat cells constitutively expressing ifn-a, some protein-stimulated, downstream isgs appear to be also constitutively expressed, but additional isg induction is nonetheless possible following viral challenge and stimulation of ifn-b (zhou et al., ; xie et al., ) . despite recent advances in molecular understanding of bat viral tolerance, the consequences of this unique bat immunity on within-host virus dynamics-and its implications for understanding zoonotic emergence-have yet to be elucidated. the field of 'virus dynamics' was first developed to describe the mechanistic underpinnings of long-term patterns of steady-state viral load exhibited by patients in chronic phase infections with hiv, who appeared to produce and clear virus at equivalent rates (nowak and may, ; ho et al., ) . models of simple target cell depletion, in which viral load is dictated by a bottom-elife digest bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. in fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals -including rabies, ebola and the sars coronavirus. bats have a suite of antiviral defenses that keep the amount of virus in check. for example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. in most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. however, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. now, brook et al. have studied this exact question using bat cells grown in the laboratory. the experiments made use of cells from one bat species -the black flying fox -in which the interferon pathway is always on, and another -the egyptian fruit bat -in which this pathway is only activated during an infection. the bat cells were infected with three different viruses, and then brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. the experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. in both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. this suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. the findings may help to explain why bats are often the source for viruses that are deadly in humans. learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. more studies are needed in bats to help these efforts. in the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats. up resource supply of infection-susceptible host cells, were first developed for hiv (perelson, ) but have since been applied to other chronic infections, including hepatitis-c virus (neumann et al., ) , hepatitis-b virus (nowak et al., ) and cytomegalovirus (emery et al., ) . recent work has adopted similar techniques to model the within-host dynamics of acute infections, such as influenza a and measles, inspiring debate over the extent to which explicit modeling of top-down immune control can improve inference beyond the basic resource limitation assumptions of the target cell model (baccam et al., ; pawelek et al., ; saenz et al., ; morris et al., ) . to investigate the impact of unique bat immune processes on in vitro viral kinetics, we first undertook a series of virus infection experiments on bat cell lines expressing divergent interferon phenotypes, then developed a theoretical model elucidating the dynamics of within-host viral spread. we evaluated our theoretical model analytically independent of the data, then fit the model to data recovered from in vitro experimental trials in order to estimate rates of within-host virus transmission and cellular progression to antiviral status under diverse assumptions of absent, induced, and constitutive immunity. finally, we confirmed our findings in spatially-explicit stochastic simulations of fitted time series from our mean field model. we hypothesized that top-down immune processes would overrule classical resource-limitation in bat cell lines described as constitutively antiviral in the literature, offering a testable prediction for models fit to empirical data. we further predicted that the most robust antiviral responses would be associated with the most rapid within-host virus propagation rates but also protect cells against virus-induced mortality to support the longest enduring infections in tissue culture. we first explored the influence of innate immune phenotype on within-host viral propagation in a series of infection experiments in cell culture. we conducted plaque assays on six-well plate monolayers of three immortalized mammalian kidney cell lines: [ ] vero (african green monkey) cells, which are ifn-defective and thus limited in antiviral capacity (desmyter et al., ) ; [ ] roni/ . (rousettus aegyptiacus) cells which demonstrate idiosyncratic induced interferon responses upon viral challenge (kuzmin et al., ; arnold et al., ; biesold et al., ; pavlovich et al., ) ; and [ ] pakit (pteropus alecto) cells which constitutively express ifn-a (zhou et al., ; crameri et al., ) . to intensify cell line-specific differences in constitutive immunity, we carried out infectivity assays with gfp-tagged, replication-competent vesicular stomatitis indiana viruses: rvsv-g, rvsv-ebov, and rvsv-marv, which have been previously described (miller et al., ; wong et al., ) . two of these viruses, rvsv-ebov and rvsv-marv, are recombinants for which cell entry is mediated by the glycoprotein of the bat-evolved filoviruses, ebola (ebov) and marburg (marv), thus allowing us to modulate the extent of structural, as well as immunological, antiviral defense at play in each infection. previous work in this lab has demonstrated incompatibilities in the npc filovirus receptor which render pakit cells refractory to infection with rvsv-marv (ng and chandrab, , unpublished results) , making them structurally antiviral, over and above their constitutive expression of ifn-a. all three cell lines were challenged with all three viruses at two multiplicities of infection (moi): . and . . between and trials were run at each cell-virus-moi combination, excepting rvsv-marv infections on pakit cells at moi = . , for which only eight trials were run (see materials and methods; figure -figure supplements - , supplementary file ). because plaque assays restrict viral transmission neighbor-to-neighbor in two-dimensional cellular space (howat et al., ) , we were able to track the spread of gfp-expressing virus-infected cells across tissue monolayers via inverted fluorescence microscopy. for each infection trial, we monitored and re-imaged plates for up to hr of observations or until total monolayer destruction, processed resulting images, and generated a time series of the proportion of infectious-cell occupied plate space across the duration of each trial (see materials and methods). we used generalized additive models to infer the time course of all cell culture replicates and construct the multi-trial dataset to which we eventually fit our mechanistic transmission model for each cell line-virus-specific combination ( figure ; figure -figure supplements - ). all three recombinant vesicular stomatitis viruses (rvsv-g, rvsv-ebov, and rvsv-marv) infected vero, roni/ . , and pakit tissue cultures at both focal mois. post-invasion, virus spread rapidly across most cell monolayers, resulting in virus-induced epidemic extinction. epidemics were less severe in bat cell cultures, especially when infected with the recombinant filoviruses, rvsv-ebov and rvsv-marv. monolayer destruction was avoided in the case of rvsv-ebov and rvsv-marv infections on pakit cells: in the former, persistent viral infection was maintained throughout the hr duration of each experiment, while, in the latter, infection was eliminated early in the time series, preserving a large proportion of live, uninfectious cells across the duration of the experiment. we assumed this pattern to be the result of immune-mediated epidemic extinction (figure ) . patterns from moi = . were largely recapitulated at moi = . , though at somewhat reduced total proportions (figure -figure supplement ). a theoretical model fit to in vitro data recapitulates expected immune phenotypes for bat cells we next developed a within-host model to fit to these data to elucidate the effects of induced and constitutive immunity on the dynamics of viral spread in host tissue ( figure ). the compartmental within-host system mimicked our two-dimensional cell culture monolayer, with cells occupying five distinct infection states: susceptible (s), antiviral (a), exposed (e), infectious (i), and dead (d). we modeled exposed cells as infected but not yet infectious, capturing the 'eclipse phase' of viral integration into a host cell which precedes viral replication. antiviral cells were immune to viral infection, in accordance with the 'antiviral state' induced from interferon stimulation of isgs in tissues adjacent to infection (stetson and medzhitov, ) . because we aimed to translate available data into modeled processes, we did not explicitly model interferon dynamics but instead scaled the rate of cell progression from susceptible to antiviral (r) by the proportion of exposed cells (globally) in the system. in systems permitting constitutive immunity, a second rate of cellular acquisition of antiviral status (") additionally scaled with the global proportion of susceptible cells in the model. compared with virus, ifn particles are small and highly diffusive, justifying this global signaling assumption at the limited spatial extent of a six-well plate and maintaining consistency with previous modeling approximations of ifn signaling in plaque assay (howat et al., ) . to best represent our empirical monolayer system, we expressed our state variables as proportions (p s , p a , p e , p i , and p d ), under assumptions of frequency-dependent transmission in a wellmixed population (keeling and rohani, ) , though note that the inclusion of p d (representing the proportion of dead space in the modeled tissue) had the functional effect of varying transmission with infectious cell density. this resulted in the following system of ordinary differential equations: we defined 'induced immunity' as complete, modeling all cells as susceptible to viral invasion at disease-free equilibrium, with defenses induced subsequent to viral exposure through the term r. by contrast, we allowed the extent of constitutive immunity to vary across the parameter range of " > , defining a 'constitutive' system as one containing any antiviral cells at disease-free equilibrium. in fitting this model to tissue culture data, we independently estimated both r and "; as well as the cell-to-cell transmission rate, b, for each cell-virus combination. since the extent to which constitutively-expressed ifn-a is constitutively translated into functional protein is not yet known for bat hosts (zhou et al., ) , this approach permitted our tissue culture data to drive modeling inference: even in pakit cell lines known to constitutively express ifn-a, the true constitutive extent of the system (i.e. the quantity of antiviral cells present at disease-free equilibrium) was allowed to vary through estimation of ": for the purposes of model-fitting, we fixed the value of c, the return rate of antiviral cells to susceptible status, at . the small spatial scale and short time course (max hours) of our experiments likely prohibited any return of antiviral cells to susceptible status in our empirical system; nonetheless, we retained the term c in analytical evaluations of our model because regression from antiviral to susceptible status is possible over long time periods in vitro and at the scale of a complete organism (radke et al., ; rasmussen and farley, ; samuel and knutson, ) . before fitting to empirical time series, we undertook bifurcation analysis of our theoretical model and generated testable hypotheses on the basis of model outcomes. from our within-host model system (equation - ), we derived the following expression for r , the pathogen basic reproduction number (supplementary file ): pathogens can invade a host tissue culture when r > . rapid rates of constitutive antiviral acquisition (") will drive r < : tissue cultures with highly constitutive antiviral immunity will be therefore resistant to virus invasion from the outset. since, by definition, induced immunity is stimulated following initial virus invasion, the rate of induced antiviral acquisition (r) is not incorporated into the equation for r ; while induced immune processes can control virus after initial invasion, they cannot prevent it from occurring to begin with. in cases of fully induced or absent immunity (" ¼ ), the r equation thus reduces to a form typical of the classic seir model: at equilibrium, the theoretical, mean field model demonstrates one of three infection states: endemic equilibrium, stable limit cycles, or no infection ( figure ) . respectively, these states approximate the persistent infection, virus-induced epidemic extinction, and immune-mediated epidemic extinction phenotypes previously witnessed in tissue culture experiments ( figure ). theoretically, endemic equilibrium is maintained when new infections are generated at the same rate at which infections are lost, while limit cycles represent parameter space under which infectious and susceptible populations are locked in predictable oscillations. endemic equilibria resulting from cellular regeneration (i.e. births) have been described in vivo for hiv (coffin, ) and in vitro for herpesvirus plaque assays (howat et al., ) , but, because they so closely approach zero, true limit cycles likely only occur theoretically, instead yielding stochastic extinctions in empirical time series. bifurcation analysis of our mean field model revealed that regions of no infection (pathogen extinction) were bounded at lower threshold (branch point) values for b, below which the pathogen was unable to invade. we found no upper threshold to invasion for b under any circumstances (i.e. b high enough to drive pathogen-induced extinction), but high b values resulted in hopf bifurcations, which delineate regions of parameter space characterized by limit cycles. since limit cycles so closely approach zero, high bs recovered in this range would likely produce virus-induced epidemic extinctions under experimental conditions. under more robust representations of immunity, with higher values for either or both induced (r) and constitutive (") rates of antiviral acquisition, hopf bifurcations occurred at increasingly higher values for b, meaning that persistent infections could establish at higher viral transmission rates ( figure ). consistent with our derivation for r , we found that the branch point threshold for viral invasion was independent of changes to the induced immune parameter (r) but saturated at high values of " that characterize highly constitutive immunity ( figure ) . we next fit our theoretical model by least squares to each cell line-virus combination, under absent, induced, and constitutive assumptions of immunity. in general, best fit models recapitulated expected outcomes based on the immune phenotype of the cell line in question, as described in the general literature (table ironically, the induced immune model offered a slightly better fit than the constitutive to rvsv-marv infections on the pakit cell line (the one cell line-virus combination for which we know a constitutively antiviral cell-receptor incompatibility to be at play). because constitutive immune assumptions can prohibit pathogen invasion (r < ), model fits to this time series under constitutive assumptions were handicapped by overestimations of ", which prohibited pathogen invasion. only by incorporating an exceedingly rapid rate of induced antiviral acquisition could the model guarantee that initial infection would be permitted and then rapidly controlled. in all panel (a) plots, the rate of induced immune antiviral acquisition (r) was fixed at . . panel (b) depicts dynamics under variably induced immunity, ranging from absent (left: r= ) to high (right: r= ). in all panel (b) plots, the rate of constitutive antiviral acquisition (") was fixed at . branch point curves are represented as solid lines and hopf curves as dashed lines. white space indicates endemic equilibrium (persistence), gray space indicates limit cycles, and black space indicates no infection (extinction). other parameter values for equilibrium analysis were fixed at: b = . , m = . , s = / , c = . special points from bifurcations analyses are listed in supplementary file . in fitting our theoretical model to in vitro data, we estimated the within-host virus transmission rate (b) and the rate(s) of cellular acquisition to antiviral status (r or r + ") ( table ; supplementary file ). under absent immune assumptions, r and " were fixed at while b was estimated; under induced immune assumptions, " was fixed at while r and b were estimated; and under constitutive immune assumptions, all three parameters (r, ", and b) were simultaneously estimated for each cell-virus combination. best fit parameter estimates for moi= . data are visualized in conjunction with br and b -" bifurcations in (r) and (b) the constitutive immunity rate of antiviral acquisition ("). panels show variation in the extent of immunity, from absent (left) to high (right). branch point curves are represented as solid lines and hopf curves as dashed lines. white space indicates endemic equilibrium (persistence), gray space indicates limit cycling, and black space indicates no infection (extinction). other parameter values for equilibrium analysis were fixed at: b = . , m = . , s = / , a = / , c = . special points from bifurcations analyses are listed in supplementary file . space corresponding to theoretical limit cycles, consistent with observed virus-induced epidemic extinctions in stochastic tissue cultures. in contrast to vero cells, the induced immunity model offered the best fit to all roni/ . data, consistent with reported patterns in the literature and our own validation by qpcr ( table ; arnold et al., ; kuzmin et al., ; biesold et al., ; pavlovich et al., ) . as in vero cell trials, we estimated highest b values for rvsv-g infections on roni/ . cell lines but here recovered higher b estimates for rvsv-marv than for rvsv-ebov. this reversal was balanced by a higher estimated rate of acquisition to antiviral status (r) for rvsv-ebov versus rvsv-marv. in general, we observed that more rapid rates of antiviral acquisition (either induced, r, constitutive, ", or both) correlated with higher transmission rates (b). when offset by r, b values estimated for roni/ . infections maintained the same amplitude as those estimated for immune-absent vero cell lines but caused gentler epidemics and reduced cellular mortality (figure ) . roni/ . parameter estimates localized in the region corresponding to endemic equilibrium for the deterministic, theoretical model (figure ) , yielding less acute epidemics which nonetheless went extinct in stochastic experiments. finally, rvsv-g and rvsv-ebov trials on pakit cells were best fit by models assuming constitutive immunity, while rvsv-marv infections on pakit were matched equivalently by models assuming either induced or constitutive immunity-with induced models favored over constitutive in aic comparisons because one fewer parameter was estimated (figure -figure supplements - ; supplementary file ). for all virus infections, pakit cell lines yielded b estimates a full order of magnitude higher than vero or roni/ . cells, with each b balanced by an immune response (either r, or r combined with ") also an order of magnitude higher than that recovered for the other cell lines ( figure ; table ). as in roni/ . cells, pakit parameter fits localized in the region corresponding to endemic equilibrium for the deterministic theoretical model. because constitutive immune processes can actually prohibit initial pathogen invasion, constitutive immune fits to rvsv-marv infections on pakit cell lines consistently localized at or below the branch point threshold for virus invasion (r ¼ ). during model fitting for optimization of ", any parameter tests of " values producing r < resulted in no infection and, consequently, produced an exceedingly poor fit to infectious time series data. in all model fits assuming constitutive immunity, across all cell lines, antiviral contributions from " prohibited virus from invading at all. the induced immune model thus produced a more parsimonious recapitulation of these data because virus invasion was always permitted, then rapidly controlled. in order to compare the relative contributions of each cell line's disparate immune processes to epidemic dynamics, we next used our mean field parameter estimates to calculate the initial 'antiviral rate'-the initial accumulation rate of antiviral cells upon virus invasion for each cell-virus-moi combination-based on the following equation: where p e was calculated from the initial infectious dose (moi) of each infection experiment and p s was estimated at disease-free equilibrium: because and " both contribute to this initial antiviral rate, induced and constitutive immune assumptions are capable of yielding equally rapid rates, depending on parameter fits. indeed, under fully induced immune assumptions, the induced antiviral acquisition rate (r) estimated for rvsv-marv infection on pakit cells was so high that the initial antiviral rate exceeded even that estimated under constitutive assumptions for this cell-virus combination (supplementary file ) . in reality, we know that npc receptor incompatibilities make pakit cell lines constitutively refractory to rvsv-marv infection (ng and chandrab, , unpublished results) and that pakit cells also constitutively express the antiviral cytokine, ifn-a. model fitting results suggest that this constitutive expression of ifn-a may act more as a rapidly inducible immune response following virus invasion than as a constitutive secretion of functional ifn-a protein. nonetheless, as hypothesized, pakit cell lines were by far the most antiviral of any in our study-with initial antiviral rates estimated several orders of magnitude higher than any others in our study, under either induced or constitutive assumptions ( table ; supplementary file ). roni/ . cells displayed the second-most-pronounced signature of immunity, followed by vero cells, for which the initial antiviral rate was essentially zero even under forced assumptions of induced or constitutive immunity ( table ; supplementary file ). using fitted parameters for b and ", we additionally calculated r , the basic reproduction number for the virus, for each cell line-virus-moi combination ( table ; supplementary file ). we found that r was essentially unchanged across differing immune assumptions for roni/ . and vero cells, for which the initial antiviral rate was low. in the case of pakit cells, a high initial antiviral rate under either induced or constitutive immunity resulted in a correspondingly high estimation of b (and, consequently, r ) which still produced the same epidemic curve that resulted from the much lower estimates for b and r paired with absent immunity. these findings suggest that antiviral immune responses protect host tissues against virus-induced cell mortality and may facilitate the establishment of more rapid within-host transmission rates. total monolayer destruction occurred in all cell-virus combinations excepting rvsv-ebov infections on roni/ . cells and rvsv-ebov and rvsv-marv infections on pakit cells. monolayer destruction corresponded to susceptible cell depletion and epidemic turnover where r-effective (the product of r and the proportion susceptible) was reduced below one ( figure ) . for rvsv-ebov infections on roni/ . , induced antiviral cells safeguarded remnant live cells, which birthed new susceptible cells late in the time series. in rvsv-ebov and rvsv-marv infections on pakit cells, this antiviral protection halted the epidemic ( figure ; r-effective < ) before susceptibles fully declined. in the case of rvsv-ebov on pakit , the birth of new susceptibles from remnant live cells protected by antiviral status maintained late-stage transmission to facilitate long-term epidemic persistence. importantly, under fixed parameter values for the infection incubation rate (s) and infectioninduced mortality rate (a), models were unable to reproduce the longer-term infectious time series captured in data from rvsv-ebov infections on pakit cell lines without incorporation of cell births, an assumption adopted in previous modeling representations of ifn-mediated viral dynamics in tissue culture (howat et al., ) . in our experiments, we observed that cellular reproduction took place as plaque assays achieved confluency. finally, because the protective effect of antiviral cells is more clearly observable spatially, we confirmed our results by simulating fitted time series in a spatially-explicit, stochastic reconstruction of our mean field model. in spatial simulations, rates of antiviral acquisition were fixed at fitted values for r and " derived from mean field estimates, while transmission rates (b) were fixed at values ten times greater than those estimated under mean field conditions, accounting for the intensification of parameter thresholds permitting pathogen invasion in local spatial interactions (see materials and methods; videos - ; figure -figure supplement ; supplementary file ; webb et al., ) . in immune capable time series, spatial antiviral cells acted as 'refugia' which protected live cells from infection as each initial epidemic wave 'washed' across a cell monolayer. eventual birth of new susceptibles from these living refugia allowed for sustained epidemic transmission in cases where some infectious cells persisted at later timepoints in simulation (videos - ; figure -figure supplement ). bats are reservoirs for several important emerging zoonoses but appear not to experience disease from otherwise virulent viral pathogens. though the molecular biological literature has made great progress in elucidating the mechanisms by which bats tolerate viral infections (zhou et al., ; ahn et al., ; xie et al., ; pavlovich et al., ; zhang et al., ) , the impact of unique bat immunity on virus dynamics within-host has not been well-elucidated. we used an innovative combination of in vitro experimentation and within-host modeling to explore the impact of unique bat immunity on virus dynamics. critically, we found that bat cell lines demonstrated a signature of enhanced interferon-mediated immune response, of either constitutive or induced form, which allowed for establishment of rapid within-host, cell-to-cell virus transmission rates (b). these results were supported by both data-independent bifurcation analysis of our mean field theoretical model, as well as fitting of this model to viral infection time series established in bat cell culture. additionally, we demonstrated that the antiviral state induced by the interferon pathway protects live cells from mortality in tissue culture, resulting in in vitro epidemics of extended duration that enhance the probability of establishing a long-term persistent infection. our findings suggest that viruses evolved in bat reservoirs possessing enhanced ifn capabilities could achieve more rapid within-host transmission rates without causing pathology to their hosts. such rapidly-reproducing viruses would likely generate extreme virulence upon spillover to hosts lacking similar immune capacities to bats. to achieve these results, we first developed a novel, within-host, theoretical model elucidating the effects of unique bat immunity, then undertook bifurcation analysis of the model's equilibrium properties under immune absent, induced, and constitutive assumptions. we considered a cell line to be constitutively immune if possessing any number of antiviral cells at disease-free equilibrium but allowed the extent of constitutive immunity to vary across the parameter range for ", the constitutive rate of antiviral acquisition. in deriving the equation for r , the basic reproduction number, which defines threshold conditions for virus invasion of a tissue (r > ), we demonstrated how the invasion threshold is elevated at high values of constitutive antiviral acquisition, ". constitutive immune processes can thus prohibit pathogen invasion, while induced responses, by definition, can only control infections post-hoc. once thresholds for pathogen invasion have been met, assumptions of constitutive immunity will limit the cellular mortality (virulence) incurred at high transmission rates. regardless of mechanism (induced or constitutive), interferon-stimulated antiviral cells appear to play a key role in maintaining longer term or persistent infections by safeguarding susceptible cells from rapid infection and concomitant cell death. fitting of our model to in vitro data supported expected immune phenotypes for different bat cell lines as described in the literature. simple target cell models that ignore the effects of immunity best recapitulated infectious time series derived from ifn-deficient vero cells, while models assuming induced immune processes most accurately reproduced trials derived from roni/ . (rousettus aegyptiacus) cells, which possess a standard virusinduced ifn-response. in most cases, models assuming constitutive immune processes best recreated virus epidemics produced on pakit (pteropus alecto) cells, which are known to constitutively express the antiviral cytokine, ifn-a (zhou et al., ) . model support for induced immune assumptions in fits to rvsv-marv infections on pakit cells suggests that the constitutive ifn-a expression characteristic of p. alecto cells may represent more of a constitutive immune priming process than a perpetual, functional, antiviral defense. results from mean field model fitting were additionally confirmed in spatially explicit stochastic simulations of each time series. as previously demonstrated in within-host models for hiv (coffin, ; perelson et al., ; nowak et al., ; bonhoeffer et al., ; ho et al., ) , assumptions of simple target-cell depletion can often provide satisfactory approximations of viral dynamics, especially those reproduced in simple in vitro systems. critically, our model fitting emphasizes the need for incorporation of top-down effects of immune control in order to accurately reproduce infectious time series derived from bat cell tissue cultures, especially those resulting from the robustly antiviral pakit p. alecto cell line. these findings indicate that enhanced ifn-mediated immune pathways in bat reservoirs may promote elevated within-host virus replication rates prior to cross-species emergence. we nonetheless acknowledge the limitations imposed by in vitro experiments in tissue culture, especially involving recombinant viruses and immortalized cell lines. future work should extend these cell culture studies to include measurements of multiple state variables (i.e. antiviral cells) to enhance epidemiological inference. the continued recurrence of ebola epidemics across central africa highlights the importance of understanding bats' roles as reservoirs for virulent zoonotic disease. the past decade has born witness to emerging consensus regarding the unique pathways by which bats resist and tolerate highly virulent infections (brook and dobson, ; xie et al., ; zhang et al., ; ahn et al., ; zhou et al., ; ng et al., ; pavlovich et al., ) . nonetheless, an understanding of the mechanisms by which bats support endemic pathogens at the population level, or promote the evolution of virulent pathogens at the individual level, remains elusive. endemic maintenance of infection is a defining characteristic of a pathogen reservoir (haydon et al., ) , and bats appear to merit such a title, supporting long-term persistence of highly transmissible viral infections in isolated island populations well below expected critical community sizes (peel et al., ) . researchers debate the relative influence of population-level and within-host mechanisms which might explain these trends (plowright et al., ) , but increasingly, field data are difficult to reconcile without acknowledgement of a role for persistent infections (peel et al., ; brook et al., ) . we present general methods to study cross-scale viral dynamics, which suggest that within-host persistence is supported by robust antiviral responses characteristic of bat immune processes. viruses which evolve rapid replication rates under these robust antiviral defenses may pose the greatest hazard for cross-species pathogen emergence into spillover hosts with immune systems that differ from those unique to bats. all experiments were carried out on three immortalized mammalian kidney cell lines: vero (african green monkey), roni/ . (rousettus aegyptiacus) (kühl et al., ; biesold et al., ) and pakit (pteropus alecto) (crameri et al., ) . the species identifications of all bat cell lines was confirmed morphologically and genetically in the publications in which they were originally described (kühl et al., ; biesold et al., ; crameri et al., ) . vero cells were obtained from atcc. monolayers of each cell line were grown to % confluency (~  cells) in -well plates. cells were maintained in a humidified ˚c, % co incubator and cultured in dulbecco's modified eagle medium (dmem) (life technologies, grand island, ny), supplemented with % fetal bovine serum (fbs) (gemini bio products, west sacramento, ca), and % penicillin-streptomycin (life technologies). cells were tested monthly for mycoplasma contamination while experiments were taking place; all cells assayed negative for contamination at every testing. previous work has demonstrated that all cell lines used are capable of mounting a type i ifn response upon viral challenge, with the exception of vero cells, which possess an ifn-b deficiency (desmyter et al., ; rhim et al., ; emeny and morgan, ) . roni/ . cells have been shown to mount idiosyncratic induced ifn defenses upon viral infection (pavlovich et al., ; kuzmin et al., ; arnold et al., ; kühl et al., ; biesold et al., ) , while pakit cells are known to constitutively express the antiviral cytokine, ifn-a (zhou et al., ) . this work is the first documentation of ifn signaling induced upon challenge with the particular recombinant vsvs outlined below. we verified known antiviral immune phenotypes via qpcr. results were consistent with the literature, indicating a less pronounced role for interferon defense against viral infection in roni/ . versus pakit cells. replication-capable recombinant vesicular stomatitis indiana viruses, expressing filovirus glycoproteins in place of wild type g (rvsv-g, rvsv-ebov, and rvsv-marv) have been previously described (wong et al., ; miller et al., ) . viruses were selected to represent a broad range of anticipated antiviral responses from host cells, based on a range of past evolutionary histories between the virus glycoprotein mediating cell entry and the host cell's entry receptor. these interactions ranged from the total absence of evolutionary history in the case of rvsv-g infections on all cell lines to a known receptor-level cell entry incompatibility in the case of rvsv-marv infections on pakit cell lines. to measure infectivities of rvsvs on each of the cell lines outlined above, so as to calculate the correct viral dose for each moi, nh cl ( mm) was added to infected cell cultures at - hr postinfection to block viral spread, and individual egfp-positive cells were manually counted at - hr post-infection. previously published work indicates that immortalized kidney cell lines of rousettus aegyptiacus (roni/ . ) and pteropus alecto (pakit ) exhibit different innate antiviral immune phenotypes through, respectively, induced (biesold et al., ; pavlovich et al., ; kühl et al., ; arnold et al., ) and constitutive (zhou et al., ) expression of type i interferon genes. we verified these published phenotypes on our own cell lines infected with rvsv-g, rvsv-ebov, and rvsv-marv via qpcr of ifn-a and ifn-b genes across a longitudinal time series of infection. specifically, we carried out multiple time series of infection of each cell line with each of the viruses described above, under mock infection conditions and at mois of . and . -with the exception of rvsv-marv on pakit cell lines, for which infection was only performed at moi = . due to limited viral stocks and the extremely low infectivity of this virus on this cell line (thus requiring high viral loads for initial infection). all experiments were run in duplicate on well plates, such that a typical plate for any of the three viruses had two control (mock) wells, two moi = . wells and two moi = . wells, excepting pakit plates, which had two control and four moi = . wells at a given time. we justify this pakit exemption through the expectation that ifn-a expression is constitutive for these cells, and by the assumption that any expression exhibited at the lower moi should also be present at the higher moi. for these gene expression time series, four -well plates for each cell line-virus combination were incubated with virus for one hour at ˚c. following incubation, virus was aspirated off, and cell monolayers were washed in pbs, then covered with an agar plaque assay overlay to mimic conditions under which infection trials were run. plates were then harvested sequentially at timepoints of roughly , , , and hr post-infection (exact timing varied as multiple trials were running simultaneously). upon harvest of each plate, agar overlay was removed, and virus was lysed and rna extracted from cells using the zymo quick rna mini prep kit, according to the manufacturer's instructions and including the step for cellular dna digestion. post-extraction, rna quality was verified via nanodrop, and rna was converted to cdna using the invitrogen superscript iii cdna synthesis kit, according to the manufacturer's instructions. cdna was then stored at ˚c and as a frozen stock at À ˚c to await qpcr. we undertook qpcr of cdna to assess expression of the type i interferon genes, ifn-a and ifnb, and the housekeeping gene, b-actin, using primers previously reported in the literature (supplementary file ) . for qpcr, ml of each cdna sample was incubated with ml of deionized water, ml of um forward/reverse primer mix and ml of itaq universal sybr green, then cycled on a quantstudio real-time pcr machine under the following conditions: initial denaturation at c for min followed by cycles of: denaturation at ˚c ( s), annealing at ˚c ( s), and extension at ˚c ( s). we report simple d-ct values for each run, with raw ct of the target gene of interest (ifn-a or ifn-b) subtracted from raw ct of the b-actin housekeeping gene in figure -figure supplement . calculation of fold change upon viral infection in comparison to mock using the d-d-ct method (livak and schmittgen, ) was inappropriate in this case, as we wished to demonstrate constitutive expression of ifn-a in pakit cells, whereby data from mock cells was identical to that produced from infected cells. after being grown to~ % confluency, cells were incubated with pelleted rvsvs expressing egfp (rvsv-g, rvsv-ebov, rvsv-marv). cell lines were challenged with both a low ( . ) and high ( . ) multiplicity of infection (moi) for each virus. in a cell monolayer infected at a given moi (m), the proportion of cells (p), infected by k viral particles can be described by the poisson distribution: p k ð Þ ¼ e Àm m k k! , such that the number of initially infected cells in an experiment equals: À e Àm . we assumed that a~ % confluent culture at each trial's origin was comprised of~ x cells and conducted all experiments at mois of . and . , meaning that we began each trial by introducing virus to, respectively,~ or cells, representing the state variable 'e' in our theoretical model. low mois were selected to best approximate the dynamics of mean field infection and limit artifacts of spatial structuring, such as premature epidemic extinction when growing plaques collide with plate walls in cell culture. six-well plates were prepared with each infection in duplicate or triplicate, such that a control well (no virus) and - wells each at moi . and . were incubated simultaneously on the same plate. in total, we ran between and trials at each cell-virus-moi combination, excepting r-vsv-marv infections on pakit cells at moi = . , for which we ran only eight trials due to the low infectivity of this virus on this cell line, which required high viral loads for initial infection. cells were incubated with virus for one hour at ˚c. following incubation, virus was aspirated off, and cell monolayers were washed in pbs, then covered with a molten viscous overlay ( % x mem/lglutamine; % fbs; % hepes; % agarose), cooled for min, and re-incubated in their original humidified ˚c, % co environment. after application of the overlay, plates were monitored periodically using an inverted fluorescence microscope until the first signs of gfp expression were witnessed (~ - . hr post-infection, depending on the cell line and virus under investigation). from that time forward, a square subset of the center of each well (comprised of either -or -subframes and corresponding to roughly % and % of the entire well space) was imaged periodically, using a cellinsight cx high content screening (hcs) platform with a x air objective (thermofisher, inc, waltham, ma). microscope settings were held standard across all trials, with exposure time fixed at . s for each image. one color channel was imaged, such that images produced show gfp-expressing cells in white and non-gfp-expressing cells in black (figure -figure supplement ) . wells were photographed in rotation, as frequently as possible, from the onset of gfp expression until the time that the majority of cells in the well were surmised to be dead, gfp expression could no longer be detected, or early termination was desired to permit hoechst staining. in the case of pakit cells infected with rvsv-ebov, where an apparently persistent infection established, the assay was terminated after + hours ( + days) of continuous observation. upon termination of all trials, cells were fixed in formaldehyde ( % for min), incubated with hoechst stain ( . % for min) (thermofisher, inc, waltham, ma), then imaged at x on the cellinsight cx high content screening (hcs) platform. the machine was allowed to find optimal focus for each hoechst stain image. one color channel was permitted such that images produced showed live nuclei in white and dead cells in black. hoechst stain colors cellular dna, and viral infection is thought to interfere with the clarity of the stain (dembowski and deluca, ) . as such, infection termination, cell fixation, and hoechst staining enables generation of a rough time series of uninfectious live cells (i.e. susceptible + antiviral cells) to complement the images which produced time series of proportions infectious. due to uncertainty over the exact epidemic state of hoechst-stained cells (i.e. exposed but not yet infectious cells may still stain), we elected to fit our models only to the infectious time series derived from gfpexpressing images and used hoechst stain images as a post hoc visual check on our fit only ( figure ; figure -figure supplements - ). images recovered from the time series above were processed into binary ('infectious' vs. 'non-infectious' or, for hoechst-stained images, 'live' vs. 'dead') form using the ebimage package (pau et al., ) in r version . for macintosh, after methods further detailed in supplementary file . binary images were then further processed into time series of infectious or, for hoechst-stained images, live cells using a series of cell counting scripts. because of logistical constraints (i.e. many plates of simultaneously running infection trials and only one available imaging microscope), the time course of imaging across the duration of each trial was quite variable. as such, we fitted a series of statistical models to our processed image data to reconstruct reliable values of the infectious proportion of each well per hour for each distinct trial in all cell line-virus-moi combinations (figure to derive the expression for r , the basic pathogen reproductive number in vitro, we used next generation matrix (ngm) techniques (diekmann et al., ; heffernan et al., ) , employing wolfram mathematica (version . ) as an analytical tool. r describes the number of new infections generated by an existing infection in a completely susceptible host population; a pathogen will invade a population when r > (supplementary file ). we then analyzed stability properties of the system, exploring dynamics across a range of parameter spaces, using matcont (version . ) (dhooge et al., ) for matlab (version r a) (supplementary file ). the birth rate, b, and natural mortality rate, m, balance to yield a population-level growth rate, such that it is impossible to estimate both b and m simultaneously from total population size data alone. as such, we fixed b at. and estimated m by fitting an infection-absent version of our mean field model to the susceptible time series derived via hoechst staining of control wells for each of the three cell lines (figure -figure supplement ) . this yielded a natural mortality rate, m, corresponding to a lifespan of approximately , , and hours, respectively, for vero, roni/ . , and pakit cell lines (figure -figure supplement ) . we then fixed the virus incubation rate, s, as the inverse of the shortest observed duration of time from initial infection to the observation of the first infectious cells via fluorescent microscope for all nine cell line -virus combinations (ranging to . hours). we fixed a, the infection-induced mortality rate, at / , an accepted standard for general viral kinetics (howat et al., ) , and held c, the rate of antiviral cell regression to susceptible status, at for the timespan (< hours) of the experimental cell line infection trials. we estimated cell line-virus-moi-specific values for b, r, and " by fitting the deterministic output of infectious proportions in our mean field model to the full suite of statistical outputs of all trials for each infected cell culture time series (figure -figure supplements - ) . fitting was performed by minimizing the sum of squared differences between the deterministic model output and cell linevirus-moi-specific infectious proportion of the data at each timestep. we optimized parameters for moi = . and . simultaneously to leverage statistical power across the two datasets, estimating a different transmission rate, b, for trials run at each infectious dose but, where applicable, estimating the same rates of r and " across the two time series. we used the differential equation solver lsoda() in the r package desolve (soetaert et al., ) to obtain numerical solutions for the mean field model and carried out minimization using the 'nelder-mead' algorithm of the optim() function in base r. all model fits were conducted using consistent starting guesses for the parameters, b (b = ), and where applicable, r (r = . ) and " (" = . ). in the case of failed fits or indefinite hessians, we generated a series of random guesses around the starting conditions and continued estimation until successful fits were achieved. all eighteen cell line-virus-moi combinations of data were fit by an immune absent (" = r = ) version of the theoretical model and, subsequently, an induced immunity (" = ; r > ) and constitutive immunity (" > ; r > ) version of the model. finally, we compared fits across each cell line-virus-moi combination via aic. in calculating aic, the number of fitted parameters in each model (k) varied across the immune phenotypes, with one parameter (b) estimated for absent immune assumptions, two (b and r) for induced immune assumptions, and three (b, r, and ") for constitutive immune assumptions. the sample size (n) corresponded to the number of discrete time steps across all empirical infectious trials to which the model was fitted for each cell-line virus combination. all fitting and model comparison scripts are freely available for download at the following figshare repository: doi: . /m .figshare. . finally, we verified all mean field fits in a spatial context, in order to more thoroughly elucidate the role of antiviral cells in each time series. we constructed our spatial model in c++ implemented in r using the packages rcpp and rcpparmadillo (eddelbuettel and francois, ; eddelbuettel and sanderson, ) . following nagai and honda ( ) and howat et al. ( ) , we modeled this system on a two-dimensional hexagonal lattice, using a ten-minute epidemic timestep for cell state transitions. at the initialization of each simulation, we randomly assigned a duration of natural lifespan, incubation period, infectivity period, and time from antiviral to susceptible status to all cells in a theoretical monolayer. parameter durations were drawn from a normal distribution centered at the inverse of the respective fixed rates of m, s, a, and c, as reported with our mean field model. transitions involving the induced (r) and constitutive (") rates of antiviral acquisition were governed probabilistically and adjusted dynamically at each timestep based on the global environment. as such, we fixed these parameters at the same values estimated in the mean field model, and multiplied both r and " by the global proportion of, respectively, exposed and susceptible cells at a given timestep. in contrast to antiviral acquisition rates, transitions involving the birth rate (b) and the transmission rate (b) occurred probabilistically based on each cell's local environment. the birth rate, b, was multiplied by the proportion of susceptible cells within a six-neighbor circumference of a focal dead cell, while b was multiplied by the proportion of infectious cells within a thirty-six neighbor vicinity of a focal susceptible cell, thus allowing viral transmission to extend beyond the immediate nearestneighbor boundaries of an infectious cell. to compensate for higher thresholds to cellular persistence and virus invasion which occur under local spatial conditions (webb et al., ) , we increased the birth rate, b, and the cell-to-cell transmission rate, b, respectively, to six and ten times the values used in the mean field model (supplementary file ) . we derived these increases based on the assumption that births took place exclusively based on pairwise nearest-neighbor interactions (the six immediately adjacent cells to a focal dead cell), while viral transmission was locally concentrated but included a small ( . %) global contribution, representing the thirty-six cell surrounding vicinity of a focal susceptible. we justify these increases and derive their origins further in supplementary file . we simulated ten stochastic spatial time series for all cell-virus combinations under all three immune assumptions at a population size of , cells and compared model output with data in . transparent reporting form data availability all data generated or analysed during this study are included in the manuscript and supporting files. all images and code used in this study have been made available for download at the following figshare dampened nlrp -mediated inflammation in bats and implications for a special viral reservoir host transcriptomics reveal antiviral gene induction in the egyptian rousette bat is antagonized in vitro by marburg virus infection kinetics of influenza a virus infection in humans type i interferon reaction 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infection including immune responses henipavirus neutralising antibodies in an isolated island population of african fruit bats support for viral persistence in bats from age-specific serology and models of maternal immunity hiv- dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time modelling viral and immune system dynamics transmission or within-host dynamics driving pulses of zoonotic viruses in reservoir-host populations r: a language and environment for statistical computing. r foundation for statistical computing establishment and maintenance of the interferoninduced antiviral state: studies in enucleated cells inhibition of herpesvirus hominis replication by human interferon biological characteristics and viral susceptibility of an african green monkey kidney cell line (vero) dynamics of influenza virus infection and pathology mechanism of interferon action immunological control of viral infections in bats and the emergence of viruses highly pathogenic to humans package desolve: solving initial value differential equations in r type i interferons in host defense niemann-pick c heterogeneity of bat cells controls filovirus tropism viruses in bats and potential spillover to animals and humans host-parasite interactions between the local and the mean-field: how and when does spatial population structure matter a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry dampened sting-dependent interferon activation in bats comparative analysis of bat genomes provides insight into the evolution of flight and immunity contraction of the type i ifn locus and unusual constitutive expression of ifn-a in bats the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord- -l ufs k authors: tomita, kengo; saito, yuna; suzuki, tokiko; imbaby, samar; hattori, kohshi; matsuda, naoyuki; hattori, yuichi title: vascular endothelial growth factor contributes to lung vascular hyperpermeability in sepsis-associated acute lung injury date: - - journal: naunyn schmiedebergs arch pharmacol doi: . /s - - - sha: doc_id: cord_uid: l ufs k vascular endothelial growth factor (vegf) is a prime regulator of vascular permeability. acute lung injury (ali) is characterized by high-permeability pulmonary edema in addition to refractory hypoxemia and diffuse pulmonary infiltrates. in this study, we examined whether vegf can be implicated as a pulmonary vascular permeability factor in sepsis-associated ali. we found that a great increase in lung vascular leak occurred in mice instilled intranasally with lipopolysaccharide (lps), as assessed by igm levels in bronchoalveolar lavage fluid. treatment with the vegf-neutralizing monoclonal antibody bevacizumab significantly reduced this hyperpermeability response, suggesting active participation of vegf in non-cardiogenic lung edema associated with lps-induced ali. however, this was not solely attributable to excessive levels of intrapulmonary vegf. expression levels of vegf were significantly reduced in lung tissues from mice with both intranasal lps administration and cecal ligation and puncture (clp)-induced sepsis, which may stem from decreases in non-endothelial cells-dependent vegf production in the lungs. in support of this assumption, stimulation with lps and interferon-γ (ifn-γ) significantly increased vegf in human pulmonary microvascular endothelial cells (hpmecs) at mrna and protein levels. furthermore, a significant rise in plasma vegf levels was observed in clp-induced septic mice. the increase in vegf released from hpmecs after lps/ifn-γ challenge was completely blocked by either specific inhibitor of mitogen-activated protein kinase (mapk) subgroups. taken together, our results indicate that vegf can contribute to the development of non-cardiogenic lung edema in sepsis-associated ali due to increased vegf secretion from pulmonary vascular endothelial cells through multiple mapk-dependent pathways. sepsis is a potentially life-threatening medical emergency that is caused by the body's extreme response to an infection. the advent of the new definition of sepsis, which has been published recently, prompts a reappraisal of organ dysfunction as the hallmark of sepsis (singer et al. ). sepsis affects every major organ, including the lung, liver, and kidney, within the body, ultimately leading to the failure of one or more organs. the respiratory system is the most affected organ of the body, and lung dysfunction is the first step in the development of multiple organ failure in septic patients. acute lung injury (ali) and its most extreme form, acute respiratory distress syndrome (ards), are the manifestations of lethal and complex respiratory dysfunction and are characterized by explosive and diffuse pulmonary infiltrates, leading to noncardiogenic alveolar edema and, ultimately, refractory hypoxemia (tsushima et al. ; matuschak and lechner ) . vascular endothelial growth factor (vegf), also known as vegf-a, is a glycoprotein originally isolated as a tumor cellsecreted vascular permeability factor (plouet et al. ) and, since then, it has long been documented that vegf serves as the prime regulator of vascular permeability (bates ) . while vegf has been subsequently shown to have additional potent mitogenic and angiogenic properties (ferrara ; sharma et al., ) , significant amounts of vegf are known to exist in the normal human lung without significant mitogenesis or angiogenesis (barratt et al. ). in the normal lung, vegf may function as a survival factor for epithelial cells and endothelial cells in a paracrine manner (gerber et al. ; mura et al. ; roberts et al. ). in the meanwhile, vegf may contribute to the development of non-cardiogenic pulmonary edema associated with ali/ards. lung-targeted overexpression of human vegf , using an adenoviral gene vector, has been shown to result in pulmonary edema and increased vascular permeability in mice (kaner et al. ) . in addition, it has been revealed that the high ventilation-induced increase in pulmonary microvascular permeability in mice can be attenuated by knockdown of vegf by short-interfering rnas (li et al. ) . moreover, pretreatment with adenovirus-encoding soluble vegf receptor (vegfr) has been found to prevent ischemia-reperfusion-induced lung injury in rats (godzich et al. ). however, a role of vegf in pulmonary vascular hyperpermeability accompanied by sepsis-associated ali is not fully understood. in the present study, by conducting in vivo and in vitro experiments using rodent models of sepsis-associated ali and human pulmonary microvascular endothelial cell line, respectively, we attempted to test the hypothesis that vegf may contribute to non-cardiogenic high vascular permeability pulmonary edema in ali associated with sepsis. all animal experimental procedures were conducted in accordance with the national institute of health guidelines on the use of laboratory animal and with approval of the care and use committee of the university of toyama. in the first series of experiments, we used the cecal ligation and puncture (clp)-induced sepsis mouse model. clp-induced sepsis is regarded as a highly clinically relevant model of polymicrobial sepsis, because it reproduces many hallmarks of sepsis occurring in human patients (hubbard et al. ) . the surgical procedure to generate clp-induced sepsis was conducted according to our previous reports (tomita et al. ; kawakami et al. ; yamashita et al. ) . in brief, male balb/c mice (sankyo lab service, tokyo japan), - weeks old, were anesthetized with - % sevoflurane by inhalation, and a middle abdominal incision was made. the cecum was mobilized, tightly ligated ( cm from the cecum tip), punctured twice with a -gauge needle, and gently squeezed to expel small amounts of feces. then, the bowel was repositioned to the peritoneal cavity, and the laparotomy site was closed with sterile suture (the skin and muscle were sutured separately). sham-operated control underwent the same procedure except for ligation and puncture of the cecum. both groups of animals were fed the same diet and water ad libitum, and housed in an environment with controlled temperature, constant humidity, and a daily -h light-dark cycle ). all animals after clp surgery were lethargic, showed lack of interest in their environment, displayed piloerection, and had crusty exudates around their eyes, as opposed to sham-operated animals that were healthy, moving freely and eating (matsuda et al. ). all mice received subcutaneous injection of . ml sterile normal saline immediately after surgery. at h after surgery, blood samples were collected and inflation-fixed lungs were harvested from mice treated with - mg/kg ketamine and mg/kg xylazine hydrochloride. in the second series of experiments, mice, under light anesthesia, were instilled intranasally with μg of lipopolysaccharide (lps) (escherichia coli :b ; list biological laboratories, campbell, ca, usa) in μl of sterile . % nacl solution. control animals received equivalent volume of vehicle saline solution. when bevacizumab, which neutralizes vegf and blocks its signal transduction through vegf receptors (papadopoulos et al. ) , was used, it was intravenously given to mice at a dose of μg at min before administration of lps. animals were euthanized at h after lps challenge. all experimental data were analyzed in a blinded fashion. the immortalized human pulmonary microvascular endothelial cell line (hpmec-st . r), which was developed by means of co-transfection of a plasmid encoding the catalytic component of telomerase and a plasmid encoding the simian virus large t antigen (krump-konvalinkova et al. ; unger et al. ) , was kindly provided by drs. c. james kirkpatrick and ronald e. unger at johannes gutenberg university (mainz, germany). hpmec-st . r cells were routinely maintained on tissue culture plastic ware in m medium (sigma-aldrich, st. louis, mo, usa) supplemented with % (v/v) heat-inactivated fetal bovine serum, μg/ml endothelial cell growth supplements (sigma-aldrich), μg/ml sodium heparin (sigma-aldrich), % (v/v) penicillin/streptomycin (nacalai tesque, kyoto, japan), and μg/ml g (nacalai tesque). cells were cultured at °c under a humidified atmosphere containing % co and % air. in hpmec-st . r cells, the presence of interferon (ifn)-γ can highly amplify the inflammatory responses to lps ). thus, cells were stimulated with μg/ml lps and ng/ml ifn-γ (r&d systems, minneapolis, mn, usa). when mitogen-activated protein kinase (mapk) inhibitors, pd ( μm; cayman chemical, ann arbor, mi, usa), sb ( μm; adipogen life sciences, epalinges, switzerland), sp ( μm; cayman chemical), and jnk-in- ( μm; merck, darmstadt, germany) were used, they were added to the medium for min before challenge with lps and ifn-γ. lps/ ifn-γ stimulation was stopped at the indicated time points by aspirating off the culture medium and then adding ice-cold pss before harvesting. blood levels of tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , and monocyte chemoattractant protein- (mcp- ) were measured by the use of commercially available enzyme-linked immunosorbent assay (elisa) kit (r&d systems, minneapolis, mn) according to the manufacturer's instructions. to measure the concentrations of vegf in serum and culture medium samples, mouse vegf quantikine elisa kit (r&d system) and human vegf duoset elisa (r&d system) were used, respectively. the plate was read on a microplate reader (nippon-intermed, tokyo, japan). assays were performed in duplicate. to estimate pulmonary microvascular permeability, igm was selected as a marker of permeability and its concentration in bronchoalveolar lavage (bal) fluid was determined immunologically by igm mouse uncoated elisa kits with plates (thermo fisher scientific, rockford, il, usa). a polyethylene catheter was inserted into the trachea, bal was performed by repeatedly infusing and removing ml of pbs, and the third drainage of effluent was kept as bal fluid. total rna was isolated from hpmec-st . r cells and lung tissues with the use of sepazol®-rna i super g (nacalai tesque) according to the manufacturer's manual. revertra ace qpcr rt master mix (toyobo, osaka, japan) was used for the reverse transcription reaction, and real-time pcr analyses were performed using powerup™ sybr® green master mix (thermo fisher scientific), as described in the manufacturers' instructions. values were normalized to the housekeeping gene gapdh according to the manufacturer's protocol (mx p real-time pcr system; agilent technologies inc., santa clara, ca, usa). additional details are described by our laboratory (kawakami et al. ; yamashita et al. ; suzuki et al. ). the pcr primers were designed as follows: forward '-tgcagattatgcggatcaaacc- ′ and reverse ′-tgcattcacatttgttgtgctgtag- ′ for vegf, forward ′-caggcccagtttctgccatt- ′ and reverse ′-ttccagctcagcgtggtcgta- ′ for vegfr , forward ′-ccagcaaaagcagggagtct gt- ′ and reverse ′-tgtctgtgtcatcggagtga tatcc- ′ for vegfr , and forward ′-tgtg tccgtcgtggatctga- ′ and reverse ′-ttgc tgttgaagtcgcaggag- ′ for glyceraldehyde- phosphate dehydrogenase (gapdh). hpmec-st . r cells were grown in -mm dish, harvested, and lysed in μl of ripa buffer ( mm tris-hcl, mm nacl, % np- , % sodium deoxycholate, . % sds, ph . ) containing protease inhibitor cocktail on ice. the lysates were centrifuged at , ×g for min at °c and the resulting supernatants were collected. the protein concentration in the remaining supernatant was measured using bca protein assay kit (nacalai tesque). blotting procedure, chemiluminescent detection, and densitometric analysis were carried out as described in our previous reports (kawakami et al. ; suzuki et al. ) . samples ( - μg of protein) were separated with % sds-page gel electrophoresis and then transferred to polyvinylidene difluoride filter membrane. the membrane was probed with the following primary antibodies: anti-human extracellular signal-regulated protein kinase (erk) / mouse monoclonal antibody ( : ; cell signaling, danvers, ma, usa), anti-human phospho-erk / (thr- /tyr- ) rabbit monoclonal antibody ( : ; cell signaling), anti-human c-jun nterminal kinase (jnk) rabbit monoclonal antibody ( : ; cell signaling), anti-human p rabbit monoclonal antibody ( : ; cell signaling), anti-human phospho-p (thr- /tyr- ) mouse monoclonal antibody ( : ; cell signaling), anti-human jnk (thr- /tyr- ) mouse monoclonal antibody ( : ; cell signaling), and anti-human gapdh chicken polyclonal antibody ( : ; emd millipore, billerica, ma, usa). irdye®-labeled secondary antibodies were purchased from li-cor bioscience (lincoln, ne, usa) and odyssey clx infrared imaging system (li-cor bioscience) was employed for primary antibody detection. gapdh was used as the loading control. results are presented as mean ± standard error. data were analyzed by the use of prism software (version ; graphpad software, san diego, ca, usa). statistical analysis was performed by student's t test or one-way analysis of variance (anova) followed by tukey's multiple-comparison test. differences were considered to be statistically significant when a p value was < . . the clp rodent model, which causes peritonitis and leads to a polymicrobial sepsis, represents an indirect insult similar to the pathogenesis of ali/ards (villar et al. ) . indeed, we have clearly demonstrated that mice - h after clp surgery display marked hypoxemia, increased lung vascular permeability, and histological damage in lungs, including wall thickening, inflammatory infiltrate, and hemorrhage (takano et al. ; oishi et al. ; imaizumi et al. ) . when blood levels of pro-inflammatory cytokines were measured using an elisa, the sham-operated control animals had extremely low levels of the cytokines examined here. the mice h after clp-induced sepsis exhibited marked elevations in blood levels of tnf-α, il- β, il- , and mcp- (c) the mrna levels of vegf, vegfr , and vegfr in lung tissues were quantified by real-time pcr. lung tissues were harvested h after surgery. values are normalized to gapdh (n = ). * p < . , ** p < . , and *** p < . vs. the sham-operated control group (fig. a) . sepsis induction by clp also resulted in a significant elevation in plasma vegf protein levels in mice (fig. b) . plasma vegf in clp mice was increased . -fold in comparison with sham-operated control animals. however, the vegf mrna level was significantly downregulated in lung tissues from mice with clp-induced sepsis (fig. c) . we further examined vegfr mrna levels in clp mouse lungs. vegf regulates vascular permeability by activating receptors, vegfr (flt- ) and vegfr (kdr/flk ) (shibuya ) . as shown in fig. c , vegfr mrna was increased and vegfr mrna was decreased in lung tissues of clp mice compared with sham-operated controls. to assess changes in pulmonary vascular permeability, bal supernatant was analyzed for igm using an elisa. intranasal challenge with lps resulted in a highly significant . -fold increase in pulmonary vascular permeability (fig. a) . treatment with bevacizumab, which neutralizes vegf and blocks vegfr signaling (papadopoulos et al. ) , significantly but partially attenuated lung vascular permeability as compared with that shown in mice challenged with lps alone. we also examined vegf mrna levels in lung tissues of mice intranasally instilled with lps. as seen in clp-induced septic mice, pulmonary mrna expression was significantly downregulated in mice challenged with lps (fig. b ). in the immortalized human pulmonary microvascular endothelial cell line hpmec-st . r, the time course of changes in gene expression of vegf and its receptors after coadministration of lps and ifn-γ were investigated. the mrna level of vegf was gradually increased, reached a maximum at - h, and then showed a return toward the baseline at h (fig. a) . the mrna level of vegfr showed a trend toward increasing throughout - h observation period (fig. b) . vegfr mrna remained virtually unchanged in a wide range of time after stimulation with lps and ifn-γ (fig. c) . when the amounts of vegf in culture media were measured by an elisa, lps/ifn-γ challenge resulted in a significant increase in the protein level of vegf (fig. b) . the mapk signaling cascades are generally thought to be important in the pathogenesis of ali/ards (newton and holden ; qian et al. ) . when activation of the three major subgroups of mapk family, erk / , p , and jnk, was assessed by increases in their phosphorylation levels, coadministration of lps and ifn-γ resulted in significant activation of all three families of mapks in hpmec-st . r cells (fig. a) . we thus examined whether expression of vegf in human pulmonary microvascular endothelial cells is regulated by mapks. when hpmec-st . r cells were treated with pd , an inhibitor of mapk kinase which is an erk / upstream activator, or sb , which is widely used as a specific inhibitor of p mapk, the lps/ifn-γinduced increase in vegf protein levels was strongly blocked (fig. b) . treatment with sp , an anthrapyrazolone inhibitor of jnk, also abrogated the vegf protein increase in hpmec-st . r cells stimulated with lps/ifn-γ (fig. b) . the striking inhibition of the lps/ifn-γ-induced vegf upregulation was similarly observed by treatment with another fig. involvement of vegf in lung vascular hyperpermeability in mice after lps administration. lps ( μg) was instilled intranasally and animals were euthanized at h after lps challenge. (a) lung vascular permeability was assessed by igm levels in bal fluid from mouse lungs (n = - ). bevacizumab ( μg) was intravenously injected to mice min before lps challenge. (b) the mrna levels of vegf in lung tissues were quantified by real-time pcr. values are normalized to gapdh (n = ). *** p < . vs. control. ## p < . vs. lps alone jnk inhibitor jnk-in- , which is far more selective for jnk than sp (brain et al. ) (fig. b ). we demonstrated in this study for the first time that vegf contributed to pulmonary vascular hyperpermeability when lps was intranasally administrated in mice. intranasal administration of lps has long been widely used as an appropriate model in which ali can be directly induced (gharib et al. ; bosmann et al. ; juschten et al. ) , and increased pulmonary vascular permeability is a critical and non-redundant pathological process involved in the ali development (herold et al., ) . we found that treatment with the vegf-neutralizing monoclonal antibody bevacizumab resulted in a significant inhibition of the increase in pulmonary vascular permeability caused by intranasal lps challenge, as evidenced by changes in igm levels in bal fluid from mouse lungs. previous reports have well established that the amounts of igm in bal fluid are related with alterations in alveolarcapillary barrier and lung vascular permeability (kantrow et al. ; matute-bello et al. ; johnston et al. ) . intriguingly, while bevacizumab is clinically used for treatment of advanced cancers of the lung, colon, brain, kidney, and others by counteracting the angiogenic effect of vegf, its ability to reduce the increase in vascular permeability associated with vegf expression may help relieve patients with the potentially serious morbidity and symptoms that accompany peritumoral edema (gil-gil et al. ) . our findings imply the active participation of vegf in non-cardiogenic high vascular permeability pulmonary edema associated with lpsinduced ali (fig. ) . however, the preventive effect of bevacizumab on lps-induced pulmonary vascular hyperpermeability was partial. our previous study has shown that treatment with n g -nitro-l-arginine, an inducible nitric oxide (no) synthase (inos) inhibitor, or diphenhydramine, a histamine h -receptor antagonist, significantly but incompletely inhibited lps-induced lung vascular permeability in mice (matsuda et al. ) , which suggests that no and histamine may also be partly responsible for mediating increased lung vascular leak following lps challenge. we thus assume that several vascular permeability molecules, including vegf, no, and histamine, can be excessively produced and thereby actually contribute to the development of pulmonary edema in sepsis-associated ali. indeed, great increases in gene and protein expression of inos and histidine decarboxylase, an enzyme that only forms histamine in mammals, have been observed in the lungs of mice after induction of sepsis with lps (matsuda et al. ) . unexpectedly, expression of vegf in lung tissues was significantly downregulated rather than upregulated in two murine models of ali, intranasal lps administration and clp-induced sepsis. this observation is consistent with a string of human studies showing a reduction in intrapulmonary vegf in the early stages of ali/ards (maitre et al. ; thickett et al. ; abadie et al. ) , although a conflicting result was reported in mice exposed to lps in a nebulization chamber (karmpaliotis et al. ) . the reduced levels of intrapulmonary vegf in ali/ards may be explained by direct injury to and clearance of epithelial type cells (medford and millar ) which are considered to be the main source of vegf in lungs (kaner and crystal ) . in this regard, caution would be required in the interpretation of the observed decrease in vegf levels in lung tissues, since the total amount of intrapulmonary vegf may be determined by the deduction of vegf released from different intrapulmonary components, such as epithelial cells and vascular endothelial cells, which can be variably affected by endotoxin. as reported in human studies (maitre et al. ; thickett et al. ; azamfirei et al. ), we found a significant rise in plasma levels of vegf in mice with clp- fig. involvement of mapk activation in vegf released from human pulmonary microvascular endothelial cells after challenge with lps and ifn-γ. hpmec-st . r cells were stimulated with μg/ml lps and ng/ml ifn-γ. (a) activation of mapks in hpmec-st . r after challenge with lps and ifn-γ. levels of phosphorylation and total expression of erk / , p , and jnk before and min after lps/ifn-γ challenge were determined by western blotting. in the top trace of each panel, typical western blots are shown. in the bottom trace, the summary of quantification of densitometric measurements as ratio of phospho-mapk relative to mapk is presented (n = - ). * p < . and ** p < . vs. unstimulated value. (b) effects of mapk inhibitors on vegf levels in hpmec-st . r cells stimulated with lps/ifn-γ for h. pd ( μm), sb ( μm), sp ( μm), or jnk-in- ( μm) was added h before lps/ifn-γ challenge. the vegf levels released from cells into the cell culture medium were measured by an elisa (n = ). ** p < . vs. control. ### p < . vs. lps/ ifn-γ alone induced sepsis. this rise in plasma vegf levels seems likely to be attributed to the release from vascular endothelial cells. we showed that stimulation with lps/ifn-γ resulted in a significant upregulation of vegf expression in human pulmonary microvascular endothelial cells at mrna and protein levels, implying that endotoxin positively regulates endothelial cell-derived vegf in a transcription manner. it should be added that neutrophils may also contribute to the increased vegf plasma levels in clp-induced septic mice, because these hemocytes have been shown to produce various vascular permeability molecules, including vegf, in certain circumstances such as inflammation (taichman et al. ; distasi and ley ) . in line with our recent report , stimulation with lps/ifn-γ significantly activated three major subgroups of mapk family, erk / , p , and jnk, in human pulmonary microvascular endothelial cells, as assessed by their phosphorylation levels. each specific inhibitor of these mapk subgroups completely blocked the increase in vegf protein levels caused by lps/ifn-γ challenge. this suggests that endotoxin upregulates vegf expression in pulmonary microvascular endothelial cells through multiple mapkdependent pathways. it is noteworthy that mapk signaling is linked to increased expression of growth factors, including vegf, in different cell types (li et al. ; pagès et al. ; rak et al. ; schrma et al. ) . vegf displays broad vascular functions, including vascular permeability, via binding to and activating its specific receptors, vegfr and vegfr (shibuya ) . in this study, in lung tissues from clp-induced septic mice, vegfr mrna was upregulated and vegfr mrna was downregulated compared with sham-operated controls. furthermore, lps/ifn-γ application showed a trend to increase vegfr mrna in human pulmonary microvascular fig. schematic diagram of the participation of vegf released from pulmonary vascular endothelial cells in noncardiogenic high vascular permeability pulmonary edema associated with lps-induced ali. see text for details endothelial cells. however, it is not clear at this time whether altered expression of vegfr can be involved as regulatory mechanisms to transduce vegf-mediated vascular hyperpermeability signals. several lines of evidence provide that vegfr may function as a decoy receptor and negatively regulate vegf functions (sato et al. ; cao ) . accordingly, what role, if any, is played by altered vegfr expression observed in this study in the pathophysiology of sepsis-associated ali awaits further study. in conclusion, the present results indicate that vegf can contribute to the development of 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sepsis-induced acute lung injury model cardioprotective and functional effects of levosimendan and milrinone in mice with cecal ligation and puncture-induced sepsis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank drs. takahiro imaizumi and takuya sakamoto for their expert help in the commencing time of this study.author contributions k.t., n.m., and y.h. conceived and designed the experiments. k.t.., y.s., t.s., and s.i. performed the experiments. k.t., y.s., and t.s. analyzed data. k.h. and y.h. wrote the article. all authors read and approved the manuscript. the authors declare that all data were generated in-house and that no paper mill was used.funding information this study was supported by grant-in-aid for young scientists ( k , k ) and for scientific research ( k , h ) from japan society for promotion of science. all animal studies were approved by the animal care and use committee of the university of toyama, which are based on the national institute of health guide for the care and use of laboratory animals and the arrive guidelines. the authors declare that they have no conflict of interest. key: cord- -yqqqe authors: ning, qin; wu, ting; su, hai-bin; ma, ke; qi, jun-ying; ni, ming; wu, di title: antiviral therapy for aechb and severe hepatitis b (liver failure) date: - - journal: acute exacerbation of chronic hepatitis b doi: . / - - - - _ sha: doc_id: cord_uid: yqqqe this chapter describes the principles of antiviral therapy, treatment strategies, medications and recommendations for aechb, hbv-aclf, hbv-related liver cirrhosis, hbv-related hcc, and liver transplantation. . severe exacerbation of chronic hepatitis b is closely related to continuous hbv replication. therefore, inhibiting hbv replication to reduce viral load may block disease progression and improve the quality of life of these patients. etv or tdf has been recommend first-line drug for the treatment of aechb. . a hyperactive immune response due to continuous hbv replication is the main mechanism for development of severe hepatitis b. in addition to comprehensive treatment, early administration of potent nucleoside analogs can rapidly reduce hbv dna concentration, relieve immune injury induced by hbv, and reduce liver inflammation and patient mortality. antiviral agents have become important in the treatment of severe exacerbation of chronic hepatitis b. . long-term antiviral treatment with nucleoside analogs can delay or reverse the progress of liver cirrhosis. virologic response, viral resistance and adverse drug reactions should be closely monitored during treatment. the treatment should be optimized for maximum effect based on each patient’s responses. . effective antiviral therapy can suppress hbv replication and reduce the incidence of hbv-related hcc. patients with hbv-related hcc should receive individualized and optimal multidisciplinary comprehensive treatment. anti-viral drugs with high efficacy, low resistance and low adverse drug reactions should be selected to improve the patient’s quality of life and prolong survival time. . methods to prevent hbv reinfection after liver transplantation include passive immunization (hbig), antiviral treatment (nucleoside analogs) and active immunization (hepatitis b vaccine). . clinical trials involving sequential combination therapy with nuc and peg-ifn have shown statistically significant decline in hbsag levels on treatment and high rates of sustained post-treatment serologic response. combination therapy with novel daa and immunotherapeutic approach may hold promise to overcome both cccdna persistence and immune escape, representing a critical step towards hbv cure. dence of hbv-related hcc. patients with hbv-related hcc should receive individualized and optimal multidisciplinary comprehensive treatment. anti-viral drugs with high efficacy, low resistance and low adverse drug reactions should be selected to improve the patient's quality of life and prolong survival time. . methods to prevent hbv reinfection after liver transplantation include passive immunization (hbig), antiviral treatment (nucleoside analogs) and active immunization (hepatitis b vaccine). . clinical trials involving sequential combination therapy with nuc and peg-ifn have shown statistically significant decline in hbsag levels on treatment and high rates of sustained post-treatment serologic response. combination therapy with novel daa and immunotherapeutic approach may hold promise to overcome both cccdna persistence and immune escape, representing a critical step towards hbv cure. ting wu and qin ning chb is a progressive disease, and its development is the interaction between hbv and the host's immune response. host's immune system eliminates hbv through cytolysis and non-cytolysis mechanism, which leads to hepatic inflammation, hepatocyte necrosis and apoptosis. continuous hepatic inflammation can lead to liver fibrosis, liver cirrhosis, liver failure, and even liver cancer gradually. hbv replication plays a key role in the disease evolution. therefore, suppressing hbv replication is crucial in treatment of chb. the development of continuous liver inflammation, cirrhosis and liver cancer is closely related to the sustainable hbv replication in chb patients. hence, inhibiting hbv replication and reducing hbv dna load is the key to block the disease progression and improve the survival quality in the treatment of chb [ ] . the fundamental goal of chb treatment is to eliminate hbv or suppress hbv permanently, thus to reduce viral pathogenicity and infectivity, relieve or inhibit hepatic necroinflammation. however, the existing antiviral drugs cannot remove the intercellular covalently closed circular dna (cccdna). therefore, the goal of antiviral treatment is to sustainably suppress the virus, reduce or prevent hepatic injury and disease progression. the short-term goal of clinical practice is sustainable hbv suppression, alt normalization and prevention from decompensated liver cirrhosis (initial response), releasing the hepatic necroinflammation and liver fibrosis during and after treatment. the long-term and ultimate goal is prevention from liver decompensation, relief or halt of the progression to liver cirrhosis or hcc, and prolonged survival period (sustained response) [ ] [ ] [ ] [ ] [ ] [ ] . hbv is difficultly to be eliminated, and there is no drug can absolutely eradicate hbv infection so far. many drugs can function against hbv, but only two classes are internationally recognized as antiviral drugs: interferon and nucleoside analogues (fig. . ). common guidelines for management of chb (table the recommendations for the treatment of hbeag positive patients with chb in guidelines (table . ) [ ] [ ] [ ] [ ] ] . the recommendations for the treatment of hbeag negative patients with chb in guidelines (table . ) [ ] [ ] [ ] [ ] ] . the recommendations for the treatment of cirrhotic patients with chb in guidelines (table . ) [ ] [ ] [ ] [ ] . the recommendations for liver biopsy in guidelines (table . ) [ ] [ ] [ ] [ ] . the recommendations for chb initial treatment options in guidelines (table . ) [ ] [ ] [ ] [ ] ] . the recommendations for chb cirrhosis initial treatment options in guidelines (table . ) [ ] [ ] [ ] [ ] . the recommendations for chb treatment duration in guidelines (table . ) [ ] [ ] [ ] [ ] ] . . antiviral treatment strategies for hbeag positive patients ( fig. . ). . antiviral treatment strategies for hbeag negative patients (fig. . ). the recommendations for management of lamivudine drug-resistance in guidelines (table . ) [ ] [ ] [ ] [ ] ] . start antiviral treatment to avoid liver transplantation entecavir and tenofovir as the first-line choice, or pegylated interferons aasld pegylated interferon-α, entecavir or tenofovir are preferred; interferon-α/pegylated interferon-α, lamivudine, adefovir, entecavir, tenofovir or telbivudine can be used; adefovir or entecavir for interferon nonresponders or patients with interferon contraindication. csld/csid pegylated interferon-α, entecavir who tenofovir and entecavir as the first-line choice ifn may be considered when hbv dna viral load and genotyping are available, or co-infection with hdv nas with low resistance first; use interferon with caution, start from small doses decompensation nas with low resistance first; interferon is forbidden the recommendations for management of adefovir drug-resistance in guidelines (table . ) [ ] [ ] [ ] [ ] ] . consensus of antiviral treatment in special populations with chronic hepatitis b was put forward by china expert committee of antiviral treatment in special population with chronic hepatitis b in . in china, hbv infection is one of the main causes of liver failure. hbv related liver failure can be further divided into acute liver failure, subacute liver failure, acuteon-chronic liver failure and chronic liver failure. nucleoside analogues can be safely used in the treatment of hbv related liver failure, and improve the prognosis of patients. nucleoside analogues treatment can improve the survival rate and reduce the incidence of complications in hbv related acute and subacute liver failure patients. therefore, nucleoside analogues treatment should be early applied in hbsag positive and hbv dna detectable patients with acute and subacute liver failure, and nucleoside analogues can quickly inhibit virus, including lamivudine, entecavir and tenofovir, are recommended for these patients. drug resistance should be monitored in long-term nucleoside analogues treatment. remaining hbv cannot be completely excluded even when hbsag and hbv dna are undetectable during the treatment, therefore the antiviral treatment should continue to hbsag seroconversion. antiviral treatment is unnecessary for anti-hbs positive patients at first visit [ ] . for patients with hbv related primary liver cancer, liver cancer resection, radiofrequency ablation and interventional therapy all can lead to hbv reactivation and liver function damage aggravation, antiviral treatment is decided depending d on liver compensatory situation. ifn-α can exhibit both anti-virus and anti-cancer effect, delay the tumor recurrence and prolong the median survival period. ifn-α should be the preference if the patients can tolerate ifn-α. if the patients have contraindications to ifn-α, lamivudine, adefovir or entecavir can be chosen depending on hbv dna loads, cirrhosis compensatory situation and kidney function. for patients undergoing hepatic arterial chemotherapy, prophylactic nucleoside analogues treatment should be given before chemotherapy. for patients with advanced liver cancer, portal vein branch thrombosis, but without contraindications to ifn-α, ifn-α treatment can extend survival period [ ] [ ] [ ] [ ] [ ] [ ] [ ] . patients awaiting liver transplantation because of hbv-related end-stage liver disease or liver cancer should be given nucleoside analogues with strong hbv inhibition and low drug-resistance, or nucleotides analogues combination treatment, in order to reduce viral load and prevent graft re-infection. lamivudine and (or) adefovir combination with hbig can be safely and effectively prevent graft re-infection, and reduce the re-infection rate to below %. hbv-associated liver transplant patients require lifelong treatment of antiviral drugs for the prevention of hepatitis b recurrence. hbsag-negative patients receiving anti-hbs positive donor liver should also receive long-term treatment of lamivudine or preventive treatment of hbig [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . according to who, elderly chb patients refers to chb patients aged ≥ years old. generally speaking, according to current guidelines, ≥ years of age is not a contraindication to antiviral therapy, so their treatment can refer to the relevant guidelines, but their desire, risks and benefits of treatment should be comprehensive evaluated. especially for the patients using ifn-α, the expected survival and liver function compensatory situation, possible side effects, underlying hypertension, diabetes, coronary heart disease, and the improvement of liver function should be comprehensive evaluated. additionally, the treatment response, side effects, blood sugar, kidney function and occurrence of liver cancer should be closely monitored during and after treatment [ ] . children chb patients are usually in the immune tolerance phase of hbv infection, hence they could not receive antiviral treatment, but should be closely followed up. fda has approved of ifn-α ( - years of age), lamivudine ( - years of age), and adefovir ( - years of age) for use in children. recommended ifn-α dose is miu/m of body surface area three times per week, and the maximum dose is miu/m total body surface area. it is showed that ifn-α effects the same in children as in adult. recommended lamivudine dose is mg/(kg day) with a maximum dose of mg/day. recommended adefovir dosage and usage are the same with adult patients [ , [ ] [ ] [ ] [ ] [ ] [ ] . mother to child transmission is the main route of transmission of hbv infection in china, in order to block the transmission of hbv, antiviral therapy in pregnant chb patients is very important. firstly, antiviral treatment should be completed before pregnancy as possible. it is recommended to consider pregnant months later after interferon or nucleoside analogs treatment. secondly, unwanted pregnancies should be terminated during ifn-α treatment because of its pregnancy toxicity. pregnancy safety of nucleosides analogs has not been proved by any clinical trials, but a large number of studies have shown that lamivudine and tenofovir were safe, so the treatment with lamivudine could continue under the premise of full communication with patients. telbivudine, adefovir or entecavir treatment may switch to lamivudine treatment. pregnant chb patients with slight alt elevation, should be monitored closely or given liver protection and symptomatic treatment, and given antiviral treatment after delivery. pregnant chb patients with poor liver function could be given lamivudine treatment after full consultations with patients and signed informed consent forms. serum hbv dna load in pregnant chb patients is the key factors of mother to child transmission, and effective antiviral treatment can significantly reduce the transmission incidence. according to the findings, lamivudine or telbivudine treatment could start in [ ] [ ] [ ] [ ] [ ] [ ] [ ] week of pregnancy to block transmission, and the withdrawal can refer to the patients with immunosuppressive agents or chemotherapy. in addition, women with husbands receiving ifn-α antiviral treatment, should only consider pregnancy months later after withdrawal. there is no evidence at present that nucleosides analogs have a negative impact on sperm and embryo, pregnancy could be taken into account under the premise of full communication with patient [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . hbv and hcv co-infection increases the incidence of severe hepatitis, liver cirrhosis and liver cancer. when co-infection, hcv may inhibit hbv generally, and different treatment should be given depending on hbv and hcv viral load and alt level (table . ) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . hbv and hiv co-infection increases hbv dna load, reduces spontaneous hbeag seroconversion rate, aggravates liver damage and increases mortality in patients with liver disease. anti-hbv regimens should combine with highly active antiretroviral therapy (haart). if anti-hbv and hiv treatment is needed, anti-hbv drugs such as tenofovir + lamivudine or tenofovir + truvada could be used in haart. if haart only contains lamivudine as anti-hbv drugs, hbv drug resistance should be closely monitored and treatment should be adjusted in time. if haart is unnecessary, adefovir, telbivudine and ifn-α can be used for anti-hbv treatment. because lamivudine, tenofovir and entecavir monotherapy induced risk of hiv drug-resistance, lamivudine, tenofovir and entecavir treatment are not recommend to these patients [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . anti-hbv treatment is critical for hepatitis b virus associated glomerulonephritis (hbv-ag). patient diagnosed with hbv-ag must start anti-viral therapy as long as hbv dna is detectable. a number of studies show that kidney disease alleviated significantly after lamivudine treatment, along with hbv dna decline and hbeag clearance. adefovir has been shown to increase serum creatinine level in some patients in clinical trials, therefore should be carefully chosen. there is not enough clinical evidence of telbivudine and entecavir treatment for hbv-ag. there is no consensus of nucleoside analogue treatment for hbv-ag currently. safety and efficacy of ifn-α and pegylated ifn-α treatment for hbv-ag have not been proved [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . elevation of hbv dna can be observed in - % of the hbsag-positive patients receiving immunosuppressive agents or cytotoxic therapy, including corticosteroids, anti-cd and anti-tnf. some patients suffer from transaminase elevation and jaundice, and severe patients develop to fulminant liver failure even death. nucleoside analogues prophylactic treatment can decrease hbv reactivation. regardless of the hbv dna level, hbsag carriers should receive nucleoside analogues antiviral treatment - weeks before immunosuppressive or cytotoxic therapy. antiviral drugs inhibit hbv dna rapid, such as lamivudine, telbivudine and entecavir, are preferred for prophylaxis. most patients cannot tolerate the recurrent aggravations induced by drug-resistance. prophylaxis decision should be made depending on baseline hbv dna load and duration of immunosuppressive therapy or cytotoxic therapy. antiviral drugs with low resistance are recommended if prophylaxis will last more than months. treatment duration: if baseline hbv dna ≤ copies/ml, treatment should be continued to months after immunosuppressive therapy or cytotoxic therapy completed; if baseline hbv dna > copies/ ml, treatment should be continued to the referring withdrawal standard of ordinary chb patients. however, ifn-α is not recommended because of the bone marrow suppression. there is no consensus of prophylactic antiviral treatment for hbsag-negative and anti-hbc-positive patients during the immunosuppressive or cytotoxic therapy. serum hbv marker and hbv dna level should be monitored [ , [ ] [ ] [ ] [ ] [ ] [ ] . hbv infection itself is not correlated with thyroid dysfunction. ifn-α can aggravate underlying autoimmune thyroid disease or induce emerging thyroid disease in some patients because of its immunomodulatory activity and direct thyroid toxicity. uncontrolled thyroid dysfunction contraindicates ifn-α antiviral therapy. patients with previous thyroid dysfunction or high titer of thyroid autoantibody (tpo-ab > iu/ml) before treatment should be monitored for thyroid function during ifn-α antiviral treatment. patients with emerging thyroid dysfunction during treatment should terminate antiviral treatment. majority of thyroid dysfunction emerged during treatment in patients without history of thyroid dysfunction is reversible, and can restore after ifn-α withdrawal [ ] [ ] [ ] [ ] . resistance to nucleoside analogues is a serious problem in chb treatment, which does make the long-term treatment strategies become a difficulty. a variety of factors may be associated with resistance to nucleoside analogues, including nucleoside analogue type, hbv dna load at initial therapy, liver fibrosis/ cirrhosis, and previous nucleoside analogues treatment. in addition, male gender, high body mass index and alcohol abuse are also the risk factors of resistance mutations in antiviral therapy. however, a growing number of studies suggest that early virological response is an important indicator to predict drug resistance [ , ] . select nucleoside analogues treatment indications reasonably. nucleoside analogues treatment is not recommended for the hbv infected patients in immune tolerant phase or non-active phase, especially those who are younger, if they do not receive immunosuppressive therapy or chemotherapy. for the chb patients with first flare, especially those who are younger, nucleoside analogues should be given with caution after fully analysis of inductions. select nucleoside analogues treatment strategies reasonably. treatment should be consulted the guideline on prevention and treatment of chronic hepatitis b in china. for patients with antiviral therapy indications, drugs with strong antiviral activity and low resistance are recommended if nucleoside analogues are chosen. the previous antiretroviral therapy, including drugs, treatment response and resistance mutations, should be understood in order to avoid nucleoside analogues with cross-resistance. furthermore, sequential monotherapy treatment should be avoided for multi-drug resistance. improve patients' compliance. prescribed time and adequate medication should be repeatedly emphasized to the patients during nucleoside analogues treatment. according to the clinical trial, more than % of cases with virologic breakthrough are resulting from poor compliance. gradual dose reduction will significantly increase the risk of resistance and is forbidden. regularly detect hbv dna load and genotypic resistance and timely adjust treatment. hbv dna load is the most important indicators of drug resistance in nucleoside analogue antiviral therapy. hbv dna levels should be monitored regularly during treatment. numerous clinical trial data show that early virological response is an important predictor of drug-resistance. therefore, aasld and easl guidelines both recommend adjusting treatment plan based on evr to improve the efficacy and reduce the incidence of drug resistance [ , [ ] [ ] [ ] [ ] . patients with normal alt and mild inflammation or fibrosis (<g s ) before treatment can stop the anti-viral treatment, and need to be monitored closely for antiretroviral therapy again promptly if aura symptoms happened. most patients with nucleoside analogues resistant, especially the decompensated cirrhotic patients, need rescue therapy timely. virologic breakthrough usually precede biochemical breakthrough, and rescue therapy before biochemical breakthrough can avoid sudden hepatitis flare and aggravation. management of lamivudine and adefovir resistance refers to guide recommendations above. add adefovir or tenofovir (the latter has not been approved by sfda, and the safety of combination of entecavir and tenofovir needs further study), or switch to ifn-α or pegylated ifn-α for patients with entecavir resistance. add adefovir or tenofovir for patients with telbivudine resistance. tenofovir resistance has not detected, if happened, entecavir, telbivudine, lamivudine or emtricitabine can be added theoretically. for lamivudine and adefovir multidrug resistance, switch to emtricitabine or combination of entecavir and tenofovir (but not been approved by sfda), or switch to ifn-α or pegylated ifn-α treatment [ , , ] . definition of refractory chb: chb patients with treatment failure or poor efficacy and chb patients have been confirmed poor efficacy by evidence-based medicine, using anti-hbv drugs, including nucleoside analogues and (or) interferon, under the existing guidelines or recommendations for various reasons and (or) factors. refractory chb patients include: ( ) patients with primary non-response, partial virological response and (or) virologic breakthrough, drug-resistant hbv mutations and clinical drug-resistant. ( ) patients with serological (hbeag) no response or partial response, namely hbeag-positive patients without hbeag loss or hbeag seroconversion after initial treatment of more than year. ( ) high baseline hbv viral load: hbeag-positive patients with hbv dna > copies/ml, hbeagnegative patients with hbv dna > copies/ml. ( ) cirrhotic (compensated and decompensated) patients and aechb patients. ( ) other patients with hbv reactivation after transplantation, immunosuppressive therapy, combination of other viral infections such as hcv and hiv, combination of metabolic/autoimmune diseases such as insulin resistance, hyperlipidemia and (or) non-alcoholic steatohepatitis and fiber cholestatic hepatitis. the present guidelines for the treatment of refractory chb involve virus strains resistant mutations and virologic breakthrough, partial virological response, cirrhosis, virus reactivation after liver transplantation, receiving immunosuppressive therapy, and combination of other viral infections (hcv and hiv). majority of the treatment recommendations are based on expert consensus, clinical experience, or clinical studies of small sample. however, for patients with serological no-response or partial response, high baseline hbv viral load, aechb, insulin resistance and non-alcoholic steatohepatitis, and fiber cholestatic hepatitis, there is no clear guidance. currently ongoing hepatitis b related clinical researches more concentrated in drug selection and optimization of treatment-naïve patients, and only a small portion and small-scale studies involve refractory chb. anti-hbv treatment was not taken into account in aecdhb in the past. it is thought that immune pathological damage is the key in the development of aechb, and hbv is just a promoter. the role of hepatitis virus in development of aechb has not been paid enough attention. with the study of aechb mechanism deep going, more and more scholars have realized that constant hbv replication induced hyperactive immune response is a major factor in exacerbation. when hbv induces hypersensitivity, a large amount of immune complexes generate and activate the immune network, leading to serious hepatocyte damage through the following mechanisms: ( ) th cells activate, release interleukin- (il- ), and mediate cytotoxicity of cytotoxic t cell (ctl), macrophages and natural killer (nk) cells; ( ) macrophages activated by hbv and endotoxin release cytokines (including tumor necrosis factor tnf-α and fgl ), inducing direct hepatocyte damage or secondary damage by microcirculation disturbance; ( ) fas ligands (fas-l) express increasingly on the surface of infected hepatocytes, conjunct to fas expressed by ctl, and induced apoptosis. antiviral therapy can quickly suppress hbv replication, reduce intercellular viral spread, and inhibit the membrane target antigen expression, so as to inhibit ctl attack and relieve hepatocyte injury and necrosis. antiviral treatment at early stage in disease is the pivot to terminate intense cellular and humoral immunity. therefore, it is advocated to start antiviral treatment for severe hepatitis patients with hbv replication. some scholars believe that viral load is an important indicator for aechb, although it does not directly related to liver damage. hbeag and hbv dna seroclearance is important for remission and positive for improving cure rate of severe hepatitis. therefore, early effective anti-viral therapy can reduce the viral load, suppress virus generation by infected hepatocytes, decrease infection of newborn hepatocytes, alleviate liver inflammation and is beneficial to liver recovery. antiviral therapy has become an effective treatment for aechb [ ] . whether antiviral therapy increase or alleviate immune response in the advanced stage of severe hepatitis had been controversial. however, recent studies reach a consensus that antiviral treatment can slow disease progression and improve recent survival rate. in most early studies, lamivudine failed to improve liver function and survival in aechb compared to placebo [ ] [ ] [ ] [ ] [ ] . however, a study from taiwan showed that lamivudine could improve survival in patients with baseline total bilirubin less than mmol/l ( mg/dl) [ ] . wong and chan reported that antiviral therapy in aechb cannot improve resent survival, but could prevent further deterioration [ ] . in early stage, there is intense immune response, high viral load, severe inflammation, and ongoing immune liver damage. viral load can affect the progression and prognosis. patients with hbv dna positive have a relative poor prognosis because of more virus antigen on hepatocyte surface activating immune injury. in middle-advanced stage, the impact of hbv dna on progression and prognosis would weaken because immune response has alleviated after self-regulation. therefore, hbv dna level in early severe chb is a significant indicator for prognosis, and antiviral therapy is essential. in middle-advanced stage, hbv dna level has little influence on the prognosis, and antiviral therapy is meaningful to prevent recurrence. in addition, lamivudine can obtain sustained virologic response, but long-term treatment may induce viral resistance and virologic breakthrough, which reduce the efficacy of antiviral therapy [ ] . most recent studies suggest that antiviral therapy for hbv dna positive patients in early severe hepatitis can postpone disease progression and improve recent survival rate [ ] . ma et al. analyzed cases of hbv-aclf retrospectively. patients added entecavir on the basis of standard medical treatment, another patients only received standard medical treatment without nucleoside analogues. the -month and -month survival rate of entecavir-treated patients were . % ( / ) and . % ( / ) respectively, and significantly higher than those of control with . % ( / ) and . % ( / ). entecavir-treated patients get a significant improved meld scores compared to control post treatment, suggesting that entecavir can postpone progression of hbv-aclf and improve recent survival [ ] . lin et al. investigated hbv-aclf cases with entecavir treatment, and concluded that entecavir can significantly increase the survival rate [ ] . hu et al. investigated the efficacy of lamivudine and entecavir on hbv-aclf. after -month treatment, survival rates are similar, but clinical improvement rate in lamivudine and entecavir group were significantly higher than basic treatment group. after -month treatment, the cumulative survival rates of lamivudine and entecavir group were . %, . % respectively and significantly higher than the basic treatment group ( %). in patients with baseline hbv dna > iu/ml, cumulative survival rate in antiviral treatment group were higher than basic treatment group. patients with pretreatment meld scores > had a lower cumulative survival rate than patients with meld scores ≤ , but obtained better response to antiviral therapy. it also showed that there was no significant difference in efficacy using entecavir and lamivudine treatment for hbv-aclf [ ] . tillmann et al. summarized cases of aechb with lamivudine treatment. it suggested that patients with lamivudine treatment had an overall survival rate without transplant of . %, but patients without lamivudine only . % [ ] . garg et al. found that tenofovir can significantly reduce hbv dna levels in hbv-aclf, improve ctp and meld score, and decrease mortality. hbv dna decrease of more than lg copies/ml after -week treatment is a predictor of good prognosis. liver failure is a common syndrome, and its incidence is increasing with the use of alcohol and growing epidemic of obesity and diabetes. liver failure is defined as inability of the liver to perform its normal, metabolic, excretory and biotransformation functions by chinese medical association [ ] . its manifestation includes coagulopathy, jaundice, hepatic encephalopathy (he) and ascites. liver failure can be divided into four classes: acute liver failure (alf), sub-acute liver failure (salf), acute on chronic liver failure (aclf), and chronic liver failure (clf) according to histopathological characteristics and the progression of disease. alf is a syndrome with liver function deterioration rapidly accompanied with a grade ii or higher he within weeks illness duration. salf onset is slowly than alf that symptoms occur within - weeks. liver failure occurred with known or unknown chronic liver disease refer to aclf. clf is defined as progressive deterioration and decompensation of liver function in patients with liver cirrhosis, mainly manifested with complications of portal hypertension. based on the severity of clinical manifestations, sub-acute and acute-on-chronic liver failure can be divided into early, middle, and end stages. early stage has severe fatigue and gastrointestinal symptoms, total bilirubin level is more than μmol/l or daily increase of total bilirubin is more than μmol/l and prothrombin activity (pta) is less than %. middle stage has stage ii he and/or ascites and pta ≤ %. end stage has refractory complications such as stage iii or higher he, hepatorenal syndrome, massive hemorrhage of the upper alimentary tract, severe infection and refractory fluid and electrolyte imbalance, pta ≤ %. otherwise, asian pacific association for the study of the liver (apasl) define aclf as a severe liver injury, leading to coagulation abnormality usually with an inr ≥ . , and any degree of mental alteration (he) in a patient with pre-existing liver disease and with an illness duration less than weeks [ ] . etiology, epidemic and precipitating factors of liver failure are different between western countries and china. non-infection factors such as alcohol and drug induced liver failure are predominant in western countries [ ] . however, hepatitis b virus (hbv) infection is the main reason to induce liver failure [ ] . we retrospective analysis the etiology of cases of liver failure came from provinces in china northern area from to and found hbv infection induced liver failure was about . % [ ] . so far, liver transplantation is the most effective way to treat hbv induced liver failure. but due to the cost and shortage of organ donor, liver transplantation can't be used widely. comprehensive treatment including supportive therapy, antiviral therapy and immunoregulation therapy is the main way to treat hbv related liver failure in china. but mortality of liver failure is high and total curative rate is only about . % because of the complicate pathophysiology in liver failure [ ] . the precise mechanism underlying the liver injury caused by hbv-aclf and the factors contributing to the progression of liver failure remain unknown. generally, virus factors, host factors, and their interaction determine the outcome of aclf. hbv dna replication is one of the key factors causing the progression from liver damage to liver failure. the hbv dna level is closely associated with the risk of hepatocellular carcinoma development, and hbv dna suppression significantly improves the prognosis of cirrhosis. current clinical guidelines advocate oral antiviral treatment in decompensated cirrhosis and sustained hbv dna suppression to reduce sequelae [ , , , ] . (liver failure) eight different hbv genotypes (a-h) have been described based on their genomic heterogeneity. many studies showed that the severity of hbv infection correlated with hbv genetype. in the asia-pacific countries, genotype b and c hbv are predominant with genotype c hbv associated with delayed hbeag seroconversion and more aggressive disease activity as compared to other hbv genotypes [ ] . patients infected with hbv genetype c have high hbv dna level and high hbeag positive rate than people infected with other hbv genetypes. these patients have low response to anti-viral therapy and progress to liver failure, particular in patients infected with genetype c . zhang et al. analyses hepatitis b patients and found that the most common hbv genetype was b and c in chronic hepatitis b patients [ ] . patients infected with genetype c was more predisposed to chronic and cirrhosis and hepatic carcinoma. genetype b and c had no influence on illness progression in acute and mild patients, but hbeag positive rate and hbv dna level were high in patients infected with genetype c. further studies showed that hbv bcp/pre c mutation was associated with hbv genetype. a t and c mutation probably associated with aclf. c was an independent prognostic factor in aclf patients [ ] . hbv dna polymerase lack proof function which can lead to viral mutation during replication. in addition, hbv exist in many quasispecies. under select press, quasispecies variance can cause the change of hbv replication, pathogenicity, antigen epitope which lead to influence immune response and viral resistance. over immune response can result in severe liver injures. hbv pro core/core, pre s, p gene and multi gene mutation were found to be associated with liver failure [ ] . one of the functions of hbeag is to induce immune tolerance. in the absence of hbeag, patients harboring pre-core mutant hbv may have a more rigorous immunological response. chronic infection with pre-core mutants has been often associated with multiple flares with interspersed asymptomatic periods. mutations at the basal core promoter (bcp) regions lead to decreased hbeag synthesis, active liver histology, and increased viral replication. these exacerbations are seen to lead to fulminant hepatic failure [ , , ] . we investigated hbv bcp a t/g a double mutation in liver failure patients [ ] . longitudinal study showed that nucleotide mutation sites were occurred more in hbv-aclf than cirrhosis patients among which nt , nt , nt and nt were associated with hbv-aclf. t mutation was found exist more in genetype b than genetype c ( . % vs . %), a/g mutation is found frequently in hbeag negative patient than positive patients ( % vs . %). these indicated that pre core/bcp mutation associated with the occurrence of liver failure and influence patients outcome. hbx is a multifunction regulatory protein which can influence gene transcription, activate signal transduction, enhance viral replication, accelerate protein degradation and regulate cell apoptosis. hbx can participate in the process of hbv pre s and bcp/pre core mutation liver failure caused by chronic hbv infection (i.e., chronic severe hepatitis b) is a common life-threatening disease in china. the pathogenesis of chronic severe hepatitis b is complex and is currently not completely understood. however, one widely accepted mechanism is the induction of cellular immune responses mediated primarily by cytotoxic t lymphocytes (ctls) and delayed-type hypersensitivity t cells. these immune responses are induced by viral protein antigens expressed in the target cell surface due to the active replication of hbv and eventually result in large areas of liver cell necrosis [ ] . the specific mechanisms may involve several factors [ ] . using a hybridization assay for hbv dna and a conventional enzyme immunoassay to measure the hbeag level, an earlier study showed significant parallel increases in serum hbeag and hbv dna levels and accumulation of intracellular viral proteins several weeks before the hepatitis flare. in addition, there was a subsequent increase in anti-hbe production and hbeag/anti-hbe immune complex formation, implicating the important role of the immune response to hbv in initiating the hepatitis flare [ ] . immunohistologic studies during the hepatitis flares have shown cd + t cells in the mononuclear cell infiltrates, strong membranous expression of human leukocyte antigen class i (hla-i), and cytoplasmic or membranous/submembranous hepatitis b core antigen (hbcag) expression [ , ] . some findings suggest that hepatitis b flares are the results of dynamic changes of the innate and adaptive immune responses with hla-i restricted, ctl mediated immune cytolysis of hbv antigen(s) expressing hepatocytes and its downstream apoptotic mechanisms [ , ] . the activated th cells release interleukin- and excite cytotoxic effects of ctls, macrophages, natural killer cells, and lymphokines [ ] . macrophages, activated by hbv and endotoxins, release various cytokines mainly with tumor necrosis factor (tnf)-a, which directly damage liver cells and also result in secondary injury of liver cells through disturbances in microcirculation. fas ligands (fas l) are highly expressed in the surface of hbv-infected liver cells and combine with fas expressed by ctls, together inducing hepatocellular apoptosis. therefore, antiviral therapy early in chronic severe hepatitis b is beneficial for suppressing intense cellular immune responses induced by hbv replication. if hbv replication is suppressed rapidly, the immune pathological responses of chronic severe hepatitis b may be reduced, thus effectively blocking the disease progression. the administration of anti-hbv therapy in chronic severe hepatitis b (acute-or subacute-on-chronic liver failure) is still undergoing research, and limited data are presently available. so far, anti hbv treatment drugs include interferon and oral antiviral drugs. interferon application is contraindicated in the treatment of liver failure due to its limited anti-viral efficacy, significant adverse drug effects, and induction of immune enhancement, which can further result in aggravation of liver damage. oral anti hbv drugs including adefovir dipivoxil (adv), lamivudine (lam), telbivudine (tbv), entecavir (etv) and tenofovir (tdf) have few adverse effects. antiviral therapy may have the advantage of shortening the replication and thereby reduce disease duration without the side effects of interferon. therefore, many studies have been carried out to find the efficacy of oral antiviral drugs on patients with liver failure. the effect of antiviral therapy in hbv related acute liver failure is controversial. some studies reported that antiviral therapy can't improve outcome because hbvdna can be eradicated spontaneously due to enhanced immune response in liver failure patients. but hbv infection is the initial factor to induce over immune response that can lead to liver damage. early treatment with antiviral therapy can inhibit hbv dna replication and attenuate immune reaction which can reduce liver damage, hepatocyte apoptosis and necrosis. tillmann et al. [ ] reported that acute hbv related liver failure patients were treated with lam and patients were treated with placebo. encephalopathy occurred in ( . %) and ( . %) patients, respectively (p = . ). it demonstrated that early use of antiviral drugs can reduce the rate of encephalopathy and mortality. in addition, a prospective study about etv on acute liver failure patients demonstrated that etv can reduce hbv dna load significantly and patients achieved anti hbsag conversion [ ] . therefore, monitor closely and early use antiviral drugs to reduce hepatocyte apoptosis and necrosis on patients with hbv related acute liver failure are very important. the objective of antiviral treatment for hbv-aclf is to reduce viral load at an appreciably high rate, thereby promoting reduction in hepatocyte cell death and improved survival outcomes by prevention of decompensation related multiorgan complications in this group of severely ill patients. several studies have delineated the fact that low pretreatment hbv dna load and a rapid decrement in viral load improves outcomes in hbv-aclf [ ] , whereas a study from india reported that a log decrease in hbv dna at week improved survival benefit in patients with hbv-aclf [ ] . antiviral therapy also promotes chances of stabilization to liver transplant time and improves transplant outcomes. studies have debated on the issue of antiviral therapy related improvement in the long term [ ] . lam decreased viral load significantly, but did not result in significant biochemical or clinical improvement compared with those patients given placebo. mortality of patients receiving nucleoside analog therapy was significantly lower than the placebo group, which indicated that antiviral therapy improved prognosis of patients with hbv-aclf if implemented as soon as possible [ ] . even in the age of effective antiviral therapy, early transfer to a transplantation facility should be considered before managing conservatively by medical means. the apasl consensus guidelines on aclf describe the value of early and prompt institution of antiviral therapy in hbv-aclf. hbv dna levels are now not an indication for commencement of antivirals in hbv-aclf reactivation, as earlier starting of such therapy, even prophylactic, has been found to have great survival benefit in the long run. from to , early and middle stage hbv-aclf patients in our hospital were recruited in a prospective study to evaluate the efficacy and safety of lam and etv [ ] . no antiviral therapy was used in control group. this study showed that lam or etv can reduce and months mortality, improve survival rate on patients with aclf-hbv. three and months cumulative survival rate of lam and etv therapy were . % and . %, % and . %, respectively, which was higher than control group ( % and %, p = . and . ). no significant different on survival rate between lam and etv (p = . ). this study also showed that meld score was an effective prognostic predict factor on patients with aclf-hbv. patients with meld score less than had a good outcome. another study also demonstrated that for hbeag-negative patients with hbv-aclf, when entecavir was added to comprehensive therapy, a meld score ≥ predicted very poor prognosis due to fatal liver failure [ ] . hu et al. made a survival analysis on hbv-aclf patients and results indicated that nucleoside analog application in early and middle stage hbv-aclf can improve survival rate and prolong patients' life. median survival time was . and . months in patients treated with and without antiviral therapy. another study indicated that patients with meld score - and hbv dna load decrease log had a better outcome than patients with hbv dna load decrease less than log within weeks treatment [ ] . in addition, mortality had no relationship with hbv dna load if meld score > . xiao et al. also demonstrated that nucleoside analog treatment is an independent prognosis predict factor in hbv induced liver failure. lam and etv had no difference. although antiviral therapy can improve patients survival rate, treatment with and without nucleoside analog had no difference in late stage liver failure patients. currently, liver transplantation is the ultimate therapeutic option for decompensated cirrhosis patients. however, liver transplantation can't be used in all patients because of the shortage of donor organs. therefore, the aim to treat decompensated cirrhosis is to decrease the occurrence of disease associated complications and the liver associated mortality rate [ ] . the natural history of decompensated hbv-related cirrhosis is affected by high hbv replication which may exist in some decompensated hbv-related cirrhosis patients [ ] . hepatic necroinflammation and fibrosis progression are improved after sustained viral suppression is achieved which can prevent decompensation in cirrhosis [ ] . oral nas treatment are strongly recommended in most clinical guidelines for decompensated hbv-related cirrhosis patients no matter what hbv dna replication level is [ , ] . yao et al. [ ] found that ctp scores was reduced more than three points in % lam treated patients with severely decompensated cirrhosis. furthermore, ctp scores was decreased to less than seven point in % of these patients, and their statuses on the united network of organ sharing waiting list changed to inactive. a randomized controlled trial in asia demonstrated less liver-related morbidity in the lam-treated patients with hbv associated advanced compensated cirrhosis compared to the untreated controls because of the reduced incidence of hepatic decompensation and lower risk of hcc. increased ctp scores were noted in . % of the patients in the lam group compared to . % of the patients in the placebo group (p = . ) [ ] . fontana et al. showed most deaths caused by liver related complications occurred within the first months in patients with decompensated hbv-related cirrhosis treated with lam. pretreatment high hbv dna replication level, serum bilirubin and creatinine were associated with -months mortality rates significantly [ ] . this finding indicates early antiviral therapy might be important. lamivudine lam is a nucleoside analogue that inhibits hbv dna synthesis which was the first oral drug to treat chronic hbv infection in . its mechanism is to compete with nature cytidine to inhibit hbv polymerase, then terminate hbv replication. chan et al. [ ] studied the effect of lamivudine in treatment of severe hepatitis-b-related acute exacerbations leading to aclf in patients as against controls. it was concluded that lamivudine had no survival benefit compared with conventional treatment in severe aggravations of chronic hepatitis b and that liver transplantation should be considered in these patients with thrombocytopenia and high bilirubin. however, another meta study [ ] analysis studies to evaluate the short-term effect of lamivudine (lmv) treatment for severe chronic hepatitis b. they found that the survival rates and pta of the test group were distinctively higher than those of the control group at weeks , , and of the treatment course. the hbv-dna negative change rate was distinctively higher throughout the weeks of lmv treatment. for patients who started lmv treatment in the middle stage, the mortality rate of the test group was lower. they concluded that lmv decreased hbv-dna levels in the serum, improved liver function in patients, and enhanced survival rate during the early and medium stages of severe chronic hepatitis b. tsubota et al. [ ] studied patients with spontaneous severe acute exacerbation treated with lamivudine, and found that lamivudine monotherapy did not prevent hepatic failure deterioration significantly, but it resulted in long-term benefits. baseline serum bilirubin, pre-existing cirrhosis and baseline pt were independent determinants of prognosis. adv is an acyclic nucleotide analogue of adenosine monophosphate. use of adefovir for hbv-aclf has been rare. in two case reports, adefovir dipivoxil failed to salvage cases of lamivudine resistance after jaundice and liver failure developed. a lower antiviral potency and the potential risk of nephrotoxicity of adv remain a problem for routine use as a first-line treatment. it is hence not advisable to use adefovir as a first-line drug in the treatment of acute severe exacerbation. but considered to the high viral resistant to lam, adv plus lam can reduce the incidence of lam resistance. the combination of adv and lam can be used in liver failure patients. patients with decompensated cirrhosis caused by lam-resistant hbv were treated with adv and hbv dna level become undetectable occurred in % of patients and ctp scores were improved [ ] . but another study focused on long time outcome found that resistance to adv was % and renal toxicity was confirmed in % of patients [ ] . etv is a cyclopentyl guanosine analogue that can inhibit hbv polymerase's function. compared with lam and adv, etv has a more potent activity against wild type hbv [ , ] . etv has been studied in in cirrhotic patients. one korea study showed ctp and meld scores were improved in patients treated with etv for month. the -year cumulative incidence of hcc was . %, and the cumulative death rate was % [ ] . many studies have been carried out to compared the efficacy of etv and lam to treat hbv related aclf. the short-term efficacy of entecavir vs lamivudine was similar and the degree of pretreatment liver failure significantly affected the outcome of treatment [ , ] . in summary, the pros and cons of lam vs etv in decompensated or severe acute exacerbation of chronic hepatitis b were etv being more effective in promoting faster viral load decrement. also the available clinical evidence suggests that clinicians treating chronic hepatitis b patients with acute hbv exacerbation or decompensated liver disease should use the most potent nucleoside analogs available [ ] . tbv is a synthetic thymidine nucleoside analogue that has potent antiviral activity against hbv. one study investigated the short-term efficacy and safety of tbv therapy in liver failure patients caused by chronic hepatitis b virus (hbv) infection [ ] . in this study, patients were treated with tbv, and the other patients were treated with lam. hbv dna levels in the tbv group fell to the lower limit of detection after the fifth week, which was more rapid than in the lam group. in addition, the total bilirubin and prothrombin time activity of the patients with tbv treatment showed a more significant improvement as compared to the patients treated with lam from the start of the fifth week. they concluded that tbv treatment is superior to lam treatment in improving the condition of patients with liver failure as a result of chronic hbv infection in the short term. but viral resistance is also a major concern. a study in decompensated cirrhosis patients with hbv infection showed genotypic resistance was developed in % of the tbv patients during the -year period [ ] . therefore, tbv used as a first-line treatment has limitations in patients with hbv-related decompensated cirrhosis. tdf is an acyclic nucleotide analogue with potent inhibition of hbv polymerase/ reverse transcriptase. in a seminal study by garg et al. [ ] , consecutive patients with aclf due to spontaneous reactivation of chronic hepatitis b were randomized to receive either tdf or placebo. the primary endpoint was survival at month. more than log reduction in hbv dna levels at weeks was associated with survival rate. the authors concluded that tdf had potent activity to reduce hbv-dna levels, improve ctp and meld scores, and reduce mortality in patients with severe spontaneous reactivation of chronic hepatitis b presenting as aclf, and that reduction in hbv-dna levels at week should be considered a desirable goal and a good predictor of survival. until now, no studies have reported viral resistance to tdf. therefore, tdf and etv can be considered to be the first-line therapy because their potent activity against hbv replication and high resistance barrier. in addition, the data about tdf to treat hbv-aclf is limited. thus, larger prospective and multicenter studies are encouraged to evaluate further the effect of tdf on shortterm mortality of patients with hbv associated aclf. there still lack multicenter, larger samples, prospective and randomized clinical trial to test the efficacy of antiviral therapy. but it is likely that antiviral therapy with nucleos(t)ide can improve patients survival rate in patients with hbv-related liver failure [ ] . therefore, antiviral therapy is reasonable to try in patients with high hbv dna replication. in recent year, lam and other nucleoside analogue have been used in severe hepatitis b wildly. the efficacy of antiviral therapy is correlated with the time to start. many clinical trials demonstrated that early antiviral treatment is important for patients with liver failure [ ] . antiviral therapy used in early and middle stage liver failure can improve patients survival rate. in patients with late stage, such as serum total bilirubin is more than μmol/l, it's rare that patients can get benefit from antiviral therapy. therefore, early use antiviral therapy can reduce viral load, inhibit viral replication, reduce new hepatocyte be infected by hbv again and alleviate liver inflammation and all of that are benefit to hepatocyte regeneration. the criteria for antiviral therapy dependent on hbv dna level in serum. some people suggested antiviral therapy should be used in patients with hbeag positive and hbv dna > copies/ml or hbeag negative and hbv dna > copies/ ml. however, take into account over immune response in liver failure patients that associated with viral eradication, even patients with hbsag positive and hbv dna undetectable should be considered for antiviral therapy. in our opinion, antiviral therapy should be used in all liver failure patients with hbv replication immediately. the feature of oral antiviral drugs should be considered when used in liver failure patients. side effects of nucleos(t)ide, including elevate ck, myopathies and lactate acidosis, can occurred during treatment. we observed the change and effect of elevated ck during the treatment of etv and lam in liver failure patients. we found that no different of ck elevation in both drugs. ck elevation was consistence with infection and hepatic renal syndrome. but long term safety of nas has not been confirmed in liver failure patients. antiviral therapy should be used for life because the nas treatment can't eradicate cccdna in the hepatocyte. some cirrhotic patients developed viral resistance to lam during long-term lam therapy which cause virologic response loss [ , ] . in antiviral treatment naïve patient, the lam resistance rate is up to % after years of continuous therapy and the annual resistance rate is up to % [ ] , compared with that etv resistance rate is less than . % in patients treated with etv at years [ ] . therefore, lam should be used with careful monitoring for the development of resistance. and etv or tdf rather than lam is recommended to be used as the first-line therapy in patients with hbv infection because of its high genetic barrier and potent activity against hbv replication [ ] . multiorgan failure occurred rapidly frequently in liver failure patients. although some patients can recover treated by comprehensive and antiviral therapy, all patients should be evaluated for liver transplantation. it's challenge to study the effect of na, how and when to use na and how to treat viral resistance to na in liver failure patients. further studies are needed to evaluate patient outcomes after antiviral therapy with na in liver failure patients. for patients with chronic hepatitis b (chb), hbv continue replication caused progression of liver disease, eventually lead to cirrhosis or hepatocellular carcinoma. the principle of treatment for hbv-related cirrhosis is a comprehensive treatment, including antiviral, anti-inflammatory, hepatoprotective treatment and anti-fibrosis, which antiviral treatment is the key point. since , listed lamivudine, there were many new ideas and concepts on treatment for hbv-related cirrhosis, liver failure. a large number of evidence-based medicine evidence suggests that sustained suppression of hbv by anti-viral treatment can reduce liver inflammation and fibrosis, reduce or delay disease progression, and ultimately improve survival rates and quality of life. internationally there are many chb antiviral therapy management guidelines, but antiviral therapy for hbv-related cirrhosis was still a hot and difficult issue in clinic. this article reviews some of the new progress and new perspectives of antiviral therapy liver cirrhosis based on the recent research. management is guided by recommendations from the american association for the society of liver disease (aasld) [ ] , european association for the study of liver (easl) [ ] , asian pacific association for the study of liver (apasl) [ ] , and society of hepatology and infectious diseases of chinese medical association (cma) have formed a consensus [ ] . guideline edition issued by the chinese medical association clearly stated that the overall goal of treatment is to chb was to suppress hbv as long as possible, relieve inflammation or necrosis of liver cells and liver fibrosis, prevent the progression of cirrhosis, reduce and prevent the occurrence of hepatic decompensation, hcc and its complications, thereby improving the quality of life and survival rate. for patients with cirrhosis, whether compensated or non-compensated, antiviral treatment can delay or reduce hepatic decompensation and hcc occurs, does not change the final outcome of end-stage liver cirrhosis. in the guideline edition issued by the chinese medical association, the goal of antiviral therapy in hbv compensated cirrhotic patients is to prevent progression of the disease to decompensated cirrhosis, end-stage liver disease, hepatocellular carcinoma. the goal of antiviral therapy in hbv-related decompensated cirrhosis is to improve the hepatic disease severity, improve the clinical symptoms and quality of life, and prolong patient's survival. for the endpoint of the antiviral treatment, the definition of each guide is slightly different. guideline edition issued by aasld explicitly mentioned these patients with compensated cirrhosis should receive long-term treatment. however, treatment maybe stopped in hbeag positive patients if they have confirmed hbeag seroconversion and have completed at least months of consolidation therapy and in hbeag negative patients if they have confirmed hbsag clearance. for these patients with decompensated cirrhosis life-long treatment is recommended. guidelines issued by american society of digestive disease [ ] recommend long-term treatment until negative hbv dna and hbsag disappears. in the guideline edition issued by easl, endpoint of antiviral treatment has even been further subdivided into "ideal endpoint", "satisfactory endpoint" and "basic endpoint", where "ideal endpoint" means to achieve hbsag clearance with/without hbsag seroconversion. patients with liver cirrhosis generally have characteristics of longer course of disease, most of mother to child transmission, the large proportion of treated patients, high complexity of quasispecies and higher risk of resistance. the clinical treatment decisions in these patients need to consider a variety of factors, including long-term treatment, delay disease progression, improve histology, low resistance rates, good safety and patient tolerance, etc. there are differences in current guidelines of countries in antiviral therapy indications for liver cirrhosis, mainly cutoff viral load. for compensated cirrhosis, apasl guideline pointed hbv dna > iu/ml, aasld guideline that the hbv dna > iu/ml or hbv dna < iu/ml but elevated alt should be treated with antiviral treatment, and easl guidelines as long as detectable hbv dna should be treated. chinese guideline point that whether normal or elevated alt, hbeag(+) and hbv dna ≥ copies/ml, hbeag negative and hbv dna ≥ copies/ml should be antiviral treated. for decompensated cirrhosis, the recommendations of the guidelines is basically the same: as long as hbv dna can be detected, even if the viral load is low, it should be treated. currently there are two types of anti-hbv drugs, including interferons and nucleos(t)ide analogues. among them, decompensated cirrhosis is a contraindication to interferon. even compensated cirrhosis, due to the risk of hepatic decompensation, the use of interferon should be very careful. patients need to be closely monitored in the clinical application process, dose adjustments, the injection interval prolonged, and assistant drug to reduce adverse reactions and other measures so that the patient can safely complete drug treatment. on the contrary, the nucleos(t) ide analogues with strong antiviral effect, ease of use, safety, and well tolerance were recommended as the preferred treatment of liver cirrhosis by the major guidelines. currently, lamivudine (lam), adefovir dipivoxil (adv), telbivudine (ldt), entecavir (etv), and tenofovir disoproxil fumarate (tdf) have been approved for chb therapy. different nucleoside drug efficacy and safety varies. cirrhotic patients should be treated with potent, fast, direct inhibition of viral replication drugs. in aasld easl and apasl guidelines, for decompensated cirrhosis nucleoside (acid) analogues with potent effect and low resistance, such as entecavir or tenofovir should be selected. the reveal-hbv study of chen [ ] has established an hbv viral load paradigm in the natural history of chb. serum hbv dna level has been shown to be significantly and independently associated with incidence of hepatocellular carcinoma (hcc) and cirrhosis. liaw et al. [ ] evaluated the effectiveness of antiviral therapy in preventing disease progression in patients with chb and advanced fibrosis or cirrhosis. this is a large-scale, multi-center, randomized, double-blind, placebo-controlled prospective study. the study has changed the concept of the world, especially the chinese doctors to treat chb and liver cirrhosis, and promote the development of antiviral treatment for chb and liver cirrhosis. results showed that -year follow-up hepatocellular carcinoma occurred in patients ( . %) who received lam and patients ( . %) who received placebo (p = . ). this study also found that lam therapy reduced the risk of hcc by %, the risk of disease progression to fibrosis/cirrhosis by %. it first confirmed that antiviral therapy with lam could delay disease progression, reduce the incidence of hcc. in the study of patients with decompensated cirrhosis has also been confirmed lam was well tolerated, could effectively stabilize or improve liver function, and delay progression of liver disease, reduce liver transplantation. hann et al. [ ] conducted a prospective, multicenter study evaluated lam in decompensated cirrhosis patients, % of whom were not waiting for liver transplantation. all patients tested hbsag(+) and % tested positive for hbeag(+) at baseline. in % of patients hbv dna levels were detectable by the branched chain dna assay. the virus in % of these patients after months treatment and in % overall became undetectable by the bdna assay. alt, bilirubin and albumin level improved throughout treatment. from at baseline to at last visit the median ctp score also improved. after a median of . months of treatment, a virologic breakthrough occurred in only % patients. treatment of lam can improve liver function in nontransplantation candidates with decompensated cirrhosis. a meta-analysis from huang et al. [ ] indicated that lam and ldt significantly decrease the mortality rate and disease severity in decompensated cirrhosis patients. eight studies (total patients) were included. data showed that lam and ldt significantly decreased the mortality rate (rr . , % ci . - . ), improved the ctp scores (mean difference − . , % ci − . to − . ). in the study of patients with liver cirrhosis showed clinical improvement after treatment with lam - months. and even in patients with clinical improvement, they may develop to hcc, therefore such patients still need early treatment, and close monitoring of hcc. it is showed good efficacy and safety in the lam treatment of hbv related compensated or decompensated liver cirrhosis. however, in the long course of lam treatment viral resistance could not be ignored. more importantly, if these patients with chronic liver disease for a long time, poor liver reserve function, not promptly be treated, condition will deteriorate or even lead to death due to viral resistance. in clinical use of lam, the proportion of viral resistance increased year by year, %, %, %, %, the first year, second year, third year, fourth year, respectively. after the occurrence of viral resistance, some patients will be worsening, and even hepatic decompensation. therefore it is emphasized that patients with compensated or decompensated liver cirrhosis by lam therapy need to be improved compliance, closely monitoring and follow-up, to be adjusted treatment based on serum hbv dna response situation. some patients with decompensated cirrhosis or high viral load should be considered an initial combined with adefovir dipivoxil. the rates of adv resistance in lam-resistant subjects with hbv chronic hepatitis and cirrhosis are reported to be . % and . % after and years, respectively [ ] . nevertheless, adv use as a rescue therapy is affected by a primary nonresponse in - % of patients. in a study of adv add-on lam rescue therapy in lamivudine-resistant patients, kim et al. [ ] reviewed patients with adv add-on rescue treatment for years. after years treatment . % patients had complete virological response. alt in . % patients normalized, hbeag seroconversion occurred in . % patients. a study from woo et al. [ ] was to determine the long-term clinical outcomes after adv rescue therapy in decompensated patients infected with lamivudine-resistant hbv. in total, patients with a decompensated state and lamivudine-resistant hbv were treated with adv at a dosage of mg/day for a median of months in this multicenter cohort study. following adv treatment, ( . %) of patients experienced a decrease in child-pugh score of at least points, and the overall end-stage liver disease score decreased from ± to ± (mean ± sd, p < . ) during the follow-up period. with adv treatment, patients ( . %) had undetectable serum hbv dna (detection limit, . pg/ml). virologic breakthrough occurred in patients ( . %) and patients had a suboptimal adv response. the overall survival rate was . % ( / ), and a suboptimal response to adv treatment was associated with both no improvement in child-pugh score (≥ points; p = . ) and high mortality following adv rescue therapy (p = . ). three years of adv treatment was effective and safe in decompensated patients with lamivudine-resistant hbv. the globe [ ] trial was one of the largest international multi-center clinical trials of ldt treatment of chb. the safety and efficacy of ldt versus lam monotherapy has been compared for years in chb patients. hbeag(+) and hbeag(−) patients were treated weeks with ldt or lam once daily. the patients treated by ldt achieved superior therapeutic response versus these treated by lam in hbeag positive ( % vs %; p < . ) and hbeag negative ( % vs %; p = . ) patients. in both the hbeag positive and the hbeag negative groups, greater hbv dna suppression and less resistance was observed in patients treated by ldt than lam. after weeks of therapy in the phase iii globe study, hbv resistance (breakthrough and resistance mutations) to ldt occurred in % of patients with hbeag(+) and % of patients with hbeag(−). after weeks of therapy, hbv dna rebound in . - . % of hbeag(+) and . - . % of hbeag(−) ldt-treated patients associated with breakthrough and resistance mutations [ ] . ldt is not active against lamivudine-resistant hbv. in the globe study, patients who failed lam therapy showed cross-resistance to ldt. ldt was relegated to second-line status in the management of chronic hbv infection due to increasing resistance over time. in aasld and easl guidelines ldt is not recommended as first-line drug for monotherapy. when necessary the combined adv or tdf is recommended. the "roadmap concept" was derived primarily from a phase iii global registration study of ldt. an optimized strategy based on the roadmap concept is supposed to improve the clinical outcomes of patients with suboptimal antiviral response. sun et al. [ ] conducted the efficacy optimization of response to telbivudine (effort) study to investigate the efficacy and safety of the roadmap strategy by adding adv to ldt for suboptimal responders. in all, hbeag(+), na naive patients with chb were randomized to the mono or optimize group. patients in the optimize group were treated with ldt for weeks. subsequently, patients with hbv dna ≥ copies/ml at weeks were added adv to weeks, while those with hbv dna < copies/ml continued monotherapy. mono group received ldt monotherapy and added adv if development of viral breakthrough. due to suboptimal response % patients in the optimize group had been added adv. in the optimize group, more patients at weeks achieved hbv dna < copies/ml versus the mono group ( . % vs . %, p < . ), and with less genotypic resistance ( . % vs . %, p < . ). combination therapy showed an additive antiviral and low resistance potency. in two groups all patients were well tolerated. patients with ldt which are suboptimal virological responders at weeks are recommended to add adv. these patients with ldt monotherapy can be benefited from combination therapy without increased side effects. chan et al. [ ] studied the safety and efficacy of ldt and lam in hbv-related decompensated cirrhosis patients. in this double-blind trial, treatment-naive patients with decompensated cirrhosis in academic hospitals were randomized ( : ) to receive ldt or lam for weeks. a modified endpoint was used hbv dna < copies/ml and alt normalization. in intent-to-treat analysis (missing = failure) response rates were . % vs . % after weeks (p = . ) and . % vs . % after weeks (p = . ) for ldt vs lam. cumulative death and hcc rates were % and % in patients with ldt, and % and % compared to lam, respectively. cumulative genotypic resistance rate were % in patients with ldt, and % compared to lam during a -year period. comparable to lam, ldt can effectively stabilize liver function and is well tolerated. however, these two drugs are not recommended as first-line drugs due to high virological breakthrough rates. international clinical trials [ , ] and independent registrational studies in china have shown that etv achieved statistically superior virological and biochemical responses compared with lam. shim et al. [ ] evaluated etv as first-line monotherapy in patients with hbv-related decompensated cirrhosis primarily treated with . mg/day etv. there was no significant differences between groups in mean hbv dna changes, rates of hbeag seroconversion or hbeag loss, the proportion of patients with alt normalization after treatment. after months of treatment, the meld score and the ctp score in decompensated patients improved significantly. in these patients, % achieved ctp class a status and % showing a decrease of ctp score p points. the cumulative death rates and hcc rates in years were % and . %, respectively. zoutendijk et al. [ ] conducted a study to investigate the effect of etv on disease progression in patients treated with etv. all patients were divided into three groups, chronic hepatitis b without cirrhosis group (n = ), decompensated cirrhosis group (n = ) and cirrhosis group (n = ). the virological response (vr) was not influenced by the severity of liver disease (p = . ). in cirrhosis group, the probability of developing clinical events was higher (hr . ( % ci . - . ), p < . ) compared to two other groups during a median follow-up of months. vr was associated with a lower probability of disease progression (hr . ( % ci . - . ), p = . ). patients with compensated and decompensated cirrhosis who have achieved vr can achieve significant clinical benefits (hr . ( % ci . - . ), p = . ). in patients with cirrhosis, virological response to etv treatment lead to a lower probability of disease progression. the association between disease progression and viral replication was reduced with a threshold of iu/ml. it suggested that complete viral suppression was essential for patients with na treatment, especially in cirrhosis patients. tenofovir disoproxil fumarate (tdf) is an acyclic adenine nucleotide analogue. tdf as a potent, oral antiviral with low resistance rates, has been recommend firstline drug for the treatment of chb in a number of international guidelines. a study [ ] evaluated the effects on chb patients with fibrosis and cirrhosis treated with tdf of at least years. ( %) patients of completed weeks treatment. % patients ( / ) had biopsy results at both baseline and weeks. % patients ( / ) had histological improvement, and % ( / ) had regression of fibrosis at weeks (p < . ). % ( / ) cirrhosis patients (ishak score or ) at baseline, % ( / ) no longer had cirrhosis (≥ unit decrease in score). the histological response and regression of fibrosis seen in this study are probably due to the potent viral suppression achieved with long-term use of tdf. liaw et al. [ ] conducted a clinical trial to observe chb patients with decompensated liver disease received either etv (n = ), emtricitabine (ftc)/ tdf (n = ), or tdf (n = ). after weeks treatment, hbv dna was < iu/ ml ( copies/ml) occurred in . % (etv), . % (ftc/tdf), and . % (tdf) of patients. alt normalization occurred in % (etv), % (ftc/tdf), and % (tdf). hbeag loss/seroconversion rate were: / % (etv), / % (ftc/ tdf), and / % (tdf). in three groups meld scores and ctp scores both improved. this study demonstrated that all nas were well tolerated in chb patients with decompensated liver disease and can effectively improve virologic and biochemical parameters. response-guided therapy study [ ] suggested continuous treatment with lam ( years) delayed clinical progression in patients with chronic hepatitis and advanced fibrosis by significantly reducing the incidence of the risk of hepatocellular carcinoma and hepatic decompensation. the biggest risk is the occurrence of viral resistance in the longterm antiviral therapy. although rescue therapy can save some patients regain virologic response, but improper treatment or less often sicker or even canceling the original clinical benefit. although treatment can save some patients regain virologic response, but improper or untimely treatment often leads to deterioration or even canceling the existing clinical benefit. therefore, in the long-term antiviral therapy in patients with cirrhosis, how to overcome and prevent resistance, maximize the clinical benefit of antiviral therapy (including histological improvement and prevent and delay disease progression) is required by clinicians to consider. response-guided therapy refers to select appropriate medication according to the baseline characteristics of patients, and based on the patient's response to treatment, especially for those who did not achieve an early virological response, timely to adjust treatment to achieve a better long-term results. response-guided therapy is a hot point in the current study of antiviral treatment for chb, it is also an important strategy and important measure for the prevention of viral resistance. some experts even believe that all of antiviral therapy will need to be optimized. response-guided therapy means optimization based on baseline characteristics and early virologic response. numerous studies have demonstrated that baseline parameters such as low viral load, high serum alt level, high inflammation activity score prompted by liver biopsy and early virological response predict better longterm effect. in keeffe's [ ] "road map concept", response-guided therapy according to virologic response at weeks has been recommended. combination therapy is an important part of response-guided therapy. combination therapy includes an initial stage combination, the combination in the course of treatment (poor response or resistance), the combination treatment for treated patients with relapse. in easl guideline it was referred that adv or tdf with lam combination treatment need to consider in patients with liver cirrhosis. in aasld guideline lam or adv was recommend initial treatment for patients with decompensated cirrhosis, but their combination is recommended to reduce the risk of resistance and rapid inhibition of virus. the combined treatment for lam+adv is the most clinically used and studied. in first-generation na lam, ymdd mutation (rtm v/i) developed in up to % of patients in years [ ] . the combination of adv dipivoxil and lam was found to lower the rates of resistance to lam and serum hbv dna levels, and fasten the rates of alt normalization in hbeag(+) patients, with similar rates of hbeag seroconversion [ ] . in china a prospective cohort study [ ] from eight medical centers was conducted to observe the effect of response-guided combination therapy with lam and adv in patients with chb. according to hbv dna level at weeks, a total of patients with chb and cirrhosis treated lam were divided into three groups: complete response group (arm a, n = , hbv dna ≤ iu/ ml), partial response group (arm b, n = , hbv dna: - iu/ml) and inadequate response group (arm c, n = , hbv dna > iu/ml). patients was added adv at week in arm c, but at week in arms a and b. at weeks undetectable rate of hbv dna and ymdd mutation rate in three arms was . %, . %, . % (p = . ) and %, . %, % (p = . ), respectively. the data showed that early complete virologic response at weeks was associated with maintained viral suppression. hbv dna level of these patients without complete virological response at week , adding adv therapy further decreased by log iu/ml. all patients achieved biochemical improvement including alt/ast decline and alb elevation. in patients with hbv dna breakthrough due to ymdd mutations, adv and lam combination therapy did not lead to further multiple drug resistance. in chb patients with compensated liver cirrhosis, continuous hbv suppression for longterm and liver function improvement could be obtained by optimized responseguided add-on therapy of lam and adv. in recent years, some new antiviral drugs have gradually entered people's field of vision. truvada is a fixed-dose combination of two antiretroviral drugs (emtricitabine and tdf) approved by the fda for anti hiv therapy. the molecular structure of ftc was similar to that of lam, and the drug resistance was also similar to lam. in a double-blind study [ ] evaluated weeks treatment of , or mg once daily doses of emtricitabine in patients with chronic hepatitis b. then these patients were followed mg emtricitabine treatment for an additional weeks. after years, % of the patients had normal alt, % seroconverted to anti-hbe and % had serum hbv dna ≤ copies/ml. eighteen percent of the patients treated with mg emtricitabine developed resistance mutations after years. emtricitabine mg once daily was chosen as the optimal dose for chb based on these data. emtricitabine was well tolerated and confirmed a potent antiviral response for up to years. in a randomized double-blind, -week trial [ ] , patients were divided ( : ) to groups given a combination of emtricitabine (ftc, mg; n = ) and tdf ( mg, ftc/tdf) or monotherapy of tdf ( mg, n = ). patients were hbeag(+) or hbeag(−), with levels of hbv dna ≥ log iu/ml and lam resistance mutations (rtm i/v ± rtl m). after weeks of treatment, . % in the ftc/tdf group and . % of patients in the tdf group had levels of hbv dna < iu/ml (p = . ). hbeag loss and seroconversion was not significant difference between groups; only patient ( . %) in the ftc/tdf group lost hbsag. no additional benefit was observed with the combination therapy of emtricitabine and tdf vs tdf monotherapy. prolonged therapy with an oral nucleoside or nucleotide can lead to the development of antiviral resistance. loss of initial response and hbv dna rebound induce the development of resistance. these patients with resistance may develop biochemical breakthrough and histologic deterioration. sometimes severe exacerbations occur due to virus resistance in patients with cirrhosis. there are many risk factors for resistance development, such as potency of the antiviral agent, pretreatment hbv dna titer, period of treatment, nucleotide antiviral therapy or oral nucleoside history, and the degree of genetic barrier to resistance to the individual drug. thus, either etv or tdf, which possess the lowest genotypic resistance, should be used as the initial therapy. patients should be evaluated in the course of treatment, and these patients with poor response should be treated combination therapy early. managing resistance recommendations vary but generally involve either adding a drug in a separate class or switching to a more potent drug within the same class. in clinical practice, most members of the panel generally avoid monotherapy in patients with resistance and either use add-on therapy with tdf or etv or switch to tenofovir/emtricitabine. however, in patients with lam resistance, there are data providing compelling reassurance that tdf monotherapy is sufficient [ ] . data suggest that tdf may also be sufficient for patients with adefovir resistance [ ] was limited. however, with newer anti-hbv agents such as etv and tdf, viral resistance in previously treatment-naïve patients is very rare and the vast majority of cases of virologic breakthrough in clinical practice are due to nonadherence [ , ] . to see treatment measures in sect. . . clinical trials and cohorts from clinical practice have shown that nas are generally well-tolerated and safe [ ] . rare serious adverse reactions includes renal insufficiency, myositis, rhabdomyolysis, lactic acidosis, etc. some of these drugs can induce impairment of mitochondrial replication with mitochondrial dysfunction or loss due to a low level of activity against the human mitochondrial dna polymerase gamma. lam has been well-tolerated and effective in patients with hbv related decompensated cirrhosis [ , ] . adefovir dipivoxil and tdf are associated with a dose-dependent renal toxicity, except for ldt, a drug that may even improve creatinine clearance [ ] . . % patients had elevation of serum creatinine ≥ . mg/dl above baseline have been reported after years of tdf therapy in chb patients [ ] . hadziyannis et al. [ ] conducted a cohort study to investigate the efficacy, safety, and resistance profile of adefovir dipivoxil treatment for up to weeks in patients with hbeag(−) chb that was lost when weeks adv treatment was discontinued. a total of hbeag(−) chb patients treated with adv for years. serum creatinine elevations ( . mg/dl above baseline) occurs in % of these patients. similarly, % of the hbeag(+) chb patients treated adv for years had reversible creatinine elevations, % developed hypophosphatemia, and % had albuminuria [ ] . there were a growing number of studies of lam and adv combination therapy in both treatment-experienced and treatment-naïve patients with lamivudine-resistant hbv. lampertico et al. [ ] conducted a study to investigate the risk of resistance in the long term of lam and adv combination therapy in lamivudine-experienced chb patients. the results showed that % of patients with lamivudine resistance developed mild nephrotoxicity. after increasing the adv-dosing interval, all these patients were able to continue combination therapy. before treatment estimated creatinine clearance and serum creatinine levels should be tested in all chb patients treated with nas. in patients with creatinine clearance < ml dosing adjustments are needed, regardless of the type of nas. etv preclinical research data showed that a higher incidence of solid tumors in animals was associated with prolonged administration of high-dose compared to placebo. however, in clinical trials prolonged administration of etv was not associated with an increased incidence of malignancy. in a clinical trials conducted by lai et al. [ ] the severity and frequency of laboratory and clinical adverse events were similar among lam-treated and etv-treated patients. furthermore no evidence of serious adverse events and mitochondrial were observed in patients with etv treatment for up to years [ ] . in recent years, in patients with liver cirrhosis in high meld score (> ) taking etv, lactic acidosis, and even death cases were reported. although rare happening, it still has to be pay more attention by the clinician, and needs for future research. ldt may cause myopathy and peripheral neuropathy. in treatment of ldt combined with peg-ifn- a, moderate-severe peripheral neuropathy may occur in patients. therefore ldt was forbidden in combination with peg-ifn. in these studies [ , ] , patients treated with ldt at years had a significantly higher incidence of severe elevations of serum creatine phosphokinase (i.e., seven times upper limit of normal) compared to patients treated with lam, . % and . %, respectively. although most of them are asymptomatic, there are still two cases of patients with ldt-induced symptomatic myopathy had to be terminated treatment. in chinese guideline, it has been stated that careful medical history investigation before treatment was needed to reduce the risk. in the course of treatment, patients with serum creatinine, ck or lactate dehydrogenase significantly increased, and accompanied by myalgia or weakness, should be immediately tested. once diagnosed with uremia, myositis, rhabdomyolysis or lactic acidosis, patients should be promptly discontinued treatment or switched to other drugs, and given the appropriate treatment. antiviral therapy with nucleos(t)ide analogue is an important means to delay or reverse progression of liver fibrosis and cirrhosis. cirrhotic patients need long-term or even lifelong antiviral treatment. all of the five antiviral agents could effectively inhibit virus replication, improve biochemical and pathological parameters in chb cirrhotic patients with good tolerance. patients should be fully assessed baseline characteristics before treatment, and closely monitored therapeutic response and adverse reactions during treatment, then to optimized treatment. according to both drug resistance and efficacy profile, etv and tdf are superior to ldt, adv, and lam, and can be recommended as the first-line drug for nuc-naïve patients with hbv related decompensated cirrhosis. finally, hbv related cirrhosis patients treated oral nuc(s) must be frequently laboratory and clinical assessed to insure medication compliance and surveillance for clinical and virological response as well as drug resistance, drug side effects, and hepatocellular carcinoma. in the world hepatocellular carcinoma is one of the most frequent malignancies: estimated , new cancer cases worldwide occurred in ( % in china alone). it is the fifth most common cancer in men ( , cases, . % of the total) and the ninth in women ( , cases, . %) [ ] . hepatocellular carcinoma is the second most common cause of cancer death worldwide, estimated to be responsible for nearly , deaths in ( . % of the total). given the very poor prognosis for liver cancer (the ratio of mortality to incidence is . ), the geographical patterns in incidence and mortality are quite similar [ ] . chronic infections with hepatitis c virus (hcv) and hepatitis b virus (hbv) are the major recognized risk factors for hcc worldwide [ ] , hbv being most common in eastern asia and hcv in mediterranean countries [ ] . current hcc treatment is the comprehensive treatment based on resection, liver transplantation, or percutaneous local ablative treatment. more and more studies indicated that after resection antiviral therapy effectively inhibit hbv replication and sequentially decrease the rate recurrence of hcc. chronic hepatitis b is the most frequent etiology of hcc. chen [ ] conducted a prospective cohort study to evaluate the relationship between mortality and past hbv dna level for years of follow-up. hbv dna level had been measured on stored samples in hbsag(+) adults from cohort entry ( - ). there was a significant increase in mortality in patients with hcc across viral load categories (p < . ). compared to the hbv undetected category, the relative risk for hcc mortality in the high viral load group was . ( % ci . - . ) and . ( % ci . - . ) in the low viral load group. the relative risk for chronic liver disease mortality were . ( % ci . - . ) and . ( % ci . - . ), respectively (p < . ). with increased follow-up time, the rr associated with high viral load did not change. in surviving cohort patients evaluated for liver disease in , the disease significantly associated with viral load. the data showed that increased mortality from hcc and cld was associated with viral load in patients infected hbv. hbv dna level may be a useful prognostic indicator in chb patients, and treatment interventions to inhibition of virus replication should be explored. the reveal-hbv study of chen [ ] assessed the relationship between risk of serum hbv dna level and hcc. from to , this prospective cohort study in taiwan enrolled participants who were hbsag(+) and - years from a community-based cancer screening program. during , person-years of follow-up and a mean follow-up of . years, there were incident cases of liver cancer and deaths. the incidence of liver cancer grew in patients with hbv dna level at baseline in a dose-response relationship ranging from . % personyears for an hbv dna < copies/ml to . % person-years for an hbv dna × copies/ml or greater. the cumulative incidence rates of liver cancer in these patients were . % and . %, respectively. after adjustment for age, sex, alcohol consumption, cigarette smoking, serum alt level, hbeag, and liver cirrhosis at baseline, the biological gradient of liver cancer by hbv dna levels were significant different (p < . ). the dose-response relationship was most prominent for hbeag(−) patients without liver cirrhosis and with normal serum alt levels at baseline. chb patients with persistent elevation of viral load had the highest liver cancer risk during follow-up. these data proved that high level of hbv dna (≥ , copies/ml) was a strong risk factor of liver cancer independent of liver cirrhosis, hbeag, and serum alt level. these data showed that the correlation between hcc and hbv dna level was more closely than alt. the current guidelines [ ] [ ] [ ] for management of chb are of the view that: for patients with chronic hepatitis b the primary goal of treatment is to permanently suppress or eliminate hepatitis b virus replication. thus hepatic infectivity and pathogenicity could be decreased, and thereby necroinflammation could be stopped or reduced. the short-term goal in clinical terms is to reduce hepatic inflammation, to prevent the development of hepatic fibrosis and decompensation, to ensure a sustained loss of hbv-dna and alt normalization. the long-term goal is to prevent progression to cirrhosis and hcc, to prevent alt flares that may lead to hepatic decompensation, and finally prolong the survival time. therefore, the ideal treatment for patients with chb should be able to effectively reduce the hbv dna level, thereby inhibit or stop the deterioration of liver disease, reduce the incidence of severe exacerbation and hcc. hbv chronic infection is a major risk predictor for the development of hepatocellular carcinoma. the hepatocarcinogenesis in chb patients has been extensively investigated, and a number of predictors relate to occurrence of hepatocellular carcinoma. the most significant predictors associated with hepatocellular carcinoma include chronic hcv and hbv infection, aflatoxin b , chronic alcohol consumption and virtually all cirrhosis-inducing conditions [ ] . for hepatocellular carcinoma in human, chronic infection of hbv was considered as the major environmental etiological factor [ ] . hbv-induced hepatocarcinogenesis can involve an array of processes, including host-viral interactions, sustained cycles of necrosis-inflammation-regeneration, viral-endoplasmic-reticulum interactions (induction of oxidative stress), viral integration into the host genome (and associated host dna deletions) and the targeted activation of oncogenic pathways by various viral proteins. a predominant risk factor for hcc is chronic active hepatitis. the mechanisms of chronic active hepatitis consist of a combination of complementary, effects, several involved in liver cell inflammation, and necrosis thus fibrosis and cytokine synthesis. the underlying chronic active hepatitis inflammatory is a major risk factor for the higher hcc occurrence in patients with progressive cirrhosis. fundamentally important information on these issues have been provided in animal models for hepadna virus infection [ ] . integration of hbv dna results continuous replication of the virus, which induces occurrence of genetic alterations. the hbv-dna sequences are integrated into cellular dna in most (approximately %) liver-tumor samples from hbsagpositive patients [ ] . hbv genome integration should be viewed as a "driver" of liver carcinogenesis. in the impact of genes, some of the other genes involved, and play an important role in the carcinogenesis process. in addition, hbv dna integration effects on host cells including cell gene deletion, chromosomal rearrangements, genomic dna copy number variation, loss of chromosomal heterozygosity, etc. [ , ] . in the host cells, the integration of hbv damage mechanisms to protect the integrity of the chromosome. hepatitis b virus x protein (hbx) exhibits pleiotropic effects on different pathways involved in intracellular signaling and transcriptional activation that modulate cell responses to protein degradation, genotoxic stress, apoptosis and cell division; these biological effects might contribute to the potential transforming activities of hbx. hbx has been confirmed to interact with p , accordingly inactivating several essential p -dependent activities, including p -mediated apoptosis transactivation properties of p , regulation of cell cycle dna repair genes and tumor suppressor genes. hbx may also play a role in tumorigenesis of hepatocellular carcinoma through modulation of angiogenesis pathway. indeed, hbx expression induces upregulation of the vascular endothelial growth factor (vegf) transcription and stabilizes hypoxia inducible factor (hif)- [ , ] . moreover, hbx causes multipolar spindle formation, chromosome segregation defects, and appearance of multinucleate cells by inducing aberrant centrosome duplication; these biological actions might be due to sequestration of the nuclear transport receptor crm in the cytoplasm [ ] . it has been frequently reported that hbx with mutations in amino acids and may be associated with the severity of chb. studies indicated that these mutations had also been detected in hcc tissue and arose before the development of hcc [ ] . other studies [ ] have pointed out that many signaling pathways have been outlined as common targets deregulated during hepatocarcinogenesis, including the wnt/β-catenin, tgf-β pathway, ras/mapk pathway, pi k/akt/mtor pathway, jak/stat pathway, pkc pathway, etc. in addition, fibrinogen-like protein (fgl ), hgf, igfs and other coagulation factors, growth factors and other angiogenesis gene may also be involved in the occurrence and development of hcc [ ] . . wnt/β-catenin pathway: the wnt signaling pathway is an evolutionary highly conserved pathway and involved in the regulation of proliferation, motility, cell/ cell interaction, organogenesis and axis formation. the accumulation and expression of β-catenin in the nucleus were decreased, and cell proliferation was suppressed followed by up-regulated gsk- β activity due to hbx induction [ ] . hbx mutants may participate in the development and progression of hcc, at least in part through the wnt- a pathway [ ] . . tgf-β pathway: tgf-β is a central regulatory factor in control of hepatocyte proliferation and death. paradoxically, either under-or overexpression seem to have deleterious consequences resulting in an increased turnover of liver cells and thereby predisposing to cancer progression [ ] . in both cases the escape from the antiproliferative, proapoptotic action of tgf-β would be a prerequisite for tumor progression. at the stage of tumor occurrence, tgf-β can promote tumor cell invasion, metastasis, but suppresses tumor growth in liver damage stage. tgf-β receptor i kinase inhibitors, blocking the tgf-β signaling pathway, show anti-tumor effect [ ] . . ras/mapk pathway: ras/mapk signaling pathway is a signal cascade waterfall reaction caused by external signal activated receptor in the cytoplasm, involving a variety of connectors, nucleotide exchange factor, small gtp binding protein. hbx retains the ability to overcome ras-induced senescence in human cells immortalized by htert, although hbx alone could neither immortalize nor transform human cells. the ability of hbx to collaborate with active ras in cell transformation may explain its role in hcc [ ] . . pi k/akt/mtor pathway: the pi k/akt/mtor protein cascade is one of major signaling pathways associated with receptor tyrosine kinases (rtks) [ ] . in nontransformed cells, a tumor suppressor pten (phosphatase and tensin homolog deleted from chromosome ), which inhibits this pathway by blocking akt activation and reversing the pi k reaction, control the pi k/akt/ mtor pathway. in almost half of the studied hccs, pten expression was reduced or absent, and hepatocyte-specific abrogation of pten expression in mice results in the development of hcc [ ] . . jak/stat pathway: the jak/stat pathway is activated by more than cytokines and growth factors and involves in multiple cell functions such as differentiation, proliferation, and apoptosis [ ] . in this pathway, the cytokines induce phosphorylation of the janus tyrosine kinases (jak , and , tyk ), followed by activation of stat - [ ] . both hbv and hcv are able to induce jak/stat pathways [ ] . in hcc, phosphorylation of jak , jak , and tyk tyrosine kinases was not detected in normal livers but increased significantly from surrounding non-neoplastic livers to hccs. activation of stat , stat , and stat was statistically higher in tumors than in respective surrounding livers, with pstat being higher in hcc with poor prognosis than hcc with better prognosis. the levels of jak/stat targets, including bcl-xl, mcl- , cyclin d , and c-myc were markedly increased in the majority of hccs [ ] . . pkc pathway: pkc isozymes have a central role in cellular signaling transduction involved in cell proliferation, differentiation, apoptosis and angiogenesis [ ] . pkc-α, pkc-δ, and pkc-ι have been found to be over-expressed in human hcc cell lines. the focus of pkc research in hcc has predominantly been on pkc-α. its expression is significantly increased in cancerous tissue and is correlated with tumor size and tnm stage. in addition, over-expression of the mrna of this isozyme has been associated with a shorter survival rate, and thus may be a marker for disease prognosis in cancer patients [ ] . . fgl : fgl could directly generate thrombin from prothrombin without activation of the conventional coagulation cascade. it was confirmed to be overexpressed in various human malignant tumors [ ] . the hfg was associated to the hypercoagulability in cancer and may induce tumor metastasis and angiogenesis via cytokine induction [ ] . fgl was overexpressed in hcc tissues and co-localized with fibrin deposition. fgl contributed to hcc tumor angiogenesis and growth in a thrombin-dependent manner, and down regulation of its expression might be of therapeutic significance in hcc [ ] . to investigate whether prevention of hcc by the hbv vaccine and to identify the predictors of hcc for vaccinated birth cohorts, a population-based study [ ] in taiwan the study shows strong evidence that the hbv vaccine reduce the incidence of hcc. those who received incomplete hbv vaccination (i.e., less than three administrations of the vaccine) during infancy and infants of hbeag-and hbsagseropositive mothers without hbig injection at birth had higher risk of developing hcc. approximately % of children with hcc born to hbeag and hbsag carrier mothers did not receive hbig at birth. improvements of the hbig injection rate within h after birth in infants of high-risk mothers should be implemented. this study has limitations in that the role of host factors, such as genetic polymorphisms, hbv genotype, virus mutation, were not studied, which could influence the interpretation of the data. a number of studies on the long-term treatment of interferon (ifn) or na for patients with hbv showed the prevention of hcc. a meta-analysis [ ] compared risk of hcc in chb patients who received ifn or na. a total of studies were included in this review. ifn treatment ( studies; n = ) showed a significantly reduced risk of hcc for patients treated by ifn compared to controls (rr . , % ci . - . ; studies) and for compensated cirrhotic patients (rr . , % ci . - . ; studies). there was no statistical heterogeneity for these comparisons. na treatment ( studies; n = ) showed a significantly reduced risk of hcc for patients treated by nas compared with controls (rr . , % ci . - . ; studies). na treatment demonstrated a more profound reduction in hcc risk of % compared to ifn which produced only a modest effect of %. this is perhaps not a surprising finding, as the viral load is found to be the most important factor leading to cirrhosis and cancer development in the liver. the more effective reduction in hcc risk may be related to the more profound effects of viral suppression of oral anti-viral agents than ifn [ ] . across subgroups there was a significantly reduced risk of hcc: hbeag(+) patients (rr . , % ci . - . ; five studies); compensated cirrhotic patients (rr . , % ci . - . ; three studies); non-cirrhotic patients (rr . , % ci . - . ; two studies); patients with drug resistance (rr . , % ci . - . ; three studies); and those without drug resistance (rr . , % ci . - . ; three studies). in subgroup analysis of ifn studies, a more significant reduction in hcc risk among those with early cirrhosis was found. the effects of ifn could be beyond its viral suppressive activities. previous studies have shown that at least ifn-α b has inhibitory activities on hepatic stellate cells (hscs) activation by suppressing the effects of il- β, tnf-α and probably inducing apoptosis of hsc [ , ] . as hscs play a central role in fibrogenesis, the effects of ifn-α on hscs are worthy of further investigation. on the other hand, the anti-cancer effect of nas, and probably ifn, was more prominent among hbeag-positive than among hbeag-negative patients. this discrepant results based on hbeag status is consistent with the fact that while hbeag(+) patients usually have a higher hbv dna level, treatment of hbeag(−) patients is more difficult and sustained virological responses are uncommon [ , ] . high risk population hbv-hcc refers to patients who are the middle-aged men with high hbv load, with hbv and hcv co-infection, with family history of liver cancer, alcoholics, and with diabetes mellitus. the long-term effect of antiviral therapy for patients with hbv showed the prevention of hcc. however, according to current national management guidelines for chb, there are still some patients without antiviral treatment. thus, some of the high risk population of hbv-hcc may lost the opportunity of early interventional treatment. current guidelines recommend liver biopsy to assess the degree of necroinflammation and liver fibrosis prior to treatment initiation in patients with increased hbv dna and/or mild elevated alt levels ( - times the uln). for patients older than years, liver biopsy should be considered. in those with "high normal" alt levels liver biopsy is strongly recommended [ ] . although liver biopsy remains the gold standard for assessing hepatic fibrosis, its use has several limitations including sampling error and intra-or inter-observer sampling variability [ ] . inadequate liver biopsy may further pose misleading histological information that precludes cirrhotic patients from antiviral treatment [ ] . in the report by tong et al. [ ] , of patients who developed hcc were diagnosed as having chronic hepatitis by histology and therefore did not fulfill the recommended treatment criteria. these patients probably had normal alt and/or intermediate hbv dna levels (between , and , copies/ml). in this report, of patients with cirrhosis who developed hcc could not be identified by the treatment recommendations. in other words, patients with cirrhosis have a significant risk of developing hcc even when their hbv dna levels are not high. patients with elevated alt between . and times the uln also was a strong risk predictor of hcc or complications [ , ] , a claim supported by a korean population study. the reveal study suggested that high hbv dna level significantly increased risk of hcc independent of liver cirrhosis, hbeag, and serum alt level. hbv dna consistently replicates and is integrated into the host genome, adding to the coexistence of metabolic disorders, inflammatory responses and oxidative injuries, which induce genetic instability and an imbalance of cell growth and apoptotic tolerance signals. these are all biologic driving forces for hcc development in chb patients. therefore, we must pay more attention to the effect of continuous hbv replication on the prognosis of patients. any antiviral drugs, if not completely clear the virus but can reduce the viral load, it may reduce the risk of patients with hcc. the risk factors for hcc include progression to cirrhosis, longer duration of hbv infection, higher serum viral load (≥ copies/ml), males, age > years, alcohol, ethnic groups native to regions of east asia and sub-saharan africa, the virus genotype (genotype a in african population or genotype c in asian population), co-infection with human immunodeficiency viruses (hiv) or hepatitis c, d, decompensated liver cirrhosis, persistent inflammation of the liver, continuous hbeag positive, and a family history of liver cancer [ ] . cirrhosis is the most important independent risk factor for hcc. up to - % of hcc occur in patients with cirrhosis. effective antiviral therapy inhibits hbv replication, reduces serum viral load and accelerates serum conversion of hbeag, which may therefore alleviate liver damage and reduce the development of cirrhosis. all the patients with the above risk factors should be received antiviral therapy. ifn-α is an immune modulator inducing antiviral, immune regulation, anti-tumor and anti-fibrosis effects. its antiviral mechanism is a complex mode of action including the destabilization of viral nucleocapsid, inhibition of viral genome transcription, activation of natural killer (nk)/nkt cells. however, disadvantages of ifn shortcomings are prominent, such as the high cost of peg-ifn, intolerance to ifn therapy in patients with cirrhosis. compared with ifn, na is safer, better tolerance for these patients. current guidelines from apasl, easl and aasld, do not provide treatment recommendations for patients with hbv-hcc. the chinese expert consensus [ ] on antiviral therapy to treat hbv/hcv-related hcc has been published in . this expert consensus indicated that promptly initiation of antiviral therapy is not only important for preventing the incidence of hcc in patients chb, but also essential for reducing hbv reactivation, improving liver function, delaying or reducing recurrence of hcc, and prolonging survival of patients with hbv-hcc after palliative and curative therapies. it puts forward the overall principle and target of antiviral therapy of hbv-hcc, and emphasizes the antiviral therapy is a part of comprehensive treatment. at present, suitable treatment for hbv-hcc is multidisciplinary comprehensive treatment. a large number of evidence-based medical evidence suggested, standard anti-hbv treatment for these patients help to improve the overall curative effect, prevent the recurrence of the tumor, and improve the os. therefore, anti-hbv therapy should be taken as a very important part of comprehensive treatment of hbv-hcc (fig. . ) . following curative liver resection for hcc, - % of postoperative death is caused by recurrent disease [ ] . high serum hbv dna levels were a strong predictor of hcc. effective control of hbv replication with antiviral therapy may lower its recurrence after liver resection. in , kubo et al. [ ] first reported the relationship between recurrence of hbv-hcc after resection and hbv dna level. later in another study [ ] he pointed out that patients with high hbv dna levels (more than meq/ml) have high risk with recurrence and poor prognosis. on the contrary, kim et al. [ ] included consecutive patients undergoing curative resection and found that, sustained hbv viremia (> log copies/ml) increased recurrence, but did not have a marked effect on survival. an et al. [ ] investigated the hbv dna changing patterns and their effects on outcome in hbv-hcc patients with resection. all patients were followed up. data from alive patients without recurrence at months were collected. the mean period of follow-up was . months and the mean age was years. for the entire population with multivariate analysis, tumor size > cm (p = . ), hbv dna > copies/ml, child-pugh class b (p = . ) at the time of resection (p = . ), and vascular invasion (p = . ) were independently risk factors of hcc recurrence. on multivariate analysis for patients, sustained hbv dna level < copies/ml was the only risk predictor for both longer survival (or . ; % ci . - . ; p = . ) and low recurrence (or . ; % ci . - . ; p = . ). that clarified that a sustained high hbv dna is among the most important risk factors of adverse outcome after liver resection of hbv related hcc. the sustained suppression of hbv dna < copies/ml strongly benefit to long-term survival and recurrence-free. kim et al. failed to show the difference in survival between the sustained viremia (> log copies/ml) and non-viremia groups despite the high recurrence rate in the sustained viremia group. the reason may be that researchers have used a higher hbv dna cut-off value (> copies/ml) to differentiate between patients with high and low viremia. in an's results, they found that a lower hbv dna level cutoff value of copies/ml is superior to > copies/ml in predicting outcomes after resection. it is therefore needed to suppress further hbv dna to a lesser level in order to obtain better clinical outcomes after surgery. studies have shown that positive hbeag was a risk predictor for recurrence of patients after resection of hcc. sun et al. [ ] evaluated the impact of hbeag on patients' survival and tumor recurrence after curative resection of hbv-hcc. all patients undergone curative resection with small hcc (⩽ cm) were divided into hbeag(+) and hbeag(−) groups. postoperative outcomes and clinicopathological factors of two groups were compared, and risk predictors for recurrence and survival were investigated. hbeag(−) patients had higher -year disease free survival rates ( . % vs . %, p = . ) and -year overall survival rates ( % vs . %, p = . ). there was no significant difference in tumor factors and operative morbidity between hbeag(+) and hbeag(−) groups, but more macronodular cirrhosis, higher serum alanine aminotransferase levels, and younger age were found in the hbeag(+) group. in patients for multivariate analysis, macronodular cirrhosis, hbeag(+) and age > years were independent risk predictors for overall survival, and multiple tumor nodules and hbeag(+) were independent predictors for disease free survival. in patients with small hcc after curative resection, hbeag(+) was a significant risk factor of early recurrence (within year). because of the adverse effects, the impact of ifn-α therapy after curative resection on recurrence of hcc and the os among patients with hbv infection are controversial. theoretically, the effect of postoperative ifn-α therapy on recurrence might be related with the direct suppression of tumor growth and metastasis, the inhibition of hbv replication, down-regulating expression of vascular endovascular growth factor (vegf), antiangiogenesis effect, and modulating some factors in tumor microenvironment. however the results of clinical trials are not the same. in recent years, more and more studies show that reasonable application of ifn-α can prevent the recurrence of the tumor and prolong the survival time of the patients. qu et al. [ ] conducted a retrospective study to investigate the impact of ifn-α therapy on survival and recurrence after curative resection in patients with hbv-hcc. of hbv-hcc patients with curative resection, patients were treated postoperative by ifn-α therapy ( million units three times every week for months). patients with postoperative ifn-α therapy had higher os rates (p = . , hr: . , % ci: . - . ). there was no significant difference in dfs rates between the two groups (p = . , hr: . , % ci: . - . ). on multivariate analysis, postoperative ifn-α therapy was an independent factor for os (p = . , hr: . , % ci: . - . ) and significantly reduced early recurrence (p = . , hr: . , % ci: . - . ). however, patients with or without postoperative ifn-α therapy had similar cumulative late recurrence rates (hr: . , % ci: . - . , p = . ). sun et al. [ ] evaluated the effects of postoperative ifn-α treatment on survival and recurrence in patients with hbv related hcc. all patients were randomized after curative resection into ifn-α treatment (n = , μg i.m. tiw for months) and control groups (n = ). if recurrence was diagnosed, treatment was terminated, these recurrence patients was managed in the same way in both groups. all clinicopathological parameters in two groups were analyzed. the median os was . months in the treatment group and . months in the control group (p = . ); the median dfs period was . versus . months (p = . ). that concluded that ifn-α therapy improved the os of hbv-hcc patients after curative resection, probably by postponing recurrence. someya et al. [ ] investigated consecutive patients with hbv cirrhosis and hcc who underwent potentially curative ablation for hcc. eleven patients received long-term ifn therapy. initial dna was high (> . log copies/ml) in patients and low (< . log copies/m) in . hcc recurrence rates in the high dna group and low dna group were . % and . % at the fifth year, and . % and . % at the tenth year, respectively (p = . ). similarly, recurrence rates after treatment of hcc in the abnormal ast group (n = ) and normal ast group (< iu/l, n = ) were . % and . % at the fifth year, and % and . % at the tenth year, respectively (p = . ). five of patients with normal ast, and of the patients with abnormal ast, received ifn-α after confirmation of tumor ablation. in the subgroup of abnormal ast, tumor recurrence rates in the untreated and ifn-α groups were . % and . % at the end of the first year, . % and . % at the second year, and . % and . % at the third year, respectively (p = . ). on multivariate analysis, ifn-α significantly reduced the recurrence rate (p = . , hr = . ) even after adjusting for background characteristics. pathogenic mechanism of hbv-hcc mainly related with the integration of hbv dna into host hepatocytes. therefore, inhibition of inflammation and viral replication can reduce the hbv dna level and the risk of hcc. after the resection the residual liver is still cirrhosis, still have a high risk of new cancer. hbv-hcc occurrence seems to have the relationship with the hbv greater than the liver repeatedly inflammation [ ] . tang et al. [ ] reported that high hbv dna levels is associated with increased risk for development of hcc. ifn has a dual role of antiviral and immune regulation. ifn as immune regulator can not only activate or mediated macrophages, nk cells and cytotoxic t lymphocyte, but also adjust the antibody. its antiviral activity includes induction of , oligonucleotide adenosine monophosphate synthetase and protein kinase. moreover, ifn also has the anti-fibrosis, anti-proliferation and anti-tumor effects. the experimental study [ ] confirmed that ifn exerts potent growth inhibitory effects on the hcc cell line plc/prf/ both in vitro and in vivo and its mode of action in this animal model system appears to be predominantly mediated by a direct antiproliferative effect on tumor cells. breitenstein et al. [ ] searched cochrane central register of controlled trials between january to october and evaluated the effects of ifn-α or -β in patients after surgical resection or ablation of hbv-hcc. seven rcts (n = patients) were included in a meta-analysis review. patients treated with ifn had a significantly decreased mortality rate than control group (rr . , % ci . - . , p < . ) and significantly reduced risk of tumor recurrence (rr . , % ci . - . , p = . ). as of the trials used ifn-α, it is interesting that the only study [ ] on ifn-β showed the largest beneficial effect on tumor recurrence. due to the small number of cases in this study, further clinical evaluation of ifn-β in the adjuvant setting of hcc treatment seems to be indicated. the rate of treatment discontinuation ranged from % to % because of the side-effects of ifn which were dose dependent and often serious. severe adverse effects of the adjuvant ifn treatment leading either to treatment disruption or dose reduction occurred in up to a quarter of the patients. in particular, work is needed to optimize the type and dosage of ifn to minimize side-effects, and to study the combination of ifn treatment with other (neo)adjuvant agents. reasonable application of nas can prevent the recurrence of hcc and prolong the survival time of the patients. a comparative nonrandomized study [ ] of postoperative antiviral treatment was conducted on hcc patients who underwent curative hepatectomy. the authors assessed the impact of antiviral therapy for patients who underwent partial hepatectomy for hbv-hcc in the immuneactive phase of hbv infection. all patients in the treatment group treated by lamivudine (lam) with or without adefovir dipivoxil (adv), while patients in control group received no antiviral treatment. at -month postoperation, the treatment group also had a significantly greater increase in residual liver volume per unit surface area following hepatectomy ( . ± . cm /m vs. . ± . cm / m ). the os rate was a significant difference between two groups. the os rates of -and -year were . % and %, respectively, for the control group, and . % and . %, respectively, for the treatment group. these results revealed that antiviral therapy with nas effectively relieved hbv-induced liver damage, improved liver function, promoted hepatocyte regeneration, and increased volume of residual liver, thus enhancing tolerance to subsequent therapy. there were no serious adverse effects to lam therapy in this study. however, the most significant problem associated with long-term therapy with lam is emergence of resistance. in this study, the emergence of ymdd mutants was observed in of patients in the lam group. administration of adv successfully controlled the virus. in a meta-analysis, wong et al. [ ] assessed whether anti-viral therapy with nas could prevent hbv-hcc patients from tumor recurrence after curative treatment. a total number of patients from cohort studies were included: patients without antiviral treatment (control group) and patients with antiviral treatment group. lam was the primary antiviral therapy in the majority of patients. patients with lam resistance was treated by either switching to entecavir (etv) or adding adv therapy. thirteen patients received etv as primary antiviral therapy. most of the antiviral therapies were started after the curative treatment of hcc. the recurrence rate of hcc in the antiviral treatment group was significant lower compared to control group ( % and %; p = . ). in the antiviral treatment group the risk of hcc was reduced by %. there were also significant differences in favour of antiviral treatment group in terms of overall mortality ( % vs. %; p < . ) and liver-related mortality ( % vs. %; p = . ). hcc patients with anti-viral therapy after curative treatment may be reduced the risk of hcc recurrence for %. besides, antiviral therapy significantly improved os, as overall mortality was reduced by %. after curative treatment of hcc, patients should be monitored regularly concerning their viral status for consideration of antiviral therapy. antiviral therapy was beneficial as it not only might reduce hcc recurrence and liver failure secondary to reactivation of hbv due to viral suppression ( % reduction in the mortality secondary to liver failure in the antiviral therapy group). after ablation, the use of ifn or nas can reduce the recurrence of hcc, improve liver function, thus enhancing tolerance to subsequent therapy and prolong the survival time of the patients. recurrence in patients with hbv-hcc after ablation was common. chung et al. [ ] assessed the correlation between viral load and intrahepatic recurrence and predictors of intrahepatic recurrence after percutaneous ablation. hbv-hcc patients undergoing percutaneous ethanol injection (pei) or radiofrequency ablation (rfa), between . and . were prospectively enrolled. a total of patients (mean age, . years; male, . %) were included. ninety patients ( . %) had serum hbv dna ≥ iu/ml. the median followup duration was . months (range, . - . ) and . % patients (n = ) experienced intrahepatic tumor recurrence. on multivariate analysis, hbeag(+) was an independent risk predictor of late recurrence (≥ year) (p = . ; hr . ) and intrahepatic recurrence (p = . ; hr . ). the afp level also significantly predicted late recurrence (p = . ; hr, . ). however, neither serum hbv dna titers nor the ablation method were correlated with intrahepatic recurrence. xia et al. [ ] conducted a study to investigate the risk factors of recurrence in patients with hbv-hcc after rfa. all patients with small hcc enrolled in this study. in patients the intrahepatic recurrence rate was . % by median follow-up of months. on univariate analysis, meld score, afp, hbv dna, precollagen iii, and hyaluronic acid were independent risk factors for recurrence. on multivariate analysis, hyaluronic acid and hbv dna were independent risk factors for recurrence. the cumulative -, -, and -year dfs rates were . %, . %, and . % in the high hbv dna group (> × copies/ml) and . %, . %, and . % in the low hbv dna group (≤ × copies/ml), respectively. that showed significant difference between the two groups (p = . ). goto et al. [ ] reported the similar results that serum hbv dna load (> . log copies/ml) were associated with the recurrence. thus reasonable antiviral therapy can improve liver function and prevent the recurrence of the tumor. lin et al. [ ] assessed the impact of ifn-α in preventing hcc recurrence. thirty eligible patients were randomized into three groups: ifn-α-continuous group (n = , ifn-α mu tiw for months), ifn-α-intermittent group (n = , ifn-α mu daily for days every month for months followed by mu daily for days every months for a further months), and control group (n = , no ifn-α therapy). the three groups were comparable in terms of demographics, laboratory data, and etiology at entry and hcc characteristics. after a median follow-up of months, % patients (n = ) in the control group and % patients (n = ) in treatment groups ( patients in the ifn-α-intermittent group and patients in the ifn-α-continuous group) developed an hcc recurrence (p = . ). cumulative hcc recurrence rates in the control groups ifn-α-intermittent, ifn-α-continuous, and were %, . %, and . % at the end of year and %, . %, and . % at the end of years (p = . ), respectively (control vs. ifn-α-continuous group, p = . ; vs. ifn-α-intermittent group, p = . ). the cumulative hcc recurrence rate of the patients treated with ifn-α and the control group was % and % at the end of year and % and % at the end of years, respectively (p = . ) if both ifn-α groups were combined. the conclusion was that hcc recurrence may be reduced by ifn-α therapy after medical ablation therapy for primary tumors. antiviral, anti-tumor and anti-angiogenesis effect of inf can effectively resist the risk factors of recurrence after ablation. some patients do not tolerate the adverse reaction of ifn, still should be treated with nas to inhibit viral replication, relieve the liver inflammation, improve liver function, enhancing tolerance to subsequent repeated ablation. yoshida et al. [ ] evaluated the efficacy of lam in hbv-hcc patients who were treated with rfa. complete ablation of hcc was achieved in patients in this study. after rfa, patients was treated by lam. there were ( %) patients with serum hbv-dna negative conversion. four patients showed alt elevation and redetection of hbv-dna. there was no difference in recurrence-free survival and overall survival between the two groups. in the lam group no specific adverse effect was observed. the conclusion was that lam for patients with hbv-hcc after rfa was safe and liver function was improved. kuzuya et al. [ ] evaluated the impact of antiviral therapy with lam on patients after initial treatment for hbv-hcc. comparison was made between patients who did not received lam therapy after treatment for hcc (control group) and patients who received at a dose of mg/day (lam group) in terms of changes in survival, hcc recurrence, and remnant liver function. there was no significant difference in cumulative recurrence rates of hcc between the two groups (p = . ). however, median ctp score at the time of hcc recurrence was significantly different; (range - ) in the control group versus (range - ) in the lam group (p = . ). in the lam group, all patients were able to receive curative treatment for recurrent hcc. in contrast in the control group, of patients were unable to receive curative optimal therapy for recurrent hcc due to deterioration of remnant liver function. in the lam group, the cumulative survival rates of patients tended to be higher than those of patients in the control group (p = . ). the conclusion is that lam therapy is beneficial for hbv-hcc patients after initial treatment because it contributes to improving remnant liver function, accordingly decreasing the probability of liver failure and increasing the possibility to receive available treatment for recurrent hcc. clinical evidence showed hbv reactivation may occur in chronic hbv carriers with tumor during chemotherapy, then followed by hepatic decompensation and various complications. hbv reactivation occurs in nearly between % and % [ ] [ ] [ ] . similarly, hbv reactivation may occur in patients with hbv-hcc after tace. some of these patients even treated with lam still occur hepatic decompensation or liver failure and eventually death, because of the delay of lam antiviral therapy, suggesting that these patients need to be treated by antiviral drugs before tace. moreover, more studies [ ] [ ] [ ] also suggested that before chemotherapy early antiviral therapy can significantly reduce the chemotherapy-induced reactivation of hbv. tace is local treatment, different from systemic chemotherapy. therefore, early antiviral treatment before tace in patients with hbv-hcc can reduce the occurrence of postoperative virus reactivation, reduce the hepatitis flare caused by hbv, and reduce the mortality of acute exacerbation of chb. in , a study about reactivation of hbv in patients with hbv-hcc undergoing tace of jang et al. [ ] was published in hepatology. in a prospective and randomized study, consecutive patients with hbv-hcc undergoing tace (cisplatin mg/m and epirubicin mg/m ) at monthly intervals were assigned to receive lam mg daily from the start of tace (preemptive group) or not (control group). during the study, . % patients ( / ) in the preemptive group and . % patients ( / ) in the control group developed hepatitis due to hbv reactivation (p = . ). in addition, there were significantly more incidences of severe grade of hepatitis (p = . ) and overall hepatitis (p = . ) in the control group. on multivariate analysis, hbv dna level > copies/ml in baseline was the only independent predictor of hepatitis due to hbv reactivation during chemo-lipiodolization (p = . ). these data demonstrated preemptive lam therapy effectively reduced hepatitis due to hbv reactivation and hepatic morbidity during tace. preemptive therapy should be considered in hcc patients with an hbv dna level > copies/ml. preemptive antiviral therapy would effectively reduce liver-related morbidity attributable to hbv reactivation and would allow more prolonged chemotherapy. this study also suggested that preemptive lam therapy decreases the severity of clinical hepatitis if it develops during tace. zhu et al. [ ] investigated the efficacy of adjuvant tace with or without antiviral therapy for hbv-hcc patients after radical hepatectomy. this study enrolled patients, of whom were treated using tace combined with antiviral therapy and using tace alone. analysis of all patients suggested that overall survival of the combination therapy group was better compared to the tace-only group (p = . ), while disease free survival was similar between the two groups (p = . ). analysis of only propensity score-matched pairs proved -year overall survival in the combination therapy group was significantly better ( . % vs. . %, p = . ) and also suggested better -year disease free survival ( . % vs. . %, p = . ). for patients after hcc recurrence, radical hepatectomy was the treatment choice for a significantly larger proportion of patients from the combination therapy group than from the tace-only group (p = . ). these data suggested that combining tace with antiviral therapy significantly improved overall survival and potentially disease free survival relative to tace alone in hbv-hcc patients. combination tace with antiviral therapy also improves remnant liver function, increasing the chance of curative resection in case of tumor recurrence. combination tace with antiviral therapy may benefit to prevent recurrence of hcc after radical hepatectomy. it has been observed that hbv reactivation occurs after the end of radiotherapy in a way similar to that after cytotoxic chemotherapy [ ] . radiotherapy to hcc can damage immune system, and cause leukocytes decreased, following by hbv reactivation. so antiviral therapy before radiotherapy for hbv dna positive patients is necessary. kim et al. [ ] evaluated the impact of three-dimensional conformal radiotherapy ( d-crt) on hbv reactivation and hepatitis exacerbation in hbv-hcc patients. this study enrolled hbv-hcc patients who underwent d-crt to the liver. group (n = ) treated lam before and during d-crt and group (n = ) did not treat with nas before d-crt. to investigate spontaneous hbv reactivation, hcc patients received no specific treatment for chb or hcc were included as a control group. the cumulative rate of radiation-induced liver disease in groups was higher than group ( . % vs. . %, p > . ). the cumulative rate of hbv reactivation was significantly higher in group ( . %) than in group ( %) or the control group ( . %) (p < . each). there was no significant difference in cumulative rate of chb exacerbation between groups ( %) and ( . %) or the control group ( . %) (p > . each). that demonstrated that hbv reactivation and consequent chb exacerbation should be considered in hbv-hcc patients after d-crt and antiviral therapy should be recommended to prevent liver function after rt. in study of huang et al. [ ] , the rate of hbv reactivation and chb exacerbation was . % ( / ) and . % ( / ), respectively. there was a relatively high rate of hbv reactivation in those patients and whose prognosis was unfavorable. the serum hbv dna level and some dosimetric parameters (normal liver volume, v , and dmean) were the prognosis factors for hbv reactivation and should been considered carefully before crt. the goal of anti-hbv therapy is to effectively reduce the hbv dna level, thereby reduce the incidence of cirrhosis and hcc. although antiviral therapy is recommended in guidelines from apasl, easl and aasld, the specific procedures are not the same. and these guidelines do not give treatment recommendations for patients with hbv-hcc. current clinical studies have confirmed that early antiviral therapy is necessary for the prevention of liver function and reduce the integration of the viral dna. although antiviral therapy inhibits viral replication, the integration of viral dna continued. there are two distinct types of hcc recurrence: tumors grown from dissemination of the primary tumor and de novo tumors arising from the "field effect" in diseased liver [ , ] . this may argue for an earlier antiviral intervention, as adjuvant therapy after the resection for the hcc patients with a high hbv dna level to prevent recurrence. anti-hbv therapy were performed in the light of the recently updated hbv treatment guidelines, on the recurrence and prognosis of hcc. to substantiate the beneficial effects of antiviral therapies, future randomized clinical trials (rcts) with longer follow-up, larger sample size, and regular hbv dna level monitoring will be needed to conduct. the molecular mechanisms of preventing tumor recurrence also need to be further studied. jun-ying qi and ming ni liver transplant is an effective treatment for hbv-related end-stage liver disease. the risk of hbv reinfection after liver transplant is the main limiting factor for long-term survival rate. combination therapy of lamivudine and hepatitis b immunoglobulin (hbig) reduced the rate of recurrence. however, considering the disadvantages of high dose hbig and high rate of lamivudine resistance, other therapies that composed by entecavir, tenofovir, or lamivudine plus adefovir, with or without hbig have been used in several liver transplant centers. other researchers have used posttransplant hbv vaccination for achieving a lasting endogenous anti-hbs antibody, yet the efficacy is still controversial. the combination hbig/nucleotide (acid) prophylaxis should be converted to oral prophylaxis within or years after liver transplantation. recently, the discovery of sodium taurocholate co-transporting polypeptide (ntcp) as the cellular receptor for hbv entry has opened up many channels of investigation, which indicate the possibility of using ntcp inhibitor in the prophylaxis of hepatitis b recurrence post lt. liver transplant (lt) is an effective treatment for hepatitis b virus (hbv)-related end-stage liver disease (such as acute or chronic liver failure, cirrhosis, hepatocellular carcinoma and so on). china has become the world's second-largest lt country as there are nearly operations annually. until the end of , there were , cases of liver transplant had been completed in china, % of which were due to hepatitis b-related liver disease, and nearly % recipients with detectable hbv dna. the risk of hbv reinfection after lt is the main limiting factor for long-term survival rate. the rate of hbv reinfection is as high as % without antiviral prophylaxis [ ] . lt recipients with recurrent hepatitis b develop an aggressive form of fibrosing cholestatic hepatitis, cirrhosis or graft failure within years [ , ] , which lead to death or re-lt. combined treatment of hepatitis b immunoglobulin (hbig) and nucleos(t)ide analogues (nas) reduced the hbv recurrence rate to - % after years of liver transplantation [ ] [ ] [ ] [ ] [ ] . [ ] . another problem in patients with hbv disease post lt is the presence of extrahepatic reservoirs of the virus that are continuous latent source of hbv recurrence [ ] . on the other hand, late recurrence is related to low anti-hbs titer or the development of hbs viral escape mutations or ymdd mutations [ ] . the strategies to prevent hbv reinfection after lt involve three stages: pre-, at and post-transplant. currently, the strategies include passive immunization (hbig), antiviral therapy (nas) and active immunization (hepatitis b vaccine). hbig was the first drug to effectively prevent hbv recurrence. limited duration of hbig therapy (< months) [ , iu iv at lt, , iu iv daily for days after lt, then iv at different intervals to maintain anti-hbs titers > iu/l] delayed but did not prevent hbv reinfection [ ] . the efficacy of this treatment seemed to be dependent on the viral load pretransplant. there was % developed recurrent hepatitis b years after transplant in patients with detectable hbv dna in serum. the recurrent rate were % in patients who were hbv dna negative pretransplant [ ] . this problem was partially resolved by using higher doses of hbig. monthly fixed doses of , iu of hbig (to keep anti-hbs levels > iu/l) or different hbig doses adjusted to maintain anti-hbs > iu/l for the first months after liver transplant significantly reduced the rate of recurrence in patients with detectable hbv dna pretransplant [ ] [ ] [ ] . however, using high doses of hbig was very expensive. lamivudine had apparent effect on hbv dna replication, decreasing the positive rate of hbv dna in patients undergoing or waiting for liver transplantation. data from north american transplant centers showed that treatment with lamivudine improved pre-liver transplantation and liver transplantation-free survival of patients awaiting liver transplantation for hbv-related cirrhosis [ ] . the early results of monotherapy of using lamivudine to prevent hbv recurrence post-lt were promising. in nine of ten survivors, hbsag and hbv dna were negative, and liver biopsy showed no evidence of recurrent by week [ ] . however, % patients re-infected with lamivudine-resistant hbv by - months post-transplant [ ] . hbig and lamivudine are different in action mechanisms. hbig is a specific passive immune agents prepared from individual plasma who has been infected by hbv or injected hepatitis b vaccine. high concentration of hbig can neutralize hbv and block its infection of hepatocytes. lamivudine is a potent inhibitor of hbv-associated dna polymerase to block hbv replication. therefore hbig and lamivudine play a complementary role to each other. combined treatment of high dose iv hbig and lamivudine had been the accepted standard prophylaxis for post-lt hbv recurrence. lamivudine was used pre-lt for reducing the viral load in the peri-lt period in most centers. hbig was used at a dose of , iu daily for the first week post-operative and then either at a fixed dose of , iu/month or with different doses to keep anti-hbs titers > iu/l [ , , [ ] [ ] [ ] . some centers had targeted anti-hbs titers > iu/l in hbv dna positive patients for - months post-lt. compared to the monotherapy of hbig or lamivudine, these combined treatments are highly effective [ , ] . however, the long-term use of hbig has many disadvantages, such as high cost, the need for injection, headache, flushing, and chest pain [ , ] . moreover, the long-term use of lamivudine induces viral resistance, which leads to a high rate of recurrence post-lt [ ] . a number of studies have shown that im hbig has similar kinetics and produces roughly equivalent trough concentrations of anti-hbs compared to iv equivalent doses of hbig but less expensive [ ] . the largest reported data of prophylaxis with using of im hbig comes from investigators in australia and new zealand [ ] . im or iu hbig daily for week from transplantation and monthly thereafter. lamivudine, mg/day, was administered to candidates waiting for transplantation with hepatitis b surface antigen (hbsag)-positive and continued posttransplantation. thirty-seven patients with positive hbsag ( patients had hepatitis b, patients had hepatitis b and c, and patient had hepatitis b, c, and d) underwent liver transplantation using this protocol. thirty-six patients were hbv dna positive. the therapy had no significant adverse events and was well tolerated. all patients were hbv dna negative and patients were hbsag negative at latest follow-up. this investigation suggested that low-dose hbig combined with lamivudine prevented recurrence of hepatitis b posttransplantation is more cost-effective. long-term results of this protocol showed that the actual rate of hbv recurrence at years was % in hbsag-positive patients underwent liver transplantation [ ] . recently, entecavir and tenofovir have been approved as the first-line regimen for the treatment of chronic hepatitis b. these new nas have replaced lamivudine as the prophylaxis of hbv recurrence post lt. according to easl clinical practice guidelines, to achieve the lowest level of hbv dna pre-lt, nas with high barrier to resistance is recommended as pre-transplant antiviral therapy for all hbsag positive patients undergoing liver transplantation [ ] . hu et al [ ] reported a lower hepatitis b recurrence rate in patients received entecavir than those received lamivudine. a total of patients were administered entecavir combined with lowdose hbig, and patients received lamivudine plus low-dose hbig in the control group. two patients in the entecavir group developed hbv recurrence with no evidence of viral resistance in the median months follow-up time. eleven patients in the lamivudine group developed hbv recurrence, three of whom were proved hbv resistance in the median months follow-up period. further analysis demonstrated that hcc at the time of liver transplantation and low anti-hbs titer post-liver transplantation were independent risk factors for hbv recurrence. perrillo et al. [ ] investigated the efficacy of entecavir combined with various hbig regimens after liver transplantation. sixty-one patients with hbv-related liver disease took . mg/day of entecavir plus various hbig regimens. in the median weeks follow-up period, only patients showed positivity hbsag but hbv dna remained undetected. na et al. [ ] showed that of recipients who received entecavir plus hbig experienced hbv recurrence after liver transplantation in the median months follow-up time. among these patients, had received lamivudine followed by entecavir. studies concerning the efficacy of tenofovir in the prophylaxis of hbv reinfection post-lt are limited. jiménez-pérez et al. [ ] reported that four patients received tenofovir plus hbig with or without entecavir for the prophylaxis of hepatitis b recurrence. no hepatitis b recurrence was observed in these four patients at months. several researchers have investigated if it was possible to stop hbig after an initial successful prophylaxis with combined hbig/lamivudine. in one largest prospective study, patients who were hbvdna negative before liver transplantation received prophylaxis with hbig/lamivudine for month after transplantation, then they were randomized to continue combination prophylaxis or lamivudine monotherapy [ ] . the early results showed that there was no recurrence case in either group by months. however, - % of the patients who were converted to lamivudine monotherapy had viral breakthrough because of lamivudine resistance in longer follow-up [ ] . an alternative choice was to change from hbig/lamivudine to a combination of antiviral drugs had a higher barrier of resistance than lamivudine. several studies indicated that this method may be more effective [ , ] . in a prospective study, of patients receiving prophylaxis with low-dose im hbig/ lamivudine months post-lt were changed to adefovir/lamivudine combination therapy, whereas the other patients continued previous prophylaxis [ ] . at a median of months post-switch, patients in both group had no recurrence. one a low titer of hbsag in serum was detected in patient in the adefovir/lamivudine group but hbv dna was negative. this change in therapy improved the life quality of patients and significantly saved the cost. more recently, a multicenter, prospective study demonstrated the results of hbig-sparing regimen combined with lamivudine plus adefovir dipivoxil initiated at the time of waiting for liver transplantation and continued post-transplantation [ ] . twenty-six patients were recruited into this study at the time of listing for transplantation. twelve patients had lam exposure before the study, but none had lamivudine resistance. to the patients who underwent transplantation, iu/day of intramuscular hbig was given immediately after transplantation for days. all transplanted patients remained alive without hbv recurrence at a median of months after transplantation. after the completion of this prospective study, the regimen was modified that no perioperative hbig was administered if the pretransplant hbv dna level < log( ) iu/ml. another patients with hbv-related liver disease underwent transplantation ( without hbig). all the patients remained alive without hbv recurrence at a median of months post-transplantation. this study indicated that combination of lamivudine and adefovir initiated at the time of listing for transplantation was safe and effective prevention of hbv recurrent without high costs and long-term hbig therapy. other researchers used posttransplant hbv vaccination for achieving a durable endogenous anti-hbs antibody. two studies reported that - % of patients achieved an anti-hbs titer > iu/l following cessation of hbig and immunization with - courses of recombinant im hbv vaccine [ , ] . however, other investigations using the same protocol have failed to replicate these results [ , ] . moreover, the low response ( - %) was reported in cirrhotic patients awaiting for lt [ ] . more recently, di paolo et al investigated the efficacy of year, monthly vaccination together with hbig plus lamivudine in lt patients. one year after vaccination, . % patients maintained anti-hbs titers more than iu/l [ ] . these results suggested that hbig can be considered as an additional strategy in the prophylaxis against hbv recurrence post lt: ( ) vaccine administration should be long-lasting (e.g. year); ( ) passive prophylaxis with hbig should preferably be maintained during the initial phase of vaccination and nas should be maintained during the entire vaccination period. lamivudine is the most widely used na to prevent hepatitis b recurrence. however, lamivudine resistance can result in hepatic decompensation and increases the rate of post-transplant recurrence. newer nas with lower resistance rates should therefore replace lamivudine in hepatitis b prophylaxis. schiff et al [ ] investigated the effect of adefovir dipivoxil as the rescue therapy in listing or post-lt patients with chronic hepatitis b and lamivudine-resistance. among listing patients, the percentage of hbv dna levels undetectable at weeks and was % and %, respectively. after weeks, liver function normalized in % and % of these patients respectively. and coagulation function normalized in % and % of these patients respectively. among post-transplantation patients, the percentage of serum hbv dna levels undetectable at weeks and was % and %, respectively. after weeks, liver function and coagulation function normalized in %, %, %, and % of these patients, respectively. among listing patients who underwent liver transplantation, prevention of graft reinfection over a median of weeks was similar among patients who did or did not receive hbig. hbsag was detected on the first test only in % and % of patients who did or did not receive hbig, respectively. serum hbv dna was detected on follow-up in % and % of patients who did or did not receive hbig, respectively. adefovir dipivoxilrelated adverse events occurred in % of patients and led to drug withdrawal. cumulative resistance rate were %, %, and % at , , and weeks, respectively. in conclusion, adefovir dipivoxil is safe and effective in prevention of graft reinfection with or without hbig for listing or post-transplant chb patients with lamivudine-resistance. more recently, one study indicated that late hbig replaced by adefovir dipivoxil (at least months post-transplant) can prevent late hbv recurrence at less cost [ ] . in a prospective open-labeled study, lamivudine plus adefovir dipivoxil given from the time of listing was well tolerated, prevented lamivudine resistance pre-transplantation and post-transplantation, regardless of the baseline hbv-dna level [ ] . the rescue therapy for patients with lamivudine or telbivudine resistance is to add adefovir or tenofovir, or change to tenofovir + emtricitabine. for patients with adefovir resistance, the approach is to add lamivudine or entecavir, or switch to tenofovir + emtricitabine. for patients with entecavir resistance, the approach is to add adefovir or tenofovir. combination therapy is still effective for some patients with multi-antiviral drugs-resistance according to evidence based medicine and clinical practice [ ] . regular monitoring and follow-up for patients post lt is also very important. items include liver function, hepatitis b markers, hbv dna quantitative, mutant, blood concentration of immunosuppressive drug and ultrasound examination. for hepatitis b recurrence patients, therapy include: support treatment, hepatocyte protection, anti hbv therapy, immunosuppressant regimen adjustment (withdrawal, reduction or change immunosuppressive agents) and liver retransplant. hepatitis b is a major cause of liver failure in asia, although the use of hbig plus lamivudine can effectively prevent hbv reinfection in liver transplantation, but the cost is high. active immunization approach is still controversial. combined hbig/ nucleos(t)ide prophylaxis should be considered to switch to oral prophylaxis at or years post-lt, particularly in patients with low hbv dna loads before antiviral therapy or hbv dna negative at lt, and in patients with liver failure due to hbv or hdv coinfection, since these patients are at lower risk of recurrence once hbig is ceased. recently, the seminal discovery of sodium taurocholate co-transporting polypeptide (ntcp) as the cellular receptor for hbv entry has opened up many channels of investigation, making hbv entry amenable to therapeutic intervention. several fda approved drugs with ntcp inhibiting activity were tested for their ability to inhibit hbv infection of the cell line [ ] [ ] [ ] . these investigations indicate the possibility of using ntcp inhibitor in the prophylaxis of hepatitis b recurrence post lt. di wu and qin ning both host and viral factors are associated with the chronicity of hbv infection. hbv has a capability of escaping the host immune responses. more importantly, the hbv genome forms a stable minichromosome, namely covalently closed circular dna (cccdna), in the nuclei of infected hepatocytes, enabling hbv to persist its infection [ ] . the goal of anti-hbv therapy is to prevent the progression of hbv-related liver disease, which may be achieved initially through sustained immunologic control over hbv, and ultimately, by completely eliminating the virus [ , , ] . however, due to the fact that hbv cccdna persists stably at a very low level even after the loss of hbsag in chronic infected patients, elimination of hbv (complete cure) is rarely achieved. it is suggested that serum hbsag could represent a surrogate marker of intrahepatic cccdna and a marker of host immune control of hbv infection. seroclearance of hbsag is found to be associated with functional remission and improved long-term clinical outcomes in patients with chronic hepatitis b, under this circumstance, even though hbv genome cannot be cleared, the host immune system is in general sufficient to control the few persisting infected hepatocytes [ ] [ ] [ ] . therefore, hbsag seroclearance with or without the appearance of hbsab (functional cure) is considered the ideal endpoint for anti-hbv therapy, representing durable immunologic control over the virus and complete suppression of hbv replication, which is the critical step towards complete cure for hepatitis b [ , , ] . nuc and ifn or its pegylated form, peg-ifn, are the only two types of approved antiviral therapeutics. as the ideal endpoint for anti-hbv treatment, hbsag loss is achieved in very few patients after long-term nuc or -week courses of peg-ifn therapy [ ] [ ] [ ] . these current standard antiviral therapies can only suppress the hbv replication and viral protein production, but cannot eliminate hbv cccdna and cure chronic hbv infection. therefore, new treatment approaches such as optimal combination therapy with the approved antivirals or emerging novel therapeutics are needed to improve rates of hbsag loss and, ideally, hbsag seroconversion. different characteristics, mechanisms of action and antiviral activities of nuc and ifn provide the possibility of combining these two types of agents for improving chances of sustained post-treatment response, thereby allowing the discontinuation of nuc without virus relapse, through harnessing both immunomodulatory and direct antiviral mechanisms [ , ] . according to the updated chinese guidelines, asian-pacific guidelines, as well as european guidelines for the treatment of chronic hepatitis b, sequential therapy with additional peg-ifn or switching to peg-ifn can be considered in chb patients who have achieved virological remission by long-term nuc treatment [ ] , though clinical trials evaluating either simultaneous or sequential combination therapy with nuc and ifn for chb patients drew different conclusions. several previous studies exploring the efficacy of simultaneous combination with peg-ifn and lam or adv have demonstrated that the therapeutic strategy led to higher rates of virological response during treatment, but did not improve durable post-treatment responses [ , , , ]. an exploratory study showed that combination treatment with peg-ifn plus adv for weeks led to remarkable decline in serum hbv dna level and intrahepatic cccdna, which was significantly correlated with reduced serum hbsag [ ] . a recent study evaluating the efficacy of combination therapy with ldt and peg-ifn in hbeag-positive chb patients have demonstrated that the combination therapy led to greater reductions in hbv dna and hbsag levels, however, it may contribute to an increased risk of unexpected severe peripheral neuropathy, combination therapy with ldt and peg-ifn should not be used [ ] . in a prospective, active-controlled randomized trial evaluated loss of hbsag in patients receiving the combination of tdf and peg-ifn for a finite duration, chb patients were randomly assigned to receive combination therapy for weeks, combination therapy for weeks followed by tdf for weeks, tdf for weeks, or peg-ifn for weeks. the study demonstrated that, . % of patients receiving -week course of combination therapy with tdf and peg-ifn had hbsag loss, which was significantly higher than those receiving tdf or peg-ifn given alone [ ] . however, it is worth noting that a prolonged followup of these subgroups of patients is required to determine the durability of treatment response and long-term benefits. although simultaneous combination of peg-ifn and nuc other than tdf may not improve sustained response rate, the optimal approach for combination treatment remains to be determine and should take into consideration the time schedule of drug administration. late breaking clinical trials have demonstrated that sequential combination therapy with nuc and ifn, either "switch" or "add-on", showed more promising results, with higher probabilities of hbeag seroconversion and hbsag loss than nuc monotherapy. an observation study has shown that the add-on of peg-ifn to a stable nuc therapy in chb patients with suppression of hbv dna, induced hbsag seroconversion in out of patients [ ] . a prospective study demonstrated that additional of peg-ifn to a long-term nuc treatment in hbeag-negative patients with undetectable hbv dna, led to a durable hbsag loss in out of patients [ ] . a global multicentered, randomized controlled trial (ares study) assessed the effectiveness of add-on peg-ifn to etv therapy in hbeag positive patients, compared to etv monotherapy, weeks of peg-ifn add-on therapy did not improve response rates (defined as hbeag loss with hbv dna < iu/ml at week ), but led to greater viral decline and appeared to prevent relapse after stopping etv, which may facilitate the discontinuation of nuc treatment [ ] . another randomized controlled trial has shown that neither etv pretreatment nor etv add-on to peg-ifn demonstrated superiority in sustained posttreatment response compared with weeks of peg-ifn alfa- a monotherapy in treatmentnaive hbeag-positive patients [ ] . a prospective, randomized controlled trial (osst study) reported that switching to -week course of peg-ifn in hbeag positive chb patients who achieved virologic remission after long-term etv treatment led to significantly increased rates of hbeag seroconversion and hbsag loss ( . %) [ ] . data from -year follow-up of these patients who received sequential therapy showed that rates of hbeag seroconversion increased from . % at the end of treatment to . % at -year post-treatment, besides, hbsag loss was durable in of patients [ ] . these results are in consistent with findings from earlier studies exploring sequential combination therapy with nuc and ifn but only tested in a small number of patients [ , ] . an exploratory study assessed the efficacy of sequential therapy in genotypes a, b, c and e chb patients with high hbv viremia, patients receiving etv alone for weeks, followed by etv plus peg-ifn for weeks, lastly only peg-ifn for weeks achieved significantly higher rates of hbeag and hbsag seroconversion than those receiving peg-ifn monotherapy for weeks [ ] . an interim analysis from new switch study demonstrated that sequential combination therapy with etv and peg-ifn for weeks in nuc-experienced hbeag positive chb patients who achieved partial responses, with hbv dna suppression and hbeag loss, led to a high rate of hbsag loss ( . %) [ ] . accumulating evidences suggest that quantitative hbsag (qhbsag) is a useful marker for guiding treatment decision, e.g. individualizing the treatment, implementing stopping rules for ending or extending ifn treatment [ ] . recently, several studies identified that hbsag loss occurred in patients with a low baseline qhbsag and high on-treatment reduction, therefore, a baseline or response-guided approach based on hbsag kinetics may help identify chb patients with the greatest chance of benefit. the osst study has demonstrated that patients undergoing long-term etv treatment with low hbsag titers (< iu/ml) and hbeag loss were suitable for sequential therapy with peg-ifn as they had a good chance of both hbsag loss ( . %) and hbeag seroconversion ( . %). patients whose hbsag levels declined to iu/ml at week of sequential therapy have the greatest chance of achieving hbsag loss. while, patients whose hbsag levels were > iu/ml at week might consider stopping peg-ifn treatment as they had a minimal chance of achieving hbeag seroconversion and hbsag loss [ ] . these findings are consistent with results from previous studies and interim analyses from the new switch study, suggesting that qhbsag identifies nuc-treated patients who are the best candidates for sequential therapy, and allows response-guided treatment [ ] [ ] [ ] . however, given the small number of patients included in the exploratory analyses, these results need to be interpreted cautiously and warrant further investigation and validation. differences in study designs and characteristics of patients make it difficult to determine the optimal combination therapy with nuc and peg-ifn for chb patients at this stage. nevertheless, we could speculate that once suppression of hbv viremia has been achieved by pretreatment with nuc, the additional use of peg-ifn would be more beneficial. these new therapeutic strategies require further investigation before being introduced into routine clinical practice. complete cure of hbv infection depends not only on the deep suppression of hbv replication, but also on the induction of durable antiviral immune response [ ] . besides the approved therapeutics, several novel therapeutic approaches including direct acting antivirals (daa) targeting different stages of the life cycle of hbv (including hbv entry, hbv genome processing, virus protein assembly, etc.) as well as immunological approaches are currently under early stage of preclinical or clinical investigation, these promising therapeutics may act in a synergistic way with currently available antiviral agents and have the potential to achieve a cure of hbv infection. specific inhibition of hbv entry may be a promising therapeutic concept to control hbv infection. a currently identified cellular receptor for hbv entry, the sodium taurocholate co-transporting polypeptide (ntcp), is an emerging target providing new research possibilities and allowing the development of hbv entry inhibitors [ ] . cyclosporine a (csa) can interfere with the binding between large envelope protein and ntcp, and thus prevent hbv entry into cultured hepatocytes [ , ] . myrcludex-b also can inhibit the binding of the hbv envelope proteins to ntcp, blocking the hbv/hepatitis d virus (hdv)'s entry, which is now under clinical development [ ] . however, these entry inhibitors can prevent new hbv infection [ , ] , but do not directly target on cccdna or eliminate the preexisting hbv infection. therefore, antiviral strategies combining entry inhibitors with anti-hbv agents might be superior to their use as monotherapy by taking advantage of synergy. therapeutic approaches targeting cccdna for hbv cure aim to directly degrade or alternatively block cccdna formation, or silence cccdna transcription. rnaguided nucleases, such as the clustered regularly interspaced short palindromic repeats (crispr)/cas [ , ] , might be the most promising strategy to target cccdna. however, the risk of undesired off-target mutagenesis and delivery constitute the major limits. histone modifications, e.g. inhibitors of histone acetyltransferase, offer great potential as therapeutic candidates for chb patients through transcriptional silencing of cccdna [ , ] . activation of ifn-a and lymphotoxin beta receptor (ltβr) has been shown to induce cytidine deaminases of the apobec family, triggering degradation of cccdna in hbv cell culture model systems. these novel strategies will make elimination of hbv a real possibility [ ] . several attempts have been made to develop capsid assembly modulators/core inhibitors, which can be divided into two main classes. the first class, including phenylpropenamides (ppas) and sulfamoylbenzamide derivatives, e.g. at- and at- , can inhibit the entry of pregenomic rna (pgrna) into the immature nucleocapsid [ , ] resulting in nucleocapsid with normal size and geometry but empty of nucleic acid. the other class, including heteroaryldihydropyrimidines (haps) antiviral compounds, e.g. bay - [ ] and nvr - , can directly inhibit the nucleocapsid formation, resulting in virus particles with abnormal size and structure. ppas and haps show synergy in vitro with nucleoside reverse transcriptase inhibitors (nrtis) and overcome resistance to nrti [ , ] , highlighting the value for developing combination therapy. post-transcriptional gene silencing by rna interference (rnai), is a new therapeutic approach to hepatitis b [ , ] . inhibiting protein production by targeting hbv messenger rna (mrna) for translational repression or degradation can impair virus replication and augment the hbv-specific immune response. several rnai-based drug candidates have currently entered early-phase clinical development for the treatment of chb, including aln-hbv and tkm-hbv [ , ] , showing clinical efficacy significant declines in hbv dna, hbsag, and cccdna levels. despite lingering concerns about delivery, the risk of resistance and safety, the rnai-based therapeutics might be promising when combined with other antiviral agents. future trials with rnai-based therapeutics in combination with ifn or other antivirals are required to determine whether these agents would act synergistically to reduce viral antigen production, activate and restore the host immune responses, and subsequently eliminate hbv infection. hbsag production and secretion is capable of altering the host immune response by inducing t cell exhaustion and tolerance to hbv, which partially mediate hbv persistence. control of hbsag secretion may help restore t cell function, suggesting the possibility of developing anti-hbv treatments targeting hbsag production and release. nucleic acid polymer (nap) could prevent hbsag release from infected hepatocytes, leading to a restoration of the immune response. newly developed hbsag release inhibitors, e.g., rep and rep [ ] [ ] [ ] , appear potent in preventing the release of hbsag in humans and thereby reducing serum hbsag levels and also potentially promoting surface antibody seroconversion. however, it remains to be seen whether these compounds may cause detrimental intrahepatocyte accumulation of hbsag. emerging exciting advances have also led to new promising approaches to attenuate hbv-induced immune impairment, such as toll like receptor (tlr) agonists [ , ] , pleiotropic cytokines [ ] [ ] [ ] , programmed cell death- (pd- ) and its ligand pd-l blockages [ ] , therapeutic vaccines [ , ] , etc. tlr agonists can activate intracellular innate pathways and stimulate both innate and adaptive immune responses. a recently developed oral active agonist of tlr- , gs- , has been shown to enhance ifn-a and isg expression and activate nk cells, t cells and b cells in animal studies [ , ] , however, early human studies have shown limited efficacy of the tlr- agonist at tolerated doses, and further research into this tlr agonist was subsequently discontinued. therapeutic cytokines play critical roles in the control of hbv infection and mediate a non-cytolytic clearance of the virus [ ] [ ] [ ] . several studies investigated the antiviral activities and therapeutic potential of cytokines including granulocyte-macrophage colony-stimulating factor (gmcsf), il- , il- , etc. a previous study has shown that hbsag vaccine in combination with lam or il- could induce antiviral immune response and consequently elimination of hbv may be achieved in chb patients [ ] . a prospective study investigated whether additional gmcsf could enhanced the immunomodulatory effect of ifn, demonstrating that the combination treatment with gmcsf and ifn was effective in patients who had previously failed ifn monotherapy [ ] . a recent prospective, randomized controlled trial (anchor study) evaluated whether sequential combination therapy with nuc, peg-ifn and gmcsf could induce hbsag loss in chb patients treated with long-term nuc and demonstrated that for patients who achieved virological suppression with nuc, this sequential combination therapy significantly increases rates of hbsag loss and hbsab appearance [ ] . the difficulties in eliminating cccdna and breaking the immune tolerance constitute the major obstacles for a cure of hbv infection. 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combination/ sequential therapy with etv, peg-ifn alpha- b and gmcsf enhanced hbsag loss and appearance of hbsab in na suppressed chb patients (the anchor a study): an interim analysis toward a cure for hepatitis b virus infection: combination therapy involving viral suppression and immune modulation and long-term outcome viral hepatitis. hbv cure-can we pin our hopes on immunotherapy? key: cord- -uf jgig authors: wang, yi; liu, li title: the membrane protein of severe acute respiratory syndrome coronavirus functions as a novel cytosolic pathogen-associated molecular pattern to promote beta interferon induction via a toll-like-receptor-related traf -independent mechanism date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: uf jgig most of the intracellular pattern recognition receptors (prrs) reside in either the endolysosome or the cytoplasm to sense pathogen-derived rnas, dnas, or synthetic analogs of double-stranded rna (dsrna), such as poly(i:c). however, it remains elusive whether or not a pathogen-derived protein can function as a cytosolic pathogen-associated molecular pattern (pamp). in this study, we demonstrate that delivering the membrane gene of severe acute respiratory syndrome coronavirus (sars-cov) into hek t, hek et, and immobilized murine bone marrow-derived macrophage (j -mφ) cells significantly upregulates beta interferon (ifn-β) production. both nf-κb and tbk -irf signaling cascades are activated by m gene products. m protein rather than m mrna is responsible for m-mediated ifn-β induction that is preferentially associated with the activation of the toll-like receptor (tlr) adaptor proteins myd , tirap, and ticam but not the rig-i signaling cascade. blocking the secretion of m protein by brefeldin a (bfa) failed to reverse the m-mediated ifn-β induction. the antagonist of both tlr and tlr did not impede m-mediated ifn-β induction, indicating that the driving force for the activation of ifn-β production was generated from inside the cells. inhibition of traf expression by specific small interfering rna (sirna) did not prevent m-mediated ifn-β induction. sars-cov pseudovirus could induce ifn-β production in an m rather than m(v a) dependent manner, since the valine-to-alanine alteration at residue in m protein markedly inhibited ifn-β production. overall, our study indicates for the first time that a pathogen-derived protein is able to function as a cytosolic pamp to stimulate type i interferon production by activating a noncanonical tlr signaling cascade in a traf -independent manner. in addition to the tlr, which can be defined as a membraneassociated prr, another set of prrs is localized at the cytoplasm and mainly includes rig-like receptors (rlrs) and nod-like receptors (nlrs) to sense viral dsrnas and bacterial cell wall components, respectively ( , ) . the rlrs consist of at least three members, including rig-i, mda , and lgp . rig-i recognizes =-triphosphate rna and short dsrna ( , ) , while mda senses long dsrna ( ) . an adaptor protein, mavs, is required for the activation of the rig-i/mda signaling pathway. the association of viral nucleic acids with mavs promotes the aggregation of mavs on the mitochondrial membrane ( ) . the "ligation" of traf with the aggregated mavs may promote the phosphorylation of irf that is required for ifn-␤ production ( ) . a recent study also shows that an endoplasmic reticulum (er)-derived adaptor protein, sting, could also function downstream of mavs to promote irf phosphorylation and the subsequent ifn-␤ response ( ) . pathogen-derived proteins such as virus-encoded proteins are frequently documented as negative regulators in subverting type i interferon (ifn-i) induction by interfering with a certain key component(s) of ifn-i activation signaling cascades. viral evolution may develop a unique strategy to inhibit host innate immunity by generating virus-derived antagonists to some key signaling molecules. the vaccinia virus encodes two toll/interleukin- (il- ) receptor (tir) domains containing proteins a r and a r, which can negatively regulate tlr signaling by two distinct mechanisms ( ) . the vaccinia virus a r inhibits tlr signaling by physically interacting with the bb loop of tir containing adaptor proteins such as myd adaptor-like (mal) and trif-related adaptor molecule (tram) to disrupt receptor-adaptor (e.g., tlr -mal and tlr -tram) interactions ( , ) . differently, the a r protein may function as a dominant negative myd to directly interact with traf and irak ( , ) . on the other hand, the vaccinia virus n l protein, another protein homologous to a r, employs a different anti-ifn-i strategy by targeting both the tbk /ib kinase (ikk) and ikk␣/ikk␤ complexes to inhibit irf and nf-b signaling, respectively ( ) . alternatively, virus may invade the cells to target the retinoic acid-inducible gene i (rig-i)-like receptor signaling pathways for the prevention of ifn-i induction. for example, the influenza virus nonstructural protein ns can sequester either the dsrna or =triphosphate rna products of viral infection which can be sensed by or directly bound to the rna helicase sensor rig-i to inhibit rig-i-mediated ifn-␤ production ( , , ) . the paramyxovirus v protein inhibits ifn-␤ induction through the blockage of mda , another rig-i-homologous cytosolic dsrna sensor ( ) . a recent study revealed that the transcriptional factor irf might be alternatively targeted and inhibited by the paramyxovirus v protein to impede ifn-␤ gene transcription ( ) . it has been demonstrated in some cases that viral proteins may function as extracellular pamps to activate the ifn-i immune response, most often through tlr (such as tlr and tlr ) signaling pathways ( ) ( ) ( ) . however, evidence is lacking in regard to whether or not a virus-derived protein can function as a cytosolic pamp. our initial study indicates that delivering the membrane gene into hek cells markedly induces type i interferon (ifn-i) production ( ) . to our knowledge, there are limited reports regarding the induction of ifn-␤ expression directly by viral structural genes. therefore, it is intriguing to know which mechanism is responsible for the severe acute respiratory syndrome coronavirus (sars-cov) m gene-mediated ifn-␤ response. in this study, we demonstrate that sars-cov m protein, rather than its mrna, activates ifn-␤ and nf-b responses through tlr-related traf -independent signaling cascades. the driving force for m-mediated ifn-␤ induction was most likely generated from the inside of the cells. using sars-cov pseudovirus as an infectious agent, we further show that single point mutation at the valine residue of m protein markedly inhibits virus-induced ifn-␤ production. overall, sars-cov m protein may stand out as a novel cytosolic pamp in mediating the ifn-␤ immune response. the sars-cov m gene stimulates beta interferon gene expression in the human embryonic kidney t cell line. the overexpression of the sars-cov m gene has been shown to upregulate the transcriptional level of ifn-␤ ( ) . to further confirm the result, using either enhanced green fluorescent protein (egfp) or poly(i:c) as a negative or positive control, respectively, we demonstrated that m gene products specifically promoted ifn-␤ production, since cotransfection with m small interfering rna (sirna) completely abolished m-mediated ifn-␤ induction at both protein (fig. a , comparing lanes and ) and mrna ( fig. b ) levels. moreover, after a -h transfection, cell supernatants were collected and assayed for the presence of ifn-␤ by enzyme-linked immunosorbent assay (elisa). figure c clearly demonstrated that delivering pcmv-myc-m into hek t cells specifically and significantly promoted the secretion of ifn-␤ into cell culture medium. in addition, the promoter sequence of ifn-␤ was placed upstream of the firefly luciferase reporter to generate the pgl -ifn-␤-luc construct. to test the specificity of m-mediated ifn-␤ induction, other viral envelope-associated genes such as the spike (s) and envelope (e) protein genes as well as the m mutant [m(v a)] from the gz isolate were also included ( ) . the result of a dual-luciferase assay using the renilla luciferase gene as a transfection control demonstrated that the sars-cov m gene rather than the s and e genes markedly increased ifn-␤ promoter activity (fig. d) , whereas the valineto-alanine alteration at residue of m protein completely abolished this induction, indicating that the specificity of m gene products played a role in this process. consistent with these results, western blotting and elisa further validated the above observation ( fig. e and f). to detect if the sars-cov m gene has a direct effect on nf-b activation, pcmv-myc-m was cotransfected with pnfb-luc, which contained five copies of nf-b recognition sites, into hek t cells. the results of the dualluciferase assay revealed that the sars-cov m gene specifically and dramatically upregulated nf-b activity compared with the controls (fig. g) . moreover, m could mediate ifn-␤ induction in both dose-and time-dependent manner ( fig. h and i) . overall, the data strongly indicated that the sars-cov m gene product was sufficient to promote ifn-␤ gene expression. the sars-cov m gene product activates the ifn-␤ signaling pathway at or upstream of tbk . to further confirm the above results, increased doses of the pcmv-myc-m gene were transiently transfected into hek et cells. the cell lysates prepared from the transfection were examined for the activation of the downstream modulator and/or effector molecules, such as tbk , irf , and nf-b. figure a clearly demonstrates that sars-cov m gene products not only enhanced the phosphorylation level of tbk but also promoted the activation of both nf-b p and irf , indicating that m gene products may stimulate ifn-␤ activation by promoting its enhanceosome activity. to further define the activation level of m-mediated ifn-␤ induction, specific sir-nas that selectively targeted either tbk ( fig. b and c) or irf ( fig. e and f) mrnas were generated. individually diminishing either tbk or irf mrna expression by sitbk or siirf sig-nificantly reversed m-mediated ifn-␤ induction ( fig. d and g, respectively), indicating that m-mediated ifn-␤ induction functions at a level at or above the signaling molecule tbk . the sars-cov m gene product preferentially activates ifn-␤ production through toll-like-receptor-related signaling pathways in hek et cells. rlr and tlr are two main prrs hek t cells. after , , , and h of transfection, dual-luciferase assays were performed to detect ifn-␤ expression. each value represents the mean Ϯ standard deviation from three independent tests. *, p Յ . ; **, p Յ . . in all data presented above, the relative luciferase activity was determined as firefly luciferase activity divided by renilla luciferase activity. the effect of tbk sirna (sitbk ) on the expression of endogenous tbk by semiquantitative rt-pcr. the increased doses of pbs/u -sitbk plasmid dnas ( , . , and g) were transiently transfected into hek et cells. total rnas or whole-cell lysates were isolated or harvested at h posttransfection. one-step rt-pcr (rt) was conducted to detect the tbk mrna expression with specific primers (upper panel), while western blotting (wb) was performed to detect tbk protein expression using specific anti-tbk antibody (lower panel). the expression of ␤-actin served as a loading control. the relative band intensity was quantitated with the image j program in comparison with the ␤-actin control. the result is representative of at least identical experiments. (c) effect of sitbk on the expression of tbk mrnas by real-time qrt-pcr analysis. total rnas isolated in panel b were subjected to qrt-pcr analysis using specific tbk primers. each value represents the mean Ϯ standard deviation from three reactions. the result is representative of at least identical experiments. (d) effect of sitbk on m-mediated ifn-␤ induction. plasmid pgl -ifn-␤-luc reporter was cotransfected with g of pcmv-myc-m or g of pcmv-myc-m plus increased doses of sitbk ( , . , and g) into hek et cells grown on a -well plate. at h posttransfection, the dual-luciferase assay was conducted to assay fold induction of m-mediated ifn-␤ expression. each value represents the mean Ϯ standard deviation from three independent tests. (e) effect of irf sirna (siirf ) on expression of endogenous ifr by semiquantitative rt-pcr. the increased doses of pbs/u -siirf plasmid dnas ( , . , and g) were transiently transfected into hek t cells. total rnas or whole-cell lysates were isolated or harvested at h posttransfection. one-step rt-pcr was conducted to detect irf expression with specific primers (upper panel), while western blotting was performed to detect irf protein expression (lower panel). the expression of ␤-actin served as a loading control. the result is representative of at least identical experiments. (f) effect of siirf on m-mediated ifn-␤ mrna expression by real-time qrt-pcr analysis. real-time qrt-pcr was performed to detect the irf mrna (isolated in panel e) expression. each value represents the mean Ϯ standard deviation from three reactions. the result is representative of at least identical experiments. (g) effect of siirf on m-mediated ifn-␤ induction by luciferase assay. plasmid pgl -ifn-␤-luc reporter was cotransfected with g of pcmv-myc-m or g of pcmv-myc-m plus increased doses of siirf ( , . , and g) into hek et cells grown on a -well plate. at h posttransfection, the dual-luciferase assay was conducted to assay fold induction of m-mediated ifn-␤ expression. each value represents the mean Ϯ standard deviation from three independent tests. recognizing the majority of extracellular and intracellular pamps. upon the ligation of a prr with its specific pamp, both rlr and tlr pathways transmit the signal to a common class of adaptors called tumor necrosis factor (tnf) receptor-associated factors (trafs) including traf /traf , traf , and traf ( , ) . to test the effect of m on rlr and tlr signaling as well as traf expression, an increased dose of pcmv-myc-m constructs was first transiently transfected into hek et cells. the results in fig. a demonstrate that the increased delivery of pcmv-myc-m into hek et cells markedly enhanced traf but not traf and traf expression, indicating that traf -mediated signaling transduction might contribute to the upregulation of ifn-␤ production. to further address how traf expression is associated with rlr and/or tlr signaling pathways, the m genetransfected hek et cells were also assayed for the expression of upstream sensors and/or signaling molecules of trafs. figure b demonstrates that no significant alteration was observed in the expression of rig-i, mda , and mavs after exogenously delivering m genes into hek et cells, indicating that the rlr signaling pathway might not be targeted by m gene products. in contrast, three adaptor proteins (myd , tram/ticam , and tirap) associated with tlrs were all upregulated (fig. c) , while the adaptor protein trif failed to be upregulated in responding to m gene overexpression (fig. d) . overall, the results indicate that tlr signaling pathways are mainly targeted by the sars-cov m gene product for the induction of ifn-␤ expression. the sars-cov m gene product promotes ifn-␤ production through toll-like-receptor-related signaling pathways in immortalized murine bone marrow-derived macrophage cells. to further confirm the above results, the pcmv-myc-m construct was also transiently transfected into j -m cells, an immortalized murine bone marrow-derived macrophage cell line established with j virus ( , ) . the delivery of the m gene product is effec-tive in stimulating the activation of both ifn-␤ and nf-b in murine j -m cells (fig. a , b, and c). figure d demonstrates that increased delivery of m gene product into j -m cells indeed promotes ifn-␤ induction through the phosphorylation of irf , nf-b p , and tbk . in accord with the results in hek et cells, the increased delivery of pcmv-myc-m into j -m cells markedly enhanced traf but not traf and traf expression (fig. e) . moreover, the m gene product did not significantly increase the protein levels of rig-i, mavs, sting, and mda ( fig. f) , indicating that the rig-i signaling pathway might not be activated in responding to exogenous delivery of the m gene product into j -m cells. in contrast, the increased delivery of the m gene into j -m cells markedly enhanced myd and tram/ ticam but not trif expression (fig. g ), indicating that tlrrelated signaling pathways might be mainly associated with m-mediated ifn-␤ induction. overall, our data in both hek et and j -m cells strongly indicate that m-mediated induction of ifn-␤ expression is likely associated with the activation of tlr-related signaling pathways. the sars-cov m gene product functions at the protein level to induce ifn-␤ production. the next question that we tried to ask was at which level (mrna or protein) the m-mediated ifn-␤ induction occurred. to address this issue directly, we created an m-stop construct by replacing the start codon aug with three in-frame tandem stop codons at the = end of the m gene (fig. a ). this expression construct can generate only mrna and no protein due to the translation failure of the mrna substrates. western blot analysis shows that the m protein synthesis was completely blocked in the m-stop construct but not the wild-type m construct (fig. b ). real-time quantitative reverse transcription pcr (qrt-pcr) analysis shows that the m-stop construct did not induce ifn-␤ production in hek t cells, indicating that m-mediated ifn-␤ production is dependent on m protein rather than m mrna (fig. c) . to directly compare m-and m-stop-induced ifn-␤ production levels, the increased doses of either m or m-stop constructs were cotransfected with ifn-␤ luciferase reporter into hek t cells. figure d clearly demonstrates that m-stop does not induce ifn-␤ production, indicating that protein translation is necessary for m-induced ifn-␤ production. to confirm this result further, the chemical inhibitor cycloheximide (chx) was used to block the protein biosynthesis in transfected hek t cells. figure e shows that addition of chx significantly inhibited and completely blocked m protein synthesis at g/ml and above g/ ml, respectively, which are directly correlated with the marked reduction and complete inhibition in ifn-␤ expression, indicating that m protein may function as a pamp to induce ifn-␤ production. if m protein indeed functions as a pamp, blocking m protein synthesis should prevent the m-mediated activation of the ifn-␤ signaling pathway. figure f clearly shows that m-stop reverses the m-mediated upregulation of the adaptor proteins myd and ticam /tram in the initiating phase of tlr signaling pathways. moreover, m-stop prevents the activation of the downstream modulator and key effectors of the ifn-␤ signaling pathway, such as tbk , irf , and nf-b p , in a dose-dependent manner (fig. f ). thus, blocking m protein translation could prevent m-mediated ifn-␤ induction by inactivating the tlrrelated signaling pathway. sars-cov m protein may function as a novel intracellular pamp to induce ifn-␤ production. one critical question remaining to be answered is whether the driving force for m protein-mediated ifn-␤ induction is generated intracellularly or extracellularly. to answer this question directly, the trap␥ gene, an endoplasmic reticulum (er)-associated gene, was cotransfected with m into hela cells. brefeldin a (bfa) was employed in the assay system to block m protein transport from the rough endoplasmic reticulum to the cell surface. indeed, the addition of bfa effectively increased the retention of m proteins in the er compartment (fig. a) . figure b shows that addition of bfa also effectively inhibited the secretion of ifn-␤ into the cell culture medium (right panel) but did not inhibit m-mediated ifn-␤ induction at either the mrna level or the protein level (left panel), indicating that the driving force for m-mediated ifn-␤ induction was indeed derived from intracellular stimulation by m proteins. reports indicate that sars-cov m protein alone can form virus-like particles (vlps) that can be secreted extracellularly. therefore, the secreted m protein might be sensed by its own or other cell prrs on the cell surface to activate ifn-i responses. to rule out this possibility, oxpapc (a tlr and tlr inhibitor) was used to block the function of the accessory proteins cd , lbp, and md that are required for tlr and tlr signaling ( ) . figure c shows that addition of oxpapc could effectively inhibit lipopolysaccharide (lps)-mediated ifn-␤ production (right panel) but did not reverse the m-mediated ifn-␤ induction (left panel), indicating that the extracellular stimulation by m pro- sars-cov m protein promotes ifn-␤ induction independently of traf . it has been well known that traf plays a critical role in tlr-mediated ifn-␤ induction, especially through tlr and tlr pathways ( , , ) . since m protein could activate the tlr pathway from inside the cells, it remained unclear whether or not this activation is in a traf -dependent or -independent manner. to address this issue directly, a specific sirna against traf was successfully constructed (fig. a) . a dual-luciferase assay was conducted to assay the effect of sitraf on m-mediated ifn-␤ induction in hek t cells. figure b clearly shows that the increased delivery of sitraf did not reverse the m-mediated ifn-␤ induction, indicating that traf might not be essential for this activation. to further confirm the above result, western blot analysis was performed to detect ifn-␤ expression in responding to the increased delivery of sitraf into hek t cells. the increased delivery of sitraf into hek t co-ip was conducted as shown in the left panel. about % input from each lysate preparation was subjected to western blot analysis using anti-ha, anti-flag, and anti-myc antibodies as probes. the rest of the lysate was first immunoprecipitated with anti-flag antibody conjugated with an affinity gel. then, the reaction products were probed with anti-ha and anti-myc antibodies. a reverse co-ip experiment was also conducted as shown in the right panel. the lysates were first immunoprecipitated with anti-myc antibody. then, the reaction products were individually probed with anti-myc, anti-flag, and anti-ha antibodies and subsequently subjected to western blotting. the result is representative of at least identical experiments. ib, immunoblotting; ns, nonspecific. (h) the m gene product does not suppress poly(i:c)-mediated ifn-␤ induction. about g/ml of poly(i:c) was cotransfected with g of either m or m(v a) plasmid along with pgl -ifn-␤luciferase reporter ( ng) plus prl-tk ( ng) into hek t cells. after h of transfection, dual-luciferase assays were performed to detect ifn-␤ expression (left panel). each value represents the mean Ϯ standard deviation from three independent tests. the statistical difference was considered to be significant at a p value of Յ . . the ifn-␤ expression was also subjected to western blotting (right panel). the relative band intensity was quantitated with the image j program in comparison with the ␤-actin control. cells failed to inactivate the activation of irf and nf-b p and did not affect the m-mediated ifn-␤ induction (fig. c) , while a result was obtained consistent with the increased delivery of si-traf into j -m cells (fig. d) . in addition, a stable hek cell line with traf knocked down by sitraf was also established (fig. e) . a dose-dependent increase in ifn-␤ production was observed with the increased delivery of m gene into si-traf stable cells (fig. f) . thus, the above data strongly indicate that traf is not required for m-mediated ifn-␤ induction. sars-cov m protein has been shown to destabilize the functional traf -tbk complex formation ( ) . however, it remains unknown whether or not m protein is able to associate with the key components of this complex. to clarify this issue directly, a coimmunoprecipitation (co-ip) experiment was conducted. plasmid tbk -flag, traf -ha, and myc-m dnas were transiently cotransfected into hek t cells. the cell lysates were first immunoprecipitated with anti-flag antibody conjugated with an affinity gel and then subsequently probed with antihemagglutinin (anti-ha) and anti-myc antibodies. the left panel of fig. g indicates that m protein was able to disrupt the physical interaction between tbk and traf , but m protein itself could not form a complex with tbk . in the reverse immunoprecipitation (ip) experiment as shown in the right panel of fig. g , m protein was unable to interact with both tbk and traf directly, indicating that m protein may modulate the tbk -traf complex formation indirectly. interestingly, in contrast to the inhibitory effect of m(v a) on poly(i:c)-mediated ifn-␤ induction, the m gene product did not affect the ifn-␤ induction stimulated by poly(i:c) (fig. h) , indicating that m has no effect on poly(i:c)induced ifn-␤ production while retaining the ability to destabilize the complex formation. one critical question remaining to be answered is whether or not m-mediated ifn-␤ induction could occur in real virus infection. it has been shown that codelivering the m, n, and s genes of sars-cov into hek cells readily produced sars-cov pseudovirus with the corona-like halo ( ) . earlier results demonstrate that valine-toalanine mutation at residue [abbreviated as m(v a)] inhibited ifn-␤ induction ( ) . we employed a sars-cov pseudovirus system to mimic the real sars-cov infection. cell lysate supernatants isolated from either m, n, and s cotransfection or m(v a), n, and s cotransfection were first incubated with -ace stable cells (fig. a) for h. then, the cell culture medium was replaced with fresh medium and further incubated at °c for another h before harvesting. the sars-cov pseudovirus vlp(s-m-n) markedly upregulated ifn-␤ expression at both rna and protein levels (fig. b) , whereas the point mutation at the valine residue of m protein completely diminished sars-cov pseudovirus-mediated ifn-␤ induction, strongly indicating that the m protein residing on sars-cov virion could specifically induce ifn-␤ production upon infecting the targeted cells. taken together, our data indicate for the first time that sars-cov m protein may function as a novel cytosolic pamp to activate ifn-␤ induction through an intracellular tlr-related signaling pathway in a traf -independent manner. pathogen-associated molecular patterns (pamps) are pathogenborne components that can be sensed by either transmembrane, endolysosomal, or cytosolic sensors known as pattern recognition receptors (prrs) ( ) . in this study, we demonstrate that the membrane protein of sars-cov significantly upregulates ifn-␤ production by activating both nf-b and tbk -irf signaling cascades. our data show that m-mediated ifn-␤ production is induced by m protein rather than m mrna, indicating that pathogen-derived protein might be able to serve as a cytosolic pamp. in addition, a mechanism study indicates that m proteinmediated ifn-␤ induction may be mainly due to the selective activation of tlr-related signaling pathways rather than the rlr signaling pathway in a traf -independent manner. overall, the current study indicates for the first time that pathogen-derived protein may function as a novel cytosolic pamp to initiate a tlrrelated traf -independent signaling pathway that subsequently promotes type i interferon (ifn-i) production. the membrane-associated prrs, such as tlr and tlr , can sense not only bacterial components but also viral coat proteins ( ) . kurt-jones and colleagues first demonstrated that the innate immune response to the fusion (f) protein of respiratory syncytial virus (rsv) is mediated by tlr plus its cofactor cd on the plasma membrane, indicating that nonbacterial components can serve as extracellular pamps ( ) . although some viral structural proteins, such as the nucleocapsid (n) from measles virus ( ) and viral ribonucleoprotein from vesicular stomatitis virus ( ) , can activate irf and tbk /ikk␦, respectively, it remains to be determined how these viral products can function as pamps and where the driving forces for their activation come from. it has been shown that the sars-cov m protein alone not only can form virus-like particles (vlps) in a viral rna-independent manner ( ) but also can induce cell apoptosis by inhibiting some key survival signal pathways, such as the akt signaling pathway ( ) . thus, the m proteins derived from these infected and apoptotic cells might be eventually secreted and released into the extracellular compartments where the tlrs (such as tlr and tlr ) on the cell surface can be potentially recognized and subsequently activated by these extracellular pamps for the induction of the ifn-i response. to clarify this possibility, we employed several approaches to test whether the driving force for m proteinmediated ifn-␤ production is generated from inside or outside the cells. we used two types of inhibitors, bfa and oxpapc, to block either the intracellular transport of m protein or the extracellular binding of m protein on tlr and tlr , respectively. our results reveal that m-mediated ifn-␤ induction is independent of m protein secretion as well as the extracellular stimulation of tlr /tlr signaling, indicating that the driving force for the m-mediated ifn-␤ induction is likely generated from inside the cells. our results may contradict some previous reports related to m-mediated ifn-i response. siu et al. demonstrated that the m protein of sars-cov negatively regulated the dsrna-induced and signaling molecule-induced ifn-i production. they also showed that m protein alone was unable to activate the ifn-i promoter activity ( ) . one study even showed that m protein negatively regulates the nf-b signaling pathway ( ) . a possible explanation for these discrepancies might be related to the m gene itself. early study has shown that m protein possesses a higher substitution rate than other structural proteins in sars-cov, and the outcome of these substitutions alters the biochemical and immunological properties of m proteins ( , ) . one amino acid alteration from valine to alanine at residue is indeed found in the m protein from the gz isolate studied by siu et al. compared with the isolate used in the current study. our functional analysis provides strong evidence to demonstrate that the valine-toalanine change at residue of m protein is sufficient to abolish m-mediated ifn-␤ induction at both the transient-transfection level and the viral infection level (fig. d and e and b and c) . therefore, this amino acid substitution in m proteins indeed affects the interaction between m protein and the prr for the subsequent ifn-i induction. it still remains elusive which cytosolic prr is responsible for m-mediated ifn-␤ induction. the m protein might be a multifaceted molecule that physically interacts with diverse intracellular sensors and signaling factors ( , ) . our data indicate that the tlr-related signaling pathway rather than the rig-i signaling cascade is responsible for m-mediated ifn-␤ induction. interestingly, we observed consistent traf reductions in both hek t and j -m cells as tested by the transiently intracellular overexpression of the m gene. traf is one of the key signaling molecules specifically responsible for ifn-i induction ( ) . data from the work of siu and colleagues have shown that m(v a) excludes the traf inclusion in the traf .tank.tbk /ikk complex ( ) . although m(v a)-mediated traf exclusion inhibits ifn-␤ induction, our study showed that m-mediated traf exclusion is independent of ligand stimulation and the disassociated traf and tbk could not complex with m protein, indicating that m protein might modulate the functions of tbk complex indirectly. interestingly, we demonstrated that m-mediated ifn-␤ induction was associated with the upregulation of the adaptor proteins myd , tirap, and ticam but not trif, which are all involved in the initiating phase of tlr signaling pathways. trif is required for both tlr -and tlr mediated ifn-␤ induction. in the activation of tlr , trif is directly recruited to the tir domain of dimerized tlr , while in the activation of tlr , trif is recruited to dimerized tlr indirectly through another tir-containing adaptor protein, ticam (also called tram). if trif is not required for m-mediated ifn-␤ induction, m-mediated ifn-␤ induction might be activated via a noncanonical tlr -related signaling cascade independently of trif and traf . in summary, the current study demonstrates for the first time that the m protein of sars-cov is able to function as a cytosolic pamp to promote ifn-␤ production by activating a tlr-related traf -independent pathway. the driving force for m-mediated ifn-␤ induction is likely generated from inside the cells rather than the extracellular binding of m proteins with the defined cell surface prrs, such as tlr . plasmid construction. plasmids pcmv-myc-m and pbs-u -sim were constructed previously ( ) . plasmids pcmv-myc-s and pcmv-myc-e were constructed by inserting s and e into the ecori and kpni sites of pcmv-myc. the mutant of pcmv-myc-m(v a) was generated by using the site-directed mutagenesis kit (takara, dalian, china). one copy of the ifn-␤ promoter sequence ( =-ctaaaatgtaaatgacata ggaaaactgaaagggagaagtgaaagtgggaaattcctctgaat agagagaggaccatctcatataaataggccatacccatggagaa aggacattctaactgcaacctttcga- =) was pcr amplified and subcloned into the kpni and xhoi sites of the luciferase reporter pgl basic (promega, madison, wi, usa) to generate the pgl -ifn-␤-luc construct. all primers were synthesized by sangon (shanghai, china). for the construction of pbs/u sitbk , the sense strand = tcgagttgcg aagccggaagtgtcctaagcttaggacacttccggcttcgcaat ttttg = and antisense strand = acgcttcggccttcacaggattc gaatcctgtgaaggccgaagcgttaaaaacttaa = were annealed and then subcloned into the ecori and xhoi sites of pbs/u . for the construction of pbs/u siirf , the sense strand = tcgagcatcggct tttgggtctgttaaagctttaacagacccaaaagccgatgtttt tg = and antisense strand = cgtagccgaaaacccagacaatttcg aaattgtctgggttttcggctacaaaaacttaa = were annealed and then subcloned into the ecori and xhoi sites of pbs/u . for construction of psilencer-sitraf , the single-strand oligonucleotides = gatccgcgagaactcctctttccctcgagggaaagagg agttctcgcaga = and = agcttctgcgagaactcctctttccc tcgagggaaagaggagttctcgcg = were annealed to double strands before being subcloned into the hindiii and bamhi sites of psilencer . -cmv neo to form the psilencer-sitraf construct. transient transfection. cells (hek et, hek t, and j -m) were transiently transfected with the plasmid dnas (pcmv-myc-m, pcmv-myc, pgl -ifn-␤-luc, or pnf-b-luc) using vigofect (vigorous biotechnology, beijing, china) according to the manufacturer's instructions. for example, about g plasmid dnas was first added to l of . % nacl. then, . l vigofect was resuspended into another l of . % nacl solution. then, the dna-nacl mixture was added to the vigofect-nacl mixture drop by drop with gentle vortexing. after a min incubation at room temperature, the reaction product was evenly distributed onto the cell culture surface of either -well plates or -mm dishes and then continuously incubated for h before harvesting. construction of the sitraf stable cells. plasmid psilencer-sitraf dnas ( g) were transfected into hek cells. after a -h transfection, the cell culture medium was replaced with fresh medium containing , g/ml g . after weeks of culture, cell colonies were picked up and expanded in a -well tissue culture plate. finally, western blot analysis was performed to detect traf expression. reverse transcription-pcr (rt-pcr) and qrt-pcr. total rnas were extracted from the cultured cells with trizol (invitrogen, carlsbad, ca, usa). the purified total rnas were treated with dnase i (qiagen, düsseldorf, germany). all primers used in the rt-pcrs are listed in table . the rt-pcr was carried out with the primescript one-step rt-pcr kit (takara biotechnology, dalian, china) according to the manufacturer's instructions. the rt-pcr was carried out in a dna thermal cycler (applied biosystems, carlsbad, ca) under the following conditions: °c for min for reverse transcription and °c for min for denaturation. the pcr conditions were °c for s, °c for s, and °c for s, repeated for to cycles; the reaction was extended at °c for min before the reaction product was stored at °c. one-step real-time quantitative rt-pcr (qrt-pcr) (takara biotechnology, dalian, china) was also performed to monitor the targeted gene expression. the primers used in qrt-pcr were also listed in table . real-time qrt-pcr was carried out with the cfx real-time pcr detection system (bio-rad laboratories, hercules, ca, usa) under the following conditions: °c for min and °c for s, and then °c for s and °c for s, repeated for cycles. the dissociation of the reaction products was conducted from °c to °c as the temperature rose at . °c per s. viral evasion and subversion of pattern-recognition receptor signalling induction and function of ifnbeta during viral and bacterial infection innate immunity to virus infection role of adaptor trif in the myd -independent toll-like receptor signaling pathway innate antiviral responses by means of tlr -mediated recognition of singlestranded rna tlr signals after translocating from the er to cpg dna in the lysosome the interferon inducing pathways and the hepatitis c virus =-triphosphate rna is the ligand for rig-i differential roles of mda and rig-i helicases in the recognition of rna viruses mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response regulation of antiviral responses by a direct and specific interaction between traf and cardif the adaptor protein mita links virus-sensing receptors to irf transcription factor activation vaccinia virus protein a r targets multiple toll-like-interleukin- receptor adaptors and contributes to virulence poxviral protein a antagonizes toll-like receptor signaling by targeting bb loop motifs in toll-il- receptor adaptor proteins to disrupt receptor:adaptor interactions viral inhibitory peptide of tlr , a peptide derived from vaccinia protein a , specifically inhibits tlr by directly targeting myd adaptor-like and trif-related adaptor molecule the poxvirus protein a r targets toll-like receptor signaling complexes to suppress host defense a r and a r from vaccinia virus are antagonists of host il- and toll-like receptor signaling poxvirus protein n l targets the i-kappab kinase complex, inhibits signaling to nf-kappab by the tumor necrosis factor superfamily of receptors, and inhibits nf-kappab and irf signaling by toll-like receptors inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus influenza c virus ns protein counteracts rig-i-mediated ifn signalling the v proteins of paramyxoviruses bind the ifninducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter inhibition of interferon regulatory factor activation by paramyxovirus v protein murine retroviruses activate b cells via interaction with toll-like receptor pattern recognition receptors tlr and cd mediate response to respiratory syncytial virus reading the viral signature by toll-like receptors and other pattern recognition receptors small interfering rna effectively inhibits the expression of sars coronavirus membrane gene at two novel targeting sites the family of five: tir-domain-containing adaptors in toll-like receptor signalling traf molecules in cell signaling and in human diseases expanding traf function: traf as a tri-faced immune regulator tumoricidal alveolar macrophage and tumor infiltrating macrophage cell lines interaction between raf and myc oncogenes in transformation in vivo and in vitro oxidized phospholipid inhibition of toll-like receptor (tlr) signaling is restricted to tlr and tlr : roles for cd , lps-binding protein, and md as targets for specificity of inhibition assembly and localization of toll-like receptor signalling complexes severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk /ikkepsilon complex generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production pattern recognition receptors and inflammation pathogen recognition by the innate immune system recognition of the measles virus nucleocapsid as a mechanism of irf- activation activation of tbk and ikkvarepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity self-assembly of severe acute respiratory syndrome coronavirus membrane protein the sars-coronavirus membrane protein induces apoptosis through modulating the akt survival pathway the membrane protein of sars-cov suppresses nf-kappab activation the m protein of sars-cov: basic structural and immunological properties persistent replication of severe acute respiratory syndrome coronavirus in human tubular kidney cells selects for adaptive mutations in the membrane protein the orphan nuclear receptor tr /nur regulates er stress and induces apoptosis via interaction with trap␥ we thank genhong cheng and hang-zi chen for providing the j -m cells and trap␥, respectively. western blot analysis. the transfected cells were lysed with a lysis buffer containing % np- , mm tris-hcl (ph . ), mm nacl, m navo , g/ml leupeptin, g/ml aprotinin, and m phenylmethylsulfonyl fluoride (pmsf). about g of cell lysate for each sample was resolved by % sds-page. after separation, the reaction products were transferred onto a hybond nitrocellulose membrane (pharmacia, st. louis, mo, usa). the transferred membrane was first probed with a primary antibody. then, a secondary antibody labeled with horseradish peroxidase was added to the reaction product and was finally visualized with an enhanced chemiluminescence (ecl) kit (santa cruz biotechnology, santa cruz, ca, usa).dual-luciferase assay. the dual-luciferase kit was purchased from promega (promega corporation, usa). cells were transfected with pgl -ifn-␤-luc or pnf-b-luc plus prl-tk at the ratio of : or : as suggested by the manual. the detection was done by following the assay protocol in the kit. the firefly and renilla luciferase activities were read with a modulus microplate multimode reader (turner biosystems, sunnyvale, ca, usa).coimmunoprecipitation (co-ip) assay. the transfected cells were lysed with a lysis buffer containing mm tris-hcl (ph . ), mm nacl, mm edta, . % triton, g/ml leupeptin, g/ml aprotinin, and mm pmsf. about % of the lysate was subjected to input analysis. flag affinity gel (sigma, usa) or anti-myc and protein g plus agarose (santa cruz biotechnology, santa cruz, ca, usa) were added to the rest of the lysate ( %) and rotated overnight at °c. the reaction products were washed times with a wash buffer and then centrifuged at , rpm for min. the ip products were resuspended in the loading buffer and subjected to western blot analysis.elisa. cell culture supernatants were harvested h after transfection. the human ifn-␤ elisa kit was purchased from bluegene (shanghai, china). the reaction was carried out by following the manufacturer's instructions. the reaction products were detected with synogen (biotek, seattle, wa, usa) under nm.generation of sars-cov pseudovirus and assay of virus-induced ifn-␤ production. the generation of sars-cov pseudovirus followed the procedure described by huang et al. ( ) . briefly, hek cells were cotransfected with either the s, m, and n genes or the s, m mutant m(v a), and n of sars-cov. after h of transfection, the culture medium and the transfected cells were collected and subjected to freezing and thawing cycles at least four times. the reaction products were centrifuged at , rpm for min. the supernatant containing vlps was then applied to hek -ace stable cells and incubated for h. then, the cell culture medium was replaced with fresh medium. after h, the infected cells were subjected to total rna isolation and preparation of whole-cell lysates as described elsewhere. sars-cov-mediated ifn-␤ expression was monitored by standard western blotting, rt-pcr, and qrt-pcr.immunostaining assay. hela cells were transiently transfected with myc-m and flag-trap␥ ( ) . after h of transfection, the cells were fixed with % formaldehyde for min on ice and then incubated in ϫ phosphate-buffered saline (pbs) containing % bovine serum albumin (bsa) for h. the cells were first incubated with primary antibody rabbit anti-flag or mouse anti-myc for h. then, the secondary antibody fluorescein isothiocyanate (fitc)-labeled goat anti-mouse or tetramethyl rhodamine isocyanate (tritc)-labeled goat anti-rabbit antibody was added, and the mixture was incubated for another h. finally, the cells were incubated with =, -diamidino- -phenylindole (dapi) and sealed with glycerol. the results were observed under an olympus fv confocal microscope.statistical analysis. all values were calculated as means Ϯ standard deviations (sds) from three independent experiments. the statistical difference between the assayed group and the standard group was subjected to student's t test. the calculated difference was considered significant at the p value of Ͻ . . key: cord- - d vxf authors: heaton, steven m.; borg, natalie a.; dixit, vishva m. title: ubiquitin in the activation and attenuation of innate antiviral immunity date: - - journal: j exp med doi: . /jem. sha: doc_id: cord_uid: d vxf viral infection activates danger signals that are transmitted via the retinoic acid–inducible gene –like receptor (rlr), nucleotide-binding oligomerization domain-like receptor (nlr), and toll-like receptor (tlr) protein signaling cascades. this places host cells in an antiviral posture by up-regulating antiviral cytokines including type-i interferon (ifn-i). ubiquitin modifications and cross-talk between proteins within these signaling cascades potentiate ifn-i expression, and inversely, a growing number of viruses are found to weaponize the ubiquitin modification system to suppress ifn-i. here we review how host- and virus-directed ubiquitin modification of proteins in the rlr, nlr, and tlr antiviral signaling cascades modulate ifn-i expression. the frontline in the cellular response to viral infection is comprised of the specific and general effectors of the innate immune system. effector molecule production is initiated by immune sentinels known as pattern recognition receptors (prrs), which screen the intra-and extracellular environment for molecular motifs uniquely associated with pathogens. prr engagement transduces pro-immune signals into the nucleus via protein signaling cascades that self-limit to mitigate autoimmunity as the infection clears (crampton et al., ) . protein posttranslational modifications (ptms) form part of this exquisite system of regulation, with ubiquitin and ubiquitin-like modifications key among them. the retinoic acid-inducible gene (rig-i)-like receptors (rlrs) and nucleotide-binding oligomerization domain (nod)-like receptors (nlrs) are intracellular prrs. the rlrs sense invasive rna produced during infection by both rna and dna viruses (schlee, ) . rlr engagement up-regulates type-i ifn (ifn-i) expression, which in turn stimulates transcription of hundreds of ifn-stimulated genes (isgs) that commit host and nearby cells to an antiviral posture. recognized for their role in antibacterial immunity, the nlrs are emerging as antiviral mediators that regulate both ifn-i and nf-κb activation. these are also activated by tlrs, a cell-specific class of extracellular and endosomal transmembrane prrs that sense a broad spectrum of pathogenic motifs. rlr, nlr, and tlr signaling proteins must be spatially and temporally coordinated for efficient immune signal transduction. ubiquitination is a ptm involving the covalent attachment of the . -kd protein ubiquitin to target proteins. ubiquitination is catalyzed by the ubiquitin-activating enzyme (e ), ubiquitin-conjugating enzyme (e ), and ubiquitin protein ligase (e ). the e largely dictates substrate specificity, with at least genes encoding putative ubiquitin and ubiquitin-like e s annotated in the human genome (li et al., ) . ubiquitin can undergo ubiquitination itself at its seven lysine residues (k /k /k /k /k /k /k ), building lysine-linked polyubiquitin chains, or its n-terminal methionine (m ), forming linear polyubiquitin chains. alternatively, ubiquitin chains may be noncovalently associated with target proteins. furthermore, ubiquitin chains may be remodeled by deubiquitinating enzymes (dubs; fig. ). the function, abundance, or subcellular distribution of proteins involved in almost every cellular process is regulated in this way, with an increasingly clear role in regulating innate immunity. viruses are obligate intracellular parasites that facilitate their own replication by manipulating the host cell environment. thus, the ubiquitin modification system presents a key manipulation target for viruses to circumvent antiviral signaling pathways. methods for this include substrate molecular mimicry, binding and blocking e -substrate pairs, expressing virally encoded e s/dubs, and hijacking host e s/dubs. additionally, a novel mechanism involving ubiquitin chain packaging into nascent virions for subsequent redeployment viral infection activates danger signals that are transmitted via the retinoic acid-inducible gene -like receptor (rlr), nucleotide-binding oligomerization domain-like receptor (nlr), and toll-like receptor (tlr) protein signaling cascades. this places host cells in an antiviral posture by up-regulating antiviral cytokines including type-i interferon (ifn-i). ubiquitin modifications and cross-talk between proteins within these signaling cascades potentiate ifn-i expression, and inversely, a growing number of viruses are found to weaponize the ubiquitin modification system to suppress ifn-i. here we review how host-and virus-directed ubiquitin modification of proteins in the rlr, nlr, and tlr antiviral signaling cascades modulate ifn-i expression. localization changes from the cytosol to distinct subcellular compartments depending on upstream signaling events (goncalves et al., ) . in promoting ifn-i signaling, tbk associates with rig-i as well as key adaptor proteins, including tank and nf-κb essential modulator (nemo; guo and cheng, ; zhao et al., ; wang et al., a) . this facilitates interactions between tbk , inhibitor of nf-κb kinase subunit ε (ikkε) and trafs, particularly traf . upon rlr activation, these proteins are colocalized at the cytosolic surface of the mitochondrial outer membrane (parvatiyar et al., ; van zuylen et al., ) , coordinated by the mitochondrial antiviral-signaling protein (mavs; also termed visa/cardif/ips- ). at rest, the rlr cascade is maintained in an inactive but tensioned state through an intricate negative feedback system involving protein expression levels, conformational changes, compartmentalization, and ptms. part of this system operates at the receptor through conformational auto-inhibition of the rig-i cards. the rig-i c-terminal repressor domain (rd) audits the cytosol for viral rna, binding of which induces a major structural rearrangement in the rd and card (saito et al., ) . conversely, mda oligomerizes along the length of rna ligands, forming immunogenic filaments that are potentiated by atp hydrolysis and interaction with lgp (peisley et al., ; bruns et al., ) . the unfurled rig-i cards undergo tetramerization upon k -linked polyubiquitination or unanchored polyubiquitin chain association (peisley et al., ) . these modifications drive mitochondrial accumulation of rig-i, promoting card-card interac- ( ) ubiquitin (ub) expresses as an inactive polyprotein, encoded by the ubb and ubc genes. dubs cleave this polyprotein into monomers that are activated by the e -activating enzyme, involving the energy-dependent adenylation of the ubiquitin c-terminal glycine. the ubiquitin-adenylate intermediate (dashed line) converts into a covalent thioester bond (solid line). ( ) ubiquitin transfers to the active site cysteine residue of an e -conjugating enzyme. ( ) the e directly or indirectly transfers the e -bound ubiquitin to a substrate acceptor residue, forming an isopeptide bond. ( ) dubs remodel ubiquitin modifications and antagonize ubiquitin-driven functional outcomes. tions with mavs and inducing its oligomerization and filamentation. the rig-i cards contain a high proportion of hydrophobic residues and are prone to aggregation, thus oligomerization and polyubiquitination may stabilize the activated cards or elicit a separate mitochondrial targeting signal. conversely, ubiquitination has no known role in mda or lgp activation. the first virus-triggered rig-i ubiquitination site described, k , depends on the e activity of tripartite motif protein (trim ; gack et al., ) . plausibly as a means of restricting escape mutant selection, this activation mechanism now appears to have evolved with partial redundancy using alternate e s. trim was recently described to modify this same site in addition to two other card residues: k and k (yan et al., ) . furthermore, these same three residues are reportedly ubiquitinated by really interesting new gene (ring) finger protein- (rnf ; also termed riplet/reul; gao et al., ) , although this is controversial ( fig. ; oshiumi et al., ) . underscoring the importance of these modifications, ubiquitin-specific protease (usp ) and ubiquitin c-terminal hydrolase are dubs that inhibit ifn-i production by removing such chains from rig-i (friedman et al., ; cui et al., ) . both trim and rnf are targets of the influenza a virus (iav) nonstructural protein (ns ), which blocks their e activity and ubiquitin-dependent rig-i activation ( fig. ; gack et al., ; rajsbaum et al., ) . iav-ns binds the central coiled-coil domain (ccd) of trim and is postulated to prevent ccd-mediated homooligomerization. although the ns -binding site on rnf is unknown, rnf and trim share a similar ring-ccd-b . /spry (sp a and ryanodine receptors) domain figure . schematic of the tlr, rlr, and nlr antiviral protein signaling cascades and modes of cross-talk. prrs (blue) screen the intracellular and extracellular environment for pathogenic motifs. ligand-activated prrs bind adaptor proteins (purple) and recruit protein kinases (yellow) and ubiquitin-protein ligases (green). these regulate immune signal transduction to transcription factors (orange) through ptm of signaling cascade proteins. other regulatory proteins (gray) support or sequester these signaling proteins. immune signaling scaffolds such as mitochondria typically coordinate these actions. activated transcription factors translocate into the nucleus and bind to promoter response elements, stimulating appropriate antiviral gene transcription. blue and green circles represent ubiquitination and phosphorylation, respectively. black arrows, activation; red lines, deactivation. organization. however, the ccds differ in size and sequence markedly, suggesting that iav-ns may bind multiple sites on trim and rnf . alternatively, given that ccds often mediate protein-protein interactions, iav-ns may sense and subvert ccd-interacting domains more broadly. notably, the iav -ns :rnf interactions observed by rajsbaum et al. ( ) were strain dependent. rnf enables rig-i card activation by trim upon ubiquitinating rd residues k and k . rnf knockdown inhibits interaction between rig -i :trim and eliminates tbk recruitment (oshiumi et al., ) , revealing an ordered functional interplay between ubiquitination and phosphorylation in coordinating rig-i activation. hepatitis c virus (hcv) ns - a protease exploits this concept by targeting rnf for proteolytic cleavage (oshiumi et al., ) . furthermore, numerous herpesviruses encode their own dubs that inhibit ifn-i expression by stripping ubiquitin modifications from activated rig-i (inn et al., b) . accordingly, hcv and herpesvirus infections are treatable with ifn (oberman and panet, ; nguyen et al., ) , although this can carry significant side effects. endogenous ifn-i expression and self-regulation may be restored by defeating such mechanisms of viral antagonism. ubiquitin in the return to homeostasis. rlr signaling is also counterbalanced and diminished through ubiquitin modifica- tion as the antiviral posture becomes unnecessary. rnf forms part of this process, ligating k -linked polyubiquitin chains to the activated card of rig-i and mda , leading to proteasome-mediated degradation of both receptors and diminished ifn-i signaling. usp is a dub that sustains rlr signaling by specifically removing such chains (wang et al., a) . in the same way, rnf ubiquitinates and degrades activated mavs (arimoto et al., ) , suggesting that rnf is an e that destabilizes proteins containing activated cards. given how commonly card-containing proteins and their homotypic interactions feature in immune signaling pathways (bouchier-hayes and martin, ) , rnf may represent a general immune signaling antagonist. conversely, the -kd repressor of the inhibitor of the protein kinase (p ripk) binds and enhances the stability of rig-i by blocking its ubiquitin-mediated degradation (now and yoo, ) . accordingly, the properties of p ripk or rnf may be exploitable in the treatment of viral infections or autoimmune disorders. the linear ubiquitin assembly complex (lub ac), containing sha nk-associated rh domain-interacting protein (sha rpin), heme-oxidized irp ubiquitin ligase l (hoil- l), and hoil- -interacting protein (hoip), was proposed to negatively regulate rlr-mediated ifn-i expression via two independent mechanisms (inn et al., a) . first, hoil- l competes with trim for rig-i card binding, abrogating the rig -i :mavs interaction. second, hoip promotes m -and k -linked polyubiquitination of trim and induces its proteasomal degradation, thereby decreasing trim -mediated activation of rig-i. if lub ac were capable of ligating k -linked polyubiquitin chains to substrates, trim would be the first example to our knowledge. another route of rlr inhibition involves tetraspanin- ubiquitination by an unknown e . during rlr activation, polyubiquitinated tetraspanin- is recruited to mavs and blocks the rlr :mavs interaction, thereby impeding recruitment of the downstream signaling apparatus (wang et al., b) . mitochondria, peroxisomes, and endoplasmic reticulum function as immune signaling platforms linking viral pattern recognition with effector molecule production ( fig. , middle). although this process remains poorly characterized, the nature and context of viral ligands detected by prrs drives accumulation of the downstream signaling apparatus to these platforms. adaptor proteins mediate this accumulation: mitochondria and peroxisomes by mavs (dixit et al., ) and endoplasmic reticulum by stimulator of ifn genes (sti ng; ishikawa and barber, ) . cyclic gmp-amp synthase (cgas) and aim -like receptors (alrs), which include aim and human ifn-inducible protein (ifi ), have also emerged as important dna virus prrs. cgas signals via sti ng and aim generates inflammasome oligomers, whereas ifi can stimulate both signaling mechanisms (diner et al., ) . rna-activated rig-i and mda colocalize with mavs, inducing its filamentation. it remains unclear why these filaments are potent inducers of downstream signaling; however, rlr cascade proteins including nemo, ikkε, and various trafs possess mavs-targeting signals (paz et al., ) . furthermore, tbk and other key rlr cascade proteins interact with these proteins but are activated only upon oligomerization. thus, steady state isolation of mavs may represent a spatiotemporal barrier that restrains innate immune signaling, overcome through coordinating these proteins into signaling complexes upon mavs multimerization. in this way, it is conceivable how immunomodulating e s/dubs may be compartmentalized together with their substrates. ubiquitin stringently regulates the mavs signalosome. the central position that mavs occupies within the rlr cascade is commensurate with the many ptms that modulate its role. to our knowledge, mavs ubiquitination has not been observed in resting cells using a variety of proteomic and biochemical approaches, indicating that mavs ubiquitination occurs specifically during viral infection. at least seven e s ubiquitinate mavs, leading to mavs degradation in almost every case, as described later in this section (fig. ) . at least five of these modify other substrates within the same cascade, highlighting mavs as a crucial locus of rlr regulation. accordingly, mavs is targeted by numerous viruses in a variety of ways; however, with the exception of hbv and severe acute respiratory syndrome-coronavirus (sars-cov; fig. ; wei et al., ; shi et al., ) , this is usually achieved by means other than manipulating mavs ubiquitination, likely given the extensive ubiquitin-mediated negative regulatory systems already in place. mavs aggregation is a key feature of rlr cascade activation, but how these aggregates are resolved during deactivation is only beginning to be clarified. in addition to ubiquitinating rig-i and enhancing its association with mavs, trim ubiquitinates mavs at lys and lys and induces its partial proteolysis (castanier et al., ) . this was proposed as a means of discharging the activated rlr signalosome from the mitochondrial recruitment platform and would begin to address how irf and other rlr signalosome components traffic correctly after activation. more recently, lys and lys were shown to be polyubiquitinated by membrane-associated ring finger protein (mar ch ), a mitochondrial membrane-bound e that effectively dissolves mavs aggregates by specifically targeting them for degradation. mar ch is an important regulator of mitochondrial fission and fusion whose expression is up-regulated during infection (yoo et al., ) . these mechanisms of mavs aggregate resolution may be nonredundant, with the trim mechanism occurring throughout the immune response and the mar ch mechanism amplifying gradually in an ifn-i negative feedback loop. complete mavs degradation is independently promoted by the e s rnf , rnf , atrophin- -interacting protein (aip ; also termed itch), smad ubiquitination regulatory factor (smurf ), and smurf (fig. ) . rnf polyubiquitinates mavs at lys and lys , whereas the adjacent residues lys and lys are polyubiquitinated by aip upon recruitment by poly(rc)-binding protein (pcbp ) or pcbp (you et al., ; zhong et al., ; zhou et al., ) . aip additionally inhibits ifn-i as well as nf-κb activation by ubiquitinating the inhibitor of apoptosis protein (ciap ), targeting it for lysosomal degradation (tigno-aranjuez et al., ) . ciap is an e that activates traf / during viral infection (mao et al., ) , revealing that aip broadly and multiply inhibits nlr-, rlr-, and tlr-mediated immune signaling. the acceptor site or sites for rnf -induced mavs ubiquitination are unknown; however, given that rnf also ubiquitinates the activated cards of rig-i and mda (arimoto et al., ) , the mavs card appears a likely candidate. nedd family-interacting protein (ndfip ) binds mavs and recruits smurf and possibly smurf , facilitating ubiquitination of unknown sites within mavs (wang et al., c; pan et al., ) . moreover, numerous trafs, including traf and traf , interact with mavs, and smurf also targets these for degradation (li et al., a) . finally, lys was reported as a single site of ifn-i-activating polyubiquitination by an unknown e , inhibiting nf-κb activation by sequestering ikkε (paz et al., ). the trafs are six multifunctional adaptor proteins that regulate both nf-κb activation and ifn-i expression via the rlr, nlr, and tlr protein signaling cascades (fig. ) . traf-mediated signaling outcomes are augmented by ubiquitin, and, excepting traf , all trafs possess a ring finger domain and multiple zinc coordination sites, features typical of ubiquitin e s. k -linked autoubiquitination at lys is a key activation mechanism of traf (lamothe et al., ; jiao et al., ) and possibly traf (habelhah et al., ) , traf (marinis et al., ) , and traf (zhong et al., ) . in vitro traf ubiquitination assays and analysis of recombinant traf Δri ng isolated from mammalian cell lysates are also consistent with an autoubiquitination activation mechanism for traf (kayagaki et al., ; zeng et al., ) . traf and traf are among the first molecules activated by mavs in the rlr pathway (fig. , middle) . furthermore, there is increasing evidence of ubiquitin-mediated cross-talk between trafs. traf promotes ifn-i expression by activating tbk /irf (parvatiyar et al., ) , whereas traf activates mitogen-activated protein kinase kinase kinase (mekk ) to activate nf-κb, which also enhances ifn-i expression (yoshida et al., ) . simultaneously, traf suppresses nf-κb by inhibiting ikk activation upon binding traf (zarnegar et al., ) , likely as a mecha-nism to skew innate immune effector molecule expression as required. inversely, the e ciap , after itself being ubiquitinated by traf , promotes traf degradation by ligating k -linked polyubiquitin chains to traf at residues k and k , thereby restoring nf-κb activation (tseng et al., ) . however, as well as degrading traf , ciap / can also activate traf by catalyzing its k -linked polyubiquitination ( fig. ; mao et al., ) . this suggests that the context-dependent ubiquitination state of ciap / determines its effect on traf . the e rnf was recently reported to ubiquitinate and activate both traf and traf . finally, the rig-i-activating e trim was reported to enhance mda -mediated nf-κb activation at the level of traf , although mechanistic details remain unclear. traf-mediated signaling is also terminated by ubiquitin in numerous ways. hsv encodes the dub ul usp, which strips k -linked polyubiquitin chains from traf to prevent downstream protein recruitment ( fig. ; wang et al., b) , possibly antagonizing ciap / -mediated ubiquitination. traf and traf are both deactivated by the dubs otubain (otub ) and otub , which remove k -linked polyubiquitin chains (li et al., b) . traf is further deactivated by the deubiquitinase duba, which removes k -linked polyubiquitin chains from traf (kayagaki et al., ) . furthermore, the e triad a redirects traf to the proteasome by ligating k -linked polyubiquitin chains (nakhaei et al., ). altogether, this constitutes a ubiquitin-dependent feedback mechanism that enables trafs to dictate the direction of immune signal transmission in a context-dependent manner. in contrast to the three rlr receptors, the nlrs have diverse expression patterns and largely under-characterized functions. the nlrs are well recognized for their roles in regulating nf-κb activation and antibacterial immunity; however, at least five members have emerging roles in antiviral immune signaling: nod , nod , nlrc (nlr family card domain-containing protein ), nlrp (nac ht, lrr, and pyd domain-containing protein ), and nlrx (nlr family member x ; fig. , right) . although nlrs recruit e s and modulate the ubiquitination of other proteins, including several in the rlr cascade, the role of ptms in nlr regulation remains under-defined. nlr regulation and innate immune signaling cross-talk. the prrs nod and nod are the best characterized nlrs. nod is expressed ubiquitously, whereas nod is expressed mainly in cells of myeloid and lymphoid origin and is up-regulated during bacterial and viral infection. the classic nod -activating ligand is bacterial muramyl dipeptide (mdp), which promotes nf-κb activation. however, nod also promotes ifn-i expression during infection by numerous rna viruses, in part through recognizing single-stranded rna (ssrna) and interacting with mavs. nod may also promote ifn-i expression during infection by particular dna viruses by an undefined mechanism (sabbah et al., ; kapoor et al., ) . accordingly, nod dysfunction leads to inefficient innate and adaptive immune responses to viral infection (lupfer et al., ) . nod features regularly in the immune signaling landscape, yet mechanisms of nod regulation and cross-talk are only beginning to be revealed. upon activation by mdp, nod is ubiquitinated by trim , leading to nod degradation and nf-κb inhibition (zurek et al., ) . nod signaling is further suppressed by aip , which ubiquitinates lys of receptor-interacting serine/threonine protein kinase (ripk ), the immediate downstream interacting partner of nod (fig. , right; tao et al., ) . conversely, nod -driven nf-κb activation is enhanced by lub ac, a negative regulator of rlr signaling, as well as x-linked iap (xiap), which respectively ligate m -and k -linked polyubiquitin chains to nod and ripk (damgaard et al., ) . these activating ubiquitin chains may be antagonized by the ubiquitin-editing enzyme a , which also disrupts ubiquitin-mediated tbk activation in the rlr signaling cascade as well as ubiquitin-mediated traf activation in the tlr cascade parvatiyar et al., ) . another mitochondrial link between the nlr and rlr signaling pathways is the ubiquitously expressed nlrx (fig. , right) , whose role in ifn-i regulation is controversial. nlrx is localized to the mitochondrial outer membrane and was reported to inhibit mavs-dependent ifn-i signaling by blocking the interaction between activated rig-i/mda and mavs, although viral replication experiments using gene knockout cells have produced conflicting results (soares et al., ) . nlrx also potentiates nf-κb signaling by promoting reactive oxygen species production during bacterial infection, linking the mitochondrial immune signaling platform with proinflammatory cytokine generation (tattoli et al., ) . nlrc was initially described to enhance ifn-γ and ifn-α expression and inhibit nf-κb and ifn-β, in the latter case through sequestering the activated effector domains of rig-i and mda (cui et al., ; kuenzel et al., ) . nlrc has also been shown to bind and inhibit tbk mediated ifn-β induction in hek- t cells, although nlrc −/− mice show relatively normal cytokine responses upon exposure to rlr-, tlr-, and nlr-activating stimuli (kumar et al., ) . still other findings indicate that the rig -i :nlrc interaction also positively regulates ifn-β expression, and this interaction is targeted by the iav-ns protein (fig. , right; neerincx et al., ; ranjan et al., ) . these disparate conclusions may reflect cell-specific differences given that nlrc is predominantly expressed in hematopoietic cells or differences between mouse and human signaling pathways, suggesting that the nlrc regulatory framework is complex. adding to this, ubiquitination plays an uncharacterized role in regulating nlrc upon lps stimulation and may be induced by nlrc overexpression (cui et al., ; kuenzel et al., ) . given the diversity of interactions that nlrc takes part in, it is likely that further ptms will be shown to regulate nlrc during viral infection. nlrp has gained prominence as another negative regulator of multiple immune signaling pathways that is more widely expressed than nlrc . nlrp was initially described to inhibit ikkα-mediated nf-κb activation (fiorentino et al., ) . upon rlr cascade activation, nlrp also inhibits irf activation by recruiting the e deltex- (dtx ) to ubiquitinate and degrade tbk (cui et al., ) , revealing yet another route for rlr/nlr cross-talk (fig. , right) . tlrs are differentially expressed in a wide range of cell populations. tlr , tlr , tlr , and tlr are expressed in endosomal vesicles, whereas tlr and tlr are expressed on the cell surface. tlr recognizes double-stranded rna, activating nf-κb-mediated proinflammatory cytokine production and strongly up-regulating tbk /irf -dependent ifn-i expression. tlr and tlr recognize ssrna, up-regulating ifn-α and proinflammatory cytokine production. tlr recognizes unmethylated cytosine-phosphate-guanine (cpg) dna, a common feature of nonmammalian genomes, and stimulates ifn-α production. tlr and tlr are activated by a variety of microbial ligands, including specific viral proteins, resulting in proinflammatory cytokine expression (fig. , left) . nf-κb activation and ifn-i up-regulation. tlr , tlr , tlr , tlr , and tlr signaling is mediated through the adaptor protein myeloid differentiation primary response gene (myd ; fig. , left). myd recruits nf-κb and ifn-i signaling components, including interleukin- receptor-associated kinase (irak ), irak , traf , and irf . activated traf ubiquitinates irf , leading to ifn-α expression (kawai et al., ) . traf also promotes k linked polyubiquitination of nemo, enabling recruitment of the tgf-β-activated kinase (tab)-tak kinase complex (tseng et al., ) . subsequent association between nemo and m -polyubiquitin chains induces tak -mediated phosphorylation of ikkα and ikkβ, priming them for full transactivation through autophosphorylation . activated ikkα phosphorylates the iκbα subunit, leading to its ubiquitination and proteasomal degradation and releasing nf-κb for nuclear translocation. furthermore, myd , irak / , and tab / are also modified and activated by k -linked polyubiquitin chains. such chains were recently described as substrates for m -polyubiquitination by hoip, resulting in hybrid chains that may connect the myd / irak and tak /ikk signaling apparatus (emmerich et al., ) . tlr signaling is mediated by tir domain-containing adaptor-inducing ifn-β (trif). trif activates traf , which promotes irf /irf activation (tseng et al., ) , and also traf , which promotes ikk activation (fig. , left; jiang et al., ) . ubiquitin regulates the myd -and trif-dependent pathways. although tlrs undergo extensive ptm, ubiquitination performs no known role in regulating tlrs directly. instead, ubiquitination modulates their downstream signaling targets, particularly myd , trif, and traf , and it is often here that viruses terminate tlr-mediated immunity. nrdp is an e that promotes ifn-i expression at the expense of proinflammatory cytokines. tbk polyubiquitination by nrdp activates tbk in trif-mediated ifn-i expression, which simultaneously k -polyubiquitinates and down-regulates myd -mediated nf-κb activation . conversely, the ubiquitin-editing enzyme a inhibits ubiquitin-mediated activation of traf , inhibiting nf-κb activation via the tlr and nlr cascades, as well as tbk , inhibiting ifn-i expression turer et al., ; parvatiyar et al., ) . additional avenues of signaling cross-talk include smurf and smurf , which degrade mavs and inhibit ifn-i activation. smurf and smurf also degrade myd , inhibiting tlr-mediated nf-κb activation (lee et al., ) . ciap is an e that, after itself being ubiquitinated by traf , targets traf for degradation (tseng et al., ) . this is important in promoting tlr -mediated signaling and cytokine production at the expense of type i ifn production . furthermore, traf itself promotes proinflammatory cytokine production at the expense of ifn-i. traf is activated by trans-autoubiquitination at k , abolition of which eliminates nemo ubiquitination and tak activation (lamothe et al., ) . this mechanism is exploited by hsv, which uses the virally encoded e infected cell polypeptide (icp ) to recruit usp to deubiquitinate nemo and traf (daubeuf et al., ). in addition, icp directly catalyzes ubiquitination and degradation of myd and tir ap (toll/interleukin- receptor domain-containing adaptor protein; van lint et al., ) . kaposi's sarcoma-associated herpesvirus (kshv) encodes replication and transcription factor (rta), an e that activates latent virus. this activation process involves suppression of antiviral cytokines, partly involving rta-catalyzed ubiquitination and degradation of myd and trif (ahmad et al., ; zhao et al., ) , thereby impairing all tlr-mediated immune signaling pathways. the final relay: irfs transmit danger signals into the nucleus tlr, nlr, and rlr ifn-i signaling converges at irf activation, the penultimate step toward ifn-i transcription. irf is constitutively expressed in most cell types, residing inactive in the cytosol until phosphorylation by tbk /ikkε within two activation clusters (ser /ser and ser / ser /ser /thr /ser ), resulting in homodimerization, nuclear accumulation, dna binding, and participation in ifnb gene transcription (lin et al., ) . ifn-β acts in an autocrine and paracrine manner upon its cognate receptor, ifn-α/β receptor (ifn ar), thereby activating jak/stat signaling and isg expression. irf expression is up-regulated in this way, which in turn activates ifna transcription and additional isg expression by a similar mechanism. ubiquitin is an irf master toggle. the irfs are among the most tightly controlled ifn-i signaling proteins through an interplay of ptms, including phosphorylation, ubiquitination, and ubiquitin-like modifications. phosphorylation of irf at ser and ser by tbk /ikkε is required for its activation (tenoever et al., ) . however, ubiquitination by traf at nearby residues lys , lys , and lys appears to be a prerequisite to this and serves as a link between the nf-κb and ifn-i activation pathways (ning et al., ) . furthermore, trim binds active irf and ligates small ubiquitin-like modifier (sumo) at two of these ubiquitination sites, lys and lys , negatively regulating virus-triggered ifn-α production (liang et al., ) , indicating that as yet unidentified dubs or desumoylating enzymes participate in regulating irf . reminiscent of irf , irf residues lys and lys accept both polyubiquitin chains and sumo, and competition between these modifications can determine the fate of ifn-i signal transduction. at steady state, the sumo-conjugating enzyme ubiquitin carrier protein (ubc ) protects irf from ubiquitin-mediated degradation by occupying these sites with sumo. alternatively, the desumoylating enzyme sentrin-specific protease (senp ) removes sumo from irf , enabling its k -linked polyubiquitination (ran et al., ) . subsequent work identified trim as an e that conjugates k -linked polyubiquitin chains to these same sites ( fig. ; wang et al., ) , triggering degradation of the active, nuclear-localized form of irf . furthermore, activated irf undergoes phosphorylation at ser . this promotes interaction with peptidyl-prolyl cis/ trans isomerase nima-interacting (pin ), a nuclear-localized protein that promotes irf degradation (saitoh et al., ) . the e recruited by pin for this purpose is unknown; however, trim is also localized to the nucleus and seems a strong candidate. irf degradation is undesirable at early stages of the innate immune response and is limited in several ways. the irf -pin interaction is inhibited by the hect (homologous to the e -ap c terminus) domain and rcc -like domain-containing protein (herc ), which ligates another ubiquitin-like protein, isg , onto irf at lys , lys , and lys , thereby sustaining irf activation (shi et al., ) . trim is a ubiquitin e described to both inhibit the irf -pin interaction and target irf for proteasomal degradation (higgs et al., ; yang et al., ) . trim reportedly also targets irf for degradation upon tlr or tlr activation (higgs et al., ) , although the trim -dependent irf /ifr ubiquitin acceptor sites remain undefined (fig. ) . jem vol. , no. several additional e s regulate irf abundance. the skp-cullin-f-box (scf)-containing complex, of which cullin (cul ) is a core component, catalyzes irf degradation as well as iκb degradation, promoting nf-κb activation ( fig. ; bibeau-poirier et al., ) . ranbp-type and c hc -type zinc finger-containing protein (rbck ) catalyzes k -linked polyubiquitination and degradation of irf during viral infection ( fig. ; zhang et al., ) . finally, the forkhead box protein o (foxo ), a regulator of insulin signaling, binds irf and promotes its degradation by recruiting an unknown e . foxo also negatively regulates irf transcription (lei et al., ) , altogether implying a link between metabolism and innate immune induction. expression of cul and the e s rbck , trim , trim , and herc is ifn-i inducible (henig et al., ) , constituting a multiply redundant negative feedback web in which ifn-i expression is self-restraining. not so fast: seizing the penultimate step toward antiviral gene transcription. irfs are a significant target of viral disruption, usually resulting in their proteasome-mediated degradation. rotavirus (rov) nonstructural protein blocks nf-κb signaling and usurps the ubiquitin modification system to redirect irf / / / to the proteasome in a strain-specific manner ( fig. ; morelli et al., ) . cells produce trace quantities of ifn-i at rest through basal activation of endogenous irf /irf , the intracellular concentration of which are regulated by the e rta-associated ubiquitin ligase (raul). kshv exploits this mechanism to diminish immune signaling, recruiting usp to deubiquitinate raul and thereby maintain raul-mediated irf /irf degradation (yu and hayward, ) . the raul-dependent ubiquitin acceptor sites on irf /irf remain unknown (fig. ) , but better characterization of the raul-irf interaction may have implications for antiviral and autoimmunity treatments. furthermore, the kshv rta protein catalyzes polyubiquitination and degradation of irf and myd (yu et al., ) . thus, kshv effectively terminates several signaling pathways at multiple stages. similar to kshv, hiv infection fails to stimulate activation of irf , endogenous levels of which are quickly reduced upon infection. underscoring the importance for hiv to disrupt early ifn-i-mediated immunity, irf degradation is independently promoted by two viral accessory proteins, viral infectivity factor (vif) and viral protein r (vpr). the e s hijacked for this purpose are unknown (fig. ) , although vif and vpr recruit scf-related components to degrade other antiviral proteins (okumura et al., ) . the innate immune signaling architecture is complex and has coevolved with the pathogens it guards against, meanwhile restraining autoimmunity through an elaborate negative feedback scheme. a cornerstone of this dynamic regulatory framework is the ubiquitin modification system, which is manipulated by viruses relevant to human disease. going forward in understanding mechanisms of infection and autoimmunity, we must address significant gaps in knowledge regarding the specificity and context-dependent regulation of e s and dubs and the consequences of ubiquitin modification. this will expose further cross-talk between the immune signaling cascades, revealing a functional and self-regulating whole. in the search for a new generation of antiviral and autoimmune treatments, we continue to learn from the pathogens that have long adapted to exploit this ready-made system of functional regulation; humans possess hundreds of specific-and general-effect e s and dubs, many of which could be harnessed for therapeutic use. kaposi sarcoma-associated herpesvirus degrades cellular toll-interleukin- receptor domain-containing adaptor-inducing β-interferon (trif) negative regulation of the rig-i signaling by the ubiquitin ligase rnf influenza a virus uses the aggresome processing machinery for host cell entry involvement of the iκb kinase (ikk)-related kinases tank-binding kinase /ikki and cullin-based ubiquitin ligases in ifn regulatory factor- degradation card games in apoptosis and immunity the innate immune sensor lgp activates antiviral signaling by regulating mda -rna interaction and filament assembly mavs ubiquitination by the e ligase trim and degradation by the proteasome is involved in type i interferon production after activation of the antiviral rig-i-like receptors ring finger protein potentiates rna virus-induced interferon-β production via enhancing the ubiquitination of traf and traf activation of the canonical ikk complex by k /m -linked hybrid ubiquitin chains a novel paad-containing protein that modulates nf-κb induction by cytokines tumor necrosis factor-α and interleukin- β the tumour suppressor cyld is a negative regulator of rig-imediated antiviral response trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i reul is a novel e ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i functional dissection of the tbk molecular network modulation of the interferon antiviral response by the tbk /ikki adaptor protein tank ubiquitination and translocation of traf is required for activation of jnk but not of p or nf-κb interferon-beta induces distinct gene expression response patterns in human monocytes versus t cells the e ubiquitin ligase ro negatively regulates ifn-β production post-pathogen recognition by polyubiquitin-mediated degradation of irf self protection from anti-viral responses-ro promotes degradation of the transcription factor irf downstream of the viral toll-like receptors the ubiquitinediting enzyme a restricts nucleotide-binding oligomerization domain containing -triggered signals linear ubiquitin assembly complex negatively regulates rig-iand trim -mediated type i interferon induction inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf sti ng is an endoplasmic reticulum adaptor that facilitates innate immune signalling toll-like receptor -mediated activation of nf-κb and irf diverges at toll-il- receptor domaincontaining adapter inducing ifn-β the kinase mst limits inflammatory responses through direct phosphorylation of the adaptor traf activation of nucleotide oligomerization domain (nod ) by human cytomegalovirus initiates innate immune responses and restricts virus replication interferon-α induction through toll-like receptors involves a direct interaction of irf with myd and traf duba: a deubiquitinase that regulates type i interferon production the nucleotidebinding oligomerization domain-like receptor nlrc is involved in ifn-dependent antiviral immune responses nlrc deficiency does not influence cytokine induction by virus and bacteria infections site-specific lys- -linked tumor necrosis factor receptorassociated factor auto-ubiquitination is a critical determinant of iκb kinase activation regulation of mda -mavs antiviral signaling axis by trim through traf -mediated nf-κb activation smad -specific recruitment of smurf e ligases mediates tgf-β -induced degradation of myd in tlr signalling foxo negatively regulates cellular antiviral response by promoting degradation of irf ubiquitin ligase smurf targets traf family proteins for ubiquitination and degradation regulation of virus-triggered signaling by otub -and otub -mediated deubiquitination of traf and traf genome-wide and functional annotation of human e ubiquitin ligases identifies mul an, a mitochondrial e that regulates the organelle's dynamics and signaling tripartite motif-containing protein is a small ubiquitin-related modifier e ligase and negative regulator of ifn regulatory factor virus-dependent phosphorylation of the irf- transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation nucleotide oligomerization and binding domain -dependent dendritic cell activation is necessary for innate immunity and optimal cd + t cell responses to influenza a virus infection virus-triggered ubiquitination of traf / by ciap / is essential for induction of interferon-β (ifn-β) and cellular antiviral response iκb kinase α phosphorylation of traf downregulates innate immune signaling putative e ubiquitin ligase of human rotavirus inhibits nf-κb activation by using molecular mimicry to target β-trcp the e ubiquitin ligase triad a negatively regulates the rig-i/mavs signaling pathway by targeting traf for degradation a role for the human nucleotide-binding domain, leucine-rich repeat-containing family member nlrc in antiviral responses meta-analysis of patients with hepatitis c virus genotype : weeks with pegylated interferon and ribavirin is superior to weeks traf and the three c-terminal lysine sites on irf are required for its ubiquitination-mediated activation by the tumor necrosis factor receptor family member latent membrane protein a protein-kinase, ifn-inducible doublestranded rna dependent inhibitor and repressor of p (prk rir) enhances type i ifn-mediated antiviral response through the stability control of rig-i protein inhibition of transcription of herpes simplex virus immediate early genes in interferon-treated human cells hiv- accessory proteins vpr and vif modulate antiviral response by targeting irf- for degradation riplet/ rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-β induction during the early phase of viral infection the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection a distinct role of riplet-mediated k -linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses smurf negatively modulates rig-i-dependent antiviral response by targeting visa/mavs for ubiquitination and degradation tax bp and a inhibit antiviral signaling by targeting tbk -ikki kinases ubiquitin-regulated recruitment of iκb kinase ε to the mavs interferon signaling adapter a functional c-terminal traf -binding site in mavs participates in positive and negative regulation of the ifn antiviral response kinetic mechanism for viral dsrna length discrimination ubiquitin in antiviral immunity by mda filaments. proc. natl. acad. sci. usa structural basis for ubiquitin-mediated antiviral signal activation by rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns protein senp negatively regulates cellular antiviral response by desumoylating irf and conditioning it for ubiquitination and degradation nlrc interacts with rig-i to induce a robust antiviral response against influenza virus infection activation of innate immune antiviral responses by nod regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp negative regulation of interferon-regulatory factor -dependent innate antiviral response by the prolyl isomerase pin master sensors of pathogenic rna -rig-i like receptors recognition of ′ triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/ traf /traf signalosome positive regulation of interferon regulatory factor activation by herc via isg modification nlrx does not inhibit mavs-dependent antiviral signalling itch k -ubiquitinates the nod binding protein, rip , to influence inflammatory signaling pathways nlrx is a mitochondrial nod-like receptor that amplifies nf-κb and jnk pathways by inducing reactive oxygen species production activation of tbk and ikkε kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity a discrete ubiquitinmediated network regulates the strength of nod signaling different modes of ubiquitination of the adaptor traf selectively activate the expression of type i interferons and proinflammatory cytokines homeostatic myd -dependent signals cause lethal inflammation in the absence of a herpes simplex virus immediate-early icp protein inhibits toll-like receptor -dependent inflammatory responses and nf-κb signaling proteomic profiling of the traf interactome network reveals a new role for the er-to-golgi transport compartments in innate immunity the e ubiquitin ligase nrdp 'preferentially' promotes tlr-mediated production of type i interferon nemo binds ubiquitinated tankbinding kinase (tbk ) to regulate innate immune responses to rna viruses usp positively regulates rig-i-mediated antiviral response through deubiquitination and stabilization of rig-i trim negatively regulates interferon-β production and antiviral response through polyubiquitination and degradation of nuclear irf herpes simplex virus ubiquitin-specific protease ul inhibits beta interferon production by deubiquitinating traf tetraspanin (tsp an ) negatively regulates retinoic acid-inducible gene i-like receptor-mediated immune signaling in a ubiquitinationdependent manner ndfip negatively regulates rig-idependent immune signaling by enhancing e ligase smurf -mediated mavs degradation the hepatitis b virus x protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein trim modulates type i interferon induction and cellular antiviral response by targeting rig-i for k -linked ubiquitination trim is essential to sustain ifn regulatory factor activation during antiviral response the mitochondrial ubiquitin ligase mar ch resolves mavs aggregates during antiviral signalling traf and mekk play a pivotal role in the rig-i-like helicase antiviral pathway pcbp mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip the ubiquitin e ligase raul negatively regulates type i interferon through ubiquitination of the transcription factors irf and irf the kshv immediate-early transcription factor rta encodes ubiquitin e ligase activity that targets irf for proteosome-mediated degradation control of canonical nf-κb activation through the nik-ikk complex pathway key role of ubc and lysine- polyubiquitination in viral activation of irf an unexpected twist to the activation of ikkβ: tak primes ikkβ for activation by autophosphorylation negative feedback regulation of cellular antiviral signaling by rbck -mediated degradation of irf kaposi's sarcoma-associated herpesvirus-encoded replication and transcription activator impairs innate immunity via ubiquitin-mediated degradation of myeloid differentiation factor the nemo adaptor bridges the nuclear factor-κb and interferon regulatory factor signaling pathways the e ubiquitin ligase rnf targets virus-induced signaling adaptor for ubiquitination and degradation negative regulation of il- -mediated signaling and inflammation by the ubiquitin-specific protease usp ubiquitin-specific protease regulates tlr -dependent innate immune responses through deubiquitination of the adaptor protein traf poly(c)-binding protein (pcbp ) mediates housekeeping degradation of mitochondrial antiviral signaling (mavs) trim negatively regulates nod by ubiquitination and proteasomal degradation ribose ′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda we are grateful to dr. sarah atkinson for critical review of the manuscript. n.a. borg is an australian research council (arc) future fellow (ft ). the authors declare no competing financial interests. accepted: december key: cord- -tm we sd authors: mossel, eric c.; sainz, bruno; garry, robert f.; peters, c. j. title: synergistic inhibition of sars-coronavirus replication by type i and type ii ifn date: journal: the nidoviruses doi: . / - - - - _ sha: doc_id: cord_uid: tm we sd nan it has been previously shown that treatment of cells with both type i and type ii ifn produces an antiviral state greater in magnitude than can be explained by additive effects alone. [ ] [ ] [ ] [ ] [ ] we sought to determine the effect of such an enhanced antiviral state on the replication of sars-cov. has been extensively examined in culture, animals, and the clinic. ifn-α, relatively ineffective in cell culture, showed suggestive but inconclusive efficacy in monkeys and sars patients. , ifn-β has most potent antiviral activity, though concentrations of greater than u/ml result in only marginal reduction of virus titer. - ifn-γ is ineffective against sars-cov in cell culture. , , to characterize the inhibitory effect of ifn-β and ifn-γ treatment on sars-cov replication, three-day viral growth assays were performed. ifn pretreated vero e cells were infected with sars-cov at a moi of . . cultures treated with u/ml of ifnor ifn-γ were significantly refractory for sars-cov urbani and hk replication (p < . ) at and hpi (figures a and b) . by hpi, however, viral titers in ifn-β-or ifn-γ-treated cultures approached levels detected in vehicle-treated groups. a potent inhibitory effect was observed when vero e cultures were treated with both ifn-β and ifn-γ. the inhibitory effect achieved with combination ifn-β and ifn-γ treatment was consistently greater than -fold at all time points tested and reached levels of greater than × -fold at hpi relative to vehicle treated vero e cells. cytopathic effect (cpe) was extensive in vehicle-treated groups infected with either sars-cov strain at hpi ( fig. a and e) , as evident by the reduced monolayer staining with crystal violet. relative to vehicle-treated and individual ifn-treated cultures, cpe is less evident in cells treated with both ifn-β and ifn-γ at hpi; monolayers appeared evenly stained with little to no visible cpe ( fig. d and h ). to determine whether this phenomenon is limited to vero e cells, additional cell lines were examined. calu- cells showed the same gradated cpe as vero e cells with little or no cpe present in cells treated with both ifn-β and ifn-γ (fig. ) . contrary to the observations of others, gross cpe does not occur in sars-cov-infected caco- cells in our hands. , as such, sars-cov-infected caco- cell monolayers remained confluent regardless of treatment. however, the cpe profile observed in calu- cells suggests that the synergistic inhibitory effect on sars-cov replication by ifn-β and ifn-γ is not vero e cell specific. it has been known for more than years that treatment of cells with type i and type ii ifn potentiates the antiviral response to levels greater than can be explained by simple additive effects. since then, the effect has been shown for a wide variety of viruses, including human cytomegalovirus, hsv- , vesicular stomatitis virus (vsv), lassa virus, and others. , , , , the mechanism of synergistic inhibition of virus replication by type i and type ii ifn has not been determined for any virus. however, it was recently shown that no, induced by a combination of ifn-γ and il- , inhibits sars-cov replication. further, it was shown in an avian system that type i and type ii ifn potentiate the antiviral response as well as the secretion of no. based on this evidence, a role for no and inos in the potentiated anti-sars-cov response induced by type i and type ii ifn cotreatment deserves consideration. the authors wish to thank dr. dr. li-kuang chen from tzu-chi university at hualien, taiwan for kindly supplying the sars-cov strain hk. this work is supported by national institutes of health grants ai (e.c.m.), ai (b.s.), and ai , ai , rr , and ca (r.f.g.) and contract no ai (c.j.p.). pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques treatment of sars with human interferons interferon-beta a and sars coronavirus replication type ii interferons the antiviral effect of interferon-beta against sars-coronavirus is not mediated by mxa protein severe acute respiratory syndrome-related coronavirus is inhibited by interferon-alpha inhibition of different lassa virus strains by alpha and gamma interferons and comparison with a less pathogenic arenavirus potentiation of interferon activity by mixed preparations of fibroblast and immune interferon alpha/beta interferon and gamma interferon synergize to inhibit the replication of herpes simplex virus type potentiation of interferon's antiviral activity by the mutually synergistic interaction of muifn-alpha/beta and muifn-gamma interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) synergistic inhibition of human cytomegalovirus replication by interferon-alpha/beta and interferon-gamma nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus chicken interferon types i and ii enhance synergistically the antiviral state and nitric oxide secretion key: cord- -f fctcru authors: wang, changlin; shan, lingling; qu, shuxin; xue, mei; wang, keliang; fu, fang; wang, lu; wang, ziqi; feng, li; xu, wanhai; liu, pinghuang title: the coronavirus pedv evades type iii interferon response through the mir- c- p/socs axis date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: f fctcru porcine epidemic diarrhea virus (pedv) is an economically important pathogen that has evolved several mechanisms to evade type i ifn responses. type iii interferon (ifn-λ), an innate cytokine that primarily targets the mucosal epithelia, is critical in fighting mucosal infection in the host and has been reported to potently inhibit pedv infection in vitro. however, how pedv escapes ifn-λ antiviral response remains unclear. in this study, we found that pedv infection induced significant ifn-λ expression in type i ifn-defective vero e cells, but virus-induced endogenous ifn-λ did not reduce pedv titers. moreover, we demonstrated that pedv escaped ifn-λ responses by substantially upregulating the suppressor of cytokine signaling protein (socs ) expression, which impaired the induction of ifn-stimulated genes (isgs) and dampened the ifn-λ antiviral response and facilitated pedv replication in vero e cells. we further showed that pedv infection increased socs expression by decreasing host mir- c- p expression. mir- c- p suppressed socs expression through targeting the ′ untranslated region (utr) of socs . the inhibition of ifn-λ elicited isgs expression by socs was specifically rescued by overexpression of mir- c- p. collectively, our findings identify a new strategy by pedv to escape ifn-λ-mediated antiviral immune responses by engaging the socs /mir- c axis, thus improving our understanding of its pathogenesis. porcine epidemic diarrhea virus (pedv), a member of the alphacoronavirus family, is an enteropathogenic coronavirus with economic importance (madson et al., ; wang et al., ; zhang and yoo, ) . pedv infection in newborn piglets is characterized by vomiting, anorexia, watery diarrhea, and dehydration (song and park, ) . the virus primarily infects small intestinal epithelial cells in vivo and causes high morbidity and mortality in piglets (li et al., ) . interferons (ifns) are the key components of innate immunity in response to viral infection . among three types of ifns (types i, ii, and iii), type iii ifn-lambda (ifn-λ) primarily acts on mucosal surfaces, including epithelial surfaces of the liver, respiratory, and gastrointestinal systems, and plays vital roles in controlling viral infection within mucosal surfaces (mordstein et al., ; pott et al., ; lazear et al., ) . we and other groups previously demonstrated that porcine ifn-λdisplays powerful antiviral activity against pedv infection in both vero e cells and porcine intestinal epithelia (li et al., (li et al., , . pedv has evolved multiple strategies to escape ifn responses, including the degradation of stat and the suppression of type i ifn production . although type i and type iii ifns have a large overlap in the spectrum of induced antiviral isg responses, recent studies demonstrated that type iii ifn is a critical non-redundant antiviral mediator of type i ifns in the gi tract and elicits a unique transcriptional profile that does not completely overlap with that induced by ifn-α (wells and coyne, ) . it is necessary to clarify how pedv evades type iii ifn following infection. unlike ample studies reporting that pedv escapes type i ifns, limited studies demonstrate that pedv escapes ifn-λ response. pedv suppresses irf -mediated type iii ifn responses by reducing the number of peroxisomes and counteracting type iii ifn response by pedv nsp endoribonuclease deng et al., ) . deng et al. showed that type i and type iii ifns exhibit different modulation in response to pedv infection and that the discrepancy of type i and type iii ifn responses is independent of pedv endoribonuclease activity (deng et al., ) , suggesting that there are distinct strategies to modify host type i and type iii ifn responses during pedv infection. because cells generally produce both type i and type iii ifns in response to viral infection, it is challenging to elucidate how viruses escape ifn-λ response separately to type i response. in this study, we used vero cells, a cell line with a defective function, to produce endogenous type i ifns. vero cells are widely used as an in vitro model to study the interactions between viruses and hosts including pedv. we and others reported that vero cells respond well to both porcine type i and type iii ifns shen et al., ; li et al., ) . ifn-λ is rapidly produced after infection and following engagement with its receptor induces ifn-stimulated gene (isg) expression to mediate antiviral activity (kotenko et al., ; dellgren et al., ; lazear et al., ) . binding of ifnλ to its receptor, which consists of two subunits, ifn-λr and il- r , leads to activation of jak and tyk , which mediates the phosphorylation of stat and stat proteins (sheppard et al., ; palma-ocampo et al., ) . the suppressor of cytokine signaling protein (socs ), a negative regulator of janus family kinase (jak) signal transducer, simultaneously binds the receptors and jaks and prevents stats from accessing the receptor kinase complex (de weerd and nguyen, ; palma-ocampo et al., ) . previous reports demonstrated that socs is an inducible negative regulator of ifn-λ-induced gene expression in vivo (blumer et al., ) . socs was also associated with denv- escape from ifn-λ response during infection (palma-ocampo et al., ) . however, the role of socs during pedv infection remains unclear. micrornas (mirnas), as important post-transcriptional modulators of gene expression, participate in modulating the host innate and adaptive immune responses in response to pathogen invasion (baltimore et al., ; gottwein and cullen, ; o'neill et al., ) . increasing evidence has shown that mirnas of viral and cellular origin can help viruses evade host immune responses by targeting critical components in the host immune system (cullen, ; sullivan et al., ; kincaid and sullivan, ) . for example, mir- c is a potent negative regulator of type i ifn signaling by targeting jak , resulting in the enhancement of prrsv infection . the mir- family is a well-studied host mirna that plays an important role in viral infection by modulating ifn signaling (zhu et al., ; liu et al., ; ma et al., ) . our previous study revealed that tgev escapes type i ifn response by engaging the ire -mir- a- p/socs / axis (ma et al., ) . the potential role of mirnas in coronavirus escape from ifn-λ response remains elusive. in this study, we showed that pedv escaped ifn-λ responses by upregulating socs expression in type i ifn-defective vero e cells. in addition, we demonstrated that pedv infection increased socs expression by decreasing the expression of host mir- c- p, which modulates socs expression by specifically targeting the ′ utr of socs . our findings identify a new strategy by pedv to escape ifn-λ-mediated host innate immune defenses. african green monkey kidney cells (vero e cells) were stocked by our laboratory and grown in dmem (gibco, gaithersburg, md, usa) supplemented with % fbs (gibco) and antibiotics ( u/ml of penicillin and µg/ml of streptomycin) at • c in a humidified atmosphere of % co . pedv-cv (genbank accession no. kt ) stocked in our laboratory was propagated in vero e cells as previously described (hofmann and wyler, ; sun et al., ) . to evaluate the anti-pedv activity of porcine ifn-λ (prosit sole biotechnology, co., ltd., beijing, china), vero e cells were pretreated with designated concentrations of ifn-λ for h and then infected with pedv (moi of . ). all of the mirna mimics, mirna inhibitors, and short hairpin rnas (shrnas) were synthesized by gene pharma (shanghai, china). the mirnas and shrnas sequences are listed in table . lipofectamine (invitrogen, carlsbad, ca, usa) or lipofectamine rnaimax (invitrogen) was used to transfect cells with plasmid dna or synthetic oligonucleotides according to the manufacturer's instructions. the cells were infected with pedv as previously described after transfection for h. the cells were harvested for quantitative real-time pcr (rt-qpcr) or treated with np- lysis buffer for western blotting after infection for h. sequences total rna isolation, reverse transcription, and qpcr total cellular rna was isolated using an rneasy mini kit (qiagen sciences, hilden, germany) according to the manufacturer's instructions. total rna was extracted and reverse transcribed as previously described (ma et al., ) . for mirna reverse transcription, cdna was prepared with a mirna first strand cdna synthesis kit (sangon biotech, shanghai, china). qpcr was conducted in triplicate with power sybr green pcr master mix reagents (takara) on a lightcycler ii system (thermo fisher scientific, waltham, ma, usa) as previously described (ma et al., ) . the mirna expression levels were normalized to the internal control of u . the sequences of rt-qpcr primers for pedv, ifn-λ, socs , ifit , isg , mxa, gapdh, mir- c, and uni-mir transcription are listed in table . the results are presented as the means ± sem from three separate trials. targetscan release . (http://www.targetscan.org) was used to predict the targets of mir- c- p. socs ′ utrs as a prospective target was cloned and constructed as previously described (ma et al., ) . to construct the monkey socs expression vector, the full-length cds region of monkey socs was amplified from vero e cellular mrna pcr and cloned into pcaggs-ha vector (clontech, mountain view, ca, usa) using ecor i and kpn i restriction sites. the ′ utr of socs (genbank: ) was amplified and inserted into the pmirglo luciferase reporter vector. the socs ′ utr mutant vector was produced by mutating five seed nucleotides using a site-directed mutagenic kit (stratagene, la jolla, ca, usa) according to the manufacturer's instructions. the constructed plasmids were verified by sequencing. the luciferase activities were tested using a dual-luciferase reporter assay system (promega, madison, wi, usa) based on the manufacturer's instructions. wild-or mutant-type socs ′ utr luciferase reporter vectors were co-transfected with mir- c- p mimics (mir- c), mimic nc (nc), mir- c- p inhibitor (mir- c-i), or inhibitor nc (nc-i) into vero e cells for h. then prl-tk was co-transfected with either mir- c, nc, mir- c-i, or nc-i for h. the cells were collected and the luciferase activity was evaluated with a dual-luciferase reporter assay system (promega). the prl-tk vector expressing the renilla luciferase gene was used as a normalization control. vero e cells were fixed with % paraformaldehyde for min at • c and permeabilized with . % triton x- for min, then blocked with blocking buffer (pbs with % fbs) for h at • c. the cells were incubated with an anti-ha monoclonal antibody (sigma-aldrich, munich, germany, : ) at • c for h, followed by labeling with an alexa fluor goat anti-mouse igg antibody (thermo fisher scientific, : ) at • c for h. ′ , -diamidino- -phenylindole (dapi, : ) was used to stain the cellular nuclei. the stained cells were visualized using an amg evos f fluorescence microscope. vero e cells were lysed with np- lysis buffer (beyotime, china) supplemented with . mm of phenylmethylsulfonyl fluoride (pmsf) (roche, indianapolis, in, usa). target proteins were separated on sds-page gels then transferred onto nitrocellulose membranes (ge healthcare, chicago, il, usa). after blocking with tbs-t containing % non-fat milk at room temperature (rt), the membranes were incubated with primary antibody at • c for h. antibodies included: β-actin (sigma-aldrich, : ) and socs (sigma-aldrich, : ). the membranes were incubated with secondary antibody goat antimouse-hrp or goat anti-rabbit-hrp, diluted at : for h at room temperature, and visualized using an ecl system (thermo fisher). the results were analyzed using imagej software. all of the data are described as the means ± the standard error of the mean (sem). graphpad prism (graphpad software, inc.) was used to analyze the data using student's t-test. each experiment was repeated three times. p-values < . were considered significant: * p < . ; * * p < . ; * * * p < . ; * * * * p < . , and ns, not significant. kinetic curve of pedv replication in vero e cells. vero e cells were inoculated with pedv at an moi of . , and the level of pedv infection compared to mock controls at , , and h was quantified by rt-qpcr and tcid . the results were obtained from three independent experiments. mean ± sem, *p < . ; **p < . ; ***p < . ; ****p < . , and ns, not significant. previous research showed that the pretreatment of porcine ifn-λ inhibits pedv infection in vero e cells and ipec-j (li et al., ) . it is well-established that pedv replicates efficiently in vero e cells. to determine whether pedv elicits an endogenous ifn-λ response in vero e cells following infection, we initially infected vero e cells with pedv at moi = . and monitored the ifn-λ expression. compared with a mock uninfected control, pedv did not increase the expression of ifnλ transcripts as observed until hpi, and then gradually induced ifn-λ expression, indicating that pedv infection elicits type iii ifn expression at the late stage of infection instead of the early stage of infection in the vero e cells (figure a) , which was consistent with the results in porcine enteroids (li et al., ) . and pedv propagated efficiently in vero e cells by quantifying viral genomes and titers (figures b,c) . the virus titer increased up to / . ml at hpi ( figure c) . interestingly, despite the increased expression of endogenous ifn-λ at the late stage of infection, the pedv virus titer did not decrease. this indicates that there are mechanisms explored by pedv to antagonize the endogenous ifn-λ isg response at the late-stage infection. pedv infection increased the expression of socs in vero e cells socs , a typical member of the socs family of proteins, is a well-known negative feedback inhibitor of jak/stat signaling pathway induced by cytokines (ma et al., ) . to explore the underlying mechanisms exploited by pedv to escape ifnλ-induced antiviral responses, we initially assessed whether pedv infection induces socs expression in vero e cells. the mrna levels of socs significantly increased following pedv infection and displayed a time-dependent response (figure a) . the induction of socs by pedv infection was further verified by socs western blotting ( figure b ). counteracted the anti-pedv activity of ifn-λ socs is a potent inhibitor of the type i and type ii ifn signaling pathway (skjesol et al., ) . we next investigated whether socs suppresses ifn-λ-mediated antiviral activity. first, we silenced endogenous socs expression by specific shrnas. socs shrnas or a non-targeting shrna (nc) were transfected into vero e cells. the efficiency of socs knockdown was confirmed by western blotting (figure a) . shsocs # and # led to a and % decrease in socs expression, respectively, compared with nc ( figure a) . silencing of endogenous socs by shsocs # or # significantly reduced pedv replication in vero e cells without the addition of exogenous ifn-λ ( figure b) . the decreased levels of pedv infection were in line with the knockdown efficiency of socs shrnas, indicating the specific effect of socs shrnas. as previously reported, exogenous ifn-λ significantly inhibited pedv infection, whereas silencing of endogenous socs by shsocs # or # further enhanced the pedv inhibition by ifn-λ in vero e cells compared with untreated ifn-λ mock control ( figure b) . the knockdown of endogenous socs by shsocs # resulted in a more than . -fold decrease in pedv titer ( figure b ) and degraded to . -fold of pedv titers with ifn-λ treatment in vero e cells (figure b) , indicating that socs knockdown increases the antiviral effects of ifn-λ. inconsistent with the viral results, socs knockdown increased the mrna levels of isg , mxa, and ifit ( figure c) . we subsequently investigated the role of socs overexpression on the anti-pedv effects of ifn-λ. the transient overexpression of socs in vero e cells was verified by ha ifa (figure d ). as expected, socs overexpression substantially elevated pedv figure | mimics or inhibitor for h, cells were pretreated with ifn-λ or dmem for h and then infected with pedv (moi = . ) and harvested at hpi for viral rna quantification and tcid . (f) the socs expression levels in vero e cells were measured by rt-qpcr at hpi at different mois. p values represent the difference from the mock-infected control for time kinetics, the socs , and mir- c- p levels. error bars, mean ± sem. (n = independent experiments). *p < . , **p < . , ***p < . , and ns, not significant. infection ( figure e ) and blunted the expression of isg , mxa, and ifit ( figure f) . thus, these data indicate that socs counteracts the anti-pedv activity of ifn-λ. modulating mir- c- p the porcine mir- family (five members: mir a-e) has been demonstrated to modulate host type i ifn response during virus infection (zhu et al., ; liu et al., ; ma et al., ) . the targetscan (http://www.targetscan. org) prediction program indicated that socs was targeted by mir- c- p through a site in the ′ utr conserved in the socs of seven representative mammals ( figure a ). to investigate whether mir- c- p is involved in modulating ifnλ signaling by directly targeting socs and downregulating endogenous socs expression, we conducted a computational analysis using targetscan release . (http://www.targetscan. org). the result showed that mir- c could directly target the site on the ′ utrs of socs ( figure a) . we cloned the predicted target sites in porcine socs ′ utr, and constructed the firefly luciferase reporter vector of porcine socs ′ utr ( figure a) . overexpression of mir- c- p, the luciferase reporter containing the socs wild-type target sequence, decreased to ∼ % relative to nc mimics, whereas the blockage of mir- c by mir- c inhibitor increased socs ′ utr luciferase activity. however, the mutation of the socs target ′ utr site of mir- c- p disrupted the effects of mir- c- p on modifying the luciferase activity in vero e cells relative to the ncs ( figure b) . these results confirmed that mir- c- p directly targets the ′ utr of socs . consistent with the luciferase results, mir- c- p overexpression reduced socs expression measured by western blotting (figure c) . conversely, blockage of endogenous mir- c- p increased the expression of socs in vero e cells compared with the nc inhibitor. to further validate the modulation of socs expression by mir- c- p during pedv infection, we examined the expression of socs in pedvinfected vero e cells with overexpression or inhibition of mir- c- p, and found that the expression pattern of socs in pedv infected vero e cells was similar to that in pedv-uninfected e cells ( figure c) . taken together, these data demonstrated that mir- c- p downregulates the expression of socs by directly targeting socs ′ utr. to verify whether pedv escape the ifn-λ antiviral signaling through mir- c- p mediated modification of socs expression, we then explored the effect of mir- c- p on pedv infection and ifn-λ antiviral signaling. transient mir- c expression reduced pedv titers and promoted ifn-λ anti-pedv activity compared with the mock control ncs, whereas mir- c- p inhibitor significantly increased pedv infection and undermined the anti-pedv activity of ifn-λ (figures d,e) . furthermore, the socs expression increased starting at hpi and substantially increased at hpi, which was inversely correlated with the kinetic expression profiles of mir- c- p ( figure f ). in agreement with the kinetics pattern of mir- c- p and socs in vero e cells, pedv infection reduced the levels of mir- c- p and increased socs expression in ipec-j starting at h post-infection (data not shown). collectively, pedv infection upregulates socs expression by modulating host mir- c- p abundance at the late stage of infection. to further verify whether pedv escape ifn-λ response through the mir- c- p/socs axis, we co-transfected mir- c- p with socs and measured the replication of pedv with or without ifn-λ treatment. socs overexpression promoted pedv replication with or without ifn-λ treatment. mir- c- p largely abolished the role of socs in promoting pedv replication, and the effect was more pronounced in the presence of ifn-λ stimulation ( figure a) . consistent with this, socs inhibited ifn downstream isgs expression such as ifit and isg expression (figures b,c) , whereas overexpression of mir- c- p abrogated the isg inhibition of socs , which was more pronounced in the presence of ifn-λ priming (figures b,c) . in summary, these data indicate that pedv escapes the response of ifn-λ through the mir- a- p/socs axis. ifn-λ is an antiviral innate cytokine induced by virus infection that plays vital roles in controlling mucosal infection (blumer et al., ) . we and other groups previously showed that ifnλ substantially inhibits pedv (li et al., ; zhang et al., ) . however, whether pedv has evolved a mechanism to counteract endogenous ifn-λ just as pedv does the type i ifn response remains unclear. in this study, we found that pedv propagated well despite the significant production of endogenous ifn-λ induced at the late stage of infection in vero e cells, indicating that pedv escaped the ifn-λ response at the late stage of infection not through suppressing ifn-λ production. we further defined the mechanism that pedv counteracted ifnλ-elicited antiviral isg responses by exploiting the mir- c- p/socs axis. pedv has evolved multiple strategies to escape type i ifn response. whether pedv exploits similar mechanisms to counteract type iii ifn remains elusive. one previous study demonstrated that pedv escaped type iii ifn by suppressing irf -mediated ifn-λ production through pedv viral nsp protein . in that study, pedv actually upregulates ifn-λ expression at h post-infection and then decreased to minimal levels of ifn-λ expression until hpi figure | mir- c- p inhibited the infection of pedv by regulating the expression of socs . (a) socs overexpression increased pedv infection and undermined the anti-pedv activity of ifn-λ. vero e cells were transfected as described with pcaggs-ha, pcaggs-socs , and mir- c- p for h, followed by incubation with porcine ifn-λ ( ng/ml) or dmem for h. the cells then were infected with pedv at an moi of . ; pedv infection was determined at hpi. (b,c) mir- c- p abolished the impairment of the overexpression of socs to ifn-λ signaling under ifn-λ-stimulated or pedv-infected conditions. e cells were treated as described in the legend for panel a. the cells were collected for rt-qpcr analysis of ifit and isg expression relative to that of gapdh. error bars, mean ± sem (n = independent experiments). *p < . , **p < . , and ns, not significant. . in agreement with this, we did not observe increased ifn-λ expression at hpi ( figure a) . they did not show the ifn-λ expression at the late stage of pedv infection. in the current study, we observed that pedv elicited substantially increased ifn-λ expression in vero e cells only after hpi (figure a) , which is consistent with the results observed in porcine enteroids following pedv infection (li et al., ) , indicating that pedv has evolved mechanisms to escape ifn-λ antiviral response instead of ifn-λ production at the late stage of infection. it is possible that pedv exploits varying strategies at different infection stages. this is also observed in other rna viruses such as influenza virus (chung et al., ) . to prevent over-activation of the ifn signaling pathways, the host evolves a few negative regulators of ifn signaling, and socs is one of the canonical inhibitors of ifn signaling (shao et al., ) . socs has been reported to be exploited by multiple viruses to abrogate ifn antiviral signaling (shao et al., ; wei et al., ; ma et al., ) . we showed that pedv significantly induced the expression of socs at the late stage of infection (figure ) . as expected, increased socs impaired the antiviral isgs expression and impaired the anti-pedv activity of ifn-λ (figure ) . this is in agreement with the results of tgev, another swine alphacoronavirus (ma et al., ) . therefore, unlike previously published studies with the modification of ifn production mediated by viral proteins such as nsp , our study found that pedv largely evades innate immunity of ifn-λ by modulating the antiviral signal of ifn-λ rather than manipulating the production of ifn-λ at the late stage of infection. mirna plays a vital role in regulating gene expression through post-transcription modification. increasing evidence demonstrates that viruses escape ifn antiviral activity for optimal infection by modifying the cellular abundance of mirna targeting vital components of the ifn response (zhu et al., ; liu et al., ; ma et al., ) . jev infection downregulates the expression of mirna mir- , which directly targets the suppressor of cytokine signaling protein (socs ) and manipulates the jak-stat signaling cascade (sharma et al., ) . the mir- family has been reported to target socs family members and manipulate the jak/stat signaling pathway (zou et al., ; ma et al., ; yuan et al., ) . in this study, we showed that pedv infection suppressed mir- c- p expression, which was conversely related to socs expression during pedv infection (figure ) . just as other members of mir- , mir- c- p specifically targeted the ′ utr of socs and inhibited socs expression (kobayashi et al., ; ma et al., ; yuan et al., ) (figure ) . however, the mechanism of pedv decreasing mir- a- p remains unclear and deserves further study. in summary, we determined that pedv escaped ifn-λ response at the late stage of infection by downregulating mir- c- p, thus increasing socs expression. therefore, unlike previously published studies with defined mechanisms such as nsp , we demonstrated that pedv escapes ifn-λ response through another pathway of the mir- c- p/socs axis. our results highlight the important role of mir- c- p in the regulation of interferon pathways during pedv infection, improve the current knowledge of pedv infection, and expand the role of micro-rna in viral infection. the datasets generated for this study are available on request to the corresponding author. pl, cw, ls, and wx designed the research studies and analyzed and interpreted the data. cw, ls, sq, mx, kw, ff, lw, zw, and lf conducted the experiments and collected the data. pl, cw, and ls drafted the manuscript. all of the authors contributed revisions. micrornas: new regulators of immune cell development and function socs is an inducible negative regulator of interferon lambda (ifnlambda)-induced gene expression in vivo construction of a transcriptomedriven network at the early stage of infection with influenza a h n in human lung alveolar epithelial cells viruses and micrornas the interferons and their receptors-distribution and regulation human interferon-lambda is a potent member of the type iii interferon family coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses viral and cellular micrornas as determinants of viral pathogenesis and immunity porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat propagation of the virus of porcine epidemic diarrhea in cell culture virus-encoded micrornas: an overview and a look to the future identification of mir- d as a novel prognostic maker of prostate cancer ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex interferon-lambda: immune functions at barrier surfaces and beyond porcine intestinal enteroids: a new model for studying enteric coronavirus porcine epidemic diarrhea virus infection and the host innate response ifn-lambda preferably inhibits pedv infection of porcine intestinal epithelial cells compared with ifn-alpha new variants of porcine epidemic diarrhea virus china microrna- c targets the interferon-alpha/beta receptor beta chain to promote type prrsv infection the coronavirus transmissible gastroenteritis virus evades the type i interferon response through ire alpha-mediated manipulation of the microrna pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/ / ) in -week-old weaned pigs lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections micrornas: the finetuners of toll-like receptor signalling interferon lambda inhibits dengue virus replication in epithelial cells ifn-lambda determines the intestinal epithelial antiviral host defense socs abrogates ifn's antiviral effect on hepatitis c virus replication japanese encephalitis virus exploits the microrna- to regulate the expression of suppressor of short communication: antiviral activity of porcine ifn-lambda against porcine epidemic diarrhea virus in vitro il- , il- and their class ii cytokine receptor il- r functional conservation of suppressors of cytokine signaling proteins between teleosts and mammals: atlantic salmon socs binds to jak/stat family members and suppresses type i and ii ifn signaling porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines expression and function of micrornas in viruses great and small analysis of protein expression changes of the vero e cells infected with classic pedv strain cv by using quantitative proteomic technique new variant of porcine epidemic diarrhea virus united states suppression of interferon lambda signaling by socs- results in their excessive production during influenza virus infection type iii interferons in antiviral defenses at barrier surfaces microrna- a inhibits the liver cell proliferation and promotes cell apoptosis through the jak/stat signaling pathway by targeting socs- in rats with sepsis microrna- c modulates type i ifn responses to facilitate porcine reproductive and respiratory syndrome virus infection by targeting jak type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp in irf signaling immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling microrna- e * suppresses dengue virus replication by promoting nf-kappab-dependent ifn production microrna- c- p ameliorates hypoxia-reoxygenation-induced tubular epithelial cell injury via hif alpha stabilization by targeting socs the authors declare that the research was conducted in the key: cord- -vox xsgd authors: deng, xufang; baker, susan c title: an “old” protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: vox xsgd here we review the evolving story of the coronavirus endoribonuclease (endou). coronavirus endou is encoded within the sequence of nonstructural protein (nsp) , which was initially identified as a component of the viral replication complex. biochemical and structural studies revealed the enzymatic nature of nsp /endou, which was postulated to be essential for the unique replication cycle of viruses in the order nidovirales. however, the role of nsp in coronavirus replication was enigmatic as endou-deficient coronaviruses were viable and replicated to near wild-type virus levels in fibroblast cells. a breakthrough in our understanding of the role of endou was revealed in recent studies, which showed that endou mediates the evasion of viral double-stranded rna recognition by host sensors in macrophages. this new discovery of nsp /endou function leads to new opportunities for investigating how a viral endou contributes to pathogenesis and exploiting this enzyme for therapeutics and vaccine design against pathogenic coronaviruses. we provide a brief outline of the major research findings related to coronavirus (cov) endoribonucleases (endou) in table . in the text below, we describe the experimental approaches that led to these findings and compare the activity of cov endou with reports of other viral and host ribonucleases. initial studies focused on identifying the products of the cov replicase polyprotein, pp ab (depicted in fig. a ). heusipp et al. used a murine monoclonal antibody ( g ) that recognizes amino acid residues - of human cov e pp ab (heusipp et al., ) . they identified a -kda polypeptide in virus-infected cells and found that this polypeptide was a product of the viral c-like proteasemediated cleavage of pp ab. this protein localizes to the perinuclear region, as detected by immunofluorescence assay, similar to other pp ab-derived polypeptides (heusipp et al., ) . shi and coworkers obtained similar findings while studying mouse hepatitis virus (mhv) (shi et al., ) . they generated a rabbit antiserum against amino acids - of mhv pp ab and found that this antiserum detected a -kda product in infected cells. they also found that this protein colocalized with de novo synthesized viral rna, and therefore postulated that this viral protein associated with the viral rna replication/ transcription machinery (shi et al., ) . later, the corresponding polypeptides that these antibodies recognized were defined as the th cleavage product of pp ab (called nsp ), counting from the aminoterminus to the carboxyl terminus of pp ab (ziebuhr et al., ; snijder et al., ) . after the outbreak of severe acute respiratory syndrome (sars) in [ ] [ ] , and once a cov had been confirmed as the etiological agent of sars, researchers intensively scrutinized cov genomic sequences to better understand this novel human pathogen. by comparative genomic characterization of cov replicases, snijder et al. reported that the c-terminus of nsp has high sequence similarity to the xenopus laevis poly(u)-specific endoribonuclease and therefore predicted that nsp possesses endou activity (snijder et al., ) . based on the available sequence information of viruses from the order nidovirales at that time, the endou was considered a nidovirus-specific marker (called nendou) (fig. b) (snijder et al., ) . the members of the family arteriviridae, including equine arteritis virus (eav) and porcine respiratory and reproductive syndrome virus (prrsv), also have endou domains within nsp . however, it was later discovered that the presence of the endou domain is not universal in all nidoviruses. nam dinh virus, the first insect nidovirus belonging to the family mesoniviridae, and roniviruses that infect invertebrates, do not encode an endou domain (nga et al., ; lauber et al., ) . these findings suggest that the endou domain may only serve as a signature for vertebrate nidoviruses, including covs and arteriviruses (fig. ). previous reviews have comprehensively summarized the biochemical and structural features of nidovirus ribonucleases (ulferts and ziebuhr, ; snijder et al., ) . here, we highlight the key experiments with respect to nidovirus endou and provide recent updates. studies performed by ivanov et al. ( ) and bhardwaj et al. ( ) first demonstrated the endou activity of sars-cov nsp in vitro (ivanov et al., ; bhardwaj et al., ) . the wild-type (wt) nsp and its mutants with alanine (ala) substitutions of the putative catalytic residues were expressed in e. coli. the recombinant nsp -wt, but not the mutants, could efficiently cleave single-stranded (ss) and doublestranded (ds) rnas. in contrast, neither ssdna nor dsdna molecules could be processed by nsp , demonstrating its predicted ribonuclease-as opposed to deoxyribonuclease-activity. blocking either the ′ or the ′ terminus of the rna substrates by covalent modifications with fluorescein or puromycin, respectively, had no effect on the rna cleavage (bhardwaj et al., ) , confirming that nsp is an endorather than an exoribonuclease. similar endou activity was detected in other cov nsp s, including human cov e, mhv, infectious bronchitis virus (ibv), and turkey cov (ivanov et al., ; bhardwaj et al., ; cao et al., ) , and in the nsp s of arteriviruses eav and prrsv (nedialkova et al., ) . hexamerization of nsp is critical for its endou activity. guarino and coworkers found that cov nsp could be present in solution as either monomers or hexamers in a protein concentration-dependent manner. the hexamer is the fully active form of endou that binds rna and executes optimal endou activity (guarino et al., ) . the residues in the n-terminal domain of nsp are critical for hexamer formation (guarino et al., ) . in addition, nsp requires divalent metal ions as a co-factor for rna cleavage and prefers mn + over mg + or other divalent cations (ivanov et al., ; bhardwaj et al., ) . addition of mn + significantly affects the protein conformation, enhances rna binding, and increases endou activity (ivanov et al., ; bhardwaj et al., ) . in contrast to cov nsp , arterivirus nsp forms dimers in solution and does not require divalent cations as a cofactor for activity in vitro (nedialkova et al., ; shi et al., ) . instead, a concentration of mn + that greatly stimulated the activity of cov nsp inhibited the endou activity of eav and prrsv nsp s. cov endou was revealed biochemically to hydrolyze the ′ end of pyrimidines, with a preference for uridylates, and release products with ′, ′-cyclic phosphate and ′-hydroxyl ends (ivanov et al., ; bhardwaj et al., ) . this finding seems to implicate a broad range of targets; however, the endou activity of cov nsp can be affected by secondary structure and modification of the rna substrate. bhardwaj et al. found that nsp preferentially cleaved unpaired uridylates in hairpin-structured rnas and that the neighboring nucleotides of targeted sites also influenced hydrolysis (bhardwaj et al., ) . on the other hand, ivanov et al. found that ′-o-ribose methyl groups present on the substrate rna blocked endou-mediated cleavage (ivanov et al., ) . these data suggest that multiple factors might limit the range of endou targets. this is reasonable because the endou activity of nsp is likely to be tightly regulated during infection in cells to avoid unwanted cleavage on viral and/or cellular targets. for example, cov nsp is a ′-o-ribose methyltransferase, whose function could theoretically block the endou-mediated cleavage of viral rnas. similar to cov nsp , arterivirus nsp also prefers ′ of uridylates for cleavage and yields products with ′, ′-cyclic phosphate ends (nedialkova et al., ) . further studies are needed to address whether the endou activity of arterivirus nsp is restricted by rna modifications or secondary structures. guarino and coworkers visualized single particles of sars-cov nsp using electron microscopy (guarino et al., ) and the same group later reported an . Å structure of nsp by cryoelectron microscopy (bhardwaj et al., ) . they reported that the nsp hexamer comprises a dimer of trimers and proposed that the rna substrate binds to the inter-trimer interface. x-ray crystal structures of sars-cov and mhv nsp s were first solved by ricagno et al. and xu et al., respectively (ricagno et al., ; xu et al., ) . these high-resolution structures define cov nsp as a separate endou family with unique folds that differ from cellular endoribonucleases (fig. a) . the monomer of sars-cov nsp consists of three domains: a small nterminal domain (ntd) (residues - , cyan), a middle domain (residues - , magenta), and a large c-terminal domain (ctd) (re-sidues - , green). the endou is located in the ctd. the monomeric structure of mhv nsp can be superimposed onto that of sars-cov nsp , except for a flexible loop structure in the middle domain of mhv nsp (fig. b ). this flexible loop is encoded by a viral rna packaging signal sequence, which is present in mhv nsp but not in sars-cov nsp (kuo and masters, ) . these structural studies demonstrated that the presence of the flexible loop did not alter the overall folding of mhv nsp relative to sars-cov nsp . in addition, through these and other structural studies (ricagno et al., ; xu et al., ; joseph et al., ; bhardwaj et al., ) , the nsp hexamer was again confirmed to be a dimer of trimers. as shown in fig. c , three monomers form a trimer and two trimers interact headto-head to form a hexamer. the ntds line up in the center of the hexamer, while the ctds face outward. this architecture allows the nsp hexamer to possess six active sites. the extensive contact between subunits through the ntd and middle domain is critical for hexamerization. the crystal structure of arterivirus nsp was also recently reported (shi et al., ; zhang et al., ) . these structural studies revealed that the monomer of nsp contains only two domains: ntd and ctd (no middle domain). the monomeric structures of cov nsp and arterivirus nsp could not be superimposed except in the ctd (catalytic domain). distinct from the hexameric structure of cov nsp , nsp monomers assembled into an asymmetric dimer (shi et al., ) . virus-encoded endoribonuclease is a genetic signature of nidoviruses that infect vertebrates. a phylogenic tree of representative nidoviruses was generated based on a conserved region of rdrp (sequnces and genbank assession numbers are available upon request). multiple sequence alignment and phylogeny analyses were conducted with the programs muscle and phyml, respectively (available at http:// www.phylogeny.fr/). the phylogenic tree was generated using dendroscope software version with default parameters. virology ( ) - since the crystallographic studies of cov nsp revealed that the active site of endou is structurally similar to that of rnase a, researchers evaluated small molecule inhibitors of rnase a for their ability to inhibit nsp activity. ortiz-alcantara et al. tested several commercially available rnase a inhibitors (benzopurpurin b, congo red, and others) and reported that their % inhibitory concentration (ic ) values for inhibiting the endou activity of sars-cov nsp ranged from . μm to μm. benzopurpurin b was shown to have a broad-spectrum activity and could inhibit nsp orthologs from mhv and ibv with ic values of . μm and . μm, respectively (ortiz-alcantara et al., ). these rnase a inhibitors had variable effects on cov replication in cell cultures. in plaque formation assays, treatment with congo red resulted in -fold and -fold reductions in mhv and sars-cov titers, respectively, while benzopurpurin b led to marginal inhibition of both covs (ortiz-alcantara et al., ) . although the impact of these rnase a inhibitors on cov replication requires more comprehensive investigation, these early results suggest that the similarity between nsp /endou and rnase a may provide a basis for exploiting small molecule inhibitors to modulate viral endou activity. due to its unique enzymatic activity and co-localization with the replicating viral rna, nsp /endou was initially thought to play an important role in virus replication. however, mhv encoding catalyticdefective endou exhibited only a subtle defect in rna synthesis and a slight reduction in viral titers (~ log) compared to wt virus when evaluated in fibroblasts (kang et al., ) . similar results were obtained for sars-cov and hcov- e nsp mutants generated using either reverse genetics or replicon systems (ulferts and ziebuhr, ) . these data indicate that the endou activity of nsp is not essential for cov replication, as was initially proposed (snijder et al., ; ivanov et al., ; bhardwaj et al., ) . intriguingly, nsp may indirectly affect virus replication through other mechanisms. ivanov et al. demonstrated that when the conserved aspartic acid (asp)- of hcov- e nsp was replaced with an ala, its endou activity was eliminated, viral rna synthesis was completely abolished, and no viable virus was recovered (ivanov et al., ) . similar phenotypes were observed for an mhv nsp mutant with an ala substitution of asp- (equivalent to asp- of hcov- e) (kang et al., ) . it is unclear how the asp residues affect viral rna synthesis. kang et al. predicted that asp- is critical for an ionic-bond network and observed that the ala substitution resulted in an insoluble protein when mhv nsp was expressed in e. coli (kang et al., ) . these data suggest that the ala substitution may prevent the nsp protein from folding correctly. since the proteins adjacent to nsp are critical replicative components, any protein-folding issue with nsp may have an effect on the proteolytic processing of the neighboring components, thereby leading to a nonviable phenotype. overall, current evidence indicates that the endou activity of cov nsp is dispensable for viral rna synthesis and virus replication in cell culture. further work is required to address any non-endou-mediated role of nsp protein in virus replication. mutagenesis of arterivirus nsp revealed pleiotropic effects of endou on the viral life cycle (posthuma et al., ; sun et al., ) . similar to cov nsp , mutations in the asp- and asp- residues (corresponding to mhv nsp asp- and asp- , respectively) in eav nsp resulted in a nonviable phenotype. compared with the mild effect of mutating the catalytic histidine (his) residues (hi-s ala and his ala) of mhv nsp , eav infectious clones with catalytic residue mutations (his ala/gln and his ala/gln) exhibited smaller plaque sizes, reduced rna synthesis, and dramatic titer reductions up to log units. other substitutions at conserved, noncatalytic residues of eav nsp resulted in an intermediate phenotype: intermediate plaque sizes and~ - log reduction in titers. similar results were obtained with the prrsv nsp mutant viruses (sun et al., ) . these data indicate that arterivirus nsp may be involved in virology ( ) - viral rna synthesis. however, similar to the asp ala mutation of mhv nsp , both asp ala and asp ala mutations in eav nsp rendered the protein insoluble, again raising the question of whether or not these mutations influence viral rna synthesis through interfering with proteolytic processing of the orf b polyprotein. the absence of an endou domain in insect nidoviruses and invertebrate roniviruses further indicates that endou activity is not required for the unique rna synthesis strategy of nidoviruses (nga et al., ; lauber et al., ) . the aforementioned studies highlight the extensive efforts of the field to investigate the characteristics of nsp /endou and its role in virus life cycle. the endou activity of cov nsp was found to play a non-essential role in viral rna synthesis and replication in immortalized fibroblast cells. recent work with different cell types and in vivo experiments revealed a novel function of nsp /endou in virus replication and pathogenesis and provided a new direction of study with respect to this "old" protein. cov nsp was first suggested to possess interferon (ifn) antagonism capabilities through ectopic expression experiments. frieman et al. used an alphavirus replication-defective vector (vrp) to screen sars-cov proteins that suppress vrp-induced ifn responses. one of the identified ifn antagonists of sars-cov was nsp (frieman et al., ) . later, arterivirus nsp was also identified as an ifn antagonist and its endou activity was found to mediate inhibition of ifn-beta induction (beura et al., ; shi et al., ) . several other studies also reported that cov nsp and arterivirus nsp inhibit cellular innate responses in ectopic expression experiments (lei et al., ; wang et al., ) . these data seem to indicate that these two proteins function as ifn antagonists; however, the endou activity of overexpressed nsp /nsp may unexpectedly affect the activities of the reporters used in these assays. we found that transfected mhv nsp reduced the signal of both ifn-reporter firefly luciferase and the internal control renilla luciferase (hackbart m, deng x, and baker s, unpublished data). a similar result was obtained with the overexpressed prrsv nsp (shi et al., ) . these observations imply that nsp /nsp may execute non-specific cleavage when ectopically expressed. since cov nsp is part of the viral replicase/transcriptase complex (rtc), it is reasonable to predict that its endou activity is tightly regulated during viral infection to avoid unwanted cleavage. in line with this prediction, we and others reported a specific, punctate, perinuclear localization of cov nsp during viral infection (heusipp et al., ; shi et al., ; deng et al., ; athmer et al., ) , while ectopically expressed nsp was distributed throughout the cytoplasm (cao and zhang, ) . hence, we advise caution when interpreting the results of overexpression studies, as the nature of the endou activity was only revealed after studying nsp /nsp in the context of viral infection. it was first discovered that the endou activity of nsp mediates the evasion of host recognition of viral dsrna by infecting primary macrophages with endou-deficient covs (deng et al., ; kindler et al., ) . infection with mhv endou-deficient mutants stimulated mouse bone-marrow derived macrophages (bmdms) to produce a remarkably high level of type i ifns during the early phase of infection compared to wt infection. this ifn response is mda -dependent, as both the ifn mrna and protein levels were not elevated in mda -deficient bmdms. moreover, the replication of endou-deficient covs was severely impaired in primary macrophages. interestingly, the ifn-induced antiviral response is not the only player responsible for this replication defect, as the titers of the endou-deficient covs were not completely restored in bmdms that lack critical genes (e.g. mda , mavs, and irf / / ) involved in the ifn response (deng et al., ; kindler et al., ) . these data suggest that other antiviral pathways may also contribute to the observed replication defect. in support of this, it was found that the infection of endou-deficient covs also activate the pkr and the oas-rnase l pathways (deng et al., ; kindler et al., ) , which both execute potent antiviral functions, discussed further below. indeed, the replication of endou-deficient covs could be partially restored in pkr/ rnase l-double knockout cells (kindler et al., ) . replication was only fully restored in type i ifn receptor-knockout macrophages, as these cells not only have a defect in ifn signaling but also express very low basal levels of pkr and oas relative to wt cells (deng et al., ; birdwell et al., ) . taken together, these studies using live viruses in primary cells effectively illustrated the ifn antagonistic properties of cov endou. as mentioned above, infection with endou-deficient covs also activates the pkr and oas-rnase l pathways in macrophages (deng et al., ; kindler et al., ) . pkr is a dsrna-activated protein kinase and serves as a dsrna sensor. activated pkr phosphorylates eukaryotic initiation factor α (eif α), resulting in inhibition of host and viral mrna translation. thus, pkr-mediated translation shutoff plays an important role in the host antiviral defense (barber, ) . the endou-deficient cov-infected macrophages exhibited increased levels of phosphorylated eif α and decreased levels of translation (deng et al., ; kindler et al., ) , indicating that pkr was activated during infection. another piece of evidence of pkr activation was that endou-deficient covs induced rapid apoptotic cell death in infectedmacrophages (deng et al., ) . it has been shown that pkr-mediated translation shutoff leads to apoptosis in macrophages (hsu et al., ) . when the endou-deficient cov-infected macrophages were treated with a pkr-specific inhibitor (c ), the level of apoptosis was significantly reduced (deng et al., ) . this result further supports the hypothesis that endou-deficient covs activate pkr. interestingly, loss of pkr expression or inhibition of its activity only partially restored the replication of endou-deficient covs in macrophages (kindler et al., ) . moreover, treatment with the pkr inhibitor did not affect ifn induction or rnase l-mediated ribosomal rna degradation in the endou-deficient cov infected-macrophages (deng et al., unpublished data) . these results imply that the infection of endou-deficient cov activates multiple host dsrna sensors independently, including mda , pkr, and oas. oas is a protein family of ′, ′-oligoadenylate ( - a) synthetases. upon activation, oas can synthesize - a, which binds to and activates rnase l. rnase l is a host ribonuclease that executes global degradation of host and viral rnas. thus, oas and rnase l constitute a potent host antiviral system. macrophages infected by the endou-deficient covs exhibited an early, rnase l-mediated degradation of ribosomal rna, demonstrating that the oas-rnase l system was activated (deng et al., ; kindler et al., ) . lack of mda expression or treatment with the pkr inhibitor did not affect virus-induced rna degradation (deng et al., ; kindler et al., ) , suggesting that the nsp -mediated blockage of oas-rnase l activation is independent of the mda -ifn and pkr pathways. loss of rnase l expression does not restore the replication of endou-deficient covs in macrophages. taken together, these results suggest again that multiple antiviral pathways, including mda -ifn, pkr, and the oas-rnase l system, were activated during infection with endou-deficient covs. the antiviral defense executed by these pathways contribute together to the replication defect of endou-deficient covs in macrophages. interestingly, some covs encode a ′, ′-phosphodiesterase (pde) (e.g. mers-cov orf b and mhv ns ) (thornbrough et al., ; zhao et al., ) . this cov pde also prevents rnase l-mediated rna degradation through digesting the - a produced by oas. thus, the presence of two antagonists of the oas-rnase l system in some cov genomes represents a functional redundancy or tissue-specific roles for these antagonists. in fact, not all covs encode a pde, and it has been reported that loss of the pde activity mitigated mhv pathogenicity in the mouse liver but not in the brain, suggesting a liver-specific effect of cov pde activity in vivo (roth-cross et al., ; zhao et al., ) . several endou-deficient covs have been evaluated in fibroblast cell lines and no marked phenotypes were obtained (mild reduction of viral rna synthesis and replication) (discussed above). we speculate that the activation of an ifn response and apoptosis in macrophages is due to high levels of basal expression of host sensors, such as mda , pkr, and oas (deng et al., ; birdwell et al., ) . indeed, when the expression of oas was induced by pre-treatment with ifn, the immortalized fibroblast cells also exhibited rna degradation upon infection with endou-deficient cov (kindler et al., ) . in line with this, without the stimulation of ifn, constitutively expressed oas and pkr in the mda -deficient macrophages is capable of sensing the endou-deficient cov infection and implementing antiviral processes (deng et al., ) . although the production of ifn is dispensable for the activation of the oas-rnase l system and pkr, type i ifn receptors or a direct signal is required for maintaining the high basal expression of these ifn-inducible genes (deng et al., ; birdwell et al., ) . this is biologically relevant because macrophages and other myeloid cells are quick responders to early virus infection, responding even before ifn is highly induced. it is unclear whether other cell types, such as epithelial cells, behave similar to macrophages, but at least mouse embryonic fibroblasts have been shown to display the nsp /endoumediated effects (kindler et al., ) . due to robust activation of antiviral responses, mhv endou-deficient mutants also exhibited a marked attenuation in vivo relative to mhv-wt. depending on the inoculation route, mhv infection can result in hepatitis or encephalitis in c /bl mice. strikingly, regardless of which infection route (intraperitoneal or intracranial injection) was used, we found that mhv endou-deficient mutants were highly attenuated (deng et al., ) . when mice were inoculated intraperitoneally using a high dose of the mutant virus, there was no detectable viral titer in the liver or spleen at day post-infection and only a minimal detection of viral mrna in mesenteric lymph nodes at day post-infection. when a sensitive encephalitis model was used, mice infected with mhv endou-deficient mutants exhibited only a transient loss of body weight and recovered completely. this significant attenuation is attributed to the loss of endou-mediated evasion of host antiviral defenses. we found that endou-deficient mutants maintained wt-level virulence in type i ifn receptor knockout (ifnar -/-) mice (deng et al., ) . similar results obtained by kindler et al. showed that the mutant virus was only detected in the organs from ifnar -/mice but not wild-type or other gene-deficient (mda -/-, tlr -/-, and mda -/-/tlr -/-) mice at day post-infection (kindler et al., ) . interestingly, even though mhv endou-deficient mutants were highly attenuated and exhibited limited replication in vivo, the pre-infected mice were protected from subsequent lethal challenges of wild-type mhv in both disease models (deng et al., ) . these results demonstrate that nsp plays an important role in virus pathogenesis and illustrate the potential use of endou-deficient covs as vaccine candidates. . . how does nsp mediate the evasion of host sensors? as an endou, it is plausible that nsp may degrade viral dsrna to prevent host recognition. previous studies of pestivirus envelope glycoprotein (e rns ) and lassa virus nucleoprotein linked viral ribonuclease activity to type i ifn antagonism through degrading viral dsrna (python et al., ; qi et al., ; hastie et al., ) , although no direct evidence for this linkage has been obtained. for cov nsp , kindler et al. detected an increased level of dsrna in endou mutantinfected cells by flow cytometry using a dsrna-specific monoclonal antibody (mab) (kindler et al., ) . this mab recognizes dsrna molecules longer than bp in length, regardless of the sequence, meaning that a long dsrna could potentially bind multiple mab molecules. to saturate the mab-dsrna binding, we tested serial concentrations of antibody but did not detect any significant change of dsrna levels by either flow cytometry or immunofluorescence analysis (deng et al., ) . the discrepancy between these two studies may be ascribed to the experimental settings in respective experiments. however, it is also possible that the methods used in these studies may not be sensitive enough to detect changes in dsrna levels if the wt-nsp produces dsrna cleavage products that are > bp, such that the uncleaved and cleaved dsrnas are indistinguishable for binding by the anti-dsrna antibody. importantly, it has been demonstrated that the endou activity of cov nsp is influenced by secondary structures and modifications of dsrna (ivanov et al., ; bhardwaj et al., ) . because these factors may limit the number of cleavage events and/or the targets of endou, nsp -mediated cleavage may produce few overall cleavage products that are < bp. consequently, nsp mediated cleavage may not reduce the level of total dsrna in the cell, but rather may hydrolyze long dsrnas into shorter cleavage products that are sufficiently short to evade recognition by host sensors (e.g. mda ) but not by the anti-dsrna antibody. it has been documented that cov dsrnas form cytoplasmic aggregated foci during replicating in cells (becares et al., ; hagemeijer et al., ) . these dsrna foci co-localize with the viral rtcs early during infection. interestingly, we noted that during the early stages of infection, dsrna foci were not co-localized with the rtcs in the endou-deficient cov-infected cells. this decrease in colocalization relative to wt infection resulted in a dispersed pattern of dsrna foci, such that some dsrnas did not appear to associate with the rtcs. it is not known whether these "free" dsrnas trigger host sensors, but this dispersed distribution of dsrna does notably coincide with early activation of the ifn response and other dsrna sensors (e.g. pkr and oas). overall, additional studies with new methods are needed to characterize the intracellular localizations and the fates of dsrna in cov-infected cells. the detailed strategy used by cov endou to evade host recognition remains enigmatic. more studies are needed to address several key questions: (i) what is the natural target of endou? (ii) how does the endou activity of nsp alter the fate of dsrna? (iii) how is this endou activity regulated to avoid unwanted cleavage events? (iv) do any interaction partners (viral or cellular) of nsp participate in regulating its endou activity (bhardwaj et al., ; athmer et al., ) ? answers to these questions will be essential for understanding the mechanism of the endou-mediated evasion of host dsrna sensors. additionally, it is possible that endou serves as a conserved antagonist in vertebrate nidoviruses. a similar phenotype has been observed in human blood-derived macrophages infected with the hcov- e nsp mutant virus (kindler et al., ) , which is a representative alphacoronavirus. whether arterivirus nsp also functions as an ifn antagonist during infection is still unknown. one obstacle to studying arterivirus nsp mutants in cell culture is that mutations of catalytic residues have been reported to severely affect viral replication even in ifn-deficient cells (posthuma et al., ; sun et al., ) . nonetheless, given the striking roles of cov endou in macrophages and in vivo, it will be important to understand the 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was essential for nsp to inhibit ifn-β induction a dimerization-dependent mechanism drives the endoribonuclease function of porcine reproductive and respiratory syndrome virus nsp unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage the nonstructural proteins directing coronavirus rna synthesis and processing nonstructural protein of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production nidovirus ribonucleases: structures and functions in viral replication the nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits nf-κb signaling by means of its deubiquitinating activity new antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp structural biology of the arterivirus nsp endoribonucleases antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus virus-encoded proteinases and proteolytic processing in the nidovirales we thank robert c. mettelman and aaron volk for assistance with editing the manuscript. our studies are supported by national institutes of health r ai (to scb). key: cord- -njpuc authors: he, xiaocui; korytář, tomáš; zhu, yaqing; pikula, jiří; bandouchova, hana; zukal, jan; köllner, bernd title: establishment of myotis myotis cell lines - model for investigation of host-pathogen interaction in a natural host for emerging viruses date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: njpuc bats are found to be the natural reservoirs for many emerging viruses. in most cases, severe clinical signs caused by such virus infections are normally not seen in bats. this indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. due to the strict protection of european bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. here, we report about the establishment and functional characterization of myotis myotis derived cell lines from different tissues: brain (mmbr), tonsil (mmto), peritoneal cavity (mmpca), nasal epithelium (mmnep) and nervus olfactorius (mmnol) after immortalization by sv large t antigen. the usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mrna patterns of immune-relevant genes after poly i:c stimulation. performed experiments indicated varying susceptibility to lyssavirus infection with mmbr being considerably less susceptible than the other cell lines. further investigation demonstrated a strong activation of interferon mediated antiviral response in mmbr contributing to its resistance. the pattern recognition receptors: rig-i and mda were highly up-regulated during rabies virus infection in mmbr, suggesting their involvement in promotion of antiviral responses. the presence of cd and cd in mmbr suggested mmbr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (cns). thus the expression pattern of mmbr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the cns controlling the lyssavirus infection. overall, the established cell lines are important tools to analyze antiviral innate immunity in m. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of m. myotis. bats belong to one of the most abundant, diverse and widely distributed mammalian groups. in the order of chiroptera which is divided into two suborders megachiroptera and microchiroptera, a total of , species have been yet described [ ] . bats evolved early and changed very little over the past million years [ ] . their wide distribution and migratory behaviour favour bats as vectors for viruses and raise concerns over their role in zoonotic diseases [ ] [ ] [ ] . among the large number of viruses detected in bats, some like hendra virus, nipah virus, severe acute respiratory syndrome coronavirus (sars), ebola virus, west nile virus were reported to be zoonotic [ , [ ] [ ] [ ] [ ] [ ] [ ] . also of the lyssaviruses, except mokola virus and ikoma lyssavirus, were detected in bats. in north america bats host rabv, whereas in europe european bat lyssavirus type and (eblv- and eblv- ) are found in different bat species [ , ] . annually, there are approximately , human deaths caused by rabies, especially in the developing countries of asia and africa [ ] . despite most human rabies deaths are associated with dog rabies, some of them can be directly linked to the contact with bats, such as out of human rabies cases were of bat origin in the americas in and a few human cases caused by eblvs were reported in europe to date [ ] [ ] [ ] [ ] . although bat associated viruses can cause severe diseases in various mammals, they seem to be less pathogenic for bats [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . after experimental infection with hendra or nipah virus, bats showed no clinical disease, while guinea pigs succumbed to the same dose of virus [ , ] . similar situation was also observed in hendra virus infection in horses and bats [ ] . lyssaviruses are the only viruses that were reported to cause clinical disease in bats [ ] . however, only a small proportion of bats develop clinical symptoms after experimental infection [ , ] . this indicates a critical difference in the development of viral disease between bats and other mammals and requires profound investigation of bat immunology and host-virus interactions. since all of identified european bats species are endangered and strictly protected, the use of animal trials for the investigation of immune mechanisms in bats is not possible. thus, development of stable cell lines for in vitro studies derived from european bat species is desirable. so far, several bat cell lines were reported in previous studies, but most of them were established from non-european bats, like tb -lu from tadarida brasiliensis, mvi/it from myotis velifer incautus, and several primary immortalized cell lines from pteropus alecto, carollia perspicillata, eidolon helvum and rousettus aegyptiacus [ ] [ ] [ ] [ ] . viral infection studies have been carried out in the fruit bat cell lines to investigate the susceptibility, infection kinetics of henipavirus as well as the host innate immunity [ , ] . however, the susceptibility to lyssavirus has not yet been examined in these cell lines. additionally, except for a brain cell line from eptesicus serotinus employed to investigate the type i interferon (ifn) response after lyssavirus infection [ ] , the use of a bat cell line as a tool for studies into lyssavirus infection in its natural reservoir host is rare. a broader variety of bat cell lines, particularly european bat cell lines from tissues of immune relevance, is therefore urgently in demand for lyssavirus-host studies. in this study, we established different cell lines from the european bat m. myotis, evaluated their susceptibility to eblv- , eblv- and rabv infection and investigated innate immune gene responses after the polyinosinic:polycytidylic acid (poly i:c) stimulation. the established m. myotis cell lines present a valuable in vitro model to study the interactions between lyssaviruses and their natural host, and to shed light on the mechanisms of resistance in bat's central nervous system (cns). ethical approval for all of the capturing and sampling were confirmed by the competent authorities in the respective federal republic of germany and czech republic. the czech academy of sciences ethics committee reviewed and approved the animal use protocol no. / in compliance with law no. / a single m. myotis male was captured in sloupsko-sosuvske caves of the moravian karst (czech republic, coordinates u . and u . ). the bat was kept to minimize stress and handling between capture and euthanasia in a clean plastic box with soft mesh to enable roosting under temperature of hibernation torpor of uc and transferred to our laboratory at veterinary and pharmaceutical sciences brno (czech republic) within a day. it was anesthetized to insensitiveness using isofluranum (isofluran, piramal healthcare, uk), and then transcription pcr (rt-pcr) using sv t specific primers [ ] . the protein expression was controlled by the immunofluorescence and western blot as described below. briefly, cells were first fixed with % paraformaldehyde and permeabilized with . % triton x. after washing with pbs, cells were stained with mouse anti-sv t monoclonal antibody (santa cruz biotechnology) and goat anti-mouse igg alexa fluor (invitrogen) as second antibody and visualized by fluorescence microscope. for western blot, the same mouse antibody was used as primary antibody and bound antibody was detected with goat anti-mouse igg peroxidase (sigma). images were developed using the ecl kit (thermo scientific pierce) according to the manufacturer's instructions. to confirm the identity of the established m. myotis cell lines derived from brain (cerebrum) (designated mmbr), tonsil (mmto), peritoneal cavity (mmpca), nasal epithelium (mmnep) and nervus olfactorius (mmnol), a m. myotis-specific pcr was developed. an nadh dehydrogenase subunit (nd ) gene (genbank accession number: dq ) from m. myotis was used as a species specific molecular marker. the genomic dna from different cell lines was isolated by dneasy blood & tissue kit (qiagen). the concentration and purity of genomic dna were determined by nanodrop (thermo). pcr was performed using a specific primer pair nd -f and nd -r (table ) and genomic dna as a template by gotaq flexi dna polymerase (promega) to get the nd fragments. pcr products were cloned into pcr . vector (invitrogen) and transformed into e. coli competent cells. plasmids were extracted from positive clones and sequenced by applied biosystems genetic analyzer (life technologies) at the friedrich-loeffler-institute, germany. to evaluate the ifn response of m. myotis cell lines and the induction of ifn mediated signaling, poly i:c was used to stimulate the cells. different cell lines were seeded in -well plates at a density ranging from . to cells/well, and cultured as described above. around hours after seeding, cells were transfected with poly i:c (sigma) at a concentration of mg/ ml by lipofectamine (invitrogen) following the manufacturer's instructions. twenty four hours post stimulation, cells were harvested into rlt buffer (qiagen) for rna extraction by an rneasy mini kit (qiagen). early after immortalization, the third passage immortalized cell lines were used to check the infectivity of rabv. cells were infected with rabv (european fox isolate, fused with green fluorescent protein, gfp) at a moi of . twenty four hours post infection (hpi), infected cells were fixed and permeabilized as described above and visualized by fluorescence microscope. mmbr and mmto cells that were infected with a serial moi of . , . , and . were harvested at hpi and used for rna extraction. to confirm the infectivity in later passaged cells, different immortalized cell lines of more than passages were infected with lyssaviruses rabv, eblv- (e. serotinus isolate) and eblv- (m. daubentonii isolate) at a moi of . . the infected cells were cultured as described above. cells were collected for rna extraction at hpi and quantitative real-time pcr (qrt-pcr) was performed on the cfx touchdetection system (bio-rad) using sensifast sybr one-step kit (bioline) according to the protocol. immunofluorescence analysis was performed on fixed cells using fitc conjugated anti-rabies monoclonal antibody (sifin) at hpi as described before [ ] . to further confirm the susceptibility, mmbr and mmnol cell lines were infected with eblv- at a moi of . to set the sensitivity at a ct value of for the inoculation dose. the viral supernatant was either changed or not changed with fresh medium at hpi, and viral replication levels were measured by qrt-pcr over hpi. qrt-pcr was introduced to measure the mrna expression levels of immune related molecules in response to poly i:c stimulation and virus infection. the selected molecules include ifn induced genes: ifn stimulated gene (isg ), isg , myxovirus resistance (mx ) and ifn induced protein with tetratricopeptide repeats (ifit ), and pattern recognition receptors (prrs): toll-like receptor (tlr ), retinoic acidinducible gene (rig- ) and melanoma differentiation-associated protein (mda+ ). all of these primers were designed based the sequence resources from our own un-published sequence database and public databases of bat species. the softwares for primers design include primer premier , online tools: http://bioinfo.ut. ee/primer - . . / and http://www.ncbi.nlm.nih.gov/tools/ primer-blast/. primers of target genes and internal control bactin were listed in table . qrt-pcr was performed on the cfx touchdetection system (bio-rad) using sensifast sybr one-step kit (bioline) according to the protocol. to assess the specificity of the pcr amplification, a melting curve analysis was performed at the end of the reaction. the relative expression levels of targets were calculated by ddct method [ ] . molecular characterization of the mmbrbecause the mmbr is derived from the cns, the target of fatal infections by lyssaviruses, a further characterization of cell type of mmbr is desired to improve the understanding of the antiviral defense in the cns. the expressions of cluster of differentiation (cd) , a marker for cells of macrophage lineage [ ] , and cd , a marker expressed in activated microglia [ ] , were investigated by rt-pcr in different cell lines. specific primers for cd , cd and internal control b-actin were listed in table . the rt-pcr was prepared according to the instructions of the one-step rt-pcr kit (qiagen). all data were presented as means s.d. statistical significant differences were analysed by one-way anova using the spss software package. five m. myotis cell lines brain (mmbr), tonsil (mmto), peritoneal cavity (mmpca), nasal epithelium (mmnep) and nervus olfactorius (mmnol) were successfully established by transformation with sv t gene integrating into the chromosomal dna. varying cell morphologies were observed in the cell lines, with mmbr, mmto, mmnep and mmnol being fibroblastic-like, and mmpca being epithelial-like (fig. a) . the mrna expression of sv t antigen was detected in all cell lines (fig. b) . protein level expression was confirmed in four of the five cell lines by immunofluorescence microscopy and western blot, respectively ( fig. c and d) . in mmto, the protein level of sv t antigen was under detectable because the transcriptional level is significantly low determined by rt-pcr (fig. b) . after immortalization, all five cell lines grew for more than passages. the identity of the cell lines was validated by a m. myotis specific pcr using nd gene as a molecular marker. a predicted -bp fragment was obtained from genomic dna of each cell line, and further confirmed by sequencing. as the first step towards the characterization of the innate immune competence of different cell lines, the permanent or inducible expression of molecules involved in cell autonomous responses was examined. the prrs: tlr , rig- and mda , display a various distribution pattern in different cell lines (fig. ) . of note, mmbr has the lowest levels of tlr , rig- and mda (fig. ) . for tlr , about -fold higher mrna levels (p, . ) were observed in mmto, mmpca, mmnep and mmnol compared to mmbr, respectively, while for mda about -fold (mmto; mmnep) or about -fold (mmpca; mmnol) (p, . ) higher expression levels were measured (fig. ) . additionally, more than times higher expression levels of rig- were shown in other cell lines compared to mmbr (p, . ) (fig. ) . further investigation focused on the expression of tlr , isg , isg and mx induced by the poly i:c stimulation (fig. ) . the obtained results indicate a -fold in mmbr, mmnep and mmnol and -fold in mmpca (p, . ) increase in the tlr expression, whilst no change in mmto (fig. a) . all of the ifn induced genes were up-regulated to different extents in different cell lines (fig. b, c, d and e) . in detail, isg expression increased from -fold in mmbr to as high as more than -fold in mmnol (p, . ) (fig. b) . the expression of isg ranged from to times more and mx from to times more in mmto and mmbr, respectively ( fig. c and d) . ifit was upregulated from to times more in mmto and mmnol, respectively (p, . ) (fig. e ). being a natural reservoir species, the main advantage of the permanent m. myotis cell lines is their susceptibility to lyssavirus infection. at an early stage of immortalization, cell lines displayed a significant susceptibility to rabv (moi of at hpi) as demonstrated by the infection with gfp fused rabv. notably, mmbr exhibited considerably lower viral load compared to the other cell lines (fig. ) . later, all passaged immortalized cell lines showed susceptibility to eblv- , eblv- and rabv in a different extent (fig. a and b) . generally, the mmbr cell line presented lower sensitivity to all three lyssaviruses (moi of . ) than the other four cell lines measured by qrt-pcr at hpi (fig. a) , and monitored by immunofluorescence at hpi (fig. b) . thus, the susceptibility could be ordered as mmnol and mmnep fully susceptible with a very high replication rate, mmpca and mmto susceptible with a much less viral replication of eblv- and , mmbr susceptible for eblv- and rabv with a very low viral replication and just single infected cells after eblv- infection (fig. b) . the different susceptibility of the cell lines to lyssavirus infection was further confirmed by the growth kinetics of eblv- in two representative models: mmbr, much less susceptible and mmnol, highly susceptible (fig. c ). to further evaluate the cell line models for study of the different susceptibility between mmbr and other cell lines, mrna expressions of prrs and ifn induced genes were investigated in mmto and mmbr after rabv infection (moi . to . ). the expression of all three prrs remained mostly unchanged in mmto, while it was significantly regulated in mmbr with -fold increased expression of tlr , about -fold increased expression of rig- and mda at moi of . (p, . ) (fig. a) . a comparable expression pattern was observed for the isg , isg , mx and ifit , which was nearly not up-regulated in mmto but displayed a dose dependent increase in mmbr along with the increase of moi, especially for isg and ifit (fig. b ). isg mrna level increased from to times, ifit from to times in the infected mmbr (p, . ). microglia are macrophage-like cells that are resident immune effector cells in the cns [ ] . they are activated in response to infection or injury and play a central role in immune surveillance and host defense [ ] . the rt-pcr results showed that cd and cd are expressed only in mmbr but not in the other four cell lines (fig. ) . this suggested mmbr is a microglia-derived cell line. cell autonomous and innate immune mechanisms are the first line defenses against viral infections. this is mediated mainly by the prrs and the machinery of the ifns and ifn induced effector molecules [ ] [ ] [ ] . viral pathogens like lyssaviruses developed evasive strategies to escape these host defenses by counteracting the ifn mediated immune responses [ ] . co-evolution of the lyssaviral evading and bat's protective mechanisms resulted in an optimal balance, which protect bats as the 'natural host' from severe clinical symptoms or death. bats, which changed very little over past million years, illustrate this phenomenon very well by the resistance to lethal diseases caused by viruses in other mammals [ , [ ] [ ] [ ] [ ] .to understand the specificity of hostpathogen interactions in 'natural host' like bats, studies in bats have to be performed. however, due to the strict protection of the endangered european bat species, in vitro models have to be used. in this study, we successfully established five m. myotis cell lines derived from neural and immune related tissues. to ensure the suitability of these cell lines to analyze virus-host cell interaction, the susceptibility to the infection as well as the presence of corresponding defensive pathways have to be confirmed. first, the existence of the viral sensors tlr , rig- and mda in these permanent cell lines suggests a capacity of these cell lines to sense a broad range of rna viruses. the increased expression of dsrna receptor tlr and ifn induced genes isg , isg , mx and ifit after stimulation with poly i:c mimicking a viral infection indicates that these cell lines can be used as effective in vitro models to study the bat's innate immune responses to virus infection [ , ] . furthermore, to serve as valuable models would be a varying susceptibility of such cell lines to infection by lyssaviruses. in the present study, different susceptibility observed in different m. myotis cell lines using eblv- , eblv- and rabv might be related to the different capacity of the cell lines to produce antiviral mediators and control the infection. moreover, the strong difference in the susceptibility to rabv infection between mmbr and other cell lines provides a unique opportunity for comparative investigations of cell autonomous and innate immune mechanisms in a reservoir host. in addition to the lyssaviruses, the other member from the rhabdoviridae family, like vesicular stomatitis virus (vsv) can also be investigated by using these models in the future studies. preliminary results indicate a correlation between the observed varying susceptibility and the ability to up-regulate the prrs and the ifn induced genes. emerging evidences have shown that prrs play pivotal roles in antiviral immunity in the cns [ ] . in the brain derived cell line mmbr, the high up-regulations of rig- and mda revealed activation of rig-i-like receptor pathway during rabv infection. as previously reported, rig- is a major prr to induce ifn in the rabv infected cells, and mda may function to sustain the ifn induction [ ] . the increased expressions of ifn induced genes: isg , isg , mx and ifit in mmbr indicate that the production of ifn was induced by activated rig- and mda . in contrast, the low expression level of tlr implies a vague involvement of tlr in anti-rabv infection immunity or resistance. it was shown that tlr participated in and benefited the rabv pathogenesis in human neuron cells [ ] . however, the roles of tlr during rabv infection in bats need further investigations. importantly, the significant expression patterns of prrs observed in presented cell line models provide an access to this issue in vitro. to reach a successful infection, the viruses must overcome the barriers of innate immune system. it was reported that ifn production and signaling pathways were antagonized in p. alecto cell lines under henipavirus infection [ ] . similarly, a recent study showed limited expressions of type i ifns and ifn induced genes during lyssaviruses infection in an e. serotinus brain cell line [ ] . a correlation between the low viral load and high expression levels of ifn induced genes in mmbr contrasts to the high viral load and a silent expression pattern of antiviral effectors in mmto, providing an evidence of a countermeasure to ifn system by lyssavirus in the peripheral tissue versus a protective mechanism to infection in the brain tissue of bats. microglial cells are one of the major cell populations in the brain tissue. additionally, comparing to neurons, they can be infected by different rabv strains to a lesser extent [ , ] . the presence of cd and cd as well as the anti-lyssavirus responses in mmbr support a microglia-like feature of mmbr in the cns. it was reported that a mouse microglia cell line can activate strong innate immunity during rabv infection [ ] . the robust immune responses of the microglia-like mmbr demonstrated a critical role of microglia in the anti-rabies defense in bat's cns. in addition to the function of microglia, the clearance of infected viruses in the cns requires systematical responses through the complex interactions of different brain resident cells. herein, the establishment and identification of a microglia-like cell model is a first step towards understanding of the complex reactions of cns in response to lyssavirus infection in the reservoir species. overall, this preliminary study using established cell lines implies that immune mechanisms that control the virus replication are present in the cns of bats. it seems that the ability to control the pathogenic rabv replication via ifn system in the cns contributes to the asymptomatic outcome in bats. in conclusion, the established immortalized cell lines from the european bat m. myotis displaying a variable susceptibility to different lyssaviruses will serve as a useful model to study virus-host interactions and antiviral resistance mechanisms in the 'natural' lyssavirus host. this study provides a preliminary insight into the antiviral innate immunity correlated to cns against neurotropic viruses infection in bats. microbat paraphyly and the convergent evolution of a key innovation in old world rhinolophoid microbats primitive 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of type i and iii interferon in response to viral infection regulation of the type i ifn induction: a current view rhabdovirus evasion of the interferon system type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity innate antiviral signalling in the central nervous system interferon in rabies virus infection tolllike receptor (tlr ) plays a major role in the formation of rabies virus negri bodies rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes global gene expression changes in bv microglial cell line during rabies virus infection we would like to thank dr. thomas müller and dr. conrad m. freuling for the eblv- and eblv- strains, dr. stefan finke for the rabv strain, matthias lenk for the prsvag plasmid, and dr. miroslav kovarik from the administration of the moravian karst protected landscape area (nature conservation agency of the czech republic) for cooperation in obtaining the m. myotis specimen. key: cord- -hkxlq os authors: anang, saumya; kaushik, nidhi; surjit, milan title: recent advances towards the development of a potent antiviral against the hepatitis e virus date: - - journal: j clin transl hepatol doi: . /jcth. . sha: doc_id: cord_uid: hkxlq os hepatitis e virus (hev) is one of the leading causes of acute viral hepatitis. it also causes acute liver failure and acute-on-chronic liver failure in many patients, such as those suffering from other infections/liver injuries or organ transplant/chemotherapy recipients. despite widespread sporadic and epidemic incidents, there is no specific treatment against hev, justifying an urgent need for developing a potent antiviral against it. this review summarizes the known antiviral candidates and provides an overview of the potential targets for the development of specific antivirals against hev. hepatitis e virus (hev) is a positive-sense, single-strand rna virus that causes acute and chronic viral hepatitis, fulminant hepatitis, acute liver failure and acute-on-chronic liver failure in infected individuals. it is known to be transmitted through the fecal-oral route, transfusion of infected blood products or through the vertical route. [ ] [ ] [ ] [ ] [ ] [ ] zoonotic transmission due to consumption of infected meat products, resulting in sporadic cases, is particularly frequent in developed countries. the disease symptoms include jaundice, nausea, vomiting, fever and sore muscles. though the infection is acute in normal individuals, it becomes chronic in immunocompromised patients, such as organ transplant recipients, individuals infected with the human immunodeficiency virus, and patients undergoing chemotherapy. [ ] [ ] [ ] [ ] [ ] [ ] the disease worsens in pregnancy, with mortality rates reaching as high as to %. , , recent reports have described extra-hepatic manifestations, such as guillain-barre syndrome, neurological amyotrophy, arthritis, pancreatitis and glomerulonephritis, in several hev infected patients. [ ] [ ] [ ] drave et al. have also demonstrated the replication of hev in human neuronal-derived cell lines. out of the eight recognized genotypes of hev, genotypes and were responsible for about . million infections in , including . million symptomatic cases, , fatalities and , still births. outbreaks of hev have been reported from different parts of the world. several parts of eastern and the central india, including orissa, chhattisgarh, maharashtra and nellore, witnessed hev outbreaks between - . [ ] [ ] [ ] [ ] many parts of africa have also been affected by frequent hev epidemics. outbreaks were also reported from egypt, uganda, sudan, ethiopia, chad, niger and kenya. an outbreak has even been reported from australia, caused by a locally-acquired hev. the recent increase in organ transplantation and exposure to the disease due to growing trade and travel has further expanded the incidence of hev infection, thereby intensifying the need for research of antivirals against hev. hev-induced acute hepatitis is usually self-limiting. the disease is generally cured in - weeks, without the need of any medication. during severe acute and chronic infections, a reduction in immunosuppressant dose or administration of pegylated-interferon-alpha (peg-ifn-a), ribavirin or a combination of both is the available therapeutic option. reduction in immunosuppressant dose helps in virus clearance in approximately % of organ transplant patients. ribavirin monotherapy has been found to be effective in the treatment of chronic hev infection. [ ] [ ] [ ] [ ] ribavirin inhibits host inosine monophosphate dehydrogenase, thereby depleting cellular gtp pools and blocking viral replication during hev infection. another possible mechanism of ribavirin action on hev is attributed to its ability to induce mutations in the viral genome (fig. ) . the g r mutation in the hev polymerase increases the replication competence of the hev genome and has been shown to confer resistance against ribavirin treatment in some patients. however, subsequent studies revealed that the g r mutation does not lead to absolute ribavirin resistance. [ ] [ ] [ ] [ ] a major limitation of ribavirin therapy is attributed to the undesirable consequences of the treatment, such as: (i) it's not being a treatment option during pregnancy, owing to its teratogenicity; (ii) its potential to cause hemolytic anemia and decreased hemoglobin, requiring direct supervision of hemolytic parameters during its application; and (iii) its potential to cause insomnia, dyspnea, lack of concentration and irritability. hev enters a permissive cell supposedly through a receptor-dependent process, aided by heparan sulfate proteoglycans (hspgs) and other unknown factors. the viral genome is released, orf gets translated and processed into different functional domains, followed by replication. multiple copies of capped (green box) genomic (g)rna and subgenomic (sg)rnas are thus produced. sgrnas synthesize viral capsid (orf ) and orf proteins. orf , grna and other viral and/or host factors mediate assembly of new virions, which are released out of the cell through an endosomal sorting complex required for transport (escrt)-dependent process involving the viral orf protein. the green asterik indicates the steps that can be targeted for antiviral development. a, b, c and d represent the unknown factors present in the viral replication complex. note that orf is present only in the case of genotype hev. known antivirals have been indicated at the appropriate steps. the mode of pegylated-interferon-alpha (peg-ifn-a) action is represented through the illustration of the inteferonalpha (ifn-a) signaling pathway. peg-ifn-a induces the production of interferon-stimulated proteins (isgs) and interferon-inducible transmemebrane proteins (ifitm), which activate the canonical antiviral signaling pathways that results in the inhibition of hev entry and/or replication. also of note is the fact that although ribavirin treatment for chronic hev-infected organ transplantation recipients is effective in the majority of cases, it does not reach a % success rate. further, in a study involving a chronic hev-infected burkitt's lymphoma patient treated with chemotherapy, months of ribavirin treatment failed to eliminate the hev. peg-ifn-a has been used in patients with liver transplant, kidney transplant, human immunodeficiency virus infection and leukemia who are chronically infected with hev. [ ] [ ] [ ] [ ] the mechanism by which peg-ifn-a clears hev is not clearly understood. however, all the types of ifn, including ifn-a (type i), ifn-g (type ii), and ifn-l (type iii) inhibit hev replication and ifn-a subtypes a and b exert the strongest antiviral activity against hev in mammalian cell culture. hev is also equipped with multiple strategies to restrict the ifn response, leading to moderate and delayed anti-hev effects in vitro and in patients treated with ifn-a. the hev x and papain-like cysteine protease domains inhibit ifn (type i) induction, while hev orf is known to inhibit ifn-a signaling by inhibiting phosphorylation of stat . , interestingly, orf also inhibits phosphorylation and nuclear translocation of stat as well as expression of its target genes in cells treated with epidermal growth factor. further studies using suitable in vivo models should decipher the significance of the crosstalk between host interferon signaling and the viral interferon restriction factors. the common side effect associated with ifn treatment is flu-like symptoms. among the more serious adverse effects are neuropsychiatric disorders, neurologic disturbances, myelosuppression, cardiovascular disorders, altered liver function, renal insufficiency and gastrointestinal manifestations. further, months of treatment with peg-ifn-a- a is reported to result in sustained virolgical response in out of chronic hev-infected liver transplant patients. in summary, peg-ifn-a treatment appears to be a promising therapeutic option against hev infection. nevertheless, additional studies involving large cohorts of patients should provide a better understanding of its therapeutic benefits. several laboratories have been focusing on identifying suitable drug targets and developing antivirals against hev. summarized below is the outcome of recent efforts to identify potent antivirals against hev. inosine monophosphate dehydrogenase is an essential enzyme in the purine biosynthesis pathway. several inosine monophosphate dehydrogenase inhibitors, such as mycophenolic acid, ribavirin and -ethynyl- -b-d-ribofuranosylimidazole- carboxamide, inhibit hev replication. , the combination of mycophenolic acid and ribavirin acts more effectively to inhibit hev replication than mycophenolic acid or ribavirin alone. further, mycophenolate mofetil, a prodrug of mycophenolic acid, exhibited frequent hev clearance in heart transplant patients, providing protection from chronification. dihydroorotate dehydrogenase and orotidine- '-monophosphate decarboxylase are essential enzymes in the pyrimidine biosynthesis pathway. dihydroorotate dehydrogenase inhibitors, such as brequinar, leflunomide and orotidine- '-monophosphate decarboxylase inhibitor -azauracil, also inhibit hev replication in mammalian cell culture models. these compounds deserve further validation as antivirals against hev. '-c-methylcytidine is a nucleoside analogue that efficiently inhibits hev replication in the cell culture system. it was also shown that '-c-methylcytidine retained anti-hev activity even after long-term exposure to the virus, implying its potential use to combat development of drug resistance. however, '-c-methylcytidine showed an antagonistic effect when tested in combination therapy with ribavirin. further in vivo evaluation of this compound should provide insights about its anti-hev effects. sofosbuvir, a prodrug of a uridine nucleoside analogue that acts as a direct-acting antiviral against hepatitis c virus (hcv) rna-dependent rna polymerase in its active form, was reported by dao thi et al. to inhibit hev genotype replication in vitro and to exert additive effect when combined with ribavirin. however, those data were not fully reproducible by wang et al. and, moreover, sofosbuvir treatment failed to clear hev viremia in an immunosuppressed patient with chronic hcv and hev without ribavirin. therefore, usage of sofosbuvir as an anti-hev therapeutic needs further validation (discussed further in the rna-dependent rna polymerase section of this manuscript). the zhang laboratory developed hev-specific ppmos and evaluated their efficacy in inhibiting viral replication. out of the four ppmos tested, ppmo hp was most effective in reducing viral replication in mammalian cell culture. ppmo hp specifically inhibits viral translation by targeting a highly conserved sequence in the start region of orf of genotype and genotype hev. treatment of cells with , and mm of ppmo hp reduced luciferase expression by . %, . % and . %, respectively, in a luciferase reporter based hev replicon system. the antiviral activity of ppmo hp was specific, dose-responsive and potent. hence, its further validation as a potential hev-specific antiviral is warranted. e has been identified as an inhibitor of hev replication in hepatocytes. e inhibits genotype hev replication by ; %, without producing any detectable cytotoxicity. interestingly, e also inhibits hcv and dengue virus replication. the mechanism by which e inhibits viral replication remains to be explored. mg is a cell permeable inhibitor of the host s proteasome complex, which is responsible for degradation of ubiquitinated proteins. it also inhibits serine and cysteine proteases with lower efficiency. it is also known to induce c-jun n-terminal kinase-dependent apoptosis, to inhibit nfkb activity and to block b-secretase cleavage. karpe et al. reported significant inhibition of hev replication-related luciferase activity in cells treated with mg . however, it was subsequently shown that journal of clinical and translational hepatology vol. | - mg also reduced the cellular rna and protein levels, indicating its effect to be nonspecific. zinc a recent report by kaushik et al. has demonstrated the antiviral activity of zinc against hev. zinc is an essential micronutrient, which plays a crucial role in multiple cellular processes. it also acts as a broad-spectrum antimicrobial against several pathogens. , zinc salts were shown to block the replication of both genotype and genotype hev by inhibiting the activity of viral rna-dependent rna polymerase in cultured human hepatoma cells. further, zinc salts did not affect virus entry into the host cell. zinc also displayed moderate cooperativity with ribavirin in inhibiting viral replication. these data indicate the possible therapeutic usage of zinc in controlling hev infection. however, considering the complexities involved in serum/plasma and intracellular zinc homeostasis, the efficacy of zinc in inhibiting hev replication in vivo remains to be evaluated. moreover, the detailed mechanism(s) underlying the inhibitory action of zinc on hev replication needs to be investigated. the following stages of the hev life cycle are potential targets for the development of specific antivirals (fig. ) . the specific receptor by which hev enters the host cell is unknown. however, it has been demonstrated that heparin sulfate proteoglycans may serve as attachment receptors to facilitate hev entry into the host cells. the hev capsid protein orf also interacts with heat shock protein and glucoseregulated protein (grp ). grp or heat shock protein may be involved in the intracellular transport of the virus. , grp has also been shown to interact with the envelope protein of the japanese encephalitis virus, facilitating its entry into the host cells. it remains to be tested whether grp and orf interaction mediates hev entry. inhibitors of receptor binding or intracellular transport of the virus are supposed to block viral life cycle at a very early stage. among the nonstructural proteins encoded by the hev orf , methyltransferase is responsible for capping of the viral genome. addition of a -methylguanosine cap at the ′terminus of the viral genome confers stability and protects the viral rna from the host innate immune effectors. uncapped hev rna is inefficient in replication. moreover, in contrast to the host methyltransferases wherein guanyltransferase donates a gmp moiety to the rna, followed by cap methylation by guanine- -methyltransferase activity; hev methyltransferase follows a reverse order, thereby restricting its activity to the viral rna. therefore, inhibition of hev methyltransferase activity appears to be a potent antiviral strategy. it is noteworthy that neplanocin a and -deaza-adenosine, the two known inhibitors of influenza virus methyltransferases, interfere with virus replication. neplanocin a is also a potent inhibitor of vaccinia virus replication. inhibitors against dengue virus methyltransferases have also been screened. direct-acting inhibitors of hev rna-dependent rna polymerase function: rna-dependent rna polymerase is the most important factor in the life cycle of all rna viruses and, therefore, rna-dependent rna polymerase inhibitors are supposed to be potent antivirals. one such antiviral against hcv is sofosbuvir, which acts by inhibiting the activity of hcv rnadependent rna polymerase. dao thi et al. indicated the effectiveness of sofosbuvir in inhibiting hev replication; however, subsequent studies failed to observe its potent inhibitory effect. [ ] [ ] [ ] nevertheless, optimization of the sofosbuvir structure that improves its inhibitory effect on hev rna-dependent rna polymerase is an attractive area of investigation. knowledge of hev rna-dependent rna polymerase structure might expedite the above study. apart from sofosbuvir-like molecules, new chemical entities should be explored to identify potent inhibitors of hev rna-dependent rna polymerase activity. other inhibitors of hev rna-dependent rna polymerase function: our earlier studies showed that the interaction between host eef a and viral rna-dependent rna polymerase is important for optimal rna-dependent rna polymerase activity. we recently reported the construction and characterization of the host-virus protein-protein interaction network of hev. using a yeast two-hybrid cdna library screening-based approach, host proteins were identified to be the direct interaction partners of g- hev rna-dependent rna polymerase and of them could also associate with g -hev rna-dependent rna polymerase. notably, host translation regulatory factors, such as eif a , eef a and eif a, directly associated with the rna-dependent rna polymerase protein of both genotype and genotype hev. further in silico analysis of the functional significance of the protein-protein interaction network revealed distinct protein-protein interaction clusters in the secondary network, representing enrichment of proteins involved in different host processes, such as translation initiation, the ubiquitin proteasome pathway and the oxidative phosphorylation pathway. depletion of the translation regulatory factors by gene silencing technique resulted in significant reduction of viral replication and pull-down studies under similar conditions revealed the assembly of a multiprotein complex consisting of the translation regulatory factors, rna-dependent rna polymerase and many other virus and host factors. remarkably, eef a was identified to be the most important host factor for maintaining the integrity of the above multiprotein complex, thereby suggesting it to be an attractive target for antiviral discovery. additionally, inhibitors against other host translation factors present in the complex such as eif a and eif a are also supposed to block viral replication. targeting a combination of direct and indirect inhibitors of rna-dependent rna polymerase function might prove to be an apt antiviral strategy against hev. inhibitors of helicase function: hev helicase is a nucleoside triphosphatase with the ability to unwind rna duplexes in the ′ to ′ direction, thus playing a role in hev replication. due to the common properties shared between the helicases encoded by viruses and their host, designing inhibitors against helicases is challenging. nevertheless, potent inhibitors of helicase encoded by the herpes simplex virus, severe acute respiratory syndrome coronavirus, hcv, dengue virus, japanese encephalitis virus, west nile virus and human papillomavirus have been reported. release of the progeny virions from infected cells leads to the infection of neighboring uninfected cells, thus amplifying the unwanted consequences. antivirals that prevent the release of the progeny virus will prevent further infection, thereby minimizing progression of the disease. release of the newly assembled virus from an infected cell is a complicated process involving multiple protein-protein interactions between the virus and host factors. a thorough understanding of such interactions will help in decoding the mechanism underlying virus release. an inhibitor of human immunodeficiency virus release has been identified, which acts by blocking the interaction between the viral gag and host tumor susceptibility gene encoded proteins. interaction between hev orf and host tumor susceptibility gene -encoded protein is also known to mediate the release of genotype hev. , an inhibitor against the above interaction may prove to be a potent antiviral against hev. apart from that, detailed investigation of hev release mechanism should identify additional targets for antiviral development. the advantages of using antivirals, particularly to cut off the disease in an infected person and providing treatment to poor responders to vaccines, such as immune-compromised patients, warrants the need for development of specific drugs against hev. the antivirals will also prove to be useful for patients with acute, chronic or fulminant hev infections. as summarized in table and fig. , a number of promising antiviral candidates have been identified through the efforts of several researchers, which should be further characterized to identify one or more potent inhibitor(s) of hev. a combinatorial therapy targeting crucial virus-encoded factors at different stages of viral life cycle as well as inhibition of virus-host interactions should be a potent antiviral strategy against hev. the recent finding of hev inhibitory activity of zinc also appears to be an attractive area for further investigation. zinc directly inhibits hev rna-dependent rna polymerase activity in vitro and displays moderate cooperativity with ribavirin in inhibiting viral replication in mammalian cell culture models of hev infection. therefore, even a moderate increase in the level of bioavailable zinc may significantly improve the therapeutic benefits when combined with ribavirin therapy. in summary, recent studies have identified multiple leads, which should be pursued further to develop a potent antiviral against hev. the dbt-rgyi grant and ramalingswamy fellowship to ms is gratefully acknowledged. sa and nk are supported by senior research fellowships from the department of science & technology and the university grants commission, government of india, respectively. the authors have no conflict of interests related to this publication. wrote all sections of the manuscript except the zinc section, and generated the figure and the table (sa), wrote the zinc section 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(review) heparan sulfate proteoglycans are required for cellular binding of the hepatitis e virus orf capsid protein and for viral infection role of heat-shock protein in hepatitis e virus capsid trafficking homology model and potential virus-capsid binding site of a putative hev receptor grp grp is an important host factor for japanese encephalitis virus entry and replication in mammalian cells virus-specific mrna capping enzyme encoded by hepatitis e virus conventional and unconventional mechanisms for capping viral mrna in vitro replication of hepatitis e virus (hev) genomes and of an hev replicon expressing green fluorescent protein the methyltransferase inhibitor neplanocin a interferes with influenza virus replication by a mechanism different from that of -deazaadenosine a potent inhibitor of s-adenosylhomocysteine hydrolase and of vaccinia virus multiplication in mouse l cells development of specific dengue virus '-o-and n -methyltransferase assays for antiviral drug screening ns a inhibitors in the treatment of hepatitis c endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype- hepatitis e virus hostvirus protein interaction network reveals the involvement of multiple host processes in the life cycle of hepatitis e virus rna '-triphosphatase activity of the hepatitis e virus helicase domain understanding helicases as a means of virus control herpes simplex virus helicase-primase inhibitors are active in animal models of human disease opportunistic intruders: how viruses orchestrate er functions to infect cells inhibition of hiv budding by a genetically selected cyclic peptide targeting the gag-tsg interaction enhanced alpha microglobulin secretion from hepatitis e virus orf -expressing human hepatoma cells is mediated by the tumor susceptibility gene a psap motif in the orf protein of hepatitis e virus is necessary for virion release from infected cells key: cord- - fmul c authors: vabret, nicolas; britton, graham j.; gruber, conor; hegde, samarth; kim, joel; kuksin, maria; levantovsky, rachel; malle, louise; moreira, alvaro; park, matthew d.; pia, luisanna; risson, emma; saffern, miriam; salomé, bérengère; selvan, myvizhi esai; spindler, matthew p.; tan, jessica; van der heide, verena; gregory, jill k.; alexandropoulos, konstantina; bhardwaj, nina; brown, brian d.; greenbaum, benjamin; gümüş, zeynep h.; homann, dirk; horowitz, amir; kamphorst, alice o.; curotto de lafaille, maria a.; mehandru, saurabh; merad, miriam; samstein, robert m. title: immunology of covid- : current state of the science date: - - journal: immunity doi: . /j.immuni. . . sha: doc_id: cord_uid: fmul c abstract the coronavirus disease (covid- ) pandemic, caused by severe acute respiratory syndrome coronavirus (sars-cov- ) has affected millions of people worldwide, igniting an unprecedented effort from the scientific community to understand the biological underpinning of covid pathophysiology. in this review, we summarize the current state of knowledge of innate and adaptive immune responses elicited by sars-cov- infection and the immunological pathways that likely contribute to disease severity and death. we also discuss the rationale and clinical outcome of current therapeutic strategies as well as prospective clinical trials to prevent or treat sars-cov- infection. the recent emergence and rapid global spread of severe acute respiratory syndrome coronavirus (sars-cov- ) and the resulting coronavirus disease (covid- ) poses an unprecedented health crisis that was declared a pandemic by the world health organization (who) on march , . the origin of sars-cov- was traced to the city of wuhan in hubei province, china, where a cluster of viral pneumonia cases was first detected, many in connection with the huanan seafood wholesale market. china reported this outbreak to the who on december st , and soon after identified the causative pathogen as a betacoronavirus with high sequence homology to bat coronaviruses using angiotensin-converting enzyme (ace ) receptor as the dominant mechanism of cell entry wan et al., b) . following a likely zoonotic spillover, human-to-human transmission events were confirmed with clinical presentations ranging from no symptoms; to mild fever, cough, and dyspnea; to cytokine storm, respiratory failure, and death. sars-cov- is also closely related to sars (retrospectively named sars-cov- ) and mers (middle eastern respiratory syndrome) coronaviruses, causing local outbreaks and zoonotic epidemics in , respectively (de wit et al., . while sars-cov- is not as lethal as sars-cov- or mers-cov (fauci et al., ) , the considerable spread of the current pandemic has brought tremendous pressure and disastrous consequences for public health and medical systems worldwide. the scientific response to the crisis has been extraordinary with a plethora of covid- studies posted in preprint servers, in an attempt to rapidly unravel the pathogenesis of covid- and potential therapeutic strategies. in response, trainees and faculty members of the precision immunology institute at the icahn school of medicine at mount sinai (priism) have initiated an institutional effort to critically review the preprint literature (vabret et al., ) , together with peer-reviewed articles published in traditional journals, and summarize the current state of science on the fast evolving field of covid- immunology. we thematically focus on the innate and adaptive immune responses to sars-cov- and related coronaviruses, clinical studies and prognostic laboratory correlates, current therapeutic strategies, prospective clinical trials, and vaccine approaches. (bouvet et al., ; chen et al., ; ivanov et al., ) , thereby resembling host mrna to promote translation, prevent degradation, and evade rlr sensing. finally, covs also encode an endoribonuclease, nsp , that cleaves ' polyuridines formed during viral replication, which would otherwise be detected by mda (deng et al., ; hackbart et al., ) . coronaviruses have evolved additional strategies to impede activation of prrs. sars-cov- n-protein prevents trim activation of rig-i . likewise, mers-cov ns a, which itself binds dsrna, impedes pkr activation (comar et al., ; rabouw et al., ) and inhibits pact, an activator of rlrs (niemeyer et al., ; siu et al., ) . additionally, mers-cov ns b antagonizes rnasel, another activator of rlrs (thornbrough et al., ) . the role of other prrs remains unclear. for example, sars-cov- papain-like-protease (plp) antagonizes sting, suggesting that self-dna may also represent an important trigger (sun et al., ) . the extent to which sars-cov- homologs overlap in these functions is currently unknown. following activation, rlr and tlrs induce signaling cascades leading to the phosphorylation of transcription factors, such as nf-kb and the interferon-regulatory factor family (irf), ultimately leading to transcription of ifn and proinflammatory cytokines. although no experimental studies have delineated the precise functions of sars-cov- proteins, proteomic studies have demonstrated interactions between viral proteins and prr signaling cascades. sars-cov- orf b indirectly interacts with the signaling adaptor mavs via its association with tom (gordon et al., ) , consistent with prior reports that sars-cov- orf b suppresses mavs signaling (shi et al., ) . furthermore, sars-cov- nsp interacts with signaling intermediate tbk , and nsp is associated with rnf /nrdp , an activator of tbk and irf (gordon et al., ) . similarly, sars-cov- m protein is known to inhibit the tbk signaling complex (siu et al., ) , as does mers-cov orf b (yang et al., ) . other proteins, including sars-cov- plp, n, orf b and orf , block irf phosphorylation and nuclear translocation (devaraj et al., ; . nf-kb is also inhibited by covs proteins. these include sars-cov- plp (frieman et al., ) and mers-cov orf b and orf (canton et al., ; menachery et al., ) . finally, sars-cov- nsp (huang et al., a; kamitani et al., ) and mers-cov nsp (lokugamage et al., ) initiate general inhibition of host transcription and translation, thus limiting antiviral defenses nonspecifically. to prevent signaling downstream of ifn release, cov proteins inhibit several steps of the signal transduction pathway that bridge the receptor subunits (ifnar and ifnar ) to the stat proteins that activate transcription. for sars-cov- , these mechanisms include ifnar degradation by orf a (minakshi et al., ) , decreased stat phosphorylation by nsp (wathelet et al., ) , and antagonism of stat nuclear translocation by orf kopecky-bromberg et al., ) . however, sars-cov- orf shares only % sequence homology with sars-cov- , suggesting this function may not be conserved. in support of this notion, sars-cov- infection fails to limit stat phosphorylation, unlike in sars-cov- infection (lokugamage et al., ) . mucosal immune responses to infectious agents are orchestrated and regulated by myeloid cells with specialized functions, which include conventional dcs (cdcs), monocyte-derived dcs (modcs), plasmacytoid dcs (pdcs), and macrophages (guilliams et al., ) . a growing body of evidence points to dysregulated myeloid responses that potentially drive the covid- hallmark syndromes such as acute respiratory distress syndrome (ards), cytokine release syndrome (crs) and lymphopenia (mehta et al., ) . flow cytometric analyses of pbmcs from symptomatic covid- patients have shown a significant influx of gm-csf-producing, activated cd + t cells and cd + hla-dr lo inflammatory monocytes (im) (giamarellos-bourboulis et al., ; zhou et al., b) . this matches single-cell transcriptomic (scrnaseq) data demonstrating cd + il- β + monocytic expansion wen et al., ) , interferon-mapk driven adaptive immune responses , and il- βassociated inflammasome signatures (ong et al., ) in peripheral blood of covid- patients, although systemic levels of il- β detected are conspicuously low (gnjatic et al., unpublished) . importantly, these immune signatures track with progression of clinical disease. scrnaseq studies performed on pulmonary tissues of patients with severe covid- disease have revealed an expansion of ims and ficolin- + monocyte-derived macrophages, at the expense of tissue-resident reparative alveolar macrophages (am) . the aforementioned study also observed signatures of ifn-signaling and monocyte recruitment that likely contribute to the rapid decline in alveolar patency and promote ards. although most of the clinical focus has been on pulmonary damage and mononuclear phagocyte (mnp) dysfunction therein, it is increasingly clear that covid- likely presents systemic challenges in other organ sites such as the ileum and kidneys. understanding the role of non-pulmonary myeloid cells in tissue-specific pathology associated with covid- will be important. while data on covid- patients continues to rapidly emerge, studies of myeloid cell dysfunction in sars-cov- and mers-cov can provide an important roadmap to understanding covid- pathogenesis ( figure ). sars-cov- infection in mouse models results in an aberrant am phenotype that limits dc trafficking and t cell activation (zhao et al., ) . additionally, ym + fizz + alternative macrophages can increase airway hypersensitivity, thus exacerbating sars-associated fibrosis (page et al., ) . further, as described above, murine sars-cov- studies have demonstrated that delayed ifn-i signaling and inflammatory monocytes-macrophages promote lung cytokine and chemokine levels, vascular leakage, and impaired antigenspecific t cell responses, culminating in lethal disease (channappanavar et al., ) . the role played by prominent ifn-producing pdcs in sars-cov- control or pathogenesis warrants investigation, as they have been shown to be critical in murine coronavirus (mhv) control (cervantes-barragan et al., ) . longitudinal studies in sars-cov- models are awaited, but initial phenotypic studies in humanized hace mice have shown the characteristic alveolar interstitial pneumonia, with infiltration of lymphocytes and monocytes and accumulation of macrophages in the alveolar lumen (bao et al., a) , which recapitulates patient findings . lastly, nonhuman primate (nhp) studies and patient data on sars-cov- have also shown that virus spike-specific igg responses can exacerbate acute lung injury due to repolarization of alveolar macrophages into pro-inflammatory phenotypes and enhanced recruitment of inflammatory monocyte via ccl and il- (clay et al., ; liu et al., ) . however, the extent to which the antibody response contributes to disease pathophysiology remains to be confirmed. the initial mode of viral pathogen-associated signal (pamp) recognition by innate cells has a major impact on downstream myeloid signaling and cytokine secretion (de marcken et al., ) . while macrophages are somewhat susceptible to mers-cov and sars-cov- infection (perlman and dandekar, ; zhou et al., ) , data do not suggest that they are infected by sars-cov- , although one study reported ace and sars-cov- nucleocapsid protein is expressed in lymph nodes and spleenassociated cd + macrophages of covid- patients producing il- . significantly elevated systemic levels of pro-inflammatory cytokine il- have been reported in several covid- patient cohorts and shown to correlate with disease severity (mehta et al., ) . increased il- can also be associated with higher levels of il- , il- , ifn-ɣ and gm-csf as seen in secondary hemophagocytic lymphohistiocytosis. in response to viral infections, mnps drive interleukin, and ifn-i and ifn-iii production, resulting in inflammasome activation, induction of pathogenic th and th cell responses, recruitment of effector immune cells and crs pathology (prokunina-olsson et al., ; tanaka et al., ) . independently, in vitro studies have demonstrated sars-cov- infection can induce intracellular stress pathways resulting in nlrp -dependent inflammasome activation and macrophage pyroptosis shi et al., ) . functional studies are required to implicate these myeloid inflammasome pathways in covid- lung pathology and to assess other immunogenic pathways such as ripk / -dependent necroptosis (nailwal and chan, ) . in conclusion, the strength and duration of myeloid interferon-stimulated gene (isg) signaling potentially dictate covid- disease severity, but rigorous studies are warranted to confirm this. lastly, more work is needed to ascertain the mechanistic role played by lung-resident and recruited granulocytes in sars-cov- control and pathogenesis (camp and jonsson, ; flores-torres et al., ) . in contrast to their early protective role, neutrophil netosis and macrophage crosstalk can drive later-stage inflammatory cascades (barnes et al., ) , underscoring the overall pathogenic nature of damagesensing host responses ( figure ) . collectively, the current knowledge of coronaviruses and sars-cov- infection, in particular, points to an inadvertent collusion involving myeloid cells in covid- pathogenesis, despite their critical role in early sensing and antiviral responses. innate lymphoid cells (ilcs) are innate immune effector cells that lack the expression of rearranged antigen receptors (tcr, bcr). the ilc family is divided into two main groups: the cytotoxic natural killer (nk) cells and the non-cytotoxic helper ilcs, which include ilc , ilc and ilc (vivier et al., ) . conventional nk cells include cd bright cd -nk cells and cd dim cd + cells, that are specialized in cytokine production or cytotoxicity, respectively. multiple studies have reported reduced numbers of nk cells in the peripheral blood of covid- patients, which is associated with severity of the disease wang et al., f; yu et al., ; zheng et al., b) . a recent scrnaseq analysis revealed a transcriptomic signature for nk cells that was equally represented in lungs from patients and healthy donors . the majority of lung nk cells are non-resident (gasteiger et al., ; marquardt et al., ) , and cxcr has been shown to mediate nk cell infiltration upon influenza infection (carlin et al., ) . in vitro, cxcr ligands (cxcl - ) are increased in sars-cov- -infected human lung tissue (chu et al., ) , and cxcr ligand-producing monocytes are expanded in the lungs of covid- patients . this suggests that the cxcr pathway might facilitate nk cell recruitment from the peripheral blood to the lungs in covid- patients ( figure ). nk cells express inhibitory and activating receptors that regulate their cytotoxicity. they are therefore able to induce the lysis of virus-infected cells that upregulate virus-derived proteins, as well as stress-inducible ligands, which are then recognized by nk cellactivating receptors, such as nkp (cerwenka and lanier, ; draghi et al., ; duev-cohen et al., ; glasner et al., ) . future studies should investigate the expression of nk receptor ligands on sars-cov- -infected cells, in order to better understand the mechanisms underlying nk cell activation in covid- disease. further, secretion of igg and igg antibodies during sars-cov- infection (amanat et al., ) may induce cd dim cd + nk cell activation through fc receptor recognition of antibodies, either bound to surface antigens expressed on infected cells or to extracellular virions as immune complexes ( figure ). this interaction might trigger both cytokine production by nk cells and lysis of infected cells through antibodymediated cellular cytotoxicity (adcc), as shown in influenza infection (von holle and moody, ) . emerging data highlight the capacity for nk-mediated adcc in response to naturally isolated sars-cov- anti-s igg that cross-reacts with sars-cov- s glycoprotein when transfected into chinese hamster ovary (cho) cells (pinto et al., ) . these findings suggest that triggering nk cell activation may not only contribute to the resolution of infection, but also contribute to the cytokine storm in ards. ex vivo nk cells from peripheral blood of covid- patients have reduced intracellular expression of cd a, ksp , granzyme b and granulysin, suggesting an impaired cytotoxicity, as well as an impaired production of chemokines, ifn-ɣ and tnf-α (wilk et al., ; zheng et al., b) . several pathways may contribute to the dysregulation of nk cells. while influenza virus infects nk cells and induces apoptosis (mao et al., ) , lung nk cells do not express the entry receptor for sars-cov- , ace , and are therefore unlikely to be directly infected by sars-cov- (travaglini et al., ) . the majority of nk cells found in human lung display a mature cd + kir + cd dim phenotype and are able to induce cell cytotoxicity in response to loss of hla class i or through fc receptor signaling, although to a lower extent than their peripheral blood counterpart (marquardt et al., ) . killer-immunoglobulin receptors (kirs) are acquired during nk cell development alongside cd (fcrγiiia) and are essential for nk cell licensing and subsequent capacity for cytolytic function (sivori et al., ) . frequencies of nk cells expressing cd and/or kir are decreased in the blood following sars-cov- and sars-cov- infection, respectively (xia et al., ; wang et al., d) . collectively, the data suggest either an impaired maturation of the nk compartment or migration of the mature, circulating nk cells into the lungs or other peripheral tissues of sars-cov- -infected patients. the immune checkpoint nkg a is increased on nk cells and cd t cells from covid- patients . nkg a inhibits cell cytotoxicity by binding the nonclassical hla-e molecule (braud et al., ; brooks et al., ) , and this interaction is strongly correlated with poor control of hiv- infection (ramsuran et al., ) . genes encoding the inhibitory receptors lag and tim are also upregulated in nk cells from covid- patients (wilk et al., ; hadjadj et al., ) . thus, increased immune checkpoints on nk cells might contribute to viral escape. additionally, covid- patients have higher plasma concentrations of il- , which significantly correlate with lower nk cell numbers . in vitro stimulation by il- and soluble il- receptor has previously revealed impaired cytolytic functions (perforin and granzyme b production) by healthy donor nk cells, which can be restored following addition of tocilizumab (il- r-blockade) (cifaldi et al., ) . tnf-α is also upregulated in the plasma of covid- patients , and ligand-receptor interaction analysis of peripheral blood scrnaseq data suggests that monocyte-secreted tnf-α might bind to its receptors on nk cells . tnf-α is known to contribute to nk cell differentiation (lee et al., ) , which includes downregulation of nkp (ivagnes et al., ) , though no effect of tnf-α or il- on nk cell-mediated adcc has been reported so far. collectively, these data suggest that cross-talk with monocytes might impair nk cell recognition and killing of sars-cov- infected cells, and antibodies targeting il- and tnf-signaling may benefit enhanced nk cell functions in covid- patients ( figure ). no studies, to date, have reported ilc , ilc , or ilc functions in sars-cov- infection. all three subsets are present in healthy lung (de grove et al., ; yudanin et al., ) . ilc s are essential for the improvement of lung function following influenza infection in mice, through amphiregulin-mediated restoration of the airway epithelium and oxygen saturation (monticelli et al., ) . however, ilc s also produce il- , contributing to the recruitment of macrophages to the lung and influenza-induced airway hyperreactivity (chang et al., ) . indeed, ilcs are involved in the polarization of alveolar macrophages, either towards a m -like phenotype (ilc and ilc ) or a m like phenotype (ilc ) (kim et al., ) . given the increased il- concentrations and the dysregulation of the macrophage compartment observed in covid- patients, the role played by ilcs in sars-cov- infection warrants further investigation. t cells play a fundamental role in viral infections: cd t cells provide b cell-help for antibody production and orchestrate the response of other immune cells, whereas cd t cells kill infected cells to reduce the viral burden. however, dysregulated t cell responses can result in immunopathology. to better understand the role of t cell responses in sars-cov- infection, the pursuit of two major questions is imperative: ( ) what is the contribution of t cells to initial virus control and tissue damage in the context of covid- , and ( ) how do memory t cells established thereafter contribute to protective immunity upon re-infection? some tentative answers are beginning to emerge. similar to earlier observations about sars-cov- infection , several current reports emphasize the occurrence of lymphopenia with drastically reduced numbers of both cd and cd t cells in moderate and severe covid- cases ( figure ) nie et al., b; wang et al., d; zeng et al., ; zheng et al., b) . the extent of lymphopenia -most striking for cd t cells in patients admitted to icu -seemingly correlates with covid- -associated disease severity and mortality diao et al., ; liu et al., b liu et al., , c tan et al., a; wang et al., d zeng et al., ; zhou et al., c) . patients with mild symptoms, however, typically present with normal or slightly higher t cell counts thevarajan et al., ) . the cause of peripheral t cell loss in moderate to severe covid- , though a phenomenon also observed in other viral infections, remains elusive, and direct viral infection of t cells, in contrast to mers-cov (chu et al., ) , has not been reported. several mechanisms likely contribute to the reduced number of t cells in the blood, including effects from the inflammatory cytokine milieu. indeed, lymphopenia seems to correlate with serum il- , il- , and tnf-α (diao et al., ; wan et al., a) , while convalescent patients were found to have restored bulk t cell frequencies paired with overall lower pro-inflammatory cytokine levels diao et al., ; liu et al., a liu et al., , b zheng et al., b) . cytokines such as ifn-i and tnf-α may inhibit t cell recirculation in blood by promoting retention in lymphoid organs and attachment to endothelium (kamphuis et al., ; shiow et al., ) . however, in an autopsy study examining the spleens and hilar lymph nodes of six patients who succumbed to covid- , chen et al. observed extensive cell death of lymphocytes and suggested potential roles for il- as well as fas-fasl interactions . in support of this hypothesis, the il- receptor antagonist tocilizumab was found to increase the number of circulating lymphocytes (giamarellos-bourboulis et al., ) . t cell recruitment to sites of infection may also reduce their presence in the peripheral blood compartment. scrnaseq analysis of bronchoalveolar lavage (bal) fluid of covid- patients revealed an increase in cd t cell infiltrate with clonal expansion . likewise, post-mortem examination of a patient who succumbed to ards following sars-cov- infection showed extensive lymphocyte infiltration in the lungs . however, another study that examined post-mortem biopsies from four covid- patients only found neutrophilic infiltration (tian et al., a) . further studies are therefore needed to better determine the cause and impact of the commonly observed lymphopenia in covid- patients. available information about sars-cov- -specific t cell immunity may serve as an orientation for further understanding of sars-cov- infection. immunogenic t cell epitopes are distributed across several sars-cov- proteins (s, n, m as well as orf ), although cd t cell responses were more restricted to the s protein (li et al., ) . in sars-cov- survivors, the magnitude and frequency of specific cd memory t cells exceeded that of cd memory t cells and virus-specific t cells persisted for at least - years suggesting that t cells may confer long-term immunity (ng et al., ; tang et al., ) . limited data from viremic sars patients further indicated that virusspecific cd t cell populations might be associated with a more severe disease course, since lethal outcomes correlated with elevated th cell (il- , il- , il- ) serum cytokines (li et al., ) . however, the quality of cd t cell responses needs to be further characterized to understand associations with disease severity. few studies have thus far characterized specific t cell immunity in sars-cov- infection. in patients recovering from mild covid- , robust t cell responses specific for viral n, m and s proteins were detected by ifn-γ elispot, weakly correlated with neutralizing antibody concentrations (similar to convalescent sars-cov- patients (li et al., ) , and subsequently contracted with only n-specific t cells detectable in about one third of the cases post recovery . in a second study, pbmcs from covid- patients with moderate to severe ards were analyzed by flow cytometry, approximately weeks after icu admission (weiskopf et al., ) . both virus-specific cd and cd t cells were detected in all patients at average frequencies of . % and . %, respectively, and very limited phenotyping according to cd ra and ccr expression status characterized these cells predominantly as either cd tcm (central memory) or cd tem (effector memory) and temra (effector memory ra) cells. this study is notable for the use of large complementary peptide pools comprising , sars-cov- epitopes (overlapping -mers for s protein as well as computationally predicted hla-i-and -ii-restricted epitopes for all other viral proteins) as antigenspecific stimuli that revealed a preferential specificity of both cd and cd t cells for s protein epitopes, with the former population modestly increasing over ~ - days after initial onset of symptoms. a caveat, however, pertains to the identification of specific t cells by induced cd and cd co-expression, since upregulation of cd by cd t cells, in contrast to cd , may preferentially capture regulatory t cells (treg) (bacher et al., ) . further analyses of s protein-specific t cells by elisa demonstrated robust induction of ifn-γ, tnf-α and il- concomitant with lower levels of il- , il- , il- , il- and il- . a third report focused on s-specific cd t cell responses in patients with mild, severe or critical covid- using overlapping peptide pools and induced cd and cd co-expression as a readout for antiviral cd t cells. such cells were present in % of cases and presented with enhanced cd , hla-dr and ki- expression indicative of recent in vivo activation (braun et al., ) . of note, the authors also detected low frequencies of s-reactive cd t cells in % of sars-cov- seronegative healthy control donors. however, these cd t cells lacked phenotypic markers of activation and were specific for c-terminal s protein epitopes that are highly similar to endemic human coronaviruses, suggesting that crossreactive cd memory t cells in some populations (e.g., children and younger patients that experience a higher incidence of hcov infections) may be recruited into an amplified primary sars-cov- -specific response (braun et al., ) . similarly, endemic cov-specific cd t cells were previously shown to recognize sars-cov determinants (gioia et al., ) . how previous infections with endemic coronavirus may affect immune responses to sars-cov- will need to be further investigated. finally, in general accordance with the above findings on the induction of sars-cov- specific t cells, using tcrseq, huang et al. and liao et al. reported greater tcr clonality of peripheral blood as well as bal t cells in patients with mild vs. severe covid- . moving forward, a comprehensive identification of immunogenic sars-cov- epitopes recognized by t cells (campbell et al., ) , as well as further studies on convalescent patients who recovered from mild and severe disease, will be particularly important. while the induction of robust t cell immunity is likely essential for efficient virus control, dysregulated t cell responses may cause immunopathology and contribute to disease severity in covid- patients (figure ). this is suggested in a study by zhou et al. which reported a significantly increased pbmc frequency of polyclonal gm-csf + cd t cells capable of prodigious ex vivo il- and ifn-γ production only in critically ill covid- patients . of note, gm-csf + cd t cells have been previously implicated in inflammatory autoimmune diseases such as multiple sclerosis or juvenile rheumatoid arthritis, and high levels of circulating gm-csf + cd t cells were found to be associated with poor outcomes in sepsis . additionally, two studies observed reduced frequencies of treg cells in severe covid- cases qin et al., ) . since treg cells have been shown to help resolve ards inflammation in mouse models (walter et al., ) , a loss of tregs might facilitate the development of covid- lung immunopathology. similarly, a reduction of γδ-t cells, a subset of t cells with apparent protective antiviral function in influenza pneumonia (dong et al., ; zheng et al., ) , has been reported in severely sick covid- patients lei et al., b) . currently, little is known about specific phenotypical and/or functional t cell changes associated with covid- . in the majority of preprints and peer-reviewed studies, there are reports of increased presence of activated t cells ( figure ) characterized by expression of hla-dr, cd , cd , cd , cd and ki- (braun et al., ; dong et al., ; guo et al., ; liao et al., ; thevarajan et al., ; yang et al., a; zheng et al., a) . generally, independent of covid- disease severity, cd t cells seem to be more activated than cd t cells thevarajan et al., ; yang et al., a) , a finding that echoes stronger cd , than cd , t cell responses during sars-cov- (li et al., ) . furthermore, in a case study of covid- patients, diao et al. showed that levels of pd- increased from prodromal to symptomatic stages of the disease (diao et al., ) . pd- expression is commonly associated with t cell exhaustion, but it is important to emphasize that pd- is primarily induced by tcr signaling; it is thus also expressed by activated effector t cells (ahn et al., ) . in addition, several studies reported higher expression of various co-stimulatory and inhibitory molecules such as ox- and cd , ctla- and tigit (zheng et al., a) , and nkg a . reduced numbers of cd + cd t cells as well as larger frequencies of pd- + /tim + cd t cells in icu patients were also reported . expression of most of these markers was found to be higher in cd than in cd t cells, and levels tended to increase in severe vs. non-severe cases, which may be due to differences in viral load. cellular functionality was shown to be impaired in cd and cd t cells of critically ill patients, with reduced frequencies of polyfunctional t cells (producing more than one cytokine) as well as generally lower ifn-γ and tnf-α production following re-stimulation with pma and ionomycin zheng et al., a zheng et al., , b . similarly, zheng et al. reported that cd t cells in severe covid- appear less cytotoxic and effector-like with reduced cd a degranulation and granzyme b (gzmb) production . in contrast, a different study found that both gzmb and perforin were increased in cd t cells of severely sick patients (zheng et al., a) . in accordance with the latter observation, when compared to a moderate disease group, convalescent patients with resolved severe sars-cov- infection had significantly higher frequencies of polyfunctional t cells, with cd t cells producing more ifn-γ, tnf-α and il- , and cd t cells producing more ifn-γ, tnf-α and cd a, respectively (li et al., ) . however, given the vigorous dynamics of acute t cell responses and potential differences in sample timing throughout disease course, these observations are not necessarily mutually exclusive. accordingly, rnaseq data by liao et al. showed that cd t cells in the bal fluid of severe covid- patients express cytotoxic genes such as gzma, gzmb, and gzmk at higher levels, while klrc and xcl are enriched in mild cases . in summary, t cells in severe covid- seem to be more activated and may exhibit a trend towards exhaustion, based on continuous expression of inhibitory markers such as pd- and tim- as well as overall reduced polyfunctionality and cytotoxicity. conversely, recovering patients were shown to have an increase in follicular helper cd t cells (t fh ) as well as decreasing levels of inhibitory markers along with enhanced levels of effector molecules such as gzm a, gzmb and perforin (thevarajan et al., yang et al., a; zheng et al., b) . collectively, these studies provide a first glimpse into t cell dynamics in acute sars-cov- infection, but any conclusions have to be tempered at this stage on account of significant limitations in many of the current investigations. the humoral immune response is critical for the clearance of cytopathic viruses and is a major part of the memory response that prevents reinfection. sars-cov- elicits a robust b cell response, as evidenced by the rapid and near-universal detection of virusspecific igm, igg and iga, and neutralizing igg antibodies (nabs) in the days following infection. the kinetics of the antibody response to sars-cov- are now reasonably well described . similar to sars-cov- infection (hsueh et al., ) , seroconversion occurs in most covid- patients between and days after the onset of symptoms, and antibody titers persist in the weeks following virus clearance (figure ) , (haveri et al., ; lou et al., ; okba et al., ; wölfel et al., ; wu et al., b; zhao et al., a) . antibodies binding the sars-cov- internal n protein and the external s glycoprotein are commonly detected (amanat et al., ; ju et al., ; . the receptor binding domain (rbd) of the s protein is highly immunogenic and antibodies binding this domain can be potently neutralizing, blocking virus interactions with the host entry receptor, ace (ju et al., ; wu et al., b) . anti-rbd nabs are detected in most tested patients (ju et al., ; . although cross-reactivity to sars-cov- s and n proteins and to mers-cov s protein was detected in plasma from covid- patients, no cross-reactivity was found to the rbd from sars-cov- or mers-cov. in addition, plasma from covid- patients did not neutralize sars-cov- or mers-cov (ju et al., ) . rbd-specific cd + igg + memory b cells were single-cell sorted from a cohort of covid- donors between days - after the onset of symptoms (ju et al., ) . from their antibody gene sequences, sars-cov- specific monoclonal antibodies were produced. the monoclonal antibodies had a diverse repertoire, relatively low or no somatic mutations, and variable binding reactivity, with dissociation constants reaching - to - , similar to antibodies isolated during acute infections. two potent neutralizing sars-cov- rbd-specific monoclonal antibodies were characterized that did not cross-react with the rbd of sars-cov- or mers-cov (ju et al., ) . together, these results demonstrate that antibody mediated neutralization is virusspecific and likely driven by binding of epitopes within the rbd. the b cell response to a virus serves not only to protect from the initial challenge, but also to offer extended immunity against reinfection. following resolution of an infection, plasma cells formed during the acute and convalescent phases continue to secrete antibodies, giving rise to serological memory. memory b cells that are also formed during the primary infection constitute the second arm of b cell memory. memory b cells can quickly respond to a reinfection by generating new high affinity plasma cells. longterm protection is achieved through the induction of long-lived plasma cells and memory b cells. there is great interest in understanding the life-span of b cell memory responses to sars-cov- . protection from reinfection has direct medical and social consequences as the world works to develop vaccination strategies and resume normal activities. in covid- patients, evidence of near universal seroconversion and the lack of substantial descriptions of reinfection point to a robust antibody response, which, along with the t cell memory response, would offer protection to reinfection. indeed, a case study of a single patient described induction of cd hi cd hi antibody secreting cells (ascs), concomitant with an increase in circulating follicular t helper cells (tfh) cells (thevarajan et al., ) , and a scrnaseq study of pbmc from critically ill and recently recovered individuals revealed a plasma cell population . in addition, igg memory cells specific to the rbd have been identified in the blood of covid- patients (ju et al., ) . consistent with the development of immunity after covid- infection, a recent study of sars-cov- infection in rhesus macaques found that two macaques that had resolved the primary infection were resistant to reinfection days later (bao et al., b) . due to the timing of this outbreak, it is not yet possible to know the nature and extent of long-term memory responses, but lessons may again be learned from other human coronaviruses. in the case of the human coronavirus e, specific igg and nabs are rapidly induced but wane in some individuals around a year after infection with some residual protection to reinfection (callow et al., ; reed, ) . the life span of the humoral response following sars-cov- infection is also relatively short, with the initial specific igg and nab response to sars-cov- diminishing - years after infection and nearly undetectable in up to % of individuals (cao et al., ; liu et al., ) . a long-term study following sars-cov- infected healthcare workers over a years period also found that virus-specific igg declined after several years, but the authors observed detectable virus-specific igg years after infection . in the case of mers-cov, antibodies were detected in of volunteers tested years after infection (payne et al., ) . igg specific to sars-cov- trimeric spike protein was detectable in serum up to days after symptom onset, but igg titers began decreasing by weeks post-symptom onset (adams et al., ) . long term protection from reinfection may also be mediated by reactive memory b cells. a study that analyzed sars-cov- s protein-specific igg memory cells at , , and months post-infection found that s-specific igg memory b cells decreased progressively about % from to months after infection (traggiai et al., ) . a further retrospective study of individuals found no evidence of circulating sars-cov- -specific igg + memory b cells years after infection (tang et al., ) . this is in contrast to the memory t cell response, which was robustly detected based on induced ifn-γ production (tang et al., ) . studies of common coronaviruses, sars-cov- and mers-cov indicate that virus specific antibody responses wane over time, and, in the case of common coronaviruses, result in only partial protection from reinfection. these data suggest that immunity to sars-cov- may diminish following a primary infection and further studies will be required to determine the degree of long-term protection (figure ) . several studies have demonstrated that high virus-specific antibody titers to sars-cov- are correlated with greater neutralization of virus in vitro and are inversely correlated with viral load in patients ( figure ) wölfel et al., ; zhao et al., a) . despite these indications of a successful neutralizing response in the majority of individuals, higher titers are also associated with more severe clinical cases okba et al., ; zhao et al., a; zhou et al., a) , suggesting that a robust antibody response alone is insufficient to avoid severe disease ( figure ). this was also observed in the previous sars-cov- epidemic, where neutralizing titers were found to be significantly higher in deceased patients compared to patients who had recovered . this has led to concerns that antibody responses to these viruses may contribute to pulmonary pathology, via antibody-dependent enhancement (ade) (figure ). this phenomenon is observed, when non-neutralizing virus-specific igg facilitate entry of virus particles into fc-receptor (fcr) expressing cells, particularly macrophages and monocytes, leading to inflammatory activation of these cells (taylor et al., ) . a study in sars-cov- -infected rhesus macaques found that anti-s-igg contributed to severe acute lung injury (ali) and massive accumulation of monocytes/macrophages in the lung . furthermore, serum containing anti-s ig from sars-cov- patients enhanced the infection of sars-cov- in human monocyte-derived macrophages in vitro (yip et al., ) . ade was also reported with a monoclonal antibody isolated from a patient with mers-cov . somewhat reassuringly, there was no evidence of ade mediated by sera from rats vaccinated with sars-cov- rbd in vitro (quinlan et al., ) , nor in macaque immunized with an inactivated sars-cov- vaccine candidate (gao et al. ) . as of now, there is no evidence that naturally developed antibodies towards sars-cov- contribute to the pathological features observed in covid- . however, this possibility should be considered when it comes to experimental design and development of therapeutic strategies. importantly, in all of the descriptions of ade as it relates to coronavirus, the fcr was necessary to trigger the antibody-mediated pathology. high-dose intravenous immunoglobulin (ivig), which may blunt ade, has been trialed in covid- patients shao et al., ) , but further studies are needed to determine the extent to which ivig is safe or beneficial in sars-cov- infection. vaccine trials will need to consider the possibility of antibody-driven pathology upon antigen re-challenge; strategies using f(ab) fragments or engineered fc monoclonal antibodies may prove particularly beneficial in this setting (amanat and krammer, ) . with the rapidly growing number of cases in the first few months, numerous reports on predictors of covid- severity with small cohorts were released. these offered clinicians and immunologists the first understanding of the clinical course and pathological processes that are associated with the novel sars-cov- infection. this section highlights key findings from those studies, with a major focus on the immune factors associated with disease risk or severity. there are currently limited known risk factors for susceptibility to covid- , although this has been evaluated in several studies. zhao et al. compared the abo blood group distribution in a cohort of covid- patients to that of healthy controls from the corresponding regions . they found blood group a to be associated with a higher risk for acquiring covid- , when compared to non-a blood groups; blood group o had the lowest risk for the infection. another study demonstrated an identical association (zietz and tatonetti, ) , and similar results have been previously described for other viruses (lindesmith et al., ) , including for sars-cov- (cheng et al., a) . several large collaborative efforts are currently underway to generate, share and analyze genetic data to understand the links between human genetic variation and covid- susceptibility and severity, the most prominent of which is the covid- host genetics initiative (covid hg.org). these studies are supported by previous observations on sars-cov- that followed the outbreak, which have identified significant associations between genetic variants and immune phenotypes (chan et al., ; wang et al., ; zhao et al., ) . although identifying such polymorphisms and their associated genes and pathways for sars-cov- will require large cohorts, several studies have already highlighted genetic polymorphisms that may potentially impact susceptibility, which remain to be tested in clinical trials. these studies have focused on genetic variants that may impact the expression or function of genes important in viral entry, namely ace (sars-cov- receptor) and tmprss (spike protein activator) (asselta et al., ; cao et al., c; renieri et al., ; stawiski et al., ) . cao et al. identified variants that are potentially expression quantitative trait loci (eqtl) of ace (i.e. they may potentially alter ace gene expression) and analyzed their frequencies in different populations (cao et al., c) . stawiski et al. listed variants that may be critical in ace binding and thereby its function, and compared the frequencies of these variants within different populations (stawiski et al., ) . while there are several limitations to these studies, the major question is whether the utility of these biomarkers is replicable in large populations with covid- clinical outcomes data and in targeted or large-scale genomic analyses that are currently underway. in addition, these studies will reveal the potential associations between genetic variants and susceptibility in a gene or loci agnostic fashion. several routine blood and serological parameters have been suggested to stratify patients who might be at higher risk for complications to aid in allocation of healthcare resources in the pandemic (table ). serologic markers from routine blood work were reported, by comparing patients with mild/moderate symptoms to those with severe symptoms. this includes different acute phase proteins, such as saa (serum amyloid protein) and c-reactive protein (crp) . interestingly, elevations in crp appear to be unique to covid- patients, when compared to other viral infections. other consistently reported markers in non-survivors are increased procalcitonin (pct) and il- levels , as well as increased serum urea, creatinine, cystatin c, direct bilirubin, and cholinesterase . overall, inflammatory markers are common in severe cases of covid- and appear to correlate with the severity of the symptoms and clinical outcome. moreover, the extensive damage that occurs in specific organs of severe covid- patients is possibly related to differences in the expression of ace ( figure ) . lymphopenia is the most frequently described prognostic marker in covid- (table ) , and it appears to predict morbidity and mortality even at early stages (fei et al., ) . tan et al. proposed a prognostic model based on lymphocyte counts at two time points: patients with less than % lymphocytes at days - from the onset of symptoms and less than % at days - had the worst outcomes in this study (tan et al., a) . wynants et al. compared predictors of disease severity across studies (> patients), highlighting crp, neutrophil-to-lymphocyte ratio (n/l) and lactate dehydrogenase (ldh) as the most significant predictive biomarkers . furthermore, a meta-analysis of covid- studies with a total of patients also attempted to identify early-stage patients with poor prognosis . the most consistent findings across the different studies were elevated levels of crp, ldh and d-dimer, as well as decreased blood platelet and lymphocyte counts zhou et al., d) . systemic and pulmonary thrombi have been reported with activation of the extrinsic coagulation cascade, involving dysfunctional endothelium and monocytic infiltration (poor et al., ; varga et al., ) ; thrombocytopenia and elevated d-dimer levels may be indicative of these coagulopathies in covid- patients with important therapeutic implications (fogarty et al., ; poor et al., ) . immunological biomarkers are particularly important, as immunopathology has been suggested as a primary driver of morbidity and mortality with covid- . several cytokines and other immunologic parameters have been correlated with covid- severity (table ) . most notably, elevated il- levels were detected in hospitalized patients, especially critically ill patients, in several studies, and are associated with icu admission, respiratory failure, and poor prognosis (chen et al., f; huang et al., b; liu et al., f) . increased il- r, il- , il- , and gm-csf have been associated with disease severity as well, but studies are limited and further studies with larger cohorts of patients are needed to indicate predictive power zhou et al., b) . conflicting results regarding il- β and il- have been reported gong et al., ; wen et al., ) . although elevated cytokine concentrations have been widely described in covid- patients, the vast majority (including il- , il- , il- , ctack, ifn-γ) do not seem to have prognostic value, because they do not always differentiate moderate cases from severe cases (yang et al., b) . this stratification was possible with ip- , mcp- , and il- ra. while there are reports that levels of il- at first assessment might predict respiratory failure (herold et al., ) , other publications with longitudinal analyses demonstrated that il- increases fairly late during the disease's course, consequently compromising its prognostic value at earlier stages . liu et al. developed a web-based tool using k-means clustering to predict prognosis in terms of death or hospital discharge of covid- patients using age, comorbidities (binary), and baseline log helper t cell count (th), log suppressor t cell count (ts), and log th/ts ratio . total t cell, helper t cell, and suppressor t cell counts were significantly lower, and the th/ts ratio was significantly higher in patients who died from infection, as compared to patients who were discharged. importantly, most serological and immunological changes observed in severe cases are associated with disease severity, but can not necessarily serve as predictive factors, as they may not have utility in early identification of patients at higher risk. discovery of truly predictive biomarkers and potential drivers of hyper-inflammatory processes requires comprehensive profiling of asymptomatic and mild cases and longitudinal studies which are limited to date. confounding variables including age, gender, comorbidities may dramatically affect associations observed. in addition, direct correlation with patient viral load will be important to provide a greater understanding of underlying causes of morbidity and mortality in covid- and the contribution of viral infectivity, hyperinflammation and host tolerance (medzhitov et al., ) . in summary, lymphopenia, increases in proinflammatory markers and cytokines and potential blood hypercoagulability characterize severe covid- cases with features reminiscent of cytokine release syndromes. this correlates with a diverse clinical spectrum ranging from asymptomatic to severe and critical cases. during the incubation period and early phase of the disease, leukocyte and lymphocyte counts are normal or slightly reduced. after sars-cov- binds to ace overexpressing organs, such as the gastrointestinal tracts and kidneys, increases in non-specific inflammation markers are observed. in more severe cases, a marked systemic release of inflammatory mediators and cytokines occurs, with corresponding worsening of lymphopenia and potential atrophy of lymphoid organs, impairing lymphocyte turnover (terpos et al., ). antivirals are a class of small molecules that function as inhibitors of one or more stages of a virus life cycle. because of similarities between different virus replication mechanisms, some antivirals can be repurposed against various viral infections. currently, most of the available antiviral drugs tested against sars-cov- are smallmolecules previously developed against sars-cov- , mers-cov, or other rna and dna viruses. a number of small molecules with known antiviral activity against other human rna viruses are being evaluated for efficacy in treating sars-cov- . the ribonucleoside analog β-d-n -hydroxycytidine (nhc) reduced viral titers and lung injury in mice infected with sars-cov- via introduction of mutations in viral rna (sheahan et al., ) . further, an inhibitor of host dhodh, a rate-limiting enzyme in pyrimidine synthesis, was able to inhibit sars-cov- growth in vitro with greater efficacy than remdesivir or chloroquine xiong et al., ) . merimepodib, a non-competitive inhibitor of the enzyme inosine- ′-monophosphate dehydrogenase (impdh), involved in host guanosine biosynthesis, is able to suppress sars-cov- replication in vitro . finally, n-( -hydroxypropyl)- -trimethylammonium chitosan chloride (htcc), which was previously shown to efficiently reduce infection by the less pathogenic human coronavirus hcov-nl , was also found to inhibit mers-cov and pseudotyped sars-cov- in human airway epithelial cells (milewska et al., ) . much of the antiviral computational and experimental data currently available for sars-cov- focus on targeting the cl or main protease (mpro). two prominent drug candidates targeting the sars-cov- mpro were designed and synthesized, by analyzing the substrate binding pocket of mpro (dai et al., ) . the x-ray crystal structures of the novel inhibitors in complex with sars-cov- mpro were resolved at . Å. both compounds showed good pharmacokinetic activity in vitro, and one exhibited limited toxicity in vivo (dai et al., ) . multiple studies also aimed to repurpose protease inhibitors to reduce sars-cov- titers. nine existing hiv protease inhibitors (nelfinavir, lopinavir, ritonavir, saquinavir, atazanavir, tipranavir, amprenavir, darunavir, and indinavir) were evaluated for their antiviral activity in vero cells infected with sars-cov- (yamamoto et al., ) and nelfinavir was the most potent at inhibiting viral replication. the coronavirus rna-dependent rna polymerase (rdrp) catalyzes the synthesis of viral rna making it essential for viral replication and a prime target for antiviral inhibitors. remdesivir, an adenosine triphosphate analog, inhibits rdrp by binding to rna strands and preventing additional nucleotides from being added, thereby terminating viral rna transcription ( figure a ) (agostini et al., ) . remdesivir has been previously shown to be effective against mers-cov and sars-cov- infections in animal models (sheahan et al., ; de wit et al., ) . similarly, a study investigated the efficacy of remdesivir treatments on rhesus macaques with sars-cov- infections (williamson et al., ) . macaques treated with remdesivir showed a reduction in lung viral loads and pneumonia symptoms, but no reduction in virus shedding. this study does provide evidence that if administered early enough, remdesivir may be effective at treating sars-cov- infections. a large number of clinical trials using experimental antiviral drugs are currently underway. a small proportion of them are aimed at repurposing existing antivirals including: arbidol (umifenovir), a broad-spectrum antiviral that blocks viral fusion; lopinavir/ritonavir (lpv/r), a combination of anti-hiv protease inhibitors; favipiravir, an rdrp inhibitor used to treat severe influenza infections (hayden and shindo, ) ; and remdesivir ( figure a ). chen et al. conducted a multicenter, randomized priority trial on patients with confirmed covid- infection to test favipiravir or arbidol . favipiravir was suggested to significantly improve symptom relief. however, the interpretation of this study is limited by a short clinical recovery window of days, only of patients with confirmed covid- , and the lack of a control group. lpv/r has previously shown efficacy in treating sars-cov- (chu et al., ) , prompting an early sars-cov- clinical trial . patients were enrolled in a trial investigating the efficacy and safety of lpv/r (n= patients), arbidol (n= ), or control (n= ) as treatment for mild-to-moderate covid- . at day of treatment, . %, . % and . % of patients had a positive to negative conversion in the lpv/r, arbidol, and control groups, respectively, with no statistical significance between groups. a randomized controlled trial (rct) with severe covid- patients did not observe a significant benefit of lpv/r either (cao et al., a) . however, a study that looked at the impact of earlier administration of (lpv/r) treatment showed that when treatment of lpv/r was started within days of symptom onset, a shorter duration of virus shedding was observed. thus, timing of lpv/r administration may be critical to its efficacy . in a multicenter clinical study assessing the compassionate use of remdesivir in severe covid- patients, patients across several countries were treated with remdesivir for days (grein et al., ) . % of the patients who received remdesivir showed clinical improvement assessed through improved oxygen-support/extubations. without a proper control group, limited conclusions can be drawn with regards to the efficacy of remdesivir from this study. the measured % clinical improvement may be in line with average clinical improvement across patients treated with standard of care . a small rct in china with severe covid- patients randomized : to remdesivir vs. placebo demonstrated no significant benefit in time to clinical improvement . almost simultaneously, preliminary results from a larger niaid rct with more than patients were announced with remdesevir to be associated with quicker time to recovery: days compared with days (ledford, ) . a non-significant benefit in mortality was also noted and the trial was stopped early to allow access to remdesivir in the placebo arm. complete safety data and full publication are awaited but this study offers encouraging results and have resulted in an fda emergency use authorization for remdesivir in hospitalized covid- patients. chloroquine (cq) and its derivative hydroxychloroquine (hcq) have gained traction as possible therapeutics for covid- . both drugs are used as antimalarial agents and as immunomodulatory therapies for rheumatologic diseases. however, the application of cq and hcq to covid- stems for their past use as antivirals (savarino et al., ) , including for sars-cov- (keyaerts et al., ; vincent et al., ) . cq and hcq interfere with lysosomal activity and have been reported to have immuno-modulatory effects. cq augments antigen processing for mhc class i and ii presentation, directly inhibits endosomal tlr and tlr , and enhances the activity of regulatory t cells (garulli et al., ; lo et al., ; schrezenmeier and dörner, ; thomé et al., a thomé et al., , b . early studies involving in vitro infection of host cells with sars-cov- demonstrated that both cq and hcq significantly impact endosomal maturation, resulting in increased sequestration of virion particles within endolysosomes. however, there has been conflicting evidence whether cq is more potent than hcq in reducing viral load yao et al., a) . notably, one group reported that treatment of infected cells with hcq before and during infection significantly reduced viral load, suggesting that combined prophylactic and therapeutic hcq use yields maximum efficacy (clementi et al., ) . to better understand host immune responses to treatment, one group compared bulk transcriptomic changes in primary pbmcs treated with hcq for hours to pbmcs from confirmed sars-cov- positive patients and controls, followed by a comparison of hcq treated primary macrophages to bal and postmortem lung biopsies from covid- patients (corley et al., ) . across all comparisons, there was minimal overlap between host differential gene expression and genes altered by in vitro hcq treatment. thus, the potential mechanistic action of hcq in the context of sars-cov- remains poorly defined. despite the apparent widespread use of hcq and cq to treat covid- ( figure b ), few controlled clinical trials have been performed so far and thus the potential benefits of these drugs for covid- remains controversial. one of the earliest trials ( - - ) was a single-arm, open label trial of mg daily hcq in covid- patients. they reported that hcq alone, or in combination with the antibiotic azithromycin (az), reduced viral load by day (gautret et al., a) . a follow up trial in patients treated with hcq + az reported that % of patients had a negative pcr result on day of treatment, and . % were discharged within days of treatment. however, it is important to note that both trials had no control arms (gautret et al., b) . rigorous statistical analyses by others that accounted for the patients excluded from the original analysis found limited evidence for hcq monotherapy (hulme et al., ; lover, ) . a double blind rrct assessed hcq monotherapy in the treatment of mild covid- (chictr ) . a total of patients were enrolled; the treatment arm received mg hcq daily over days. by day , patients who received hcq had clinical resolution on average one day earlier than controls; no patients progressed to severe disease compared to patients in the control arm. in a smaller rct treated patients with mild covid- (nct ) with mg hcq for days, there were no significant differences in the number of patients with negative pcr results on day (all but one positive), median duration of hospitalization, time to fever resolution, or progression of disease on chest ct . the largest rct to date enrolled patients with mild covid- across centers in an open label trial of hcq + standard of care (chictr ). there were no significant differences between groups in conversion to negative sars-cov- rt-pcr result on day or rate of symptom resolution; there were significantly more adverse events in the hcq arm, though largely non-serious; they reported some evidence for faster normalization of c-reactive protein in the patients who received hcq plus standard of care, but this finding was not significant (tang et al., b) . a metaanalysis including most of the studies described here found no clinical benefits to patients receiving standard of care plus an hcq regimen (shamshirian et al., ) . two studies have assessed hcq efficacy in severe covid- . in a prospective study of patients who had received mg hcq over days with az on days - , there were several patients with worsening clinical status and one death; / patients had a positive pcr result on day ( molina et al., ) . an ongoing double blind rct of patients with severe covid- (nct ) randomized patients into high dose hcq ( mg x/d for days) or low dose ( mg/day for days) treatment groups (borba et al., ) . recruitment into the high dose arm was halted prematurely due to poor safety outcomes. there was no significant difference in negative pcr results on day or need for mechanical ventilation on day . taken together, the clinical trials performed thus far to evaluate the efficacy of hcq ± az for covid- have not demonstrated clear evidence of clinical benefit in patients with severe disease. a search of clinicaltrials.gov on april , found clinical trials investigating hcq. this number is rapidly growing, indicating the heightened interest in this therapeutic and pressing need for evidence-based recommendations. because of their anti-inflammatory activity, corticosteroids (cs) are an adjuvant therapy for ards and cytokine storm. however, the broad immunosuppression mediated by cs does raise the possibility that treatment could interfere with the development of a proper immune response against the virus. a meta-analysis of , patients with mers-cov, sars-cov- , or sars-cov- infection found that cs treatment was associated with higher mortality . a more recent meta-analysis of only sars-cov- infection assessed , patients and found no mortality difference associated with cs treatment, including in a subset of patients with ards (gangopadhyay et al., ) . other studies have reported associations with delayed viral clearance and increased complications in sars and mers patients (sanders et al., ) . in fact, the interim guidelines updated by the who on march , advise against giving systemic corticosteroids for covid- (world health organization, a) . yet, new data from covid- are conflicting. one group reported no significant difference in time to viral clearance between patients who received methylprednisolone orally (mild disease) or iv (severe) and those who did not . retrospective studies from groups in china report that patients who were transferred to the icu were less likely to have received cs and that patients with ards who received methylprednisolone had reduced mortality risk . in contrast, another retrospective analysis found that patients who received cs were more likely to have either been admitted to the icu or perished, although the cs treated group also had significantly more comorbidities . a smaller observational study of patients found no association between corticosteroid treatment and time to viral clearance, length of hospital stay, or symptom duration (zha et al., ) . a larger study of adjuvant cs in patients with critical covid- found no association with -day mortality; subgroup analysis of patients with ards found no association between treatment with cs and clinical outcomes . they also found that increased dosage was significantly associated with increased mortality risk. a retrospective review of patients, of whom received iv methylprednisolone, found that early, low-dose administration significantly improved spo and chest ct, time to fever resolution, and time on supplemental oxygen therapy (wang et al., h) . others have published perspectives in support of early and short-term, low dose administration based on anecdotal evidence, but not clinical trials. most of the current data on cs use in covid- are from observational studies, and support either modest clinical benefit or no meaningful effects. larger rcts are necessary to understand the risks and benefits of cs for these patients; there are trials evaluating various corticosteroids registered on clinicaltrials.gov as of april , . one of the first defenses of the human body against rna viruses like sars-cov- is the release of type i and iii ifns. it is important to note that type i ifn (ifnα/β) receptors are ubiquitously expressed, so ifnα/β signaling can result in not only antiviral effects, but also in the activation of immune cells that potentially exacerbate pathogenesis. in contrast, type iii ifn (also known as ifnλ) signals mainly in epithelial cells, as well as in a restricted pool of immune cells. because type iii ifns have immunomodulatory functions, subsequent signaling could induce a potent antiviral effect without enhancing pathogenic inflammation (andreakos et al., ; prokunina-olsson et al., ) . recently, there has been a growing interest in the potential therapeutic impact of modulating the ifn response to disable covid- pathogenesis. before the current pandemic, groups have studied the role of ifns in other betacoronavirus infections. one study of patients with sars-cov- infection described unresolved elevated type i ifns and ifn-stimulated genes (isgs) in those with poor outcomes (cameron et al., ) . others report that exogenous type i ifn does not improve outcomes when given with ribavirin in patients with mers-cov infection (arabi et al., ) , suggesting that the role of ifn as a therapeutic or prophylactic option may be strain-or species-specific (sheahan et al., ) . interestingly, a recent study by mount sinai virology groups revealed that type i ifn signaling is impaired in the early response to sars-cov- ; in vitro, sars-cov- may be more susceptible to type i ifn than sars-cov- (blanco-melo et al., ) . based on additional evidence that ifn responses to betacoronaviruses are altered as compared to other respiratory viruses (blanco-melo et al., ; channappanavar et al., ; okabayashi et al., ) , trials of ifn-i/iii administration have been initiated (nct , nct ). hyperinflammatory responses and elevated levels of inflammatory cytokines, including interleukins (il)- , , and , have been shown to correlate with covid- severity diao et al., ; gong et al., ; moore and june, ; wan et al., a; xu et al., b) . the drivers of this cytokine storm remain to be established, but they are likely triggered initially by a combination of viral pamps and host danger signals. the heterogeneous response between patients suggests other factors are involved, possibly including the sars-cov- receptor, ace (hirano and murakami, ) . several studies have begun to report the cellular programs that may contribute to the cytokine storm detected in covid- patients. one group reported that in the context of generalized lymphopenia, certain subsets of cd t cells that express gm-csf and il- are more abundant in severe covid- patients than in covid- patients who do not require intensive care . reports that other major proinflammatory cytokines (tnf-α, ifn-ɣ, il- ) and chemokines (ccl , ccl , ccl ) are elevated underscore a potentially pathogenic t h / program in covid- (diao et al., ; giamarellos-bourboulis et al., ) . histological and single-cell analyses identified monocytes/macrophages as other potent sources of inflammatory cytokines in covid- cytokine storm giamarellos-bourboulis et al., ; law et al., ; moore and june, ; zhou et al., b) . studies of other betacoronavirus infections, including sars-cov- and mers-cov, have also identified similar hyperactivation of monocytes, macrophages, and dendritic cells as a driver of cytokinemediated immunopathology in humans chien et al., ; huang et al., c; konig et al., ; wang et al., ; wong et al., ; xu et al., b; zhou et al., b) . following preliminary reports of il- as a critical cytokine in covid- -associated cytokine release syndrome (crs), monoclonal antibodies that target the il- signaling pathway have been proposed as therapeutic candidates (moore and june, ) ( figure c ). the commercial anti-il- r antibodies tocilizumab (actemra) and sarilumab (kevzara), and the anti-il- antibody siltuximab (sylvant), are now being tested for efficacy in managing covid- crs and pneumonia in ongoing clinical trials (table ). to date, only one group has reported preliminary results from a cohort of covid- patients treated with a single administration of tocilizumab ( mg, iv), along with lopinavir, methylprednisolone, and oxygen therapy (chictr ) . the single observation study found recuperated lymphocyte counts in / patients and resolution of lung opacities in / patients on chest ct; / patients were discharged. all patients experienced an improvement in symptoms, and no subsequent pulmonary infections were reported. a second report described an association between use of tocilizumab and reduced likelihood of icu admission and mechanical ventilation. still, in declining patients with severe covid- pneumonia, this retrospective study did not report significant improvement in mortality on weighted analysis (roumier et al., ) . nevertheless, these studies are encouraging but like other treatment approaches, larger rcts are needed. in addition to the il- signaling pathway, other cytokine-/chemokine-associated elements, including il- r, gm-csf and the chemokine receptor ccr , have been proposed as potential targets for blockade to manage covid- crs ( figure c ). finally, complement activation was shown to be over-activated in lungs of covid- patients. although results from the randomized trial are not yet published, anti-c a monoclonal antibody therapy showed benefits in two critically-ill covid- patients . while vaccines are being developed to educate a person's immune system to make their own nab against sars-cov- , there is interest in using adoptive transfer of nab as a therapeutic approach ( figure d ). this strategy has already proven to be effective against sars-cov- (cao et al., ; ho et al., ; ter meulen et al., ; sui et al., ; zhu et al., ) and mers-cov (forni et al., ; jia et al., ; ying et al., ) . in the case of sars-cov- , these efforts are primarily centered on identifying nab made during natural infections or generating nab through animal vaccination approaches. patients who have recovered from sars-cov- infection are one potential source of nabs (ju et al., ; walls et al., ; wölfel et al., ; ye et al., ; yuan et al., ) . in an effort to obtain these nabs, scientists sorted rbd specific memory b cells and cloned their heavy and light variable region to express recombinant forms of the corresponding antibodies (ju et al., ; ye et al., ) . four of the antibodies produced in these studies ( b , d , p c- f p c- f ) showed high neutralizing potential in vitro, and all inhibited ace /rbd binding. successful antibody-mediated neutralization of sars-cov- seems to be dependent on the inhibition of ace /rbd binding. however, ye et al. showed that nearly all antibodies derived from serum of recovered patients bound to s and rbd, with only actually inhibiting ace /rbd binding . of note, a sars-cov- derived neutralizing antibody ( d ) and a single chain antibody against sars-cov- (n ) have also been shown to neutralize sars-cov- without inhibiting ace /rbd binding. thus, blocking this interaction may not be a prerequisite for an effective sars-cov- nab. the generation of a hybridoma producing a monoclonal nab against sars-cov- provides the potential for a therapeutic ab that can be directly administered to patients to block ongoing infection and potentially even as a prophylactic ( figure d ). sars-cov- and sars-cov- consensus sequences share about % identity (tai et al., ) . thus, a wide range of sars-cov- nabs have been tested for crossreactivity with sars-cov- , as they could help speed up the development of potential covid- treatments. in a recent study, antibodies were isolated from the memory b cells of an individual who recovered from sars-cov- infection. while out of isolated antibodies could bind sars-cov- s protein, one of them (s , see table ) also neutralizes sars-cov- (pinto et al., ) . the combination of s with a weakly neutralizing antibody that could bind another rbd epitope led to enhanced neutralization potency. in addition, cr (table ) was found to bind sars-cov- rbd , but this antibody did not neutralize sars-cov- . computational simulations identified amino-acids that could be modified on cr to enhance its binding affinity with sars-cov- rbd (corrêa giron et al., ) , potentially augmenting its neutralization potential. animal models represent another tool to generate nabs against sars-cov- (table ) . in one study, the authors developed a protocol to synthetize human nanobodies, smaller antibodies that only contain a heavy variable (vh) chain as first described in camelids (figure d ). another antibody isolated from camelids immunized with sars-cov- and mers-cov s proteins then fused to a human fc fragment showed neutralization potential against sars-cov- (vhh- -fc) (wrapp et al., ) . genetically modified mice with humanized antibody genes can also be used to generate therapeutic monoclonal antibodies, as successfully experimented against ebola virus (levine, ) . similar studies are now focused on the use of sars-cov- or derivatives to generate highly effective nab in animal models, which can be directly given to infected patients, and efforts are already underway with estimates of clinical trials of pooled antibody cocktails beginning in early summer by regeneron. finally, another approach to nab development is to fuse ace protein and the fc part of antibodies as they would bind rbd and potentially be cross reactive among other coronaviruses ( figure d ). indeed, an ace -fc as well as an rbd-fc have been shown to neutralize both sars-cov- and sars-cov- in vitro. although recombinant nabs could provide an effective treatment, they will require a significant time investment to develop, test, and bring production to scale before becoming widely available to patients. a faster strategy consists of transferring convalescent plasma (cp) from previously infected individuals that have developed high titer nabs that target sars-cov- ( figure d ). despite the current lack of appropriately controlled trials, cp therapy has been previously used and shown to be beneficial in several infectious diseases such as the influenza pandemic (luke et al., ) , h n influenza (hung et al., ) , and sars-cov- (arabi et al., ) . thanks to the development of serological tests (amanat et al., ; cai et al., ; xiang et al., b; zhang et al., d) , recovered covid- patients can be screened to select plasma with high antibody titers. some studies and case reports on cp therapy for covid- have evaluated the safety and the potential effectiveness of cp therapy in patients with severe disease (ahn et al., ; duan et al., ; pei et al., ; shen et al., ; zhang et al., b) (table ). these studies were neither controlled nor randomized, but they suggest that cp therapy is safe and can have a beneficial effect on the clinical course of disease. further controlled trials are needed to determine the optimal timing and indication for cp therapy. cp therapy has also been proposed for prophylactic use in at-risk individuals, such as those with underlying health conditions or health care workers exposed to covid- patients. the fda has approved the use of cp to treat critically ill patients (tanne, ) . determining when to administer the cp is also of great importance, as a study in sars-cov- patients showed that cp was much more efficient when given to patients before day day of illness (cheng et al., b) , as previously shown in influenza (luke et al., ) . this study also showed that cp therapy was more efficient in pcr positive, seronegative patients. the amount of plasma and number of transfusions needed requires further investigation (table ) . overall, cp therapy seems to be associated with improved outcomes, and appears to be safe, but randomized clinical trials are needed to confirm this. several clinical trials are currently in progress worldwide (belhadi et al., ) the devastating effects of the pandemic spread of sars-cov- in a globally naïve population has resulted in unprecedented efforts to rapidly develop, test, and disseminate a vaccine to protect against covid- or to mitigate the effects of sars-cov- infection. although vaccination has a long and successful history as an effective global health strategy, there are currently no approved vaccines to protect humans against coronaviruses (andré, ) . previous work after the sars-cov- and mers-cov epidemics has provided a foundation on which many current efforts are currently building upon, including the importance of the s protein as a potential vaccine. diverse vaccine platforms and preclinical animal models have been adapted to sars-cov- , facilitating fast-moving and robust progress in creating and testing sars-cov- vaccine candidates. a number of vaccine candidates are already being tested in clinical trials and more are continuing to progress towards clinical testing. since sars-cov- first emerged, the s protein has been favored as the most promising target for vaccine development to protect against coronavirus infection. this particular viral protein has important roles in viral entry and in stimulating the immune response during natural infection and in vaccination studies of both sars-cov- and mers-cov (du et al., ; song et al., ; zhou et al., ) , which has also been confirmed for sars-cov- . the s protein has been found to induce robust and protective humoral and cellular immunity, including the development of nabs and t cell-mediated immunity (du et al., ) . in animal models, correlates of protection against sars-cov- infection appear to be induction of nabs against the s protein, although antibodies to other proteins have been detected such as those against nucleoprotein (n) and orf a (qiu et al., ; sui et al., ) . nabs are also believed to protect against infection by blocking receptor binding and viral entry, which has been shown with pseudovirus-based neutralization assays nie et al., a) . studies of sars-cov- indicate that t cell responses, which were targeted to the s protein after natural infection, included cd + t cell responses against the membrane (m) and n proteins, may also be a correlate of protection and that memory t cell responses can persist even years after infection (li et al., ; ng et al., ) . rbd-specific antiviral t cell responses have also been detected in people who have recovered from covid- , further validating its promise as a vaccine target (braun et al., ; dong et al., ) . although the antibodies targeting the rbd of the s protein have greater potential for providing cross-protective immunity, other fragments of the s protein and additional viral proteins have been investigated as target epitopes, especially for t cells. researchers have taken advantage of the genetic similarity between sars-cov- and sars-cov- and mers-cov and bioinformatics approaches to rapidly identify b and t cell potential epitopes in the s and other proteins, with many studies providing data regarding antigen presentation and antibody binding properties and one study looking into the predicted evolution of epitopes (ahmed et al., ; baruah and bose, ; bhattacharya et al., ; fast et al., ; grifoni et al., ; lon et al., ; zheng and song, ) . while the s protein has been found to be the most immunodominant protein in sars-cov- , the m and n proteins also contain b and t cell epitopes, including some with high conservation with sars-cov- epitopes . for sars-cov- and mers-cov, animal studies and phase i clinical trials of potential vaccines targeting the s protein had encouraging results, with evidence of nab induction and induction of cellular immunity martin et al., ; modjarrad et al., ) . these findings are being translated into sars-cov- vaccine development efforts, hastening the progress drastically. the who provided a report earlier in april that reported sixty-three vaccine candidates in preclinical testing and three in clinical testing (world health organization, b) . a recent search on may , , on clinicaltrials.gov revealed ten registered vaccine candidates (table ). the university of pittsburgh is also looking to move their microneedle array vaccine candidate containing a codon-optimized s subunit protein into clinical trials . sanofi and glaxosmithkline (gsk) have recently reported their intent to collaborate and bring together sanofi's baculovirus expression system, which is used to produce the influenza virus vaccine, flublok, to create an s protein vaccine adjuvanted with gsk's as . the purified inactivated sars-cov- virus vaccine candidate (picovacc) of sinovac biotech ltd. will also be starting a clinical trial in china after finding that their candidate protected rhesus macaques from viral challenge without signs of detectable immunopathology . although some of these vaccine candidates are based on platforms that have been used or tested for other purposes, there remain questions regarding their safety and immunogenicity, including the longevity of any induced responses, that will require continual evaluation. although the development of a vaccine to protect against sars-cov- infection has progressed at an unprecedented rate and produced an impressive volume of candidates for testing, many challenges lie ahead. the prior knowledge gained after sars-cov- was first discovered in and the subsequent emergence of mers-cov in provided a significant jumpstart, but the progress of sars-cov- vaccine development has already far outstripped the point of the blueprint created before covid- became a pandemic. while a variety of platforms are simultaneously being innovated or adapted, they each have strengths and limitations, many of which relate to the delicate balance between safety and immunogenicity. many shortcuts have been taken and will continue to be taken due to the urgency of the ongoing covid- pandemic, but significant concerns need to be addressed. one such concern involves the accumulating data supporting the initial assessment that covid- is disproportionately severe in older adults. in conjunction with the large body of work related to immune-senescence, these findings indicate that vaccine design should take into consideration the impact of aging on vaccine efficacy (nikolich-Žugich, ). furthermore, questions remain regarding the possibility of antibody-dependent enhancement of covid- , with in vitro experiments, animal studies, and two studies of covid- patients supporting this possibility (cao, ; tetro, ; zhao et al., a) . assuming vaccine candidates are identified that can safely induce protective immune responses, additional major hurdles will be the production and dissemination of a vaccine. for some types of vaccines, large-scale production will not be as much of an issue and infrastructure already in place to produce current good manufacturing practice (cgmp)-quality biologics can be repurposed, but this will only be applicable to a subset of the candidates (thanh le et al., ) . in order to address the urgent need and stem the covid- pandemic, regulatory agencies need to continue to support rapid testing and progression of vaccine candidates, companies need to disseminate important findings directly and openly, and researchers need to investigate correlates of protection using in-depth immune monitoring of patients with a broad range of clinical presentations and clinical trial participants. the newly announced accelerating covid- therapeutic interventions and vaccines (activ) is designed to bring together numerous governmental and industry entities to help address this need. the rapid spread of sars-cov- and the unprecedented nature of covid- has demanded an urgency in both basic science and clinical research, and the scientific community has met that call with remarkable productivity. within months, there has been a significant generation of scientific knowledge that has shed some light on the immunology of sars-cov- infections. studies of past coronavirus outbreaks, involving sars-cov- and mers-cov, have provided a foundation for our understanding. the pathology of severe cases of covid- do indeed resemble certain immunopathologies seen in sars-cov- and mers-cov infections, like crs. however, in many other ways, immune responses to sars-cov- are distinct from those seen with other coronavirus infections. the emerging epidemiological observation that significant proportions of individuals are asymptomatic despite infection, not only reflects our current understanding that sars-cov- has a longer incubation period and higher rate of transmission than other coronaviruses, but also speaks to significant differences in the host immune response. therefore, it is imperative that immune responses against sars-cov- and mechanisms of hyperinflammation-driven pathology are further elucidated to better define therapeutic strategies for covid- . here, we reviewed the recent literature and highlighted hypotheses that interrogate mechanisms for viral escape from innate sensing; for hyper-inflammation associated with crs and inflammatory myeloid subpopulations; for lymphopenia marked by t cell and nk cell dysfunction; and for correlates of protection and their duration, among others. still, additional studies are needed to address how these immune differences across patients or between different types of coronavirus infections dictate who succumbs to disease and who remains asymptomatic. existing studies of sars-cov- and mers-cov and ongoing studies of sars-cov- will likely provide a robust framework to fulfill that unmet need. overview of innate immune sensing (left) and interferon signaling (right), annotated with the known mechanisms by which sars-cov- and mers-cov antagonize the pathways (red). [based on data from preliminary covid- studies and earlier studies in related coronaviruses] il- , il- β and ifn-i/iii from infected pulmonary epithelia can induce inflammatory programs in resident (alternate) macrophages while recruiting inflammatory monocytes as well as granulocytes and lymphocytes from circulation. sustained il- , and tnf-ɑ by incoming monocytes can drive several hyperinflammation cascades. inflammatory monocyte-derived macrophages can amplify dysfunctional responses in various ways (listed in top left corner) . the systemic crs-and shlh-like inflammatory response can induce neutrophilic netosis and microthrombosis, aggravating covid- severity. other myeloid cells such as pdcs are purported to have an ifn-dependent role in viral control. monocyte-derived cxcl / / might recruit nk cells from blood. preliminary data suggest that the antiviral function of these nk cells might be regulated through cross-talk with sars-infected cells and inflammatory monocytes. a decrease in peripheral blood t cells associated with disease severity and inflammation is now well documented in covid- . several studies report increased numbers of activated cd and cd t cells which display a trend towards an exhausted phenotype in persistent covid- , based on continuous and upregulated expression of inhibitory markers as well as potential reduced polyfunctionality and cytotoxicity. in severe disease, production of specific inflammatory cytokines by cd t cells has also been reported. this working model needs to be confirmed and expanded on in future studies to assess virus-specific t cell responses both in peripheral blood and in tissues. in addition, larger and more defined patient cohorts with longitudinal data are required to define the relationship between disease severity and t cell phenotype. abbreviations: il, interleukin; ifn, interferon; tnf, tumor necrosis factor; gm-csf, granulocyte-macrophage colony-stimulating factor; gzma/b, granzyme a/granzyme b; prf , perforin. virus-specific igm and igg are detectable in serum between and days after the onset of symptoms. viral rna is inversely correlated with neutralizing antibody titers. higher titers have been observed in critically ill patients, but it is unknown whether antibody responses somehow contribute to pulmonary pathology. the sars-cov- humoral response is relatively short lived, and memory b cells may disappear altogether, suggesting that immunity with sars-cov- may wane - years after primary infection. the gastrointestinal tract, kidneys and testis have the highest ace expressions. in some organs, different cell types have remarkably distinct expressions, e.g. in the lungs, alveolar epithelial cells have higher ace expression levels than bronchial epithelial cells; in the liver, ace is not expressed in hepatocytes, kupffer cells nor endothelial cells, but is detected in cholangiocytes, which can explain liver injury to some extent. furthermore, ace expression is enriched on enterocytes of the small intestine compared to the colon. ace , angiotensin-converting enzyme ; bnp, b-type natriuretic peptide; crp, creactive protein; il, interleukin; n/l, neutrophil-to-lymphocyte ratio; pt, prothrombin time; aptt, activated partial thromboplastin time. a. rdrp inhibitors (remdesivir, favipiravir), protease inhibitors (lopinavir/ritonavir), and anti-fusion inhibitors (arbidol) are currently being investigated in their efficacy in controlling sars-cov- infections. b. cq and hcq increase the ph within lysosomes, impairing viral transit through the endolysosomal pathway. reduced proteolytic function within lysosomes augments antigen processing for presentation on mhc complexes and increases ctla expression on tregs. c. antagonism of il- signaling pathway and of other cytokine-/chemokine-associated targets has been proposed to control covid- crs. these include secreted factors like gm-csf that contribute to the recruitment of inflammatory monocytes and macrophages. d. several potential sources of sars-cov- neutralizing antibodies are currently under investigation, including monoclonal antibodies, polyclonal antibodies, and convalescent plasma from recovered covid- patients. abbreviations: gm-csf, granulocyte-macrophage colony-stimulating factor; cq, chloroquine; hcq, hydroxychloroquine; rdrp, rna-dependent rna polymerase. tai, w., he, l., zhang, x., pu, j., voronin, d., jiang, s., zhou, y., and du, l. 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immunogenicity of a recombinant adenovirus type- vector-based ebola vaccine in healthy adults in sierra leone: a single-centre, randomised, double-blind potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is enriched in specific cell subsets across tissues testing the association between blood type and covid- infection, intubation, and death. medrxiv. lymphocyte count predicted the disease severity and the outcomes of hospitalized patients prognostic value was confirmed in numerous studies decreased continuously in non-surviving patients were reported to be more likely to develop severe illness and to require intensive care unit (icu) admission on admission a risk factor for short-term progression of patients with moderate pneumonia to severe pneumonia confirmed to be of prognostic value in covid- in several studies even in early stages, crp levels were positively correlated with lung lesions and reflected disease severity confirmed in numerous studies predicted the risk of acute myocardial injury ldh (lactate dehydrogenase) higher in severe cases than in mild cases we apologize to all authors whose work we could not cite due to space limitations. trainee (phd and md/phd or postdocs) and faculty contributing authors are listed in alphabetical order. illustrations by jill k gregory and used with permission of ©mount sinai health system. we would like to acknowledge funding sources including fastgrant (mm), nci cancer center support grant supplement (mm, rms) burroughs wellcome fund (rms), nih director's early independence award (rms) and nih r ai (nv, nb, bdg) purpose ro uti ne bl oo dw or k predicted severity independently of other variables . elevated levels and disseminated intravascular coagulation are found in non-survivors . identified patients at risk for acute cardiac injury . other coagulation parameters such as fibrin degradation product levels, longer prothrombin time and activated partial thromboplastin time, were also associated with poor prognosis (tang et al., a) . saa (serum amyloid protein) saa was proposed to be used as an auxiliary index for diagnosis as it was elevated in % of the patients in a small cohort . nt-probnp (n terminal pro b type natriuretic peptide) nt-probnp was an independent risk factor of in-hospital death in patients with severe covid- . high platelet-to-lymphocyte ratio is associated with worse outcome (qu et al., ) . thrombocytopenia is associated with poor outcome and with incidence of myocardial injury in covid- (liu et al., h; shi et al., ) . im mu nol ogi cal cd +, cd + and nk cell counts lower cd +, cd + and nk cells in pbmc correlated with severity of covid- (nie et al., b) . validated by several studies zheng et al., b) . pd- and tim- expression on t cells increasing pd- and tim- expression on t cells could be detected as patients progressed from prodromal to overtly symptomatic stages (diao et al., ) .expression was higher in infected patients versus healthy controls and in icu versus non-icu patients in both cd and cd t cells . phenotypic changes in peripheral blood monocytes the presence of a distinct population of monocytes with high forward scatter (cd b+, cd +, cd +, cd +, cd +, cd +, cd + which secrete il- , il- and tnf-alpha) was identified in patients requiring prolonged hospitalization and icu admission . cd +cd +il- + monocytes are increased in icu patients . ip- , mcp- , and il- ra ip- , mcp- , and il- ra were, among examined cytokines, the only ones that closely associated with disease severity and outcome of covid- in a study by yang et al. (yang et al., b) . associated with disease severity (hospitalization and icu admission) and poor prognosis (chen et al., f; huang et al., b; liu et al., b . increase levels were associated with higher risk of respiratory failure (yao et al., b) . il- positively correlated with disease severity (chen et al., d; gong et al., ) , with severe cases showing the highest il- levels. increased in severe or critical patients as compared to mild patients zhou et al., d ) without a statistically significant difference between severe and critical cases . associated with disease severity in a study that, amongst other cytokines, also associated ferroprotein levels, pct levels, and eosinophil counts with covid- severity . il- β cd +il- β+ monocytes are abundant in early recovery patients as shown in a single-cell rna-seq analysis and thought to be associated with cytokine storm . il- β did not correlate with disease severity in a cross-sectional study with mild, severe and critical patients . il- il- was associated with impaired lung lesions , but some reports point to a potential mediator effect . in modeling immune cell interaction between dc and b cells in late recovery covid- patients, il- was found to be important in b cell production of antibodies, which suggests its importance in recovery . gm-csf (granulocytemacrophage colony-stimulating factor) gm-csf+ifn-γ+ t cells are higher in icu than non-icu patients, cd +cd +gm-csf+ monocytes are higher in covid- patients as compared to healthy controls .il- and ifn-γ il- and ifn-γ levels were shown to be increased in severe cases . anti-sars-cov- antibody levels prolonged sars-cov- igm positivity could be utilized as a predictive factor for poor recovery . higher anti-sars-cov- igg levels and higher n/l were more commonly found in severe cases . between and days after hospital admission.-body temperature normalized within days in of patients -clinical improvement -viral loads became negative within d after the transfusion -neutralizing antibody titers increased severe patients ( - yo)median . dpo.-disappearance of clinical symptoms after d -chest ct improved -elevation of lymphocyte counts in patients with lymphocytopenia.-increase in sao in all patients -resolution of sars-cov- viremia in patients increase in neutralizing antibody titers in patients (ahn et al., ) key: cord- - ksgl t authors: li, liang; xue, mei; fu, fang; yin, lingdan; feng, li; liu, pinghuang title: ifn-lambda mediates antiviral protection against porcine epidemic diarrhea virus by inducing a distinct antiviral transcript profile in porcine intestinal epithelia date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ksgl t type iii interferon-lambda (ifn-λ) plays a critical role against infection, particularly in mucosal infection in the respiratory and gastrointestinal tract. our study and other previous studies have shown that porcine ifn-λ more efficiently curtails the infection of porcine epidemic diarrhea virus (pedv) in the intestine epithelia than type i ifn, whereas ifn-λ exerts a more potent effect than ifn-λ . however, the underlying mechanism remains elusive, and in particular, the transcriptional profile induced by ifn-λ has not been reported. here, to resolve the mechanism responsible for the disparity between ifn-λ and type i ifn in anti-mucosal virus infection, we compared the transcription profiles induced by the two ifns in porcine intestinal epithelial (ipec-j ) cells by rna-seq. our results showed that the pretreatment of ipec-j cells with ifn-λ resulted in the differential expression of genes. in contrast, ifn-α only modified the expression of genes, and of these genes were also observed in the response to ifn-λ . a transcriptional enrichment analysis indicated that ifn-λ or ifn-α regulates multiple cellular processes and that ifn-λ activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, than ifn-α. furthermore, we verified the rna-seq results through an rt-qpcr analysis of ipec-j cells and porcine enteroids. moreover, transient expression of the porcine rsad and mx genes among the top genes induced by ifn-λ significantly inhibited pedv infection. collectively, the data showed that ifn-λ induces a unique transcriptional profile that does not completely overlap with that induced by ifn-α and strongly elicits a set of genes responsible for the antiviral activity of ifn-λ . these findings provide important knowledge regarding the elicited isgs of type i and iii ifns in restricting porcine intestinal viral infection. type iii interferon-lambda (ifn-λ) plays a critical role against infection, particularly in mucosal infection in the respiratory and gastrointestinal tract. our study and other previous studies have shown that porcine ifn-λ more efficiently curtails the infection of porcine epidemic diarrhea virus (pedv) in the intestine epithelia than type i ifn, whereas ifn-λ exerts a more potent effect than ifn-λ . however, the underlying mechanism remains elusive, and in particular, the transcriptional profile induced by ifn-λ has not been reported. here, to resolve the mechanism responsible for the disparity between ifn-λ and type i ifn in anti-mucosal virus infection, we compared the transcription profiles induced by the two ifns in porcine intestinal epithelial (ipec-j ) cells by rna-seq. our results showed that the pretreatment of ipec-j cells with ifn-λ resulted in the differential expression of genes. in contrast, ifn-α only modified the expression of genes, and of these genes were also observed in the response to ifn-λ . a transcriptional enrichment analysis indicated that ifn-λ or ifn-α regulates multiple cellular processes and that ifn-λ activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, than ifn-α. furthermore, we verified the rna-seq results through an rt-qpcr analysis of ipec-j cells and porcine enteroids. moreover, transient expression of the porcine rsad and mx genes among the top genes induced by ifn-λ significantly inhibited pedv infection. collectively, the data showed that ifn-λ induces a unique transcriptional profile that does not completely overlap with that induced by ifn-α and strongly elicits a set of genes responsible for the antiviral activity of ifn-λ . these findings provide important knowledge regarding the elicited isgs of type i and iii ifns in restricting porcine intestinal viral infection. the surface epithelia of the mucosa are the major entry site of most pathogens in the host and serve as a first line of defense against invading pathogens. one of the most important antiviral cytokines in the host is interferons (ifns), which perform key roles in inhibiting viral infection ( , ) . the ifn family is categorized into three different types: type i ifn (ifn-α/β), type ii ifn (ifn-γ), and type iii ifn (ifn-λ). type ii ifn, which is primarily produced by t cells and natural killer cells, exerts limited direct antiviral activity and plays a key role in modulating the host immune response ( ) , whereas type i ifns (α/β) and the more recently discovered type iii ifns induce a strong antiviral state in responsive cells and play crucial roles in controlling viral infection ( ) ( ) ( ) ( ) ( ) . although type i ifns have generally been thought to be a key element against systemic infections, recent research has shown that ifn-λ plays a critical role in mucosal infections, such as enteric infection ( , ) . unlike type i ifns that are secreted by a wide range of different cell types upon stimulation, type iii ifns are primarily produced by epithelial cells, nk cells, and dendritic cells (dcs) ( , ( ) ( ) ( ) . ifn-λ acts primarily on the mucosal epithelium, which might result in fewer side effects compared with type i ifn treatment ( ) . these features make ifn-λ a potentially superior antiviral therapeutic candidate against local mucosal infection ( ) . although the receptors for type i and iii ifns are different, the binding of both type i and iii ifns to their corresponding receptors stimulates a janus kinase (jak)-signal transducer of transcription (stat) pathway, and the stimulation of this pathways subsequently drives the transcription of ifnstimulated genes (isgs) and prompts cells toward an antiviral status ( ) . consistent with the similarity of the induced signaling pathways, the spectrum of genes elicited by the two types of ifns show a high overlap ( ) . however, recent studies have demonstrated that type iii ifns are critical non-redundant antiviral mediators of type i ifns in the gi tract ( ) . to date, numerous studies in humans or mice have taken advantage of rna-seq or chip assays to show that ifn-λ and ifn-α elicit distinct downstream signaling events, even though many genes are induced by both type i and iii ifns ( , ) . mice with type i ifn or iii ifn receptor knockout experience more severe viral intestinal infections, but ifnl −/− mice show higher viral loads and more serious clinical symptoms than ifnar −/− mice ( , ) . studies conducted by pott et al. showed that intestinal epithelial cells exhibit stronger responses to ifn-λ compared with ifn-α/β in vivo ( , ) . a comprehensive understanding of the unique signaling profiles of type i and iii ifns has become increasingly important for understanding host-virus interactions and the development of ifn-λ therapeutics. however, thus far, no direct comparative analyses of the transcriptional profiles induced by porcine type i vs. type iii ifns in swine intestinal epithelia have been performed. the piglet diarrhea caused by enteric coronavirus porcine epidemic diarrhea virus (pedv) is a highly contagious disease characterized by watery diarrhea, dehydration, and causes up to % mortality in neonatal piglets. we and other research groups previously reported that porcine ifn-λ results in better suppression against pedv infection compared with ifn-α and that ifn-λ more efficiently inhibits pedv than ifn-λ ( ) ( ) ( ) . however, the mechanisms underlying the difference among ifn-λ , ifn-λ , and ifn-α in inhibiting enteric coronavirus remain less clear. previous studies have largely focused on the gene profiles induced by human or mouse ifn-λ and ifn-α, but the ifn-λ -and ifn-α-elicited genes have not been compared. in this study, we comprehensively compared the transcriptional profiling of ifn-λ -and ifn-α-induced genes in a porcine intestinal epithelial cell line (ipec-j ) and verified the rna-seq results by reverse transcriptase quantitative pcr (rt-qpcr) in vitro, and further confirmed the transcriptional profile difference in crypt-derived porcine enteroids. the intestinal porcine epithelial cell line j (ipec-j ; kindly provided by dr. anthony blikslager, north carolina state university, raleigh, nc, usa) was maintained in dulbecco's modified eagle's medium nutrient mixture f- (dmem/f ) supplemented with antibiotics ( units/ml penicillin and µg/ml streptomycin), . mm hepes (gibco, usa), and % heat-inactivated fetal bovine serum (fbs) (gibco). african green monkey kidney cells (vero e ) were grown and maintained in dmem supplemented with antibiotics ( units/ml penicillin and µg/ml streptomycin) and % heat-inactivated fbs (gibco). pedv strain cv of genotype (genbank accession no. kt ) was maintained at the harbin veterinary research institute of the chinese academy of agricultural sciences, harbin. the biological antiviral activity of e. coli-derived recombinant porcine ifn-lambda was prepared in our laboratory and evaluated in mdbk cells using a recombinant vesicular stomatitis virus (vsv) with a gfp reporter as described previously ( , ) . the weight-activity unit (u/ml) of samples was calculated using porcine prokaryotic-derived ifn-α ( . × u/mg) (prosit sole biotechnology, co., ltd., beijing, china) as a reference. porcine intestinal crypts were prepared from specific pathogenfree piglets using previously described protocols ( ) . in brief, the intestine was flushed with cold pbs with antibiotics ( units/ml penicillin and µg/ml streptomycin), cut into mm segments, and washed with cold pbs with antibiotics until the supernatant was clear. the washed intestinal pieces were suspended in ml of gentle cell dissociation reagent (stemcell, canada) and shaken at rpm for min to disassociate the crypts at room temperature (rt). the pellets of the intestinal pieces were suspended in ml of cold pbs with . % bovine serum albumin (bsa) and antibiotics (pen-strep) and passed through a -µm cell mesh. the crypt pellets were harvested by centrifugation at × g at • c for min and resuspended in ml of cold dmem/f . after counting, the intestine crypts were resuspended in µl of intesticult organoid growth medium (stemcell, canada) and µl of matrigel (bd biosciences, usa) per crypts and seeded into a -well plate at crypts per well. the plate was incubated at • c for min until the matrigel solidified. the plate was filled with complete intesticult organoid growth medium and then incubated at • c in a % co incubator. the culture medium was exchanged every - days. the institutional animal care and use committee of the harbin veterinary research institute approved all the protocols related to the animal experiments performed in this study. expanded d enteroids were recovered from the matrigel after - days of growth by the addition of ice-cold dmem/f medium, transferred into -ml tubes, and centrifuged at × g at • c for min. the pellet of enteroids was incubated in . % trypsin (gibco) for min at • c and dissociated by repeated pipetting to obtain a single-cell suspension. dmem-f with % (v/v) fbs was added into the single-cell suspension, and the mixture was centrifuged at × g for min. the cell pellets were resuspended in complete intesticult organoid growth medium at rt and seeded at enteroids per well in a matrigel-precoated -well plate. after differentiation for about - days, planar monolayers of d enteroids were ready for use in experiments. total cellular rna was extracted using the simply p total rna extraction kit (bioflux, china) according to the manufacturer's instructions. total rna ( µg) was reverse-transcribed to cdna using the primescript tm ii first-strand cdna synthesis kit (takara, china). the synthesized cdna was subjected to qpcr performed in triplicate using a lightcycler r ii real-time pcr instrument (roche, switzerland) and sybr green pcr mix (life technologies, usa) according to the manufacturer's instructions. all the data were acquired and analyzed using lightcycler r ii software . based on the cycle threshold ( ct) method ( ) . gapdh served as the internal control. the amplification efficiency of qpcr primers ranged from . to . %. the primers used in this assay were designed using primer premier software and are listed in table . three biological replicates of each of the three groups, namely, untreated ipec-j cells (mock control), ipec-j cells treated with ifn-λ ( , ng/ml) for h, or ipec-j cells treated with ifn-α ( , ng/ml) for h, were prepared for rna sequencing. total rna was purified using the trizol reagent according to the manufacturer's instructions (thermo fisher scientific, usa). the total rna from each sample was quantified and qualified using an agilent bioanalyzer (agilent technologies, usa), a nanodrop instrument (thermo fisher scientific, inc.), and a % agarose gel. one microgram of total rna with a rin value > was used for subsequent library construction. next-generation enzyme mix to repair both ends and add a da tail through one reaction, and the product was subjected to t-a ligation to add adaptors to both ends. size selection of adaptor-ligated dna was then performed using an axyprep mag pcr clean-up kit (axygen), and fragments of ∼ bp (with an approximate insert size of bp) were recovered. each sample was then amplified by pcr for cycles using the p and p primers carrying sequences that can anneal during bridge pcr with a flowcell and the p primer carrying a six-base index to allow multiplexing. the pcr products were cleaned up using the axyprep mag pcr clean-up kit (axygen), validated using an agilent bioanalyzer (agilent technologies, usa), and quantified with a qubit . fluorometer (invitrogen, usa). libraries with different indices were then multiplexed and loaded on an illumina hiseq instrument (illumina, usa) according to the manufacturer's instructions. sequencing was performed using a × -bp paired-end (pe) configuration and image analysis and base calling were conducted using hiseq control software (hcs) + olb + gapipeline- . (illumina) provided with the hiseq instrument. rna-seq was performed and analyzed using genewiz (suzhou, china). the analysis of the microarray data to identify differentially expressed genes was performed using edger software. the analysis starts with the count, and the data are standardized by tmm and then subjected to differential expression analysis. we selected | log (fold change) (logfc) | > and fdr < . as the screening criteria for the identification of differentially expressed genes. ipec-j or vero e cells were fixed with % paraformaldehyde for min at rt. the fixed cells were permeabilized with . % triton x- for min at rt and then blocked with blocking buffer (pbs with % fbs and % skim milk) for min at • c. the cells were then incubated with pedv n protein antibody at • c for h and then labeled with alexa fluor goat anti-mouse igg antibody (thermo fisher scientific, usa) at • c for h. dapi (sigma, usa) was used for the staining of cellular nuclei. the stained cells were visualized using an amg evos f fluorescence microscope (thermo fisher scientific, usa). the porcine rsad or mx coding region was amplified by rt-pcr, and cdna was prepared using the primescript tm ii st strand cdna synthesis kit (takara, china). rsad or mx was then amplified using a pair of primers specific for porcine rsad or mx (table ) , respectively. the pcr products were purified, digested, and cloned into pcaggs-ha through ecor i and kpn i. construction of the prsad /pmx -ha expression plasmid was confirmed by sequencing. vero e cells were grown in -well plates to - % confluence and then transfected with the prsad /pmx -ha expression plasmid using the attractene transfection reagent (qiagen), and the expression of prsad /pmx was verified by anti-ha ifa. graphpad prism software version was used to analyze the experimental results. the results are expressed as scatter plots in which the mean is indicated by a line. the differences between groups were compared using an unpaired mann-whitney test. p < . was considered significant, and the p-values are expressed as follows: * p < . , * * p < . , * * * p < . , and * * * * p < . . the inhibition of pedv in ipec-j cells by exogenous ifn-λ is superior to that achieved with ifn-α according to our study and other previous studies ( ) ( ) ( ) , ifn-λ , ifn-λ , and ifn-α all significantly inhibit pedv infection, but ifn-λ shows the strongest antiviral activity against pedv. to confirm this disparity in the antiviral activities of ifn-λ and ifn-α against pedv, ipec-j cells were primed with ifn-α ( , ng/ml) or ifn-λ ( , ng/ml) for h and then infected with pedv (moi = ). consistent with our previous results, both ifn-α and ifn-λ substantially suppressed pedv infection in ipec-j cells, as demonstrated by measurements of the viral genomes and titers by rt-qpcr ( figure a ) and tcid titration (figure b) , respectively. ifn-α reduced the number of pedv genomes by -fold, whereas ifn-λ decreased the number of pedv genomes by approximately -fold. the virus titers were consistent with results of pedv genomes: pedv titers decreased and -fold after pretreatment with ifn-α or ifn-λ , respectively. the inhibition of pedv infection by both ifnα and ifn-λ was further verified by pedv n protein ifa ( figure c) . the virus-infected cells were significantly decreased after pretreatment with ifn-α or ifn-λ , whereas the number of pedv-infected cells in the ifn-λ -pretreated group was significantly decreased, as demonstrated by only a few sporadically distributed cells, compared with those in the ifnα-pretreated group. thus, ifn-λ restricted pedv infection in ipec-j cells more effectively than ifn-α, regardless of the quantification of viral rna, infectivity, or viral protein (figure ). to assess the underlying mechanisms of the disparity between the anti-pedv responses induced by ifn-α or ifn-λ , we performed an rna-sequencing (rna-seq) analysis of total cellular rna isolated from ipec-j cell cultures stimulated with ifn-α or ifn-λ for h and ifn untreated ipec-j mock control. this duration of stimulation ( h) was selected based on the efficacy of viral inhibition after exposure to ifn-λ and ifn-α (figure ) ( ) . each of the triple replicate yielded more than . × clean reads and has more than % q (%), indicating the reliability of the rna-seq data. the ifn-λ stimulated cells showed a total of differentially expressed genes, including upregulated genes and downregulated genes, compared with the mock control, whereas ifn-α only upregulated the expression of genes among the total of differentially expressed genes and reduced the expression of four genes (figure a) . the number of ifn-λ -modified genes was approximately -fold higher than that of ifn-αmodified genes. these results indicate that the intestine epithelia respond better to ifn-λ than to ifn-α. we further grouped the corresponding genes through supervised partitioning clustering and combined the differentially expressed genes induced by ifn-λ and ifn-α to obtain the heat map ( figure b ) and the venn map ( figure c ). ifn-λ yielded different gene expression profiles compared with that induced by ifn-α, even though these showed substantial overlap ( figure b) . one hundred ten genes were upregulated in both the ifn-λ -and ifn-α-treated cells, whereas and genes were uniquely upregulated in the presence of ifn-λ and ifn-α, respectively ( figure c) . none of the coexpression genes were downregulated in both the ifn-λ -and ifn-α-treated cells, whereas and genes were only downregulated in the presence of ifn-λ and ifnα, respectively. these results demonstrated that ifn-λ induces a unique gene transcriptional profile in the intestine epithelia compared with ifn-α. to further clarify the functional consequences of the gene profiles elicited by either ifn-λ or ifn-α, we performed a gene ontology (go) enrichment analysis using a database established by the gene ontology consortium, which aims to define and describe the functions of genes and proteins in various species (figure ) ( ) . among the genes upregulated by ifn-λ , and genes ( . and . % of the total genes, respectively) were associated with a binding function and catalysis, respectively, and these two functions account for the main molecular functional changes. the dominant functions of the ifn-α-regulated genes are cellular transporter activity and nucleic acid-binding transcription factor activity, and these functions were associated with genes ( . % of total genes) and genes ( . % of total genes), respectively. the analysis of cellular components revealed that differentially expressed genes in the ifn-λ -treated group affected the cell part, and genes associated with this cellular component were differentially expressed in the ifn-α-treated group. with respect to biological processes, the ifn-λ -treated group included , , , , and differentially expressed genes associated with the cellular process, the single-organism process, biological regulation, the metabolic process, and response to stimulus, respectively. the pattern obtained for the ifn-α-treated group was similar to that of the ifn-λ -treated group, and the differentially expressed genes were also concentrated on the following five functions: the cellular process, the single-organism process, the biological regulation, the metabolic process, and the response to stimulus. however, the numbers of differentially expressed genes after ifn-α treatment were notably lower than those obtained after ifn-λ treatment. these results indicated that both ifn-λ and ifn-α are involved in the regulation of multiple cellular processes in ipec-j cells, such as cellular components and molecular functions, but ifn-λ exerts more potent effects than ifn-α. to further investigate the function of genes specifically induced by ifn-λ , we extracted the transcriptional profiles of ifn-λ and ifn-α regarding biological processes (p < . ) and combined them based on the -log (p) values to obtain a heat map that showed the enrichment of biological processes ( figure s ). the data demonstrated that in ipec-j cells, ifn-λ stimulation triggered more biological reaction processes than ifn-α, and these processes mainly involved the modulation of metabolic processes, including cellular metabolic processes, macromolecular metabolic processes, and primary metabolic regulation. taken together, these results indicate that the differentially expressed genes induced by ifn-λ are involved in more intracellular biological processes than those induced by ifn-α. to explore the clustering of the ifn-induced differential expression genes in anti-viral signaling pathways, we performed a kegg enrichment analysis of the differentially expressed genes using kobas . . the kegg enrichment analysis revealed that the differentially expressed genes induced by ifn-λ involved in much broader signal pathways compared with ifn-α though they both primarily modified the nf-κb signaling pathway, the jak-stat signaling pathway, the phosphoinositide- -kinase-akt (pi k-akt) signaling pathway, the mitogen-activated protein kinase (mapk) signaling pathway, and the cgmp-pkg signaling pathway ( figure a) . to determine the signaling pathways involving the differentially expressed genes induced by ifn-λ or ifn-α, we combined the differentially expressed genes and analyzed their expression patterns ( figure b) . the results showed that the differentially expressed genes were most abundantly involved in the jak-stat signaling pathway, followed by the pi k-akt signaling pathway and the mapk signaling pathway, which is consistent with the finding that the jak-stat signaling pathway primarily mediates ifn-induced antiviral responses. among the proteins encoded by the differentially expressed genes, sos , pik ca, jak , il- , socs , il- b, stat , il ra , and socs play a major role in the jak-stat signaling pathway, and kras, pik ap , prkaa , enssscg , enssscg , and map k play a major role in the pi k-akt signaling pathway. to predict the protein interactions between differentially expressed genes, we extracted the union of differentially expressed genes and introduced these into the web-based tool string (http:// www.string-db.org/) to generate protein-protein interaction networks ( figure c) . the results of the string analysis indicated that jak , stat , pten, pik ca, irs , kras, and il st are closely related to other proteins and displayed significant differential expression compared with the ifn-αinduced proteins, which play critical roles in the innate immune response induced by ifn. collectively, the results showed that the ifn-λ -induced differentially expressed genes are involved in more signaling pathways, particularly those associated with innate immunity, than those induced by ifn-α, which indicates that ifn-λ exhibits stronger antiviral activity in intestinal epithelial cells. to further elucidate the mechanisms underlying the antiviral activity discrepancy between ifn-λ and ifn-α, we focused on the top ifn-λ -induced genes (fold change compared with the mock control) and divided them into three subgroups (classical isgs, weakly ifn-λ -induced genes, and strongly ifn-λ -induced genes) as in a previous study ( ) . the expression of the top genes in all three subgroups induced by ifn-λ was significantly higher compared with that induced by ifn-α ( figure ) . the isgs in the classic isg subgroup are primarily the classical isgs induced by ifn reported in the literature ( , ) . the levels of isgs in this subgroup induced by ifn-λ were from -to -fold higher than those induced by ifnα. the weakly ifn-λ -induced gene subgroup contained genes, including three unknown genes (being denoted un , un , and un ). the analysis of this subgroup showed that both ifn-λ and ifn-α induced a more than -fold increase in the expression of isgs compared with mock control. the ifn-λ -induced expression levels of ifit , oasl, oas , and gbp , which are important innate immunity factors, were fold higher than those induced by ifn-α. the expression of of the genes in the strongly ifn-λ -induced gene subgroup was strongly induced by ifn-λ , whereas ifn-α did not induce a substantial increase in expression (< -fold compared with the mock control). interestingly, radical s-adenosyl methionine domain containing (rsad ), a multifunctional protein with broad antiviral activity that can inhibit both dna and rna viruses, is the top gene upregulated by ifn-λ ( ) . in contrast, ifn-α did not substantially upregulate rsad expression. protein interaction prediction analysis of differentially expressed genes by string database. the relevant genes were input into string (http://www.string-db. org/), the active interaction sources were selected as homology, and the experimentally determined interaction and database annotated were used as predictive conditions for protein interaction prediction. the thickness of the line represents combined score, which is the combined score of the prediction results of the three prediction conditions, indicating the strength of data support. several other genes (ifit , parp , and gbp ) in this subgroup are also important innate viral restriction factors ( , ( ) ( ) ( ) . thus, ifn-λ induces a stronger antiviral innate immune response compared with ifn-α. to verify the unique antiviral gene expression profile induced by ifn-λ that was detected by rna-seq, as shown in figure , we randomly selected three genes from each group to be verified by rt-qpcr. the analysis of the three subgroups confirmed that ifn-λ most substantially upregulated the expression of isgs (figure ) . specifically, ifn-λ upregulated the expression of large distinct classes of isgs in the classical isg subgroup, such as mx , isg , and ifit , the isg fold changes induced by ifn-λ were up to nearly -fold higher than those induced by ifn-α ( figure a ). the analysis of the weakly ifn-λ induced gene subgroup showed that the expression of oasl, oas , and isg (a) was induced by both ifn-λ and ifnα to significantly different levels. the oasl and oas fold changes induced by ifn-λ were up to -fold greater than those induced by ifn-α ( figure b ). more substantial fold changes in the expression of isgs in the strongly ifn-λ -induced gene subgroup were obtained with ifn-λ compared with ifn-α, which resulted in only slight changes in expression ( figure c ). the differences between groups were compared using an unpaired mann-whitney test. p < . was considered significant, and the p values are expressed as follows: *p < . , **p < . , ***p < . , and ****p < . . rsad , plac , and ifit were more substantially upregulated by ifn-λ than by ifn-α. collectively, the in vitro rt-qpcr results confirmed that ifn-λ induces stronger isg expression compared with ifn-α. enteroids derived from intestinal crypt stem cells, which mimic the diverse cellular nature and physiological activity of the small intestine while also maintaining the genetic identity of the host, constitute a unique ex vivo model for studying the intestine ( , ) . to further confirm whether the ifn-λ induced gene expression profile in enteroids was the same as that in ipec-j cells, we stimulated enteroids with ifn-λ or ifn-α under the same conditions as ipec-j cells and evaluated their gene expression by rt-qpcr. in all three groups, the expression pattern of the genes induced by ifn-λ and ifn-α in porcine enteroids was consistent with that found in the ipec-j cells (figure ) . in the classical isg subgroup, the upregulated levels of mx and ifit elicited by ifn-λ were ∼ -to -fold higher than those induced by ifn-α ( figure a ). in contrast, the upregulated levels of oasl and oas , which belong to the weakly ifn-λ -induced gene subgroup, induced by ifn-λ were -fold higher than those induced by ifn-α, and the fold change difference was moderate ( figure b) . for the strongly ifn-λ -induced gene rsad and plac , just as we observed in the other subgroups, enteroids were or were not primed with ifn-λ ( , ng/ml) or ifn-α ( , ng/ml) for h, and the total rna from the enteroids was extracted and used for rt-qpcr. the differences between groups were compared using an unpaired mann-whitney test. p < . was considered significant, and the p values are expressed as follows: *p < . , **p < . , ***p < . , and ****p < . . ifn-λ significantly induced higher expression than ifn-α does ( figure c ). in summary, ifn-λ induces higher expression of isgs than ifn-α in porcine enteroids, which suggests that ifn-λ exerts a greater effect in gastrointestinal epithelial cells than ifn-α. pmx and prsad inhibit pedv infection in vero e cells we selected mx (classical isg) and rsad (strongly ifn-λ induced gene) among the top genes induced by ifn-λ to evaluate the antiviral effect of the ifn-λ -induced expression of isgs against pedv infection. we cloned porcine mx or rsad into the eukaryotic expression vector pcaggs-ha, and the transient expression of pmx or prsad in vero e cells was verified by ifa (data not shown). as expected, pmx or prsad transient overexpression significantly inhibited pedv infection in vero e cells, as demonstrated by measuring the viral rna (figures a,c) and pedv n protein expression by ifa (figures b,d) . thus, these data indicate that the differentially upregulated pmx or prsad serves as an important ifn-λ elicited antiviral host factor against pedv. type i and type iii ifns, which establish a cellular antiviral state and restrict viral infection in the host, are key players at the earliest stages of innate immunity against viral infection. despite the similarities between the effects of the two types of ifns, increasing evidence demonstrates that each class of ifn is essential for antiviral host defense and is not functionally redundant ( ) . a previous study in mice demonstrated that the role of ifn-λ in functionally redundant intestinal viral infections cannot be compensated by ifn-α/β ( ) . therefore, clarification of the induction of cell-specific ifn signaling profiles is essential for understanding the non-redundant roles of ifn-λ and ifn-α in viral infection at the tissue and organism levels. in this study, we found that the transcriptional profile induced by type iii ifn in the intestinal epithelia the differences between groups were compared using an unpaired mann-whitney test. p < . was considered significant, and the p values are expressed as follows: *p < . , **p < . , ***p < . , and ****p < . . was unique compared with that induced by type i ifn. compared with ifn-α, stimulation with ifn-λ not only resulted in higher levels of most isgs but also substantially increased diversity in the gene profiles involved in various cellular functions. type i and iii ifns each signal through different heterodimeric complex receptors to initiate multiple downstream signaling pathways. in addition to activation of the jak-stat signaling pathway, ifns also activate the pi k and mapk signaling cascades ( , ) . the combined use of these signaling pathways by ifns and many other cytokines might help explain the different roles of ifns in regulating the antiviral and immune responses in a variety of environments and locations. we have very limited knowledge on the pathways activated by ifn-λ, which are important for understanding the immune-modulating activities of ifn-λ ( ) . in this study, we found that ifn-λ not only activates the classical antiviral response jak-stat signaling pathway but also primarily activated the nf-κb signaling pathway, the camp signal pathway, the pi k-akt signaling pathway, and mapk signaling pathway (figure ) . ifn-λ activated much more signaling pathways in epithelia than ifn-α, and this finding might provide new insights into the mechanism through which ifn-λ modulates other cellular functions beyond its direct antiviral activity. ifn-λ largely stimulated wnt signaling pathway in iec compared with ifn-α, which plays critical roles in maintaining the homeostasis of intestinal epithelia in vivo ( ) . further studies of different signaling pathways induced by ifn-λ or ifn-α will help dissect the complex interaction of ifn-induced signaling pathways with similar or overlapping intracellular signaling pathways stimulated by other cytokines. most somatic cells can induce and respond to type i ifns, but certain specialized cells, such as those in the mucosal epithelia, selectively produce and respond to type iii ifns during various virus infections. we demonstrated that the porcine intestinal epithelia respond to both type i and iii ifns, even though these ifns induced different isg expression levels. type iii ifns comprise four functional and highly homologous subtypes, namely, ifn-λ , ifn-λ , ifn-λ , and ifn-λ , in humans. our study and other studies have revealed differences in antiviral activity among different ifn-λ subtypes ( , , ) . ifn-λ is superior or equal to ifn-λ in terms of its antiviral activity in the intestinal epithelia ( ) and the immortalized liver cell line hepg in vitro ( ) . the different kinetics and magnitude of isg induction after stimulation might account for the variation in the antiviral activity among different types or subtypes ifns. previous studies that attempted to identify the gene profiles induced by type iii ifns primarily focused on the comparison of ifn-λ with type i ifn ( ) , and most of these studies were performed in immortalized human or mouse cell lines. the gene transcription profile induced by ifn-λ , particularly the gene transcription profile induced by ifn-λ in porcine intestinal epithelial cells, has not been reported. here, we performed comparative analyses of the transcriptional profiles induced by ifn-λ and ifn-α in porcine intestinal epithelia cells. more importantly, in this study, we verified the rna-seq results in primary swine crypt-derived enteroids, an in vitro system that well recapitulates the complicated cellularity of the gi tract, which exhibits a potent response to both type i and iii ifns ( , , ) . the differential expression in ipec-j or enteroids detected by qpcr is not related to the selection of housekeeping gene gapdh since we observed the same pattern when using another housekeeping gene actin (data not shown). therefore, studying the differences among ifn-λ -and ifn-α-induced gene expression in enteroids is more realistic and clinically significant. the different antiviral activities of ifn-λ and ifn-α are likely due to the different degrees of isg induction by these two cytokines, and ifn-λ induced increased isg expression. the functions of some of the genes that were strongly induced by ifn-λ have been reported, but the functions and mechanisms of more genes remain unclear. rsad , a host viral restrictor gene, showed the highest upregulated levels after ifn-λ rather than ifn-α stimulation, whereas mx was the most highly upregulated gene after ifn-α stimulation. a previous study demonstrated that porcine rsad effectively inhibited csfv replication in vitro, and this effect might occur via the interaction of rsad with csfv e protein in the cytoplasm ( ) . our study found that rsad transient expression also substantially curtailed pedv infection in vero e cells in vitro, but the mechanism is unclear. mx , an ifn-induced gtpbinding protein ( , ) , reportedly inhibits the replication of lentivirus hiv- , simian immunodeficiency virus (siv), and equine infectious anemia virus (eiav) in vitro ( , ) . the function of porcine mx has been poorly studied. we confirmed that porcine mx inhibits pedv infection in vero e cells. we also discovered some unknown genes that were differentially expressed in response to ifn-λ or ifn-α stimulation, and this finding might provide a foundation for further elucidation of the different mechanisms of action of ifn-λ and ifn-α. in summary, we compared the transcriptional profiles of ifn-λ and ifn-α in ipec-j cells and identified a unique set of genes that were strongly induced by ifn-λ compared with ifn-α. the transcriptional enrichment analysis indicated that ifn-λ or ifn-α is involved in the regulation of cellular processes, such as cellular components and molecular functions, in ipec-j cells, and that ifn-λ activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, compared with ifn-α. ifn-λ preferentially upregulates antiviral genes in epithelial cells compared with ifn-α. these results indicate that ifn-λ selectively targets small intestinal epithelial cells and induces a non-redundant antiviral host response at the enteric mucosal site. all datasets generated in this study are included in the manuscript/supplementary material. the animal study was reviewed and approved by the harbin veterinary research institute of the chinese academy of agricultural sciences, harbin. written informed consent was obtained from the owners for the participation of their animals in this study. pl, ll, and lf designed the research studies and analyzed and interpreted the data. ll, mx, ff, and ly conducted experiments and acquired data. ll and pl drafted the manuscript and all authors contributed revisions. funding support for this work was provided by grants from the national key r&d program of china ( yfd ) and the national natural science fund ( ). the content is solely the responsibility of the authors and does not necessarily represent the official views of the funding resources. ifn-lambda decreases murid herpesvirus- infection of the olfactory epithelium but fails to prevent virus reactivation in the vaginal mucosa type iii interferons in antiviral defenses at barrier surfaces cell type-specific signaling in response to interferon-gamma interferon-lambdas: frontline guardians of immunity and homeostasis in the respiratory tract interferon lambda genetics and biology in regulation of viral control. front immunol lambda interferon is the predominant interferon induced by influenza a virus infection in vivo interferon-lambda: a potent regulator of intestinal viral infections. front immunol type iii interferons in viral infection and antiviral immunity interferon lambda protects the female reproductive tract against zika virus infection induction and function of type i and iii interferon in response to viral infection interferon induction and function at the mucosal surface ifn-alpha receptor- upregulation in pbmc from hcv naive patients carrying cc genotype possible role of ifn-lambda interferon-lambda: immune functions at barrier surfaces and beyond type iii interferon (ifn) induces a type i ifn-like response in a restricted subset of cells through signaling pathways involving both the jak-stat pathway and the mitogen-activated protein kinases distinct and overlapping genomic profiles and antiviral effects of interferon-lambda andalpha on hcv-infected and noninfected hepatoma cells identification of a predominantly interferon-lambda-induced transcriptional profile in murine intestinal epithelial cells commensal microbes and interferon-lambda determine persistence of enteric murine norovirus infection type iii interferon-mediated signaling is critical for controlling live attenuated yellow fever virus infection in vivo diverse intracellular pathogens activate type iii interferon expression from peroxisomes ifn-lambda determines the intestinal epithelial antiviral host defense porcine intestinal enteroids: a new model for studying enteric coronavirus porcine epidemic diarrhea virus infection and the host innate response ifn-lambda preferably inhibits pedv infection of porcine intestinal epithelial cells compared with ifn-alpha type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp in irf signaling establishment of a stable cho cell line with high level expression of recombinant porcine ifn-beta analyzing real-time pcr data by the comparative c(t) method gene ontology: tool for the unification of biology. the gene ontology consortium a family of ifn-gamma-inducible -kd gtpases protects against bacterial infection porcine viperin protein inhibits the replication of classical swine fever virus (csfv) in vitro decreased ifit expression promotes gastric cancer progression and predicts poor prognosis of the patients ifit is a restriction factor in rabies virus pathogenicity structure, function and inhibition of poly(adp-ribose)polymerase, member (parp ) regulation of type i interferon responses sustained in vitro intestinal epithelial culture within a wnt-dependent stem cell niche human interferon-lambda is a potent member of the type iii interferon family expression of ifnlr on intestinal epithelial cells is critical to the antiviral effects of interferon lambda against norovirus and reovirus cdna structures and regulation of two interferon-induced human mx proteins human mxb protein, an interferon-alpha-inducible gtpase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope equine myxovirus resistance protein restricts lentiviral replication by blocking nuclear uptake of capsid protein the interferon-inducible mxb protein inhibits hiv- infection the authors thank dr. xiangxi pei (northeast agricultural university, harbin, china) for assistance and advice in rna-seq data analysis. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material figure s | the heat map of ifn-λ and ifn-α transcriptional profile biological process enrichment analysis. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © li, xue, fu, yin, feng and liu. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - lpe hg authors: kageyama, y.; aida, k.; kawauchi, k.; morimoto, m.; ebisui, t.; akiyama, t.; nakamura, t. title: jinhua qinggan granule, a chinese herbal medicine against covid- , induces rapid changes in the plasma levels of il- and ifn-γ date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: lpe hg background: currently, effective vaccines or specific therapeutic agents against covid- are not available. however, in china, traditional chinese herbal medicines have provided therapeutic benefit to patients with covid- . jinhua qinggan granule (jhqgg) is a chinese multi-herbal formula previously developed for the treatment of h n influenza and has been encouraged for patients clinically suspected of covid- during medical observation. however, the immunological mechanism for the efficacy of jhqgg has not been confirmed. objectives: we thus examined whether the administration of jhqgg affects hematological and immunological measures in healthy individuals. method: we enrolled healthy volunteers, all of whom tested negative for antibodies to sars-cov- . peripheral blood samples were collected h after oral administration of jhqgg and subjected to hematological, biochemical, and cytokine tests. results: jhqgg rapidly induced a significant decrease in the plasma level of il- and an increase in the plasma level of ifn-{gamma}. conclusions: our finding suggests that the therapeutic efficacy of jhqgg against covid- is, in part, associated with its rapid immunomodulatory activity. there have been no specific vaccines or drugs proven to be clinically effective against covid- . however, in china, the majority of covid- patients have been treated with a combination of traditional chinese and modern western medicine [ ] [ ] [ ] . the use of several chinese herbal formulas is encouraged for the treatment of covid- in the latest version of the diagnosis and treatment protocol released by the national health commission of china. one of them is jinhua qinggan granule (jhqgg), which was formulated specifically for the symptoms of h n influenza [ ] . previous preclinical studies showed that jhqgg reduces pulmonary lesions and mortality in mice infected with the h n influenza virus [ ] . a clinical study also demonstrated that jhqgg reduces the duration of fever and alleviates respiratory symptoms of patients with h n influenza [ ] . on the basis of its therapeutic efficacy for influenza, jhqgg has been recommended for patients clinically suspected of covid- during medical observation. in a randomized controlled trial using mild cases in wuhan, combined administration of jhqgg with western medicine significantly ameliorated respiratory symptoms and relieved psychological anxiety compared with the administration of western medicine alone [ ] [ ] [ ] . however, the pharmacological mechanism underlying the efficacy of jhqgg has not been confirmed. we thus examined whether jhqgg administration affects hematological and immunological measures in healthy individuals. this is an open-label, single-arm study to obtain a clue to the pharmacological action of jhqgg. we enrolled a total of healthy volunteers ( males, females; ages - years; mean age [sd], . [ . ] years), all of whom tested negative for igm and igg antibodies to sars-cov- . individuals were excluded if they had current infectious, inflammatory, or immune-related diseases. jhqgg was kindly provided by hugh wang, juxiechang (beijing) pharmaceutical co., ltd. the subjects were instructed to take a packet ( g) orally min after lunch. this dose is known to be effective for the treatment of influenza [ ] . peripheral blood samples were obtained h after the administration. hematological and biochemical tests were outsourced to srl, inc. (tokyo, japan). plasma all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint cytokines were quantified using v-plex proinflammatory panel human kit (meso scale diagnostics) and human il- elisa kit (abcam). all collected data (n = ) were subjected to the statistical analysis using a two-tailed paired t-test. hematocrit and mean corpuscular volume changed marginally within the normal ranges (table ) , but there were no significant differences in other measures of complete blood count and blood biochemistry between pre-and post-administration. notably, in blood cytokine analysis, the plasma levels of il- and ifn-γ were significantly decreased and increased, respectively, compared with those in pre-administration (il- , . vs. . , % ci: - . to - . , p = . ; ifn-γ, . vs. . , % ci: . - . , p = . ). the plasma il- was decreased in ( . %) out of subjects, whereas the plasma ifn-γ was increased in ( . %) out of subjects (fig. ) . we also found a relatively large decrease in the il- level; however, the difference was not statistically significant ( vs. , % ci: - - . , p = . ). dysregulated cytokine production is a hallmark of patients with covid- . elevated blood levels of il- , soluble il- receptor (sil- r), il- , il- , il- , and tnf-α have been observed especially in severe cases [ ] [ ] [ ] [ ] . in particular, il- plays pivotal roles in the exacerbation of covid- . evidence is accumulating that an aberrant increase in il- leads to complex immune dysregulation caused by sars-cov- infection. the blood il- level is positively correlated with the severity and mortality in covid- patients [ ] [ ] [ ] . ifn-γ is a central mediator of antiviral immunity with an ability to directly interfere with viral replication. in addition, ifn-γ is indirectly involved in viral clearance through potentiating the action of ifn-α/β, activating th -dependent immune responses, and enhancing the mhc class i pathway [ ] . compared to healthy individuals, covid- patients have significantly lower numbers of cd + t, cd + t, and nk cells with reduced capacity to produce ifn-γ, which causes the slightly decreased expression of ifn-γ [ , ] . notably, the reduced cytotoxic potential of cd + t, cd + t, and nk cells is il- -dependent [ ] and severe covid- cases have a higher il- /ifn-γ ratio than moderate cases [ ] . in conclusion, jhqgg can down-and up-regulate the plasma levels of il- and ifn-γ, respectively, immediately after oral administration. the rapid immunomodulatory effects of jhqgg may be able to remedy the immune dysregulation observed in covid- patients all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint and thus provide therapeutic benefit to mild to severe cases as well as asymptomatic or suspected cases. we thank hugh wang [juxiechang (beijing) pharmaceutical co., ltd.] for generously providing us with jhqgg. this study was carried out in accordance with the code of ethics of the world medical association (declaration of helsinki). all procedures were approved by the ethics committees of the takanawa clinic (approval number: - ). a signed informed consent form was obtained from each participant prior to inclusion in this study. all experimental procedures and data analyses were conducted by investigators who were blinded to the subjects' clinical information using a de-identified dataset. this study has been registered on under the trial number umin . this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. ebisui contributed to the conception and design of the study, contributed to data acquisition, analysis, and interpretation, and critically revised the manuscript for important intellectual content. tetsu akiyama contributed to the conception and design of the study and critically revised the manuscript for important intellectual content. tsutomu nakamura contributed to the conception and design of the study, contributed to data analysis and interpretation, and drafted the manuscript. all authors gave final approval of the submitted version of the manuscript and agree to be accountable for all aspects of the work. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cao x. covid- : immunopathology and its implications for therapy. nat rev all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint high-density lipoprotein; ldl: low-density lipoprotein; crp: c-reactive protein; ifn: interferon; il: interleukin; tnf-α: tumor necrosis factor-α; sd: standard deviation; ci: confidence interval. *p < . , **p < . . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . pre post all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint traditional chinese medicine for covid- treatment traditional chinese medicine in the treatment of patients infected with -new coronavirus (sars-cov- ): a review and perspective the important role of polysaccharides from a traditional chinese medicine-lung cleansing and detoxifying decoction against the covid- pandemic complementary and alternative medicine is expected to make greater contribution in controlling the prevalence of influenza herbal medicine for the treatment of coronavirus disease (covid- ): a systematic review and meta-analysis of randomized controlled trials efficacy and safety of integrated traditional chinese and western medicine for corona virus disease (covid- ): a systematic review and meta-analysis the clinical benefits of chinese patent medicines against covid- based on current evidence treating influenza patients of windheat affecting fei syndrome by jinhua qinggan granule: a double-blinded randomized control trial. zhongguo zhong xi yi jie he za zhi clinical features of patients infected with novel coronavirus in wuhan clinical and immunological features of severe and moderate coronavirus disease complex immune dysregulation in covid- patients with severe respiratory failure imbalanced host response to sars-cov- drives development of covid- viral and host factors related to the clinical outcome of covid- molecular mechanisms of ifn-γ to up-regulate mhc class i antigen processing and presentation impaired immune cell cytotoxicity in severe covid- is il- dependent high il- /ifn-γ ratio could be associated with severe disease in covid- patients key: cord- -x xo t authors: theresine, maud; patil, neha d.; zimmer, jacques title: airway natural killer cells and bacteria in health and disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: x xo t natural killer (nk) cells are innate lymphoid cells at the interface between innate and adaptive immunity and mostly studied for their important roles in viral infections and malignant tumors. they can kill diseased cells and produce cytokines and chemokines, thereby shaping the adaptive immune response. nowadays, nk cells are considered as a strong weapon for cancer immunotherapy and can for example be transduced to express tumor-specific chimeric antigen receptors or harnessed with therapeutic antibodies such as the so-called nk engagers. whereas a large body of literature exists about the antiviral and antitumoral properties of nk cells, their potential role in bacterial infections is not that well delineated. furthermore, nk cells are much more heterogeneous than previously thought and have tissue-characteristic features and phenotypes. this review gives an overview of airway nk cells and their position within the immunological army dressed against bacterial infections in the upper and predominantly the lower respiratory tracts. whereas it appears that in several infections, nk cells play a non-redundant and protective role, they can likewise act as rather detrimental. the use of mouse models and the difficulty of access to human airway tissues for ethical reasons might partly explain the divergent results. however, new methods are appearing that are likely to reduce the heterogeneity between studies and to give a more coherent picture in this field. historically, human natural killer (nk) cells have mostly been harvested from and studied in peripheral blood (pb), which is an easy way to access them, and where they usually represent - % of all lymphocytes ( ) ( ) ( ) . two different subsets have been initially described, called cd bright cd − (up to % of pb nk cells) and cd dim cd bright (at least % of pb nk cells). phenotypic and functional (cytokine production for the former and cytotoxic activity for the latter) characteristics distinguish both populations ( ) ( ) ( ) . however, things are not that simple, as four additional subpopulations have been identified, which are (i) cd bright cd dim , (ii) cd dim cd − , (iii) cd − cd bright and finally (iv) cd dim cd dim ( ) , the latter still being almost systematically overlooked in the literature ( ) . human nk cell functions are governed by a balance between the messages transmitted through inhibitory receptors (ir), such as kir, cd /nkg a, ilt , tigit, and activating receptors (ar), such as particularly nkg d and the natural cytotoxicity receptors (ncr) nkp , nkp , and nkp ( ). when stimulated, nk cells exert natural cytotoxic activity against tumor cells and virally infected cells, antibody-dependent cellular cytotoxicity (adcc) toward antibody-coated target cells via the fcγ receptor cd , and cytokine and growth factor production ( , ) . most of the ligands of the ir are represented by human leukocyte antigen (hla) class i molecules, so that target cells lacking those molecules in part or in total, become killed by the nk cells. the ir nevertheless have another important function, as they are responsible for nk cell education. indeed, before a developing nk cells becomes functional, its self-specific ir must interact with their ligands expressed by cells in their micro-environment ( , ) . nk cells without such ir, which can represent up to % of all pb nk cells, remain uneducated, and hyporesponsive ( , ) . however, they can be activated under certain conditions, such as some viral infections ( ) . a hot topic in the nk cell field is of course their potential use as immunotherapeutic anticancer agents. to reach this aim, several approaches are studied, and for example the chimeric antigen receptor (car) nk cells, which allow the specific targeting of a tumor antigen ( ) , or the use of multi-specific antibody constructs directed simultaneously at several nk cell ar and tumor surface molecules ( ), appear as particularly promising. it has also been discovered that nk cells, which had been previously considered as exclusively innate immune cells, can develop a memory-like behavior ( ) . finally, nk cell metabolism, which appears to be different between educated and uneducated cells, is extensively studied ( , ) . another aspect that has changed our view on nk cells in recent years is the observation of a broad heterogeneity of this population. not only are there many subsets in pb based on the clonal distribution of several ir, immature, partly mature and completely mature fractions based on the relative expression of cd , cd and the ir nkg a and kir ( ) , conventional and adaptive nk cells ( , ) , but there are also heterogeneous aspects between pb and various tissues ( , ) . very recent data by dogra et al. ( ) suggests a model in which the phenotype, the degree of maturity and the functions of nk cells are closely dependent on the anatomic location, with no influence of age and gender. it is quite difficult to find a substantial amount of references regarding upper airway nk cells. in human, the articles were mostly reporting on the investigation of nk cells in chronic rhinosinusitis, an inflammatory state of the mucosa of the nose and the sinuses ( ) with a significant impact on quality of life. two different forms, one with nasal polyps and one without nasal polyps, are distinguished ( , ) . bacterial pathogens are considered as one of the etiological factors in this disease ( ) . however, as the bacteriology of ethmoidal biopsies was the same regardless of the presence or absence of polyps, niederfuhr et al. questioned the bacterial role in the pathogenesis of the polyps as well as a systematic antibiotic treatment ( ) . in a study of patients, further subdivided into those responding and those resistant to treatment, and healthy controls, kim et al. investigated exclusively pb nk cells. the authors demonstrated that the pb nk cells from the patients had decreased effector functions compared to the healthy controls, with the treatmentresistant individuals being most severely affected ( ) . the recent manuscript by kaczmarek et al. ( ) reported not only on pb nk cells, but also on those from nasal mucosa and from nasal polyps. however, the exploitation of the material was limited to cd − cd + cd + events, which excluded the population of cd bright cd − nk cells that might be numerically well represented in these tissues. the phenotypic investigations of this subset in the nose revealed a predominance of relatively immature, cd + nk cells. furthermore, the ar nkg d was expressed at lower frequencies (lower percentages of nkg d + cells) and lower density of expression in the nasal mucosa and the polyps compared to pb (populations negative for nkg d were identified in the tissues). finally, the percentage of nk cells among lymphocytes (mean: %) was significantly higher in the polyps than in pb ( ) . okada et al. published a paper about nk cells in the nasal mucosa of the mouse on the c bl/ background ( ) , in which they showed that these nk cells were more immature (according to the relative levels of expression of cd and cd b) and phenotypically more activated (reduced expression of cd l, higher percentage of cd + cells) than those from spleen and lung. around % expressed the tissue residency and activation marker cd and one third of those also cd . the pattern of expression of the ly receptor family was different between the three tissues. functionally [cd a staining and interferon (ifn)-γ production], nasal nk cells appeared to be hyporesponsive compared to their spleen, but not their lung counterparts ( ) , which might be related to the possibility that the fraction of cd + nk cells was not sufficient to significantly activate the global nk cell population in the chosen experimental readouts. although this dataset is interesting per se, it should not be ignored that casadei and salinas ( ) , in a review about different animal models of nasal infections and immunity, cited several anatomic (functional vomeronasal organ in contrast to human, no waldeyer's ring) and physiologic (macrosmatic, no coughingsneezing reflex, lower sensitivity to human viruses) limitations of the mouse in this context, so that such results should always be considered with care before extrapolating to the human situation. lung nk cells have recently been extensively reviewed in this journal ( , ) , so that a summary of their most important features might be sufficient. lungs are constantly exposed to microparticles from the environment. particularly, as the mucosal lung epithelium is at the interface between the outside world and the organism, it can become the entry site for infectious pathogens, be they bacterial, fungal, or viral in nature. therefore, an extensive and sophisticated local immune response is waiting to be triggered at this anatomic location, and human nk cells, which represent around - % of lung lymphocytes ( ) , are a part of it. the work of marquardt et al. has established that most human lung nk cells represented the circulating subset and had the mature cd dim cd bright phenotype ( ) . they expressed more frequently the differentiation marker cd as well as educating kir than blood nk cells from the same donors but were relatively hyporesponsive upon stimulation with hla class i-negative target cell lines. in addition, however, a putative tissue-resident subset (around % of all lung nk cells), further subdivided into relatively immature cd bright cd − and cd dim cd − cells ( , ) , expressed the tissue residency markers cd , cd a, and cd . these cells were characterized in detail again by marquardt et al. ( ) , who showed that they were functional, especially after stimulation with the cytokine interleukin (il)- and displayed a unique transcriptional profile. several subpopulations could be distinguished based on the relative expression of cd a and cd ( , ) . natural killer cells have likewise been investigated in mouse lungs, particularly by wang et al. ( ) and michel et al. ( ) . both groups found that lung nk cells were more mature than those from the spleen ( ) or other organs ( ) according to the relative expression of cd and cd b. whereas the former authors described a higher expression level of the ir cd /nkg a and a lower level of the ar nkg d, the second paper could confirm this data only regarding nkg d in terms of mean fluorescence intensities. lung nk cells proliferated less, degranulated less (cd a assay) and were less cytotoxic than splenic nk cells ( ) , but these functions were rapidly up-regulated upon bacterial lung infection ( ) . this suggests that at homeostasis, lung nk cells are inhibited to avoid damage to normal autologous cells, but that they can quickly intervene in case of an infectious insult ( ) . michel et al. showed in in vitro co-culture systems that both spleen and lung macrophages could significantly up-regulate the cytotoxic activity of lung nk cells through a contact-dependent mechanism ( ) . regarding the homeostatic situation, research in recent years has revealed that in contrast to the older view of the lungs as sterile organs, a lung microbiota is present in the lower airways which exerts significant effects in health and disease, although it is not as abundant as in the gut ( ) ( ) ( ) ( ) . the term "microbiota" refers to all the microorganisms present, namely bacteria, fungi, protozoans, and viruses ( ) , but here we will only consider the role of bacteria. six phyla are predominantly represented in the lower airways: firmicutes, proteobacteria, bacteroidetes, actinobacteria ( , ) , acidobacteria, and fusobacteria ( ) . this microbiota is supposed to be transient in healthy donors and to be established from micro-aspiration and inhalation ( ) and its composition at any given time point submitted to the parameters of bacterial arrival, bacterial removal, and local immune responses ( , ) . in this way, an equilibrium state is reached that depends also strongly on the gut microbiota through various bacterial metabolites and contributes to the maintenance of homeostasis in the lower airways (gut -lung axis) ( ) ( ) ( ) . everything that disturbs this balance, such as some medications and particularly antibiotics, increases in nutrients (high fat diet, low fiber diet), cigarette smoke, infectious agents, chronic inflammation, can disturb the gut as well as the lung microbiota and lead to a state of dysbiosis, characterized by an increased number of airway bacteria and a change in its composition. the dysbiosis is profoundly linked to several severe lung diseases [asthma, chronic obstructive pulmonary disease (copd), infections, cancer] ( ) ( ) ( ) ( ) ( ) ( ) ( ) . natural killer cells have, to our knowledge at least, not been investigated in detail in the context of a normal lung microbiota to date. as most lung nk cells are not activated nor tissueresident (as illustrated by their negativity for cd ), they might not react very strongly to a normal microbiota. however, as they are expressing several bacteria-specific toll-like receptors (tlrs) that signal in the presence of bacterial pathogens ( ) , it might be conceivable that they could also mount an immune response toward microbiota components and that this would contribute to homeostasis. the overall immunosuppressed state of lung nk cells at baseline would help to avoid aggression of harmless and useful bacteria and of uninfected autologous cells ( ) . yang et al. ( ) , as well as fuchs and colonna ( ) , discuss data claiming that at steady state, alveolar macrophages secrete immunosuppressive cytokines which keep nk cells in respect. this is in contrast with the results of michel et al. ( ) , discussed above. however, the macrophages and dendritic cells (dc) switch to pro-inflammatory cytokine production in case of a bacterial or viral infection and thereby activate the nk cells. this entity is the third cause of mortality in the united states of america ( ) and worldwide ( ) and is in most cases the consequence of prolonged cigarette smoking ( ) . it is characterized by airflow obstruction, emphysema, recurrent infections ( , ) , chronic inflammation, and overproduction of mucus ( ) . acute exacerbations significantly limit the quality of life of the patients ( , ) . the exacerbations are in principle caused by viral or bacterial infections, the latter most frequently due to haemophilus influenzae, streptococcus pneumoniae, and moraxella catarrhalis ( ) . pseudomonas aeruginosa is another bacterium frequently involved and one of the most dangerous ones, based on its highly pathogenic properties ( ) , and its remarkable level of resistance to antibiotics. natural killer cells have been investigated in human copd as well as in animal models of this disease. motz et al. demonstrated that exposure of pulmonary leukocytes to viral pathogenassociated molecular patterns (pamp) induced higher functional properties (degranulation measured with the cd a assay, and ifn-γ production) ex vivo in chronic cigarette smoke exposed than in non-exposed c bl/ mice ( ) . interestingly, bacterial pamp appeared to be less efficient in this model, as among five molecules tested, only fsl- (bacterial lipopeptide, tlr / agonist) and lipopolysaccharide (lps, tlr ligand) increased the percentage of ifn-γ + nk cells above the one of the non-exposed mice. in contrast, other papers reported that nk cell functions are diminished in copd ( ) . it has been further repeatedly demonstrated that in copd or relevant animal models, nk cell cytotoxic activity is increased relative to non-copd smokers and healthy individuals ( , ) . based on the model of lung nk cell hypo-responsiveness at baseline, cigarette smoke and even more the inflammatory state of the lower airways in copd would activate the alveolar macrophages and induce their production of pro-inflammatory cytokines. these would, in turn, unleash the nk cells and increase their cytotoxic activity, cytokine and chemokine expression, leading to a further aggravation of the inflammation and the clinical status of the patients. indeed, in accordance with this concept, freeman et al. ( ) showed that cd + cells (in fact a mixture of nk cells and cd + t lymphocytes) isolated from lung parenchymal samples of non-copd smokers and copd patients with a smoking history, although similar in terms of frequencies between the cohorts, had a different cytotoxic activity toward autologous lung epithelial cells. the cd + lymphocytes from the copd patients were more cytotoxic than the cells from the non-copd smokers, in an experimental setup without additional stimulation. the target cells were supposed to be mostly epithelial cells based on their positivity for cd , their size, and their negativity for the hematopoietic cell marker cd . the cytotoxicity was measured as the percentage of annexin v + target cells after the co-culture with the effectors and was around % in most samples. this was not a lot, but the nk cells and cd + t cells were not otherwise activated. most of the parenchymal lung nk cells were cd + cd + and the minor rest cd + cd − ( ). another study was provided by the same group in ( ) . it showed that isolated, purified lung nk cells induced apoptosis in autologous epithelial cells. this time, the mean level of cytotoxicity was rather high compared to the previous paper, and it was very significantly stronger in copd patients than in non-copd smokers. the nk cells, but not the target cells, determined this increased cytotoxic activity, because k , a hla class i-negative myeloid leukemia cell line used as the standard nk cell target, was also lysed more efficiently by copd nk cells than by their non-copd counterparts. the authors confirmed their data in a mouse model and then showed that the nk cells were primed by dc via trans-presentation of il- ( ), a phenomenon first described in by andreas diefenbach and his group ( ) . this would nicely explain the higher level of nk cell cytotoxicity observed in copd. along the same line, okamoto et al. administered il- and il- to normal mice and observed a lethal effect within days, selectively involving the lungs, with a profound interstitial infiltration of lymphocytes dominated by nk cells ( ) . high levels of various cytokines and chemokines were found in serum and lungs. depletion of the nk cells by antibodies completely abrogated the lethal injury, which is a convincing demonstration of the potentially destructive power of nk cellactivating cytokines and nk cells themselves ( ) . this work was intended as a contribution to the elucidation of the pathogenesis of interstitial pneumonia, but similar mechanisms, in the presence of high levels of pro-inflammatory cytokines in bacterial infections, might contribute to copd. in human cancer patients, administration of high dose il- induced a vascular leakage syndrome where the so-called lymphokine activated killer cells (equivalent to highly activated nk cells) destroyed endothelial cells, causing a generalized edema, and damaging several organs ( ) . hodge et al. demonstrated a higher number of nk cells in the bronchoalveolar lavage fluid (balf) of copd patients (the cohort was composed of current smokers and of ex-smokers) than in healthy smokers ( ), a higher content of the cytolytic molecule granzyme b and, most importantly, a significantly increased cytotoxic activity against k . they also found a reduction in the percentage of balf nk cells expressing cd (which they consider as ir, although it is more a chaperone molecule for the true ir nkg a). nevertheless, this indirect measure of a down-regulation of nkg a could indicate that it contributes to the higher nk cell cytotoxic activity observed in copd ( ) . recently, osterburg et al. presented a multiparameter flow cytometry study of pb nk cells from copd patients compared with smokers and never smokers ( ) . in contrast to those, copd patients and smokers highly expressed the maturation marker cd as well as the ar nkp and nkp (normally only present on activated but not on baseline nk cells), but lower levels of cd . certain nk cell subpopulations were indicative of prior exacerbations ( ) . the ar nkg c, which is significantly present only on adaptive nk cells from human cytomegalovirus (cmv)-infected individuals, was not differentially expressed in pb of copd patients with a smoking history and healthy volunteers, but present on a higher percentage of nk cells in the patients with the most frequent exacerbations and the most reduced lean mass ( ). a relationship with the bacterial burden cannot be excluded in this context, as there might be a correlation between the viral reactivations and the bacterial colonization, contributing together to the higher number of exacerbations. most of the papers discussed above investigated the nk cell cytotoxicity toward autologous cells or conventional nk target cells, but what about a potential direct bacterial killing? nk cells, upon appropriate stimulation, release cytolytic molecules called perforin, granzymes and, in human but not in mice, granulysin, which have an additive or synergistic cytolytic effect toward bacteria ( ) . they can form pores in the target cell walls and thereby eliminate the microorganisms, but in addition they are able to eliminate some types of eukaryote cells infected by bacteria ( , , ) . furthermore, in addition to the direct effect, nk cells are embedded in the immunological network and react (through an increased cytotoxic activity and cytokine production) to the immune cells and the cytokines/chemokines in their environment ( ) , which is strongly shaped in case of a bacterial infection ["cellular crosstalk" ( ) ]. data about chronic rhinosinusitis, nasal polyposis and copd are summarized in table . as mentioned above, this ubiquitous gram-negative pathogen is part of those colonizing the lower airways in copd, but it is also a major problem in cystic fibrosis and in nosocomial infections, with a high morbidity and mortality ( ) . the role of nk cells in the host defense against this bacterium has been quite extensively studied by the team of michael t. borchers ( , ) in consequences of the indicated diseases on nk cell phenotype and functions. copd, chronic obstructive pulmonary disease; balf, bronchoalveolar lavage fluid; and ifn-γ , interferon-gamma. mouse models. in the chronologically first work, outbred cd- mice were intranasally infected with the p. aeruginosa laboratory strain pao ( ) and evaluated h later. the findings can be summarized as follows: (i) the infection increased the expression of ligands for the ar nkg d, present on almost all nk cells but also on a subpopulation of cd + t lymphocytes, in vivo; (ii) similarly, these ligands increased in an infected human lung epithelial cell line in vitro; (iii) the inhibition of the ar nkg d with a monoclonal antibody significantly reduced the clearance of p. aeruginosa from the lungs; (iv) antibody-mediated nkg d blockade down-regulated the amount of the cytokines il- β, ifn-γ and tumor necrosis factor (tnf)-α and in addition of nitric oxide; and finally, (v) the same experiment also revealed a threefold reduction of epithelial cell damage, measured as shedding of these cells into the balf ( ) . the latter point brings us again to the recurrent theme of lung cell damage that can be induced by activated nk cells, whereby it would have to be determined if this is beneficial (elimination of infected cells by nk cells) or deleterious (exaggerated damage to the epithelium). the follow-up paper ( ) then presented a conditional mouse model with an inducible expression of nkg d ligands on lung epithelial cells. here, the bacterial clearance was significantly higher in those mice that overexpressed the nkg d ligands. moreover, the survival up to h post-infection and the level of phagocytosis were significantly increased in the latter group. similarly, in in vitro experiments, where the nk cells were stimulated with lps, the percentage of nk cells producing ifnγ was much higher in the mice with the increased expression of nkg d ligands. as expected, this percentage dropped (but was not completely abolished) in nk cells from infected mice treated with an anti-nkg d antibody ( ) . however, the p. aeruginosa-derived exotoxin a, which in combination with il- α may induce a dangerous inflammatory state with tissue damage in the host, has also been shown to inhibit nk cell cytotoxic activity against k , even in the presence of usually stimulating cytokines such as il- ( ). the inhibition was almost complete with a high dose of the toxin and still partial with a low dose ( ) . the effector cells were not purified nk cells but peripheral blood mononuclear cells (pbmc), so that an indirect effect on the nk lymphocytes might play a role in this readout. furthermore, pedersen and kharamzi described already in that the p. aeruginosa-derived alkaline protease and elastase inhibited nk cell cytotoxic activity against k , presumably due to a reduction in the effector-target conjugate formation ( ) . in addition, these molecules strongly reduced the binding of an anti-cd (called leu- at that time) antibody ( ) . this group of pathogenic gram-negative bacteria is composed of several species, of whom some are dangerous for cystic fibrosis patients, as they are highly resistant to multiple antibiotics ( ). li et al. ( ) investigated the interaction between burkholderia cenocepacia and nk cells, and first demonstrated that the nklike leukemia cell line yt ( ) , as well as primary purified human nk cells, significantly reduced the number of living bacteria (measured as cfu) after a co-incubation of to h. the results were confirmed with life cell imaging techniques and bacterial uptake of propidium iodide (pi). the authors then wanted to know if the killing activity was contact-dependent or not, and first showed that yt cells bound the fluorochromelabeled bacteria. then, they could demonstrate that a direct contact was needed for the killing activity, as nothing happened to the bacteria when they were separated from the nk cells by a porous membrane, allowing passage of soluble molecules but not of cells ( ) . most bacteria remained extracellular and were not taken up by the yt cells. killing was almost completely abrogated after treatment with strontium chloride (srcl ), which is known to deplete nk cells from their cytotoxic granules ( ) . finally, it was established that src family kinases were activated in yt cells after the contact with b. cenocepacia ( ) . this is a very nice demonstration that nk cells are able to directly kill certain extracellular bacterial species through nk cell -bacteria contact, although the precise mechanism is still unknown. other possible mechanisms of nk cell-mediated elimination of bacteria are the lysis of intracellular pathogens within the infected cells and the activation of other immune cells, and particularly of macrophages, via nk cell-derived cytokines (such as ifn-γ) ( ) , and most likely also the killing of bacteria-infected cells expressing ligands for nk cell ar. this is another gram-negative pathogen which poses a major problem due to its frequent causative involvement in nosocomial infections (particularly in pneumonia) and the steady increase of strains multi-resistant to antibiotics ( ). chalifour et al. ( ) demonstrated that the outer membrane protein a (kpompa) from this microorganism, known to signal via tlr , induced ifn-γ and α-defensin (an antimicrobial peptide) synthesis and release in human nk cells. in the mouse, both nk cell-derived ifn-γ ( ) and il- ( ) have been described to be necessary for bacterial clearance ( , ) . in the paper from xu et al., it was nicely shown with genetic controls and depletion experiments that the immune defense against this pathogen indeed deeply involved nk cells and that a subset of them produced il- . the nk cells had a conventional and mature phenotype (less cd + , more klrg + ) distinct from other innate lymphoid cells (ilc) ( ). ivin et al. focused on the fact that ifn-γ production by nk cells, likewise necessary for the elimination of the bacteria through a network with alveolar macrophages, was dependent on the nk cell-intrinsic stimulation by type i ifn, in turn induced by k. pneumoniae ( ) . in contrast to the crucial role of nk cell-derived cytokines, their granzymes (a and b) , one of the constituents of the lytic granules, did not seem to play a major role in this model ( ) . however, this does not rule out that in human, granulysin and perforin together might have a cytotoxic effect on these bacteria. in the case of helicobacter pylori, responsible for chronic gastric inflammation with the potential to lead to ulcers or cancer, preincubation with fixed bacteria increased the cytotoxic activity of nk cell-enriched pbmc toward k and other tumor target cells, as well as the release of ifn-γ ( ). furthermore, rudnicka et al. showed that the bacterial glycine acid extract induced nk cell expansion and ifn-γ production, whereas the lps from the same bacteria inhibited these parameters, and instead favorized the apparition of il- -producing nk cells ( ) . although this might just marginally be relevant for nk cells in the lungs, it nevertheless shows to which extent these cells can react to bacteria and how the latter try to manipulate them. legionella pneumophila, the agent of legionnaires' disease, is replicating intracellularly in macrophages. here again, nk cell production of ifn-γ, induced probably through direct tlr messages ( ) one of the most frequent culprits in community-acquired pneumonia is s. pneumoniae. regarding the role of nk cells in this infection, their beneficial or detrimental action depended on the pathogen's serotype ( ). thus, the control of serotype depended on nk cells, as demonstrated by baranek et al. in a mouse model ( ). these authors investigated the consequences of a defect in the transcriptional cofactor four-and-a-half lim-only protein (fhl- ) on nk cells in general and on pneumococcal infection particularly. it had been previously established that ifn-γ was, once more, the crucial factor in host defense in this context, and that nk cells were one of its major producers ( ) . in the spleen and the lungs of fhl- knockout (ko) mice, the number of nk cells and their expression of the ar nkg d and nk . (cd c) were down-regulated and a negative effect of the deficiency on nk cell maturation was observed. mortality to s. pneumoniae lung infection was strongly increased in the ko mice but could be rescued by the adoptive transfer of wildtype nk cells. finally, the authors showed that ifn-γ production by nk cells was severely reduced and that less neutrophils were recruited to the lungs of the ko animals ( ). the role of the mostly immunosuppressive cytokine il- in dampening the immune response to pneumococcal infection was shown in , when van der poll et al. administered the pathogen intranasally together with il- and observed early mortality and reduced levels of the pro-inflammatory factors ifn-γ and tnf. conversely, all this was restored when the mice were pre-treated with an anti-il- antibody ( ). these results were very recently confirmed by clark et al. ( ) , who worked with il- reporter and il- -ko mice to observe that s. pneumoniae induced il- production by nk cells (around % of total lung nk cells) with a negative effect on animal survival, and that the bacterial burden was diminished in the lungs of the ko mice compared to wildtype animals. nk cell depletion in the latter induced a strong reduction in the bacterial lung counts and in il- . furthermore, il- -deficient mice had significantly more neutrophils and monocytes in the infected lungs. finally, the virulence protein spr from s. pneumoniae was identified as the il- -inducing factor ( ) . none of these papers investigated the potential balance between the pro-inflammatory and anti-inflammatory effects of ifn-γ and il- , respectively, on the outcome of this infection, which would anyhow have been technically challenging. one might suppose that il- is there to down-regulate an overwhelming immune response that would damage lung tissues, but on the other hand, it might also be counterproductive to dampen it too much and thus to lose control over the pathogens ( ) . other groups have described that human as well as mouse nk cells could produce and release il- ( , ), although, according to perona-wright et al., this only occurred in the case of a systemic, but not a localized, pulmonary infection (with the gram-negative bacterium yersinia pestis) ( ) . in the case of systemic infections with listeria monocytogenes and y. pestis, approximately % of blood nk cells became il- + , and the cytokine was produced by a nk cell subset circulating in blood prior to the infection ( ) . before studying s. pneumoniae ( ), clark et al. had already shown that l. monocytogenes elicits il- production by nk cells via the virulence factor p (with, as a consequence, an inhibition of the recruitment and the activation of myeloid cells) in a mouse model of systemic infection, where the lungs were not further investigated ( ) . another frequently encountered nosocomial and multiresistant infectious agent is staphylococcus aureus. small et al. could demonstrate the fundamental role of nk cells in the response to these bacteria in the case of mouse lung infections ( ) , as (i) nk cell numbers in the airways increased; (ii) in vitro contact with products from the pathogens activated nk cells; (iii) co-culture of nk cells with alveolar macrophages increased the phagocytic activity of the latter, (iv) il- -ko mice were much more susceptible to the infection than wildtype mice, whereas they had much more neutrophils and macrophages in the lungs; and (v) nk cell depletion rendered even wildtype mice highly sensitive, despite a conserved il- production ( ). these findings demonstrate indeed once again the important role of nk cells in immune defense against extracellular bacteria. in accordance with this model, zhao et al. showed that particular matter, associated epidemiologically with enhanced numbers of lung infections, diminished the amount of nk cells migrating to rat lungs in case of infection with s. aureus, whereas adoptive nk cell transfer restored a vigorous nk cell response ( ) . in ex vivo experiments, nk cells improved, as in the previous study, the phagocytosis of the pathogens by alveolar macrophages. it is well known that after influenza, recovering patients are very susceptible to bacterial superinfection, notably by s. pneumoniae and s. aureus ( ) . the contribution of nk cells to this phenomenon was demonstrated in a mouse model of h n influenza virus infection followed by intratracheal instillation of s. aureus. the sequentially double-infected mice were much more susceptible to the infection (weight loss, survival rate) than those receiving pbs or bacteria only. this went hand in hand with severe changes in the histopathological aspect of the lungs and a marked reduction of local nk cell numbers and tnfα + nk cells. furthermore, the concentrations of tnf-α and of the chemokines ip- and mip- α were diminished in the balf. adoptive transfer of naive nk cells could restore the immune response. the nk cells needed tnf-α to perform their antibacterial effect and this was organized via an interaction with alveolar macrophages and increased phagocytosis ( ) . the conclusion that might be drawn from all these papers is that nk cells are very important, at least in mouse models, for the immune response to and the defense against pulmonary infections due to gram-positive bacteria, with, on the other hand, a detrimental influence of these lymphocytes in case they produce too much immunosuppressive factors [the same old story ( ) ]. mycobacterium tuberculosis is the agent of tuberculosis (tb), an infectious disease that puts a high burden on the populations in developing but also in developed countries and increasingly shows resistance to conventional antibiotics. it latently infects about % of the total population and becomes clinically apparent in ten million patients per year, according to estimations from the world health organization (who) ( , ) , rendering it a major public health issue. as recently reviewed by cong and wei ( ) , nk cells could interact with this intracellular pathogen through the ar nkp , nkp , and nkg d, as well as tlr . although they became activated under these conditions, they seemed to play only a negligible protective role, according to junqueira-kipnis et al. ( ) . these authors showed in a mouse model that lung nk cells augmented in number within the first weeks after exposure to aerosols containing the mycobacteria and up-regulated cd , ifn-γ and perforin. however, their depletion did not at all change the kinetics of the infection. human pb nk cells likewise up-regulate ifn-γ after contact with m. tuberculosis ( ) . barcelos et al. ( ) compared pb nk cells in cohorts of patients with active tb, isolated tuberculin + skin tests, and tuberculin − healthy donors. they found a different subset distribution according to the cohorts, with putative tb-exposed but-resistant individuals (defined as those with a positive tuberculin test) having overall less nk cells but an increased percentage of cd − cd + , cd + cd − and especially cd bright cd −/+ nk cell subsets compared to the other two donor groups. in contrast, tb patients displayed lower frequencies of cd + cd + cells. the authors speculated that this different subset distribution might have been related to the resistance or sensitivity to active tb, but of course, as the cells stem from pb, this dataset might have to be interpreted with some caution. surprisingly, however, roy chowdhury et al. ( ) , by following a cohort of adolescents from an endemic region in south africa, could demonstrate with mass cytometry and functional experiments, that latent tb was associated with increased responses of pb nk cells, with a particular role for the ar cd . indeed, the percentages of nk cells among total living cells, of total cd + cells, of granzyme b + and of perforin + cells were significantly higher in patients with latent tb than in healthy, non-infected donors. in addition, adcc (mediated via cd ) against p target cells was also higher in latent tb. by following further cohorts, the authors found that the percentage of pb nk cells was dynamically regulated during latency, progression of the disease and responses to antibiotic medication. this level of nk cells in pb even correlated inversely with inflammation in the lungs of patients with active tb ( ) . such observations push nk cells again at the forefront of immune defenses in tb and at a possible role in the maintenance of latency. with a similar cohort-based approach, harris et al. ( ) evaluated nk cell phenotype and functions in individuals with latent tb compared with healthy controls. furthermore, participants were separated in infected and non-infected in a tb-endemic region in kenya and a healthy volunteer cohort from the united states. among the three groups, the persons from the united states had the significantly lowest percentage of cd − cells, which are known to expand in chronic human immunodeficiency virus (hiv) infection and other viral diseases and to be dysfunctional ( ) . the kenyan volunteers displayed, among cd dim nk cells, a higher expression of granzyme b and of the non-mhc class i-specific ir tigit, with the highest levels found in the healthy cohort. furthermore, these individuals had an increased expression of the ar nkp . within the cd bright subpopulation, the kenyan participants showed increased expression of nkg d but again decreased levels of nkp , compared to the cohort from the united states. functionally, degranulation (cd a assay), ifnγ production (intracellular flow cytometry) and cd expression were compared between the three cohorts after co-culture with k cells (evaluation of natural cytotoxicity) and p mouse cells plus anti-mouse antibody (evaluation of adcc). whereas for the latter parameter, no significant differences were observed, frequencies of cd + , cd a + , and ifn-γ + nk cells turned out to be significantly higher in the united states study population, such as if an environment endemic for tb would impact the "missing self "-recognition capacities of nk cells ( ) . the same regional discrepancies were observed after stimulation of total pbmc with three different antigen extracts from m. tuberculosis, and the reactivity to these antigens was shown to be at least partially dependent on the presence of il- and il- , supposed to be derived from accessory cells. conradie et al. ( ) described that the level of activation of pb nk cells (frequency of cd + and cd + hla-dr + events) allowed, among other parameters, to discriminate between m. tuberculosis-induced immune reconstitution syndrome, hiv infection and co-infection with both pathogens. although these three papers suggest some influence of m. tuberculosis on pb nk cells, it is not clear yet to which extent nk cells really intervene in the immune defense against this pathogen that persists in the lungs. an investigation on tuberculous pleurisy ( ) revealed a large predominance of cd bright cd − nk cells in the pleural fluid, and an apoptotic effect of soluble factors from this environment predominantly on cd + nk cells. m. tuberculosis induced ifn-γ production from cd bright nk cells in the absence of monocytes, t cells and b cells, leaving open the possibility of a direct productive interaction between the bacteria and the nk cells. lai et al. ( ) presented a work on nontuberculous mycobacterial lung infections, which means due to other mycobacterial species, such as mycobacterium abscessus and mycobacterium kansasii. as the latter become more and more prevalent in developed countries, these authors performed a study in c bl/ mice that were infected intratracheally with m. kansasii. they found that nk cell depletion increased bacterial burden, mortality, and pathogenetic postinfectious changes (macrophage phagocytosis, dc activation, cytokine production, and development of granuloma). the same observations were made in ifn-γ-ko animals and restored after transfer of wildtype nk cells. these cells were also the most important producer of ifn-γ in this model ( ) . lai et al. further cited papers that had demonstrated a similar protective effect of ifn-γ produced by nk cells in the infections with bordetella pertussis, francisella tularensis, and chlamydia muridarum in mouse models of respiratory infection. previous publications by the same group had shown that nk cells can directly lyse m. tuberculosis and m. kansasii via the cytotoxic proteins granulysin and perforin in a contactdependent manner disrupting mycobacteria cell wall integrity ( ) , and that in some patients with mycobacterial infections, anti-ifn-γ autoantibodies were detected ( ) . the killing process involved signaling through nkg d and ncr as well as map kinases, suggesting that similar mechanisms are involved for the killing of bacteria and of eukaryotic target cells ( ) . this is potentially a very important observation, as it strongly suggests that both conventional cytotoxic mechanisms and cytokine production might be relevant in anti-mycobacterial defense. some studies were also performed on mycobacterium bovis bacillus calmette-guérin (bcg), an attenuated mycobacterial strain used as an anti-tuberculous vaccine ( ) . for example, it was demonstrated in vitro that cd bright nk cells reacted to this microorganism by proliferation and ifn-γ production, whereas their cd dim counterparts better up-regulated the cytolytic proteins perforin and granzyme a ( ), all of which was largely expected based on what is known about the functional specialization of these two nk cell subsets ( ) ( ) ( ) . in a mouse in vivo model, where bcg was directly administered (intratracheally) into the lungs, nk cell-mediated production of ifn-γ rapidly increased in the first days after infection, similarly to the number of lung nk cells ( ) . after nk cell depletion, the reduction of body weight was less pronounced compared to non-depleted mice, whereas the bacterial load remained identical. importantly, inflammation and injury of the pulmonary structures was much less pronounced in the nk cell-depleted animals, suggesting a pathogenic role for these lymphocytes. indeed, the level of pro-inflammatory cytokines and chemokines was also reduced in the absence of nk cells, and the percentages of ifn-γ + cd + and ifn-γ + cd + t cells was significantly increased in these mice. bacillus calmette-guérin-infected macrophages up-regulated nkg d ligands, which induced their lysis via this receptor-ligand interaction. finally, the blocking of nkg d with a monoclonal antibody restored the survival of the macrophages and the t cell-mediated immune response ( ) . it is difficult to make a coherent synthesis of all these observations on nk cells and mycobacteria, but it is nevertheless quite appealing that again positive, negative and neutral aspects are described, which may vary according to the models and the experimental setup. this shows that nk cells still hide a lot of secrets regarding their function in anti-mycobacterial infections as well as in bacterial pathogenesis overall. a summary of the relationships between the bacteria discussed and nk cells is presented in table . these are obligate intracellular pathogenic bacteria that are responsible for several types of human and mouse diseases. various studies dedicated to this type of microorganisms illustrated the concept that nk cells usually do not respond as a pure population as may be the case for in vitro experiments, but that in vivo they are part of a tightly controlled immune network composed of cells, cytokines, chemokines and exosomes. thus, in mouse models, nk cells influenced the interaction between dc, t helper (h) and th t lymphocytes in c. muridarum lung infection ( ) , modulated the balance between th and th t cells and t regulatory cells (treg) in the same type of infection ( ) , and again positively regulated the interactions between dc and t lymphocytes against chlamydophila pneumoniae ( ) . in all these situations, nk cells exerted a protective and disease-controlling effect via their influence on the bridge between innate and adaptive immune responses. with the ambitious aim to experimentally investigate the famous "hygiene hypothesis, " han et al. ( ) studied mice infected with c. muridarum and rendered allergic to ovalbumin (ova). they observed that prior infection could inhibit at least certain parameters of allergy. however, nk cell depletion partly suppressed the "beneficial" effect of the lung infection. adoptive transfer of nk cells from infected mice inhibited partially the development of an allergic response in non-infected recipients. nk cell-devoid mice coherently produced more th type cytokines ("pro-allergic" th cytokines, il- , and il- ) than ifn-γ ("anti-allergic" th cytokine). a detrimental effect of nk cells had been shown for the immune response to the respiratory rodent pathogen mycoplasma pulmonis, related to the human infectious agent mycoplasma pneumoniae. indeed, in a quite complicated experimental setup, bodhankar et al. demonstrated that nk cell depletion interfered positively with the development of a protective adaptive immunity after nasal-pulmonary immunization with bacterial antigens ( ) . this could be explained because nk cells shaped the t cell cytokine response toward more il- , il- , and il- but away from ifnγ production. wurzel et al. presented large cohort studies of children with protracted bacterial bronchitis (pbb) and mild bronchiectasis, associated or not with human adenovirus co-infection ( , ) . besides typical socio-economic and clinical factors, an elevated nk cell number relative to the values of healthy children of the same age was observed in the pb of diseased children in general and with adenovirus species c particularly. nk cell phenotype and function were not further investigated. recently, sim et al. ( ) made the important discovery that the hla-c-specific activating kir ds did recognize a conserved bacterial peptide presented by hla-c, and more precisely by hla-c * : . the sequence of the peptide required for this recognition was a "rare" self-peptide, but the epitope of interest is conserved in the recombinase a (reca) of many bacterial species (more than according to the authors' claims), most of them belonging to serious human pathogens, such as helicobacter pylori, brucella, campylobacter jejuni, and chlamydia trachomatis ( ) . interestingly, activation of resting nk cells via kir ds alone was sufficient to induce degranulation and cytokine production, whereas all other known ar, except cd , need at least one co-activating molecule engaged at the same time ( ) . there was, furthermore, an inverse correlation between the frequency of the kir ds full length gene and the hla-c * : allele. thus, it appears that the kir family is not only involved in nk cell licensing and in multiple disease associations, but also, most likely, in antibacterial defense. this paper received an accompanying commentary by peter parham, which places the findings in the broader context of kir and hla class i molecules ( ) . to sum up, nk cells might be directly activated by various bacteria via contact-dependent mechanisms whose modes of functioning are still unknown, via tlr, via kir ds , or more indirectly via the up-regulation of ligands for their ar, such as nkg d, by infected cells. they might also react to cytokines released into the microenvironment by antigen-presenting cells (macrophages, dc). dietert et al. published a plea for the histopathological evaluation of the consequences of different infectious lung diseases in mouse models and described the pathogen-specific features characteristic for each of them ( ) . indeed, many variations were observed between the infecting microorganisms, be they bacterial or viral in nature. the authors emphasized that histopathology remains the "most conclusive and practical read out" for the evaluation of the effects of the various infectious models on mouse lungs. although this is true, more "modern" and state-of-theart methods are being developed and are about to enter the laboratories, as a consequence of general scientific and technical progress but also of the " r" approach regarding experiments with animals. in , the team of hans clevers described the generation of human airway organoids derived from surgical material or from balf ( ) . these were long-term proliferating structures that recapitulated a normal airway with different types of cells that are physiologically present in vivo. the beauty of the system per se was already an accomplishment, but it could be used for the study of various lung diseases, such as cystic fibrosis, cancer, or viral infections ( ) . therefore, it is likely that bacterial infections could similarly be investigated in this system, and the data obtained would probably be more relevant to human pathology than the mere mouse models (and save the life of many mice by the way). the same year, ross et al. ( ) published a review on the "ex vivo human lung." they worked with donor lungs not retained for transplantation, extracted primary cells from them and developed an "ex vivo-perfused single human lung" that would allow the investigation of different lung diseases. the system seems at first sight less elegant than the lung organoids and is maybe also more limited in the spectrum of possible pathologies that can be investigated. the advantage would be that an entire, complete organ is available and not just an organoid. yet another option is the "alveolus-on-a-chip, " developed by deinhardt-emmer et al. ( ) . it was a three-dimensional structure with an air phase and a liquid phase, where endothelial cells, epithelial cells and macrophages could be co-cultured. in the presented work, a primary influenza virus infection, followed by a s. aureus superinfection, were investigated, and it was shown that the endothelium was seriously damaged under these conditions. likewise, single cell transcriptomics is a powerful tool that can reveal huge amounts of details about all kinds of immune cells, and among them nk cells, as exemplified by lung cancerinfiltrating immunocytes in human and mouse ( ) . one aspect of nk cells is their putative potential for a dual role as "pro-inflammatory" and "regulatory" effectors, which might be mediated by different subsets ( ) . our group has previously touched the problem that nk cells are in fact a double-edged sword, meaning that they might have sometimes beneficial but sometimes rather deleterious effects ( ) . this has again become clear throughout this review, although the models and studies presented and discussed were all but homogeneous. it might be expected that this will change in the coming years if more and more teams will use the organoid and organ on-a-chip technologies and go into various "omics." overall, given the current and justified hype for nk cells as efficient agents for cancer immunotherapy, it would be difficult to convince the nk community that their favorite cells might also have a dark side. we emphasize that several methods to improve nk cell antitumoral efficiency, such as particularly car-nk cells ( ) and nk cell engagers, recently described by the vivier group ( ) , should be sufficient to stand up for the use of nk cells in this indication. however, what about the therapeutic indication of nk cells in infectious diseases in general and in the lungs particularly? due to the current covid- pandemic, this question has gained increased interest ( , ) , and in addition, most of the papers discussed here that describe an influence of nk cells on the disease course in the airways conclude with the statement that nk cells should be targeted in respiratory infections. but it has clearly been shown that these lymphocytes can have detrimental side effects and cause significant damage to the airways, at least in viral diseases ( , ) . available literature does not give clear indications regarding bacterial pathogenesis, but the issue was already discussed in , with the question if nk cells are angels or devils in bacterial infectious diseases ( ) . this problem is, in our opinion, not yet resolved and a lot of research work will be necessary in the field, keeping in mind that the number of multi-resistant bacterial strains is increasing at a terrifying rate and that alternatives to antibiotics must be discovered and developed. finally, a general problem in the field and a caveat to many of the presented studies is the difficulty of distinguishing nk cells reliably form ilc , which also produce ifn-γ as a signature cytokine and have a partially overlapping phenotype. a high plasticity within the ilc family renders even possible the conversion of nk cells, in certain microenvironments, into ilc like cells ( ) ( ) ( ) . however, whereas both nk cells and ilc require the transcription factor t-bet for their development and function, nk cells need and express eomes in addition. ilc are preferentially located in tissues and are very rare in peripheral blood, in contrast to nk cells. thus, one can be confident that the studies discussed here that worked with blood (human) and blood or spleen (mouse) nk cells really investigated nk cells and not ilc . for tissue-based studies, the differences might be more blunted, although ilc are considered as non-cytotoxic cells ( ) ( ) ( ) . these difficulties are in line with the increasing number of "new" cell types that are currently discovered [for example mr t cells ( ) ], as a consequence of the ever growing diversification and performance 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model single-cell transcriptomics of human and mouse lung cancers reveals conserved myeloid populations across individuals and species regulatory nk cells suppress antigen-specific t cell responses multifunctional natural killer cell engagers targeting nkp trigger protective tumor immunity flattening the covid- curve with natural killer cell based immunotherapies the promise and peril of natural killer cell therapies in pulmonary infection innate lymphoid cells: diversity, plasticity, and unique functions in immunity nk cells and ilcs in tumor immunotherapy plasticity of innate lymphoid cell subsets mr -restricted t cells are unprecedented cancer fighters the authors would like to thank prof. dr. markus ollert, md, the director of the department of infection and immunity of the luxembourg institute of health, for his continuous support. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © theresine, patil and zimmer. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -o ep b authors: carolan, louise a.; butler, jeff; rockman, steve; guarnaccia, teagan; hurt, aeron c.; reading, patrick; kelso, anne; barr, ian; laurie, karen l. title: taqman real time rt-pcr assays for detecting ferret innate and adaptive immune responses date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: o ep b the ferret is an excellent model for many human infectious diseases including influenza, sars-cov, henipavirus and pneumococcal infections. the ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. however, the range of reagents available to measure the ferret immune response is very limited. to address this deficiency, high-throughput real time rt-pcr taqman assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (ifnα, ifnβ, ifnγ, il α, il β, il , il , il , il , il , il p , il , granzyme a, mcp , tnfα), as well as four endogenous housekeeping genes (atf , hprt, gapdh, l ). these assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to ng rna per real time rt-pcr reaction) and to select the most appropriate housekeeping genes. using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. these new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states. ferrets are an outbred population widely used to study influenza virus infection (belser et al., ; laurie et al., ; rockman et al., ; hurt et al., ) as well as a range of other diseases, including sars-coronavirus (cov) (reviewed in (roberts et al., ) ) and henipaviruses, such as infection with hendra virus and nipah virus (bossart et al., ; pallister et al., pallister et al., , geisbert et al., ) . the anatomical and physiological similarity between human and ferret lungs also enables ferrets to be used as a model to study lung carcinomas (reviewed in baric et al., ) . recently, absence of the cystic fibrosis transmembrane conductance regulator (cftr) was associated with spontaneous disease induction in the lung and pancreas in ferrets, showing similar pathology to that of cystic fibrosis in humans (sun et al., , reviewed in keiser and engelhardt, ) . the broad utility of this model is highlighted by the use of ferrets to study pneumococcal transmission, reproductive biology and human fetal brain development (reviewed in baric et al., ) . while the ferret is a good model for human respiratory virus infections, reagents to identify ferret leukocytes and immune mediators are limited. studies have identified cross-reactive antibodies that recognize populations of ferret leukocytes, such as cd , cd ␤, cd and cd , and cytokines ifn␥, tnf␣, il and il (rutigliano et al., ; martel and aasted, ; pillet et al., ) . cloning and sequencing of ferret cytokine genes have enabled molecular approaches targeting the corresponding mrnas (von messling et al., ; danesh et al., ; nakata et al., ; ochi et al., ; qin et al., ) . expression of cytokine and chemokine genes has been assessed in ex vivo samples following infection of naïve or vaccinated ferrets with influenza virus or sars-cov by microarray analysis fang et al., ; rowe et al., ) . cytokine and chemokine gene profiles have also been assessed ex vivo and in in vitro epithelial cultures using sybr green real time rt-pcr assays (svitek and von messling, ; cameron et al., ; danesh et al., danesh et al., , svitek et al., ; kim et al., ; fang et al., ; hamelin et al., ; kobinger et al., ; rowe et al., ; kang et al., ; von messling, , ; pillet et al., ; huang et al., ; maines et al., ; belser et al., ; zeng et al., ) . taqman chemistry incorporates target-specific fluorescent labeled probes enabling multiple genes can be assessed in a single real time pcr reaction (giulietti et al., ) . to date, taqman real time rt-pcr assays have only been developed for a smaller number of ferretspecific gene targets (nakata et al., ; suguitan et al., ) . to enable a broader characterization of the immune response in the ferret model, we developed a panel of taqman assays to detect mrna of fifteen ferret cytokines, chemokines and immune mediators (ifn␣, ifn␤, ifn␥, il ␣, il ␤, il , il , il , il , il , il p , il , granzyme a, mcp- , tnf␣) and four housekeeping genes (atf , gapdh, l and hprt). the cytokine and chemokine profile induced by stimulation of ferret leukocytes with mitogens or influenza virus was also assessed to investigate the relevance of the ferret immune response to human infection studies. sequences for cytokine, chemokine and housekeeping genes of multiple species were obtained from genbank (http://www. ncbi.nlm.nih.gov/genbank) and aligned. regions of conservation were identified and primers were designed using primerselect (dnastar lasergene , madison, usa) or primerexpress (applied biosystems, california, usa) to amplify the region from ferret cdna. cloned genes were sequenced and taqman real time pcr primers and probes designed using primerexpress. all oligonucleotide primers and probes used in this study, including those previously published, are listed in table . primers for ifn␣ were designed to amplify multiple subtypes ( - ) (easlick et al., ; hillyer et al., ) . lyophilized oligonucleotide primers were synthesized by geneworks (adelaide, australia) and dissolved in nuclease-free water (promega, madison, usa) at m. all taqman ® mgb tm probes were synthesized by applied biosystems with a reporter dye (either fam, ned or vic) and a non-fluorescent quencher (nfq). adult male and female ferrets (weight - g) were purchased from independent breeders and housed at csl limited (victoria, australia) using services provided under a support services agreement. serum samples were tested by hemagglutination inhibition assay to ensure seronegativity (titer < ) to currently circulating influenza strains before use. experiments using ferrets were conducted with approval from the csl limited/zoetis australia animal ethics committee, in accordance with the australian government national health and medical research council australian code of practice for the care and use of animals for scientific purposes (nhmrc, ). a/tasmania/ / (a(h n )pdm ) influenza virus was passaged in the allantoic cavity of embryonated hen's eggs and stored in aliquots at − • c. to heat inactivate, virus was incubated at • c for min. retropharyngeal lymph nodes were collected from naïve ferrets and placed in rpmi- aq media tm (sigma-aldrich, new south wales, australia) supplemented with % (v/v) fetal calf serum (interpath services, victoria, australia), mm l-glutamine (safc biosciences, usa), u/ml penicillin/ g/ml streptomycin (sigma-aldrich) (complete-rpmi). single cell suspensions were made by mashing the tissue and passing through a sterile m cell strainer (bd, san jose, usa). cell suspensions were washed twice then resuspended in complete-rpmi. lymph node cells from each ferret ( × per well) were plated in a -well plate in ml complete-rpmi with or without l of live or heat-inactivated virus ( tcid ) or g/ml concanavalin a (cona), phytohaemagglutinin (pha-p), lipopolysaccharide (lps), ionomycin (iono) or phorbol -myristate -acetate (pma) (all from sigma-aldrich) in duplicate or triplicate. cell cultures were incubated at • c in % co in a humidified incubator for the indicated periods. total rna was extracted from cultured cells using the rneasy ® mini kit (qiagen, victoria, australia) according to the manufacturer's instructions. briefly, cells from a single well were pelleted and resuspended in l rlt buffer. the sample was vortexed and then run through a qiashredder column. rna was extracted from the supernatant using the animal cells spin protocol, without on-column dnase digestion and eluted with a l volume. rna purity was assessed (a /a ) using a nanodrop spectrophotometer (thermo scientific, massachusetts, usa). rna was stored at − • c. for removal of genomic dna, ng rna was incubated with units dnase i (rnase-free) (new england biolabs, massachusetts, usa) in dnase reaction buffer (final volume ) at • c for min. the reaction was terminated by the addition of edta (final concentration mm, sigma-aldrich) and incubation at • c for min. cdna was generated using the superscript iii first strand synthesis system for rt-pcr (invitrogen, california, usa) with random hexamer primers, according to the manufacturer's instructions. rnaseh treatment was performed. a simultaneous reaction without the reverse transcription enzyme was performed in parallel to generate a '-rt' control. the reaction volume resulted in ng initial rna/l final cdna preparation for culture samples, except at points indicated in the text, where ng initial rna/l final cdna preparation was used. cdna standards were generated in parallel with cdna test samples for each experiment. cdna standards were prepared using rna pooled from a range of test samples within each experiment, with at least l of ng initial rna/l final cdna standard prepared. ten-fold and two-fold serial dilutions (five dilutions of each) were prepared and used for standard curve generation for efficiency calculations. cdna was stored at − • c. table oligonucleotide primer and probe sequences used in this study. primers and probes developed for the taqman real time rt-pcr assay in this study are highlighted in bold italics. primers (and probes) from other published real time rt-pcr studies, taqman and sybr green, are indicated. primers used to clone inserts for plasmid controls are also indicated, and referenced as appropriate. amplification of gene-specific products was performed using platinum ® taq dna polymerase high fidelity (invitrogen) according to the manufacturer's instructions. reactions were run on a s tm thermal cycler (bio-rad laboratories pty ltd., new south wales, australia). total rna was extracted from stimulated ferret cell cultures, reverse transcribed and ferret genes amplified by conventional and taqman real time pcr, using primers indicated in table . both cdna and 'no rt' control samples were run to ensure the specificity of the amplification. the matrix influenza gene was amplified from rna extracted from virus isolate a/california/ / (a(h n )pdm ). the products were agarose gel-purified using the qiaquick gel extraction kit (qiagen) according to the manufacturer's instructions. purified dna was quantified and ligated into pgem ® -t easy vector (promega) (ifn␥, il , il , il , il , il , il p , il , mcp , tnf␣, atf , hprt, gapdh, l , matrix) or pcr tm blunt-topo ® vector (invitrogen) (ifn␣, ifn␤, il ␣, il ␤, granzyme a) according to manufacturer's instructions. ligation reactions were transformed into one shot ® top chemically competent e. coli (invitrogen) by heat shock according to the manufacturer's protocol. positive transformants were identified by pcr using m primers (promega) and gene-specific primers. plasmid dna was isolated from bacterial cultures using the qiaprep spin miniprep kit (qiagen) according to the manufacturer's instructions. the generation of oligonucleotide dimers for each taqman primer pair was assessed using power sybr ® green pcr master-mix (applied biosystems) with melting curve analysis, according to the manufacturer's instructions. primers which resulted in oligonucleotide dimer generation were redesigned and retested. a comparison between primer pairs was also performed using power sybr ® green pcr mastermix without a melting curve, according to the manufacturer's instructions. all other real time pcr assays were performed using the taqman ® fast universal pcr master mix ( ×), no amperase ung (applied biosystems), according to the manufacturer's instructions. one microliter cdna sample was assayed per reaction. each reaction consisted of cycle of • c for s, followed by cycles of • c for s and • c for s. real time pcr runs for each gene included cdna standards ( -fold and -fold dilutions, in duplicate), dna plasmid controls ( -fold dilutions; dilutions), no template control and test samples. when test samples were run over multiple plates for a single gene, dna plasmid controls were included on each plate and the same mastermix preparation was used for all pcr plates. dna plasmid controls were reproducible (< ct difference) between plates. all real time pcr reactions were run on an applied biosystems fast real-time pcr system using the fast system software, version . . . (applied biosystems). data were analyzed using fast system software, version . . . except for plasmid dna efficiency calculations which were determined using fast system software version . . . the efficiency of each gene amplification was calculated by plotting the average ct (y-axis) against the logarithm of the input amount of rna/l cdna (x-axis). both a -fold dilution series and a -fold dilution series were used for each gene and rna table ferret genes amplified in this study and sequence similarly to other published ferret experimental and predicted sequences by blastn. features of the blastn alignments are indicated. note that the aligned sequences are divided into sequences submitted from laboratory-derived experimental data as well as sequences from the ferret genome which have been predicted and designated using the gene prediction tool. genbank accession # generated for this study the efficiency of each reaction was determined using a -fold dilution standard curve. cytokine and chemokine (black circles) and housekeeping (white circles) genes are shown. the mean efficiency for all genes is indicated by the horizontal line. the reaction efficiency (e) is indicated with the corresponding % efficiency, with the ideal value 'e = ' and the acceptable range ( . - . ), indicated. (c) one or two primer/probe sets were combined in a real time pcr reaction and assayed against each of the single target genes. (d) one primer/probe set was assayed against the target gene in a pool (four to seven plasmids) or alone. (e) one or two primer/probe sets were combined in a real time pcr reaction and assayed against the target gene in a paired pool. (c-e) all samples were run in triplicate, with mean and standard deviation indicated. *p < . . set. real time pcr efficiency (e) = ( − /slope ) for -fold dilution series (pfaffl, ) and ( − /slope ) for -fold dilution series. % real time pcr efficiency = (e − ) × . if the standard deviation for the efficiencies determined using -fold and -fold dilution series fell within %, the average efficiency was used in all calculations. if the standard deviation was > %, the efficiency calculated using -fold dilutions was used. the geometric mean of the efficiencies for the indicated genes was used for the housekeeping gene efficiency. the gene stability of housekeeping genes was calculated using genorm in qbase+ version . (biogazelle) (vandesompele et al., ) . the fold change of expression of a gene was calculated using relative quantitation with kinetic pcr correction (pfaffl, ) . fold change = (e target ) ct target (control−test) /(e hkp ) ct hkp (control−test) where 'hkp' was the geometric mean of all housekeeping genes for each data point as indicated in figure legends and ' ct' = ct control -ct test. "undetermined' data points in cytokine gene expression were assigned a ct of " " to enable calculation of fold change and are indicated in figure legends. the control sample for cultures was unstimulated cells cultured in complete-rpmi. the ability to perform duplex real time pcr was assessed using two tailed t-test. data were analyzed using r software (version . . ( - - )) (team, ) . expression of target genes in cultured cells was compared using one-way anova and tukey's post hoc test (only differences to unstimulated cells are indicated). the mean of duplicate or triplicate wells was calculated and used as a single value for each ferret. *p < . , **p < . , ***p < . . . . development of a two-step taqman real time pcr assay to measure expression of ferret immune mediator and housekeeping genes cytokine, chemokine and housekeeping genes were amplified from total mrna generated from cultures of ferret lymph node cells using primers targeting conserved regions of each gene. all table comparison of the sensitivity of taqman and sybr green real time pcr assays. all reactions were performed with the same plasmid standard, except in cases where the appropriate plasmid standard encompassing both primer sets was not available, and a cdna sample was used instead. the same samples were used to compare the taqman assays in this study with either previously published taqman assays or previously published sybr green assays for an individual gene. genes cloned in this study were sequenced and the sequences run in blastn whereby they matched other published and predicted sequences for the corresponding ferret gene on genbank, especially those derived from the ferret genome project (di palma et al., ) ( table ) . sequences generated in this study were also translated and all sequences aligned to known sequences of the corresponding proteins from other species in genbank (data not shown). all primer/probe sets for the taqman real time pcr assay (table ) were tested to ensure specificity and optimum efficiency using plasmid dna standards. the absence of non-specific amplicons was verified for all primer/probe sets (fig. a) . the % efficiency of each primer/probe set was consistent and between and % which is equivalent to an efficiency of . - . (fig. b) , indicating that the template doubled after each cycle during exponential amplification. the correlation co-efficient for all samples was between . and . (data not shown). the sensitivity of the primer/probe sets for each gene was consistent across the different taqman assays, with all genes detected down to . pg dna plasmid (average . rna copies), except gapdh, which was detected down to . pg dna ( . rna copies) ( table ). the limits of detection were consistent for previously published taqman primer/probes sets (nakata et al., ) (table ) . there was no change in the sensitivity of the assay when primer/probes were mixed in the indicated duplex real time pcr reactions (fig. c) , or when plasmid samples were prepared in pools (fig. d) , or a combination of both duplex real time pcr reactions and sample pools (fig. e) , except for l , which was less sensitive when assayed with other genes (fig. e) . to enable comparison between samples from multiple ferrets, the expression of all genes was determined as a fold change, using the calculation for relative quantification to housekeeping gene(s), with kinetic pcr efficiency adjustment (pfaffl, ) . as this equation relies on consistency between samples in the rna quantity assayed, and the optimum reaction efficiency, as well as minimal fluctuation in the expression of housekeeping genes (endogenous controls) (peters et al., ; mane et al., ; bruder et al., ) , these parameters were optimized using rna extracted from cultured ferret lymph node cells stimulated with mitogens or with influenza virus. as the rna quantity varied between samples (range . - . g/well, fig. a ), we first determined the minimum amount of rna for use in the taqman real time rt-pcr assay. the reproducibility of both reverse transcription (fig. b) and real time pcr (fig. c ) steps was assessed separately for samples with different amounts of rna. a minimum of ng total rna produced highly reproducible results for both reverse transcription and real time pcr steps (standard deviation < . ct) and the ct value was able to detect gene expression ( fig. b and c) where ct (cycle threshold) is the cycle number at which the fluorescent signal of the reaction crosses the threshold to exceed background level. a set of cdna standards was generated from the mitogenand influenza virus-stimulated ferret lymph node cells and the reaction efficiency for all genes determined (examples of graphs and calculations for efficiency are shown in fig. a , compilation of efficiencies for all genes is shown in fig. b and c) . although the reaction efficiencies clustered for each of the ferret lymph node cdna preparations (mitogen or virus stimulation in fig. b and c, respectively), the efficiencies using the -fold dilutions of cdna samples were more broadly spread than for the plasmid preparations (compare -fold dilution in fig. b and c with fig. b) . furthermore, analysis of the -fold dilutions indicated some variability due to low level cytokine mrna expression (such as for il ␣, ifn␣). use of -fold dilutions of the cdna standards enabled the efficiency to be determined and data points overlaid with the data points generated by the -fold dilution series, suggesting this approach is acceptable to determine cytokine levels (fig. a) . to minimize the potential effect of initial sampling variability, a calculation of gene stability was performed for housekeeping genes (vandesompele et al., ) . assessment of the stability of expression of housekeeping genes following stimulation with different mitogens (fig. a ) or influenza virus (fig. b ) demonstrated that expression of some housekeeping genes, such as gapdh, was affected more than others and this effect was specific to different stimuli (compare fig. a to b). as gapdh was more variable in expression than the other housekeeping genes; atf , l and hprt following mitogen stimulation, gapdh was excluded and a combination housekeeping genes was used as a reference (fig. c ). in contrast, following virus stimulation, all housekeeping genes were relatively stable and thus all housekeeping genes were able to be used as references (fig. d) . these data suggests that reaction efficiencies and the most stable housekeeping genes should be determined for a type of sample set of cdna under analysis to be able to accurately calculate gene expression. to test the ability of ferret leukocytes to produce mrna cytokines and chemokines, lymph node cells from naïve ferrets were cultured with various mitogens known to activate t and b lymphocyte and macrophage/monocyte responses (fig. ) and with live or heat inactivated influenza virus (fig. ) and the cytokine and chemokine expression profiles were determined (summarized in (cona) or phytohaemagglutinin (pha), which act by cross linking t cell receptors via sugars on the surface of human t lymphocytes (chilson and kelly-chilson, ) , induced similar cytokine profiles, increasing expression of il , il , il , il , il , granzyme a, tnf␣ and ifn␥, most with high fold changes, consistent with effective stimulation of t lymphocytes (fig. ) . pha also induced expression of il p mrna. the addition of con a or pha significantly reduced the expression of mcp- and ifn␣ in their respective cultures (fig. ) . ionomycin and phorbol -myristate -acetate (pma) are mitogenic for lymphocytes as they bypass surface receptors and activate cellular responses by increasing intracellular calcium and directly activating protein kinase c, respectively (nishezuka, ; al wabel et al., ) . culture with ionomycin significantly increased mrna expression of il , il and ifn␥. pma significantly increased levels of tnf␣ and il ␤. expression of ifn␣ and il p mrnas was significantly reduced upon culture with ionomycin or pma (fig. ) . lipopolysaccharide (lps) is a potent activator of naïve and mature b lymphocytes (andersson et al., ; smith et al., ) , monocytes and macrophages. stimulation with lps induced significant levels of il and a small increase in expression of il . no expression of ifn␤ was detected in any culture stimulated with mitogen. real time pcr assays performed as duplex pcrs with the same primer pairs as shown in fig. e detected upand down-regulation of the same cytokines as assays using single primer/probe sets (data not shown). real time assays run with (vandesompele et al., ) . the least stable gene is shown on the left, the most stable gene on the right. -fold less cdna ( ng rna/l cdna) for il , ifn␥, l and atf detected the same fold differences (data not shown). culture of ferret leukocytes with live or heat inactivated a(h n )pdm virus for h induced different cytokine profiles from those stimulated by mitogens (fig. ) . live a(h n )pdm virus induced a significant reduction in expression of il ␣, il ␤, il and mcp , as well as large increases in expression of granzyme a, ifn␣ and ifn␥, whereas heat inactivated virus induced negligible effect on cytokine expression. note that cultures assayed at and h also showed similar results (data not shown). fig. . gene expression in mitogen-stimulated ferret lymph node cells. lymph node cells from four naïve ferrets were stimulated with the indicated mitogens for h, then rna was isolated, reverse transcribed and assayed by real time pcr using ng initial rna/reaction. each data point represents the average fold change of duplicate or triplicate culture wells compared to the geometric mean of l , hprt and atf housekeeping genes. ifn␤ was not detected. horizontal bars indicate the mean for each group; dotted line indicates no fold change. statistical difference in fold-change compared to cells with no stimulation is indicated. in this study, we describe a series of real time taqman rt-pcr assays that can be used to characterize the expression of cytokines and chemokines of the innate and adaptive immune response in ferrets. the sequences of the ferret genes cloned in this study and used for design of the primers and probes, aligned with other previously published sequences as well as with the predicted sequences from the ferret genome project (di palma et al., ) . previous studies have predominantly utilized sybr green real time rt-pcr and required a minimum quantity of ng initial rna per reaction for detection of a single ferret cytokine or chemokine (svitek et al., ; . here we have demonstrated that ng total rna is sufficient in a taq-man real time rt-pcr assay to detect most cytokine and chemokine mrnas. by multiplexing reactions with probes using different fluorochromes, samples with low amounts of rna, such as ferret respiratory samples (suguitan et al., ) , may still be analyzed using this assay without loss of sensitivity, providing a significant sample and cost reduction. in our study we showed that a minimum of duplex assays could be used, mixing primers and probes specific for a housekeeping gene and a cytokine/chemokine gene or two cytokine/chemokines genes. with further optimization, a higher number of targets may be able to be multiplexed. real time pcr results can be reported in copy number or as fold change both compared to a control, depending on whether absolute or relative quantification is required (giulietti et al., ; pfaffl, ) . absolute quantification requires dna or in vitro transcribed rna standards to be included in each reaction, whereas relative quantification can be achieved by including a set of cdna standards generated from the sample of interest, or no standards at all, if the efficiency of each reaction is identical (pfaffl, ) . as it is difficult to maintain the stability of a large number of rna standards (giulietti et al., ) , absolute quantification was not used in this study. furthermore, the necessity for a standard curve on each assay plate would increase the number of plates required for an experiment, which may become impractical when assaying a large number of test samples. calculation of fold change relative to housekeeping gene(s), with kinetic pcr efficiency adjustment, incorporates corrections for reverse transcription efficiency and pcr efficiency, as well as individual sample addition, by using normalizer housekeeping genes. in our study, although initial assays using dna plasmids demonstrated that the efficiencies of all primer/probe sets in the taqman real time pcr assays were consistent, data from cultures of ferret lymph nodes and our preliminary data with ferret respiratory samples (not shown) indicate that the efficiency of reactions clusters for each set of cdnas and appropriate housekeeping genes need to be determined for each sample type. the importance of careful assessment of suitable housekeeping genes for real time pcr has also been demonstrated for human, canine and ferret tissues (peters et al., ; mane et al., ; bruder et al., ) . as real time pcr measures the ct in the exponential phase of the pcr, it has been argued that the effect of differences in pcr efficiency is minor (giulietti et al., ) . however, we anticipate this taqman assay will be useful for various ferret samples, particularly nasal washes or bronchoalveolar lavages, and respiratory tissues in virus-infected ferrets, which may have low amounts of rna, and variable levels of expression of cytokine and chemokine genes. given the variability in the cellular composition of samples from different sites (e.g. lung tissue compared to lymph node), incorporation of corrections for reverse transcription and pcr efficiency and housekeeping gene variability are necessary in these samples. the cytokine profiles induced by stimulation of lymph node cells from naïve ferrets with mitogens were consistent with those reported in studies using human and other animal leukocytes. stimulation of ferret lymph node cells with cona induced similar profiles to cultures of mouse splenocytes (candolfi et al., ) , human pbmcs (al wabel et al., ; yaqoob and calder, ; radke et al., ) , woodchuck pmbcs (menne et al., ) and calf cd + and cd + pbmcs (tanaka et al., ) stimulated with cona. of interest, cona has been shown to induce expression of il ␤ and low levels of il ␣ in human pbmcs (yaqoob and calder, ), but we did not detect increases in either cytokine in ferret lymph node cells. we also used the other published primers for detecting il ␤ rowe et al., ) with our samples but could not detect expression (data not shown). increased levels of tnf␣, il and ifn␥ were reported following culture of human pbmcs with pha (godoy-ramirez et al., ; anderson and teuber, ) , and we obtained similar results using ferrets cells. ionomycin and pma have been shown to increase production of il , il and ifn␥ in human pbmcs (jung et al., ) and il , il , ifn␥ and tnf␣ in ferret bal, splenocytes or pbls (rutigliano et al., ; martel and aasted, ); all of these genes were also upregulated in this current study following stimulation of ferret leukocytes with either ionomycin or pma. lps is a potent stimulator of il ␣ and ␤, il , and tnf␣ from human pbmcs (al wabel et al., ; yaqoob and calder, ; matera et al., ; coch et al., ) and il and tnf␣ from mouse pbmcs and macrophages (kawai et al., ) . similarly, il ␣ and ␤, il , and il , but not tnf␣, were increased in lps-stimulated ferret lymph node cultures. a similar study in which ferret pbmcs were cultured with lps detected expression of il , ifn␥, il and tnf␣ by taqman real time rt-pcr at earlier timepoints, suggesting the kinetics of the response are important (nakata et al., ) . we also assessed tnf␣ expression using previously published taqman primers and probe (nakata et al., ) and sybr green primers rowe et al., ) with our samples and the profiles were consistent with those obtained with the taqman assay designed in this study (data not shown). stimulation of ferret lymph node cultures with live influenza virus resulted in upregulated expression of ifn␥ and granzyme a, and these markers of t lymphocyte (and nk cell) activation, are also upregulated following stimulation of human pbmcs with influenza virus (forbes et al., ; vanders et al., ) . il was not detected in ferret cells, although it has been detected by flow cytometry in human cells following in vitro influenza virus stimulation (scheible et al., ; guérin-el khourouj et al., ) . increased expression of type i interferons (also seen upon virus stimulation in human pbmcs (forbes et al., ) ) and a corresponding decrease in mcp expression was induced in ferret cells following exposure to influenza virus. this inverse relationship has also been reported in mice co-infected with bacteria and influenza virus and is suggested to be due to type i interferon-mediated suppression of macrophages (nakamura et al., ) . this may also explain the reductions in il and il in ferret lymph node cells cultured with virus as both these mediators would be typically produced by macrophages in the lymph node. the significant down-regulation of ifn␣ and variable expression of mcp- and il p in cultures stimulated with mitogens may also be due to altered activation of cells of the innate immune system. the magnitude and differential patterns of gene expression detected here indicate 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) avian influenza virus acts as a virulence factor in a host-specific manner in mammals disease phenotype of a ferret cftr-knockout model of cystic fibrosis early cytokine mrna expression profiles predict morbillivirus disease outcome in ferrets severe seasonal influenza in ferrets correlates with reduced interferon and increased il- induction differential cytokine gene expression in cd + and cd + t cell subsets of calves r: a language and environment for statistical computing. r foundation for statistical computing alterations in inflammatory, antiviral and regulatory cytokine responses in peripheral blood mononuclear cells from pregnant women with asthma accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes receptor (slam [cd ]) recognition and the v protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus cytokine production by human peripheral blood mononuclear cells: differential sensitivity to glutamine availability tropism and infectivity of influenza virus, including highly pathogenic avian h n virus, in ferret tracheal differentiated primary epithelial cell cultures the authors are grateful for advice provided by dr wa-chin boon, dr john roiniotis, professor paul hertzog and mr steve vander hoorn. the authors are also grateful for technical assistance provided by biocsl limited animal house staff. the authors also acknowledge the contribution of gifts of reagents from dr liyen loh and ms sarina camuglia. the melbourne who collaborating centre for reference and research on influenza is supported by the australian government department of health. key: cord- -ym jsir authors: eisenächer, katharina; steinberg, christian; reindl, wolfgang; krug, anne title: the role of viral nucleic acid recognition in dendritic cells for innate and adaptive antiviral immunity date: - - journal: immunobiology doi: . /j.imbio. . . sha: doc_id: cord_uid: ym jsir abstract dendritic cells which are located at the interface of innate and adaptive immunity are targets for infection by many different dna and rna viruses. dendritic cell subpopulations express specific nucleic acid recognition receptors belonging to the toll-like receptor family (tlr , , , ) and the cytosolic rna helicase family (rig-i, mda , lgp ). activation of dendritic cells by viral dna and rna via these receptors is essential for triggering the innate antiviral immune response and shaping the ensuing adaptive antiviral immunity. this review will summarize our current knowledge of viral nucleic acid recognition and signaling by toll-like receptors and rna helicases focusing on recent evidence for their specific functions in antiviral defense in vivo. dendritic cells (dcs) , which are present in the tissues of all organs as well as in the circulation and in lymphatic organs, are targets for infection by many different viruses. viruses entering the body through epithelial surfaces or directly via the blood stream encounter dcs at early time points during infection. dcs are unique in their capacity to migrate from the periphery to lymphoid organs or from the blood stream into lymphatic and peripheral tissues. virus-infected dcs can therefore transport viruses to other parts of the organism and thus contribute to the spreading of the pathogens to many organs during systemic infection. however, dcs are also equipped with a range of receptors (for example toll-like receptors, tlrs) that recognize conserved molecular patterns of viruseseither viral proteins or viral nucleic acids with specific distinguishing features (lund et al., ; krug et al., a, b; schulz et al., ) . triggering of these pattern recognition receptors in virus-infected dcs initiates the innate antiviral immune response, which is essential for limiting further viral dissemination (dalod et al., ; krug et al., a) . the dcs themselves produce inflammatory cytokines and antiviral interferons in response to viral infection. in addition dcs express ligands for activating receptors on the surface of natural killer (nk) cells leading to activation of this innate antiviral effector cell type (andoniou et al., ) . dcs are experts in the processing and presentation of viral antigens on mhc class i as well as crosspresentation on mhc class ii. the antigen presenting function of dcs is critically involved in the generation of efficient adaptive immunity against viruses (belz et al., ) . dc activation by viruses via pattern recognition receptors provides the nd and rd signals required for the priming of naı¨ve-specific t lymphocytes and their differentiation into effector t cells. dc subpopulations fulfill specific tasks in the generation of antiviral immune responses. plasmacytoid dcs (pdc), which have also been described as natural interferon producing cells (perussia et al., ; ronnblom et al., ) represent a specialized subset of dcs characterized by their ability to express large amounts of type i interferons and interferoninduced genes in response to viruses as well as synthetic tlr and tlr ligands (cella et al., ; krug et al., a krug et al., , siegal et al., ) . in response to many viruses pdcs produce -to fold more type i interferon on a single cell basis than other cell types such as conventional dc or macrophages (colonna et al., ) . in many viral infections pdcs have been shown to release the first wave of type i interferons and thus support subsequent steps of antiviral immunity, including nk cell activation, th cell and ctl differentiation (dalod et al., (dalod et al., , krug et al., a; cervantes-barragan et al., ) . it has been shown in vivo and in vitro however, that cells other than pdcs (for example conventional dcs, macrophages and non-immune cells), also significantly contribute to type i ifn responses during viral infection despite the lower amount of type i interferon produced per cell barchet et al. ; krug et al., a) . in contrast to other dc populations, pdcs are less efficient in presenting antigens. several studies report that pdcs are capable of presenting antigens to t cells and triggering t cell proliferation and differentiation to effector cells (boonstra et al., ; dalod et al., ; salio et al., ; schlecht et al., ) . however, initial priming of t cell responses in many viral infections relies on conventional dcs, especially the cd a + dcs in the murine system (belz et al., ) . in addition it has been reported recently that pdcs activated via tlrs may also induce regulatory t cells (moseman et al., ) , which may inhibit the expansion and differentiation of antiviral t lymphocytes to prevent overstimulation of the immune system. what is the molecular basis for the rapid and robust interferon production in pdcs? pdcs recognize viruses via tlrs, particularly via the endosomally located tlr , tlr and tlr . downstream signaling of tlr / and tlr leads to the activation of irf , which is the central transcription factor for expression of type i interferon and interferon inducible genes (honda et al., b) . in contrast to most other cell types pdcs show constitutive expression of irf which is required for the rapid and potent production of the full range of type i ifns (izaguirre et al., ; kerkmann et al., ) . pdc are unique in that ifn-a expression is induced by ligands of tlr / and tlr via a supramolecular complex formed between myd , traf , irak / and irf , which leads to direct activation of the constitutively expressed irf only in this specialized cell type (honda et al., ; kawai et al., ) . the cytoplasmatic rna helicases rig-i or mda do not contribute to viral recognition in pdcs, because the response of pdcs from mice deficient in rig-i or the essential signaling adaptor of rig-i and mda to rna viruses is comparable to that of wild-type mice . it has been demonstrated in several studies that pdcs recognize herpes simplex virus type and (hsv- and - ) (krug et al., b; lund et al., ) , murine cytomegalovirus (mcmv) (krug et al., a) and also recombinant replication-deficient adenovirus (basner-tschakarjan et al., ) , via tlr , whereas influenza virus (barchet et al., ; diebold et al., ) , vesicular stomatitis virus (vsv), newcastle disease virus (ndv) and sendai virus (sev) (lund et al., ) are recognized by tlr in pdcs. many viruses that enter pdcs and trigger tlrs are prevented from expressing viral genes and replicating their genome. thus, the activation of the type i ifn response in pdcs most often is not counteracted by viral immune evasion strategies. accordingly, active viral replication is not required for the induction of type i ifn production in pdcs for many enveloped viruses, for example influenza virus or herpes viruses (krug et al., b; lund et al., ) . following endocytic uptake it is assumed that these viruses are retained in acidified endosomal compartments, where tlr and tlr are localized, so that the viral particles are degraded and viral nucleic acids come into contact with tlr and tlr . a recent report, however, clearly demonstrates that cytosolic viral replication and a transport mechanism involving autophagy is necessary for triggering the tlr -dependent response to other rna viruses, such as vsv or sev in pdcs (lee et al., ) . after infection of pdcs with these rna viruses, viral replication intermediates in the cytosol are internalized in autophagic vesicles which are then directed towards the tlr -containing lysosomes, where recognition of the viral rna occurs (lee et al., ) . the toll-like receptors are a family of type i transmembrane glycoproteins characterized by the extracellular leucine-rich-repeat domain and the cytoplasmatic tir domain for downstream signaling, which is homologous to the tir domain of the il- and il- receptors. tlr expression is predominantly found in various immune cells like dendritic cells, macrophages, b cells and some types of t cells. moreover tlrs can be expressed by non-immune cells such as fibroblasts and epithelial cells. among the tlr family a subgroup of mainly intracellularly localized tlrs (tlr , , , ) can be differentiated from tlrs which are expressed on the cell surface (tlr , , , , , ) . whereas surfaceexpressed tlrs are mainly involved in the recognition of bacterial and fungal cell wall components as well as some viral proteins, the intracellular/endosomal tlrs have the capacity to detect microbial nucleic acids, particularly viral dna and rna. apart from activating the nfkb and mapk signaling pathways leading to inflammatory cytokine and chemokine production as well as costimulatory molecule expression, the intracellularly localized nucleic acid recognition receptors tlr , , and specifically trigger type i interferon production via myd -and trif-dependent signaling pathways. tlr binds double-stranded (ds) rna which is found in dsrna viruses, such as reovirus, or is generated during replication of single-stranded (ss) rna viruses such as west nile virus (wang et al., ) or respiratory syncytial virus (rudd et al., ) or as a by-product of symmetrical transcription of viral dna, for example from herpes viruses. another ligand for tlr is poly(i:c) which is a synthetic dsrna mimicking viral infection (alexopoulou et al., ) . studies in tlr À/À mice identified poly(i:c) and dsrna as ligands for tlr , which induce type i ifn and proinflammatory cytokine production. up-regulation of tlr expression by type i ifn amplifies the response to tlr ligands. tlr is expressed in conventional dc (cdcs) subpopulations and macrophages as well as nonimmune cells, such as fibroblasts and epithelial cells, and the cellular localization of tlr varies between different cell types. in conventional dcs tlr is thought to be localized in intracellular vesicular structures (matsumoto et al., ) . high expression of tlr is seen in the subset of cd a + cd À dc which phagocytose apoptotic cells, including dying cells that are infected by rna viruses (edwards et al., ) . in other cell types such as fibroblasts or epithelial cells tlr is expressed on the cell surface (matsumoto et al., ) . epithelial expression of tlr can be found in many different organs including the airways as well as the gastrointestinal and urogenital system. furthermore, strong expression of tlr is detectable in the brain suggesting a specific role in response to viral infections of the central nervous system. despite this wide expression pattern tlr does not appear to be essential for the initial antiviral immune response in several mouse models of viral infection (edelmann et al., ; lopez et al., ) . downstream signaling of tlr is unique among all tlrs, because it is entirely myd -independent and is mediated by the cytosolic tir-domain containing adaptor protein trif. trif is recruited to the cytoplasmatic tir domain of tlr and interacts with a set of different signaling molecules and kinases which in turn initiate activation of nfkb or irf and irf leading to type i interferon induction. interaction of trif with traf allows complex formation with the non-canonical ikks-tbk and ikke leading to the activation of irf and irf , which form homo-or heterodimers upon phosphorylation and are then translocated to the nucleus to induce type i ifn and ifn inducible gene expression sharma et al., ; yamamoto et al., ) . nfkb activation by tlr ligands is mediated in two waysvia association of trif with rip or via interaction of trif with traf , which in turn activates tak . both rip and tak mediate activation of canonical ikks (ikka, ikkb) resulting in ikb degradation and nfkb translocation to the nucleus (meylan et al., ; sato et al., ) . the role of tlr in antiviral immunity remains unclear so far. most studies did not find an essential role of tlr for the generation of effective antiviral immune responses. tlr -deficient mice are as susceptible to reovirus, vsv and lcmv infection as wild-type mice and there was no significant difference in generating specific cd and cd t cell responses to these viruses (edelmann et al., ) . interestingly, however, virally induced cns injury was improved by tlr -deficiency suggesting that inflammatory responses mediated by tlr are at least partially responsible for a breakdown of the blood-brain barrier which facilitates and enhances virus entry into the brain. tlr -deficient mice survived viral cns infection with west nile virus (wang et al., ) , which is characterized by meningitis and encephalitis induced by inflammatory mediators, for longer time periods than wild-type mice. similarly, in murine influenza a virus infection tlr -mediated inflammatory responses in the lung contribute significantly to host morbidity and lethality. despite higher pulmonary viral loads tlr À/À mice showed less inflammation and better survival (le goffic et al., ) . interestingly, human lung epithelial cells express proinflammatory cytokines including il- and il- upon infection with influenza a virus in a tlr dependent manner (le goffic et al., ) , suggesting that tlr -mediated inflammatory responses may also contribute to influenza virus-induced lung pathology in humans. the role of tlr in mcmv infection is more controversial. whereas edelmann et al. ( ) claimed no impairment in antiviral response to mcmv in tlr À/À mice, another publication suggested a higher susceptibility to mcmv in the absence tlr (tabeta et al., ) . in this study tlr deficiency results in higher splenic viral titers compared to wild-type mice. similar observations were made when infecting trif lps /lps mice with mcmv (hoebe et al., ) . the increased viral load was accompanied by a decreased cytokine response which mostly affected type i ifn and to a lesser extent il p and ifn-g produced by nk and nkt cells (tabeta et al., ) . crosspriming occurs most efficiently in the specialized subpopulation of cd a + dcs, which have a high capacity for internalization of apoptotic cells. tlr which is expressed at high levels in cd a + dcs (edwards et al., ) plays a critical role for crosspriming of ctls directed against viruses that do not infect dcs directly. cd a + dcs are activated by viral rna contained in internalized virally infected apoptotic cells and crosspresent viral antigens to specific cd + t cells in a tlr -dependent manner (schulz et al., ) . toll-like receptor tlr has been described to recognize bacterial dna or synthetic oligodesoxyribonucleotides (odn) containing specific unmethylated cpg sequence motifs. these can also be found in the genome of dna viruses. viruses recognized by tlr are hsv type and krug et al., b) , mcmv (krug et al., a; lund et al., ; tabeta et al., ) , adenovirus (basner-tschakarjan et al., ) and baculovirus (abe et al., ) . recognition of hsv or mcmv by tlr of pdcs results in robust induction of type i interferon and inflammatory cytokines (krug et al., a, b; lund et al., ) . cpg odn class a, b and c have been designed to trigger primarily type i ifn response (a) or costimulatory molecule expression and inflammatory cytokine production (b) or both (c) in pdcs. cpg-b and cpg-c additionally trigger b cell stimulation via tlr (krug et al., ; verthelyi et al., ; hartmann et al., ) . induction of type i ifns by tlr ligands in pdcs depends on retention of the ligand-receptor complex within early endosomes at a ph value between . and . (guiducci et al., ) . delivery of tlr ligands to late endosome with lower ph values (o . ) impairs the induction of type i ifns and promotes inflammatory cytokine and costimulatory molecule expression in pdcs (guiducci et al., ) . early endosomal retention is achieved by using cpg-a, which forms aggregates or by transfecting cpg-b with cationic liposomes or delivering cpg dna in the form of immune complexes (kerkmann et al., ; means et al., ; guiducci et al., ) . tlr ligands delivered within herpes virus particles also seem to be retained long enough in the early endosomal compartment to trigger type i ifn responses efficiently. upon ligand binding the tir domain of tlr recruits myd which forms a supramolecular complex with traf , irak , irak and irf (honda et al., ; kawai et al., ) . irf becomes activated upon phosphorylation which results in homodimerization of irf or heterodimerization of irf and irf . these dimers then translocate to the nucleus and induce expression of type i interferon and interferon inducible genes. several proteins were implicated in the phosphorylation of irf including irak- , ikka, and a precursor of osteopontin (hoshino et al., ; shinohara et al., ; uematsu et al., ) . lack of irak abolishes ifn-a production and irf activation in response to tlr , tlr and tlr ligands . irf plays the role of a ''master regulator'' in the induction of type i interferon and ifn inducible genes in response to viruses (honda et al., a) . the currently accepted two step model of positive feedback regulation of type i interferon gene expression consists of an initial phase with activation of irf expressed constitutively at low levels, formation of irf -homodimers or irf /irf -heterodimers and induction of small amounts of ifn-b/ifn-a and chemokines. during the second phase secreted type i ifns signal via type i ifn receptor in an autocrine and paracrine manner. downstream signaling of the type i ifn receptor induces a strong up-regulation of irf production leading to full expression of type i ifn genes (positive feedback loop). although pdcs constitutively express irf at higher levels than other cells, they also depend on further up-regulation of irf by type i ifn signaling to be able to sustain high level production of type i ifns honda et al., b) . in contrast to pdcs, type i ifn production in myeloid dcs in response to dna viruses is mediated by both tlr -dependent and tlr /myd -independent pathways. downstream signaling of tlr in murine myeloid dcs shows differences compared to pdcs. in myeloid dcs irf plays a crucial role in the downstream signaling of tlr . irf À/À mice show an impaired induction of ifn-b, inos and il- p upon stimulation with tlr ligand cpg-b, whereas type i interferon response of pdcs is not affected by lack of irf (negishi et al., ; schmitz et al., ) . what is the role of tlr for antiviral immunity in vivo? several studies have described increased susceptibility of tlr À/À mice to systemic murine cytomegalovirus (mcmv) infection (tabeta et al. ; krug et al., a; delale et al., ) . we could show that tlr -dependent recognition of mcmv by pdcs and conventional dc is clearly involved in the innate immune response to mcmv. in the very early phase of systemic mcmv infection pdcs and conventional dcs release the first wave of type i interferons and inflammatory cytokines including il- . the expression of these cytokines peaks between and h postinfection (dalod et al., ; orange and biron, b) and triggers non-specific nk cell activation at this time point. control of viral replication and clearance of the virus from infected organs during the first week of mcmv infection relies mostly on nk cells, which produce ifn-g (mediated by il- and il- ) and kill infected cells (orange and biron, a) . we found a significant reduction in ifn-a and il- serum levels in tlr -and myd -deficient mice at h after infection. however, ifn-a serum levels comparable to those of wild-type mice could be observed in tlr -and myd -deficient mice at later time points suggesting the existence of an additional tlr /myd -independent type i ifn induction mechanism with delayed kinetics. loss of the early ifn-a peak is due to an impaired function of pdcs in tlr À/À mice as mcmv recognition and type i ifn production are tlr dependent in this cell type. pdc depletion in vivo led to markedly reduced ifn-a levels at h after infection reflecting their almost exclusive role for ifn-a production at this early time point. in contrast to ifn-a, il- production was severely impaired in tlr -and myd -deficient mice at all time points reflecting the requirement of tlr for il- responses to mcmv in all dc subpopulations. ifn-g production by nk cells which is induced by il- and il- was also significantly reduced in the absence of tlr or myd . these defects in the innate immune response to mcmv correlated with higher viral titers in spleen and liver of tlr À/À and myd À/À mice on the c bl/ background reflecting increased susceptibility to mcmv in the early phase of the infection krug et al., a) . a recent report showed that pdcs which are recruited to the vaginal mucosa after local infection with hsv- are activated to produce type i ifn in a tlr dependent manner, thus reducing local viral replication and pathology (lund et al., ) . in two mouse models of local infection with hsv- (footpad or eye infection), however, we did not find a significant difference in viral titers between tlr À/À and myd À/À mice and wildtype mice (krug et al., b) . the requirement for tlr in the innate immune response to herpes viruses in vivo is influenced by the site of virus entry and the recruitment of dc subpopulations to the infected tissue. all of the described in vivo studies show that in addition to tlr other pattern recognition receptors and signaling pathways responding to herpes virus infection must exist, which can partially compensate for the lack of tlr and other myd -dependent receptors (krug et al., b; hochrein et al., ; lund et al., ) . the cytosolic dna receptor which has been described recently (stetson and medzhitov, ; ishii et al., ; takaoka et al., ) may also be involved in the tlr independent component of the immune response to herpes virus infections. toll-like receptor and tlr and tlr are both located on the x chromosome and are homologous to each other. gu-rich ssrna sequences of viral or host origin, poly-u rna and specific sirna sequences are ligands for both receptors, whereas synthetic imidazoquinoline derivatives have been designed to specifically activate tlr or tlr or both receptors (heil et al., ; hemmi et al., ; diebold et al., ; jurk et al., ; hornung et al., ; gorden et al., ) . tlr is expressed in human and murine pdcs, conventional dcs and b cells, whereas tlr seems to be functional mainly in human monocytes and myeloid dcs. the role of tlr in the murine immune system remains to be elucidated. similar to what has been described for tlr , activation of tlr and tlr by specific ligands occurs in the acidified endosomal compartment. the myd -dependent signal transduction pathway downstream of tlr and is very similar to tlr -mediated signaling. several viruses are recognized by pdcs in a tlr dependent manner including influenza virus (diebold et al., ; barchet et al., ) , newcastle disease virus (ndv) , vesicular stomatitis virus (vsv) (lund et al., ) , coronaviruses (cervantes- barragan et al., ) and rna viruses (heil et al., ; beignon et al., ) . tlr does not seem to play an essential role in the innate immune response to rna virus infection in vivo. we could not find a significant difference between myd À/À and wild-type mice in the susceptibility to intranasal influenza virus infection and type i ifn response to intravenously injected influenza virus was only partially reduced in the myd -deficient animals (barchet et al., ) . at high viral doses type i ifn response to systemic vsv infection is tlr dependent and mediated by pdcs, whereas at lower doses tlr -independent type i ifn induction pathways play the major role. a recent study demonstrates an essential function of tlr for the innate immune response to systemic coronavirus infection in mice (cervantes-barragan et al., ) . pdcs, but not conventional dcs are capable of producing significant amounts of type i ifn in response to the rapidly replicating coronaviruses and this response is entirely tlr -dependent. pdc depletion led to abrogation of type i ifn response and increased disease severity (cervantes- barragan et al., ) . it can be concluded that tlr (and possibly tlr in the human system) contribute to the initiation of the antiviral immune response against viruses which specifically target pdcs. in addition the ubiquitously expressed rna helicases provide protection against rna viruses in all cell types (see below). depending on the route and kinetics of infection, cellular tropism, viral entry and replication as well as immune evasion mechanisms of individual virus strains one or the other viral rna recognition pathway may dominate the innate antiviral immune response in vivo. as suggested by the murine in vivo studies there is considerable redundancy in tlr-dependent and tlr-independent viral pattern recognition mechanisms also in the human system. irak- -deficient patients are prone to invasive infection with pneumococci, but are resistant to natural viral infection (picard et al., ) . irak -deficient pbmc do not respond to any of the myd -dependent tlr ligands, but are capable of type i ifn production in response to many dna and rna viruses. there also seems to be considerable redundancy between different tlrs, because in contrast to the irak -deficient patients, which show no significant defects in antiviral responses (picard et al., ) , individuals with autosomal recessive deficiency in the intracellular protein unc- b have an impaired antiviral type i ifn response and are susceptible to hsv- encephalitis (casrouge et al., ) . unc- b-deficiency prevents signaling in response to ligands of the endosomally localized tlr , tlr , tlr and tlr . in addition, the unc- b protein, which is localized in the endoplasmic reticulum, is required for efficient crosspresentation and mhc class ii presentation of exogenous antigens . therefore, unc- b-deficiency may affect innate and adaptive antiviral immune responses at the same time. no human deficiencies or mutation in the rig-i/ mda pathway of rna virus recognition have been described so far. however, the critical role of this major pathway for innate antiviral immune responses in humans is greatly supported by the existence of viral immune evasion strategies, which, for example, are employed by hepatitis c virus and paramyxoviruses and are directed against several signaling molecules in this pathway (see below). in addition to the tlrs, a new family of viral pattern recognition receptors consisting of rna helicases retinoic acid inducible gene i (rig-i), melanoma differentiation antigen (mda ) and laboratory of genetics and physiology (lgp ) -was discovered and characterized in the last years. these molecules, which are localized in the cytosol, bind specific rna molecules derived from the genome of different rna viruses and, with the exception of lgp which does not signal itself, trigger a signaling cascade leading to the production of type i ifns and of proinflammatory cytokines in response to viral infection. retinoic-acid-inducible protein i (rig-i) is a dexd/ h box-containing rna helicase that was originally identified as an enhancer of type i ifn expression in response to dsrna poly (i:c) (yoneyama et al., ) . dexd/h box rna helicases are defined by their ability to unwind dsrna using their intrinsic atpase activity. in addition to the c-terminal helicase domain rig-i contains two caspase recruitment domains (card) at its n terminus (zhang et al., ) . cards are found in a number of caspases, but also in other proteins such as nod and nod , involved in sensing intracellular bacterial products (inohara and nunez, ) . the functions of rig-i have been analyzed in detail in the first publication by yoneyama et al. ( ) using deletion constructs of full length rig-i. overexpression of the n-terminal region of rig-i (delta rig-i) comprising the two tandem cards is sufficient to induce irf and nfkb activation even in the absence of a dsrna stimulus or viral challenge. the mutant of rig-i lacking the card domain is not capable of transmitting signals. this n-terminally truncated molecule even has a dominant negative effect since expression of this domain alone prevents activation of irf by dsrna transfection or ndv infection (yoneyama et al., ) . since cards are known to engage in homophilic protein-protein interactions in other signal transduction pathways, it was therefore likely that the cards of rig-i interact with other card containing molecules to activate downstream signaling molecules. the adaptor protein that links rig-i to the activation of tbk /ikke and ikkb was identified and functionally characterized by four independent groups and designated as ifn-b promoter stimulator (ips- ) , mitochondrial antiviral signaling protein (mavs) , virus-induced signaling adapter (visa) (xu et al., ) and card adapter inducing ifn-b (cardif) (meylan et al., ) , respectively. ips- contains an n-terminal card domain that interacts with the tandem card domains of rig-i and a c-terminal hydrophobic transmembrane (tm) domain that localizes it to the outer mitochondrial membrane . deletion analyses have shown that both the card and the transmembrane domain are essential for the function of ips- . the mitochondrial localization of ips- is essential for its activity because deletion of the tm domain, which leads to cytosolic expression of the protein, abolishes the signaling function of ips- . the binding of dsrna to the helicase domain of rig-i likely induces a conformational change that exposes the n-terminal card domains to recruit its signaling adaptor ips- . this interaction subsequently induces ifn-a/inf-b and ifn-induced antiviral effector mechanisms that suppress virus replication (meylan et al., ) . in a recent publication saito et al. reported that an internal repressor (or regulatory) domain (rd) at the c-terminus controls rig-i multimerization and ips- interaction during virus infection and rna binding (saito et al., ) . expression of the c-terminal rd domain, encompassing the amino acids - of the rig-i protein, prevents signaling to the ifn-b promoter and increased cellular permissiveness to hepatitis c virus, whereas deletion of the rig-i rd results in constitutive signaling. saito et al. suggest a model of rig-i autoregulation and signaling predicting that in resting cells the c-terminal rd mediates a conformation of rig-i that masks the cards from signaling. once the cell is infected with virus, rig-i activation and signaling occurs in a stepwise manner involving dsrna binding and conformational changes that subsequently facilitate self-association and interaction with the ips- signaling adaptor. these conformational changes comprising displacement of the rd and unmasking of the cards for signaling via ips- might be triggered by atp hydrolysis. saito et al. also identified an analogous rd within the c terminus of lgp suggesting that lgp might inhibit rig-i through their rd interactions. lgp is a close relative of rig-i, which lacks the cards, but is capable of binding dsrna . thus, lgp may act as a postinduction repressor of rig-i signaling . from several studies it is known that rig-i is essential for antiviral responses to a specific set of rna viruses belonging to flaviviridae, paramyxoviridae, orthomyxoviridae and rhabdoviridae sumpter et al., ; yoneyama et al., ) . since rna is a fundamental entity of most living organisms and is found in abundance in host cells, a molecular pattern must exist that enables rig-i to discriminate between viral rna and host rna species in the cytoplasm of infected cells to prevent constant induction of type i ifns which would lead to autoimmune responses. two recent studies have identified features of viral rna that are structurally different to host rna. hornung et al. ( ) demonstrated that ssrna synthesized in vitro acquires a -triphosphate moiety which is crucial for ifn production in host cells. furthermore, they demonstrated that rna isolated from rabies virus (rv) infected cells is a potent ifn inducer, whereas rna from non-infected cells and dephosphorylated rna isolates abrogated this ifn response. the -phosphorylation status and the absence of a -methyl-guanosine cap provided by the viral polymerase is critical for recognition by rig-i. taken together, the results of hornung et al. ( ) show that rig-i directly recognizes -triphosphate single stranded or double stranded rna independently of viral replication. in the second study, pichlmair et al. ( ) found that influenza a virus, which is known to be sensed by both rig-i and tlr , did not generate dsrna upon infection of bone-marrow-derived dendritic cells. moreover, they observed that influenza virus ssrna, which is uncapped and bears -triphosphates, associated with and activated rig-i. ips- functions as critical link between viral detection by rig-i and the downstream signaling events leading to interferon production. this receptor-adapter interaction results in the activation of the noncanonical kinases tbk /ikke, which in turn induces dimerization of phosphorylated irf and irf and translocation to the nucleus where they activate the transcription of type i ifn genes . coimmunoprecipitation experiments suggest that ips- interacts with tbk and recruits endogenous irf in a virus-inducible manner (xu et al., ) . recently, traf was shown to be critically involved in ips- -mediated ifn-a production and antiviral responses through a direct interaction between the traf domain of traf and a traf interaction motif within ips- (saha et al., ) . there is evidence that the tbk /ikke adaptor protein tank plays a role in ips- -traf -mediated activation of tbk /ikke (guo and cheng, ) . rip and fadd are additional molecules that have been reported to be required for type i ifn production in response to dsrna (balachandran et al., ) . another branch of ips- signaling leads to the activation of the ikk complex resulting in activation of nfkb that controls the expression of genes encoding inflammatory responses, but also expression of ifn-b. previous work showed that ips- activates a nfkbdependent reporter construct in cultured cells (matsuda et al., ) . one of the key signaling proteins in the nfkb pathway is traf , an essential upstream regulator of the ikk complex. ips- binds to traf upon overexpression in mammalian cells and in a yeasttwo hybrid screen xu et al., ) . xu et al. ( ) reported that endogenous ips- also interacts with traf . in addition, they showed that ips- fails to activate nfkb in the absence of traf . seth et al. ( ) reported that virus-mediated induction of the ifn-b gene is not abolished in traf deficient cells and that a mutant ips- protein lacking the traf binding domain is still capable of inducing ifn-b. thus, traf seems to be required for nfkb activation but not ifn-b induction downstream of ips- which is mainly mediated by tbk /ikke. in vitro studies performed with primary cells obtained from rig-i knockout mice confirmed that rig-i plays an essential role in eliciting immune responses against specific negative strand and positive strand rna viruses such as ndv, sev, vsv, japanese encephalitis virus (jev) and influenza virus in various cell types with the exception of pdcs . the experiments demonstrated that type i ifn production by rig-i deficient fibroblasts and conventional dcs is severely impaired in response to these rna viruses. in contrast, pdcs lacking rig-i show normal type i ifn responses to ndv or vsv for example, as the pdc response to these viruses is mediated by tlr . the studies performed so far indicate that the rna helicases rig-i and mda as well as their common signaling adaptor ips- are dispensible for viral triggering of type i ifn responses in pdcs . mda is another dexd/h-box-containing rna helicase that is involved in the sensing of intracellular dsrna and the induction of type i ifn in response to rna viruses (andrejeva et al., ) . it is the closest relative of rig-i, exhibiting and % amino acid homology in the n-terminal card and c-terminal helicase domain, respectively. it was reported in a previous publication, that mda , which was then called helicard, is cleaved by caspases upon induction of apoptosis, thereby separating the card domains from the c-terminal helicase domain, which localizes to the nucleus where it is involved in dna degradation and nuclear remodelling during apoptotic cell death (kovacsovics et al., ) . furthermore, mda has been implicated in the regulation of the growth and differentiation of melanoma cells (kang et al., ) . mda is ubiquitously expressed in low abundance and similarly to rig-i and lgp its expression is induced by type i ifn. like rig-i, mda interacts with the adapter protein ips- upon ligand binding leading to activation of protein kinases that subsequently activate transcription factors irf , irf and nfkb, respectively. it was found recently by sato et al. ( ) that in contrast to rig-i, the c-terminal regulatory domain of mda did not exert a repressor function on mda signaling, which was also not inhibited by lgp . mda overexpression in cell lines even at low levels induces ifn-b promoter activity in the absence of ligand binding. thus, mda expression which is upregulated in response to type i ifn signaling may function as an amplifier of type i ifn production even in the absence of specific mda ligands. although rig-i and mda are similar proteins inducing synthesis of type i ifn via the same signaling pathway, they are specialized in the recognition of different viruses. in vitro studies performed with embryonic fibroblasts and conventional dcs derived from mda knockout mice have shown that mda is specifically required for the recognition of intracellular poly (i:c) dsrna (but not in vitro transcribed ppp-rna) and picornaviruses such as encephalomyocarditis virus (emcv), theiler's virus and mengo virus, whereas rig-i was not necessary for the response to poly (i:c) or picornaviruses gitlin et al., ) . in addition, it was shown that mda might also play a role in the measles virus (mv) induced activation of ifn-b mrna synthesis since a cells transfected with mda showed a strong activation of the ifn-b promoter upon infection with mv. in contrast, the virus did not enhance reporter gene activity in cells that overexpressed rig-i (berghall et al., ) . mda was not required for recognition of vsv, ndv, jev, sev and influenza virus, indicating that rig-i was the predominant pattern recognition receptor for these viruses. it is currently unknown which specific viral rna motifs in picornaviruses are recognized by mda and how ligand specificity is determined on the molecular level. the in vivo relevance of the rig-i and mda pathways for innate antiviral immune responses was addressed by the generation of knockout mice. the specific susceptibility of mda -deficient mice to encephalomyocarditis virus (emcv) infection was shown in two studies gitlin et al., ) . in a direct comparison with ifnar À/À and myd À/À mice kato et al. ( ) could show that survival upon emcv infection is as dramatically reduced in mda À/À mice as in ifnar À/À mice. myd -deficient mice were only slightly more susceptible to emcv infection than wildtype mice. survival after emcv infection was not affected by rig-i-or tlr -deficiency. studies in the ips- knockout mouse confirmed the dramatic reduction in the innate immune response to emcv infection, which correlated with increased viral titers and decreased survival, in the absence of the central downstream signaling adaptor of mda and rig-i (kumar et al., ) . only few in vivo experiments with rig-ideficient mice have been published so far, because for unknown reasons most of the rig-i-knockout mice (on a /c bl/ mixed background) died in utero or within a few weeks after birth . after crossing with icr mice healthy rig-i À/À mice were obtained and compared with littermate controls in the jev and vsv infection models. rig-i À/À mice were more susceptible to jev and vsv infection than wildtype mice and this correlated with reduced type i ifn responses. myd -dependent type i ifn production induced by jev and vsv played only a minor role in these infection models . in accordance with these results ips- -deficient mice succumbed rapidly to vsv infection (sun et al., ) . as pointed out by the study of sun et al. ( ) , type i ifn responses to systemic vsv infection were not entirely abrogated in ips- -deficient mice, suggesting that tlr -dependent pdc-mediated type i ifn release at least partially compensates for the dysfunctional rna helicase pathway, but this compensatory mechanism cannot prevent death after infection with high doses of vsv. the rna helicase pathway as target for viral immune evasion strategies viruses have adapted strategies to evade or inhibit key elements of antiviral immunity. a number of viral proteins inhibit host innate immune responses, prevent viral antigen presentation and abrogate induction of cell death. since the cytoplasmic rig-i/mda system is critical for host defense against rna viruses, the signaling cascades induced by these sensors are also targeted by viruses. various proteins encoded by rna viruses have been shown to antagonize the cytoplasmic rna helicase pathways. ifn antagonists of negative strand rna viruses can interfere with this pathway by hiding their rna (influenza a virus ns protein) or binding to the dsrna receptor (paramyxovirus v proteins) or preventing activation of downstream factors such as irf (ebola virus vp , rhabdovirus p), respectively. the non-structural protein (ns ) of the influenza viruses is a dsrna-binding protein that acts as an ifn antagonist (garcia-sastre et al., ) . by binding to dsrna ns disguises the viral dsrna pattern from the cytoplasmic receptors and inhibits ifn-a/inf-b induction via irf (garcia-sastre, ; talon et al., ) . rig-i was recently demonstrated to be essential for the induction of ifn-b by influenza virus in murine cells. furthermore, it was observed that ns colocalizes with rig-i (mibayashi et al., ) , suggesting that ns forms a complex with rig-i and ips- during viral infection, resulting in inhibition of further downstream signaling. respiratory syncytial virus (rsv) specifically interferes with irf activation and ifn-b response: two viral proteins of rsv, ns and ns , are involved in blocking the pathway leading to irf phosphorylation, although the activation of nfkb and ap- is unaffected (bossert et al., ) . irf phosphorylation by tbk was identified as target of the rabies virus phosphoprotein p (brzozka et al., ) . recently it was shown that the ny- hantavirus g cytoplasmic tail inhibits rig-i-and tbk -directed interferon responses (alff et al., ) . the v proteins of paramyxoviruses target mda , but not rig-i. the v proteins of this diverse group of viruses bind mda via their highly conserved cysteinerich c-terminal domain. this suggests that paramyxoviruses use this interaction to reduce the amount of ifn released by infected cells (andrejeva et al., ; childs et al., ) . furthermore, it was shown that mda is also a target of picornaviruses since mda is degraded in poliovirus-infected cells. interestingly, mda is not directly cleaved by virus-encoded proteinases. degradation of mda in poliovirus-infected cells occurs in a proteasome-and caspase-dependent manner and correlates with the induction of apoptosis in poliovirus-infected cells. poliovirus-induced mda cleavage attenuates the production of type i ifn, thereby allowing higher levels of viral replication and dissemination in the host (barral et al., ) . hepatitis c virus (hcv) encodes the protease ns / a which targets ips- by cleaving it at position cys- , thereby dislocating it from the mitochondrial membrane and thus abrogating further downstream signaling to type i ifn and nfkb-dependent target gene expression meylan et al., ) . interestingly, ips- was also found to be localized to the cytosol and not the mitochondria in liver tissue from patients chronically infected with hcv (loo et al., ) . inhibitors of the hcv ns / a, which have originally been designed to inhibit hcv replication, are able to prevent ips- cleavage and restore the rig-imediated innate immune response to hcv . therefore, ns / a inhibitors which are already being tested in clinical trials may have therapeutic potential for chronic hepatitis c infection. the fact that rna viruses have developed so many effective strategies to interfere with the rna helicase pathway of viral recognition during coevolution with their host provides further proof for the central role of this pathway in antiviral immune defense. involvement of the toll-like receptor signaling pathway in the induction of innate immunity by baculovirus recognition of double-stranded rna and activation of nf-kappab by toll-like receptor the pathogenic ny- hantavirus g cytoplasmic tail inhibits rig-i-and tbk- -directed interferon responses interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter a fadddependent innate immune mechanism in mammalian cells dendritic cells respond to influenza virus through tlr -and pkrindependent pathways mda- is cleaved in poliovirus-infected cells adenovirus efficiently transduces plasmacytoid dendritic cells resulting in tlr -dependent maturation and ifnalpha production endocytosis of hiv- activates plasmacytoid dendritic cells via toll-like receptor-viral rna interactions cutting edge: conventional cd alpha+ dendritic cells are generally involved in priming ctl immunity to viruses the interferoninducible rna helicase, mda- , is involved in measles virus-induced expression of antiviral cytokines flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing t helper type and cell development: dependency on antigen dose and differential toll-like receptor ligation nonstructural proteins ns and ns of bovine respiratory syncytial virus block activation of interferon regulatory factor identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein p interferes with phosphorylation of interferon regulatory factor herpes simplex virus encephalitis in human unc- b deficiency plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon control of coronavirus infection through plasmacytoid dendritic cell-derived type i interferon mda- , but not rig-i, is a common target for paramyxovirus v proteins interferon-producing cells: on the front line in immune responses against pathogens interferon alpha/beta and interleukin responses to viral infections: pathways regulating dendritic cell cytokine expression in vivo dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta myd -dependent and -independent murine cytomegalovirus sensing for ifn-alpha release and initiation of immune responses in vivo viral infection switches non-plasmacytoid dendritic cells into high interferon producers innate antiviral responses by means of tlr -mediated recognition of single-stranded rna does toll-like receptor play a biological role in virus infections? toll-like receptor expression in murine dc subsets: lack of tlr expression by cd alpha+ dc correlates with unresponsiveness to imidazoquinolines ikkepsilon and tbk are essential components of the irf signaling pathway identification and characterization of viral antagonists of type i interferon in negative-strand rna viruses influenza a virus lacking the ns gene replicates in interferondeficient systems essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus synthetic tlr agonists reveal functional differences between human tlr and tlr properties regulating the nature of the plasmacytoid dendritic cell response to toll-like receptor activation modulation of the interferon antiviral response by the tbk /ikki adaptor protein tank rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells species-specific recognition of single-stranded rna via toll-like receptor and small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway herpes simplex virus type- induces ifn-alpha production via toll-like receptor -dependent and -independent pathways identification of lps as a key transducer of myd -independent tir signalling irfs: master regulators of signalling by toll-like receptors and cytosolic patternrecognition receptors role of a transductional-transcriptional processor complex involving myd and irf- in toll-like receptor signaling spatiotemporal regulation of myd -irf- signalling for robust type-i interferon induction irf- is the master regulator of type-i interferon-dependent immune responses sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr -triphosphate rna is the ligand for rig-i ikappab kinase-alpha is critical for interferon-alpha production induced by toll-like receptors and nods: intracellular proteins involved in inflammation and apoptosis a toll-like receptorindependent antiviral response induced by double-stranded b-form dna comparative analysis of irf and ifn-alpha expression in human plasmacytoid and monocyte-derived dendritic cells functional and therapeutic analysis of hepatitis c virus ns / a protease control of antiviral immune defense human tlr or tlr independently confer responsiveness to the antiviral compound r- mda- : an interferoninducible putative rna helicase with double-stranded rna-dependent atpase activity and melanoma growthsuppressive properties cell type-specific involvement of rig-i in antiviral response differential roles of mda and rig-i helicases in the recognition of rna viruses interferonalpha induction through toll-like receptors involves a direct interaction of irf with myd and traf ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction activation with cpg-a and cpg-b oligonucleotides reveals two distinct regulatory pathways of type i ifn synthesis in human plasmacytoid dendritic cells spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferonalpha induction by cpg-a in plasmacytoid dendritic cells overexpression of helicard, a card-containing helicase cleaved during apoptosis, accelerates dna degradation identification of cpg oligonucleotide sequences with high induction of ifn-alpha/beta in plasmacytoid dendritic cells tlr -dependent recognition of mcmv by ipc and dc generates coordinated cytokine responses that activate antiviral nk cell function herpes simplex virus type activates murine natural interferon-producing cells through toll-like receptor essential role of ips- in innate immune responses against rna viruses detrimental contribution of the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells autophagy-dependent viral recognition by plasmacytoid dendritic cells hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity viral and therapeutic control of ifn-beta promoter stimulator during hepatitis c virus infection tlr-independent induction of dendritic cell maturation and adaptive immunity by negative-strand rna viruses toll-like receptor -mediated recognition of herpes simplex virus- by plasmacytoid dendritic cells recognition of single-stranded rna viruses by toll-like receptor cutting edge: plasmacytoid dendritic cells provide innate immune protection against mucosal viral infection in situ large-scale identification and characterization of human genes that activate nf-kappab and mapk signaling pathways subcellular localization of toll-like receptor in human dendritic cells human lupus autoantibody-dna complexes activate dcs through cooperation of cd and tlr rip is an essential mediator of toll-like receptor -induced nfkappa b activation cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus intracellular pattern recognition receptors in the host response inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus human plasmacytoid dendritic cells activated by cpg oligodeoxynucleotides induce the generation of cd +cd + regulatory t cells evidence for licensing of ifngamma-induced ifn regulatory factor transcription factor by myd in toll-like receptor-dependent gene induction program an absolute and restricted requirement for il- in natural killer cell ifn-gamma production and antiviral defense. studies of natural killer and t cell responses in contrasting viral infections characterization of early il- , ifn-alphabeta, and tnf effects on antiviral state and nk cell responses during murine cytomegalovirus infection a leukocyte subset bearing hla-dr antigens is responsible for in vitro alpha interferon production in response to viruses rig-i-mediated antiviral responses to single-stranded rna bearing -phosphates properties of human natural interferon-producing cells stimulated by tumor cell lines the rna helicase lgp inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i deletion of tlr alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection regulation of antiviral responses by a direct and specific interaction between traf and cardif regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp cpg-matured murine plasmacytoid dendritic cells are capable of in vivo priming of functional cd t cell responses to endogenous but not exogenous antigens toll/il- receptor domain-containing adaptor inducing ifn-beta (trif) associates with tnf receptor-associated factor and tank-binding kinase , and activates two distinct transcription factors, nf-kappa b and ifn-regulatory factor- , in the toll-like receptor signaling murine plasmacytoid dendritic cells induce effector/memory cd + t-cell responses in vivo after viral stimulation interferon-regulatory-factor controls toll-like receptor -mediated ifn-beta production in myeloid dendritic cells toll-like receptor promotes cross-priming to virus-infected cells identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf triggering the interferon antiviral response through an ikk-related pathway osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells the nature of the principal type interferon-producing cells in human blood recognition of cytosolic dna activates an irf -dependent innate immune response regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i the specific and essential role of mavs in antiviral innate immune responses toll-like receptors and as essential components of innate immune defense against mouse cytomegalovirus infection the unc b mutation d disrupts exogenous antigen presentation and signaling via toll-like receptors , and dai (dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response activation of interferon regulatory factor is inhibited by the influenza a virus ns protein interleukin- receptorassociated kinase- plays an essential role for toll-like receptor (tlr) -and tlr -mediated interferon-{alpha} induction human peripheral blood cells differentially recognize and respond to two distinct cpg motifs toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis visa is an adapter protein required for virus-triggered ifn-beta signaling role of adaptor trif in the myd -independent toll-like receptor signaling pathway the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity an rna helicase, rhiv- , induced by porcine reproductive and respiratory syndrome virus (prrsv) is mapped on porcine chromosome q key: cord- - td efjw authors: zhou, yanrong; wu, wei; xie, lilan; wang, dang; ke, qiyun; hou, zhenzhen; wu, xiaoli; fang, ying; chen, huanchun; xiao, shaobo; fang, liurong title: cellular rna helicase ddx is involved in transmissible gastroenteritis virus nsp -induced interferon-beta production date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: td efjw transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus (cov) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. unlike most covs that antagonize type i interferon (ifn) production, previous studies showed that tgev infection induces ifn-i production both in vivo and in vitro. however, the underlying mechanism(s) remain largely unknown. in this study, we found that tgev infection significantly facilitated ifn-β production as well as activation of the transcription factors ifn regulatory factor (irf ) and nuclear factor-kappab (nf-κb) in porcine kidney (pk- ) cells. screening of tgev-encoded proteins demonstrated that non-structural protein (nsp ) was the most potent ifn-β inducer and induced ifn-β production mainly by activating nf-κb but not irf . further analysis showed that nsp interacted with ddx , a member of the dexd/h helicase family. knockdown of ddx by specific small interfering rna (sirna) significantly decreased nsp -induced ifn-β production and nf-κb activation. furthermore, tgev-induced ifn-β production and ifn-stimulated gene (isg) expression were decreased in cells transfected with ddx -specific sirna, indicating the vital role of ddx to tgev-induced ifn-β responses. in summary, our data revealed a potential coactivator role of host rna helicase ddx to the induction of ifn-β response initiated by tgev and demonstrated that nsp is an important ifn inducer among the tgev-encoded proteins. transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus (cov) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. unlike most covs that antagonize type i interferon (ifn) production, previous studies showed that tgev infection induces ifn-i production both in vivo and in vitro. however, the underlying mechanism(s) remain largely unknown. in this study, we found that tgev infection significantly facilitated ifn-β production as well as activation of the transcription factors ifn regulatory factor (irf ) and nuclear factor-kappab (nf-κb) in porcine kidney (pk- ) cells. screening of tgev-encoded proteins demonstrated that non-structural protein (nsp ) was the most potent ifn-β inducer and induced ifn-β production mainly by activating nf-κb but not irf . further analysis showed that nsp interacted with ddx , a member of the dexd/h helicase family. knockdown of ddx by specific small interfering rna (sirna) significantly decreased nsp -induced ifn-β production and nf-κb activation. furthermore, tgev-induced ifn-β production and ifn-stimulated gene (isg) expression were decreased in cells transfected with ddx -specific sirna, indicating the vital role of ddx to tgev-induced ifn-β responses. in summary, our data revealed a potential coactivator role of host rna helicase ddx to the induction of ifn-β response initiated by tgev and demonstrated that nsp is an important ifn inducer among the tgevencoded proteins. keywords: transmissible gastroenteritis virus, non-structural protein , ddx , interferon-beta, innate immune response, pattern-recognition receptors introduction transmissible gastroenteritis virus (tgev) is a member of the alphacoronavirus genus within the family coronaviridae in the order nidovirales. tgev infection mainly causes acute enteric disease characterized by lethal watery diarrhea, severe dehydration, and high mortality in suckling piglets less than weeks old, which has led to severe economic losses in the global swine industry since gene name sirna sequence hddx (sense) ggagcuucugauaauuggagguguu hddx (anti-sense) aacaccuccaauuaucagaagcucc pddx (sense) gaaagaccuuggucuggcauuugaa pddx (anti-sense) uucaaaugccagaccaaggucuuuc the first outbreak in in ilinois, usa ( , ) . tgev contains a single-stranded, positive-sense rna genome of about . kb ( ) , including at least nine open reading frames (orfs). two slightly overlapping orfs, orf a and orf b, located at the ' two-thirds of the viral genome, encode a replicase complex that is proteolytically processed into non-structural proteins (nsp to ) ( ). structural proteins, nucleocapsid (n) protein, membrane (m) glycoprotein, spike (s) glycoprotein, a small envelope (e) glycoprotein, and accessory proteins a, b, and are encoded by genes located at the ′ end ( ) . as early as , the presence of high levels of type i interferon (ifn-i) activity in the digestive tract of tgev-infected newborn piglets was first observed ( ) . thereafter, charley et al. reported that tgev infection in human or bovine peripheral blood mononuclear cells also induced high ifn-α production ( ) . subsequently, bosworth et al. demonstrated that ′, ′-oligoadenylate synthetase (oas), a well-known ifn-stimulated gene (isg), was increased in tgev-infected pigs ( ) . our previous quantitative proteomics analysis revealed that tgev infection induced canonical ifn-i signaling through janus kinase signal transducer and activator of the transcription (jak-stat ) pathway, and eight tested isgs, including ifninduced protein with tetratricopeptide repeats (ifit ), ifit , ifit , oas , oas , mx , mx , and isg were upregulated after tgev infection ( ) . these early results indicated that tgev infection activated the ifn-i pathway in vitro and in vivo. however, the underlying mechanism(s) utilized by tgev to induce ifn-i, and especially which viral protein(s) contribute to it, remain largely unclear. the ifn-i response is a well-known innate immune reaction that occurs in response to virus infection and considered as an important bridge between innate and adaptive immunity. nuclear factor-kappab (nf-κb) and ifn regulatory factor (irf ) are two critical transcription factors for the regulation of ifn-i production. secreted ifn-i then stimulates the jak-stat signaling pathway to induce the expression of numerous isgs, which collaborate to regulate the replication of virus ( ) . many viruses antagonize ifn responses to benefit their propagation, and some viruses such as human immunodeficiency virus-type ( ), vesicular stomatitis virus (vsv) ( ), influenza a virus (iav) ( ) , encephalomyocarditis virus ( ), reovirus ( ), herpes simplex virus (hsv ) ( ), respiratory syncytial virus ( ), newcastle disease virus ( ) , and sendai virus (sev) ( ) initiate innate immune responses. different viruses employ different mechanisms to regulate innate immune responses. for example, hsv induces ifn-α/β production through toll-like receptor (tlr ), dna-dependent activator of ifn regulatory factors (dai), and ifn-inducible ( ) . for iav infection, at least tlr , tlr , retinoic acid-inducible gene i (rig-i), and pyrin domain-containing (nlrp ) are responsible for the detection of iav and subsequent innate immune responses ( ) . elucidating the mechanisms through which viruses regulate innate immune responses will help us understand the interactions between virus and host. this study sought to identify tgev-encoded protein(s) involved in the induction of ifn-β production. our results revealed that tgev nsp was the best inducer of the ifn-β pathway among the tgev-encoded proteins. mechanistically, nsp activates nf-κb but not irf , and it interacts with rna helicase ddx , which in turn activates ifn-β production. porcine kidney (pk- ) cells and hek- t cells were cultured in dulbecco's modified eagle's medium (invitrogen, carlsbad, ca, usa) supplemented with % fetal bovine serum in a humidified incubator with °c/ % co . tgev strain wh- (genbank accession no. hq ) was propagated and titered in pk- cells. recombinant vsv-expressing green fluorescent protein (vsv-gfp) was generously provided by prof. zhigao bu from the harbin veterinary research institute, china. rabbit polyclonal antibodies against p , irf , phosphorylated irf (p-irf ), and ddx were purchased from abclone (wuhan, china). rabbit polyclonal antibody against phosphorylated p (p-p ) was purchased from cell signaling technology (beverly, ma, usa). anti-β-actin antibody was purchased from beyotime (nantong, china). mouse monoclonal antibodies (mabs) against hemagglutinin (ha) and flag were purchased from medical and biological laboratories (mbl, nagoya, japan). mab against tgev n protein was prepared by our laboratory. horseradish peroxidase (hrp)-conjugated goat anti-rabbit antibody and hrp-conjugated goat anti-mouse antibody were purchased from mbl. alexa fluor -conjugated donkey anti-rabbit igg, alexa fluor -conjugated donkey anti-mouse igg, and alexa fluor -conjugated donkey antimouse igg were obtained from santa cruz biotechnology inc. (santa cruz, ca, usa). expression plasmids of tgev-encoded proteins used in this study were constructed by rt-pcr amplification from the genomic rna of tgev strain wh- and cloned into expression vector pcaggs-ha. the details of primers used for pcr clone are available on request. the p gene was derived from human rela cdna and cloned into pegfp-c vector. the ddx expression plasmid was constructed by rt-pcr amplification from the cdna of pk- cells and cloned into pcmv-tag b vector. luciferase reporter plasmids p -luc (ifn-β-luc), × prdiii/i-luc (referred to as irf -luc), × prdii-luc (referred to as nf-κb-luc), and the internal control plasmid prl-tk have been described previously ( ) . small interfering rna (sirna) targeting ddx or negative control sirna (sinc, invitrogen) were each transfected at a final concentration of nm. the sirna sequences used here are listed in table . np- , nm phenylmethylsulfonyl fluoride], and protein concentration was measured and adjusted. for each immunoprecipitation, µg of cell lysate protein was incubated with µg of indicated antibody and µl of protein a/g-agarose (beyotime) overnight at °c. after three washes with ml lysis buffer, precipitates were subjected to % sds-page and subsequently analyzed with immunoblot analysis using the indicated antibodies. porcine kidney (pk)- cells were fixed with % paraformaldehyde for min followed by permeabilization with pre-cooled methanol for min, blocking with % bovine serum albumin for min, incubated with the indicated primary antibodies for h, followed by staining with specific alexa fluor-conjugated secondary antibodies for h. the cells were subsequently stained with ', -diamidino- -phenylindole (beyotime) for min. after washing with pbs, fluorescent images were obtained using an olympus fv laser scanning confocal microscope (olympus, japan). total rnas were extracted using trizol reagent (invitrogen). real-time rt-qpcr was performed using sybr green real-time pcr master mix (toyobo biologics, osaka, japan) in the abi prism sequence detection system (applied biosystems). individual transcripts in each sample were assayed three times. the fold change in gene expression relative to normal was calculated using the delta-delta cycles to threshold (ΔΔct) method. primers ( table ) were designed using primer express software (version . ; applied biosystems, carlsbad, ca, usa). all experiments were performed at least three times with reproducible results. data are presented as the mean ± sd. statistical analysis was performed using one-way anova without interaction terms followed by dunnett's for multiple comparisons. all animal experiments were approved by the hubei administrative committee for laboratory animals (permission number ) and complied with the guidelines of hubei laboratory animal welfare and ethics of hubei administrative committee of laboratory animals. previous studies demonstrated that tgev infection potently induced ifn-α ( , , ) , as well as ifn-β in ipec-j and swine testicular (st) cells ( ) ( ) ( ) . however, whether tgev infection induces ifn-β in pk- cells remains unknown. to explore the effect of tgev on ifn-β, dual luciferase assays were performed. pk- cells were transfected with ifn-β-luc and prl-tk. after the induction of ifn-i is reliant on the co-regulation of transcription factors irf and nf-κb. to investigate the potential mechanism(s) involved in the ifn-β production by tgev infection, the effect of tgev on irf and nf-κb promoters were also tested. as displayed in figures c,d , tgev infection also upregulated irf and nf-κb promoter activity dosedependently, indicating that irf and nf-κb are involved in the ifn-β production by tgev infection. because of the high sensitivity of vsv-gfp to ifn, vsv-gfp expression is commonly monitored for ifn detection. to further evaluate the ifn-β response in tgev-infected cells, a vsv-gfp-based ifn detection assay was performed. pk- cells were infected or mock infected with sev or tgev (moi = . , . , . ). then, cell supernatants were collected, uv-irradiated, and then transferred onto fresh pk- cells. after h, cells were infected with vsv-gfp and observed under a fluorescence microscope at hpi. as a positive control, supernatants collected from sev-treated cells suppressed vsv-gfp replication prominently compared with the negative control group (mock). in accordance with the results of dual luciferase assays described above, tgev infection limited the replication of vsv-gfp in a dose-dependent manner (figure e) . these results suggested that tgev infection increased ifn-β production. transmissible gastroenteritis virus encodes non-structural proteins (nsp - ), four structural proteins (n, m, s, e), and three accessory proteins (orf , orf a, orf b) . to identify the key viral protein(s) involved in ifn-β induction, an ifn promoterreporter system was employed to screen tgev-encoded proteins for their relative capacities to activate the ifn-β promoter. hek- t cells were cotransfected with ifn-β-luc, prl-tk, and different tgev protein expression vectors. as shown in figure a , nsp was the most significant inducer of ifn-β production. furthermore, similar to sars-(coronavirus) cov ( ), tgev m glycoprotein also potently mediated ifn-β induction. because nf-κb and irf are necessary transcription factors for ifn-β production, we examined the effects of all tgev-encoded proteins on irf and nf-κb promoter activity. interestingly, nsp upregulated an approximately . -fold change in nf-κb promoter activity, but only an approximately . -fold change in irf promoter activity (figures c,e) . m glycoprotein induced a higher fold change in irf , but a lower fold change in nf-κb compared with nsp . because m glycoprotein has been investigated previously ( ), we focused on nsp . to confirm the ability of nsp to induce ifn-β production, large-scale screen experiment was also conducted in pk- cells, a permissive cell line of tgev infection. as shown in figures b,d,f, nsp was also a potent ifn inducer that mainly induced activation of nf-κb but not irf promoter in pk- cells. to confirm the above large-scale screen results, increasing doses ( . , . , . µg) of pcaggs-ha-nsp and ifn-β-luc, irf -luc or nf-κb-luc together with prl-tk were cotransfected into hek- t cells. in line with the results in figure , nsp enhanced the activation of ifn-β and nf-κb in a dose-dependent manner (figures a-c) . interestingly, nsp induced the activation of nf-κb to a greater extent than that of irf , indicating nf-κb has a fundamental role in nsp induced ifn-β activation. similar results were also obtained in pk- cells (figures d-f) . nf-κb and irf activation are characterized by the phosphorylation and subsequent translocation of p (nf-κb subunit) or irf to the nucleus, respectively ( ) ( ) ( ) . next, we investigated the role of nsp in the phosphorylation of p and irf . hek- t cells were transfected with increasing amounts of pcaggs-ha-nsp , and cell lysates were examined for the expression levels of p-p or p-irf and total p or irf at h post-transfection. as shown in figure g , nsp overexpression had no significant effect on the amount of p , irf , and p-irf ; however, markedly increased p phosphorylation levels, indicating the activation of nf-κb, rather than irf is associated with nsp -induced ifn-β production. therefore, the subcellular location of p was further investigated after nsp overexpression. as shown in figures h,i , ectopic expression of nsp resulted in the nuclear translocation of overexpressed p in pk- cells ( figure h ) and endogenous p in hek- t cells (figure i ). early studies suggested that nsp of cov ibv and sars-cov interacts with host protein ddx ( ) . therefore, we investigated whether tgev nsp also interacts with ddx by co-ip. hek- t cells were cotransfected with pcaggs-ha-nsp and pcmv-tag b-ddx . because ddx is a dexd/h-box helicase and is associated with rna metabolism, the lysates were treated with rnase to avoid the effect of rna on the co-ip experiment. as shown in figure a , flag-tagged ddx was coprecipitated by ha-tagged nsp , indicating the interaction between nsp and ddx . reversed ip with flag antibody further confirmed this interaction ( figure b) . to test whether the colocalization of nsp and ddx occurs, a prerequisite for the interaction, pk- cells were transfected with pcaggs-ha-nsp or empty vector and fixed at h posttransfection. ha-tagged nsp protein was detected with mouse anti-ha antibody, and ddx was detected with rabbit anti-ddx antibody. the results revealed that nsp was colocalized with ddx and distributed both in the cytoplasm and nucleus (figure c) , which further confirmed the interaction between tgev nsp and ddx . because nsp activates ifn-β and interacts with ddx , we investigated whether ddx is involved in nsp -induced ifn-β production. synthesized sirna targeting human ddx (hsiddx ), which efficiently decreases the expression of endogenous ddx mrna ( figure a ) and protein (figure b) , was selected. next, hek- t cells were transfected with hsiddx or sinc, followed by co-transfection of ifn-β-luc or nf-κb-luc, prl-tk, along with pcaggs-ha-nsp or empty vector. the results revealed that knockdown of ddx significantly decreased nsp -induced promoter activity of ifn-β ( figure c ) and nf-κb ( figure d) . moreover, mrna expression levels of nsp -induced ifn-β were downregulated values are the mean ± sd of three independent tests. **p < . or ***p < . compared with empty vector group. (g) hek- t cells were transfected with increasing quantities ( , , µg) of pcaggs-ha-nsp or empty vector for h, and then subjected to immunoblotting with antibodies specific for endogenous irf , phosphorylated irf (p-irf ), p , or p-p . anti-ha mouse antibody was used to confirm the expression of nsp . β-actin expression was used as a loading control. the ratio of phosphorylated/total p and phosphorylated/total irf was analyzed using imagej software. (h) pk- cells were cotransfected with plasmids encoding ha-tagged nsp protein or empty vector together with plasmids encoding egfp-tagged p protein. then, the cells were fixed and immunostained with anti-ha monoclonal antibodies (mabs) to observe the nuclear translocation of overexpressed p using confocal microscopy. (i) hek- t cells were transfected with plasmids encoding ha-tagged nsp protein or empty vector. then, the cells were fixed and immunostained with anti-p antibody to observe the nuclear translocation of endogenous p using confocal microscopy. by ddx deficiency (figure e) , suggesting the involvement of ddx in ifn-β induction by nsp . to determine whether ddx is involved in tgev-induced ifnβ activation, we designed three pairs of sirnas targeting porcine ddx (psiddx ) and selected one with the best knockdown efficiency as demonstrated by rt-qpcr ( figure a ) and western blot assay (figure b) , for subsequent experiments. pk- cells were cotransfected with ifn-β-luc or nf-κb-luc, prl-tk, together with psiddx or sinc. at h post-transfection, cells were infected with tgev for h, followed by dual luciferase assay. ddx depletion had no effect on the basal activity of ifn-β and nf-κb promoter, but significantly decreased tgev-induced activation of ifn-β and nf-κb (figures c,d) . we also detected the expression level of p-p in tgev-infected pk- cells when ddx was silenced. as shown in figure e , knockdown of ddx reduced tgev-induced p phosphorylation. these results suggested that ddx is associated with tgev-induced ifn-β and nf-κb activation. interferon-i initiates a series of signaling cascades through the jak/stat pathway, resulting in the expression of numerous isgs ( ) . furthermore, nf-κb activation plays a pivotal role in regulating the transcription and expression of many proinflammatory cytokines. because ddx is involved in tgev-induced ifn-β and nf-κb activation, theoretically, it should have an impact on tgev-induced isg and pro-inflammatory cytokine expression. the expression levels of some isgs (ifit , ifit , ifit ) and proinflammatory cytokines (il- , il- ) in ddx -knockdown cells were analyzed after tgev infection. as expected, ddx depletion inhibited the expression of ifit , ifit , ifit , as well as il- and il- to some degree, compared with that in cells transfected with sinc (figures f-j) . the innate immune response characterized by the synthesis of ifn and proinflammatory cytokines is the first line of antiviral defense. multiple studies have reported the involvement of covs in the regulation of innate immune responses. the majority of covs decreased dsrna-mediated ifn-β production, including porcine epidemic diarrhea virus (pedv) ( ), severe acute respiratory syndrome coronavirus (sars-cov) ( ) , and infectious bronchitis virus (ibv) ( ) . interestingly, mouse hepatitis virus (mhv)-induced ifn-α/β and established an antiviral state in plasmacytoid dendritic cells and macrophages, but failed to produce ifn in neurons, astrocytes, and hepatocytes ( , ) , indicating that mhv induction of ifn-α/β is cell type dependent. a previous study showed that tgev-induced ifn-α secretion in vitro and in vivo ( ) . here, we found that tgev infection increased the production of ifn-β in a dose-dependent manner in pk- cells, in line with previously reported data ( ) . the significant roles of many cov proteins in the regulation of innate immune response have been identified. for example, nsp , nsp , nsp , papain-like protease (plpro), orf b, orf , ( ) . this indicated the potential effect of tgev nsp on ifn-β induction, which is in line with our data and further confirms our conclusion using a reverse genetic system. although nsp was identified as a key ifn-β activator among tgev-encoded proteins, it remains unclear which prr(s) is involved in its detection and induction of ifn-β production. indeed, viral proteins, similar to viral nucleic acids or replication intermediates, can in some cases also function as pamps, specifically recognized by certain host prrs, such as tlr and tlr , to modulate the ifn responses during viral infection ( , ) . for example, the m glycoprotein of sars-cov has been reported to function as a novel cytosolic pamp to promote ifn-β production by activating a non-canonical tlr signaling cascade ( ) . in addition to the four major prr groups reported previously, including tlrs, rig-i-like receptors, nod-like receptors and cytoplasmic dna receptors ( ) , multiple dexd/h-box helicases, such as ddx , ddx , ddx , and ddx , were reported recently to act as prrs and sense viral pamps to activate the nf-κb signaling pathway and induce ifn-β production ( ) ( ) ( ) ( ) ( ) . previous study revealed that ddx , ddx , and dhx form a complex with the adaptor molecule trif to sense dsrna in dendritic cells ( ) . in this paper, ddx interacted with tgev nsp in a rna-independent manner and enhanced both tgevand nsp -induced activation of ifn-β responses. however, the direct interactions between nsp and ddx or dhx were not observed. we speculated that nsp may be sensed by ddx / ddx /dhx complex by interacting with ddx . these results suggest that nsp may be recognized as pamp by ddx , (e) pk- cells were transfected with psiddx or sinc, and h later, cells were mock infected or infected with tgev (moi = . ) for h. then, the cells were collected for western blot assay with specific antibodies against p , p-p , ddx , or tgev n protein, using β-actin expression as a loading control. the ratio of phosphorylated/total p was analyzed using imagej software. (f-j) pk- cells were treated as described for (e) and collected at hpi. cell rnas were extracted for rt-qpcr to examine the mrna expression levels of ifit (f), ifit (g), ifit (h), il- (i), and il- (j). the mrna expression levels were normalized to porcine gapdh transcripts. values are the mean ± sd of three independent tests. *p < . or **p < . compared with the sinc group. which triggers an antiviral response. further studies are required to investigate this in more detail. ddx is a dexd/h helicase family member composed of the dead-box and related deah, dexh, and dexd protein family and is involved in multiple cellular processes of rna metabolism ( , ) . besides these traditional roles, it appears that multiple proteins of the dexd/h-box helicase family are associated with viral components and/or have alternative effects on viral propagation ( ) ( ) ( ) . for instance, ddx shows antiviral functions against vaccinia virus, denv, and hbv ( - ), but is of benefit for hcv and hiv infection ( , ) . in addition, ddx interacts with the nsp of venezuelan equine encephalitis virus and enhances viral multiplication ( ) . the interaction of ddx with human immunodeficiency virus type rev protein is involved in the regulation of virus replication ( ) . in the present study, the knockdown and ectopic expression of ddx demonstrated that ddx had antiviral activity against tgev replication (data not shown). interestingly, earlier studies also showed an interaction between ddx with coronavirus (ibv and sars-cov) nsp , and in contrast to tgev, this interaction might enhance the replication of ibv ( ) . this difference suggests that ddx is not likely to be a general target against cov infection. furthermore, it should be noted that the effect of ddx on progeny tgev production was moderate (data not shown). difference from other covs, such as sars-cov and mers-cov which antagonize ifn-i, tgev infection induces ifn-i production, and a most recent paper showed that poly(i:c)induced ifn-i responses could only inhibit tgev replication in the early infection stage, but failed in the late infection stage ( ) . they also demonstrated that the activation of ifn-i responses by tgev infection cannot inhibit viral replication. our results are consistent with the conclusion proposed by zhu and colleagues. in addition, it is surprising that the expression levels of ifn-β are paralleled with the increase of viral rna during tgev infection. these may explain why the effect of ddx on tgev replication was moderate accompany with its significant role in ifn-β induction by tgev. however, more studies are required to investigate the complex interaction between tgev and ifns. in conclusion, our data demonstrate that tgev infection induces ifn-β production and nsp is the most significant ifn-β inducer among the tgev-encoded proteins. nsp interacts with cellular dexd/h helicase ddx to activate ifn-β in a nf-κb dependent manner, and ddx is associated with tgev-induced 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genome-wide, proteomic, and molecular studies ddx dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids viral targeting of dead box protein reveals its role in tbk /ikkepsilon-mediated irf activation dead-box rna helicase ddx x inhibits denv replication via regulating type one interferon pathway understanding the interaction of hepatitis c virus with host dead-box rna helicases requirement of ddx dead box rna helicase for hiv- rev-rre export function venezuelan equine encephalitis virus non-structural protein (nsp ) interacts with rna helicases ddx and ddx in infected cells ddx is an rna-dependent atpase involved in hiv- rev function and virus replication key: cord- -gd y ggj authors: zhang, can; lu, long-feng; li, zhuo-cong; zhou, xiao-yu; zhou, yu; chen, dan-dan; li, shun; zhang, yong-an title: grass carp reovirus vp represses interferon production by degrading phosphorylated irf date: - - journal: fish shellfish immunol doi: . /j.fsi. . . sha: doc_id: cord_uid: gd y ggj grass carp reovirus (gcrv) is an efficient pathogen causing high mortality in grass carp, meanwhile, fish interferon (ifn) is a powerful cytokine enabling host cells to establish an antiviral state; therefore, the strategies used by gcrv to escape the cellular ifn response need to be investigated. here, we report that gcrv vp inhibits host ifn production by degrading the transcription factor ifn regulatory factor (irf ). first, overexpression of vp inhibited the ifn production induced by the polyinosinic-polycytidylic acid (poly i:c) and mitochondrial antiviral signaling protein (mavs), while the capacity of irf on ifn induction was unaffected. second, vp interacted with rlrs but did not affect the stabilization of the proteins in the normal state, while the phosphorylated irf activated by tbk was degraded by vp through k -linked ubiquitination. finally, overexpression of vp remarkably reduced the host cellular ifn transcription and facilitated viral proliferation. taken together, our results demonstrate that gcrv vp suppresses the host ifn response by targeting phosphorylated irf for ubiquitination and degradation. grass carp reovirus (gcrv), which belongs to the genus aquareovirus of the family reoviridae, has caused severe epidemic outbreaks of hemorrhagic disease and has an extremely high mortality rate among grass carp (ctenopharyngodon idella) [ ] . at present, known gcrv strains can be divided into three distinct subtypes based on sequence comparisons and analysis. the representative strains of the three groups are gcrv- (gcrv-i), gcrv-hz (gcrv-ii), and gcrv- (gcrv-iii) [ ] . the most commonly isolated strain is gcrv-Ⅱ [ ] . gcrv is a double-stranded rna (dsrna) virus and the genome consists of segments (termed s -s ) encased in a multilayered icosahedral capsid shell [ , ] . the protein sequence comparison showed that the similarity among the three groups is < %, so the functions of the encoded proteins are likely diverse. for instance, s of gcrv-i has been found to encode a non-structural protein (ns , ns ) [ , ] . s of both gcrv-ii and gcrv-iii are codified as a fiber like protein, which are predicted to be kda (vp ) and kda (vp ) [ , ] . in recent years, great progress has been made in the pathogenesis of gcrv and the host response to gcrv infection. for example, by using high-throughput methods such as transcriptomic and proteomic analyses, many immune-related genes have been identified as being involved in a gcrv infection [ ] . overexpression of mxs blocks the replication of gcrv and delays the cpe induced by gcrv infection [ ] . in crucian carp, viperin confers cells significant protection against gcrv infection [ ] . interferon (ifn) response plays an essential role in protecting the host against virus infection [ , ] . the host possesses conserved pathogen recognition receptors (prrs) that can sense viral rnas and trigger multiple intracellular signaling pathways, including the retinoic acid-inducible gene i (rig-i)-like receptor (rlr) pathway, which eventually results in the production of ifn to set up the antiviral state [ , [ ] [ ] [ ] . the specific rlr pathway is as follows: upon binding with viral rna, the n-terminal caspase recruiting domain (card) of the rlr family (including rig-i and melanoma differentiation-associated gene (mda ), interacts with another card-containing protein, mitochondrial antiviral signaling protein (mavs, also called visa, ips- , and cardif) [ ] [ ] [ ] [ ] . then, the signal transmits to the downstream mediator of ifn regulatory factor (irf ) activation (mita, also known as sting, eris, or myps) and tank binding kinase (tbk ) [ ] . subsequently, activated tbk phosphorylates irf / are translocated to the nucleus and trigger the transcription of ifn [ , ] . similar to mammals, fish also possess a conserved rlrs signaling pathway. for example, mavs or mita plays an important role in ifn activation in zebrafish (danio rerio), which can significantly decrease the probability of viral infection [ , ] . overexpression of grass carp tbk induces the expression of irf and ifn-stimulated genes (isgs), and inhibits the replication of gcrv [ ] . irf in zebrafish and grass carp also exhibits a critical role in ifn activation [ ] . however, viruses have evolved a multitude of elaborate strategies to escape the host immune response. one such mechanism is to blunt ifn production. irf is a key transcription factor that regulates ifn induction in response to viral infection [ , ] , most signals ultimately converge on irf , so it is targeted by viruses as a major negative regulatory target to decrease the ifn response and facilitate viral replication. for instance, porcine reproductive and respiratory syndrome (prrs) utilizes the nsp protein to inhibit irf expression, thereby down-regulating ifn production and counteracting the host antiviral state [ ] . the c pro of seneca valley virus (svv) reduces irf and irf protein expression and phosphorylation via its protease activity, thus blocking ifn transcription to escape the host immune response [ ] . kaposi's sarcoma-associated herpesvirus (kshv)-encoded viral interferon regulatory factor (virf ) interacts with irf , resulting in the inhibition of irf dimerization and ultimately suppressing ifn production [ ] . as a highly virulent pathogen that causes severe hemorrhagic disease and tremendous mortality in fish, gcrv likely has strategies to negatively regulate or evade the host immune response. in our previous study, the gcrv vp attenuated mita phosphorylation by acting as a decoy substrate of tbk , thus reducing ifn production and facilitating viral replication. to further investigate the immune evasion strategies of gcrv, we further revealed that gcrv vp is associated with irf and promotes the k -linked ubiquitination and degradation of irf , thereby inhibiting ifn expression and accelerating viral proliferation. these results will lay a foundation for further studying host-virus interactions among lower vertebrates. human embryonic kidney (hek) t cells were provided by dr. xing liu (institute of hydrobiology, chinese academy of sciences) and were grown at °c in % co in dulbecco's modified eagle's medium (dmem; invitrogen) supplemented with % fetal bovine serum (fbs, invitrogen). epithelioma papulosum cyprini (epc) cells and grass carp ovary (gco) cells were maintained at °c in % co in medium (invitrogen) supplemented with % fbs. gcrv (strain , gcrv-ii) was a gift from lingbing zeng (yangtze river fisheries research institute, chinese academy of fishery sciences). because gcrv-ii cannot cause a cytopathic effect (cpe) but can propagate in gco cells, the cultured media with gco cells infected with gcrv-ii for days were harvested and stored at − °c until used. the open reading frame (orf) of gcrv vp (kc . ) was generated by pcr and then cloned into pcdna . (+) (invitrogen), pcmv-myc (clontech), or pcmv-ha vectors (clontech), respectively. the orfs of grass carp mavs (kf . ), mita (jn . ), tbk (jn . ), irf (kt . ), and irf (ky . ) were cloned using the vectors previously described [ ] for subcellular localization, the orf of vp was inserted into pegfp-n vector (clontech). the orfs of mavs, mita, tbk , irf , and irf were also inserted into pcs -mcherry vector (clontech). the expression plasmids for flag-drtbk , ha-drirf , ha-drirf , and myc-drirf were described previously [ ] . for promoter activity analysis, ifn pro-luc construct was generated by insertion of corresponding ′-flanking regulatory region of ifn promoter (gu . ) into pgl -basic luciferase reporter vector (promega, madison, wi). the isre-luc plasmid in the pgl -basic luciferase reporter vector (promega) was constructed as described previously [ ] . the renilla luciferase internal control vector (prl-tk) was purchased from promega. the primers including the restriction enzyme cutting sites used for plasmid construction are listed in supplemental table i . all constructs were confirmed by dna sequencing. epc cells were seeded into -well plates overnight and co-transfected with various constructs at a ratio of : : (mavs/irf , ifn pro/isre-luc, and prl-tk expression vectors). the prl-tk was used to normalize the expression levels of the transfected plasmids. an empty vector pcdna . (+) was used to maintain equivalent amounts of dna in each well. the transfection of poly i: c was performed at h after post-transfection, and cells were harvested at h after poly i: c transfection. at h post transfection, the cells were washed with phosphate-buffered saline (pbs) and lysed for measuring luciferase activity by a dual-luciferase reporter assay system, according to the manufacturer's instructions (promega). the results are representative of more than three independent experiments, each performed in triplicate. transient transfections were performed in epc cells seeded in -well or -well plates by using x-tremegene hp dna transfection reagent (roche) according to the manufacturer's protocol. for the antiviral assay using -well plates, epc cells were transfected with . μg pcdna . (+)-vp or the empty vector. at h post-transfection, cells were infected with svcv at a multiplicity of infection (moi = . ). after or d, supernatant aliquots were harvested for detection of virus titers, the cell monolayers were fixed by % paraformaldehyde (pfa) and stained with % crystal violet for visualizing cpe. for virus titration, μl of culture medium were collected at h post-infection, and used for plaque assay. the supernatants were subjected to -fold serial dilutions and then added ( μl) onto a monolayer of epc cells cultured in a -well plate. after or h, the medium was removed and the cells were washed with pbs, fixed by % pfa and stained with % crystal violet. the virus titer was expressed as % tissue culture infective dose (tcid /ml). results are representative of three independent experiments. total rnas were extracted using the trizol reagent (invitrogen). cdna was synthesized using a goscript reverse transcription system (promega), according to the manufacturer's instructions. quantitative real-time pcr (qpcr) was performed with fast sybr green pcr master mix (bio-rad) on the cfx real-time system (bio-rad). pcr conditions were as follows: °c for min and then cycles of °c for s, °c for s, and °c for s. the β-actin gene was used as an internal control. primer sequences are listed in supplemental table i . the relative fold changes or relative mrna of level were calculated by comparison to the corresponding controls using the −ΔΔct method. three independent experiments were conducted for statistical analysis. in transient transfection and co-ip experiments, we used hek t cells instead of epc cells because of their high transfection efficiency. the hek t cells seeded in -cm dishes overnight were transfected with μg flag-mavs/tbk /mita/irf /irf and μg myc-vp . at h post transfection, the medium was removed carefully, and the cell monolayer was washed twice with ml of ice-cold pbs. then the cells were lysed in ml of radioimmunoprecipitation lysis buffer ( % nonidet p- , mm tris-hcl, ph . , mm nacl, mm edta, mm naf, mm sodium orthovanadate [na vo ], mm pmsf, . % sodium deoxycholate) containing protease inhibitor mixture (sigma-aldrich) at °c for h on a rocker platform. the cellular debris was removed by centrifugation at , ×g for min at °c. the supernatant was transferred to a fresh tube and incubated with μl of anti-hemagglutinin (ha)-agarose beads or anti-flag affinity gel (sigma-aldrich) overnight at °c with constant agitation. these samples were further analyzed by immunoblotting. immunoprecipitated proteins were collected by centrifugation at , ×g for min at °c, washed three times with lysis buffer, and resuspended in μl of × sds sample buffer. the immunoprecipitates and whole-cell lysates (wcls) were analyzed by ib with the indicated abs. the cells were lysed using a ripa lysis buffer containing % sds and denatured by heating for min. the supernatants were diluted with lysis buffer until the concentration of sds was decreased to . %. the diluted supernatants were incubated with μl anti-myc affinity gel (sigma-aldrich) overnight at °c with constant agitation. these samples were further analyzed by immunoblotting (ib). immunoprecipitated proteins were collected by centrifugation at ×g for min at °c, washed three times with lysis buffer and resuspended in μl × sds sample buffer. immunoprecipitates or wcls were separated by % sds-page and transferred to polyvinylidene difluoride (pvdf) membrane (bio-rad). the membranes were blocked for h at room temperature in tbst buffer ( mm tris-hcl, mm nacl, . % tween , ph . ) containing % nonfat dry milk, probed with the primary abs indicated on the figures at an appropriate dilution overnight at °c, washed three times with tbst, and then incubated with secondary abs for h at room temperature. after three additional washes with tbst, the membranes were stained with the immobilon western chemiluminescent horseradish peroxidase (hrp) substrate (millipore) and detected by using an image quant las system (ge healthcare). abs were diluted as follows: anti-β-actin (cell signaling technology) at : , , anti-flag/ha (sigma-aldrich) at : , , anti-myc (santa cruz biotechnology) at : , , and hrp-conjugated anti-rabbit igg or anti-mouse igg (thermo scientific) at : , . results are representative of data from three independent experiments. transfected hek t cells were lysed as described above, except that the phosphatase inhibitors (na vo and edta) were omitted from the lysis buffer. protein dephosphorylation was carried out in μl reaction mixtures consisting of μg of cell protein and u of cip (sigma-aldrich). the reaction mixtures were incubated at °c for min, followed by immunoblot analysis. epc cells were plated onto coverslips in six-well plates and transfected with the indicated plasmids for h. then the cells were washed twice with pbs and fixed with % paraformaldehyde (pfa) for h. after being washed three times with pbs, the cells were stained with ′, -diamidino- -phenylindole (dapi) ( μg/ml; beyotime) for min in the dark at room temperature. finally, the coverslips were washed and observed with a confocal microscope under a × oil immersion objective (sp ; leica). luciferase and qpcr assay data are expressed as the mean ± standard error of the mean (sem). error bars indicate the sem (n = , biologically independent samples). the p values were calculated by one-way analysis of variance (anova) with dunnett's post hoc test (spss statistics, version ; ibm). a p value < . was considered statistically significant. previously, our study has demonstrated that gcrv vp reduces mita phosphorylation and blocks ifn production, thus escaping the host immune response. given that one virus should possess multiple strategies to elude host defense mechanisms, other immune escape mechanisms of gcrv should be identified. here, to further investigate the other strategies used by gcrv to combat the host, other constructs of gcrv segments were employed for luciferase experiments in vitro, and the s -encoded protein (vp ) exhibited the potential to inhibit host ifn activation. upon infection with gcrv, the viral s gene was total rnas were extracted to examine the transcriptional levels of cellular ifn and isg by qpcr. the relative transcription levels were normalized to the transcription level of the β-actin gene and are represented as fold induction relative to the transcription level in control cells, which was set to . data were expressed as mean ± sem, n = . asterisks indicate significant differences from control (*, p < . ). all experiments were repeated for at least three times with similar result. increased significantly in gco cells, which indicated that the cells were successfully infected with gcrv (fig. a) . four type i ifns (ifn -ifn ) have been identified in grass carp, but only ifn can be significantly activated by poly i:c, a mimic of viral rna [ ] (data not shown). thus, grass carp ifn was used in this study. as shown in fig. b , poly i:c stimulation induced the upregulation of ifn transcripts; however, such induction was significantly impeded by the overexpression of vp . moreover, vp also blocked the poly i:c-activated expression of isg (fig. c) . these data indicate that gcrv vp serves as a negative regulator to interfere with host ifn production. fish rlr factors are efficient for triggering ifn production [ ] . consequently, grass carp rlr constructs and ifn promoter (ifn pro) were employed in the following studies. as shown in fig. a , the overexpression of mavs and irf upregulated the activation of ifn pro, and the activation of ifn pro induced by mavs was inhibited by co-transfection with vp . however, the ectopic expression of vp did not affect the irf -stimulated ifn pro activity. in the host ifn response, the isre motif is considered the binding site of isgs that responds to transcriptional factors. after co-transfection with vp and isre-luc and rlr factors, the upregulation of isre activity activated by mavs but not irf was reduced by vp (fig. b) . collectively, these results suggest that vp decreases ifn production via negatively regulating mavs. to further explore the function of vp , whether vp interacts with rlrs at the protein level was investigated. hek t cells were co-transfected with myc-vp and flag-tagged rlr factors, including mavs, tbk , mita, irf , and irf . the results showed that most of the anti-flag ab-immunoprecipitated protein complexes were recognized by the anti-myc ab, which suggests that vp associates with tbk , mita, irf , and irf but not mavs (fig. a) . next, the subcellular location of vp was monitored in epc cells. confocal microscopy revealed that the vp -egfp signal was mainly distributed in the cytoplasm (fig. b) . we co-transfected dsred-mavs, dsred-mita, dsred-tbk , dsred-irf , or dsred-irf with vp -egfp. a red signal from tbk , irf , and irf was observed in the cytosol and almost overlapped with the green signal from vp (fig. c-g) . taken together, these data suggest that vp is located in the cytosol and associates with rlr factors. to investigate the regulatory mechanism of vp on the rlr axis, we examined the effect of vp on rlr molecules at the protein level. mavs-, tbk -, mita-, irf -, and irf -ha expression vectors were cotransfected with myc-vp or an empty vector. as shown in fig. a , overexpressed vp did not cause any obvious change in rlr molecules at the protein level. given that irf / phosphorylation is indispensable for mediating the ifn response, whether the phosphorylation of irf / is influenced by vp needs to be clarified. first, the function of grass carp tbk was investigated. as shown in fig. b and c, both irf and irf caused a band shift and exhibited higher mobilities when they were co-transfected with tbk -flag in t cells. subsequently, the cell lysates were incubated with cip. as expected, the band shifts disappeared, demonstrating that irf and irf are also phosphorylated by tbk in grass carp. then, we evaluated whether irf / phosphorylation would be impaired by the overexpression of vp . as shown in fig. d and e, the amount of irf was dramatically reduced by the overexpression of vp in a dose-dependent manner; in contrast, vp had minimal effects on the irf level. similar results occur in zebrafish ( fig. f and g) . these results suggest that vp specifically promotes the degradation of irf . protein degradation is one of the main strategies involved in modulating protein functions in biological processes and there are two main systems for protein degradation: ubiquitin proteasome and autophagosome pathways. to identify the degradation pathway of irf , the cells were treated with indicated inhibitors. the vp -mediated degradation of irf was completely inhibited by the proteasome inhibitor mg but not -ma, which is an autophagosome pathway inhibitor (fig. a) . since ubiquitination is an important process during proteasome-dependent degradation, we further determined whether the degradation of irf was due to ubiquitination. hek t cells were transfected with flag-tbk , myc-irf , ha-vp , and ha-ub in the presence or absence of mg . following the immunoprecipitation of myc-irf , ib revealed that vp potentiated the ubiquitination of irf (fig. b) . k and k , the lysines at positions and of ubiquitin linked with polyubiquitin chains, are two canonical polyubiquitin chain linkages. given that k -linked polyubiquitin chain modification leads to the targeting of proteins for proteasome recognition and degradation, whereas k -linked polyubiquitin chain modification enhances the stability of target proteins [ ] [ ] [ ] , we chose to investigate whether vp promoted the k -or k -linked ubiquitination of irf . to achieve this goal, plasmids expressing ubiquitin mutants and retaining only a single lysine residue either k (ubiquitin-k ) or k (ubiquitin-k ) were used. as shown in fig. c , immunoprecipitation and ib indicated that vp promoted irf ubiquitination with wild-type ubiquitin and ubiquitin-k but not with ubiquitin-k . the above results indicate that vp induces the k -linked ubiquitination of irf , which is recognized and subsequently degraded by the proteasome pathway. to determine whether vp interferes with the cellular ifn response to facilitate virus proliferation, epc cells were transfected with vp or the empty vector and infected with svcv. total rnas were extracted and monitored by qpcr. as shown in fig. a and b, the expression of the ifn transcript in the cells that overexpress vp was reduced compared to their levels in the control cells and the reduced expression of host vig was also observed. moreover, more cpe was observed in the vp group at d post-infection (fig. c ). this was confirmed by the titer of svcv, which had significantly increased ( , -fold) in the vp -overexpressing cells compared to the control cells (fig. d) . these data demonstrate that vp suppresses the cellular ifn response and enhances the capacity of svcv to replicate. as in mammals, fish ifn plays a critical role in the host immune response when defending against viral infection. however, aquatic viruses still cause high mortality in the cultured fish industries. the immune evasion mechanisms involved in the pathogenesis of aquatic viruses remain poorly understood. here, we report that gcrv vp interacts with irf and promotes the k -linked ubiquitination and degradation of irf , which suppresses the host ifn response. gcrv, the first viral pathogen identified from aquatic animals in china in , causes a serious epidemic of hemorrhagic disease, which results in extremely high mortality among grass carp [ ] . the gcrv virion consists of dsrna genome segments surrounded by multiple concentric protein capsids [ ] . the segments encode seven structural proteins and five nonstructural proteins. five of these proteins (vp -vp and vp ) form the core layer [ ] . the functional exploration of non-structural proteins among the segments remains unclear. we have reported that vp prevents the fish ifn response by attenuating the phosphorylation of mita for viral evasion [ ] . further study in this manuscript has revealed that vp interacts with irf and promotes the ubiquitination and degradation of irf . many viruses have evolved strategies to elude the host immune system and one or several host immune molecules may be involved in this evasion because of viruses' relatively limited genome capacity [ ] . rlrs toll-like receptor (tlr)-and nod-like receptor (nlr)-mediated immune responses are the first line of defense against pathogenic microbes [ ] . the ifn system is a particularly critical component in the host response, including the induction of ifn, ifn-mediated signaling, and the amplification of the ifn response [ ] . irf is a critical component of the intrinsic immune system [ ] . although various prrs activate different signal pathways, most signals transduction ultimately converge on irf . in addition, irf is induced by viral infection and is essential for sustained transcriptional activation of ifn genes [ ] . in response to host defend mechanisms, viruses have evolved various strategies to inhibit the activation of irf , including inhibiting irf dimerization and altering irf modification (sumoylation, phosphorylation, or ubiquitination) [ ] [ ] [ ] [ ] . in fish, antiviral effects of irf has been reported in crucian carp, zebrafish, and grass carp [ , ] . following gcrv infection, host induces a series of antiviral immune responses, then stimulates the transcription of a broad range of isgs including irf , which in turn establish an antiviral state within the cells to eliminate viral infection [ ] . however, viruses have established multiple mechanisms to counteract host antiviral state [ ] . so far, the relationships between fish viruses and irf are not well known. in the present study, gcrv vp induced the degradation of irf , resulting in the reduction of ifn expression. these results demonstrate that fish virus also possesses the function to antagonize host ifn response. ubiquitination, which is a reversible covalent modification, plays an important role in regulating the stability, activity, and localization of target proteins [ ] . many viruses have taken advantage of ubiquitination to target host proteins and change the proteins' original state in immune signaling pathways. for example, the n-terminal protease fragment (npro) of the bovine viral diarrhea virus (bvdv) blocks the binding of irf to dna and targets irf for proteasomal degradation, thus blocking ifn production [ ] ; severe acute respiratory syndrome coronavirus (sars-cov) papain-like protease (plpro) inhibits the tlr -mediated ifn induction by removing the polyubiquitination of traf and traf [ ] ; the n protein of svcv suppresses fish ifnϕ expression by degrading mavs in an ubiquitination-proteasome manner [ ] . here, our results reveal that gcrv vp represses ifn production by inducing the k -linked ubiquitination and degradation of irf . in conclusion, the current study reveals a potential vital mechanism used by vp of gcrv to negatively regulate irf through the ubiquitination-proteasome degradation pathway. these data shed light on the novel manner of immune evasion utilized by gcrv. further studies are needed to ascertain the functions of other proteins of gcrv in immune evasion, which will promote an in-depth understanding of gcrv pathogenesis and provide ideas for preventive strategies. we declare no financial and commercial conflicts of interest. fig. . vp dampens the cellular ifn response and facilitates svcv proliferation. (a and b) epc cells seeded into -well plates overnight were infected with svcv (moi = ). after h, total rnas were extracted to examine the transcriptional levels of cellular epcifn and epcvig by qpcr. the relative transcription levels were normalized to the transcription level of the β-actin gene and are represented as fold induction relative to the transcription level in control cells, which was set to . data were expressed as mean ± sem, n = . asterisks indicate significant differences from control (*, p < . ). (c and d) epc cells seeded in -well plates overnight were transfected with . μg pcdna . (+)-vp or pcdna . (+) vector. at h post-transfection, cells were infected with svcv (moi = . ) for h. (c) then, cells were fixed with % pfa and stained with % crystal violet. (d) culture supernatants from the cells infected with svcv were collected, and the viral titer was measured by standard tcid method. all experiments were repeated for at least three times with similar results. 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ubiquitination by mindbomb e ubiquitin protein ligase is mediated by the mitochondrial antiviral signaling protein complete genomic sequence of a reovirus isolated from grass carp in china grass carp reovirus vp targets fish mita to abrogate the interferon response the primary function of rna binding by the influenza a virus ns protein in infected cells: inhibiting the '- ' oligo (a) synthetase/rnase l pathway type i ifn modulates innate and specific antiviral immunity irf : activation, regulation, modification and function irf- is the master regulator of type-i interferon-dependent immune responses virus infection triggers sumoylation of irf and irf , leading to the negative regulation of type i interferon gene expression vaccinia virus protein c is a virulence factor that binds tbk- adaptor proteins and inhibits activation of irf and irf kshv-encoded viral interferon regulatory factor (virf ) interacts with irf and inhibits interferon alpha production human parainfluenza virus type v protein inhibits traf -mediated ubiquitination of irf to prevent tlr -and tlr -dependent interferon induction molecular characterization and transcription regulation analysis of type i ifn gene in grass carp (ctenopharyngodon idella) functions of the two zebrafish mavs variants are opposite in the induction of ifn by targeting irf mda induces a stronger interferon response than rig-i to gcrv infection through a mechanism involving the phosphorylation and dimerization of irf and irf in cik cells type interferons and the virus-host relationship: a lesson in detente ubiquitylation in innate and adaptive immunity the npro product of bovine viral diarrhea virus inhibits dna binding by interferon regulatory factor and targets it for proteasomal degradation sars coronavirus papain-like protease inhibits the tlr signaling pathway through removing lys -linked polyubiquitination of traf and traf we thank dr. fang zhou (institute of hydrobiology, chinese academy of sciences) for assistance with confocal microscopy analysis and dr. feng xiong (china zebrafish resource center, institute of hydrobiology, chinese academy of sciences) for rna sample extraction. supplementary data to this article can be found online at https:// doi.org/ . /j.fsi. . . . key: cord- -oer lxxr authors: onodi, fanny; bonnet-madin, lucie; karpf, léa; meertens, laurent; poirot, justine; legoff, jérome; delaugerre, constance; amara, ali; soumelis, vassili title: sars-cov- induces activation and diversification of human plasmacytoid pre-dendritic cells date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: oer lxxr several studies have analyzed antiviral immune pathways in severe covid- patients. however, the initial steps of antiviral immunity are not known. here, we have studied the interaction of isolated primary sars-cov- viral strains with human plasmacytoid pre-dendritic cells (pdc), a key player in antiviral immunity. we show that pdc are not permissive to sars-cov- infection. however, they efficiently diversified into activated p -, p -, and p -pdc effector subsets in response to viral stimulation. they expressed checkpoint molecules at levels similar to influenza virus-induced activation. they rapidly produced high levels of interferon-α, interferon-λ , il- , ip- , and il- . importantly, all major aspects of sars-cov- -induced pdc activation were inhibited by hydroxychloroquine, including p - and p -pdc differentiation, the expression of maturation markers, and the production of interferon-α and inflammatory cytokines. our results indicate that pdc may represent a major player in the first line of defense against sars-cov- infection, and call for caution in the use of hydroxychloroquine in the early treatment of the disease. severe acute respiratory syndrome-coronavirus- (sars-cov- ) is the third zoonotic coronavirus that emerged in the last two decades. sars-cov- is the causative agent of coronavirus disease that appeared in late in wuhan, hubei province in china (nandakumar, ; sheahan and frieman, ) . sars-cov- became rapidly pandemic, and infection have now been detected in countries and territories, and is responsible for approximatively million confirmed cases and , deaths as of of june (who weekly update).  sars-cov- infection may lead to a diversity of clinical presentations, ranging from asymptomatic or mild "flu-like" syndrome, to severe and life-threatening acute respiratory failure. disease aggravation usually occurs after to days following initial symptoms (tang et al., ) . at this late stage, three main factors were shown to contribute to the progression and severity of the infection (tang et al., ) : ) viral persistence was evidenced in the lung and systemic circulation, although it is not constant (tang et al., ) , ) an excess production of pro-inflammatory cytokines, such as il- b and il- (tay et al., ; arnaldez et al., ) , ) a defect in type i interferon (ifn) production, especially in critically ill patients (tay et al., ; acharya et al., ) . although these abnormalities were confirmed in several studies, their origin and underlying mechanisms remain mostly unknown. in particular, it is not known whether an imbalance between inflammatory cytokines and type i ifn occurs early in the disease, at the stage of the primary infection, and whether the virus itself may be responsible. to fill this gap of knowledge, it becomes essential to investigate the early innate immune response to sars-cov- . among the immune cells that are involved in innate anti-viral immunity, plasmacytoid predendritic cells (pdc) play a particularly important role as the major source of type i ifn in response to viral infection (liu, ) . pdc can sense a large array of viruses including the coronaviruses murine hepatis virus (mhv) and the middle east respiratory syndrome coronavirus (mers) (scheuplein et al., ; cervantes-barragan et al., ) , and respond by producing innate cytokines, including all forms of type i ifns (α and β), type iii ifn, and inflammatory cytokines, in particular tnf-α and il- (liu, ; yin et al., ; gilliet et al., ) . however, different viruses may induce different cytokine patterns (thomas et al., ) , possibly creating an imbalance between ifn versus inflammatory cytokine response. additionally, some viruses were shown to subvert pdc functions through different mechanisms not necessarily related to productive infection. this is the case for hiv, which may induce pdc apoptosis in vitro (meyers et al., ) and pdc depletion in vivo (soumelis et al., ; meera et al., ) . human hepatitis c virus can inhibit ifn-α production by pdc through the glycoprotein e binding to bdca- (florentin et al., ) . human papillomavirus induces very low ifn response in pdc (bontkes et al., ) , which may be due to impaired tlr- and - signaling (hirsch et al., ) . whether sars-cov- induces efficient pdc activation, or may interfere with various biological pathways in pdc is currently unknown. in this study, we have systematically addressed the interactions between clinical sars-cov- isolates and primary human pdc in order to reproduce the early stages of the infection. we showed that pdc are resistant to productive infection with sars-cov- strains but still mount substantial ifn responses upon viral challenge. interestingly, pdc responded to sars-cov- by a complete activation program, including diversification into effector subsets, production of type i and type iii ifn, as well as inflammatory cytokines. we also showed that hydroxychloroquine, an antimalarial drug proposed for treatment of covid- patients (das et al., ; mahévas et al., ) , inhibits sars-cov- -induced pdc activation and ifn production in a dose-dependent manner. our results establish pdc as a potential key player in innate immunity to sars-cov- , and raise caution regarding pharmacological manipulation that could inhibit pdc effector functions. in order to efficiently recapitulate sars-cov- -pdc interactions, we used two strains of sars-cov- primary isolates. their viral genome sequences were nearly identical with , % identity. sequence comparison with reference strain wuhan-hu- (ncbi accession number nc_ . ) showed that both strains contain a subset of mutations (c t; c t; a g and g t), characteristic of the gh clade based on gisaid nomenclature. human primary pdc were purified from healthy donor peripheral blood mononuclear cells (pbmc) by cell sorting. first, we asked whether sars-cov- was able to induce pdc activation, and diversification into ifnproducing and/or t cell stimulating effectors, as we previously described for influenza virus a (flu) (alculumbre et al., ) . after hours of culture, sars-cov- activated pdc efficiently diversified into p (pd-l + cd -), p (pd-l + cd + ), and p (pd-l + cd + ) pdc subsets, similar to flu stimulation ( fig a) . p -, p -, and p -pdc were all significantly induced by sars-cov- and flu, as compared to medium control ( fig b) . in parallel, we observed a sharp decrease in non-activated p -pdc (pd-l -cd -) (fig a and b) . sars-cov- -induced pdc activation was comparable with magnetically-versus facs-sorted pdc (fig s a and s b ), confirming that both methods are suitable for subsequent experiments. all main findings were confirmed on at least three independent experiments using facssorted pdc, with a protocol that excluded as-dc, a rare dendritic cell (dc) subset that shares some markers and functional features with pdc (villani et al., ) , based on cd , cd and axl expression ( fig s a) . pdc activation and diversification was observed with two independent primary sars-cov- strains (fig c) , which both induced similar proportions of p -p subsets. pdc diversification was also observed by co-culturing of pdc with sars-cov- -infected vero e cells with a similar efficiency than free sars-cov- ( fig s c) . sars-cov- improved pdc viability as compared to medium condition ( fig d) , which is compatible with subsequent effector functions. next, we asked whether sars-cov- -induced pdc activation was dependent on productive infection. we first checked whether pdc express at their cell surface ace , the major sars-cov- entry receptor (hoffmann et al., , ) . no significant expression was detected using an anti-ace -specific antibody, as compared to a low and high expression on vero e and t-ace cell lines, respectively ( fig e) . the ability of pdc to replicate sars-cov- was then assessed. human pdc were challenged with sars-cov- strain _ at moi of , and cultured for h, h or h. our results showed that pdc were refractory to sars-cov- infection, as evaluated by quantifying ) the intracellular production of the nucleoprotein antigen (n) (fig f) , or the accumulation of viral rna in sars-cov- -infected cells (fig s d) , and ) the release of infectious progeny virus in the supernatants of infected cells using plaque assays ( fig g) . as positive control, the permissive vero e cells produced high level of the n antigen, increased viral rna overtime ( fig s d) , and high viral titers following sars-cov- incubation ( fig g) . similar results were obtained with pdc isolated from three independent donors ( fig s e) . overall, these results show that pdc are resistant to sars-cov- infection, and are efficiently activated by the virus independently of ace expression. their viability was not affected by sars-cov- challenge. activating immune checkpoints play a key role in t cell stimulation, and serve as surrogate markers of dc differentiation (guermonprez et al., ) . we first assessed the dose-dependent effect of sars-cov- on cd expression and subset diversification. cd was induced in a dose-dependent manner by sars-cov- at moi . to (fig a) . this was accompanied by an increase in p -pdc subset, and a slight decrease in p -pdc ( fig b) . a detailed phenotypic analysis was subsequently performed on pdc after and hours of culture with sars-cov- ( fig c and fig s a) . diversification was observed at both time points, with a slight increase in p -pdc at hours ( fig s a) . p -and p -pdc significantly upregulated cd , cd , ccr , and ox l, as compared to non-activated p -pdc, in both sars-cov- and flu conditions ( fig c) . pd-l , and cd l, an integrin that promotes lymph node homing, were both higher on p -and p -pdc ( fig c) . expression of checkpoint molecules persisted at h, especially the higher cd and cd expression on p -pdc ( fig s b) . a key and defining function of pdc is their ability to produce large amounts of type i ifn (gilliet et al., ) . we measured the production of several cytokines at the protein level after hours of culture. both sars-cov- and flu induced high levels of ifn-α and ifn-λ , both being critical anti-viral effector cytokines ( fig a) . ifn-α levels following sars-cov- activation reached up to ng/ml, indicating a very efficient activation. the chemokine ip- was also significantly induced ( fig a) , possibly due to an autocrine ifn loop (blackwell and krieg, ) . inflammatory cytokines il- and il- were comparably induced by sars-cov- and flu ( fig a) . however, tnf-α levels were marginally induced by sars-cov- as compared to flu activation ( fig a) . cytokine production was maintained after hours of viral activation ( fig b) . secreted protein levels were similar to h levels for most cytokines. interestingly, ifn-α levels raised by -fold between h and h for one donor ( fig a and b) , indicating the possibility of increased production. such strong ifn producer suggests a potential virus controller. because the oropharyngeal mucosa is an entry site for sars-cov- , we aimed at validating our results using pdc purified from tonsils. sars-cov- induced a marked diversification of tonsillar pdc into all three activated subsets ( fig s c) . tonsillar pdc efficiently produced ifn and inflammatory cytokines in response to sars-cov- ( fig s d) . overall, our results establish sars-cov- as a very efficient inducer of type i and type iii ifn responses. inflammatory cytokines were induced at similar level than flu activation, without any significant imbalance that would be suggestive of excessive inflammatory response. given that sars-cov- did not infect pdc, and did not interfere with pdc activation, we asked whether pharmacological agents could modulate pdc diversification and cytokine production. hydroxychloroquine (hcq) is known to inhibit endosomal acidification which may diminish pdc activation (kuznik et al., , ; sacre et al., ) . additionally, it is being tested in several clinical studies as a potential treatment for covid- (das et al., ; mahévas et al., ) . hence, we addressed its role in sars-cov- -induced pdc activation. following hours of culture, we found that hcq inhibited pdc diversification in response to sars-cov- , which is similar to the decrease observed with flu, used as a positive control ( fig a) . in particular, p -and p -pdc differentiation were almost completely inhibited ( fig b) . inhibition of sars-cov- -induced pdc diversification by hcq was dosedependent ( fig s a) . the significant decrease in p -pdc was paralleled by a decrease in cd , cd , and ccr expression (fig c and d) . ox -ligand expression was not significantly affected by hcq (fig c and d) . however, hcq inhibited the appearance of an ox -ligand high pdc population ( fig s b and s c ), which may impact subsequent t cell activation. last, we assessed the effect of hcq on innate pdc functions. we measured cytokine production after hours of sars-cov- -induced pdc activation in the presence or absence of hcq. we found that ifn-α and λ levels were decreased by hcq (fig e) . this was also the case for il- and il- , with a much lesser impact on ip- ( fig e) . together, these results show that hcq inhibits sars-cov- -induced pdc diversification and cytokine production. type i ifns are critical cytokines that control viral replication. several chronic viral infections were associated to poor type i ifn responses (lee et al., ; snell et al., ; marsili et al., ; dolganiuc et al., ) . in covid- patients, decreased serum levels of type i ifn were associated with severity in late stage infection, and increased viral load (tay et al., ; acharya et al., ) . this raised the question as to whether sars-cov- was intrinsically capable of inducing a robust ifn response, or on the contrary could interfere with ifn production and other antiviral immune pathways. in this study, we have used primary sars-cov- isolates and human primary pdc in order to increase the relevance to a naturally occurring infection. our results demonstrate that sars-cov- is a strong ifn inducer by efficiently stimulating primary pdc. viral sensing was independent of the expression of the ace entry receptor or the ability of the virus to replicate in pdc. however, the precise molecular mechanisms involved remain to be investigated. both type i and type iii ifns were induced at high levels upon sars-cov- stimulation. this strongly suggests that the defects observed in critically ill covid- patients are acquired during disease evolution through secondary events, not necessarily associated to direct effect of the virus. possible mechanisms could be related to inflammatory cytokines, such as tnf, and endogenous glucocorticoid response, both being able to promote pdc apoptosis ( (abe and thomson, ) . however, additional mechanisms may be involved and need to be explored in the context of severe covid- . an excessive production of inflammatory cytokines, such as il- β, il- and tnf, was associated to covid- severity (tay et al., ; arnaldez et al., ; vabret et al., ) . their cellular source and the underlying mechanisms are currently unknown. our results indicate that sars-cov- -induced pdc activation promotes a balanced production of innate cytokines, including large amounts of type i and type iii infs, without any significant excess in inflammatory cytokines. this suggests that pdc activation may not be a causal factor of covid- aggravation. in support to that, recent studies indicated that endothelial cells may be a target of sars-cov- infection, and could be at the origin of the systemic and multi-organ production of inflammatory cytokines (pons et al., ) . bronchial epithelial cells could also be involved in the production of high levels of il- , which were not detected in the serum and in pbmc transcriptomic studies in severe covid- (wilk et al., ) . this supports the view of pdc as being protective through an early and efficient production of antiviral cytokines, with later defects due to currently unknown mechanisms, associated with late stage aggravation. on the contrary, nonprofessional innate immune cells such as endothelial cells and bronchial epithelial cells may be involved in the secondary worsening of covid- through the excessive and uncontrolled production of inflammatory cytokines. several therapeutic approaches have been explored and are currently being tested in clinical trials on covid- patients (vabret et al., ; tay et al., ) . these include antiviral agents (yang et al., ) , immune-modulatory molecules, such as glucocorticoids (fernández cruz et al., ) , and anti-inflammatory molecules, such as hcq (touret and de lamballerie, ; lecuit, ) . this latter drug was additionally shown in in vitro studies to interfere with sars-cov- replication (wang et al., ) . continued efforts in mapping and dissecting immune effector pathways to sars-cov- will be of major importance in order to design efficient treatment strategies adapted to each patient and stage of the infection. buffy coats from healthy human donors were obtained from etablissement français du sang, paris, saint-antoine crozatier blood bank. peripheral blood mononuclear cells (pbmcs) were isolated through ficoll density gradient centrifugation (ficoll-paque; ge healthcare). pdc were isolated through a first step of pdc magnetic sorting (human plasmacytoid dc enrichment kit; stemcell), and subsequent flow cytometric sorting on the basis of live, lineage -(cd , cd , cd , cd , cd and cd ), cd c -cd + , and cd -cd cells to a % purity. due to some logistic issues, alternatively frozen pbmcs from etablissement français du sang, paris, saint louis blood bank, were thawed and placed at °c for h for cell recovery. sars-cov- viruses were isolated from nasopharyngeal swab specimens collected at service de virologie (hospital saint louis, paris). samples were centrifugated at , x g for min then filtered using a . μm filter, and diluted : with dmem- % (dmem supplemented with % fbs, % penicillin-streptomycin, % glutamax and mm hepes). vero e cells were seeded in -well cell culture plate ( , cells/well), and incubated at °c with μl of inoculum and observed daily for cytopathogenic effects (cpe) by light microscopy. substantial cpe were seen at - hours post inoculation. culture supernatants were then collected, clarified by centrifugation, filtered using a . μm filter and kept at - °c. we confirmed sars-cov- replication by rt-qpcr and whole viral genome sequences were obtained by next generation sequencing using illumina misseq sequencers. strains sequences have been deposited in the global initiative of sharing all influenza data (gissaid) database with accession id epi_isl_ ( _ ) and epi_isl_ ( _ ). all viruses belong to the gh clade. sars-cov- strains were further propagated on vero e in dmem- % (dmem supplemented with % fbs, % penicillin-streptomycin, % glutamax and mm hepes). viruses were passaged three times before being used for experiments. for the last passage, viruses were purified through a % sucrose cushion by ultracentrifugation at , x g for hours at °c. pellets were resuspended in hne x ph . (hepes mm, nacl mm, edta . mm), aliquoted and stored at - viruses titer was ascertained by plaque assays in vero e cells and expressed as pfu per ml. cells were incubated for hour with -fold dilution of viral stocks. the inoculum was then replaced with avicel . % mixed at equal volume with dmem supplemented with % fbs, % glutamax, mm mgcl , . % of nahco , and incubated days at °c before plaque counting. vero cells were plated ( , cell per well) in p -well plates hours before being incubated with sars-cov- diluted in dmem- %. freshly purified pdc were seeded in p -well plates ( , cells per well) and incubated with sars-cov- diluted in pdcs culture medium (rpmi medium with glutamax, % of fbs, % of mem neaa, % of sodium pyruvate, and % of penicillin/streptomycin). at , and hour post-inoculation, vero cells were trypsinized and transferred to p -well plates. to quantify infectious viral particle released in the supernatants of infected cells, pdc and vero cells were inoculated with sars-cov- as described above and incubated at °c for -hour. at indicated time points, supernatants were collected and kept at - °c. virus titer were then determined by plaque assay on vero e cells as described above. vero e and pdc were inoculated with sars-cov- as described above. at the indicated time points, cells were washed thrice with pbs. vero e were further incubated with trypsin . % for min at °c to remove cells surface bound particles. total rna was extracted using the rneasy plus mini kit (qiagen) according to manufacturer's instruction. cdnas were generated from ng total rna by using the maxima first strand synthesis kit following manufacturer's instruction (thermo fisher scientific). amplification products were incubated with unit of rnase h for min at °c, followed by min at °c for enzyme inactivation, and diluted fold in dnase/rnase free water. real time quantitative pcr was performed using a to sort pdc, cells were stained with zombie violet or buv fixable viability dye pdc cytokine production of ifn-α , il- , il- , ip- and tnf-α, was measured in culture supernatants using bd cytometric bead array (cba), according to the manufacturer's protocol, with a pg/ml detection limit. acquisitions were performed on a lsr fortessa (bd biosciences), and cytokine concentrations were determined using fcap array software (bd biosciences). the concentration of secreted ifn-λ was measured by enzyme-linked immunosorbent assay (elisa) (r&d systems, duoset dy ), according to the manufacturer's instructions. the manufacturer reported no cross-reactivity nor interference with ifn-α, ifn-β a, il- rβ, ifn-λ and λ , and il- rα. the optical density value (od) of the supernatants was defined as its absolute od value, minus the od absorbance from blank wells. the detection limit was pg/ml and all samples were run in duplicates. statistical analyses were performed with one-way anova, kruskall wallis's test with dunn's multiple comparison post-test or mann whitney's test, in prism 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by and stimulate human plasmacytoid dendritic cells key: cord- - zrmgxq authors: bergman, scott j.; ferguson, mckenzie c.; santanello, cathy title: interferons as therapeutic agents for infectious diseases date: - - journal: infectious disease clinics of north america doi: . /j.idc. . . sha: doc_id: cord_uid: zrmgxq this article explains the rationale for development of interferons as therapeutic agents, and describes commercial products available today. it also provides a summary of studies that have been performed with interferons for use as exogenous biological response modifiers in viral infections. overall, the best data exist for treatment of viral hepatitis b and c, for which interferons are a cornerstone of therapy. although infections with human papillomavirus and common cold viruses sometimes respond favorably to interferons, their outcomes are far from ideal. finally, the role of interferons as vaccine adjuvants is still being explored but could be promising. replication in vitro at concentrations as low as pg/ml-the development of ifns as clinically useful drugs has been largely disappointing. this fact can be attributed partly to their short half-life in vivo and their extensive side effects. in fact, many symptoms of viral infections such as influenza can be blamed on endogenous ifn release. the adverse effects prevalent at therapeutic doses include fever, myalgia, and headache, dubbed "flulike symptoms," along with bone marrow suppression leading to leukocytopenia and thrombocytopenia, plus central nervous system manifestations including depression. ifns have been studied for the treatment or prevention of herpes zoster, herpes simplex, and cytomegalovirus infections, but the successful development of acyclovir and ganciclovir gave clinicians safer and more effective alternatives for dealing with these viruses. , ifns can also be used in the treatment of multiple sclerosis and certain cancers, but this article reviews the therapeutic applications of ifns for infectious diseases, focusing on viral infections. ifns are not absorbed orally because of their large amino acid sequence, which is susceptible to the proteolytic enzymes in the digestive tract. however, ifn-a is readily absorbed after both intramuscular and subcutaneous injection. this rapid absorption combined with a short half-life means that frequent injections are needed to maintain adequate concentrations in the body. both commercially available ifn-a products in the united states have now been chemically attached to polyethylene glycol (peg) to enhance their half-life and make once-weekly dosing possible. this coupling not only makes administration easier, but also reduces side effects by having a predictably lower peak concentration of the exogenous cytokine. both pegylated inf-a a (pegasys) and ifn-a b (peg-intron) are obtained from escherichia coli by recombinant methods. these agents consist of naturally occurring small proteins with molecular weights of , to , da. each is considered a first-line option for the treatment of chronic hepatitis c virus (hcv) infection in combination with ribavirin. more details on this use and others are described later in this article. along with the list of additional indications approved by the food and drug administration shown in table , ifn-a was shown to be an effective treatment for the symptoms of an aggressive case of chronic active epstein-barr virus, but did not eliminate infection entirely. therefore, additional studies would need to be performed before recommendation for this use. human leukocyte derived ifn-an (alferon n) injection contains a spectrum of a ifns, and is only approved for the treatment of refractory or recurring condylomata acuminata in adult patients. a low-dose oral version is in development for use in the treatment and prevention of influenza. both versions have been studied against human immunodeficiency virus (hiv)- infection, but with little success. , ifn alfacon- (infergen) is considered the synthetic "consensus interferon" because it contains a nonnatural sequence of ifn-a amino acids all chosen for the highest activity against viral hepatitis. to date, no pegylated formulation of this product has been brought to market. all the a ifns include a black-box warning in their prescribing information about how their use .may cause or aggravate fatal or life-threatening neuropsychiatric, autoimmune, ischemic and infectious disorders. patients should be monitored closely with periodic clinical and laboratory evaluations. therapy should be withdrawn in patients with persistently severe or worsening signs and symptoms related to side effects. in many, but not all cases, these resolve after stopping therapy. , ifn-b a (avonex or rebif) and ifn-b b (betaseron) are recombinant proteins with and amino acids, respectively. these b ifns have antiviral and immunomodulatory properties too, but their use at this time is limited to treatment of multiple sclerosis, not infections. ifn-g b (actimmune) injection is used regularly for the prevention of infections in patients with chronic granulomatous disease along with antibacterials and antifungals. its mechanism of action for this purpose is not entirely known, but long-term studies show a definite benefit. ifn-g can also be used as a salvage therapy for mycobacterial infections, but is not routinely used for treatment of this or other infections. topical imiquimod % (aldara) and . % (zyclara) creams do not have inherent antiviral activity alone, but instead induce ifn-a, ifn-b, and ifn-g plus tumor necrosis factor (tnf)-a through toll-like receptors (tlrs). local application to external genital and perianal warts results in an immunomodulatory response that stimulates cytokines, which have antiviral action and cause a reduction in both viral load and wart size. chronic infection with hepatitis b virus (hbv) and hcv affects over million people worldwide. [ ] [ ] [ ] chronic viral hepatitis is a leading cause of cirrhosis, liver transplantation, and hepatocellular carcinoma. with the development of a vaccination series for hepatitis b in the mid- s, along with increased public education and awareness, acute infection rates of both hbv and hcv in the united states have declined steadily. hbv is a double-stranded dna virus whereas hcv is a single-stranded rna virus, both of which are capable of significant morbidity and mortality in chronic infection. the exact mechanisms of hepatic injury from hbv and hcv infection are not completely understood. because asymptomatic carriers with normal liver transaminases exist, it is likely multiple immune-mediated mechanisms result in hepatocyte damage as opposed to the virus itself being directly cytotoxic. following acute viral infection, the innate immune response initiates formation of nk cells, followed by virus-specific cd t cells and cd cytotoxic t lymphocytes. nk cells stimulate production of ifn-a/b and promote cellular clearance of viral proteins through disruption of the replication process. following successful clearance, either spontaneously or by treatment with ifn, peripheral cytotoxic t lymphocytes and cd t-cell response persists. chronic infection is likely a result of failed innate and adaptive immunity. specifically, chronic infection with hcv has been associated with impaired t-cell and nk-cell response. [ ] [ ] [ ] [ ] genetic factors also likely influence progression of disease and predisposition to adverse effects. although an abundance of research has investigated the immune response in relation to chronic viral hepatitis, many areas of uncertainty still exist. standard ifn-a, the first approved ifn for viral hepatitis, lacked several desirable pharmacokinetic properties. the addition of peg created an ifn that has a slower rate of absorption, reduced elimination, and a longer half-life, necessitating less frequent dosing and fewer adverse effects. furthermore, the peg moiety results in reduced immunogenicity and sterically hinders the antigenic binding site. , although pegylated ifn has replaced standard ifn-a in treatment of chronic hbv and hcv, as many as % to % of patients still fail to respond to treatment. successful response depends on many factors including but not limited to viral genotype, viral load, and degree of liver fibrosis. chronic hepatitis b and c are treated similarly with peginterferon (pegifn); however, only pegifn-a a is fda-approved in the united states for treatment of hbv. both pegifn products are administered as subcutaneous injections once weekly for durations up to weeks, dependent on viral genotype and early viral response for treatment of hcv. pegifn-a b is dosed based on body weight ( . mg/kg once weekly) whereas pegifn-a a is a fixed dose ( mg/wk). ribavirin is used in combination with pegifn for treatment of hcv. the exact mechanism of action of ribavirin as an adjunctive antiviral agent in hcv is not completely understood. , some studies have proposed ribavirin to act as an ifn-stimulated gene inducer to improve second-phase viral decline. protease inhibitors (boceprevir and telaprevir) are recently approved adjunctive oral agents for the treatment of chronic hcv with pegifn and ribavirin. to date, all studies of protease inhibitors have been conducted in patients with hcv genotype , and have shown an increase in sustained virologic response (svr) rates particularly for patients previously unresponsive to ifn therapy. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the use of ifn for the treatment of chronic hbv and hcv has represented a mainstay of treatment for several decades. the specific mechanisms behind the antiviral effects of ifn for hepatitis are complex. ifn-stimulated genes are induced by ifn and disrupt viral replication. hundreds of ifn-stimulated genes are thought to exist. viperin, isg and protein kinase r (pkr) are just a few of the most commonly cited. it is also highly possible that ifn-stimulated genes work synergistically to produce antiviral activities. , a lack of pkr can lead to an environment conducive to hcv replication, though it may not be a good predictor of exogenous ifn response. the study of ifn-stimulated genes and their role in determining who responds to ifn therapy has been evaluated in several studies. , , additional studies of ifnstimulated gene expression are needed to clarify which are directly involved in successful viral response, in what capacity they affect response, and whether pharmacotherapy directed at induction of ifn-stimulated genes can help improve treatment response. chronic hbv infection can be successfully treated with ifn monotherapy. loss of viral dna and antibody formation are successful outcomes associated with ifn treatment. the mechanism of ifn antiviral activity varies depending on hepatitis be antigen (hbeag)-positive or hbeag-negative disease. in hbeag-positive patients, an immune response is stimulated by ifn whereas in hbeag-negative disease, ifn acts directly as an antiviral. hbeag-negative disease tends to be more difficult to treat, and is associated with a longer duration of disease and a higher likelihood of complications such as cirrhosis. several oral nonnucleoside reverse transcriptase inhibitors are also available for treatment of hbv (entecavir, tenofovir, adefovir, lamivudine, and telbivudine). although ifn is still considered a first-line alternative and provides the advantage of defined treatment duration rather than potentially lifelong administration, these oral agents are often used in therapy because of their ease of use and reduced number of side effects associated with treatment. the ability of hcv to evade the host immune response has produced a complex rna virus capable of lingering infection, ultimately resulting in opportunities for increased risk of transmission and complications from advanced liver disease. much of the research regarding the use of ifn for chronic viral hepatitis has focused on use in hcv. following treatment with ifn, a decline in hcv rna occurs over several phases. a rapid inhibition of rna production within the first to days of treatment is followed by a second, slower phase associated with clearance of infected cells. , , the interferons for infectious disease immune response to endogenous ifn produced by innate immunity and that administered exogenously can differ in terms of antiviral activities based on the phase of viral decline. studies have shown that response to ifn-based treatment for hcv may be affected by differences in ifn signaling and induction. it is likely that hcv has mechanisms to avoid recognition by the innate immune response, and as such inhibits the ability of hcv-infected cells to generate ifn. , , early studies conducted in nonresponders to current therapy showed wide genetic diversity, with many showing no common traits to predict nonresponse to ifn therapy. , , however, in several major studies were published associating a singlenucleotide polymorphism (snp) just upstream from interleukin- b gene (il b) with ifn response in patients with hcv genotype . [ ] [ ] [ ] [ ] [ ] additional evidence points to the fact that the il b polymorphism is also linked to spontaneous clearance of hcv. , the il b variant encodes for ifn-l , a type iii ifn belonging to the interleukin (il)- superfamily, which function in a manner similar to type i ifns, resulting in ifn-stimulated gene induction. [ ] [ ] [ ] the genome-wide association study conducted by ge and colleagues evaluated more than treatment-naïve hcv genotype patients, the majority of whom originated from the ideal study. results from logistic regression showed that the il b polymorphism was a stronger predictor of svr than baseline viral load, ethnicity, or degree of fibrosis. further research in this area is needed to clearly identify a future role for genotype testing and further clarify whether it may influence response to therapy in other hcv genotypes. a multicenter, randomized, controlled study by mangia and colleagues analyzed caucasian patients with hcv genotype (n ) and (n ). out of % of patients who achieved rapid virologic response (rvr), il b genotype was not associated with svr, whereas in those patients who did not achieve rvr a significant difference in svr was noted based on il b genotype. at this time genotype testing for il b is not routinely recommended for all hcv patients planning to undergo treatment, but it may be in the future. if done, it should not be used as the only factor when choosing a treatment strategy. the complexity of viral defense mechanisms and subsequent effect on the host response has led not only to development of chronic infections but also to a lack of a viable vaccine. hcv viral polymerase lacks a proofreading capability, creating a more diverse target for vaccine development. additional challenges include the lack of a suitable animal model to mimic a human environment and medium for viral growth. one of the major limitations to ifn therapy is adverse effects. malaise, gastrointestinal effects, neuropsychiatric effects, neutropenia, and anemia can all limit the effectiveness of treatment by necessitating dosage reductions or treatment discontinuation. for newer ifn therapies to be successful, they must induce an antiviral response while at the same time limiting adverse effects. albinterferon is a new ifn therapy currently in development for the treatment of chronic hcv. this product is a combination of ifn-a b fused to recombinant human albumin. one of the advantages with this product is that it only requires once or twice monthly dosing. not much is known at this time about the immunomodulating effects of albinterferon in hcv. it has been shown to have similar svr and adverse event rates to traditional pegifn when used in combination with ribavirin. [ ] [ ] [ ] research into ifn-l as an agent to treat hcv has also been initiated. it is hypothesized that l ifns may be associated with less adverse effects than ifn-a because ifn-l receptors are primarily found in hepatocytes. , specifically, research into new investigational pharmacotherapy in the form of pegylated il- (ifn-l ) in patients with hcv genotype who relapsed following traditional treatment with peg-ifn-a and ribavirin appears promising. , both ifn-l and ifn-l share a common receptor and have a similar sequence identity. a -week, open-label study conducted in patients with chronic hcv genotype was designed to assess pegifn-l in combination with ribavirin. it was a dose escalation study conducted in parts. parts and evaluated patients who relapsed following treatment with ifn-a, and part included treatment-naïve patients. in part , pegifn-l monotherapy ( . mg/kg or mg/kg) was administered subcutaneously every weeks or weekly. in parts and , a range of pegifn-l dosages ( . mg/kg, . mg/kg, . mg/kg, or . mg/kg) were administered weekly in combination with ribavirin twice daily ( mg if weight < kg and mg if weight ! kg). the primary outcomes were safety and tolerability. pharmacokinetics and viral load reduction were evaluated as secondary end points. commonly reported adverse effects with pegifn-l included fatigue ( %), nausea ( %), myalgia ( %), and headache ( %). most adverse events were mild or moderate in severity. four patients ( %) experienced treatment-related toxicity and required doses to be withheld. one patient experienced grade thrombocytopenic purpura and another patient had elevated alanine aminotransferase, aspartate aminotransferase, and bilirubin levels. both events were considered to be related to treatment with pegifn-l. aminotransferase elevations occurred most often in patients who received high-dose ( mg/kg) pegifn-l monotherapy. no clinically relevant decreases in absolute neutrophil count occurred. also, hemoglobin values remained consistent with known effects in patients who received ribavirin therapy. viral activity decreased in the majority of patients who relapsed with previous treatment, with of patients achieving at least a greater than -log reduction in hcv rna. six of treatment-naïve patients achieved a similar reduction in viral load and achieved undetectable hcv rna levels. kinetic data showed a linear relationship between dose and exposure independent of body weight, which may prompt future research to evaluate a fixed dose of pegifn-l. larger, longer, controlled, and blinded studies of ifn-l as a viable treatment option in hcv are needed to define its place in therapy and benefits over existing ifn therapy. studies in other hcv genotypes are also needed. in addition, with the advent of protease inhibitors, more research will be necessary to evaluate how direct antivirals and il b genotyping interact in guiding treatment decisions. adjunctive therapy with agents that induce or restore ifn-stimulated gene expression has recently been evaluated in patients with hcv. s-adenosylmethionine (same) given orally was evaluated in an open-label study in patients with chronic hcv, genotype who were considered nonresponders to previous ifn and ribavirin treatment. same was administered at a dose of mg twice daily in combination with pegifn-a a ( mg/kg weekly) and weight-based ribavirin ( mg if weight < kg and mg if weight ! kg). the primary outcome was change in firstphase and second-phase viral decline. treatment response and ifn-stimulated gene expression were also evaluated after up to weeks of treatment. results showed significant improvement in second-phase viral decline assessed at weeks. svr was also evaluated; however, this study was not powered to detect differences in virologic response rates. furthermore, at the time of publication not all patients had reached weeks post treatment, so the full effects on svr were not fully known. the addition of same showed greater induction of ifn-stimulated genes, including viperin, myxovirus resistance protein, and isg , compared with control. adverse effects noted with same were mild and mostly related to gastrointestinal upset, likely as a result of lactose in the tablet preparation. additional research is aimed at investigating structure-activity relationships, and preliminary pharmacokinetic studies on oral ifn inducers that act on tlrs in the treatment of hcv. upper respiratory tract infection in the form of "the common cold" can be caused by a variety of viruses including rhinovirus, coronavirus, influenza, parainfluenza, respiratory syncytial virus, adenovirus, coxsackie, and echovirus families among others. symptoms may include rhinorrhea, nasal obstruction, cough, fever, and sore throat. the disease is usually mild and self-limited, but several trials have addressed treatment or prevention of the common cold with therapeutic agents. ifns were once one of the most popular prospects for this purpose, but the minor benefit that was derived from them was counteracted by the adverse effects inflicted. an early double-blind trial with ifn-a b intranasal drops did demonstrate that with use for several days before experimentally induced rhinovirus infection, common cold symptoms were significantly fewer in study participants compared with placebo-drop users. administration of the drops times daily was superior to a higher dose given once daily at preventing infection. short-term use was well tolerated, but obviously it is not realistic for everyone to use intranasal drops times daily throughout the entire cold season. in an attempt to prevent natural infection during the period of increased acute respiratory tract virus activity, a twice-daily nasal spray was studied in volunteers over days. there was a significant decrease in the number of rhinovirus infections noted, but not in any other types of viral respiratory tract infections including parainfluenza. adverse events with the ifn formulation were common in this placebocontrolled trial. during the first week alone, % of participants receiving ifn spray reported nosebleeds. this number increased to % by the end of the study. providing ifn prophylaxis for family members of those infected with common cold viruses is a more targeted approach to therapy. several studies have addressed the usefulness of ifn nasal sprays in this scenario. seven days of use did significantly reduce rhinovirus infections in different trials when compared with placebo for both individuals ( . % vs . %) and their families ( . % vs . %, both p<. ), but not in other studies when lower doses were given for a shorter -day course. [ ] [ ] [ ] [ ] overall, the intranasal dose of ifn needed to protect against upper respiratory tract infection appears to cause significant unwanted effects. infection with coronavirus and respiratory syncytial virus has also been an object of investigation for ifn-a b nasal sprays, but with little success. , a study of intranasal human lymphoblastoid ifn-an (wellferon) suggested lower prophylactic activity for influenza than it did for rhinovirus. because results of prophylactic trials with ifns for common cold viruses were not favorable, use in the treatment of infection seemed a logical application for this biological response modifier. although some benefit was originally seen with twice-daily ifn-a b intranasal drops for treatment of experimentally induced rhinovirus, no advantage was clear when an intranasal spray was used once daily for days to treat natural infection. increased rates of blood in the mucus were again noted for participants receiving the intervention, and the ifn group experienced more secondary complications requiring prescription of antibiotics. the investigators concluded that intranasal ifn was ineffective for treating the common cold and was associated with clinically significant side effects. similar trials with ifn-b-serine and ifn-g formulations, although initially positive, have shown equally disappointing clinical results. [ ] [ ] [ ] [ ] even though the prospects of further study on ifns for upper respiratory tract infection appear limited, one modern trial did demonstrate an added benefit of intranasal ifn-a b in combination with an antihistamine (chlorpheniramine) and nonsteroidal anti-inflammatory drug (ibuprofen) at reducing common cold symptoms, showing that at least one group is still interested in studying the topic. investigators have also recently begun research on an alternative therapeutic approach for rhinovirus infections using the ifn and tnf-a inducer, imiquimod. application of this intranasal cream in primates has shown promising results in terms of enhancing cytokine response, but human trials have not yet been published. human papillomaviruses (hpvs) are now known to be the cause of cervical cancer and are also responsible for genital warts. hpvs are nonenveloped, double-stranded dna viruses that invade mucosal and epithelial tissues during sexual contact with an infected partner. it is estimated that more than % of the sexually active american population has been or will be infected with hpv at one point in their lives. when hyperproliferation of infected cells occurs, this can lead to genital warts or cancer of the cervix, vagina, vulva, and penis, among others. there are more than different types of hpv and approximately of them infect genital mucosa. fifteen carcinogenic types of hpv have been identified, but of them are associated with % of cervical cancers. two vaccines have recently been introduced that prevent infection with these most common high-risk types of hpv, and . one of these vaccines can also induce protection against the most prevalent hpv types that have a low risk of malignancy, but instead cause genital warts: hpv- and hpv- . hpv has the ability to persist in stratified epithelia for decades because of mechanisms that avoid immune eradication. ifn plays a large role in this cycle. ifns are normally secreted by keratinocytes, but hpv reduces their expression. introduction of low-level ifn can actually increase early gene transcription and hpv replication, which may explain why use of the agent therapeutically has had mixed results. overall outcomes have been positive more often for cases of genital warts than reduction of hpv lesions associated with cancers. a study comparing the in vitro activity of ifn-a b and ifn-an on oncogenic hpv- , hpv- , and hpv- b demonstrated that increasing concentrations did not always correlate with a stepwise inhibition of hpv replication. meanwhile, a meta-analysis recently analyzed locally used and systemic ifn for genital warts. seven randomized studies of ifn intralesional injection or topical gel met criteria for inclusion, and overall there was a benefit in complete response rates over placebo ( . % vs . %, relative risk . , % confidence interval . - . ). however, there was no difference in outcomes for trials comparing systemic ifns with placebo. in comparison, clearance of genital and perianal warts occurs in % of patients with the topical ifn inducer imiquimod, usually after to weeks of use. the % imiquimod cream (aldara) should be applied to affected areas times a week for up to weeks, whereas the newer . % cream (zyclara) can be applied once daily for as little as weeks to treat external genital warts caused by hpv. systemic ifn therapy may be useful when hpv affects areas of the body other than the anogenital region. successful treatment with systemic pegifn-a and a topical retinoid has been reported for mucosal carcinomas from epidermodysplasia verruciformis, a genetic abnormality leading to persistent and widespread hpv infection of the skin. recurrent respiratory hpv infection has also been effectively treated with ifn-a ( of patients), although it had no effect on viral load or replication. a -year follow-up of patients treated with ifn-a for recurrent respiratory papillomatosis confirmed better response rates for hpv- than hpv- , which had a higher likelihood of malignant transformation. for recurrent conjunctival papilloma, topical plus systemic or intralesional ifn has been effective with partial excision. , the rapid resolution of significant hpv-associated warts on the hand, foot, and face has also occurred in an hiv-infected patient on antiretrovirals while being treated for hepatitis c with pegifn-a b and ribavirin. case reports of treatment with the topical ifn inducer, imiquimod, have shown promise for its use in focal epithelial hyperplasia (heck disease), a rare disorder caused by specific types of hpv ( , , , and ) affecting oral mucosa primarily in children. in addition, imiquimod % cream has been used successfully in the treatment of plantar warts, a smoother, flatter manifestation of hpv- , hpv- , and hpv- on the foot. of interest, the oral h -antagonist cimetidine, along with reducing stomach acid, also induces production of ifn-g and il- , which eliminates viral warts in some patients. in the future the improved application of more effective topical ifns may become a reality, which could provide a valuable treatment for hpv infections without the systemic side effects of current injectable formulations. adjuvants (adjuvare, latin for "to help") are substances that augment the immunogenicity of an antigen when mixed with the antigen for use in a vaccine. adjuvants ( ) stimulate granuloma (which is a macrophage-rich mass), ( ) enhance costimulatory signals, ( ) stimulate nonspecific lymphocyte production, ( ) prolong the antigen concentration in a site for lymphocyte exposure, and ( ) induce cytokines. , research in vaccine development has shown that one of the most promising uses of ifns is as an adjuvant with specific antigens in prophylactic vaccines. toporovski and colleagues provide a current review of the use of ifn-a, ifn-b, ifn-g, and ifn-l in vaccine studies that focus primarily on murine, avian, porcine, and nonhuman species. regardless of the species, the use of ifns as adjuvants seems to improve the efficacy and safety of most vaccines while providing the immunomodulatory effect of stimulating the t-helper response. in humans, ifn-a, predominantly produced by plasmacytoid dendritic cells, plays a large role in the body's immune response against viruses. it induces plasma cell differentiation from b cells causing an increase in the serum level of influenzaspecific immunoglobulins, and channels antigen-presenting cells (apcs) to the site of infection. most research on ifn-a adjuvant activity and its subsequent use in approved vaccines seems to indicate that it is a potent adjuvant. when mixed with the influenza vaccine and injected intramuscularly, it is a highly effective adjuvant. oromucosal administration of recombinant ifn-a, like that of natural oromucosal ifn production, has been shown to provide immunity against viral infection and tumor cell growth. nonresponders low responders to a previous vaccine showed an improved immunoglobulin response with a recombinant ifn-a and hbv vaccine. although research is also focused on the other classes of ifns as adjuvants, thus far they have not yielded results as promising as that of ifn-a. the use of ifn-b has yielded mixed results; ifn-g has been used primarily in dna vaccines; and even less is known about the use of ifn-l in vaccines. nevertheless, the use of ifns as adjuvants shows great promise in augmenting vaccine efficiency, and should continue to be a top priority in the development of vaccines. ifns have been tested repeatedly against infectious diseases, but injections are used mostly for the treatment of viral hepatitis c and prevention of infections in patients with chronic granulomatous disease clinically. intralesional ifn and topical inducers are effective in reducing the manifestations of genital warts, but they do not eliminate cancer-causing hpv from the body. ifn has not proved to be consistently effective for treatment of respiratory tract infections from the common cold or influenza viruses, and prophylactic use is not currently feasible. the severity and quantity of adverse effects from systemic ifn therapy make it unattractive for many uses. several infections, including herpes simplex, herpes zoster, cytomegalovirus, and even viral hepatitis b have other effective pharmacologic treatments. ifn has been successfully used as a vaccine adjuvant, and further research may allow for its additional use for this application in the future. mim's medical microbiology goodman & gilman's the pharmacological basis of therapeutics role of endogenous biological response modifiers in pathogenesis of infectious diseases interferons at age : past, current and future impact on biomedicine immunoglobulins, vaccines or interferon for preventing cytomegalovirus disease in solid organ transplant recipients interferon: mechanisms of action and clinical value interferon-alpha therapy for chronic active epstein-barr virus infection: potential effect on the development of t-lymphoproliferative disease fda authorizes alferon ldo clinical study for treatment and prevention of influenza a multicenter, randomized, controlled trial of three preparations of low-dose oral alpha-interferon in hivinfected patients with cd counts between and cells/mm( ). division of aids treatment research initiative (datri) study group vitro activity of interferon-alpha n (alferon n) against hiv- . interscience conference on antimicrobial agents and chemotherapy nj: pegasysÒ (peg-interferon alfa- a) nj: peg-intronÒ (peginterferon alfa- b) a controlled trial of interferon gamma to prevent infection in chronic granulomatous disease. the international chronic granulomatous disease cooperative study group long-term interferon-gamma therapy for patients with chronic granulomatous disease biological response modifiers as adjunct treatment for refractory localized mycobacterium avium complex infections fact sheet: world health organization viral cancers: world health organization 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protease inhibitor of hcv antiviral effects and safety of telaprevir, peginterferon alfa- a, and ribavirin for days in hepatitis c patients antiviral activity of telaprevir (vx- ) and peginterferon alfa- a in patients with hepatitis c sch , a novel hepatitis c virus protease inhibitor, plus pegylated interferon alpha- b for genotype nonresponders telaprevir with peginterferon and ribavirin for chronic hcv genotype infection telaprevir and peginterferon with or without ribavirin for chronic hcv infection the interferon inducible gene: viperin identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus the induction of type i interferon production in hepatitis c-infected patients a treatment algorithm for the management of chronic hepatitis b virus infection in the united states: update effect of ribavirin on hepatitis c viral kinetics in patients treated with pegylated interferon s-adenosyl methionine improves early viral responses and interferon-stimulated gene induction in hepatitis c nonresponders the interferon inducing pathways and the hepatitis c virus genetic variability of hepatitis c virus before and after combined therapy of interferon plus ribavirin a new era of hepatitis c therapy begins genetic variation in il b predicts hepatitis c treatment-induced viral clearance il b is associated with response to chronic hepatitis c interferon-alpha and ribavirin therapy genome-wide association of il b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c genetic variation in il b and spontaneous clearance of hepatitis c virus hepatitis c pharmacogenetics: state of the art in pharmacogenetics of hepatitis c therapy snipping away at hepatitis c. hepatology an il b polymorphism determines treatment response of hepatitis c virus genotype or patients who do not achieve a rapid virologic response albinterferon alfa- b dosed every two or four weeks in interferon-naive patients with genotype chronic hepatitis c safety and antiviral activity of albinterferon alfa- b dosed every four weeks in genotype / chronic hepatitis c patients albinterferon alfa- b was not inferior to pegylated interferon-alpha in a randomized trial of patients with chronic hepatitis c virus genotype or albinterferon alfa- b was not inferior to pegylated interferon-alpha in a randomized trial of patients with chronic hepatitis c virus genotype phase b study of pegylated interferon lambda with or without ribavirin in patients with chronic genotype hepatitis c virus infection design and optimisation of orally active tlr agonists for the treatment of hepatitis c virus infection the common cold alpha -interferon for the common cold intranasal interferon alpha for prevention of rhinovirus infection and illness intranasal interferon-alpha b for seasonal prophylaxis of respiratory infection prophylactic efficacy of intranasal alpha -interferon against rhinovirus infections in the family setting 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of interferon alpha in human volunteers and treatment of patients with human papillomavirus infections new york: w.h. freeman and co advances in vaccine adjuvants interferons as potential adjuvants in prophylactic vaccines plasmacytoid dendritic cells induce plasma cell differentiation through type i interferon and interleukin adjuvant activity of interferon alpha: mechanism(s) of action oromucosal interferon therapy: marked antiviral and antitumor activity randomized comparative trial of interferon-alpha versus placebo in hepatitis b vaccine non-responders and hyporesponders key: cord- -h iv crq authors: sumino, kaharu; tucker, jennifer; shahab, muhammad; jaffee, katy f.; visness, cynthia m.; gern, james e.; bloomberg, gordon r.; holtzman, michael j. title: antiviral ifn-γ responses of monocytes at birth predict respiratory tract illness in the first year of life date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: h iv crq background: viral respiratory tract infections are the leading cause of acute illness during infancy and are closely linked to chronic inflammatory airway diseases later in life. however, the determinants of susceptibility to acute respiratory tract infections still need to be defined. objective: we investigated whether the individual variation in antiviral response at birth determines the risk for acute respiratory tract illness in the first year of life. methods: we studied children who were enrolled in a birth cohort study of inner-city children with at least parent with allergy or asthma. we cultured cord blood monocytes and assessed ifng and ccl mrna production at hours after inoculation with respiratory syncytial virus. we also monitored the frequency of acute respiratory tract illness at -month intervals and analyzed nasal lavage samples for respiratory tract viruses at the time of illness during the first year. results: respiratory tract infection was reported for % of subjects, and respiratory tract viruses were recovered in % of symptomatic children. we observed a wide range of antiviral responses in cord blood monocytes across the population. furthermore, a decrease in production of ifng (but not ccl ) mrna in response to respiratory syncytial virus infection of monocytes was associated with a significant increase in the frequency of upper respiratory tract infections (r = − . , p < . ) and the prevalence of ear and sinus infections, pneumonias, and respiratory-related hospitalizations. conclusion: individual variations in the innate immune response to respiratory tract viruses are detectable even at birth, and these differences predict the susceptibility to acute respiratory tract illness during the first year of life. viral respiratory tract infections are a common cause of early childhood illness. most of these infections are short-lived and selflimited, but some can be severe enough to require hospitalization. indeed, viral respiratory tract infections are associated with % of all mortality in children less than years of age. in addition to the morbidity of the acute infection, viral respiratory tract infections with wheezing are strong indicators of subsequent asthma. , therefore predicting those infants at risk for respiratory tract infections is an important first step in preventing acute and chronic respiratory disease. previous studies have identified a variety of potential risk factors for viral lower respiratory tract infections during the first year of life. these factors include day care attendance, number of siblings, small lung size, exposure to tobacco smoke, low birth weight, and premature birth. [ ] [ ] [ ] [ ] infections caused by respiratory syncytial virus (rsv) are particularly implicated in acute illness and chronic lung disease in the first years of life. however, the majority of rsv infections in infants occur without any known risk factors. thus we still do not understand the precise mechanism for the wide variation in susceptibility to severe respiratory tract infections among children in these settings. one possible explanation for the range of susceptibility to viral respiratory tract infection in early childhood is that there are definable variations in the antiviral response, such as a congenital deficiency in the innate immune response that can be detected even at the time of birth. a central ingredient of the innate immune response to respiratory viruses is the system for interferon production and signaling. in that regard a decrease in ifn-g production from cord blood mononuclear cells (cbmcs) stimulated by pha or allergens has been associated with increased risk for acute respiratory tract illness during infancy. , perhaps more relevant to viral infection, the lack of a detectable ifn-g response to rsv in cbmcs was associated with decreased wheezing in the first year of life, but a detectable response was only found in a third of subjects, and therefore predictive power was limited. therefore in the present study we developed alternative methods to determine whether the innate immune response of virus-infected cbmcs could predict the later development of respiratory tract illness. we used rsv to activate cbmcs based on the well-established association of rsv infection with subsequent childhood asthma. , , however, to monitor the innate immune response to rsv, we determined the induction of the genes encoding ifn-g and the remarkably virus-responsive chemokine ccl based on sensitive and quantitative methods for mrna from we also monitored interferon signal transduction by tracking the level of signal transducer and activator of transcription (stat) activation in response to ifn-b stimulation. in both cases we used cultured/adherent cbmcs to select for monocytes versus the mixed cell population that included t cells in previous studies. we assessed whether each of these immune end points could predict the development of respiratory tract illness during the first year of life in a prospective birth cohort of children at high risk for asthma and allergic disease. the experimental matrix led to the unexpected finding of rsv-induced ifng gene expression in monocytes as a predictor of subsequent viral respiratory tract illness. we analyzed cord blood samples from newborns enrolled in the urban environment and childhood asthma (ureca) study. this group represents a subset of the children enrolled at the st louis site, which in turn was a subset of the total number of children enrolled at the baltimore, boston, and new york city sites between february and march , as described previously. [ ] [ ] [ ] subjects were required to have at least parent with allergic rhinitis, eczema, and/or asthma and to reside in an area with greater than % of the residents below the poverty level, as well as being born at weeks' gestation or later. at the st louis site, a small number of children without an allergic parent (n ) were also recruited for comparison. after enrollment, all subjects were monitored for any episodes of acute respiratory tract illness over the next year along with quarterly assessments of respiratory (and nonrespiratory) tract illness and wheezing by questionnaire. nasal lavage samples were obtained when a caregiver reported an acute respiratory tract illness and at the time of a -year follow-up visit. the washington university human research protection office approved the study protocol. cord blood samples were collected in the delivery room, and cbmcs were isolated by means of density gradient centrifugation with accuspin tubes (sigma, st louis, mo) within hours of collection, as described previously. , when sufficient amounts of sample were available (ie, in / subjects), the cells were resuspended in rpmi medium with % fbs, mmol/l l-glutamine, and mmol/l nonessential amino acids to a final concentration of per milliliter and plated in -well lab-tek chambers ( ml per well; nunc a/s, roskilde, denmark) for the real-time pcr assay and in -well lab-tek chambers ( ml per well) for the stat activation assay, as described below. nonadherent cells were removed after hours, and adherent cells were cultured for days with media changes on days , , and and removal of additional nonadherent cells. at the end of the cell-culture period, the adherent cells were greater than % positive for cd immunostaining as a marker of monocytic lineage and therefore designated as cord blood monocyte cultures. the approach avoided purification methods (eg, magnetic bead selection or fluorescence-activated cell sorting) that modify the cell membrane or cell-culture methods (eg, growth factor supplementation) that promote full differentiation and polarization and thereby aimed to obtain cells of the monocyte lineage that were representative of naive lung tissue monocytes and macrophages (the target for viral respiratory tract infection in vivo). on culture day , cord blood monocytes were infected with rsv (a strain) at a multiplicity of infection of . or an equivalent amount of uvinactivated respiratory syncytial virus (rsv-uv). cellular rna was isolated immediately and hours after inoculation with the rnaeasy mini kit (qiagen, valencia, calif) and transcribed to cdna by using the high-capacity cdna reverse transcription kit (applied biosystems, foster city, calif). single-target quantitative real-time pcr was used to monitor ifng and ccl mrna and rsv rna levels. for ifng and ccl mrna, primers were obtained from applied biosystems (hs _a and hs _a ). for rsv rna, primers f ( -tccctacggttgtgatcgataga- ) and r ( -tgatgggaagtagtagtgtaaagttggt- ) and probe t ( -aggtaatacagccaaatc- ) targeting the viral l gene were based on the sequence of rsv strain crd (genbank accession no. dq ). for glyceraldehyde- -phosphate dehydrogenase (gapdh) mrna, primers f ( -cagccgagccacatccctcagacaccat- ) and r ( -ctttaccagagttaaaagcagccctggtgacca- ) and probe t ( -aggtcggagtcaaccgatttggtcgtattg- ) were used. plasmids encoding ccl and ifng (origene, rockville, md) and a portion of the rsv l gene (nt - ) and gapdh gene sequence (genbank accession no. nm_ ) were used to generate rna standards. rt-pcr was performed by using taqman real-time pcr master mix with ml of sample cdna in accordance with the manufacturer's protocol (applied biosystems). all data for gene copy number was normalized to gapdh level. in a subset of cord blood samples (n ) with an adequate number of cells, we also assessed interferon signal transduction by monitoring the level of stat phosphorylation in response to interferon stimulation. the corresponding cbmcs were processed as described above and serum starved on day of culture. on culture day , the cells were incubated with ifn-b ( u/ml) for minutes. cell lysate was harvested after treatment with cell lysis buffer (cell signaling, danvers, mass). the level of total stat was determined by using elisa (invitrogen, carlsbad, calif), and phosphorylated stat (tyr ) levels were determined by using a sandwich elisa (pathscan phospho-stat , cell signaling). nasal lavage samples were obtained during acute respiratory tract illnesses during the first year of life and at the -year follow-up visit. for illness samples, a respiratory symptom scorecard was completed as described previously. , when the score indicated a moderate-to-severe respiratory tract illness, the site staff obtained a nasal lavage sample within hours. all nasal lavage samples were processed for identification of respiratory tract viruses by using a pcr-based assay, as described previously. descriptive data were expressed as percentages, means sds, or medians with interquartile ranges for nonnormally distributed data. to test differences between specific groups, x or fisher exact tests were used to compare categorical variables, whereas unpaired t tests were used to compare continuous variables. appropriate log transformations were made to the data to yield an approximately normal distribution. for nonnormally distributed data, the wilcoxon rank sum tests (mann-whitney u tests) were used to compare groups. each measurement was standardized as the ratio over control to minimize variability in day-to-day experiments as follows: d ifng mrna response to rsv ifng mrna copies with rsv/ ifng mrna copies without rsv, d ccl mrna response to rsv ccl mrna copies with rsv infection/ccl mrna copies with no rsv infection, and rsv level was expressed as the rsv rna copy number with infection minus the value for no infection. spearman correlations were calculated to test for associations between variables. we examined the possibility of confounding by the following variables: sex, breast-feeding, maternal smoking during pregnancy and the first year of the child's life, an overall sum of bedroom allergen exposure (mus musculus, blattella germanica, and felis domesticus), and exposure to endotoxin (recombinant factor c assay) and ergosterol. we evaluated whether any of these variables were associated with the outcomes under analysis, and we found only that maternal smoking during the first year of life was related to the pneumonia outcome and the sum of bedroom allergen exposures was related to sinus infections. however, in logistic models controlling for these variables, the odds ratios changed by less than % when adjusted, and therefore we present unadjusted relationships here. all statistical tests were -tailed, and p values of less than . were considered statistically significant. statistical procedures were conducted with both sas . (sas institute, inc, cary, nc) and r . . software. we processed all cord blood samples that contained an adequate number of cells, representing of the total of children who were enrolled at the st louis site of the ureca cohort. among the newborns, % of the babies were african american, the mean age of the mother at the time of delivery was . years, and % of the infants had at least parent with asthma (table i) . subjects were reported to have an average of . upper respiratory tract infections (colds), . wheezing illnesses, and an all-cause hospitalization rate of % during the first year of life. the basic demographics and first-year outcomes for this group of children were not significantly different from those of the remaining group of children who were not part of the present analysis (see table e in this article's online repository at www.jacionline.org). we collected nasal lavage samples during symptomatic respiratory episodes (n ) and at routine -year follow-up visits (n ). respiratory tract viruses were detected in % of the symptomatic episodes and in % of the routine visits (table ii) . human rhinovirus was found with the highest frequency and rsv with the next highest frequency in subjects with symptomatic episodes, but both could also be detected in asymptomatic subjects. together, the findings indicate that the majority of respiratory tract illnesses during the first year of life are associated with detectable levels of respiratory tract viral pathogens, but asymptomatic infants also have a high rate of apparent viral carriage. the distribution of values for rsv-induced ifng and ccl mrna, rsv rna, and stat activation showed a broad range among the subject population, suggesting a degree of individual variation among subjects for each of these responses (table iii) . this finding further suggests that the differences in responses might translate to variable degrees of protection against viral infection. we next investigated whether individual variation in antiviral response was associated with the development of respiratory tract fig ) . there was no correlation between ifng response and available measures that might be associated with a subject's atopy (ie, number of parents with asthma, number of parents with hayfever, and presence of parental asthma at the time of initial screening). the relationship to respiratory tract infections was selective for the ifng response because we found no association between ccl response, rsv titer, or stat activation with the frequency of upper respiratory tract infections (r . , p . ; r . , p . ; and r . , p . , respectively). we found no association of ifng response, ccl response, rsv titer, or stat activation with the frequency of wheezing episodes in the first year of life (r . , p . ; r . , p . ; r . , p . ; and r . , p . , respectively). we also investigated whether there was an association of antiviral responses with the occurrence of infections at other respiratory and nonrespiratory sites (ie, sinus, ear, croup, and stomach), as well as any association with the number of reported hospitalizations for respiratory tract illness. we found that ifn-g responses to rsv in cord blood monocyte cultures were inversely related to the frequency of ear infections (p . ), sinus infections (p . ), pneumonias (p . ), and respiratory-related hospitalizations (p . , fig ) . we found no differences in ifn-g responses between those who did and did not have croup, ''stomach flu,'' or unexplained fevers (fig and data not shown) . together, these findings reinforce the association of a decreased ifn-g response to rsv with the development of increased viral respiratory tract infections in the first year of life. in this study we provide evidence that a decreased antiviral interferon response at the time of birth is selectively associated with an increase in acute respiratory tract infections in the first year of life among infants at high risk for asthma and allergic disease. in support of this relationship between antiviral response and respiratory tract infection, we show that ( ) rsv-driven induction of ifng mrna production in cord blood monocytes is variable among infants at birth; ( ) decreased levels of rsv-induced ifng mrna in cord blood monocytes are associated with a significant increase in the frequency of upper respiratory tract infections, as well as the prevalence of ear infections, sinus infections, pneumonias, and respiratory tract illnesses requiring hospitalization; ( ) levels of rsv-induced ccl mrna expression (another highly inducible antiviral system) and interferon-driven activation of stat (the downstream target of the interferon receptors) are not associated with this phenotype for subsequent illness; ( ) symptomatic respiratory tract illnesses were frequently associated with detectable levels of respiratory viral pathogens; and ( ) levels of rsv-induced ifng mrna were not linked to other types of infections, such as croup or stomach flu, which are caused by other types of pathogens. together, these findings provide for a close relationship between rsv-driven ifng mrna production and the development of viral respiratory tract illness and in turn suggest the possibility that a decrease in this type of response might lead to an increase in this type of illness. the present findings offer distinct insights from those obtained previously. for example, others found that pha-and allergenstimulated ifn-g production in cbmcs was inversely correlated with the frequency of viral respiratory tract infection in the first year of life. , however, these studies likely measured the responsiveness of t cells because nonadherent cells were not eliminated and t-cell mitogen and antigen were used for stimulation. moreover, the effect of viral infection itself was not assessed in these studies. other studies examined the capacity of pbmcs to produce ifn-g during rsv-induced illness in children, but in this case cells were activated with phorbol -myristate -acetate-ionomycin and cross-linking antibodies to t-cell costimulatory receptors, and the response was localized to cd t cells. similarly, others again made no attempt to purify cells and then stimulated cells with t-cell mitogen and did not separately quantify ifng mrna levels. the same approach was taken in previous reports of an association between decreased ifn-g production from cbmcs and an increased risk of allergic sensitization and recurrent wheezing. , , here again, this might reflect the focus on t-cell production of ifn-g and the proposed role of t h versus t h cytokines in the development of atopy and asthma. after these studies, we have come to better recognize the critical role of the innate immune system in controlling viral infection and postviral asthma. in particular, the interferon and monocyte-macrophage systems are required for protective immunity against respiratory tract viruses, such as rsv, [ ] [ ] [ ] and these same systems are capable of driving postviral asthma independent of the adaptive immune system, at least in experimental models. [ ] [ ] [ ] therefore the present approach was designed to directly assess the innate immune response to viral infection and was done so by using the relevant cell type (purified monocytes) and stimulus (rsv infection), as well as more specific and sensitive methods (real-time quantitative pcr) than applied previously. the upshot is the first evidence that the monocyte ifng gene also serves as a marker and might even participate in the susceptibility to infection during infancy. this unexpected finding provides a new lead for control of the antiviral response because the previous view was that ifng gene expression was silenced in the monocyte lineage and was only active in lymphoid cells. in that regard we note that ifn-g production is generally attributed to natural killer (nk) cells, nkt cells, and t cells, whereas monocytes and macrophages are solely a target of ifn-g action. indeed, studies of atopic disease in infancy often focus exclusively on t-cell production of ifn-g. this circumstance is also likely due to the lower levels of ifn-g produced by monocytes and macrophages under conditions used in previous studies. here we are able to measure ifn-g production by using a sensitive assay for the corresponding mrna. whether this level of interferon production has functional consequences is uncertain, but its utility as a biomarker for susceptibility to viral infection proved quite useful. in contrast, it appears that the monocytemacrophage lineage is critical for host defense against respiratory tract viruses (including rsv). in particular, lung macrophages are charged with clearance of infected cells without dying themselves, and this protection derives from an antiapoptotic survival function of the chemokine ccl . if this function is lost (eg, in mice that are ccl deficient), the host is more susceptible to viral respiratory tract infection. other work suggests that ccl is also needed to direct dendritic cell traffic in the face of viral infection. each of these observations are consistent with those linking ccl promoter gene polymorphisms to susceptibility to severe rsv-induced bronchiolitis. nonetheless, we did not find that rsv induction of ccl gene expression was significantly associated with viral respiratory tract infection rates in the first year of life. it is still possible, however, that ccl production at the level of the lung macrophage would be predictive of susceptibility to infection, especially given the heterogeneity of monocyte-macrophage populations in the circulation and the lung. our study was conducted in a population selected for high prevalence of atopic disease. however, we do not expect that ifng response or viral susceptibility is attributable to atopy. indeed, we found no association of the ifng response with the available measures associated with atopy in our subject group because full evaluation of atopic status in our subjects was not yet performed at year of age. similarly, others found no association between lower respiratory tract illness in the first year of life and the occurrence of parental atopy or subject eczema. these findings suggest that viral susceptibility can be independent of atopy, and certainly there is evidence that this can be the case in experimental models of viral respiratory tract infection. however, it will require a nonatopic cohort, follow-up of the present cohort, or both to formally test the relationship between virus-induced ifn-g production in monocytes, viral susceptibility, and atopic status in human subjects. the molecular mechanism for rsv induction of ifng gene expression still needs to be defined. thus the pathogen recognition receptor system is responsible for mediating viral induction of the various forms of ifn-a, ifn-b, and ifn-l, and rsv is remarkably effective in blocking the induction (and signaling) of these interferon species. , by contrast, the pathogen recognition receptor system of toll-like receptors and retinoic acid inducible gene i (rig-i)-like receptors (rlrs) does not appear to regulate ifng gene expression or signaling. instead, ifng gene expression is subject to a distinct type of positive and negative regulation at pretranscriptional, transcriptional, and posttranscriptional levels, at least in the case of lymphoid (nk, nkt, and t) cells. however, these regulatory mechanisms have not been studied in monocytes or in response to rsv in any cell type. we could not define this regulatory mechanism with such limited human samples, but our work should open this new field of research. rsv is the most common cause of serious respiratory tract illness during infancy, and severe rsv-induced bronchiolitis is linked to subsequent wheezing illness/asthma. furthermore, paramyxoviral infection is established as a high-fidelity experimental model of asthma. moreover, rsv and related paramyxoviruses are easily detected in monocytes and macrophages in the lung. hence we chose rsv for our study of newborns and their monocytes, unaware of course that the subsequent results would predict respiratory tract illness associated with rhinovirus. with such a severe limitation in cell sample size, we were unable to test multiple viruses, but an analysis of the response to other types of viruses (including rhinovirus) and viral strains (including other rsv types) would be a valuable goal in the future. in summary, we report that congenital variations in the innate immune response might predict the susceptibility to acute respiratory tract illness during the first year of life. our effort uncovered evidence that the ifng mrna monocyte response to virus rather than t-cell response to mitogen/allergen might be linked to the development of viral infections and eventually postviral asthma. we were able to define this relationship despite a relatively small sample size. sample availability for complex immunologic analysis served to limit the number of ureca participants that could be studied. however, the demographic characteristics of our cohort were no different from those of the overall group, suggesting that we studied a representative sample of subjects. in addition, our study included mostly african american children, and therefore the result might not be generalized to children with other racial backgrounds. interestingly, others have recently found that interferon-stimulated genes (eg, pyhin ) are also linked to the development of asthma, particularly in subjects of african descent. our findings are also consistent with observations of decreased ifn-g production in response to rsv in pbmcs from older children and adults with allergic asthma. , thus these early events in infancy might carry over to a similar deficit in antiviral defense in later life. together, the findings suggest that a full analysis of interferon production pathways might provide key insights into the susceptibility to viral respiratory tract infection and subsequent chronic obstructive lung diseases, such as asthma. 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conflict of interest: k. sumino has received research support from the national institutes of health (nih). j. e. gern is on the scientific advisory board for and owns stock options in v biosciences; has consulted for centocor, boeheringer ingelheim, glaxosmithkline, biota, medimmune, and theraclone; and has received research support from astrazeneca. g. r. bloomberg has received research support from the nih/national institute of allergy and infectious diseases. m. j. holtzman has consulted for and received research support from hoffman-la roche and forest laboratories. the rest of the authors declare that they have no relevant conflicts of interest. clinical implications: individual variations in the immune response to respiratory tract viruses are detectable at birth, and these differences predict the susceptibility to acute respiratory tract illness during first year of life. key: cord- -dbexsugq authors: wu, yang; zhang, hongling; shi, zhaorong; chen, jianfei; li, mingwei; shi, hongyan; shi, da; guo, longjun; feng, li title: porcine epidemic diarrhea virus nsp antagonizes interferon signaling by rna degradation of tbk and irf date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: dbexsugq porcine epidemic diarrhea virus (pedv) causes a porcine disease associated with swine epidemic diarrhea. the type i interferon (ifn-i or ifn α/β) is a key mediator of innate antiviral response during virus infection. different antagonistic strategies have been identified and determined as to how pedv infection inhibits the host’s ifn responses to escape the host innate immune pathway, but the pathogenic mechanisms of pedv infection are not fully elucidated. our preliminary results revealed that endogenous tank-binding kinase (tbk ) and interferon regulatory factor (irf ), the key components in the ifn signaling pathway were downregulated in pedv infected ipec-j cells by itraq analysis. in this study, we screened nsp as the most important viral encoded protein involved in tbk and irf reduction. endoribonuclease (endou) activity has been well determined for coronavirus nsp . three residues (h , h , and k ) of pedv nsp were identified as critical amino acids for pedv endou but not d , which was not well correlated with published results of other coronaviruses, such as severe acute respiratory syndrome virus (sars-cov). moreover, pedv nsp can directly degrade the rna levels of tbk and irf dependent on its endou activity to suppress ifn production and constrain the induction of ifn stimulated genes (isgs), by which pedv antagonizes the host innate response to facilitate its replication. collectively, these results have confirmed that pedv nsp was capable of subverting the ifn response by the rna degradation of tbk and irf . porcine epidemic diarrhea (ped) is caused by porcine epidemic diarrhea virus (pedv), which has a positive-strand rna genome of kb in length in the genus alphacoronavirus, family coronaviridae and order nidovirales [ ] [ ] [ ] . the disease is characterized by severe enteritis, vomiting, watery diarrhea, dehydration, and a high mortality rate among swine [ ] . two-thirds of the genome (orf a and b) encode a large replicase polyprotein, whereas the remainder of the genome encodes for structural proteins and accessory proteins [ , ] . after pedv virus entry into the host cells, orf a codes for a large polyprotein a (pp a), while orf b is expressed as the pp ab fusion protein via the ribosomal frameshifting. by the proteinase activity of nsp (a papain-like protease) and nsp (a main protease), these polyproteins are proteolytically processed to nonstructural proteins (nsp to ) , which mediate the replication of the viral rna genome and synthesis of a nested set of subgenomic mrnas [ ] . in late , a newly emerging pedv variant was reported with more virulence and higher mortality in suckling piglets, compared to the classical pedv that was first discovered in europe in [ ] . to date, the newly emerging pedv variant is recognized as the major infectious pathogen for swine diarrhea-associated diseases in swine-raising farms in china and it has spread to other countries worldwide, causing a high number of pig deaths and significant economic impacts [ ] [ ] [ ] [ ] [ ] . during viral infection, the innate immune response is activated, leading to the induction of the type i interferon (ifn-i or ifn α/β). ifn-i is the potent cytokine of critical importance in controlling viral infections and priming adaptive immune responses [ ] . following production, ifn-i initiates a positive feed-back loop by binding to their cognate receptors on the cell surface in an autocrine and paracrine manner [ , ] and activating jak protein tyrosine kinases (jak and tyk ) which phosphorylate signal transducers and activators of transcription stat and stat . stat and stat together with interferon regulatory factor (irf ) form a transcription factor complex termed ifn-stimulated gene factor (isgf ). then, isgf is translocated into the nucleus and binds to the ifn-stimulated response elements (isre) to induce the expression of ifn-stimulated genes (isgs), which establish an antiviral state [ , ] . however, many viruses, including coronaviruses, have evolved mechanisms to evade the host immune system [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . previous studies suggest that pedv can restrain host innate immune response by different strategies, such as by degradation or cleavage of key factors essential the ifn signaling pathway [ , ] , competitive interaction between viral encoded proteins and modulators for ifn production [ , ] , or localization changes of antiviral components [ ] . whether there are other mechanisms utilized by pedv to circumvent the host response remains unclear. our previous results have demonstrated that differentially expressed proteins were identified in pedv infected ipec-j cells by the analysis of isobaric tags for relative and absolute quantitation (itraq). we identified differentially expressed cellular proteins, of which eight were upregulated and downregulated. these differentially expressed proteins were involved in apoptosis, signal transduction, and stress responses. in our analysis, tbk and irf , two important modulators in the activation of the interferon signaling pathway were downregulated post pedv infection. in this study, we screened the pedv viral proteins involved in the reduction of tbk and irf expression. among the identified viral proteins, nsp was recognized as the most effective viral protein contributing to the reduction of both tbk and irf expression after the co-transfection of tbk or irf with individually encoded pedv proteins in vitro. it was confirmed that the two major well-known degradation systems, namely the ubiquitin-proteasome system or autophagy, were not involved in pedv nsp mediated reduction of tbk and irf . in contrast, pedv nsp was capable of suppressing tbk and irf expression by endoribonuclease-dependent degradation of tbk and irf rna. this resulted in a decrease of ifn and isg production, resulting in pedv host innate immune escape. ipec-j cells (porcine small intestine epithelial cell clone j ; atcc), vero e (african green monkey kidney cell line; atcc), and hek cells (human embryonic kidney epithelial cells; atcc) were cultured in dulbecco's minimum essential medium (dmem) (life technologies, usa) supplemented with % heat-inactivated fetal bovine serum (fbs) (gibco, usa), u/ml penicillin, µg/ml streptomycin at • c in an incubator with % co (thermo scientific, usa). pedv strain cv (genbank accession number kt ) was prepared and titrated as previously described [ ] . vesicular stomatitis virus that expresses the green fluorescence protein (vsv-gfp) was preserved in harbin veterinary research institute, harbin, and stored at − • c. the full-length sequence of tbk and irf were constructed into the pcaggs-ha vector to obtain recombinant plasmids, pcaggs/ha-tbk and pcaggs/ha-irf , respectively. the recombinant pcaggs plasmids containing individual pedv viral protein (nsp - , nsp - , s, e, m, and n) with a flag fusion tag were kindly provided by prof. yue wang from harbin veterinary research institute. mutagenesis of the pedv nsp constructs (h a, h a, d a and k a) were performed by using site-directed mutagenesis kit (takara, china). recombinant prokaryotic expression plasmids were obtained following cloning of the individual gene of pedv nsp and its mutant into the ecor i and xho i sites of pgex- p- plasmid vector (ge healthcare life sciences). recombinant pgem-t/tbk and pgem-t/irf plasmids were generated to serve as dna templates for an rna transcription assay in vitro by amplifying the full length sequence of tbk or irf into pgem-t easy vector by the t-a ligation method. the specific primers used for the construction of target plasmids are listed in table and all the constructed plasmids were verified by dna sequencing. the listed antibodies were used in this study including tbk rabbit monoclonal antibody (mab) (cell signaling technology), irf rabbit mab (cell signaling technology), and phospho-irf (ser ) ( d g), rabbit mab (cell signaling technology), anti-flag mouse mab (sigma), anti-ha mouse mab (sigma), irdye-conjugated secondary antibody (li-cor biosciences) , and β-actin mouse mab (sigma). monolayers of vero e and ipec-j cells were infected with pedv strain cv at multiplicity of infection (moi) of . for h at • c. unbound virus was removed, and cells were maintained in complete medium for various time points until samples had been harvested. some cell samples were treated with proteasome inhibitor mg (sigma) at the concentration of µm, autophagy inhibitor -methyladenine ( -ma, sigma) at mm, or carrier control dmso during some transfection assays as previously described [ ] . hek cells were transfected with indicated plasmids using x-tremegene transfection reagent according to manufacturer's instruction (roche, usa). at h post transfection, cell samples were collected and lysed in ripa buffer (beyotime, nantong, china) for the western blot analysis of targeted proteins. immunofluorescence assays (ifa) were performed as described previously with slight modification [ ] . briefly, hek cells were co-transfected with tbk , irf together with nsp , nsp mutants, or empty vector control followed by the collection of supernatants for each treatment at h post transfection. ipec-j cells were treated with the collected supernatants with three-fold dilution for h followed by inoculation of vsv-gfp at moi of . . the fluorescence was visualized at hpi with an olympus inverted fluorescence microscope equipped with a camera. western blot analysis was performed as previously described with a slight modification [ ] . treated samples were lysed in radioimmunoprecipitation assay (ripa) buffer (haigene, china) containing protease inhibitor cocktail and phosphatase inhibitors (roche, switzerland), separated by sds-page under reducing conditions, and transferred onto a pvdf membrane (merck millipore, usa). after blocking, the membranes were incubated with a primary antibody and then incubated with an appropriate irdye-conjugated secondary antibody (li-cor biosciences, lincoln, ne). the membranes were scanned using an odyssey instrument (li-cor biosciences) according to the manufacturer's instructions. linearized dna was prepared by digestion with restriction endonuclease sal i prior to in vitro transcription to produce rna of defined length. in vitro transcribed rna of tbk and irf were generated from recombinant pgem-t/tbk or pgem-t/irf plasmid as template, respectively, using the ribomax™ large scale rna production systems (promega, usa). transcribed rna was purified by removal of the dna template and proteases following transcription reaction as the manufacturer's instruction (promega, usa) and stored for nuclease assay at - • c. for protein expression, individual plasmid of pgex- p- -pedv nsp , pgex- p- -pedv nsp mutant derivatives (h a, h a, d a, and k a), or pgex- p- empty vector was transformed to escherichia coli bl (de ) cells, respectively. the glutathione s-transferase (gst) fusion proteins were expressed following isopropyl-β-d-thiogalactopyranoside (iptg) inductions and purified by affinity chromatography using glutathione immobilized to a sepharose matrix per the manufacturer's instruction (ge healthcare life sciences, usa). the endoribonuclease activity assay was done as previously described [ ] . briefly, nuclease reactions contained µg of purified wild-type pedv nsp protein, pedv nsp mutant protein, or gst tag protein as control, and µg tbk or irf rna transcribed and purified in vitro. reactions were performed in mm hepes-koh (ph . )/ mm nacl/ mm mncl / mm dtt. following incubation at • c for h, the reactions were extracted using phenol-chloroform-isoamyl alcohol and analyzed by agarose-formaldehyde gel electrophoresis. for northern blot, total rna was harvested by using trizol reagent (invitrogen, usa) and analyzed by agarose-formaldehyde gel electrophoresis. rnas were transferred to a . -µm nylon membrane and probed with biotin-labeled dna probes generated with the specific primers (table ) using the north south tm biotin random prime dna labeling kit (thermo scientific). the membrane was imaged an odyssey instrument (li-cor biosciences) followed by incubation with irdye -conjugated streptavidin. quantitative rt-pcr analyses were carried out as described previously with a slight modification [ ] . at indicated time points post transfection or pedv infection, total rna was extracted from cells and subjected to quantitative rt-pcr using specific primers as listed in table . relative gene quantification was performed by the (-delta delta c(t)) method [ ] . collected virus samples were frozen and thawed three times and clarified by centrifugation at × g for min prior to titration. tcid assays were performed in vero e cells following the method of reed & muench as previously described [ ] . briefly, cell monolayers were inoculated with serial dilutions of each virus stock and incubated for days prior to observation of the presence of cytopathic effect. variables are expressed as mean ±s.d. data were statistically analyzed by using graphpad prism v . software. statistical analyses were performed using student's t test. a p value of < . was considered significant. ipec-j cells were infected with pedv or left untreated as control and cell samples were collected at h and h for itraq analysis as previously described [ ] . our preliminary results revealed that endogenous tbk and irf were downregulated at h and h in pedv infected ipec-j cells by itraq analysis (data not shown). subsequently, ipec-j cells were infected with pedv at moi of . and the mrna levels were determined by quantitative pcr. as shown in figure a , tbk mrna levels were significantly decreased at h and h post infection (hpi). in contrast, irf mrna levels were first increased at hpi and then decreased at hpi, indicating no obvious changes in irf mrna levels following pedv infection ( figure b) . to further confirm the results, hek cells were inoculated with pedv at moi of . and cell samples were collected for endogenous tbk and irf detection at indicated time points post infection. consistent with the results from ipec-j cells by itraq, endogenous tbk and irf were evidently reduced at hpi and hpi in hek cells ( figure c ). these findings suggest that a reduction of endogenous tbk and irf may be achieved by downregulating the mrna transcriptional levels of tbk and irf post pedv infection. to explore which viral protein contributes to the reduction of tbk and irf , hek t cells were co-transfected with tbk or irf and each pedv encoded protein. at h post transfection, cell samples were collected and lysed for detection of tbk or irf expression. several viral proteins were involved in the reduction of tbk or irf expressions to varying extents, e.g., nsp , nsp , and nsp for tbk and nsp , nsp , nsp , and nsp for irf , among which nsp can evidently downregulate the expression of either tbk or irf compared with other viral proteins (figure a,b) . moreover, endogenous tbk and irf were also reduced followed by the ectopic overexpression of nsp at h post transfection ( figure c ). therefore, we mainly focused on pedv nsp as the research target in this study to investigate its role in modulating tbk and irf expressions. table . three independent experiments were performed in triplicate, and values are the means ± sd for all three experiments. *, p < . . (c) hek cells were inoculated with pedv at moi of . for h and h followed by verification of endogenous tbk and irf proteins by western blot analysis. within eukaryotic cells, there are two major intracellular protein degradation pathways: the ubiquitin-proteasome system and autophagy [ ] . the proteasomal degradation pathway has high selectivity and the proteasome generally recognizes ubiquitinated substrates [ ] . by contrast, autophagy is a highly conserved process for degrading redundant cellular components by encircling them with membrane followed by a fusion of the vesicle with lysosomes [ ] . therefore, to determine the mechanism that might be responsible for the depletion of tbk and irf by nsp , the expression levels of tbk and irf proteins were examined in cells treated with a protease inhibitor mg [ , ] . as shown in figure a ,c, treatment with mg cannot block the downregulation of tbk and irf in hek t cells with co-transfection of tbk or irf together with nsp , thus not suggesting the proteasome-mediated degradation of tbk and irf by nsp . additionally, we tested the possible role of autophagy in the reduction of tbk and irf by treating cells with -ma, which is commonly used to inhibit autophagy [ , ] . we observed that -ma treatment did not inhibit tbk or irf downregulation in hek t cells co-transfected with tbk or irf along with nsp ( figure b,d) . these data indicate that downregulation of tbk and irf by nsp is not through the ubiquitin-proteasome system and autophagy. coronavirus nsp has been reported as a uridine-specific endoribonuclease and nuclease activities as well as crystal structure have been well identified as previously described [ , [ ] [ ] [ ] [ ] . four inactive mutants of sars-cov nsp , including h a, h a, d a, and k a, have been identified to lose the cleavage activity for substrate rna due to loss of endoribonuclease activity, indicating that these four residues were critical for maintaining the nuclease activity of sars-cov nsp [ ] . based on amino acid sequence alignment of pedv nsp with other coronavirus orthologs as well as xendou from x. laevis, the four mentioned residues were also conserved in pedv nsp and were denoted in red with the corresponding position in pedv nsp sequence below ( figure e ). here, we asked whether tbk or irf downregulation by pedv nsp is dependent on its endoribonuclease activity. to determine whether the amino acid residues are also required for pedv nsp endonuclease activity, four mutants (h a, h a, d a, and k a) were constructed by mutating corresponding residue of pedv nsp to alanine. hek t cells were co-transfected with tbk or irf together with nsp or constructed mutants following the detection of tbk and irf by western blot. it was demonstrated that nsp and d a mutant can obviously reduce the expression levels of tbk and irf post transfection. in contrast, the reduction of tbk or irf expression was blocked when hek t cells were co-transfected with tbk or irf and the remaining mutants (h a, h a, and k a), indicating that residues of h , h , and k but not d are critical for the endoribonuclease activity of pedv nsp ( figure f,g) . . pedv-induced reduction in tbk and irf expression is due to pedv nsp endoribonuclease activity, but not proteasome or autophagy-mediated mechanisms. (a,c) hek t cells were co-transfected with nsp (flag tagged) and tbk or irf (ha tagged) and were treated with the proteasome inhibitor mg ( µm) or carrier control dmso. detergent lysates were collected and subjected to reducing sds-page and immunoblotting with anti-flag and ha antibodies. (b,d) hek t cells were co-transfected with nsp (flag tagged) and tbk or irf (ha tagged) and were treated with -ma ( mm) for further culture. at h post transfection, cell lysates were subjected to blotting with corresponding antibody. (e) alignments of the nsp orthologs from several coronaviruses and x. laevis endou. the amino acid sequence of the pedv nsp (pedv, kt ) was aligned with orthologs of severe acute respiratory syndrome coronavirus (sars, kf ), murine hepatitis virus (mhv, kf ), avian infectious bronchitis virus (aibv, nc_ ) and xenopus laevis (xendou, bc ) using dnastar software. gaps in the sequence alignment are denoted by hyphens. residues with red are the conserved residues critical for nsp activity as previously described. the number below the red residue indicates its corresponding position at pedv nsp protein, respectively. (f,g) four mutants (h a, h a, d a and k a) of pedv nsp were constructed by mutating the corresponding conserved residue mentioned in (e) into alanine using site-directed mutagenesis kit. hek t cells were then co-transfected with tbk or irf and wild-type pedv nsp , the constructed nsp mutant (h a, h a, d a or k a) or empty vector. at h post transfection, cell samples were subjected to immunoblotting with antibodies to flag, ha or β-actin (loading control). combined with the previous results, we hypothesized that pedv nsp contributed to reduction of tbk and irf expression by targeted mrna level degradation in an endou activity dependent manner. to test this hypothesis, hek t cells were co-transfected with tbk or irf and pedv nsp as well as the constructed mutants followed by quantitative analysis of tbk or irf mrna level with the primers listed in table . as shown in figure a , the relative mrna level of tbk was significantly more decreased in pedv nsp and d a transfected cells than in other mutants and empty vector transfected cells, which suggested that pedv nsp can reduce tbk expression by downregulating the tbk mrna levels dependent on its endou activity. similar results were obtained that nsp can reduce irf expression by decreasing the irf mrna levels in an endou dependent manner ( figure b ). in addition, the mechanism was further verified by northern blot assay using the specific probe as designed in table , following co-transfection with tbk or irf and pedv nsp as well as the constructed mutants in hek t cells. consistent with the quantitative results by real time pcr assay, tbk or irf rna was evidently more reduced in nsp and d a mutant transfected cells than in mutant h a, h a, k a, or empty vector control transfected cells ( figure c,d) . these data demonstrate that pedv nsp can reduce tbk and irf expression by the targeted degradation of tbk and irf mrna. table . three independent experiments were performed in triplicate, and values are the means ± sd for all three experiments. *, p < . . (c,d) hek t cells were co-transfected with tbk or irf and pedv nsp expression plasmid (pedv nsp , h a, h a, d a and k a) or empty vector. at h post transfection, rna were extracted from the collected cell samples followed by rna detection by northern blot assay using the designed specific probes (table ) as described in the materials and methods. we next investigated whether pedv nsp can directly degrade mrna of tbk and irf . to this end, wild-type and four mutant versions of pedv nsp were produced as gst-tagged proteins and purified under mild conditions by the addition of reduced glutathione to the elution buffer as the manufacture's instruction (ge healthcare life sciences). as shown in figure a , recombinant wild-type pedv nsp and four mutants (h a, h a, d a, and k a) were purified successfully at the expected molecular weight of kda by sds-page analysis. the tbk and irf sequences were amplified by pcr and subsequently cloned into the pgem-t easy vector containing a t rna polymerase promoter upstream of the multiple cloning region to construct the recombinant plasmids of pgem-t/tbk and pgem-t/irf , respectively. in vitro-transcribed rnas of tbk and irf were synthesized from the constructed recombinant dna templates (pgem-t/tbk and pgem-t/irf ) by the ribomax™ large scale rna production systems (promega, usa). the gst-purified pedv nsp proteins were incubated with the generated tbk and irf mrna as substrate in presence of mn + , a known cofactor for the endoribonuclease activity. [ , ] . it was revealed that synthesized tbk and irf mrna were effectively reduced post incubation with wild-type pedv nsp and d a mutant, but not with the other mutant derivatives (h a, h a, and k a) , demonstrating the pedv nsp can directly degrade tbk and irf mrna dependent on its endoribonuclease activity ( figure b,c) . (b,c) targeted rna was transcribed and purified in vitro as described in the materials and methods. endoribonuclease activity assay was performed by incubating the transcriptional rna (tbk or irf ) with purified recombinant pedv nsp proteins (pedv nsp , h a, h a, d a or k a) or gst tag protein as a control at • c for h followed by rna detection of tbk or irf rna as described in the materials and methods. type i ifns are transcriptionally regulated, and are induced following recognition of pathogen components during infection. tbk and irf are the key effectors during viral infections to induce ifn production [ ] . following stimulation with virus components including dsrna, irf becomes phosphorylated by the serine-threonine kinases tank-binding kinase- (tbk ) or the inducible iκb kinase (ikk-i/ikkε) [ , ] . irf- then dimerizes, translocates into the nucleus, and combines with the co-activator cbp/p to activate the expression of ifnβ [ ] . to determine whether pedv nsp modulates the phosphorylated irf , cells were co-transfected with tbk and irf along with pedv nsp as well as its mutant derivatives (h a, h a, d a, and k a) and then collected to examine the phosphorylated irf levels by western blot. as anticipated, the tbk stimulation led to the irf phosphorylation in empty vector transfected cells. however, the irf phosphorylations were significantly inhibited in nsp and d a mutant transfected cells compared to the remaining mutants (h a, h a, and k a) transfected cells, suggesting that pedv nsp impeded irf expression as well as irf phosphorylation dependent on its endou activity ( figure a ). vesicular stomatitis virus (vsv) is frequently used for the assessment assay of ifn activity [ ] . to determine the role of nsp in regulation of ifn production, hek t cells were co-transfected with tbk and irf together with wild-type nsp , individual nsp mutant or empty vector and cell supernatants were collected at h post co-transfection for determining the status of ifn secretion. ipec-j cells were treated with the supernatant followed by inoculation of vsv-gfp at moi of . . fluorescence was visualized at h post infection with an olympus inverted fluorescence microscope equipped with a camera. vsv-gfp infection was evident in treatments with supernatant from nsp and d a mutant transfected cells, and conversely vsv-gfp infection was obviously inhibited in treatments with supernatant from cells transfected with the remaining mutants (h a, h a, and k a) or empty vector, demonstrating that nsp was an antagonistic protein in ifn production ( figure b ). moreover, we continued to investigate the effects of collected supernatant on pedv infection in vero e and ipec-j cells. vero e cells were treated with the individual collected supernatant as mentioned above prior to pedv infection, cell samples were then collected and subjected to virus titration by tcid assay. pedv infection was significantly enhanced in cells treated with supernatant from wild-type nsp and d a mutant transfected cells than that from mutant h a, h a, k a or empty vector transfected cells ( figure c ), confirming that pedv nsp can evade the host antiviral response by antagonizing ifn production. meanwhile, a pedv infection assay was further performed in ipec-j cells, a target cell line for pedv infection in vivo. similar results were obtained in that pedv nsp can facilitate pedv infection, based on pedv genomic quantitation by real-time pcr assay instead of by tcid assay due to its low susceptibility to ipec-j cells ( figure d ). these data collectively demonstrate that nsp can promote pedv infection by limiting ifn secretion dependent on its endoribonuclease activity. ifn-i is the key innate immune cytokine produced by cells to trigger antiviral function [ , ] . therefore, we assessed the effect of nsp on the ifn mediated antiviral response signaling pathway. here, hek cells were co-transfected with tbk and irf together with wild-type nsp , nsp mutants, or empty vector control and cells samples were collected to investigate the effects of nsp on induction of innate antiviral molecules. quantitative rt-pcr showed that mrna levels of immune related molecules, such as ifnβ, tnfα, oas , isg , isg , and isg , were significantly disrupted by nsp and mutant d a post transfection compared to that by empty vector control. however, the disruptions were impeded by the other nsp mutants that impaired the endoribonuclease activities (figure ) , suggesting that pedv nsp restrains cellular antiviral activity and thus facilitates pedv infection. table . the results are representative of three independent experiments (mean ± sd). *, p < . . the p value is calculated using student's t-test. the host innate immune system is the first line of defense against virus invasion through production of ifns as well as various other cytokines. innate immune responses are activated through host pattern recognition receptors (prrs), which recognize pathogen-associated molecular patterns [ ] . ifns exert antiviral effects through inducing the expression of hundreds of isgs [ , , ] . however, during coevolution with their host, viruses always evolve diverse strategies to escape and even inhibit host ifn responses [ , , [ ] [ ] [ ] [ ] , ] . pedv has acquired multiple mechanisms that avoid the action of ifn by preventing the binding of viral products to cellular sensors. it was revealed that pedv n protein antagonized ifn production by preventing tbk from interaction with irf [ ] . of the several known viral evasion strategies, the cleavage of crucial innate immune molecules, including adaptors, kinases, and transcriptional factors, are considered to be a particularly powerful way for viruses to escape the innate immune response. for example, the c-like protease of pedv and porcine delta coronavirus (pdcov), disrupts type i ifn signaling by cleaving the nf-κb essential modulator (nemo) [ , ] . in addition, pdcov nsp antagonizes type i ifn signaling by cleaving stat , an essential factor for ifn responses [ ] . furthermore, the ubiquitination and deubiquitination are highly regulated post-translational modification processes in modulating the antiviral innate immune response. within the cells, polyubiquitination plays several different roles depending upon the attachment position on the target proteins, and linked polyubiquitin chains regulate the proteasomal degradation of target proteins [ , , ] . multiple ubiquitin ligases and ubiquitin-binding scaffold proteins contribute to the positive regulation of the ifn response, such as rig-i, traf , traf , and tbk . previous studies have indicated that pedv plp significantly inhibits the ubiquitination of rig-i and sting, which is essential for the activation of type i ifn signaling [ ] . meanwhile, the proteasomal degradation of target proteins for the ifn response can also be achieved by viruses through the removal of k polyubiquitin chains [ ] [ ] [ ] . pedv-induced stat degradation inhibits type i interferon signalling in a proteasome-dependent manner [ ] . however, viruses are not just limited to the mentioned strategies to antagonize ifn responses. in this study, we first identified that pedv nsp was capable of subverting the ifn response by the rna degradation of tbk and irf , which differentiated from the strategies utilized by other coronavirus orthologues previously described. the functions of nsp of coronaviruses (nsp in arteriviruses), an endoribonuclease encoded by nidoviruses, have received more attention. previous studies showed that the nsp encoded by sars-cov [ ] , mhv [ , ] , pedv [ ] , pdcov [ ] , and the nsp encoded by prrsv [ , ] can antagonize antiviral innate immune responses by utilizing the different mechanisms involved, e.g., by mediating the evasion of viral dsrna by host for mhv and hcov- e [ , ] , by suppressing both mavs and rig-i expression for prrsv [ ] , by impairing the activation of transcription factor nf-κb for pdcov [ ] , or by inhibiting mavs-induced apoptosis for sars-cov [ ] . pedv nsp of is a -residue polypeptide that results from the cleavage of pp ab at sites nlq↓gle and qlq↓ase by the main protease nsp . several recent studies have focused on the structural and functional characterization of coronavirus nsp due to its potential importance as a drug target. it has been reported that the endou activity of pedv nsp is not required for pedv replication in vero cells. however, the endou activity is involved in the suppression of host ifn response in epithelial cells and macrophages in vitro, and subsequently can facilitate pathogenesis development in vivo by enhancing viral replication and shedding [ ] . although previous studies have reported pedv nsp as a key virulence factor that suppressed ifn responses in vitro and facilitated pedv replication, the underlying mechanism remains unknown. in this study, we found that endogenous tbk and irf were downregulated post pedv by previous itraq assay and western blot analysis. nsp was selected as the investigation candidate due to its evident effect on tbk and irf reduction post co-transfection with each viral encoded protein. it was exhibited that pedv nsp was capable of downregulating the expression of tbk and irf proteins by the degradation of the rna of tbk and irf in an endoribonuclease activity dependent manner, while residues of h a, h a, and k a were critical for the endoribonuclease activity of pedv nsp , but not d a. the reason that results in these differences remains unclear. whether these structure differences result in different mechanisms used to antagonize ifn production remains a subject of further study (figure ) , elucidating a novel antagonistic mechanism utilized by pedv to counter the antiviral response. in summary, our data reveal that pedv nsp acts as an ifn antagonist to inhibit immune response by the rna level degradation of tbk and irf , key ingredients involved in the ifn signaling pathway dependent on its 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degradation induction of otud by viral infection promotes antiviral responses through deubiquitinating and stabilizing mavs early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication coronavirus nonstructural protein mediates evasion of dsrna sensors and limits apoptosis in macrophages porcine deltacoronavirus nsp antagonizes interferon-beta production independently of its endoribonuclease activity porcine reproductive and respiratory syndrome virus nsp antagonizes type i interferon signaling by targeting irf nonstructural protein of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production mavs-mediated apoptosis and its inhibition by viral proteins this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we want to thank jin tian for his excellent technical assistance on this project. the authors declare no conflict of interest. key: cord- -b sbvy g authors: marques neto, lázaro moreira; kipnis, andré; junqueira-kipnis, ana paula title: role of metallic nanoparticles in vaccinology: implications for infectious disease vaccine development date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: b sbvy g subunit vaccines are safer but less immunogenic than live-attenuated vaccines or whole cell inactivated vaccines. adjuvants are used to enhance and modulate antigen (ag) immunogenicity, aiming to induce a protective and long-lasting immune response. several molecules and formulations have been studied for their adjuvanticity, but only seven have been approved to formulate human vaccines. metallic nanoparticles (menps), particularly those containing gold and iron oxides, are widely used in medicine for diagnosis and therapy and have been used as carriers for drugs and vaccines. however, little is known about the immune response elicited by menps or about their importance in the development of new vaccines. there is evidence that these particles display adjuvant characteristics, promoting cell recruitment, antigen-presenting cell activation, cytokine production, and inducing a humoral immune response. this review focuses on the characteristics of menps that could facilitate the induction of a cellular immune response, particularly t-helper and t-helper , and their potential functions as adjuvants for subunit vaccines. (tlr agonists) are immunomodulatory molecules, capable of generating a th response ( ) . there is a demand for safe adjuvants capable of inducing efficient cellular immunity, especially th and th , to be used against tuberculosis, leishmaniasis, malaria, and other diseases caused by intracellular microorganisms ( , ) . the majority of molecules with this type of adjuvanticity (th driven) are related toward the response of danger receptors to trigger inflammation, thus safety and tolerance could be major barriers that prevent their use in human vaccines ( ) . however, comparing alum and cpg/dna adjuvants in human trials, only common adverse effects, including local site reaction, flu-like symptoms and headache were observed when cpg/dna was used ( ) . also, verstraeten et al. ( ) , analyzing more than , individuals, who received vaccine-containing as , observed that only common side effects occurred. nanoparticles (nps) are classically described as structures smaller than nm and can be classified, based on their composition, as polymeric, inorganic, liposomes, immunostimulating complexes, virus-like particles, emulsions, or self-assembled proteins ( ) . they are made of different materials and differ in size, shape, and surface properties; interactions with biological systems, therefore, are varied, with several applications in modern medicine. in vaccinology, they are classically thought to have delivery and deposit properties. however, many nps have been shown to stimulate immune responses, including cell recruitment, activation of antigen (ag)-presenting cells (apcs), and induction of cytokine and chemokine release. the development of nanostructures and nanoadjuvants may therefore offer alternatives to currently used adjuvants once studies establish ways for them to elicit innate immune response and support the development of adaptive immune response in the context of vaccine formulations ( ) . metallic nanoparticles (menps) are relatively non-biodegradable, have rigid structures, and possess simple synthesis methodology. many have been studied for their immunological properties ( ) . however, there are still gaps in understanding the immune response generated by nps, especially menps. few studies have compared nps of different types and there is no standardization among published methodologies, which hampers comparisons of immunostimulatory characteristics. several important characteristics, therefore, have not been well studied. for example, how chemical and physical properties (including material composition, size, shape, surface charge, and hydrophobicity) impact vaccine immune response ( ) . this review focuses on the use of menps in formulations against infectious diseases, aiming to assess progress of their use in vaccinology and their possible applications as adjuvant. the immune response generated by menp-formulated vaccines table summarizes the articles that report the use of menps as part of vaccine formulations against infectious diseases and the immune responses they elicited. a range of immune responses is required to fight a diverse group of microorganisms. the type of protective immune response can be simplistically divided based on the type of microorganism: extracellular bacteria and toxin, intracellular bacteria, viruses, fungi, and protozoa. among the vaccines targeting extracellular bacteria and toxin, two were formulated with lipopolysaccharide (lps) in glycopeptide ag. the use of glycoantigen and lps can trigger an intense response through tlr and b cell receptor activation; the presence of gold nps (aunps) may have minimal influence on this response. however, in the work of gregory et al. ( ) and torres et al. ( ) , the use of aunps in the formulation generated a different response, improving anti-lps immunoglobulin g (igg) response, decreasing bacterial burden, generating a more efficient humoral response, and improving animal survival, showing that aunps may influence immune response and protection. using protein ag, barhate et al. ( ) formulated a vaccine using aunps and toxoid ag and demonstrated that their formulation could induce a mucosal and systemic igg and iga response. when co-administered with asparagus racemosus extract, a botanically derived adjuvant, the response was further enhanced ( ) . dakterzada et al. ( ) developed a vaccine against pseudomonas aeruginosa using the flagellin subunit and aunps that elicited an igg response comparable to that induced by freund adjuvant. flagellin is a tlr agonist but the recognition and signaling is structure dependent. this study, however, used only the - aa from flagellin and its ability to activated tlr could not be maintained ( ) . gregory et al. ( ) used an f yersinia pestis ag conjugated to aunps that induced an ab response with higher igg a associated with higher levels of interferon gamma (ifnγ), suggesting activation of th cells. among the studies that used menps in vaccine formulation, only one targeted intracellular bacteria (listeria monocytogenes). the protective immune response against intracellular bacterial infections requires th activation and, therefore, apcs activation and ag presentation through major histocompatibility complex ii (mhc ii). to generate a th response, an aunp and listeria ag formulation were used in different strategies. although the authors tested direct vaccination, when dendritic cells (dc), in vitro loaded with aunp plus listeria ag, were adoptively transferred to a naïve animal, they induced th , cd +, and natural killer (nk) cells that provided better protection against l. monocytogenes than the traditional vaccine approach ( ) . in evaluating vaccines developed with menps against viral infections, niikura et al. ( ) ( ) also evaluated the addition of cytosine and guanine linked by phosphodiester unmethylated (cpg/ dna) and found that it improved ab levels and animals' survival rates. another important feature of studies by niikura et al. ( ) and chen et al. ( ) was the use of various np sizes and the demonstration that all different np shapes were capable of inducing a humoral response. the levels of ab were size dependent, but the results were inconsistent: the first study found that a nm sphere was the most efficient ab inducer and the second study found that the nm and nm spheres performed best. a special case of the use of menps was the use of nickelfunctionalized nanolipoprotein particles (ninlps) by yan et al. ( ) and wadhwa et al. ( ) in combination with hiv ag. ninlps are nanometer-sized nanolipoprotein particles with nickel incorporation into their surface in order to induce polyhistidine tagged proteins adsorption ( ) . they demonstrated that specific igg (igg and igg a) levels were greater than those obtained when alum was used in the formulation. fischer et al. ( ) used truncated wnv envelope protein ag and found that a single dose vaccination induced a superior anti-wnv igg response and improved protection against a wnv challenge ( ) . these responses were associated with nickel functionalization, described as a hapten, and triggered responses through activation of human tlr and intracellular transduction signals through myeloid differentiation primary response (myd- ), nuclear factor-κb (nf-κb), and mitogenactivated protein kinase (mapk), inducing pro-inflammatory responses [tumor necrosis factor (tnf)-α and interleukin (il)- ] ( , ) . for protozoan infections, parween et al. ( ) , using plasmodium falciparum merozoite surface protein subunit and aunps, evaluated the humoral immune response (igg , igg a, igg b, and igg ) and found an intense igg response compared with the alum formulation ( ). kaba et al. ( ) , using p. berghei circumsporozoite protein and aunps, generated long-lasting protective immunity with th that produced il- and mixed high avidity igg /igg a (th /th ) ( ) . in other studies, these authors replaced ag with p. falciparum circumsporozoite protein; vaccination was shown to induce protective cytotoxic (cd +) cells, high avidity ab titers, and specific effector memory, central memory, and long-term central memory cd + t cells in draining lymph nodes, spleen, and liver ( ) . this response was shown to be generated by dc cross-presentation, which had delayed fusion and interaction of endosomes with lysosomes caused by the aunp formulation ( ) . finally, pfmsp was used with dextrancoated iron oxide nps (ionps) and was capable of inducing a humoral response in two animal models (mouse and monkey). this response was also shown to inhibit parasite growth by - % ( ). most studies evaluated immunogenicity through measurement of the humoral immune response. according to their findings, the use of nps was efficient in inducing an ab-based response. based on heavy chain structure, there are five types of ab, each with a different role: igg, igm, iga, igd, and ige. igg and iga can be subdivided as igg , igg , igg , igg , iga , and iga based on additional small differences in their heavy chain. with regard to vaccination, humoral immunity is especially important in responding to infection by extracellular pathogens, toxins, protozoa, and viruses. its importance is associated with the biological activities of immunoglobulins, including microorganism opsonization and phagocytosis; complement activation ( ); toxins and microorganism neutralization ( ) ; and mast cells and basophil activation ( , ) . in addition, immunoglobulins can help target cytotoxicity against infected cells (ab-dependent cell cytotoxicity of cd t cells and nk). in some cases, however, the pathogens have the ability to evade the humoral system or can even use immunoglobulins as a way to facilitate cell invasion, as in the cases of mycobacterium tuberculosis and leishmania spp. ( , ) . the studies described above clearly show that menps (gold, iron, and nickel) can be used for vaccine development. different menps were used in conjunction with several ag for distinct microorganisms and showed the ability to generate humoral and cytotoxic responses. although the generation of igg a and ifn-γ shown in some studies are indicators of th responses using menps as adjuvant, further research is needed to specifically assess the role of different menp vaccines in th induction. to understand the possible uses of menps as platforms for vaccines against infectious diseases, analysis is needed of the impact of different physicochemical characteristics of nps on the innate immune response (figure ) . several strategies have included menps as vaccine platforms, involving menps of different materials (including gold, iron oxide, and nickel); shapes (including spheres, cubes, rods, and disks); sizes (from nm to over nm); and types of coating [e.g., citrate, chitosan, dextran, or cetyltrimethylammonium bromide/ -styrenesulfonic acid-comaleic acid (ctab/pss-ma)]. the material from which an np is made has a direct influence on the functions of apcs; gold nps (aunps) have been most commonly used in vaccinology ( table ) . the most recent studies involving aunps demonstrate the effects of gold sodium thiomalate on macrophage function, showing lysosomal enzyme inhibition and reducing phagocytosis ( ) . similar effects were seen in macrophages of several origins, which, when stimulated with aunps, showed diminished bactericidal activity against staphylococcus aureus ( ) and low or absent cytokine production il- , il- , and tnf-α ( , ) . moreover, when splenocytes were stimulated with lps, the addition of aunp reduced il- and tnf-α release ( ) . some of these results raise the concern on the use of aunps as adjuvants, since these immunomodulatory properties can act inhibiting the generation of th . however, the response to aunps is also correlated with other physicochemical characteristics that will be discussed below, which may be tailored to improve immunostimulatory or immunomodulatory capacity. iron oxide nanoparticles have also been used as adjuvants. iron is an important ion in the homeostasis of all cells and in generating immune responses to several microorganisms. the effect of ionps phagocytosis have been explored in several studies, for example, m macrophages after exposure to ionps induced reactive oxygen species (ros), but after h induced il- production ( ) . the use of ionps in balb/c mice demonstrated the immunomodulatory capacity of this np by diminishing splenocyte cytokine production (il- and ifn-γ) ( ) as well as suppressing the response to pancreatic ag in diabetic mice ( ) . sindrilaru et al. ( ) , however, showed that macrophages, under iron overloaded conditions, became unrestrained m (with an incomplete switch to m macrophages) and produced more tnf-α, which impaired wound healing and had an important role in the immunopathology of chronic venous leg ulcers. consequently, ionp response seems to have direct correlation with time and dose, once iron overload seems to be a requisite to developed pro-inflammatory response and this aspect must be evaluated to avoid the inhibition of the desired immune response. other critical characteristics are the shape and size of nps, which have a direct impact on vaccine efficiency, ag load capacity, and interaction with cells (phagocytes and apcs). these characteristics have been studied in different nps; shah et al. ( ) published a review focusing on the impact of size for alum, oil-in-water, emulsion, polymeric particles, and liposome adjuvanticity, but did not evaluated menps. in the studies reviewed here, np sizes range from nm nanospheres to nm nanocapsules. two authors have evaluated the impact of size and shape for menps ( table ) : chen et al. ( ) evaluated differences in immune response based on aunp sizes (ranging from to nm nanospheres) and found that and nm were the most drained np ( ); niikura et al. ( ) went further and, using four different shapes of np ( nm sphere, nm sphere, cube, and rod), showed that ab responses and tnf-α were directly correlated with the specific np surface area (the ratio of the total surface area per single np volume). furthermore, nm spheres appear to be the most efficient in generating immune responses (il- and il- ) and granulocyte macrophage colony-stimulating factor production. surface charge and hydrophobicity are additional important np characteristics for immune response induction and are directly influenced by np functionalization (chemical modification of nps surface by adding or replacing functional groups) and coating (ag) ( ) . most studies used citrate-coated nps, but dextran and ctab/pss-ma have also been used; all three result in negatively charged (anionic) particles. only one np, revised here, used positive charged (cationic) functionalization [( ); table ]. the higher hydrophobicity of aunp was shown to activate the innate immune system (tnf-α secretion) ( ) . although the surface charge of other non-metallic nps has been studied ( ) , to our knowledge the studies using menps did not address the other characteristics associated with immune response induction. for non-metallic nps, it appears that a positive charge signified a greater ability to induce immune responses than a negative charge. interestingly, negatively charged non-metallic nps were associated with ag-specific tolerance ( ) . further studies are needed to investigate whether or not the charge imputed by np coating influences the immune response. though the size and shape of menps had little to no impact on the innate response elicited, coating modifications may improve the capacity of these molecules to influence immune responses. finally, it is important to note that the majority of adjuvant characteristics were evaluated using non-metallic nps. t-helper cells are associated with immunity against intracellular pathogens and the secretion of ifn-γ, which, in turn, is essential for the activation of mononuclear phagocytes, including macrophages, resulting in enhanced phagocytic activity ( ). th cells (il- a and il- f producer cells) are associated mainly with stimulation and chemotaxis of neutrophils to the site of inflammation. however, their function goes beyond this and includes the targeting of various cells types, including nonlymphoid cells and the stimulation of cytokine, chemokine, and prostaglandin production. another characteristic of these cells is their memory effector subset, which is maintained in mucosal tissues for extended periods. this subset has high plasticity and is able to transform into th or th phenotypes depending on the cytokine milieu at mucosal sites. this diversity of function and actuation make th cells very important in defense against several microorganisms, mainly those acquired through mucosal routes ( , ) . t-helper and th cells have their own distinct sets of functions and differentiation factors. both cell types require t cell receptor downstream activation by ag presentation cells through mhc ii and co-stimulatory molecules ( ) . consequently, cytokine release during ag presentation is correlated with the type of adaptive immune response generated. while th differentiation requires stimulation by il- , th generation requires transforming growth factor-β and il- . however, this generation is influenced by other factors and how menp are involved in the possible induction of th or th will be discussed below. in this review, only one study investigated the development of the direct th (type t helper cell) and th response. using a listeria ag, rodriguez-del rio et al. ( ) showed that in contrast to advax™ adjuvant alone, a combination of nm aunps and advax™ was capable of inducing the highest th response. pusic et al. ( ) immunized mice with ionps covered with rmsp , a p. falciparum merozoite ag, and showed that after immunization (intramuscular, subcutaneous, or intraperitoneal), production of il- was greater than that of ifn-γ, suggesting a predominant th response (although the cellular immune response was not directly evaluated). the first major determinant in generating th and th populations is the route of vaccine administration, which dictates the cell dynamic and initial response to the vaccine. for example, mohanan et al. ( ) , in a cross-sectional study using a liposome plus ag (ova) vaccine formulation, compared intradermal (high igg ; intermediate igg ; and ifn-γ), intralymphatic (high igg , igg , and ifn-γ), intramuscular (high igg ; intermediate igg and ifn-γ), and subcutaneous (high igg ; low igg and ifn-γ) routes of administration ( ) . the predominant th response to administration through the intradermal route was most likely due to the cooperation between langerhans cells, the primary innate immune response cells and keratinocytes that may also be stimulated by the formulation. these elicited the production of cytokines and chemokines that helped in the activation of other apcs ( ) . the early phase of vaccination is characterized by recruitment of neutrophils and monocytes to the site of inoculation. both cell types can also act as apcs, delivering ag-specific and co-stimulatory signals to t cells. their collaborative endeavors have been found to modulate (positively or negatively) the activity of different effector t cell subsets ( , ) . neutrophils are the first cell lineage to migrate to inflammation sites and, when stimulated, they produce cytokines and chemokines that will attract and activate other cell types. for example, neutrophils were shown to be an important inducer of th and th cells ( ) , but their role in cytokine secretion is much broader ( ) . moreover, signals may elicit different function in neutrophils and therefore, influence the quality of t cell responses. for example, aunps have been described as capable of inducing neutrophil extracellular traps, which act as damage-associated molecular patterns and stimulate immune system through dna receptors such as tlr ( ) . upon stimulation by nps (tio -titanium dioxide-and alum), duffin et al. ( ) demonstrated neutrophil influx to the lungs and also induced production of il- . silver nps were also shown to be capable of interacting with neutrophils, inducing apoptosis of these cells, and inducing caspase- \ caspase- partially dependent il- β secretion ( ) . in another study, cobalt and nickel nps were shown to induce higher nitric oxide, tnf-α, and cxcl chemokine production, by human peripheral blood neutrophils, than titanium nps (tio np) ( ) . nonetheless, tio nps also induced polymorphonuclear cell activation through phosphorylation of several proteins, including p mapk and extracellular signal-regulated kinases- / (erk- / ), which were associated with increased neutrophil life-span and production of several cytokines and chemokines ( ) . classically, apcs, macrophages, and dcs act at the site of vaccine inoculation by sensing foreign agents, through tlrs and other receptors, and triggering inflammation. apcs play a key role in the initiation, maintenance, and selectivity of inflammation, through their three major functions: endocytosis, ag presentation, and production of various cytokines and growth factors ( ) . the main family of pattern recognition receptors in microbial recognition is the tlrs, part of the family of transmembrane proteins, which affect the transcription of genes involved in inflammatory and immune response-enhancing cellular activities such as phagocytosis, endocytosis, cytotoxic functions, and cytokine production ( , ) . the adjuvants most frequently used for the induction of th and th responses are tlr agonists, such as as , cpg/ dna, and others. menps seems to have capacity to induce the expression of toll-like receptors, such as tio nps and zirconium oxide nps that have been described to enhance tlr , tlr , and tlr expression in macrophages ( ) and tlr and tlr in mouse liver cells ( ) . zinc oxide nps (plus ova) generated an inflammatory response in balb/c mice and also improve tlr- , - and - expression, followed by activation of src family kinases ( ) . consequently, tio nps and ionps were shown to induce dc upregulation of co-stimulatory molecules (mhc ii, cd ) ( , , ) , which can also be related to tlr stimuli pathways. however, none of these works demonstrate the direct interaction of nps with tlr (using knock-out mice, agonists, or antagonist molecules) thus, this interaction must be further studied. the next step in the generation of adaptive responses is the tailoring of cytokine secretion by apcs at immunological synapses, which will guide the development of the response. several nps have been reported to trigger cytokine and chemokine production, which may be used as biomarkers for immunotoxicity ( ). among those described, tio nps were used in mimetic systems composed of blood vein endothelial component (including pbmc) and was reported to trigger pro-inflammatory cytokines (il- , ifn-γ, and tnfα) ( ); zinc oxide nps were shown to be preferentially associated with monocytes and, when used in pbmc, induced ifn-γ, tnf-α, and il- cytokine production ( ); aunp-stimulated bone marrow-derived dc produced il- , tnf-α, and ifn-γ ( ) ; and ionps were shown to induce the activation of apcs with an increase of il- , tnf-α, ifn-γ, and il- , as well as chemokines. the response generated by ionps, however, was weaker than that generated by the positive control lps which may be beneficial in controlling possible side effects ( ) . the generation of a cellular response associated with protection against intracellular pathogens is the ultimate goal of vaccination. however, the direct effects of nps on cellular responses have been evaluated in only a few studies. tio nps were shown to activate and induce proliferation of naïve cd + t cells and to generate a pronounced th response with ifn-γ and tnf-α production, associated with pro-inflammatory cytokine production (il- , il- a, il- b) and dc maturation (cd + and cd + expressions increase). schanen et al. ( ) hypothesized that the oxidative capacity of an np could impact the response and trigger pro-inflammatory (oxidant capacity) or anti-inflammatory (antioxidant capacity) responses. this oxidant effect could control ros generation and thus control downstream pro-inflammatory effects while antioxidants prevent the initiation of the innate immunity in lps-stimulated macrophages ( ) . this study was, however, conducted with mitogens (non-specific stimuli) and not with vaccine stimuli, but nevertheless serves as a warning about the direct action of nps, not only on the innate immune system but specifically on t cells. there is enough evidence to suggest that menps are not only particulate formulations but also immunostimulatory molecules with several studies demonstrating their capacity to generate humoral and cytotoxic responses. menps clearly have figure | metallic nanoparticles adjuvanticity and its prediction capacity to generate t-helper (th ) and th responses. to generate a cellular immune response, the np must be able to be recognized by the host innate immune response and stimulate a sequence of events that will lead to the release of a specific milieu of cytokines and better antigen presentation (bottom arrow). in the top arrow is the immune response elicited by metallic nanoparticles to aid th and th generation. nf-κb, nuclear factor kappa b; ccl, chemokine ligand; cxcl, chemokine (c-x-c motif) ligand; gm-csf, granulocyte macrophage colonystimulating factor; ifn, interferon; il, interleukin; m-csf, macrophage colony-stimulating factor; myd, myeloid differentiation factor; tcr, t cell receptor; th, t-helper cell; tlr, toll-like receptor; tnf, tumor necrosis factor. immunostimulatory capacity and can induce several reactions in all phases of vaccine development. these capabilities correlated with np physicochemical characteristics such as size, charge, and hydrophobicity, but there are several gaps in our understanding of their mechanism of actions and how they may lead to adjuvanticity, immunomodulation, or tolerance to the ag formulated with nps. there are also evidence of menp being capable of help in the generation of th and th ; figure presents an overview of the generation of these cells subsets and the possible role of menp in this induction. ln designed the review and wrote the first draft. aj-k edited the first draft and critically reviewed the manuscript. ak edited the first draft and critically reviewed the manuscript. all authors read and approved the final version of the manuscript and agreed to submission. this work is part of ln phd thesis at biotechnology and biodiversity graduate program from capes. this work was funded by fapeg (grant number - ) and cnpq (grant number: / - ). ln received a phd fellow from capes, and apjk (# / - ) and ak (# - - ) received a productivity research fellow from cnpq. key roles of adjuvants in modern vaccines novel adjuvant formulations for delivery of anti-tuberculosis vaccine candidates landmark 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tio( ) nanoparticles the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -viy y authors: burrack, kristina s.; morrison, thomas e. title: the role of myeloid cell activation and arginine metabolism in the pathogenesis of virus-induced diseases date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: viy y when an antiviral immune response is generated, a balance must be reached between two opposing pathways: the production of proinflammatory and cytotoxic effectors that drive a robust antiviral immune response to control the infection and regulators that function to limit or blunt an excessive immune response to minimize immune-mediated pathology and repair tissue damage. myeloid cells, including monocytes and macrophages, play an important role in this balance, particularly through the activities of the arginine-hydrolyzing enzymes nitric oxide synthase (nos ; inos) and arginase (arg ). nitric oxide (no) production by inos is an important proinflammatory mediator, whereas arg -expressing macrophages contribute to the resolution of inflammation and wound repair. in the context of viral infections, expression of these enzymes can result in a variety of outcomes for the host. no has direct antiviral properties against some viruses, whereas during other virus infections no can mediate immunopathology and/or inhibit the antiviral immune response to promote chronic infection. arg activity not only has important wound healing functions but can also inhibit the antiviral immune response during some viral infections. thus, depending on the specific virus and the tissue(s) involved, the activity of both of these arginine-hydrolyzing enzymes can either exacerbate or limit the severity of virus-induced disease. in this review, we will discuss a variety of viral infections, including hiv, sars-cov, lcmv, hcv, rsv, and others, where myeloid cells influence the control and clearance of the virus from the host, as well as the severity and resolution of tissue damage, via the activities of inos and/or arg . clearly, monocyte/macrophage activation and arginine metabolism will continue to be important areas of investigation in the context of viral infections. tissue -resident and monocyte-derived macrophages are innate immune cells that play a key role in normal tissue homeostasis, presentation of foreign and self antigens following infection or injury, pathogen clearance, and resolution of inflammation and wound healing. depending on the microenvironment, macrophages can be programed to adopt a variety of proinflammatory, regulatory, resolving, and immunosuppressive activation phenotypes, particularly in vivo. these activation states exist as a complex continuum of overlapping phenotypes; however, macrophage subsets with distinct functions have been defined ( ) . macrophages are considered m -polarized when stimulated by ifn-γ or toll-like receptor (tlr) ligands, such as lipopolysaccharide (lps), to express inducible nitric oxide synthase (inos; nos ) and produce nitric oxide (no). nos enzymes metabolize l-arginine to citrulline and no. no is a short-lived gaseous messenger with physiological and pathological effects. nanomolar concentrations of no, generated by endothelial nos and neuronal nos, are important for maintaining homeostasis, regulating vasodilation, and for the aggregation, recruitment, and adhesion of platelets to the vascular endothelium. inos generates micromolar levels of no that modulates various pathophysiological processes and is important for killing intracellular pathogens ( ) . in contrast, m -polarized macrophages result following stimulation of cells with a variety of stimuli, including type cytokines such as interleukin (il)- or il- . m -polarized macrophages express a distinct l-arginine-metabolizing enzyme, arginase (arg ), which hydrolyzes l-arginine to l-ornithine and urea. l-ornithine can be further metabolized to polyamines, which participate in a variety of fundamental cellular functions (e.g., proliferation, cell membrane transport), and l-proline, which is an essential component of collagen. in addition to playing important roles in defense against extracellular parasites and tissue repair, arg expression and activity in myeloid cells have emerged as a major regulator of innate and adaptive immune responses ( ) . other m -like suppressive or anti-inflammatory macrophages include myeloid-derived suppressor cells (mdscs) and tumor-associated macrophages (tams). mdscs are considered to be an immature population of myeloid cells, including both monocyte-like (gr- /ly- c + ) and neutrophil-like (gr- /ly- g + ) populations, associated with tumors or infections that suppress proinflammatory responses ( , ) . depending on the context, mdscs have been shown to mediate their suppressive activity via no-and/or arg -dependent mechanisms. importantly, macrophages are not permanently programed, but are considered "plastic" -that is, macrophages have been shown to change activation phenotypes depending on the local environment. although the m /inos and m /arg division is generally appropriate, arg can be induced in m -like macrophages under certain conditions. thus, due to the spectrum of activation states for macrophages, a framework for macrophage-activation nomenclature was recently suggested ( ) . in an attempt to avoid confusion in this review, we focused on the specific effects of the l-arginine metabolizing enzymes inos or arg on the pathogenesis of viral infections, noting other activation markers where appropriate. increasing evidence suggests that myeloid cell programing, inos, and arg contribute to the pathogenesis of numerous virus infections, suggesting that therapies that target these cells and pathways may be beneficial for the treatment of some virus diseases. in this review, we highlight recent studies of viral infections where myeloid cell polarization -resulting in expression of inos or arg -contribute to viral control or the development of chronic virus infection and mediate the resolution of tissue damage or cause immunopathology. no has antimicrobial activity against a number of bacteria, parasites, and fungi ( , ) . additionally, no has been shown to have direct antiviral effects in vitro and/or in vivo against several viruses, including dna viruses such as herpes simplex virus type- (hsv- ), ectromelia virus (ev), and vaccinia virus (vv) ( , ), as well as some rna viruses such as vesicular stomatitis virus (vsv) ( ), japanese encephalitis virus (jev) ( ) , dengue virus (denv) ( ) , and coxsackievirus ( table ) ( ) ( ) ( ) ( ) . there are several advantages of using no as an antiviral agent. for instance, unlike complement and antibody, no can readily pass through cellular membranes into neighboring cells as well as some viruses. additionally, no is likely to act on a variety of both viral and virally exploited cellular targets, inhibiting viral replication as well as limiting the capacity of viruses to develop resistance. lastly, the effect of no is independent of immune recognition of the infected cell, in contrast to that of antiviral lymphocytes, which could be important in virusinfected cells where expression of mhc class i molecules may be downregulated and in some virally infected tissues such as the brain where expression of mhc class i and ii molecules is limited. in initial studies in vitro, inhibition of ev, vv, and hsv- replication in mouse raw . macrophages and in primary mouse macrophages following ifn-γ treatment was shown to be largely dependent on no production ( , ) . additionally, pharmacologic inhibition of nos or genetic deletion of nos resulted in increased viral titers and mortality following ev infection in mice ( , ) . moreover, no affects several events in the late stages of the life cycle of vv, including viral dna replication, viral protein synthesis, and virion maturation in vitro ( ) . these studies provided some of the first evidence that macrophage-produced no has direct antiviral effects. in addition to inhibiting hsv- replication in vitro, macrophage-derived no has been shown to have anti-hsv properties in vivo. in a mouse model of hsv- -mediated corneal disease, inos was highly induced in the trigeminal ganglion (tg) of hsv- -infected mice, and its expression was markedly reduced in mice depleted of macrophages ( ) . depletion of macrophages prior to hsv- infection resulted in markedly reduced inos expression and higher viral loads in the tg of infected mice ( , ) , suggesting that macrophages were the main source of inos expression in the affected tissues following hsv- infection and that no had important anti-hsv- properties in vivo. consistent with these data, inhibition of nos activity resulted in increased viral loads in the tg ( ) . additional studies showed that f / + gr- + inflammatory monocytes were recruited to the eye via an ifn-α-driven ccl gradient and restricted hsv- replication in that tissue via no production ( ) . it was further shown that no production by f / + macrophages in the brains of hsv- -infected mice blocked viral replication in a partially tlr -and tlr -dependent mechanism ( ) . finally, following footpad inoculation, hsv- -infected nos −/− mice displayed a delayed clearance of virus from the dorsal root ganglia (drg) and exhibited an increase in the frequency of virus reactivation in drg ( ) . the reactivity of no and its higher oxides and nitrosothiol products ( ) makes it likely that a variety of molecular targets are involved in its antiviral action. it has been shown that no can inhibit ribonucleotide reductase ( , ) , a rate-limiting enzyme in dna synthesis, and no can lead to the deamination of mammalian and bacterial dna ( , ) , which may be important antiviral mechanisms. indeed, hsv- encodes its own ribonucleotide reductase and although it is not required for hsv- replication in vitro, it is necessary under conditions where the intracellular pool of deoxynucleotides is limited ( , ) . thus, by inactivating this cellular and/or viral enzyme, no may halt virus replication by directly inhibiting viral dna synthesis. in addition to hsv- , treatment of primary human cells with an no donor following infection with human cytomegalovirus (hcmv), a beta-herpesvirus, resulted in a significant reduction of early and late viral protein expression ( ) . consistent with these in vitro data, nos −/− mice ( /sv/ev x c bl/ f ) exhibited increased viral titers and mortality following infection with murine cmv (mcmv; smith vr strain) ( ) . nitric oxide has also been shown to have antiviral properties on a chicken herpesvirus, marek's disease virus (mdv), which can cause t cell lymphomas in chickens: addition of no-generating compounds inhibited viral replication in chicken fibroblasts ( ) . additionally, the treatment of chickens with an inhibitor of inos increased the level of mdv replication in vivo ( ) . further studies demonstrated that no production was limited to chickens that were genetically resistant to tumor development following mdv infection or to chickens that were vaccinated before being inoculated with mdv ( ) . thus, no appeared to be produced in both types of resistance to tumor development in marek's disease, either acquired after vaccination or genetic. together, these findings suggest a role of no in the protective immune mechanisms against marek's disease, possibly through its activity on viral replication. finally, studies with hbv, a hepadnavirus associated with acute and chronic hepatitis, demonstrated that hbv replicated to higher levels in the livers of hbv-transgenic nos −/− mice than control transgenic mice, and transgenic nos −/− mice had increased frontiers in immunology | antigen presenting cell biology liver disease ( ) . it was further demonstrated that no production by mononuclear cells, most likely macrophages, in the liver mediated most of the antiviral activity resulting from ifn-γ production by virus-specific t cells ( ) , suggesting an antiviral role for macrophage-derived no following hbv infection in mice. in addition to dna viruses, macrophage-derived no also exerts antiviral effects against a number of rna viruses. inhibition of jev, a mosquito-transmitted flavivirus that causes encephalitis in humans, in ifn-γ-activated raw . macrophages in vitro correlated with no production, and ifn-γ-activated raw . macrophage-mediated inhibition of jev replication in murine neuroblastoma n cells was no-dependent ( ) . moreover, inhibition of nos activity led to increased mortality in jev-infected mice ( ) . in terms of its mechanism of action, no was found to inhibit jev rna synthesis, viral protein accumulation, and virus release from infected cells in vitro ( ) . these data suggest that no may be directly or indirectly inhibiting viral enzymes and/or other www.frontiersin.org cellular components required for viral replication, and this may subsequently block viral protein synthesis. additionally, no may interfere with the release and/or maturation of virions. monocyte/macrophage-derived no may also block replication of denv, another mosquito-transmitted flavivirus. infection with denv resulted in increased levels of no in patients with dengue fever, the classic form of the disease ( ) . additionally, inos expression was induced in cd + monocytes from a subset of acutely infected individuals ( ) . it was further shown that ex vivo infection of human monocytes with denv- resulted in increased inos expression, and inhibition of inos activity led to increased denv antigen detection in these cells ( ) . moreover, treatment of c / mosquito cells with an no donor resulted in reduced denv-positive cells ( ) . these data suggest that denv replication is susceptible to no-mediated inhibition. consistent with this, nos −/− mice were shown to be more susceptible to denv infection, resulting in more severe disease and increased lethality in mouse models of denv- and denv- infection ( , ) . it was further demonstrated that, following denv infection in vivo, il- and il- induced ifn-γ production, resulting in inos expression and no production, which contributed to viral control ( , ) . in addition to monocyte/macrophage-derived no, a recent study demonstrated that platelets isolated from patients with dengue fever had increased l-arginine transport and increased no production compared to platelets from healthy controls ( ) . however, no has anti-aggregatory properties, and mendes-ribeiro et al. ( ) found that dengue patients exhibited decreased collagen-induced platelet aggregation, consistent with the vascular leak and hemorrhagic manifestations of dengue hemorrhagic fever/dengue shock syndrome (dhf/dss), thus establishing an association between reduced platelet aggregation, enhancement of the l-arginine-no pathway, and dhf/dss ( ) . in contrast, getts and colleagues showed that experimentally abrogating no activity during west nile virus (wnv) encephalitis, a related flavivirus, in no-competent mice at a specific, relatively late time point prolonged survival of infected mice, while pharmacological inactivation throughout disease did not ( ) . combined, these data suggest that although during denv infection ifn-γ-induced no production has a role in antiviral defense, it is likely that dysregulation of the il- / -ifn-γ-no axis leads to immune-mediated damage in certain flavivirus infections. along these lines, it has also been shown that treatment of mice with a nos inhibitor increased mortality rates following sindbis virus (sinv) infection ( ) , suggesting a protective role for no during this particular cns infection. however, sinv replication in the brain was unaffected. furthermore, treatment of neuroblastoma cells with no donors had little effect on sinv replication but increased cell viability ( ) . these data suggest that no protects mice from fatal sinv-induced encephalitis by a distinct mechanism that does not directly involve the inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication. nitric oxide also plays an antiviral role during cns infection with reovirus. infection of neonatal mice with the prototypic neurotropic reovirus strain (t a) induced inos expression in brain areas demonstrating reovirus antigen expression and associated virus-induced injury ( ) . reovirus also induced inos expression following in vitro infection of primary neuronal and glial cultures. reovirus was shown to infect a subpopulation of microglial cells in vitro ( ) , suggesting that direct virus interaction may induce inos in this specialized population of macrophages. treatment of neuronal cultures with an no donor inhibited viral replication whereas a nos inhibitor increased viral growth ( ) , suggesting inos has the potential to exert antiviral activity in vivo. finally, coxsackievirus infection has been shown to induce expression of inos in macrophages infiltrating the hearts of infected mice ( ) . treatment of wt mice with a nos inhibitor and infection of nos −/− mice resulted in more severe coxsackievirus-induced pancreatitis and myocarditis, elevated viral loads in tissues, and decreased survival compared to wt mice following coxsackievirus b (cvb ) infection ( , , ) . similarly, nos −/− mice infected with coxsackievirus b exhibited decreased survival and delayed viral clearance compared to wt mice ( ) . these data suggest an antiviral effect of no against coxsackievirus infection. consistent with this, it was demonstrated that no inhibits the a and c proteinases of cvb in vitro ( ) . additionally, cvb -infected outbred mice showed significantly reduced signs of myocarditis after treatment with no donors ( ) . despite its protective capacity during some viral infections, no can also contribute to immunopathology. the pathological effects of no are likely due, at least in part, to oxidative damage caused by the interaction of no with oxygen radicals such as the superoxide anion radical o − and hydrogen peroxide (h o ). for example, although addition of an no donor to virusinfected mdck cells reduced influenza a and b viral burden in vitro ( ) , treatment of mice with inhaled no (ino) did not decrease the viral load of influenza a (mouse-adapted h n strain)-infected mice; in fact, prophylactic treatment with ino resulted in enhanced weight loss and decreased survival following infection ( ) , suggesting a pathogenic role for no. consistent with this, chickens, which show a high level of mortality and associated pathology following avian influenza infection, had higher levels of inos expression in the lungs compared with h n influenza-infected ducks, which show relatively minor symptoms following influenza infection ( ) . additionally, akaike and colleagues ( ) found evidence of the production of peroxynitrite, which is generated through the reaction of no and o -, in the lungs of influenza a (mouse-adapted h n strain)-infected mice. moreover, inhibition of nos resulted in enhanced survival and decreased pneumonia, but not decreased viral loads, in influenzainfected mice ( , ) , suggesting that no was contributing to pathogenesis rather than having direct antiviral effects. nos −/− mice also survived a lethal dose of influenza a virus (pr/ / strain) infection with little histopathologic evidence of pneumonitis; however, in these studies no infectious virus was detected in nos −/− mice at day after infection ( ) . the enhanced viral control in nos −/− mice was shown to require the activity of ifn-γ ( ), with nos −/− mice also producing increased virusspecific igg a antibody titers ( ) . additionally, genetic deletion frontiers in immunology | antigen presenting cell biology of nos or pharmacologic inhibition of nos enhanced survival of mice inoculated with the highly pathogenic (non-mouse-adapted) influenza virus strain, although mice exhibited similar viral loads to control mice in lung tissue at the peak of viral replication ( ) . influenza infection in vitro was shown to induce apoptosis, and a reduction in influenza-mediated apoptosis was noted in cells treated with a nos inhibitor ( ) . similarly, fewer apoptotic cells were found in the lungs of influenza-infected nos −/− mice, suggesting that no mediates cell death following influenza infection ( ) . the cellular source of inos/no following influenza infection in mice was shown to be ccr + inflammatory monocytes that accumulate in the lungs: ccr −/− mice survived a lethal challenge of influenza infection (pr/ / strain) and had significantly reduced accumulation of inos-expressing macrophages in the lung, with no associated increase in viral titers or dissemination ( ) . it was also recently shown that a subset of monocyte-derived dendritic cells (dcs), described as tnf-α/inos-producing dcs (tipdcs), accumulate in greater numbers during the course of lethal versus sublethal influenza infections, suggesting a pathogenic role for this subpopulation of myeloid cells ( ) . interestingly, though, aldridge et al. ( ) found that the tipdcs also stimulated a local, protective cd + t cell response in the virusinfected respiratory tract, indicating both protective and pathogenic roles for these cells in influenza infection. it was further shown that partially compromising tipdc recruitment via treatment with pioglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor-γ (ppar-γ), was protective against lethal influenza challenge ( ). pioglitazone treatment led to a reduction in the levels of ccl (mcp- ) and mcp- in the bal fluid of influenza-infected mice ( ) . pioglitazone has also been shown to reduce the production of a wide range of proinflammatory molecules, including inos ( ), providing further evidence for the importance of no production by monocyte-derived cells in the pathogenesis of influenza infection. pharmacologic inhibition of nos using l-nmma also decreased pneumonitis and increased survival following intranasal infection of cba/j mice with hsv- , despite a -fold increase in viral titers in the lung at day after inoculation ( ). in contrast, treatment of balb/c mice with a different nos inhibitor [aminoguanidine (ag), administered intranasally] resulted in enhanced pneumonitis, viral titers, and mortality following infection with a different strain of hsv- ( ) . thus, the precise role of no in hsv- pneumonitis remains to be determined. no and other ros/rns were also shown to be pathogenic in the brains of mice with herpes encephalitis: inos was induced in cd b + resident microglia following intranasal infection with hsv- , and oxidative and nitrative damage was found in the brains of infected animals ( ) . a common neurological complication of hiv infection in the developed world is sensory neuronal injury accompanied by inflammation, which is clinically manifested as disabling pain and gait instability. feline immunodeficiency virus (fiv) infection of cats, which causes similar neuroinflammation together with immunosuppression in cats, resulted in induction of inos and stat- , which were predominantly produced by macrophages, in drg ( ) . additionally, inhibition of nos resulted in reduced nitrotyrosine and prevented neuronal injury in fiv-infected drg cultures in vitro ( ) . these data suggest that lentivirus infection contributes to axonal and neuronal injury through a mechanism involving m macrophage immune activation mediated by stat- and inos activation. in addition to these studies, infection of mice with the retrovirus lp-bm , which causes profound immunodeficiency, induces cd b + gr- + ly- c + mdsc-like cells that inhibit both t-and b-cell responses in an inos/no-dependent but arginase-independent fashion ( ) . this study identified an important -and only recently appreciated -role for inosexpressing myeloid cell-mediated suppression of b cell responses in retrovirus infection. the oxidative effects of no have also been shown to inhibit immune cells, particularly t cells. this phenomenon has been appreciated for a number of years in the context of tumors ( ), where myeloid suppressor cells can inhibit the anti-tumor t cell response via the effects of no in addition to other mechanisms ( , ) . in a similar manner, it has been shown that no can inhibit the antiviral immune response. mcmv clearance from balb/c mice is predominantly cd + t cell-mediated. a recent report showed that mcmv infection in balb/c mice induced cd b + ly- c hi inflammatory monocyte recruitment from the bone marrow to infected tissues that was dependent on ccr signaling ( ) . this recruitment was shown to inhibit antigen-specific cd + t cell activation, expansion, and cytotoxic activity via no production, thus facilitating viral persistence ( ) . in a similar fashion, no may contribute to a defective immune response following infection of mice with an attenuated neurotropic coronavirus (rj . strain of mouse hepatitis virus). rj . infected wt mice exhibited mild acute encephalitis, followed by a non-lethal, chronic demyelinating disease ( ) . in marked contrast, rj . infection of mice that transgenically express ccl in the brain (ccl tg) ineffectively cleared virus and rapidly succumbed to the infection ( ). ccl tg mice mounted a dysregulated immune response, characterized by increased accumulation of inos-expressing macrophages and microglia as well as regulatory t cells, but decreased arg expression ( ) . these data suggest that persistent ccl overexpression establishes and sustains an immunological milieu that may predispose mice to a defective immune response to a typically minimally virulent virus. arginase activity is important for wound healing and tissue regeneration through the production of polyamines and proline ( ) . in the context of some viral infections, arginase activity and m macrophage activation have been shown to be beneficial for tissue repair following virus-induced damage. for instance, resolution of severe respiratory syncytial virus (rsv)-induced bronchiolitis in mice is mediated by m macrophages that counteract cyclooxygenase (cox)- -induced lung pathology ( , ) . arg was induced in the lungs of rsv-infected mice, and its induction was shown to be il- rα-dependent ( ) . additionally, wt macrophages adoptively transferred into rsv-infected il- rα −/− mice restored the www.frontiersin.org m phenotype in the lungs and decreased lung pathology ( ) . it was further shown that the lipoxogenase pathway was important for m macrophage activation and lung resolution following rsv infection ( ) . most recently it was demonstrated that treating mice with agents that sustain arg expression (e.g., il- /anti-il- immune complexes) limited rsv-induced lung pathology ( ) . consistent with a pathogenic role for inos/no following influenza infection (described above), it was recently shown that the presence of airway bacteria polarize alveolar macrophages into a m phenotype, thus limiting influenza-mediated lethal lung inflammation. wang and colleagues ( ) demonstrated that priming with staphylococcus aureus, which commonly colonizes the upper respiratory mucosa, attenuated influenzamediated lung injury via tlr signaling that recruited peripheral ccr + cd b + monocytes into the alveoli ( ) . these monocytes polarized alveolar macrophages into a m phenotype characterized by high arg as well as ym , fizz , and il- expression ( ) . it was further shown that s. aureus-primed m alveolar macrophages inhibited inflammatory cell recruitment to the lung, including neutrophils, nk cells, and cd t cells ( ) . s. aureus-primed m alveolar macrophages also expressed higher levels of the inhibitory ligand pd-l ( ) , suggesting that expression of a combination of anti-inflammatory cytokines and inhibitory ligands could be the mechanisms by which s. aureusprimed m alveolar macrophages limit influenza-mediated lung inflammation. as discussed above, coxsackievirus b (cvb ) infection causes myocarditis in human beings as well as in male balb/c mice. although female mice do not develop severe myocarditis, both male and female mice have comparable numbers of infiltrating macrophages and viral titers in the heart following cvb infection ( ) . the macrophages infiltrating the heart in male mice were skewed toward a m phenotype characterized by high expression of inos ( ) as well as m -associated cytokines such as ifn-γ and il- ( ) . additionally, inhibition of nos resulted in increased viral titers and higher mortality in cvb -infected mice ( ), consistent with an antiviral role for no during cvb infection (see above). however, in contrast to male mice, the heart-infiltrating macrophages in female mice were skewed toward a m phenotype characterized by high expression of arg as well as il- and il- ( ). moreover, adoptive transfer of ex vivo-programed m macrophages significantly increased myocarditis in both male and female mice. strikingly, transfer of m -programed macrophages into susceptible male mice alleviated myocardial inflammation by modulating the local cytokine profile from a m to m phenotype and promoting peripheral regulatory t cell (treg) differentiation ( ) . using different variants of cvb , one that caused myocarditis in c bl/ mice and one that did not, it was additionally shown that the myocarditic variant induced a m macrophage phenotype ( ) . in contrast, the amyocarditic variant induced a m macrophage phenotype, which was also associated with the activation of nkt cells that promoted a treg response ( ) . the ability of nkt cells to suppress myocarditis was shown by adoptive transfer of purified nkt cells into nkt knockout (jα knockout) mice infected with the myocarditic cvb variant, which inhibited cardiac inflammation and increased treg response ( ) . cardiac virus titers were equivalent in all mouse strains indicating that nkt cells did not participate in control of virus infection ( ) . thus, although no appears to have antiviral properties against cvb , these data indicate an important role for arg -expressing m macrophages in controlling cvb -induced myocarditis. as a consequence of their co-evolution with their hosts, viruses have developed numerous strategies to evade the host immune system and ensure their own replication and survival. recent studies have identified a new evasion strategy for viruses: exploitation of the host's anti-inflammatory, wound repair response to promote chronic infection. two strains of lcmv -armstrong (arm) and clone (c )have been studied for decades as models for acute and chronic infections ( ) . infection of mice with the arm strain leads to a robust cd + t cell response that rapidly clears the virus ( ) , whereas infection with c results in t cells with impaired functionality, enabling the virus to persist ( ) . it was recently demonstrated that c infection led to an enhanced and sustained expansion of cells that resembled mdscs ( ) . these suppressive myeloid cells inhibited t cell proliferation ex vivo via an inos/no-dependent but arg -independent mechanism. another study, however, found that arg -expressing immunoregulatory antigen presenting cells induced during c infection suppressed t cell responses ( ) . most recently, it was demonstrated that t cell responses were improved -resulting in clearance of the normally chronic c infection -when either myeloid cells or t cells lacked il- production ( ) . overall, these data demonstrate the importance of inos/arg -expressing myeloid cells in viral persistence. similar to lcmv c infection, it was recently demonstrated that infection of mice with the arthritogenic alphaviruses ross river virus (rrv) and chikungunya virus (chikv) resulted in the induction of arg in macrophages in the infected and inflamed musculoskeletal tissues ( ) . it was further shown that genetic deletion of myeloid cell arg resulted in enhanced viral control in inflamed muscle tissue and reduced tissue pathology following rrv infection in mice ( ) , suggesting an important role for arg -expressing macrophages in the persistence of these chronic viruses. infection of mice with theiler's murine encephalomyelitis virus (tmev) results in persistent virus infection in the cns, which contributes to the development of a demyelinating disease that has similarities with multiple sclerosis. bowen and olson ( ) showed that cd b + ly- c + cells infiltrated the cns following infection and were the dominant cell type during the innate immune response. depletion of the cd b + ly- c + cells via administration of an anti-gr- ab resulted in reduced development of demyelinating disease and enhanced virus-specific cd + and cd + t cell responses ( ) . additionally, tmev-infected, anti-gr- ab-treated mice had decreased myelin-specific cd + t cell responses compared to control ab-treated mice during the demyelinating disease at a later time post-infection ( ) . although the expression of arg was not investigated in this study, tmevinfected mice had elevated expression of il- in the brain and spinal cord ( ), suggesting a role for this cytokine in the suppression of antiviral t cell responses, potentially through the effects of arg . interestingly, a role for the modulation of arginine metabolism in viral control versus persistence along with associated disease has recently been demonstrated for the tumor-inducing, chickenspecific herpesvirus mdv. we mentioned above that mdv was vulnerable to the antiviral properties of no, with inos being induced in genetically resistant chickens and in vaccinated chickens ( ) . in contrast, mdv induced strong macrophage arginase activity in cell extracts from adherent monocytes from genetically susceptible chickens, but not in chickens that were resistant to marek's disease, either genetically or acquired after vaccination ( ) . together, these data suggest that in the case of marek's disease, the state of resistance versus sensitivity to disease was correlated with a reciprocal balance of nos versus arginase activities in macrophages. this phenomenon of arg -mediated t cell suppression has also been recognized in human viral infections. arg mrna and protein levels were elevated in hcv-infected liver cell lines in vitro and in hcv-infected liver samples compared with paired hepatocellular carcinoma samples from the same patients or with uninfected liver tissues ( ) . additionally, the number of mdscs in chronic hcv patients correlated with levels of plasma hcv-rna ( ). cai et al. ( ) also found that mdscs from patients with chronic hcv infection suppressed t cell function via an arg -dependent mechanism. an additional study found that more pbmcs from chronic hcv patients expressed the phenotypic markers of mdscs than pbmcs from healthy controls, and these cells expressed increased levels of p phox , a component of the nadph oxidase complex ( ) , suggesting a role for ros in mdsc-mediated suppression. consistent with this, cd + mononuclear cells co-cultured with hcv-infected hepatocytes or hcv core protein suppressed t cell proliferation in a ros-dependent manner ( ) . overall, these data suggest that multiple mechanisms -including arginine metabolism and ros -may be at play in myeloid cell-mediated suppression of anti-hcv t cell responses. it has been suggested that prolonged immune activation during chronic virus infections, such as hcv and hiv, provides an environment that drives viral replication and disease progression ( , ) . moreover, immune activation can drive an anti-inflammatory response to limit immunopathology, which can be characterized by the presence of m -like macrophages. indeed, similar to hcv infection, a role for arginase and m polarized mdsc-like cells has been identified in the suppression of antiviral t cell responses following hiv infection. individuals with detectable hiv- infection showed an increase in the frequency of cd + cd + cd + monocytes, which are thought to be precursors of m macrophages, when compared to seronegative or hiv- -infected persons with undetectable viral loads, and monocyte frequency correlated positively with hiv- viremia and negatively with cd + t cell counts (in patients with counts < cells/µl) ( ) . furthermore, qin and colleagues ( ) observed elevated levels of mdscs, defined as hla-dr −/low cd b + cd +/high cd + cd − cells, in the peripheral blood of hiv- -seropositive subjects compared with healthy controls, and these mdscs suppressed t cell responses in an arg -dependent manner. moreover, pbmcs from hivseropositive patients exhibited increased levels of arginase activity ( ) . cloke and colleagues ( ) found that increased arginase activity correlated with lower cd + t cell counts, and this association was abrogated following antiretroviral treatment ( ) . additionally, exposure of pbmcs to hiv gp expanded t cellsuppressive mdscs in vitro ( ) . these data point to a direct role for arginase-expressing mdsc-like cells in the suppression of anti-hiv t cell responses. consistent with that, individuals co-infected with hiv and leishmania parasites had increased arginase activity in pbmcs and plasma compared with leishmania-only infected individuals, even though leishmania infection alone results in increased arginase activity ( ) . in addition, the parasite load in the spleen was significantly higher in co-infected patients ( ) . the arginase-expressing cells were identified as low-density granulocytes ( ) . these results suggest that increased arginase might contribute to the poor immune responses and disease outcome characteristic of patients with leishmania and hiv co-infection. hepatitis b virus (hbv) infection is another common chronic viral infection, with estimates as high as million chronically infected humans ( ) . bility and colleagues ( ) recently developed a humanized mouse model with both a human immune system and human liver cells, named the a /nsg-hu hsc/hep humanized mouse model, to study the pathogenesis of hbv infection. following hbv infection, the mice developed persistent hbv infection as well as chronic hepatitis and liver fibrosis ( ) . the liver disease was associated with a high level of infiltrating human macrophages with a m -like activation phenotype ( ) . similarly, m -like macrophage accumulation was seen in chronic hbv-infected patients, and m -like macrophage induction in the liver was associated with accelerated liver fibrosis and necrosis in patients with acute hbv-induced liver failure ( ), suggesting a role for m macrophages in persistent hbv infection. additionally, patients with acute hbv infection had increased serum levels of arginase, and this serum inhibited ifn-γ production by cd + t cells ( ) . das et al. ( ) also found decreased l-arginine levels in the circulation of chronic hbv patients with marked liver inflammation (> alt) and increased arginase activity in liver extracts taken directly ex vivo from patients with chronic hbv compared with those from patients with other types of liver pathology ( ) . they further showed that cd + t cells from chronic hbv patients, regardless of their antigen specificity, exhibited less il- but not ifn-γ or tnf-α production and impaired proliferation following tcr-dependent stimulation, indicating an aberrant antiviral t cell response in chronic hbv infection ( ) . in the a /nsg-hu hsc/hep humanized mouse model, hbv-infected mice had impaired liver t cell responses, and m macrophages were associated with t cells in the liver ( ) . expression of the tcr signaling molecule cd ζ was reduced in both peripheral and intrahepatic cd + t cells from chronic hbv patients; similarly, cd was also downregulated on cd + t cells from high viral load hbv patients ( ) . downregulation of the cd ζ molecule has previously been shown to occur in the arginine-depleted tumor microenvironment. consistent with this, in vitro transfection of cd ζ and cd restored il- production and supplementation of l-arginine partially restored cd ζ expression and t cell proliferation ( ) . these data suggest a role www.frontiersin.org for arginase activity and arginine depletion in the impairment of anti-hbv t cells functions. in the absence of inkt cells, influenza a (pr/ strain) infection was shown to induce the expansion of cd b + gr- + mdscs in the lungs of mice, which suppressed influenza-specific t cell and antibody responses through the activity of both arginase and nos, resulting in higher viral titers and increased mortality ( ) . adoptive transfer of inkt cells reversed this phenotype; mice had an increased survival rate, reduced viral titers, and increased virusspecific immune responses, suggesting a novel immunomodulatory role for inkt cells during influenza virus infection ( ) . moreover, these authors identified that influenza infection in humans induced the expansion of cd b + myeloid cells with suppressive activity that could be reduced by inkt cell activation or the inhibition of arginase and nos activity. similarly, it was recently shown that highly pathogenic h n and h n influenza virus infection induced the accumulation of cd b + gr- + cells and the expression of arg in the lungs ( ) , further supporting a role for m -polarized mdsc-like cells in promoting viral persistence and immunopathology. helminth infection induces the expression of type cytokines and is associated with m macrophage activation, as determined by arg , fizz , and ym expression. indeed, osborne and colleagues ( ) found that arg , fizz , and ym were highly induced in the ileum of mice infected with the helminth trichinella spiralis (ts). interestingly, they further showed that co-infection of mice with ts and murine norovirus (mnv) resulted in decreased frequencies and numbers of mnv-specific cd + and cd + t cells within the small intestine and spleen as well as decreased polyfunctionality of these t cells, compared to ts-only infected mice ( ) . additionally, the defective t cell responses were associated with increased viral loads in the double-infected mice compared to the mono-infected controls ( ) , suggesting that ts-elicited m -activated macrophages inhibited the antiviral t cell response to mnv. lastly, neutralization of ym , a chitinase-like molecule, in co-infected mice partially restored antiviral immunity and was associated with enhanced control of viral replication ( ) . these data point to a new mechanism by which arg -expressing macrophages inhibit antiviral responses. cumulatively, these data are reminiscent of macrophages found in tumors (e.g., mdscs, tams) that have been shown to suppress anti-tumor t cell responses via a variety of no-and/or arg -dependent mechanisms ( , ) . indeed, in a mouse model of human papillomavirus (hpv)-induced cancer, arg -expressing cd b + f / + macrophages infiltrated the tumors and inhibited t cell responses, including virus-specific t cells, by suppressing t cell proliferation and promoting a regulatory phenotype ( ) . moreover, depletion of the tumor-infiltrating macrophages resulted in reduced tumor growth and increased tumor infiltration by virus-specific cd + t cells ( ) . thus, increasing evidence points to a direct role for arginase-expressing m -polarized cells in the suppression of antiviral t cell responses and the persistence of a variety of important pathogenic viruses. in addition to the actions of inos and arg , mdsc-like cells can employ other mechanisms to promote chronic viral infections, which were recently reviewed by goh and colleagues ( ) . in contrast to some parasitic infections where m macrophages limit th cell-mediated immunopathology, m -polarized macrophages have been shown to promote immunopathology in some viral infections. for example, it was recently demonstrated that sars-cov infection of mice induced suppressive alveolar macrophages that inhibited the induction of antiviral t cell responses, a phenotype that was reversed by the adoptive transfer of activated bone marrow-derived dcs into mice prior to virus infection ( ) . additionally, sars-cov-infected mice lacking hematopoietic stat- expression were shown to have greater weight loss and lung pathology, and this was associated with the activation of m macrophages ( ) . to further test the role of m macrophages in enhanced pathogenesis following sars-cov infection, the authors generated stat- /stat- double knockout mice due to the established role for stat- in driving m macrophage activation in response to il- /il- stimulation. stat- /stat- double knockout mice, which reversed the upregulation of m macrophages observed in stat- -deficient mice, had reduced lung disease and prefibrotic lesions ( ) . these data support the notion that m macrophages contribute to sars-cov pathogenesis. in another example, mice deficient in the ifn-γr exhibit more severe disease following infection with murine gammaherpesvirus- (mhv- ), including interstitial and intra-alveolar fibrosis that is reminiscent of idiopathic pulmonary fibrosis (ipf) in human beings. in this model, alveolar macrophages were recruited to the lungs of mhv- -infected ifn-γr −/− mice, were associated with areas of fibrosis, and exhibited a m -polarized phenotype characterized by the expression of fizz , ym , and arg ( ) . additionally, lung tissue from patients with ipf showed increased expression of arg in alveolar macrophages compared with normal lung ( ) . these results suggest that virus-induced upregulation of arg could be mediating lung fibrogenesis. mhv- infection in ifn-γr −/− mice also resulted in fibrosis in lymphoid tissues such as the spleen, which is a site of latent mhv- infection, and the liver ( , ) . similar to the lung, mhv- infection in the absence of ifn-γr signaling induced a m macrophage response in the spleen, characterized by high arg expression along with fizz and m /th cytokines such as il- , resulting in fibrotic disease in the spleen ( ) . moreover, depletion of t cells prevented mhv- -mediated fibrosis in ifn-γr −/− mice ( ) , suggesting that m macrophages were further driving th activation to possibly create a m /th cytokine-induced cycle, resulting in the exaggerated pathology. in contrast to ifn-γr −/− mice, inos was induced in the spleen of mhv- -infected wt mice ( ) , indicating an important role for ifn-γ in inducing a m -associated immune response to control gamma-herpesvirus infection and limiting arg -mediated immunopathology. macrophages and other myeloid cells have marked phenotypic heterogeneity, as a result of distinct cellular differentiation programs, distribution in tissues, and responsiveness to various endogenous and exogenous stimuli. indeed, macrophages have well-established roles in development, tissue homeostasis, coordinating the adaptive immune response and inflammation, as well as directing tissue resolution and repair following damage -processes that are often modulated via the actions of the arginine-hydrolyzing enzymes nos and arg . we have highlighted a number of viral infections in which these enzymes have a beneficial effect: no has antiviral properties against a variety of viruses, and arginase activity can mediate tissue repair and regeneration following a viral insult (table ) . however, no production can also result in immunopathology in some virus infections, and the suppressive functions of arg -expressing macrophages can promote immunopathology. additionally, some viruses have exploited the immune-suppressive properties of inos-and/or arg -expressing macrophages to evade the immune response, particularly the antiviral t cell response, resulting in chronic viral infections. clearly, inos-and/or arg -mediated responses are important in many viral infections. thus, there is the potential to develop the means to selectively stimulate or inhibit either m or m responses to mediate viral clearance or repair tissue damage. due to the overlap in immunosuppressive mechanisms of inos-and/or arg -expressing suppressor cells, therapeutic strategies under development to limit the immunosuppressive effects of myeloid cells in cancer may be beneficial in treating persistent/chronic virus infections. however, as described above, inos and arg activity can be both beneficial and detrimental during certain viral infections. therefore, further research is needed to define the molecular and tissue-specific mechanism(s) by which inos and arg influence the clearance of viral pathogens as well as the injury and repair of tissues. in addition, a better understanding of the pathways regulating macrophage polarization (specifically inos and/or arg induction and activity), macrophage trafficking, and the precise effects of inos and arg activity on other immune cells 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nitric oxide donors inhibit the coxsackievirus b proteinases a and c in vitro, virus production in cells, and signs of myocarditis in virus-infected mice molecular basis of "suppressor" macrophages. arginine metabolism via the nitric oxide synthetase pathway selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice massive expansion of antigen-specific cd + t cells during an acute virus infection viral persistence alters cd t-cell immunodominance and tissue distribution and results in distinct stages of functional impairment macrophage and t cell produced il- promotes viral chronicity innate immune cd b+gr- + cells, suppressor cells, affect the immune response during theiler's virus-induced demyelinating disease clinical significance and functional studies of myeloid-derived suppressor cells in chronic hepatitis c patients myeloid suppressor cells induced by hepatitis c virus suppress t-cell responses through the production of reactive oxygen species macrophage polarization and hiv- infection contribution of immune activation to the pathogenesis and transmission of human immunodeficiency virus type infection hepatitis b virus infection hpv tumor associated macrophages suppress antitumor t cell responses myeloid-derived suppressor cells: the dark knight or the joker in viral infections? murine gammaherpesvirus-induced fibrosis is associated with the development of alternatively activated macrophages work in dr. morrison's laboratory is supported by nih-niaid grants u ai and r ai . kristina s. burrack was supported by nih-niaid training grant t ai . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- - rzibpbl authors: eng, yi shin; lee, chien hsing; lee, wei chang; huang, ching chun; chang, jung san title: unraveling the molecular mechanism of traditional chinese medicine: formulas against acute airway viral infections as examples date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: rzibpbl herbal medicine, including traditional chinese medicine (tcm), is widely used worldwide. herbs and tcm formulas contain numerous active molecules. basically, they are a kind of cocktail therapy. herb-drug, herb-food, herb-herb, herb-microbiome, and herb-disease interactions are complex. there is potential for both benefit and harm, so only after understanding more of their mechanisms and clinical effects can herbal medicine and tcm be helpful to users. many pharmacologic studies have been performed to unravel the molecular mechanisms; however, basic and clinical studies of good validity are still not enough to translate experimental results into clinical understanding and to provide tough evidence for better use of herbal medicines. there are still issues regarding the conflicting pharmacologic effects, pharmacokinetics, drug interactions, adverse and clinical effects of herbal medicine and tcm. understanding study validation, pharmacologic effects, drug interactions, indications and clinical effects, adverse effects and limitations, can all help clinicians in providing adequate suggestions to patients. at present, it would be better to use herbs and tcm formulas according to their traditional indications matching the disease pathophysiology and their molecular mechanisms. to unravel the molecular mechanisms and understand the benefits and harms of herbal medicine and tcm, there is still much work to be done. it is common to initiate the therapy of orthodox western medicine by fitting the pharmacologic characteristics of a drug, including pharmacokinetic and pharmacodynamic effects, to the disease pathophysiology. physicians further validate the clinical application according to evidence-based medicine (ebm); however, this is not the case for tcm. tcm developed in ancient china, and at that time, physicians managed diseases with herbs only by clinical experience, without any knowledge of disease pathophysiology, nor the pharmacologic activities of herbs, to say nothing of the molecular mechanisms of herbs. to unravel the molecular mechanism of tcm formulas, it would be better to understand how physicians prescribe them first. tcm includes herbal therapy, acupuncture, massage, and dietary therapy. in the current work, tcm will be simply defined as herbal and dietary therapies. tcm is widely popular in east asia and forms the kampo medicine in japan, as well as traditional korean medicine; importantly, traditional medicines form the mainstream of healthcare in these countries. in ancient china, several famous tcm textbooks summarized the clinical experiences of using tcm formulas against various diseases, including endemic diseases, and each formula has its indication, including specific symptoms of patients. this is quite different from using tcm formulas based on the yin-yang theory (two opposite, but complementary forces) and five elements theories (everything in the world can be classified into the natural five elements. these elements promote as well as feedback each other to keep everything in balance). kampo medicine in japan classified tcm theories into ancient formula sect and recent formula sect for prescribing tcm formulas. the physicians of the ancient formula sect (a-physicians) use the indications of formula formed before formula can have active molecules that are pharmacologically different from that ingredient or the tcm formula. obversely, the pharmacological activities of a tcm formula may differ from those of their active ingredient or active molecules of ingredients. as a consequence, unraveling the molecular mechanism of a tcm formula needs comparison of the pharmacological activities between the formula, its ingredients, and the active molecules in the ingredients. the amount of most bioactive compounds in the herbs is very low. combination of herbs to form a tcm formula can further decrease their concentrations. is it possible that herbs and tcm formulas can be effective in this low concentration of bioactive compounds? is it possible that little amount of bioactive molecules can cause interactions? in orthodox western medicine, vitamins of little amount can show their clinical effects. the molecular mechanisms are the key, instead of their amount. in the real world practice, herbs and tcm formulas are bioactive. several side effects of tcm formulas have been reported [ ] [ ] [ ] [ ] [ ] [ ] [ ] that raise the safety issue of tcm formula. natural products and tcm formulas might not be safe; however, some a-physicians suppose that the side effects may come from the misuse of tcm formulas without fulfilling their indications, while r-physicians consider the side effects developing from the misinterpretation of the disharmony. most tcm physicians do not agree that these side effects come from tcm formulas themselves, although with the same indications or disharmony, it is common to find that some patients respond well while others do not-while some patients develop side effects, some show responses opposite to the in vitro pharmacological effects. for example, panax quinquefolius prolongs thromboplastin time, prothrombin time (pt) and thrombin time in vitro [ ] . however, in combination therapy with warfarin, panax quinquefolius actually decreases seral concentration of warfarin and has shortened inr in a clinical trial [ ] . with therapy using panax ginseng, one can develop thrombosis [ ] , bleed [ , ] , or remain without any particular response [ ] . several factors may affect the molecular mechanisms and subsequent clinical effects of tcm formulas, including individual gene-based response, composition and amount of active molecules in tcm formulas, complex interactions, and appropriateness of use of tcm formulas. individual genetic basis is unique to metabolize tcm formulas and produces different responses. from the results of pharmacokinetic study of gan-lu-siao-du-yin, a tcm formula (submitted data), the blood concentrations of structurally-related index molecules, baicalin and baicalein, wogonin, and wogonoside, are highly variable between participants. the different patterns of blood concentrations support the unique pharmacokinetic profile based on individual genes. different concentrations of active molecules may affect the pharmacologic activities. therefore, individual gene-based metabolism could be one of the major factors affecting the molecular mechanisms and subsequent clinical effects of tcm formulas. to provide insights into action mechanisms of tcm formulas, metabolomic technologies might be helpful. a metabolomics integrative approach accepts a 'top-down' strategy to express the function of organisms through terminal symptoms of metabolic network and will gain a revolution in understanding of the holistic concept of tcm [ , ] . such technologies have been used to investigate the biological mechanisms of different syndromes of patients by studying the functional activities of the human body from a system-wide perspective. for example, the overall biological characterization of the urine of psoriasis patients with tcm blood stasis syndrome was performed to investigate the therapeutic metabolomic mechanism of the optimized yinxieling formula [ ] . in addition, metabolomics have been considered a powerful tool in diagnosis and treatment of primary dysmenorrhea by supporting information on changes of metabolites and changes in endocrinal, neural, and immune pathways [ ] . the xiang-fu-si-wu formula has been demonstrated to affect some significant perturbations in sphingolipid and glycerophospholipid metabolism as well as steroid hormone biosynthesis to make the metabolic discrepancy return to the normal level [ ] . tcm formulas are complex with numerous active chemical molecules in variable amounts. among these molecules with different pharmacologic activities, it is unclear which one mainly accounts for the clinical effect of a tcm formula, as the most abundant molecule might not be the most important one for a specific activity. the amount of an active molecule can be easily changed in different batches of product, or by different agriculture and collection of medicinal plants, therefore, understanding the molecular mechanisms of a tcm formula requires analysis not only of the mechanisms of the tcm formula as a whole, but those of individual active molecules and ingredient respectively as well. understanding the molecular mechanisms of an active molecule can facilitate its development into an investigational new drug (ind). meanwhile, unraveling the molecular mechanisms of a tcm formula can help to validate its traditional use and avoid its misuse and side effects. to keep a relatively constant amount of active molecules and pharmacologic activities, several things should be paid attention to, including use of right specie, use of right part of a plant, and use of a plant harvested in the right season. both use of closely related but wrong species and use of wrong part of herbs might lead to different active molecules with different pharmacologic activities, and various clinical effects and side effects. plants harvested in different seasons might contain variable amounts of active molecules thereby affecting their activities. some active molecules are secondary metabolites of plants against physical, chemical, or biological stimulants. active molecules of some plants can vary from year to year and place to place. therefore, confirmation of its authenticity is the cornerstone. in addition to this, fingerprints of the active molecules are needed to confirm the authenticity of a plant or a formula and to confirm the amount of active molecules via high-performance liquid chromatography (hplc) or liquid chromatography coupled with mass spectrometry (lcms). this is highly necessary for quality control and efficacy assessment. to have quality control of the products of tcm formulas, good manufacturing practice (gmp) procedure should be followed to avoid (a) inadequate processing that might lead to different chemical compositions of the final product; (b) inadequate storage conditions or prolonged storage that might lead to microbial contamination and early decay of the active molecules; and (c) adulteration of formulas and accidental contamination creating serious uncertainty in quality. adulteration is a plant or formula containing active pharmaceuticals or other bioactive agents for the purpose of claiming better efficacy or broader indications. accidental contamination is the plant raw materials containing heavy metals or other toxic substances from the manufacturing process due to ecological collapse. lead, mercury, and arsenic contamination in traditional chinese herbs has been reported [ ] [ ] [ ] [ ] . . complex interplays between herb-drug, herb-food, herb-herb, herb-microbiome, and herb-disease in the clinical practice of orthodox medicine, the more drugs used, the more adverse drug reaction (adr) occurred [ ] . this is commonly caused by drug-drug interactions. tcm formulas are mixtures of several ingredients. each ingredient has several bioactive compounds, so a tcm formula has numerous bioactive compounds. thinking of dozens or even hundreds of active molecules in a tcm formula been taken at once implies that the probability of drug interaction could be high. such interactions may be found between herb and drug, herb and food, herb and herb, herb and microbiome, and even between herb and disease. for example, in herb-drug interaction, scutellariae baicalensis is a common ingredient in tcm formulas. s. baicalensis contains baicalin, a flavonoid, as one of its major molecules. interactions between s. baicalensis and drugs are found due to baicalin affecting metabolic enzymes of drugs, displacing plasma protein binding, and regulating various transporters involved in the pharmacokinetics [ ] . baicalin may inhibit the expression and hydroxylation activity of cyp a in the liver to change the pharmacokinetics of drugs [ ] . co-administration of extract of s. baicalensis, and mefenamic acid, a kind of nsaid, can potentiate its anti-inflammatory effect [ ] . co-administration of baicalin and rosuvastatin, a hmg-coa reductase inhibitor commonly used to reduce serum cholesterol level, might find reduced plasma concentration of rosuvastatin in certain patients with certain genomes [ ] . as for herb-food and herb-food-drug interactions, baicalin can potentiate the antioxidant activity of β-carotene, which is a terpenoid of red-orange color, abundant in plants and fruits [ ] . the intakes of flavonoid-rich foods and beverages, containing baicalin and rutin, might compete with the binding site of calcium channel blockers on human serum albumin to affect their clinical effects [ , ] . by contrast, baicalin and rutin will increase the binding affinity of curcumin on human serum albumin to change its bioavailability [ ] . herbs may also interact with each other. for example, baicalin and berberine are important coexisting molecules of the combination of s. baicalensis and coptidis chinensis. berberine, but not baicalin, can increase glucose consumption. co-administration of berberine and baicalin had a synergetic effect on glucose utilization [ ] . additionally, tcm herbs are commonly used as food supplements and dietary therapy. foods have been reported to modify the intestinal microbiome [ ] . commensal microbiota have been thought to be involved in the development of the innate and adaptive immunity, nutrient metabolism of humans, and protection from the overgrowth of intestinal pathogens [ ] . herbs can change pharmacokinetics of drugs by intestinal microbiota [ ] . the intestinal microbiome is metabolically active to play an important role in the absorption of certain active molecules and change their bio-availabilities, particularly in those containing glycosidic linkages [ , ] . therefore, change of intestinal microbiome by herbs-containing foods may affect human health care and drug therapy. as for interactions between disease and herb, more absorption of active molecules of maxing shigan decoction (mxgst), including liquiritin, glycyrrhizin, amygdalin, prunasin, ephedrine, pseudoephedrine, and methylephedrine, can be found in rsv pneumonia-infected rats vis-à-vis normal rats by reducing the clearance rates of these active molecules [ ] . there are highly complex interactions between herbs and drugs, foods, herbs, microbiome, and diseases, and most of these complex interactions are not completely discovered or remain unseen, just like the submerged part of an iceberg. therefore, there are still insufficient data to completely understand the molecular mechanisms, pharmacokinetics, pharmacodynamics, and interactions of tcm formulas. acute airway infections, including acute bronchitis, viral pneumonia, and acute exacerbation of chronic obstructive pulmonary disease (copd), are commonly caused by viruses of different families, including rhinovirus, influenza, and parainfluenza virus, enteroviruses, coronavirus, adenovirus, respiratory syncytial virus, etc. [ , ] . these viruses infect epithelia, produce inflammation, induce immune response, and cause symptoms. from the viewpoint of pathophysiology, tcm formulas used to manage airway viral infections need to have antiviral activity against such viruses listed above, and/or to induce antiviral cytokines, and/or anti-inflammatory effect, and/or to relieve symptoms commonly presented in airway infections ( figure ). to simplify the molecular mechanisms and to correlate the pharmacologic activities with their clinical effects, five formulas of a-physicians will be used as examples against airway infections: ge-gen-tang (ggt; table ) [ ] has been reported to be effective in the treatment of common colds, chronic sinusitis, allergic rhinitis, and pneumonia. the indication to use ggt is patients with symptoms of headache, fever without sweating, and particularly stiffness of neck and shoulders. ggt can successfully reduce various symptoms of dogs infected with common cold viruses [ ] . ggt has antiviral activities against respiratory syncytial virus (rsv) [ ] and influenza virus [ ] . ggt can reduce the mortality of influenza virus-infected mice [ ] . among its active molecules, uralsaponins from glycyrrhiza uralensis [ ] and procyanidin from cinnamomum cassia [ ] may effectively inhibit the replication of the influenza virus. allicin in ginger (zingiber officinale) and coumarin in cinnamomum cassia might have the activity to inhibit influenza neuraminidase [ ] . paeonol and , , , , -penta-o-galloyl-β-d-glucopyranose from paeonia lactiflora show antiviral activity against rhinovirus [ ] . of its anti-inflammatory effects, ggt can suppress the interleukin- α (il- α) production induced by interferons (ifn) in influenza [ ] . ggt was found to decrease cigarette smoking-(cs-) and lipopolysaccharide (lps)-induced elevated counts of inflammatory cells, and expression of inflammatory cytokines and proteins (il- , tnf-α, inos, and cox- ) [ ] . ggt can stimulate il- and ifn-β to counteract viral infection [ ] , and can also enhance the phagocytic activity of macrophages [ ] . among its active molecules, paeoniflorin, a major constituent of paeonia lactiflora, exerts anti-inflammatory and immunomodulatory effects by balancing the function of th /th [ ] . glycyrrhizin, a major constituent of glycyrrhiza uralensis, ge-gen-tang (ggt; table ) [ ] has been reported to be effective in the treatment of common colds, chronic sinusitis, allergic rhinitis, and pneumonia. the indication to use ggt is patients with symptoms of headache, fever without sweating, and particularly stiffness of neck and shoulders. ggt can successfully reduce various symptoms of dogs infected with common cold viruses [ ] . ggt has antiviral activities against respiratory syncytial virus (rsv) [ ] and influenza virus [ ] . ggt can reduce the mortality of influenza virus-infected mice [ ] . among its active molecules, uralsaponins from glycyrrhiza uralensis [ ] and procyanidin from cinnamomum cassia [ ] may effectively inhibit the replication of the influenza virus. allicin in ginger (zingiber officinale) and coumarin in cinnamomum cassia might have the activity to inhibit influenza neuraminidase [ ] . paeonol and , , , , -penta-o-galloyl-β-d-glucopyranose from paeonia lactiflora show antiviral activity against rhinovirus [ ] . of its anti-inflammatory effects, ggt can suppress the interleukin- α (il- α) production induced by interferons (ifn) in influenza [ ] . ggt was found to decrease cigarette smoking-(cs-) and lipopolysaccharide (lps)-induced elevated counts of inflammatory cells, and expression of inflammatory cytokines and proteins (il- , tnf-α, inos, and cox- ) [ ] . ggt can stimulate il- and ifn-β to counteract viral infection [ ] , and can also enhance the phagocytic activity of macrophages [ ] . among its active molecules, paeoniflorin, a major constituent of paeonia lactiflora, exerts anti-inflammatory and immunomodulatory effects by balancing the function of th /th [ ] . glycyrrhizin, a major constituent of glycyrrhiza uralensis, suppresses nuclear factor-kappa b (nf-κb) via the phosphoinositide -kinase (pi k) pathway, inhibits the production of nitric oxides (no), prostaglandin e (pge ), and reactive oxygen species (ros), and reduces the protein and mrna levels of inducible no synthase (inos) and cyclooxygenase- (cox- ) [ ]( figure ). meanwhile, glycyrrhiza polysaccharide, isolated from glycyrrhiza uralensis, significantly induces no production and inos transcription in peritoneal macrophages [ ] . however, this study design uses intraperitoneal injection to show the induction of no [ ] , instead of the traditional oral route. polysaccharides will be normally digested in the gastrointestinal tract into monosaccharide, so that polysaccharides can hardly reach intraperitoneal macrophages in the regular oral route, so this pharmacologic activity has been questioned. isoliquiritigenin, a flavonoid from glycyrrhiza uralensis, inhibits nf-κb activation to suppress inflammatory response [ ] . cinnamaldehyde, from cinnamomum cassia, inhibits the secretion of pge , il- β and tumor necrosis factor-α (tnf-α), and the activation of nf-κb to show the anti-inflammatory effect [ ] [ ] [ ] . particularly, e-cinnamaldehyde and o-methoxy-cinnamaldehyde down-regulate no and tnf-α production to show anti-inflammatory activity [ ] . although it can mediate antiviral activity, tnf-α plays only a minor role in clearance of various airway viruses; rather, it is the major contributor of t-cell-mediated lung injury [ ] . for enhancement of antiviral immunity, puerarin, an isoflavonoid from pueraria lobate, increased ifn-γ [ ] . -gingerol ( -g), the main bioactive component of ginger (zingiber officinale), increases ifn-γ and il- , but decreases il- and transforming growth factor-β (tgf-β ) levels [ ] . on the contrary, gingerol was also reported to suppress t cell response and inhibit ifn-γ synthesis [ ] (figure ). these conflicting data may come from different study designs and raise questions about the actual pharmacological activity in humans. (ros), and reduces the protein and mrna levels of inducible no synthase (inos) and cyclooxygenase- (cox- ) [ ] (figure ). meanwhile, glycyrrhiza polysaccharide, isolated from glycyrrhiza uralensis, significantly induces no production and inos transcription in peritoneal macrophages [ ] . however, this study design uses intraperitoneal injection to show the induction of no [ ] , instead of the traditional oral route. polysaccharides will be normally digested in the gastrointestinal tract into monosaccharide, so that polysaccharides can hardly reach intraperitoneal macrophages in the regular oral route, so this pharmacologic activity has been questioned. isoliquiritigenin, a flavonoid from glycyrrhiza uralensis, inhibits nf-κb activation to suppress inflammatory response [ ] . cinnamaldehyde, from cinnamomum cassia, inhibits the secretion of pge , il- β and tumor necrosis factor-α (tnf-α), and the activation of nf-κb to show the antiinflammatory effect [ ] [ ] [ ] . particularly, e-cinnamaldehyde and o-methoxy-cinnamaldehyde downregulate no and tnf-α production to show anti-inflammatory activity [ ] . although it can mediate antiviral activity, tnf-α plays only a minor role in clearance of various airway viruses; rather, it is the major contributor of t-cell-mediated lung injury [ ] . for enhancement of antiviral immunity, puerarin, an isoflavonoid from pueraria lobate, increased ifn-γ [ ] . -gingerol ( -g), the main bioactive component of ginger (zingiber officinale), increases ifn-γ and il- , but decreases il- and transforming growth factor-β (tgf-β ) levels [ ] . on the contrary, gingerol was also reported to suppress t cell response and inhibit ifn-γ synthesis [ ] (figure ). these conflicting data may come from different study designs and raise questions about the actual pharmacological activity in humans. ma-huang- tang (mht; table ) [ ] have been reported to be effective in the treatment of influenza, upper respiratory tract infection, acute and chronic bronchitis, and asthma [ , ] . the indication to use mht is patients with chills, fever without sweating, headache, shortness of breath, and joint pain. clinically, mht can effectively reduce fever and flu symptoms, including myalgia, headache, arthralgia, fatigue and cough, in patients with seasonal influenza type a [ ] . among its active molecules, l-ephedrine from ephedra sinica and amygdalin from prunus armeniacae, possess the antitussive effect [ ] , so co-administration of ephedra sinica and prunus armeniacae shows a better antitussive effect than use singly [ ] . mht clearly shows anti-inflammatory activity via suppressing the no/pge pathway [ ] , reducing inflammatory cells infiltration and reducing pro-inflammatory cytokine, including tnf-α, il- β, and il- , in lung experiments [ ] . in an acute bronchial asthma mice model, mht can also mitigate the pathological changes of acute asthma-like syndrome through inhibition of the toll-like receptor (tlr ) pathway [ ] . mht can modulate th /th cytokines via decreasing il- & il- and increasing ifn-γ levels. mht can inhibit th cells [ ] , and decreases il- , il- , tnf-α, cd +, cd + t cell levels (th response), but increases il- , ifn-γ, and cd + t cell levels (th response) to increase cd +/cd + ratio [ ] . among its active ingredients, ephedra sinica inhibits pge biosynthesis, reduces ige-mediated histamine release, reduces the mrna or protein levels of il- β, il- , tnf-α, cox , and nf-κb [ ] and inhibits complement activation of both classical and alternative pathways [ ] . additionally, ephedra sinica can directly activate both alpha-and beta-adrenergic receptors to reduce bronchial mucosal edema and to dilate the bronchus respectively [ , ] . to understand the molecular mechanism, several active molecules have been identified. ephedrannin a and b, from ephedra sinica, effectively suppressed the transcription of tnf-α, il- β, and nf-κb, and the phosphorylation of p mitogen-activated protein (map) kinase to exert their anti-inflammatory actions on lps-stimulated macrophages [ ] . glycyrrhiza uralensis and cinnamomum cassia contain active molecules mediating anti-inflammatory and immunomodulatory effects mentioned in the ggt section. for its antiviral activity, mht was initially thought to inhibit airway viruses through inducing antiviral ifn. however, herbacetin from ephedrine alkaloid-free extract of ephedra sinica might have anti-influenza activity similar to its extract containing ephedrine and pseudoephedrine [ ] . the study of ephedra sinica is relative rare owing to it containing ephedrine and pseudoephedrine, which are illegal in several countries. glycyrrhiza uralensis and cinnamomum cassia also have active antiviral constituents mentioned in the ggt section ( figure ). ma-xing-gan-shi- tang (mxgst; table ) [ ] , a similar formula to ma-huang-tang, is effective against influenza virus infection [ ] . mxgst has only one ingredient different from that of mht, i.e., using gypsum instead of cinnamomum cassia, so their pharmacologic effects and active molecules are similar, except that gypsum possesses a more powerful anti-pyretic effect by decreasing the pge level in the hypothalamus [ ] . co-treatment with ephedra sinica and gypsum can have synergistic effects to manage fever and asthma than single use [ ] . mxgst add-on therapy may improve pulmonary function indicies, such as forced expiratory volume in one second (fev ), forced vital capacity (fvc), and fev /fvc in patients with acute exacerbation of copd [ ] . additionally, at higher cumulative doses, mxgst might reduce the incidence of pneumonia and protect against admission [ ] . the indication to use mxgst is patients with fever, cough with yellow and sticky sputum, chest pain, and shortness of breath. mxgst has been found to have antitussive and anti-pyretic effects in an lps-induced hyperthermia rat model [ ] . mxgst has bronchodilation effect mediated by stimulation of β -adrenoceptors in pigs [ ] and can block acetyl-cholinergic and histaminergic receptor-induced bronchial contraction in rats [ ] , reduce neutrophilic inflammation [ ] , and in a copd rat model, can decrease il- , il- , and tnf-α, but increase ifn-γ [ ] . these effects may be beneficial to manage airway viral infections with cough. the molecular mechanism of mxgst is summarized (figure ). molecules , , x; doi: www.mdpi.com/journal/molecules ma-xing-gan-shi- tang (mxgst; table ) [ ] , a similar formula to ma-huang-tang, is effective against influenza virus infection [ ] . mxgst has only one ingredient different from that of mht, i.e., using gypsum instead of cinnamomum cassia, so their pharmacologic effects and active molecules are similar, except that gypsum possesses a more powerful anti-pyretic effect by decreasing the pge level in the hypothalamus [ ] . co-treatment with ephedra sinica and gypsum can have synergistic effects to manage fever and asthma than single use [ ] . mxgst add-on therapy may improve pulmonary function indicies, such as forced expiratory volume in one second (fev ), forced vital capacity (fvc), and fev /fvc in patients with acute exacerbation of copd [ ] . additionally, at higher cumulative doses, mxgst might reduce the incidence of pneumonia and protect against admission [ ] . the indication to use mxgst is patients with fever, cough with yellow and sticky sputum, chest pain, and shortness of breath. mxgst has been found to have antitussive and antipyretic effects in an lps-induced hyperthermia rat model [ ] . mxgst has bronchodilation effect mediated by stimulation of β -adrenoceptors in pigs [ ] and can block acetyl-cholinergic and histaminergic receptor-induced bronchial contraction in rats [ ] , reduce neutrophilic inflammation [ ] , and in a copd rat model, can decrease il- , il- , and tnf-α, but increase ifn-γ [ ] . these effects may be beneficial to manage airway viral infections with cough. the molecular mechanism of mxgst is summarized (figure ). xiao-qing-long- tang (xqlt; table ) [ ] is one of the most common prescriptions used against allergic rhinitis [ ] . xqlt, at higher cumulative doses, might reduce the incidence of pneumonia and protect against admission [ ] . xqlt with/without ma-xing-gan-shi-tang (mxgst) is the most frequently prescribed tcm formula for copd [ ] , and is also commonly used in the treatment of patients with respiratory diseases, such as common cold, flu, bronchitis, asthma, bronchiectasis, and emphysema. the indication to use xqlt is patients with cough, watery rhinorrhea or watery sputum, but without thirst. to manage airway viral infection, xqlt is effective against human respiratory syncytial virus (rsv) infection by preventing viral attachment, internalization, syncytial formation, and by stimulating ifn-β secretion [ ] , and has been proven beneficial against influenza virus in vivo through the augmentation of antiviral iga antibody [ , ] . xqlt can reduce the airway inflammation with the decrease of eosinophils count, the ovalbumin (ova)-specific immunoglobulin e (ige) antibody, and histamine release [ ] [ ] [ ] , can also modulate th /th balance thereby reducing il- and restoring ifn-γ levels [ , ] . among its active molecules, schisandrin a, a bioactive lignin of schisandra sphenanthera, inhibits the il β-induced inflammation via suppression of mitogen-activated protein kinase (mapk) and nf-κb signal pathways [ ] , and can also inhibit the nf-κb, mapk and pi k/akt pathways, partially mediated by the activation of the nuclear factor erythroid -related factor /heme oxygenase- (nrf /ho- ) pathway to manage inflammatory and oxidative disorders caused by over-activation of macrophages [ ] . schisandrin b, another bioactive lignin of schisandra sphenanthera, increases the expression of nrf and ho- and blocks the activation of nf-κb induced by lps to suppress the production of vascular cell adhesion molecule (vcam- ), intercellular adhesion molecule (icam- ), tnf-α, and il- expressions in human umbilical vein endothelial cells (huvecs) [ ] . α-cubebenoate, isolated from schisandra chinensis, can block the increase of il- β and il- during inflammation [ ] , and inhibit lps-induced expression of inos and cox- [ ] . oral polysaccharide from schisandra chinensis showed antitussive effect in a guinea pig model [ ] . the active molecules of common ingredients, including ephedra sinica, cinnamomum cassia, paeonia lactiflora, glycyrrhiza uralensis, and zingiber officinale, have several pharmacologic activities mentioned in the above sections. most of these activities aim at inhibiting inflammation induced by airway infection, however, lectin from pinellia ternate may activate macrophages, induce neutrophil migration, cytokine release, ros overproduction and the activation of the nf-κb signaling pathway to produce pro-inflammatory activity [ ] (figure ). therefore, interactions, including both synergistic and antagonistic between active molecules in a tcm formula could be so complex as to affect the final effects. from more than two thousand years ago in china and japan, ye-gan-ma-huang- tang (ygmht; table ) [ ] has been used to manage flu-like symptoms. a meta-analysis shows that ygmht can improve the total effective rate, fev , and asthma control test (act) score of refractory asthmatic patients [ ] . when combined with salbutamol aerosol, ygmht can obviously improve their pulmonary functions, including act score, pef, fev , and fev % predicted value. the indication to use ygmht is patients with chill and fever, hyperinflation of lung, cough with stridor and rales associated with frothy or whitish sputum [ ] . ygmht has been reported effective against enterovirus infection, including coxsackievirus [ ] and ev [ ] . it can regulate the serum levels of tnf-α, il- , and il- to show clinical effect in management of cough and variant asthmatic symptoms in children [ ] . additionally, a modified ygmht (also named san-long-ping-chuan-decoction; slpcd) can significantly inhibit airway inflammation, reduce inflammatory cells in bronchoalveolar lavage fluid (balf), and decrease the serum total ige levels [ ] . slpcd can significantly down-regulate the mrna expression levels of th cytokines (il- , il- , il- , and il- ) and up-regulate those of th cytokines (il- and ifn-γ) in lungs of asthmatic mice [ ] . for this anti-inflammatory activity, aster tataricus can protect from lps-induced acute lung injury mainly through inhibiting the release of inflammatory cells (wbc, macrophage, neutrophil, lymphocyte), regulating the pro-inflammatory cytokines (il- β, il- , tnf-α), and attenuating the pulmonary edema [ ] . among its active molecules, irigenin, a major active constituent of belamcanda chinensis, can reduce no and pge production by decreasing the mrna and protein expression of inos and cox- , respectively, as well as by suppressing nf-κb activation [ ] (figure ). molecules , , x; doi: www.mdpi.com/journal/molecules from more than two thousand years ago in china and japan, ye-gan-ma-huang- tang (ygmht; table ) [ ] has been used to manage flu-like symptoms. a meta-analysis shows that ygmht can improve the total effective rate, fev , and asthma control test (act) score of refractory asthmatic patients [ ] . when combined with salbutamol aerosol, ygmht can obviously improve their pulmonary functions, including act score, pef, fev , and fev % predicted value. the indication to use ygmht is patients with chill and fever, hyperinflation of lung, cough with stridor and rales associated with frothy or whitish sputum [ ] . ygmht has been reported effective against enterovirus infection, including coxsackievirus [ ] and ev [ ] . it can regulate the serum levels of tnf-α, il- , and il- to show clinical effect in management of cough and variant asthmatic symptoms in children [ ] . additionally, a modified ygmht (also named san-long-ping-chuan-decoction; slpcd) can significantly inhibit airway inflammation, reduce inflammatory cells in bronchoalveolar lavage fluid (balf), and decrease the serum total ige levels [ ] . slpcd can significantly down-regulate the mrna expression levels of th cytokines (il- , il- , il- , and il- ) and up-regulate those of th cytokines (il- and ifn-) in lungs of asthmatic mice [ ] . for this anti-inflammatory activity, aster tataricus can protect from lps-induced acute lung injury mainly through inhibiting the release of inflammatory cells (wbc, macrophage, neutrophil, lymphocyte), regulating the pro-inflammatory cytokines (il- β, il- , tnf-α), and attenuating the pulmonary edema [ ] . among its active molecules, irigenin, a major active constituent of belamcanda chinensis, can reduce no and pge production by decreasing the mrna and protein expression of inos and cox- , respectively, as well as by suppressing nf-κb activation [ ] (figure ). to unravel the molecular mechanism of tcm formulas, several issues need to be solved. many pharmacologic activities are obtained from in vitro and animal studies. could these activities be extrapolated into humans? there are so many active molecules with various pharmacologic activities in a tcm formula or herbs. are these molecules specific for that specific activity of a tcm formula or herbs? could they reach a minimal serum level to exert that particular pharmacologic activity in humans? active molecules may have the same activity with different potency. the most abundant one may not be the most important one for a particular activity. is it possible that there might be unidentified active molecules that actually account for a particular pharmacologic activity? several pharmacologic activities of a tcm formula cannot find a corresponding active molecule. is it possible that interactions between active molecules, e.g., synergism, but not active molecules themselves, account for a specific activity? is it possible that new active molecules are generated during preparation of the tcm formula? the genetic basis will affect the drug pharmacokinetics. is there a specific gene or single nucleotide polymorphism (snp) largely affecting the pharmacokinetic profile? does publication bias exist so that undesired pharmacologic effects are not published? is it possible that a particular gene is prone to a specific adr of a tcm formula or herbs? could a specific adr come from interactions between specific active molecules? several tcm formulas clearly show that some ingredients are not active in managing their traditional applications. could these inactive ingredients be omitted? all these questions need many studies to determine valid answers. at this moment, it would be better to use herbs and tcm formulas according to their traditional indications and the disease pathophysiology by matching their molecular mechanisms. as the world population has continued to age, the elderly have become associated with chronic diseases requiring multiple medications. increasing dissatisfaction with orthodox medicine and/or preference for alternative therapists and/or naturopathy has meant people continue to seek alternatives to maintain or improve their health, and this has spurred the growth of complementary and alternative medicine (cam) therapies. the validity of pharmacologic studies, safety issues, and validation of clinical effects of herbal medicine and tcm are limitations that should be highly considered; however, most clinicians are not familiar with herbal medicine and tcm so they do not recommend herbal therapies. more effort should be placed on unraveling the molecular mechanisms in order to solve the above issues. the reasons that limit clinicians from becoming more familiar with herbal medicine and for not recommending herbal therapies are explored below. herbal medicine and tcm form a part of complementary and alternative medicine (cam). however, evidence-based research in the field of cam therapies is still limited. there is a wrong perception that a naturally derived product is relatively safe. it is highly important to identify both usefulness and safety of cam and integrate these health approaches with orthodox medicine through rigorous scientific investigation to improve health care. it is also important for medical educators and providers to recognize the trend, the evidence, the benefits, and the risks of herbal medicine and tcm to educate clinicians for appropriate patient management and education. tcm studies published in chinese are usually not translated into english. the terminology of tcm is also difficult to translate, particularly those used by r-physicians, so medical education of herbal medicine and tcm has been neglected worldwide, except in germany and china. with their increasing use since the s, understanding the molecular mechanisms, benefits, and limitations by physicians has become increasingly important to monitor their benefits and harmfulness. most of the evidence supporting clinical claims of herbs and natural products come from studies of inadequate design that do not provide tough evidence of the effects. relatively few well-designed studies support their clinical claims. sometimes, adequately powered, double-blinded, placebo-controlled clinical trials come to conclusions against previous reports, such as echinacea for upper respiratory infection [ ] , or ginkgo for dementia or mild cognitive impairment [ ] . additionally, the amounts of most bioactive compounds in herbs and tcm formulas are very low. is it possible that the clinical effects of herbs and tcm formulas can be effective with such a small amount of bioactive compounds? one study discussed the clinical effect of ma-huang-tang (mht) against seasonal influenza [ ] . mht showed an equivalent clinical effect as neuraminidase inhibitors. however, not every tcm formula can provide such evidence. several tcm formulas, such as ma-xing-gan-shi-tang (mxgst), ge-gen-tang (ggt), and xiao-qing-long-tang (xqlt), are among the top ten most common tcm prescriptions for patients with upper respiratory tract infections (urtis) in taiwan [ ] . they are commonly used for urtis, but more research is required to validate their clinical effects and mechanisms. without tough clinical evidence and clear molecular mechanisms, physicians tend to avoid herbal therapies. most clinical claims of herbal therapies are based on bench studies that do not possess external validity to support their conclusions; for example, using animal-derived cancer cell lines to study antiviral effects in humans; using cancer cell lines to study physical changes; using intraperitoneal injection to study oral medications; and using high-dose pharmacological designs to study physiological responses, etc. therefore, there is still much space for improvement of our understanding of the mechanisms of herbal medicine. herbs are pharmacologically active and can positively or negatively affect patient's health. with increasing use of herbs as dietary supplements and alternative therapy, there is an increased risk of negative impacts, such as adverse effects and interactions. herbal medicine is among the most common causes of drug-induced liver injury (dili) [ ] . additionally, several adverse effects of commonly used herbs have been reviewed, including hypoglycemic or hyperglycemic effect, hypolipidemic or hyperlipidemic effect, hormonal activities, hypertensive, cardioactive [ ] , and hepatotoxic effects [ ] . some of them are serious even in recommended dosage, such as ephedra alkaloids (derived from ephedra sinica or ma huang) [ ] [ ] [ ] . however, most of the molecular mechanisms causing side effects are unknown. plants containing pyrrolizidine alkaloids can lead to hepatotoxicity and venoocclusive disease, possibly by the accumulation of toxic metabolites produced via the cytochrome system [ ] . some herbal treatments containing aristolochic acid (aa), including aristolochia fangchi, aristolochia debilis sieb. et zucc., aristolochia manshuriensis, aristolochia debilis, can cause aa nephropathy requiring renal replacement therapy [ , ] ; additionally, aa is associated with urothelial cancers [ ] . although most of the above issues have been identified, the unrecognized issues are just the tip of the iceberg. tcm formulas have numerous bioactive compounds to form a kind of cocktail therapy. however, the amounts of each bioactive compound in herbs and tcm formulas are very low. this may raise questions about the likelihood of their interactions with others during administration in a decoction. ginseng, a common natural product used among adults [ ] , has about ginsenosides as its major bioactive compounds [ ] , although the actual amount of each ginsenoside is small. after oral administration, ginsenosides are metabolized and transformed by intestinal microbiota. diet can markedly influence the transformation of ginsenosides. they exert pharmacologic effects in animals, and also show various clinical effects in randomized controlled trials, including several side effects and drug interactions [ ] , so small amounts of bioactive compounds do have clinical effects and side effects, which might come from the individual pharmacological activity of bioactive compounds or their synergism. the side effects may also come from individual unfavorable bioactivity or from interactions. additionally, herbal medicine and tcm therapy are commonly used in combination with orthodox medicine by patients, sometimes unknown to their doctors. the complex and unknown interactions, including herb-drug, herb-herb, herb-food, herb-microbiome, and herb-disease interactions, make use of herbal medicine more complicated and requiring frequent monitoring. the mechanisms of these interactions can be divided into molecular mimicry and pharmacologic interactions, such as pharmacodynamic and pharmacokinetic interactions. for example, with molecular mimicry, several herbs naturally has coumarin or salicylate analogue that may potentiate the bleeding risk of warfarin and salicylate, respectively. for pharmacodynamic interactions, ephedra sinica (ma huang) should not be used with sedative or anti-hypertensive agents. for pharmacokinetic interactions, st john's wort and several tcm formulas have been noted to affect the cyp system of liver, which may increase or decrease the effects of other drugs [ ] . additionally, the herb-drug interaction may be individualized, e.g., in combination with warfarin, panax ginseng may cause thrombotic event [ ] , bleeding [ , ] , or neither [ ] . currently, most of the molecular mechanisms of these identified interactions are not fully understood, to say nothing of the unrecognized interactions. quality uncertainty impacts the reproducibility of clinical efficacy and safety of commercially available natural products. variability of the quality of natural products may come from a lack of standardization of manufacturing, including misidentification of authenticity, inadequate processing, inadequate storage, adulteration of formulas, and accidental contamination [ ] . most of those quality problems can be gradually solved by following the who guidelines on good agriculture and collection practices for medicinal plants [ ] and botanical drug development guidance for industry of fda [ ] . several health benefits of herbal medicine and tcm are claimed; for example, herbs and tcm formulas, including those discussed above, are believed to have anti-oxidative activities helpful against several diseases. this idea is based on reactive oxygen and nitrosative species (ros/rns) as metabolic byproducts that can cause damage to cellular macromolecules; thus, many diseases can be triggered by oxidative stress under high levels of ros/rns. these diseases include cancer, inflammation, and degenerative diseases. oxidative stress causes damage either with an overwhelming production of ros/rns or under insufficient levels of antioxidants or repair mechanisms, so blocking the generation of ros/rns might prevent and/or manage these diseases [ ] . however, ros/rns are also signaling molecules for several physiological functions, including regulation of vascular tone, control of ventilation, and erythropoietin production, etc. actually, ros-mediated responses may protect against oxidative stress [ ] . additionally, ros/rns may play a dual role in different diseases, i.e., ros/rns might contribute to, or counteract, the disease progression. it remains unclear that more dosage of antioxidants is not better and may even worsen a medical condition [ ] . there are insufficient data to establish the ability of tcm to decrease ros/rns levels and establish its effects on the disease, and this affects the interpretation of any claims of benefit. to validate such claims of the benefits of herbal medicine and tcm, much work remains to be done. only when these limitations can be minimized, can the molecular mechanisms of herbs and tcm formulas be understood. consequently, clinicians can help patients by giving adequate prescriptions and suggestions to minimize the harmfulness and maximize the benefits to healthcare. herbal medicine, including tcm, is commonly used worldwide. herbal remedies contain numerous active molecules to form a kind of cocktail therapy. these active molecules may interact with each other to affect the therapeutic effects and produce side effects. understanding their pharmacological effects, interactions, side effects, clinical effects, and the underlying molecular mechanisms is very important to provide benefits, but avoid harm, to the patient. to unravel the molecular mechanisms, much work remains to be done. the abc clinical guide to herbs trends in alternative medicine use in the united states, - : results of a follow-up national survey trends in the use of complementary health approaches among adults: united states complementary and alternative medicine use among adults and children: united states current state of phytotherapy 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(japanese herbal) medicine "sho-seiryu-to (xiao-qing-long-tang)" on airway inflammation in a mouse model schisandrin a inhibits the il- beta-induced inflammation and cartilage degradation via suppression of mapk and nf-kappab signal pathways in rat chondrocytes schisandrin a suppresses lipopolysaccharide-induced inflammation and oxidative stress in raw . macrophages by suppressing the nf-kappab, mapks and pi k/akt pathways and activating nrf /ho- signaling schisandrin b inhibits lps-induced inflammatory response in human umbilical vein endothelial cells by activating nrf anti-septic activity of alpha-cubebenoate isolated from schisandra chinensis identification of a novel anti-inflammatory compound, alpha-cubebenoate from schisandra chinensis antitussive activity of the schisandra chinensis fruit polysaccharide (scfp- ) in guinea pigs models pinellia ternata lectin exerts a pro-inflammatory effect on macrophages by inducing the release of pro-inflammatory cytokines, the activation of the nuclear factor-kappab signaling pathway and the overproduction of reactive oxygen species meta-analysis on shegan mahuang tang for refractory asthma clinical observation on therapeutic effect of traditional chinese medicine granules made by formula of shegan mahuang decoction for patients with asthma yakammaoto inhibited human coxsackievirus b (cvb )-induced airway and renal tubular injuries by preventing viral attachment, internalization, and replication yakammaoto inhibits enterovirus infection by reducing viral attachment, internalization, replication, and translation effect of modified shegan mahuang decoction on cytokines in children patients with cough and variant asthma antiasthmatic effects of sanglong pingchuan decoction through inducing a balanced th /th immune response network pharmacology-based investigation of protective mechanism of aster tataricus on lipopolysaccharide-induced acute lung injury inhibitory effects of irigenin from the rhizomes of belamcanda chinensis 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of herbal remedies rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including chinese herbs chinese herbs nephropathy: a clue to balkan endemic nephropathy? kidney int aristolochic acid as a probable human cancer hazard in herbal remedies: a review panax ginseng and panax quinquefolius: from pharmacology to toxicology progress in cytochrome p enzyme in toxicity of traditional chinese medicines. chin the state pharmacopoeia commission of the people's republic of china. pharmacopoeia of the people's republic of china who guidelines on good agricultural and collection practices (gacp) for medicinal plants drug development guidance for industry positive or negative actors? biomolecules free radicals in the physiological control of cell function the authors would like to thank teachers of the center for languages and culture of kaohsiung medical university for the technical support. the authors declare no conflict of interest. key: cord- -zfpcuhpy authors: doménech, ana; miró, guadalupe; collado, victorio m.; ballesteros, natalia; sanjosé, leticia; escolar, elena; martin, sonsoles; gomez-lucia, esperanza title: use of recombinant interferon omega in feline retrovirosis: from theory to practice date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: zfpcuhpy type-i interferons (ifns) are cytokines that have non-specific antiviral activity, participating mostly in innate defense mechanisms. their administration has been proposed to treat several viral and immunomediated diseases as an immunomodulatory therapy. due to its availability, recombinant human interferon-alpha (rhuifn-α) has been studied in relation to feline retrovirosis, both in vitro and in vivo. however, ifns are species-specific and antibodies have been shown to develop in response to the high rhuifn-α doses necessary for an effective therapy. a recombinant feline ifn has been developed, which has been characterized as interferon-omega (rfeifn-ω), designed to overcome these problems. nonetheless, very few studies have been undertaken to evaluate its efficacy in cats naturally infected with fiv or felv. in an initial study, we here demonstrated that rfeifn-ω can dramatically improve the clinical condition of infected cats, and induce improvement of hematologic parameters. minor changes or no change was observed for hypergammaglobulinemia, cd /cd ratio, proviral load, viremia and rt activity, suggesting that the overall effect of ifn was on innate immunity. more studies are needed in order to better understand its in vivo mechanisms. feline retroviruses, notably feline leukemia virus (felv) and feline immunodeficiency virus (fiv), induce chronic infections which eventually lead to the progressive weakening of cats and the presence of various clinical signs. in advanced stages of the disease, the immune suppression established may contribute to the death of the animal (review in hartmann, ; sellon and hartmann, ) . treatment of animal retroviral diseases is usually based on supportive and symptomatic therapy, such as rehydration when needed, control of secondary infections with antibiotics, antiparasitic and antifungal drugs, among others, while the administration of antiviral drugs is uncommon (hartmann, ; sellon and hartmann, ; dunham and graham, ) . most of the antivirals used for feline retrovirosis are the same as used in human medicine, including azt (zidovudine), ribavirin, zalcitabine and foscarnet, singularly or in combination. the use of these drugs in cats has several disadvantages: doses and protocols are not well established, and they can be toxic for animals as well as producing secondary effects (caney, ; hartmann, ; sellon and hartmann, ; dunham and graham, ) . as these infections are accompanied by a wide array of clinical signs (anemia, gingivitis, anorexia, secondary respiratory infections, tumors, etc.), there is no preferred curative treatment. thus, a reasonable - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.vetimm. . . alternative for treatment is the use of immunomodulators, particularly type-i interferons that have an additional antiviral effect. the aim of the present work was both to review the literature on the possibility of using interferonomega (ifn-) for treating feline retrovirosis, and to describe the results of a preliminary study conducted on cats infected either by felv or fiv, observing clinical, biopathological and virological effects. innate immunity plays a role in protection against retroviral infections (lehner et al., ) , and includes both intracellular innate antiviral factors, and extracellular factors, particularly interferon. interferons (ifns) are cytokines with important multiple biological functions. interferons are classified into type-i and type-ii ifns (pestka et al., ) . type-i ifns are produced by virusinfected cells and have non-specific antiviral activity on adjacent non-infected cells. thus, they are known as "viral ifns" and are associated with innate immunity. these interferons include ifn-␣, ifn-␤, and ifn-, among others. each them has a general mechanism of action based on interaction with specific cell surface receptors and the subsequent induction of expression of interferon-stimulated genes (sen, ; pestka et al., ) . these cytokines also induce anti-proliferative and anti-inflammatory responses, and therefore, can also participate in adaptive immune responses (gerlach et al., (gerlach et al., , . thus the administration of type-i ifn has been proposed as a treatment for several viral diseases as immunomodulatory therapy (truyen et al., ; collado et al., ) . these products have been used empirically and quite successfully in feline medicine, without a profound knowledge of their molecular mechanisms. the efficacy of human ifn-␣ (huifn-␣) in the feline clinic was the first to be evaluated, as it was the first one commercially available (recombinant huifn-␣( a), rhuifn-␣( a), roferon ® ), as well as being the one with the highest in vitro antiviral effect. even though clinical improvement and lengthening of the life expectancy of infected cats was observed, several disadvantages soon became apparent, such as: its activity in vivo may be lower than expected as cytokine activity is often species-restricted (it would have less effect on feline cells than on human cells); when injected, the higher doses ( , u/kg/day) that are able to induce adequate serum levels may lead to the development of specific neutralizing antibodies that block the active ingredient müller, ) and may have adverse effects (caney, ) . these disadvantages could be overcome with the administration of a species-specific feline ifn (feifn). several subtypes of recombinant feifn-␣ (rfeifn-␣) have been described that could have potential benefits for treating chronic viral infections in cats, given their in vitro antiviral activity (wonderling et al., ) . however, to date no rfeifn-␣ is available for clinical use, although a recombinant feline interferon omega (rfeifn-) is commercially available and used with relative success in feline viral infections of various etiologies. ifn-, a type-i ifn secreted by virus infected leukocytes, was identified by hauptmann and swetly ( ) and is one of the more recently characterized interferons (adolf, ) . it was initially described in humans and is encoded by multiple ifn-or ifn--like genes, which are present across mammalian groups, including cats (roberts et al., ) . like other type-i ifns, ifn-has speciesrestricted biological activity in vitro. it is able to bind to the same type-i ifn receptor complex as other type-i ifns and therefore exerts similar antiviral, antiproliferative and immunomodulatory effects (adolf, ) . however, its antigenic structure is distantly related to ifn-␣, -␤ and -␥, as it does not cross-react with antibodies against them (adolf, ) . the rfeifn-that has been developed (nakamura et al., ; ueda et al., a) has a - % homology to human ifn-␣ . its amino acid sequence consists of amino acid residues and an n-glycosylation site at amino acid position . this recombinant ifn has been characterized as omega on the basis of its amino acid identity and the processing pattern of the n-terminal sequence (ueda et al., a; adolf, ) . it has a proven antiviral effect, both in vitro (mochizuki et al., ; truyen et al., ; ohe et al., ) and in vivo against canine and feline parvovirus, herpesvirus, calicivirus, coronavirus and rotavirus (truyen et al., ; de mari et al., ; ishida et al., ; paltrinieri et al., ) . in addition, the pharmacokinetic properties of rfeifn-are comparable to those of human interferons in that it does not have a residual accumulation in the body (ueda et al., b) . it has been licensed for use in veterinary medicine (virbagen ® , virbac) in europe, japan, australia, new zealand and mexico, although there are few clinical studies that support its use, and its in vivo molecular mechanisms are not well understood. in general, an improvement in clinical signs has been described, justifying its use in infections in which other treatments are not fully effective, such as retroviral diseases. the in vitro effects of ifn-i on human retroviruses have been extensively studied. generally, results from most studies have shown that retroviral protein synthesis is not affected, and suggest that ifn affects the latter stages of the viral cycle, preventing correct assembly or the release of viral particles (review in gómez-lucía et al., ) . unfortunately, few studies have focused on the effect of this cytokine on feline retroviruses (rogers et al., ; jameson and essex, ; yamamoto et al., ; tanabe and yamamoto, ; collado et al., ) . in reviewing the literature, only collado et al. ( ) have compared the effect of rhuifn-␣( a) (roferon ® ) and rfeifn-(virbagen ® ) on persistently felv-infected feline cells. their results have indicated that, as with other retroviruses, rfeifn-affects the felv cycle at the posttranscriptional level, as protein synthesis was not altered, while rt activity (used to estimate the number of infectious particles) decreased in a dose-dependent manner. given its ic , rfeifn-appeared to be around - times more potent than rehuifn-␣( a) in inhibiting rt. in addition, the study revealed that ifns induced a decrease in the viability of felv-infected cells, enhancing apoptosis in infected cells that were treated. the significance of this effect on cell viability and its in vivo impact is, at present, unknown. in the case of fiv, similar experiments with rhuifn-␣( a) and rfeifn-in infected feline cells have shown comparable results to that of felv (tanabe and yamamoto, ; collado, unpublished data) . the ic of rfeifnsuggests that it is ca. times more effective than rhuifn-␣( a) in inhibiting fiv rt (collado, unpublished data) . collectively, results from treatment with rhuifn-␣( a) and rfeifn-of felv-or fiv-infected cells have demonstrated that these ifns apparently do not inhibit feline retrovirus gene expression, but suppress the processing or assembly of viral proteins and/or the release of virions in the late stages of maturation. in summary, a more profound antiviral effect of rfeifnover rhuifn-␣( a) was observed in vitro against felv and fiv, which, in part, may be due to the species-specificity of type-i interferon and the homologous nature of rfeifnand feline cell lines. it is difficult to predict the possible clinical applications of interferons based on in vitro studies due to the variety of physiological conditions present in vivo and the myriad of individual responses by treated animals. nevertheless, the results of the in vitro studies seem to suggest that, although both rfeifn-or rhuifn-␣( a) may be of prophylactic and therapeutic clinical value, rfeifn-would likely be more effective in vivo. additionally, type-i ifns have immune modulating properties concurrent with antiviral activity (which are not measured in vitro) as proposed by other researchers (gerlach et al., ) , and thus an enhanced effect of rfeifn-may be expected in vivo. due to the therapeutic possibilities of interferon against feline retrovirosis, several studies have been undertaken with different type-i ifns and protocols, although results, to date, are inconclusive. in initial experiments, rhuifn-␣ was injected into felv-infected cats, singularly or in combination with azt zeidner et al., ) , and viral antigenic load (antigenemia) was observed to decrease. however, this beneficial response had a limited duration as cats developed antibodies against human ifn . to reduce the development of antibodies, lower doses of ifn were administered orally ( - iu/kg body weight), resulting in longer survival rates during some studies, with clinical and analytical improvement noted (steed, ; cummins et al., ; weiss et al., ) ; in others although there was no improvement (mccaw et al., ) . similarly, oral treatment of clinically sick fiv-infected cats with low doses of natural huifn-␣ resulted in an extended survival period and improved clinical signs (pedretti et al., ) . in spite of the clinical benefits of huifn-␣ treatment, felv-and fiv-infected cats became persistently viremic or had a reduced viral replication only during the treatment period (mccaw et al., ; pedretti et al., ) . in addition, low oral doses of ifn-␣ were ineffective with regard to lymphocyte depletion, with no relevant variations in the cd /cd ratio during treatment in naturally-infected felv cats (riondato et al., ) . these low doses even failed to maintain the balance between cd + and cd + cells in fiv-infected cats (riondato et al., ; pedretti et al., ) . moreover, the altered cd /cd ratio had no evident correlation with clinical condition (pedretti et al., ) . however, a correlation was found between clinical condition and leukocyte counts, which suggested that huifn-␣ treated cats undergo a strength-ened innate immune response with better control against opportunistic infections (pedretti et al., ) . after the synthesis and commercialization of the recombinant feline ifn-molecule, most studies in feline medicine have focused on its application. presently, it is the only interferon that the european medicine agency (http://emea.europa.eu) has registered in europe for feline medicine. it has been approved for the treatment of parvovirosis in dogs from one month of age, and felv and/or fiv infections in non-terminal cats from the age of weeks (http://emea.europa.eu). yet very few studies have analyzed the effect of rfeifn-in naturally-infected cats with felv and/or fiv. one of the most extensive studies undertaken evaluated this interferon in a multicentric, double-blind, placebo-controlled trial in felv-infected or felv/fiv co-infected cats with associated clinical signs (de mari et al., ) . cats were treated subcutaneously with rfeifn-( u/kg/day) daily for five consecutive days in three series (day , , ). an improvement in clinical signs and decrease in the mortality rate was observed after treatment as compared to control animals. there was also an increase in the leukogram and in the rbc count in anemic cats (de mari et al., ) . however, as no virological (viremia or proviral loads) or immunological (such as cd /cd ratio) parameters were measured throughout the study, it is difficult to conclude whether or not these effects were due to its immunomodulatory or antiviral activities. since ifn-has biological properties similar to ifn-␣, it is possible that the clinical benefits observed in treated cats corresponded to the activation of their innate immune system for an improved control of environmental pathogens, as compared with an improvement in their viremic state. further studies are necessary in order to discover answers to these questions. only one controlled clinical trial has been performed in asymptomatic fiv-infected cats, using both low ( u/cat/day orally for six weeks) and high ( u/kg/day by subcutaneous injection for five days) doses of rfeifn-. no improvements in laboratory parameters such as proviral load, cd + t-cell counts and total white blood cell counts (wbcs) were observed (caney et al., ) . to our knowledge, no clinical studies have been performed on the effect of this interferon on symptomatic fiv-infected cats. veterinary practitioners who treat felv-and/or fivinfected cats usually do not know when the cat was infected, or the stage of the disease. cats may have one or more clinical signs, or may be totally asymptomatic. practitioners may not know the immune status of the animal, its cd /cd ratio or the proviral load. thus, treatment with rfeifn-may produce different results than expected, ranging from no visible effects to an apparent clinical improvement. a big concern for the clinical veterinarian is whether this improvement correlates with a true improvement of the infection, i.e., a decrease in viremia or the proviral load of the cat, or whether the infection may revert when treatment of rfeifn-is suspended. treatment is also hampered by the cost of the product. the company recommends three cycles of treatment for five consecutive days, on days , and . as there is usually clinical improvement, oftentimes, owners consider that the cat has been cured, and suspend treatment before its completion. another issue for practitioners is deciding when treatment should be repeated, when no analytical values are available. for these reasons, we undertook a preliminary study on the use of rfeifn-in randomly selected household cats that were brought to the complutense university veterinary clinic hospital of madrid, spain. eleven naturally-infected cats were treated (four infected by felv, felv + ; seven infected by fiv, fiv + ), with ages ranging between months and years, including both male ( / ) and female ( / ), most of them ( / ) of mixed breed, and with different regimes of roaming. they were treated for eight weeks with commercial rfeifn-(virbagen ® , virbac, france) following the protocol suggested by de mari et al. ( ) . ten other cats (five felv + and five fiv + ), eight of them female, and housed in a cattery, were left untreated and used as controls. the date of infection was unknown in all cases; presumably, the cats were in different stages of the disease, since they included three asymptomatic fiv + cats, and ten cats ( felv + and fiv + ) with six or more different retrovirus-associated clinical signs (racs) of various degrees of severity (clinical score ≥ , severe disease). the most frequent disorders were anorexia, apathy, pallor and oral lesions. blood samples were taken before the beginning of treatment (v ), and two weeks after completion (v ), with equivalent sampling dates in untreated cats. three treated cats were brought for follow-up examination until month (v ). blood sampling, hemogram, leukogram, biochemical profile, electrophoretogram, and cd /cd determination were conducted as described previously (miro et al., ) . felv p and fiv p proteins were evaluated using commercial petchek kits developed by idexx (westbrook, me, usa), and rt activity was measured with kits provided by cavidi tech (uppsala, sweden). quantitative pcr (qpcr) was conducted using the procedures described by pinches et al. ( ) and leutenegger et al. ( ) . results were statistically analyzed using statgraphics. differences in the anova and chi-square values of p < . were considered significant. animal handling, treatment, reagent manipulations and data collection were all carried out in compliance with guidelines for good clinical practice, and good laboratory practice of the animal welfare committee of the veterinary clinical hospital and the complutense university (madrid), and the experimental procedures were approved by the institutional animal care and use committee of the complutense university. after treatment with rfeifn-(at v and for the subsequent months), an evident clinical improvement was observed in all sick treated cats, especially those with a clinical score (cs) of ≥ (table ) . such an improvement was not observed in untreated cats (p < . ). asymptomatic cats or those with mild disease remained stable. this clinical improvement agrees with data from de mari et al. ( ) and from studies with rhuifn-␣ (pedretti et al., ) . one treated and one untreated-felv + cat, each with table changes in the clinical score (cs) and analytical parameters between v (immediately before treatment) and v (two weeks after treatment). n-n n-n n-n n-n n-n n-n ↑-↑ (u) felv n-n n-n n-n n-n n-n n-n ↑-n (f) fiv n-n n-n n-n n-n n-n n-↓ (u) fiv n-n n-n n-n n-n n-n n-n ↑-↑ (u) fiv n-n n-n n-n n-n n-n n-n ↑-n (f) neg. = (u) c- fiv n-n n-n n-n n-n n-n n-n ↑-n (f) n-n n-n neg. neg. ↑ (u) c- fiv n-n n-n n-n n-n n-n n-↑ the cs was rated as described previously (collado et al., submitted for publication) . for the erythrogram, leukogram, electrophoretogram, and cd /cd ratio the situation in v and v are provided as follows: n, within normal limits; ↓, below normal limits; ↑, above normal limits. for the viral parameters the change from v to v is provided as follows: ↓, ≥ % decrease; ↑, ≥ % increase; =, stable. (f), favorable change; (u) unfavorable change. pcv, packed cell volume; hgb, hemoglobin concentration; rbc, erythrocyte counts; leuko, leukocyte counts; ntr, neutrophil counts; lymph, lymphocyte counts; electroph., electrophoretogram; ␥-s, gammaglobulin concentration; nd, not determined; neg., below the detection limit of the kit. very severe initial disease (cs = and cs = , respectively) died between v and v . treated cats that initially presented erythrogram and leukogram values outside the norm showed improvement by v . this agrees with previous reports that ifn was effective on anemic cats (de mari et al., ) . however, one treated fiv + cat had developed lymphocytosis, and another, lymphopenia by v (table ). the three cats that were examined at v did not have a worse clinical or hematologic condition (data not shown). most untreated cats initially had normal erythrogram and leukogram values. however, in three non-treated cats there was an unfavorable change in several hematologic parameters, although the values of neutrophil counts improved sporadically in two other non-treated cats. initially, cats had hypergammaglobulinemia, with a low a/g ratio in all felv + cats and in two fiv + cats. a significant decrease in gammaglobulin concentrations (p < . ) was observed at v in five treated cats ( fiv+ and felv+), but it was not sustained as cats followed till v were again hypergammaglobulinemic shortly after the v period (data not shown). gammaglobulin levels in untreated cats in which this parameter had initially been abnormally high always remained above normal limits. in the rfeifn-treated cats, the cd /cd ratio decreased in one felv + cat and in two fiv + cats. an improvement in the ratio was observed in two of them, but in another three, the cd /cd ratio did not increase or decrease (table ) . riondato et al. ( ) observed no correlation between cd /cd ratio variations and clinical signs, as sick and healthy cats had very similar ratios, which agrees with our results. proviral load was detected in all animals. only one of the treated fiv + cats decreased its initial proviral load by v , but was still within the detection limits. no treated or untreated cat became negative. in one treated-felv + and one treated-fiv + cat the proviral load increased, though they did not display worsened clinical condition. as regards to viremia, only felv + cats were positive for rt and capsid protein. in these cats, the response to interferon was variable (table ) . one of the treated felv + cats worsened in all the viral parameters, but its cs decreased. caney et al. ( ) did not observe an effect of rfeifn-on the proviral load. to date, the in vivo mechanism of type-i ifns on retroviral infections is unknown. in a murine model with the friend murine leukemia retrovirus, the immunomodulatory effect of ifn-␣ seemed to be more important than the antiviral effect through the induction of antiviral enzymes (gerlach et al., ). in our preliminary study, rfeifninduced an evident improvement of the clinical condition of treated cats, as well as of the hemogram parameters. the cd /cd ratio improvement was inconsistent, and the virological parameters, such as proviral load and viremia were not seen to be affected by treatment. this suggests that rfeifn-possibly lacks antiviral effects in vivo, but rather may have an immunomodulatory effect. taking these results into consideration, it would seem advisable to treat retrovirus-infected cats with interferon when they have clinical signs, as they would benefit from its effects in improving their clinical 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immunity in cats with spontaneous parvovirus infection: consequences of recombinant feline interferonadministration low-dose interferon-␣ treatment for feline immunodeficiency virus infection interferons, interferon-like cytokines, and their receptors diagnosis of feline leukaemia virus infection by semiquantitative real-time polymerase reaction effects of interferon-alpha (ifn-␣) therapy on peripheral blood lymphocyte subsets from fiv and fev naturally infected cats the evolution of the type i interferons cat interferon inhibits feline leukaemia virus infection in cell culture feline immunodeficiency virus infection viruses and interferons improved survival of four cats infected with feline leukemia virus after oral administration of interferon feline immunodeficiency virus lacks sensitivity to the antiviral activity of feline ifn-␥ a study of the antiviral activity of interferon-omega (ifn-) against selected canine and feline viruses homogeneous production of feline interferon in silkworm by replacing single amino acid code in signal peptide region in recombinant baculovirus and characterization of the product pharmacokinetic properties of recombinant feline interferon and its stimulatory effect on , -oligoadenylate synthetase activity in the cat low-dose orally administered alpha interferon treatment for feline leukemia virus infection cloning, expression, purification, and biological activity of five feline type i interferons a feline retrovirus induced t-lymphoblastoid cell-line that produces an atypical ␣ type of interferon alpha interferon ( b) in combination with zidovudine for the treatment of presymptomatic feline leukaemia virus-induced immunodeficiency syndrome the authors are indebted to david bruhn (usa) for his experienced editorial assistance. this work was supported with spanish agl - /gan, and bsch-ucm gr / (ucm ). v.m. collado is grantee of the spanish ministry of science and technology. all authors declare no financial or commercial conflict of interest. key: cord- -or in m authors: nguyen, khue g.; vrabel, maura r.; mantooth, siena m.; hopkins, jared j.; wagner, ethan s.; gabaldon, taylor a.; zaharoff, david a. title: localized interleukin- for cancer immunotherapy date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: or in m interleukin- (il- ) is a potent, pro-inflammatory type cytokine that has long been studied as a potential immunotherapy for cancer. unfortunately, il- 's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. early clinical trials in the mid- 's showed that systemic delivery of il- incurred dose-limiting toxicities. nevertheless, il- 's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. the development of strategies which maximize il- delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. diverse il- delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. several localized il- delivery approaches have recently reached the clinical stage with several more at the precipice of translation. taken together, localized delivery systems are supporting an il- renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. this review begins with a brief historical account of cytokine monotherapies and describes how il- went from promising new cure to ostracized black sheep following multiple on-study deaths. the bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for il- -based cancer immunotherapies. advantages and limitations of different delivery technologies are highlighted. finally, perspectives on how il- -based immunotherapies may be utilized for widespread clinical application in the very near future are offered. interleukin- (il- ) is a potent, pro-inflammatory type cytokine that has long been studied as a potential immunotherapy for cancer. unfortunately, il- 's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. early clinical trials in the mid- 's showed that systemic delivery of il- incurred dose-limiting toxicities. nevertheless, il- 's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. the development of strategies which maximize il- delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. diverse il- delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. several localized il- delivery approaches have recently reached the clinical stage with several more at the precipice of translation. taken together, localized delivery systems are supporting an il- renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. this review begins with a brief historical account of cytokine monotherapies and describes how il- went from promising new cure to ostracized black sheep following multiple on-study deaths. the bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for il- -based cancer immunotherapies. advantages and limitations of different delivery technologies are highlighted. finally, perspectives on how il- -based immunotherapies may be utilized for widespread clinical application in the very near future are offered. keywords: interleukin- (il- ), cancer immunotherapy, cancer vaccine, cytokine delivery system, localized delivery, intratumoral administration overview of il- -based immunotherapies a brief history of cytokine and il- immunotherapies since the discovery of an "endogenous pyrogen, " now known as il- , in , scientists have anticipated the use of exogenous cytokines to manipulate a patient's immune system in an effort to control malignant neoplasms ( ) . early obstacles to cytokine-based immunotherapy centered on difficulties achieving reproducible manufacture of a sufficient and pure supply of cytokines for clinical trials. in the early s, recombinant dna technology and advances in the biochemical characterization of proteins combined to overcome this hurdle. finally, in , ifn-α broke through as the first cytokine to win fda approval as a single agent cytokine therapy for cancer ( ) . since then, hundreds, if not thousands, of studies have evaluated more than cytokines against a range of preclinical tumor models. a number of promising cytokines, including gm-csf, il- , tnfα, ifn-γ, and il- , subsequently entered clinical trials as single agents but failed to provide clinical benefit. currently, only of + identified cytokines are approved as single agent immunotherapies for a limited number of indications ( table ) . another fda approved cancer immunotherapy, talimogene laherparepvec (t-vec; imlygic tm ) is an oncolytic herpes simplex virus that uses gm-csf expression as an immune enhancer ( ) . perhaps the greatest disappointment in cytokine immunotherapy development thus far is il- . il- is a potent, pro-inflammatory cytokine produced by antigen presenting cells typically in response to microbial pathogens. it is comprised of two subunits, p and p , that are linked by three disulfide bridges to form a p heterodimer ( ) ( ) ( ) ( ) . il- is chiefly responsible for the induction and enhancement of cell-mediated immunity. among its diverse functions, il- has been shown to: (i) induce t h cell differentiation; (ii) increase activation and cytotoxic capacities of t and nk cells; and (iii) inhibit or reprogram immunosuppressive cells, such as tumor associated macrophages (tams) and myeloid-derived suppressor cells (mdscs) ( ) ( ) ( ) ( ) ( ) ( ) . il- also induces the production of large amounts of ifnγ which itself is cytostatic/cytotoxic ( , ) , anti-angiogenic ( , ) and can upregulate mhc i and ii expression on tumor cells for enhanced recognition and lysis ( ) . not surprisingly then, il- has demonstrated remarkable antitumor effects against a range of malignancies in preclinical studies ( ) ( ) ( ) ( ) . these effects are largely dependent on cd + t cells, nk cells, and nk t cells ( , , ) . in clinical studies, il- has been evaluated as an experimental treatment for numerous malignancies ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . unfortunately, the efficacy of il- at tolerated doses has been minimal ( , , ) . atkins and colleagues were the first to employ il- immunotherapy in a clinical trial ( ) . this phase i study enrolled patients, including with renal cancer and with melanoma, to investigate intravenous administration of recombinant hil- (rhil- ). one melanoma patient experienced a transient complete response and one renal cancer patient had a partial response ( ) . subcutaneous rhil- was employed in a separate pilot study that enrolled advanced melanoma patients ( ) . in this study, a fixed dose of rhil- ( . µg/kg) was given to patients on days , , and for two sequential cycles of days. no partial or complete responses were reported. minor tumor shrinkages involving some subcutaneous metastases and hepatic metastases were observed ( ) . in yet another early melanoma study, the administration of il- was found to induce a striking peripheral burst of hla-restricted ctl precursors directed to autologous tumors and to multiple immunogenic tumor-associated antigens ( ) . significantly, the infiltration of cd + t cells with an effector-memory phenotype was identified in posttreatment metastatic lesions, but not in pretreatment metastatic lesions of three patients ( ) . il- has also been shown to induce productive antitumor responses against cutaneous t cell lymphoma variants ( ) , aids-related kaposi sarcoma ( ) , and non-hodgkin's lymphoma ( ) . although il- has demonstrated robust antitumor activity in preclinical studies and potent immune-stimulating potential in humans, systemic administrations of il- have been shown to be exceedingly toxic. in one phase ii trial, a maximal dose of . µg/kg per day resulted in severe side effects in out of enrolled patients and the deaths of two patients ( ) . interestingly, the same dose of . µg/kg il- per day was found to be well-tolerated in patients that were enrolled in a previous phase i study. a change in dosing schedule accounted for the differences in toxicity between the phase i and phase ii trials. in the phase i trial, a single tester dose of il- was administered week before a multiple-dose regimen. the tester dose was found to blunt the toxicity induced by subsequent doses ( ) . overall, severe toxicities in early clinical trials, including on-study deaths ( ) due to frequent systemic injections of il- , together with disappointing clinical responses in large phase studies ( , ) , dampened enthusiasm for il- -based immunotherapy. the disappointing antitumor responses in clinical trials raised the possibility that il- is simply less active in humans. however, the severe toxicities outlined above indicate that il- has potent biological activity in humans. another possibility for the limited clinical efficacy is insufficient delivery of il- to the tumor microenvironment in humans. il- , like most cytokines, functions locally through paracrine and autocrine mechanisms. the ideal targets of il- immunotherapy are not lymphocytes in circulation, but rather immune cells within the tumor and nearby lymph nodes, including activated but exhausted t cells, nk cells, tams, and mdscs. therefore, maximizing the amount of il- that reaches the tumor seems critical for a robust antitumor response. we and others have noted that il- immunotherapeutics would be more effective and less toxic if delivered and maintained in the tumor through the use of novel delivery technologies. there are five benefits of local, persistent il- delivery. the first is enhanced spatiotemporal distribution of il- compared to systemic delivery. the failure of il- -based immunotherapies to achieve widespread clinical success may be at least partially attributed to the inability of a tolerated dose of systemically administered il- to reach therapeutic concentrations within human tumors. in mice, implanted or induced tumors are disproportionately large. for example, a . -g tumor (∼ . cm ) comprises % of the body weight of a -g mouse. a comparably sized tumor in a kg ( lbs) human would weigh . kg ( . lbs). furthermore, rapidly growing murine tumors are highly vascularized relative to their human counterparts ( , ) . taken together, a significantly larger fraction of a systemically administered il- dose can be expected to reach the tumor in a mouse compared to a human. localized delivery strategies, on the other hand, are capable of enhancing il- concentrations in the tumor microenvironment by one or more orders of magnitude ( ) ( ) ( ) ( ) . a second benefit of localized il- delivery is the ability to generate systemic antitumor immunity from a locally initiated intravenous injection ( , ) talimogene laherparepvec (t-vec) unresectable advanced melanoma intratumoral injection ( ) * subcutaneous and intramuscular injections are local deliveries. however, they result in systemic cytokine distribution and are utilized in the clinic as systemic therapies. immune response. as cancer metastasizes and becomes a "systemic" disease, conventional wisdom has suggested that metastases must be treated with systemic therapies such as i.v. administered chemotherapies or immune checkpoint inhibitors. however, systemic delivery increases the frequency of adverse events through off-target interactions. for instance, systemic il- therapy has the potential to cause activation and/or differentiation of all circulating t cells whereas activation/differentiation of only tumor-specific or tumor antigen-experienced t cells is preferred. fortunately, a growing mountain of evidence demonstrates that localized il- can generate systemic, adaptive immunological memory capable of controlling anestic tumors, inhibiting metastases, and preventing tumor recurrences ( ) ( ) ( ) . in particular, local administration of il- has been shown to activate or reactivate tumor infiltrating cd + t cells, improve antigen presenting machinery and subsequently cause the expansion of tumor-specific cd + effector t cells. this often leads to enhanced infiltration of contralateral untreated tumors ( ) . research from our own lab demonstrated that local/intravesical administration of il- eliminated untreated flank tumors only when a primary orthotopic bladder tumor was treated. these data indicated that t cells must be educated with antigens from a primary tumor in order to find and eliminate abscopal tumors. similarly, intratumoral injections of il- neoadjuvant to resection have been shown to inhibit metastases by multiple groups in a t cell, nk cell, or ifn-γ dependent manner ( , ) . taken together, the convincing evidence demonstrating that localized il- can induce abscopal immunity renders systemic il- delivery unnecessary even for the treatment of metastatic disease. third, as mentioned above, il- is a pleiotropic cytokine with context dependent consequences. when il- is administered systemically, it induces rapid increases in pro-inflammatory cytokines, such as ifn-γ, tnf-α, and il- ( ). this "cytokine storm" combined with rapid decreases in peripheral blood lymphocytes, monocytes, and neutrophils can be lethal ( ) . however, when controlled locally, pleiotropic cytokines have the potential to engage multiple antitumor effector mechanisms. for instance, il- increases the activation and cytolytic capacity of cd + t cells and nk cells and induces the production of ifn-γ. ifn-γ, in turn, may kill tumor cells directly, inhibit angiogenesis ( ) ( ) ( ) ( ) , and stimulate nk cells, ctls ( , ) , and macrophages ( ) while upregulating mhc i and ii molecules ( ) on the surfaces of tumor cells. fourth, high levels of locally administered il- can reverse tumor-supporting immunosuppression. the immunosuppressive tumor microenvironment is a major hindrance to the clinical efficacy of all cancer immunotherapies. in fact, the cancer vaccine literature teaches that the majority of patients in clinical studies are able to mount significant antigen-specific t cell responses, yet few patients experience clinical benefit ( ) ( ) ( ) . similarly, the extraordinary activity of car t cells against hematologic malignancies becomes less than ordinary against solid tumors. many solid tumors lack the chemokines and inflammation necessary to recruit cytotoxic t cells ( ) . moreover, dense tumor stroma prevents t cell penetration while immunosuppressive factors released by tumor cells, suppressor t cells and tams can cause t cell anergy. regarding the latter, many tumors bathe in a cocktail of immunosuppressive factors such as tgfβ, il- , ido, and larginase. fortunately, high intratumoral concentrations of il- can cause apoptosis and elimination of cd + cd + foxp + suppressor t cells in tumors ( ) . in addition, the tumor suppressive phenotype of tams can be converted to a cytotoxic, antitumor phenotype in the presence of localized il- ( ) . finally, il- has been shown to modulate and alter the suppressive activities of tumor-associated mdscs ( ) . lastly, and perhaps most importantly, activation of t cells in the presence of il- can not only enhance ctl function, but also reduce negative regulatory mechanisms such as pd- /pd-l signaling and autocrine ifnγ-induced apoptosis. this "protective" effect has been observed mostly in the cellular immunotherapy literature. standard protocols for ex vivo expansion of tumor infiltrating lymphocytes for adoptive cell therapy (act) traditionally used high dose il- to facilitate t cell proliferation ( ) . the inclusion of il- in conditioning/expansion media has been explored recently because it had been shown previously to result in optimal t cell priming ( ) . indeed, adoptive transfer of tumor-specific cd + t cells primed ex vivo in the presence of il- resulted in enhanced antitumor responses ( , ) , increased persistence of infused t cells ( , ) , as well as increased expression of il- rα (cd ], icos, ox , granzyme b, and ifnγ ( ) . importantly, cytotoxic t lymphocytes (ctls) stimulated with il- were more effective in controlling tumors following adoptive transfer than ctls stimulated with ifnα ( ) . il- -stimulated t cells expressed lower levels of pd- and higher levels of ifnγ and il- compared to ifnα-stimulated t cells ( ) . il- conditioning caused downregulation of ifnγr with a concomitant decrease in susceptibility to ifnγ-induced apoptosis of tumor-infiltrating cd + t cells ( , ) . il- delivery strategies can be divided into three general approaches. the first involves fusion of a targeting moiety to il- in order to facilitate accumulation in a tumor following a systemic injection. the most common of class of fusion molecules are immunocytokines, which involve linking a tumor binding antibody fragment to a cytokine. the second approach involves delivery of genetic material encoding il- directly to the tumor or a tissue of interest. this category can be further divided based on the type or method of gene delivery. plasmids, mrna, viruses, and transduced cells are all capable of expressing and delivering il- after a local injection. the third major approach involves controlled release of recombinant il- protein from a sustained delivery system. here, the cytokine delivery system is injected or implanted directly in a tumor or tissue of interest. the remainder of this section will present and discuss the most relevant preclinical and clinical data pertaining to each il- delivery strategy. a summary of current clinical trials utilizing localized il- delivery is presented in table . as mentioned above, immunocytokines are part or whole cytokines that have been engineered to contain antibody fragments or other targeting moieties. these "targeted" cytokines are administered systemically but are expected to accumulate within tumors at higher levels compared to non-targeted cytokines. various tumor-related features have been targeted by immunocytokines including: ( ) tumor antigens which are overexpressed or uniquely expressed by tumor cells; ( ) cryptic extracellular matrix epitopes found only in tumors; and ( ) neovasculature markers as tumors require angiogenesis for growth. developments in immunocytokines are discussed below. the pan-carcinoma antigen, epithelial cell adhesion molecule (epcam), is highly expressed by cancer cells of epithelial origin such as colon, prostate, breast, and lung carcinomas. huks-il- is an immunocytokine of il- fused to the fc fragment of a humanized antibody that recognizes epcam. in a murine prostate cancer model, huks-il- was found to suppress experimental metastases in scid mice reconstituted with activated human t and nk cells lymphocytes ( ) . another immunocytokine, hu . -il- , which is irrelevant in this system due to its targeting of ganglioside gd , was found to be somewhat less effective than huks-il- , although differences in antitumor activity were not statistically significant ( ) . dual immunocytokines in which both il- and il- were fused to the huks / antibody fragment were found to eliminate epcam-expressing llc flank tumors following intratumoral (i.t.) injection ( ) . interestingly, a mixture of huks-il- and huks-il- was less effective than the dual immunocytokine if delivered i.t., but exhibited similar in antitumor activity if administered i.v. ( ) . the epidermal growth factor receptor her /neu is overexpressed in roughly a third of breast and ovarian cancers, with high expression correlating with poor prognosis. trastuzumab, a monoclonal antibody targeting her , has been approved for the treatment of certain breast cancers for more than years. a mouse single chain il- fused to an anti-her /neu igg (mscil- .her .igg ) retarded the growth of ct -her /neu tumors in immunocompetent mice ( ) . a direct comparison demonstrated that mscil- .her .igg and free il- induced similar activities against ct -her /neu tumors ( ) . follow up studies revealed that mscil- .her .igg also displayed robust antitumor activity against mc /her /neu and d f /e tumors ( , ) . a more recent study revealed that disruption of the heparin binding domain in the mscil- .her .igg immunocytokine, reduced il- bioactivity ( ) . this result was consistent with recent studies showing that heparin and heparan sulfate bind to and enhance the activity of il- ( ) ( ) ( ) ( ) ( ) . while eliminating heparin binding reduces il- activity, we speculate that this reduction could be counterbalanced by an enhancement in tumor targeting as the il- immunocytokine may no longer bind to ubiquitous sulfated glycosaminoglycans in non-targeted tissues. mesothelin is a differentiation antigen that is highly expressed in a number of human cancers including mesotheliomas, pancreatic and lung adenocarcinomas, and ovarian and breast carcinomas. to direct il- to mesothelin expressing cancer cells, a scfv, called ss , that specifically binds to mesothelin was fused to the p subunit of a single-chain il- ( ) . human peritoneal mesotheliomas established in nude mice were significantly inhibited by i.p. injections of il -ss ( ) . that these studies were successful in nude mice seems to imply a prominent role for nk cells in this model. ca - is a cancer antigen that is expressed in about half of human ovarian cancers ( ) . a scfv of the b monoclonal antibody that binds to ca - was fused to mil- ( ) . systemically (i.v.) administered b scfv-mil- was found to inhibit the growth of subcutaneously implanted id ovarian tumors more effectively than non-targeted mil- ( ) . cd is expressed by activated lymphocytes and thus serves as a useful target for several types of lymphoma. a cd -targeted il- fusion protein was developed for cd + hodgkin's lymphoma therapy ( ) . the immunocytokine was found to induce activation of t and nk cells and secretion of proinflammatory cytokines resulting in enhanced cytotoxicity of cd + mc cells. interestingly, a cd -targeted il -il fusion protein outperformed targeted il- or il- alone in all in vitro measures. the dual cytokine construct induced regression of cd + but not cd -mc tumors in vivo ( ) . whether the dual cytokine fusion protein was better than the single cytokine constructs is not known as the latter were not evaluated in vivo. many solid tumors overexpress extracellular matrix (ecm) which serves as transport barrier to the penetration of therapeutics and immune cells. in ecm-rich solid tumors, it may frontiers in immunology | www.frontiersin.org targeting ecm proteins instead of cancer cells, therefore, is a promising strategy to encourage immunocytokine accumulation in tumors. there are two immunocytokines, hubc -il and il- -l , that have been developed to target the splice variant extra domain b (ed-b) of fibronectin, which is highly expressed in tumor tissues but undetectable in normal adult tissues with the exception of endometrium ( ) . bc- is a monoclonal antibody that recognizes the ed-b isoform, thus a hubc -il immunocytokine has been constructed from two molecules of il- fused to each of the igg heavy chains of humanized bc- . systemic administration of hubc -il was found to eliminate experimental pc metastases and suppress the growth of multiple human tumor lines in immunocompromised mice more effectively than il- alone ( ) . a phase i trial evaluated the safety of weekly infusions of as (hubc -il ) in renal carcinoma and malignant melanoma patients ( ) . the maximum tolerated dose (mtd) was found to be µg/kg. in contrast, the mtd of twice weekly i.v. il- was previously found to be . µg/kg ( ) . dose limiting toxicities, including fever, fatigue, and elevated transaminase levels, were consistent with known toxicities of il- ( ) . the second ed-b targeted immunocytokine, il- -l , is comprised of the ed-b-binding l scfv and il- ( ) . l -targeted cytokines have been shown to selectively accumulate in tumors following i.v. administration ( , ) . intravenous administration of il- -l every h was found to control the growth of primary c colon adenocarcinomas, f teratocarcinomas as well as experimental pulmonary c metastasis ( ) . biodistribution studies confirmed that a greater percentage of the injected dose of il- -l was found in tumors as compared to an il- -fusion negative control. il- -l also demonstrated synergistic antitumor activity when combined with l -tnfα ( ) . most recently, il- was fused to the collagen-binding proteoglycan lumican and mouse serum albumin (msa), to create il -msa-lumican ( ) . lumican binds to collagen types i and iv, components of the thick fibrotic capsule surrounding tumors and perivascular basement membrane, respectively. in mice bearing established subcutaneous flank b f tumors, treatment with il -msa-lumican resulted in prolonged tumor control and longer survival. significant weight loss was observed following il -msa compared to il -msa-lumican treatment, indicating that collagen targeting may reduce systemic toxicities of il- . finally, the combined treatment of lumican-msa-il and il -msa-lumican potentiated anti-pd- increasing survival in multiple models and completely protecting cured mice from live tumor rechallenge ( ) . another collagen-binding immunocytokine comprised of the a cbd of von willebrand factor fused to both subunits of il- was also recently developed ( ) . systemic (i.v.) administration of this cbd-il was found to accumulate in emt mammary carcinomas at significantly higher levels and induce higher rates of complete tumor regression against -week old b f and emt tumors compared to il- ( ) . inclusion of the cbd resulted in a - -fold decrease in plasma half-life despite the larger size of cbd-il . the distributions of cbd-il and il- in normal tissues following i.v. injection were surprisingly similar although typical sites of collagen targeted drugs, e.g., bone and skin, were not examined. most importantly, although elevated liver enzymes were observed, levels following cbd-il at an impressive dose of µg/mouse were similar to µg/mouse of il- ( ) . in general, the key advantage of systemically administered immunocytokines is their ability to preferentially accumulate within a site of disease, e.g., a tumor. however, immunocytokines retain complete cytokine activity in circulation which allows them to interact with circulating lymphocytes and induce similar cytokine-induced toxicities as parental cytokines ( ) . one clever strategy has been developed to reduce adverse effects associated with systemic il- by separating the targeted delivery of the p and p subunits. this split-immunocytokine approach involves first delivering a bivalent p -based antibody fusion protein (f -p s-f ). f binds to the alternatively spliced extra domain a (ed-a) domain which is present on the subendothelial extracellular matrix of tumor neovasculature ( ) . after allowing time for binding and clearance of unbound f -p s-f , a subsequent administration of p , which has no activity by itself, interacts with p to recover il- activity. quantitative biodistribution investigation in f teratocarcinomas bearing mice showed that both targeted subunits accumulated in the tumor ( ) . furthermore, the recombined subunits displayed robust il- activity in terms of ifnγ production and stat phosphorylation ( ) . tumor necrosis is a common feature of most advanced solid tumors. approaches to target dna strands that become uniquely exposed in necrotic foci are under investigation. the monoclonal antibody, chtnt- , recognizes single-stranded dna ( ) . a fusion between the variable heavy chain of chtnt- and hil- forms the necrosis-targeting immunocytokine, chtnt- /hil- ( ) . chtnt- /hil- was retained in a subcutaneous tumor after i.v. injection and resulted in a significant inhibition of du prostate tumors in human pbl-engrafted scid mice ( ) . another necrosis-targeting il- , capitalizes on the specificity of the nhs antibody for ssdna and dsdna ( , ) . nhs-il is comprised of the full length nhs antibody fused to single-chain il- molecules. systemic administration of a murine analog, nhs-muil , has been shown to delay the growth of mc -cea+ colorectal carcinomas in cea.tg mice ( ) . furthermore, tumorbearing mice treated with nhs-muil developed cd + t cell responses against an endogenous tumor antigen, p e. in vivo imaging studies have shown that nhs-muil accumulated in flank tumors following a s.c. injection ( ) . subcutaneous administrations of nhs-muil were also recently shown to provide significant reductions in orthotopic mb luc bladder tumors ( ) . tumor control was associated with a noticeable reduction in markers of immunosuppression, e.g., mdscs, macrophages and tumor-associated tgf-β ( ) . the combination of nhs-muil with avelumab, an anti-pd-l antibody, resulted in improved control of both mc and mb flank tumors with higher frequencies of cd + t cells and enhanced t cell activation compared to either agent alone ( ) . against orthotopic emt- mammary tumors (∼ mm ) the combination of nhs-muil and avelumab induced complete regression in of mice ( ) . the same treatment was shown to delay, but not completely regress, the growth of - mm established emt- tumors. importantly, nhs-muil plus avelumab was shown to induce protective immunity as all cured mice resist an emt- tumor challenge but not a t challenge. furthermore, treatments enhanced cytotoxic nk and cd + tcell proliferation, t-bet expression, plasma cytokine levels, and innate and adaptive immune genes ( ) . combining nhs-il with fcil- or il- mab resulted in improved antitumor immunity, increased survival, and longterm remission in sarcoma-bearing mice ( ) . fcil- is a fusion of interleukin- and an fc fragment while il- mab is a fusion of il- and a monoclonal antibody against il- , mab . separately, the combination of nhs-il with local tumor irradiation was shown to increase treatment efficacy ( , ) . in preparation for first-in-human clinical trials, a comparative oncology study in client-owned dogs with melanoma revealed that s.c. injections of nhs-il- induced transient increases in serum ifnγ and il- . two of dogs in a dose escalation cohort experienced a partial response while of dogs had increased levels of tumor-infiltrating cd + t cells ( ) . nhs-il is currently in phase i clinical studies either as a monotherapy (nct ) or in combination with avelumab (nct ). in the former study, nhs-il induced transient lymphopenia and elevated liver transaminases, but was otherwise well-tolerated with a mtd of . µg/kg ( ) . no objective tumor responses were observed, however, of patients experienced stable disease. immune assays revealed that nhs-il treatment increased nk cell frequencies and broadened the tcr diversity of tumor-infiltrating t cells ( ) . systemically administered immunocytokines can significantly reduce but are unlikely to completely avoid il- -related toxicities. as mentioned above, in circulation, immunocytokines will interact with immune cells and induce signaling outside of the tumor. in addition, all targeting moieties are susceptible to non-specific binding and distribution in normal, untargeted tissues. for example, radiolabeled nhs has been found in all major tissues in mice for - days after i.v. administration ( ) . furthermore, substantial amounts of il- -l were found in the livers of treated animals, likely leading to hepatotoxicity ( ) . on-target/off-tissue specific binding may create additional concerns. in the case of nhs-targeting moieties, cancer patients have high levels of circulating cell-free dna that is shed from tumors ( ). it is not clear how circulating dna impacts nhs targeting. in the case of neovasculature targeting moieties, angiogenesis is a normal process of wound healing and promotes collateral circulation for atherosclerotic blood vessels. disrupting non-cancerous angiogenesis could induce hypertension and cardiac ischemia which are among the adverse events associated with anti-angiogenic agents, such as bevacizumab. moreover, the potential immunogenicity of a nonendogenous immunocytokine is another factor that may limit therapeutic potential. as non-native proteins, immunocytokines could contain immunogenic epitopes against which an immune response, likely an antibody response, could be raised. antiimmunocytokine antibodies could induce pharmacological abrogation, therapeutic alteration, or hypersensitivity reactions ( ) . because of the potential for anti-immunocytokine antibodies, novel immunocytokines should be engineered to minimize the presence of immunogenic epitopes. overall, although immunocytokines remain capable of inducing il- -related adverse events, the use of targeting moieties may improve biodistribution enough to expand the therapeutic window of il- -based immunotherapies. intratumoral (i.t.) injections of dna and rna encoding il- have the potential to localize and sustain the production of il- in the tumor microenvironment. nucleic acids are much easier to produce, purify and manipulate than recombinant cytokines. however, mammalian host cells are not easy to transfect and typically require chemical, physical, or electrical assistance to achieve reasonable transfection rates. this section will highlight progress in nucleic acid-based approaches both preclinically and clinically. around the same time that recombinant il- was failing in clinical trials, a limited number of preclinical and clinical studies explored i.t. injection of plasmid dna encoding il- (pil- ) as a potentially less toxic approach. preclinically, i.t. pil- inhibited but did not eliminate b melanomas ( ) . in this study, il- was not detected in the serum following i.t. injection. in another study involving gray horses with metastatic melanoma, i.t. pil- resulted in detectable levels of pil- in the serum for up to h ( ). however, it is not clear if systemic dissemination of pil- resulted in significant systemic increases in serum il- or ifnγ as these were not measured ( ) . in a phase i/ii trial of intralesional injections with pil- , of and of patients with stage iv malignant melanoma experienced clinical and local responses, respectively ( ) . in a phase i/ib study, i.t. pil- was found to reduce the size of treated lesions by at least % in of malignant melanomas and renal cell carcinomas ( ) . pil- injections were welltolerated as no patient in either study experienced a significant treatment-related adverse event. despite successful safety studies, the use of naked pil- for cancer immunotherapy has not progressed, mostly likely due to poor transfection efficiency. the application of pulsed, high electric fields to facilitate cellular uptake and expression of genes has been a part of the molecular biologist's toolbox for decades. intratumoral injection of pil- immediately followed by electroporation, referred to here as pil- +ep, has been explored in several murine tumor models ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . as expected, the benefit of adding electroporation was immediately apparent as one early study showed pil- alone had no effect on b f tumor growth while pil- +ep significantly inhibited tumors and extended survival ( ) . importantly, the increase in antitumor efficacy was not associated with an increase in systemic il- levels ( ) . among the more notable responses in other early preclinical studies, nearly half of mice bearing established b f melanomas experienced complete tumor regression following weekly treatments with pil- +ep ( ) . in a follow up study, pil- +ep induced tumor regression in up to % of mice, whereas i.t. injections of pil- alone delayed but could not eliminate b f primary tumors ( ) . cured mice displayed protective immunity as of rejected a b f challenge ( ) . in the sccvii squamous cell carcinoma (scc) model, complete regressions were observed in % of mice following pil- +ep ( ). furthermore, of cured mice resisted a tumor challenge containing five times the original dose of tumor cells ( , ) . against bjmc murine mammary adenocarcinomas, ct murine colon adenocarcinomas and renca renal cell carcinomas, pil- +ep significantly suppressed, but did not eliminate implanted tumors ( , ) . against murine sa- fibrosarcomas, pil- +ep suppressed tumor growth and induced complete regression in % of treated mice with of becoming resistant to tumor rechallenge ( ) . in this study, il- and ifnγ were detected in the serum of treated mice, however, no side effects were observed ( ) . abscopal responses have been documented in several studies. against bilateral sa- tumors, pil- +ep treatment consistently eliminated primary, treated tumors, while slowing the growth of secondary, untreated tumors ( ) . similarly, pil- +ep treatment of mh hepatocellular carcinomas, inhibited both treated and untreated tumors while preventing spontaneous pulmonary metastases ( ) . a recent study using bilateral b tumors demonstrated that an optimized pil- +ep protocol ( ) was capable of regressing treated lesions while inhibiting the growth of contralateral untreated tumors ( ) . injecting pil- directly into tumors is important as multiple studies have confirmed that i.t. pil- +ep treatments were significantly more effective than either peritumoral or intramuscular (i.m.) routes ( , , , ) . the i.m. route also resulted in significantly more il- and ifn-γ in the serum ( , ) . in terms of mechanism, multiple studies agreed that pil- +ep treatment was associated with increased t cell infiltration, increased ifnγ expression and decreased angiogenesis ( , , , , , ) . these findings are consistent with known antitumor mechanisms of il- . more recent mechanistic studies have focused on changes in immune cell phenotype and function. for example, b f tumor regression following pil- +ep was mediated via the perforin/granzyme lytic pathway while antigen-specific cd + t cell responses were directed against tyrosinase-related protein epitope trp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ( ) . in another study, pil- +ep-induced elimination of b f tumors was associated with increased tumor infiltration and polarization of macrophages toward an m phenotype ( ) . another group found that pil- +epinduced antitumor responses against b f tumors were correlated with a reduction in pd- expression on cd + and cd + t cells ( ) . yet, another group found that the treatment of bilateral b f tumors induced a unique population of cd + effector t cells with low pd- expression in both untreated tumors and systemically ( ) . this finding suggested that a subset of cd + effectors generated by pil- +ep may be protected or "armored" against checkpoint-mediated exhaustion ( ) . a unique feature of pil- +ep immunotherapy is the potential to manipulate electric field parameters to enhance transfection, and therefore, efficacy. an exploration of electric field parameters demonstrated that pil- +ep-induced cures ranged from to % in b f tumor-bearing mice depending on pulsing conditions ( ) . about half of these cured mice resisted a b f rechallenge ( ) . by further enhancing electric field intensity and/or pulse length, it is possible to directly kill tumor cells and release tumor antigens via irreversible electroporation. in one recent study, partial-irreversible electropermeabilization (pire) administered after peritumoral electrotransfection with pil- caused complete regression of about - % of treated b f tumors ( ) . two of cured mice completely resisted tumor rechallenge while the remaining two experienced delayed tumor growth from the rechallenge. this pil- plus pire approach was found to delay, but not eliminate distant, untreated tumors in about half of the mice ( ) . there have been several attempts to enhance antitumor activity through the incorporation of additional cytokineencoding plasmids. of note, two studies have demonstrated that ep using a combination of il- and il- plasmids outperformed il- alone in terms of antitumor activity ( , ) . the rationale to combine these two cytokines is wellsupported given that il- and il- synergize to enhance th responses and ifn-γ production. however, adverse events are also enhanced as systemic co-administration of recombinant il- and il- proteins leads to lethal toxicity in mice ( ) . in one study, addition of pil- to pil- increased serum il- and ifn-γ levels for at least days after ep although no inflammation was observed in liver, lung, and intestine samples ( ) . in a second study, pil- did not increase il- -induced serum ifn-γ, but intratumoral ifn-γ was significantly higher ( ) . several notable canine clinical studies have explored pil- +ep in dogs with naturally occurring tumors. in one such study, pil- +ep resulted in a - % reduction in mast cell tumor volume ( ) . treated nodules displayed increases in leukocytic inflammation and decreases in the number of malignant mast cells ( ) . in beagles with canine transmissible venereal tumors (ctvts), pil- +ep induced complete regression of all treated lesions ( ) . contralateral untreated tumors were also significantly inhibited. serum il- levels peaked days after treatment; however, relevant blood chemistries, i.e., liver and kidney enzymes, as well as cell counts were not different from those of control dogs ( ) . another study investigated pil- +ep for the treatment of canine oral malignant melanoma (omm). there were no differences in the percentages of helper cd + and cd + cells before and after treatment, while t reg frequencies declined from . to . %. one month post treatment, the objective response rate was % ( / ) but by the end of the observation period, all but one of the dogs developed progressive disease ( ) . a more recent study in dogs with a range of spontaneous cancers, demonstrated that pil- +ep induced immunostimulatory and anti-angiogenic effects ( ) . administration of three pil- +ep treatments every other day caused significant systemic toxicities, including anemia and thrombocytopenia. after switching to a weekly schedule, treatments were well-tolerated, however, all treated tumors continued to progress ( ) . several human clinical trials, mainly against advanced melanoma, have investigated the safety and efficacy of pil- +ep. in a phase i study, patients with stage iii or iv melanoma received i.t. pil- +ep (six µs, , v/cm pulses) on days , , and during a single -day cycle. fifty three percent of patients experienced a systemic response, defined as either stable disease or regression of untreated lesions, following pil- +ep ( ) . most notably, of patients showed complete regression of all metastases. no grade or higher adverse events were observed and neither il- nor ifnγ was detectable in serum samples. in a follow up phase ii study, patients with in-transit or m a melanoma were treated with up to four -week cycles of pil- +ep as described above ( ) . intermediate results revealed an objective response rate of % with % complete responses. the treatment was found to increase nk cell levels both intratumorally and systemically ( ) . no grade / drug-related adverse events were noted. a subsequent analysis of clinical samples revealed that responses were associated with increased intratumoral infiltration of cd + t cells ( ) . in addition, t cell receptor beta chain (tcrβ) sequencing revealed a focusing of the tcr repertoire following treatment. however, there were no differences in t cell clonality between responders and non-responders to pil- +ep ( ) . a second study from the same trial, nct , found a complete response rate of up to . % and a best overall response rate of . % in patients with stage iii/iv melanoma ( ) . nearly half of these patients experienced regression of at least one anenestic lesion. an analysis of transcripts in melanoma biopsies found increases in t cell trafficking, immune activation, and antigen presentation ( ) . genes associated with adaptive resistance, e.g., pd-l , tgfβ, and trail, were also increased ( ) . recent results from a safety study in patients with locoregional merkel cell carcinoma (mcc) and patients with metastatic mcc received or up to cycles of pil- +ep, respectively ( ) . the overall response rate in the metastatic mcc cohort was % ( / ). of patients with measurable untreated lesions, experienced abscopal regressions. in addition, patients experienced clinical responses lasting and + months, respectively. two of the locoregional mcc patients, all of whom were treated with definitive surgery after pil- +ep, were recurrence-free at + and + months, respectively ( ) . serum il- levels were not measured, but treatments were well-tolerated, and no serious adverse events were observed. lipoplexes, polyplexes, and lipopolyplexes are complexes of lipids, polymers, and lipids plus polymers, respectively, with dna. these complexes are under investigation to enhance the delivery and transfection efficiency of plasmids encoding genes of interest, including pil- . numerous studies have explored a range of different materials to create novel pil- complexes ( ) ( ) ( ) ( ) ( ) . studies demonstrating antitumor efficacy following local or targeted delivery of pil- complexes are highlighted below. polyethyleneimine (pei), a highly cationic polymer that readily complexes with negatively charged dna, has been widely used to enhance gene delivery. pei protects dna from degradation in vivo, encourages interaction with negatively charged cell membranes, and enhances release from lysosomes by acting as proton sponge ( ) . pei:il- complexes were shown to transfect lung tissue following delivery via nebulization ( ) . this approach led to production of il- in the lungs which was not detectable in the plasma of treated mice ( , ) . weekly or twice weekly administration of aerosolized pei:il- was found to suppress or eliminate experimental pulmonary metastases of saos- human osteosarcomas in athymic nude mice ( ) . recent attempts to enhance uptake and il- production have focused on modification of pei with tetraiodothyroacetic acid (tetrac) which binds the α v β integrin receptor that is overexpressed in some tumors ( ) or diethylene triamine penta-acetic acid (dpta) which can reduce the surface charge of pei:il- complexes ( , ) . as of this writing, no in vivo data using either modification have been published. polyvinylpyrridilone (pvp) is another cationic polymer that readily complexes with dna. twice weekly i.t. injections of pil- /pvp complexes were found to eliminate and % of - mm renca and ct tumors, respectively ( ) . most mice that were cured of a primary tumor rejected a subsequent rechallenge ( ) . in a follow-up study, pil- /pvp was found to be more effective than pifnα/pvp in controlling preclinical tumors, while the combination of pil- /pvp and pifnα/pvp synergized to eliminate % of renca and % of ct tumors ( ) . in both studies, cd + t cells but not cd + t cells were identified as primary effectors. complexation with poly-a-( -aminobutyl)-l-glycolic acid (paga), a biodegradable polyester, enhanced transfection efficiency of pil- and expression of il- in vitro and in vivo ( , ) . however, t cell infiltration of injected ct colon adenocarcinomas and antitumor activities following repeated injections of paga/pil- and naked pil- were similar ( ) . encapsulation of pil- in nanoparticles comprised of poly-(d,l-lactic-co-glycolic acid) (plga) and , -dioleoyl- -(trimethylammonium) propane (dotap) demonstrated complete regression of up to % of established heterotopic bnl hepatocarcinomas following a single i.t. injection ( ) . importantly, treatment with encapsulated pil- was more effective than treatment with nanoparticles with pil- adsorbed to the surface ( ) . long term expression of inflammatory cytokines could be a concern as both il- and ifnγ were detected in the serum for up to days after treatment ( ) . a similar, so-called dmp nanoparticle, comprised of dotap and methoxy-poly(ethylene glycol)-poly(lactide) (mpeg-pla), has been developed to facilitate gene delivery ( ) . complexation with pil- resulted in inhibition of ct tumors with no signs of systemic toxicity, as determined by appearance, body weight, fecal output, and urinary excretion ( ) . a slightly different dmp nanoparticle that uses polycaprolactone (pcl) instead of pla, inhibited the growth of intraperitoneal c colon carcinomas and ll/ lewis lung carcinomas ( ) . in addition to delaying tumor growth, dmp/il- particles resulted in high il- gene expression and t cell infiltration although the treatment regimen consisted of daily or every other day treatments ( ) . plasmids complexed with mannosylated chitosan (mc) are under development as a method to target mannose receptors on i.t. dcs. chitosan is a linear co-polymer of β-linked dglucosamine and n-acetyl-d-glucosamine. it is primarily derived from the structural polysaccharide, chitin, found in shells of crustaceans. i.t. injection of mc/pil- complexes elicited modest growth delay of ct tumors, which was associated with increased tumor cell apoptosis and decreased angiogenesis ( ) . lipopolymers, which incorporate a lipid tail on a polymer backbone, are also under exploration for non-viral gene delivery. water soluble lipopolymers (wslp) comprised of cholesterol conjugated to pei have been developed to increase cell membrane permeability and reduce pei-mediated toxicity ( ) . wslp/p cmvmil- dna complexes inhibited the growth of ct tumors and improved survival following a single i.t. injection ( ) . however, the antitumor efficacies of wslp/pil- complexes and naked pil- appeared similar. in a later study, wslp/p cmvmil- complexes injected every days outperformed single injections and multiple injections with naked pil- or pei/pil- complexes ( ) . the vast majority of injected wslp/p cmvmil- was found in the tumor for up to h with small but increasing accumulation in the liver and blood ( ) . subsequent studies demonstrated that i.t. wslp/p cmvmil- significantly suppressed the growth of primary and metastatic t , tsa, and emt- mammary carcinomas ( , ) . polytraxane (prx) is a composite molecule made of polyethylene glycol (peg) and cationic cyclodextrin (cd) that self assembles with dna into a spherical particle ( ) . a -arm configuration, rather than a linear configuration, had a higher accumulation in mc -luc tumors and lower accumulation in the lungs following systemic delivery. the -arm prx/pil- also produced higher levels of il- and significantly slowed mc -luc tumor growth after five i.v. injections of the complex starting days after tumor implantation. ( ) systemic injections of prx/pil- complexes induced moderate lymphopenia, but no elevation of liver enzymes ( ) . pil- complexed with a polyethyleneglycolpolyethylenimine-cholesterol (ppc) lipopolymer was shown to inhibit t and sccvii tumors ( ) and increase survival in mice with intracranial gl gliomas ( ) following localized injections. of note, although the i.p. route is frequently used to deliver drugs systemically, i.p. injections of pmil- /ppc for treatment of id ovarian carcinomas resulted in high levels of il- and ifn-γ in ascites but low levels in serum ( ) . in this study, i.p. pmil- /ppc was well-tolerated with no significant changes in serum chemistries ( ) . in a phase i study, phil- /ppc was administered i.p. to women with chemo-resistant recurrent ovarian cancer ( ) . escalating doses of phil- /ppc were well-tolerated with no dose-limiting toxicities. five of the treated patients reported a serious adverse event, however, only one was possibly related to the phil- /ppc ( ) . similar to preclinical studies, no detectable increase in serum il- was found following phil- /ppc treatment ( ) . a subsequent phase ii study in patients with platinum-resistant recurrent ovarian cancer demonstrated similar safety following weekly i.p. phil- /ppc, however, with no objective clinical responses observed ( ) . a different phase i trial evaluated the safety of phil- /ppc in ovarian cancer patients when combined with carboplatin and docetaxel chemotherapy ( ) . while there were no dose limiting toxicities, grade adverse events included manageable abdominal pain and cytokine release syndrome. two of patients experienced complete response while of experienced a partial response ( ) . a more recent phase i trial combined weekly i.p. pil- /ppc with i.v. pegylated liposomal doxorubicin in patients with persistent or recurrent platinum-resistant ovarian or peritoneal cancers ( ) . although increased levels of il- , ifn-γ, and tnf-α were found in peritoneal fluid, no dose limiting toxicities were observed and a maximum tolerated dose was not reached. the best partial response ( . %) and stable disease ( . %) rates were found at the highest dose ( mg/m ) of pil- /ppc ( ) . pil- complexed with a cationic lipid (+/-)-n-( -hydroxyethyl)-n,ndimethyl- , -bis(tetradecyloxy)- propanaminium bromide/dioleoylphosphatidylethanolamine (dmrie/dope), and injected i.t. was found to inhibit and eliminate ct and renca tumors while protecting up to and %, respectively, of mice from rechallenge ( ) . interestingly, a direct comparison between naked pil- and dmrie/dope/pil- revealed no difference in antitumor activity ( ) . polyphosphazene particles were modified with hydrophobic n,n-diisopropylethylenediamine (dpa) and hydrophilic monomethoxy poly-(ethylene glycol) (mpeg) to create weakly cationic particles to complex with pil- ( ) . mpeg/pil- polymersomes delayed ct tumor growth when administered i.v. body weights were unaffected by mpeg/pmil- treatments. tumor il- levels steadily increased, reaching about pg/g tumor on day , while serum il- concentration remained on average about pg/ml after day and continued to day . the concentration of ifn-γ in the tumor reached a maximum of pg/g tumor on day , while serum levels of ifn-γ slightly increased from day to day , reaching a concentration of only pg/ml. while mpeg/pmil- polymersomes did not affect cd + cd + cells in the tumor, there was a -fold increase of cd + cd + cells and significant increases of cd − nk . + and cd + nk . + cells in the tumor. in another study, all-trans-retinoic acid (atra) was incorporated in cationic liposomes and complexed with pil- ( ) . atra was previously found to increase the expression of tnf receptor and mediate apoptosis of lung cancer cells via tnfα ( , ) . i.v. injections of atra-cationic liposome/pil- reduced lung nodules and extended survival compared to cationic liposome/pil- treatment in an experimental pulmonary metastasis model using c cells expressing luciferase ( ) . concentration of il- in the lungs reached a maximum of pg/mg protein at h post injection, while levels of il- in the spleen and liver were significantly lower and nearly eliminated by h. interestingly, the incorporation of atra reduced liver enzymes levels and thus hepatic toxicity suggesting a possible anti-inflammatory role ( ) . recently, mrna delivery platforms have received tremendous attention, most notably as front running vaccines against sars-cov- . mrna, like dna, can encode an unlimited number of proteins and polypeptides. although mrna-based platforms are less stable than dna-based platforms, mrna can be protected from digestion through encapsulation in polymeric or lipidbased micro-or nanoparticles. a key advantage of mrna is their ability to directly translate encoded proteins in the cytoplasm. in contrast, dna must first translocate to the nucleus to be transcribed to mrna before translation in the cytoplasm. regarding the use of mrna to deliver il- locally, a recent study demonstrated that weekly i.v. delivery of lipid nanoparticles (lnp) loaded with mrna encoding il- reduced tumor burden in a myc-driven transgenic mouse model of hepatocellular carcinoma (hcc) ( ) . the tumor inhibition and extended survival were attributed to increased infiltration of cd + cd + cd + immune cells and not suppression of myc ( ) . similarly, moderna/astrazeneca has developed, medi , an lnp formulation with il- mrna. medi is currently in phase i clinical trials for intratumoral injection of advanced solid tumors in combination with durvalumab ( table ) ( ) . in preclinical studies, a single intratumoral injection of mrna encoding murine il- (mil- ) increased ifnγ expression and genes associated with a th response in mc tumor-bearing mice ( ) . when combined with anti-pd-l , enhanced t cell infiltration and expanded tumor-specific t cell subsets were observed ( ) . in another phase i clinical trial, sanofi and biontech are testing sar (bnt ), an mrna platform encoding a cocktail of il- sc, il- sushi, ifnα, and gm-csf for intratumoral injection as a monotherapy and in combination with cemiplimab ( ) . to our knowledge, no preclinical data with sar have been disclosed. in general, nucleic acid-based il- delivery approaches are limited by variable transfection rates as well as unregulated production of gene products. in other words, it is easy to control the amount of pil- or il- mrna delivered but it is not easy to control the dose of recombinant il- that each subject receives. variable transfection rates may be responsible for conflicting reports on whether pil- +ep does ( - , , , ) or does not ( , , ) produce significant increases in serum il- and ifn-γ. fortunately, lethal il- -related toxicities have not been reported in the any of the preclinical or clinical studies detailed above. nevertheless, the potential for severe il- -related adverse events caused by continued and/or unregulated production of il- remains. strategies to enhance safety and efficacy, either by localizing gene-based il- though incorporation of an anchoring or binding domain or by incorporation of an inducible safety switch to turn off il- production, will help improve the therapeutic window of promising nucleic-acid-based il- delivery technologies. another potential limitation that must be considered, is any type of adverse reaction against components of the delivery vehicle or against the nucleic acid vector itself. regarding the former, foreign delivery components have the potential to induce immune responses which could influence il- delivery. most notably, it has been reported that about in humans have circulating anti-peg antibodies ( ) . the high prevalence of anti-peg antibodies could limit the efficacy of any peg-based delivery vehicle. regarding the immune responses against il- vectors, nucleic acids, particularly dna that is found in the cytoplasm have the potential to stimulate the cgas-sting pathway, which may induce its own inflammatory response. thus, studies utilizing nucleic acid-based delivery must take care to decouple the effects of il- from sting activation. lastly, although an exceedingly rare event, dna vectors could become integrated within a cell's genome. depending on the site, such integration could have deleterious or even transforming effects. given that only transient il- expression is desirable, strategies capable for preventing integration, like the use of circular instead of linearized plasmids, should be preferred. adenoviruses, herpes simplex viruses, semliki forest viruses, poxviruses, and other viral vectors have been engineered to express biologically active il- . these engineered viruses injected directly into a tumor are able to infect cancer cells and induce expression of il- within the tumor microenvironment. furthermore, many viruses have the unique ability to selectively lyse cancers cells after infection. such oncolytic viruses take advantage of defective cell cycle and interferon signaling pathways that are hallmarks of cancer cells but not normal cells ( ) . oncolytic viruses can kill compromised cancer cells in a variety of ways from direct virus-mediated cytotoxicity to indirect destruction of tumor-feeding blood vessels ( ) . the following sections discuss the progress and limitations of il- encoding viral vectors. adenoviruses are the most well-studied among the il- expressing vectors ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in preclinical studies, i.t. injections of adenoviruses encoding il- (ad-il- ) have mediated regressions of murine colorectal carcinomas ( ) ( ) ( ) , breast carcinomas ( , , ) , prostate carcinomas ( , ) gliomas ( , ) , bladder carcinomas ( ) , fibrosarcomas ( , ) , laryngeal squamous cell carcinoma ( ) , hepatomas ( ) and hepatocellular carcinomas ( , ) medullary thyroid carcinomas ( ) , thyroid follicular cancer ( ) , and ewing's sarcoma ( ) . among the more robust responses, ad-il- induced complete regression of subcutaneous neuro- a neuroblastomas in nearly half of mice receiving a single i.t. injection ( ) . mice becoming tumor-free also rejected a subsequent tumor rechallenge ( ) . similar results were found against ct colon adenocarcinomas with more than three-fourths of mice completely eliminating their tumors and all cured mice rejecting a tumor rechallenge ( ) . the antitumor immune response was mediated primarily by cd + t cells. impressively, of mice with bilateral tumors experienced complete regression of an untreated tumor ( ) . against - rat medullary thyroid carcinomas, i.t. injection of adtcpmil- caused complete regression of more than % of treated tumors ( ) . all cured rats rejected a tumor rechallenge while separate experiments showed that treatment of a single tumor resulted in inhibition of a distant untreated tumor ( ) . while liver infection following i.t. adtcpmil- injection was documented, no toxicity was observed ( ) . a follow up study found similar antitumor and abscopal responses against rat thyroid follicular cancer ( ) . in the pymt-derived transplanted mammary carcinoma model, adenoviral vectors encoding il- induced complete regression in % and partial regression in % of mice ( ) . ten of tumor-free mice completely rejected a tumor rechallenge. in contrast with similar studies using ad vectors expressing il- , no obvious toxic side effects due to admil- were noted ( ) . in another difficult model, a single i.t. injection of ad. / .crgd-mil p resulted in > % long term survival of mice with intracranial gl gliomas ( ) . in a murine model of ewing's sarcoma (tc ), twice weekly i.t. injections of ad.mil- significantly delayed treated tumors as well as untreated tumors ( ) . ad.mil- also induced complete regression in all treated mice bearing heterotopic mb bladder carcinomas ( ) . although body weights were not affected, serum ifn-γ levels due to i.t. ad.mil- were maximal from (∼ , pg/ml) to days (∼ , pg/ml) post injection ( ) . in a useful comparison against other cytokines, one study demonstrated that ad-ifn-γ had no greater antitumor activity than an empty ad vector, whereas admil- induced complete regressions of p mastocytomas in > % of treated mice ( ) . similarly, ad-gm-csf inhibited the growth of frtl-tc rat thyroid tumors, however, adil- was found to be much more effective ( ) . adil- also generated systemic immunity capable of inhibiting the growth of distant tumors ( ) . an adenoviral vector expressing a single chain il- (scil- ) was developed to enhance bioactivity over the native heterodimeric form ( ) . long-term tumor-free survival was observed in up to % of rats with established mh- a hepatocellular carcinomas following i.t. infections with ad.scil- ( ) . all tumor-free mice were protected from tumor rechallenge. however, despite i.t. injections, il- and ifn-γ were detected in the serum of treated mice ( ) . an oncolytic adenovirus expressing single-chain il- (ad-dhscil ) was more effective than non-replicating (nononcolytic) adenoviruses expressing il- at controlling liver metastases of pancreatic cancer in hamsters ( ) . however, il- levels in serum and non-tumor tissues were similar ( ) . in clinical studies, i.t. ad.il- was well-tolerated with no dose-limiting toxicities in a phase i trial in patients with advanced pancreatic, colorectal, or primary liver malignancies ( ) . serum ifnγ levels peaked day after ad.il- administration and was likely responsible for the grade adverse events observed. one patient had a partial response and % of patients experienced stable disease. four of assessable patients experienced increases in tumor infiltrating cd + and cd + cells. delayed-type hypersensitivity tests with inactivated adenovirus indicated that all patients developed an immune response against the adenovirus ( ) . despite robust antitumor immune responses, interest in ad-il- waned in the mid- s due to the aforementioned lethal toxicities associated with systemic administration of ril- and the inability of ad-il- , even if administered intratumorally, to prevent systemic dissemination of the cytokine. in recent efforts to mitigate systemic il- dissemination and associated toxicities, two strategies have been developed. the first involves engineering il- to prevent its dissemination. this has been accomplished either by anchoring il- to the surface of tumor cells via fusing a transmembrane domain or glycosylphosphatidylinositol (gpi)-anchored signal sequence to the cytokine ( , ) or by deleting the n-terminal signal peptide and thus preventing il- secretion ( ) . using the former technology, i.t. injection of an adenoviral vector encoding membrane-anchored il- (ad/scil- -b tm) eliminated the majority of primary ct tumors and suppressed the growth of untreated contralateral tumors ( out of mice), in which complete regression occurred in out of mice ( ). importantly, negligible il- was found in the circulation of mice treated with ad/scil- -b tm. the second technology deletes the signal peptide from the p subunit of il- to prohibit il- secretion ( ) . newly designed oncolytic adenoviral vectors with three genes deleted, i.e., triple deletion (td), and encoding either wild-type il- (ad-td-il- ) or a non-secreting il- (ad-td-nsil- ) were evaluated in syrian hamster models of pancreatic cancer. six i.t. injections of either ad-td-il- or ad-td-nsil- were found to eliminate subcutaneous hpd nr tumors in all mice ( ) . against peritoneally disseminated shpc tumors and orthotopic hap-t pancreatic tumors, ad-td-nsil- outperformed ad-td-il- in terms of overall survival ( ) . most importantly, ad-td-nsil- resulted in significantly lower serum il- levels and reduced systemic inflammatory cytokine expression ( ) . the second strategy to mitigate systemic il- dissemination involves conditional expression of il- . the rheoswitch therapeutic system r (rts) is an ecdysone receptor-based gene regulation platform in which a transcription factor becomes activated only in the presence of a synthetic small molecule ligand ( ) . an adenoviral vector encoding the rts switch and mil- (ad-rts-mil- ) and controlled by the oral activator, veledimex (vdx) was recently shown to extend survival in mice bearing intracranial gl gliomas ( ) . intratumoral ad-rts-mil- plus oral vdx was found to induce il- expression in a dose-dependent manner. local il- expression correlated with increases in tumor-infiltrating lymphocytes. il- and ifnγ were detected in the sera of treated animals, albeit at an order of magnitude lower than levels found in tumors ( ) . several clinical studies utilizing the regulatable ad-rts-il- platform are underway ( table ) . recent results from a phase i study in patients undergoing resection of recurrent highgrade glioma demonstrated that vdx induced il- expression in a dose-dependent manner ( ) . likewise, the frequency and severity of adverse events, including grade cytokine release syndrome, also increased with vdx dose. demonstrating the advantage of the inducible system, all serious adverse events were reversible with vdx discontinuation. interestingly, the use of corticosteroids negatively impacted survival ( ) . at the optimal dose, and in the absence of corticosteroids, the median overall survival of . months was encouraging ( ) . while the localized injection of ad-rts-hil- in the resected tumor bed induced il- expression in a recurrent tumor microenvironment, systemic dissemination of il- and its resultant toxicities could not be avoided. herpes simplex viruses (hsvs) are another family of viruses that have been widely explored for localized il- delivery. wild-type hsv are cytolytic and thus must be significantly attenuated or rendered replication-incompetent to avoid systemic infection. injection of replication-incompetent hsv-il- into established hepatomas prior to partial hepatectomy inhibited the engraftment of an intraportal tumor cell challenge in preclinical studies ( ) . importantly, no changes in serum il- were detected in treated buffalo rats ( ) . in order to capitalize on their lytic potential, hsvs have been engineered, through deletion or mutation of genes responsible for viral replication such that only cancer cells are lysed. not surprisingly, these oncolytic hsvs (ohsvs) have been shown to exhibit greater antitumor potential than their replicationincompetent, parental counterparts. for instance, treatment of established hepatomas with a non-cytokine encoding ohsv exhibited significant antitumor activity which was further increased with an il- insert ( ). the ohsv-il- treatment was also more effective than ohsv at protecting animals from a tumor rechallenge ( ) . similar antitumor responses to ohsv and increased efficacy with ohsv-il- were observed against flank ct and sccvii tumor models ( ) ( ) ( ) . a single i.t. injection was able to delay the growth of established sccvii tumors whereas multiple injections induced complete regressions in of treated mice ( ) . in contrast, multiple injections of ohsv-gm-csf cured only half of treated mice ( ) . in the ct model, ohsv-il- inhibited or eliminated injected mm tumors while inhibiting non-injected, contralateral tumors ( , ) . when low dose mil- was added to i.t. ohsv-il- , both treated and untreated ct tumors were eliminated in more than two-thirds of mice ( ) . the importance of t cells in this model was established as ohsv-il- had no antitumor effect in ct -bearing nude mice ( , ) . intraperitoneal administration of ohsv-il- also increased survival in misiir-tag mice bearing ovarian carcinomas ( ) . treatment was associated with tumor antigen-specific cd + t cell infiltration ( ) . against flank sarc- and sarc- sarcomas, ohsv-il- extended survival compared to saline injections ( ) . although there was no difference is survival between control and il- encoding ohsv, the ohsv-il- was found to increase tumor-infiltrating effector t cells while decreasing immunosuppressive mdsc and t reg populations ( ) . in an interesting comparison of cytokines, ohsv-il- was more effective at inhibiting flank prostate tumors, tramp-c and pr - , than ohsv encoding gm-csf (ohsv-gm-csf) which was no more effective than non-cytokine encoding ohsv ( ) . only of mice treated with ohsv-il- exhibited an increase in serum il- days after treatment ( ) . ohsv-il- also significantly outperformed ohsv-gm-csf in a model of ct metastasis ( ) . in addition to localizing il- , i.t. injections may be key in assuring safety as intrasplenic injections of ohsv-il- induced concerning increases in il- and ifnγ in both serum and liver specimens ( ) . frequent and worthwhile targets of ohsv-il- immunotherapy are the various forms of brain cancer. in one early study, ohsv-il- was found to extend survival and cure approximately one-fourth of mice bearing -day-old intracranial neuro- a neuroblastomas ( ) . treatment was associated with an influx of cd + and cd + t cells as well as macrophages. the inclusion of il- was critical, as median survivals of mice treated with the parental, non-cytokine encoding ohsv or saline were similar ( ) . likewise, ohsv-il- but not non-cytokine ohsv was effective at extending survival against intracerebral mouse glioblastomas ( ) . in an intracranial c glioma model, ohsv-il- was significantly more effective than ohsv at extending survival and curing mice ( ) . in a model of breast cancer metastasizing to the brain, i.t. ohsv-il- modestly extended the survival of mice bearing intracranial sck tumors ( ) . intracerebral injections of ohsv-il- in owl monkeys resulted in neither histopathological changes in brain tissue nor clinical evidence of toxicity, as assessed by changes in temperature, neurologic performance, feeding or social behavior, or weight ( ) . in addition to encoding cytokines, hsvs can be engineered to target cancer-associated antigens. four i.t. injections of a her -targeted ohsv-il- was significantly more effective at inhibiting both day and day tumors than the non-cytokine encoding parental hsv (day : / ohsv-il- vs. / hsv becoming tumor free; day : / ohsv-il- vs. / hsv becoming tumor free) ( ) . immune responses to ohsv-il- included elevated levels of ifnγ, il- , granzyme b, tbet, and tnfα as well as th polarization and nk activation ( ) . this her- targeted ohsv-il- was also found to induce complete remission in more than one-fourth of treated mice bearing orthotopic high grade gliomas (hgg) expressing her ( ) . cured mice were protected from hgg rechallenge regardless of her expression ( ) . this is an important finding given the heterogeneity of her expression among and within tumors. the clinical precedence for hsv has been established with talimogene laherparepvec, which encodes for gm-csf and is approved for intratumoral injection in patients with advanced, non-resectable melanoma. a clinical grade preparation of hsv- encoding hil- (m ) induced no adverse clinical signs after intracerebral injection in non-human primates ( ) . a phase clinical trial exploring the safety of m in patients with recurrent or progressive glioblastoma multiforme, anaplastic astrocytoma, or gliosarcoma is currently recruiting [nct ]. semliki forest virus (sfv) is an alphavirus that was first isolated from mosquitos in uganda and has a broad range of hosts, making it ideal for translation ( ) . in addition, modified sfv can produce higher levels of recombinant protein than retroviral vectors, and expresses protein more stably than adenoviral vectors ( ) . sfv is also less pathogenic in humans, and induces apoptosis of tumor cells at the end stage of virulence ( ) . sfv encoding il- (sfv-il- ) was found to extend the survival of mice with established orthotopic gliomas ( ) . because sfv infection induces apoptosis, uptake of infected tumor cells by dendritic cells in the presence of il- is posited as a potential mechanism of enhanced antitumor activity. in a b brain tumor model, the same group demonstrated a prolonged median survival by days when immunizing with sfv-il- pulsed dendritic cells, compared to a retroviral vector encoding il- ( ) . in a woodchuck hcc model, induced by chronic infection with woodchuck hepatitis virus (whv), using a single intratumoral treatment with sfv-enhil- , which included separate injections at different sites of one tumor, partial tumor regressions of up to % occurred in out of animals ( ) . although all tumors regrew after treatment, injections of sfv-enhil- resulted in a favorable safety profile with only transient reductions in body weight ( ) . in a transgenic mouse model of spontaneous hcc, i.t. sfv-il- treatments achieved % survival for at least days ( ) . interestingly, sfv-il- , which induces transient infection and expression of il- , was found to be more effective and less toxic than long-term il- expression induced by a plasmid encoding il- which was delivered hydrodynamically ( ) . i.t. sfv-il- inhibited tumor growth and extended survival in mice bearing orthotopic t mammary carcinomas ( ) . when administered neoadjuvant to resection and combined with attenuated salmonella (lvr ) as a post-surgery adjuvant, an impressive % long-term tumor free survival was achieved ( ) . while there is consensus on its benefits, the mechanism of sfv-il- -induced tumor regression is debated. initial studies performed in a subcutaneous b melanoma model did not find a significant decrease in tumor regression when using t cell-deficient nude or nk cell-deficient beige mice. rather, this group suggested that inhibition of angiogenesis mediated by ifnγ production causes massive tumor necrosis ( ) . a decade later, the same group further supported this conclusion using inos deficient mice that express high levels of vegf ( ) . the effect of il- was even more pronounced, with fewer tumor vessels in inos −/− mice than in wild-type mice given the same treatment with sfv-il- ( ) . however, they did find immunohistochemical evidence that nk cell activation and recruitment is correlated with murine endothelial cell death ( ) . more recently, the efficacy of sfv-il- was found to be dependent on type i interferons produced by macrophages and dendritic cells ( ) . furthermore, the type i interferon receptor, ifnar, is necessary for il- -dependent cd + t cell expansion ( ) . different routes of administration were compared using a subcutaneous p model and demonstrated that i.t. injection of sfv-il- was superior in producing ifnγ compared to either s.c. or i.v. routes ( ) . importantly, none of the sfv-il- injections resulted in increased serum levels of ifnγ ( ) . the combination of sfv-il- with monoclonal antibodies or adjuvants is another strategy that has been studied. by administering sfv-il- and anti-cd to provide costimulation to t cells, survival was improved in both s.c. b -ova and s.c. tc- models with a maximum longterm survival of % in both models ( ) . in the bilateral b -ova model, % of treated and % of untreated tumors experienced complete regression with sfv-il- viral particles and anti-cd ( ) . cd + t cells were crucial for this tumor regression and sfv-il- increased the ratio of cd t/t regs compared to anti-cd alone. a subsequent paper further demonstrated that sfv-il- induces pd-l expression on b -ova cells and therefore combined pd- /pd-l blockade with the viral construct ( ) . this combination significantly enhanced survival compared to individual components using b -ova and mc models, with long-term survival > % ( ) . modifications to improve the performance of sfv-il- vectors have also been explored. an enhanced (sfv-enhil- ) vector with separate promoters for each subunit of il- increased il- expression -fold over the single promoter construct ( ) . one i.t. injection of viral particles using either the original or enhanced constructs resulted in > % longterm tumor free survival of mc tumor-bearing mice ( ) . lower doses of sfv-enhil- induced tumor regression more efficiently than sfv-il- , although the enhanced vector induced higher levels of serum il- ( ) . a separate enhanced sfv vector with x higher gene expression, called psfv -e-il , has also been developed ( ) . this vector also included separate promoters for p and p with an additional capsid enhancer to drive enhanced expression of the recombinant protein. two helper vectors with instructions for structural proteins were cotransfected with the enhanced sfv to produce virus-like particles (vlps). intratumoral injections in subcutaneous k-balb and ct tumors caused complete regression and furthermore inhibited primary tumor growth and reduced metastases in a heterotopic t model ( ) . in an effort to limit undesirable virus-mediated cytotoxicity for intracranial applications, sfv vlps expressing il- have been developed ( ) . low dose ( × vlps) treatment decreased orthotopic rg rat glioma volumes by % and extended survival by a little more than % over vehicle controls. high dose ( × vlps) treatment resulted in enhanced tumor reduction but also caused cns inflammation, necrosis, and treatment-related death ( ) . the broad infectivity of sfv-based vectors is a major limitation to their use in cns applications. vaccinia virus (vv), which was used extensively during the eradication of smallpox, has a large capacity for gene insertion, a wide host range and high gene expression efficiency. vv expressing il- (vv-il- ) has been found to inhibit, but not eliminate, c gliomas in nude mice following i.t. injection ( ) . low vv-il- doses, - pfu, and high vv-il- doses, - pfu, resulted in similar levels of tumor inhibition ( ) . all doses above pfu resulted in cytokine-associated toxicities punctuated with a % mortality rate in the high dose group ( ) . similar tumor inhibition and mortality were found in a subsequent publication which also correlated high plasma levels of ifnγ and tnfα with toxicity ( ) . vv-il- and il- expressing vv (vv-il- ) exhibited similar inhibition of c gliomas ( , ) however, against ae mesotheliomas, vv-il- cured % of mice compared to only % of mice treated with vv-il ( ) . recently, an oncolytic vv-il- was shown to increase lymphocytic infiltration and inhibit llc and b f tumors with - % complete responses ( ) . antitumor efficacy was enhanced by the addition of an il- expressing vv (vv-il ) as well as immune checkpoint inhibitors ( ) . the combination of vv-il- and vv-il did not affect mouse body weight ( ) and appears to be better tolerated than previous vv-il- treatments which utilized different vv strains ( , ) . a recombinant canarypox virus (alvac) encoding il- has been shown to induce complete regression of ts/a mammary carcinomas after six i.t. injections, whereas a single i.t. injection of alvac-il- had no effect on tumor growth ( ) . of the mice that were rendered tumor-free, % were protected from a contralateral rechallenge. antitumor activity was driven by il- as the alvac vector itself displayed very limited tumor inhibition. in a phase i study in patients with unresectable metastatic melanoma, i.t. injections of alvac-il- resulted in increased intratumoral il- mrna expression in only of patients ( ) . three patients experienced modest increases in serum il- and ifnγ levels and no dose-limiting toxicities were observed. one patient had a complete response of an injected lesion and an uninjected in-transit metastases ( ) . in a similar phase i study performed by the same group, only of evaluable patients exhibited an increase in intratumoral il- mrna after alvac-il- administration ( ) . attenuated measles viruses (mevac) have been used safely as a vaccine to prevent measles in billions of people. mevac is another type of oncolytic virus which makes it an attractive potential cancer therapy. mevac encoding il- (mevac fmil- ) achieved complete regression of carcinoembryonic antigen (cea) expressing mc tumors in % of mice treated i.t. ( ) . mev encoding anti-pd-l or igg-fc induced complete regressions in and % of treated mice, respectively. antitumor activity was cd + t cell dependent and correlated with high levels of intratumoral ifnγ. all mice experiencing complete regressions following mevac fmil- also complete rejected tumor rechallenge ( ) . a follow up study demonstrated that mevac fmil- was more effective at controlling tumors than mev encoding il- in two murine tumor models ( ) . against mc -cea tumors, mevac fmil- eliminated tumors in all treated mice whereas mev-il induced complete tumor regressions in % of mice ( ) . effects appear to be model dependent as treatment of b tumors expressing human cd with mev-il- and mev-il only modestly extended survival ( ) . vesicular stomatitis virus (vsv) is another potent oncolytic virus under exploration for cancer therapy. vsvs engineered to express il- (vsv-il- ) were recently evaluated in an orthotopic floor of mouth model of sccvii murine squamous cell carcinoma ( ) . multiple i.t. injections of vsv-il- were more effective at eliminating tumors and induced higher longterm survival rates ( vs. %) compared to non-cytokine encoding vsv ( ) . moloney murine leukemia virus (momlv) is a positive sense, single-stranded rna (ssrna) retrovirus. a momlv expressing il- was engineered to display an anti-erbb scfv against the her receptor ( ) . eight daily i.t. injections of -day-old flank mbt tumors was found to result in inhibition and complete regression in about one-fifth of mice treated with the her targeted momlv-il- ( ) . a hybrid viral vector which pairs the high infectivity of adenoviruses with the high gene expression of sfv has been engineered to specifically infect and express il- in hepatocellular carcinoma cells ( ) . in buffalo rats bearing orthotopic mca-rh , the adv-sfv hybrid vector expressing il- induced complete regression in of treated animals. while the non-hybrid sfv-il- induced complete regression in % of treated rats, treatment was accompanied by elevated liver enzymes indicating hepatotoxicity ( ) . in contrast, liver enzyme levels following administration with the tissue-specific, hybrid vector were no different than levels in saline-treated control rats ( ) . newcastle disease virus (ndv) is an oncolytic, enveloped, negative-sense, single-stranded rna virus. ndvs have been engineered to express checkpoint inhibitor molecules and checkpoint inhibitor-cytokine conjugates ( ) . although the antitumor activity of i.t. ndv expressing il- alone was not tested, i.t. injections of ndv-anti-cd -mil- plus i.p. anti-ctla- or ndv-antipdl -mil- plus i.p. anti-ctla- resulting in complete responses in or %, respectively of mice bearing day-old b f flank tumors ( ) . the same treatments of one of two bilateral tumors resulted in and % complete responses, respectively, of untreated tumors ( ) . a major benefit of virus-based il- delivery is high transfection rates, particularly when compared to non-viral vectors. however, virus-based delivery is limited in several aspects. first, neutralizing immune responses including anti-viral antibody production ( , , ) and dth responses ( ) may preclude repeated injections with the same vector. however, the extent to which anti-viral immunity limits repeated injections is unclear. mice previously exposed to a viral vector demonstrated a . -fold reduction in intratumoral transgene expression. this level of reduction did not affect antitumor activity; however, the potential exists for reduced antitumor immune responses when lower doses of virus are used ( ) . on the positive side, viral dissemination and transgene expression in non-targeted liver tissue was reduced by more than , -fold in mice pre-exposed to the viral vector ( ) . secondly, viral il- delivery requires transfection of host cells, which has been shown to be highly heterogeneous due to inherent susceptibility or previous exposure to related vectors ( ) . in an extreme example of susceptibility, a dose of adcmvmil- that was found to be lethal in % of treated c bl/ mice did not result in a single death in the balb/c strain ( ) . livers of c bl/ mice exhibited much higher levels of adenovirus transduction ( ) . the current coronavirus outbreak further illustrates the point that some individuals appear to be more susceptible than others to viral infections. third, although clinical trials have shown that recombinant viruses are tolerated, viral dissemination, and infection in liver cells following i.t. injection were reported at an alarming degree ( ) ( ) ( ) . even when small volumes ( - µl) of recombinant adenoviruses were injected i.t., transgene expression was found in the liver, intestine, spleen, kidney, and brain. as much as % of the injected adv infected non-targeted normal tissues ( ) . similar levels of transgene expression was found in the liver and tumor days after an i.t. injection ( ) . fourth, some viral vectors, such as momlv and mev, can insert their genetic information into an infected cell's genome causing undesirable mutations and uncontrolled longterm expression of il- . the benefits of using such integrating vectors must be weighed against these risks as other viruses, such as adv, hsv, vsv, and ndv, are non-integrating and induce transient expression ( ) . lastly, and most relevant to the topic of localizing il- , even local injections of viruses encoding il- are not able to prevent il- dissemination ( , , , , ) . consequently, antitumor responses and il- -mediated adverse events are strongly correlated with virus dose. similar to plasmidbased il- delivery, novel strategies that anchor il- to an injection site or prevent its secretion appear best suited to break this correlation and widen the therapeutic window of virusencoding il- . dendritic cells ( , ) , fibroblasts ( , ) , macrophages ( , ) , tumor cells ( , ) , and mesenchymal stem cells (mscs) ( ) ( ) ( ) have all been engineered to express il- . transduced dcs, fibroblasts, tumor cells, and macrophages have been directly injected into tumors, while mscs have been injected systemically for migration to tumor beds ( , ) . a much larger number of preclinical and clinical studies have used il- -transduced tumor cells for s.c. immunization. these studies are not covered in this review as il- in this context is functioning as a vaccine adjuvant following injection of transduced cells in healthy, non-cancerous s.c. tissue rather than a strategy to localize il- to a tumor. dendritic cells and macrophages have been engineered to express a variety of cytokines including il- . in addition to supplying the tumor microenvironment with il- , transduction of these antigen presenting cells increases their ability to induce robust antitumor immune responses. following i.t. injection, dcs and macrophages are capable of capturing tumor antigen, migrating to draining lymph nodes, presenting antigen and priming tumorspecific t cell responses. transfection of antigen presenting cells with il- helps polarize t cells to a th phenotype and facilitate robust tumor-specific ctl responses ( ) . in an early preclinical study, bone marrow-derived dendritic cells (bmdcs) expressing il- were more effective than dcs expressing il- in controlling the growth of b f melanomas and generating tumor-specific ctl activity ( ) . il- -transduced bmdcs were also found to significantly suppress established mca fibrosarcomas, b melanomas, d adenocarcinomas, and - bma prostate carcinomas following a single i.t. injection ( , , ) . in one study, % of treated mca tumors were completely rejected. tumor-free mice also rejected a subsequent mca rechallenge ( ) . the growth of contralateral, non-treated tumors was also suppressed, implying that systemic tumor-specific immunity can be induced following i.t. injection. il- -transduced bmdcs were substantially more effective than il- -transduced fibroblasts or non-transduced bmdcs at inducing tumor-specific ctl activity and ifnγ production by draining lymph node cells and splenocytes ( ) . enhanced trafficking of dcs to regional lymph nodes provides a major advantage over non-migratory cells such as il- transduced tumor cells or fibroblasts. dcs transfected with adenovirus to express il- (dc-adcmvil- ) were found to eliminate ct and mc colon adenocarcinomas in - % and % of mice, respectively ( , ) . mice experiencing complete regression exhibited tumor-specific ctl activity and rejected tumor rechallenge. i.t. injections of adcmvil- -infected fibroblasts or allogeneic dc-adcmvil- were not effective ( ) . in a similar study, dc-adcmvil- reversed immune suppression mediated by tbj neuroblastomas and induced complete tumor regression ( ) . dcs engineered to co-express both il- and il- displayed synergistic antitumor activity that was greater than either dcencoding cytokine alone ( ) . all mice experienced complete regression of cms tumors while treated metha tumors were substantially reduced ( ) . while serum ifn-γ levels reached pg/ml days after dc administration, no toxicities were noted ( ) . bmdcs transfected by simian virus (sv ) to express il- or il- demonstrated complete regression of ct colon adenocarcinomas in or % of mice, respectively ( ) . there was no synergy between il- and il- when delivered together. interestingly, maturation of dcs with lps prior to i.t. injection impaired antitumor activity, presumably due to reduced antigen loading in the tumor. however, this hypothesis was not evaluated ( ) . transfection of bmdcs isolated from cms sarcoma-bearing mice with an adenoviral vector expressing il- was shown to restore antigen-presenting and allostimulatory functions ( ) . direct injections of the rescued bmdcs were shown to eliminate intrahepatic cms tumors and induce protective immunity ( ) . dcs can also be isolated from mouse spleens prior to transduction with an il- encoding vector. a recent study demonstrated that splenic dcs overexpressing il- can inhibit the growth of melanomas in mice ( ) . il- -transduced macrophages are less well-studied, however, like dcs, macrophages have antigen presenting capabilities that can be enhanced by il- . a single i.t. injection of peritoneal exudate-derived macrophages transduced by admil- induced suppression of primary and metastatic - bma orthotopic prostate tumors ( ) . serum il- levels peaked days after injection and remained elevated for at least weeks. antitumor activity was correlated with increased leukocytic infiltrate and cytolytic activity ( ) . the admil- -transduced macrophages were found to migrate to draining lymph nodes following i.t. injection. similar to some viral constructs, to reduce the potential for toxicity, il- expression can be placed under the control of an inducible promoter. one inducible system consists of cassettes including gal -ecr;vp -rxr and il- . first, gal -ecr and vp -rxr are expressed under the constitutive ubiquitin c promoter. the heterodimerization of gal -ecr and vp -rxr are triggered by addition of small-molecule ligand, e.g., pharmacologically inert diacylhydrazine. gal -ecr-vp -rxr heterodimers bind to a synthetic promoter containing gal binding site and induce the expression of il- ( ) . dcs with inducible il- demonstrated inhibition of renca and metha tumors following i.t. administration ( ) . combining il- expressing dcs with either il- -or ifnα expressing dcs did not improve antitumor efficacy ( ) . the ability to turn cytokine expression "on" and "off " is an attractive feature which may potentially allow for better control over cytokine delivery. to date, only one clinical study has been published. in a phase i study, autologous dendritic cells transfected to express il- were administered i.t. to patients with metastatic gastrointestinal carcinomas ( ) . of the treated patients, one experienced a partial response and two experienced stable disease. serum ifnγ, but not il- , levels were elevated following treatment. four instances of transient grade lymphopenia were observed, but no grade toxicities were observed. in vitro il- production was highly variable, most likely due to differences in susceptibility of dendritic cell infection with adenoviruses encoding il- ( ) . fibroblasts expressing il- caused the elimination of -day mca tumors in - % of mice after a single peritumoral injection ( ) . in addition, local il- -mediated regression was also able to control tumors on the contralateral flank. as has been shown in other systems, i.t. injections were more effective than distant s.c. injections at controlling tumor growth. repeated injections of il- -engineered fibroblasts were welltolerated, with no abnormalities detected in liver and renal function tests ( ) . a similar peritumoral implantation of alginate encapsulated nih t fibroblasts expressing il- was found to inhibit ct tumor growth ( ) . in a phase i study, peritumoral injections of il- -transduced autologous fibroblasts induced transient tumor reductions in four of nine patients with disseminated cancers ( ) . treatment with fibroblasts capable of secreting up to , ng of il- per day was well-tolerated ( ) . mesenchymal stem cells (mscs) are multipotent adult stem cells that divide and differentiate in order to repair skeletal tissues, such as bone, cartilage, and muscle. as such, mscs are predisposed to traffic from bone marrow to a wound site based on expression of soluble factors and unique receptors in damaged tissues. tumors can express many of the same factors and receptors which results in mscs trafficking to, and infiltrating tumors following systemic injections ( ) . thus, using il- transduced mscs as a "trojan horse" to target tumors and deliver il- is a promising approach. in one preclinical study, ad.il- -transduced mscs inhibited the growth of tc human ewing sarcomas in mice ( ) . one potential concern about il- -expressing mscs (msc/il- ) is their tropism for some normal tissues. in the above study, msc/il- were found in tumor, liver, lung and spleen tissues but not kidney, heart, or brain tissues days after i.v. injection ( ) . in a different study, high levels of msc/il- were found in the tumor but not in the liver, lung, and spleen days after administration ( ) . earlier time points were not presented. il- levels were high in the tumor; however, significant serum il- levels were found to persist for at least weeks after msc/il- administration ( ) . these high levels of intratumoral il- resulted in the inhibition of - renal cell carcinomas in nude mice ( ) . in a study that illustrates the importance of tropism, mscs that were non-virally transfected with pil- significantly inhibited b f lung metastases but not subcutaneous b f tumors following i.v. administration ( ) . only when administered i.t., were msc/il- cells able to inhibit s.c. b f tumors ( ) . despite the tumor-homing potential of mscs, published data suggest that msc/il- may be most effective at treating tumors in tissues where mscs have a natural tropism, e.g., lung, liver and bone ( ) . several other studies have favored local over systemic administration of msc/il- to treat various tumors. intracranial gl glioma-bearing mice treated with i.t. ucb-msc-il m generated from human umbilical cord blood experienced significantly prolonged survival ( ) . specifically, % of ucb-msc-il m treated mice survived more than days post tumor implantation whereas all mice treated with mscs expressing gfp succumbed within days ( ) . cured mice were completely protected from ipsilateral and contralateral tumor rechallenge ( ) . in another study, bone marrow derived mscs were transfected with a lentivirus to express mil- ( ) . these lenti-mil- -mscs reduced the volume of h and metha ascites and increased survival when administered i.p. and days after tumor inoculation ( ) . no pathological abnormalities were noted on biopsies taken from internal organs of lenti-mil- -msc treated mice ( ) . human mscs transduced with a retroviral vector expressing a modified il- (mscs/il- m) inhibited b f melanomas following five i.t. injections over weeks ( ) . safety was implied as no il- was detected in the serum following treatment. interestingly, the antitumor activities elicited by i.t. injections of mscs/il- m and il- plasmid dna were similar ( ) . in a rare comparison of cytokine delivery technologies, i.t. injections of mscs transfected with rad/il- m inhibited b f primary and metastatic tumors more effectively than i.t. injections of rad/il- m alone ( ) . despite the homing feature tsa mammary carcinoma cells transduced to secrete il- were found to cure of mice bearing -day established tsa tumors following local injection ( ) . when tumors were allowed to establish for seven days prior to treatment, antitumor efficacy dropped, with only of mice experiencing tumor regression. local delivery of l gliosarcoma cells engineered to express il- ( l-il ) significantly prolonged the survival of rats challenged intracranially with wild-type l tumors ( ) . similar results were observed whether the l-il treatment was given at the same time or days later than the tumor inoculation. the authors noted that only mice receiving the delayed treatment were protected from tumor rechallenge ( ) . natural killer (nk) cells and chimeric antigen receptor (car) t cells have also been transduced to produce il- . in one recent study, primary nk cells isolated from c bl/ mice and transduced to express il- (nk il− ) increased mhc class i expression in ova-transfected melanoma cells (b m ) compared to mock plasmid transduced nk cells ( ) . combination treatment studies involving tumor-specific cytotoxic t lymphocytes (ctls), pegylated il- , and activated nk il− revealed that only groups containing activated nk il− demonstrated statistically significant prolonged survival over mock constructs, untreated, and ctl alone controls in b -ova tumor bearing mouse models. in particular, nk il− plus il- treatment increased survival by . days, while nk il− plus ctl and il- prolonged survival for . days. nevertheless, all mice eventually succumbed to the disease and no stable tumor regressions were reported ( ) . car t cells have shown remarkable efficacy against hematologic malignancies but have yet to achieve clinical benefit against solid tumors. engineering car-t cells to express il- is under investigation to improve antitumor efficacy against solid tumors. in one study, i.v. infusion of il- expressing anti-vegfr car t cells induced curative regressions in - % of mice in five different tumor models including - -day-old b f melanomas, mca- sarcomas, mc colorectal adenocarcinomas, mc - sarcomas or - days old ct colon adenocarcinomas ( ) . t cells singularly transduced with either anti-vegfr- or il- were markedly less effective ( ) . because constitutive systemic expression of il- can be toxic, inducible il- expression strategies have been developed. by using an nfat-responsive promoter, il- could be expressed only after tcr engagement ( ) ( ) ( ) . this modification reduced toxicity but required lymphodepletion to achieve durable tumor regressions ( ) . the efficacy of car t cell therapy in general is dependent upon lymphodepletion via chemotherapy or radiotherapy. in an attempt to eliminate the need for lymphodepleting preconditioning, cd car t cells expressing il- (cd /il- ) have been investigated ( , ) . indeed, treatment with cd /il- car t cells significantly enhanced the survival of cd -el tumor-bearing mice vs. treatment with cd car t cells ( vs . % survival at day after tumor inoculation) ( ) . similarly, second generation cd /il- car t cells produced a % long-term survival rate in mice with established a b cell lymphomas. without the il- insert, all mice treated with cd car t cells succumbed to disease ( ) . h - z/il- car t cells, which are specific for the muc- ecto antigen and secrete il- , controlled skov human ovarian carcinomas in scid-beige mice following i.p. infusion or weeks after tumor implantation ( ) . specifically, ∼ or % of mice treated or weeks, respectively, after tumor implantation with h - z/il- car t cells survived for days. in contrast, ∼ % and % of mice treated or weeks, respectively, after tumor implantation with h - z car t cells, not containing the il- gene, survived for days ( ). glypican- (gpc )-specific car t cells with inducible expression of il- (gpc - z-nfat-il- car t) significantly inhibited or eliminated established plc/prf/ and huh- human hccs ( ) . the il- secreting car t cells resulted in decreased t reg infiltration. serum ifnγ and il- levels were elevated although neither pathological changes to critical organs nor significant loss in body weight were observed ( ) . it should be noted, however, that these studies were performed in immunocompromised mice which is not a reliable model for immune-related adverse events. from a clinical standpoint, administrations of il- -transduced cells have been well-tolerated, with patients experiencing only transient symptoms of lymphopenia, fever, and malaise; however, significant, durable clinical responses have been elusive. from a manufacturing perspective, the transduction of allogeneic and xenogeneic cells is easier and more reproducible. however, these cells are rapidly recognized and rejected by the host immune system. thankfully, recent advances in autologous cell transduction due to the emergence of car t cell immunotherapy has eliminated many concerns related to technical feasibility and patient heterogeneity ( ) . local administration of recombinant il- protein is the most direct and most quantifiable strategy in terms of ensuring the accuracy and reproducibility of a delivered dose. however, recombinant cytokines disseminate rapidly from local injection sites into the systemic circulation ( ) . thus, in order to maintain high levels of recombinant il- in the tumor microenvironment while minimizing systemic exposure, sustained delivery technologies, capable of locally releasing proteins and cytokines over extended periods of time following direct injection, are under development ( ) . the encapsulation of pharmaceuticals in polymeric microspheres for sustained delivery has been explored for several decades ( , ) . the use of microspheres to deliver cytokines is a more recent trend. synthetic polymers, such as poly-lactic coglycolic acid (plga), poly-caprolactone (pcl), and poly-lactic acid (pla), that have been widely explored for drug delivery, are being adapted to encapsulate and deliver various cytokines including il- ( , ( ) ( ) ( ) ( ) ( ) . human il- -loaded pcl:pla microspheres, when co-delivered with human peripheral blood lymphocytes (pbls), were shown to prevent engraftment of human tumors in scid mice ( ) . peg-il- also suppressed tumor engraftment and growth, but not as effectively as il- -loaded microspheres. although high levels of il- and ifn-γ were found in sera after administration of il- -loaded microspheres, this localized delivery strategy was more effective at preventing tumor engraftment than repeated i.p. injections of il- ( ) . similarly, a single i.t. injection of il- -loaded pcl:pla microspheres outperformed bolus injections of il- in the inhibition of human head and neck tumor tissue xenografts containing human pbls ( ) . il- -loaded microspheres completely suppressed engraftment in % of mice, whereas il- alone slowed but could not eliminate tumor growth ( ). in the above xenograft studies, antitumor activity was mediated by cd + t cells and cd + natural killer cells ( , ) . in yet another study, lung tumor xenografts using non-disrupted human lung tumor tissue were completely suppressed when injected with il- -loaded pla microspheres week after implantation ( ) . mechanistically, quiescent cd + effector memory t cells present within the tumor microenvironment were reactivated upon exposure to high local levels of il- ( ) . the proliferation of reactivated cd + cells and their production of ifn-γ facilitated tumor destruction ( ) . in murine tumor models, i.t. injections of il- -loaded pla microspheres eliminated up to % of line- lung alveolar carcinomas ( ) . in contrast, i.t. injections of free il- induced a complete response in only one of five treated mice. interestingly, mice experiencing complete tumor regression following microsphere administration were more resistant to tumor rechallenge than mice vaccinated with either live or irradiated line- cells co-injected with il- -loaded microspheres ( ) . in other studies, i.t. injections of il- loaded pla microspheres significantly reduced the incidence and number of spontaneous pulmonary tumor nodules ( , ) . when administered neoadjuvant to tumor resection, il- -loaded microspheres inhibited local recurrence, reduced pulmonary metastases, and improved disease-free survival as compared to resection alone ( ) . a similar study using more advanced line- tumors, which were , - , mm at study onset, demonstrated that neoadjuvant i.t. injections of il- -loaded microspheres prevented both local recurrence and pulmonary metastases better than surgery alone ( ) . the addition of gm-csf-loaded pla microspheres enhanced antitumor activity and survival. however, more cytokines were not always better, as the addition of low dose il- to neoadjuvant immunotherapy with il- -and gm-csf-loaded microspheres abrogated antitumor immunity ( ) . a theme common to nearly all il- -based immunotherapies was that il- -loaded microspheres induced cd + t cell and nk cell activation, including increases in cytolytic function and ifn-γ expression. in-depth effector mechanisms are elegantly described in a series of papers by egilmez and kilinc et al. ( , , ( ) ( ) ( ) ( ) . in addition to inducing a strong effector response, a potentially more important feature of i.t. injected il- -loaded microspheres is their potential to reverse immunosuppression in the tumor microenvironment. for example, a single i.t. injection of il- -loaded microspheres with and without gm-csf-loaded microspheres induced apoptosis and elimination of tumor-infiltrating cd + cd + foxp + t suppressor cells ( , ) . il- -loaded microspheres were also shown to reprogram immunosuppressive tumor-infiltrating macrophages (tims) and tumor-associated macrophages (tams) to a proinflammatory, antitumor phenotype ( , ) . microsphere platforms are well-suited to explore codelivery of multiple cytokines. the combination of il- -and tnfα-loaded pla microspheres was shown to be more effective than il- and gm-csf-loaded microspheres at eliminating mt- mammary carcinomas ( ) and b melanomas ( ) . nearly and % of mt- tumor-bearing mice were rendered tumor-free following i.t. injections with dual cytokine loaded microspheres of il- /tnfα and il- /gm-csf, respectively ( ) . dual cytokine loaded microspheres of il- /tnfα also induced increases in tumor-specific cytokine release from effector cells as compared to microspheres containing either cytokine alone ( ) or the combination of il- and gm-csf ( , ) . a single i.t. injection of il- -and il- -loaded pla microspheres outperformed injections of either cytokine-loaded microsphere alone in terms of delaying the growth of b melanomas ( ) . unlike previous studies demonstrating the elimination of % of line- tumors ( ) and impressive reductions in metastatic lesions ( , ) , il- -loaded microspheres had no impact on either primary b tumor growth or pulmonary metastases ( ) . interestingly, the triple combination of il- /il- /tnfα loaded in microspheres was found to reduce antitumor activity in the t model ( ) . in her- /neu transgenic mice, which develop spontaneous, multifocal mammary carcinomas, il- -and gm-csf-loaded microspheres injected intratumorally caused complete regression of primary tumors in up to % of mice ( ) . unfortunately, responses were temporary, and all tumors recurred. interestingly, chronic immunotherapy with cytokine-loaded microspheres could not control tumor burden due to a progressive decline in the intensity of antitumor t cells and an increase in tumor-infiltrating cd + cd + foxp + t suppressor cells with repeated injections ( , ) . subsequent studies have shown that administration of cyclophosphamide can block the postimmunotherapy increase in t suppressor cells ( , ) . recently, intratumoral delivery of il- microspheres (il- ms) in combination with stereotactic body radiation (sbrt) resulted in remarkable tumor regression in several types of preclinical pancreatic ductal adenocarcinoma mouse models ( ). this study showed that intratumoral levels of ifnγ were enhanced following the treatment of sbrt/il- ms, which in turn increased cd + t cell activation. sbrt/il- ms treatments also established systemic tumor immunity that was confirmed by antitumor effects on liver metastases ( ). chitosan (cs) is a bioadhesive polysaccharide derived primarily from the exoskeletons of crustaceans. it is nontoxic, biodegradable, and non-immunoreactive ( ) with an established safety profile in humans. co-formulation of il- in cs (cs/il- ) has been shown to increase the i.t. residence of il- from - days to - weeks ( ) . cs is believed to hinder il- diffusion through viscous and electrostatic interactions. the sustained presence of cs/il- induced complete regression of - % of mice bearing mc , panc , ct , e , and mb tumors ( , ) . administration of cs/il- neoadjuvant to t tumor resection resulted in complete clearance of lung metastases and long-term survival in about two-thirds of mice. in contrast, all mice treated with surgery alone died within days and mice receiving il- without cs neoadjuvant to resection had a median survival of days ( ). cured mice rejected a subsequent challenge with live t cells, but not live renca cells illustrating the specificity of the antitumor immune response. in another demonstration of systemic tumorspecific immunity from localized il- , % of mice bearing both flank and orthotopic mb bladder tumors were able to clear the flank tumor following intravesical administration of cs/il- ( ) . in contrast, mice with only a flank tumor, did not benefit from intravesical cs/il- in a naïve bladder. these data indicate that intravesical cs/il- induced systemic tumor-specific immunity only when il- was localized in or near a tumor. in a subsequent study, orthotopic bladder tumor regression following intravesical cs/il- immunotherapy was associated with a rapid infiltration of macrophages and granulocytes followed by increases in cd + and cd + effector t cells along with a reduction of immunosuppressive t regs and mdscs ( ) . importantly, local administrations of cs/il- did not result in significant toxicity, such as elevated liver enzymes, commonly observed following regular, free il- injections ( ) . a polysaccharide gel matrix, designated f gel, is comprised of % deacetylated poly-n-acetyl glucosamine (p-glcnac) coformulated with lactate salt. p-glcnac gels were evaluated as slow-release cytokine depots for gm-csf, il- , and il- ( ) ( ) ( ) ( ) ( ) . in particular, i.t. injections of il- in p-glcnac gel delayed the growth of ova-transfected ae malignant mesotheliomas ( ) . interestingly, gm-csf and il- depots had no impact on mesothelioma growth, although only a single i.t. injection was given. the p-glcnac gel by itself induced a strong, transient inflammatory response which was thought to be due to a foreign body reaction or toxic species that may have been contained within the gel ( , ) . human il- has been encapsulated in multilamellar liposomes for sustained release after i.t. injection ( ) . antitumor activity on xenografts of human lung tissues indicated that liposomal encapsulation is a promising approach capable of eliminating tumor cells and inducing lymphocyte infiltration weeks after i.t. injection. liposomal leakage and/or il- liposome dissemination could be problematic following delivery of large doses ( µg/mouse) as significant sustained levels of il- and ifn-γ were found in sera of treated mice ( ) . capitalizing on the tumor disruption caused by cationic liposomes, il- -and tlr ligand monophosphoryl lipid a (mpl)-loaded cationic liposomes were injected i.t. to treat t tumors. this combination produced an abscopal effect which arrested growth in both treated and distal tumors but did not result in tumor elimination ( ) . to date, sustained release technologies for recombinant il- have not been evaluated in clinical studies. while sustained release technologies are designed to maximize il- concentrations in the tumor microenvironment, some amount of delivered il- can be expected to reach the systemic circulation. however, due to the infrequency of administration for these long-acting platforms, they are unlikely to induce the same lethal toxicities that were observed following the frequent systemic il- administrations in early clinical trials ( ) . nevertheless, it will be essential to perform rigorous pharmacokinetic and toxicology studies prior to clinical translation of any sustained recombinant il- platform. a possible limitation of the microsphere encapsulation technology is the use of organic solvents during the encapsulation process. organic solvents can denature cytokines immediately or adversely affect long term storage. one study indicated that cytokines lose about half of their bioactivity during microencapsulation ( ) . loss of bioactivity appears to be cytokine dependent ( ) . specific activity of il- after encapsulation in pla microspheres was %. however, more than % of the bioactivity of il- was lost when pla/il- microspheres were stored for days ( ) . in addition, because many commonly used biodegradable polymers are comprise of acidic monomers, polymer degradation often forms highly acidic microenvironments that may inactivate cytokines ( ) . a number of techniques are under investigation to either raise ph ( ) or stabilize encapsulated cytokines ( ) . a common limitation of liposomal carriers is their inherent leakiness, and therefore the choice of lipid and modifications to stabilize the bilayer are critical for sustained release. high temperature phase-transition lipids, such as dspc, increase the retention of cargo and enhance drug circulation time. incorporation of cholesterol into the lipid bilayer increases the rigidity and further decreases leakage of interior cargo ( ) . another major limitation of liposomes is opsonization by serum proteins and clearance by phagocytes of the reticuloendothelial system (res). pegylation can reduced res clearance, however, peg can sterically inhibit ligand-cell interaction ( ) . if administered i.t., loss of tumor targeting is likely not detrimental. while some localized il- immunotherapies have shown limited efficacy in clinical studies, a long-awaited breakthrough leading to widespread application of localized il- as a monotherapy does not appear imminent. however, there are a number of near-term opportunities to combine localized il- with traditional or experimental cancer management approaches. and in fact, most contemporary experimental therapies for aggressive or advanced cancers involve combination approaches. thus, near future opportunities involving translatable combination approaches are explored in the following sections. the majority of cancer patients with solid tumors undergo tumor resection as part of their treatment. surgery, by itself, is unable to prevent recurrence and/or metastasis which is responsible for out of cancer-related deaths. patients at high risk of recurrence receive adjuvant chemotherapy and/or radiotherapy following resection. while chemotherapy and radiotherapy may help reduce the risk of recurrence, they induce life-altering side effects and fail in a significant fraction of patients. as a result, most metastatic cancers arise from previously treated non-metastatic disease ( ) ( ) ( ) . administration of localized il- to the tumor microenvironment prior to surgery has the potential to reduce recurrence rates and/or eliminate occult metastases through the induction of systemic, tumor-specific immunity. as mentioned above, our own investigation demonstrated that neoadjuvant i.t. injections of cs/il- prior to resection can improve overall survival from to % in a highly metastatic t breast carcinoma model ( ) . cured mice demonstrated tumor-specific immunity via tumor rechallenge, ctl activity, and elispot assays. if proven to be safe, localized il- can be administered neoadjuvant to any solid tumor resection, not just in high risk patients. in fact, at the time of surgery, it is likely that a significant fraction of patients already has occult metastases that cannot be detected via radiographic imaging. the median primary breast tumor size for which % of patients had micrometastases was found to be only . cm ( ) . while most immune-oncology trials focus on developing therapies for metastatic disease, an approach, such as neoadjuvant localized il- , capable of preventing metastasis in the first place, may be a more effective strategy to reducing cancer mortality. the combination of il- -based immunotherapy and chemotherapy ("biochemotherapy") is also under exploration. several clinical studies have shown significant clinical benefit when certain cytotoxic drugs are combined with il- ( , , ) . there are three distinct advantages for combining chemotherapy with il- immunotherapy. first, chemotherapy provides a debulking benefit with the elimination of some or most of the tumor. second, some chemotherapeutics can deplete immunosuppressive cells. cyclophosphamide has been shown to deplete t regs while -fluorouricil and gemcitabine have been shown to deplete mdscs. third, some chemotherapies cause upregulation of t cell-attracting chemokines in the tumor ( ) . for example, melanoma-bearing mice treated with temozolomide, cisplatin, or dacarbazine increase expression of ccl , cxcl , and cxcl ( ) . the timing of chemotherapy relative to local il- immunotherapy is expected to be critical. unlike the neoadjuvant resection approach mentioned above, chemotherapy should be administered prior to il- therapy to avoid elimination of beneficial proliferating immune cells. chemotherapy may synergize with il- by reducing tumor burden and creating a source of tumor antigen for in situ vaccination. in fact, certain chemotherapies can induce an immunogenic cell death that is capable of priming an antitumor immune response ( ) . to date, several examples demonstrate the promise of chemotherapy plus local il- delivery. antitumor efficacy of pil- /ppc is improved with paclitaxel, cyclophosphamide, or taxol/paraplatin ( , , ) . the antitumor effect of il- lipoplexes against intracranial glioma was substantially improved when combined with fda-approved carmustine (bcnu) wafers ( ) . finally, a phase- clinical trial found that intraperitoneal il- gene therapy in combination with intravenous pegylated liposomal doxorubicin resulted in greater clinical benefit relative to the chemotherapeutic alone ( . % vs. - . %) in patients (n = ) with platinum-resistant epithelial ovarian cancer ( ) . standard-of-care radiotherapy of inoperable tumors can be administered prior to local il- delivery. radiation-induced tumor cell death can supply a source of tumor antigens, while radiation has been shown to upregulate co-stimulatory molecules on the surface of tumor cells which makes them more susceptible to immune-mediated killing ( ) . the addition of tumor irradiation after pil- +ep was found to improve antitumor activity ( ) . similarly, radiotherapy improved the antitumor activity of i.t. oncolytic adenoviruses expressing il- and gm-csf ( ) . it has also been found that radiotherapy can induce infiltration of mscs in the tumor site. through a combination of radiation and il- -expressing mscs, both primary tumor growth and metastases were reduced in hca-i and hepa - hepatoma models ( ) . in another study, radiation along with an i.t. injection of an il- -encoding adenoviral vector greatly reduced tumor growth and metastasis compared to either treatment alone in a bnl-p hepatocellular carcinoma model, while a near-complete abscopal response occurred in a ct colorectal cancer model ( ) . an abscopal response was also induced in a fully humanized rhabdomyosarcoma xenograft model. mice with bilateral tumors were treated with a combination of single-tumor radiation and systemic nhs-il , which resulted in significantly lower tumor burdens than either individual treatment ( ) . cryoablation, radiofrequency ablation (rfa), microwave ablation (mwa), embolic microspheres and high intensity focused ultrasound (hifu) are routinely used in the clinic to destroy some operable and many inoperable solid tumors. irreversible electroporation is another ablative technology in clinical and preclinical development ( ) ( ) ( ) ( ) . all of these strategies have the potential to induce in situ vaccinations through the release of tumor antigen, although abscopal responses or robust anti-tumor immune responses are rare. among the ablation strategies, cryoablation appears distinct from most other ablation approaches that kill via high temperatures and result in coagulation, enzyme inactivation, and antigen denaturation. cryoablation is thought to mostly preserve tumor antigen structure ( , ) and has been shown to induce greater levels of tumor antigen uptake by dendritic cells compared to rfa ( ) . in addition, cryoablation also induces higher levels of inflammatory cytokines, such as il , il , and tnfα, compared to rfa and mwa ( , ) . perhaps most importantly, cryoablation was recently shown to induce expansion of oligoclonal t cells both in tumor tissues and in peripheral blood of kidney cancer patients ( ) . the addition of localized il- to the above ablation strategies may enhance tumor control as well as antitumor immunity. our recent preclinical data, demonstrated that the combination of cryoablation and cs/il- could eliminate multiple tumor types and induce abscopal immunity in a bilateral flank model ( ) . ongoing studies are aimed at determining mechanisms of systemic tumor control. immune checkpoint inhibitors are ineffective against noninflamed, "cold" tumors. localized il- induces profound intratumoral infiltration that can be expected to synergize with checkpoint inhibitors. indeed, the addition of anti-pd- or anti-ctla- improved the antitumor activity of l -il- in multiple murine tumor models ( ) . similarly, systemic anti-ctla- in combination with intratumoral il- induced synergistic antitumor responses in gl- glioblastoma and sma- astrocytoma models ( ) . also, as mentioned in a previous section, i.t. injections of vv-il- and vv-il enhanced the responsiveness of poorly immunogenic tumors to checkpoint inhibition ( ) . finally, a recent phase ii study of i.t. pil- +ep and systemic pembrolizumab in patients with non-infiltrated melanomas resulted in a % objective response rate with % complete responses ( ) . correlative studies demonstrated increased antigen cross-presentation along with enhance t cell infiltration and activity leading to higher than expected clinical responses ( ) . il- enhances cytotoxic t and nk cell activity while reversing tumor-induced immunosuppression, inhibiting angiogenesis, increasing lymphocyte trafficking and antigen presentation either directly or through induction of ifnγ ( ) . based on these diverse mechanisms as well as its profound antitumor activity in numerous preclinical studies, il- is arguably one of, if not, the most potent antitumor cytokine evaluated to date. unfortunately, the much-anticipated clinical translation of il- -based immunotherapies suffered a tremendous setback in the late s/early s due to severe clinical toxicities associated with systemic il- injections. through the development of several clever approaches to localize il- to the tumor microenvironment while limiting systemic exposure, il- -based immunotherapies are making a comeback. several clinical studies evaluating localized il- have been initiated ( table ) with more on the way. while each delivery strategy has limitations, the approaches reviewed above may retain enough il- in the tumor while eliminating the need for frequent systemic injections. by reducing the potential for il- -mediated toxicities associated with systemic injections, localized il- can expand the therapeutic window and finally allow il- to fulfill its considerable potential. as exemplified by the current immune-oncology clinical trial landscape, combination approaches appear to be most effective for accelerating clinical impact. several promising preclinical combination approaches with localized il- are likely to be translated in the near future. there is unprecedented interest in finding immunomodulators that can enhance lymphocytic infiltration in order to improve the efficacy of checkpoint inhibitors. localized il- , based on its ability to drive th responses, enhance cytolytic activity, and protect t cells from pd- /pd-l exhaustion and ifnγ-induced apoptosis may be an ideal partner for checkpoint inhibitors. if safety concerns are assuaged, localized il- could be used in earlier stage cancer patients as a neoadjuvant to resection. for tumors that are inoperable, combining localized il- with ablation may help increase local as well as distant tumor control. with the interesting combination approaches, as well as the uptick in il- -based immunotherapies in clinical trials, there is reason to believe that localized il- may play a major role in cancer immunotherapy in the near future. studies on the pathogenesis of fever. ii. characterization of fever-producing substances from polymorphonuclear leukocytes and from the fluid of sterile exudates anticancer cytokines: biology and clinical effects of interferon-alpha , interleukin (il)- , il- , il- , and il- melanoma: therapeutic options with recombinant interferons results of treatment of patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin- therapy high-dose recombinant interleukin therapy for patients with metastatic melanoma: analysis of patients treated between and ( ) talimogene laherparepvec improves durable response rate in patients with advanced melanoma coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor identification and purification of natural killer cell stimulatory factor (nksf), a cytokine with multiple biologic effects on human lymphocytes cloning of cdna for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on t and natural killer cells cloning and expression of murine il- il- rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo the role of interleukin- on modulating myeloid-derived suppressor cells, increasing overall survival and reducing metastasis interleukin- : a master regulator immunological mechanisms of intravesical chitosan/interleukin- immunotherapy against murine bladder cancer. oncoimmunology differential effects of il- on tregs and non-treg t cells: roles of ifn-gamma, il- and il- r il- triggers a programmatic change in dysfunctional myeloidderived cells within mouse tumors antiproliferative effects of four different cytokines on renal carcinoma cell lines ifn-gamma induces apoptosis in ovarian cancer cells in vivo and in vitro inhibition of angiogenesis in vivo by interleukin inhibition of tumor-induced angiogenesis by the administration of recombinant interferon-gamma followed by a synthetic lipid-a subunit analogue (gla- ) cellular responses to interferon-gamma interleukin- in anti-tumor immunity and immunotherapy intratumoral immunotherapy of established solid tumors with chitosan/il- effect of interleukin on tumor induction by -methylcholanthrene therapeutic effect of interleukin on mouse haemangiosarcomas is not associated with an increased antitumour cytotoxic t-lymphocyte activity the anti-tumor activity of il- : mechanisms of innate immunity that are model and dose dependent antitumor and antimetastatic activity of interleukin against murine tumors phase i evaluation of intravenous recombinant human interleukin in patients with advanced malignancies pilot study of subcutaneous recombinant human interleukin in metastatic melanoma phase i trial of subcutaneous recombinant human interleukin- in patients with advanced renal cell carcinoma interleukin- therapy of cutaneous t-cell lymphoma induces lesion regression and cytotoxic t-cell responses phase i study of intraperitoneal recombinant human interleukin in patients with mullerian carcinoma, gastrointestinal primary malignancies, and mesothelioma phase study of the intravesical administration of recombinant human interleukin- in patients with recurrent superficial transitional cell carcinoma of the bladder a phase ii trial of interleukin- in patients with advanced cervical cancer: clinical and immunologic correlates. eastern cooperative oncology group study e e nk and cd + t cell-mediated eradication of poorly immunogenic b -f melanoma by the combined action of il- gene therapy and - bb costimulation intratumoral recombinant human interleukin- administration in head and neck squamous cell carcinoma patients modifies locoregional lymph node architecture and induces natural killer cell infiltration in the primary tumor activity of subcutaneous interleukin- in aids-related kaposi sarcoma phase ii clinical trial of interleukin- in patients with relapsed and refractory non-hodgkin's lymphoma and hodgkin's disease peripheral burst of tumor-specific cytotoxic t lymphocytes and infiltration of metastatic lesions by memory cd + t cells in melanoma patients receiving interleukin after initial setback, il- regaining popularity interleukin : still a promising candidate for tumor immunotherapy? effects of single-dose interleukin- exposure on interleukin- -associated toxicity and interferon-gamma production evaluation of recombinant human interleukin- in patients with recurrent or refractory ovarian cancer: a gynecologic oncology group study randomized multicenter phase ii trial of subcutaneous recombinant human interleukin- versus interferon-alpha a for patients with advanced renal cell carcinoma selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal patterns of vasculature in mouse models of lung cancer are dependent on location direct intratumoral injection of an adenovirus expressing interleukin- induces regression and long-lasting immunity that is associated with highly localized expression of interleukin- co-delivery of antigen and il- by venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects right time and place for il : targeted delivery stimulates immune therapy new insights into il- -mediated tumor suppression gene therapy of cancer based on interleukin neoadjuvant immunotherapy with chitosan and interleukin- to control breast cancer metastasis intravesical chitosan/interleukin- immunotherapy induces tumorspecific systemic immunity against murine bladder cancer characterization of abscopal effects of intratumoral electroporation-mediated il- gene therapy neoadjuvant intratumoral cytokine-loaded microspheres are superior to postoperative autologous cellular vaccines in generating systemic anti-tumor immunity repeated administrations of interleukin (il)- are associated with persistently elevated plasma levels of il- and declining ifn-gamma, tumor necrosis factor-alpha, il- , and il- responses transcriptionally active stat is required for the antiproliferative effects of both interferon alpha and interferon gamma ifn-gamma regulates donor cd t cell expansion, migration, and leads to apoptosis of cells of a solid tumor ifn-gamma-mediated upmodulation of mhc class i expression activates tumor-specific immune response in a mouse model of prostate cancer ifn-gamma-mediated inhibition of tumor angiogenesis by natural killer t-cell ligand, alpha-galactosylceramide interferon: a cytotoxic t lymphocyte differentiation signal il- and ifn-gamma are two necessary lymphokines in the development of cytolytic t cells gamma interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin , and urokinase genes, which are controlled by short-lived repressors interferon and major histocompatibility complex genes: a model to analyse eukaryotic gene regulation? placebo-controlled phase iii trial of patient-specific immunotherapy with mitumprotimut-t and granulocyte-macrophage colony-stimulating factor after rituximab in patients with follicular lymphoma evaluation of ipilimumab in combination with allogeneic pancreatic tumor cells transfected with a gm-csf gene in previously treated pancreatic cancer cancer immunotherapy comes of age cancer immunotherapy strategies based on overcoming barriers within the tumor microenvironment reversing tumor immune suppression with intratumoral il- : activation of tumor-associated t effector/memory cells, induction of t suppressor apoptosis, and infiltration of cd + t effectors use of tumor-infiltrating lymphocytes and interleukin- in the immunotherapy of patients with metastatic melanoma. a preliminary report il- priming during in vitro antigenic stimulation changes properties of cd t cells and increases generation of effector and memory cells synergy of brief activation of cd t-cells in the presence of il- and adoptive transfer into lymphopenic hosts promotes tumor clearance and anti-tumor memory ex vivo interleukin- -priming during cd + t cell activation dramatically improves adoptive t cell transfer antitumor efficacy in a lymphodepleted host ex vivo conditioning with il- protects tumor-infiltrating cd + t cells from negative regulation by local ifn-gamma cutting edge: il- and type i ifn differentially program cd t cells for programmed death re-expression levels and tumor control switching on of the proliferation or apoptosis of activated human t lymphocytes by ifn-gamma is correlated with the differential expression of the alpha-and beta-chains of its receptor clinical trial listed in this table meet the following four criteria: ) involve a delivery technology; ) be administered locally, i.e. intratumoral or peritumoral; ) use il- as an effector and ) demonstrate antitumor activity of il- . clinical trials listed in this table include: ) nhs-il for solid tumors ) a first-in-human dose escalation and expansion study to evaluate intratumoral administration of sar as monotherapy and in combination with cemiplimab in patients with advanced solid tumors antibody-il- fusion proteins are effective in scid mouse models of prostate and colon carcinoma metastases bifunctional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer a single-chain il- igg antibody fusion protein retains antibody specificity and il- bioactivity and demonstrates antitumor activity mechanism of antitumor activity of a single-chain interleukin- igg antibody fusion protein (mscil- .her .igg ) cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal her /neu expressing tumors long-term immunity elicited by antibody-cytokine fusion proteins protects against sequential challenge with murine mammary and colon malignancies amino acid residues involved in the heparin-binding activity of murine il- in the context of an antibody-cytokine fusion protein modulation of interleukin- activity in the presence of heparin molecular mechanisms of heparin-induced modulation of human interleukin bioactivity species-specific differences in heparin-induced modulation of il- family cytokines efficient production and purification of recombinant human interleukin- (il- ) overexpressed in mammalian cells without affinity tag novel immunocytokine il -ss (fv) inhibits mesothelioma tumor growth in nude mice prognostic value and characterization of the ovarian cancer-specific antigen ca - construction, expression, and function of b scfv-mil- , a fusion protein that attacks human ovarian carcinoma an il -il -antibody fusion protein targeting hodgkin's lymphoma cells potentiates activation of nk and t cells for an anti-tumor attack expression of the extra domain b of fibronectin, a marker of angiogenesis, in head and neck tumors hubc -il , an immunocytokine which targets edb-containing oncofetal fibronectin in tumors and tumor vasculature, shows potent anti-tumor activity in human tumor models a phase study of as , a novel antibody-cytokine fusion protein, in patients with malignant melanoma or renal cell carcinoma phase i trial of twice-weekly intravenous interleukin in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain ifn-gamma induction is associated with clinical response an engineered antibody-interleukin- fusion protein with enhanced tumor vascular targeting properties enhancement of the antitumor properties of interleukin- by its targeted delivery to the tumor blood vessel extracellular matrix engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy enhancement of the antitumor activity of interleukin- by targeted delivery to neovasculature synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin and to tumor necrosis factor alpha anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy collagen-binding il- enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours a dose-escalation and signal-generating study of the immunocytokine l -il in combination with dacarbazine for the therapy of patients with metastatic melanoma targeted reconstitution of cytokine activity upon antigen binding using split cytokine antibody fusion proteins a new chemically modified chimeric tnt- monoclonal antibody directed against dna for the radioimmunotherapy of solid tumors the immunocytokine nhs-il as a potential cancer therapeutic characterization of a phage display-derived human monoclonal antibody (nhs ) counterpart to chimeric tnt- directed against necrotic regions of solid tumors temporal changes within the (bladder) tumor microenvironment that accompany the therapeutic effects of the immunocytokine nhs-il enhanced antitumor effects by combining an il- /anti-dna fusion protein with avelumab, an anti-pd-l antibody combination therapy with nhs-muil and avelumab (anti-pd-l ) enhances antitumor efficacy in preclinical cancer models cancer-targeted il- controls human rhabdomyosarcoma by senescence induction and myogenic differentiation tumortargeted il- combined with local irradiation leads to systemic tumor control via abscopal effects in vivo. oncoimmunology enhanced binding of necrosis-targeting immunocytokine nhs-il after local tumour irradiation in murine xenograft models defining the pharmacodynamic profile and therapeutic index of nhs-il immunocytokine in dogs with malignant melanoma first-in-human phase i trial of a tumor-targeted cytokine (nhs-il ) in subjects with metastatic solid tumors immunogenicity to biotherapeutics -the role of anti-drug immune complexes tumor regression of human and murine melanoma after intratumoral injection of il- -encoding plasmid dna in mice in vivo induction of interferon gamma expression in grey horses with metastatic melanoma resulting from direct injection of plasmid dna coding for equine interleukin intratumoral injection of dna encoding human interleukin into patients with metastatic melanoma: clinical efficacy intratumoral injection of il- plasmid dna-results of a phase i/ib clinical trial effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation il- plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous b .f melanoma il- gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma evaluation of toxicity following electrically mediated interleukin- gene delivery in a b mouse melanoma model regression of tumor growth and induction of long-term antitumor memory by interleukin electro-gene therapy administration route-and immune cell activation-dependent tumor eradication by il electrotransfer intratumoral delivery of interleukin expression plasmids with in vivo electroporation is effective for colon and renal cancer radiosensitizing effect of intratumoral interleukin- gene electrotransfer in murine sarcoma in vivo electrogene transfer of interleukin- inhibits tumor growth and lymph node and lung metastases in mouse mammary carcinomas local and systemic antitumor effect of intratumoral and peritumoral il- electrogene therapy on murine sarcoma electroporation-mediated interleukin- gene therapy for hepatocellular carcinoma in the mice model improving therapeutic efficacy of il- intratumoral gene electrotransfer through novel plasmid design and modified parameters systemic administration of interleukin- can restore the antitumor potential of b melanoma-draining lymph node cells impaired at a late tumor-bearing state intratumoral electroporation of il- cdna eradicates established melanomas by trp ( - )-specific cd + ctls in a perforin/granzyme-mediated and ifn-gamma-dependent manner: application of trp ( - ) peptides gene electrotransfer of plasmid-encoding il- recruits the m macrophages and antigen-presenting cells inducing the eradication of aggressive b f murine melanoma il- gene electrotransfer triggers a change in immune response within mouse tumors pre-clinical investigation of the synergy effect of interleukin- gene-electro-transfer during partially irreversible electropermeabilization against melanoma in vivo electroporation-mediated transfer of interleukin- and interleukin- genes induces significant antitumor effects against melanoma in mice combination of il- and il- of electro-gene therapy synergistically inhibits tumor growth coadministration of interleukin- and interleukin- induces a fatal inflammatory response in mice: critical role of natural killer cell interferongamma production and stat-mediated signal transduction electrogene therapy with interleukin- in canine mast cell tumors electroporation-mediated il- gene therapy in a transplantable canine cancer model a combination of electrochemotherapy, gene electrotransfer of plasmid encoding canine il- and cytoreductive surgery in the treatment of canine oral malignant melanoma intratumoural interleukin gene therapy stimulates the immune system and decreases angiogenesis in dogs with spontaneous cancer phase i trial of interleukin- plasmid electroporation in patients with metastatic melanoma intratumoral plasmid il electroporation therapy in patients with advanced melanoma induces systemic and intratumoral t-cell responses intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients intratumoral delivery of plasmid il via electroporation leads to regression of injected and noninjected tumors in merkel cell carcinoma integrin-targeted nanocomplexes for tumour specific delivery and therapy by systemic administration conjugation of poly(amidoamine) dendrimers with various acrylates for improved delivery of plasmid encoding interleukin- gene preparation and characterization of gelatin nanoparticles containing pdna encoding il- and their expression in ct- carcinoma cells preparation and characterization of chitosan/beta-cyclodextrin nanoparticles containing plasmid dna encoding interleukin- efficient gene delivery by egf-lipoplexes in vitro and in vivo polyethylenimine: a versatile, multifunctional nonviral vector for nucleic acid delivery aerosol gene therapy with pei: il- eradicates osteosarcoma lung metastases eradication of osteosarcoma lung metastases following intranasal interleukin- gene therapy using a nonviral polyethylenimine vector tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding il- gene enhanced delivery of plasmid encoding interleukin- gene by diethylene triamine penta-acetic acid (dtpa)-conjugated pei nanoparticles delivery of plasmid encoding interleukin- gene into hepatocytes by conjugated polyethylenimine-based nanoparticles intratumoral delivery of il- gene by polyvinyl polymeric vector system to murine renal and colon carcinoma results in potent antitumor immunity combination of interleukin and interferon alpha gene therapy induces a synergistic antitumor response against colon and renal cell carcinoma biodegradable polymer-based interleukin- gene delivery: role of induced cytokines, tumor infiltrating cells and nitric oxide in anti-tumor activity soluble biodegradable polymer-based cytokine gene delivery for cancer treatment in vivo targeted gene delivery by cationic nanoparticles for treatment of hepatocellular carcinoma modified nanoparticle mediated il- immunogene therapy for colon cancer local and systemic delivery of interleukin- gene by cationic micelles for cancer immunogene therapy mannosylated chitosan nanoparticle-based cytokine gene therapy suppressed cancer growth in balb/c mice bearing ct- carcinoma cells intratumoral delivery of p cmvmil- using water-soluble lipopolymers tumor regression by repeated intratumoral delivery of water soluble lipopolymers/p cmvmil- complexes combination of local, nonviral il gene therapy and systemic paclitaxel treatment in a metastatic breast cancer model local, non-viral il- gene therapy using a water soluble lipopolymer as carrier system combined with systemic paclitaxel for cancer treatment development of self-assembled multi-arm polyrotaxanes nanocarriers for systemic plasmid delivery in vivo synthesis and application of a non-viral gene delivery system for immunogene therapy of cancer a safety and efficacy study of local delivery of interleukin- transgene by ppc polymer in a model of experimental glioma treatment of disseminated ovarian cancer using nonviral interleukin- gene therapy delivered intraperitoneally phase-i clinical trial of il- plasmid/lipopolymer complexes for the treatment of recurrent ovarian cancer a phase ii trial of intraperitoneal egen- , an il- plasmid formulated with peg-pei-cholesterol lipopolymer in the treatment of persistent or recurrent epithelial ovarian, fallopian tube or primary peritoneal cancer: a gynecologic oncology group study phase i trial of a formulated il- plasmid in combination with carboplatin and docetaxel chemotherapy in the treatment of platinum-sensitive recurrent ovarian cancer a phase i trial of intraperitoneal gen- , an il- plasmid formulated with peg-pei-cholesterol lipopolymer, administered with pegylated liposomal doxorubicin in patients with recurrent or persistent epithelial ovarian, fallopian tube or primary peritoneal cancers: an nrg oncology/gynecologic oncology group study intratumoral injection of interleukin- plasmid dna, either naked or in complex with cationic lipid, results in similar tumor regression in a murine model cationic polyphosphazene vesicles for cancer immunotherapy by efficient in vivo cytokine il- plasmid delivery enhanced growth inhibition of metastatic lung tumors by intravenous injection of atra-cationic liposome/il- pdna complexes in mice retinoic acid receptors and cancers all-trans-retinoic acid upregulates tnf receptors and potentiates tnf-induced activation of nuclear factors-kappab, activated protein- and apoptosis in human lung cancer cells lipid nanoparticles that deliver il- messenger rna suppress tumorigenesis in myc oncogene-driven hepatocellular carcinoma visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (plga) microspheres a study of medi in sequential and concurrent combination with durvalumab in subjects with advanced solid tumors abstract : medi , a novel il- mrna therapy for intratumoral injection to promote t<sub>h</sub> transformation of the patient tumor microenvironment a first-in-human dose escalation and expansion study to evaluate intratumoral administration of sar as monotherapy and in combination with cemiplimab in patients with advanced solid tumors analysis of pre-existing igg and igm antibodies against polyethylene glycol (peg) in the general population viruses as anticancer drugs oncolytic virotherapy adenovirus-mediated interleukin- gene therapy for metastatic colon carcinoma construction of a double recombinant adenovirus vector expressing a heterodimeric cytokine: in vitro and in vivo production of biologically active interleukin- adenovirus-mediated expression of interleukin- induces natural killer cell activity and complements adenovirus-directed gp treatment of melanoma lung metastases construction and characterization of a recombinant adenoviral vector expressing human interleukin- intranasal therapy with an adenoviral vector containing the murine interleukin- gene eradicates osteosarcoma lung metastases antitumor mechanism of intratumoral injection with il- -expressing adenoviral vector against il- -unresponsive tumor the antitumor effects of adenoviralmediated, intratumoral delivery of interleukin require endogenous il- induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin- regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin- role of nk and t cells in il- -induced anti-tumor response against hepatic colon carcinoma adenovirusmediated interleukin- gene therapy for prostate cancer: suppression of orthotopic tumor growth and pre-established lung metastases in an orthotopic model attenuation of the glucocorticoid response during ad il- adenovirus vector treatment enhances natural killer cell-mediated killing of mhc class i-negative lncap prostate tumors depletion of myeloid-derived suppressor cells during interleukin- immunogene therapy does not confer a survival advantage in experimental malignant glioma in situ adenoviral interleukin gene transfer confers potent and longlasting cytotoxic immunity in glioma eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cdnas for the il- heterodimer and its inhibition by the il- p subunit homodimer a single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin induces remarkable anti-tumor and anti-metastatic activity in mice with meth-a fibrosarcoma growth suppression of human laryngeal squamous cell carcinoma by adenoviral-mediated interleukin- enhanced antitumor effect of combined replicative adenovirus and nonreplicative adenovirus expressing interleukin- in an immunocompetent mouse model adenovirus-interleukin- -mediated tumor regression in a murine hepatocellular carcinoma model is not dependent on cd -restricted natural killer t cells gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin effective gene therapy for medullary thyroid carcinoma using recombinant adenovirus inducing tumor-specific expression of interleukin- gene therapy of a rat follicular thyroid carcinoma model with adenoviral vectors transducing murine interleukin- intratumor murine interleukin- gene therapy suppressed the growth of local and distant ewing's sarcoma neuroblastoma regression and immunity induced by transgenic expression of interleukin- high frequency of specific cd + t cells in the tumor and blood is associated with efficient local il- gene therapy of cancer thyroid cancer immuno-therapy with retroviral and adenoviral vectors expressing granulocyte macrophage colony stimulating factor and interleukin- in a rat model low-dose adenoviral immunotherapy of rat hepatocellular carcinoma using single-chain interleukin- treatment of pancreatic cancer with an oncolytic adenovirus expressing interleukin- in syrian hamsters phase i trial of intratumoral injection of an adenovirus encoding interleukin- for advanced digestive tumors membrane-tethered proteins for basic research, imaging, and therapy tumor therapy applying membrane-bound form of cytokines re-designing interleukin- to enhance its safety and potential as an anti-tumor immunotherapeutic agent cancer immunotherapy using a membrane-bound interleukin- with b - transmembrane and cytoplasmic domains the rheoswitch system for inducible up-and down-regulation of gene expression regulated intratumoral expression of il- using a rheoswitch therapeutic system((r)) (rts((r))) gene switch as gene therapy for the treatment of glioma regulatable interleukin- gene therapy in patients with recurrent highgrade glioma: results of a phase trial neoadjuvant interleukin- immunogene therapy protects against cancer recurrence after liver resection in an animal model neoadjuvant treatment of hepatic malignancy: an oncolytic herpes simplex virus expressing il- effectively treats the parent tumor and protects against recurrence-after resection interleukin secretion enhances antitumor efficacy of oncolytic herpes simplex viral therapy for colorectal cancer angiogenesis inhibition by an oncolytic herpes virus expressing interleukin cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma in situ cancer vaccination: an il- defective vector/replication-competent herpes simplex virus combination induces local and systemic antitumor activity augmentation of antitumor immune responses by multiple intratumoral inoculations of replicationconditional hsv and interleukin- il- expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice newly characterized murine undifferentiated sarcoma models sensitive to virotherapy with oncolytic hsv- m enhanced therapeutic efficacy of il- , but not gm-csf, expressing oncolytic herpes simplex virus for transgenic mouse derived prostate cancers cytokine-secreting herpes viral mutants effectively treat tumor in a murine metastatic colorectal liver model by oncolytic and t-cell-dependent mechanisms engineered herpes simplex virus expressing il- in the treatment of experimental murine brain tumors multifaceted oncolytic virus therapy for glioblastoma in an immunocompetent cancer stem cell model increased efficacy of an interleukin- -secreting herpes simplex virus in a syngeneic intracranial murine glioma model preclinical evaluation of oncolytic deltagamma( ) . herpes simplex virus expressing interleukin- for therapy of breast cancer brain metastases preclinical evaluation of a genetically engineered herpes simplex virus expressing interleukin- a fully-virulent retargeted oncolytic hsv armed with il- elicits local immunity and vaccine therapy towards distant tumors eradication of glioblastoma by immuno-virotherapy with a retargeted oncolytic hsv in a preclinical model evaluation of the safety and biodistribution of m , an attenuated herpes simplex virus type expressing hil- , after intracerebral administration to aotus nonhuman primates the molecular pathogenesis of semliki forest virus: a model virus made useful? semliki forest virus vectors engineered to express higher il- levels induce efficient elimination of murine colon adenocarcinomas induction of a therapeutic antitumor immunological response by intratumoral injection of genetically engineered semliki forest virus to produce interleukin- marked enhancement of antitumor immune responses in mouse brain tumor models by genetically modified dendritic cells producing semliki forest virus-mediated interleukin- semliki forest virus expressing interleukin- induces antiviral and antitumoral responses in woodchucks with chronic viral hepatitis and hepatocellular carcinoma short-term intratumoral interleukin- expressed from an alphaviral vector is sufficient to induce an efficient antitumoral response against spontaneous hepatocellular carcinomas neoadjuvant administration of semliki forest virus expressing interleukin- combined with attenuated salmonella eradicates breast cancer metastasis and achieves long-term survival in immunocompetent mice transfer of the murine interleukin- gene in vivo by a semliki forest virus vector induces b tumor regression through inhibition of tumor blood vessel formation monitored by doppler ultrasonography the anti-angiogenic activity of il- is increased in inos-/-mice and involves nk cells strict requirement for vector-induced type i interferon in efficacious antitumor responses to virally encoded il immunotherapy with recombinant sfv-replicons expressing the p a tumor antigen or il- induces tumor regression immunotherapeutic synergy between anti-cd mab and intratumoral administration of a cytopathic semliki forest virus encoding il- virotherapy with a semliki forest virus-based vector encoding il synergizes with pd- /pd-l blockade regression of mouse tumours and inhibition of metastases following administration of a semliki forest virus vector with enhanced expression of il- semliki forest virus-mediated gene therapy of the rg rat glioma low-dose vaccinia virus-mediated cytokine gene therapy of glioma evaluation of cytokine toxicity induced by vaccinia virus-mediated il- and il- antitumour immunotherapy cytokine-armed vaccinia virus infects the mesothelioma tumor microenvironment to overcome immune tolerance and mediate tumor resolution intratumoral expression of il- and il- using an oncolytic virus increases systemic sensitivity to immune checkpoint blockade canarypox virus-mediated interleukin gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity intratumoral administration of a recombinant canarypox virus expressing interleukin in patients with metastatic melanoma phase i study of the intratumoral administration of recombinant canarypox viruses expressing b . and interleukin in patients with metastatic melanoma oncolytic measles virus encoding interleukin- mediates potent antitumor effects through t cell activation. oncoimmunology immunological effects and viral gene expression determine the efficacy of oncolytic measles vaccines encoding il- or il- agonists interleukin- expression enhances vesicular stomatitis virus oncolytic therapy in murine squamous cell carcinoma enhancement of antitumor activity of gammaretrovirus carrying il- gene through genetic modification of envelope targeting her receptor: a promising strategy for bladder cancer therapy increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector engineering newcastle disease virus as an oncolytic vector for intratumoral delivery of immune checkpoint inhibitors and immunocytokines immunotherapy of established tumors in mice by intratumoral injection of an adenovirus vector harboring the human il- cdna: induction of cd + t-cell immunity and nk activity effect of viral dose on neutralizing antibody response and transgene expression after aav vector re-administration in mice pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing il- but inhibits virus dissemination genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin- systemic vector leakage and transgene expression by intratumorally injected recombinant adenovirus vectors systemic dissemination of viral vectors during intratumoral injection in vivo cancer gene therapy with a recombinant interleukin- adenovirus vector gene therapy leaves a vicious cycle intratumoral delivery of adenovirus-mediated interleukin- gene in mice with metastatic cancer in the liver preclinical toxicology of oncolytic adenovirus-mediated cytotoxic and interleukin- gene therapy for prostate cancer induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin- induces complete regression of established murine transplantable colon adenocarcinomas fibroblasts genetically engineered to secrete interleukin can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo cancer immunotherapy of established tumors with il- . effective delivery by genetically engineered fibroblasts therapeutic effects of gelatin matrix-embedded il- gene-modified macrophages in a mouse model of residual prostate cancer macrophages transduced with an adenoviral vector expressing interleukin suppress tumor growth and metastasis in a preclinical metastatic prostate cancer model antitumor efficacy of adenocarcinoma cells engineered to produce interleukin (il- ) or other cytokines compared with exogenous il- paracrine delivery of il- against intracranial l gliosarcoma in rats expression of interleukin- by adipose-derived mesenchymal stem cells for treatment of lung adenocarcinoma. thorac cancer therapeutic potential of human mesenchymal stem cells producing il- in a mouse xenograft model of renal cell carcinoma anti-tumor activity of mesenchymal stem cells producing il- in a mouse melanoma model enhancement of antitumor immunity against b melanoma tumor using genetically modified dendritic cells to produce cytokines route of administration influences the antitumor effects of bone marrowderived dendritic cells engineered to produce interleukin- in a metastatic mouse prostate cancer model augmented anti-tumor effect of dendritic cells genetically engineered by interleukin- plasmid dna upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin- interleukin- transduced dendritic cells induce regression of established murine neuroblastoma intratumoral delivery of dendritic cells engineered to secrete both interleukin (il)- and il- effectively treats local and distant disease in association with broadly reactive tc -type immunity intratumoral injection of dendritic cells transduced by an sv -based vector expressing interleukin- induces curative immunity mediated by cd + t lymphocytes and nk cells injection of il- gene-transduced dendritic cells into mouse liver tumor lesions activates both innate and acquired immunity intratumoral injection of dendritic cells overexpressing interleukin inhibits melanoma growth conditional interleukin- gene therapy promotes safe and effective antitumor immunity therapeutic effect of intratumoral administration of dcs with conditional expression of combination of different cytokines intratumoral injection of dendritic cells engineered to secrete interleukin- by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas continuous release of interleukin from microencapsulated engineered cells for colon cancer therapy interleukin gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase i study concise review: mesenchymal stem cell tumorhoming: detection methods in disease model systems murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin- gene delivery into ewing sarcoma tumors reversal of tumor growth by gene modification of mesenchymal stem cells using spermine-pullulan/dna nanoparticles gene therapy of intracranial glioma using interleukin -secreting human umbilical cord blood-derived mesenchymal stem cells mesenchymal stem cells genetically modified by lentivirus-mediated interleukin- inhibit malignant ascites in mice the effects of mesenchymal stem cells injected via different routes on modified il- -mediated antitumor activity adoptive transfer of natural killer cells promotes the anti-tumor efficacy of t cells local delivery of interleukin- using t cells targeting vegf receptor- eradicates multiple vascularized tumors in mice il- release by engineered t cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression improving adoptive t cell therapy by targeting and controlling il- expression to the tumor environment tumor-targeted t cells modified to secrete il- eradicate systemic tumors without need for prior conditioning cd car t cells expressing il- eradicate lymphoma in fully lymphoreplete mice through induction of host immunity il- secreting tumor-targeted chimeric antigen receptor t cells eradicate ovarian tumors in vivo armored inducible expression of il- enhances antitumor activity of glypican- -targeted chimeric antigen receptor-engineered t cells in hepatocellular carcinoma chitosan solution enhances the immunoadjuvant properties of gm-csf sustained release technology and its application in environmental remediation: a review designing hydrogels for controlled drug delivery recent advances in hydrogel based drug delivery systems for the human body neoadjuvant therapy with interleukin- -loaded polylactic acid microspheres reduces local recurrence and distant metastases in situ tumor vaccination with interleukin- -encapsulated biodegradable microspheres: induction of tumor regression and potent antitumor immunity cytokines delivered by biodegradable microspheres promote effective suppression of human tumors by human peripheral blood lymphocytes in the scid-winn model synergistic effect of intratumoral il- and tnf-alpha microspheres: systemic antitumor immunity is mediated by both cd + ctl and nk cells intratumoral delivery of encapsulated il- , il- and tnf-alpha in a model of metastatic breast cancer antitumor immune response of human peripheral blood lymphocytes coengrafted with tumor into severe combined immunodeficient mice interleukin- delivered by biodegradable microspheres promotes the antitumor activity of human peripheral blood lymphocytes in a human head and neck tumor xenograft/scid mouse model human cd + t cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of il- to kill tumors in situ by indirect effects of ifn-gamma human cd + effector memory t cells persisting in the microenvironment of lung cancer xenografts are activated by local delivery of il- to proliferate, produce ifn-gamma, and eradicate tumor cells transient activation of tumor-associated t-effector/memory cells promotes tumor eradication via nk-cell recruitment: minimal role for long-term t-cell immunity in cure of metastatic disease cancer immunotherapy with interleukin and granulocyte-macrophage colony-stimulating factor-encapsulated microspheres: coinduction of innate and adaptive antitumor immunity and cure of disseminated disease dichotomous effects of ifn-gamma on dendritic cell function determine the extent of il- -driven antitumor t cell immunity central role of tumorassociated cd + t effector/memory cells in restoring systemic antitumor immunity activated cd + t-effector/memory cells eliminate cd + cd + foxp + tsuppressor cells from tumors via fasl mediated apoptosis central role of ifngamma-indoleamine , -dioxygenase axis in regulation of interleukin- -mediated antitumor immunity rapid release of cytoplasmic il- from tumor-associated macrophages is an initial and critical event in il- -initiated tumor regression intratumoral il- and tnf-alpha-loaded microspheres lead to regression of breast cancer and systemic antitumor immunity generation of a tumor-specific systemic response after intratumoral injection of il- and il- -loaded polylactic acid microspheres il- + gm-csf microsphere therapy induces eradication of advanced spontaneous tumors in her- /neu transgenic mice but fails to achieve long-term cure due to the inability to maintain effector t-cell activity chronic immune therapy induces a progressive increase in intratumoral t suppressor activity and a concurrent loss of tumor-specific cd + t effectors in her- /neu transgenic mice bearing advanced spontaneous tumors chronic chemoimmunotherapy achieves cure of spontaneous murine mammary tumors via persistent blockade of posttherapy counterregulation effect of chitosan properties on immunoreactivity intravesical immunotherapy of superficial bladder cancer with chitosan/interleukin- characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-n-acetyl glucosamine-based polymer matrix sustained release of granulocyte-macrophage colony-stimulating factor from a modular peptide-based cancer vaccine alters vaccine microenvironment and enhances the antigen-specific t-cell response paracrine release of il- stimulates ifn-gamma production and dramatically enhances the antigen-specific t cell response after vaccination with a novel peptide-based cancer vaccine intratumoral poly-n-acetyl glucosamine-based polymer matrix provokes a prolonged local inflammatory response that, when combined with il- , induces regression of malignant mesothelioma in a murine model using poly-n-acetyl glucosamine gel matrix to deliver il- with anti-schistosomasis vaccination evaluation of the biocompatibility of a chitosan scaffold in mice biocompatibility evaluation of crosslinked chitosan hydrogels after subcutaneous and intraperitoneal implantation in the rat il- delivered intratumorally by multilamellar liposomes reactivates memory t cells in human tumor microenvironments adjuvant cationic liposomes presenting mpl and il- induce cell death, suppress tumor growth, and alter the cellular phenotype of tumors in a murine model of breast cancer cytokine immunotherapy of cancer with controlled release biodegradable microspheres in a human tumor xenograft/scid mouse model characterization of cytokine-encapsulated controlled-release microsphere adjuvants stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide) biodegradable microand nanoparticles as long-term delivery vehicles for interferon-alpha challenges in development of targeted liposomal therapeutics advances and challenges of liposome assisted drug delivery surgery for cancer: a trigger for metastases chemotherapy-induced metastasis in breast cancer radiotherapy targeting cancer stem cells "awakens" them to induce tumour relapse and metastasis in oral cancer biological behavior of human breast cancer micrometastases a novel synergistic combination of cyclophosphamide and gene transfer of interleukin- eradicates colorectal carcinoma in mice a phase i clinical trial of adoptive t cell therapy using il- secreting muc- (ecto) directed chimeric antigen receptors for recurrent ovarian cancer the next challenge in cancer immunotherapy: controlling t-cell traffic to the tumor chemotherapy induces intratumoral expression of chemokines in cutaneous melanoma, favoring t-cell infiltration and tumor control immunogenic effects of chemotherapy-induced tumor cell death radiation-induced effects and the immune system in cancer a novel combination treatment of armed oncolytic adenovirus expressing il- and gm-csf with radiotherapy in murine hepatocarcinoma irradiation-induced localization of il- -expressing mesenchymal stem cells to enhance the curative effect in murine metastatic hepatoma combination of radiation and interleukin eradicates large orthotopic hepatocellular carcinoma through immunomodulation of tumor microenvironment the role of irreversible electroporation (ire) for locally advanced pancreatic cancer: a systematic review of safety and efficacy irreversible electroporation reverses resistance to immune checkpoint blockade in pancreatic cancer systematic review of surgical and percutaneous irreversible electroporation in the treatment of locally advanced pancreatic cancer high-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity immunologic response to cryoablation of breast cancer thermal ablation of tumours: biological mechanisms and advances in therapy synergy between in situ cryoablation and tlr stimulation results in a highly effective in vivo dendritic cell vaccine changes in interleukin- beta and after hepatic microwave tissue ablation compared with radiofrequency, cryotherapy and surgical resections image-guided thermal ablation of tumors increases the plasma level of interleukin- and interleukin- characterization of the cryoablation-induced immune response in kidney cancer patients abscopal immunity achieved via in situ vaccination using a novel combination of cryoablation and interleukin- the antibody-based delivery of interleukin- to solid tumors boosts nk and cd + t cell activity and synergizes with immune checkpoint inhibitors intratumoral il- combined with ctla- blockade elicits t cell-mediated glioma rejection phase ii trial of il- plasmid transfection and pd- blockade in immunologically quiescent melanoma revisiting interleukin- as a cancer immunotherapy agent abbreviations a-nk il− or activated nk il− , activated primary natural killer (nk) cells transduced to express il- adcmvil- or adcmvmil- , recombinant defective adenovirus expressing il- or mil- under control of a cmv promoter adenoviral vector expressing murinedil- protein under the control of the hcmv promoter and sv polyadenylation sequences; ad-dhscil , adenovirus with replication dependent on hypoxia-inducible factor (hif) activity expressing single-chain il- ; ad-il- or adil- or ad.il- or admil- or ad.mil- inducible adenoviral vector engineered to express il- under the control of the rheoswitch therapeutic system r (rts) gene switch adtcpmil- , recombinant replication-defective adenoviral vector expressing murine interleukin- (mil- ), driven by a modified calc-i promoter oncolytic triple-deletion (td) adenoviral vector encoding wild-type il- oncolytic triple-deletion (td) adenoviral vector encoding non-secreting il- antibody-dependent cellular cytotoxicity; ad.scil- , adenoviral vector expressing single-chain il- ad -ycd/muttksr rep-hil , a replication-competent oncolytic adenovirus encoding the murine pro-inflammatory cytokine interleukin- (il- ) gene and two suicide fusion genes, a yeast cytosine deaminase (ycd) and a mutant form of herpes simplex virus type thymidine kinase / .crgd-mil p , a replication-deficient double targeted ad backbone-based vector carrying a chimeric ad / fiber with integrin-binding rgd motif incorporated in its ad knob domain expressing murine il- p adenoviral vector encoding membrane-anchored murine single-chain il- with b - transmembrane and cytoplasmic domains atra-cationic liposome/il- pdna, pil- complexed with cationic liposomes incorporating all-trans-retinoic acid chimeric antigen receptor; cbd, collagen binding domain; cbd-il- , collagen-binding immunocytokine comprised of a cbd of von willebrand factor fused to both subunits of il- chtnt- /huil- , necrosis-targeting immunocytokine comprised of huil- fused to the variable heavy chain of chtnt- antibody dendritic cells transfected with adenovirus expressing il- under control of the cmv promoter enzyme-linked immune absorbent spot fda, food and drug administration; gm-csf, granulocyte-macrophage colony-stimulating factor glypican- -specific car t cells with nfat-inducible expression of il- herpes simplex viruses (hsv) encoding il- ; hubc -il , immunocytokine comprised of two molecules of il- fused to each of the igg heavy chains of humanized bc- antibody immunocytokine comprised of il- fused to the fc fragment of humanized ks antibody; hu- . -il- , immunocytokine comprised of il- fused to gd targeting hu dmp/il- , pil- complexed with dmp cationic micelles -dimyristyloxypropyl)-n,n-dimethyl-( -hydroxyethyl)ammonium bromide/dioleoyl phosphatidylethanolamine (dmrie/dope) cationic lipids; il- , interleukin- ; il- m, modified il- ; an n-glycosylation mutant of il- at asn ; il -il , immunocytokine comprised of il- and il- fused to anti-cd scfv antibody immunocytokine comprised of il- fused to l scfv antibody; il -msa, il- fused to mouse serum albumin (msa) il- fused to mouse serum albumin (msa) and lumican, a collagen-binding proteoglycan immunocytokine comprised of p subunit of single-chain il- fused to ss , a mesothelin-binding single-chain variable fragment lenti-mil- -mscs, bone-marrow derived mscs transfected with lentivirus to express il- mc/pmil- , pmil- complexed with mannosylated chitosan (mc) myeloid-derived suppressor cells mevac fmil- , attenuated measles viruses (mevac) encoding il- histocompatibility complex; momlv-mil- , moloney murine leukemia virus (momlv) expressing il- ; mpeg/pmil- , polymersomes comprised of pmil- complexed with polyphosphazene particles modified with dpa and mpeg; mscs, mesenchymal stem cells; mscil- .her .igg , mouse single-chain il- fused mesenchymal stem cells (mscs) expressing il- ; l -mil , immunocytokine comprised of murine il- fused to l scfv antibody; nhs-il , necrosis-targeting immunocytokine comprised of two single-chain il- molecules fused to fulllength nhs antibody; nhs-muil , murine analog of nhs-il ; nk cells a biodegradable polyester; pbl, peripheral blood lymphocytes; pd- , programmed cell death protein ; pd-l , programmed death-ligand ; pei:il- , il- complexed with polyethyleneimine (pei); pil- enhanced sfv vector with x higher gene expression of recombinant il- protein; rad/il- m, recombinant adenovirus expressing il- m; rndv-anti-cd -mil- , newcastle disease virus (ndv) expressing immunocytokine comprised of anti-cd antibody fused to mil- ; rndvanti-pdl -mil- , newcastle disease virus (ndv) expressing immunocytokine comprised of anti-pdl antibody fused to mil- ; rsfv/il , recombinant semliki forest viruses (sfv) encoding il- ; rvsv-il , recombinant vesicular stomatitis virus (vsv) encoding il- ; rvv-mil- severe combined immunodeficient; scil- , single-chain il- ; sfv-il , semliki forest viruses encoding il- sfv-il- , semliki forest viruses encoding il- tumor associated macrophages; tlr, toll-like receptor; tnf, tumor necrosis factor; ucb-msc-il m, human umbilical cord blood-derived mesenchymal stem cells (ucb-mscs) expressing il- m vv-il- , vaccinia virus (vv) expressing il- il- expression plasmid complexed with water-soluble lipopolymers car t cells which are specific for the muc- ecto antigen and secrete il- ; b scfv-mil- , immunocytokine comprised of mil- fused to scfv of b antibody key: cord- - p yr n authors: nan title: poster exhibition date: - - journal: hepatol int doi: . /s - - - sha: doc_id: cord_uid: p yr n nan background: biliary atresia (ba) is one of the most common causes of neonatal cholestasis and the most frequent hepatic cause of death in early childhood. the incidence rate of ba is higher in asian countries, occurring in approximately of , (asian countries ) to of , (european countries) live births. early identification and prompt intervention is very important. to im prove the early diagnosis, we used proteomic technology to screen serum biomarker for ba. methods: two-dimensional electrophoresis ( -de) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (maldi-tof-ms) were employed to screen serum biomarkers specific to ba sera from idiopathic neonatal hepatitis. after pretreatment including albumin and immunoglobulin (igg ) depletion, sera were subjected to -de and there after image analysis. the differentially expressed protein spots were identified by maldi-tof-ms. result: from optimized -de gel images, thirty-four spots were differentially expressed and identified by maldi-tof-ms to be eight proteins. overall, kininogen variant was under expressed and alpha- -b-glycoprotein, leucine-rich alpha- -glycoprotein , 'sp , ', a bg protein, vitamin d-binding protein/group specific component , apolipoproteina-, aqgv were over expressed in ba group com pared to idiopathic neonatal hepatitis. conclusion: -de based serum proteome analysis can be useful in detecting protein expression alteration and new discovered biomarkers might be an aid in the diagnosis of ba, though further validation is needed. s. somani , a. somani , a. jain , v. dixit suvidha, navjeevan hospital, ims, bhu, varanasi, india background: a variety of autoreactive antibodies are detected in patients with chronic liver disease. this prospective, nonrandomized study was undertaken to evaluate the nature & prevalence of various autoantibodies in patients with chronic liver disease of diverse etiologies. methods: study population included patients ( % males), who met defined criteria for chronic liver disease. detailed clinical, laboratory and sonographic evaluation was done. sera were tested for asma, anti-lkm type , ama, apa, ana, by standard methods. p< . was considered significant. results: among various etiologies for chronic liver disease, hepatitis b was most common ( %), followed by alcohol ( %), autoimmune hepatitis in %, hepatitis c ( %) and miscellaneous ( %). % of patients were labeled as cryptogenic after detailed investigations. ana (> / ) was positive in % of definite aih, % of hcv related cld but at titer of > / , . % of hcv related cld & % of probable aih were found positive. asma (> / ) was positive in % of hbv related cld, % of alcohol related cld, % of definite aih, % of probable aih, % of hcv related cld but asma in titer of > / was positive only in % oh definite aih. apa was detected in . % of cryptogenic cld, . % of hbv related cld & % of alcohol & probable aih related cld each. ama was detected in % of cryptogenic, hbv, aih (definite) & hcv related cld each, and % of alcohol related cld & % of pbc. conclusions: apart from aih there is high prevalence of ana & sma in hcv related cld while other antibodies has low prevalence in non-aih related clds. this study also suggests that prevalence of various autoantibodies should be borne in mind while considering the diagnosis of cld especially of mixed etiology. conclusions: oa infusion did not lower ammonia levels or improve survival. results: the mortality ( . %) of patients in lamivudine group with meld score from to was lower than that ( . %) of control group ( = . , p= . ). univariate analysis showed that mortality was significantly related to age (p= . ), meld score (p= . ), treatment method (p= . ), pretreatment hbv dna load (p= . ), the decline of hbv dna load during therapy (p= . ) and encephalopathy (p= . ). in multivariate analysis, in patients with meld scores - , treatment method (p= . ), pretreatment hbv dna load (p= . ), decline of hbv dna load during therapy (p= . ) and encephalopathy (p= . ) were independent predictors of mortality; for meld scores above , only meld score (p= . ) was independent predictive. conclusions: lamivudine treatment significantly decreases the month's mortality of patients with meld score - , and a low viral load pre-treatment and quick decline of hbv dna load are good predictors for the survival of lamivudine treatment. background/aims: early identification of patients with fulminant hepatic failure (fhf) who need a liver transplantation is very important. to construct a prediction model for early diagnosis and prognosis of fhf, we studied dynamics of metabolic profiles using a d-galactosamine/lipopolysaccharide (galn/lps)-treated mouse model. methods: balb/c mice were used to construct fhf model and sacrificed for blood collection at , , and hour after treatment, respectively. levels of plasma metabolites were quantified using gas chromatography/time-of-flight mass spectrometry and data were processed using partial least squares discriminant analysis (pls-da). results: distinct clustering differences were observed and h after treatment between survival and dead groups. at h, plasma levels of some metabolites differed significantly between survival, dead and control groups. ketogenesis and the tca cycle were inhibited in both survival and dead groups, but in dead group, the urea cycle was also inhibited and glycolysis was elevated. pls-da indicated that principal component weighting was greatest for plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate. the y-predicted scatter plot in pls model assigned samples to survival or dead groups using an apriori cutoff of . with % sensitivity and specificity. similar results were observed in fhf patients with different outcomes. pe association between polymorphisms in the interleukin- gene promoter and hepatitis b-related acute liver failure conclusions: the pls model based on metabonomics analysis can be used to predict outcomes well, and plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate may constitute a set of markers for early diagnosis and prognosis of fhf. il- promoter are associated with the susceptibility to hepatitis b-related alf in the chinese population. il- a- c may be a regulatory polymorphism that affects gene regulation. hepatocyte cell death in aclf: mechanism and significance -an immunohistochemical study. p. sakhuja , a. rastogi , s. s hissar , a. singh , a. kumar , r. gondal , s.k. sarin gb pant hospital, institute of liver and biliary sciences, new delhi, india background: acute on chronic liver failure (aclf) is defined as acute hepatic insult complicated within weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. caspases play an essential role in apoptosis. cox- is an inducible immediate early gene responsible for the release of prostaglandins during inflammatory response. we studied the immunohistochemical expression of cox- and caspase- in liver tissue to assess their role in pathophysiology and in predicting outcome of aclf. method: a retrospective analysis of liver biopsies with clinical diagnosis of aclf was undertaken. patients were divided into two groups a and d based on clinical outcome (alive/died respectively). immunohistochemical analysis for cox- and caspase was performed on and cases respectively and scored from - as per intensity and distribution. score - indicated high intensity with focal to diffuse distribution, and was considered significant. results: etiology of acute liver failure was viral or alcoholic. increased expression of caspase was observed in / cases in group d and none of the cases in group a(n= ) (p= . ). increased expression of cox- was observed in / cases in group d and none of the cases in group a (n= ) (p= . ). conclusion: increased immunoreactivity of caspase in liver biopsies of patients of aclf may indicate worse prognosis and its important role in the pathophysiology of aclf. immunostaining for caspase is useful for assessment of prognosis and possibility of anti-apoptotic and anti-fibrotic therapies in future. conclusion: hhgf expression vector (pcmv-hhgf) has been successfully constructed and repeated hydrodynamic injections can promote sustained and high expression of hhgf in vivo. j.h. kim , k.w. kim asan medical center, seoul, korea background: splenic artery embolization (sae) is performed to increase hepatic arterial flow or to decrease portal venous flow in recipients of liver transplantation (lt). thus, the purpose of this study was to estimate sae effect on the basis of changes in caliber of related vessels and splenic volume on pre-sae and serial post-sae ct scans in lt recipients. methods: between and , among lt recipients who underwent sae and serial follow-up ct, with no compounding factor that may obscure sae effect were included in this study. they underwent ct before and after ( week, month, and year) sae. a radiologist retrospectively measured diameters of ca, cha, sa, sv and splenic volume on serial ct scans. their diameters and splenic volume on each ct were compared with those on the prior and pre-sae ct. the difference was compared using repeated-measures anova tests. results: cas decreased between week and month after sae (p<. ), but were stable before week and after month. chas increased within week (p<. ) but decreased between week and month (p<. ) and remained stable after month. compared with pre-sae ct, chas were larger for month after sae. sas continuously decreased for year (p<. ). svs decreased for month (p<. ) and remained stable after month. compared with pre-sae ct, sas and svs were smaller from week after sae and on. splenic volume continuously decreased for year except a period between week and month. conclusion: the increase of hepatic arterial flow persists for month after sae, but returns to baseline thereafter. the decrease of portal flow may lasts for at least year after sae. poster exhibition -cholangioca and other liver neoplasm poster session, hall b background: now, rfa has becoming an important practice of hcc therapy. in this study, we evaluated whether rfa therapy for metastatic liver tumor has a beneficial effect on patients' survival. methods: forty six patients were treated by rfa for metastatic liver tumor from july through february in our hospital, of the , patients were metastasis either form colon or stomach cancer. these patients were analyzed in this investigation. cumulative survival rate from initial rfa therapy was calculated by kaplan-meier method. predictive factors for survival were identified using cox proportional hazard regression model. results: the mean age of the patients were . (range, - ) . the mean size of the tumor is mm (range, - mm) and the numbers of tumor foci are . nodules range, . the survival rates of patients treated by rfa were . % at years and . % at years in colon cancer, . % at years and . % at years in gastric cancer. in this series of patients, primary cancer: colon (p= . odds ratio . %ci . - . ), younger patients ( ) (p= . odds ratio . %ci . - . ) and multiagent chemotherapy (p= . odds ratio . % ci . - . ) were significantly correlated with better survival. conclusion: the survival of patients treated by rfa for metastatic colon cancers had better survival than those of gastric cancers. in addition, good indication of rfa is for metastatic colon cancers, younger patients and has to be treated by multiagent chemotherapies. utility of contrast enhanced ultrasonography with sonazoid in radiofrequency ablation (rfa) for liver metastasis e. goto , s. shiina , r. tateishi , r. masuzaki , k. enooku , t. sato , j. imamura , t. goto , y. sugioka , h. ikeda , , h. yoshida , m. omata department of gastroenterology, university of tokyo, department of clinical laboratory, tokyo, japan background & aims: contrast enhanced ultrasonography (ceus) with sonazoid is effective for liver metastasis because enhance defect in kupffer imaging is well delineated. the aim of this study is to investigate the detection ability of ceus and the utility of sonazoid in rfa for metastasis liver tumors. material & methods: from january to december , a total of liver metastatic nodules in patients ( colon cancer, breast cancer, gastric cancer, islet cell tumor, and others) admitted to receive rfa were studied. the detection ability of liver metastasis was compared between ceus and conventional us using enhanced ct as reference standard. the mean numbers of treatment session of rfa were compared between patient treated with ceus assistance and historical controls matched for size and number of tumors. results: the detection rate was . % with conventional us and . % with ceus (p= . ). nodules in patients were not detected by conventional us and detected after injection of sonazoid. in addition, nodules in patients were detected not by ct but only by ceus. the mean number of session was . ± . as compared to . ± . in the historical controls (p< . ). conclusions: ceus with sonazoid is useful for detection of liver metastasis. sonazoid is an excellent supportive agent in rfa of liver metastasis. background/aims: carcinogenesis of intrahepatic cholangiocarcinoma (icc)-associated liver fluke infection accumulated genetic and epigenetic alterations. cholangiocarcinoma cell line (kku-m ) is adenosquamous carcinoma which rare variants and not commonly found in icc. however, interactions of liver fluke-associated icc proceed to genetic alterations in adenosquamous carcinoma that have been not elucidated. objectives: to analyze the whole genome-wide genetic alterations in kku-m using microarray comparative genomic hybridization. methods: dna of kku-m and matched-sex reference were differentially labeled with fluorescence dries (cy and cy ) and mixed together with cot- dna. the mixture was hybridized on array with spotting , human bacterial artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log ratio (cy /cy ) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m , which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on q . , q . , q . , p tel, and p . , whereas homozygous deletions were detected on q , q , q , q - , q . , q , q , q - q , p - p , p , q . , q . - q . , q . , q . and q . . conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log ratio (cy /cy ) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m , which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on q . , q . , q . , p tel, and p . , whereas homozygous deletions were detected on q , q , q , q - , q . , q , q , q - q , p - p , p , q . , q . - q . , q . , q . and q . . conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. acknowledgements: this work was supported by faculty of medicine, kku, thailand (grant no. i background/aims: we studied the clinical efficacy of arterial chemoinfusion therapy through an implanted port system for patients with intrahepatic cholangiocarcinoma (icc). thirty patients with unresectable icc or intrahepatic recurrence of icc after surgery were studied. comparison was made between patients who received arterial chemoinfusion therapy through an implanted port system with adriacin and lecithin-added lipiodol emulsion in patients and -fluorouracil ( -fu) in patients. eighteen patients were treated without port system. results: disease was stable in patients with adriacin and lecithin-added lipiodol emulsion and in patients with -fu. disease was progressed in patients with -fu. the mean survival period was . months in patients with adriacin and lecithin-added lipiodol emulsion, . months in patients with -fu, and . months in patients without port system (p= . , p= . ). conclusion: arterial chemoinfusion therapy through an implanted port system is useful for patients with intrahepatic recurrence of icc after surgery. pe s. kaur , t. kaur department of biophysics, panjab university, chandigarh. india background: a number of dietary factors have been involved in the pathogenesis of cholelithiasis. cholesterol overfeeding is the primary means of inducing supersaturated bile and cholesterol gallstones in animal models.aim of the study was to investigate the rate of epithelial cell death and proliferation in gallbladder during gallstones formation. methods: balb/c mice was divided into two groups control in this group animals were fed normal chow diet, high fat diet group in this group ( % cholesterol, . % sodium cholate, % butter fat and % coconut oil) mixed with chow diet was fed to the mice for weeks. cell apoptosis and proliferation was assayed in gallbladder epithelial cells. histological analysis of gallbladder sections were done with hematoxylin and eosin staning. results: mice fed high fat diet had apoptotic as well as necrotic epithelial cells. rate of proliferation was enhanced after and hrs in mice fed high fat diet group as compared to the control group. the histopathological section of control gallbladder has normal morphology whereas gallbladder wall thickness was markedly increased; epithelial cells appeared more elongated in mice fed high fat diet. conclusion: results obtain show that high fat diet markedly induced biliary epithelial cell proliferation and biliary epithelial cell apoptosis. it has been determined that when there is an injurious stimulus that leads to apoptosis, it is later followed by reparative proliferation and when there is no injurious stimulus, apoptosis occurs late in the course as part of remodeling. background: obstructive jaundice can be caused by malignancy. the treatment can be drainage by biliary stenting. in advanced malignant jaundice, the stent placement is often difficult. objective: to evaluate the success rate of malignant obstructive jaundice evaluation of ercp and success rate of stent placement. methods: retrospective study based on data of ercp from october until july . results: we evaluated patients who has done ercp examination, ( , %) patients have clinical diagnosis of obstructive jaundice. there were ( , %) male and ( , %) female, age range - (median age was ). there were no malignancy in ( , %) patients; malignancy in ( , %) patients and ( %) patients need further evaluation.. from patients, ( %) patients attempted to have stent placement, ( , %) patients do not and ( , %) patients have no data. we done descriptive study on patients attempted to have stent placement, ( , %) patients succeed in stent placement whereas ( , %) failed. malignancy was showed to be a factor of stent failure (malignancy: fail and success ( , %) vs non malignancy: fail and success ( , %)). conclusion: ercp can identify the cause of obstructive jaundice in % patients. the success rate of stent placement was , %. the success rate of biliary stenting in malignant obstructive jaundice was , % whereas in non-malignant cases was , %. papillary carcinoma was the most frequent cause of malignant obstructive jaundice. background: in hydatid disease of the liver cystobiliary fisula (cbf) constitutes an entity characterized by the occurrence of a life-threatening cholangitis with increased morbidity. aim: to study the different diagnostic and therapeutic aspects of cystobiliary fistula in hydatid disease of the liver. patients and methods: fourteen patients with complicated cysts were divided into groups; group a: nine patients presented with cholangitis, and group b: five patients had history of jaundice. in all patients, the diagnosis of cbf was confirmed by erc (endoscopic retrograde cholangiography). preoperative endoscopic sphincterotomy (es) was done in group a with retrieval of hydatid daughter cysts. seven patients (subgroup a ) were subsequently submitted to surgery entailing endocystectomy in and hepatic resection in two. the remaining patients in group a (subgroup a ), were managed by endoscopic therapy only. patients of group b (n= ), were not submitted to preoperative es and were subsequently managed by hepatic resection in one patient and endocystectomy in four. results: there was no mortality in the studied group. postoperative bile leak occurred in four cases in group b. in contrast, none of the patients who were submitted to preoperative es (subgroup a ) had bile leak. all patients received albendazole treatment. conclusion: erc is important in confirming the diagnosis of cbf. also, therapeutic erc has a place in the treatment algorithm of cbf as it was found to be a safe and a reliable therapeutic alternative especially in high risk patients for surgery. v. singh , g. singh , g.r. verma , v. gupta , s. ghosh , r. gupta , r. kapoor , n. sharma , a. bhalla , s.k. mahi background: endoscopic palliation in malignant hilar biliary obstruction requires ercp. however, contrast injection leads to cholangitis. recently, contrast-free metal stenting with or without mrcp has shown encouraging results. however, mrcp and metal stents are costly. there have been no reports on the use of air cholangiography in these patients. methods: we prospectively studied the role of air cholangiogaphy assisted unilateral plastic stenting in these patients. results: ten patients with unresectable malignant hilar biliary obstruction were studied. air cholangiography detected type ii obstruction in and type i in patients which is similar to mrcp. all patients underwent unilateral plastic stenting. a successful endoscopic drainange was achieved in % patients. cholanngitis occurred in none and there was no -day mortality. no major complications were observed. conclusion: air cohlangiography assisted plastic stenting in these patients is a safe and effective method of palliation. however, it requires a larger study. introduction: a description of igg -related sclerosing cholangitis (igg -sc) without pancreatic lesion has recently been reported. in addition to imaging, diagnosis relies on findings of elevated serum igg and immunodetection of invading igg -positive cells. here we report a case of igg -sc with only slight common bile duct abnormalities and normal pancreatic findings. case study: a -year-old man suffering from cephalalgia, general malaise and muscle ache was admitted to our hospital. his blood examinations on admission revealed eosinophilia, mild anemia, liver dysfunction and an igg level of mg/dl (igg mg/dl). although ercp did not reveal typical stenosis or irregularities of the bile duct wall, visualization of peripheral bile ducts was slightly impaired. echography revealed thickening of the intrahepatic bile duct and gallbladder walls as well as adenopathy. due to a gradual increase in pleural effusion and a progression of anemia, oxygenation was begun on the seventh day of illness. based on the combination of eosinophilia, elevated serum igg levels, image findings and a negative result for helminth, igg -sc was suspected. liver biopsy was performed on the ninth day of illness and steroid therapy was initiated, after which symptoms and laboratory findings improved. the igg -positive plasmocytic infiltrate present around the portal region at the time of biopsy disappeared within eight months of treatment. summary: this case displayed two unusual features that are not generally observed with igg -sc: complications due to hemolytic anemia, and destruction of the peripheral bile duct with little damage to the common bile duct. introduction: various systemic diseases have been reported to be associated with igg . although steroids are effective in the treatment of igg -related diseases, there are some reports on relapses with their treatment, and cases are often difficult to differentiate from malignant diseases. we encountered a case of autoimmune pancreatitis with sclerosing cholangitis (aip-sc), in whom ca - was elevated with episodes of exacerbation and an elevated serum igg concentration. igg staining was also useful for the diagnosis. case study: an -year-old woman noticed tumors beneath the bilateral jaw and was found to have an elevated level of ca - ( ) seven years previously. her left submandibular gland was removed and diagnosed as sclerosing sialadenitis. four years previously, she was diagnosed as having diabetes mellitus complicated by a recurrence of ca - ( ) elevation and liver dysfunction. cholangiocarcinoma was suspected based on ercp, but was not confirmed by histologic findings of bile duct biopsy. elevated igg and other test results established the diagnosis of aip-sc, so steroid therapy was initiated, after which symptoms and laboratory findings improved. this recurrence of ca - elevation ( ) was diagnosed as a relapse of aip-sc based on an increased igg level and histologic findings. summary: some papers have reported that igg -positive cells are found in liver tissue in this disease, but such cells were not detected in the liver specimens in our case. this might be because intra-liver sites may have differed in the degree of morbidity, and long-term steroid therapy might have suppressed inflammation in the liver tissue. s. kaur , t. kaur department of biophysics, panjab university, chandigarh, india background: cholelithiasis, a gallstone disease is major cause of morbidity affecting millions of people throughout the world. aim of the present study was to investigate the predisposing factors that lead to the formation of gallstones. methods: the study was carried out on gallstones, bile and serum of patients. gallstones and bile were divided into three groups' cholesterol, pigmented and mixed gallstones. blood of the patients was divided into two groups with gallstones and without gallstones patients. trace elements and various biochemical estimations were carried out. clinical history of the gallstones patients was recorded from the hospital records. results: trace elements analysis in bile and gallstones showed that calcium is the main element in all the three types of stones. iron was the main element in mixed gallstones. in pigmented gallstones magnesium and zinc were the major trace elements. liver function tests and lipid peroxidation levels in sera were significantly increase whereas, antioxidant enzymes concentrations in sera were significantly decreased in patients with gallstones. clinical history of the gallstones revealed the cases had jaundice, diabetes mellitus and estrogen replacement therapy respectively. conclusion: results suggest that trace elements in gallstones and bile as well as clinical history of patients with chronic cholelithiasis could be the underlying factor in the pathogenesis of gallstones. the concentration of products derived from the free radicals reactions increases with degree of inflammation. such a condition increases risk of bile saturation which would further contribute to the progress of gallstones formation. background and aims: diseases of the biliary tree and gallbladder are being described with increasing frequency among patients with the acquired immunodeficiency syndrome (aids).therefore there is a need to do a research about the risk factors of gallbladder diseases in hiv/aids patients. so it can be useful to clinicians to predict the possibility of a patient having gallbladder disease and consider the options of further plans. the aim of this study was to find the prevalence and varieties of gallbladder diseases in hiv/aids patients. methods: a cross sectional study was performed in patients with hiv/aids who visited ciptomangunkusumo hospital, jakarta. the risk factors (route of transmision,cd ,arv,hepatitis) and clinical presentations were studied.ultrasonography examinations were performed to detect gallbladder annormalities. results: patients with hiv/aids match the study criteria. there were gallbladder abnormalities in ( . %) subjects, which ( . %) had acalculous cholecystitis and ( . %) had cholecystitis with cholelithiasis. on bivariate analysis, there was a significant association between abdominal pain, jaundice and the use of arv to gallbladder abnormalities (p = . ; . ; . ; . ). however, there was no association between age, sex, transmision route of hiv, hepatitis and cd to gallbladder abnormalities. conclusion: hiv/aids patients are susceptible to opportunistic gallbladder infection. acalculous cholecystitis is the most frequently encountered gallbladder abnormalities of hiv/aids patients in this study. poster exhibition -hbv poster session, hall b long-term stopping therapy t.b. trung , p.h. phiet university medical center, hochiminh city, vietnam background: among the approved nucleos(t)ide analogues therapies for chronic hepatitis b, lamivudine was used widely, sometime inappropriate in practice due to high safe and low price but lamivudine is associated with the highest rate of drug resistance. objectives: the aim of the study was to determine the ymdd variants after long-term stopping treatment in lamivudine-resistant patients using more sensitive technique. methods: blood samples from lamivudine resistant patients were collected after long-term stopping therapy. the ymdd variants are detected using technique pcr restriction fragment length polymorphism (pcr-rflp) at hcmc university medical center results: after stopping lamivudine treatment months ( - months) ymdd mutants were detected in ( , %) of patients. among them ( . %) had the most important m v/i mutant, ( %) had accompanying l m mutant. it means that once drug resistant mutants have been selected, they are archived for the long time even if treatment is stopped. many of patients have the features characterized for the patients in immune tolerance phase (young age, hbeag positive, normal alt). the treatment of this group is not strongly recommended due to low efficacy and high risk of drug resistance. conclusion: the most important m v/i mutant was still detected with significant portion of the virus population after long-term stopping therapy in lamivudine resistant patients. the options of retreatment for this patients when necessary are limited due to cross-resistance. the management of chronic hepatitis b should be followed strickly the recommendations of specialized association to avoid this problem. background/aim: whether liver stiffness measurement (lsm) using transient elastography is reliable to assess liver fibrosis in the settings of severe acute exacerbation of chronic hepatitis b (chb) is uncertain. methods: we prospectively recruited consecutive patients with severe acute exacerbation of chb (alanine aminotransferase or alt > x upper limit of normal). the relationship of alt levels and lsm were serially assessed and liver biopsy was performed after alt normalization. results: eleven patients ( male, median age years) were followed up for weeks; patients received anti-viral therapy. overall, lsm was positively correlated with alt levels (r= . , p< . ). at initial presentation, the median serum alt and lsm was ( - ) iu/l and . ( . - . ) kpa. a progressive reduction in lsm was observed during subsequent visits in parallel with the reduction of alt levels. even after the normalization of alt at week , lsm of patients continued to drop at week . at the last visit, the median alt was ( - ) iu/l and lsm was . ( . - . ) kpa. among the patients who had liver biopsy performed at week , patients had f fibrosis (lsm . - . kpa) and patient had f fibrosis (lsm . kpa). conclusions: lsm using transient elastography may misdiagnose liver cirrhosis in patients suffering from severe acute exacerbation of chronic hepatitis b. lsm should be assessed after normalization of alt levels in order to accurately assess the degree of fibrosis. h.c. lai , s.w. lai , k.f. liao , c.s. liu , t. lin , c.c. lin china medical university hospital, taichung, taiwan background: in , chronic liver disease was the seventh leading cause of death in taiwan. hepatitis b and hepatitis c are two major causes of chronic liver disease in taiwan. the purpose was to investigate the seroepidemiology of hepatitis b surface antigen (hbsag) and hepatitis c virus (hcv) antibody in taiwan. method: this was a hospital-based cross-sectional study. we analyzed viral hepatitis data from subjects who received health checkups at one medical center in taichung from to . all subjects were divided into three age groups, including - , - and . this study emphasized the prevalence of hbsag and hcv antibody by gender and age. the statistical analysis was performed by t test and . result: there were men ( . %) and women ( . %). the mean age was . (standard deviation . , range - ). the overall prevalence of hbsag was . %, with statistically significant difference(ssd) between gender ( . % for men vs . % for women, p < . ). the prevalence of hbsag was decreased with age in men, with ssd (p < . ), and also decreased in women, without ssd (p = . ). the overall prevalence of hcv antibody was . %, without ssd between gender ( . % for men vs . % for women, p = . ). the prevalence of hcv antibody was increased with age both in men and in women, with ssd (p < . ). conclusion: we hope this study can provide the epidemiological data for further studies of hepatitis b and hepatitis c virus infection in taiwan. s.m. wu , x. zhou wuhan medical treatment center, center for gene diagnosis, zhongnan hospital, wuhan university, china e-selectin is revealed to facilitate leukocyte adhension to the endothelium and migration into inflamed tissue in inflammatory diseases. chronic hepatitis b virus infection is regarded as a chronic inflammatory process. to examine the possible involvement of e-selectin in the etiology of chronic hbv infection, we analyzed two polymorphisms of e-selectin and determined the plasma souble e-selectin levels in patients with chronic hbv infection and controls. the frequency of c allele of the a c polymorphism was significantly increased in patients with lc campared with controls. no significant positive association was observed between the g t polymorphism and chronic hbv infection. but in patients with lc, divided according to the child-pugh classification, the frequency of t allele was of significant difference between child'class a and class b plus c. plasma levels of soluble e-selectin were significantly increased in patients with chronic hepatitis and liver cirrhosiscompared with controls. in the liver cirrhosis group, levels of se-selectin were significantly decreased from child' class a to class c. in each group, patients with c allele of the a c polymorphism showed higher soluble e-selectin levels than those with a allele. this is the first report describing the association between e-selectin polymorphisms and hbv-related hepatic fibrosis. our data showed the a c polymorphism of e-selectin gene is associated with disease progression in patients with hbv infection and controls the expression of plasma soluble levels, the g t polymorphism may be related to fibrotic severity in patients with liver cirhosis. background: chronic hepatitis b (chb) patients with high serum hbv-dna and normal serum alanine aminotransferase (alt) levels might be considered for treatment if histopathological findings show fibrosis stage or more. however, to our knowledge there is no recommendation with regard to the therapeutic agents for this group of patients. objective: this study was aimed to evaluate the efficacy of nucleoside analogues (entecavir or telbivudine) in treating chronic hepatitis b patients with high serum hbv-dna and normal serum alt levels. patients and method: this was an open-label study in chb patients with high level serum hbv-dna levels between january and october . patients were included if they showed normal serum alanine aminotransferase (alt) level at two measurements within a -month interval and had fibrosis stage > on liver biopsy specimens. patients were treated with entecavir . mg/day or telbivudine mg/day. the primary endpoint was the reduction or undetectable of serum hbv-dna at week and week of treatment, while the secondary endpoint was hepatitis b e antigen (hbeag) seroconversion. results: during a -year period, chb patients with high level serum hbv-dna with normal alt two times with months interval underwent a liver biopsy. twenty-eight ( . %) of pts showed fibrosis stage on histological findings (metavir score). twelve of these patients received nucleoside analogues, ( . %) of them were men. patients' median age was (range: - ) years. there were patients with stage- , patients with stage- and patient with stage- fibrosis. eleven ( . %) patients had genotype b virus. at baseline, the mean serum alt level was + . u/l and mean hbv-dna level was . x iu/ml, ranging from . x to . x iu/ml. six patients received entecavir and the other six received telbivudine therapy. undetectable hbv-dna was achieved by ( . %) patients at week- and ( . %) patients at week- of treatment. one patient who had the highest hbv-dna level had viral load reduction to . x iu/ml at week- of treatment. two out of patients with positive hbeag achieved hbeag seroconversion at week- of treatment. conclusion: this preliminary study has shown that nucleoside analogues might be considered in the treatment for chronic hepatitis b patients with high serum hbv-dna and normal serum aminotransferases levels. j. chen , x.j.. wu , y. wang , g.q. wang department of infectious diseases, peking university first hospital, beijing, china background: the dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death- (pd- ) and its ligands, pd-l /pd-l , are new members of cd /b family, as co-stimulatory molecules expressing on t cells and antigen present cells (apcs). their engaging can downregulate the t cells function, including proliferation, cytokines secretion and cytotoxicity. in periphery blood, pd- was upreguated on virus specific-t cells, leading to the impairment of t cells. blocking the pd- /pd-l can improve the function of t cells. methods and patients: patients with chronic hepatitis b (chb) were treated by pegylated ifn - b (pegintron from schering-plough, once a week, . or g/kg/weight). the periphery blood were taken at weeks, weeks, weeks, and weeks. periphery blood mononuclear cells (pbmc) were isolated from fresh heparinized blood by ficoll-hypaque (density: . g/l) density gradient centrifugation. then the cells were incubated with apc-conjugated anti-pd- antibodies. the pd- expression on lymphocytes was detected by flow cytometry (fcm). results: the pd- expression on lymphocytes at weeks was . ± . %, at weeks was . ± . %, at weeks was . ± . %, at weeks was . ± . % (p< . ). conclusion: treatment with ifn - b can downregulate the pd- expression on lymphocytes and may partially restore the function of t cells. to investigate the effects of nucleoside analogs therapy in hepatitis b related acute-on-chronic liver failure, we treated hbv related acute-on-chronic liver failure patients with entecavir. as control, the remaining were not treated with nucleoside analogues. results show the survival rate of entecavir therapy group has no significantly difference with none-treated group (p> . ). although entecavir greatly reduced hbv replication during different therapy times (p< . ), the meld score and liver function (alt, albumin, bilirubin, prothrombin time) had no significant changes (p> . ). further more, we analyzed the meld score and liver function in different hbv-dna level patients .no significantly difference was observed (p> . ). there is no significant correlation between hbv-dna level and meld score in different therapy times (p> . ).the hbv-dna level between patients with over months and less than months survival patients showed no significant difference either (p> . ). however, meld score and some parameters of liver function (albumin, bilirubin, prothrombin time) showed significant difference (p< . ). these results suggest hbv-dna loading may not be a direct factor to increased liver injury and suppression of hbv replication may not reduce the severity of liver failure in hbv related acute-on-chronic hepatitis. s. firdoos , u. adeeb , a. mehmood , m. gill islamabad specialists clinic, islamabd, pakistan background: before the availability of etv, it was common to use adv for treatment of chronic hepatitis b patients. primary nonresponse and suboptimal response is a common problem with adv treatment. methods: we wanted to study the outcomes of entacavir therapy in this subset of patients. study was conducted between april to april . we enrolled chb patients who had non response to - weeks of mg adv therapy. non response and suboptimal response was defined as non dimunition of at least one log of hbvdna from baseline after weeks of therapy and persistence of log after weeks of therapy respectively.they were switched to mg entacavir before breakfast daily for at least months.they had serial alt cbc and hbvdna measured every weeks. results: out of patients male and were female. only patients were hbeag(+).mean hbvdna level prior to adv exposure was . log copies/ml.mean duration of exposure to adv was weeks. patients lost to f/u.we did intention to treat analysis. out ( %) patient has, undetectable level of hbvdna after weeks of therapy labelled as group . out of ( %) had hbvdna level reduced by a mean of log copies/ml labelled as group .on week treatment analysis all patients from group was hbvdna undetectable, additional patients from group had undetectable hbvdna. conclusion: entacavir therapy results in rapid suppression of hbvdna levels in majority of patients with primary nonresponse or partial non response to adv therapy. background: except for serum alt level, baseline factors predictive of therapeutic response to lamivudine in patients with hbeag-positive chronic hepatitis b remain largely unknown. we thus studied the influence of pre-therapy viral factors on end-of-treatment responses to lamivudine therapy. methods: a total of treatment-naïve hbeag carriers who had pre-therapy serum alt level> xuln and received lamivudine for months reimbursed by the national health insurance were prospectively enrolled. hbeag seroclearance and combined hbeag seroclearance, alt normalization as well as undetectable hbv dna at the end of therapy were defined as primary and secondary endpoint, respectively. the pre-therapy viral factors including viral load, genotype, precore stop codon (pc)/ basal core promoter (bcp) status, and pre-s deletion were determined to correlate with therapeutic endpoints. results: the frequency of patients with detectable pc mutation (g a), bcp mutation (a t/g a), and pre-s deletion at baseline was . %, . %, and . %, respectively. after completing -month lamivudine therapy, overall hbeag seroclearance rate was . %. patients with hbeag seroclearance had a higher prevalence of baseline pc mutation than those without ( . % vs, . %, p= . ). by multivariate analysis, the odds ratio of patients with pc mutation to develop hbeag seroclearance was . (p= . ). in addition, the presence of pc mutation also correlated with the combined response. conclusions: for hbeag-positive chronic hepatitis b patients with serum alt> xuln, pc mutation could predict a higher hbeag seroclearance rate at the end of -month lamivudine therapy. the efficacy of adefovir dipivoxil against all patterns of lamivudine resistant hepatitis b d.j. kim , y.d. park , y.g. kwon , h.g seo daegu fatima hospital, kunngpook national university hospital, daegu, korea background: our aim was to evaluate the efficacy of adefovir dipivoxil (adv) and determine patient-dependent or laboratoroy variables that are predictive of hbeag loss and ivr for hepatitis b patients resistant to lamiduvine. also we evaluated the activity of adv against all patterns of lamivudine-resistant hbv. method: hbv-infected patients with lamivudine resitance received adv for months. quantitative hbv dna, hbeag/anti hbeag, alt was checked every - months. the hbv polymerase of patients were sequenced for baseline samples to determine the presence of lamivudine resistance mutations. result: there is no significant difference in all patterens of hbv mutation about hbv dna reduction at w, w, w. there is no significant difference in all patterens of hbv mutation about alt normalization at w, w, w. conclusion: adefovir dipivoxil demonstrated similar potent anti-hbv efficacy regardless of the different patterns of lamivudine-resistant hbv mutations. g. novelli , m. rossi , v. morabito , f. pugliese , p. berloco la sapienza university, rome, italy background: hepatitis b (hbv)-related end-stage liver disease is one of the most common indication for liver transplantation (lt). a number of patients dying while on the waiting list or removed because of being too ill is progressively increasing. we valued the possibility to improve the model end-stage liver disease (meld) of patients awaiting liver transplantation using a albumin dialysis: molecular adsorbent recirculating system (mars). methods: we treated patients ( male and female) with a mean age . . inclusion criteria: serum bilirubine > mg/dl, meld , inr > . , encephalopathy grade ii. all patients were treated with mars mean ± . hr cycles and mean treatments (range - ). all patients received standard medical treatment in addition to mars dialysis. the patient survival was valued at six months. results: we obtained a significant change of cytokines levels as interlukine (p< . ) and tumor necrosis factor alfa (p< . ) in association with an improvement of kidney, hepatic and hemodynamic parameters. at the end of mars treatments we observed a significant reduction of meld score (p< . ). the results of meld show a rebound effect between the end of treatment and the follow up at six months without returning at starting values (p< . ). twenty patients lived and dead for clinical complications. conclusion: the improved meld score with mars gave patients on lt waiting list more time of survival, thus allowing them more opportunity for liver transplantation. entecavir for treatment of lamivudine-refractory patients chronic hepatitis b h.t. dat , p.t.t. thuy medic medical centre, ho chi minh, vietnam lamivudine treatment is associated with frequent development of resistant hepatitis b virus. this incidence especially is higher in longer time of treatment and loss of treatment benefit. entercavir is a new antiviral agent shown its high efficacy even in cases of mutations with lamivudine resistance. in this study, we evaluate the efficacy, the safety of entercavir in treatment of lamivudine-refractory patients chronic hepatitis b. sixty chronic hepatitis b patients with evidence of lamivudine resistance were randomly divided into two groups in proportion of : . group i (n= ) used entecavir mg/day, group ii (n= ) used lamivudine mg/day. treatment time was weeks. histology, alt, hbvdna were evaluated in the end of the treatment. age, sex, alt, hbvdna, genotype, hbeag were analyzed to evaluate their influences to the treatment. the results have showed hbvdna< copies/ml in entecavir group . % vs. % lamivudine group (p< . ). hbvdna negative in entecavir group was . % and incidence of seroconversion of hbeag was . %. alt was normal in entecavir group . % vs. . % in lamivudine group (p< . ).histologic improvement in entecavir group was . % vs. . % in lamivudine group (p< . ). patients with hbeag negative, genotype b, low viral load were shown better results. entecavir was shown to be efficacious in treatment for chronic hepatitis b patients experienced with lamivudine resistance. entercavir is safe, with almost no side effects. factors such as hbeag negative, genotype b, low viral load seems to be better in response to treatment. recurrence or mutation of entecavir resistance should be studied further in future. j.m. kim , s.k. hwang , b.h. choe department of pediatrics, kyungpook national university hospital, daegu, korea backgrounds: by analyzing the characteristics of children with chronic hepatitis b who have lost hbsag by long-term lamivudine treatment, the selection of target patients could be relevantly predictable in the treatment of chronic hepatitis b in children. methods: a total of hbeag positive children (< y-o) were recruited who have visited kyungpook national university hospital from mar. , to may , . they were treated with lamivudine for at least months. hbeag seroconversion occurred during lamivudine treatment in out of children. they were divided into hbsag clearance and non-clearance group. parameters influencing treatment results were analyzed according to hbsag loss. result: thirteen out of the ( . %) patients with hbeag seroconversion were classified as hbsag clearance group, while ( . %) as non-clearance group after lamivudine treatment. twenty five of patients with hbeag seroconversion were under years old, in ( / , %) of whom hbsag loss occurred as well. twenty four of patients were over years old, in ( / , . %) hbsag loss occurred, that showed significantly difference (p-value= . , or: . , ci: . - . ) compared to younger group. age was significantly lower in hbsag clearance group ( . ± . years) than non-clearance group ( . ± . years) (p= . ), but no difference was observed in other parameters. anti-hbs appeared in patients. conclusion: in the treatment of hbeag positive chronic hepatitis b with lamivudine, age was significantly lower in hbsag clearance group than non-clearance group. background: dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death- (pd- ) and its ligands, pd-l /pd-l , are members of cd /b family, was reported to transfer inhibitory signal, leading to the dysfunction of t cell. background: hepatitis b viral mutants can emerge in patients as a result of selection pressure from either immune response or treatment options. mutations of hbsag allow mutant virus to propagate in the presence of a neutralizing immune response, while wild-type virus in reduced to undetectable levels. methods: immunohistochemical analysis of tissue samples from patients with chronic hepatitis b (chb), acute hepatitis b (ahb) patients and health controls was performed. results: pd- was positively expressed on lymphocytes infiltrating the portal area.pd-l expression was the same as pd- ,also expressed in interlobular.pd-l expressed on kupffer cells and dendritic cells.pd- -,pd-l -,and pd-l -positive cells express index of chb patients were much more than that of health controls and ahb patients(p . ).between groups in chb,the expression rate increase with the disease progression (p . ). methods: chronic hepatitis b patients with both positive for hbsag and hbsab were studied.serological markers of hbv were detected by elisa and microparticle enzyme immunoassay. hbv dna levels were determined by fluorescent quantitative pcr, s gene fragments were directly sequenced, liver function was analyzed by automatic biochemistry analyzer au . correlation test was conducted to evaluate their dependablity. conclusion: overexpression of pd- and pd-l within liver might be involved in inhibiting the immune response and be a mechanism of chronicity in hbv infection. results: the level of hbsag and hbsab was . ± . s/n and . ± . miu, respectively. hbv dna was detectable in patients. fifty-one mutations of s gene were detected in patients, and the relating amino acid substitution was at the sites of , , , , , , , , , , and . eight ( . %) out of mutations were located at the "a" determinant region in patients, while no mutation was found at the sites of , and . however, the mutation did not affect hbv replication. hbv dna was positive correlated with hbeag. conclusions: change in hbsag antigenicity due to s gene resulted in concurrent hbsag and hbsab. the existence of hbsab did not affect hbv replication. the damage of liver failure in those patients was slight. background: hbv infection is common in bangladesh. we often encounter young patients incidentally detected with hbeag negative chronic hepatitis b (chb) in our clinical practice. however the characteristics of these patients is yet to be studied in this country. the aim of this study was to study the characteristics of young bangladeshis incidentally detected with hbeag negative chb. methods: we did percutaneous liver biopsies of chb patients aged between to years. they were all hbeag negative with persistently normal or raised serum alt values. we did pre-core mutation (pcm) study in patients who were randomly selected. results: % patients had significant necro-inflammation (hai-ni > ), while significant fibrosis (hai-f > ) was seen in . %. serum alt (cut off u/l) was raised in . %, while high hbv dna load (> copies/ml) was observed only in . %. pcm was negative in all . conclusion: although chb patients between - years of age are supposed to be in immune clearance phase, which is characterized by low hbv dna and hbeag positivity, the study shows that hbeag negative chb is an entity that can also be seen in this age group and a significant percentage of such patients may have considerable hepatic involvement. this challenges our current concept about immune clearance state of hbv infection, although much larger study is needed to draw any specific conclusion. background: hbv infection is common in bangladesh, but characteristics of young patients incidentally detected with chronic hepatitis b is yet to be studied in this country. methods: we did percutaneous liver biopsies of chb patients aged between to years. results: significant necro-inflammation (hai-ni > ) was seen in . % patients with hbeag positive and % patients with hbeag negative chb, while significant fibrosis (hai-f > ) was seen in . % and . % patients in these two groups respectively. serum alt (cut off u/l) was raised in % hbeag positive and . % hbeag negative patients, while in these two groups % and . % patients respectively had high hbv dna load (> copies/ml). conclusion: hbeag negative chb is an entity that can also be seen in young population. a significant percentage of both hbeag positive and negative patients may have considerable hepatic involvement. profile of hbeag +ve chronic hbv infection in bangladesh m. mahtab , s. rahman , f. akbar , f. karim , a. shrestha , m. khan , m. kamal bangabandhu sheikh mujib medical university, toshiba general hospital, dhaka, bangladesh background: inactive hbv carriers constitute the major reservoir of hbv. present management guidelines provide inadequate treatment modalities. they are recommended for regular check-up; treatment is only recommended when patients exhibit evidence of liver damage. this is due to lack of information about their extent of liver damage. aim of this study was to assess extent of liver damage in hbeag +ve patients, unaware of their infection. methods: in this retrospective study, records of hbeag +ve chb patients from our pool of chb patients were reviewed. they were tested for hbsag, hbeag, hbv dna, anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: . % ( / ) patients were males and . % ( / ) females. they were between - years of age. alt was raised > times unl in % ( / ). . % ( / ) patients had high hbv dna (> copies/ml), while low hbv dna (< copies/ml) was seen in . % ( / ) . in high hbv dna group, significant necro-inflemmation (hai-ni > ) was seen in . % ( / ) and significant fibrosis (hai-ni > ) in . % ( / ) . figures were . % ( / ) and . % ( / ) respectively in low viral load group. none tested positive for hcv infection. conclusion: study indicates that machinery should be developed to characterize undetected hbv carriers in developing countries by conducting multi-center clinical studies. we have shown that considerable number of patients, unaware of their hbv infection, suffer from progressive liver damage. the overall strategy of management of chronic hbv infection should also be revisited. high viral load does not necessarily represent significant liver damage in patients with chronic hbv infection in bangladesh m. mahtab , s. rahman , f. akbar , f. karim , a. shrestha , m. khan , m. kamal bangabandhu sheikh mujib medical university, toshiba general hospital, dhaka, bangladesh background: in general, it is assumed that patients with chronic hepatitis b virus (hbv) infection with high viral load exhibit increased liver damages. treatment guidelines also emphasize on reducing viral load. these observations were mainly accumulated from developed countries. > % chronic hbv carriers live in the developing nations, but little is known about relationship between hbv viral load and extent of liver damage in these countries. in this study, we addressed this issue. methods: in this retrospective study we reviewed records of chb patients from our pool of patients. all had high hbv dna (> copies/ml). . % ( / ) were hbeag +ve and . % ( / ) hbeag -ve. they were alsotested for anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: . % ( / ) hbeag +ve patients with high hbv dna had non-significant hepatic necro-inflammation (hai-ni < ); this figure was . % ( / ) in hbeag -ve patients. non-significant hepatic fibrosis (hai-f < ) was observed in . % ( / ) and . % ( / ) in hbeag +ve and -ve patients respectively. none tested positive for hcv. conclusion: correlation doew not exist between viral load and liver damage in chb in bangladesh. many with both hbeag +ve and -ve chb with high hbv dna do not have significant hepatic necro-inflammation and fibrosis. further study may be needed to find out influence of other factors on liver damages in chb in bangladesh. most of these patients have not been characterized and treatment modalities have not been defined for them. background/aims: expression of intrahepatic hepatitis b core antigen (hbcag) is related to the immunopathogenesis of hepatitis b virus (hbv) infection. the role of hbv genotype and basal core promoter (bcp) mutation in expression of hbcag was investigated. methods: seventy hbeag-positive chronic hepatitis patients (genotype b in and c in ; bcp t /a mutation in ) were enrolled. clinical, virologic and histologic features were compared with regard to localization and expression of intrahepatic hbcag. the effects of hbv genotype and bcp t /a mutation on the expression of hbcag were further evaluated by in vitro assays. results: cytoplasmic, mixed cytoplasmic/nuclear, and nuclear localization of intrahepatic hbcag were found in ( . %), ( . %) and ( . %), respectively. fifty-eight ( . %) of these patients expressed a high level of hbcag. in multivariate analysis, cytoplasmic localization of hbcag correlated only with low serum viral load (p= . ) and bcp mutation (p= . ). high expression level of hbcag also correlated with high serum viral load (p= . ) and bcp wild-type sequence (p= . ). in vitro assays supported that hbv bcp mutant had lower subcellular expression of hbcag compared with bcp wild-type strain. conclusions: hbv bcp mutation and viral load but not genotype contributes to the expression of intrahepatic hbcag. hepatitis b virus (hbv) genotypes show distinct geographical distributions and virological and clinical differences. in some of genotypes, specific substitutions and mutations have been described in association with hepatitis b e (hbe) protein expression and viral replication. in this study, genetic characteristics of hbv genotype e (hbv/e) were investigated using clinical samples obtained from hepatitis b e antigen (hbeag)-positive, and anti-hbe-positive asymptomatic carriers (ascs) in west-africa. full-genome analysis of isolated hbv strains revealed strong association between precore (pc) mutation and hbeag to anti-hbe seroconversion. furthermore, using partial genome sequences, correlation among hbeag/anti-hbe status, viral load and key mutations were analyzed. the data showed that pc mutation is associated with hbeag seroconversion and enhanced viral replication efficiency. comparison between hbv/e and hbv/d strains reveals these two genotypes to have an identical sequence in their core-promoter-upstream and basic core promoter (curs/bcp) regions. it has been known from the previous phylogenetic studies, that hbv/d and hbv/e cluster together in trees reconstructed on x and precore/core orfs. in addition, this study, demonstrates that in spite of the high sequence similarity of curs/bcp region, the seroconversion-related mutation patterns are different between hbv/e and hbv/d in asc. further studies are needed to clarify the clinical significance of the regulatory sequence similarity between hbv/e and hbv/d. necro-inflammation and fibrosis p. siddappa , p. kar , b. das , r. gondal , m. asim maulana azad medical college, icpo, new delhi, india background: chronic hepatitis b(chb) is an important cause of morbidity and mortality. methods: pilot study involving patients of chb, were equally randomized to receive either adefovir or lamivudine for months. quantification of serum and hepatic hbv dna levels by real time pcr and liver biopsy done at start and end of months. results: after months there was significant and comparable reduction in serum and hepatic hbv dna viral load and liver biopsy showed significant reductions in hai scores in both the groups. serum alt which was elevated to or more times normalized in both the groups. in the adefovir group patients became hbeag negative and patients who were hbeag negative at the start of therapy remained so. in the lamivudine group one patient became hbeac negative and patients who were negative at the start of therapy remained so. in the adefovir group patients became hbv dna (qualitative test) and in the lamivudine group patients became hbv dna negative. there was strong correlation between serum and hepatic hbv dna levels both before and after the completion of therapy. conclusion: both the drugs bring about biochemical, histological and serological improvement with significant reduction in viral load in serum liver after months without complete clearance of virus. there was not enough evidence to show therapeutic advantage of one drug over the other. the serum and hepatic hbv dna levels correlate well with eachother before and after treatment. aim: assessing efficacy and safety of treatment of chronic hepatitis b in children with pegylated ifn. materials and methods: children ( boys and girls) aged - years with chb treated with peg-ifn alfa- a, g/m /week during weeks, hbeag-positive and hbeag-negative children, previously treated with recombinant interferon. no child had liver disease greater than grade , stage . serum hbv dna was quantified at baseline, tw , ("rvr") tw , tw (etr) and w (svr) with rt pcr method (roche taqman). alt activity, haematology and adverse events were monitored. results: after weeks treatment median hbv dna level decreased from . x iu/ml at baseline to . x iu/ml (p< . ). "rvr" -undetectable hbv dna at tw was observed in / children and associated with lower pretreatment alt levels < iu and pretreatment viral load < iu/ml. all children with "rvr" were hbeag-negative pretreatment. at tw and tw seven children including all with "rvr" had undetectable hbv dna. children achieved svr (undetectable serum hbv dna in w ), among them with "rvr". in / children with "rvr" hbsag disappearance was observed since tw . leukopenia was reported in children, thrombocytopenia in . no adverse events were observed following dose modifications. conclusions: . peg-ifnalfa- a is a good therapeutic option for children with chb, in particular with hbeag-negative chb . low pretreatment viral load and "rvr" seem to be predictive factors of efficient therapy. control by investigating the sanitizing modes among appliances used in the public service places (psp) and hbsag among appliances and practitioners worked in those places. methods: beauty parlors, barber shops and bathing centers selected by stratified randomization sampling, workers were investigated in questionnaire. the hbsag in appliances of psp and employee was detected by ria. results: the rate of hbsag among appliances of psp was . %. the rate of hbsag in large-, medium-and small-sized appliances was . %, . % and . %. the rate of hbsag has different( = . p . ). the rate of hbsag among appliances of beauty parlors, barbering shops and footbath inns was . %, . % and . %. different appliances had different rate of hbsag, such as the rate of acne needle and the forceps was . % and . %. the positive of hbsag amongworkers in psp was . %. the rate of hbsag among workers in large-, medium-and small-sized psp was . %, . % and . %. the rate of hbsag among workers in beauty parlors, barbering shops, footbath inns and bathing centers was . %, . %, . % and . %. the hbsag rate among workers was different in different works, the rate was higher in tattoo workers ( . %), pedicures workers ( . %), massagists ( . %). conclusions: it is important to enhance the sanitizing management in psp and improve workers kap) of hepb. and we should promote health education to enhance the knowledge of hepatitis b control and build up supervision consciousness. background: integration of hepatitis b virus (hbv) dna into host chromosomes is often found in chronic liver disease and hepatocellular carcinoma, which is likely an early event of hbv-related carcinogenesis. however, the molecular mechanism of integration remains unclear. here we describe a potential mechanism of hbv integration and identify that ku and ku , the gatekeepers of non-homologous end-joining (nhej) repair pathway, can serve as targets for anti-hepatitis virus integration. methods: using i-sce endonuclease-based system, we induced a dna double-strand break (dsb) in human hepatoma cell line huh- . the cells were then incubated with serum from patients with chronic hbv infection. pcr amplification and direct sequencing were used to detect the inserted sequence in the site of dsb. finally, we employed taqman-based real-time pcr assay to quantify the integrated hbv dna and evaluate the effects of shrna on hbv integration. result: when huh- were exposed to viral serum and incubated for several days, hbv dna was detected in integrated form at the exact site of dna damage. furthermore, small interference rna (sirna) targeted against gatekeeper genes for nhej can down-regulate nhej repair and even the frequency of hbv integration. conclusion: thus, this project provided us with the first direct evidence that dna double-strand breaks are potential targets for hbv integration. the study has also shown that shrnas targeted against gatekeeper genes for nhej can regulate the frequency of hbv integration. objective: to screen proteins of human pancreas cdna library interacting with hbsag protein. methods: the library was amplifed, purified and evaluated, and then the puried library plasmids were transformed into yeast strain y . the reconstructed plasmid pgbkt -hbsag was transformed into yeast strain ah and screened on the nutrient deficiency medium sd/-trp. the transformed ah mated with y containing the library plasmid. the diploid yeast cells were plated on nutrient deficiency medium sd/-trp/-leu/-his/-ade and sd/-trp/-leu/-his/-ade containing x--gal for selecting. the plasmids in diploid yeast cells were extracted and electrotransformed into e.coli dh . the plasmids in dh were extracted, sequenced and analyzed by bioinformatic methods. results: sixteen proteins interacting with hbsag were founded. conclusions: these results show that hbsag protein may be related with metabolism of glucose and lipid. comparison of the sensitivity and specificity of the elecsys ® hbsag ii assay with other available assays in china for detection of hbsag j.d. jia , l. wei , x.x. zhang , y.l. mao , l.l. wang , z.l. gao , j.l. hou , j. zhang , w. melchior , w. van der helm , beijing friendship hospital, beijing, china, beijing people hospital, beijing, china, ruijin hospital, shanghai, china, beijing hospital, beijing, china, west china hospital, chengdu, china, guangzhou, china, guangzhou nanfang hospital, guangzhou, china, shanghai public health clinical centre, china, roche diagnostics ltd, rotkreuz, switzerland, conclusions: in this patient population the prevalence of hbsag positive and anti-hcv were much higher than reported in community studies. genotypes and accounted for most of hcv. these very high rates of viral hepatitis in a hospital setting challenge to healthcare providers in terms of patient management as well as caregiver's prevention. hepatitis b is one of the major diseases of mankind that kills about one million persons each year in the world. accoring to primary study about % of iranian population is chronic hbv carriers. among iranian cirrhotics, - % has evidence of exposure to hbv and - % is carriers. because increase demand of blood transfusion, high blood dependent patients and long term window period of hbv infection, any controlling hbv infection program in blood donors can enhance the blood safety and public health. pe in this descriptive study included all the blood donors that referred to dezful blood transfusion center during - . all the blood donors screened for hbs ag by using enzyme immuno assay and repeatedly reactive (r.r) samples confirmed by hbc-ab or confirmatory (neutralization) tests. the data analyzed by using spss . . we found that in the first year . % were repeatedly reactive and . % confirmed. the results for other years as the followed: . %(r.r) and . % confirmed and in the last . % (r.r) and . % confirmed. the repeated blood donors increase in this period ( . %, . % and . % respectively). aim: we aimed to evaluate the cost-effectiveness of telbivudine versus entecavir with reference to lamivudine by roadmap model. methods: decision analysis model was used to study the incremental cost-effectiveness ratios (icer), i.e. the additional cost (in usd) required to achieve undetectable hbv dna or hbeag seroconversion for a patient at years in america and hong kong. entecavir was used as a continuous monotherapy. lamivudine and telbivudine would be shifted to entecavir if hbv dna was detectable at month and continued otherwise with drug resistance treated by add-on adefovir. weighted event rates based on previous reports were estimated for analysis. according our study, although the prevalence was higher than other region in our province, the hbv prevalence showed good decrease after stablishment strategies such as of repeated blood donor recruitment , improvement the donor selection and other educational programs . good following up those strategies to enhance the blood safety recommended. results: telbivudine was generally cheaper than entecavir to achieve an incremental case of undetectable hbv dna from lamivudine at years. entecavir was least effective and most costly for hbeag seroconversion. conclusions: telbivudine is a cost-effective alternative to entecavir particularly when its cost is low in hong kong. h. tang , g.l. zhang , y.x. li , r.q. tian , m. liu , x. li tianjin life science research center, tianjin medical university, tianjin , china micrornas (mirnas) are single-stranded noncoding rnas of to nucleotides that play critical roles in a wide spectrum of biological processes. we investigated whether the mirnas-silencing machinery influences hbv replication or antigen expression. on the basis of elisa and mtt, the effect of mirnas on the hbsag expression and cell proliferation was examined. three micrornas efficiently inhibited hbsag expression without significant effect on the proliferation of hepg . . cells compared to lacz control. subsequently, bioinformatics analysis were used to predict targets for the three mirnas, and the prediction results were conformed by cdna microarray analysis. the target region in hbv genome and the 'utr region of one cellular gene were identified by fluorescent reporter assay, semi-quantitative rt-pcr and western blot. the results demontrated that mirna may play an important role in replication and gene expression of hbv. hepatitis b virus (hbv) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. although considerable progress has been made, the pathogenesis of hbv infection is still elusive. there's an urgent need to elucidate the mechanisms of hbv-host interactions, to discover novel biomarkers for diagnosis and prognosis and to develop therapeutic targets for anti-hbv treatment. herein, we applied a two-dimensional gel electrophoresis and maldi-tof/ms based comparative proteomics approach to globally analyze the host response to hbv by using an inducible hbv-producing cell line hepad . of the differentially expressed proteins identified, glucose regulated protein (grp ) was one of the most striking proteins elevated by hbv replication, which was confirmed by real-time pcr and western blotting. knockdown of grp expression by rna interference resulted in a significant increase of both intracellular and extracellular hbv virions in hbv-transfected hepg cells. reversely, grp overexpression led to hbv suppression. the expression levels of hepatitis b surface antigen (hbsag) and hepatitis b e antigen (hbeag) were determined by enzyme linked immunosorbent assay (elisa). immunofluoresce further revealed a positive correlation between the expression levels of grp and hbsag in both hbv-transfected hepg cells and hbv-infected human liver tissues. altogether, these data demonstrate for the first time that grp is an endogenous anti-viral factor in hbv-transfected hepg cells and may serve as a potential prognostic indicator of viral status in anti-hbv therapies. background/aims: to evaluate the predictors of response to long-term treatment of adefovir dipivoxil (adv) in patients with emerging lamivudine (lam)-resistant hepatitis b e antigen (hbeag)-positive chronic hepatitis b (chb) patients. methods: one-hundred-thirty-four lam-resistant hbeag-positive chb patients were treated with adv for a median of . months (range, - months), following lam therapy for a median of . months (range, - months). patients ( . %) were switched from lam to adv monotherapy, ( . %) were switched to adv with month of lam overlap therapy, and ( . %) were switched to adv with months of lam overlap therapy. the influence of baseline parameters on treatment response to adv in patients with lam-resistant hbeag positive chb was analyzed. result: during the follow-up period, ( . %) of patients achieved complete response, defined as normalization of alt level, negative hbv dna by a digene hybrid capture assay and achievement of hbeag loss. sixteen ( . %) patients achieved hbeag seroconversion. twenty-eight ( . %) patients developed adv-related mutations during adv treatment. in multivariate analysis, virological response at months (or= . , % ci: . - . , p= . ), defined as serum hbv dna levels less than log copies/ml or a reduction in serum hbv dna levels greater than log copies/ml after months of adv therapy, independently predicted complete response. conclusions: virological response at months was the strongest predictor of adv response in lam-resistant hbeag-positive chb patients. background/aims: to explore the effects of hbv dna level hbv genotype/subgenotype on the pathogenesis of severe liver diseases in chongqing. methods hbv dna level was analyzed in patients with severe liver diseases in retrospect,and hbv genotype/subgenotype hbv dna level and hbeag were determined in patients with hepatocellular carcinoma (hcc,n= ), liver cirrhosis(lc, n= ),chronic hepatitis b(chb, n= ) and acute on chronic liver failure(aclf, n= ). results hbv level from high to low with chb were lc, aclf and hcc in turn(p . ). hbv genotype was mainly genotype b.the rate of genotype b and c were . % and . respectively in hcc patients, . % and . in lc patients, % and in chb patients, % and in aclf patients. the percentage of genotype b/c in aclf patients was higher in compared with other groups. but the distribution of hbv genotype among groups was not statistically different(p . ).subgenotypes of genotype b were almost ba but one. subgenotypes of genotype c were mainly ce in chongqing area, and there was no statistical difference among the groups (p . ). conclusion: hbv dna level seems not to be a determining factor at end point of severe liver disease. both genotype b and c of hbv can lead to severe liver diseases, and there are more mixed infections by different genotypes in aclf. the efficacy of switching to entecavir (etv) monotherapy in japanese lamivudine (lvd)-experienced patients. background: this study aims to determine the efficacy of switching to . mg etv daily in chronic hepatitis b (chb) patients previously treated with lvd. method: retrospective analysis of chb patients (n= ) previously on mg lvd daily and switched to . mg etv daily. results: lvd-experienced patients were divided into three groups based on hbv viral load at time of switching to etv (< . log copies/ml; . - . log copies/ml and > . log copies/ml). detection of lvd-resistant virus at the time of switching was higher in the group with hbv dna . log copies/ml ( % in both . - . and > . log copies/ml groups versus % in < . log copies/ml group) and was higher in patients treated with lvd for years ( % versus % for patients on < year of lvd). a year after switching to . mg etv daily, hbv dna undetectable rates were % ( / ), % ( / ) and % ( / ) for < . , . - . and > . log copies/ml groups, respectively. alt normalization occurred in more than % patients at the end of the first year of switching to etv for all three patient groups. only one patient in the . - . log copies/ml group, who had lvd-resistant mutants at the time of switching, developed etv resistance during follow-up. conclusion: switching from lvd to etv maintains or improves viral suppression and alt normalization, especially in patients with viral load < . log copies/ml. background/aims: we investigated the association between on-treatment hbsag decline and sustained response in patients treated with pegasys±lamivudine. methods: hbsag levels were measured retrospectively pre-treatment and at weeks , , and using the abbott architect hbsag assay in sera from patients ( % asian) treated with pegasys alone ( g qw; n= ) or combined with lamivudine ( mg qd; n= ) alone for weeks as part of a large multinational trial. response was measured months post treatment. results: more patients treated with combination therapy had > log decline in hbsag from baseline to week ( figure) . hbsag level < iu/ml at week was associated with higher rates of response to pegasys±lamivudine months post treatment ( figure) . data comparing hbsag and hbv dna as on-treatment predictors of response will be presented. conclusion: on-treatment hbsag monitoring may be useful for predicting response in patients treated with pegasys. y. wakui , j. inoue , y. ueno , t. shimosegawa division of gastroenterology, tohoku university graduate school of medicine, sendai, japan background/aim: chronic hepatitis b patients are clinically treated with nucleot(s)ide analogues and ifn-. nucleot(s)ide analogues have problems including drug resistance in continuous treatment, and ifn-has disadvantages of limited effectiveness and side effects. therefore, novel antiviral drugs are still needed. in this study, the suppressive effect to the replication of hbv was examined in vitro by using bezafibrate and rosiglitazone, which are ligands of peroxisome proliferator activated receptor (ppar) and , respectively. methods: the cytotoxicity of bezafibrate and rosiglitazone to hepg cells was examined with mts assay, and the concentration of % cytotoxicity (cc ) was calculated. hepg cells were transiently transfected with the plasmid containing . -fold hbv genome of a genotype b strain. after hours of transfection, rosiglitazone and bezafibrate was added to the cells. using the medium at day after the addition of drugs, hbv dna was quantified with real-time pcr. results: the cc of bezafibrate and rosiglitazone in hepg cells were m and m, respectively. the amount of hbv-dna in the medium was decreased when the density of bezafibrate was over m, but the density demonstrated considerable cytotoxicity. in contrast, rosiglitazone of m, which showed no cytotoxicit, decreased the amount of hbv dna. the % effective concentration (ec ) was calculated to be . m. conclusions: in this study, it was suggested that the replication of hbv was inhibited by rosiglitazone of the density without cytotoxicity. the mechanism is uncertain and being investigated now. q. zheng center for liver diseases, the first affiliated hospital, fujian medical university, fuzhou, p. r. china background: the objective of this study was to evaluate the early virologic response for prediction of achievement of hbeag seroconversion and hepatitis b virus (hbv) dna negativity after two years of lamivudine treatment in chronic hepatitis b (chb) patients. methods: in this retrospective study, adult patients with chronic hepatitis b ( hbeag-positive and hbeag-negative) were treated with lamivudine ( mg/day), and followed-up up to months. response and resistance to the treatment were assessed during the treatments with lamivudine. results: it was found that gender, age, baseline levels of alt and hbv dna, serum hbv dna at week (p = . , or = . ) were closely related to the achievement of hbeag seroconversion, undetectable hbv dna level and emergence of drug resistance after years of lamivudine treatment. hbeag positive patients with baseline serum hbv dna in - copies/ml and serum hbv dna copies/ml at week showed high response rate of alt normalization rate ( . %), undetectable hbv dna rate ( . %), hbeag seroconversion rate ( . %), as well as low drug resistance rate ( . %) after years of treatment. similarly, hbeag negative patients with serum hbv dna copies/ml at week could achieve high -year response rate of alt normalization rate ( . %), undetectable hbv dna rate ( . %), and low drug resistance rate ( . %). conclusion: serum hbv dna copies/ml at -week provide the best prediction of -year lamivudine treatment response. background/aims: unlike oral antivirals, a finite course of (peg)interferon can induce sustained post-treatment response in patients with chronic hepatitis b (chb), with increasing rates of hbsag clearance observed in patients who respond during post-treatment follow-up. hbsag clearance is considered to be the closest outcome to a cure, being associated with improved histological outcome, reduced incidence of hcc and increased survival. methods: in a randomised multinational study, patients (hbeag-negative) received µg pegasys+placebo (n= ); µg pegasys+ mg lamivudine (n= ); or mg lamivudine (n= ) for weeks, and were assessed months post-treatment. from this initial study, of those who had received pegasys±lamivudine and patients who had received lamivudine monotherapy participated in a long-term observational study to investigate post-treatment response. hbsag clearance at yearly post-treatment follow-up visits up to years post-treatment was analysed. results: hbsag clearance in patients treated with pegasys±lamivudine increased post-treatment ( % at year to %, %, % and . % at years , , and ). at year , pegasys-treated patients ( . %) had cleared hbsag compared with ( . %) of lamivudine-treated patients (p= . ). / pegasys-treated patients had anti-hbsag (hbsag seroconversion). detailed analysis of the -year follow-up data will be presented. conclusion: the ability of a finite course of pegasys to induce sustained response with increasing hbsag clearance rates in responders during post-treatment follow-up supports its use as first-line therapy in hbeag-negative patients with chb. background/aims: recent studies suggest that quantification of hbsag levels early during treatment can be used to predict post-treatment response to pegasys. elecsys ® hbsag ii (roche) is a sensitive assay for the detection of hbsag. this assay can be used for the quantification of hbsag levels using a simple dilution algorithm. we compared results obtained using the elecsys ® hbsag ii method with those of a commonly used quantification assay. methods: hbsag levels obtained using the elecsys ® hbsag ii assay were compared with those obtained using the abbott architect ® hbsag assay for a total of samples from patients infected with hbv genotypes a (n= ), c (n= ), d (n= ) and f (n= ). samples were diluted : in diluent provided by the manufacturer. samples with hbsag levels > iu/ml were retested at a final dilution of : . samples with hbsag levels < . iu/ml were retested undiluted. results: overall, hbsag levels measured with the two assays correlated well (r = . ) over a wide range ( - x iu/ml). discrepancies in hbsag levels >± % were reported for a minority of the samples (n= ), mainly distributed evenly above and below the ideal line (n= ). in the four low titre (range - x iu/ml) samples with greatest discrepancy elecsys ® underestimated values (in two cases by > %). conclusion: the elecsys ® hbsag ii assay provides a simple and reliable means for determining hbsag levels. this simple assay format could be used to provide useful information during on-treatment monitoring of hbsag levels in patients with chronic hepatitis b undergoing therapy. conclusions: hbv/a has been increasing in chb patients in japan as the consequence of ahb, spreading in the younger generation through promiscuous sexual contacts, thrust by an inclination of hbv/a to induce chronic hepatitis. the spread of hbv/a infection in japan should be prevented by universal vaccination programs. introduction: chronic viral hepatitis is common in end-stage renal disease (esrd), from endemic hepatitis b (chb) and nosocomial hepatitis c (chc). reduced outcomes post-renal transplant were reported thus chb and chc cirrhosis became contraindications to listing. however, these predated effective anti-viral therapies. we reviewed outcomes of patients with chronic viral hepatitis following assessment for renal transplantation. methods: prospective database of esrd patients with viral hepatitis referred for renal transplantation was reviewed. results: patients were assessed. patients underwent kidney transplantation. two were cirrhotic and had liver/kidney transplantation; both died within months. were non-cirrhotics, of whom are alive. / have functioning allografts; predictors were normal alt and low viral load. of the non-transplanted, had cirrhosis; / received anti-virals. mortality was % - liver-related ( hepatoma, bacterial peritonitis, sepsis - inactive cirrhosis); non-liver related ( cerebral, haemorrhage, renal - inactive cirrhosis). / surviving cirrhotics received anti-virals. in non-transplanted non-cirrhotics, mortality was %; % of survivors had inactive disease. chb patients received lamivudine; adefovir (lamivudine resistance). chc patients received ifn-based therapy. conclusion: excellent outcomes are achieved in esrd patients with chb/chc post-renal transplant, in absence of cirrhosis. normal alt/non-detectable viral load can predict graft function. however, cirrhosis is associated with high mortality on dialysis whereas non-cirrhotics with inactive disease do well. the role of kidney transplantation in cirrhotics with suppressed viral replication needs to be reassessed. the truncated hbc interferes with replication of hepatitis b virus j.c. han , , x.b. pan , , l. wei , , k. deng , institute of hepatology, peking university people's hospital, china hepatitis b virus (hbv) capsids play an important role in production of progeny virus and other elements of the virus life cycle. misdirection of capsid assembly and formation of aberrant particles may be an effective approach to interfere with virus replication. hbv capsids can be assembled in vivo and vitro from the dimeric hbv core protein (hbcag). the interaction of single and dimeric hbcag with some truncated hbcag is verified in vitro. the truncated hbcag consists of the first amino acids and lacks the c-terminal, -residue rna-binding domain. method: we transiently transfected hepg . . with pcdna . hbc by fugene .after and h, hbvdna, hbeag and hbsag in culture supernatant were detected and cell subjected to southern blot and immunofluorescence analysis. result: the level of hbsag and hbeag had gentle change, we found that hbvdna decreased at h after transfection( copies/ml p< . ) ,but replication intermediates obviously decreased from h. some positive signal of hbcag located around the nuclear and conglomerated in cytoplasm compared to the control. conclusion: the truncated hbc can inhibit replication of hepatitis b virus. misdirection of capsid assembly and formation of aberrant particles could be an important cause. y.p. li , r.c. li objective: to assess the long-term efficacy of recombinant yeast derived hepatitis b vaccine in infant s born to hbsag and hbeag carrier mother. methods: a total of neonates born to hbsag, hbeag both positive mothers were vaccinated with , , g doses of recombinant yeast derived hepatitis b vaccine by , , and months schedule. they were all followed for years after the primary vaccination. results: twelve infant s ( . %) become hbsag positively conversed in year after primary vaccination ,and the positive rate of hbsag in - year was . %- . % , . % of child in no/ lowly respond become hbsag positively. at the ninth year, the positive rates of anti-hbs were % above. anti-hbs positive rates and immunity level were higher at - year old by repetition immunity than others. conclusion: the recombinant yeast derived hepatitis b vaccine have good immunogenecity and long-term protective efficacy to hbv interruption of perinatal transmission , a booster dose seems necessary in aged - years to the mother with hbsag and hbeag.it is high risk tobecome hbsag positively in the baby of norespones to hepatitis b vaccine. chb patient group-initiated programme to improve awareness, adherence and treatment outcomes in asia pacific n. leung founding chairperson of asiahep background: worldwide, over million people live with chronic hepatitis b (chb); million in asia pacific. regional survey data from , patients in countries showed a lack of knowledge and understanding of chb, its severity and impact on quality of life. this initiative aims to coordinate patient groups in the region and devise programmes to improve knowledge and healthcare outcomes. methods: the patient groups met in hong kong in may and identified common needs to: ( ) improve educational resources; ( ) raise awareness; ( ) increase diagnostic yield; and ( ) enhance treatment compliance through education about the need for sustained viral suppression to reduce long-term complications. results: a patient engagement programme was developed for people with newly diagnosed or known chb. the programme comprises: -detailed information about chb -a health-tracking tool for self-monitoring of blood tests and treatment progress -detailed information for carers/family -a patient-physician communications video (including role-play) -mobile phone text messages providing advice and compliance/appointment reminders conclusion: this programme was developed to address the needs of patients and clinicians. improved knowledge and long-term support, particularly for patients on antiviral medication, is expected to improve quality of life. the programme encourages clinicians and patients to develop enduring therapeutic partnerships to promote optimal outcomes. acknowledgement: the chb patient group meetings and the patient engagement programme are supported by an unrestricted educational grant from glaxosmithkline. serum hbv rna level reflects the potency of nucleos(t)ide analogue y.w. huang , , k. chayama , , m. tsuge , , s. takahashi , , t. hatakeyama , , m.y. lai , , h.l. you , j.t. hu , c.j. liu , , p.j. chen , , d.s. chen , , s.s. yang , j.h. kao , liver unit, cathay general hospital medical center, background and aims: serum hbv rna is detectable in patients treated with lamivudine (lmv) or entecavir (etv) (hatakeyama, and huang, ) . the aim of this study was to determine the clinical significance of serum hbv rna levels in patients treated with nucleos(t)ide analogues of different potency. methods: serum hbv rna was serially determined in patients treated with nucleos(t)ide analogues for to weeks ( with adefovir (adv), with lmv, and with etv). serum hbv rna was quantified by reverse transcription of hbv nucleic acid extract with subsequent real-time pcr. results: hbv rna was detectable in patients as follows: of in adv ( %), of in lmv ( %), and of in etv ( %) (p = . ). mean log serum hbvdna levels at baseline were . ± . for adv, . ± . for lmv, and . ± . for etv, which were comparable between less potent adv and most potent etv (p = . ). during antiviral therapy, peak log hbv rna level of patients with etv was significantly higher than that of those with adv or lmv ( . ± . vs. background: in the phase iii clinical trials, clevudine mg for months showed potent antiviral activity along with a marked post-treatment antiviral effect. the objective of this study is to compare the anti-hbv activity of combination of clevudine and vaccine over clevudine alone in chronic hepatitis b (chb) patients in a randomised way. methods: the patients are received clevudine for weeks and then combination of clevudine and vaccine for another weeks or clevudine alone for weeks. eligible patients were treatment-naïve hbeag(+) chb patients with hbv dna levels , copies/ml. the primary endpoint is the proportion of patients with hbeag loss. preliminary results are presented here. results: thirty-one patients have completed week visits and from them, patients ( in clevudine alone and in combination group) have completed week visits. at week , % of patients had hbeag loss. at week , % in clevudine alone and % in combination group ( months on combination after clevudine monotherapy) had hbeag loss. at week , % of patients had negative hbv dna by amplicor pcr (< copies/ml). at week , all of patients in both groups had negative hbv dna by pcr and % in clevudine alone and % in combination group had normal alt. conclusion: clevudine demonstrated good serologic response as well as significant viral suppression and alt normalization. with this data, we conclude that combination therapy of clevudine and vaccine for short period does not show the superiority over clevudine alone. background/aims: to determine the reasonable number of clones for hbv quasispecies analysis. methods: chronic hepatitis b patients were enrolled with hbvdna levels range from ~ log copies/ml. hbvdna was extracted. hbv reverse transcriptase (rt) gene encompassing the overlapping surface s gene was amplified by polymerase chain reaction, then cloned and sequenced. ten positive clones for each sample were sequenced in the first group, and then additional ten positive clones were sequenced in other groups until up to thirty clones. the characteristics of hbv quasispecies including shannon entropy and genetic distance were calculated. results: the shannon entropy and genetic distance of clones group was higher than those of and clones group, either in rt gene or in s gene (p< . ). while the shannon entropy and genetic distance of clones group showed on difference with those of clones group, neither in rt gene nor in s gene (p> . ). the number of different quasispecies detected in clones group was higher than that of and clones group (p< . ). the shannon entropy and genetic distance in three different clones group had no correlation with hbv dna levels (p> . ). conclusion: although the number of different quasispecies detected was increased with the augmentation of clone number, the quasispecies characteristic didn't changed significantly when the clone number more than . the information contained in clones per sample could well represent the quasispecies characteristics. the clone number was not necessary modulated according to different hbv dna levels. background: recent studies reported that basal core promoter mutation (a t and g a) was associated with more aggressive progression of liver disease from inactive carrier to active hepatitis, and eventually to liver cirrhosis and hcc. but the effect of the double mutations on the activity of enhancer ii/basal core promoter is still uncertain. objectives: to evaluate the influence of nt a/t and nt g/a mutations on hbv enhancer ii/basal core promoter activity. methods: the pcr fragments of hbv enhancer ii/basal core promoter (nt to nt ) from the serum-derived genotype b hbv dnas of one hbv carrier aged and one hbv related hepatocellular carcinoma patient aged were introduced into the pgl -basic-vector from promega via restriction sites of xho i and hind iii. the nt a to t and t to a, the nt g to a and a to g mutations were carried out by genetailor site-directed mutagenesis system from invitrogen. the promoter activity was evaluated by comparing firefly luciferase measurement with renilla luciferase as the internal control using the dual-luciferase reporter assay system from promega. results: the luciferase reporter assay results indicated that the t to a combined with a to g mutations increase (p< . ) while the a to t combined with g to a mutations decrease (p< . ) the hbv enhancer ii/basal core promoter activity significantly. conclusions: associated with increased risk of hepatocellular carcinoma, a t and g a double mutations of hepatitis b virus reduce the enhancer ii/basal core promoter activity. background/aims: a substantial proportion of chronic hepatitis b (chb) patients with mildly elevated alanine aminotransferase (alt) have significant fibrosis. we evaluated the factors associated with significant fibrosis and clinical outcomes in these patients. methods: one hundred five chb patients with alt less than two times the upper limit of normal underwent liver biopsy. multiple clinical, biochemical and virologic variables were evaluated to determine the predictors of significant fibrosis and progressive liver disease. results: there were patients in the low normal alt group, in the high normal alt group, in the low elevated alt group, and in the high elevated alt group. fifty eight patients ( . %) had significant fibrosis ( stage ) and ( . %) had significant inflammation ( grade ). the age, platelet count and grade of inflammation were factors associated with significant fibrosis. progressive liver disease was observed in ( . %) of the followed-up patients. the stage of fibrosis, alt group and antiviral therapy were significant predictive factors for progressive liver disease. conclusion: liver biopsies should be recommended in patients over years with mildly elevated alt levels, and antiviral therapy should be considered in patients with significant fibrosis to prevent progressive liver disease. background: four nucleos(t)ide analogues (nas) are currently approved for the treatment of hbv infection in china. however, long-term benefits are limited by the emergence of drug-resistant viruses. methods: patients accepted the examination based on physician's instruction. hbv reverse transcriptase gene was amplified from serum via nested pcr and sequenced directly. results: well-recognized drug-resistant mutations were detected in of , patients. in patients receiving na monotherapy, corresponding drug-resistant mutations were detected in / for lamivudine (lam), / receiving adefovir (adv), / for entecavir (etv), and / for telbivudine (l-dt). the mutations were detected in / patients receiving kinds of sequential/combined usages of the nas. m i ( %), m v+l m v l ( %), and m i+l m ( %) were identified as major mutant patterns of lam monotherapy. n t a substitution was the dominant adv-resistant mutation. t substitution was the dominant etv-resistant mutation always accompanied with lam-resistant mutation. l-dt-resistant mutation was m i l m exclusively. adv-resistant mutation was frequently seen in lam-resistant patients receiving adv sequential therapy rather than those receiving adv add-on therapy. controversial lam/adv-resistant mutations including a t, v a, q s and i v were detected in some patients singly or with the well-recognized drug-resistant mutations. interestingly, the drug-resistant mutations were also observed in a few of patients naïve to nas. conclusions: the exploration of hbv drug-resistant mutation profile in large clinical samples furthers our understanding of hbv drug-resistant status in china with implications for administrating anti-hbv therapy more reasonably. toll-like receptor (tlr) , tlr and cd +cd +cd low/-regulatory t cells correlate with hepatitis b virus infection y. zhang , j.q. lian , c.x. huang , x. wei , j.p. wang , p.z. wang , x.f. bai center of infectious diseases, tangdu hospital, the th military medical university, xi'an, china background: tlrs play a crucial role in sensing and initiating innate antiviral response and tregs actively suppress immune response, contributing to viral persistence and chronic tissue damage. in this study, we determined tlr and expression and treg frequency, as well as their function in the effect of hbv infection. methods: tlr and tlr expression on monocytes and circulating cd + cd + cd low/-tregs were determined by flow cytometry in ahb, chb, asc and nc. spearman correlation was performed to investigate associated variables on treg or tlrs. pbmcs were stimulated with hbeag or hbcag and the tlrs profile was examined. result: tlr expressions were up-regulated in chb and asc, while tlr were increasingly expressed in ahb and asc. treg frequency in chb was significantly higher than that in nc. in chb, the increased tlr negatively correlated with hbv dna loads and treg frequency negatively correlated with tlr expressions. tlr was up-regulated after hbeag stimulation in both nc and chb. conclusion: increased tregs may be associated with chb and there might be possible interactions between hbeag, tlr signaling and the innate immune response, which may partially explain the mechanism of hbv infection induced immuno-tolerance. ( . ± . ) . hbv-dna was quantitatively determined by polymerase chain reaction (pcr) technique, and hbv genotype was determined by pcr microwave gene chip technique. antiviral efficacy was assessed using measuring the following scales: the alt normalization rate, hbv-dna negative conversion rate and the hbeag/anti-hbe seroconversion rate. results: among serum specimen, hbv genotype distribution was genotype c, genotype b, and genotype non-b or c respectively. in genotype b, alt normalization rate was . %( cases), hbv-dna negative conversion rate was . %( cases) and the hbeag/anti-hbe seroconversion rate was . %( cases). in genotype c, alt normalization rate was . % ( cases), hbv-dna negative conversion rate was . %( cases) and the hbeag/anti-hbe seroconversion rate was . %( cases). the efficacy of adefovir dipiroxil showed no significant differences between genotype b and c in the treatment of chronic hepatitis b p> . . conclusion: adefovir dipiroxil is an effective antiviral drug. hbv genotype is irrelevant to the antiviral efficacy of adefovir dipiroxil in treatment of patients with chronic hepatitis b. the effect of anti-hbv drugs on albumin and bilirubin levels, and platelet count h. yoshida , h. taniguchi , r. nagano , k. sakitani , e. seki , t. serizawa , y. ito , h. mizuno , y. mitsuno , r. nakata , m. omata japanese red cross medical center, university of tokyo, japan background/aim: we assessed the efficacy of anti-hbv drugs on the liver function. methods: patients with hbv-related disease followed at our center between and were enrolled. lamivudine ( mg), lamivudine ( mg) +adefovir ( mg), or entecavir ( . mg) was administered to the patients with detectable hbv dna and elevated alt. liver function (alt, alb, and t.bil) and platelet count were observed. alt, alb, t.bil, and platelet count of treated group at pretreatment, year , and year were compared with untreated group. results: eighty six patients with positive hbsag were enrolled between jan and dec . seven patients ( acute infection, overlap infection with hcv, lost of follow up) were excluded. in total patients were followed up for a median follow up of (range - ) months. of patients, received anti-viral treatment. twenty one patients were treated with lamivudine, with lamivudine+adefovir, and with entecavir. the mean of levels of pre-treatment-year -year were alt: - - (u/l), alb: . - . - . (g/dl), t.bil: . - . - . (mg/dl), and plt: . - . - . (x mcl) respectively. markers of untreated group (n= ) (at baseline-year -year ) were alt: - - (u/l), , t. bil: . - . - . (mg/dl), and plt: . - . - . (x mcl) respectively. although all of four markers in treated group were significantly worse than untreated group at baseline, all of four markers did not showed significant difference from untreated group at year . conclusion: treatment with anti-hbv drugs showed the efficacy not only transaminase levels, but also on albumin, bilirubin, and platelet count improvement-improvement of "hepatic reserve" which is valuable for prevention of cirrhosis. background: currently, hbeag-negative chronic hepatitis b(chb) is increasing. but there are still controversial on the treatment of hbeag-negative chb with alt ×uln. we have investigated the clinical efficacy of nucleotide analogues(nas) in the treatment of hbeag-negative chb with alt ×uln. methods: the data of patients who were treated by nas for more than years and with alt ×uln (n= ) , alt ×uln(n= ) and alt ×uln(n= ) were collected, and w and w virologic response, w and w complete response, virologic breakthrough and clinical resistance were analyzed. results: compared with the base line, hbv dna level in all three groups were significantly decreased (p . ), and there was no significant difference between alt ×uln group and alt ×uln group. the viral load was significant decreased in alt ×uln group at w, w and w (p< . ). virologic response at w and w complete response at w and w was . %, . % , . % and . % respectively in alt ×uln group and was . %, . %, . % and . % respectively in alt ×uln group. there was no significant difference between alt ×uln group and alt ×uln group. virologic response at w and w and complete response at w were significant decreased (p< . ) in alt ×uln group. there was no significant difference among the three groups in virologic breakthrough and clinical resistance. conclusion: hbv replication can be satisfactory inhibited by nas in hbeag-negative chb patients with alt ×uln, which suggests that in these patients the indication of alt is different from hbeag-positive patients. quantitative hbeag assay as a predictive factor of hbeag seroconversion induced by peg-ifn - a therapy to hbeag-positive chronic hepatitis b y.y. zhu , j. dong , y.t. chen , j. chen , j.j. jiang liver diseases research center, the first affiliated hospital of fujian medical university, fuzhou, fujian, china rp, background: to find predictive factor for hbeag seroconversion in the treatment of hbeag-positive chronic hepatitis b (chb) by peg-ifn - a. methods: hbeag-positive chb patients were given peg-ifn - a treatment for weeks. clinical data were collected every months. receiver operator characteristic (roc) curve was employed to calculate positive predictive value (ppv), negative predictive value (npv), sensitivity and specificity. results: sixty-five patients completed peg-ifn - a therapy. among them, ( . %) were found hbeag seroconversion and ( . %) were found hbeag loss at cessation of therapy. none of age, gender, alt level and hbv dna load at baseline had relationship with hbeag seroconversion. hbeag level of baseline was correlated to hbeag seroconversion, with p value as . (table ) . according to roc curve, supposed auc as . and p value as . , the ppv, npv, sensitivity and specificity of hbeag level as at week were . , . , . and . , respectively. supposed auc as . and p value as . , the ppv, npv, sensitivity and specificity of hbeag level as at week were . , . , . and . , respectively. the hbeag level (s/co) and decreased degree (percentage) at week and week were significant related to hbeag seroconversion (table ) . conclusion hbeag level at baseline and at th and th week and its decreased degree (percentage) during the treatment course could be used as predictive factor for hbeag seroconversion. background: it is well documented that perinatal transmission is the major cause of chronic hbv infection in china. the aim of this study was to evaluate the efficacy of interruption of hbv intrauterine infection with hepatitis b immunoglobulin (hbig) in pregnant women with hbeag positive. methods:: a prospective randomized controlled trial was adopted. each subject in the trial group ( cases) was given iu hbig intramuscularly every weeks from -week of gestation, while each subject in the control group ( cases) received placebo in the same way. the cord blood of newborns were collected for detecting hbsag, hbeag and hbv-dna. results: for newborns, hbeag positive rate in trial group was . %( / ).hbeag positive rate in control group was . %( / ). there was significant difference in hbeag positive rate of newborns between the two groups( p < . , rr = . ). hbv-dna positive rate in trial group was %( / ). hbv-dna positive rate in control group was . %( / ). there was significant difference in hbv-dna positive rate of newborns between the two groups( p < . , rr = . ). hbv-dna load of cases of newborns in trial group was lower than that of their mothers(t = ,p = . ). there was no significant difference in hbv-dna load between women and their newborns after delivery in control group (t = . ,p > . ). conclusion: it is effective and safe to prevent hbv intrauterine infection with hbig from the (th) wk in pregnant women with hbeag positive. ), especially, the cirrhosis and hcc cases obviously more in both hbeag and anti-hbe patients are negative than hbeag-negative but anti-hbe positive patients (. . % vs . %; . %vs . %, p ) .the prevale nce of pre-core g a mutate have no significant difference regardless of hbv serum marker status or the state of illness. conclusion: recent years the hbeag-nagative chronic hepatitis b patients are gradually increasing in yunnan province. while the hbeag disappear but no anti-hbe serum transfer, and the virus still active replication -it may be a crucial phase determined the diseases outcome, which should be pay more attention by physicians. the clinical significant of pre-core g a mutate remain unknow. efficacy of interferon for chronic hepatitis b patients with normal or paranormal alt z. liu , j.z. guo , y.j. lin , y.j. zhang , z.w. lang beijing ditan hospital, beijing, china background: we reported interferon treatment for cases with normal or paranormal alt but in which liver histologic exam showed g - and/or s - . methods: patients were male with an average age of years.mean alt was . iu/l and hbv dna level was ~ log copies/ml. two patients were hbeag positive; one patient was both negative for hbeag and anti-hbe and one patient was anti-hbe positive. liver biopsy showed g ~g and s ~s respectively. patients were treated with ifn-alpha, liver biopsy was repeated after year.only one patient had received combination therapy with ifn and adefovir after months treated ifn monotherapy and liver biopsy was taken after . years. results: all patients got normal alt after year treatment. hbv dna was undetectable in patients. patients with initial positive hbeag cleared. but patients still were anti-hbeag negative.liver biopsy showed change fromg s - to g s - in patient; fromg s to g s in patient and no change in the other patients. conclusions: though alt and hbv dna improved after year treatment, histological improvement is not satisfying. patient's improvement in liver histology may be due to seroconversion before treatment and adding adefovir after months of interferon therapy. after months of combination therapy we did liver biopsy again. the other patients were hbeag negative, but hbeab were also negative, liver biopsy was taken year later without combination of nucleoside analogs. evaluation of long term efficacy of hepatitis b vaccination r.c. li , j. gong , j.y. yang objective: to evaluate the long term effectiveness of preventive hbv infection and to monitor the incidence of hepatitis b in children to see possible impact on the program of long an that was launched in . methods: ( ) set up a surveillance systemof hepatitis ,to evaluate the possible impact on incidence of hepatitis b. ( ) to serologically evaluate the effect of the program, a stratified random sampling of subjects in birth cohorts was recruited for long term follow up at the age - years. ( ) cross-sectional seroepidemiolgical survey was carried out in the county in before the program and years later. hbsag , anti-hbs and anti-hbc were tested by ria. results: the average coverage of hepatitis b vaccine was . %. at years after vaccination, the seropositivity for hbsag in population of - years has decreased from . % to . %, the annual effectiveness was . %. hbv accumulated infection rate was . %, protective rate was . %. the incidence of acute hepatitis b was . per , in population aged - years , it decreased by . % as compared with the incidence of . per , in same age group in - . conclusion: mass hepatitis b vaccination program in long an county has proved to be effective in control of hbv chronic infection and incidence of acute hepatitis b. background and aims: although the evolution of viral quasispecies may be related to the pathological condition of disease, little is known about this in hepatitis b virus (hbv), especially during hbeag seroconversion. methods: nucleotide sequences of hbv precore/core genes from time points were analyzed in four cohorts of chronic hepatitis b, interferon-induced seroconverters (is, n= ), interferon non-responders (in, n= ), spontaneous seroconverters (ss, n= ) and non-seroconverters (sn, n= ), followed during months on average. only patients with genotype c were used. viral diversity was then estimated after nucleotide genetic distance was assessed and phylogenetic trees were constructed. results: analysis of nucleotide sequences showed that the nucleotide genetic distance of seroconverters (is and ss; x - substitutions/site and . x - subsititutions/site, respectively) was similar to that of non-seroconverters (in and sn; both x - substitutions/site) before seroconversion. compared to that of nonseroconverters (in and sn; substitutions/site and . x - substitutions/site, respectively) the viral diversity of seroconverters (is and ss; x - substitutions/site and x - substitutions/site, respectively) was significantly higher after seroconversion (p< . ) and it was higher after seroconversion in seroconverters compared with that berore seroconversion (p< . ) while it almost didn't change in non-seroconverters irrespective seroconversion. phylogenetic trees also showed that complex trees appeared in secoconverters and relatively simple in nonseroconverters. conclusions: the distinctly higher viral diversity after seroconversion in hbeag seroconverters could be related to increased hbv-specific t-cell responses and escape mutant which arise from stronger selective pressure caused by host immune activity. adefovir dipivoxil mg (adv) resistance at yrs in chinese hbeag+ve chronic hepatitis b (chb) j.l. hou , y.z. wang , x.q. zhou , j.q. niu , y.m. wang , h. wang , y.m. mao , k.f. barker nanfang hospital, guangzhou, prc, jinan infectious disease hospital, jinan, prc, ruijin hospital, shanghai, prc, st hospital of jilin university, changchun, prc, xinan hospital, chongqing, prc, people's hospital, beijing, prc, renji hospital, shanghai, prc, glaxosmithkine r&d, london, uk background: long term adv provides clinical and histological improvement in chb, but may lead to emergence of treatment associated resistant mutations. we report on adv resistance data from chinese hbeag positive subjects treated for years. methods: hbeag positive chb subjects were randomized in an initial weeks controlled adv study (with a weeks placebo period in half of patients) and then offered open label adv treatment for a further weeks. a total of , , , and subjects completed the st , nd , rd , th and th yr, respectively. at the end of each year samples were analysed from those subjects with protocol-defined hbv dna breakthrough for the rtn t or rta v adv mutations associated with resistance. sera from subjects with breakthrough were analysed at all subsequent yearly timepoints whenever possible. results: at the end of the st yr, none of the subjects with hbv dna breakthrough had either mutation. sera were available for analysis from , , and subjects with viral breakthrough at the end of the nd , rd , th and th yr, respectively, with new mutations identified in , , and subjects at the same timepoints. of the cumulative subjects at the th yr analysis had rtn t, had rta v, and had both mutations. conclusion: treatment with adv in chinese hbeag positive chb subjects for up to yrs resulted in a cumulative rate of . % ( / ) adv resistance-associated mutations with hbv dna breakthrough. background and aims: to evaluate the predictive significance of rapid virologic response (rvr) for achieving an end-of-treatment virologic response (er) or hbeag seroconvertion and the predicting indicator of nonresponse (nr). methods: patients with chronic hepatitis b were treated with adv and prospectively observed to weeks. we assessed the values of virus load reduction at weeks , , and weeks to predict the er and hbeag seroconversion. the association between less reduction of viral load at and weeks and nonresponse was also analyzed. results: of etv-treated patients enrolled in etv- , met criteria for inclusion into year etv treatment analyses. the proportion of patients achieving efficacy endpoints through years of etv therapy is presented the table. results: after weeks of therapy, serum hbv dna levels decreased with a median . ± . log copies/ml. twenty-three( . %) of patients had er. twenty-six ( . %) patients achieved hbeag seroconversion. hbv dna < log copies/ml at week predict both er and hbeag seroconversion. hbv dna> log copies/ml at weeks but decline to < log copies/ml at weeks or weeks both can predict er and hbeag seroconversion. less than log hbv dna reductions at weeks might predict nr. conclusions: the majority of patients experienced durable serum hbv dna suppression ( %) and alt normalization ( %) after years etv therapy. conclusions: the virologic response within weeks could be useful for prediction of er and hbeag seroconversion of adefovir therapy. failing to evr might not predict nr. objectives: to determine the accuracy of hbcigm in diagnosing ahb and the correlation between hbcigm and liver inflammation (alt), bilirubin & biosynthetic functions (albumin,pt). result: a total of patients were included: in patients, adv was added on lam (add-on therapy), and in patients, lam was switched to adv (switch therapy). during . months of follow-up, patients developed adv resistance (rta v and/or rtn t) and all had undergone switch therapy. the cumulative probability of adv resistance at the th month was . %. although add-on therapy induced no adv resistance, it failed to show significant superiority over switch therapy (p= . ). in multivariable analysis, female (odds ratio [or], . ; % confidence interval [ci], . - . ; p= . ), liver cirrhosis (or, . ; % ci, . - . ; p= . ), and age > yr (or, . ; % ci, . - . ; p= . ) were independent risk factors of adv resistance. methods: a retrospective cross-sectional study involving patients with hbcigm positivity between june -december , satisfying the definition for ahb and chbf,and fulfilling the exclusion criteria was performed. hbcigm test were done by using microparticle enzyme immunoassay (meia) and results were expressed as an index value.hbcigm positivity was defined as index value of > . results: patients were positive for hbcigm and fulfilled the criteria( ahb, chbf).hbcigm was significantly higher in ahb compared with chbf(median . vs . ;p < . ).the hbcigm arbitary index value of . was highly sensitive( %) and specific( %) in diagnosing ahb with high accuracy(auroc . ; % ci: . - . ;p< . ).among patients in both groups, there was a weak, but significant negative correlation between hbcigm and pt above control(r = - . ,p = . ).however, among patients with chbf,the negative correlation between hbcigm and pt above control was moderately strong(r = - . ,p = . ).there was also a weak, but significant positive correlation between hbcigm and albumin in with chbf(r = + . ,p = . ). conclusion: adv add-on therapy developed no adv resistance during the observation period. therefore, add-on therapy is recommended to lam-resistant chb patients with genotype c who have any risk factors for development of adv resistance: female, liver cirrhosis, and age > yr. hbcigm-hepatitis b core igm antibody ahb-acute hepatitis b,chbf-chronic hepatitis b flare alt-alanine transaminase,pt-prothrombin time pe detection of emerging drug resistance mutations associated with major approved hbv antivirals using a novel line probe assay (lipa). j. doutreloigne , f. shapiro , r. maertens , e. van assche , e. sablon hepatitis diagnostics unit, innogenetics nv, belgium background: in study etv- , etv demonstrated superior virologic, histologic and biochemical benefit compared to lamivudine (lvd). this study (etv- /- ) presents efficacy and safety results for patients who received years continuous etv treatment. background/aims: an increasing number of antiviral drugs are being used to treat chronic hepatitis b virus (hbv)-infected patients. however, induced viral escape mutants -some potentially cross-resistant -lead to viral non-responsiveness and treatment failure. effective treatment strategies must therefore take possible drug resistance (dr) into account with respect to monitoring and selection of alternative drugs. we evaluated the use of an updated inno-lipa hbv dr v +v reverse hybridization assay versus sequence analysis to detect resistance mutations. methods: the study evaluates etv-treated nucleoside-naïve hbeag (+) patients who completed etv- and enrolled into etv- with a treatment gap days. the proportion of patients with hbv dna < copies/ml, alt normalization, hbeag loss or hbeag seroconversion was evaluated at week . background: etv resulted in improved liver histology compared to lvd at year. histologic data for patients on etv for a median of years is evaluated. methods: clinical samples (from untreated hbv patients or treated with different antivirals; hbv genotypes a-h) were tested for mutations with the lipa assay and sequencing. for lipa, samples were extracted with the qiaamp® dna blood mini kit (qiagen), and then tested on the lipa strips. sequencing-derived reference data were subjected to phylogenetic analysis (kodon version . applied maths, neighbour joining, with kimura- parameter). sequential samples from patients were evaluated as well. methods: etv-treated patients completing etv- or etv- received etv ( . mg daily) in etv- . primary endpoints included -point decrease in knodell necroinflammatory score, no worsening of knodell fibrosis score and improvement in ishak fibrosis score (ifs) ( -point decrease) vs. baseline. secondary endpoints included proportions with hbv dna< copies/ml, alt normalization, and ifs normalization in patients with advanced fibrosis/cirrhosis. results: quasi-perfect concordance (> . %) was obtained between the two assays for the samples tested. no indeterminate results were observed. for one sample, lipa provided additional information (wild-type/mutant mix), whereas sequencing showed only wild type. for sequential samples, lipa was clearly able to detect emerging treatment-resistance mutations associated with viral breakthrough. results: etv treatment led to significant histological improvements and improved ifs in % ( / ) and % ( / ) of patients respectively. of patients with baseline fibrosis/cirrhosis (ifs ), all demonstrated -point improvement in ifs (median change of - ). conclusions: lipa accurately detects the complex quasispecies nature of hbv and can help unravel the dynamics of emerging hbv resistance during treatment with different antiviral drugs. like its predecessor, it is useful for the monitoring and early detection of drug resistance. conclusions: long-term etv therapy in nucleoside-naïve chb patients results in durable virologic suppression, continued histologic improvement and regression of fibrosis/cirrhosis. precore (pc, g a) and basal core promoter (bcp, a t and g a) mutations of hbv are important for predicting the risk of hepatocellular carcinoma (hcc). we developed a new mass spectrometry-based assay using restriction fragment mass polymorphism (rfmp) to detect a and t /a mutations, and applied it to analyze their clinical significance in type b liver diseases (n= ), including hccs, liver cirrhosis (lc), chronic hepatitis b (chb), and hbsag-positive with low level viremia (inactive hbsag carrer, ihc). we devided patients into major groups according to the presence of wild (w) or mutant (m) genes in bcp/pc regions; w/w, w/m, m/w and m/m gene types. each proportion was . %, . %, . % and . %, respectively. mixed infection (x) was also found as minority; w/x, m/x, x/w, x/m and x/x. disease distributions (hcc, lc, chb and ihc) in each group were as follows; [w/w (n= )] . %- . %- . %- ; [w/m (n= )] - . %- . %- %; [m/w (n= )] . %- . %- %- . %; [m/m (n= )] . %- . %- . %- . %. these results suggest that, in korea where only genotype c has been identified, bcp dual mutation is predominant (> . %), while bcp wild alone is only . %. especially, a mutation alone without bcp mutation (w/m type) is uncommon, while bcp mutation alone without a mutation (m/w type) is most common. it might be suggested that prognosis of wild type in bcp and pc region (w/w type) is much better than that of m/w or m/m types. background/aims: entecavir is a potent inhibitor of hbv dna polymerase, which has been shown to be safe and effective for the treatment of chronic hepatitis b (chb) patients. the aim of this study was to evaluate the virologic, biochemical, and serologic responses of entecavir through year in chb patients. methods: from may to october, we reviewed patients (mean age ± years, male:female= : ) who were diagnosed as chb patients (hbeag (+) ). forty-seven patients ( . %) had been treated with . mg of entecavir and ( . %) with mg of entecavir, respectively. mean follow-up period was ± weeks. hbv dna was quantified by bdna assay with a lower limit of detection of , copies/ml. results: median hbv dna levels before therapy was . log copies/ml and the median decreases from baseline in hbv dna were - . , - . , - . , - . , and - . log copies/ml at (n= , p< . ), (n= , p< . ), (n= , p< . ), (n= , p< . ), and (n= , p = . ) weeks of follow-up, respectively. at baseline, overall median alt was iu/l and the proportions of patients with normal alt were %, %, %, %, %, and % at baseline (n= ), (n= ), (n= ), (n= ), (n= ), and weeks (n= ) after entecavir therapy, respectively. thirteen cases ( . %) of hbeag seroconversion were noted. background: hepatitis b virus (hbv) infection is a major risk factor for the progression of liver diseases. because its clinical course varies, it is difficult to detect the predictive factor for the prognosis of patients with hbv infection. the aim of the present study was to determine the risk factors for the occurrence of hcc. methods: a total of patients who tested positive for hepatitis b surface antigen and were referred to chiba university hospital between february and march were included in the study, and their following characteristics were analyzed: age, gender, the status of hbeag, alt, hbv-dna level, and plt. result: hcc was detected in cases during the follow-up period ( . ± . years). multivariate analysis revealed that age [compared with young patients: odds ratio (or) = . , % confidence interval (ci) = . - . ] and plt level (compared with patients with low plt level: or = . , % ci = . - . ) were the predictive factors for hcc occurrence. in patients with age more than years, the hbv-dna level (compared with < . log copies/ml: or = . , % ci = . - . ) and plt level (or = . , % ci = . - . ) were the predictive factors for hcc occurrence. conclusion: advanced age and low plt level were the risk factors for hcc occurrence in patients with hbv infection irrespective of the plt level at baseline. in patients with age more than years, viral load was also a risk factor for hcc. results: before treated by lps, the total mapk p level of pbmcs have no significant difference among the healthy control group, different stage groups with hbv infection, however, after treated with lps, the phosphorylated mapk p (ptpy / ) in healthy control group are significant elevate than hbv infected groups( . ± vs . ± . , p< . ). in the two groups which hbsag, hbv dna are positive, alanine aminotransferase elevate than normal and hbsag positive, but hbv dna lower under the detect limited level, after treated by lps the ptpy / although lower than healthy control yet, but significant elevated than themselves before treated by lps( . ± . vs . ± . , . ± . vs . ± . ; p< . ).otherwise, in the group of both hbsag and hbv dna are positive, but alt is normal, before and after treated by lps, the level of ptpy / have no significant difference. conclusion: mapk p is a important signal transduction pathway which involving in inflammation and immune response, especially, mapk p activated up-regulate the ifn-gamma mrna. according to the result shown, we propose a hypothesis, hbv infection and virus active replication inhibit the mapk p activated, consequent on host immunotolerance and hbv persistence, thus, mapk p may be as a potential therapeutic target to break immnotolerance and establish host anti-viral states. van der helm siriraj hospital, bangkok, thailand, ramathibodi hospital, bangkok, thailand, phyathai hospital, bangkok, thailand, singapore general hospital, singapore, national university hospital, singapore, changi general hospital, singapore, cheil general hospital, korea, hwasun jeonnam university hospital, korea, st mary hospital, korea, roche diagnostics ltd, rotkreuz, switzerland background/aims: hepatitis b virus (hbv) surface antigen (hbsag) is one of the most important markers for diagnosis of acute and hbv infection. high sensitivity of hbsag assays can reduce the diagnostic window during course of disease. in addition, the presence of hbv mutants may be affected by the performance of the hbsag kit. therefore, the technical performance of the elecsys ® hbsag ii assay was explored, using samples (including recombinant mutants), at multiple sites in three countries. methods: nine hbsag screening centers in thailand, korea and singapore compared the sensitivity of elecsys ® hbsag ii assay with that of their routine testing procedure -abbott architect ® ( centers), abbott axsym ® ( center) and bayer advia ® centaur hbsag assays ( center) using preselected seroconversion panels (n= ), recombinant hbv mutant panels (n= ) and routine clinical practice samples (n= , ). results: the sensitivity of elecsys ® in seroconversion samples was equivalent to the architect ® assay, but more sensitive than the axsym ® and advia ® centaur assays ( vs and vs positive bleeds, respectively). there was concordance between the elecsys ® and architect assay results with respect to potentially cross-reactive samples ( . %). the elecsys ® and architect ® assays detected all recombinant mutant samples, whilst axsym ® and advia ® centaur failed to detect three and nine samples, respectively. conclusion: elecsys ® hbsag ii assay was not only highly sensitive and specific when compared with established hbsag screening assays, but also reliably detected hbsag mutants. therefore, this attractive assay is suitable for hbv diagnosis and assessing safety of blood products. background: recent studies have shown a higher rate of adefovir-resistant mutation in lamivudine-resistant chronic hepatitis b (chb) patients treated with switch-to therapy than those treated with add-on therapy. we compared the clinical efficacy of adefovir monothrapy and lamivudine-adefovir combination therapy in lamivudine-resistant chb. methods: a prospective cohort study was performed in patients with lamivudine-adefovir combination therapy and patients with adefovir monotherapy for lamivudine-resistant chb over months. result: biochemical response was achieved in patients ( . %) treated with combination therapy and in patients ( . %) treated with monotherapy (p= . ). virologic response was observed in patients ( . %) in combination therapy and in patients ( . %) in monotherapy (p= . ) and treatment periods for virologic response was significantly shorter in patients with combination therapy than in monotherapy ( . ± . months vs. . ± . month, p= . ). cumulative rate of virologic response was significant higher in patients with combination therapy than monotherapy (p= . ). hbeag loss was found in patients ( . %) in combination therapy and patients ( . %) in monotherapy (p= . ). biochemical breakthrough was found in patients ( . %) with monotherapy significantly more frequent than patients ( . %) with combination therapy (p= . ). genotypic resistance to adefovir was developed in patient ( . %) in combination therapy and patients ( . %) in monotherapy conclusion: to achieve a complete virological response and reduce the risk of adefovir-resistant mutants in lamivudine-resistant chb patients, adefovir in combination with lamivudine is preferable. background/aims: adefovir dipivoxil (adv) effectively inhibits both wild-type and lamivudine (lam)-resistat chonic hepatitis b virus (chb) replication. the aims of this study were to determine the factorts associated with antiviral effect of adv in lam-resistant chb. methods: one hundred-eighteen lam-resistant chb patient ( . % hbeag-positive) were treated with adv plus lam (n= ) or adv monotherapy (n= ) for a mean of . months. restriction-fragment mass polymorphism analysis was used for detection ymdd and adv mutants. results: fifty-eight patients ( . %) achieved complete response(cr) defined as hbv-dna levels < copies/ml and alt normalization. twenty-eight patients ( . %) achieved initial vilologic response(ivr) defined as hbv-dna levels < copies/ml within the first month of treatment. ( . %) of hbeag-positive patients exhibited hbeag loss and % seroconverted to anti-hbe ab. five ( . %) patients developed adv-related mutations. factors associated with ivr were pretreatment level of alt (p= . ), ast (p= . ), pretreatment hbv dna level (p= . ), hbeag negativity (p= . ) and hbeab positivity (p= . ). factors associated with cr were ivr (p= . ), hbeab positivity (p= . ), pretreatment level of alt (p= . ), ast (p= . ) and y-glutamyl transferase (p= . ). age, sex, presence of liver cirrhosis, pretreatment hbv dna level and the type of ymdd mutants were not related to an cr during adv treatment. conclusions: adv therapy achieved cr in more than % of lam-resistant chb. factors associated with cr were ivr, hbeab positive status, high base line alt, ast, ggt levels. q.j. sheng , y. ding , x.g. dou department of infectious disease, shengjing hospital affiliated to china medical university, shenyang, , china objectives: to study a kinetics of hepatitis b virus during -week and -week of treatment with enticavir (ent); to compare the detecting results of hbvdna levels from different detection reagents. methods: thirty-seven cases of chronic hbv infections were selected randomly, treated with daily dose of ent . - . mg ( . mg for nucleoside-naïve patients, . mg for lamivuding-refractory patients). evaluation indexes: serum hbvdna, hbv serological markers, and liver function tests. hbvdna levels were measured by pcr assay, using both domestic reagents and roche cobas amplicor,. the lower limits of measure level of hbvdna were copies/ml and copies/ml, respectively. results: mean baseline of hbvdna was . log copies/ml for detection using domestic reagents and . log copies/ml for that using roche cobas amplicor, (p> . ). the ratios of cases with undetectable (< copies/ml) hbvdna at week- and week- were . % and . %, respectively. the ratios of cases with undetectable (< copies/ml) hbvdna at week- were . %. among the cases whose hbvdna were lower than copies/ml(using domestic reagents), the ratio of hbvdna lower than copies/ml(using roche cobas amplicor) was . %. conclusions: ent can suppress hbv dna rapidly no matter the patients with alt elevation or not. there is a concordance on hbvdna levels detection between domestic reagents and roche cobas amplicor. background the study on the effect of nucleoside analogue therapy on the quantity of hepatocellular cccdna and tdna and sera hbv dna, hbsag to probe reliable marks for evaluation of therapy endpoint. methods the quantity of hepatocellular cccdna and tdna and sera hbvdna were assayed by fq-pcr, and sera hbsag by elisa in chb patients over years nucleoside analogue therapy satisfied the china criteria of therapy endpoint (therapy group)and chb patients without antiviral therapy and sera hbvdna< copies/ml(control group). results: the quantity of hepatocellular cccdna, tdna and sera hbvdna, hbsag in therapy group were lower than that of control group,but low level hepatocellular cccdna in therapy group could be detected. conclusion: long term nucleoside analogue therapy may consume hepatocellular cccdna with decreasing of hepatocellular tdna and sera hbvdna, hbsag; although the patients have satisfied the china criteria of therapy endpoint, low level of hepatocellular hbvcccdna were detected, cessation of therapy may cause relapse. peptides that lead nuclear entry of nucleocapsid of hepatitis b virus in hepg . . cells x.b. pan , , l. wei , , j.c. han , , k. deng , peking university hepatology institute, peking university people's hospital background: the nuclear entry of nucleocapsid is a key step for the hbv life cycle and the formation of covalently closed circled dna (cccdna). it has been supposed that the carboxyl-terminal arginine-rich domain of the core protein contains a signal for nuclear localization (nls). whereas hbcag was primarily distributed in cytoplasm and no marked cccdna was detected in hepg . . cells. methods: we designed peptides containing a cell-penetrating sequence (rrrrrrr) and a nucleocapsid binding sequence (gsllgrmkga) with/without a classic nuclear localization sequence (pkkkrkv) and these sequences were linked by a soft linker acp. hepg . . cells were treated with the peptides at levels of m, m and m for days. results: compared with that of control cells, the results showed hbv dna levels in culture medium decreased at least log both in m of peptide rrrrrrracpgsllgrmkga treatment group and rrrrrrracpgsllgrmkgaacppkkkrkv treatment group; whereas hbsag and hbeag increased at . + . folds and . + . folds respectively. the signal strength of cytoplasmic hbcag increased at about . -fold in both groups. in rrrrrrracpgsllgrmkgaacppkkkrkv treatment group, nuclear hbcag increased about . -fold and obvious cccdna signal was detected by southern blot. conclusion: our results implied that the nls of core protein likely does not expose to surface of nucleocapsid in hepg . . cells, the artificial peptide containning nls binds to the nucleocapsid and leads nuclear entry of nucleocapsids and then facilitates the formation of cccdna. our study presents a tool for study on cccdna formation and nuclear entry of nucleocapsid. b. tang , j. xia , y.m. wang , h.f. wang liver failure treatment and research center, rd military hospital, the dept. aims: to establish a reliable real-time fluorescence quantitative (rfq) pcr method to quantify hbv cccdna, basing on lightcycler system and taqman probe. methods: hbv genotypes a-g were aligned to obtain a conserved sequence, crossing rcdna gap, which was used to design cccdna primers and taqman-mgb probe. also another pair of primers for quantify hbv total dna (tdna) was designed, utilizing the same probe. to increasing specificity, we added plasmid-safe atp-dependent dnase (psad) digestion step just before cccdna pcr amplification. a standard curve from standard plasmid samples, from . × to . × copies, was created to examine our system. hbv cccdna samples with known-amount were quantified by creating standard curve from standard samples. results: the standard curve had clear log-phase and excellent parallelism, which means nice and equal amplification efficiency in all reaction capillarys. the slope (regression coefficient) of standard curve was - . , mean square error was . and regression coefficient was - . . all of these key indexes measured up. in tests, we got right results if the starting templates of cccdna copies were between ~ copies. the range was superior to commercial hbv kits. by quantifying samples containing different amounts of cccdna and rcdna, digested or undigested, psad digestion would eliminate rcdna molecules, leaving cccdna molecules untouched. the test specificity was maintained up to : , ratio of cccdna:rcdna. conclusions: the rfq-pcr based on lightcycler system for hbv cccdna quantification is reliable, sensitive, with high specificity and low cost. background: to study the changes of toll-like receptor (tlr) on dendritic cells derived from peripheral blood mononuclear cells(modc) and its role in the pathogenesis of chronic hepatitis b(chb) and chronic severe hepatitis b(cshb). methods: the expressions of tlr on modc were stimulated by poly i:c, and then were determined by flow cytometry in healthy controls, patients with chb and patients with cshb.the level of interferon ifn-was determined by elisa. the differences of expression of tlr on modc and serum ifn-among the three groups of study subjects were determined by student-t test.the correlation between tlr and ifn-were determined by linear correalation test. results: the values of mean fluorescence intensity(mfi) of tlr on modc of the healthy controls, patients with chb and cshb were . ± . , . ± . ,and . ± . .the serum ifn-(pg/l) of respective groups was . ± . , . ± . and . ± . . there was a gradual decrease of these values from the group of healthy controls to the group of patients with chb and cshb .significant positive correlations between tlr and serum ifn-were found. conclusion: tlr may have a role in the pathogenesis of chb and cshb. background/aim: to evaluate the efficacy and safety of entecavir treatment in patients with hbeag-positive chronic hepatitis b who had not previously received a nucleoside analogue. methods: fifty-five patients received -week entecavir . mg/d therapy. serum hbv dna load was measured with quantitative real-time-pcr. alanine aminotransferase (alt) activity, hbeag, anti-hbe-antibodies, hbv dna level in serum were evaluated at baseline, week , , and during therapy. evaluation of safety and tolerance was based on clinical adverse events and laboratory analyses. results: hbv dna levels declined sharply by around log copies/ml during the first two weeks, with a highly significant reduction (p< . ) at week and thereafter, as compared to those at baseline; %, % and % of the patients had undetectable serum hbv dna levels at week , and respectively. highly significantly decreasing serum alt (p< . ) occurred during the first weeks of the study. at week , alt levels were normalized in % of the patients. hbeag seroconversion (hbeag negative, hbeab positive) was achieved in . % and . % of patients by and week. at the end of th and th weeks, complete response (alt normalization and hbv dna and hbeag loss) was observed in % and %, respectively. there was no evidence of drug resistance or adverse effect in chb patients treated for up to weeks. conclusion: entecavir treatment through weeks was well tolerated and resulted in continued benefit for patients with hbeag-positive chronic hepatitis b. aim: to assess the associations of single nucleotide polymorphisms of the mxa gene promoter and sustained treatment response of chronic hepatitis b or c patients with interferon treatment by meta-analysis of individual dataset from all studies published till date. methods: to clarify the impact of mxa gene promoter polymorphisms on sustained treatment response of chronic hepatitis b or c patients with interferon treatment, we performed a meta-analysis of the published data from eight studies comparing the frequencies of mxa gene promoter polymorphisms at nt - g/g, - g/t, - t/t and nt - c/c, - c/a , - a/a alleles in individuals with interferon treatment. as we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. results: the sustained treatment response rate was higher in patients with the nt - g/t and nt - c/aalleles in the mxa promoter snp . the meta-analyses yielded summary estimatesodds ratio (or) were . [ %ci ( . , . ), p < . ] and . [ %ci ( . , . ), p = . ] of the nt - g/t and nt - c/a alleles, respectively. conclusion: mxa gene promoter polymorphisms at nt - g/t and nt - c/a may be useful as a marker to predict the sustained treatment response of chronic hepatitis b or c patients with interferon treatment, and further investigation regarding their real significance is warranted in a large series of patients. background: to determine whether hbv with the same characteristics causes dissimilar mutations in different hosts. methods: full-length hbv genome was amplified and linked with pmd t vector. positive clones were selected by double-restriction endonuclease digestion (ecori and hindiii) and pcr. twenty seven clones were randomly selected from an asymptomatic mother [at two time points: ( d) and ( mo)] and her son [ (s)]. bioeditor, clustal x and mega software were used to perform phylogenetic and mutational analysis. potential immune epitopes were determined by the stabilized matrix method (smm), smm-align method and emini surface accessibility prediction. result: all of the sequences were genotype c, the inner-divergence for the mother and son was %- . %. specific nucleotides differed from the other pubished genotype c isolates were co-exist in the mother and her son. aa - deletion in pres was the dominant mutation in the mother ( / ). the t/ a double mutation existed in all clones of the mother, of them were also coupled with g a mutation, but none were found in the son. bp deletion starting at nucleotide was the major mutation ( / ) in the son, which caused seven potential hla class i epitopes and one b cell epitope deletion, and produced a presumptive new start codon, downstream from the original one of the p gene. conclusion: the son was infected hbv from his mother, and discrepant mutation occurred in the mother and her son during infection. background/aims: nucleos(t)ide analogues have been recognized as an effective treatment for chronic hepatitis b. this randomized, double-blind trial compared the efficacy and safety of telbivudine and lamivudine after -week therapy in patients with compensated chronic hepatitis b in taiwan. methods: we analyzed taiwanese patients from globe trial receiving telbivudine mg (n= ) or lamivudine mg (n= ) once daily for weeks. the primary efficacy endpoint was therapeutic response with serum hbv dna < log copies/ml and either hepatitis b e antigen (hbeag) loss or alanine aminotransferase (alt) normalization. results: the therapeutic response at week was . % in telbivudine group versus % in lamivudine group (p= . ). more patients with telbivudine achieved nondetectable serum hbv dna (< copies/ml) (p= . ) and alt normalization (p= . ) at the end of treatment. the cumulative resistant rate was significantly lower in those with telbivudine treatment (p= . ). the rate of hbeag seroconversion was comparable in both groups (p= . ). although a lower percentage of patients in lamivudine group ( . %) reported adverse events than those in telbivudine group ( . %), the difference was not significant. conclusions: telbivudine demonstrates a significantly greater efficacy and a lower resistant rate than lamivudine in treatment of chronic hepatitis b in taiwan. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf-- gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf-- gene promoter and chronic hbv infection. method: meta-analysis was done of case-control studies in relation to tnf-- gene promoter, involving a total of chronic hbv infection cases and controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that - a heterozygotes (ga) had % decreased risk of developing chb with a borderline significance (or = . ; % ci: . - . ; p = . ). for the - a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = . ; % ci: . - . ; p = . ; and or = . ; % ci: . - . ; p = . , respectively) ( table ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with - variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf-- a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. aim: to define the potential role of pd- /pd-lpathway in different hbv infection status; we examined the expression of pd- on cd + t cells in pbmc of patients with chb and aehb infection. methods: the pd- level on cd + t lymphocytes and the number of hbv specific cd + t lymphocytes in patients and healthy controls were analyzed by flow cytometry. pcr was used to measure the serum hbvdna levels. results: the level of pd- expression on cd + t cells in chb patients was higher than that in aehb patients and healthy individuals. compared to aehb patients, lower frequency of hbv-specific cd + t cells was detected in chb patients. there was an inverse correlation between the strength of hbv-specific cd + t-cell response and the level of pd- expression. besides, there was a significant positive correlation between hbv viral load and the percentage of pd- expression on cd + t cells in chb and aehb subjects. however, pd- expression was not associated with alt levels. conclusion: our results confirm previous reports that hbv specific cd + t-cell response in the peripheral blood is more intense in patients with aehb than in chb with persistent viral infection. moreover, there is a negative correlation between the level of pd- and the intensity of virus specific cd + t cell response. observed in . % of patients with a mean age of . ± . . the ethnic composition was . % chinese; . % malay; % indigenous sabahans; . % indigenous sarawakians; . % indians and . % others. chinese patients were on average, older (mean . ± . years), indians patients had higher mean alanine transaminase and indigenous sarawakian patients had the highest rate of cirrhosis (p< . ). during the study period, . % of patients were on treatment and they were significantly older than those who were not on treatment (mean age . ± . vs . ± . ). lamivudine was the first agent used in . % of cases. conclusions: in malaysia, chb remains a public health issue and significantly afflicts males in the productive age groups and of chinese ethnicity. the observed differences among ethnic groups could point to different disease severity which needs to be addressed in the local treatment guideline and policy. background/aims: liver stiffness measurement (lsm) has been validated for predicting fibrosis stage in patients with chronic hepatitis c. however, studies on lsm for chronic hepatitis b (chb) are few, and the relationship between histologic findings and liver stiffness needs to be further elucidated. this study was conducted to assess the association of histologic activity on liver stiffness in addition to fibrosis in patients with chb methods: thirty three patients who had taken liver biopsy and lsm at korea university ansan hospital between march and october were enrolled. necroinflammatory activity and fibrosis stage were assessed by metavir system. activity, fibrosis, and the sum of both score were included for the correlation analysis with lsm results: among patients, ( . %) were male, and median values were as follows: age, ( ~ ); ast, iu/l ( ~ ); alt, iu/l ( ~ ); total bilirubin, . mg/dl ( . ~ . ); lsm, . kpa ( . ~ . ). fibrosis stages were f in ( . %), f in ( . %), f in ( . %), and f in ( . %) patients. spearman correlation coefficient with lsm were . (p= . ) for activity, . (p< . ) for fibrosis stage, and . (p< . ) for the sum of activity and fibrosis. in linear regression analysis, only the sum of activity and fibrosis remained to be significant. conclusions: not only fibrosis but also activity was an important factor for determining lsm for chb. it would be more appropriate to consider both activity and fibrosis for interpretation of lsm in patients with chb background: hbeag seroconversion is a key goal of chb therapy. hbeag kinetics may predict hbeag seroconversion during treatment. we aim to develop a robust hbeag quantitative method as the value of hbeag quantitation is undefined and data is limited. methods: we evaluated two commercially available qualitative hbeag assays (abbott architect, siemens centaur) for their linear range and validated them against paul-ehrlich institute (pei) standards. hbeag levels were determined from samples of untreated and telbivudine-treated chb patients. results: as a pre-requisite for quantitative use, the linear range for the architect ( . - peiu/ml) and centaur ( . > peiu/ml) assays were defined. architect was selected for further investigation. hbeag levels of untreated patients (mean hbv-dna . log copies/ml, mean alt . iu/ml) varied from . to . peiu/ml (median . peiu/ml). in patients (mean hbv-dna . log copies/ml, alt iu/ml) treated with telbivudine for weeks, baseline hbeag levels varied from . to peiu/ml (median . peiu/ml). after weeks of telbivudine treatment, median hbeag level was . peiu/ml, with % decline from baseline (median decline . peiu/ml, range . - . peiu/ml). individual hbeag decline from baseline varied but occurred in all patients and was not correlated to baseline or decline from baseline hbvdna. conclusion: hbeag quantitation is feasible and robust with architect hbeag assay. hbeag decline occurred in all telbivudine-treated patients, and was not correlated to hbvdna. whether the magnitude of hbeag decline is predictive of future hbeag seroconversion merits further investigation. experience from the combined globe (nv- b- /cldt a ) and (nv- b- ) study clinical safety database. c. avila , r. laeufle , w.b. bao novartis pharma ag, fabrikstrasse , basel, switzerland, novartis pharma ag, basel, switzerland, novartis pharma, east hanover, us background: creatine phosphokinase (ck) is a commonly used marker of muscle damage and is elevated by many factors (e.g. exercise, injury, drugs). normal ck levels are affected by muscle mass and elevated levels are described during the natural course of chb. % of patients in the globe study had pretreatment grade - ck elevations. methods: we reviewed data from this combined study clinical safety database, and describe the experience of ck elevation and its relationship to adverse event reports of muscle related symptoms. results: the frequency of new onset of grade - ck elevations in telbivudine-treated patients (combined database itt population) was . % ( / ), . % ( / ), . % ( / ) and . % ( / ) from weeks - , - , - and - respectively. the frequency of grade - ck elevations for all patients from week - was . % ( / ). the majority of grade - ck elevations were asymptomatic, rarely resulted in discontinuation or interruption, spontaneously declined within or visits and were not associated with more frequent muscle-related adverse events. cumulative data from this combined database showed no relationship of the degree of increased ck to acute or persistent muscle disease. conclusion: ck elevations are associated with hbv disease and were also common during the globe and trials and were not predictive of the development of muscle related symptoms. onset of muscle-related symptoms should prompt clinical and treatment review, including concomitant medications. backgrounds: leptin plays a crucial role in the regulation of energy balance and body weight control by activating the long form of the leptin receptor (ob-rl). epidemiologic studies showed that obesity is one of the factors associated with hbv related hepatocellular carcinoma. methods: huh cells were transiently transfected with . copies of hbv-replicon plasmid. after h, cells were harvested and total rna of the cells were extracted and reverse-transcribed into cdna. long form and short form leptin receptor (ob-rl, ob-rs) mrna transcription levels were assayed by real-time pcr respectively. and mrna transcription levels and protein expression of ob-rl and ob-rs in hepg . . cells were also detected. results: after transfected by . copies hbv-replicon plasmid, the mrna transcription level of ob-rl was inhibited significantly (**p< . ), but the mrna transcription level of ob-rs did not change, and the ob-rl protein expression was reduced. in hepg . . cells, the mrna transcription level of ob-rl was also significantly lower than the mrna transcription level of ob-rl in hepg cells, while the mrna transcription of ob-rs in hepg . . and hepg cells didn't show significant difference. besides, the protein expression level of ob-rl in hepg . . was also lower than it in hepg cells. conclusion: hbv replication down-regulated the expression of long form leptin receptor in cell cultures, which could in part explain the clinical observation of obesity in association with development of serious sequelae in hbv infections. the results of entevavir treatment in patients with chronic hepatitis b s. kose , g. akkoclu , m. turkeri , a. gozaydin the ministery of health tepecik training and research hospital, izmir purpose: we evaluated the short and long term effectiveness of entecavir. patients and methods: those patients had received diagnosis of chronic hepatitis b. their pretreatment transaminases, hbsag, anti-hbs, hbeag, anti-hbe, hbv-dna were checked and a liver biopsy and a resistance test for lamivudine (lam) and adefovir (adv) were performed. a total of patients who were taking entecavir for at least weeks were included in the study. findings: the biochemical and virologic response were observed in . % at and months and in % at months. in hbeag positive patients who had received therapy previously, the biochemical response was observed in . % at and months and in % at months. the virologic response in . % at , in . % at , and % at months. posttreatment hbeag seroconversion did not develop. in hbeag negative patients the biochemical and the virologic responses were observed in . % at months and % and months, respectively. in hbeag negative patients had received therapy previously, the biochemical response was observed in . % at , in % at and months. the virologic response in . % at , in % at and at months. conclusion: in our study, a higher therapy-response rate was achieved, especially in hbeag negative patients. in hbeag positive patients biochemical and virologic response rates were high. background/summary: patients with liver disease are known to have a higher prevalence of glucose intolerance. preliminary studies suggest that viruses can be an additional risk factor for the development of diabetes mellitus. individuals with type ii diabetes have an increased prevalence of cirrhosis, and a proportion of patients with acute and chronic liver disease develop diabetes mellitus. there is now emerging epidemiological data to suggest that hepatitis c virus (hcv) infection may also contribute to the development of diabetes reported to be higher than expected compared with the general population. while these investigations suggest an epidemiological association between hcv infection and diabetes, large controlled studies are required to observe association between hbv infection and diabetes. the present study was designed to study the relative proportion of diabetes mellitus in patients suffering from hepatitis b virus (hbv) infection. background: in easl , we reported significant liver disease among hbeag negative patients with serum hbv-dna level < log copies/ml (i.e. . x log iu/ml) and alanine aminotransferase (alt) < iu/l. this reflects fluctuating nature of these levels. the aim of this retrospective study is to demonstrate the frequency of fluctuation among this group of chb patients. methods: clinical records of hbeag negative treatment naïve chb patients with at least one serum hbv-dna < log copies/ml were reviewed. results: there were ( . % male, median age . years) chb patients with negative hbeag and hbv-dna < log copies/ml (roche cobas amplicor pcr assay, lod< copies/ml). had serial hbv-dna measurements within years; of them ( . %) had increase serum hbv-dna level by > log copies/ml; patients had associated serum alt elevation from normal (normal range < iu/l), had persistent normal alt, and one had persistently raised alt. ( . %) had serum hbv-dna level decrease by > log copies/ml. another patients had hbv-dna levels fluctuating within log copies/ml. hbeag negative patients with single hbv-dna measurement showing < log copies/ml had serial serum alt measurements within years. ( . %) patients had intermittent / persistently raised alt; while ( . %) patients had persistently normal alt. conclusions: hbeag negative chb is common among chinese. serial serum hbv-dna and alt measurements are necessary to detect fluctuating levels and progressive liver disease that may require antiviral therapy. background: to determine the best vaccination strategy, a model that reflects the country-specific infection profile is needed. methods: a model was built in order to obtain the age-specific infection frequency q(t) for neonates(n), infants(i), children(c), and adults(a). the infected group can either become hbsag(+) or anti-hbs(+)* based on f(t). q(t) can be found from p(t) = [q(t) x ( -f(t)) x crs + q(t) x f(t) x ( -cras)], where p(t) represents the proportion of the late anti-hbs(+)** group and crs/cras denote natural conversion of hbsag/anti-hbs. to test the model, cross-sectional serologic marker data in korea were used. because f(t), crs, and cras were known(f(n)= . , f(i)= . , f(c)= . , f(a)= . , crs= . , cras= . ), in order to determine q(t), only p(t) values were needed, which were evaluated from logistic modeling using the glm() function of s-plus. results: the infection frequencies during neonate, infant, children, and adult periods in non-vaccinees were . %, . %, . %, and . %, respectively. each group's likelihood of infection compared to adults was then: neonates . times more likely, infants . times, and children . times, making a strong case for neonatal and infantile vaccination for the studied region. conclusions: the hbv infection model can be used for determining the most cost-effective strategy for hb vaccination in nations where longitudinal data are not available. and where longitudinal data are available, it can be used to determine the appropriate time of transition of vaccination strategy to maintain cost-effectiveness. the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b x.c. pan , f. yang , m. chen objective: to investigate the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b. methods: patients with hbeag positive chronic hepatitis b were recruited and receiving telbivudine treatment for months. before and during months of treatment , flow cytometry was used to detect the proportion of peripheral blood tregs; real-time pcr was used to detect the levels of hbv dna in surum, markers of hepatitis b virus infection were detected by elisa assay and levels of alanine aminotransferase in serum were measured. results: the proportion of peripheral blood tregs in patients with chb was significantly higher than that in healthy controls and decreased over or months of treatment to a level comparable to that of healthy controls. after months of treatment, the rate of alt normalization in patients which the proportion of peripheral blood tregs was unreduced was significantly lower than that in patients which the proportion of peripheral blood tregs was reduced (p< . ). , or months of telbivudine treatment resulted in negative hbeag in ( %) patients, ( %) patients or ( %) patients respectively. within months of treatment, ( %) patients seroconverted from hbeag to anti-hbe , in which the proportion of peripheral blood tregs had decreased to a level comparable to that of healthy controls over or months of treatment. conclusion: during antiviral treatment with subsequent reduction of the viral load or alt levels, the proportion of tregs decreased to a level similar to that of normal healthy controls. in addition, seroconversion from hbeag to anti-hbe was prone to be established in patients which the proportion of tregs decreased quickly at the early phase of antiviral treatment with telbivudine. background: in china a part of patients with alt < . ×uln and hbv dna > copies/ml will advance into hepatic cirrhosis even hepatoma. so these patients should not only be monitored but also be treated. this study was made to determine the safety and efficacy of combining therapy of pegylated interferon alpha a (peg-ifn - a) and entecavir in treating naive patients with alt < . ×uln and hbv dna> copies/ml. methods: nine patients with hbsag positive over months and alt< . ×uln hbv dna > copies/ml were taken as research subjects. before treatment,liver biopsy was used to assess histological damage. patients were treated with peg-ifn - a g /week for weeks, and in the first weeks entecavir . mg/day was applied, then it was stopped. results: liver biopsy showed that patients had mild inflammation. after weeks' treatment , hbv dna level in all patients decreased to less than copies/ml, and after weeks' treatment( weeks after entecavir was stopped)hbv dna in all patients was less than copies/ml. normal alt was seen in all patients after weeks' treatment and weeks' treatment. none of the patients had peripheral neuropathy with combining treatment. conclusions: . bulk of patients with alt < . ×uln and hbv dna> copies/ml had mild inflammation and need treatment. combing treatment of peg-ifn and entecavir was safe and effective to this group. it proved that it was safe for patients to stop treatment with entecavir after short time use. background & aims: quantification of serum hbv dna levels is important to monitor viral replication in chronic hepatitis b (chb) patients. both abbott realtime hbv and roche cobas amplicor hbv monitor are updated fully automatic commercial assays for hbv dna quantification. the aim of this study is to compare the performance of these two assays on the hbv dna quantification in chb patients. methods: serial serum samples from chb patients were collected at the baseline and at days , , and and weeks , , and after the commencement of therapy. genotype was determined by sequence alignment. abbott and roche assays were employed for hbv dna extraction and quantification according to the instructions of manufactories. results: hbv dna quantification results of abbott assay was significantly correlated with those of roche assay (r= . , p< . ). for genotype c, the difference in hbv dna levels [median (range): . (- . - . ) log units] measured by these two assays was significantly higher than that for genotype b [ . (- . - . ) log units, p< . ]. moreover, the difference in serum hbv dna levels after weeks antiviral treatment [ . (- . - . ) log units] measured by these two assays was significantly higher than that in baseline serum hbv dna levels [ . (- . - . ) log units, p< . ]. conclusion: the quantification results of abbott realtime hbv showed a good correlation with those of roche cobas amplicor hbv. but the performances of these two assays have significant difference in the quantifications of serum hbv dna levels in genotype c patients and in patients after weeks antiviral therapy. background: guidelines suggest hepatitis b virus (hbv) vaccination to all hepatitis c virus (hcv) infected patients and healthcare workers. we attempted to find out hbv vaccination status in our hcv infected population, and healthcare workers. methods: prospective survey of consecutive hcv infected patients and also doctors and paramedical staff in our hospital. results: major sources of viral infection in study patients ( males; average age years -range to yrs) were reused syringes ( pts). twenty had a household member infected with hcv. twenty were co-infected with hbv. eighty five of hcv infected patients were not vaccinated.against hbv. twenty five of them ( %) had financial reasons and patietns ( %) had lack of awareness. out of doctors, and did not know about their hbv and hcv status respectively, but none was known to have either of these infections. four ( %) were not vaccinated against hbv. out of paramedical staff, was hcv positive, each were unaware of their hbv and hcv status, and remaining were negative for these markers. thirteen of them ( %) were not vaccinated against hbv. conclusion: thirty eight percent hcv infected patients were infected by reuse of syringes. eighty five percent were not vaccinated against hbv, out of which % had no awareness about it, whereas % could not financially afford it. a significant number of paramedical staff and some doctors were also not vaccinated background: early prediction of efficacy could decrease unnecessary interferon exposure of patients with chronic hepatitis b. methods: a multi-center clinical study. patients were injected interferon alpha b million iu subcutaneously every other day for weeks and -week follow-up was followed. results: patients ( male) were enrolled, . ± . years old. hours after administration, hepatitis b virus (hbv) load decreased significantly ( . ± . log copies/ml, p< . ) from baseline ( . ± . log copies/ml). hbv load was . ± . and . ± . log copies/ml at week and , respectively. at the two points upwards, complete response rate was . %( / ) and . %( / ), partial response rate was . %( / ) and . %( / ), respectively. at week and , hbv dna levels of complete responder and partial responder were lower than those of non-responder (p< . ) at week . at baseline, on hour , day , , week , and , hbv dna levels of complete responder were lower than those of non-responder at week (p< . ). multiple linear regression showed that baseline hbv dna was the independent variables to predict the response at week and . conclusions: interferon alpha b was effective in treating patients with hbeag positive chronic hepatitis b. it could decrease the hbv dna level rapidly. early hbv dna levels were predictive to response at the end of treatment and follow-up. baseline hbv dna level was the independent predictor of the response at the end of treatment and follow-up. aim: to investigate features of pd- expression on peripheral tcells and pd-l expression in liver in chronic hepatitis b (chb) patients in immune clearance phase. methods: pd- expression on total peripheral t cells were evaluated by using flow cytometry. immunostaining was performed according to the envision chemmate methods. the degree of pd-l expression was scored and assessed according to the percentage and staining intensity of positive cells. results: compared to health control, the percentage of total peripheral t cells expressed pd- was elevated in chb with repeatedly increasing alt level. no specific association between the percentage of pd- positive and the mean fluorescence intensity mfi of pd- expression on total t cells with serum viral load were found. but alt level was correlated with the mfi of pd- expression on total cd +t cells significantly. pd-l is up-regulated on hepatocytes by viral infection, and high expressed in fibrosis section. conclusion: the mfi of pd- on cd +t cells plays important role in regulating the immune-host interaction in chb in immune clearance phase. and pd- expression on t cells is correlated with high immune inflammatory refection. aim: to study the quantity, characteristic of hbv-specific t-cell and the extent of liver damage in chronic hepatitis b (chb) patients with different hbeag status. methods: chb patients were enrolled and divided into two groups according to the hbeag status, and the liver damage index were analyzed. the frequency and foxp expression of cd + cd + regulatory t cells (treg) were measured, as well as the frequency and phenotypic molecules expression of hbv-pentamer+ t-cell. hbv specific t-cell responses including cellular proliferation and ifn-production, with or without anti-pd-l and/or anti-ctla- blocking, were also observed. results: the demographic characters, serum alt, ast levels, the frequency and foxp expression of cd + cd + treg were similar, while the serum hbv dna levels were higher in hbeag+ patients (p < . ). the liver necroinflammation was comparatively more severe in hbeag-patients (p = . ), but the median percentage of liver cirrhosis was much higher in hbeag+ patients (p < . ). the difference of hbv-specific t-cell frequency was not significant between two groups, while the expression levels of pd- and ctla- on hbv-specific cd t cells were significantly higher in hbeag+ patients (p both < . ). combined using of anti-pd-l and anti-ctla- mab significantly increased the cellular proliferation in either hbeag+ or hbeag-patients, but only markedly enhanced the ifnproduction in hbeag+ patients. conclusion: hbeag persistency could probably induce higher expression of pd- and ctla- on the hbv-specific t cells and result in t-cell impairment, high hbv dna load and high percentage of liver cirrhosis in hbeag+ chb patients. hepatocyte apoptosis in patients with chronic hepatitis b y. liu , k. wang background: to investigate the relationship between hepatocyte apoptosis and the level of inducible nitric oxide synthase (inos) in hepatic tissue in the patients with chronic hepatitis b chb . methods: we observed cases with chb and normal controls. transferase-mediated-utp-biotin nick-end labling ( tunel) technique was used to detect apoptosis cells and immunohistochemical staining were also performed to investigate the expression of inducible nitric oxide synthase (inos) in biopsy samples .the serum level of alt hbv-dna grading of necroinflammatory activity and staging of fibrosis were also assessed. results: hepatocytes in all chb liver tissues were positively stained by tunel in various degree. in contrast, control tissues did not show dna fragmentation. a significant correlation was seen between apoptosis index (ai) and necroinflammatory grading ((r= . , p= . ) and serum inos level r= . , p= . . it did not correlate with fibrosis stage and serum alanine aminotransferase level. conclusion: the oxidative stress.in patients with chb may reflected the apoptosis of hepatocyte. apoptosis involves in liver injury of chb,but with no significant correlation to serum level of alt. objectives: to investigate the genotype-dependent development of lamivudine resistance in hepatitis b virus (hbv). methods: patients with chronic hepatitis b who had been treated with lamviudine for more than year, and become lamviudine resistance were analysed for the hbv genotypes and cumulative rate of rt region mutant with standard dna sequencing technology. results: among the patients, patients were infected with hbv genotype b (hbv/b)( . %), and with genotype c (hbv/c)( . %). in the hbv/b patients, / ( . %) were of subtype ba, and / ( . %) were of of subtype bj. the cumulative type and ymdd mutation rates in patients with genotype c were showed as l m+m v ( / , . %) > l m+m i ( / , . %) > m i ( / , . %), while in patients with genotype b as l m+m v / ( . %) > m i( / , . %), none of l m+m i. conclusions: our results indicated that in patients with lamivudine resistance, hbv genotype c (hbv/c) were higher than genotype b (hbv/b). in both genotypes the combined mutations ( + sites) were found more than the single site, showed some significance for monitoring lamivudine resistance. background & aims: il- , a novel identified inhibitory cytokine specifically produced by regulatory t cells (tregs), is an ebi -il- heterodimer encoded by epstein-barr-virus-induced gene (ebi ) and interleukin- alpha (il ). the aim of the study is to determine the expression levels of il- in peripheral blood mononuclear cells (pbmcs) of chronic hepatitis b (chb) patients in different phases. methods: a total of treatment naïve chb patients, including in immune-tolerant phase [group , alt: ( - ) u/l, serum hbv dna: . x ( . x - . x ) copies/ml] and in immune-clearance phase [group , alt: ( - ) u/l, serum hbv dna: . x ( . x - . x ) copies/ml] were enrolled in the study. the relative mrna expression levels of ebi , il and foxp were determined by semi-quantitative pcr. results: the significant correlations were observed between the expression of ebi and il (r= . , p< . ), ebi and foxp (r= . , p< . ), il and foxp (r= . , p< . ). the relative expression levels of ebi and il in pbmcs were significantly higher in group when compared with those in group ( . ± . vs . ± . and . ± . vs . ± . , p< . , respectively). furthermore, the relative expression levels of ebi and il in group were significantly correlated with alt levels (r= . , r= . , p< . , respectively), but not with serum hbv dna levels. conclusions: the expression levels of il- in pbmcs were significantly higher in chb patients in immune-clearance phase than that in immune-tolerant phase. increased il- expression levels were associated with liver injury. background: there are a number of oral antivirals approved for chronic hepatitis b. lamivudine, the first oral nucleoside analog, is associated with increased rates of drug resistance with prolonged use--from % at one year to % at three years. therefore, an alternative or add-on treatment is necessary. adefovir, an oral nucleotide analog, is used either in combination with lamuvudine or as monotherapy in lamivudine-resistant chronic hepatitis b. we did a meta-analysis to compare the efficacy of adefovir in combination with lamivudine versus adefovir alone in the treatment of lamivudine-resistant chronic hepatitis b infection. methods: a comprehensive literature search was performed using the following databases: medline, cochrane, and embase. a total of randomized controlled trials were retrieved and analyzed. outcomes measured were virologic response, biochemical response and resistance rates. results: meta-analysis on virologic response showed that combination treatment with adefovir and lamivudine is as effective as adefovir monotherapy (or . , % ci . - . , p= . ). likewise, in terms of biochemical response, both regimens were equally effective (or . , % ci . - . , p= . ). one study showed statistically significant increase in adefovir resistance rate in the monotherapy arm compared to combination arm (p= . ) after the first year of therapy. conclusion: in patients with lamivudine-resistant chronic hepatitis b infection and compensated liver disease, adding adefovir to lamivudine is as effective as switching to adefovir alone in terms of virologic and biochemical response. r. safadi , q. xie , y.g. chen , y.k. yin , l. wei , s.g. hwang south korea, bnai zion medical center, haifa, israel, beijing friendship hospital, beijing, china, novartis pharma ag, basel , switzerland background: ldt produces greater viral suppression than lam. we investigated whether patients receiving lam can benefit from switching to ldt. methods: hbeag positive and negative persistently viraemic patients (median hbv dna . (ldt), . (lam) log copies/ml) and lam treated for - months, were randomized to either switch to ldt or continue lam. we report the benefit of ldt switch assessed by primary treatment failure (tf, < log hbv dna decline) and viral breakthrough (vb, > log above nadir). results: % ( / ) of the ldt switch and % ( / ) continuing lam patients had pre-existing m mutations at screening. tf was % (ldt) versus % (lam, p< . ). in patients with > weeks prior lam treatment, tf was % (ldt) versus % (lam). % ldt tf ( / ) was associated with resistance at screening versus % lam tf. in ldt switch with < weeks prior lam, no ldt tf occurred versus % lam. in hbeag positive, tf occurred in % (ldt) versus % (lam). among hbeag positive with > weeks prior lam treatemtn, vb was % (ldt) versus % (lam, p< . ). differences were not significant for hbeag positive with > weeks lam or for hbeag negative regardless of duration of prior lam treatment. conclusions: early switch to ldt is associated with better virological outcomes in these patients. persistent viraemia for > months on lam treatment is associated with a high risk of tf and vb. for these patients, genotypic analysis is recommended prior to screening. objective: the aim of this study is to evaluate the proper endpoint in the treatment of chronic hepatitis b with antivirals by investigating the viral rebound ratio after one year's nucleosides or (three months) sustained treatment with lamivudine, adefovir, entecavir, or interferon when viral response and seroconversion response have been finished . methods: eag positive chronic hepatitis b naïve patients with alanine aminotransferase (alt) more than uln were assigned to receive mg of lamivudine, mg of adeforvir, or . mg of entecavir once daily, respectively. patients in the interferon group were administrated with , , iu of a interferon on every other day, and the therapeutic duration lasted for another three months after eag-ab seroconversion appeared. hbv dna and eag-ab in the serum were tested during the off-treatment period of months. results: thirty four patients in lamivudine group of cases got eag-ab seroconversion after treatment with ± months of average duration, and the viral rebound ratios in the off -treatment an months follow up period were . / and . / , respectively. in adeforvir group were / and . / . in enticavir group were / and . / . in interferon group was . / in the off-treatment months follow up period. conclusions: we conclude that eag-ab seroconversion in the treatment of eag positive chronic hepatitis b patients is the goal but not an endpoint of therapy physicians should aim at. to gain everlasting effect, longer duration of treatment may be needed. background: universal hepatitis b(hb) vaccination of hbsag negative people (especially infants) is widely recommended and practised. objective: to assess whether there is robust evidence of protective efficacy to back such practice. methods: this cochrane review included randomised trials identified from six databases through detailed electronic searches. trials comparing hb vaccine versus placebo/another vaccine, in hbsag negative persons were included without any restrictions. the primary outcome was hb infection (developing hbsag or anti-hbc). robustness of evidence was assessed through comparison of available-case analysis versus intention-to-treat(itt) analysis using four different models: (i)assuming unfavourable event for all missing data, (ii)assuming favourable outcome for all missing data, (iii)best-case-scenario and (iv)worst-case-scenario results: twelve trials were eligible among citations; all were methodologically poor (high risk of bias). data from four trials could be included in meta-analysis. efficacy of vaccination varied with the type of data analysis. available-case analysis suggested efficacy in reducing risk of developing hbsag (rr= . ; %ci= . - . ;n= ) and anti-hbc (rr= . ; %ci= . - . ;n= ). itt analysis results varied depending on the model chosen (table) , but liberal approaches suggested high efficacy, whereas conservative approaches did not. the available evidence on efficacy of hb vaccination in hbsag negative people is not robust; there are serious limitations in quality and quantity. background: open-label rollover study (etv- ) assessed histologic improvement in chb patients on at least years etv therapy. methods: % nucleoside-naïve patients and % lamivudine (lvd)-refractory patients from etv- and etv- studies, respectively, entered etv- study and received etv at . / mg for greater than weeks. improvement in knodell necroinflammatory (ni) score and knodell fibrosis score at weeks and were studied. results: at week , % of nucleoside-na ve patients and % of lvd-refractory patients achieved hbv dna < copies/ml. furthermore, % of nucleoside-na ve patients and % of lvd-refractory patients had normalized alt levels. mean platelet counts in both naïve and lvd-refractory patients were improved at weeks and compared with baseline. conclusions: naïve and lvd-refractory chb patients showed significant improvement in liver histology after year etv therapy, and improved dna and serum alt levels. results: . percent of patients were younger than years old, . percent were older than in this study. . % patients' mothers were hbsag positive. high levels of serum hbv dna were founded in all patients, > copies/ml were . %. only cases ( . %) whose liver inflammation grade were g , the rest patients were mild inflammation, in which g were cases ( . %), g were ( . %); there were patients ( . %) had no signifecant liver fibrosis, the rest cases ( . %) had different fibrosis, among those s were cases ( . % , s were . % , s were . % , none of patients had cirrhosis. the fibrosis stages of higher alt level were markedly severer than lower alt in patients with normal alt p < . . conclusions: most of patients with chronic hepatitis b virus in immune tolerant phase present mild inflammation in liver, part of them have already appeared fibrosis, so some patients determinated by clinics are actually not in immune tolerant phase. although alt testing are in the normal range, but the possibility of liver fibrosis is increased in patients with relative higher alt level, so liver pathology should be recommended to judge illness correctly. background/aims: hepatitis b virus infection (hbv) is a global health problem. in bangladesh, - % of people are hbsag positive. this study was carried out to evaluate the efficacy and safety of peginterferon alfa- a in chronic hepatitis b patients. methods: a total of patients with chronic hepatitis b, ( . %) were hbeag positive (group a) while ( . %) were hbeag negative (group b) were included in this study after meeting the following criteria: age to years, hbsag positive for more than months, serum hbv-dna was > log( ) copies/ml and alt more than two times the upper normal limit. they were given peginterferon alfa- a ( microgram once weekly) for weeks and followed for an additional weeks. results: after weeks of follow-up, the percentage of patients with normalization of alanine aminotransferase levels or hbvdna levels below , copies per milliliter was significantly higher in hbeag positive patients ( percent and percent, respectively) than among hbeag negative patients ( percent and percent). loss of hepatitis b surface antigen occurred in patients in group a, as compared with patients in the group b (p< . ). adverse events including pyrexia, fatigue, myalgia, headache and haematologic abnormalities were similar in both groups. conclusions: patients with hbeag positive chronic hepatitis b had significantly higher rates of response, sustained for weeks after the cessation of therapy, with peginterferon alfa- a. background: the effect of hepatitis b vaccination on individuals with isolated anti-hbc in endemic areas is not clear. we investigated the prevalence of individuals positive for anti-hbc only and their antibody response after hepatitis b vaccination in a single healthcare center. methods: the study included , healthcare workers. after screening for hbsag and anti-hbs, the individuals negative for both hbsag and anti-hbs were examined for anti-hbc and were vaccinated with a recombinant hepatitis b vaccine at , , and months. the serum anti-hbs level was measured after the vaccination. results: of the subjects, ( females) were negative for both hbsag and anti-hbs. forty ( . %) subjects had isolated anti-hbc, including more males ( . % vs. . %) and older people ( . ± . vs. . ± . years), compared with individuals negative for all of the viral markers. the anti-hbs seroconversion rate and anamnestic response in the individuals with isolated anti-hbc after the first vaccine injection were % and . %, respectively. in the persons who were negative for all hepatitis b viral markers, the seroconversion rate after the first vaccination was . %. the anti-hbs seroconversion rate did not differ between the isolated anti-hbc positive individuals and those negative for all hepatitis b markers ( . % vs. . %) after the full course of vaccination. conclusions: serum hbsag and anti-hbs tests are sufficient for screening before hepatitis b vaccination, especially in healthcare workers. objective: to understand the quantity and distribution of cd + mature dendritic cells in patients with hepatitis b virus in immune tolerant phase. methods: there were immune tolerant phase patients with hepatitis b virus infection (fibrosis stages were s ), immune clerance phase patients, non-active status patients and healthy controls involved in our research. the quantity and distribution of cd + mature dendritic cells in liver were determined by immunohistochemical staining. result: the liver inflammation grades were between g -g in patients who in inmmune tolerant phase and non-active status, moreover, patients in immune clerance phase were between g -g . there were a small amount of cd + dendritic cells in healthy liver tissue, scattered in portal areas and hepatic lobules. the quantity and distribution of cd + dendritic cells in patients who in inmmune tolerant phase and non-active status were similar to the healthy, and the quantity were no difference among them p . .the number of cd + cells in patients of immune clerance phase was significant increased compared with other groups, there were differences among them p . , the cd + cells mainly distributed in portal areas infiltrated with inflammatory cells and hepatic lobules with inflammatory necrosis. conclusion: cd + mature dendritic cells are involved in liver immune response in patients of inmmune clerance phase, is likely to related to hepatitis b virus clearance. lack sufficient mature dendritic cells may be one of the mechanisms of immune tolerance. background: local hospitals provide obstetric services including antenatal care to women normally living in the mainland china, whose prevalence of hepatitis b carrier is unknown. objectives: compare prevalence of hbv carrier of pregnant women from the mainland china with local counterparts and discuss the implications of results. materials and methods: antenatal serological results were retrieved from corporate laboratory information system databases. pregnant women from the mainland china were identified by a specific set of temporary-allocated identity number during january -october . results: pregnant local residents and pregnant women from the mainland china underwent antenatal serological tests for hepatitis b surface antigen. positive hepatitis b surface antigen results were more frequent in pregnant women from the mainland china ( . %) than in local pregnant women ( . %) (p< . ). discussion: because infected pregnant women can transmit the hepatitis b virus to the infant at delivery, specific management could entail maternal medication, injection of hepatitis b immune globulin to the infant at birth and immunization later on. however, early repatriation to the mainland china, which is common, will make completion of immunization program difficult. these babies will be at a higher risk to be infected by hbv, particularly when breast-fed by hbv carriers. their return to hong kong later will dilute the effects of local immunization program. the volume of work derived from the provision of obstetric services to women from the mainland chinese is larger with regard to medication, counseling and immunization for babies born to hbv carriers. immunosuppressive or anticancer therapy k. hirano , t. kodani , s. sato , y. narita , t. kikuchi , t. genda , k. iijima , k. ogawa , t. ichida background/aim: we compared the prevention of hbv reactivation in (hbsag)-positive patients with hbsag-negative patients who were positive for antibody to (anti-hbc) and/or (anti-hbs) undergoing immunosuppressive, anticancer or molecular target therapy. methods: from sep to nov hbsag-positive patients and anti-hbc and/or anti-hbs-positive patients were enrolled in this study. we compared with groups about background disease, age, blood examination, and nucleoside analogues. results: in hbsag-positive patients mean age were . ± . years old, median ast levels were ( - ) iu/l, and median alt levels were ( - ) iu/l for ( %) haematological disease and ( %) collagenosis disease. in anti-hbc and/or anti-hbs-positive patients mean age were . ± . years old, median ast levels were ( - ) iu/l, median alt levels were . ( - ) iu/l for ( %) haematological disease and ( %) collagenosis disease. serum hbv-dna levels > . log copies/ml were ( %), . ~ . were ( %), < . were ( %) in hbsag-positive patients, and serum hbv-dna levels < . were all cases in anti-hbc and/or anti-hbs-positive patients. ( %) of hbsag-positive patints received nucleoside analogues ( lam and etv), and ( %) of anti-hbc and/or anti-hbs-positive patients received nucleoside analogues ( lam and etv). mean duration of treatment for . months in hbsag-positive patients, and for . months in anti-hbc and/or anti-hbs-positive patients, the resistance virus occurred to ( %) of hbsag-positive patients treated with lam for collagenosis disease more than two years. conclusion: when hb carriers of collagenaous disease undergoing immunosuppressive therapy required the nucleoside analogues more than two years, we recommended treatment to prevent hbv reactivation with etv. background: adefovir dipivoxil is used for the initial treatment of chronic hepatitis b or rescue treatment of lamivudine-resistant chronic hepatitis b, and exhibits excellent antiviral activity. however, the presence of resistance to adefovir dipivoxil was more frequently in lamivudine-resistant chronic hepatitis b patients than in lamivudine-naïve patients during adefovir dipivoxil monotherapy. but the rate of adefovir resistance related mutations is little known in lamivudine-resistant patients before adefovir dipivoxil treatment. the aim of this study was to investigate the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus in lamivudine-resistant patients not treated with adefovir dipivoxil. methods: the existence of adefovir resistance-related mutations was examined in lamivudine-resistant chronic hepatitis b patients with breakthrough hepatitis and antiviral-naïve chronic hepatitis b patients. both polymerase chain reaction restriction fragment length polymorphism (pcr-rflp) and directly sequencing of pcr product were used to detect resistant viruses. results: rta t mutants were detected in only two sera of lamivudine-resistant patients, while none in the antiviral-naïve chronic hepatitis b patients. there was no rtn t detected in the two groups. conclusion: our results suggest that the rta mutant virus were present in a few lamivudine-resistant chronic hepatitis b patients before they have been treated with adefovir dipivoxil, but the rtn t mutant was not detected in any of the two groups. the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus was low in such lamivudine-resistant patients before adefovir dipivoxil treatment. objective: in this study, we tried to detect and identify the special protein of hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. to find new opinion on the developing of chronic liver disease. methods: the sera of health adult, hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma were respectively detected by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (seldi-tof-ms). the arrays of every group were analysised by clustering analysis and to establish disease predictive model. then the sample was eluted with different ph tris, trypsinization on-chip, mass determination and peptide database comparison. results: according cm chip we find protein with obviously deviation (p< . ) among hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. clustering analysis for the data from seldi-tof-ms confirmed differentially expressed proteins. then we developed disease predictive mathematic models ( decision tree model, dt model ) with average validity up to . . the da protein peak was identified to be chondroitin sulfate synthase (chss ), which is a potential molecule involved in the pathologic process and a potential serum marker for the hbv related hepatic diseases as well. conclusions: our results suggest that seldi-tof-ms is a usefull technique for differential expressed proteins screening and analysis in hbv related chronic liver disease. chss may be useful during the developing of hbv related chronic liver disease. backgound: clevudine is a new nucleoside analogue with potent antiviral activity in chronic hepatitis b patients. however, the efficacy and safety of clevudine in cirrhotic patients are not well recognized. this study was conducted to evaluate the early virologic and biochemical response rate as well as safety of clevudine in cirrhotic patients with chronic hbv infection. methods: patients with chronic hbv infection who visited korea university ansan hospital and guro hospital between may and may were included. patients had chronic hepatitis b (group a) and had liver cirrhosis (group b). early virologic response was defined as hbv dna less than iu/ml at week . early biochemical response was defined to be normalization of alt (< iu/l) at week . result: pretreatment hbv dna levels were higher in group a compared with group b ( . log iu/ml vs . log iu/ml, p= . ). pretreatment alt levels were not significantly different between the two groups ( iu/l vs iu/l, p= . ). the rate of early virologic response was significantly higher in group b compared with groups a ( . % vs %, p= . ). the rate of early biochemical response were not significantly different in both groups ( % vs . %, p= . ). conclusion: clevudine is considered to be safe and effective in cirrhotic patients with chronic hbv infection as well as chronic hepatitis b patients. long term safety and efficacy need to be evaluated in the future. objective: the aim of this study was to evaluate the role of nucleos(t)ide analogues against hbv reactivation in immunosuppression. methods: non-active hbsag carriers suffering from cancer, autoimmune diseases and needing the treatment of immunosuppressants or cytotoxic chemotherapy were enrolled in the study. the outpatients or in-patients from april to july were enrolled. the nucleos(t)ide analogues were used in cancer patients - weeks before chemotherapy, and the duration lasted - months according to patients' compliance after completion of chemotherapy. patients with other diseases used nucleos(t)ide analogues in - months before using glucocorticoids or other immunosuppressive agents, and continued to use for - months after accomplishing the course of immunosuppressant treatment. the characheristics and clinical manifestations about hbv reactivation were investigated. results: of the thirty two patients in prospective group, twenty two patients suffered from cancer, eight patients suffered from idiopathic thrombocytopenic purpura, two patients suffered from chronic nephritis. the amount of hbv dna was detected in the first, third, sixth and th month after the use of nucleos(t)ide analogues. after chemotherapy or immunosuppressant treatment, only . % ( / ) of them suffered from hbv reactivation, which presented with hbv dna positive and abnormal liver function. conclusion: non-active hbsag carriers would appear potential incidence of hbv reactivation during use of chemotherapy or immunosuppressant. nucleos(t)ide analogues could be used in early phase as prophylaxis for reactivation of hepatitis b in immunosuppression and to improve clinical prognosis. background: hbv therapies are evolving toward combination antivirals. this study evaluated the combination of clevudine (clv), a potent nucleoside analog, with tenofovir dipivoxil (tdf). methods: a phase i, single-arm, multi-dose study in healthy adult volunteers to evaluate pharmacokinetic and safety interactions between clv and tdf. subjects received days of clv mg followed by days of clv mg +tdf mg. pk profiles were obtained on days , and . clv auc and cmax were compared on days and . day tenofovir pk was compared to historical data. safety assessments were conducted throughout. results: subjects were enrolled ( m/ f); completed the study. the mean (range) age was y ( - ) and body mass index (kg/m ) was . ( . - . ). aes were reported by subjects, with aes reported during clv-only dosing and aes reported during clv+tdf dosing. aes included nausea ( ) and pharyngolaryngeal pain ( ). the majority of the aes were mild. there were no clinically significant changes in ecgs or laboratory parameters. comparisons of clv auc and cmax on day and revealed no significant impact of tdf upon the plasma clv exposure (d /d auc ratio= . , d /d cmax ratio= . ). there is no significant effect of clv on tenofovir when comparing auc and cmax of tdf to historical values. conclusion: safety and pharmacokinetic results demonstrate that clv and tdf may be safely co-administered, supporting the further study of this drug combination for the treatment of chronic hbv infection. s. kuznecovs , , i. kuznecovs , k. jegina , g. kuznecova background: dolichyl (dol), the main lipid intermediator of dolichyl phosphate cycle (dpc) has been reported to be elevated in urine of patients with multidrug resistance in cancer. drug resistance poses a major threat to nucleoside analogue-based therapies for chronic hbv infection. methods: with focus on a risk predictor for susceptibility to the development of hbv drug resistance the present study was carried out to estimate urinary levels of dol in chronic hbv infection. the samples obtained every week before and during the course of treatment from patients with hbv. the occurrence of exacerbations of chronic hbv were registered for years. dol in urine was assayed by hplc method. results: the normal amounts of dol in healthy persons urine (n= ) are , + , mkg/mmol creatine. during the period of observation ( %) of patients treated with nucleoside analogue-based therapies were diagnosed with exacerbations due to resistance of hepatitis b virus to antiviral drugs. from this group of hbv patients ( %) have had elevated urinal dol excreation ( , ± , g/ml vs . , ± , g/ml, p< . ) in more than months of observation. conclusion: there is a reason to suggest that elevated urinal dol detected in patients with exacerbations during hbv treatment may evidence of possible defect of host mechanism of drug resistance development to nucleoside analogue-based therapies. the interest drawn to the employment of dol as a predictor for exacerbation of chronic hbv is explained by the role of dpc in p-glycoprotein regulation in human hepatocytes. background: a significant proportion chb patients treated with adv have a suboptimal response, increasing the risk of disease progression and development of resistance. we report clinical results from patients who either failed or relapsed following adv therapy and were subsequently switched to etv. methods: study etv- was a randomized, open-label study comparing antiviral efficacy of etv ( . mg/day) vs adv ( mg/day) in nucleoside-naïve hbeag-positive patients. after up to weeks of treatment in etv- , patients treated with adv ( suboptimal responders) rolled over into study etv- ( . mg/day). hbv dna viral suppression and safety was evaluated during weeks of etv treatment. results: at entry to etv- , the median hbv dna was . log copies/ml. median exposure to etv ( . mg) in etv- was weeks and patients currently remain on study therapy. at week , the mean reduction in hbv dna was . log copies/ml and / ( %) reached hbv dna levels < copies/ml. nine patients have achieved week and all have achieved hbv dna < copies/ml and / ( %) had hbv dna levels < copies/ml. no patients experienced virologic breakthrough on etv. the safety profile of etv in adv-treated patients remained consistent with the previously reported experience. conclusions: the majority of patients who were suboptimal responders or virologic rebounders following adv treatment in study etv- , experienced rapid reductions in hbv dna levels when switched to etv. hbv dna levels continued to decline to undetectable levels with weeks of etv treatment. y. li , , t. han tianjin third central hospital, aim:to quantify hepatitis b virus (hbv) total dna and covalently closed circular dna (cccdna) in liver biopsies and sera which from chronic hepatitis b(chb) liver cirrhosis of hepatitis b(lc) and hepatitis b relevance hepatocellular carcinoma(hcc) patients, and analysis hbv replication under the circumstances of different diseases. methods:total hbv dna and cccdna in serum and liver biopsy samples were measured in chb lc and hcc patients by the real-time pcr assay. results: the levels of total hbv dna in serum,intrahepatic total hbv dna, intrahepatic cccdna, as well as the proportion of intrahepatic cccdna in total hbv dna decreased progressively in chb,lc and hcc ,moreover chb had significantly higher levels of total hbv dna in serum and liver biopsy samples than lc (log [total serum hbv dna] p = . ;log [total intrahepatic hbv dna] p = . ); chb and lc had significantly higher levels of intrahepatic cccdna and the proportion of intrahepatic cccdna in total hbv dna than hcc(p < . ); cccdna couldn't be detect in all patients'serum. in chb ,the levels of serum's total hbv dna,intrahepatic total hbv dna and cccdna in hbeag-positive group had significantly higher than the hbeag-negative group(p < . ) ,but in lc only intrahepatic total hbv dna had statistical difference between hbeag-positive and negative group (p= . ) , no statistical difference between hbeag-positive and negative group in hcc. conclusions: the replication activity of hepatitis b virus in chb,lc were higher than hcc, hbv reproduction reduced significantly in hcc. duplication of hbv in lc was lower than chb but had no statistical difference. the levels of hbv reproduce in hbeag-positive group was higher than hbeag-negative group of all three desease. background: clevudine is a pyrimidine analogue with potent and sustained antiviral activity against hbv in the week therapy. the present study assessed the efficacy and viral resistance of week clevudine therapy in patients with chronic hepatitis b. method: a total of patients ( hbeag positive and hbeag negative) who were received clevudine mg once daily for weeks were included in this analysis. serum hbv dna was quantified by real time pcr assay. result: at week , median reductions of serum hbv dna from baseline were . log iu/ml ( . log iu/ml for hbeag positive and . log iu/ml for hbeag negative) and . % of patients showed undetectable serum hbv dna (< iu/ml) ( . % for hbeag positive and . % for hbeag negative). the normalization of alt levels (< iu/l) was achieved in . % ( . % for hbeag positive and . % for hbeag negative). . % of hbeag positive patients showed hbeag loss or seroconversion. hbv dna negativity at week was associated with hbeag negativity (p = . ), hbv dna < , iu/ml at weeks and (p = . and . , respectively). two hbeag positive patients showed viral breakthrough with m i mutation during week. conclusion: clevudine therapy in patients with chronic hepatitis b showed potent virologic responses at week , especially in those with hbeag negativity and complete early virologic response (hbv dna < , iu/ml at weeks and ). but clevudine resistance can occur in hbeag positive patients. background: serum alanine aminotransferase (alt) activity, the variable most commonly measured to assess hepatic disease, fails to identify many patients with hepatic injury. current standards for "normal" alt level were defined by using populations that included persons with subclinical liver disease. there is no study regarding normal level of alt and its modulating factors in healthy thai people. objective: to definitions of normal ranges for serum alt level in thai people. design: prospective observational study setting: phramongkutklao hospital and army institute of pathology pramongkutklao medical center(a.i.p.), bangkok, thailand participants: persons who were first-time blood donors from august through december were negative for anti-hepatitis c virus(hcv), negative hbsag(hbv), and had no contraindications to donation. measurements: univariate and multivariate analyses examined associations between clinical and laboratory factors and alt levels. normal ranges for alt were computed from the population at lowest risk for liver disease. results: serum alt activity was independently related to body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. normal ranges for serum alt level in thai people upper limits for men u/l and for women . u/l. conclusion: in men serum alt is strongly associated with body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. the normal range of alt should be defined for male and female separately. background: the open-label rollover study etv- was conducted after etv phase ii clinical study etv- for nucleoside-na ve adult chb patients in japan. in this analysis, we report etv long-term efficacy and safety in patients who were switched from -week lvd treatment to etv therapy. methods: ninety-seven percent ( / ) of lvd-treated patients from etv- were rolled over into etv- treated with . mg of etv. thirty patients completed weeks of etv therapy and were evaluated for hbv dna level, alt normalization, hbeag seroconversion, resistance and safety. results: comparing to baseline before switching to etv, after week of etv treatment, the proportion of patients achieving undetectable hbv dna (< copies/ml) increased from % to %. increases were also observed for alt normalization ( % to %) and hbeag seroconversion ( % to %). three patients had detectable hbv dna at week after etv treatment and samples from two were tested for resistance. neither demonstrated substitutions associated with etv or lvd resistance. five patients had grade - laboratory abnormalities, including increased ast/alt and increased lipase levels. conclusions: switching patients from lvd therapy to etv resulted in increased proportions of patients achieving hbv dna suppression, alt normalization and hbeag seroconversion, with no evidence of etv resistance. etv was well tolerated during treatment. backgrounds/aims: hepatitis b virus (hbv) reactivation in patients undergoing chemotherapy hampers an adequate administration of cytotoxic agents and even causes an treatment failure. prophylaxis failure occasionally results from viral breakthrough or withdrawal flare. the aims of this study were to identify predictors of anti-viral prophylaxis failure and to determine the optimal strategy for anti-viral prophylaxis. methods: cancer patients with positive hbsag who underwent cytotoxic chemotherapy in a tertiary medical center from january to june were included. prophylactic lamivudine was started with initiation of chemotherapy, continued during the chemotherapy, and discontinued within months after the completion of chemotherapy. all patients were followed up even after withdrawal of lamivudine. results: patients were enrolled. twenty-nine patients ( . %) had hematologic malignancies and eighty-six ( . %) had solid tumors. median follow-up duration was . months and twenty-six patients ( . %) experienced the prophylaxis failure: viral breakthrough ( patients, . %), withdrawal flare ( patients, . %). ymdd mutation developed in four patients. withdrawal flare occurred at a median . months after discontinuation of lamivudine. using log-rank test and cox multi-variate analysis, our results showed that the type of underlying malignancies (hr . , % ci, . - . ; p= . ) and baseline hbv dna titer (hr . , % ci, . - . ; p= . ) were significant independent risk factors for antiviral prophylaxis failure. conclusion: cancer patients with high viral load of hbv and hematologic malignancies may need more prolonged and potent anti-viral prophylaxis to avoid interruption or delay of chemotherapy. back ground: the usefulness of hepatitis b virus (hbv) dna and hbv core-related antigen (hbcrag) was evaluated for timing hepatitis flare after viral breakthrough or withdrawal of antiviral treatment in chronic hepatitis b. method: a total of events of hbv reactivation due to withdrawal of lamivudine (lam) or emergence of mutants resistant to lam or adefovir dipivoxil (adv) virus were analyzed in patients with chronic hepatitis b ( men, median age years [range: - ]). they were followed monthly for serum alt, hbv dna and hbcrag before, during and after the treatment. result: high alt flare (alt > iu/ml) after viral breakthrough or withdrawal was related with baseline hbeag positivity (p= . ), hbcrag level at hbv dna elevation (p= . ) and duration from hbcrag elevation to salvage therapy (p= . ). in multivariate analysis, hbcrag > . log u/ml (or . , %c.i. . - . , p= . ) and salvage therapy after weeks from hbcrag elevation (or . , %c.i. . - . , p= . ) were selected as related factor with high alt flare. after appearance of resistant-virus or withdrawal of lam or adv therapy, hbv dna re-elevated without increase of hbcrag, then hbcrag elevated with hbv dna. re-elevations of alt occurred in of the ( %) events. in of the ( %) events, alt re-elevated within weeks from the start of hbcrag increase. conclusion: hbcrag was useful for timing the re-elevation of alt after hbv dna re-elevation induced by drug-resistant virus or withdrawal of lam or adv therapy. background and objectives: the aim of the study was to observe the efficacy of a patient's therapy for switching lamivudine + placebo to adefovir dipivoxil (adv), and modeling the viral dynamics. methods: the studied object was a chinese chb patient with lamivudine mutation. used the lvd + placebo for weeks' therapy. then switched adv for another weeks. after that stopping the treatment and following up for weeks. based on our modified basic virus infection model, we introduce a personalized model consisting of four variables: x, y, v, e , representing uninfected cells, infected cells, free virus, and ctl cells, respectively. results: selected the model parameters, the simulation data of hbv dnas of our model are good in agreement with the clinical ones. observe that after weeks' treatment cessation, the benefit (hbv dna < copies/ml) for suppressing hbv replication can still be kept. numerical simulation show that if the patient's immune functions can be kept after therapy stops, it needs years to replace all infected cells by normal ones. conclusion: for lvd mutation patients, lvd+ placebo to avd therapy scheme may help patients to suppressing hbv replication. further researches are promising. acknowledgments: this work is jointly supported by the nnsf of china background: g-a- pre-core mutants (p-c-mt) cause hbeag-negative chronic hepatitis (chb) in genotype d infected mediterranean adults. we studied their emergence during chronic hbv infection in children. methods: eighty consecutive hbsag carriers ( / males/females, age y, range . - y) with vertical ( %) or intra-familial ( %) transmissions were followed-up for . y (range - y). hbv genotype and hbeag status were determined at the admission, hbeag/anti-hbe every years thereafter. during the follow-up, hbv-dna was measured in sera ( - sera/patient) (cobas-amplicor, roche); p-c populations were characterized by direct sequencing (ds), by oligo-hybridization (oha) and allele-specific-pcr (as-pcr) with %, % and . % sensitivities, respectively. results: seven children were genotype a and d; ( . %) were hbeag-positive. fifty-five ( . %) underwent hbeag/anti-hbe seroconversion (median age y, range . - y). baseline hbv-dna (cp/ml) was lower in seroconverters ( . + . vs , + , , anova p= . ). ds/oha p-c-mt were . % at the admission and . % after follow-up; as-pcr p-c-mt . % and % respectively. after seroconversion ( . %) became inactive carriers, ( . %) lost hbsag ( genotype d/p-c-wt); p-c-mt had chb. hbv-dna (cp/ml) was lower in p-c-wt than in p-c-mt inactive carriers ( . + . vs . + . ; anova p= . ). conclusions: in genotype-d infected children p-c-mt is selected progressively after hbeag/anti-hbe seroconversion to become predominant in hbeag-negative chb. early and efficacious immune control of hbv replication avoids p-c-mt selection and leads to hbsag loss. , a, , d, k, u, m, n , k ,k and k . here k , k , and k are the rate of ctl production and dead, killing virus, respectively. results: the patients with hbv dna levels less than copies/ml were reported in . % ( / ). a patient whose hbv dna levels were higher than copies/ml can keep treatment benefits even stopping the therapy for over ten weeks. the simulation data of our model are in agreement with the patient's hbv dna data. our simulation also shows that it needs to spend about years for clearing all infected cells. conclusion: the simulation result implies that some chinese patients may need long term's therapy to clear all infected cells. patients' ctls assays are needed to confirm the effectiveness of the personalized modeling, and help doctors to decide whether stop the drug treatment even patients' hbv dnas are higher than undetectable levels. background/aim: recent reports have shown that programmed death (pd- ) expression is associated with t cell exhaustion and persistent viral infection. we studied longitudinally chronic hepatitis b(chb) patients undergoing treatment with nucleos(t)ide analogues or pegylated interferon-(peg-ifn-) in - weeks to determine the relationship between pd- expression levels on t cells and early reduction of viral load induced by treatment. methods: our investigations were focused on three points: baseline (time point , t ), treatment weeks - (time point , t ) and treatment weeks - (time point , t ). pd- expression on total cd and cd t cells in chb patients during antiviral therapy was detected by flow cytometry. serum hepatitis b virus (hbv)-dna load was measured by real time polymerase chain reaction. results: between t and t , pd- expression on total cd (p< . ) and cd t cells (p< . ) dropped concurrently with treatment-induced hbv-dna decline(p< . ). between t and t , however, only the hbv-dna levels reduced significantly (p< . ). conclusion: early suppression of hbv replication induced by antiviral treatment results in a significant decrease in pd- expression on total cd and cd t cells in chb patients. c. zhao , , w. zhang , , x.c. tian , c.y. fang , , h.j. lu , , p.y. yang , , y.m. wen , background/aims: hepatitis b virus (hbv) is still regarded as one of the major causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. the interactions between hepatitis b surface antigen (hbsag) and host cells still remain largely unknown and need to be explored in detail. methods: differential protein expression profiles of hepg -s-g ( stably expressing hbsag cell line) and hepg -neo-f ( control cell line) were compared using two dimensional gel based differential proteomic approach. cell proliferation assay and survival assay were used for further studies on the candidate protein. results: compared with the control down regulation of proteins and up regulation of proteins were found in hepg -s-g cell. all these regulated proteins were identified by ms/ms and could be fell into several categories including metabolism-associated, immune-response-related, protein modification, signal transduction and others. among them, a group of proteins in putative pathways associated with apoptosis were found out and discussed, including glucose-regulated protein kd (grp /bip), heterogeneous nuclear ribonucleoprotein (hnrnp), far upstream element-binding protein (fusebp), rho gdp dissociation inhibitor (gdi), cystatin b and some scaffold proteins. grp , an important chaperone protein involved in multiple functions in host cells, was consistently decreased in hepg -s-g and in huh cell transiently transfected with hbsag expression plasmid. decreased crp inducing by hbsag or blockage of rnai consistently led to the less resistance to staurosporine-induced cell death. conclusions: these results revealed a possible pathogenesis induced by hbsag via grp . background/aim: to evaluate the efficacy of adefovir dipivoxil alone and in combination with lamivudine in treating patients with lamivudine-refractory hbeag-positive chronic hepatitis b. methods: eighty-five hbeag-positive patients who had received lamivudine treatment for various periods and had a lamivudine-resistant liver function abnormality, documented ymdd mutations and persistent viremia were randomized to adefovir dipivoxil mg, lamivudine mg, or addition of adefovir dipivoxil to ongoing lamivudine daily. the primary efficacy measure was virological response. the secondary efficacy measure was serological response (hbeag loss rate and hbeag seroconversion rate) and alt normalization rate. results: after weeks of therapy, mean reduction of hbv-dna level, the percentage of patients with hbv-dna lower than log copies/ml and the percentage of patients with hbv-dna level decrease of more than log copies/ml in patients of adefovir dipivoxil/lamivudine and adefovir dipivoxil monotherapy groups were significantly higher than those in patients of lamivudine group ( . , . log copies/ml vs. . log copies/ml, . %, . % vs. . %, . %, . % vs. . %; p < . , respectively). at the end of weeks, mean reduction from baseline in serum hbv-dna level at was . , . , and . log copies/ml in the lamivudine, adefovir dipivoxil/lamivudine, and adefovir dipivoxil groups, respectively. alt normalization rates were significant hihger in adefovir dipivoxil/lamivudine and adefovir dipivoxil recipients than those in lamivudine recipients ( %, % vs. %, p < . , respectively). a similar pattern was observed in hbeag loss among three groups. conclusions: adefovir dipivoxil is an effective treatment option for patients with lamivudine-refractory hbeag-positive chronic hepatitis b. aim: to find the prevalence of hbv virologic flare as defined by hbv dna viral load of > , iu/ml in inactive chronic hepatitis b (hbv dna less than , iu/ml), hbeag-negative patients who have not received any treatment and to identify if there are any predictors that can predict virologic flare. methods: we retrospectively analyzed medical records of the patients who have attended hepatitis clinic, siriraj hospital from january , to february , . the patients were eligible if they were naïve to any treatment and hbv dna less than iu/ml at entry. co-infection with hiv and/or hepatitis c virus were excluded. hbv dna measurement determine by roche amplicor ® (detection limit of iu/ml). hbv virologic flare was defined as hbv dna more than iu/ml during follow up period. result: there were patients with mean follow up time was days with annual prevalence of hbv virologic flare of . , . , and . % for the first, second and third year of follow up, respectively. initial hbv dna level was the only predictor that can predict reactivation. no patients with hbv dna at entry below detection limit developed flare and the patients with hbv dna above iu/ml had times higher chance to develop flare during follow up. conclusion: hbv dna flare is not uncommon in inactive chronic hepatitis b patients. most of the virologic flares occur in the first year. the most important predictor or virologic flare is higher hbv dna at beginning. background: multi-drug resistant hbv developed with multiple antiviral agents. there existed difficulty in dealing with multi-drug resistant hbv. methods: retrospective analysis of consecutive patients who exhibited chronic hepatitis b associated with multiple drug-resistant mutations to lamivudine and adefovir during antiviral treatment. multiple drug-resistant mutations were detected in those patients by dna direct sequencing. result: before multiple drug-resistant hbv emerged, patients accept sequential antiviral therapy, patients accept na monotherapies. there were cases of rta t/v rtm v/i mutation, cases of m v/i +n t mutation, cases of a t/v+m v/i +n t mutation, case of l m+a t/v mutation. cases received rescue therapy of interfronand hbv dna level of cases decreased; other cases received combination treatment and hbv dna level of cases decreased. conclusion: the main reason of multiple drug-resistant mutations was sequential antiviral therapy. another reason may be pre-exist drug-resistant mutation before nucleoside or nucleotide analogue treatment. de novo combination of antiviral agents should be recommended. combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant hbv. interfron may be one choice for hbv of multiple drug-resistant mutations. background: to furnish basis for an accurate evaluation of hbeag negative chronic hepatitis b (e chb), the present study studies the clinical features and hepatic pathology, and analyzes the relation between the data and the grade and stage of hepatic pathology in e chb. methods: a study is performed in chinese e chb patients ( men and women; mean age sd, . . years). the relationship between the clinical features and the grade and stage of hepatic pathology was analyzed by spearman's rank correlation test or kruskal-wallis test by applying stata . software. result: negative correlation is shown between the grade and leucocyte count methods: patients with hbeag-positive compensated chb with hbv dna > log copies/ml, serum alt x uln were divided two groups:one treated with telbivudine and the other treated with entecavir. results: baseline characteristics were well balanced between treatment groups. at wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively . (p . ), the rates of hbeag negative were respectively, the rates of hbeag seroconversion were respectively; at wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively (p . ), the total rates of hbeag negative were respectively, the total rates of hbeag seroconversion were . . respectively(p . ).no adverse reactions were found in both groups conclusion: there was no significant difference in hbv dna undetectable rates between two nucleotide analogs in short-term ( weeks).the telbivudine group has better effect in hbeag seroconversion rate than the entecavir group in early stage,but no statistical significance. j.w. song , , z. xin , j.x. tang , l. yao , b. wu sun yat-sen university, zhuhai sinochips biosci. co.,ltd., china background: to develop a equipment free, and can be widely used in clinical practice biosensor-based microarray for hepatitis b virus pre-c/bcp mutation assay. methods: a thin film optical biosensor were applied for amplification the microarray signal in situ. and hbv sites , , , and were selected as the targets and the microarray were be fabricated. the mutated plasmids contained , , , and sites and hbv sera were be tested in our study and all the plasmids and sera pcr products were be assayed by really time pcr and sequencing. results: the biosensor based microarray signal can be easily record by digital camera or even by the naked eyes and the detection signal for positive discriminated from negative were sharply contrasted as whole yes or no and it looks be significantly superior to classical microarray technique; . the sensitivity of the detection limitation of sera hbv load is x e copies/ml with % reproducibility. the concordance index of times negative and mutated plasmids were %(kappa= . ). . sera samples of hbv> e load and sera of hbv negative tested by pcr fragment sequencing were showing very good agreement between sequencing with our biosensor based microarray and the concordance index kappa was . . conclusion: our biosensor-based microarray for pre-c/bcp mutation assay were a both sensitive and accurate method. and its advantages of equipment free, sharply contracted signal of positive vs negative and easily be perform in testing were make it be a promised assay for clinical application. objective: to investigate the frequencies of cd + cd + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b, we assayed the differences among hbsag-positive and healthy subjects by flow cytometry. the results might offer some experimental evidence to explain the high rates of hbv persistent infection in vertical transmission of hbv from hbv-infected mothers. methods: newborns born from hbsag positive mothers were recruited , healthy subjects being used as a control group. the cord blood and peripheral blood of mothers were collected respectively .frequencies of cd + cd + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b were analyzed by flow cytometric analysis. result: the number of cd + cd + regulatory t cells/pbmcs in the cord blood of newborns born from hbsag positive mothers . ± . significantly exceeded that in normal controls . ± . ,p . ;and newborns born from hbsag positive mothers presented a much higher fraction of cord blood cd + cd + /cd + . ± . than those in normal controls . ± . ,p p= . p . . conclusions: the results indicate that the proportion of cd + cd + regulatory t cells in hbsag positive mother cord blood was higher than those of healthy cord blood. objective: to study the clinical features of chronic severe hepatitis b with negative hepatitis b e-antigen (hbeag) and positive hepatitis b e-antigen (hbeag) methods: a total of in-patients with chronic severe hepatitis b were recruited into the study and divided into two groups according to the hbeag status. the serological chemistry data, hepatitis b virus (hbv) dna quantification data were detected, and morbility of cirrhosis, its complications and prognosis were also studied. results: of the in-patients, ( . %) patients were hbeag-negative. ( . %) patients were hbeag-positive. the ratio of hbeag-positive patients was significantly higher than that of hbeag-positive patients (p< . ).the average age of hbeag-negative patients was older than that of hbeag-positive patients (p= . ). the serum hbv dna level of hbeag-negative patients was significantly lower than that of hbeag-positive patients ( . ± . ) vs( . ± . ) log copies/ml (p< . ).the ratio of patients who had a serum hbv dna level less than log copies/ml in hbeag-negative patients was significantly higher than that in hbeag-positive patients ( . % vs . % ,p= . ). there was no significant difference in serological chemistry data, morbility of cirrhosis and its complications on infections, ascites, hepatoencephalopathy, gastrointestinal hemorrhage, as well as prognosis of the patients between those two groups. conclusions: the study suggested that serological chemistry data, morbility of complications and prognosis of the disease of hbeag-negative patients mimics that of hbeag-positive patients. the hbeag-negative patients had a higher level of age, while a lower level of serum hbv dna. to reduce the incidence of liver failure, more frequent monitoring and earlier antiviral therapy prone to be reasonable for chronic hepatitis b patients with negative hepatitis b e-antigen. background: the emergence of lam-resistant virus greatly limits the efficacy of therapy and induces the liver injury. the aims of this study were to assess the related factors of lam-resistant mutation in hbeag positive chb patients. methods: thirty-five patients carrying lam-resistant with hbeag positive were enrolled in this study. all of them underwent percutaneous liver biopsy, histological findings and had detectable viral load. age, viral load, levels of alt, types of mutation and hbv genotype was monitored. result: the median year of mutation found was months. . % were genotype c and . % were genotype b. the mutation of l i, l v, g l, l m, m v and m i were detected. the emergence rates were . %( / ), . %( / ), . %( / ), %( / ), . %( / ), . %( / ) respectively. the rate of patients with two or three mutation were much more than one or four mutation. . % patients were found to have significant histological findings, even had established cirrhosis. two had no histological finding. one had rtl i and rtm i. the other had rtl v, rtl m and rtm v. the number of resistant mutation has no significant finding with histological finding, basic alt level and basic viral load. conclusions: the emergence rate of l m, m v and m i were higher than that of l i, l v, g l in hbeag positive chb patients with lam-resistance. most of them have two or three lam-resistant mutation regardless of histological finding severity, level of basic alt and viral load. we must select the efficacious method to treat the patients with lam-resistant. objective: to investigate the therapeutic efficacy of foscarnet sodium in the treatment of patients with severe chronic hepatitis b. methods: forty four patients were randomly divided into foscarnet sodium treatment and placebo groups.each group consisted of patients, patients in foscarnet sodium group were treated with foscarnet sodium twice daily . g given by intravenous infusions ,in addition to general therapy for days.the other cases were treated without any form of antiviral therapy as control.all patients were followed up for months.the hbv markers, quantification of hbv-dna, serological chemistry data were measured at baseline , during therapy period and the end of follow-up period . results: clinical symptoms were improved in two groups patients, meanwhile alanine aminotransferase (alt) and total serum bilirubin (tbil) decreased. compare alt and tbil at the end of trentment, there were no significant differences between the two groups (p> . ). in foscarnet sodium treatment group, the level of serum hbv-dna descreased from ( . ± . ) log copies/ml to( . ± . ) log copies/ml (p< . ), the rate of hbv-dna descrease of more than two log was . % / . in the control group, the level of serum hbv-dna descreased from( . ± . ) log copies/ml to ( . ± . )log copies/ml, the rate of hbv-dna descrease of more than two log was . % / .a comparison of serum hbv-dna showed significant differences between the two groups(p< . ) conclusion: foscarnet sodium administered can inhibit hbv replication in treating severe chronic hepatitis b.it can rapid lower the level of serum hbv-dna obviously.but the relapse rate was . % in foscarnet sodium treated at the end of follow up period objective: evaluation of efficacy and safety of five years trail of entecavir for chronic hepatitis bpatients failed with lamivudine therapy in the chongqing area. methods: thirty-two eligible patients were enrolled who had documented lvd failure.in the double-blind phase,patients were randomized( : )to etv . mg/d (n= )and placebo (n= ) for weeks.in the open-lable phase ,patients received etv . mg/d for weeks.hbv-dna level,liver function tests,hbv serology and safety assessments were conducted. results: the mean reduction in hbv dna levels at week was logl copies ml in etv group compared to . logl copies ml in placebo group(p< ). the mean of hbv dna levels after weeks of etv treatment decreased to . logl copies m the proportion of hbv dna< log copy/ml raised from at baseline to . % at week ,to . % at week ,to % at week ,and raised to . % at week .there were two patients with hbsag seroconversion and four patients with hbeag seroconversion at the end of study. the mean of alt became normal at week and remained normal throughout week .there was one patient who had a severe adverse event during the trail. conclusion: the findings from this study demonstrated the antiviral activity and safety of etv in adults with chb who have failed lvd pe showing delayed response on t cells as increased on day .the mrna expression of il- and il- showed no response to hbv vaccine but highly regulated in tt after day (p= . , . ).myd andtraf (p= . )upregulated in hbv vaccine group followed by of ifn-( . )no change of ifn found in tt conclusions: i) hbv vaccine stimulates innate response by day which potentiates further cascade,peripheral dendritic cells plays significant role in generating immune flare follows myd pathway and releases ifn-.ii) whereas t cells marjory involved in tt showed delayed immune response.iii) identification of key factors at different time points may prove to be a novel model to study the initial events after vaccination. objective: to compare th /th cytokines' dynamic change and its clinical significance in hepatitis b e antigen-positive patients treated with telbivudine. methods: twelve hepatitis b e antigen-positive patients treated with telbivudine.the blood sample was collected at baseline, week , week , week , week and week and stored at - c; serum il- , il- , il- , il- , tnf-and ifn-were tested at each time point by cytometric bead array (cba), compare th /th cytokines' dynamic change at different time point in each group and compare th /th cytokines' dynamic change cross four different groups: complete response, partial response, non-response and break through . results:the level of th type cytokines in complete response group are obviously higher than the group of partial response non-response and breakthrough,but the level of th type cytokines are lower than the group of partial response, non-response and breakthrough. conclusions: th /th cytokines is essential for the regulation of the immune function of the body. after treated with telbivudine, the level of th -type cytokines in the complete response group increased significantly, while the level of th cytokines declined trend. a. soamni , s. somani , a. jain , v. dixit navjeevan hospital, suvidha, background: chronic infection with hepatitis b virus causes spectrum of manifestations ranging from asymptomatic carrier state (often inactive with low replication) to the development of cirrhosis-related complications.the characterization of asymptomatic state has not been done in this part of the country, which forms important objective of present study. methods: incidentally detected asymptomatic hepatitis b surface antigen positive (idahs) subjects having hbsag positivity for > month presenting to our liver clinic were enrolled after appropriate consent. detailed clinical, laboratory and sonographic evaluation was done. they were divided into two groups according to presence or absence of e antigen. group a -hbeag + (n= ) group b -hbeag -(n= ) results: most of our patients ( %) were young adults ( - years) with male to female ratio of . : . approximately half of our patients were detected during routine medical checkup, followed by family screening of contacts. most of our patients were asymptomatic, and fatigue was most common symptom found in %. all demographic and biochemical parameters other than ast & alt were comparable in both groups. among hbeag negative ( %) subjects, hbv dna level > copies/ml was found in %. subjects with positive hbeag as compared to non-replicative infection (antihbe positive and hbv dna negative) had more frequent elevation of transaminase levels ( % versus %, p< . ). antihbe antibody was positive in all hbeag negative subjects. mean age of seroconverted (antihbe positive) individuals was a decade older than hbeag positive. conclusion: from our study we can suggest that ongoing liver disease is present in approximately one-thirds of incidentally detected asymptomatic hepatitis surface antigen positive subjects previously referred to as carrier state. hbsag testing should be mandatory in all routine medical checkup and family and sexual contacts of index case should be screened. background and objectives: this research was carried out to determine the prevalence of hbcab among the hbsag negative first-time blood donors who had referred to khorramabad and borujerd centers for blood donation. materials and methods: this study was established on a descriptive cross-sectional basis in which hbsag test (elisa) was primarily performed on all of the donors having referred to khorramabad and borujerd blood centers; then, out of all those referred subjects, who were first-time and hbsag negative, were selected for furthur investigation. the information concerning age, gender, job, blood transfusion, and hbv vaccine injection was included in the questionnaire of the study. hbcab (total & igm) and hbsab tests were performed on the selected donors. data were collected and finally the prevalence rate of hbcab was determined. results: the results of the study showed that out of hbsag-negative first-time blood donors, only were hbcab+, from which were hbcab (total)+, and were hbcab (igm)+. were both hbsab+ and hbcab+, and were seropositive only for hbsab. conclusions: it was demonstrated that the first-time blood donors who are seronegative for hbsag marker will be easily identified through hbcab test if they are in the so-called core window period of the virus. meanwhile, this group of donors have been implicated as high-risk for transfusiontransmitted hbv infection. so, detecting this marker will remarkably reduce the chance of latent cases of hbv infection and help promote blood safety. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf-- gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf-- gene promoter and chronic hbv infection. method: meta-analysis was done of case-control studies in relation to tnf-- gene promoter, involving a total of chronic hbv infection cases and controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that - a heterozygotes (ga) had % decreased risk of developing chb with a borderline significance (or = . ; % ci: . - . ; p = . ). for the - a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = . ; % ci: . - . ; p = . ; and or = . ; % ci: . - . ; p = . , respectively) ( table ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with - variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf-- a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. method: hbv transgenic mice were randomly divided into physiologic saline group and matrine injection group. another normal mice at the same species and age with hbv transgenic mice were regarded as the normal group. the mice in matrine injection group were administrated at dosage of . mgkg - d - by intraperitoneal for days. the mice in physiologic saline control group and normal group were administrated normal saline with the same volume at same time. the contents of hbv dna in serum and liver were quantitated by pcr. and the spleens were separated for cultivating dendritic cells. the surface molecules of dendritic cells were tested by flow cytometry. ifn-mrna and tnf-mrna in liver were tested by rt-pcr. result: there was no significant difference of the serum hbv-dna level between physiologic saline and matrine injection groups. the content of serum hbv-dna after treatment showed a significant decrease in two groups. the content of serum hbv-dna in matrine injection dropped significantly as compared with that in the physiologic saline group. but there was no significant difference in the content of hbv-dna in liver between physiologic saline and matrine injection groups. the expression level of mhc-ii on dendritic and hepatic ifn-r mrna and tnf-a mrna showed a significant decrease in hbv transgenic mice than normal mice. in comparison with physiologic saline group the expression level of them in matrine injection group showed a significant increase. conclusion: matrine injection was effective on depressing hbv-dna in hbv transgenic mice. its antiviral action may be achieved through regulating mhc-ii on dcs surface and promoting the production of antiviral factor such as ifn-and tnf-. purpose: to stimulate non-specific immune response capacity as the main content of the study to explore the hbv-dna and non-specific immune responses in the relationship between the low response capability, methods: cases of asymptomatic carriers, double-blind, randomized into mycobacterium fu , lamivudine and traditional chinese medicine for the treatment group, mycobacterium fu with traditional chinese and lamivudine with traditional chinese medicine were in the control group, a total of weeks of treatment, follow-up six months after the termination of treatment. results: different treatment of hbv -dna effect of the existence of significant differences; p> . , the performance of different types of asymptomatic carriers negative rate of hbv-dna there is a significant difference; p> . , as well as the performance of the different types of asymptomatic carriers continued application a treatment plan presented hbv-dna rebound rate there is a significant difference; p> . , conclusion: hbv-dna and non-specific immune responses in response to the lower capacity, anti-hbv therapy is not associated with non-specific immune response capacity or improve is the anti-hbv drugs alone can not solve the asymptomatic carriers in anti-hbv therapy where the cause of the problem, solve the asymptomatic carriers in the anti-hbv treatment although the need for anti-hbv drugs with non-specific immune activation synchronous drugs on the basis of the joint application , but the simultaneous combination of two drugs rather than as a result of hbeag and hbv-dna can hbeag-positive asymptomatic carriers receive hbv-dna negative effect of the results. background: adefovir dipivoxil (adefovir) effectively inhibits both wild-type and lamivudine (lam)-resistant hepatitis b virus (hbv) replication and resistance to this drug is infrequent compared with lam. in this study, we tried to identify factors affecting the emergence of resistant mutants after adefovir monotherapy in lam-resistant chronic hepatitis b (chb) patients. methods: the subjects were chb patients with lam-resistance who had received adefovir for more than months (range - months). the initial viral response (ivr) was defined as hbv dna < . log copies/ml. the adefovir resistant mutant was assayed at baseline and every months during adefovir administration. results: ivr was observed in % of patients. the cumulative emergence rates of adefovir resistance were . % at months, . % at year, . % at years and % at years. in univariate analysis, factors contributing to the emergence of adefovir resistant virus were baseline hbv dna > log copies/ml (p= . ) and ivr (p< . ). the presence of precore mutation and type of ymdd mutants were not related. in multivariate analysis, only ivr was an independent factors affecting the emergence of adefovir resistant virus (p< . ). conclusion: ivr is a useful predictor for emergence of adefovir resistant mutants after adefovir monotherapy in lam-resistant chb patients. for ivr-negative patients, the change of therapeutic options such as add-on lam or switch to other drugs should be considered because of the high incidence of the emergence of adefovir resistant mutants. background: elevated hbv dna is strongly associated with the risk of disease progression. this study investigated the early viral suppression effects of etv and lvd in nucleoside-naïve chinese patients with active hbeag (+) chb. methods: this open-label study was conducted in major hospitals in china. at study entry all patients had hbv dna levels copies/ml, elevated alt ( . - xuln) and compensated liver function. patients received either . mg etv or mg lvd daily. hbv dna measurements were taken at baseline and at weeks , , and during treatment, using roche cobas amplicor assay (llod copies/ml). results: a total of patients were enrolled; / etv patients and / lvd patients completed weeks of treatment. at baseline, mean hbv dna levels were . . in etv group and . . log copies/ml in lvd group (p< . ). the mean change in hbv dna from baseline (log copies/ml) was - . ± ngo's/funding-agencies representative at apasl -conference need to address-this-issue. we ngo-representatives from developing-nations need exposure to research treatments used by european/american experts. do we all failed in addressing socio-economic issues of cancer-sufferers? we need to address these socio-economic issues of affected population in resource-poor-nations. background: a garlic derivative s-allylcysteine (sac) has anti-cancer effect in human prostate and colon cancers. we aimed to investigate the effect of sac and combination of chemo-drug on tumorigenesis and metastasis of liver cancer. methods: the orthotopic liver tumor model using a metastatic liver cancer cell line mhcc l labeled with luciferase gene was applied. sac was given at day after tumor implantation at mg/g/day, or mg/g/day combined with low dose cisplatin for weeks. tumor growth and metastasis were monitored by xenogen in vivo imaging system. hepatic stellate cell (hsc) activation and tumor-associated macrophage (tam) in the tumor tissue were detected by -sma and ed /ed staining. tumor micro-vessel density (mvd) and apoptosis were also analyzed. in vitro functional tests including proliferation assay, cell cycle analysis and apoptosis analysis were performed. results: tumor growth was inhibited by sac combined with cisplatin treatment at different time points accompanied by lower incidence of lung metastasis compared with other groups. the observation of xenogen ivis was confirmed by histopathological examination. the hsc activation by -sma staining in the liver tumors was suppressed by sac and cisplatin treatment accompanied with less tam infiltration. consistent with in vivo study, in vitro functional study also demonstrated that sac not only induced cell cycle arrest, apoptosis, and inhibited tumor cell proliferation, but also sensitized the anti-cancer effect of cisplatin. conclusion: sac treatment inhibited liver tumor growth and metastasis by inhibiting tumor cell proliferation, inducing apoptosis and sensitization of chemotherapy. background: anti-angiogenic therapy would be a promising approach against hepatocellular carcinoma (hcc). although a sorafenib has survival benefits in patients at advances stages of hcc, there seem to be several serious concerns to employ this agent for chemoprevention against hcc. branched-chain amino acid (bcaa) reportedly inhibits the incidence of hcc in patients with insulin resistance (ir). however, the possible mechanism is still obscure. the aim of the current study was to examine the effect of bcaa on hepatocarcinogenesis under the condition of ir, especially in conjunction with angiogenesis. methods: the effect of bcaa on the development of liver enzyme-altered pre-neoplastic lesions and angiogenesis in the obese diabetic otsuka long-evans tokushima fatty rats was examined. we also performed an in-vitro study to elucidate the possible mechanisms involved. result: treatment with bcaa markedly inhibited the glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions along with suppression of neovascularization in the liver. the hepatic expression of the vascular endothelial growth factor (vegf), a potent angiogenic factor, was also attenuated. bcaa treatment significantly suppressed the glucose-and insulin-induced in-vitro angiogenesis in the presence of vegf. these results indicate that bcaa exerted a chemopreventive effect under the condition of ir via suppression of vegf-mediated angiogenesis. conclusion: since bcaa is widely used in the clinical practice for patients with chronic liver diseases, this agent may represent a new strategy for chemoprevention against ir-based hcc in the future. background: krüppel-like factor (klf ) is a member of transcription factors. whether and how klf signaling pathways contribute to hepatocellular carcinoma (hcc) development and progression is unknown. this study investigated role of klf in hepatocellular carcinoma cell line hcclm proliferation, invasiveness and epithelial to mesenchymal transition (emt). methods: the expression of klf in different liver cell lines was detected by quantitative real-time pcr and immunocytochemistry. we used small interfering rna (sirna) to down-regulate klf expression in hcclm . the change of proliferation and invasive ability of klf down-regulated hcclm was investigated by mtt reduction assay and trans-well invasive assay respectively. the change of proliferation, invasiveness and emt related gene in klf down-regulated hcclm was evaluated by quantitative real-time pcr. result: klf protein expressed predominantly in the nuclei of cancer cells and its expression is positively correlated with metastatic potential of these cell lines. hcclm has the highest klf level. decreased klf expression can notably inhibit the proliferation (p< . , n= ), mobility and invasiveness of hcclm (p< . , n= ). we found that the mrna level of n-cadherin, fibronectin and vimentin is much higher than that of e-cadherin in hcclm . the expression of cyclin d , focal adhesion kinase (fak) and fibroblast markers including n-cadherin and fibronectin was obviously suppressed in klf down-regulated hcclm . conclusion: klf plays an important role in the process of hcclm proliferation, invasiveness and emt. background: insulin resistance (ir) has shown to play an important role in the progression of chronic liver diseases, including liver fibrosis development and hepatocellular carcinoma. the aim of this study was to elucidate the possible mechanisms of ir on the liver fibrosis development and hepatocarcinogenesis using obese diabetic otsuka long-evans tokushima fatty (oletf) rats. methods: to induce liver fibrosis, . ml/kg of pig serum was injected twice a week for weeks in the oletf and leto rats. in the hepatocarcinogenesis model, glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions were induced by a single injection of mg/kg of diethyl nitrosamine (den). we also performed in-vitro studies to examine the mechanistic insights. results: the liver fibrosis development and gst-p-positive pre-neoplastic lesions were both markedly accelerated in oletf. in the fibrosis experiment, -smooth muscle actin-positive activated hepatic stellate cells (hsc) also increased in oletf along with augmentation of the hepatic collagen content and transforming growth factor-b . in the den model, the neovascularization was up-regulated in oletf almost in parallel with the pre-neoplastic lesions development and a potent angiogenic factor, the vascular endothelial growth factor. our in-vitro study showed that both glucose and insulin stimulated the proliferation of the activated hsc and augmented the neovascularization. conclusion: these results indicated that the ir status directly accelerated the liver fibrosis development and hepatocarcinogenesis at least partly through the stimulation of activated hsc proliferation and hepatic neovascularization, respectively, in the rat. n. wakui , t. ikehara , r. takayama , m. takahashi , k. shiozawa , h. nagai , m. watanabe , k. ishii , k. iida , y. igarashi , y. sumino case: a years old man diagnosed with chronic hepatitis c regularly visited our hospital. in april of , ultrasonography revealed a tumor mm in diameter in s of the liver and another tumor mm in diameter in s / of the liver. the patient was hospitalized for further examination. computer tomography (ct) revealed that the tumor localized in s / presented a pattern of hypervascular hepatocellular carcinoma (hcc). for the tumor localized in s , the following were revealed. ) contrast-enhanced ultrasound findings: a tumor vessel passed from outside the tumor to the center of the tumor in the early vascular phase, then radiated in a wheel-like shape at the center of the tumor; parenchymal phase perfused imaging in the area produced a similar imaging obtained from the area surrounding the liver. ) ct: no tumor was detected. ) spio-mri (t weighted imaging): iso-low intensity images were obtained. although these imaging findings indicated fnh, the patient was hcv positive. in order to disprove the possibility of hcc, a biopsy was performed on the tumor at s in the liver. the resulting diagnosis was well-differentiated hcc. discussion: until now, a characteristic finding of fnh has been spoke-like vasculariation, which is considered diagnostically quite important. however, some recent cases of hcc have been reported to present fnh-like vascularization. from now on, when evaluating a tumor that presents spoke-like vascularization underlining chronic hepatitis, the possibility of hcc should be considered and a close examination may be needed. chronic infection with hcv is problem .clinical management of chronic hcv depend on extent liver fibrosis .liver biopsy gold stander an invasive procedure responsible for severe complications and sample variability interpretation. serum biomarkers for inflammation/fibrosis investigated to wave liver biopsy. diagnostic accuracy panel of non-invasive serum biomarkers for hepatic fibrosis (fibrosure , apri score, forn's score) versus liver biopsy. hcv patients subjected for: apri, forn's , fibrosure scores pcr quantitative hcv-rnaliver functions .lipid profile cbc . ultrasound guided liver biopsy. forns score; auroc ( . ) with % ci( . - . ) for(fof ) vs. (f f f ) while( . )with % ci( . - . )for(fof f ) vs.(f f ). cutoff(> . )sensitivity for significant fibrosis(f f f )and extensive fibrosis (f f )were ( %) and with low specificity ,with accuracy( %) and ( %)respectively.-apri score; auroc( . )with % ci( . - . )comparing(f f ) vs.(f f f )while was( . )with % ci( . - . )for( f f f )vs.(f f ).cutoff(< . ) had low sensitivity and specificity( %)with accuracy( %)for significant fibrosis and( %)for extensive fibrosis.-fibrosure(fibro-acti test); showed best auroc( . )in different fibrotic stages with % ci ( . - . ).cutoff(> . ) sensitivity( %)for significant fibrosis and( %)for extensive fibrosis while specificity( %)in all fibrotic stages. the ppv ( %)for significant and extensive fibrosis .npv and accuracy( %, %)respectively for significant fibroses,while it was ( %) for extensive fibrosis respectively.significant correlation between liver biopsy and fibro-test(p . )and acti-test(p . ).significant correlation between liver biopsy hepatitis activity score and apri (p . )and forns score (p . ). conclusion: forns score wasn't considered since does not discriminate between significant and extensive fibrosis. low sensitivity of apri prohibtes detection of minmal fibrosis and allow undetermined results. fibrosure classified all cases of chronic hcv sufficient to wave liver biopsy pe introduction: hcc is the th common cancer. global increase of hepatitis b and c infection, the incidence of hcc steadily increasing. egypt seroprevalence of hcv in nile delta - %. afp had limited sensitivity % and specificity % for small hcc. gpc- oncofetal protein over expressed in hcc. evaluating validity of glypican- as early detector of hcc.: healthy controls and hcv positive patients: patients chronic hepatitis c virus infection. patients compensated cirrhosis [child-pugh class a and b]. patients decompensated cirrhosis [child-pugh class c]. patients hcc. liver functions: alt, ast, bilirubin(t), albumin, gt.tumor markers: afp and gpc- .viral markers: hcv antibodies, hbs ag and hbc ab. the median value of gpc- in hcc, dc, cc significantly higher than chronic hepatitis and control groups. no significant correlation between afp and gpc- . auroc of afp . & auroc of gpc- . . the diagnostic sensitivity of afp ( ng/ml) % with ppv . %. the specificity % with npv . %. while the diagnostic sensitivity of gpc- ( ng/ml) % with ppv %. the specificity . % with npv %. combined serial approach of afp and gpc- improved specificity to . %. conclusion:gpc- although a serological test for early detection of hcc, showed limited specificity, where detected in different stages of chronic liver disease,it is oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc- improve prediction accuracy of hcc patients in those showing seronegativity to afp. results: patients with hcv infection (n= ) were significantly older (mean age, years) than patients with dual virus (n= , years) and hbv infection (n= ; years) (p< . ). the male-to-female ratio for hbv, dual virus and hcv group was . , . and . , respectively (p< . ). patients in the hbv group more often had higher total tumor volume (mean, cm ) than the dual virus group ( cm ) and hcv ( cm ) group (p< . ). no significant differences of the severity of liver cirrhosis, performance status, cancer staging and tumor cell differentiation were noted among the three groups. patients in the hcv group had a significantly poor survival in comparison to the hbv group only in the subset of patients with small tumor volume (< cm ) in the cox proportional hazards model (relative risk: . , p= . ). conclusions: dual hbv and hcv virus infection does not accelerate the speed of hcc formation in patients with chronic hepatitis b, and appears to have a modified course of carcinogenesis pathway diverted away from the biological behavior of hbv and hcv infection. background: patients presenting with hcc is not infrequent in our clinical practice. the aetiology vary ranging from hbv, hcv, nash and alcohol. the aim of this study was to see the aetiology of hcc in bangladeshi patients. methods: in this retrospective study, records of patients who attended our opd between july to august were reviewed. patients having hepatic sol and/or heterogeneous echotexture of liver on usg and/or ct scan were included. diagnosis of hcc was confirmed at usg guided fine needle aspiration cytology with or without elevated serum afp (> ng/ml). results: of the patients, % ( / ) had hbv infection. hcv infection was diagnosed in % ( / ). nash was responsible for % ( / ) cases, alcohol in % ( / ), while in the rest % ( / ) cases no specific aetiology could be established. conclusion: the study shows that hbv is the commonest cause for hcc in bangladesh followed by hcv. background: the aim of this study was to determine whether the hepatitis b virus (hbv) dna viral load and antiviral therapy is associated with hepatocellular carcinoma (hcc) recurrence. methods: this retrospective study involved patients who underwent hepatic resection or radiofrequency ablation for initial hcc curative treatment. the patients were divided into four groups. fifteen patients with low serum hbv dna levels ( log copies/ml) at the time of initial hcc treatment received antiviral therapy (lamivudine, adefovir, dipivoxil, entecavir) before hcc appeared (pre antiviral therapy group; pre-tg). thirty-four had low serum hbv dna levels without antiviral therapy (low virus group; lvg). fourteen had high serum hbv dna levels and received antiviral therapy after hcc appeared (post antiviral therapy group; post-tg). thirty patients had high serum hbv dna levels without antiviral therapy (high virus group; hvg). results: the cumulative hcc recurrence rates at years in the hvg, lvg, pre-tg, and post-tg groups were . %, . %, . %, and . %, respectively. there were significant differences in the hcc recurrence rates between the hvg and lvg groups (p = . ), and between the hvg and pre-tg groups (p = . ). the recurrence rate was lower, though not significantly, in the post-tg group than in the hvg group (p = . ). conclusions: not only hbv dna viral load but also antiviral therapy is associated with hcc recurrence. antiviral therapy before hcc appears is important for patients with high serum hbv dna levels to prevent hcc recurrence. background/aims: few reports have described methods for predicting prognosis in unresectable hepatocellular carcinoma (hcc) patients, especially those treated by repeated transcatheter arterial chemoembolization (tae). to determine risk factors for death and determine prognosis in patients treated with repeated-tae, we evaluated clinical data. methodology: we retrospectively analyzed clinical parameters of unresectable hcc patients treated with repeated-tae from january to december . tae was repeated when recurrence was diagnosed by tumor marker elevation and/or dynamic computed tomography findings. factors affecting survival were evaluated using multivariate analysis after univariate analysis. next, we combined the score for each significant factor into a single prognostic score, after which the results were compared with jis and clip score methods. results: multivariate analysis revealed that bilobular hcc, alpha-fetoprotein ( ng/ml), tumor invasion of the portal vein, tumor size ( cm), and albumin (< . g/dl) were related to poor prognosis, using those factors, we developed a new prognostic scoring system. the % survival period was . months for all subjects, while it was . , . , . , . , and . months for those with scores of , , , , and or over, respectively (p< . ), using our new system. clip score was not useful to predict prognosis, while jis score was better. however, subjects with jis scores of and were difficult to differentiate. conclusion: our scoring system was easy to perform and the results showed that repeated-tae was effective for unresectable hcc with a score of or less. local ablative therapies and intrahepatic pressure c. kawamoto , a. yamauchi , k. kaneko , n. miyagi , k. kani , t. aoyama , k. yakabi saitama medical center, saitama medical university, japan background: some of the unexpected recurrence observed after radiofrequency ablation (rfa) might be caused by increased intratumoral pressure. the present study examined the relationship between local ablative therapies and intrahepatic pressure. methods: a. basic study: under general anesthesia, laparotomy was performed on pigs. a leveen needle and a percutaneous ethanol injection (pei) needle were inserted into the liver and intrahepatic pressure was monitored using an invasive blood pressure monitor. ablation was performed as follows: . rfa. ) single-step method: after fully deploying the electrode, the power was initially applied at w, then increased in increments of w/min until power roll-off. ) multi-step method: the array was deployed in steps. at each step, the power was fixed at w until power roll-off. . pei. injection of ethanol ( ml). b. clinical study: we examined the multi-step rfa and pei for hcc. under local anesthesia, intratumoral pressure was monitored. . rfa. patients with a mean tumor size of . ± . mm were studied. . pei. in patients with a mean tumor size of . ± . mm, to ml of ethanol was injected per session. results : a. basic study: the intrahepatic pressures were: single-step method, . ± . mmhg; multi-step method, . ± . mmhg; and pei, . ± . mmhg. b. clinical study: intratumoral pressure was . ± . mmhg for rfa and . ± . mmhg for pei. conclusion: these results suggest that consideration of intrahepatic pressure is crucial in local ablative therapies. background: a late evening snack (les) is recommended for liver cirrhosis. however, no clinical study has evaluated the nutrition status and the effect of les in cirrhotic patients with hepatocellular carcinoma (hcc). we investigated the effect of les undergoing hepatic arterial infusion chemotherapy (haic) in patients with hcc. method: nineteen patients with hcc were enrolled. ten patients were les group, and nine were control group. in the les group, the patients received les supplementation with a branched-chain amino acid (bcaa)-enriched nutrient mixture. in the control group, the patients received ordinary food. there were no significant differences in relation to age, gender, etiology, child-pugh scores, tumor stage, clinical responses to haic between two groups. blood biochemical data, nutrition status using an indirect calorimeter were evaluated at before and at the end of chemotherapy. results: the non-protein respiratory quotient (nprq) and molar ratio of branched-chain amino acid to tyrosine (btr) were significantly improved in the les group but not in the control group. there were no significant differences in the area under the concentration curve for glucose between before and the end of chemotherapy in two groups. background & aims: hepatocellular carcinomas (hccs) often show hypoor mixed vascularity, and the prognosis of these relatively hypovascular hccs is not fully elucidated. cytokeratin (ck) expression profiles may also be useful prognostic indicators, and specifically ck may reflect metastastic potency in hccs. this study was to assess the prognostic implication of tumor vascularity and its relation to ck expression in hcc patients. methods: a total of patients who underwent surgical resection for hcc were enrolled. tumor vascularity was evaluated according to arterial enhancement pattern on ct scans and ck expression was evaluated using tissue microarray methods. clinicopathologic data were analyzed using kaplan-meier and cox proportional hazard model. results: during follow-up period, ( . %) patients experienced tumor recurrence. forty-five patients ( %) had hypovascluar tumor at the time of diagnosis, and they showed significantly higher positivity for ck expression (p= . ) and shorter disease-free survival (p= . ) than patients with hypervascular hccs. in addition, recurred tumors in these patients showed more frequently hypovascular pattern than in patients with hypervascular hccs (p= . ). hypovascularity at initial diagnosis and microvascualr invasion were independent poor prognostic factors predicting survival. following treatment of recurred hccs, hypovascular tumors showed poor response to transarterial chemoembolization (tace), which resulted in shorter overall survival than hypervascular tumors (p= . ). conclusions: these results demonstrate that tumor hypovascularity in hccs is associated with positive ck expression, early tumor recurrence, poor tace response and poor survival. therefore, tumor vascularity may also be a prognostic indicator in hcc patients. background: hepatic stellate cells (hscs) transdifferentiate to become extracellular matrix-producing myofibroblasts during liver injury. myofibroblasts can also promote invasion and metastasis of hepatocellular carcinoma(hcc). in this study, we determine gene expression changes in two different models of hscs activation and investigate whether induction-activated hscs(ihscs) gene expression changes are different from culture-activated hscs(ahscs). methods: hscs were isolated by density centrifugation and exposed to conditioned medium from rat hcc cell lines c f. twenty-seven thousands and one hundred gene expression between quiescent hscs(qhscs), ahscs and ihscs was analyzed by microarray and confirmed by real-time rt-pcr and western blot. results: sixteen hundreds and seventy-one probe sets were differentially expressed in ahscs, including genes that encode proinflammatory factors, adhesion molecules, cell surface receptors, signaling transduction and immune factors. seven hundreds and eleven probe sets were differentially expressed in ihscs. induction-activated hscs showed specific gene expression patterns including raf , rac , adam , wnt , mmp- and tnf, suggesting that hcc cells can specifically induce hscs activation. induction-activated hscs might play a important role in invasion and metastasis of hcc. conclusions: induction-activated hscs gene expression patterns are different from ahscs. culture-activated hscs does not properly regulate gene expression in hscs, suggesting that ihscs may be considered the model for the study of hscs biology in hcc. background: hepatocellular carcinoma (hcc) is a hypervascular tumor, and angiogenesis is important for tumor growth. ephrin receptors are related with vascular system development and the polymorphism of ephb in the carcinogenesis of digestive tract has been reported. our aim was to examine the polymorphsims of ephb with the occurrence of hepatocelluar carcinoma in korean population. methods: genomic dna was extracted from patients with hepatocellular carcinoma (hcc), healthy subjects. ephb polymorphism was determined by polymerase-chain reaction-based assays, and the association with hcc was investigated. results: with regard to ephb polymorphism, a/a genotype at rs , t/t genotype at rs , a/a genotype at rs , t/t genotype at rs and g/g genotype at rs were significantly associated with hcc but these were not associated with clinical characteristics of hcc. conclusions: five out of seven polymorphisms on ephb gene were statistically associated with hcc, in the korean population. therefore, more studies of ephb gene polymorphisms including various risk factors should be performed to use as genetic markers of hcc occurrence. background: we aimed to compare the results of hepatectomy for hcc in patients older than years old with those for younger patients. methods: clinicopathological data and outcomes for elderly patients and younger patients with hcc who underwent hepatectomy between and were retrospectively compared. results: although postoperative delirium was more common in the elderly group, there were no significant differences between the groups with regard to operative morbidity, hospital death, disease-free survival, and overall survival. the overall recurrence rate was significantly higher in the elderly patients with alcohol abuse than in younger patients with alcohol abuse. multivariate analysis revealed that preoperative alcohol abuse was a prognostic factor for elderly patients. conclusions: elderly patients with preoperative alcohol abuse should be followed closely, even after r surgery, because alcohol abuse is strongly correlated with postoperative recurrence and worse survival. background: little is known about the effect of transfusing fresh frozen plasma on the outcome after hepatectomy for hepatocellular carcinoma. methods: among patients who underwent curative resection between and , patients had perioperative transfusion with whole blood or packed red blood cells and fresh frozen plasma (group a), while patients were only transfused with packed red cells (group b), patients were only transfused with fresh frozen plasma (group c), and patients had no transfusion (group d). results: group c had significantly fewer postoperative complications and a shorter hospital stay than group a. preoperative coagulation was significantly worse in group c. survival was significantly better in groups c and d than in group a. conclusions: perioperative transfusion of fresh frozen plasma improves clotting factors without an adverse influence on the survival of patients with liver dysfunction undergoing resection of hepatocellular carcinoma. background: this study investigated risk factors for postoperative liver failure after resection of hepatocellular carcinoma to detect markers that could identify candidates for hepatectomy. methods: perioperative risk factors for liver failure after hepatectomy were analyzed in patients with hepatocellular carcinoma. results: liver failure occurred postoperatively in patients, of whom died. the hyaluronate/gsa-rmax ratio was a risk factor for postoperative liver failure by univariate analysis and was the only risk factor according to multivariate analysis. all patients who died had a hyaluronic acid/gsa-rmax ratio mg min/dl. conclusions: to reduce postoperative liver failure, preoperative planning should employ various measures of the hepatic functional reserve, including tests of both parenchymal and nonparenchymal liver function. the hyaluronate/gsa-rmax ratio can predict liver failure after hepatectomy, and a ratio greater than mg min/dl is a relative contraindication to liver resection. the patient was a -year old japanese man with chronic hepatitis c(ch-c) who achieved a sustained virological response(svr) to interferon(ifn) therapy. as a result the liver functions were normalized and the histological findings of the liver also improved. however, years after svr, mild liver dysfunction was noticed along with a marked increase of tumor markers. several modalities revealed huge liver tumors about cm in greatest diameter in the left lobe invading the bile ducts and another tumor about cm diameter in segment v. we performed liver biopsy and confirmed that this tumor was well-differentiated hepatocellular carcinoma (hcc). only mild fibrosis development could be observed in the adjacent non-cancerous lesions. we successfully treated these tumors with transcatheter arterial chemoembolization and stereotactic radiosurgery. recent studies revealed that the risk of developing hcc still exists even after svr. since most of hcc that develop in patients with svr are usually detected within years, several investigators speculate that hcc is already present but too small to be detected at the time of completion of ifn therapy. this speculation is not the case in our patient, since svr was achieved years ago and no hcv-rna could be detected when hcc appeared. therefore, another possible mechanism should be considered. an annual follow-up with strict surveillance program for hcc should be performed for more than years after the completion of ifn therapy. background/aims: in order to investigate the role and importance of oxidative stress as to carcinogenecity of hepatocellular carcinoma (hcc) we analyze the expression of -hydroxydeoxyguanosine ( -ohdg) in the liver tissue of the hcc patients with and without hepatitis viral marker. methods: patients undergoing hepatic resection for the first hcc from to were enrolled into the study. only the cases that took no alcohol or small amount of alcohol were enrolled. cases were negative for hepatitis b surface antigen (hbsag) and antibody to hepatitis c virus (hcvab) (nbnc group). were positive for hbsag and negative for hcvab (b group). were positive for hcvab and negative for hbsag and antibody to hepatitis b core antigen (c group). staining with hematoxylin and eosin (h&e) and berlin-blue, and immunohistochemical staining for -ohdg were performed using the non cancerous liver regions. the degree of -ohdg immunostaining was expressed as the labeling index, which means the percentage of positive hepatocytes per hepatocytes. results: the labeling index of -ohdg for nbnc group is . (± . ), significantly lower (p= . ) than that for b group . (± . ), and also lower (p= . ) than that for viral group (b group and c group)( . ± . ). the labeling index of -ohdg had no correlation with grading, staging, fatty and iron deposit among all cases. conclusions: there is possibility that oxidative stress might not associate with the carcinogenesis of hcc in some cases without hepatitis viral infection. background: no effective chemopreventive agent has been approved against hepatocellular carcinoma (hcc) yet. since neovascularization plays a pivotal role in hcc, an angiostatic agent is considered as one of the promising approaches. recently, it has reported that vitamin k (vk) and angiotensin-converting enzyme inhibitor (ace-i) exert anti-angiogenic activity. the aim of the current study was to elucidate the combination effect of the clinically used vk and ace-i on cumulative recurrence after curative treatment, especially in consideration of neovascularization. methods: vk (menatetrenone; mg/day) and/or ace-i (perindopril; mg/day) were administered for to months after the curative therapy for hcc. the cumulative recurrence and several indices were analyzed. results: a -month follow-up revealed that the combination treatment with vk and ace-i markedly inhibited the cumulative recurrence of hcc in association with suppression of the serum level of vascular endothelial growth factor (vegf); a central angiogenic factor. the serum level of lectin-reactive a-fetoprotein was also suppressed almost in parallel with vegf. these beneficial effects were not observed with single treatment of vk or ace-i for months. conclusions: the combination treatment of vk and ace-i may suppress the cumulative recurrence of hcc after the curative therapy, at least partly through suppression of the vegf-mediated neovascularization. aim: the aim of this study was to clarify the cilnicopathologic features and management of hepatocellular carcinoma (hcc) patients surviving more than years after hepatectomy. materials & methods: retrospective study was carried out on hcc patients who underwent curative hepatectomy between and . clinicopathologic factors in -year survivors and patients who died within years were compared. the prognostic factors affecting survival were examined among the -year survivors. results: there were patients who survived for more than years after initial hepatectomy, and of those patients survived for more than years after hcc recurrence. the overall -, -, -and -year survival rates were . %, . %, . %, and . % respectively. in multivariate analysis, absence of underlying cirrhosis, solitary tumor, alfa-fetoprotein less than ng/ml, and absence of microscopic vascular invasion were favorable independent factors associated with -year survival. negative hepatitis c virus antibody status was favorable independent factor associated with longer disease-free interval and survival after tumor recurrence. multimodal treatments such as repeat hepatectomy or percutaneous ablation led to improved survival after recurrence, compared with the survival after transarterial chemoembolization (p<. ). conclusions: the results suggest that patients without underlying cirrhosis who have a solitary hcc that does not demonstrate vascular invasion or high afp levels might survive for longer than years after the initial hepatectomy. close follow-up and multimodal treatment could contribute to prolongation of survival in such patients, even if cancer recurrence occurs. the history of the use of carbon ion radiotherapy (cirt) for treating hepatocellular carcinoma (hcc) goes back to , when clinical trials were initiated at the national institute of radiological sciences. we have already reported that cirt used for the treatment of hcc is safe and effective, and that it causes only minor liver damage. in a phase ii clinical trial, the local control and cumulative overall survival rates were % and % at years, respectively. however, the patients with tumor adjacent to the gastrointestinal tract are thought to be ineligible for cirt because of the high risk of radiation injury of the digestive organs. in order to extend the indication of cirt, we have challenged the cirt for such patients under the use of spacers. a case was a -year-old female with cm tumor in segment . in radiological findings, the tumor revealed typical enhancement pattern for hcc, and was near the ec junction. she had been judged ineligible for hepatectomy because of the high retention rate of indocyanine green. she could undergo the . gye/ -fraction cirt after the placement of gore-tex soft tissue patch under the laparoscopic procedure. up to the present date, no adverse effect due to the spacer has been occurred, and an apparent anti-tumor effect has been observed. this method seems to have a promising efficacy for extension of the indication of cirt to the patients with tumors adjacent to the gastrointestinal tract. background: previously we reported that high ubiquitination was marker of human hepatocellular carcinoma. on the basis of these finding, we firstly analyzed the effect of bortezomib(proteasome inhibitor) on human hcc cell line. we also reported that hhm/dip /gcip was early marker for human hepatocarcinogenesis. hhm was suggested to be a new tumor suppression gene, but the mechanism was not well confirmed. we analyzed change of hhm signal by bortemib. method and result: we used hcc cell line (huh , hlf, hepg ) . the inhibitory effect of bortezomib was evaluated using mtt assay. nm bortezomib significantly inhibited proliferation of hcc cell line. the inhibitory effect by nm bortezomib was similar with m cisplatin. on the other hand, bortezomib has no inhibit effect in isolated hepatocyte from rat. in this condition, we analyzed the expression of cyclin d , phospho-rb and hhm in hcc cell line by western blot analysis. expression of cyclin d , phospho-rb decreased, but hhm was increased with time. next we analyzed cell cycle by facs. bortezomib induced hcc cell line into cell cycle arrest in g /m. the transcriptional activity of hhm was also activated by bortezomib administration using ptimer-promoter-hhm plasmid. conclusion: bortezomib has specific anti-proliferative effect on hepatocellular carcinoma. the induction of hhm by bortezomib might be related with cell cycle arrest. bortezomib will be a useful drug for hcc. neovascularization is required for carcinogenesis of non-alcoholic steatohepatitis: experimental and clinical study m. kitade , h. yoshiji , r. noguchi , k. kaji , t. namisaki , y. aihara , h. background/aim: non-alcoholic steatohepatitis (nash) may progress to liver cirrhosis, and finally hepatocellular carcinoma. recent study suggested that development of hepatic angiogenesis correlates the risk for hepatocarcinogenesis in liver cirrhosis patient. we therefore examined the role of angiogenesis in the hepatocarcinogenesis of nash in both experimental and human study. methods: as an experimental nash model, zucker (z) rats, which naturally develop leptin receptor mutations, and their lean littermate (l) rats were fed a choline-deficient, amino acid-defined (cdaa) diet. in human study, patients with nash-related cirrhosis or pre-cirrhosis, regarded as high risk group of hepatocarcinogenesis, and with simple fatty liver (fl) were enrolled and underwent clinico-pathological examinations. immunohistochemical analysis of -hydroxy- -noneal ( -hne) and cd were employed for detection of reacrive oxidative stress (ros) and angiogenesis in the liver tissues, respectively. results: in experimental nash model, both groups showed marked steatohepatitis by feeding cdaa diet. in sharp contrast, the development of glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions and hcc could be observed only in the l-rats. the hepatic neovascularization was also significantly increased only in the l-rats. in human study, both nash and fl exerted a marked elevation of ros. in sharp contrast, significant development of hepatic neovascularization was observed only in nash, whereas almost no neovascularization could be observed in fl. conclusion: in conclusion, these results suggested that neovascularization might play a important role in hepatocarcinogenesis in nash. background: paternally expressed gene (peg ), which was an imprinted gene with an active paternal allele but silent maternal allele, was highly expressed in a great majority of hepatocellular carcinoma(hcc). the aim of this study was to generate transgene mice expressing peg in the liver under the control of mouse albumin (alb) promoter and study the integration, transcription, expression of peg gene in the transgenic mice methods: the linearized bp transgene fragments, which contained alb promoter and structural gene of peg , were microinjected into fertilized eggs of mice. then manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. all the newborn mice were screened and identified by pcr detecting genomic dna in tail tissue. as the transgene was driven by the alb promoter, we examined its expression in the liver of transgenic mice by rt-pcr and western blotting. results: the transgene fragment was microinjected into the male pronucleus of fertilized oocytes. the injected eggs were implanted into oviducts of pseudo-pregnant foster mothers, of which mice became pregnant and give birth to offspring. of them died from unknown reason. among the offspring, were identified to carry peg cdna as demonstrated by pcr, and peg transgene could be expressed successfully in the liver of the established transgenic mice. the ratio of transgene integration were . % ( / ) by pcr. conclusions: the peg transgenic mouse model should be valuable for studying the in viro function of this imprinted gene in hcc. background/aims: brivanib alaninate is the l-alanine ester prodrug of bms- , an oral selective dual inhibitor of vascular endothelial growth and fibroblast growth pathway receptors. it is being developed in treating hepatocellular carcinoma (hcc), a disease highly prevalent in asia-pacific region. this analysis investigated whether bms- exposure was different between asian and non-asian subjects. methods: a population pharmacokinetic (ppk) model was developed with data collected in subjects ( non-asian, asian) with advanced and metastatic solid tumors (including hcc) from clinical studies. potential effects of the following covariates on model parameters were examined: age, gender, race, and baseline body weight. model-based simulation was performed to examine bms- exposure in asian and non-asian patients following brivanib doses of mg qd (phase iii dose). results: the ppk of bms- was characterized by a -compartment model with first-order absorption and elimination. clearance was found to slightly increase with body weight (p< . ). however, effects of age, gender and race on clearance were not statistically significant. the median of apparent clearance in asian was . % lower than that of non-asians, which was adequately explained by % lower body weight in asians. there was substantial overlap in steady-state bms- auc of asian and non-asian patients, simulated based on their observed body weight distributions in these patient groups. conclusions: bms- pk can be adequately described by a linear -compartment model; exposures in asian and non-asian subjects are similar following brivanib doses of mg qd. background/aims: hepatic resection is the standard treatment for hepatocellular carcinoma. in some patients with multiple hcc, one-block resection can not be feasible due to either the tumor location or the reserved liver function. in this study, we attempted to analyze the outcome of multiple-site resection or combined resection and rfa in patients with multiple hcc. the prognostic factors for postoperative survival were also investigated. methods: among patients who received resection from january to august , patients had a radiologically detected multiple hcc. patients with multiple hcc were divided into: group a, patients treated with one-block resection (n= ) and group b, patients with multiple-site resection or combined resection and rfa (n= ). results: in group b, received multiple-site resection and underwent combined resection and rfa. the clinicopathological variables and postoperative complication rate were not significantly different between the two groups. the -year disease-free survival rates for group a and b were . % and . %, respectively (p= . ). the overall survival rates were also not significantly different ( . % vs. . %, p= . ). the multivariate analysis revealed that radiological tumor number , edmondsons-steiner grade (iii-iv) and indocyanine green retention rate at minutes> % were adverse prognostic factors for overall survival. conclusions: active treatments including multiple-site resection and combined resection and rfa showed similar treatment outcomes compared with one-block resection in patients with multiple hcc. the prognosis after treatment was associated with tumor number, tumor grade and icg r . background: nasopharyngeal carcinoma (npc) is endemic to southern china. mortalities are mostly associated with secondary metastases. novel treatments for npc metastases are thus urgently needed. we aim to test the efficacy of a physiologically stable gold compound, gold (iii) meso-tetraarylporphyrin a (gold- a), in treating intrahepatic npc metastasis in athymic mice. methods: twenty million of c - human npc cells were injected into the livers of athymic mice to induce primary tumors. gold- a was administrated by intraperitoneal injection. survival times, tumor volumes and degrees of metastasis of the animals were evaluated. intratumoral microvessel density was determined by immunohistochemical staining for cd . tube formation by ms mouse endothelial cells were conducted with an in vitro angiogenesis assay kit. gene expression level was determined by semi-quantitative reverse transcription-polymerase chain reaction. cell proliferation was performed by methylthiazolyldiphenyl-tetrazolium bromide assay. result: gold- a prolonged the survival and inhibited intrahepatic and lung metastasis of the tumor-bearing animals. the compound induced tumor tissue necrosis and reduced tumor microvessel formation. in in vitro studies, gold- a inhibited tube formation and proliferation of ms cells, and downregulated the expression of stanniocalcin (stc ), which plays roles in angiogenesis. furthermore, our preliminary data showed that overexpression of stc in ms cells rescued cells from gold- a-induced death. conclusion: gold- a is a novel anticancer agent that prolongs survival of the npc metastases-bearing mice. it inhibits intrahepatic and lung metastasis in vivo and inhibits angiogenesis in vitro, in part via downregulation of stc . tbx is a transcriptional repressor that is important for embryonic development. overexpression of tbx was found in a large variety of cancers, including breast cancer, ovary cancer, cervical cancer, lung cancer, bladder cancer and liver cancer. tbx promote carcinogenesis by bypass cellular senescence via suppression of p arf . our resent studies revealed that two key motifs composed of + residues are essential for its transcriptional repression. based on this finding, we designed a set of peptides to block its transcriptional repression activity and tested their antiviral effects. we found that tat-tagged peptides (taps) effectively transduced hepatoma hepg and bel cells at almost % efficiency and inhibited cell growth in a dose dependent manner. further studies revealed that the tap treated cells underwent up-regulate apoptosis via suppression of p arf both at mrna and protein levels, demonstrating the potential of novel taps for anti-hcc treatment in the future. safety and long-term outcomes of radiofrequency ablation therapy in elderly and cirrhotic patients with hepatocellular carcinoma k. kakisaka , h. kuroda , k. kasai , y. takikawa , k. suzuki iwate medical university background and purpose: a tendency of the aging in patients with hepatocellular carcinoma (hcc) is predominantly seen in japan. in fact, the mean age of patients with hcc in our institute in was . years old, while that in was . years old. it is not still remained whether the percutaneous radiofrequency ablation (rfa) therapy in elder patients with hcc is safety and equal in therapeutic usefulness compared to the non-elder patients with hcc. subjects and methods: two hundred six cirrhotic patients with hcc ( tumor nodules) received rfa therapy curative intent since august, were enrolled. we divided all patients into two groups: over years (elder group: n= ) and under years (non-elder group: n= ), and compared the patient's characteristics, tumor factors and survival rate and causes of death in two groups. results: the characteristics of patients, tumor factors, cumulative survival rate and recurrence rate were not revealed in two groups. although in elder group two patients complicated aspiration pneumonia and respiratory depression due to sedation under rfa respectively, total occurrence rate of complications did not differ between two groups. conclusion: rfa therapy is safety and effective even in elder patients with hcc, although their care is necessary to prevent any complications which are often occurred during the rfa therapy. background and purpose: the aim of this study is to evaluate whether administration of the branched-chain amino acid (bcaa) enriched nutrient (namely, aminoleban en, ostuka pharmaceutical company, japan) might improve protein-energy malnutrition (pem) status and quality of life (qol) in cirrhotic patients with hcc receiving rfa therapy. subjects and methods: thirty-five cirrhotic patients with hcc who had received rfa therapy from october to october in our institute were randomized into two groups: diet with supplementation of aminoleban en (en group: patients, kcal/day) and diet only (control group; patients). the total intakes of calories ( - kcal/kg) and protein ( . - . g/kg) were equal between tow groups. the primary end point was event-free survival rate (development of liver cancer, rupture of esophageal varices, or progression of hepatic failure) and second end points were serum albumin levels and the health-related qol by shortform- questionnaire (sf- ). results: total intakes of calories and protein were similar during the one year after rfa. no significant differences in event-free survival rate were seen between two groups. however, decreased serum albumin levels and one (general health perception) of domains in sf- were significantly improved in en group compared to the control group. conclusion: supplementation of bcaa-enriched nutrient may improve the impaired liver function and qol after rfa therapy. large scale prospective study should conduct to confirm these results near the future. backgrounds and aims to investigate the effects of selective cox- and cox- inhibitor on proliferation and apoptosis of hcc cell. methods hep b and snu cells were treated with ns- and sc- . mtt assay, caspase / activity assay and tunel assay were performed. cox protein and mrna expression were measured by western blot and real time rt-pcr. results in hep b cell line, cox- , cox- ( , , um) and combination ( + , + , + um) treatment after hr showed a significant dose dependent inhibitory effect on cell growth (p< . ). cox- , cox- ( um) and combination ( + , + um) treatment after hr significantly increased caspase / activity (p< . ) and induced apoptosis (p < . ). however, the combination treatment could not showed a additive effect to cox- or cox- inhibitor (p> . ). in snu cell line, cox- inhibitor and combination treatment showed a inhibitory effect on cell growth (p < . ) similar to hep b cell line but any of treatment could not induce apoptosis significantly (p > . ). in cox protein and mrna expression, snu cell line showed significant cox- predominency (p= . ) but hep b cell line showed cox- predominency (p= . ). conclusions in hcc cells, no additive effect of the combination treatment of cox- and cox- inhibitors could be anticipated. the apoptosis inducing effect of cox inhibitor could be different between hcc cell lines. more studies for the mechanism of different response to cox inhibitor between cell lines is needed. background: the aim of this study was to determine the maximum tolerated dose and recommended dose of combination chemotherapy with mitoxantrone and uracil/tegafur (uft) (phase i part), and to clarify its efficacy (tumor response, overall survival, and progression free survival) and safety in patients with advanced hepatocellular carcinoma (hcc) at the recommended dose (phase ii part). methods: patients eligible for study had histologically confirmed, chemo-naïve advanced hcc, who were unsuitable for resection, local ablation therapy or transcatheter arterial chemoembolization. the therapy consisted of mitoxantrone dosages ( , and mg/m /day) intravenously on day and oral administration of uft mg/m on day through day . the treatment was repeated every four weeks if there was no evidence of tumor progression or unacceptable toxicity. results: a total of patients were entered into the study. all had a good ecog performance status score of - . in phase i part, dose limiting toxicities occurred in all three patients (two patients: grade neutropenia, one patient: grade creatinine elevation) given mitoxantrone at dosage of mg/m /day, and the recommended mitoxantrone dosage was mg/m /day. among patients administered at the recommended dosage, one patient ( . %) achieved a partial response, patients ( . %) had stable disease and patients ( . %) had progressive disease. one-year survival proportion, median survival and median progression free survival were . %, . months and . months, respectively. the most common toxicities were grade - leucopenia ( . %) and neutropenia( . %). conclusion: mitoxantrone mg/m with uft mg/m /day is recommended dose. this regimen is generally well tolerated, but appears to have little activity for advanced hcc. these findings do not support its use in practice, and further trials with this regimen in patients with advanced hcc are not recommended. the study assessed the benefits of -d reconstruction of spiral ct scans for the diagnosis of and surgical guidance to large liver tumors or tumors at the hepatic hilum. we retrospectively analyzed cases of children with such tumors treated in past years.the patients were examined by -d reconstruction using slice spiral ct. in cases, the volume of tissue removed exceeded / the entire volume of the liver. in cases, the excised tissue represented less than / of the total liver volume, but the location of the tumor was adjacent to major hepatic vessels. pathological diagnoses included hepatoblastoma (n = ), hepatocellular carcinoma (n = ), mesenchymal hamartoma (n = ), teratoma (n = ) and adenoma (n = ). all children had curative resections with tumor-free microscopic margins. -d ct imaging can provide high quality images and accurate location of the tumors. it could help the surgeon identify the tumor borders accurately and devise a safe surgical strategy. with its help the surgeon could identify vital hepatic blood vessels before operation, and can avoid massive hemorrhaging during operation. background: to investigate the association between c- t polymorphism of transforming growth factor (tgf)- gene and hbv-related hepatocellular carcinoma (hcc). methods: patients with hbv infection ( cases were hbv carriers, cases were hcc) and healthy volunteers were enrolled. the polymorphism of tgf- gene c- t was identified by polymerase chain reaction-restriction fragment length polymorphism method. the concentrations of plasma tgf- were measured by enzyme linked immunosorbent assay (elisa). tgf- mrna expression was quantified by real-time pcr. a recombinant construct containing - c>t variant as promoter and cat as reported gene was transfected into hepg cells. the reporter gene cat was detected with elisa. results: the ct genotype at position - of tgf- gene prevailed in all three groups, the frequency of genotype cc and allele c at - in hcc were significantly higher than those of the hbv carriers and controls. the plasma tgf- concentration among the three genotypes did not show any significant difference in three groups. however, both the tgf- concentration and liver mrna levels were statistically higher in patients with cc genotype than in those with tt genotype in the hcc group. reporter gene cat was elevated when hepg were transfected with - c-cat recombinant construct compared to that with - t-cat one (p< . ) conclusion: the presence of c allele at position - may play an important role in the development of hbv-related hcc through influencing tgf- expression both at mrna level and protein level. background: to assess diagnostic value of n-glycan markers in identifying hepatocelluar carcinoma (hcc) from liver fibrosis after hbv infection. methods: a total of cases of hbv related liver fibrosis (n= ) and hcc (n= ) patients as well as matched healthy controls (n= ) were recruited. routine liver function and tumor markers were detected by automatic biochemistry or immunological analyzer. n-glycome of serum protein was profiled by dna sequencer-assisted fluorophore-assisted carbohydrate electrophoresis with a capillary electrophoresis-based abi sequencer. results: the abuncance of a single agalacto biantennary glycan (ng a f, peak ) was increased in liver fibrosis and decreased in hcc, while that of a branching triantennary glycan (na fb, peak ) was decreased in fibrosis and increased in hcc. the efficacy of the log ratio of above two n-glycan abundance [log (p / )] was similar to afp in differentiation hcc from fibrotic patients. with logistic regression analysis, the accuracy and sensitivity of the diagnostic model combining afp with n-glycan analysis(cscore b) were increased - % compared to afp. log(p / ) was even more powerful in monitoring the progresison of hcc with the specificity improved % and accuracy improved % compared to that of afp. besides, log(peak / ) was correlated well with other tumor markers and tnm stages. conclusions: the log ration of the abundance of a branching triantennary glycan (na fb, peak ) to a single agalacto biantennary glycan (ng a f, peak ) and the model combining afp with n-glycome markers are promising in hcc diagnosis and progression monitoring. the low incidence of tumor seeding and post-procedure bleeding after radiofrequency ablation (rfa) of hepatic tumors has been attributed to the use of thermocoagulation of the tract, which results in necrosis, upon electrode withdrawal. however, different investigators use different techniques with no experimental evidence of the effectiveness of a particular technique. objective: we aimed to compare the necrotic zone produced using different electrode withdrawal techniques. methods: eighteen tract ablation zones were created in ex vivo porcine livers by withdrawing an internally-cooled rfa electrode (cool-tip radiofrequency system, valleylab) - mm/second using energy-dependent ( vs. vs. vs. watts) and temperature-dependent ( vs. c) techniques. horizontal mathematical modeling suggests an impractical number of radiofrequency ablation (rfa) zones needed in order to ablate a medium-large hepatic tumor. however, overlapping rfa zones may increase the necrotic diameter disproportionately to that deduced from single ablation alone. objectives: to compare the necrotic diameter in single (group ), dual overlapping (group ) and dual non-overlapping (group ) ablation. methods: single (n= ) and dual (overlapping n= ; non-overlapping n= ) ablation zones were created in ex vivo porcine livers using cool-tip rfa electrodes. necrotic diameter was measured at the midpoint (maxd) of the single and the two distinct rfa zones of the dual ablation groups and compared with the necrotic diameter at the tip of the second ablation (maxd-o), corresponding to the point of overlap in group . the rfa electrode was withdrawn . and . cm before re-ablating for group and group , respectively. results: despite no difference in end-rfa temperature between groups (group = . + . cvs.group = . + . cvs.group = . + . c; p= . ), maxd was significantly greater (p= . ) in group ( . + . cm) as compared to group ( . + . cm) and group ( . + . cm), with no difference between group and group (p= . ). further proof of synergism between two overlapping ablations is that the maxd-o in group ( . + . cm) was larger than maxd of group (p= . ) and group (p= . ), and was similar to maxd of group (p= . ). conclusions: overlapping two rfa zones results in incremental increase in necrotic diameter compared to single and dual non-overlapping ablation. this may explain the discrepancy in the number of ablation zones needed between clinical and mathematical modeling studies. background: hepatocellular carcinoma (hcc) is the fourth most common cancer worldwide, main etiological factors being chronic infections with hepatitis b and c viruses. the present study was undertaken to evaluate the association of glutathione-s-transferase (gst) t and m null genotypes and microsomal epoxide hydrolas e(mephx) polymorphisms with hepatitis virus related hcc risk in indian population. subjects and methods: three groups of subjects were considered viz. control (n= ), chronic viral hepatitis (n= ) and hcc (n= ). pcr-rflp was used for this polymorphic study. genotype distributions between categories were compared using the test; odds ratios (ors) and % ci were calculated to express the relative risk. results: presence of gstm null genotype significantly (p< . ) decreased the risk for hcc development among chronic viral hepatitis subjects. however, gstt null genotype was associated with an increased risk for hcc by . and . times among control and hepatitis subjects respectively. in case of mephx, tyr his and his his genotypes significantly (p< . ) reduced the risk of hcc development in both viral hepatitis and control subjects. in case of mephx exon genotypes, arg arg imposed an approximate fold risk for hcc development in the two groups. combination of heterozygous mutant genotypes at mephx exons & also imposed around fold risk (non-significant) for hcc. conclusions: polymorphic forms of gst and mephx share an association with viral related hcc risk in indian population and should be further evaluated as the candidate genes to determine individual susceptibility for viral related hcc. background : the association between type diabetes mellitus (dm ) and hepatocarcinoma (hcc) has been identified in the last ten years. methods: to clarify the temporal relationship between dm and hcc and the possible effects of antidiabetic therapy on hcc risk, we recruited patients with hcc compared with control subjects without liver diseases and cirrhotic patients. results: prevalence of dm was . % in hcc, . % in cirrhotic and . % in control group. in univariate and multivariate analysis, the odds ratio (or) for hcc in diabetic patients were respectively . (ci . - . ; p < . ) and . (ci . - . ; p= . ). or in univariate analysis were higher in male than in female patients. in . % of the patients dm pre-exists the diagnosis of hcc from a mean time of . months. moreover, the insulin treatment was more frequent in diabetic hcc patients than controls and we report an or for hcc of . (ci . - . ; p= . ) in patients treated with insulin or sulfonylureas, and an or of . (ci . - . ; p= . ) in patients treated with metformin. conclusion: our study confirms that male patients with type diabetes mellitus have a significantly increased risk of hcc independently of other cofactor such as hbv, hcv and alcoholic abuse. dm is a pre-existing disease in most hcc patients and suggests that insulin and sulphonylurea treatments in dm are associated with an increased risk of hcc development, while metformin may have a protective effect. background & aims: over the last few years, techniques that allow systematic analysis of chromosome aberrations at a genome-wide level were applied to hcc. the purpose of this study is to apply gene loss expression profiling in the attempt to discover new related genomic regions not revealed by loh or cgh, and search the new tumor suppression genes for hcc. methods: primary hcc and corresponding non-tumor liver tissues were obtained from surgery. serologically, cases were with hepatitis b virus infection and cases were with hepatitis c virus infection. four non-viral infected tissues from four patients receiving surgical resection for hepatic adenoma or focal nodular hyperplasia.affymetrix genechip, u a, was used to compare the loss and gained gene expression in liver needle biopsy samples (n= ). results: after adjusting by chromosome arm length, p, p, p, q and q showed higher gene loss-expression ratio (>= loss / cm) in the comparison between normal samples and tumor samples; q, p and q showed higher gene loss-expression ratio in the comparison between tumor and non-tumor tissues. more than genes showed different loss expression level in this study. for example, cd was loss expression in all non-tumor samples comparing to four normal samples. ficolin and ficolin were loss expression in hcc samples with hbv infection and with hcv infection, respectively. conclusion: our results revealed the potential tumor suppression genes and the genomic region they harbored. further study is needed to validate the observation. background/aims: hepatocellular carcinoma is common malignancy in human, accounting for million deaths in the world annually. caspase , as an initiator caspase, is involved in the induction of apoptosis. survivin, a novel inhibitor of apoptosis is related to the ability to inhibit caspases and involved in critical steps of onset and progression of hcc with unfavorable prognosis. methods: to explore the possibility that the epigenetic alteration of caspase and survivin genes is implicated in the development and progression of hcc, promoter methylation of two genes was analyzed in cases of primary hcc by methylation specific pcr. the relationship between immunohistochemical expression of gene products and proliferative/apoptotic indices, and clinicopathologic parameters was also investigated. results: the methylation of caspase ( . %, / ) and survivin ( . %, / ) demonstrated a negative correlation with immunohistochemical expression of capsase ( . %, / ) and survivin ( . %, / ) (p= . and p= . respectively). methylation of caspase and immunohistochemical expression of its gene product was significantly correlated with apoptosis (p= . and p= . ). survivin nuclear immunoreativity revealed significantly correlated with proliferative activity of tumor cells (p= . ). by survivial analysis, the negative caspase expression and positive survivin expression showed worse prognosis in hcc, that was statistically insignificant (p> . ). conclusion: in conclusion, caspase and survivin may contribute an important regulatory mechanism for tumor cell proliferation and apoptosis, and may be prognostic predictors in hcc. injection was recently reported to be effective against hcc with pvi, though the therapy is not always applicable for the patients with arterial abnormality. therefore we tried combination therapy of transcatheter arterial cisplatin embolization and radiation, and will report the effectiveness and toxicity of the therapy. methods: the combined therapy was conducted in hcc patients with pvi. transcatheter arterial embolization with mg/kg cisplatin powder (ia call) was performed against intralobar lesions, followed by external radiation targeted for pvi ( gy in gy fractions). the following variables were evaluated with the survival rate: gender, age, viral etiology, child's class, performance status, and location of pvi. results: one ( %) patient showed complete response and another two ( %) partial response. two ( %) showed no change, and one ( %) showed progress of disease. the survival rates at six months among overall patients were . %. adverse events were limited to nausea and appetite loss. one of the patients with partial response underwent curative resection, and is still alive without any recurrence for days. conclusions: the combination therapy of cisplatin embolization and radiation is safe, effective and also feasible to the patients with arterial abnormality. this therapy is suggested to be a useful alternative therapy for the patients with extensive pvi. recently, the injection port has been used for hepatic arterial infusion chemotherapy (hai) in japan. hai is usually used for the treatment of multifocal bilobar tumors of the liver or hccs combined with portal vein tumor thrombosis (pvtt), not amenable to tace. this study examined the efficacy and toxicity of repeated hepatic hai using lipiodol suspension mixed with cisplatin powder. methods: from april to september , patients with inoperable advanced hcc were enrolled in this study. all received cisplatin powder ( mg) and lipiodol ( ml) suspension, with an intervening weeks interval. the drugs were delivered from an injection port. patients had hcc with pvtt, and had hcc without pvtt. patients with liver function of child grade a, of grade b, and of grade c were enrolled. result: the mean number of hai given during the follow-up period was . times. we found complete response in case, partial responses in , no change in , and progressive disease in . the overall response rate was . %. the -year survival rate was . % and the -year survival rate was . %. although patients had cisplatin-induced anaphylaxis, no severe adverse events (hepatic failure and renal failure) were observed. conclusion: chemo-lipiodolization using cisplatin powder delivered via an injection port provides some clinical benefits without severe adverse events in patients with far advanced hcc. background: recently, the antitumor efficacy of angiogenesis inhibitors is expected in the treatment to hepatocellular carcinoma. the gene expression relevant to the vascularization, which is a target of these inhibitors, has a difference according to each case and it is thought that it influences the therapeutic effect of them. however, there are still few reports of mrna expression of vascular endothelial growth factor (vegf) receptors in hcc. methods: the relative mrna level of vegf and its receptors (kdr and flt- ) was analyzed using quantitative rt-pcr in patients with hcc. matched samples of hcc (t) and non-tumor liver tissue (nt) were obtained by fine needle ( gauge) biopsy. results: gene expression level of vegf and flt- was significantly higher in hcc than nt (vegf; p< . , flt- ; p< . ). according to the clinicopathological findings, gene expression level of vegf and kdr in hcc was significantly high in hypervascular hcc compared to hypovascular hcc (vegf; p= . , kdr; p= . ). additionally, flt- tended to be expressed higher in hypervascular hcc than hypovascular hcc (p= . ). moreover, gene expression level of vegf, kdr and flt- tended to be higher in advanced-stage hcc than early-stage hcc. conclusion: not only vegf but kdr and flt- were highly expressed in hypervascular and advanced hcc. aims: fibrinogen-like protein /fibroleukin (fgl ) has been reported to play a vital role in the pathogenesis in mhv- (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo-and xeno-graft rejection by mediating "immune coagulation". fgl functions as an immune coagulant with the ability to cleave prothrombin to thrombin directly. therefore, this study was designed to examine the role of fgl in tumor development. methods: tumor tissues from patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (hcc) model on nude mice (established from high metastasis hcc cell line mhcc lm ) were obtained. results: hfgl was detected in tumor tissues from out of patients as well as tumor tissues collected from human hcc nude mice. hfgl was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, nk cells, and cd + t lymphocytes and vascular endothelial cells. hfgl mrna was localized in cells that expressed hfgl protein. fibrin (nogen) co-localization with hfgl expression was determined by dual immunohistochemical staining. in vitro, il- and ifn-increased hfgl mrna by - folds and protein expression in both thp- and huvec cell lines. one-stage clotting assays demonstrated thp- and huvec cells expressing hfgl had increased procoagulant activity following cytokines stimulation. conclusion: the hfg contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction. . the therapy was either terminated at the end of the first cycle in cases with progressive disease, or continued for at least cycles, when responses to treatment were evaluated by eastern cooperative oncology group criteria. results: of patients treated (male, %; median age, years), % had child-pugh a, and % had b. % had either metastasis or vascular invasion. % had metastasis and % had vascular invasion. on the basis of independent assessment, three ( . %) patients achieved a complete response, thirteen ( . %) had a partial response, and ( . %) had stable disease. there was no grade / drug related toxicities. median overall survival was . months. conclusion: combination therapy of ifn + -fu has modest efficacy in hcc. background: amt is a mixture of approved pharmaceuticals in low therapeutic doses (human insulin and chlorpheniramine) and herbal components (aqueous camomile extract). preclinical and phase i data in healthy volunteers showed a favourable safety profile for amt. this pilot study should examine efficacy and safety of amt in the patients with advanced hepatocellular carcinoma (hcc). methods: thirteen patients with advanced hcc (tnm stage iii-iv), who did not respond to existing therapy, were treated with i.m. amt at . ml/kg up to a maximum volume of ml twice daily for - months. primary study objectives: clinical benefit response (cbr). secondary objectives: safety of amt, tumor response according to who-recist criteria, quality of life (qol) and iimmunomodulatory effects. the effects were evaluated by cytokine production of pbmcs before and after the treatment. results: there were no significant safety issues. four and patients showed positive and stable responses for cbr, respectively. tumor response was pr, sd and pd. even in the patients with pd, and patients showed positive and stable responses for cbr. qol data showed clear improvement. immune monitoring demonstrated effects of amt on the functional immune parameters in about half of patients. in the patients with pr, histological examination showed tumor necrosis and many lymphocytes including plasmacytes infiltrating in the tumor. conclusion: these results suggest that a promising rate of patients with advanced hcc respond clinically to the amt treatment without significant safety issue and amt has some immuno modulatory capacities. background/aims: dysplastic nodules are important due to premalignant potential. the aim of this study was to evaluate the electron microscopic findings of liver dysplastic nodule in patients with liver cirrhosis. methods: a total of patients (mean age: ± years old, male ) with dysplastic nodules which suspected as malignant nodule (mean size . ± . cm) was enrolled from cases of liver cirrhosis undergone ultrasonography-guided biopsy from december to january . the etiologies of liver cirrhosis were as follows; alcohol ( patient), hepatitis b virus ( ), and hepatitis c virus ( ). results: hepatocytes showed rosette formation of regenerative hepatocyte or degeneration. the nucleus was round or oval shaped and the nucleus membrane was irregular. the nucleolus was prominent and clear, the mitochondria were crowded to one side in the cytoplasm with megamitochondria. glycogen granules and lipofuscin pigments were abundant. sinusoid formation was poorly developed and collagen fiber bundles were increased. the hepatocytes of rosette formation and bile ductules cell made of canal of hering, which was dilated and microvilli was decreased. the number of canal of hering was , which was composed of . ± . with hepatocyte and . ± . with bile ductule cell, respectively. there was no oval cell in the canal of hering, which was relatively well developed. schwann cells were clustered together in nerve plexus. therefore, these electron microscopic findings showed that dysplastic nodule was similar to early hepatocellular carcinoma. conclusions: this study showed that dysplastic nodule in liver cirrhosis is nearly identified to early hepatocellular carcinoma. dcp is an important risk factor for recurrence after radiofrequency ablation of single hepatocellular carcinoma -< cm in diameter r. kuromatsu , a. takata , n. fukushima , s. sumie , m. nakano division of gastroenterology, department of medicine, kurume university school of medicine background and aims: the aim is to analyze the risk factors for local recurrence + intrahepatic metastasis after radiofrequency ablation (rfa) and hepatic resection (hr) for single hepatocellular carcinoma (hcc) < cm in diameter. methods: between and , patients with single nodule < cm in diameter and child-pugh grade a were treated by hr and rfa, and recurrence rate and survival rate using kaplan-meier method, and important risk factors for recurrence using cox's proportional-hazards regression model were analyzed. factors used for multivariate analyses were age, gender, viral marker, tumor diameter, afp, afp-l , dcp, and platelet count. results: mean age was years old, m/f ratio was / , hr/rfa was / , and mean observation period was days. five-year survival rates, and -year local recurrence-free + intrahepatic metastasis-free rates were not significant between hr group and rfa group ( / %, / %). in rfa group, the only independent risk factor for local recurrence-free + intrahepatic metastasis-free survival was dcp (p= . ). tumor diameter was not significant for recurrence. in hr group, there was no risk factor for recurrence. in pathological analyses of hr group, dcp had a tendency to associate with microvascular invasion (p= . ). conclusions: rfa was effective for hcc < cm in diameter and dcp < mau/ml. hepatic resection should be selected for single hcc with dcp > mau/ml even though hcc < cm in diameter. background: radiofrequency ablation (rfa) is now a common treatment for small hepatocellular carcinoma (hcc). however, critical complications after rfa such as rapid intrahepatic dissemination have been reported. in this study, we investigated the method how to estimate the malignant potential of small hcc by dynamic ct before rfa. methods and results: firstly, hccs less than cm in diameter were analyzed. those tissues were classified into groups as followed, small nodular type with indistinct margin (type e), simple nodular type (type ), simple nodular type with extranodular growth (type ), confluent multinodular type (type ). in the type and groups, portal invasion over vp were observed more frequently than those in the type and e groups. at the next step, these hccs were classified into above-mentioned types by two radiologists according to the shape of early stain or defect of delay phase of dynamic ct before operation. the accorded rate was % between those classifications. next, patients, which had solitary hcc less than cm in diameter and treated with rfa, were classified into those types by dynamic ct before rfa. the recurrence rate and prognosis of those patients were examined. in the type and groups, the recurrence rate was higher and significant worse prognosis was showed than those in type and e group . conclusion: it was suggested that hcc with type and might process higher malignant potential and rfa should be carefully performed on those types of hcc. background/aims: hypoxia-inducible factor- (hif- ) is the central transcriptional factor in the cellular response related to various aspects of cancer biology, including proliferation, survival, angiogenesis, and extracellular matrix metabolism to hypoxia. il- became known to replace hif- functions in the other cancer cell lines. the aim of this study was to evaluate whether il- may induce angiogenic factors without hif- by inflammation signal of hypoxic condition. methods: hif- knockdown cell lines of hcc (huh and hepg ) were constructed by rna interference tools, and cultured under normoxia ( %o , hours) and hypoxia ( %o , hours) conditions. following transfection, the amounts of hif- , il- , angiogenic factors and matrix metalloproteinase (mmp) were examined using rt-pcr and western blotting, respectively. results: the expression of hif- , angiogenic factors, mmp, il- was markedly enhanced in wild types that were cultured under hypoxia, and the hypoxic induction of angiogenic factors and mmp was partially blocked in hif- knockdown hcc cell lines. nf-b inhibitor suppressed angiogenic effects by blocking il- activity. conclusion: these data suggest that il- induced tumor angiogenic factors in hif- knockdown hcc cell lines. background there were some reports that liver caner related to the levele of sera hbvdna our research focused on the relationship between the quantity of hepatocellular hbv cccdna , tdna and liver cancer. methods the samples included the liver tissue of chb patients (chb group) and the para-liver cancer tissue of primary liver cancer patients (phc group) the quantity of hepatocellular hbvcccdna, tdna were assayed by fq-pcr in both groups. result: the quantity of hepatocellular hbv cccdna in chb group was . ± . copys/cell higher than phc group( . ± . copys/cell), p= . ; the quantity of hepatocellular hbv tdna in chb group was . ± . copys/cell higher than that of phc group( . ± . copys/cell),p= . .; conclusion: the quantity of hepatocellular hbvcccdna, tdna can not be used as predictors of liver cancer for hepatitis b patients. hepatic cancer predominantly occurs in males. this is almost a commonsense to most of us. but the detailed mechanisms underlying such phenomenon are still not well-known. the average age of liver cancer patients are about - years old. so most female patients have udergone pregnancy at least one time. pregnancy is a very important event before or during the development of liver cancer in females. in this special period, not only sex hormones secrete in a strange manner, but also immune system functions in a special module which is very different from normal. so it is urgent to investigate the impact of process of pregancy on the development of hepatic cancer. in this study, female sd rats are randomly divided into two groups: pregance group and controll group. rats in both groups are injected iv diethylnitrosomine(a chemical carcinogen). in pregnance group, rats are raised together with male rats in : ratio( female, male) to make every rats undergo pregnancy. while in controll group, rats are coupled with spermaduct-ligated male rats. the size and amount of hepatic cancers in pregancy group are smaller and less than those in controll group. the survial rate is also significantly higer than that in controll group. we conclude that the process of pregnancy exerts an inhibitory role in the development of chemical induced hepatic cancer in rats. acknowledgement: this project was sponsed by the national natural science foundation of china. the number of the grant is : the use of alpha-fetoprotein measurement in detection of recurrent hepatocellular carcinoma after living donor liver transplantation n. yamashiki , , y. sugawara , , s. tamura , r. tateishi , h. yoshida , j. kaneko , y. matsui , n. kokudo , , m. omata organ transplantation service, department of gastroenterology, university of tokyo, department of surgery, university of tokyo background: the recurrent hepatocellular carcinoma (rhcc) after liver transplantation (lt) can occur in to % of transplant recipients despite with a careful patient selection. for the surveillance of rhcc, frequent measurement of alpha-fetoprotein (afp) and annual ct scan is commonly used. however, the usefulness of afp is not clear. we report the update of our experience using our surveillance protocol. methods: between and march , adult living donor lt were performed at the university of tokyo. among them, recipients with hcc in their explanted liver were subjected to analysis. we used monthly measurement of afp and des-gamma carboxy prothrombin (dcp) with annual dynamic ct scan. results: met milan criteria pre-operatively and did not. were incidental hcc. rhcc was experienced in patients at ( - ) months after lt. recurrence sites were graft ( ), lung ( ), bone ( ), and multiple organs ( ). rhcc was first suspected from elevation of tumor markers in ; afp in , dcp in , and both afp and dcp in . rhcc was confirmed with ct scan ( ) or mri ( ) in ( - ) months after the first sign of rhcc. when the cutoff level of afp> ng/ml was used, the sensitivity and specificity for rhcc were % and %. six cases were treated surgically of which two achieving prolonged survival. conclusions: although the confirmation of the rhcc sites required multiple imaging studies, afp measurement was useful as for the first sign of rhcc. purpose: to evaluate the therapeutic effect of heated ( c) lipiodol via hepatic artery administration in vx rabbit liver cancer model. materials and methods: thirty male new zealand white rabbits were randomly divided into groups with rabbits for each group. vx carcinoma cells were surgically implanted into the left liver lobe. the tumors were allowed to grow for weeks, and studies were performed until the diameter of tumors detected by ultrasonograph reaching to cm. under the anaesthesia, transcatheter hepatic arterial embolization was performed and doxorubicin-lipiodol ( ºc) ( ml), lipiodol ( ºc) ( ml) and control (physiological saline ( ºc) ( ml)) were injected into hepatic artery of the different groups. one week later, the volume of tumor was measured by ultrasonograph again. the serum of all rabbits was collected before injection and at and days after injection and the level of aspartate aminotransferase (ast) was checked. the survival period of groups of rabbits after treatment was also recorded. during the last course of their disease, the rabbits were given some analgetics to relieve suffering. results: the tumors' growth rate in lipiodol ( ºc) background/aims: hepatocellular carcinoma (hcc) is one of the male-dominant cancers, and hepatitis c virus (hcv) is one of the causes of hcc. it was reported that androgen receptor (ar) is expressed in hcc and its surrounding tissues. androgen signaling and ar may be involved in hepatocarcinogenesis. in this study, we investigated whether hcv interacts with androgen signaling in human hepatocytes. methods: hcv protein expression vectors were co-transfected with ar-expression vectors and ar-responsive element-driven reporter vector into immortalized human hepatocytes (ihhs) and human hepatoma cell lines. kinase inhibitors were used to examine the activation of the akt, mapk, and jak/stat pathways. real-time pcr and western blotting were performed. cell culture grown hcv (hcvcc) were also used, and angiogenesis was evaluated by tubule formation assays in human coronary microvascular endothelial cells in the presence of -androgen- -ol- -one. results: hcv enhances ar-responsive gene expression in the presence of androgen. hcv core protein has the strongest effects and induced ar activation associated with jak/stat signaling. hcvcc enhances vegf mrna expression and angiogenesis. conclusions: hcv core protein is an enhancer in androgen signaling and can be expected to play an important role in hcv-related hepatocarcinogenesis. background: to evaluate the therapeutic outcomes and the toxicity of the combination of arsenic trioxide and the chinese traditional jianpiliqi (jplq) formula in the treatment of advanced hepatocellular carcinoma (hcc). methods: patients with advanced hcc, not suitable for resection but with normal major organ functions, were enrolled to receive a therapeutic regimen consisting of intravenous arsenic trioxide ( mg / m ) administration from days - , and an oral administration of jplq formula twice daily from days - . each cycle was composed of days and treatment could expand up to cycles before evidences of intolerable toxicity or disease progression. result: one patient had partial response, one had minor response, showed stable disease and ( . %) had disease progression. total disease control rate was . %, median survival time was . months ( - . ms), and time to progression was . months ( - . months). the incidences of grade - abdominal distention and nausea/vomiting were . % and . %, respectively. increases in ggt occurred in patients ( grade , grade , and grade ) and increases in serum creatinine in patients ( grade and grade ), respectively. conclusion: compared with the single arsenic trioxide treatment reported in past literature, treatment by arsenic trioxide combined with jplq showed modestly higher anti-tumor activity and tolerable toxicity in patients with advanced hcc; its manageable toxicity and increased tumor response rate may offer a better treatment regimen, and deserve further investigation. aim: to investigate the effect of osteopontin (opn) expressions down-regulated by rna interference (rnai) on the invasion and metastasis of human hepatocelluar carcinoma (hcc). methods: hcc cell line (hcc-lm ) was transfected with the chemically synthesized small interfering rna (sirna) in study arm and with non-specific sirna in control arm. real-time pcr and western blotting were used to quantify the mrna and opn protein levels. the malignant phenotypes including cellular growth rates, colony formation and matrigel invasion activities of the hcc cell line were analyzed. results: in study arm opn mrna expressions decreased % and opn protein decreased % compared to those of blank arm. the number of formed colonies and migrating numbers of the cells in vitro decreased significantly ( . % and . % respectively) in study arm compared to these of blank controls (p< . ). the parameters in the control arm did not differ from those of the blank arm (p> . ). conclusion: the specific sirna was able to reduce opn expressions at both the mrna and protein levels and significantly diminished the invasiveness of hcc cells. methods: the expressions of mif and vegf in hcc and adjacent tissues were detected from patients. specific sirna targeting mif gene was synthesized, and transfected into the hcc cell lines (plc and hepg ) in study group and non-specific sirna was used in controls. the mrna and protein expressions of mif and vegf were examined by pcr and western blot. results: mif and vegf mrnas were overexpressed in the hcc tissues compared with adjacent tissues (rq= . ± . and . ± . , p . ). the mrna and protein expressions of mif and vegf of hcc cell lines significantly decreased in study group compared with controls (p . ). vegf mrna levels decreased . %± . %; . %± . % in plc, and . %± . %; . %± . % in hepg cells when disposed with sirna nm and nm. vegf protein levels also significantly reduced in study group p . . conclusions mif and vegf mrnas were overexpressed in the hcc tissues in vivo, and mif sirna was able to knock down the expressions of mif and vegf in hcc cell lines in vitro. y.y. li, y.c. zhang, y.j. zhou, y.m. wei aim: to identify tumor-associated genes by constructing transcription profiles of pure hepatocellular carcinoma (hcc) tissues and normal liver tissues with the combination of laser capture microdissection and microarray. methods: hcc cells and normal liver cells from resection samples of patients were laser capture microdissected. micro-rna was isolated from them for linear amplification then crna was tested with whole genome microarray. differentially expressed genes were screened. results: the quality control of this technique was satisfactory with rna integrity number> , a /a ratio for crna measurement= . ~ . and good pictures for microarray. compared with normal liver tissues, hcc had differentially expressed genes, with being up-regulated and being down-regulated genes respectively. among the top ten ranked up-and down-regulated genes (total ), genes were known as hcc differentially expressed genes, , known as other tumors expressed genes previously. four unknown tumor related genes (depdc b, aspm, fcn and bbox ) were detected in this study. conclusion: the combination with laser capture microdissection and microarray was effective in screening the differentially expressed genes of hcc. background/objective: young patients present with large hcc on initial presentation are not uncommon. our aim is to study the computed tomography(ct) imaging of hcc and the clinical features of this special group of patients. methods: hcc patients had ct imaging of liver peformed in a three year period, patients had ct imaging peformed at the time of initial hcc diagnosis in our centre and were selected. they were divided into three age groups: young patients with age </= year-old(group ), age to (group ), age > (group ) to study imaging and clinical factors. univariate and multivariate analysis by cox regression model done to look for prognostic predictive factors. results: infiltrative tumour in ct scans, symptomatic presentation, child's and tm staging are prognostic factors in hcc. conclusion: young hcc patients have larger infiltrative tumour in initial ct scans and more being symptomatic. age is not an independent prognostic factor. aim: to investigate the expression change of nk cells receptor nkg d from human peripheral blood in patients with primary carcinoma of liver and study the relationship between nkg d expression and cytotoxicity of nk cells. background/aims: lens culinaris agglutinin-reactive alpha-fetoprotein (afp-l ) is a specific protein produced by hepatocellular carcinoma(hcc), which is more valuable than afp in the diagnosis of hcc. aptamers are oligonucleotide ligands binding to target molecules sensitively and specifically, which are screened from a great capacity of synthetically oligonucleotide library by systematic evolution of ligands by exponential enrichment (selex). our aims were to select the aptamers against afp-l from a self-designed ssdna library for potential application in diagnosis of hcc. methods: a random ssdna library and its corresponding primers were designed and synthesized. aptamers against afp-l were selected by selex. individual aptamers were separated by polymerase chain reaction-single strand conformation polymorphism (pcr-sscp) analysis and characterized. results: a ssdna library of nucleotides with random nucleotides in middle were designed and used for the selection. the binding rate of library against afp-l was increased from . % to . % after round selection. seven aptamers (s to s ) were isolated, and their sequences in random region and secondary structures were different from each other. all aptamers could bind afp-l in a different extent, and the dissociation constants of s and s are nmol/l and nmol/l. conclusions: aptamers for afp-l are successfully screened out and could bind afp-l specifically. methods: flow cytometry was used to determine the number of nk cells and the expression of nk cells receptor nkg d from human peripheral blood in patients with case primary carcinoma of liver case hepatitis b cirrhosis case hepatitis b and healthy cases and enzyme mark instrument was used to detect cytotoxicity of nk cells in all cases. results: killing rate of nk cell for k cell,nkg d expression level of nk cells, and the number of nk cells in the patients with primary carcinoma of liver decreased significantly p< . compared with those in the healthy subjects and hepatitis b group ,and decreased a little compared with those in the hepatitis b cirrhosis (p> . ).the activity of nk cells showed a obvious positive-correlation with the number of nk cell and expression level of nk cell receptor nkg d. conclusion: the cytotoxicity of nk and the nkg d expression of nk cells decreased significantly from human peripheral blood in patients with primary carcinoma of liver .the activity of nk cells is closely related to the nkg d expression level of nk cells. enhancing the nkg d expression level of nk cell may provide a new idea for adoptive immunotherapy of primary carcinoma of liver. and alpha-fetoprotein afp in serum and tissues for primary hepatic cancer(phc). methods: sixty-six phc and cirrhotic patients were enrolled. in phc patients,male /female was : , age was . ± . .of them, patients were defined as stage a-a. in cirrhotic patients, male /female was : , age was . ± . . serumgpc was detected using elisa. serum afp was detected using electrochemiluminescence. the hepatic expressions of gpc and afp were measured using immunohistochemistry in phc and cirrhotic patients. results: the cutoff value of afp diagnosis for phc was g / l or more, afp positive in phc patients was . % ( / ); the cutoff value of gpc diagnosis for phc was ng / l or more, gpc positive was in . % ( / ), p = . . in a-a stage phc patients,the positive of gpc and afp was . % ( / ), ( / ), respectively,p = . . in serum afp negative or positive patients, the positive of gpc was . % ( / ) , . % ( / ), respectively,p = . . the relationship between gpc with age, sex, child-pugh grade, hbv infection, tumor size and metastasis were not observed.the positive expression of gpc and afp in hepatocellular carcinoma tissue was . % ( / ), . % ( / ), respectively, p = . . neither gpc ,nor afp in the paracarcinomatous and cirrhotic tissue, was expressed. conclusions: diagnosis of glypican- protein for primary hepatic cancer is superior to afp.gpc can be regared as a early marker to diagnosis phc. objective: to investigate the effects and the possible mechanism of curcumin on the proliferation and the invasion of human hepatocellular carcinoma in vitro and in vivo. methods: hcclm -rfp cell lines were maintained in dmem medium supplemented with % fetal bovine serum. the fluorescent areas of hcclm -rfp were photographed daily and repeated in consecutive days after curcumin treatment for obtaining cell growth curves. the cell morphologic changes were also observed. cell invasion experiment was performed with boyden chamber array. the rfp-expressing human hcc xenograft model in nude mice was established to study the anti-tumor effects of curcumin. the ctc was detected by facs. the expression of cyclind and mmp- was detected by sybr green real-time pcr. results: after incubation with m, m and m curcumin respectively for , and hours, the growth of hcclm -rfp was significantly inhibited and some morphologic changes were observed. the mean tumor size in nude mice treated with curcumin since day were significantly less than those of the control group(p . ). the mean metastasis area of lung and the number of ctc in curcumin group on day were remarkably less than in the control group(p . ). the mrna levels of cyclin d p . and mmp- p . in curcumin group on day were significantly lower than in the control group. conclusion: curcumin can inhibit the proliferation and invasion of hcclm cell line not only in vitro but also in vivo mainly by down-regulating the expression of cyclin d and mmp- in mrna levels. phosphorylated erk is a potential predictor of sensitivity to therapy with sorafenib in hepatocellular carcinoma -evidence from in vitro study z. zhang , y.h. wang background: sorafenib is the first agent that has demonstrated an improved overall survival benefit in advanced hepatocellular carcinoma (hcc) and thus sets the new standard for the first-line treatment of advanced hcc. however, it remains unresolved to predict the drug sensitivity in treating hcc with sorafenib. pretreatment perk level has been shown to be associated with favorable response to such therapy in a phase preclinical study, indicating that perk may be a potential biomarker for treatment of hcc with sorafenib. methods: the effects of sorafenib and -fluorouracil on cell proliferation were evaluated by cell viability assay in four types of hcc cell line (smmc- , mhcc -l, mhcc -h and hcclm ), with different metastatic potential and basal perk expression. levels of perk expression were determined by immunocytochemical analysis and quantification, along with western blot analysis. correlation analysis was carried out between the ic values of drugs and mean optical density values of perk. results: the basal perk levels increased stepwise in cell lines in accordance with their metastatic potential. sorafenib inhibited erk phosphorylation at a concentration between and m dose-dependently, while no changes were observed after -fu treatment. correlation analysis between the ic values and mod values of perk revealed that the effects of sorafenib were significantly correlated with basal perk levels (spearman r=- . , p= . ). on the other hand, the resistance to -fu were significantly associated with basal perk expression in these hcc cell lines (spearman r= . , p= . ). conclusions: in this vitro study, perk was confirmed to be a useful biomarker predictive of sensitivity in treating hcc with sorafenib. the raf/mek/erk pathway may be involved in invasion, metastasis and drug resistance to traditional chemotherapy in hcc. background: to investigate the dynamic expression of igf-ii and igfbp- and its alteration of bcl- in hcc. methods: hcc models were induced with -faa on male sd rats. morphological changes of livers were observed and the dynamic changes of liver or serum igf-ii, igfbp- , and bcl- were quantitatively analyzed by elisa. the expression and distribution of liver igf-ii were observed by immunohistochemistry. result: hepatocytes from granule-like degeneration to a typical hyperplasia to hcc and the progressing increasing of the levels of hepatic igf-ii after rats induced by -faa. the levels of igf-ii in hepatoma and sera were significantly higher than any of other groups. the positive relationship of igf-ii was found between liver and sera (p< . ). the igfbp- levels in hepatoma were significantly lower than that in other groups (p< . ) and the progressing increasing of the levels of hepatic bcl- expression during the course. the levels of bcl- in hepatoma tissues were significantly higher than those in normal and degeneration ones. the immunohistochemistry evidences indicated the positive expression and hepatocyte distribution of bcl- in rat hepatoma. conclusion: hepatic igf-ii, igfbp- and bcl- may participate in hepatocyte canceration and accelerate the occurrence and development of hcc. the expression of igf-ii and igfbp- could be useful molecular markers for early diagnosis and prognosis of hcc. background: this study was done to assess the etiological role of hepatitis b virus (hbv), hepatitis c virus (hcv) and aflatoxin b (afb ) in development of hepatocellular carcinoma (hcc) in bangladesh. it was also investigated whether alpha-feto protein (afp) and protein induced by vitamin k absence or antagonist ii (pivka-ii) has any diagnostic advantage over each other methods: fifty five histologically proven hcc patients were tested for serological markers of hepatitis b and hepatitis c, and afb -dna adduct. during the diagnosis, they were also investigated for liver function tests, afp pivka-ii. results: out of fifty five hcc patients, ( . %) were found positive for serological markers of hbv, ( %) for hcv and ( %) for both. eight cases ( . %) were negative for the markers of hbv and hcv. however, none had afb -dna adduct above normal range. both pivka-ii and afp is strong marker for hcc with satisfactory level of sensitivity and specificity; but pivka-ii is more sensitive ( . %) and afp is more specific ( %). conclusions: hbv and hcv is the major etiological agent responsible for the development of hcc in bangladesh. background: to investigate the influences on the malignant transformation of hepatocytes through the intervention of nf-b activation pathway. method: hcc models were induced with -faa on sd rats, thalidomide was administered intragastrically and rats were sacrificed fortnightly interval to the twelfth week. morphological changes were observed by he staining. nf-b expressions were detected by ihc. the relationship between nf-b expression and pathological characteristics in hcc and non-hcc were analyzed. results: rat hepatocytes showed vacuole-like denaturations at the early stages, then dysplastic nodules appeared at middle stage, and finally progressed to tubercles of cancerous nest, all of which were highly differentiated hcc. thalidomine can repress the morphologic change of liver cells. there were only punctiform denaturations at the early and middle stage; nodosity hyperplasy and minority atypical hyperplasia were found at the finally stage. the ihc results demonstrated that nf-b level was significantly higher than those in normal ones, and the nf-b level of livers in hcc was higher than those in thalidomide group. an increasing tendency of nf-b was found from normal to hcc. nf-b in hcc were significantly higher than those in nc. the nf-b levels with thalidomide intervence raised first and decreased later. nf-b expressions in hcc were higher than that in their non-cancerous tissues. no positive relationship presented between nf-b expression and histological differentiation grade or the number of tumor, and size of tumor. conclusion: decrease nf-b expression can inhibit hcc development and nf-b is expected to be a new molecular target of hcc therapy. method: the cellular distributions of vegf expression in hcc tissues were investigated by immunohistochemistry. the levels of total rna and vegf were quantitatively detected in hcc, their paracancerous, and distal cancerous tissues, respectively. simultaneitily, serum vegf were analyzed in patients with chronic liver diseases for clinical values. results: the positive expression showed palm-yellow or palm-brown granules and distributed in hepatocyte plasma of hccs. the incidence of vegf was . % in hcc tissues, . % in non-encapsulated hccs, and . % in hccs with extrahepatic metastasis, respectively. no significant difference was found between hepatic vegf and hcc diameter or differentiation degree. the specific concentration (pg/mg liver) of vegf expression was significantly higher (p< . ) in hcc than their paracancerous or distal cancerous tissues, respectively. the circulating vegf was abnormally elevated in hcc. if the cut off values was more than pg/ml, the incidence of serum vegf was . % in hcc, . % in chronic hepatitis, and % in liver cirrhosis, respectively. the combined vegf and afp can increase positive rate up to . % for hcc. conclusion: the vegf overexpression is a useful marker for vascular invasion and metastasis of liver tumors. background: hepatocellular carcinoma (hcc) represents a major health problem world wide. it accounts for % of all primary liver cancers and is the fifth most common malignancy ( ). objectives: evaluation of radiofrequency thermal ablation versus transarterial hepatic chemoembolization with the effect of viscum (fraxini ) on tumour recurrence. methods: patients with hcc were enrolled in the study. group include patients and were treated with radiofrequency thermal ablation ( patients of them received viscum by subcutaneous route for years). group included patients with hcc and were treated by tace ( patients of them received viscum subcutaneously for years). results: group patients showed total ablation in % with persistant inactivity during years follow up. group did not show significant difference from group as regards relapse rate nor the performance status. complications as nausea, vomiting, fever, jaundice, and elevation of transaminases were significantly more encountered with tace. viscum did not significantly arrest tumour recurrence. conclusion: non surgical patients with hcc can achieve curative treatment with radiofrequency with minmal side effects. tace is a palliative treatment option for large hcc. a new technique had been attempted to increase the field of radiofrequency ablation of expandable electrode needles in the treatment of hepatic neoplasms much larger than the routinely covered size of - cm according to the needle size overcoming the technical difficulties usually met with in the overlapping balls technique due to the hyperechoic focus that develops at the needle tip making reinsertion difficult and inaccurate. in this technique, two or three needles were inserted from the start into the mass with accurate estimation of the exact field of ablation of each needle trying to cover the whole extent of the mass before application of radiofrequency waves. patients were included in the study, all presented with hepatic neoplastic mass lesion that range in size between and cm in its maximum diameter. all had a pretreatment helical (triphasic) ct study for accurate delineation of the whole extent and vascularity of the mass. two needles were sufficient to cover the whole extent of the mass in patients ( %) while in the remaining patients ( %) three needles were necessary. the procedure was done under general anathesia and ultra sound guidance, patients tolerated procedure well with smooth recovery. no major complications. follow up spiral (triphasic) ct was done weeks after ablation revealed percentage of tumour necrosis of % or more in patients ( %), - % in patients ( %) while in the remaining four patients ( %) the percentage was - % necrosis. in conclusion this technique should be considered in the treatment of hepatic masses larger than the usual field of the needle. results: the median value of gpc- in hcc, dc, cc was significantly higher than chronic hepatitis and control groups. no significant correlation found between afp and gpc- . auroc of afp was . & auroc of gpc- was . . the diagnostic sensitivity of afp ( ng/ml) was % with ppv . %. the specificity was % with npv . %. while the diagnostic sensitivity of gpc- ( ng/ml) was % with ppv %. the specificity was . % with npv %. combined serial approach of afp and gpc- improved the specificity to . %. conclusion:gpc- although it is a serological test for early detection of hcc, it showed limited specificity, where it is detected in different stages of chronic liver disease, as it is an oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc- may improve the prediction accuracy of hcc patients in those showing seronegativity to afp. outcome of inoperable hepatocellular carcinoma patients receiving transarterial chemoembolization: retrospective analysis in an asian regional hospital w.m. yip , k.f. li , k.k. li , m.l. szeto background: hepatocellular carcinoma (hcc) is a common cancer worldwide causing substantial mortality. although surgical resection is a form of curative treatment in hcc, only a minority of patients is suitable for this treatment and the postoperative recurrence remains high. transarterial chemoembolization (tace) is a treatment option for inoperable hcc and it was proven by randomized control trials that tace can prolong survival in selected patients. the aim of this study is to evaluate the survival and the prognostic factors in patients with advanced hcc treated by tace. methods: seventy four patients with inoperable hcc diagnosed from january to december were analyzed retrospectively in this study. only patients with unresectable hcc or who refused operation were included. patients with advanced cirrhosis, extrahepatic metastasis or previously treated hcc were excluded. multiple host, tumor and treatment variables were analyzed in order to evaluate the predictive factors of favorable response to treatment and better survival. results: the median survival of the study patients was . days. the cumulative survival rates at year, year and year were . %, . % and . % respectively. by multivariate analysis, superselective cannulation performed in tace (hazard ratio: . , % ci: . - . , p= . ), embolization with gelfoam (hazard ratio: . , % ci: . - . , p= . ), treatment interval more than days (hazard ratio: . , % ci: . - . , p= . ), child-pugh grade b (hazard ratio: . , % ci: . - . , p= . ), and pre-treatment serum fp level (hazard ratio: . , % ci: . - . , p= . ) were independent predictors of survival. conclusions: survival of patients with inoperable hcc is still grave despite treatment. this study provided information in predicting the survival of patients with inoperable hepatocellular carcinoma treated by transarterial chemoembolization. result: age < , total bilirubin (tb) < . mg/dl, albumin (alb) . g/dl, prothrombin time (pt) %, platelet counts (plt) . /mm , single nodule, and type of treatment (surgery or local ablation therapy) were linked to increased survival at univariate analysis of clip - hcc patients. of clip - hcc patients, tb < . mg/dl, alb . g/dl, des-gamma-carboxy prothrombin (dcp) < mau/ml, absence of vascular invasion, and type of treatment were correlated with survival. the following factors were related to survival by multivariate analysis: clip - hcc patients; age, alb, single nodule, and absence of vascular invasion, clip - hcc patients; age, tb, alb, alpha-fetoprotein (afp) < ng/ml, dcp, absence of vascular invasion, and type of treatment. conclusion: age, albumin, vascular invasion were significant predictors of survival both clip - and clip - hcc patients. clip - hcc patients: single nodule; clip - hcc patients: lower levels of tumor markers and patients receiving promising treatment had a better chance of prolonged survival. the role of gross classification as the predictor of microvascular invasion in hepatocellular carcinoma. s. sumie , r. kuromatsu , k. okuda , e. ando , a. takata , n. fukushima , m. sata background; the presence of microvascular invasion (mvi) as the risk factor in hepatocellular carcinoma (hcc) is controversial. the aim of this study was to determine the outcomes and predictive factors after hepatic resection for hcc with mvi. methods; one hundred and ten patients who underwent curative resection for hcc were included in this retrospective study. the risk factors of these patients for recurrence-free and disease-specific survival were investigated, and the clinicopathological factors predicting the presence of mvi were also evaluated. result; multivariate analysis showed that cirrhosis and mvi were identified as independent risk factors for recurrence-free survival. the -year recurrence-free survival rates for patients with and without mvi were . % and . %, respectively. multivariate analysis showed that the number of tumors, presence of mvi, and im were identified as independent predictors of disease-specific survival. the -year disease-specific survival rates for patients with and without mvi were . % and . %, respectively. by univariate analysis, mvi was significantly associated with greater tumor size, gross classification, histological grade, and intrahepatic micrometastasis (im). gross classification proved to be the only independent predictive factor for mvi by multiple logistic regression analysis. the gross classification could be evaluated by preoperative imaging diagnosis. conclusion; mvi is strongly associated with recurrence and survival in hcc patients after curative resection. furthermore, gross classification of hcc can be helpful in predicting the presence of mvi. background: hcc is a common cause of cancer morbidity and mortality. pxd is a novel, low molecular weight, histone deacetylase inhibitor. this phase i study aims to determine dose limiting toxicity (dlt) and maximum tolerated dose (mtd). methods: patient eligibilities include unresectable disease, ecog , adequate organ functions. pxd was given intravenously on day - every weeks; dose levels were: (level ), (level ), (level ) and mg/m /day (level ). dlts are defined as grade hematological toxicity or grade / non-haematological toxicity during cycle (according to nci ctc v ), or treatment delay > weeks. the mtd is defined as the dose below which > of or > of patients experiencing dlt. results: patients were entered; level ( ), level ( ), level ( ) and level ( ). grade / / toxicities in cycle included: raised alt / / , diarrhea / / , abdominal distension / / , anaemia / / . a total of cycles were administered; overall grade / / toxicities: raised alt / / , bilirubinaemia / / ; cardiac ischaemia / / ; diarrhoea / / , abdominal distension / / , anaemia / / ; variceal haemorrhage / / ; hypercalcaemia / / ; hyperkalaemia / / ; hyponatraemia / / ; infection / / ; liver dysfunction / / ; muscle weakness / / ; abdominal pain / / ; prolonged qtc / / ; syncope / / ; seizure / / . there were sd and pd. conclusion: at the maximum dose of mg/m /day, mtd has not been reached. pxd is very well tolerated. sponsor: the division of cancer treatment and diagnosis, national cancer institute, usa. tumor thrombus (pvtt) is prone to be produced in the portal vein near the main tumor nodule for hepatocellular carcinoma (hcc) patients and its molecular mechanism is still unclear. in this study, we first established a hcc cell line named csqt- from resected tumor thrombus in portal vein in a patient with histopathologically proved to be a moderately differentiated hepatocellular carcinoma . this cell line was composed of polygonal shaped cells and its peaks of the chromosome number was and . study on stem cell biology in this cell line suggests that cd cells represent about one fourth of the tumor cell population and cd (+) cells possess a greater colony-forming efficiency, higher proliferative output, and greater ability to form tumor in vivo. with this cell line model and resected tumor thrombi specimen, we also studied the different expression of proteins in primary tumor and tumor thrombus and found proteins expressed differentially between primary tumor and the pvtt. from these proteins, annexinv, prx , cycb were selected for further analysis to find potential biomarkers of pvtt in hepatocarcinogenesis. for clinical study, we recommended a new tumor thrombus type system ( type i iv) according to anatomic features of portal vein and tumor thrombus of hcc developing modes, then evaluate this type system to predict prognosis of hcc patients. the retrospective data of hcc patients with pvtt underwent resection shows that the y, y, y overall survival rates were . , . and . for type i, . , . and . for type ii, . , . and . for type iii, . , and for type iv, respectively, suggests tumor thrombus type system may be helpful to determine treatments and prognosis of hcc patients with pvtt. polyprenol could decrease the risk of hepatocarcinogenesis in hbv g. kuznecova , , s. kuznecovs , , i. kuznecovs , background: over-expression of p-glycoprotein (pgp) is associated with liver cancer development from hbv . glycoprotein synthesis in malignant tissues is limited by dolichyl phosphate (dolp). the aim of the present study was to investigate the effect of polyprenol (pp) which provides a dolp substitute in regulation of n-glycosylation on pgp over-expression in the development of liver cancer in hbv infection. methods human hepatocytes, infected with hbv and human hepatocarcinoma hep b cell line were used. pgp was assessed by an immunohistochemical technique. dolp fractions were analysed by hplc methods. results it is confirmed that plasmatic membrans of hepatocytes cells contain , - , % of pgp (the total protein amount) as a resistance marker. hbv infected cells differ from normal hepatocytes in pgp content by - times and hep b cells differ by - times the study showed -fold dolp decrease in hbv infected cells and -fold dolp decrease in hep b cells. the investigations demonstrate that the situation can be changed by treatment with dolp and pp. the dolp concentration in hbv infected hepatocytes was returned to the normal level. it is established that dolp in the concentration - m aid - -fold reducing pgp in membranes of hbv infected cells. background: metastasis is one of the most complicated and major pathological processes responsible for poor prognosis of hepatocellular carcinoma. snail was recently highlighted as a critical transcriptional factor for tumor metastasis. method & result: real time rt/pcr and western blot analysis demonstrated that snail mrna and protein, respectively, were induced by -otetradecanoylphorbol- -acetate (tpa) in hepatoma cell hepg . blockade of gene expression of snail by antisense oligodeoxynucleotide and/or sirna technique can prevent not only the tpa-triggered emt/cell migration and growth inhibition of hepg but also tpa-induced down-regulation of e-cadherin and up-regulation of p ink b. moreover, the tpa-triggered promoter activation of p ink b was also prevented. on the other hand, two of the hepg clone overexpressing snail, namely s and s , had a scattered fibroblastic morphology and acquired higher motility than parental hepg . also, the proportion of g /g phase of s and s was higher than that of parental hepg , consistent with the longer doubling time of both cells. semiquantitative rt/pcr analysis demonstrated a greatly elevated gene expression of snail accompanied with decreased e-cadherin and increased p ink b in both snail-overexpressing cells. on the transcriptional level, p ink b promoter activity was . -fold higher in s as compared with parental hepg . furthermore, electrophoretic mobility of dna fragments encompassing proximal p ink b promoter can be retarded by incubation of nuclear extract of s . conclusion: our results demonstrated that snail play diverse trans-regulatory roles in hepg . notably, we suggested that snail may upregulate p ink b gene expression by directly activating its promoter. a. schmitt-graeff , r. fischer , m. grosse-perdekamp , o. skalli universityhospital freiburg , louisiana state university health sciences center, shreveport background/aims: synemin is an intermediate filaments (if) protein which affects the motility of several cell types by modulating the dynamic properties of alpha-actinin and f-actin. we have previously shown that synemin is expressed in resident hepatic stellate cells (hsc) and myofibroblasts (mf) in hepatic inflammation and fibrosis. in the present study we evaluated systematically the expression of synemin in a large cohort of western european hepatocellular carcinoma (hcc). methods: single and double immunolabelin for alpha-smooth muscle actin (sma), vimentin, cd , cd , cd , cea, cd , cellular retinol-binding protein (crbp- ) and synemin were performed on paraffin-embedded hccs and controls. results: synemin-positive hscs/ mfs were a hallmark of non-neoplastic fibrotic liver tissue at the border to the neoplastic lesion but were absent from normal controls. tumour cell plates of the trabecular and pseudoglandular types of hcc were covered by scattered synemin-positive cells outlining sinusoidal structures. a subpopulation of these cells showed features of pericytes while others resembled endothelium. this pattern correlated with the degree of differentiation and was not observed in poorly differentiated hccs which generally contained rare intratumoral mfs. conclusion: the presence of synemin-positive hscs/mfs in the vicinity of hccs suggests a possible contribution of mesenchymal cells to the promotion of liver carcinogenesis. since synemin expression is linked to motility, a migration of this cell type into the tumour and a differentiation in vascular mural cells may be implicated in sinusoidal remodeling and the expansion of the neoplastic population. a.s. butt , a. ahmed , s. hamid , w. jafri , h. ali shah aim: to estimate the prevalence of viral marker negative hcc and to compare the clinical, biochemical, histological, radiological characteristics and initial treatment response among patients with viral marker negative and viral marker positive hcc. methods: medical records of patients diagnosed to have hcc visiting aga khan university hospital, karachi during january to december were reviewed. patients were divided in to nbnc-hcc(those who have negative hbsag and anti-hcv antibody)and viral hcc(those who have positive hbsag and anti-hcv antibody)group. results: out of patients ( . %) had nbnc-hcc. over all mean age was . ± . years and . % were males. the proportion of hcc detected under surveillance was significantly smaller in nbnc-hcc group(p . ). there was no difference in distribution of age, gender, bmi, child score, bilirubin, serum albumin, prothrombin time and alfa feto protein in both groups. however, patients with viral-hcc were found to be more thrombocytopenic( . ± . vs. . ± . ,p< . ) and had hepatopulmonary syndrome. on liver biopsy greater proportion of moderate to poorly differentiated hcc was observed in nbnc group( . %vs. . %,p< . ). hcc measuring cm in diameter( . %vs. . %, p . ), non -solitary hcc(p . ) and portal thromboses(p . ) were strongly demonstrated in nbnc-hcc group. involvement of right hepatic lobe and extra hepatic tumor spread was greater in nbnc-hcc group but that difference was not statistically significant. out of patients who underwent for liver transplantation( . %),tace( . %),resection( %),ethanol ablation( %) and chemotherapy( %), poorer responses were observed in nbnc-hcc group (p . ). conclusion: hcc secondary to nbnc-cirrhosis is not uncommon. patients with nbnc-hcc tended not to be under surveillance that leads to diagnoses at more advanced stage and poor prognosis. background: hepatocellular carcinoma is a common malignancy in asia and is related to the high prevalence of chronic viral hepatitis. we examined the clinical features, treatments and survival rates in asian americans with hcc. methods: retrospective cohort study of hcc patients who presented to the ucla liver cancer center in los angeles, california, usa from september to december . results: two hundred and seventeen of ( %) hcc patients were male, % and % had hbv and hcv infection respectively, and % had cirrhosis. hcc patients detected by surveillance had smaller tumor sizes, more within the milan and ucsf criteria, lower hcc tokyo system scores and had improved , , year overall patient and disease free survival rates compared to hcc patients who presented with symptoms (p< . to p< . ). by multivariate analysis, independent predictors of patient survival were tumor volumes greater than cm (hr . , p= . ), afp per unit log increase (hr . , p= . ), hcc tokyo score per unit increase (hr . , p< . ), liver transplantation (hr . , p< . ), hepatic resection (hr . , p< . ), rfa (hr . , p< . ), tace (hr . , p= . ), and hepatitis b infection (hr . , p= . ). factors associated with disease free survival were age per year increase (hr . , p= . ), meld per unit increase (hr . , p= . ), liver transplantation (hr . , p< . ), and hepatic resection (hr . , p< . ). conclusion: hbv and hcv infection accounts for the majority of hcc in asian americans. hcc detected by surveillance resulted in treatments which improved overall patient and disease free survival. treatment of small ( cm) hcc tumours can be achieved by surgical resection and complete eradication always correlates with good patient's outcome, with low local recurrence and high survival rates. indeed, surveillance program for the early detection of small hcc tumour is imperative to facilitate curative treatment, and hence better survival. discovery of new blood-based biomarkers is obligatory and vimentin is a distinct novel small hcc tumour marker herein identified using proteomics. experimental design: a total of liver tissues were evaluated by -de analysis. differentially expressed proteins were unequivocally identified by maldi-tof/tof and validation of the best candidate from protein to gene levels. indirect elisa assay was developed to detect soluble vimentin from serum samples. results: vimentin was significantly over-expressed in small hcc tumours compared to non-malignant controls and maintained expression in > cm tumours using -de analysis. blind verification displayed over-expression of vimentin in both transcripts and proteins levels. soluble vimentin was significantly detected at high level in small hcc as well as in overt hcc tumours. receiver operating characteristic analysis showed vimentin exhibited . % sensitivity and . % specificity in detecting small hcc at a cutoff of ng/ml. combined diagnostic performance of soluble vimentin and serum afp increases the detection sensitivity and specificity to . % and . %, respectively. conclusion: in this context, over-expression of vimentin is associated with the favourable cm sub-class of hcc thus may potentially be used as an effective serum-based diagnostic marker for cancer surveillance in high-risk cirrhotic patients. purpose: our recent comparative oncogenomic analysis in mouse model has identified yap (yes associated protein) as a novel oncogene in hcc. however, its clinical significance is unknown. in this study, we aimed to investigate the clinical values of yap as an independent prognostic marker in hcc. experimental design: a total of hcc cases with retrospective clinicopathologic and follow-up data were recruited in this study. both tumor and adjacent non-tumor tissues were examined for immunoreactivity of yap expression by immunohistochemistry. clinicopathologic features and yap expression were investigated with pearson test. hcc-specific disease free survival and overall survival with yap expression were analyzed by kaplan-meier curves and log-rank test. cox regression was used to test the independence and magnitude of the effects. results: yap was found over-expressed in hcc ( . %) with nuclear expression pattern. positive yap immunoreactivity was significantly correlated with worse tumor differentiation grade (p= . ) and high serum alpha-fetoprotein (afp) level > ng/ml (p< . ). kaplan-meier plot and cox regression showed that yap was an independent predictor for hcc-specific disease free survival (hazard ratio, . ; % ci, . - . ; p= . ) and overall survival (hazard ratio, . ; % ci, . - . ; p= . ). conclusions: yap expression in hcc is correlated with tumor differentiation and serum afp level. it served as an independent prognostic marker for hcc. background: integrative analysis of global protein and mrna expression patterns could help researchers to understand cancer cell physiology without the need of any prior hypothesis. methods: we used a d-page approach to profile and compared the global protein expression profiles of hepatitis b virus-related hcc tissues, adjacent non-tumor liver tissues, normal liver tissues and hcc cell lines. subsequently, we established the bioinformatic tools for integrative analysis of gene expression and protein expression data. we compared the dysregulated protein list and the dysregulated gene lists obtained by meta-analysis of microarray gene expression data from research centers in different countries. results: we identified proteins dysregulated in hcc. hierarchical clustering analysis revealed that there was a progressive change of protein expression patterns from normal liver, adjacent non-tumor liver tissues, hcc tissue, then to hcc cell lines. according to the biological functions, the differential proteins could be classified into various groups, including heat shock protein, chaperone, kinase substrate, cell signaling, apoptosis regulation, transcription regulation, free-radical scavenger and metabolic enzyme. ontology analysis of the genes with consistent dysregulations at both mrna and protein levels identified specific pathways down-regulated during the progression of hcc. the inhibition of those pathways provides new insights in the hepatcarcinogensis and treatment strategies. results: in pre-s/surface regions, hcc patients had higher frequencies of pre-s deletions, amino acid substitutions at codon , , and in pre-s genes, at the start codon in pre-s genes, and at codon in surface genes. but they had a lower frequency of amino acid substitution at codon in pre-s genes than those without hcc. in bcp/precore regions, hcc patients had higher frequencies of c or g , a /t , t , and a than those without hcc. multivariate analysis showed that pre-s deletions, i t in surface gene, t /a , and a were independent factors for hcc. the hbv with a complex mutation pattern (pre-s deletion, t /a , and a ) rather than a single mutation was associated with hcc. patients with combined mutations of t /a and pre-s deletion, t /a and a , pre-s deletions and a , and t /a , pre-s deletions and a had a . , . , . , and . fold increased risk of hcc, respectively, compared to patients with wild-type at both or three genomic regions. conclusions: pre-s deletions, i t in surface gene, t /a , and a were independent factors for hcc. combination of these viral mutations appeared increasing hcc risks. high peritumoral expression of placental growth factor in hepatocellular carcinoma is a poor factor for survival after curative resection h.x. xu , x.d. zhu , p.y. zhuang , w.zhang , h.chuan sun background/aims: angiogenesis plays a significant role in the metastasis and recurrence of hepatocellular carcinoma (hcc). placental growth factor (plgf), which is one member of the vascular endothelial growth factor family, may have prognostic values in patients after curative resection of hcc. methods: expression of plgf was assessed by immunohistochemistry in tissue microarray containing paired peritumoral liver tissue and tumor from patients underwent hepatectomy for histologically proved hcc. prognostic values of plgf and clinicopathological factors were evaluated. result: plgf staining was mainly on the cytoplasm of tumor cells or hepatocytes. the mean integrated optical densities of peritumoral and intratumoral density of plgf were . ± . and . ± . respectively. peritumoral plgf density was significantly higher than that in tumor (p< . ), and this result was also validated in another cohort of patients by quantitative real-time reverse transcription-pcr (p= . ). intratumoral density of plgf was not correlated with common clinicopathological factors (eg, tnm stage, tumor size, microvascular invasion, intra-hepatic metastasis) or overall survival (os) (p= . ) and time to recurrence (ttr) (p= . ). however, peritumoral density of plgf, which was correlated with tumor size (p= . ) and intrahepatic metastasis (p= . ), was a prognostic factor for both os (p= . ) and ttr (p= . ). in multivariate analysis, peritumoral expression of plgf was also an independent prognostic factor for os (p= . , rr: . % ci: . - . ) and ttr (p= . , rr: . % ci: . - . ). conclusion: peritumoral expression of plgf in hcc patients is an independent risk factor for survival and recurrence, and may be a target of anti-angiogenic therapy in preventing post-operative recurrence. purpose: to further research rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection for treatment of advanced hepatocelluar carcinoma (hcc). methods: cases were treated with transhepatic artery chemoembolization (tace) + radiofrequency ablation (rfa) + introportal vein chemotherapy (pvc) + percutaneous ethanol injection (pei) (four combined group) and this method was compared with cases that were performed tace + pei (two combined group) . the serum level of afp was measured respectively after and months, ct scan and color doppler ultrasound were measured after treatment for six months. results: the serum level of afp declined in two groups after months. for treatment after six months, afp in four combined groups was rose lower than two combined group (x = . , p< . ). ct and doppler ultrasound examination, four combined groups was superior than two combined groups to the control in tumor shrinkage (x = . , p< . ) and blood supply x = . , p< . ), relapse and mortality are also less. conclusions: rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection is a safe, effective combined method and has less complication in treatment of advanced hcc. poster exhibition -hcv poster session, hall b on the average, hepatitis c virus infects . % of the population worldwide. in egypt, the prevalence rates reach % in some areas. ability of the virus to persist in about - % of infected individuals is related to the virus higher mutation rate. six major hcv genotypes have been identified. genotype seems to be confined to the middle east and central africa. extra hepatic syndromes have been reported in up to / of hcv patients. we aim in this study to determine the relationship between viral genotypes and specific extra hepatic haematological disease in patients with chronic hepatitis c. the study group included selected patients with chronic hepatitis c having various haematological problems. we studied hepatitis c virus genotypes using rt-pcr. we found among patients , genotype ( %) and patients genotype a ( %). patients ( . %) were diagnosed as chronic hepatitis c with associated thrombocytopenia, patients ( . %) were diagnosed mixed essential cryoglobulinemia(mec), patients ( . %) were diagnosed non-hodgkin's lymphoma, and patients ( %) were aplastic anemia. positive serum cryoglobulins level was found in patients ( . %).no significant correlation was found between the level of viraemia and specific haematologic disease, biochemical liver markers or liver enzymes (p> . ). we did not find correlation between hcv genotype and specific extrahepatic haemological disorder in hcv infected patients. several environmental, genetic and immunological factors may contribute in disease progression. results: in the targeted area shops of barbers were successfully interviewed and total questionnaires were filled by both groups. the mean age were found in both groups of barbers (n= ) and clients (n= ), . years. the both groups showed that there are no any drugs which can protect us from diseases. both of the groups were not vaccinated for hepatitis b diseases. regarding the care providers the barbers replied that they prefer registered medical practitioners and the clients generally prefer the hakeems. those who knew hepatitis as liver disease, were ( . %), out of barbers only ( . %) were knowing about hepatitis-b&c, when we enquired about routes of hbv& hcv transmission only ( %) replied correct routes of transmission in both groups. about hbv vaccination ( . %) were aware, only ( . %) were vaccinated against hbv. % barbers claimed for disinfection of instruments before shaving ( . %) claimed for use of new blades. in the sero-surveillance the hbv found was very low and hcv became epidemic ( . % - . %) respectively. conclusion: the both groups need awareness for transmission. the use of new blade for the clients reduces the burden of hbv and hcv. the study highlights the roles of male sex, older age, and genotype b in the progression from chc infection to hcc. patients with higher hcv viral load potentially tend to develop hcc; however, hcc occurrence could be prevented using antiviral treatment. these two points need to be clarified further by a larger study population with longer follow-up period. an approach have recently been described that retroviral vectors encoding t cell receptor (tcr) genes are used to redirect the specificity of normal peripheral blood lymphocyte (pbl)-derived t cells to recognize the tumor antigens. the therapy in which t cells have been genetically modified with tcr genes to recognize hcv would represent a novel approach for the treatment of hcv infections and hcv-related malignancies. we have previously shown that hcv+ liver transplant patients that have received hla disparate liver allografts have hcv reactive t cells of host origin in their peripheral blood that are restricted by the donor hla molecules. initial studies indicate that the tcrs expressed by hcv reactive t cell clones from these patients have relatively high affinity for their ligands. we have cloned and expressed two tcrs which mediate recognition of the - and - epitopes from the hcv ns protein. the results indicate that these tcr transduced t cells can recognize the wild type epitopes, as well naturally occurring mutant variants of these epitopes. most importantly, the tcr transduced t cells could also recognize hcv+ hepatocellular carcinoma cells. these data suggest this high affinity hcv-specific tcr might have potential new immunotherapeutic implications. background: factors associated with svr in patients without an rvr remains unclear. methods: hcv- ( for and weeks, separately) and hcv- ( for weeks, for weeks) patients were randomized to peginterferon-alpha- a and ribavirin for analysis. results: multivariate analysis showed that treatment duration and a complete evr were the strongest independent factors associated with an svr. a higher svr rate and a lower relapse rate were observed in the standard regimen group than in the abbreviated group in patients who had a cevr (table ). the best levels of viral loads in predicting cevr at week were < iu/ml (table ) . conclusion: it was crucial to achieve a cevr with adequate treatment duration in patients who failed to achieve an rvr. our aim was to evaluate the impact of some biochemical, histological and viral factors on both evr and svr in patients with genotype chronic hepatitis c (chc) treated with peginterferon plus ribavirin. patients and methods: we evaluated retrospectively naïve patients with chc treated with peginterferon plus ribavirin at standard weight-based doses for weeks. biopsies were assessed for inflammatory activity and fibrosis. steatosis was categorized by the proportion of hepatocytes per low-power field with fatty changes: > %, > - %, - %, > %. biopsies were also assessed for stainable iron using the brissot scoring system. all patients were evaluated for metabolic syndrome (ms) using the ncep-atp iii criteria. results: evr was achieved in / pts ( . %) while svr occurred in / ( . %). after adjusting for sex and age, independent factors that negatively interfered with both evr and svr were: fibrosis score, steatosis, iron score, homa-ir index and viral load. after excluding the patients with ms criteria (n= ), evr was observed in / ( . %) and svr in / ( . %). factors that independently influenced both evr and svr were: fibrosis score, steatosis, iron score and viral load. conclusion: fibrosis, steatosis and iron scores, as well as viral load are independent parameters that can affect both evr and svr in genotype chc patients, regardless the presence of ms. if ms is present, high homa-ir index can also additionally impair viral response. issue/argument: asia has rising cases of hepatitisb/c. alcohol/food-habits cause high prevalence in rural/tribal areas. lack of monitoring/follow-up complicates management. vaccines emerge as hope. clinical-trials of vaccines debated-issue. design of hepatitis-vaccine-trials in developing-countries complex ethical-issue. we focus on controversies identified in international/regional/local cme/pharma programs as vulnerability of volunteers to exploitation by foreign/local research-groups/funding-agencies. critical task is protect interests of vaccine-subjects in face of substantial-risks. determine if hepatitis-vaccine-volunteers will have access to treatment during trial. access to vaccine-trial-outcome. interaction with seniors th apasl-congress from developed-countries will give voice to such burning-issues. methodology: researchers/pharmaceuticals/govt-policy planners need to develop forum to solve these problems. ngo's can play pivotal role. obligation on part of researchers to create mechanisms to offset anticipated risks of participation in controversial, risky vaccine-development. conclusion: counselling/right to withdraw from trial be made basic guideline. apart from monetary aspects unsuspected adverse reactions/deaths be properly evaluated/monitored. researchers need to evolve policy-guidelines to overcome barriers as variation in interpretation of essential ethical ideas, legal-system-differences, educational/economic-status. need to develop common consensus between research-community/pharma sector to reduce suffering of hepatitis-affected patients community. recommendations: researchers/ngos should come together at th apasl-congress platform to form workgroup to settle these issues. we shall raise our this burning issue & present hepatitis-prevention-advocacy plan of our ngo graphically to apasl- participants. results: % patients expressed that alternative-medicines-rx most important factor to cope with hepatitis. higher scores of qol (anova p < . ) correlated with alternative-medicines-rx. our ngo-initiative suggests that over % patients will need well trained specialist for home-based-care unit. conclusions: life-span/qol of hepatitis-sufferers depends on appropriate-palliative-care. ngo-personals should be trained in palliative-care-services. our data is being used for palliative care advocacy. field of spiritual/psycho-social/community support is fertile ground for further investigations. such use of complimentary indian medicinal plant extracts needs further evaluation in a large group in multicentre trial. treatment with adacolumn in patients with hepatitis c related who have undergone kidney transplantation: preliminary study g. novelli la sapienza university introduction: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype b. patients underwent five hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at rd month follow up we observed a significant decrease in plasma hcv-rna in patients (p< . ) associated with attenuation of inflammatory phase (p< . ) and variations in immunomodulation. only one patient presented altered cd + and cd + where positive was observed at rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. background: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype b. patients underwent five hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at rd month follow up we observed a significant decrease in plasma hcv-rna in patients (p< . ) associated with attenuation of inflammatory phase (p< . ) and variations in immunomodulation. only one patient presented altered cd + and cd + where positive was observed at rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: the treatment was found to be safe without hemodynamic or infective complications. considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. m. sharaf-eldin , h. el batae , n. abd el-ghaffar , w. rasheed tanta faculty of medicine , egypt., national research centre, cairo, egypt aim: we aimed to characterize serum cytokine levels of interleukin- beta (il- ) and interleukin - (il- ) in hcv infected patients & in patients with hepatocellular carcinoma (hcc) in comparison to control group and their possible use as markers of disease progression. patients and methods: sixty patients were divided into three groups: group i: twenty hcv infected patients without cirrhotic changes. group ii: twenty hcv infected patients with liver cirrhosis (lc). group iii: twenty hcv infected patients with hcc and healthy subjects as control group. all patients and control group were subjected to biochemical and serological tests, anti hcv, hcv (rt-pcr) and cytokines measurements of serum il- & serum il- levels. results: showed a high statistically significant elevated serum il- and il- levels in patients with chronic hcv infection in comparison to control group. highly statistically elevated levels of il- and il- in liver cirrhosis and higher levels were found in hcc group in comparison to control group. the levels of il- and il- increased significantly in hcv infected patients as the disease progress. conclusion: serum il- , and il- levels are elevated in patients with hepatitis c-related liver diseases, especially in lc and hcc patients. their levels reflect hepatic dysfunction better than liver inflammation parameters; accordingly, we may use serum il- and il- as markers for liver disease progression in hcv-infected patients instead of invasive techniques. atsushi tanaka , naoko hanawa , mitsuhiko aiso , yoriyuki takamori , hajime takikawa teikyo university school of medicine background and aim: pegylated interferon (peg-ifn) therapy is not indicated for many cases with hcv-related cirrhosis due to various adverse effects. however, patients with hcv cirrhosis are at high risk for development of hepatocellular carcinoma (hcc). thus we have introduced low-dose peg-ifn treatment for patients for compensated hcv cirrhosis. patients and methods: selection criteria for low dose peg-ifn is ) compensated hcv-related cirrhosis, and ) either the elderly (> ) or presence of thrombocytopenia (< . x / l). we have treated patients who met these criteria with low-dose peg-ifn, consisting of either peg- a g/ - w or peg- b . g/kg/w+ribavirin mg/d. [results] twenty patients with compensated hcv cirrhosis (all patients genotype b) have been treated with low-dose peg-ifn (peg- a: , peg - b+rib: . the age, platelet counts (x / l), and alt (iu/l) of patients at baseline were . ± . , . ± . , and . ± . respectively. all patients were well tolerated. low-dose peg-ifn has been continued . ± . weeks on average. although viral response was not detected, biological response (br), defined as maintenance of alt within normal range, was obtained in patients ( / = %). of note, neither development of hcc nor decompensated cirrhosis was observed in these br cases. by contrast, hcc and decompensation developed in and patients respectively among patients who failed to achieve br. conclusion: low-dose peg-ifn treatment was safe and well tolerable, and could potentially prevent hcc or decompensation in patients with liver cirrhosis when br was obtained. aims: to study the efficacy of peginterferon and ribavirin in treating chronic hepatitis c (chc) with genotype a in hong kong chinese. methods: to assess sustained virological response (svr) (serum hcvrna< iu/ml) at -months follow-up. results: nine patients with genotype a chc (included from jan to dec ) received peginterferon and ribavirin. mean age: (range - ). mean alt before treatment: iu/l (range - iu/l). seven patients had liver biopsy performed, only one showed stage - fibrosis and others showed active hepatitis without advanced fibrosis. mean serum hcv-rna: . x iu/ml(range . x - . x iu/ml). six patients had received peginterferon alfa- b ( . mcg/kg/week), other received peginterferon alfa- a ( mcg/week). ribavirin dosage ranged from mg- mg/day depending on body weight and baseline haemoglobin. treatment durations were - weeks in patients, - weeks in patients as one showed rapid virologic response at week and the other was intolerant to side effect of peginterferon. eight patients had early virologic response at week and one had > log drop of hcvrna. eight patients had end-of-treatment response. eight patients ( . %) achieved svr at end of follow-up. two patients who received only - weeks of combination therapy also achieved svr. the one who failed to achieve svr was at older age of and had advanced fibrosis. conclusions: the efficacy of pegylated interferon and ribavirin in treating chinese patients with chronic hepatitis c genotype a can achieve high sustained virologic response rate of . %. t. bharati , , p. kar , a. mohammad , k. mariappan , j. annamalai , r. introduction: hcv is a recognized cause of hcc. information on hcv genotypes in hcc are scanty in india. methods: a total of hcc cases from delhi, hcc cases from madurai and cases of chronic hepatitis without hcc were controls in the study. rt-pcr for hcv rna and genotyping were carried out in all the cases results: in group-i, hcv rna was positive in . % hcc cases in which genotype was found in . % genotype was observed in . % hcc cases. whereas . % cases remained nontypable. in group-ii, hcv rna was positive in . % hcc cases, with genotype in . % cases, genotype in . % cases and genotype in . % cases. however, . % cases remained nontypable. out of the control cases, were ch and were cirrhosis. in ch group, hcv rna was positive in . % cases in which, genotype was detected in . % cases whereas genotype was observed in . % cases . however . % cases remained nontypable. in cirrhosis group, hcv rna was positive in . %cases. genotype was found in . % cases. while genotype was present in . % cases and . % cases remained nontypable. conclusion: genotype in delhi and genotype in madurai were predominant. in hcc cases. our study demonstrates that no particular hcv genotypes were associated with hcc and genotype did not appear to influence the development of hcv-associated hcc. background/aims: the standard treatment for chronic hepatitis c infected with hcv genotype- is a combination of pegylated interferon alfa and ribavirin for a weeks. it is unclear if weeks treatment is possible for patients showing a rapid virologic response (rvr) without compromising the sustained virologic response (svr) in korea. method: between june and july , among patients chronically infected with the hcv genotype- (hcv- ) who were treated with pegylated interferon alfa subcutaneously once weekly plus ribavirin (weight-based), consecutive paients who had low pretreatment viral load ( . x copies/ml) and rvr were treated for weeks and then followed up for weeks. the hcv rna was quantitatively assessed pretreatment, at weeks of treatment and was qualitatively assessed at weeks of treatment, the end of treatment ( weeks), weeks after end of treatment. rvr was defined as undetectable hcv rna at the weeks. results: baseline characteristics of patients was as followed; age ( - years:mean years), bmi ( - kg/m²:mean . kg/m²), hcv rna titer ( . - . × copies/ml:mean . × copies/ml), alt ( - iu/l:median iu/l). among the patients, all patients ( %) had sustained virologic response (svr). conclusions: hcv- infected patients with a low baseline hcv rna concentration ( . × copies/ml) who had hcv rna negative at week of treatment may be treatment for weeks without compromising sustained virlolgic response. however, an additional trial will be needed to optimize the treatment duration. background/aims: acute hepatitis c (ahc) has a high chronicity rate of up to ~ % if it is not treated. although the good treatment response to pegylated interferon (peg-interferon) therapy has reported, there is not definite guideline to treat of ahc in korea yet. the aim of our study was to investigate the clinical course and treatment outcome of ahc in single center of korea. methods: we performed a retrospective analysis of patients who were diagnosed with ahc during the period from may to december . the diagnosis of ahc was based on seroconversion to anti-hcv antibody or the clinical and biochemical diagnostic criteria satisfactory to ahc and on the presence of hcv rna in first serum sample. the spontaneous resolution was defined as loss of hcv rna in serum for -month in untreated group, and in treatment group, the sustained virological response (svr) was defined as a index of treatment success. results : thirteen of thirty-five patients were treated, six of thirty-five were untreated and observed clinical course, and sixteen patients were not followed up after diagnosis. in treatment group, nine of thirteen ( %) acquired svr, and two of six ( %) showed spontaneous resolution in untreated group. ten of thirteen treatment patients used conventional interferon, and another three patients used peg-interferon. conclusion : compared with untreated group, there was higher svr rate in treatment group ( % vs. %). so early interferon treatment in acute hepatitis c should be considered. background: this study was conducted to identify predictors of thyroid dysfunction and to determine whether virologic factors or treatment response affect thyroid dysfunction development during peginterferon (pegifn) therapy in chronic hepatitis c patients. methods: sixty chronic hepatitis c patients treated with pegifn - a or - b in combination with ribavirin from st july to th july were included in this study. treatment responses were evaluated and thyroid functions were assessed every weeks. results: seventeen patients ( . %) experienced thyroid dysfunction during treatment, and that occurred more frequently in women and in patients with a lower body mass index (bmi). the proportion of patients with a high viral load (a serum hcv rna titer > , iu/ml) was significantly higher in the thyroid dysfunction group rather than in the euthyroid group( . % vs. . %, p= . ). among patients with hcv genotype , the rate of sustained virologic response was lower, and relapse occurred more frequently in the thyroid dysfunction group than in the euthyroid function group during pegifn-based therapy(svr, p= . ; relapse, p= . ). the female gender and the high viral load were independent predictors of thyroid dysfunction in multivariable analyses (female, or . , p= . ; high hcv rna titer, or . , p= . ) . conclusion: the risk of thyroid dysfunction during pegifn therapy for chronic hepatitis c was found to be higher for women and for those with a low bmi and a high viral load. background: in peginterferon alpha b (peg-ifn b) and ribavirin (rbv) combination therapy for weeks for patients with chronic hepatitis c, it is still difficult to predict which patients will achieve sustained viral response (svr) at the completion of this therapy. aim: to predict svr and non-svr (relapse) at the end of this combination therapy by determining changes of serum hyaluronic acid (ha) levels. methods: eighteen patients were enrolled and their serum ha levels were measured before therapy, and after the st, nd, rd, and last trimesters during therapy. results: eleven patients achieved svr and became relapsers. all patients showed higher ha levels in the st trimester than the pretreatment levels. in the svr group, of ( . %) patients in the nd, of ( . %) in the rd, and of ( . %) in the last trimester showed lower ha levels than the pretreatment levels. by contrast, in the relapser group, none in the nd, of ( . %) in the rd, and of ( . %) (p< . ) in the last trimester showed lower ha levels than the pretreatment levels. this study revealed that as the -week therapy went on, ha levels were more likely to fall below the pretreatment levels by the last trimester in patients achieving svr. however, ha levels of relapsers tended to continuously be above the pretreatment levels. conclusion: determination of changes of serum ha levels during peg-ifn b and rbv therapy predicts svr and non-svr at the completion of this therapy. results: the most frequent lymphomas were with high malignancy ( %), intermediate ( . %) and low degree ( . %). cryopathy was negative ( . %). the presence of viral markers was performed soon as possible after the nhl diagnosis, at the same time ( . %) or during the first year of evolution ( . %). the prevalence of the hcv infection was %, comparable to the one in the control patients group ( . %), admitted in a gastroenterological clinic. on the other hand, this prevalence is significantly increased compared to the one in the general romanian population ( . %). the patients with nhl and hcv infection belonged especially to the low and intermediate malignancy degrees; the survival was influenced by the malignancy degree and not by the presence of hcv infection. the prevalence of hbv infection in the tested patients was . %, being lower than that of hcv infection ( . % vs. %, p = . ) but comparable to the one in the general population ( . % vs. . %, p = . ). conclusions: the prevalence of hcv infection in the patients having nhl was %, comparable to the one in the control group, but significantly increased compared to the one in the general population, leaving open the issue of a causal relationship between hcv infection and nhl. iron hepatic overload and hepatitis c d. damian , m. grigorescu , m.d. grigorescu , t. zaharie third medical clinic, cluj napoca, romania aim: evaluation of the prevalence and the degree of iron loading and the relationships with the clinical, biological and morphological changes. method: patients with chronic hepatitis c were included, to whom we tested the blood iron level. in order to evaluate the hepatic iron accumulation we performed the perls staining, using a qualitative analysis and a semiquantitative scoring system (deugnier). results: from a total of patients, . % presented increased blood iron level (p = . ). the evaluation of the liver iron loading was performed in patients, some having normal blood iron level (n = ) and others (n= ) increased (p = . ). the stainable iron was observed in patients. the iron loading was usually low, the deposits were observed mostly at the sinusoid cells and the hepatocyte and less in the portal spaces, usually as a pale staining or of small, nonmerging granules. the total iron score deugnier was low. the increased blood iron correlated with the alt and ggt levels, the necroinflamatory activity and fibrosis. no correlationships between stainable iron and increased blood iron. the presence of liver iron accumulation only correlated with the fibrosis degree. conclusions: of the patients to whom we tested the blood iron, . % had increased levels. the perls staining was positive in % of the patients. the iron loading was mainly low, with a more frequent distribution in the sinusoid cells and in the hepatocytes and correlated only with the stage of fibrosis. response patients whose hcv rna became negative at - weeks t. ide , t. arinaga , k. ogata , i. miyajima , k. kuhara , r. kuwahara , m. background/aim: chronic hepatitis patients whose hcv rna became negative at weeks of peg-interferon/ribavirin treatment achieved excellent svr(sustained viral response) rate of almost %. however, in patients whose rna became negative after weeks, the svr rate is very low. since many patients became rna negative at - weeks, it is important to clarify the characteristics of the patients. material and method: among patients, became rna negative at - weeks and the therapy completed (total - weeks). the characteristics were analyzed by using sex, age, weight, bmi, alt, gtp, hemoglobin, platelet counts, ccr, hyaluronic acid, the mutation of hcv core region (aa , ) and interferon sensitivity determining region, adiponectine, home-ir, rna dynamics, dose and the treatment period. result: the svr rate was . %( / ). because all patients of non svr were female, we compared these patients and female svr. the platelets counts were low in non svr (non svr . ± . (x /mm ) vs svr . ± . (p< . )). the mean dose of ribavirin was lower ( ± mg/day) in non svr (p< . ) than in svr ( ± ). conclusion: as for the characteristics of the patients whose hcv rna became negative at - weeks but became non svr, female, low platelet count and low dose of ribavirin were important factors. in the patients who received reduced ribavirin doses, the idea to increase the ribavirin dose and to maintain it are necessary. (ex, use mg and mg alternately) pe background: chronic hepatitis c virus (hcv) infection poses a challenge for a growing number of infected patients who exhibit disease complications, including cirrhosis, hepatocellular carcinoma, and liver failure in china. the combination treatment of peginterferon alpha (peg-ifn alpha) plus ribavirin (rbv) is recommended as a standard care for hcv infections, which can improves hepatic markers and eradicates the virus in about % of patients. however, a significant number of patients do not respond to therapy or relapse following treatment discontinuation. several viral, hepatic, and patient-related factors influence response to therapy. methods: in our clinical practice, a total of interferon-naïve patients ( % male; median age years) with chronic hepatitis c include cirrhotic patients (no genotyping) received peg-ifn alpha- a mcg/week plus rbv - mg/day for weeks and follow up weeks. results: show that the patients have more rvr and evr rate ( % and . % respectively). while the svr (undetectable hcv-rna weeks after treatment completion) rate is only . % in conclusion: comparing with the data of clinical trail, the rvr, evr and eotr were higher, while svr was the same in chinese patient with chronic hepatitis c patients received the combination therapy of peg-ifn plus rbv. the reason of high relapse was still unknow. although optimal duration of retreatment and benefits and safety of maintenance therapy have not been determined, an extended duration is likely needed, even for the patients who achieved evr. s. nakamoto , f. imazeki , k. background/aims: recently amino acid (aa) substitutions in hepatitis c virus (hcv) core region (double wild (dw); arginine at aa , leucine at aa ) were reported to be associated with sustained virological response (svr) in a combination therapy of peginterferon and ribavirin. we evaluated the viral factors influencing treatment response. methods: nucleotide sequences of core region were determined directly in patients with genotype and high viral load ( kiu/ml) treated with peginterferon-alpha b and ribavirin for weeks. rapid virologcal response (rvr) was defined as more than log decrease of hcv-rna during the first four weeks of therapy and early virological response (evr) as that during the first weeks. svr was defined as negative hcv-rna months after the end of treatment and non-virological response (nvr) as less than log decrease of hcv-rna during the treatment. results: dw at aa and was shown in / ( %) patients with rvr and in / ( %) with non-rvr (p= . ), in / ( %) with evr and in / ( . %) with non-evr (p= . ), in / ( %) with svr and in / ( %) with non-svr (p= . ), and in / ( %) with nvr and in / ( %) with non-nvr (p= . ). in multiple logistic regression analysis, dw was significantly associated with rvr, evr, svr and nvr. conclusions: dw at aa and in hcv core region was closely associated with virological response in a combination therapy of peginterferon and ribavirin. medicine and hepatology, henry dunant hospital, athens, greece, biometrics, ist gmbh, mannheim, germany, background: among patients with chronic hcv treated with pegylated interferon and ribavirin, the highest sustained virologic response (svr) rates are achieved in patients with a rapid virological response (rvr). here we investigate how the time taken to become hcv rna undetectable influences the probability of relapse during untreated follow-up. methods: data from patients treated for weeks with peginterferon alfa- a ( kd) µg/week plus ribavirin / mg/day were included in the intent-to-treat analysis. response was classified as rvr, complete early virological response (cevr) slow responder and non-evr. results: there was a correlation between the time required to become hcv rna undetectable and the relapse rate after stopping treatment. patients with an rvr had the lowest relapse rate ( %); this increased among patients with slower responses. conclusion: there was an inverse correlation between the time taken to achieve a virologic response and the probability of relapse. background: rapid virologic response (rvr; hcv rna < iu/ml) at week of treatment with pegylated interferon plus ribavirin can be used to predict the probability of achieving an svr. patients with detectable hcv rna at week have a lower probability of achieving an svr than those with an rvr; further subdivision of these patients may be useful in predicting outcomes. methods: we conducted a retrospective analysis including genotype patients treated for weeks with peginterferon alfa- a ( kd) g/week and ribavirin / mg/day. patients were categorized as rvr and non-rvr. those without an rvr were further subdivided into detectable but unquantifiable, , , or < log drop in hcv rna. the proportion of patients with undetectable hcv-rna at week and achieving an svr was calculated within each category. results: rvr and non-rvr patients had an % and % rate of svr respectively. among non-rvr patients, rates of svr depended on the categorical response at week : detectable but unquantifiable hcv rna, %; log drop in hcv rna, %; log drop, %; log drop, %; and < log drop, %. independent of week response, undetectable hcv rna at week was also highly predictive of svr. conclusions: patients achieving an rvr have high rates of svr. among patients who do not achieve an rvr a more precise prediction of svr can be achieved by considering the extent of viral load reduction at week and week . retrospective japanese validation study of fibrotest and actitest in patients with chronic hepatitis c. n. nagata , t. mine background: fibrotest (ft) and actitest (at) are biochemical markers of fibrosis and activity for use as a non-invasive alternative to liver biopsy in patients with chronic hepatitis c virus. the aim of this study was to perform a validation study the discordances between ft and at(ft/at) and liver biopsy in patients with chronic hepatitis c in japan. methods: serum samples of chronic hepatitis c patients sended at - °c at the biochemistry department of pitié salpetrière hospital were analysed between july and august . ft/at components were assessed on thawed sera for patients. from patients had liver biopsy at the moment of serum analysis. liver biopsy fibrosis and activity scores were assessed by a pathologist in japan according to metavir scoring system. for each individual test-ft/at the following statistical analysis were performed result: ft observed auroc for the diagnosis of advanced fibrosis was . and after adjustment according to the prevalence of different stages of fibrosis the auroc was . . this difference could be explained by the non-homogenous distribution of different stages of fibrosis (low prevalence of extremes stages of fibrosis -f and f -and high prevalence of adjacent intermediate stages -f and f ). the observed auroc of ft for the diagnosis of precirrhosis and cirrhosis was . and the observed auroc of at for the diagnosis of moderate to severe activity was . . conclusion: these results are similar to those observed in all independent validations worldwide. background: accurate monitoring of hcv-rna level throughout anti-hcv therapy is key factor for predicting sustained virological response (svr). real-time detection polymerase chain reaction (rtd-pcr) based methods are sensitive, have wide dynamic range of quantification and carryover contamination caused by classical pcr. aim: to compare rtd-pcr based assays; cobas ampliprep/cobas taqman (cap/ctm) and recently developed abbott realtime hcv for hcv rna quantification and measurements differences by assays in different genotypes. methods: in total, serum samples were used including, , , , , and with genotypes b, a, b, a and respectively were tested quantatively for hcv-rna by cap/ctm and abott realtime. results: good correlation between two assays as overall (r= . ) with correlation coefficient (r) in genotypes b, a, b, a ranged between . to . and least in genotype (r= . ). mean differences between cap/ctm and abott realtime was significnat in genotypes b and . significantly hcv-rna genotype underestimation by cap/ctm ( . + . log iu/ml) than abbott realtime ( . + . log iu/ml; p= . ). in genotype b, significantly higher hcv-rna measurement by cap/ctm ( . + . log iu/ml) than abbott realtime ( . + . log iu/ml, p= . ). two hcv genotype samples showed measurement differences (cap/ctm minus abbott realtime) of - . and - . log iu/ml. studying genotype sequences within utr , target for cap/ctm rt-pcr amplification, revealed nucleotide polymorphisms at positions a , a , t , a , and a . conclusion: different measurement efficiency by commonly used cap/ctm in different genotypes compared to abbott realtime. new york, new york, usa, vertex pharmaceuticals, cambridge, ma, usa, duke clinical research institute , duke university, durham, nc, usa background: prove is a placebo-controlled study of subjects with genotype chronic hepatitis c randomized to weeks of peginterferon-alfa- a ug/week (p) plus ribavirin - mg/d (r) (pr , n= ), or regimens of mg q h telaprevir (tvr) with pr: tvr/pr for wks followed by pr for wks (t /pr , n= ), wks (t /pr , n= ) or wks (t /pr , n= ). the impact of african american race (aa) and bridging fibrosis on sustained virologic response (svr) was examined. methods: subjects with cirrhosis were excluded from study. fibrosis was categorized as mild/minimal, portal, or bridging from biopsy within years. itt analysis was performed. results: overall, svr was achieved by % of subjects in the pr group, % in t /pr group, % in t /pr group, and % in t /pr group. subgroup analyses indicated svr was improved with tvr/pr (tvr/pr arms pooled) vs pr alone in aa subjects ( % ( / ) vs % ( / )), and in subjects with bridging fibrosis ( % ( / ) vs. % ( / )). adverse events leading to discontinuations were more frequent in the tvr/pr groups ( % vs. %). rashes, gastrointestinal events and anemia were more common in the t/pr arms, and rashes were more frequently severe ( % vs %). conclusions: tvr-based treatment for or weeks was associated with an increase in svr rates compared to pr . subgroups with impaired response to standard peg-ifn/rbv therapy appeared to benefit from the addition of telaprevir. adverse events leading to discontinuation were more frequent in tvr-based regimens. background and aims: induction of type i ifns is a core issue in antiviral responses and must be tightly controlled. the protein kinase tbk is critically involved in virus-triggered type i ifn signaling. in previous studies, an alternatively spliced isoform of tbk , termed tbk s, was identified to be induced in both human and mouse cells. bound to rig- , it is able to disrupt the interaction between rig-i and visa. this study was designed to observe the expression of tbk s in hcv-infected patients. methods: total rna was extracted from samples of peripheral blood mononuclear cells obtained from hcv patients, hcv patients treated with ifn-/ribavirin and healthy controls, and subjected to real -time pcr using the primer-probe sets for human tbk s, tbk and ifn-genes. results: the tbk s expression was significantly elevated in hcv-infected patients, while treatment of hcv-infected patients with ifn-/ribavirin resulted in down-regulation of tbk s to the normal level. conclusions: the study strongly supports the idea that expression of tbk s is correlated with hcv infection, and indicates that tbk s may play an important role in the regulation of hcv infection.this work was supported by nsfc( , , background: infringement of iron metabolism is one of fibrosis progressing factors during diffuse liver diseases. the interrelation between the syndrome of iron overload (sio) and svr achievement is studied during chronic hcv infection treatment. methods: patients with chronic hcv infection (genotyping: - ; + - ; - ; - ; - ) are investigated. sio criteria: iron increase-more than mkmol/l, ferritin -more than mkmol/l, percent of transferriny saturation with iron (%tf) -more than %. results: sio revealed in patients ( . %): patients - genotype ( assotiative with a diabetes) and patients-genotype ( -in combination with liver steatosis and obesity). venipuncture series were done up to getting ferritin referential parameter values before therapy beginning. rvr: sio - patients, normal metabolism - ; evr: and , svr: and relatively. nonresponding patients (sio) had steatosis and diabetes, hereditary hemochromatosis (c y/h d) is verified in case. increase of ferritini values and %tf during therapy and positive hla-a and hla-b is registered in nonresponding patients. conclusions: sio in hla-a , hla-b and c y/h d positive patients is independent predictor of nonresponse during peginterferon alpha- a ( kd)" ribavirin treatment. a.p. srivastava , g. dogra , s. sachdeva , n. nigam , a. chakravarty dr. rml hospital, new delhi (india)- , maulana azad medical college & associated hospitals, new delhi, india background: hepatitis c virus (hcv) has emerged as a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. genotyping and assessment of viral load in hcv patients are vital for designing therapeutic strategies. we aimed to determine the pattern of hcv genotypes and its association with viral load and biochemical profile. methods: hcv rna positive patients were included in the present study attending the medical-opd and wards of dr rml hospital, a tertiary care hospital in new delhi during - . hcv genotyping was carried out by restriction fragment length polymorphism (buoro et al ) followed by the type specific primers from the core region (ohno et al ) . viral load estimation was carried out by taqman real time pcr system using previously described method (martell et al ) . result: . % of cases were having genotype ( a, b, f & i) followed by genotyping ( a & b) in . % and genotype in . %. there was no statistical significant difference seen in the biochemical profile between the three groups of genotypes. genotype one was associated with a significantly higher viral load as compared to the genotypes three and two. parentral mode of transmission was accounted for the % of all the infected cases. conclusion: hcv genotypes and accounted for % of our cases. the genotype is associated with higher degree of disease severity as assessed by viral load. also two unusual subtypes i and f were identified from this geographical region. a.p. srivastava , g. dogra , s sachdeva , n. nigam background: the development and resolution of an inflammatory process is regulated by a complex interplay among cytokines that have pro and anti-inflammatory effects. regulatory mechanisms that control the production of cytokines include genetic polymorphism in particular promoter/leader region. polymorphisms may directly or indirectly affect the binding of transcriptional factors, consequently increasing or decreasing the production of mrna, thus regulating cytokine production. we aimed to determine the polymorphism of tumor necrosis factor-alpha (tnf-alpha) and interleukin- (il- ) genes in chronic hepatitis c patients. methods: hcv rna positive patients were included in the present study conducted during - . healthy controls were also included. genomic dna was extracted by using q a amp dna blood kit protocol according to manufacture's instruction and desired fragment was amplified by using the primer's of vidigal et al . result: genotyping of - -promoter variant of tnf-alpha was performed by pcr. polymorphism in the tnf-alpha (g/g, g/a and a/a allele) was different between hcv patients and healthy controls. il- variants (c/t, c/c) were more frequent among hcv patients as compared to healthy controls. conclusion: genetic polymorphism analysis on il- promoter have indicated that distribution pattern of il- polymorphism was significantly different between controls and hcv patients. furthermore, polymorphism in promoter region of tnf-alpha (- ) was found, though the difference was not significant. since this is a preliminary study, we believe that our findings may stimulate further research on larger number of patients. introduction: the assessment of liver fibrosis provides useful information not only for diagnosis but also for therapeutic decision. although liver biopsy is the gold standard for fibrosis assessment, it is invasive and may have some risks, this has led to the development of non-invasive biochemical markers of liver fibrosis. fibro-test which have five parameters used for the quantitative assessment of liver fibrosis. our aim is to validate the performance of fibro-test in an independent cohort of patients with chronic hepatitis c genotype . methods: subjects were patients with chronic hepatitis c genotype . all biopsies were scored using metavir system by two independent pathologists. fibro-test was done with (biopredictive, houilles, france) for the assessment of liver fibrosis. sensitivity, specificity, ppv and npv were measured for distinguishing between different degrees of severity of fibrosis. results: patients ( male and female) age ranged - years, liver biopsy showed % (f ), % (f ), % (f ), % (f ), % (f ). the efficacy of fibrotest is . %, sensitivity . %, specificity %, positive predictive value % and negative predictive value %. conclusion: fibrofast has a low performance in assessment in fibrosis in chronic hepatitis c genotype . introduction: liver biopsy is the reference method for assessing liver fibrosis. however, it is invasive, costly and has some limitations. european liver fibrosis (elf) markers have shown to be accurate in assessing liver fibrosis in a range of chronic liver disorders. our aim is to test the performance of elf markers in an independent cohort of patients with chronic hepatitis c genotype . methods: subjects were patients with chronic hepatitis c genotype . all biopsies were staged for fibrosis using metavir system by two independent pathologist. elf markers were done by (diagnostic & operations, england) and fibrosis scores were derived using the published elf algorithm. the area under the curve (auc) for receiver operator characteristic curves was measured along with sensitivity and specificity, positive (ppv) and negative (npv) predictive values for distinguishing between different stages. results: patients ( male and female), age was ranged - years, liver biopsy showed % (f ), % (f ), % (f ), % (f ) and % (f ). elf markers had no correlation with fibrosis score where r = - . , p = . , aucs: . , specificity . %, sensitivity only . %, ppv: only . %, npv: . % and efficacy . %. conclusion: the performance of elf marker is low and can not be used for assessment of fibrosis in chronic hepatitis c genotype . background: the prove trial is a randomized, placebo-controlled study that assessed the safety and efficacy of mg q h telaprevir (tvr) combined with g/week peg -ifn alfa- a (p) ± - mg/day ribavirin (r) in chronic hcv genotype -infected treatment-naïve patients without cirrhosis. methods: overall, patients received tvr + pr for weeks (t /pr ; n= ), tvr + pr for weeks then pr for weeks (t /pr ; n= ), tvr + p for weeks (t /p ; n= ), or to pr for weeks (pr ; n= ). primary endpoint: sustained virologic response (svr, undetectable hcv-rna weeks post-treatment). results: baseline characteristics were well balanced across groups. numerically higher svr rates were observed in patients receiving t /pr ( %; p= . for difference vs. pr ) than t /pr ( %), t /p ( %) or pr ( %). relapse rates were lower in the t /pr group ( %) than the t /pr ( %), t /p ( %) and pr ( %) groups. the relapse rate in patients receiving t /pr with -week and -week undetectable hcv-rna was % ( / ). the aes occurring more frequently with the t/pr regimen were pruritus, rash, asthenia, nausea and anemia. in the t/pr arms, patients discontinued due to rash, discontinued due to pruritus, and patients due to anemia. conclusion: these results showed that a telaprevir-based regimen led to significantly higher svr rates than pr, and indicate that this regimen could shorten the overall treatment duration from weeks to weeks for most patients infected with hcv genotype . a.c. cardoso , c. stern , r. moucari , n. giuily , p. bedossa , p. marcellin hopital beaujon background/aim: this study evaluated the effect of the response (svr) to therapy on fibrosis stage, as assessed by ls, in patients with advanced fibrosis (f ) or cirrhosis (f ). methods: hcv patients with f or f who received interferon-based treatment were studded. ls was assessed after treatment (median delay of months, - ) in patients with or without svr. correlations between ls and clinical and treatment characteristics were analyzed. results: patients were included: male gender ( %), mean age ( ± years), diabetes ( %), mean bmi ( ± kg/m ), genotype ( %). % had svr. ls was performed - , - , > years following treatment. by linear regression, the median of the ls was independently associated with svr (p= . ) and diabetes (p= . ). svr patients had lower ls ( . kpa; range . - ) than non svr patients ( . kpa; range . - ) (p< . ). among the svr patients the median ls was lower when the delay between ls and the end of treatment was longer ( . , . , . ) (p= . ). on the opposite, among the non-svr patients the median ls was not significantly different (p= . ). the median of liver stiffness was higher in patients with diabetes (p= . ). bmi and dyslipidemia did not influence the median of the ls. conclusion: in patients with advanced fibrosis or cirrhosis, ls was lower in patients with svr and decreased with time while it was higher and did not decrease in non-svr patients. ls could be important for assessment of fibrosis stage during the post-treatment follow-up. to study peripheral blood and intrahepatic natural killer (nk) cells in patients with chronic hepatitis c in relation to disease activity and severity of hepatic fibrosis. patients & methods: fifteen untreated patients with histologically-proven chronic hepatitis c, and matched healthy subjects. the nk cells and natural killer t (nkt) cells were identified in fresh whole blood samples using two-color flow cytometric assay as cd -cd + and cd + cd + positive cells. immunohistochemical staining of liver biopsies taken from all patients was done using monoclonal antibody against cd for detection of nk cells and rabbit polyclonal antibody against smooth muscle actin (sma) for identification of activated hepatic stellate cells (hscs). results: patients with chronic hepatitis c showed significant decreases in the percentages of nk cells and nkt cells in peripheral blood. a negative correlation was found between serum hcv rna levels and the percentages of peripheral blood nk cells and the intensity of intrahepatic nk cells. the percentages of circulating nk cells and nkt cells and the intensity of intrahepatic nk cells were inversely correlated with the metavir fibrosis stage and the steatosis grade, and also with the intensity of intrahepatic activated hscs. conclusion: patients with chronic hepatitis c had significant deficiency in circulating nk and nkt cells as well as in intrahepatic nk cells. this may provide a possible mechanism for the suppression of innate immunity against hcv. background: hcv infection is the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. the virus is classified in six genotypes and more subtypes, which are related distinct with antiviral therapies reply. in brazilian amazon, epidemiologist's studies in blood donors had pointed high frequency of genotype ( %) followed by genotypes ( %) and ( %). however, epidemiological research in populations of risk to the infection still is scarce. aim: to determine hcv genotypic frequency in blood donors, patients with blood transfusions multiples, patients in hemodialysis and drugs users in the state of pará, brazilian amazon. methods: using real time pcr and nucleotide sequencing followed phylogenetic analysis had been gotten viral diagnosis and genotyping. results: in blood donors, hcv distribution was constituted by genotypes ( . %) and ( . %). in multitransfunded patients occurs maximum prevalence of genotype ( %), probably reflect of genotype specific transmission of blood donors population. on the other hand, in hemodialysis patients had been detected genotypes ( . %), ( . %) and ( . %), result of a bigger diversity of transmission routes (transfusional, interfamilial, nosocomial, etc) . in drug users occurs the biggest frequency of genotype ( . %) with prevalence of genotype ( . %), suggesting that the sharing of abuse machinery is allowing strains diffusion of genotype . conclusions: the genotype possesses the biggest frequency in different population. moreover, through hcv genotypic frequency if it detached the contribution of transmission distinct routes indicated by previous epidemiologists researches. virol. ). we established real-time polymerase chain reaction (pcr) assays for the easy detection of these hcv mutations. methods: plasmids p-core-w, including wild type hcv core coding region ( r and l), and p-core-m, including mutant type hcv core ( q/h and m), were constructed by cloning and pcr-based mutagenesis for control vector of wild type core and that of mutant core, respectively. using serially diluted forms of these vectors, sybr green-based real-time pcr detections with mutation-specific primers were performed. results: analysis of known scalar concentrations of references indicated that the detection limits of these methods were at least copies, copies, copies, and copies of -wild, -mutant, -wild, and -mutant, respectively. each primer could clearly distinguish the difference between p-core-w and p-core-m at the same copy numbers. concerning substitution , the ratios : , : , : , : , and : of p-core-w versus p-core-m could be distinguished. on the other hand, for substitution , the ratios : , : , : , : , : , and : could be distinguished, confirming the sensitivity and specificity of the assay. conclusions: this method could represent a useful alternative for the detection of genotype b hcv core amino acid substitutions and and be reliably applied for rapid screening. efficacy and tolerability of hcv treatment in asian patients according to age and genotype at a tertiary centre in western australia n. saroj , n. kontorinis , t. lorenzo , m. marion , s.l. chen , w. cheng , royal perth hospital, centre for international health, curtin introduction: race and ethnicity can influence efficacy and tolerability to treatment in hcv. the higher response rate in asians is thought to be associated with better adherence and tolerability. objectives: ( ) to evaluate the adherence according to age and genotype ( ) to assess the effect of age on treatment efficacy ( ) background: hepatitis c virus (hcv) infection is a major health problem. there is huge regional variation in its prevalence and genotypic distribution. voluntary blood donors are thought to have somewhat lesser prevalence than the rest of the community. reliable statistics are not available for the entire country, particularly for the rural areas. it is important to know local situation and rationalize use of limited resources. methods: retrospective study of the records of patients attending the free liver clinic (flc) of our hospital located in a rural area of pakistan, and those screened for hcv infection prior to voluntary blood donation. results: patients at flc ( out of [ %; males %] were found to have higher chances of being reactive for hcv antibodies as compared to voluntary blood donors ( / [ %]; p = . ; or . - % ci = . - . ). out of a total of hcv reactive patients, ( %) were found to be positive on hcv rna testing. out of a total of typeable genotypes, ( %; % ci = . - . , estimated odds = . ) were infected with a single genotype, and only patients ( %) were infected with genotype , either alone (n= ) or in combination with a. conclusions: one out of every people tested in our flc is seropositive for hcv, and % of "healthy" voluntary blood donors have the same results. genotype is very rare in our region. s.a. batool , s.z. abbas department of gastroenterology, muhammad hospital, mirpurkhas, pakistan background: hepatitis c viraus (hcv) infection is common in our region. data is not available on success rates of conventional interferon (inf) based products here. we attempted to find out the dominant genotype, and to determine the success rate of conventional inf-based treatment in eradicating hcv. methods: retrospective case series study of hcv infected patients' records treated with different brands of inf. results: / ( %) of all patients tested were positive for hcv antibodies. hcv-rna was tested by pcr for patients, of which ( %) turned out to be positive. genotype type was the dominant genotype -found in / ( %) patients. men and women were treated with various brands of inf with the same manufacturer's brand of ribavirin. the overall etr achieved was / ( %) - / ( %) men and / ( %) women. / ( %) of genotype achieved etr. there was no significant difference in average ages for those who achieved good etr and those who did not ( years each). the etr achieved by different brands ranged from % to %. svr was achieved by / patients. conclusions: % of all people tested positive for hcv antibodies, of which about % had evidence of active hcv infection. etr achieved by different brands averaged %. this was % in female sex, although age did not appear to be a factor in determining a favourable etr. patients & methods: a total of consecutive diabetic patients of either sex were evaluated for hcv and hbv infection by using enzyme linked immunosorbant assay (eliza- ) along with serum alt levels. on the basis of this test, the patients were divided into two groups, sero +ve and sero -ve. different variables were: age, sex, bmi, area of residence (rural or urban), type and duration of dm, smoking, literacy and alt. results: males . % and females . %. age ranged from to . majority were married ( . %), from rural area ( . %), had type- dm ( . %), normal weight ( . %), normal alt( . %) and non-smokers ( . %). seroprevalence for hcv, hbv and both were . %, . % and . %. two groups were made, sero +ve and sero -ve. raised alt ( . %) was significant (p< . ) factor while all others variables were insignificant (p> . ). conclusion: hbv and hcv infections are more prevalent in dm with increased alt levels. while hcv infection is more common than hbv in patients with dm. hepatitis c virus (hcv) envelope proteins (e and e ) mediate the entry of virus into host cells by binding to its cellular receptors and resulting in the fusion of the viral membrane with host cell membrane. the expression and secretion of biologically active envelope proteins in vitro have proven to be a difficult task due to the high degree of glycosylation and the existence of hydrophobic domains within these sequences. in order to obtain glycosylated, correctly-folded hcv envelop proteins in large quantities, we optimized the dna sequences of hcv envelop proteins by substituting the encoded sequence with human preferable codons and expressed them in human embryonic kidney (hek) cells. both proteins were detected intracellularly, with a small portion secreted into supernatant. in order to enhance secretion, truncated forms of envelop proteins including e tm, e - , e - were also expressed. both full-length and truncated forms of envelop proteins were glycosylated and expressed at high level. in addition, we also expressed the codon-optimized hcv receptors cd and claudin- in cells. by comparing the expression level of codon-optimized sequences and the sequences that were obtained from cdna library by pcr, we found that codon-optimization enhance protein expression significantly in cells. these results not only lay solid foundation for further research concerning the mechanism of hcv entry, including the optimal ph and right protein conformation for fusion, cell types that permit viral entry; but also potentiate a useful cell model for testing antiviral agents. background: prolactin (prl) is an immunoregulatory hormone secreted from lymphocytes, however, prl induction in relation to hepatitis c virus (hcv) infection has not been elucidated. methods: serum prl levels were measured in both subjects of our hcv cohort study and male patients of the hospital, who were chronically infected with hcv. furthermore, serum prl levels were compared in male patients before and after interferon therapy. we measured expression of prl mrna level in pbmcs in male patients, and also investigated prl mrna of pbmcs collected from healthy men that stimulated by hcv produced by huh . cells in vitro. result: serum prl levels were significantly higher in the hcv-infected subjects than in the controls (p< . ). they were significantly higher in hcv-infected male subjects than in the controls (p< . ). serum prl levels were significantly higher in male patients than in the controls (p< . ). serum prl levels decreased significantly after interferon therapy in patients with sustained virological response to therapy (p< . ). the levels of prl mrna in pbmcs derived from hcv-infected patients were significantly higher in male patients than in the controls (p< . ). conclusion: the high levels of prl expression are associated with hcv infection in carriers. background and objectives: hepatitis c virus (hcv) is a major cause of chronic liver hepatitis, cirrhosis, and hepatocellular carcinoma.current clinic standard therapy is interferon alpha (ifn-) combination with ribavirin, but this treatment is associated with adverse effects and often fails to induce a sustained response. until recently, development of a hcv cell culture system (hcvcc) provides a suitable tissue culture system to study the complete hcv life cycle. in this study, we tested the effect of ifn omega (ifn-)-a member of type interferon on hcv compared with ifnbased on hcv b replicon and hcvcc. methods: we compared ifn-and ifn- a effects on hcv rna replication and protein expression, as measured by ribozyme protection assay and western blot. we also compared the intracellular protein level of phosphorylated signal transducer and activator of transcription (p-stat ) treated with different interferon type and concentration with western blot analysis. results: hcv rna and protein level were inversely related with ifnconcentration and compared with ifn- a, at the same concentration, the hcv rna and protein levels treated with ifn-were lower than that treated with ifn- a p . .also based on the hcv rna analysis, ec of ifn-was folds lower than ifn- a. ifns increased intracellular p-stat level at a dose dependent manner and compared the same concentration of ifn-and ifn- a, p-stat protein level was higher in ifn-treated group p . . conclusions: these results demonstrate distinct antiviral effect of ifncompared with ifn- a and this difference maybe partly caused by the stronger stimulation of ifn receptor . outstanding antiviral activity of ifnmay be useful for developing new hcv treatment strategies. background & aim: hepatitis b virus (hbv) infection with undetectable levels of hepatitis b surface antigen (hbsag) is called an occult infection, which although has been described among subjects with chronic hepatitis c liver disease in the western world, it's prevalence and clinical significance are still ambiguous in the indian subcontinent. materials and methods: we investigated hbv-dna pcr in serum samples of hbsag negative subjects with chronic hcv-related liver disease, and apparently healthy volunteers negative for hbsag and anti-hcv as control. results: serum samples found positive by at least two independent pcr assays were considered hbv dna positive. hbv-dna was detected among hcv-related chronic liver disease (cld) patients ( . %), which was higher (p = . ) as compared with the control volunteers ( . %). it was more frequent ( . %) in anti-hbs negative/anti-hbc positive patients than in anti-hbs/anti-hbc positive ( %, p < . ). hcv rna by qualitative pcr was significantly (p < . ) higher in occult hbv compare to non-occult. hcv genotype b was predominantly associated with occult hbv ( %), especially among subjects with hepatocellular carcinoma (hcc) (p< . ) as compared to non-occult hbv cases. though not significant, frequency of occult hbv infection was higher than healthy controls and hcv b genotype was significantly associated in patients with hcc. conclusion: this study suggests that in all hbv-endemic areas, the possibility of occult hbv in patients with hcv should be considered and hbv-dna should be performed. j. zhao , , w.d. cai , l. chen , y.x. gan , m.l. he , x.r. wang background: besides hiv and syphilis, hepatitis c virus (hcv) is also rapidly spread among men who have sex with men (msm). this study was designed to identify the prevalence of these sexual transmitted diseases in msms in shenzhen, china. methods: a cross sectional study was conducted by using time location sampling method from april to july, . msm participants (including male sex workers) were recruited and finished guided self-administered questionnaires (or interviews if they have difficulty in reading or understanding) in venue-date-time randomly selected from active venues. results: results were analyzed using spss. blood samples were collected for hiv, syphilis and hcv test. participated msms were between the age of to years ( . ± . ) with a majority of - years ( . %). most of them finished junior high school education ( . %). . % had high level of knowledge on modes of transmission and prevention. likewise, . % msms have ever sold sex to men, . % of them were self identified as gay, . % as bisexual. . % msms had multiple male sexual partners and . % msms always used condom. . % of them had sex with women in the past month, and the condom use rate decline to . % during both male and female sex. hiv positive rate is of . % and syphilis for . %, hcv is only found in cases ( . %). conclusions: a greater number of the participants have both male and female sex partners. this survey shows that hcv infection rate is still low among msms in shenzhen, although the hiv and syphilis rate is high and continuing increased in the past few years. the change of insulin sensitivity in hepatitis c patients with normal insulin sensitivity s.g. park , y.k. cho , j.w. lee , j.w. yun , h.j. kim , w.k. jeon , b.i. background: hepatitis c virus (hcv) infection is associated with a high prevalence of diabetes mellitus (dm). insulin resistance (ir) is known to play a crucial role in the development of dm in chronic hepatitis c (chc) patients. we prospectively investigated the change of insulin sensitivity in chc patients during -year period, and analyzed factors significantly associated with ir. methods: subjects consisted of non-cirrhotic chc patients with normal alanine aminotransferase (alt) and normal insulin sensitivity (chc group), and healthy control group of subjects matched by age, sex, body mass index and life styles. we compared initial baseline insulin sensitivity, metabolic parameters and incidence rate of ir at the end of follow up period in both groups. the change of insulin sensitivity and metabolic parameters and development of ir was analyzed, and factors associated with development of ir were evaluated. results: ir developed in . % of chc patients and . % of normal individuals (p< . ). hcv infection per se and genotype were independent risk factors of ir. initial fasting glucose - mg/dl, fasting insulin uiu/ml, homa-ir . - . were significantly associated with development of ir in chc group. conclusions: hcv infection is independent risk factor of ir. even if chc patients with normal insulin sensitivity, careful monitoring for ir is necessary. prevalence of viral hepatitis c in latvia i. tolmane , , b. rozentale , , j. keiss , f. arsa sa infectology center of latvia, riga stradin's university background and aim: viral hepatitis c (vhc) because of its prevalence and clinical course has become one of the most actual infectious diseases in the world. to date chronic hepatitis c affects over million individuals worldwide. chronic vhc is a leading cause of cirrhosis and hepatocellular carcinoma. the aim of this study was to investigate how many residents of latvia, that are over years of age have been exposed to vhc (anti-hcv prevalence) and how many are infected at the moment (hcv-rna prevalence). until now such research has not been performed in latvia. methods: from the register of general practitioners there were randomly selected gp's from different regions of latvia, persons over years of age were selected out of each gp register and tested for anti-hcv with screening test (elisa). in case of positive result antibodies were confirmed with western-blot reaction and person was tested for hcv-rna (pcr). results: in total person was invited by general practitioners for the test and persons responded (response rate . %). confirming test (western-blot) was positive in participants and out of which hcv rna test was positive in patients. conclusions: there are . % of people exposed to hepatitis c virus in latvia and . % are infected with hepatitis c virus, respectively, infected persons per thousand individuals. genetic variation in the ikk/nf-b pathway and the live fibrosis progression in chronic hepatitis c r. sho , k. ishii , r. ishii , h. watanabe , k. sugahara , y. nishise , k. okumoto , t. saito , s. kawata , a. fukao department of public health, department of gastroenterology, yamagata university faculty of medicine background/aims: i b kinase/nf-b (ikk/nf-b) signaling pathway is thought to play critical roles in liver inflammation and fibrogenesis. we carried out a haplotype-based association study to examine the contribution of common genetic variations in the genes encoding nf b inhibitor kinase alpha and beta (ikbka and ikbkb; the major components of ikk/nf-b pathway) to the progression of live fibrosis in chronic hepatitis c. methods: based upon the common single nucleotide polymorphisms (snps; minor allele frequency(maf) . ) and linkage disequilibrium (ld) information derived from the hapmap, we selected and tag snps from ikbka, and ikbkb, respectively, for genotyping. by using melting curve analysis, snps were genotyped in chronic hepatitis c patients, including patients with hepatocellular carcinoma. association between common genetic variations in ikbka/ikbka and platelet count (plt) was tested by both genotype-and haplotype-based approaches. results: we succeeded in genotyping a total of tag snps that efficiently capture common variation across the kb-block of ikbka and the kb-block of ikbkb. for each of genes tested, haplotypes were found in population studied. all snps were in hardy-weinberg equilibrium, but no significant association was observed between any single tag snp or haplotype and decreased plt in patients analyzed. conclusions: our data suggest that it is unlikely that polymorphisms within the ikbka and ikbkb genes are involved in the progression of live fibrosis in chronic hepatitis c. further studies on genetic variations in other nf-b-related genes in chronic hepatitis c are needed. background: hepatitis c virus infection is a major burden after liver transplantation. the effective treatment for patients who underwent liver transplantation has not been well established. management of these patients is the most challenging task. cyclophilins are essential host factors for hcv replication. we report here the efficacy of divided administration of ifn plus cyclosporine a in the treatment of chronic hepatitis c patients who failed peg-ifn or ifn combined ribavirin. patients and method: we prospectively included patients (median age, ) with genotype b and, failures to combination ifn plus ribavirin or combination pegylated ifn plus ribavirin. the present treatments consisted of an induction therapy, an intensified therapy and a maintenance therapy. the induction therapy comprised intravenous mu ifn every hours for the first days, . mu ifn every hours for the next days and mu ifn every hours for the following weeks, totaling mu of ifn . the intensified therapy was induction therapy shortened to weeks. the maintenance therapy comprised of pegylated ifn b and ribavirin. csa was given times daily during the induction and the intensified therapies. ribavirin was given twice daily during the maintenance therapy. results: the end treatment response and sustained virological response rate of the present study were % ( / ) and % ( / ), respectively. the relapse rate was %( / ). non-responders was % ( / ). all adverse effects were completely reversible. the treatment protocol was well tolerable. conclusion: we concluded that our protocol should be effective in failures to the previous combination therapies. host factor targeting treatment will become a promising treatment option. cyclophilin targeting treatment is a promising new anti-hcv treatment k. inoue , t. watanabe , s. yoshiba background: hepatitis c virus (hcv) is the most common cause of chronic liver disease. however, the efficacy of currently available treatments is limited. we recently reported the effects of combined interferon-/cyclosporin a treatment. cyclophilins are associated with hcv replication and bind cyclosporin a. which cyclophilins are closely associated with hcv replication remains controversial. in this study, several cyclophilins were found to be essential host factors for hcv replication and hcv replication was rescued by overexpression of cyclophilin a in the presence of cyclosporin a. methods: we evaluated the effect of cyclosporin a and its analogues on the replication of hcv in vitro using several types of hcv replicon. the gene expression of representative cyclophilins and pin- was knocked down using small interfering rna (sirna) to identify cyclophilins associated with hcv replication. the specificity of the effect of sirna was confirmed by western blot analysis. the effect of overexpression of cyclophilins on hcv replication in the presence of cycloporin a was also studied. results: cyclosporin a and its analogues suppressed hcv replication in a dose dependent manner. cyclophilin f, cyclophilin lc and cyclophilin lc as host factors which are closely associated with hcv replication, in addition to the previously reported cyclophilin a. knockdown of chclophilin b showed little effect on hcv rna replication. cyclophiln-dependent hcv replication varied among the three hcv replicon cell-lines used. overexpression of cyclophilin a rescued hcv replication in the presence of cyclosporin a. conclusions: these findings suggest several cyclophilins are essential host factors for hcv rna replication. thus potent cyclophilin inhibitors have the potential to be anti-hcv drugs. background/aims: hepatitis c virus (hcv) genotypes - have a worldwide distribution. types a and b are predominant in northern europe and north america, and in southern and eastern europe and japan, respectively. type is endemic in south asia and is variably distributed in different countries. genotype in egypt, genotype in central and south america and genotype is common in china, japan and south east asia. in pakistan a is the commonest genotype, which is associated with the most favorable outcome regarding end treatment response and sustained virological response after weeks of therapy. the aim of this study is to find out hcv genotypes in newly diagnosed chronic hepatitis c patients. methods: this observational study was conducted in chronic hepatitis c patients. all patients had raised alt levels for last months, had positive polymerase chain reaction (pcr) for hcv rna by real time method and liver biopsy was done in all patients under national program for prevention and control of hepatitis during year - . genotyping was done on roche genotyping kit. data was analyzed by spss . results: out of patients, . % (n= ) were genotype a. . % (n= ) were genotype b. . % (n= ) were genotype a. n= had genotype b. . % (n= ) had mixed genotype ( a, b/ a, b, a, b). conclusion: majority ( . %) of chronic hepatitis c patients were genotype a which is associated with favorable outcome after weeks of interferon and ribavirin therapy and only . % had genotype a in this cohort. s.t. zhou , y. zhao , f.j. zhang background/aims: as human immunodeficiency virus (hiv) infected children who are receiving antiretroviral therapy (art) are living longer in china, comorbidities of hepatitis b virus (hbv) and hepatitis c virus (hcv) coinfection should be carefully considered when making management decisions. however, the coinfection rate of either hbv or hcv is unknown in hiv-infected children in china. we evaluated the seroprevalence of hbv and hcv in the china national pediatric art cohort of hiv-infected patients. methods: patients were selected from hiv infected children medically eligible for art who were enrolled into the china national pediatric art cohort since . interviews, medical assessment, serology for hbsag, anti-hcv antibody, transaminase levels, and hiv serostatus and cd counts at baseline of patients were obtained. results: of hiv-infected children were hbsag seropositive ( . %; %ci: . %- . %), and of children were anti-hcv antibody seropositive ( . %; %ci: . %- . %). only age was associated with hbv coinfection. multivariate analysis revealed that children infected with hiv through contaminated blood or transfusion of blood products were . times more likely to be anti-hcv antibody positive than those infected with hiv through other routes. and children from central china provinces, henan, anhui, shanxi, and hubei were . times more likely to be hcv seropositive. conclusion: the high seroprevalence of hbv and hcv coinfection in hiv-infected children attending china national pediatric art cohort calls for routine screening for hepatitis viral coinfection and modification of the management of hiv-infected children in china. background: bms- is a first-in class and highly selective hepatitis c virus (hcv) ns a inhibitor with picomolar in vitro potency against genotypes a and b. in a sad study with healthy subjects, bms- was safe, well-tolerated, and had a pharmacokinetic profile suggestive of once-daily dosing. methods: the objectives of this randomized, double blind, placebo-controlled, sad study were to evaluate the safety, tolerability, antiviral effect and pharmacokinetics of bms- in patients with genotype chronic hepatitis c (chc). treatment naïve or experienced patients were randomized to receive , , or mg of bms- or placebo. results: all bms- single doses were well tolerated and had a safety profile similar to that of placebo. following oral administration, bms- was readily absorbed with dose proportional exposures over the studied dose range. the mean terminal half-life of bms- was approximately hours. mean decline in hcv rna hours after a single , and mg dose of bms- was . log (range . to . log ), . log (range . to . log ) and . log (range . to . log ), respectively. the mg dose resulted in a mean decline of . log (range . to . log ) hours after dosing, which was maintained at hours. conclusions: single doses of up to mg of bms- were safe and well tolerated in patients chronically infected with hcv genotype . bms- produced a robust decline in hcv rna and has a pharmacokinetic profile that potentially supports once-daily dosing. background: the global infection rate of hcv is approximately %, and nearly . % in china. only %- % of patients with genotype b can achieve sustained virological response (svr) after antivirus therapy, nearly half of them experienced treatment failure. the study aimed to determine hcv- b sequence evolution in patients experienced treatment failure during and after therapy, and further analyze relations between the mutations and treatment outcome. methods: patients with genotype b accepted antiviral treatment of ifn plus ribavirin for weeks, and long-term follow-up after therapy. patients experienced treatment failure were further analyzed (one for relapser, another for nonresponder). sera were reserved at baseline, w, w and -year after therapy. hcv-rna was extracted. hcv full-length orf was amplified by rt-nested-pcr and sequencing. result: of the patients achieved svr ( . %). from sequence alignments of relapser at baseline and w, we find that p , ns a and ns a have higher mutation rate both in nucleotide and amino acid level ( . % and . %, . % and . %, . % and . %, respectively). but there is no significant difference in the alignments of w and -year after therapy, the mutation rate is lower. mutation rates of the non-responder among baseline, w, w and -year after therapy are very low. conclusion: antivirus effect is correlated with specific hcv sequences in chronic hepatitis c, mutations in hcv non-structure protein p , ns a and ns a have important impacts on treatment outcome in ifn-based therapy. background: the results of antiviral therapy for hepatitis c (hcv) have improved recently with the use of peg-interferon (peg-ifn)/ribavirin therapy. however, age of patients are concerned because of side effects and safety. as we known, a few studies have targeted therapy in elder with chronic hcv. aim: we reviewed the results of interferon based antiviral therapy in the elderly with chronic hcv at our institution. methods: patients were defined as elderly if they were years and elder who received therapy for hcv. the prescribed treatment duration, end of treatment response were mention. the data recorded included laboratory tests, adverse events (ae), dose modification, and withdrawal rate of therapy. results: of chronic hcv patients treated with peg-ifn/ribavirin between nov and feb . patients were older than years old. the mean age of the elder patients was . ± . years old. were male and were female. histological studies showed with cirrhosis. almost all patients had experienced ae/side effects. the most common abnormalities were anemia and neutropenia. therapy was discontinued in % ( / ). the rate of dose modification was % ( / ) patients who received weeks therapy. transaminases were normalized in % ( / ) after weeks treatment and sustained in % ( / ) one year later. conclusion: the elder patients are more at risk of developing ae while on treatment. most patients should be discontinued or decreased dosage of medication. however, the elder patients with chronic hcv can be treated successfully. background: in the general population the incidence of interstitial lung disease is estimated to be . % and has also been reported with the use of interferons. the higher reporting rate of ip in japan has created interest and warrants further investigation. methods: using both data from randomized clinical trials (ex-japan) and the roche world-wide safety database (advent), the frequency of ip was estimated in patients treated with peginterferon alfa- a ± ribavirin. ip was defined as: interstitial lung disease, alveolitis, pulmonary fibrosis, pneumonitis and pulmonary toxicity. results: one case of ip was reported among the patients included in the clinical trials ( . %). in the advent database considering the estimated , patients with cumulative exposure to peginterferon alfa- a ( , in japan and , us/row) the reported cases of ip represent a rate of . % with a proportional reporting ratio (prr) of . (p< . ). of these cases, were reported in japan (prr . ; p< . ), in the usa (prr . ; p= . ) and row (prr . ; p= . ) representing reporting rates of . % in japan and . % in the usa and row. japanese patients with reported ip were older ( versus - years) and were more likely to have been treated with peginterferon alfa- a monotherapy ( % versus - %). furthermore, the yearly incidence rate has remained unchanged. conclusions: the apparently higher rate of ip reported in japan may result from differences in patient demography, diagnostic criteria and treatment patterns. the overall incidence of ip remains low. background: hepatitis c virus (hcv) infection carries a significant risk for development of insulin resistance (ir) and/or diabetes (dm). recently, retinol-binding protein (rbp ) has been reported as a protein contributing to ir. this study aimed to assess the different expression of serum rbp between chronic hcv infection (chc) patients and non-chc controls. methods: serum rbp was measured in treatment-naïve chc patients and its correlation with the homeostasis model assessment of insulin resistance index (homa-ir), liver histology, virology and metabolic factors was investigated. patients were stratified into different stages of glucose tolerance by oral glucose tolerance test. another sex-and age-matched non-chc adults served as the controls. results: the mean rbp level of controls tended to be higher than that of chc patients ( . ± . vs . ± . g/ml, p= . ). the mean rbp level of igt control-group subjects was . ± . g/ml, which was significantly higher than that of ngt ( . ± . g/ml, p< . ) and dm controls ( . ± . g/ml, p< . ). in contrast, the mean rbp level ( . ± . g/ml) of dm/chc patients was not significantly different from that of ngt/chc ( . ± . g/ml, n= ) and igt /chc ( . ± . g/ml, n= ) patients. amongst chc patients, there was a significant decreasing linear trend of rbp dependent of both histological grading and staging progression, whilst a significant increment of homa-ir was found. conclusion: serum rbp is dysregulated in chc patients. introduction: sustained viral response (svr) in hepatitis c treatment with interferon alfa and ribavirin is affected by adherence and compliance due to severe myalgia, fatigue-anxiety and disturbed sleep. pregabalin, an orally effective gabasergic drug is not metabolized via cytochrome p and is used in fibromyalgia and fatigue-anxiety syndromes without hepatic toxicity.this study evaluates the addition of pregablin to standard agents in achieving svr by reducing side events. methods: thirty patients with chronic hepatitis c {mean age - years, male: female - : ,genotype(g) (n= ), g (n= ), fibrotic score f - (n= ) and f (n= ), mean bmi > kg/m , initial viral load > , iu/ml} were randomized to pregablin mg (n= ) or duloxetine mg (n= ) both orally daily with interferon alfa a mcg sq once a week and ribavirin mg daily for weeks. myalgia anxiety scale, modified quality of life score -evaluated at entry and tri-monthly. all were tested for rapid viral response, early viral response and end treatment viral response and svr. results: at the end of weeks, in the pregablin arm, ( . %) completed the therapy without interruption, one stopped due to excessive somnolence. duloxetine arm - ( . %) completed with interruptions, ( . %) withdrew from the trial due to side events, one left the country. ( . %) achieved svr in pregablin arm and ( . %) with duloxetine. conclusions: pregablin may be considered with ifn and rbv for better adherence and compliance in achieving svr in treatment of chronic hepatitis c. larger randomized studies are needed to confirm the findings. in this study we extended this treatment approach to on treatment nonresponders (defined as having detectable hcv-rna after at least weeks of soc). methods: so far, pts. hcv-rna pos. after weeks of soc ( male, female, genotype : ; genotype a: , with cirrhosis) participated in this protocol; were treatment naïve pts, relapser to two previous therapies ( and weeks). mg/kg/d sil was given for days, soc was continued. hcv-rna was quantified by taqman (roche diagnostics, usa) at monthly intervals on standard treatment and weekly after starting sil. results: all patients received at least weeks of soc, at week had a log drop < , two patients had detectable but unquantifiable hcv-rna (< iu/ml). after days of sil all had undetectable hcv-rna, in one hcv-rna increased to iu/ml and recived after a second course of sil. .all patients are still on soc and are hcv-rna negative. conclusion: sil iv. is an effective "rescue treatment" for on treatment nonresponders to full dose of peginterferon/ribavirin combination therapy. poster exhibition -imaging modalities poster session, hall b background: levovist-enhanced ultrasonography using subtractions makes it possible to depict the perfusion of hyperechogenic nodules. our institution performs sonazoid-enhanced ultrasonography using a toshiba aplio that is set to a ps low images, as generally recommended. the resulting images, however, are difficult to evaluate the kind of staining image that is obtained from a hyperechogenic nodule. these staining images were then compared to advanced dynamic flow (adf) images of a hyperechogenic nodule recorded using levovist-enhanced ultrasonography. methods: the subjects were five nodules who had undergone sonazoid-enhanced ultrasonography. two patients had experienced a recurrence of hcc after tace, while three patients had a hyperechogenic nodule of hcc that had never been treated. one patient with hcc after tace was imaged at a ps low. the second patient with hcc after tace and the three patients with hcc showing a high echoic nodule, were imaged using adf. results: in the patients with hcc after tace, the remaining tumor was difficult to observe in both the vascular phase and the kupffer phase taken at a ps low. in the other patients, however, images taken using adf clearly showed the residual tumor. also, with regard to the findings from the perfused images obtained from the three patients with hyperechogenic nodules of hcc, the hcc was more easily detectable in the adf images than in those taken at a ps low. conclusion: hyperechogenic perfused nodules are easier to identify in images taken using adf than in images taken using ps low. y. komorizono , t. shibatou , k. sako nanpuh hospital background: this study aimed to evaluate the usefulness of sonazoid enhanced radiofrequency ablation under real-time virtual sonography (rvs) guidance in a series of patients with hepatocellular carcinoma (hcc). method: twenty-five patients with a solitary hcc tumor measuring < = . cm in greatest dimension were enrolled in this study. eight patients received an initial treatment, seven also received an additional treatment for local recurrent tumors, and the remaining ten had distant recurrent tumors. all patients were easy to scan by multiple detector ct (mdct), but not by conventional ultrasound ( conclusions: the combination of the rvs system with sonazoid-enhanced us appears to have a high potential for use on patients that are difficult-to-scan by us examinations for percutaneous radiofrequency ablation. background & aims: contrast enhanced ultrasonography (ceus) with sonazoid can be expected to be useful not only for detection of tumor but also for us guided ablation therapy because kupffer imaging lasts for long time. the aim of this study is to investigate the usefulness of sonazoid in rfa for hcc. material & methods: a total of hcc nodules in patients admitted to receive rfa were studied. the detection ability of hcc was compared between ceus and conventional us using dynamic ct as reference standard. the effectiveness in the treatment was assessed by comparing the mean numbers of treatment session of rfa in patient treated with ceus assistance and that in historical controls matched for tumor and background conditions. results: the detection rate was . % in conventional us and . % in ceus (p= . ). sixty-nine nodules in patients were not detected by conventional us and detected after injection of sonazoid. the mean increase in detected tumor number with contrast enhanced us were well correlated with serum albumin level (p= . ). ceus was not superior to conventional us in patients with low albumin level. the mean number of session was . ± . as compared to . ± . in the historical controls (p= . ). conclusions: ceus with sonazoid is useful for detection of tumor in patients with well-conserved hepatic reservoir. the decrease in the mean number of sessions compared to historical controls suggested that sonazoid is an excellent supportive agent in rfa treatment of hcc. direct measurement of peri-operative change in portal blood flow and pressure is difficult in human. in the present study, computational simulation of pre-and post-operative portal blood flow and pressure was performed using computational flow dynamic (cfd) software in patients with primary liver cancers. methods: patients with fibrotic or non-fibrotic livers were analyzed. according to preoperative md-ct, mesh models of portal branches were constructed. cfd software (fluent . , fluent inc.) was employed for flow simulation. on the fluent . , changes in flow dynamics in the remnant portal branches were simulated by virtual cutting of an interested portal branch. the simulation was also performed days after the operation using dicom data obtained at that time. results: relative increase in blood flow in each remnant portal branch was not uniform throughout the liver in each patient. the sudden increase in portal pressure just after the virtual cutting of interested portal branch was almost normalized by day in non-fibrotic liver according to the flow simulation, while the increase in fibrotic liver did not return to the pre-operative values by day . these results suggest that responsive dilatation of remnant portal branches and subsequent regional regeneration could normalize the sudden increase in portal pressure after surgery in non-fibrotic livers, while the mechanism is impaired in fibrotic livers. discussion: computational flow dynamic simulation is useful to analyze the differences in the peri-operative portal flow dynamics and liver regeneration between non-fibrotic and fibrotic livers. aim: to determine if roi analysis can characterize washout in hepatocellular carcinoma (hcc) better than visual analysis. methods: surgically proven hccs from a single institution were studied. the patients' gender, age, date of scan, date of surgery were recorded. patients with pre-operative triphasic (n= ) and quadriphasic ct scans (n= ) were included. a representative section containing the lesion was selected for each case. the hu change between the precontrast and arterial (huabsolute hypervascularity) and the hu change between the peak attenuation and late portovenous phases (huabsolute washout) were recorded. cases were deemed positive if the hu change was more than the standard deviation ( hu). this was compared against visual analysis to determine if our method would increase sensitivity of ct for hcc. results: the mean patient age was . years (range to years); there were males and females. the mean duration between surgery and the scan was . days (range to days). peak enhancement was seen in the early portal venous phase in . % cases. the mean huabsolute washout was . hu (range - - ). roi analysis detected / cases ( . %). this was . % more than visual assessment, which detected / cases. this was statistically significant (p= . ). conclusion: visual assessment of lesion density is subjective. quantitative measurement of lesion attenuation changes between scan phases is a simple and objective method that is more sensitive than visual assessment in determining lesion washout. background: abdominal ultrasonogram(usg) is a common available diagnostic tool to screen and follow up for hepatocellular carcinoma(hcc). but it has been reported that the specificity of ultrasonogram is high but the sensitivity of it is insufficient. we investigated the characteristics of hccs that was missed in the usg but was detected in the ct. methods: total patients who were diagnosed with hcc between december, and february, , were enrolled and analysed retrospectively. all patients were performed with a usg prior to a spiral ct. the period between usg and spiral ct was limited within month. we investigated age, gender, cause(hbv, hcv, alcohol), the size of hcc(the length of long diameter), stage(modified uicc), child-pugh grade, cirrhosis, tumor number, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns), and the tumor location at segement as the possible related factors. results: the mean period between usg and spiral ct was . ± . days. the diagnostic accuracy rate to hcc was . %( / ). there was no interobserver variation. in analysis of associated factors, there was no statistical significance in age, gender, cause(hbv, hcv, alcohol), stage(modified uicc), child-pugh grade, cirrhosis, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns) (p > . ). there was statistical correlation in the tumor size less than cm, the solitary tumor and location at segement . (p > . ). conclusion: tumor size less than cm, solitary lesion and location at segment are significant factors to miss hccs in usg diagnosis. s. somani , a. somani , a. jain , v. dixit suvidha, navjeevan hospital, background: histopathological examination is required in the evaluation of various liver diseases for both diagnosis and prognosis. earlier blinded percutaneous liver biopsy was done commonly but now there are various studies suggesting that sonographic guided percutaneous liver biopsy could be more precise and safer. our aim was to compare the safety and diagnostic utility of sonographic guided versus blind percutaneous liver biopsy. methods: it was a retrospective single center study done between june and may . trucut liver biopsy needle was used in all patients. demographic, clinical and histological characteristics between the two groups were evaluated. insufficient biopsy was defined as a sample with less than portal spaces. we reviewed the type of complications and if hospitalization was required, or any mortality related to the procedure. results: out of liver biopsies done in this period after excluding patients we included patients, in group a( %, blind approach) and in group b ( %, sonographic guided approach). mean age was ± . years and male: female ratio was . : . biopsy was sufficient in % in group a and % in group b (p < . ). minor complications occurred in % in group a and % in group b which was not significant. major complications occurred in . % in group a and . % in group b which was statistically significant. mortality was . % in group a and . % in group b which was statistically significant. conclusion: our study suggest that sonographic guided percutaneous liver biopsy is superior in the diagnosis of liver diseases in all aspects when compared to blind approach as it is more safe, has more diagnostic utility with significantly less complications and mortality. poster exhibition -liver fibrosis poster session, hall b background/aims: hmg-coa reductase inhibitors have been shown to reduce hepatic stellate cell proliferation and collagen production and decrease oxidative stress and hepatic vascular tone in cirrhotic patients. therfore, the aim of the present study was to examine whether the lipid lowering agents atorvastatin (ato) or rosuvastatin (ros) would prevent experimentally-induced acute or chronic hepatic damage in rats. methods: liver cirrhosis was induced by thioacetamide (taa, mg/kg, i.p.) twice a week, for weeks. acute damage was induced by two consecutive taa injections ( mg/kg in a h interval). rats were treated concurrently with taa only or taa and either ato or ros daily by nasogastric gavage. another group was treated with taa+pentoxifyline (ptx), an agent with known antifibrotic effect through a different mechanism and served as positive control. results: presented in the conclusions: the lipid lowering agents used in our study had no effect on the development of acute or chronic hepatic damage in rats or on oxidative stress induced by taa. purpose: the development of hepatic fibrosis in patients with chronic liver disease increases the risk of liver cancer. the present study was conducted to determine whether an easily performed myocardial examination technique can be applied to the assessment of hepatic fibrosis. strain rate imaging is a new method based on tissue doppler imaging (tdi). the usefulness of strain rate imaging in assessing the degree of hepatic fibrosis was evaluated. this time, it mede comparative study with fibroscan in cases. methods: strain rate imaging was performed using a diagnostic ultrasound system (aplio tm , toshiba medical systems corporation, tochigi, japan) in a total of subjects: in the chronic hepatitis group, in the cirrhosis group, and in the normal control group. tdi-q, the tissue doppler analysis software installed in the aplio system, was used for analysis. measurement was performed five times from the epigastrium, with the roi size set to mm and the derivative pitch to mm. results: (i): both scores were largely reproducible among the different laboratories. however, compared to the histological findings, the error ratio was % for all results calculated by fibrotest and actitest. (ii): calculated scores varied among f ( %), f ( %), f -f ( %), and f ( %) (fibrotest), as well as a /a ( %), a ( %), a -a ( %), and a ( %) (actitest). results: the mean strain value was . in the chronic hepatitis group, . in the cirrhosis group, and . in the normal control group.the correlation was not thought to be fibroscan. conclusion: the results of the present study suggest that this noninvasive method permits quantitative assessment of the degree of hepatic fibrosis to be performed easily and in a short time. it is expected that the accuracy of the strain rate imaging method in determining the degree of hepatic fibrosis will be improved when it is used in combination with histological examination. conclusion: despite reproducibility of fibro-and actitest results among the six laboratories, large scale investigation (n= ) displayed increasing variability of the results depending on interlaboratory differences that were still in a quality controlled, analytically acceptable range. furthermore, calculated scores coincided with histological findings only in less than % of all cases. thus, the diagnostic accuracy of these tests seems low, if histology is accepted as gold standard. background: current knowledge attributes connective tissue growth factor (ctgf/ccn ) a crucial role in enhancing tgf-actions during hepatic fibrogenesis. recently, we demonstrated that caffeine leads to an upregulation of ppar in hepatocytes, thus sensitizing these cells to the well known inhibitory effect of -deoxy- , -prostaglandin j ( -d-pgj ) on ctgf expression. however, upregulation of the receptor alone is not sufficient per se, its physiological ligand -d-pgj is required for exerting its inhibitory effect on ctgf synthesis. aim and methods: this study compares serum concentrations of -d-pgj in caucasian patients with fibrotic liver diseases (n= ), caucasian controls (n= ) and caucasian non-liver disease sick (n= ), as well as of chinese patients with hepatocellular carcinoma (n= ) and chinese healthy controls (n= ) in order to characterize their suitability for therapeutic approaches with ppar inducing (i.e. ctgf inhibitory) drugs such as caffeine. results: presented data show that caucasian patients with ongoing hepatic fibrogenesis (mean . ± . µg/l) display impressingly higher serum concentrations of -d-pgj than healthy probands (mean . ± . ) and caucasian patients with non-liver disease (mean . ± . µg/l). similar results are found in chinese patients with fully developed hcc (mean . ± . µg/l) compared to chinese healthy controls (mean . ± . µg/l). we identified the predictors of tumor recurrence using cox-regression model. introduction: non-invasive, i.e. serum-based multiparametric panels of biomarkers have been proposed for the diagnostic assessment of liver fibrosis. aims/methods: (i) haptoglobin, alt, ggt, alpha -macroglobulin, apolipoprotein a and bilirubin in sera of patients with histological proven fibrosis (f -f , a -a ) were determined in different quality-controlled laboratories. interlaboratory variations of the calculated fibrotest score for staging and actitest score for grading (both biopredictive tm ), and their error ratios compared to biopsy results were calculated. (ii) the variability of obtained fibrotest/actitest scores depending on differential combinations of the allowed analyt-specific maximum/minimum permissible values as determined by the external quality control of the german association of laboratory medicine was determined and the frequency distribution of the results calculated. results: a total of patients (mean age, . ± . years; male, . %) were included. median follow-up duration was . months (range, . - . ) and patients ( . %) experienced local tumor recurrence during the observational period. multivariable analyses showed that low p /ms level (relative risk, . ; % confidence interval [ci], . - . ; p= . ) and serum alpha-fetoprotein level > ng/ml (relative risk, . ; % ci, . - . ; p= . ) were independent risk factors for tumor recurrence. patients with p /ms level < . revealed . -fold ( % ci, . - . ; p= . ) increase in the risk of recurrence after adjustment for serum alpha-fetoprotein level, as compared to those with p /ms level > . . however, tumor size, child-pugh score, and hepatitis b virus dna level failed to significantly affect the time-to-recurrence. conclusion: our study suggests that lower p /ms value, which means more severe liver fibrosis, is an independent predictor for hcc recurrence after rfa. background/aims: despite of its high prevalence, osteoporosis is an underestimated complication of liver cirrhosis. the aims of this study is to prove the prevalence of osteoporosis and osteopenia in patients with liver cirrhosis and to identify the principal risk factors associated. methods: the prevalence of osteoporosis and osteopenia was studied in patients with alcoholic or viral liver cirrhosis who were admitted to the institute of gastroenterology and hepatology, cnuh between march and september . osteoporosis and osteopenia was evaluated by measuring their bone density using dual energy x-ray absorptiometry (dexa) at lumbar spine and femoral head. the variables taken into consideration were: sex, body mass index (bmi), presence of cholestasis, severity and duration of liver disease. results: total patients (male and female , respectively) were estimated for association of liver disease and osteoporosis. of these, patients were estimated for bone density of lumbar spine and neck of femur by dual x-ray absorptiometry (dexa). morning blood samples were taken for hormonal and biochemical analysis from all patients. among patients, patients ( %) were found to have osteopenia or osteoporosis. there was no statistically significant correlation between age, bmi, severity and duration of liver disease, pth, vitamin d, alp and igf- . conclusion: there is high prevalence rate of osteopenia or osteoporosis in liver cirrhosis. although the causes of osteopathy are heterogeneous, the early diagnosis and treatment of osteopathy in patients with liver cirrhosis is important. background: to build and to evaluate mathematical models for predicting liver fibrosis progression by using conventional laboratory indicators in chronic hepatitis b. methods: liver biopsy and routine laboratory tests were performed in patients with chronic hepatitis b. using multiple logistic regression to analyze evidently relevant indicators, then the predicting models were built and analyzed by roc curve. results: after spearman analysis, factors such as age, platelet count(plt), international rate(inr), total bilirubin(tbil), albumin(alb), aspartate aminotransferase (ast), gamma glutamyltranspeptidase (ggt), total bile acid(tba) and cholinesterase(che) were found to be correlated with liver fibrosis p . . three models (s , s , s= , respectively) were built by plt, inr, alb, ggt and che, which were independent predictors after multiple logistic regression analysis.finally, fibrosis score (fs) was calculated to predict different liver fibrosis stages. roc curve analysis revealed that the auc of fs was . in model (s ), . in model (s ) and . in model (s= ) fig .the cut-off fs in model was at . with . % sensitive, . % specificity and the accuracy was . %. the cut-off fs in model was at . with . % sensitive, . % specificity and the accuracy was . %. the cut-off fs in model was at . with . % sensitive, . % specificity and the accuracy was . %. conclusions: the predicting models, built by using conventional laboratory indicators, have fairly well value for diagnosing hepatic fibrosis or hepatocirrhosis in chronic hepatitis b. background: to investigate the effect of liver cirrhosis on the development of atherosclerosis in the rabbits chronically fed with high fat diet. methods: normal male new zealand white rabbits were randomly divided into four groups: a control group, a high fat diet group, a carbon tetrachloride (ccl ) group and a complex group. pathologic changes in ascending aortas and livers were observed. the levels of serum alanine aminotransferase alt , lipid, c-reactive protein (crp) were also determined. results: significant hepatic steatosis, inflammation and fibrosis could be observed in the three treatment groups; while atherosclerosis and typical arteriosclerotic plaques in ascending aortas could only be observed in the two high fat diet groups. compared with the control group, serum alt and lipid levels in ccl group were increased significantly (p< . ), but no difference of arterial intima-media thickness (imt) and i/m ratio between these two groups. the levels of serum alt, lipid, crp and imt in two high fat diet groups were significantly increased compared with the control group (p< . ). the level of serum alt in the complex group was significant higer than that in the high fat diet group, but the i/m ratio was just opposite (all p< . ), and there was no difference of imt between the two groups. conclusions rabbits treated with ccl can elevate serum lipid levels, but can not induce atherosclerosis. though the activity of liver inflammation was aggravated in the complex model group, it has no effect on atherosclerosis possibly partly because of malnutrition. higher values of liver stiffness in males with mild chronic hepatitis c c. stern , a.c. cardoso , r. moucari , a.d. pumpo , n. giuily , p. bedossa , p. marcellin hopital beaujon background/aim: liver stiffness (ls) measured by fibroscan (echosens) is a noninvasive method to assess liver fibrosis in patients with chronic liver diseases. we evaluated the impact of factors on ls results in mild chronic hepatitis c (chc). methods: chc patients with metavir fibrosis stage at liver biopsy and a reliable ls exam were eligible. all patients had no prior antiviral treatment. the ls values were compared to clinical and biochemical data. results: patients were included with the following characteristics: mean age , male gender ( %), mean bmi . , median ls . kpa ( . - . ), diabetes ( %), genotype ( %), metavir activity a ( %), a ( %), steatosis at biopsy % ( %), mean glucose . , abnormal alt ( %), abnormal ggt ( %), homa ( . ). the ls values were associated wtih male gender (median . in males vs . in females) (p= . ), bmi (p= . ), alt (p= . ), ggt (p= . ) and glucose levels (p= . ). no association was found between ls and activity stage (p= . ) or steatosis (p= . ). in the linear regression, the only factor independently associated with higher ls was gender (p= . ). in men, higher ls was related to levels of alt (p= . ), but not to necro-inflammation grade (p= . ). in women, ls was not associated with alt levels, but with bmi (p= . ) and ggt levels (p= . ). conclusion: in patients with mild chc, liver stiffness values are higher in males. these results suggest that different cut-off for fibrosis stage should be proposed according to gender. aims: to investigate the effects of shuanghu qinggan granule(sqg) on prevention and treatment of hepatic fibrosis induced by carbon tetrachloride in rats. methods: sd rats were divided into groups, normal control groupamodel groupb, sqg largec , middlec small dose groupsc and silymarin positive contrast groupd. the rats of bc c c d were injected with carbon tetrachloride for weeks. the rats of c c c were then administered with sqg for weeks. the rats of d were then administered with silymarin for weeks. results the liver structure of rats of b was severely damagedlarge amount of liver cells became obviously degeneratedand hepatic veins were clearly congested. the hepatic cells fatty degeneration and infiltration of inflammatory cells in rats of c c c d reduced significantly. there was no fiber hyperplasia in liver tissues of rats of c c c d. blood serum ha cp p levels in rats of b were significantly higher than those in ac c c d. conclusion: sqg has remarkable therapeutic effects on rats with hepatic fibrosis induced by carbon tetrachloride, the higher the dosage of sqg was, the more effective the results would be. conclusions: none of sophisticated biomarkers had value in addition to readily available laboratory data for the prediction of significant fibrosis in hbeag positive patients. two markers out of sophisticated biomarkers provide additional diagnostic information in hbeag negative patients. before new biomarkers are accepted, their superiority to routine laboratory data should be meticulously appraised. objective: to evaluate the efficiency and safety of "tinmax" hb- herbal compound (cpd) in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. methods: a double-blind randomized method was employed. patients of hepatofibrosis or cirrhosis post hepatitis b were separated into study group ("tinmax" hb- group) and control group (natural vitamin group) by randomized method. the course was weeks. patients visited once every weeks and the last visit at weeks after the cessation of treatment. part of patients had liver biopsy before and after treatment. before, during the course and at the end of therapy, clinical symptoms and physical signs were evaluated, hepatic function, and serum markers of hepatofibrosis (such as hyaluronate acid, laminin, serum type iii procollagen and collagen iv) were tested, and ultrasound evaluation was performed. results: patients enrolled in the evaluation. patients completed the evaluation according to the protocol. patients had liver biopsy twice, from the study group and from the other one. at the end of therapy, the total effective rate of hepatofibrosis in histopathology is . % in the study group, much higher than that of . % in the control group (p< . ). the total effective rate of serum markers of hepatofibrosis at the end of therapy in the study group was . %, much higher than that of . % in the control group (p< . ). the total effective rate of non-invasion markers of hepatofibrosis at the end of therapy in the study group was . %, much higher than that of . % in the control group (p< . ). the drugs of adverse event had not happened in both groups. conclusion: "tinmax" hb- herbal compound (cpd) is effective and safe in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. w.h. sha , xiaohui zeng , yuyuan li gi department, first municipal hospital of guangzhou, guangzhou aim: to investigate the clinical value of serum indices for hepatic fibrosis in chronic liver diseases. methods: competitive radioimmunoassay was used to determine the serum level of collagen type ( c), laminin (ln) and hyaluronic acid(ha) in patients with different severity degree of chronic liver diseases, and in healthy subjects. results: the serum levels of c, ln, and ha in the patients with liver diseases increased to different extent, compared with those in the healthy subjects. of which the highest of c, ln, and ha were found in the patients with primary carcinoma of liver or hepatocirrhosis and the serum level of ha is highlight. the combination detection of serum c, ln, and ha is more valuable than single index. conclusion: joint detection of serum c , ln, and ha is of higher significance in clinical diagnosis and prognosis of hepatocirrhosis, and is also available for successive observation on the development of liver diseases. aims: to investigate the mechanism of fuzheng huayu decoction (fzhy) on hepatic stellate cells (hscs) activation relating to tgf- signal transduction pathway. methods: hscs were isolated from normal rats by in situ pronase/collagenase perfusion followed by density gradient centrifugation. at day after isolation, cells were stimulated with pm tgf- for h, then incubated with % fzhy pharmacological serum or m t r -i inhibitor (sb- ) for h. protein expression of -sma, smad was assayed by immunofluorescent stain; total protein expression of -sma, t r -i, smad / and nuclear expression of smad was analyzed by western blotting. results: fzhy pharmacological serum significantly decreased expression of -sma, t r -i, and inhibited smad nuclear expression and translocation in tgf- stimulated hscs. conclusions: fuzheng huayu decoction can prevent hscs activation through tgf- signaling transduction pathway in hscs, which may be the important molecular pharmacological mechanism of fuzheng huayu decoction action against liver fibrosis. background: fatty liver disease has become a health problem related to metabolic syndrome worldwide although its molecular pathogenesis has remained further studied and it is unclear whether advanced fibrosis induced by steatohepatitis will regress when diet is controlled. aim of this study is ) to study the involvement of endoplasmic reticulum stress (er stress) in the occurrence of seatohepatitis and ) to obtain the evidence of resolution of fibrosis by changing the diet. methods: non-alcoholic steatohepatitis with advanced fibrosis was produced in rats by giving methionine-choline-deficient diet (mcdd) for weeks. methionine-choline-control diet (mccd) instead of mcdd was given for the last weeks in an experimental group. fibrosis and inflammation was determined by several tissue stainings. gene expression related to fibrosis and inflammation was determined by immunoblotting and real-time pcr. expression of caspase- , caspase- , and glucose-regulated protein was evaluated to clarify the presence of er stress aim against liver fibrosis relating to hypoxia and angiogenesis regulation. methods: the rats were divided into normal, model, sa-b and perin control group. rats in sa-b and perindopril group were administrated with sa-b and perindopril respectively. liver fibrosis was induced by ip dimethylnitrosamine (dmn) for w. fibrosis degree was observed by sirius red staining. col-i protein expression was analyzed by western blot; col-i , vcam- , icam- , hif- and vwf expression in liver tissue was checked by immunohistochemistry; gelatinase activities in liver tissue were detected by gelatin zymography and in situ flourescent zymography. result: compared to normal group, col-i, hif- , icam- , results: ) changing the diet from mcdd to mccd triggered the reduction in fat in hepatocytes, the decrease of inflammatory gene expression and oxidative stress, and the regression of fibrosis accompanied by the disappearance of activated stellate cells and macrophages. ) immunohistochemistry, immunoblotting, and rt-pcr analysis all indicated the occurrence of er stress in steatohepatitis while it recovered immediately after changing the diet from mccd to mcdd. vwf protein expression and gelatinase activity in liver tissue were increased obviously in model group, while sa-b and perindopril treatment significantly decreased these protein expressions and gelatinase activity. conclusions: this simple experiment clearly shows that the changing diet from steatohepatitis-causing mcdd to mccd triggers the resolution of inflammatory and fibrotic reaction in the liver, suggesting that food intake is a very important factor for controlling the state of fat and pathology of the liver. er stress is involved in the process. background: liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix proteins, which is a characteristic of most types of chronic liver disease. under injury conditions, hepatic stellate cells (hscs) are activated to transdifferentiate into myofibroblasts, which are capable of secretion of many connective tissue elements, especially collagens i, iii, and iv. gynostemma pentaphyllum is a popular folk medicine that has been used for treatment of hepatitis in asia. gypenosides are the major saponins derived from g. pentaphyllum. in previous study, gypenosides have hepatoprotective and anti-fibrotic activities in rat chronic liver injury induced by ccl , and anti-proliferative effect in rat isolated hscs. methods: in cultured hscs model, we detected type procollagen protein and mrna by western blot and rt-pcr. result: we found that g. pentaphyllum inhibited type procollagen protein expression in % at hours. furthermore, g. pentaphyllum also inhibited type procollagen and mrna expression in % and % respectively. in addition to transcriptional inhibition, we found that g. pentaphyllum also enhanced the degradation rate of type procollagen protein. base on the effect of enhancing protein degradation, we used some protease inhibitors like ca- me, z-fa-fmk, aebsf, tpck and tlck to identify the potential target of g. pentaphyllum. on the other hand, in the ubiquitin-proteasome system analysis, we quantified the change of some target proteins of proteasome in the presence or absence of g. pentaphyllum. conclusion: g. pentaphyllum reduced type procollagen protein by inhibiting transcription and enhancing protein degradation. aim: excessive oxidative stress in diabetic patients has been implicated in the pathology and complication of liver. the present study was designed to examine whether ginger has a direct hepatoprotective effect in diabetic cases. methods: wistar strain albino rats were selected for this study. the rats were divided into groups: (i) control, (ii) ginger treated ( mg/kg b.w. orally, days) (iii) diabetic ( mg/kg b.w., i.p.) and (iv) diabetic + ginger treatment. the lipid metabolic profiles such as total cholesterol, triglycerides, phospholipids and lipid peroxidation as stress markers and histopathological studies were carried out to assess the damage in hepatic tissue. results: ginger treated diabetic rats demonstrated significant reduction in glucose levels as compared to the nontreated diabetic animals. diabetic rats have shown increased total cholesterol, triglycerides, phospholipids and lipid peroxidation content in hepatic tissue compared to control, indicate prevailing of oxidative stress and alterations in fatty acid metabolism in these rats. further, degenerative changes of hepatic cells in diabetic group are minimized to nearness in structure by administration of ginger as evinced by histopathological examination. conclusion: we summarize that the hypolipidemic and antioxidant compounds present in ginger may be useful in delaying the complicated effects of diabetes. this results also reveal that ginger possess hepatoprotective properties in diabetic cases. anti-fibrotic action m. naime , s. ali hamdard university rhizomes of valeriana jatamansi (family, valerianaceae) have long been used in indian subcontinent by the traditional healers for the treatment of various diseases. this study provides experimental evidence suggesting the therapeutic effect of the crude extract of rhizomes on rat liver fibrosis, and demonstrates its antiproliferative role. crude extract ( % ethanolic) at a dose level of mg/kg body weight was administered to rats to study the effect on biochemical and other markers of liver fibrosis. administration of the extract for weeks could bring down elevated the levels of biochemical markers of liver injury, and modulate several other biochemical responses. morphology and hisopathological examination cooroborated with the biochemical changes, and indicated partial reversal of fibrosis. dpph assay confirmed the antioxidant property of the extract, which is suggested to be due to -ionone, -sitosterol and other chemical constituents. further, treatment could restore depleted glutathione level, inhibit lipid peroxidation, and inhibited elevated xanthine oxidase activity in fibrosis. the study also reports anti-tumour promotion activity of the extract as evident by a significant decrease in [ h]-thymidine incorporation by hepatic dna in extract treated rats. results suggest that v. jatamansi extract has curative effect and can partially reverse biochemical and histological changes associated with liver fibrosis. with chronic hepatitis c c. wongjitrat , s. chainuvati , a. manuyakorn , s. aroonparkmongkol , t. tanwandee mahidol university, background: leptin is a peptide hormone that mainly regulates food intake, energy expenditure and reproductive function. leptin also releases from activated hepatic stellate cells and may have a role in regulation of fibrogenesis and inflammation. in human chronically infected by hcv, the role of leptin-associated fibrosis of the liver is still unclear. there is no data in thai patients chronically infected by hcv regarding leptin level and its correlation with hepatic histology and fibrosis.the purpose of this study was to evaluate the relationship between leptin level and severity of liver fibrosis in thai patients chronically infected by hcv. methods: sixty-six patients ( men, women) with chronic hcv infection and liver biopsy was done within months were enrolled. fasting blood samples were obtained and serum leptin levels were measured by elisa. bmi, blood sugar, liver function test, lipid profile, hcv rna viral load and hcv genotype were also measured and related to histological findings. results: mean serum leptin levels were significantly higher in women than in male. there was a significantly correlation between serum leptin and bmi (r = . , p < . ). leptin levels were not associated with hepatic fibrosis (r = . , p = . ) and necroinflammation (r = . , p = . ). steatosis was significantly associated with severe necroinflammation (r = . , p = . ), but not fibrosis (r = . , p = . ). conclusions: these findings failed to demonstrate correlation of serum leptin and hepatic fibrosis in thai patients chronically infected with hcv. background and aim: liver cirrhosis is one of the leading causes of mortality in our country as well as in our region. even though deterioration of glucose metabolism and existence of insulin resistance in liver cirrhhosis has been well documented in many studies, it is still unclear how insulin resistance mechanism develops. the aim of the present study is to assess insulin resistance, cytokines and crp levels in patients with liver cirrhosis and control subjects. in additon, we aimed to investigate the relation of insulin resistance in liver cirrhosis with such parameters as age, sex, etiology, child-pugh classification, spleen size, tnf-?, il- ?, il- res, il- , il- , il- , crp and hs-crp. material and method: a total of patients with cirrhosis of different etilogy ( male, female) were included into the study. as controls, ( male and female) subjects were taken. the two groups were compared with each other in terms of glucose, insulin, c-peptid, homa-ir, tnf-?, il- ?, il- res, il- , il- , il- , crp and hs-crp levels. in the second part of our study, the liver cirrhosis group was divided into two subgroups: patients with homa-ir value > . as insulin resistance positive, and those with homa-ir value > . as insulin resistance negative. these two groups, i.e. , homa-ir positive and homa-ir negative, were compared in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il- ?, il- res, il- , il- , il- , crp and hs-crp levels. results: in liver cirrhosis group, glucose, insulin, c-peptid, homa-ir, tnf-?, il- res, il- , crp and hs-crp levels were determined to be significantly higher than controls. between patients with homa-ir positive and negative, however, statistically no significant difference was found in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il- ?, il- res, il- , il- , crp and hs-crp levels, but il- level was seen to be significantly low in patient homa-ir positive. conclusion: in patients with liver cirrhosis, the levels of glucose, insulin, c-peptid, homa-ir, tnf-?, il- res, il- , crp and hs-crp increase with respect to normal population. determination of increased homa-ir level in liver cirrhosis supports the view that insulin resistance develops in liver cirrhosis as reported in related studies. in the study, it was also determined that the mechanism of insulin resistance development occurs independent of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il- ?, il- res, il- , il- , crp and hs-crp levels. the determination of statistically lower level of il- in patients with homa-ir positive with respect to those with homa-ir negative does not indicate similarity with the studies carried out earlier. ) in patients ( . %) than in controls( %)in group i hla-b significantly increased in patients ( %)as compared to controls ( %) . in group ii hla -b significantly higher in patients ( . %)than controls ( %) also hla-aw significantly higher ( . %) in patients than controls ( . %).in group iii hla-aw significantly increased in patients ( . %) compared to controls.no significant association between hla antigens and cases with hbv or hcv infection. conclusion: the significantly high association of hla-aw and hla-b in patients with hepatic schistosomiasis as compared to normal controlstogether with the lack of any association with active intestinal schisto . antigens predispose to liver affection.individuals possessing hla-aw appear to be more prone to severeform of liver disease background: atp b mutation is one of the factors that result in cholestasis and progress to chronic liver disease, but has never been reported in the mainland china before. the aim of this study was to elucidate the role of atp b mutation in mainland chinese patients with progressive intrahepatic cholostasis and low ggt. methods: children who presented with progressive intrahepatic cholostasis and low ggt were admitted in a tertiary pediatric hospital in eastern china. abcb gene was analyzed firstly to exclude bsep deficiency. afterwards, all the encoding exons and their flanking areas of atp b gene were sequenced in the remaining patients in whom only one or no mutations of abcb were found. results: mutations of atp b gene were found in patients. i n had been reported in taiwanese patients with pfic , and the others were novel. p t and ivs + t g were linkage and found in of patients, including homozygote and heterozygote. liver biopsy had been performed in patients with atp b mutations and with abcb pe mutations. variety portal fibrosis was showed in patients with atp b mutations and patients with abcb mutations. giant cell transformation was detected in one patient with atp b mutations and patients with abcb mutations. laboratory of exercise biochemistry, taipei physical education college, jhongcheng rd., no. , sec. , taipei city- , taiwan, roc background/aim: generation of reactive oxygen metabolites are depends on the consumption of oxygen and their cumulative effects may be different from lean to obese population. this study was designed to investigate the deleterious effects of oxidants on hepatic antioxidant defence system in lean and obese rats under hypoxic condition. methods: zucker rats lean ( ± gms) and obese ( ± gms) were divided into control and acute hypoxia groups. the acute hypoxia treatment was performed in a hypoxic chamber at % oxygen consumption. objectives: to compare the diagnostic value of morning urine copper to zinc (copper/zinc) ratio and hour urinary copper excretion in wilson's disease (wd) children. results: in the results, acute hypoxia caused a significant (p< . ) decrease in major antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px), glutathione reductase (gr) and glutathione (gsh) content in lean groups when compare to their controls. the decrease in the activities of all antioxidant enzymes was also noticed in obese group with hypoxia treatment. however, this decrease was not significant in case of cat, gsh-px activities and gsh content. the mda levels (lipid peroxidation marker) were higher in obese rats compare to lean rats. methods: morning urine and hour urine were collected from patients over three years age who were hospitalized in a tertiary pediatric liver service. each patient was re-evaluated according to wd scoring system, and was assigned to one of the three groups: wd, suspecting wd, and non-wd. , , and cases were assigned to wd, suspecting wd, and non-wd respectively. urine copper and zinc concentration was determined simultaneously by using inductively coupled plasma mass spectrometry. conclusions: the higher hepatic mda values observed in obese rats indicate that accumulation of free radicals may be more in obese rats thus leads to promote the lipids oxidation. from this study it is concluded that decrease of antioxidant enzymes (except gr) with acute hypoxia treatment were more in lean group compared to with that of obese group. results: the morning urine copper/zinc ratio and hr urinary copper excretion correlated well (r= . , p < . ). the median of morning urine copper/zinc ratio, hr urine copper/zinc ratio, h copper excretion, and h zinc urinary excretion were . , . , . and . in wd group, and . , . , . and . in the non-wd group respectively. the differences of morning urine copper/zinc ratio, hr urine copper/zinc ratio, and h copper excretion were significant (z-value - . , - . and - . respectively, all p values < . chd l is a recently discovered oncogene localized at q , one of the most frequently amplified chromosomal regions in hcc. herein, by yeast-two hybrid assay, we demonstrate that the anti-apoptotic ability of chd l is associated with its interaction with nur , a critical member of a p -independent apoptotic pathway. as the first cellular protein identified to bind nur , chd l inhibits the nucleus-to-mitochondria translocation of nur , and subsequently hinders the release of cytochrome c and the initiation of apoptosis ( figure ). further study found that c-terminal macro domain of chd l is responsible for the interaction with nur , and a chd l mutant lacking residues - failed to interact with nur and prevent nur -mediated apoptosis. we also find that chd l confers cellular chemoresistance to drugs that induce apoptosis via the nur -mediated pathway, which may lead to the identification of new therapeutic targets for hcc treatment. background/aim: accumulation of oxidative damage to proteins, lipids and mitochondria could increase with advancing of age. the current study was aimed to test the hypothesis that swimming exercise training could revert the age dependent oxidative damages in liver. methods: sprague-dawley rats of young ( months) and old ( months) were divided into four groups; young control (n= ), young exercise (n= ), old control (n= ) and old exercise (n= ). minutes of swimming exercise was given to the exercise group for a period of two weeks. results: the estimated antioxidant enzyme activities including, superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px) and glutathione reductase (gr) were decreased with age and significantly (p< . ) increased with exercise training. however, elevated protein carbonyls and mda levels were noticed in old animals, which indicate that old liver had greater accumulated oxidative damages. the significant drop in protein carbonyl content and increase in mitochondrial succinate dehydrogenase (sdh) activity was observed with response to swim training in old rats. conclusions: this data implied that swim exercise training could revert the oxidative damages in liver. this was also proven by enhanced antioxidant enzyme status with response to exercise training in old rats. to sum-up these results it is cleared that age induced detrimental effects to the liver might be reversed by regular swimming exercise training in old rats. results: peg was expressed in l /peg (fig. ) . peg accelerated the growth of l . after treatment with mm h o for h, the inhibitory rate of l /peg cells was . %; the chromosomal condensation and ladder-like dna fragmentation were not observed (fig. ) . methods: hepg or hepg . . were co-cultured with jurkat cells, with blocking test by adding anti-pd- antibody. the pd- expression was detected by flow cytometry (fcm); cytokines in culture supernatant in blocking groups and controls were measured by enzyme-labeled immunosorbent assay (elisa); cytotoxic test of t cells were measured by methyl thiazolyl tetrazolium (mtt). conclusions: over-expression of peg can significantly promot l proliferation and ameliorate apoptosis-inducing effects of h o on l . results: the pd- expression on jurkat cells was induced by hepatoma cells, the expression rate were . ± . % (by hepg ) and . ± . % (by hepg . . ), respectively. the cytokines il- level ( . ± . pg/ml), inf-level ( . ± . pg/ml) and il- level ( . ± . pg/ml) in culture supernatant of blocking groups were significant higher than that of controls (il- , . ± pg/ml, inf-, . ± . pg/ml and il- , . ± . pg/ml, respectively. p < . ). the cytotoxic test (od value) was markedly higher in blocking group ( . ± . ) than that of control group ( ± . p< . ) . conclusion: the pd- expression on lymphocytes can be induced by hepatoma cells, and cytokines expression and cytotoxic test were recovered by blocking pd- /pd-l interaction. background: hedgehog (hh) pathway is well known as a positive regulator for tissue construction( during development) and reconstruction (in adults). our aim to observe the expression change of hh pathway on rat hepatic regeneration . materials and methods: adult male sprague-dawley rats underwent approximately % partial hepatectomy (ph) or sham operation (so). liver specimens were collected at , , , , , , , and h after ph or so. hedgehog expression was determined in mrna level by rt-qpcr as well as in protein levels via immunohistochemical staining and western-blotting. results: so treatment did not induce remarkable changes in hedgehog expression; however, the level of transcript for hedgehog was significantly upregulated after ph. we found sonic hedgehog(shh )and glioblastoma (gli - ) mrna expression in the regenerating liver arrive at its peak at as early as h and returned to its physiologyical level h later. it is similar to the change of proteins (shh and gli ) .as seen from immunohistochemistry experiments; shh protein was expressed uniquely in regenerating hepatocytes. similarly, ph induced over expression for shh protein occurred from h with a peak level at h after surgery. but gli protein mainly located in nucleus and no significantly changes in the phrase of liver regeneration. conclusion: hedgehog pathway may play a role in the activation of hepatic proliferation during liver regeneration induced by physiological stress or pathological states, such as ph. background: to investigate whether peg , an imprinted gene with an active paternal but silent maternal allele, was involved in hydrogen peroxide (h o ) induced cellular apoptosis. methods: peg gene was stable transfected into l . cellular gene expression was determined by rt-pcr, western blot and immunocytochemistry. cell proliferation was analyzed by mtt. after treatment with different concentrations ( - mm) of h o , cell proliferation inhibition rate was measured by mtt. morphological changes of apoptotic cells were determined by hoechst staining, dna fragmentation was observed by agarose gel electrophoresis. hua tang , xiao-yan tang , min liu , xin li tianjin life science research center, tianjin medical university, tianjin , china we determined how afp modulates the proliferation of hepatoma cells. a recombinant adenovirus expressing sirna against afp (adv-afpsirna) was created and found that it reduced expression of afp specifically in hepatoma cells, and markedly inhibited the proliferation of hepatoma cells in vitro. local treatment using adv-afpsirna caused significant repression of the growth of hepatoma derived hepg cells in xenograft in nude mice. knockdown of afp resulted in an obvious delay in the g /s transition of cell cycle, but did not affect apoptosis in hepg cells, as analyzed by flow cytometry and tunel assay. also, differential expressions of some genes related to the cell cycle, including skp , cyclin d , csk and ebag were identified by microarray and rt-pcr in hepg cells and hepg cells with knocked down afp. these results suggest that endogenous afp is a critical determinant of the growth of hepatoma cells. hematopoietic stem cell (cd +) therapy can improve liver function in patients with cirrhosis. these cells can be mobilized into peripheral blood using granulocyte colony stimulating factor (gcsf). this study was undertaken to assess feasibility and safety and of gcsf induced cd + cell mobilization and its impact on liver function in patients with cirrhosis. patients with liver cirrhosis (cryptogenic or alcoholic with m abstinence) with cpt > and < , and splenic diameter < cm were included. gcsf injection was given for days ( mcg/kg/dose). baseline & day- cd + counts in peripheral blood were done by flow cytometry. follow up was weekly for weeks and then monthly. cpt was compared at baseline and months. patients (median age y, range - y, males; etiology: alcohol, cryptogenic; median cpt , range - ) were included. cd + cell counts at baseline and day were ( - ) and ( - ) respectively (median, range). side effects were fever in , allergic reaction in and increase in splenic size in (excluded). in follow up, patients died ( , & m after therapy, after olt), lost to follow-up. patients showed improvement in - ) at -month follow-up. gcsf treatment is safe and yields adequate cd + cells in peripheral blood. in short term it results in improvement in liver function in patients with cirrhosis pe molecular cloning and transcriptional analysis of kctd gene promoter b. pi , j.s. wang , m.f. han , y.y. zhou , x.j. liu , x.p. luo , q. ning department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, department of pediatrics, tongji hospital, tongji medical college, huazhong university of science and technology aim: our previous work has shown that high expression of kctd , a potassium channel associated gene, correlated with the disease severity of patients with severe chronic hepatitis b(schb). to further understand the gene transcription and regulation, kctd promoter was cloned and gene transcription was studied. methods: a full length of isolated promoter and series of ' truncated promoter of kctd gene was subcloned into the luciferase report vector pgl -basic to form the promoter-report constructs. the kctd promoter-report construct upstream of the luciferase report gene was cotransfected with constructs expressing hbv x,c and s protein respectively or stimulated with cytokines (il- , ifn and tnf ) in t cells to investigate kctd gene regulation upon both viral factors and host cytokines. result: a bp kctd segment upstream of atg translation start site was evidenced to contain potential regulative domains. an important regulation site located between - bp and - bp upstream of atg translation start site. based upon the luciferase activity assay, il- was able to upregulate the transcription of kctd whereas there was no effect from neither hbv viral proteins nor ifn and tnf . conclusion: here we first successfully cloned the full length promoter of kctd . il- significantly enhanced the transcription of kctd , a gene which has been shown to be involved in t cell activation and disease severity of schb from our group. this work was supported by nsfc( , , ) istanbul university, cerrahpasa medical school, marmara university, okmeydani teaching hospital, background: the treatment in chronic hepatitis c virus (hcv) is not highly effective, and cost, duration, and side effects are challenging. predicting favorable factors of response to treatment would make it possible to give it only responsive patients. recent studies report more conclusive results about the role of apoptosis in inflammation and fibrosis seen in chronic viral hepatitis. hepatocyte damage in hcv is mediated by cytotoxic t-cells. apoptosis primarily developed by the interaction between fas antigen on hepatocyte and fas ligand on t-cell corresponds to a main mechanism for hepatocyte damage. methods: in this study, we aimed to detect any relationship between apoptotic markers (fas, fas ligand, fas-associated death domain, caspases , , and , insitu apoptosis) in liver biopsy taken before the treatment and response to the treatment of interferon+ribavirin. additionally, any relationship between these parameters and the other ones predicting the response to therapy including alt level, viral load, genotype, and gender were studied. results: the study includes the patients in centers managing chronic hcv infection. all parameters were studied in patients. study results revealed that histological activity index is correlated with cd staining density, caspase intensiveness, and portal and parenchymal fas ligand scores. fibrosis is also seen to be correlated with the same parameters. apoptotic parameters of the responsive cases were not significantly different from unresponsive ones. conclusion: apoptotic parameters studied in the liver tissue is associated with inflammation and fibrosis, however these parameters may not predict the response to the treatment. s. gao , d. xi , j.w. guo , c.l. zhu , x.p. luo , q. ning department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, department of pediatrics, tongji hospital, tongji medical college, huazhong university of science objective: this study was designed to explore the opportunity of microrna interference technique in the inhibitory application of human fgl , human fas and tnfr expression. methods: the eukaryotic expression plasmids of human fgl , fas and tnfri genes were constructed and have successfully expressed hfgl , hfas and htnfri protein. mirna expression plasmids of hfgl , hfas and htnfri complimentary to the sequence responsible for hfgl , hfas and htnfri respectively were also constructed, meanwhile an irrelevant mirna plasmid was used as control. by respective cotransfection of p-hfgl mirna and pcdna . -hfgl , p-hfasmirna and pcdna . -hfas, p-htnfri mirna and pcdna . -htnfri expression construct into t cells, the inhibition of hfgl , hfas and htnfri expression were analyzed by quatitative real time pcr and western blot. results: the experiments showed the significantly inhibitory effect of p-hfgl mirna on hfgl , p-hfasmirna on hfas and p-htnfri mirna on htnfri expression at h post-transfection both at rna level and at protein level, as well in t cell lines the inhibitory efficiency reached as high as . % for hfgl , . % for hfas and % for htnfri, respectively. conclusions: the study demonstrated the constructs of p-hfgl mirna, p-hfasmirna and p-htnfri mirna successfully interfered their target genes expression in vitro, which provides the foundation for further investigation of these constructs' application in vivo and further more as a therapeutic strategy for a targeting intervention in the diseases which the gene fgl , fas and tnfri contribute to. this work was supported by nsfc , , ; cb , cb pe influence of the id on the anti-tumor activity of histone deacetylase inhibitor in hepatocellular carcinoma cells r. tsunedomi , , s. harada , n. iizuka , m. oka dept. of digestive surgery and surgical oncol., yamaguchi univ. , research fellow of the japan society for the promotion of science for young scientists background: our recent study revealed that levels of the inhibitor of dna binding/differentiation (id ) were associated with the progression of hcv-related hepatocellular carcinoma (hcc) and can affect susceptibility of hcc cells to histone deacetylase (hdac) inhibitors. we here aimed to investigate how and whether id expression affected on the anti-tumor activity of sodium butyrate (nab), one of hdac inhibitors. methods: two hcc cell lines, hle and huh- , were used for gene targeting experiments. the id over-expressing and knockdown cells were subjected to mts assay to evaluate the susceptibility to nab. time-course of the expressional change of bcl- and bcl-xl genes after nab administration was measured by real-time rt-pcr. result: upregulation and downregulation of id levels in hcc cells resulted in decreased and increased susceptibility to nab, respectively. we observed that after nab administration, the id expression was induced gently, the bcl- expression was greatly increased immediately, and the bcl-xl expression was decreased to less than half once and then recovered. these increase and recovery of the expression of anti-apoptotic genes were inhibited in the id knockdown cells. in the id overexpressing cells, the bcl- expression was more upregulated than mock-transfected cells. conclusion: in hcc cells, id influences the susceptibility to the hdac inhibitor by regulating the expression of anti-apoptotic genes caused by the hdac inhibitor stimulus. we suggest that id could serve as a fascinating marker predictive of response to hdac inhibitors. the role of zinc finger protein in liver cancer m.m.y. waye , , t.l. yeung , , j.l. zhu , the chinese university of hong kong, croucher laboratory for human genomics background/aims: the aim of this research project is the characterization of a krüppel zinc finger protein, zinc finger protein (znf ) using hepatocellular carcinoma as a disease model. zinc finger protein (znf ), also named as only zinc fingers protein (ozf), is a kda nuclear zinc finger protein consisting solely of ten c h zinc finger motifs of the krüppel type. like most of krüppel proteins, it is assumed to be the transcription factor and involved in the regulation of gene expression. the znf gene is amplified in - % of pancreatic carcinomas and overexpressed in more than half of the tumors including liver cancer. it is thus proposed that overexpression of the znf may contribute to the development or progression of hepatocellular carcinoma. methods: we used flow cytometry, microarray, green fluorescent recombinant protein, rt-pcr site-directed mutagenesis, and transfection to study the effect of expression of znf . results: our results shown that znf was over-expressed in two human hcc cell lines hepg and hep b. expression profiles of znf over-expressing shown that genes related to the p tumor suppressor activity or dna damage, repair response and control were deregulated upon overexpression of znf . conclusions: znf is possibly involved in liver carcinogenesis by affecting dna repair and cell cycle control upon induced dna damage. background: in the present study, we reported the establishment of a real-time monitor system for directly observing the catalytic, kinetic characteristics of dnazyme - in vitro cleavage on the target rna molecules as well as for rapid, accurate, high-throughout evaluation of varied dnazymes on their counterpart rna molecules. methods: dnazyme named dz-hcv- specific to hepatitis c virus (hcv) orf aug were designed and synthesized. dz-hcv-mis- with mismatched substrate-recognition domains, dz-hcv-mut- with mutant catalytic domains, antisense oligonucleotide ason and nonsense oligonucleotide nson were synthesized respectively as controls. a chimeric oligonucleotide of nt containing both rna and dna bases was designed and synthesized as the substrate: ' fam-gt agaccgugcaccaugagcacgaaucct-bhq ', corresponding to the - nt (underline) of hcv genome(gi: ) the reporter fam/bhq was incorporated at the ' and ' end, respectively. under simulated physiological conditions ( ), kinetic characterization of rna-cleaving dnazyme was analyzed in a real-time way. factors that influencing dnazyme cleavage were analyzed. results: dz-hcv- specific to hcv orf aug could cleave target rna at a•u site, a continuous change of fluorescence intensity was monitored. while the control oligonucleotides couldn't cleave rna, there were no change of fluorescence intensity. factors that influencing dnazyme cleavage concluded different substrate-recognition domain, mg + concentration and ph. conclusion: a real-time monitoring system for kinetic characterization of rna-cleaving dnazyme was successfully established in the first time. the study on the apoptosis of hepatoma cells synergeticly induced by plasmid-mediated anti-angiogenesis and immunopotentiation therapy p.y. li , q. zhang , y. chang , j.s. lin , d.a. tian background: angiogenesis is improtant to hepatoma and decreasing of host immunity promotes the development of tumor. we want to study the effect and mechanism of apoptosis of mice implanted hepatoma cells induced by eukaryotic plasmid-mediated anti-angiogenesis and immmunopotentiation therapy. methods: mouse endostatin eukaryotic plasmid (pseces) and mouse il- (interleukin ) eukaryotic plasmid (pmil- ) were extracted and purified from e. coli. h hepatoma cells were inoculated into the leg muscle of mice, which was divided into four groups and injected with pseces, pmil- , pseces+pmil- or pcdna . naked plasmid dna respectively into implantation sites repeatedly. tumor formation and its weight was evaluated. tumor microvessel density, tumor infiltrating lymphocytes and apoptosis of tumor cells were assayed by cd staining, he staining and tunel assay respectively. results: inoculated mice received pseces, pmil- injection formed tumor slowly with less microvessel density, more tumor infiltrating lymphocytes in the latter and more tumor apoptosis cells in both groups compared with pcdna . injection. there were much more tumor apoptosis cells in pseces+pmil- group ( . ± . per × microscope field p< . ) than any other single plasmid injection group ( × microscope field: pseces . ± . , pmil- . ± . , pcdna . . ± . ). conclusion: tumor cells of implanted hepatoma in mice could be synergeticly induced to apoptosis by eukaryotic plasmid-mediated anti-angiogenesis and immunotherapy through inhibiting tumor angiogenesis and promoting tumor lymphocytes to infiltrate, by which mice implanted hepatoma was inhibited. ( , , , , ng/ml) in a serum-free medium for h. cell proliferation was measured by brdu incorporation analysis, untreated wb-f cells were taken as controls. after treatment with wnt a ( ng/ml) for h, subcellular localization and protein expression of -catenin in wb-f cells treated and untreated with wnt a were examined by immunofluorescence staining and western-blot analysis. cyclind mrna expression was determined by semi-quantitative reverse-transcript polymerase chain reaction (rt-pcr). mrna levels of some phenotypic markers (afp, ck- , alb) and two hepatic nuclear factors were measured by rt-pcr. expressions of ck- and afp protein were detected by western-blot analysis. results: wnt a promoted proliferation of wb-f cells. stimulation of wb-f cells with recombinant wnt a resulted in accumulation of the transcriptional activator -catenin, together with its translocation into the nuclei, and up-regulated typical wnt target gene cyclind . after d of wnt a treatment in the absence of serum, wb-f cells retained their bipotential to express several specific phenotypic markers of hepatocytes and cholangiocytes, such as afp, ck- following activation of the canonical wnt signaling pathway. conclusion: the canonical wnt signaling pathway promotes proliferation and self-renewal of rat hepatic oval cells. the expression level of bid and other pro-and anti-apoptotic proteins were detected by immunoblotting. results: hbx/ showed the most sensitive towards dox treatment, and truncated bid (tbid) was also only detected in this cell line. the level of bax was also increased in hbx/ cells. conclusions: the carboxy-terminal of hbx may enhance the processing of bid into tbid, which may contribute to increased sensitivity of the cell towards the dox treatment. cell homeostasis were performed with concentrations of oxysterol ( x - - - m) faraway from the physiological and/or pathological one ( . and x - m). in our study, we asked the effects of oxysterols ( k and ' s) on hepatoma cell lines homeostasis. to this purpose we used concentrations similar to those described in physiological or pathological conditions. sub-physiological ( - m) to pathological ( - m) oxysterol ( k and ' s) concentrations were used to stimulate hepg cells. a surprising pro-proliferative effect of ' s at sub-physiological ( - m) concentration was observed. this behaviour was confirmed by the synergic increase of erk / levels. facs analysis revealed an early progression of cells in s phase at the lowest concentration of ' s, while all the remaining concentrations of the two studied oxysterols induced a weakly accumulation of cells in g /m phase. apoptosis was absent at all concentration used, except for the highest one ( - m). at this point we asked if cells didn't undergo apoptosis but acquired a senescent profile. effectively, both k and ' s, at all concentration used (except for - m), induced cell senescence (revealed by sa-ß-gal staining and sirt and p over-expression). in conclusion the two oxysterols analyzed have different and in same case opposite effects on hepatocellular line. the main effect is surely the senescence induction, but it is important to highlight the proproliferative effects of ' secosterol at low concentration. mortalin, a member of hsp family protein, has been shown to play an important role in hepatocellular carcinoma (hcc). it has been reported that mortalin is binding to the c-terminal of p , which acts as a safety guard and is a commonly mutated gene in hcc. in this study mortalin was silenced by specific shrna in plc/prf/ , a hcc cell line constitutively expressing p ser , and normal liver cells miha, and we found that suppression of mortalin can selectively trigger the mitochondria mediated apoptosis pathway by p dependent way in plc cells. tunel staining positive cells were only found in the plc cells mortalin knockdown group, and apoptosis associated protein, such as p , bax, bcl-xl, cleaved-caspase , have been screened by western blot after transfection. quantitative-pcr data also showed that p mrna level are upregulated about folds in mortalin knockdown group compared with the control groups in liver tumor cells. two p inhibitors, pft-and pft-, which can reverse this apoptosis was applied to demonstrate p dependent way. in summary, knockdown mortalin can selectively kill liver cancer cells through reactive apoptosis by sensitizing mutant p in plc cells, but had no effect on normal cells. the clinical application of this study suggested that motalin specific shrna might be a potential anti-cancer drug for hcc. background: nafld can proceed to nash and are at risk of cirrhosis and hcc. aim was to study profile of bangladeshi nafld patients. methods: patients with nafld were included. of them . % were males and . % females. patients were between - years of age. they presented with dull right upper abdominal ache and/or incidental detection of raised alt/ast and/or fatty liver on ultrasonography. all tested negative for hepatitis b and c. none had history of alcohol. all underwent per-cutaneous liver biopsy for histopathology. they were also tested for dm, dyslipidaemia, insulin resistance, hypothyroidism and hepatitis c. their bmi and bp were recorded. results: . % had nash. . % of them were males and rest . % females. . % had nafl. of them % each were males and females. majority had nash. . % were obese and . % had dyslipidaemia. . % had hypertension, . % insulin resistance and % were diabetics. . % had hypothyroidism. none had hepatitis c. alt was raised in % and ast in %. although all patients with nash did not have elevated alt, it was raised in majority, contrary to ast, which was normal in most. conclusion: majority nash patients in bangladesh are obese. other leading causes of nash include dyslipidaemia, hypertension and insulin resistance. some nash also had diabetes and hypothyroidism. this study also reveals that elevated alt in patients with nafld is suggestive of fibrosis, although normal serum alt does not exclude nash. the study further suggests that alt is superior to ast in predicting nash. background: non-alcoholic fatty liver disease is prevalent in obese patients. liver biopsy remains the best diagnostic tool for confirmation. we tried to find out the correlations of laparoscopic parameters with histology and laboratory data. besides, we also evaluated the effectiveness of laparoscopy in liver disease diagnosis. methods: in the period of one year and five months, morbidly obese patients submitted to laparoscopic bariatric surgeries at our institutions were prospectively studied. results: laparoscopic parameters of significant correlations with histologic steatosis, inflammation and fibrosis were summarized in table . besides, important parameters with relationships to laboratory data were summarized in table department of internal medicine, seoul national university hospital gangnam healthcare center, seoul, south korea, department of internal medicine and liver research institute, seoul national university college of medicine, seoul, south korea background/aims: hepatic fibrosis is associated with poor prognosis in non-alcoholic fatty liver disease (nafld). recently, many non-invasive fibrosis markers have been studied to overcome the limitations of liver biopsy. among them, bard score and guha's simple panel are easy to use in clinical practice. in this study, we evaluated the efficacy of bard score and guha's simple panel as a noninvasive fibrosis marker in korean nafld patients. methods: data from patients with biopsy-proven nafld in seoul national university hospital from to were used. bard score and guha's simple panel were calculated by using clinical and biochemical data and were compared with the histological fibrosis stages. results: stage fibrosis were found in patients, stage in , stage in , stage in and stage in . the relationship between fibrosis stage and bard score ( = . , p < . ) was statistically significant. all patients with advanced fibrosis (stage - ) had bard score greater than . mean values from original guha's simple panel for no fibrosis were not different between the patients with and without fibrosis. however, after adjusting coefficients by logistic regression analysis, the differences in mean values became statistical significant (p < . ). conclusions: our data suggest that bard score may be effective for detecting high risk patients for advanced fibrosis, and modification of coefficients within the guha's simple panel may be needed to use as a fibrosis marker in asian nafld patients. s.k. mohan , s. subramaniam , s. subramaniam assistant professor, department of biochemistry, saveetha medical college & hospital, saveetha university, t.n, india., consultant, department of biochemistry, apollo hospitals, chennai, t.n, india. , department of biochemistry, apollo first med hospitals, chennai, t.n, india. background: non-alcoholic fatty liver disease (nafld) covers a spectrum of liver diseases from simple fatty infiltration to progressive fibrosis. non-alcoholic steato hepatitis (nash) is a severe form of nafld and progresses to the end stage of liver disease. it is becoming the leading cause for referral to liver clinics in most areas. the prevalence of nafld in indian population is estimated around - %. the nafld has the potential to progress to hepatocellular carcinoma or liver failure, both events that ultimately lead to early death. aim: to evaluate the combination of inter cellular adhesion molecule - (icam - ), adiponectin and type-iv collagen, a new biomarker profile for nash in patients with nafld. methods: patients with nafld and age & sex matched normal healthy individuals as controls were selected for this study. levels of serum icam - , adiponectin, type-iv collagen, lipid profile and liver function test parameters were estimated in patients and compared with controls. results: serum icam - & type -iv collagen levels were significantly increased in patients with nash among the nafld patients compared to controls. the serum adiponectin levels were significantly reduced in patients with nash among the nafld patients compared to controls. compared to liver function test parameters and lipid profile levels, nash profile has got positive negative predictive value among the nafld patients. conclusion: in patients with nafld, nash profile test -a simple, noninvasive and reliable to predict the presence or absence of nash. background/aim: oxidative stress and cytokines plays an important role in the pathogenesis of nonalcoholic fatty liver disease (nafld). aim of study was to assess lipid peroxidation, serum levels of transforming growth factor-( tgf-) and tumor necrosis factor-( tnf-) in patients with nafld and compare it with patients of chronic viral hepatitis (cvh) and healthy controls (hc). methods: lipid peroxidation was studied by estimating plasma malondialdehyde (mda) levels as per the methodology described by buege and aust and tgf-& tnf-levels were measured by elisa kits (ray biotech, usa, & diaclone, uk) in the stored sera in biopsy proven patients with nafld (m: , f: , mean age: . ± . yrs), patients with cvh ( m: , f: , mean age: . ± . yrs) and hc (m: , mean age: . ± . yrs). results: there was no difference in mean plasma mda levels amongst patients with nafld ( . ± . mol/l), cvh ( . ± . mol/l) and hc ( . ± . mol/l). serum tgf-levels between nafld ( . ± . ng/ml) and cvh ( . ± . ng/ml) patients and hc ( . ± . ng/ml) were also comparable. though patients with cvh ( . ± . pg/ml) and nafld ( . ± . pg/ml) had higher levels of tnf-than hc ( . ± . pg/ml), the difference was not significant statistically. conclusion: lipid peroxidation, tgf-and tnf-need to be studied in a larger number of patients with nafld. background/aim: burnt out nonalcoholic fatty liver disease (nafld) may be responsible for cirrhosis and hepatocellular carcinoma (hcc) in the absence of other causes. aim of this study was to evaluate the surrogate markers of nafld in patients with cryptogenic cirrhosis (cc) and cryptogenic hcc (chcc). methods: sixty five patients with cc and patients with chcc were analyzed for the presence of abnormal body mass index (bmi) and type diabetes mellitus (dm aim: to investigate the relation of phosphatidylethanolamine n-methyltransferase pemt gene g a single nucleotide polymorphism (snp) with the susceptibility to nonalcoholic fatty liver disease nafld . methods the genotypes and allele frequencies of pemt exon snp g a were analyzed by using pcr-rflp in nafld patients and controls. results: the g to a variation of the pemt gene g a snp was significantly higher in nafld group compared with controls. the frequencies of gg ga and aa genotypes were . . and . in nafld and . . and . % in controls (p= . . the a allele of the pemt gene was significantly more frequent in nafld group ( . %) than that ( . %) in controls p= . .there were significant differences in serum levels of cholesterol, triglyceride, hdl-c and ldl-c between gg and ga/aa genotypes p < . . n. assy , , g. lipez , s. korem , m. grozovski sieff hospital, safed, israel, technion institute, faculty of medicine, haifa, israel, ort braude college, karmiel, israel background: previous studies reported increase in serum protein c and decrease in serum paraoxonase levels in patients with non alcoholic fatty liver diseases (nafld). conclusion people with pemt gene g a snp were more susceptible to develop nafld aim: ) determine whether there is a relationship between nafld, protein c and paraoxonase levels in quiescent and in regenerating rats fatty liver ) determine the effect of isa on hepatic "protein c" and paraoxonase mrna. pe methods: forty-eight sd rats were treated with fructose enriched diet (fed), or fed with metformin ( mg/kg/d), fed with rosiglitazone ( mg/kg/d), or the combination of both drugs for wks. % phx was performed at wk . protein c, paraoxonase mrna expressions, lipids, mda were measured before and hours after phx. results: hepatic "protein c" mrna was higher in rats with fatty liver than control rats (+ %, p< . ) whereas hepatic paraoxonase mrna was lower in rats with fatty liver than control rats (- %, p< . ). hepatic protein c and paraoxonase mrna increased in rats with fatty liver in regeneration (+ %, p< . , and + %, p< . respectively). the combination of metformin and rosiglitazone decreased hepatic protein c expression at hours after phx by - % (p< . ) and increase paraoxonase mrna by + % (p< . ). serum paraoxonase correlates with serum protein c (r=- . ), mda (r= . ), background: non-alcoholic steatohepatitis (nash) is a type of non-alcoholic fatty liver disease (nafld), and may progress to hepatic fibrosis and cirrhosis. the pathogenesis of nash remains unclear. the aim of this study was to explore the arginase change in the progress of steatohepatitis in rats. methods: male sd rats weighing - g were obtained. twenty animals were randomly divided into two groups. in the model group, five animals were fed with high lipid forage that includes % cholesterol and % lard for weeks, five were fed for weeks, while the control group ate normal foods. the animals were sacrificed after weeks. the animals were sacrificed after weeks and weeks. liver and blood serum were collected while the serum levels of alt, ast, tg and tc were measured. the pathology of liver was observed by he staining. western blot was used to investigate the expression of arginase in control and model group. tg (r=- . ). conclusion: hepatic "protein c" mrna levels are high at baseline, up regulated during liver regeneration and decrease after treatment with (isa) whereas hepatic paraoxonase mrna levels are low at baseline, up regulated during liver regeneration and increase after treatment with isa. results: vacuolization were observed extensively in hepatic cells in the model group after weeks and weeks of high-fat diet. it is demonstrated that rats fed with high-cholesterol food are indeed fatty liver models. western blot showed that the level of arginase ii increase in the liver of model group rats as compared to the control group. furthermore, the level of arginase was higher in liver samples obtained from model rats that were weeks on a fat diet as compared to rats that were only weeks on the same diets. conclusion: the level of arginase ii was altered in the progress of non-alcoholic steatohepatitis in rats suggesting that arginase ii is putative biomarkers and may represent new targets in the development of therapeutic strategies against fatty liver disease hepatic fibrosis and cirrhosis. methods: c bl /j mice were fed with mcd diet to induce hepatic fibrosis and rosiglitazone was given in treated group. effect of rosiglitazone was assessed by comparison of the severity of hepatic fibrosis in liver sections, expression of mmp- / , timp- / mrna and protein detected by rt-pcr and western blot respectively. the ethanolic extract of fructus schisandrae chinensis decreased hepatic triglyceride level in mice fed with a high fat/cholesterol diet results at week , fibrosing nash models showed severe hepatic steatosis, infiltration of inflammation and fibrosis, which is associated with down-regulated mmp- / mrna and protein, up-regulated timp- / mrna and protein. rosiglitazone significantly reduced mcd-induced fibrosis by induced mmp- / expression and reduced timp- / expression by activating ppar . s.y. pan , z.l. yu beijing university of chinese medicine, hong kong baptist university effects of the ethanolic extract of fructus schisandrae chinensis (etfsc) on serum and liver lipid contents were investigated in mice fed with normal diet or high fat/cholesterol diet for or days. single dose of etfsc ( or g/kg/day, i.g.) increased the serum triglyceride (tg) level ( and %, respectively), but decreased hepatic total cholesterol (tc) level ( and %, respectively) in normal mice. the hypertriglyceridemia produced by etfsc was suppressed by the co-administration of fenofibrate. the induction of hypercholesterolemia by high fat/cholesterol diet caused significant increases in serum and hepatic tc levels (up to %) and hepatic tg levels (up to %) in mice. etfsc treatment ( or g/kg/day for days, i.g.) significantly decreased the mouse hepatic tg level (by %) and slightly increased the hepatic index (by %). whereas fenofibrate treatment ( . g/kg/day for days, i.g.) significantly lowered the hepatic tg level (by %), it significantly elevated the hepatic index (by %) in hypercholesterolemic mice. the results indicate that etfsc treatment can invariably decrease hepatic tg in hypercholesterolemic mice, suggesting its potential use for fatty liver treatment. aim: to investigate the influence of multiple gene polymorphisms in the susceptibility of nafld. methods: the data of single nucleotide polymorphisms (snps) in nafld patients who had at least one of the genetic variations at the sites of tnf-- , adiponectin - and leptin- were analyzed. the genotypes were determined by using pcr-rflp. our previous studies showed that the variations of these sites increased the susceptibility of nafld. results: the prevalence of nafld in adiponectin variation alone group (n= ) was . %; in tnf-alone group (n= ) . %; in leptin alone group (n= ) . % (p> . ). in comparison with the above groups with single snp, the prevalence of the groups with two gene variations of tnf-plus adiponectin ( . %, n= ) increased significantly (p< . ). however the prevalence of other two groups i.e. adiponectin plus leptin ( . %, n= ) and tnf-plus leptin ( . %, n= ) did not differed significantly from those of groups with single snp (p> . ). the prevalence in the group with three gene variations ( . %) differed significantly from all (p< . ) except that of tnf-plus adiponectin group (p> . ). the metabolic features of the nafld patients in the groups mentioned above were not different significantly (p> . ). conclusion: nafld is a polygenic disease. multiple gene polymorphisms may, but not always, increase the susceptibility of nafld. chronic hepatitis b patients with nonalcoholic fatty liver disease r.d. zheng , c.r. xu , j. chen , b.f. chen southeast hospital background: to investigate clinical pathological characteristic in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). methods: we measured fasting blood glucose, insulin, triglyceride, cholesterol, alanine aminotransferase (alt), aspartate aminotransferase (ast) in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). and we detected hepatitis b virus marker, hbv-dna, counted body mass index, insulin resistance index and observed pathological characteristic. all these patients with diagnosis were confirmed by clinical and pathological evidence. result : the body mass index, homeostatic model assessment (homa) of insulin resistance, fasting blood glucose, insulin, triglycerides, cholesterol, were significantly higher in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld) than hbeag negative chronic hepatitis b patients. but the alanine aminotransferase (alt), aspartate aminotransferase (ast), hbv dna levels were significantly lower in hbeag negative chb patients with nafld than in hbeag negative chronic hepatitis b patients. histologic features in hbeag negative chronic hepatitis b(chb) patients with nonalcoholic fatty liver disease (nafld) are in zone predominate macrovesicular steatosis and mild inflammatory infiltrate in portal region. conclusion: the hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease, whose hepatic steatosis changes are mainly caused by the metabolic factors. to carry out liver biopsy selectively for the patients with hbeag negative chronic hepatitis b having metabolic factors, which is helpful for early diagnosis in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). aims: to investigate the preventive effect of cordyceps sinensis and its possible mechanism on apoptosis of nafld. methods: rats were randomly divided into basic diet group (b group), pathologic group (nash group) and cordyceps sinensis group(cs group).the latter two groups were administered with high-fat diet to establish nafld animal models. cs group were treated with cs at the th week after high fat diet. rats were sacrificed at the end of the th week. biochemical examination were used to detect superoxide dismutase (sod) of liver tissue. hepatocyte apoptosis was assessed in each group using the tunel assay and immunohistochemistry for activated bax bcl- caspase- and nf-kb p . results: ( ) compated with the b group, severe hepatosteatosis, inflammative necrosis and local fibrigenisis were showed in liver of nfsh group. sod lever was significantly decreased (p< . ) and tunel-positive cells were significantly increased (p< . ). immuunohistochemistry test demonstrated active bax caspase- was increased (p< . ) while no apparent change was observed in bcl- . ( ) in cs group, only diffusive steatosis but not inflammation or fibrosis was found. sod lever was increased than that of nash group (p< . ). tunel-positive cells and active bax caspase- were significantly decreased (p< . p< . ) that those of nash group. bcl- and nf-kb p were increased (p< . ) than those of nash group. conclusions: hepatocyte apoptosis is a prominent feature of nafld. cordyceps sinensis may be useful as an antiapoptosis theraphy in this syndrome through increasing activity of sod, decreasing express of bax and increasing express of bcl- and nf-kb p . background: non-alcoholic steatohepatitis (nash) is a leading cause of chronic liver disease. insulin-sensitizing , anti-inflammatory and anti-fibrotic effect of thiazolidinediones support their use in the treatment of nash. we aimed to evaluate the efficacy of thiazolidinediones in the treatment of nash. methods: we have identified randomised clinical trials, evaluating the efficacy of thiazolidinediones versus placebo in nash, through medline, embase, ami, cochrane central register of controlled trials. data were abstracted from each study and disagreements were resolved by consensus. dichotomous outcomes were reported as relative risk with % confidence interval based on fixed-effects model. results: we included three trials, two evaluating pioglitazone and another rosiglitazone. a total of patients were involved in the analysis. thiazolidinediones was noted to improve liver function tests. it was effective in the reduction of steatosis among patients with nash (rr . , % ci . - . ). it was found to be beneficial in improving ballooning necrosis (rr . , % ci . - . ). it was also found to improve lobular inflammation (rr . , background: it is well known that the weight reduction is effective for alt normalization in patients with non-alcoholic fatty liver disease (nafld). the necessary condition for alt normalization is still unclear. to clarify the necessary and sufficient condition for alt normalization, we investigated the effects of body fat decrease in nafld patients by body composition analyzer. methods: forty-six nafld patients ( male, female, mean age . ± . years old) with abnormal alt levels were evaluated. the volume of skeletal muscle, body fat and bmr were examined by using the body composition analyzer (in body ; biospace co. ltd., tokyo japan). all patients were received an individualized diet consultation by dietician every weeks for months. daily energy was bmr (basal metabolic rate) x . kcal and protein was . - . g per ideal body weight. result: twenty-eight of patients ( . %) were achieved normal alt level. in alt normalized group, the body weight and fat loss were . ± . kg, . ± . kg ( . ± . %body fat) respectively. on the other hand, in cases with alt remained abnormal level, the body weight and fat loss were . ± . kg, . ± . kg ( . ± . %body fat). conclusion: our results demonstrate that the fat loss of kilograms or more was necessary to normalize alt level in nafld patients. a. somani , s. somani , a. jain , v. dixit navjeevan hospital, suvidha, background : nafld is often clustered within families and the causes include both genetic and environmental factors. family studies done thus far have been limited by small sample size. to examine the familial patterns , we performed a prospective study to see (a) whether nafld is more common in first degree relatives (b) genetically determined risk factors associated for clustering. methods: first degree relatives of histologically confirmed nafld patients and spouses (controls) were included after excluding other causes of fatty liver. those having raised transaminases > months or sonographic examination consistent with fatty liver, had undergone liver biopsy for histological confirmation. they were divided into three groups. group i patients group ii first degree relatives group iii spouses results: nafld was more prevalent among first degree relatives then spouses ( % and %, p< . ). anthropometric measurements, systolic and diastolic blood pressure, lipid profile and liver function tests were comparable in three groups. homa-r was similar in group i and ii (p= . ), but was significantly different in group i and iii (p= . ) and group ii and iii (p= . ) respectively. metabolic syndrome was present in > % of patients and were comparable in three groups except for fasting glucose > , which was present in %, % and % of patients in group i, ii and iii respectively. majority (> %) of our patients among groups i, ii and iii were having only steatosis while nash was present in %, % and % of patients. a. somani , v. dixit , a. jain , s. somani navjeevan hospital, ims, bhu, varanasi, suvidha background: normal levels of alanine aminotransaminase (alt) have been demonstrated in nafld patients. alt levels are also modulated by age, gender, bmi, fasting glucose, and serum triglyceride levels. we performed a prospective study of patients with histologically confirmed nafld and having alt < . times and compared them with those having raised alt to determine (a) clinico-pathologic features of nafld patients with normal alt (b) to observe any differences between them. methods: patients with fatty liver on sonography had under gone biopsy for histological confirmation after excluding other causes of fatty liver. participants were divided into two groups (a) those having alt > . times normal (n= ) (b) those having normal alt (n= ) results: mean age was comparable with slight male predominance. there were significant differences in anthropometric measurements like bmi (p= . ) and whr ( . ± . and . ± . , p= . ). mean bp, lipid profile, fasting glucose, insulin, and homa r were comparable. there were significant differences in both mean ast ( . ± and . ± . , p= . ) and alt ( ± . and . ± . , p= . ) levels. metabolic syndrome was present in > % of patients and individual components were comparable except for increased waist circumference which was significantly more in those with raised alt ( . % and . %, p< . ). majority of our patients were having only steatosis, while nash was present in ( . % and . %, p< . ) of patients. conclusion: nafld can exist in patients with normal alt values. although more work is needed to determine who should be screened for nafld and how such individuals should be evaluated, this study is a step toward the identification and characterization of nafld patients with normal alt. we can suggest that patients having metabolic syndrome or insulin resistance, despite having normal alt, should be screened for nafld. also alt values should be adjusted for variables like bmi to appropriately screen nafld patients. background: scientific evidence has demonstrated that traditional chinese medical (tcm) approaches and products can be beneficial for managing non-alcoholic fatty disease (nafld), but few rigorous criteria of patterns of tcm therapy are available to guide practitioners in deciding the cam interventions. objectives: to evaluate criteria of patterns of tcm therapy for the management of nafld identified by biomedicine. methods: literature research, clinical epidemiological investigation and mathematical statistics were employed to make information collecting tables and to establish database. descriptive analysis, factor analysis, and cluster analysis were involved. results: ( ) background/aim: serum uric acid level has been suggested to be associated with factors that contribute to the metabolic syndrome. the aim of this study was to investigate the association of serum uric acid level with nonalcoholic fatty liver disease (nafld). methods: a cross-sectional study was performed among the employees of zhenhai refining & chemical company ltd., ningbo, china. results: the study included subjects ( men) with a mean age of years. the prevalence rate of nafld and hyperuricemia was . % and . %, respectively. nafld patients had significantly higher level of serum uric acid than controls ( . ± . vs. . ± . mol/l; p < . ). the prevalence rate of nafld was significantly higher in the subjects with hyperuricemia than those without hyperuricemia ( . % vs. . %; p < . ), and the prevalence rate increased along with serum uric acid levels (p value for trend < . ). multiple regression analysis showed that hyperuricemia was associated with increased risk for nafld (odds ratio [or]: . , % confidence interval [ci]: . - . ; p < . ). conclusion: serum uric acid level is significantly associated with nafld, and increased serum uric acid level is an independent risk factor for nafld. background: development of fatty liver is believed to be an early and reversible consequence of excessive alcohol consumption. however, the cellular and molecular events in the early development of alcoholic liver diseases (ald) and the contributory effects of a high fat diet are not fully understood. methods: this study was designed to quantify specific enzymatic and cytokinetic activity as well as the development of hepatic steatosis in a rat model of alchohol-induced liver injury without high fat diet. results: ethanol-fed rats exhibited high blood ethanol levels ( . + . %) and significant increases in serum alt ( . + . unit/l), ast ( . + . unit/l), and alp ( + . unit/l) when compared with control rats (p< . , respectively). histopathological examination found unevenly raised knodell scores ( . + . in the ethanol-fed livers vs. . + . in control), which were characterized by scattered hepatocyte ballooning, portal inflammation and collagen fiber deposition. however, typical steatosis lesions were absent. qpcr demonstrated up-regulation of genes in the ethanol-fed livers, including hepatocyte metabolism enzymes/receptor (adh , p< . ; cytochrome p e , cyp e , p< . ; gsta , p< . ; ppar , p< . ), and genes coding for pro-inflammatory cytokines (il- , p< . vs. control livers; tnf-p< . ; tgf -, p< . ; rantes p< . ), ecm components and proteinases (collagen- , p< . ; sma, p< . ; mmp - , p< . and timp- , p< . ). conclusion: chronic administration of ethanol to rats without high fat diet productively induces alcohol hepatitis in the absence of fatty liver, suggesting that alcohol hepatitis may precede steatosis in the development of ald. the aim of the present study was to evaluate the changes of several cytokines associated with inflammatory liver disease and liver regeneration by molecular adsorbent recirculating system (mars) in aclf patients versus patients treated with medical standard therapy (smt) that presented alcoholic liver disease etiology and similar model end-stage liver disease (meld). methods: mars group: fifteen ( male and female) patients were treated with mars® (gambro). five patients were excluded by study.the number of mars applications was about , the length of applications was about h. smt group: fifteen patients ( male and female) were treated medical standard therapy such as prophylaxis against bacterial infections, albumin and fresh plamsa and judicious use of diuretics. three patients were excluded by the study. the patients were valued during days from inclusion with a survival follow up a three months. results: mars group: we observed a significant changes in levels of il- (p< . ), il- (p< . ), il- (p< . ) and tnf-alfa (p< . ) in association with improvement of hepatic growth factor (p< . ). the patient's survival at three months was %. smt group: we observed only a significant changes in il- (p< . ) and tnf-alfa (p< . ). the patient's survival at three months was %. conclusion: the mars liver support device has corrective effects on disturbed pathophysiology of aclf and may be used to enhance spontaneous recovery or as bridge to transplant. a study of protective effect of centella asiatica in -methyl- phenyl- , , , -tetrahydropyridine (mptp)-induced liver injury n. haleagrahara , s. chakravarthi , p. kumar international medical university, malaysia background: centella asiatica has been used for centuries as a medicinal herb for wound healing, memory enhancement, cancer, vitality, respiratory ailments, psoriasis and eczema, revitalizing connective tissue, burn and scar treatment, skin infections, arthritis, rheumatism, periodontal disease, varicose veins, hypertension, sedative, anti-stress, anti-anxiety, aphrodisiac, and as immune booster. results: ppc significantly reduced hepatocyte damage, hepatitis, and hepatic fibrosis, but did not affect steatosis. phosphorylation of apoptosis signal-regulating kinase , p mitogen-activated protein kinase, and protein kinase c, as well as activation of nuclear factor-kappa b, were markedly suppressed by ppc. these effects were likely a consequence of decreased oxidative stress through down-regulation of reactive oxygen species (ros)-generating enzymes, including cytochrome p e , acyl-coa oxidase, and nadph oxidases, in addition to restoration of ethanol-induced increases in toll-like receptor and cd . ppc also decreased the pro-apoptotic proteins bax and truncated bid, thus inactivating mitochondrial permeability transition. furthermore, ppc suppressed overexpression of transforming growth factor- and hepatic stellate cell activation, which retarded hepatic fibrogenesis. conclusion: ppc exhibited anti-inflammatory, anti-apoptotic, and anti-fibrotic effects on ald as a result of inhibition of alcohol-induced ros production. background: dysctamnus dasycarpus has used for the promotion of health in south korea. but, there were rare a report concerning the hepatotoxicity. we report cilinical features of liver injury by dysctamnus dasycarpus. method: eighteen patients diagnosed as acute toxic hepatitis by dysctamnus dasycarpus in chungnam national university hospital between january and arpil was enrolled. toxic hepatitis was diagnosed by rucam score ( ). the medical records were reviewed, retrospectively. result: eleven patients ( %) were female and the mean age was . . most common symptom was jaundice. initial laboratory findings were as follows(mean value): wbc /ul, hemoglobin . g/dl, platelet × /l,alt iu/l, total bilirubin . mg/dl, alkaline phosphatase u/l, ggt u/l, prothrombin time(inr) . . the mean hospitalization was . days. peak laboratory findings were as follows: alt iu/l, total bilirubin mg/dl. recovery time of each biochemical finding was as follows: alt days, total bilirubin . days. recovery rates of alt and total bilirubin were . % and . %, . % and . % at weeks, weeks, respectively. the main biochemical pattern of hepatotoxicity was hepatocellar ( . %) type. prednisolone was prescribed in six patients. progressive anemia and thrombocytopenia were detected in one patient diagnosed as pure red cell aplasia. other one patient had prolonged jaundice ( days). but, all patients had recovered without sequelae. conclusion: in south korea, liver injury by dysctamnus dasycarpus was more frequent in women. the main pattern of hepatotoxicity was hepatocelluar type. most patients had prolonged icteric phase and hospitalization. patients were recovered by supportive management after drug cessation or prednisolone therapy. in korea, traditional medicine that is based on the use of herbal medicine developed from a long time ago. however, clinical study of the herbal medicine is not conducted in a structured manner. we report three cases of toxic hepatitis caused by the intake of dictamnus albus. the first patient, a year old woman was admitted due to nausea after ingestion of liquor containing dictamnus albus for months. total bilirubin was . mg/dl ast/alt / , iu/l on admission. liver biopsy observed hepatocyte necrosis and cholestasis. the elevated bilirubin and transaminase returned to normal weeks later after cessation of dictamnus albus. the second patient, a year old man was admitted due to jaundice after ingestion of boiling dictamnus albus for months. total bilirubin was . mg/dl ast/alt , / , iu/l on admission. liver biopsy observed pericellular fibrosis and necrosis. the bilirubin decreased slowly compared to the transaminase and normalized months later after cessation of dictamnus albus. the third patient, a year old man was admitted due to jaundice after ingestion of liquor containing dictamnus ablus for month. total bilirubin was . mg/dl ast/alt , / , iu/l the hepatocyte necrosis was observed by liver biopsy. the elevated bilirubin and transaminase levels normalized month later after cessation of dictamnus albus. all patients had negative viral markers and non-specific ultrasonographic findings. the above mentioned three cases demonstrate that liver may have been damaged by dictamnus albus, which indicated clinical characteristics. background/aims: cmili poses a diagnostic challenge as no tests are available to confirm the causality. the aims of this study were ) to evaluate clinical features and patterns of cmili and ) to assess the likelihood of causality among patients with liver impairment and exposure to chinese medicine (cm) by a multidisciplinary approach. method: between / and / , patients who had liver derangement and cm or proprietary cm exposure within six months managed in the united christian hospital were studied. clinical features and the cm were reviewed by a multidisciplinary team involving a hepatologist, a toxicologist and cm experts. literature search of relevant herbs in chinese and western journals were performed. cm samples or residue were sent to toxicology laboratory for analysis to look for any toxic constituents, adulterant or contaminant. the likelihood of causality was ranked by various experts independently and disagreements were settled by a consensus meeting. results: there were forty-six cases of suspected cmili, nineteen cases with alternative causes of liver diseases were excluded. twenty-seven cases of cmili proceeded to detailed analysis. median age of patients was ( - ) with female predominance. the median duration of cm exposure to presentation was ( - ) days. majority of them ( %) had hepatocellular liver injury pattern. one case of adulteration with nsaid and erroneous substitution of herb was identified respectively causality were classified as unlikely, possible, probable and highly probable in , , and patients respectively. conclusion: a multidisciplinary approach allows systemic evaluation of suspected cmili. mouse model i. nassar , t. pasupati , i. segarra , j.p. judson international medical university, kuala lumpur, malaysia background: imatinib, a selective tyrosine kinase inhibitor, exhibits drug interactions with other drugs that are metabolised via the cytochrome p pathway. acetaminophen, a widely used analgesic and anti-pyretic drug is also metabolised via p pathway. this study aimed to evaluate the nature of hepatotoxicity after co-administration of imatinib and acetaminophen in a preclinical mouse model. methods: four groups of male icr mice ( - g) were used. the mice were administered either saline solution orally, imatinib mg/kg orally (control), acetaminophen mg/kg intraperitoneally (positive control) or co-administered imatinib mg/kg and ip acetaminophen mg/kg (study group). the mice (n= per group) were fasted overnight, dosed respectively and sacrificed at pre-determined time intervals of , minutes, , , , and hours and liver samples obtained by dissection. h&e stained liver sections ( µm thick) were histopathologically analysed. results: the liver samples showed reversible cell damage like feathery degeneration, microvesicular fatty change, sinusoidal congestion and pyknosis, with both imatinib and acetaminophen, administered separately. the damage increased gradually with time, peaked at hours and then resolved completely by hours. liver samples showed irreversible damage (cytolysis, karyolysis and karyorrhexis) when both drugs were administered concurrently, the damage increased with time and had not resolved after hours duration. conclusion: co-administration of acetaminophen and imatinib increased the hepatoxicity caused by acetaminophen and imatinib to become irreversible. this may be due to the fact that both drugs are metabolised by the cytochrome p pathway in the liver. background: a higher risk of antituberculosis drug (att) induced hepatotoxicity has been reported in indian subcontinent compared to the western counterparts. slow acetylator genotype of n-acetyltransferase (nat ) and ci genotype of cytochrome p e (cyp e ) gene are two known risk factors associated with this disease. cyp e gene encodes a rifampicin inducible enzyme which increases hepatotoxicity. therefore slow acetylation of isoniazid and simultaneous use of rifampicin may augment the toxicity of isoniazid. objectives: to analyze the allelic distribution of nat and cyp e gene in patients of pulmonary tuberculosis who developed att induced hepatitis materials and methods: the study included cases of pulmonary tuberculosis ( ) and att induced hepatitis ( ). polymorphism of nat and cyp e gene was studied by pcr-rflp method in both these groups. results: occurrence of att hepatotoxicity was . %. there was a higher prevalence of slow acetylator genotype particularly nat * /* and nat * /* in patients with hepatotoxicity compared to patients without hepatotoxicity ( . % vs . %, p value < . ). no association of cyp e rsai polymorphism could be considered with att hepatotoxicity. however, drai c/d genotype of cyp e appears as a risk factor for predicting the occurrence of antituberculosis drug induced hepatitis (or . , p value < . ). conclusion: the study demonstrates that patients with slow acetylator genotype particularly nat * /* and nat * /* and heterozygous mutant c/d genotype of cyp e gene are predisposed to develop antituberculosis drug induced hepatotoxicity. regular monitoring of clinical and biochemical profile may be considered in these patients when they receive antituberculosis treatment. background: drug-induced liver injury is the most common adverse drug reaction. we often use two kinds of diagnostic scales to evaluate suspected patients. however, we still can't diagnose accurately without the direct drugs history and the pathological evidence. methods: twenty-seven drug-induced liver injury cases with liver biopsies from to were reviewed retrospectively by maria and japanese scale. result: there were . % of cases with increasing eosinophils. herbs ( . %) were the most common suspected drug and unknown drugs intake history ( . %) were described in these cases. the high possibility and possibility were . %, . % by maria scale and . %, . % by japanese scale, respectively (p= . ). conclusions: japanese scale seems more sensitive than maria scale in these cases. however, there are still some definite cases ignored as low possibility due to absence of obvious drug using history. early treatment and suspected drugs prohibition interferes the outcomes of the two diagnosis systems and lead to a false result. it is still a clinical challenge without strong drug using history or pathological evidence of liver biopsies to diagnose the drug-induced liver injury quickly and accurately. background: previous study suggested that oxidative stress may be an important mediator of methamphetamine-mediated tissue injury. the study was to examine the mechanism of antioxidant activity and methamphetamine-mediated liver injury. materials and methods: the days old male sprague-dawley rats were subcutaneous injected daily with methamphetamine ( mg/kg body weight) for , , and days. control group received equal volumes of vehicle. the liver tissues were extracted to measure the activities of sod, catalase, glutathion reductase (gr), and glutathione peroxidase (gpx), and the level of glutathione. western blot were used to measure the expression of rho and phosphor-ezrin-radixin-moesin (p-erm). results: compared with vehicle group, treated with methamphetamine for and days, the activities of liver sod, gpx, and catalase were significantly decreased. in and days group, the activities of antioxidant enzymes of methamphetamine-treated liver was not different from that of vehicle group. the levels of glutathione production also had the same trend. the activities of gpx and catalase on vehicle group gradually reduced following the days of treatment. however, administration of methamphetamine resulted to a lower activity of catalase through the treated days. there was no difference on the activity of gr between vehicle and methamphetamine group. the expression of rho and p-erm were also increased by methamphetamine treated for days. conclusion: these results suggested the methamphetamine lead to liver remodeling via decreased antioxidant activity. finally, the situation of mechanism needs taking in advantage discussion. background: to observe intervening effects of preventive and theraptical treatment of radix sophorae tonkinensis's polysaccharides(rstp) on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: kunming mice intoxicated with anit mg/kg orally and treated with rstp mg/kg for days before anit exposure and for days after anit exposure respectively, the general condition,mortality rate and serum alt activity are obeverated. result: it was found that by preventive treatment the general condition and mortality rate were improved, serum alt activity reduced.by therapeutic treatment,the general condition deteriorated,mortality rate and serum alt activity increased. conclusion: the preventive treatment of rstp reduce the liver damage due to increasing the anti-stress ability such as the antioxidant capacity,its therapeutic treatment increase the injuried liver damage due to increasing the non-specific immune response and aggregating the preexisting liver inflammation. background: the product's instruction pointed out that in some patients polyphenolic acids' salt from salvia miltiorrhiza(ppas-sm) may lead to a temporary increase in serum alt activity.so we observe effects of ppas-sm on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: hours after intoxicated with anit mg/kg orally, kunming mice were treated with ppas-sm , , mg/kg/days for days orally, then serum alt activity was measured. result: all doses of ppas-sm led to rise of serum alt activity in mice, most obvious in group of high dose.but the general situation and mortality rate did not increase significantly. conclusion: ppas-sm lead to rise of serum alt activity in mice with damaged liver.the auther suggests as a double-edged sword,the antioxidant ppas-sm may have a prooxidative effect in some condition too. *this project was supported by grants from shanghai municipal education commission under high school high-tech characteristic development programme (no smec finance ( ) ) pe a. somani , a. jain , v. dixit , s. somani navjeevan hospital, ims, bhu, varanasi, suvidha introduction: hepatic encephalopathy, a complex neuropsychiatric syndrome secondary to acute liver failure, chronic parenchymal liver disease or portal-systemic shunting, may possibly develop through mediators of endotoxin and tumor necrosis factor-alpha (tnf-). several studies have shown that serum levels of (tnf-) are significantly elevated in patients with acute and chronic liver diseases, where these elevations are independent of the etiology of the underlying disease. it has been shown that plasma levels of tnf-correlate with the severity of hepatic encephalopathy (he) in fulminant hepatic failure. however, still there is very few published data regarding the relationship between serum levels of tnf-and the presence or severity of he in patients with chronic liver failure. methods: the aim of this study is to determine the relationship between serum levels of tnf-and clinical grades of he in patients with chronic liver failure. this prospective study included consecutive male patients with alcoholic cirrhosis in various clinical grades of he (according to west haven criterion). detailed clinical, biochemical and sonographic examination was done in all patients. circulating levels of tnf-was measured using solid-phase elisa. results: the mean±sem values of serum tnf-at presentation in patients with mhe (n= ), grade (n= ), grade (n= ), grade (n= ), and grade (n= ) were . ± . , . ± . , ± . , . ± . , and ± . pg/ml, respectively. significant positive correlation was found between serum levels of tnf-and severity of he (correlation coefficient = . ). conclusion: from the present study we can suggest that there is significant relationship between tnf-and he in patients with alcoholic cirrhosis and it could be involved in its pathogenesis. background: acute hepatitis a (aha) is one of the most common infectious diseases and usually a self-limiting disease. although extrahepatic manifestations are not common, a few cases associated with acute renal failure (arf) have been reported. methods: we reviewed clinical features of aha patients complicated with arf (group a) and compared with non-complicated aha patients (group b). medical records of patients with aha were reviewed between january and december . we experienced patients ( . %) with arf associated aha. result: there were no differences between group a and group b in sex ratio and age. the peak value of alt (median: iu/l vs iu/l, p< . ), alkaline phosphatase (median: iu/l vs iu/l, p= . ), prothrombin time (inr, median . vs . , p< . ) was significantly higher in group a than b. nine patients ( . %) recovered completely with hemodialysis ( patients, . %) and only conservative management ( patients, . %), while patient underwent liver transplantation and patient died due to fulminant hepatic failure. there were patients who underwent kidney biopsy. two patients were diagnosed as acute tubular necrosis and patient as acute interstitial nephritis and iga nephropathy. conclusion: aha patients with arf had higher alt and more prolonged prothrombin time. the prognoses were poorer than those without arf. however, arf patients with nonfulminant aha had a good prognosis with a proper treatment and should not be confused with hepatorenal syndrome. background/aims: to investigate the hev infection among different animals and people with special profession, and to analyse the genotype of hev isolated in this study. methods: serum and fecal samples were collected from various animals and people with special profession in the south suburbs of beijing. hev antigen and anti-hev antibody were detected by das-elisa. hev rna was extracted from fecal samples and amplified by rt-npcr. the nucleotide sequence homology and phylogenetics of hev strains isolated from swine were analysed. results: the anti-hev antibody positive rate of adult swine, cow, sheep and younger swine were . % ( / ), . % ( / ), . % ( / ) and . % ( / ), respectively. the hev antigen positive rates of adult swine, cow, sheep and younger swine were . % ( / ), . % ( / ), . % ( / ) and . % ( / ), respectively. the hev antigen and anti-hev antibody positive rate of professional group was . % ( / ) and . % ( / ) respectively. the hev rna positive rate of fecal samples from younger swine was . %( / ). of samples were hev rna positive by pcr with primers of hev orf and orf . the sequence analysis of the samples showed that there were groups designated as bj- ( / ) and bj- ( / ). the nucleotide homology of bj- and bj- was %. phylogenetic analysis of hev orf indicated that both of them belonged to genotype d. conclusion: phylogenetic analysis of hev orf indicated that hev isolated in the south suburbs of beijing belonged to genotype d. bracops hospital brussels , st. jan hospital, bruges , chu brugmann, chu sart tilman, liège , gent university hospital , zna middelheim, antwerp hepatitis delta virus is a subviral satellite requiring hepatitis b virus to propagate, usually leading to severe, chronic liver disease. as data on epidemiology and management practice of hdv infection in belgium are lacking, a retrospective and prospective, multi-centric questionnaire-based registry is performed in . results of patients are reported. background/ aims: hepatitis a is an acute infectious disease that is transmitted by fecal-oral root. because the incidence of hepatitis a has been increased in gwangju and chonnam province of korea recently, hepatitis a patients in chonnam national university hospital employees had been increased. so we investigated the seroprevalence of igg anti-hav in hospital empolyees less than years old. methods: we analysed seroprevalence of anti-hav igg from , hospital employees (men: , women: ) . serum alt and bilirubin at admission were , , iu/l and . . mg/dl, respectively. these levels were elevated up to , , iu/l and . . mg/dl, respectively. ana was positive in patients ( . %). age, duration from peak-alt day, duration from peak-bilirubin day, alt level, and peak-bilirubin level were not different between ana(-) patients and ana(+) patients. in the while, sex, duration from symptom-onset day, and bilirubin level, and peak-alt level were significantly different. in ( %) of patients with positive ana, ana was followed after month and ana became negative in patients ( . %). among patients with positive ana after month, titer decreased from the baseline in patients, showed no interval change in , and increased in . conclusions: positive ana result is not rare in patients with acute hepatitis a. it is considered that ana transiently appear during the course of acute hepatitis a and then, disappear with the improvement of acute hepatitis. ( ), ( ). the clinical data such as sex, admission period, ast, alt, total bilirubin, prothrombin time, crp, alt normalization time did not show difference. just wbc and gtp were higher on group. the older age patients were more on group. the patients admitted mainly on april, may, june, july ( %) on while admitted even on past years. conclusion: acute hepatitis a ptients is increasing. it is occurring in older age people and mainly on specific period. the more concern to prevention should be needed. background: we analyzed the ' non-translated region ( 'ntr), non-structural proteins b and c of hepatitis a virus (hav) genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems, in order to align all of our data and examine whether genomic differences in hav are responsible for the range of clinical severities. methods: our accumulated hav strains of 'ntr (nt and ), entire b and c from japanese patients with sporadic hepatitis a, consisting of patients with fulminant hepatitis (fh), with severe acute hepatitis (ahs), and with self-limited acute hepatitis (ah), in whom the sequences of all regions were available, were subjected to phylogenetic analysis. results: fh patients had fewer nucleotide substitutions in 'ntr, had a tendency to have more amino acid (aa) substitutions in b, and had fewer aa substitutions in c, than ah patients. four fh and ahs with higher viral replication were located in the near parts of the phylogenetic trees, indicating the association between the severity of hepatitis a and genomic variations in 'ntr, b and c of hav. conclusions: our study suggests that genetic variations in some parts of hav might cooperatively influence replication of the virus, and thereby affect virulence. viral factors should be considered and examined when discussing the mechanisms responsible for the severity of hepatitis a. aims: the incidence of acute viral hepatitis a in adults is increasing very much in south korea, . the aim of this study was to the clinical features and course in daejoen and its surrounding area. methods: forty seven patients admitted as acute viral hepatitis a in chungnam national university hospital between january and june were enrolled. the medical records were reviewed, retrospectively. results: the mean age was . . common occupations were company employee and studuents. most common symptom was jaundice. presumptive infection sources were raw fish or shellfish and raw meat. initial laboratory findings were as follows(mean value): wbc /ul, hemoglobin . g/dl, platelet × /l, ast iu/l, alt iu/l, total bilirubin . mg/dl, alkaline phosphatase u/l, ggt u/l, prothrombin time(inr) . . hospitalization was . days. peak laboratory findings were as follows: alt iu/l, total bilirubin . mg/dl. leukopenia (< /ul) and thrombocjtopenia (< × /l) were ocurred in sixteen and six patients, respectively. recovery time of each biochemical finding was as follows: alt . days, total bilirubin days. recovery rates of altand total bilirubin were . % and %, . % and % at weeks, weeks after diagnosis, respectively. prolonged jaundice ( days) was detected in one patient. all patients were recovered by supportive management. conclusions: in south korea, acute viral hepatitis a was more prevalent in young adults, recenlty. presumptive infectious sources were raw fish or guangxi center for disease prevention and control shellfsh and raw meat. if it can not change the food style that many korean enjoy raw seafood, vaccination for adults must be considered to prevent it. objective: to assess the safety and immunogenicity of a new inactivated hepatitis a vaccine (vero cell). pe methods: subjects were selected in gongcheng city of guangxi zhuang autonomous region, and the clinical trail was carried out according to the random, double-blind and parallel principle from january to august, . after vaccination by , schedule, adverse events of the subjects were observed, the seroeonversion rate and geometric mean titer (gmt) were tested by the competitive inhibition elisa. results: after immunization, the systemic and local reaction rates of adults were . % and . %, which was no significantly statistical difference compared with control group, . %and . %; while the rates of children were . and . %%, and no significant statistical difference compared with control group, . %and . %. one month after first dose of vaccination, the seroconversion rates of children and adults were . % and . %, and one month after second dose of vaccination, the rates were all %, the gmts of children and adults were miu/m and miu/ml, which was significant statistical difference in children compared with control group, miu/ml and miu/ml, respectively. methods: igg anti-hav was measured in a total of subjects under the age of , who visited hanyang university seoul and guri hospitals between january and may . results: fig. shows the relatively low positive rates of the antibody in ages of to and the lowest rates of . % and . % in the age group of to , following the ages of to with rates of background: some viruses encode proteins that affect their cap-independent internal ribosomal entry site (ires)-mediated translation and their replication. it was recently reported that hepatitis a virus (hav) proteases interact with intracellular dsrna-induced retinoic acid-inducible gene (rig-i)-mediated signaling, but it remained unknown whether hav proteins have any effects on hav ires-independent translation. in this study, we investigated the effects of hav non-structural proteins on their ires-mediated translation using a reporter assay. pe methods: the bicistronic reporter constructs, termed psv -hm -ires, psv -a -ires, psv -a -ires, psv -f -ires, and psv -f -ires, contain the sv promoter that controls the expression of a bicistronic message coding for renilla and firefly luciferases separated by hav ires, and are derived from strain hm , acute convalescent hepatitis clones a , a , fulminant hepatitis clones f , f , respectively. human hepatoma cell lines were co-transfected with psv -hav-ires and each hav protein-expression vector. luciferase activity was determined h after transfection. were from other countries within asia, africa, middle east, and eastern europe. patients of a wide age range were affected by hepatitis delta (mean age . , median . , range - ). ( %) of were co-infected with hcv. hepatitis b virus (hbv) dna was detectable in ( %) patients and negative in ( %) patients. all hepatitis delta patients were extracted from a prior study conducted by this collaboration. there were , chronic hbv carriers. ( . %) were hbv/ hcv/hdv infected. ( %) of patients carried a diagnosis of cirrhosis compared to ( %) of chronic hbv patients. ( %) hcv co-infected patients had evidence of cirrhosis while ( %) patients did not. conclusion: individuals with hbv/hdv co-infection have higher rates of cirrhosis. individuals with hbv/hcv/hdv infection have rates of cirrhosis significantly higher than individuals with either chronic hbv infection or hbv/hdv co-infection. testing for hdv should be performed in all patients, especially those with advanced liver disease or high risk behavior. clinical characteristics were compared between the patients with significant endoscopic findings (group a) and without such findings (group b). peak ast and alt level were higher in group a (p< . ). there were no statistical differences in age, gender, comorbidity, and etiology of acute hepatitis between group a and group b conclusion: significant endoscopic findings were found in considerable proportion of patients with acute hepatitis. severity of acute liver injury was associated with significant upper gastrointestinal endoscopic findings. in patients with severe acute hepatitis who complain of upper gastrointestinal symptom, esophago-gastro-duoenoscopy should be performed. background: in japan, hepatitis e virus (hev) testing is not allowed as routine one. to study the role of hev testing, we checked sera of the patients diagnosed as etiology-obscure acute liver injury. methods: we have seen cases of acute liver injury from january through december in our hospital and cases of them were etiology-obscure. in cases, were retrospectively tested for hev-igm, hev-iga and hev-rna (rt-pcr) by direct sequence method on stored sera taken at the time of presentation. result: two of cases ( . ) were positive for both hev-igm and hev-iga and one case was positive for hev-rna. in cases of acute liver injury, the cause of virus was cases ( . %) and unknown was cases ( . %). hev was occupied in . % in all cases and . % in the cases caused by virus. one of the two cases had been misdiagnosed as "drug induced hepatitis". hev of genotype was detected in one case and its nucleotide sequences of hev showed quite a high degree of similarity to the reported one at closed city in the same year. conclusion: hev is not rare in japan and the hev testing can reverse the diagnosis of acute liver injury. hev testing sould be used as routine one for acute liver injury. association of progesterone receptor gene with hepatitis e disease severity in pregnancy p.d. bose , b. das , a. kumar , p. kar maulana azad medical college, background/aims: incidence of fulminant hepatic failure (fhf) in hepatitis e is high in pregnancy particularly during rd trimester when there is an altered status of hormone and immunity. progesterone receptor (pr) up regulation provides fetal protection via immunosuppression but lower immune status in pregnancy may add to the disease severity. till now, no data is available whether pr can play any role in hepatitis e disease severity during pregnancy. progins, a haplotype of pr consisting of -bp insertion in intron g together with point mutations in exons and is associated with increased stability and higher transcriptional activity. the aim of the study is to analyze pr mutation (progins) and m rna expression in hepatitis e virus infected pregnant women with avh and fhf. methods: a total of avh and fhf cases were studied. blood and placental tissue were collected from the medicine and gynecology wards of lnjp hospital, new delhi. cases were screened for acute viral markers by commercially available elisa kit. extraction of dna from blood and rna from placental tissue was done by qiagen kit. mutation in pr was detected by pcr-rflp. semiquantitative rt-pcr for pr expression was performed in placental tissue using beta-actin as internal control. results: pr mutation (progins) was significantly more in fhf compared to avh ( . % vs . %, p value< . ). protein expression was found higher in progins carriers. conclusion: progesterone receptor mutation (progins) may have a role in the hepatitis e disease severity in pregnant women. results: the hepatitis e was predominantly sporadic, some patients superinfected with other viral hepatitis, especially hepatitis b. in the old patients, jaundice lasted longer and the length of stay was longer, the incidence of complication was higher than the young men. the incidence of complication in the superinfected group was higher than the simple infection. the transaminase in the simple infection group was obviously raise than superinfected with liver cirrohsis. methods: liver sample were paraform-glutaral fixed, paraffin-embedded, sectioned and immunohistochemical stained, and positive samples were selected for histological analysis and rt-pcr detection. result: positive rate of hev immunohistochemistry ranged from % to % (fig. ) . hepatocyte degeneration, scattered singled karyopyknosis, lymphocytic infiltrate, hyperplasia of bile canaliculus at the portal area and fibrous connective tissue hyperplasia been observed during histological analysis (fig. ) , and two genotype hev which closely related to many strain isolated from patients with sporadic acute hepatitis been detected. conclusion: the patients infected with hepatitis e of young men were frequently. jaundice lasted long in the old patients, the incidence of complication was higher in the superinfected men and the old men. conclusion: additional public-health concerns might be placed on pork safety and the risk of hev infection via the consumption of undercooked pork products. poster session, hall b aim: esophageal varices (ev) recurs frequently after endoscopic variceal ligation (evl) or endoscopic injection sclerotherapy (eis). we retrospectively investigated risk factors for early recurrence of ev after endoscopic treatment. methods: we treated patients with ev, who had no past history of ev, at ehime prefectural central hospital from october to june . of those, ( %) were observed for at least months after treatment and enrolled. we divided them into rupture cases at initial endoscopic treatment [(bleeding group; n= ( %)], and cases with preventive evl or eis performed [preventive group; n= ( %)]. all received periodic upper endoscopy examinations to confirm recurrence or no recurrence of ev. results: recurrence of ev occurred in of all subjects and the average period after treatment was . ± . months. the recurrence rate was significantly higher in the bleeding group ( / ) as compared to the preventive group ( / ) (p= . ). there was a significant relationship between recurrence of ev and hepatic reserve function (child-pugh a+b, c; / , / respectively; p= . ). in logistic multi-variant analysis, ev rupture at initial treatment and child-pugh c were risk factors for recurrence. in contrast, age, sex, hepatocellular carcinoma, portal tumor thrombosis, continuous alcohol consumption, therapeutic modality (evl or eis), number of treatment sessions, and operator experience did not have a significant relationship with recurrence. conclusion: in cases with ev rupture at initial treatment or child-pugh c, the risk for early recurrence must be considered and patients carefully observed in follow-up examinations. endoscopic cyanoacrylate injection: less oil for less ectopic embolism c.z. li , l.f. cheng , z.q. wang , f.c. cai , q.y. huang , e.q. linghu general hospital of chinese pla background and aim: endoscopic injection sclerotherapy with n-butyl- cyanoacrylate (nbca, histoacryl) has been reported to be effective for hemostasis of bleeding gastric varices, but occasionally the gel flows to other organs and causes ectopic embolism. the present study aimed to determine whether less amount of iodized oil preload in nbca injection helps in decreasing ectopic embolism. methods: from january to april , different methods of endoscopic nbca injection, "sandwich method" and "modified sandwich method" (in which iodized oil preload was minimized), were applied on gv cases, to evaluate if decrease of iodized oil preload resulted in less ectopic embolism. results: altogether cases of ectopic embolism occurred in the whole group ( . %), including cases of splenic infarction, case of transient paralysis and case of minor infarction of the lung. the modified sandwich method showed some superiority over original method in decreasing ectopic embolism ( / vs. / , p= . ). less cough during procedure was also found with the modified method ( / vs. / , p= . ). conclusions: less amount of iodized oil preload in endoscopic nbca injection is beneficial to decrease ectopic embolism. background: portal hypertension is closely associated with serious complications of liver cirrhosis which contribute to bad prognosis. hepatocellular carcinoma (hcc) and low serum sodium (sna) are manifestations of end-stage liver disease (esld) and are associated with poor survival in decompensated cirrhosis patients. therefore, we aimed to determine the relationship between hepatic venous pressure gradient (hvpg) and the development of hcc or low sna in decompensated alcoholic cirrhosis patients. methods: child-pugh scores, meld scores, and hvpg at baseline, and the development of low sna (sna < meq/l) or hcc during follow-up were analyzed prospectively in patients with decompensated alcoholic liver cirrhosis. the predictive values of different risk factors for the progression to the esld were investigated by multivariate analysis and the kaplan-meier method results: twenty-four patients developed hcc during the follow-up period. in the multivariate analysis, only baseline hvpg> mmhg was an independent predictive factor for the development of hcc (relative risk (rr)= . , p< . ). those with hvpg > mmhg showed a significantly shorter time for the development of hcc on kaplan-meier analysis. twenty patients developed low sna during follow-up. initial hvpg was also an independent predictive value for the development of low sna in the multivariate analysis (rr= . , p< . ). those with hvpg> mmhg also showed significantly shorter times for the development of low sna on kaplan-meier analysis. conclusions: in decompensated alcoholic cirrhosis, hvpg may be a useful predictive factor for the development of hcc and low sna, both of which are characteristic of esld and poor prognosis. the effectiveness of the treatment of octreotide on chylous ascites after liver cirrhosis d.x. zhou , , h.p. hu , background: octreotide is a crucial drug used for treating patients with chylous ascites; however, there have been few reports related to octreotide that are being used in cirrhotic patients. thus, this thesis is designed to determine the effects of octreotide on patients with chylous ascites after liver cirrhosis. methods: eight patients were diagnosed with chylous ascites, on the basis of laboratory findings on ascites samples, between january and may . octreotide was given to the six patients, while the remaining two were treated as a control. all patients had persistent peritonea drainage with the quantity and quality of the drainage fluid observed once every other day. all the necessary care was individually given to the patients during the therapy results: all patients properly received combined therapy including low fat and sodium diet, and diuretic and peritoneal drainage. the volume of the peritoneal drainage was reduced to zero in one of the six patients who received octreotide therapy, while the other five had the drainage volumes decreased from ml to ml with a clear appearance and negative qualitative analysis of chyle for those two patients who did not receive octreotide therapy, the conditions of peritoneal drainage seldom changed both from the qualitative and quantitative aspects. conclusion: octreotide, along with combined therapy, can rapidly relieve portal hypertension and reduce fat absorption from intestinal mucosa. it appears to be an effective therapy available for the treatment of chylous ascites caused by liver cirrhosis. albumin < g/l were the best predictors of large varices. a model using these predictors in a validation cohort study is planned. background-aim: cirrhosis is associated with raised acute phase proteins (app), irrespective of infection. it is, however, unclear whether their values differ significantly or whether a particular app might be more indicative of infection, and these questions were addressed in our study. methods: we measured serum crp, fibrinogen, ferritin, haptoglobin, -microglobulin, c , c , and c inhibitor in consecutive, cirrhotic patients, on admission. all patients were investigated according to a standard protocol for infection. child-pugh scores (cps) were calculated. results of app were expressed as means sem and compared with the mann-whitney test. results: ( , %) patients, median age years, (cps: a= ; b= ; c= ), were diagnosed with infection (spontaneous bacterial peritonitis= ; pneumonia= ; septic shock= ; extensive cellulitis= ; listeria monocytogenes meningitis= ; viral infection= ), while ( %) patients, median age years, (cps: a= ; b= ; c= ), showed no infection. although most app values were raised, there was no statistically significant difference between patients with or without infection, or among different cps groups, except for crp, which was significantly more raised in patients with infection (p< . ). this difference remained even after cps a cases in the non-infection group were excluded from analysis. interpretation: a significantly raised crp in cirrhosis would seem to be independent of cps staging and should prompt a thorough work up to exclude infection. by contrast, the discriminating power of all other app in the face of possible infection is negligible. the predictive value of crp towards infection is under investigation prospectively. although bleeding from ectopic varices such as duodenal, jejunal, ileal, colonic, and rectal varices is less common, it can also cause life-threatening problem, which is often difficult to diagnose and treat successfully. here we present a novel endoscopic approach for hemorrhagic rectal varices using endoscopic injection sclerotherapy with ligation (eisl). patients and methods: in - , we performed endoscopic treatment in patients with portal hypertensive varices. among those, four cases of hemorrhagic rectal varices were treated with the combined evl and sclerosing technique. the etiology of portal hypertension included oen idiopathic portal hypertension and three hcv cirrhosis. all patients had a history of prior abdominal surgery or endoscopic treatment for gastro-esophageal varices. results: hemostasis was obtained easily by the evl initially. furthermore, to avoid recurrent bleeding, the patients underwent endoscopic varicerography injection sclerotherapy (evis) using % ethanolamine oleate with iopamidol and the feeding vein was sclerosed successfully with no major complication occurred during the entire course of the treatment. conclusions: it is important to recognize the possibility of ectopic varices as a cause of gastro-intestinal haemorrhage especially in patients with a history of variceal therapy or abdominal surgery. the eisl technique is useful to control the initial and recurrent bleeding from rectal varices. t. hirano , t. okada , j. yamanaka , y. iimuro , n. kuroda , k. oh , y. yoshida , j. fujimoto aim: interferon (ifn) therapy is a powerful treatment for hcv-related hepatitis and is known to decrease the incidence of progression of hepatocellular carcinoma (hcc). however, thrombocytopenia is a common side effect of ifn treatment, often leads to discontinuance without insufficient therapeutic effect. in this study, we investigated the efficacy and safety of laparoscopic splenectomy ( ) in reversing thrombocytopenia in patients with hepatitis c cirrhosis and portal hypertension. patients and methods: out of patients who underwent ls in our department during aug and december , patients associated with portal hypertension. among these patients, three patients had hcc, and they were simultaneously underwent partial hepatectomy after splenectomy. platelet count, operative time, blood loss, complications and length of stay were calculated. results: thirteen patients underwent laparoscopic splenectomy; their mean age was years (range to years). six patients were child's class a and seven patients were class b. mean operative time was minutes (range to minutes). blood loss was little, and none required transfusion with packed red cells. a hand-assisted laparoscopic technique was used in four cases ( . %). average length of stay was . days. there have been no major complications during follow-up. platelet counts improved from a preoperative mean of /ul ( to ) to /ul ( to ) postoperatively. six patients are ongoing ifn treatment without remarkable thrombocytopenia. conclusion: laparoscopic splenectomy is safe and in patients with portal hypertension and thrombocytopenia. it may allows these patients by reversing thrombocytopenia. background: hepatic encephalopathy (he) is a significant cause of mortality in advanced cirrhosis patients. l-acyl-carnitine has been suggested as an alternative treatment for patients with he patients. to assess the clinical efficacy of acetyl-l-carnitine in the treatment of hepatic encephalopathy in cirrhotic patient, especially in diminishing the recurrence and reduction serum ammonia level. methods: we performed a randomized placebo-controlled, cross-over study. we administered acetyl-l-carnitine to group during months first then placebo during later months, and administering acetyl-l-carnitine to group alternatively. results: between january and february , thirty two selected cirrhotic patients were enrolled in this study. following randomization, the patients were divided into two groups (group = , group = ). during administering acetyl-l-carnitine period, serum ammonia level was decreased significantly in both groups significantly (p= . , vs. p= . respectively). however, during administering placebo period, serum ammonia level changes were not significant. in group , the first recurrence cases of hepatic encephalopathy were more than group (group = , group = ), and the first recurrences were occurred during first months in all groups. conclusion: our study demonstrates that acetyl-l-carnitine administration reduced serum ammonia level, but not definitely diminishing the recurrence of hepatic encephalopathy. sodium (na + ) and water retention are the most common abnormalities in cirrhotic patients and the magnitude varies from patients to patients. aim: to assess the relationship between the meld score and urinary excretion of na+ in non-azotemic cirrhotic patients. methods: fifty four cirrhotic patients with ascites and normal serum creatinine (< . mg/ml) were admitted and placed on a low sodium diet ( g/day), while all diuretics were withdrawn for days. the electrolytes (na + , k + , na + / k + ) were measured in a random urine and both the volume and na + concentration of urine collected for h after administration of furosemide mg i.v. were determined. results: table. conclusions: the meld score was significantly correlated with the degree of impairment of urinary na+ excretion. the ratio of na + /k + in a random urine specimen and furosemide-induced na+ excretion reflect the degree of impaired natriuresis in non-azotemic cirrhotic patients with ascites. background: portal hypertensive gastropathy (phg) is common finding in patients with liver cirrhosis and portal hypertension. despite portal hypertension remains the crucial trigger for the development of phg, the relationship between portal hypertension and phg has not been widely investigated. methods: fifty-three cirrhotic patients ( males, mean age years) who were performed hepatic vein catheterization between november and august were prospectively included in this study. the degree of phg was assessed according to the third baveno international consensus workshop, and classified three degrees as no, mild and severe. the hepatic venous pressure gradient (hvpg=whvp-fhvp) measurements were performed by triplicate in each case, and results were given as arithmetic means of the three determinations. result: hvpg values did not differ between the patients without phg ( . ± . mmhg) and those with phg ( . ± . , p= . ), nor between those with mild ( . ± . mmhg) or severe phg ( . ± . mmhg, p= . ). the degree of phg and hvpg did not differ regarding the etiology of the cirrhosis(p= . , p= . ) nor regarding the child pugh classification(p= . , p= . ). no correlations were found between the degree of phg and child pugh score, age, with or without ascites, albumin, bilirubin, creatinine, meld score and the degree of gastroesophageal varices. conclusions: our data show that the presence and the severity of phg does not correlate with the degree of hvpg, and that correlate with esophageal varices in patients with liver cirrhosis. introduction: phlebosclerotic colitis is a rare form of ischemic colitis characterized by the thickening of the colonic wall due to fibrous degeneration of the submucosal layer and fibrotic sclerosis of the venous wall. there are a few reports those this entity might be related to portal hypertension with disturbed venous return from the colon and mesentery. case description: a -year old man with alcoholic liver cirrhosis presented with right lower abdominal pain/tenderness and bloody diarrhea. a colonoscopy revealed multiple circumferential ulcerations in the transverse colon and the scope could not get through the ascending colon due to luminal stenosis, showing histologic finding of ulcerative inflammation with inflammed granulation tissue. abdominal computed tomography demonstrated liver cirrhosis with splenomegaly, multiple portosystemic venous collaterals, diffuse vascular engorgement and the wall thickening of right proximal to mid ascending colon with increased density in the surrounding fatty tissue. a follow-up colonoscopy performed one month later showed still remained multiple ulcerations in the transverse colon and could not further advance to ascending colon. superior mesenteric angiography revealed no main branch occlusion but pooling at the venous phase on ascending colon. a right hemicolectomy was performed because of the colonic obstruction. gross findings on operation showed thickening of the cecum and ascending colon. microscopic examination showed fibrous thickening in the submucosa, abundant neurovascular bundles in the mesentery and several intravascular hyaline thrombi of the mesenteric vessels. here we report the first case of early stage of phlebosclerotic colitis in a cirrhotic patient in korea. spontaneous bacterial peritonitis (sbp) is one of the severe complications in advanced cirrhotic patients with a high mortality rate. although a more rapid diagnosis should lead to the better survival, it takes several days to detect the causal bacteria from ascitic fluid cultures. furthermore, despite the use of sensitive methods, ascitic fluid cultures were negative in more than % of patients with suggestive clinical manifestations of sbp. therefore, diagnosis of sbp is based on the polymorphonuclear leucocytes (pmn) cell count in the ascitic fluid. the hybrizep kit (fuso pharmaceutical industries, osaka, japan) detects the dna of bacteria that have been phagocytized in neutrophils and macrophages, using in-situ hybridization method within one day. here we present a case of the patient for whom the hybrizep kit was used to detect the causal pathogen of sbp. a -year-old man had been admitted for the treatment of ascites and esophageal varices. one week after the admission, he complained abdominal pain and fever. because the pmn cell count in ascites fulfilled the criteria of sbp ( /mm ), we started an empirical antibiotic therapy without waiting for a result of the culture, and his symptoms improved within a few days. on the following day of the onset, in situ hybridization showed the positive signals by the ek probe, which detected the genomeic dna of e.coli species. however, the ascitic fluid culture was negative. this case suggested that the hybrizep kit was useful for the rapid diagnosis of sbp with high sensitivity. background: it has not been known that the hemodynamic effect of a portal hypertension for splenomegaly or esophageal and gastric variceal formation. this study was performed to access the parameters of doppler ultrasonography associated with splenomegaly or varices in patients with cirrhosis. patients and methods: from may to may , cirrhotic patients were performed the doppler ultrasonography. of these patients were accessed the severity of varices endoscopically. the three dimensional volume of spleen was measured from a length, width and thickness on sonography. results: the splenic volume ( . ml vs . ml, p= . ) and blood flow of main portal vein ( . cm/s vs . cm/s, p= . ) were statistically significant different in alcoholic ( / ) and non-alcoholic ( / ) cirrhosis groups. the splenic volume ( . ml vs . ml, p= . ), damping index ( . vs . , p= . ), and blood flow of main portal vein( . cm/s vs . cm/s, p= . ) were statistically significant different in esophageal variceal groups ( / ) and non-esophageal variceal groups( / ). the only splenic volume ( . ml vs . ml, p= . ) were statistically significant different in gastric variceal groups ( / ) and non-gastric variceal groups ( / ). the hemodynamic parameters venous ammonia and cff at baseline and after one month of treatment with lactulose. mhe diagnosed by abnormal psychometry and/or p erp.response defined by normalization of abnormal test parameters. results: mhe diagnosed in ( %) patients. of patients ( %) had both abnormal psychometry and p erp whereas ( %) alone had abnormal psychometry, ( %) had abnormal p erp.cff was < hz in ( %) patients. mhe recovered in % with treatment and cff > hz was seen in ( %) of patients. cff sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy before and after treatment is shown in table. conclusions: critical flicker frequency is a simple and accurate test without any age or literacy dependence for the diagnosis and recovery of patients with mhe. background/aims: endoscopic injection of n-butyl- -cyanoacrylate (histoacryl) is an effective treatment of varix bleeding. but nontarget embolizations and septicemia are unwanted complications. we evaluate the risk factors for complications. methods: thirty-three patients with esophageal or gastric varix bleeding received endoscopic histoacryl therapies ( procedures). baseline varix size, ctp score were checked. serum leukocyte, blood culture and body temperatures were repeated checked within one week after procedure. average volume of histoacryl per each session was . ml, and dilution volume ratio of histoacryl/lipiodol was / or / . results: average of ctp score was . ± . . three cases of septicemia were correlated with ctp score rather than session frequency or injection volume. two cases of systemic embolizations (pulmonary and splenic arterial embolism) were correlated with high lipiodol dilution ratio ( / ) and lipiodol volume rather than histoacryl volume or ctp score. conclusion: ctp score, lipiodol volume and dilution ratio of histoacryl/lipiodol were significant risk factors for complications. detection of circulating toll-like receptor and and cd +cd + regulatory t cells in patients with hbv-related liver cirrhosis x.q. wang , y. zhang , x.f. bai , j.q. lian background : to detect circulating cd + cd + regulatory t cells and toll-like receptor(tlr) and tlr expression on the peripheral blood mononuclear cells (pbmcs) of patients with hbv-related liver cirrhosis (lc), and to explore the correlation between them. methods: pbmcs isolated from lc patients , chronic hepatitis b (chb) patients and normal controls(nc) were stained with fluorescent labeling anti-tlr -pe, anti--tlr -apc, anti--cd -fitc monoclonal antibodies and anti-cd -percp anti-cd -fitc anti-cd -pe. samples were collected and detected of three-color immunofluo rescence by flow cytometry. results: the expression of tlr and tlr were significantly up-regulated in patients with lc than those in the controls.the expression of tlr was significantly increased in patients with lc than those in patients with chb, but there were no differences of tlr expression between lc and chb.treg/cd + t cells were significantly increased in patients with chb than those in patients with nc and lc, but there were no differences between lc and nc. there were no correlation between the expression of tlr ,tlr and treg in patients with lc . the expression of tlr and tlr on pbmcs in patients with lc were positive correlation.the expression tlr and hbv dna level were negative correlation in patients with lc. conclusion: the expression of tlr and tlr were up-regulated on pbmcs in patients with lc. it seems to be expression of tlr and tlr invovlved in the pathogenesis of lc. evaluation of c-phenylalanine breath test for the measurement of hepatocyte function in patients with chronic liver disease z.j. bao , d.k. qiu , , x. ma , , g.s. zhang , y.q. huang , z.p. fan , , s.m. yin huadong hospital, fudan university, renji hospital, shanghai jiao tong university school of medicine, background: the objective is to investigate whether the c-phenylalanine breath test(pbt) would be useful for the evaluation of hepatic function in patients with chronic hepatitis b, liver cirrhosis and minimal hepatic encephalopathy (mhe). methods: l- [ - c] phenylalanine was administered orally in a dose of mg to patients with liver cirrhosis, with chronic hepatitis b and healthy subjects. the pbt was measured at different time points ( , , , , , , , min) to obtain the values of delta over baseline, percentage co exhalation rate and cumulative excretion (cum). the relationships of the cumulative excretion with the c-%dose/h and blood biochemical parameters were investigated. results: the c dose h - at and min combined with the cumulative excretion at and min showed correlations with the chronic liver diseases, especially child-pugh score and mhe or not. and the data showed correlations with serum albumin hemoglobin platelet and child-pugh score. prothrombin time, total and direct bilirubin were significantly increased, while serum albumin, hemoglobin and platelet, the cumulative excretion at and min values decreased by degrees in healthy controls, child-pugh a, b, and c patients (p< . ). similar results of pbt were in the patients with and without mhe, while only prothrombin time prolonged and total bilirubin increased (p< . ). conclusions: the pbt can be used as a non-invasive assay to evaluate hepatic function in patients with liver cirrhosis and mhe. the % c dose h- at min, % c dose h- at min and cumulative excretion at min may be the key value for determination at a single time-point. branched chain amino acids in improving survival and decreasing risk of liver failure among cirrhotic patients: a meta-analysis h. flores , e.l. ang , n. iv estanislao philippine general hospital background: the state of a patient's nutritional status greatly affects disease outcome. among cirrhotic patients, approximately - % are in a state of protein-energy malnutrition. hence, adequate nutritional support is essential to improve their general medical condition and long term prognosis. several studies have shown than branched chain amino acids (bcaa) may be of benefit for this purpose. it is the aim of this study to evaluate the effectiveness of diet plus bcaa compared to diet alone in improving survival and in decreasing liver failure among cirrhotic patients. methods: pubmed, cochrane, and embase search was done for articles which compared the clinical effects of bcaa supplementation versus diet alone among patients with liver cirrhosis. the following free-text terms and mesh words were used -"branched chain amino acids", "amino acids, branched chain", "bcaa", "liver cirrhosis", "cirrhosis", "randomized controlled trials'" and "meta-analysis". after critical appraisal of the included studies, a random effects model using odds ratio was used to synthesize the results (revman . ). results: rcts were included for analysis with a total study population of . combination of the studies showed a significant decrease in the risk of liver failure (or . , % ci . - . , p= . ) and a trend towards benefit in improving survival (or . , % ci . - . , p= . ). conclusions: the overall trend appears to show benefit in the use of branched chain amino acids for patients with cirrhosis with respect to liver failure and survival. background: the proxisome prolifrator-activated receptor gamma (ppar ) is a member of the nuclear hormone receptor superfamily that is involved in the control of inflammation, carcinogenesis and gastric ulcer. on the other hand, the frequency of gastrointestinal ulceration is higher in cirrhotic patients compared with the normal population. the present study was designed to investigate the effect of specific ppar ligand, pioglitazone, on the mucosal lesions induced by ethanol in cirrhotic rats and the possible involvement of nitric oxide in the pioglitazone effect. methods: cirrhosis was induced by surgical ligation of bile duct and sham-operated rats served as controls. both cirrhotic and sham rats were kept for days after the operation. different groups of sham and cirrhotic animals received saline, or , or mg/kg pioglitazone, daily during last days of the fourth week after the surgery. another groups of bdl or groups of sham rats received l-name, a non selective inhibitor of nitric oxide synthase, alone or along with mg/kg pioglitazone for days. on day , rats were killed hour after ethanol administration and the area of gastric lesions was measured. results: the ethanol-induced gastric mucosal damage was significantly more sever in cirrhotic rats than sham-operated ones (p < . ). pretreatment with pioglitazone dose dependently attenuated gastric lesions induced by ethanol in both sham and cirrhotic rats, but this effect was more significant in cirrhotic ones. concurrent treatment of l-name and pioglitazone decreased the ulcer index in bdl rats more than the groups that received l-name or pioglitazone alone. conclusion: we conclude that chronic treatment with pioglitazone exerts a potent gastroprotective effect on the stomach ulcers of cirrhotic rats probably due to inhibition of nitric oxide synthase. inhibition of phosphodiesterase -a novel therapeutic strategy for portal hypertension l. halverscheid , p. deibert , b. pannen , r. schmidt , m. roessle , w. kreisel university hospital duesseldorf, germany, university hospital freiburg, germany introduction: the no-cyclic gmp system is a key factor in the regulation of splanchnic and hepatic blood flow and may be a target for medical treatment of portal hypertension. clinical data have shown that inhibitors of phosphodiesterase (pde ) lower portal pressure in cirrhotics. methods: we monitored in rats the effects of the pde inhibitors vardenafil and sildenafil on systemic and hepatic hemodynamic parameters up to minutes after the drug. the drugs were administered intravenously into the tail vein at (group a), (group b), and g/kg body weight (group c). . % nacl was the control. n = for each group. results: the most prominent changes were observed in the vardenafil b group: mean arterial and portal venous pressure decreased (- %, - %), as well as portal venous, hepatic arterial, and systemic vascular resistance (- %, - %, - %). portal venous and sinusoidal flow increased (+ %, + %). in the vardenafil c and sildenafil b and c groups there was an increase of portal venous flow by - %, an increase of sinusoidal flow by - %, and a decrease of portal venous resistance by about %. there was a trend for reduction of portal venous pressure. conclusions: vardenafil and sildenafil influence portal hemodynamics in the rat. portal venous flow increases by - %, portal venous resistance decreases by > %. dependent on the dose, portal venous pressure decreases significantly. these data yield further evidence that pde inhibitors may be a novel therapeutic option for portal hypertension. groups of liver cirrhosis e. havrilyuk lviv national medical university introduction: rupture of esophageal varicose resulting in posthemorrhagic anemia is a common life-threatening complication of liver cirrhosis. but it is not clear, why the other patients, having the same degree of sclerosis and histologic activity index, die from hepatocellular failure or other reasons. aims & methods: autopsy cases performed in lviv regional hospital in - were analyzed. screening of slides with liver tissue allow to select cases ( , %) with cirrhosis (complete and incomplete), which are examined in order to evaluate the frequency of lethal portal hypertension complications in the different etiologic groups of liver cirrhosis. results: according to the etiologic factor the following groups of liver cirrhosis were examined: alcoholic disease ( , %), viral hepatitis ( , %), nonalcoholic steatohepatitis ( , %), secondary biliary cirrhosis ( , %), cardiac sclerosis ( , %), combined lesions ( , %) and cryptogenic cirrhosis ( , %). analysis shows that in cases ( , %) patients die from cirrhotic complications (hepatocellular failure, jaundice, portal hypertension) and only in cases ( , %) -from posthemorrhagic anemia caused by rupture of esophageal varicose. in the latter cases correlation between the etiologic types of cirrhosis is almost the same, as in the main group and only alcoholic lesions ( %) and biliary cirrhosis ( , %) are more frequent. conclusion: analysis shows that development of lethal complications of portal hypertension can not be explained only by etiologic factor. probably additional stimuli are more important for morphogenetical variants of cirrhotic transformation. patients with viral cirrhosis k. mumtaz , s. ahmed , h. ali shah , s. hamid , w. jafri background and aims: increased nitric oxide (no) production is incriminated in the pathogenesis of arterial vasodilation and hyperdynamic circulatory state in non cirrhotic models of portal hypertension (pht). we investigated the relative roles of constitutive nos (enos) and inducible nos (inos) isoforms in the development of rabbit models of endotoxemia induced portal hypertension (eipht) methods: eipht was induced by chronic injection of lipopolysaccharide via an indwelling cannula placed in the gastrosplenic vein of rabbit and maintained for months. the concentration of no, expression of nos (enos and inos) mrna and protein was measured in eipht and sham operated control animals. results: rabbits with eipht compared with controls had raised portal pressure (in mmhg- . ± . vs . ± . ;p< . ; mo; . ± . vs . ± . ; p< . , mo; . ± . , vs . ± . ;p< . ), arterial hypotension (in mmhg- . ± . vs . ± . , p< . , mo; . ± . vs . ± . ,p< . , mo; . ± . vs . ± . , p< . , mo), splenomegaly (in g- . ± . vs . ± . , mo; . ± . vs . ± . , mo; . ± . vs . ± . , mo), normal liver functions and preserved hepatic architecture at , and mo. serum levels of no as well as the no were significantly elevated in eipht rabbits as compared to the controls. the expression of enos, at the level of mrna, was significantly increased in eipht rabbits consistent with increased levels of expression of inos as compared to the controls. the enos but not inos protein expression was elevated in eipht than control rabbits. conclusion: vascular dysfunction in the splanchnic circulation during the development of endotoxemia induced portal hypertension is predominantly characterized by enos and partly by inos gene up-regulation. liver cirrhosis contributed to the immunocompromised status by shedding the membranous tnfrii t.n. lin , c.h. chao , i.s. sheen , y.p. ho , w.t. chen , c.j. lin , c.t. yeh , c.y. lin liver research unit, linkou medical center, chang gung memorial hospital, chang gung university, taoyuan, taiwan background & aim: patients with decompensated liver cirrhosis (dlc) were regarded as immunocompromised, reflected by high incidence of bacterial infection. paradoxically, the proinflammatory cytokine like tnfincreased significantly in patients with dlc even in the face of this immunocompromised status. on the other hand, regulatory t cell (treg cell) is believed to play an important role in inhibiting immune responses, including innate immune responses like blockade of tnf-effect through soluble tnfrii. here, we studied the role of treg cells and tnfrii in patients with decompensated liver cirrhosis. patients and methods: healthy volunteers and cirrhotic patients were enrolled. the percentage of treg cells were enumerated by flow and serum levels of il- , tgf-and tnf-by elisa. results: the percentage of treg cells increased significantly in patients with dlc associated with increased serum levels of il- and tgf-. in addition, these treg cells were mainly memory type reflected as high cd ro. furthermore, the tnfrii expression increased significantly on these treg cells of dlc. interestingly, these membranous tnfrii on treg cells could be shed-off. lastly, we found the serum soluble tnfrii concentration increased significantly in patients with dlc when compared with normal volunteers. conclusion: our results demonstrated memory treg cells with high tnfrii expression increased significantly in patients with decompensated liver cirrhosis that could possibly blocked the biological effect of tnf-by shedding membranous tnfrii and contributed to the immunocompromized status of dlc. background: portal pressure measured as hepatic venous pressure gradient (hvpg) correlates with severity of portal hypertension and the development of complications. hvpg measurement is invasive. recently, liver stiffness measurement has been shown to correlate with liver biopsy and helps predict outcome in chronic liver disease patients. this study was conducted with the aim to study the correlation between portal pressure as measured by hvpg and liver stiffness as measured by fibroscan among patients with portal hypertension due to various causes. methods: between august and september , consecutive patients with portal hypertension were included and were subjected to hvpg measurement and fibroscan (echosens, france). results: of the patients with portal hypertension, both hvpg and liver stiffness were measurable in [ ( %) males; mean age . ( . ) years]. the etiological distribution was hbv related cirrhosis in patients, hcv cirrhosis in , cryptogenic cirrhosis in , alcoholic cirrhosis in , hbv and alcoholic cirrhosis in and primary extra-hepatic portal vein obstruction in . the mean hvpg and liver stiffness of this group were . ( . ) mm hg and . ( . ) kpa respectively. there was a strong positive correlation between hvpg and liver stiffness [r = . ; p = . ]. conclusions: non-invasive measurement of liver stiffness correlates well with invasive measurement of portal pressure. liver stiffness measurement could be used as a prognostic indicator to predict the severity of portal hypertension. endpoints were rate of rebleeding and mortality till day after inclusion and to see for any adverse events. results: the bleeding was stopped in all patients ( %). rebleeding till day was observed in ( %) patients ( each in group a and b). total patients ( %) died ( each in both groups) due to rebleeding. transfusion needs were higher in group a ( . ± . versus . ± . , p<. ). serious adverse effects leading to treatment discontinuation were not seen in any patients in both groups. conclusion: prolonging terlipressin treatment did not confer any significant decrease of mortality or bleeding recurrence. however transfusion requirements were significantly decreased in patients receiving prolonged treatment. serious adverse effects leading to treatment discontinuation are rare. poster exhibition -miscellaneous poster session, hall b background: it is important to know the prevalence of atp b gene mutations of different geographical areas to justify the local screening strategies for wilson disease (wd). materials: eleven unrelated lithuanian families, including wd patients were tested. genomic dna was extracted from whole venous blood using a salt precipitation method. firstly, semi-nested pcr technique was used to detect the c. c>a (p.h q) mutation. patients not homozygous for c. c>a (p.h q) mutation were further analyzed. the exons of the wd gene were amplified in a thermal cycler. direct sequencing of the amplified pcr products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer. results: total of wd patients (mean age . years; range - ; male/female, / ) presented with hepatic disorders and their first degree relatives were studied. some of wd patients in addition to hepatic symptoms have had extrahepatic disorders (haemolytic anaemia ; fanconi syndrome ; neurophsychiatric and behavioural disorder ). twelve of ( . %) wd patients have had c. c>a (p.h q) mutation, of them in both chromosomes, were presented as compound heterozygotes with additional c. - delggtttaaccat, c. delc or c. g>a (p.r q) mutation. for one patient with liver cirrhosis and psychiatric disorder no mutations were found. out of first degree wd relatives ( . %) were heterozygous for c. c>a (p.h q) mutation. conclusion: c. c>a (p.h q) missense mutation is characteristic for lithuanian wd patients. even . % of wd patients with hepatic presentation of the disease are homozygous or compound heterozygote for this mutation. background: diabetic dyslipidemia is a crucial problem of diabetic patients with inadequate control. we investigated the relationship between glutamic-pyruvic transaminase (gpt) and high-density lipoprotein cholesterol (hdl-c) in diabetic patients. methods: with informed consents, we recruited outpatients with diabetes at a hospital in rural area in taiwan in [ ] [ ] [ ] [ ] . anthropometric measures, blood tests and urine screening were examined in diabetic patients. results: overall, there were diabetic patients aged - years enrolled in this study and ( . %) of them had low hdl-c. diabetic patients with the highest quintile of gpt had higher average of body mass index (p< . ), diastolic blood pressure (p = . ), but lower average of hdl-c (p< . ) compared with diabetic patients with the lowest quintile of gpt. the prevalence of obesity ( . % vs. . %, p< . ) and low hdl-c ( . % vs. . %, p< . ) were higher in diabetic patients with highest quintile of gpt than in diabetic patients with lowest quintile of gpt. in the multivariate logistic regression, diabetic patients with highest quintile of gpt had higher odds ratio (or) of low hdl-c compared with diabetic patients with lowest quintile of gpt (or = . , % confidence interval [ci] = . - . ). the corresponding or of low hdl-c in patients aged years and older was . ( % ci = . - . ). conclusion: high gpt is one of factors associated with low hdl-c in diabetic patients. the effect of desferrioxamine as supplement to cefotaxime in the treatment of spontaneous bacterial peritonitis na. seda , m. el-hamamsy , r. el-wakil , m. al azizi ain shams specialized hospital, faculty of pharmacy,ain shams university, faculty of medicine,ain shams university, faculty of pharmacy ,ain shams university background: oxidative damage lead to cell damage, organ dysfunction and death in sepsis. desferrioxamine (dfx), an antioxidant iron chelators. the aim was to assess the efficacy of desferrioxamine supplemented to cefotaxime in the treatment of spontaneous bacterial peritonitis (sbp) in cirrhotic patients. methods: thirty patients divided into two groups: group i (n= ) with sbp and receiving cefotaxime ( g iv every hours) alone and group ii (n= ) with sbp receiving cefotaxime ( g iv every hours) with desferrioxamine ( mg im twice daily).all patient were monitored for seven days, their vital organs were screened and their ascitic fluid was assessed completely including microbiological investigations. results: the concomitant administration of desferrioxamine with cefotaxime significantly at (p< . )and(p< . ) improved the therapeutic outcome and the cure rate after days of treatment as compared to patients using cefotaxime only. conclusions: desferrioxamine can improve the therapeutic outcome by preventing iron-induced organ damage and inhibiting bacterial growth. oligella ureolytica is a gram-negative, nonfermenting rod that is infrequently recovered from clinical specimens and is most commonly isolated from the urine of patients with chronic indwelling urinary catheters or other urinary drainage systems. bacteremia due to this organism is an extraordinary finding. we describe here a case of oligella ureolytica being detected in the blood of a patient with decompensated cirrhosis. a -year-old male man was admitted to hospital with -month duration of debility, poor appetite and abdominal distension. decompensated cirrhosis was diagnosed based on clinical findings such as hepatic face, ascites, edema of lower limbs and icteric sclera. laboratory results showed positive serum anti-hcv and high serum hcv rna level. the patient received a therapeutic regimen of pegylated interferon alpha a plus ribavirin after being admitted to hospital. during hospital stay, a fever of -day duration with shivering occurred to the patient. three blood cultures were drawn, which all grew oligella ureolytica in pure culture. the organism was identified by the viteck compact (biomerieux, france). additional tests for identification resulted positive for nitrate reduction and urea hydrolysis, strongly positive for phenylalaninedeaminase activity and showed no growth at . c. tests for nitrite reduction and motility resulted negative. the organism was resistant to amikacin, cefoperazone, levofloxacin, piperacillin/tazobactam, trimethoprimsulfamethoxazole, aztreonam, cefotaxime, piperacillin and was susceptible to gentamicin, imipenem, meropenem, and netilmicin. a -day of combined therapeutic regimen with cefminox and isepamicin was administered to the patient. within days, the patient became afebrile. background: endoscopic ultrasound (eus) is often performed in patients with unexplained liver tests to assess the gallbladder, bile ducts and pancreas. an unremarkable eus exam and negative hepatology workup often leads to a liver biopsy. eus may provide histopathologic evaluation of the liver in these cases under direct, real-time visualization. aim: to assess the feasibility and efficacy of eus guided core biopsy of the liver in a porcine model. methods: female pigs were used and live procedures were performed under general anesthesia. a linear echoendoscope was used and the liver identified endosonographically. transgastric core biopsies of the liver were obtained with a gauge quick-core ultrasound biopsy needle (wilson-cook) and sent for histopathologic evaluation. live animals were euthanized at the end of the procedure and necropsy performed. results: core biopsies of the liver biopsy were obtained in animals ( cadaver and live anesthetized). a total of thirteen needle passes were made (mean . ; range - needle passes per animal) and a visible core of tissue obtained. the maximum length of liver tissue obtained was mm and considered adequate for assessment as more than one such specimen could be obtained. microscopic evaluation confirmed liver tissue. no complications were noted. necropsy did not show any evidence of bleeding, perforation or damage to surrounding structures. conclusion: eus-guided liver biopsy is feasible and can be performed at the time of routine echoendoscopic exam in select patients undergoing eus examination for abnormal liver tests. background: as the common indexes, alt, ast and plt play an important role in disease diagnosis, treatment and prognosis. many researchers suggested that there was inflammatory changes and fibrosis in chronic hepatitis b and c patients whose alt level was persistently normal. a large sample investigation showed that the serum level of alt in healthy persons is lower than the normal reference value. this study re-evaluated the normal serum level of alt, ast and plt. methods: people were enrolled in the study between sep. and oct. . the platelet count and serum alt and ast levels were measured. frequencies, one-sample kolmogorov-smirnov test and nonparametric tests were used to analyze the difference between age groups, male and female, glucose groups, cholesterol groups and triglyceride groups. result: in the five groups, there is significant difference in alt and ast levels between male and female. in group , the alt and ast levels showed a significant difference between different age groups, between different glucose groups and triglycide groups. in the three groups the plt level is significantly different between male and female, and the serum level in male is higher than female. there is significant difference between different age groups conclusion: the serum levels of alt, ast and plt are all significantly different between male and femal. there is significant difference between different genders and age groups for plt. the serum level of plt is higher than the reference value. background/aims: myeloproliferative disorders (mpd) (like polycythemia vera, essential thrombocythemia and primary myelofibrosis) are responsible for % cases of hepatic venous thrombosis (hvt) and % of portal venous thrombosis (pvt) in western series. latent form of mpd lacks the characteristic blood picture and may be classified as idiopathic thrombotic disorder. a point mutation at val phe of janus kinase tyrosine kinase gene (jak v f mutation) occurs in high proportion of the patients with mpd. this non-invasive test with high positive predictive value is now considered to be essential for diagnosis of various mpd. this test may be useful in diagnosing latent form of mpd in splanchnic venous thrombosis methods: patients with confirmed pyogenic liver abscesses admitted from to in our institution were included. there were men and women ranging in age from to years. the medical records were reviewed for clinical, laboratory and radiographic characteristics. results: among patients, ( . %) experienced at least one complication. there were pulmonary (pleural effusion, pneumonia, empyema) complications, septic shock, acute renal failure, abscess rupture, pseudomembranous colitis, and pericardial effusion. the predictive factors for its complications were: systemic inflammatory response syndrome (sirs, factors), thrombocytopenia ( , /ml), hypoalbuminemia ( . g/dl), elevated ast or alt (> iu/l), hyperbilirubinemia ( > . mg/dl), k. pneumonia, air within abscess cavity (p< . ). conclusions: the incidence of complications in the pyogenic liver abscess was . %. the various predictive factors of complication should be monitored carefully. further large scaled study should be warranted. background/aims: hepatic iron deposition is a common feature in chronic hepatitis c (ch-c), however, whether it could enhance the progression of fibrosis or not is controversial. the aim of this study was to evaluate the status and significance of hepatic iron deposition in the korean patients with chronic hepatitis c. methods: untreated, ch-c patients who underwent liver biopsy were included. the hepatic iron was assessed by scheuer's scoring system, and activity, fibrosis, and steatosis were scored by a pathologist in a blind manner to the clinical features. clinical and laboratory data including serum iron indices, virological, biochemical results were analyzed to search for significant factors associated with hepatic iron deposition. results: hepatic iron staining was positive in ( %). among patients with hepatic iron deposition, serum levels of ferritin (p= . ) and -fetoprotein (p= . ), and body mass index(bmi) (p= . ) were significantly elevated. there was no significant association between the degree of hepatic iron deposition and fibrosis stage (p= . ), although elevated levels of serum hyaluronic acid (p= . ), -glutamyl transpeptidase (p= . ), and prothrombin time (p= . ) were associated with advanced fibrosis. conclusions: hepatic iron deposition in asian-pacific ch-c patients seemed to be neither frequent nor related to hepatic fibrosis, but related to obesity. therefore, phlebotomy might not commonly applicable to this area. further studies on the pathogenic role of iron in ch-c in asian-pacific countries are warranted. a late stage of progressive hepatic fibrosis characterized by distortion of the hepatic architecture, necrosis of hepatocytes and the formation of regenerative nodules contributes to cirrhosis. limitations like organ donors shortage, high cost, absence of proliferation in cultured hepatocytes, inherent risks of infection, rejection in xenogenic cells and other socio-economical complications emerges advanced regenerative human hepatic stem cells(hhpscs) transplantation. hhpscs are located in the ductal plates in fetal and canals of hering in adult livers [schmelzer et al.( ) ]. hepatoblasts, in turn, give rise to the hepatocytic and biliary lineages, the hepatocytes and cholangiocytes [schmelzere etal( ) ].hhpscs express cd (epcam)marker. scjelzer etal demonstrated that during embryogenesis % of the epcam positive cells had hepatoblast phenotype. in animal study, on transplantation of freshly isolated hhpscs in scid mice results in mature liver tissue expressing human-specific proteins. recently, we(aleem etal )have shown clinical improvement in study in patients with crigler-najjar syndrome, biliary atresia using hhpscs infusion. in the present study we transplanted hepatic progenitors to five subjects of end stage liver cirrhosis with meld score > . hhpscs were sorted using macs with cd antibody microbeads and infused through hepatic artery via femoral artery catheterization, a safe procedure provided portal pressure to monitor cell infusion route in order to prevent vascular thrombosis. all the patients showed improvement clinical and functional biochemical parameters after first month of cell infusion. ascites was decreased and changed encephalopathy grade into normal level was observed. meld score system falling to normal level from > to < after infusion. the aga khan university patients. the apri of . in combination with a cut-off ha of ng/ml can best detect patients with moderate to severe fibrosis (stages - ). it has a ppv of . %. also, for patients without moderate to severe fibrosis, the test is hardly ever positive (specificity of . %). but the apri of . in combination with different ha as cut-off points is not possible to detect patients with no or mild fibrosis. objectives: terlipressin is used in esophageal variceal bleed (evb) along with endoscopic band ligation (ebl) for days (uc). due to its high cost, it was stopped < days (sc) who could not afford & were stable after achieving hemostasis with ebl. we retrospectively assessed the efficacy of sc vs uc of terlipressin for control of evb and length of stay. conclusion: the apri of . in combination with ha ng/ml as cut-off points to predict patients with moderate to severe fibrosis (stages - ) is an easy and accurate method. methods: patients with evb who had achieved hemostatsis with ebl from jan -dec were included. all were managed on standard protocol on hospital variceal bleeding pathway. the course of terlipressin as sc or uc was based on patient's inability to afford the cost of hospitalization and terlipressin. the efficacy of terlipressin in the control of evb was defined based on baveno iii criteria. results: total of patients were admitted during the study period. out of them, received uc & sc of terlipressin. the base line characteristics were comparable except younger age in sc. there were re-bleed ( %) in uc and ( %) in sc terlipressin group. the length of stay was shorter in sc group. ( . ± . vs . ± . days). conclusions: sc seems as effective as uc terlipressin in the control of evb after initial control of hemostatsis with ebl and may reduces the length of hospital stay. rcts are needed to assess this as all stable patients may not need to continue terlipressin for hours. background/aims: to analyze the relationship between conventional laboratory results and death risk in patients with esophageal varices bleeding due to cirrhosis (cevb), and establish a simple model for timely predicting death rsik of the patients. outcome of patients with gastro-oesophageal bleeding in a tertiary center j. wat, w.h. li, m.t. cheung methods: the medical documents of cevb patients were reviewed retrospectively and the data were collected. univariate and multivariate logistic regressions were performed, in which the discharged results (survival or death) as dependent variable and the results of liver function, kidney function, serum electrolytes and blood cell analysis as independent variables. the multivariate regression equation was as the model for the prediction of patient outcome and its predictive performance was evaluated. objectives: to determine the rebleeding rate, mortality and long term survival in cirrhotic patients presented with acute gastro-oesophageal variceal bleeding. method: this is a retrospective review of adult patients who were admitted to our hospital with the diagnosis of acute gastro-oesophageal variceal bleeding for the first time regardless of their underlying causes for cirrhosis. the study period was from january -october . data were collected from our hospital computer system and records. results: in univariate regression, the significant positive variables for death outcome were dbil, akp, k, wbc and plt, and the significant negative variables were tp, ap, a/g, na, cland ca + . the variables entered the multivariate regression are alt, tbil, dbil, gp, a/g, cr, na + , cl -, ca + , wbc, hb, plt. the sensitivity, specificity and accuracy of the regression model for predicting death of cevb patients were . %, . % and . %. results: a total of patients were included in this study, with male and female. their mean age was . . the initial failure rate in endoscopic haemostasis was . %. the -day and -week mortality rates were . % and . %, respectively. poor child's grading, multiple columns of oesophageal varices, high grade of varices, failed initial endoscopic haemostasis, presence of inoperable hcc, low platelet count on admission, and short duration from index bleed to rebleed were factors associating with increased risk of -week mortality (p < . ). mean duration from index bleed to first rebleed was . months. poor child's grading and presence of inoperable hcc were associated with both early or multiple rebleed (p < . ). overall, . % of our patients developed rebleed before their variceal eradication. -year survival in patients with child's a, b and c were %, %, and %, respectively (log rank test p . ). conclusions: the liver function, kidney function, serum electrolytes and blood cell analysis are generally independent factors for cevb patient death risk, especially dbil, a/g and ca + . the established model shows a excellent predictive performance. an imbalance in plasma amino acids of advaced cirrhotic patients impairs the maturation of dendritic cells via mtor/s k signaling pathway e. kakazu , y. ueno , y. kondo , k. fukushima , m. shina , j. inoue , k. tamai , m. ninomiya , t. shimosegawa division of gastroenterology, tohoku university hospital conclusion: although endoscopic haemostasis is an effective treatment modality; rebleeding is still commonly seen among patients with poor child's grading and inoperable hcc. this will result in significant bleeding-related death and poor overall survival. further advancement in treatment strategies for this group of patients are required to improve their outcome and prognosis. background: we have demonstrated that extracellular branched-chain amino acids (bcaas), especially valine, regulate the maturation and function of monocyte-derived dendritic cells (j immunol. : ) . however, it is not clear whether an imbalance in plasma amino acids of advaced cirrhotic patients influence the function of dendritic cells (dcs). methods: we used human pbmcs and cd c+dcs in this study. we made two mediums: a serum free culture medium consistent with the average concentration of the plasma amino acids from a healthy volunteer (n= ) was defined as the healthy control medium (hcm); whereas that from advanced cirrhotic patients (n= ) was defined as the advanced cirrhotic active hepatitis b replication is defined as hbeag + or hbv dna > fibrosis was scored according to the metavir system. alt levels were characterized as being normal, < x normal, and > x normal. conclusions: pe frequency of portal hypertensive gastropathy and its non-invasive predictors in patients with viral methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent for screening egd for varices in last years was reviewed. phg was defined endoscopically by using mccormack classification. noninvasive markers such as spleen/platelets ratio, meld score and child score of all the patients who underwent for egd were recorded. results: out of patients ( . %) were males. out of ( . %) patients who had phg, ( . %), ( %) and ( . %) had mild, intermediate and severe phg respectively. higher proportion of esophageal varices ( . %) was present among those who have phg (p< . ). ( . %) with phg has child score of . meld score > and were seen in . % and . % of patients with phg, respectively. platelet/spleen ratio was . ± in patients with phg as compared to methods: retrospective analysis of cirrhotics undergoing surveillance endoscopy was undertaken assessing for oesophageal varices. clinical, biochemical and radiological indices were analysed. results: cirrhotics underwent surveillance endoscopy during the study. childs pugh scoring (cps) to assess prognosis of liver disease showed cps a( %), cps b( %) and cps c( %). % were male albumin ( g/l vs g/l significant factors on multivariate analysis were albumin (p= . ) and platelet count (p= . ) conclusion: in our cohort there were significant biechemical and radiological differences in differentiating patients with large varices (grade - ) on surveillance endoscopy aims and objectives: to study the frequency of ev in patients with cirrhosis due to viral etiology and its correlation with different non-invasive markers. methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent screening egd for varices in last years was reviewed. ev were divided in two grades (small and large) as proposed in consensus development workshop. noninvasive markers such as spleen/platelets ratio, meld and child turcotte pugh (ctp) scores of all patients were recorded. results: out of patients, ( . %) were males on multivariate analysis ctp score of (or . , p< . ), meld score > (or . , p< . ) and platelet/spleen ratio (or . , p= . ) were found as significant predictors of large ev. conclusion: the frequency of ev is high in viral cirrhosis patients on screening egd. meld score> , ctp score and spleen/platelets ratio can be used as non-invasive predictors of large ev. pe treatment outcome and prognostic factors of spontaneous bacterial peritonitis and culture negative neutrocytic ascites in patients with hepatitis b virus-related thirty-seven ( . %) patients had sbp while ( . %) had cnna. except the higher proportion of renal failure at admission in patients with sbp than cnna ( . % vs. . %, p= . ), no significant difference in the clinical and laboratory data related to liver and renal function was observed. overall mortality during hospitalization was higher in patient with sbp than that of cnna evl was repeated every -weeks till varicial eradication.bb dose was titrated to achieve a resting heart-rate of bpm or a maximum dose mg/d or when side-effects began to appear.primary end-points were rebleed and death.secondary end-points were complications as a result of evlor beta-blocker,variceal recurrence afterevl,and decrease in variceal grade inbb limb. results: patients (median age [range - ] yrs, males %) were included (evl arm[n= ]and bb arm[n= ]. median grade of varices was iii(range ii to iv) nitric oxide synthase isoforms play distinct roles in the evolution of hyperdynamic state in endotoxemia induced portal hypertension m.r. rizvi , m. shahid institute of genomics and integrative biology, mall road, delhi , department of physiology a seconderc was performed after two or more years to assess the progression of the disease. results: ehpvo patients(median age [range - ]yr, males %) were studied.history of present or previous jaundice was present in %,ascites % and pain %. on erc, % had portal biliopathy.the type of bile duct involvement was categorized as: type b( %) and type ( %).the pattern of involvement included indentations( %) and dilatation and strictures( %). %of the patients had bile duct stone and % had history of cholangitis.the mdian bilirubin was . (range . - . ) mg/dl and median serum alkaline phosphatase (range - ) iu/l.all patients were treated endoscopically by endoscopic stone extraction, dilations with/without stenting. ( %) patients underwent second erc after a median interval of (range - )months.the type of involvement progressed, % patients developed type- involvement compared to % at the baseline (p=ns).indentations progressed to develop strictures, from % to %.the frequency of new bile duct stones per year was %(p= ns). conclusions: portal biliopathy is very common in ehpvo, often remaining asymptomatic.however,it is slowly progressive leading to development of biliary strictures objective: to investigate the mechanisms of angiotonin ii (angii)-induced ca ( +)-independent pathways mediated by rho kinase in hepatic stellate cells (hscs) various vasoactive drugs that reduce portal pressure are used in treatment of esophageal variceal bleeding alongwith endoscopic treatment. terlipressin use decreases both, recurrent bleeding and mortality. it is given usually for - days, nevertheless there is very little data comparing different time periods. our aim was to compare the efficacy and safety of -days versus days of terlipressin treatment in bleeding esophageal varices. methods: out of patients who presented with variceal bleeding, were randomized to receive terlipressin mg hrly, i.v. daily for first days and placebo for next days (group a) and to receive terlipressin mg hrly, i.v. daily for days (group b). both groups were both age and sex matched. (svt) {consisting of hvt and pvt}. there is no such data from india a comparative study of male vs background: mucosal lesions are frequently observed.gender differences are expected due to food habits, nature of job, mobility related to work, consumption of alcohol, tobacco etc. material and methods: procedure done in m & ( . %) f& duo ( . %) m & %)f.varices ( ) eso varices ( . %) m & ( . %) f & gastric varices (. %) m & (. %) f .growth ( )eso growth (. %) m & (. %) f & stomach growth ( . %)m & (. %) f & laryngeal (. %)m. eso monoliasis ( ) (. %) m & (. %) f.polyp ( ) eso polyp (. %) m & (. %) f & gastric polyp (. %)m & (. %) f & moniliasis and ulcers are more common in female lee department of internal medicine, gyeongsang national university school of medicine background/aims: although the pyogenic liver abscess is a common intraabdominal inflammatory disease, this complications are not rare. however, reports dealing with this complications are not good enough and results are often variable. the aim of this study was to identify the predictive factors of complication in the pyogenic liver abscess. pe effects of saikosaponinsd (ssd) on expression of c-myc and pcna in experimental hepatocarcinoma of all rats were killed in the th week, then general conditions of rats were recorded, the serum alt akp ggt afu was detected and pathological examination was made. the expression of pcna and c-myc were tested by immunohistochemistry. results: he staining showed that rats were induced to hepatocellular carcinoma,the results of liver function in the th week displayed that alt akp ggt and afu of all groups were increased than that of normal control group conclusion: ssd can inhibit development of hepatoma induced by den, possibly by down-regulating the expression of pcna and c-myc protein lethal endotoxic shock was induced by single endotoxin (e.coli) mg/kg injection into abdominal cavity. ppc in % gs was given via tail vein at ml/ g ( . mg/kg) h and h before endotoxin. rats' behavior and h survival were recorded, venous blood taken for ast and alt, liver preserved for he staining and liver/body weight ratio l/b and liver wet/dry weight ratio (w/d) calculated. intercellular adhesion molecule- (icam- ) expression in liver tissue was observed with -step immunohistochemistry assay. results: rats of ns and ppc group demonstrated similar normal activity, histology and other characters (p> . ), while lps group showed sag and less water-intak and severe inflammation in liver including inflammatory cell accumulation, parenchymal cells edema and tissue exudation. p+l group turned tired but could drink water the role of insulin resistance, adipokine and cytokine pro-inflammatory in non-alcoholic fatty liver disease n. ratnasari , , s. anam , p. bayupurnama , , s. maduseno , , s. nurdjanah , dr sardjito general hospital yogyakarta indonesia, background: non-alcoholic fatty liver disease (nafld) is a benign disease during - years period, with %- % survival. nafld can progress to fibrosis, cirrhotic and cancer of the liver. the etiopathogenesis of nafld is still unknown, however genetic and environment factors are predicted. objective: to know the role of insulin resistance, adipokine and cytokine pro-inflammatory on nafld. methods: the cross sectional study was performed on general check-up population at dr. sardjito general hospital yogyakarta, indonesia. the study was begun from january until november at internal medicine outpatient department. inclusion criteria: adult, alcohol consumtion g/day, metabolic syndrome patients, and healthy subjects. exclusion criteria: the diseases with increasing liver enzymes (hbv, hcv, ischemic hepatitis, congestive liver), a "bright liver" on ultrasound examination (malnutrition, rapid weight decreased, post gut surgery on obesity patients, and drug induced). based on liver ultrasound subjects were devided into steatosis group and non-steatosis group. data were analyzed by t-test and non-parametrical test. results: subjects that were enrolled the study steatosis ( . %) and non-steatosis ( . %) and the subjects who completed cytokine and adipokine examination were steatosis and non-steatosis. there were significantly different on homa -ir and adiponectin level in steatosis group (homa-ir . ± . vs. . ± . , p= . ; adiponectin . ± . vs. . ± . , p= . ). there were not significantly different on tnf-, il- , leptin and visfatin level (p> . ). conlusions: there were significantly different on homa-ir and adiponectin level in nafld patients compared non-nafld patients. background: to optimize management of nonalcoholic fatty liver disease (nafld), a simple screening tool is necessary. in this study, we aimed to devise a simple index that reflects the presence of nafld in the korean population. methods: a cross-sectional study was conducted on , health check-up subjects at a healthcare center ( , cases with nafld versus age-and sex-matched controls). study subjects were randomly assigned to a derivation cohort or a validation cohort. an index reflecting the presence of nafld was derived in the derivation cohort and validated in the validation cohort. results: multivariable analysis indicated that body-mass index (bmi), serum alanine aminotransferase (alt) to serum aspartate aminotransferase (ast) ratio, sex, and the presence of diabetes mellitus were independent predictors of nafld. using these variables, a formula was derived using a linear regression model: nafld index (nafldi) = ×alt/ast ratio +bmi (+ , if female; + , if diabetes mellitus).nafldi had an area under receiver-operating curve of . ( % confidence interval, . - . ). at a value < . , nafldi ruled out nafld with a sensitivity of . % and a negative likelihood ratio of . , and at a value > . , nafldi detected nafld with a specificity of . % and a positive likelihood ratio of . . in the validation cohort, the predictive power of nafldi was maintained at similar levels.aim: since nash could progress to liver cirrhosis and hepatocellular carcinoma, it is important to correctly diagnose between nash and simple steatosis (ss). the aim of this study was to determine the prevalence of nash among nafld patients and to clarify differences in clinical features between nash and ss. subjects and methods: thirty-one patients with nafld showing abnormalities in serum transaminase (ast and /or alt > iu/l) were enrolled (sex: male , female ; mean age: . yrs, mean body mass index: . ), after obtaining informed consent. differential diagnosis between nash and ss was performed histologically according to the matteoni classification and clinical features were compared. results: among the patients with nafld, % and % were diagnosed with ss and nash, respectively. no significant differences in the sex, mean age and bmi were seen between nash and ss groups. the levels of ast, alt, homeostasis model assessment-insulin resistance (homa-ir) and hyaluronic acid were significantly elevated in nash patients compared to ss patients. no significant differences in serum levels of adiponectin, as well as the rates of occurrence of diabetes, hypertension and hyperlipidemia were observed between the two groups. conclusion: the prevalence of nash in nafld patients was about %. nash patients showed higher levels of serum transaminase, homa-ir and hyaluronic acid, compared to ss patients. a large-scale biochemical study is required to accurately diagnose nash patients and confirm these results. conclusion: nafldi was a simple, efficient screening tool for nafld that could be utilized for selecting individuals for liver ultrasonography and for determining the need for lifestyle modifications.pe aim: this study was conducted to evaluate the hepatoprotective effects of the centella asiatica extract in -methyl- phenyl- , , , -tetrahydropyridine (mptp)-induced liver injury in rats. methods: sprague dawley rats were treated with alcohol extract of centella asiatica orally in two doses ( and mg/kg/day) for months along with intraperitoneal injection of mptp ( ml/kg). biochemical parameters such as serum total protein, albumin and marker enzymes were estimated. histopathological studies of liver were also carried out to confirm the biochemical changes.results & discussion: mptp -induced hepatotoxic effects were evident by a significant (p < . ) increase in the serum marker enzymes and a decrease in the total serum protein and albumin. administration of extract of centella asiatica effectively inhibited these changes in a dose-dependent manner; maximum effect was with mg/kg. histopathological examination of liver tissue corroborated well with the biochemical changes. hepatic steatosis, hydropic degeneration and necrosis were observed in mptp-treated group, while there was a significant reduction in these changes in the treatment group. conclusion: centella asiatica extract exhibited hepatoprotective action against mptp induced liver injury. further optimization of tuberculosis chemotherapy requires a comprehensive evaluation of the effects of antitubercular drugs on metabolic processes in organism.wistar albino male rats, body weight (b.w.) of - g, were divided into three groups: group i received pyrazinamide per os at a dose of mg/kg b.w./day, whereas group ii received a dose of mg/kg b.w./day, in both groups it was given for days; the control group was composed of intact animals. the contents of free amino acids were determined using an amino acid analyzer - (czech republic). the study of the effects of pyrazinamide administered in different doses on the liver contents of free amino acids showed the largest number of changes at a dose of mg/kg b.w./day. the content of free amino acids at the level of amino acids and total sum of amino acids significantly differed from controls. part of these changes could be regarded as compensatory answer of organism to this drug action. further pyrazinamide dose increasing caused exhaustion of liver adaptive possibilities. the study of the influence of pyrazinamide on liver contents of free amino acids allows to fully estimate the effects of this substance on metabolic processes in this organ. moreover, the effect of pyrazinamide on the majority of free amino acids in the liver is dose-dependent. background: taiwan is an endemic area of hbv and hcv infection, chemotherapy for lymphoma patients who has been hbv infection, may induce serious clinical sequela due to reactivation of hbv. this study want to clarify the difference of the chemotherapy induced hepatitis between hbv and hcv carrier in lymphoma patients. methods: from july, to july , non-hepatocellular carcinoma patients were enrolled, ( . %) cases were lymphoma, in these lymphoma patients, ( . %) cases have been hbv infected, and ( . %) cases have no hbv or hcv infected. ( . %) cases have been infected with hcv. all patients received chemotherapy with the regimen of chop or r-chop. liver function , viral markers, hbv dna, hcv rna were checked before and after chemotherapy. results: hepatitis happened in ( . %) lymphoma patients, ( . %) cases were in hbv infected patients, ( %) cases were in non-hbv infected patients, cases of hcv infected patients suffered from hepatitis. hbv infected hepatitis patients hbv dna elevated more than log as before chemotherapy. non of the hcv infected patient has elevated of hcv rna after chemotherapy. the mortality rate in hbv infected patient is %. no mortality in hcv infected patients after chemotherapy. conclusions: .high rate of hbv reactivation and mortality in chemotherapeutic lymphoma patients who has been hbv infected. screening of hbv viral markers among candidates for cancer chemotherapy is mandatory, especially in lymphoma patients. large number and prospect study for chemotherapy induced hepatitis in hcv carrier are needed. background: excessive drinking leads to social, psychological, physical and other problems. this study investigated the epidemiology of ald and analyses the associated risk factors. methods: from , residents , blood samples were collected. alcohol consumption and the impact of alcohol on liver function, blood lipids, blood pressure and bmi and mcv have been evaluated. results: the drinking rate and average daily alcohol intake was . % and . ± . g respectively. the total alcohol intake was . ± . kg and the average drinking age was . ± . years. the average -gt, ast, alt, mcv, chol, tg, ldl-c, hdl-c and bp increased gradually with increase in alcohol intake. the population ald prevalence was . %. the prevalence of ald among the drinking population and the alcoholic population was . % and . % respectively. conclusion: chol, -gt, ast, alt, and mcv were highly correlated with daily alcohol intake which closely related to the occurrence of ald. n. tanaka , w. okiyama , t. aoyama background: alcoholic liver disease (ald) is one of the leading causes of cirrhosis and yet efficient therapeutic strategies are lacking. polyenephosphatidylcholine (ppc), a major component of essential phospholipids, prevented alcoholic liver fibrosis in baboons. however, its precise mechanism remains uncertain. we examined the effects of ppc on ald using peroxisome proliferator-activated receptor (ppara)-null mice treated with an ethanol-containing diet, which showed pathological features similar to human ald. methods: male ppara-null mice were pair-fed a lieber-decarli control or % ethanol-containing diet with or without ppc at a clinically comparable dose ( mg/kg/day) for months. o. parkash , a. almas , s.h. ali shah , w. jafri , s. hamid , j. akhtar aga khan university hospital karachi .hdv-hbv co-infection presents mostly as moderate to advanced liver disease. background: liver injury due to dengue infection is not uncommon. acute liver injury is a severe complicating factor in dengue, predisposing to life-threatening hemorrhage, dic and encephalopathy. results: there were no significant differences of demographic features and laboratory parameters such as peak serum alt, total bilirubin and creatinine between groups. however, peak ast was higher in superinfected group than control group (median: , iu/l vs iu/l, p= . ,). additionally, the peak serum albumin levels, prothrombin time and platelet counts were lower in superinfected group than control group (median: . mg/dl vs . mg/dl, p= . , . % vs . %, p= . and x /mm vs x /mm , p< . , respectively). of superinfected group, patients were followed over months after resolution of aha. interestingly, serum hbv-dna levels decreased significantly over months following resolution of aha, then rebounded subsequently (median: - . , - . , - . , . and . log copies/ml at , , , and months, respectively).methods: the overlapping fragments of hev isolate swgx were amplified with reverse-transcription nested polymerase chain reaction (rt-npcr) and the ' and ' ends of viral genome were amplified with rapid amplification of cdna ends (race). the pcr products were cloned and sequenced. the phylogenetic analysis of swgx was performed.result: the genome of swgx consisted of , nucleotides, excluding the poly (a) tail of residues. the genome contained three open-reading frames (orfs), orf- encoding amino acids, orf- encoding amino acids and orf- encoding amino acids. the full-length genomic sequencing showed that swgx strain shared similarity with all known hev genotype , and isolates by . % to . %, and with an identity of . % to . % among genotype hev isolates, and a high nucleotide identity as % with chinese guangxi human strain lz- .conclusions: acute hav super-infection may suppress hbv-dna replication in chronic hbv carriers and chronic hepatitis b, although the suppressive effect did not seem to sustain longer than months. conclusion: the swine hev strain swgx was phylogenetically close to the human hev strain lz- , both from the same region in south china. therefore it was concluded that hev sub-genotype b might have existed in south china at least for years and now it was prevalent both in local human and swine, which also strongly supported the zoonosis hypothesis of hepatitis e. associated with splenic volume were damping index (r= . , p= . ) and blood flow of portal vein (r=- . , p= . ). conclusions: the splenomegaly in portal hypertension was more frequent in non-alcoholic groups, and associated with a damping index and a blood flow of portal vein. the measurement of blood flow of portal vein, damping index, and splenic volume by a doppler sonography can be helpful to predict the varices.z.q. zhang , j. cao , w. lu , l.g. shi objective: to appraise the clinical efficacy of simple non-invasive models of ast-to-alt ratio (aar), ast-to-platelet ratio index (apri), spleen-to-platelet ratio index (spri), age-platelet index (api), age-spleen-to-platelet ratio index (aspri) for predicting hepatitis b associated cirrhosis. methods: patients and patients were diagnosed pathologically as non-cirrhosis and cirrhosis, respectively. the simple non-invasive models were calculated as described originally. spss . was used for statistical analyses.background: to reveal the microrna (mirna) expression profile of the hepatic fibrosis inducing cells, rat hepatic stellate cells (hscs), during in vitro activation. results: the areas under roc curve of aar, apri, spri, api, aspri for predicting the cirrhosis were . , . , . , . , . , respectively which were larger than those under the diagnosis reference line (p . , . , . , . , . , respectively).methods: the hscs were isolated from male sd rats by in situ perfusion and density-gradient centrifugation. the quiescent and activated hscs, which were harvested at day and , respectively, were then subjected to immunocytochemical staining (desmin and -sma), oil red o staining and quantitative rt-pcr (desmin, -sma, albumin, cd , cd and cytokeratin- ). after extraction and labeling, the hy -labeled cellular rna samples and hy -labeled reference pool rna samples were mixed pair-wise and hybridized to the lna mercury microarray. differentially expressed mirnas were filtered and randomly verified by stem-loop rt followed by quantitative pcr.conclusion: all of the simple non-invasive models of aar, apri, spri, api, aspri can be used for predicting hepatitis b associated cirrhosis; and aar has the most practical efficacy for predicting hepatitis b associated cirrhosis. results: both the purity and the total activation of hscs were validated. global analysis of the mirna expression profile based on quiescent and activated hscs demonstrated differentially expressed mirnas. among these, mirnas were up-regulated more than -fold in activated hscs as compared to that in quiescent hscs, while mirnas were less than the threshold level ( . -fold) during the hsc activation. furthermore, the expression of mir- , b, , , and had been proved. background/aims: only limited patients with chronic hepatitis b virus (hbv) infection will develop liver cirrhosis, and no effective methods to precisely predict ones who will develop cirrhosis. we try to establish a model to predict the patients with the risk of cirrhosis development basing on a clinical epidemiological factor survey. z.q. zhang , w.y. bao , w. lu , l.g. shi methods: cirrhosis patients with hbv markers (case group) and asymptomatic hbsag carriers (control group) were recruited and inquired by researchers with a specific designed questionnaire including items. a multivariate logistic regression analysis were conducted to establish a predictive model, in which two third patients selected randomly as model sample and another / patients as validating sample, key factors screened out as variables. the predictive performance of the model was evaluated. objective: to explore the practical significance of the peripheral blood corpuscle counts for prediction of hepatitis b associated cirrhosis. methods: and male patients with chronic hepatitis b were pathologically diagnosed as non-cirrhosis and cirrhosis. peripheral blood corpuscle counts were measured by coulter ac•t diff hematology analyzer. results: red blood cell (rbc), platelet (plt), neutrophil (n) counts in cirrhosis were significantly lower than those in non-cirrhosis; and lymphocyte, mid-cell counts were similar to those in non-cirrhosis. the areas under the roc curves of rbc, plt, n counts for prediction of cirrhosis were . , . , . respectively; according the optimal cut-off determined by the roc curves, the sensitivity, specificity, positive predictive value, negative predictive value, accuracy of rbc, plt, n counts for prediction of cirrhosis were . - . method: pbmcs of active chb patients under pyg-interferon treatment were analyzed for their th , treg and pdc by flow cytometry. they were determined by cd /il- for th- , cd /cd /foxp for treg and cd /cd /cd -for pdc. pbmc were collected every weekly during the treatment until end of therapy (week ) and every weekly until the end of follow-up (week ). alt was quantified at every time of the pbmc collection. ifn-gamma release cells were analyzed by elispot to hbv-core and s ag.background: alpha fetoprotein (afp) is a well-recognized tumor marker for hcc; elevated level of afp is found in at least % of hcc. other liver diseases such as cirrhosis and chronic hepatitis are also related with an elevated level of afp. the regulation of afp gene expression has been relatively less studied although the gene has been suggested to play a role in hcc development. in this study, we tried to identify genetic variations in afp gene and analyze its effect on serum afp level and possible hcc progression.results: in parallel with decline in alt for the first weeks, we found decline in both pdc (r= . , p= . ) and elispot (r= . , p= . for hbv-core ag; r= . , p= . for hbv-s ag). although there is trend that th- decline with treg decline, but they are not statistically significant differences in the same period. there is no significant difference between the svp and non-svp patients.methods: direct dna sequencing was carried out to sequence afp promoter and bp upstream and downstream of afp coding regions in dna samples isolated from hcc subjects and controls respectively. for each samples serum afp levels were determined using commercially available elisa kits. conclusions: under pegyintron treatment, pdc, ifn-gamma change in the same trend of alt during weeks of treatment. it implied that pdc take regulatory effects on ifn-gamma releasing cells and be very tightly related to alt. there wasn't a significant difference in both treg and th- , which implies that treg and th might be of important cells in keeping the stability of the immune system.results: a total of snps were detected in the afp genomic region analyzed, including known snps and one novel snp. among the identified snps, the c>g nucleotide change in the position - bp upstream of afp transcriptional start site showed a significant association with hcc (p < . ) and a decrease in afp gene expression level. conclusion: our preliminary results indicated a possible association between serum afp expression and - g allele. the identified snp is located in afp promoter region with possible binding sites for known transcription factors, such as tfiid, coup, apf and nfiii. - °tail-suspension (ts) rats were used as the model to simulate the physiological effects of weightlessness. thirty-two wistar male rats were randomly divided into groups: control for d ( d con), d con, ts for d (ts d) and ts d. histopathological changes of testicle of the rat were observed by he stain. localization and expression of ar and hsp in testicle of rat were observed comparatively by means of immunohistochemistry, and the density of ar and hsp immunoreactivety in four groups were compared. results: signal molecules mrna level are shown in the table (** p< . , * p< . ). tnfa, il , il and il were higher in group , and than those in group . ifna was higher in group than that in other groups. there are no significant difference in infc, il , and il . there was a positive correlation between tnf and myd in group , tnf and nfkb in group and . results: obvious pathological lesions presented in testicle of ts d and ts d rat. germinal epithelium irregularity and malformed spermatozoa were found in seminiferous tubules. degeneration and necrosis of germinal epithelium appeared in testicle of ts d and ts d rat. ar immunoreactive cell density in the ts d and ts d groups were significantly decreased compared with the in-phase normal control groups ( p < . ). while hsp immunoreactive cell density in the ts d and ts d rats were significantly increased than those of control rats( p < . ), and in testicular interstice or extracellular there were very strong ehsp immunoreactive positive staining signals. the results indicate that ground simulated weightlessness induced by d- d tail-suspension in rats can lead to the serious injury , depressed expression of ar and enhanced expression of hsp in testicle. despite the absence of any serologic marker of hbv recurrence, however, it remains unknown whether there is occult reinfection in the liver graft. we aimed to detect and quantify the presence of intrahepatic hbv dna in the liver grafts of patients who remain seronegative for hbsag for more than year after liver transplantation. materials and methods: liver biopsy and blood samples were obtained from patients who had been receiving nucleoside analogue prophylaxis alone and remained persistently seronegative for hbsag for at least year (median . months, range . to . months) after liver transplantation for chronic hepatitis b. quantitative polymerase chain reaction was performed to detect and quantify total and covalently-closed circular (ccc) hbv dna in the liver (lowest detection limit, copies/ml), serum and pbmc. direct sequencing was used for hbv quasispecies screening. results: liver biopsy was performed and intrahepatic hbv dna as measured by quantitative real-time pcr was detectable in of recipients. donors anti-hbc status before liver transplant was significantly related to the presence of intrahepatic hbv dna in the recipient's study biopsy (p= . ). donor intrahepatic hbv cccdna levels correlated with recipient post-liver transplant intrahepatic hbv cccdna levels (p= . ). hepatitis b virus sequencing results and phylogenetic analysis revealed that hbv reinfection in two recipients were of donor origin, four recipients were of recipient origin and four recipients were of both donor and recipient origins. conclusions: our findings demonstrate the presence of occult hbv reinfection with persistence of hbv dna in liver allografts despite long term nucleoside analogue prophylaxis after liver transplantation, suggesting the need to continue indefinite antiviral therapy. the use of liver grafts from anti-hbc-positive donors might increase the risk of occult hbv reinfection. both donor and recipient hbv dna could contribute to occult hbv reinfection in liver transplant recipients. aim: to construct one noninvasive assessment model consisting of routine laboratory data to predict both significant fibrosis and cirrhosis among patients with chronic hepatitis b(chb). methods: we have retrospective analyzed consecutive patients with chronic hepatitis b who underwent percutaneous liver biopsy. we calculated sensitivity, specificity, positive predictive value(ppv), and negative predictive value(npv) of an apri . in combination with different hyaluronic acid(ha) cut-off points medium (acm). we stimulated pbmcs or dcs under hcm and acm, and evaluated the function. results: after adding the stimulants under hcm, the cd and cd expression of dcs from cirrhotic patients (lc) were lower than those from healthy contorols (hc). in both hc and lc, the cd and cd expression of dcs stimulated under acm was lower than that under hcm. the il- production in acm was lower than that in hcm. the expression of cd , which is related to amino acid transport, was not different between hcm and acm. however, dcs cultured in acm expressed lower levels of phospho-p s k than those cultured in hcm. finally, we ascertained that the ifn gamma production by pbmcs was significantly decreased under acm.conclusions: an imbalance in plasma amino acids of advanced cirrhotic patients suppresses the maturation of dcs via mtor/s k signaling pathway. key: cord- -fe sacjj authors: butler, j. e.; lager, k. m.; golde, william; faaberg, kay s.; sinkora, marek; loving, crystal; zhang, y. i. title: porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic date: - - journal: immunol res doi: . /s - - - sha: doc_id: cord_uid: fe sacjj porcine reproductive and respiratory disease syndrome (prrs) is a viral pandemic that especially affects neonates within the “critical window” of immunological development. prrs was recognized in and within a few years became pandemic causing an estimated yearly $ , economic loss in the usa with comparative losses in most other countries. the causative agent is a single-stranded, positive-sense enveloped arterivirus (prrsv) that infects macrophages and plasmacytoid dendritic cells. despite the discovery of prrsv in and the publication of > , articles, the control of prrs is problematic. despite the large volume of literature on this disease, the cellular and molecular mechanisms describing how prrsv dysregulates the host immune system are poorly understood. we know that prrsv suppresses innate immunity and causes abnormal b cell proliferation and repertoire development, often lymphopenia and thymic atrophy. the prrsv genome is highly diverse, rapidly evolving but amenable to the generation of many mutants and chimeric viruses for experimental studies. prrsv only replicates in swine which adds to the experimental difficulty since no inbred well-defined animal models are available. in this article, we summarize current knowledge and apply it toward developing a series of provocative and testable hypotheses to explain how prrsv immunomodulates the porcine immune system with the goal of adding new perspectives on this disease. north american-like (prototype vr- ). the virus is a member of the family arteriviridae in the order nidovirales, which includes lactate dehydrogenase-elevating virus of mice (ldv), simian hemorrhagic fever virus (shfv), equine arterivirus (eav) and the recently described wobbly possum disease virus (wpdv) [ , ] . as the name implies, except for wpdv that appears to be only neurologic, they are associated with some form of vasculitis. the virus can be transmitted across the placenta to infect the fetus [ , ] despite the fact that the porcine placenta is impermeable to maternal antibodies [ ] . prrs is the number one disease problem in major swine producing areas around the world. it is estimated to cost the industry million dollars a year just in the usa with proportional losses recognized in other countries. this is attributed to the remarkable ability of prrsv to: ( ) infect swine at all stages of production, ( ) be shed in the semen of boars for extended periods of time, ( ) be easily transmitted between farms, ( ) tolerate a high mutation rate, and ( ) negatively modulate the host's immune response. prrs has been a troubling disease because of its persistence and because [ years of research has failed to produce an efficacious vaccine. this has been somewhat surprising since eav infections are resolved in - days and a number of efficacious vaccines are available [ ] . the rapid resolution of eav is reminiscent of the pattern of sterilizing immunity seen with porcine influenza even in germfree (gf) piglets, so it is not simply a case of neonatal incompetence. rather, prrsv is more similar to ldv in which both the virus and the antibody response persist in mice [ ] . as implied by its name, prrs causes two separate pathologies: fetal abortion and respiratory disease in young and older pigs. there is some evidence that prrsv replicates predominately in the thymus, which results in thymic atrophy [ , , ] . this feature separates prrsv from both eav and ldv. while this is especially pronounced with highly pathogenic strains (hp-prrsv) [ , ] , it is not necessarily the case for all isolates. more than , papers have been published on prrs, nearly all of which describe studies using conventional animals [ , , [ ] [ ] [ ] . most initial studies focused on adaptive immunity, although it is well recognized that viral infection also affects the innate immune system [ ] . few studies have focused on immune dysregulation by prrsv, but recent work describes how prrsv can suppress innate immunity (''the innate immune response to prrsv'' section). murtaugh and genzow propose that ''identification of the viral structures that elicit the protective immunity in pigs and factors that modulate the efficacy of protection in vivo is essential to rational development of immunological tools to prevent and control prrs.'' this focus is very important but as general guderian advised hitler in ''if what you are doing is not working, try something different'' [ ] . what is lacking in prrsv research is a greater effort to determine the mechanisms, whereby the virus modulates the porcine immune response. in this review, we describe testable hypotheses to explain how this virus modulates the host immune system. both prrsv and ldv are immune modulatory and although not retroviruses, may have more in common with hiv than eav. ldv elevates igg levels in mice with little production of virus-specific antibodies [ , ] , which is almost identical to what is seen in isolator piglets infected with prrsv [ ] (''the effect of age, rearing, complement and the role of mucosal immunity'' section). polyclonal b cell activation is often associated with autoimmunity and is common to a number of viral infections that are genetically unrelated to the arteriviridae [ ] . many viral infections such as bovine viral diarrhea virus [ ] interfere with ''normal'' immune processes, which prolong the replication window for the viruses and thus increase the opportunity for contagious spread. thus, virus classification may be a poor predictor of the effect of a virus on the immune system. with rare exception, interference with the immune response is not the cause of death; good parasites rarely kill their host. rather, secondary bacterial infections are more likely to cause death in prrsv-infected conventional animals [ , , , ] . renukaradhydad et al. [ ] showed that coinfection with prcv (porcine respiratory coronavirus) reduced nk cell function more than prrsv alone and dual infection caused more pathology [ ] . likewise, prrs decreased the efficacy of siv vaccination and increased clinical disease [ ] , and mycoplasma hyopneumoniae infection significantly prolonged and increased the severity of prrs [ ] . as implied in the name of the disease, the clinical manifestations of prrs involve reproductive failure in sows and respiratory disease in young and growing pigs. historically, field reports described ''uncomplicated'' prrsv infections in young pigs as a mild-to-moderate pneumonia recognized clinically as an increased respiration rate at rest that would become labored with exertion. these observations were readily demonstrated experimentally. reproductive failure, which became the hallmark sign of prrs, included abortion ''storms'' and a sudden increase in dead fetuses and weak-born pigs that would affect most of the sows in the herd. in experimental sow infections during late gestation, fetal death and weak-born pigs are a predictable outcome, but prrsv-induced abortions are uncommon. the course of clinical disease following prrsv infection has been well chronicled. in the hundreds of animal experiments that have been reported since , it has become clear that there is considerable variation in clinical responses. most of this is attributed to the use of different prrsv isolates, and collectively, it appears that the isolates from the early s are less pathogenic than isolates from the late s and certainly much less pathogenic when compared to asian hp-prrsv. although differences in viruses may be a major factor in clinical variability, differences do occur when using the same virus under similar conditions suggesting that the host is also an important variable. fortunately, there is considerable knowledge and expertise in prrsv genetics to allow this to be further tested (''prrs the virus'' section). at this time, variation in clinical response is attributed to genetics, age, and coinfections [ ] . based on early field reports and experimental data, swine become more resistant to clinical disease with age, and boars and sows exhibit fewer clinical signs. this is not completely accurate since there is growing evidence that as prrsv mutates overtime, it may gain in virulence. why adults are more resistant to clinical disease and more likely to resolve the disease with vn antibodies [ ] is unclear, but it may reflect the less well-developed immune system of neonates (fig. ) . likewise, how the virus develops a chronic infection in the boar and is shed in the semen for extended periods of time is not known. current swine husbandry practices are almost completely dependent on the use of artificial insemination resulting in a population of boar studs that may supply semen to tens of thousands of sows. this practice dramatically magnifies the danger of using prrsv-contaminated semen. similarly, the concentration of sows in large buildings certainly contributes to possible horizontal transmission of virus and subsequent clinical and economic affects. at a cellular level, prrsv antigens and nucleic acids have been demonstrated in cells of the monocyte and dendritic cell lineage in a variety of organs. prrsv in the lung is often associated with lesions; however, the presence of virus and lesions is less frequent in other organs. the observations support a tropism of the virus for the lung, which could lead to pneumonia. however, when compared to other swine pathogens, the presence of prrsv in the lung and other organs seems minimal in relationship to clinical disease. one explanation for this may be that the pathogenic mechanism(s) of prrsv is(are) not necessarily a simple cytolytic effect on a tissue with influenza a that infects airway epithelia. instead, prrsv may just affect a smaller group of cells that have important regulatory controls, which could lead to a variety of diseases most likely those of hematopoietic/lymphoid tissues. the behavior of good parasites like viruses is to cause a delay in their eviction to allow for reproduction and transfer of their offspring to another host. others may revert to a low virulence state and continue to survive in the host. viruses such as those in the herpes family that are persistent for life have all evolved mechanisms that dysregulate the immune system. few investigative groups have seriously focused on immune dysregulation during prrsv infections. a great many viruses foil antigen presentation by interfering with mhc expression. rapid reduction of mhc adaptive immunity fig. the critical window of immunological development. neonates are vulnerable during this period since their adaptive immune system is undeveloped, and they depend on innate and passive immunity. within this period, healthy gut colonization takes place which drives the development of adaptive immunity and both oral tolerance and immune homeostasis develop. in some mammals, passive maternal antibodies are provided in utero as well as post-natally through suckling. the colors are a result of blending overlapping events. modified from butler and sinkora [ ] class i surface expression is a common feature of viral infections and is seen with foot-and-mouth disease virus [ ] . in epstein barr virus (ebv) infection, degraded peptides from the ebna- nuclear antigen are not degraded, and so, these peptides are not presented [ ] . something similar happens with presentation of peptides derived from a -kda transcription factor in human cytomegalo virus (hcmv) [ ] . while the complex mechanism in these two examples is incompletely understood, there is better data for several other herpes viruses that inhibit the tap complex. tap is required for the transport of cytosolic peptides (including those derived from a virus) across the er. this step is required in their eventual presentation to cd t cells. tap inhibition is found in herpes infection of swine, dogs, and cattle but not in rodents or lagomorphs [ ] . an adenovirus protein (e ) retains degraded peptides in the er and thus also prevents their presentation to t cells [ ] . in hcmv, several gene products target mhc i for proteasome degradation [ ] . in hiv, the nef and vpu proteins downregulate expression of surface mhc i [ ] . in both human and bovine papilloma viruses, the gene product e is believed to interfere with the processing of cellular proteins and could thus affect presentation of peptides [ ] . viruses may also interfere with mhc ii expression that is induced by ifn [ ] . viral infection also disrupts cell cycling and interferes with cytokine and chemokine production and also cytokine action. the list of examples is long but in general, il- , il- , both type i and ii interferons are affected. as reviewed above, interference with innate cytokine synthesis may be especially important. these effects have been reported for a wide variety of viruses including pox viruses, herpes viruses, adenoviruses, and others. this further indicates that immune dysregulation is widespread among viral infection and that many families are involved indicating that it is a feature of the type of particular pathogens and but not their place in phylogeny. viral gene products also interfere with effector functions of the immune system. for example, they can interfere with apoptosis, and in swine, fmdv has been shown to inhibit the natural killer (nk) cell response to infection [ ] . it is known that adenoviruses can cause lysosomal degradation of fas that is part of the complex used by cytotoxic t cells and nk cells to induce apoptosis of virus-infected cells [ , ] . more than viral genes affect this part of the anti-viral defense [ ] . infecting viruses may also interfere with virus neutralization. the mechanism of viral neutralization has been a matter of conjecture for[ years. do neutralizing antibodies bind those viral epitopes that prevent their recognition by the receptors on potentially permissive cells or do they inhibit the fusion of the viral membrane with the endocytic membrane? if it is simple blocking, multiple antibodies appear to be needed since as many as % of such viral epitopes must be antibody bound to prevent infection [ , ] . is simple blocking by antibodies enough or is help needed from an immune complex? in the case of eav, adding fresh serum as a source of complement, greatly increased the effectiveness of vn. covalent binding of c and c can facilitate clearance by cells that express complement receptors. in addition to merely facilitating clearance, complement-containing immune complexes can augment b cell activation [ ] , whereas igg complexes without complement can downregulate b cell responses through crosslinking to fccriib [ ] . non-neutralizing antibodies may also act as a trojan horse in facilitating virus uptake through fccrs, a process dubbed as antibody-dependent enhancement that can increase infectivity - fold [ ] . recently, attention is being given to another immunosuppressive player in cancer and persistent viral infection. myeloid-derived suppressor cells (mdsc) were first described from a mouse model of lung cancer in which these cells inhibited t cell proliferation [ ] . these cells function through reactive oxygen species (ros), inos and arginase- [ ] . acting through ros, tcr can become nitrated preventing peptide binding [ ] . ros-dependent suppression of cd ? and cd ? t cells by mdsc in hcv infections [ ] . current understanding suggests that mdsc also inhibit nk cell function. mdsc suppression is also known for hiv, vsv, and vaccinia [ ] . since prrsv can be persistent, a role for mdsc should not be ignored. if viral neutralization is complement dependent, viruses that interfere with this mechanism can prolong their replication time in the host. there is evidence that vaccinia, cowpox, and variola secrete proteins that block c convertase action [ , ] . while the mechanism involved is unclear, herpes viruses can also inhibit complement activation [ , ] . it has been known for some time that many viruses that cause persistent infection including ldv and prrsv are strong polyclonal b cell activators and often lead to the appearance of autoantibodies, a symptom that the preimmune repertoire has been expanded [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . tumorigenic viruses like ebv that target b cells give rise to elevated levels of monoclonal antibodies not directed to ebv [ ] . in these cases, immunoglobulin (igg) levels are a poor indicator of the anti-viral response. the innate immune response to prrsv host innate immune responses play a key role against early viral infection. host pattern recognition receptors for rna viruses include rig (retinoic-acid-inducible gene)-i-like receptors (rlrs) and toll-like receptors (tlrs) [ , ] . activation of rlr and tlr signaling pathways leads to activation of interferon regulatory factor (irf- ), irf , and nf-jb, followed by induction of type i ifns (i.e., ifna and b) and expression of inflammatory cytokines. type i ifns are critical to innate immunity against viral infections and play an important role in the stimulation of adaptive immune response [ , ] . prrsv is sensitive to type i ifns, and the sensitivity is confirmed in vivo. pigs that were inoculated with recombinant adenovirus for ifn-a expression and challenged with prrsv day later had reduced lung lesion and delayed viremia and antibody response [ ] . the presence of ifn-a at the time of infection alters innate and adaptive immune responses to prrsv [ ] . prrsv appears to inhibit synthesis of type i ifns in pigs, while swine transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) induced high level of ifna [ , ] . ifn-a could not be detected in the lungs of pigs in which prrsv actively replicated. it was estimated that the ifn-inducing capacity of prrsv is at least -fold lower than that of prcv [ ] . prrsv infection of pulmonary alveolar macrophages (pams) does not lead to ifn-a production [ ] . plasmacytoid dendritic cells (pdcs) are thought to be the major source of ifn-a in vivo. prrsv also fails to induce porcine pdcs to produce ifn-a, while pseudorabies virus (prv), swine influenza virus (siv), and tgev stimulated the pdcs to synthesize ifn-a [ , ] . however, nf-jb activation occurred in the presence of prrsv. loving et al. [ ] showed that prrsv replicated in monocyte-derived dcs but not lung dcs and that dc response to prrsv was merely limited to ifn-b transcription but no ifn-alpha transcription. prrsv replication in marc- cells significantly inhibits the doublestranded rna-induced type i ifn transcription [ ] . the prrsv proteins that are found to be antagonists of ifn induction include nsp , nsp , nsp , and n (see review [ ] ). nsp has been studied in more detail than the others. nsp is self-cleaved into nsp a and nsp b subunits, both of which mainly localize in the cell nucleus and dramatically inhibit ifn-b expression [ ] . beura et al. [ ] showed that nsp b inhibited double-stranded rna (dsrna)-induced irf phosphorylation and nuclear translocation. however, kim et al. [ ] showed that nsp inhibited irf association with creb-binding protein (cbp) in the nucleus but had no effect on irf phosphorylation and nuclear translocation. the discrepancy is possibly because an nsp b that is -residue longer than its authentic form was used in the beura's study. another possible reason is that different prrsv strains were used. nsp inhibits ifn induction by blocking irf activation, and the ovarian tumor (otu) protease domain interferes with the nf-jb signaling [ ] . nsp also inhibits the antiviral function of isg by the deubiquitinase activity of the out domain [ ] . nsp , an endonuclease, is also an ifn antagonist [ ] . the ifn antagonizing activity is not restricted to nonstructural proteins. nucleocapsid (n) protein inhibits ifn-b induction by interfering with dsrna-induced irf activation [ ] . the multiple components of nsps interfere with ifn induction. the nsps are early proteins, and n is a late one, which may play roles at different stages of viral replication. prrsv interferes not only with ifn induction, but also with ifn-activated signaling. ifns bind to their receptors on cell surface and activate jak/stat signaling, resulting in the expression of ifn-stimulated genes (isgs) [ ] . prrsv inhibits the ifn-activated jak/stat signal transduction and isg expression in both marc- and pam cells [ ] [ ] [ ] . prrsv replication in marc- cells suppresses jak/stat signaling stimulated by addition of ifn-a [ ] . prrsv infection of pam cells also blocks jak/stat signaling, while a vaccine strain ingel-vac prrs mlv has little effect, possibly due to its less efficient replication in the primary cells [ ] . nsp b inhibits the jak/stat signaling via inducing the degradation of karyopherin-alpha (kpna , also called importin-alpha ), which is known to mediate the nuclear import of stat [ ] . prrsv infection of marc- cells also reduces kpna expression. besides nsp b, other prrsv proteins including nsp , nsp , gp , and n were also found to be able to inhibit ifn signaling [ ] . prrsv field isolates have variable suppressive effect on ifn-a induction in pam cultures, and the suppression was found at post-transcriptional stage [ ] . this is not unexpected as prrsv strains are divergent in genomic sequences (''prrs the virus'' section). prrsv infection of monocyte-derived dendritic cells (mo-dc) induces the transcription of ifn-a/b but no detectable ifn-a in culture supernatant, suggesting a blockage at post-transcriptional stage [ ] . prrsv infection of marc- cells inhibits ifn expression by interfering with the rlr signaling pathway [ ] . a variety of type and prrsv were found to stimulate ifn-a secretion by pdc via tlr- pathway, and the effect did not require live virus [ ] . the suppressive effect on pdc was thought to be strain dependent. a novel isolate, a mc , induced ifns in both marc- and pam cells, and virus replication was needed for ifn induction [ ] . type ifns and isgs were detected in a mc -infected cells. a mc infection of pigs resulted in higher level neutralizing antibody than a mlv vaccine strain that is highly homologous in sequence [ ] . variable effect on ifn signaling among prrsv strains was also found [ ] . among six prrsv strains (vr- , ingelvac prrs mlv, vr- , nvsl - , mn , and lelystad) tested, all but mn inhibited ifn signaling in marc- cells, and all but mlv and nvsl blocked the ifn activation in pams. nsp b from the six strains were cloned, and all but mlv nsp b inhibited ifn signaling when overexpressed [ ] . there is good agreement that prrsv infections are not resolved rapidly in piglets, e.g., not in - days, in contrast to infections with swine influenza, fmdv, or eav in horses [ , , ] . further, the carrier state may exist for up to days [ ] , and viral rna can be detected out to dpi [ , ] . antibodies to prrsv can be detected as early as week after infection [ ] (fig. ), yet viral neutralizing (vn) antibodies are not usually detected prior to weeks [ , ] (fig. ) . maximum titers may not be reached until - weeks dpi, and the peak titers are usually modest [ , ] . igg antibody levels appear to peak at - dpi in piglets but persist at lower levels thereafter [ ] . some reports indicate that viremia and viral replication can persist even in the presence of vn antibodies [ , ] , and viremia can be resolved before vn antibodies are detected [ , , ] . in the case of prrsv, ldv, and eav, gp is considered the most important neutralizing epitope in vn [ , [ ] [ ] [ ] . focus has been on the hydrophilic ectodomain of gp [ ] . however, gp has numerous glycosylation sites that might influence the avidity and specificity of antibodies to gp . in general and because of the high frequency of mutation in rna viruses, there is considerable variation in gp among various strains of prrsv (''prrs the virus'' section). thus, the concept of the dependence of antibodies to gp for vn is complicated. using recombinant polypeptides, li and murtaugh [ ] showed that the titer of antibody to the gp ectodomain did not correlate with the vn antibody titer. vane et al. [ ] used peptide-specific antisera to show that the largest number of antigenic sites was associated with gp and no neutralizing targets were associated with either gp or m. using chimeric viruses, lu et al. [ ] showed that gp and m were not responsible for tissue tropism. furthermore, other studies have shown that viremia is resolved before vn antibodies appear [ ] (fig. ) and animals are protected from the european variant without them [ ] . evidence suggests that recognition may depend on strain variants/types. mabs to gp recognize the european variant but not the north american variant [ ] . in spite of these often contradictory reports, the bulk of the evidence supports the view that vn neutralizing antibodies are important for protection [ , , , ] . unfortunately, the mechanism of vn for prrs has not been researched. as regards vn antibodies to prrsv, there are some concerns about work already published. one concern is the amount of data available and from what experimental animal group they was obtained. if vn depends on labor intensive culture studies, it is likely that data currently available are from a few time points and a few animals. whatever viral epitopes or whole virus variants are used, a high throughput microtiter system should be adapted. it would be a shame if the current belief in poor vn activity is a consequence of selected and limited sampling. one can also question the methods used. in most studies, vn is tested using a lab strain virus and marc cells to which the virus has become adapted in vitro. this is a valid assay for the cell line and the prrsv strain used but does it test whether neutralization has occurred in vivo in infected animals in which different target cells and virus variants are interacting? the failure of swine to develop a sterilizing immune response has raised the issue of whether this virus produces fig. in piglets, the appearance of neutralizing antibodies is delayed, but other antibodies appear shortly after infection. from lopez et al. [ ] suppression or tolerance [ ] . some have reported the presence of cd ? cells with a suppressor phenotype (cd ? cd ? foxp ? ) after infections with prrsv [ , ] . silva-campa et al. [ ] showed that porcine cells with the treg phenotype make il and tgfb, confirming their analogous function to those in mice. it is known that pulmonary dendritic cells can induce tolerance through il- [ ] . however, in a three virus study using isolator piglets, an increase in cd cells with a suppressor phenotype was not associated with prrs [ ] . few studies have experimentally tested whether prrsv is functionally immunosuppressive while many show inhibition of type i interferons by prrsv (''the innate immune response to prrsv'' section). if tregs in conventional animals are functional, they appear not to interfere with the antibody response to klh in prrsv-infected pigs [ ] . the thymic atrophy caused by prrsv can result in subnormal levels of double-positive thymocytes drives t cell development and loss of peripheral cd cells [ , ] . some coinfection studies suggest that prrsv can interfere with protective responses to other viruses (''history and discovery of the causative virus'' section), which is supported by extensive field reports of synergy between prrsv infections and endemic infections within herd. infections with asian hp-prrsv elevate a large number of cytokines associated with both innate and adaptive immunity, both pro-inflammatory and otherwise [ ] . this ''cytokine storm'' suggests that prrsv affects many pathways leading to innate and adaptive responses or their suppression. an element in the kinetics of prrsv infection is the age of the host. klinge et al. [ ] showed that prrsv antibodies are detected at the same time in infected piglets and adults, yet viremia is immediate and resolved in sows, but develops late and remains persistent in piglets (fig. ). the delayed increase in viremia in piglets is correlated with a delay in the infection-induced increase in il- ; the increase in this suppressive cytokine seems correlated with viral replication, but not the time of infection. the muchcited viral persistence seems to be a feature of piglets since, except for boars, the virus does not persist in swine infected later in life [ ] (fig. ) . furthermore, the presence of vn antibodies in older pigs is correlated with elimination of the virus [ ] . by contrast isolator piglets appear much more susceptible to b cell immune dysregulation (''response to prrsv infection in germfree piglets'' section) and prrsv is most immune dysregulatory during the critical window of immunological development before immune homeostasis has been established ( fig. ) . figure shows that viremia persists in piglets but not in adults. figure shows that antibodies detected by elisa appear early but the appearance of those with vn activity is delayed. this could reflect a difference in sensitivity between elisa-based assays and vn assays. early protection to all infections depend on innate immunity which then raises the question of whether persistence of viremia in piglets (fig. ) reflects suppression of innate responses in piglets (''the innate immune response to prrsv'' section). while this may initially be critical, there is still too little information to conclude that the adaptive immune response is not impaired. there are reports that the amnestic antibody response to prrsv is poor or absent [ ] , yet little is known about t helper and memory cells in response to prrsv infection. t cell recognition of viral epitopes has been described [ , ] , but a tetramer assay system for these epitopes has not been developed for prrsv. despite the fact that so many viruses interfere with class i presentation, little attention has been given to prrs. overall, there is insufficient information as to whether the b cell or the t cell systems are most affected by prrsv and about the extent to which one or the other is impaired. the genetic variability of prrsv (''prrs the virus'' section) could also be a major player in the puzzle that has confounded investigators for [ years. hard evidence for escape mutants during infection is lacking but heterologous challenge studies indicate immunity to one strain does not confer immunity to all [ ] . in conventional herds, persistence might be due to re-infection with extrinsic variants for which crossprotection is absent. a particularly useful observation comes from so-called herd closure [ , ] . this essentially involves immunizing adult animals in a virus-free herd and then isolating them from exposure to outside animals. that these animals remain prrsv-free suggests that: ( ) vaccinated adult swine can develop sterilizing immunity if isolated from other animals and ( ) escape mutants are unable to establish a re-infection in such herds. however, these experiments have not been performed with asian hp-prrsv or with very young piglets whose immune system is just developing (fig. ). more than vaccines have been developed for prrs, although no single product has been totally successful [ ] . these vaccines and their efficacy are the subject of another review (k.m. lager, submitted). the functional, cellular response in adaptive immunity is characterized by the activation and expansion of antigenspecific, mhc-restricted cytotoxic t lymphocytes (ctl). in general, this is the primary effector function and most efficient immunity against viruses in mammalian species as university of iowa immunology ( ) : - because ctl kill virus-infected cells and arrest the generation of new viral particles. the role of this aspect of the immune response in prrsv infection is poorly understood. costers et al. [ ] published that induction of virusspecific ctl in prrsv-infected swine is very weak and slow to develop. they analyzed this by using prrsvinfected autologous cells as targets of ctl killing. by comparison, these authors show a strong response of similar pigs infected with pseudo rabies virus (prv) in ctl assays using prv-infected target cells. in chronic viral infections, the regulatory element ppp r d plays a significant role in ctl dysfunction [ ] . other in vivo studies have not tested for the predicted prrsv epitopes that would induce ctl responses [ ] and have used nonswine animal models. this complicates interpretation of the small literature available on this subject. furthermore, analysis of ctl induction is complicated by the nature of this effector function. experimentally, ctl killing is measured by analysis of these cells killing virus-infected cells in vitro in an antigen-specific, mhc-restricted manner. in most cases, the virus also kills the virus-infected cells. provided it is allowed by the in vitro system, killing takes days to occur. thus, new approaches are needed. the role of c/d t cells in prrs is unclear. several reports describe that c/d cells are affected by prrsv and other viral infections [ , , ] . the latter shows that c/d t cells behave similarly to cytotoxic and nk cells. in isolator piglets, only the subset of cd ? cd ? c/d t cells was increased, which is the only subset is known to be cytotoxic [ ] . the paucity of information at this point is insufficient to construct a meaningful hypotheses regarding the role of c/d t cells in prrs. however, depleting them in vivo using mabs could determine whether they play a role in either disease resolution or pathology. prrsv affects lymphocyte development in thymus prrsv infection can cause an acute lymphopenia, thymic atrophy, and lymphadenopathy associated with the presence of prrsv antigen in the thymus. thus, development of a protective, adaptive immune response to prrsv may be impaired because prrsv infection negatively impacts circulating and developing lymphocyte populations, and reconstitution of the peripheral lymphocyte pool can be impaired. lymphopenia appears soon after infection [ , , , ] and follows an influx of macrophage-like cells in the thymus and secondary lymphoid organs that contain prrsv [ , ] . there is also a loss of immature t cells in the thymus [ , , ] accompanied by significant lymphadenopathy [ , , - , , ] . it seems important to connect these observations to understand how prrsv affects the development of prrsv-specific immunity. the two mechanisms on which the animal relies to return balance to the circulating t cell pool are thymopoiesis and homeostatic proliferation of peripheral cells [ ] . homeostatic proliferation, or expansion of the existing peripheral t cell pool, is the primary means for reconstitution following peripheral depletion. in mice, both peripheral memory t cells and naïve t cells undergo homeostatic proliferation, though at different rates (fast vs. slow, respectively) and with differing signal requirements (mhc, il- , etc.). naïve t cells undergo slow homeostatic proliferation in secondary lymphoid organs (such as lymph nodes) that is dependent on il- and self-peptide:mhc presentation by an apc [ ] . this type of proliferative recovery has been implicated in autoimmunity because of preferential expansion of t cells with greater specificity and stronger avidity for self, which has been observed following administration of lymphodepleting drugs [ ] . prrsv infection has been shown to result in production of autoantibodies [ , , ] , which may be related to the expansion of autoreactive t cells and/or the failure of the pre-immune repertoire to diversify (''response to prrsv infection in germfree piglets'' section). memory t cells can proliferate outside secondary lymphoid organs, and the signal does not require mhc contact. collectively, the noted lymphadenopathy associated with prrsv infection may be the result of homeostatic proliferation of peripheral t cells, and possibly b cells, to repopulate the peripheral pool. if lymphoid hyperplasia is the result of homeostatic proliferation, it requires determining why the cells do not egress from the lymph node. in addition to proliferation of existing t cells, newly developed thymic emigrants can contribute to restoring the peripheral pool to a normal level following a lymphopenicinducing event. however, reports indicate a loss of t cells in the thymus following prrsv infection [ , ] . development of t cells in thymus is well described in textbooks, and at a certain stage, cd ? cd ? cells (double-positive,dp) interact with cortical thymic epithelial cells (ctec) to scan for positively selecting antigens. positive selection occurs when the t cell receptor has an intermediate affinity/avidity interaction with self-peptide presented by mhc on the ctec. positively selected cells then commit to the cd or cd lineage (single-positive, sp) and rapidly relocate to the medulla where they sample antigen presented by medullary tecs (mtec) and/or dendritic cells. these dp cells should not be confused with those dpc cells in the periphery of normal pigs [ ] . medullary tecs are unique in the expression of autoimmune regulator (aire) gene, which controls the expression of tissue-restricted antigens. tissue-restricted antigens (i.e., self-proteins) are picked up by neighboring thymic medullary dendritic cells for presentation to developing sp t cells, which drives t cell selection. if a high affinity/avidity signal through the t cell receptor at this stage is received, cells die by negative selection to prevent release of autoreactive cells into the periphery, which is referred to as central tolerance [ ] . mature naïve t cells, presumably those that only recognize foreign antigen, are then released into the periphery. various groups have shown a population of macrophage-like cells in the thymus stains for prrsv antigen by immunohistochemistry [ , , ] . in addition, reports have highlighted the negative impact of prrsv infection on thymic cellularity [ , ] , primarily as a loss of cd / cd dp cells in the thymus of prrsv-infected pigs [ ] . the loss of developing t cells in the thymus likely affects the number and nature of newly developed t cells exiting the thymus during prrsv infection. the presentation of prrsv antigens in the thymus may also induce tolerance (loss of naïve cells that would recognize prrsv antigen) and provide a mechanism for the reported increase in regulatory t cells after prrsv infection [ ] . these data together give support to the notion that infection of apcs in the thymus has a detrimental effect on the development of naïve t cells, and this likely has a negative impact on the development of a protective immune response to clear the virus from the pig. some of the lymphopenia that occurs shortly after birth may reflect the rapidly expanding blood volume but whatever the cause, it is not due to a selective depletion of t cells [ ] . in young pigs, prrsv induces a reduction in circulating lymphocytes early after infection, but not in age-matched controls (c. loving, pers com). since the decrease in circulating lymphocytes occurs before obvious phenotypic changes in the thymus, the lymphopenia is: ( ) not due to thymus infection by prrsv, ( ) an effect by prrrv on the peripheral t cell compartment, or ( ) a red herring in the quest to understand how prrsv dysregulates the piglets immune system. it is unclear if the drop in circulating lymphocytes is related to the lymphadenopathy observed later in the infection, but could be a compensatory attempt to repopulate the peripheral lymphocyte pool. response to prrsv infection in germfree piglets ''isolator piglets'' are recovered by caesarian surgery and reared in germfree isolators [ , ] . these animals have not encountered gut flora, which drives development of adaptive immunity through stimulation of toll-like receptors [ , ] (fig. ) . furthermore, they obtain no passive maternal antibody in utero and receive no colostrum that could protect them from pathogens or interfere with immune responsiveness [ ] . finally, isolator piglets have no exposure to other pathogens or to other strains of prrsv. the response of isolator piglets is intrinsic and not modulated by other pathogens, subclinical infections, maternal antibodies, or exposure to other environmental factors. these piglets provide the best in vivo opportunity to identify the direct in vivo effects of prrsv on the neonatal immune system. isolator piglets can also be considered as ex vivo fetal piglets and, therefore, a good model to study prrsv-infected fetuses. since the adaptive immune system is not developed in fetuses, their intrinsic response is either innate or driven by fetal infections that promote development of adaptive immunity (fig. ) . rna viruses are often sensed by intracellular by toll-like receptors which sense either positive or negative single-stranded rna or double-stranded rna (a recognized adjuvant) generated as part of viral replication. these molecules can drive development of adaptive immunity as shown with swine influenza [ ] . fetal piglets are immunocompetent as early as days of gestation (dg) [ ] and have lymph nodes, an active bone marrow, ig gene class-switch recombination has occurred, and the ileal peyer's patches are especially well developed. while some changes are likely to occur between dg and birth (dg ), these have not been identified. when fetuses are confronted with prrsv, they respond in the same manner as isolator piglets [ ] (see below). studies using prrsv-infected isolator piglets [ , , , ] have revealed a number of features about the immune response to prrsv that may provide clues as to how this virus modulates the host immune system. immediately obvious is hypergammaglobulinemia, lymphoid adenopathy, and the appearance of autoantibodies [ ] (fig. ) . polyclonal b cell activation, hypergammaglobulinemia, and the appearance of autoantibodies are also seen in infections by unrelated viruses [ ] . polyclonal b cell activation is also a feature on ldv infection in mice, a related arterivirus that is also persistent [ ] . autoantibodies in prrsv-infected isolator piglets to golgi proteins [ ] are also a feature of ldv infections [ , ] and may be in part due to the site of morphogenesis of arteriviruses [ ] . in addition to hypergammaglobulinemia and autoimmunity, prrsv-infected isolator piglets exhibit abnormal antibody repertoire and b cell development. measured as a repertoire diversification index, the values are in the range of . , not significantly greater than for fetal piglets or sham control isolator piglets but - fold less than sivinfected isolator piglets and conventionally reared piglets (pic; fig. a ). sequence analyses revealed that the cdr binding sites of the ig from prrsv-infected piglets are even more hydrophobic than in newborns and sham controls while those for siv and pic are shifted to the hydrophilic region (fig. b) [ ] . hydrophobic binding sites are incompatible with antibodies that recognize glycoproteins and are a feature of the pre-immune antibody repertoire [ ] . in these animals, b cell differentiation is extremely rapid and cells representing the activated b cell stage are nearly undetectable indicating that b cells rapidly become plasma cells [ ] . comparative cellular studies of isolator piglets infected with prrsv and siv failed to reveal any evidence of immune suppression, i.e., lack of evidence for elevation of fox p cd ? , cd ? t cells. however, cells with a suppressor phenotype were observed in parallel studies using pcv -infected piglets [ ] in which functional immune suppression has been reported [ ] . accepting the fact that the effect of a viral, bacterial, or fungal infection in germfree reflects a direct effect of the pathogen, our data suggest that dysregulation of b cell differentiation is one of the principal feature of neonatal infections with prrsv during the critical window (fig. ) . (figs. , ) . by contrast, adult animals make good vn antibodies and eliminate the infection [ ] . some additional support comes from studies using homologous variants [ ] . osorio et al. [ ] demonstrated that passively administered ig-containing vn antibodies obtained from convalescent sows could provide sterilizing immunity in piglets although a follow-up study showed that while viremia was ablated, viral replication persisted in some tissues [ ] . in the same studies, passive administration of non-neutralizing anti-prrsv serum had little effect although the mechanism of vn was not described. it would be wise to know whether active complement was also transferred. since prrsv is a respiratory infection, it would also seem important to know whether passive antibodies would have reached the respiratory tract. it is known that parenteral and oral vaccination of the sow generates passive antibodies that are protective against tgev [ , ] . these and other studies support the view that effective antibodies were made by adults [ , , , ] . tgev is a gastrointestinal infection, so ingestion of passive maternal antibodies, via milk and colostrum, has access to the site of infection. by analogy to ww ii: ''you need to stop them on the beaches.'' the respiratory tract, especially the upper portion, is the domain of the mucosal immune system. thus, parenterally administered passive antibodies to prrsv are unlikely to reach mucosal sites. this may explain why follow-up studies by lopez et al. [ ] showed that virus still replicated in some tissues. the differences among result obtained using isolator versus conventional piglets might provide clues as to the nature of the apparent neonatal immune dysregulation. while lymph node adenopathy and some thymic atrophy are common to both groups, the extraordinary hypergammaglobulinemia of all isotypes and b cell expansion has only been consistently reported for gf isolator piglets (fig. ) . this may in part be due to the fact that investigators who studied conventional piglets rarely measure ig levels in serum or bal. such measurements in conventional piglets would be difficult to interpret since conventional piglets would have ingested maternal ig through suckling. conventional piglets used in these studies would be from prrsv-free herds, so very little of the ingested and absorbed ig would be prrsv specific and therefore not protective. this may explain why the extent of the disease is similar. both groups of animals make virusspecific antibodies but because of the extraordinary hypergammaglobulinemia seen in isolator piglets, and because absorbed ig are from prrs-free sows, only a tiny proportion would be virus specific [ ] . however, knowing how many cells are virus-specific relative to other viral infections would be a much more useful parameter for comparing both groups. the b cell clonal analysis done with isolator piglets showing selected expansion of the pre-immune repertoire has not been performed in studies of conventional piglets. the opposite is true for cytokine studies. however, cytokine studies in conventional piglets might be misleading because of undetected secondary infection or the effect of regulatory elements in colostrum or the impact of gut colonization [ ] . while the impact of normal gut flora can impact cytokine levels in conventional animals, investigators typically compare their data to control littermates raised in the same environment, so this should play little role. however, the lack of gut colonization of isolator piglets might be in part responsible for the differences in the degree of hypergammaglobulinemia, since elements received via colostrum could establish immune homeostasis which might dampen polyclonal b cell activation and proliferation [ ] . in limited studies, no differences were found between isolator piglets colonized with benign escherichia coli and their colonization-free littermates [ ] . however, studies in mice and rabbits indicate that all colonizers are ''not created equal'' [ ] , so results obtained using only e. coli could be misleading. difference in the innate immune response in isolator versus conventional piglets has not been reported. in summary, prrsv infections that result in fetal abortion, b cell dysregulation in isolator piglets suggests that piglets are more susceptible during the critical window of immunological development (fig. ). since siv infections are rapidly resolved even in gf piglets, it suggests that age-related neonatal immune incompetence cannot alone explain the persistence of prrsv. this would appear to shift blame to active immune dysregulation. while siv is quickly evicted, one must remember it infects primarily epithelial cells, not cells of the hematopoietic/ immune system. thus, siv infections would theoretically provide less opportunity for immune dysregulation of the developing neonatal immune system. in any case, investigators need to be careful about assuming that what happens in piglets, also happens in adults. the prrsv genome as indicated previously, prrsv is a member of the family arteriviridae, in the order nidovirales, which also includes university of iowa immunology ( ) : - the viral families of coronaviridae, inclusive of coronavirinae and torovirinae, and roniviridae [ ] . the nidovirales order (latin: nested set) contains viruses with similar genomic organization and replication strategy. the arterivirion contains a polyadenylated molecule of singlestrand, positive-sense rna (which is itself infectious) that varies in length for prrsv ( , - , bp) and eav ( , - , bp), but not as yet in complete published genomes for shfv ( , bp) and wpdv ( , bp). the particles are roughly spherical with an average virion diameter of nm and consist of a helical nucleocapsid surrounded by a lipid bilayer containing several proteins [ , ] . all arteriviruses replicate in alveolar macrophages of their respective host, apart from wpdv, for which the host cell type is not known. except for wpdv, which was only recently genetically characterized [ ] , each individual arterivirus species consists of many diverse genomes. prrsv has been most studied in terms of host pathogenesis. there are two recognized prrsv genotypes: type or european-like (prototype lelystad) and type or north american-like (prototype vr- ) [ ] . the two main genotypes share approximately % nucleotide identity, but each may vary more than % in nucleotide sequence. the genome length of type ( , - , bp) not only differs from type ( , - , bp), but discrete sections of the genomes are different as well. prrsv rna includes a untranslated region (utr) of - (type ) or - (type ) followed a large replicase gene of variable length processed into at least recognized nonstructural proteins (nsp a, b, ( tf, n), - a, b- ) by self-encoded proteases. the proteases include papain-like protease (plp) a and plp b in nsp , plp in nsp , and a serine protease (sp) in nsp [ , , ] . presently, most of the cleavages have been defined using eav. plp a and b, and plp cleave once cotranslationally, directly downstream of the respective enzyme. sp completes the remaining cleavages. nsp harbors the core rna-dependent rna polymerase (rdrp), nsp is a helicase, and nsp contains a mn ?dependent rnase that cleaves at u stretches (nendou) and is involved in rna replication [ ] . downstream of the replicase gene is overlapping open reading frames (orfs) enumerated as orf encoding for glycoprotein (gp) , orf b encoding non-glycosylated envelope protein e, orf encoding gp , orf encoding gp , orf a encoding non-glycosylated protein a, orf encoding gp , orf encoding the non-glycosylated membrane protein m, and orf encoding the nucleocapsid protein n. since these orfs overlap, mutations to one coding sequence may affect adjacent orfs. they are transcribed as a nested set of at least six subgenomic rnas (sgrnas) in infected cells. all of the downstream orfs encode structural proteins [ , ] . as mentioned above, type prrsv differs in the length of most structural orfs when compared to type viruses. a remarkable feature of the prrsv genome has been the rate of mutational diversification. it has been estimated that prrsv rna may have evolved at a higher rate ( - /site/ year) than other rna viruses ( - - - /site/year) [ ] although another investigator estimates the rate is similar to other rna viruses [ ] . the frequency of mutation includes not only simple mutation, but also is accounted for by a high rate of recombination [ ] [ ] [ ] [ ] . it is estimated that there now exist as many as four major subtypes of type prrsv, based on orf and orf phylogeny [ , ] . even more subtypes, as many as nine, have been identified for type prrsv when based on orf . the husbandry of commercial swine, with large numbers of hogs from different source herds and artificial insemination with boar stud semen, is believed to have accelerated the evolution of prrsv [ ] . there is also ample evidence that two or more prrsv strains may infect an individual pig [ , ] . the combination of husbandry with genetic mutation and recombination between different viral strains has made the study of prrsv evolution challenging. the major envelope proteins of prrsv consist of gp and m [ , ] . gp forms a heterodimeric complex with m linked by a disulfide bond [ ] . both gp and m are thought to traverse the viral envelope three times and have only a small extravirion domain and a longer intravirion domain, much as was shown for ldv and eav [ , ] . gp is the most variable structural protein, and the predicted ectodomain after signal sequence cleavage is approximately residues [ , ] . within these amino acids, two hypervariable regions surround a quite conserved region, which contains the completely conserved cysteine disulfide-linked to m and two potential n-glycosylation sites [ , ] . the conserved domain has been shown to harbor a neutralization domain, and the n-terminal sequence has been termed a decoy epitope that is not neutralizing [ , , , [ ] [ ] [ ] [ ] [ ] . however, since the conserved domain is surrounded by complex oligosaccharides, it is shielded from neutralizing antibodies [ , ] . the m protein, which is believed to act as glue to bring all virion components together, has also been implicated in neutralization [ ] [ ] [ ] . in addition, two of the minor glycoproteins (gp and gp ) have also been shown to harbor neutralizing epitopes [ , , [ ] [ ] [ ] [ ] [ ] . as shown for eav, gp :gp :gp are thought to be disulfidelinked heterotrimers on the extravirion of prrsv and are thought to be in very low amounts compared to gp [ , ] . although the minor glycoproteins may play a role in neutralization of some or all prrsv strains, there is little else known about the viral functions these proteins perform in prrsv [ ] [ ] [ ] . the phosphorylated n protein encapsidates the rna genome, probably in a helical conformation [ , ] , and is most likely involved in capsulation and budding from the endoplasmic reticulum as was shown for eav [ ] . the swine host synthesizes the most antibodies to the abundant n protein, which are non-neutralizing [ ] . replicase proteins that have been shown to induce high levels of antibody are nsp , nsp , and nsp [ ] . nsp has also been shown to harbor many b cell epitopes from different prrsv strains [ , [ ] [ ] [ ] and has recently been shown to be incorporated into the virion [ ] . several infectious clones of prrsv have been produced [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most of the clones were developed using type viruses. these infectious clones represent only a fraction of the variability seen in the field, but are extremely useful in probing the genome for dispensable regions [ , , , ] , insertion of foreign genes to develop diva viruses [ , , ] , investigation of structurefunction relationships [ , , , [ ] [ ] [ ] [ ] , examination of host virulence [ , , [ ] [ ] [ ] , and/or the probing of host response [ , , ] . there are also several studies using chimeric viruses, either within or between certain arteriviruses. some chimeric studies have led to the conclusion that the minor glycoproteins, not gp , are important for tropism in cell culture [ , [ ] [ ] [ ] and that the m protein is also not involved [ ] . other investigators have explored combining different regions of type prrsv with type to examine viability [ , ] or to explore the effect of nglycosylation differences between strains [ ] . in an attempt to develop broader crossneutralizing antibody, researchers have mixed regions of the prrsv genome from different strains, creating a panel of chimeric viruses to explore changes in the virus as well as the swine host antibody response [ ] . the same investigators used this technique to attenuate a strain of prrsv [ ] . lastly, researchers have attempted to define regions of the prrsv genome responsible for attenuation/virulence [ , ] or to act as vaccines [ ] . these studies have led to the knowledge that it appears that attenuation, as well as virulence, is multifactorial, involving two or more regions that can differ based upon the lineage of virus used for study. the main lesson learned from these studies is that each strain of prrsv, derived from field isolates or those with defined mutations, harbors individual characteristics that influence the specific pathogenesis seen. these characteristics include viral replication rate, the amount of specific subgenomic messages, the relative ability to process viral replicase proteins, the amount of n-glycans displayed on the virion, the amount of each individual viral protein, the relative interaction rate between viral proteins, and the relative ability of each strain to inhibit type i interferon and to induce humoral and cellular immunity. added to these viral causes of pathogenic differences under defined clinical conditions are the host response to each individual viral strain, host genetics, climate effects, and herd immunity, among other factors. the need for new experimental tools and approaches advances in science have mostly succeeded because the experiments employed were focused on testing a specific hypothesis and because they were designed so that the number of variables was minimized. naturally, this is much more difficult in biology because of the complexity of living systems and because many variables are unknown when the study begins. the image that emerges from the cumulative literature on prrs is that many: (a) represent a category that is often derogatorily referred to as fishing expeditions, i.e., exploratory research, (b) are repetitious of other work already done or represents near re-publication of the same work in another journal, and (c) are noncomparative studies. the work appears to be driven by the pressure to produce a vaccine, not to understand how prrsv modulates the immune system. the combination of swine and prrs offers a particular challenge to immunologists. prrsv does not replicate in mice, there are no practical inbred strains of swine, immunological reagents are limited, and producing stable cells lines has proven to be difficult. most studies have been done using conventionally reared piglets, which represents a complex model as illustrated in the following hypothetical example. consider pigs infected with prrsv and noninfected controls. since pigs are outbred, difference in responses can be genetic. if they are conventional, each animal in each group has not had the same experience since it may have a different mother, and its passive immune experience could differ in terms of colostral regulatory factors obtained and their dosage. suckling patterns differ within a litter giving rise to the often used ''hind teat'' syndrome. if you split the litter, you must then move some piglets to surrogate mothers, which introduces another set of variables. gut colonization plays university of iowa immunology ( ) : - an important role in development of adaptive immunity [ , ] , and colonizers do not have an equal effect [ ] . colonization typically occurs by contamination at the birth canal and thereafter by contact with the mother through suckling or contact with her feces. assuming that each newborn piglet in each experimental group encounters the same environmental experience is extremely difficult to prove. all of these assumes they have the same living conditions and have no contact with other animals that not part of the study. the ''closed herd'' studies cited earlier is an example of how this latter aspect can be properly controlled. conventional animals almost invariably contact other microorganism, some that are pathogens and some that are merely commensals. while experimenters may control for serious pathogens, they typically do not control for subclinical infection or for differences in the make-up and effect of benign colonizers. all of these may affect how a young pig responds to an experimental infection with prrs or a prrs vaccine. the literature shows that animals studied differ in age and there appears to be an age factor in their immune responsiveness and in the persistence of the virus (''the effect of age, rearing, complement and the role of mucosal immunity'' section). if the purpose of a study is to understand how a virus affects the immune system, conventional piglets are probably a poor choice. if on the other hand, the goal is only to test a vaccine under farm conditions, then the approach is fine. after all, the sabin and sauk vaccines and many successful bacterial vaccine before them prevented the spread of many horrible diseases but it would take decades to understand the etiology of the disease and just why these vaccines worked. the story of prrs is more like the story of hiv; the old time vaccine recipes do not work, and so, it is now time to understand the etiology of the viral infection and how it interferes with its immune-based eviction. while there is no mouse model for prrs, there is a mouse model for ldv. the superficial similarities in outcome are such that one wonders why the ldv model has not been used more for prrsv given the vast number of immunological reagents that are available for mouse immunology. assuming that for other reasons, ldv is not a good model, then perhaps the next approach would be to compare how siv, prrsv, and fmdv affect the porcine response in a controlled in vivo setting such as the isolator piglet. one glance at the literature reveals that compared to their counterparts in mainstream immunology/virology, those in the veterinary field are at a disadvantage. one obvious problem is the lack of reagents for work on the swine immune system. however, the literature also suggests an apparent reluctance to employ some of the -year-old technologies already available. notably, simple assays like quantification of igs are rarely used, as are immunohistochemical assays that measure ig-containing cells and elispots that measure isotypic distributions, antigenspecific b cells, and cytokine secretions. while elispot and pcr assays have been used in prrs research, neither of these methods provide data on where the cells responsible are located within the geography of the organs studied. refining these to single cells in situ assays as used in other species would provide more useful information. single cell sorting and recovery of rna by micromanipulation are also available. given the many studies done in conventional piglets that refer to the lack of vn early in development of prrsv infection, why there are no assays to determine the mechanism of vn to test if complement is required or if antibody affinity is important is puzzling. likewise for a disease that affects the respiratory tract, the lack of studies on the mucosal/local immune response to prrsv is conspicuous. while using more controlled in vivo studies can help to understand prrs, they cannot address questions about what prrsv does at the cell and molecular level. without in vitro studies, it will be difficult to understand how prrsv affects the host immune system. as mentioned above, the lack of stable cell lines presents a real problem. this can partially explain why there are no mixed culture studies to determine whether mhc i is downregulated by prrsv and how infected macrophages or the virus itself affects t and b cells and their interactions. even a question still exists as to the exact cell population that can be infected. for example, does prrsv infect lymphocytes or only macrophages/dendritic cells? if this should occur, lymphocytes are present at all different stages of development, and if a particular viral receptor is needed, it may not be present at all times during lymphocyte differentiation. since porcine cell lines immortalized at each stage of lymphocyte development are not available, the question is more difficult to answer. it may also be dangerous to use only laboratory strain for infection studies and only established cell lines to which the strain has been adapted. for example, marc cells used to propagate prrsv do not show downregulation of type ifn, while this is not true for pdcinfected in vivo. to address whether the remarkable polyclonal b cell proliferation seen in gf isolator piglets is the direct effect of the virus, studies involving t-b cell interactions or contact between b cells and infected macrophages are needed. the same applies to cytokines: what cells are making which cytokines and where are these cells histologically located since cytokines typically act at short distances? especially useful for these studies would be engineered prrsv mutants lacking the ability to make certain gene products. the wealth of information on the prrsv genome, the many variants, and engineered mutants, provide a rich resource of research material (''prrs the virus'' section). in the last two decades, which covers the same period in which prrs has been studied, tetramer assays to quantify t cell specificity and involvement have become well established and can now be used with some limitation for cattle and swine. studies that concern innate immunity are already being conducted in vitro (''the innate immune response to prrsv'' section). perhaps the best way to determine how prrsv modulates or dysregulates the immune system is to start with fetal and neonatal animals since the pandemic nature of prrs appears developmentally linked. that the effectiveness of neonatal vaccines is age-dependent is no surprise to any immunologist and forms the basis for the timing of childhood vaccination schemes. while for prrsv and other viruses that cross the placenta, studying the fetal immune response would be wise, but quite impractical. fortunately, in swine and other artiodactyls, newborns are essentially ex vivo fetuses since they can be reared in gf isolators in which maternal regulatory factors and the effects of gut colonization are absent [ , ] . given the experimental ''cleanliness'' of using isolator piglets (''response to prrsv infection in germfree piglets'' section), why they are so seldom used is surprising. first, there is a matter of expense which is not trivial. second is the rather subjective view that isolator piglets are artifacts because they do not reflect the farm experience and environment. so what is the purpose of prrs research: to simulate the farm experience and produce a vaccine ''in the blind'' or to first understand how the virus affects the host? if the former is successful, the latter usually becomes mute. unfortunately, the latter does not seem to be the case for prrs since the virus was identified [ years ago and the disease has not been controlled. one argument favoring isolator piglets is their use as a model for fetal piglets that are aborted after in utero infection. the most compelling argument for the use of isolator piglets to understand how the virus dysregulates the immune system is that it minimizes the number of variables, always a feature of good experimental design. finally, if prrs is primarily a persistence problem in neonates, the use of isolator piglets automatically confines studies to the critical window of immunological development (fig. ) . all studies in biology must grapple with what is ''normal.'' eviction of the virus shortly after infection might be considered ''normal'', while those that are not might be ''abnormal.'' this reasoning is certainly open to discussion. from a practical position, this is a good starting point if the goal is to understand how certain infectious agents affect the immune system. good experiments cannot be done in a vacuum. a glance of the literature shows that many experimental studies compare virus-infected piglets only with noninfected controls. this overlooks the possibility that the changes observed are common to all viral infections including suppression of nk function, interference with class i presentation, and polyclonal b cell activation. rather, experiments need to be designed in a manner to identify ''prrs-specific'' immune dysregulatory factors. a number of those done in studies on innate immunity have been done comparatively (''the innate immune response to prrsv'' section). coinfection studies are really relevant. for example, renukaradhyad et al. [ ] showed that while prcv reduced nk activity by %, dual infection with prrsv reduced this - %. in nearly all coinfection studies, there was an increase in disease [ , , ] as might be expected resulting in increased morbidity and mortality. it would be surprising if coinfection did not result in more pathology and perhaps a delayed/depressed immune response. thus, such studies would seem unreliable in the identification of virulence factors of prrsv. there are also parallel studies using siv, pcv , fmdv, and tgev to distinguish ''normal'' versus ''abnormal.'' however, these viruses have different cell tropism. are there any other porcine virus that infect macrophages and are eliminated in - days? there is also the issue of virulence. in the case of prrsv, one expects the degree of immune dysregulation to parallel the degree of virulence. hp-prrsv is more virulent because it kills the host in a shorter time or produces more severe clinical symptoms. does it also cause more severe immune dysregulation? if not, then assuming all events seen with vaccine strains of prrsv are due to immune dysregulation could lead in the wrong direction. the purpose of this review was to allow individual specialists to review their area of expertise and then to ask each to contribute a subhypothesis. we then assembled these separate views into global hypothesis. our goal was to especially provide new investigators with a number of testable hypotheses that could explain how prrsv dysregulates the neonatal porcine immune system. prrsv suppresses innate immunity, which delays adaptive immune responses prrsv infection in pigs leads to delayed production and low titer of neutralizing antibodies [ ] as well as weak cell-mediated immune response [ ] . we hypothesize that the suppression of innate immunity can be an important contributing factor to the modulation of host immune responses because type i ifns promote antigen presentation and natural killer cell functions, enhance antibody production of b cells, and play an important role in the differentiation of both cd ? and cd ? t cells. the prrsv interference with the innate immunity is at multiple levels, from ifn induction, ifn-activated signaling to activity of isgs. therefore, viral-mediated suppression of innate immunity not only inhibits early host defense against the infection, but also interrupts the development of adaptive immunity, especially in the young pigs. this may explain why young pigs develop more severe disease and poorer protective immune response during the critical window of development (fig. ) . therefore, we would suggest comparative studies using siv and tgev to determine at the cytokine/cellular level, if prrsv-infected pams or pdcs alter the signal to t and b cells or even developing thymocytes. using the ifn-inducing prrsv strain a mc could add to the value of the model. we further hypothesize that given the divergence of prrsv strains in sequences and clinical features that experiments utilize various strains and engineered mutants. since type i ifns are proinflammatory, the proper amount at the right site and time may be protective, whereas extreme elevation could result in damaging inflammation. a typical example is that hp-prrsv induces high-level ifn-a, but causes high mortality in pigs [ ] . polyclonal b cell activation resulting in hyperplastic lymph nodes packed with ig-containing cells (igcc) is a hallmark of prrsv-infected isolator piglets. this is paralleled by hypergammaglobulinemia in which de novo-synthesized ig levels can increase as much as , -fold in weeks postinfection although \ % of these are virus specific [ , ] (fig. ) . we assume that the same type of immune dysregulation occurs in conventional piglets, although it may be masked by the high concentration of absorbed passive ig that increase serum ig levels to [ mg/ml. the extraordinary hypergammaglobulinemia simultaneously occurs as b cells rapidly differentiate to plasma cells in a manner in which the intermediate stage of activated b cells (cd ? cd -) is virtually absent [ ] . future studies in both conventional and isolator piglets need to confirm or reject the observation that a very small proportion of specific antibodies characterizes the response to prrsv. if confirmed, it would lend support to the view that rapid b cells differentiation allows little time for diversification of the antibody repertoire. this can be tested after pcr recovery and cloning of the rearranged vdj from various tissues. using labeled probes specific for the nonmutated cdr and cdr regions of the seven porcine vh genes, a repertoire diversification index (rdi) can be calculated as described previously and shown in fig. [ , ] . since the rdi is largely a measure of the degree of somatic hypermutation, it indirectly tests whether gc formation and function have been normal. it would be nice to confirm this in conventional piglets and adult swine, but the data would be uninterpretable since conventional piglets and adult swine have been antigenized through contact with other microorganisms, and changes could not be ascribed to prrsv. suspicion about abnormal gc activity might also explain the findings of mulupuri et al. [ ] . they used in vitro restimulation assays to suggest that there is a poor memory b cell response to prrsv. work by raymond and rowland [ ] identified gc in newborn prrsv-infected piglets using a mab to cdw that has not been validated in swine. the gc and memory cell questions need to be pursued using better reagents and better experimental designs. the delay in development of vn antibodies in prrsvinfected piglets while the anti-viral response continue to rise (fig. ) might be because early antibodies are: ( ) complement dependent for vn, ( ) of low affinity, ( ) specific for non-neutralizing epitopes, or ( ) of the wrong antibody isotype. alternatively, the differences between iddex elisa titers and vn merely reflect differences in assay sensitivity. in a single study, the addition of fresh serum did not improve vn to ldv, but it did improve the efficiency of vn to eav in horses suggesting that vn is complement dependent in horses but not in mice [ ] . this is a simple assay and should be done with sera from prrsv-infected swine. a most likely possibility is that antibody affinity is too low in neonates to perform as effective vn antibodies. in the case of denge virus, at least % of the neutralizing epitopes must be bound by antibodies for vn to occur [ ] . immunochemists over the last years have developed a plethora of methods to determine antibody affinity. most of these were developed to study antibody interactions with defined haptens. these studies established a number of very important principles including the observation that avidity, i.e., the staying power of an antibody, was determined by the ratio of the on-rate to the off-rate. thus, some ''quick and dirty'' methods have surfaced based on the principle that antibodies that remain bound in the presence of denaturants like urea or guanidine hcl are used [ ] , which are of high affinity. using this procedure, the relative affinity of a non-vn serum could be compared to that from adult swine that has vn capacity. should the experiments designed to test the role of complement or antibody affinity give negative results, another approach would be to test the specificity of early antibodies for certain viral epitopes. as reviewed in ''humoral responses of conventional animals'' section, vn antibodies to the lelystad virus preferentially recognize gp . assuming gp is the critical epitope, and affinity has been ruled out; it might suggest that antibodies to gp appear late during infection or that gp is poorly expressed on the virions used in the assay. once bound, the fate of the virus-antibody complex can also depend on the isotype of the antibody, which brings us to the fourth possibility. multivalency such as with pentameric igm can compensate for intrinsic binding site affinity and, therefore, perform much better than nonpolymeric igg so that early igm should provide good vn activity. the subclass of the igg antibody can also play a functional role in the effectiveness of complement-mediated vn. in swine, igg is the most totipotent igg based on its motifs for complement and fccr binding [ ] . however, actual functional comparisons have not been carried out. igg is expressed very early in fetal and newborn piglets but after antigen exposure, other igg subclasses, especially igg replace igg [ , ] . during the period in which vn has been typically measured (fig. ) , there is at least tenfold more igg than igm present, and thus, igg is most likely the antibody in serum that is being measured in current vn tests. to determine which subclass of igg is involved would be extremely difficult. first, all commercially available mabs to swine igg are more or less pan specific [ ] . even if such reagents were available, those which bind the virus would almost certainly be a mixture, so most probably antibodies of all subclasses involved, albeit probably dominated by igg . perhaps the only way to truly test the effector function of the different igg subclass antibodies seems at this point unjustifiable. this would require construction of chimeric antibodies for each subclass each with a binding site that recognizes a neutralizing epitope of prrsv akin to the method we have described for expression and recovery of individual porcine igg subclass proteins [ ] . confirmation of this subhypotheses might explain the initial ineffectiveness of the humoral response to prrsv during the critical window, but it does not explain why the extraordinary b cell expansion occurs and what force is driving this event. these require other subhypotheses and experiments to test them. we hypothesize that prrsv infects a population of antigen-presenting cells that migrate to or are constituent in the thymus of fetal or newborn animals, e.g., tecs, macrophages, and pdc that are engaged in thymocytes development and compromises proper t cell development. the interaction of thymocytes with these infected apcs might result in cytokine production/transcription and other protein transcription, which is abnormal compared with agematched controls. furthermore, the emerging t cell populations could be tested for their ability to recognize peptides derived from prrsv or a control antigens like ovalbumin. contrived in vitro systems should be developed to determine whether t cells developed in prrsv-infected thymi can provide t cell help for antibody responses, activation of macrophages, or can behave as ctls. we propose that the role of ctls in prrsv infection is fundamentally different in the infection of neonatal pigs compared to adults. we propose that the ability of pigs infected in utero or shortly after birth to mount any ctl response against prrsv is compromised by the impaired development of ctl precursors due to reduction of thymic selection. further, t cell selection that does occur could suffer from prrsv antigens being seen as self-antigen, as a result of infection of thymic cells involved in t cell selection. contrarily, in animals infected with prrsv as adults, ctl precursors have developed normally, and even though the infection impairs innate immunity, the presence of virus-infected cells eventually could lead to a protracted development of a moderate ctl response. further, we propose that the dysregulation of b cell function favors expansion of cd helper t cells not those required for induction of ctls. this could also contribute to or be the sole cause of the protracted development of antiviral ctl responses in adult animals. we describe below techniques to test theses hypotheses. first, we can use live, virulent virus in the short (hours long) assays to detect ctl killing. alternatively, avirulent strains of the virus can be used as surrogates, allowing the cell death to be solely a result of ctl killing of the target cell. in other circumstances, viral proteins can be delivered to target cells artificially, by vectors for instance [ ] . since the ctl are from an infected animal and the autologous cells (or mhc matched target cell line) are given the vector expressing viral proteins, the measure of killing is now attributable to the ctl, as there is no live virus. a dominating concept of the immunopathogenesis of prrsv infection is the immunosuppression or dysregulation of the adaptive immune response. as with many livestock studies, there is a body of work describing the antibody response but little analysis of ctls. the single report of ctl function describes a basic analysis of a single strain of virus and concludes there is a low-level ctl response that is protracted in the kinetics of development [ ] . a better understanding of ctl biology in prrsv infection will require a more sensitive assay for ctl function. using tools available today, class i mhc tetramers can be designed and tested to track ctl development and function. for instance, cd a (lamp a) is an integral membrane protein that lines the vesicles that contain the granules that mediate killing by nk cells and ctls. these granules are released by the vesicle membrane fusing with the cell membrane and releasing the contents. as a consequence, cd a is now detected on the cell surface. so, a tetramer-positive, cd a expressing cell is a prrsv-specific ctl that has just killed a virus-infected cell. so, not only is the cell phenotype determines, i.e., prrsv-specific cd t cells but also whether these cells function as ctls. another possibility to explain the decrease in ctls might be the action of mdsc [ , ] . these macrophages accumulate at the site of chronic viral infections and tumors and suppress ctls. therefore, highly infected sites such as thymus, lung, and certain lymph nodes [ , , , ] may harbor these cells. since prrsv targets macrophages, could their infection result in differentiation of myeloid cells to mdsc? with these tools, hypothesis testing can determine whether ctls are efficiently induced, induced but not functional, develop early but are rapidly downregulated, develop late, etc. elevation of p expressing, cd ? , cd ? treg populations reported in prrsv-infected isolator pigs is controversial (''humoral responses of conventional animals'' section). however, if class ii sla tetramers could be used to focus on the prrsv reactive cells in that population exclusively, this antigen-specific population may be highly induced, but masked by the present methods of analysis. however, given the evidence available, a more likely hypothesis is that the normal, t cell differentiation is dysregulated as reflected in the apparent dysregulation of helper t cells that promote excessive b cell proliferation while preventing prrsvspecific ctls from expanding that become activated to kill virus-infected cells. the opportunity to manipulate the prrsv genome provides the opportunity to test whether certain viral genes/proteins are responsible for immune dysregulation. nsp is the most variable protein in the virus, subject to insertion/deletion(s) compared to the prototype type strain, vr- . the fact that the nsp protein is an early protein and also a structural component of virions [ ] suggests that it may be in contact with host macrophages and dcs, and stimulators derived from those and other host cells. it also possesses that a key protease, plp , whose ability to downregulate ifn-a and can act to deubiquinate proteins is well established, has a key role in the viral replication cycle by cleaving the nsp / junction. lastly, this protein is the largest protein of the virus. a prior in vivo study has shown that a specific deletion of aa in nsp of strain vr- resulted in virus (vr- d ) with replication kinetics in -week-old swine about log lower than the parent strain, while other deletions elsewhere in nsp had a more dramatic effect on viral replication (''prrs the virus'' section). it was also shown that swine inoculated with vr- d had no delay in onset of antibodies to the nucleocapsid protein. what was intriguing was that these same animals showed a delay in serum ifn-c and a significant decrease in lymph node enlargement over that seen with vr- . unfortunately, no comparison was completed on the thymic tissue or any other immune response measurement. these prior studies must now be examined using more virulent prrsv strains, and we must delineate the amino acids responsible for immune evasion. two strains that we will develop deletion mutants for and test our hypothesis are type strains mn- and asian hp-prrsv. one can begin by deleting the nucleotides of these more virulent viruses that represent the same region as vr d . however, other regions of nsp may serve to evade immune responses. only the hypervariable regions (aa - ; aa - of vr- ) of the respective viruses have been shown to be mutable, so work should concentrate on those areas and make successive deletions based on nsp secondary structural predictions in the infectious clones of the parent viruses. once developed, these mutants will be used in in vivo studies with conventional and isolator piglets and in in vitro studies. since infected mq and pdcs fail to secrete ifna [ , ] , they would also poorly stimulate the antiviral state, so the first event is to compromise the first line of defense (innate immunity), which would allow spread of the virus. second, the ifna-deficient infected mq may then present to peripheral t cells in lymph nodes and without normal levels of il- from dcs and pdcs, would not favor a th profile and differentiation to ctls. thus, a major element in adaptive antiviral immunity is impaired. rather these events favor a th profile that might cause proliferation of cd helper cells at the expenses of tregs and cd ctls. the suggestion that infected mq and pdcs could induce apoptosis of thymocytes might indicate they could have the same effect on the peripheral t cell compartment. this could create a lymphopenic state. the increase in il- suggests suppression that could account for the increase in tregs [ ] and may be derived from mdsc [ ] . the elevation of tregs might be a delayed event, which would have been overlooked by sinkora et al. [ ] who worked only with isolator piglets. it is still difficult to accept that if adaptive immunity is forced to a th profile, it explains the polyclonal b cell activation and runaway b cell proliferation. the third event is that these infected mq, cdcs, and pdcs move to the developing thymus as apcs where they interact with dp thymocytes in the medulla that for reasons unknown, resulting in atrophy of dp thymocytes. together with help from thymic epithelial cells (nurse cells), prrsv may be therefore recognized as a self-antigen so surviving thymocytes could enter the periphery and recognize prrsv as self, as reported by the wieland for anti-golgi antibodies. in fact, the vasculitis that is a feature or arterivirus infections may be due to self-antibodies that coat the vascular as shown by lemke et al. [ ] . while the loss of dp thymocytes might lead to the loss of emerging t cells and in t cell lymphopenia, there is little evidence to support this. however, the quality and quantity of emerging cd , cd , and tregs might be altered as described above for the peripheral t cell compartment. without functional tregs, activated b cells may initially proliferate out of control as suggested from sinkora et al. [ ] . could an abundance of selfreactive th cells, some of which may crossreact with prrsv, be sufficient to drive rapid differentiation to plasma cells or perhaps il- from infected mq? alternatively, gc may not form or are abnormal, so there is little selection and the resultant plasma cells show little repertoire diversification (fig. ) and therefore poor affinity to viral epitopes so that few which are strongly virus specific. while prrsv-specific vn antibodies can control the peripheral spread of the virus, ctls are needed to eliminate virus-infected cells. in most viral infections, pdcs secrete il- that promotes th cells that can also activate mq to kill their intracellular parasites/viruses. if chronically infected tissues are infiltrated by mdsc, such t cells may be inhibited [ ] . in any case, since events in the thymus might reduce the number of peripheral th helpers, the infection would persist. perhaps of greatest effect is that if the number of virus-specific peripheral cd cells is low, there would be fever potential ctls to attack the infected mq. not trivial is that most scenarios described for ctl involve killing of epithelial cells like in siv. in the case of prrsv, it would involve the killing of infected mq. how easy is that? r d i fig. a antibody repertoire diversification measured as a repertoire diversification index (rdi). prrs = isolator piglets infected with prrsv; gf = germfree controls; c/ v = isolator piglets colonized with benign e. coli or infected with siv; pic = young, helminth-infected conventionally reared pigs (pic). b hydropathicity profiles calculated from sequence analysis of the hcdr region of ig from prrsv-infected piglets compared to pic animals (top) and compared to newborns (bottom). the numbers in parentheses indicate the number of sequences examined. hydrophobic hcdr regions i and ii are a feature of an undiversified pre-immune repertoire whereas region iii is characteristic of a diversified repertoire. from butler et al. [ ] adult model while what we have written above might explain the impact of prrsv on neonates, the literature we have reviewed suggests that a separate model is required for the situation in adult swine. while we may be dealing with one disease at the cellular/molecular level, we may be dealing with two disease models at the organismal level as regards the immunological perspective: one for adults and one for neonates. for all sorts of reasons, we believe that immune homeostasis is developing during the critical window of immune development (fig. ) when most piglets are prrsv infected. when an adult pig is considered, they have already properly developed their t cell repertoire and compartment. that means they have normal levels of cd cells that are potential ctls. likewise, they have th cells to form gc and tregs to prevent uncontrolled b cell expansion. as a result, adult animals mount effective immune responses with vn antibodies and ctls that resolve the disease, regardless of whether the innate response continues to be compromised since host protection is now heavily dependent on de novo adaptive immunity (fig. ) . in fetal and newborn piglets, innate immunity probably plays the major role in immune defense but after development of adaptive immunity, it become compensatory, not primary. this most likely explains why studies like those of robinson et al. [ ] show that prrs is resolved in adults, presumably by both vn antibodies and ctls. thus, the host adaptive response override the negative effect of prrsv on innate immunity in adult animals. this suggests that the principal impact of prrsv is on the fetus and the neonate during the critical window and is thereafter not a serious threat to adults. from the position of vaccinologists, it would seem wise to supply neonatal vaccinates with the ingredients that would promote immunocompetence as summarized in fig. . all of the events described for fetal/neonatal and adult animals are relevant to the common vaccine version of prrsv. however, is the effect of hp-prrsv merely a quantitative difference or does it have a qualitative effect? namely, does hp-prrsv primarily target the thymus so its greatest impact is on t cell cells development? since hp-prrsv has a greater effect than vaccine strain, prrsv on post-natal lymphopenia suggests that hp-prrsv also acts in the periphery. as previously described, failure to produce vn antibodies could be epitope dependent, so that differences between animals with and without vn antibodies could be epitope specificity, not a difference in affinity regardless of the mechanism of vn. the beauty of prrrv genetics is that a large number of variant are available and others can be engineered (''prrs the virus'' section). the availability and expertise of the investigators in this area provide an unusual opportunity for the experimental design of studies to determine how certain viral genes affect immune dysregulation and how epitopes differs in their ability to stimulate protective immune responses. testing the global hypothesis the working hypothesis offers numerous opportunities for testing. exactly, how each step in the scheme is tested is left to the ingenuity of the investigators. suffice to say there is a great need to know the cytokine, co-stimulatory molecule expression and signaling features of prrsvinfected macrophages when acting as apc versus noninfected macrophages both in thymus and in the periphery. do these ifna-impaired macrophages preferentially or inappropriately stimulate certain t cell subsets or do they promote differentiation of mdsc? using engineered mutants, one might determine what genetic features of the virus are responsible for any aberrant signaling. the core protein of hcv promotes mdsc differentiation [ ] . such ''defective mutants'' might also be the basis for future vaccines. likewise, it would be wise to know what signaling events are aberrant in thymocytes from prrsvinfected animals. as the runaway b cell proliferation still lacks an explanation, it would seem important to know whether infected macrophages can explain that part of the puzzle. testing for germinal center formation, antibody affinity and the complement dependence of vn are relatively straightforward. the issue of tregs should be resolved. are those with a suppressor phenotype functionally suppressive? could it be the lack 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envelope proteins in the backbone of genotype vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras the isolator piglet: a model for studying the development of adaptive immunity dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study negative impact of porcine reproductive and respiratory syndrome virus infection on the efficacy of classical swine fever vaccine the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load antibody repertoire development in fetal and neonatal pigs. xiii. ''hybrid vh genes'' and the preimmune repertoire re-visited antibody repertoire development in fetal and neonatal piglets. xxi. vh usage remains constant during development in fetal piglets and postnatally in pigs exposed to environmental antigen adaptation o a commercial elisa to determine the igg avidity in sweep experimentally and naturally infected with neospora caninum porcine igg: structure, genetics and evolution linkage haplotype for igg and iga subclass genes antibody repertoire development in fetal and neonatal piglets. xvii. igg subclass transcription revisited with emphasis on new igg resolution of an immunodiagnostic dilemma: heavy chain chimeric antibodies for species in which plasmacytomas are unknown induction of foot and mouth disease virus (fmdv) specific cytotoxic t cells killing by vaccination acknowledgments the authors acknowledge and thank nancy wertz for her help in assembly of the manuscript and to dr. eric nelson for scientific review of the manuscript. key: cord- -ofp tupv authors: kühl, a.; pöhlmann, s. title: how ebola virus counters the interferon system date: - - journal: zoonoses public health doi: . /j. - . . .x sha: doc_id: cord_uid: ofp tupv zoonotic transmission of ebola virus (ebov) to humans causes a severe haemorrhagic fever in afflicted individuals with high case‐fatality rates. neither vaccines nor therapeutics are at present available to combat ebov infection, making the virus a potential threat to public health. to devise antiviral strategies, it is important to understand which components of the immune system could be effective against ebov infection. the interferon (ifn) system constitutes a key innate defence against viral infections and prevents development of lethal disease in mice infected with ebov strains not adapted to this host. recent research revealed that expression of the host cell ifn‐inducible transmembrane proteins – (ifitm – ) and tetherin is induced by ifn and restricts ebov infection, at least in cell culture model systems. ifitms, tetherin and other effector molecules of the ifn system could thus pose a potent barrier against ebov spread in humans. however, ebov interferes with signalling events required for human cells to express these proteins. here, we will review the strategies employed by ebov to fight the ifn system, and we will discuss how ifitm proteins and tetherin inhibit ebov infection. the ebola virus (ebov) is an enveloped, negativestranded rna virus of the filovirus family. infection of humans with ebov causes ebola haemorrhagic fever. at present, neither antivirals nor vaccines are available to combat this lethal disease, and ebov is classified as a category a priority pathogen (niaid, ) . four ebov species have been defined (kuhn, ) , zaire ebolavirus (zebov), sudan ebolavirus (sebov), côte d'ivoire ebolavirus (ciebov) and reston ebolavirus (rebov), and a fifth species has been proposed, bundibugyo ebolavirus (bebov) (towner et al., ) . african fruit bats are a natural reservoir of the second filoviral genus, marburg virus (marv), and have also been proposed as a natural reservoir of ebov (leroy et al., ) . african fruit bats may transmit the virus to humans either directly or via an intermediate host (groseth et al., ) . outbreaks of summary zoonotic transmission of ebola virus (ebov) to humans causes a severe haemorrhagic fever in afflicted individuals with high case-fatality rates. neither vaccines nor therapeutics are at present available to combat ebov infection, making the virus a potential threat to public health. to devise antiviral strategies, it is important to understand which components of the immune system could be effective against ebov infection. the interferon (ifn) system constitutes a key innate defence against viral infections and prevents development of lethal disease in mice infected with ebov strains not adapted to this host. recent research revealed that expression of the host cell ifn-inducible transmembrane proteins - (ifitm - ) and tetherin is induced by ifn and restricts ebov infection, at least in cell culture model systems. ifitms, tetherin and other effector molecules of the ifn system could thus pose a potent barrier against ebov spread in humans. however, ebov interferes with signalling events required for human cells to express these proteins. here, we will review the strategies employed by ebov to fight the ifn system, and we will discuss how ifitm proteins and tetherin inhibit ebov infection. zebov, sebov, ciebov and bebov have been recorded in africa and were associated with case-fatality rates of up to % in larger outbreaks. rebov has been detected in swine in the philippines and is believed to be apathogenic for humans with an intact immune system (barrette et al., ; hartman et al., ) . the determinants accounting for the differential pathogenicity of the different ebov species are poorly understood. the ebov genome encodes three non-structural (sgp, ssgp, d-peptide, as discussed later) and seven structural proteins (fig. ) . the n protein (np), the l protein, viral protein (vp ) and vp are associated with the viral rna, forming a ribonucleoprotein (rnp) complex. the l protein has a polymerase function and is essential for genome replication and transcription, and these processes are regulated by vp and vp (dolnik et al., ) . the minor matrix protein, vp , contributes to nucleocapsid formation, while the major matrix protein, vp , facilitates budding of progeny particles from infected cells (huang et al., ; hartlieb and weissenhorn, ; dolnik et al., ) (fig. ) . the structural glycoprotein, gp , , mediates binding and infectious entry into host cells (kawaoka, ) . innate defences of the host are crucial to successfully fight ebov and other acute infections. the interferon (ifn) system is an integral part of the innate immunity against viral infections. sensor molecules of the ifn system recognize viral components and induce signalling that triggers expression of ifns (baum and garcía-sastre, ) . subsequent binding of ifn to ifn receptors on uninfected cells activates signal transducer and activator of transcription (stat)-dependent signalling, which commandeers the cell to express ifn-stimulated genes (isgs), which can function as antiviral effectors molecules (schindler et al., ; sadler and williams, ) . ebola virus strains lethal in humans were found to be unable to produce fatal disease in adult mice. however, when essential components of the ifn system were inactivated in mice, fatal disease was observed (bray, ) . similarly, adaptation of ebov to efficient replication in adult mice (bray et al., ) resulted in the generation of viruses with mutations allowing efficient interference with components of the murine ifn system . thus, the ifn system is generally capable of restricting filovirus spread and pathogenesis. however, several ebov proteins are well adapted to block processes essential for the establishment of a vigorous ifn response in human cells, potentially explaining why the ifn system frequently fails to protect humans from lethal ebov infection, as discussed below. the molecular pathways leading to expression of ifninduced antiviral effector molecules, and thus to the transition of cells into an antiviral state, are well characterized (baum and garcía-sastre, ) . however, the antiviral effector molecules induced by ifn and the molecular mechanisms underlying their antiviral action are incompletely understood. recent, groundbreaking studies attempted to close this gap (schoggins et al., ) and identified novel isgs, among them the tetherin and ifninduced transmembrane proteins (ifitms) (neil et al., ; van damme et al., ; brass et al., ). tetherin exhibits an unusual topology and restricts release of several enveloped viruses and filovirus-like particles from infected cells, while ifitm proteins inhibit infection by filoviruses and other enveloped viruses at the stage of viral entry, as discussed below. in the present review, we will summarize current knowledge on ebov interference with the ifn system. in addition, we will discuss how tetherin and ifitms block filovirus infection. the interferon system constitutes a major innate defence against infections by viruses and other pathogens. the components and signalling pathways of the ifn system have been described in several recent reviews (garcía-sastre and biron, ; baum and garcía-sastre, ; liu et al., ) and are only briefly summarized here. three classes of ifns have been defined according to the receptors bound by these cytokines. type i ifn, including ifna and ifnb, are produced by many cell types as a direct result of viral infection. ifnc, the only type ii ifn, is generated by activated t cells and nk cells. type iii ifns, which include ifnk - , are incompletely characterized, but are believed to regulate the antiviral response. the ifn system integrates two major sensor and signalling networks. in cells exposed to viruses, receptors for pathogen-associated molecular patterns sense the presence of the invading pathogens. these receptors are located in the cytoplasm, like retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated gene (mda- ), or in the extracytoplasmic space, like toll-like receptor (tlr)- and tlr- / / , and, upon pathogen recognition, induce ifn regulatory factor (irf)- -and irf- dependent signalling cascades that lead to the expression of type i ifns (fig. ) . secreted type i ifns then bind to cells expressing the type i ifn receptor, which consists of two subunits, ifna receptor (ifnar ) and ifnar . ligand binding to ifnar and ifnar triggers receptor dimerization and signalling. stat and stat are integral components of the signalling pathway induced by ifnars (fig. ) . homodimers of stat bind to ifncactivated sites (gas), while stat /stat heterodimers recognize ifn-stimulated response elements, resulting in the transcription of isgs, many of which have antiviral activity, like the well-characterized myxovirus resistance guanosine triphosphatases (mx gtpases) (haller et al., ; sadler and williams, ) . however, the full spectrum of isgs has only been recently characterized (schoggins et al., ) , and novel isgs that target discrete steps in the viral life cycle, like ifitms and tetherin, have been identified, as discussed below. vp is the smallest of the seven ebov-encoded structural proteins and constitutes the minor matrix protein relative to the major matrix protein vp (han et al., ) . it is required for the assembly of fully functional nucleocapsids (huang et al., ; hoenen et al., ) and contributes to the budding of virus-like particles (vlps) (han et al., ; licata et al., ) . furthermore, vp can shut down the host's ifn-a/b and ifn-c recognition of viral pathogen-associated molecular patterns (pamps) by prrs (purple) in infected cells initiates a signalling cascade including adaptor molecules (blue) and kinases (green), which activate transcription factors (red) that induce the expression of type i ifns and isgs (left panel). binding of ifn-a/b and ifn-c to their receptors induces phosphorylation (by jak /tyk ), dimerization and nuclear translocation of stat transcription factors, which induce the expression of several isgs such as tetherin, ifitm, pkr and others (right panel). ifn, interferon; tlr, toll-like receptor; rig-i, retinoic acid-inducible gene i; mda- , melanoma differentiation-associated gene ; myd , myeloid differentiation primary response protein ; trif, tir domain-containing adaptor-inducing ifn-b; ips- , ifn-b promoter stimulator ; traf , tumour necrosis factor receptor-associated factor ; irak, interleukin- receptor-associated kinase; ikke, ijb kinase e; tbk , tank-binding kinase ; irf, ifn regulatory factor; nf-jb, nuclear factor j light chain enhancer of activated b-cells; isg, ifn-stimulated gene; ifnar, ifn-a receptor; ifngr, ifn-c receptor; jak , janus-activated kinase ; prr, pathogen recognition receptor; tyk , tyrosine kinase ; stat, signal transducer and activators of transcription. a. kü hl and s. pö hlmann response to viral infection (reid et al., ) . for this, vp inhibits the nuclear translocation of the transcription factor stat (reid et al., ) (fig. ) , a key component of the ifn-induced signalling pathway controlling the expression of isgs, as discussed below. upon activation, stat is tyrosine-phosphorylated (py-stat ) and either heterodimerizes with py-stat for type i ifn signalling or homodimerizes with py-stat for type ii ifn signalling. the dimerization leads to the exposition of a nuclear localization signal (nls) in stat , which is recognized by the nuclear import adaptor protein importin a, specifically importin a , a and a (karyopherin a , a , a ; np- subfamily of importins). importin a is then bound by the nuclear import factor importin b (sekimoto et al., ) , which facilitates import of stat dimers into the nucleus ( fig. ) . however, in the presence of vp , which like stat binds to importins a , a and a (reid et al., (reid et al., , , the recognition of the py-stat nls by these importins is disrupted, and py-stat is not imported into the nucleus. blockade of nuclear transport of py-stat is not because of a global inhibition of nuclear import, as vp does not bind to importin a (karyopherin a ; rchi subfamily), a or a (karyopherin a , a ; qip subfamily) (reid et al., ) and does not affect nuclear import mediated by these factors. mutational analyses revealed that vp binds to amino acids - in importin a , which constitute armadillo repeat (arm) and comprise part of the binding site for py-stat (reid et al., (reid et al., , . predictions on vp structure offer insights into the mechanisms potentially underlying vp inhibition of importin a. ). thus, it was suggested that vp , like importin a, importin b and exportins (nuclear export factors important for recycling of importin a to the cytoplasm), belongs to the arm family of proteins. furthermore, it was posited that binding of vp to importin a mimics that of exportin, which also targets arm repeat . as a consequence, vp may block release of the autoinhibitory nls of importin a, and this would inhibit binding of other proteins to the nls binding site of importin a, including py-stat . alternatively, targeting the arm repeat of importin a by vp may block binding of py-stat because of competitive inhibition . in vp , the amino acid residues and - were found to be crucial for the inhibition of ifn-b-induced signalling and py-stat accumulation in the nucleus (mateo et al., ) . the lack of inhibition of ifn-b-induced signalling by a vp mutant with amino acid exchanges at these positions correlated with absence of importin a binding, highlighting that binding of vp to importin a is essential for ifn antagonism (mateo et al., ) . the ifn system can protect immune-competent mice from lethal ebov infection (bray, ; mahanty et al., ) . adaptation of zebov to lethal infection of mice was associated with mutations in vp and np . however, both wild-type vp and vp of the mouse-adapted (ma) strain were able to bind to human and mouse np- importins and to disrupt the interaction with py-stat (reid et al., ) . similar findings were documented for vp of rebov, which is believed to be non-pathogenic for humans, and it was shown that zebov, rebov and ma vp can suppress ifn-b-induced gene expression (reid et al., ) . thus, alterations in vp interference with the ifn response might not account for the acquisition of virulence of ma zebov in mice and for the lack of virulence of rebov in humans, respectively. the ebov-encoded protein vp fulfils several functions important for viral amplification. as essential polymerase cofactor, vp is involved in the formation of the ebov rnp complex and therefore crucial for transcription and viral replication (mühlberger et al., ) . furthermore, it is required for nucleocapsid assembly (huang et al., ) . consequently, interference with expression of vp attenuates viral growth and virulence (enterlein et al., ; hartman et al., a; prins et al., b) . additionally, vp blocks multiple steps of the innate antiviral defence (fig. ) , such as the signalling pathways leading to the expression of type i ifns and type i ifn-induced genes, double-stranded (ds) rna-dependent protein kinase (pkr) translation inhibition and rna silencing cárdenas et al., ; feng et al., ; haasnoot et al., ) . the key role of vp in the defence against innate immunity is exemplified by studies demonstrating that vp mutated viruses are unable to block the ifn-dependent induction of antiviral factors (cárdenas et al., ; hartman et al., a,b) . moreover, vp impairs the maturation of dendritic cells and thus prevents the establishment of an adaptive immune response (jin et al., ) , an important feature of ebov haemorrhagic fever pathogenesis (hartman et al., a) . the ability of vp to antagonize the type i ifn response was identified by basler et al. ( ) who showed that vp can functionally replace the influenza a virus (fluav) non-structural protein (ns ) protein, a viral type i ifn antagonist. vp was found to inhibit the virus-induced activation of type i ifn promoters by targeting the transcription factor irf- . in contrast, activation of ifn promoters through exogenous ifn was not modulated by vp . upon activation by viral infection, irf- is phosphorylated by cellular kinases such as tank-binding kinase (tbk- ) and ijb kinase epsilon (ikke) (prins et al., ). subsequently, ifr- dimerizes and translocates into the nucleus where it activates transcription of isgs (fig. ) . vp blocks irf- phosphorylation and therefore subsequent nuclear accumulation of irf- cárdenas et al., ) . for this, vp binds to ikke and tbk- and inhibits substrate binding and kinase activity (prins et al., ) . mutational studies revealed that a n-terminal coiledcoil domain in vp mediates homooligomerization, which was found to be essential for ifn antagonism exhibited by the c-terminus of the protein (reid et al., ) . several amino acid residues (r , k , r , k , r ) in the central basic patch of the c-terminal ifn inhibitory domain (iid) of vp were shown to be required for inhibition of virus-induced activation of ifn-regulated promoters and production of ifn-b (cárdenas et al., ; prins et al., b) , with r being of particular importance (hartman et al., ; cárdenas et al., ) . mutations in the iid attenuate viral growth in cell lines and the c-terminal basic patch residues are highly conserved between filoviruses, highlighting their importance for the function of vp (hartman et al., ; prins et al., b) . notably, fig. . interference of ebola virus vp with the expression of ifn and isgs. transcription of the ebov genome results in the production of dsrna intermediates, which are recognized by the cytoplasmic pathogen recognition receptor (prr) rig-i. single-stranded rna is recognized by the endosomal prrs tlr- and tlr- . activation of these prrs induces signalling cascades leading to the expression of type i ifns. in addition, dsrna is recognized by the rna interference machinery that facilitates rna degradation and thereby suppresses the expression of viral genes. vp is able to block several of the above-described processes. because of its dsrna-binding domain, it is able to sequester dsrna from recognition by rig-i and to protect it from degradation through the rna-induced silencing complex. furthermore, it inhibits the phosphorylation of irf- and therefore irf- dimerization, translocation into the nucleus and the expression of ifn-induced genes. in addition, vp is able to induce su-moylation of irf- and irf- , which interferes with dimerization and nuclear translocation. ifn, interferon; tlr, toll-like receptor; trbp, hiv- trans-activation response rna-binding protein; pact, protein activator of pkr; rig-i, retinoic acid-inducible gene i; myd , myeloid differentiation primary response protein ; trif, tir domain-containing adaptor-inducing ifn-b; ips- , ifn-b promoter stimulator ; traf , tumour necrosis factor receptor-associated factor ; irak, interleukin- receptor-associated kinase; ikke, ijb kinase e; tbk , tank-binding kinase ; irf, ifn regulatory factor; isg, ifn-stimulated gene; pkr, dsrna-dependent protein kinase; ifitm, ifn-induced transmembrane protein. the disruption of the irf- inhibitory activity of vp does not influence the function of vp in viral replication and transcription , as amino acid residues essential for polymerase cofactor function of vp differ from those required for ifn antagonism (prins et al., a) . sequences in the c-terminus of vp resemble the dsrna-binding domain of fluav ns , which is important for ifn antagonism (donelan et al., ; hartman et al., ) . indeed, vp is able to bind dsrna, but not single-stranded (ss) rna or dsdna and mutations in the central basic patch of iid, which abrogate ifn antagonism were found to be incompatible with dsrna binding (cárdenas et al., ; leung et al., ; prins et al., b) . the structure of vp iid in complex with dsrna has been determined at the atomic level (kimberlin et al., ; leung et al., ) , and the protein was found to display a unique fold compared with known dsrna-binding proteins, including that of fluav ns (leung et al., ) . interestingly, vp displays a bimodal dsrna-binding strategy. upon dsrna recognition, vp builds asymmetric dimers; one monomer binds the dsrna phosphate backbone, whereas the other binds terminal nucleotides of the dsrna molecule (kimberlin et al., ; leung et al., ) . this dsrna-binding mode of vp dimers seems to mimic that of rig-i, a key cellular sensor of dsrna, and vp and rig-i might occupy overlapping binding sites on dsrna (kimberlin et al., ; leung et al., ) . these findings suggest that vp may sequester dsrna from recognition by rig-i and potentially other dsrna-binding molecules such as mda- or dicer (kimberlin et al., ; . in addition, differences in the structural organization and dsrna binding of zebov and rebov vp were identified, which might contribute to the differential pathogenicity of these viruses in humans (kimberlin et al., ; leung et al., ) , although it needs to be noted that both viruses are highly pathogenic in macaques. vp promotes sumoylation of ifr- via pias ifr- but not irf- is critical for the production of type i ifns (honda et al., ) , and a recent study shows that vp inhibits irf- by promoting its sumoylation (chang et al., ). thus, vp forms a complex with irf- and pias (the small ubiquitin-like modifier (sumo) e ligase protein inhibitor of activated stat) and promotes irf- sumoylation via pias (chang et al., ) (fig. ) . this study also revealed that vp displays an ifn inhibitory activity independent of its ability to recognize dsrna, and this activity was mapped to the n-terminus, which is essential for interactions with irf- and pias . the interferon-inducible dsrna-dependent protein kinase (pkr) recognizes dsrna produced in the context of infection by rna viruses and dna viruses encoding opposite open reading frames from which overlapping mrna sequences are generated (george et al., ). subsequently, it phosphorylates the eukaryotic translation initiation factor a (eif a), which results in the arrest of protein synthesis from cellular and viral mrnas (garcía et al., ) . in the context of ebov infection, it was noted that the expression of vp and eif- a phosphorylation inversely correlate and that autophosphorylation of pkr is disrupted in the presence of vp (feng et al., ) . thus, vp inhibits pkr activation (fig. ) . in fact, zebov was shown to not only block, but to reverse activation of pkr (schumann et al., ). feng et al. ( suggested that vp inhibition of pkr might not involve interactions of these proteins and might not depend on dsrna binding by vp . instead, a role of the n-terminal sequences in vp was suggested. subsequent work by schumann et al. ( ) indicates that mutations in the iid can be sufficient to relief the block to pkr activation imposed by vp and that prk antagonism by vp is therefore functionally separate from dsrna binding and irf- inhibition. vp inhibits rnai by targeting the components of the rna-induced silencing complex rna interference allows cells to specifically recognize and destroy viral rna and thus to combat viral infection. the observation that several viruses encode rnai silencing suppressors (rss), like the ns protein of fluav or the tat protein of the human immunodeficiency virus type (hiv- ), underlines the potency of this cellular defence mechanism (bivalkar-mehla et al., ). haasnoot et al. ( ) demonstrated that ebov vp is an rss, which inhibits shrna-mediated suppression of reporter gene expression and rescues production of tat-defective hiv- from suppression by rnai. the rss activity of vp was dependent on its dsrna-binding capability (haasnoot et al., ) , suggesting that vp may sequester and protect dsrna from recognition by dicer, a central molecule of the rnai pathway (fig. ) . however, a subsequent study demonstrated that vp interacts with components of the rna-induced silencing complex (risc) independent of the presence of sirna (fabozzi et al., ) . specifically, vp was found to interact with the two dsrna-binding proteins hiv- trans-activation response rna-binding protein (trbp) and protein activator of pkr (pact), which are part of the risc complex, but not directly with dicer (fabozzi et al., ) . in addition, evidence was presented that also vp and vp act as rss, and vp like vp was shown to bind to components of the risc complex, while no such interactions were seen for vp , which might employ a different strategy to inhibit rnai (fabozzi et al., ) . the tetherin protein (hm . , bst- , cd ) was identified as a type i interferon-inducible host cellular factor, which restricts release of progeny hiv- particles from infected cells (neil et al., ; van damme et al., ) . tetherin is counteracted by the hiv- accessory protein vpu (viral protein u), which allows efficient hiv- release form tetherin-expressing cells (neil et al., ; van damme et al., ) . the antiviral action of tetherin is not limited to hiv- ; several recent studies found that tetherin restricts release of vlps and progeny particles of several enveloped viruses, including members of the retroviridae, arenaviridae, paramyxoviridae, filoviridae, rhabdoviridae and herpesviridae families (jouvenet et al., fig. . countermeasures of ebola virus against ifn-induced antiviral proteins. ebola virus infection commences with viral uptake and transport of virions into endosomal compartments, where the ebov-gp is activated by cathepsins. subsequently, gp mediates fusion of the viral envelope with the endosomal membrane, allowing release of the viral genome into the cytosol. subsequently, the viral genome is transcribed into mrnas and replicated. viral proteins are translated and transported to the cellular membrane for assembly of new particles, which eventually bud from the host cell membrane. virus entry is inhibited by the ifn-induced ifitm proteins. the exact step that is blocked by ifitms remains to be defined. the protein kinase r, which senses dsrna intermediates of viral replication, phosphorylates the translation initiation factor eif a, which results in the arrest of translation of viral and cellular mrnas. the tetherin protein tethers budding ebov-like particles to the cell surface and is counteracted by ebov-gp. gp, glycoprotein; vp, viral protein; l, rna-dependent rna polymerase; np, nucleoprotein; ifitm, ifn-induced transmembrane protein; pkr, dsrna-dependent protein kinase; ebov-gp, ebola virus glycoprotein. ; mansouri et al., ; sakuma et al., ; pardieu et al., ; radoshitzky et al., ; weidner et al., ; watanabe et al., ; yondola et al., ) (fig. ) . the broad spectrum antiviral action of tetherin is intimately linked to its unusual structural organization. tetherin encodes a short cytoplasmic domain at its n-terminus, followed by a single transmembrane (tm) domain, an extracellular coiled-coil region and a c-terminal glycosylphosphatidylinositol (gpi) anchor (kupzig et al., ) . thus, the protein spans the membrane twice and is able to insert one end into the cellular membrane and the other end into the viral envelope. by this mechanism, tetherin retains budding virions at the surface of the infected cell and inhibits their transmission to new target cells (perez-caballero et al., ; hammonds et al., ). an artificially constructed tetherin, which displays the same topology as the original molecule, but is assembled from completely unrelated sequences, is able to inhibit virus spread as well, confirming that it is tetherin's unusual architecture that is responsible for tethering virions to cells (perez-caballero et al., ) . antagonism of tetherin is not limited to vpu. hiv- uses its envelope (env) protein to counteract tetherin (le tortorec and neil, ) , while the simian immunodeficiency virus (siv) uses either the accessory protein nef or vpu or env, depending on the origin of the virus (gupta et al., ; sauter et al., ) . the kaposi's sarcoma-associated herpes virus encodes the e ubiquitin ligase k as a tetherin antagonist (mansouri et al., ) . for the ebov, the gp , serves as tetherin antagonist (kaletsky et al., ). this ability of gp , is conserved between the gps of the different ebov species (zaire, sudan, côte d'ivoire and reston), the proposed species bebov and the second filoviral genus marv (kaletsky et al., ; lopez et al., ; radoshitzky et al., ; kühl et al., a,b) . the ebov glycoprotein and hiv- vpu employ different strategies to counteract tetherin the ebov glycoprotein (ebov-gp , ) is the only viral surface protein and mediates viral entry into the host cell that requires binding of gp , to the endosomal membrane protein niemann-pick c (npc ) (carette et al., ; cô té et al., ) . ebov-gp , is synthesized as a precursor protein, which is post-translationally cleaved by subtilisin-like proteases into its two subunits gp and gp (volchkov et al., ) . the large and heavily glycosylated, extracellular domain gp mediates attachment to the host cell; the smaller tm unit gp facilitates fusion of the viral envelope with the membrane of host cell endosomes (takada et al., ; wool-lewis and bates, ) . a recent study demonstrated that gp , also inactivates one of the cell's innate defences against infection, the tetherin protein (kaletsky et al., ) . vpu allows hiv- to evade tetherin by mediating cell surface downregulation and relocalization of tetherin into intracellular compartments (van damme et al., ) . in addition, vpu facilitates degradation of tetherin in lysosomal or proteasomal compartments goffinet et al., goffinet et al., , mangeat et al., ; mitchell et al., ; dubé et al., ) . in contrast, no evidence for downregulation, relocalization away from the cell surface or degradation of tetherin was observed in the presence of ebov-gp , or marv-gp , (kaletsky et al., ; lopez et al., ; radoshitzky et al., ; kühl et al., a) . furthermore, ebov-gp , is active against tetherin homologues from different monkeys, in accordance with the ability of ebov to infect several non-human primates (kühl et al., a) . in contrast, tetherin antagonism by vpu is limited to tetherin of human, chimpanzee and gorilla origin, and these species are infected by hiv- and vpu encoding siv, respectively (goffinet et al., ; jia et al., ; mcnatt et al., ). thus, vpu and ebov-gp , have a different specificity for tetherin and employ different strategies to counteract tetherin. tetherin interacts with the gp subunit of the ebov-gp , four different proteins are expressed from the ebov-gp gene: (i) the primary transcript from the gp gene is sgp, a soluble, secreted form of the gp (volchkov et al., ; sanchez et al., ) , which has an anti-inflammatory property (wahl-jensen et al., ) . (ii) a soluble d-peptide is released upon proteolytic processing of sgp by furin (volchkova et al., ) . (iii) the full-length, membrane-bound form gp , , which is incorporated into the virions, is produced by rna editing of the primary transcript (volchkov et al., ; sanchez et al., ) . (iv) furthermore, a small soluble gp (ssgp) has recently been identified, but the function of this protein is at present unknown (mehedi et al., ) . in addition, the tnfa converting enzyme induces shedding of gp , d from the surface of infected cells by cleaving gp , near the tm domain (dolnik et al., ) . all different forms of the gp might play a role in combating the host's immune system as, for example the shedded form of gp might be able to act as a decoy for neutralizing antibodies (dolnik et al., ) . however, neither sgp nor gp , d are able to counteract tetherin (kaletsky et al., ). in addition, a gp , mutant that is retained in the er failed to counteract tetherin, suggesting that the correct localization of the ebov-gp , at sites of viral budding is crucial for tetherin antagonism (kaletsky et al., ) . indeed, because of the expression of the gpi-anchor, tetherin is localized to lipid rafts, which are used by hiv- and ebov as platforms for budding (ono and freed, ; bavari et al., ) . vpu and tetherin interact via their tm domains, and the interaction is critical for tetherin antagonism . in contrast, the sequences of tetherin's cytoplasmic tail (ct) and tm domain do not determine counteraction by ebov-gp , . thus, tetherin chimeras in which the tm region and the n-terminus of tetherin were exchanged against similar domains of the transferrin receptor type (tfr) displayed antiviral activity and were counteracted by ebov-gp , (lopez et al., ) . nevertheless, zebov-gp , is able to efficiently coimmunoprecipitate human tetherin (kaletsky et al., ) , and the gp subunit of ebov-gp , was shown to be critical for the interaction (kühl et al., a) . which domain of tetherin interacts with zebov-gp remains to be determined, and, because of topological constraints, the tm unit and the ct are interesting candidates. however, the tm and ct have been shown to be irrelevant for tetherin counteraction by zebov-gp , (lopez et al., ) , and the importance of tetherin binding for tetherin inhibition by ebov-gp , remains to be determined. the ebov-gp , might relocalize tetherin within the plasma membrane or interfere with the structural integrity of tetherin tetherin and vp , which is essential for ebov budding, colocalize at the plasma membrane and release of vp based vlps is inhibited by tetherin (jouvenet et al., ) . it is thus possible that the ebov-gp , rescues the block to particle release by interfering with the integrity of tetherin at filoviral budding sites (kühl et al., a) . alternatively, the extracellular, heavily glycosylated ebov-gp subunit might interfere with the formation of the 'tetherin-clamp' between the cellular and the viral membrane, because of steric hindrance. finally, ebov-gp , might relocalize tetherin within the plasma membrane, thereby excluding it from membrane domains used by ebov for budding. indeed, it was observed that tetherin is excluded from plasma membrane sites positive for gp , in ebov-infected cells (radoshitzky et al., ) , lending support to the idea that ebov-gp , blocks tetherin's antiviral action by inducing its mislocalization within the plasma membrane. regardless of the mechanism underlying tetherin counteraction by ebov-gp , , it remains to be determined whether endogenous tetherin can reduce ebov release from infected cells, as modest effects were observed in one study (kühl et al., a) but not in another applying a substantially higher multiplicity of infection (moi) (radoshitzky et al., ) , and might thus restrict viral spread in the infected host. in addition, it will be interesting to determine whether african fruit bats, the potential natural reservoir of ebov, encode a tetherin-like protein and whether this tetherin homologue restricts ebov spread. the ifitm , and were recently discovered as inhibitors of host cell entry of several enveloped viruses, including ebov (fig. ) . ifitms , and are ubiquitously expressed in human cells and tissues upon exposure to type i (a) and type ii (c) ifn, and homologous proteins are present in many vertebrates (siegrist et al., ) . ifitm proteins were shown to play a role in early development, cell adhesion and control of cell growth (siegrist et al., ) . their antiviral activity was discovered in a sirna screen designed to identify host cell factors modulating fluav infection (brass et al., ). ifitm was identified as a potent inhibitor of host cell entry of fluav and members of the family flaviviridae, west nile virus and dengue virus serotype , but not hepatitis c virus (brass et al., ; jiang et al., ) , and the induction of ifitm expression was shown to be largely responsible for the blockade to fluav entry imposed by treatment of target cells with ifns (brass et al., ). ifitm and were also shown to restrict viral infection although to a lower extent, and the antiviral activity of ifitms was conserved between human proteins and murine orthologues (brass et al., ) . thus, ifitms are novel ifn-induced antiviral effector proteins, which could modulate viral spread in humans and animals. ifitms restrict viral entry into host cells while antiviral activity of ifitms was initially reported for influenza and flaviviruses (brass et al., ; jiang et al., ) , subsequent studies showed that ifitms inhibit entry of additional enveloped viruses such as vesicular stomatitis indiana virus (vsiv), severe acute respiratory syndrome coronavirus (sars-cov) and filoviruses huang et al., ) , which, very much like fluav and flaviviruses, depend on endo-/ lysosomal acidic ph for host cell entry. inhibition of filoviruses and sars-cov was demonstrated employing lentiviral vectors pseudotyped with the respective viral gps and with replication competent virus (huang et al., ) , but the inhibitory efficiency was modest. in contrast to fluav, ifitm showed the most prominent antiviral effects against replication competent ebov and marv, while inhibition by ifitm was less efficient (huang et al., ) . entry of marv and ebov was reduced by the expression of several ifitm orthologues of mouse and chicken, although with different efficacies compared to the human proteins (huang et al., ) . depletion of ifitm by shrna was sufficient to rescue entry of fluav pseudotypes, whereas a knockdown of both, ifitm and ifitm , was required to increase entry of ebov and marv pseudotypes (huang et al., ) . finally, a different study also detected inhibition of hiv- infection (lu et al., ) , which does not depend on low ph, and it is at present unclear which structure or process shared by the viruses listed above for host cell entry is targeted by ifitms. determinants of the antiviral activity of ifitm proteins domains and modifications important for antiviral activity of the ifitms have been identified, although most studies were not conducted in the context of filovirus infection. abrogation of s-palmitoylation of ifitm by mutation of crucial cysteine residues inhibited ifitm clustering in membrane compartments as well as the antiviral activity of ifitm against fluav (yount et al., ) . the sites of s-palmitoylation are highly conserved in members of the ifitm protein family, suggesting a general role in the antiviral activity. in addition, the sequence of the n-terminus is distinct for the different ifitms as ifitm is lacking an n-terminal amino acid stretch present in ifitm and . part of the n-terminus might be crucial for their different antiviral activity (siegrist et al., ) . indeed, the characterization of ifitm /ifitm chimeras revealed that sequences within the n-and c-terminus of ifitm are important for antiviral activity against vsiv . furthermore, lu et al. ( ) could show that only ifitm- and ifitm- inhibit the hiv- life cycle at the step of viral entry, and that deletion of the n-terminal region abrogated this ability. ifitm , in contrast, needs an intact intracellular domain to inhibit hiv- replication, whereas the n-and c-terminus are dispensable (lu et al., ) . what is known about the mechanism underlying ifitm dependent inhibition of host cell entry of ebov and other viruses? one possibility is that ifitms interfere with receptor expression. however, ifitm proteins do not interfere with the level of surface expression of sialic acids and ace , the receptors for fluav and sars-cov, respectively (brass et al., ; huang et al., ) . ifitm expression is compatible with fluav access to low ph compartments, and inhibition of sars-cov by ifitms could be rescued by forcing the virus to fuse with the plasma membrane instead of an internal membrane (huang et al., ) . thus, viruses might reach internal compartments in ifitm expressing cells in which fusion of viral and compartment membrane could normally occur, but membrane fusion might be blocked by ifitms. one possibility could be that ifitms interfere with the activity of cathepsins, ph-dependent endo-/lysosomal proteases essential for proteolytic activation of sars-cov and filoviruses in vitro (chandran et al., ; simmons et al., ) . no appreciable decrease of cathepsin activity was observed in ifitm expressing cells (huang et al., ) . the endosomal membrane protein npc has recently been reported as an essential host cell factor for ebov entry (carette et al., ; cô té et al., ) . according to the model of cô té et al., npc expression or activity is required for endosomal fusion after cathepsin-mediated processing of gp , . it is conceivable that ifitms interfere with npc expression or activity. furthermore, it is possible that a step in the filovirus life cycle different from membrane fusion could be affected by ifitms, as it was reported that ifitm and inhibit hiv- entry, whereas ifitm acts later in the viral life cycle by suppressing gag translation (lu et al., ) . thus, the mechanisms underlying inhibition of filoviruses and other viruses remain to be defined, including the possibility that ifitms require cellular cofactors to exert their antiviral effects. in sum, ifitms inhibit filovirus entry into host cells, and the level of constitutive ifitm expression might shape the choice of early target cells in filovirus infection. further research is needed to define the basal expression of ifitms in filovirus target cells and to elucidate the mechanism by which the different ifitm proteins restrict filovirus infection. the ifn system can potently restrict ebov spread and pathogenesis in infected mice, but is tuned down by viral proteins in infected humans. vp and vp play key roles in suppressing the ifn response by preventing nuclear translocation of stat and by targeting irf- and irf- , respectively. tetherin and ifitm proteins are novel isgs, which could suppress ebov infectious entry into target cells (ifitms) and release of ebov particles from infected cells (tetherin). efficient suppression of ebov release has so far only been demonstrated with vlps and remains to be shown with infectious ebov. for this, the sequences in gp , , which facilitate tetherin antagonism, need to be identified and altered, which should render ebov susceptible to inhibition by tetherin. the gp subunit is an excellent candidate for these endeavours (kühl et al., a) . the mechanism underlying ebov inhibition by ifitms is at present unknown, and its elucidation might require the identification of potential interaction partners of gp , and ifitms in host cell endosomes. in addition, it will be interesting to determine to which extent basal (in the absence of ifn) expression of ifitms and tetherin impact ebov spread. a recent study demonstrated that basal tetherin expression is broader than initially appreciated and raised doubts concerning the sole regulation of tetherin expression by ifns (erikson et al., ) . moreover, the role of the ifn system in the ebov infection of the potential reservoir host, fruit bats (leroy et al., ) , is of high interest. thus, ebov infection of these animals does not seem to induce disease (swanepoel et al., ; hayman et al., ) , and it is tempting to speculate that the bat ifn system efficiently controls viral replication. finally, the interesting insights into ebov interference with the ifn system open new avenues to the development of filovirus inhibitors. proof of concept comes from studies with hiv- , which demonstrated that stabilization of antiviral effectors molecules of the ifn system or inhibition of viral ifn antagonists by small molecules can suppress viral spread (cen et al., ; jiang et al., ) . the authors have no potential conflicts to declare. the ebola virus vp protein functions as a type i ifn antagonist the ebola virus vp protein inhibits activation of interferon regulatory factor induction of type i interferon by rna viruses: cellular receptors and their substrates differential recognition of viral rna by rig-i lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses viral rna silencing suppressors (rss): novel strategy of viruses to ablate the host rna interference (rnai) defense system the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus the role of the type i interferon response in the resistance of mice to filovirus infection a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever ebola virus vp protein binds doublestranded rna and inhibits alpha/beta interferon production induced by rig-i signaling ebola virus entry requires the cholesterol transporter niemann-pick c small molecular compounds inhibit hiv- replication through specifically stabilizing apobec g endosomal proteolysis of the ebola virus glycoprotein is necessary for infection ebola zaire virus blocks type i interferon production by exploiting the host sumo modification machinery small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ectodomain shedding of the glycoprotein gp of ebola virus filoviruses: interactions with the host cell a recombinant influenza a virus expressing an rna-bindingdefective ns protein induces high levels of beta interferon and is attenuated in mice vpu directs the degradation of the human immunodeficiency virus restriction factor bst- /tetherin via a {beta}trcp-dependent mechanism antagonism of tetherin restriction of hiv- release by vpu involves binding and sequestration of the restriction factor in a perinuclear compartment molecular determinants of ebola virus virulence in mice vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice in vivo expression profile of the antiviral restriction factor and tumor-targeting antigen cd /bst- /hm . /tetherin in humans ebolavirus proteins suppress the effects of small interfering rna by direct interaction with the mammalian rna interference pathway the vp protein of ebola virus inhibits the antiviral effect mediated by double-stranded rna-dependent protein kinase pkr the dsrna protein kinase pkr: virus and cell control type interferons and the virus-host relationship: a lesson in detente tipping the balance: antagonism of pkr kinase and adar deaminase functions by virus gene products hiv- antagonism of cd is species specific and involves vpu-mediated proteasomal degradation of the restriction factor antagonism of cd restriction of human immunodeficiency virus type (hiv- ) particle release and depletion of cd are separable activities of hiv- vpu the ecology of ebola virus simian immunodeficiency virus envelope glycoprotein counteracts tetherin/bst- /cd by intracellular sequestration the ebola virus vp protein is a suppressor of rna silencing interferoninduced mx proteins in antiviral host defense immunoelectron microscopic evidence for tetherin/bst as the physical bridge between hiv- virions and the plasma membrane biochemical and functional characterization of the ebola virus vp protein: implications for a role in virus assembly and budding filovirus assembly and budding a c-terminal basic amino acid motif of zaire ebolavirus vp is essential for type i interferon antagonism and displays high identity with the rna-binding domain of another interferon antagonist, the ns protein of influenza a virus reverse genetic generation of recombinant zaire ebola viruses containing disrupted irf- inhibitory domains results in attenuated virus growth in vitro and higher levels of irf- activation without inhibiting viral transcription or replication inhibition of irf- activation by vp is critical for the high level of virulence of ebola virus whole-genome expression profiling reveals that inhibition of host innate immune response pathways by ebola virus can be reversed by a single amino acid change in the vp protein ebola and marburg hemorrhagic fever long-term survival of an urban fruit bat seropositive for ebola and lagos bat viruses infection of naive target cells with virus-like particles: implications for the function of ebola virus vp irf- is the master regulator of type-i interferon-dependent immune responses the assembly of ebola virus nucleocapsid requires virion-associated proteins and and posttranslational modification of nucleoprotein distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus. plos pathog species-specific activity of siv nef and hiv- vpu in overcoming restriction by tetherin/bst identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections the vp protein of ebola virus impairs dendritic cell maturation induced by virus and lipopolysaccharide broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein how ebola virus infects cells ebolavirus vp uses a bimodal strategy to bind dsrna for innate immune suppression the ebola virus glycoprotein and hiv- vpu employ different strategies to counteract the antiviral factor tetherin comparative analysis of ebola virus glycoprotein interactions with human and bat cells filoviruses. a compendium of years of epidemiological, clinical, and laboratory studies bst- /hm . is a raft-associated apical membrane protein with an unusual topology antagonism to and intracellular sequestration of human tetherin by the human immunodeficiency virus type envelope glycoprotein fold prediction of vp protein of ebola and marburg viruses using de novo fragment assembly fruit bats as reservoirs of ebola virus structure of the ebola vp interferon inhibitory domain structural basis for dsrna recognition and interferon antagonism by ebola vp contribution of ebola virus glycoprotein, nucleoprotein, and vp to budding of vp virus-like particles new developments in the induction and antiviral effectors of type i interferon ebola virus glycoprotein counteracts bst- /tetherin restriction in a sequence-independent manner that does not require tetherin surface removal the ifitm proteins inhibit hiv- infection protection from lethal infection is determined by innate immune responses in a mouse model of ebola virus infection hiv- vpu neutralizes the antiviral factor tetherin/bst- by binding it and directing its beta-trcp -dependent degradation molecular mechanism of bst /tetherin downregulation by k /mir of kaposi's sarcoma-associated herpesvirus ebolavirus vp binding to karyopherins is required for inhibition of interferon signaling species-specific activity of hiv- vpu and positive selection of tetherin transmembrane domain variants a new ebola virus nonstructural glycoprotein expressed through rna editing vpu antagonizes bst- -mediated restriction of hiv- release via beta-trcp and endo-lysosomal trafficking comparison of the transcription and replication strategies of marburg virus and ebola virus by using artificial replication systems tetherin inhibits retrovirus release and is antagonized by hiv- vpu plasma membrane rafts play a critical role in hiv- assembly and release the ring-ch ligase k antagonizes restriction of kshv and hiv- particle release by mediating ubiquitindependent endosomal degradation of tetherin tetherin inhibits hiv- release by directly tethering virions to cells ebola virus protein vp impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk- basic residues within the ebolavirus vp protein are required for its viral polymerase cofactor function mutations abrogating vp interaction with double-stranded rna render ebola virus avirulent in guinea pigs infectious lassa virus, but not filoviruses, is restricted by bst- /tetherin homooligomerization facilitates the interferon-antagonist activity of the ebolavirus vp protein ebola virus vp binds karyopherin alpha and blocks stat nuclear accumulation ebola virus vp proteins inhibit the interaction of npi- subfamily karyopherin alpha proteins with activated stat interferon-inducible antiviral effectors inhibition of lassa and marburg virus production by tetherin the virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing tetherin-driven adaptation of vpu and nef function and the evolution of pandemic and nonpandemic hiv- strains jak-stat signaling: from interferons to cytokines a diverse range of gene products are effectors of the type i interferon antiviral response ebola virus vp antagonizes pkr activity through its c-terminal interferon inhibitory domain extracellular signal-dependent nuclear import of stat is mediated by nuclear pore-targeting complex formation with npi- , but not rch the small interferon-induced transmembrane genes and proteins ebola virus counters the interferon system a. kü hl and s. pö hlmann inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry experimental inoculation of plants and animals with ebola virus a system for functional analysis of ebola virus glycoprotein newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda. plos pathog. , e gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases processing of the ebola virus glycoprotein by the proprotein convertase furin delta-peptide is the carboxy-terminal cleavage fragment of the nonstructural small glycoprotein sgp of ebola virus effects of ebola virus glycoproteins on endothelial cell activation and barrier function influenza virus is not restricted by tetherin whereas influenza vlp production is restricted by tetherin interferon-induced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines budding capability of the influenza virus neuraminidase can be modulated by tetherin palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm we thank t.f. schulz for support. key: cord- -ayek zbo authors: har-noy, michael; or, reuven title: allo-priming as a universal anti-viral vaccine: protecting elderly from current covid- and any future unknown viral outbreak date: - - journal: j transl med doi: . /s - - - sha: doc_id: cord_uid: ayek zbo background: we present the rationale for a novel allo-priming approach to serve the elderly as a universal anti-virus vaccine, as well serving to remodel the aging immune system in order to reverse immunosenescence and inflammaging. this approach has the potential to protect the most vulnerable from disease and provide society an incalculable economic benefit. allo-priming healthy elderly adults is proposed to provide universal protection from progression of any type of viral infection, including protection against progression of the current outbreak of covid- infection, and any future variants of the causative sars-cov- virus or the next ‘disease x’. allo-priming is an alternative approach for the covid- pandemic that provides a back-up in case vaccination strategies to elicit neutralizing antibody protection fails or fails to protect the vulnerable elderly population. the allo-priming is performed using activated, intentionally mismatched, ex vivo differentiated and expanded living th -like cells (allostim(®)) derived from healthy donors currently in clinical use as an experimental cancer vaccine. multiple intradermal injections of allostim(®) creates a dominate titer of allo-specific th /ctl memory cells in circulation, replacing the dominance of exhausted memory cells of the aged immune system. upon viral encounter, by-stander activation of the allo-specific memory cells causes an immediate release of ifn-ϒ, leading to development of an “anti-viral state”, by-stander activation of innate cellular effector cells and activation of cross-reactive allo-specific ctl. in this manner, the non-specific activation of allo-specific th /ctl initiates a cascade of spatial and temporal immune events which act to limit the early viral titer. the release of endogenous heat shock proteins (hsp) and damp from lysed viral-infected cells, in the context of ifn-ϒ, creates of conditions for in situ vaccination leading to viral-specific th /ctl immunity. these viral-specific th /ctl provide sterilizing immunity and memory for protection from disease recurrence, while increasing the pool of th /ctl in circulation capable of responding to the next viral encounter. conclusion: allo-priming has potential to provide universal protection from viral disease and is a strategy to reverse immunosenescence and counter-regulate chronic inflammation (inflammaging). allo-priming can be used as an adjuvant for anti-viral vaccines and as a counter-measure for unknown biological threats and bio-economic terrorism. har-noy and or j transl med ( ) : pneumococcal pneumonia, shingles and hepatitis a/b. successful prophylactic vaccination mechanisms provide protection through eliciting neutralizing antibodies to prevent viral entry into cells. however, this strategy does not provide protection against antigenic shift or drift variants of the original virus [ , ] . currently, there are at least three known variants of the sars-cov- virus [ ] . in addition, pathological viruses are intracellular and not always accessible to antibodies. for this reason, neutralizing antibody vaccines have not been effective against a number of complex viruses, including hiv, hcv, cmv, zika, rsv, dengue and sars/mers. for the same reason, convalescent serum prophylaxis and treatment may not be able to confer sterilizing immunity or memory. these sophisticated viruses may require an effective cellular immune response for sterilizing immunity [ ] [ ] [ ] [ ] [ ] . without sterilizing cellular immunity, there can be viral recurrence as has been reported with covid- [ ] . many efforts are underway to develop anti-viral vaccines which elicit protective cellular immunity [ ] , but these have not yet been successfully translated to demonstrate clinical benefit [ ] . the age-related functional decline in cellular immunity (immunosenescence) makes the elderly less able to mount a cellular immune response to vaccination, making this population more vulnerable to morbidity and mortality associated with viral diseases and less likely to respond to an anti-viral vaccine. in addition, elderly also suffer detrimental effects on their immune function due to chronic inflammation, known as "inflammaging" [ ] . inflammaging is correlated with comorbidities such as cancer, arthrosclerosis, neurodegenerative diseases (e.g., alzheimer's and parkinson's disease) all which increase the likelihood of serious progression of viral infection. in addition, the aging of the structure and function of the lungs contributes to increased incidence of pneumonia, acute respiratory distress syndrome (ards) and sepsis in the elderly after respiratory viral infection. the remodeling of the senescent immune systems of the elderly through allo-priming is proposed as a method to restore cellular immune function in this population. the ability to restore functional cellular immunity to the elderly can increase responsiveness to viral infections, including covid- and any future emergent novel virus. in essence, an elderly immune system modulated by allo-priming would potentially respond to viral infection in a similar manner to the immune response of younger individuals, resulting in less serious disease. the immunomodulation of the elderly immune system to function more like a youthful immune system should also restore responsiveness to any current or future viralspecific vaccines. a more balanced immune system in the elderly can also counter-regulate inflammaging, providing broad ranging health benefits to the elderly and to society [ ] . the allo-priming concept is designed to prime the immune systems of healthy elderly adults to create high titers of allo-specific th /ctl memory cells which can become activated upon encounter with any virus (bystander activation) [ ] . the by-stander activation of allo-specific th /ctl memory cells upon viral encounter causes the release of interferon-gamma (ifn-ϒ), which creates an "anti-viral state" [ ] . ifns create an antiviral state in both virus-infected cells and uninfected, bystander cells, by inducing a program of gene transcription that interferes with multiple stages of viral replication cycles through various mechanisms [ ] . the ifn-ϒ release from by-stander activated allo-specific th /ctl memory cells activates innate effector cells (e.g., nk, nkt and macrophages) which in turn release additional ifn-ϒ, sustaining the anti-viral state. these activated innate effector cells lyse viral infected cells, controlling acute viral burden. by-stander activation of resident allo-specific ctl can also cross-react to lyse viral infected cells [ ] . the lysis of viral infected cells by activated innate effector cells and cross-reactive allo-specific memory ctl releases "danger signals" [ ] and heat shock proteins (hsp) [ ] which chaperone viral antigens (e.g., grp , hsp ) [ , ] into the microenvironment, creating the conditions for "in situ vaccination", which leads to development of viral-specific cellular immunity. antigen presenting cells (apc), such as dendritic cells (dc), engulf and process released hsp-chaperoned viral antigens. processing in the context of danger signals and ifn-ϒ, causes the maturation of immature dc to il- + dc [ , ] . il- production acts to further increases ifn-ϒ, sustaining the anti-viral state and upregulating mhci, mhcii and co-stimulatory molecules on apc [ , ] . the mature dc migrate to regional lymph nodes and display viral antigens on mhc i and mhc ii with co-stimulator cd / expression, leading to viral-specific th /ctl expansion. the ifn-ϒ upregulates mhci on viral infected cells so these cells can be recognized and killed by viral-specific ctl [ ] . these viral-specific ctl can then orchestrate a sterilizing immune response and later differentiate into memory cells that provides protection from re-infection. thus, allo-antigenic priming can modulate the systemic immune balance and provide a pool of non-exhausted allo-specific th /ctl memory cells capable of rapidly responding to viral infection. the non-specific activation of these allo-specific memory cells upon viral encounter causes release of ifn-ϒ. the release of ifn-ϒ orchestrates the sequential activation of immune cells resulting in formation of an antiviral state, innate elimination of invading viruses and development of a viral-specific effector response and memory. each time this cascade is initiated by viral encounter, the virus elimination serves as a booster to the original allo-antigenic priming. the new viral-specific th /ctl memory cells resulting from viral elimination join the resident memory allo-specific th /ctl resulting from the allo-antigenic priming, increasing the titer of th / ctl memory cells in circulation. encounter with each new virus will non-specifically activate all previous nonexhausted memory cells and support the immune cascade that eliminates the new virus. memory th /ctl to each new virus, in turn, increases the titer of th /ctl memory cells primed to respond to a subsequent viral encounter. creating a memory th /ctl immune response to alloantigen thus provides protection against an unrelated viral infection through the immune mechanism known as "heterologous immunity". heterologous immunity occurs when by-stander activation of immune memory cells to one virus alters the immune response to, and the course of infection of, an unrelated virus encountered later [ , ] . in this manner, healthy adults can be provided pan-viral protection against viral infections, including sars-cov- , influenza a/b and any future variants and unknown novel viruses that may emerge. this self-amplifying process can provide long-term disease mitigation and universal protection against known and unknown viral infections for the elderly. in order to effectuate the anti-viral and anti-aging immune mechanisms, the allo-priming must elicit allospecific th /ctl immunity. injection of alloantigen alone can elicit humoral and th responses, and multiple injections can result in tolerance to the allo-antigens. many factors, both intrinsic and extrinsic to the allograft, can influence the nature and magnitude of the allorejection immune response, including the nature of the allograft, the site of the body where it is placed, and the immunological status of the recipient [ ] . to assure consistent differentiation of allo-specific th /ctl memory after allo-antigen priming, regardless of host immune status and age, a bioengineered, patented, immune cell called "allostim ®" is used. allostim ® is living, activated, intentionally mismatched, ex vivo expanded and differentiated allogeneic th -like cells derived from healthy donors. injection of undifferentiated allogeneic immune cells or non-activated allogeneic immune cells were not able to elicit allo-specific th memory, while priming with activated allogeneic th like allostim ® cells elicited dominant th immunity and had both a protective effect and a therapeutic effect in a cancer model [ , ] . allostim ® cells are administered intradermally (id) in an activated state. to assure the injected cells are activated when administered, they are prepared with anti-cd /anti-cd -coated microbeads attached. the activated allostim ® cells are formulated as a frozen dosage form at × cells/ml in plasmalyte a containing % human serum albumin and % dsmo. individual doses of . ml are thawed and administered intradermally every - days for up to five total doses. this priming schedule is sufficient to provide high titers of circulating allo-specific th /ctl. allostim ® has been evaluated in phase i/ii, phase ii and phase iia clinical trials in usa and thailand in chemotherapy-refractory metastatic solid tumor patients. phase iib pre-registration and phase iib/iii randomized, controlled registration clinical trials are currently being prepared for launch in third line metastatic colorectal cancer and advanced/ metastatic hepatocellular carcinoma in usa and asia, respectively. in these human clinical trials, allostim ® has demonstrated ability to down-regulate checkpoint molecules in the tumor microenvironment, cause system-wide infiltration of effector t-cells and nk cells into metastatic lesions (conversion from "cold" to "hot") and has consistently demonstrated a tail of between and % of long-term survivors in chemotherapy-refractory metastatic disease with a good safety profile [ , ] . activated allostim ® express high density cd l and type cytokines, including ifn-ϒ, tnf-α and gm-csf. allostim ® has been shown to modulate the immune systems of heavily pre-treated metastatic cancer patients (which resemble the senescent immune systems of the elderly) and has been used in protocols which were designed to elicit the same anti-tumor mechanism of allogeneic stem cell transplant procedures (graft vs tumor or "gvt") without the need for a matched donor, chemotherapy conditioning or risk of gvhd [ , , [ ] [ ] [ ] [ ] [ ] . allogenic cells are highly immunogenic and are rejected even by immunocompromised hosts [ ] . the rejection of the mis-matched cells eliminates risk of gvhd side-effects of allogeneic cell administration. multiple id injections of allostim ® amplify the titer of allo-specific th /ctl cells in circulation and a portion of these cells differentiate into long lasting memory cells. these allo-specific th /ctl memory cells provide a functional pool of immune cells that are capable of nonspecific (by-stander) activation [ ] upon encounter with virus [ , ] resulting in immediate release of ifn-ϒ. har-noy and or j transl med ( ) : the production of proinflammatory cytokines by allostim ® , including ifn-γ and tnf-α, initially activates host nk cells to reject the allogeneic cells, due to missing self-mhc i on the cd + allostim ® cells. the nk cell compartment is highly stable in terms of function and phenotype in the elderly [ ] and can therefore readily reject mismatched allogeneic cells. in subsequent id allostim ® injections, the production of ifn-ϒ and expression of cd l by allostim ® activates macrophages that reject the allostim ® [ ] . the rejection of the allogeneic cells by nk cells or macrophages in the skin results in release of endogenous hsp and damp danger signals [ ] , which in the context of cd l and ifn-ϒ expressed by allostim ® result in dc maturation to il- + dc phenotype and their migration to draining lymph nodes to interact with cognate t-cells to elicit allo-specific th /ctl immunity [ ] . the current covid- viral pandemic has led to high number of deaths worldwide. this is the third serious coronavirus (cov) outbreak in less than years, following sars in - and mers in . elderly people account disproportionately to the morbidity and mortality associated with cov infection. vaccines have been the main global strategy to minimize the impact of viral infections, but no successful vaccines have yet been developed for these previous highly virulent covs. in any event, elderly adults generally respond poorly to antiviral vaccines [ ] [ ] [ ] . this creates a high unmet medical need for a vaccine to provide protection to the elderly from the current covid- pandemic and protect from any future pandemics. while healthy younger adults generally present with more mild symptoms in response to viral infection, the elderly are slow to respond and are susceptible to higher viral titers and chronic viral infection which leads to progression to severe symptoms, especially in the setting of respiratory viral infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the delayed and poorly effective responsiveness of elderly to viral infection is due, in large part, to compromised cellular immunity [ ] [ ] [ ] related to "immunosenescence" [ ] [ ] [ ] . elderly individuals have a significantly delayed innate immune response to viral infection and the adaptive immune response that follows is often sub-optimal [ ] . the delayed innate immune response results in accumulation of high viral titers which, in turn, serves to elicit a late over-active immune response which is correlated with a more severe and often lethal clinical course [ ] . the acute high viral levels that are achieved in elderly as a result of late innate immune response also increases the virulence of the virus, by increasing chances of humanto-human spread of infection. there is a dysregulation in the th /th -system in the elderly, which is dominated by th -functions [ , ] . memory th /ctl cells in elderly adults are diminished and functionally exhausted [ ] [ ] [ ] [ ] . memory cells of the elderly become exhausted due to lifelong antigenic challenge which can be as a result of chronic viral infection (e.g., cmv, ebv, hbv, hcv) [ , ] . exhausted memory cells are unable to be non-specifically activated upon viral encounter. this defect in cellular immunity in the elderly limits the availability of heterologous immune mechanisms to react to acute viral infection, which contributes to a slower and often inadequate innate response to viral infection. elderly also have limited t-cell repertoires [ , ] which inhibits their ability to develop viral-specific adaptive t-cell immune responses, both prophylactically (by vaccination) [ ] , and in situ (as a result of adaptive immune clearance of infection). this suppressed cellular immunity together with the aging of the respiratory organs makes elderly more susceptible to pathological effects of viral infection, especially effects of respiratory viral infection [ ] . the immune defects of the elderly make this population less likely to benefit from vaccinations as preventative measures against infectious diseases [ ] . the current inability to provide elderly with protection from viral infection has an enormous economic impact on society [ , ] . the medical and economic impact of pandemics which disproportionately affect the elderly will be exacerbated as the proportion of the adult population increases globally. the current projections indicate that by , the group of years and older will represent . % of the population [ ] . therefore, there is an urgent unmet need to develop strategies to reverse immunosenescence and to develop methods which provide protection of the elderly population from viral epidemics. the progressive age-related dysregulation and decline of the immune system is known as "immunosenescence". in the aging immune system, there is an accumulation of memory cells as a result of chronic stimulation with repeated clinical and subclinical infections [ ] . this chronic stimulation causes these memory cells to become exhausted and no longer able to function, contributing to an increased susceptibility of elderly to infectious pathogens [ , ] . the aging immune system is also characterized by a decline in the numbers of naive t cells, an imbalance in th /th cell subsets, and a decrease in t cell receptor (tcr) repertoire [ , , ] . a cellular immune response with release of th (type ) cytokine profile, including ifn-ϒ, tnf-α and il- , is known to be protective against most viral infections [ ] . ifn-ϒ in particular has been shown to play an essential role in clearance of viral infection [ ] . modulating the immune system of elderly individuals through alloantigen priming to provide high titers of non-exhausted th / ctl memory cells that can be non-specifically activated upon encounter with any virus to cause release type cytokines may provide an immediate anti-viral immune response upon viral exposure and could also reinstate responsiveness to viral vaccines [ ] . the delay in ifn responses to virus infection in the elderly is due to active suppression by viral proteins and immunosenescence [ ] , which allows virus to reach high titers early in infection, leading to immunopathology, t-cell apoptosis and progression to lethal pneumonia [ , ] . the immediate release of ifn-ϒ upon acute viral infection corrects the problem of delayed immune responsiveness and creates an early "anti-viral state" [ , ] . establishment of the anti-viral state with early release of ifn-ϒ provides a crucial initial line of defense against viral infection [ ] . the anti-viral state is normally induced through early release of type i/iii ifn from viral infected cells [ ] . however, many viruses, including coronavirus, can suppress production of type i/iii ifn as an immune avoidance mechanism [ ] [ ] [ ] [ ] . the strategy of providing a pool of non-exhausted memory cells which produce type ii ifn (ifn-ϒ) upon viral encounter, overcomes the viral escape. ifn-ϒ creates an anti-viral state even in type i/iii ifn resistant cells [ ] . further, ifn-ϒ mobilizes innate effector cells that can respond rapidly to eliminate viral infection [ ] and also produce ifn-ϒ. thus, early ifn-ϒ response to viral infection through allo-priming provides a rapid means of control over viral infection in the elderly. chronic inflammation in the elderly is known to be associated with increased susceptibility to cancer [ , ] , atherosclerosis [ ] and neurodegenerative disorders [ , ] . the modulation of the elderly immune system that occurs by allo-priming can also have the effect of counter-regulating resident chronic inflammation [ , ] which causes "inflammaging" resulting in reversing of immunosenescence [ ] . thus, allo-priming protocols may not only provide universal anti-viral protection for the elderly, but may also serve as an "antiaging" mechanism to protect from co-morbid diseases of aging. allo-priming is predicted to reduce the severity of clinical symptoms upon encounter with virus. clinical features of respiratory viral infections in humans vary from a first subset that experience mild flu-like symptoms which subside over a few days; to a second subset that experience moderate to severe flu-like symptoms associated with high fever, hypoxemia and progression to pneumonia-like symptoms. these symptomatic patients are at high risk to progress to acute respiratory distress syndrome (ards), sepsis, multiple organ failure and death. the subset with mild flu-like symptoms is associated with younger healthy individuals, while the subset associated with more severe symptoms is mostly associated with elderly adults that are frail and/or present with co-morbidities. the progression to more serious symptoms can occur even when there is a decline in virus titers [ ] connected to a late over-exuberant immune response. the initial delayed immune response leads to high viral titers which is followed by an exaggerated late immune response and a "cytokine storm". this sequence of events is believed to be responsible for the severe symptoms related to respiratory virus infections in the elderly. during pathogenic virus infection, a cytokine storm leads to excessive inflammatory infiltrates and virusinduced tissue destruction which contributes to morbidity and mortality. the timing and source of the ifn in the cytokine storm can affect the clinical course of viral disease. high serum levels of ifn-ϒ late in the course of viral infection is correlated with more severe disease [ ] and immunopathology [ ] , whereas early ifn release results in lower viral titers and less severe disease. these observations support the prediction that early activation and release of ifn-ϒ from non-exhausted allospecific memory th /ctl will correct this problem and avoid progression to severe disease. the type of cytokine storm associated with severe disease is caused by cytokines released by activated monocytes, producing a storm of ifn-α, ccl- , il- , tnf-α, ccl- , ccl- , cxcl- , il- α, and ifn-γ. this monokine cytokine storm is directly correlated with morbidity and mortality [ ] . the majority of fatalities associated with cytokine storm also developed bacterial pneumonia and sepsis. it is recognized that a major cause of respiratory failure is coexistent bacterial pneumonia leading to ards. ards is characterized by damage to the endothelial-epithelial barrier of the alveoli, resulting in fluid leakage and accumulation in the alveolar lumen inhibiting gas exchange. the elderly have a pre-disposition to develop progressive respiratory compromise and sepsis. in fact, sepsis is considered a disease of aging [ ] and is among the top causes of icu admissions of the elderly. the features of sepsis-induced immunosuppression, independent of age, share many of the same characteristics of immunosenescence. during the last decades, there has been a significant increase in incidence of sepsis in patients over years of age [ ] . many respiratory virus patients that progress to develop pneumonia and ards [ , ] later die due to sepsis. the overactive immune response to sepsis, including monokine "cytokine storm" results from over activated m macrophages in response to tissue damage [ ] . the response to sepsis is similar between old and younger patients. however, the mortality is much higher in older patients [ ] . the difference is related to the dysregulated immune systems of the elderly. the elderly are unable to turn down the over-activated monocyte response, leading to disease progression, while regulatory mechanisms in the young counter-act and prevent the consequences of monocyte over-activation. the dysregulated immune system of the elderly is related to the increased activity of myeloid-derived suppressor cells (mdsc) [ , ] . mdscs are potent inducers of immunosenescence [ ] , sepsis-acquired immunodeficiency [ ] as well as cancer metastasis and progression [ ] . mdsc are positively correlated with il- and negatively correlated with ifn-ϒ [ , ] . immunosenescence causes an imbalanced homeostatic regulation of innate and adaptive immune responses, diminishing the host capacity to rapidly restore balanced immune functions. thus, combined effects of ageinduced immunosuppression, delayed innate immune responses [ ] , exaggerated late immune responses due to altered homeostasis [ ] , all combine to make the post-viral infection period in the elderly have a longer duration. as a result, there are increased levels of viral propagation, higher incidences of tissue injury, and increased progression to ards and septic shock in the elderly. accordingly, allo-priming enabling the early release of ifn-ϒ in response to viral infection can prevent the differentiation of mdsc and counter-regulate resident mdsc immunosuppression in the elderly [ ] . this will have the effect of remodeling the elderly immune system in a manner which will prevent the severe symptoms of viral infection and progression to ards and sepsis. highly pathogenic human covs pose a substantial threat to public health. there are basically two groups of patients upon exposure to cov, with the majority developing a short duration of clinical symptoms and the minority experiencing severe disease characterized by pneumonia and ards. the elderly are disproportionately vulnerable to severe disease due to immunosenescence and comorbidities. it is likely that covs will continue to cross species and cause additional outbreaks in the future. therefore, even if a vaccine is developed for prevention of covid- , it would not protect against the next outbreak of a novel virus. therefore, there is a need to develop novel strategies, not only to control the current pandemic, but also to be prepared to prevent the next pandemic. strategies to develop a vaccine to elicit neutralizing antibodies to sars-cov- are technically challenging due to the conformational hiding of the receptor binding domain (rbd) and the ability of this virus to transfer cell-to-cell in syncytia and infect target cells in a ace -and protease-independent manner by pinocytosis, limiting environmental exposure needed for antibody neutralization activity. development of an anti-viral vaccine against complex viruses, such a sars-cov- , is likely to require a potent and broad t-cell response to overcome viral escape mechanisms related to humoral immunity. a vaccine that elicits a robust cellular immune response requires identification of conserved viral epitopes and effective processing and presentation of viral antigens by apc on mhci and mhcii in conjunction with costimulatory signals, as well as a diversity in the naive t cell repertoire. in addition, ctl memory cells resulting from vaccination will require infected cells to present the selected viral antigens in the vaccine on mhci. selection of common viral epitopes which present on mhc i (and mhc ii) for vaccine development is technically difficult and cov infection causes the down-regulation of mhc i on infected cells, making infected cells invisible to ctl. since the elderly lack naïve t-cells that are necessary for development of ctl with broad specificity to viral antigens, elderly would be less likely to respond efficiently to any future vaccine which targets cellular immunity. the rejection of allografts is one of the most conserved and powerful immune mechanisms, making priming with alloantigens an ideal candidate for use in vaccination of the elderly. allostim ® is in use under a us fda cleared ind and has been shown to readily be rejected by heavily pre-treated, immunosuppressed metastatic cancer patients. a phase i/ii clinical trial protocol for use of allostim ® in healthy elderly adults in currently under review by us fda. upon clearance, clinical trials could be initiated to evaluate this allo-priming concept in short order. for proof-of-concept, longitudinal pbmc samples are proposed to be collected pre-and post-allo-priming. the pbmc are to be pulsed with a panel of inactivated viruses or recombinant viral proteins to determine if they will non-specifically activate the allo-specific th /ctl memory cells. the supernatants from the viral-pulsed pbmc are proposed to be evaluated in live virus cytolytic plaque assays to determine if viral propagation is suppressed. elderly persons are particularly susceptible to progressive viral disease and have delayed immune responses to viral infection due, at least in part, to immunosenescence, th immune bias, decreased diversity of naïve t-cells and high frequencies of exhausted memory cells from chronic inflammation (e.g., cmv). this results in a natural loss of the ability to mount effective innate and adaptive cellular immune responses to invading pathogens. the delayed and suppressed cellular immune response in the elderly enables viruses to become widely established and the resulting increased viral titers causes dys-regulated immune responses that can lead to a late, over-active immune response which can progress to pneumonitis, multiple organ failure and death. the key to an effective natural innate immune response that can limit the course of virus infection is the early production of type i/iii interferons (ifn) and subsequent switch to type ii ifn (ifn-ϒ) production as the immune response matures. successful viral infections become established due, in large part, to delayed innate immune responses and viral-mediated suppression of type i/iii ifn production, allowing for rapid viral propagation which eventually results in dys-regulated immune responses which are correlated with tissue destruction, co-infection, multiple organ failure and death. to prevent accumulation of high viral burden in the elderly upon viral infection, allo-priming provides a mechanism whereby a ready pool of de-novo primed t-cells are in circulation that can respond rapidly to viral infection by producing ifn-ϒ. the presence of non-exhausted th /ctl memory immune cells will modify the elderly immune character by providing a th re-balancing mechanism in the memory cell compartment. this is accomplished through the creation of a high titer of polyclonal, allo-specific, non-exhausted, memory th /ctl t-cells through intradermal injections of activated allogeneic th -like cells (allostim ® ). the allo-specific memory cells resulting from the priming are programmed to produce ifn-ϒ upon activation. ifn-ϒ has a direct anti-viral effect on cells infected with virus and can also protect uninfected cells from infection. ifn-ϒ creates the same anti-viral environment as innate release of type i/iii ifn does in an effective natural innate immune response. these allo-specific memory cells are capable of crossreacting with foreign viral antigens and can be readily activated non-specifically by environmental stimuli such as cytokines and foreign pathogens. the memory pool of allo-specific immune cells in primed individuals can also be re-activated by additional intradermal or intravenous infusion of allostim ® allogeneic cells upon first symptoms of viral disease. the by-stander effect of allo-specific th /ctl memory t-cell activation and ifn-ϒ production is predicted to elicit protective effects on cells in the respiratory tract and generate rapid immune-mediated viral clearance as well as condition the microenvironment in infected tissues (e.g., lung epithelial cells) for an in situ vaccination leading to viral-specific immunity and memory that is specific for the invading virus. the heterologous immune effect can amplify the pan-viral protection upon each viral encounter, providing the possibility of long-term protection. the proposed alloantigen priming strategy can also be used in conjunction with any viral-specific vaccines. co-injection of allo-specific and viral specific antigens could accelerate the adaptive cellular immune response to a known virus and serve as a means to enhance response to vaccines in the elderly. subsequent injection of allogeneic cells will stimulate an allo-rejection memory recall response which can non-specifically activate resident viral-specific cells elicited by vaccination. this dual activation provides a mechanism to upregulate mhci on infected targets, making the memory cells elicited by viral-specific vaccination able to identify and kill viral infected targets. the combination approach also has the advantage of overcoming the narrow immunity conferred by a single peptide vaccine by incorporating the in situ vaccination mechanism to elicit broad viral-specific cellular immune responses. this allo-priming mechanism can also be used as a counter-measure to bio-terrorism and bio-economic terrorism. individuals that have been primed with alloantigen could be treated with an emergency injection of alloantigen to activate innate immunity and initiate the cascade of immune events leading to clearance of an unknown pathogen. alloantigen could be provided in syringes for emergency id injection upon concern for bioweapon exposure or first occurrence of symptoms, much in the same manner as epinephrine is provided to prevent anaphylactic shock. while finding methods to treat and prevent covid infection is of urgent priority, it is also important to consider that the current pandemic is the third major outbreak of a novel coronaviral infection in humans within the past years. currently there are no registered vaccines or means of therapeutic protection against the prior sars or mers outbreaks available anywhere in the world. this is despite considerable efforts from experts worldwide to develop vaccines. the same technical obstacles preventing development of specific vaccines and treatments for these past cov outbreaks likely also exist for development of a vaccine for the current covid- viral pandemic. however, even if an 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and increased citations maximum visibility for your research: over ready to submit your research ? choose bmc and benefit from ifn-gamma regulates survival and function of tumor-induced cd b+ gr- high myeloid derived suppressor cells by modulating the anti-apoptotic molecule bcl a publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. mhn conceived of the allopriming concept and wrote the manuscript. ro reviewed and edited the manuscript. both authors read and approved the final manuscript. not applicable. not applicable. not applicable. not applicable. mhn is the founder of immunovative therapies, ltd. and mirror biologics, inc which own patent rights to allostim ® . key: cord- -b re bky authors: zhang, qingzhan; shi, kaichuang; yoo, dongwan title: suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: b re bky type i interferons (ifn-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. we found that the ifn-β production was significantly suppressed during pedv infection in cells. to identify viral ifn antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. of pedv nonstructural proteins (nsps), nsp , nsp , nsp , nsp , nsp and nsp were found to inhibit the ifn-β and irf promoter activities. the sole accessory protein orf , structure protein envelope (e), membrane (m), and nucleocapsid (n) protein were also shown to inhibit such activities. pedv nsp did not interfere the irf phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of irf and creb-binding protein (cbp) by degrading cbp. a further study showed that the cbp degradation by nsp was proteasome-dependent. our data demonstrate that pedv modulates the host innate immune responses by degrading cbp and suppressing isgs expression. porcine epidemic diarrhea (ped) is a highly contagious acute enteric disease characterized by vomiting, watery diarrhea, and severe dehydration of up to - % mortality in suckling piglets (song and park, ; sun et al., a; debouck and pensaert, ; junwei et al., ) . ped was first reported in england in feeder and fattening pigs during s (wood, ) , and reemerged in asia since with greater virulence and economic losses li et al., ; puranaveja et al., ; yang et al., ) . in the us, pedv appeared for the first time in and severely affected most pig-producing states marthaler et al., ; mole, ; stevenson et al., ) . the causative agent is porcine epidemic diarrhea virus (pedv), which belongs to the alphacoronavirus genus in the family coronaviridae (http://ictvonline.org/virustaxonomy.asp). pedv is an enveloped virus with a single-stranded positive-sense rna genome of approximately kb in length with the -cap and the polyadenylated tail. the pedv genome is arranged with orf a, orf b, s, orf , e, m, n, in order with both termini flanking with the -and -untranslated regions (utrs) (duarte et al., ) . orf a codes for the large polyprotein pp a, while orf b is always expressed as a fusion protein pp a/b with pp a through a ribosomal frameshifting. pp a and pp a/b are further processed to nonstructural proteins, nsp through nsp . orf codes for an accessory protein which is likely an additional nonstructural protein, whereas s, e, m and n genes code for four structural proteins (song and park, ) . during viral infection, the sensing of foreign nucleic acids in the cytosol leads to the activation of an innate immune response to produce type i interferons (ifn-α/β) and establishes an antiviral state. the type i ifns and ifn-mediated response provide a first line of defense against viral infection. the host innate immune system deploys the pattern-recognition receptors (prrs) to sense and respond to the pathogen-associated molecular patterns (pamps) of virus (kawai and akira, ) . this recognition triggers the activation of retinoic acid-inducible gene i (rig-i) or melanoma differentiation gene (mda ), which further binds to the mitochondrial adapter protein mavs/ips- and recruits tnf receptor-associated factor / (traf and traf ). traf activates iκb kinase (ikk)-related kinases such as tank-binding kinase (tbk ) and ikkε for phosphorylation of interferon regulatory factors and (irf /irf ) and type i ifn production (fitzgerald et al., ; sharma et al., ) . traf leads to tank activation, followed by nf-kb activation and cytokine production (rajsbaum and garcia-sastre, ) . upon tbk activation, phosphorylated irf undergoes homodimerization and unveils the nuclear localization signal leading to the nuclear translocation, where it forms a complex with the transcription co-activator creb (camp responsive element binding)-binding protein (cbp)/p (dragan et al., ; lin et al., ; panne et al., ) . the irf -cbp/p complex further binds to the positive regulatory domain (prd) i-iv regions of the ifn-β promoter to assemble the enhanceosome together with nf-κb and other factors to turn on the transcription of type i ifn genes (honda and taniguchi, ) . the irf -cbp/ p interaction is crucial for ifn transcription. following production and secretion, ifn molecules bind to the cell surface receptors and trigger the activation of janus kinase-signal transducers and activators of transcription (jak-stat) signaling cascade. phosphorylated stat and stat associate to form a heterodimer, which in turn recruits the ifn-regulatory factor (irf ) to form the ifn-stimulated gene factor (isgf ). isgf translocates to the nucleus and induces genes regulated by ifnstimulated response elements (isre), resulting in expression of hundreds of antiviral genes and establishment of an antiviral state (stark and darnell, ) . in turn, many viruses have evolved to counteract the host innate immune defense and such viral functions are often redundant. for nidoviruses, eleven and six viral proteins have been described as ifn antagonists for severe acute respiratory syndrome coronavirus (sars-cov) and porcine reproductive and respiratory syndrome virus (prrsv), respectively kindler and thiel, ; shi et al., ; sun et al., b; totura and baric, ) . for betacoronaviruses, nsp has been reported as a multifunctional viral antagonist for innate immune response (huang et al., b; narayanan et al., ; wang et al., ) . for pedv, the viral modulation of innate immune signaling is poorly understood. pedv infects vero cells, but these cells are type i ifndeficient due to a chromosomal deletion (desmyter et al., ). in the present study, we identified marc- cells as a suitable line of cells for pedv infection and for study of innate immune modulation. we showed that pedv suppressed the type i interferon production and isgs expression in these cells, and identified nsp , nsp , nsp , nsp , nsp , nsp , e, m, n and orf as the viral ifn antagonists. we showed that pedv nsp caused the cbp degradation by the proteasome-dependent pathway. the cbp degradation is a novel mechanism of coronavirus nsp for ifn suppression and our study provides a new insight into the immune modulation and evasion strategy of pedv. pedv replicates in the cytoplasm of villous epithelial cells of the small and large intestines (debouck and pensaert, ; sueyoshi et al., ) . the viral antigen is also detectable in the macrophages that infiltrated the lamina propria (lee et al., ) . histological studies showed that pedv replicates in the porcine respiratory tract in vivo and transformed alveolar macrophages ( d ) in vitro (park and shin, ) . vero cells are widely used for pedv for diagnosis, virus isolation, and research, but these cells are type i ifn-deficient due to the chromosomal deletion (desmyter et al., ) . to study a possible regulation of innate immune signaling by pedv, various cell lines were examined for susceptibility. cells were infected with pedv at an moi of . in various trypsin concentrations and cpe was examined daily for up to days. in vero and marc- cells, apparent cpe of multinucleation was observed by h post-infection ( fig. a and b, left panel). trypsin activates the cleavage of s protein and induces membrane fusion to trigger infection (park et al., ; wicht et al., ) . pedv infection was characterized by syncytia formation (hofmann and wyler, ) and infection foci were visualized by anti-pedv m and n antibodies, indicating the susceptibility of both cell types for pedv infection ( fig. a and b, middle and right panel). the viral proteins were detected using specific antibodies by western blot and the specific bands were corresponding to the m and n proteins, further confirming the productive infection of these cells by pedv (fig. c ). the optimal trypsin concentration for pedv propagation was μg/ml and μg/ml for vero and marc- cells, respectively. marc- cells have been used to study type i ifn signaling of porcine arterivirus (kim et al., ; overend et al., ; patel et al., ) , and thus infection of these cells one μg of each of the cloned genes was transfected to hela cells in -well plates, and protein expression was determined by immunofluorescence (b) and western blot (c) for each gene using anti-flag antibody. (d and e) regulation of poly(i:c)-induced ifn-β promoter activity by individual pedv proteins. hela cells were seeded in -well plates and co-transfected with pifn-β-luc along with individual pedv genes and prl-tk at a ratio of : : . . since the expression levels of nsp and nsp were low, three-times more plasmids were transfected for these genes to ensure the comparable level of protein expression. prrsv nsp α (p-nsp α) is a known type i ifn suppressor, and the ifn-suppression of its mutant p-nsp α(m) was lost. both constructs were included as controls. at h post-transfection, cells were stimulated with poly (i:c) ( . μg/ml) for h and the luciferase activities were measured. the reporter experiments were repeated three times, each time in triplicate. asterisks indicate the statistical significance. statistical analysis was performed by student's t test using gst as a control. n p o . , nn po . and nnn p o . . (f) vsv-gfp bioassay. the cell culture supernatants for ifn-β promoter luciferase assays were collected and diluted serially by -folds up to : . fresh marc- cells were grown in -well plates and incubated with each dilution of supernatants for h, and then infected with vsv-gfp at an moi of . for h. vsv replication was measured by monitoring the fluorescence by gfp expression using fluorescent microscopy. data were presented as log sample dilution folds. (g) inhibition of irf promoter activation by pedv proteins. the ifn antagonists were further examined for irf activities by luciferase reporter assays. statistical analysis was performed by student's t test using gst as a control. by pedv allowed us to study ifn modulation and signaling cascade. to determine whether pedv infection antagonized the type i ifn response, ifn-β mrna was determined in virus-infected cells. marc- cells were infected with pedv and stimulated with poly (i:c) followed by qrt-pcr for ifn-β mrna using total rna. as shown in fig. a , pedv infection did not induce the level of ifn-β mrna expression whereas poly(i:c) alone induced the ifn-β gene expression effectively, indicating the suppression of ifn-β response by pedv. to further evaluate the ifn-β response in pedv-infected cells, a dual luciferase assay was performed. the results showed the suppression of ifn-β promoter activity in pedv-infected cells upon poly(i:c) stimulation (fig. b) , demonstrating the modulation of ifn production by pedv infection. irf was additionally examined for its role for pedv-mediated ifn-β suppression. the irf promoter activity was found to be inhibited (fig. c ). the suppression of ifn-β production was confirmed by bioassay using vsv-gfp. vsv is sensitive to type i ifn treatment and thus commonly used for ifn bioassays. culture supernatants were collected from pedv-infected cells and were uv-irradiated, followed by incubation with marc- cells and infection with vsv-gfp. vsv grew normally (fig. d) , whereas it did not grow with supernatants collected from poly(i:c)-treated cells for up to : dilution. vsv-gfp also grew normally with supernatants from both pedv-infected cells with or without poly(i:c) stimulation, confirming the suppression of type i ifn production by pedv. the viral ifn antagonism is often redundant, and at least viral proteins have been identified as ifn antagonists for sars-cov (kindler and thiel, ; shi et al., ; totura and baric, ) . to identify such proteins for pedv, we cloned pedv genes representing nsps through , and structural genes for s, e, m, and n including the orf accessory protein gene (fig. a ). among these, nsp is a small oligopeptide generated from pp a when ribosomal frameshifting does not occur and so was not included in this study. each gene was inserted into the pxj expression vector with the flag tag at either n-or c-terminus, and examined for ifn suppression. the protein expression of cloned genes was examined by immunofluorescence (fig. b ) and western blot (fig. c ) using anti-flag antibody. all genes were expressed as anticipated. hela cells were then co-transfected with an individual gene along with pifn-β-luc and prl-tk, and reporter assays were conducted. prrsv nsp α (p-nsp α) is known as an ifn-β suppressor, and its cystine mutant p-nsp α(m) (c s) is an ifn suppression revertant (han et al., ; song et al., ) , and so they were included as positive and negative controls, respectively. poly(i:c) upregulated the ifn-β transcription in cells expressing pxj , gst, and p-nsp α (m), while p-nsp α suppressed the ifn-β promoter activity as expected ( fig. d and e). of the nsps, nsp , nsp , nsp , nsp , nsp and nsp were shown to down-regulate the ifn-β activity (fig. d ). for structural proteins, e, m, and n were found to suppress the ifn induction (fig. e) , and orf was additionally identified as an ifn suppressive protein (fig. e ). the findings from the reporter assays were validated by vsv-gfp bioassays. the dilution corresponding to % of cells exhibiting gfp expression was determined as the end-point inhibition. for pxj , gst, and nsp , gfp expression was evident and their end-point inhibitions were determined as : (fig. f ). in contrast, the viral proteins identified as the luciferase suppressors showed an apparent inhibition of vsv-gfp replication and their end-points were determined to be : to : (fig. f ). these titers represent -to fold lower than those of controls. taken together, these data demonstrate that pedv has the ability for ifn suppression, and nsp , nsp , nsp , nsp , nsp , nsp , orf , e, m and n are the viral ifn antagonists. to determine the target for ifn inhibition, the irf pathway was examined for individual viral proteins using the irf luciferase reporter constructs. upon stimulation, the irf -dependent luciferase expression was reduced by nsp , nsp , nsp , nsp , nsp , nsp , orf , e, m, and n, comparing to those of pxj and gst (fig. g ). this suggests that the irf signaling pathway was interfered by these viral proteins for the suppression of the ifn-β production. pedv nsp antagonism is a nuclear event sars-cov is a betacoronavirus and its nsp triggers inhibition of type i ifn induction and downstream signaling, host mrna decay and cleavage, and inhibition of protein translation (huang et al., b; lokugamage et al., ; narayanan et al., ; tanaka et al., ) . transmissible gastroenteritis virus (tgev) is an alphacoronavirus and its nsp inhibits host protein expression (huang et al., a) . nsp of alphacoronavirus and betacoronavirus lacks the overall sequence similarity (narayanan et al., ) , and thus alphacoronavirus nsp may have a distinct basis for its biological function. since nsp appeared the most potent suppressor in our study on pedv, nsp was chosen to study the molecular basis for the ifn suppression. the subcellular localization was first examined by confocal microscopy in transiently expressing cells. the nsp distribution was evident in the both nucleus and cytoplasm ( fig. a) , which is consistent with tgev nsp (narayanan et al., ) . co-expression of nsp with either the endoplasmic reticulum or mitochondrial marker showed the site for cytoplasmic nsp in the endoplasmic reticulum (fig. b ). quantitative rt-pcr was conducted to evaluate ifn-β suppression in nsp -gene transfected cells. the expression of nsp significantly suppressed the ifn-β mrna transcription (fig. a) , further validating the nsp antagonism against ifn-β production. subsequently, the ifnmediated antiviral gene expression was examined for isg and isg by qrt-pcr. pedv nsp reduced the poly(i:c)-stimulated mrna levels of both isg (fig. b ) and isg (fig. c ), indicating the suppression of ifn signaling by nsp . the suppression of ifn-β, irf , and nf-κb activations raises a possibility that nsp may target a component of the rig-i like receptor (rlr) signaling pathway. to examine this premise, nsp was co-expressed with one of the main components in the rlr signaling pathway, and ifn luciferase activities were determined at h post-transfection. the over-expression of ips- , or irf led to the robust activation of the ifn-β promoter as anticipated, whereas the activation was significantly inhibited by nsp ( fig. a fig. . subcellular localization of pedv nsp . hela cells were seeded on slides in -well plates and transfected with pedv nsp gene (a), or co-transfected with nsp and pdsred -er or pdsred -mito (b). at h post-transfection, cells were fixed and permeabilized with triton x- . cells were then incubated with rat anti-flag mab for h, followed by alexa fluor -conjugated goat anti-mouse (green) secondary antibody to visualize nsp . the er and mitochondrial proteins were fused with the er and mitochondria targeting sequence (clontech) and so directly visualized (red). nuclei (blue) were stained with dapi. images were collected using a zeiss lsm- meta confocal laser-scanning microscope and processed with the lsm image browser (zeiss). irf is a resident protein in the cytoplasm. when stimulated, it is phosphorylated and homodimerized, leading to the translocation to the nucleus (dragan et al., ) . to determine whether pedv nsp targeted the irf -dependent pathway, the irf phosphorylation was first examined. nsp -gene transfected cells were stimulated with poly(i:c), and the irf phosphorylation was examined by western blot. as anticipated, the poly(i:c) stimulation led to irf phosphorylation in pxj -transfected cells, and similarly, in nsp -expressing cells, the irf phosphorylation was evident and comparable to that of control (fig. a , top panel, lane ), suggesting that pedv nsp exerts its suppression downstream of the irf phosphorylation. thus, the nuclear translocation of irf was next examined. prrsv nsp α is known not to block the irf nuclear localization, and so was used as a control in this study. endogenous irf was normally diffused and distributed in the cytoplasm, but translocated to the nucleus when stimulated by poly(i:c) (fig. b, second panel) . similarly to prrsv nsp αexpressing cells (fig. b , fourth panel), irf also localized normally in the nucleus after stimulation in pedv nsp -expressing cells (fig. b, bottom panel) , suggesting that the ifn suppression by pedv nsp may be a nuclear event. the irf nuclear translocation in nsp -expressing cells was further confirmed by cell fractionation and western blot analyses. while irf was phosphorylated and localized in the nucleus after stimulation, pedv nsp did not inhibit the irf phosphorylation and nuclear translocation (fig. c ), further indicating that the nsp -mediated ifn suppression was a nuclear event. interruption of irf and cbp association by nsp after nuclear translocation, an irf dimer associates with the creb-binding protein (cbp). this complex then binds to the prd i-iii . disruption of irf -mediated ifn signaling by nsp . hela cells were seeded in -well plates and co-transfected with pmavs/ips- (a) or pirf (b) along with the nsp gene, prl-tk, and ifn-β-luc reporter for h. cells were harvested to measure the firefly and renilla luciferase activities. relative luciferase activity was defined as a ratio of the firefly luciferase to renilla luciferase activities. data are presented as mean value standard deviation from three independent experiments. statistical analysis was performed by student's t test. n po . , nn p o . , and nnn po . . immunofluorescence staining for irf nuclear translocation by nsp . hela cells were transfected with the nsp -expressing plasmid ( μg/well) in -well plates for h and stimulated by poly(i:c) for h. cells were fixed and incubated with rabbit anti-irf pab and rat anti-flag mab for h. prrsv nsp α does not inhibit the irf nuclear localization and was used as a control. alexa fluor -conjugated goat anti-rabbit and -conjugated goat anti-mouse secondary antibodies were used to visualize irf (red) and viral nsp (green), respectively. nuclei (blue) were stained with dapi. yellow arrows indicate irf localization in the nucleus in the absence of nsp expression. white arrows indicate irf localization in the nucleus in nsp -expressing cells. (c) phosphorylation and nuclear localization of irf by nsp . hela cells were transfected with the pedv nsp gene for h, then stimulated with poly(i:c) for h. cells were lysed for nuclear-cytoplasmic fractionations and subcellular distribution of irf and pirf . hsp was used as a cytosolic protein marker and parp was used as a nuclear protein marker. regions of the ifn-β promoter to assemble the basal transcription machinery complex together with nf-κb and other transcription factors to turn on the transcription of type i ifn genes (honda and taniguchi, ) . thus, the irf -cbp/p interaction for the assembly of enhanceosome is crucial for ifn expression. since pedv nsp did not block the irf phosphorylation and nuclear translocation in our study, it was hypothesized that nsp might disrupt the formation of enhanceosome in the nucleus. to address this, the irf / cbp association was first examined in nsp -expressing cells. cells were transfected with the nsp gene and stimulated with poly(i:c) followed by co-immunoprecipitation using anti-irf antibody and immunoblot with anti-cbp antibody. in unstimulated cells, cbp was undetectable due to the absence of irf /cbp association (fig. a , left lane), but irf /cbp association became evident upon stimulation (fig. a, middle lane) . in nsp -expressing cells however, the association of irf and cbp disappeared even upon stimulation (fig. a , right lane) and the detectable level of irf remained unchanged ( fig. a, second panel) . absence of the association of cbp/irf may occur when nsp binds to either irf or cbp, or when irf is unstable in the presence of nsp . since pedv nsp was found to be a nuclear protein (figs. a, b and b, c), nsp in the nucleus might interact with either irf or cbp. however, neither the interaction between irf and nsp , nor between cbp and nsp was observed by co-immunoprecipitation in our study. irf was also stable in the presence of nsp ( fig. a and c) , indicating that the absence of irf / cbp association was not due to the instability of irf . interestingly, the level of cbp was found to decrease in nsp -expressing cells (fig. a) , leading us to investigate the degradation of cbp by nsp . some viruses including htlv, adenovirus, and an orthormyxovirus thogoto interact with cbp to modulate type i ifn induction, suppress protein expression, or promote virus infection (ferrari et al., ; jain et al., ; jennings et al., ; wurm et al., ; zhang et al., ) . degradation of cbp has been described for the porcine arterivirus prrsv as a strategy for ifn antagonism . since the level of cbp was found to decrease in pedv nsp -expressing cells (fig. a) , cbp expression was validated in pedv-infected cells by co-staining using anti-cbp antibody and anti-pedv m pab. in uninfected cells, cbp was predominately localized in the nucleus in marc- and vero cells (fig. b, yellow arrows) . in contrast, cbp was depleted in virus-infected cells (fig. b , white arrows), demonstrating that the cbp was degraded by pedv. we further sought to study whether the cbp degradation by pedv was mediated by nsp protein. cbp was exclusively nuclear in control cells, whereas it was depleted in nsp -expressing cells (fig. a) . prrsv nsp α is known to degrade cbp in the nucleus , and in prrsv nsp αexpressing cells, cbp was significantly depleted (fig. a ). the cbp degradation was quantified by examining the ratio of nsp expressing cells showing cbp degradation out of the chosen number of nsp -expressing cells (fig. b ). approximately % of prrsv nsp α-expressing cells showed more than % reduction of cbp, which is in consistent with the previous report (han et al., ) . for pedv nsp -expressing cells, approximately % cells showed more than % reduction of cbp, while no cbp reduction was observed in control cells. this finding was confirmed by western blot. in pedv nsp -expressing cells, cbp degradation was evident compared to that of control cells (fig. c , top panel, lane ). to eliminate a possibility that the reduction of cbp might be due to the short half-life of cbp, cyclohexamide (chx) treatment was conducted (fig. d) . at h post-transfection, cells were treated with chx to shut down the new protein synthesis for indicated times followed by western blot. in nsp -expressing cells, cbp reduction was evident at the beginning of chx treatment, and further decreased by h post-treatment. the cbp degradation was complete by h post-treatment, whereas nsp and β-actin remained stable (fig. d) . together, our data show that pedv nsp was the viral protein contributing to the cbp degradation. unlike prrsv nsp α, pedv nsp does not contain a proteinase activity, and no direct interaction between cbp and nsp was identified in our study. it is thus unlikely that cbp would be a direct substrate of pedv nsp . therefore, it was of interest to examine whether the cbp degradation was a proteasomedependent process. the treatment with mg blocked the cbp degradation by nsp . as little as μm of mg was sufficient to inhibit the cbp degradation, and μm was able to restore the cbp level back to the control level (fig. e) . to eliminate the cbp degradation by nsp was cell-type specific, we further tested the cbp degradation by nsp in pig intestinal epithelial cell line (ipec-j cells), which reported to be susceptible to pedv (zhao et al., ) . cbp degradation in nsp -expressing cells was evident comparing to control cells (fig. f, top panel) . additionally, the cbp degradation by nsp was also blocked by mg treatment in ipec-j cells (fig. f, bottom panel) . this study indicates that the cbp degradation by pedv nsp was proteasome-dependent in the nucleus. the innate immune system is the first line of host defense in response to viral infection. it initiates the production of type i ifns and proinflammatory cytokines through the recognition of pamps by prrs and establishes antiviral states which are highly effective on resisting and controlling infections. in turn, many viruses have developed strategies to counteract the host innate immune response to establish productive infection. previous studies have shown that pedv infection fail to induce the ifn-β promoter activation and that plp (papain-like proteinase ) of pedv antagonizes the ifn response by deubiquitinating rig-i and sting (xing et al., b) . the pedv n protein suppresses the irf and nf-κb activities and antagonizes the ifn-β production by disrupting the interaction between irf and tbk (ding et al., ) . on the contrary, a recent study shows that pedv infection induces nf-κb activation in intestinal epithelial cells with the n protein as the activator (cao et al., b) . in the present study, we have identified marc- as pedv permissive cells, and used these cells as a model to study the innate immune modulation for pedv. we have shown the suppression of type i ifn production by pedv, which is consistent with the recent finding in iecs (cao et al., a) . we also have identified multiple viral proteins responsible for this suppression. we have further determined pedv nsp as the viral component promoting cbp degradation in the nucleus via the proteasome-dependent pathway. many viruses in the order nidovirales are able to modulate the host innate response, which plays an important role for their pathogenesis. in the family arteriviridae, equine arteritis virus suppresses type i ifn production in equine endothelial cells (go et al., ) , and prrsv also suppresses ifn production (albina et al., ) . prrsv is susceptible to type i ifns in cells and the suppression of type i ifn varies for different isolates (albina et al., ; lee et al., ; overend et al., ) . mouse hepatitis virus (mhv), which is a betacoronavirus, induces a high level of ifn-α secretion by plasmacytoid dendritic cells (pdcs) during infection (cervantes-barragan et al., ) . however, other cell types infected by mhv such as macrophages, microglia, and oligodendrocytes produce extremely low-levels of type i ifns (li et al., ; roth-cross et al., ; zhou and perlman, ) . the mhv ns protein is dispensable for virus replication in cells but is required for induction of hepatitis in mouse (schwarz et al., ) . the , -phosphodiesterase (pde) activity of ns mediates the cleavage of , -oligoadenylate and prevents the activation of rnase l, while enhancing viral growth and pathogenesis, thus ns is a viral ifn antagonist (zhao et al., ) . sars-cov, which is another member virus in the genus betacoronavirus, impairs the ifn response in virus-infected cells, and an ifn therapy has been suggested to be efficacious for sars patients (cinatl et al., ; spiegel et al., ) . mers-cov is also a betacoronavirus, and both mers-cov and sars-cov do not induce a pronounced ifnresponse in polarized airway epithelial cells (calu- ), alveolar adenocarcinoma cells (a ) and human monocyte-derived macrophages (lau et al., ; zhou et al., ; zielecki et al., ) . even though the acute infection of tgev induces a highlevel of ifn-α in newborn pigs (la bonnardiere and laude, ) , protein counteracts the host antiviral response and influences viral pathogenesis (cruz et al., , . the a protein of an alphacoronavirus feline infectious peritonitis virus is a type i ifn antagonist (dedeurwaerder et al., ) . type i ifns of chickens inhibits viral replication and respiratory illness of the gammacoronavirus infectious bronchitis coronavirus (ibv) (pei et al., ) . ibv delays the ifn response and the a and b accessory proteins have been identified as the ifn antagonists (kint et al., ) . thus, modulation of type i ifn response seems to be a common evasion strategy of viruses in the order nidovirales. we have shown in the present study the direct evidence that pedv indeed downregulates type i ifns production during infection. pedv suppresses the ifn-β and irf activities. since irf is a key element in the production of type i ifns, our finding leads to a hypothesis that pedv modulation of type i ifns production targets the irf signaling pathway. interestingly, pedv normally activates the nf-κb activity in vero e cells (xing et al., b) . a recent study confirms that pedv infection in intestinal epithelial cells induces nf-κb activation (cao et al., b) . in that study, nuclear localization of p increases by pedv after h through h. however, activation of nf-κb during viral infection is generally an early event. for prrsv, nf-κb is activated min after infection (fu et al., ) . thus, how pedv modulates nf-κb activation during early time of infection needs to be further investigated. we have identified at least ten viral ifn antagonists and all ten proteins inhibit the irf activity. whether these ifn antagonists modulate the nf-κb activity needs to be further investigated. the pedv n protein suppresses sendai virus-induced nf-κb activity in a dose-dependent manner (ding et al., ) . in other study, n protein activates nf-κb in intestinal epithelial cells (cao et al., b) . a possible explanation is that the nf-κb activation may be time-dependent and cell type-dependent. together, the irf signaling is likely the target by pedv for type i ifns modulation. at least eleven viral proteins have been identified as ifn antagonists for sars-cov (kindler and thiel, ; shi et al., ; totura and baric, ) , whereas ten proteins have been identified for pedv in our study. thus, coronaviruses seem to arm with multiple antagonists. a possible explanation for such a functional redundancy is that coronavirus genomes are the largest rna known to biology and undergo continuous genetic evolution. when a functional mutation occurs in a major antagonist, other antagonists may complement the function to ensure efficient replication and adaptation in hosts. for sars-cov, nsp is a multifunctional protein with the suppressive activity for ifn and blocks the phosphorylation of stat and degrades host cell mrna (totura and baric, ) . sars-cov nsp and nsp works as exoribonuclease and endoribonuclease, respectively, thus specific digestion of dsrnas and the consequent removal of rna-pamps may lead to an inadequate activation of ifn response (kindler and thiel, ) . sars-cov nsp contains -omethlytransferase activity and modifies the cap of viral rnas in order to evade the detection by the host immune system (totura and baric, ) . the sars-cov m protein impedes the formation of traf Á tank Á tbk /ikkϵ complex for suppression of type i ifn production (siu et al., (siu et al., , . the plp domain of sars-cov nsp negatively modulates type i ifn pathway and functions as a viral deubiquitinase. in our study, the full length pedv nsp indeed inhibit the ifn activity. all ten antagonists identified for pedv correspond to the respected antagonists of sars-cov. the corresponding proteins of pedv may share the similar motifs and functions with those of sars-cov. additionally, sars-cov encodes several accessory proteins. they are nonessential for viral replication but function as innate immune antagonists. for pedv, orf is the sole accessory protein, and a previous report shows that orf functions as an ion channel protein and is relevant to infectivity and pathogenicity (wang et al., ) . orf is nonessential for viral replication in vitro as shown by targeted rna recombination . in our study, orf is a potent ifn antagonist. the viral antagonists may target different pathways of the host innate immune signaling and their synergistic effects may shut down the host innate immune response more efficiently during the course of infection. cbp is a histone acetyltransferase and plays a key role in transcription regulation. the cbp/p coactivators interact with hundreds of transcription factors including stats, c-myc, pias , p , nf-κb, and irf family (bedford and brindle, ; goodman and smolik, ; long et al., ) . for ifn expression, the assembly of an enhanceosome consisting of nf-κb, irfs, atf /c-jun, and the architectural protein hmg i(y) is required in response to virus infection. the ifn enhanceosome recruits cbp/p for synergistic activation of transcription (merika et al., ) . some viruses modulate the cbp activity for viral evasion. two distinct regions in the simian virus t antigen can independently alter the levels and loading of cbp/p transcripts onto polysomes for cell immortalization and transformation (robles et al., ) . african swine fever virus nuclear protein a l inhibits the expression of tnf-α by displacing the cbp/p coactivators (granja et al., ) , and herpes simplex virus (hsv- ) icp protein recruits activated irf and cbp/p to the nuclear foci, which may result in reduced transcription of ifn-β and inhibition of the host response (melroe et al., ) . hsv- vp protein inhibits nf-κb activation and interferes the recruitment of irf to cbp to block the ifn-β production (xing et al., a) . the ml protein of thogoto virus interferes with irf function without blocking its nuclear translocation but interrupts the association of irf with cbp (jennings et al., ) , which is similar to the function of pedv nsp . the ml protein was later found to interact with the rna polymerase ii transcription factor iib (tfiib), however, this interaction hardly interferes the host general gene expression but strongly suppresses both the irf -and nf-κbregulated promoter activities (vogt et al., ) . thus, it is hypothesized that the virus-mediated cbp degradation may play a specific and key role for ifn modulation with a little impact on general cellular gene transcriptions. the degradation of cbp is a novel strategy for ifn modulation and has been extensively studied in the family of arteriviridae, especially for prrsv . for prrsv nsp α, cbp degradation is associated with the zinc-finger motif and is likely the key mechanism for ifn suppression (han et al., ) . for pedv, nsp is the most potent ifn suppressor among all viral antagonists without affecting the irf phosphorylation and nuclear localization. in line with this, pedv infection depletes the endogenous cbp. furthermore, pedv nsp disrupts the association of cbp-irf and degrades cbp in a proteasome-dependent manner. sars-cov nsp inhibits type i ifn production, induces host mrna degradation, and suppresses host protein translation (narayanan et al., ) . however, the domains of sars-cov nsp responsible for suppression of host gene expression and type i ifn production are absent in pedv nsp (huang et al., b; narayanan et al., ) . even though nsp s of alphacoronavirus and betacoronavirus share similar functions, they lack an overall sequence similarity and neither conserved motifs nor domains exist in viruses of alphacoronaviruses. thus, it is plausible that nsp of alphacoronaviruses may have a distinct function regulating host innate immune responses and gene expression. tgev nsp suppresses protein translation in cells and cell-free extracts. however, the suppression of protein translation by pedv nsp may not be a general event since the β-actin shows the similar level of expression after infection and transfection. the lack of association of cbp-nsp and irf -nsp suggests that the cbp degradation by nsp is an indirect event that needs to be further determined. similar to tgev nsp , the subcellular localization of pedv nsp is nuclear-cytoplasmic. the sequence of pedv nsp does not harbor any known nuclear localization signal, and thus nsp may piggy-bag a nuclear protein to enter the nucleus. the proteasome-dependent cbp degradation seems a unique viral tactic utilized to inhibit ifn-β production. it is of interest to study whether this is a common evasion strategy for coronaviruses. cbp localizes in the pml nuclear bodies, which are discrete nuclear foci that are disrupted in acute promyelocytic leukemia (boisvert et al., ; doucas et al., ; lamorte et al., ) . the pml nuclear bodies dynamically colocalize with numerous proteins including cbp, pml, p , rb, sp , daxx, eif e, and sumo (jensen et al., ) . upon inhibition of proteasome activity, pml, sp , ebna- , sumo- , and the s proteasome subunit move to the nucleolus, suggesting that proteasomal degradation occurs at the nuclear loci (boddy et al., ) . hausp, the ubiquitin-specific hydrolase in the pml nuclear bodies, removes ubiquitin moieties from proteins prior to proteasomal degradation (everett et al., ) . thus, pml nuclear bodies may represent the sites where ubiquitinated proteins are processed by enzymes such as hausp prior to degradation in the nucleolus (st-germain et al., ) . valproic acid, a histone deacetylase inhibitor, could induce cbp degradation through the ubiquitin-proteasome pathway, while increasing the colocalization of cbp with ubiquitin nuclear speckles and with pml nuclear bodies (st-germain et al., ) , suggesting that pml nuclear bodies may be the sites for the ubiquitin-dependent degradation of cbp. it is of interest to examine whether pedv nsp promotes ubiquitination of cbp for degradation in the nucleus and whether this degradation associates with pml nuclear bodies. pedv infects vero cells and marc- cells. porcine amino peptidase n (papn) has been identified as the major cell entry receptor for pedv (li et al., ; nam and lee, ) . transient expression of papn confers pedv non-permissive canine kidney cells (mdck) to be permissive for pedv infection. papn also increases the pedv infectivity in porcine small intestine epithelial cells (iecs) (cong et al., ) . the respiratory tract may support pedv infection in pigs and the virus infects and replicates in transformed alveolar macrophages ( d ) in vitro (park and shin, ) . primate apn or receptor-independent pathways in vero and marc- cells may complement the function of papn for pedv infection (taguchi and matsuyama, ) . in summary, we have shown the suppression of type i ifn production by pedv and have identified specific viral ifn antagonists. among these antagonists, nsp is the most potent protein and functions to degrade cbp in the nucleus. our data provides a novel insight into the understanding of the immune evasion strategy of pedv. hela cells (nih aids research and reference reagent program, germantown, md) and marc- cells (kim et al., ) reagent was purchased from invitrogen (carlsbad, ca). qiaamp viral rna mini kit and rneasy mini kit were purchased from qiagen (venlo, limburg). power sybr green pcr master mix was purchased from life technologies (carlsbad, ca). alexa fluor conjugated (goat anti-rabbit, red) and -conjugated (goat antimouse, green) secondary antibodies and pierce™ ecl western blotting substrate were purchased from thermo scientific (waltham, ma). the firefly luciferase genes were used as reporters with its expression under the control of various promoters as indicated below. the plasmid pifn-β-luc contains the entire ifn-β enhancer-promoter. the plasmid p  irf -luc contains four copies of irf binding region prd i-iii of the ifn-β promoter. pifn-β-luc and p  irf -luc were obtained from dr. stephan ludwig at heinrich-heine-universität, düsseldorf, germany (ehrhardt et al., ) . the renilla luciferase plasmid prl-tk (promega) contains the herpes simplex virus thymidine kinase (hsv-tk) promoter and was included in all experiments to serve as an internal control. active stimulator pmavs/ips- was obtained from dr. j. shisler (university of illinois, urbana, il). pirf was kindly provided by dr. b. gotoh (university of fukui, fukui, japan). pdsred -er and pdsred -mito were purchased from clontech. plasmids with the flag tag for expression of nsp through nsp , and the s, s , s , orf , e, m and n genes were cloned from the viral genomic rna by standard reverse transcription and pcr techniques using indicated primers (table ) . twenty-three viral genes were amplified and cloned into the eukaryotic expression vector pxj using indicated restriction enzymes. the nsp to nsp , orf , and n genes were expressed as fusion proteins with the n-terminal flag tag, and the s, s , s , e, and m genes were expressed as fusion proteins with the c-terminal flag tag to avoid the functional disruption of the signal sequence. the constructs were confirmed by sequencing, immunofluorescence, and western blot. prrsv nsp α and its cystine mutant p-nsp α(m) (c s) are described elsewhere (han et al., ; song et al., ) . hela cells were seeded in -well plates and grown to % confluency prior to transfection. individual viral protein genes, luciferase reporters, and prl-tk as an internal control were transfected at a ratio of : : in a total of . μg/well using lipofectamine according to the manufacturer's instruction (invitrogen). at h post-transfection, cells were stimulated by transfection with . μg/well of poly(i:c) for h. cells were then lysed and luciferase assays were performed using the dual luciferease assay system according to the manufacturer's instructions (promega). values were normalized using the renilla luciferase activity as the internal control and presented in fold-changes. three independent assays were performed with each assay in triplicate. total rna was extracted from hela or marc- cells using rneasy mini kit according to the manufacturer's instructions (qiagen). the rna was treated with dnase i to remove contaminating genomic dna. reverse transcription (rt) reaction was performed with mg of total rna using random primers and m-mlv reverse transcriptase (invitrogen). sybr green real-time pcr was conducted in the abi real-time pcr system according to the manufacturer's instructions (life technologies). the real-time pcr primers for ifn-β, isg , isg and β-actin were listed in table . for each sample, the β-actin gene was amplified and used as an internal control. specific amplification was confirmed by sequencing pcr products. the threshold cycle for target genes and the difference between their c t values (Δc t ) were determined. the relative transcript levels of target gene are equal to À ΔΔct threshold method (livak and schmittgen, ) and are shown as fold changes relative to the respective untreated control samples. hela cells were seeded in -well plates and transfected with μg of plasmid. at h post-transfection, cells were stimulated by transfection with μg of poly(i:c) for h. supernatants were harvested for bioassay. for pedv, marc- cells were infected with pedv at an moi of for h prior to poly(i:c) stimulation. supernatants from virus-infected cells were uv-irradiated for min to remove infectivity prior to bioassay. the supernatants were then serially diluted by -fold. marc- cells were freshly grown in -well plates and incubated with μl of each dilution for h. cells were then infected in μl of vsv-gfp at pfu/ ml for h and gfp expression was examined by inverted fluorescence microscopy (nikon eclipse ts ,  ). each dilution was examined twice in triplicate each. indirect immunofluorescence assay (ifa) and confocal microscopy cells were seeded on coverslips and transfected with plasmids or infected with pedv. for transfection of hela cells, total mg of individual plasmids were transfected for h using lipofectamine table primers used for the cloning of pedv nonstructural and structural genes (pedv strain usa/colorado/ ). restriction enzyme recognition sequences are underlined. the flag tag is italicized and underlined. primers used for relative quantitative real-time rt-pcr. ifn-β-f gatttatctagcactggctgg ifn-β-r cttcaggtaatgcagaatcc isg -f caccgtgttcatgaatctgc isg -r ctttatttccggcccttgat isg -f cctccttgggttcgtctaca isg -r ggctgatatctgggtgccta β-actin-f atcgtgcgtgacattaag β-actin-r attgccaatggtgatgac according to the manufacturer's instructions (invitrogen). cells were then either treated with poly(i:c) for h or ifn-β for min. cells were fixed with % paraformaldehyde in pbs overnight at °c and permeabilized using . % triton x- for min at room temperature (rt). after blocking with % bsa in pbs at rt for min, cells were incubated with a primary antibody in pbs for - h. cells were then washed three times with pbs and incubated with alexa fluor -labeled anti-mouse secondary antibody, or alexa fluor -labeled anti-rabbit secondary antibody (thermo scientific) for h at rt in the dark. cells were incubated with dapi for min at rt for nuclear staining. after washing with pbs, cover slips were mounted on microscope slides using fluoromount-g mounting medium (southern biotech, birmingham, al), and visualized by fluorescence microscopy (nikon eclipse ts ). confocal microscopy was conducted as described elsewhere (kannan et al., ) . hela cells were seeded in -well plates to % confluency and transfected with μg/well of nsp plasmid for h. cells were stimulated with μg of poly(i:c) for h and fractionated using the nuclear/cytosol fractionation kit (biovision, milpitas, ca) with minor modifications. briefly, cells were washed with cold pbs and collected using cell scrapers in ml of cold pbs. cell pellets were resuspended in μl ceb-a buffer and incubated on ice for min. after addition of ceb-b, tubes were vortexed and incubated on ice for min. the cell lysates were then centrifuged at °c min at , g and supernatants were collected as the cytosolic fraction. the cell pellets were suspended in neb buffer and vortexed for s and repeated times every min. the nuclear pellets were finally centrifuged for min at °c , g and kept the supernatants as the nuclear fraction. cells were harvested in ripa buffer [ mm tris (ph . ), mm nacl, mm edta, mm phenylmethanesulphonyl fluoride (pmsf), . % sds, . % sodium deoxycholate, % np- ] containing the proteinase inhibitors cocktail (promega). cells were frozen-thawed, collected in the pre-cold tubes, and centrifuged to remove insoluble components. total protein concentration was determined using pierce bca protein assay kit (thermo scientific). equal amounts of proteins were resolved by sds-page and blotted to pvdf membranes (millipore). after blocking with % nonfat dry milk in tbst ( . % tween- ) for h, membranes were incubated with a primary antibody in tbst containing % nonfat dry milk overnight at °c, followed by washing and incubation with horseradish peroxidase (hrp)-conjugated secondary antibody for h at rt. the membrane was visualized using pierce ecl western blotting substrate (thermo scientific) and images were taken by fluorchem™ r system according to the manufacturer's instructions (proteinsimple). co-immunoprecipitation (co-ip) was performed as described previously with modifications (kim et al., ) . gene-transfected cells were lysed in lysis buffer [ mm tris (ph . ), mm nacl, mm na vo , mm pmsf, mg/ml leupetin, % np- , % glycerol] supplemented proteinase inhibitors cocktail (promega). cell lysates were clarified by centrifugation at °c for min at , g. supernatants were transferred to fresh tubes and incubated with either flag-or irf -antibody at °c overnight, followed by incubation with protein g agarose beads (fast flow, millipore) at °c for h. pellets were collected by centrifugation and washed for five times. the final pellets were eluted with laemmli sample buffer (bio-rad) and were subjected to western blot. student's t-test was used for all statistical analyses. asterisks indicate the statistical significance. *po . , **p o . and ***p o . . interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus is histone acetylation the most important physiological function for cbp and p pic , a novel ubiquitin-like protein which interacts with the pml component of a multiprotein complex that is disrupted in acute promyelocytic leukaemia the transcription coactivator cbp is a dynamic component of the promyelocytic leukemia nuclear body porcine epidemic diarrhea virus inhibits dsrna-induced interferon-β production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway porcine epidemic diarrhea virus infection induces nf-kappab activation through the tlr , tlr , and tlr pathways in porcine intestinal epithelial cells control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in china isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states role of interferons in the treatment of severe acute respiratory syndrome porcine aminopeptidase n mediated polarized infection by porcine epidemic diarrhea virus in target cells alphacoronavirus protein modulates host innate immune response coronavirus gene counteracts host defenses and modulates virus virulence experimental infection of pigs with a new porcine enteric coronavirus, cv orf -encoded accessory protein a of feline infectious peritonitis virus as a counteragent against ifn-alpha-induced antiviral response defectiveness of interferon production and of rubella virus interference in a line of african green monkey kidney cells (vero) porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf and tbk modulation of creb binding protein function by the promyelocytic (pml) oncoprotein suggests a role for nuclear bodies in hormone signaling mechanisms of activation of interferon regulator factor : the role of c-terminal domain phosphorylation in irf- dimerization and dna binding genome organization of porcine epidemic diarrhoea virus rac and pak are upstream of ikk-epsilon and tbk- in the viral activation of interferon regulatory factor- the disruption of nd during herpes simplex virus infection correlates with the vmw -and proteasome-dependent loss of several pml isoforms adenovirus small e a employs the lysine acetylases p /cbp and tumor suppressor rb to repress select host genes and promote productive virus infection ikkepsilon and tbk are essential components of the irf signaling pathway porcine reproductive and respiratory syndrome virus induces interleukin- through the nf-kappab signaling pathway equine arteritis virus does not induce interferon production in equine endothelial cells: identification of nonstructural protein as a main interferon antagonist the viral protein a l inhibits tnf-alpha expression through a cbp/p transcriptional coactivators pathway degradation of creb-binding protein and modulation of type i interferon induction by the zinc finger motif of the porcine reproductive and respiratory syndrome virus nsp alpha subunit modulation of innate immune signaling by nonstructural protein (nsp ) in the family arteriviridae propagation of the virus of porcine epidemic diarrhea in cell culture irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors alphacoronavirus transmissible gastroenteritis virus nsp protein suppresses protein translation in mammalian cells and in cell-free hela cell extracts but not in rabbit reticulocyte lysate sars coronavirus nsp protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp -induced rna cleavage porcine reproductive and respiratory syndrome virus nonstructural protein antagonizes beta interferon expression by targeting the nf-kappab essential modulator myocyte enhancer factor (mef)- plays essential roles in t-cell transformation associated with htlv- infection by stabilizing complex between tax and creb thogoto virus ml protein suppresses irf function pml protein isoforms and the rbcc/trim motif cloning and sequence analysis of the n gene of porcine epidemic diarrhea virus ljb/ the hepatitis e virus open reading frame product interacts with microtubules and interferes with their dynamics toll-like receptors and their crosstalk with other innate receptors in infection and immunity enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein in marc- and hela cells to sense or not to sense viral rna -essentials of coronavirus innate immune evasion activation of the chicken type i interferon response by infectious bronchitis coronavirus high interferon titer in newborn pig intestine during experimentally induced viral enteritis localization of nascent rna and creb binding protein with the pml-containing nuclear body delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment detection of porcine epidemic diarrhea virus by immunohistochemistry with recombinant antibody produced in phages porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes porcine aminopeptidase n is a functional receptor for the pedv coronavirus manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda new variants of porcine epidemic diarrhea virus virus-dependent phosphorylation of the irf- transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method severe acute respiratory syndrome coronavirus protein nsp is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation activation of smad transcriptional activity by protein inhibitor of activated stat (pias ) complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/ from the united states recruitment of activated irf- and cbp/p to herpes simplex virus icp nuclear foci: potential role in blocking ifn-beta induction recruitment of cbp/ p by the ifn beta enhanceosome is required for synergistic activation of transcription contribution of the porcine aminopeptidase n (cd ) receptor density to porcine epidemic diarrhea virus infection severe acute respiratory syndrome coronavirus nsp suppresses host gene expression, including that of type i interferon, in infected cells coronavirus nonstructural protein : common and distinct functions in the regulation of host and viral gene expression recombinant swine beta interferon protects swine alveolar macrophages and marc- cells from infection with porcine reproductive and respiratory syndrome virus an atomic model of the interferon-beta enhanceosome receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion porcine epidemic diarrhea virus infects and replicates in porcine alveolar macrophages porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat /stat nuclear translocation chicken interferon type i inhibits infectious bronchitis virus replication and associated respiratory illness chinese-like strain of porcine epidemic diarrhea virus viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia two independent regions of simian virus t antigen increase cbp/p levels, alter patterns of cellular histone acetylation, and immortalize primary cells murine coronavirus nonstructural protein ns is not essential for virus replication in transformed cells triggering the interferon antiviral response through an ikk-related pathway sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk /ikkepsilon complex nonstructural protein alpha subunit-based inhibition of nf-kappab activation and suppression of interferon-beta production by porcine reproductive and respiratory syndrome virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor involvement of pml nuclear bodies in cbp degradation through the ubiquitin-proteasome pathway the jak-stat pathway at twenty emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an immunohistochemical investigation of porcine epidemic diarrhoea outbreak of porcine epidemic diarrhea in suckling piglets china interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus soluble receptor potentiates receptorindependent infection by murine coronavirus severe acute respiratory syndrome coronavirus nsp facilitates efficient propagation in cells through a specific translational shutoff of host mrna sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon the interferon antagonist ml protein of thogoto virus targets general transcription factor iib pedv orf encodes an ion channel protein and regulates virus production nsp proteins of group i and sars coronaviruses share structural and functional similarities proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture an apparently new syndrome of porcine epidemic diarrhoea the htlv- -encoded protein hbz directly inhibits the acetyl transferase activity of p / cbp herpes simplex virus -encoded tegument protein vp abrogates the production of beta interferon (ifn) by inhibiting nf-kappab activation and blocking ifn regulatory factor to recruit its coactivator cbp the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from human t-cell leukemia virus type tax modulates interferon-alpha signal transduction through competitive usage of the coactivator cbp/p antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j cells mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus key: cord- -cdnkk ou authors: gabor, kristin a.; stevens, chad r.; pietraszewski, matthew j.; gould, travis j.; shim, juyoung; yoder, jeffrey a.; lam, siew hong; gong, zhiyuan; hess, samuel t.; kim, carol h. title: super resolution microscopy reveals that caveolin- is required for spatial organization of crfb and subsequent antiviral signaling in zebrafish date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: cdnkk ou understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. caveolae are integral to numerous signaling pathways and these membrane domains have been previously implicated in viral entry but not antiviral defense. this study shows, for the first time, the importance of spatio-temporal regulation of signaling receptors and the importance of the regulation of clustering for downstream signaling. a novel mechanism for virus evasion of host cell defenses is demonstrated through disruption of clusters of signaling molecules organized within caveolin-rich domains. viral infection leads to a downregulation in caveolin- b (cav- b), disrupting clusters of crfb , a zebrafish type i interferon receptor (–r) subunit. super-resolution microscopy has enabled the first single-molecule imaging of crfb association with cav- b-containing membrane domains. strikingly, downregulation of cav- b, the major protein component of caveolae, caused crfb clusters to disperse. dispersal of crfb clusters led to a suppressed antiviral immune response both in vitro and in vivo, through abrogation of downstream signaling. this response strongly suggests that crfb organization within cav- b-containing membrane domains is critical for ifn-mediated antiviral defense and presents a previously undescribed antiviral evasion strategy to alter ifn signaling and the antiviral immune response. the structure and organization of cellular membranes play important roles in a wide range of biological processes. caveolae are specialized membrane nanodomains with a distinct v-shaped morphology in the membrane. caveolae may act as signaling platforms by allowing signaling molecules to cluster together within their ordered domains, facilitating interactions among the components [ ] . critical cellular processes associated with caveolae include signal transduction, cholesterol homeostasis, and adaptive immune signaling [ , , , , , ] . caveolin- (cav- ) serves as one of the structural components of caveolae and also functions as a scaffolding protein that recruits signaling molecules to caveolae [ ] . clustering of proteins in caveolae provides an environment and a mechanism for controlling probabilities of protein interaction and modulating the efficiency of signal transduction. for example, epidermal growth factor receptor (egfr) has been shown to interact with caveolin [ ] and changes in receptor clustering may provide a mechanism for regulating egfr signaling [ ] . in addition, caveolae are exploited by some viruses to initiate infection [ , ] , whereas other viruses enter cells without the involvement of caveolae [ , , , , , ] . for example, damm et al. [ ] observed that when introduced to cells devoid of caveolae, sv exploits an alternative, cav- -independent pathway in the absence of caveolae. ewers et al. [ ] , used transmission electron microscopy to demonstrate that sv induced the formation of membrane invaginations in the absence of caveolar coats. it has also been determined that ebola virus can fully infect cell types lacking caveolae [ ] and that sars coronavirus entry was mediated by a clathrin-and caveolae-independent mechanism [ ] . type interferon (ifn) is crucial for initiation of the innate response to viral infection, and knockout studies in mice have shown that disruption of this response renders the host more susceptible to infection. other studies have demonstrated that mice lacking a functional ifn receptor (ifn-r) were unable to cope with an array of viral infections, including vaccinia virus, vesicular stomatitis virus (vsv), and semliki forest virus [ ] . ifn-r knockout mice were highly susceptible to infection with vsv due to high levels of viral replication [ ] . the relationship between cell membrane organization and the antiviral immune response is largely unexplored. one of the primary antiviral responses is the generation of ifn, and only recently has the role of lipid rafts in interferon production come under scrutiny [ ] . ifn-r and caveolin- have both been found in detergent-resistant membrane (drm) fractions [ ] , but have not been observed with sufficient spatial resolution to determine their nanoscale distribution in intact cell membranes. such evidence could provide critical insights into the spatial and temporal organization of antiviral receptors and nanodomains. it has previously been shown that zebrafish infected with snakehead rhabdovirus (shrv) produce an ifn response [ , ] , leading to the binding of ifn to its cognate receptor, ifn-r. the zebrafish ifn-r complex has been recently identified as cytokine receptor family members crfb , crfb , and crfb , which constitute crfb /crfb and crfb /crfb heterodimers [ , ] . the jak-stat signal transduction pathway is activated upon ifn binding to the receptor and culminates in the expression of ifn-stimulated response element (isre) driven ifn-stimulated genes (isgs) [ ] . the ifn pathway and ensuing antiviral response of zebrafish is similar to that described in mammalian systems [ , , , ] . many properties of membrane domains cannot be understood solely from drm studies [ ] , because drms isolated from cells may not correspond precisely to preexisting rafts in living cells [ ] . the small size of caveolae and the spatial proximity of proteins prohibit direct visualization of the dynamic interaction between the host cell membrane nanodomains and antiviral receptors using conventional light microscopy. fluorescence photoactivation localization microscopy (fpalm) ( figure s ) [ , ] is a novel, super resolution technique that extends the resolution of optical microscopy below the diffraction limit, which is on the order of nm, allowing for spatial resolution on the scale of - nm [ , , ] . three-dimensional fpalm has achieved a lateral resolution of nm and nm axially [ ] . fpalm is well suited for investigation, at the single molecule level, of the highly complex molecular structures and mechanisms underlying biological processes. few investigators have focused on the role of caveolae in the immune response. our results suggest that crfb interacts with caveolae and that caveolae may be critical for maintaining spatial organization and clustering of crfb molecules. the present study demonstrates a novel role for cav- b-containing membrane domains in the zebrafish response to viral infection. we demonstrate that upon virus infection, cav- is downregulated, circumventing the host antiviral ifn response. in vivo knockdown studies showed that disruption of the ifn response by cav- depletion renders the host more susceptible to infection. using fpalm, we show that cav- b-containing membrane domains corral crfb molecules together and that this clustering of crfb is critical for a robust antiviral immune response. in addition, we determined that the membrane protein cav- is responsible for maintaining the crfb clustering and that the functional consequence of cav- depletion is crfb dispersion and abrogation of downstream signaling. by gaining an under-standing of the complex dynamics of membrane domains and the mechanisms through which viruses modulate their function, we will better understand how viruses evade host antiviral mechanisms and can implement this knowledge to develop more targeted therapeutics. we investigated the membrane localization of the crfb subunit of the zebrafish ifn-r complex, the components of which are necessary for a functional ifn response in the zebrafish [ ] . to test whether crfb localizes to cav- b-containing membrane domains, fpalm was used to simultaneously image crfb -dendra and cav- b-pamcherry h after transfection of zebrafish liver (zfl) cells. zfl cells express endogenous cav- b and crfb mrna ( figure s a ), as do rat liver cells [ ] , liver sinusoidal cells [ ] and primary rat hepatocytes [ ] . figure illustrates that at the surface of a single cell, crfb colocalizes with cav- b. acquisition conditions and more details about fpalm imaging and analysis are described in the methods section and figure s . these data were acquired in the absence of ligand stimulation and show that clusters of cav- b molecules are in very close proximity to crfb molecules, and in many instances overlap within the estimated spatial resolution of the technique (, nm). to quantitatively explore this result, pair correlation analysis was performed for crfb and cav- b ( figure c ). pair correlation between the two species had a g(r) value greater than one, which implies that the two molecules are not randomly distributed, and instead, are colocalized. additionally, when cav- b is knocked down in zfl cells using a previously characterized morpholino oligonucleotide (mo) [ ] , the clustering of crfb is significantly decreased, with a more random distribution than observed in controls ( figure d ). the observation of colocalization between cav- b-containing membrane domains and crfb molecules led to the investigation of whether cav- plays a role in the antiviral response to virus infection, since ifn is a critical component of the innate immune response. the roles of both cav- a and cav- b in zebrafish development have been previously revealed using mo knockdown technology [ ] . further, the presence of caveolae in zebrafish has been confirmed via electron microscopy [ ] . compared to cav- a, cav- b in the zebrafish is more similar to cav- b in human and mouse, and in previous studies the two isoforms have been shown to have non-redundant roles [ ] . our studies revealed that although cav- a gene expression was also downregulated after shrv infection ( figure s a ), the effect was not as pronounced, nor was it as long lasting as the downregulation of cav- b gene expression ( figure a) . furthermore, when compared to controls, knockdown of cav- b resulted in greater mortality than knockdown of cav- a after shrv infection ( figure s b ), leading us to focus our subsequent studies on cav- b. in embryos infected with shrv, early cav- b gene expression was shown by quantitative rt-pcr to be significantly dampened at , , and hpi, with a . -, . -, and . -fold decrease in transcript levels compared to controls, respectively (figure a , p, . ). in order to confirm that during viral infection general suppression of all host gene expression did not occur, zebrafish bactin primers were used to normalize the initial quantity of rna, as previously described [ ] . to confirm these results, the s housekeeping gene was also used to normalize the gene expression in the rt-pcr experiments. the s gene has been previously characterized in the zebrafish and shown to be stable during development and across tissue types [ , , ] . the s gene was selected due to its high, relatively stable expression levels. if viral infection globally affected gene expression, then s would also be influenced. however, similar results (data not shown) were obtained when s primers were used to normalize the quantity of rna. knockdown of cav- b with mo in embryos has been characterized previously and a reduction in cav- b protein levels, as well as a reduction in the number of caveolae domains, was demonstrated [ , ] . we performed knockdown experiments as described [ , ] after confirming the amount of mo required to knock down cav b. figure d demonstrates that at hpi in control mo-injected embryos cav- protein was still detected, while in cav- b mo-injected embryos no cav- protein was found. western blot analysis of age-matched shrv infected embryos detected endogenous cav- protein at lower levels than in control mo embryos, but greater than in the cav- b mo samples. this result shows effective knockdown of cav- b protein expression by morpholino injection (figure d ). these experiments were performed using whole embryo lysates, and so in order to more thoroughly understand the results, we sought to identify tissues in which cav- b is expressed. we examined cell typespecific pools of cdna from adult zebrafish. of particular interest, cav b expression was detected in liver, kidney, lymphocyte, and myeloid lineages ( figure s c ). in addition, cav- b and crfb are also expressed in the liver tissue of embryos when infection studies were performed ( figure s b ). the cav- b mo was used to determine whether the observed disruption of cav- b gene expression would alter the host's susceptibility to virus infection [ ] . morphant and control embryos were monitored for survival rates and viral burden. in the absence of virus, knockdown of cav- b did not affect embryo survival. kaplan-meier curves [ ] were constructed showing survival of cav- b morphants compared to controls after viral challenge, and revealed that cav- b morphant embryos exhibited a significant increase in mortality (p = . ) compared to the controls (figure b ). uninfected control morphants had low levels of mortality, similar to that of uninjected controls. cav- b morphants showed increased mortality throughout the first three days post infection. after just hpi, over % of the cav- b morphants had succumbed to the infection compared to , % of control infected embryos. controls and cav- b morphants that were uninfected both had , % or greater survival rates. since the adaptive immune response is not fully developed in zebrafish at the developmental stage selected for these studies [ , , ] , the results are due solely to perturbation of the innate immune response. we sought to determine whether the increase in mortality was a result of increased incidence of viral entry due to cav- knockdown, or reduced ability of morphant embryos to clear the infection. preliminary studies suggested that shrv does not utilize cav- b-containing membrane domains as a means of entry in vitro ( figure s ) ; therefore entry of virus should not be affected by cav- knockdown. viral burden assays were conducted to determine if disruption of cav- b mediated viral entry after infection with shrv. from - hpi, no significant increase in viral burden was observed between control and cav- b mo embryos, which were infected at hpf (and therefore h after mo injection). however, by - hpi, a significant increase in caveolin- is critical for antiviral signaling plos one | www.plosone.org viral burden (figure c ) was measured. cav- b morphants showed a -fold and . -fold increase in viral titer compared to controls at and hpi, respectively ( figure c ). the data were significant (two-way anova, p, . ) and correlated with the increased mortality shown at hpi and hpi in the cav- b morphant embryos. if cav- b-containing membrane domains are being used as a platform for immune signaling through the ifn-r pathway, knockdown of cav- b should dissipate antiviral signals, such as gene expression of stat and subsequent induction of isre. transcript levels of stat were assessed at h in both control mo and cav- b mo embryos with and without shrv infection. a . -fold ( . ) decrease was observed in control mo embryos and a . -fold ( . ) decrease was observed in cav- b mo embryos ( figure a) . to compare the effect of cav- b depletion on the antiviral response to pathogen, we examined control mo cells, cav- b mo cells, and control cells after shrv infection in an isre promoterdriven luciferase assay. zfl cells were transfected with cav- b mo or standard control mo, along with an isre luciferase construct [ ] , and subsequently exposed to shrv ( . moi for h) ( figure b ). cav- b depletion by cav- b mo in zfl cells is shown in figure s . shrv infected cells displayed a significant decrease in isre activity compared to control mo samples (twotailed student's t test, p, . ). similarly, depletion with cav- b mo also resulted in a significant decrease in isre activity compared to control mo samples (two-tailed student's t-test, p, . ), mimicking the effect of shrv infection. a greater reduction in isre activity was observed in either shrv-infected or cav- b mo cells when compared to control mo infected cells, a finding that is consistent with the decrease in stat gene expression shown in figure a . we examined whether disrupted ifn signaling resulting from cav- b depletion was due to dispersal of crfb molecules corralled by cav- b-containing membrane domains, or to effects on other antiviral components that could exist within cav- bcontaining membrane domains. covalent crosslinking studies were performed using bis(sulfosuccinimidyl) suberate (bs ) reagent with zfl cells that were transfected with either cav- b mo or standard control mo and subsequently crosslinked. the crosslinking reagent was employed to ''rescue'' the dispersal of crfb that results from cav- b disruption. if cav- b depleted cells with crosslinked crfb molecules were able to produce an antiviral response, this would indicate that cav- b depletion and subsequent dispersal of receptor molecules was directly responsible for the abrogated antiviral response. fpalm imaging demonstrated that infection of zfl cells with virus resulted in dispersion of crfb molecules ( figure ). similar numbers of crfb molecules are seen in the uninfected cell ( , ) compared to the infected cell ( , ) , which indicates that there is no overall loss of surface crfb as a result of infection. these results demonstrate that virus infection leads to dispersal of ifn receptors. control mo with crosslinking treatment yielded crfb molecules that remained clustered together, while cav- b depletion without crosslinking treatment yielded crfb molecules that were dispersed ( figure ). pair correlation analysis quantitatively confirmed the result of our fpalm images, showing that with cav- depletion and crosslinking treatment, the receptors remained clustered (figure d ). a parallel experiment was conducted in zfl cells that were transfected with control mo, cav- b mo, or crfb /crfb / crfb mo (all subunits of the ifn-r) in order to measure the induction of antiviral genes downstream from the ifn-r. polyinosinic-polycytidylic acid (poly(i:c)) was used in another experiment to mimic an infection and to stimulate the production of ifn by the immune system. poly(i:c) is a synthetic analog of double stranded rna (dsrna) which is associated with viral infection. it is recognized by pattern recognition receptors (prr) [ , ] and leads to the induction of type i ifn and inflammatory cytokines. cells were crosslinked with bs , exposed to poly(i:c), or both crosslinked with bs and exposed to poly(i:c) (figure a ). depletion of cav- b using the mo in zfl cells is demonstrated in figure s . time points shown correlate with the time of an additional experiment was performed with cells transfected together with both mo and cav- b plasmid to rescue the effect of the cav- b knockdown. cells were subsequently infected with shrv ( figure b ). transcripts of mxa were measured by quantitative rt-pcr. mxa was chosen because its transcripts are produced solely from the ifn-a/b pathway and not the ifn-c pathway [ ] . as expected, upon either poly(i:c) exposure ( figure a ) or shrv infection (figure b ) cav- b mo samples displayed decreased mxa expression compared to controls (second group of bars, gray), as did crfb /crfb /crfb mo samples (second group of bars, black). to rescue the effects of cav- b depletion, cells were also transfected with cav- b plasmid which resulted in detection of low levels of mxa in the absence of shrv (figure b ). cells were then infected with shrv and mxa gene expression was measured. with either crosslinking or cav- b rescue, mxa gene expression remained comparable to that of control cells (figure a and b, fourth group of bars in each, gray bars). we have thus demonstrated that when the depletion of cav b is rescued, the expression of mxa did not decrease. by developing a more thorough understanding of the mechanisms of the antiviral immune response, we hope to find clues that will aid the development of new therapeutics and vaccine adjuvants capable of augmenting the immune system and providing more effective protection to the host. this study demonstrates an entry-independent mechanism for virus evasion of host cell defenses through disruption of clusters of signaling molecules organized within cav- b-containing membrane domains. upon viral infection, cav- b was downregulated (figure a and d), leading to a decrease in the number of cav- b-containing caveolin- is critical for antiviral signaling plos one | www.plosone.org membrane domains [ ] . we report here the first nanoscale visualization of crfb association with cav- b-containing membrane domains in intact cells and demonstrate the dramatic effect that depletion of cav- b-containing membrane domains has on the antiviral response. the use of fpalm enabled imaging of the clustering and subsequent dispersal of crfb following cav- knockdown. the primary focus of the present studies was to investigate the potential abrogation of the antiviral response. the data show that in cav- b knockdown cells, crfb molecules are dispersed and cav- b-containing membrane domains are disrupted during viral infection, leading to impairment of the antiviral response. this suggests that intact caveolin domains may be crucial for proper clustering and function of crfb . receptor dispersal from cav- knockdown suppressed the antiviral immune response by disrupting downstream signaling, indicating that crfb organization within cav- b-containing membrane domains is critical for ifnmediated antiviral defense. the functional consequences of cav- depletion were shown by direct observation with fpalm of crfb clustering and identification of the membrane protein responsible for maintaining this clustered state. the crfb subunit of the zebrafish ifn receptor complex has been reported to heterodimerize with crfb [ ] . levraud et al. [ ] , assessed several candidates of the crfb family as likely members of the ifn receptor complex and found that knockdown of crfb and crfb have a dramatic effect on zebrafish ifn responsiveness. the authors postulated that the two should be designated as the heterodimer subunits of the ifn receptor. we have considered this interesting question in light of our current findings. we would like to know where crfb is localized and whether or not this receptor subunit also clusters or relies upon cav b-containing domains for a robust ifn response. such tantalizing questions are currently being investigated. type i ifn belongs to a class of cytokines that play a crucial role in the innate immune response to viral infection [ , ] . molecular patterns such as viral double stranded rna are detected by prr [ , ] , resulting in production of ifn and antiviral proteins. in zebrafish, as in mammals, ifn molecules interact with the ifn-r subunits [ , ] , which exist as heterodimeric complexes [ , ] . the janus kinase and signal transducer and activator of transcription (jak-stat) signaling pathway is highly conserved evolutionarily, and it is believed that in zebrafish, stat transduces signals through a classical jak-stat pathway [ ] . briefly, the jak-stat pathway becomes active upon ifn binding to ifn-r, but two discrete ifn pathways activate jak-stat: ifn-a/b and ifn-c. it is possible to measure components upstream from isre or mxa, such as stat phosphorylation, but nonspecific contributions from the ifn-c pathway can occur, making it difficult to discriminate between the jak-stat contributions of the two pathways. it has been hypothesized that the ifn-c response is attenuated due to reduced levels of stat in the ifn-r knockout [ ] . to investigate whether a reduced level of stat gene expression also contributed to a dampened ifn response in our current studies, stat transcripts were measured by qrt-pcr after cav- b depletion. a decrease in stat gene expression was observed at hpi (figure a ) as a result of cav- b depletion. we further demonstrate that crfb is dispersed and that downstream mxa signaling can be restored by maintaining crfb clusters ( figure ). this indicates that the clustering of crfb is critical for an antiviral response. cav- bcontaining membrane domains appear to corral the receptor molecules, thus providing an environment conducive to efficient signal transduction. previous studies in murine embryonic fibroblasts demonstrated that type i ifn receptors (ifn-r) and type ii ifn receptors (ifngr and ifngr ) were associated with caveolae domains after drm isolation [ ] . in contrast, ifnar and ifngr distribution in hela cells showed that only ifngr complexes could be found in drms after stimulation [ ] . the significance of protein associations with lipid rafts must therefore be reevaluated and interpreted with caution. in our studies, we use zfl cells, in which the endogenous expression levels of cav- b have been confirmed (data not shown). localization of ifn receptors in cav- b-containing membrane domains by microscopy is likely to yield less equivocal results than biochemical drm isolation. our study yields images through the use of fpalm, which circumvents the resolution limit imposed by optical diffraction in conventional light microscopy. membrane structure and organization are important for many signaling processes. in this study, crfb clustering in cells was examined and the results provided insights into the dynamic behavior of this receptor. super-resolution imaging with fpalm showed that crfb clustering is mediated by cav- b-containing membrane domains and that disruption of such domains results in dispersal of receptor molecules. the consequence of crfb dispersal was a dampened antiviral response. studies were performed using either poly(i:c) stimulation or shrv infection, both of which will induce ifn, the ligand that will bind to crfb and stimulate it. it is important to note that the crosslinking reagent is non-specific and may impact cellular function because it crosslinks everything on the cell surface, not just cav- or crfb molecules. we tested for non-specific crosslinking effects by also depleting crfb / / and demonstrated that nonspecific induction of mxa due to crosslinking of other cellular components does not occur. we also rescued crfb clustering through exogenous plasmid expression of cav- b and found that after shrv infection, expression levels of mxa remained essentially equal to controls (figure b ), similar to results seen in figure a . mxa was measured because it is a selective and quantitative indicator of antiviral activity that is produced through induction of the ifn pathway. taken together our data demonstrate that viral infection is exacerbated due to the reduced ability of cav- b morphant embryos to clear the infection resulting from the dispersal of crfb and subsequent decrease in stat gene expression, isre activation, and mxa induction. our results contrast with the conclusions of many studies demonstrating that viruses use caveolae as a method of entry [ , , ] , but not as a means to alter the host immune response. others have observed virus infections that do not use caveolae as a method of entry [ , , , ] , but did not necessarily study the role of caveolae in the antiviral immune response. we took a novel approach and discovered that shrv downregulates cav- expression to disrupt the host antiviral response. many viruses in a range of species have developed mechanisms to target and evade the ifn system [ , , ] . the studies outlined here reveal that viruses can escape the antiviral immune response by downregulating cav- b protein levels, leading to a disruption of antiviral signaling through dispersion of ifn-r and abrogation of downstream signal transduction. we assessed the virus induced downregulation of cav- b compared to the morpholino depletion of cav- b and found that viral infection alone is enough to decrease cav- b protein levels and dampen isre activity (figures d and , respectively). taken together, these studies support the hypothesis that cav- b-containing membrane domains provide the local environment for interaction of critical antiviral receptor molecules. additionally, these studies have demonstrated that cav- b is responsible for maintaining crfb clusters and have shown the functional consequences of cav- knockdown. from these observations, it is postulated that cav- b-containing membrane domains increase crfb signaling efficiency by concentrating receptor molecules so that proteins remain at the site of signaling. there have been several studies of immunity in zebrafish that demonstrate similarities to immune function in higher vertebrates [ , , , , , ] . in addition, a publically available microarray database (european bioinformatics institute's gene expression atlas, part of the european molecular biology laboratory; http://www.ebi.ac.uk/gxa/) was used to identify downregulation of cav- in humans after infection with viruses such as hsv and hiv. our identification of a cav- binding domain in human ifn-r, together with the high degree of functional conservation between the immune system of zebrafish and higher vertebrates, suggests that our studies are relevant to immunity in higher vertebrates. fpalm studies provided critical insight into the mechanisms of viral evasion and modulation of membrane domains that are critical to the host immune response to virus infection. understanding the complex mechanisms through which viruses modulate immune function should provide insight into a range of potential targeted antiviral therapies. zebrafish used in this study were handled in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the protocol was approved by the institutional animal care and use committee (iacuc) at the university of maine (protocol number: a - - ). iacuc approved guidelines for zebrafish care were followed using standard procedures (www.zfin.org). cell culture. epc (epithelioma papulosum cyprinid) cells originated from carp epidermal herpes virus-induced hyperplastic lesions [ ] . epc cells have a broad sensitivity for fish viruses and are commonly used for isolation, propagation, and diagnostic assays for fish viruses. epc cells were maintained at uc, % co in minimum essential medium (mem) (gibco-invitrogen, carlsbad, ca) supplemented with % heat-inactivated fetal bovine serum (gibco-invitrogen, carlsbad, ca) and antibiotics. zfl (zebrafish liver) cells were derived from normal adult zebrafish liver [ ] . they display an epithelial morphology. ghosh et al demonstrated that the cells exhibit properties in culture that are associated with liver cell function in vivo. zfl cells were maintained at uc, % co in ldf culture medium ( % leibovitz's l- medium, % dulbecco's modified eagle's medium, and % f- medium) supplemented with heatinactivated fetal bovine serum. expression plasmids. a modified pegfp-n plasmid (clontech) containing pa-mcherry in place of megfp [ ] was digested with xmai and noti (new england biolabs) to linearize the plasmid. cav- b was cloned from a dpf zebrafish cdna library and psti and xmai sites were added by polymerase chain reaction. dendra-crfb was generated using a dendra -ha construct [ ] in which psti and xmai restriction sites were added to crfb by polymerase chain reaction. crfb was subsequently inserted between psti and xmai, replacing ha from the vector. the final constructs were purified by endotoxin free miniprep (omega). cell transfection. zfl cells were transfected by nucleofection according to the manufacturer's protocol (lonza). cells were transfected h prior to fixation. for fixation, cells were removed from the incubator and rinsed three times in dulbecco's pbs (biowhittaker lonza, walkersville, md), and incubated at room temperature for min in % paraformaldehyde (sigma-aldrich). immersion water and pbs were both irradiated for , min by w uv-lamp to reduce background fluorescence. during measurements, uv-bleached dulbecco's pbs was used as the imaging medium. luciferase assay. luciferase assays were performed in a manner similar to that described previously [ , ] . the ifn stimulated regulatory element (isre)-reporter vector isre-luc was provided by r. medzhitov (yale university, new haven, ct) [ ] . prior to transfection, cells were allowed to reach - % confluence in a t flask, at which point they were resuspended in buffer sf (lonza) at cells/ ml and mixed with a total of ng of indicated plasmid dna, of either the pb luciferase or pgl -ifn reporter construct, and . ng of prl-cmv renilla luciferase internal control construct. cells were then electroporated using the amaxa -well shuttle (lonza) using program ew- . cells were then plated at cells/well, in triplicate, using fresh medium and incubated at uc for hours prior to . mg/ml polyinosinic-polycytidylic acid (poly(i:c)) exposure for hr. since poly(i:c) resembles the rna of infectious viruses, it was used to mimic an infection and stimulate the immune system to produce ifn and other cytokines. following poly(i:c) exposure or shrv infection, cells were lysed and firefly and renilla luciferase activity was measured using the dual-luciferase reporter assay system (promega). two experiments were performed with three replicates per experiment. the mean of the three replicates was taken for each experiment, and the standard deviation of the means was taken to generate the sem. relative luminescence units (rlu) were measured in a glo-max luminometer (promega). zebrafish care and maintenance. wild-type (strain ab) fish were maintained in the zebrafish facility at the university of maine, orono. the zebrafish facility is maintained according to the institutional animal care and use committee (iacuc) standards. fertilized eggs were collected in petri dishes at the onecell stage before the start of experiments and raised in egg water ( mg/ml instant ocean sea salts) at uc. the translation blocking cav- mos were previously published by fang [ ] and are targeted to the atg start sites of cav- a and cav- b mrnas. cav mos and control mo were injected at the same concentration. the cav- a mo sequence is -tcccgtccttgtatccgctagtcat- and the cav- b mo sequence is -ttcgttgatgctgtcgttatccatt- . mos were microinjected into zebrafish embryos at ng/ embryo during the - cell stage. injected embryos subsequently developed in egg water at uc. all crfb mos were previously published [ , ] . crfb mo is a translation blocking mo with the sequence -cagtgtat-gatgatgatgtcttcat- . crfb mo is a splice blocking mo with the sequence -ctatgaatcctcacctaggg-taaac- . crfb mo is a translation blocking mo with the sequence -cagggcacactcctccatgatccgc- . snakehead rhabdovirus (shrv) was propagated in epc cells. cells were infected at a multiplicity of infection (moi) of . . the supernatant was then collected and filtered to obtain purified virus at a titer of . % tissue culture infectious doses (tcid )/ml. for infection and imaging experiments, cell monolayers at , - % confluency were infected h prior to fixation and imaging. shrv infection at . moi proceeded for h at uc before cells were overlain with additional growth medium for another h ( h total infection time). wild-type and caveolin-deficient zebrafish embryos were infected by static immersion hours post fertilization (hpf) for hours with tcid /ml shrv or maintained as uninfected controls. twenty fish were collected at hr post infection (hpi) for each treatment and homogenized in minimum essential medium (gibco-invitrogen, carlsbad, ca), with mg/ml gentamycin. the homogenate was frozen at uc before the tcid assay. tcid is a type of virus quantification method. this endpoint dilution assay enables us to determine how much virus is needed to produce a pathological change (observed as cytopathic effects, or cpe) in % of inoculated cells in culture. cpe (i.e. infected cells) was manually observed and recorded for each virus dilution. for our experiments, supernatants previously frozen at uc were thawed to be used in tcid assays and subsequently monitored for cytopathic effects (cpe). after seven days, cpe was determined and the tcid /ml of the virus was calculated according to the reed-muench formula [ ] . virus infection in cell culture experiments was also performed with shrv propagated in epc cells. for these studies, cells were infected at an moi of . . the virus was allowed to adsorb for h. subsequently, virus was removed and regular cell culture media was replaced. dual luciferase assays were performed as described after hpi. total rna was extracted after cav- b mo-injected and control mo injected fish were infected with shrv by static immersion for hr. viral samples were collected at , , and hpi by homogenizing fish from each treatment, per time point in trizol reagent (invitrogen, carlsbad, ca) and subsequently stored at uc. rna was extracted according to the manufacturer's protocol. reverse transcription reactions were performed as previously described [ ] to synthesize cdna. quantitation of mxa was carried out using an i-cycler iq detection system (bio-rad laboratories, hercules, ca). the cycling parameters used were chosen as described previously [ ] . fluorescence measurements were made at each cycle during the annealing step and the copy number was determined based on a standard curve using the icycler software. the value for each sample was normalized to the corresponding b-actin value to determine relative copy number. fold inductions were calculated by dividing the copy number in the virus infected samples by the uninfected samples at the same time point. to identify the cell lineages in which cav- is expressed, zebrafish tissues were dissected into trizol (invitrogen, carlsbad, ca) and total rna was purified in preparation for qpcr. lymphoid and myeloid cells (frozen pellets) isolated from zebrafish kidneys and purified by fluorescence-activated cell sorting were generously provided by dr. david traver (university of california, san diego, ca) [ , , ] and resuspended in trizol for rna purification. total rna from tissues ( ug) and sorted cells ( ug) was reverse transcribed (superscript tm iii reverse transcriptase, invitrogen) and subjected to thermal cycling with gene-specific primers and titanium tm taq dna polymerase (clontech, mountain view, ca). expression of cav a and cav b isoforms was detected using primers previously described [ ] and cycles with an annealing temperature of uc. pcr conditions and primer sequences for detecting myeloperoxidase (mpx), tcra and b-actin expression were described previously [ ] . liver tissue was isolated for detection of cav- b, crfb , l-fabp, and b-actin expression in the liver of a hpf embryo according to previously published methods [ ] . total rna from liver tissue was extracted and cdna synthesized as described above. pcrs were analyzed by gel ( % agarose) electrophoresis. embryos were prepared in a manner similar to that published previously [ ] . embryos were collected, egg water removed, and flash-frozen in a slurry of dry ice prior to storage at uc. for use, frozen embryos were solubilized in ripa lysis buffer (pierce, rockford, il) and halt protease/phosphatase inhibitor cocktail (thermo scientific). embryonic zebrafish were incubated on ice for minutes prior to centrifugation at , g for minutes at uc. supernatants were collected as whole cell lysates. zfl cells were transfected with control morpholino (mo) or cav- b mo to knock down the expression of cav- b. samples were taken at and h post transfection (hpt), corresponding to the time points used to perform experiments shown in figure . for sampling, cells were centrifuged at g for minutes at uc. cells were washed twice in dpbs (biowhittaker lonza, walkersville, md) prior to storage at uc. for use, cell pellets were solubilized in ripa lysis buffer (pierce, rockford, il) and halt protease/phosphatase inhibitor cocktail (thermo scientific). cells were incubated on ice for minutes prior to centrifugation at g for minutes at uc. supernatant was collected for use as the soluble fraction. to determine protein concentrations, a bradford assay was performed using the bio-rad protein assay dye reagent (bio-rad laboratories, hercules, ca). equal volumes of total cell lysate were solubilized in lysis buffer, boiled for minutes, and fractionated by sds-page gel electrophoresis. fractionated proteins were transferred to a nitrocellulose membrane by electrophoresis, blocked with % non-fat dry milk, and immunoblotted with the anti-human cav- polyclonal antibody ( : dilution, bd transduction laboratories). cav- protein was visualized using horseradish peroxidase conjugated secondary antibody (santa cruz biotechnology) and the supersignal chemiluminescence system (pierce, rockford, il). membranes were re-probed with antibody against b-actin to control for protein loading. cells were transfected via nucleofection with control mo, cav- b mo, or combined crfb /crfb /crfb mo and allowed to recover/adhere to cell culture plates for hr prior to bis(sulfosuccinimidyl)suberate (bs ) cross-linking treatment. cross-linking reactions were performed according to the manufacturer's procedures (pierce, rockford, il). cells were subsequently washed times with ml dpbs and replenished with media and returned to the incubator for hr post exposure (hpe) to bs . for cells that were exposed to mg/ml poly(i:c) (invitrogen), treatments were initiated hpe to bs reagent and proceeded for hr. following exposures, rna samples at sequential time points were taken with trizol according to the manufacturer's procedures. rna extractions, cdna synthesis, and quantitative rt-pcr were performed as described above. in normal fluorescence microscopy, many of the fluorescent molecules are visible at the same time and their images are blurred together by diffraction. since diffraction blurs objects smaller than - nm, important biological details can be obscured. fpalm circumvents diffraction by limiting the number of visible/fluorescent molecules visualized at once. rather, many small subsets of fluorescently labeled molecules within a sample are imaged separately, such that each molecule is distinct. this is achieved by optical control of molecular transitions between bright and dark states. by limiting the numbers of emitting molecules and activating a subset of molecules, and then imaging and photobleaching them and repeating this process for many subsets of molecules, coordinates of thousands of molecules can be obtained [ , ] . this iterative process is repeated until sufficient molecules have been localized and the structure of the sample is revealed. the positions of the single molecules can be determined (localized) with a precision better than diffraction limited resolution. the fpalm image is generated by plotting the positions of the localized molecules. single color fpalm imaging and analysis. single color fpalm imaging and analysis were performed as described earlier [ , , ] . a nm diode laser (bcl- - , crystalaser,reno, nv) was used to activate labeled molecules in the sample, while a nm (lrs- -nm- - , laserglow, toronto, canada) diode laser was used to read out active molecules. both beams were focused at the back aperture of a / . na waterimmersion objective lens (uplapo w, olympus, melville, ny) to produce widefield illumination at the sample. fluorescence from the sample was collected by the objective, separated from laser light by a dichroic mirror (t lp, chroma technology, rockingham, vt), bandpass filtered (et / m, chroma), and imaged by an emccd camera (ixon+ du dcs-bv, andor scientific, south windsor, ct) operated at an em gain of and frame rate of , . hz. the camera was controlled using solis software (andor). additional achromatic lenses (f = + mm and f = + mm, newport corporation, irvine, ca), arranged as a telescope, were mounted in the detection path to provide additional magnification and to produce an effective camera pixel size of , nm. a motorized filter wheel (fw , thorlabs, newton, nj) containing neutral density filters provided control over the activation intensity to maintain a density of visible molecules of , per mm or less. cells were selected for fpalm imaging by exciting ( / , chroma) the sample with an hg lamp and searching for green fluorescence (bandpass-filtered, hq / m, chroma) to locate cells transfected with crfb -dendra . during post acquisition analysis, each frame of an image series (typically , frames) was background subtracted and positive intensity peaks with at least one pixel above a minimum threshold were fitted to a twodimensional gaussian to determine the x and y coordinates, amplitude (i ), e radius (r ), and an offset. fitted values of i and r were then used to calculate the number of detected photons. fits that yielded n and r consistent with that expected for a single molecule were recorded for further analysis. for each localized molecule the localization precision was calculated using the standard analytical equation from the literature, including an additional % [ ] . lateral drift of the sample stage has been characterized previously [ ] and was assumed to be negligible over the duration of these experiments, compared to the estimated lateral resolution of , - nm. all analysis was performed using custom software written in matlab (mathworks, natick, ma). two-color fpalm imaging and analysis. two-color imaging of fixed zfl cells transfected with pamcherry-cav and dendra-crfb was performed at room temperature using the geometry employed in [ ] . a dichroic mirror (z rdc, chroma) and emission filters (ff - - - , semrock and et / m, chroma for transmitted and reflected wavelengths, respectively) were mounted in the detection path between the dichroic mirror and the electron multiplying charge-coupled device camera (emccd) (ixon+du dcs-bv, andor technology, south windsor, ct). illumination of the sample was achieved by placing a lens f = + mm) (thorlabs, newton, nj) near the rear epi-illumination port of an inverted microscope (ix , olympus america, melville, ny) to focus the beams to the secondary (back) focal plane of a , . na water-immersion objective lens (uplapo w, olympus). a nm diode laser (bcl- - , crystalaser) was used for photoactivation and a nm laser was used for readout. frames were acquired at . hz (em gain ) with the emccd camera. images were acquired using labview software (national instruments corporation, austin, tx). analysis for two color imaging was an extension of standard fpalm analysis, described above and previously reported [ , ] . analysis was performed using matlab software (mathworks, inc. natick, ma) as follows: raw frames containing the two spatially separated images were background subtracted, then correlated and superimposed for localization. each localized molecule is identified by a, the ratio of emission in the red detection channel divided by the sum of the intensity in both red and yellow channels [ ] . the species of each localized molecule was assigned as either dendra -crfb (or dendra -shrv for figure s a ) or pamcherry-caveolin using a range of a values determined numerically (typically . - . for dendra and . - . for pamcherry), such that the error in assignment to either species was , %. pair correlation calculations. pair correlation analysis and calculation was performed similar to methods previously described by the hess laboratory [ ] . single color pair correlation. coordinates obtained from fpalm imaging were used to calculate pair correlation functions. localizations of the same molecules in consecutive frames were removed from the data set by linking molecules in the i th frame to molecules (i+ ) th frame that were separated by less than times the median localization precision. the positions of linked molecules were then averaged for use in pair correlation calculations. values of g(r). indicate correlation between species while g(r), indicates anti-correlation. for uniform distribution of molecules, g(r) = is expected. calculated values of g(r) were fitted to the analytical correlation function [ ] , including a constant offset, where a is the amplitude and r is the correlation length, and g is a number. two color pair correlation. prior to pair correlation calculation using coordinates obtained from two-color fpalm analysis, duplicate localizations of the same molecule in consecutive frames were removed as described above. the crosscorrelation, g(r), of species a with species b was calculated from: where n (i) b (r) is the number of molecules of species b that lie within a dr = nm-thick circular shell of radius r from the i-th molecule of species a, a r is the area of the shell of radius r (dr/ ), n a is total number of species a used in the summation over index i, and r b is the average density of species b. the summation was performed only over molecules of species a that were more than a distance d from the edge of the cell and the imaged region of interest, where d is the maximum length scale of interest for pair correlation analysis such that edge corrections were not required. figure . at hpt there is a marked decrease in cav- protein expression compared to control cells, while at hpt a slight decrease in cav- protein expression is still observed. membranes were re-probed with antibody against b-actin to control for protein loading. caveolins, a family of scaffolding proteins for organizing ''preassembled signaling complexes'' at the plasma membrane caveolin- alpha and - beta perform nonredundant roles in early vertebrate development caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis the caveolin proteins caveolin- -deficient mice show defects in innate immunity and inflammatory immune response during salmonella enterica serovar typhimurium infection the multiple faces of caveolae model systems, lipid rafts, and cell membranes crowded little caves: structure and function of caveolae interaction of a receptor tyrosine kinase, egf-r, with caveolins. caveolin binding negatively regulates tyrosine and serine/threonine kinase activities nanoscale imaging of epidermal growth factor receptor clustering: effects of inhibitors caveolar endocytosis of simian virus reveals a new two-step vesicular-transport pathway to the er emerging themes in lipid rafts and caveolae assembly of endocytic machinery around individual influenza viruses during viral entry folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection clathrin-and caveolin- -independent endocytosis: entry of simian virus into cells devoid of caveolae sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway characterization of rotavirus cell entry gm structure determines sv -induced membrane invagination and infection antiviral defense in mice lacking both alpha/beta and gamma interferon receptors antiviral protection by vesicular stomatitis virus-specific antibodies in alpha/ beta interferon receptor-deficient mice cross talk between interferon-gamma and -alpha/beta signaling components in caveolar membrane domains molecular and functional analysis of an interferon gene from the zebrafish, danio rerio cloning and characterization of an mx gene and its corresponding promoter from the zebrafish, danio rerio the two groups of zebrafish virus-induced interferons signal via distinct receptors with specific and shared chains identification of the zebrafish ifn receptor: implications for the origin of the vertebrate ifn system signaling through the jak/stat pathway, recent advances and future challenges immunology and zebrafish: spawning new models of human disease the zebrafish as a model organism to study development of the immune system use of detergents to study membrane rafts: the good, the bad, and the ugly lipid rafts, detergent-resistant membranes, and raft targeting signals ultra-high resolution imaging by fluorescence photoactivation localization microscopy imaging biological structures with fluorescence photoactivation localization microscopy dynamic clustered distribution of hemagglutinin resolved at nm in living cell membranes discriminates between raft theories three-dimensional sub- nm resolution fluorescence microscopy of thick samples distribution and localization of caveolin- in sinusoidal cells in rat liver caveolin- mediates endotoxin inhibition of endothelin- -induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells effect of insulin on caveolinenriched membrane domains in rat liver characterization of snakehead rhabdovirus infection in zebrafish (danio rerio) characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment caveolin- is required for lateral line neuromast and notochord development mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity zebrafish as a model for infectious disease and immune function development and maturation of the immune system in zebrafish, danio rerio: a gene expression profiling, in situ hybridization and immunological study evidence for evolving toll-il- receptor-containing adaptor molecule function in vertebrates recognition of double-stranded rna and activation of nf-kappab by toll-like receptor differential roles of mda and rig-i helicases in the recognition of rna viruses toll-like receptor signalling regulation of the type i ifn induction: a current view innate immune recognition of viral infection toll-like receptors and type i interferons type i interferon receptors: biochemistry and biological functions the jak/stat pathway in model organisms: emerging roles in cell movement functional crosstalk between type i and ii interferon through the regulated expression of stat interfering with interferon receptor sorting and trafficking: impact on signaling endocytosis via caveolae involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells internalization of echovirus in caveolae innate recognition of viruses mechanisms of inhibition of the host interferon alpha/ beta-mediated antiviral responses by viruses inhibition of interferon-mediated antiviral responses by influenza a viruses and other negative-strand rna viruses animal models of human disease: zebrafish swim into view some properties of the epithelioma papulosum cyprini (epc) cell line from carp cyprinus carpio derivation and characterization of a zebrafish liver cell line photoactivatable mcherry for high-resolution two-color fluorescence microscopy nanoscale imaging of molecular positions and anisotropies a simple method of estimating fifty percent end points structural characteristics of zebrafish orthologs of adaptor molecules that associate with transmembrane immune receptors transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants developmental and tissue-specific expression of nitrs tissue micromanipulation in zebrafish embryos precise nanometer localization analysis for individual fluorescent probes nanoscale imaging of molecular positions and anisotropies superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells photochromic rhodamines provide nanoscopy with optical sectioning statistical mechanics of phase transitions we thank dr. paul millard, dr. con sullivan (tif) key: cord- -jl zhg authors: khalaf, khalil; papp, natalia; chou, jadzia tin-tsen; hana, doris; mackiewicz, andrzej; kaczmarek, mariusz title: sars-cov- : pathogenesis, and advancements in diagnostics and treatment date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: jl zhg the emergence and rapid spread of sars-cov- in december has brought the world to a standstill. while less pathogenic than the – sars-cov, this novel betacoronavirus presents a global threat due to its high transmission rate, ability to invade multiple tissues, and ability to trigger immunological hyperactivation. the identification of the animal reservoir and intermediate host were important steps toward slowing the spread of disease, and its genetic similarity to sars-cov has helped to determine pathogenesis and direct treatment strategies. the exponential increase in cases has necessitated fast and reliable testing procedures. although rt-pcr remains the gold standard, it is a time-consuming procedure, paving the way for newer techniques such as serologic tests and enzyme immunoassays. various clinical trials using broad antiviral agents in addition to novel medications have produced controversial results; however, the advancement of immunotherapy, particularly monoclonal antibodies and immune modulators is showing great promise in clinical trials. non-orthodox medications such as anti-malarials have been tested in multiple institutions but definitive conclusions are yet to be made. adjuvant therapies have also proven to be effective in decreasing mortality in the disease course. while no formal guidelines have been established, the multitude of ongoing clinical trials as a result of unprecedented access to research data brings us closer to halting the sars-cov- pandemic. coronaviruses are widely known virulent pathogens affecting mammalian and avian species. previously, six globally distributed species of the virus have been identified to cause illness in humans. they are: human coronavirus oc (hcov-oc ), human coronavirus hku (hcov-hku ), human coronavirus e (hcov- e), human coronavirus nl (hcov-nl ), severe acute respiratory syndrome coronavirus (sars-cov), and middle east respiratory syndrome coronavirus (mers-cov) ( table ) . sars-cov first emerged in [ ] [ ] in china, presenting as an atypical pneumonia with febrile state, headaches, and a marked cough that may rapidly deteriorate into respiratory failure. the sars-cov outbreak was the first to be declared a pandemic, and ultimately infected , persons in countries, with a mortality rate of % ( ). epidemiological analysis posited an infectious zoonotic agent, and further serological studies identified the intermediate host to be the palm civet (paguma larvata). spillover from animals to humans was hypothesized to be via the horseshoe bat (genus rhinolophus). an adaptation of the sars-cov virus was later proven to be due to interspecies dwelling ( ) . a combination of ribavirin and pulse prednisolone showed positive results early in the outbreak and was adopted as the standard protocol. eventually, ribavirin was demonstrated to have increased cytotoxicity and lacked efficient antiviral action in vitro, and while pulse prednisolone showed efficacy in managing critically ill patients, its high dose administration was associated with disseminated fungal infections ( ) . later, the combination of interferon-alpha (inf-α ) and corticosteroids was found to yield a better prognosis, but did not prove beneficial for patients in the late stage of the disease ( ) . protease inhibitors produced an outcome similar to that of inf-α ( ) . a novel coronavirus later named mers-cov emerged in saudi arabia in . similar to sars, infected patients presented with a variety of clinical courses, from mild upper respiratory symptoms to fulminant pneumonia and multi-organ system failure. phylogenic and sequencing studies have proposed a bat origin, and surface protein modification was found to be derived from the intermediate hosts, dromedary camels. from to , more than , confirmed cases were reported, mainly in middle eastern countries, with a mortality rate of % ( ) . as with sars-cov, no definitive protocol was implemented, and most patients were treated with supportive therapy to preserve organ integrity ( ) . in a cohort study conducted by omrani et al., a treatment protocol involving ifn-α and ribavirin was initiated, and while survival improved significantly at days after treatment, this was not seen at day , necessitating further treatment options ( ) . sporadic outbreaks occur to this day, with patients undergoing largely supportive therapy. the sars-cov- outbreak began in december in wuhan, china where clusters of atypical pneumonia yielded evidence of a novel strain of coronavirus. as of august , , , , cases had been reported worldwide, with a fatality rate of %. although this novel virus is less severe than the first sars-cov outbreak, human-to-human transmission remains very high and the number of cases continues to rise exponentially in major urban areas, highlighting the urgent need to develop new containment, diagnostic, and treatment protocols. as time is of the essence, the pathogenic mechanism cannot be studied with the rigor and comprehension otherwise expected, and the opportunities to compare therapeutic protocols are few. origin of the virus during the initial outbreak, liu et al. obtained samples of blood, bronchoalveolar lavage fluid, and anal and oral swabs from patients in wuhan, china suffering from a pneumonialike illness resembling the - sars-cov. a pan-cov pcr was performed on samples from five patients, and were positive for a pathogen from the coronaviridae family. this was compared with a full-length sequence of viral rna from a bat coronavirus (bat-covratg ), and demonstrated . % similarity. thus, it is probable that the bat is the main reservoir of the novel coronavirus. identification of the intermediate host is an essential step in controlling the spread of disease, and became a priority for research teams. unfortunately, this was complicated by the many species of wild animals sold at the huanan seafood market, where the first cases were reported to have had contact. in , a sars-cov-like pathogen known to be widely distributed in the malayan pangolin samples was discovered. the receptorbinding domain (rbd) present on the spike protein (s) is a crucial determinant in host range, as its interaction with the host receptor is responsible for the infection. rbd sequences from bat-covratg , pangolin-sars-like cov and the novel sarslike pathogen were aligned. ninety three percentage similarity was demonstrated between the novel sars-like pathogen and the pangolin sars-like cov, and % similarity was demonstrated between the novel sars-like pathogen and the bat-covratg . thus, on the basis of the rbd, the pangolin-sars-like cov is determined to be more likely than the bat-covratg to infect humans, making this the possible intermediate host ( ) . xiao et al. conducted another study in which the pangolin-sars-like cov was isolated and amino acid sequence was compared to sars-cov- . this yielded , . , . , and . % similarity with the s, m, e, and n proteins, respectively, of the novel sars-cov, strengthening the previous assumption that the pangolin was the intermediate host ( ) . pathogenic classification is used to determine whether the pathogen is new or recurring in order to best implement safety and treatment protocols. while serological reactivity to viral proteins had been the mainstay of viral classification in the past, the process today now depends on replicated protein sequences. the international committee on taxonomy of viruses (ictv) maintains a study group for each viral family ( ) . after analysis, the novel virus was assigned to the order nidovirales on the basis of the following domains: polyprotein protease ( clpro), catalytic domain of rna polymerase (rdrp), nidovirus-associated rdrp (niran), zinc binding domain (zbd), and helicase (hel ) ( ) . subsequent next generation sequencing and phylogenic analysis placed the novel pathogen within the subgenus sarbecovirus of the genus betacoronavirus ( ) ( table ) . like other coronaviruses, the viral envelope is composed of a lipid bilayer derived from host cellular material, and the structural proteins are spike proteins composed of a trimeric glycoprotein projecting from the envelope like a crown. cryo-electron microscopy was used to compare the structural differences between the spike (s) proteins of sars-cov and sars-cov- . unlike the previous sars-cov which possessed a single arginine targeted by a trypsin protease, sars-cov- possesses an s /s protease cleavage site which is recognized by furin proteases. neuman et al. demonstrated the high level of protein organization and interaction within the envelope: the s protein was shown to be aligned with the npc, a crucial component of viral organization in protein-protein interactions that ensures high specificity and effectiveness for host invasion ( ) . sars-cov- is an enveloped virus with a , bp positivesense non-segmented rna genome. the non-structural proteins (nsps) are described in table . the remainder of the genome is responsible for accessory and structural proteins such as s, m, e and n. host cell recognition is the first and most essential step in viral pathogenesis. studies on the - sars-cov outbreak uncovered key interactions between the spike (s) protein, the rbd and angiotensin-converting enzyme (ace- ). due to the previously mentioned similarities between sars-cov- and its predecessor, a viral infectivity study was performed using hela cells with and without the ace- receptor, which showed that only cells possessing the ace- receptor were infected ( ) . the spike trimer is a class i fusion protein; upon infection the spike is cleaved by host proteases at the s /s site for division of the two domains. the s domain possesses the rbd which recognizes and binds the ace- receptor in a prefusion state. structural rearrangement of the s protein subsequently exposes a furin cleavage site on the s domain which enables viral entry by means of fusion after the s domain is shed ( ) . viral replication was hypothesized to occur via a process called autophagy: an evolutionary cellular process in which cytoplasmic proteins create isolation membranes surrounding materials destined for degradation ( ) . evidence of coronavirus autophagy was first demonstrated by prentice et al., who showed that in coronavirus mouse hepatitis viruses, atg -induced autophagy was required for the formation of double membrane vesicles (dmv) and for replication ( ) . another study confirmed the use of atg dependent autophagy of betacoronaviruses through nsp ( ). chen et al. previously showed that both sars-cov and mers-cov employ the use of papain-like proteases (plpro) to induce the formation of autophagosomes, but their fusion to lysosomes is inhibited which promotes the replication process ( ) . however, other studies have demonstrated that key autophagy proteins atg or atg were not required for coronavirus mouse hepatitis or sars-cov viral replication ( ) . reggiori et al. also yielded comparable results showing that the replication and release processes are not dependent on autophagy, but that the dmv's were coated with (lc )-i (non-lipidated microtubule associated protein i light chain ), thus showing the importance of this protein for viral replication ( ) . a recent study by benveunto et al. has shown that sars-cov- induced mutations within the nsp protein, which in turn induced autophagosome formation by inhibiting phagosomelysosome fusion ( , ) . sars-cov- may take advantage of this autophagy mechanism, as the acidic ph required for endosome maturation also functions to release the virion at the appropriate site. once all necessary proteins are translated and replication has taken place, virion assembly occurs in the golgi apparatus before release from the cell ( ) . symptoms attributed to sars-cov- shereen et al. determined a mean incubation period of days in the first patients testing positive for sars-cov- ( ). the time from infection to death varied from to days, with a mean of days ( ) . in january of , huang et al. examined all patients exhibiting pneumonia-like symptoms in order to determine the clinical features of sars-cov- ; importantly, this study did not discriminate between previously healthy patients and patients suffering from chronic illnesses. data was collected from the positive cases, and analysis yielded the following: fever was the first and most common primary symptom ( %), followed by dry cough ( %), lymphopenia ( %), dyspnea ( %), and fatigue/myalgia ( %). less common symptoms included sputum production ( %), headache ( %), hemoptysis ( %), and diarrhea ( %) ( ) . a recent systematic review of studies spanning nine countries disputes the above-mentioned data. in this analysis by grant et al., the most common symptoms were as follows: fever ( %), dry cough ( %), fatigue ( %), anosmia ( %), and difficulty breathing ( %) ( ) . another study by clemency et al. assessed the likelihood that sars-cov- infection presents with symptoms. the positive likelihood ratio of having symptoms with disease from highest to lowest was as follows: anosmia and ageusia, followed by loss of appetite, fever, muscle pain, fatigue, dry cough, productive cough, diarrhea, difficulty breathing, and sore throat ( ) . the stability of sars-cov- on various surfaces was compared to that of its predecessor sars-cov, and it was found that both viruses exhibit similar stability. the differences in epidemiological spread are therefore most likely to be dependent upon other factors such as high viral load and viral shedding in asymptomatic persons. the study is highly suggestive of an aerosolized pathogen as it remains viable and infectious in droplet form for up to h ( ). while resembling the previous sars-cov outbreak, this data confirms that sars-cov- is far more transmissible. kinetic studies employed to compare the two strains showed that sars-cov- binds to the ace- receptor with a -to -fold higher affinity than sars-cov, suggesting that this could be another main factor explaining the ease of transmission ( ) . to further support this hypothesis, computational structural predictions were established to differentiate between the rate of association between the ace- receptor and the spike protein of both sars-cov and sars-cov- . by incorporating the computer simulation into a mathematical model, it was shown that the novel pathogen has slower binding capacity than sars-cov, consistent with the varying life cycles of the two strains. in other words, the longer incubation period of sars-cov- was associated with the slower interaction between the spike protein and the ace- receptor. this allows for maintenance of an increased viral load in the body, thus contributing to increased human-to-human transmission ( ) . the renin-angiotensin-aldosterone system (raas) is responsible for inducing a coordinated cascade regulating both cardiovascular and renal functions. angiotensin-converting enzyme (ace) is responsible for converting angiotensin i to angiotensin ii, with the latter functioning as a pro-sympathetic molecule throughout the body to regulate blood pressure. wakahara et al. measured ace and ace- expression under various conditions. in addition to the ace- role in local regulation of raas through the conversion of angiotensin ii into vasodilators and anti-trophic peptide, it acts and is expressed in synergy with ace concentrations and is believed to possess counter-regulatory effects to ace ( ) . as the expression of the ace- receptor is vital for viral entry, it is essential to establish where this gene is expressed throughout the body to identify possible routes of infection and/or the extent of organ damage during infection. zou et al. employed the dataset systems of single-cell rna sequences from different body tissues to identify organs expressing the ace- gene in high concentrations. the symptoms reported to be associated with covid- (dyspnea, cardiac injury, kidney failure, diarrhea) were compared to ace- expression on target cells. it is on this basis that the organs at high risk of damage during viremia were recognized to be the lungs, heart, kidney, and upper respiratory tract ( ) . a minority of patients is seen to present with gastrointestinal involvement, and a further study by wong et al. on patients in wuhan demonstrated this complaint in % of their cohort. the virus was detected in stool by means of rt-pcr. staining of the n protein showed presence of the virus in the cytoplasm of duodenal, rectal and gastric epithelium thus indicating another possible mode of transmission and another sign of possible infection ( ) . zhao et al. also compared the symptoms and disease characteristics of covid- patients with that of other pneumonias, indicating that liver damage was more prominent in covid- patients. however, it was not clear whether liver enzyme elevation was due to drug toxicity or viral shedding ( ) . the ace- -receptor mapping experiment, while important, provides little information on the broad expressions of the receptor, as ace- concentration may differ due to various clinico-pathologies such as hypertension, diabetes, heart failure, smoking and kidney injury ( , ) . remuzzi and remuzzi in italy demonstrated that two-thirds of patients who died from sars-cov- were elderly, diabetic or suffered from cardiovascular disease ( ) . their first line drug of choice was angiotensin receptor blockers (arbs). ferrario et al. demonstrated in that selective blockade of angiotensin ii synthesis or activity has an up-regulatory effect of ace- receptors in the kidneys and heart ( ) . this ace- upregulation in the aforementioned comorbidities could therefore be responsible for the severity of infection in these patients. guo et al. demonstrated that in sars-cov- -positive patients, . % had an increase in troponin-t and crp levels that correlated with myocardial injury. sars-cov- patients who presented with an increase in cardiac markers exhibited a . % mortality rate compared to those with normal cardiac markers ( . % mortality rate). sars-cov- genomic material has also been isolated from cardiac biopsies. while the pathophysiologic development of myocardial injury is not yet fully understood, it is believed to be caused by either cytokine storm or direct myocardial damage through viral integration ( ) . to further understand the organ damage and hemostatic changes, han et al. assessed the change in coagulation proteins in both covid- patients and healthy patients. they found that antithrombin levels were lower in covid- patients, whereas fibrinogen, fibrinogen/fibrin degradation products and d-dimer were high ( ) . a recent cohort study by wichmann et al. reported a possible connection between covid- and incidence of thromboembolism. autopsies of covid- patients revealed the incidence of deep vein thrombosis (dvt) in % of cases, and a third of patients suffered a fatal pulmonary embolism. further studies should be conducted to further elucidate the formation of thromboembolism in the course of covid- ( ) . cd , also known as emmprin (extracellular matrix metalloproteinase inducer) is a highly glycosylated transmembrane glycoprotein shown to be involved in facilitating sars-cov endocytosis. this presents an alternative route of viral invasion ( ) . like the ace-receptor, cd is also present in various tissues such as lymphoid ( ) , testes, liver, cerebral, and renal tissues, as well as cardiac and skeletal muscle ( ) . in addition to enabling viral entry, cd has been linked to other pathologic conditions such as cancer ( , ) , heart disease ( ) and alzheimer's disease ( ) . furthermore, the speed of invasion may be increased as cd has been shown to be upregulated upon t-cell activation ( ) . wang et al. has since shown that the spike protein possesses high affinity for cd , thus mediating viral invasion and facilitating viral replication. it acts as a negative regulator of the immune system which may contribute to the significant cd + decline during covid- ( ) . under hypoxic conditions, this receptor is involved in the regulation of cell proliferation and apoptosis. the cd receptor, a marker of early-stage disease, can be useful for the assessment of pathological progression because it is often expressed on the surface of activated regulatory t cells ( ) . lung epithelium is the largest area of the body in constant contact with the external environment, and is responsible for processing inhaled air containing large volumes of bacteria and viruses. the immune response to sars-cov was widely studied, and sars-cov- is believed to induce the same responses. innate immunity is the primary countermeasure against infection, and is maintained by a variety of cell types found in airway epithelium, including dendritic cells, innate lymphoid cells and alveolar macrophages. the protective signaling cascade begins as the pattern recognition receptors (prrs) of innate cells recognize pathogen-associated molecular patterns (pamps) of viruses. after recognition, type i and iii interferon (ifn) and other proinflammatory cytokines undergo transcription. autocrine and paracrine signals in both healthy and infected cells subsequently ensure the translation of these cytokines ( ) . passive evasion refers to any mechanism of immune avoidance that does not directly interfere with the host immune system. many non-structural proteins (nsp) are translated in order to shield viral genomic material from the human immune system, as shown in table . nsp , , and are believed to function as modifiers of the intracellular membranes of organelles, where the virus may safely replicate. another passive mechanism is the conformational change of the viral mrna through either the addition of a '-cap (via formation of n- -methyltransferase in nsp ), or a '-o-methyl-transferase as with nsp , which marks the viral mrna to be of host origin. in another example, nsp induces endoribonuclease activity against viral mrna at certain stages of its development to avoid detection ( ) . nsp was shown to inhibit host mrna translation through the activation of exonucleases. these induce degradation of the host genome and prevent the ifn signal transduction that is necessary to activate viral clearance. nsp expresses plpro, which greatly resembles host cellular de-ubiquitinase action. this induces disruption of ubiquitin-mediated degradation and may also inhibit the innate immune response ( ). as with the previous outbreak, immune clearance of sars-cov- relies mostly on the activity of th cells via the extensive secretion of il- , il- , ifn-γ, and tnf-α/β. this cytokine microenvironment activates macrophages and causes cytotoxic t-cell proliferation, initiating pathogen clearance. following degradation and the presentation of viral antigens on apcs, the humoral response acts to limit future infections through antibody neutralization ( ) . in the lungs, alveolar macrophages are the first cells to make contact with microorganisms. their main function is to destroy any invaders without overstimulating the adaptive immune system, since foreign pathogens are ever-present. protective measures employed by alveolar macrophages include the surface expression of cd and tgf-β receptors, which function as negative regulators of dendritic cells and t cells ( , ) . this inactivation is exploited by sars-cov, leading to an increase in viral load. additionally, law et al. demonstrated that macrophages and dendritic cells are targeted by sars-cov, resulting in a very low expression of antiviral cytokines. this inhibits apoptosis and hinders the bridging of the innate and adaptive immune systems, leading to lymphopenia ( ) . in a cohort study by qin et al. of patients with confirmed sars-cov- infection, lymphopenia and an increase in neutrophil-to-lymphocyte ratio was routinely observed among infected patients and was especially evident in severe cases ( ) . it has been shown that these two parameters are indicative of systemic inflammation, and were associated with the worst prognosis ( , ) . because qin and colleagues did not observe any changes in either cd + or b cells, it was then hypothesized that sars-cov- plays a major role in indirectly damaging lymphocytes, with the release of proinflammatory cytokines and chemokines that results in the consumption of the lymphocytes necessary to prevent innate overactivation ( ). qin et al. also noted that the increase in neutrophil-to-lymphocyte ratio was accompanied by an increase in procalcitonin, indicating bacterial co-infection ( ). zhao et al. showed that mice depleted of alveolar macrophages and subsequently infected with a mouse-adapted sars-cov strain quickly developed activation of dendritic cells, which in turn activated cytotoxic t cells and initiated viral clearance ( ) . while most viral infections end with eradication and development of immunological memory, adaptive immunity does on occasion fail to develop adequately. callow et al. demonstrated in that patients previously infected with human coronavirus e showed a decline in antibody concentration and were capable of being re-infected after year ( ) . other studies involving mers-cov have come to the same conclusion, determining that the concentration of virusneutralizing antibody was dependent upon disease severity ( , ) . it is important to note that the failure of adaptive immunity could be due to either insufficient antibody response or decrease in t-cell durability ( , ) . appropriate adaptive immunity requires early cd + and cd + responses. in the case of sars-cov- , viral evasion of the innate immune system leads to an increase in cytokine production and late cd +/cd + response, which then leads to pathogenic inflammation in patients with high viral loads. due to rapid and unopposed sars-cov- replication, cd + t lymphocytes are quickly activated to differentiate into th cells and are responsible for releasing pro-inflammatory cytokines il- , gm-csf, and ifn-γ. gm-csf activates to further produce inflammatory monocytes (cd + and cd +) which release more il- . this disrupts the homeostasis of the immune system leading to cytokine storm ( ) . sars-cov- -related hyperinflammation involves very high levels of il- -β, il- , and tnf-α ( ). sars-cov- is thought to bind to the toll-like receptor (tlr), activating inflammasomes and resulting in the cleavage of pro-il- β to form il- β, a mediator of inflammation, fever and lung injury ( ) . the pathological immune response has a wide variety of clinical presentations, from mild symptoms to pulmonary failure and death. extensive lung damage is associated with neutrophil and macrophage infiltration while cd + and cd + count in peripheral blood are simultaneously lowered ( ) . croft et al. performed serological analyses in both healthy and sars-cov- -positive individuals. the cd + t cells in infected individuals demonstrated very high levels of cd , cd , and cd , indicating a hyperactive state compared to the healthy group. further analysis showed high levels of the t-cell activation marker ox on cd + cells, which has been proven to be crucial for cell expansion, survival and cytokine production in both t cells and innate cells ( ) . it is important to note that expression levels of ox also varied depending on the severity and progression of the disease, and may possibly be a marker of poorer prognosis. cd + t cells were also found in a hyperactive state, with higher expressions of cd , cd , and cd . in various deteriorating icu patients, there was an increase in expressions of pd- and tim- in both cd + and cd + t cells, indicating immune exhaustion ( , ) . aside from exhaustion antigens such as pd- , bellesi et al. ( ) revealed upregulated expression of the cd antigen on both cd + and cd + t cells. when cd apoptosis-related antigen is observed to be highly expressed, this is accompanied by a lower cd + and cd + count, which may partially explain the loss of lymphocytes in sars-cov- -positive patients. the loss of naive cells appears to be particularly important in this context. complement activation can be initiated via the alternative pathway, classical pathway or lectin pathway. innate immunity recognizes pathogen-associated molecular patterns (pamps) in host cells and initiates a destructive response. the mannosebinding lectin (mbl) pathway has been shown to be the first line of defense against sars-cov ( ). it is composed of pattern recognition molecules associated with serine protease from the mbl-associated serine proteases (masps), which circulate as zymogens and become active after recognition, binding to carbohydrate-based pamps ( ) . to further understand the triggers of the excessive immune response to sars-cov- , gao et al. showed that as with sars-cov, masp- contact with the n protein led to cleavage of c and c into c a/c b and c a/c b, respectively, and the mbl pathway proceeded as normal. none of the other pathways triggered complement activation ( ) . due to the high infectivity and severity of the virus, the world health organization (who) has been prompted to develop new tools to ensure the fast and accurate diagnosis of the viral infection. many governments have given an unprecedented level of freedom to nominated laboratories to establish reliable diagnostic tests. the increased demand for rapid testing has fueled research in sequencing, characterizing, and understanding sars-cov- , in order to definitively diagnose it in human samples. as of august , the recommended sampling sites are as follows: the upper respiratory tract (nasopharynx, oropharynx, anterior nares), lower respiratory tract (sputum, bronchoalveolar lavage, pulmonary tissue biopsy) as well as urine, feces or whole blood. according to centers for disease control (cdc) guidelines, material from the upper and lower respiratory tracts are preferred, and should be sampled if possible. material should be collected by an experienced specialist to ensure high standards, and should be tested as soon as possible; exceptions are serum samples which can be stored for up to - weeks, to be referenced in future follow-up ( ) . the overwhelming number of infected cases compounded with the shortage of healthcare personnel has led to the prioritization of testing of affected individuals. due to limited access and resources, the cdc has been compelled to define specific criteria for testing all -ncov patients under investigation (pui) . in order to be tested, the pui needs to manifest with: -"fever and symptoms of lower respiratory illness (cough, difficulty breathing) after days of travel to wuhan city or contact with a -ncov pui within the last days, " or -"fever or symptoms of lower respiratory illness (cough, difficulty breathing) after contact with a patient with a confirmed case of sars-cov- infection within days" ( ) . the introduction of restrictions not only guarantees good quality patient care and prioritizes acute and severe cases, but maintains the integrity of an already strained healthcare system. the high volume of cases demands fast and efficacious testing regimes for a variety of settings, including the hospital. depending on the type of technology and the personnel available, a few specialized diagnostic protocols have proven to be useful in the field. sars-cov- is an rna virus which sheds detectable genetic material in almost all excretions of an infected individual. this material can be detected by a simple nucleic acid test which is capable of identification and characterization of nucleotide sequences. in blood, sputum and other samples, the amount of genetic material is very sparse thus necessitating an additional amplification step in order to reach a particular detection threshold. this method is known as the nucleic acid amplification test (naat). another technique currently available is the polymerase chain reaction (pcr). real-time polymerase chain reaction (rrt-pcr) is the gold-standard molecular technique for detection of sars-cov- viral rna in all recommended samples. it is a primary diagnostic test that targets the following sequences that code for structural viral proteins: spike (s), membrane (m), envelope (e), nucleocapsid (n), and rnadependent rna polymerase (rdrp). the available literature suggests that the spike protein (s) may be the primary pivot in intracellular interactions with host cells, thus demonstrating high immunogenic character ( ) . high infectivity of sars-cov- has compelled the cdc to publish rrt-pcr primers and probes together with all relevant literature for public access ( ) . such advances in research were possible thanks to past experience with the previous betacoronavius epidemic. primer design was based on the nucleotide sequences that matched sars-cov and mers-cov with to % accuracy ( ) . the wide availability of protocols has accelerated progress in research and diagnostic measures. nevertheless, it should be noted that the high mutation rate and large genetic variability of the virus may negatively affect the performance of the assay, and may lead to an increasing number of false-negative results ( ) . additionally, the difficulty of the assay, complexity of the logistic analysis, and protocol duration ( min to a few hours) ( ) confer some limitations to this diagnostic tool ( ) . the full rna extraction protocol should be implemented in a biosafety cabinet at bsl- security level by trained and skilled personnel. it is recommended that none of the samples be heat-treated before rna extraction, which means that samples pose a high risk of infection to laboratory technicians ( ) . false-positive results may also be obtained in cases where the amount of viral material in a collected sample is too low for detection ( ) . based on a published summary report by the fda, the analytical sensitivity of rt-pcr is % with a limit of detection of . cp/ul. specificity showed no crossreactivity with the most common pathogens: bacterial (legionella pneumophilia, mycobacterium tuberculosis and m. pneumoniae, and streptococcus pneumoniae and s. pyogenes) and viral (adenovirus, parainfluenza, rhinovirus, rsv, etc.). the only detected cross-reactivity corresponded to the severe acute respiratory syndrome coronavirus (sars-cov), but this is not surprising since the n target gene in sars-cov- demonstrates more than % genetic similarity to other betacoronaviruses ( ) . the diagnostic protocol formulated by the cdc clearly states the process of confirmation of sars-cov- infection, though the criteria depend on the area where the pui is being diagnosed. in areas where the concentration of sars-cov- virus is high, a single positive naat result is required to diagnose the patient. in areas with low viral circulation more than one of the following is required: -positive naat for at least two different targets on the sars-cov- genome (one specific for the virus) - positive naat for betacoronavirus (sars-cov, mers-cov, sars-cov ) with sars-cov- genome sequencing. it is indicated that the sequenced fragment must be longer or different from the fragment used in the naat assay ( ) . in cases where all tested controls are positive, with all sars-cov- markers below growth thresholds, the naat result is negative. however, cases with high clinical suspicion may still produce a negative rt-pcr result. this may be due to a number of factors, such as poor quality and handling of specimens, specimen collection too early or late in the disease course, contamination, and/or technical errors ( ) . in other words, the lack of a positive result does not exclude covid- disease. in such instances identification of the infected individual is based on clinical observation, patient history and epidemiological information. given the number of tests done to date, in the case of discordant opinions, re-sampling and use of different markers is advised ( ) . the limitations of naat diagnostic techniques for sars-cov- have generated increasing demand for quicker and simpler tests based on serology. rt-pcr, while very reliable and precise, is only capable of identifying the presence of viral load, and cannot inform on the state or progress of the infection in a given patient. in contrast, serological testing and enzyme-based testing measure immunological responses to the virus, allowing for differentiation between exposed asymptomatic, acutely or mildly sick, and recovered cases. additionally, it is able to quantify the number of cases within a short period of time, and this is crucial for modeling the population-scale of infection, determining the level of prophylaxis, and has implications for the development of a vaccine against sars-cov- . there are many immunoassay techniques currently approved or pending. their versatility and creativity augment the number of ways with which one can detect the pathogen in a studied sample. they can be divided into serologic tests and enzyme-based immunoassays. serologic tests exploit the natural responses of the human immune system, while enzyme-based immunoassays detect the antigen with specifically manufactured monoclonal antibodies. all tests in this category detect either the antigen shed by the virus or the antibody that is produced by b-cells in response to the pathogen. the rapid diagnostic test (rdt) is a serologic type of diagnostic tool that allows for detection of the target after - min ( ) . it is a qualitative lateral flow type assay that resembles the standard rapid chromatographic test used for the detection of beta-hcg hormone in the urine of pregnant women. as a field-based test, it requires a drop of blood or other body fluid for identification of igg and/or igm antibodies, or the viral antigen itself ( ) . antigen based-rdts target the structural viral proteins (s, n, etc.) mainly found in secretions of the upper respiratory tract, and react to the antibodies that are produced - days after the initial infection. sufficient time is required for the concentration of antibodies to reach the threshold of detection ( ) . it is also advised to measure igg and igm titer baselines before or during the first few days after exposure ( ) . the non-governmental organization find recognizes cemarked rapid tests ready for use in the field ( , ) . another report by john hopkins center for health and security lists a few rdts currently approved for diagnostic use worldwide, but the antibody-rdt lateral flow assay by cellex inc. is the only rdt approved in the united states. this test was developed in the usa and china, with a sensitivity and specificity equal to . and . %, respectively ( ) . the statistical significance was determined from studies in two chinese hospitals on sars-cov- -positive patients and sars-cov- -negative patients ( ) . the test is currently available for purchase with a disclaimer from the fda that the test alone cannot be used for definitive diagnosis. in regards to the april who statement concerning immunoassays, the antibody-/antigen-detecting rdts are only to be used for research purposes ( ) . according to recent data, these diagnostic tests do not produce results with appropriate reliability. bruning et al. ( ) claims that immunodiagnostic testing for influenza infections in cases with a viral load comparable to sars-cov- showed test sensitivity that varied between and % ( ) . also, due to their abrupt development, the majority of available rapid tests appear not to have been properly validated by relevant institutions and may pose a serious risk to diagnostic efficacy. the simplicity of the rdt means it is neither capable of quantifying the number of antibodies and therefore the phase of infection, nor is it able to ascertain whether these antibodies are part of long-term immunity to the virus. as of august , it is unknown whether exposure imparts immunity after recovery. a day or more delay in diagnosis is a serious diagnostic limitation and may not be of increased benefit to acute-state patients. another type of serologic testing for sars-cov- is a neutralization assay. it is a valuable method that detects antibodies capable of clearing the infection. the procedure consists of the infection of a special type of cell (e.g., veroe ) with sars-cov- , followed by incubation for - days at variable concentrations. during this time cells are titrated with human serum antibodies in order to detect the quantity of antibodies necessary to stop viral replication ( ) . a study performed by zhou et al. demonstrated that : - : dilutions of igg-positive sample were capable of neutralizing tcid ( % tissueculture-infective dose) in sars-cov- cultures ( ) . other than this result, it remains a relatively novel area of research, and information regarding its usefulness in the diagnosis of covid- is sparse ( ) . table lists a number of exemplary testing kits currently available for diagnostic use. enzyme-linked immunosorbent assay (elisa) is currently one of the most popular commercially used enzyme based immunoassays. as indicated by the name, it is an assay using an enzymatic reaction to indicate a positive diagnostic result. plates covered with viral antigens, prepared by the manufacturer, are incubated with the patient's serum. in cases where the specific igg and igm antibodies are present, the antibody-antigen binding complex will be visualized through an enzymatic reaction (colorimetric change, fluorescence, etc.) the whole procedure may take up to h and requires a large sample of blood, plasma, or serum. it is both a qualitative and quantitative type of assay, thus has great potential for future diagnoses of sars-cov- infections. in a study of sars-cov- -specific antibody responses in covid- patients, nisreen et al. used elisa to demonstrate that severe viral infection resulted in higher antibody levels. additionally, they claim that igg seroconversion can be confirmed by applying the same technique in the second week of symptom onset ( ) . another team proved that the accuracy of elisa is dependent on the timing of the infection. on days - after symptom onset, elisa showed < % positive rate; the rate rapidly increased beyond that time frame for both igm and igg antibodies. liu et al. evaluated immunoassays for detection of antibodies against sars-cov- , and demonstrated that viral protein s (spike)-based igm elisa had statistically higher sensitivity than n (nucleocapsid)based elisa (p < . ) in detecting igm antibodies. it is suspected that the level of immunogenicity of the s protein is the reason for the relatively higher specificity compared to the n protein ( ) . based on this data, the antigen-based elisa will be a valuable diagnostic method for the fight against covid- . currently, the ngo find lists different immunoassay kits already commercialized, with additional test kits in development ( ) . with supplemental research, these have the potential to become a powerful rapid diagnostic tool for the hospital setting. however, due to their complexity they, unlike rdts, may not be used in the field. to reiterate, there is an insufficient amount of evidencebased research regarding elisa's specificity and sensitivity for use in diagnostic confirmation, to be comparable with rrt-pcr assays. a lesser known enzyme-based immunoassay that is nevertheless noteworthy is the chemiluminescent immunoassay (clia). very similar to elisa, it uses enzyme-labeled antibodies which activate an enzymatic reaction upon contact with their target. the photon of light emitted-luminescencecan then be quantified and directly corresponded to the volume of reagents. the benefit of this technique is its high sensitivity and the possibility of enhancing the reaction to allow for a larger threshold in samples with higher substrate concentration. clia can be used to detect versatile targets including igm, igg, and iga, and there seems to have been a recent increase in trust for this technique among clinicians. a systematic review by bastos et al. compared lfia, elisa, and clia tests, and the results suggest that clia exhibits the highest sensitivity and specificity for igm and igg in patients with covid- ( ) . however, bastos and colleagues also demonstrated that due to excessive discrepancies in test rests, none of the three techniques tested were reliable enough to be recommended for large-scale diagnostic purposes. after the - sars outbreak, chandwani and shuter conducted an in vitro study using an engineered prototype of sars-cov to test lopinavir/ritonavir, protease inhibitors indicated for dual-therapy prophylaxis and treatment of hiv- ( ). lopinavir has higher potency but is less bioavailable, thus co-administration with ritonavir, which additionally inhibits cytochrome p a, leads to prolongation of its action ( ) . they showed that this dual therapy inhibited viral cytopathic activity. furthermore, chu et al. conducted a non-randomized trial in , placing two groups of patients on different regimens: the first group received ribavirin, a nucleoside inhibitor, in dual therapy with a reducing dose of corticosteroids; and the second group received lopinavir/ritonavir/ribavirin. it was shown that treatment group two had a decrease in viral load, fewer nosocomial infections and an increase in circulating lymphocytes, indicating a favorable outcome ( ) . after the emergence of sars-cov- , a randomized trial was conducted involving severe cases. ninety nine of these patients received lopinavir/ritonavir while received standard supportive care. detectable viral load in both groups was the same, and time to improvement after lopinavir/ritonavir was only decreased by day, as compared to the group receiving standard care. the authors concluded that the treatment benefits of lopinavir/ritonavir were not established for severe illness ( ) . similarly, an open-label randomized control trial by cao et al. (chictr ), involving severe sars-cov- cases, compared lopinavir/ritonavir treatment with standard care alone, and they showed that the antivirals yielded no clinical benefits. further trials are thus recommended to establish any possible benefits for patients suffering from less severe illness ( ) . favipiravir, another rdrp inhibitor, is a broad antiviral with a mechanism of action that is hypothesized to either incorporate within viral rna leading to chain termination, or bind to a conserved region of the rdrp and prevent nucleotide addition ( ) . interferon-alpha (ifn-α) is an antiviral that binds to interferon receptors and activates signal modulators (jak / ). the phosphorylated interferon receptor binds to the signal modulators, resulting in immune modulation and antiviral protein transcription. in an open-label control study conducted by cai et al., the antiviral activity of favipiravir + ifn-α was compared to that of lopinavir/ritonavir + ifn-α in patients with confirmed sars-cov- infection. they demonstrated that patients receiving favipiravir + ifn-α exhibited faster viral clearance and radiological improvement, compared to the other study group. although yielding positive results, this was not a double-blind placebo-controlled randomized study, and more trials must be implemented before definitive conclusions can be drawn ( ) . umifenovir is a broad spectrum antiviral possessing dual properties: direct antiviral and virucidal action, as well as demonstrating virustatic effect through impedance of various stages of the viral life cycle ( ) clinical trials-mechanism of action unknown. uprifosbuvir, setrobuvir, balaprevir, '-c-methylcytidine, valopectibine bms- , mk , r , r , idx- , yak, psi- and psi- apparent in the umifenovir group, and no side effects were observed in either group ( ) . an open-label randomized control trial (chictr ) was conducted by chen et al. in which adult sars-cov- patients were administered either favipiravir or umifenovir. they showed that favipiravir significantly improved symptoms associated with cough and pyrexia. however, no clinical benefit could be observed when comparing viral clearance between the two therapies ( ) . after isolating the genomic sequence of sars-cov- , in particular that pertaining to rna dependent rna-polymerase (rdrp), elfiky showed that the rdrp of sars-cov- exhibits . % similarity with that of sars-cov. an rdrp model was engineered from the ncbi nucleotide protein database using the sars-cov rdrp genome as a template to test several antiviral drugs. to ensure high reliability of the model, a nucleotide comparison between the rdrp model and the sars-cov- rdrp was established, and was shown to yield . % homology. the study sought to establish which of compounds (table ) could bind to rdrp active sites and elicit inhibitory activity. drug docking site analyses were interpreted using protein-ligand-interaction-profiler (plip). five fda-approved drugs showed very high affinity for rdrp, and could prove to be beneficial against sars-cov- . additionally, three drugs from the clinical trial by elficky showed high affinity for rdrp, and these include idx- , setrobuvir and yak. drug side effects and toxicity are yet to be disclosed ( ) . additionally, one antiviral drug that sparked interest was remdesivir. in a small cohort study, a day course of remdesivir was administered to patients with severe infection, and clinical improvement was observed in %. fewer clinical benefits were observed in patients who received invasive ventilator support, and % of the patients, especially those who received invasive ventilation, died after the treatment course. multiple factors impede the accurate measurement of remdesivir efficacy and these include preexisting conditions and duration of intubation. as a result, any clinical benefits need to be further investigated in future placebo-controlled trials ( ) . in a double blind placebo-controlled randomized trial (ntc ) biegel et al. administered remdesivir to hospitalized sars-cov- patients presenting with lower respiratory tract involvement to assess its efficacy and safety. remdesivir was shown to be superior to placebo in decreasing recovery time ( ) . however, a multicenter, randomized, placebo-controlled trial conducted by wang et al. to assess the efficacy of remdesivir in patients with severe sars-cov- (ntc ) showed that remdesivir was of no clinical benefit compared to placebo ( ) . lastly, a multicenter analysis involving a double-blind placebo-controlled trial (ntc ) was implemented to assess the efficacy and safety of remdesivir in the treatment of hospitalized sars-cov- patients. preliminary results from this trial showed that patients receiving remdesivir had a faster time to recovery and a lower mortality rate when compared to the placebo group, and it is on the basis of this that the fda issued authorization of remdesivir for emergency use on may , . interferons (ifns) are important cytokines with critical antiviral activity. infected innate immune cells produce ifn which enable the jak-stat pathway, leading to recruitment of more nk cells and macrophages. as with sars-cov and mers-cov, nsp of sars-cov- exhibits anti-ifn activity by targeting proteins of the jak-stat signaling pathway ( ) . ifn inhibition has been correlated to disease severity ( ) . furthermore, ifn treatment has already proved effective against ss-rna viruses, and is widely used in the treatment of hcv and hbv. zhang et al. combined ifn-α and ifn-γ in a study conducted in vivo and in vitro. this combination demonstrated synergy, and smaller dosages were required than in ifn monotherapy. this suggests that reduction in unwanted side effects may be possible with the use of dual therapy ( ) . it was shown that sars-cov- evades detection from cytosolic rig-i and mda , preventing the activation of ifn-i and the subsequent stimulation of innate cells. it is at this point that the importance of toll-like receptors (tlrs) should be emphasized. negishi et al. demonstrated that viruses that evade cytosolic safeguards can be inhibited by tlr- action. tlr- functions to activate ifn-ii, which in turn elicits an antiviral response ( ) . the use of tlr- agonists in mice by shahabi nezhad et al. showed promising results; they were able to increase levels of ifn-α/β/γ, which compensates for the inhibitory actions of sars-cov on signaling pathways ( ) but further research is needed to determine if this may result in toxic overstimulation of the immune system ( ) . in an open-label randomized phase ii trial by hung et al., a triple combination of ifn-β- b, lopinavir/ritonavir and ribavirin was administered to covid- patients with mild to moderate disease. this combination proved to be safe and superior to lopinavir/ritonavir + ribavirin, with patients showing alleviation of symptoms, shortened duration of viral shedding, and shortened hospital stay. these promising results mean that future trials utilizing ifn-β- b are warranted ( ) . anti-c a-antibody, eculizumab, and bevacizumab the discovery of the sars-cov- specificity to masp- of the mbl pathway opened the potential for prophylactic treatment against cytokine-mediated lung damage. it was previously shown in the literature that acute lung injury due to viral infection could be prevented by the use of anti-c a-antibody treatment ( ) , and on the basis of this, gao et al. used a recombinant c a-antibody in an open-label trial involving severely ill patients the outcome of the first two recipients of the monoclonal antibody was described. both patients showed improved oxygen saturation, increased lymphocyte count, decrease in inflammatory proteins, improvement in liver function, and alleviation of pneumonia. although the use of anti-c a antibody shows great promise, the trial is still ongoing and the final efficacy is yet to be disclosed ( ) . another trial (ntc ) is currently assessing the potential of eculizumab to reduce mortality in patients. in a similar mechanism of anti-inflammatory action, eculizumab inhibits c cleavage thus preventing the release of c a. for the treatment of ards, a clinical trial (nct ) is comparing the therapeutic potential and side effects of the monoclonal antibody bevacizumab for critical covid- patients. by targeting vascular endothelial growth factor (vegf), an angiogenic factor, bevacizumab may prevent the disruption of the vascular barrier that causes edema and lung injury ( ) . a preliminary study conducted by wang et al. ( ) identified potential antibodies with the capacity to neutralize sars-cov- . the s protein ectodomains of both sars-cov and sars-cov- were expressed in hek- t cells using plasmid transduction. similarly, hek- t cells were transfused with plasmids containing sars-cov / in s protein subdomains tagged with either the mice or human fc portion of igg. h l mice antibodies were produced through gradual immunization with the sars-cov s protein ectodomain. the spleen and lymph nodes were then harvested to produce hybridomas. of the samples, only one of the hybridomas ( d ) exhibited cross-reactivity sars-cov and sars-cov- s proteins. the chimeric antibody was reformatted and expressed as a fully human igg. it was shown that this novel igg tightly binds the conserved rbd of the sars-cov- s protein in infected cells and neutralized the virus in veroe cells. this represents the very first human monoclonal antibody that is able to fully neutralize sars-cov- ( ) . this cross-neutralizing antibody, due to a conserved epitope region on the spike protein, could be the key to preventing future betacoronavirus outbreaks. d has recently completed phase i clinical trials (nct ) to establish dosing in hospitalized patients with sars-cov- under longterm follow up. now undergoing phase ii trials (nct ), the monoclonal antibody will be tested on ambulatory patients, with results estimated to be available in september. as stated earlier, the ace- receptor is crucial for viral attachment and entry. an in vitro experiment conducted by monteil et al. exposed veroe cells to varying concentrations of plaque forming units (pfu) from sars-cov- in the presence and absence of human recombinant soluble ace- (hrsace- ). the infection was inhibited h after introduction of the virus. the experiment was repeated using human capillary organoids and human kidney organoids, and the same inhibitory actions of hrsace- were observed. it is important to note the dosedependent nature of this inhibitory action. hrs-ace- has already undergone phase i and ii clinical trials for the treatment of ards ( ) , and monteil et al.' findings suggest that hrs-ace- could be a potential therapeutic agent against sars-cov- and phase ii trials (nct ) are currently underway in various european countries ( ) . in an open label trial by the recovery collaborative group, , hospitalized patients received either low dose dexamethasone or standard care alone by random assignment. it was observed that the group of patients on mechanical ventilation who received dexamethasone exhibited lower mortality rates compared to those receiving standard care alone ( ) . while other studies further support the role of glucocorticoids in the reduction of mortality ( ) some reported conflicting results and showed no clinical benefit or even harm to the patient ( ) . a systematic review of studies concluded that while critically ill patients are more likely to benefit from glucocorticoid therapy, their use was associated with increased mortality as it resulted in longer hospital stays and increased tendency toward serious nosocomial infections ( ) . clinical trials are currently ongoing to assess the risk vs. benefit for the use of glucocorticoids in the treatment of covid- . cytokine storm remains the main cause of acute lung injury and organ damage in the course of sars-cov- infection. it is chiefly caused by gm-csf and il- . il- receptors exist in two forms: the soluble form (sil- ) and the membrane bound (mil- ). to initiate pro-inflammatory action, il- binds to sil- and the complex binds to gp to complete signal transduction. due to this, the therapeutic use of the il- antagonist tocilizumab, which binds to both sil- and mil- , was suggested. xu et al. qualified critical patients in a trial with tocilizumab. clinical symptoms, radiological findings and laboratory values all improved after treatment, and patients were successfully discharged ( ) . in an open label study by morena et al., however, critically ill sars-cov- patients were treated with tocilizumab. all patients presented with decreased oxygen saturation and an increase in plasma il- . while positive results were observed with tocilizumab rapidly decreasing inflammatory markers, no clinical benefit was reported as patients quickly developed life-threatening bacterial and fungal infections ( ) . jordan et al. conducted another study administering critical patients with a single dose of tocilizumab. this resulted in significant decrease in inflammatory proteins and reduction in fever. twenty two patients receiving mechanical ventilation were able to be extubated and vasopressors discontinued. two patients died, and the authors report that these patients were already in severe septic shock due to sars-cov- -related pneumonia, and were unresponsive to vasopressors. four patients did not respond to the medication and had poorer outcomes. while the results are promising and in line with the findings by xu et al., limitations of this work include the lower-thanrecommended dose of tocilizumab, chosen by the authors due to drug shortage, and the absence of a control group ( ) . a placebo-controlled randomized clinical trial is still necessary before recommendations can be made. historically, il- has been shown to act as a pro-inflammatory cytokine with actions on several immune cells ( , ( ) . convalescent plasma has been employed and has shown promise in the treatment of sars, mers and influenza ( ) ( ) ( ) . in a study by duan et al., severely ill patients were transfused with ml of convalescent plasma harvested from donors who have recovered from sars-cov- and had antibody titers above : . within to days, symptoms had disappeared in all patients, and radiological investigation showed improvement after seven days. viral load was undetectable by rt-pcr in patients and no adverse side effects were detected ( ) . shen et al. treated five covid- patients with convalescent plasma. all five patients were critically ill and intubated, presenting with ards and pneumonia and experiencing high viral load despite treatment with antivirals. the outcome was largely positive: ards was resolved in four patients within days, and three patients were able to be weaned off mechanical ventilation within weeks. three patients were discharged while two remained stable in recovery ( ) . joyner et al. recently made an assessment of the safety of convalescent plasma in , covid- patients. the incidence of serious adverse events including transfusion reactions, cardiac events and thrombotic events was low. mortality rates were shown to be higher in critical patients receiving mechanical ventilation or those in septic shock. the conclusion drawn from the study provided evidence that the administration of convalescent plasma in a hospital setting was safe and that early administration is more likely to reduce fatality rates ( ) . it has been previously shown that sars-cov induces viroporin production in the host cell membrane to facilitate virion release. viroporin a has been associated with nlrp (nod-like r family, pyrin domain ) inflammasome activation ( ) , which induces the production of pro-inflammatory cytokines such as il- -β and il- via the gasdermin d (gsmd) pathway ( , ) . like sars-cov, sars-cov- induces the production of large amounts of il- -β ( ) . on the basis of this, a possible treatment could be il- , a member of the il- family. when placed with activated peripheral mononuclear cells, il- demonstrates suppressive and anti-inflammatory effects through the inhibition of il- , il- , and tnf production ( ) . another member of the il- family is il- . both in vitro and in vivo studies showed that il- acts as a negative regulator of inflammation, aiding in the protective actions exhibited by tgfβ on dendritic cells and thus attenuating the t cell response ( ) . both il- and il- could potentially be valuable in the treatment of covid- ( ) . baricitinib is a janus kinase (jak) / inhibitor used to treat rheumatoid arthritis. jak / inhibition prevents activation of pro-inflammatory cytokines such as gm-csf, il- , il- , il- , and il- . adaptor-related protein complex (ap )-associated protein kinase i (aak ) induces receptor-mediated endocytosis. baricitinib has been shown to have very high affinity for aak , thus could feasibly inhibit both cytokine storm and viral entry to the cell ( ) . in a small cohort study, titanji et al. administered baricitinib and hydroxychloroquine to patients with moderate to severe covid- . twelve of the patients recovered, and vitals and inflammatory markers were seen to improve after baricitinib was initiated. two patients however developed serious bacterial or fungal infections due to prolonged icu stay ( ) . a phase iii double blind placebo-controlled randomized trial involving baricitinib (nct ) is currently ongoing to assess the efficacy and safety of the drug as a potential immune inhibitor preventing cytokine storm and viral entry. sars-cov- is the most structurally and genetically similar to sars-cov, thus findings from monoclonal studies on sars-cov have been utilized to target the shared aspects between the strains. the monoclonal antibodies shown in table are engineered to specifically bind to different domains on s or s . though none have progressed to clinical trials, they mechanism of action r binds s and prevents interaction with ace- ( ) synergistic action; bind s and prevent interaction with ace- ; prevent immune escape ( , ) f g f g m bind linear epitope of s ; bind conformational epitope of s ; inhibit interaction of s with ace- ( ) binds hdr domain of s and prevents interaction of receptor in vitro ( ) binds s and prevents interaction with ace- ( ) monoclonal antibodies studied for the treatment of sars-cov- function igg that binds to a conserved region in the rbd of the s protein and fully neutralizes sars-cov and sars-cov- ( ) anakinra il- -inhibitor used to reduce effects of the cytokine storm in sars-cov- ( ) show promise in vitro and in vivo against sars-cov and sars-cov- ( ) . the immunomodulatory potential of mesenchymal stem cells (msc) was first identified by luk thromboembolism as a result of endothelial injury in the course of infection is a serious and fatal complication in critically ill patients ( ) ( ) ( ) . tang et al. enrolled covid- patients with severe disease for a study in which patients who received low molecular weight heparin for a week or more. these patients were associated with better prognosis than the patients who were not administered anticoagulant therapy ( ) . helms et al. followed icu patients in a multicenter prospective cohort study. thromboembolic events were observed in . % of patients despite prophylactic and therapeutic use of anticoagulants. the authors noted that pulmonary embolism was diagnosed a few days after icu admission and was more common in ards patients. they conclude that although other papers have reported the effectiveness of heparin, a higher anticoagulant target should be implemented with other anticoagulants such as anti-xa ( ) . the pathogenesis of thromboembolism in the course of covid- is still unclear. one of the proposed pathways implicates ards: the profound hypoxemia and vasoconstriction may lead to vascular occlusion ( ) . another proposed mechanism is that unlike in a healthy lung, a diseased lung is unable to maintain the balance between fibrinolysis and coagulation, thus resulting in decreased action of tissue plasminogen activators (tpa) ( ) . more randomized clinical trials are currently ongoing to assess the efficacy of various anticoagulants. in a pilot study by fowler et al., critically ill septic patients were given either a vitamin c infusion or a placebo. a dramatic decline in inflammatory markers was observed in the vitamin c group, and their sequential organ failure assessment (sofa) score was decreased compared to the placebo group. there were also no adverse events observed with the vitamin c group ( ) . in , fowler et al. published on the use of vitamin c vs. placebo in septic patients with ards, and they concluded that the vitamin c infusion did not improve either inflammatory markers or organ dysfunction score ( ) . several clinical trials are ongoing verifying the benefits of vitamin c in the treatment of covid- . widely used as an antimalarial drug, hydroxychloroquine has been shown to possess broad antiviral action, including effectiveness against hiv- and influenza type a and b. its antiviral activity has been tested on sars-cov- . in preventing the glycosylation of the ace- receptor, hydroxychloroquine effectively prevents viral entry ( ) . furthermore, it has been shown to alkalinize the organelle, which serves to prevent the formation of mature endosomes required to shield the virus from immune cells, and for replication ( ) . apart from its broad antiviral activity, hydroxychloroquine has proven to be adequately anti-inflammatory, interfering with nlrp activation and impairing the production of pro-inflammatory cytokines, specifically il- -β ( ) . in an open-label nonrandomized clinical trial by gautret et al., sars-cov- -positive patients were divided into three groups: group receiving hydroxychloroquine, group receiving hydroxychloroquine + azithromycin (antibiotics were given at the discretion of the physician to prevent opportunistic infections), and group not receiving hydroxychloroquine. the primary outcome in group and was viral clearance within to days, but greater results were achieved when hydroxychloroquine was combined with azithromycin ( ) . azithromycin, a bacteriostatic agent, has shown antiviral activity against zika virus, ebola virus ( ) and rsv. its antiviral mechanism of action, with regards to rsv, is hypothesized to be in decreasing the expression of fusion proteins in airway epithelium ( ) . in a study conducted by million et al., patients suffering from early sars-cov- infection were treated with hydroxychloroquine and azithromycin; this combination proved to efficaciously reduce the viral load and was deemed safe with minimal risk of complications ( ) . chen et al. also showed that hydrochloroquine was safe and efficacious in the treatment of mild disease ( ) . the efficacy of hydrochloroquine with or without azithromycin has since been disputed. in a recent open-label randomized control trial, the efficacy and safety of hydroxychloroquine + standard care vs. standard care alone was assessed by wei et al. in patients with mild to moderate covid- . the group receiving hydroxychloroquine was shown to suffer more adverse effects, and there was no observed clinical benefit compared to patients given standard care alone ( ) . molina et al. also concluded that while hydroxychloroquine proved to be beneficial in past studies, the combination of hydroxychloroquine and azithromycin for severe sars-cov- infections resulted in inadequate viral clearance, and the clinical benefits previously seen in patients with mild to moderate illness were not observed in hospitalized patients with severe disease ( ) . this broad spectrum antiparasitic agent has been shown to possess antiviral activity. in vitro studies indicate that ivermectin prevents non-structural proteins of both dengue virus ( ) and hiv- ( ) from interacting with the importin α/β on the host cell, which prevents viral integration. it has been shown that the sars-cov nucleocapsid (n) protein integrates with the nucleus and nucleolus, and prevents cytokinesis of the host cell via importin-α/β . the exact role of the n protein in the cell cycle is not known, but it is postulated that this structural protein enters the nucleolus to promote viral replication and encourage suitable conditions for viral packaging ( ) . an in vitro experiment by caly et al. showed that a single dose of ivermectin administered to inoculated vero cells effectively controlled viral replication within to days, which may prove beneficial for newly infected patients. it was hypothesized that like in other viruses, ivermectin's antiviral action against sars-cov- is derived from the inhibition of importin-α/β . ( ) . alam et al. treated mild to moderately ill patients with a combination of ivermectin and doxycycline. symptom improvement was observed after h following treatment, and no side effects were noted. this study however makes no conclusion on the efficacy and safety of this therapy as it is not a place-controlled randomized clinical trial ( ) . similarly, caly et al.' results, while promising, cannot be applied until safety margins have been established in further clinical trials. this glycopeptide antibiotic used in the treatment of grampositive bacterial infections has been shown to possess antiviral activity against ebola, mers, sars and hiv- . it is believed that teicoplanin interferes with endosome formation through alkalization. cleavage of the s protein by cathepsin in the late endosome is inhibited, which in turn prevents the release of viral rna ( ) . baron et al. showed that the cathepsin l sequence is conserved in sars-cov, suggesting that teicoplanin could be a key treatment in patients who are diagnosed early with sars-cov- ( ) . there have been tremendous advancements in the field of immunotherapy since the development of chimeric antigenreceptor t cells (car-t). these cells differentiate from our basic t-cells by overcoming t-cell control safeguards and therefore express fewer exhaustion markers pd- , tim , and lag . furthermore, they are capable of differentiating into terminal effector t-cells responsible for pathogen and tumor destruction. car-t cell treatment has already shown great advances in oncology, inducing long-term remission in patients suffering from acute lymphoblastic leukemia ( ) . it is currently being investigated for the treatment of viral infections such as hiv- , hbv, and hcv. development of car-t cells specific to hiv- infection has now entered clinical trials ( ) . crispr-cas (clustered regularly interspaced short palindromic repeats), a mechanism evolved to protect against bacteriophages has shown great promise as a genetic editing tool. in vitro studies have used lentiviral vectors consisting of cas (crispr-associated proteins) and sgrna specific ccr (single guided rna responsible for the detection of the genome of interest) against cd + t cells susceptible to hiv- infection. the viral vector was able to disrupt the ccr gene in the cd + cells thus inhibiting hiv- entry ( ) . in a novel study, the use of the crispr-cas system against human lung epithelium infected with sars-cov- yielded positive results: crispr-cas was able to be transfected in the lung epithelium to degrade the virus ( ) . this method was shown to possess protective actions against the known pathogenic coronaviruses. gene editing and car-t cell therapy open a new frontier in future treatment modalities. the rapid spread of sars-cov- poses a threat of global proportions. time is of the essence, and the discovery of accurate diagnostic methods and treatment protocols are imperative in preventing further spread of this pathogen. similarities between sars-cov- and its predecessor have formed the framework upon which diagnostic and treatment approaches to the novel virus are based. rt-pcr primers, based on sars-cov and mers-cov, have proven to be highly sensitive and specific, though not without their flaws. time consuming and prone to producing false negative results, this has led to the employment of more efficient testing methods such as serologic tests and enzyme-based assays, capable of quantifying infected patients on a large scale. to date, there are still no therapies specifically targeting sars-cov- . while many fda-approved antivirals on the market have had success in patients presenting with differing degrees of illness severity, the development of specific antivirals remains an area of active research. on the other hand, immunotherapy has been shown to be effective, particularly with the discovery of hrs-ace- and other promising immune modulators, the development of the d monoclonal antibody capable of neutralizing sars-cov- , as well as msc therapy. the non-traditional use of anti-malarial agents had previously showed great promise but have now proven to lack adequate antiviral action and have been associated with severe complications. until guidelines are updated following the multitude of ongoing clinical trials, standard care remains the main treatment modality. rigorous research with regards to this pandemic not only adds to the scientific literature, but is critical for public health policy surrounding future outbreaks. only with collaborative research efforts and dissemination of knowledge may we interrupt exponential transmission of disease and maintain human losses at the minimum. kk and jc: conceptualization. kk, np, and dh: investigation and resources. kk, np, jc, and dh: writing-original draft preparation. kk, jc, and mk: writing-review and editing. jc and mk: visualization. mk and am: supervision and project administration. all authors contributed to the article and 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dose-escalation trial car t cells beyond cancer: hope for immunomodulatory therapy of infectious diseases ccr gene disruption via lentiviral vectors expressing cas and single guided rna renders cells resistant to hiv- infection development of crispr as a prophylactic strategy to combat novel coronavirus and influenza key: cord- -sxux ujo authors: vatner, ralph e.; janssen, edith m. title: sting, dcs and the link between innate and adaptive tumor immunity date: - - journal: mol immunol doi: . /j.molimm. . . sha: doc_id: cord_uid: sxux ujo cancer and the immune system are intimately related. much of the bulk of tumors is comprised of stromal leukocytes with immune functions, which serve to both promote and inhibit tumor growth, invasion and metastasis. the t lymphocytes of the adaptive immune system are essential for tumor immunity, and these t cells are generated by cross-priming against tumor associated antigens. dendritic cells (dcs) are essential in this process, serving as the cellular link between innate and adaptive immunity. as a prerequisite for priming of adaptive immune responses, dcs must take up tumor antigens, process them and present them in the context of the major histocompatibility complex (mhc). dcs also serve as sensors of innate activation signals from cancer that are necessary for their activation and effective priming of cancer specific t cells. here we discuss the role of dcs in the sensing of cancer and in priming the adaptive response against tumors. furthermore, we present the essential role of the stimulator of interferon genes (sting) signaling pathway in producing type i interferons (ifns) that are essential in this process. cancer and the immune system are intimately related. much of the bulk of tumors is comprised of stromal leukocytes with immune functions, which serve to both promote and inhibit tumor growth, invasion and metastasis. the t lymphocytes of the adaptive immune system are essential for tumor immunity, and these t cells are generated by cross-priming against tumor associated antigens. dendritic cells (dcs) are essential in this process, serving as the cellular link between innate and adaptive immunity. as a prerequisite for priming of adaptive immune responses, dcs must take up tumor antigens, process them and present them in the context of the major histocompatibility complex (mhc). dcs also serve as this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). sensors of innate activation signals from cancer that are necessary for their activation and effective priming of cancer specific t cells. here we discuss the role of dcs in the sensing of cancer and in priming the adaptive response against tumors. furthermore, we present the essential role of the stimulator of interferon genes (sting) signaling pathway in producing type i interferons (ifns) that are essential in this process. dendritic cells (dcs) are a heterogeneous population of professional antigen-presenting cells (apcs) from hematopoietic origin that serve as a key link between the innate and adaptive immune systems, and are the lynchpin in generating anti-tumor immunity (steinman and idoyaga, ) . dcs initially develop in the bone marrow from a flt expressing common myeloid precursor shared with monocytes and macrophages (macrophage-dc progenitor, mdp) which gives rise to dc-restricted progenitors that migrate to the lymphoid organs. development of specific subsets is dictated by transcriptional regulatory programs, sensing of growth factors (e.g. gm-csf and flt l), and cues from the local environment. consequently, the various dc subtypes display distinct transcriptional programs, morphology, phenotype, and function in different tissues under different conditions. here we provide a brief overview of the three main dc populations; blood borne and lymphoid dcs, tissue dcs, and monocyte-derived dcs that encompass the inflammatory dc populations involved in tumor immunity. in-depth coverage of dc development, lineages and subtypes has been excellently reviewed elsewhere (hashimoto et al., ; del rio et al., ; randolph et al., ; merad et al., ; chopin et al., ; satpathy et al., ; moore et al., ; moore and anderson, ; liu and nussenzweig, ) . dcs are generally separated into two major groups within the blood and lymphoid tissues of mice and humans; plasmacytoid dcs (pdcs) and conventional dcs (cdcs). the cdcs are further divided into subsets based on their presumed development from myeloid or lymphoid precursors and their expression levels of cd α, cd and cd b. due to technological advancements in omics and multi-parameter functional and phenotypic assessments, this rudimentary division has been shown to be too limited to properly capture the true variety of dc populations. currently, there is a movement to divide the various subtypes of conventional dcs into two main categories, cdc and cdc , based on a combination of lineage and expression of transcription factors and surface markers. while this approach to classification is a significant improvement, it has been primarily developed in mice and does not fully translate to the human system. however, clear genomic and phenotypic patterns exist in both mouse and human dc populations, giving confidence in these tools for identification and subdividing of the major dc populations. require transcription factors irf , id and batf for their development. they generally express mrna for most toll like receptors (tlrs) except tlr and tlr and are characterized by high tlr expression (edwards et al., ) . human cdc also express cd c, hla-dr, xcr , cd , clec a, dec- and tlr , and batf has been implied in human cdc development (poulin et al., ; poulin et al., ) , while irf might affect both cdc and cdc development (hambleton et al., ; salem et al., ) . like mouse cdc , human cdc have the greatest capacity for cross-presentation within the human dc populations (poulin et al., ; crozat et al., ; jongbloed et al., ) . in contrast to the cdc population, mouse and human cdc strongly promote cd + t cell responses and have relatively poor capacity to cross-present cell-associated antigens to cd + t cells. mouse and human cdc express several overlapping markers, including cd c, mhcii, cd b and cd a (signal regulatory protein sirpa). mouse cdc often express cd while human cdc are further identified by cd c/bdca expression. mouse cdc have been shown to require irf , eb , relb and notch for their development, the role for these factors in human cdc development is not known. dcs-pdcs are factories for type i ifn, playing an important role in the immune response to infection due to their capacity to produce large amounts of type i ifn upon engagement of tlr and tlr . mouse pdcs express relatively low levels of cd c and mhcii and high levels of pdca (cd ), b , cd , and siglech. human pdc also have reduced cd c and hla-dr levels and co-express bdca (cd ), bdca (cd ), and cd . both human and mouse pdc development requires the transcription factor e - and its target gene, spi-b (sasaki et al., ; schotte et al., ; cisse et al., ; reizis, ) . although functionally relatively similar, human pdcs are able to cross-present cell-associated antigens in contrast to mouse pdcs, which show very poor capacity to do so (hoeffel et al., ; villadangos and young, ) . a large percentage of cdcs reside in peripheral tissues and non-lymphoid organs where they help maintain tissue homeostasis, promote immune tolerance, and serve as first responders to infectious and neo-plastic insults. while these cells are generally less well characterized than their blood and lymphoid associated counterparts, they play an important role in antitumor responses as they represent the first dc populations present on site in the tumor microenvironment. (merad et al., ; haniffa et al., ; kashem et al., ) . mouse and human langerhans cells (lcs) reside in the epidermis and share several markers (cd c, mhcii/hla-dr, cd b, cd a, langerin, epcam, and e-cadherin). mouse lcs further express cd , while human lcs express cd a and cd c. studies in mice indicate that although lcs share many characteristics with cdc, they are resistant to ionizing radiation and self-renew in situ. lcs have been shown to cross-present cell-associated antigens in vitro, but their capacity to do so in vivo has not been established (igyarto and kaplan, ) . the two other principal mouse skin dc subsets are cdc -like cd + cd b lo langerin + dcs and cdc -like cd − cd b hi langerin − dcs, which reside in the dermis. the cd + dermal dcs have the capacity for cross-presentation of cell-associated and particulate antigens (henri et al., ; romani et al., ; bedoui et al., ) . in humans, bdca- expression is found on most of the dermal dcs with the majority of cells co-expressing cd c and cd a. interestingly, similarly to mice, the cdc -like bdca- + dermal dcs demonstrate superior cross-presentation of soluble antigens as compared with other dc populations (haniffa et al., ) . other tissue dcs-similar to the skin, various dc populations have been identified in the intestine. the mouse lamina propria contains major populations, cd + xcr + cd b − cd − dcs that are related to the batf , ifr and id dependent cdc and cd + xcr − cd b + cd + dcs that are related to the irf and notch dependent cdc . in humans, analogous populations have been identified, and are divided based on expression of cd + xcr + cd + clec a + cd − and cd − xcr − cd − clec a − cd + . a smaller, less studied cd − xcr − cd b + / cd a + population can also be found in both mouse and humans, but its function is poorly understood (bekiaris et al., ) . cdc and cdc like cells are also found in the lung, with cd + langerin + xcr + clec a + dc displaying potent capacity for cross-presentation compared to their cdc − like cd b + cd + counterparts. cd + /cd + and cd b/ cd + dc populations have been identified in both human and murine kidney and liver, but there is currently limited information on their functional properties (rogers et al., ; rahman and aloman, ) . several of these tissue-associated cdc can migrate to the draining lymph nodes through the lymphatic system and are called tissue-migratory cdc. the phenotype of these dc therefore strongly correlates with the phenotype of their tissue resident counterparts. in addition to these steady state dcs, there are several populations of monocyte-derived dc (modc) which differ from cdc in that they are derived from monocytes and not pre-dcs, migrating into tissues from the periphery. generally, inflammatory modc arise at the site of inflammation and the local inflammatory cues prompt their development and functional capacities. inflammatory modcs have been found in the skin of patients with atopic dermatitis (inflammatory dendritic epithelial cells (idec)), psoriasis (tnf and inos producing, tip dcs), and synovial fluid of arthritis patients (wollenberg et al., ; segura and amigorena, ) . these types of modc express cd c, mhcii, cd b, ly c, and cd (mouse) and cd c, hla-dr, bdca , cd a, fcer , cd , cd a, cd and cd b (human). the development of both human and mouse inflammatory modcs seems to depend on transcription factor zbtb and growth factor m-csf (segura and amigorena, ) . while in vivo cross-presentation of cell-associated antigens has not been well established by this subset, in vitro generated human and mouse dcs can efficiently crosspresent cell-associated and soluble antigens. relative composition and functionality of tidcs is strongly dictated by the tumor microenvironment, and can be highly dynamic, depending on factors such as tumor growth rate, vascularization and hypoxia, accumulation of other leukocytes, and cytokines and other soluble factors secreted by neoplastic cells. the frequency of tidcs correlates with favorable prognosis and is inversely correlated with tumor pathologic stage in a variety of cancers (karthaus et al., ; ladanyi et al., ; reichert et al., ) . due to their role in initiating anti-tumor immunity, there is a negative selective pressure hampering the accumulation of dcs by tumor-secreted mediators that inhibit dendropoiesis, promote dc apoptosis, and accelerate dc turnover (esche et al., ; pirtskhalaishvili et al., ; peguet-navarro et al., ; kiertscher et al., ) . moreover, the tumor and its microenvironment actively exploit various mechanisms to suppress proinflammatory functions of infiltrating dcs and even induce dcs to suppress immune responses, and promote immune tolerance, tumor vascularization, and metastases (ma et al., ; ma et al., ; tran janco et al., ; chaput et al., ) . in order to generate tumor-specific t cell responses, dcs must endocytose tumor antigens, process them and present them in the context of mhc along with co-stimulatory molecules and cytokines. the mhc/peptide complex of tumor antigens provides the first of three signals needed for optimal t cell activation. the mhc/peptide complex is recognized by its cognate t cell receptor (tcr) on naïve t cells in the draining lymphoid tissue, and the strength of t cell activation correlates with the mhc/peptide complex density on the dc as well as the tcr affinity for these complexed peptide antigens. the second signal derives from interactions of membrane associated co-stimulatory molecules in the synapse of t cells and dcs. this signal is mediated by co-stimulatory molecules and their ligands, including cd -cd /cd , cd lcd , - bbl/ bb, and slam-slam associated protein, as well as molecules that provide negative signaling such as pdl and pdl (reviewed in (chen and flies, ) ). in the absence of sufficient co-stimulation (signal ), t cells become anergic and fail to respond upon subsequent encounters with their cognate mhc/ peptide complex. the third signal dictates the phenotype and function of the primed t cell, and is mediated by paracrine signaling by soluble cytokines. these can be pro-inflammatory (e.g. il- and type i ifns) or anti-inflammatory (e.g. tfg-β and il- ), and help determine expression of chemokine receptors and integrins that facilitate t cell homing to specific target tissues. by providing these three signals, tidcs induce tumor specific immunity mediated by cd + t cells, which can help eliminate primary and metastatic tumors, and protect against cancer recurrence. however, tidcs often have functional defects which impair their ability to prime anti-tumor immune responses and can even suppress anti-tumor immunity and facilitate tolerance. these functional deficits range from decreased capacity for phagocytosis, reduced antigen processing capacity, abnormal motility and decreased expression of mhci, mhcii, proinflammatory cytokines, and co-stimulatory molecules such as cd , cd and cd (stoitzner et al., ; diao et al., ; diao et al., ; ataera et al., ; chiba et al., ) . tidcs also often have increased expression of inhibitory mediators, including the cytokines il- and tgf-β, ido, inos, arginase, and the inhibitory b family members pdl and pdl . one of the central dogmas of immunology is that endogenous antigens are efficiently presented in the context of mhci and exogenous antigens are presented in the context of mhcii. intriguingly, some dcs are able to take up exogenous antigens in certain contexts (e.g. cell associated or particulate), process them, load them on mhci, and present them on the cell surface through a process called cross-presentation (compeer et al., ; burgdorf et al., ; bevan, ; bevan, ) . this is the prevailing mechanistic hypothesis explaining how tidcs present tumor antigens in the context of mhci, providing signal for priming naïve cd + t cells in the draining lymph nodes. several pathways for cross-presentation have been identified. the cytosolic pathway closely resembles the classic pathway for presentation of endogenous peptides on mhci, in which endogenous proteins are cleaved by the proteasome, generating peptides that are subsequently translocated into the er lumen by tap½ for further processing and loading onto newly synthesized mhci molecules (amigorena and savina, ; joffre et al., ) . antigens cross-presented by this mechanism are taken up by dcs where they escape endosomes, entering the cytosol by an unknown mechanism where they are then cleaved by the proteasome. in contrast to the cytosolic pathway, cross-presentation through the vacuolar pathway does not require tap½ and seems to result from antigen processing and loading of peptide antigens onto mhci in the endocytic compartments. a third mechanism involving phagosome-er fusion has also been demonstrated in dcs (guermonprez et al., ; ackerman et al., ; houde et al., ) . this is a sort of hybrid between the cytosolic and vacuolar pathways in which phagosomes combine with the er and acquire antigen processing proteins such as mhci and tap½. antigens escape these phagosomes where they are cleaved by the proteasome, generating peptides that are transported back into the er-phagosome where they are further processed and loaded on mhci. shown to be more capable to cross-present cell-associated antigens than other populations. while most dc populations can cross-present materials under targeted experimental conditions, optimal presentation of (tumor) cell-associated antigens seems to be limited to a few populations of cdc s (faure et al., ; nierkens et al., ; thacker and janssen, ) . several mechanisms have been uncovered that explain the improved capacity for cross-presentation of cell-associated antigens by these cdc s. these include increased capacity to take up cell-associated materials, alterations in trafficking and acidification of endocytosed materials, and alterations in expression of components of the antigen processing machinery or its intracellular trafficking. dcs are able to recognize and endocytose dying cells by means of receptors that discriminate between "don't eat me" signals provided by healthy cells (including cd that is recognized by cd and cd ), "find-me" and "eat-me" signals provided by specific entities on dead and dying cells (reviewed in (medina and ravichandran, ; elliott and ravichandran, ; bratton and henson, ; liu et al., ) ). the "find-me" signals have been suggested to include atp, uridine 'triphosphate (utp), sphingosine -phosphate (s p), lysophosphatidyl choline, and fractaline/cx cl . depending on the manner of cell death, the dying cells will expose "eatme" signals on a continuum of dynamic and distinct cellular changes. phosphatidyl serine (ptdser), an early "eat-me" signal, can be bound directly by receptors such as tim- , tim- , tim- and bai or indirectly, using bridging molecules mfge , gas , protein s. additional "eat-me" signals have been shown to result from changes in expression of icam- , cq , oxidized lipids and alterations in glycosylation patterns. in the last decade, several new receptors and pathways that promote internalization of dying cells have been identified and it has become clear that the expression of these receptors is associated with distinct dc populations. specifically, expression of dec , clec a and axl/lrp / ranbp by cdc has been linked to the uptake of dead and dying cells (shrimpton et al., ; sancho et al., ; ramirez-ortiz et al., ; zelenay et al., ; subramanian et al., ) . intriguingly, use of specific internalizing receptors has been shown to affect subsequent endosomal trafficking, with receptors that promote uptake of smaller particles associated with a slower endosomal acidification rate. the delayed endosomal acidification would reduce proteolytic degradation and preserve antigenic structures, thereby providing a longer time-span where antigens can escape into the cytosol for processing (compeer et al., ; thacker and janssen, ; ackerman and cresswell, ; basha et al., ) . cdc already display an increased propensity for delayed endosomal/lysosomal fusion compared to other dc populations, and one can easily envision that different combinations of dc populations and endocytic receptor usage will greatly affect the efficacy of a specific dcs in their capacity to cross-present cell-associated antigens in the context of mhci. dcs form the cellular link between innate and adaptive anti-tumor immunity, but only in the context of the appropriate maturation signals. the maturation status of the dc is critically important to the induction of t cell responses as it dictates the expression levels of mhc and membrane-associated and soluble co-stimulatory molecules (lutz and schuler, ) . immature dcs reside in the periphery and have great capacity for endocytosis. they express low levels of mhc class i and ii, and membrane associated co-stimulatory molecules including cd , cd and cd . upon maturation, dcs reduce expression of ccr and increase expression of ccr which facilitates homing to the draining lymphoid tissues. during migration to the lymphatics, dcs process the endocytosed antigens for presentation in mhc class i and ii molecules, and increase the expression of mhci/ii and activating costimulatory molecules while acquiring the capacity to produce proinflammatory cytokines and chemokines that will facilitate their interaction with antigen-specific t cells. in contrast to normal tissue dcs, these essential functions are often impaired in tidcs, which can maintain their immature phenotype. this can contribute to tumor tolerance, and has been suggested to result from decreased capacity to respond to normal maturation signals as a result of immune suppressive mediators produced by the tumor and its microenvironment. several pathways for dc maturation have been identified, with only some of them relevant to sterile inflammation and cancer, although there is significant overlap between pathogen sensing signals and signals that mediate sterile inflammation. most research has been performed in the context of infectious pathogens which has led to the identification of pathogen-associated molecular patterns (pamps) that engage specific pattern recognition receptors (prrs) on the dc surface and in intracellular vesicles. each dc subset has its own unique expression pattern of pprs, causing these dc populations to respond differently upon engagement of the same ppr, providing highly modulated nuance to the immune response (segura et al., ) . there are several families of prrs, including the toll-like receptors (tlrs) and c-type lectin receptors that mainly recognize extracellular stimuli, and cytosolic prrs, including rig-i-like receptors (rlrs) and nod-like receptors (nlrs) that are critically important for the sensing of intracellular pathogens (kawai and akira, ; loo and gale, ; elinav et al., ; osorio and reis e sousa, ; kawai and akira, ; pandey et al., ) . as maturation of dc by prr signaling has been covered by various other reviews, we will provide a summary of different maturation signals, with emphasis on the sting and type i ifn pathway. tlrs were the first prrs to be identified and in addition to sensing pamps, they can also detect certain markers of sterile, lytic cell death, implicating them in tumor immunity in addition to infectious processes. the tlrs are membrane bound receptors that include at least (mouse) and (human) proteins that recognize a wide range of pamps and even some danger-associated molecular patterns (damps), a broader term including endogenous molecules secreted by cells undergoing stress, or released during tissue damage and cell death that signal inflammation. prokaryotic products recognized include lipoproteins (tlr½/ ), lipopolysaccharide (tlr ), and flagellin (tlr ) on the cell surface membrane. the intracellular vesicle-associated tlrs recognize dsrna (tlr ), single stranded rna (ssrna, tlr / ) and dna (tlr ). depending on the type of tlr, its signaling requires one or several of the adapter molecules myd , trif, tirap and tram, followed by activation downstream mediators which ultimately induce the translocation of nfκb or ifn-regulatory factors (irfs), including irf , irf , and irf (kawai and akira, ; kawai and akira, ) . these receptors can induce the rapid transcription of inflammatory cytokines, including type i ifn, il- , tnfα, and il- . non-mammalian carbohydrates such as mannose and acetylglucosamine form one such class of pamps recognized by c-type lectin receptors (osorio and reis e sousa, ) . these receptors are clustered into an activating group signaling through immunoreceptor tyrosine-based activation (itam) like motifs and itambinding fcrg adaptor molecules, and an inhibitory group which mediate their effect through immunoreceptor tyrosine-based inhibition (itim)-motifs. while important in sensing many bacteria, they are less relevant to innate immune sensing of cancer. : nod-like receptors (nlrs) encompass group of cytosolic innate sensors that predominantly recognize bacterial cell components. upon engagement with their cognate ligands, nlrs oligomerize into a caspase- -activating scaffold known as the inflammasome. active caspase- subsequently functions to cleave the proinflammatory il- family of cytokines into their bioactive forms. the aim -like receptor (alr) or p family contains several sensors of cytoplasmic dna, including ifi and aim . like the nlrs, aim functions in an inflammasome that complexes with caspase- and promotes the cleaving of the proinflammatory il- family of cytokines. interestingly, while having some homology with aim , the ifi (mouse p ) binds dsdna, but does not form an inflammasome, and signals via sting for the induction of type i ifns and other inflammatory mediators. retinoic acid inducible gene (rig-i, or ddx ) like receptors are another family of prrs comprised of dexd/h box rna helicases that play an important role in antiviral immunity that detect viral associated rna (loo and gale, ) . this family encompasses cytosolic proteins rig-i that binds ′-triphosphorylated rna and short double stranded rna (dsrna), and melanoma differentiation associated protein (mda ) that recognizes long dsrna. rig-i and mda signal via their caspase activation and recruitment domain (card) to the mitochondrial antiviral signaling domain (mavs, also known as cardif or ips- ) which subsequently stimulates tbk , ikk and the irf/nfκb pathways resulting in the induction of inflammatory cytokines. additional studies indicate that, for specific viruses, the rig-i pathway converges with the sting pathway potentiating the anti-viral response. recently, sting has been identified as an important regulator of immunity by mediating type i ifn production in response to cytosolic dna (burdette and vance, ; ishikawa et al., ) . sting is a protein sensor of cytosolic dna anchored in the er. while initial studies suggested that sting may sense dna structures directly, it is now widely accepted that other cytosolic dna binding molecules upstream of sting are required for the activation of the pathway. the cyclic-gmp-amp synthase (cgas) is considered the predominant dna sensor in this pathway. upon binding to dna in the cytosol, cgas catalyzes the synthesis of cyclic gmp-amp (cgamp) from guanosine triphosphate (gtp) and adenosine triphosphate (atp) (ablasser et al., ; civril et al., ) . cgamp functions as a second messenger that binds to and activates sting, resulting in its trafficking from the er to the golgi and on to perinuclear endosomes where it mediates subsequent signaling via phosphorylation of tank-binding kinase (tbk ) and irf , which induces transcription of type i ifns and other inflammatory genes (fig. ) . while the cgas-cgamp pathway is considered the dominant pathway, there is evidence that other sensors recognize cytosolic dna associated with viral infection and use the sting-tbk-irf pathway to induce type i ifn responses. among them, dna-dependent activator of ifn-regulatory factor (dai), dexd/h-box rna helicase ddx , dna dependent protein kinase (dna-pk), and ifn-γ-inducible protein (ifi in humans and ifi /p in mouse) have been implied. damps-as with the immune response to pathogen infections, dcs must be appropriately activated in order to prime t cell responses against cancer. however, uptake of dying cells is generally considered to be a tolerogenic event in order to maintain tissue homeostasis and prevent development of autoimmune disorders. dcs that have phagocytosed dying cells have been reported to produce the immune-suppressive cytokines tgf-β and il- , and are significantly inhibited in their capacity to produce pro-inflammatory cytokines and upregulate co-stimulatory molecules. one mechanism for sensing cancer and developing inflammation in a sterile environment involves the release of endogenous molecules that serve as damps, which can signal through the same prrs that sense pathogen derived molecules. damps encompass cellular components and factors that are either present before death and released by dying cells or generated as a result of cellular stress. examples include atp which may engage the nod/ nlrp inflammasome, hmgb which has been suggested to signal through tlr , filamentous actin which is recognized by clec a, as well as heat shock proteins and uric acid (zhang et al., ) . normally intracellular and out of the reach of dcs under physiological conditions, these damps serve as a danger signal when released into the extracellular milieu by stressed or dying cancer cells. when damps are accessible to dcs, their presence signifies the loss of integrity of the cell membrane, suggestive of tissue damage caused by noxious origins such as infection, mechanical injury, or hypoxia. in a cancer vaccination animal model, tlr and its downstream signaling adaptor myd have been implicated in the immune response to cancers induced by radiation and certain chemotherapies, however in other models tlr /myd was not found to be necessary or sufficient for the development of an immune response to cancers. more recently, type i ifn production and signaling through the sting pathway has been identified as having an essential role in the development of anti-tumor immunity in response to tumor derived dna. protection of the integrity of genetic information encoded in dna has been a primary prerogative of living organisms since their inception, and sensing threats in the form of dna damage through radiation exposure, oxidative stress, and viral infection is essential to maintaining and continuing the line of genetic information. cells have maintained this potential, especially in apcs such as dcs, which respond to dna damage and cytosolic dna by producing type i ifn. in addition to the recognition of dsdna, several reports suggest that sting can promote type i ifn production upon rna-virus infection through its interaction with the rna sensing pathway components rig and mavs (nazmi et al., ; sun et al., ) . however, evidence that the sting pathway in dcs is activated by rna upon uptake of dead or dying cells is not available. type i ifn signaling in dcs has been shown to promote their capacity to prime and crossprime t cells in several ways. type i ifns are cytokines with potent activity on dc phenotype and function through their effects on all three signals needed to generate a robust t cell response. type i ifns are a collection of ifnα proteins, as well as ifnβ, ifnκ, and ifnω, all of which signal through the common interferon (α and β) receptor (ifnar). essentially all nucleated cells express ifnar, a heterodimer including the two distinct subunits ifnar and ifnar . binding of ifns to their receptors leads to dimerization of the two subunits, resulting in auto phosphorylation of tyrosine kinase (tyk ) and janus activated kinase (jak) that activate the signal transducer and activator of transcription (stat) and (theofilopoulos et al., ) . the activated stat proteins dimerized and rapidly translocate to the nucleus where they bind ifn-stimulated response elements (isres), initiating the transcription of hundreds of ifn-sensitive genes (isgs). type i ifn facilitates cross-priming by dcs through a variety of mechanisms. it promotes antigen persistence through the reduction of the endosomal-lysosomal acidification rate, thus facilitating the storage of exogenously acquired cell-associated antigens in rab + and rab + compartments (thacker and janssen, ; lorenzi et al., ) . furthermore, type i ifn promotes transcription and translation of the immunoproteasome subunits β i (lmp ), β i (mecl- ), and β i (lmp ) (shin et al., ; shin et al., ) , which modifies the peptidase activity of the constitutive proteasome, changing the repertoire of peptides for binding and presentation on mhci. in addition, ifnα treatment promotes localization of mhci molecules to antigen storage compartments in dcs, enhancing antigen presentation (spadaro et al., ) . in addition to these effects on antigen persistence, processing and loading, type i ifn also increases overall surface expression levels of mhci and mhcii, as well as the membrane associated co-stimulatory molecules cd , cd , and cd . moreover, type i ifn can serve as a signal cytokine, enhancing cd + t cell priming through its signaling within t cells (agarwal et al., ; curtsinger and mescher, ; xiao et al., ). table ). in murine models, type i ifn seems to be required for anti-tumor immunity. dunn et al. demonstrated that mice that lacked ifnar were more susceptible to carcinogeninduced tumors, and mice treated with anti-ifnar antibodies were much more susceptible to transplanted tumor outgrowth than mice given isotype controls (dunn et al., a; dunn et al., b) . fuertes et al. discovered spontaneous antitumor cd + t cell responses were defective in mice lacking either ifnar or stat , a transcription factor immediately downstream of ifn signaling (fuertes et al., ) . studies of mice lacking ifnar specifically on dcs or lacking batf -dependent dc populations demonstrated that the antitumor responses were dependent on cdc -like dcs (diamond et al., ; hildner et al., ; klarquist et al., ) . is not possible to be certain of the role of type i ifn in the immune response to human cancers, but there is strong correlative data suggesting its importance. in human patients with metastatic melanoma, metastatic lesions infiltrated with t cells are characterized by an ifn gene signature (fuertes et al., ; harlin et al., ) . this inflamed phenotype is associated both with inhibitory factors that suppress t cell function and with response to immunotherapy, including checkpoint inhibitors targeting ctla- and pd- (ji et al., ; topalian et al., ) . a type i ifn gene signature in the primary tumor at diagnosis has also been found to be associated with improved prognosis and response to anthracycline chemotherapy in women with breast cancer (sistigu et al., ) . these associations are supported by studies in murine models, demonstrating that breast cancer is more likely to metastasize to bone when the irf pathway is blocked (bidwell et al., ) . this pattern also holds in studies of cancer vaccines, where a similar gene signature has been found to correlate with improved clinical responses. in aggregate, these findings support the relevance of type i ifn responses in human patients, and in fact motivated much of the work highlighting the importance of ifn in the animal models (ulloa-montoya et al., ) . signaling through the sting pathway is an essential precursor to generating the type i ifn needed for both spontaneous and treatment induced cancer immune responses. dna sensing by sting is the upstream link that triggers type i ifn production by dcs and facilitates effective cross-priming of tumor specific cd + t cells. this dna is likely derived from stressed and dying cancer cells, and is introduced into the dc cytosol through a yet unknown mechanism where it binds to cgas, which initiates sting mediated type i ifn transcription (fig. ) . the importance of this pathway has been demonstrated in a variety of tumor models, including colon carcinoma, melanoma, and lymphoma. (woo et al., ) . sting was also required for spontaneous rejection of an immunogenic sarcoma, and the authors nicely explore the mechanism, showing that the sting pathway is activated in host dcs as a result of sensing tumor derived dna (woo et al., ; woo et al., ) . these findings have been extended to other tumor models, such as colitis induced cancer, in which sting deficient mice have increased susceptibility to colon cancer oncogenesis in response to inflammation (zhu et al., ; ahn et al., ) . in a mouse model that spontaneously develops intracranial glioma, a loss of function mutation in sting results in reduced production of type i ifn by myeloid cells with impairment of tumor control by the immune system (ohkuri et al., ) . dna damage is the likely stimulus for activating sting and transcription of type i ifn, as a breast cancer model defective in dna damage repair is found to have accumulation of dna in the cytosol and activation of an ifn response in a sting dependent manner (parkes et al., ) . these tumors with impaired dna damage repair are grown in mice, they develop a t cell infiltrate compared with the wild type breast cancer tumors. approaches have now been shown to require the dc-sting-ifn pathway for their effects. radiation therapy is a prime example of this. ionizing radiation has long been known to selectively kill cancer cells and has been used in cancer treatment for local tumor control for over a century. cytotoxicity through damage of genomic dna is thought to be the principal mechanism for the effects of radiotherapy, but more recently evidence has emerged that implicates the immune system in tumor control mediated by radiation (apetoh et al., ; burnette and weichselbaum, ; burnette et al., ; lee et al., ) . furthermore, focal irradiation of tumors activates dcs and induces cross-priming of tumor specific t cells that mediate systemic anti-tumor immunity (demaria et al., ; gupta et al., ; lim et al., ; lugade et al., ; weichselbaum et al., ) . this radiation induced anti-tumor immune response is dependent on sting and its upstream mediator cgas (deng et al., a) . tumors in mice lacking these genes are not controlled by an otherwise effective dose of radiation, their dcs do not produce type i ifn in response to radiation, and there is a defect in cross-priming of tumor specific cd + t cells after radiotherapy. to radiation therapy, many antineoplastic chemotherapy agents induce cytotoxicity by damaging chromosomal dna. fibroblasts treated with ara-c undergo dna damage with nuclear dna transported into the cytosol which can be sensed by sting, triggering a type i interferon response (lan et al., ) . chemotherapy agents such as etoposide can also have direct affects on dcs causing dna damage and transport of dna to the cytosol which also elicits a sting-dependent type i interferon response. different classes of chemotherapy agents induce differing types of dna damage, and one can speculate that chemotherapeutic agents that support the formation of ligands for the sting pathway would provide clinical benefits by facilitating anti-tumor immunity. however, there is currently little known on the induction of sting ligands and type i ifn inducing capacity of the main dna damaging chemotherapy agents. , recent studies indicate that not all of the effects of sting activation promote tumor immunity and protection. sting agonists can also promote oncogenesis, for example mice treated cutaneously with , -dimethylbenz[a]anthracene (dmba) develop inflammation and skin cancer which is mediated by sting (ahn et al., ) . sting can also modify the tumor microenvironment to become more tolerogenic promoting tumor progression and metastasis through induction of ido which promotes treg function and immune tolerance of tumors by cytokines such as il- and tgf-β (lemos et al., ; huang et al., ) . at this point it is not clear why or under which circumstances sting and type i ifn promotes or suppresses anti-tumor immunity, although chronic inflammation is a well accepted stimulus of oncogenesis, and one can speculate that the complex tumor micro-environment with its various regulatory components such as tregs, myeloid derived suppressor cells, and macrophages may help determine the effects of ifn stimulation. in recent years, pharmacologic agents that activation the sting pathway have been used to mimic and augment the role of sting mediated production of type i ifn in anti-tumor immunity. administration of sting ligands (c-di-gmp, dmxaa, cgamp, cdn) significantly improve anti-tumor responses and animal survival in a diverse array of mouse models of solid and blood borne cancers (deng et al., a; baird et al., ; chandra et al., ; corrales et al., ; curran et al., ) . dmxaa is a sting agonist with a multi-faceted effect on the tumor microenvironment, and treatment of b melanoma with dmxaa augments tumor-specific cd + t cell responses which effectively immunize and protect mice against future tumor challenge . similar results are seen in mouse models of prostate and breast cancer as well as sarcoma (baguley and ching, ) . in light of these promising results from the animal models, dmxaa was tested in a clinical trial of patients with non-small cell lung cancer. unfortunately, dmxaa does not bind to human sting and thus was unable to activate this pathway, resulting in a negative trial (lara et al., ; gao et al., ) . another sting agonist, the cyclic diguanylate nucleotide (cdn) c-di-gmp, is effective in the treatment of t mammary carcinoma. a synthetic cdn agonist mlrr-s cda has high affinity for human and murine sting and stimulates type i ifn production in human pbmcs . this agent induces immune mediated tumor rejection of melanoma, breast and colon carcinoma in mice . cdn based sting agonists have also been used in conjunction with the gvacs cancer vaccine along with a pd antagonist, which resulted in an additive effect on tumor immunity (fu et al., ) . this opens new avenues for additional novel therapies including cd ("don't eat me" signal) blockade which has been shown to be critically dependent on the ifn/sting pathway (liu et al., ) . as described above, radiotherapy induces dna damage that is sensed by tidcs through the sting pathway, and there are currently many pre-clinical and several clinical studies utilizing radiation to stimulate anti-tumor immunity. since radiotherapy alone rarely induces an anti-tumor immune response that results in systemic control of metastatic disease, the therapeutic effects of radiation on the innate and adaptive immune system have been largely explored in combination with other immunotherapy modalities. the initial studies demonstrating the synergistic effect of radiation combined with immune modulators were performed by demaria and formenti. their group observed improved tumor control within the irradiated field and objective responses in distant sites of disease using flt ligand or antibody antagonists to the ctla- checkpoint receptor in breast cancer and colon cancer models (demaria et al., ; demaria et al., a; dewan et al., ; demaria et al., b) . similar findings in other animal models combining radiation with pd- inhibitors, dc vaccines, and other immune modulators have been observed, and further mechanistic studies have been performed by multiple groups (deng et al., b; filatenkov et al., ; sharabi et al., a; sharabi et al., b; vatner et al., ; yoshimoto et al., ) . one study even combined radiation therapy with a pharmacological sting agonist and found improved tumor control and anti-tumor immunity, suggesting that radiation is providing a unique ingredient to the production of anti-tumor immunity above and beyond sting activation (baird et al., ) . these animal studies have been translated successfully into preliminary clinical trials of patients with metastatic cancer, and there are many ongoing trials testing this in patients with a variety of malignancies. most of these trials combine radiation with an immune checkpoint inhibitor of ctla- or pd- with the goal of priming tumor specific t cells with radiation, and boosting that t cell response with the checkpoint inhibitors to overcome the suppressive microenvironment. preliminary results appear promising, but mature results from these trials are pending (golden et al., ) . with radiotherapy there are always questions about the optimal dose to use and how many treatments to deliver (fractionation) for activating sting and initiating an immune response. this is an active question in the field, however there are few preclinical studies and even fewer clinical trials comparing different doses and fractionation treatment schedules. some of the original studies by the demaria group explored dose and fractionation, finding that gy delivered in three fractions of gy resulted in more robust systemic tumor control compared with gy in five fractions or a single fraction of gy (dewan et al., ) . a recent study by this group explored the mechanistic underpinnings of why a single larger fraction of gy of radiation is not as effective as three fractions of gy, and found that both treatments resulted in transport of dsdna to the cytosol of tumor cells where it was sensed by cgas, which activated sting and type i ifn transcription (vanpouille-box et al., ) . however, the larger dose of radiation induced the three prime exonuclease (trex ), which degraded the cytosolic dna, limiting sting activation and the ifn response. studies by other groups comparing single fraction treatment with fractionated treatment have shown conflicting results, however most of these utilized single fraction treatment doses < gy, the apparent threshold for activation of trex . beyond dose and fractionation, there are other questions that remain about the optimal application of radiation therapy for stimulating sting mediated tumor immunity. some of these questions include the importance of the anatomical location of the target lesion, whether it is better to treat the primary tumor or metastatic sites, the effect of radiation dose rate, and the effect of different modalities and energies of ionizing radiation (e.g. photon vs. proton vs. electron). these questions remain to be worked out. the role of the immune system in combatting cancer has been a topic of speculation and debate over the past century, with mounting experimental evidence in support of this function of the immune system. however, it was not until recent years with the success of cancer immunotherapy applied to patient care that cancer immunity has received widespread acceptance. how the immune system is alerted to the presence of cancer has been one of the central questions in cancer immunology. dcs are the link between the innate and adaptive immune systems, only priming t cell responses against cellular antigens in the context of an inflammatory signal. there has been some debate regarding the nature of this inflammatory signal in the response to tumors, but recent evidence summarized in this review support the hypothesis that damaged dna released by cancer cells is the stimulus for generating tumor immunity. this dna gains access to the cytoplasm of dcs where it is recognized by cgas which triggers sting and production of the type i ifns necessary for effective crosspriming of cd + t cells against tumor antigens. the primacy of dna sensing as a stimulus for dc activation in response to cancer helps inform an ontological question in cancer immunology. cancer has historically been viewed as pathogenic process separate from the host organism. even the concept of "tumor" and "host" suggests an otherness to cancer. this thinking naturally led to a search for the danger signals produced in response to cancer, and broadened our understanding of innate immunity as a means to detect self from non-self through pamps into a more generalized view as a means of detecting danger from the absence of danger through damps. the fact that dcs are sensing damaged dna rather than a damp associated with lytic cell death places the immune response to cancer more in the realm of assaults on the integrity of the genome rather than an exogenous pathogen. although much of the mechanism for sensing of cancer derived dna via the sting pathway in dcs has been elucidated, many questions remain. one such question relates to how tumor dna taken up by dcs gains access to the dc cytosol. it may be more than coincidence that dc populations that are efficient in cross-presentation and that exhibit delayed endosomal acidification seem to be explicitly implicated in the sting dependent induction of type i ifn. one can envision a mechanism for dna escape from endosomal compartments analogous to the manner by which endocytosed proteins escape into the cytosol where they are processed into peptides for presentation on mhci. another unanswered question relates to the nature of the direct upstream signaling molecules that bind cytosolic dna and engage sting, as these could be targeted for therapeutic benefit. it is also intriguing how sting activation promotes anti-tumor immunity in some models, while in others it suppresses the immune response to the tumor, promoting tolerance. this is a most exciting time for tumor immunology, as we are seeing the scientific understanding of cancer translated into human clinical trials. the application of therapies targeting the sting pathway to mimic and enhance the endogenous immune response to cancer is currently underway, and results from these trials will help enhance our understanding of cancer immunity, offering a hopeful future for enlisting the immune system in the fight against cancer. the sting signaling pathway. cytosolic dna is primarily sensed by cyclic-gmp-amp synthase (cgas), which generates cyclic gmp-amp (cgamp) from guanosine triphosphate (gtp) and adenosine triphosphate (atp). cgamp functions as a second messenger that binds to and activates sting, resulting in its trafficking from the er to the golgi and on to perinuclear endosomes where it mediates subsequent signaling via phosphorylation of tank-binding kinase (tbk ), irf , and nf-κb, which induce transcription of type i ifns and other inflammatory genes. ddx , zbp , dna-pk, and ifi can also act as sensors of cytosolic dna with similar downsream effects on sting. page mol immunol. author manuscript; available in pmc september . sting-mediated induction of type i ifn in dcs upon sensing of tumor dna. dna damaging therapy results in the induction of cell death, nucleosome release, mitochondrial damage that are endocytosed by dcs. upon escape of endosomal tumor-derived dna into the cytosol, tumor-dna can interact with cgas which results in the induction of cdn and the activation of sting. downstream signaling from sting induces pro-inflammatory genes, including type i ifns. in parallel, protein escape from endosomal degradation promotes cross-presentation in mhc class i. immune responses dependent on type i ifn, sting and damp (dna). cgas produces a '− '-linked cyclic dinucleotide second messenger that activates sting cellular mechanisms governing cross-presentation of exogenous antigens early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens gene regulation and chromatin remodeling by il- and type i ifn in programming for cd t cell effector function and memory inflammation-driven carcinogenesis is mediated through sting diverse roles of sting-dependent signaling on the development of cancer intracellular mechanisms of antigen cross presentation in dendritic cells toll-like receptor -dependent contribution of the immune system to anticancer chemotherapy and radiotherapy murine melanomainfiltrating dendritic cells are defective in antigen presenting function regardless of the presence of cd cd regulatory t cells immunomodulatory actions of xanthenone anticancer agents radiotherapy combined with novel sting-targeting oligonucleotides results in regression of established tumors mhc class i endosomal and lysosomal trafficking coincides with exogenous antigen loading in dendritic cells cross-presentation of viral and self antigens by skin-derived cd + dendritic cells intestinal dendritic cells in the regulation of mucosal immunity cross-priming for a secondary cytotoxic response to minor h antigens with h- congenic cells which do not cross-react in the cytotoxic assay cross-priming silencing of irf pathways in breast cancer cells promotes bone metastasis through immune escape apoptotic cell recognition: will the real phosphatidylserine receptor(s) please stand up? sting and the innate immune response to nucleic acids in the cytosol spatial and mechanistic separation of crosspresentation and endogenous antigen presentation radiation as an immune modulator the efficacy of radiotherapy relies upon induction of type i interferondependent innate and adaptive immunity sting ligand c-di-gmp improves cancer vaccination against metastatic breast cancer the janus face of dendritic cells in cancer molecular mechanisms of t cell co-stimulation and co-inhibition tumor-infiltrating dcs suppress nucleic acid-mediated innate immune responses through interactions between the receptor tim- and the alarmin hmgb transcription factor e - is an essential and specific regulator of plasmacytoid dendritic cell development structural mechanism of cytosolic dna sensing by cgas antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation direct activation of sting in the tumor microenvironment leads to potent and systemic tumor regression and immunity the xc chemokine receptor is a conserved selective marker of mammalian cells homologous to mouse cd alpha+ dendritic cells sting pathway activation stimulates potent immunity against acute myeloid leukemia inflammatory cytokines as a third signal for t cell activation development and functional specialization of cd + dendritic cells ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated combining radiotherapy and immunotherapy: a revived partnership immune-mediated inhibition of metastases after treatment with local radiation and ctla- blockade in a mouse model of breast cancer sting-dependent cytosolic dna sensing promotes radiation-induced type i interferon-dependent antitumor immunity in immunogenic tumors irradiation and anti-pd-l treatment synergistically promote antitumor immunity in mice fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-ctla- antibody type i interferon is selectively required by dendritic cells for immune rejection of tumors recruitment and differentiation of conventional dendritic cell precursors in tumors tumors suppress in situ proliferation of cytotoxic t cells by promoting differentiation of gr- (+) conventional dendritic cells through il- a critical function for type i interferons in cancer immunoediting interferon-gamma and cancer immunoediting toll-like receptor expression in murine dc subsets: lack of tlr expression by cd alpha+ dc correlates with unresponsiveness to imidazoquinolines regulation of the antimicrobial response by nlr proteins the dynamics of apoptotic cell clearance tumor's other immune targets: dendritic cells long-lasting crosspresentation of tumor antigen in human dc ablative tumor radiation can change the tumor immune cell microenvironment to induce durable complete remissions sting agonist formulated cancer vaccines can cure established tumors resistant to pd- blockade host type i ifn signals are required for antitumor cd + t cell responses through cd {alpha}+ dendritic cells structure-function analysis of sting activation by c[g( ', ')pa( ', ') p] and targeting by antiviral dmxaa local radiotherapy and granulocyte-macrophage colony-stimulating factor to generate abscopal responses in patients with metastatic solid tumours: a proof-of-principle trial erphagosome fusion defines an mhc class i cross-presentation compartment in dendritic cells radiotherapy promotes tumor-specific effector cd + t cells via dendritic cell activation irf mutations and human dendritic-cell immunodeficiency human tissues contain cd (hi) cross-presenting dendritic cells with functional homology to mouse cd (+) nonlymphoid dendritic cells human skin dendritic cells in health and disease chemokine expression in melanoma metastases associated with cd + t-cell recruitment dendritic cell and macrophage heterogeneity in vivo cd + cd + dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of langerhans cells batf deficiency reveals a critical role for cd alpha+ dendritic cells in cytotoxic t cell immunity antigen crosspresentation by human plasmacytoid dendritic cells phagosomes are competent organelles for antigen cross-presentation cutting edge: dna sensing via the sting adaptor in myeloid dendritic cells induces potent tolerogenic responses antigen presentation by langerhans cells sting regulates intracellular dna-mediated, type i interferondependent innate immunity an immune-active tumor microenvironment favors clinical response to ipilimumab cross-presentation by dendritic cells human cd + (bdca- )+ dendritic cells (dcs) represent a unique myeloid dc subset that cross-presents necrotic cell antigens deciphering the message broadcast by tumor-infiltrating dendritic cells antigen-presenting cells in the skin the roles of tlrs, rlrs and nlrs in pathogen recognition toll-like receptors and their crosstalk with other innate receptors in infection and immunity tumors promote altered maturation and early apoptosis of monocyte-derived dendritic cells sting-mediated dna sensing promotes antitumor and autoimmune responses to dying cells density of dc-lamp(+) mature dendritic cells in combination with activated t lymphocytes infiltrating primary cutaneous melanoma is a strong independent prognostic factor dnase a deficiency uncovers lysosomal clearance of damaged nuclear dna via autophagy randomized phase iii placebo-controlled trial of carboplatin and paclitaxel with or without the vascular disrupting agent vadimezan (asa ) in advanced non-small-cell lung cancer therapeutic effects of ablative radiation on local tumor require cd + t cells: changing strategies for cancer treatment sting promotes the growth of tumors characterized by low antigenicity via ido activation antitumor activity of cgamp via stimulation of cgas-cgamp-sting-irf mediated innate immune response type i interferons induced by radiation therapy mediate recruitment and effector function of cd (+) t cells origin and development of dendritic cells cd blockade triggers t cell-mediated destruction of immunogenic tumors immune signaling by rig-i-like receptors type i ifns control antigen retention and survival of cd alpha (+) dendritic cells after uptake of tumor apoptotic cells leading to cross-priming local radiation therapy of b melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity tumor associated regulatory dendritic cells dendritic cells in the cancer microenvironment do not let death do us part: 'find-me' signals in communication between dying cells and the phagocytes the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting dendritic cell development: a choose-your-own-adventure story transcriptional priming of intrathymic precursors for dendritic cell development sting mediates neuronal innate immune response following japanese encephalitis virus infection antigen cross-presentation by dendritic cell subsets: one general or all sergeants? sting contributes to antiglioma immunity via triggering type i ifn signals in the tumor microenvironment protective role of sting against gliomagenesis: rational use of sting agonist in anti-glioma immunotherapy. oncoimmunology , e myeloid c-type lectin receptors in pathogen recognition and host defense microbial sensing by toll-like receptors and intracellular nucleic acid sensors. cold spring harbor perspect activation of sting-dependent innate immune signaling by s-phase-specific dna damage in breast cancer gangliosides from human melanoma tumors impair dendritic cell differentiation from monocytes and induce their apoptosis cytokine-mediated protection of human dendritic cells from prostate cancer-induced apoptosis is regulated by the bcl- family of proteins characterization of human dngr- + bdca + leukocytes as putative equivalents of mouse cd alpha+ dendritic cells dngr- is a specific and universal marker of mouse and human batf -dependent dendritic cells in lymphoid and nonlymphoid tissues dendritic cells and liver fibrosis the scavenger receptor scarf mediates the clearance of apoptotic cells and prevents autoimmunity migration of dendritic cell subsets and their precursors the number of intratumoral dendritic cells and zeta-chain expression in t cells as prognostic and survival biomarkers in patients with oral carcinoma regulation of plasmacytoid dendritic cell development dendritic cells and macrophages in the kidney: a spectrum of good and evil langerhans cells and more: langerin-expressing dendritic cell subsets in the skin functional characterization of the human dendritic cell immunodeficiency associated with the irf (k e) mutation identification of a dendritic cell receptor that couples sensing of necrosis to immunity spi-b is critical for plasmacytoid dendritic cell function and development transcription factor networks in dendritic cell development the ets transcription factor spi-b is required for human plasmacytoid dendritic cell development inflammatory dendritic cells in mice and humans differential expression of pathogen-recognition molecules between dendritic cell subsets revealed by plasma membrane proteomic analysis radiation and checkpoint blockade immunotherapy: radiosensitisation and potential mechanisms of synergy stereotactic radiation therapy augments antigen-specific pd- -mediated antitumor immune responses via cross-presentation of tumor antigen virus-induced type i ifn stimulates generation of immunoproteasomes at the site of infection proteasome activator and antigen-processing aminopeptidases are regulated by virus-induced type i interferon in the hepatitis c virus-infected liver the cd + dendritic cell subset dec- ): a recognition receptor for apoptotic and necrotic self cancer cell-autonomous contribution of type i interferon signaling to the efficacy of chemotherapy ifn-alpha enhances cross-presentation in human dendritic cells by modulating antigen survival, endocytic routing, and processing features of the dendritic cell lineage inefficient presentation of tumor-derived antigen by tumor-infiltrating dendritic cells an axl/lrp- /ranbp complex mediates dc efferocytosis and antigen cross-presentation in vivo coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling cross-presentation of cell-associated antigens by mouse splenic dendritic cell populations type i interferons (alpha/beta) in immunity and autoimmunity safety, activity, and immune correlates of anti-pd- antibody in cancer tumor-in-filtrating dendritic cells in cancer pathogenesis predictive gene signature in mage-a antigen-specific cancer immunotherapy dna exonuclease trex regulates radiotherapy-induced tumour immunogenicity antigen-presentation properties of plasmacytoid dendritic cells cgas is essential for the antitumor effect of immune checkpoint blockade radiotherapy and immunotherapy: a beneficial liaison? immunomorphological and ultra-structural characterization of langerhans cells and a novel, inflammatory dendritic epidermal cell (idec) population in lesional skin of atopic eczema sting-dependent cytosolic dna sensing mediates innate immune recognition of immunogenic tumors the sting pathway and the t cell-inflamed tumor microenvironment programming for cd t cell memory development requires il- or type i ifn radiotherapy-induced anti-tumor immunity contributes to the therapeutic efficacy of irradiation and can be augmented by ctla- blockade in a mouse model the dendritic cell receptor dngr- controls endocytic handling of necrotic cell antigens to favor cross-priming of ctls in virus-infected mice the dendritic cell receptor clec a binds damaged cells via exposed actin filaments cutting edge: sting mediates protection against colorectal tumorigenesis by governing the magnitude of intestinal inflammation key: cord- -b v c c authors: de lang, anna; baas, tracey; teal, thomas; leijten, lonneke m; rain, brandon; osterhaus, albert d; haagmans, bart l; katze, michael g title: functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov–infected macaques date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: b v c c the pathogenesis of severe acute respiratory syndrome coronavirus (sars-cov) is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. using functional genomics, we analyzed early host responses to sars-cov infection in the lungs of adolescent cynomolgus macaques (macaca fascicularis) that show lung pathology similar to that observed in human adults with sars. analysis of gene signatures revealed induction of a strong innate immune response characterized by the stimulation of various cytokine and chemokine genes, including interleukin (il)- , il- , and ip- , which corresponds to the host response seen in acute respiratory distress syndrome. as opposed to many in vitro experiments, sars-cov induced a wide range of type i interferons (ifns) and nuclear translocation of phosphorylated signal transducer and activator of transcription in the lungs of macaques. using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. our studies emphasize that the induction of early ifn signaling may be critical to confer protection against sars-cov infection and highlight the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of sars. infection with sars-cov causes lower respiratory tract disease with clinical symptoms that include fever, malaise, and lymphopenia [ ] . approximately %- % of sars patients require management in intensive care units, and the overall fatality rate has approached %. interestingly, children seem to be relatively resistant to sars, but the reason for this restriction is not known [ ] [ ] [ ] . the clinical course of sars follows three phases [ , ] . in the first phase, there is active viral replication and patients experience systemic symptoms. in the second phase, virus levels start to decrease while antibodies, which are effective in controlling infection, increase. however, pneumonia and immunopathological injury also develop in this phase. ultimately, in the third phase, fatal cases of sars progress to severe pneumonia and acute respiratory distress syndrome (ards), characterized by the presence of diffuse alveolar damage (dad) [ , ] . it has been hypothesized that the pathological changes are caused by a disproportional immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such as cxcl (ip- ), ccl (mcp- ), interleukin (il)- , il- , il- , il- b, and interferon (ifn)-c [ ] [ ] [ ] [ ] [ ] [ ] . these in vivo data have been confirmed with in vitro experiments, demonstrating that sars-cov infection induces a range of cytokines and chemokines in diverse cell types [ ] [ ] [ ] [ ] [ ] [ ] . in contrast, production of type i ifns seems to be inhibited or delayed by sars-cov in vitro [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, no ifn-a or ifn-b has been detected in the sera of sars patients or in lungs of sars-cov-infected mice [ ] [ ] [ ] . recent in vitro studies demonstrated that type i ifn inhibition or delay may be orchestrated by sars-cov proteins orf b, orf , and n [ ] . the inhibition of ifn production would benefit sars-cov replication, since pretreatment of cells with ifn before sars-cov infection efficiently prevents replication in these cells [ , [ ] [ ] [ ] [ ] . furthermore, prophylactic treatment of macaques with pegylated ifn-a reduces sars-cov replication in the lungs [ ] . although ifn production was absent in clinical samples, gene and protein expression profiles in these patients were likely impacted by clinical treatments and concurrent preexisting disease. in addition, most if not all virus-host response information is from clinical blood/sera samples that were taken relatively late during infection-little is known about what happens early during infection. animal studies are of great value to decipher the host's initial innate immune response, without confounding clinical treatment (steroid and mechanical ventilation) or underlying co-morbidity. in order to elucidate early host responses during the acute phase of sars-cov infection, we infected cynomolgus macaques with sars-cov and used macaque-specific microarrays and real-time (rt)-pcr techniques to study host gene expression profiles. adolescent cynomolgus macaques infected with sars-cov develop dad similar to sars patients, but clear most of the virus in the lungs by day [ ] . because sars-cov replicates predominantly in the lower respiratory tract of macaques, the virus infects a range of cells, including type and type pneumocytes, that are different from those analyzed in vitro. the ability to simultaneously examine virus replication and host response gene expression profiles in the lungs of these animals during the acute phase of sars offers the opportunity to further unravel the pathogenesis of sars. six cynomolgus macaques were inoculated with sars-cov strain hku- and lung tissues were collected at day (n ¼ , a and b) or day (n ¼ , a- d). no lesions or clinical symptoms were detected on day after sars-cov infection, whereas on day , three out of four monkeys were lethargic, with one of these animals showing mildly labored breathing. pathological changes at day post infection included dad, characterized by flooding of the alveoli with edema fluid, infiltration of neutrophils, damage to the alveolar and bronchial epithelia, and occasional type pneumocyte hyperplasia, as described earlier [ ] . four mock-infected animals were included in the study to serve as a reference for host response without viral challenge and to examine outbred inter-animal variation. our previous experience with a/ texas/ / influenza virus demonstrated that viral mrna was detected in representative samples of the lung rather than throughout the whole lung [ ] . based on this experience, the level of infection in separate lung samples was evaluated using rt-pcr. sars-cov mrna was detected in all animals, and pieces out of the total of lung pieces from infected animals contained high levels of virus, while the three remaining pieces of lung contained very low levels of virus (; - logs lower, figure a) . no viral rna could be detected in the samples from the mock-infected animals. for gene expression experiments, lung samples from sars-cov-infected animals were compared to a reference lung sample from mockinfected animals. the three samples with lower virus levels ( a-low, a-low, and d-low) were analyzed individually so as not to dilute the gene expression of pooled pulmonary samples with higher sars-cov levels and also to potentially further define pulmonary infection. samples from animals with high viral mrna levels showed greater gene expression changes (; , genes day , ; genes day ) compared to samples from animals with low levels of viral mrna (; genes), indicating a response of lung tissue to the virus ( figure b) . additionally, the two day animals showed higher numbers of differentially expressed genes than the day animals. in contrast, gene expression analysis of the separate mock samples revealed limited differentially expressed genes. in order to examine how gene expression would be influenced by presence of virus, timing after inoculation, and individual animal variation, global expression profiling was performed. hierarchical clustering methods were used to order rows (genes) and columns (samples) to identify groups of genes or samples with similar expression patterns [ , ] . these data were plotted as a heat map in which each matrix entry represents a gene expression value (figure a ). red corresponds to higher gene expression than that of the controls; green corresponds to lower gene expression. this analysis yielded , genes with day samples on one side of the heat map and day samples on the other side of the heat map, indicating an influence of timing after inoculation. there are two major roots to the hierarchical dendrogram, with the larger root composed of all the day samples and the three day samples with the highest virus levels. the smaller root is composed of the remaining day samples with the lowest sars-cov levels. although transcriptional profiling shows some variation when comparing samples from the same animal, the underlying gene expression is similar with a reduction in fold change in the ''low'' samples. these comparisons suggest that both individual animal variation and the ''asynchronous'' nature of the infection in the animals' lungs are factors involved in determining transcription of cellular genes. to validate that the host response from infected animals comprises a stronger transcriptional profile than individual variation from mock-infected animals, differential gene expression patterns in the separate mock samples were investigated, but only genes were differentially expressed ( figure b ). these results suggest that underlying basal levels of gene transcription do not confound expression levels after infection. even in a basal state, some low-level lung-to-lung variations were identified within the same animal but not enough to disrupt segregation of lung pieces based on mock-infected animals. severe acute respiratory syndrome coronavirus (sars-cov) infection causes a progressive atypical pneumonia. in typical cases, largely confined to adult and elderly individuals, acute respiratory distress syndrome develops, and admission to an intensive care unit is required. although these complications can be fatal, most sars patients recover, suggesting that protective immune responses are operational. in this study, we simultaneously examined virus replication and host-response gene expression profiles in macaque lungs during the acute phase of sars to gain more insight into the early events that take place after sars-cov infection. we show that a strong host response is induced in the lungs of sars-cov-infected macaques, illustrated by the induction of several pathogenic cytokines and chemokines. interestingly, antiviral pathways are activated as well, demonstrated by the presence of phosphorylated signal transducer and activator of transcription (stat ) transcription factors throughout the lung, but not in sars-cov-infected cells. a subset of cells was shown to produce interferon-b, a cytokine involved in the resistance to many viral infections and able to activate stat . activation of this antiviral pathway upon sars-cov infection may be an important escape route of the host to withstand the devastating effects of sars-cov. different time points after inoculation, a venn diagram was generated with each set (circle) holding to the parameters of an absolute fold change . and p , . in at least two animals ( figure a ). the day set contained , genes and the day set contained genes. when examining host responses that were similar throughout the course of the infection, the intersection of the day and day sets indicates that genes show shared responses. the heat map of these genes is shown in figure b . if more stringent criteria were used to find common responses in all six animals, using the , genes from the day set and the genes that are differentially expressed in all day animals, a subset of genes was identified. this subset included ifnstimulated genes (isgs), like ifits, mx , gbp , and g p , and also various chemokines and cytokines, such as cxcl (ip- ), ccl (mcp- ), il- , and il- ( figure s ). these same cytokines and chemokines have been reported to be upregulated in human sars cases [ ] [ ] [ ] [ ] . this set also included cathepsin l (ctsl), which has been shown to be required for sars-cov entry into a cell [ ] . even though only genes were commonly regulated in all animals, indicated with blue bars in figure b , the heat map highlights that the other genes show similar expression trends. both sets of commonresponse genes showed similar functionality: cellular growth and proliferation, cell death, cellular movement, immune response, and cell-to-cell signaling. next, we analyzed genes that were differentially expressed exclusively on either day or day in order to find signature gene expression patterns for each day. genes identified as unique responses at day ( genes) and at day ( genes) in the venn diagram showed unique functionality ( figure c ). the gene expression profile at day shows a prominent innate host response to viral infection; top functional categories on day are the immune response, the hematological system, and the immune and lymphatic system. genes like ifn-c, ccl (mip- -b), csf , il a, and tnf are included in these categories. at day , a smaller panel of unique differentially expressed genes that play a role in cell cycle, cellular assembly, and dna repair were identified like ccnb , ccne , cdca , cenpa, chaf a, and prc . in order to investigate genes that are most strongly regulated after sars-cov infection, genes included in the venn diagram ( figure a ) that also held to an absolute fold change . were queried ( figure s ). from this set, genes that were involved in the immune response and lung repair processes were used to generate a heat map ( figure ) . a number of genes that have been reported to be up-regulated in sars patient sera, such as ccl (mcp- ), cxcl (ip- ), il- , and il- , were strongly (; -fold) induced in all animals. many cell cycle and matrix genes indicative of tissue repair processes were also highly differentially expressed at day (e.g., anln, areg, cdc , cdkn , cks , fosl , and kif c). likewise, tissue factor pathway inhibitor (tfpi ), an anticoagulant, was strongly up-regulated during infection in all animals (averaging ; -fold), as well as plscr , ser-pine (pai ), and thbs , all genes involved in procoagulation and platelet activation, were induced. concomitant expression of tfpi with these pro-coagulation genes might function as an inhibitory response to restrain the activation of the coagulation pathway during acute inflammation. surprisingly, expression of diverse ifn-a genes and expression of ifn-b was up-regulated ; to -fold in the day samples. furthermore, ifn-c, a type ii ifn, was efficiently transcribed on day after sars-cov infection (; -fold). other genes associated with the induction of ifns like ddx (rig-i), irf- , and signal transducer and activator of transcription (stat ), were also highly induced (; fold). up-regulation of type i ifns in these sars-cov infected macaques is remarkable, since sars-cov inhibits ifn production in many in vitro studies. we did not detect induced ifn-b mrna expression using ma cells or caco cells and the sars-cov-hku virus (unpublished data). not only ifns, but also several ifn-responsive genes (e.g., g p , gbp / , ifi/ifits, mx / , isg , and oas / /l) were highly transcribed, showing a persistent activation of the innate immune response. furthermore, suppressor of cytokine signaling (socs ) is induced at the onset of infection, presumably to establish negative feedback to attenuate cytokine signaling. of note, ifit (isg /ifi ), often used to gauge ifn induction, was up-regulated an average of ; fold. to further explore some of the pathogenic and antiviral pathways that are induced after sars-cov infection, we investigated the transcription of various cytokines, chemokines, ifns, isgs, and transcription factors involved in the jak/stat pathway. as can be seen in figure a , a wide range of chemokines and cytokines are differentially expressed after sars-cov infection in macaque lungs, especially on day after infection. besides previously mentioned chemokines, we detected monocyte chemotactic protein genes like ccl (mcp- ) and ccl (mcp- ), but also ccl (eotaxin), a chemotactic protein for eosinophils. in the samples with low sars-cov mrna levels, the induction of chemokines is less evident, suggesting that the presence of these molecules is restricted to areas in the lung where virus is present. furthermore, sars-cov-infected macaques showed a stronger induction of ifns ( unique genes) and isgs ( unique genes) on day than day and when virus was present at high levels. note that besides ifn-a, ifn-b, and ifn-c, the ifn-ks (il- , il- a, il- b), which are type i ifns, were induced in samples with high sars-cov levels. in the absence of viral rna, no ifns, but interestingly, a number of isgs ( unique genes) were detected, suggesting paracrine stimulation ( figure b ). differential expression of a selection of strongly upregulated genes, cxcl (ip- ), il- , il- , and ifn-b, was confirmed using rt-pcr ( figure ). in accordance with the microarray data, the rt-pcr data showed that cxcl (ip- ), il- , il- , and ifn-b were all expressed at levels that were approximately times higher in the sars-cov-infected animals at day than in the uninfected control animals and were still elevated on day after infection. as can be seen in figure , the induction of ifn-b was strongly correlated to the presence of virus (r spearman ¼ . , p , . ). for cxcl (ip- ), il- , and il- the correlation is less evident, which is not surprising since these cytokines can be induced by other factors than the virus itself. in order to visualize the host response in the lungs of sars-cov-infected macaques, ifn-b production and translocation of phosphorylated stat was studied using immunohistochemistry. in the lungs of the sars-cov-infected macaques, a modest number of cells stained positive for ifnb at day post infection, whereas no ifn-b-positive cells could be detected in mock-infected macaques ( figure a - c). notably, most of the cells that stained positive for ifn-b were located very close to blood vessels, but not in the alveoli where most sars-cov antigen-positive cells (mainly type pneumocytes at day post infection) are located. to examine whether the ifns that are produced in the lungs of these sars-cov-infected macaques are biologically active and able to induce stat phosphorylation and translocation, lung sections of the infected macaques were stained with antibodies against phosphorylated stat . as shown in figure d and e, no phosphorylated stat could be detected in the lungs of pbs-infected macaques, while in the lungs of sars-cov-infected macaques, cells with phosphorylated stat in their nucleus were abundantly present. subsequently, the same pieces of lung from sars- . genes were included if they met the criteria of a -fold change or more (p . ). a two-of-nine strategy allowed samples to cluster together if profile similarities existed based on timing of inoculation (n ¼ samples for day ). (b) the number after pbs refers to the animal (i.e., pbs ), while the number after the dash refers to the lung piece (i.e., pbs - ). thirty-eight genes are displayed with an absolute fold change . and p , . in at least two animal samples. up-regulated genes are indicated in bold underline. only one gene, hla-dqa , was down-regulated . . no up-regulated genes met these criteria in mock-infected animals. separate mock samples (i.e., pbs - ) were compared to the total mock pool. doi: . /journal.ppat. .g cov-infected macaques at day were double stained for phosphorlylated stat and sars-cov (figure f) . notably, phosphorylated stat was not detected in the nucleus of sars-cov-infected cells (type pneumocytes), while cells directly adjacent to these sars-cov-infected cells stained for phosphorylated stat in many, but not all, foci containing sars-cov-positive cells. thus, type i ifns are produced in the lungs of sars-cov-infected macaques, and are able to activate the jak/stat pathway. however, translocation of stat does not occur in sars-cov-infected pneumocytes. although recent studies indicate that the sars-cov orf protein is able to inhibit nuclear translocation of stat in vitro, this was not demonstrated in experiments using infectious sars-cov [ ] . in order to assess whether sars-cov inhibits phosphorylation and translocation of stat , ma cells were infected with sars-cov for h and then either fixed directly or treated with type i ifn. cells infected with sars-cov, but not treated with ifn, stained positive for sars-cov (unpublished data), but lacked staining for phosphorylated stat , indicating that sars-cov or other soluble mediators are not able to induce stat phosphorylation ( figure ). after treatment of the ma cells with ifn, phosphorylated stat could be detected in the nucleus of most cells, but not in the nucleus of sars-cov-infected cells (figure ). this demonstrates that sars-cov inhibits the translocation of phosphorylated stat to the nucleus, confirming our in vivo data. besides inhibiting translocation of phosphorylated stat , sars-cov also seems to reduce stat phosphorylation, as the majority of sars-covinfected cells contained low levels of phosphorylated stat in their cytoplasm. pathogenic viruses escape the antiviral action of the ifn system by inhibiting both ifn production and signaling pathways. here, we report that even though production and signaling of type i ifns is inhibited by sars-cov in vitro as well as in sars-cov-infected cells in vivo, high levels of type i ifns are induced in the lungs of sars-cov-infected macaques. these ifns are able to activate stat , followed by the transcription of numerous isgs. using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. our results emphasize the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of sars-cov infection in cynomolgus macaques. to our knowledge, this study represents the first functional genomics investigation of sars-cov infection in cynomolgus macaques. all experimental animals showed signs of infection because viral mrna could be detected in random samples from the lung, indicating that the virus had spread throughout the whole lung at the time of necropsy. furthermore, pathological examination of sars-cov-infected macaques at day post infection revealed multifocal dad [ ] . unlike % of humans with sars, which are mainly restricted to the elderly, adult macaques used in this study do not succumb to sars-cov infection. however, the sars-cov-induced pathology in these macaques likely resembles the pathological changes seen in the majority of human sars patients that recover from the disease. although none of the current animal models has fully reproduced all features of sars, the most important aspects of this disease are observed in experimentally infected macaques, providing valuable insights into the initial innate immune response after infection without confounding clinical treatment or underlying co-morbidity. using macaque-specific microarrays, we were able to observe that with early infection, high levels of viral mrna corresponded to a strong cellular host response. this strong host response is dominated by genes involved in the immune response and includes a wide range of genes corresponding with what is seen in human ards. during the acute phase of human ards, activated neutrophils and macrophages enter the alveoli and produce a number of cytokines and chemokines such as il- , il- , and cxcl (ip- ) [ ] , as were found in the lungs of our sars-cov-infected macaques. researchers have postulated that these genes also predict adverse sars patient outcome [ ] . during the chronic phase of human ards, type pneumocytes start to proliferate and differentiate in order to repair the damaged lung. at day , [ , ] . we also detected a strong presence of genes involved in the coagulation pathway, including tfpi , serpine , and timp . the idea of a pro-coagulation profile mimics the clinical-pathological observations of sars patients that showed unusually disseminated small vessel thromboses in the lungs [ , ] . additionally, cathepsin l was up-regulated in all sars-cov-infected macaques. induction of this gene after sars-cov infection is quite interesting because cathepsin l is an endosomal protease that is necessary for sars-cov to infect a cell [ ] . remarkably, sars-cov infection in macaques leads to a strong transcription of ifns. not only ifn-a, ifn-b, and ifnk (all type i ifns), but also ifn-c, a type ii ifn, were all highly up-regulated, especially on day after infection. the expression of ifn-b, which strongly correlated to the amount of virus present, continued throughout day and was confirmed using immunohistochemistry; ifn-b-positive cells could be detected in the lungs of the sars-cov-infected macaques. the induction of ifn-b in these sars-covinfected macaques is surprising, because several reports have shown that sars-cov inhibits or delays type i ifn production in a number of cell types [ ] [ ] [ ] [ ] [ ] , ] . for example, sars-cov blocks a step in the activation of irf- , a transcription factor that is required for ifn-b induction [ ] . in addition, the sars-cov proteins orf b, orf , and nucleocapsid have been shown to function as ifn antagonists, as has the sars-cov nsp gene that prevents the production of sendai virus-induced ifn-b in cells [ , ] . interestingly, it was recently shown that plasmacytoid dendritic cells (pdcs) are able to produce ifn-a and ifn-b after sars-cov infection, while conventional dcs did not produce these type i ifns [ ] . pdcs are known for their ability to produce very high amounts of ifn-a and ifn-b and are considered firstline sentinels in immune surveillance in the lung [ ] [ ] [ ] [ ] [ ] . we speculate that the ifn-b-producing cells detected in the lungs of sars-cov-infected macaques are pdcs. future studies may address the nature of these ifn-producing cells once technical difficulties in detecting pdcs in macaque tissues have been tackled. these studies may also shed light on whether decreasing numbers of pdcs observed in clinical blood samples from human sars patients are caused by sequestering of pdcs by the lungs, destruction of pdcs by sars-cov, or destruction or suppression of pdcs by steroid treatment [ ] . when ifns are produced, they bind to their receptors on the cell membrane, after which stat , a key member of the jak/stat pathway, is phosphorylated and subsequently translocated to the nucleus, followed by the production of a wide range of ifn-stimulated genes. in vitro, sars-cov inhibited translocation of stat to the nucleus, and phosphorylation of stat was strongly reduced. however, the inhibition of stat phosphorylation was not absolute because cells with low levels of phosphorylated stat in their cytoplasm were also detected. in accordance with our data, kopecky-bromberg et al. recently showed that the sars-cov protein orf is able to inhibit stat translocation [ ] . this strategy is not unique to sars. other viruses have been shown to be able to block signaling of ifns by affecting phosphorylation and/or translocation of the stat proteins. for example, measles virus v protein inhibits translocation of stat , but does not affect phosphorylation, whereas measles virus p protein blocks both of these processes [ ] . other paramyxoviruses, like rinderpest virus, nipah virus, hendra virus, and mumps virus, as well as flaviviruses like west nile virus and japanese encephalitis virus, are able to block activation of stat and stat [ ] [ ] [ ] [ ] . inhibition of stat phosphorylation is not always complete. for example, sendai virus suppresses tyrosine phosphorylation of stat during the early stages of infection, but this block becomes leaky after a couple of hours with phosphorylated stat accumulating in the cytoplasm [ ] . in contrast to these in vitro data, we observed phosphorylated stat in the nuclei of numerous cells in the lungs of sars-cov-infected macaques, indicating that these cells had been activated by the ifns produced in the lung. however, phosphorylated stat was not detected in sars-covinfected cells. the observations made in this study indicate that sars-cov-infected macaques produce ifns in response to virus infection and are further capable of activating the stat pathway in cells surrounding the sars-cov-infected cells. the importance of ifns in controlling sars-cov infection has been suggested in several animal studies. mice clear sars-cov in the absence of nk cells, t cells, or b cells, suggesting that innate immune responses are sufficient to limit sars-cov infection in these animals [ ] . indeed, stat knock out mice, which are resistant to the effects of ifns, to some extent show a worsening of pulmonary disease and an increase in viral replication in the lungs compared to normal mice after infection with sars-cov [ ] . although ifn treatment was not conducted in sars-cov infection mouse studies, prophylactic treatment of macaques with pegylated ifn-a protects type pneumocytes from infection with sars-cov [ ] . in addition, potent antiviral activity is observed in vitro when cells are treated with ifns before they are infected with sars-cov [ , , ] . although we cannot determine the effect of neutralizing ifn-b in sars-covinfected animals, based on the experiments utilizing recombinant ifns in these animals, we postulate that type i ifns are partly responsible for the relatively mild clinical symptoms that are seen in sars-cov-infected macaques. in addition, a recent study again demonstrated the importance of ifns in viral infections, as macaques infected with the highly pathogenic and fatal influenza virus showed limited induction of type i ifns (only ifna reached fold changes . ) and delayed induction of isgs, while macaques infected with the low-pathogenic k influenza virus showed a strong induction of these antiviral molecules early during infection [ ] . notably, ifn-b was not up-regulated (absolute fold change , ) in any of the influenza virusinfected animals, even in those animals that recovered, unlike sars-cov-infected macaques that showed a very strong presence of ifn-b. in conclusion, our study demonstrates that cynomolgus macaques can be infected with sars-cov, as indicated by presence of viral mrna at different locations throughout the lung at day and day , with gross pathology becoming noticeable at day . furthermore, we show that infection of cynomolgus macaques with sars-cov leads to a strong immune response, including the induction of various cytokines and chemokines, resembling the host response seen in human sars patients. strikingly, despite the fact that sars-cov infection blocks the production of ifns in vitro, type i ifns are strongly induced in the lungs of sars-cov- infected macaques. the production of ifn early during infection leads to widespread activation of stat and the production of isgs. this suggests that, although sars-cov blocks ifn signaling in infected cells, locally produced ifns are capable of activating non-infected cells and possibly can prevent infection of these cells. thus, sars-cov infection in macaques leads to the differential activation of both pathogenic and antiviral signaling pathways in vivo, and the outcome may be determined by the relative contribution of these signaling pathways. were infected intratracheally with tcid sars-cov (hku- ) as described earlier [ ] . virus stocks were generated in vero e cells that were defective in ifn production. two animals were euthanized on day after infection and four animals were euthanized on day . in addition, four animals were mock (pbs) infected and euthanized on day , serving as a negative control group. one lung from each monkey was fixed in % formalin for histopathology and immunohistochemistry while the other was used for real-time pcr and microarrays. lung samples were randomly excised from three different lung areas (cranial, medial, caudal) and stored in rnalater (ambion, http://www.ambion.com/). sixteen pieces of lung were taken from the sars-cov-infected animals, two to three pieces of lung per animal. twelve pieces of lung were taken from the mock-infected animals, three pieces of lung per animal. individual lung samples in rnalater were transferred to trizol reagent (invitrogen, http:// www.invitrogen.com/), homogenized using polytron pt tissue grinders (kinematica, http://www.kinematica.ch), and then processed to extract rna. all experiments were executed under a biosafety level , and approval for animal experiments was obtained from the institutional animal welfare committee. oligonucleotide microarray analysis. infected macaque lung samples were co-hybridized with a reference mock-infected macaque lung sample on macaque oligonucleotide arrays containing viral probes, corresponding to viruses, and , rhesus probes, corresponding to ; , rhesus genes. the reference mock-infected sample was created by pooling equal mass quantities of total rna extracted from the individual lung pieces from mock-infected animals. an agilent bioanalyzer was used to check the purity of the total rna prior to crna probe production with the agilent low rna input fluorescent linear amplification kit (agilent technologies, http://www.agilent.com/). arrays were scanned with an agilent dna microarray scanner, and image analysis was performed using agilent feature extractor software (agilent technologies). each microarray experiment was done with two technical replicates using dye reversal [ ] . all data were entered into a custom-designed database (expression array manager) and analyzed with resolver . (rosetta biosoftware, http://www.rosettabio.com/) and spotfire deci-sionsite for functional genomics (spotfire, http://www.spotfire.com/). in our data analysis, genes were selected to be included for transcriptional profile based on two criteria: a greater than . % probability of being differentially expressed (p . ) and an expression level change of -fold or greater. ingenuity pathway analysis (ingenuity systems, http://www.ingenuity.com/) was used to functionally annotate genes according to biological processes and canonical pathways. in accordance with proposed miame standards, primary data are available in the public domain through expression array manager at http://expression.microslu.washington.edu/ expression/index.html [ ] . quantitative real-time rt-pcr. rt-pcr was performed to detect sars-cov mrna and to validate cellular gene expression changes as detected with microarrays. each reaction was run in triplicate using taqman x pcr universal master mix (applied biosystems, http:// www.appliedbiosystems.com/) with primers and probe specific for the sars-cov nucleoprotein gene [ ] , or for macaque cellular genes (sequences shown in table ). differences in gene expression are represented as the fold change in gene expression relative to a calibrator and normalized to a reference, using the Àddct method [ ] . gapdh (glyceraldehydes- -phosphate dehydrogenase) or s rrna were used as endogenous controls to normalize quantification of the target gene. the samples from the mock-infected macaques were used as a calibrator. immunohistochemistry. formalin-fixed, paraffin-embedded lung samples from sars-cov-infected and mock-infected macaques were stained for sars-cov, phosphorylated stat , and ifn-b using mouse-anti-sars-nucleocapsid (clone ncap , mouse igg b; imgenex, http://www.imgenex.com/), mouse-anti-phospho-stat (clone st p- a , mouse igg a-j; zymed laboratories, http://www. invitrogen.com/), and rabbit-anti -ifn-b (chemicon, http://www. chemicon.com/), respectively. after deparaffinization, antigen retrieval was performed using a citrate buffer for the sars-cov and stat staining. no antigen retrieval was performed when staining for ifn-b. goat-anti-mouse igg a hrp, goat-anti-mouse igg b ap (southern biotech, http://www.southernbiotech.com/), and anti-rabbit igg-hrp (dako, http://www.dako.com/) were used as secondary antibodies. signals were developed with fast red and dab (sigma, http://www.sigmaaldrich.com/) and counterstained with mayer's hematoxylin. in vitro sars-cov and stat staining. ma cells (african green monkey foetal kidney cells, ecacc) were cultured in eagle's minimal essential medium (emem; cambrex, http://www.cambrex. com/) supplemented with mm glutamine, % non-essential amino acids and % foetal bovine serum. cells were seeded in -well plates and infected with sars-cov (moi . ), and h after infection, selected wells were treated with universal type i ifn ( , u/ml, sigma) for min at c. subsequently, cells were fixed with % neutral-buffered formalin and treated with % ethanol. sars-cov-infected cells were visualized using purified human igg from a convalescent sars patient (csl), followed by staining with an antibody to human igg, linked to alexa fluor (invitrogen). phosphorylated stat was visualized using mouse-anti-phospho-stat (zymed), followed by staining with a fitc-linked antibody to mouse igg. lung pathology of fatal severe acute respiratory syndrome severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update clinical presentations and outcome of severe acute respiratory syndrome in children clinical picture, diagnosis, treatment and outcome of severe acute respiratory syndrome (sars) in children clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study the severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome an interferongamma-related 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acute respiratory distress syndrome early enhanced expression of interferon-inducible protein- (cxcl- ) and other chemokines predicts adverse outcome in severe acute respiratory syndrome the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclincyclin-dependent kinase complex and blocks s phase progression in mammalian cells lung pathology of severe acute respiratory syndrome (sars): a study of autopsy cases from singapore severe acute respiratory syndrome coronavirus nsp protein suppresses host gene expression by promoting host mrna degradation control of coronavirus infection through plasmacytoid dendritic-cellderived type i interferon the nature of the principal type interferon-producing cells in human blood different roles for human lung dendritic cell subsets in pulmonary immune defense mechanisms plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon plasmacytoid dendritic cells in immunity characterization of myeloid and plasmacytoid dendritic cells in human lung longitudinal alteration of circulating dendritic cell subsets and its correlation with steroid treatment in patients with severe acute respiratory syndrome tyrosine in the measles virus phosphoprotein is required to block stat phosphorylation inhibition of interferon signaling by the new york strain and kunjin subtype of west nile virus involves blockage of stat and stat activation by nonstructural proteins mumps virus v protein antagonizes interferon without the complete degradation of stat blocking of interferoninduced jak-stat signaling by japanese encephalitis virus ns through a protein tyrosine phosphatase-mediated mechanism rinderpest virus blocks type i and type ii interferon action: role of structural and nonstructural proteins sendai virus c protein impairs both phosphorylation and dephosphorylation processes of stat resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat aberrant innate immune response in lethal infection of macaques with the influenza virus statistical design and the analysis of gene expression microarray data minimum information about a microarray experiment (miame)-toward standards for microarray data analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method we thank s. smits for her assistance with sars-cov infections and rna isolations. author contributions. adl, tb, ado, blh, and mgk conceived and designed the experiments. adl, tb, tt, lml, and br performed the experiments. adl, tb, and blh analyzed the data. adl, tb, ado, blh, and mgk wrote the paper.funding. this work was supported by the us national institutes of health, r grant hl - a , and by the european union, grant sp- -ct- - .competing interests. the authors have declared that no competing interests exist. key: cord- -ntq f ix authors: mao, he-ting; wang, yan; cai, juan; meng, jun-ling; zhou, yu; pan, yu; qian, xiao-ping; zhang, yu; zhang, jun title: hace negatively regulates virus-triggered type i ifn signaling by impeding the formation of the mavs-traf complex date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ntq f ix during virus infection, the cascade signaling pathway that leads to the production of proinflammatory cytokines is controlled at multiple levels to avoid detrimental overreaction. hace has been characterized as an important tumor suppressor. here, we identified hace as an important negative regulator of virus-triggered type i ifn signaling. overexpression of hace inhibited sendai virus- or poly (i:c)-induced signaling and resulted in reduced ifnb production and enhanced virus replication. knockdown of hace expression exhibited the opposite effects. ubiquitin e ligase activity of the dead mutant hace /c a had a comparable inhibitory function as wt hace , suggesting that the suppressive function of hace on virus-induced signaling is independent of its e ligase activity. further study indicated that hace acted downstream of mavs and upstream of tbk . mechanistic studies showed that hace exerts its inhibitory role on virus-induced signaling by disrupting the mavs-traf complex. therefore, we uncovered a novel function of hace in innate immunity regulation. to initiate an effective antiviral response, rna viruses are recognized by pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and rig-i-like receptors (rlrs), and then trigger multiple signaling pathways to promote the production of proinflammatory cytokines, including type i ifns [ ] [ ] [ ] [ ] . aberrant overreaction may lead to proinflammatory diseases. therefore, the intensity and duration of the signaling are finely modulated at multiple steps of the signaling cascades [ , ] . in recent years, we have had great interest in the identification of the essential regulators in this signaling pathway. this will provide potential therapeutic intervention and targets for infection, inflammation or autoimmune diseases in the future. rlrs are cytosol sensors, which include rig-i, melanoma differentiation factor (mda ) and laboratory of genetics and physiology (lgp ) [ , ] . all three rlrs possess a dexd-box rna helicase domain for rna binding [ ] . except lgp , both rig-i and mda also contain a caspase recruitment domain (card) that is indispensable for downstream protein-protein interactions. upon viral infection, the activated rig-i undergoes self-dimerization and structural changes that permit the card domain of rig-i to interact with the card domain of downstream essential adaptor protein mavs (also known as ips- /cardif/visa) [ ] [ ] [ ] [ ] . mavs has a transmembrane domain (tm), that guides it to the outer mitochondrial membrane. besides, mavs contains three traf-interacting motifs (tim), two included in the n-terminal proline-rich region (pro), the other one located in the c-terminal region [ , ] . upon rna virus infection, the downstream tumor necrosis factor (tnf) receptor-associated factors (trafs) are recruited to mavs, and the mavs complex is formed [ , ] . this is a crucial step to initiate type i ifn signaling. traf , , and are all mavs binding partners through different tim. the mavs-traf complex provides the essential platform for downstream tbk -dependent irf or irf activation. traf bridges the upstream mavs and downstream kinase tbk and assembles the active mavs-traf -tbk signaling complex [ , ] . therefore, the regulation on the mavs-traf signalosome may be very important for the pathway. hace (hect domain and ankyrin repeat-containing e ubiquitin protein ligase ) is a hect-type ubiquitin e ligase. the functions of hace have not been fully understood. until now, the identified ubiquitinated substrates of hace include active rac [ , ] , optineurin (optn) [ ] and rab gtpases [ ] . the catalytic cysteine (c ) of hace is essential for its e ligase activity [ , , ] . mutation of c to serine or alanine will abolish its e ligase activity. hace gene is located on chromosome q , a prominent tumor-suppressor region [ , ] . the tumor suppressive function of hace is also characterized. hace is downregulated in multiple cancer types due to allelic loss or promoter methylation, such as wilms' tumor, gastric cancer, lymphoma, hepatocellular carcinoma, breast cancer, neuroblastoma, advanced colorectal cancer, etc. [ ] [ ] [ ] [ ] [ ] [ ] [ ] . hace -deficient mice developed spontaneous, late-onset cancer [ ] . re-expression of hace in human tumor cells directly abrogates in vitro and in vivo tumor growth, which is dependent on its e ligase activity. the mechanical analysis for its growth control shows that hace modulates the expression level of cyclin d , then reducing cell cycle progression [ ] . moreover, in breast cancer, hace ubiquitinates and promotes the degradation of rac , then leading to impaired rac signaling [ ] . in contrast, hace deficiency results in enhanced rac signaling, contributing to breast cancer progression [ ] [ ] [ ] . in lung cancer, hace ubiquitinates optn and targets it for autophagic degradation. the hace -optn axis synergistically suppresses the growth and tumorigenicity of lung cancer cells [ ] . moreover, hace is also involved in other biological processes or pathological conditions. for example, hace mediates resistance to oxidative stress [ ] . hace regulates golgi membrane fusion in cells [ ] . it has protective roles in the pathology of neurodegenerative diseases, such as huntington disease [ ] . it also provides cardiac protection in response to hemodynamic stress [ ] . however, the functions of hace in immune responses are not investigated. in recent years, ubiquitination has been reported as an important post-transcriptional modification to control the duration and intensity of antiviral immune responses [ ] . both hect and ring domain e ubiquitin ligases are identified as essential regulators in this pathway. for example, rnf is reported to ubiquitinate and degrade mda- , rig-i and mavs [ ] . the hect domain containing ubiquitin ligase aip can ubiquitinate and degrade mavs in collaboration with pcbp [ ] . our group previously showed that smurf promotes the ubiquitination and degradation of mavs, as well [ ] . in the search for unknown ubiquitin e ligases involved in antiviral signaling, some ubiquitin e ligases were used for the dual reporter luciferase assay. then, hace was suggested as a potential candidate in the regulation of this pathway. in this study, we demonstrate for the first time that hace contributes to negative regulation of the virus-induced type i ifn signaling via disrupting the mavs-traf complex. hace suppressed virus-induced type i ifn signaling independently of its ubiquitin e ligase activity. this study highlights the importance of hace in the modulation of virus-induced type i ifn response. hek t and hek cells were cultured with high-glucose dmem (life technologies, new york, ny, usa) medium plus % heat-inactivated new-born bovine serum and supplemented with antibiotics ( u/ml penicillin, µg/ml streptomycin). cells were grown at ˝c in a humidified atmosphere with % co . mouse anti-flag (m ) (sigma-aldrich, st. louis, mo, usa), mouse anti-hemagglutinin (ha) (merck millipore, darmstadt, germany), anti-gapdh (bioworld, atlanta, ga, usa), anti-hace (abcam, cambridge, uk) and anti-gfp (neobioscience, shenzhen, china) were from the indicated manufacturers. mammalian expression plasmids for human ha-tagged hace and flag-tagged rac were constructed by inserting the open reading frame of hace or rac into the n terminal ha or flag-tagged prk vector. the mammalian expression plasmid for hace /c a was constructed by site-directed mutagenesis. all of these vectors were verified by sequencing. pcdna -flag-tbk was a gift from tom maniatis. pef-bos-flag-rig-i was a gift from takashi fujita. pcdna -flag-mavs was a gift from zhijian chen. the prl-tk-renilla luciferase plasmid was from promega (madison, wi, usa). ifn-β and isre luciferase reporter plasmids were provided by hong-bing shu. all small interfering rnas (sirnas) (gene-pharma, shanghai, china) were transfected by permute (ucallm, jiangsu, china) at nm according to the manufacturers' instructions. to determine the efficiency of protein knockdown, at h post-transfection, cells were harvested, lysed and immunoblotted with rabbit anti-hace ab. the sequences of the individual sirnas were as follows: nonspecific control, -uucuccgaacgugucacgu- ; hace # , -uauagcgcugaugucaaca- ; hace # , -ggucuguuucugaacuacu- [ ]. the luciferase assay was performed as described [ ] . cells ( . ˆ ) were seeded on -well plates and transfected the next day using vigofect (vigorous biotechnology, beijing, china) with ng isre luciferase reporter, or ifn-β reporter and ng prl-sv plasmid, or with indicated plasmids. in the same experiment, when necessary, an empty control plasmid was added to ensure that each transfection received the same amount of total dna. then, h after transfection, cells were infected with sev at the multiplicity of infection (moi) of or transfected with poly (i:c) (invivogen, san diego, ca, usa) using lipofectamine (invitrogen, carlsbad, ca, usa) for h, and luciferase activity was measured with the dual-luciferase reporter assay system (promega) according to the manufacturer's instructions. firefly luciferase activity was normalized based on renilla luciferase activity. all reporter assays were performed in duplicate and repeated at least three times. the representative results are shown in each figure. total rna was isolated using trizol reagent (life technologies). cdna was synthesized using a reverse transcription system (promega) according to the manufacturer's instructions. quantitative real-time polymerase chain reaction (pcr) was carried out with the power sybr green pcr master mix (bio-rad, berkeley, ca, usa). each reaction was in duplicate. the amounts of hifnb were amplified using the following primers: ifnb -f: -attgcctcaaggacaggatg- and ifnb -r: the cells were infected by vsv-gfp virus at moi of . then, the vsv genome was quantified by real-time pcr using the following primers: vsv-f: -acggcgtacttccagatgg- ; vsv-r: -ctcggttcaagatccaggt- . hek t cells were transfected with indicated plasmids for h. then, the cells were lysed in lysis buffer containing a proteinase inhibitor mixture (roche, indianapolis, in, usa) and pmsf. cell lysates were incubated with µg/ml anti-ha ab or anti-flag or control ig (igg) and protein a-sepharose (ge healthcare, ge healthcare, calbiochem, sweden) and resolved by sds-page. the blot was then probed with anti-flag or anti-ha ab. irdye -conjugated anti-igg or hrp-conjugated anti-igg was used as a secondary ab, and proteins were identified using the odyssey imaging system or detected by the ecl assay. statistical analysis was carried out with spss . . all data are shown as the mean˘sd. the mean values from each group were compared by student's t-test. in all tests, p-values of less than . were considered statistically significant. by a small-scale screening of unknown ubiquitin e ligases in the regulation of virus-induced type i ifn signaling by the dual-luciferase reporter, we identified hace as a potential negative regulator in this pathway. then, we tried to systematically investigate whether hace is indeed involved in the regulation of virus-induced ifn signaling. as shown in figure a , overexpression of hace inhibited sev-induced activation of both isre (an interferon stimulated response element) and ifn-β promoter in a dose-dependent manner in hek t cells. in addition, activation of the isre promoter primed with the synthetic rna duplex poly (i:c) was also inhibited by overexpression of hace ( figure b) . to further support these results, the amount of ifnb was measured at various time points by reverse transcription (rt)-pcr during the twelve hours of infection by sendai virus. hace suppressed sev-induced transcription of endogenous ifnb gene ( figure c ). vsv is another representative rna virus for rig-i signaling studies. it is easy to detect the virus replication. consistent with the suppressive function of hace on virus-induced signaling, the replication of vsv was enhanced when hace was overexpressed ( figure d ). these data together suggested that hace negatively regulates virus-induced type i ifn signaling. next, to investigate the functions of endogenous hace on virus-induced type i ifn signaling, we knocked down the expression of hace in hek cells. two sirna oligos against hace were used, and the knockdown efficiency was monitored. as shown in figure a , both # and # sirna oligos can remarkably reduce the expression of endogenous hace in hek cells. compared to control cells, knockdown of hace expression augmented sev-induced ifnb gene transcription ( figure b ) and inhibited vsv replication ( figure c ). thus, hace is a negative regulator in virus-induced type i ifn signaling. statistical analysis was carried out with spss . . all data are shown as the mean ± sd. the mean values from each group were compared by student's t-test. in all tests, p-values of less than . were considered statistically significant. by a small-scale screening of unknown ubiquitin e ligases in the regulation of virus-induced type i ifn signaling by the dual-luciferase reporter, we identified hace as a potential negative regulator in this pathway. then, we tried to systematically investigate whether hace is indeed involved in the regulation of virus-induced ifn signaling. as shown in figure a , overexpression of hace inhibited sev-induced activation of both isre (an interferon stimulated response element) and ifn-β promoter in a dose-dependent manner in hek t cells. in addition, activation of the isre promoter primed with the synthetic rna duplex poly (i:c) was also inhibited by overexpression of hace ( figure b) . to further support these results, the amount of ifnb was measured at various time points by reverse transcription (rt)-pcr during the twelve hours of infection by sendai virus. hace suppressed sev-induced transcription of endogenous ifnb gene ( figure c ). vsv is another representative rna virus for rig-i signaling studies. it is easy to detect the virus replication. consistent with the suppressive function of hace on virus-induced signaling, the replication of vsv was enhanced when hace was overexpressed ( figure d ). these data together suggested that hace negatively regulates virus-induced type i ifn signaling. next, to investigate the functions of endogenous hace on virus-induced type i ifn signaling, we knocked down the expression of hace in hek cells. two sirna oligos against hace were used, and the knockdown efficiency was monitored. as shown in figure a , both # and # sirna oligos can remarkably reduce the expression of endogenous hace in hek cells. compared to control cells, knockdown of hace expression augmented sev-induced ifnb gene transcription ( figure b ) and inhibited vsv replication ( figure c ). thus, hace is a negative regulator in virus-induced type i ifn signaling. hace has been documented to act as a ubiquitin e ligase. so far, only a few ubiquitinated substrates have been identified, including rac , optineurin (optn) and rab gtpases [ , , ] . the catalytic cysteine (c ) is indispensable for its e ubiquitin ligase activity [ , , ] . then, we tried hace has been documented to act as a ubiquitin e ligase. so far, only a few ubiquitinated substrates have been identified, including rac , optineurin (optn) and rab gtpases [ , , ] . the catalytic cysteine (c ) is indispensable for its e ubiquitin ligase activity [ , , ] . then, we tried to determine whether the e ligase activity of hace is required for the inhibition of virus-induced type i ifn signaling. it is well-known that the catalytic cysteine (c ) of hace is indispensable for its e ligase activity [ ] . therefore, we constructed a mutant hace in which the amino acid cysteine was mutated into alanine ( figure a) . consistent with the previous report, wt hace can promote the degradation of rac , whereas the hace /c a mutant lost the ability to degrade rac , indicating the lost e ligase activity of hace /c a ( figure b ). reporter assays showed that sev-induced or poly (i:c)-induced activation of isre and ifn-β promoter activities were inhibited by hace /c a to a similar degree as wt hace (figure c,d) . these data indicate that hace inhibits virus-induced type i ifn induction independently of its e ligase activity. upon viral infection, recognition of viral rna by rig-i induced a downstream signaling cascade, including mavs, tbk , irf [ ] . we next sought to determine a step within the signaling pathway that hace targets. as shown in figure , isre and ifn-β promoter activity were activated by transfection of an active form of rig-i (rig-in), mavs, tbk and an activated form of irf (irf / d), respectively. co-expression of hace inhibited rig-in, mavs-induced isre or ifn-β reporter activation ( figure a,b) . on the other hand, it had no apparent effects on tbk or irf - d-induced isre or ifn-β reporter activation. these data suggested that hace acted downstream of mavs and upstream of tbk in the virus-induced signaling. hek t cells were coexpressed with rac and increasing amounts of ha-tagged wt hace or hace /c a. twenty-four hours after transfection, the cells were harvested and detected by western blot; (c) hace /c a still has the ability to inhibit sev-induced isre or ifn-β activation. hek t cells were seeded on -well plates and were transfected the next day with mock control, hace or hace /c a expressing vector, together with isre or ifn-β reporter vector. twenty-four hours later, cells were infected with sev or left uninfected for h before luciferase assays were performed; (d) hace /c a inhibited isre and ifn-β promoter activation by transfected poly (i:c) in hek t cells. data are representative of at least three independent experiments. * p < . ; *** p < . . upon viral infection, recognition of viral rna by rig-i induced a downstream signaling cascade, including mavs, tbk , irf [ ] . we next sought to determine a step within the signaling pathway that hace targets. as shown in figure figure a,b) . on the other hand, it had no apparent effects on tbk or irf - d-induced isre or ifn-β reporter activation. these data suggested that hace acted downstream of mavs and upstream of tbk in the virus-induced signaling. as suggested by figure , hace modulated the virus-induced signaling downstream of mavs and upstream of tbk . then, we tried to investigate the exact mechanisms underlying the suppressive function of hace . we coexpressed hace with mavs, traf and tbk . unexpectedly, hace cannot interact with either mavs or tbk . it interacted with traf ( figure a ,b). hace is a hect ubiquitin e ligase. then, we tested whether hace can promote the degradation of traf . as shown in figure c , hace cannot degrade traf . this is consistent with the results above ( figure c ,d) that the hace /c a mutant has a comparable inhibitory function to wt hace on virus-induced signaling. it has been reported that the mavs and traf complex is essential for virus-induced signaling [ , ] . then, we detected the impact of hace expression on the mavs-traf complex. with the increase of the expression level of hace , the formation of the complex of mavs-traf was severely impaired ( figure d ). these data indicate that hace may negatively regulate the virus-induced signaling by disrupting the mavs-traf complex. seeded on -well plates and transfected the next day with indicated signaling molecules, ifn-β or isre luciferase reporter and prl-sv plasmid and increasing doses of hace for h. the luciferase activities were quantified by normalizing with renilla luciferase activities. data are representative of at least three independent experiments. * p < . ; ** p < . ; *** p < . . in the present study, we provide evidence for the first time that hace is an important negative regulator of virus-induced type i ifn signaling. additionally, this suppressive function is e ligase activity independent. hace plays its suppressive role downstream of mavs and upstream of tbk . co-immunoprecipitation assays showed that hace did not interact with mavs or tbk . unexpectedly, it binds traf , which interacts with mavs and forms a platform for rna virus signaling. hace does not promote the degradation of traf . this is consistent with the data that hace negatively regulates the virus-induced signaling independent of the e ligase activity. further studies shown that hace can disrupt the mavs-traf complex. this provides a mechanistic explanation for the suppressive function of hace on virus-induced innate immune response. hace is a hect-type e ligase. the most studied function of hace is its involvement in tumor development. this function is e ligase dependent [ ] . hace can also function in an e ligase independent manner. for example, hace can repress the transcriptional activity of rarα and rarβ . mutation of the putative catalytic cysteine (c ) does not alter the repressive effect of hace on the transcriptional activity of rarβ [ ] . hace can mediate p -dependent selective autophagic turnover of ubiquitinated proteins. this process is achieved by protein-protein interaction through its ankyrin repeat domain and is independent of its e ligase activity [ ] . under stress conditions, hace cleared the ubiquitinated proteins in an e ligase activity independent manner. here, we demonstrated that hace suppressed virus-induced signaling independently of its e ligase activity, suggesting that hace exerts diverse biological functions by different mechanisms. several viral or cellular negative regulators may hijack traf or the traf complex to mediate immune evasion. herpes simplex virus ubiquitin-specific protease ul deubiquitinates traf [ ] . a few sars coronavirus proteins are identified as viral negative regulators, which target the traf signalosome [ ] . sars coronavirus papain-like protease binds and disrupts the sting-traf -tbk complex [ ] . sars coronavirus m protein or open-reading frame- b impedes the formation of the traf /tank/tbk /ikkε complex or the mavs/traf /traf complex, respectively [ ] . besides, some studies reported endogenous physiological negative regulators that target traf or the mavs-traf complex. the linear ubiquitin assembly complex (lubac) downregulates virus-mediated ifn induction by targeting nemo for linear ubiquitination. then, linear ubiquitinated nemo is associated with traf and disrupts the mavs-traf complex, which inhibits ifn activation [ ] . mip-t , a ciliary protein, is also a traf binding protein, which acts as a cellular inhibitor in virus-induced ifn production. mip-t impedes the formation of multiple traf signaling complex, such as the mavs-traf complex, the traf -tbk or traf -ikkε complex [ ] . the ubiquitin e ligase triad a targets traf for degradation to negatively regulate the rig-i signaling [ ] . here, we elucidate a novel role of hace in virus-induced signaling, which also targets the central mavs-traf complex. the interaction of proteins with traf family members were mediated by the traf interaction motif. we also analyzed the structure of hace and found that there is a potential tim between amino acid to amino acid (fkplellwh); we mutated central amino acid ple to aaa, and then performed the luciferase assays. the results showed that this mutant lost the ability to suppress virus-induced signaling, indicating that the suppressive function of hace is dependent on the complete traf interaction motif. further studies will focus on the link of the spontaneous mutation of hace with inflammation or tumor development, which will be intriguing. in this study, we identified a novel function of hace on virus-induced type i ifn signaling, which targets the mavs-traf complex and impedes the assembly of the mavs-traf complex. activation of host pattern recognition receptors by viruses innate recognition of viruses innate immune response to viral infection interferon induction by rna viruses and antagonism by viral pathogens regulation of rig-i-like receptor signaling by host and viral proteins activation and regulation of pathogen sensor rig-i. cytokine growth factor rev recognition of viruses by cytoplasmic sensors recognition of viral nucleic acids in innate immunity rig-i-like receptors and negative-strand rna viruses: rlrly bird catches some worms ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-beta signaling identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus regulation of antiviral responses by a direct and specific interaction between traf and cardif a functional c-terminal traf -binding site in mavs participates in positive and negative regulation of the ifn antiviral response the e ubiquitin-ligase hace catalyzes the ubiquitylation of active rac ubiquitylation of active rac by the e ubiquitin-ligase hace ubiquitylation of autophagy receptor optineurin by hace activates selective autophagy for tumor suppression ubiquitylation and activation of a rab gtpase is promoted by a β ar-hace complex the e ligase hace is a critical chromosome q tumor suppressor involved in multiple cancers hace : a novel repressor of rar transcriptional activity constitutional translocation breakpoint mapping by genome-wide paired-end sequencing identifies hace as a putative wilms tumour susceptibility gene differential expression of a novel ankyrin containing e ubiquitin-protein ligase, hace , in sporadic wilms' tumor vs. normal kidney hace is a tumor suppressor gene candidate in natural killer cell neoplasms hace , a potential tumor suppressor gene on q , is not involved in extranodal natural killer/t-cell lymphoma pathophysiology aberrant methylation of the hace gene is frequently detected in advanced colorectal cancer methylation of the hace gene is frequently detected in hepatocellular carcinoma methylation of hace in gastric carcinoma loss of the e ubiquitin ligase hace results in enhanced rac signaling contributing to breast cancer progression the tumour suppressor hace controls cell migration by regulating rac degradation rac in human breast cancer: overexpression, mutation analysis, and characterization of a new isoform hace reduces oxidative stress and mutant huntingtin toxicity by promoting the nrf response the ubiquitin ligase hace regulates golgi membrane dynamics during the cell cycle hace -dependent protein degradation provides cardiac protection in response to haemodynamic stress smurf negatively modulates rig-i-dependent antiviral response by targeting visa/mavs for ubiquitination and degradation negative regulation of the rig-i signaling by the ubiquitin ligase rnf pcbp mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip e ligase wwp negatively regulates tlr -mediated innate immune response by targeting trif for ubiquitination and degradation immune signaling by rig-i-like receptors mammalian hect ubiquitin-protein ligases: biological and pathophysiological aspects herpes simplex virus ubiquitin-specific protease ul inhibits beta interferon production by deubiquitinating traf sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex we are grateful to takashi fujita for the rig-i expressing vector, tom maniatis for the tbk and ikkε vector and zhijian chen for the mavs (visa) vector. we also thank hong-bing shu, hong tang the authors declare no conflict of interest. key: cord- -q rky r authors: stewart, cameron r.; deffrasnes, celine; foo, chwan hong; bean, andrew g. d.; wang, lin-fa title: a functional genomics approach to henipavirus research: the role of nuclear proteins, micrornas and immune regulators in infection and disease date: - - journal: roles of host gene and non-coding rna expression in virus infection doi: . / _ _ sha: doc_id: cord_uid: q rky r hendra and nipah viruses (family paramyxoviridae, genus henipavirus) are zoonotic rna viruses that cause lethal disease in humans and are designated as biosafety level (bsl ) agents. moreover, henipaviruses belong to the same group of viruses that cause disease more commonly in humans such as measles, mumps and respiratory syncytial virus. due to the relatively recent emergence of the henipaviruses and the practical constraints of performing functional genomics studies at high levels of containment, our understanding of the henipavirus infection cycle is incomplete. in this chapter we describe recent loss-of-function (i.e. rnai) functional genomics screens that shed light on the henipavirus–host interface at a genome-wide level. further to this, we cross-reference rnai results with studies probing host proteins targeted by henipavirus proteins, such as nuclear proteins and immune modulators. these functional genomics studies join a growing body of evidence demonstrating that nuclear and nucleolar host proteins play a crucial role in henipavirus infection. furthermore these studies will underpin future efforts to define the role of nucleolar host–virus interactions in infection and disease. paramyxoviruses (order mononegavirales) are single-stranded rna viruses of negative polarity that can cause diseases in humans (rabies, measles virus, mumps virus, respiratory syncytial virus, human parainfluenza virus, ebola virus) and animals (newcastle disease virus, canine distemper virus, borna disease virus). the family paramyxoviridae is divided into two subfamilies (paramyxovirinae and pneumovirinae), with hendra virus (hev) being the foundation member of the genus henipavirus in the subfamily paramyxovirinae. the discovery of the hev and nipah virus (niv) had a striking impact on our understanding of paramyxovirus biology. henipaviruses have a much wider host range and a significantly larger genome than other paramyxoviruses, and to date are the only biosafety level (bsl)- agents within the family. with mortality rates of human infection between and %, hev and niv are among the most deadly viruses known to infect humans. hev emerged in in the brisbane suburb of hendra, queensland, australia, where it caused an outbreak of severe respiratory disease in horses that led to the natural death or euthanasia of out of affected animals. two people who had close contact with the infected horses were infected and one of these patients died (murray et al. ) . extensive sampling demonstrated that australian mainland flying foxes (family pteropodidae, genus pteropus) were seropositive for neutralising antibodies against hev (young et al. ) , while the virus was subsequently isolated from flying fox uterine fluid and urine (halpin et al. ) , providing strong evidence for australian mainland flying foxes as the hev reservoir. sporadic hev incidents occurred in horses between and , with events identified. an alarming number of hev incidents ( in total) occurred between and , with of those occurring in alone, highlighting the unpredictable nature of hev outbreaks. seven human cases of hev disease have been observed, four of which resulted in fatal disease. all recorded cases of hev transmission to humans have occurred directly from affected horses. the horses are believed to have acquired hev infection following direct exposure to secretions from flying foxes. more recently, the decline of reported human cases of hev infection is potentially due to the development of a vaccine to inhibit hev disease in horses (middleton et al. ) . niv was first identified during a disease outbreak on the west coast of peninsular malaysia in late . commercial pig farmers suffered disease characterised by febrile encephalitis that was linked to mild respiratory and neurological disease in pigs (mohd nor et al. ; from the centers for disease control and prevention ) . nucleotide sequencing demonstrated the virus was closely related to hev, whilst fruit bats of the pteropodidae family, pteropus genus, were confirmed as the natural reservoir (yob et al. ) . epidemiological evidence suggested that human infections were caused by transmission from pigs which likely had prior contact with fruit bats (update: outbreak of nipah virus-malaysia and singapore ). by mid- , cases of human infection were reported in singapore, where abattoir workers developed niv infection associated with contact with pigs imported from malaysia. this initial outbreak of niv in malaysia resulted in human cases reported with deaths. since , niv outbreaks have been reported almost every year in selected districts of bangladesh (hossain et al. ; luby et al. a) . unlike hev, human-to-human transmission of niv has been documented (luby et al. b) , including in a hospital setting. an increasing focus on flying foxes as viral reservoirs has led to the discovery of new henipaviruses. the genus was expanded in upon the isolation and characterisation of cedar virus (cedpv), isolated from bat urine samples from a flying fox colony in cedar grove, south east queensland. cedpv shows a remarkably similar genome organisation to hev and niv, antigenic cross-reactivity of the nucleocapsid protein between henipaviruses, and shares the same predominant entry receptor molecule, ephrin-b (marsh et al. ). however, a critical difference between cedpv and hev and niv is that the cedpv p gene lacks coding capacity for the immune antagonising v protein, whilst the cedpv p protein shows an impaired capacity to bind and inhibit ifn signalling via signal transducer and activator of transcription (stat) and stat (lieu et al. ) . accordingly, cedpv infection induces a robust type i interferon (ifn) response in human cells in vitro and does not cause clinical disease in ferret and guinea pig models of disease. such findings highlight the importance of immune evasion in the context of henipavirus pathogenicity and demonstrate the diverse range of pathogenicity within the same genus. in addition to these three viruses, the henipavirus genus is likely to be expanded in the future to accommodate the discovery and characterisation of emerging viruses from bats and other reservoirs. west african fruit bats harbour neutralising antibodies against hev and niv in particular, demonstrating a wider geographical range for henipaviruses not limited to pteropid bats (hayman et al. ). furthermore, a novel henipa-like virus, mojiang paramyxovirus, was isolated from rats in the yunnan province of china in and may have caused fatal disease in three individuals (wu et al. ) . alarmingly, a recent study looking at bat and human serum samples from cameroon found that - % of human samples were seropositive for henipaviruses, and that this was almost exclusively among individuals who reported butchering bat meat, providing the first evidence of human henipavirus spillover infections in africa (pernet et al. ). there are currently no licensed therapies to treat human cases of henipavirus infection. therefore, gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new antiviral therapies. viruses rely on the cell host machinery for completion of their infection cycle and therefore have adapted to interact with or exploit host molecules. retroviruses, most dna viruses, and many orthomyxoviruses replicate their genomes in the host nucleus. conversely, most positive-sense single-stranded viruses such as picornaviruses and flaviviruses and negative-sense, single-stranded viruses such as filoviruses, rhabdoviruses, and paramyxoviruses are perceived as cytoplasmic viruses and therefore are believed to not have a nuclear stage in their life cycle, replicating their genome entirely in the cytoplasm (lamb and parks ) . however, proteins of some of these viruses can traffic into nuclear compartments during infection (peeples ; yoshida et al. ; ghildyal et al. ; monaghan et al. ; wang et al. ) and this movement is sometimes critical for efficient infection (wang et al. ). this evidence indicates that the host nucleus may play a significant role in the infection cycle of henipaviruses and that the dynamics of virus-host interactions that occur in the nuclear compartments is an understudied area of molecular biology and virology. furthermore, since important discoveries in cell biology often follow studies of how viruses exploit normal host machinery, investigations into these nuclear interactions may reveal interesting novel insights into the cell biology of the mammalian nucleus. with this in mind, functional genomics provides a powerful and unbiased approach to study these biological questions. functional genomics refers to the development and application of global (genome-wide or system-wide) experimental approaches to assess gene function by making use of the information and reagents provided by sequenced genomes (hieter and boguski ) . a wide range of laboratory techniques can be considered as functional genomics, including genome interaction mapping (at the dna level), microarrays, transcriptomics and serial analysis of gene expression (sage) (at the rna level), yeast hybrid systems and affinity chromatography and mass spectrometry (at the protein level) and loss-of-function studies such as mutational studies, rna interference (rnai) and clustered regularly interspaced short palindromic repeats (crispr) studies. functional genomics has demonstrated much power in its ability to dissect the dynamic interplay between host and viral factors during a virus infection, paving the way for novel drug targets. for instance, a haploid genetic screen resulted in the discovery of the once elusive entry receptor for ebola virus (carette et al. ). there have been many full-or partial-genome rnai screens of host-virus interactions, including orthomyxoviruses (brass et al. ; hao et al. ; karlas et al. ; konig et al. ; shapira et al. ), retroviruses (zhou et al. ; konig et al. ; brass et al. ) and flaviviruses (ang et al. ; sessions et al. ). until recently, such information was lacking for henipaviruses, and perhaps surprisingly, for paramyxoviruses generally. functional genomics screens can be technically challenging, laborious and involve the use of robotics and advanced imaging equipment. consequently there are technical and practical challenges to performing high-throughput screens at higher levels of containment. hev and niv are classified at bsl- agents due to their association with lethal human disease and the absence of preventive measures and effective treatments to combat infections. bsl- facilities feature additional precautions to protect workers from infections and prevent exposure, such as infectious work being conducted within class ii biosafety cabinets, limited access by secure, locked doors, hepa filtration of laboratory air, and additional primary containment (positive pressure air suits or class iii biosafety cabinets). due to these limitations, previous genome-wide screens for bsl- viruses used surrogate viruses, such as pseudotyped particles, and have been performed under bsl- conditions (kouznetsova et al. ; kleinfelter et al. ) . functional genomics have been employed to study henipavirus infection. for instance, the entry receptor of hev and niv, ephrin-b , was identified by microarray analysis of infection-permissive and infection-resistant cell lines (bonaparte et al. ) . transcriptomics and proteomics have been utilised to uncover key differences in cellular responses to hev infection in hev disease-susceptible (human) and disease-resistant (bat) cells, and suggest that activation of apoptosis pathways via the innate immune pathway may contribute to the tolerance of henipaviruses by flying foxes (wynne et al. ). here we largely focus on findings from two recent rnai screens to identify protein-coding genes and host-encoded micrornas impacting the henipavirus infection cycle in human cells. not only can these findings be compared to published rnai screens of hostvirus interactions, but the identification of host genes required for infection (as opposed to those that are merely differentially expressed during infection) may deliver new targets for the development of antiviral therapies. the large number of hev incidents in australia from to prompted researchers at our laboratory to establish the capability to perform genome-wide rnai screens at bsl- . central to this work was the development of a recombinant hev expressing the renilla luciferase construct, which allowed for high throughput and rapid measurement of virus infection (marsh et al. ). this recombinant virus was shown to be lethal in the ferret model of henipavirus disease and exhibited a pathogenesis profile comparable to the wild-type virus. functional genomics at high containment also required the establishment of protocols and/or safe work procedures for the operation and decontamination of liquid handling robots. a genome-wide analysis of host protein-coding genes required for henipavirus infection involved a primary screen assaying , protein-coding genes, followed by a secondary deconvolution screen and a tertiary screen determining whether screen results obtained using recombinant hev could be recapitulated using wild-type hev and niv (deffrasnes et al. ) . applying a robust z score normalisation method often used to interpret sirna screen results (birmingham et al. ; zhang et al. ) , and genes were identified that promoted or suppressed hev infection, respectively, without adversely impacting cell numbers. at the completion of the primary screen, proviral genes were selected based on rank for the secondary deconvolution screen. by this measure, high-and medium-confidence genes (> standard deviations from mean mock values for / or / , or / sirnas, respectively) were identified as being required for hev infection. the apparent reliance of henipavirus infection on the nuclear or nucleolar host proteins was particularly striking, as over % of high confidence hits localise in the nucleus or nucleolus, with many involved in ribosome biogenesis (table ) . the nucleus is the site of gene expression and dna transcription into mrna, and houses the early steps of the rnai pathway. the nucleus is separated from the cell cytoplasm by the nuclear envelop which contains nuclear pores and import/export proteins allowing the passage of small molecules such as mrna. nuclear import/export proteins such as xpo and kpna , which are required for trafficking of larger molecules like proteins, were identified by rnai screen as required for henipavirus infection (deffrasnes et al. ) . the nucleolus is a highly dynamic structure and has increasingly been shown to play a critical role in virus-host interactions (rawlinson and moseley ; xu et al. ). the nucleolus contains three regions composed of the fibrillar centre (fc) in the middle, surrounded by the dense fibrillary component (dfc) and the granular component (gc). this membrane-less structure contains a high concentration of proteins and rnas and is the site of ribosomal rna (rrna) synthesis and ribosome production but is also a multifunctional structure in eukaryotic cells. cell cycle progression, stress response, genetic silencing, regulation of apoptosis, cell migration and invasion are all functions associated with the nucleolus or partly regulated in this compartment (rawlinson and moseley ; xu et al. ; pederson ). fibrillarin is the main nucleolar protein responsible for the chemical modification of ribosomal rna (rrna). this - kda ′-o-methyltransferase transfers methyl groups from its substrate, the s-adenosylmethionine (sam), to the -hydroxyl groups of ribose target in rrna. fibrillarin has also been shown to methylate glutamine residue of the human histone h a, weakening its binding to the fact (facilitator of chromatin transcription) complex and impacting chromatin remodelling and rdna transcription by rna pol i (tessarz et al. ) , which points at an additional role for fibrillarin in ribosome biogenesis and translation. pre-ribosome processing, modification of pre-rrna rpl a component of the s ribosomal subunit sp transcriptional regulation xpo nuclear export fibrillarin itself is methylated on several arginine residues by protein arginine n-methyltransferase (prmt ), which is thought to influence its activity (rodriguez-corona et al. ) . expression levels of fibrillarin have been shown to be regulated by p through direct binding to fibrillarin intron . abnormal levels of fibrillarin have been detected in p -inactivated cancer cells and a decrease in p levels has been associated with an increase in fibrillarin expression, and conversely an increase in p expression results in decreased fibrillarin expression (marcel et al. ) . high levels of fibrillarin lead to changes in the rrna methylation pattern, diminished translation fidelity and increase in ires-mediated translation of some cancer genes. moreover, ribosome biogenesis is often dysregulated and over-activated in cancer cells that have a decreased or absent p expression (marcel et al. ) . in its n-terminal region, fibrillarin contains a glycine-and arginine-rich region (the gar domain) enabling interaction with cellular and viral proteins, and acting as a nucleolar retention signal. its c-terminal region (mtase) contains multiple rna-binding domains, a catalytic site allowing for fibrillarin methyltransferase function, and is the site for nop / interaction. fibrillarin is a part of at least one nucleolar ribonucleoprotein (snornp) complex comprising the nop , nop and . k nucleolar proteins. x-ray data have suggested that the methylation of rrna requires the formation of this complex with involvement of four fibrillarin molecules interacting with different regions of the target rrnas. the yeast equivalent of fibrillarin, nop , has been more extensively studied than the human counterpart but fibrillarin is a well-conserved protein in most organisms, reinforcing the notion that all post-transcriptional processes involving fibrillarin such as chemical modification (methylation) of rrna, pre-rrna cleavage and ribosome assembly are essential for proper cellular functioning (rodriguez-corona et al. ) . in eukaryotes, ribosome biogenesis involves numerous nucleolar proteins and accessory factors, around ribosomal proteins, many small nucleolar rnas (snornas), three rna polymerases (rna polymerase i, ii and iii) and four different species of rrnas. the process of assembly of elongation-competent s ribosomes is divided into three major steps: ( ) ribosomal dna (rdna) transcription into precursor rrnas (pre-rrnas), ( ) processing of pre-rnas into mature rrnas, and then ( ) assembly of rrnas with ribosomal proteins into functional ribosomes. in the nucleolus, the rna polymerase i (rna pol i) is responsible for transcribing the s, . s and s rrna from a single polycistronic pre-rrna, while rna pol iii transcribes the s rrna in the nucleus (xue and barna ) . the pre-rnas are then cleaved and modified during the pre-rrna processing phase. all ribosomal proteins (rp) are transcribed in the cytoplasm by rna pol ii and then translated before migrating to the nucleolus. these rp, along with nucleolar proteins such as fibrillarin and rpl a, are responsible for modifying the rrnas (ribose ′-o-methylation, pseudouridylation, etc.) with the activity of more than snornas guiding the process in a site-specific manner. the main nucleolar protein involved in rrna modification is fibrillarin, which methylates more than sites essential for ribosome biogenesis and stability. although these post-transcriptional modifications are crucial for ribosome functions, their roles are not yet fully understood. in eukaryotes, the large s subunit of ribosomes is made of the s, . s, and s rrna along with multiple large subunit ribosomal proteins (rpl), while the small s subunit is made of the s rrna along with multiple small subunit ribosomal proteins (rps). the two subunits are assembled in the nucleolus into the s ribosomes before being transferred into the cytoplasm. deffrasnes and colleagues showed that sirna-mediated knockdown of fibrillarin expression dramatically reduced hev protein production and viral genome replication but did not impact viral fusion, and that fibrillarin catalytic activity was essential to henipavirus infection. on the other hand, overexpression experiment did not lead to an increase in viral titers, suggesting that a simple reduction or increase in overall ribosome production is unlikely to explain the reliance of henipaviruses on fibrillarin activity (deffrasnes et al. ). the requirement of fibrillarin and several other proteins from the ribosomal biogenesis pathway for henipavirus infection points a reliance on translation for efficient infection. however, while we tend to view ribosomes as homogenous, new studies reveal a more heterogeneous nature of ribosomes due to differences in the ribosomal proteins recruited, post-translational modifications of rrna and rrna composition. moreover, ribosomal proteins have been found to have additional functions outside of their primary roles in ribosomes and to be involved in other nucleolar functions such as regulation of cell proliferation, tumorigenesis and dna damage response (xu et al. ; xue and barna ; au and jan ) . in eukaryotes, most messenger rna (mrna) harbour a ′ -methylguanosine cap structure and a ′ poly(a) tail, which are both required for canonical, cap-dependent translation. a cap-independent translation mechanism also utilised by a subset of host proteins is called internal ribosome entry site (ires)-mediated translation. it is believed that most genes translated via an ires are related to stress response, cell proliferation, cell death/survival, and that ires-mediated translation happens when the canonical cap-dependent translation is inhibited either by the host reaction to environmental factors, damage, stress or infections. however, a group recently suggested that thousands of human genes are translated via this cap-independent mechanism, representing a -fold increase in the number of sequences previously associated with this translation pathway (weingarten-gabbay et al. ) . recently a new type of translation has been described in vesicular stomatitis virus (vsv)-infected cells. this non-canonical cap-dependent protein translation involves the ribosomal protein rpl acting as a constituent of the large subunit of ribosomal complexes and suggests a novel ribosome-specialised translation initiation pathway benefiting viral mrna translation (lee et al. ). translations of viral proteins from several other mononegaviruses, including the paramyxoviruses measles virus (mev) and newcastle disease virus (ndv), and a subset of cellular transcripts, are also rpl -dependent. how henipavirus mrnas are translated is not fully understood. whilst the rpl -dependent form of cap-dependent translation remains to be characterised in detail, one could speculate that fibrillarin, like rpl , acts a novel initiation factor for henipavirus mrnas. the fact that depleting cells of fibrillarin did not impact synthesis of influenza a viral proteins (which occurs via the canonical cap-dependent pathway) suggests that henipavirus mrna translation occurs via a non-canonical pathway, perhaps used by a subset of cellular transcripts. such a concept would allow henipavirus protein synthesis to proceed in an environment where viruses may induce cellular translation shutdown in order to suppress host antiviral immune responses. there are several reports of paramyxoviruses blocking canonical translation pathways, including the mev n protein binding to the eukaryotic initiation factor (eif -p ) (sato et al. ), whilst the p and v proteins of simian virus (sv ) limit activation of the double-stranded rna (dsrna)-dependent protein kinase (pkr) to limit both host and viral protein translation (gainey et al. ). similar to sv , sirna-mediated depletion of pkr results in increased hev growth (robust z score . ), consistent with the notion that shutdown of host protein translation inhibits henipavirus infection. if future studies do indeed demonstrate a role of fibrillarin in influencing the synthesis of ribosome subtypes required for viral protein translation, this may explain the targeting of fibrillarin by several viral proteins. fibrillarin binds the hev matrix (m) protein during the early stages of infection, whilst the hiv-tat protein has been reported to bind fibrillarin and u snorna, both required for pre-rrna processing, and this interaction reduces the pool of cytoplasmic ribosomes (ponti et al. ) . intriguingly, the nucleoprotein of porcine reproductive and respiratory syndrome virus, the non-structural protein (ns ) of a h n influenza virus (melen et al. ) and the non-structural protein b of the severe acute respiratory syndrome coronavirus (yuan et al. ) all bind and co-localise with fibrillarin in the nucleolus; however, the reasons for this binding are yet to be determined. many negative strand viruses encode viral proteins that localise in the nucleus and/or nucleolus at some point in their infection cycle [reviewed in (rawlinson and moseley ; hiscox ; oksayan et al. ; flather and semler ; watkinson and lee ) ]. within the paramyxoviridae, nuclear localisation of matrix (m) protein has previously been described for ndv (peeples ) , sendai virus (sev) (yoshida et al. ), human respiratory syncytial virus (ghildyal et al. ) , hev (monaghan et al. ) and niv (wang et al. ) . during the early stages of henipavirus infection or when expressed ectopically (monaghan et al. ; wang et al. ) , the hev and niv m proteins traffic through the nucleolus to the cytoplasm. it has been recently shown that nuclear traffic is required for the henipavirus m protein to coordinate viral budding. the henipavirus m protein is a structural protein that mediates viral assembly and budding (liljeroos and butcher ; takimoto and portner ; eaton et al. ). indeed, for both hev-m and niv-m, overexpression of these proteins alone is sufficient to trigger viral-like particles (vlps) that bud into the supernatant. wang and colleagues ( ) demonstrated that mutation of niv-m nuclear localisation signals (nls) or nuclear export signals (nes) blocks nuclear/cytoplasmic traffic and impairs viral budding. furthermore, a highly conserved lysine residue in the nls (k ) serves two functions: its positive charge mediates niv-m nuclear import, while is also a potential site for monoubiquitination which regulates niv-m nuclear export. niv infection (deffrasnes et al. ). this raises the question: do henipavirus m proteins traffic through the nucleolus for other reasons? the multi-faceted roles of paramyxovirus proteins in replication-specific roles and various cellular processes, particularly immune evasion, would suggest so. to explore whether m binds to host proteins associated with infection efficiency, results from the genome-wide rnai hev screen were cross-referenced against a proteomics study by pentecost and colleagues cataloguing host proteins that bind hev-m and niv-m, among other paramyxovirus m proteins (pentecost et al. ) . that study revealed that the henipavirus m interactome spans hundreds of host proteins, with interactions with nuclear pore complex proteins, nuclear transport receptors and nucleolar proteins particularly prevalent. interestingly, niv-m and hev-m interactomes show notable overlap to other paramyxovirus m proteins, including sev and ndv, with over % of the proteins found in any single interactome also found in the interactomes of one or more of the other three viruses (pentecost et al. ) . whilst the binding of fibrillarin to hev-m was demonstrated by co-immunoprecipitation assays (deffrasnes et al. ) and this was not observed by proteomics, interactions were observed between hev-m and numerous nucleolar proteins such as nop (pentecost et al. ) which forms a complex with fibrillarin, supporting a functional interaction between fibrillarin and hev-m. the relative hev growth (presented as robust z scores) in cells depleted of the hev-m-binding host proteins is shown in fig. a . of the candidates assayed, hev-m binds to protein-coding genes that have a large impact (robust z score − or ! ) on hev infection, roughly evenly distributed between proviral ( ) and antiviral ( ) candidates. designating all candidates genes with z scores < as proviral and genes with z scores > as antiviral, host proteins that bind hev-m appears to be pro-and antiviral at approximately equal ratios with a slight enrichment of proviral genes ( proviral candidates vs. antiviral candidates). an assessment of whether the relative abundance of hev-m-host protein interactions indicated a likelihood of that host protein adopting a proviral or antiviral function was also carried out (fig. b) . the relative abundance of host proteins within the proteomics dataset is represented as the normalised spectral abundance factor (nsaf), with higher nsaf values presenting more abundant interactions. plotting nsaf values against robust z scores demonstrates that host proteins that bind hev-m with high abundance (nsafe scores between and ) were more proviral ( candidates) than antiviral ( candidates, z score sums: proviral . , antiviral . ). these candidates are listed in table and include several ribosomal proteins, further implicating m in host translation. akin to fibrillarin, the critical role of host molecules in henipavirus infection and pathogenesis can be inferred by their specific targeting by viral proteins. this is particularly true in the context of immune evasion, as the innate antiviral immune response is a known target for several henipavirus proteins. the henipavirus genome contains six transcriptional units, n, p, m, f, g and l, coding for nine proteins (eaton et al. ). the p gene alone codes for at least four of the proteins: p, w, v and c . all four of these proteins are involved in modification of the immune response in the host cell, through inhibition of the type i interferon (ifn) responses [reviewed in (audsley and moseley ) ]. intracellular detection of pathogen-associated molecular patterns (pamps) is mediated by membrane-bound toll-like receptors (tlrs) or cytoplasmic retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) and nucleotide-binding oligomerisation domain containing (nod)-like receptors (nlrs). engagement of these receptors with their agonists results in the activation of complex signalling pathways culminating in the production of cytokines and anti-microbial compounds. a critical component of this response is the type i ifn system, which induces a local antiviral state upon detection of viruses or intracellular bacteria or molecules associated with their replication (schoggins and rice ). genes with z scores < were designated proviral, while genes with z scores > were designated antiviral. values represent the sum of all the z scores. it should be noted that genes were excluded from analysis due to ambiguous gene identification listings in the proteomics study, whilst the silencing of additional gene targets resulted in cell death that prevented the measurement of virus growth. b plot of the z score of hev-m-binding proteins (x-axis) and relative abundance of hev-m interactions, represented by normalised spectral abundance factor (y-axis) viral replication is typically detected by tlrs and / in endosomal compartments (alexopoulou et al. ; lund et al. ) , whilst rig-i and/or melanoma differentiation-associated gene (mda ) recognise short or long viral dsrna intermediates in the cytosol (yoneyama et al. ; triantafilou et al. ) . tlr activates the tir-domain-containing adapter-inducing ifn-b (trif) (matsumoto et al. ) , whilst rig-i/mda interact via their caspase recruitment domains (cards) with mavs (mitochondrial activated signalling protein) (seth et al. ) to induce signalling. activation of trif or mavs promotes recruitment of multiple cytosolic effectors, resulting in the phosphorylation and dimerisation of interferon regulatory factor (irf) or liberation of nf-jb from its inhibitory complex. these transcription factors then shuttle into the nucleus to form part of a large multiprotein complex that binds to the promoter region of ifn-b and initiates transcription (honda and taniguchi ) . the c-terminus of the hev v protein binds and sequesters mda , thereby impairing ifn-b transcription in response to double-stranded rna (andrejeva et al. ) . this binding appears to be conserved amongst most paramyxoviruses including niv, sv and mumps virus (childs et al. ). intriguingly, rig-i is not targeted by paramyxovirus v proteins, and perhaps consistent with this, the genome-wide rnai screen suggested that depleting cells of mda increased hev infection (robust z score . ), whilst targeting rig-i had very little impact (z score − . ). similar to the nlr cytoplasmic antiviral immune responses, tlr -dependent antiviral signalling is also inhibited by henipaviruses, with the w protein localising to the nucleus via the importin molecules kpna and kpna to block irf -responsive promoter activation by virus and intracellular dsrna (shaw et al. ). transfecting niv-w into cells in a dose-dependent manner sequesters inactive irf in the nucleus, thus depleting the pool of available irf for phosphorylation and activation. from the genome-wide screen, the impact of down-regulating tlr (z score . ) and irf ( . ) was a moderately antiviral phenotype. the best-characterised target of henipavirus immune evasion is the stat proteins, critical signalling molecules in the context of type i ifn cytokine production conferring the antiviral state [reviewed in (platanias ) ]. the binding of type i ifn (ifn-a and ifn-b) and type ii ifn (ifn-c) to their respective receptor complexes leads to the phosphorylation and association of stat and stat heterodimers (for type i ifn signalling), or stat homodimers (type ii ifn). this prompts the formation of stat -stat -irf (ifn-regulatory factor ) complexes that translocate to the nucleus and bind ifn-stimulated response elements (isres) in dna to initiate transcription of ifn-stimulated genes (isgs). whilst there are hundreds, potentially thousands of isgs that collectively confer antiviral immunity, very few isgs have been functionally characterised in the context of henipavirus infection. one isg, cholesterol hydroxylase (ch h), inhibits infection by niv and a range of other rna viruses by blocking membrane fusion between host and viral membranes (liu et al. a) . consistent with this observation, ch h blocked hev infection in the genome-wide rnai screen (robust z score . ). henipaviruses, like other paramyxoviruses, generate multiple alternative mrnas from the p gene locus-p, v and w (thomas et al. ) . a fourth protein, c, is generated by alternate translation initiation site selection from all these mrnas and does not share sequence homology to the other proteins. the p, v, and w proteins share amino acids in their n termini and all three proteins bind to stat and stat via this n-terminal region (ciancanelli et al. ; rodriguez et al. ) . virus-host interactions in this context prevent stat / phosphorylation and activation, and lead to their sequestration in high molecular weight complexes (rodriguez et al. ; rodriguez et al. ; shaw et al. ) . interestingly, the sirna-mediated inhibition of stat increased hev infection in the genome-wide screen, but inhibition of stat did not (robust z scores of . and − . ). this preliminary observation suggests that stat activity may have a greater impact on henipavirus infection than stat , and may implicate type ii in antiviral immunity against henipaviruses. although the role of henipavirus p gene products in immune evasion is well-established, a recent study demonstrates the surprising ability of niv-m to antagonise the antiviral type i ifn response (bharaj et al. ) . the study by bharaj and colleagues shows that niv-m binds to and targets the e -ubiquitin ligase trim for degradation. trim catalyses the synthesis of unanchored polyubiquitin chains that are used as a substrate for the activation of ikb kinase-e (ikke), which phosphorylates irf and activates irf -dependent transcription of type i ifn, and tnf-a. trim targeting by niv-m occurs in the cytoplasm via an unknown mechanism not involving the proteasome or the lysosome, and requires nuclear/cytoplasmic trafficking of niv-m. similar to viral budding, this function of m is dependant on nuclear traffic, as k mutants of niv-m do not target trim for degradation. the study expands our understanding of immune antagonism and highlights the potential purpose of henipavirus m protein nuclear trafficking. . although far less frequent, mirna binding may also cause an increase in target mrna translation and thus up-regulation of protein expression (vasudevan et al. ). in terms of target complementarity, mirnas do not require perfect base pairing (tenoever ). as a result, one mirna has the potential to regulate a surprisingly broad network of genes (skalsky and cullen ; zhang et al. ) , with certain mirnas found to have binding sites located on several hundred different mrna sequences (guo and steitz ) . despite the potential for widespread impacts, studies have described the effects of mirna gene regulation on protein expression levels as generally 'subtle' (tenoever ) or 'typically relatively mild' (selbach et al. ). this is due to the fact that, in general, mirnas do not entirely silence but rather moderately repress translation and, hence, effectively fine tune rather than knock out gene expression (baek et al. ). the role of mirnas in the infection cycle of rna viruses is becoming increasingly apparent. certain mirnas may promote virus replication by directly interacting with the viral genome or, alternatively, by down-regulating the expression of host genes that suppress virus infection (skalsky and cullen ; roberts et al. ) . inhibiting specific 'proviral' mirnas, therefore, may have a direct negative impact on the viral life cycle (janssen et al. ) or alternatively render the intracellular environment unfavourable for virus replication (stewart et al. ) . in an example of the latter, mir- a has been found to promote hev infection by repressing ring finger protein , a negative regulator of nf-ĸb activity (stewart et al. ) . furthermore, inhibiting mir- a has been found to significantly reduce hev replication in vitro (stewart et al. ). on the other hand, mir- is an example of a mirna that promotes hepatitis c virus (hcv) replication by directly interacting with the viral genome-this activity is the basis of the first mirna inhibitor drug to enter phase ii clinical trials (janssen et al. ; wilson and sagan ) . the functional genomics platform established as part of the screen of protein-coding genes associated with hev infection was recently adapted to study the impact of host-encoded mirnas on hev growth (foo et al. ) . the screen involved the use of synthetic mirna mimics and inhibitors targeting micrornas. mimic and inhibitor screens identified and micrornas, respectively, that promoted hev infection, and and micrornas, respectively, that inhibited virus infection. a major finding from this study was that all four members of the mir- family (-a to -d) promote infection by hev and niv. infection promotion was primarily mediated via the ability of mir- to significantly enhance henipavirus-induced membrane fusion. cell signalling receptors of ephrins, namely epha and epha , were identified as novel negative regulators of henipavirus fusion. the expression of these receptors, as well as ephb , was suppressed by mir- overexpression, suggesting that simultaneous inhibition of several ephs by the mirna contributes to enhanced infection and fusion. to our knowledge, this study represented the first evidence of a host-encoded mirna promoting virus cell entry. previous studies have reported that members of the mir- family are involved in different aspects of immune regulation (hutchison et al. ; galicia et al. ; zietara et al. ) . specifically, mir- has been found to play a central role in the regulation of b cell differentiation and t cell selection, maturation and sensitivity (sun et al. ) . for instance, induction of mir- a has been found to occur at the cd (+)-cd (+) double-positive stage of t cell development, inhibiting the expression of cd , bcl- and t cell receptor-all involved in positive selection and t cell maturation (neilson et al. ). in addition, mir- c has been found to suppress cd + t cell activation by targeting interleukin (il- ) (sun et al. ; xue et al. ). in addition, mir- a expression levels have been shown to correlate with pro-inflammatory signals (e.g. il- b, il- and tnf-a) in blood and various tissues of humans with chronic inflammation, as well as in the blood of lps-treated mice (xie et al. ) . consistent with the notion that mir- expression is immune-responsive, levels of mir- were up-regulated in the biofluids of ferrets and horses infected with hev, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. the study of both mirnas and protein-coding genes associated with hev infection allows an assessment whether genes required for virus infection (i.e. proviral genes) are regulated by mirnas that inhibit virus infection (i.e. antiviral micrornas). multiple members of the let- mirna family inhibited hev infection. there are mature let- sequences in humans, with multiple roles described, including negative regulation of tumorigenesis (shi et al. ; esquela-kerscher and slack ) . in a transcriptome-wide study in hela cells, genes significantly down-regulated by let- b at either the mrna level, protein level or both, included fourteen validated genes required for wild-type hev infection, including akt (selbach et al. ). furthermore, six proviral genes contain putative let- b binding sites in their ′ utr (akt , c orf , eif s , hmga , ifitm and serpinh ), as identified by diana-mirextra (alexiou et al. ) . collectively, these data suggest that let- mirnas inhibit hev by suppressing host proteins required for virus infection. cross-referencing results from the protein-coding screen study showed that the majority of verified target genes for mir- and mir- - mirnas (proviral in the mirna screen) were predominately antiviral, demonstrating a level of congruency between mirna and protein-coding gene screens. in contrast to let- , all six members of the mirna precursor mir- family (mir- , - a, - b, - a, - b and - ), part of the oncogenic mir- - polycistron, strongly promoted hev infection. interestingly, other mirnas of the mir- - cluster with distinct "seed" families (based on sequence identity at positions - )-mir- , mir- and mir- ) did not impact virus replication to a similar extent. the mir- - cluster is a known oncogene locus-it is amplified in b cell lymphomas (ota et al. ) and accelerates tumour development in a mouse b cell lymphoma model (he et al. ) . members of the mirna precursor mir- family are expressed in almost all human tissues (liang et al. ). in addition, mir- a and - b are expressed in peripheral blood mononuclear cells (pbmcs), platelets and exosomes derived from peripheral blood (hunter et al. ) . henipaviruses are dangerous pathogens and control of disease caused by these viruses will critically rely on the development of new antiviral therapeutics and vaccination strategies. currently, there is requirement for renewed research into the host immune responses to henipavirus infection and how competent immune responses may fight disease. a major challenge is to ascertain the molecular mechanisms of virus replication and immunity associated with protection to infection. the improved knowledge of functional genomics approaches and immune response to viral infection means that we now have the tools to further progress our understanding and knowledge. nevertheless, this must be implemented to develop advanced infection control approaches. the diana-mirextra web server: from gene expression data to microrna function recognition of double-stranded rna and activation of nf-kappab by toll-like receptor the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda- , and inhibit its activation 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cellular development subcellular trafficking in rhabdovirus infection and immune evasion: a novel target for therapeutics identification and characterization of a novel gene, c orf , as a target for q -q amplification in malignant lymphoma the nucleus introduced differential detergent treatment allows immunofluorescent localization of the newcastle disease virus matrix protein within the nucleus of infected cells evidence for ubiquitin-regulated nuclear and subnuclear trafficking among paramyxovirinae matrix proteins evidence for henipavirus spillover into human populations in africa mechanisms of type-i-and type-ii-interferon-mediated signalling the hiv tat protein affects processing of ribosomal rna precursor the nucleolar interface of rna viruses the role of micrornas in viral infection identification of the nuclear export signal and stat-binding domains of the nipah virus v protein reveals mechanisms underlying interferon evasion nipah virus v protein evades alpha and gamma 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their regulation viruses, micrornas, and host interactions promotion of hendra virus replication by microrna a role of mir- family in regulating vascular inflammation and immunity molecular mechanism of paramyxovirus budding rna viruses and the host micro rna machinery glutamine methylation in histone h a is an rna-polymerase-i-dedicated modification two mrnas that differ by two nontemplated nucleotides encode the amino coterminal proteins p and v of the paramyxovirus sv visualisation of direct interaction of mda and the dsrna replicative intermediate form of positive strand rna viruses update: outbreak of nipah virus-malaysia and switching from repression to activation: micrornas can up-regulate translation ubiquitin-regulated nuclear-cytoplasmic trafficking of the nipah virus matrix protein is important for viral budding nipah virus matrix protein: expert hacker of cellular machines comparative genetics. systematic discovery of cap-independent translation sequences in human and viral genomes hepatitis c virus and human mir- : insights from the bench to the clinic novel henipa-like virus, mojiang paramyxovirus, in rats proteomics informed by transcriptomics reveals hendra virus sensitizes bat cells to trail-mediated apoptosis ) mir- a and inflammation: mirna homeostasis response to inflammatory stimuli in vivo the role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity human activated cd (+) t lymphocytes increase il- expression by downregulating microrna- c specialized ribosomes: a new frontier in gene regulation and organismal biology nipah virus infection in bats (order chiroptera) in peninsular malaysia the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses membrane (m) protein of hvj (sendai virus): its role in virus assembly serologic evidence for the presence in pteropus bats of a paramyxovirus related to equine morbillivirus nucleolar localization of non-structural protein b, a protein specifically encoded by the severe acute respiratory syndrome coronavirus robust statistical methods for hit selection in rna interference high-throughput screening experiments progress in microrna delivery genome-scale rnai screen for host factors required for hiv replication critical role for mir- a/b- in agonist selection of invariant natural killer t cells key: cord- -r nlef h authors: teuber, g.; berg, t.; naumann, u.; raedle, j.; brinkmann, s.; hopf, u.; zeuzem, s. title: randomized, placebo‐controlled, double‐blind trial with interferon‐α with and without amantadine sulphate in primary interferon‐α nonresponders with chronic hepatitis c date: - - journal: j viral hepat doi: . /j. - . . .x sha: doc_id: cord_uid: r nlef h in primary interferon‐α (ifn‐α) nonresponders with chronic hepatitis c, retreatment with ifn‐α has only limited efficacy with sustained response rates below %. therefore, the aims of the present study were to compare the efficacy and safety of ifn‐α alone or in combination with amantadine sulphate in nonresponders to previous ifn‐α monotherapy. fifty‐five ifn‐α nonresponders with chronic hepatitis c (mean age: . years) received ifn‐α miu thrice weekly for weeks followed by miu thrice weekly for additional weeks. amantadine sulphate (n= ) or a matched placebo (n= ) was given orally twice daily for weeks. because of a low initial response rate at week ( / patients) and a high breakthrough rate ( / patients) after ifn‐α dose reduction in week , a virological end‐of‐treatment response with undetectable serum hcv‐rna (< copies/ml) was achieved in only five patients (ifn‐α/amantadine sulphate, one patient; ifn‐α/placebo, four patients). after weeks follow‐up a sustained virological response was observed in only two patients receiving ifn‐α and placebo. health‐related quality‐of‐life analysis showed a substantial improvement of the profile of mood states (poms) scale concerning the subscales fatigue (p < . ) and vigor (p < . ) in patients receiving combined ifn‐α/amantadine sulphate treatment compared with those treated with ifn‐α alone. ifn‐α/amantadine sulphate combination therapy was well tolerated without any serious adverse events. in conclusion, retreatment with ifn‐α and amantadine sulphate does not increase the low sustained virological response rates of ifn‐α therapy in primary ifn‐α nonresponders with chronic hepatitis c, but may lead to a sustained improvement of health‐related quality‐of‐life. hepatitis c virus (hcv) infection frequently leads to chronic hepatitis and may progress to liver cirrhosis and possibly hepatocellular carcinoma [ ± ] . hcv-related end-stage liver disease is currently one of the leading indications for liver transplantation. treatment with interferon-a (ifn-a) alone leads only to a sustained viral clearance in approximately ± % of patients [ , ] . the addition of ribavirin to standard interferon-a treatment improves the sustained virological response rate to approximately % in previously untreated patients [ , ] . several treatment strategies have been investigated in patients with chronic hepatitis c not responding to previous interferon-a monotherapy. however, retreatment of previous interferon-a nonresponders with standard regimens of interferon-a alone ( ´ ± miu/week s.c.) or in combination with ribavirin ( ± mg/day orally) showed only limited therapeutic ef®cacy with sustained viral response rates below % [ , ] . amantadine ( -aminoadamantanamine sulphate) is a tricyclic, symmetric amine with an antiviral activity against toga-, myxo-, arena-,¯avi-and coronaviruses [ ± ] . the drug has been studied in detail in the in¯uenza a virus infection and the antiviral activity was found to be related to inhibition of viral uncoating and viral budding by interaction with the viral m protein [ ± ] . the antiviral properties of amantadine have led to clinical trials evaluating the potential therapeutic role of amantadine alone or in combination with interferon-a in patients with chronic hepatitis c [ ± ] . for retreatment of previous interferon-a summary. in primary interferon-a (ifn-a) nonresponders with chronic hepatitis c, retreatment with ifn-a has only limited ef®cacy with sustained response rates below %. therefore, the aims of the present study were to compare the ef®cacy and safety of ifn-a alone or in combination with amantadine sulphate in nonresponders to previous ifn-a monotherapy. fifty-®ve ifn-a nonresponders with chronic hepatitis c (mean age: . years) received ifn-a miu thrice weekly for weeks followed by miu thrice weekly for additional weeks. amantadine sulphate (n ) or a matched placebo (n ) was given orally twice daily for weeks. because of a low initial response rate at week ( / patients) and a high breakthrough rate ( / patients) after ifn-a dose reduction in week , a virological end-of-treatment response with undetectable serum hcv-rna (< copies/ml) was achieved in only ®ve patients (ifn-a/amantadine sulphate, one patient; ifn-a/placebo, four patients). after weeks follow-up a sustained virological response was observed in only two patients receiving ifn-a and placebo. health-related quality-of-life analysis showed a substantial improvement of the pro®le of mood states (poms) scale concerning the subscales fatigue (p < . ) and vigor (p < . ) in patients receiving combined ifn-a/amantadine sulphate treatment compared with those treated with ifn-a alone. ifn-a/amantadine sulphate combination therapy was well tolerated without any serious adverse events. in conclusion, retreatment with ifn-a and amantadine sulphate does not increase the low sustained virological response rates of ifn-a therapy in primary ifn-a nonresponders with chronic hepatitis c, but may lead to a sustained improvement of health-related quality-of-life. nonresponders with chronic hepatitis c with the combination of interferon-a and amantadine, only data from a few uncontrolled studies with inconsistent response rates are available [ , ] . therefore, the aims of the present study were to evaluate the therapeutic ef®cacy, tolerability and health-related quality of life of interferon-a a (ifn-a a) plus amantadine sulphate in comparison with ifn-a a plus placebo in patients with chronic hepatitis c not responding to previous ifn-a treatment. patients with chronic hepatitis c not responding to previous ifn-a monotherapy with a minimal total ifn-a dose of miu and a treatment duration of at least weeks were eligible for enrolment when they met all of the following inclusion criteria: ( ) nonresponse to previous ifn-a monotherapy with persistence of serum hcv-rna and a treatment-free interval of at least weeks; ( ) elevated alanine aminotransferase (alt) levels; ( ) a positive anti-hcv test; ( ) detectable serum hcv-rna; ( ) compensated liver disease; ( ) leukocyte count p /ll, platelets count p /ll; and ( ) aged between and years. demographic, biochemical, serological and virological pretreatment characteristics of the enrolled patients are summarized in table . exclusion criteria were coinfection with hepatitis b virus or human immunode®ciency virus types and , concomitant autoimmune disease, clinically signi®cant cardiovascular, metabolic, renal, haematological, rheumatological, neurological or psychiatric disease, organ grafts, systemic infections, bleeding disorders, anaphylactic reactions, a history of neoplastic disease within the last years, systemic immunosuppressive treatment, average daily intake of alcohol > g, drug abuse within the previous year, pregnancy and lactation period. liver biopsy was performed in all patients ± months before the initiation of the primary ifn-a therapy. according to the study protocol, additional liver biopsies before retreatment were not required for enrolment in the present study. in the present prospective, randomized, double-blind, placebo-controlled trial, which was conducted at the university hospitals of berlin and frankfurt, germany, eligible patients were randomly assigned to treatment with either the combination of ifn-a a plus amantadine sulphate (n ) or ifn-a a plus placebo (n ). randomization was performed with a random number generator in ®xed blocks of four with a ratio of : . all patients were treated with miu ifn-a a (roferon a Ò , hoffmann la-roche ag, grenzach-wyhlen, germany) thrice weekly subcutaneously for weeks followed by miu ifn-a a thrice weekly subcutaneously for additional weeks (fig. ) . a daily dose of mg amantadine sulphate (infex Ò , merz + co. gmbh & co, frankfurt/m., germany) or matched placebo were given orally twice daily for weeks. treatment was continued only in patients with undetectable serum hcv-rna (amplicor hcvä, roche diagnostic systems, branchburg, nj; lower detection limit: copies/ml) at treatment week [ ] . the follow-up period was weeks in all patients. clinical examination, haematological and biochemical tests were performed within ± weeks before treatment, at initiation of treatment, every weeks for the ®rst weeks of treatment, and every weeks thereafter until the end of treatment. during the follow-up period patients were evaluated , and weeks after the end of treatment. serum hcv-rna was determined in all patients quantitatively before treatment (amplicor monitor hcv tm , version . , roche diagnostic systems, branchburg, nj) and thereafter qualitatively at treatment weeks , , and , and at the end of the follow-up period. genotyping was performed by a reverse hybridization assay (inno lipa hcv ii, innogenetics, gent, belgium) [ ] . informed written consent was obtained from all patients before enrolment. the study was approved by the local ethics committees for medical research of the participating study centres and performed according to the declaration of helsinki, the german drug law, and the ich guidelines of`good clinical practice'. individual emotional and psychological states were determined at baseline, at treatment week , and at the end of the weeks follow-up period by a german adapted and validated pro®le of mood states (poms) scale assessing four factor scores for depression, fatigue, vigor and anger [ , ] . at the same time points quality of life was evaluated by an`everyday life' questionnaire [ , ] . this german-validated questionnaire, related to the sf- health survey, assesses the following six subscales of health-related quality-of-life for subjective health: body (e.g. make demands on body, concentrate on a task), mind (e.g. cope with illness, accept oneself), everyday life (e.g. solve daily problems, personal hygiene), social activity (e.g. get along with family, count on partner's help), zest for life (e.g. enjoy life) and medical treatment (e.g. believe in success of treatment). sum scores were determined for every patient. missing items were replaced by the mean for the nonmissing items of the subscales. however, missing questionnaires were not replaced. the primary ef®cacy endpoint of the present study was de®ned as a sustained virological response with undetectable serum hcv-rna by qualitative serum hcv-rna at the end of the follow-up period. secondary ef®cacy parameters included the virological response at treatment weeks , and as well as the corresponding biochemical response de®ned as a normalization of alt. as an additional secondary ef®cacy parameter changes of hrqol during and after treatment were evaluated. based on an intent-to-treat analysis, primary ifn-a nonresponders, who received at least one dose of the study medication, were included in the primary statistical analysis. virological response rates of the two treatment arms were compared with a one-tailed fisher's exact test on a signi®cance level of a %. corresponding biochemical response rates were analysed with a two-tailed mann±whitney u-test (a . ). statistical procedures were performed using sas procedures (version . ). for the evaluation of hrqol, the alt, alanine aminotransferase; ast, aspartate aminotransferase; c-gt, c-glutamyltransferase. *mean sd. normal reference ranges: ± u/l for alt, ± u/l for ast, ± u/l for c-gt, and ± . lmol/l for bilirubin. àclassi®cation according to desmet et al. [ ] . differences of the sum scores between the two treatment groups at treatment weeks and , and at the end of follow-up were compared with a two-tailed wilcoxon rank sum test (a . ) using statxact . an initial virological response with undetectable serum hcv-rna (< copies/ml) at treatment week was achieved in ®ve of ( %) and in eight of ( %) patients treated with ifn-a a and amantadine sulphate or placebo, respectively ( table ). according to the protocol the antiviral therapy was discontinued at treatment week in patients because of detectable serum hcv-rna at treatment week . because of breakthrough events during treatment weeks ± , a virological end-of-treatment response was found only in one of ( %) patients receiving combined ifn-a a/amantadine sulphate treatment and in four of ( %) patients on ifn-a a and placebo. at the end of follow-up, a sustained virological response was observed in no patient treated with ifn-a a and amantadine sulphate and in two patients treated with ifn-a a plus placebo. initial biochemical responses with alt values within the normal range at treatment week were comparable in both groups ( % for each treatment arm, table ). a sustained according to the study protocol antiviral treatment was discontinued at week in of patients with detectable serum hcv-rna at treatment week . therefore, healthrelated quality of life during treatment was evaluated in all patients at treatment week . assessment of health-related quality of live during treatment revealed a deterioration of the mean of all four factor scores of the poms scale in the patients treated with ifn-a plus placebo and in three of the four factor scores (depression, fatigue and anger) in patients treated with combined ifn-a/amantadine sulphate (fig. a) . the extent of the observed impairment of the poms scale was less pronounced for the subscales depression and fatigue in patients treated with ifn-a a plus amantadine sulphate. at the end of follow-up, a sustained improvement of the mean factor scores for depression (p n.s.), fatigue (p . ), and vigor (p . ) in comparison with baseline levels was observed only in patients receiving combined treatment with ifn-a a plus amantadine sulphate. irrespective of treatment, the evaluation of the`everyday life' questionnaire at treatment week showed an impairment in the means of most subscales in our patients compared with the corresponding pretreatment scores (fig. b) . at the end of the follow-up period, a sustained improvement in the means of all subscales was observed only in patients receiving treatment with ifn-a plus amantadine sulphate. in the group of patients treated with ifn-a plus placebo the means of all subscales at the end of the follow-up period was worse than the corresponding pretreatment scores. adverse events (n ) occurred in a similar frequency in both treatment arms and were mostly related to ifn. the spectrum and frequency of the observed adverse events in relation to treatment are listed in detail in table . serious adverse events were not observed in this study. however, one patient treated with ifn-a a plus placebo was discontinued prematurely from antiviral treatment at week due to depression. a slight decrease of mean leukocyte and thrombocyte count was found in most patients irrespective of the treatment group from baseline to treatment week ( . . / nl to . . /nl, and /nl to /nl, respectively). at the end of the follow-up period leukocyte and thrombocyte counts returned to baseline levels with no signi®cant differences between the treatment groups. hyperthyroidism developed in one patient treated with ifn-a a alone and in ®ve patients receiving ifn-a a plus amantadine sulphate. in one patient, diabetes mellitus developed and in another patient diabetes mellitus was aggravated during treatment. both patients received ifn-a a plus amantadine sulphate. during the last decade ifn-a therapy has been established as the standard treatment for chronic hepatitis c. sustained . with respect to the low sustained response rates to retreatment with ifn-a alone or in combination with ribavirin in ifn-a nonresponder, the need for the development of alternative, effective retreatment regimens in this subgroup of patients became apparent. in a published pilot study, a bene-®cial therapeutic effect of six months amantadine retreatment in ifn-a nonresponders with chronic hepatitis c was reported with a sustained biochemical and virological response in four of patients ( %) after a week followup period [ ] . the postulated antiviral effect of amantadine was supported in vitro by a dose-dependent decrease of hcv-rna in the supernatant of isolated blood mononuclear cells from patients with chronic hepatitis c cultured with amantadine [ ] . however, in vivo determination of hepatitis c viral load failed to show a direct synergistic antiviral effect of amantadine sulphate during combined ifn-a/amantadine treatment in previously untreated patients with chronic hepatitis c [ ] . subsequently, mainly uncontrolled clinical trials of amantadine monotherapy in small cohorts of nonresponders with chronic hepatitis c showed con¯icting results with sustained virological response rates varying from % to %. as indicated by a recently published pilot study, the combination of ifn-a with amantadine may lead to an improvement of virological response rates in nonresponders with chronic hepatitis c [ ] . the present study is the ®rst randomized, double-blind, placebo-controlled trial evaluating the ef®cacy of retreatment with ifn-a plus amantadine sulphate compared with ifn-a alone in previous ifn-a nonresponders with chronic hepatitis c. considering the demographic, biochemical, virological and histological patients' characteristics, signi®cant differences between both treatment groups were not present. the sustained virological response rates were similar in both treatment groups. thus, with respect to the results of the present controlled study, the addition of amantadine sulphate to ifn-a does not improve the sustained virological response rates in nonresponders with chronic hepatitis c. these results con®rm a larger controlled trial in treatment naive patients with chronic hepatitis c which also showed no bene®cial virological effect of the addition of amantadine sulphate to ifn-a treatment in these patients [ ] . in contrast, a recently published study in nonresponders, treated with ifn-a, ribavirin and amantadine or ifn-a and amantadine, could demonstrate increased virological response rates in nonresponders with chronic hepatitis c who received amantadine [ ] . however, these data have to be con®rmed by larger randomized, double-blind, placebo-controlled trials in nonresponders with chronic hepatitis c. despite a similar number of adverse events in both treatment groups, additional analysis of the individual emotional and psychological state as well as the evaluation of health-related quality of live revealed an improved outcome in patients treated with the combination of ifn-a plus amantadine sulphate compared with those patients receiving ifn-a alone. in the poms scale all four subscores deteriorated during treatment with ifn-a alone in comparison with the corresponding baseline levels and remained impaired during the follow-up period. the combined retreatment with ifn-a plus amantadine sulphate led to a smaller reduction of three of the four subscores, i.e. depression, vigor, and fatigue during treatment and interestingly, to a sustained improvement of the corresponding subscores in comparison with baseline levels at the end of the week follow-up period. a similar improvement during amantadine sulphate treatment has also been shown for patients with central nervous disease, including multiple sclerosis and parkinson's disease [ ] . in the present study, analysis of the sf- healthrelated`everyday life' questionnaire showed a sustained improvement of all six subscores in the group of patients treated with ifn-a plus amantadine sulphate at the end of the follow-up period in comparison with the corresponding baseline scores while ®ve of six subscores remained impaired in patients treated with ifn-a alone. a sustained improvement for the poms scale and the sf- health-related everyday life' questionnaire has also been reported in a previously published study in naive patients with chronic hepatitis c receiving ifn-a/amantadine sulphate combination therapy compared with those treated with ifn-a alone [ ] . in summary, retreatment with ifn-a in combination with amantadine sulphate does not increase the low sustained virological response rates of ifn-a monotherapy in primary ifn-a nonresponders with chronic hepatitis c, but may lead to a sustained improvement of health-related quality of life. the recently suggested improved antiviral effect of triple retreatment with ifn-a, ribavirin and amantadine in previous ifn-a nonresponders has to be con®rmed in larger randomized, double-blind, 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antiviral therapy as a new option for patients with interferon nonresponsive chronic hepatitis c fatigue therapy in multiple sclerosis: results of a double-blind, randomized, parallel trial of amantadine, pemoline, and placebo key: cord- -lodjcj c authors: zhang, xuming; hinton, david r.; cua, daniel j.; stohlman, stephen a.; lai, michael m.c. title: expression of interferon-γ by a coronavirus defective-interfering rna vector and its effect on viral replication, spread, and pathogenicity date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: lodjcj c abstract a defective-interfering (di) rna of the murine coronavirus mouse hepatitis virus (mhv) was developed as a vector for expressing interferon-γ (ifn-γ). the murine ifn-γ gene was cloned into the di vector under the control of an mhv transcriptional promoter and transfected into mhv-infected cells. ifn-γ was secreted into culture medium as early as hr posttransfection and reached a peak level (up to u/ml) at hr posttransfection. the di-expressed ifn-γ (de-ifn-γ) exhibited an antiviral activity comparable to that of recombinant ifn-γ and was blocked by a neutralizing monoclonal antibody against ifn-γ. treatment of macrophages with de-ifn-γ selectively induced the expression of the cellular inducible nitric oxide synthase and the ifn-γ-inducing factor (igif) but did not affect the amounts of the mhv receptor mrna. antiviral activity was detected only when cells were pretreated with ifn-γ for hr prior to infection; no inhibition of virus replication was detected when cells were treated with ifn-γ during or after infection. furthermore, addition of ifn-γ together with mhv did not prevent infection, but appeared to prevent subsequent viral spread. mhv variants with different degrees of neurovirulence in mice had correspondingly different levels of sensitivities to ifn-γ treatmentin vitro,with the most virulent strain being most resistant to ifn-γ treatment. infection of susceptible mice with de-ifn-γ-containing virus caused significantly milder disease, accompanied by more pronounced mononuclear cell infiltrates into the cns and less virus replication, than that caused by virus containing a control di vector. this study thus demonstrates the feasibility and usefulness of this mhv di vector for expressing cytokines and may provide a model for studying the role of cytokines in mhv pathogenesis. ). resistance to ifn-g may lead to incomplete viral clearance and contribute to the establishment of persis-interferon-g (ifn-g) is a pleiotropic cytokine produced tent infection (moskophidis et al., ) . by contrast, ifnby activated cd / and cd / t cells and natural killer g is also involved in inflammatory processes. ifn-g incells (trinchieri and perussia, ; pestka and langer, duces the expression of many other inflammatory cyto- ; ijzermans and marquet, ) , which exerts both kines, such as interleukin- (il- ) and tumor necrosis antiviral and immunomodulatory effects. these include factor (tnf), and acts synergistically with these cytokines the activation of mononuclear phagocytes, enhancement (wong and goeddel, ) . the multitude of immunoof the generation of oxygen-free radicals, modulation of modulatory effects of ifn-g makes it a particularly interclass i and ii major histocompatability complex (mhc) esting cytokine for studying viral pathogenesis. in the antigen expression, and promotion of differentiation of central nervous system (cns), no cells constitutively exboth t and b cells (for reviews, see references by pestka press ifn-g. during encephalomyelitis, for example as and langer, ; benveniste, ) . it plays an ima result of mouse hepatitis virus (mhv) infection, actiportant role in the early phase of many viral infections vated nk cells and t cells which pass through the blood- (wheelock, ; wong and goeddel, ; leist et al., brain barrier into the cns express ifn-g (bukowski et ; klavinskis et al., ; feducchi and carrasco, al., ; pearce et al., ) . in addition to its effects on ; ramsey et al., ; heise and virgin iv, ; mononuclear cells, ifn-g acts upon cells of the cns, rodriguez et al., ) , inhibiting the replication of a varisuch as astrocytes, microglia, and macrophages (benety of viruses prior to activation of antiviral effector cytoveniste, ) . toxic t lymphocyte (ctl) or antibodies. because of its mhv, a murine coronavirus, causes a variety of disantiviral activity, ifn-g has been implicated in virus cleareases in rodents, such as hepatitis, enteritis, and neuroance and resolution of viral infection (ramshaw et al., logical diseases, depending on the viral strain (cheever et al., ; gledhill and niven, ; ishida et al., ) . lination (stohlman et al., ; lai and stohlman, ) . may allow studies of the interaction between mhv and the host's immune system by expressing immunoregula-the dl variant derived from the parental jhmv causes an acute, fulminant, necrotizing encephalomyelitis with tory proteins at the foci of viral infection. minimal or no demyelination. by contrast, the neuroattenuated variant . -v- derived from dl produces a nonfa-materials and methods tal encephalomyelitis with extensive demyelination virus and cells (fleming et al., (fleming et al., , wang et al., ) . disease outcome also depends on the genetic background, the the following virus strains were used in this study: the developmental stage, and the immunological status of neuropathogenic mhv strain jhm isolate (dl), which is the host. previous studies have shown that immunocoma large plaque variant derived from the parental jhm petent mice infected with mhv exhibited increased exstrain (stohlman et al., ) ; the small plaque variant pression of a number of cytokines, including il- , il- , ds (stohlman et al., ) ; the neutralization-escape mu-tnf-a, and ifn-g, in the cns at the time of viral cleartant . -v- (fleming et al., ; wang et al., ) , and ance (pearce et al., ) . however, the role of these strain a , which is both neurotropic and hepatotropic. cytokines in mhv pathogenesis is not fully understood. the murine astrocytoma cell line (dbt) (hirano et al., for example, it has been suggested that ifn-g may not ) and j . macrophage cell line (obtained from be necessary for induction of the mhc class i molecules the american type culture collection) were used for in on neural cells in vivo (pearce et al., ) , a prerequisite vitro experiments. dbt cells were also used for plaque to ctl-mediated clearance (stohlman et al., ) . howassay. ever, ifn-g treatment ameliorates mhv-induced disease (smith et al., ) , suggesting that either the antiviral plasmid construction role or the immunomodulatory role of ifn-g is a critical a previously constructed plasmid p cat (liao and component of mhv infection. lai, ) , which contains the plasmid bluescript (pro-mhv contains a single-strand, positive-sense rna gemega) sequence with a cat gene inserted behind an ig nome of kb (lee et al., ) . it undergoes rapid recombisequence in the disse cdna (makino et al., a) , was nation, probably due to its large rna genome and the used as the basic di vector. for cloning the murine ifnspecial properties of its rna-dependent rna polymerase g gene into the di vector, a cdna fragment containing . similarly, defective interfering (di) rnas are the complete ifn-g gene (kindly provided by dr. j. a. frequently generated in mhv-infected cells. recently, re-frelinger, university of rochester) was generated by combinant di rnas have been developed which can replipolymerase chain reaction (pcr) using a pair of primers. cate in the presence of a helper mhv (makino et al., a, the sense primer ( -taactagtaatctaatctaa- ; van der most et al., ) . we have modified an mhv actttaaggaatgaacgctacacact- ) contains a re-di rna and developed an expression vector. this di rna striction enzyme spei site (underlined), the coronavirus contains both the -and the -ends, an internal region of intergenic sequence (in boldface), and the first nucleothe parental mhv genome (makino et al., b) , and an tides of the ifn-g open reading frame (orf). the intergenic (ig) sequence, which is a recognition signal for antisense primer ( -tcagaattcaatcagcagcgasubgenomic mrna transcription, followed by an exoge-ctcct- ) contains the last nucleotides of the ifn-g nous gene. upon transfection of this di rna into mhv-orf and a restriction enzyme ecori site (underlined). infected cells, a subgenomic mrna is synthesized and the after restriction enzyme digestion of the pcr products inserted gene expressed. this system has been used to with spei and ecori, a . -kb cdna fragment was puriexpress the chloramphenicol acetyltransferase (cat) profied by low-melting-point agarose gel electrophoresis tein and the coronavirus structural protein hemagglutinin/ and directionally cloned into the spei and ecori sites of esterase (he) in mhv-infected cells (liao and lai, ; p cat, resulting in pde-ifn-g (fig. a) . the resulting liao et al., ) . these proteins are expressed only in construct contains the ifn-g gene placed behind the ig infected cells during virus replication, thus providing some sequence between genes and (ig ) of mhv. degree of targeted gene expression. furthermore, the expressed he protein can be incorporated into virus particles, rna transcription and transfection and the expression can be detected in serial virus passages (liao et al., ) . thus, this di rna expression plasmid dna (pde-ifn-g) was linearized with xbai, and rna was transcribed in vitro using t rna polymer-system provides an alternative to an infectious full-length cdna clone, which is still not available, for studying the ase according to the manufacturer's recommended procedure (promega). rna transfection was carried out molecular biology and pathogenesis of coronaviruses. in the present study, we have used this di rna system using the dotap method (boehringer-mannheim) as described previously (zhang et al., ) . briefly, mono-to express the murine ifn-g gene. the expressed ifng exhibited antiviral activity, prevented virus spread in layers of dbt cells grown at approximately % confluence in -mm petri dishes were infected with mhv at vitro, and altered viral pathogenesis in mice. this system de-ifn-g rna. following centrifugation at g for min, supernatants were tested for ifn-g using a sand-cells were washed with phosphate-buffered saline (pbs) and covered with ml of prewarmed eagle's minimum wich elisa as previously described (cua et al., ) . r - a (anti-ifn-g) (american type culture collection) essential medium (mem) containing % newborn calf serum (intragen). five to ten micrograms of in vitro tran-serum-free hybridoma supernatant was used to coat well plates. biotinylated xmg- . (anti-ifn-g) was ob-scribed rnas were mixed slowly with ml of dotap (boehringer-mannheim) in hbs buffer ( mm hepes; tained from pharmingen. avidin-peroxidase and o-phenylenediamine (opd) were obtained from sigma chemical mm nacl; ph . ), and incubated at room temperature for min. the mixture was then added to the cell co. recombinant ifn-g (rifn-g) (zymogen) was used as elisa standard, and the concentration of ifn-g is re-culture. the final concentration of dotap was mg/ml. ported in international units per milliliter (u/ml). enzyme-linked immunosorbent assay (elisa) for ifn-g mhv replication in the presence of ifn-g to quantitate expression of ifn-g, medium was collected at , , , , , and hr posttransfection from dbt cells were seeded at a concentration of cells per well into -well plates and incubated for hr dbt cells infected with jhm or a and transfected with at Њ in mem containing % newborn calf serum. j . extension. pcr products were analyzed by agarose gel electrophoresis. cells were seeded at a concentration of cells per well into -well plates and incubated for hr at Њ in dulbecco's modified mem (dmem) containing % dot blot analysis fetal calf serum. cells were treated with various concen-rt-pcr products were quantitated using the dot blot trations of the di-expressed ifn-g (de-ifn-g) or rifn-g method previously described (murphy et al., ; cua and infected with viruses at an m.o.i. of , . , . , or et al., ) . briefly, pcr-amplified cdna ( ml) was . . after virus adsorption for hr, the respective medenatured in ml of denaturing solution ( . n naoh dium with or without ifn-g was added and the cells were and mm edta) for min and neutralized by the incubated for the indicated periods of time. addition of an equal volume of m tris-hcl, ph . . samples were transferred to a nylon membrane via a isolation and detection of intracellular mrnas minifold i dot blot apparatus (schleichel and schuell), to study the effects of ifn-g treatment on the expresand the wells were washed with ssc ( . % sodium sion of cellular genes [inducible nitric oxide synthase chloride, . % sodium citrate). membranes were air-dried (inos), interferon-g-inducing factor (igif), and mhv reand the cdna was fixed using a stratalinker uv oven ceptor (mhvr)], macrophage cells (j . ) were grown (stratagene). following prehybridization [ % ssc, to % confluence in -mm petri dishes and then treated . % sodium dodecyl sulfate (sds), . mg/ml salmon with medium from cells expressing de-ifn-g or de-cat, sperm dna] at room temperature for min, p-labeled both of which had been irradiated with uv to inactivate specific probes (table ) were added. following hybridhelper virus. at and hr after treatment, cells were ization at Њ, the membranes were washed three times collected and intracellular rna was isolated as dewith ssc containing . % sds for min, air dried, scribed previously (zhang et al., ) . to determine the and scanned on an ambis radioanalytic imaging system effects of mhv infection on the expression of cellular (ambis systems). total counts of each duplicate sample genes, j . cells were infected with mhv-jhm virus at for inos, igif, and mhvr at each time point were noran m.o.i. of . at hr after ifn-g treatment. rna was malized to the control hprt. the blots were further autoisolated at hr postinfection. the rna samples were radiographed. used for synthesis of cdnas by reverse transcription (rt) with random priming hexamers (boehringer-mannheim). mice to detect individual genes, cdna pools were subjected to pcr amplification using gene-specific primers (table c bl / mice were purchased at weeks of age from the jackson laboratory. mice were infected with ). the gene encoding the housekeeping enzyme hypoxanthine phosphoribosyltransferase (hprt) was used as pfu of a expressing de-ifn-g or de-cat. preliminary experiments showed no difference in virus replication in an internal control. the pcr was performed for cycles under the following condition: Њ for min for denatur-the cns comparing parental a and a virus containing the de-cat vector. ation, Њ for min for annealing, and Њ for min for virus titers in the cns were determined by homogenization of half of the brain in pbs followed by plaque assay on monolayers of dbt cells as previously described (stohlman et al., ) . the remaining half of the brains were fixed in clark's solution ( % ethanol, % glacial acetic acid), embedded in paraffin, and stained with hematoxylin and eosin to examine the extent of encephalitis or with the immunoperoxidase method (vectastain abc kit; vector laboratories, burlingame, ca) using the anti-nucleocapsid monoclonal antibody j. . . (fleming et al., ) to determine the percentage of cuture medium from dbt cells infected with jhm virus and transfected virus-infected cells. with either de-ifn-g or de-cat rna was harvested at various time points posttransfection, and virus titers were determined by plaque assays. expression of ifn-g using an mhv di rna vector cell metabolism prior to infection or it may be that interferon acts at an early stage of viral replication. the murine ifn-g gene was cloned into the mhv di to distinguish these possibilities, the culture medium rna vector (liao et al., ) under the control of the harvested from jhm-infected and de-ifn-g-transfected mhv ig sequence. the resulting rna, de-ifn-g rna, cells late in infection was used to infect dbt cells. this was transfected into mhv-infected cells, and the producmedium contained not only jhm virus but also ifn-g tion of ifn-g in the culture medium was detected by ( u/ml) (fig. ) . therefore, ifn-g was present through-elisa. as shown in fig. b , when mhv-jhm was used out the infection, beginning with the initiation of viral as helper virus, ifn-g was secreted into the medium ( infection. no significant differences in virus titer released u/ml) as early as hr posttransfection and increased from the de-ifn-g-and de-cat-infected cells were dewith time. at hr posttransfection, when cell monotected (both yielded approximately pfu/ml) (data not layers were completely lysed, the amount of ifn-g shown). thus, ifn-g has little antiviral effect even when reached approximately u/ml. when a was used present at the initiation of viral infection. as helper virus, the production of ifn-g was detected at in view of the known mechanisms of action of ifn-a u/ml at hr posttransfection and reached a maximum and -b, whose antiviral activities require preadsorption (approximately u/ml) earlier (at hr posttransfecto cells prior to viral infection (bianzani and autonelli, tion) (fig. c) , consistent with the observation that a ), we examined the effects of pretreatment of cells replicates faster than jhm. these results indicated that with ifn-g prior to infection. for this study, the culture mhv di vector can be used for the production of a semedium from jhm-infected and de-ifn-g-transfected creted cytokine during mhv infection in vitro. cells was uv-irradiated to inactivate infectious virus and then used as a source of ifn-g to pretreat dbt cells. twenty-four hours later, cells were infected with jhm or replication in vitro a virus at m.o.i.'s ranging from . to . in the continual presence of de-ifn-g. virus titers were deter-ifn-g exerts multiple biological functions both in vitro and in vivo (trinchieri and perussia, ; pestka and mined at hr postinfection. as shown in fig. a , de-ifng exhibited a slight inhibitory effect on jhm replication langer, ), but its effects on coronavirus infections have not been extensively examined. we first determined (approximately log reducation in virus titer), when an m.o.i. of . was used; similar results were obtained whether di-expressed ifn-g had antiviral effects on helper viral replication. virus titers in the medium of dbt with a virus (fig. a) , suggesting that pretreatment of cells with ifn-g prior to viral infection induces an antiviral cells infected with jhm and transfected with de-ifn-g rna were determined at various time points after infec-state. this inhibitory effect was less pronounced when higher m.o.i.'s were used (data not shown), suggesting tion and compared to de-cat rna-transfected cells. figure shows that the virus titers in the presence of de-that the observed antiviral activity was weak and could be overcome by a higher virus titer. ifn-g were lower by approximately half a log compared to cultures transfected with the de-cat rna. this differ-to further establish that the antiviral effect was due to the specific effects of ifn-g, the uv-inactivated de-ifn-ence was small but reproducible, suggesting that ifn-g exerts at most a weak antiviral effect. the absence of g preparation was preincubated for hr with a hamster neutralizing monoclonal antibody specific for rifn-g. significant anti-viral effect of ifn-g in this system could be due to the requirement for interferon to modify host antiviral effects were completely blocked by this treat- the uv-irradiated supernatants were used either as a source of ifn-g or as a control (cat) to pretreat cells for hr, and the cells were then infected with either jhm or a at an m.o.i. of . . after virus adsorption, cells were incubated with the same supernatants for hr, and the virus titers in culture medium at hr postinfection were determined by a standard plaque assay. (b) neutralization assay of ifn-g. both uv-irradiated supernatants (ifn-g and cat) were incubated with mg/ml of a hamster anti-ifn-g neutralizing monoclonal antibody for hr at room temperature prior to being used for pretreatment of cells. subsequent procedures were the same as in (a). ment (fig. b ), demonstrating that ifn-g, but not the repli-log , similar to the data obtained with dbt cells. thus, the absence of strong antiviral effects of ifn-g is not cation of the di vector itself, was responsible for the antiviral activity. these combined results suggest that due to nonresponsiveness of cells to ifn-g. ifn-g has a weak antiviral effect, which was evident only di rna-expressed ifn-g prevents virus spread when cells were pretreated with ifn-g prior to infection. the relatively weak antiviral effects of ifn-g also could the results described above indicated that antiviral be due to the possibility that dbt cells do not respond effects of ifn-g could be demonstrated only when cells well to ifn-g. since it is known that macrophages are were pretreated with ifn-g before viral infection and particularly sensitive to ifn-g treatment (ijzermans and when a low m.o.i. was used. they suggested the possibil-marquet, ), we further determined the inhibitory efity that ifn-g could prevent virus spread, if virus initially fects of ifn-g on mhv replication in an mhv-susceptible infects only a small number of cells. to establish an in macrophage cell line (j . ). j . cells were previtro model for studying the potential effects of ifn-g in treated with various concentrations of rifn-g for hr preventing virus spread, uv-irradiated culture medium before and throughout virus infection. as shown in fig. from de-ifn-g-transfected cells, which contained ifn-g , both a and jhm were inhibited by rifn-g by to at u/ml, was mixed with a very low titer of jhm virus at approximately one infectious particle in each well of a -well plate. cells were observed for cytopathic effects daily for days and the number of fusion plaques was counted. results of these experiments are presented in table . the number of plaques increased more slowly when the de-ifn-g was present (for example, from plaque on day to plaques on day ), as compared to those in the control wells, in which diexpressed cat preparation was used (i.e., from plaque on day to plaques on day and too numerous to count by day ) (table ) . initially, the plaque sizes in the presence of ifn-g were indistinguishable from those of the control wells (data not shown); however, by day or postinfection, while all plaques in the ifn-g-treated cultures remained of uniform size, plaques in the absence of ifn-g became numerous and heterogeneous it has been suggested that ifn-g induces a number no. of plaques c on of cellular proteins and enzymes which either act as t cells (okamura et al., ) . mhvr is a member of virus. one milliliter of each culture medium was then mixed with jhm the biliary glycoprotein (bgp)/carcinoembryonic antigen virus and added to the cell monolayers, so that an average of pfu per well was present. (cea) family and serves as a receptor for mhv infection b each sample was quadruplicated in wells of a -well plate. (williams et al., ) . treatment of cells with di-exc plaques were counted in the liquid medium using a light micropressed ifn-g for hr increased the expression of inos scope. and igif mrnas. mhv infection did not affect the expresd uc, uncountable due to extensive cytopathic effects and detachment of cells. due to the rapid spread of progeny virus before ifn-g exhibited its antiviral effect (data not shown). similar results were obtained when various concentrations of rifng ( , , and u/ml) were used, suggesting that u/ml rifn-g is sufficient to prevent virus spread in vitro (data not shown). sensitivity of different jhm variants to ifn-g treatment in vitro was assessed in an effort to determine whether the ifn-g sensitivity correlates with the pathogenicity of the virus in vivo. three jhm variants with different degrees of neurovirulence were used: dl (ld - pfu), ds (ld - pfu), and . -v- (ld - , pfu) (stohlman et al., (stohlman et al., , fleming et al., fleming et al., , . dl causes little demyelination and infects predominantly neurons whereas variant . -v- causes extensive demyelination and infects predominantly glial cells with a particular tropism for oligodendrocytes. variant ds causes less demyelination than variant . -v- . dbt cells pretreated with ifn-g ( u/ml) for hr were infected, and the same concentrations of ifn-g were maintained throughout the infection. at hr postinfection, culture medium was collected and virus titer determined by plaque assay. as shown in fig. , a reduction of approximately . log in pathogenicity in vivo, groups of c bl/ mice were infected with pfu of a virus containing either de-ifn-g small numbers of perivascular and subarachnoid mononuor de-cat. preliminary experiments showed no difference clear cells, the brains of the de-ifn-g-expressing group in virus replication in cns between mice infected with pashowed widespread meningomyeloencephalitis with promrental a virus and those infected with a -de-cat (data inent perivascular cuffs, infiltration of mononuclear cells not shown). at days postinfection, four mice in each group into the parenchyma, and subarachnoid infiltrates (fig. ) . were sacrificed and the brains were examined for mhv this result supports the immunostimulatory effects of ifntiter and histological changes. the remaining mice in each g. although this experiment used only a small number of group were monitored daily for survival. table shows that mice, the data suggest that expression of immunomodulathere was approximately . log less virus in the cns of tory molecules from the di vector can alter the pathogenemice infected with a expressing de-ifn-g vector comsis of mhv-induced disease. pared to the mice infected with a expressing de-cat vector. correspondingly, all the mice infected with de-ifng-expressing a survived the entire -day observation the molecular basis for the relative ifn resistance of different mhv strains is not yet known. previous studies this study demonstrates that the mhv di rna system have shown that the neutralization-escape mutant . -vcan be utilized as a vector to express the ifn-g gene of jhm strain has a single nucleotide mutation at posiand that the ifn-g protein is translated and secreted tion of the s gene, which results in a leucine to from infected cells as a biologically active molecule. phenylalanine substitution (wang et al., ) . whether these data represent the first successful attempt to exthis single mutation affects the sensitivity of the virus to press a mammalian cellular gene product using a coro-ifn-g remains unclear. in lymphocytic choriomeningitis navirus di rna vector. thus far, we have demonstrated virus, resistance of various virus strains to ifn-a/b or the feasibility of this di rna system for expressing a ifn-g in vitro correlates with their ability to establish prokaryotic bacterial gene cat (liao and lai, ) , a persistent infections in adult immunocompetent mice viral structural protein gene he (liao et al., ) , and (moskophidis et al., ) . one possibility is that ifn the mammalian cellular gene ifn-g (this report). these resistance allows enhanced viral replication and spread, studies showed a broad range of usage of this di rna facilitating exhaustion of antiviral ctl, thereby resulting system for expressing various genes of interest. in virus persistence. whether mhv utilizes a similar currently, an infectious, full-length cdna clone of mhv mechanism to modulate its infection in mice is an inter-rna is not available; therefore, it is difficult to unequivoesting issue. correlation between ifn resistance and cally elucidate the mechanism of pathogenesis of mhv viral pathogenicity has also been documented for meaat the molecular level. the development of a di rna sles virus, adenovirus, and herpes simplex virus type i expression system thus provides an alternative ap- (carrigan and kehl-knox, ; su et al., ; kalvakoproach, allowing the expression of both viral and cellular lanu et al., ) . genes to be manipulated. further, this system allows the in vitro experiments showed that the di-expressed expression of heterologous gene products at the site of ifn-g had inhibitory effects on virus spread from initially viral replication. this system has an advantage over the infected cells to neighboring uninfected cells. the inhibipassive administration of cytokines for studying viral tory effect was more pronouced at a lower m.o.i., which pathogenesis, since cytokines usually have a short halfapparently allowed sufficient time for ifn-g to activate life, making it difficult to maintain high local concentraan antiviral state in adjacent uninfected cells. pretreattions at the site of infection. one drawback of the di ment of cells (astrocytoma and macrophages) with ifnsystem, however, is its limited expression. the di rna g is required to induce an antiviral state (figs. and ) , cannot be packaged beyond the fourth passage in vitro consistent with previous findings from studies of primary (data not shown). we have attempted to increase retenmouse macrophages (lucchiari et al., ) and other tion of the di rna via incorporation of a packaging signal. target cells (lewis, ) . expression of both inos and however, the expression level of the gene product was igif mrna in macrophages was induced by ifn-g. howreduced; no significant retention was found (lin and lai, ever, whether these molecules mediate the antiviral ef- ). nevertheless, our data indicated that, during the fects of ifn-g is not clear. recently, it was demonstrated first several passages, the expression level of ifn-g was that inos expression did not play a significant role in such that a sufficiently high level of ifn-g can be mainthe pathogenesis of the mhv oblv strain (lane et al., tained locally at the beginning of viral infection. ). nevertheless, we can conclude from our study the virulence of several mhv variants correlates with that the antiviral effects of ifn-g are not mediated by their resistance to ifn-g treatment, suggesting that ifndown-regulation of mhvr. the precise mechanism of the g may play a role in the pathogenesis of mhv. an earlier antiviral effects of ifn-g will require additional studies, study analyzed the effects of ifn-g during jhm infection as there appears to be discordance between the antiviral using passive transfer of an anti-ifn-g-antibody (smith effects of no in vivo and its effects in vitro (lane et al., et al., ) . this treatment significantly enhanced virus ). replication and resulted in a higher mortality with de- the alteration of a neuropathogenesis by de-ifn-g creased survival times. ifn-g treatment of macrophages provides further support for the significance of ifn-g from a/j mice rendered them partially resistant to mhv in mhv infection. inhibition of ifn-g action by passive infection, whereas the macrophages from susceptible transfer of antibody (smith et al., ) enhanced virus balb/c mice did not respond to ifn-g, suggesting that replication and increased mortality, suggesting that local the resistance of mice to mhv infection involves the production of ifn-g by infiltrating leukocytes is a critical sensitivity of macrophages to ifn-g (lucchiari et al., component of the host response to mhv infection. in ; vassao et al., a,b) . ifn-g was also shown to our experiments, the production of ifn-g by de-ifn-g be more effective than ifn-a/b in inducing an antiviral resulted in an exaggeration of the host response with state in macrophages infected with mhv (vassao et al., more prominent encephalitis, improved viral clearance, a). these reports support the notion that ifn-g may and decreased mortality. the increased encephalitis may, in turn, induce local cytokine production and ctl play a role in mhv infection. the complete , - . sequence ( kilobases) of murine coronavirus gene encoding the -sequence as an upstream cis-acting element for coronavirus sub- . genomic mrna transcription a murine virus (jhm) causing disseminated encephalomyelitive-interfering rna as an expression vector: the generation of a tis with extensive destruction of myelin. i. isolation and biological pseudorecombinant mouse hepatitis virus expressing hemagglutiproperties of the virus deletion mapping of a mouse hepatiduced th responses in experimental allergic encephalomyelitis tis virus defective interfering rna reveals the requirement of an (eae)-resistant mice: th -mediated suppression of autoimmune disinternal and discontiguous sequence for replication acquired interferon and tumor necrosis factor exert a synergistic blockade on immunity of a/j mice to mouse hepatitis virus infection: dependence the replication of herpes simplex virus defectiveruses: analysis using monoclonal antibodies to jhm (mhv- ) virus. interfering particles of murine coronavirus: mechanism of synthesis virology primary structure and translation of a defective-interfering navirus jhm selected with monoclonal antibodies experimental demyelination induced by coronavidefective-interfering rna results from intergenic site insertion isolation and characterization of two plaque morphology variants of the jhm neurotropic strain mouse hepatitis virus-specific cytotoxic t lymphocytes protect from lethal infection without eliminating virus from the central ingitis virus to alpha/beta interferon and to gamma interferon detection of in vivo expression of interleukin- using a semi-quantitative polymerherpes simplex virus type i strain is associated with heightened sensitivity to alpha/beta interferon immune interferon: a pleiotropic lymphokine with multiple effects cloning of a new cytokine that induces ifn-g production by dation of coronavirus defective interfering rnas a genetic analysis of macrophage activation and specific antibodies in relation cytokine induction during t-cell-mediated clearance of mouse hepatitis virus from neurons in vivo the astrocyte is a target cell in mice persistently infected with mouse hepatitis virus, strain interferons and their actions. annu. of genetic heterogeneous mouse populations to mouse hepatitis virus infection sequence analysis of the spike protein gene of murine coronavirus variants: study of as effector molecules in the resolution of virus infection expression of cytokines by recombinant vaccinia viruses: a model leukocytes by phytohemagglutinin hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins tumour necrosis factor a and b inhibit virus replication and synergize with interferons coronavirus leader the role of gamma interferon in infection of susceptible mice with murine coronavirus, mhv-jhm gledhill, a. w., and niven, j. s. f. ( ) . latent virus as exemplified by activity. altogether, these data demonstrated that ifn-g mouse hepatitis virus (mhv). vet. rev. annotat. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] plays a critical role at least early in a infection. the haller, o. ( ) . inborn resistance of mice to orthomyxoviruses. curr.longer-term consequences of ever, cannot be definitively determined from this study heise, m. t., and virgin, iv, h. w. ( ) . key: cord- -j ot lt authors: ahmed-hassan, hanaa; sisson, brianna; shukla, rajni kant; wijewantha, yasasvi; funderburg, nicholas t.; li, zihai; hayes, don; demberg, thorsten; liyanage, namal p. m. title: innate immune responses to highly pathogenic coronaviruses and other significant respiratory viral infections date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: j ot lt the new pandemic virus sars-cov- emerged in china and spread around the world in < months, infecting millions of people, and causing countries to shut down public life and businesses. nearly all nations were unprepared for this pandemic with healthcare systems stretched to their limits due to the lack of an effective vaccine and treatment. infection with sars-cov- can lead to coronavirus disease (covid- ). covid- is respiratory disease that can result in a cytokine storm with stark differences in morbidity and mortality between younger and older patient populations. details regarding mechanisms of viral entry via the respiratory system and immune system correlates of protection or pathogenesis have not been fully elucidated. here, we provide an overview of the innate immune responses in the lung to the coronaviruses mers-cov, sars-cov, and sars-cov- . this review provides insight into key innate immune mechanisms that will aid in the development of therapeutics and preventive vaccines for sars-cov- infection. severe acute respiratory syndrome coronavirus (sars-cov- ) reportedly emerged at a live animal market in the chinese city of wuhan is currently causing a pandemic and negatively affecting global health ( ) ( ) ( ) . there are more than million confirmed sars-cov- infections with an mortality rate that widely varies by country and region ( ) . even in industrialized countries, sars-cov- led healthcare systems approach the brink of collapse by overwhelming their capacity and straining resources. governments and local leaders ordered the shutdown of cities, regions, countries leading to massive disruptions in the local and global economy. unlike previous coronavirus (cov) infections, the rapid global spread, high transmission rate, longer incubation time, and disease severity of sars-cov- requires a better understanding of the epidemiology and immunopathogenesis of this viral outbreak in order to learn from this experience and to manage future pandemics. sars-cov- is a highly pathogenic cov ( ) (case-fatality rate of . - . %) ( , ) that is related to severe acute respiratory syndrome cov (sars-cov) (case-fatality rate of - %) and the middle east respiratory syndrome cov (mers-cov) (case-fatality rate of . %), see also table ( , ) . sars-cov, sars-cov- and mers-cov target the lower respiratory system, causing respiratory illnesses, including severe pneumonia, acute lung injury (ali) and acute respiratory distress syndrome (ards) ( , ) . sars-cov- infection results in higher hospitalization rates in the elderly (> ) and persons with pre-existing conditions including hypertension, diabetes and obesity compared to rates among younger populations without pre-existing conditions ( table ) ( , ) . in addition to an age disparity, males with covid- appear to have higher risk for worse outcomes and death ( , ) . epidemiological research of the sars and mers infections also showed that males had a higher mortality rate than females ( ) ( ) ( ) . while sars-cov- is a novel coronavirus, several important insights have already been made about its basic mode of transmission. virus particles are inhaled in respiratory droplets expelled from the airways of infected individuals. angiotensinconverting enzyme (ace ), expressed on the ciliated bronchial cells, endothelial cells, and type i and ii alveolar cells, is the host receptor for cell entry into the respiratory tract by both sars-cov- and sars-cov ( table ) ( ) ( ) ( ) ( ) . the spike protein (s) of cov is responsible for the entry of the virus into the target cell (figure ) ( , ) . ace is a type i transmembrane metallocarboxypeptidase that plays a vital role in the renin-angiotensin system (ras) ( , ) , which in turn is critical in maintaining blood pressure homeostasis as well as fluid and salt balance in mammals. ace is found in vascular endothelial cells, in the renal tubular epithelium, and in leydig cells of the testes ( ) . studies have shown that ace is expressed in gastrointestinal (gi) tissues, making it a potential site for harboring sars-cov ( ) . this may be one of the reasons for gi pathology reported in some patients with covid- and viral shedding in stool. in contrast, mers-cov uses dipeptidyl-peptidase (dpp ) as an entry receptor, which is expressed on endothelial cells and some epithelial tissues ( table ) ( , ) . accumulating data suggest that the innate anti-viral response and adaptive immunity may contribute to a cytokine storm leading to systemic hyper inflammation and exacerbation of the disease in patients with (a) comorbidities (b) older than years of age (c) of the male sex. the exact role of the innate immune system in disease pathogenesis and prevention between the sexes and the impact of age is not fully elucidated. in addition, the potential dysregulation of the innate immune response by sars-cov and sars-cov- is not completely understood which warrants further research. the cells of the airway epithelium are the first line of defense, providing a mechanical barrier (mucociliary escalator) that expels particles and pathogens via cilia, mucus, and induced coughing ( , ) . this barrier includes cells of the pulmonary epithelium and tissue-resident macrophages and dendritic cells (dcs). the macrophages and dcs express pattern recognition receptors (prrs) that can detect molecules from pathogens (pathogen-associated molecular patterns-pamps) or molecules released from damaged cells (damage or danger-associated molecular patterns-damps) ( ) ( ) ( ) . in the lung, there are two populations of macrophages, alveolar and interstitial macrophages ( ) . in addition to these macrophages, dcs play a vital role in facilitating the host defenses against respiratory diseases ( ) ( ) ( ) . dcs can be divided into plasmacytoid (pdc) and myeloid types (mdc) ( ) ( ) ( ) . macrophages and the two dc subtypes trigger antiviral responses by generating a substantial amount of type i interferon and these cells play important roles in immune surveillance in the airways and the distal lung ( , ( ) ( ) ( ) ( ) ( ) . during steady state, the dc density in lung associated tissue declines from the trachea to the alveoli ( ) while the representation of cells in macrophage compartment seems more complex ( ) . if a virus infects airway epithelial cells, the viral rna would be sensed via intrinsic innate receptors, including rig- , mda , nlrp inflammasome, and the rna sensing tlrs and . in the case of influenza a infection, triggering the prrs causes a strong induction of type interferon (ifn) in epithelial cells ( , ) . in other viral infections, such as respiratory syncytial virus (rsv), alveolar macrophages are the predominant source of type ifns ( ). furthermore, respiratory epithelial cells and lung macrophages are capable of secreting a broad range of chemokines like il- , macrophage inflammatory protein- (mip- ), rantes and cytokines including tnf-α, il- , il- β that influence the types of immune cells being recruited to the area in response to acute viral infections ( , ) . macrophages, depending on their polarization status of either m or m , and in a similar way as airway epithelial cells, can further elicit a th or th response ( , , ) . in the case of influenza virus infection, the magnitude of epithelial cell response can be proportional to the amount of virus which result in paracrine induction of ifn λ ( ) . not only can airway epithelial cells produce a large array of cytokines/chemokines in response to an acute viral infection, but, depending on the magnitude of prr engagement and the combination of pamps and damps triggered, these epithelial cells can modulate the type of chemokines/cytokines produced and influence the influx of innate and adaptive immune cells ( , ) . the response to different viral infections is generally similar, however, the response can be tailored in timing, magnitude and the induced gene signatures in response to each virus ( ) . unlike rsv and mers-cov, which both productively infect alveolar multiple cell types in the lower respiratory tract were found to be infected, including type i alveolar epithelium, macrophages, and putative cd + oct- + stem/progenitor cells in human lungs ( ) ( ) ( ) ciliated bronchial epithelial cells and type ii pneumocytes ( , ) un-ciliated bronchial epithelial cells and type ii pneumocytes ( ) ( ) ( ) infect mostly human type i and type ii pneumocytes and alveolar macrophages ( ) respiratory, nasal, corneal and intestinal epithelial cells ( ) club cells, ciliated cells, type i and type ii alveolar cells ( ) ciliated epithelial cells of the upper and lower respiratory tract ( ) the ciliated cells of the human airway epithelium are the main target, it also infects basal cells ( ) and immune cells, such as macrophages, b cells cd + and cd + t cells ( ) upper and lower airways epithelial cell ( no specific antiviral drugs ( ) antiviral drugs may be a treatment option ( ) no antiviral agents symptomatic treatment supportive care ( ) there are no approved antiviral medications ( ) ace macrophages ( , ) , seasonal influenza and sars-cov usually lead to non-productive infections in these cells ( ) . in addition, sars-cov infection of primary monocytes yielded little virus, likely due to the suppressive effects of ifn-α ( ) . thus, the initial cell type(s) involved in propagating a viral infection intensifies the complexity of the immune response. another key factor that determines the magnitude of the immune response is the induction and rate of cell death. although related, mers-cov induces widespread cell death when compared to sars-cov in human bronchial epithelial cell cultures ( ) . however, the sars-cov open reading frame a (orf a) protein can induced necrotic cell death in a variety of cell lines ( ) . the same orf a protein can activate the nlrp inflammasome, leading to activation of nf-κb and elevated secretion of active il- β in cell culture ( ) . cytokine ( ), with slightly different cytokine/chemokine profiles. this delay in cytokine induction was confirmed in another study using the same epithelial cell lines ( ) as well as in human alveolar type ii cells ( ) . in both cell lines and primary alveolar type ii cells, sars-cov induced ifn-β, ifn-λ, cxcl , cxcl , il- , ip- , and tnf-α ( , ) . mers-cov did not induce ifn-β but induced higher level of il- transcript in cell culture. however, no difference in il- production was observed between sars-cov and mers-cov at h post-infection ( ) . this was confirmed in-vivo using a non-human primate model comparing the immune responses to sars-cov infection between young adult cynomolgus macaques ( - yrs) and older macaques ( - yrs) ( ) . interestingly, the high induction of il- was observed on a transcript level in the older animals, while the younger once showed higher levels of ifn-β transcript ( ) . in all animals, the expression of ifn-β was inversely correlated with the pathology score, supporting the role of ifn-β in controlling disease severity ( ) and introducing a potential area of research to define age disparity observed in patients infected with sars-cov- . both older age and male sex are important factors associated with high mortality of sars-cov and sars-cov- infection ( , ) . many viruses have evolved to disrupt and subvert the immune responses. a common virus that is well-known to affect the lower airway and counteract the immune response is rsv ( , ) . the rsv genome encodes non-structural proteins ns and ns that can block type ifn production and signaling in cell cultures ( ) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat and stat signaling in cell lines ( ) , whereas mers-cov m protein also suppresses type ifn by inactivating irf ( ) , leading to the low expression of ifn-β. in contrast to reports in epithelial cell lines or primary alveolar type ii cell culture and observations in non-human primates, sars-cov nucleocapsid (n) protein and membrane (m) protein, as well as nsp , can suppress ifn response via various mechanisms in cell lines ( ) ( ) ( ) . to bridge the dichotomy of inhibition of ifn signaling in cell lines, and the ifn expression in vivo, cells recruited by the infection need to be considered as a potential source. as previously discussed, infected epithelial cells via paracrine signaling to neighboring cells and resident macrophages, secrete chemokines and cytokines to attract other immune cells. in general, monocytes/macrophages are recruited by ccl (mip- a), ccl (mcp- ), and neutrophils are recruited by il- (cxcl ), cxcl , and cxcl chemokines, all of which can be secreted by airway epithelial cells ( , , ) . both monocytes and neutrophils are also recruited by complement fragment (anaphylatoxin) c a (figure ) . both influenza and sars virus can induce acute lung injury (ali) which is accompanied by high levels of c a, leading to the influx and activation of innate immune cells ( ) (figure ) . serum samples and lung tissue of sars patients showed high-level expression of cxcl (ip- ), which is also found to be induced by sars-cov in the epithelial cell line calu- ( ) . significant neutrophil, macrophage and cd t-cell infiltration can be found in the lung of sars patients by immunohistochemistry ( , , ) . in addition to post-mortem lung histology analysis in patients with sars-cov, experiments using rhesus macaques infected with sars-cov found monocyte and macrophage recruitment. the accumulation of pathogenic inflammatory monocytemacrophages (imms) was also observed in a sars-cov mouse model. the accumulation of imms resulted in heightened lung inflammatory cytokine/chemokine levels, extensive vascular leakage, and impaired virus-specific t cell responses ( ) . a strong infiltration of cd and mac positive monocytes/macrophages were found in the human and animals lung samples ( , ) . macrophages further stimulate fibroblasts to deposit extracellular matrix leading to pulmonary fibrosis ( ) , which was also observed in patients who recovered from sars ( , ) . autopsy samples acquired from patients with sars-cov- patients contained viral nucleocapsid protein (np) positive cd + macrophages in the capillaries of the spleen and in lymph nodes, indicating that sars-cov- might migrate into the spleen and lymph nodes through macrophages. this study also found that cd + macrophages appear to mediate sars-cov- into these tissue sites, contributing to virus dissemination ( ) . similar to sars-cov- , sars viral particles and genomic sequences were detected in monocytes, macrophages as well as within different organs of sars patients ( ) . sars-cov was shown to infect both immature and mature human monocyte-derived dcs by electron microscopy and immunofluorescence. the detection of negative strands of sars-cov rna in dcs suggests viral replication, but no increase in viral rna was observed ( ) . as mentioned above, there was no perceivable increase to sars-cov replication in primary monocytes ( ) . another study looked at sars-cov and mers-cov replication in human macrophages, human monocyte-derived macrophages, and dendritic cells (mddcs) and found that both viruses replicated poorly. mers-cov induced ifn-λ , cxcl , and mxa mrnas in both macrophages and mddcs, however, sars-cov was unable to induce such responses ( ) . interestingly, depletion of inflammatory monocyte-macrophages in the mouse model partially protected from lethal sars infection ( ) . these data suggest that monocytes, macrophages and dendritic cells have essential roles in cov infection. the severity of disease and the response to these viruses seems to be dependent on the induced cytokine/chemokine profiles and the amplification of the immune response by the recruited cells. growing evidence of dysregulation of an innate anti-viral response originates from studies using clinical samples ( ) and murine models ( , , ) . in addition to dysregulated cellular responses, the complement system may play an important role in sars-cov- infection (figure ) . evidence comes from sars infected patients who had lower levels of mannan binding lectin (mbl) in serum compared to healthy controls ( ) . the sars patients with a higher frequency of mbl gene polymorphisms were associated with lower serum levels or deficiency of mbl ( ) . it is still unknown if this is also true for covid- patients, which requires further investigation. in cell culture experiments sars-cov was able to bind and activate the complement cascade and block viral infection ( ) . preliminary findings in a limited number covid- patients found significant deposits of the membrane attack complex (mac), c d and mbl-associated serine protease (masp) in the microvasculature indicating sustained, systemic activation ( ) . the sars-cov- spike protein was co-localized with c d and mac ( ) . in a non-peer reviewed publication by gao et al., mers-cov, sars-cov and sars-cov- n protein are able to bind to masp- leading to enhanced complement activation ( ) (figure ) . in a later phase of the infection, the complement system might be also triggered via antibodies bound to the virus (classic activation pathway, figure ). this excessive complement activation might play a role in multi organ damage in severe covid- cases ( ) . in a mers-cov mouse model the blockade of the c a-c ar axis alleviated not only lung damage but also spleen damage ( ) . mice treated with a monoclonal antibody to c a showed reduced lung infiltration of cd + cells and significantly lower cytokine levels of il- β, tnf-α, inf-γ and il- ( ) . complement blockade might be an important way to curb part of the immune dysregulation associated with covid- . overall, we need to look closer at the role of the complement system, the recruited innate immune cells and their combined role in pathogenesis, viral clearance and the eventual resolution of the infection. the most abundant leukocytes, neutrophils, play a critical role in clearing viral infections. neutrophils, attracted by chemokines/cytokines released by tissue-resident macrophages and dcs, swarm to the site of infection. they recognize and release bioactive compounds, including cytokines, chemokines and ros, as well as nos in the very early phase of the infection ( , ) . neutrophils modulate other innate and adaptive immune responses via cytokine/chemokine release and cell death and, therefore, can ameliorate or exacerbate disease progression. neutrophils infiltrate tissues infected by cov, including sars-cov, rat coronavirus (rcov), and mouse hepatitis virus (mhv). a high neutrophil count in the blood of sars patients at the time of hospital admission is associated with a poor prognosis ( , ) . gao et al. suggested that patients with sars-cov- pneumonia can be stratified by neutrophil to lymphocyte ratio (nlr) and age ( ) . patients older than years of age and having an nlr ≥ . had more severe illness, so rapid access to the intensive care unit is required ( , ) . experiments in mice showed that sars-cov disease severity in older mice correlated with increased pulmonary inflammation and influx of neutrophils ( , ) . infection of rats with rcov could lead to neutrophil infiltrating into the respiratory tract early after inoculation, followed by the recruitment of macrophages and lymphocytes ( ) . infection of mice with a neurotropic murine cov (mhv-jhm) showed infiltration of neutrophils into the brain as early as the first day after inoculation, which then promoted the recruitment of other types of inflammatory cells into the brain, likely through the loss of the blood-brain barrier integrity ( ) . gene expression analysis in experimentally infected rhesus macaques with mers-cov revealed the recruitment of neutrophils into infected lung tissue ( , ) . angiotensin-converting enzyme inhibitors (ace-is) could serve as a potential risk for fatal covid- through the upregulation of ace ( ) and may provide a direct linkage to neutrophils and disease progression. investigators found that ace modulates il- -mediated neutrophil influx by impacting stat activity ( ) . animal models used to study the pathogenesis of sars-cov- have revealed important roles of neutrophils in infection and confirmed findings observed in patients. a new aspect in sars-cov- infection is the potential role of neutrophil extracellular traps (nets). the process of net formation is a specific type of cell death that can be triggered under inflammatory conditions ( , ) , such as gm-csf+c a, il- , ifn-α+c a or other tlr response mediators; all conditions present in severe sars-cov- infection ( , ) . the net formation has been observed in covid- patients and may contribute to thrombotic complications in covid- patients ( , ) . microvascular injury and thrombosis have been reported in covid- patients, increasing the likelihood that neutrophil net formation might play a role ( , , ) . net formation was reported to be involved in clot formation and thrombosis and can further increase inflammation ( , ) . therefore, neutrophils can attract a second wave of immune cells to the site of infection by cytokine/chemokine secretion as well as via netosis ( , ) , which included monocytes and natural killer cells. on the other hand aggregated nets were reported to limit inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation ( ) . despite the presence of neutrophils in sars-cov- -infected tissues, their role in the clearance and/or immunopathology of the viral infection remains unclear. future studies on the responses of neutrophils to sars-cov- -infection or infected cells in vitro may elucidate the role of neutrophils in the pathogenesis of respiratory cov infections. natural killer (nk) cells are a heterogenic immune cell subset that acts promptly to combat viral infections. they produce significant amounts of ifn-γ, kill virus-infected cells, provide direct support to other innate immune cells, and aid in the adaptive immune response to counter viruses. nk cells express activating receptors that detect viral antigens, enabling the destruction of infected cells ( ) ( ) ( ) ( ) . lectin-like receptor cd and killer immunoglobulin-like receptors, such as cd b, regulate the function of nk cells. a study of patients with sars explored the relationship of the number of nk cells and the expression level of their immunoglobulin-like receptor cd b in the peripheral blood to the severity of sars ( ) . the overall count of nk cells and cd + nk cells and the percentage of cd + nk cells in patients with sars were significantly lower than counts in healthy subjects ( ) . a separate study that analyzed lymphocytes and lymphocyte subsets in a cohort of patients with sars observed reduced nk cell counts in patients ( %) ( ) . clinical reports reveal that children appear to have a milder form of sars-cov- , with peripheral blood lymphocyte levels remaining in the normal range, suggesting less immune dysfunction following the disease ( ) . this could be attributed to healthy children expressing lymphocytes, especially nk cells, in a greater quantity compared to healthy adults ( ) . interestingly, previous studies found rapid and significant restoration of lymphocyte subsets including, nk cells, in peripheral blood in patients recovering from the initial stages of sars infection ( ) . although the primary mechanism for the decrease in nk cells and other subsets during disease onset remains unknown, their contribution to sars-cov- needs further study especially from a treatment perspective. innate lymphoid cells (ilcs) are a family of innate immune cells that include ilc , ilc and ilc . although ilc facilitates lung repair after injury, the role of ilcs during respiratory viral infection is not clearly defined ( ) . evidence for the potential involvement of ilc cells in the lung during viral infection was reported in a murine model ( ) . this study found a rapid accumulation of ilc cells in the lung after an influenza virus infection, however their initial contribution to exacerbation of the disease was limited ( ) . a recent study identified an interaction between ace -expressing sars-cov- target cells and ilcs in the colon ( ) . thus, elucidating the role of ilc subsets will be important in understanding the pathogenesis of sars, sars-cov- and mers infections. there is distinct evidence indicating an important role of ifns in sars and other cov infections ( , ) . the sera of patients with sars revealed the presence of high levels of il- , il- , infγ, ccl , cxcl , and il- and products of interferon stimulated genes ( , ) . high expression levels of isgs such as cd , ifnar , and ifngr and ifn-stimulated chemokines cxcl and ccl were observed in another cohort of sars patients and were correlated with the severity of pathogenesis ( ) . significant upregulation of cxcl gene expression was observed in the severe phase of patients who died from sars. this data is corroborated by studies in patients with mers that found upregulation of cxcl in the serum of patients who developed pneumonia ( ) . cxcl and infα were also correlated with severity of disease ( ) . the importance of ifn signaling in response to cov infection has been well-demonstrated in several knockout mouse models. type i, ii, and iii ifn signaling deficient mice have increased susceptibility to mouse-adapted sars-cov strains ( , ) . studies using mice lacking the ifnar and ifnlr or stat identified higher sars-cov replication in the lungs and delayed virus clearance ( , ) . another study with mers-cov in mice expressing the human dpp receptor showed a role for the ifnar in viral clearance and lung inflammation ( ) . these mouse models suggest an important role of ifn response for cov clearance. this quick expanding medical literature is very suggestive of an important role of ifn responses for cov control and clearance. many viruses have evolved to disrupt and subvert the immune response. rsv counteracts the immune response ( , ) ; as discussed earlier, the rsv genome encodes non-structural proteins (ns and ns ) that are able to block type ifn production and signaling in cell cultures ( ) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat and stat signaling in cell culture lines ( ) . covs have developed several ways to escape from innate immune pressure. mers-cov m protein suppresses type ifn by blocking the irf activation ( ) , explaining the low expression of ifn-β. in various cell lines, sars-cov nucleocapsid (n) protein, membrane (m) protein, as well as nsp , were reported to suppress ifn response ( , , ) . the nucleocapsid protein (n) of sars-cov interferes with the function of irf . although it does not form a complex with rig-i or mda , rna binding activity at the initial recognition stage of viral rna potentially contributes to immune evasion ( , ) . aside from the hcov, structural proteins, accessory, and non-structural proteins (nsp) are involved in innate immunity modulation. in both sars-cov and mers-cov, host mrna endonucleolytic cleavage is promoted by nsp protein, which modifies capped non-viral rnas ( , ) . nsp in sars-cov prevents host mrna translation by interacting with the s subunit of the ribosome; in turn, transcription and translation of viral rna is given preference over the host mrna ( ) . another study found that additional sars-cov nsp residues interfered with ifn-dependent signaling ( ) . ifn production is affected by nsp proteins in sars-cov and mers-cov. these proteins have both papain-like protease (plpro) and a plp domain, and the plpro domains in both sars-cov and mers-cov downregulate mrna levels of ccl , infβ, cxcl , and other pro-inflammatory cytokines ( ) . the suppression of ifn responses by sars-cov plpro is due to the inhibition of phosphorylation of ifn-regulatory factor (irf ) and its subsequent translocation to the nucleus where it enhances ifn gene transcription ( ) . mers-cov plpro also suppresses rig-i and mda and antagonizes ifn induction ( , ) . in hcov- e and sars-cov suppression of ifn responses, the key molecule is a adp-ribose- -monophosphatase macrodomain encoded within nsp ( ) . accessory proteins are not key in viral replication; however, in human cov, this group of proteins are involved in diverse cellular signaling, including cell proliferation, apoptosis, and ifn signaling ( ) . by downregulating phosphorylation and nuclear translocation of irf , open reading frame orf b and - interfere with ifnβ synthesis and prevent ifnβ-induced activation of ifnstimulated response element (isre) in the promoter of isg in sars-cov ( ) . in cells transfected with orf a, - b, and - of mers-cov, ifnβ promoter-driven luciferase activity is significantly reduced, and it may follow a similar pattern of suppression of irf nuclear translocation ( ) . therefore, the suppression of signaling events in infected immune and airway epithelial cells, as well as the magnitude of suppression due to elevated expression levels of these accessory proteins, needs to be further elucidated to understand delayed or hyperimmune responses and cytokine storm that occurs in cov infection. in addition to revealing our unpreparedness of handling a worldwide pandemic by a viral infection, covid- exposed our lack of understanding of the pathogenesis of diseases as well as the host immunity. the interaction of the host innate immune system and other factors including age, sex, and pre-existing conditions need further investigation regarding disease severity and morbidity of sars/mers and covid- . disease severity and its related progression are further associated with dysregulation of multiple components of both innate and adaptive immune responses leading to a cytokine storm and severe pathology. for the development of a therapeutic intervention, it is vital to elucidate the interplay among the different layers of the innate immune response and their relation to the clinical factors associated with increased morbidity and mortality in cov infection. investments in basic science research are needed to help elucidate the roles of different immune cells, and their contribution to disease severity; it will pave the way to prevent or abrogate cov outbreaks and potentially new viruses. nl, td, dh, zl, and nf performed the literature search, analyzed the literature, and wrote the manuscript. ha-h, bs, yw, and rs performed the literature search and wrote the manuscript. all authors contributed to the 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antiviral signaling mers-cov papain-like protease has deisgylating and deubiquitinating activities regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the adp-ribose- ''-monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus e mediate resistance to antiviral interferon responses accessory proteins of sars-cov and other coronaviruses the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © ahmed-hassan, sisson, shukla, wijewantha, funderburg, li, hayes, demberg and liyanage. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -bjs oliw authors: jin, yilin; jia, kuntong; zhang, wanwan; xiang, yangxi; jia, peng; liu, wei; yi, meisheng title: zebrafish trim promotes innate immune response to rgnnv infection by targeting card and rd regions of rig-i for k -linked ubiquitination date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: bjs oliw rig-i-like receptors (rlrs) play important roles in response to virus infection by regulating host innate immune signaling pathways. meanwhile, the rlr signaling pathway is also tightly regulated by host and virus to achieve the immune homeostasis between antiviral responses and virus survival. here, we found that zebrafish trim (zbtrim ) functioned as a positive regulator of rlr signaling pathway during red spotted grouper nervous necrosis virus (rgnnv) infection. post-rgnnv infection, zbtrim expression was obviously inhibited and ectopic expression of zbtrim led to enhanced expression of rlr signaling pathway-related genes. overexpression and knockdown analysis revealed that zbtrim promoted zebrafish rig-i (zbrig-i)-mediated ifn signaling and inhibited rgnnv replication. mechanistically, zbtrim bound to zbrig-i; in particular, the spry domain of zbtrim interacted with the tandem caspase activation and recruitment domains ( card) and repressor domain (rd) regions of zbrig-i. zbtrim promoted the k polyubiquitination of card and rd regions of zbrig-i. furthermore, zbtrim -mediated zbrig-i activation of ifn production was enhanced by k -linked ubiquitin, indicating that zbtrim -mediated zbrig-i polyubiquitination was essential for rig-i-triggered ifn induction. in conclusion, these findings reveal a novel mechanism that zbtrim positively regulates the innate immune response by targeting and promoting the k -linked polyubiquitination of zbrig-i. the innate immune system recognizes pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) as against microbial pathogen invasion ( ). retinoic acid inducible gene-i (rig-i)-like receptors (rlrs), as intracellular prrs, composed of rig-i, mda , and lgp , recognize non-self signatures of viral rnas in the cytosol of cells. after activated by viral rna, rig-i and mda recruited the downstream adaptor molecule, mavs, to their n-terminal caspase-recruitment domains (cards). then, tumor necrosis factor receptor-associated factors (traf) and tank-binding kinase /iκ-b kinase ε interacted with mavs, which in turn leads to the phosphorylation and cytoplasm-to-nucleus translocation of interferon (ifn) regulatory factor (irf ), and the activation of type i ifn. subsequent ifns activated a variety of ifn-stimulated genes (isgs) to limit the virus replication ( ) . nervous necrosis virus (nnv) is a non-enveloped, singlestranded rna virus belonging to the family nodaviridae. increasing evidence has shown that nnv can infect more than fish species and causes mass mortalities of infected fish worldwide ( ) . it has been revealed that rlrs respond in vivo or in vitro to the stimulation of nnv and possess capacities in the induction of ifns and isgs in a variety of fish species. for example, in zf cells, expression of rlrs was significantly enhanced post-nnv infection and rig-i knockdown significantly restrained group ii type i ifn activation ( ) . our previous studies also suggested that rlr signaling pathway was activated during red spotted grouper nervous necrosis virus (rgnnv) infection in sea perch and its key components possessed anti-rgnnv activities ( , ) . however, regulation mechanisms of rlr signaling pathway during rgnnv infection is still unclear. rlr-mediated antiviral signaling pathway is tightly regulated at multiple steps in the signaling cascade. several studies demonstrated that posttranslational modifications, including ubiquitination, isgylation, and phosphorylation, were important mechanisms that regulated the rlr signaling pathway, of which ubiquitination was a key regulatory mechanism for rlr pathway ( ) . for instance, rnf negatively regulated rlr signaling pathway by targeting rig-i ( ) . mda and mavs were targeted for k -linked ubiquitination by trim and rnf , respectively, which induced mda and mavs degradation and rlrs signal termination ( , ) . trim e ubiquitin ligase induced the k linked ubiquitination of rig-i, which activated rlr signaling pathway to elicit host antiviral innate immunity ( ) . trim , an ifn-inducible e ligase, is associated with all kinds of cellular processes, such as the immune response, cancer, and so on ( ) . it is becoming evident that trim has a dual role in rig-i regulation, since trim not only induces k -linked ubiquitination of rig-i to positive regulate rlr signaling activation but also negatively regulates rig-i activation through inhibiting hla-f adjacent transcription degradation, a negative regulator of rig-i-mediated inflammatory response ( ) . multiple fish trim homologs have been reported, including rhodeus uyekii ( ), epinephelus coioides ( ) , and larimichthys crocea ( ) . increasing evidence showed that fish trim was involved in antiviral immunity and played a pivotal role in rlr antiviral signaling pathway ( ) . however, the mechanism by which fish trim regulates rlr signaling pathway has not been explored. in the present study, zebrafish trim (zbtrim ) was involved in rgnnv infection and was identified as a positive mediator of rlr signaling pathway by binding to and ubiquitinating the caspase activation and recruitment domain ( card) and repressor domain (rd) regions of rig-i, which is different with the findings in mammals. our findings reveal a novel mechanism of trim to activate rlr signaling pathway and will help to develop new treatments for viral nervous necrosis disease. all procedures with zebrafish were approved by the ethics committee of sun yat-sen university and the methods were carried out following the approved guidelines. zebrafish wild-type ab line was purchased from china zebrafish resource center. fish were raised with h darkness and h light at • c and were fed with commercial pellets twice a day. all embryos were obtained by natural spawning and staged as previously reported ( ) . zbe cells derived from zebrafish embryos were cultured at • c as previously described ( ) . hek t cells were cultured in dmem (invitrogen) enriched with % fbs (invitrogen) at • c under a humidified atmosphere of air containing % co . rgnnv was propagated in zbe cells and stored at − • c until use. anti-flag (m ), anti-myc (m ), anti-his (m l), and anti-ha antibodies (m ) were purchased from abmart. anti-α-tubulin (ab ) and anti-gfp antibodies (g ) were purchased from abcam and sigma, respectively. goat anti-rabbit igg-hrp, goat anti-mouse igg-hrp, alexa fluor -labeled goat anti-mouse igg, and alexa fluor -labeled goat anti-rabbit igg secondary antibodies were purchased from invitrogen. for in vitro infection, zbe cells were challenged with rgnnv [multiplicity of infection (moi) = ] for , , and h, respectively. subsequently, rna from cells was extracted to detect the expression of zbtrim mrna by quantitative realtime pcr (qrt-pcr). for in vivo infection, rgnnvs ( tcid /ml) were injected into the egg yolk of embryos at the single-cell stage in the experimental group. in the mock group, embryos were injected with dmem. a total of nl of solution was microinjected into each embryo using a microinjector. rna from zebrafish embryo was extracted to detect the expression of zbtrim mrna by qrt-pcr at h post-injection. knockdown of zbtrim by sirna zbtrim sirna ( ′ -gaatccagttgaagagaaa- ′ ) and control sirna were synthesized by ribobio company (guangzhou, china). zbe cells were transfected with zbtrim sirna or control sirna according to the manufacturer's protocol using lipofectamine as previously described ( ) . twenty-four hours after transfection, zbe cells were infected with rgnnv (moi = ) for h and total rnas were extracted for qrt-pcr analysis. the orf of zbtrim (genbank accession no. nm . ) was sub-cloned into pcmv-flag or pcmv-myc vectors (invitrogen) to generate recombinant plasmid pcmv-flag-zbtrim or pcmv-myc-zbtrim , respectively. full-length zbrig-i and zbrig-i deletion mutant cdnas encoding amino acids - (zbrig-i- card), - (zbrig-i- card), - (zbrig-i-rd), and - [zbrig-i-( card+rd)] were inserted into the pegfp-n vectors. full-length zbrig-i was inserted into the pet- a(+) (clontech) vector to generate recombinant plasmid pet- a(+)-zbrig-i. zbtrim deletion mutant zbtrim -spry and zbtrim -spry were generated using the pcmv-flag-zbtrim plasmid as a template. primers used for amplifying these genes are listed in table s . ha-k ub plasmid was purchased from rebio (shanghai, china). rna extraction and cdna synthesis were performed using trizol (invitrogen) and primescript tm st strand cdna synthesis kit (takara) according to the manufacturer's instructions. qrt-pcr analyses of zbtrim , zbrig-i, rna dependent rna polymerase (rdrp), rlr signaling pathway related genes (mavs, traf , irf , and ifn ), and isg were performed as previously described ( ) . relative expression levels of target genes were normalized to s rrna using − ct methods. data represent the mean ± sd from three independent experiments, each performed in triplicate. primers sequences used for qrt-pcr are listed in table s . hek t cells, pre-seeded in -well plates, were transfected with ng of pgl -drifn -pro-luc plasmid or pgl -basic empty vector with ng of prl-tk vector (promega) together with pcmv-myc-zbrig-i or pcmv-myc and pcmv-flag or pcmv-flag-zbtrim ( ng per well) for h. then, cells were incubated with poly i:c for h and lysed. luciferase activities were measured using the dual-luciferase reporter assay system (promega). relative luciferase activities were expressed as the ratio of firefly to renilla luciferase activity. the results were the representative of three independent experiments in triplicate. hek t cells, pre-seeded in -well plates, were transfected with ng of pgl -drifn -pro-luc plasmid or pgl -basic empty vector with ng of prl-tk vector (promega). meanwhile, pcmv-myc-zbtrim , mutant zbrig-i or empty control plasmids were co-transfected. after being incubated with poly i:c for h, cells were lysed for luciferase assay as described above. at least three independent experiments were performed. hek t cells, seeded on glass cover slips, were transfected with pcmv-myc-zbtrim and pcmv-flag-zbrig-i plasmids. twenty-four hours post-transfection, cells were washed with pbs three times and fixed with prechilled methanol and then permeabilized using % triton x- in pbs for min and blocked with % normal goat serum for min at room temperature (rt). cells were incubated with anti-myc and anti-flag antibodies for min at rt. finally, cells were washed with pbs and incubated with the appropriate alexa fluor or conjugated secondary antibodies for h. after cell nucleus was stained with hoechst , cells were observed by a confocal microscope (zeiss, germany). co-ip and western blotting experiments were performed as described previously ( ) . hek t cells in -cm flasks were co-transfected with µg of different plasmid combinations for h. then, the cells were lysed on ice with lysis buffer for min and were immunoprecipitated with the indicated antibodies. the precipitated samples and whole-cell lysates (input) were analyzed by immunoblotting with the indicated antibodies. escherichia coli bl (de ) cells were transformed with pet- a(+)-zbrig-i or pet- a(+) plasmids, respectively. then, cells were grown in ml of lb medium (beyotime) containing . mm isopropyl- -thio-β-d-galactopyranoside (iptg) (sigma) at • c overnight with shaking at rpm. cells were pelleted by centrifugation at , rpm for min and lysed in ml of lysis buffer ( mm sodium phosphate, ph . , mm nacl, and . % tween- ) (beyotime) via sonication on ice. the sonicated mixture was centrifuged at , rpm at • c for min, and then the supernatant was affinity-purified with dynabead his-tag magnetic beads (invitrogen) according to the manufacturer's instruction. his pull-down assays were performed as described previously with some modifications ( ) . his-zbrig-i-magnetic beads were incubated with the lysates of hek t cells transfected with pcmv-flag-zbtrim or pcmv-flag empty vectors on a roller, respectively. after incubation at • c overnight, the magnetic beads were washed three times with lysis buffer to remove unbound his-zbrig-i and then analyzed via western blotting using anti-flag or anti-his antibodies. his tag protein alone was served as a negative control. ubiquitination assays were performed as described previously with some modifications ( ) . hek t cells, pre-seeded in -cm flasks overnight, were co-transfected with µg of different plasmid combinations. cells were lysed at h after transfection, and then gfp-zbrig-i mutants were immunoprecipitated with anti-gfp antibodies as described above. immunoprecipitates or input were analyzed by immunoblotting with the indicated antibodies. all statistics were calculated using spss version . differences between control and treatment groups were assessed by one-way anova. p < . is considered statistically significantly different. p < . was considered highly significant. as shown in figure a , mrna level of zbtrim was downregulated within h after rgnnv infection. meanwhile, we also investigated the expression of zbtrim in rgnnvinfected zebrafish embryos at h, and the results were concordant with zbe cells (figure b) . these data indicated a potential role of zbtrim in innate immune response to rgnnv infection. in mammals, trim has been suggested to promote ifnβ production by functioning as a key upstream activator of rig-i to activate the rlr signaling pathway ( ) . to investigate whether zbtrim regulated rlr signaling pathway in zebrafish, the expression of several rlr signaling pathwayrelated genes was measured in zbtrim overexpressing zbe cells. as shown in figure a , overexpression of zbtrim markedly enhanced the expression of rig-i, mavs, traf , irf , ifn , and isg during rgnnv infection. similar results were detected in zbtrim overexpressing zbe cells treated with poly i:c ( figure b) . furthermore, coexpression of zbtrim with zbrig-i induced a dosedependent increase in ifn activation compared with the zbrig-i overexpression alone (figure c) . overexpression of zbtrim dose-dependently inhibited rgnnv replication ( figure d) . on the contrary, knockdown of zbtrim using sirna increased the level of rdrp in rgnnv-infected zbe cells (figures e,f) . all these results demonstrate that zbtrim is a positive regulator of rlr signaling pathway and functions as an antiviral factor during rgnnv infection in zebrafish. to elucidate the mechanism by which zbtrim participates in rlr signaling pathway in zebrafish, the interaction of zbtrim and zbrig-i was investigated. we co-expressed myc-zbtrim and flag-zbrig-i plasmids in hek t cells, and immunofluorescence imaging showed zbtrim and zbrig-i colocalized in the cytoplasm of hek t cells (figure a) . co-ip against the myc tag revealed that zbtrim could interact with full-length zbrig-i but not with flag-vector ( figure b) . his pull-down analysis showed that zbtrim was directly bound to zbrig-i ( figure c ). all these data suggest that zbtrim interacts with zbrig-i. to identify the region involved in the zbrig-i/zbtrim interaction, firstly, zbrig-i deletion mutants [pegfp-zbrig-i- card, pegfp-zbrig-i- card, pegfp-rig-i-rd, and pegfp-rig-i-( card+rd)] were constructed and cotransfected with flag-zbtrim in hek t cells ( figure a) . zbrig-i- card, zbrig-i- card, and zbrig-i-rd could bind to zbtrim individually (figures b-d) ; however, zbrig-i-( card+rd) failed to co-precipitate with zbtrim ( figure e) . these results indicate that zbrig-i binds to zbtrim through its n-terminal card region and the c-terminal rd region. furthermore, we constructed two truncations of zbtrim (zbtrim -spry and zbtrim -spry) co-transfected with zbrig-i- card or zbrig-i-rd in hek t cells, respectively ( figure f) . we found that the spry domain of zbtrim interacted with card and rd regions of zbrig-i (figures g-j) . collectively, these results indicate that the spry domain of zbtrim is responsible for its interaction with card and rd regions of zbrig-i. to investigate whether the e ligase activity of zbtrim is involved in the regulation of zbrig-i, the ubiquitination of zbrig-i was tested in zbtrim overexpressing cells. we found that zbtrim markedly promoted the k polyubiquitination of zbrig-i ( figure a) . furthermore, hek t cells were transfected with flag-tagged zbtrim , zbrig-i deletion mutants [gfp-zbrig-i- card, gfp-zbrig-i-( card+rd), and gfp-zbrig-i-rd], and ha-tagged k ubiquitin, and our results showed that zbtrim obviously enhanced the ubiquitination of zbrig-i- card and zbrig-i-rd (figures b,c) , but not zbrig-i-( card+rd) (figure d) . these data suggest that zbtrim ubiquitinates both n-terminal card and c-terminal rd regions of zbrig-i. it has been reported that ubiquitination of rig-i by trim is vital for ifn signaling. thus, the effect of zbtrim -mediated zbrig-i ubiquitination on zbrig-i's ifn-inducing activities was assessed. our results showed that ectopic expression of zbrig-i- card and zbrig-i-rd could enhance ifn promoter activity (figure a) , and this activation was markedly enhanced by zbtrim overexpression (figures b,c) . furthermore, overexpression of k -linked ubiquitin dose-dependently increased the promotion effect of zbtrim on zbrig-i- card and rd mediated ifn promoter activation (figures b,c) . these data confirm the importance of zbtrim -mediated k ubiquitination in the n-terminal card region and c-terminal rd region of zbrig-i for zbrig-i-mediated ifn induction. rlr signaling pathway plays crucial roles in recognizing viral infections and initiating the antiviral immune response. rig-i, as an important component of rlr signaling pathway, can detect viral dsrnas in the cytoplasm and induce type i ifn production and the secretion of pro-inflammatory cytokines to suppress virus spread during virus infection ( ) . multiple studies have demonstrated that the ubiquitination of rig-i plays an important role in the rig-i-mediated antiviral signaling pathway. for instance, trim , trim , and mex c positively regulate rig-i-mediated signaling by targeting rig-i for k linked polyubiquitination ( , ) . trim , well-known as an ubiquitin e ligase and an isg e ligase, is widely involved in the regulation of innate immunity ( , ) . in mammals, previous reports showed that trim enhanced rlrs antiviral pathway by binding viral rna-activated rig-i to induce its k -linked polyubiquitination and subsequent ifns and isgs production ( ) . in teleost fish, several trim homologs were reported to play a pivotal role in innate immunity ( , ) ; however, the mechanisms by which fish trim modulates the innate immune response against viruses remain elusive. here, we found that zbtrim positively regulated rlr signaling pathway and facilitated zbrig-i-mediated ifn promoter activation, and overexpression of zbtrim inhibited rgnnv infection, indicating the conservative antiviral properties of trim in fish and mammals. several reports showed that trim was involved in the regulation of antiviral innate immunity by targeting rig-i ( , ) . the mammal rig-i protein contains two n-terminal cardlike domains, a c-terminal rd region and an rna helicase region ( ) . in zebrafish, rig-ia (an insertion variant of rig-i) and rig-ib (the typical rig-i) were identified as two transcripts of rig-i, and overexpression of rig-ib in cultured fish cells, but not rig-ia, activated zebrafish type i ifn and induced antiviral response ( ) . thus, in this report, we investigated the interaction of zbtrim and zbrig-i (rig-ib), and our results showed that zbtrim was directly associated with zbrig-i and especially the card or rd region of zbrig-i was sufficient for its interaction with zbtrim . trim is characterized by an n-terminal region containing a catalytic ring domain, one or two b-box domains, a coiled-coil dimerization domain, and a c-terminal spry domain ( ) . among these domains, spry was associated with protein-protein interactions and/or rna binding ( ) . gack et al. reported that the c-terminal spry domain of trim interacted with the first card of rig-i, but not the helicase region and rd of rig-i, and this interaction delivered the k -linked ubiquitin moieties to the second card of rig-i, which facilitated the dimerization of rig-i and subsequent interaction with mavs to induce antiviral signal transduction ( ) . we further investigate whether the spry domain of zbtrim was responsible for its interaction with zbrig-i. unlike previous studies, we found that the spry domain of zbtrim interacted not only with card but also with rd regions of zbrig-i. in non-infected cells, rd covered the rna-binding and helicase domains and cards folded over one another, which made rig-i to exist in an auto-repressed conformation. upon virus infection, viral rnas interacted with the rd and the helicase domain of rig-i, which in turn exposed the cards for mavs interaction, thereby triggering antiviral responses ( , ) . considering the interaction between rd of rig-i and viral rnas, we speculated that the interaction of zbtrim and zbrig-i rd might inhibit zbrig-i sensing of viral rnas. meanwhile, it has been known that card domains of rig-i are widely involved in its interaction with other proteins, such as mavs, trim , and virus proteins ( , , ) . thus, the interaction of zbtrim and zbrig-i rd might also make room for other proteins to bind to card of zbrig-i, zbtrim , and other proteins and will work cooperatively in regulation of rlr signaling pathway. the differences between the findings for zbtrim and trim in mammals indicate that zbtrim may regulate rlr signaling pathway in various ways. ubiquitination is a vital post-translational modification for the modulation of rig-i activity. several e ubiquitin ligases that mediate k -linked ubiquitination of rig-i for its activation have been identified. for instance, mex c overexpression caused the k -linked ubiquitination of rig-i- card but not rig-i- card, and lysines , , and of rig-i were required for rig-i ubiquitination by mex c ( ) . rnf mediated the k -linked polyubiquitination of rig-i-rd, and lysines and residues of rig-i were crucial for rnf -mediated ubiquitination ( ) . in contrast to rnf , trim mediated the k -linked polyubiquitination of rig-i- card, but not rig-i- card, and the lysine residue of rig-i was critical for efficient trim -mediated ubiquitination and the ability of rig-i to activate antiviral signal transduction ( ) . our results indicated that zbtrim mediated k -linked polyubiquitination of both card and rd regions of zbrig-i, which is distinct from the findings in mammals that trim only targeted and promoted the k -linked polyubiquitination of rig-i card. in addition, our reporter analysis showed that overexpression of zbrig-i- card led to the activation of ifn promoter, which is similar with other reports ( ) . overexpression of zbrig-i-rd also resulted in the activation of ifn promoter. furthermore, k -linked ubiquitin is essential for the zbtrim -mediated enhancement of zbrig-i card and rd-dependent ifn promoter activation. zbrig-i possessed capacities in the induction of ifns and isgs to enhance the antiviral response ( ) . taken together, these findings suggest that zbtrim mediated ubiquitination of card and rd regions of zbrig-i is crucial for its antiviral innate immune response. however, due to the lack of trim or rig-i-knockout zebrafish, we cannot assess the impact of the zebrafish trim /rig-i pathway at the in vivo level. a recent study demonstrated that zebrafish rnf also interacted with and ubiquitinated zbrig-i ( ) . further studies will be performed to determine the precise architecture of the zebrafish trim /rnf /rig-i protein complex and the mechanism by which zbtrim and zbrnf worked together to regulate ubiquitination of zbrig-i. it was known that several virus proteins could positively or negatively regulate rlr signaling pathway by targeting its key components or regulatory proteins ( ) . for instance, paramyxovirus v proteins interacted with the rig-i/trim regulatory complex and inhibited rig-i signaling ( ) . influenza a virus ns protein bound to trim to block ubiquitination of the rig-i ( ) . severe acute respiratory syndrome nucleocapsid inhibited trim -mediated rig-i ubiquitination, causing the inhibition of ifn production ( ) . the rgnnv genome encodes a structural (capsid protein, cp) and a nonstructural (rna-dependent rna polymerase, rdrp) protein ( ). huang et al. reported that rdrp from ognnv induced ifn by activating irf , the key regulatory component of rlrs-ifn signaling ( ) , indicating that rdrp might be a positive rlr signaling pathway. whether rdrp targets the key components of rlr signaling pathway to exert its positive regulation role is a question that deserves further research. in addition, some mirnas could target critical regulatory proteins of rlr pathway for immune evasion ( , ) ; whether rgnnv infection-related mirna was also involved in the regulation of rlr signaling pathway needs to be further investigated. in summary, zbtrim is identified as a positive regulator of rlr signaling pathway by targeting zbrig-i. the spry domain of zbtrim is required for its interaction with card and rd regions of zbrig-i. zbtrim promotes k polyubiquitination of both zbrig-i card and rd regions, which subsequently induces the activation of downstream signaling event via mavs and thereby inhibits viral infection (figure ) . these findings represent a new mechanism underlying the regulation of rlr signaling pathway. all datasets generated for this study are included in the article/supplementary material. the animal study was reviewed and approved by the ethics committee of sun yat-sen university. shaping the landscape of host immunity regulation of rlr-mediated innate immune signaling -it is all about keeping the balance understanding the interaction between betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy rig-i specifically mediates group ii type i ifn activation in nervous necrosis virus infected zebrafish cells identification and characterization of the melanoma differentiation -associated gene in sea perch, lateolabrax japonicus characterization and expression analysis of laboratory of genetics and physiology gene in sea perch rnf suppresses antiviral type i interferon production by targeting rig-i cards to mediate rig-i degradation trim is a negative regulator of mda -mediated type i interferon production the e ubiquitin ligase rnf targets virus-induced signaling adaptor for ubiquitination and degradation trim ringfinger e ubiquitin ligase is essential for rig-i-mediated antiviral activity trim in the regulation of the antiviral innate immunity. front immunol ubiquitin-like modifier fat attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform molecular characterization of tripartite motif protein (trim ) involved in erα-mediated transcription in the korean rose bitterling rhodeus uyekii ring domain is essential for the antiviral activity of trim from orange spotted grouper the two trim isoforms were differentially induced in larimichthys crocea post poly (i:c) stimulation. fish shellfish immun stages of embryonic development of the zebrafish establishment of a cell line with high transfection efficiency from zebrafish danio rerio embryos and its susceptibility to fish viruses mandarin fish caveolin interaction with major capsid protein of infectious spleen and kidney necrosis virus and its role in early stages of infection interferon regulatory factor from sea perch (lateolabrax japonicus) exerts antiviral function against nervous necrosis virus infection the eukaryotic translation initiation factor subunit e binds to classical swine fever virus ns a and facilitates viral replication ubiquitination is essential for avibirnavirus replication by supporting vp polymerase activity rig-i-like receptor regulation in virus infection and immunity pivotal role of rna-binding e ubiquitin ligase mex c in rig-i-mediated antiviral innate immunity trim modulates type i interferon induction and cellular antiviral response by targeting rig-i for k -linked ubiquitination the ubiquitin-specific protease usp promotes rig-i-mediated antiviral signaling by deubiquitylating trim mechanism of trim catalytic activation in the antiviral rig-i pathway paramyxovirus v proteins interact with the rig-i/trim regulatory complex and inhibit rig-i signaling structural features of influenza a virus panhandle rna enabling the activation of rig-i independently of '-triphosphate higher antiviral response of rig-i through enhancing rig-i/mavs-mediated signaling by its long insertion variant in zebrafish trim/rbcc, a novel class of 'single protein ring finger' e ubiquitin ligases structure and function of the spry/b . domain proteins involved in innate immunity structural insights into rna recognition and activation of rig-i-like receptors the structural basis of ' triphosphate double-stranded rna recognition by rig-i c-terminal domain the e ubiquitin ligase trim attenuates antiviral immune responses by targeting mda and rig-i. cell rep identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection involvement of zebrafish rig-i in nf-kappab and ifn signaling pathways: insights into functional conservation of rig-i in antiviral innate immunity retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) in fish: current knowledge and future perspectives rnf is a positive regulator of ifn expression and involved in rig-i signaling pathway by targeting rig-i. fish shellfish immun influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination protein a from orange-spotted grouper nervous necrosis virus triggers type i interferon production in fish cell downregulation of microrna mir- a by enterovirus inhibits rig-i-dependent innate immune response microrna- a feedback inhibits rig-i-dependent type i ifn production in macrophages by targeting traf , irak , and irak yj and kj performed all experiments with assistance from yx, wz, pj, and wl analyzed data. kj and my conceived the study and designed experiments. kj and my wrote the manuscript. all authors read and approved the final manuscript. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © jin, jia, zhang, xiang, jia, liu and yi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -vgbrwegt authors: charley, b.; lavenant, l. title: characterization of blood mononuclear cells producing ifnα following induction by coronavirus-infected cells (porcine transmissible gastroenteritis virus) date: - - journal: research in immunology doi: . / - ( ) -j sha: doc_id: cord_uid: vgbrwegt abstract porcine blood mononuclear cells (pbmc) were shown to produce interferon-α (ifnα) following incubation with cells infected by a coronavirus, transmissible gastroenteritis virus. monoclonal antibodies (mab) with specificities for leukocyte subsets and major histocompatibility complex (mhc) antigens were used to characterize ifnα producer cells. the production of ifnα was found to be a function of non-phagocytic, non-adherent, non-t, non-b, cd + (and to a lesser extent cd +) mhc-class-ii-positive cells. furthermore, addition of anti-mhc (class ii) mab during pbmc incubation with virus-infected cells reduced ifn yields, suggesting that masking of these surface antigens alters pbmc responsiveness to ifn induction. introduction peripheral blood leukocytes have been shown to produce interferon alpha (ifna) upon induction by viruses, bacterial products or tumour cells (dianzani and capobianchi, ) . in contrast to ifn beta, for which the critical inducing factor appears to be viral rna, a different mechanism seems to be involved in induction of ifna. thus, inactivated virus particles, virus-infected cells, mycoplasmas and cell membranes are capable of inducing ifna (lebon et ai., ; hughes and blalock, ; goblet al., ; capobianchi et al.., ) , which suggests that membrane interactions between the inducer structure and the leukocyte membrane is sufficient to trigger ifna produc-tion. in the case of transmissible gastroenteritis virus (tgev), a coronavirus which induces acute diarrhoea and intense ifn synthesis in newborn piglets (la bonnardiere and laude, ) , we have previously shown that early ifn~ production could result from exposure of non-immune pig lymphocytes to virus-infected cells. moreover, experiments in which ifn c production was inhibited by monoclonal antibodies (mab) directed at some epitopes of the tgev glycoprotein e suggested that a defined domain of this transmembrane viral protein played a key role in the ifn t induction process (charley and laurie, ) . the nature of ifn~ producer cells (ipc) is not clear, since several cell types appear to be involved depending upon the inducer or the induction protocol (dianzani and capobianchi, ) . thus, among non-adherent human mononuclear cells, "nuli" (non-t, non-b) lymphocytes and large granular lymphocytes (lgl) were shown to produce ifn c upon exposure to herpes, influenza or dengue virus (peter et ai., ; kirchner et al., ; lebon et al., ; djeu et al., ; kurane et al., ) . more recent reports have indicated that a common phenotypic feature for murine and human ipc in response to different stimuli is their surface expression of mhc class ii molecules (abb et al., ; reiss et al., ; perussia et al., ; kurane and ennis, ; hughes and blalock, ; capobianchi et al., ; oh et al., ; fitzgerald-bocarsky et al., ; sandberg et al., a) . in the present report, we analysed the nature of lymphocyte subpopulation(s) responsive to ifna induction by tgev-infected cell monolayers by using cell-depletion experiments with mab plus complement. in addition, ifn induction-blocking experiments conducted with anti-mhc (sla, swine leukocyte antigens) class ii mab without complement suggest a functional role for these membrane molecules in the ifn induction process. porcine peripheral blood mononuclear cells (pbmc) were obtained from heparinized blood collected from -to -month old animals. phagocytic cells were depleted by carbonyl iron ingestion (salmon, ) before pbmc isolation by ficoll density centrifugation on msl (density . , eurobio, paris). pbmc were suspended in rpmi- medium supplemented with ° o foetal calf serum. ifn = interferon. ipc = ifnqt producel cell. lgl = large granular lymphocyte. mab = monocional antibody. = major histocompatibility complex. pbmc = peripheral blood mononuclear cell, sla = swine leukocyte antigen. tgev = transmissible gastroenteritis virus. anti-pbmc _.'nab msa (anti-cd ), - - (anti-b), - - (anti-cd ) were kindly provided by j. lunney (usda, beltsville, md, usa); / (anti-cd ) mab was kindly provided by u. koszinowski (tiibingen, frg). mab - - (anti-sla class i) and msa (anti-sla class ii) were provided by j. lunney. mab th a (anti-sla class ii) was purchased from vmrd (pullman, wa, usa). other anti-sla class ii mab (th b, h a and th a) were kindly provided by w. davis (pullman, wa, usa) (table i). these antibodies were used for cell-depletion experiments as ascitic fluids, excepts for msa which was used as hybridoma cell supernatant. pbmc were incubated for min with mab and complement at °c as described previously (charley et al., ) : briefly, one-month old rabbit serum served as a source of complement at a final dilution of / and mab were used at a final dilution of / . the percentage of dead cells was determined by trypan blue dye exclusion and ceils were resuspended in the initial volume, i.e. without readjustment of the viable cell concentration, before being used in the assays. pbmc were induced to produce ifn t by overnight incubation on tgev-induced, glutaraldehyde-fixed cell monolayers as described previously (charley and laude, ) : briefly, pig kidney cells were plated in -well microplates, infected by the coronavirus tgev for h, then fixed with . % glutaraldehyde ( h at °c) and stored with % glycine. monolayers were washed before addition of pbmc ( izl/well at x /ml). supernatants were collected after h of incubation at °c and assayed for ifn activity. various dilutions of dialysed mab preparations, as indicated in "results", were added during overnight incubation of pbmc with tgev-infected monolayers or with virus particles. alternatively, pbmc were pretreated with various amounts of dialysed mab hammerberg and schurig, pescovitz et al, jonjic and koszinowski, pescovitz et al, davis et al., hammerberg and schurig, vmrd, inc., pullman wa davis et ai., davis et al, davis et al, mab preparations for min at °c, washed to remove unbound material, resuspended at × /ml and incubated overnight on t~ev-infected monolayers. supernatants were assayed for ifn. log dilutions of pbmc supernatants were assayed for ifn on bovine mdbk cells using vesicular stomatitis virus as a challenge (la bonnardi re and laude, ) . a standard porcine ifna was included in each assay. this standard was calibrated on mdbk ce!ls with the human international reference ifn b / (nih, bethesda, md, usa). in our results, u is equivalent to ilu of human ifn. we have previously shown that phagocyte-depleted porcine pbmc could secrete ifn t following incubation on tgev-infected glutaraldehyde-fixed cell monolayers (charley and laude, ) . antibody plus complement depletion experiments were conducted to characterize the ipc nature. when pbmc were pretreated with rabbit serum as a source of complement, they produced high amounts of ifn ( +_ u/ml in different experiments). table ii shows the effect of treatment with various anti-lymphocyte mab and complement on ifn production. since ifn assays are performed on log dilutions of pbmc supernatants, any reduction lower than % was considered as negligible. in fact, pretreatment of pbmc with anti-t or anti-b cell mab plus complement did not alter ifn ~ production" ifn titres obtained were ~c~p~t~vc~y equa~ to and ~o oi-"~ titres obtained with complement-treated pbmc (table ii) . in contrast, pretreatment with / (anti-cd ) mab and, no. = number of experiments. viable cells = ° o -(% dead cells after effect of mab + complement-% dead cells with complement alone). ifn production is expressed as % of ifn produced by complement-pretreated pbmc. table iii shows that pretreatment by anti-sla class i mab and complement, which destroyed almost all pbmc, completely abolished ifn production. pretreatment by anti-sla class ii mab th a plus complement markedly lowered ifn production, along with the lysis of o pbmc. these experiments therefore indicated that porcine ipc were largely sla-class-ii-positive cells. at °c), washed and incubated overnight on tgev-infected glutaraldehyde-fixed cell monolayers. ifn activity was assayed in cell supernatants. during cell-depletion experiments, controls performed in the absence of complement indicated that most mab used had no direct inhibitory effects on ifn production. however, two preparations of anti-sla class ii mab (th a and msa ) could directly block ifn induction when added during co-incubation of pbmc with i'gev-infected fixed monolayers or tge alone ( independent experiments). thus, rii a mab used as an ascitic fluid could hlhibit up to . o ifn production at a final ig concentration of ,. /ml ( fig. la) . msa hybridoma culture supernata,~t blocked up to ifn production at a final ig concentration of . [~g/ml ( fig. ib) . three other ascitic fluids, directed at sla class ii, also showed dose-dependent ias~hition of ifn production ( fig. ). in addition, the latter blocking experiment was achieved by pretreatment of pbmc with mab, followed by washing of cells before induction on tgev-infected fixed-cell monolayers, which argues for a direct effect of mab on pbmc as opposed to an antibody effect on infected cell monolayers. in addition, such inhibiting effects could not be related to toxic effects of mab on pbmc (not shown). ifn induction by other viruses such as newcastle disease virus (ndv), sendai virus and types a and b myxoviruses was also blocked by co-incubation of pbmc with th a mab (not shown). we have previously shown that phagocyte-depleted pbmc incubated with tgev-infected cells are rapidly induced to secrete ifna (charley and laude, ) . this ifn~ induction seems to result from membrane interactions between pbmc and a defined domain of the viral glycoprotein e expressed on the surface of infected cells (charley and laude, ). in the present paper, cell-depletion experiments by mab and complement-dependent lysis indicate that porcine ifna producer cells (ipc) are mostly msa -(cd or pan-t)-and - - -(b) cells, whereas depletion of cd + cells, and to a lesser extent cd ÷ cells reduced ifn yields by and ° , respectively (table ii) . in addition, ipc are also sla class i ÷ and class ii ÷ cells. furthermore, the addition of anti-sla class ii mab reduces ifn~t production. our present data on the nature of porcine ipc are in agreement with several reports about the characterization of human ipc. thus, following induction by different viruses including herpes virus, influenza virus, dengue virus or cytomegalovirus, as well as by mycoplasma membranes, human mononuclear leukocytes producing ifn~ were described as non-adherent, non-phagocytic, non-t,non-b cells (trinchieri et al, ; kirchner et al, ; peter et al, ; lebon et al, ; abb et al, ; djeu et al, ; perussia et al, ; kurane et al. ). human ipc were generally shown to lack natural killer function (lebon et al, ; abb et al., ; fitzgerald-bocarsly et al., ) , but to express mhc class ii antigens (abb et al., ; perussia et al., ; capobianchi et al., ; oh et al., ; fitzgerald-bocarsly et al, ) . a recent report using combined immunocytochemistry ,~o.,u,,a,~t ,,t on numan rt~mt; stimulated by hsvinfected cells clearly showed that ipc lacked antigens typical of t and b lymphocytes, but expressed hla-class ii antigens (sandberg et al., a) . the same laboratory observed that ipc also expressed cd antigens (sandberg et al, b) . the expression of mhc class ii antigens led to the hypothesis that human ipc could be dendritic cells (fitzgerald-bocarsly et al, ) . however, hla-dr + cells could recently be divided into two separate subsets: a loosely adherent population meeting the functional criteria of dendritic cells but distinct from ipc which were non-adherent (chehimi et ai., ) . our data therefore indicate that porcine ifna-producing cells in response to tgev-infected cells have the same properties as human ipc: porcine ipc are non-phagocytic, non-adherent (salmon et al., ) , non-t,non-b, mltc-clas~-ii-positive and cd + cells. preliminary experiments using dna-rna in situ hybridization suggested that porcine ifna-mrnacontaining cells were infrequent (around / pbmc; buseyne and charley, unpublished observations) as already described for human ipc (goblet al., ) . interestingly, one might hypothesize that a defined, albeit infrequent cell population exhibiting unusual phenotypic features could produce ifna in response to a wide range of ifn inducers. in order to further analyse these cells and their mode of activation, it will be necessary to use positive selection procedures such as cell sorting (sandberg et al., b) . blocking experiments conducted with anti-sla-class ii msa mab used as hybridoma culture supernatant revealed a reduction in ifn yield of more than ° ( fig. ). comparable inhibition was obtained with other anti-sla class ii mab used as ascitic fluids. this inhibition is not observed when virus-infected cells are pretreated with mab, then washed before pbmc are added. however, when pbmc are pretreated with mab, then washed before induction, ifn yield is still reduced, which argues for a direct effect of anti-sla class ii antibodies on pbmc. in addition, pbmc are not lysed by such treatments. therefore, these results suggest that masking of mhc class ii antigens on the pbmc membrane reduces their responsiveness to ifn induction. this anti-sla class-ii-ab-mediated blockage is observed when pbmc are induced by tgev, sendai, ndv or influenza virus. a similar observation was reported by capobianchi et al. ( ) : pretreatment of human pbmc by anti-hla-dr antibody reduced ifn t yield after induction with mycoplasma membranes. similarily, ia antigens were shown to bind cell surface glycoproteins responsible for ifn induction (hughes et al., ) . therefore, our findings suggest that, in addition to mycoplasmas and cell surface glycoproteins, viruses could also interact with mhc class ii antigens to induce ifn t synthesis. the fact that masking of mhc class ii could reduce pbmc responsiveness to several ifn inducers suggests that these surface antigens are not virusspecific receptors on lymphoid cells. in fact, anti-sla class ii mab did not block tgev replication in susceptible pig kidney cells (data not shown). the actual functional role of mhc class ii molecules in the ifn t induction process remains to be clarified" they could represent broadly reactive recogni-t~u. ~t, m.tu~e~ tto~c to omd utllcrcnt ifn-inducing components (as suggested by hughes et al., ) . alternatively, mhc class ii molecules could be involved in post-recognition events, such as internalization or recycling of membrane structures, which seem to precede activation of ifn c synthesis (lebon, ) . experiments are in progress to further define the nature of virus lymphocyte interactions leading to ifn t production. des cellules sanguines mononucl es du porc produisent de l'interf ron cx (ifn~x) h la suite de leur incubation avec des cellules infect es par le coronavirus get (gas-troent rite transmissible). les cellules productrices d'interf ron ont t caract~ris es l'aide d'anticnrps monoclonaux (acm) sp~cifiques des sous-populations leucocytaires et des antigbnes du complexe majeur d'histocompatibilit (cmh). les cellules productrices d'lfn t sont des cellules non phagocytaires, non adh&entes, ni t, ni b, cd ÷ (pour une moindre part cd +) et cmh-classe-ii÷. de plus, l'addition d'acm dirig s contre les antig nes de classe ii du cmh, au m ange d'incubation cellules mononucl es plus cellules infect es par le virus get, r duit la production d'ifn~, ce qui sugg re que le masquage de ces antig nes de surface modifie la capa-cit des cellules mononucl es/l r pondre aux signaux inducteurs d'ifn t. mots-cli~s" lnterf ron alpha, lymphocyte, cmh, coronavirus; anticorps monoclonaux, virus de la gastroent rite transmissible du pore. phenotype of human alpha interferonproducing leucocytes identified by monoclonal antibodies membrane interactions involved in the induction of interferon-alpha by mycoplasma pneumoniae inhibition of transmissible gastroenteritis coronavirus (tgev) multiplication in vitro by non-immune lymphocytes induction of alpha interferon by transmissible gastroenteritis dendritic cells and ifn-g-producing cells are two functionally distinct non-b,non-monocytic hla-dr + cell subsets in human peripheral blood the development and analysis of species-specific and cross-reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species mechanism of induction of alpha interferon positive self regulation of cytotoxicity in human natural killer cells by production of interferon upon exposure to influenza and herpes viruses human mononuclear cells which produce interferon-alpha during nk (hsv-fs) assays are hla-dr-positive cells distinct from cytolytic naturel killer effectors different induction patterns of mrna for ifn-a and -b in human mononuclear leukocytes after in vitro stimulation with herpes simplex virus-infected fibroblasts and sendai virus characterization of monoclonal antibodies directed against swine leukocytes ia antigen: a murine b lymphocyte receptor for transformed cell induction of interferon- t/b interferon-inducing transformed cell surface glycoproteins: purification by ia antigen affinity chromatography monoclonal antibodies reactive with swine lymphocytes.-l. antibodies to membrane structures that define the cytolytic t lymphocyte subset in the swine studies of the producer cell of interferon in human lymphocyte cultures induction of interferon-a from human lymphocytes by autologous, dengue virus-infected monocytes induction of interferon alpha and gamma from human lymphocytes by dengue virus-infected cells high interferon titer in newborn pig intestine during experimentally induced viral enteritis inhibition of herpes simplex virus type-l-induced interferon synthesis by monoclonal antibodies against viral glycoprotein d and by lysosomotropic drugs human lymphocytes involved in ~-interferon production can be identified by monoclonal antibodies directed ,o cooperation between cd (leu- lb) + nk ceils and hla-dr + cells in natural killing of herpesvirus-infected fibroblasts a leukocyte subset bearing hla-dr antigens is responsible for in vitro interf,~ron production to viruses preparation and characterization of monoclonal antibodies reactive with porcine pbl human peripheral null lymphocytes.-ii. producers of type interferon upon stimulation with tumor cells, herpes simplex virus and corynebacterium parvum interferon production by cultured murine splenocytes in response to influenza virus-infected cells surface markers of porcine lymphocytes and distribution in various lymphoi'd organs atural killer (nk) activity and interferon (ifn) production by a fraction of spleen and blood lymphocytes in swine porcine cells i i characterization of the blood mononuclear leucocytes producing alpha interferon after stimulation with herpes simplex virus in vitro, by means of combined immunohistochemical staining and in situ rna-rna hybridization cd antigen are expressed on the interferon- t-producing cells induced by herpes simplex-infected cells antiviral activity induced by culturing lymphocytes with tumor-derived or virus-transformed cells. identification of the antiviral activity as interferon and characterization of the human effector lymphocyte subpopulation we are grateful to dr. j. lunney (beltsville, md, usa), dr. u. koszinowski (tiibingen, frg) and dr. w. davis (pullman, wa, usa), who kindly provided antiporcine lymphocyte subsets and anti-sla monoclonal antibodies. we also thank dr. f. blecha (manhattan, ks, usa) for revising the manuscript. key: cord- -cd w o authors: whitman, lucia; zhou, haixia; perlman, stanley; lane, thomas e. title: ifn-γ-mediated suppression of coronavirus replication in glial-committed progenitor cells date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: cd w o the neurotropic jhm strain of mouse hepatitis virus (jhmv) replicates primarily within glial cells following intracranial inoculation of susceptible mice, with relative sparing of neurons. this study demonstrates that glial cells derived from neural progenitor cells are susceptible to jhmv infection and that treatment of infected cells with ifn-γ inhibits viral replication in a dose-dependent manner. although type i ifn production is muted in jhmv-infected glial cultures, ifn-β is produced following ifn-γ-treatment of jhmv-infected cells. also, direct treatment of infected glial cultures with recombinant mouse ifn-α or ifn-β inhibits viral replication. ifn-γ-mediated control of jhmv replication is dampened in glial cultures derived from the neural progenitor cells of type i receptor knock-out mice. these data indicate that jhmv is capable of infecting glial cells generated from neural progenitor cells and that ifn-γ-mediated control of viral replication is dependent, in part, on type i ifn secretion. inoculation of the neurotropic jhmv strain of mouse hepatitis virus (a positive-strand rna virus and a member of the coronaviridae family) into the cns of susceptible strains of mice results in an acute encephalomyelitis. the resulting infection is characterized by widespread viral replication in astrocytes, microglia, and oligodendrocytes with relatively few infected neurons knobler et al., ; parra et al., ; perlmane et al., ) . jhmv infection of the cns induces localized expression of pro-inflammatory factors that precedes and accompanies the activation and recruitment of immune cells into the cns. during the acute disease phase, infiltrating virus-specific cd + t cells control viral replication by two different effector mechanisms: ifn-γ secretion controls viral replication in oligodendrocytes, while a perforin-dependent mechanism promotes viral clearance from astrocytes and microglia (lin et al., ; parra et al., ) . while a robust and effective cell-mediated immune response is generated in response to jhmv infection, virus persists within the cns and is associated with the development of an immune-mediated demyelinating disease similar to the human demyelinating disease ms. during this stage, both t cells and macrophages are important in amplifying disease severity by contributing to myelin damage (cheever et al., ; perlman et al., ) . stem cells and neural precursors represent attractive sources for the generation of remyelination-competent cells since they can readily amplify and differentiate into oligodendrocyte committed cells (ben-hur et al., ; brustle et al., ) . stem cell-derived glial precursors have been shown to myelinate following transplantation into the myelin-deficient rat (brustle et al., ) , and neural precursor-derived glial-committed progenitors (ben-hur et al., ; keirstead et al., ) have been shown to remyelinate following transplantation into regions of acute experimental demyelination (keirstead et al., ) . more recently, intracerebroventricular or intrathecal implantation of neural precursors into rodents with eae, an autoimmune model of demyelination, resulted in the migration of transplanted cells into white matter and improved clinical outcome (ben-hur et al., ; pluchino et al., ) . while implantation of myelin-competent cells has shown to be effective in promoting remyelination in animal models of demyelination initiated by either infiltration of autoreactive lymphocytes or injury, there is limited information available with regards to the ability of these cells to enhance demyelination resulting from viral infection. we believe this is an important and clinically relevant question as the etiology of ms remains enigmatic although viruses have long been considered potential triggering agents for initiating disease (gilden, ; olson et al., ) . therefore, evaluating potential cellreplacement strategies for inducing remyelination in viral models of neurologic disease may yield insight into whether this method of (totoiu et al., ) . moreover, remyelination was not associated with dampened t cell infiltration into the cns as has recently been reported following nsc transplantation in mice with eae (aharonowiz et al., ; einstein et al., ; hardison et al., ) . having demonstrated that engraftment of glial cells promotes remyelination following jhmv-induced demyelination, we next were interested in addressing several interrelated issues including i) if glial cells derived from neural precursor cells were susceptible to infection and ii) how infection may be controlled within this population of cells. we believe these are relevant questions within the context of studying animal models of viral-induced demyelination as cells are being transplanted into the cns in which a persistent virus is present. therefore, analyzing the susceptibility of cellular progeny derived from neural precursor cells to viral infection is important in that these cells may represent important viral reservoirs in the face of persistent infection. understanding consequences of infection and how replication may be controlled within these cells will provide insight into understanding host defense mechanisms of implanted cells as well as potential relevance to disease outcome. the relevance of this is further highlighted by the fact that while previous studies have demonstrated that jhmv is able to infect and replicate within glial cells (dubois-dalcq et al., ; lavi et al., ; rempel et al., ) , the fate of neural progenitor cells as well as cells derived from this population to viral infection is not well characterized. in the present study, we demonstrate that primary cultures of glia derived from neural progenitor cells are susceptible to jhmv infection and support viral replication. additionally, while ifn-β production is dampened in response to viral infection, treatment with recombinant mouse ifn-γ inhibits jhmv replication. the ifn-γ-mediated antiviral effect is dampened in experiments using cells derived from type i ifn receptor-deficient mice (ifnar−/−) indicating a role for type i ifn signaling in limiting jhmv replication in glia-committed progenitor cells. therefore, these findings provide, to our knowledge, the first demonstration that glia-committed cells derived from neural precursors are susceptible to jhmv infection as well as identify a potential mechanism responsible for controlling viral replication. the in vitro culture of neural progenitor cells dissected from the striatal region of the brains of day postnatal c bl/ mice resulted in the generation of numerous neurospheres (fig. a ) (hardison et al., ; totoiu et al., ) . after the mature neurospheres were plated on an adherent matrix and incubated in growth medium, the majority of the cells exhibited oligodendrocyte morphology characterized by extensive arborization (fig. b) . immunocytochemical staining confirmed the morphology results indicating that ∼ % of the cells differentiated into oligodendrocytes (determined by galc staining) (fig. c ). the remaining cells had differentiated into either astrocytes (∼ %, gfap-expression) or neurons (b %, map staining) (fig. d ). these differentiated neural progenitor cultures were used for the subsequent studies examining jhmv susceptibility to infection. jhmv is able to infect and replicate in differentiated neural progenitor cultures as demonstrated by increasing viral titers measured at , , and h p.i. (fig. a) . immunocytochemistry revealed viral antigen distributed extensively throughout the monolayer (fig. b ). in addition, jhmv infection resulted in cytopathic effects by h p.i. characterized by wide-spread syncytia formation (fig. c ). these findings indicate that differentiated cells derived from neural progenitors are susceptible to jhmv infection and are capable of supporting replicating virus which results in extensive cytopathology. previous studies by parra et al. ( ) demonstrated that ifn-γ has an important role in controlling jhmv replication within oligodendrocytes of persistently infected mice. therefore, we next determined whether ifn-γ was capable of inhibiting jhmv replication following infection of differentiated neural progenitor cells. as shown in fig. a , treatment of jhmv-infected cells with recombinant mouse ifn-γ inhibited viral replication at ( % reduction, p b . ), ( % reduction, p b . ), and h p.i. ( % reduction, p b . ) compared to media-treated controls. moreover, the ifn-γ-mediated inhibition of jhmv-replication was concentration-dependent; titration of ifn-γ resulted in diminished antiviral effects (fig. b ). the pretreatment of cultures with ifn-γ ( u/ml) resulted in a significant (p b . ) reduction in viral titers at h p.i. when compared to cultures incubated with ifn-γ following infection (fig. c ). immunocytochemistry revealed that ifn-γ treatment of differentiated neural progenitor cultures limited the extensive cytopathic effects (figs. d and e) observed in untreated cells (fig. f ) as characterized by diminished syncytium formation. together these data indicate that ifn-γ activates differentiated neural progenitors to inhibit jhmv replication, which correlates with muted cytopathology. it is known that ifn-γ is capable of inducing expression of the non-elr chemokines cxcl and cxcl in numerous cell types including resident cells of the cns such as astrocytes and microglia (bhowmick et al., ; majumder et al., ; vanguri and farber, ) . moreover, in vivo astrocytes have been shown to express cxcl and cxcl mrna transcripts during the acute response to jhmv infection and in vitro cultured astrocytes are capable of expressing cxcl mrna transcripts (lane et al., ; liu et al., liu et al., , . neither cxcl nor cxcl are detectable in differentiated neural progenitor cultures in response to jhmv infection at or h p.i. (figs. a and b) . in contrast, ifn-γ treatment of infected cultures resulted in measurable levels of both cxcl and cxcl at and h p.i. (figs. a and b) . levels of cxcl were dramatically higher (∼ , pg/ml at h) compared to cxcl levels (∼ pg/ml at h) suggesting differential promoter sensitivities to ifn-γ treatment or altered stability at either rna or protein levels. to assess the importance of ifn-γ-mediated production of cxcl and cxcl in the inhibition of jhmv replication, neural progenitor cells were isolated from cxcl −/− mice and mice deficient in the signaling receptor for cxcl and cxcl , cxcr (cxcr −/− mice). the neural progenitor cells from deficient mice were differentiated in vitro, infected with jhmv and treated with ifn-γ. as we observed in wildtype mice, such treatment resulted in a significant reduction in viral titers at h p.i. compared to infected cells incubated with medium alone (figs. c and d). therefore, the ifn-γ-mediated anti-viral effect observed occurs independently of either production of cxcl , and cxcl or cxcr signaling. type i ifn (ifn α and β) exhibit potent antiviral activity and have recently been shown to be important in controlling jhmv replication in vivo (ireland et al., ) . therefore, we next evaluated production of type i ifn from differentiated progenitor cultures following jhmv infection. as shown in fig. a , jhmv infection did not result in detectable levels of ifn-α/β at either or h p.i. however, ifn-γ treatment of jhmv-infected cultures resulted in expression of ifn-α/β that was elevated compared to treatment with ifn-γ alone (fig. a) . further, direct treatment with either recombinant mouse ifn-α or ifn-β of jhmv-infected cultures resulted in a dramatic reduction in viral replication compared to media treatment (fig. b) . ifn-β exhibited ∼ % greater reduction in viral replication compared to ifn-α treatment indicating a more potent antiviral activity associated with ifn-β signaling (fig. b) . next, progenitor derived glial cultures were generated from type i ifn receptor-deficient mice (ifn-r−/−) mice and treated with ifn-γ following jhmv infection. as shown in fig. c , such treatment did result in a reduction in viral replication (p b . ) compared to media-treated controls by h p.i. however, viral replication was reduced, on average, by only % in ifn-r−/− cells compared to n % reduction in wildtype cells (figs. a and c). therefore, these data indicate that the ifn-γ-mediated antiviral effect is diminished in the absence of type i ifn signaling indicating that one mechanism by which ifn-γ promotes control of jhmv replication within glial-derived progenitors is through induction of type i ifn. the findings put forth in this paper provide, to our knowledge, the first demonstration that jhmv is capable of infecting and replicating within primary cultures of glia derived from neural progenitor cells. these findings are distinct from earlier studies (dubois-dalcq et al., ; lavi et al., ; rempel et al., ) showing that primary neural cultures are susceptible to viral infection, as we have allowed for differentiation of glial cells from neural progenitor cells into defined glia populations. in addition, we have demonstrated that ifnγ treatment of jhmv-infected cultures suppresses jhmv replication and this is dependent, in part, on secretion of ifn-i. the importance of ifn-i in defense following viral infection of the cns has been documented in several animal models. infection of mice in which ifn-i is genetically silenced or signaling blocked results in uncontrolled proliferation of west nile virus (samuel and diamond, ) , sindbis virus (burdeinick-kerr et al., ; byrnes et al., ) , and semliki forest virus (fragkoudis et al., ) . the cellular source of ifn-i production is controlled by viral tropism and the model system employed. for example, neurons are a primary source of ifn-i in response to west nile virus infection and these cells also represent a prominent cellular target for viral infection and replication (samuel and diamond, ) . similarly, robust cytokine production, including ifn-β, is observed following infection of astrocyte cultures with theiler's murine encephalomyelitis virus (tmev) (palma et al., ) . in the case of jhmv infection, emerging evidence highlights the importance of ifn-i in protection of the cns in response to infection. bergmann and colleagues (ireland et al., ) recently demonstrated increased mortality correlating with wide-spread jhmv dissemination throughout the parenchyma including expanded cell tropism with neurons infected in mice deficient in ifn-i receptor (ifnr−/−). additional support for an important role for ifn-i in host defense in response to infection with mouse coronaviruses are derived from studies that demonstrate increased disease severity following anti-ifn antibody treatment (lavi and wang, ) and enhanced resistance following treatment with recombinant ifn-β (matsuyama et al., ; smith et al, ) . further support for mouse coronaviruses in initiating ifn-i production following experimental infection of mice are provided by studies from cervantes- barragan et al. ( ) indicating that peripheral infection with a hepatotropic strain of mhv (mhv-a ) results in increased ifn-i production by plasmacytoid dendritic cells. while it is clear that ifn-i is produced in vivo in response to mouse coronavirus infection and participates in effective host defense, the molecular signals regulating expression on a cellular basis are less well characterized. indeed, ifn-β is not produced in response to jhmv infection of fibroblasts but this is not dependent on the absence of intracellular double-stranded rna or deficiencies in ifn-β signaling (roth-cross et al., ) . impaired ifn-β production within jhmvinfected cultures correlated with impaired translocation of transcription factors irf- and irf- into the nucleus of infection cells (versteeg et al., ; zhou and perlman, ) . in addition, it may be possible that double-stranded rna generated during the course of jhmv infection is not accessible to cellular pattern recognition receptors (ppr) such as rig-i, mda- , and tlr- (zhou and perlman, ) . while the molecular mechanisms associated with inhibited ifn-β production have not been completely defined, the mhv nucleocapsid protein has been suggested to be an ifn-i antagonist (ye et al., ) . data provided in the current study demonstrate that jhmv infection of differentiated progenitor cells resulted in muted expression of ifn-i and virus was able to replicate in an unrestricted manner. in addition, secretion of cxcl and cxcl was also impaired following jhmv infection of differentiated progenitor cultures and this is in contrast to previous findings indicating robust chemokine expression following infection of primary cultures of astrocytes. these results support the earlier hypothesis that within certain host cell populations, double-stranded rna generated during the course of jhmv replication may not be accessible to ppr and this impacts secretion of ifn-i and non-elr chemokines cxcl and cxcl . moreover, since the majority of progenitor cells (∼ - %) differentiate into oligodendrocyte progenitor cells (opc), it is possible that oligodendrocytes are unable to synthesize either cxcl or cxcl in response to jhmv infection. treatment of progenitor cultures with ifn-γ resulted in reduced jhmv replication and this highlights the importance of this cytokine in host defense following jhmv infection. additionally, these data support and extend studies by stohlman and colleagues (gonzalez et al., parra et al., ) that have demonstrated ifn-γ is critical in controlling jhmv replication in oligodendrocytes in vivo. infection of ifn-γ−/− mice with jhmv highlighted a critical role for this cytokine in controlling viral replication within oligodendrocytes (parra et al., ) . additionally, transgenic mice expressing a dominant-negative ifn-γ-receptor specifically on oligodendroglia demonstrated that ifn-γ is required for inhibiting viral replication (gonzalez et al., . the findings put forth in the present study clearly indicate that ifn-γ suppresses jhmv replication in glial-committed progenitor cells derived from neural precursors. moreover, the ifn-γ-mediated antiviral effect is not dependent on secretion of cxcr -binding chemokines. although the in vivo mechanism(s) by which ifn-γ evokes an antiviral response have not yet been defined, our data suggest that production of ifn-i by ifn-γtreated glia may contribute to viral control. further support for type i ifn in controlling jhmv replication within oligodendrocyteenriched cultures is provided by the demonstration that treatment of jhmv-infected oligodendrocytes with either recombinant ifn-α or ifn-β resulted in n log decrease in viral titers with ifn-β having a much greater anti-viral effect compared to ifn-α in controlling replication. neurotropic viruses are capable of infecting and replicating within opc (dietrich et al., ; levine et al., ; mock et al., ) . for example, human herpesvirus (hhv ) is capable of infecting and replicating within the human oligodendrocytes and suggested to be involved in the pathogenesis of both acute and chronic inflammatory demyelinating diseases (dietrich et al., ; mock et al., ) . hhv infection of human opc cultures results in formation of multinucleated syncytia and elevated expression of galc (dietrich et al., ) . opc proliferation was also impaired in hhv -infected cultures and infected cells and suggests that infection in vivo may have longlasting effects on precursor cell properties. these findings are interesting in that remyelination is relatively slow in jhmv-infected mice yet opc are present within and surrounding areas of on-going demyelination. this suggests that the ability of opc to successfully remyelinate axons is impaired and/or an environment that is conducive for promoting remyelination is not available. having demonstrated that jhmv is capable of infecting and replicating within primary cultures of opc indicates that these cells are susceptible to infection in vivo. therefore, it is interesting to speculate that early infection of neural progenitor cells impacts either generation of opc and/or the ability of opc to successfully remyelinate demyelinated axons at later stages of infection. we are currently addressing these possibilities. the neurotropic strain jhmv ( . v- ) of mouse hepatitis virus (mhv) was used for all experiments described here (fleming et al., ) . wild type mice for progenitor cell isolation, c bl/ mice (on the h- b background), were purchased from the national cancer institute (frederick, md). additional mouse strains used for progenitor cultures, cxcl −/−, cxcr −/−, and ifn-i receptor deficient (ifnar−/−) (c bl/ h- b background), were bred in the university of california, irvine animal facility. the animal protocols and procedures used for these studies were reviewed and approved by the institutional animal care and use committee of the university of california, irvine. neural progenitor cells were cultured as previously described (totoiu et al., ) . in brief, striata from to postnatal day mice were dissected, triturated and dissociated in . % trypsin-edta. the resulting single cell suspension was cultured for - days in ml serum free media (dmem:f supplemented with b supplement, × insulin-transferrin-selenium-x supplement, × penicillin-streptomycin and t ) with ng/ml human recombinant epidermal growth factor (egf; sigma-aldrich) (ben-hur et al., ) . media was replaced on days , , and ; culture supernatant and floating clusters were removed, centrifuged at × g for min and resuspended in fresh media with egf. after one week, cells had proliferated into numerous free-floating spheres. after one week, cell spheres were transferred to matrigel (bd bioscience, bedford, ma) coated flasks (use thin coat method, : dilution) at a low density. individual cells spread out from the attached spheres and formed a monolayer with to days. once the monolayer formed, cells were trypsinized, counted and plated into four chamber imaging slides (nalgene-nunc international, rochester, ny), -well plates or t flask (costar, corning, ny) previously coated with matrigel (bd biosciences). cells were allowed to equilibrate for an additional - days before viral infection or staining procedures were done. for all experiments shown, jhmv was added to cultures at a multiplicity of infection (moi) of . pfu/cell. virus was allowed to adsorb for h, cultures were washed with pbs and replaced with ml of fresh medium. recombinant mouse ifn-γ, ifn-α, and ifn-β cytokines were purchased from cell sciences (canton, ma). viral titers in supernatants of infected cultures were determined on dbt astrocytoma cells at defined time points post-infection (p.i.) (hirano et al., ; lane et al., ) . to assess differentiation potential, cells were grown on matrigel coated imaging slides for a total of days, fixed in % paraformalde-hyde (fisher scientific, fair lawn, nj) for min and immunofluorescence staining was performed using standard protocols. imaging chambers were blocked with % normal goat serum (ngs) (vector laboratories, burlingame, ca) for h at room temperature. primary antibodies (polyclonal rabbit anti-galc, chemicon, : dilution in % ngs; monoclonal mouse anti-map , sigma, : dilution in % ngs; polyclonal rabbit anti-gfap, invitrogen, : dilution in % ngs) or blocking solution (negative control, % ngs in pbs) were applied to chambers overnight at °c on rocker. slides were rinsed three times with pbs and fluorescent-conjugated secondary antibody (alexa , goat anti-rabbit or goat anti-mouse igg h + l, : dilution in % ngs; invitrogen) was applied and incubated for min at room temperature. slides were rinsed three times in pbs, and nuclear staining was with hoechst ( μg/ml in pbs, molecular probes, eugene, or) for min. cell quantification was conducted using an olympus bx- microscope, × magnification. the percentage of immunopositive cells was determined by dividing the total number of immunopositive cells by the total number of hoechst-positive cells in five images from each chamber, and averaging the results from three different chambers per marker. each -chamber imaging slide had one no-primary control chamber and three stained chambers for each of the markers mentioned above. only immunopositive cells with a hoechst-positive nucleus were counted. distribution of viral antigen in cultures was determined by immunoperoxidase staining, as specified by the manufacturer (vectastain-abc kit and dab peroxidase substrate kit; vector laboratories). imaging chambers were blocked with triton containing blocking buffer (bb), ( . % triton x- and % ngs normal goat serum in pbs), for h at room temperature. the anti-jhmv mab j. . ( : dilution in bb) specific for the carboxyl terminus of the viral nucleocapsid (n) protein was applied and incubated overnight at ϒc. slides were rinsed three times in pbs and secondary antibody (biotinylated goat-anti-mouse igg h + l, vector laboratories, : dilution in bb) was applied and incubated for . h at room temperature. slides were rinsed three times in pbs and counterstained with hematoxylin (bergmann et al., ; fleming et al., ; walsh et al., ) . differentiated neural progenitor cells were infected with mhv, and levels of ifn were measured using a bioassay based on inhibition of vsv growth in l cells. supernatants were harvested and exposed to uv light to inactivate infectious virus. l cells infected with pfu vsv were treated with dilutions of supernatants or recombinant murine ifn-β (pbl biomedical laboratories, piscataway, nj) at min post-infection (p.i.). titers of vsv were determined on vero cells. ifn levels were calculated based on standard curves generated with recombinant ifn-β. opc culture supernatants were used to measure chemokines cxcl and cxcl . elisas were performed using the duoset mouse cxcl and cxcl elisa kit (r & d systems, minneapolis, mn), as specified by the manufacturer. statistically significant differences between groups of mice were determined by student's t test and p values of b . were considered significant. neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis growth and fate of psa-ncam+ precursors of the postnatal brain transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis perforin-mediated effector function within the central nervous system requires ifn-gamma-mediated mhc up-regulation induction of ip- (cxcl ) in astrocytes following japanese encephalitis embryonic stem cell-derived glial precursors: a source of myelinating transplants viral-induced neurodegenerative disease synergistic roles of antibody and interferon in noncytolytic clearance of sindbis virus from different regions of the central nervous system control of sindbis virus infection by antibody in interferon-deficient mice control of coronavirus infection through plasmacytoid dendritic cell-derived type i interferon a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin infection with an endemic human herpesvirus disrupts critical glial precursor cell properties cell tropism and expression of mouse hepatitis viruses (mhv) in mouse spinal cord cultures transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv- ) virus pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies the type i interferon system protects mice from semliki forest virus by preventing widespread virus dissemination in extraneural tissues, but does not mediate the restricted replication of avirulent virus in central nervous system neurons infectious causes of multiple sclerosis expression of a dominant negative ifn-gammareceptor on mouse oligodendrocytes inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination transplantation of glial-committed progenitor cells into a viral model of multiple sclerosis induces remyelination in the absence of an attenuated inflammatory response utility of mouse cell line dbt for propagation and assay of mouse hepatitis virus type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells polysialylated neural cell adhesion molecule-positive cns precursors generate both oligodendrocytes and schwann cells to remyelinate the cns after transplantation selective localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy dynamic regulation of alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease a central role for cd (+) t cells and rantes in virus-induced central nervous system inflammation and demyelination the protective role of cytotoxic t cells and interferon against coronavirus invasion of the brain coronavirus mouse hepatitis virus (mhv)-a causes a persistent, productive infection in primary glial cell cultures reactions of oligodendrocyte precursor cells to alpha herpesvirus infection of the central nervous system mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis the t cell chemoattractant ifn-inducible protein is essential in host defense against viral-induced neurologic disease expression of mig (monokine induced by interferon-gamma) is important in t lymphocyte recruitment and host defense following viral infection of the central nervous system regulation of human ip- gene expression in astrocytoma cells by inflammatory cytokines protective effects of murine recombinant interferon-beta administered by intravenous, intramuscular or subcutaneous route on mouse hepatitis virus infection infection of murine oligodendroglial precursor cells with human herpesvirus (hhv- ) -establishment of a murine in vitro model a virus-induced molecular mimicry model of multiple sclerosis infection with theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via nf-kappab activation: potential role for the initiation of demyelinating disease ifn-gamma is required for viral clearance from central nervous system oligodendroglia coronaviruses: hepatitis, peritonitis, and central nervous system disease injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis viral induction of central nervous system innate immune responses inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival intranasally administered alpha/beta interferon prevents extension of mouse hepatitis virus, strain jhm, into the brains of balb/cbyj mice remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the mhv model of multiple sclerosis ifn and virus-inducible expression of an immediate early gene, crg- /ip- , and a delayed gene, i-a alpha in astrocytes and microglia group coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition expression of cxc chemokine ligand from the mouse hepatitis virus genome results in protection from viral-induced neurological and liver disease mouse hepatitis coronavirus a nucleocapsid protein is a type i interferon antagonist mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna this work was supported by the national multiple sclerosis society grants (t.e.l.) and (s.p.) and the national institutes of health grant ns to t.e.l. key: cord- -fod xkd authors: summerfield, artur; mccullough, kenneth c. title: the porcine dendritic cell family date: - - journal: dev comp immunol doi: . /j.dci. . . sha: doc_id: cord_uid: fod xkd considering the pivotal roles played by dendritic cells (dcs) in both innate and adaptive immune responses, advances in the field of porcine immunology dc biology have recently progressed rapidly. as with the more extensively studied murine and human dcs, porcine dc can be generated from bone marrow haematopoietic cells or monocytes, and have been analysed in various immunological and non-immunological tissues. both conventional dc (cdc) and plasmacytoid dc (pdc) have been characterized. the function of porcine monocyte-derived dc has not only been characterized in terms of antigen presentation and lymphocyte activation, but also their response to various ligands of pattern recognition receptors. these have been characterized in terms of the induction of dc maturation and pro-inflammatory, th -like or th -like cytokines secretion. porcine pdc most effectively sense virus infections and are characterized by their capacity to produce large quantities of ifn-α and the pro-inflammatory cytokines tnf-α, il- and il- . as such, the dc family as a whole is a powerful ally in the host battle against pathogen attack. nevertheless, dc in particular tissue environments or under particular stimuli can down-regulate immune response development. this is not only important for preventing over-activation of the immune system and also for ensuring tolerance against self or “friendly” substances including food components, but may also be used as a mechanism of pathogens to evade immune responses. dendritic cells (dcs) are a heterogeneous group of potent antigen-presenting cells (apcs) with the unique capacity to prime naive t-cell responses [ ] . in order to fulfil their role as sentinels of the immune system, they express several families of specialized pattern recognition receptors (prrs) for particular pathogenassociated molecular patterns (pamps). these include toll-like receptors (tlrs), nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), retinoic acid-inducible gene i (rig-i)like receptors (rlrs) and c-type lectin receptors (clrs) all reacting directly with pathogen components [ ] [ ] [ ] . in addition, many other receptors exist such as fc receptors and activated complement component receptors, reacting with complexes of pathogen antigen with antibody or complement. another element essential to dc biology is their migratory behaviour in response to chemokine gradients. dcs are the main cellular element controlling t lymphocyte activation and regulation. furthermore, they are involved in b-cell responses, possibly through the delivery of native antigen to b lymphocytes [ ] [ ] [ ] , but certainly through the production of b-cell stimulatory factors important for b-cell proliferation, differentiation and isotype switching [ , ] . dcs also play a functional role for nk cell activation [ , ] , and are in a two-way communication with neutrophils [ ] . understanding dc biology requires the consideration of the heterogeneity and independence of dc functional plasticity. an important element therein is the functional and phenotypic differentiation of ''conventional dc'' (cdc) from ''plasmacytoid dc'' (pdc). the term cdc summarizes all dc subsets with ''professional antigen presenting'' function, while pdc represent the ''professional interferon-a producers'' [ ] . indeed, pdc are also referred to as ''natural interferon producing cells'' (nipc), a functional entity first described years ago [ ] . however, more recent studies have demonstrated that also pdc have important antigen-presenting functions and that the two dc subsets complement each others by having a distinct regulation of mhc class i-and ii-dependent antigen presentation [ ] [ ] [ ] . furthermore, dcs show a high level of heterogeneity, particularly in the specialized roles of various dc subsets dependent on their tissue localization and local immunological environment, which guides their function (table ) . one general functional consideration of all dc subsets is their critical roles as immunological sentinels. being strategically located at sites of pathogen entry, such as mucosal surfaces and considering the pivotal roles played by dendritic cells (dcs) in both innate and adaptive immune responses, advances in the field of porcine immunology dc biology have recently progressed rapidly. as with the more extensively studied murine and human dcs, porcine dc can be generated from bone marrow haematopoietic cells or monocytes, and have been analysed in various immunological and nonimmunological tissues. both conventional dc (cdc) and plasmacytoid dc (pdc) have been characterized. the function of porcine monocyte-derived dc has not only been characterized in terms of antigen presentation and lymphocyte activation, but also their response to various ligands of pattern recognition receptors. these have been characterized in terms of the induction of dc maturation and proinflammatory, th -like or th -like cytokines secretion. porcine pdc most effectively sense virus infections and are characterized by their capacity to produce large quantities of ifn-a and the proinflammatory cytokines tnf-a, il- and il- . as such, the dc family as a whole is a powerful ally in the host battle against pathogen attack. nevertheless, dc in particular tissue environments or under particular stimuli can down-regulate immune response development. this is not only important for preventing over-activation of the immune system and also for ensuring tolerance against self or ''friendly'' substances including food components, but may also be used as a mechanism of pathogens to evade immune responses. ß elsevier ltd. all rights reserved. dermal layers, dc can rapidly interact with pathogen pamps resulting in cell activation, pathogen uptake and degradative processing of the pathogen (antigen). a critical element therein is the concomitant uptake of antigen with pamp recognition. the latter represents the ''danger'' signalling which the dc requires to be activated. in the absence of the ''danger'' signal, the dcs tend to function more as tolerogenic dc, inducing lymphocyte anergy. such processes are involved in responses to self-antigens and tolerance of food antigens. following interaction with a pathogen (and its pamps), cdc endocytic activity-particularly macropinocytosis-is enhanced during the first h after stimulation, followed by down-regulation as the dc mature. the latter relates to a prolonged capacity for efficient presentation of the endocytosed and processed antigen [ ] . with completion of the dc maturation process, important biological changes occur to the dc. chemokine receptor expression is modified, enabling migration to the inductive sites of the adaptive immune system. for example, the ccr and ccr receptors for inflammatory chemokines such as ccl and ccl are down-regulated, while the ccr receptor for ccl and ccl is up-regulated [ ] . the latter provides dc with the signal for entry into the inductive sites of the adaptive immune system such as the lymph node. dc activation also results in an increase of cell surface mhc and co-stimulatory molecule expression-such as cd and cd -as well as the production of immunoregulatory and/or inflammatory cytokines. mhc class ii antigenic peptide complexes are stabilized on the cell surface to ensure efficient stimulation of t-cell responses. overall, the induced functional changes are usually associated with increased t-lymphocyte stimulatory capacity, although this depends on the stimulus received [ ] . clearly, the interaction of microbial pathogens with dc can provide insight into the pathogenesis of and defence against infectious diseases. moreover, the central role of dc in immune defence development makes them a prime target for vaccines and immunotherapies. recent advances in porcine immunology have allowed the characterization of the porcine dc system in this direction (as witnessed by the identified cell types shown in table ). this has also been possible through the availability of antibodies against cell surface markers classified in three international swine cd workshops [ ] [ ] [ ] and studies using crossreactive antibodies-summarized in table . by such means, rapid advancement in the current knowledge on porcine apc and the diversity of porcine dc function has been forthcoming. the subsequent sections of this review will present these advancements. similar to other species, porcine dc can be generated by stimulating blood monocytes with interleukin- (il- ) and granulocyte macrophage-colony-stimulating factor (gm-csf). after - days of culture, non-adherent or loosely adherent cells with dendritic morphology can be harvested [ , ] . generation of monocyte-derived dc (modc) in vitro can also employ a gm-csf/ ifn-a combination. this can prove more potent than the gm-csf/ il method when seeking dc for restimulating virus-specific cytotoxic t-cells [ ] . addition of ifn-a to the gm-csf/il- cocktail also influences the dc, resulting in an enhanced t-cell a two subsets. b variable, two subsets with some animals (see also fig. ). c can be negative in mucosal tissue [ ] . d not determined. e four subsets based on cd a/cd ri expression and dependent on localization in lp, pp and mln [ ] . f negative on lp dc [ ] . g two subsets: cd a + cd + cd + and cd a À cd À cd À . stimulatory capacity in mixed leukocyte cultures [ ] . in fact, cytokine modulation is important for manipulating the type of dc generated. tgf-b permits the generation of cells with langerhans cell characteristics [ ] , while pamps modulate mrna expression levels for particular tlrs [ ] . bautista et al. [ ] have also demonstrated that il- can be replaced by il- for the generation of modc. phenotypically, porcine modc are characterized as cd + cd + cd + cd / + cd a + and mhc class ii + [ , , [ ] [ ] [ ] (table ) . from a comparative immunological point of view, it was unexpected to find cd , because human cd is considered to be a typical monocyte/macrophage rather than a dc marker [ ] . nevertheless, modc from other species such as cat, cattle and dog have also been shown to express cd [ ] . of the other markers, the cd a was expected due to its classification as the porcine swine workshop cluster (swc ) antigen expressed on cells of the myelomonocytic lineage [ ] . it is expressed on many monocytic and granulocytic cells quite early during their differentiation [ ] . functionally, this marker represents the signal regulatory protein alpha (sirp-a). altogether, the co-expression of cd a and cd along with relatively high levels of both cd / and mhc class ii represent phenotypic characteristics of porcine modc but no marker clearly differentiating them from monocyte-derived macrophages has been identified. porcine modc generated with gm-csf and il- relate to human modc in that they are in an immature state, and represent a convenient cell culture model to study the dc maturation process. akin to their human counterparts, porcine modc up-regulate cd / , mhc classes i and ii, and t-cell stimulatory activity upon maturation, while inflammatory chemokine receptors such as ccr and macropinocytic activity are down-regulated [ , , , [ ] [ ] [ ] . porcine dc, which phenotypically and functionally resemble modc, can also be generated from bone marrow haematopoietic cells (bmhc), stimulated with gm-csf plus tnf-a, or gm-csf alone, for - days-bmdc [ ] . addition of stem cell factor to the gm-csf/tnf-a cocktail increases the yield of dc obtained [ ] . in contrast to gm-csf, flt ligand (flt l) stimulation induces the differentiation of both cdc and pdc, similar to the situation with human and mouse bmhc [ ] (guzylack-piriou and summerfield, unpublished data). furthermore, flt l-induced bmhc-derived cdc phenotypically and functionally differ from gm-csf-derived dc either generated with monocytes or bmhc. flt l induces the differentiation of cd À cdc which are more sensitive to stimulation by tlr /tlr , tr , tlr , tlr and tlr ligands in terms of cytokine responses and maturation [ ] (guzylack-piriou and summerfield, unpublished data). this may relate to the central role of flt l in the generation of dc from a clonogenic bmhc dc precursor [ ] . porcine blood is similar to human blood in representing an important source of apc. several populations have been identified, including monocytes, dc precursors and fibrocytes. all of these express cd a, and in a model of antigen presentation of inactivated foot-and-mouth disease virus (fmdv) to t lymphocytes the activation was dependent on the presence of cd a + cells [ , ] . similar results were also obtained with classical swine fever virus (csfv) and protein antigens derived from this virus [ , ] , as well as with tetanus toxoid [ ] . separating the cd a + pbmc into a major population of cd + monocytes and a minor population of cd À cells showed that the latter contained the cd À blood cdc along with the cd + pdc (table ; [ ] ). it is notable that although the blood cdc and pdc differ phenotypically (cd expression), they are similar in their lack of cd expression, which distinguishes them from in vitro generated modc. however, this simple discrimination is not absolute. the composition of blood apc is more complex, as summarized in fig. . accordingly, it is necessary to appreciate each of the blood apc in turn, to obtain a better understanding of their roles and function in immune defences. porcine blood carries a subpopulation of pbmc, which are cd a + cd À cd À , with characteristics of cdc-high levels of mhc class ii and cd / ( fig. and table ), nonadherence and potent t-cell stimulatory capacity [ ] . after in vitro culture, these cells strongly up-regulate mhc and co-stimulatory molecules, and their dendritic morphology becomes clear. based on these characteristics, we have proposed that this population contains the precursors of blood cdc. these cells have variable expression of cd and cd and can be differentiated from monocytes by lower levels of cd a ( fig. and [ ] ). although the majority of porcine blood monocytes are cd À cd À cd + cd + cd a + (table and fig. ), a detailed analysis of the monocytic population has identified at least four subsets based on cd and cd [ ] . it is currently not clear how the blood cdc population phenotypically defined as cd a+cd À cd À cell relates the cd À cd + monocytic cell. cd is proposed as a macrophage marker, which is up-regulated during the differentiation of monocytes to macrophages [ ] but down-regulated if monocytes are induced to differentiate into dc by gm-csf/il- [ ] . this would argue for two distinct subsets of cells, a point which is not surprising considering the diversity of the dc family, but still requires further clarification (see also dominguez et al., this volume). based on the frequent expression of the cd r marker on dc in the mucosa [ , ] , this marker has also been proposed for identification of cdc in the blood and other organs as a cd a + cd r + population [ , ] . however, this definition may not be sufficient as no functional studies have been described, and it does not consider that a subset of monocytes also express cd r [ ] . although several cell types can produce type i ifn upon viral infection, pdc are particularly adept at secreting very high levels of type i ifns [ ] . representing less of . % of the pbmc [ ] , porcine pdc were identified through their ifn-a responses to transmissible gastroenteritis virus (tgev) [ ] . charley and lavenant [ ] originally described them as being non-adherent, non-t, non-b, cd +, mhc-class-ii positive cells. subsequently, they went on to describe their ontogeny [ , ] and migration into lymphoid tissue after viral challenge in vivo [ , ] . recently, using the ovine and porcine models, it has been demonstrated that pdc also migrate in the afferent lymph of cannulated animals [ ] . phenotypically, porcine pdc can be clearly identified as cd a low cd high cd À cd À within the peripheral blood population (table ). they express no or low levels of the t-cell markers cd , cd and cd , as well as no cd [ ] , but can carry low levels of cd and moderate levels of cd (fig. ). related to their interaction with immune complexes porcine pdc express fc receptors including cd (fig. , [ ] ) and cd [ ] . in contrast to porcine pdc, ovine pdc do not express cd and cd a but are characterized by high expression of cd rb [ ] . porcine pdc appear to be the major dc population producing high quantities of ifn-a and tnf-( in response to cpg motifs [ ] . this clearly relates to the human dc system, placing the porcine dc system in line with that of humans and therefore distinct from the murine model [ ] . certainly related to their human and mouse counterparts is their ability to respond to many viruses by the production of large quantities of ifn-a (see below). with the major site for pathogen entry being through the mucosa of the respiratory and digestive tract, it is not surprising that most lymphocytes are in the mucosalassociated lymphoid tissues (malt). consequently, it is essential to regulate the mucosal immune responses against harmless microorganisms and food antigens in contact with the mucosae. this is a major role played by mucosal dc and is reflected by their anatomical localization in the mucosal tissues. they can mediate tolerance to self-antigens and harmless entities, while acting as sentinels sensing the danger posed by invading pathogens and deleterious entities. the first report on porcine mucosal dc described a putative dc in peyer's patches (pp) as mhcii + cell lacking t-and bcell markers [ ] . in the lamina propria (lp) of the small intestine the presence of an mhcii+cd a + cd r + cd + dc subset was demonstrated [ ] . bimczok and colleagues [ ] further char-acterized dc in other immunological sites of the gut and proposed four phenotypically distinct subsets of dc based on the expression of cd r and cd a. lp dc are mainly cd a + cd r +, pp dc are mainly cd a + cd r À in subepithelial domes and cd a À cd r À in interfollicular regions, and mesenteric lymph node (mln) dc are mostly cd a À cd r + . interestingly, only the cd r + dc subsets were present in lymph, suggesting that dc migration to mln originates largely from the lp. with respect to antigen sampling a rare population of lp dc extending cytoplasmic processes between enterocytes have been described [ ] . in the pp, it appears that antigen transfer from m cells to dc is an important process, as many dc in the subepithelial dome have been demonstrated to be adjacent to m cells [ ] . in contrast to the lack of cd a on many dc in the inductive interfollicular areas of the mln and pp, cd a is expressed on porcine dc is at peripheral sites of pathogen entry and antigen contact such as the skin, lp and pp subepithelial dome [ , , ] . this would indicate a possible down-regulation of this molecule during the maturation and migration process, and would be supported by the observed expression of cd a on modc, bmdc generated using either gm-csf or flt l, as well as circulating blood cd a + blood apc can be differentiated into cd + monocytic cells and cd À dc. two major subpopulations of cd À monocytic cells can be defined based on cd expression (depicted light blue; see also dominguez et al., this issue). furthermore, a small cd + cd + cd + subset with unknown function can be identified (dark blue). porcine blood dc express relatively low levels of cd a and lack cd and cd . while cdc are cd À (green), pdc express high levels of cd (red). (b) expression of cd , mhc class ii, cd , cd , cd and cd on cd a high cd À monocytes (light blue dots), cd a high cd + monocytic cells (dark blue dots), cd a low cd À cdc (green dots) and cd a low cd + pdc (red dots). a representative animal is shown. comparison of five different spf pigs of the similar age ( - month old) revealed high variability in the expression levels of mhc class ii, cd , cd and cd on the dc subpopulations. dc [ , , ] . nevertheless, with in vitro maturation studies using modc, bmdc as well as blood dc [ , , , , ] no loss of cd a was observed indicating that additional factors would be required for this process. an alternative explanation would be that cd a À dc represent lymph node tissue resident dc which are phenotypically distinct from in vitro generated dc. in humans, rat, cattle and sheep, the expression of cd a differentiates functionally distinct dc subsets. while cd a + dc are more stimulatory for t cells, it is possible that the cd a À subsets is specialized in the phagocytosis of apoptotic cells [ ] [ ] [ ] [ ] [ ] . future studies are required to clarify such functional differences in the pig. dcs from the porcine upper respiratory tract have also been described. in the tracheal mucosa many dc are located above the basal membrane and inside the epithelial layer where they form a dense network with many cytoplasmic processes probably related to their important role as immunological sentinels in this organ [ ] . the majority of these cells co-express cd and mhc class ii but not cd r . jamin et al. [ ] recently described putative dc in the tonsils by co-expression of cd r and cd (dc lamp) or coexpression of cd r and cd a. nevertheless, from this study it is unclear whether a cd a À dc would exist in this organ. it is also not yet clear how cd a is expressed in dc of non-mucosal lymphoid tissue. several functional properties have been assigned to mucosal dc originating from the gut including the capacity to imprint the mucosal homing receptors a b integrin and ccr on t and b lymphocytes, the secretion of cytokines of the mucosal microenvironment such as il- and tgf-b, the promotion of t regulatory and th rather than th responses, and the induction of iga secretion [ ] [ ] [ ] [ ] [ ] . considering that the capacity of gut dc to produce retinoic acid (ra) is a requirement for many of these functions and that gut dc are likely to be themselves under the influence of ra derived from gut epithelial cells, we have tested whether porcine modc can acquire the function of gut mucosal dc. after treatment of porcine modc with ra the dc acquired the capacity to promote a b integrin and ccr on t lymphocytes, to secrete tgf-b and to promote iga responses [ ] . we have extended these studies and also demonstrated that ra induces the expression of retinaldehydrogenase, a rate-limiting enzyme in the synthesis of ra. the drug-mediated inhibition of this enzyme in ra-treated dc abrogated their capacity to promote mucosal homing receptors expression on lymphocytes (saurer and summerfield, unpublished data). this underlines the important role of tissue-specific factors in governing dc function. nipc/pdc have been identified as ifn-a-positive cells by immunohistochemistry in the intestinal epithelial layer, the lp, near the pp and in the mln early after infection with transmissible gastroenteritis virus (tgev) [ ] . since this was associated with high levels of serum ifn-a and only few ifn-a producing cells were identified in other organs it appears that during an enteropathic virus infection ifn-a would almost exclusively originate from gut pdc triggered locally. the rapid ifn-a response of pdc as early as h after infection would imply that pdc are present in mucosal tissue fulfilling their role as sentinels. this relates to the recently identified ccr expression and migration of mouse pdc to the small intestine under steady-state conditions [ ] and also to the presence of pdc identified as cd a + cd + cells in tonsils and mln of healthy pigs [ ] . bautista et al. have isolated dc migrating from porcine skin explants. phentotypically these cells resemble modc in terms of cd a co-expression with cd and the high levels of mhc class ii and cd / (table ) . moreover, isolated skin-derived dc show variable expression of cd r , cd and cd [ ] . immunohistochemical analysis of porcine thymic tissue has shown dc to be large cells located in the medullary and the corticomedullary regions, as evidenced by the presence of surrounding hassall's corpuscles. porcine thymic dc have also been partially purified and characterized [ ] . they too relate to modc and skin dc in their cd , cd a and mhc class ii expressions, but additionally express cd (table ) , which can also be found on blood dc (fig. ) . croizet and colleagues [ ] demonstrated the value of the porcine model for characterizing dc from non-lymphoid organs, such as the thyroid dc. besides the more ''classical'' apc, a relatively recent addition has been described. fibrocytes are a blood-derived cell population with fibroblastoid morphology, which is distinct from dc. fibrocytes have been described for mice, humans and pigs [ , , ] , and represent . - % of nucleated cells in peripheral blood. they express cd and cd , which would suggest a haematopoietic origin, possibly myeloid. the presence of cd , cd and cd a on porcine fibrocytes [ ] , and the differentiation of human fibrocytes in vitro from a blood-derived cd + population [ ] support this hypothesis. porcine fibrocytes originate from a cd + pbmc subpopulation [ ] . fibrocytes are important during wound healing, rapidly entering sites of injury together with inflammatory leukocytes [ ] . they are also an important source of cytokines and chemokines important for t lymphocyte and dc development. these include il- , il- , macrophage-colony-stimulating factor, macrophage inflammatory protein- a, mip- b and monocyte chemoattractant protein- [ ] . it has been suggested that fibrocytes may play an early role in the induction of antigen-specific immunity [ , ] , in particular the activation of t helper lymphocytes [ ] . porcine fibrocytes were also shown to be potent apc, relating to their expression of mhc class ii, cd and cd /cd , as well as their endocytic activity [ ] . these cells activate cd + t cells, but also efficiently stimulate virus-specific cd + ctl. in fact, fibrocytes are effective at low ratios with t lymphocytes, ratios at which modc are less efficient [ ] . porcine fibrocytes also respond to tlr ligands by producing large quantities of il- [ ] . these tlr ligands include lps (tlr -ligand), lipopeptide (diacylated form recognized by tlr /tlr heterodimers; triacylated form recognized by tlr / tlr heterodimers), and tlr ligands [ ] . the importance of dc within the innate immune system is due to their recognition of pamps through their prrs. tlr play a central role in this concept of innate immune recognition. this results in a robust cytokine and chemokine response, along with dc activation and maturation, all essential for adaptive immune response development (fig. ) . a simplified concept is that antigen presentation in the absence of dc activation leads to tolerance or shortlived immune responses without immunological memory development [ ] . the current evidence shows that concomitant recognition of the antigen and a ''danger'' signal is essential for the dc to promote adaptive immune defence development [ ] . with self and food antigens, the absence of the ''danger'' signal ensure that the dc involved are tolerogenic. for efficacious immune defence development, the cell subsetspecific recognition of pamps plays a critical role, resulting in the dc-derived cytokines necessary for both the innate response and the strength and quality of the adaptive response. in addition to phenotypic differences, it is also in this area of pamp recognition that species-dependent differences are observed. consequently, it is important to understand how porcine apc recognition of pamp compared with human apc, and to determine the distribution of tlr within the porcine dc system. in fig. , a schematic overview of the functional specialization and cytokine production of porcine cdc and pdc is represented. the current knowledge on the cytokine responses of modc is also included in the overview provided by table . one of the important responses induced by tlr ligation on dc is the induction of maturation. as a simple read-out of this process increased levels of surface molecules involved in antigen presentation such as mhc class ii, cd and cd / are often used. nevertheless, the interpretations of such results alone do not permit definitive conclusions on the maturation status of a dc in terms of t-cell stimulatory activities [ ] . porcine modc as a model of cdc relate to those from other species in their response to tlr ligands by upregulation of mhc and cd / (table ). these include tlr ligands pam cys lipopeptide, pseudomonas opri lipoprotein, lipoteichoic acid (lta) and peptidoglycan [ , ] , tlr ligands such as synthetic doublestranded (ds) rna polyinosinic-polycytidylic acid (polyic) [ , , , ] , tlr ligand lipopolysaccharide (lps) [ , , ] and the tlr ligands r and polyuridylic acid [ ] . similar to human dc, both porcine modc and blood cdc have been reported to be unreceptive to the tlr ligand cpg-odn, when analysed for mhc class ii and cd / expression [ ] . nevertheless, modc express tlr mrna [ , ] , and cpg-odn can induce the mrna of tlr , tlr , ifn-g and il p , as well as increased levels mhc class ii as well as cd / [ ] . such discrepancies between the studies may have been caused by sequence differences in the odn employed or may reflect differences in the responsive status of the cells employed. for example, the relatively modest increase of cd / and mhc class ii expression on modc after tlr stimulation is synergistically enhanced in the presence of ifn-a [ ] . in this study it was also shown that a full phenotypic maturation only occurred during antigen presentation to t lymphocytes. we have also observed that in contrast to modc, flt -ligand derived bmdc are clearly more responsiveness to a range of tlr ligands including tlr , , , and (guzylack-piriou and summerfield, unpublished data). tlr ligation can induce cytokine mrna species for il- , il- p , il- and ifn-g in dc, although this depends on the tlr ligand employed [ ] . while polyic, lps, lta, and cpg induced the th promoting cytokines il and ifn-g, the th -like cytokines il- and il- were induced only by lps and lta (table ) . raymond et al. [ ] also demonstrated that similar to human and mouse dc, the cytokine response of porcine dc can be modulated by cytokines. for example, il- p mrna can be enhanced with tnf-a, il- and ifn-g and reduced with il- treatment of dc. along the same line, the th /th cytokine profile will depend on the type of antigen encountered [ ] . at the protein level modc have been reported to produce tnf-a and il- after stimulation with poly(ic), lps, opri and pam cys (both tlr ligands), while production of il- and il- has been difficult to detect by elisa [ , , , ] . related to the stimulation by these specific tlr ligands is the cytokine response of modc to heat-inactivated actinobacillus pleuropneumoniae in terms of il- , il- and il- secretion [ ] . modc also produce type i ifn after stimulation with poly(ic) and mrna transfection [ , , ] . the latter is dependent on the secondary structure of the mrna molecule forming dsrna structures [ ] . it is important to note that modc maturation and cytokine production is highly variable and subject to immunomodulation. another example for this is the observation that ra has a potent synergistic effect on il- secretion induced by tlr ligands [ ] . this relates to the fact that the dc family has a high diversity to deal with the varying environments and signals it receives in vivo from the local tissue environment and from pathogens. with in vitro analyses, it is only possible to reproduce a small fraction of this diverse scene. therefore, what we are observing in vitro is true for the conditions being created, but can not be taken as a general rule for all dc subsets under all conditions in vivo. as with cdc, the pdc can also upregulate mhc class ii and costimulatory molecules after in vitro culture [ ] and when stimulated with tlr ligands [ ] . however, the notable trait of pdc is their ability to produce inf-a similar to the human and mouse immune system, porcine pdc are the most potent producers of inf-a after stimulation with certain pamps. these pamps are the cpg type a motifs [ ] and tlr / ligands such as r [ ] . such characteristics show a particularly close relationship to the human dc system [ ] ; pdc respond to tlr and ligands, whereas cdc such as modc tend to recognize more tlr - ligands. in contrast, murine dc and macrophages also respond well to tlr ligands, although it is again the pdc which produce large quantities of ifn-( [ ] . dependent on the stimulus, porcine pdc also produce large quantities of tnf-a, il- and as mentioned above il- [ , ] . it is this il- induction, which distinguishes porcine pdc from their human counterparts [ ] . the dc family is particularly effective at sensing viruses through cytosolic and endosomal prr, which detect viral nucleic acid. important cytosolic receptors include the dsrna-dependent protein kinase r (pkr) and the rlr's rig-i and mda- [ ] . generally, such receptors recognize viral replication occurring in situ-being cytosolic, these prr will detect the intermediates, which are also usually cytosolic. while rig-i can sense triphosphorylated singlestranded rna, mda- appears to be more specific for dsrna, but the fine specifity within the helicase system is not entirely clear [ ] . although these receptors are ubiquitously expressed, their important role in the interaction of cdc with viruses has been demonstrated [ ] . this is different with the endosomal prr, all members of the tlr family including tlr , , and , which are expressed only on cells playing a specialized role in innate immune responses such as dc. tlr represents a receptor for dsrna, tlr and for single-stranded rna and tlr for cpgmotif containing dna. tlr will not only sense viruses with dsrna genomes but also endocytosed dsrna replicative intermediates produced during the replicative cycle of singlestranded rna viruses, released from dying cells in the vicinity. as summarized in table , the studies with porcine cdc and pdc demonstrate that similar to human and mouse, pdc produce high levels of ifn type i to most viruses studied including classical swine fever virus, fmdv, influenza virus, lentiviral vectors (lv), pseudorabies virus (prv) and tgev, while such responses are absent or weak with cdc. the only exceptions described to date are the porcine circovirus , which can suppress ifn response in pdc [ ] , and sendai virus which is a potent inducer of ifn type i in cdc [ ] . an important basis for the responsiveness of pdc to viruses is the expression of endosomal tlr and tlr , which can be triggered by dna and rna viruses respectively independently of viral replication [ ] . in addition, the constitutive expression of irf , the master regulator of type i ifn, represents a unique feature of pdc [ , ] . a good example showing the importance of pdc recognition of viruses is the response seen with csfv. this monocytotropic, haemorrhagic rna virus replicates efficiently in cdc without apparently inducing their activation or maturation [ ] . the lack of dc activation is not due to the absence of a trigger, because the virus generates a dsrna intermediate in infected modc theoretically capable of stimulating rlrs. csfv actively prevents the cdc response to dsrna through its non-structural n pro protein targeting the irf- pathway [ ] , on which cdc depend for induction of ifn [ ] . in contrast, in pdc csfv will induce ifn-( production [ ] . as mentioned above, these cells do not rely on irf- due to their high levels of constitutive irf- [ ] . the production of large quantities of ifn-( in the serum of csfv-infected pigs [ , ] presumably originating from pdc [ ] indicates the importance of pdc for systemic ifn-( responses, particularly when cdc activities are impaired by the pathogen. in fact, a number of rna viruses encode proteins interfering with cellular antiviral machinery and therefore prevent activation of cdc [ ] . also with porcine reproductive and respiratory syndrome virus (prrsv), a productive infection of cdc has been observed [ ] [ ] [ ] [ ] . this apparently does not result in the secretion of type i ifn, although ifn mrna is induced, indicating a block of the ifn system at the translational level [ ] . whether prrsv can activate pdc has not yet been described. in addition to the tlr-recognition of viral pamps, pdc possess other receptors to sense viruses. this has been demonstrated for tgev, an rna-genome coronavirus, which can activate pdc using a surface receptor interacting with the viral m glycoprotein [ ] . use of inactivated virus and subunit structures has demonstrated that this activation is independent of viral nucleic acid [ ] , excluding a role for tlr and tlr . moreover, mutation of the glycosylation site in the m protein yields a virus incapable of inducing pdc, but still capable of replication [ ] . also studies in our own laboratory support the conclusion of triggering through a cell surface receptor-inhibitors of endosomal acidification such as chloroquine and bafilomycin prevent cpg-induction, but not tgev-induction of ifna production by pdc [ ] . similar observations have been made with other viral proteins such as hiv-derived gp and human pdc [ ] . although porcine pdc like human pdc are highly efficient producers of ifna and effective at sensing virus infections, not all viruses will activate pdc so efficiently. nonenveloped viruses such as fmdv-an rna virus of the picornaviridae related to poliovirus and rhinoviruses-are less efficient to activate pdc. it should be noted that these viruses either fail to replicate in dc, or produce only an abortive infection [ ] [ ] [ ] . nevertheless, pdc can be efficiently activated by fmdv under particular conditions. in the presence of opsonizing factors such as virus-specific immunoglobulin (ig), which mediate fcgrii-enhanced uptake of virions, pdc activation was observed-an event dependent on the presence of intact and active viral rna [ ] . such an activity would provide important antiviral innate defences at a time early post-vaccination when the adaptive response had begun producing specific antibody, but is inadequate to protect the host from disease or virus replication. this function of pdc would have an additional advantageassisting cdc in promoting the development of an efficacious adaptive immune defence for protecting the host. fmdv can also infect cdc including skin dc and modc [ , ] , which results in low levels if ifn-b secretion. just as in vitro analyses cover only a proportion of likely events in vivo, focusing on phenotypic modulation of dc and cytokine profiles will reveal only part of the story. during immune defence development, these modulations of the dc serve a purpose beyond the direct attack on the pathogen by the dc. that additional purpose is promoting the functional interaction with the adaptive immune system. the interaction between t lymphocytes and dc is a bilateral process. on one side there is the central role of dc in presenting antigen and stimulating t cells. this has been shown in several models including mixed leukocyte reactions [ , ] , superantigen presentation [ ] as well as antigen specific t-cell restimulation [ , , , , ] (see also table ). on the other side, t lymphocytes provide important signals to dc. with porcine modc cultures we have observed that after the co-culture of cytokinematured dc with t cells in the presence of antigens, a further upregulation of mhc class ii expression was obtained reaching levels clearly above those obtained with any stimuli in the absence of tcells [ ] . interestingly, tnf-a pre-treatment of the dc was as efficient as tnf-a/ifn-a cocktails to sensitize the dc for this process. these in vitro observations indicate that modc maturation is a regulated multistep process. from a practical point of view this means that although tnf-a alone is not sufficient to induce modc maturation, the t-cell responses induced by tnf-a-treated dc can compensate to provide stimuli reaching those obtained with more potent maturation cocktails such as tnf-a/ifn-a or tlr ligands [ ] . moreover, the t-lymphocyte activity provides an additional advantage in that cocktails such as tnf-a/ifn-a, as well as those combined with tlr can have the drawback of ''exhausting'' the dc. similar to other species, it has been demonstrated that dc can modulate the type of t-cell response induced. for example, the th /th profile will be influenced by the type of antigen presented as well as by the cytokine environment [ ] . another example of such modulation is the observation that cholera toxin-treated dc have the capacity to suppress t-cell proliferation [ ] . this was associated with decreased mhc class ii expression and increased il- secretion of the dc and was reversible by addition of tnf-a suggesting an immunoregulatory process. prrsv has also been described by several authors to modulate dc and monocytes towards reduced t-cell stimulatory capacity after in vitro infection [ , ] . also here reduced expression of mhc and costimulatory molecules together with increased il- levels have been described [ ] [ ] [ ] ] . such studies are valuable to understand viral pathogenesis but care must be taken to avoid any contamination of the cultures with mycoplasma as modc cultures apparently efficiently support mycoplasma growth, which can result in a potent antiproliferative activity [ ] . dc not only determine the type of t-cell response but also their homing characteristics. as described in section ''mucosal dc'', modc treated with ra promote a b and ccr expression, representing essential gut homing receptors [ ] . classically, b lymphocytes equipped with their surface ig receptor will recognize native unprocessed antigen and would only require t-cell help for clonal expansion and differentiation into antibody producing cells. in this sense, b cells should show only an indirect requirement for apc such as cdc. nevertheless, apc produce a number of cytokines, which have a direct stimulatory effect on b cells. these are classical cytokines such as il- , il- and ifn-(/b, but also more recently identified cytokines such as the b-cell activating factor (baff) and a proliferation-inducing ligand (april)-members of the tumour necrosis factor superfamily [ ] . in addition, several studies also demonstrated the ''delivery'' of native unprocessed antigen by dc to b cells [ ] [ ] [ ] . our own studies with porcine dc in an in vitro model of fmdv-specific ig synthesis demonstrated an important direct role of apc's during antigenspecific restimulation of immune b lymphocytes. purified b cells produced virusspecific ig only in the presence of tcells and apc. monocytes and modc but not pdc supported b-cell differentiation into antibody-secreting cells. while il- could replace t-cells, addition of baff could compensate for a lack of apc. in fact, blocking of baff receptor abrogated the apc-derived help for bcell responses [ ] . in addition, dc also influence isotype switching. as mentioned above, ra-treated modc promote virus-specific iga secretion in vitro [ ] . not surprisingly, similar to the interaction with t lymphocytes, the interplay of dc with b lymphocytes is also bilateral. an important role is certainly played by the ''mé nage à trois'' of fcr expressed on dc, antibody and antigen. this permits an amplification of antigen uptake and presentation, and can also mediate efficient cross-presentation of antigen for stimulation of mhc class i-restricted t-cell responses. furthermore, the fcrsystem sensitizes dc for inflammatory and antiviral cytokine responses. one example is the response of pdc to fmdv mentioned above (table ). these pdc only respond by producing ifn-( to fmdv when the virus is complexed with antibodies [ ] . another example is with csfv, where pdc sensitized with cytophilic antibodies show enhanced ifn-( production in response to lower virus quantities than when no cytophilic antibodies are present [ ] . in both cases fcgrii is involved. recent advances in the characterization of the porcine immune system, particularly in porcine dc biology, have permitted the use of the porcine model for many immunological studies. although the library of reagents for such studies is still restricted compared to that for mouse and human studies, knowledge of porcine immunology is well advanced. with the unveiling of the sequence for the porcine genome, there will be clear advantages for using the pig. in particular, the modc model has a number of advantages and applicability. large numbers of dc can be generated without killing the animal. for example, a typical figure of - million dc can be generated using monocytes isolated from ml of blood. the facility to repeat blood sampling with the pig enables the use of these dc in antigen presentation assays, to monitor autologous tcell responses in immunization experiments with outbred animals [ ] . moreover, with the modc being in an immature state, another advantage is the possibility to study dc maturation in response to cytokines, tlr ligands and infections. considering that dc are a rare cell type within the leukocyte populations, the large size of the pig, its lymphoid organs and the availability of larger volumes of blood together with repeated samplings offer an advantage allowing considerable immunological progress. in addition to this facility of recovering large volumes of blood regularly from the same animal, cannulation procedures for porcine lymph vessels at both peripheral and mucosal sites are now available. this approach allows us the much sought ability of studying dc migrating from peripheral sites over periods of several days [ , ] , a procedure not possible with humans and certainly cumbersome with mice considering their size and the quantities of material obtained. moreover, the pig is more closely related to the human-both genetically and physiologically-when compared to mice. this is reflected by immunological similarities such as the prr and their cellular distributions [ , ] . finally, the advantages which porcine immunology has to offer have gained a further boost with the advent of novel technologies such as rna interference (rnai) [ ] , which can be combined with the generation of transgenic pigs [ ] . localization of distinct peyer's patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (mip)- alpha, mip- beta, and secondary lymphoid organ chemokine myeloid c-type lectins in innate immunity cooperation of toll-like receptor signals in innate immune defence signaling pathways downstream of patternrecognition receptors and their cross talk dendritic cells interact directly with naive b lymphocytes to transfer antigen and initiate class switching in a primary t-dependent response critical role of itim-bearing fcgammar on dcs in the capture and presentation of native antigen to b cells cell surface recycling of internalized antigen permits dendritic cell priming of b cells 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characterization of swine leukocyte differentiation antigens summary of the first round analyses of the third international workshop on swine leukocyte differentiation antigens porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties functional and phenotypic characterization of distinct porcine dendritic cells derived from peripheral blood monocytes fibrocytes are potent stimulators of anti-virus cytotoxic t cells characterisation of porcine monocyte-derived dendritic cells according to their cytokine profile toll-like receptor, mhc ii, b and cytokine expression by porcine monocytes and monocytederived dendritic cells in response to microbial pathogenassociated molecular patterns il- replaces il- in development of monocyte derived dendritic cells (modc) of swine differentiation of porcine dendritic cells by granulocytemacrophage colony-stimulating factor expressed in pichia pastoris in vitro differentiation of porcine blood cd À and cd + monocytes into functional dendritic cells dendritic cells in different animal species: an overview a porcine cell surface receptor identified by monoclonal antibodies to swc is a member of the signal regulatory protein family and associates with protein-tyrosine phosphatase shp- porcine bone marrow myeloid cells: phenotype and adhesion molecule expression cholera toxin promotes the generation of semi-mature porcine monocyte-derived dendritic cells that are unable to stimulate t cells efficacy and functionality of lipoprotein opri from pseudomonas aeruginosa as adjuvant for a subunit vaccine against classical swine fever double-stranded secondary structures on mrna induce type i interferon (ifn alpha/beta) production and maturation of mrna-transfected monocytederived dendritic cells c-kit positive porcine bone marrow progenitor cells identified and enriched using recombinant stem cell factor summerfield a. toll-like receptor and myd knockdown by lentivirus-mediated rna interference to 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transmissible gastroenteritis virus (tgev) infected piglets plasmacytoid dendritic cells migrate in afferent skin lymph fcgammarii-dependent sensitisation of natural interferon-producing cells for viral infection and interferon-alpha responses type-a cpg oligonucleotides activate exclusively porcine natural interferon-producing cells to secrete interferonalpha, tumour necrosis factor-alpha and interleukin- of men, mice and pigs: looking at their plasmacytoid dendritic cells isolation and characterisation of pig peyer's patch dendritic cells phenotype and distribution of dendritic cells in the porcine small intestinal and tracheal mucosa and their spatial relationship to epithelial cells characterization and functional analysis of skin-derived dendritic cells from swine without a requirement for in vitro propagation plasmacytoid dendritic cell activation by foot-and-mouth disease virus requires immune complexes role of natural interferon-producing cells and t lymphocytes in porcine monocyte-derived dendritic cell maturation a discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to t cell areas of mesenteric lymph nodes dendritic cell heterogeneity in vivo: two functionally different dendritic cell populations in rat intestinal lymph can be distinguished by cd expression identification of two distinct populations of dendritic cells in afferent lymph that vary in their ability to stimulate tcells enrichment for a cd hi sirp-subset in lymph dendritic cells from the upper aero-digestive tract bidirectional negative regulation of human t and dendritic cells by cd and its cognate receptor signalregulator protein-alpha: down-regulation of il- responsiveness and inhibition of dendritic cell activation mucosal dendritic cells gut lymphocyte migration: we are halfway 'home' generation of gut-homing iga-secreting b cells by intestinal dendritic cells small intestine lamina propria dendritic cells promote de novo generation of foxp treg cells via retinoic acid reciprocal th and regulatory t cell differentiation mediated by retinoic acid in vitro induction of mucosa-type dendritic cells by all-trans retinoic acid ccr is a homing receptor for plasmacytoid dendritic cells to the small intestine dendritic cells enriched from swine thymus coexpress cd , cd and major histocompatibility complex class ii and actively stimulate alloreactive t lymphocytes culture of dendritic cells from a nonlymphoid organ, the thyroid gland: evidence for tnfalphadependent phenotypic changes of thyroid-derived dendritic cells circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair the peripheral blood fibrocyte is a potent antigen-presenting cell capable of priming naive t cells in situ peripheral blood fibrocytes: differentiation pathway and migration to wound sites responsiveness of fibrocytes to toll-like receptor danger signals regulated production of type i collagen and inflammatory cytokines by peripheral 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immune response induction innate immune responses against foot-and-mouth disease virus: current understanding and future directions interferon-alpha production by swine dendritic cells is inhibited during acute infection with footand-mouth disease virus porcine reproductive and respiratory syndrome (prrs) virus-specific interferongamma(+) t-cell responses after prrs virus infection or vaccination with an inactivated prrs vaccine identification of classical swine fever virus protein e as a target for cytotoxic t cells by using mrnatransfected antigen-presenting cells porcine reproductive and respiratory syndrome virus (prrsv) infects mature porcine dendritic cells and upregulates il- production mycoplasma contamination and viral immunomodulatory activity: dendritic cells open pandora's box efficient transgenesis in farm animals by lentiviral vectors phenotype of porcine monocytic cells: modulation of surface molecule expression upon monocyte differentiation into macrophages molecular cloning and characterization of a novel cd gene from the pig innate immune responses following emergency vaccination against foot-andmouth disease virus in pigs the fusarium toxin deoxynivalenol disrupts phenotype and function of monocyte-derived dendritic cells in vivo and in vitro dendritic cells harbor infectious porcine circovirus type in the absence of apparent cell modulation or replication of the virus key: cord- -x dq sy authors: wan, dongshan; jiang, wei; hao, junwei title: research advances in how the cgas-sting pathway controls the cellular inflammatory response date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: x dq sy double-stranded dna (dsdna) sensor cyclic-gmp-amp synthase (cgas) along with the downstream stimulator of interferon genes (sting) acting as essential immune-surveillance mediators have become hot topics of research. the intrinsic function of the cgas-sting pathway facilitates type-i interferon (ifn) inflammatory signaling responses and other cellular processes such as autophagy, cell survival, senescence. cgas-sting pathway interplays with other innate immune pathways, by which it participates in regulating infection, inflammatory disease, and cancer. the therapeutic approaches targeting this pathway show promise for future translation into clinical applications. here, we present a review of the important previous works and recent advances regarding the cgas-sting pathway, and provide a comprehensive understanding of the modulatory pattern of the cgas-sting pathway under multifarious pathologic states. pattern-recognition receptors (prrs) serve as innate cellular sensors of danger signals, such as pathogen-associated molecular patterns (pamps) or danger-associated molecular patterns (damps), and yield cellular-stress response. dna molecules are vital genetic components within cells, which are compartmentalized restrictively into specific regions. the occasionally misplaced dna is degraded rapidly by scavenger cells and extracellular or intracellular ribonucleases. aberrant accumulation of dna is relevant to tissue damage ( ) . in , several research teams discovered a new protein on the endoplasmic reticulum (er) which can be activated by immune-stimulatory dna (isd) and initiate type-i interferon (ifn) responses, which was named "stimulator of interferon genes" (sting, also known as mita, eris) ( ) ( ) ( ) . sting does not bind to dna directly, and bacteria-derived cyclic di-guanylate monophosphate (c-dgmp) or cyclic di-adenosine monophosphate (c-damp) were confirmed to be ligands for sting ( , ) . subsequently, it was found that some dna sensors can facilitate sting activation, such as interferon gamma inducible protein (ifi ) ( ) . however, sting activation could not be fully explained by the upstream factors/ligands that had been found. it was postulated that an unknown upstream regulator might be responsible for sting activation. in , wu and sun found that cyclic guanosine monophosphate-adenosine monophosphate (cgamp) was a novel secondary messenger serving as a ligand of sting ( ) . beside it, they purified a new protein named "cyclic-gmp-amp synthase" (cgas) that had cytosolic dna-sensing ability and can synthesize cgamp ( ) . also, they found that the cgas-cgamp-sting pathway was indispensable for host anti-viral immunity ( ) . their work filled in the gaps missing from upstream of sting. stimulator of interferon genes or cyclic-gmp-amp synthase is expressed widely in a broad spectrum of cells including immune, non-immune, cancer cells ( ) . mounting evidence has demonstrated that the cgas-sting pathway is important for mediating cellular immune sensing, and shows particular responses pattern to the isd distinguished from other nucleotide-sensing pathways. it is also regulated delicately by several molecules or feedback loops to maintain cellular homeostasis. nevertheless, cgas-cgamp-sting pathway itself has distinctive or even opposing effects under different conditions. in this review, we cover the roles of cgas-sting pathway in cellular type-i ifn immune response, and several cellular processes including autophagy, survival and senescence. we also summarize the literature on intrinsic cellular mechanisms modulating cgas-sting pathway as well as its cross-regulations with other dna-sensing pathways. moreover, the inflammationmodulation capacities of this pathway in infectious disease, inflammation and cancers have been elucidated too, and a pervasive pattern of this pathway has been described, which could provide a plausible explanation of the contradictory findings of studies. finally, current or prospective therapeutic strategies targeting the pathway, and issues that need to be addressed in the future, are discussed. cyclic-gmp-amp synthase belongs to the structurally conserved cgas/dncv-like nucleotidyltransferase (cd-ntases) superfamily. the latter is expressed universally in prokaryotes and eukaryotes, and can use purines or pyrimidines selectively as substrates for the production of linear or cyclic di-or even tri-nucleotide compounds, which act as secondary intracellular messengers ( ) . cyclic-gmp-amp synthase is distributed mainly in the cytosol (also nucleus in some specific conditions) ( ) . generally speaking, cgas is activated upon the recognition of b-type double-stranded dna (dsdna) without sequence-specificity but not a-type dsdna or rna ( , ) . hybrid dna:rna or stemlike single-stranded dna (ssdna) are also low-affinity ligands for cgas ( , ) . after binding with ligands, cgas undergoes an allosteric structural change, and subsequently catalyzes its substrates guanosine triphosphate (gtp) and adenosine triphosphate (atp) to produce a mixed phosphodiester-linked cyclic dinucleotide: g( - )pa ( - ) p cgamp (abbreviated as , -cgamp or cgamp) ( ) . cgas also catalyze the synthesis of linear dinucleotides such as amp- -atp, gmp- -gtp, and amp- -gtp as intermediate products ( ) . there are two major dsdna-binding sites on opposite sides of the catalytic pocket: a and b site. site a is the primary contact surface for dsdna, whereas site b is complementary, binding another dsdna. it allows for cgas to the formation of a : cgas:dsdna complex structure directed into two orientations with dsdna at least bp ( ) ( ) ( ) (figure a) . increased numbers of back-to-back dimers of cgas hold the two dsdna molecules together and permit successive recruitment of cgas which, consequently, forms a n: cgas:dsdna higherordered "ladder-like" oligomerization, with cgas arrayed "head to head/tail to tail" ( , ) . the dna-binding protein hu, mitochondrial transcription factor a (tfam), or bacterial high mobility group box protein (hmgb ) can bend the dsdna into a u-shaped structure and, thus, facilitate binding of cgas dimers to the same strand as it travels in opposite directions ( ) (figure b) . human cgas, unlike mouse cgas, is prone to formation of this ladder-like network with long dsdna, because of the human-specific residues k and l . these two dsdna-interfacing residues of site a loosen the interaction of dsdna with cgas, leading to dsdna curving and allowing more convenient binding for the next adjacent cgas ( , ) ( figure c) . finally, accumulated cgas-dsdna complexes can go through a liquid-phase separation and condense into gellike droplets as a reaction unit ( figure d) . this conformation requires a sufficiently long dsdna strand to form multivalent interaction positions, also requires the function of the n-terminal tail of cgas and a recently discovered dsdna-binding site in the catalytic domain of cgas (site c) ( , ) . meanwhile, the n-terminal tail of cgas mediates cgas localization onto the membrane by binding to phosphatidylinositol , bisphosphate (pi ( , ) p ) and prevents liberation of cgas and oligomerization, but can release cgas during cell stress ( ). the structure of cgas determines long strand dsdna (> - , bp) could potentially stimulate the enzyme activity and cgamp production of cgas ( ). the ability of human cgas to discriminate long dsdna strands from shorter dsdna may contribute to the specific sensing and recognition of the "danger dna" of pathogens, necrotic cells or cancer cells rather than irrelevant shorter dsdna, thereby enhancing the immunity against them specifically. double-stranded dna is restricted into the nucleus or mitochondria and is rarely present in the cytoplasm. extrinsic dsdna from pathogens such as viruses, bacteria, transcellular vesicles or rupture of dying cells can be internalized into the cytosol in several diverse ways ( - ). these extrinsic dsdna sources are engulfed by endosome through phagocytosis and digested immediately by dnaseii when fusing with lysosomes ( , ). however, some escaping mechanisms under certain conditions could help protect them from being degraded. for example, antimicrobial peptide ll could efficiently transports self-dna from endosome into cytosol of monocytes ( ). cell oxidative stress can lead to phagosomal acidification delay and probably release endosome context including dsdna owing to increased membrane permeability ( , ). the intrinsic self-dsdna can also be segregated inaccurately and released into the cytosol ( , ). for example, genomic dna (gdna) injury as a result of genotoxic stress and dna self-instability or replication errors leads to double-strand breaks (dsbs) and can be repaired by several ways ( ). impaired mediators of dna-damage repair response mediators, such as ataxia telangiectasia mutated (atm)-rad , poly adpribose polymerase (parp) and breast cancer / (brca / ) multiple cgas molecules can bind two double-stranded dnas (dsdna) to form a n: cgas:dsdna higher-ordered "ladder-like" oligomerization. mitochondrial transcription factor a (tfam) can bend the dsdna into a u-shaped structure and promote polymerization. (c) cgas can recognize b-type dsdna. in humans, the cgas dna-interfacing residue of site a loosens the interaction of dsdna to curve dsdna away for more convenient binding with next adjacent cgas. cgas can catalyze gtp and atp to synthesize cyclic guanosine monophosphate-adenosine monophosphate (cgamp). the n-terminal tail binds to the cell membrane, associating with phosphatidylinositol , -bisphosphate [pi ( , )p ]. (d) accumulation of cgas-dna complex goes through a liquid-phase separation and condenses into gel-like droplets. are associated with persisting dsbs and accumulation of cytosolic dna ( - ). extra-nuclear micronuclei formation during mitosis is a source of cytosolic dsdna caused by dsbs ( , ). followed by homologous recombination repair of collapsed replication forks, dna cleavage by methyl methanesulphonate (mms) and ultraviolet-sensitive (mus ) also lead to cytosolic dsdna presenting ( ). furthermore, manually cre/loxp recombination technology can induce dsdna damage during dna cleavage, which results in the accumulation of cytoplasmic dsdna ( ). in normal cellular mitotic processes, chromosomal dna can be exposed to the cytoplasm, while it is hard to bind and trigger cgas ( ). in addition, mitochondrial dna (mtdna) is also a considerable ligand of cgas and can be released into the cytosol under mitochondrial stress or dysfunction of proteins which participates in maintaining mitochondrial operations ( , , ) (figure a) . cells have several types of nucleases to restrict cytosolic dna to avoid cgas activation. for example, three-prime repair exonuclease (trex also known as dnaseiii) is a cytosolic dna exonuclease which removes unprotected dsdna from the cytosol ( ) . rnaseh locates to the nucleus and specifically degrades the rna in rna:dna hybrids participating in dna replication ( ) . dnaseii is a lysosomal dnase which degrades undigested dna in endosomes or autophagosomes to prevent their entry into the cytoplasm ( ). sam domain and hd domain-containing protein (samhd ) is characterized as a dntpase and restricts reverse transcription of the rna virus ( , ) . samhd can also stimulate the exonuclease (but not the endonuclease) activity of mre to degrade nascent cdns (including cgamp) might be exported by some ways. (c) inflammatory signaling mediated by the cgas-sting pathway. after sensing dna, cgas produces cgamp and extracellular cdns, promoting stimulator of interferon genes (sting) to undergo dimerization. sting can exit from the endoplasmic reticulum (er), and be translocated from the er to the er-golgi vesicle, and arrives at the golgi. sting and tank binding kinase (tbk ) can be oligomerized and cluster at the golgi. the sting-tbk /iκb kinase (ikk) signalosome forms a scaffold to phosphorylate interferon regulatory factor (irf ) and inhibitor of nf-κbα (iκbα). then, dimerized irf and the activated canonical nf-κb p /p complex can be translocated into the nucleus as transcription factors to promote transcription of type-i ifn. (d) autophagy initiation and degradation. sting activation on er triggers er stress and mechanistic target of rapamycin complex (mtorc ) dysfunction. er stress and mtorc dysfunction can stimulate the unc- like autophagy activating kinase (ulk ) complex and beclin -phosphatidylinositol -kinase catalytic subunit type (pi kc ) complex. autophagy-related protein (atg ) and light chain (lc ) are associated with genesis and elongation of the autophagosome. after autophagy initiation, cgas-sting is ubiquitinated and binds with p . then, they are packaged into autophagosomes and terminally sorted to lysosomes (bold arrows represent main signaling pathways, thinner arrows represent regulatory signaling pathways, and dashed arrows represent bypass or suspicious pathways). ssdna, and start dna-repair responses at stalled replication forks ( ) . depletion of samhd leads to the cleaving of nascent ssdna by the activity of mre endonuclease and cytosolic translocation of gdna ( ) . deficiency of any of these nucleases can lead to accumulation of self-dna in the cytoplasm, thereby activating the cgas-sting pathway against dna molecules ( ) (figure a ). the production of asymmetrically linked , -cgamp catalyzed by cgas has the highest affinity for sting to promotes sting dimerization ( , ) . cgamp as a second messenger can be also transferred among cells in several ways to pass danger signaling of frontiers in immunology | www.frontiersin.org cytosolic dna. intercellular gap junction consists of two docking hexamer channels formed by different connexins, which allows many small molecules, including cgamp, to pass bi-directionally through cells. and intercellular transfer of cgamp through gap junction is largely dependent on connexin ( ) ( ) ( ) . additionally, cgamp can be packaged into virons and pre-notify newly infected cells ( , ) . cell fusion is a distinct manner for intracellular transmission of the human immunodeficiency virus (hiv); cgamp also enter membrane-fused bystander cells in this way ( ) . extracellular vesicles such as exosomes can contain cgamp along with viral dna, host gdna or mtdna, and mediate cells communication ( , ) . there were no evidences that cgamp could be pumped out to extracellular space by a channel/transporter. however, it was found that slc a can transmit cyclic dinucleotides (cdns) into cell plasma ( , ) . notably, ectonucleotide pyrophosphatase/phosphodiesterase family member (enpp- ) can degrade extracellular cgamp ( ) (figure b ). besides triggering sting, these exogenous cgamp can directly bind to cgas and prompt its activation as well ( ) . after binding to cgamp, the "lid" region of the sting dimer undergoes a conformational change that converts sting from an inactive "open" formation to an active "closed" formation. following that, the sting dimer translocates from the er to perinuclear er-golgi intermediate compartment (ergic) vesicles, finally arriving at the golgi to form punctuate structures with downstream molecules ( , ) . er-retention of sting caused by mutations results in reduced ifn signaling ( , ) . the translocon-associated protein β (trapβ) recruited by inactive rhomboid protein (irhom ) initially forms the trap translocon complex that mediates sting exit from the er ( , ). they both assist cytoplasmic coat protein complex-ii (copii) to drive er-vesicle formation and carry the sting complex to the golgi ( , ) . trafficking sting can bind directly to and be phosphorylated by tank binding kinase (tbk ) dimer or iκb kinase (ikk) complex ( , , ) . the c-terminal tail (ctt) of sting is a linear unfolded segment, which determines the optimization of combination specificity. sting ctt in mammals tends to bind tbk , whereas in fish it tends to activate nuclear factor-kappab (nf-κb) signal ( ) . the sting phosphorylation site ser in the ctt cannot reach the kinase-domain active site of its directly bound tbk , instead can reach the kinase-domain active site of the next adjacent tbk binding with another sting and be phosphorylated, while tbk phosphorylate each other ( , ) . hence, sting and tbk can aggregate on the golgi to form the sting signalosome. clustering sting undergoes palmitoylation and full activation ( ) . it is also possible for sting-ikk to cluster and form the sting signalosome in this manner. the sting-tbk /ikk signalosome produces a scaffold to phosphorylate interferon regulatory factor (irf ) or inhibitor of nf-κbα (iκbα). activated irf undergo dimerization ( ) . the activation of iκbα leads to its polyubiquitination and degradation by the proteosome, thereby eliminating its inhibition of nf-κb. there is also evidence suggesting that nf-κb activation might not require sting trafficking from the er ( ) . then, the dimerized irf or activated nf-κb p /p (p is also known as rela) complex are translocated into the nucleus as transcription factors and bind to the promoter of type-i ifn to aid the transcription of type-i ifn ( , , ) . meanwhile, activation of nf-κb p /relb can prevent recruitment of p and inhibit the p /p signal ( ) (figure c ). expressed type-i ifn can propagate among cells in paracrine or autocrine manners. the binding of ifnα/β with its receptor triggers janus kinase (jak) and signal transducer and activator of transcription (stat) pathways, then induce transcription of type-i ifn-stimulated genes (isgs), which have ifn-sensitive response elements (isres) in their -untranslated regions (utrs) ( ) . irf can also bind partially to several isres alone ( ) . herein, the expression of some isgs including interferon-induced protein with tetratricopeptide repeats (ifit) and pro-inflammatory cytokines such as tumor necrosis factor α (tnfα), interleukin (il)- , c-x-c motif chemokine ligand (cxcl ) and c-c motif chemokine ligand (ccl ) is increased remarkably by the cgas-sting pathway ( ) . furthermore, cgas and sting are both isgs, suggesting a positive feedback loop in spreading of the ifn signal ( , ) . stimulator of interferon genes activation on the er also triggers an er stress response with an "unfolded protein response (upr) motif " on the c-terminus of sting, which leads to and er stress-mediated autophagy ( , ) . sting-tbk activation and er stress also induce mechanistic target of rapamycin complex (mtorc ) dysfunction ( ) . er stress or reduced mtorc signaling activates unc- -likeautophagy activating kinase (ulk ) complex and the beclin- -class iii phosphatylinositol -kinase (pi kc also known as vps ) complex, which promotes initiation of the classical autophagy path ( ) . cgas can also interact directly with the autophagy protein beclin- -pi kc complex and trigger autophagy ( ) . furthermore, cgas-dsdna polymer can form a liquid-phase condensate (as mentioned above), which could theoretically be an initiator of autophagy ( ) . after autophagy initiation, autophagy-related protein (atg ) undertakes the genesis of the autophagosome along with light chain (lc ) undergoing lipidation, thereby resulting in elongation of the autophagosome ( ) . lc can also be recruited directly by ergic-loading sting and bypass the classical autophagy pathway ( , ) . cgas-sting-mediated autophagy can spread to the whole cell and help the elimination of intracellular microorganisms, subcellular organelles or misfolded proteins, as well as the er itself that loads the sting signalosome ( - ) ( figure d ). cgas-sting-mediated autophagy is also indispensable for removing cytosolic dna and inflammatory signaling factors to restrict the inflammatory response raised by the pathway itself ( ) . excessive signaling of the autophagy cascade can lead to irreversible apoptosis termed "autophagic cell death" ( ) . consequently, oligomerized cgas or sting undergoes ubiquitination and is packaged into autophagosomes with the help of p , to be terminally sorted into lysosomes ( , , , ) . cgas or sting is digested immediately in the autophagolysosome after transient activation of downstream signaling ( , , , ) . autophagy functions as a negative feedback loop which ensures transient cgas-sting signaling and avoids consistent over-reaction of the pathway. thus, impairment of autophagy may give rise to destructive inflammatory diseases ( ). we cataloged factors in the literature that could potentially up-or down-regulate expression of cgas/cgamp/sting in pre-translational or post-translational stages (tables , ) . the regulatory mechanisms of tbk , irf, and nf-κb in signaling pathways associated with expression of type-i ifn are outside the scope of this review. pyhin family member absent in melanoma (aim ) is a cytoplasmic dsdna sensor. it can recruit apoptosis-associated speck-like protein containing a card (asc) by its pyhin domain and form the aim inflammasome. the inflammasome activates caspase- , which activates il- and trigger pyroptosis ( ) . the aim pathway could counteract the cgas-sting pathway ( ) . first, cgas is a target for caspase- cleavage ( ) . second, gasdermin d activated by caspase- can lead to potassium ion (k + ) efflux which inhibits cgas ( ) . conversely, the cgas-sting pathway can trigger the aim inflammasome or nlr family pyrin domain containing (nlrp ) by several means, and the process lags behind canonical ifn signaling ( , ) . in this way, the inhibitory nucleic-acid sensor nlr family card domain containing (nlrc ) can counteract sting by binding and occupying it, but viral dna as a possible nlrc ligand can reverse its occupation of sting ( ) (figure a) . another pyhin family member, ifi , is a dna sensor located in the nucleus. ifi can bind to viral dna sequences or damaged chromatin dna and be translocated to the cytoplasm to recruit sting cooperatively with tnf receptor associated factor (traf ) and p ( , ) . several studies have shown that ifi (which can stimulate the phosphorylation and recruitment of sting and tbk ) is required for the full response of sting ( , ) ( figure b) . conversely, cgas can partially enter the nucleus and interact with ifi to promote its stability ( ) . therefore, it is inferred that during viral infection, ifi can facilitate recognition of decapsidated viral dna in the nucleus, while cgas in the cytoplasm engages with viral gene transcription products ( , ) . however, sting signaling can trigger ifi degradation by tripartite motif-containing (trim ) ubiquitination ( ) . tlr is also an important prr for multiple pamps ( ) . tir domain-containing adaptor-inducing ifnβ (trif) is downstream of several subtypes of tlrs (including tlr ). trif may be responsible for interacting with sting and helping the dimerization of sting ( ) . during viral infection, the tlr -myeloid differentiation primary response (myd )-irf / pathway is necessary for mouse monocytes recruitment to lymph nodes, whereas the sting pathway is necessary for local production of type-i ifn ( ) . however, sting signaling can induce suppressor of cytokine signaling (socs ) expression, which can negatively regulate myd activity ( ) (figure c ). oxidized mtdna can be released into the cytoplasm during cell stress elicited by hypoxia, viral infection and mitochondrial damage, etc.; oxidized mtdna is resistant to degradation by the cytosolic nuclease trex ( ) . in addition, mtdna accompanied with tfam (a mtdna-binding protein that can bend mtdna) is also a reasonable target for recognition by cgas ( , ) . however, during regulated cell death (as represented by apoptosis), it undergoes mtdna release but has certain mechanisms to ensure a minimal cgas-stingmediated immune response. mitochondrial outer membrane permeabilization (momp) activation, which is executed by bcl- -associated x protein (bax) and bcl- antagonist or killer (bak), is a highly controlled conserved process in regulated cell figure | interaction of the cgas-sting pathway with other dna-sensing pathways and its role in cell survival. (a) absent in melanoma (aim ) pathway and pyroptosis and necroptosis. aim can be triggered by cgamp and form an inflammasome, consequently triggering interleukin (il)- production and pyroptosis. stimulator of interferon genes (sting) trafficking to the lysosome ruptures the lysosome membrane, resulting in k + efflux and activation of the nlrp inflammasome, leading to pyroptosis. cyclic-gmp-amp synthase (cgas) and interferon regulatory factor (irf ) can be a target for caspase- cleaving. gasdermin d can lead to k + efflux and inhibition of cgas. (b) interferon gamma inducible protein (ifi ). ifi can be transported to the cytoplasm to help to recruit sting and tank binding kinase (tbk ). ifi as a pyhin family protein may form the inflammasome only in theory. (c) toll-like receptor (tlr) pathway. tir domain-containing adaptor-inducing ifnβ (trif) may be responsible for helping the dimerization of sting. sting signaling can induce suppressor of cytokine signaling (socs ) expression, which negatively regulates myd activity. (d) apoptosis. mitochondrial outer membrane permeabilization (momp) formed by bax/bak induced by mitochondrial stress can release oxidized mitochondrial dna (mtdna) and cytochrome c into the cytosol. oxidized mtdna is a suitable ligand for cgas recognition and is resistant to dnaseiii (trex ) degradation. cytochrome c binds to apoptotic protease-activating factor (apaf ) and initiates the formation of an apoptosome cooperatively with caspase- to activate caspase- , which can induce apoptosis. caspase- can cleave cgas. death. bak and bax activated by apoptosis signals cooperatively form a pore-like conformation on the mitochondrial outer membrane, leading to a permeability change of outer and also inner membranes ( , ) . consequently, the mitochondrial matrix, including cytochrome c and oxidized mtdna-tfam, is released into the cytoplasm ( , ) . cytochrome c binds to apoptotic protease-activating factor (apaf ) and initiates the formation of the apoptosome cooperatively with caspase- , which further triggers the intrinsic apoptosis program ( ) . in vivo and in vitro studies have shown that an absence of caspase- is associated with greater release of type-i ifn ( , ) . this occurs because caspase- and its downstream caspase- can cleave cgas and irf to restrain deleterious inflammation ( ) (figure d) . the cgas-sting pathway can also initiate programmed cell death. activation of sting enhances phosphorylation and activation of receptor interacting serine/threonine kinase (rip ) and mixed lineage kinase domain-like pseudokinase (mlkl). proapoptotic bcl binding component (puma), a member of bh -only family, is subsequently activated in a rip /mlkl-dependent manner, which promotes leakage of mtdna by momp ( , ) . activated irf can bind directly to bax to form irf /bax complex and induce apoptosis ( ) . excessive cgas-sting-mediated autophagy signaling can cause "autophagic cell death" and prevent malignant transformation of cells through dna damage ( , ) . sting trafficking to the lysosome can broaden permeabilization of the lysosome membrane, thereby rupturing the lysosome and releasing its contents, resulting in "lysosomal cell death (lcd)". lcd further triggers k + efflux and nlrp activation, ultimately resulting in pyroptosis ( , ) (figure d) . moreover, stimulating sting-dependent type-i ifn and tnfα signals simultaneously can lead to necroptosis of tumor cells ( , ) . cell senescence is recognized as a permanent arrest of the cell cycle, and is common in aging, immunity, ontogenesis and infectious defense ( ) . it lacks a specific biomarker but can be identified by the expression of several antiproliferative molecules (representatively rb-p andp -p pathway) ( ) . during senescence, changes in the nuclear structure and loss of the nuclear lamina protein disrupt the integrity of the nuclear envelope, leading ultimately to dna damage and cytoplasmic chromatin fragments ( ) . cellular senescence can be accelerated by accumulation of cytoplasmic chromatin in turn ( ) . these senescent cells produce the senescence-associated secretory phenotype (sasp), which shapes an inflammatory microenvironment ( ) . the cgas-sting pathway has been reported to be involved in the recognition of cytoplasmic chromatin fragments from senescence-related dna damage, and mediate the expression of sasp genes ( ) ( ) ( ) ( ) . along with these actions, the expression of trex and dnaseii is inhibited by dna damage through the inhibition of e f/dp (a potential transcription factor of trex and dnaseii) ( ) . for hematopoietic stem cells (hscs), dna damage can promote excessive secretion of type-i ifn in the hsc niche and activate p pathway, both of which can lead to long-term senescence and exhaustion of hscs ( , ) . hscs expressing a circular rna named "cia-cgas" in the nucleus, however, are protected from this exhaustion as a result of cia-cgas having stronger affinity than that of self-dna, which prevents it from being sensed ( ) . it implied a novel target to manipulate the immune environment in bone marrow and help for finding treatment approaches for hematopoiesis-based diseases, such as aplastic anemia. utilizing cellular senescence to restrain tumor growth is discussed below. cgas-sting signaling has an essential role in defense against a broad spectrum of intracellular dna and rna viruses ( , , ) . hiv is a typical rna retrovirus: there is neither dsdna in its genome, nor production of nucleic acids ( ) . nevertheless, cgas can detect the presence of hiv. rna:dna hybrids synthesized during reverse transcription that can be sensed by cgas explain (at least in part) this phenomenon ( ) . cgas may be triggered by endogenous dna broken and released during hiv infection as well theoretically. however, some studies found the new mechanisms. the early reverse-transcription production of hiv- can flank short stem loops with paired base, which lead to the production of y-type dna containing unpaired guanosines that can activate cgas well ( ) . moreover, nucleolus protein non-pou domain-containing octamer-binding protein (nono), as a sensor of capsid components of hiv, can help cgas to be translocated to the nucleus and assist cgas to sense hiv dna accompanied by polyglutamine-binding protein (pqbp ) ( , ) . the assistance proffered by nono in assisting cgas to sense dna is also associated with its role in constructing a ribonuclear complex with dna-dependent protein kinase (dna-pk) subunits around hexamethylene bisacetamide-inducible protein (hexim ), termed as "hexim -dna-pk -paraspeckle components-ribonuclear protein complex (hdp-rnp), " which also has a role in repair of dna damage and transduction of genotoxic signals ( ) . this complex is also required to accompany cgas-pqbp in sensing dna virus, such as kaposi's sarcoma-associated herpes virus ( ) . in addition, during virus infection, sting activation can lead to global suppression of translation in cells, which restricts viral replication ( ) . compared with hiv- , hiv- is less infective because it can infect dendritic cells (dcs) and elicit an anti-virus immune response. as a result, hiv- can cross-protect against hiv- ( ). this phenomenon has been attributed to the fact that hiv- (instead of hiv- ) can encode protein vpx, which overcomes the samhd restriction of dntp in dcs ( , ) . hiv- can infect dcs via vpx presentation, nevertheless, hiv- still cannot be fully sensed and induce an efficient immune response owing to certain escape mechanisms. whether it is hiv- or hiv- , a completely robust ifn response is required at pre-and post-integration sensing stages ( ) . cgas in dcs can detect reverse-transcribed cdna of hiv- before and after integration, whereas hiv- sensing is after genome integration owing to its capsid protection ( , ) . it was suggested that during initial infection by hiv- , nucleotides are recruited into the intact capsid through the hexamer pores on the hiv- capsid. therefore, the capsid-coated hiv- virus prevents the encapsidated reverse-transcription production from being sensed by the cytosolic nucleic-acid sensors ( ) . hiv- capsids can be ubiquitinated and then degraded by the host e ubiquitin ligase function of trim , which leads to detection of viral dna, meanwhile hiv- could use some host protein like cyclophilins to evade the sensing ( , ) (figure a) . similarly, other viruses also have evasion mechanisms to escape cgas-sting pathway surveillance (table ) . therefore, identifying and preventing such viral-evasion factors could be a viable means to design novel anti-viral drugs. cgas-sting pathway is responsible to protect against intracellular or extracellular bacterial infection (especially hsv, herpes simplex virus; cmv, cytomegalovirus; irhom , inactive rhomboid protein ; trapβ, translocon-associated protein β; kshv, kaposi's sarcoma-associated herpes virus; hdp-rnp, hexim -dna-pk-paraspecklecomponents-ribonuclear protein complex; lana, latency-associated nuclear antigen; virf , viral interferon regulatory factor ; plpro, papain-like protease; lc , light chain ; mtor, mechanistic target of rapamycin; traf , tnf receptor associated factor ; hcv, hepatitis c virus; hpv, human papilloma virus; hbv, hepatitis b virus; hiv, human immunodeficiency virus; nf-κb, nuclear factor-κ b. intracellular infections). cdns (e.g., c-dgmp, c-damp, and cgamp) produced by bacteria are essential for the regulation of bacterial function, such as biofilm formation, colonization, and reproduction ( , ) . as ligands for sting, cdns can bind directly to and activate sting independently of cgas, which contributes to several immune responses from bacteria ( ) . usually, bacteria can enter or be engulfed by the cell through the endophagosome and be sequestered from the cytosolic sense receptor. some bacteria, such as mycobacterium tuberculosis (mtb), can survive in vacuoles, resulting in an insufficient cellular immune response to defend against it ( ) . in contrast, the esx- secretion system of the mycobacterium can translocate the phagosomal vacuolar matrix including bacterial genome molecules into the cytoplasm and trigger the cgas-sensing pathway ( ) . for other bacteria, such as legionella pneumophila or and brucella abortus, the host guanylate binding proteins (gbps) facilitate rupture of phagosome vacuoles and are indispensable for controlling their infection ( , ) . autophagy signaling mediated by cgas/sting is also involved in microorganism clearance mentioned above ( , ) . bacteria have evolved strategies to confront this pathway too. bacterial phosphodiesterase cdnp produced by mtb or group-b streptococci can degrade cdns ( , ) . cpsa (a type of mtb lytr-cpsa-psr domain-containing protein) can prevent autophagy responses for eliminating pathogens ( ) . chlamydia trachomatis inclusion membrane proteins can maintain the stability of the inclusion membrane and avoid inclusion lysis (leading to pathogen antigens leaking out and being detected by the host cell) ( , ) . yersinia outer protein j (yopj) deubiquitinates sting and impedes the formation of the sting signalosome ( ) . the cgas-sting pathway activation even impedes the elimination of listeria monocytogenes because bacterial dna can be packaged into evs and transferred into t cells, where it induces apoptosis of t cells ( , ) . several protozoans, such as toxoplasma gondii and malaria parasites, have an intracellular period in their lifecycle. t. gondii could engage cgas-sting exclusively ( ) . however, irf activation inducing isg expression promotes t. gondii development independently of ifn expression ( ) . p. falciparum can target erythrocytes, lacking a nucleus and unable produce ifn, but infected erythrocytes can secrete evs containing parasitic gdna to monocytes and trigger cgas ( ) . the immune system is regulated by a complicated network. disorder of immune signaling can elicit non-infectious inflammatory or autoimmune diseases. excessive, uncontrolled production of type-i ifn can lead to a spectrum of inflammation diseases termed "type-i interferonopathies, " which have some common manifestations ( ) . cgas-sting is the one of main sources of type-i ifn, acts as a cellular immune-sensing signaling axis, and is involved in type-i interferonopathies. stimulator of interferon genes -associated vasculopathy with onset in infancy (savi) is a typical sting-related hereditary inflammatory type-i interferonopathy, and is manifested by interstitial lung disease, dermatomyositis and arthritis. its pathology is featured by leukocytoclastic vasculitis and microthrombotic angiopathy of small dermal vessels ( , ) and patients can also suffer from lymphopenia ( ) ( ) ( ) ( ) . the etiology of savi is a gain of function (gof) mutant in sting which leads to constitutive sting activation without cdns stimulation ( ) . currently, several mutant amino acids residues have been found in or close to the dimerization domain (v m, n s, g e, v l, and v m) ( , , , ) , as well as r g, r s, r q, and c y in the cgamp-binding domain ( ) . other types of type-i interferonopathies, such as systemic lupus erythematosus (sle) and aicardi-goutières syndrome (ags), have relationships with defective clearance of cytosolic nucleic acids caused by congenital dysfunction of trex , rnaseh , and samhd . sle is a heterogeneous autoimmune disease which has prominent type-i and also -ii ifn signatures ( ) . ags comprises some systemic autoimmune syndromes overlapping with sle, and can be classified as a "lupuslike disease" ( ) . additionally, ags also causes severe developmental neurological disorders, including cerebral calcifications, encephalopathy and cerebral atrophy. systemic lupus erythematosus is a representative model for elucidating the mechanism of type-i interferonopathies. in sle, the level of self-dna which is packaged into apoptosis-derived membrane vesicles along with the level of anti-dsdna antibody is increased in the serum of patients ( ) . a study revealed increasing levels of isgs (including cgas/sting) as well as the cgamp-detected ratio in peripheral-blood mononuclear cells of sle patients ( ) . as innate immune cells, dcs have essential roles in antigen presentation, cytokine secretion, and priming the adaptive response of immune cells ( ) . plasmacytoid dcs (pdcs) can internalize and recognize self-dna and they are the main source of type-i ifn in serum during sle ( ) . ifnα/β is essential for complete function of immature pdcs ( ) . ifnα/β and il- can induce mitochondrial oxidative stress in dcs and decrease atp production, which blocks proton-pump function and increases ph of lysosomes. this process inhibits mitochondrial degradation and blocks mtdna clearance, which engages the cgas-sting pathway ( , ). moreover, monocytes may sense mtdna through cgas-sting and differentiate into dcs ( , ). neutrophil extracellular traps (nets) are complexes released by neutrophils exposed to stimuli or autoantibody immune complexes. nets comprise extracellularly released chromatin, myeloperoxidase enzymes, and also oxidized mtdna. in lupus-like diseases, nets can be induced by ifnα/β and may play a major part in priming pdcs ( , ) . all mechanisms stated above contribute to a more aggravated type-iifn response and exacerbate disease. a similar phenomenon can be observed in savi, ataxia telangiectasia (at) and artemis deficiency ( ) . however, compared with savi, dcs in sle can prime t-cell maturation significantly and increasing secretion of pro-inflammatory cytokines, such as il- and tnfα can also lead to activation of adaptive immunity ( figure a) . the cgas-sting pathway can mediate systemic inflammation as well as autoimmune activation. however, it is also involved in the local inflammation of multiple tissues. with regard to ischemic myocardial infarction (mi), cardiac macrophages can sense dying ruptured cells and lead to fatal post-mi cardiac inflammation, which is reversed by ablation of cgas/sting/irf ( , ) . in a non-alcoholic steatohepatitis (nash) model induced by a methionine-and choline-deficient or high-fat diet, lipotoxicity can cause mitochondrial damage and up-regulate sting/irf expression in hepatocytes, which in turn promotes lipid accumulation and inhibits glycogen synthesis. all above bring out hepatic inflammation and hepatocytes apoptosis ( ) . in this model, mice with deficiency of sting presents alleviated insulin resistance and lower levels of low-density lipoprotein in serum, and also decreased hepatic inflammation and fibrosis/steatosis, in which hepatic macrophages/kupffer cells may take a big part ( , ) . lipotoxicity can induce p to be phosphorylated through the cgas-sting-tbk pathway, which causes aggravated protein inclusions in hepatocytes and it indicates that p could be a biomarker for nash prognosis ( ) . mtdna-dependent inflammation induced by lipotoxicity also occurs in adipose tissue and endothelial cells of blood vessels, which contributes to tissue inflammation, insulin resistance, and cardiovascular diseases ( , ) . in traumatic brain injury (tbi), local injury initiates breakdown of the blood-brain barrier and global neuroinflammation ( ) . sting expression is up-regulated in tbi and can lead to increased expression of pro-inflammatory cytokines and enlargement of secondary injury ( ) . reduced autophagy-associated protein expression induced by sting may contribute to the dysfunction of autophagy and dampen the elimination of necrotic tissue, thereby intensifying inflammation ( ) . during silicosis, silica can yield cytosolic dsdna release and engage cgas-sting, which activates dcs and macrophages to cause severe lung inflammation. it also leads to death of epithelial cells through the nlrp pathway and pulmonary fibrosis ( ) . similarly, mtdna release in renal tubule cells has been found to be associated with acute kidney injury by cytotoxic drugs and chronic renal fibrosis ( , ) . neurodegenerative diseases are correlated with local inflammation ( ) . in the central nervous system (cns), microglia is considered to be the main source of cgas-sting-dependent ifn expression ( ) . in neurodegenerative diseases, levels of the marker of microglia activation-cluster of differentiation (cd ), and pro-inflammatory cytokines are increased ( ) . a significant feature of parkinson's disease (pd) is the neuronal loss of cerebral nuclei (especially dopaminergic neurons in the substantianigra). serine/threonine-protein kinase pink and e ubiquitin-protein ligase parkin are ubiquitinrelated factors that take part in removing damaged mitochondria by autophagy, and their dysfunction lead to the early onset of pd ( ) . parkin −/− and pink −/− mice, following exhaustive exercise, show inflammation and loss of dopaminergic neurons, which can be rescued by loss of sting ( ) . similarly, at is a genetic disease caused by missense mutation of a dna-repair protein: atm. at patients usually show neurodegenerative defects (especially ataxia) complicated with telangiectasia on their eyes or body, deficiency of adaptive immune cells, and predisposition to cancer ( ) . nevertheless, up-regulation of expression of type-i ifn can also be found in at patients and mice, causing them to be prone to autoimmune diseases ( , , ) . this syndrome is related to p -mediated senescence but also the chronic inflammation mediated by the cgas-sting pathway which engages cytosolic uncombined broken gdna caused by atm dysfunction ( ) . in addition, accumulation of cellular mtdna occurs in age-related macular degeneration characterized by retinal pigmented epithelium (rpe) degeneration. this can trigger chronic inflammation by cgas-sting pathway, in which nlrp inflammasomes, inflammatory/apoptotic caspases are also involved ( , ) . with regard to other diseases in which adaptive immune cells prime, cgas-sting has a different role. multiple sclerosis (ms) is a local inflammatory disease of cns. ms is characterized by over-reactive microglia, infiltration of self-reactive t cells, demyelization of nerve fibers and hyperplasia of gliocytes. autoantibodies against proteins expressed in immune-privileged regions of cns also contribute to its pathogenesis ( ) . in ms, ifnα/β can attenuate disease severity effectively. this implies a protective role for type-i ifn in cns, which is considered to counteract the pro-inflammatory ifnγ ( ) . using experimental autoimmune encephalitis (eae) as a ms model, sting was found to be indispensable for amelioration of type-i ifnmediated neuroinflammation, and it could be induced by a conventional anti-viral drug ganciclovir ( ) . ultraviolet (uv) radiation is a factor inversely related to the morbidity of ms ( ) . it was found that uv-b irradiation can recruit inflammatory monocytes and produce type-i ifn in a sting-dependent manner ( ) . all above indicate that cgas-sting-ifnα/β pathway may have a beneficial effect on some cns inflammatory diseases such as ms. a possible reason for the observed effect above is due to indoleamine , -dioxygenase (ido), which can catabolize tryptophan (trp) oxidatively. trp withdrawal or trpoxidative catabolites can interact with general control non-derepressible (gcn ) and mtor, of which both can control cellular aminoacid metabolism and suppress t helper (t h ) cells immunity ( ) . ifnα/β is a potent ido inducer to suppress proliferation of cd + t h cells and promote differentiation of foxp + regulatory t (t reg ) cells, which are believed to suppress cnsspecific autoimmunity ( , ) . in addition, dna released from dying cells can be internalized directly by t cells and sensed by cgas-sting pathway, which leads to enhancement of the t h transcription factor gata but suppression of the t h transcription factor t-bet. consequently, this process polarizes naive t cells toward t h differentiation ( ). studies mentioned above may (at least in part) explain why the cgas-sting signal is a negative regulator of ms. the inhibitory role of cgas-sting in inflammation is also attributed to its apoptosis-triggering role. in some subtypes of savi and mouse models, apoptosis of blood-vessel endothelial cells or bronchial epithelial cells and leucopenia can be observed (especially t-cell lymphopenia) ( , , ) . when the sting signal is stimulated, apoptosis occurs more frequently in normal or cancerous t cells ( ) . also, bone-marrow chimeras and gene-knockout studies have shown that t cells defect in savi are not associated with type-i ifn signaling or cgas ( , ) . localization of sting at the golgi can cause delay of t-cell mitosis and reduced proliferation independently of irf and tbk ( ) . furthermore, a "upr motif " on the c-terminus of sting can cause er stress and upr, resulting in ca + overloading and t-cell death ( ) . a controversial view is that b cells express sting variously and may undergo apoptosis through this way ( , , ) . however, simultaneous signaling by sting and the b-cell antigen receptor can promote b-cell activation and antibody production independently of type-i ifn ( ) (figure b) . as for some diseases with inflammatory responses involved, the acute phase of pancreatitis causes dying acinar cells to produce free dsdna, which activates cgas-sting signaling in macrophages, and exacerbates inflammation severity ( ) . however, in the chronic phase of pancreatitis, cgas-sting activation decreases pancreatic inflammation, which may be mediated by limiting t h response ( ). for gut mucosal immunity, transient stimulation of sting could strengthen the function of antigen-presenting cells (apcs) and promote t h and t h immune responses against microbes ( ) . chronic sting signaling, however, elicits an il- response to control the inflammation and avoids inflammatory enterocolitis such as bowel disease ( ) . sting knockout mice present reduced numbers of goblet cells, a decreased ratio of commensal versus harmful bacteria and compromised t reg cells in the gut, making it prone to enterocolitis ( ) . chromosomal instability (cin) is an intrinsic feature of cancer, and results spontaneously from errors in chromosome segregation during the mitosis of cancer cells. cin can also be induced manually by radiotherapy or chemotherapy, which causes dsbs. it results in micronuclei formation outside the nucleus, of which rupture brings out irrepressible accumulation of cytosolic self-dna and engages cgas ( , , ). however, normal mitotic processes involve exposure of chromosomal dna to the cytoplasm, but this cannot initiate a substantial inflammatory reaction or apoptosis because nucleosomes can suppress dsdna-cgas binding in a competitive manner ( ). an appropriate immune response against tumors via a type-i ifn plays an indispensable part in limiting tumors and prolonging host survival ( ) . it was found sting-deficient mice are prone to developing several types of cancer and have poor survival under a tumor burden, whereas stimulation of sting can elicit robust immunity to tumors ( ) ( ) ( ) . a mechanism is many cancer cells expressing cgas can recognize cytosolic dna and produce cgamp to stimulate secretion of type-i ifn through sting ( , ) . excessive expression of trex in cancer cells, which can be induced by radiotherapy, attenuates this progression ( ) . cgas-sting can also promote senescence of cancer cells through the p -p pathway ( ) . cgas-sting-mediated autophagy contributes to autophagic cell death if mitotic crisis occurs to avoid transformation of cancer cells ( ) . melanoma cells can also transfer cgamp produced by them to proximal non-cancerous host cells through gap-junction channels and activate sting in these cells, which contributes to the recruitment of tumor-infiltrating immune cells such as natural killer (nk) cells ( , ) . expression of the nk cellspecific ligand nkg d retinoic acid early transcript (rae ) on cancer cells is highly up-regulated by sting once nk cells permeate into tumor tissue ( ) . the activation of sting in the endothelium within the tumor microenvironment (tme) could contribute to the remodeling of tumor vasculatures, and may have positive effects on tumor regression ( ) . dendritic cells are the main source of type-i ifn in several types of tmes and are dependent on sting signaling ( ) . more preferentially than macrophages, infiltrating dcs take up tumor-derived dna or cgamp from dying cell fragments by phagocytosis ( , , , , ) . moreover, cancer cells can package dna into exosomes and transfer dna to dcs ( ) . produced cgamp by cancer cells can also be transferred to dcs through forming gap junction ( ) . by activating cgas-sting signal in dcs, cd α + subtype dcs secret chemokines such as ccl and cxcl and crossprime infiltrating anti-tumor cd + t cells ( , , ( ) ( ) ( ) . in contrast, numbers of immune-suppressing cells such as t reg cells, myeloid-derived suppressor monocytes and m macrophages have been reported to be decreased ( , , ) . expression of il- /il- rα complex is up-regulated in myeloid cells with the help of sting/type-i ifn and promotes tumor regression ( ) . tumor cells can evade intrinsic cellular surveillance in different ways. in various cancer cell lines, cgas, sting, tbk , and irf are mutated frequently and their decreased expressions are also related to the high level of methylation ( ) . sting expression has been shown to be suppressed by the alternative lengthening of telomeres (alt) pathway, which is responsible for prolonging the telomere length and maintaining the proliferation of tumor cells ( ) . a hypoxic environment in tumor cells can lead to accumulation of lactic acid and is associated with the inhibition of tumor-conditional dcs and reduced expression of ifn signaling molecules ( ) . in breast cancer, functional up-regulation of expression of human epidermal growth factor receptor (her ), a ligand-independent receptor tyrosine kinase (rtk), can arrest the expression of rac-alpha serine/threonineprotein kinase (akt ) (a key factor in the mtor pathway), which is reported to inhibit the activation of cgas and tbk ( , ) . patients with lung adenocarcinoma have a low probability of survival if they have reduced expression of cgas ( ) . thus, expression of the cgas-sting and dna-damage marker histone γh ax in tumor cells could be considered as independent prognostic factors to predict therapy response and clinical outcome, and could be superior to that of traditional markers like immunogenic cell death and t cells number ( ) . however, some scholars have arrived at opposite conclusions. when dsbs occur in cancer cells, cgas can be relocated to the nucleus and obstruct the formation of the parp -timeless complex, thereby inhibiting homologous recombination repair and maintaining cin, which potentiates tumor evolution ( , ) . it has also been reported that cgas recognizing cin activates non-canonical nf-κb signaling and potentiates cellular metastasis programs ( ) . furthermore, sting −/− mice are resistant to skin carcinogenesis in a , -dimethylbenz(a)anthracene (dmba)-treatment model. it has been demonstrated that when dmba-induced nuclear dna leaks into the cytoplasm, sting can induce chronic inflammatory stimulation that contributes to cancer development ( ) . during brain metastasis, cgamp transferred to bystander cells (e.g., astrocytes) can also produce ifnα and tnfα in the tme but, in this context, it will support tumor development and chemoresistance ( ) . coordinating with myeloid cells penetrating into the tumor, myeloidderived suppressor cells can also be recruited through the c-c chemokine receptor type (ccr ) ( ) . another study found that microparticles yielded by tumor cells can turn macrophages into the m type through cgas-sting-tbk , contrary to previous findings ( ) . immune-system interactions with tumor cells are complicated. the effect of cgas-sting on cancer is dependent on the type of tumor, host immune state, activated cell types, therapeutic intervention, and the magnitude of cgas-sting activation. like inflammation generated by cgas-sting, a time-dependent inflammatory anti-tumor response mediated by cgas-sting may be present. temporary activation of cgas-sting in innate immune cells could enhance the anti-tumor effect, whereas sustained activation of cgas-sting might induce immune tolerance of the tumor. more investigations are necessary to ascertain the exact role of cgas-sting in oncology, and elucidate the specific advantages and adverse effects of targeting the cgas-sting pathway in cancer therapy ( figure ). considering the pivotal role of the cgas-sting pathway in infection, inflammation and cancer, positive modulation of the pathway signaling is a promising way to enhance the immune state and restrict microorganisms or heterogeneous cells, whereas negative modulation can control aberrant inflammation. radiotherapy or chemotherapy drugs such as cisplatin or cyclophosphamide can induce dsbs and micronuclei, then trigger the cgas-sting pathway to enhance tumor immunogenicity ( ) ( ) ( ) . in addition, parp inhibitors such as olaparib have promising effects on cancer cells lacking brca because of their cooperative dna-repair functions ( ) . although cgas activation is inhibited by nucleosomes, taxol can induce mitotic cell-cycle arrest and sustain divided chromatin in the cytosol to activate the cgas-sting pathway slowly, and accumulation of signaling could stimulate apoptosis of cancer cells ( ). inhibitors of topoisomerase or used conventionally as chemotherapy drugs trigger minor damage to dna and accumulation of cytosolic dna, which can engage cgas and enhance the anti-tumor or anti-infection responses of cells ( ) ( ) ( ) . cgas-sting is essential on anti-tumor immune checkpoints therapies. for example, blockade of cd -signal regulatory protein α (sirpα) signaling on dcs can activate nadph oxidase (nox ) and increase the ph in phagosomes along with incomplete degradation of mtdna, which can trigger cgas-sting ( ) . sting deficiency in mice abrogates the anti-tumor effect of cd blockade ( ) . a similar phenomenon also can be seen in anti-programmed cell death- (pd- ) therapy ( ) . there is greater infiltration of ifnγ + cells and cd + t cells and pd- /pd- ligand (pd-l ) expression in tme treated by sting-ligand derivatives ( ) . therefore, in several types of tumors, combined administration of a sting agonist and immune-checkpoint antibody could elicit a more curative outcome compared with one therapy alone ( , ) . viruses can infect cells lacking cgas-sting more effectively, and have higher oncolytic activity compared with virus therapy alone. hence, the use of oncolytic viruses such as talimogene laherparepvec is beneficial for treating tumors with low expression of cgas/sting. sting expression can be regarded as a prognostic measurement for such therapy ( ) . some artificial analog molecules of cdns, such as , dimethylxanthenone- -acetic acid (dmxaa) and -carboxymethyl- ( h) acridone (cma) can bind the cdn pocket of mouse-specific sting dimers and promote conformational transition of sting from inactive "open" to an active "closed" state ( , ) . dmxaa showed convincing efficacy in restraining tumors in mice ( ) . however, dmxaa is restricted in activating mouse-specific sting but not humanspecific sting, which could be an explanation for the failure of dmxaa in treating non-small-cell lung cancer patients in a phase-iii clinical trial (nct ) ( ) . nonetheless, with three substitutions (g i, q i, and s a), human sting can also be induced by dmxaa to undergo conformational transition ( ) . another new compound, amidobenzimidazole, has been found to be an agonist of sting without lid closure and has potential therapeutic value ( ) . cyclic dinucleotides and their derivates that can stimulate sting directly are candidate adjuvants to restrain tumors. intratumoral administration of c-damp, c-dgmp, or cgamp analogs alone or combined with other adjuvants or tumor antigens have shown anti-tumor effects ( , ) ; phase-i or ii clinical trials (nct , nct , and nct ) of dithio-(rp,rp)-c-damp (known as adu-s ) are ongoing ( ) . to avoid the degradation and ensure maximal phagocyte internalization of cdns, endosomolytic nanoparticles have been designed to package and deliver cdns. for example, ph-sensitive nanoparticles (e.g., stingnanoparticles) can release their contents if located in acidic endosomal environments ( ) . for treatment of type-i interferonopathies, lessons can be taken from the treatment of canonical autoimmune disease such as sle, but there are several differences. for example, it was found that corticosteroid pulse therapy, γ-immunoglobulins, disease-modifying anti-rheumatic drugs, anti-cd , and some immunosuppressants (e.g., methotrexate) have limited efficacy against savi ( , ) . jak inhibitors such as ruxolitinib, tofacitinib and baricitinib that reduce type-i ifn downstream signaling have shown therapeutic value against savi, but further verification of their efficacy is needed ( ) . moreover, novel immune therapies, such as anti-ifnα and anti-ifnar immunoglobin, are in clinical trials for sle. these could also be tested against savi in the future ( ) . pharmaceutically screening has revealed that some antimalaria drugs, such as suramin, have an inhibitory effect on cgas by blockade of interaction between dna and cgas ( ) . in addition, novel small molecules such as ru or g can occupy the enzymatic pocket of species-specific cgas to abrogate cgamp synthesis ( , ) . recently, a study found that aspirin can acetylate cgas at three lysine residues and block cgas activity ( ) . with regard to sting, the cyclopeptide astin c can block irf recruitment onto the sting signalosome ( ) . the molecule h- can block the palmitoylation of human-sting ( ) . all of these agents are potential candidates for alleviating type-i interferonopathies. the cgas-sting pathway is primarily responsible for the modulation of immune response in cells when facing cytosolic dsdna challenge. moreover, it is complicatedly cross-regulated by other cellular processes or cellular signaling networks. the exact fundamental mechanism of the pathway in cells and the effect on the whole organism in specific states is not completely clear and requires further investigation. in conclusion, the cgas-sting pathway has dichotomous roles in the immune system. in general, cgas-sting-type-i ifn signaling can promote the innate immune response in myeloid cells but alleviate the adaptive immune response exerted by t cells and b cells. cgas shows high expression in apcs such as macrophages and dcs, but sting is expressed in most cells ( ) . cgas-sting signaling corresponding to cytoplasmic dsdna in apcs can boost innate and adaptive immunity transiently. in this situation, the dna challenge signal is limited to only macrophages and dcs. their pro-inflammatory and antigenpresenting functions to adaptive immune cells are promoted in the short-term. if the signal spreads to other bystander cells, such as t cells, b cells, local resident cells by means of cgamp transfer, or just aberrant sting activation by gof, it causes apoptosis in bystander cells or adaptive immune cells and immune tolerance in the long-term. therefore, it is reasonable to conclude that the intrinsic function of the cgas-sting pathway is essential for the innate immune system responses of the host immediately after pathogen invasion or abnormal cell appearing. once the challenge persists, the cgas-sting pathway controls the adaptive immune system to avoid chronic, detrimental inflammatory reactions or autoimmune diseases. the inflammatory response exists universally in almost all physiologic and pathologic progressions. cgas-sting is pivotal in modulating cellular inflammation, so it is promising to extend our conception of the cgas-sting pathway onto more diseases with inflammatory responses involved, especially cns-based diseases such as stroke, in which the inflammatory reaction exists but was recognized less. moreover, for targeting the cgas-sting pathway for therapeutic purposes, drugs should be optimized to augment the desirable effect and prevent its unwanted effects. for example, to eliminate tumor cells or infectious agents, agonists of cgas-sting would be a rational option if designed to target apcs exclusively but not t cells or b cells. in this scheme, the antitumor immune response is enhanced while avoiding apoptosis of adaptive immune cells and infiltration of immune suppressor cells. also, most research on the cgas-sting pathway has focused on its ifn-expressing role but overlooked autophagy and cell-death roles, which are also main downstream signaling of the pathway. therefore, some drugs, such as emricasan, are potential apoptosis inhibitors that may have a complementary effect on ameliorating apoptosis of blood-vessel endothelial cells or bronchial epithelial cells, and lymphopenia, in savi. until now, studies of the cgas-sting pathway have been done mainly in the laboratory but it has large space to be explored in clinical or translational fields. additionally more prrs and cellular immune-surveillance pathways may remain to be discovered to piece together the molecular puzzles of the cell. dw drafted the manuscript and drew the figures. wj and jh conceived and revised the review. frontiers in immunology | www.frontiersin.org recognition of endogenous nucleic acids by the innate immune system sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf transcription factor activation an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response sting is a direct innate immune sensor of cyclic di-gmp ifi is an innate immune sensor for intracellular dna cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects an expression atlas of human primary cells: inference of gene function from coexpression networks bacterial cgas-like enzymes synthesize diverse nucleotide signals structural mechanism of cytosolic dna sensing by cgas structure of human cgas reveals a conserved family of second-messenger enzymes in innate immunity cytosolic rna:dna hybrids activate the cgas-sting axis sequence-specific activation of the dna sensor cgas by y-form dna structures as found in primary hiv- cdna cgas produces a - -linked cyclic dinucleotide second messenger that activates sting the catalytic mechanism of cyclic gmp-amp synthase (cgas) and implications for innate immunity and inhibition the cytosolic dna sensor cgas forms an oligomeric complex with dna and undergoes switch-like conformational changes in the activation loop cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization structure of the human cgas-dna complex reveals enhanced control of immune surveillance cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders dna-induced liquid phase condensation of cgas activates innate immune signaling human cgas catalytic domain has an additional dna-binding interface that apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production obsessive-compulsive disorder is associated with broad impairments in executive function: a meta-analysis ribonuclease h mutations induce a cgas/sting-dependent innate immune response restriction by samhd limits cgas/sting-dependent innate and adaptive immune responses to hiv- host restriction factor samhd limits human t cell leukemia virus type infection of monocytes via sting-mediated apoptosis samhd acts at stalled replication forks to prevent interferon induction cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp connexin-dependent transfer of cgamp to phagocytes modulates antiviral responses cancer-cell-intrinsic cgas expression mediates tumor immunogenicity viruses transfer the antiviral second messenger cgamp between cells transmission of innate immune signaling by packaging of cgamp in viral particles cgas-mediated innate immunity spreads intercellularly through hiv- envinduced membrane fusion sites priming of dendritic cells by dna-containing extracellular vesicles from activated t cells through antigen-driven contacts cells infected with herpes simplex virus export to uninfected cells exosomes containing sting, viral mrnas, and micrornas slc a is an importer of the immunotransmitter cgamp slc a transports immunoreactive cyclic dinucleotides hydrolysis of -cgamp by enpp and design of nonhydrolyzable analogs cgas facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity activation by translocation from the er is associated with infection and autoinflammatory disease atg a controls dsdna-driven dynamic translocation of sting and the innate immune response irhom is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting tmed potentiates cellular ifn responses to dna viruses by reinforcing mita dimerization and facilitating its trafficking autophagy induction via sting trafficking is a primordial function of the cgas pathway phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation sting specifies irf phosphorylation by tbk in the cytosolic dna signaling pathway modular architecture of the sting c-terminal tail allows interferon and nf-kappab signaling adaptation structural basis of sting binding with and phosphorylation by tbk a conserved plplrt/sd motif of sting mediates the recruitment and activation of tbk activation of sting requires palmitoylation at the golgi ubiquitination of sting at lysine controls irf activation non-canonical nf-kappab antagonizes sting sensor-mediated dna sensing in radiotherapy immunomodulatory functions of type i interferons dna-binding landscape of irf , irf and irf dimers: implications for dimer-specific gene regulation attenuation of cgas-sting signaling is mediated by a p /sqstm -dependent autophagy pathway activated by tbk positive feedback regulation of type i ifn production by the ifn-inducible dna sensor cgas positive feedback regulation of type i interferon by the interferon-stimulated gene sting sting-mediated disruption of calcium homeostasis chronically activates er stress and primes t cell death sting senses microbial viability to orchestrate stress-mediated autophagy of the endoplasmic reticulum chronic innate immune activation of tbk suppresses mtorc activity and dysregulates cellular metabolism mtor signaling in growth, metabolism, and disease crosstalk between the cgas dna sensor and beclin- autophagy protein shapes innate antimicrobial immune responses a brief history of autophagy from cell biology to physiology and disease autophagy in the renewal, differentiation and homeostasis of immune cells sting directly activates autophagy to tune the innate immune response mycobacterium tuberculosis is protected from nadph oxidase and lc -associated phagocytosis by the lcp protein cpsa extracellular m. tuberculosis dna targets bacteria for autophagy by activating the host dna-sensing pathway upr, autophagy, and mitochondria crosstalk underlies the er stress response dnase a deficiency uncovers lysosomal clearance of damaged nuclear dna via autophagy self-consumption: the interplay of autophagy and apoptosis trafficking-mediated sting degradation requires sorting to acidified endolysosomes and can be targeted to enhance anti-tumor response the dna inflammasome in human myeloid cells is initiated by a sting-cell death program upstream of nlrp aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc antagonism of the sting pathway via activation of the aim inflammasome by intracellular dna inflammasome activation triggers caspase- -mediated cleavage of cgas to regulate responses to dna virus infection gasdermin d restrains type i interferon response to cytosolic dna by disrupting ionic homeostasis a noncanonical function of cgamp in inflammasome priming and activation viral dna binding to nlrc , an inhibitory nucleic acid sensor, unleashes sting, a cyclic dinucleotide receptor that activates type i interferon non-canonical activation of the dna sensing adaptor sting by atm and ifi mediates nf-kappab signaling after nuclear dna damage nuclear ifi induction of irf- signaling during herpesviral infection and degradation of ifi by the viral icp protein ifi is required for dna sensing in human macrophages by promoting production and function of cgamp ifi and cgas cooperate in the activation of sting during dna sensing in human keratinocytes cgas-mediated stabilization of ifi promotes innate signaling during herpes simplex virus infection viral dna sensors ifi and cyclic gmp-amp synthase possess distinct functions in regulating viral gene expression, immune defenses, and apoptotic responses during herpesvirus infection sting-mediated ifi degradation negatively controls type i interferon production assembly and localization of toll-like receptor signalling complexes sting requires the adaptor trif to trigger innate immune responses to microbial infection sequential activation of two pathogen-sensing pathways required for type i interferon expression and resistance to an acute dna virus infection cross-regulation of two type i interferon signaling pathways in plasmacytoid dendritic cells controls anti-malaria immunity and host mortality oxidative damage of dna confers resistance to cytosolic nuclease trex degradation and potentiates sting-dependent immune sensing mitochondria and cell death: outer membrane permeabilization and beyond mitochondrial inner membrane permeabilisation enables mtdna release during apoptosis apoptotic caspases prevent the induction of type i interferons by mitochondrial dna apoptotic caspases suppress type i interferon production via the cleavage of cgas, mavs, and irf signalling strength determines proapoptotic functions of sting puma amplifies necroptosis signaling by activating cytosolic dna sensors autophagic cell death restricts chromosomal instability during replicative crisis induction of necroptotic cell death by viral activation of the rig-i or sting pathway tnfalpha and radioresistant stromal cells are essential for therapeutic efficacy of cyclic dinucleotide sting agonists in nonimmunogenic tumors cellular senescence: aging p ink a induces an age-dependent decline in islet regenerative potential autophagy mediates degradation of nuclear lamina extranuclear dna accumulates in aged cells and contributes to senescence and inflammation senescenceassociated secretory phenotypes reveal cell-nonautonomous functions of oncogenic ras and the p tumor suppressor cytoplasmic chromatin triggers inflammation in senescence and cancer downregulation of cytoplasmic dnases is implicated in cytoplasmic dna accumulation and sasp in senescent cells innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence cgas is essential for cellular senescence dna-damage-induced type i interferon promotes senescence and inhibits stem cell function bacterial c-di-gmp affects hematopoietic stem/progenitors and their niches through sting a circular rna protects dormant hematopoietic stem cells from dna sensor cgas-mediated exhaustion pqbp is a proximal sensor of the cgas-dependent innate response to hiv- nono detects the nuclear hiv capsid to promote cgas-mediated innate immune activation p induces formation of neat lncrna-containing paraspeckles that modulate replication stress response and chemosensitivity hexim and neat long non-coding rna form a multi-subunit complex that regulates dna-mediated innate immune response stingdependent translation inhibition restricts rna virus replication inhibition of hiv- disease progression by contemporaneous hiv- infection samhd is the dendritic-and myeloid-cell-specific hiv- restriction factor counteracted by vpx reshaping of the dendritic cell chromatin landscape and interferon pathways during hiv infection the capsids of hiv- and hiv- determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hiv- activation of innate immunity depends strongly on the intracellular level of trex and sensing of incomplete reverse transcription products hiv- uses dynamic capsid pores to import nucleotides and fuel encapsidated dna synthesis restriction of hiv- and other retroviruses by trim hiv- evades innate immune recognition through specific cofactor recruitment cyclic di-gmp: second messenger extraordinaire cyclic gmp-amp signalling protects bacteria against viral infection a bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis tuberculosis pathogenesis and immunity mycobacterium tuberculosis differentially activates cgas-and inflammasome-dependent intracellular immune responses through esx- brucella abortus triggers a cgas-independent sting pathway to induce host protection that involves guanylate-binding proteins and inflammasome activation beneficial effects of yogasanas and pranayama in limiting the cognitive decline in type diabetes group b streptococcus degrades cyclic-di-amp to modulate stingdependent type i interferon production inhibition of innate immune cytosolic surveillance by an m. tuberculosis phosphodiesterase absence of specific chlamydia trachomatis inclusion membrane proteins triggers premature inclusion membrane lysis and host cell death the chlamydia trachomatis inclusion membrane protein cpos counteracts sting-mediated cellular surveillance and suicide programs yersinia yopj negatively regulates irf -mediated antibacterial response through disruption of sting-mediated cytosolic dna signaling intracellular bacteria engage a sting-tbk -mvb b pathway to enable paracrine cgas-sting signalling sting-dependent type i ifn production inhibits cell-mediated immunity to listeria monocytogenes induction of interferon-stimulated genes by irf promotes replication of toxoplasma gondii malaria parasite dna-harbouring vesicles activate cytosolic immune sensors type i interferon in rheumatic diseases activated sting in a vascular and pulmonary syndrome inherited sting-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations stimulator of interferon genes-associated vasculopathy with onset in infancy: a mimic of childhood granulomatosis with polyangiitis severe combined immunodeficiency in stimulator of interferon genes (sting) v m/wild-type mice sting-associated vasculopathy develops independently of irf in mice disease-associated mutations identify a novel region in human sting necessary for the control of type i interferon signaling personalized immunomonitoring uncovers molecular networks that stratify lupus patients aicardi-goutieres syndrome and the type i interferonopathies apoptosisderived membrane vesicles drive the cgas-sting pathway and enhance type i ifn production in systemic lupus erythematosus expression of cyclic gmp-amp synthase in patients with systemic lupus erythematosus. arthritis rheumatol the role of dendritic cells in autoimmunity human plasmacytoid dentritic cells elicit a type i interferon response by sensing dna via the cgas-sting signaling pathway interferon priming is essential for human cd + cell-derived plasmacytoid dendritic cell maturation and function interleukin- beta induces mtdna release to activate innate immune signaling via cgas-sting induction of dendritic cell differentiation by ifn-alpha in systemic lupus erythematosus neutrophil extracellular traps enriched in oxidized mitochondrial dna are interferogenic and contribute to lupus-like disease netting neutrophils are major inducers of type i ifn production in pediatric systemic lupus erythematosus type i ifnrelated netosis in ataxia telangiectasia and artemis deficiency cytosolic dna sensing promotes macrophage transformation and governs myocardial ischemic injury irf and type i interferons fuel a fatal response to myocardial infarction activation of the sting-irf pathway promotes hepatocyte inflammation, apoptosis and induces metabolic disorders in nonalcoholic fatty liver disease expression of sting is increased in liver tissues from patients with nafld and promotes macrophage-mediated hepatic inflammation and fibrosis in mice sting-mediated inflammation in kupffer cells contributes to progression of nonalcoholic steatohepatitis lipotoxicity induces hepatic protein inclusions through tank binding kinase -mediated p /sequestosome phosphorylation sting-irf triggers endothelial inflammation in response to free fatty acid-induced mitochondrial damage in diet-induced obesity priming the inflammatory pump of the cns after traumatic brain injury stingmediated type-i interferons contribute to the neuroinflammatory process and detrimental effects following traumatic brain injury sting-dependent sensing of self-dna drives silica-induced lung inflammation mitochondrial damage and activation of the sting pathway lead to renal inflammation and fibrosis mitochondrial damage causes inflammation via cgas-sting signaling in acute kidney injury how neuroinflammation contributes to neurodegeneration sensing of hsv- by the cgas-sting pathway in microglia orchestrates antiviral defence in the cns chronic neurodegeneration induces type i interferon synthesis via sting, shaping microglial phenotype and accelerating disease progression the roles of pink , parkin, and mitochondrial fidelity in parkinson's disease parkin and pink mitigate sting-induced inflammation atm and the molecular pathogenesis of ataxia telangiectasia autoimmune phenomena in ataxia telangiectasia mitochondrial dna has a pro-inflammatory role in amd cgas drives noncanonical-inflammasome activation in age-related macular degeneration multiple sclerosis type i/ii interferon balance in the regulation of brain physiology and pathology activation of the sting-dependent type i interferon response reduces microglial reactivity and neuroinflammation modulation of the immune system by uv radiation: more than just the effects of vitamin d? ultraviolet b irradiation causes stimulator of interferon genes-dependent production of protective type i interferon in mouse skin by recruited inflammatory monocytes. arthritis rheumatol engineering dna nanoparticles as immunomodulatory reagents that activate regulatory t cells activation of the sting adaptor attenuates experimental autoimmune encephalitis nucleic acid sensing by t cells initiates th cell differentiation sting-associated lung disease in mice relies on t cells but not type i interferon hierarchy of clinical manifestations in savi n s and v m mouse models intrinsic antiproliferative activity of the innate sensor sting in t lymphocytes agonist-mediated activation of sting induces apoptosis in malignant b cells b cell-intrinsic sting signaling triggers cell activation, synergizes with b cell receptor signals, and promotes antibody responses sting signaling promotes inflammation in experimental acute pancreatitis habtezion a. sting signalling protects against chronic pancreatitis by modulating th response stingactivating adjuvants elicit a th immune response and protect against mycobacterium tuberculosis infection sting-dependent signaling underlies il- controlled inflammatory colitis the cytosolic sensor sting is required for intestinal homeostasis and control of inflammation the multifaceted role of chromosomal instability in cancer and its microenvironment immunogenic cell death in cancer and infectious disease sting contributes to antiglioma immunity via triggering type i ifn signals in the tumor microenvironment sting-dependent cytosolic dna sensing mediates innate immune recognition of immunogenic tumors suppression of sting associated with lkb loss in kras-driven lung cancer dna exonuclease trex regulates radiotherapy-induced tumour immunogenicity growing tumors induce a local sting dependent type i ifn response in dendritic cells tumorderived cgamp triggers a sting-mediated interferon response in nontumor cells to activate the nk cell response rae ligands for the nkg d receptor are regulated by sting-dependent dna sensor pathways in lymphoma sting activation reprograms tumor vasculatures and synergizes with vegfr blockade dendritic cells but not macrophages sense tumor mitochondrial dna for cross-priming through signal regulatory protein alpha signaling exosomes shuttle trex -sensitive ifn-stimulatory dsdna from irradiated cancer cells to dcs host type i ifn signals are required for antitumor cd + t cell responses through cd {alpha}+ dendritic cells sting-dependent cytosolic dna sensing promotes radiation-induced type i interferondependent antitumor immunity in immunogenic tumors targeting dna damage response promotes antitumor immunity through sting-mediated t-cell activation in small cell lung cancer intratumoral sting activation with t-cell checkpoint modulation generates systemic antitumor immunity sting signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion il- is a component of the inflammatory milieu in the tumor microenvironment promoting antitumor responses suppression of sting signaling through epigenetic silencing and missense mutation impedes dna damage mediated cytokine production extrachromosomal telomere repeat dna is linked to alt development via cgas-sting dna sensing pathway downregulation of membrane trafficking proteins and lactate conditioning determine loss of dendritic cell function in lung cancer her recruits akt to disrupt sting signalling and suppress antiviral defence and antitumour immunity akt kinase-mediated checkpoint of cgas dna sensing pathway dna damage predicts prognosis and treatment response in colorectal liver metastases superior to immunogenic cell death and t cells chromosomal instability drives metastasis through a cytosolic dna response inflammation-driven carcinogenesis is mediated through sting carcinomaastrocyte gap junctions promote brain metastasis by cgamp transfer host stingdependent mdsc mobilization drives extrinsic radiation resistance tumor cellderived microparticles polarize m tumor-associated macrophages for tumor progression parp inhibition enhances tumor cell-intrinsic immunity in ercc -deficient non-small cell lung cancer parp inhibitor efficacy depends on cd (+) t-cell recruitment via intratumoral sting pathway activation in brca-deficient models of triple-negative breast cancer radiotherapy combined with novel sting-targeting oligonucleotides results in regression of established tumors topoisomerase inhibition promotes cyclic gmp-amp synthasedependent antiviral responses topoisomerase ii inhibitors induce dna damage-dependent interferon responses circumventing ebola virus immune evasion cgas/sting axis mediates a topoisomerase ii inhibitor-induced tumor immunogenicity cd blockade triggers t cell-mediated destruction of immunogenic tumors sting activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity a sting agonist given with ox receptor and pd-l modulators primes immunity and reduces tumor growth in tolerized mice magnitude of therapeutic sting activation determines cd (+) t cellmediated anti-tumor immunity recurrent loss of sting signaling in melanoma correlates with susceptibility to viral oncolysis species-specific detection of the antiviral small-molecule compound cma by sting bindingpocket and lid-region substitutions render human sting sensitive to the species-specific drug dmxaa the sting agonist dmxaa triggers a cooperation between t lymphocytes and myeloid cells that leads to tumor regression randomized phase iii placebo-controlled trial of carboplatin and paclitaxel with or without the vascular disrupting agent vadimezan (asa ) in advanced non-small-cell lung cancer design of amidobenzimidazole sting receptor agonists with systemic activity sting ligand c-di-gmp improves cancer vaccination against metastatic breast cancer endosomolytic polymersomes increase the activity of cyclic dinucleotide sting agonists to enhance cancer immunotherapy efficacy of the janus kinase / inhibitor ruxolitinib in the treatment of vasculopathy associated with tmem -activating mutations in children suramin potently inhibits cgamp synthase, cgas, in thp cells to modulate ifnbeta levels small molecule inhibition of cgas reduces interferon expression in primary macrophages from autoimmune mice development of human cgas-specific small-molecule inhibitors for repression of dsdnatriggered interferon expression the cyclopeptide astin c specifically inhibits the innate immune cdn sensor sting targeting sting with covalent small-molecule inhibitors trim -mediated monoubiquitination of cgas for cytosolic dna sensing the ubiquitin ligase trim regulates innate immune responses to intracellular doublestranded dna the e ubiquitin ligase rnf facilitates the cgas-mediated innate immune response rnf temporally regulates virus-triggered type i interferon induction by two distinct mechanisms the e ubiquitin ligase amfr and insig bridge the activation of tbk kinase by modifying the adaptor sting trim inhibits cgas degradation mediated by selective autophagy receptor p to promote innate immune responses p inhibition provides anti-dna virus immunity by regulation of usp phosphorylation and sting activation usp recruits usp to promote innate antiviral response through deubiquitinating sting/mita the deubiquitinase cyld is a specific checkpoint of the sting antiviral signaling pathway sumoylation promotes the stability of the dna sensor cgas and the adaptor sting to regulate the kinetics of response to dna virus senp potentiates cgas activation by relieving sumo-mediated inhibition of cytosolic dna sensing g bp promotes dna binding and activation of cgas zcchc is a cosensor of cgas for dsdna recognition in innate immune response manganese increases the sensitivity of the cgas-sting pathway for double-stranded dna and is required for the host defense against dna viruses tmem is a binding partner and regulator of sting-mediated inflammatory signaling in macrophages the erassociated protein zdhhc is a positive regulator of dna virus-triggered, mita/sting-dependent innate immune signaling association of abnormal elevations in ifit with overactive cyclic gmp-amp synthase/stimulator of interferon genes signaling in human systemic lupus erythematosus monocytes s k-sting interaction regulates cytosolic dna-mediated activation of the transcription factor irf glycogen synthase kinase beta regulates irf transcription factor-mediated antiviral response via activation of the kinase tbk trim short isoform preferentially promotes dna and rna virus-induced production of type i interferon by recruiting gsk beta to tbk lsm a plays a critical role in antiviral immune responses by regulating mita level in a cell-specific manner trim promotes dna virus infections by inhibiting innate immune response trim alpha is a negative-feedback regulator of the intracellular dna and dna virustriggered response by targeting sting the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita usp negatively regulates antiviral responses by deubiquitinating sting oligoadenylatesynthetase-family protein oasl inhibits activity of the dna sensor cgas during dna virus infection to limit interferon production interactome and proteome dynamics uncover immune modulatory associations of the pathogen sensing factor cgas sensing of bacterial cyclic dinucleotides by the oxidoreductase recon promotes nf-kappab activation and shapes a proinflammatory antibacterial state the ca( +) sensor stim regulates the type i interferon response by retaining the signaling adaptor sting at the endoplasmic reticulum nitro-fatty acids are formed in response to virus infection and are potent inhibitors of sting palmitoylation and signaling nlrc , a member of the nlr family of proteins, is a negative regulator of innate immune signaling induced by the dna sensor sting ppm a regulates antiviral signaling by antagonizing tbk -mediated sting phosphorylation and aggregation ptpn / -mediated dephosphorylation of mita/sting promotes its s proteasomal degradation and attenuates innate antiviral response nlrx sequesters sting to negatively regulate the interferon response, thereby facilitating the replication of hiv- and dna viruses nlrx promotes immediate irf -directed antiviral responses by limiting dsrnaactivated translational inhibition mediated by pkr mitigating sox -potentiated immune escape of head and neck squamous cell carcinoma with a sting-inducing nanosatellite vaccine mediates hypoxia-induced immunosuppression by repressing cgas nrf negatively regulates sting indicating a link between antiviral sensing and metabolic reprogramming kdm histone demethylases repress immune response via suppression of sting herpes simplex virus abrogates the cgas/sting-mediated cytosolic dna-sensing pathway via its virion host shutoff protein, ul herpes simplex virus tegument protein vp abrogates cgas/sting-mediated antiviral innate immunity species-specific deamidation of cgas by herpes simplex virus ul protein facilitates viral replication evasion of the sting dna-sensing pathway by vp / of herpes simplex virus herpes simplex virus gamma . protein inhibits sting activation that restricts viral replication human cytomegalovirus protein ul inhibits dna sensing of cgas to mediate immune evasion type i interferon production by inactivating the dna sensor cgas without affecting sting essential role of hcmv deubiquitinase in promoting oncogenesis by targeting anti-viral innate immune signaling pathways human cytomegalovirus tegument protein ul inhibits sting-mediated signaling to evade antiviral immunity the herpesviral antagonist m reveals differential activation of sting-dependent irf and nf-kappab signaling and sting's dual role during mcmv infection human cytomegalovirus-encoded us targets mavs and sting signaling to evade type i interferon immune responses inhibition of cgas dna sensing by a herpesvirus virion protein cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas modulation of the cgas-sting dna sensing pathway by gammaherpesviruses sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex poxviruses evade cytosolic sensing through disruption of an mtorc -mtorc regulatory circuit viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling zika virus elicits inflammation to evade antiviral response by cleaving cgas via ns -caspase- axis denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus ns b protein targets cgas for degradation and prevents mitochondrial dna sensing during infection dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity hepatitis c virus ns b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dnasensing pathways hiv- /siv vpx targets a novel functional domain of sting to selectively inhibit cgas-stingmediated nf-kappab signalling we thank k. wood from barrow neurological institute for discussions and editing. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © wan, jiang and hao. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -xms su w authors: rahmani, hamid; davoudi-monfared, effat; nourian, anahid; khalili, hossein; hajizadeh, nooshin; zarei jalalabadi, narjes; reza fazeli, mohammad; ghazaeian, monireh; saeed yekaninejad, mir title: interferon β- b in treatment of severe covid- : a randomized clinical trial date: - - journal: int immunopharmacol doi: . /j.intimp. . sha: doc_id: cord_uid: xms su w in this study, efficacy and safety of interferon (ifn) β- b in the treatment of patients with severe covid- were evaluated. among an open-label, randomized clinical trial, adult patients (≥ years old) with severe covid- were randomly assigned ( : ) to the ifn group or the control group. patients in the ifn group received ifn β- b ( mcg subcutaneously every other day for two consecutive weeks) along with the national protocol medications while in the control group, patients received only the national protocol medications (lopinavir/ritonavir or atazanavir/ritonavir plus hydroxychloroquine for - days). the primary outcome of the study was time to clinical improvement. secondary outcomes were in-hospital complications and -daymortality. between april and may , , patients were enrolled and finally patients in each group completed the study. time to clinical improvment in the ifn group was significantly shorter than the control group ([ ( - ) vs. ( - ) days respectively, p= . , hr= . ; % ci: . - . ]). at day , the percentage of discharged patients was . % and . % in the ifn and control groups respectively (or= . ; % ci: . - . , p= . ). icu admission rate in the control group was significantly higher than the ifn group ( . % vs. . %, p = . ). the duration of hospitalization and icu stay were not significantly different between the groups all-cause -day mortality was . % and . % in the ifn and control groups respectively (p = . ). ifn β- b was effective in shortening the time to clinical improvement without serious adverse events in patients with severe covid- . furthermore, admission in icu and need for invasive mechanical ventilation decreased following administration of ifn β- b. although -day mortality was lower in the ifn group, further randomized clinical trials with large sample size are needed for exact estimation of survival benefit of ifn β- b. coronavirus disease (covid- ) was reported from wuhan for the first time in late december . causing severe acute respiratory syndrome coronavirus (sars-cov- ) [ ] , it rapidly spread throughout the world to the extent that the world health organization (who) stated it as pandemic in march [ ] . until july , , more than million confirmed cases of covid- were reported worldwide. furthermore, more than . deaths were recorded [ ] . until now, there is no definite antiviral treatment for covid- and attempts continue for finding effective treatments worldwide. however, from the beginning of the pandemic, various treatments such as antiretrovirals, anti-malaria agents, favipiravir, remdesivir, and corticosteroids, immunoglobulin and cytokine blockers as adjunctive therapies were suggested for the treatment of covid- [ ] . except for the remdesivir which has had acceptable results, the efficacy of other drugs has not been significant on the outcomes of the patients with covid- [ ] [ ] [ ] [ ] [ ] . interferons (ifns) have a key role in defense against viral infections as a component of innate immune system [ ] . invitro activity of ifn β has been shown against severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) [ ] [ ] [ ] [ ] . although ifn β was used less than ifn α for the treatment of sars-cov and mers-cov in human studies, it was effective in the treatment of mers-cov in retrospective studies and case series [ ] [ ] . the efficacy of ifn β- b is being assessed in the treatment of mers in a randomized clinical trial [ ] . according to the presence of this evidence, ifn β was considered as a promising option for the treatment of in this open-label, randomized clinical trial, efficacy and safety of ifn β- b in the treatment of patients with severe covid- were assessed. this open-label, randomized clinical trial was designed to evaluate the efficacy and safety of ifn β- b in the treatment of patients with covid- adult patients (≥ years old) with positive pcr and clinical symptoms/signs of pneumonia (including dyspnea, cough and fever), peripheral oxygen saturation (spo ) ≤ % in ambient air or arterial oxygen partial pressure to fractional inspired oxygen (pao /fio ) < or spo /fio < and lung involvement in chest imaging were included. these criteria indicated severe form of the disease [ ] . at baseline, patients with serious allergic reactions to ifn, history of suicide thoughts and attempts, alanine amino transferase (alt)> × the upper limit of the normal range, uncontrolled underlying diseases such as neuropsychiatric disorders, thyroid disorders, cardiovascular diseases and also pregnant and lactating women were not included. recruitment was considered during the first -hour of the hospital admission. during the study period, patients who received less than doses of ifn β- b were excluded. if patients were discharged before fulfilment of the treatment course, the treatment was applied at home. eligible patients were recruited in the ifn group or the control group according to the permuted block randomization. patients in the ifn group received ifn β- b along with the national protocol medications, while in the control group, patients received only the national protocol medications. ifn β- b (ziferon®, zist daru daneh co., iran) was administrated as mcg subcutaneously every other day for two consecutive weeks. the national protocol consisted lopinavir/ritonavir ( / mg bd) or atazanavir/ritonavir ( / mg daily) plus hydroxychloroquine ( mg bd in first day and then mg bd) for - days. other supportive cares such as fluid therapy, stress ulcer prophylaxis, deep vein thrombosis, treatment of electrolyte disorders and antibiotic therapy were considered according to the hospital protocols. the duration of the study was two weeks. a -week follow-up period was considered for all patients. patients' demographic data, baseline diseases, symptoms at the time of disease presentation, vital signs and laboratory data at the time of hospital admission were recorded. patients were daily monitored in terms of changes in the vital signs, hemodynamic parameters, oxygenation status, laboratory data and treatment strategies. clinical status of the patients was assessed by the six- category ordinal scale at days , , and of the randomization [ ] . need for supplemental oxygen therapy and also invasive or non-invasive respiratory supports were evaluated regularly. time to clinical improvement was considered as primary outcome of study. clinical improvement was defined as improvement of at least two points from the baseline status on the six-category ordinal scale [ ] . this scale contains the subsequent categories: ( ) death ( ) hospital admission requiring invasive mechanical ventilation ( ) hospital admission, requiring non-invasive positive pressure ventilation ( ) hospital admission, requiring oxygen ( ) hospital admission, not requiring oxygen ( ) discharge. secondary outcomes were clinical status of patients at day , and , icu admission and intubation rates, length of hospitalization and icu stay, and -day mortality. side effects related to ifn therapy and other adverse events during the study period were monitored and recorded as the safety outcomes. categorization of adverse events was done according to the common terminology criteria for adverse events (ctcae), national institutes of health and national cancer institute, . also serious complications during the hospitalization course such as acute respiratory distress syndrome (ards), nosocomial infections, septic shock, acute kidney injury (aki) and acute hepatic injury (ahi) were considered. continuous variables are demonstrated as median (interquartile range (iqr)) and categorical variables as frequencies and percentages. continuous variables were compared between the groups by mann-whitney u test. the fisher's exact test was applied for comparison of categorical variables. the hazard ratio (hr) and % ci for clinical improvement were estimated by cox proportional hazards regression analysis. the effect of ischemic heart disease, lymphocyte count, aspartate aminotransferase (ast) and c-reactive protein (crp) on the primary outcome was evaluated by the adjusted cox regression models as potential confounding factors. time to clinical improvement was estimated by kaplan-meier plot and compared with a log-rank test. all statistical analysis was done by spss software (version . ). time to clinical improvement was estimated to be approximately days and sample size was calculated by following equation: power= . according to the above equation, at least patients in each group were expected to make a difference of days in time to clinical improvement with power of %. patients were randomly recruited ( : ) to the ifn group or the control group. the method of randomization was the permuted block randomization ( patients per block). a biostatistician who was not involved in patients' care did this process. a total of patients were screened. of them, patients did not have the eligibility criteria of study and patients were referred from another hospital. three and four patients withdrew the consent during the study in the ifn group and control groups, respectively. four patients did not adhere to ifn injection after second or third dose. also three patients in the control group were enrolled in another trial. finally, patients in each group completed the study ( figure ). the median (iqr) age of patients was ( - ) years and . % of them were male. no significant difference in terms of the patients' demographic data was detected between the groups. the most common comorbidities were hypertension, diabetes mellitus and ischemic heart disease. dyspnea, fever and cough were the most frequent symptoms at the time of hospital admission. the median (iqr) time from onset of the symptoms to hospital admission was ( - ) and ( - ) days in the ifn group and control groups respectively. the time from onset of the symptoms to randomization was not statistically significant between the groups. all of patients required respiratory support at the time of randomization. oxygenation through facemask was required for more than percent of patients. none of the patients in both groups were intubated at baseline ( the ec values for remdesivir and lopinavir were determined as . and . µm respectively. the cc values of ifns, remdesivir and lopinavir were > . iu/ml, > µm and µm respectively. among ifns, the most reductive effects on viral load belonged to ifn β- a and ifn β- b. however, ifn β- b showed highest potency and selectivity index against sars-cov- [ ] . in a randomized clinical trial, and patients were recruited in the combination and control groups respectively. patients in the combination group received ifn β- b, lopinavir/ritonavir and ribavirin while those in the control group received only lopinavir/ritonavir. the primary outcome was defined as the time to reach a negative rt-pcr of respiratory secretions for sars-cov- . the time to resolution of the symptoms was considered as one of the secondary outcomes. the median time to achieving a negative rt-pcr was significantly shorter in the combination group compared to the control group ( vs. days). moreover, resolution of the symptoms occurred notably faster in the combination group than the control group ( vs. days) [ ] . similar with our study, ifn β- b was started in the viral phase of covid- i.e. within first days of onset of the symptoms. in our study median time from onset of the symptoms to randomization was days. in both studies, first dose of ifn β- b was administered within to hours of hospital admission. initiation of antiviral agents as soon as possible following onset of the symptoms is critical in control of viral replication and prevention of tissue viral invasion. the efficacy of antivirals significantly decreased after establishment of the cytokines release phase in covid- [ ] [ ] . due to resource limitations, evaluation of viral clearance was not possible in our study. and patients were assigned to the ifn and control groups respectively. in-hospital mortality was considered as the primary outcome of study. the mortality rate was statistically significant in the control group than the ifn group ( . % vs. . %) [ ] . retrospective design and lack of matching of the groups in terms of receiving other antivirals should be considered when interpreting the results. in a case series, characteristics and outcomes of five patients with severe covid- , who were treated with ifn β- b, lopinavir/ritonavir and hydroxychloroquine, were described. the antiviral regimen applied for these patients was similar to our study. treatment was successful in patients while clinical status of patients deteriorated during the treatment course. all patients received corticosteroids. furthermore, all patients were initially admitted in another hospital and later transferred to the referral hospital [ ] . . %). early administration of ifn β- a significantly reduced the mortality rate compared with late administration [ ] . absence of follow-up pcr and chest imaging along with the small sample size were the major limitations of the study. our study suffered from some limitations. follow up chest imaging or virological assessment was not possible due to resources limitations, therefore the effect of ifn β- b on viral clearance was not determined. small sample size did not allow accurate estimation of survival benefit of ifn β- b. in conclusion, ifn β- b was effective in shortening the time to clinical improvement without serious adverse events in patients with severe covid- . furthermore, icu admission rate and need for invasive mechanical ventilation significantly reduced by administration of ifn β- b. although compared with the control group, ifn β- b reduced duration of hospitalization, length of icu stay, intubation rate and -day mortality were not statistically different between the groups. further randomized clinical trials with enough sample size are needed to accurately estimate survival benefit of ifn β- b. in patients with severe covid- : -as add-on therapy, ifn β- b shortened the time to clinical improvement -ifn β- b significantly increased the discharge rate at day -ifn β- b reduced overall -day mortality -ifn β- b related adverse effects were mild and did not cause treatment interruptions review of the novel coronavirus (sars-cov- ) based on current evidence who declares covid- a pandemic johns hopkins coronavirus resource center home page pharmacologic treatments for coronavirus disease (covid- ): a review observational study of hydroxychloroquine in hospitalized patients with covid- a trial of lopinavir-ritonavir in adults hospitalized with severe covid- effectiveness and safety of glucocorticoids to treat covid- : a rapid review and meta-analysis clinical efficacy of intravenous immunoglobulin therapy in critical patients with covid- : a multicenter retrospective cohort study the antiviral effect of interferonbeta against sars-coronavirus is not mediated by mxa protein interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) interferon-beta a and sars coronavirus replication inhibitors of middle east respiratory syndrome coronavirus in cell-based assays ifn-α a or ifn-β a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia and the miracle trial group. treatment of middle east respiratory syndrome with a combination of lopinavirritonavir and interferon-β b (miracle trial): study protocol for a randomized controlled trial management and treatment of covid- : the chinese experience analysis of an ordinal endpoint for use in evaluating treatments for severe influenza requiring hospitalization type interferons as a potential treatment against covid- heightened innate immune responses in the respiratory tract of covid- patients imbalanced host response to sars-cov- drives development of covid- broad-spectrum host-based antivirals targeting the interferon and lipogenesis pathways as potential treatment options for the pandemic coronavirus disease (covid- ). viruses triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial covid- : towards understanding of pathogenesis covid- infection: the perspectives on immune responses clinical evaluation of ifn beta b in covid- pneumonia: a retrospective study five severe covid- pneumonia patients treated with triple combination therapy with lopinavir/ritonavir, hydroxychloroquine, and interferon β- b interferon beta- a as a candidate for an open-label single-arm clinical trial subcutaneous administration of interferon beta- a for covid- : a non-controlled prospective trial efficacy and safety of interferon beta- a in treatment of severe covid- : a randomized clinical trial table -baseline characteristics of patients parameter; median (iqr) or n (%) we would like to thank the nurses and other staffs of imam khomeini hospital complex for their kind supports and also ms. ava khalili for english proofreading the manuscript. the authors did not receive any fund for this work. there is no conflict of interest for authors to declare. key: cord- -bbvlowyo authors: sang, eric r.; tian, yun; gong, yuanying; miller, laura c.; sang, yongming title: integrate structural analysis, isoform diversity, and interferon-inductive propensity of ace to predict sars-cov susceptibility in vertebrates date: - - journal: heliyon doi: . /j.heliyon. .e sha: doc_id: cord_uid: bbvlowyo the current new coronavirus disease (covid- ) has caused globally near . / million confirmed deaths/infected cases across more than countries. as the etiological coronavirus (a.k.a. sars-cov ) may putatively have a bat origin, our understanding about its intermediate reservoir between bats and humans, especially its tropism in wild and domestic animals are mostly unknown. this constitutes major concerns in public health for the current pandemics and potential zoonosis. previous reports using structural analysis of the viral spike protein (s) binding its cell receptor of angiotensin-converting enzyme (ace ), indicate a broad potential of sars-cov susceptibility in wild and particularly domestic animals. through integration of key immunogenetic factors, including the existence of s-binding-void ace isoforms and the disparity of ace expression upon early innate immune response, we further refine the sars-cov susceptibility prediction to fit recent experimental validation. in addition to showing a broad susceptibility potential across mammalian species based on structural analysis, our results also reveal that domestic animals including dogs, pigs, cattle and goats may evolve ace -related immunogenetic diversity to restrict sars-cov infections. thus, we propose that domestic animals may be unlikely to play a role as amplifying hosts unless the virus has further species-specific adaptation. findings may relieve relevant public concerns regarding covid- -like risk in domestic animals, highlight virus-host coevolution, and evoke disease intervention through targeting ace molecular diversity and interferon optimization. erupting in china last december, the novel coronavirus disease has become a worldwide pandemic and caused near . million confirmed deaths and million infected cases across countries by the end of may [ , ] . the etiological virus, designated as severe acute respiratory syndrome coronavirus (sars-cov ) has been identified [ ] and related to the viruses previously causing sars or middle east respiratory syndrome (mers) in humans in and , respectively [ ] . these three human-pathogenic coronaviruses putatively evolve from bat coronaviruses, but have different animal tropisms and intermediate reservoirs before transmission to humans [ , ] . as civet cats and camels were retrospectively determined as reservoirs for sars and mers respectively, there is no conclusion about what animal species passing sars-cov to humans [ , ] . investigations indicated that canivora animals including raccoon dogs, red foxes, badgers and minks as well swine, at a less extent, are susceptible to sars virus infections [ , ] . although the viral nucleic acids and antibodies to mers were detectable in multiple ruminant species including sheep, goat, and donkeys, the virus inoculation studies did not result in a productive infection for mers disease in these domestic ruminants, nor in horses [ , ] . as a group of obligate pathogens, viruses need to engage cell receptors for entering cells and race with the host immunity for effective replications and spreading to initiate a productive infection [ ] . in this context, the spike proteins protruding on the coronavirus surface are responsible for cell receptor binding and mediating viral entry [ , , ] . for example, mers-cov adopts the dipeptidyl peptidase (dpp , a.k.a cd ) and sars-cov uses angiotensin-converting enzyme (ace ) as primary receptors for cell attachment and entry [ , , , , , ] . several groups have reported that sars-cov uses the same ace receptor as sars-cov, but exerts higher receptor affinity to human ace , which may ascribe to the efficacy of sars-cov infection in humans [ , ] . after cell attachment via the receptor binding domain (rbd) in the n-terminal s region of the s protein, the c-terminal s region thus engages in membrane fusion. further cleavage of s from s by a furin-like protease will release and prime the virus entering the receipient cells. several furin-like proteases, especially a broadly expressed trans-membrane serine protease (tmprss ), are adopted for priming sars-cov entry [ , ] . compared with sars-cov, studies showed that sars-cov spike protein also evolutionarily obtains an additional furin-like proteinase cleavage site within the s /s junction region for efficient release from the cell surface and entry into the cells [ , , , ] . because tmprss is widely expressed, the tissue-specific expression of ace has been shown to determine sars-cov cell tropism in humans [ , ] . namely, human nasal secretory cells, type ii pneumocytes, and absorptive enterocytes are ace -tmprss double positive and highly permissive to sars-cov infection [ , ] . for cross-species animal tropism, the potential infectivity of sars-cov in both wild and domestic animals raises a big public health concern after the prevalence of sars-cov infections in humans [ , ] . this concern involves two aspects: ( ) screening to identify the animal species that serve as a virus reservoir originally passing sars-cov to humans; and ( ) the existing risk of infected people passing the virus to animals, particularly domestic species, thus potentially amplifying the zoonotic cycle to worsen sars-cov evolution and prevalence [ , ] . by diagnosis of animals that in close contact with covid- patients or screening of animal samples in some covid- epidemic zones, studies detected that domestic cats and dogs could be virally or serologically positive for sars-cov [ , , , , , , ] , as was a reported infection in a zoo tiger [ ] . using controlled experimental infection of human sars-cov isolates, several studies demonstrated that ferrets, hamsters, domestic cats and some non-human primate species are susceptible to human sars-cov strains [ , , , , , , , ] . obviously, it is impractical to test sars-cov susceptibility experimentally in all animal species. by adoption of a structural simulation based on published structures of the viral s-rbd/ace complex, studies have predicted a broad spectrum of vertebrate species with high potential for sars-cov susceptibility, which, if true, entails unexpected risks in both public and animal health, and warrants further critical evaluation [ , , ] . ace is a key enzyme catalyzing angiotensin (agt) further conversion into numeral active forms of agt - , which are hormonal mediators in the body's renin-angiotensin system (ras) [ , ] . thus, ace plays a regulatory role in the blood volume/pressure, body fluid balance, sodium and water retention, as well as immune effects on apoptosis, inflammation, and generation of reactive oxygen species (ros) [ , ] . in this line, the expression of ace is also inter-regulated by immune mediators pertinent to its systemic function. multiple physio-pathological factors, including pathogenic inflammation, influence on ras through action on ace expression [ , , ] . interferon (ifn) response, especially that mediated by type i and type iii ifns, comprises a frontline of antiviral immunity to restrict viral spreading from the initial infection sites, and therefore primarily determines if a viral exposure becomes controlled or a productive infection [ ] . most recent studies indicated that sars-cov evolved viral antagonisms against ifn responses; however, the viral infections was significantly inhibited in vitro or at the early phase in vivo using human ifn-α, ifn-β or type iii inf-λ, indicating that ifns are potential anti-covid prophylactics [ , , ] . several recent studies revealed that human ace gene behaves like an interferon-stimulated gene (isg) and is stimulated by a viral infection and ifn treatment; however, mouse ace gene is not [ , , ] . therefore, to determine the cell tropism and animal susceptibility to sars-cov , the cross-species ace genetic and especially epigenetic diversity in regulation of ace expression and functionality should be evaluated [ , , , , , , , , ] . in this study, through integration of structural analysis and key immunogenetic factors that show species-dependent differences, we critically refine the sars-cov susceptibility prediction to fit recent experimental validation [ , , , , , , , , , ] . along with showing a broad susceptibility potential across mammalian species based on structural analysis [ , , ] , our results further reveal that domestic animals including dogs, pigs, cattle and goats may evolve previously unexamined immunogenetic diversity to restrict sars-cov infections. the amino acid sequences of ace proteins and dna sequences of the proximal promoters of each ace genes were extracted from ncbi gene and relevant databases (https://www.ncbi.nlm.nih.gov/gene). ace genes and corresponding transcripts have been well annotated in most representative vertebrate species. in most cases, the annotations were double verified through the same gene entries at ensembl (http s://www.ensembl.org). the protein sequences were collected from all non-redundant transcript variants and further verified for expression using relevant rna-seq data (ncbi geo profiles). the proximal promoter region spans~ . kb before the predicted transcription (or translation) start site (tss) of ace or other genes. the protein and dna sequences were aligned using the multiple sequence alignment tools of clustalw or muscle through an embl-ebi port (https://www.ebi.ac.uk/). other sequence management was conducted using programs at the sequence manipulation suite (http://www.bioinformatics.org). sequence alignments were visualized using jalview (http://www.ja lview.org) and megax (https://www.megasoftware.net). sequence similarity calculation and plotting were done using sdt . (http://web.cbio.uct.ac.za/~brejnev). other than indicated, all programs were run with default parameters. the phylogenic analysis and tree visualization were performed using megax and an online program, evoview. the evolutionary history was inferred using the neighbor-joining method. percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( , replicates) was also performed. the evolutionary distances were computed using the p-distance method and in units of the number of amino acid differences per site. other than indicated, all programs were run with default parameters as the programs suggested. the structure files of human ace protein and its interaction with sars-cov s-rbd were extracted from the protein data bank under the files of m and m j. the residual mutation and structure simulation were performed using ucsf chimera and pymol available at http s://www.cgl.ucsf.edu/chimera/ and https://pymol.org/, respectively. structural visualization were using pymol. the binding affinity energy (Δg), dissociation constant (kd) and interfacial contacts between s-rbd and each ace were calculated using a prodigy algorithm at https://bi anca.science.uu.nl/prodigy/. the regulatory elements (and pertinent binding factors) in the~ . kb proximal promoter regions were examined against both human/animal tfd database using a program nsite (version . , at http: //www.softberry.com). the mean position weight matrix (pwm) of key cis-elements in the proximal promoters were calculated using pwm tools through https://ccg.epfl.ch/cgi-bin/pwmtools, and the binding for expression confirmation, several sets of rna-seq data from ncbi gene databases, and one of ours generated from porcine alveolar macrophages (bioproject with an accession number of srp ), were analyzed for verification of the differential expression of ace genes in most annotated animal species. especially, the expression of porcine ace isoforms and relevant other genes in the porcine lung macrophage datasets. significantly differentially expressed genes (degs) between two treatments were called using an edger package and visualized using heatmaps or bar charts as previously described [ ] . the phylogenic tree of major identified ace orthologs/variants from different species was built with a neighbor-joining approach and visualized using an evoview program under default parameter setting. the prediction of sars-cov susceptibility is based on the sequence similarity of each ace to human ace in the s-rbd binding region and simulated using a published human ace -rbd structure ( m j) and refers to two recent publications using similar procedures but different structural models [ , ] . compared with the currently available experimental data, incongruence of the predicted sars-cov susceptibility is clearly demonstrated in pangolin, ferret, tiger, cat and horseshoe bat, indicating that some other factors besides ace -rbd affinity should be considered. figure . prediction of sars-cov susceptibility in major livestock species based on the conservation of key interacting residues and binding capacity between the viral spike (s) protein on the host ace receptor. (a) sars-cov- uses the cell receptor, angiotensin-converting enzyme (ace ) for entry and the serine protease tmprss and furin for s protein priming. (b) as tmprss is broadly expressed and active with a furin-like cleavage activity, the affinity adaption of the s receptor binding domain (rbd) and ace receptor determines the viral permissiveness. the contacting residues of human ace (a distance cutoff . Å) at the sars-cov- rbd/ace interfaces are shown, and the contacting network involves at least residues in ace (listed in the table cells and referred to the aligned residual positions in human ace ) and residues in the sars-cov- rbd (blue circles with residue labels), which are listed and connected with black lines (indicating hydrogen bonds) and red line (represents salt-bridge interaction). the cross-species residual identity (%) of these interacting residues in ace are listed in a broad range ( - %) [ , , ] . (c) we also detected several short ace isoforms (underlined) in the domestic animals including dog, pig, goat and cattle, which have an n-terminal truncation spanning - key residues in the contacting network to s-rbd but keeping the enzyme active sites (indicated by yellow triangles), thus resulting in little engagement by the viral s protein and predicting an unexpected evolutionary advantage for relieving potential covid- risk caused by the viral engagement and functional distortion on the classical long ace isoforms in these animal species. the ncbi accession numbers of the ace orthologs are listed as in figure . human ace -rbd dog-ace l-rbd table . predicƟon of binding affinity energy (Δg), dissociaƟon constant (kd) and interfacial contacts of the sars-cov s-rbd with ace orthologs of major livestock species simulated using the human ace /cov -rbd structure ( m j). . most residues involved in binding are highlighted as magenta (ace ) or orange (s) sticks and labeled as one-letter amino-acid codes plus residual numbers in bold or regular font respectively for s or ace residues. the dotted/ blue lines indicate intermolecular salt bridge or hydrogen bonds between interacting residues (generated and visualized with ucsf chimera and pymol from protein data bank file m j). (b) to (d) rbd interaction with the simulated structures of ace long isoforms from the dog, pig and cattle, respectively. amino acid exchanges in ace from another species compared with human ace are highlighted in red. (e) prediction of binding affinity energy (Δg), dissociation constant (kd) and interfacial contacts of the sars-cov s-rbd with ace orthologs of major livestock species. most domestic animals ace including that from mouse and rat (species known not to be susceptible to sars-cov ) have a binding affinity (Δg) at - . to - . kcal/mol that is within the range ( . - . kcal/mol) between the rbd and the ace from the known susceptible species (underlined in the left part of the table), indicating that some other factors, especially those from genetic divergence and natural immunity, contribute to the sars-cov susceptibility of different animal species. figure . detection of several short ace isoforms (ace -s) in the domestic animals including dog, pig, goat and cattle. (a) in contrast to most splicing isoforms such as in cats and humans, which share a common proximal promoter and encode ace proteins with similar sequences containing all key rbd-interacting residues, these short ace -s isoforms in domestic animals truncate for (cattle/goat ace -s) or (dog/pig ace -s) residues at their n-termini compared with human ace or the long ace isoforms in these species, thus destroying - key residues in the contacting network to s-rbd but retaining all enzyme active sites (yellow triangles in the blue ace domain bar). this results in little chance to be engaged by the viral s protein binding and predicts an unexpected evolutionary advantage to relieve potential covid- risk caused by the viral engagement and functional distortion on the classical long ace isoforms in these animal species. (b), (c) and (d) paired structural comparison between the human ace structure ( m ) with each simulated ace -s structure from pig (b), dog (c) and cattle/goat (d). human ace structure are in green, and each compared animal ace -s structure in magenta. the n-terminal residues of both compared structures are in cyan (arrows indicating n-termini of the ace -s isoforms) and shared c-termini are in red. the key s-interacting residues in human ace are shown in blue sticks. in general, all ace -s orthologs, particular the porcine, show high structural similarity to the human ace except the n-terminal truncations. . results and discussions . . vertebrate ace orthologs share an functional constraint but experience intra-species diversification in livestock with unknown selective pressure sequence comparison among ace orthologs across representative vertebrate species shows a pairwise identity range at - % ( figure a and supplemental fig. s and excel sheet), which is - % higher than the average value generated through a similarity analysis at - % on gene orthologs at a genome-wide scale [ ] . this indicates that ace exerts a similar and basic function cross-species, consistent with its systemic and regulatory role as a key enzyme in ras, an essential regulatory axis underlying the body circulatory and execratory systems in vertebrates [ , , ] . a comparison of evolutionary rates of major genes within ras including angiotensinogen (agt), ace, and several receptors of the processed angiotensin hormones showed that ace actually evolves slightly faster than ace [ , and unpublished data] . this implies that ace may bear pressure for ras adapting evolution per a species-dependent physiological and pathological requirement [ , , ] . this evolutionary adaptability of ace genes is demonstrated by the existence of numerical genetic polymorphisms [ ] and several transcript isoforms particularly in humans and major livestock species ( figure b and supplemental fig. s and excel sheet). we identified (and verified by rna-seq data analysis) four transcripts of ace isoforms in humans ( figure b ) that primarily differ in the c-terminal residues within the collectrin domain. particularly, - short ace isoforms were identified in dogs, pigs, cattle, and goats in addition to the longer ace consensus to the human's (designated as -s or -l, respectively after the animal common names in figure b and thereafter). these livestock ace -s isoforms have a - residual truncation at their n-terminal peptidase domains, which also span the region interacting with sars-cov spike protein. the selective mechanisms driving the evolution of these short ace isoforms in livestock are unknown, but may relate to previous pathogenic exposure or unprecedented physio-pathological pressure. to support this reasoning, short ace isoforms are detected in both domestic bos taurus and hybrid cattle, but not in the wild buffalo and bison; and ace isoforms from each species are generally paralogous and sister each other within a clade in the phylogenic tree ( figure b ). phylogenic analysis of vertebrate ace orthologs/paralogs reveals a general relationship aligning to the animal cladistics ( figure b) . in this context, homologs from the fish, frog and chicken conform to a primitive clade. all ungulate homologs form into parallel clades next to each other. the homologs from the glires, primates and carnivores cluster into a big clade (marked with yellow triangle node), which contains all the sars-cov susceptible species that have been verified via natural exposure or experimental infections ( figure b , marked with red/orange circles). we examined and merged several previous studies about the prediction of sars-cov susceptibility in vertebrates based on the simulated structural analysis of s-rbd-ace complex [ , , ] . as numerous vertebrate species were predicted to be high or low potential ( figure b , labeled as red h or green l) for sars-cov susceptibility, incongruence between the predicted sars-cov susceptibility and infected validation is apparent in pangolin, ferret, tiger, cat and horseshoe bat, indicating that some other factors besides ace -rbd affinity should be considered [ , , , ] . we, therefore, refined the prediction matrix to include the rbd-binding evasion of some ace orthologs identified in major livestock species and the interferon-stimulated ace expression in priming sars-cov infections [ , , , ] . several recent studies have elegantly demonstrated the structural interaction of the viral s protein or its rbd in complex with human ace receptor [ , ] . showing that the contacting residues at the rbd/ace interface (figure a ) involve at least residues in ace ( figure b , listed in the table cells and referred to the aligned residual positions in human ace ) and residues in the sars-cov- rbd ( figure b , blue circles with residue labels above the table) [ , , ] . the cross-species residual identity (%) of these interacting residues in ace are dispersed in a broader range ( - %) than the whole ace sequence identity rate at - % [ ] , indicating a faster evolution rate of this virus-interacting region. notably, the s-binding region spans a large part of the n-terminal peptidase domain, thus s-binding may competitively block a majority of active sites to inhibit the physiological action of ace ( figure c ). using a similar structural analysis procedure [ , ] , we modeled the ace structures of animal species of interest and simulated their interaction with sars-cov s-rbd based on a published rbd-human ace structure (protein data bank file m j) [ ] . figure demonstrates the s-rbd interaction with the simulated structures of ace long isoforms from the dog, pig and cattle, respectively. the major changes of the rbd-ace interacting interfaces are from the residual exchanges in ace from other species compared with human ace ( figure b - d, highlighted in red). in addition, the exchange of n t (in pigs) and n y (in cattle and sheep) would destroy the n-glycosylation site in human ace . ace from goat (supplement fig. s ) exhibits identical amino acid exchanges as in cattle in the rbd-ace interfacial contacts. in contrast, when compared with human ace , ace from cats (supplement fig. s ) conserves all relevant glycosylation sites in human ace [ , ] . we also calculated the interfacial contacts using parameters of protein-protein interaction including the predictable binding affinity energy (Δg), dissociation constant (kd) and number of different interfacial contacts within the s-rbd and ace contact. although the exact numbers may differ from the previous reports [ ] , they provide a very comparable matrix generated using the same algorithm ( figure e ) [ ] . data show that the ace of most domestic animals, including that from mouse and rat (the species known to be unsusceptible to human sars-cov ) have a binding affinity (Δg) at - . to - . kcal/mol. this is within the binding affinity range ( . - . kcal/mol) between the rbd and the ace from known susceptible species ( figure e , underlined in the left part of the table). this indicates that other factors, conceivably from genetic divergence and/or natural immunity, also contribute to sars-cov susceptibility in animal species. therefore, an effective prediction matrix should include the critical immunogenetic factors to further determine virus susceptibility in addition to the sequence/structural similarity of ace receptors (figure and fig. s ) [ , , ] . we detected several short ace isoforms in the domestic animals including dog, pig, goat and cattle that have an n-terminal truncation spanning - key residues in the contacting network to s-rbd but retain the enzyme active sites ( figure a ). most of the splicing isoforms from ace genes such as in zebrafish, cats and humans, share a common proximal promoter and encode ace proteins containing all key rbdinteracting residues [ , ] . however, these short ace -s isoforms in domestic animals truncate for (cattle/goat ace -s) or (dog/pig ace -s) residues at their n-termini compared with the long ace isoforms in the same species (figure and fig. s ). therefore, these short ace isoforms destroy - key residues in the contacting network to s-rbd but likely retain ace enzymatic function in ras. paired structural comparison between the human ace structure (extracted from m ) with each simulated ace -s structure from the pig, dog, and cattle/goat, reveals that all these ace -s orthologs from domestic animals, particularly the porcine one, show high structural similarity to the human ace except for the n-terminal truncations ( figure b- d ). this indicates that these short ace isoforms in domestic animals have little chance to be engaged by the viral s-binding, and predict an unexpected evolutionary advantage to allay potential covid- risk resulting from viral engagement and functional distortion on the classical long ace isoforms in these animal species [ , ] . sars-cov infection induces a weak ifn response but a production of high amount of inflammatory cytokines including interleukin (il)- and chemokine cxcl in most severe covid- patients [ , , , ] . studies of sars and mers showed that these pathogenic coronaviruses share similar viral antagonisms including the endoribonuclease (endou) encoded by nonstructural protein (nsp ), which directly blunts cell receptors responding to viral dsrna and in turn weaken the acute antiviral response [ ] . several recent studies revealed that sars-cov seems more cunning in not only evading or antagonizing but also in exploiting the ifn response for efficient cell attachment [ , , , ] . as a key enzyme in ras, the expression of ace gene has been primarily investigated for physiological response to circulatory regulations, and a response to pathological inflammation is also expected [ , , ] . however, the expression of human ace gene was highly responsive to both viral infection and host ifn response, i.e. human ace gene seems an unstudied ifn-stimulated gene (isg) [ , ] . surprisingly, the isg propensity of ace genes is species-dependent, for examples, the mouse ace gene is less ifn responsive, which may partly explain the mouse insusceptibility to sars-cov infection [ ] . to categorize the different ifn-inductive propensity of ace genes in vertebrates, particularly in major livestock species, we profiled the regulatory cis-elements and relevant transcription factors in the proximal promoter regions of each ace genes ( . kb before tss or atg). figure illustrates major regulatory cis-elements located in ace genes from major livestock animals and several reference animal species. data show that animal ace gene promoters are evolutionally different in containing ifn-or virus-stimulated response elements (isre, prdi, ifrs, and/or stat / factors) and cis-elements responsive to proinflammatory mediators. all these cis-elements recruit corresponding transcription factors (tf) to mediate differential ace responses to antiviral ifns and inflammation that is associated with covid- disease [ , , ] . we discover that ace genes obtain species-different isg propensity responsive to ifn and inflammatory stimuli. in most (if not all) of the sars-cov susceptible species, the ace genes obtained the ifn-responsive elements between the typical robust and tunable ifn-stimulated genes (isg) [ ] . in general, the robust isgs (isg as an example here) are stimulated in the acute phase of viral infection and play a more antiviral role; in contrast, the later responsive tunable isgs (irf as an example) contribute more to anti-proliferation of ifn activity [ ] . in addition, unlike the promoter of the short ace isoforms in cattle and goats, which share most common promoter regions with their paralogous long isoforms, the short ace isoforms of dogs (dog-s) and pigs (pig-s) have distinct proximal promoter regions (and different ifn responsivity) to the paralogous long ace isoforms (figures and ) . results indicate that the short ace isoforms in pigs and dogs diversify from their long paralogs at both the levels of genetic coding and epigenetic regulation to adapt to some evolutionary pressure, such as that from pathogenic interaction (figure ) [ , ] . . . matrix scores of interferon-inductive elements in ace gene promoters correspond to sars-cov susceptibility the position weight matrix (pwm) stands as a position-specific scoring model for the binding specificity of a transcription factor (tf) on the dna sequences [ ] . using pwm toolsets online (https://ccg.epfl. ch/cgi-bin/pwmtools), we evaluate mean pwm of key cis-elements in the proximal promoters of ace genes that containing binding sites for canonical ifn-dependent transcription factors, which include isre/stat, irf . irf / and irf , as well as c/ebp representing a core transcription factor for pro-inflammation. these ifn-dependent transcription factors, particularly irf / and isre/stat for ifn stimulation, are differentially enriched in the promoter regions of ace genes in a species-dependent way. higher enrichment of isre/stat / and/or irf / binding sites are detected in most sars-cov /covid susceptible species (indicated with solid orange or red circles, respectively). in contrast, the pwm for irf and c/ebp, which regulate inflammation, are less differential in ace promoters from different animal species, indicating that ace genes are more universally regulated by inflammation than that by the viral infection or ifn-induction in a species-dependent way ( figure ). as compared with the promoters of a typical human robust isg and tunable irf genes, this data indicate that ace genes (particularly the primate ones) are not typical robust or tunable isgs as represented by isg or irf , but respond differently to viral infection (through irf / ) or ifn auto-induction (via isre/stat) in a species-dependent manner ( figure ) [ ] . higher enrichment of isre/stat / and/or irf / corresponds to sars-cov susceptibility in experimentally validated mammalian species especially primates, but not to the phylogenically distant species such as zebrafish, which has very low potential for sars-cov susceptibility due to the high disparity of ace structures (figure and fig. s ). in addition, the proximal promoters of the pig and dog ace -s genes differ much in their ifn-responsive elements to most ace promoters in mammalians (figures and ). however, they are phylogenically sister to the ace promoters from the primitive vertebrates (frog, chicken and zebrafish) (figure , phylogenic tree). this indicates that the expression of these short ace isoforms is more conservative than the long ace paralogs, which represent a more recent evolution obtaining ace epigenetic regulation by ifn-signaling ( figure ) [ ]. studies show that affinity adaption of the viral s-rbd and ace receptor determines the cellular permissiveness to the virus [ , , ] . sars-cov not only adapts a high binding affinity to human ace for cell attachment, but also antagonizes host antiviral interferon (ifn) response and utilizes ifn-stimulated property of human ace gene to boost spreading [ , , , ] . in addition to structural analysis of simulated s-rbd-ace interaction, we propose that several immunogenetic factors, including the evolution of s-binding-void ace isoforms in some domestic animals, the species-specific ifn system, and epigenetic regulation of ifn-stimulated property of host ace genes, contribute to the viral susceptibility and the development of covid- -like symptoms in certain animal species [ , , , ] . a computational program in development that incorporates this multifactorial prediction matrix and in vitro validation of sars-cov susceptibility in major vertebrate species will be necessary to address public concerns relevant to sars-cov infections in animals (figure ). it will also lead to the development of better animal models for anti-covid investigations [ ] . in addition, several ifn-based therapies for treatment of covid have been proposed and are in the process of clinic trials [ , , , ] . considering the viral stealth of ifn-stimulated property of human ace , a timely and subtype-optimized ifn treatment should be delivered than a general injection of typical human ifn-α/β subtypes [ , , , ] . in this line, domestic livestock like pigs and cattle have a most evolved ifn system containing numerous unconventional ifn subtypes. some of these unconventional ifn subtypes, such as some porcine ifn-ω exert much higher antiviral activity than ifn-α even in human cells and most ifn-λ retaining antiviral activity with less pro-inflammatory activity, could be utilized for developing effective antiviral therapies [ , ] . in summary, a predication matrix, which integrates the structural analysis of s-rbd-ace interfacial interface and the species-specific immunogenetic diversity of ace genes, was used to predict the sars-cov susceptibility and fit current knowledge about the infectious potential already validated in different animal species (figure ). more extensive validation experiments are needed to further improve this prediction matrix. our current results demonstrate several previously unstudied immunogenetic properties of animal ace genes and imply some domestic animals, including dogs, pigs and cattle/goats, may obtain some immunogenetic diversity to confront sars-cov infection and face a less figure . scores of mean position weight matrix (pwm) of key cis-elements in the proximal promoters of ace genes that containing binding sites for canonical ifndependent transcription factors, which include isre/stat / , irf , irf / and irf , as well as c/ebp as a core transcription factors for pro-inflammatory response. these ifn-dependent transcription factors, particularly irf / and isre/stat critical for ifn stimulation, are differentially enriched in the promoter regions of ace genes in a species-dependent way. especially, increased enrichment of isre/stat / and irf / binding sites are detected in the sars-cov /covid susceptible species (indicated with solid orange or red circles, respectively). in contrast, the pwm for irf and c/ebp, which regulate inflammation, are less differentially enriched in ace promoters from different animal species. the promoters of a typical human robust interferon-stimulated gene (isg) and irf (a typical tunable isg) are used as references. higher enrichment of isre/stat / and irf / corresponds to sars-cov susceptibility in experimentally validated animal species and humans. abbreviations: c/ebp, ccaat/enhancer binding protein; irf, interferon-regulatory factor; isre, interferon-sensitive response element; stat, signal transducer and activator of transcription; pwm, position weight matrix. the pwm tools are used through https://ccg.epfl.ch/cgi-bin/pwmtools. covid risk than previously thought. however, immediate biosecurity practices should be applied in animal management to reduce animal exposure to the virus and prevent potential species-specific adaptation ( figure ) . for livestock breeding programs that targeting disease resistance to respiratory viruses, the genetic and epigenetic diversity of ace genes as well antiviral isgs are highly recommended [ , , , ] . in conclusion, sars-cov evolves to fit well with human (and nonhuman primates) ace receptor through the structural interfacial affinity, immunogenetic diversity and epigenetic expression regulation, which results in a highly infectious efficacy [ , , , , , , ]. most mammalian animals, especially those that belong to glires, primates and carnivores, have a higher potential for sars-cov susceptibility but in a species-different manner based on the existence of s-binding-void ace isoforms and the difference of the ifn-inductive propensity of the major ace genes. most ungulate animals appear have a low susceptibility potential with horses and sheep having a high potential (figure ) . current development of ifn-based anti-covid therapies should consider the isg property of human ace gene to optimize for timely application using a highly-antiviral subtype that potentially have less inflammatory (or even anti-inflammatory) activity [ evolution of the ifn complex and functional diversity in domestic animals (such as pigs and cattle) provides a natural model for optimizing ifn antiviral regulation and therapy development [ , ] . eric r. sang, yongming sang: conceived and designed the experiments; performed the experiments; analyzed and interpreted the data; wrote the paper. yun tian: performed the experiments; analyzed and interpreted the data; wrote the paper. yuanying gong: contributed reagents, materials, analysis tools or data; wrote the paper. laura c. miller: conceived and designed the experiments; contributed reagents, materials, analysis tools or data; wrote the paper. covid- dashboard by the center for systems science and engineering (csse) at johns hopkins university (jhu) a familial cluster of pneumonia associated with 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covid- and emerging viral infections: the case for interferon lambda heliyon xxx (xxxx) xxx the authors declare no conflict of interest. supplementary content related to this article has been published online at https://doi.org/ . /j.heliyon. .e . key: cord- -oldy gta authors: barriocanal, marina; carnero, elena; segura, victor; fortes, puri title: long non-coding rna bst /bispr is induced by ifn and regulates the expression of the antiviral factor tetherin date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: oldy gta many long non-coding rnas (lncrnas) are expressed in cells but only a few have been well characterized. in these cases, lncrnas have been shown to be key regulators of several cellular processes. therefore, there is a great need to understand the function of more lncrnas and their regulation in response to stimuli. interferon (ifn) is a key molecule in the cellular antiviral response. ifn binding to its receptor activates transcription of several ifn-stimulated genes (isgs) that function as potent antivirals. in addition, several isgs are positive or negative regulators of the ifn pathway. this is essential to ensure a strong antiviral response and a later return of the cell to homeostasis. as the isgs described to date are coding genes, we sought to determine whether ifn also regulates the expression of long non-coding isgs. to this aim, we used rna sequencing to analyze the transcriptome of control and huh cells treated with ifnα . the results show that ifn-treatment regulates the expression of several unknown non-coding transcripts. we have validated two lncrnas upregulated after treatment with different doses of type i ifnα in different cells or with type iii ifnλ. these lncrnas were also induced by influenza and vesicular stomatitis virus mutants unable to block the ifn response, but not by several wild-type lytic viruses tested. these lncrna genes were named lncisg and lncbst as they are located close to isgs isg and bst , respectively. interestingly, inhibition experiments showed that lncbst is a positive regulator of bst . therefore lncbst has been renamed bispr, from bst ifn-stimulated positive regulator. our results may have therapeutic implications as lncbst /bispr, but also lncisg and their coding neighbors, are increased in cells infected with hepatitis c virus and in the liver of infected patients. these results allow us to hypothesize that several lncrnas could be activated by ifn to control the potency of the antiviral ifn response. the interferon (ifn)-mediated innate immune response provides a potent defense against pathogens ( ) . upon invasion, pathogenassociated molecular patterns (pamps) are detected by specific receptors in the cells. these can be located on the surface of the cell, as in the case of toll-like receptors (tlrs), or intracellularly, as in the case of the retinoic acid-inducible gene i (rig-i). pamp recognition triggers a series of signaling cascades that lead to the production and secretion of type i ifn. type i ifn (ifnα, ifnβ, and others) binds to ifn receptors present on the surface of all cell types and activates janus-activated kinase/signal transducer and activator of transcription (jak/stat) signaling. this gives rise to the nuclear translocation of the stat /stat /irf (ifn regulatory factor ) complex that binds ifn-stimulated response elements (isre) in the promoters of ifn-stimulated genes (isgs) and activates their transcription. a similar response is induced by type iii ifn (ifnλ) upon binding to its receptor ( , ) . in contrast, type ii ifn or ifnγ, produced by cells of the immune system, binds to the widely expressed ifnγ receptor ( , ) leading to nuclear translocation of stat homodimers, which bind to gamma-activated sequences (gas) in the promoter of immunoregulatory genes. ifn-stimulated genes are antiviral factors, positive regulators of the ifn pathway (stat and and irf ) or negative regulators that help ifn-induced cells to return to cellular homeostasis (socs and ups ) ( ) ( ) ( ) ( ) ( ) ( ) . among antiviral genes, there are factors that function to increase cell sensitivity to pamps (oas and pkr) or true antiviral effectors that block viral entry (mx, ifitm, and trim), virus replication, translation and stability (ifit, oas, pkr, and isg ), or viral release (viperin and tetherin/bst ) ( ) . while most ifn-induced factors known to date are proteins, ifn also activates the expression of several micrornas that contribute to the antiviral state or to the control of ifn response ( ) . few studies have been performed to address whether ifn could also regulate expression of long non-coding rnas (lncr-nas) ( ) ( ) ( ) . in recent years, viral infection has been reported to be able to induce the expression of cellular lncrnas. this has been shown for infection with enterovirus, influenza, hiv, hepatitis b and c viruses, and the sars coronavirus ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (carnero et al., in preparation) . the lncrna signature found after infection www.frontiersin.org should be a mixture of transcripts induced by the virus and transcripts that respond to the cellular antiviral pathways activated by the infection. in fact, activation of tlrs by pamps induces the expression of several lncrnas. tlr signaling leads to the activation of lncrna-cox , which regulates the expression of genes related to the immune system ( ) . activation of tlr results in increased neat , which increases the expression of genes such as il ( ) . tlr controls il b-erna and il b-rbt lncr-nas whose downregulation diminishes il b and accumulation of lps-induced rnas ( ) . likewise, the lps-induced inflammatory response is controlled by lnc-il r ( ) . innate activation also induces linc /thril, which controls tnfα and other genes involved in the immune response ( ) . in turn, tnfα induces lethe, a pseudogene that responds to nfκb and reduces inflammation by inhibiting nfκb dna binding activity ( ) . lncrna responses are also critical for the functionality of dendritic cells, cd + and cd + t-cells ( ) ( ) ( ) ( ) . thus, nest lncrna controls ifnγ locus in cd + t-cells leading to decreased salmonella enterica pathogenesis ( , ) . these studies illustrate the interest in identifying novel lncr-nas and elucidating their function and regulation. lncrnas are thought to be at least as numerous as protein-coding genes, but only a few are well characterized ( ) ( ) ( ) ( ) . lncrnas are transcripts similar to mrnas but with poor coding potential. they are more cell type-specific, less expressed, and less well conserved than mrnas ( , ) . interestingly, lncrnas are cell regulators that can function in cis, co-transcriptionally, or in trans. some control the expression of coding genes located in the same genomic region. therefore, the genomic location of lncrnas can provide hints as to their functionality. they can be sense or antisense (when overlapping with one or more exons of another transcript in the same or in the opposite strand, respectively); intronic (when derived from an intron of another transcript); divergent or bidirectional (when they share a promoter with another transcript in the opposite strand and therefore are co-regulated); or intergenic (when they are independent, located in between two other genes). several mechanisms are involved in the regulation of neighboring or antisense genes by lncrnas. these include transcriptional activation or interference, recruitment of chromatin modifiers and remodelers, regulation of imprinting, editing, splicing or translation, and stability ( ) ( ) ( ) ( ) ( ) . to address the issue of whether ifn could also regulate expression of lncrnas, which may play key roles in the antiviral response, we analyzed the transcriptome of cells treated or not with ifnα by rna sequencing (rnaseq). in this analysis, we identified two lncrnas upregulated in response to ifn in different cell lines. interestingly, these lncrnas are expressed from positions in the genome divergent from the well-characterized isgs isg and bst . therefore, we have called them lncisg and lncbst . these lncrnas and their coding counterparts are also induced in cells infected with mutants of influenza or vesicular stomatitis viruses (vsv) that fail to block the ifn response. surprisingly, they are also induced in culture cells infected with hepatitis c virus (hcv) and in the liver of patients with hcv infections. finally, according to hugo regulation, we have renamed lncbst bispr, from bst ifn-stimulated positive regulator, as we show that inhibition of lncbst expression by rnai leads to decreased levels of bst mrna, providing a new layer of regulation of the ifn response. the huh cell line, derived from a human hepatocarcinoma, was provided by dr. chisari's lab (scripps research institute, la jolla, ca, usa). a cells, from human non-small cell lung carcinoma, were kindly provided by estanislao nistal (cima, university of navarra, spain). human liver samples with or without hcv infection were obtained from the biobank of the university of navarra under approval from the ethics and scientific committees. liver tissue sections were snap frozen and stored at − °c. the clinical data from hcv-infected subjects are shown in table s in supplementary material. cells were grown in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) and % penicillin-streptavidin and maintained at °c in a % co atmosphere. twenty-four hours before treatment with ifn, huh and a cells were seeded in six-well plates. then, , , , , , or units/ml of ifnα (sicor biotech, lithuania) were used in a final volume of ml. huh cells were also treated with ng/ml of il b/ifn-λ (r&d systems) in a final volume of ml. for treatment with ruxolitinib (selleckchem), cells were seeded out h before and treated with . µm ruxolitinib in a final volume of ml. one hour after treatment media were discarded and replaced by media containing units/ml ifnα. cells were harvested for rna extraction at the indicated times post-treatment. sirnas targeting lncbst /bispr were designed using iscore designer and rna scales ( , ) and purchased from dharmacon. the lncbst /bispr sirnas targeted the sequence gacuagugugagcaacaaa. for cell transfection with sir-nas, lipofectamine reagent (invitrogen) was used according to manufacturer's instructions. cells were seeded h before transfection. for each well of a six-well plate, pmoles sirna were used. the sirna was mixed with µl optimem. furthermore, µl lipofectamine were mixed with µl optimem media and incubated for min. then, lipofectamine and sirna solutions were mixed and incubated for - min at room temperature. after incubation, half of the volume of the cell media was discarded and , , or µl of the lipofectamine mixture were added to each well of , , or -well plates, respectively. six hours post-transfection the media from the cells was discarded and substituted with dmem media enriched with % fbs and antibiotics. hepatitis c virus jfh- was obtained from an initial viral stock from the genotype a jfh- plasmid (pjfh- ) previously described by wakita et al. ( ) . the virus was amplified as described ( ) . influenza virus strain a/pr / wt (pr ), a mutant lacking ns (∆ns ), vsv-gfp, and the mutant m r frontiers in immunology | molecular innate immunity were kindly provided by estanislao nistal (cima, university of navarra, spain) ( ) ( ) ( ) , semliki forest virus (sfv) was a gift from cristian smerdou (cima, university of navarra, spain), and adenovirus serotype (ad ) was amplified as previously described ( ) . vsv-egfp titration was performed in quadruplicates on a cells. the supernatant from infected cells was collected and : serial dilutions were performed. cells were seeded h before infection in -well plates and infected with µl of each dilution. twenty-four hours after infection, gfp expression was visualized by microscopy and used to determine the titer. cells were infected with hcv at a multiplicity of infection (moi) of . , with vsv at a moi of and with a moi of of influenza a, ∆ns , ad , and sfv. in the case of the lytic viruses, we used a moi of or as this causes cytopathic effects at h (for vsv, influenza and sfv) or h (for ad) in huh or a cells. after infection, the virus was removed and fresh medium was added to the cells. cells were harvested for rna extraction at the indicated times post-infection. two million huh cells were incubated in µl of cytoplasmic buffer ( mm tris hcl ph . , mm edta, and % np ) for min at °c. then, cells were centrifuged for min at g and the supernatant was used to isolate cytoplasmic rna. the pellet was washed with cytoplasmic buffer and centrifuged as before. the supernatant was discarded and the pellet was used to isolate the nuclear rna. rna from nuclear and cytoplasmic fractions was isolated with maxwell research system (promega). human tissue was homogenized using the ultra-turrax dispersing machine (t basic ika-werke) ( ) . total rna from the tissue was extracted in ml trizol (sigma-aldrich) and recommendations of the supplier were followed ( ) . dnase (fermentas) treatment was performed to eliminate dna from the samples before rt-pcr reactions. rna was extracted from cells with the maxwell research system from promega following the manufacturer's recommendations. rna concentration was measured using nanodrop spectrophotometer. the quality of the rna was analyzed by bioanalyzer (agilent technologies). reverse transcription (rt) was performed as described ( ) . the reaction was performed in the c touch thermal cycler from bio-rad. the samples were incubated at °c for min, then at °c for s and then immediately cooled to °c. qpcr was performed in the cfx real-time system from bio-rad as described ( ) . the results were analyzed with bio-rad cfxmanager software. gapdh levels were evaluated in all the cases as a reference. only the samples with similar gapdh amplification were analyzed further. the primers used are listed in table s in supplementary material and were designed with the primer program . rna of excellent quality, as determined by bioanalyzer (agilent technologies) was treated with the ribo-zero rrna removal kit http://frodo.wi.mit.edu (epicenter) to deplete from ribosomal rna. library preparation with truseq rna sample preparation kit (illumina) and sequencing was performed at the embl genomics core facility (genecore) in an illumina hiseq . sequences were paired-end, bases long, and strand specific. rnaseq data are available at the ncbi gene expression omnibus (geo) data repository . rna sequencing data analysis was performed using the following workflow: ( ) the quality of the samples was verified using fastqc software; ( ) the preprocessing of reads was performed by elimination of contaminant adapter substrings with scythe and by quality-based trimming using sickle; ( ) the alignment of reads to the human genome (hg ) was performed using the tophat mapper ( ); ( ) transcript assembly and quantification using fpkm of genes and transcripts was carried out with cufflinks ( ); ( ) the annotation of the gene locus obtained was performed using cuffmerge with gencode v as reference; and ( ) differential expression analysis was performed using cuffdiff ( ) . genes were selected as differentially expressed using a p-value threshold of . . further analysis and graphical representations were performed using an r/bioconductor ( ) . reads from all the differentially expressed sequences were visualized in the integrative genomics viewer (igv) ( , ) and the sequences were compared to the ensembl and encode databases and searched for in the genome browser from ucsc for more information ( , ) . candidates were divided into coding, non-coding (according to ucsc classification), or non-assigned, when the transcription of the sequence had not been annotated in the databases. functional enrichment analysis of gene ontology (go) categories was carried out using a standard hypergeometric test ( ) . biological knowledge extraction was complemented through the use of ingenuity pathway analysis (ingenuity systems) , with a database that includes manually curated and fully traceable data derived from literature sources. open reading frame finder (ncbi) was used to evaluate the length of all probable open reading frames (orfs) in lncisg and lncbst /bispr. coding potential was assayed with the coding potential assessment tool (cpat) ( , ) and by searching the lncipedia database ( ) for the presence of our candidates in the pride archive ( ) or in lists of transcripts associated with ribosomes ( , ) . phylogenetic codon substitution frequencies (phylocsf) were also used to predict the coding potential of lncisg and lncbst /bispr ( ). statistical analysis of the rnaseq data has been already described. remaining analysis was performed using graph-path. statistical significance of infected versus non-infected samples was calculated using a two-tailed non-parametric mann-whitney t -test or with a two-tailed students t -test when the samples followed a normal distribution according to the shapiro-wilk test. welch's correction was applied for samples with heterogeneous variance. for correlation studies, a two-tailed non-parametric spearman analysis was used. p values lower than . were deemed as significant. to identify lncrnas that respond to ifn, we treated huh cells with units/ml of ifnα for days. these conditions serve to induce the expression of well-known isgs such as gbp , irf , bst , oas, or isg ( ) . in addition, this treatment induces an antiviral effect, as hcv-infected huh cells treated with units/ml of ifnα , show decreased levels of viral proteins and viral genomes compared to untreated infected cells (data not shown). finally, the rna isolated from huh cells treated with units/ml of ifnα for days was used to hybridize an agilent array. analysis of the array showed that well-characterized isgs such as mx , stat , irf , isg , bst , and several members of the gbp, oas, and ifi families were upregulated with a very high statistical significance (b > ) ( ) . ingenuity analysis of the data showed that ifn signaling was the pathway with the highest enrichment followed by other antiviral responses. the microarrays were used to identify lncrnas regulated by ifnα ( ) . however, an array will only evaluate the expression levels of the transcripts that hybridize to probes spotted in the array. in the case of the lncrnas, the array used only addresses the expression of regions described as long intergenic noncoding rnas (lincrnas). however, it has been estimated that there could be as many lncrna genes as coding genes, and some authors consider that the number of lncrnas could be as high as ( , ) . therefore, to achieve a more complete identification of lncrnas that respond to ifn, we analyzed the transcriptome by rnaseq. rna isolated from control cells or huh cells treated with ifn as described, was sequenced after ribodepletion. around million reads were obtained per sample. analysis was performed using a bioinformatic workflow that includes tophat and cufflinks as described in the methods section. the analysis showed that, among the genes upregulated in response to ifn, there were several isgs such as mx , isg , bst , or members of the ifi and oas families ( figure a and table s in supplementary material). ingenuity analysis showed that ifn signaling is a top canonical pathway (p = . × − ), the top upstream regulator is ifnα (p = . × − ), and cell signaling and infectious and inflammatory diseases are among the main functions. the expression of~ coding genes was altered by ifn (table s in supplementary material). the rnaseq analysis also showed that the expression levels of many regions that do not correspond to coding genes were also significantly modified in response to ifn ( figure b) . out of the putative non-coding genes whose expression was significantly altered, half were upregulated (table s in supplementary material). all candidates where visualized using igv ( figure s in supplementary material) ( , ) . we also paid special attention to altered sequences located close to well-known isgs and to genomic regions that were highly expressed and deregulated in response to ifn. eight candidates that fulfill at least one of these two criteria were chosen for further validation ( table and figure s in supplementary material). to validate the eight candidates chosen, we treated huh cells with different doses of ifnα . rna was isolated from the cells at , , , or h post-treatment and the expression levels of the candidates were evaluated by quantitative rt-pcr (qrt-pcr) (table ; figure ). all the candidates were induced after ifn-treatment from to more than -fold. however, many of the candidates were detected at very low cycles in the pcr amplification. a closer examination of their sequences indicated that they contained repetitive sequences or sequences similar to mitochondrial or ribosomal rnas that could have led to an erroneous alignment of the rnaseq reads to the human genome. we believe that, even when the oligonucleotides used for amplification were specific, a partial homology to other sequences could allow cross-amplification and thus increased possibilities of misleading results. these candidates were not studied further. we focused on two lncrnas with no repetitive sequences whose expression was highly upregulated in response to ifn (table ; figure ). interestingly, database analysis showed that they are expressed from positions in the genome located close to isg and bst , both of which are well-characterized isgs. this may have functional relevance as some lncrnas have been described to regulate the expression of neighboring genes. therefore, we originally named these lncrnas after their neighbor, lncisg and lncbst . later, lncbst was renamed bispr to follow hugo regulations. when we evaluated the expression of these lncrnas and their neighboring transcripts, we observed that both were strongly upregulated at early times in response to ifn (figure a) . furthermore, they responded to ifnα doses as low as units/ml. these are similar levels to those found in the sera of some hcv patients ( ) . the induction was also observed at late times post-ifn-treatment. to evaluate further the robustness of the effect www.frontiersin.org of ifn on these lncrnas, we tested whether they also respond to ifnλ, a type iii ifn. huh cells treated with ifnλ for , , , , or h also showed increased levels of lncisg , lncbst /bispr, and their neighbors ( figure b) . in this case, all the transcripts showed a higher upregulation at later times post-ifnλ treatment. viruses activate the ifn response by several mechanisms. therefore, they have evolved to block ifn production and the activation of the ifn pathway. the molecular mechanisms involved in this ifn blockade have been characterized for many viruses. thus, for instance, ns protein from influenza virus and matrix protein from vsv are key factors in controlling ifn in infected cells ( ) ( ) ( ) ) . we sought to check whether lncisg and lncbst /bispr were induced by the physiological ifn induced by an influenza virus that lacks ns . therefore, we evaluated the expression of these lncrnas in cells infected with an influenza wild-type virus or a ns mutant. we also included cells infected with other rna viruses such as sfv and hcv or dna viruses such as adenovirus. all these viruses have developed mechanisms to block the cellular antiviral response and, with the exception of hcv, lead to a lytic infection. different times post-infection were evaluated. the last point was collected when the cytopathic effect was apparent. this occurred at h post-infection in the case of influenza and sfv or h post-infection, in the case of adenovirus. hcv-infected cells were collected at and h postinfection. the results showed that at later times post-infection with the influenza virus lacking ns , there was increased expression of lncisg , lncbst /bispr, and their neighboring coding transcripts (figure a) . this increase was not observed in cells infected with wild-type influenza virus, or with other wildtype lytic viruses, suggesting that the induction may be mediated by ifn. most lncrnas are tissue-specific. to determine whether lncisg and lncbst /bispr respond to infection only in huh cells or whether this effect is specific for influenza viruses, we infected alveolar epithelial a cells with vsv-gfp wild-type virus or with a m r matrix mutant that fails to control ifn. we chose a , because lung cells serve as the primary site for productive infection of vsv and many respiratory viruses ( ) . infection with the wild-type virus did not increase the expression of lncbst /bispr or bst (data not shown). however, a cells infected with the vsv mutant m r for , , or h did show increased levels of lncisg , lncbst /bispr, isg , bst , and other isgs such as gbp ( figure b) . surprisingly, infection with hcv also increased the expression of lncisg , lncbst /bispr, and other isgs, including isg , bst , and irf ( figure a and data not shown). to determine whether these genes were also upregulated in infected patients, we used liver samples from hcv-negative and hcv-positive donors. after quantification of the rna levels, we observed a significant increase in lncisg , lncbst /bispr, isg , and bst in hcvinfected patients compared to controls (figure a ). with the number of patients evaluated, a significant correlation was not found between expression levels and infection with a particular genotype of hcv, presence of hcv-induced hepatocellular carcinoma (hcc), liver cirrhosis, or with a particular cirrhosis stage. therefore, there were no significantly different levels of these transcripts in hcv-infected livers without hcc compared with the peritumoral tissue of hcv-infected livers with hcc. although most of the samples belong to patients that are still alive, no significant correlation was observed between the levels of the evaluated transcripts and survival post-diagnosis. finally, we performed correlation studies to analyze whether in the patients, the expression level of lncisg or lncbst /bispr correlates significantly with the expression level of their neighboring coding genes. the results show a highly significant positive correlation between lncisg and isg or lncbst /bispr and bst (figure b ). the experiments performed so far suggest that a general correlation could exist between the expression of lncisg and isg or lncbst /bispr and bst . each lncrna and its neighboring coding gene have similar induction patterns in response to ifn or to viral infection (compare their levels in figures and ) . furthermore, the levels of each coding/non-coding pair correlate significantly in patient samples (figure b) . to analyze this in more detail, we performed correlation studies of the coding/noncoding pairs in all the samples evaluated in figures and . the results show that the correlation of each pair was highly significant ( figure s in supplementary material). this suggests that they could be co-regulated, and therefore, they could share similar functions. however, expression of lncisg and lncbst /bispr also correlated significantly with the expression of other isgs such as oas, gbp , or irf (data not shown). to obtain more information on the relationship between the coding/non-coding pairs, we searched several databases. lncisg and lncbst /bispr genes are in head-to-head orientation with their coding neighbors ( figure s in supplementary material) and they could share the same promoter. this is based on the following facts: (i) the distance between the two genes is < bp, a cut-off for bidirectional promoters ( , ) ; (ii) there is a single dnase hypersensitivity region located between the genes, and (iii) polymerase ii (pol ii) chipseq analysis of k cells shows a single peak covering the h k ac region between both genes. interestingly, the peaks observed for pol ii chipseq are increased at min or h post-treatment with ifnα or ifnγ. finally, the promoter regions contain conserved isre sites and binding sequences for irf , irf , and irf . to discriminate whether lncisg and lncbst /bispr are induced directly by the jak/stat signaling pathway or by a secondary wave of the ifn response, we evaluated the expression of these lncrnas and their coding neighboring genes in huh or a cells incubated or not with the jak/stat inhibitor ruxolitinib. expression of gbp , a bona fide isg, was also evaluated as a positive control (figure ) . the results show that the levels of gbp , bst , and lncbst /bispr are significantly reduced liver samples from hcv-negative and hcv-positive donors were used to quantify the levels of lncisg , lncbst /bispr, isg , bst , and gapdh mrnas. statistical significance was calculated using a two-tailed non-parametric mann-whitney t -test for lncbst /bispr, isg , and lncisg and with a two-tailed students t -test with welch's correction for bst , which follows a normal distribution according to the shapiro-wilk test. (b) expression levels observed for lncisg , lncbst /bispr in patient samples were compared to the expression levels of their coding neighbors isg and bst , respectively. a correlation analysis was performed and statistical significance was calculated using a two-tailed non-parametric spearman analysis. www.frontiersin.org in the presence of ruxolitinib, indicating that their expression is stat-dependent. levels of bst and lncbst /bispr were also reduced in cells treated with sirnas targeting stat or by inhibition of irf , a transcription factor that acts downstream of ifn (data not shown). this indicates that bst and lncbst /bispr respond to stats but also to other transcription factors induced by ifn. these results agree with the possibility that bst and lncbst /bispr share a bidirectional promoter. in contrast, the effect of the jak/stat pathway on isg and lncisg expression was less robust. treatment with ruxolitinib decreased the expression of isg and lncisg , but in the latter, this effect was only observed in a cells. no effect on isg or lncisg expression was observed with a milder inhibition of stat or inhibition of irf using rna interference (data not shown). thus, although isg is induced very rapidly after ifntreatment, we do not observe a strong regulation of isg or lncisg by the stat pathway under the conditions used. in fact, it has been reported that a major regulator of isg is irf , a transcription factor activated in response to pamps, but also a downstream effector of the ifn response ( ) . we evaluated the coding capacity of lncisg and lncbst /bispr bioinformatically. orf finder (ncbi) was used to determine all possible orfs in these lncrnas ( figure s in supplementary material). the analysis shows that all putative orfs are shorter than amino acids. only two orfs could be translated according to their poor susceptibility to nonsense mediated decay. however, these orfs have non-consensus kozak sequences at the initiation codon and therefore a poor coding capacity. then, we evaluated the coding potential of lncisg and lncbst /bispr with the cpat ( , ) ( figure a) . cpat uses a model built with orf size and coverage together with codon (ficket score) and hexamer (hexamer score) usage bias. according to this program, lncisg and lncbst /bispr are non-coding as they have a coding probability of . and . , respectively, much lower than . , used as a threshold with the highest sensitivity and specificity to differentiate between coding and non-coding transcripts in humans figure | lncisg , lncbst /bispr have poor coding potential and accumulate preferentially in the nucleus. (a) bioinformatic analysis of the coding potential of lncisg and lncbst /bispr. results obtained from cpat and lncipedia. two transcripts have been evaluated for lncbst /bispr. lncisg and lncbst /bispr have a coding probability and a coding label of "non-coding rnas" according to these analyses. "lists" indicated the number of times that these transcripts have been found in the pride archive or in lists containing ribosome-associated rnas published by lee or bazzini. see the text for other details. (b) subcellular localization of lncisg and lncbst /bispr. huh cells were mock-treated or treated with units/ml of ifnα and divided into nuclear and cytoplasmic fractions. rna was isolated from each fraction and used to evaluate the expression levels of lncisg and lncbst /bispr by qrt-pcr. malat , gapdh, and isg mrna was also quantified and used as a reference to calculate the relative levels of each transcript and as a control to evaluate the subcellular fractionation. the ratio of cytoplasmic to nuclear levels is shown. the experiment was performed three times and each value shows the average of three replicas from a representative experiment. error bars indicate standard deviations. ( ) . lncisg and lncbst /bispr were also described as noncoding in lncipedia ( ) . this lncrna database shows that these lncrnas are not found in the pride archive, a database for proteomic data, or in lists of transcripts associated with ribosomes in ribosome profiling experiments ( ) ( ) ( ) . further, lncisg and lncbst /bispr were also described as non-coding by the analysis of phylocsf, which uses multiple alignments to calculate the frontiers in immunology | molecular innate immunity phylogenetic conservation score and determines whether a multispecies nucleotide sequence alignment is likely to represent a protein-coding region ( ) . finally, we evaluated the subcellular localization of lncisg and lncbst /bispr in huh cells mock-treated or treated with units/ml of ifnα. rna was isolated from nuclear or cytoplasmic fractions and quantified by qrt-pcr. we found that the coding gapdh or isg mrnas accumulate preferentially in the cytoplasm while the nuclear rnas malat or u are preferentially nuclear (figure b data not shown) . similarly, lncisg and lncbst /bispr, compared to mrnas, accumulate preferentially in the nucleus. this result, together with the bioinformatic analyses, strongly suggests that lncisg and lncbst /bispr are non-coding rnas. to address the role of lncbst /bispr, we used rna interference. huh cells treated or not with ifn, were transfected with sir-nas targeting lncbst /bispr and rna expression was evaluated by qrt-pcr. the results show that expression of lncbst /bispr was decreased compared to cells transfected with control sirnas ( figure a) . surprisingly, inhibition of lncbst /bispr also led to decreased levels of bst mrna. expression of lncisg , isg , gbp or expression of genes located in the genome close to bst or lncbst /bispr, such as gtpbp or mvb a, was not affected (figure a and data not shown). to determine whether this was a general phenomenon, we transfected the sirnas targeting lncbst /bispr into a cells infected or not with the vsv m r mutant or treated with ifn. similarly to what has been observed in huh cells, the sirna that targets lncbst /bispr leads to decreased levels of lncbst /bispr and bst mrna while the levels of isg mrna are not significantly affected (figure b) . similar results were observed with a different sirna targeting lncbst /bispr. rna sequencing analysis of human cells treated with ifnα and controls has allowed the identification of lncrnas induced in response to ifn (figure ) . analysis of the rnaseq data shows that several of the upregulated genes are well-known coding isgs such as isg or oas ( figure a , table s in supplementary material). ingenuity analysis confirms the enrichment of genes involved in the ifn response among the regulated factors. we have used rnas from similar ifn-treated and control cells to hybridize expression arrays ( ) . comparison of the datasets obtained in the analysis of the array and the rnaseq shows that only coding genes were identified in both studies, including oas, isg , mx , and some members of the ifi family. generally, overlap between microarray and rnaseq analysis is not high ( ) . furthermore, the overlapping decreases with sequencing depth and when low fold-changes or low abundance genes are analyzed. ( ) . this is because sequencing of low transcript abundances is characterized by high variance, which impedes their identification in rnaseq analysis. we believe that this may explain the poor correlation found between the array and the rnaseq datasets. in fact, we have determined by qrt-pcr that some ifnrelated low abundance transcripts are detected only in the array analysis. these are early responders to ifn, which increased only marginally days after ifn-treatment, when the analysis was performed. therefore, we believe that some lncrnas induced early post-ifn-treatment may have not been identified in our analysis. interestingly, in the process of writing this manuscript, a paper www.frontiersin.org was accepted describing the identification of ifn-induced lncr-nas by rnaseq in samples treated with ifn for short times ( ) . we believe that this study will be complementary to our work. together, the datasets should contain lncrnas regulated at early and later times post-ifn-treatment. similarly, the lack of correlation between the microarray and rnaseq datasets also indicates that they can complement each other. we have identified two lncrnas whose expression is highly upregulated in response to different doses of ifnα (table ; figure a ) or ifnλ (figure b) . our results show that induction of these lncrnas by ifnα seems faster than that observed for ifnλ. we cannot rule out the possibility that a fast response to ifnλ may also be observed when higher doses are used. encode analysis of polymerase ii binding to the promoters of these lncr-nas also shows that they may be induced by treatment with ifnα and ifnγ ( figure s in supplementary material). these lncrnas have been named lncisg and lncbst /bispr after their neighboring genes, which play a key role in the antiviral ifn response. our molecular and bioinformatic analyses strongly suggest that lncisg and lncbst /bispr are indeed lncrnas, as they accumulate preferentially in the nucleus of ifn-treated or untreated cells ( figure b ) and they have poor coding potential ( figure a and figure s in supplementary material). in general, the upregulation of lncisg and lncbst /bispr mimics that of their coding counterparts (figures - ) . in fact, analysis performed with all the expression data obtained in our studies, shows a highly significant correlation between lncisg and isg and between lncbst /bispr and bst . significant correlations also exist between these lncrnas and other ifn-induced genes such as oas, gbp , or irf ( figure s in supplementary material and data not shown). this may reflect the fact that all these genes are induced by ifn with a similar kinetics. in the case of the lncrnas and their coding counterparts, correlation of the expression may result from their transcriptional co-regulation. experimental and bioinformatic analyses indicate that bst and lncbst /bispr are bona fide isgs strongly induced by the jak/stat pathway in response to ifn (figure and figure s in supplementary material). furthermore, expression of bst , isg , and their neighboring non-coding genes is induced by downstream effectors of the ifn response. these studies allow us to suggest that lncisg /isg and lncbst /bispr/bst may share bidirectional promoters. other ifn-induced gene pairs may also be co-regulated by bidirectional promoters as these are enriched in stat binding ( ) . bidirectional promoters often couple genes involved in the same process, allowing for coordinated temporal and environmental responses ( , ( ) ( ) ( ) ( ) ( ) . non-coding rnas generated from bidirectional promoters may have functional roles that affect the bidirectional promoter, the neighboring protein-coding gene, or more distal genes ( ) . these effects could lead to activation or repression of the expression and could be mediated by either the transcription process itself or by the produced ncrna transcript ( ) . in this study, we show that post-transcriptional inhibition of lncbst /bispr leads to reduced levels of bst mrna (figure ) . therefore, lncbst /bispr should increase transcription or stability of its coding neighboring gene. our results demonstrate that this regulation seems specific for bst , as lncbst /bispr downregulation does not affect the expression of genes located nearby, which has been described for compact genomes ( ) . moreover, inhibition of lncbst /bispr does not affect expression levels of other ifn-related genes such as isg or gbp (figure and data not shown). we anticipate that inhibition of lncbst /bispr, and therefore of bst , could impact the antiviral effects of ifn. bst is also named tetherin, as it inhibits viral budding by using anchors that trap virions on the cell membrane ( ) ( ) ( ) . several enveloped viruses have been shown to be susceptible to the action of bst /tetherin and have evolved to develop evasion strategies ( ) . interestingly, hiv, influenza, hcv, and vsv are among the susceptible viruses and could be used to test the antiviral role of lncbst /bispr ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in fact, we show that lncbst /bispr and bst are induced after infection with hcv or influenza and vsv mutant viruses that activate the ifn response (figure ) . upregulation of lncbst /bispr, lncisg , and their coding neighbors was also observed in patients infected with hcv compared to controls (figure ) . similarly, a significant upregulation of lncbst /bispr and isg was also detected in human tlymphocytes infected with hiv compared to controls (data not shown). a non-significant increase in bst and lncisg was also observed in these samples. this leads to the possibility that interference with these factors could have therapeutic relevance. it is unclear why cells infected by these viruses, which employ several viral proteins to block the ifn pathway, show activation of these ifn response genes ( ) . in the case of hcv, it has been previously shown that patients with chronic hcv infections express isgs, including high levels of isg ( ) ( ) ( ) . in fact, hcv has evolved to use some isgs for viral replication ( , ) . this is the case for isg . isg is an ubiquitin-like protein that attaches to its targets in a process called isgylation ( , ) . protein isgylation may result in increased or decreased functionality depending on the target ( ) . isg preferentially conjugates newly synthesized proteins affecting more strongly viral proteins or cellular proteins translated into ifn-induced cells ( ) . viruses such as influenza, hiv, or vsv are susceptible to the action of isg ( , ) . in the case of hcv, a pro-hcv role for isg has been reported ( , ) . isg has been shown to negatively regulate rig-i and thus to inhibit the signaling process leading to ifn induction that affects hcv replication ( ) . furthermore, isg expression in the liver of chronically infected patients is considered a negative predictive biomarker of the ability of the patients to respond to ifn therapy ( ) ( ) ( ) (figure ) . in our study, we cannot address whether lncisg , bst , or lncbst /bispr are markers for the susceptibility of hcv patients to respond to ifn-treatment, as the hcv patients that we have studied are non-responders to ifn. we believe that, similar to lncbst /bispr, lncisg could affect the expression of isg or other genes. this lncrnamediated control has also been described for a lncrna located close to the isg viperin, which has been shown to regulate the levels of many ifn-inducible genes ( , ) . further experiments will be required to address the role of lncisg and to decipher the molecular mechanisms that allow the control exerted by lncbst /bispr on bst . we believe that these studies may be important to better understand the ifn response and its pro or antiviral functions on hcv and other viruses. frontiers in immunology | molecular innate immunity regulation of type i interferon responses dynamic expression profiling of type i and type iii interferon-stimulated hepatocytes reveals a stable 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modification the interferon stimulated gene functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses isg , a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment negative feedback regulation of rig-imediated antiviral signaling by interferon-induced isg conjugation the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein a regulation of interferon-stimulated gene bst by a lncrna transcribed from a shared bidirectional promoter barriocanal contributed to the writing of the manuscript; victor segura was in charge of all the bioinformatic analyses; and puri fortes conceived the project and the required experiments, provided the budget, interpreted the data, and wrote the manuscript. we thank nerea razquin and celia prior for excellent technical assistance, paul miller for editorial work, estanis nistal, cristian smerdou, and rafael aldabe for influenza and vsv viruses, sfv, and hcv, respectively. we also thank pablo gastaminza for the huh cells sensitive to hcv infection used in all the experiments, esther larrea for ifnα and primers for isgs, elizabeth guruceaga for the initial studies on rnaseq data and ruben hernandez for helpful comments on the manuscript. we would like to thank patients for the generous donation of samples and virginia villar and the biobank of the university of navarra for their mediation. this work was supported by grants from ministerio de ciencia e innovacion bio / , and saf - , feder funding, funds from the "ute project cima" and by the project rnareg [csd - ], funded by the ministry of science and innovation under the program consolider ingenio . a manuscript from dr. valadkhan's laboratory has been accepted for publication that describes similar results ( ) . the supplementary material for this article can be found online at http://www.frontiersin.org/journal/ . /fimmu. . / abstract the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the review editor saba valadkhan declares that, despite having collaborated on a publication in the last years with authors puri fortes, marina barriocanal, and elena carnero, the review process was handled objectively. key: cord- -hh h t authors: tseng, chin-kai; hsu, sung-po; lin, chun-kuang; wu, yu-hsuan; lee, jin-ching; young, kung-chia title: celastrol inhibits hepatitis c virus replication by upregulating heme oxygenase- via the jnk mapk/nrf pathway in human hepatoma cells date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: hh h t background and purpose: celastrol, a quinone methide triterpene isolated from the root extracts of tripterygium wilfordii, can greatly induce the gene expression activity of heme oxygenase- (ho- ) to achieve disease prevention and control. ho- induction was recently shown to result in anti-hcv activity by inducing type i interferon and inhibiting hepatitis c virus (hcv) ns / a protease activity. the aim of the present study is to evaluate the anti-hcv activity of celastrol and characterize its mechanism of inhibition. methods: the anti-hcv activity of celastrol was evaluated using the hcv subgenomic replicon and hcvcc infection systems. the anti-hcv mechanism of celastrol targeting ho- expression was clarified using specific inhibitors against several signaling pathways. the transcriptional regulation of celastrol on target gene expression was determined using promoter-based reporter activity assay. the synergistic effect of celastrol and a numbers of clinically used anti-hcv drugs was determined via a drug combination assay. results: celastrol inhibited hcv replication in both the hcv subgenomic and hcvcc infection systems with ec values of . ± . and . ± . μm, respectively. celastrol-induced heme oxygenase (ho- ) expression promoted antiviral interferon responses and inhibition of ns / a protease activity, thereby blocking hcv replication. these antiviral effects were abrogated by treatment with the ho- -specific inhibitor snmp or silencing of ho- expression by transfection of shrna, which indicates that ho- induction contributes to the anti-hcv activity of celastrol. jnk mitogen-activated protein kinase and nuclear factor erythroid -related factor (nrf ) were confirmed to be involved in the inductive effect of celastrol on ho- expression. celastrol exhibited synergistic effects in combination with interferon-alpha, the ns a inhibitor daclatasvir, and the ns b inhibitor sofosbuvir. conclusion: celastrol can serve as a potential supplement for blocking hcv replication. targeting the jnk/nrf /ho- axis presents a promising strategy against hcv infection. hepatitis c virus (hcv) is an enveloped single-stranded positive-sense rna virus belonging to the hepacivirus genus of the flaviviridae family (brass et al., ) . the genome of the virus includes base pairs encoding structural proteins (c, e , e and p ) and nonstructural proteins (ns , ns , ns a, ns b, ns a and ns b) (brass et al., ) . hcv infection is a risk factor of chronic liver diseases, including cirrhosis, fibrosis and hepatocellular carcinoma. hcv-infected patients have been estimated to number up to million worldwide (alter, ) . in the past, the standard of care (soc) for hcv infection is treatment with a combination of pegylated interferon (peg-ifn-a) and ribavirin (rbv); however, the efficacy of this treatment is only %e % in patients infected with genotype hcv (ghany et al., ) . as well, this soc therapy is associated with several adverse effects, such as anemia, headache, fatigue and depression (schoggins et al., ) . in , telaprevir and boceprevir, the first daas were approved by the us food and drug administration (fda) and found to exhibited higher rates of sustained virologic responses (svrs) in patients infected with genotype hcv when combined with peg-ifn-a, which provided a new soc for the treatment of chronic hcv infection (kiser et al., ) . despite the increasing efficacy of the treatment modes developed for hcv infection, however, undesirable side effects continue to be observed during therapy (kiser et al., ) . the fda recently approved the two-agent combo harvoni and the fouragent combo viekira pak for distribution; these treatments could achieve svrs up to % in patients infected with genotype hcv (koretz, ) . unfortunately, while these antiviral agents achieve higher rates of svr while avoiding the adverse effects often induced by ifn (koretz, ) , they also exist the risk to reactivate the hepatitis b virus (hbv) in treating patients with hcv and hbv co-infection (yeh et al., ) . moreover, these two drugs relatively more expensive than other drugs. thus, development of a more safety and cost-effective substitute for treatment is an important endeavor. celastrol is a quinone methide triterpene isolated from tripterygium wilfordii, a medicinal plant used to treat a range of illnesses including inflammation, swelling, fever, sores, and pain in india, japan, china, korea, and other asian countries (ju et al., ; lee, j.y. et al., ; youn et al., ) . celastrol is a meal supplement that is widely used in herbal medicine because of its diverse biological activities, which include anti-inflammation, anti-cancer, hepatoprotection, and anti-microbial properties (ju et al., ; lee et al., ) . several reports have demonstrated that celastrol exhibits inhibitory effects on human immunodeficiency virus (youn et al., ) , dengue virus (yu et al., a) , and other severe acute respiratory syndrome-associated coronaviruses (ryu et al., ) . celastrol can induce heme oxygenase- (ho- ) gene expression via nuclear factor erythroid -related factor (nrf ) activation to prevent circulatory failure prevention and protect against myocardial ischemia (der sarkissian et al., ) . ho- induction was recently shown to result in anti-hcv activity by inducing type i interferon and inhibiting hcv ns / a protease activity (lehmann et al., ; zhu et al., ) . ho- is an inducible and rate-limiting enzyme that degrades heme to produce biliverdin, carbon monoxide (co) and ferrous iron (fe þ) via the heme catabolic pathway (maines, ) . the enzyme and its metabolites exhibit protective effects against oxidative stress-induced tissue damage upon exposure to multiple stimuli such as viral and bacterial products including lipopolysaccharides, cytokines, oncogenes, mitogens, and various growth factors (farombi and surh, ; paine et al., ) . upon hcv infection, down-regulation of ho- expression has been observed in hcv-infected patients and hcv core protein expression although the biological role of ho- suppression and the detailed mechanism of hcv against ho- expression were unclear (abdalla et al., ) . ho- is a detoxifying enzyme that is mainly regulated by nrf (shan et al., ) . activation of ho- expression occurs when nrf binds to the antioxidant response element (are) of the ho- promoter region. by contrast, bach suppresses ho- expression by competing with the are binding site (shan et al., ) . under normal conditions, nrf is sequestered in the cytoplasm by kelchlike ech-associated protein (keap )-mediated proteasome degradation (shan et al., ) . upregulating nrf /are-dependent ho- expression to achieve anti-inflammatory and anti-oxidative stress functions is mediated by several host factors, including the mitogene-activated protein kinase (mapk) molecules p mapk, extracellular signal-regulated kinase and (erk / ), and c-jun nterminal kinase (jnk) (paine et al., ) . in this study, our data revealed that celastrol significantly inhibits hcv replication, and that the anti-hcv effect of celastrol was attenuated by the ho- specific inhibitor tin mesoporphyrin (snmp) or ho- gene expression silencing. celastrol-mediated ho- induction contributed to the anti-hcv action through inducing antiviral ifn response and inhibiting hcv ns protease activity. moreover, the jnk/nrf / are axis was the critical pathway involved in celastrol-mediated ho- induction against hcv replication. combinations of celastrol and ifn-a, sofosbuvir or daclatasvir were tested to determine their ability to enhance of anti-hcv activity. human hepatoma (huh- ), ava (huh- cells containing hcv genotype b subgenomic rna replicon cells) (blight et al., ) , and huh . /j /jfhemcviresrlucneo (huh- cells harboring hcv genotype a subgenomic rna and renilla luciferase reporter gene) obtained from apath llc (st. louis, mo) were maintained in dulbecco's modified eagle's medium containing % fetal bovine serum, % non-essential amino acids, and % antibioticeantimycotic in a % co atmosphere at c. huh- . cells stably expressing the pef/jfh -rz/n plasmid were grown to produce cell culture-derived infectious hcv particles (hcvcc), and the conditional medium was collected to harvest viral particles according to a protocol described previously (kato et al., ) . celastrol (pubchemcid: ) was purchased from fusol-material co., ltd (tainan, taiwan). ifn-a- a (roferon © -a) was purchased from roche ltd. an ho- specific inhibitor [tin mesoporphyrin (snmp)], and mapk-specific inhibitors (sp , sb , and pd ) were purchased from sigma (st. louis, mo). daclatasvir and sofosbuvir was purchased from shanghai haoyuan chemexpress co., ltd. the final concentration of dmso in all reactions was maintained at . %. ava cells were seeded in -well plates at a density of  cells per well and treated with celastrol at the indicated concentrations. after days incubation, the cell viability was determined by the celltiter aq ueous one solution cell proliferation assay (promega, madison, wi, usa) according to the manufacturer's instructions. pho- -luc (hill-kapturczak et al., ) and p xare-luc (liao et al., ) vectors were used to measure the transcriptional activity of ho- and nrf , respectively. pisre-luc, a reporter vector containing firefly luciferase under the control of an ifn-stimulated response element (isre), was used to measure ifn response-dependent transcriptional activity (stratagene, agilent technologies, ca, usa). all cloned dna fragments were verified by dna sequencing. western blotting was performed as described previously (lee et al., ) . in brief, mg of cell lysates was analyzed by sds-page, and then transferred to a pvdf membrane. the membranes were probed with anti-hcv ns b ( : ; abcam, cambridge, ma, usa), anti-ho- ( : ; abcam), anti-nrf ( : ; genetex, ca, usa), anti-phospho-erk / ( : ; cell signaling technology, inc. danvers, ma, usa), anti-phospho-p ( : ; cell signaling), anti-phospho-jnk ( : ; cell signaling), anti-erk / ( : ; cell signaling), anti-p ( : ; cell signaling), or anti-jnk ( : ; cell signaling)antibody. a loading control was determined using anti-gapdh antibody ( : ; genetex). rna isolation and quantitative real-time rt-pcr (qrt-pcr) were performed as described previously (lee et al., ) . relative mrna levels were determined by normalization against the cellular endogenous glyceraldehyde- -phosphate dehydrogenase (gapdh) gene. the primers used in the study are listed in table . to evaluate the transcriptional regulation of ho- , nrf , or ifn response by celastrol, the ava cells were seed on the -wells plates at a density of  cells per well. after h incubation, the . mg of promoter-driven firefly luciferase plasmids, including pho- -luc, p xare-luc, or pisre-luc, were respectively transfected into pre-seeded ava cells using t-pro™ transfection reagent (ji-feng biotechnology co., ltd. taiwan), according to the manufacturer's instructions. the transfected cells were then treated with celastrol at various concentrations for days. cell lysates were prepared for the luciferase activity assay and western blotting using the bright-glo luciferase assay system (promega, madison, wi, usa). the cell-based ns / a activity assay was performed as described previously (lee et al., ) . in brief, the huh- cells were seed on the -wells plates at a density of  cells per well. after h incubation, the cells were co-transfected with the ns / a protease reporter vector peg(ded ab)seap and ns / a expression vector pns / a, followed by celastrol treatment with or without snmp or days. the . mm telaprevir treatment server as the positive control. supernatants were harvested for the seap activity assay using the phospha-light assay kit. each transfection mixture contained . mg of the firefly luciferase expression vector (pfluc) as a transfection control for normalization against seap activity. cells were seeded in -well plates at a density of  cells/ well and treated with various concentrations of celastrol for days. the cell culture medium was harvested to measure ifn-a concentration using a human ifn-a elisa kit (uscnk life science inc., usa) according to the manufacturer's protocol. absorbance was detected at nm using an epoch microplate spectrophotometer. ava cells were seeded in a -cm dish at a density of .  cells per -cm dish, and treated with increasing concentrations of celastrol for days. the cells were then collected for preparation of a nuclear fraction as described previously (lee et al., ) . ava cells were treated with serially diluted celastrol ( , . , . , or . mm) in combination with serially diluted ifn-a ( . , , , or u/ml), telaprevir ( . , . , . , and . mm) sofosbuvir ( , , , or nm), or daclatasvir ( . , , and pm). after days of incubation, hcv rna levels were determined by qrt-pcr. multiple drug combination data were analyzed using calcusyn ™ software (biosoft, cambridge, uk) which compares single and multiple drug dose effects and determines the combination index (ci) value. the effect of a multiple drug combination is presented as antagonism (ci > ), additivity (ci ¼ ), or synergism (ci < ). data were presented as the mean ± standard deviation of at least three independent experiments. statistical significance was analyzed using student's t-test. a significant difference was considered as *p < . . celastrol is a quinone methide triterpene (fig. a) possessing anti-denv activity (lee et al., ) . to discover whether celastrol exhibits anti-hcv activity, we first treated hcv subgenomic replicon ava cells with celastrol at increasing concentrations for days. western blotting assay and qrt-pcr analysis were performed to determine hcv protein and rna levels under celastrol treatment, respectively. the cytotoxic effect of celastrol on ava cells was also tested using the mts assay. the results indicated that celastrol dose-dependently reduced hcv protein synthesis (fig. b) and rna replication with a % effective concentration (ec ) of . ± . mm (fig. c ) without cytotoxicity at effective antiviral concentrations ( % cytotoxicity concentration: cc ¼ . ± . mm) (fig. d ). based on the results collected from hcv replicon, the selectivity index of celastrol against hcv replication approximates . the jfh replicon and hcvcc infectious assay was performed to confirm the anti-hcv activity of celastrol. here, huh . /j /jfhemcviresrlucneo replicon cells and jfh infected huh- cells were treated with increasing concentrations of celastrol for days. as shown in fig. e and f, qrt-pcr analysis revealed that celastrol dose-dependently reduced hcv rna levels and fully inhibited hcv replication at a concentration of . mm. celastrol was recently shown to induce on ho- gene expression for antiviral activity (youn et al., ) . we first examined whether celastrol could induce ho- expression by determining ho- promoter activity, as well as ho- rna and protein levels, in ava cells in the presence of celastrol. ava cells were transfected pho- -luc containing a firefly luciferase gene driven by the ho- promoter. then, the transfected-cells were treated with celastrol at increasing concentrations for days and subjected to luciferase activity assay. the results indicated that celastrol significantly induced the ho- promoter activity in a concentrationdependent manner ( fig. a) . as expected, the ho- rna and protein levels were also dose-dependently induced by celastrol ( fig. b and c). to investigate whether celastrol-induced ho- expression is involved in the anti-hcv activity of celastrol, ava cells were cotreated with a fixed concentration of celastrol and increasing concentrations ho- specific inhibitor snmp for days. western blotting assay was preformed to determine the restorative effect of snmp on hcv protein synthesis upon celastrol treatment. as shown in fig. d , celastrol inhibited hcv protein synthesis compared with non-celastrol treated cells (lanes and ) by contrast, snmp treatment dose-dependently attenuated the suppressive effect of celastrol on hcv protein synthesis (lanes e ). as expect, the ho- specific shrna mediated ho- gene silencing also can attenuate the suppressive effect of celastrol on hcv protein synthesis (fig. e) . these results reveal that celastrol inhibited hcv replication is correlated with ho- induction. the results of qrt-pcr analysis indicated that ifn-a- and ifn-a- rna levels were gradually induced by celastrol (fig. a) . by contrast, the inductive effect of celastrol on ifn-a- and ifn-a- rna levels was significantly attenuated by ho- inhibitor snmp in a concentration-dependent manner (fig. b) . we next measured ifn-a protein secretion levels in the culture medium after celastrol treatment. as expected, elisa results indicated that ifn-a protein secretion levels were gradually induced by celastrol after days of treatment (fig. c ). an antiviral effect on cells may be attributed to the interaction of ifn-a and cell surface ifn-a receptors, leading to the activation of isre and upregulation of downstream antiviral genes (yu et al., b) . to evaluate whether celastrol-induced ifna could activate downstream antiviral genes, isre activity and the expression of three critical ifn-mediated antiviral genes, including - -oligoadenylate synthetase e (oas e ), were measured. ava cells were transfected with isre-driven firefly luciferase reporter plasmid followed by treatment with celastrol for days. luciferase assay results indicated that the isre promoter activity was increased by approximately . e . -fold by celastrol (fig. a ). oas e gene expression levels were significantly upregulated approximately . ± . , . ± . , and . ± . -fold by celastrol, respectively (fig. b) . another proposed anti-hcv action of ho- induction is inhibition of ns / a protease activity (zhu et al., ) . therefore, we performed a cell-based hcv protease assay to examine the ability of celastrol to target hcv ns / a protease activity. huh- cells were co-transfected with peg(de ab)seap reporter and hcv pns / protease expression plasmids, followed by celastrol treatment for days. as shown in fig. a , hcv ns / a protease activity decreased by approximately . e -fold in comparison with that of the control by celastrol. by contrast, the inhibitory effect of celastrol on hcv ns / a protease activity was dose-dependently attenuated by ho- inhibitor snmp (fig. b) . these data indicate that targeting hcv ns / a protease is an alternative anti-hcv action of celastrol. nrf functions as an important upstream regulator in the mediation of ho- expression by binding to the are response element (farombi and surh, ) . to investigate whether celastrol-induced ho- induction is mediated by nrf activation, we first investigated nrf nuclear translocation. ava cells were treated with increasing concentrations of celastrol for days, after which the total cells lysate and nuclear fraction were harvested and subjected to western blotting assay. as shown in fig. a , total nrf protein levels were not affected by celastrol (upper panel). by contrast, nuclear nrf protein levels significantly accumulated upon celastrol treatment at a concentration of . mm, which is the effective dosage against hcv replication. we then examined the nrf -mediated are activation caused by celastrol. here, ava cells were transfected with are-driven firefly luciferase reporter plasmid. the transfected-cells were treated with increasing concentrations of celastrol for days. as expected, are-driven firefly luciferase activity was elevated by celastrol in a concentrationdependent manner (fig. b ). considering these results, the nrf -are-ho- axis can be concluded to be strongly associated with the anti-hcv activity of celastrol. activation of the mapk signaling cascade, which includes p , erk / , and jnk, has been reported to be involved in ho- induction resulting in anti-hcv activity (huang et al., whether mapks are involved in anti-hcv effect of celastrol, ava cells were treated with . mm celastrol for e min. the phosphorylation levels of p , erk / , and jnk were then measured by western blotting with phospho-specific antibodies. ava cells were incubated with celastrol for days, and the total cell lysate and cellular nuclear fraction were harvested. total nrf (t nrf ) and nuclear nrf (ne nrf ) levels were analyzed by western blotting using anti-nrf antibody. lamin b served as the internal control for the nuclear fraction, and gapdh served as internal control for the total cell lysate. (b) celastrol increases nrf -mediated are transactivation. ava cells were transfected with . mg of p xare-luc followed by celastrol treatment for days, and then harvest to analyze their luciferase activity. here, luciferase activity was used to represent isre activity. error bars denote the mean ± sd of three independent experiments. *p < . . as shown in fig. a , the jnk phosphorylation levels were elevated by celastrol in a time-dependent manner compared with that at the time point of min. by contrast, celastrol showed no significant effect on erk/ / or p phosphorylation at any time point. to clarify the role of jnk in the ho- induction of celastrol, we used specific inhibitors against erk / (pd ), jnk (sp ), and p (sb ), to measure ho- rna expression. as shown in fig. b , ho- rna expression levels were induced by celastrol compared with non-celastrol treated cells and the jnk inhibitor sp significantly reduced the ho- inductive effect of celastrol. by contrast, the erk / inhibitor pd and p inhibitor sb showed no significant effect on celastrol-induced ho- induction. these results suggest that the anti-hcv effect of celastrol is associated with jnk-mediated ho- induction in ava cells. . . celastrol synergistically or additively inhibits hcv replication when combined with ifn-a, sofosbuvir, daclatasvir or telaprevir to determine whether celastrol can enhance the anti-hcv activity of several of clinically used anti-hcv drugs, such as ifn-a, the ns / a inhibitor telaprevir, the ns b inhibitor sofosbuvir (lam et al., ) and the ns a inhibitor daclatasvir (lee et al., a) . ava cells were co-treated with each drug and celastrol at various concentration ratios for days. the synergistic effect of each combination was evaluated by calcusyn . as described by chou and talalay ( ) . the ci values for the ec , ec , and ec of ifn-a ranged from . to . , those of telaprevir ranged from . to . , sofosbuvir ranged from . to . and daclatasvir is ranging from . to . (table ) . no significant cytotoxicity was observed in any combination treatment, as assessed by a colorimetric mts assay (data not shown). these findings reveal that celastrol may serve as a dietary supplement for enhancing the therapeutic effect of clinically used anti-hcv drugs. the type i ifn system presents important innate immunity to effectively block virus replication (jones et al., ; morrison et al., ) . hcv infection has been reported to inhibit antiviral ifn responses that facilitate virus replication by promoting the hcv ns / a protease-mediated cleavage of mitochondrial antiviralsignaling protein (mavs)/tir-domain-containing adapterinducing interferon-b (trif) (gokhale et al., ) . in the present study, we found that a natural product celastrol could effectively inhibit hcv ns / a protease activity and enhance ifn-mediated antiviral gene expression through ho- induction (figs. e ) . the ho- metabolite biliverdin has been proven to be a blocker of hcv ns / a protease activity. therefore, the regulatory effect of celastrol on the mitochondria-mediated inf signaling pathway against virus replication presents promising prospects for future investigations. our data revealed that nrf- -mediated ho- induction contributed to the anti-hcv activity of celastrol based on the accumulation of nuclear nrf- and enhancement of nrf- binding activity on the are response element (fig. ) . given that the activation of nrf nuclear translocation is regulated by keap dependent ubiquitination and bach , a competitor of nrf for binding to are in the ho- promoter region (maines, ) , future studies should be performed to determine whether celastrol alters the expression levels of keap- or bach for regulating ho- induction. knowledge in this area will help provide alternative targets for screening anti-hcv agents. we further found that the ho- inductive effect of the celastrol was also associated with jnk activity (fig. ) . however, several kinases are involved in jnk activation, including mitogen-activated protein kinase kinase (mkk ), mkk and mixed-lineage kinases (mlks) (wasserman et al., ) . future studies should examine the effect of celastrol on the kinases involved in jnk activation comprehensively describe the relationship between celastrol and ho- induction. several studies have indicated that celastrol exhibits anti-inflammatory activity by inhibiting of nuclear factor-kb (nf-kb) and downstream cycloxygenase- (cox- ) (ding et al., ; joshi et al., ) . on the basis of earlier findings on a promising tactic against hcv infection via down-regulation of nf-kb-mediated cox- expression lee et al., b) , we propose that the inhibitory effect of celastrol on nf-kb-mediated cox- expression may, at least in part contribute to its anti-hcv activity. hence, more work is necessary to elucidate additional signaling pathways involved in the anti-hcv activity of celastrol. drug combination therapy is considered to be a promising approach to increase therapeutic efficacy and decrease drug resistance in comparison with mono-drug therapy (hajj et al., ) . the two-agent combo harvoni and the four-agent combo viekira pak, which could achieve the svrs of up to % in patients infected with genotype hcv. however, the low fidelity of hcv polymerase during viral replication may lead to the emergence of drug resistance, which poses a major challenge in treating hcv infection (lee et al., ) . targeting host factors could be an alternative strategy to eliminate drug resistance in hcv therapeutic regimens because the mutation rate of the host genome is lower than that of the rna virus genome (liao et al., ) . in this study, we showed that celastrol can be considered as a suitable candidate for hcv therapy to minimize the risk of drug resistance by targeting host ho- signaling pathway and synergistically inhibiting hcv replication in combination with clinically used drugs against different viral targets (table ) . further studies are warranted to clarify the potential clinical relevance of our findings. in summary, the data indicated that celastrol efficiently inhibited hcv replication via the induction of the jnk/nrf /ho- axis, which may represent a therapeutic target for the future development and discovery of anti-hcv drugs. celastrol exhibited synergetic effects on anti-hcv activity in combination with ifn, sofosbuvir or daclatasvir. these results reveal that celastrol may serve as a dietary supplement for enhancing therapeutic effects of the anti-hcv drugs currently available. down-regulation of heme oxygenase- by hepatitis c virus infection in vivo and by the in vitro expression of hepatitis c core protein epidemiology of hepatitis c virus infection efficient initiation of hcv rna replication in cell culture aqueous extract of the edible gracilaria tenuistipitata inhibits hepatitis c viral replication via cyclooxygenase- suppression and reduces virus-induced inflammation quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors celastrol protects ischaemic myocardium through a heat shock response with up-regulation of haeme oxygenase- celastrol, an inhibitor of heat shock protein beta potently suppresses the expression of matrix metalloproteinases, inducible nitric oxide synthase and cyclooxygenase- in primary human osteoarthritic chondrocytes american association for the study of liver diseases, diagnosis, management, and treatment of hepatitis c: an update hepatitis c virus. strategies to evade antiviral responses combination of acamprosate and baclofen as a promising therapeutic approach for parkinson's disease an internal enhancer regulates heme-and cadmium-mediated induction of human heme oxygenase- mechanistic link between the anti-hcv effect of interferon gamma and control of viral replication by a ras-mapk signaling cascade dengue virus inhibits alpha interferon signaling by reducing stat expression celastrol modulates inflammation through inhibition of the catalytic activity of mediators of arachidonic acid pathway: secretory phospholipase a group iia, -lipoxygenase and cyclooxygenase- celastrol ameliorates cytokine toxicity and pro-inflammatory immune responses by suppressing nf-kb activation in rinm f beta cells production of infectious hepatitis c virus of various genotypes in cell cultures review and management of drug interactions with boceprevir and telaprevir acp journal club: review: telaprevir, boceprevir, simeprevir, or sofosbuvir improves response in hcv type genotype and subtype profiling of psi- as a nucleotide inhibitor of hepatitis c virus. antimicrob the hepatitis c virus ns a inhibitor (bms- ) alters the subcellular localization of the ns a non-structural viral protein anti-hepatitis c virus activity of acacia confusa extract via suppressing cyclooxygenase- andrographolide exerts anti-hepatitis c virus activity by up-regulating haeme oxygenase- via the p mapk/nrf pathway in human hepatoma cells celastrol blocks binding of lipopolysaccharides to a toll-like receptor /myeloid differentiation factor complex in a thiol-dependent manner cinnamaldehyde inhibits the tumor necrosis factor-alpha-induced expression of cell adhesion molecules in endothelial cells by suppressing nf-kappab activation: effects upon ikappab and nrf the heme oxygenase system: update dengue virus co-opts ubr to degrade stat and antagonize type i interferon signaling signaling to heme oxygenase- and its anti-inflammatory therapeutic potential regulation of hepatitis c virus replication and gene expression by the mapk-erk pathway sars-cov clpro inhibitory effects of quinone-methide triterpenes from tripterygium regelii a diverse range of gene products are effectors of the type i interferon antiviral response role of bach and nrf in up-regulation of the heme oxygenase- gene by cobalt protoporphyrin a novel c-jun n-terminal kinase (jnk)-binding protein wdr is recruited to stress granules and mediates a nonclassical jnk activation oxidative stress induces anti-hepatitis c virus status via the activation of extracellular signal-regulated kinase reactivation of hepatitis b in patients of chronic hepatitis c with hepatitis b virus infection treated with direct acting antivirals celastrol ameliorates hiv- tat-induced inflammatory responses via nf-kappab and ap- inhibition and heme oxygenase- induction in astrocytes celastrol inhibits dengue virus replication via up-regulating type i interferon and downstream interferon-stimulated responses schisandrin a inhibits dengue viral replication via upregulating antiviral interferon responses through stat signaling pathway biliverdin inhibits hepatitis c virus nonstructural / a protease activity: mechanism for the antiviral effects of heme oxygenase we are grateful to dr. charles rice (rockefeller university and aapth, lcc, usa) for kindly supporting con b replicon plasmid, human hepatoma cell; huh- and hcv subgenomic replicon containing cell line; ava , huh . /j /jfhemcviresrlucneo hcv replicon cells and dr. t. wakita (national institute of infectious diseases, japan) for providing the jfh plasmid. key: cord- -zzys e authors: ryan, elizabeth j.; harenberg, anke; burdin, nicolas title: the canarypox-virus vaccine vector alvac triggers the release of ifn-γ by natural killer (nk) cells enhancing th polarization date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: zzys e we investigated the mechanism by which alvac activates innate immunity. combining alvac with protein antigens significantly augmented antigen-specific igg a responses; this was dependent on the presence of bioactive interferon (ifn)-γ. immuno-depletion of nk cells prior to alvac immunisation abrogated ifn-γ production indicating that they are the main cellular source of early ifn-γ in vivo. murine bone-marrow derived dendritic cells (bmdcs) cultured in the presence of alvac secreted high levels of the chemokines cxcl and ccl and up-regulated expression of the maturation markers cd , cd and cd . therefore, we conclude that alvac acts as an adjuvant through a mechanism requiring nk cell derived ifn-γ, dc activation and chemokine secretion. adjuvants improve the adaptive immune response to coinjected antigen by activating innate immunity [ ] . in spite of intense research, there is still a limited number of adjuvants available for human use, in particular to enhance the induction of the polarized th response required for optimum protective immunity to viral infection and for cancer therapy [ , ] . vaccination strategies based on the canarypox-virus vector alvac have shown potential in both infectious disease [ , ] and cancer [ ] . alvac, a large dna virus whose replication is confined to avian hosts, has a good safety profile in humans [ ] . despite its attenuated nature, alvac does cause inflammation at the site of injection as evidenced by infiltration of neutrophils [ ] . furthermore, previous reports show that alvac possesses adjuvant activity [ , ] . in this study, we confirm that alvac acts as an adjuvant, enhancing antigen-specific igg a to co-injected protein antigen in mice. intra-muscular (i.m.) injection of alvac increases levels of circulating ifn-␣ and ifn-␥ shortly after injection. alvac enhanced antigen-specific igg a responses to co-injected protein antigen in ifn-␣ receptor knockout (ifn-␣r −/− ) mice but not ifn-␥r −/− mice, indicating the importance of bioactive ifn-␥ in mediating the adjuvant effect of alvac. immuno-depletion of nk cells dramatically impaired the ability of alvac to induce circulating ifn-␥, indicating that these cells are the major producer of this cytokine in vivo. a previous study has shown that alvac triggers tnf-␣ production by human dcs [ ] . as the interaction between nk and dcs is important for the induction of the innate immune response [ ] we investigated the effect of alvac exposure on murine bmdc regarding maturation and cytokine production. we show that under the experimental conditions we used, low levels of apoptosis were detected, and that alvac induces the maturation of dcs (up-regulation of cd , cd and cd expression) and the production of low levels of inflammatory cytokines (il- , tnf-␣ and il- ). however, a marked increase in the secretion of the chemokines cxcl and ccl was observed. therefore, this leads us to suggest that alvac can act as a th polarizing adjuvant by inducing local inflammation [ ] , resulting in dc maturation and chemokine production which in turn causes the recruitment of ifn-␥ secreting nk cells. understanding the mechanism of adjuvant action of alvac, will allow more effective targeting of the desired immune response by taking advantage of the fact that alvac can be manipulated to encode a variety of immunomodulatory transgenes, e.g. gm-csf [ ] or il- [ ] that can further enhance the activation of nk cells or dcs. six to eight week old female balb/c byj mice (charles river, les oncins, france) or /sv, ifn-␣ receptor knockout and ifn-␥ receptor knockout (b + k universal, hull, uk) were used. animals were housed and experiments carried out in an accredited facility under the guidelines of sanofi-pasteur's local ethics committee. in this study we used alvac-hiv, vcp (sanofi-pasteur, marcy l'etoile, france) a recombinant canarypox vector that contains the genes for hiv- envelope gp (strain mn) linked to the trans-membrane portion of hiv- gp (strain lai), the hiv- lai gag gene encoding for the entire gag protein, the sequence of the pol gene encoding the protease, and a synthetic polynucleotide encompassing several known human ctl epitopes from the nef and pol gene products. it also contains sequences encoding the e l and k l vaccinia virus proteins in the c site. alvac placebo (sanofi-pasteur) contains a mixture of mm tris-hcl buffer ph . , virus stabilizer and freeze-drying medium. both alvac and placebo were re-constituted in sterile . % saline prior to injection. recombinant hiv tat protein from the hiv- iiib isolate and glycoprotein b (gb) from cytomegalovirus (cmv) were purified from bacterial culture at sanofi-pasteur. all vaccines used in this study were clinical grade and were verified to be free of lipopolysaccharide (lps) and other impurities. groups of balb/c, /sv, ifn-␣r knockout, or ifn-␥r knockout mice were immunised i.m. in the quadriceps following anaesthesia with imalgene (merial, lyon, france) with either gb ( g/dose), tat ( g/dose), alone or in combination with placebo or alvac ( / human dose). mice were boosted at day and serum and spleen samples taken at day . anti-tat and gb antibody titres and specific cellular responses were then analysed as described below. maxi-sorp elisa plates (nunc, wiesbaden, germany) were coated with g/ml of either tat or gb in pbs overnight at • c. plates were blocked with % non-fat milk (gibco, paisley, uk) at • c for h, after washing serial dilutions of serum samples and standards were added for h at • c. plates were then washed and the detection antibody, goat-anti-mouse igg-hrp (jackson immunoresearch laboratories, inc., west grove, pa) was added for a further hour at • c. plates were washed, and a tetramethlybenzidine substrate solution added (tebu bio-labs, le perray-en-yvelines, france), the enzyme reaction was stopped with the addition of phosphoric acid ( m), and od values at nm were determined. antibody titres were calculated from the linear regression curve of a standard specific serum originating from the same animal species as the samples, present on each elisa plate (softmax software tm , molecular devices, sunnyvale, ca). the titre of the standard had been previously determined in independent experiments. multiscreen hts plates (millipore, bedford, ma) were coated overnight at • c with anti-ifn-␥ capture monoclonal antibody (mab) (clone no. r - a , bd pharmingen, san diego, ca), washed and blocked. responder spleen cells were added at a concentration of × cells/well in rpmi (gibco) supplemented with % fcs (hyclone, logan, ut), penicillin-streptomycin (gibco), mm lglutamine (gibco), and mm ␤-mercaptoethanol (gibco). cells were stimulated in triplicate with g/ml of or mer tat or gag peptides (neosystem, strasbourg, france), medium alone as negative control and pma (sigma-aldrich, st. louis, mo)/anti-cd (bd pharmingen) as positive control. alvac specific immune responses were measured by adding p cells (mouse lymphoblast-like mastocytoma cell line) that had been infected with a multiplicity of infection (moi) of of parental virus (cppp) overnight. cultures were incubated overnight at • c/ % co . the following day, detection using mab ifn-␥-biotin (clone no. xmg . , bd pharmingen), followed by streptavidin-hrp (southern biotech, birmingham, al) was visualized using -amino- -ethylcarbazole (aec) substrate (sigma-aldrich). ifn-␥ producing spots were counted using an automated elispot reader equipped with spot tm software (microvision instruments, evry, france). alvac's ability to induce early circulating ifn-␥ following i.m. injection was evaluated using an in vivo ifn-␥ detection assay as described by the manufacturer (bd pharmingen). briefly, prior to i.m. injection with alvac or placebo groups of balb/c mice were given an intraperitoneal (i.p.) injection with g/dose of a biotin-ifn-␥ mab, serum samples were removed at various time-points and frozen at − • c until analysis of the ifn-␥ mab complex by elisa. in some experiments, nk cells were depleted using antiasialo gm polyclonal antibody (cedarlane laboratories, ontario, canada) i.p. at day − and day at g/dose. control mice received the same dose of non-specific rabbit igg (caltag laboratories, burlingame, ca). the capture ifn-␥ mab with either placebo or alvac were injected as described and serum samples were collected at h postimmunisation, and frozen at − • c until analysis by elisa. spleens were removed from both control and treated animals and the efficacy of the nk cell depletion measured by evaluating the % of spleen cells expressing the nk cell marker cd b by flow cytometry using a specific mab, clone no. dx (bd pharmingen). secretion of cytokines/chemokines was analyzed by elisa using commercially available matched pairs of mabs; murine cxcl and cxcl (r&d systems, minneapolis, mn); murine il- , tnf-␣, il- , ifn-␥, il- p , il- , and human il- and il- p (bd pharmingen, san diego, ca). the limit of sensitivity for detection was pg/ml for all elisa assays. murine ifn-␣ was detected in serum samples using a commercially available elisa kit as instructed (endogen, rockford, il). the detection range for this assay was . - pg/ml. in some experiments cytokine determinations were performed using the cytometric bead array (cba) mouse inflammation kit from bd pharmingen as described by the manufacturer. samples were acquired on a bd facscalibur and analysed with cellquest pro tm software. the detection range for all cytokines by this method was - pg/ml. bone-marrow was flushed from the femurs and tibiae of balb/c byj mice using cold complete rpmi. cells were washed and re-suspended in complete rpmi supplemented with gm-csf ( ng/ml, r&d systems). on day fresh media containing gm-csf was added to the adherent cells. on day , cells were washed, phenotyped by flow cytometry and if > % of the cells were cd c + they were then used for experiments. bmdcs were cultured at a concentration of × cells/ ml in complete rpmi for h in the presence of alvac at a moi of . and , or the equivalent volume of placebo. cells cultured in medium alone were used as a negative control and cells cultured with a toll like receptor (tlr) agonist (sanofi-pasteur) at a concentration of . g/ml, as a positive control. cells were washed, re-suspended in pbs with % fcs and . % nan ; labelled with mabs specific for cd , cd , cd , cd , cd c, cd , cd , and cd , with the appropriate isotype controls (all from bd pharmingen). cells were acquired using a facscalibur (bd) and results analyzed using cellquest pro tm software (bd). cell death analysis of either human peripheral blood mononuclear cells (pbmcs) or murine bmdcs was carried out using the tacs annexin-v-fitc apoptosis detection kit as described by the manufacturer (r&d systems). all results are presented as mean ± s.e.m., unless otherwise indicated. differences between the means of multiple groups in an experiment were analysed using one-way anova analysis of variance. in the case where only two groups are being considered results were analysed using a two-tailed independent variable student's t test. all analysis was performed using statview software (sas, cary, nc). p-values < . were considered significant. groups of balb/c mice were immunised i.m. with either antigen alone (tat or gb) or in combination with placebo or alvac. mice received a booster immunisation after weeks, and were sacrificed weeks later in order to investigate antigen-specific immune responses. titres of antigen-specific igg and igg /igg a were determined by elisa. the levels of gb specific igg observed in the group who received gb and alvac were higher than both the group that received gb alone, and the group immunised with gb and placebo (fig. a) . a similar increase in antigen-specific igg was observed when alvac was co-injected with another antigen, tat (fig. b) . looking at the particular subclasses of igg, the addition of alvac to the antigens tat or gb did not cause an increase in the levels of antigen-specific igg ( fig. c and d) . however, a -fold increase was observed in the levels of anti-gb igg a in mice that received gb and alvac compared to mice that received gb alone (fig. e) . a similar augmentation of tat-specific igg a was observed in the groups of mice that received tat and alvac compared to those that received either tat alone or tat and placebo (fig. f) . these results clearly show that alvac can act as an adjuvant augmenting antigen-specific igg a responses to co-injected antigen. next, we investigated the effect of alvac on the cellular immune response to co-injected antigen. groups of balb/c mice were immunised with tat alone, tat and placebo or tat and alvac and weeks after a booster immunisation the mice were sacrificed and their spleen removed to analyse the cellular immune responses. the spleen cells were restimulated in vitro and the numbers of antigen-specific ifn-␥ secreting cells were determined by elispot (fig. g ). the number of tat-specific ifn-␥ secreting cells in the groups of mice that received tat alone or tat and placebo were not significantly different from background levels (fig. g) . however, tat-specific ifn-␥ secreting cells were detectable in the group that received tat together with alvac. ifn-␥ secreting cells specific for the vector itself (cppp) and for gag (encoded by alvac) were detected in all groups that received alvac (fig. g) . these data show that alvac augments both humoral and cellular type responses to coinjected antigen. two major signalling pathways participate in the innate immune response to viral dna, namely, the nuclear factor-b (nf-b)-dependent pathway leading to inflammatory cytokine secretion (including il- and ifn-␥) and the type i ifn-dependent pathway inducing ifn-␣ and ifn-regulated genes [ ] . therefore, we wished to determine if ifn-␣ and/or ifn-␥ were produced in vivo following an injection of alvac. groups of balb/c mice were immunised simultaneously by the i.m. route with / th of the human dose of alvac and i.p. with a biotin-conjugated anti-ifn-␥ mab, and subsequently the mice were bled at various time-points after immunisation. levels of the ifn-␥:mab complex were determined by elisa. significant quantities of ifn-␥ were detectable in the circulation of mice h after immunisation ( fig. a) . the ifn-␥:mab complex continued to accumulate in the circulation of the alvac immunised mice up to h post-immunisation ( fig. a) . the anti-ifn-␥ antibody is stable for up to h in vivo. using / th of the human dose resulted in less circulating ifn-␥ than / th of the human dose, illustrating the effect is dose-dependent ( fig. a) . in order to detect circulating ifn-␣ groups of balb/c mice were immunised i.m. with / th of the human dose of alvac and then bled at various time-points, and the levels of ifn-␣ in the serum were determined by elisa. a low and transient amount of ifn-␣ could be detected at h in the serum of alvac immunised mice (fig. b) . in summary, alvac immunisation induces a low amount of ifn-␣ and significant amounts of ifn-␥ with very different kinetics. ifn-␣ being early and transient and ifn-␥ appearing later but more sustained. however, as we used an in vivo capture assay to determine the circulating amounts of ifn-␥ (stable ifn-␥:mab complex) and a straightforward elisa approach to detect the relatively labile ifn-␣, we cannot draw any conclusions about the relative importance of the two cytokines in the adjuvanticity of alvac from these data. at various time points serum samples were prepared from these mice and frozen at − • c until analysis. levels of the complex of mab + ifn␥ were determined by an elisa using a standard curve generated using recombinant ifn-␥ that had been pre-incubated with biotinylated mab. results are expresses as mean cytokine concentration ± standard deviation. * p < . , *** p < . and ns = not significant. (b) in a separate experiment groups of balb/c mice ( per group) were immunised with placebo or alvac ( / human dose) at time . serum samples were removed at various time-points and frozen at − • c until analysis. levels of serum ifn-␣ were determined by elisa in triplicate on serum pools from each group. results are expressed as the mean cytokine concentration ± standard error. as we demonstrated that alvac could induce both innate ifn-␣ and ifn-␥ (albeit in different amounts and with varied kinetics), we wished to address the question whether the induction of one or both of these cytokines were crucial for the adjuvant activity of alvac. in order to do this we immunised groups of wild type ( /sv), ifn-␣r −/− and ifn-␥r −/− mice with either gb and placebo or gb and alvac and then evaluated the gb specific immune responses. this approach enabled us to observe if alvac maintained its adjuvant properties in the absence of bioactive ifn-␣ or ifn-␥. anti-gb specific igg levels were similar in all groups of mice irrespective of mouse genotype or vaccine (fig. a) . anti-gb specific igg levels were marginally reduced in the groups that received gb and alvac compared . the animals were boosted at day and serum samples taken at day . anti-gb specific igg (a), igg (b) and igg a (c) titres were determined by elisa. results represented are the mean ± standard deviation of mice per group, *** p < . and ns = not significant. the anti-gb specific igg :igg a ratio was determined (d). a value of indicates that equal amounts of antigen-specific igg and igg a was produced, values greater than indicate that more anti-specific igg than igg a, while values less than one signify that more igg a than igg . with those that received gb and placebo in each of the genotypes (fig. b) . however, a clear augmentation of gb specific igg a was observed in both /sv and ifn-␣r −/− mice that received gb and alvac compared to gb and placebo. in contrast, no significant increase in anti-gb igg a was observed in ifn-␥r −/− mice upon the addition of alvac to the protein antigen (fig. c) . these data indicates that although alvac induces both innate ifn-␣ and ifn-␥ production in vivo, ifn-␣ is dispensable for the augmentation of specific igg a to co-injected antigen. nk cells are a major cellular source of the ifn-␥ produced during the first phase of the immune response to infection [ ] . in order to determine their role in the innate immune response to alvac we depleted nk cells from balb/c mice and then measured the ability of these mice to produce ifn-␥ in response to an i.m. injection of alvac. nk cell depletion was achieved using two doses of a polyclonal anti-asialo-gm ab, one given days before and the second together with the alvac injection. the efficacy of this depletion is illustrated by the lack of cd b (a nk cell specific marker in balb/c mice) expressing cells in the spleens of anti-asislo-gm treated mice compared with mice treated with an isotype control antibody (fig. b) . groups of mice, either nk cell depleted or not, were immunised with placebo or alvac and were given a simultaneous i.p. injection of biotin-anti-ifn-␥. after days, the mice were sacrificed and their serum analysed for the levels of circulating ifn-␥:mab complex by elisa. we found that the induction of circulating ifn-␥ by alvac was significantly reduced following nk cell depletion (fig. a ). this indicates that nk cells fig. . alvac induction of circulating innate ifn-␥ that is dependent upon the presence of cd b + nk cells. (a) nk cells were depleted from balb/c mice using polyclonal anti-asialo gm antibody. mice were injected i.p. with g of antibody at day − and day , rabbit igg was used as an isotype control. on day biotinylated anti-ifn-␥ at g/dose was given i.p. to all animals, and then the various groups were immunised i.m. with either placebo or alvac at / human dose. serum samples were prepared at day and the levels of the circulating ifn-␥:antibody complex were determined by elisa. values shown are the mean ± standard deviation of mice per group, *** p < . and ns = not significant. (b) treatment with anti-asialo-gm ab reduced the % cd b + cells in the spleens of the treated mice from . % (± . ) in the control treated group to . % (± . ) in the anti-asialogm treated group (the average of mice/treatment group ± standard deviation). spleen cells were isolated from mice per group and the expression of cd b determined by flow cytometry using a pe-labelled specific mab. quadrants were determined by the staining of the isotype control antibodies; representative facs plots of per treatment are shown. are the primary source of the ifn-␥ secreted in response to alvac within the first h of immunisation. activated dcs augment ifn-␥ production by nk cells [ ] . therefore, we wished to investigate if the exposure to alvac activates dcs. in order for alvac to act as an effective vaccine vector it must infect the host cells in order to produce the antigens which are integrated in its genome. however, here as we are examining the adjuvant effect of alvac we are interested in investigating the immuno-modulatory effects of exposing cells to alvac without them necessarily becoming infected. in addition in vitro infection protocols using low or no serum conditions and a high moi have been shown to induce apoptosis in the target cells [ ] . preliminary experiments showed that a moi of . to did not induce significant cell death in murine bone-marrow derived dcs (bmdcs) over a culture period of h (data not shown). murine bmdcs were cultured for h at a concentration of cells/ml in complete rpmi supplemented with % fcs alone or with the addition of alvac with a moi of . , the equivalent volume of placebo or with a tlr agonist as a positive control. cells were then stained with fluorescently labelled mabs specific for the co-stimulatory molecules cd , cd and cd and their expression levels were analysed by flow cytometry. the percentage of bmdcs expressing co-stimulatory molecules (cd , cd and cd ) was significantly increased following exposure to alvac compared to medium alone or placebo (fig. a-c) . however, the enhancement of costimulatory molecule expression induced by alvac was weaker than that observed following the stimulation of bmdcs with a tlr agonist (fig. a-c) . nonetheless, these data illustrate that exposure to alvac activates dcs. activated dcs secrete inflammatory cytokines and chemokines to recruit and activate the cells of both the innate and adaptive immune response. in response to alvac low amounts of the pro-inflammatory cytokines il- , tnf-␣ and il- p are produced by dcs, and in general the amounts are -fold less than those induced by the tlr agonist used as a positive control in these experiments (fig. d-f) . however, the levels of the chemokines cxcl and ccl produced by bmdcs following incubation with alvac exceeded or were equivalent to those induced by the tlr agonist ( fig. a and b) . cxcl secretion by bmdc was not detected in response to alvac (fig. c ). in order to reap the optimum benefit from therapeutic immunisation strategies in cancer or chronic viral infections, vaccines will need to be formulated in such a way as to augment the th effector arm of the immune response. in this report, we show that alvac can act as a th polarizing adjuvant in a mouse model (fig. g) . we show that bioactive ifn-␥ but not ifn-␣ is crucial for this adjuvant effect, as antigenspecific igg a responses to co-injected protein are enhanced in ifn-␣r −/− mice but not ifn-␥r −/− mice (fig. c) . immuno-depletion of nk cells revealed that they are an important source of the innate ifn-␥ produced in response to alvac injection in vivo (fig. a) . furthermore, we show that stimulation of murine bmdcs with alvac in vitro can induce dc maturation (fig. a-c) and chemokine secretion (particularly cxcl and ccl , fig. a and b) . additionally, alvac injection resulted in an enlargement of the local draining lymph node (popliteal) following i.m. injection (data not shown). therefore, we can conclude that alvac injection activates the innate immune response enhancing the induction of th responses to co-injected antigen. the recombinant alvac (vcp ) that we use in this study has been shown to be well tolerated in clinical trials [ ] . previous reports have shown that alvac can act as an adjuvant [ , ] . in particular, boudet et al. [ ] showed that purified alvac in either an infectious or non-infectious form had an adjuvant effect when co-administered with the recombinant hiv antigen, gp ; resulting in an increase of antigen-specific igg in both mice and guinea pigs. hutchings et al. [ ] furthered this by showing that this adjuvant activity was common to other poxviruses (nyvac, fowlpox (fp ), as well as alvac). the poxviruses: nyvac, fowlpox (fp ), alvac and to a lesser extent, adenovirus all enhanced the immune response to co-injected recombinant hepatitis b surface antigen (hbsag) [ ] . interestingly, the combination of the non-recombinant alvac vector with hbsag induced the strongest antigen-specific cellular response in the draining lymph node compared to the responses resulting from including the other poxvirus vectors investigated [ ] . two major tlr mediated signalling pathways participate in the response to pathogens, the nuclear-factor b (nf-b)-dependent pathway leading to the secretion of il- and other inflammatory cytokines and the type i ifn pathway which synergises with the nf-b pathway for optimal il- p secretion [ ] . we show that injection of alvac results in an increase in the levels of both circulating ifn-␣ and ifn-␥ ( fig. a and b) . in order to determine if the induction of one or both of these cytokines was crucial for the adjuvant effect of alvac, we immunised wild type ( /sv), ifn-␣r −/− and ifn-␥r −/− mice with gb (cmv) either with or without alvac. both the /sv and ifn-␣r −/− mice that received gb and alvac produced higher titres of gb specific igg a when compared to the groups that received gb and placebo (fig. ) , indicating that bioactive ifn-␣ is dispensable for the adjuvant effect of alvac. a study investigating the immunogenicity of adenovirus vectors demonstrated that although type i ifn signalling is an important component of adenovirus-mediated dc maturation, it was dispensable during the generation of transgene product specific cd + t cell responses [ ] . therefore, it appears that although ifn-␣ is induced by alvac and may well play a role in augmenting the innate immune response, it is not required for its' adjuvant effect. activated nk cells can secrete large amounts of ifn-␥ in response to pathogens or activated dcs [ ] . in order to determine if the ifn-␥ produced in response to immunisation with alvac ( fig. a) was produced by nk cells, we immunised nk cell depleted mice with alvac and then determined the levels of circulating ifn-␥ (fig. a) . the ifn-␥ produced in response to alvac in the nk cell-depleted mice was significantly lower than that observed in mice treated with the isotype control indicating that nk cells are a significant source of the ifn-␥ produced. it has been recently reported that alvac preferentially infects human cells of the monocytic lineage and was infact unable to infect human nk cells in vitro [ ] . our data indicates that in a mouse model, nk cells do play a crucial role in mediating the inflammatory response to alvac. further studies with human cells in vitro or ex vivo are required to determine the role of nk cells in the innate response to alvac following vaccination in man. recently, it has become clear that there is an important bidirectional dialogue between dc and nk cells (reviewed by [ ] ). tlr-dependent microbial stimuli typically associated with th responses confer on dcs the ability to activate nk cells [ ] to secrete ifn-␥ directly or induce nk cells the ability to lyse immature dcs [ ] . the discovery that human nk cells express tlr and tlr suggested the existence of tlr-based nk cell-dc crosstalk [ ] . this might be relevant in explaining how nk cells and dcs can be activated simultaneously by the same pathogen and build an efficient innate immune response, which amplifies the downstream adaptive th response [ ] . a previous report showed that both mature and immature human dcs could be successfully infected with alvac [ ] . many alvac infected immature dcs rapidly underwent apoptosis, and the endocytosis of infected, dead or dying dcs by uninfected immature dcs was observed. concurrently, a sub-population of alvac-exposed dcs matured. maturation was driven by the tnf-␣ secreted following exposure to alvac and partially by the ingestion of infected cell debris [ ] . we investigated the effect of exposing murine bmdcs to alvac in vitro and found that exposure for h to a moi of . (conditions that did not induce a significant amount of apoptosis) did activate dcs as evidenced by their up-regulation of the co-stimulatory molecules cd , cd and cd (fig. ) . additionally, exposure to alvac induced murine bmdcs to secrete significant quantities of the chemokines cxcl and ccl (fig. ). cxcl (ip- ) is chemotactic for t cells [ ] and nk cells [ ] expressing cxcr . while ccl (mcp- ) plays a role in the recruitment of monocytes/macrophages by interacting with ccr . recent studies with cxcl neutralising antibodies or cxcl −/− mice demonstrate that the absence of functional cxcl results in increased mortality and reduced t cell infiltration within the brains of mice infected intracerebrally with a murine coronavirus [ , ] , illustrating the importance of this chemokine in co-ordinating a protective immune response against a viral pathogen. ccl 's interaction with ccr is an important factor promoting early inflammatory responses and effective local antiviral defense [ ] . the ability of alvac to induce dcs to secrete chemokines, such as cxcl may provide the signal required for nk cells to migrate to dc-rich areas bringing them in close proximity enabling direct cell-cell contact, thereby resulting in downstream th responses. we found that there was a significant increase in both size and cellularity in the local draining lymph node following injection with alvac (data not shown). using flow cytometric analysis of the lymph node cells we could not determine a change in the % of any cell type (data not shown). these data in conjunction with previous work by boudet et al. demonstrate that alvac can trigger an inflammatory response, induce the migration of neutrophils to the site of inoculation within h of administration [ ] and enlargement of the local draining lymph node. therefore, we now propose the following mechanism for the adjuvanticity of alvac: alvac causes inflammation, and pmn recruitment to the site of injection [ ] . this, in turn, causes the activation of local dcs (as demonstrated by increased co-stimulatory molecule expression). these cells may then migrate to the local draining lymph node. here, dc secretion of chemokines including ccl and cxcl cause an influx of cells including nk and t cells. the interaction of the dc and nk cells in the local lymph node triggers ifn-␥ secretion by the nk cells that results in an increase in th cells specific for the co-administered protein. when these cells re-circulate to the b cell rich areas of the spleen they enhance isotype switching to igg a. innate immune induction of the adaptive immune response towards the rational design of th adjuvants immunological foundations to the quest for new vaccine adjuvants safety and immunogenicity of a high-titered canarypox vaccine in combination with rgp in a diverse population of hiv- -uninfected adults: aids vaccine evaluation group protocol a safety and immunogenicity of an hiv subtype b and e prime-boost vaccine combination in hiv-negative thai adults recombinant viruses as a tool for therapeutic vaccination against human cancers modulation of the antibody response to the hiv envelope subunit by co-administration of infectious or heat-inactivated canarypoxvirus (alvac) preparations novel protein and poxvirus-based vaccine combinations for simultaneous induction of humoral and cell-mediated immunity canarypox virus-induced maturation of dendritic cells is mediated by apoptotic cell death and tumor necrosis factor alpha secretion the dialogue between human natural killer cells and dendritic cells comparative studies of avipox-gm-csf versus recombinant gm-csf protein as immune adjuvants with different vaccine platforms intratumoral administration of a recombinant canarypox virus expressing interleukin in patients with metastatic melanoma a type i interferon autocrine-paracrine loop is involved in toll-like receptor-induced interleukin- p secretion by dendritic cells induction and regulation of ifns during viral infections safety and immunogenicity of alvac vcp and recombiwith prolonged highly active antiretroviral therapy dendritic cell maturation, but not cd + t cell induction, is dependent on type i ifn signaling during vaccination with adenovirus vectors the small subset of cd brightcd -natural killer cells is selectively responsible for both cell proliferation and interferongamma production upon interaction with dendritic cells comparative analysis of tropism between canarypox (alvac) and vaccinia viruses reveals a more restricted and preferential tropism of alvac for human cells of the monocytic lineage tlrdependent activation stimuli associated with th responses confer nk cell stimulatory capacity to mouse dendritic cells cpg and double-stranded rna trigger human nk cells by toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells tlr / -mediated activation of human nk cells results in accessory cell-dependent ifn-gamma production neutralization of the chemokine cxcl reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis cxc chemokine ligand controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells ifngamma-inducible protein (ip- ; cxcl )-deficient mice reveal a role for ip- in effector t cell generation and trafficking monocyte chemoattractant protein- and ccr interactions are required for ifnalpha/beta-induced inflammatory responses and antiviral defense in liver key: cord- -ebkwv zo authors: bodmer, bianca s.; fiedler, anna h.; hanauer, jan r.h.; prüfer, steffen; mühlebach, michael d. title: live-attenuated bivalent measles virus-derived vaccines targeting middle east respiratory syndrome coronavirus induce robust and multifunctional t cell responses against both viruses in an appropriate mouse model date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: ebkwv zo cases of middle east respiratory syndrome coronavirus (mers-cov) continue to occur, making it one of the who´s targets for accelerated vaccine development. one vaccine candidate is based on live-attenuated measles virus (mv) vaccine encoding the mers-cov spike glycoprotein (mers-s). mv(vac )-mers-s(h) induces robust humoral and cellular immunity against mers-s mediating protection. here, the induction and nature of immunity after vaccination with mv(vac )-mers-s(h) or novel mv(vac )-mers-n were further characterized. we focused on the necessity for vector replication and the nature of induced t cells, since functional cd (+) t cells contribute importantly to clearance of mers-cov. while no immunity against mers-cov or mv was detected in mv-susceptible mice after immunization with uv-inactivated virus, replication-competent mv(vac )-mers-s(h) triggered robust neutralizing antibody titers also in adult mice. furthermore, a significant fraction of mers cov-specific cd (+) t cells and mv-specific cd (+) t cells simultaneously expressing ifn-γ and tnf-α were induced, revealing that mv(vac )-mers-s(h) induces multifunctional cellular immunity. the middle east respiratory syndrome coronavirus (mers-cov) is a member of the coronaviridae family and emerged in in the kingdom of saudi arabia (zaki et al., ) . coronaviruses typically cause mild infections of the upper respiratory tract, but already in , the severe acute respiratory syndrome cov (sars-cov) with a mortality rate of about % among infected patients was introduced in the human population. sars-cov spread world-wide and caused more than diagnosed infections, but was contained within a year after its emergence (http://www.who.int/csr/sars/country/table _ _ / en/). in contrast, infections with mers-cov are ongoing for more than years, with laboratory-confirmed cases distributed over countries with at least deaths that were reported to the who by november (http://www.who.int/emergencies/mers-cov/en/). this apparent case-fatality rate of % is of grave concern, because epidemic spread as has been observed for sars-cov could result in a disastrous death toll. mers-cov has been introduced zoonotically by transmission from dromedary camels to human patients (alagaili et al., ; haagmans et al., ; reusken et al., a) and serological studies indicate wide-spread and early distribution among this animal host (alagaili et al., ; reusken et al., b) . therefore, a continuous risk of transmission especially to persons in close contact to camels is evident. fortunately, the human to human transmission rate has remained low. aside of individuals with regular contact to camels, only health care workers or relatives of mers-cov patients have a considerable risk of infection (alraddadi et al., ; drosten et al., ) , but still at a modest level. nonetheless, the high case fatality rate, the recurrent outbreaks of mers-cov infections, and especially the risk of virus adaptation potentially resulting in epidemic or even pandemic spread make the development of an effective vaccine against mers-cov an international priority. the efficacy of several vaccine candidates has been demonstrated in different animal models up to even dromedary camels (reviewed in (okba et al., ) ). one of these candidates, mv vac -mers-s(h) (malczyk et al., ) , is based on the measles virus (mv) vaccine platform technology (mühlebach, ) , and encodes the mers-cov spike protein (s) as an additional antigen in the backbone of recombinant mv vac (del valle et al., ) resembling vaccine strain moraten that is authorized and in use in the us since . this candidate induces both robust humoral and functional cellular immuneresponses against mers-cov. moreover, mers-cov viral load and inflammation of the lung were significantly reduced in challenged mice that had been vaccinated with mv vac -mers-s(h), before (malczyk et al., ) . while these experiments provided proof of concept for efficacy of this vaccine candidate, further mechanistic insights into the nature of the induced t cell responses remain to be elucidated. these are of special interest, since it has been shown that t cells are essential for clearance of the infection (coleman et al., ; zhao et al., ) : depletion of cd + t cells increased overall inflammation, bronchiolar inflammation, lymphocyte infiltration, and pleuritis at day post-infection in mice (coleman et al., ) , while mers cov-susceptible mice depleted of all t cells were unable to clear the virus . as an alternative to the spike glycoprotein, conserved (internal) structural proteins such as the nucleocapsid protein n are of special interest as putative target of anti-viral t cell responses to be triggered by future mers vaccines . therefore, we have also generated and characterized mers-cov n protein-encoding vaccine candidates based on the mv vac vaccine platform, in this study. to further characterize the induction of mers cov-specific immune responses, we first analyzed the necessity for viral replication for the induction of mers cov-and mv-specific immune responses using the highly immunogenic mv vac -mers-s(h) vaccine candidate. in addition, we characterized the functionality of cd + and cd + t cell responses in juvenile ( - week old) and adult ( months of age) mice using flow cytometry and functional assays. vero (african green monkey kidney) (atcc# ccl- ) and t (atcc crl- ) cell lines were purchased from atcc (manassas, va, usa) and cultured in dulbecco's modified eagle's medium (dmem, biowest, nuaillé, france) supplemented with % fetal bovine serum (fbs; biochrom, berlin, germany) and mm l-glutamine (l-gln; biochrom). jawsii dendritic cells (atcc crl- ) were purchased from atcc and cultured in mem-α with ribonucleosides and deoxyribonucleosides (gibco brl, eggenstein, germany) supplemented with % fbs, mm l-gln, mm sodium pyruvate (biochrom), and ng/ml murine gm-csf (peprotech, hamburg, germany). dc . murine dendritic cells (shen et al., ) were cultured in rpmi containing % fbs, mm l-gln, % non-essential aminoacids (biochrom), mm hepes (ph , ), and μm -mercaptoethanol (sigma-aldrich, steinheim, germany). cells were cultured at °c in a humidified atmosphere containing % co for a maximum of months of culture after thawing of the original stock. the codon-optimized gene encoding mers-cov-n (genebank accession no. jx ) flanked with aatii/mlui binding sites in plasmid pma-rq-mers-n was obtained by gene synthesis (invitrogen life technology, regensburg, germany). the antigen and the immediate early cytomegalovirus (cmv) promoter (martin et al., ) were inserted into plasmids p(+)br-mv vac -atu(p) (del valle et al., ) or p(+)mv vac -gfp(h) via mlui/aatii and sfii/sacii, respectively, to generate p(+)polii-mv vac -mers-n(p) or p(+)polii-mv vac -mers-n (h). for construction of lentiviral transfervectors encoding mers-cov-n, the orf of mers-n was amplified by pcr with primers encompassing flanking restriction sites nhei/xhoi and template pma-rq-mers-n. details on primers and pcr are available upon request. pcr products were cloned into pcr . -topo (invitrogen life technology) and fully sequenced. intact antigen orf was cloned into pcscw gluc-ires-gfp (hewett et al., ) using nhei/xhoi restriction sites to yield pcscw -mers-n-ires-gfp. lentiviral vectors were produced and used for the generation of antigen-expressing dendritic cell lines as described, before (malczyk et al., ) . in short, hiv- -derived vectors were generated using a standard plasmid system and the transfer vector plasmid pcscw -mers-n-ires-gfp by pei transfection. subsequent purification after harvest of transfected t cells yielded virus stocks used to transduce dc cell lines, which were single cell-sorted by facs and selected for antigen expression. mers-n encoding vaccine candidates mv vac -mers-n(p) and mv vac -mers-n(h) were rescued as described (malczyk et al., ; martin et al., ) . single syncytia were picked and overlaid onto % confluent vero cells cultured in -well plates and harvested as "passage " (p ) by scraping and freeze-thaw cycle of cells at the time of maximal infection. subsequent passages were generated as described for the following viruses. mers-n encoding vaccine viruses in p were used for characterization, viruses in p for vaccination. mers-s encoding vaccine virus mv vac -mers-s(h), and control virus mv vac -atu(p) (malczyk et al., ) were also used in p for vaccination. both as well as mv vac -gfp(p) and mers-cov (isolate emc/ ) (zaki et al., ) used for neutralization assays were propagated and titrated on vero cells by the method of spearman and kaerber (hubert, ; kärber, ) . mv vac -mers-s(h) was inactivated by uv-irradiation using a cl- uv crosslinker (uvp, cambridge, uk). μl of virus suspension in -well-plates on ice were exposed to uv light of nm at cm distance from the uv source of , × μj/cm for min. inactivation of virus was controlled by incubation of vero cells with a control aliquot inactivated, in parallel. all virus stocks were stored in aliquots at − °c. cells were lysed and immunoblotted as previously described (funke et al., ) . a rabbit anti-mers-cov serum ( : ) was used as primary antibody for mers-cov-n and a rabbit anti-mv-n polyclonal antibody ( : , ) (abcam) for mv-n detection. a donkey hrp-coupled anti-rabbit igg (h&l) polyclonal antibody ( : , ) (rockland, gilbertsville, pa) served as secondary antibody for both. peroxidase activity was visualized with an enhanced chemiluminescence detection kit (thermo scientific, bremen, germany) on amersham hyperfilm ecl (ge healthcare, freiburg, germany). all animal experiments were carried out in compliance with the regulations of german animal protection laws and as authorized by the rp darmstadt. six-to -week-old or months old ifnar -/--cd ge mice (mrkic et al., ) deficient for type i ifn receptor and transgenically expressing human cd were inoculated intraperitoneally (i.p.) with × tcid of recombinant viruses or uv-inactivated vaccine preparations on days and either on day or . mice were bled on days , , and post initial infection (p.i.). serum samples were stored at − °c. mice were euthanized on days , , or p.i., and splenocytes were harvested for assessment of cellular immune responses. quantification of vnts was done as described, before (malczyk et al., ) . in brief, mouse sera were serially diluted in -fold dilution steps in dmem in duplicates. a total of pfu of mv vac -gfp(p) or tcid of mers-cov (strain emc/ ) were mixed with diluted sera and incubated at °c for h. virus suspensions were added to × vero cells seeded h prior to assay in -well plates and incubated for days at °c. vnts were calculated as the reciprocal of the highest mean dilution that abolished infection. murine gamma interferon (ifn-γ) enzyme-linked immunosorbent spot (elispot) assays (ebioscience, frankfurt, germany) were performed according to the manufacturer's instructions using multiscreen immunoprecipitation (ip) elispot polyvinylidene difluoride (pvdf) well plates (merck millipore, darmstadt, germany). × isolated medium inoculated mice served as mock control. vnts were calculated as the highest dilution abolishing infectivity. dots represent single animals (n = ); horizontal lines represent mean per group. the y-axis starts at the detection limit; all mice at the detection limit had no detectable vnt. (g) secretion of ifn-γ after antigen-specific re-stimulation of splenocytes harvested days post prime immunization and after co-culture with jawsii (left) or dc . (middle) dendritic cells transgenic for mers-n (black) or untransduced controls (nc, white). (right) to analyze cellular responses directed against mv, splenocytes were stimulated with μg/ml mv bulk antigen (mv bulk) or left unstimulated (sham). the reactivity of splenocytes was confirmed by cona treatment ( μg/ml). tcid , tissue culture infectious dose ; one-way-anova with tukey multiple comparison. *: p < , ; **: p < , ; ***: p < ; ****: p < , . splenocytes were co-cultured with different stimuli in μl rpmi + % fbs, mm l-gln, and % penicillin-streptomycin for h. for re-stimulation of mers n-specific t cells, splenocytes were co-cultivated with × jawsii, dc . dendritic cells, or clones of either cell line encoding mers-n. on the other hand, splenocytes were stimulated with μg/ml mv bulk antigen (serion immunologics, würzburg, germany), μg/ml mers s-derived peptide s (biosynthesis inc., lewisville, tx, usa, (channappanavar et al., ) ), or μg/ml siinfekl control peptide (sin) of ovalbumin (aa - ) (invivogen, san diego, ca, usa), as appropriate. for general t cell stimulation, μg/ml concanavalin a (cona, sigma-aldrich, st. louis, mo, usa) was used. as negative control, splenocytes were left untreated. after h, cells were removed from the plates, and plates were incubated with biotin-conjugated anti-ifn-γ antibodies and avidin-hrp according to the manufacturer's instructions. -amino- ethyl-carbazole (aec; sigma-aldrich) substrate solution for development of spots was prepared according to the manufacturer's instructions using aec dissolved in n,n-dimethylformamide (merck millipore) and used for peroxidase-dependent staining, afterwards. spots were counted using an eli.scan elispot scanner (ae.l.vis, hamburg, germany) and elispot analysis software eli.analyse v . (ae.l.vis). for flow cytometry-based determination of cytokine expression by intracellular cytokine staining (ics), splenocytes of vaccinated mice were isolated, and × splenocytes per mouse were cultivated in μl rpmi + % fbs, mm l-gln, × non-essential amino acids (biochrom), mm hepes, % penicillin-streptomycin, μm βmercaptoethanol, μg/ml brefeldin a (sigma-aldrich), and one of the stimuli also used for elispot analysis. for general t cell stimulation, . μg/ml tetradecanoylphorbol acetate (tpa, sigma aldrich) and . μg/ml ionomycin (iono, sigma-aldrich) were used as positive control, and only medium was used as negative control. splenocytes were stimulated for h at °c. subsequently, cells were stained with fixable viability dye efluor (ebioscience), cd -pe ( : ) (bd, franklin lakes, nj, usa), cd -fitc ( : ) (bd), and, after permeabilization with fixation/permeabilization solution (bd) and perm/ wash buffer (bd), stained with ifn-γ-apc ( : ) (bd) and tnf-α-pe-cy ( : ) (bd). cells were fixed with ice-cold % paraformaldehyde (pfa) in pbs and analyzed via flow cytometry using an lsrii sorp flow cytometer (bd) and fcs express software (de novo software, glendale, ca, usa). since the nucleocapsid protein (n) of cov is quite conserved, it is regarded as an appropriate target to induce anti-viral t cells. therefore, mers-cov n was chosen as an alternative antigen to be expressed by the recombinant mv vaccine platform. full-length mers-n was cloned into two different additional transcription units (atus) either behind p (post p) or h (post h) cassettes of measles vaccine strain mv vac genome, and virus clones were successfully rescued and amplified in vero cells with titers of up to × tcid /ml. the essential verification of antigen expression by western blot analysis of vero cells infected with the mv vac -mers-n vaccines revealed expression of the n antigen with only little impact of the genomic position of the transgene cassette (fig. a) , while growth kinetics showed no impairment of virus replication compared to the respective mv vac -gfp(p) control virus ( fig. b, c) . to test the efficacy of the mv vac -mers-n candidate in vivo, genetically modified ifnar -/--cd ge mice were chosen, since they are the prime small animal model for analysis of mv-derived vaccines (mrkic et al., ) . thus, mice per group were inoculated via the intraperitoneal (i.p.) route on days and with each time × tcid of mv vac -mers-n(p), mv vac -mers-n(h), or empty control virus mv vac -atu(p). medium-inoculated mice served as negative controls. days or four days after boost immunization, sera or splenocytes of immunized mice were sampled, respectively (fig. d) . as expected, all mice immunized with recombinant mv (including the control virus) developed high mv virus neutralizing titers (vnt) ( - vnt, fig. f ). little evidence for induction of neutralizing antibodies against mers-cov was found in all mice, as expected for the intra-particular antigen (fig. e) . no vnts against mv or mers-cov were detected in control mice inoculated with medium alone. to analyze splenocytes of animals vaccinated with mv vac -mers-n (h) or control animals inoculated with medium or mv vac -atu(p) by elispot assay for antigen-specific ifn-γ secretion, the antigen-specific t cells were re-stimulated in vitro by syngeneic murine dc cell lines (jawsii and dc . ), which had been genetically modified by lentiviral vector transduction to stably express mers-n protein and thereby to present the respective t cell epitopes on mhc. single cell clones were derived by flow cytometric sorting of single gfp-positive cells. antigen expression by transduced dcs was verified by western blot analysis (data not shown). elispot assays using splenocytes of vaccinated animals in co-culture with jawsii-mers-n or dc . -mers-n revealed about ifn-γ secreting cells per × splenocytes after immunization with mv vac -mers-n(h) (fig. g) , which was significant over controls. additionally, cellular immune responses targeting mv antigens were detected upon stimulation with mv bulk antigens in vaccinated mice that had received any recombinant virus, as expected. however, mv bulk antigens stimulated about - ifn-γ secreting cells per × splenocytes of mv vaccinated animals, as described, before (malczyk et al., ) . finally, splenocytes of all mice revealed a similar basic reactivity to unspecific t cell stimulation, as confirmed by similar numbers of ifn-γ secreting cells upon cona treatment (fig. g) . thus, the generated mv-based vaccine platform expressing mers-n induces significant mers n-specific cellular immune responses, as desired. in any case, humoral and cellular responses induced by vaccine candidate mv vac -mers-s had been considerably higher in previous analyses under similar conditions (malczyk et al., ) . therefore, further characterization of anti-mers-cov immunity induced by mv vac -derived vaccines proceeded with this mers-s encoding vaccine candidate, which yielded approximately -fold higher numbers of reactive t cells after vaccination. since the mers vaccine candidate mv vac -mers-s(h) induced robust protective humoral and cellular immune responses in ifnar -/--cd ge mice (malczyk et al., ) , we were interested in the necessity of viral replication of this life-attenuated vaccine for the induction of mers cov-specific immunity. for these analyses ifnar -/--cd ge mice were chosen as the animal model, again, since these mice are the standard animal model for analysis of mv-derived vaccines (mühlebach, ) , their genetic composition is compatible with an established mers-cov challenge model , as shown, before (malczyk et al., ) , and their size allows housing under regularly available conditions opposed to dromedary camels, the only know natural host of mers-cov, to date. as all morbilliviruses, the mv-based vaccine virions are highly cellassociated, and transfer of antigenic protein within the vaccine preparation cannot be excluded. therefore, we vaccinated these mv-susceptible mice with either × tcid of live or of the same formulation and quantity uv-inactivated mv vac -mers-s(h) in a primeboost regimen ( fig. a) . mv vac -atu(p), which does not encode any additional antigen, was included as vector control. blood was drawn from naïve mice on day before vaccination, and on days and post-immunization. serum samples were tested for their ability to neutralize mv vac -gfp(p) (fig. b , d, f) or mers-cov (fig. c, e, g) . sera of naïve mice had no neutralizing antibodies against either virus (fig. b, c) . after the first immunization, both live virus preparations induced neutralizing antibodies against mv, with mv vac -atu(p) triggering significantly higher titers ( - vnt) than mv vac -mers-s(h) ( - vnt). after the second immunization, anti-mv vnts increased to titers of - in both cohorts. in contrast, only one out of four animals in the uv-inactivated vaccine group had a borderline neutralizing antibody titer of after the first immunization, and another animal had a titer of after the boost. while mv vac -atu(p) and the uv-inactivated mv vac -mers-s(h) vaccine did not induce neutralizing antibodies against mers-cov above background levels over the course of the experiment, the group vaccinated with live mv vac -mers-s(h) developed titers around after the first immunization and - (mean of ) after the boost. taken together, these data reveal that replication of the vaccine is necessary to induce functional antibody responses against mv and the additional antigen mers-s. to assess the capacity of the different mv vac -mers-s(h) vaccine preparations to induce mers-cov s-specific cellular immune responses, splenocytes of mice, which had already been tested for humoral responses ( fig. a) , were isolated and analyzed days after immunization for antigen(ag)-dependent ifn-γ secretion using elispot assay. the isolated splenocytes were re-stimulated with mers-s immunodominant peptide s (channappanavar et al., ) or mv bulk antigen (mv bulk) to analyze mv-specific cellular immune responses. ovalbumin-derived siinfekl-peptide (sin) served as peptide negative control, or cells were left untreated (mock). stimulation with concanavalin a (cona) was used to confirm general t cell reactivity in splenocyte preparations (fig. h) . while splenocytes of all mice responded to cona with to spots per × splenocytes, only those from animals vaccinated with live mv vac -mers-s(h) could be stimulated with mers s-specific peptide s reaching mean values of spots per × splenocytes. in contrast, splenocytes of the uv-inactivated group or control virus mv vac -atu(p) could not be restimulated to secrete ifn-γ. furthermore, only replication-competent vaccine viruses induced mv-specific cellular immune responses in vaccinated mice. re-stimulation with mv bulk ag induced a mean of and spots per × splenocytes for mv vac -mers-s(h) or mv vac -atu(p) vaccinated mice, respectively. consequently, replication of the vaccine candidate is essential to induce both arms of the immune system with responses against mv as well as the additional mers-s antigen. usually, - weeks old juvenile mice are used for our mers-cov neutralizing antibodies in sera of (b, c) naїve mice, or in sera of mice after (d, e) prime-or (f, g) boost-immunization. one-way anova with tukey multiple comparison. * : p < , ; * *: p < , ; ***: p < ; ****: p < , . (h) secretion of ifn-γ after antigen-specific re-stimulation of splenocytes. ifn-γ elispot analysis using splenocytes of mice vaccinated on days and with indicated vaccines isolated days after boost immunization and after incubation with indicated stimuli (mers-s peptide s , mv bulk antigen (mv bulk), immunodominant ovalbumin-derived siinf-ekl-peptide (sin) as a peptide negative control) or untreated (mock). the reactivity of splenocytes was confirmed by concanavalin a (cona) treatment ( μg/ml). the number of cells per × splenocytes represent the amount of cells expressing ifn-γ upon re-stimulation. dots represent individual animals, horizontal bars mean. one-way anova with tukey multiple comparison. ****: p < , . b.s. bodmer et al. virology ( ) - immunization studies. to evaluate if there is an age-dependent change in vaccination efficacy, approximately months-old mice were vaccinated with mv vac -mers-s(h) in a prime-boost vaccination scheme with weeks between prime and boost vaccination (fig. a ). mice were sacrificed at day post-immunization, and splenocytes were re-stimulated with mv-antigens or mers-s peptide s . we found that reactive ifn-γ-secreting t cells were also specifically induced in mice of this age (fig. b) . a mean of spots per × splenocytes was detected upon re-stimulation with mv bulk antigen, whereas spots per × splenocytes were induced by re-stimulation with the mers s-derived peptide s , illustrating that mv-and mers-cov-specific cellular immune responses are effectively induced in adult mice. to gain more detailed insights in the quality of the observed t cell responses, we further characterized the responsive t cell populations by flow cytometry, determining the expression of cd + and cd + surface markers as well as ifn-γ and tnf-α upon re-stimulation with s or mv bulk antigen. as a positive stimulus for t cell activation tetradecanoylphorbol-acetate and ionomycin (tpa/iono) were used. exocytosis of cytokines was blocked by addition of brefeldin a ( μg/ ml) during stimulation. cells were permeabilized, labelled, and fixed for flow cytometry. the gating strategy excluded duplicates (not shown), selected for living cells (fig. a, upper panel) , and separated cd + and cd + t cells (fig. a, lower panel) . selected cd + t cells were then analyzed for their expression of ifn-γ (fig. b left panel) , tnf-α (fig. b middle panel) , or both ( fig. b right panel) as exemplarily shown for splenocytes re-stimulated with mers-s peptide s . likewise, cd + t cells expressing ifn-γ (fig. c, left panel) , tnf-α (fig. c, middle panel) , or both (fig. c , right panel) are depicted after re-stimulation with mv bulk antigen. vaccination with mv vac -mers-s(h) induced a significant amount of mers s-specific cd + t cells expressing either ifn-γ (fig. d, left panel) or tnf-α (fig. d, middle panel) , with means of . % and . % of total positive cells, respectively. among those, a significant fraction of cells revealed to be multifunctional, with a mean of . % of all cd + cells or % of the tnf-α − responsive cells being positive for both cytokines (fig. d, right panel) . moreover, vaccination induced a significant fraction of vector-specific cd + t cells expressing ifn-γ (fig. e, left panel) , or tnf-α (fig. e , middle panel) upon re-stimulation with mv bulk antigen. among those, multifunctional cd + t cells expressing both cytokines were induced with a mean of about . % (fig. e, right panel) . to conclude, vaccination with mv vac -mers-s(h) induces not only ifn-γ or tnf-α expressing t cells directed against mers-cov and mv, but also a significant fraction of multifunctional cytotoxic t cells specific for mers-s and cd + t cells specific for mv antigens, illustrating that a broad and robust mers-covspecific immune response is induced by vaccination with mv vac -mers-s(h). in this study, we aimed to understand the induction of immunity and the functionality of induced t cell responses after vaccination with mv vac -mers-s(h), a vaccine candidate that induces protective immunity against mers-cov in an appropriate animal model. in parallel, we generated and tested also alternative mv-based vaccine candidates expressing mers-cov n protein as conserved t cell antigen. mv vac -mers-n vaccine candidates indeed induced significant antigen-specific cellular immune responses in vaccinated transgenic mice, revealing that also mers n-expression by recombinant mv may have a useful role to combat mers-cov. since the immune responses induced by mers-s expressing candidates had been nevertheless considerably higher, we proceeded with s-expressing vaccine virus to characterize the induction of anti-mers-cov immunity by mv-based vectors. using mv vac -mers-s(h), robust anti-mers cov immune responses were induced also in older mice, while replication of the vaccine vector was necessary to induce either arm of adaptive immunity against vector or pathogen. furthermore, vaccination with mv vac -mers-s(h) triggered significant numbers of multifunctional mers s-specific cd + t cells and mv-specific cd + t cells, simultaneously producing ifn-γ and tnf-α upon stimulation with respective antigens. since not only numbers, but also the quality of the induced mers cov-specific t cell responses might be relevant for protection against mers-cov, it is quite encouraging to see that approx. % of ifn-γ reactive cd + t cells also expressed tnf-α, whereas in reverse % of tnf-α-reactive cd + t cells co-expressed ifn-γ upon stimulation with the immune-dominant mers-s peptide. this induction of multifunctional t cells is quite in accordance with previous studies, since the potential of mv during natural infection or the recombinant mv to induce multifunctional, antigen-specific t cells has already been demonstrated. infection of macaques with wild-type mv induces polyfunctional t cells specific for mv proteins with increasing numbers of cells secreting il- , tnf-α, as well as ifn-γ over time (nelson et al., ) , and polyfunctional t cells directed against mv-h can be expanded from pbmc of human donors (ndhlovu et al., ) . likewise, hiv-vaccine candidates mv -f , which encode gag, rt, and nef of an hiv- clade b or a clade c strain as foreign antigen, induce antigenspecific multifunctional t cells simultaneously expressing ifn-γ, tnf-α, and il- in mice and also macaques (stebbings et al., (stebbings et al., , . while the combination of ifn-γ and tnf-α indicates functional t cells fig. ) were re-stimulated and subjected to intracellular staining (ics) for ifn-γ and tnf-α and stained for extracellular t-cell markers cd and cd for flow cytometry analysis. (a -c) gating strategy for analysis of cd + or cd + t-cells expressing ifn-γ or tnf-α within splenocytes stimulated with (b) s peptide or (c) mv-bulk ag. duplicates (not shown) and dead cells (a) were excluded from analysis. (b, c) cd + and cd + cells were separately subjected to analysis for ifn-γ-(left panels), tnf-α-(middle panels) or double-positive cells (right panels). quantification of flow cytometry data of (d) cd + -and (e) cd +positive cells after incubation with indicated stimuli (mers s-specific peptide s , mv bulk ag (mv bulk), immunodominant ovalbumin-derived siinf-ekl-peptide (sin) as a peptide negative control, or untreated cells (mock); reactivity of splenocytes was confirmed by tetradecanoylphorbol-acetate and ionomycin (tpa/iono) treatment ( μg/ml). dots represent individual animals, horizontal bars mean. repeated-measures one-way anova with tukey multiple comparison. *: p < , . with higher potency, in general, expression of il- is a sign of the induction of cd + memory t cells (williams et al., ) . in our study, the strong correlation of ifn-γ and tnf-α expression thus indicates a high functionality of induced t cell responses. extension of the antibody panel for il- detection could yield further insight into the durability of these t cell responses induced by the mv vaccine platform in future studies. such multifunctional cd + t cells specific for mers-cov may become especially important, since mouse studies have shown that cd + t cells are crucial for clearance of mers-cov infection (coleman et al., ; zhao et al., ) . noteworthy, for other viral infections such as human immunodeficiency virus (hiv), modified vaccinia virus ankara (mva), or cytomegalovirus (cmv) the amount of just ifn-γ producing t cells does not correlate with ctl killing effectivity, but the multifunctionality of antigen-specific t cells inversely correlated with viral load (betts et al., ; lichterfeld et al., ; precopio et al., ; sandberg et al., ) , further underlining the importance of multifunctionality. besides these cellular immune responses, also considerable humoral immunity was induced in vaccinated animals, here. the mean vnt was somewhat lower than expected (malczyk et al., ) , but still quite high. an alternative vaccine candidate derived from modified vaccinia virus ankara, mva-mers-s, revealed protection in dromedary camels, the natural host for mers-cov (haagmans et al., ) . passive immunotherapy with dromedary immune sera significantly reduced mers-cov titers in lung tissue of challenged mice, starting with a vnt of (zhao et al., ) . neutralizing antibody titers in reconvalescent plasma of human patients diagnosed with mers were determined by microneutralization tests in two previous studies, and were on average at (arabi et al., ) or . (zhao et al., ) . furthermore, a prnt titer of at least just was required to lower virus titers by more than . log in mice challenged after transfer of human reconvalescent plasma (zhao et al., ) . these titers were exceeded in this study. in addition, mv vac -mers-s(h) induced higher anti-mers-s titers in c /bl mice than mva-mers-s in balb/c mice, when comparing studies that used similar virus titers for vaccination (malczyk et al., ; volz et al., ) , thus indicating an at least comparable efficacy. thus, also vnt determined here indicate efficacy and were anyway not statistically different from those published before for mv vac -mers-s(h) (malczyk et al., ) . nevertheless, the exact correlates of protection for mers-cov remain to be determined in future studies, since it will be essential to evaluate the efficacy of the different vaccine candidates against this most important benchmark. in contrast, uv-inactivated mv vac -mers-s(h) did not induce any antibodies able to neutralize mers-cov or mv. while neutralizing antibodies can in principle also be induced by inactivated vaccines or proteins, e.g. full-length or truncated mers-s protein in combination with adjuvant (wang et al., ) . obviously, the amount of mers-s antigen within the mv vac -mers-s(h) vaccine formulation or the adjuvant effect of the inactivate were not sufficient during application. therefore, replication of the mv-derived mers vaccine candidate is necessary for the induction of immune responses both against vector and antigen of interest in vaccinated animals. indeed, the induction of cellular immunity is usually more efficient by de novo expression of antigen after immunization. consequently, the application of a replication competent vaccine platform is justified here to robustly induce potent responses of both arms of the adaptive immune system. these powerful immune responses were not only induced in juvenile mice - weeks of age, but also in adult mice older than half a year of age. this is quite of interest, since adult vaccinees are also the target group for vaccination in response to the mers-cov outbreak, as defined in the target product profile by the who (http://www.who.int/ blueprint/what/research-development/mers_cov_tpp_ .pdf). remarkably, mv vaccine strain virus encoding chikungunya virus (chikv) antigens was already tested in a phase i clinical trial in adult human vaccinees ( - years old) (ramsauer et al., ) . these adult test subjects all developed significant humoral immunity against chikv, despite their adult age and most interestingly also independent from measles pre-immunity. taken together, these study shows that mv vac -mers-s(h) induces surprisingly high numbers of multifunctional t cells specific for mers-s also in adult test subjects, as a result from replication of the recombinant vector. therefore, high quality cellular immune responses are induced in addition to the robust antibody responses by this vaccine candidate, further qualifying mv vac 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effector molecules in human cd + t lymphocytes cloned dendritic cells can present exogenous antigens on both mhc class i and class ii molecules immunogenicity of a recombinant measles-hiv- clade b candidate vaccine immunogenicity of a recombinant measles hiv- subtype c vaccine protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein evaluation of candidate vaccine approaches for mers-cov interleukin- signals during priming are required for secondary expansion of cd + memory t cells isolation of a novel coronavirus from a man with pneumonia in saudi arabia recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses rapid generation of a mouse model for middle east respiratory syndrome passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection this work was supported by the german center for infection research (dzif; ttu . ). the authors would like to thank vivian scheuplein, jürgen schnotz, and daniela müller for excellent technical assistance. the authors are indebted to ron fouchier for providing mers-cov strain emc/ , kenneth rock for dc . cells, roberto cattaneo for providing the pb(+)mvvac construct, and urs schneider for providing the polii rescue system originally used to generate and to rescue recombinant mv vectors. the authors would further like to thank bakhos tannous for providing pcscw gluc-ires-gfp. moreover, the authors would like to thank veronika von messling for critically reading the manuscript. key: cord- -e rpryrh authors: tomasello, elena; pollet, emeline; vu manh, thien-phong; uzé, gilles; dalod, marc title: harnessing mechanistic knowledge on beneficial versus deleterious ifn-i effects to design innovative immunotherapies targeting cytokine activity to specific cell types date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: e rpryrh type i interferons (ifn-i) were identified over years ago as cytokines critical for host defense against viral infections. ifn-i promote anti-viral defense through two main mechanisms. first, ifn-i directly reinforce or induce de novo in potentially all cells the expression of effector molecules of intrinsic anti-viral immunity. second, ifn-i orchestrate innate and adaptive anti-viral immunity. however, ifn-i responses can be deleterious for the host in a number of circumstances, including secondary bacterial or fungal infections, several autoimmune diseases, and, paradoxically, certain chronic viral infections. we will review the proposed nature of protective versus deleterious ifn-i responses in selected diseases. emphasis will be put on the potentially deleterious functions of ifn-i in human immunodeficiency virus type (hiv- ) infection, and on the respective roles of ifn-i and ifn-iii in promoting resolution of hepatitis c virus (hcv) infection. we will then discuss how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the subtypes and dose of ifn-i produced, (ii) the cell types affected by ifn-i, and (iii) the source and timing of ifn-i production. finally, we will speculate how integration of this knowledge combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments in patients. specifically, we will discuss how induction or blockade of specific ifn-i responses in targeted cell types could promote the beneficial functions of ifn-i and/or dampen their deleterious effects, in a manner adapted to each disease. type i interferons (ifn-i) were the first cytokines discovered, over years ago, based on their potent anti-viral effects ( , ) . ifn-i play a crucial, non-redundant role in vertebrate anti-viral defenses ( ) ( ) ( ) . ifn-i also mediate protective effects in other physiopathological contexts, including cancer ( ) and multiple sclerosis (ms) ( ) . on the contrary, ifn-i responses can be deleterious in a number of circumstances, including bacterial or fungal infections ( - ), many autoimmune diseases ( ) , and, paradoxically, certain chronic viral infections ( ) ( ) ( ) . it is only recently that an integrated picture has emerged of the cellular and molecular mechanisms regulating the production of ifn-i and underlying their functions. much knowledge was gained recently on another class of potent innate anti-viral interferons, the lambda, or type iii ifns (ifn-iii). we will review knowledge on ifn-i/iii (ifns) and discuss how it could be harnessed to develop innovative therapeutic strategies aimed at surgically tuning ifn activity toward protective responses in a manner adapted to each disease. we will focus on ifn-α/β/λ because they are the best characterized ifns and already used therapeutically. recent reviews are covering information on other ifn-i subsets including ifn-ε, which is produced at mucosal sites and promotes local anti-viral defenses ( , ) . dendritic cells (dcs) are rare heterogeneous mononuclear phagocytes functionally characterized by their unique efficacy for antigen-specific activation of naïve t lymphocytes. dcs are sentinel cells of the immune system, able to sense and integrate a variety of danger input signals for delivery of output signals instructing the activation and functional polarization of effector immune cells. in mammals, five major dc subsets exist, which differ in their expression of innate immune recognition receptors (i r s) and in their functional specialization: monocyte-derived dcs (modcs), langerhans cells, cd b + dcs, xcr + dcs, and plasmacytoid dcs (pdcs) ( ) . a recurrent theme of this review will be the intricate relationships between ifns and dcs, since these cells can be a major source and/or target of these cytokines under various conditions. www.frontiersin.org the first section will synthesize current knowledge on ifn production and protective anti-viral functions. the i r s and downstream signaling pathways responsible for ifn-i production during viral infection will be listed. the roles of different cell types for this function will be discussed. the two main mechanisms through which ifn-i promote anti-viral defense will be reviewed, succinctly for direct anti-viral effects and in greater details for immunoregulatory functions. the second section will focus on the detrimental functions of ifn-i. selected diseases will be discussed to illustrate how different, and sometimes opposite, processes underlie deleterious ifn-i responses depending on the physiopathological contexts. ifn-i induction of unbridled inflammatory responses causing lethal tissue damage will be discussed as a major pathological mechanism during bacterial encounters secondary to influenza infection or in some autoimmune diseases. inappropriate functional polarization of immune responses by ifn-i will be discussed as one potential cause for enhanced susceptibility to bacterial or fungal infections. the complex and disputed role of ifn-i in chronic viral infections will be reviewed, with emphasis on the physiopathology of the infections by human immunodeficiency virus type (hiv- ) and human hepatitis c virus (hcv), with an outlook for the development of novel immunotherapeutic strategies to combine with anti-viral drugs. the third section will recapitulate how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the source and timing of ifn-i production, (ii) the cell types affected by ifn-i, and (iii) the signaling pathways activated by ifn-i. in the last section, we will speculate how integration of all the knowledge discussed before combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments, based on induction or blockade of specific ifn-i responses in targeted cell types. this"activity-by-targeting"concept is based on the design of novel "immuno-ifns" consisting in covalent association between a mutated ifn-i with decreased affinity for its receptor and an antibody with high avidity for a molecule specifically expressed on target cell types ( ) . this design ensures lack of activity of the immuno-ifns on all cell types but those targeted, contrary to previous strategies using ifns with close to maximal potency that were still able to mediate strong off-target effects despite their coupling to cell-type specific antibodies and/or their local delivery. type i interferons expression is not detectable under steady state conditions in vivo using classical methods such as gene expression analysis by rt-pcr or protein titration by elisa or bioassays. however, mice deficient for the expression of the alpha chain of the ifn-i receptor (ifnar ) harbor alteration in the ontogeny or functions of various cell types ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . hence, extremely small or localized but functionally relevant quantities of ifn-i must be produced under steady state conditions ( ) . indeed, the existence of steady state responses to ifn-i in various organs in vivo was demonstrated by using reporter mice expressing the firefly luciferase under the control of the promoter of ifnb ( ) or of mx ( ) , a canonical ifn-i-stimulated gene (isg). steady state ifn-i responses are promoted by gut commensals ( ) . early and transiently after many viral infections, large amounts of ifns can be detected, in blood and spleen in the case of systemic infections or locally in the case of confined infections. ifn induction during viral infections results from the detection of specific danger signals by specialized i r s. this includes the detection of pathogen-associated molecular patterns as well as the sensing of stress signals or damage-associated molecular patterns ( , ) . based on the nature and intracellular location of the danger signals that induce the production of the cytokines, the cellular sources of ifns during viral infection can be classified in two main groups. infected cells often contribute to ifn production as a response to their sensing of endogenous viral replication, or consecutive to the metabolic stress induced during massive translation of viral structural proteins, or as a result of plasma membrane perturbations upon viral entry. specific subsets of uninfected cells can also significantly contribute to ifn production upon engulfment of material containing viral-derived nucleotide sequences and sensing of these molecules in endosomes by specific i r s. all sensing pathways leading to ifn induction converge on the activation of interferon response factors or (irf / ), which are the master transcription factors inducing ifn genes. most cell types constitutively express irf but not irf or only at low levels. irf expression requires ifn-i stimulation. ifn-β can directly be induced by irf . all but one of the ifn-α subtypes require irf for their induction. hence, ifn-β secretion promotes its own production and that of ifn-α in an autocrine manner ( , ) . this positive feedback loop strongly amplifies ifn production during viral infections, promoting fast and widespread induction of cell-intrinsic anti-viral defenses in uninfected cells to prevent virus dissemination. other feedback loops tightly regulate ifn-i production positively or negatively. this section reviews different mechanisms controlling ifn production and how they could play different roles in host/virus interactions. different innate immune recognition receptors are involved in sensing various types of viral nucleic acids in distinct categories of cells during viral infections, which may promote different types of anti-viral defenses. for each selected sensor shown, the types of viral nucleic acids recognized and the downstream signaling cascade induced are represented in a simplified, schematic manner. the potential specific role of each cell type in anti-viral defenses is also indicated at the bottom of each panel. (a) potentially all types of infected cells can detect endogenous viral replication through cytosolic sensors triggering their local production of ifn-β/λ to control viral replication in an autocrine and paracrine fashion in infected tissues. (b) uninfected xcr + dcs selectively produce high levels of ifn-λ and ifn-β upon engulfment of materials containing dsrna and the consecutive triggering of tlr in endosomes. the receptor of ifn-λ is mostly expressed by epithelial cells. hence, xcr + dcs might be involved in inducing local ifn responses in virally infected epithelial tissues. since xcr + dcs are especially efficient at producing ifn-iii upon hcv stimulation, they might contribute to local or systemic ifn production during infection with this virus, to promote ifn-λ-mediated protection of hepatocytes. uninfected xcr + dcs and other uninfected cells may produce some ifn-β upon engulfment of materials containing ssrna and the consecutive triggering of tlr in endosomes. the contribution of this pathway to anti-viral defense is not well understood yet, in part because mouse tlr is deficient for this function. (c) uninfected pdcs selectively produce high levels of all subsets of ifns upon engulfment of materials containing ssrna or cpg dna and the consecutive triggering of tlr / in endosomes. however, pdcs seem to be activated for this function only in lymphoid tissues. hence, pdc might contribute to systemic ifn production during blood-borne viral infections or as a failsafe mechanism activated upon abnormal widespread dissemination of a viral infection once it has escaped local confinement at its portal of entry. cm, cell membrane; nm, nuclear membrane. particular nucleotide sequences or tertiary structures, their signaling pathways and their physiological significance have been recently reviewed ( , ) . cytosolic rna sensors encompass dexd/h helicases among which the retinoic-acid-inducible gene (rig)-i-like receptors (rlrs) have been the most studied, namely rig-i and melanoma differentiation associated gene (mda ). rig-i recognizes rna with a -ppp or -pp ( ) (uncapped) moiety, or double-stranded rna (dsrna), both structures being present in viral, but not in cytosolic eukaryotic, rna molecules. mda might specifically recognize long dsrna fragments. both rig-i and mda contain a dexd/h box-containing rna helicase domain, and caspase recruitment domains (card / ), which bind to mitochondrial anti-viral signaling protein (mavs). rna/rlr/sting molecular complexes initiate a signaling cascade leading to irf / -dependent induction of ifns (figure ). other dexd/h helicases can promote ifn-i production in dcs, www.frontiersin.org although their physiological roles for in vivo immune defenses against viral infections remain to be established ( ) . cytosolic dna sensors able to induce ifn-i (mostly ifn-β) and ifn-iii encompass molecules belonging to different protein families, including dexd/h helicases, the inflammasome component ifn-γ-inducible protein (ifi ) , the z-dna binding protein (zbp ), and the cyclic gmp-amp (cgamp) synthase (cgas) ( , ) . most of the cytosolic dna sensors activate sting and lead to irf / -and nfκb-dependent induction of ifn-β and ifn-iii. many cell types express zbp and are able to produce ifn-i upon triggering of this molecule, including macrophages, dcs, and fibroblasts following an hsv- infection ( , ) . upon dna binding, cgas catalyzes the production of cgamp. cgas is critical for the detection of lentiviruses including hiv- / ( , ) and can contribute to sensing of, and protection against, other rna viruses, including in vivo in mice ( ) . cgamp also acts as a secreted second messenger signal alerting uninfected cells to directly induce their expression of intrinsic immune anti-viral defenses. the cgas/sting/irf signaling cascade and the irf transcription factor are "master" inducers of cell-intrinsic immunity able to control the replication of most dna and some rna viruses at least in part independently of ifns ( ) . infected cells become a factory for production of viral particles. hijacking of the translation apparatus of the host cell for massive production of viral structural proteins leads to an overload of the capacity of the er for correct folding of newly synthesized proteins. er overload induces a homeostatic response of the cell, the unfolded protein response (upr). upr aims at restoring normal er functions by inhibiting translation. upr activation in infected cells contributes to prevent viral replication, including through inhibition of the production of viral proteins, promotion of ifn-i production, and induction of cell suicide ( ) . toll-like receptors (tlrs) are among the first and best characterized i r s. tlrs are transmembrane proteins with a leucine-rich repeat extracellular domain involved in ligand recognition and an intracellular toll/interleukin- receptor domain essential for signaling ( ) . among the nine tlrs conserved between mouse and human, tlr , tlr , tlr , and tlr are located in endosomes where they can detect the abnormal presence of nucleic acids such as occurs upon endocytosis of virions or of virally infected cell material. tlr recognizes dsrna, tlr / ssrna, and tlr dna sequences containing unmethylated cytidinephosphate-guanosine (cpg) motifs. tlr fine specificity and signaling pathways have been reviewed recently ( ) and are summarized in figure . we will discuss the expression patterns and functions of endosomal tlrs with regards to ifn production in uninfected specialized immune cell types, pdcs and xcr + dcs. plasmacytoid dcs uniquely produce very large amounts of ifns in response to in vitro stimulation with many viruses, without being infected ( ) . ifn-i mrnas represent up to % of all mrnas in pdcs at the peak of their activation ( ) . in vitro, upon exposure to influenza virus, herpes virus type , cytomegaloviruses, or vesicular stomatitis virus, individual pdcs produce - times more ifns than total pbmcs, monocytes, modcs, cdcs, neutrophils, and fibroblasts ( ) ( ) ( ) ( ) ( ) ( ) . however, in vitro, high molarity infection of cdcs with certain viruses unable to inhibit ifn-i production in their target cells can also induce massive ifn-β secretion ( ) . pdcs produce high levels of all subtypes of ifns, contrary to many other cell types including infected cells, which often preferentially produce ifn-β ( , ) . in vivo, pdc depletion during systemic viral infections leads to over % decrease of ifn-i production, while the total number of pdcs producing ifn-i (< , in one mouse) is much lower than the total number of infected cells ( ) ( ) ( ) ( ) ( ) ( ) . this shows that in vivo also individual activated pdcs produce much more ifn-i/iii than most other cell types, including virus-infected cells. the professional ifn-producing function of pdcs largely results from their high constitutive and selective expression of irf , tlr , and tlr (figure ) . these molecules are pre-associated in readyto-signal complexes located in specialized endosomes specific to pdcs ( , ) . pdcs must also be equipped for efficient sensing and up-take of virions and virus-infected cells. the corresponding cell surface i r s remain to be identified. selective expression of tlr in xcr + dc endows them with a unique ability to produce very high amounts of ifn-β and ifn-iii upon stimulation with dsrna or hcv irrespective of their own infection. xcr + dcs are very potent for antigenspecific activation of cd + t cells, in particular through crosspresentation of exogenous antigens that they have captured from other cells and processed for association with class i major complex histocompatibility (mhc-i) molecules ( ) . in mice, xcr + dcs are crucial for the initiation of protective adaptive immune responses against tumors and a variety of viruses ( ) . mouse and human xcr + dcs constitutively and selectively express high levels of tlr (figure ) . they produce large amounts of ifn-iii and ifn-β upon stimulation with a synthetic mimetic of dsrna, polyinosinic:polycytidylic acid (polyi:c) ( , ) . human xcr + dcs uniquely respond to stimulation with hcv by producing large amounts of ifn-iii in a tlr -dependent manner ( , ) , irrespective of their own infection. positive feedback loops. in addition to irf induction, other positive feedback mechanisms exist to amplify the production of ifns rapidly after initiation of a viral infection as illustrated by the following selected examples. ifns induce the expression of many cytosolic rna/dna sensors and of tlr . this broadens the spectrum of host's cell types able to detect endogenous viral replication for ifn induction. induction of oasl by ifns in human cells enforces rig-i signaling, counteracting viral immune frontiers in immunology | microbial immunology evasion genes interfering with this sensing pathway ( ) . the ifninducible ribonuclease l (rnasel) generates viral and cellular rna degradation products, which engage rlrs for amplification of ifn production ( , ) . the ifn-inducible protein kinase r (pkr) stabilizes ifn-i mrna ( ) . to prevent unbridled responses deleterious for the host, ifn activity must be tightly controlled including during viral infections. several negative feedback loops exist to terminate ifn production, after anti-viral defenses have been activated. the isg ubiquitin specific peptidase (usp ) binds to ifnar , preventing it from recruiting signal transducer and activator of transcription (stat ). ifns induce the expression of tam receptor tyrosine kinases in dcs, monocytes, and macrophages. tam receptors associate and signal in part through ifnar . they activate the suppressors of cytokine signaling- / (socs- / ). socs inhibit tlr and rlr signaling, thereby terminating ifn production ( ) . tam receptor ligands, gas and pros, bind phosphatidylserine on dying cells and are produced by activated dcs and monocytes/macrophages. thus, ifn induction of tam inhibitory receptors on uninfected phagocytic immune cells could limit their propensity to produce the cytokines upon engulfment of dying virally infected cells. ifns induce tetherin on most cell types. pdcs express a receptor for tetherin, leukocyte immunoglobulin-like receptor, subfamily a (with tm domain), member (lilra ). lilra triggering on pdcs inhibits their production of ifn-i. hence, through lilra engagement by tetherin, pdcs can monitor their efficacy at inducing an antiviral gene expression program in neighboring cells through ifns, and timely terminate their ifn production. how positive and negative feedback loops integrate in time and space to promote optimal kinetics and intensity of ifn production in order to efficiently control viral infection without causing severe immunopathology is not completely understood. positive feedback loops may occur very rapidly after initiation of viral infection to allow rapid secretion of high levels of the cytokines for fast and strong induction of anti-viral cell-intrinsic immunity. negative feedback loops occur likely later to terminate the response and thus avoid chronicity of cytokine production and its ensuing deleterious effects. pdcs do not constitute the major source of ifn production upon local infections by several viruses in the lung or in the female reproductive tract. pdcs are dispensable for resistance against these infections ( , , ) . during pulmonary infection by newcastle disease virus (ndv), ifn-i are produced locally in the lungs mainly by infected alveolar macrophages. lung pdcs do not express the cytokines ( ) . selective depletion of lung alveolar macrophages leads to systemic dissemination of ndv, and to a strong activation of pdcs for ifn-i production specifically in the spleen. even in the case of systemic viral infections such as caused by intravenous injection of ndv or intraperitoneal injection of mouse cytomegalovirus (mcmv), pdc ifn production is confined to the spleen. it is not detected in other organs even those with high viral replication ( , ) . hence, splenic pdcs are especially prone to high level ifn production upon systemic acute viral infections. pdcs located in non-lymphoid organs, in particular mucosal barrier tissues, appear to be inhibited for ifn production. thus, ifn production by infected cells serves as first line of defense to block virus replication at its portal of entry in the body. ifn production by uninfected pdcs might constitute a failsafe mechanism mainly activated in the spleen when viral infection gets systemic ( ) . under these conditions, to promote health over disease, the benefits for the host of producing high circulating levels of ifns in order to induce widespread cell-intrinsic anti-viral defenses might prevail over the deleterious effects that this could cause on certain cell types or tissues. indeed, pdcs are required for protection against hsv- and hsv- in mice only in systemic but not local infections ( ) . this observation is consistent with the crucial role of pdcs for protection of mice against systemic infection by mouse hepatitis virus (mhv), a fast replicating coronavirus ( ) . conflicting results have been obtained on the role of pdcs during intranasal influenza infection ( , ( ) ( ) ( ) . a possible explanation is that pdc ifn production contributes to resistance to highly pathogenic influenza strains that might systemically spread from the lung early after infection, even if at low levels. another intriguing observation is that ifns are critical for host resistance to mcmv and that pdcs are the major source of ifns in this infection model but are dispensable for virus control ( ) . studies are ongoing to understand this apparent paradox. patients bearing genetic mutations disrupting endosomal tlr signaling do not appear to suffer from life-threatening viral infections ( , ) , contrary to patients impaired in ifnar signaling ( , ) . a notable exception is the specific susceptibility to severe herpes virus encephalitis in individuals' deficient for tlr signaling ( , ) . however, contrary to extracellular tlr, endosomal tlr have evolved under strong purifying selection in human beings ( ) . hence, while pdcs and endosomal tlr might have been required for protection of our species against viral infections in the past, this appears not to be the case anymore perhaps due to improved social, hygiene, and health care in modern society ( ) . attesting to the importance of ifns for anti-viral defense in vertebrates, many mammalian viruses encode immune evasion genes specifically inhibiting the production of ifns in infected cells ( , ) . pdcs or xcr + dcs might be essential for ifndependent host protection against these viruses, because they are spared from the intracellular functions of viral immune evasion genes ( ) . to the best of our knowledge, mcmv does not encode for immune evasion genes inhibiting ifn production. however, mcmv manipulates ifn-i responses through specific inhibition of stat functions in infected cells. thus, pdcs might be dispensable for resistance against systemic mcmv infection due to sufficient levels of ifn production by infected cells locally in all infected tissues. hepatocyte responses to ifn-iii appear to play a www.frontiersin.org critical role in human resistance to hcv. in infected hepatocytes, hcv induces the expression of cellular micrornas binding to ifn-iii mrna and leading to its degradation. uninfected xcr + dcs produce high levels of ifn-iii in vitro upon hcv stimulation ( , ) . hence, during acute hcv infection in vivo, xcr + dc may be a strong and early source of ifn-iii not subjected to virus immune evasion strategies, therefore, contributing to protect naturally resistant individuals. in secondary lymphoid organs, a subset of macrophages is critical for the clearance of viruses from the lymph ( ) . these macrophages are located on viral entry routes, near to subcapsular sinuses where the afferent lymph drained from non-lymphoid tissues flows. contrary to other subsets of macrophages, subcapsular sinus macrophages are highly susceptible to viral infection, because they constitutively express only low levels of effector molecules of cell-intrinsic anti-viral immunity and because their responses to ifns are inhibited by their high constitutive expression of usp . subcapsular sinus macrophages rapidly become infected by viruses incoming from the lymph and produce large amounts of ifns. this altruistic suicide prevents virus dissemination to other adjacent cell types and promotes the induction of innate and adaptive anti-viral immunity ( ) . upon instruction by ifns, cells express a wide variety of viral restriction factors, whose combined action blocks pathogen invasion by interfering with the different stages of viral life cycle (figure a ). this has been extensively reviewed recently ( ) and will only be described succinctly here. virus fusion with host cell membrane can be blocked by cholesterol- hydrolase (ch h) that inhibits sterol biosynthesis. some viruses enter cells by escaping from endosomes/lysosomes, which can be blocked by interferon inducible transmembrane (ifitm) proteins. virus uncoating can be blocked by tripartite motif (trim) proteins, such as trim α, which bind to hiv- capsid thus promoting its degradation, and by myxoma resistance gtpases, mx , and mx , which efficiently trap viral structural proteins at an early stage following virus entry into the cell. mx inhibits a number of viruses, including influenza virus through sequestration of its nucleocapsid. mx associates with host cyclophilin a and hiv- capsid protein. virion assembly can be blocked at transcriptional, translational, and posttranslational levels. the adenosine deaminase acting on rna (adar ) and the apolipoprotein b mrna editing enzyme, catalytic polypeptide-like (apobec) deaminases induce viral rna destabilization and hypermutation ( , ) . the sterile alpha motif and histidine-aspartic domain (hd) containing protein (samhd ) blocks reverse transcription by hydrolyzing dntps ( ) . adar , apobec, and samhd functions have been mainly studied in infections by hiv- and other retroviruses. the , -oligoadenylate synthase (oas) proteins, the ifn-induced proteins with tetratricopeptide repeats (ifit), and pkr inhibit viral and host protein translation by using complementary mechanisms ( ) . the major post-translation modification induced by ifns is the binding of the ubiquitinlike modifier isg to several viral and host proteins, a process called isgylation. most of the known isgylated proteins are targeted for degradation, with few exceptions that are on the contrary stabilized like irf ( ) . finally, the egress and budding of virions of many enveloped viruses can be inhibited by tetherin or by viperin ( ) . many anti-viral isgs have been functionally characterized only recently, largely thanks to large-scale screening approaches. they display a variable degree of viral specificity ( , ) that might inversely relate to the extent of their side effects on host cells ( figure b ). anti-viral effectors acting on a broad spectrum of viruses often target key metabolic pathways that are also crucial for host cell functions. this is the case for the control of cholesterol metabolism by ch h ( ) or of protein translation by pkr, oas, or ifits ( ) . other anti-viral restriction factors such as mx may specifically target one molecule of a very restricted set of viruses with no apparent side effects on host cells. some anti-viral isgs target specific functions critical for only a restricted array of viruses and might similarly exert side effects only on a subset of host cell types. for example, samhd inhibits retrovirus replication through dntp depletion, which might more specifically affect proliferating host cells. hence, the infected host must balance the intensity, breadth, and location of isg induction to circumvent viral replication while preventing life-threatening damages to vital cell types or tissues. one of the mechanisms contributing to this balance is translational control of the expression of isgs, especially those with pro-apoptotic or anti-proliferative functions ( ) . while many anti-viral isgs are transcriptionally activated in most ifn-stimulated cells, their translation can be specifically blocked in uninfected cells by cellular microrna. this inhibition is relieved upon cell infection through negative regulation of the function of the rna-induced silencing complex. hence, ifn stimulation of uninfected cells prepares them for rapid and strong induction of cell-intrinsic anti-viral defenses upon viral infection while avoiding their unnecessary exposure to the toxic effects of certain isgs. further knowledge on the functions and the dynamic regulation of isgs is essential to develop novel therapeutic strategies against viral infections aiming at modulating ifn responses to promote their protective anti-viral cell-intrinsic functions over their deleterious toxic effects. a better understanding of the immunoregulatory effects of ifns will also help. type i interferon can modulate the functions of a broad spectrum of immune cells ( figure a ). we will review this knowledge, focusing on the functions of dcs, nk cells, t cells, and b cells, since they are involved in the control of most viral infections. we will discuss the hypothesis that dcs play a central role in ifn-i orchestration of innate and adaptive immunity for the induction of optimal anti-viral defenses (figure ) . during viral infections and cancer immunosurveillance, ifn-i constitute one of the most important input signal acting on dcs to promote their delivery of appropriate output signals to t cells, b cells, and nk cells for protective immunity ( figure a ). dcs deliver three types of signals to activate and functionally polarize t cells. signal is the triggering of the t cell receptor by viral peptide-mhc complexes. signal is the triggering of activating t cell co-stimulation receptors such as cd or cd by the cd / and cd co-stimulation molecules expressed on dcs. signal corresponds to cytokines, which can promote t cell proliferation and acquisition of specific effector functions. under steady state conditions, most dcs are in an immature state characterized by low level expression of mhc-ii (signal ) and co-stimulation molecules (signal ) and by the lack of production of t cell-activating www.frontiersin.org cytokines (signal ). upon activation, including early after viral infections in vivo, dcs up-regulate their expression of signal and activating signal and secrete t cell-activating cytokines. this process is called dc maturation. gene expression profiling of dcs stimulated by microbial stimuli identified a core set of genes upregulated in mature dcs irrespective of the stimulus they receive, irrespective of the subset they belong to, and conserved across evolution ( ) . most of these genes are induced during dc maturation in part through cell-intrinsic ifn-i signaling ( ) . consistently, cell-intrinsic ifnar signaling in dcs is required in many circumstances for the induction of protective immunity, including efficient cd t cell responses during viral infection or tumor development ( ) ( ) ( ) , th responses upon polyi:c injection independently of il- or ifn-γ effects ( , ) , as well as follicular helper t cell and humoral responses ( , ) . mechanistically, ifn-i promote dc immunogenicity for efficient t cell activation through a variety of effects ( figure b) . it drives dc up-regulation of signal in vivo during viral infections ( ) and boosts their capacity to cross-present antigens for increased delivery of signal to cd t cells ( ) ( ) ( ) . it shapes their delivery of activating signal , in particular inducing il- and promoting or inhibiting il- depending on experimental conditions ( , ) . finally, it is necessary to induce their metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, which fuels the increased needs in energy and the expansion of the intracellular organelles required for the production and proper intracellular routing of the signal and proteins ( , ). selective inactivation of ifnar on cdcs compromises mouse resistance to mcmv and mhv infections ( , ) . in contrast, ifnar expression is not required on nk cells for protection against mcmv and on pdcs, t cells, and b cells for early control of mhv replication ( , ) . although cell-intrinsic ifn-i signaling in nk cells can promote their activation ( ) (figure a) , ifn-i-induced il- trans-presentation by dcs plays a more prominent role for this function in many conditions including in vivo during mcmv infection ( , ) ( figure c) . cell-intrinsic ifn-i signaling in cd t cells ( ), cd t cells ( , ) , and b cells ( ) can also contribute to their efficient activation and functional polarization (figure ). this depends on experimental settings. cd t cell-intrinsic ifn-i responses are crucial for mounting efficient cytotoxic cd t cell responses against lcmv but are less critical against vaccinia virus and vesicular stomatitis virus ( , , ) . mechanistically, cell-intrinsic ifn-i signaling in cd t cells can promote their survival during their antigen-induced proliferation ( ) . cell-intrinsic signaling in dcs and cd t cells may act in a synergistic manner. indeed, conditional inactivation of ifn-i responsiveness was required to occur simultaneously in both of these two cell types to dramatically affect cd t cell expansion upon vaccination with a modified ankara vaccinia virus ( ) . in summary, ifn-i generally play a crucial, beneficial, role in immune defenses against viral infections, both through the induction of cell-intrinsic anti-viral defenses and through the orchestration of innate and adaptive immunity. however, if these responses are not properly regulated, they can contribute to diseases as we will now discuss. a frequent side effect of ifn-i treatment against cancer or chronic viral infections is the induction of autoimmune reactions. consistently, isg expression is a hallmark of many spontaneous systemic or tissue-specific autoimmune diseases, including systemic lupus erythematosous (sle), sjogren's syndrome, psoriasis, and other skin disorders ( ) . the dysregulation of ifn-i responses observed in patients with these autoimmune diseases likely results from both genetic and environmental factors. genome-wide association studies show that polymorphisms in genes involved in ifn-i responses strongly correlate with increased susceptibility to many autoimmune diseases ( ) . diverse environmental factors can also contribute to the onset of autoimmune diseases. microbial infections often precede first clinical manifestations of autoimmune diseases. whether infections ( ) and/or alterations in the commensal microbiota of the affected barrier tissues ( , ) are the cause or rather the consequence of autoimmunity is still matter of debate. infection-or dysbiosis-induced tissue damages and unbridled ifn-i responses can contribute to initiate autoimmune reactions. gender is another prominent factor affecting susceptibility to autoimmune diseases. women are more prone to autoimmunity, which may result from endocrine regulation of ifn-i responses. pdc ifn-i production is enhanced in human and mouse females, due at least in part to cell-intrinsic enhancement of tlr / responses by the female hormone estradiol ( ). in autoimmune diseases, different mechanisms could operate to initiate the dysregulation of immune responses leading to a vicious circle of reciprocal activation between innate ifn-i responses and adaptive self-reactive lymphocyte responses (figure ) . adaptive immune cells are educated to spare "self." this occurs through negative selection of potentially autoimmune b and t cells during their development in the bone marrow or thymus, respectively, a process called central tolerance. self-reactive b or t cells that have escaped this pruning can be either deleted or functionally inactivated once they have egressed in secondary lymphoid organs or non-lymphoid tissues, a process called peripheral tolerance. in some individuals, polymorphisms in genes involved in the promotion of central or peripheral tolerance lead to a higher number, diversity, and/or responsiveness of self-reactive lymphocytes in the periphery, in particular of b cells secreting anti-dna or anti-rnp antibodies ( , ) . mammalian dna or rna are poor inducers of pdc ifn-i induction under normal conditions. however, pre-existing anti-dna or anti-rnp autoantibodies can break this innate tolerance of pdc. indeed, antibodies binding to self nucleic acids can protect them from degradation and compact them into nanoparticles that are very effective for the induction of ifn-i in pdc (figure ) . dna-containing immune complexes (ics) are frequently found in the serum of sle patients (sle-ics) and can activate pdc ifn-i production ( ) . in turn, pdc ifn-i activate cdcs, monocytes ( ) , and b cells, leading to a vicious circle of reciprocal activation between dcs and frontiers in immunology | microbial immunology figure | a simplified model of the deleterious role of ifn-i in several autoimmune diseases. when exposed to different kinds of injuries (microbial infection, commensal microbiota dysbiosis, chemical or physical insults), healthy tissues can undergo cell damage and death. these events induce the release of apoptotic bodies encompassing self rna or dna. neutrophil recruitment and activation in inflamed tissues can also constitute a potent source of self nucleic acids, through the release of neutrophil extracellular traps (net). self rna or dna can associate with cationic peptides (e.g., ll ) as shown in psoriatic patients or with inflammatory molecules (e.g., high mobility group box , hmgb ) to generate nanoparticles that are extremely efficient for ifn-i production by pdc and eventually other cell types. pdc can also be efficiently activated for ifn-i production by immune complexes (ics) generated by the association between self nucleic acids and auto-antibodies as frequently found in the serum of systemic lupus erythematosus patients. ifn-i promote the differentiation and/or the maturation of antigen-presenting cells, in particular different subsets of dc. activated dc can then present self-antigens for activation of auto-reactive t cd + cells, including follicular helper lymphocytes, which in turn activate auto-reactive b cells for auto-antibody secretion, leading to a vicious circle of reciprocal activation between innate and auto-reactive adaptive immune cells. idc, immature dc; mdc, mature dc; mo-dc, monocyte-derived dc. see main text for further details. self-reactive lymphocytes and to the exacerbation of autoimmune responses (figure ) . certain infections or dysbiosis of the commensal microbiota of the affected barrier tissues could promote chronic production of host amphiphatic peptides able to combine with eukaryotic dna or rna, likely released from dying cells, thus forming pdc-activating nanoparticles. indeed, in psoriatic skin, both a high expression of ll and a massive infiltration of pdcs is observed ( ) (figure ) . hence, to treat many autoimmune diseases, novel therapeutic strategies could be designed to target dysregulated pdc ifn-i production or b cell activation by ifn-i. one of the most common complications of primary infections by many respiratory viruses, in particular influenza virus, is a lifethreatening pneumonia due to secondary pulmonary infections by bacteria, such as streptococcus pneumoniae, staphylococcus aureus, or haemophilus influenza ( , ) . these pathologies affect especially infants, elderly, and immunocompromised patients. retrospective studies indicate that secondary bacterial pneumonia was highly recurrent in lung tissues isolated from patients who died during last century influenza pandemics, independently of antibiotic availability ( , ) . influenza virus induces high ifn-i responses in human beings and mice. in both hosts, secondary bacterial infections are lethal only when they occur in a limited time window following primary viral infection ( - days), around the peak of ifn-i responses, before complete virus clearance. mouse models of viral/bacterial coinfections are being used to dissect disease mechanisms ( ) . ifnar -deficient mice appear more resistant to secondary pulmonary bacterial infections, showing that ifn-i responsiveness contributes to disease ( ) . similarly, after lymphochoriomeningitis virus (lcmv) infection, wild-type but not ifnar -deficient mice are more susceptible to lpsinduced septic shock ( ) . several mechanisms may contribute to the detrimental role of ifn-i in secondary bacterial infections ( figure ) . early during viral infection, ifn-i decrease the host ability to control bacterial replication, by dominantly polarizing immune responses toward anti-viral functions, simultaneously inhibiting the development of the types of immune responses required for protection against most bacterial infections. ifn-i can inhibit the production of chemokines required for the recruitment to the respiratory tract of antibacterial effector innate immune cells, in particular neutrophils or monocytes/macrophages ( , ) (figure ) . depending on the experimental models used, ifn-i can on the contrary induce a ccr -dependant recruitment of classical monocytes ( ) . in infected tissue, ifn-i might skew the functional polarization of resident or infiltrating monocytic cells toward immunosuppression, because it does limit their antibacterial functions by inhibiting their il- production ( ) ( ) ( ) while it might promote their production of il- and nitric oxygen intermediates. the exact nature of infiltrating monocytic cells is not clear and could correspond to activated classical monocytes, modcs, monocyte-derived macrophages, or myeloidderived suppressor cells (mdscs). the boundaries between these putatively different cell types are currently ill-defined ( ) . these cells could fuel local replication of monocyte/macrophage-tropic bacteria ( ) , be immunosuppressive ( ) or contribute to local immunopathology ( ) . the role of ifn-i on monocytes/macrophages is complex and will require further investigations to determine when it is protective versus deleterious and what the underlying mechanisms are. depending on the context, ifn-i can either promote or inhibit the induction of th cytokines such as il- and ifn-γ, and myeloid cell responses to ifn-γ ( , ( ) ( ) ( ) . ifn-i can also polarize cd t cell responses toward th at the expense of th , while the th -type cytokines il- a and il- are required for host defense against pulmonary www.frontiersin.org bacteria by inducing the production of anti-microbial peptides and of tissue repair molecules ( figure ) ( ) ( ) ( ) . ifn-i may not only affect host resistance to bacterial infection, but also host tolerance, i.e., the ability of the host to tolerate a given burden of pathogen without undergoing excessive tissue damages ( , ) . hence, to counter ifn-i deleterious effects during secondary bacterial infections, it will be important to better delineate the respective contribution of lung tissue tolerance modulation and of immune-mediated resistance weakening. another well documented example of deleterious effects of ifn-i due to their inappropriate functional polarization of immune responses is the enhanced susceptibility to fungal infections of patients with genetically determined hyperactive ifn-i responses, as exemplified in the hereditary disease chronic mucocutaneous candidiadis (cmc) (figure ) ( ) . patients with cmc have a significant deficit in th cd t cells, at least in part as a consequence of altered responsiveness to il- or il- . several stat mutations were identified in patients with autosomal dominant cmc. gain-of-function stat mutations were found to hard wire cd t cell responses to cytokines toward stat signaling, compromising their stat -dependent ability to produce il- upon il- or il- stimulation. this was associated to induction of a global ifn-i transcriptomic signature in blood ( ) . deleterious ifn-i effects on immunity to candida might not only occur in cmc patients but also in other types of individuals upon secondary fungal infections occurring shortly after a primary viral infection, likewise to the situation discussed above for secondary bacterial infections. indeed, polyic induced ifn-i abrogate innate immunity to systemic candidiasis in mice ( ) , and ifnar-deficient mice can be more resistant to candida infection under certain experimental settings ( ) . however, the role of ifn-i in the modulation of the ability of immunocompetent hosts to control fungal infection is disputed ( , ) . the inhibition of th responses by ifn-i could be protective in at least one important human pathology, ms (figure ) . ms represents a striking exception to the previously discussed detrimental role of ifn-i in autoimmune diseases. indeed, a large proportion of ms patients have low serum ifn-i activity and low isg levels. these ms patients present a significant reduction of ms relapse upon ifn-β administration ( ) . the underlying mechanisms are not yet completely unraveled. however, in the experimental autoimmune encephalitis mouse model of ms, th responses bear a major contribution to nervous system damages and are inhibited by the il- and il- induced upon ifn-i administration ( ) . in summary, ifn-i responses can be deleterious in autoimmunity by promoting a vicious circle of reciprocal activation between innate immune cells and auto-reactive cd t or b lymphocytes. ifn-i responses can also be deleterious upon secondary bacterial or fungal infections in the lung or the kidneys occurring shortly after a primary viral infection, by compromising the recruitment of anti-microbial innate effector cells and/or by preventing the proper functional polarization of immune responses. we will now discuss how ifn-i responses can also compromise host immune defenses against certain viruses and promote chronic infections. different lcmv strains such as armstrong and clone- (cl ), respectively, lead to acute versus chronic infections in mice. a hallmark of chronic lcmv infection is the loss of the proliferative potential and effector functions of anti-viral cd t cells, a process called exhaustion. exhausted cd t cells are characterized by a high expression of the inhibitory receptors pd- , ctla , and lag- ( ) . in vivo blockade of these inhibitory receptors can reverse t cell exhaustion and allow resolution of the chronic infection ( ) . ifn-i and isgs are induced early after infection with all strains of lcmv, albeit to lower levels with those leading to chronic infection. this early ifn-i production is critical to limit viral replication ( ). in models of acute infection, ifn-i responses rapidly return to normal, undetectable, levels, before viral replication is completely controlled. in contrast, isg induction is maintained in chronic infection, including the expression of pd- ligands on apcs and of the immunosuppressive il- cytokine, consistent with a prolonged expression of ifn-i albeit at low levels ( , ) . in vivo neutralization of ifn-i by antibody administration promoted resolution of chronic lcmv cl infection, allowing the frontiers in immunology | microbial immunology restoration of functional anti-viral cd t cell responses at least in part through cd t cell-and ifn-γ-dependent mechanisms ( , ) . during persistent lcmv cl infection, chronic low level ifn-i production polarizes cd t cell responses toward t follicular helper (tfh) rather than th functions. thus, chronic ifn-i responses promote enhanced anti-viral b cell responses but facilitate cd t cell exhaustion due to deficient cd t cell help, therefore contributing to host failure to prevent chronic infection ( ) . strikingly, establishment of chronic infection by lcmv cl could also be prevented by early administration of two shots of a high dose of exogenous ifn-i, at days and post-lcmv inoculation. this treatment allowed viral clearance by rescuing anti-viral cd t cell from exhaustion ( ) . altogether, these studies show that the timing and duration of ifn-i production during viral infections is critical in determining how this response will impact the balance between the virus and the host. an early and robust but transient production of ifn-i promotes strong induction of cell-intrinsic viral restriction mechanisms as well as adequate polarization of adaptive anti-viral immune responses, which combined effects lead to viral clearance. in contrast, if the production of ifn-i is too low and/or too late, both viral replication and low ifn-i responses become chronic, their combined action leading to induction of immunosuppressive effects and to inadequate functional polarization of cd t cells. this results in cd t cell exhaustion and maintenance of chronic infection. chronic viral replication and cd t cell exhaustion is also a hallmark of hiv- infection. we will now discuss the complex and disputed role of ifn-i in this disease. both in hiv- infection and in its most relevant animal model, infection of non-human primates with simian immunodeficiency virus (siv), disease progression after the acute phase of the infection is associated with high and chronic expression of isgs while ifn-i production is inconsistently detected ( ) ( ) ( ) . in contrast, the individuals that do not progress toward disease despite persistent high viral loads show much lower immune activation, in particular low isg expression, after the acute phase of the infection ( ) ( ) ( ) ( ) . hence, chronic low levels of ifn-i are associated to disease progression independently of the level of viral replication. therefore, an outstanding question still open for a better understanding of the physiopathology of hiv- infection is whether chronic ifn-i responses are merely a marker of progression, or whether they are implicated in driving disease development. in addition to mechanisms similar to those uncovered in the mouse model of chronic lcmv infection, during hiv- infection other effects of ifn-i could promote a vicious circle of reciprocal activation between chronic viral replication and sustained, deleterious immune responses (figure ). very early after hiv- infection, in most individuals, ifn-i production might be too weak or too late to induce a combination of cell-intrinsic defense mechanisms and of immune responses efficient enough to prevent later establishment of chronic infection. on the contrary, as demonstrated in the case of the mouse model of lcmv infection, ifn-i responses could favor cd t cell exhaustion, either by direct cell-intrinsic effects on cd t cells (figure , ) or by contributing to deprive them from cd t cell help (figure , ) . several effects of ifn-i might compromise anti-hiv- th responses or more generally contribute to the global depletion of cd t cells. these mechanisms include functional polarization of anti-hiv- cd t cells toward tfh rather than th responses, cxcl production leading to enhance recruitment of memory cd t cells to the sites of viral replication where they fuel chronic viral replication with new hiv- target cells (figure , to ) , direct pro-apoptotic and anti-proliferative effects on cd t cells (figure , ), as well as trail induction on pdcs licensing them for killing cd t cells irrespective of their infection (figure , ) ( , ) . altogether, these mechanisms entertain chronic viral replication and continuous depletion of cd t cells, leading to the dramatically enhanced susceptibility to opportunistic infections (figure , ) characteristic of the acquired immunodeficiency syndrome (aids) (figure , ) . other lines of evidences have been reported to support a deleterious role of pdc activation during hiv- infection. women undergo faster hiv- disease progression than men with similar viral loads, which may result in part from the highest ifn-i production of women's pdcs including in response to hiv- stimulation ( ) . pdc recruitment and activation in the vaginal mucosa of female macaques early after local siv inoculation contribute to attract and activate cd t cells, which can then be infected and promote virus dissemination from its portal of entry ( ) . however, in vivo blockade of pdc ifn-α production by administration of tlr / -antagonistic oligonucleotides early after siv infection of macaques did not decrease t lymphocyte activation, which suggests that additional sources of ifn-i likely contribute to the immune dysfunction observed in siv/hiv- infections. targeting dysregulated ifn-i responses during hiv- infection might represent an interesting adjuvant therapeutic strategy to highly active antiretroviral treatments. administration of ifn-i in the non-pathogenic siv infection model of sooty mangabeys was not sufficient to switch it into a pathogenic model. no cd t cell depletion ensued, no hyperactivation of immune responses were observed. viral loads were even significantly decreased. however, this could be consistent with the positive impact of early and high dose ifn-i administration in chronic lcmv infection ( ) . indeed, during the review process of this manuscript, it was reported that, early during primary siv infection in the pathogenic rhesus macaque model, a high dose injection of ifn-i was protective while neutralization of endogenous ifn-i was deleterious. in contrast, in the same animal model, prolonged ifn-i administration accelerated disease development in the chronic stage of the infection ( ) . in mice with a humanized immune system, pdc depletion strongly decreased isg induction and enhanced viral replication both in the acute and chronic phases of hiv- infection. however, pdc depletion during chronic infection decreased infection-induced t cell apoptosis and increased t cell numbers in lymphoid organs ( ) . these results further emphasize the dual role of ifn-i and pdcs in the physiopathology of hiv- infection. a strong and transient production of ifn-i early after infection benefits the host by lowering the set-point of viral replication during chronic infection. sustained production of low levels of ifn-i during chronic infection contributes to immune dysregulation and cd t cell depletion. further studies will be necessary to examine whether complementing standard-of-use antiretroviral drugs with pdc www.frontiersin.org depletion, ifn-i neutralization, or selective inhibition of t cell responses to ifn-i could yield additional benefits to chemotherapy in non-human primates during chronic siv infection. ifn-i administration has been used for many years to treat another human chronic viral disease, hcv infection. roughly, half of the patients do not show sustained virological responses (svr). the treatment causes severe side effects in many individuals. new chemotherapeutic drugs very potent at blocking hcv replication in vivo have recently become available. hence, whether ifn-i administration still constitutes a viable treatment against chronic hcv infection is being questioned ( , ) . we will now discuss this issue. chronic hcv infection is the main cause of liver cirrhosis and hepatocellular carcinoma. there is currently no vaccine against hcv. the most common therapy for chronic hcv patients is the administration of recombinant pegylated ifn-α (peg-ifn-α) combined with the anti-viral drug ribavirin. however, because of ifnar pleiotropic expression, ifn-α administration induces severe side effects including flu-like syndrome, fever, fatigue, myalgia, and nervous depression ( figure a ) ( ) . moreover, only about half of treated patients harbor svr ( ) . prior-totreatment high hepatic isg expression is a negative predictor of svr upon peg-ifn-α therapy. high isg expression in untreated patients likely results from chronic but low ifn-i production triggered by persistent hcv replication. indeed, hepatocytes from non-responder patients were found to be infected at a greater frequency and to exhibit dampened antiviral and cell death responses ( ) . what the cellular sources of ifn-i production are and why they persist only in non-responder patients still remain to be established. in chronic hcv infection, cytotoxic effector lymphocytes may contribute to the development of hepatocarcinoma by causing low level but sustained hepatocyte death and renewal. in contrast, local production of ifn-γ in the liver by nk and t lymphocytes could promote resistance to disease through non-cytolytic control of viral replication. as discussed previously for lcmv and hiv- , low chronic production of endogenous ifn-i in hcv patients could compromise both innate and adaptive anti-viral immune responses. chronic exposure to ifn-i could dampen the ability of frontiers in immunology | microbial immunology utr of ifnl mrna to promote their degradation. the favorable ifnl allele associated with responsiveness to peg-ifn-α treatment may allow endogenous expression of sufficient levels of ifnl for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes. this process is, however, hampered by the limited expression of the receptor for this cytokine (ifnλr ) in these patients. peg-ifn-α treatment might promote resolution of the infection by inducing ifnλr in these patients, potentiating their response to their endogenous production of ifnl . in the patients that do not respond to peg-ifn-α treatment, endogenous levels of ifnl are insufficient for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes, due to the degradation of the corresponding mrna in infected hepatocytes. in these patient's hepatocytes, however, ifnλr is already expressed to high levels prior to treatment due to their high endogenous ifn-i responses. administration of exogenous ifn-λ might cure these patients. see main text for further details. nk and cd t cells to produce ifn-γ ( , ) and promote cd t cell exhaustion ( ) . it could also induce an antagonist form of cxcl , a chemokine required for recruitment to the liver of anti-viral nk and cd t cell effectors ( ) . it may also polarize monocytes toward immunosuppressive functions ( ) . therefore, better understanding ifn-i effects in hcv infection is critical to improve care of both responders and non-responder patients to peg-ifn-α. for responder patients, the issue is to modify the treatment to favor beneficial antiviral and immunoactivating effects over side effects strongly affecting patient's quality of life (figure a ). this might be achieved by specific delivery of ifn-i to targeted cell types as discussed later. for non-responder www.frontiersin.org patients, the issue is to understand the mechanisms underlying treatment failure to determine whether alternative therapies could be designed (figure a) . genome-wide association studies identified various single nucleotide polymorphisms (snp) in the gene encoding il- b/ifn-λ , one of the ifn-iii, as well in its and non-coding regions ( ) ( ) ( ) ( ) . one snp, called rs , is located kb upstream of the ifnl gene. patients harboring the cc genotype have a favorable prognosis to ifn-i treatment. patients with the tt genotype are at high risk of treatment failure ( , ) . in europeans, the favorable cc genotype is the major, most common, ifnl allele. the unfavorable tt snp is the minor allele. the frequency of these alleles is reversed in africans. the favorable allele allows escape of ifnl mrna from degradation by cellular microrna induced upon hcv infection ( ) . until recently, ifnl genotypes and hepatic isg expression were considered as independent predictors of response to peg-ifn-α treatment in hcv patients ( ) . here, we propose a potential explanation, which integrates both factors in a relatively simple model ( figure b ). our main hypothesis is that efficient control of hcv infection depends on hepatocyte response to ifn-λ rather than ifn-α. this is supported by reports that ifn-λ induces a stronger and more sustained isg expression in hepatocyte cell lines in vitro ( ) , and that polyi:c-induced control of hcv replication in humanized liver in chimeric mice is correlated to the induction of ifn-λ but not ifn-i in human hepatocytes ( ) . responder patients harboring the favorable ifnl allele preventing the degradation of the corresponding rna in infected cells might express significant levels of endogenous ifn-λ , although this is disputed. however, they express only low levels of ifn-λr , which limits ifnλ efficiency ( figure b ) ( ) . how these patients benefit from peg-ifn-α treatment could be that it induces ifn-λr expression on hepatocytes thus boosting endogenous ifn-λ effects ( ) . in contrast, high isg-expressing non-responder patients harboring the unfavorable ifnl allele might not express enough ifn-λ for virus control. however, they do express ifn-λr as a result of their endogenous production of ifn-i. hence, peg-ifn-α might be ineffective in these patients because they already express ifn-λr but fail to produce endogenous ifn-λ due to the degradation of its mrna in infected hepatocytes (figure b) . these patients may be good candidates for peg-ifn-λ therapy, currently undergoing clinical development. since the expression of ifn-λr is mainly restricted to epithelial cells, melanocytes, and hepatocytes, some of the side effects related to ifn-i treatment might be strongly attenuated in peg-ifn-λ therapy. however, as ifn-i are key to induce anti-viral immune responses, it will be critical to determine whether, beside viral clearance, peg-ifn-λ therapy can also induce long-term immune protection against hcv. ifn-i transduce intracellular signals through a single receptor, ifnar, but via a multitude of downstream signaling pathways. the janus activated kinase (jak)/stat pathway was the first to be identified ( ) . ifnar is composed of two distinct subunits, ifnar and ifnar , which are constitutively associated with members of the jak family, tyrosine kinase (tyk ) and jak , respectively ( ) . the binding of ifn-i to their receptor leads to the phosphorylation of jak and tyk , which in turn induce the phosphorylation and activation of the stat proteins ( ) . different stat complexes can form upon triggering of ifnar (figure ) . a transcriptional complex that forms in most conditions of ifn-i stimulation and induces the expression of many molecules of cell-intrinsic anti-viral immunity is interferonstimulated gene factor (isgf ), a heterotrimer composed of pstat , pstat , and irf ( ) (figure ) . following its translocation into the nucleus, isgf binds to isre regulatory sequences in target genes. many molecules playing a key role in the function of innate or adaptive immune leukocytes are also induced by isgf , including cd , cd , or il- in dc, and granzyme b in nk cells. isgf is generally composed of stat phosphorylated on tyr and ser and of stat phosphorylated on tyr . however, alternative isgf complexes have been described in various contexts which could participate to the diversity of ifn-i effects ( ) . the pstat homodimer also plays a prominent role in cell-intrinsic ifn-i-dependent gene induction. it binds ifnγactivated sequences (gas) and controls the expression of many pro-inflammatory molecules ( ) . pstat homodimers can form upon stimulation with either ifn-i or ifn-γ. many gasregulated genes can be induced by either cytokines. depending on cell types, jak signaling downstream of ifnar can lead to the activation of virtually all stat proteins and to their combinatorial association into a variety of complexes with different affinities for specific gas elements ( ) ( ) ( ) (figure ) . this diversity contributes to ifn-i induction of different transcriptional programs in distinct cell types ( ) . stat complex formation depends in part on the relative abundance of stat molecules in the cell ( ) . while stat , stat , stat , and stat can be activated in most cell populations, stat and stat are mainly activated in lymphocytes ( ) . for example, quiescent nk cells express more stat than stat , leading to constitutive association of ifnar to stat in these cells. hence, quiescent nk cells mount pstat homodimer-dependent responses to ifn-i stimulation, including ifn-γ production and t-bet-driven proliferation (figure ) ( , ) . changes in stat levels can also occur upon the differentiation/activation of a given cell type and lead to a shift in its functional response to the cytokines ( ) . upon activation, nk cells decrease their expression of stat and increase that of stat , shifting their ifn-i response from stat -dependent in a quiescent state to stat dependent in pre-activated cells. this translates into opposite ifn-i effects on ifn-γ production and proliferation for quiescent versus pre-activated nk cells ( ) . however, this outcome can be modulated by simultaneous exposure to other cytokines such as il- or il- / . a reverse stat -to-stat shift occurs in dc during their maturation, shifting their functional responses from inhibition to activation of il- production in response to combined stimulations with ifn-i and cd l ( ) . this frontiers in immunology | microbial immunology ifn-i binding to ifnar triggers the phosphorylation of tyk and jak , which in turn phosphorylate a variety of stat proteins. activated stats are able to form complexes, as homo-or hetero-dimers. the heterodimer stat -stat binds to a third partner, ifn-regulatory factor (irf ), in order to form the isgf complex. this complex translocates into the nucleus and binds to specific regulatory sequences, ifn-stimulated response elements (isre), to activate the expression of many interferon-stimulated genes (isgs). in particular, isgf induces most, if not all, of the isgs encoding effector molecules of cell-intrinsic anti-viral defenses such as oas or mx . alternative jak/stat pathways include the formation of stat or stat homodimers, which may drive different functional responses to ifn-i. stat homodimers bind to ifnγ-activated sequences (gas) in the promoter of certain isgs, which may promote inflammatory, anti-proliferative, and pro-apoptotic responses. stat homodimers also bind to gas but promote ifn-γ production and pro-proliferative responses. enables mature dc to efficiently activate cd t cells. other yet unknown mechanisms control the formation of different stat complexes in distinct cell types. the nature and dynamics of the signaling pathways triggered by ifn-α or -β were evaluated in bulk cultures of human blood leukocytes using flow cytometry ( ) or high throughput mass cytometry ( ) . a diversity of phosphorylation patterns of stat / / was observed upon ifn-i stimulation. ifn-α activation induced phosphorylation of stat , stat , and stat in most cell types, peaking at min ( ) . ifn-β-induced stat phosphorylation was found to be poor in b cells as compared to monocytes and t cells ( ) . however, the underlying mechanism remains to be identified since b cells did not express lower amount of ifnar or stat or enhanced levels of the inhibitory socs molecule. the high stat activation in monocytes led to their induction of ifn-i-dependent pro-apoptotic genes while this was not the case in b cells. these results strikingly differ from those obtained in the other study upon ifn-α stimulation, where stat phosphorylation was on the contrary lower in cd + monocytes and was prolonged in b cells and nk cells ( ) . the differences between these two studies might have resulted from the use of different subsets and doses of ifn-i. in any case, both studies consistently reported that cd t cells showed the highest activation of stat . all cd t cells but not all cd t cells activated stat and for a longer time ( ) . ifn-β activation of stat was delayed in cd t cells and b cells as compared to cd t cells and monocytes ( ) . different stat complexes may lead to distinct transcriptional programs linked to different biological functions (figure ) . more systematic studies are needed to understand this complexity. besides changing stat levels between cell types or www.frontiersin.org activation states, the processes controlling differential formation of stat complexes downstream of ifnar triggering remain to be identified. in addition to jak/stat signaling, other pathways can be activated downstream of ifnar, including those involving the phosphatidylinositol -kinase (pi k), mitogen-activated protein kinases (mapk), and the crk adaptor molecules ( , ) . this leads to the activation of other transcription factors such as irf, nf-κb, or pu. , which contribute to orchestrate cell responses to the cytokines by regulating both distinct and overlapping sets of genes as compared to stat ( , ) . in summary, ifnar signals through a remarkable diversity of pathways, including but not limited to diverse combinations and kinetics of stat phosphorylations. this explains at least in part the diversity of ifn-i effects, including their induction of opposite responses depending on the physiopathological contexts and/or the nature of the principal responding cell types ( , ) . ifn-iii induce the same signaling pathways as ifn-i, although they engage a different heterodimeric receptor, composed of the il- ra and il- rb chains and preferentially expressed on epithelial cells including hepatocytes. in mice and human beings, numerous ifn-i subtypes exist. functional and population genetic analyses showed that these ifn-i subtypes significantly differ in their functions ( ) ( ) ( ) ( ) ( ) ( ) . hence, one of most extraordinary feature of ifn-i biology is how ifn-i subtypes can elicit so many pleiotropic and diverse functions by interacting with the same receptor complex ( ) . both ifnar and ifnar are required for the initiation of ifn-i-dependent signals, as mice deficient in either one are highly susceptible to viral infections ( , ) . the assembling of the ifnreceptor ternary complex is a two-step process. first, a binary complex is formed by the binding of one side of the ifn molecule to ifnar . then, a single binary complex interacts with ifnar via the other side of the ifn molecule. the stability of the ternary complex will be determined in part by the association and dissociation kinetics between the cytokine and the two receptor chains, as well as by ifnar expression levels since the cell surface concentrations of the receptor subunits are relatively low. hence, both the affinity of ifn-i subsets for ifnar and the amounts of ifn-i, ifnar , and ifnar will regulate their biological effects ( figure a ) ( , ) . cell membrane density of ifnar and ifnar is also involved in differential ifn-β-versus ifn-α-induced functional activities, such as anti-proliferative function ( ) . a variety of cell-intrinsic parameters can also impact the lifetime of the ifn-receptor ternary complex, such as the rate of endocytosis/degradation/recycling of signaling complexes, and negative isg regulators such as usp that decrease the affinity of ifn-ifnar binding ( , ) . based on a definition of a prototypic cytokine-receptor binding module and by analogy with the epo receptor system, ifn-i subtypes were originally postulated to form ternary complexes of differing architectures, resulting in distinct geometry and assembling of intracellular signaling components ( ) . experimental evidence rejected this hypothesis. rather, the differential activities of ifn-i subtypes are determined by the stability of the ligand/receptor ternary complex ( , ) . differential affinities of the ifn-i subtypes for ifnar and ifnar extracellular domains generate subtype-specific signaling cascades and biological outcomes ( figure a ) ( , ) . crystal structure of ternary ifn-i/ifnar /ifnar complex illuminated the biochemical complexity of ifn-i interaction with their cognate receptors ( ) . the main conformational features of ifn-i/ifnar /ifnar ternary complexes are conserved among the different ifn-i, but are quite different from the other cytokine receptors ( , ) . in the formation of the binary ifn-i/ifnar complex, ifn-i ligand discrimination resides on differential energetics during the interaction of anchor points with ifnar , shared by all ifn-i, as well as on key amino acid substitution among ifn-i subtypes ( ) . ifnar then performs major conformational changes to interact with ifn-i associated in the binary complex, thus displaying an optimized functional plasticity ( ) . these differences in the chemistry of ifn-i subtype interaction with ifnar and ifnar thus explain the different affinities of ifn-α versus ifn-β within ternary complex and their differential activities ( ) . the functions regulated by ifn-i strongly depend on the main responding cell types ( figure b ). this has been studied in vitro by examining the functional consequences of the stimulation of different cell types with ifn-i, and in vivo by determining the contribution of cell-intrinsic ifn-i responses of different cell types to resistance or susceptibility to various diseases. an emerging concept is the central role of dc responses to ifn-i for induction of protective immunity against viral infections or tumors (figure ) . the development of mutant mice allowing conditional genetic inactivation of ifnar in a cell-type specific manner using the cre-lox system ( ) has been instrumental in accelerating our understanding of how different cell types respond to ifn-i in vivo and what their respective contribution is to protective or deleterious ifn-i responses. this has been investigated most extensively in viral infections ( , , , , ) but also in cancer ( , ) , bacterial infections ( ), autoimmunity ( , ) , sepsis ( ), or inflammatory diseases ( ) . efforts are being pursued to better understand which cell types respond to ifn-i in a manner promoting protective versus deleterious effects in different physiopathological settings. that knowledge will considerably help to develop novel strategies to modulate ifn-i functions for promoting health over disease. the development of mutant mice allowing conditional genetic inactivation of stat , stat , and stat ( - ) will help better understanding how different signaling pathways in different cell types determine the outcome of ifn-i response in vivo in various conditions. this knowledge might lead to the development of strategies aiming at targeting a given cell type with a specific subset of ifn-i, or in the presence of antagonists of certain signaling pathways, to surgically tune ifn-i responses in vivo toward the most desirable outcome. frontiers in immunology | microbial immunology for example, the affinity of ifn-β for ifnar is -times higher than that of ifn-α , and ifnβ is much more potent in inhibiting cellular proliferation or (continued ) ifn-i can also determine distinct functional outcomes. for example, during viral infections, early and transient high levels of ifn-i promote protective dc and t cell responses, while delayed, chronic and low level ifn-i production compromises host immune defenses and promotes chronic viral infections. within a given cell type, the outcome of ifn-i stimulation also depends on time of exposure to these cytokines relative to other modulatory signals (timing relative to other stimuli). for example, in naïve cd t cells, tcr signaling prior to ifn-i stimulation leads to increased expression of stat and promotes ifn-γ production and proliferation, while ifn-i stimulation prior to tcr triggering leads to stat -dependent anti-proliferative and pro-apoptotic effects. the formation of specific stat complexes is a highly dynamic process. it depends not only on the cell type but also on its specific state at the time it sees ifn-i. hence, major parameters controlling the effects of ifn-i in a given cell type also include its microenvironment ( figure c ) and the timing of its exposure to the cytokines both in terms of duration of the stimulation and of previous activation history ( figure d) . the tam receptor ligand gas is expressed within tumor cells in various solid cancers ( , ) . elevated gas expression is of bad prognosis in different cancers ( , ) . in a mouse model of ovarian cancer, early during tumorigenesis tumor-infiltrating dcs were found to be immunogenic and promote antitumor immunity, but they were later altered in the course of tumor development to acquire immunosuppressive properties beneficial to the tumor ( ) . one may thus hypothesize that expression of tam soluble ligands in certain tumors and of tam receptors on tumor-infiltrating dcs might contribute to dampen dc response to ifn-i and therefore facilitate their polarization by the tumor microenvironment into immunosuppressive cells (figure c) . acute versus chronic exposure to ifn-i can lead to strikingly opposite effects on a given cell type ( , , ) . in addition to duration, the time when a cell is exposed to ifn-i can also dramatically impact its functional response, depending on its previous activation history ( figure d) . in vitro stimulation of dcs with ifn-β can lead to opposite outcomes depending whether it occurs simultaneously to, or after, tnfα-induced maturation. ifn-β polarizes dcs toward th induction in the former case, and toward il- -secreting t cells in the latter case. these opposite effects result at least in part from the differential expression of il- / by dcs ( ) . similarly, ifn-i effect on the functional polarization of cd t cells is strongly modulated by the other cytokines present in the lymphocyte microenvironment at the same time ( ) . ifn-i can also mediate opposite effects on cd t cells depending whether it occurs before or after cognate engagement of the t cell receptor. indeed, while cd t cells have the potential to respond to ifn-i by inducing both stat -and stat -dependent genes, this depends upon their activation history. naïve cd t cells respond mostly to ifn-i through stat signaling, leading to the inhibition of their proliferation and eventually to the induction of their apoptosis. however, cognate triggering of the t cell receptor causes a decrease in stat and an increase in stat expression in cd t cells. this leads to a shift of their ifn-i response from stat -to-stat signaling, resulting in the promotion of their proliferation and ifn-γ production. during lcmv infection, this mechanism promotes stat -dependant expansion of anti-viral cd t cells, but stat -dependant inhibition of naïve cd t cell proliferation ( ) . since the late s the clinical potential of ifn-i for the treatment of patients suffering of viral infection or cancer diseases has been widely acknowledged ( ) . today, this expectation is tempered because ifn-i treatment can induce severe side effects and sufficient doses cannot be administered in patients. therefore, there is a strong need to create tuned ifn molecules devoid of side effects. based on our current understanding of ifn-i responses as reviewed above, many parameters could be tuned individually or in a combined manner to modulate ifn-i activity to promote their beneficial effects over the deleterious ones in a number of diseases. these parameters include modifying the affinity of ifn-i for its receptor, playing with the local quantity/concentration of ifn-i and with the duration of its delivery, and modulating the nature of the cells that are responding to ifn-i. we will discuss here novel strategies being developed to deliver ifn-i to, or block ifn-i responsiveness of, a specific target cell type in vivo (figure ). if ifn-i-induced side effects are a consequence of the pleiotropic nature of ifn-i, and if the bioactivities mediating deleterious effects have some degree of independence from those mediating beneficial effects, one could mutate the ifn-i molecules in order to skew their activity toward a desired bioactivity. indeed, introducing key mutation in ifn-α allowed increasing its affinity to ifnar by a factor of . accordingly, this ifn-α mutant is times more potent in inhibiting cell proliferation, but as potent as wt ifn-α in inducing an anti-viral state ( ) ( ) ( ) . hence, it is possible to tune ifn activity by modifying its binding to ifnar. however, translating such an approach for the design of molecules for clinical application is severely hampered by the poor understanding we have on the ifn-i bioactivities mediating the side effects. furthermore, we are far from having established the list of bioactivities that could be differentially modulated by changing the stability of the ifn-i/ifnar complex. we know more about the frontiers in immunology | microbial immunology cell types that mediate beneficial versus deleterious ifn responses in various diseases. hence, we will now discuss strategies aimed at focusing ifn activity to specific cell types to promote health over disease. several strategies have been developed to specifically target ifns on tumor cells, tumor-infiltrated immune cells or infected tissues. these strategies include intra-lesional injection ( , ) , adenoviral-mediated gene transfer ( ) ( ) ( ) , engineered tumorinfiltrating monocytes ( ) , and fusion of ifns with a cleavable protecting shell ( ) . another strategy to increase cytokine accumulation within the tumor or infected tissue is antibody-mediated targeting of cytokine delivery, where a cytokine moiety is fused to an antibody directed against a specific cell surface marker (figure ) . the fusion molecule retains both antigen-binding and ifn-i bioactivities, and is enriched at the targeted site upon in vivo injection ( ) ( ) ( ) ( ) . when targeted to human cd , ifn-i inhibited the proliferation of lymphoma cells engrafted in immunodeficient mice ( ) . an ifn-i targeted to a tumor antigen can also amplify the therapeutic effect of the antibody by acting on tumor-infiltrated dcs, thus increasing antigen cross-presentation and antitumor cytotoxic t cell responses ( ) . on non-targeted cells, the antibody conjugation negatively impacts ifn-i potency, but only modestly ( , , ) (figure a) . fusion molecules generally retain full ifn-i biological activity on the cells expressing the antibody target ( figure b) . hence, this difference only leads to a modest ratio between the ifn-i specific activity measured on target and non-target cells (figure b) . such a targeting efficiency is definitely too low to reduce the toxic effect of ifn-i administration, because it will not specifically focus ifn-i activities on "beneficial cells" without stimulating "deleterious cells." the engineering of immuno-ifn-i must be improved to reach the very high targeting efficacy required to significantly diminish the treatment side effects. we recently reported an innovative strategy reaching this goal ( ) . it is based on the postulate that the antibody moiety of an immuno-ifn-i stabilizes the ifn-i/receptor-complex by avidity. it also takes into account the fact that the biological potency of an ifn-i is proportional to the stability of the ifn-i/receptor complex up to a certain threshold beyond which increasing the stability does not increase its potency ( , ) . ifn-α and ifn-β are used in most immuno-ifn-i studies. they have evolved to retain close to maximal potency. hence, their targeting by an antibody that only provides a modest gain in terms of biological potency. however, it is expected that decreasing the affinity of the ifn-i for its receptor, by introducing a mutation, would increase the targeting effect of the antibody (figures c,d) . this is indeed the case. using an ifn-i with a single point mutation that dramatically decreases its affinity for ifnar ( figure c ) allows engineering immuno-ifns that are up to -fold more potent on cells expressing the antibody target ( figure d) . the three log targeting efficiency of these novel types of immuno-ifns is found for various activities measured in vitro or in vivo when delivered in mice. if the toxic side effect experienced by the patients treated with ifn-i is due to systemic ifn-i activity, this targeting technology may find considerable clinical applications since such engineered immuno-ifns are virtually inactive while "en route" and are activated only after binding of the fused antibody to the desired target. it remains to define the useful targets according to pathologies, for example, tumor cells themselves and professional cross-presenting xcr + dcs for cancer ( , , ) , or hepatocytes for chronic hcv infection. to treat autoimmune diseases, novel therapeutics targeting ifn-i have been developed, including two ifn-α-neutralizing monoclonal antibodies currently in clinical trials (sifalimumab and rontalizumab) ( , ) . however, long-term systemic neutralization of ifn-i activity may increase susceptibility to viral infection and tumor development. alternative strategies are needed to specifically inhibit ifn-i deleterious effects in these diseases without globally compromising ifn-i anti-viral and www.frontiersin.org anti-tumoral functions. the sequential nature of the assembling of the ifn-i/receptor complex opens the possibility to design ifn-i antagonists specifically targeting the cell subsets responsible for ifn-i deleterious effects. an ifn-α carrying a single amino acid substitution that blocks the ifn-i/ifnar interaction engages ifnar in a complex, which cannot bind ifnar ( ) . since the binary ifn-i/ifnar complex is devoid of any ifn-i activity, such mutant behaves as a potent ifn-i antagonist. when linked to an antibody specific for a cell surface marker, the antagonistic activity of the mutant ifn-i should be significantly reinforced specifically on the cells expressing the target. hence, it should be possible to design and construct targeted antagonists that inhibit responsiveness to endogenous ifn-i specifically on the cell subsets on which the cytokines act to promote autoimmunity or severe side effects, leaving the other cells fully responsive. for example, in chronic hcv patients treated with peg-ifn-α, one of the most deleterious side effects is nervous depression, which might be prevented by co-administration of an ifn-i antagonist specifically targeting neurons or other cells of the central nervous system. in the last decade, several major technological breakthroughs and the generation of novel animal models have remarkably advanced our understanding of the mode of action of ifns. in vitro high throughput screening allowed systematically studying the functions of isgs by ectopic expression or knock-down. advance biophysical investigation of the interactions between ifn-i and the ifn-i receptor allowed to rigorously investigate the mechanistic basis for the differential bioactivities of ifn-i subtypes. the analyses of the responses of different cell types to ifns or to viral infection, in vitro but also in vivo in various pathologies, demonstrated that ifn-i often mediate beneficial versus deleterious roles by acting on different cell types. from integrative analysis of these data, a picture is now emerging suggesting that it will be possible to segregate protective from deleterious ifn-i effects, based (i) on their differential induction depending on ifn-i subsets or on the magnitude/timing of ifn-i production, (ii) on their conditioning in different tissues, (iii) or on their occurrence in different cell types. hence, innovative immunotherapeutic treatments are being designed to tune ifn-i activity toward desired effects in order to promote health over disease in a manner adapted to each physiopathological condition. in particular, a proof-of-concept has been made in vitro that it will be possible to target ifn-i activity on given cell types or tissues to administer to patients sufficiently high doses of the cytokine at the site of interest while limiting unwanted effects in other tissues or cell types. the next steps will be to demonstrate efficacy of this strategy in vivo in preclinical animal models. importantly, to foster the development of 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neurotropic virus enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus interferon-stimulated genes: a complex web of host defenses protein kinase pkr and rna adenosine deaminase adar : new roles for old players as modulators of the interferon response dna deamination mediates innate immunity to retroviral infection samhd host restriction factor: a link with innate immune sensing of retrovirus infection a diverse range of gene products are effectors of the type i interferon antiviral response the transcription factor stat- couples macrophage synthesis of -hydroxycholesterol to the interferon antiviral response reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells plasmacytoid, conventional, and monocyte-derived dendritic cells undergo a profound and convergent genetic reprogramming during their maturation crosspriming of cd + t cells stimulated by virus-induced type i interferon type i interferon is selectively required by dendritic cells for immune rejection of tumors host type i ifn signals are required for antitumor cd + t cell responses through cd {alpha}+ dendritic cells dendritic cells require a systemic type i interferon response to mature and induce cd + th immunity with poly ic as adjuvant direct type i ifn but not mda /tlr activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly ic stimulation type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo johansson-lindbom b. type i interferon signaling in dendritic cells stimulates the development of lymph-noderesident t follicular helper cells differential responses of immune cells to type i interferon contribute to host resistance to viral infection a type i interferon autocrine-paracrine loop is involved in toll-like receptorinduced interleukin- p secretion by dendritic cells tlrdriven early glycolytic reprogramming via the kinases tbk -ikkvarepsilon supports the anabolic demands of dendritic cell activation type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection direct action of type i ifn on nk cells is required for their activation in response to vaccinia viral infection in vivo dendritic cells prime natural killer cells by trans-presenting interleukin cutting edge: the direct action of type i ifn on cd t cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection type i interferons act directly on cd t cells to allow clonal expansion and memory formation in response to viral infection direct stimulation of t cells by type i ifn enhances the cd + t cell response during cross-priming cutting edge: enhancement of antibody responses through direct stimulation of b and t cells by type i ifn cd t cells specific for lymphocytic choriomeningitis virus require type i ifn receptor for clonal expansion innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd t cells for clonal expansion and memory formation concomitant type i ifn receptor-triggering of t cells and of dc is required to promote maximal modified vaccinia virus ankara-induced t-cell expansion antiviral immune responses: triggers of or triggered by autoimmunity? is chronic plaque psoriasis triggered by microbiota in the skin? genetic dysbiosis: the role of microbial insults in chronic inflammatory diseases the tlr-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor alpha signaling accumulation of peripheral autoreactive b cells in the absence of functional human regulatory t cells cvid-associated taci mutations affect autoreactive b cell selection and activation human lupus autoantibody-dna complexes activate dcs through cooperation of cd and tlr ifn priming is 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population innate and adaptive interferons suppress il- alpha and il- beta production by distinct pulmonary myeloid subsets during mycobacterium tuberculosis infection host-directed therapy of tuberculosis based on interleukin- and type i interferon crosstalk inflammation. -hydroxycholesterol suppresses interleukin- -driven inflammation downstream of type i interferon dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny cytomegalovirus impairs antiviral cd + t cell immunity by recruiting inflammatory monocytes tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection influenza a inhibits th -mediated host defense against bacterial pneumonia in mice influenza a virus exacerbates staphylococcus aureus pneumonia in mice by attenuating antimicrobial peptide production role of tissue protection in lethal respiratory viral-bacterial coinfection disease tolerance as a defense strategy gainof-function human stat mutations impair il- immunity and underlie chronic mucocutaneous candidiasis type i interferon inhibits interleukin- production and inflammasome activation type i interferons promote fatal immunopathology by regulating inflammatory monocytes and neutrophils during candida infections ifnalpha/beta signaling is required for polarization of cytokine responses toward a protective type pattern during experimental cryptococcosis interferonbeta production via dectin- -syk-irf signaling in dendritic cells is crucial for immunity to c. albicans aberrant type i interferon regulation in autoimmunity: opposite directions in ms and sle, shaped by evolution and body ecology janus-like effects of type i interferon in autoimmune diseases coregulation of cd + t cell exhaustion by multiple inhibitory receptors during chronic viral infection type i interferon suppresses de novo virus-specific cd th immunity during an established persistent viral infection timing and magnitude of type i interferon responses by distinct sensors impact cd t cell exhaustion and chronic viral infection distinct transcriptional profiles in ex vivo cd + and cd + t cells are established early in human immunodeficiency virus type infection and are characterized by a chronic interferon response as well as extensive transcriptional changes in cd + t cells chronic cd + t-cell activation and depletion in human immunodeficiency virus type infection: type i interferon-mediated disruption of t-cell dynamics host genes associated with hiv- replication in lymphatic tissue global genomic analysis reveals rapid control of a robust innate response in siv-infected sooty mangabeys nonpathogenic siv infection of african green monkeys induces a strong but rapidly controlled type i ifn response downregulation of robust acute type i interferon responses distinguishes nonpathogenic simian immunodeficiency virus (siv) infection of natural hosts from pathogenic siv infection of rhesus macaques comparative transcriptomics of extreme phenotypes of human hiv- infection and siv infection in sooty mangabey and rhesus macaque hiv turns plasmacytoid dendritic cells (pdc) into trail-expressing killer pdc and down-regulates hiv coreceptors by toll-like receptor -induced ifn-alpha plasmacytoid dendritic cells express trail and induce cd + t-cell apoptosis in hiv- viremic patients sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv- glycerol monolaurate prevents mucosal siv transmission type i interferon responses in rhesus macaques prevent siv infection and slow disease progression plasmacytoid dendritic cells suppress hiv- replication but contribute to hiv- induced immunopathogenesis in humanized mice chronic hepatitis c: future treatment host-targeting agents in the treatment of hepatitis c: a beginning and an end? the interferons: years after their discovery, there is much more to learn immune responses to hcv and other hepatitis viruses interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness altered interferon-alpha-signaling in natural killer cells from patients with chronic hepatitis c virus infection natural killer cells are polarized toward cytotoxicity in chronic hepatitis c in an interferon-alfa-dependent manner coexpression of pd- , b , cd and klrg on exhausted hcv-specific cd + t cells is linked to antigen recognition and t cell differentiation evidence for an antagonist form of the chemokine cxcl in patients chronically infected with hcv monocyte activation by interferon alpha is associated with failure to achieve a sustained virologic response after treatment for hepatitis c virus infection genetic variation in il b and spontaneous clearance of hepatitis c virus genetic variation in il b predicts hepatitis c treatment-induced viral clearance a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus the favorable ifnl genotype escapes mrna decay mediated by aurich elements and hepatitis c virus-induced micrornas distinct and overlapping genomic profiles and antiviral effects of interferon-lambda and -alpha on hcv-infected and noninfected hepatoma cells targeted induction of interferon-lambda in humanized chimeric mouse liver abrogates hepatotropic virus infection ifn-lambda receptor expression is induced in chronic hepatitis c and correlates with the ifn-lambda genotype and with nonresponsiveness to ifn-alpha therapies how cells respond to interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins regulation of type i interferon responses stat and irf : beyond isgf . jakstat ( ) single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators major differences in the responses of primary human leukocyte subsets to ifn-beta critical role for stat activation by type interferons in the interferongamma response to viral infection immunomodulatory functions of type i interferons high basal stat balanced by stat induction to control type interferon effects in natural killer cells type interferon induction of natural killer cell gamma interferon production for defense during lymphocytic choriomeningitis virus infection changing partners at the dance: variations in stat concentrations for shaping cytokine function and immune responses to viral infections dendritic-cell maturation alters intracellular signaling networks, enabling differential effects of ifn-alpha/beta on antigen cross-presentation mechanisms of type-i-and type-ii-interferon-mediated signalling type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors complex modulation of cell type-specific signaling in response to type i interferons alternate interferon signaling pathways ifn-beta induces serine phosphorylation of stat- in ewing's sarcoma cells and mediates apoptosis via induction of irf- and activation of caspase- usp -based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine cxcl expression differential activity of type i interferon subtypes for dendritic cell differentiation evolutionary genetic dissection of human interferons the receptor of the type i interferon family interferons as biomarkers and effectors: lessons learned from animal models protection against progressive leishmaniasis by ifn-beta multifaceted activities of type i interferon are revealed by a receptor antagonist receptor density is key to the alpha /beta interferon differential activities structural and dynamic determinants of type i interferon receptor assembly and their functional interpretation the type i interferon receptor: structure, function, and evolution of a family business differential receptor subunit affinities of type i interferons govern differential signal activation structural linkage between ligand discrimination and receptor activation by type i interferons distinct and nonredundant in vivo functions of ifnar on myeloid cells limit autoimmunity in the central nervous system type i interferons protect t cells against nk cell attack mediated by the activating receptor ncr lymphadenopathy in a novel mouse model of bartonella-induced cat scratch disease results from lymphocyte immigration and proliferation and is regulated by interferon-alpha/ beta cytosolic rig-i-like helicases act as negative regulators of sterile inflammation in the cns expression of type i interferon by splenic macrophages suppresses adaptive immunity during sepsis myeloid type i interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions stat activation is responsible for il- -dependent t cell proliferation through preventing apoptosis: generation and characterization of t cell-specific stat -deficient mice generation of mice with a conditional stat null allele conditional stat ablation reveals the importance of interferon signaling for immunity to listeria monocytogenes infection loss of stat from mouse mammary epithelium results in an increased neu-induced tumor burden inactivation of stat in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation meta-analysis of microarray data identifies gas expression as an independent predictor of poor survival in ovarian cancer axl and growth arrest-specific gene are frequently overexpressed in human gliomas and predict poor prognosis in patients with glioblastoma multiforme gas expression identifies high-risk adult aml patients: potential implications for therapy ovarian cancer progression is controlled by phenotypic changes in dendritic cells ifnalpha activates dormant haematopoietic stem cells in vivo timing of ifn-beta exposure during human dendritic cell maturation and naive th cell stimulation has contrasting effects on th subset generation: a role for ifn-beta-mediated regulation of il- family cytokines and il- in naive th cell differentiation combinatorial flexibility of cytokine function during human t helper cell differentiation regulating type ifn effects in cd t cells during viral infections: changing stat and stat expression for function interferons at age : past, current and future impact on biomedicine inquiring into the differential action of interferons (ifns): an ifn-alpha mutant with enhanced affinity to ifnar is functionally similar to ifn-beta an interferon alpha mutant optimized by phage display for ifnar binding confers specifically enhanced antitumor activities the stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities intra-lesional low-dose interferon alpha a therapy for primary cutaneous marginal zone b-cell lymphoma intratumoral injection of interferon-alpha and systemic delivery of agonist anti-cd monoclonal antibodies synergize for immunotherapy delivery of interferon alpha using a novel cox -controlled adenovirus for pancreatic cancer therapy the efficacy of radiotherapy relies upon induction of type i interferondependent innate and adaptive immunity a trial of intrapleural adenoviral-mediated interferon-alpha b gene transfer for malignant pleural mesothelioma tumortargeted interferon-alpha delivery by tie -expressing monocytes inhibits tumor growth and metastasis targeting cytokines to inflammation sites livertargeting of interferon-alpha with tissue-specific domain antibodies antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation targeting ifn-alpha to b cell lymphoma by a tumor-specific antibody elicits potent antitumor activities targeting the tumor microenvironment with interferon-beta bridges innate and adaptive immune responses targeted delivery of interferon-alpha via fusion to anti-cd results in potent antitumor activity against b-cell lymphoma targeted delivery of interferon-alpha to hepatitis b virus-infected cells using t-cell receptor-like antibodies variations in the unstructured c-terminal tail of interferons contribute to differential receptor binding and biological activity safety and pharmacodynamics of rontalizumab in patients with systemic lupus erythematosus: results of a phase i, placebo-controlled, double-blind, dose-escalation study safety profile and clinical activity of sifalimumab, a fully human anti-interferon alpha monoclonal antibody, in systemic lupus erythematosus: a phase i, multicentre, double-blind randomised study mutation of the ifnar- receptor binding site of human ifn-alpha generates type i ifn competitive antagonists the studies performed in the laboratories are supported by funding from inserm, cnrs, aix-marseille university, the labex mabimprove, and the european community's seventh framework programme fp / - (grant agreement for gilles uzé, european research council starting grant agreement number for marc dalod including salary support to emeline pollet and thien-phong vu manh). we thank past and present laboratory members for their contribution to studies on dcs or ifns. we apologize for not quoting certain studies because of space limitations. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -gs k authors: jacques, alexandre; bleau, christian; turbide, claire; beauchemin, nicole; lamontagne, lucie title: a synergistic interferon-γ production is induced by mouse hepatitis virus in interleukin- (il- )/il- -activated natural killer cells and modulated by carcinoembryonic antigen-related cell adhesion molecules (ceacam) a receptor date: - - journal: immunology doi: . /j. - . . .x sha: doc_id: cord_uid: gs k the production of interferon-γ (ifn-γ) by infiltrating natural killer (nk) cells in liver is involved in the control of mouse hepatitis virus (mhv) infection. the objectives of this study were to identify the mechanisms used by mhv type to modulate the production of ifn-γ by nk cells during the acute hepatitis in susceptible c bl/ mice. ex vivo and in vitro experiments revealed that nk cells, expressing carcinoembryonic antigen-related cell adhesion molecules (ceacam) a (the mhv receptor), can produce a higher level of ifn-γ in the presence of both l -mhv and interleukin- (il- )/il- . the synergistic production of ifn-γ by nk cells depends on viral replication rather than viral fixation only, because it is inhibited or not induced in cells infected with ultraviolet-inactivated viruses and in cells from ceacam a(−/−) mice infected with virulent viruses. the synergistic ifn-γ production involves the p mitogen-activated protein kinase (mapk) rather than the extracellular signal-regulated kinase- / mapk signalling pathway. however, the signal triggered through the engagement of ceacam a decreases the production of ifn-γ, when these molecules are cross-linked using specific monoclonal antibodies. these results suggest that control of acute hepatitis by ifn-γ-producing nk cells may depend on both production of il- and il- in the liver environment and viral infection of nk cells. natural killer (nk) cells are an important arm of innate immunity against virus-infected cells, bacteria and tumour cells, and exert direct cytotoxicity functions as well as indirect antiviral functions through the secretion of interferon-c (ifn-c). the role of intrahepatic nk cells in the outcome of human viral hepatitis is not fully understood. nk cells are known to act as a first line to control many viral infections such as herpes simplex virus type , epstein-barr virus, human herpesvirus and murine cytomegalovirus. , however, human hepatitis c virus has developed strategies to evade the detection and elimination by nk cells, as is also suggested by the increased numbers of nk cells in the livers of patients during the immunotolerance phase of chronic hepatitis b virus infection. the liver is enriched in nk and nkt cells, which play major roles in the control of viral hepatitis infections by limiting viral replication via an ifn-c-dependent mechanism. recently, several studies have demonstrated the importance of the cytotoxic activities of nk cells in preventing the establishment of chronic human hepatitis c virus infections. , natural killer cells are rapidly recruited from the bone marrow and spleen during viral infections. interleukin- (il- ), il- , il- and ifn-c enhance the cytotoxic activity of nk cells and further increase the production of ifn-c by these cells. , moreover, the production of ifn-c by nk cells stimulated with il- and il- is dependent on the activation of the immunoreceptor tyrosine-based activation motifs (itams) and is regulated through the p and extracellular signal-regulated kinase- / mitogenactivated protein kinase (erk- / mapk) pathways. , however, little is known about the mechanism involved in the efficient production of antiviral ifn-c by intrahepatic nk cells during the acute phase of viral hepatitis. mouse hepatitis virus (mhv) is an excellent model for studying the immunological disorders associated with viral the production of interferon-c (ifn-c) by infiltrating natural killer (nk) cells in liver is involved in the control of mouse hepatitis virus (mhv) infection. the objectives of this study were to identify the mechanisms used by mhv type to modulate the production of ifn-c by nk cells during the acute hepatitis in susceptible c bl/ mice. ex vivo and in vitro experiments revealed that nk cells, expressing carcinoembryonic antigen-related cell adhesion molecules (ceacam) a (the mhv receptor), can produce a higher level of ifn-c in the presence of both l -mhv and interleukin- (il- )/il- . the synergistic production of ifn-c by nk cells depends on viral replication rather than viral fixation only, because it is inhibited or not induced in cells infected with ultraviolet-inactivated viruses and in cells from ceacam a )/) mice infected with virulent viruses. the synergistic ifn-c production involves the p mitogen-activated protein kinase (mapk) rather than the extracellular signal-regulated kinase- / mapk signalling pathway. however, the signal triggered through the engagement of ceacam a decreases the production of ifn-c, when these molecules are cross-linked using specific monoclonal antibodies. these results suggest that control of acute hepatitis by ifn-c-producing nk cells may depend on both production of il- and il- in the liver environment and viral infection of nk cells. keywords: ceacam a; coronavirus; hepatitis; interferon-c; interleukin- / interleukin- ; mitogen-activated protein kinase; natural killer cells hepatitis. the hepatotropic mhv serotype induces acute or chronic hepatitis according to the strain, age and immune status of the mouse. moreover, mhv can replicate in hepatocytes, liver sinusoidal endothelial cells (lsec) and kupffer cells (kc), leading to virus-induced cell death, resulting in a fulminate hepatitis in susceptible c bl/ mice, and their death within - days postinfection (p.i.). we have previously reported that the development of hepatitis in pathogenic l -mhv -infected c bl/ mice is related to a decrease in splenic and myeloid nk cells because of the formation of syncytia and their subsequent apoptosis. moreover, in vivo depletion of nk cells during mhv infections enhances virus replication, which in turn leads to a more pronounced hepatitis. in contrast, no or low decreases in nk cells were detected in mice infected with attenuated virus variants, , suggesting a protective role of nk cells in hepatitis. interferon-c is known to play a protective role against the hepatitis induced by various mhv serotypes. , very few studies have targeted the efficiency of nk cell functions during mhv infection. recently, it was reported that enhanced protection in mhv-cxcl -infected mice correlated with increased ifn-c production by infiltrating nk cells within brain and liver. furthermore, the addition of il- and il- in mice susceptible to mhv infection led to an increase in ifn-c production in the liver and better control of the infection, although the ifn-c-producing cells involved were not identified. infection of susceptible cells by mhv depends on the fixation of viral surface proteins to a receptor, identified as the carcinoembryonic antigen-related cell adhesion molecule a (ceacam a). it was shown that ceacam homotypic interactions between nk cells and various target cells inhibit nk cell cytotoxicity. in addition, the engagement of ceacam a leads to the inhibition of t-cell proliferation and ifn-c production by nkt cells. in contrast, toll-like receptor-dependent activation of nuclear factor-jb upregulates the expression of ceacam a, whereas ifn-c downregulates it, so decreasing the permissivity of susceptible cells to mhv infection. , cea-cam a is expressed at the surface of hepatic cells including lsec, kc, hepatocytes and b and nk cells, but is scarce or not expressed by naive cd + or cd + t cells. [ ] [ ] [ ] [ ] it was reported that the p and erk- / mapk pathways were activated in peritoneal macrophages infected with mhv within the first min of infection, suggesting that this effect occurred in response to the fixation of the virus to its receptor. it was also suggested that the replication of the mhv-a virus was dependent on the activation of p but not the erk- / mapk pathway, without further identifying the replication step involved. in this respect, the effect of the ceacam a engagement by mhv surface proteins and the further activation of the p or erk- / mapk pathways on the antiviral function of nk cells remain unknown. the fact that ceacam a )/) mice are resistant to a mhv-a infection suggests the absolute requirement of ceacam a for viral infectivity. in this work, we demonstrate a synergistic production of ifn-c by nk cells in the presence of both mhv and il- /il- , involving viral replication and the p mapk signalling pathway, but decreased by the engagement of ceacam a. wild-type c bl/ mice were purchased from charles river laboratories (st constant, qc, canada). ceacam a knockout (ceacam a )/) ) mice were generated by dr n. beauchemin as previously described. the animals, certified as mhv -free by the manufacturer, were housed under hepa-filtered air (forma scientific, marietta, oh). female mice between and weeks of age were used in all the experiments. the study was conducted in compliance with the regulations of the animal committee of the université du québec à montreal (uqam). the pathogenic l -mhv virus is a cloned substrain isolated from the liver of infected dba mice and propagated in l cells as previously described. the pathogenic properties of the l -mhv virus were assessed regularly. groups of three or six c bl/ mice, according to the experiments, were infected by the intraperitoneal route with % tissue culture infective dose (tcid ) of l -mhv . mock-infected mice received a similar volume of rpmi- (gibco laboratories, grand island, ny). after hr of infection, the mice were anaesthetized by intraperitoneal injection using ketamine hydrochloride ( mg/kg; vetrepharm canada inc., belleville, on, canada) and xylazine ( mg/kg; bayer inc., toronto, on, canada). mice were bled by section of the portal vein and aortic artery. the livers were harvested following exsanguination and intrahepatic mononuclear cells (mncs) were isolated as described by watanabe et al. the continuous mouse fibroblast l cell line was grown in rpmi- supplemented with l-glutamine ( mm), antibiotics (penicillin u/ml and streptomycin mg/ml) (gibco laboratories) and % fetal calf serum (fcs). l cells were used for viral production. intrahepatic mncs were isolated from the liver of mice from each experimental group as previously described. briefly, the liver was pressed through a lm cell strainer (falcon scientific co., montreal, qc, canada) which was then washed with ml rpmi- supplemented with % fcs. the cell suspension was then deposited on ml fcs to allow debris sedimentation; the top layer was then recovered and centrifuged for min at g. the supernatants were collected for quantification of cytokines by enzyme-linked immunosorbent assay (elisa) after been passed through a Á lm filter (sarstedt inc., montreal, qc, canada). the cell suspension was deposited on the top of a discontinuous percoll gradient [ % and % percoll in phosphate-buffered saline (pbs)] (amersham pharmacia, uppsala, sweden; gibco laboratories) and centrifuged for min at g. the mncs were collected at the interface of the % and % percoll layers and washed with rpmi- supplemented with % fcs and finally adjusted to Á · or Á · cells/ml. myeloid mncs were isolated from femurs of uninfected mice. briefly, the femurs were soaked in % ethanol for - min and washed subsequently with rpmi- supplemented with % fcs. the femurs were then excised from the surrounding muscle tissues. the marrow was flushed using a syringe filled with rpmi- supplemented with % fcs and fitted with a needle calibre g / (becton dickinson & co., franklin lakes, nj). the cell suspension was thereafter purified on a lymphoprep gradient (cedarlane, hornby, on, canada), washed and adjusted to cells/ml. intrahepatic or myeloid nk cells were eliminated by positive selection using the cd b (dx or pan nk) antibody coupled to microbeads (stemcell technologies, vancouver, bc, canada). total myeloid mncs, purified dx + cells or 'dx -depleted' mncs were resuspended in rpmi- supplemented with % fcs and adjusted to cells/ml. in all experiments, the cell viability, ranging from % to %, was assayed by a trypan blue exclusion test. intrahepatic mncs were isolated from six mock-or l -mhv -infected c bl/ mice after hr of infection and were seeded in -well plates at concentrations of Á · cells/ml in rpmi- supplemented with % fcs. recombinant murine il- (ril- ) or ril- (bio-source, montreal, qc, canada) were added to a final concentration of Á and ng/ml, respectively. the cells were then incubated at °, under % co for hr. the supernatants were collected for ifn-c quantification by elisa. the mncs were isolated from the livers or bone marrows from uninfected wild-type or ceacam a )/) c bl/ mice and cells were thereafter seeded in -well plates at a concentration of cells/ml in rpmi- supplemented with % fcs. recombinant il- or ril- was added to a final concentration of Á or ng/ml, respectively. in some experiments, sb and u , specific p and erk- / mapk inhibitors (calbiochem, san diego, ca), were added to a final concentration of lg/ml. different concentrations of sodium stibogluconate (ss) were also added during min to inhibit the shp- phosphatase. a monoclonal murine anti-ceacam a antibody (agb , produced in rat and affinity-purified on a hitrap protein g column) was also added at a concentration of lg/ cells. the cells were infected with a Á - Á multiplicity of infection of infectious l -mhv or l -mhv treated for hr with ultraviolet (uv) light and then incubated at °, under % co for hr. the supernatants were collected for ifn-c quantification by elisa. percent of intrahepatic nk . + tcr-bcells from in vivo mock-or l -mhv -infected c bl/ mice were determined by double immunolabelling. the intrahepatic mncs were isolated and cells were resuspended in ml pbs and further incubated on ice in the presence of a cd /cd blocker (pharmingen, toronto, on, canada) for min. thereafter, the cells were incubated for min with lg fluorescein isothiocyanate-conjugated nk . [clone pk ; mouse immunoglobulin g aj (igg aj); pharmingen] and lg phycoerythrin-conjugated t-cell receptor-b (tcr-b; clone h - ; armenian hamster igg k ; pharmingen). the cells were then washed in pbs and fixed overnight at °in pbs, ph Á containing % formaldehyde (fisher scientific co., montréal, qué, canada). flow cytometric analyses were performed on a fluorescence-activated cell flow cytometer (facscan) with cell quest software (becton-dickinson, moutain view, ca). ten thousand cells were analysed per sample and the percentages of nk . + tcr-bcells were determined by a multiparametric analysis. interferon-c, il- and il- levels produced in supernatants from ex vivo or in vitro infections or in liver extracts were determined using mouse il- (p ), mouse il- and mouse ifn-c bd opteia elisa sets (bd biosciences, mississauga, on, canada). for in vivo and ex vivo studies, statistical analyses were performed using an analysis of variance (anova) test. for in vitro studies, statistical analyses were performed using a student's t-test. all statistical comparisons were calculated with graphpad prism . software (graphpad software inc., la jolla, ca). error bars represent standard errors and a value of p < Á was considered significant. mhv infection synergizes the production of ifn-c by intrahepatic mncs in the presence of il- and il- to verify the ability of intrahepatic mncs from l -mhv -infected mice to produce ifn-c during the acute phase of hepatitis, intrahepatic mncs from six mock-or l -mhv -infected c bl/ mice were isolated after hr of infection and activated ex vivo with ril- /ril- for hr. levels of ifn-c were then evaluated in cell supernatants. as shown in fig. (section i), unstimulated intra-hepatic mncs isolated from l -mhv -infected mice produced slightly more ifn-c than cells from mockinfected mice (p < Á ). addition of ril- and ril- , however, increased the production of ifn-c by mncs from both l -mhv -and mock-infected mice (p < Á ). to verify if the viral infection is involved in the production of ifn-c by intrahepatic mncs, these cells were isolated from uninfected c bl/ mice and infected in vitro with the l -mhv for hr in the presence of ril- , ril- or both. the ifn-c released in the cell supernatants was then quantified. as shown in the fig. (section ii, a), l -mhv infection of intrahepatic mncs induced low but significant ifn-c production (p < Á ). addition of ril- (p < Á ), ril- (p < Á ) or ril- /ril- (p < Á ) to uninfected intrahepatic mncs further increased the ifn-c production (fig. , section ii a and ii b) . production of ifn-c by intrahepatic mncs, however, synergistically increased when the cells were simultaneously treated in vitro with both the l -mhv and cytokines (p < Á for all treatments when compared with the uninfected treated cells) (fig. , section ii a and ii b) . these results indicate that il- and il- act in synergy with the l -mhv on one or more susceptible cell types present in the intrahepatic mnc culture from uninfected mice to produce ifn-c. nk cells are responsible for the production of ifn-c in response to a combined treatment with the l -mhv and il- /il- it was previously demonstrated that the production of ifn-c in the liver in response to il- administration depends on nk cells. however, intrahepatic nk cells were recruited in the liver from the bone marrow or spleen in l -mhv infected mice. , the number of nk cells expressing the ceacam a receptor was significantly higher in the bone marrow when compared with the spleen or the liver, and so bone marrow mncs were then used as a source of nk cells. mncs from bone marrow were isolated and nk cells were thereafter purified with an anti-dx antibody coupled to microbeads. total myeloid mncs, purified dx + cell preparations ( % purity) and dx depleted cell preparations (< % dx + cells remaining) were then infected in vitro with l -mhv in the presence of ril- and ril- . as shown in table , low ifn-c was produced by total myeloid mncs infected with l -mhv or treated only with ril- /ril- . however, addi- tion of these cytokines to l -mhv -infected myeloid mncs induced a synergistic production of ifn-c when compared with untreated l -mhv -infected cells (p < Á ) or with uninfected ril- /ril- -treated cells (p < Á ). no significant ifn-c was produced by uninfected or l -mhv -infected dx -depleted myeloid mncs, treated or not with ril- and ril- , suggesting that nk cells were the only cells involved in the production of ifn-c. purified dx + cells produced ifn-c when stimulated with ril- /ril- (p < Á compared with untreated cells) and responded in a synergistic manner when infected with l -mhv (p < Á compared with ril- /ril- -treated cells). these results indicate that nk cells are involved in the synergistic production of ifn-c induced both by the l -mhv and the cytokines. to verify if the production of ifn-c by l -mhv -infected nk cells, treated with il- and il- is initiated by the viral replication, cells from ceacam a )/) mice (the specific mhv receptor) were compared with those from wild-type c bl/ mice. myeloid mncs from wild-type and cea-cam a )/) c bl/ mice were infected in vitro with l -mhv for hr in the presence of ril- /ril- . the ifn-c levels in supernatants were further quantified. results revealed that no ifn-c was induced by myeloid mncs from ceacam a )/) mice when treated with ril- / ril- infected or not with l -mhv (table ). in addition, no viral proteins were detected in myeloid nk cells from ceacam a )/) mice following a double immunolabelling using anti-dx and anti-mhv antibodies, indicating that no viral replication occurred in these cells. the failure of ceacam a )/) cells to produce ifn-c suggests that viral fixation or viral replication is essential for this response. the role of viral replication was then verified using uv-inactivated viruses. myeloid mncs from wildtype c bl/ mice were infected with infectious or uvinactivated l -mhv in the presence of ril- /ril- . levels of ifn-c were thereafter determined in supernatants at hr p.i. as shown in fig. (section iii a) , production of ifn-c strongly decreased when uv-inactivated l -mhv was added to cells in the presence of ril- /ril- (p < Á ). the absence of viral translation in myeloid nk cells infected with the uv-inactivated viruses was also confirmed by a double immunolabelling using anti-dx and anti-mhv antibodies, because no intracellular viral proteins were detected. however, when bone marrow mncs were treated with ril- /ril- for hr before infection with the virus, the synergistic ifn-c production failed to occur (data not shown), indicating that the cellular signalling pathway(s) involved in this response may be simultaneously activated by cytokines and the first steps of viral replication. the p mapk pathway, rather than erk- / mapk, is involved in synergistic ifn-c production in nk cells the p and erk- / mapk pathways are mainly involved in the production of ifn-c by nk cells when stimulated by il- and il- . , moreover, unidentified early steps of mhv replication also depend on activation of the p and erk- / mapk pathways, , whereas ceacam a engagement predominantly induces the erk- / mapk pathway, so verifying if the ifn-c response depended on the p and/or erk- / mapk pathways. total bone marrow mncs and purified dx + cells ( % purity) from wild-type c bl/ mice were treated for hr with specific inhibitors of p (sb ) and erk- / (u ) mapk and thereafter infected with l -mhv in the presence of ril- and ril- . the ifn-c levels were then quantified hr p.i. as shown in table , production of ifn-c by l -mhv infected total bone marrow mncs treated with ril- and ril- significantly decreased in the presence of the p mapk inhibitor (p < Á ), when compared with l -mhv -infected cells treated only with the cytokines. such an effect was conserved in the purified myeloid dx + cell fraction (p < Á for the p mapk inhibitor). the erk- / mapk inhibitor did not induce any effect. it is known that cross-linking or homotypic interactions between ceacam a are essential to induce an inhibitory response in nk or nkt cells. , we therefore studied if the cross-linking of the anti-ceacam a monoclonal antibody (agb ) could provide this effect. we had verified first that the specific antibody agb did not induce or inhibit by itself the synergistic ifn-c production in the presence of ril- /ril- only (results not shown). then, agb was coated on -well plates for hr after which myeloid mncs from wild-type c bl/ were seeded and infected with l -mhv in the presence of ril- /ril- . the ifn-c levels were then quantified after hr p.i. as shown in fig. (section iii b) , the synergistic ifn-c production was inhibited by coated agb , suggesting that cross-linking of ceacam a can downregulate the synergistic response induced by l -mhv in the presence of il- /il- or compete with the viral fixation to the ceacam a receptor (p < Á when compared with l -mhv treatment plus ril- /ril- ). to further support the hypothesis of the engagement of ceacam a in the downregulation of the ifn-c response, bone marrow mncs from wild-type c bl/ mice were treated with different concentrations of a shp- phosphatase inhibitor (ss), which is involved in the inhibitory signalling pathway of ceacam a but not in viral replication, before infection with l -mhv in the presence of ril- /ril- . the ifn-c levels were thereafter quantified after hr p.i. as shown in fig. (section iii c) , production of ifn-c increased when myeloid cells were treated with the shp- phosphatase inhibitor at concentrations ranging from to lg/ml (p < Á at and lg/ml, p < Á at lg/ml). this result supports the inhibitory role of ceacam a rather than neutralization of viral fixation. we have recently demonstrated that the level of hepatitis in mice infected with l -mhv depends on viral permissivity of kc and lsec, involved in the production of intrahepatic il- and il- and the subsequent ifn-c production by nk cells. intrahepatic ifn-c, il- and il- levels were then evaluated by elisa tests in liver extracts from six c bl/ mice infected with tcid of the l -mhv at hr p.i. the intrahepatic ifn-c was not induced in livers from l -mhv -infected mice at hr p.i. compared with mock-infected mice (< Á pg/ml in both mice). this absence of ifn-c production may be related to the inability of hepatic cells to produce adequate levels of il- and il- . effectively, the intrahepatic il- was not induced in l -mhv infected mice after days of infection compared with mock-infected mice (< Á pg/ml in both mice). furthermore, intrahepatic il- levels decreased in l -mhv -infected mice during the acute hepatitis (mockinfected mice: ± pg/ml; l -mhv -infected mice: ± pg/ml) (p < Á ). these cytokine decreases were also observed as early as hr p.i. (results not shown). in addition, we have previously reported that nk cells from c bl/ mice support viral replication and subsequent apoptosis. to verify if the decrease of ifn-c production is related to apoptosis of intrahepatic nk cells during the acute phase of the viral hepatitis, three c bl/ mice were infected with tcid of l -mhv and intrahepatic mncs were thereafter isolated at different times p.i. intrahepatic mncs were then labelled with fluorescein isothiocyanate-conjugated anti-nk . and phycoerythrin-conjugated anti-tcr-b antibodies and analysed by fluorometry. two distinct cell populations were observed according to the forward/side scatter (fsc/ssc) parameters. normal lymphoid cells were characterized by normal fsc and ssc parameters (tunel+ cells < Á ± Á %), whereas apoptotic lymphoid cells, characterized by lower fsc and higher ssc parameters, were confirmed by a tunel test ( Á ± Á % positive cells). intrahepatic nk . + tcr-bcells from l -mhv infected mice underwent apoptosis from hr p.i. (mock-infected mice: Á ± Á %; l -mhv -infected mice: Á ± Á %; p < Á ) to hr p.i. (mock-infected mice: Á ± Á %; l -mhv -infected mice: Á ± Á %; p < Á ) and peaking at hr p.i. (mock-infected mice: Á ± Á %; l -mhv -infected mice: Á ± Á %; p < Á ). in this study, we report that l -mhv , in the presence of il- and il- , induces a synergistic ifn-c production by nk cells from ceacam a +/+ mice. this effect was shown to be dependent on viral replication, and was under the control of the p mapk but not the erk- / mapk signalling pathway. however, the signal triggered through the engagement of ceacam a inhibits the synergistic production of ifn-c. in this work, we have demonstrated that mhv can induce a synergistic ifn-c response, both in intrahepatic and myeloid mncs, in the presence or absence of il- / il- . this property was mainly the result of nk (dx + ) cells because it was lost when nk cells were depleted, whereas purified nk cells exhibit this synergistic ifn-c response. it was important to demonstrate the role of nk cells in the production of ifn-c, since cd + t cells, or nkt cells, which are also presents in the liver and/or the bone marrow, can be other sources for ifn-c in response to il- /il- treatment. , however, neither cd + nor cd + t cells expressed ceacam a, or have a very low level of expression, and can be infected by l -mhv . on the other hand, b cells, which may be infected by mhv , are not known to be ifn-c-producing cells. we have previously reported that nk cells are a new cell target for mhv. these cells can express the viral ceacam receptor. in this context, the synergistic production of ifn-c by nk cells in the presence of a coronavirus requires ceacam a. effectively, nk cells from ceacam a )/) mice do not produce ifn-c in a synergistic manner in response to il- /il- and l -mhv . the absence of a synergistic response of cells from cea-cam a )/) mice did not result from functional anomalies of nk cells because no significant differences in the intrahepatic lymphoid cell subpopulation, or in nk cell functions, have been observed in cells from ceacam a )/) mice when compared with wild-type c bl/ mice. moreover, no maturation defects of nk cells have been detected in the bone marrow of ceacam a )/) mice compared with wild-type c bl/ mice (unpublished observations from dr beauchemin's team). however, our results suggest that l -mhv may induce this synergistic ifn-c production independently of ceacam a, but requires this molecule to enter into nk cells. this hypothesis is also supported by the involvement of viral replication rather than viral fixation, as demonstrated by the use of uv-inactivated mhv and the p mapk inhibitor. in addition, ceacam a receptor does not directly trigger the production of ifn-c by nk cells, as the addition of a specific anti-ceacam a antibody (agb ) did not induce production of this cytokine. agb binds an area between the first and second immunoglobulin domains of the ceacam a. , however, experiments using plates bound with anti-ceacam a agb antibodies, or the shp- inhibitor, revealed that engagement of ceacam a receptors activates the shp- signalling pathway, which in turn leads to the inhibition of ifn-c production by nk cells in the presence of l -mhv . the ceacam a receptor exists under two isoforms constituted either of a long inhibiting cytoplasmic domain, which contains immunoreceptor tyrosine-based inhibitory motifs (itims), or a short activating cytoplasmic domain, which does not possess itims or itams. ortaldo et al. have recently demonstrated that il- and il- override the inhibitory mechanisms of the ly- inhibitory receptors containing itims, so enabling ifn-c production. these authors have nevertheless noticed that cross-linking of activating ly receptors which possess itams is essential for a response to il- and il- . ceacam a receptor has already been associated with the inhibition of the ifn-c production by human nkt cells. in our study, ceacam a may play an inhibitory role, by cross-linking or homotypic interactions, in ifn-c production by nk cells. on the other hand, inhibition of shp- , which is associated with itims, increased the production of this cytokine. ceacam a may therefore be involved in the modulation of an il- / il- -dependent ifn-c pathway in nk cells. we can hypothesize that the high number of viral s proteins produced by mhv -infected hepatic cells may bind to ceacam a molecules expressed at the nk cell surface attenuating the secretion of ifn-c. our results indicate that the synergistic response of ifn-c in l -mhv -infected nk cells, in the presence of ril- and ril- , is completely dependent on the p , but not the erk- / , mapk pathway. the p mapk pathway therefore links the viral infection in the nk cells with activation mediated by ril- and ril- . the production of ifn-c by nk cells stimulated with il- and/ or il- is dependent on the activation of itams and is regulated through the p and erk- / mapk pathways. , pretreatment of nk cells with il- for min is sufficient for ifn-c induction. simultaneously, the p mapk pathway is essential for the first steps of mhv replication, whereas the erk- / mapk pathway is predominantly activated by ceacam a. we have observed that uv-inactivated l -mhv fails to induce ifn-c production compared with the infectious l -mhv , indicating that a rna-dependent phase of the viral replication, rather than viral fixation to ceacam a receptor, is involved in the synergistic ifn-c production. the uv-inactivated virus may bind to the ceacam a receptor and be internalized, but cannot induce the translation of viral messenger rna. effectively, banerjee et al. have demonstrated that uv-inactivated mhv failed to activate mapks, whereas activation of p mapk and c-jun terminal kinase are essential for phosphorylation of the translation initiation factor e involved in the enhancing of translation rates of cap-containing messenger rna. an absence of synergistic ifn-c production was observed between l -mhv and ril- /ril- added at an interval of hr p.i. this result reinforces the implication of the synergistic production of ifn-c by nk cells occurring at an early stage in viral replication. recently, barr et al. have reported a rapid and transient activation of nk cells and ifn-c production following infection with herpes simplex virus type , resulting from a release of il- by dendritic cells. we cannot exclude the possibility that the loss of synergistic production of ifn-c when nk cells were pretreated with ril- /ril- for hr before infection with l -mhv may be consequent to impaired expression of ceacam a. indeed, ifn-c reduces the expression of ceacam a, as reported by vassao et al. consequently, nk cells may become resistant to a subsequent fixation of mhv , which in turn would affect ifn-c production. during the acute phase of viral infection, recruitment of nk cells from the bone marrow or spleen to the affected organ is generally observed. several studies have demonstrated that myeloid or splenic nk cells are essential during acute hepatitis, as these cells determined antiviral protection. , , , for the induction of an efficient antiviral response by recruited nk cells, the intrahepatic microenvironment must be adequate to activate them. hepatic nk cells normally colocalize at sites expressing viral antigen and ifn-c during infection. this environment may allow nk cells to receive cell-to-cell or cell-tomatrix contact signals that favour an il- -dependent ifn-c secretion, as nk cells are differently activated by il- depending on their localization. however, recruited nk cells in liver require activation by adequate levels of il- and il- to become high producers of ifn-c. it is known that liver kc produced il- and il- following various stimuli and activated intrahepatic nk and nkt cells to produce ifn-c. the results that we have provided are in agreement with a previous in vivo study, reporting that treatment with ril- and ril- of susceptible mice creates an anti-mhv state by significantly increasing ifn-c production. however, il- and il- were not induced in livers from l -mhv -infected c bl/ mice, preventing the synergistic ifn-c production by recruited nk cells. in e Ó blackwell publishing ltd, immunology, , e -e addition, the recruited intrahepatic nk cells from spleen and bone marrow may be infected by l -mhv produced by infected lsec or kc, leading to a virus-induced apoptosis phenomenon. the susceptibility of c bl/ mice to acute hepatitis reflects an inefficient innate antiviral response by low ifn-c production because of deficient il- /il- production and apoptosis of nk cells. the apoptosis of intrahepatic recruited nk cells, such as previously reported for spleen and bone marrow, [ ] [ ] [ ] is an additional evading mechanism of the innate antiviral defence. our results suggest that intrahepatic nk cells from resistant mice, as a/j or c h mice, would produce an efficient ifn-c response in the presence of adequate intrahepatic il- and il- levels. on other hand, hepatic nk cells also exert their functions in a tolerant environment and indeed, immunosuppressive cytokines such as transforming growth factor-b and il- determine the levels of ifn-c inhibition. however, we have recently observed that these immunosuppressive cytokines decrease in the liver of l -mhv -infected mice but not in mice infected with attenuated kc + lsecand kc -lsecvirus variants. the inability of intrahepatic nk cells to produce ifn-c may be related to their apoptosis, which is dependent on viral permissivity of kc and lsec to mhv replication. in fact, viral infection of kc and lsec may favour the viral infection of nk cells when these cells are in contact. these observations suggest that il- /il- produced in vivo by kc may be essential to preserve the antiviral functions of nk cells, and the integrity of lsec favours the survival of nk . cells recruited to the liver. however, the synergistic production of ifn-c occurred during the first steps of viral replication when the most of kc and lsec were not yet infected. further work is in progress to identify the step of viral replication involved in the synergistic ifn-c production by nk cells and the role of l -mhv -infected hepatocytes, kc and lsec in the intrahepatic functions of nk and nkt cells. nk cells and nkt cells in innate defense against viral infections interleukin- as an activator of natural killer cell-mediated antiviral response pathogenesis of murine cytomegalovirus infection in natural killer cell-depleted mice natural killer cells: primary target for hepatitis c virus immune evasion strategies different composition of intrahepatic lymphocytes in the immune-tolerance and immune-clearance phase of chronic hepatitis upregulation of major histocompatibility complex class i on liver cells by hepatitis c virus core protein via p and tap impairs natural killer cell cytotoxicity natural killer cells inhibit c virus expression tlr ligandinduced accumulation of activated splenic natural killer cells into liver stabilization of ifn-gamma mrna by mapk p in il- and il- -stimulated human nk cells regulation of itam positive receptors: role of il- and il- the virulence of mouse hepatitis virus , as evidenced by permissivity of cultured hepatic cells toward escaped mutants murine viral hepatitis involves nk cell depletion associated with virusinduced apoptosis natural killer cell depletion enhances virus synthesis and virusinduced hepatitis in vivo intrahepatic endothelial and kupffer cells involved in immunosuppressive and nk/nk-t cell disorders in the viral acute hepatitis virus specificity of the antiviral state induced by ifn gamma correlates with resistance to mhv infection in vivo depletion of interferon-gamma leads to susceptibility of a/j mice to mouse hepatitis virus infection evidence for differential roles for nkg d receptor signalling in innate host defense against coronavirus-induced neurological and liver disease arginine metabolism during macrophage autocrine activation and infection with mouse hepatitis virus cd a interactions between human melanoma and nk cells: a novel class i mhcindependent inhibitory mechanism of cytotoxicity pivotal role of ceacam protein in the inhibition of activated decidual lymphocyte functions pathogenic neisseria trigger expression of their carcinoembryonic antigen-related cellular adhesion molecule (ceacam : previously cd a) receptor on primary endothelial cells by activating the immediate early response transcription factor, nuclear factor-jb down-regulation of bpg viral receptor by interferon-c is related to the antiviral state and resistance to mouse hepatitis virus infection b lymphocyte and macrophage expression of carcinoembryonic antigen-related adhesion molecules that serve as receptors for murine coronavirus morphological analysis of mouse hepatitis virus a -induced pathology with regard to viral receptor expression biliary glycoprotein (cd a), a cell adhesion molecule of the immunoglobulin superfamily, on human lymphocytes: structure, expression and involvement in t cell activation biliary glycoprotein (bgp) expression on t cells and on a natural-killer-cell sub-population murine hepatitis virus strain induces the macrophage prothrombinase fgl- through p mitogenactivated protein kinase activation murine coronavirus replication-induced p mitogen-activated protein kinase activation promotes interleukin- production and virus replication in cultured cells beauchemin n. ceacam a )/) mice are completely resistant to infection by murine coronavirus mouse hepatitis virus a deletion of the carcinoembryonic antigen-related cell adhesion molecule (ceacam ) gene contributes to colon tumor progression in a murine model of carcinogenesis heterogeneity in evolutive patterns of inbred mice infected with a cloned substrain of mouse hepatitis virus type details of an isolation method for hepatic lymphocytes in mice mouse hepatitis viral infection induces an extrathymic differentiation of the specific intrahepatic alpha beta-tcr intermediate lfa- high t-cell population compartmental differences in nk cell responsiveness to il- during lymphocytic choriomeningitis virus infection cd a (ceacam ) expression by mouse natural killer cells ceacam a (cd a) promotes human monocyte survival via a phosphatidylinositol -kinase-and akt-dependent pathway biliary glycoprotein (bgpa, cd a, ceacam ) mediates inhibitory signals tl a synergizes with il- and il- to enhance ifn-gamma production in human t cells and nk cells il- time-dependently modulates th /th cytokine production by ligand-activated nkt cells low-virulent mouse hepatitis viruses exhibiting various tropisms in macrophages, t and b cell subpopulations, and thymic stromal cells mouse hepatitis virus pathogenicity expressed by a lytic viral infection in bone marrow . + mu + b lymphocyte subpopulations carcinoembryonic antigen (cea) inhibits nk killing via interaction with cea-related cell adhesion molecule biliary glycoprotein expression during embryogenesis: correlation with events of epithelial differentiation, mesenchymal-epithelial interactions, absorption, and myogenesis redefined nomenclature for members of the carcinoembryonic antigen family expression of ifn-c upon triggering of activating ly d nk receptors in vitro and in vivo: costimulation with il- or il- overrides inhibitory receptors a role for plasmacytoid dendritic cells in the rapid il- -dependent activation of nk cells following hsv- infection imbalanced intrahepatic expression of interleukin , interferon gamma, and interleukin in fulminant hepatitis b cutting edge: selective il- requirements for induction of compartmental for ifn-c responses during viral infection the liver as a crucial organ in the first line of host defense: the roles of kupffer cells, natural killer (nk) cells and nk . ag + t cells in t helper immune responses different modes of il- and tgf-b to inhibit cytokine-dependant ifn-c production: consequences for reversal of lipopolysaccharide desensitization this work was supported by a grant from nserc-canada (ll) and the canadian institutes of health research (nb). a. jacques was supported by a nserc fellowship from the canadian government. the authors thank dr tatiana scorza (université du québec à montréal) for revising this manuscript and her critical comments. all the authors have no potential conflicts of interest, including financial interests in any company or institution that might benefit from the publication of this work. key: cord- -i pic o authors: boris, bonaventure; antoine, rebendenne; de gracia francisco, garcia; marine, tauziet; joe, mckellar; valadão ana luiza, chaves; valérie, courgnaud; eric, bernard; laurence, briant; nathalie, gros; wassila, djilli; mary, arnaud-arnould; hugues, parrinello; stéphanie, rialle; olivier, moncorgé; caroline, goujon title: a genome-wide crispr/cas knock-out screen identifies the dead box rna helicase ddx as a broad antiviral inhibitor date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: i pic o genome-wide crispr/cas knock-out genetic screens are powerful approaches to unravel new regulators of viral infections. with the aim of identifying new cellular inhibitors of hiv- , we have developed a strategy in which we took advantage of the ability of type interferon (ifn) to potently inhibit hiv- infection, in order to create a cellular environment hostile to viral replication. this approach led to the identification of the dead-box rna helicase ddx as an intrinsic inhibitor of hiv- . depletion of endogenous ddx using sirna or crispr/cas knock-out increased hiv- infection, both in model cell lines and in physiological targets of hiv- , primary cd + t cells and monocyte-derived macrophages (mdms), and irrespectively of the ifn treatment. similarly, the overexpression of a dominant-negative mutant of ddx positively impacted hiv- infection, whereas wild-type ddx overexpression potently inhibited hiv- infection. the positive impact of endogenous ddx depletion on hiv- infection was directly correlated to an increase in viral dna accumulation. interestingly, proximity ligation assays showed that ddx , which can be mainly found in the nucleus but is also present in the cytoplasm, was in the close vicinity of hiv- capsid during infection of primary monocyte-derived macrophages. moreover, we show that ddx is also able to substantially decrease infection with other retroviruses and retrotransposition of long interspersed elements- (line- ). finally, we reveal that ddx potently inhibits other pathogenic viruses, including chikungunya virus and severe acute respiratory syndrome coronavirus (sars-cov- ). over the past years, a growing list of cellular proteins with various functions have been identified as capable of limiting different steps of hiv- life cycle (doyle et al., ; ghimire et al., ) . lentiviruses have generally evolved to counteract the action of these so-called restriction factors. however, type interferons (ifns) induce, through the expression of interferonstimulated genes, an antiviral state particularly efficient at inhibiting hiv- when cells are preexposed to ifn (ho et al., ; bednarik et al., ; coccia et al., ; baca-regen et al., ; goujon and malim, ; cheney and mcknight, ) . the dynamin-like gtpase mx , and, very recently, the restriction factor trim a, have both been shown to participate in this ifninduced inhibition (goujon et al., a; kane et al., ; ohainle et al., ; jimenez-guardeño et al., ) . with the hypothesis that additional hiv- inhibitors remained to be identified, we took advantage of the hostile environment induced by ifn to develop a wholegenome screen strategy in order to reveal such inhibitors. the development of crispr/cas as a genome editing tool in mammalian cells has been a major breakthrough, notably with the generation of pooled single guide (sg)rna libraries delivered with lentiviral vectors (lvs), allowing high-throughput screens at the whole-genome scale (shalem et al., , doench, ) . we used the genome-scale crispr knock-out (gecko) sgrna library developed by feng zhang's laboratory shalem et al., shalem et al., , to generate cell populations knocked-out for almost every human gene in the t g glioblastoma cell line. this model cell line is both highly permissive to lentiviral infection and potently able to suppress hiv- infection following ifn treatment (supplementary information, si figure ). the screen strategy is depicted in figure a . t g cells were first modified to stably express cas and a high number of t g-cas cells were then transduced with lvs coding sublibraries a or b, the two halves of the gecko library. a low multiplicity of infection (moi) was used to avoid multiple integration events and increase the probability to express only sgrna per cell. deep sequencing analysis of the gecko cell populations showed more than % coverage for both libraries (≥ reads for , and , sgrna-coding sequences out of gecko populations were subjected to type ifn treatment in order to induce the antiviral state and, h later, incubated with vsv-g-pseudotyped, hiv- based lvs coding for an antibiotic resistance cassette. two days later, the cells successfully infected despite the ifn treatment were selected by cell survival in the presence of the corresponding antibiotic. in order to enrich the population with mutants of interest and to limit the presence of false-positives, two additional rounds of ifn treatment, infection and selection (using different antibiotics) were performed ( figure a ). as expected, the cells enriched after each round of the screen became less refractory to hiv- infection following ifn treatment (si figure ) . . the gecko populations were then exposed to ifn for h and challenged with hiv- -based lvs coding for an antibiotic resistance gene. after selection by antibiotic addition, the surviving cells (i.e. efficiently infected despite the ifn treatment) were amplified. in total, the gecko population underwent three successive rounds of ifn treatment, infection and selection using lvs coding for different resistance cassettes. the genomic dnas of the initial gecko populations and the three-time selected populations were extracted, the sgrnacoding sequences were amplified by pcr and sequenced by next generation sequencing (ngs). b. the candidate genes were identified using the mageck computational statistical tool (li et al., ) . mageck establishes a robust rank aggregation (rra) score for each gene based on the sgrna enrichment and the ctrl ifnar mx wars ddx cldn reep ikbip topbp copg fgf spryd rdh dctpp prlr sun cxorf tmem a kcn lsm loc pkd ndpc kiaa mir- vstm a calu smarca dnajb fam a etnppl rhoc mir- - cxorf ccser slc a or n uba hspa b siae krt adra a lin tectb cdrt tceal trerf cd hsp aa ctnna plac l relb bsnd gecko population versus selected population number of sgrnas targeting the same gene. here, genes belonging to the ifn-response pathway (indicated in blue) and ddx (in red) are represented (together with their respective rank into brackets) for the independent screens (the results of which were merged in the analysis). c. t g/cd /cxcr /cas /firefly ko populations were generated for the best candidate genes of each screen. the control (ctrl) condition represents the mean of four negative control cell populations (i.e. expressing different non-targeting sgrnas) and ifnar and mx ko cell populations were used as positive controls. ko cell populations were pre-treated with ifn and infected with hiv- renilla. the cells were lysed h post-infection and the two luciferase signals were measured (renilla signals were normalized to internal firefly control). the ifn inhibition (corresponding to the ratio of the untreated / ifn-treated conditions) was calculated and sets at % inhibition for the average of the negative ctrl populations. a representative experiment is shown (mean and standard deviation from technical duplicates). to identify the genes of interest, the differential sgrna abundance between the starting gecko populations and the enriched ( -times selected) populations was analysed by ngs. , and , different sgrnas were identified (≥ reads) for screens a and b, which represented , % and % of the sgrnas present in the initial gecko population a and b, respectively. the mageck algorithm, which assigns a robust ranking aggregation (rra) score, was used to rank the gene candidates from each screen ( figure b ). for both screens, we observed a positive enrichment for genes (rra score > , ), with the best hits being ifnar , jak and stat ( figure b ). all the crucial mediators of the type ifn signalling cascade were present among the top hits in both screens (with the notable exception of stat ), validating our approach and confirming the identification of relevant genes. interestingly, most of the other positively selected genes displayed unknown functions or functions that were a priori unrelated to the ifn response pathway or to innate immunity. of note, very little overlap was observed between the two independent screens, performed with two different sub-libraries. however, a poor overlap between independent screens has been observed before and does not preclude obtaining valid data (doench, ) . therefore, the top candidate genes from each independent screen were selected for further validation. as a first validation step, the sequences of the most enriched sgrna for each gene were chosen and cloned into the lentiguide-puro vector . t g-cas cells expressing hiv- cd and cxcr receptors, as well as the firefly luciferase as internal control (t g cas /cd /cxcr /firefly cells), were transduced with the sgrna-expressing lvs to generate individual ko populations. four irrelevant, non-targeting sgrnas, as well as sgrnas targeting ifnar and mx , were used to generate negative and positive control populations, respectively. the ko cell populations were pre-treated with ifn and infected with an hiv- reporter virus expressing the renilla luciferase reporter and bearing hiv- envelope (nl - /nef-ires-renilla, hereafter called hiv- renilla). infection efficiency was analysed h later ( figure c ). as expected, ifnar and mx ko fully and partially rescued hiv- infection from the protective effect of ifn, respectively (goujon et al., a; bulli et al., ; xu et al., ) . the ko of two candidate genes, namely wars and ddx , allowed a partial rescue of hiv- infection from the ifn-induced inhibition, suggesting a potential role of these candidate genes. ddx is a member of the dexd/h box family of rna helicases with rna chaperone activities (uhlmann-schiffler et al., ) and, as such, retained our attention. indeed, various dead box helicases, such as ddx , ddx and ddx , are well-known to regulate hiv- life cycle (gringhuis et al., ; sithole et al., sithole et al., , soto-rifo et al., ; williams et al., ; yedavalli et al., ) . however, to our knowledge, the impact of ddx on hiv- replication had never been studied. in order to validate the effect of ddx ko on hiv- infection in another model cell line, two additional sgrnas were designed (sgrna- and - ) and used in parallel to the one identified in the gecko screen (sgddx - ) (figure a ). u -mg/cd /cxcr cells were used here, as we previously extensively characterized the ifn phenotype in these cells (goujon et al., a) . control and ddx ko cell populations were treated or not with ifn for h prior to infection with increasing amounts of hiv- renilla. ddx depletion improved hiv- infection with all three sgrnas used, confirming that endogenous ddx had a negative impact on hiv- infection. interestingly, the increase in infection efficiency induced by ddx ko was observed irrespectively of the ifn treatment. ddx is not known to be an isg (interferome database and our previous study (goujon et al., a) , geo accession number: gse ), which we confirmed in a number of cell types (si figure ). the fact that the ifn-induced state is at least partially saturable (si figure ) explains why an intrinsic inhibitor of hiv- , which is not regulated by ifn, could be identified by our approach: removing one barrier to infection presumably rendered the cells generally more permissive and, in this context, ifn had less of an impact. sgddx - , and sgddx - ) and different non-targeting sgrnas, respectively (for the ctrl condition, the average of the data obtained with the four cell populations is shown). cells were pre-treated or not with ifn h prior to infection with hiv- renilla ( ng p gag ) and the ratio of renilla/firefly activity was analysed, as in ) and the infection efficiency was measured by p gag intracellular staining followed by flow cytometry analysis. when indicated, the cells were treated with µm zidovudine (azidothymidine, azt) and lamivudine ( tc) reverse transcription inhibitors for h prior to infection. d. blood monocytes from healthy donors were isolated, differentiated into mdms, and transfected with non-targeting sirnas (sictrl and sictrl ) or sirnas targeting ddx (siddx - and siddx - ). two days after transfection, mdms were infected with a ccr tropic version of nl - -renilla ( ng p gag ). infection efficiencies were monitored h later by measuring renilla activity. the relative luminescence results from experiments performed with cells from different donors are shown. e. cd + t cells were isolated from peripheral blood mononuclear cells, activated with il- and phytohemagglutinin, and electroporated with cas -sgrna rnp complexes using two non-targeting sgrnas (sgctrl and sgctrl ) and five sgrnas targeting ddx (sgddx - , - , - , - , - ) two days later. four days after electroporation, the activated cd + t cells were infected with nl - renilla for h. relative infection efficiencies obtained with cells from three independent donors are shown. ddx protein levels were determined by immunoblot and actin served as a loading control (a representative experiment is shown in order to confirm ddx 's effect on hiv- infection with an independent approach, we used different sirnas to knockdown ddx expression. we observed that depleting ddx with sirnas (with > % efficiency both at the mrna and protein levels, figure b ) improved hiv- infection efficiency by to -fold when using an hiv- renilla reporter in u -mg/cd /cxcr cells, irrespectively of the presence of ifn ( figure b , right panel). of note, wild-type hiv- infection was also impacted by ddx silencing, as shown by capsid (p gag ) intracellular staining h post-infection ( figure c ). we then investigated whether ddx had an impact in hiv- primary target cells. in mdms, we observed that hiv- infection was increased by about fold following ddx silencing ( figure d ), whereas ddx mrna abundance was decreased by only % in these cells using sirnas (si figure ). as the sirna approach did not work in our hands in primary t cells, we used electroporation of pre-assembled cas -sgrna ribonucleoprotein complexes (rnps) to deplete ddx in primary cd + t cells ( figure e ). highly efficient depletion of ddx was obtained with all sgrnas as compared to the sgctrls ( figure e , bottom panel) and this depletion increased hiv- infection by -to -fold, showing a role of ddx as an intrinsic inhibitor of hiv- in primary cd + t cells. having established that endogenous ddx had an impact on hiv- infection, we then analysed the consequences of ddx overexpression. an irrelevant control (firefly) or ddx were ectopically expressed in u -mg/cd /cxcr and the cells were challenged with hiv- renilla ( figure f ). ddx ectopic expression induced a substantial inhibition of hiv- infection (about -fold decrease in infection efficiency in comparison to the control) ( figure f ). we then tested a mutant version of ddx that is unable to hydrolyse atp and may supposedly act as a dominant negative, ddx k e (granneman et al., ; rocak, ) (si figure ) . interestingly, the expression of ddx k e mutant increased hiv- infection by -fold, reminiscent of what we observed with ddx depletion. altogether, these data showed for the first time that endogenous ddx is able to intrinsically inhibit hiv- infection. in order to determine the step of hiv- life cycle affected by ddx , we first analysed viral entry with a blam-vpr assay (cavrois et al., ) . consistent with the observation that vsv-gpseudotyping did not bypass ddx -mediated inhibition of infection (si figure ), we observed that ddx silencing did not impact hiv- entry (si figure ) . we then quantified hiv- dna accumulation over time in ddx -silenced and control cells. ddx depletion increased by -to -fold the accumulation of early and late reverse transcript products ( figure a , b and c), as well as integrated provirus and -ltr circles at h post-infection ( figure d and e). more than % knockdown was achieved with both sirnas targeting ddx ( figure f ). these data suggested that ddx rna helicase could inhibit the reverse transcription process and/or impact the stability of hiv- genome, leading to a decrease in viral dna accumulation. we hypothesized that if that was the case, ddx should be found in close proximity to hiv- reverse transcription complexes during infection. in agreement with this, proximity ligation assay (pla) performed on mdms infected with hiv- showed that ddx could indeed be found in close vicinity of capsid ( figure f and g). we next examined the ability of ddx to inhibit infection by a range of primate lentiviruses including laboratory-adapted strains of hiv- , hiv- -transmitted founder strains, hiv- and simian immunodeficiency virus derived from the rhesus macaque (sivmac). tzm-bl cells were transfected with ddx -targeting or scramble sirnas and infected with vsv-g-pseudotyped lentiviruses. infection efficiencies were monitored after h by measuring β-galactosidase activity ( figure h ). ddx depletion increased infection levels with all the tested hiv- strains to the same extent than what was observed with hiv- nl - (i.e. -to -fold). hiv- rod and sivmac infection efficiencies were also slightly improved in the absence of ddx (by about -fold). the analysis was then extended to two non-primate lentiviruses, the equine infectious anaemia virus (eiav) and feline immunodeficiency virus (fiv), using gfp-coding lvs derived from these viruses in comparison to hiv- and hiv- lvs (si figure ). ddx antiviral activity appeared less potent on hiv- lvs compared to replication-competent, full-length hiv- , which might suggest that viral components, absent in lvs, could be playing a role in ddx -mediated hiv- inhibition. nevertheless, ddx depletion appeared to increase hiv- , hiv- and fiv lv infection to the same extent, i.e. by about -fold, whereas eiav infection was less impacted by ddx (si figure ). we extended this study to the gammaretrovirus murine leukaemia virus (mlv) and observed that ddx depletion led to an increase in infection with gfp-coding mlv vectors ( figure i ). these results strongly support a general antiviral activity of ddx against retroviruses. ddx can be found in the cytoplasm but is predominantly located in the nucleus (si figure ; uhlmann-schiffler et al., ; zyner et al., ) . considering that ddx showed a broad activity against retroviruses and seemed to act at the level of reverse transcription, we sought to investigate whether ddx could inhibit retrotransposons. long interspersed nuclear elements (line)- are non-ltr retrotransposons, which have been found to be active in the germ line (branciforte and martin, ; ergün et al., ; trelogan and martin, ) and in some somatic cells (belancio et al., ; muotri et al., ; rangwala et al., ) . interestingly, ddx was identified among the suppressors of line- retrotransposition through a genome-wide screen in k cells, although not further characterized (liu et al., ) . to confirm that ddx could inhibit line- retrotransposition, hek t cells were co-transfected with two different, gfpexpressing line- plasmids (rps or lre ) or an inactive line- (jm ) together with a ddx -or a control (firefly)-expressing plasmid ( figure j ). gfp-line- retrotransposition was quantified by flow-cytometry days post-transfection (moran et al., ) . because the gfp cassette is cloned in antisense and disrupted by an intron in this reporter system, gfp is only expressed after line- transcription, splicing and orf p-mediated reverse-transcription and integration into the host genome (moran et al., ) . considering that most line- replication cycles lead to truncations and defective integrations (gilbert et al., ) , gfp expression derived from a new integration is a relatively rare event and, as expected, the percentage of gfp+ cells observed was very low ( figure j ) (figure ) . strikingly, ddx depletion did not have an impact on iav or vsv replication ( figure a and b), thereby confirming that manipulating ddx expression did not have a broad and unspecific impact on target cells. however, depletion of endogenous ddx increased infection with zikv, chikv and sars-cov- , and had a particularly high impact on the latter two (up to log and log increase in infection efficiency in ddx -depleted cells in comparison to control cells, for chikv and sars-cov- , respectively, figure d and f). of note, silencing efficiency was similar in the two types of target cells used here ( figure e and g). interestingly, ddx was recently identified as a potential inhibitor of sars-cov- replication in a whole-genome crispr/cas screen in simian vero e cells (wei et al., ) , supporting our observations that endogenous ddx potently inhibits the replication of this highly pathogenic coronavirus. taken together, our data showed that ddx is a broad inhibitor of viral infections, albeit presenting some specificity. further work is now warranted to explore in depth the breadth of ddx antiviral activity. . in contrast, our study revealed broad activity of endogenous ddx among retroviruses and retroelements, which was observed in various cell types, including primary cd + t cells. interestingly, our pla assays showed a close proximity between ddx and hiv- capsid, which is a viral protein recently shown to remain associated with reverse transcription complexes until proviral dna integration in the nucleus (burdick et al., ; dharan et al., ; peng et al., ) . this observation could suggest a direct mode of action on viral ribonucleoprotein (rnp) complexes. interestingly, we observed that ddx was able to inhibit viruses from other families, which possess different replication strategies, including sars-cov- and chikv. however, ddx did not have an impact on all the viruses we tested, reminiscent of broad-spectrum antiviral inhibitors such as mxa, which show some specificity (haller et al., ) . ddx is known to be a non-processive helicase, which also possesses rna annealing activities and the ability to displace rna-binding proteins from single-stranded rnas (uhlmann-schiffler et al., ) . moreover, ddx binds g-quadruplexes (zyner et al., ) , which are secondary structures found in cellular and viral nucleic acids and involved in various processes, such as transcription, translation and replication (fay et al., ; ruggiero and richter, ) . all these known activities of ddx would be consistent with a potential role in rnp remodeling (uhlmann-schiffler et al., ; will et al., ) . nonetheless, further investigation will be needed to determine whether ddx acts directly by altering viral rnps, and, if that's the case, what are the determinants for viral rnp recognition. in conclusion, this work highlights the importance of understanding the mechanism of action of ddx rna helicase and its contribution to the control of rna virus replication, an understanding which may contribute to the development of future antiviral interventional strategies. plasmids were a gift from prof. f. zhang (addgene # , # , and # , respectively ). lvs coding for sgrnas targeting the candidate genes and bearing hiv- nl - , iiib and hiv- proviral clones have been described (adachi et al., ; simon et al., ; schaller et al., ) , as well as the transmitted founder hiv- molecular clones ch .t, ch .c, rejo.c (gifts from prof. b. hahn, (ochsenbauer et al., ) ) and hiv- rod and sivmac (ryan-graham and peden, ; gaddis et al., ) . pblam-vpr and padvantage have been described (cavrois et al., ) . gfp-coding hiv- based lv system (i.e. p . hiv- gag-pol, pmd.g, and gfp-coding minigenome), and hiv- , fiv, and eiavderived, gfp coding lvs, as well as mlv-derived, gfp coding retroviral vectors have all been described (naldini et al., ; bainbridge et al., ) , (o'rourke et al., ; saenz et al., ) . the line- plasmid rps-gfp pur (prps-gfp), rps-gfp jm pur (pjm ) and plre -gfp were developed by prof. kazazian's lab (moran et al., ; ostertag et al., ; goodier et al., ) . was added at u/ml for - h prior to virus infection or rna extraction, and azt and tc (aids reagent program) at µm for h prior to infection. sang, under agreement n° pler - . peripheral blood mononuclear cells (pbmcs) were isolated by centrifugation through a ficoll® paque plus cushion (sigma-aldrich). primary human cd + t cells and monocytes were purified by positive selection using cd and cd microbeads, respectively (miltenyi biotec), as previously described (goujon et al., a) . hiv- renilla and nl - hiv- were produced by standard pei transfection of hek t. when indicated, pmd.g was cotransfected with the provirus at a : ratio. the culture medium was changed h later, and virus-containing supernatants were harvested h later. viral particles were filtered, purified by ultracentrifugation through a sucrose cushion ( % weight/volume in tris-nacl-edta buffer) for min at °c and , rpm using a sw ti rotor (beckman coulter), resuspended in serum-free rpmi or dmem medium and stored in small aliquots at - °c. β-lactamase-vpr (blam-vpr)-carrying viruses, bearing the wild-type env, were produced by cotransfection of hek t cells with the nl - /nef-ires-renilla provirus expression vector, pblam-vpr and padvantage at a ratio of : : . , as previously described (cavrois et al., ) . viral particles were titrated using an hiv- p gag alpha-lisa kit and an envision plate reader (perkin elmer) and/or by determining their infection titers on target cells. wild-type and/or vsv-g pseudotyped-hiv- , target cells were plated at . x cells per well in -well plates or at x cells per well in -well plates and infected for - h before lysis and renilla (and firefly) luciferase activity measure (dual-luciferase® reporter assay system promega) or fixation with % paraformaldehyde (pfa)-pbs, permeabilization (perm/wash buffer, bdbiosciences) and intracellular staining with the anti-p gag kc -fitc antibody (beckman coulter), as described previously (goujon and malim, ) . for tzm-bl assays, the bgalactosidase activity was measured using the galacto-star™ system (thermofisher scientific). (corman et al., ) . blam-vpr assay for hiv- entry. these assays were performed as described previously (goujon and malim, ) . briefly, pnl - or ptopo- ltr (generated by ptopo cloning of a -ltr circle junction amplified from nl - infected cells, using ohc and u -reverse primers into pcr™ . -topo™) were diluted in ng/ml of salmon sperm dna to create dilution standards used to quantify relative cdna copy numbers and confirm the linearity of all assays. proximity ligation assay. the proximity ligation assays were performed using the duolink® in situ detection reagents (sigma-aldrich, duo ). for this, mdms were plated in -well plates with coverslips pre-treated with poly-l-lysin (sigma-aldrich) and infected with µg p gag of hiv- nl - (ba-l env) or mock-infected. h later, the cells were fixed with % paraformaldehyde in pbs x for min, washed in pbs x and permeabilized with . % triton x- for min. after a couple of washes in pbs x, either ngb buffer ( mm nh cl, % goat serum and % bovine serum albumin in pbs) or duolink® blocking solution was added for h. cells were incubated with ag . mouse anti-capsid antibody obtained from the national institutes of health (nih) aids reagent program (# ) and anti-ddx rabbit antibody (hpa , sigma-aldrich) diluted in ngb buffer or in duolink® blocking solution for h. after washes in pbs x, the cells were incubated with the duolink® in situ pla® probe anti-rabbit minus (duo ) and duolink® in situ pla® probe anti-mouse plus (duo ) for h at °c. after washes in pbs x, the ligation mix was added for min at °c. after washes in pbs x, the cells were incubated with the amplification mix for min at °c. finally, the cells were washed twice with pbs x and stained with hoechst at µg/ml for min, washed again and the coverslips mounted on slides in prolong mounting media (thermofisher scientific). zstack images were acquired using an lsm confocal microscope (zeiss) using a x lens. pla punctae quantification was performed using the fiji software (schindelin et al., ) . briefly, maximum z-projections were performed on each z-stack and the number of nuclei per field were quantified. then, by using a median filter and thresholding, pla punctae were isolated and quantified automatically using the analyse particles function. to obtain a mean number of dots per cell, the number of pla dots per field were averaged by the number of nuclei. for representative images, single cells were imaged using a lsm confocal microscope coupled with an airyscan module. processing of the raw airyscan images was performed on the zen black software. immunoblot analysis. cell pellets were lysed in sample buffer ( mm tris-hcl, ph . , . % sds, % glycerol, . % bromophenol blue, % β-mercaptoethanol), resolved by sds-page and analysed by immunoblotting using primary antibodies specific for human ddx (hpa , sigma-aldrich) and actin (mouse monoclonal a , sigma-aldrich), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence (bio-rad). images were acquired on a chemidoc™ gel imaging system (bio-rad). we have described previously iav nanoluciferase reporter virus generation (doyle et al., ) . stocks were titrated by plaque assays on mdck cells. iav challenges were performed in serum- zikv production and infection. the nanoluciferase expressing zikv construct has been described (mutso et al., ) . the corresponding linearized plasmid was transcribed in vitro using the sp mmessage mmachine™ (thermofischer scientific) and hek t cells were transfected with the transcribed rna. after days, supernatants were harvested, filtered and stock titers were determined by plaque assays on vero cells. for infections, . x cells per well in -well plates were infected, at the indicated mois. h after infection, cells were lysed and nanoluciferase activity was measured using the kit nano glo luciferase assay (promega). chikv production and infection. the gaussi luciferase coding chikv construct has been described (pohjala et al., ) . the linearized plasmid coding chikv genome was transcribed with the t mmessage mmachine kit (thermofischer scientific) and x hek t were transfected with - µg of transcribed rna, using lipofectamine (thermofischer scientific). after h, supernatants were harvested, filtered and viruses were then amplified on baby hamster kidney (bhk ) cells. stock titers were determined by plaque assays on vero cells. for infections, . x cells per well in -well plates were infected at the indicated mois. h after infection, cells were lysed and gaussia luciferase activity was measured using the pierce™ gaussia luciferase flash assay kit (thermofischer scientific). the sars-cov- betacov/france/idf / isolate was supplied by pr. sylvie van der werf and the national reference centre for respiratory viruses hosted by institut pasteur (paris, france). the virus was amplified in vero e cells (moi , ) in serum-free media supplemented with , µg/ml l- -p-tosylamino- -phenylethyl chloromethylketone (tpck)-treated trypsin (sigma-aldrich). the supernatant was harvested at h post infection when cytopathic effects were observed (with around % cell death), cell debris were removed by centrifugation, and aliquots stored at - c. viral supernatants were titrated by plaque assays in vero e cells. typical titers were . plaque forming units (pfu)/ml. infections of a -ace cells were performed at the indicated multiplicity of infection (moi; as calculated from titers obtained in vero e cells) in serum-free dmem and % serum-containing dmem, respectively. the viral input was left for the duration of the experiment and cells lysed at h post-infection for rt-qpcr analysis. the datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request. requests for material should be addressed to caroline goujon or olivier moncorgé at the corresponding address above, or to addgene for the plasmids with an addgene number. b.b. and cg designed the study, analysed the data and wrote the manuscript. b.b. and c.g. and mageck analyses, respectively. all authors have read and approved the manuscript. the authors have no conflicts of interest to declare in relation to this manuscript. supplementary production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone alpha interferoninduced antiretroviral activities: restriction of viral nucleic acid synthesis and progeny virion production in human immunodeficiency virus type -infected monocytes in vivo gene transfer to the mouse eye using an hiv-based lentiviral vector; efficient long-term transduction of corneal endothelium and retinal pigment epithelium inhibition of human immunodeficiency virus (hiv) replication by hiv-trans-activated alpha -interferon somatic expression of line- elements in human tissues developmental and cell type specificity of 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replication of hiv ddx potentiates hiv- transcription as a co-factor of tat the dead-box helicase ddx substitutes for the cap-binding protein eif e to promote compartmentalized translation initiation of the hiv- genomic rna tightly regulated, developmentally specific expression of the first open reading frame from line- during mouse embryogenesis ddx p--a human dead box protein with rna chaperone activities the dead box protein ddx p modulates the function of aspp , a stimulator of apoptosis genome-wide crispr screens reveal host factors critical for sars-cov- infection characterization of novel sf b and s u snrnp proteins, including a human prp p homologue and an sf b dead-box protein identification of rna helicases in human immunodeficiency virus (hiv- ) replication -a targeted small interfering rna library screen using pseudotyped and wt hiv- fastq screen: a tool for multi-genome mapping and quality control role of mxb in alpha interferon-mediated inhibition of hiv requirement of ddx dead box rna helicase for hiv- rev-rre export function genetic interactions of g-quadruplexes in humans indirect immunofluorescence analysis of endogenous ddx in primary mdms. mdms were fixed, endogenous ddx and the nuclei were visualized using ddx -specific antibodies and hoechst staining, respectively, and confocal microscopy we wish to thank tom doyle and chad swanson for their useful comments on the manuscript, key: cord- - k p authors: yu, chengjun; kang, lian; chen, jiadong; zang, na title: evaluation of safety, efficacy, tolerability, and treatment-related outcomes of type i interferons for human coronaviruses (hcovs) infection in clinical practice: an updated critical systematic review and meta-analysis date: - - journal: int immunopharmacol doi: . /j.intimp. . sha: doc_id: cord_uid: k p background: there is no vaccine or specific antiviral treatment for hcovs infection. the use of type i interferons for coronavirus is still under great debate in clinical practice. materials and methods: a literature search of all relevant studies published on pubmed, cochrane library, web of science database, science direct, wanfang data, and china national knowledge infrastructure (cnki) until february was performed. results: of the identified articles, only studies were included in the final analysis. comorbidities and delay in diagnosis were significantly associated with case mortality. type i interferons seem to improve respiratory distress, relieve lung abnormalities, present better saturation, reduce needs for supplemental oxygen support. type i interferons seem to be well tolerated, and don’t increase life threating adverse effects. data on ifns in hcovs are limited, heterogenous and mainly observational. conclusions: current data do not allow making regarding robust commendations for the use of ifns in hcovs in general or in specific subtype. but we still recommend type i interferons serving as first-line antivirals in hcovs infections within local protocols, and interferons may be adopted to the treatments of the sars-cov- as well. well-designed large-scale prospective randomized control trials are greatly needed to provide more robust evidence on this topic. coronaviruses are single -stranded and positive -sense rna viruses. among both sars-cov and mers-cov caused outbreaks affecting multiple countries, severe disease, and global threatening, for its widespread infectivity, rapid progress, high variance and mortality rate, and nonspecial treatment, somewhat the same as sars-cov- in wuhan, china . as for treatments, currently there is no defined primary remedy, vaccination or prophylaxis. nowadays, treatments for such cases range from supportive treatment (including fluid balance, nutrition support, invasive ventilation, renal replacement therapy, vasopressors, corticosteroids, immunoglobulins, etc.) to antiviral treatment, or both [ ] [ ] [ ] [ ] [ ] . the specific antiviral treatments were interferons (ifn), ribavirin, lopinavir, and other related antiviral agents. clinically, the use of specific antivirals, especially the utility of ifns, is still under great debate, for its efficacy, safety, and treatment-related adverse effects. initial in vitro investigations demonstrated type i interferons (ifn-α, ifn-β) to inhibit replication of sars coronavirus (sars-cov) . based on previous studies, morgenstern et al. investigated the combination effect of ifn-β and ribavirin to prevent sars-cov, and yield potential benefits of the ribavirin plus ifn-β for the treatment of sars . illuminated by the possible antiviral treatment for sars, several in vitro studies determined a possible efficacious effect of ifn-α b and ribavirin in the treatment of mers-cov infection , . subsequently, the same investigators further examined the efficacy of these drugs in an animal study (macaques), hours after they were inoculated with mers-cov with favorable outcomes could make contributions to increase survival rate, improve oxygen saturation and associated with a more rapid resolution of pyrexia or radiographic lung opacities and respiratory improvements - , or even prophylaxis efficacy , . a review of such anecdotal experiences is greatly needed for the more rational use of type i ifns for coronavirus. therefore, we conducted this updated systematic review and meta-analysis to recapitulate relevant studies to evaluate the safety, efficacy, tolerability and treatment-related outcomes of type i ifns for coronavirus infection in clinical practice, with expectation to provide more robust evidence whether ifns should be served as first-line agents for coronavirus infection, including the sars-cov- . this study was performed in accordance with prisma (preferred reporting items for systematic reviews and meta-analyses) . the systematic literature search of databases was conducted by two independent reviewers on february, . these articles that contained relevant information on ifn and coronavirus were initially searched on pubmed, cochrane library, web of science database, science direct, wanfang data, and china national knowledge infrastructure (cnki), without time period, language, and region restriction. a mesh terms search and keywords search were combined. the references of the included studies and reviews were also manually searched. we used the following search terms using the boolean operators: # "interferon" or "ifn" or "antivir*" or "drug effect" or "drug ther*" or "combination drug ther*" and # "coronavirus" or "middle east respiratory syndrome: or "mers-cov" or "mers virus*" or "sars" or "severe acute respiratory syndrome" or "sars-cov" ( ) clinical trials regarding type i ifn (ifn-α, ifn-β) solely or combinationally for the treatment of coronavirus infections or prophylaxis; ( ) human studies, regardless of randomized controlled trial (rct), case-control studies, observational study, cohort studies or case series; ( ) compared the treatment outcomes of ifn and other remedies (supportive treatment only, corticosteroids, or between ifns). ( ) in vitro studies or animal models; ( ) cellular, molecular, histological, or pathological mechanism studies or hypothesis; ( ) pharmaceutical mechanism or toxicology hypothesis addressing ifn or related agents on coronavirus; ( ) other antiviral therapies that do not include type i ifn; ( ) repeated studies, staged trials or studies without comparison information; ( ) reviews, comments or letters. two investigators independently reviewed the electronically and manually retrieved articles. after screening the titles and abstracts, potentially relevant studies were selected, and a full-text review was performed. all disagreements were solved by discussion or, still unsolved, by a third supervisor. each included article was thoroughly reviewed, and the following baseline information were extracted (table ) : first author, publication year, region, study type, participants, diagnostic method of coronavirus, data collection method, time from admission to treatment start, time from diagnosis to treatment start, primary endpoints, and treatment-related adverse effects. in addition, the study design, treatment plan (including ifn dosage, frequency and duration), main findings and conclusions were extracted in detail in table . data on total mortality rate, -day survival, -day survival, -month survival, transferring rate to intensive care unit (icu), required intubation and mechanical ventilation, resolution of pyrexia, and respiratory improvement (days) were recorded for possible meta-analysis. for better understanding of severity and case mortality rate of coronavirus, we divided these patients into critically ill patients and mild ill patient. critically ill defined as coronavirus-infected patients with other severe comorbidities, respiratory distress or failure, directly or indirectly transferred to icu, needing intubation, mechanical ventilation, or extracorporeal membrane oxygenation (ecmo), when admitted to primary treatment. mild ill patients defined as these real-time polymerase chain reaction (rt-pcr) or other laboratory confirmed coronavirus infected asymptomatic or otherwise laboratory well patients. dichotomous variables were analyzed using review manager version . (cochrane collaboration, oxford, united kingdom) and the mantel-haenszel method. the crude ors and their % confidence intervals (cis) were calculated. for continuous variables, mean difference (md) with % ci was applied. the single-rate meta-analysis was performed using stata . software (stata corporation, college station, texas, usa), which assigned a weight to each study based on both withinstudy variance and between-study heterogeneity. heterogeneity of these manuscripts was tested using both the chi-square test (with a low p-value indicating high heterogeneity, and p-value ≥ . indicating low heterogeneity) and i index statistics ( % indicating no inter-study heterogeneity) . when i was < %, the fixed effects model was applied; otherwise, the random effects model was applied . in all analysis, p-value less than . was considered significant. the initial database search yielded articles ( figure ). in addition, six articles were added by manual searching from retrieved study lists and relevant reviews, and two papers added by expert suggestion. after eliminating duplicate articles, titles and abstracts were screened. after comprehensively screening full texts, only studies complied with the eligibility criteria and were included at last. among these, three were rcts , , one of which has not published yet , four were retrospective cohort studies , , , , four were case series , , , , one prospective cohort study , one open-label preliminary study , one open-label prospective randomized study , and one retrospective observational study . turner et al. firstly explored whether the prophylactic recombinant ifns could decrease cov- e catch rate or reduce the severity of coronavirus cold symptoms in in a well-design randomized placebo-control study . they recruited absolutely healthy volunteers for participants. in their study, they found that the cold-catch rate, the mean nasal symptom score, the mean total symptom score, and the mean number of days with total symptom score ＞ were much lower in ifns prophylaxis group than placebo group, all reached significant difference ( table ) on account of insufficient data, inconsistent initial study design, and complexity of human bodies and case variance, statistical synthesizing was impossible regarding abovementioned parameters ( table ) . as for total mortality rate, we investigated the variance between critically and mild ill patients. on the basis of our analysis, the mortality rate was . % ( % confidence interval: . %- . %, i = . %) and . % ( % confidence interval: . %- . %, i = . %) in critically and mild ill coronavirus-infected patients. both presented high heterogeneity and the random effect model was used (figure ). our study systematically investigated the application of type i interferons for hcovs infection in clinical practice. according to our review, ifns mainly acted a vital role in rapid resolution of lung abnormalities, respiratory improvements, better oxygen saturation, reduced needs for supplemental oxygen support, and less of an increase in creatine kinase level, which are indispensable for advanced life support and further increase survival. in the meantime, several adverse effects were detected, including drop in platelet, drop in hemoglobin, rise in lipase or bilirubin, and emergency of pancreatitis (only one critically ill case at terminal phage of disease), but these treatment-related outcomes couldn't rule out the effects of other agents like ribavirin, and still need further investigation . these side effects were not life threating, and much easier to solve compared with respiratory distress, intractable hyoxemia, or rapid progress of renal or hepatic failure. the tolerability of type i ifns was acceptable, and no premature discontinuation of ifn secondary to adverse effects was found in all case. apart from remedy effect of ifn in coronavirus infection described above, we also found the prophylaxis efficacy of ifn in coronavirus infection , , which increased and enhanced the utility of ifn in clinical practice. al-tawfiq et al. reported their experience of five critically ill patients that were all died of multi-organ failure after treatment of ifn plus ribavirin and concluded that combination antivirals may not contribute to mers-cov-infected patients , as preclinical data suggested. in addition, vast majority of adverse effects were reported by them. we think this conclusion may be not objective. they included only critically ill patients with multiple comorbidities, all under mechanical ventilation and, most importantly, diagnosed late in admission. the mortality rate was significantly related with comorbidities, like chronic renal failure, diabetes mellitus, coronary artery disease, hypotension, elevated creatinine, anemia, etc., and age more than -year , . what's more, severity of illness was the greatest predictor of reduced survival in the multivariate analysis . as for adverse effects, this couldn't absolutely ascribe to ifn alone, critically ill patients may suffer from respiratory abnormality, internal environment disturbance, and other disease-related complications. beyond this, some side effects, at least drop in hemoglobin level, was found related with ribavirin, for its temporal toxicity , . cheng et al. concluded from their research that even with steroid therapy alone, the mortality rate appeared to be low when compared with conservative treatment for pneumonia caused by sars-cov, and the combination of an effective antiviral and steroid was associated with a better outcome . the same results from omrani et al., a retrospective cohort study, ifn plus ribavirin have a decreased mortality rate than supportive treatment only, and didn't significantly increase adverse effects. apart from antiviral therapy, management should primarily focus on strict lungprotective ventilation . our analysis indicated that the overall mortality rate of coronavirus-infected critically ill patients was about . %, and . % in mild ill patients, in accordance with imran khalid's conclusion that delay in remedy would increase mortality . but this caculated mortality rate may be higher that its actual level, for publication bias. as a consequence, early dignosis and intervention would greatly improve outcomes . this also suggested us paying attention to early screen of close contacts and suspected patients of such disease was equally crucial. clinically, combination of ifn and ribavirin are reletively widely adopted to coronavirus onfection, though lack of robust evidence . one well-designed randomized placebo-control trial regarding effects of recombinant ifn-β b plus opinavir/ritonavir was registed in and still pending completion . in this rct, primary and secondary outcomes are mortality in the icu, mortality in the hospital and -day mortality, -day mortality, sequential organ failure assessment scores at baseline and on study days , , , , and . this seems to be the best conceived trial to determine the efficacy of antivirals in coronavirus infection. we are looking forward to the successful administration of this clinical trial, and calling for largescale prospective randomized studies to assess the role of antivirals for the treatments of coronavirus, to better guide clinical practice. in conclusion, type i interferons seem to improve respiratory distress, relieve lung abnormalities, present better saturation, reduce needs for supplemental oxygen support. type i interferons seem to be well tolerated, and don't increase life threating adverse effects. we still recommend type i interferons serving as first-line antivirals in coronavirus infections within local protocols, with timely administration and monitoring of adverse events. and interferons may be used to treat sars-cov- infected patients. well-designed large-scale prospective randomized control trials are greatly needed to provide more robust evidence on this topic. no financial or nonfinancial benefits have been received or will be received from any table the study designs, treatment strategies, and outcomes of included studies for evaluation of safety, efficacy, tolerability, and treatment-related outcomes of interferon for coronavirus infection in clinical practice the severe acute respiratory syndrome screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study. clinical microbiology and infection : the official publication of the clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and meta-analysis isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus from patients with pneumonia in china ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome coronavirus in children clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection a hospital outbreak of severe acute respiratory syndrome in guangzhou inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs ribavirin and interferon-beta synergistically inhibit sarsassociated coronavirus replication in animal and human cell lines inhibition of novel β coronavirus replication by a combination of interferon-α b and ribavirin mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment treatment with interferon-α b and ribavirin improves outcome in none. the study was supported by the foundation of children's hospital of chongqing overseas retureness (cx ). not required. key: cord- - eatplc authors: wang, yongjin; shi, huiling; rigolet, pascal; wu, nannan; zhu, lichen; xi, xu-guang; vabret, astrid; wang, xiaoming; wang, tianhou title: nsp proteins of group i and sars coronaviruses share structural and functional similarities date: - - journal: infect genet evol doi: . /j.meegid. . . sha: doc_id: cord_uid: eatplc the nsp protein of the highly pathogenic sars coronavirus suppresses host protein synthesis, including genes involved in the innate immune system. a bioinformatic analysis revealed that the nsp proteins of group i and sars coronaviruses have similar structures. nsp proteins of group i coronaviruses interacted with host ribosomal s subunit and did not inhibit irf- activation. however, synthesis of host immune and non-immune proteins was inhibited by nsp proteins at both transcriptional and translational levels, similar to sars coronavirus nsp . these results indicate that different coronaviruses might employ the same nsp mechanism to antagonize host innate immunity and cell proliferation. however, nsp may not be the key determinant of viral pathogenicity, or the factor used by the sars coronavirus to evade host innate immunity. viral infections are sensed by the host innate immune system through toll-like receptors and retinoic acid-inducible gene-i-like receptors, which trigger innate immune signal transduction, leading to production of type i interferons (ifns), and hundreds of proinflammatory cytokines that suppress viral spread (kawai and akira, ) . in return, viruses have evolved at least three mechanisms to evade or antagonize host innate immunity. one is blocking ifn induction. host innate immunity is initiated by recognition of cellular rna sensors and viral rna, which in turn interacts with the adaptor ips- to activate the ifn transcription factor irf- and nf-kb through kinases tbk- /ikki and ikka/b (kawai and akira, ) . many viruses encode proteins to block the ifn induction pathway. examples include the influenza a virus ns protein (lu et al., ; garcia-sastre, ) , the reovirus sigma protein (jacobs and langland, ) , the ebola virus vp protein (cardenas et al., ) , the poxvirus e l protein (xiang et al., ) , the herpes simplex virus us protein (poppers et al., ) and the murine cytomegalovirus m and m proteins (valchanova et al., ) . the second mechanism is interfering with ifn-activated signaling, mainly through interacting with the janus kinases jak- , tyk- , stat- and stat- to block the jak-stat signaling pathway. examples include the ebola virus vp protein (reid et al., ) , the paramyxovirus c and v proteins (didcock et al., ; gotoh et al., ) , and the rabies virus p protein (brzozka et al., ) . the third method is inhibiting the specific antiviral proteins that mediate the antiviral state. viral dsrna-binding proteins are mostly studied for their capability of preventing activation of pkr or the - oas/rnasel system, as demonstrated by the reovirus sigma (imani and jacobs, ) , the herpesvirus us protein (poppers et al., ) , the poxvirus e l (langland and jacobs, ) , the cytomegaloviruses dsrnabinding proteins (hakki and geballe, ) , and the influenza virus ns protein (min and krug, ) . coronaviruses are important human and animal pathogens that are divided into three groups based on serological criteria. the group ii coronaviruses severe acute respiratory syndrome coronavirus (sars-cov) and mouse hepatitis coronavirus (mhv) encode a number of proteins that antagonize host innate immunity. the orf b and orf proteins inhibit both ifn synthesis and signaling (kopecky-bromberg et al., ) . the nucleocapsid protein inhibits nfkb promoter and ifn synthesis (kopecky-bromberg et al., ; ye et al., ) . the papain-like protease interacts with irf- and inhibits its phosphorylation and nuclear translocation (devaraj et al., ) . the a protein causes endoplasmic reticulum stress, and antagonizes ifn responses and innate immunity (minakshi et al., ) . finally, the m protein associates with rig-i, tbk , ikkepsilon, and traf to inhibit the activation of irf- /irf- the nsp protein of the highly pathogenic sars coronavirus suppresses host protein synthesis, including genes involved in the innate immune system. a bioinformatic analysis revealed that the nsp proteins of group i and sars coronaviruses have similar structures. nsp proteins of group i coronaviruses interacted with host ribosomal s subunit and did not inhibit irf- activation. however, synthesis of host immune and non-immune proteins was inhibited by nsp proteins at both transcriptional and translational levels, similar to sars coronavirus nsp . these results indicate that different coronaviruses might employ the same nsp mechanism to antagonize host innate immunity and cell proliferation. however, nsp may not be the key determinant of viral pathogenicity, or the factor used by the sars coronavirus to evade host innate immunity. ß elsevier b.v. all rights reserved. transcription factors (siu et al., ) . nsp is another intensively studied viral protein. the nsp proteins of sars-cov and mhv promote host mrna degradation and suppress host gene expression (kamitani et al., ; zust et al., ; narayanan et al., ) . nuclear magnetic resonance (nmr) analysis revealed that sars-cov nsp has a novel b-barrel structure mixed with ahelixes (almeida et al., ) . more recently, sars-cov nsp was found to block host translational machinery function by binding to the ribosome small subunit (kamitani et al., ) . these studies suggest that coronavirus nsp is a major virulence and pathogenicity factor (kamitani et al., ; wathelet et al., ; zust et al., ) . little is known about the biological function of group i coronavirus nsp proteins. in this study, we conducted a comparative study of nsp proteins from groups i and ii coronaviruses. we compared models for nsp proteins of hcov- e and hcov-nl with models of sars-cov. we analyzed the interaction between nsp proteins and cellular proteins by coimmunoprecipitation (co-ip). finally, we systematically investigated the mechanism by which group i coronavirus nsp proteins suppress host protein synthesis, including in the innate immune system. the cell line was maintained at c in a % co incubator in dulbecco's modified eagle medium (dmem; gibco) supplemented with % fetal bovine serum (excell bio, new zealand). newcastle disease virus (ndv) was harvested from chicken embryos. plasmids pcdna-sarsnsp , pcdna- ensp and pcdna-nl nsp were constructed by reverse-transcription pcr amplification of nsp genes from sars-cov strain tor , hcov- e strain atcc vr- and hcov-nl strain amsterdam i genomic rna and inserted into pcdna . vector (invitrogen, carlsbad, ca) under the control of the human cytomegalovirus (cmv) promoter. an ha tag was added at the c-terminus for all constructs. plasmids pgl . , pgl . and pgl . , which contain renilla luciferase reporter genes driven by the sv , herpes simplex virus thymidine kinase (hsv-tk) and cmv promoters, respectively, were from promega (madison, wi). plasmids pgl -hifnb-p and pgl -hisg -p were constructed by pcr amplification of the human ifn-b gene promotor region (À to + ) or the human interferon stimulated gene (isg ) promotor region (À to + ) from genomic dna, and cloned into pgl . (promega). structures for the nsp proteins of hcov- e and hcov-nl were computed with modeller software (marti-renom et al., ) using the solution structure of nsp - from the sars-cov (pdb code hsx) as a template structure (almeida et al., ) . sequence alignment of the nsp proteins from hcov- e, hcov-nl and sars-cov was performed with the program clustalw and refined manually. cells in six-well plate were transfected with ug of nsp expressing plasmids or pcdna . control plasmid (with ha tag) using lipofectamin (invitrogen). cells were harvested h after transfection and washed three times with phosphatebuffered saline (pbs). half the cells were treated with native lysis buffer (promega) for min on ice, and centrifuged, and the supernatant was used for immunoprecipitation using anti-ha antibody-coupled agarose beads (pierce) according to the manufacturer's instructions. eluted proteins were separated by % sds-page followed by immunoblotting analysis using anti-s polyclonal antibodies (genscript). proteins from the other aliquot of cells were separated by % sds-page followed by immunoblotting using anti-ha or anti-b-actin antibodies (abcam, hongkong). transfection of cells was as above for h, followed by ndv infection for h. ifn-b levels in cell supernatants were measured using a human ifn-b enzyme-linked immunosorbent (elisa) kit according to the manufacturer's instructions (pbl interferonsource, nj). cells were washed three times with pbs. proteins were separated by % sds-page gel and immunoblotted with anti-irf- or anti-phospho-irf- (ser ) antibodies (cell signaling, beverly, ma). transfection of cells was as above for h, followed by ndv infection for h. alternatively, cells were cotransfected with nsp -expressing plasmids or pcdna . control plasmid together with pgl -hifnb-p or pgl . for h. cell rna was extracted using a cell total rna isolation kit (axygen), followed by dnase i (fermentas) treatment for min at c to remove genomic dna. rna was reverse transcribed using a primescript first-strand cdna synthesis kit (takara, dalian, china), and sybr green-based quantitative real-time pcr was performed in a cfx machine (bio-rad) using a perfect real-time pcr kit (takara). primers were listed in table lipofectamine (invitrogen) was used to transfect cells with plasmids pgl -hifnb-p or pgl -hisg -p, and stable cell lines were selected with . mg/ml g (invitrogen) and subcloned. the stable cell lines were transfected with nsp expressing plasmids for h and then challenged with ndv for h or were co-transfected with nsp -expressing plasmids and poly (i:c) (sigma) for h. cells in microplates were disrupted with ml of passive lysis buffer (promega) and firefly luciferase activity measured using a dual-luciferase reporter assay system (promega) and a synergy hybrid multi-mode microplate reader (biotek, winooski). alternatively, normal cells were cotransfected with nsp -expressing plasmids with pgl . , pgl . or pgl . for h, and renilla luciferase activity was measured. data were analyzed with the paired student's t-test assuming that the values followed a gaussian distribution. a p-value of < . was considered significant. the three-dimensional structure of group i coronavirus nsp has not been previously solved. to evaluate the general fold of these proteins, we built models for nsp proteins of hcov- e and hcov-nl based on the solution structure of nsp - from the sars-cov (almeida et al., ) . sequence alignment showed only % identity and % similarity between the hcov- e and sars-cov nsp proteins. similarly, the nsp proteins of hcov-nl and sars-cov share only % identity and % similarity (fig. a) . nevertheless, a careful inspection of several nsp regions revealed conserved hydrophobic clusters counterbalancing the regions of low identity and low similarity. thus, % of the hydrophobic residues of hcov- e nsp , and % of the hydrophobic residues of hcov-nl nsp are conserved, or replaced by other hydrophobic residues, in the sars-cov nsp (fig. a) . these conserved or similar hydrophobic residues are not randomly positioned. most are found within, or very close to the regular secondary structure elements of nsp - of sars-cov (fig. a) . moreover, the hydrophobic residues crucial for the original b-barrel of sars-cov nsp - (almeida et al., ) are conserved or replaced by other hydrophobic residues in the hcov- e and hcov-nl nsp proteins ( fig. a and c) . the hydrophobic cluster analysis identified stronger signatures of the nsp fold, centered on the secondary structure elements. modeling of the hcov- e and hcov-nl nsp proteins led to three-dimensional structures that conserved the regular secondary structure elements of the unique b-barrel of sars-cov nsp - , while the loops linking the secondary structures were less similar (fig. b) . intriguingly, hcov- e nsp contains five cysteine residues, four of which are potentially exposed to solvent. taking into account the distances separating the cysteines, formation of disulfide bridges is unlikely. taken together, these findings reinforce the hypothesis that hcov- e and hcov-nl nsp proteins fold in a manner similar to sars-cov nsp - , and suggest that these proteins have similar structural and functional relationships. sars-cov nsp suppresses host cell protein synthesis by binding to the host cell ribosomal s subunit (kamitani et al., ). based on the bioinformatics results, we examined whether nsp proteins of group i coronaviruses also interacted with the ribosomal proteins. ha-tagged nsp proteins of hcov- e, hcov-nl and sars-cov were expressed in cells, and immunoprecipitated with anti-ha antibody. immunoblotting with anti-s antibody detected a band of $ kda from all three nsp proteinexpressing cells, which corresponds to the predicted size for the ribosomal protein s . the nsp proteins were detected using anti-ha antibody. the s and nsp proteins were not detected in mocktransfected cells. as controls, b-actin was detected in all cell lysates ( fig. a) . we next examined whether the activation of the ifn transcriptional factor irf- was inhibited by nsp proteins. innate immune signal transduction was stimulated by ndv infection in cells transfected with plasmids-expressing nsp from hcov- e, hcov-nl or sars-cov, or with a control plasmid. immunoblotting with anti-phospho-irf- (ser ) antibody showed a consistent, homogenous band for the phosphorylated irf- protein in cells expressing the three nsp proteins, and in mock-transfected cells, but not in negative control cells that were not stimulated with ndv. moreover, no obvious inhibition effect was observed in nsp -transfected cells compared to the mock-transfected cells. expression of irf- and b-actin proteins was consistently detected in all cell lysates (fig. b ). [ ( f i g . _ ) t d $ f i g ] we further investigated whether group i coronavirus nsp proteins inhibit ifn-b and luciferase transcription. nsp -plasmids were transfected into cells, and ifn-b or luciferase transcription was stimulated by ndv or ifn-b promoter-or cmv promoterdriven plasmids, followed by quantification of ifn-b mrna by real-time pcr. as shown in fig. , the mrna levels for cell ifn-b, ifn-b promoter-driven firefly luciferase or cmv promoter-driven renilla luciferase were inhibited by . - dct values by nsp proteins from hcov- e, hcov-nl and sars-cov. suppression of host protein synthesis by sars-cov nsp protein, including in the innate immune system, has been seen in several studies (kamitani et al., ; zust et al., ; narayanan et al., ) . since the group i coronavirus nsp proteins also interact with the cellular translational machinery, we examined the influence of hcov- e and hcov-nl nsp proteins on host immune and non-immune protein synthesis. luciferase reporter assays showed that synthesis of the innate immune promoter ifn-band isg -driven genes was suppressed by - -folds in hcov- e and hcov-nl nsp -expressing cells (fig. a) . synthesis of non-immune promoter-driven genes, including for sv , hsv-tk and cmv promoters, was inhibited to a similar extent by the two group i coronavirus nsp proteins (fig. b) . in contrast, sars-cov nsp suppressed promoter activity by only - -folds by all assays (fig. ) . titration of the released ifnb proteins in cell supernatant by elisa revealed similar results. the ifn-b levels in coronavirus nsp -expressing cells were - -folds lower than in control cells. (fig. c) . studies on nsp proteins of group ii coronavirus sars-cov and mhv revealed a novel mechanism for interaction between host and viral proteins. group i coronaviruses also encode nsp proteins of only about amino acids, with relatively high conservation within the group, but with a high degree of polymorphism with sars-cov nsp , which has amino acids. however, the nmr analysis revealed that the segment from residue to of sars-cov nsp determined its core structure (almeida et al., ) , and this corresponded to the overall length of the group i coronavirus nsp proteins. therefore, we built models for the nsp proteins of hcov- e and hcov-nl , two low-pathogenic group i human coronaviruses, for comparison with nsp from the highly pathogenic sars-cov. careful inspection of the sequence alignment showed that most of the structurally important hydrophobic residues are conserved or similar for nsp proteins from hcov- e, hcov-nl and sars-cov. computational modeling demonstrated overall similarity in the three-dimensional nsp structures from the two group i coronavirus and sars-cov. bioinformatics suggest that group i coronavirus and sars-cov nsp proteins might have similar biological functions. recent studies revealed that sars-cov nsp suppresses host protein synthesis by interacting with the ribosomal s subunit (kamitani et al., ) . in this study, we demonstrated that the group i coronavirus nsp proteins also bound to the ribosomal s subunit, as shown by co-ip. further analysis showed that activation of irf- was not affected, but synthesis of host immune and nonimmune proteins was potently suppressed by group i coronavirus nsp proteins. it seemed that synthesis of these proteins was more strongly inhibited at translational level than transcriptional level. these results indicate that group i coronaviruses have evolved a mechanism strikingly similar to sars-cov for antagonizing host cell proliferation and innate immunity using nsp . our results are in general consistent to that of kamitani et al. ( ) , who firstly reported that sars-cov nsp inhibited ifn-b mrna transcription and ifn-b promoter-driven luciferase protein synthesis. however, in our study, the inhibition of ifn-b mrna transcription and luciferase protein synthesis was less strong than that reported by kamitani et al. ( ) , most probably due to different expression levels of nsp . in their study, the sars-cov nsp was driven by a chicken b-actin/rabbit b-globin hybrid promoter (ag promoter) in pcaggs vector, which is known for its robust expression in eukaryote cells. in this study, nsp was driven by a cmv promoter, which was probably less efficient than the hybrid ag promoter. most human coronaviruses are low-pathogenic viruses that often cause mild lower respiratory tract infections like common cold (thiel and weber, ) . however, sars-cov infection appears highly pathogenic and causes severe pneumonia and acute respiratory distress syndrome, characterized by the presence of diffuse alveolar damage (weiss and navas-martin, ) . surprisingly, few viral particles are isolated from lung tissues of sars-cov infected patients, but levels of inflammatory cytokines and chemokines are greatly elevated in the lung. a hyperinflammatory response is presumed to be the key determinant of the high pathogenicity of sars-cov, rather than rapid viral spread (de lang et al., ) . the specific mechanism for sars-cov pathogenicity is not known, but a number of viral coding proteins may be involved (weiss and navas-martin, ) . the nsp protein of sars-cov is defined as a major pathogenicity factor (kamitani et al., ; wathelet et al., ) . however, our results showed that the nsp proteins from low-pathogenic coronaviruses suppressed host protein synthesis more strongly than nsp from sars-cov, indicating that the nsp protein is actually a virulence factor for facilitating viral spread, but is not a major determinant in coronavirus pathogenicity. although an engineered sars-cov with a mutated nsp was greatly attenuated in an animal model (wathelet et al., ) , caution should be taken in developing this mutant virus as human vaccine because of the pathogenicity factors still present in the virion. sars-cov and mhv fail to induce or induced only weak and delayed innate immune responses (spiegel et al., ; spiegel and weber, ; roth-cross et al., ; zhou and perlman, ) . the underlying mechanism for this phenomenom is not fully understood, but nsp is proposed to be a major factor in the ability of the viruses to antagonize host innate immunity (kamitani et al., ; wathelet et al., ; zust et al., ; narayanan et al., ) . however, surprisingly, activation of host innate immunity and induction of type i ifn were observed for the group i coronavirus tgev (la bonnardiere and laude, ; charley and laude, ; charley and lavenant, ) . we also found that group i coronaviruses hcov- e steadily activated ifn transcription factors, and infected cells produced robust ifn-b (unpublished data) . this study clearly showed that the hcov- e and hcov-nl nsp proteins also potently suppressed host innate immune protein synthesis like nsp from sars-cov, implying that group ii coronaviruses do not employ nsp to evade host innate immunity. the mechanism underlying the different innate immune responses for the two 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a nucleocapsid protein is a type i interferon antagonist mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna coronavirus non-structural protein is a major pathogenicity factor: implications for the rational design of coronavirus vaccines key: cord- - f o authors: dhariwal, jaideep; edwards, michael r; johnston, sebastian l title: anti-viral agents: potential utility in exacerbations of asthma date: - - journal: curr opin pharmacol doi: . /j.coph. . . sha: doc_id: cord_uid: f o asthma is the most common chronic respiratory disease and its prevalence is on the increase. respiratory viral infections in early life have been suggested to increase the risk of development of asthma in later life and virus infection remains the single greatest precipitant of asthma exacerbations. the development of effective anti-viral treatments remains a key target for therapeutic intervention. here we discuss the role of respiratory viral infection in asthma exacerbation and highlight current and potential anti-viral agents and their mechanisms of action. anti-viral agents: potential utility in exacerbations of asthma jaideep dhariwal , , , , michael r edwards , , and sebastian l johnston , , , asthma is the most common chronic respiratory disease and its prevalence is on the increase. respiratory viral infections in early life have been suggested to increase the risk of development of asthma in later life and virus infection remains the single greatest precipitant of asthma exacerbations. the development of effective anti-viral treatments remains a key target for therapeutic intervention. here we discuss the role of respiratory viral infection in asthma exacerbation and highlight current and potential anti-viral agents and their mechanisms of action. asthma is a heterogeneous airway disease characterised by airway inflammation, airway hyperreactivity, reversible bronchoconstriction and airway remodelling. patients experience shortness of breath, fluctuations in normal breathing patterns, and also periodic episodes of wheeze and cough. asthma is treated with inhaled corticosteroids, with and without other therapies including short or long acting bronchodilators. asthma exacerbations (aes) are the major cause of morbidity, mortality and healthcare costs associated with asthma [ , ] , and are generally defined as worsening of the above symptoms accompanied by a drop in lung function prompting a gp consultation or visit to the emergency room. in extreme cases, ae can require oral corticosteroid therapy, supplemental oxygen and may result in death. respiratory virus infections account for at least % of exacerbations in adults and children [ ] [ ] [ ] [ ] and among respiratory viruses human rhinoviruses (rvs) are by far the most common viruses associated [ , , ] . the importance of respiratory viruses as triggers of ae has therefore made them a target for therapeutic intervention. in this review we discuss the potential of two therapeutic approaches, one targeting host factors that may induce natural anti-viral immunity, such as the addition of an anti-viral cytokine, manipulation of the host's immune response such as administration of a vaccine, and secondly targeting the virus itself; including small molecule inhibitors of virus replication, and virus specific immunotherapy. these approaches are summarised in figure . because of the overwhelmingly important role viruses play in ae, we argue that now is the time to carefully re-consider anti-viral interventions for ae. respiratory virus infections are triggers of ae. viruses such as rvs, respiratory syncytial virus (rsv), seasonal influenza a viruses, metapneumoviruses, coronaviruses and bocaviruses may all trigger ae in adults and children. atypical bacteria mycoplasma pneumoniae (m. pneumoniae) and chlamydophila pneumoniae (c. pneumoniae) are also common respiratory pathogens associated with aes in both adults and children [ ] [ ] [ ] . the major viruses associated with aes are rvs, accounting for approximately % of all ae in all ages [ , ] . rvs are members of the picornaviridae, and are positive sense ssrna viruses with genomes of . - . kb and can be divided into major and minor groups based on receptor utilisation. major group rvs bind icam- while minor group rvs bind the ldl receptor. rvs may also be classified based on nucleotide sequence identity (rv-a, rv-b, rv-c). the rv-c group [ , ] have unique sequences at the icam- and ldl receptor binding sites, suggesting they use a unique, currently unknown receptor [ ] . rvs represent a diverse group of viruses with serotypes known and an estimated further or so group c viruses. rv-c may cause more severe aes, although how this occurs is currently unknown [ ] . in the northern hemisphere, rv infection precipitates an increase in emergency room admissions due to aes [ ] , known as the 'asthma epidemic.' this occurs in the third week of september, after children return to school, highlighting that school age children are vectors for rv infection and their crucial role in aes [ ] . major and minor group rv mouse models of rv infection [ , ] and rv induced exacerbations of airway allergen challenge [ , ] have also recently been developed. these animal studies mirror the human data gathered to date and support the idea that rv infection augments airways inflammation caused by allergen sensitisation and challenge, providing further evidence that respiratory viruses such as rv exacerbate asthma. recent studies have reported that impaired innate immunity to virus infections is important in the pathogenesis of aes. reduced capacity to induce type i interferons (ifns) ifn-a, ifn-b or type iii ifns, the ifn-ls upon challenge with respiratory viruses or the dsrna mimetic polyic in bronchial epithelial cells (becs), bronchoalveolar lavage (bal) macrophages, dendritic cells (dcs) and peripheral blood mononuclear cells (pbmcs) from persons with asthma have recently been described [ , , , , , , , , ] . importantly, deficient ifn-l was also strongly related to virus load, and ae pathogenesis and severity in vivo [ ] . the mechanism responsible for impaired ifn-a, ifn-b and ifn-l remains poorly understood. however, the above studies advocate a role for ifn therapy in ae. recently, a phase ii placebo controlled trial of inhaled ifn-b in poorly controlled adult atopic asthmatics was performed [ ] . inhaled ifn-b, started at the reporting of a clinical cold, showed promise, reducing rates of ae in this group and increasing lung function. virus load was studied in only a few patients, and showed trends for lower virus loads in treated patients. therefore, inhaled ifn-b improves ae rates and associated symptoms, most likely due to a direct anti-viral activity. it is also possible that ifn-b could modulate additional processes, such as respiratory anti-viral approaches may ( ) prevent viruses acting in a synergistic or additive manner with allergens, and cause immune deviation to a th rather than th immune response. anti-viral approaches may be ( ) agents that prevent virus infection or replication at mucosal surfaces and prevent epithelial damage, inflammation, mucous production, activation of macrophages and attraction of neutrophils into the lung, which promote further inflammation and damage. acting as an antagonist of th immunity. recent studies have shown that ifn-b and type iii ifn-l have potent th antagonistic activity [ , ] , suggesting that inhaled ifn-b may deliver a benefit on two levels in atopic asthma, firstly reducing virus replication and hence virus driven inflammation, and secondly dampening the th responses to allergens. further studies with inhaled ifn-b in ae are eagerly anticipated. the keto-macrolide telithromycin had previously shown efficacy in ae in adult asthmatics in a phase iv clinical trial [ ] . the mechanism responsible for this beneficial effect is unknown, and telithromycin could have been merely acting as an anti-inflammatory agent. in cell based assays, azithromycin, but not the closely related erythromycin or telithromycin, was shown to have anti-viral activity to rv by inducing ifn and interferon stimulated genes (isgs) [ ]. this was a previously unknown property of azithromycin, and has since promoted the further investigation of azithromycin in phase iv clinical trials in ae which are currently ongoing. the number of viruses implicated in the aetiology of asthma and their associated antigenic diversity has thus far limited development of effective vaccines. there are a number of potential rsv vaccine candidates although none are currently licensed for use [ ] [ ] [ ] [ ] . the creation of an effective rsv vaccine was significantly affected following the use of formalin inactivated rsv (fi-rsv) vaccine in the s which led to increased morbidity and enhanced respiratory disease following infection with live virus and the subsequent deaths of children [ , ] . this phenomenon was felt to be as a result of induction of th immune responses by the vaccine [ , ] . a new development in the creation of vaccines has been the use of tlr ligands as adjuvant agents. one recent study using monophosphoryl lipid a (mpla), a derivative of bacterial lps, incorporated with rsv virosomes demonstrated an enhanced th response with increased production of ifn-g and decreased il- in animals immunised with vaccine and then challenged with virus [ ]. the mpla adjuvanted vaccine conferred similar protection from live rsv virus as fi-rsv vaccine but with no evidence of enhanced respiratory disease. in addition the use of mpla led to enhanced immunogenicity of the rsv vaccine with production of higher affinity antibodies. the only vaccines commercially available and recommended for use are against influenza where the annual vaccine has been shown to play a key role in the prevention of virus infection and its associated morbidity [ ] . this preventive approach may well have advantages over treatment of acute viral infections where current treatment options are limited. as rv infections are implicated in the vast majority of virus induced aes they are perhaps the most attractive target for a respiratory vaccine. however, there are > serotypes of rv and unlike influenza there is limited epidemiological information regarding the most important circulating serotypes [ ] . humans are infected with rv in early life and recurrently through life and most adults have antibodies to multiple rv strains, complicating human study of antibody responses. improved diagnostic and molecular techniques have recently allowed the identification of the rv-c group further highlighting the difficulty in selecting specific serotypes for vaccine generation [ ] . a major hurdle to the understanding of antibody production following virus infection has been a scarcity of animal models that have allowed us to study both asthma exacerbations and the subsequent effects of immunisation in greater detail [ ] . a recent paper describing a novel mouse model of rv infection and immunisation has allowed study of rv mediated induction of antibody responses [ ] . this paper demonstrated the generation of strong cross-serotype igg responses to the rv capsid protein vp and that multiple infections were necessary to induce neutralising antibodies [ ] . another group have also recently shown that use of a recombinant vp protein was able to generate neutralising antibodies displaying cross-reactivity to distantly related rv strains [ ] . these studies suggest that efforts to develop rv vaccines may be worth re-visiting. using small molecule inhibitors of rv infection, replication or release was a popular theme in the s and s for the treatment of the common cold. despite approaches showing some promise in common cold studies, the use of these anti-virals has yet to be examined in virus induced ae. small molecule anti-virals offer the advantages of being cost effective (small molecule production and validation), and their selectivity and safety is relatively straightforward to establish. they have disadvantages in that they may be limited to specific virus types, may select for escape mutants over time and may suffer from toxicity or have side effects with continual use. the anti-rv agent pleconaril was used in randomised, placebo-controlled, phase ii clinical trials as a treatment of the common cold [ ] . pleconaril prevents uncoating of most serotypes of rvs. pleconaril was tested as a therapeutic agent, with infected individuals beginning therapy - . days after experiencing clinical colds. pleconaril showed significant improvement in mean symptoms scores and decreases in mean duration of illness. despite promising initial results, pleconaril was abandoned as a treatment due to side effects. the rv c protease inhibitor ruprintrivir was tested in a double blind, placebo-controlled phase ii trial of experimental rv challenge [ ] . ruprintrivir was designed to bind irreversibly to the rv c active site. as a prophylaxis, ruprintrivir reduced mean total symptom score, viral titre and nasal secretions but not the incidence or frequency of clinical colds. as a therapeutic treatment, ruprintrivir also reduced symptom scores, nasal secretions and viral titre. the soluble icam derivative tremacamra was tested in randomised double-blind placebo-controlled studies both as a therapeutic and prophylactic intervention to rv challenge [ ] . tremacamra showed promise as a therapy, reducing the frequency of colds, total symptom score, nasal mucus weight, and virus induced inflammation. quercetin is a polyphenol which has a range of properties some of which are anti-viral. quercetin is thought to inhibit phospho-inositol- -kinase inhibition and inhibition of viral endocytosis, rv and poliovirus protease activity, and rna polymerase activity of some rna viruses. in a mouse model of rv infection, quercetin if given during infection reduced virus titre and improved lung function. however, if given daily for days finishing hours before rv infection, quercetin had little effect on virus replication and lung function [ ] . development of all these drugs was abandoned for various reasons. however, considering the role of viruses in ae, and the available human and mouse models of virus induced ae, there has never been a better time to trial small molecule anti-virals as therapies for ae. the future is likely to witness further studies of anti-virals as a potential treatment for virus-induced ae. the recombinant monoclonal antibody (mab) palivizumab has been licenced for use in human rsv immunoprophylaxis since [ ] . it acts against an epitope in the a region of the rsv fusion protein and has been shown to reduce the rate of hospitalisations in high riskinfants when used prophylactically [ ] . there is a scarcity of evidence regarding the role of palivizumab in the treatment of acute rsv disease with evidence predominantly limited to case reports and small retrospective studies. a single dose of mg/kg in children intubated with respiratory failure due to rsv was shown to reduce rsv concentration in tracheal aspirates [ ] and palivizumab has been shown to be well tolerated in adult stem cell transplant recipients [ ] . however, there is a need for further large scale studies to assess the role of palivizumab as therapy in rsv infection. motavizumab (medi- , medimmune) is a second generation mab developed from palivizumab by affinity maturation [ ] . in comparison to palivizumab, it has been shown to bind to rsv f protein -fold better and have an approximately -fold improvement in neutralisation of rsv in vitro [ ] as well as being able to reduce pulmonary rsv titres up to -fold lower than equivalent doses of palivizumab [ ] . motavizumab is able to inhibit viral replication in the upper respiratory tract making it a potentially attractive therapy for treatment of rsv infection [ ] . a study comparing motavizumab with palivizumab for rsv prophylaxis showed that motavizumab treatment resulted in % fewer rsv related lower respiratory tract infections needing medical attention [ ] . however, this study also documented an increase in cutaneous hypersensitivity reactions and motavizumab is not currently licenced for use in humans. there has been a growing body of work directed towards the generation of mabs against influenza. two recently described mabs, fi v and pn-sia , have been shown to be broadly neutralising against both group and influenza a subtypes [ , ] . fi v antibody was generated from single cell culture of plasma cells from individuals following natural influenza a infection or vaccination and passive transfer of this mab was protective in both mice and ferrets against h , h and h subtypes [ ] . pn-sia was identified from a single healthy donor who had a negative history for influenza in the preceding decade and it too demonstrated neutralising activity against group and subtypes [ ] . both fi v and pn-sia act on regions near the ha stem and have identified new mechanisms underlying virus-host interaction and new areas of interest in development of anti-viral therapies [ , ] . there are currently no mabs available for use against rv. current treatment options for ae are limited and have developed little in recent years. furthermore, these treatments do not address the cause of the exacerbations, nor specific mechanisms involved in their pathogenesis. new clinical studies are needed to further our understanding of the mechanisms of virus induced ae so that targets for development of novel approaches to prevention and therapy can be identified. anti-viral therapies may be a source of these new therapies; this review has highlighted potential therapies that target the virus or boost host response to the virus. the latter approach is based on recent studies that show an impairment in the ability of the asthmatic host to raise an effective anti-viral immune response, and there are several potential ways to restore this. alternatively, the virus itself may be targeted, using specific anti-virals or immunotherapy. we believe the future will likely see greatly increased study of anti-viral therapies in one form or another for treatment/prevention of virus induced ae. the global burden of asthma: executive summary of the gina dissemination committee report study comparing direct health care costs between moderate/severe asthmatics with and without exacerbations. demonstrated that moderate severe asthmatics who had exacerbations had higher total and asthma related health care costs frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study respiratory viruses and exacerbations of asthma in adults personal exposure to nitrogen dioxide (no ) and the severity of virus-induced asthma in children community study of role of viral infections in exacerbations of asthma in - year old children the september epidemic of asthma exacerbations in children: a search for etiology chlamydia pneumoniae immunoglobulin a reactivation and airway inflammation in acute asthma chronic chlamydia pneumoniae infection and asthma exacerbations in children the effect of telithromycin in acute exacerbations of asthma sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution paper examining diversity of human rhinovirus (hrv) by completing the genome sequences for all known serotypes (n = ) including newly discovered hrv-c clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus c paper detailing development of a sinus organ culture and reverse genetics system allowing study of hrv-c in vitro, demonstrating a unique cell attachment site for effects of atazanavir/ritonavir or fosamprenavir/ ritonavir on the pharmacokinetics of rosuvastatin mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation paper describing the first mouse model of rv infection human rhinovirus b exposure induces phosphatidylinositol -kinase-dependent airway inflammation in mice rhinovirus infection of allergen-sensitized and -challenged mice induces eotaxin release from functionally polarized macrophages peripheral blood mononuclear cells from patients with bronchial asthma show impaired innate immune responses to rhinovirus in vitro study suggesting increased susceptibility of asthmatics to rv infection as a consequence of impaired production of ifna and other inflammatory cytokines atopic phenotype in children is associated with decreased virusinduced interferon-alpha release asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus study demonstrating deficient innate immune response in bronchial epithelial cells from asthmatic individuals. in particular, deficiency of ifn-b in asthma shown to facilitate virus replication and cytolysis. addition of exogenous ifn-b in vitro restored this deficit impaired virus-induced interferon-alpha release in adult asthmatic patients role of deficient type iii interferon-lambda production in asthma exacerbations paper demonstrating a previously unknown mechanism of susceptibility to rv infection in asthmatics as a result of deficient ifn-l production double-stranded rna induces disproportionate expression of thymic stromal lymphopoietin versus interferon-beta in bronchial epithelial cells from donors with asthma impaired innate interferon induction in severe therapy resistant atopic asthmatic children anti-viral agents in asthma dhariwal pathogenesis of respiratory syncytial virus vaccine-augmented pathology respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine respiratory synctial virus infection in balb/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant th -like cytokine pattern a potential molecular mechanism for hypersensitivity caused by formalin-inactivated vaccines efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis meta-analysis of effectiveness of influenza vaccines, highlighting lack of evidence of protection in adults over and need for new vaccines with increased efficacy and effectiveness rhinovirus infections and immunisation induce cross-serotype reactive antibodies to vp study characterising the generation of antibody responses to rv following infection and immunisation of mice. identified potential of vp based antigens combined with adjuvants in generation of successful antibodymediated vaccine design and development antibodies induced with recombinant vp from human rhinovirus exhibit cross-neutralisation oral pleconaril treatment of picornavirusassociated viral respiratory illness in adults: efficacy and tolerability in phase ii clinical trials phase ii, randomized, double-blind, placebocontrolled studies of ruprintrivir nasal spray -percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers efficacy of tremacamra, a soluble intercellular adhesion molecule , for experimental rhinovirus infection: a randomized clinical trial quercetin inhibits rhinovirus replication in vitro and in vivo study demonstrating quercetin, a plant flavinoid, possesses anti-rhinoviral effects. the authors show that quercetin inhibits viral infection at multiple stages, including endocytosis and viral protein synthesis humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. the impact-rsv study group phase evaluation of the respiratory syncytial virus-specific monoclonal antibody palivizumab in recipients of hematopoietic stem cell transplants development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins study identifying a broadly neutralising monoclonal antibody with potential for use against all influenza a viruses a human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes the authors describe a human monoclonal antibody able to neutralize pandemic influenza subtypes as well as other subtypes with pandemic potential key: cord- -eff le g authors: eng, vik ven; wemyss, madeleine a.; pearson, jaclyn s. title: the diverse roles of rip kinases in host-pathogen interactions date: - - journal: semin cell dev biol doi: . /j.semcdb. . . sha: doc_id: cord_uid: eff le g receptor interacting protein kinases (ripks) are cellular signaling molecules that are critical for homeostatic signaling in both communicable and non-communicable disease processes. in particular, ripk , ripk , ripk and ripk have emerged as key mediators of intracellular signal transduction including inflammation, autophagy and programmed cell death, and are thus essential for the early control of many diverse pathogenic organisms. in this review, we discuss the role of each ripk in host responses to bacterial and viral pathogens, with a focus on studies that have used pathogen infection models rather than artificial stimulation with purified pathogen associated molecular patterns. we also discuss the intricate mechanisms of host evasion by pathogens that specifically target ripks for inactivation, and finally, we will touch on the controversial issue of drug development for kinase inhibitors to treat chronic inflammatory and neurological disorders, and the implications this may have on the outcome of pathogen infections. pathogen infection initiates multiple innate immune signaling pathways via the stimulation of pattern recognition receptors (prrs) with pathogen-associated molecular patterns (pamps). the outcomes of pamp-prr signaling are diverse, and often dependent on pathogenspecific virulence factors as well as host factors including, cell type, species, existing gene polymorphisms or pre-existing co-morbidities. nevertheless, the signal transduction that follows pathogen recognition elicits numerous defence mechanisms, including the induction of inflammatory cytokine, chemokine and interferon production, activation of programmed cell death pathways, and inevitably an adaptive immune response, all of which contribute to pathogen control and elimination [ ] . there are seven members of the receptor interacting protein kinase (ripk) family, ripk - , some of which have emerged as critical effectors of immunity to infection with a diverse array of bacterial, viral and protozoal pathogens. structurally and functionally, all members of the ripk family share a homologous serine-threonine kinase domain (kd) with a catalytic site [ ] , with ripk boasting additional tyrosine kinase activity [ ] . ripk has a c-terminal death domain (dd), and an intermediate domain (id) which harbors a rip homotypic interaction motif (rhim). ripk contains a c-terminal card (caspase activation and recruitment domain) and an id. ripk has a c-terminal rhim domain alongside the n-terminal kd. ripk and ripk are characterized by c-terminal ankyrin repeats, and ripk and ripk each harbor a leucine-rich repeat (lrr) motif, ankyrin repeats, a ras-of-complex (roc) domain followed by a c-terminal of roc (cor) domain, and a wd domain. to date, the most studied ripks in relation to inflammation of host immune responses are ripk , and , with ripk following steadily behind. this rapidly moving field has brought the biochemical functions of ripks to the forefront of cellular immunity. here, model pathogens including listeria monocytogenes, shigella flexneri, mycobacterium tuberculosis, influenza a virus (iav) and cytomegaloviruses (cmv) have been instrumental in dissecting the role of ripks in host immunity. furthermore, research over the past decade has revealed multiple pathogenic mechanisms of bacteria and viruses that specifically target ripks or their downstream signaling networks for inactivation. despite the volume of research on ripks and host responses to infection, there is a long way to go in understanding the physiological role of each ripk and their functional domains in specific infection settings. this is partly due the fact that ripk and ripk are implicated in pro-survival transcriptional pathways as well as cell death, but also due to the ongoing discovery of novel virulence mechanisms of pathogens. in this review, we will provide an up to date account of ripkmediated host responses to bacterial and viral infections as well as the mechanisms pathogens have evolved to evade ripk signaling outcomes. importantly, we have focused on studies that have utilised in vitro or in vivo infections with pathogenic organisms to stimulate physiological immune responses, rather than those that have used purified or synthetic stimulants, for example lipopolysaccharide (lps) or polyinosinic: polycytidylic acid (poly i:c). we note that although we were not able to discuss all relevant pathogens, including protozoal organisms, we have generated a comprehensive list of pathogens, the associated ripk signaling pathways and relevant references in table . ripk regulates inflammation in response to death receptors (dr), including tumour necrosis factor receptor (tnfr ), tnf-related apoptosis-inducing ligand receptors and (trailr / ) and fas [ ] [ ] [ ] , as well as toll-like receptor (tlr) and tlr [ , ] (fig. ) . ripk , although best known for its role in necroptosis (discussed later), can act in concert with ripk to engage in pro-inflammatory, non-cell death signaling [ ] . here we will focus on research that indicate direct ripk and/or ripk involvement in pathways specifically described to be cell death-independent during host infection. west nile virus (wnv) and zika virus (zikv) are medically important arboviruses that cause severe neurological disease in humans [ , ] . control of wnv requires robust neuroinflammation and infiltration of peripheral leukocytes into the central nervous system (cns), mediated by ripk and ripk [ , ] . daniels et al. [ ] demonstrated that ripk − /− and ripk kinase-dead (ripk k a/k a ) mice have suppressed tlr-induced chemokine expression, as well as reduced recruitment of t lymphocytes and myeloid cells into the cns, resulting in accelerated virus-induced mortality. similarly, direct zikv infection in the cns of ripk − /− , ripk k a/k a and dai − /− mice induces rapid mortality and elevated viral titres within the brain [ ] . pharmacological blockade of ripk also enhances zikv replication in human neuroblastomas. this intrinsic neuronal cell defect results from disrupted dai/ripk-mediated transcription of immunoresponsive gene (irg ) required for itaconate production. itaconate inhibits succinate dehydrogenase (sdh) activity and subsequently reprograms neuronal metabolism into an antiviral state [ ] [ ] [ ] . as such, these results highlight the contribution of ripk / in defence against neurovirulent viruses, independently of cell death. cmvs are large double-stranded dna viruses that have the capacity to encode viral effectors that directly manipulate components of host immune pathways. murine cmv (mcmv) expresses the m protein, which interacts with ripk through its catalytically inactive ribonucleotide reductase (rr) domain and inhibits ripk ubiquitylation [ ] . this process blocks pathways dependent on polyubiquitylated ripk , including tnf-induced nf-ĸb and p mapk activation, as well as tlr -induced nf-ĸb activation, thus facilitating immune evasion. m is also shown to bind ripk , ripk and dai via its n-terminal rhim domain, which interferes with dai-induced nf-ĸb signaling [ ] . similarly, human cmv (hcmv) is able to inhibit ripk -mediated nf-ĸb signaling through synergistic function of its encoded ul and ul proteins [ ] . during the late stages of infection, ul cleaves the k -polyubiquitin chains on ripk , thus preventing tnf-induced nf-ĸb activation [ , ] . ul supports this process by enhancing ul -ripk interaction and re-localization of ripk to the cytoplasmic virion assembly complex. conversely, rip kinases can also be utilised to promote viral propagation. following japanese encephalitis virus (jev) infection, ripk gene expression is upregulated in primary neurons of wild-type (wt) mice, which corresponds to heightened viral titres [ ] . rna-sequencing of infected brain tissues revealed ripk − /− mice upregulate a number of ifn-stimulated genes (isgs), independently of ripk and mlkl phosphorylation. silencing of the isg, ifi l, in both wt and ripk − /− neurons increases viral rna levels, suggesting that the elevated ripk assists with jev immune evasion. [ ] chlamydia muridarum gram-negative ripk activates -mouse, human [ , ] citrobacter rodentium gram-negative ripk , ripk inhibits espl mouse [ ] enteropathogenic ripk -regulated type i ifn signaling is also shown to be crucial in anti-iav immunity. indeed, infected ripk − /− mice exhibit increased pulmonary viral load and enhanced immunopathology and mortality [ ] . this susceptibility is largely attributed to defective ifn production from infected ripk -/macrophages, independent of iav-associated cell death outcomes. specifically, ripk controls type i ifn signaling at the transcriptional level, where virus-induced upregulation of ripk disrupts ripk -mavs interaction and reduces ifn-β mrna expression, which facilitates iav persistence [ , ] . interestingly, the increased ripk also activates protein kinase r (pkr) that stabilises ifn-β transcripts [ ] , thus elevating ifn-β production and type i ifn protection [ ] . this is presumed to have been counter-evolved by hosts in response to viral evasion and represents a potential avenue for therapy against pathogenic iav. several other viruses are known to modulate ripk / signaling in non-cell death pathways to benefit pathogenesis. for example, the common cause for infectious mononucleosis, epstein-barr virus (ebv), encodes a latent membrane protein (lmp ) that binds both ripk and ripk , and recruits e ligases to regulate protein ubiquitylation [ ] . consequently, lmp promotes ripk polyubiquitylation, while ripk polyubiquitylation is suppressed through yet undefined means, which switches cell fate from necroptosis to survival. human immunodeficiency virus type (hiv- ) protease (pr) cleaves ripk with high specificity in human t lymphocytes, resulting in failure to activate nf-ĸb [ ] . conversely, coxsackievirus b (cvb) employs ripk to promote autophagic flux for viral assembly [ ] , and as such, silencing of ripk in human intestinal epithelial cells restricts early viral replication [ ] . yersinia pestis, the etiological agent of bubonic plague, produces an array of yersinia outer protein (yop) virulence effectors that are translocated into host cells via a type iii secretion system (t ss) to alter host cell signaling [ ] . infection of macrophages with y. pestis results in tnf, il- and il- β secretion, mediated through a ripk and caspase- dependent, but ripk -independent mechanism [ ] . as a result, ripk deletion in murine myeloid cells causes defective cytokine release, associated with decreased iĸbα degradation and nf-ĸb nuclear translocation. in the absence of caspase activity, y. pseudotuberculosis is also shown to induce ripk /ripk /trif-mediated synthesis of ifn-β in macrophages, similar to klebsiella pneumoniae [ ] . this process requires the kinase activity of both ripks but operates independently of mlkl-mediated necroptosis and caspase- apoptosis. to counter host inflammation, y. pestis encodes yopj, which acetylates mapks, ikkβ and tak within their activation loops, thus dampening host mapk and nf-ĸb signaling [ ] . legionella pneumophila is an environmental organism and accidental human pathogen that causes legionnaires' disease, a severe form of acute pneumonia [ ] . following infection, l. pneumophila effector mavc (also known as lpg ) deamidates the human ubiquitin-conjugating enzyme e n (ube n), which disrupts ube n-mediated formation of lys polyubiquitin chains [ ] . this prevents ripk polyubiquitylation, thus suppressing downstream nf-ĸb signaling in infected cells in vitro. given the presence of mavc homologues in the cycle inhibiting factor (cif) effector family of t ss pathogens [ , ] , there may be similar routes for ripk inhibition in other bacterial contexts. bacterial effectors can also directly manipulate ripks. enteropathogenic escherichia coli (epec) encodes an arginine glycosyltransferase, nleb , that modifies ripk following infection of human cells [ , ] . this activity inhibits the recruitment of ubiquitinated ripk to tnfr , thus disrupting the assembly of complex i required for tnf-induced nf-ĸb signaling [ ] . although transient expression of nleb inhibits nf-ĸb activation [ ] , nleb alone is not sufficient to inhibit nf-ĸb-mediated il- production during epec infection in vitro [ ] , as multiple effectors are required to achieve this outcome [ ] . apoptosis is a common host response to infection that helps restrict the replicative niche of pathogens, and facilitates phagocytosis and antigen presentation through the production of apoptotic bodies [ ] . many pathogens however, have evolved mechanisms to manipulate apoptotic processes to benefit survival and virulence (fig. ). the execution of apoptosis is heavily reliant on host caspases, thus most large dna viruses encode effectors for inhibiting caspase activity [ ] , however, few viral factors target ripks directly. herpes simplex virus (hsv) contains numerous proteins that can interact with apoptotic signaling components, including icp and icp encoded by hsv- and hsv- respectively [ ] . these proteins possess a c-terminal rr domain that inhibits caspase- function, thus suppressing tnf and fas-induced apoptosis [ ] . curiously, this c-terminal rr, and not its n-terminal rhim-like domain, also binds ripk and prevents poly(i:c)-induced apoptosis in human cells [ ] . moreover, icp and icp can disrupt the rhim-dependent interaction between trif and ripk , which may contribute to the disabling of apoptosis. although the role of this cell death inhibition in viral pathogenesis has not been examined, it appears hsv has a strategy for managing dsrna mediated apoptosis. similarly, initiation of iav-induced pan-optosis (pyroptosis, apoptosis, necroptosis) requires dai sensing of viral rna through its second zα domain [ , ] , though dai is recently reported to also detect iav nucleoprotein (np) and polymerase subunit (pb ), as a collective unit of viral ribonucleoprotein [ , ] . upon stimulation, dai associates with ripk , which recruits ripk , fadd and mlkl to form a "ripoptosome" complex [ , ] . here, the apoptotic arm operates under fig. . inflammatory pathways mediated by ripk / in response to pathogen sensing. upon stimulation with tnf, tnfr recruits tradd and ripk via the dd, then subsequently traf / and ciap / to form a membrane-bound pro-inflammatory signaling complex [ , ] . ubiquitylation of ripk by ciap / and lubac enables recruitment of tak and the ikk complex, which promotes activation of mapk and canonical nf-κb signaling [ ] [ ] [ ] [ ] . ligation of tlr / reinforces these pathways through rhim-mediated interactions between trif and ripk , with subsequent ripk ubiquitylation continuing to drive nf-κb activation [ , ] . tlr -trif interactions can also induce type i ifn signaling via recruitment of ripk and ripk , which activates tbk and ikkε to promote nuclear translocation of irf [ ] . in response to dsrna sensing by cytosolic rig-i or mda , mavs-ripk interactions additionally drive irf and nf-ĸb transcription pathways [ ] [ ] [ ] . finally, cytosolic dsdna sensing by dai/zbp promotes nf-ĸb induction via rhim-mediated recruitment of ripk , with ripk kinase activity also required for synergistic activation [ , ] . virulence factors that interact with these pathways are indicated, and have been discussed in text the main body of text. a ripk -fadd-caspase- axis, in a manner independent of ripk kinase activity [ ] . neither caspase inhibition through z-vad nor fadd deletion in mouse fibroblasts rescues cell viability, but pre-treatment of these cells with ripk kinase inhibitors grants significant protection, thus highlighting the importance of ripk in regulating the parallel cell death pathways. surprisingly, dai also triggers ripk -independent apoptosis, where dai engages ripk directly to recruit fadd and caspase- , which deploys an alternative, delayed apoptotic cell death [ , ] . as inhibition of necroptosis does not affect iav control, while mlkl-/-fadd-/-mice exhibit marked susceptibility to iav-induced lethality [ ] , it signifies that ripk / -mediated apoptosis is a major form of host protection against iav infection. in response to yopj-mediated abrogation of inflammation during yersinia infection [ ] , infected macrophages engage tlr /trif and tnfr to induce apoptotic cell death [ ] . both cell death pathways occur in a non-redundant manner and rely on ripk -mediated activation of caspase- for downstream signaling [ , ] . the ripk -dependent apoptosis provides a cell-extrinsic signal required for programmed cell death pathways regulated by ripk / . following the tnfr -mediated assembly of pro-inflammatory complex i, deubiquitinases (cyld or a ) remove polyubiquitin chains from ripk to terminate inflammation and enable downstream death signaling [ , ] . ripk (with ripk ) interacts with fadd and pro-caspase- to form complex iib (ripoptosome) [ , ] , and can initiate extrinsic apoptosis [ , ] . active caspase- facilitates repression of necroptosis and nf-ĸb signaling by cleaving ripk and ripk [ ] [ ] [ ] . in the absence of caspase activity, ripk and ripk oligomerize to form complex iic (necrosome) that phosphorylates mlkl and induces necroptosis [ ] [ ] [ ] . tlr-driven trif-ripk interactions can also promote ripoptosome formation [ ] , while trif-ripk phosphorylates mlkl for necroptosis [ ] . following nucleic acid sensing, rhim interactions between dai/zbp and ripk induces ripoptosome formation or direct phosphorylation of mlkl [ , ] . however, the resulting necroptosis can be suppressed by ripk rhim [ , ] . dai/zbp -ripk complexing also promotes nlrp inflammasome activation and death via pyroptosis [ , ] . virulence factors that interact with these pathways are indicated, and have been discussed in the main body of the text. cytokine production by inflammatory monocytes and neutrophils [ ] . consequently, ripk k a/k a mice orally infected with y. pseudotuberculosis exhibit decreased caspase- staining within the mesenteric lymph nodes, as well as reduced levels of il- -producing monocytes and tnf-producing neutrophils. these mice are unable to restrict bacterial replication and systemic dissemination, resulting in rapid mortality. necroptosis has largely been studied in the context of antiviral responses, often as an alternate form of cell death for restricting viral replication. necroptosis is a form of regulated lytic cell death that operates independent of caspases, and requires both ripk and mlkl function (fig. ) . it is important to note that although the studies discussed below have specified necroptosis as the specific cell death outcome involved in the infection process, some have only documented ripk dependence but have not directly shown involvement of mlkl or pmlkl. therefore, we have noted whether experimental evidence has been provided for dependence on mlkl within the text. mouse fibroblasts infected with vaccinia virus (vv), which is a poxvirus strain that encodes the caspase inhibitor b r/spi , are shown to have resistance to tnf-induced apoptosis but increased sensitivity to ripk -dependent necroptosis [ , ] . vv-infected wt mice exhibit extensive inflammation and necrotic tissue damage within the liver, associated with formation of ripk -ripk complexes [ ] . ripk − /− mice fail to initiate virus-induced necroptosis, resulting in elevated viral titres and mortality [ , ] . the requirement for ripk kinase activity remains unclear however, as groups have reported either increased [ ] or unchanged [ ] vv loads within the liver and spleen of infected ripk d n/d n mice. vv also encodes e l, which contains an n-terminal z-dna binding domain that competes with dai to inhibit dai/ripk necroptosis [ , ] . administration of an e l mutant lacking this domain in mouse fibroblasts and human epithelial cells results in increased ifn-induced necroptosis, featuring increased mlkl phosphorylation, and fails to elicit disease in wt mice [ ] . similarly, mcmv encodes a viral inhibitor of caspase- activation (vica), which blocks dr-induced apoptosis but sensitises cells to necroptosis [ , ] . to combat this, mcmv expresses the viral inhibitor of rip activation (vira), that binds ripk and ripk through its n-terminal rhim domain [ ] . vira is a potent inhibitor for mcmv-triggered necroptosis that is dependent on dai/ripk signaling [ ] . mcmv mutants lacking vira or its rhim domain are severely attenuated in wt mice, but unaffected in ripk − /− and dai − /− mice [ ] [ ] [ ] . phosphorylation of mlkl occurs within hours during in vitro infection with vira rhim mutant mcmv, and is reliant on ie -mediated transcription of its genome [ ] . briefly, hcmv also elicits dai-mediated production of ifns and tnf-induced necroptosis in a ripk-regulated manner [ , , ] . notably, the tnf-driven cell death is inhibited by hcmv-encoded ie through a yet unknown process that occurs after ripk activation and mlkl phosphorylation, thus distinguishing the immunosuppressive strategies of hcmv from its murine counterpart [ ] . hsv- and hsv- modulate immune responses in a manner similar to mcmv. they carry icp and icp respectively, which expresses an nterminal rhim domain that mediates ripk and ripk interaction, leading to anti-necroptotic signaling in human cells, but pro-necroptotic death in mice [ ] [ ] [ ] . expression of icp / or hsv challenge in human cells causes competitive rhim binding that prevents ripk -ripk necrosome formation and subsequent tnf-induced mlkl phosphorylation and necroptosis [ , ] . in contrast, icp interaction with ripks in mouse cells promotes ripk -ripk complex formation for mlkl recruitment and execution of necroptosis, independent of dr, dai and tlr signaling [ , ] . this ripk -dependent necroptosis is crucial for controlling hsv- propagation, as ripk − /− mice present with markedly elevated susceptibility to infection and death, not seen in wt mice. as mentioned previously, human γ-herpesvirus ebv binds ripk and ripk through the c-terminal activation region of its encoded lmp effector, which prevents ripk -ripk complex formation in human nasopharyngeal cells [ ] . moreover, lmp promotes k -linked polyubiquitylation of ripk that forms the scaffolding required for tnf-induced nfĸb signaling [ ] , while inhibiting k -linked polyubiquitylation of ripk that typically supports necrosome assembly [ , ] . these post-translational modifications drive a switch from necroptotic death to a pro-survival cell fate, as indicated by the suppressed ripk and mlkl phosphorylation following ebv infection in tnf-induced necroptotic cells [ ] . as opposed to targeting the rhim sequences of ripks, bean poxvirus (bav) and cotia poxvirus (cotv) carry viral mlkl-like proteins (vmlkl) that block necroptosis by interacting with the ripk kinase domain [ ] . in both human and mouse epithelial cells, vmlkl and ripk bind via a pseudokinase to kinase domain interface, such that it overlaps the site typically engaged by cellular mlkl. in particular, this process in human cells prevents ripk interaction, but drives ripk phosphorylation despite pharmacological kinase inactivation of both ripk and ripk [ ] , which suggests that vmlkl alters ripk in a manner that nulls kinase inhibitor treatment or promotes phosphorylation by another yet unknown kinase. however, in mouse cells, vmlkl inhibits ripk phosphorylation, thus preventing ripk activation and subsequent catalytic activity [ ] . regardless, vmlkl is capable of sequestering both mouse and human ripk upon expression, which disrupts downstream tnf-induced necroptosis. in contrast to dna viruses, the mechanisms surrounding manipulation of necroptosis by rna viruses are less explored. the hiv- -encoded pr, previously described to inhibit nf-ĸb activation, also disrupts ripk -ripk interaction in cd + t cells via ripk cleavage [ ] . this may contribute to necroptosis suppression, but no study has confirmed this observation. additionally, necroptosis is implicated in the proliferative defect of hiv-specific cd + t lymphocytes in patients with progressive infection [ ] . this defect is successfully reversed following pre-treatment of antigen-stimulated cd + t cells with necrosis inhibitor necrox- or ripk silencing. however, as mlkl dependence was not explored in this study, it would be appropriate to address this in further studies targeting necroptosis for hiv therapy. coxsackieviruses are the etiological agent of hand, foot and mouth disease [ ] . coxsackievirus a (ca ) infection of human cells triggers upregulation of ripk via its non-structural protein (nsp)- d, which increases subsequent necrotic death [ ] . this cell death can be inhibited by nec- , which dramatically reduces virus production, suggesting that necroptosis is required for ca infection. it should be specified that although the study attributes necroptosis to ca pathogenesis, no changes in mlkl nor pmlkl expression was found. in contrast, at late stages of human intestinal epithelial cell infection with coxsackievirus b (cvb), nsp- c ( c pro ) proteolytically cleaves ripk and disrupts necroptosis, as determined by lack of hmgb release [ ] . intriguingly, the cleaved ripk interacts with ripk to induce a non-necrotic death [ ] , implying that cvb manipulates ripk to redirect the host cell into a more favourable but yet undefined death pathway. respiratory syncytial virus (rsv) triggers necroptosis without direct modulation of ripk / . rsv causes bronchiolitis in children, typically characterised by airway epithelial cell (aec) death and massive cytokine release [ ] . in response to infection, primary human aecs exhibit increased levels of pripk , pmlkl and hmgb release, which correlates with increased necroptotic death [ ] . pharmacological inhibition of ripk and mlkl reduces viral titres, suggesting that necroptosis promotes viral persistence. these results are recapitulated in a mouse model using pneumonia virus of mice (pvm). here, inhibition of ripk or mlkl lowered viral loads and prevented severe bronchiolitis, also seen in ripk k a/k a mice [ ] . the necrosome is also reported to trigger neutrophil extracellular traps (net) release in rsv-stimulated human neutrophils or neutrophils co-incubated with rsv-infected aecs [ ] , which facilitates rsv containment. the topic of viral respiratory illness has gained much attention recently due to the emergence of the hypervirulent severe acute respiratory syndrome coronavirus (sars-cov- ) responsible for covid- . in an effort to identify potential therapeutic targets, gordon et al. [ ] have mapped the interactions between sars-cov- proteins and human proteins. amidst the phenomenal set of results, ripk was shown to associate with the viral nsp- , which is inferred to be an rna polymerase. perhaps unsurprisingly, the sars-cov has been shown to interact with ripk via its open reading frame (orf)- a protein (sars a) to promote sars a oligomerization and subsequent necrotic death in human lung cells [ ] . this process operates independently of ripk kinase activity and mlkl [ ] , with sars a likely replacing the latter as the necroptotic executioner due to its ability to function as an ion channel following membrane insertion [ ] . however, sars a also reduces ripk and mlkl phosphorylation [ ] . infection with a sars a-deletion mutant rescued mice from virus-induced mortality, suggesting an antiviral role for necroptosis in sars-cov infection [ ] . compared to viral pathogens, far fewer bacterial effectors have been identified to directly interact with ripks for necroptosis modulation. epec employs a cysteine protease, espl, that directly cleaves the rhim domain of ripk and ripk , which prevents mlkl membrane complex formation during infection [ ] . this inhibits tnf and tlr / -induced necroptosis in vitro. furthermore, mice orally challenged with an espl deletion mutant of the epec-like mouse pathogen, citrobacter rodentium, exhibit attenuated bacterial colonization in the intestine [ ] . this suggests that espl-mediated blockade of necroptosis contributes to bacterial persistence. staphylococcus aureus is an opportunistic pathogen that causes pneumonia and bacteraemia in immunosuppressed patients. its poreforming toxin (pft) induces necroptosis in both human and murine pulmonary macrophages, and as such, inhibition of ripk or mlkl in vitro, or genetic deletion of ripk in vivo, significantly reduces cytotoxicity and improves s. aureus clearance in the lungs [ ] . other pft-carrying bacterial pathogens, such as serratia marcescens, streptococcus pneumoniae, listeria monocytogenes and uropathogenic e. coli (upec) also trigger ripk /ripk /mlkl-dependent necroptosis in macrophages, highlighting necroptosis as a promising target for pft-associated disease intervention [ ] . in contrast, in models of skin infection or sepsis, inhibition of ripk or mlkl results in exacerbated disease, due to excessive il- β-induced inflammation [ ] . this is not seen in ripk − /mice, likely due to ripk influence on inflammasome activation. these results indicate tissue-specific roles for ripks during s. aureus infection. similarly, salmonella enterica serovar typhimurium (s. typhimurium) triggers necroptosis in macrophages to benefit bacterial survival. this process is dependent on initial type i ifn signaling following injection of s. typhimurium in mice, as well as pathogen-mediated caspase- inactivation, which enables ripk recruitment to ifnar for phosphorylation and subsequent association with ripk [ ] . the resulting necroptotic cell death facilitates macrophage elimination, leading to compromised pathogen control. alternatively, ro et al. [ ] reported that microrna (mir)- upregulation following s. typhimurium infection drives necroptotic macrophage death. transfection of mir- in vitro induces ripk and ripk activation and subsequent necroptosis, which is partly inhibited following nec- treatment. it is worth noting that although the authors describe the mode of cell death in both of the above s. typhimurium infection studies as necroptosis, no experimental evidence showing mlkl phosphorylation or dependence on mlkl presence was provided. thus far, studies on mycobacterial-induced necroptosis in macrophages have been largely divisive. mycobacterium tuberculosis triggers ripk -dependent necroptosis in both human and murine macrophages in a pathway reliant on reactive oxygen species (ros) production and the mitochondrial bcl- family member protein b-cell lymphoma-extra large (bcl-xl) [ , ] . the ensuing macrophage death is suggested to assist bacterial pathogenesis, as ripk − /− macrophages display enhanced restriction of bacterial replication in vitro and in vivo. however, despite increased mlkl expression, pmlkl is not detected in infected macrophages, suggesting that the signaling process utilises an alternative executioner or is non-necroptotic. this is further complicated by results from stutz et al. [ , ] , which argue that deletion of mlkl or ripk does not rescue macrophages from death during m. tuberculosis infection. in fact, the macrophage population and bacterial burden in infected ripk -/mice are indistinguishable from wt controls. further research is required to ascribe a role for necroptosis in mycobacterial infections. host cell death and inflammation can also be induced independently of death receptors through inflammasome signaling, as depicted in fig. . ripk and ripk are primarily involved in alternative inflammasome activation pathways, which remain largely unexplored. a number of studies have used purified pathogen components such as lps to investigate the outcomes of ripk / -mediated inflammasome signaling, which appear to be largely dependent on cell type, stimulus, and the availability or functional activity of certain host signaling proteins [ , [ ] [ ] [ ] [ ] [ ] . here we will discuss ripk and ripk involvement in inflammasome signaling during pathogen-specific infections. vesicular stomatitis virus (vsv) is a rhabdovirus that causes vesicular lesions on the mucosa of livestock [ ] . vsv-infected mice exhibit nlrp inflammasome activation, characterised by elevated levels of cleaved caspase- , as well as il- β and il- secretion [ ] . here, ripk complexes with ripk following stimulation of a yet unidentified rna sensor, which enables ripk -mediated activation of dynamin-related protein (drp ). subsequent translocation of drp to the mitochondria promotes aberrant fission and ros production in both mouse and human cells, thus activating nlrp inflammasome. this drp -mediated inflammasome signaling is reported to also occur in response to dengue virus [ ] and swine influenza virus infection [ ] . some studies contradict these observations [ ] [ ] [ ] , likely due to different experimental models, therefore additional research is necessary to conclusively define this form of nlrp signaling. yersinia is capable of inducing caspase- processing and cell death following infection of mouse macrophages [ , , ] . this is driven by yopj-mediated suppression of tak , which enables ripk , fadd and caspase- recruitment, and subsequent activation of caspase . the resulting cell death exhibits significant ripk /caspase- -mediated gsdmd cleavage, implicating pyroptosis in this process [ ] . notably, nlrp and caspase- / are not involved in this pyroptotic pathway, but instead activated following potassium efflux to promote il- β processing and release. these observations illustrate an alternative nlrp -independent mechanism for caspase- and gsdmd activation during yersinia infection. ripk (also known as rip , rick and cardiak) is an essential scaffold for signal transduction via the nucleotide-binding oligomerization domain (nod) proteins, nod and nod [ ] [ ] [ ] [ ] , and is thus frequently implicated in innate inflammatory responses to pathogens. nod proteins are cytosolic pattern-recognition receptors (prrs) that activate pro-inflammatory and antimicrobial responses when exposed to pathogen associated molecular patterns (pamps). nod recognizes Ɣ-d-glutamyl-mesodiaminopimelic acid (ie-dap) from gram-negative bacteria and some gram-positive bacteria, whereas nod recognizes a conserved component of bacterial peptidoglycan (pgn) consisting of muramyl dipeptide (mdp) from both gram-positive and gram-negative bacteria [ ] [ ] [ ] [ ] (fig. ) . these stimulatory agents are released from bacteria upon cell wall fragmentation in bacterial killing, bacterial division, or they can be co-injected into host cells with virulence proteins by bacterial secretion systems [ ] . alternative mechanisms of nod/ripk activation will be discussed in context within the review. to date, much of the research on nod /nod /ripk signaling mechanisms has relied on purified pgn components as a cellular stimulus. this has been useful for the identification of cellular mechanisms, however can have limitations when considering the physiological role of signaling mediators in the context of pathogen infection, especially when many pathogens encode virulence factors that can inactivate innate immune signaling. although many studies have assessed mechanisms of nod signaling in bacterial control, especially in the context of autophagy, this review will focus on studies with direct experimental evidence of ripk involvement, and where live bacterial agents have been utilised. fig. . ripk regulation of nod and nod signaling in response to pamp sensing. upon activation by bacterial peptidoglycan components, nod and nod oligomerize and interact with ripk via homotypic card-card interactions [ ] . once engaged, ripk is activated by autophosphorylation, then ubiquitylated by e ligases including xiap and lubac, which further activates both nf-κb and mapk pathways and promotes pro-inflammatory cytokine production [ ] . alternatively, traf can interact with ripk following nod / ligation, redirecting signaling to tbk and ikkε to promote downstream ifn production [ ] . other roles of ripk include mediating interactions between nod / and key autophagy protein atg l , which enables autophagic bacterial clearance following nod sensing of peptidoglycan or bacterial omvs [ ] [ ] [ ] . virulence factors that interact with these pathways are indicated, and have been discussed in the main body of the text. some of the earliest studies that characterised the role of ripk in host responses to infections utilised l. monocytogenes, the causative agent of listeriosis. initial studies showed macrophages from ripk − /− mice were defective in nf-κb signaling and produced significantly less pro-inflammatory cytokines than wt macrophages following infection [ , ] . furthermore, ripk − /− mice were unable to control l. monocytogenes infection due to decreased nf-κb activation, and impaired ifn Ɣ production in t helper (th ) and natural killer (nk) cells. overall this suggests that ripk plays an important role in both innate and adaptive immunity to infection [ , ] , and that ripk was mediating these responses via nod / and not directly via tlr activation [ ] . ripk has since been shown to play a role in controlling a diverse array of bacterial pathogens, particularly those with an intracellular lifestyle ( table ) . single nucleotide polymorphisms (snps) in ripk increase susceptibility to mycobacterial infections, for example, multibacillary leprosy caused by m. leprae [ ] , and m. tuberculosis infections within the western chinese han population [ ] . although the mechanisms underlying susceptibility are unclear, m. tuberculosis activates nod /ripk , which stimulates the activity of irf to induce transcription of type i ifns [ ] . furthermore, ifnγ production by th cells induces macrophage maturation and anti-mycobacterial molecules important for resistance against mycobacterial infections [ , ] . zhang et al. [ ] suggested ripk and nod may regulate ifnγ which could explain increased susceptibility to mycobacterial infections in those with ripk polymorphisms. streptococcus pneumoniae (pneumococcus) is a an opportunistic pathogen associated with pneumonia, ear infections, sinus infections, meningitis and bacteremia [ ] . nod /ripk are critical for anti-inflammatory signaling in response to the purified pneumococcal cell wall (pncw) of s. pneumoniae. pncw induces intensive inflammatory responses by macrophages and dendritic cells during systemic infection in a tlr -dependent, nod /ripk -independent manner [ , ] . this inflammation is critically modulated by il- [ ] , as il- deficiency increases mortality in s. pneumoniae infection in vivo [ ] . curiously, this il- production is tlr , nod and ripk -dependent, whereby ripk or nod -deficient bmdms have compromised il- production in response to pncw [ ] . although the mechanism is not clear, this suggests that there is cell-specific and stimulus-specific crosstalk between tlr and nod /ripk pathways [ ] . during l. pneumophila infection, ripk mediates nf-κb activation independently of tlr/myd activation, but in response to bacterial factors (likely pgn) delivered directly into the host cell cytosol by the legionella t ss [ ] . in vivo studies have shown that ripk -deficiency results in poor neutrophil recruitment into the lung, a significant reduction in proinflammatory cytokine and chemokine secretion, and increased bacterial burden [ ] . while one study suggested only nod was involved in these responses in ripk − /− mice [ ] , another implicated both nod and nod [ ] . overall, it appears nod signaling plays a role in ripk -mediated responses to l. pneumophila, however it doesn't rule out involvement of other signaling pathways initiated by tlr , il- r or il- r [ ] . borrelia burgdorferi is the causative agent of lyme disease, a tickborne infection that causes multi-systemic illness [ ] . early studies showed that ripk expression is increased in astrocytes and microglia exposed to borrelia spirochetes [ , ] . subsequent work showed that peritoneal macrophages from ripk − /− mice exhibit a significant reduction in il- β, il- and il- production following stimulation with heat-killed borrelia, compared to wt mice [ ] . here, through an unknown mechanism, uptake and degradation of borrelia in lysosomes introduces pgn to the cytosol and stimulates nod /ripk -mediated nf-κb activation and inflammatory cytokine production [ ] . ripk induces antibacterial autophagic responses by signaling between nods and the autophagy factor atg l [ ] (fig. ) . mutations in atg l disrupt an inhibitory interaction with nod and consequently increase the activation of ripk [ ] . excessive ripk activation has been reported in pediatric crohn's disease (cd) [ , ] and there is a strong link between resident intestinal bacteria and cd pathogenesis; therefore, it has been proposed that ineffective bacterial clearance due to impaired anti-bacterial autophagy is an important contributor to the pathogenesis of this chronic inflammatory disease [ , ] . autophagy has an essential role in innate immunity and the elimination of pathogens that have escaped into the cytoplasm, as it forms a double-layered membrane that envelopes cytosolic bacteria for degradation via fusion with lysosomes [ ] [ ] [ ] . the invasive gastrointestinal pathogen, shigella flexneri, is a major cause of morbidity and mortality in children under years in developing countries [ ] , and is an emerging sexually transmitted infection of men who have sex with men [ ] . s. flexneri induces nf-κb and jnk activation in a nod /ripk -dependent manner to limit bacterial replication in intestinal epithelial cells [ ] . although early studies did not investigate mechanisms of s. flexneri killing, it was pivotal in understanding the mechanisms of nod/ripk -mediated autophagy in future infection studies. the gastric pathogen helicobacter pylori is subject to degradation by autophagy via the nod /ripk signaling axis. h. pylori infection has long been implicated in the development of gastric cancer [ ] [ ] [ ] , and recently, polymorphisms in ripk were found to be associated with increased susceptibility to gastric cancer in japanese populations [ ] where the prevalence of this disease is very high. indeed, nod − /− mice are highly susceptible to h. pylori infection [ ] , and mechanistically, irving et al. [ ] demonstrated that in gastric epithelial cells, ripk mediates nod -dependent il- production and autophagosome formation in early endosomes in response to h. pylori-derived outer membrane vesicles (omvs) containing pgn. overall, nod /ripk signaling protects against h. pylori infection and subsequent malignancies. nod /ripk -mediated autophagy aids in the control of a number of pathogens including l. monocytogenes, s. typhimurium and shigella spp. [ ] . l. monocytogenes undergoes autophagosomal degradation in phagocytic cells in mouse bmdms and in vivo via erk activation, in a process mediated by tlr , nod and ripk [ ] . in s. typhimurium infected intestinal epithelial cells, nod /ripk is required for autophagy induction [ ] . this in vitro model of salmonella infection demonstrated a dual role for ripk tyrosine kinase activity in nod -dependent autophagy through activation of p mapk and indirect repression of pp a phosphatase activity [ ] . in addition to pgn stimulation, there is increasing evidence that nod/ripk signaling can be activated by pathogen-induced modifications to the host actin cytoskeleton [ ] [ ] [ ] [ ] . cytoskeletal dynamics are mediated via a balance of active gtp-bound and inactive gdp-bound forms of small rho gtpases [ ] . salmonella and shigella spp. utilise t ss to translocate bacterial effector proteins into host cells, and manipulate host cytoskeletal proteins and innate immune responses [ ] . shigella infection induces the recruitment of gef-h , a guanidine exchange factor (gef) for the small rho gtpase rhoa, to the site of invasion to promote host cell entry. following invasion, the shigella effectors ipgb and ospb induce ripk -dependent nf-κb activation mediated by the interaction of recruited gef-h with nod [ ] . the s. typhimurium effector sipa drives nod /nod /ripk dependent nfκb activation via an unknown mechanism [ ] , whereas, sope, functions as a gef for the small rho gtpases rac and cdc . in this setting, rac and cdc interact with the nod /ripk signaling complex in the absence of pgn to mediate nf-κb-dependent inflammation [ ] . in addition, the escherichia coli cytotoxic necrotising factor (cnf ) activates the small rho gtpase rac , which then interacts with ripk and ripk to induce a potent inflammatory response, independent of nod / [ ] . many other bacterial pathogens have also been shown to induce changes to rho gtpases [ ] , overall highlighting the role of pathogen-induced small rho gtpase activation in nod /ripk -mediated inflammation. pathogen-activated endoplasmic reticulum (er)-stress also drives nod/ripk -induced inflammation [ ] . the intracellular pathogen brucella abortus induces er stress via the ire ⍺ pathway of the unfolded protein response (upr). ire ⍺ acts as a receptor that is stimulated upon binding of the brucella t ss effector vcec to the er chaperone bip, and subsequently recruits traf to activate nod /ripk -mediated nf-κb activation [ ] . although the precise mechanism is not yet established, the intracellular pathogen chlamydia muridarum also induces nod /-nod /ripk signaling in response to er stress in vitro [ ] , however in vivo studies have shown that ripk deletion has a limited effect on chlamydial infection in terms of bacterial burden, immune responses and pathology [ ] . given that many pathogens including iav [ ] and hcmv [ ] activate er-stress followed by induction of the upr, it could be likely that nod/ripk signaling has an underappreciated role in host immunity via this pathogen-induced mechanism. regulation of ripk -mediated inflammatory responses to infection is dependent upon the deubiquitinating enzyme cyld [ ] . during in vitro infection of mouse bone marrow-derived macrophages (bmdms) with l. monocytogenes, cyld binds and deubiquitylates ripk , resulting in decreased activation of nf-κb and erk / signaling. thus, inhibition of ripk by cyld leads to impaired pathogen control due to a reduction in antimicrobial responses including pro-inflammatory cytokine production, ros and nitric oxide (no) production. another recent study used kinase inhibitors to demonstrate functional specificity of the kinase domain of ripk in controlling bacterial pathogens. wehi- is a potent inhibitor of ripk that specifically targets serine/threonine kinase activity [ ] and pre-treatment of cd β + monocytes with this inhibitor significantly reduces tnf production during in vitro infection with l. monocytogenes [ ] , suggesting a role for the serine/threonine kinase activity of ripk in protection against bacterial infection. ripk signaling may also be regulated via the formation of riposomes, which are high molecular weight cytoplasmic complexes comprised of ripk [ , ] . ellwanger et al. [ ] showed riposomes form post-nf-κb activation, and suggest that sequestration of ripk in these complexes may act to dampen ripk signaling. intriguingly, riposomes form in the cytosol of epithelial cells upon invasion with s. flexneri, suggesting the pathogen may actively prevent ripk signaling via an unknown process. given that inhibition of xiap was shown to promote deposition of ripk in riposomes, it may be that shigella encodes a virulence factor that targets xiap for cleavage or degradation, or actively inhibits xiap-designated sites of ubiquitylation on ripk to inhibit inflammatory signaling [ ] . it is now established that nod and nod respond to viral infections, thus participating in the coordinated host defense against viruses [ ] [ ] [ ] [ ] . activation of nod , nod and ripk during viral infection depends on type i ifn, synthesized as a result of activation of other prrs. one of the first studies to assess the role of nod /ripk activation in viral infection showed that ripk was critical for dampening inflammasome activation during h n iav infection [ ] . here, ripk − /− mice were highly susceptible iav infection, whereby enhanced nlrp inflammasome activation and increased il- secretion were potent drivers of disease progression and mortality in vivo. negative regulation of inflammasome activity by ripk is dependent its kinase-mediated activation of the mitophagy inducer, ulk . thus nod and ripk respond to iav infection by promoting ulk phosphorylation and inducing mitophagy, which dampens inflammasome activation and il- production. in addition, both nk cells and cd + t cells isolated from iav-infected ripk -/mice are highly activated and exhibit increased ifn-γ production despite the total numbers of these cells being similar in wt mice. these results indicate that increased il- in ripk -/mice subsequently leads to increased ifn-γ production from innate and adaptive cell populations [ ] . hepatitis b and c virus (hbv and hcv) are associated with the development of hepatocellular carcinoma [ ] . nod /ripk signaling is activated by the viral polymerase ns b of hcv [ ] , thus deletion of ripk in heparg cells expressing ns b, results in significantly reduced mapk activation, proinflammatory cytokine production, and ifnβ production. in hbv infection, the hepatitis b e-antigen (hbeag) inhibits ripk expression and also interacts with ripk in hepg cells in vitro, resulting in inhibition of nod /ripk -mediated nf-κb activation and subsequent il- production [ ] . these studies highlight the importance of ripk in controlling chronic hepatitis infections. to date, the only known pathogens to directly target ripk during infection are hiv- and the primary etiologic agent of periodontal disease, porphyromonas gingivalis. the hiv- protease pr cleaves ripk within the n-terminus, although its outcomes in infection have not been tested [ ] . similarly, p. gingivalis infection of human aortic endothelial cells results in rapid direct cleavage of ripk , and is dependent upon the lysine-specific protease, gingipain (kgp) [ ] . given the mounting evidence for pathogens targeting rip kinases, it would not be surprising if other pathogens were found to specifically inactivate ripk in future studies. there is currently relatively little published on ripk - and their role in host responses to pathogen infection, however ripk is emerging as an important mediator of immunity to intracellular pathogens. ripk has a well-described role in cellular differentiation, but also mediates proinflammatory cytokine production in keratinocytes via direct stimulation of irf [ ] . furthermore, overexpression of ripk leads to nf-κb and mapk activation [ ] . although no study has reported a direct role for ripk in host responses to pathogens, it would not be surprising to find ripk mediates inflammation during infection. ripk on the other hand, has no reported association with innate or adaptive immune responses in mammals. pathogenic variants of ripk are one of the most prominent genetic causes of parkinson disease (pd) [ ] , whereas ripk variants have been shown to have no association with the development of pd. as for infection, the only reported data for ripk is in relation to bovine viral diarrhoea virus (bvdv), where ripk is downregulated in pbmcs infected with bvdv- [ ] . ripk however, has been shown to play a role in numerous cellular processes associated with pathogen control, including vesicular trafficking, microtubule binding, autophagy and mitophagy [ ] . one of the first studies to examine the role of ripk in innate immunity found that ripk contributes to the restriction of s. typhimurium by macrophages in vitro [ ] . this was supported in vivo as ripk − /− mice are more susceptible to s. typhimurium intraperitoneal infection, exhibiting decreased il- β secretion, reduced neutrophil infiltration, high bacterial load in the peritoneal cavity and overall increased mortality [ ] . mechanistically, this study showed ripk forms a complex with the nlrc inflammasome in a kinase dependent manner, which then promotes inflammasome activation and restriction of bacterial growth during infection [ ] . similarly, ripk -/mice are more susceptible to oral infection with l. monocytogenes than wt mice [ ] . this study showed that ripk is highly expressed in lysozyme-positive paneth cells and myeloid cells within the lamina propria of the ileum, suggesting ripk plays a protective role at the intestinal mucosa. this is further supported by the fact that mutations in ripk are associated with increased severity of inflammatory bowel disease [ ] . similar to ripk , polymorphisms in ripk are associated with the development of multibacillary leprosy caused by m. leprae [ , , fig. . cellular responses to bacterial infection mediated by ripk (lrrk ). sensing of lps by tlr promotes localization of ripk to endosomal membranes [ , ] . here, ripk can be exploited by m. tuberculosis (mtb) to promote bacterial replication, as ripk recruits rubicon to the endosome, where this complexes with pi k to prevent further phagosome maturation [ ] . following priming by prrs, detection of s. typhimurium bacterial components such as flagellin (or type iii secretion system rod proteins) by naip family members induces nlrc activation [ ] . in contrast to ripk 's role in mtb infection, kinase-dependent interactions between ripk and nlrc promote efficient inflammasome assembly and aid downstream restriction of bacterial growth [ ] . ] and mtb infection [ ] . a recent study identified a mechanism whereby ripk negatively regulates phagosome maturation in macrophages by controlling rubicon/pi k activity on phagosomes in a kinase dependent manner, resulting in impaired immune responses and promotion of mtb replication [ ] (fig. ) . contradictory to the requirement of ripk for the control of salmonella and listeria infection, ripk -deficiency in macrophages or mice results in improved control of mtb infection, which supports a specific role for ripk in mycobacterial control via the regulation of degradative pathways [ ] . ripk − /− mice exhibit increased transcription of type ii ifn, but decreased transcription of type i ifn during mtb infection [ ] , and ripk -deficient macrophages fail to induce type i ifn in vitro when infected with mtb [ ] . mechanistically, ripk regulates type i ifn gene expression by maintaining mitochondrial homeostasis [ ] . given that production of type i ifns during mtb infection have been shown to promote disease [ ] , this may explain why mtb infection is limited in the absence of ripk . thus, ripk acts as a regulator of early clearance of mtb and given its function is kinase dependent, there may be therapeutic potential for ripk -specific kinase inhibitors in tuberculosis. sensing of lps by tlr promotes localization of ripk to endosomal membranes [ , ] . here, ripk can be exploited by m. tuberculosis (mtb) to promote bacterial replication, as ripk recruits rubicon to the endosome, where this complexes with pi k to prevent further phagosome maturation [ ] . following priming by prrs, detection of s. typhimurium bacterial components such as flagellin (or type iii secretion system rod proteins) by naip family members induces nlrc activation [ ] . in contrast to ripk 's role in mtb infection, kinase-dependent interactions between ripk and nlrc promote efficient inflammasome assembly and aid downstream restriction of bacterial growth [ ] . ripk , , and have emerged as critical mediators of inflammation and innate immunity in response to multiple diverse pathogens. not surprisingly, many pathogens have evolved highly specific mechanisms to either directly or indirectly target ripk signaling networks to benefit replication and survival, and have thus provided invaluable knowledge on the physiological role of ripk signaling in the context of infection. one of the major challenges that remain in the field of host-pathogen interactions is the consistency of experimental conditions, whereby factors including genetic background of animal models, specific pathogen strains (lab adapted vs currently circulating clinical isolates), cell types (primary, site specific vs immortalised carcinoma cell lines) and the use of inhibitors (e.g. nec- vs nec- stable) heavily influence experimental outcomes. the more unified this becomes globally, the more reliable the data will become. finally, ripks are currently under critical review as potential therapeutic targets, as dysregulation of ripk signaling is closely associated with hyperinflammation and pathology. multiple studies are investigating various classes of kinase inhibitors for ripk - [ ] , however given the importance of kinase-dependent cell-death responses to infection, it is critical that we understand the impact of these therapeutic interventions on infection outcomes before introduction to the clinic. the same goes for the recently identified small molecule therapy, proteolysis-targeting chimeras (protacs), for the selective degradation of ripk [ ] ; given the importance of ripk in detection of multiple intracellular pathogens, what would be the effect on infection outcome? overall, there has been significant progress made in the field of ripk biology, and continued efforts on this front will help to bolster our understanding of host-pathogen interactions and potential therapeutic development for infectious diseases. to this end, we must continue to develop a comprehensive understanding the of ) the biochemical function of each ripk domain and the role they play in response to specific pathogens, and ) biochemical mechanisms of virulence factors in currently circulating pathogenic organisms. we, the authors declare no competing interests. pattern recognition receptors and inflammation rip kinases as modulators of inflammation and immunity inhibition of rip 's tyrosine kinase activity limits nod -driven cytokine responses the death domain kinase rip mediates the tnf-induced nf-kappab signal the death domain kinase rip is essential for trail (apo l)-induced activation of ikappab kinase and c-jun n-terminal kinase rip: a novel protein containing a death domain that interacts with fas/apo- (cd ) in yeast and causes cell death rip mediates the trif-dependent toll-like receptor -and -induced nf-κb activation but does not contribute to interferon regulatory factor activation rip is an essential mediator of toll-like receptor -induced nfkappa b activation ripk in cell death and inflammation: the good, the bad, and the ugly tradd-traf and tradd-fadd interactions define two distinct tnf receptor signal transduction pathways induction of tnf receptor i-mediated apoptosis via two sequential signaling complexes the role of the death-domain kinase rip in tumournecrosis-factor-induced activation of mitogen-activated protein kinases activation of ikk by tnfalpha requires site-specific ubiquitination of rip and polyubiquitin binding by nemo vucic, c-iap and c-iap are critical mediators of tumor necrosis factor alpha (tnfalpha)-induced nf-kappab activation diverse ubiquitin linkages regulate rip kinases-mediated inflammatory and cell death signaling peli facilitates trif-dependent toll-like receptor signaling and proinflammatory cytokine production kinase activities of ripk and ripk can direct ifn-β synthesis induced by lipopolysaccharide differential roles of mda and rig-i helicases in the recognition of rna viruses rig-i rna helicase activation of irf transcription factor is negatively regulated by caspase- -mediated cleavage of the rip protein identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf receptor-interacting protein homotypic interaction motif-dependent control of nf-kappa b activation via the dnadependent activator of ifn regulatory factors dai/zbp recruits rip and rip through rip homotypic interaction motifs to activate nf-kappab molecular mechanisms of neuroinflammation and injury during acute viral encephalitis zika virus pathogenesis: from early case reports to epidemics ripk restricts viral pathogenesis via cell death-independent neuroinflammation the nucleotide sensor zbp and kinase ripk induce the enzyme irg to promote an antiviral metabolic state in neurons krebs cycle reimagined: the emerging roles of succinate and itaconate as signal transducers differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded rna viruses inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus rip -interacting protein cooperative inhibition of rip -mediated nf-κb signaling by cytomegalovirus-encoded deubiquitinase and inactive homolog of cellular ribonucleotide reductase large subunit cleavage specificity of the ul deubiquitinating protease activity of human cytomegalovirus and the growth of an active-site mutant virus in cultured cells ripk promotes jev replication in neurons via downregulation of ifi l ripk interacts with mavs to regulate type i ifn-mediated immunity to influenza a virus infection protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity epstein-barr virus encoded latent membrane protein suppresses necroptosis through targeting ripk / ubiquitination autophagy during viral infection -a doubleedged sword rip regulates autophagy and promotes coxsackievirus b infection of intestinal epithelial cells cell death programs in yersinia immunity and pathogenesis caspase- and rip kinases regulate bacteria-induced innate immune responses and cell death yopj family effectors promote bacterial infection through a unique acetyltransferase activity, microbiol molecular pathogenesis of infections caused by legionella pneumophila discovery of ubiquitin deamidases in the pathogenic arsenal of legionella pneumophila glutamine deamidation and dysfunction of ubiquitin/nedd induced by a bacterial effector family cycle inhibiting factors (cifs): cyclomodulins that usurp the ubiquitin-dependent degradation pathway of host cells pathogen blocks host death receptor signalling by arginine glcnacylation of death domains a type iii effector antagonizes death receptor signalling during bacterial gut infection the type iii effectors nlee and nleb from enteropathogenic e. coli and ospz from shigella block nuclear translocation of nf-kappab p a type iii effector protease nlec from enteropathogenic escherichia coli targets nf-κb for degradation apoptotic cell-derived extracellular vesicles: more than just debris the tumour suppressor cyld negatively regulates nf-κb signalling by deubiquitination deubiquitination and ubiquitin ligase domains of a downregulate nf-kappab signalling leverkus, ciaps block ripoptosome formation, a rip /caspase- containing intracellular cell death complex differentially regulated by cflip isoforms the ripoptosome, a signaling platform that assembles in response to genotoxic stress and loss of iaps ripk contributes to tnfr -mediated ripk kinase-dependent apoptosis in conditions of ciap / depletion or tak kinase inhibition tnf-alpha induces two distinct caspase- activation pathways cleavage of rip inactivates its caspase-independent apoptosis pathway by removal of kinase domain cleavage of the death domain kinase rip by caspase- prompts tnf-induced apoptosis activation of a pro-apoptotic amplification loop through inhibition of nf-κb-dependent survival signals by caspase-mediated inactivation of rip the rip /rip necrosome forms a functional amyloid signaling complex required for programmed necrosis mixed lineage kinase domain-like protein mediates necrosis signaling downstream of rip kinase mixed lineage kinase domain-like protein mlkl causes necrotic membrane disruption upon phosphorylation by rip apoptosis induced by the toll-like receptor adaptor trif is dependent on its receptor interacting protein homotypic interaction motif toll-like receptor -mediated necrosis via trif, rip , and mlkl zbp : innate sensor regulating cell death and inflammation zbp and tak : master regulators of nlrp inflammasome/pyroptosis, apoptosis, and necroptosis (panoptosis) ripk counteracts zbp -mediated necroptosis to inhibit inflammation ripk inhibits zbp -driven necroptosis during development innate immune priming in the absence of tak drives ripk kinase activityindependent pyroptosis, apoptosis, necroptosis, and inflammatory disease the panoptosome: a deadly protein complex driving pyroptosis, apoptosis, and necroptosis (panoptosis), front targeting apoptosis pathways in infections the suppression of apoptosis by α-herpesvirus the ribonucleotide reductase r subunits of herpes simplex virus types and protect cells against tnfα-and fasl-induced apoptosis by interacting with caspase- the ribonucleotide reductase r subunits of herpes simplex virus and protect cells against poly(i ⋅ c)-induced apoptosis the zα domain of zbp is a molecular switch regulating influenza-induced panoptosis and perinatal lethality during development dai senses influenza a virus genomic rna and activates ripk -dependent cell death zbp /dai is an innate sensor of influenza virus triggering the nlrp inflammasome and programmed cell death pathways zbp /dai ubiquitination and sensing of influenza vrnps activate programmed cell death ripk activates parallel pathways of mlkl-driven necroptosis and fadd-mediated apoptosis to protect against influenza a virus cell-extrinsic tnf collaborates with trif signaling to promote yersinia-induced apoptosis ripk -dependent apoptosis bypasses pathogen blockade of innate signaling to promote immune defense induction of necrotic-like cell death by tumor necrosis factor alpha and caspase inhibitors: novel mechanism for killing virus-infected cells phosphorylation-driven assembly of the rip -rip complex regulates programmed necrosis and virus-induced inflammation a role for tumor necrosis factor receptor- and receptorinteracting protein in programmed necrosis and antiviral responses cutting edge: ripk kinase inactive mice are viable and protected from tnf-induced necroptosis in vivo rip kinase activity is critical for skin inflammation but not for viral propagation identification of a conserved motif that is necessary for binding of the vaccinia virus e l gene products to double-stranded rna inhibition of dai-dependent necroptosis by the z-dna binding domain of the vaccinia virus innate immune evasion protein differential function and expression of the viral inhibitor of caspase -induced apoptosis (vica) and the viral mitochondria-localized inhibitor of apoptosis (vmia) cell death suppressors conserved in primate and rodent cytomegaloviruses a cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase- activation cytomegalovirus m cell death suppression requires receptor-interacting protein (rip) homotypic interaction motif (rhim)-dependent interaction with rip dai/zbp /dlm- complexes with rip to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vira the ribonucleotide reductase r homolog of murine cytomegalovirus is not a functional enzyme subunit but is required for pathogenesis virus inhibition of rip -dependent necrosis murine cytomegalovirus ie -dependent transcription is required for dai/zbp -mediated necroptosis human cytomegalovirus induces the interferon response via the dna sensor zbp suppression of rip -dependent necroptosis by human cytomegalovirus herpes simplex virus suppresses necroptosis in human cells rip /rip binding to hsv- icp initiates necroptosis to restrict virus propagation in mice direct activation of rip /mlkl-dependent necrosis by herpes simplex virus (hsv- ) protein icp triggers host antiviral defense the ubiquitin-modifying enzyme a restricts ubiquitination of the kinase ripk and protects cells from necroptosis viral mlkl homologs subvert necroptotic cell death by sequestering cellular ripk hiv- protease cleaves the serine-threonine kinases ripk and ripk dysfunctional hiv-specific cd + t cell proliferation is associated with increased caspase- activity and mediated by necroptosis coxsackievirus a induces necroptosis for viral production advances in understanding respiratory syncytial virus infection in airway epithelial cells and consequential effects on the immune response rsv infection promotes necroptosis and hmgb release by airway epithelial cells respiratory syncytial virus induces the classical ros-dependent netosis through pad- and necroptosis pathways activation a sars-cov- protein interaction map reveals targets for drug repurposing sars-coronavirus open reading frame- a drives multimodal necrotic cell death severe acute respiratory syndrome-associated coronavirus a protein forms an ion channel and modulates virus release role of severe acute respiratory syndrome coronavirus viroporins e, a, and a in replication and pathogenesis espl is a bacterial cysteine protease effector that cleaves rhim proteins to block necroptosis and inflammation toxin-induced necroptosis is a major mechanism of staphylococcus aureus lung damage pore-forming toxins induce macrophage necroptosis during acute bacterial pneumonia necroptosis promotes staphylococcus aureus clearance by inhibiting excessive inflammatory signaling type i interferon induces necroptosis in macrophages during infection with salmonella enterica serovar typhimurium salmonella-induced mir- enhances necroptotic death in macrophage cells via targeting rip / susceptibility of mycobacterium tuberculosis-infected host cells to phospho-mlkl driven necroptosis is dependent on cell type and presence of tnfα bcl-x(l) mediates ripk -dependent necrosis in m. tuberculosisinfected macrophages necroptotic signaling is primed in mycobacterium tuberculosis-infected macrophages, but its pathophysiological consequence in disease is restricted is receptor-interacting protein kinase a viable therapeutic target for mycobacterium tuberculosis infection? toward targeting inflammasomes: insights into their regulation and activation human monocytes engage an alternative inflammasome pathway caspase- blocks kinase ripk -mediated activation of the nlrp inflammasome ripk promotes cell death and nlrp inflammasome activation in the absence of mlkl differential requirement for the activation of the inflammasome for processing and release of il- beta in monocytes and macrophages vesicular stomatitis virus transmission: a comparison of incriminated vectors rna viruses promote activation of the nlrp inflammasome through a rip -rip -drp signaling pathway platelets mediate increased endothelium permeability in dengue through nlrp -inflammasome activation swine influenza virus induces ripk /drp -mediated interleukin- beta production caspase- scaffolding function and mlkl regulate nlrp inflammasome activation downstream of tlr rna viruses promote activation of the nlrp inflammasome through cytopathogenic effectinduced potassium efflux the mitochondrial phosphatase pgam is dispensable for necroptosis but promotes inflammasome activation in macrophages caspase- mediates caspase- processing and innate immune defense in response to bacterial blockade of nf-κb and mapk signaling pathogen blockade of tak triggers caspase- -dependent cleavage of gasdermin d and cell death rick/rip /cardiak mediates signalling for receptors of the innate and adaptive immune systems rip is a novel nf-kappab-activating and cell death-inducing kinase the kinase activity of rip determines its stability and consequently nod -and nod -mediated immune responses rick/rip mediates innate immune responses induced through nod and nod but not tlrs an essential role for nod in host recognition of bacterial peptidoglycan containing diaminopimelic acid nod detects a unique muropeptide from gram-negative bacterial peptidoglycan nod is a general sensor of peptidoglycan through muramyl dipeptide (mdp) detection host recognition of bacterial muramyl dipeptide mediated through nod . implications for crohn's disease type iv secretion-dependent activation of host map kinases induces an increased proinflammatory cytokine response to legionella pneumophila a ripk inhibitor delays nod signalling events yet prevents inflammatory cytokine production nod , rip and irf play a critical role in the type i interferon response to mycobacterium tuberculosis atg l and nod interact in an autophagy-dependent antibacterial pathway implicated in crohn's disease pathogenesis the immune receptor nod and kinase rip interact with bacterial peptidoglycan on early endosomes to promote autophagy and inflammatory signaling the protein atg l suppresses inflammatory cytokines induced by the intracellular sensors nod and nod in an autophagy-independent manner involvement of receptor-interacting protein in innate and adaptive immune responses ripk polymorphisms and susceptibility to tuberculosis in a western chinese han population genetic dissection of immunity to mycobacteria: the human model control of human host immunity to mycobacteria streptococcus pneumoniae: transmission, colonization and invasion the tlr -myd -nod -ripk signalling axis regulates a balanced pro-inflammatory and il- -mediated antiinflammatory cytokine response to gram-positive cell walls cutting edge: recognition of gram-positive bacterial cell wall components by the innate immune system occurs via toll-like receptor cell wall-mediated neuronal damage in early sepsis interleukin- plays a key role in the modulation of neutrophils recruitment and lung inflammation during infection by streptococcus pneumoniae nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized streptococcus pneumoniae the pattern recognition receptors nod and nod account for neutrophil recruitment to the lungs of mice infected with legionella pneumophila nod and nod regulation of pulmonary innate immunity to legionella pneumophila lyme disease: review characterization of nucleotide-binding oligomerization domain (nod) protein expression in primary murine microglia functional expression of nod , a novel pattern recognition receptor for bacterial motifs, in primary murine astrocytes recognition of borrelia burgdorferi by nod is central for the induction of an inflammatory reaction inflammatory signaling by nod-ripk is inhibited by clinically relevant type ii kinase inhibitors activation of nod -mediated intestinal pathway in a pediatric population with crohn's disease digesting the genetics of inflammatory bowel disease: insights from studies of autophagy risk genes autophagy in intracellular bacterial infection autophagy, immunity, and microbial adaptations autophagy in immunity against intracellular bacteria morbidity and mortality due to shigella disease study extensively drug-resistant shigellosis in australia among men who have sex with men card /nod mediates nf-kappab and jnk activation by invasive shigella flexneri association between infection with helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation helicobacter pylori infection and the risk of gastric carcinoma helicobacter pylori infection and the development of gastric cancer association between receptor interacting serine/threonine kinase polymorphisms and gastric cancer susceptibility nod responds to peptidoglycan delivered by the helicobacter pylori cag pathogenicity island amino acid starvation induced by invasive bacterial pathogens triggers an innate host defense program tlr and rip pathways mediate autophagy of listeria monocytogenes via extracellular signalregulated kinase (erk) activation a dual role for receptorinteracting protein kinase (rip ) kinase activity in nucleotide-binding oligomerization domain (nod )-dependent autophagy the salmonella type iii effector ssph specifically exploits the nlr co-chaperone activity of sgt to subvert immunity gef-h mediated control of nod dependent nf-kappab activation by shigella effectors manipulation of small rho gtpases is a pathogeninduced process detected by nod a salmonella virulence factor activates the nod /nod signaling pathway the small gtpase rho: cellular functions and signal transduction assembly, structure, function and regulation of type iii secretion systems pathogen-derived effectors trigger protective immunity via activation of the rac enzyme and the imd or rip kinase signaling pathway nod and nod : beyond peptidoglycan sensing nod and nod signalling links er stress with inflammation stimulation of the cytosolic receptor for peptidoglycan, nod , by infection with chlamydia trachomatis or chlamydia muridarum influenza induces endoplasmic reticulum stress, caspase- -dependent apoptosis, and c-jun n-terminal kinase-mediated transforming growth factor-β release in lung epithelial cells human cytomegalovirus induces apoptosis in neural stem/progenitor cells derived from induced pluripotent stem cells by generating mitochondrial dysfunction and endoplasmic reticulum stress receptor-interacting protein kinase- inhibition by cyld impairs antibacterial immune responses in macrophages xiap controls ripk signaling by preventing its deposition in speck-like structures structural basis of rip activation and signaling viral infection augments nod / signaling to potentiate lethality associated with secondary bacterial infections molecular cloning and functional analysis of nucleotide-binding oligomerization domain (nod ) in olive flounder, paralichthys olivaceus activation of innate immune antiviral responses by nod a physical and regulatory map of host-influenza interactions reveals pathways in h n infection receptor interacting protein kinase -mediated mitophagy regulates inflammasome activation during virus infection a clinical review of viral hepatitis simonin, nod participates in the innate immune response triggered by hepatitis c virus polymerase hepatitis b virus e antigen physically associates with receptor-interacting serine/threonine protein kinase and regulates il- gene expression pathogen-mediated proteolysis of the cell death regulator ripk and the host defense modulator ripk in human aortic endothelial cells ripk activates an irf -mediated proinflammatory cytokine response in keratinocytes rip (dik/pkk), a novel member of the rip kinase family, activates nf-kappa b and is processed during apoptosis for the courage-pd consortium, t.f.p.s.d. c, the international parkinson's disease genomics, c, frequency of loss of function variants in lrrk in parkinson disease transcriptome analysis reveals differential immune related genes expression in bovine viral diarrhea virus- infected goat peripheral blood mononuclear cells (pbmcs) lrrk at the interface of autophagosomes, endosomes and lysosomes lrrk is involved in the ifngamma response and host response to pathogens lrrk promotes the activation of nlrc inflammasome during salmonella typhimurium infection commensal bacteria direct selective cargo sorting to promote symbiosis the kinase lrrk is a regulator of the transcription factor nfat that modulates the severity of inflammatory bowel disease a missense lrrk variant is a risk factor for excessive inflammatory responses in leprosy association of the lrrk genetic polymorphisms with leprosy in han chinese from southwest china meta-analysis of human gene expression in response to mycobacterium tuberculosis infection reveals potential therapeutic targets lrrk is a negative regulator of mycobacterium tuberculosis phagosome maturation in macrophages lrrk maintains mitochondrial homeostasis and regulates innate immune responses to mycobacterium tuberculosis genome-wide expression profiling identifies type interferon response pathways in active tuberculosis leucine rich repeat kinase and innate immunity membrane recruitment of endogenous lrrk precedes its potent regulation of autophagy molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases inhibitors targeting ripk /ripk : old and new drugs extended pharmacodynamic responses observed upon protac-mediated degradation of ripk induction of necroptotic cell death by viral activation of the rig-i or sting pathway depletion of ripk in hepatocytes exacerbates liver damage in fulminant viral hepatitis reovirus activates a caspase-independent cell death pathway viral rna at two stages of reovirus infection is required for the induction of necroptosis the nod/rip pathway is essential for host defenses against chlamydophila pneumoniae lung infection receptor-interacting serine/threonine kinase -and -dependent inflammation induced in lungs of chicken infected with pasteurella multocida leishmania braziliensis subverts necroptosis by modulating ripk expression ripk -ripk -mlkl-associated necroptosis drives leishmania infantum killing in neutrophils ripk and pgam control leishmania replication through distinct mechanisms involvement of nod in the innate immune response elicited by malarial pigment hemozoin transcriptomic alterations in trypanosoma cruzi-infected cardiac myocytes we would like to acknowledge medina pell for her assistance with assembling the manuscript. key: cord- -n x z authors: zelaya, hortensia; alvarez, susana; kitazawa, haruki; villena, julio title: respiratory antiviral immunity and immunobiotics: beneficial effects on inflammation-coagulation interaction during influenza virus infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: n x z influenza virus (ifv) is a major respiratory pathogen of global importance, and the cause of a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given its high capacity to change antigenically, acquired immunity is often not effective to limit ifv infection and therefore vaccination must be constantly redesigned to achieve effective protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatory-mediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation–coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. influenza virus (ifv) is a major respiratory pathogen of global importance, and the cause of a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given its high capacity to change antigenically, acquired immunity is often not effective to limit ifv infection and therefore vaccination must be constantly redesigned to achieve effective protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatory-mediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation-coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. keywords: immunobiotics, influenza virus, inflammation, coagulation, respiratory immunity introduction influenza virus (ifv) is a member of the orthomyxoviridae family that contains a negative-sense, single-stranded, segmented rna genome protected by a capsid of viral ribonucleoproteins. this virus is categorized into subtypes based on the expression of hemagglutinin (ha) and neuraminidase on the surface of the viral envelope. influenza is a highly contagious viral infection that has a substantial impact on global health and ifv is a major respiratory pathogen that causes a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given the high capacity of ifv to change antigenically, acquired immunity is often not effective to limit infection and therefore vaccination must be constantly redesigned to achieve protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatorymediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation-coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. the first barrier that protects the host against ifv infection is the respiratory epithelium through its capacity to recognize the viral attack. when ifv successfully overcomes the respiratory barrier constituted by the mucus layer and the ciliar movement, it mediates its attachment and internalization into respiratory epithelial cells to start its replication ( ) . during the viral attack, several pathogen-associated molecular patterns (pamps) are exposed and recognized by pattern-recognition receptors (prrs) expressed in respiratory cells (figure ). it is now well established that the most important prrs involved in the recognition of ifv are the toll-like receptor (tlr)- and tlr and the rna recognition protein rig- ( ) . tlr is expressed in endosomes and is able to recognize viral double-stranded rna (dsrna) that is produced during viral replication; while endosomal tlr and cytoplasmic rig-i recognize single-stranded rna (ssrna). rig-i signals through mitochondrial antiviral signaling protein. the pamps-pprs interaction leads to the activation of several signaling pathways that induce the activation of nuclear factor κb (nf-κb) and interferon (ifn) regulatory factor (irf ) and the production of type i and iii ifns and inflammatory cytokines ( ) . type i ifns, especially ifn-β, produced and released during the earlier stages of ifv infection are key to develop an antiviral state in the respiratory tract. it was reported that human bronchial epithelial cells release preformed ifn-β in response to ifv challenge inducing a protective role ( ) . ifns produced by infected cells are able to act in a paracrine or autocrine manner activating their receptors (ifnar) and increasing the expression of hundreds of genes that counteract viral replication. functional genomic studies have identified several of the ifn-induced factors that have important roles in controlling ifv replication ( ) including the ifn-inducible transmembrane proteins , , and ( ), mx proteins ( ) , and ′, ′-oligoadenylate synthetase (oas)-rnaasel system ( ) . proinflammatory cytokines and chemokines produced as a result of tlr and rig-i activation during ifv infection are also important for the generation of the respiratory antiviral innate immune response. infection of epithelial cells by ifv increases the expression of tnf-α, il- , il- , ccl (mip- ), ccl (rantes), ccl (mip- α), and cxcl (ip- ) ( ). the production of these cytokines is complemented by activity of inflammasomes that induce the activation of caspase- and promote the generation of the active forms of il- β and il- (figure ) . ifv has been shown to activate mainly the nlrp inflammasome which is essential for the protection against the virus since several studies demonstrated that mice lacking nlrp or caspase- have decreased il- β and il- secretion and increased mortality after ifv challenge ( ) ( ) ( ) . the proinflammatory cytokines and chemokines are responsible for the activation of resident immune cells such as innate lymphoid cells, alveolar macrophages, and dendritic cells (dcs) as well as for the recruitment of neutrophils, macrophages, and lymphocytes into the respiratory tract ( , ) (figure ) . respiratory cells infected with ifv express ha on their surface that is important for its recognition by nk cells ( ) . it was established that ha expressed by the infected cells is recognized by nkp and pkp receptors of nk cells that then mediated the lysis of ifv-infected cells ( ) . macrophages activated during ifv infection produce ifns, il- , tnf-α, and nitric oxide synthase that amplify the inflammatory response. in addition, macrophages limit the viral spread by the elimination of apoptotic-infected cells and through phagocyte-mediated opsonophagocytosis of ifv ( ) . the production of proinflammatory cytokines during the generation of the respiratory innate immune response against ifv also conditions the adaptive immune response, which includes the production of virusspecific systemic and mucosal antibodies as well as the induction of specific t cell responses ( ) . after exposure to ifv there is an activation of antibody responses in the respiratory tract. upper airway exposure results primarily in an iga response while the contact of ifv with the deep lung induces an increased production of pathogen-specific igg ( ) . following exposure to ifv in the airways there is an antigen uptake and processing by dcs, activation of cd + th cells, and generation of iga-producing plasma cells that populate airway lamina propria. secretory iga has a noninflammatory protective function since these antibodies can bind to virus without activating complement or stimulating the release of inflammatory mediators by innate immune cells ( , ) . iga prevents ifv from adhering to the epithelial surface by inducing viral agglutination, and masking adhesion epitopes. in the deep lung, when ifv reach the alveolar space, there is a differentiation and expansion of antibody-secreting plasma cells that are committed to the production of igg. induction of neutralizing respiratory and serum igg antibodies is a key event in the defense against influenza infection since igg prevents systemic spread ( ) . influenza infection in the lungs also activates the cellular adaptive immune response by stimulating the production of ifn-γ by th cells that effectively activate cd + t cells and macrophages, which clear virus and infected cells from the lungs ( ) . therefore, during uncomplicated influenza, adaptive immune response ultimately results in clearance of ifv from the lungs through the activity of virus-specific antibodies and cd + and cd + t cells. the gut microbiome, which is defined as the collective group of microorganisms and their associated genes within the intestinal tract, is considered as a key player in the modulation of host intestinal immune responses ( , ) . in fact, the impact of gut commensal bacteria on the innate and adaptive immune responses to enteric pathogens has been recognized conclusively ( ) ( ) ( ) . however, the effect of gut microbiome on the immune responses in distal mucosal sites and its impact in the outcome of respiratory infections has recently been exposed. in this regard, some studies have demonstrated an important role for intestinal microbiota in maintaining respiratory antiviral immunity against ifv ( , ) . iwasaki and colleagues observed that commensal bacteria within the gut, especially gram-positive bacterial populations, had an important role in supporting an appropriate immune response to ifv infection in the respiratory tract ( ) . the work demonstrated that oral antibiotic treatments impaired the resistance of mice to the intranasal infection with ifv as noted by the elevated lung viral titers when compared to non-antibiotictreated animals. results indicated that gut gram-positive bacteria provided protection by triggering an adequate inflammatory response through inflammasomes activation. in antibiotictreated mice, synthesis of pro-il- β, pro-il- , and nlrp was impaired even at the steady state. in addition, depletion of grampositive bacterial populations in the gut resulted in an alteration of the distribution and activation of respiratory dcs at steady state as well as in a diminished dcs migration from the lung to the draining lymph nodes, resulting in reduced activation of cd + and cd + t cells after influenza challenge ( ) . alteration of respiratory dcs activities also correlated with impaired expansion of influenza-specific b cells and reduced influenza-specific antibodies. by using germ-free and antibiotic-treated mice challenged with ifv, abt et al. ( ) showed that the absence or the alteration of intestinal microbiota induced an exacerbated weight loss, a greater drop in blood oxygen saturation, increased mortality, and elevated lung viral titers indicating a weaker ability to resist influenza. even more, germ-free and antibiotic-treated mice infected with ifv experienced higher epithelial cell necrosis, peribronchiolar inflammation, severe bronchiole epithelial degeneration, and epithelial hyperplasia when compared to conventional animals ( ) . interestingly, those effects were observed when both the pr strain and the x -gp virus, a less pathogenic strain of ifv that causes minimal mortality and morbidity in conventional mice, were used. consistent with the work by ichinohe et al. ( ) , germ-free and antibiotic-treated mice challenged with ifv had an impaired adaptive immune response as shown by the lower influenza-specific antibodies (serum igm and igg), fewer number of ifv-specific t cells present in lungs, as well as a reduced capacity of specific t cells to produce effector cytokines such as tnf-α, mip- α, il- , and ifn-γ ( ) . moreover, authors demonstrated that the alterations of adaptive immune responses were related to defects in the early innate immune response mediated by macrophages. in fact, transcriptional profiling and computational analyses of macrophages from antibiotic-treated mice indicated a reduced expression of antiviral genes including ifnb, tnfa, il b, irf , mx , and oas a when compared to immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december | volume | article conventional mice. in addition, functional assays of macrophages from antibiotic-treated mice demonstrated that those cells had a defective response to type i ifns and an impaired capacity to limit ifv replication ( ) . the cellular and molecular mechanisms through which the gut microbiome and their derived signals maintain and modulate immune responses in distal mucosal sites are poorly understood. two possible mechanisms that are not mutually exclusive have been proposed to explain this beneficial effect of the gut microbiome. one possibility is that distal mucosal and peripheral immune cells are directly exposed to bacterial products that activate prrs in the steady state and help to maintain the normal immune tone. there is evidence that bacterial products from gut commensals such as peptidoglycan can be absorbed and circulate throughout the host and help to modulate the normal development of immune cells ( ) . in line with this hypothesis, iwasaki and colleagues speculated that bacterial products from gut commensals trigger prrs to stimulate immune cells systemically and that factors released by those cells supported steady-state production of pro-il- β, pro-il- , and nlr proteins. this idea was sustained by their observation that intestinal injection of tlr ligands restored immune responses to ifv in antibiotic-treated mice ( ) . another possibility is that commensal bacteria may indirectly influence systemic and distal mucosal immune responses through immune factors released from the intestinal mucosa including cytokines, chemokines, and grow factors. these research works demonstrated that the gut microbiome provides signals to sustain antiviral innate immune defense mechanisms in the respiratory tract allowing robust and efficient effector responses upon challenge by viral pathogens such as ifv. therefore, the role of the gut microbiome in regulating respiratory antiviral immunity represents an exciting area of research that could help to provide the scientific basis for the development of novel prevention strategies for lung infectious diseases. however, several questions need to be answered to identify new alternatives to improve antiviral respiratory defenses by modulating the microbiota. how the different microbial species from the gut microbiota influence the common mucosal immune system? which prrs are activated by the gut microbiota to functionally modulate antiviral immunity locally and in distal mucosal sites? which cellular functions are modulated by the microbiota after prr activation? has the microbiota the ability to influence immune responses to other respiratory viruses? are similar immune mechanisms activated by the microbiota in high-risk populations (infants, elderly, immunocompromised hosts) in which respiratory viral infections are more frequent and severe? is it possible to beneficially modulate antiviral respiratory defenses by orally administering selected microorganisms with immunomodulatory capacities? research from the last years has provided some answers for the last question. the first studies that assessed the capacity of immunobiotics to favorably modulate the immune response against ifv focused on the humoral immunity ( table ) . yasui et al. ( ) reported that the oral administration of bifidobacterium breve yit improved the production of anti-ifv igg antibodies in serum of ifv-infected mice. the yit strain reduced viral titers, improved the survival rate, and decreased the severity of the symptoms associated to the influenza infection. similarly, it was shown that orally administered non-viable lactobacillus pentosus b ( ) or viable lactobacillus brevis kb ( ) were able to improve the levels of respiratory specific iga and igg antibodies of mice challenged with ifv. moreover, the improved humoral response induced by these strains correlated with significant reduction of viral titers, body weight loss, and a decrease of the alterations of physical conditions induced by ifv. more recently, kikuchi et al. ( ) demonstrated a beneficial effect on the outcome to ifv infection related to an improved respiratory humoral response in lactobacillus plantarum ayatreated mice. in addition, the work proposed a mechanism for the distal immunomodulatory activity induced by orally administered immunobiotics. authors showed that l. plantarum aya fed to mice impacted in peyer's patches (pps) inducing an activation of antigen presenting cells (mainly cd b + dcs) and increasing the production of il- . those changes promoted an igm-to-iga class switch recombination, the differentiation of iga + b cells into plasma cells, and improved the production of mucosal iga in both the intestine and the respiratory tract. those studies show that immunobiotics are capable to modulate the production of systemic and mucosal antibodies against influenza and therefore, to enhance the humoral immune response (figure ) . however, the precise mechanism by which orally administered immunobiotics induce iga production in distant mucosal sites remains unclear. it was also demonstrated that immunobiotics are able to improve cellular immune response against ifv (figure ) . in this regard, it was reported that orally administered lactobacillus casei shirota improved the outcomes of ifv infection of aged ( ) and infant mice ( ) by increasing systemic and respiratory nk cell activity and improving the production of ifn-γ and tnf-α by respiratory lymphocytes. both studies also demonstrated that ifv titers were significantly reduced in aged and infant mice treated with the shirota strain ( , ) . similar to the mechanism proposed to explain the improvement of humoral response, it was postulated that immunobiotic l. casei shirota stimulated th cells and nk cells in pps and induced a mobilization of those cells to lungs and respiratory-associated lymphoid tissues where they produced ifn-γ and enhanced the antiviral defenses. several other studies corroborated these findings by showing similar effects for orally administered lactobacilli ( , ) . immunobiotic lactobacillus strains (l. gasseri tmc , l. rhamnosus gg, or l. plantarum cc ) beneficially modulated nk cells activity and th response against ifv, diminished virus titers and reduced lung pathological changes ( , ) ( table ) . more recently, kawahara et al. ( ) described the improvement of respiratory antiviral response by an orally administered bifidobacteria strain. it was shown that bifidobacterium longum mm- increased respiratory nk cell activity and ifn-γ production resulting in improved clinical symptoms, reduced mortality, and decreased virus titers after ifv challenge. research work has also demonstrated that nasal administration of immunobiotics is an interesting alternative to improve cellular response against influenza infection ( - ) ( table ) . hori et al. ( ) observed that balb/c mice nasally treated with non-viable l. casei shirota had increased levels of il- , ifnγ, and tnf-α in mediastinal lymphoid nodes and lungs. this improved cellular respiratory immunity correlated with a beneficial clinical outcome to ifv challenge. similar observations were performed with nasally administered l. pentosus s-pt ( ) and l. rhamnosus gg ( ). other recent studies have also demonstrated the ability of immunobiotics to improve respiratory innate antiviral defenses in the respiratory tract (table ; figure ) . it was reported that orally administered non-viable l. plantarum l- improved protection against ifv by increasing type i ifn production ( ). the work clearly demonstrated that the increased production of ifn-β induced by the immunobiotic strain correlated with the reduction of viral loads in lungs as well as the improved survival of infected mice. more recently, it was shown that l. gasseri sbt enhanced survival rates and reduced lung viral titers in mice infected with ifv ( ). interestingly, authors observed that the lung expression of the antiviral genes mx and oas a was enhanced in l. gasseri sbt -treated mice and that the inflammatory response triggered by ifv was differentially regulated inducing a lower inflammatory damage ( ). our group has also reported a beneficial regulation of the ifv-triggered inflammatory response by immunobiotics. lung damage induced by ifv is known to be produced by virus replication as well as the uncontrolled inflammatory response that is characterized by a hypersecretion of proinflammatory cytokines, especially tnf-α, il- β, and il- ( ). the adequate production of inflammatory factors is necessary to protect against ifv infection together with an appropriate regulation with anti-inflammatory cytokines to prevent the damage of lung tissue. thus, the proper balance of cytokines is a key factor in determining the outcome of ifv infection. in this regard, we observed that orally ( ) or nasally ( ) administered immunobiotic l. rhamnosus crl differentially regulated the levels and kinetics of inflammatory cells and cytokines in mice after ifv challenge. in our experimental model, we observed increased levels of respiratory tnf-α, il- , neutrophils, and macrophages in crl -treated mice early after the challenge with ifv. later, proinflammatory cytokines and infiltrated cells started to decrease in immunobiotic-treated animals in contrast to control mice, in which those parameters continued increasing. the trend toward lower inflammatory factors and cells registered later during ifv infection in l. rhamnosus crl -treated mice correlated with a reduced severity of pulmonary damage when compared to control mice ( , ). chen et al. ( ) also investigated the ability of orally administered enterococcus faecalis kh to beneficially modulate the innate immune response to influenza infection. authors observed that kh strain protected c bl/ mice against ifv as observed by the reduced mortality, weight loss, and lung viral titers. as expected, ifv enhanced the levels of proinflammatory mediators in the respiratory tract including il- , tnf-α, ifn-γ, il- β, il- , and mcp- while the treatment with e. it is not clear how immunobiotics initiates the cross-talk with the immune system in order to modulate the respiratory antiviral immunity. it is not known exactly which prrs are activated by immunobiotics in the intestinal or respiratory mucosa to functionally modulate antiviral immunity locally and in distal mucosal sites, respectively. neither it has been determined with exactitude which cellular functions are modulated by immunobiotics immediately after prr activation. research from the last decade has demonstrated that the immunomodulatory effects of probiotic bacteria are the consequence of complex interactions between several bacterial molecules and host receptors located in different immune and non-immune cells ( , ). it has also been shown that the immunomodulatory properties of immunobiotics are dependent on the strains. therefore, studies carried out with certain strains cannot be easily extrapolated to other bacteria, even those of the same genus and species ( , ). consequently, it is still necessary to carry out deeper studies to find out the molecular mechanisms by which immunobiotics beneficially influence the respiratory antiviral immunity. the studies mentioned before showed the potential of immunobiotics to be used for the reduction of the incidence and severity of ifv infections. however, in addition to deepening the knowledge of their mechanisms of action, several other points should be considered for the efficient application of immunobiotics in humans. for example, it is necessary to determine the best time as well as the most appropriate route for their administration. immunobiotics used as components of functional foods can be included in diets on a regular basis and thus help to improve respiratory defenses, especially in high-risk populations and during the seasons with the highest incidence of respiratory infections occurs. in this sense, in a randomized controlled trial we demonstrated that l. rhamnosus crl (administered in a yogurt formulation) improved mucosal immunity and reduced the incidence and severity of intestinal and respiratory infection in children ( ). hence, the incidence of infectious events was reduced from % in the placebo group to % in the group that received the probiotic yogurt. furthermore, there was also a significant reduction in the occurrence of indicators of disease severity such as fever and the need for antibiotic treatment in children receiving the probiotic yogurt. this immunobiotic yogurt (yogurito ® ) has been included into official national nutritional programs in argentina and is given daily to children at schools in several provinces thanks to the government actions. epidemiological studies in the schools receiving the immunobiotic product have shown a reduction in the incidence of infections and in the associated school absenteeism (alvarez et al., unpublished results). on the other hand, as mentioned earlier the nasal administration of immunobiotics is more efficient than the oral administration to enhance respiratory immunity. this route of administration poses a practical disadvantage considering that the treatments with immunobiotics showed favorable results when they were used before the infectious challenges. in this way, it would be necessary to predict the exact moment in which the viral pathogen will be in contact with the host in order to carry out the prophylactic immunobiotic treatment. this option could be used for example during a school or work outbreak in which cases of respiratory infections occur and it is desired to prevent or reduce the severity of infections in asymptomatic individuals. for an intervention of these characteristics, it would be also important to determine the exact time after the contact with the virus in which it is possible to administer immunobiotics to achieve the beneficial effect. in a recent study, percopo et al. ( ) have defined this as "the window of opportunity. " the work evaluated the effect of the nasal administration of live or inactivated l. plantarum ncimb in a mice model of severe respiratory infection with the pneumonia virus of mice (pvm) and found that immunobiotic treatment promoted full survival from acute pvm infection when administered within day after virus challenge ( ) . similar studies would be of value in ifv infection models. another point of interest is related to the duration of the improvement of respiratory defenses after the last immunobiotic administration. our studies have showed that the immunomodulatory effect of some nasally administered immunobiotics persisted for at least days (villena et al., unpublished results) . other studies have also reported short-term protection after nasal treatment with different immunobiotic strains ( ). interestingly, garcia-crespo et al. ( ) found that adult mice primed nasally with l. plantarum ncimb or lactobacillus reuteri f were completely protected against lethal pvm infection and that protection persisted for at least months after the initial priming. these findings open an interesting challenge in the study of immunobiotics to improve the defenses against ifv, since it would be very useful to establish the duration of the protective effect for each strain and treatment, since in the majority of cases these long-term studies were not taken into account. ifv infections often result in mild to moderate lung infection; however, life-threatening disease can occur. it has been demonstrated that the most severe disease outcomes are associated with secondary bacterial pneumonia caused primarily by staphylococcus aureus or streptococcus pneumoniae ( ) . taking into account the high incidence of viral infections and the frequency of associated secondary bacterial infections which contribute to aggravate the health status of the host and reduce its chance of recovery, various approaches for preventing and treating influenza and secondary bacterial pneumonia are been investigated. a wide range of antibiotics and anti-inflammatory drugs has been tested in mice [reviewed in ref. ( ) ]. it would be of interest to evaluate the potential beneficial effect of immunobiotics on these circumstances. in this regard, preliminary studies from our laboratory showed that nasally administered l. rhamnosus crl is able to improve survival, reduce bacterial cell counts in lung and blood, and limit lung inflammatory damage caused by s. pneumoniae infection in mice produced after the infection with ifv or respiratory syncytial virus (rsv) (villena et al., unpublished results) . these results opened an interesting topic for future investigations. finally, it would be also of interest to investigate whether immunobiotic treatments may influence other physiological systems involved in the defenses against viral respiratory infections such as the coagulation system. our group has made some progress in this regard, as mentioned below. coagulation is an extremely ordered process that involves the interaction of three key components: endothelial cells (ecs), platelets, and coagulation factors. tissue injury that activates ecs typically initiates coagulation that is characterized by the binding of platelets to activated ecs and the formation of the platelet plug. almost simultaneously, tissue factor (tf) released by ecs result in factor x activation, which induces thrombin and the generation of fibrin strands to strengthen the platelet plug leading to a stable platelet-fibrin clot. all these processes are tightly regulated by anticoagulant and fibrinolytic mechanisms to avoid thrombotic and/or haemorrhagic complications. a key role has been attributed to ecs in the temporal and special regulation of coagulation activation. resting ecs avoid the inappropriate plug formation by controlling platelet adhesion and activation and generating several anticoagulant factors providing a non-thrombogenic barrier ( , ) . once activated or injured, ecs expose collagen to blood, increase platelet binding and aggregation, reduce the expression physiological anticoagulant factors, increase the expression of tf and von willebrand factor, and suppress the fibrinolytic activity ( , ) . all these changes in the hemostatic system facilitate thrombosis in the infected or inflammated tissue. both hemorrhagic and thrombotic complications have been described during ifv infection. influenza is able to cause pulmonary hemorrhage and edema related to coagulopathy or induce uncontrolled thrombosis through an over-activated coagulation (figure ) ( , ) . animal models have helped to explain the mechanisms by which ifv infection activates coagulation and key role has been attributed to tf. it was described that ifv activates coagulation by enhancing tf production, thrombin generation and fibrin deposition in c bl/ mice ( ) . in a mice model of ifv infection, it was recently shown that wild-type animals increased lung tf expression and activation of coagulation but presented alveolar hemorrhage ( ) . moreover, selective deletion of tf in epithelial cells from lung significantly reduced tf expression after ifv infection and had higher alveolar hemorrhage and immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december | volume | article reduced survival than controls. on the contrary, deficiency of tf in either respiratory myeloid cells or ecs did not enhanced alveolar hemorrhage or modified survival of ifv-infected mice ( ) . these results indicate that an appropriate modulation in the production of tf in the lung during ifv infection is necessary to maintain tissue hemostasis avoiding hemorrhage and excessive fibrin deposition. production of tf by lung epithelial cells will be required to maintain alveolar hemostasis during ifv infection, while excessive release of tf by macrophages and ecs would contribute to pathology and lung tissue injury ( , ) . it is considered that ecs may play an important role in the pathogenesis of ifv. influenza infection is able to induce alveolar edema and pulmonary hemorrhage through the alteration of ecs via several mechanisms, including direct damage and loss of tight junctions and apoptosis ( ) . in addition, recognition of damageassociated molecular patterns such as hmgb or oxidized phospholipids through tlr activates ecs to drive lung injury ( ) . direct stimulation of tlr by viral rna also results in the upregulation of tf and the downregulation of thrombomodulin (tm) in ecs ( ) . at the same time, the inflammatory activation of ecs leads to the activation of the coagulation cascade. inflammation caused by ifv infection increases various proinflammatory cytokines such as tnf-α, il- β, and il- that induce the secretion of tf by ecs and monocytes ( ) . in addition to their roles in coagulation, activated proteins such as thrombin, fxa, and fviia also enhance the inflammatory response. the inflammatory potentiating abilities of coagulation factors are mediated through their activation of protease-activated receptors (pars) that are expressed in platelets, ecs, macrophages, and respiratory epithelial cells ( ) . the tf/thrombin/par- pathway has been associated to the promotion of a deleterious innate inflammatory response to ifv infection in mice ( , ) . therefore, both the hyper-inflammatory response and the aberrant activation of coagulation, which are potentiated with each other, are involved in severe influenza pneumonia and are key events that have to be controlled in order to reach a favorable resolution of the infectious process. considering the importance of the coagulative response in the outcome of influenza infection and the ability of immunobiotics to beneficially influence the immune response to this respiratory pathogen, we wonder whether some immunobiotic strains would be able to beneficially modulate the immuno-coagulative response triggered by ifv. for this purpose, we performed challenge-infection experiments in mice and evaluated the influence of viable and non-viable immunobiotic l. rhamnosus crl strain on the respiratory immuno-coagulative response induced by ifv ( , ) . our data demonstrated that oral administration of l. rham nosus crl to mice significantly reduced lung viral titers and tissue damage after the challenge with ifv ( ). we later explored the capacity of nasally administered l. rhamnosus crl , alive or heat killed, to reduce the influenza burden of disease ( ). those treatments induced a significant decrease in ifv titers in lungs, lessened pulmonary damage, and increased survival. interestingly, a similar effect was achieved with the nasal administration of viable and non-viable crl strain. moreover, the nasal route was more efficient than the oral administration to protect mice against ifv infection ( , ). the protective effect achieved by the immunobiotic strain was related to its ability to modulate the respiratory antiviral immune response, particularly to its capacity to improve the levels of ifn-γ and ifn-β in the respiratory tract (figure ) . type i ifns trigger the activation of the jak-stat pathway and increase the expression of antiviral genes. in addition, ifn-γ is produced by immune cells, especially th cells, and it further improves antiviral immune response by inducing activation of nk cells and macrophages. therefore, the modulation of type i ifns and ifn-γ would be responsible of the reduction of viral loads in ifv-infected mice previously treated with the crl strain, similarly to other immunobiotic strains as mentioned before ( table ) . we demonstrated that the crl strain increased the levels of gut cd + cd + ifn-γ + t cells, induce a mobilization of these lymphocytes into the lung and enhanced the respiratory production of ifn-γ and the activity of local antigen presenting cells ( , , ) . it was also noted that nasal administration was more effective than the oral route to increase pulmonary cd + cd + ifn-γ + t cells ( , ). the mechanism by which nasally administered viable or heat-killed l. rhamnosus crl improves ifn-γ + t cells population is not clear. however, our studies support the possibility that the immunobiotic strain l. rhamnosus crl impact in the nasalassociated lymphoid tissue or bronchial-associated lymphoid tissue producing an innate imprinting in antigen presenting cells that contribute to the enhanced number and activity of cd + cd + ifn-γ + t cells. our studies also showed that immunobiotic treatments were able to beneficially modulate the activation of coagulation during respiratory viral infection, an effect that was not reported before ( , ). then, our studies were the first in demonstrating a beneficial modulation of the immune-coagulative response during respiratory trl activation and ifv infection induced by immunobiotic microorganisms (figure ) . although ifv is an ssrna virus, it generates dsrna replication intermediates that activate tlr and contribute to the initiation of the antiviral respiratory immune response. in fact, ifv triggers type i ifn secretion through tlr recognition in immune (myeloid dcs or macrophages) and non-immune (fibroblasts or pneumocytes) cells ( ) . challenge-infection experiments with respiratory viruses in tlr −/− mice showed that tlr does not modify the clearance of viral pathogens but it is relevant for the modulation of the lung inflammatory response ( , ) . it was showed that wild-type mice mount a robust inflammatory response in the lung after ifv infection and that this process is significantly diminished in tlr −/− animals ( ) . tlr −/− mice showed a longer survival when compared wild-type animals and this effect was associated with a reduction of inflammatory cells recruitment and lower levels of inflammatory factors in the respiratory tract. other in vivo studies also demonstrated that tlr activation by poly(i:c) enhanced proinflammatory cytokines and immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december | volume | article antiviral factors expression ( ), altered vascular permeability ( ) , and incremented the levels of d-dimers indicating that coagulation and fibrinolysis were triggered. in line with these findings, it was observed that the levels of d-dimers in tlr −/− mice were significantly lower than in wild-type animals after poly(i:c) administration ( (neutrophils and macrophages) and proinflammatory mediators (il- β, tnf-α, il- , and il- ) in the respiratory tract. moreover, tlr activation also induced an increase in tf expression and thrombin-antithrombin complex (tatc) levels in the lung while it reduced tm expression. these inflammatory-coagulative modifications were accompanied by respiratory tissue alterations and impairment of lung function ( , ) . of interest, we demonstrated that orally ( ) or nasally ( ) administered immunobiotics before the challenge with poly(i:c) differentially modulated the inflammatory-coagulative response. l. rhamnosus crl was able to reduce and increase the expression of tf and tm, respectively, after the respiratory activation of tlr . thus, the crl strain significantly diminished coagulation activation in blood and in the respiratory tract after the nasal stimulation with poly(i:c). we also evaluated pulmonary coagulation during ifv infection ( , ). the respiratory virus induced activation of coagulation in the lungs of infected mice as demonstrated by the increased levels of respiratory tatc. these procoagulant changes were related to alterations in the expression of tm and tf in lungs. our findings are in line with previous studies in humans and animal models of influenza infection demonstrating increased lung fibrin deposition and enhanced numbers of intravascular thrombi in the respiratory tract ( , , ) . we demonstrated that immunobiotic treatment is able to significantly diminish the activation of coagulation in ifv-challenged mice. in fact, lower levels of respiratory tatc and a reduced expression of tf was observed in l. rhamnosus crl -treated mice infected with ifv when compared to controls ( , ). as mentioned before, ifv promote a procoagulant state directly through its capacity to infect ecs and monocytes stimulating the expression of tf ( , ) . in addition, ifv induce activation of coagulation indirectly by the enhancement of proinflammatory factors such as il- ( , ) . therefore, the ability of immunobiotics to modulate the ifv-triggered immune-coagulative response could be explained by their direct influence on viral replication related to the enhancement of the antiviral state in the respiratory mucosa, and indirectly through the modulation of the inflammatory response. considering this last point, we performed experiments using anti-il- r blocking antibodies in order to evaluate the role of the regulation of the inflammatory response in the reduction of coagulation activation. results showed that il- is important for the regulation of coagulation induced by the immunobiotic l. rhamnosus crl ( ). blocking of il- r abolished the capacity of the crl strain to change the expression of tm and tf in the lungs. this was in line with our previous studies evaluating the ability of l. rhamnosus crl to confer protection against inflammatory damage induced by tlr activation or rsv infection, which showed that il- is a key factor for the reduction of lung injury ( ) . additionally, it was demonstrated that lethal disease caused by ifv infection is prevented by il- administration through the reduction of lung immunopathology ( ) . moreover, tf expression and procoagulant activity of macrophages and ecs are reduced by il- ( , ) . therefore, we demonstrated that immunobiotic administration induce an early increase in the levels of tnf and il- in the respiratory tract after poly(i:c), rsv, or ifv challenge, while the levels of those proinflammatory factors are significantly reduced later during infection ( , , ) . the early increase of proinflammatory mediators and the augmented levels of ifn-γ explain the ability of l. rhamnosus crl to diminish viral replication while the improved production of il- would lead to a beneficial modulation of the immune-coagulative response which results in a reduced severity of lung damage. it has been suggested that respiratory viral infections increase the risk of venous thromboembolism and ischemic heart disease through ecs perturbation, coagulation activation, reduction of anticoagulant factors, and inhibition of fibrinolysis ( ) ( ) ( ) . then, our studies suggest that immunobiotics could be an interesting alternative not only to reduce the incidence and/or severity of respiratory viral infections, but in addition to reduce the risk of atherothrombotic alterations associated to respiratory viral infections. research from the last decade has clearly demonstrated that beneficial microorganisms are able to modulate respiratory tract immunity and promote the resolution and lessen the severity of respiratory infections caused by pathogens such as ifv. studies in animal models have demonstrated that orally or nasally administered immunobiotics are able to improve protection against ifv by three main mechanisms. first, immunobiotics increase the respiratory antiviral state by their capacity to improve levels of type i ifns, the number and activity of antigen presenting cells, nk cells, cd + ifn-γ + t, and iga + b lymphocytes, as well as the levels of systemic and mucosal specific antibodies. second, immunobiotics beneficially modulate the ifv-triggered respiratory inflammatory response by inducing changes in the levels and kinetics of proinflammatory factors and immunoregulatory cytokines such as il- that allow the clearance of virus with a minimal inflammatory lung tissue damage. finally, as demonstrated by our recent research works, immunobiotics modulate lung immune-coagulative response triggered by tlr activation or ifv infection, mainly by downregulating lung tf and restoring tm levels. studies in animal models suggest that immunobiotics would influence principally the innate immune response, modulating in that way the early antiviral inflammatory response and the subsequent cellular and humoral immune responses. therefore, immunobiotics would have mainly an adjuvant effect. however, the exact molecular mechanisms by which immunobiotics differentially modulate the innate antiviral immune response against ifv remain to be elucidated. additionally, a growing number of studies in humans have examined the effect of immunobiotics on the incidence and severity of ifv infection. considering the impact of immunobiotics in the innate immune response clinical studies have evaluated principally their potential adjuvant effects on ifv vaccination ( table ) . although mechanistic studies have not been addressed in depth, there is promising evidence for beneficial effects of immunobiotics on human respiratory health and resistance against ifv. these observations might be helpful to propose new influenza a penetrates host mucus by cleaving sialic acids with neuraminidase the 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influenza-like illness and immunological response to influenza virus key: cord- -rv npz z authors: an, dong; guo, yongli; bao, jun; luo, xiuxin; liu, ying; ma, bo; gao, mingchun; wang, junwei title: molecular characterization and biological activity of bovine interferon-omega date: - - journal: res vet sci doi: . /j.rvsc. . . sha: doc_id: cord_uid: rv npz z bovine interferon-omega (boifn-ω ) gene was amplified from bovine liver genomic dna, which encodes a -amino acid protein containing a -amino acid signal peptide. analysis of the molecular characteristics revealed that boifn-ω evolving from ifn-ω, contained four cysteine residues and five alpha helices, showing that boifn-ω presented the typical molecular characteristics of type i interferon. boifn-ω exhibited antiviral and antiproliferative activities, which exerted a protective effect against vsv in several mammalian cell lines, as well as against bev, ibrv, and bvdv in mdbk cell. moreover, boifn-ω was shown to be highly sensitive to trypsin, but remaining stable despite changes in ph and temperature. additionally, boifn-ω induced the transcription of mx , isg , and isg genes, as well as the expression of mx protein in a time-dependent manner. these findings will be useful to further study boifn-ω in host's defence against infectious diseases, particularly viral infections. furthermore, results will facilitate further research on the bovine interferon family. interferons (ifns), which are produced in response to viral infections, contribute to host defence by establishing an antiviral state in target cells wherein viral replication is blocked or impaired as a result of the synthesis of a number of enzymes interfering with cellular and viral processes (staeheli, ; sen, ) . type i ifns are a family of cytokines with pleiotropic activities including inhibition of viral replication and cell proliferation, as well as activation of the immune system (stark et al., ) . their administration has been proposed as an immunomodulatory therapy (domenech et al., ) to treat several viral and immunomediated diseases. type i ifns, including ifn-α, ifnβ, ifn-ω, ifn-δ, ifn-τ, ifn-ε, ifn-ν, and ifn-κ, are produced by virus-infected cells and exert nonspecific antiviral activities on adjacent non-infected cells (krause and pestka, ) . they display a general mechanism of action based on their interaction with specific cell-surface receptors and the subsequent induction of ifn-stimulated genes (isgs) expression, thereby encoding direct antiviral effectors or molecules with the potential to positively and negatively regulate ifn signaling and other host responses, including enzymes, signaling proteins, chemokines, antigen-presenting proteins, transcription factors, and apoptotic proteins (der et al., ) . overall, a number of isgs that act to enhance pathogen detection and innate immune signaling (schneider et al., ) , such as isg , isg , and gtpase mx , have been shown to function as antiviral effectors. the mx gene product, which is one of the first described inhibitors of virus entry, is broadly inhibitory and acts prior to genome replication at an early postentry step of the virus life cycle (schneider et al., ) . isg demonstrates numerous antiviral functions, including inhibition of virus release, lysis of both viral and host proteins, and immunomodulatory cytokine-like properties in its unconjugated form (schoggins, ) . isg , which is induced in response to type i ifns, dsrnas, and viruses (terenzi et al., ) , has been implicated in antiviral actions of ifns against west nile virus and lymphocytic choriomeningitis virus (wacher et al., ) . ifn-ω, which was first discovered by hauptmann and swetly (hauptmann and swetly, ) , is a type i ifn secreted by virus-infected leukocytes, that are encoded by multiple ifn-or ifn-like genes present across mammalian groups, including cats, porcine and rabbit (hauptmann and swetly, ; roberts et al., ; charlier et al., a; mege et al., ) . like other type i ifns, ifn-ω presents species-restricted biological activity in vitro and can bind to the same type i ifn receptor complex as other type i ifns, thus exerting similar antiviral, antiproliferative, and immunomodulatory effects (adolf, ) . however, its antigenic structure is distantly related to ifn-α, -β, and -γ, as it does not cross react with antibodies against them (adolf, ) . interferon-ω has been used for antiviral treatment in humans and many other mammalian species (tiefenthaler et al., ; hagelstein et al., ; gil et al., ; leal et al., ; martin et al., ) . although human ifn-ω (huifn-ω) can exert in vivo antitumor effects in several models of human cancer (horton et al., ) , feline ifn-ω (feifn-ω) shows a proven antiviral effect, both in vitro (mochizuki et al., ; ohe et al., ; litzlbauer et al., ) and in vivo leal et al., ; martin et al., ; horton et al., ; mochizuki et al., ; ohe et al., ; litzlbauer et al., ) , against canine and feline parvovirus, herpesvirus, calicivirus, coronavirus, and rotavirus, and has been licensed for use in veterinary medicine (virbagen®, virbac) in europe, japan, australia, new zealand and mexico (domenech et al., ) . research on boifn-ω subtypes was not much reported, in , boifn-ω was reported to have biological activation, and can sufficiently prevent luteolysis, without being deleterious to embryonic survival (rodriguez et al., a) ; in , boifn-ω has been characterized and can be used as a candidate antiviral therapeutic reagent (luo et al., ) . the newfound boifn-ω , which presents a potential as a novel antiviral therapeutic agent, exerts antiviral activity against vsv in several mammalian cell lines and a protective effect against bovine virus that including bev, ibrv and bvdv. in this study, we present a novel type i ifn named boifn-ω on the basis of the location in the bovine genome. boifn-ω demonstrates typical characteristics of type i ifn, exerts antiviral activity in several mammalian cell lines, and displays low cytotoxicity in vitro. additionally, boifn-ω can induce the transcription of mx , isg , and isg genes, as well as the expression of mx protein in a time-dependent manner. the current findings will be useful to further study boifn-ω in host's defence against infectious diseases, particularly viral infections, and should facilitate research on the bovine interferon family. liver was collected from holstein cows in a dairy farm in harbin, heilongjiang in northeast china. madin-darby bovine kidney (mdbk) cells, primary bovine testicular (bt) cells, primary embryo bovine lung (bl) cells, feline kidney (f ) cells, madin-darby canine kidney (mdck) cells, baby hamster syrian kidney (bhk- ) cells, porcine kidney (pk- ) cells and rabbit kidney (rk- ) cells were preserved in our laboratory. vesicular stomatitis virus (vsv) was purchased from the china institute of veterinary drug control. bovine intestinal virus (bev), bovine infectious bovine rhinotracheitis virus (ibrv), bovine viral diarrhea virus (bvdv) were preserved in our laboratory. rabbit polyclonal antibody (pab) against boifn-ω were prepared and preserved in our laboratory (luo et al., ) . rabbit pab against mx (gtx ) and gapdh (gtx ) were purchased from genetex (ca, usa). hrp-conjugated goat anti-rabbit igg was purchased from zsgb (beijing, china). genomic dna extracted from bovine liver (sambrook and green, ) was used as the pcr template. degenerate primers boifnws and boifnwa (table ) designed according to the alignment of boifn-ω subtypes, were used as the pcr primers with the following thermal profile: initial denaturation at °c for min, amplification cycles of °c for s, °c for s, and °c for s, followed by a final extension at °c for min. the pcr products obtained were cloned into the pmd -t cloning vector (takara, japana) and sequenced. sequences analysis was performed on the dnastar program (dnastar inc., usa). the obtained sequences were identified by blast (http://blast.ncbi.nlm.nih.gov/blast.cgi) and the putative amino acid sequence was compared with its counterparts of other animals by using the program clustalx. signal peptide was predicted with signalp (http://www.cbs.dtu.dk/services/signalp/). the glycosylation sites were analyzed by netglycate . server (http://www.cbs.dtu.dk/ services/netglycate/) online. multiple alignments and phylogenetic tree were constructed with clustalx and mega . using upgma method, and the availability of the branch stem was verified with bootstrap replicates. secondary structure elements were predicted using the algorithms available from nps (http://www.npsa-pbil.ibcp.fr). pet a (invitrogen, ca, usa) was used as the expression vector. express primers boifn-ω s and boifn-ω a (table ) containing the bamh i and xho i sites were designed. mature boifn-ω gene was cloned into the pet- a vector to generate pet a-boifn-ω , which contains an n-terminal his tag for facilitating protein purification. recombinant plasmid was transformed into escherichia coli rosetta (de ) lys cells and then recombinant protein his-boifn-ω was induced with iptg (sigma-aldrich, st. louis, mo), the recombinant his-boifn-ω was analyzed through % reduced sds-page. the whole cells were collected and treated with ultrasonic, supernatants and sedimentations were isolated by centrifuge and analyzed by % reduced sds-page, which were used to identify the solubility of his-boifn-ω . then the recombinant protein his-boifn-ω was purified using a nickel-chelated column (genscript, nanjing, china) according to the manufacturer's instructions. the purified protein was renatured through dialysis with tge ( mm tris-hcl, . mm edta, mm nacl, % glycerinum, ph . ) under a urea gradient to reduce the concentration from m to m. after denaturation and renaturation, soluble homogeneous protein was obtained and concentration was quantified with the bradford protein assay kit (beyotime, shanghai china) according to the instructions. first, titers of viruses described above were determined by an endpoint dilution assay and the titers were expressed as the tissue culture infectious dose (tcid ) per milliliter using the reed-muench method . virus titers were calculated by determining the dilution giving % of wells containing cells that displayed cytopathic effect. antiviral activity was determined with a standard cytopathic effect assay (rubinstein et al., ) with some modifications (boue et al., ; bracklein et al., ; rodriguez et al., b; shao et al., a) . briefly, the monolayers cells were seeded in well plates, then inoculated with four-fold serial diluted boifns. after overnight cultivation, tcid viruses was added to each well and the plate was then re-incubated under the conditions of °c in a humidified % co atmosphere for - h. eight wells without the viruses were table sequences of the primers. sequences( ′- ′) the restriction enzyme sites that were introduced in primers are underlined. used as the cell controls, and the other eight wells without boifns were used as the virus controls. one antiviral activity unit ( u) was defined as a % reduction in the virus induced destruction of the cell monolayer. based on the antiviral activity measured on mdbk cells, u represents ng, . ng and ng of boifn-ω , boifn-ω and boifn-αa respectively. to check whether this antiviral activity is specific, the neutralization antiviral activity of boifn-ω was measured according to the method as previously described (shao et al., b) with some modifications. mdbk cells were treated with boifn-ω ( u) for h and -fold serial dilutions of anti-boifn-ω rabbit serum for h at °c. then, all cells were challenged with tcid of vsv. the preimmune rabbit serum was used as negative control. the antiproliferative activity of boifn-ω against mdbk cells was determined by mtt assay in vitro (luo et al., ) with some modification. the monolayers of mdbk cells were seeded in -well plates and treated with boifn-ω , boifn-ω or boifn-αa at °c for h. then, μl of mtt ( mg/ml) was added to each well, and the plates were further incubated at °c for h. the culture medium was removed, and μl of dmso was added to each well followed by agitation of the plate on an orbital shaker for min. light absorbance of the reaction wells was measured at nm. boifn-ω was combined with trypsin at a final concentration of . % and incubated in the water bath at °c for h. then antiviral activity of boifn-ω in the mdbk/vsv system was determined. the samples without treatment were used as control (shao et al., b) . boifn-ω were adjusted to ph . using hcl and incubated at °c for h, and then the treated samples were reverted to the original ph ( . ) (cheng et al., ; zhao et al., ) . antiviral activity was determined in the mdbk/vsv system. the untreated samples were used as control. boifn-ω were separately incubated in water bath at °c, °c, and °c for h, then the treated samples were cooled down in an icebox. antiviral activity was determined in the mdbk/vsv system. the untreated samples were used as control. mdbk cells were seeded in six-well plates and treated with boifn-ω for , , , , , and h. subsequently, the cells total rna was prepared using rnaprep pure cell/bacteria kit (tiangen, beijing, china), and then reverse transcribed into cdna using a reverse transcription system (promega, usa). quantitative real-time pcr (qrt-pcr) was carried out by using an abi real-time pcr system with sybr premix ex taq (takara). the pcr was set up under the following thermal cycling conditions: °c for s, followed by cycles of °c for s, °c for s. the qrt-pcr was performed with the following primers: bogapdhs and bogapdha, bomx- s and bomx- a, boisg s and boisg a, boisg s and boisg a (table ) . gapdh was used as the internal reference, the transcription of mx , isg and isg mrna were analyzed with the method −ΔΔct (livak and schmittgen, ). mdbk cells were seeded in six-well plates and treated with boifn-ω for , , , , , and h. subsequently, cells were lysed in mammalian protein extraction reagent (thermo, ma, usa). lysates were collected and separated by sds-page, and then transferred onto biotrace pvdf membrane followed by blocking the membrane with % skimmed milk in pbst at °c for h. after incubating the membrane with pab against mx- ( : dilution) and gapdh ( : dilution) simultaneously at °c for h, the membrane was washed three times with pbst before incubation with goat anti-rabbit igg antibodies at : , dilution in pbst at °c for h. after three times washing with pbst, the membrane was developed with western blot ecl plus (beyotime, beijing, china) in the dark. data were presented as means ± standard deviations. student's ttest was used for statistical comparisons. statistical significance was expressed as the following p values: p ≤ . (*); p ≤ . (**). the complete boifn-ω included an open reading frame of nucleotides, which encoded a mature peptide consisting of amino acids and a signal peptide consisting of amino acids. a bp mature boifn-ω gene was then amplified (fig. a) . boifn-ω contained four cysteine residues at positions , , , and and one arginine (arg) at position (fig. b) , which is the conserved sequence among ifn-ω. the three-dimensional structure of boifn-ω included five alpha helices labeled a to e (fig. c) . only one putative o-glycosylation site was found at amino acid residue of mature boifn-ω . a phylogenetic tree was constructed to determine the evolutionary position of boifn-ω . subsequently the phylogenetic relationships among bovine, sheep, human, chimpanzee, rhesus monkey, porcine, and feline ifn-ω types showed that boifn-ω was clustered more closely with sheep ifn-ω ( fig. a) . moreover, the amino acid alignment of boifn-ω with other ifns showed that boifn-ω . % identical to huifn-ω, . % identical to boifn-ω , . % identical to poifn-ω , . % identical to boifn-α , and . % identical to boifn-β (fig. b) . sds-page analysis revealed that the recombinant protein his-boifn-ω was mainly expressed as an inclusion body exhibiting an apparent molecular weight of kda (fig. a) . a purified protein his-boifn-ω band was then observed after purification (fig. b ). boifn-ω exerted a protective effect against vsv infection in mdbk, bl, bt, bhk , f , rk- , and pk- cells, but not in mdck cells, while also exerting a protective effect against bev, ibrv, and bvdv (table ) . unlike boifn-αa, boifn-ω exerted a protective effect in mdck-vsv and bhk -vsv systems. additionally, the antiviral activity of boifn-ω measured by the mdbk-vsv system was completely abrogated by pab against boifn-ω at a dilution of : . when, the antibody dilution was : , the blocking activity was almost non-existent, whereas no blocking activity was observed with the addition of pre-immune rabbit serum (data not shown). mtt assay showed that boifn-ω , boifn-ω and boifn-αa exerted dose-dependent antiproliferative effects in mdbk cells (fig. ) . the antiproliferative activity of boifn-ω and boifn-ω was significantly different form that of boifn-αa at a concentration of μg/ml (p ≤ . ) but was only slightly different when the concentrations were lower than ng/ml. moreover, the antiproliferative activity analysis showed that boifn-ω exerted low cytotoxicity in mdbk cells. boifn-ω completely lost its antiviral activity when treated with . % trypsin, and this finding demonstrated that boifn-ω was highly sensitive to trypsin. after acid treatment, boifn-ω retained its antiviral activity which was not significantly different from that of the untreated sample; this result confirmed that boifn-ω was characteristically stable at ph (fig. a) . after treatment at °c, °c, and °c, the antiviral activities of boifn-ω decreased by . , . , and . , respectively, with no significant difference from that of the control group. this finding illustrated that boifn-ω was not sensitive to temperature (fig. b ). to assess the effect of boifn-ω on type i ifn response, the transcription of isgs (including mx- , isg and isg ) was quantified. quantitative real-time pcr analysis showed that the transcription of mx- , isg and isg increased sharply at h in boifn-ω -treated mdbk cells and sustained high expression levels until h, and the expression of h was significantly (p ≤ . ) than that of h (fig. a) . immunoblotting analysis showed that the mx- protein was detected as a single major protein band of approximately kda, whereas the amounts of mx- protein gradually increased from to h after treatment. in addition, mx- protein expression was much higher at h than at h (fig. b ). gapdh was used as an endogenous control, and a kda protein band was detected in all the samples. ifns are a family of regulatory proteins with antiviral, antitumour, and multiple immunomodulatory effects. bovine type i ifn contains the purified boifn-ω was adjusted to ph . , °c, °c, °c and then finally reverted to the original ph ( . ) or cooled down in an icebox. afterwards, antiviral activities were separately analyzed in mdbk-vsv system. after acid and high temperature treatment, boifn-ω retained its antiviral activity with no significant difference from that of the control group. - ifn-α, - ifn-β, - ifn-τ, and - ifn-ω subtypes (including pseudogenes) (velan et al., ; chaplin et al., ; capon et al., ) , which are located on chromosome (ryan and womack, ) . since the discovery of ifn-ω by hauptmann and swetly, ifn-ω has been gradually found in human, feline, equine, porcine, rabbit, and several cattle species (sambrook and green, ; himmler et al., ; charlier et al., b; yang et al., ) . with fluorescence in situ hybridization technology, bovine ifn-ω was found to locate on chromosome q (sen, ) . in this study, a novel bovine ifn-ω was cloned from bovine liver, which was named as boifn-ω on the basis of its position in the bovine genome. the molecular characteristics of boifn-ω were analyzed using bioinformatics software. boifn-ω displayed the highest similarity to bovine ifn-ω at . % identity. phylogenetic analysis revealed that boifn-ω was closely related to boifn-ω and sheep ifn-ω, suggesting that the ifn-ω gene may have originated from gene duplication in a common ancestor during evolution. cys- , cys- , cys- and cys- of mature boifn-ω formed two disulfide bonds crucial for its activity, which is conserved in some type i ifns (wetzel, ) . to explore the biological activities of boifn-ω , antiviral and antiproliferative activities were examined with the recombinant protein his-boifn-ω . the results indicated that boifn-ω exerted a considerable antiviral activity against vsv infection in mdbk, bl, bt, bhk , f , rk- , and pk- cells, but not in mdck cells, whereas boifn-αa, boifn-ε, and boifn-ω demonstrated no antiviral activity against vsv infection in bhk- cells (sambrook and green, ; bracklein et al., ) . boifn-ω also exerted a protective effect against bev, ibrv, and bvdv in mdbk cells. similar to boifn-ω , boifn-ω displayed low cytotoxicity in mdbk cells. in addition, boifn-ω was verified to be highly sensitive to trypsin, insensitive to temperature, and stable at ph . given the substantial antiviral activity and low cytotoxicity of boifn-ω , it is a potential candidate for a novel, effective therapeutic agent. isgs take on a wide range of activities. many isgs control viral, bacterial, and parasitic infections by directly targeting pathways and functions required during pathogen life cycles (lehner et al., ) . boifn-ω can induce the time-dependent transcription of isgs (including mx- , isg and isg ). the transcription of mx- increased h after boifn-ω treatment, and peaked h thereafter. this event was consistent with the expression of mx- protein. the transcription of isg and isg showed a trend similar to that of mx- . given that isgs were downstream in the ifn signaling pathways, boifn-ω can obviously induce isgs expression in a time-dependent manner, suggesting that boifn-ω can further inhibit viral replication and exert antiviral activity. in this study, the characterization and biological activity of boifn-ω were analyzed. boifn-ω presented moderate biological activity, low cytotoxicity, and stable physicochemical characteristics, as well as exerted antiviral activity on several mammalian cell lines and a protective effect against vsv, bev, ibrv, and bvdv. additionally, boifn-ω can activate the expression of the isgs gene of ifn signaling pathway downstream. overall, our study on boifn-ω enriched the current knowledge about the bovine ifn-ω family. no competing financial interests exist. human interferon omega-a review antiviral and antiluteolytic activity of recombinant bovine ifn-omega obtained from pichia pastoris activity of feline interferon-omega after ocular or oral administration in cats as indicated by mx protein expression in conjunctival and white blood cells two distinct families of human and bovine interferon-alpha genes are coordinately expressed and encode functional polypeptides cloning and biologic activities of a bovine interferon-alpha isolated from the epithelium of a rotavirus-infected calf cloning and structural analysis of four genes encoding interferon-omega in rabbit cloning and structural analysis of four genes encoding interferon-omega in rabbit characterization of the porcine alpha interferon multigene family identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays use of recombinant interferon omega in feline retrovirosis: from theory to practice oral recombinant feline interferon-omega as an alternative immune modulation therapy in fiv positive cats: clinical and laboratory evaluation molecular cloning and characterization of a novel bovine ifn-epsilon antiviral potential of interferon-omega on hepatitis b virus replication in human hepatoma cells a novel class of human type i interferons molecular cloning and expression in escherichia coli of equine type i interferons antitumor effects of interferonomega: in vivo therapy of human tumor xenografts in nude mice analysis of isgs transcription and mx- protein expression in mdbk cells after boifn-ω treatment. (a) transcription analysis of isgs in mdbk cells after boifn-ω treatment. quantitative real-time rt-pcr was used to detect the changes in mx , isg and isg transcription levels. mdbk cells without boifn-ω treatment were used as control, while gapdh was used as the internal reference ). (b) expression analysis of mx- protein in mdbk cells after boifn-ω treatment. mdbk cells without boifn-ω treatment were used as control, while gapdh was used as the internal reference. lane m: easysee western marker lane - : cell lysates collected at , , , , and h after treatment with boifn-ω evolution of the class cytokines and receptors, and discovery of new friends and relatives monitoring acute phase proteins in retrovirus infected cats undergoing feline interferon-omega therapy the emerging role of innate immunity in protection against hiv- infection oral and subcutaneous therapy of canine atopic dermatitis with recombinant feline interferon omega analysis of relative gene expression data using realtime quantitative pcr and the −ΔΔct method characterization and antivirus activities of a novel bovine ifn-omega treatment of canine parvoviral enteritis with interferon-omega in a placebo-controlled challenge trial the porcine family of interferon-omega: cloning, structural analysis, and functional studies of five related genes inhibitory effects of recombinant feline interferon on the replication of feline enteropathogenic viruses in vitro sensitivity of fcv to recombinant feline interferon (rfeifn the evolution of the type i interferons the bovine ifn-omega is biologically active and secreted at high levels in the yeast pichia pastoris the bovine ifn-omega is biologically active and secreted at high levels in the yeast pichia pastoris convenient assay for interferons type i interferon genes in cattle: restriction fragment length polymorphisms, gene numbers and physical organization on bovine chromosome molecular cloning: a laboratory manual interferon-stimulated genes: a complex web of host defenses interferon-stimulated genes: roles in viral pathogenesis viruses and interferons characterization of the biological activities and physicochemical characteristics of recombinant bovine interferon-alpha characterization of bovine interferon alpha : expression in yeast pichia pastoris, biological activities interferon-induced proteins and the antiviral state how cells respond to interferons distinct induction patterns and functions of two closely related interferon-inducible human genes, isg and isg a comparison of the antiproliferative properties of recombinant human ifn-alpha and ifn-omega in human bone marrow culture bovine interferon alpha genes. structure and expression coordinated regulation and widespread cellular expression of interferon-stimulated genes (isg) isg- , isg- , and isg- in the central nervous system after infection with distinct viruses assignment of the disulphide bonds of leukocyte interferon cloning and characterization of a novel feline ifn-omega characterization and virus-induced expression profiles of the porcine interferon-omega multigene family china agriculture research system (cars- ), the synergetic innovation center of food safety and nutrition in northeast agricultural university, the sub-project of national th -year support key projects ( bad b , bad b ) and the key technologies research and development program of heilongjiang province (ga b ), support this study. key: cord- -hv gwvdi authors: allam, gamal; alsulaimani, adnan a.; alzaharani, ali k.; nasr, amre title: neonatal infections in saudi arabia: association with cytokine gene polymorphisms date: - - journal: cent eur j immunol doi: . /ceji. . sha: doc_id: cord_uid: hv gwvdi in recent years, many studies have reported potential associations between cytokine gene polymorphisms and the development, course, and outcome of sepsis, often with apparently conflicting results. the objective of this study was to investigate single nucleotide polymorphism (snp) in the interleukin (il)- β – t/c, il- – g/c, tumor necrosis factor α (tnf-α) – g/a, and interferon γ (ifn-γ) + a/t genes for their possible association with susceptibility to early onset sepsis (eos) in saudi newborn infants. a total of newborn infants aged - days were consecutively enrolled onto the study having met the inclusion criteria (as per the research protocol). dna was extracted from filter papers using the chelex- method. the cytokines snp were genotyping using taqman ’ nuclease allelic discrimination. for cytokine measurements we used the commercially available enzyme-linked immunosorbent assay (elisa) kit. our results show that the circulating il- β, il- , tnf-α, and ifn-γ were significantly (p < . ) elevated in eos patients compared to suspected and sepsis-free control groups; and il- β – c, il- – g, tnf-α – g, and ifn-γ + a alleles were associated with eos in saudi infants. in conclusion, analysis of cytokines concentrations and snp for the four tested genes can be used as a predictor of sepsis outcome in newborns. sepsis is a host condition of systemic inappropriate inflammatory response to the invasion of microorganisms [ ] . although there are many advances in the development of antibiotics and there has been an explosion of knowledge about the inflammatory response, sepsis still causes one million deaths each year ( % in the first week of life) [ ] . currently, it is accepted that inappropriate host inflammatory responses or inappropriate defence mechanisms play important roles in the development of sepsis [ ] [ ] [ ] . cytokines play vital roles in the regulation of host immune response, and altered expressions of cytokines are proven to be involved in the development of sepsis [ , ] . several risk factors for sepsis development have been identified [ ] , but the cause of basic differences in susceptibility between individuals and populations remains unclear [ ] . in recent years, several groups have provided accumulating evidence indicating that the genetic background of the host influences the susceptibility and prognosis of sepsis [ ] [ ] [ ] . previous studies have suggested that the variations in the genes encoding cytokines are involved in the modulation of inflammatory responses and are responsible for inter-individual differences in the susceptibility to sepsis [ ] . recent, epidemiological studies suggest that some single nucleotide polymorphisms (snp) in the genes encoding inflammatory cytokines may influence the course and outcome of sepsis [ , [ ] [ ] [ ] . exploration of host genetic markers with prognostic value for sepsis might be helpful in managing the neonates who would be most likely to benefit from aggressive antibiotic treatment, and they put the challenges involved into perspective [ ] . therefore, identification of newborns that are at high risk of developing sepsis could help us develop some effective prevention strategies. tumour necrosis factor α (tnf-α), interleukin (il)-lβ, and il- are proinflammatory cytokines, which have been reported to play important roles in the inflammatory response and in the pathogenesis of sepsis syndrome [ ] . the study of il- β and tnf-α, cytokines that are synthesised at the beginning of the inflammatory cascade, has given differing results. some studies have reported an increase in the concentrations of these proinflammatory cytokines in systemic circulation in septic newborns [ ] [ ] [ ] [ ] [ ] , while others demonstrated similar or even lower levels in infected newborns compared to healthy newborns [ ] [ ] [ ] [ ] . such discrepancies in results among different studies could explain the different outcomes of sepsis seen in neonates [ ] . therefore, the variable of clinical evolution of neonatal sepsis is related to the individual variability in cytokine production and intensity of inflammation [ , ] , which may be due to the variation of the genetic background of the patients [ ] . results of different published studies addressing the association between the snp tnf-α - and development of sepsis in adults and neonates are contradictory. one of the studies suggested that the tnf-α - a allele has been associated with increased susceptibility to septic shock and mortality from septic shock in adults [ ] . however, this association has not been consistently observed [ , ] . in a study by hedberg et al. [ ] the septic patients presenting the aa/ga genotypes had a mortality rate from sepsis that was three times greater than that seen among those presenting the gg genotype. moreover, a meta-analysis study has confirmed an increased risk of sepsis in carriers of the a allele for the tnf-α - g > a snp [ ] . regarding il- β gene polymorphisms, previous studies indicate that snps of il- β may be associated with a poor prognosis from sepsis [ , ] . however, other studies did not find any association between il- β - polymorphisms and development of bronchopulmonary dysplasia (bpd) in preterm infants [ ] . on the other hand, genetic variation within the regulatory part of the il- gene may affect the incidence and outcome of sepsis [ , ] . an association of the il- - genotype with sepsis in preterm infants has been reported. harding et al. reported a higher incidence of the il- - gg genotype in infants who developed septicaemia [ ] . similarly, ahrens et al. reported that the il- - gg genotype was more frequent in infants with sepsis compared to infants without infection [ ] . consequently, genetic variability in the regulatory and coding regions of inflammatory cytokine genes may influence the susceptibility and/or outcome of sepsis. we reported in a previous study that a high level of c-reactive protein (crp) was associated with early onset sepsis (eos) infection in neonates living in saudi arabia, and those babies carrying the a-allele were associated with high levels of circulating crp [ ] . because il- β, il- , and tnf-α appear before crp in eos infection [ ] [ ] [ ] [ ] , such cytokine gene polymorphisms may provide useful biomarkers for the screening of patients at risk, as well as in the identification of those most likely to benefit from specific therapeutic choices [ ] . therefore, the primary aim of the current study was to investigate snps in the il- β - t/c (rs ), il- - g/c (rs ), tnf-α - g/a (rs ), and ifn-γ + a/t (rs ) genes for their possible association with susceptibility to eos in saudi newborn infants. the second aim was to evaluate the association between such snps in gene promoter and the circulatory level of corresponding cytokines. a prospective cohort (cross-sectional) study was carried out over six months, from march to august , in the neonatal intensive care unit (nicu), king abdel aziz specialist hospital (kaash), taif city, kingdom of saudi arabia (ksa). a total of newborn infants aged - days were included in this study. patients were enrolled if they met the following inclusion criteria for participation in the study: ± weeks gestation, an admission to the nicu at kaash for at least hours, fever (≥ . °c measured consecutively on two occasions at least six hours apart), and symptoms or signs of sepsis as described previously [ , ] . the subjects were classified into three different groups according to nasr et al. [ ] . group i is the control group, in which babies have no symptoms or signs of sepsis and a negative blood culture. controls babies consecutively enrolled to this study, who were admitted to the newborn nursery unit for routine check up within the first two days after birth. group ii is the suspected group of neonates, which includes any infant with signs or symptoms of sepsis, chorioamnionitis, and those born to mothers who had intrapartum temperature ≥ . °c or prolonged rupture of membrane ≥ hours. it is important to note that blood cultures for this group were negative. once infection was confirmed by means of a positive blood culture, the infant was put into group iii, which is the early onset sepsis (eos) group. babies were clinically examined during their stay in the nicu. once sepsis was confirmed, blood samples were taken within - hours after birth. babies suspected of having sepsis infection, before proven positive culture, were treated by clinicians (neonatologist) with double antibiotics (ampicilin and gentamicin) until the baby became symptom free or finished their course of antibiotic. full blood count was routinely taken. all investigations and treatments were provided free of charge. newborns were weighed, and those with low birth weight were assessed clinically to differentiate between intrauterine growth restriction and low birth weight using the dubowitiz scoring system. before pharmacological treatment was started, μl of blood were collected on filter paper (schleicher & schuell; n° tm) for dna amplification and quantification of cytokines. two mls of venous blood was collected in edta vacutainer ® tubes (becton dickinson, meylan, france) for bacterial culture. dna was extracted from filter papers using chelex- , which was stored at - °c. μl from peripheral blood or discs of the same size from filter paper were incubated overnight in ml of . % saponin in pbs at °c, and they were then washed for - minutes in ml pbs at °c. the discs or the pellets, were boiled in μl of % chelex- in water for minutes, and the dna was collected in supernatants after centrifugation at × g for minutes [ ] . the following locations were investigated in this study: il- β - t/c (rs ), il- - g/c (rs ), tnf-α - g/a (rs ), and ifn-γ + a/t (rs ) genes. genotyping was performed with taqman ® ' nuclease allelic discrimination (assay by design/demand, applied biosystems, foster city, ca) as described previously [ ] [ ] [ ] . interleukin β, il- , tnf-α, and ifn-γ levels were estimated by using a high-sensitivity sandwich elisa kit (abcam ® , cambridge, uk) according to the manufacturer's instructions. briefly, a monoclonal antibody specific for each cytokine (il- β, il- , tnf-α, and ifn-γ) was coated onto the wells of the microtitre plates provided. samples and standards of known concentrations were applied into the plates. after the incubation period, the biotinylated monoclonal antibodies specific for il- β, il- , tnf-α, and ifn-γ were added and incubated for one hour. after washing, the enzyme streptavidin-hrp that binds the biotinylated antibody was added and incubated for one hour at room temperature. a tmb substrate solution was added, and the plates were incubated in the dark for minutes at room temperature. the reaction was stopped by using n hcl, and the absorbance was measured at nm using a microplate reader (biotech, ca, usa). cytokine concentrations were determined by reference to standard curves construction. the distribution of cytokines (il- β, il- , tnf-α, and ifn-γ) genotype, allele frequencies and cytokine (il- β, il- , tnf-α, and ifn-γ) concentrations was analysed using spss version . (spss, inc., chicago, il, usa). using fisher's exact test, each snp was tested to determine if the population under investigation showed deviation from genotype frequencies expected under hardy-weinberg equilibrium (hwe) proportions [ ] . tests were performed separately in the cases and controls. logistic regression analyses were performed to assess associations of genotype (independent variable). associations were quantified using odds ratios [or] with % confidence intervals (ci), which when they do not cross . are defined as statistically significant. the (il- β, il- , tnf-α, and ifn-γ) heterozygote genotypes were used as a reference in the analyses because they were the most frequent genotype in the sepsis-free controls. using the same software, we performed an overall comparison of allele frequency using a × test. the differences in cytokine (il- β, il- , tnf-α, and ifn-γ) concentrations between different study groups were analysed using kruskal-wallis test and the p-value was corrected for ties. logistic regression analyses were performed to assess the association between the cytokine (il- β, il- , tnf-α, and ifn-γ) serum levels in individuals with different cytokine genotypes. linkage disequilibrium (ld) analysis was performed in fstat version . . . to find any haplotype association within the study groups. this study received ethical clearance from the ethical committee of the college of medicine, taif university, ksa. informed consent was obtained from the neonates' parents/legal guardians, who participated in the study after we provided them with adequate information on the objectives and benefits of the project. the general characteristics of the study population are presented in table . in group i, individual had no symptoms for early onset sepsis (eos) with negative blood culture at a mean ± standard deviation (sd) age of ± . days. group ii, suspected of eos: patients had a clinical symptom of eos with negative blood culture at a mean ± sd age of ± . days. group iii (eos): patients had a clinical symptom with positive blood culture at a mean ± sd age of ± . the age and sex were matched between the three groups and were not significantly different (p > . , table ). newborns with sepsis had significantly higher serum levels of pro-inflammatory cytokines (il- β, il- , tnf-α, and ifn-γ) compared to both suspected and sepsis-free control groups (overall p value < . , table ). as shown in table , neonates with eos had significantly higher serum levels of il- β, il- , tnf-α, and ifn-γ compared to the suspected group (p value < . , . , . , and . , respectively). genotype frequencies for the candidate snps among the study groups with and without sepsis are shown in table . all polymorphisms analysed in this study were found to be in hwe (data shown in table ). the il- β cc genotype and allele were associated with eos compared to suspected patients by unadjusted analysis; for cc genotype: or = . , % ci = ( . - . ), p value < . ; and for c allele, or = . , % ci = ( . - . ), p value < . (table ). the il- gg and cc genotypes were associated with the eos patients compared to suspected patients; for gg genotype: or = . , % ci = ( . - . ), p value < . ; and for cc genotype: or = . , % ci = ( . - . ), p value = . (table ). patients carrying the g allele of the il- were associated with eos compared to suspected patients; or = . , % ci = ( . - . ), p value = . (table ). however, no significant association was found in eos patients carrying the c allele of il- compared to the suspected group. on the other hand, patients carrying the tnf-α gg genotype and g allele were significantly associated with eos compared to the suspected group; for gg genotype: or = . , % ci = ( . - . ), p value = . ; and for the g allele: or = . , % ci = ( . - . ) , p value = . (table ). the ifn-γ aa genotype and allele were associated with eos when compared to suspected patients by unadjusted analysis; for aa genotype: or = . , % ci = ( . - . ), p value = . ; and for a allele: or = . , % ci = ( . - . ), p value < . (table ). in addition, we investigated if the cytokine snps were in the ld. our results indicated that cytokine snps were not in the ld (data not shown) due to the small sample size and the limited risk of losing false negative results. in order to investigate if the cytokine (il- β, il- , tnf-α, and ifn-γ) snps affected the circulatory level of the corresponding cytokine (il- β, il- , tnf-α, and ifn-γ), the association between the snps and the levels were analysed. as shown in table , there was a significant association between il- β cc genotype and the higher concentration of circulating il- β cytokine compared to heterozygote il- β tc genotype [or= . , % ci = ( . - . ), p value < . ]. the il- gg genotypes were associated with higher level of circulating il- cytokine compared to individuals carrying the heterozygous il- gc genotype [or = . , % ci = ( . - . ), p value < . (table ) ]. individuals carrying the tnf-α aa genotype were significantly associated with lower level of circulating tnf-α cytokine compared to individuals carrying the tnf-α ga [or = . , % ci = ( . - . ), p value = . (table )]. there was a significant association between the individuals carrying ifn-γ aa genotypes and the higher concentration of ifn-γ cytokine [or = . , % proinflammatory cytokines play an important role in the immune response, pathogenesis of sepsis, and organ dysfunction [ ] . single nucleotide polymorphisms in cytokine genes may explain, at least to some extent, the variability of the clinical course observed in sepsis and infections. the majority of previous studies related to snps in sepsis were performed on adults [ ] . however, data from neonate populations with sepsis are poor, and developmental differences that affect inflammation and immune responses make it difficult to extrapolate data from adult studies to newborn populations [ , ] . the current study focused on analyses of snps in four genes (il- β - t/c, il- - g/c, tnf-α - g/a, and ifn-γ + a/t) that may play a critical role in, or are associated with, inflammatory response and sepsis severity, in order to identify possible predictive mechanisms for sepsis risk stratification. interleukin β is thought to be one of the key mediators in the pathogenesis of sepsis syndrome [ , ] . data of the present study clearly showed that newborns with eos had significantly higher serum levels of il- β compared with suspected and sepsis-free groups. this finding supports previous studies that have demonstrated an elevated level of il- β in plasma of septic neonates when com-pared with both healthy infants and infants with clinically suspected but not confirmed sepsis [ , ] . therefore, several studies have evaluated the role of this cytokine in the early diagnosis of neonatal sepsis [ ] [ ] [ ] . in addition, our data clearly showed a significant association between il- β cc genotype and higher concentration of circulating il- β cytokine compared to heterozygote il- β tc genotype. also, the il- β cc genotype and allele are associated with eos as compared to suspected patients. previous data indicates that snps of il- β may be associated with a poor prognosis from sepsis [ , ] . however, other studies did not find any association between il- β (c t) polymorphisms and outcome of sepsis [ , ] . kang et al. [ ] concluded that il- β- and il- β- polymorphisms are not associated with the development of bronchopulmonary dysplasia in preterm infants. this discrepancy may be explained by differences in the definition of sepsis and its severity, studied il- β snps, and in the general characteristics of the enrolled subjects, including ethnicity. interleukin plays an important role in the development, pathogenesis, and outcome of sepsis and septic shock. data from the present investigation clearly demonstrated that neonates with eos showed a highly significant table . logistic regression analysis of cytokines (il- β, il- , tnf-α and ifn-γ) levels (pg/ml) in relation to cytokines (il- β, il- , tnf-α and ifn-γ) genotypes polymorphism in the combined study population serum level of il- compared to suspected and sepsis-free groups. this finding is in agreement with previous studies that reported that il- is excessively elevated in septic neonates when compared with both healthy infants and infants with clinically suspected but not confirmed sepsis [ ] . many investigators have demonstrated that levels of circulating il- correlate with severity of sepsis and may predict outcome [ , , , ] . interleukin appears to be ideal for detecting early-onset neonatal infection with a high degree of sensitivity and specificity [ , , , ] . since il- is a very early marker that appears before crp, it may be useful in the monitoring of patients with high risk of developing infection. genetic variation within the regulatory part of the il- gene may affect the incidence and outcome of sepsis [ ] . our data showed that the il- gg genotypes are associated with eos when compared to suspected patients, and il- - g allele is associated with eos. however, no significant association was found between il- - c allele and eos. in addition, the il- gg genotypes are associated with higher levels of circulating il- cytokine compared to individuals carrying the heterozygous il- gc genotype. this result is in accordance with previous studies suggesting that individuals with il- - c allele have significantly lower plasma concentrations of il- [ ] , and this allele is associated with reduced il- plasma levels in newborns [ ] . bennermo et al. [ ] revealed that the il- - g allele is functional in vivo with increased inflammatory response. similar results were found by studies that included caucasian infants and showed that there was a risk of neonatal sepsis associated with the snp il- - gg [ , ] . however, contradictory results were seen by reiman et al. [ ] and baier et al. [ ] in studies that included very-low-birth-weight (vlbw) infants of different ethnicities; they showed that the il- - c allele was associated with increased incidence of sepsis. on the other hand, other studies did not detect any association between the snp il- - and neonatal sepsis [ ] [ ] [ ] . such contradictions in data between various studies could be attributed to the differences in study design, ethnicity, and gene-to-gene and gene-to-environmental interactions [ ] . in our study, we reported that the il- - g allele is associated with higher levels of circulating il- and eos. consequently, il- gene polymorphisms g- > c could be predictors of risk of development and/or predictors of the severity of sepsis in the saudi newborn population. tumor necrosis factor α is recognised as a primary mediator in the pathophysiology of sepsis and septic shock (reviewed in [ ] ). data from the current study showed that serum levels of tnf-α were significantly higher in the eos group compared to suspected and sepsis-free control groups. this result is in accordance with previous studies that demonstrated an elevated serum level of tnf-α on day of neonatal sepsis, and the role of tnf-α as an early diagnostic marker in neonatal sepsis has been evaluated [ ] [ ] [ ] [ ] . with regards to the snp in tnf-α gene promoter, the data presented here indicate that patients who are carriers of the tnf-α gg genotype and g allele are significantly associated with eos patients, compared to the suspected group. furthermore, individuals carrying the tnf-α aa genotypes are significantly associated with lower levels of circulating tnf-α cytokine, compared to individuals carrying the tnf-α ga genotype. these results are in parallel with the findings of previous studies that have demonstrated a pattern of protection that was conferred by the tnf-α - ga genotype against both sepsis mortality and acute respiratory distress syndrome (ards) outcome in a paediatric population [ ] . similarly, in the adult population, another study has found an association between snp tnf-α - a allele and protection against ards [ ] . however, several studies have reported an association between snp tnf-α - g/a and sepsis outcome in the adult and paediatric population [ , , , , ] , while other studies did not show any association between the snp tnf-α - g/a and increased risk of sepsis [ , , ] . such discrepancies in results among different studies could be explained by the variations in study design, enrolled subjects, ethnicity, and gene-to-gene and gene-to-environmental interactions [ ] . in addition, azevedo et al. [ ] speculated that - g > a and - g > a snps in the tnf-α gene promoter seem to work in opposite directions, and could in fact reflect a different impact depending on age. data from this study clearly showed high serum ifn-γ levels in eos patients, compared to the suspected and sepsis-free control group. high levels of ifn-γ could lead to side-effects such as tachycardia, myalgia, malaise, leucopaenia, and weakness [ ] . interferon γ enhances lps-induced mortality and increases levels of lps-induced circulating tnf-α [ ] ; consequently, anti-ifn-γ antibodies protected against lps-and escherichia coli-induced mortality [ ] . in addition, ifn-γ was shown to be a mediator of tnf-α-induced lethality [ ] . therefore, ifn-γ could play an important role in sepsis development and outcome. our results demonstrated that the ifn-γ aa genotype and a allele are associated with eos when compared to suspected patients. there is a significant association between individuals carrying the ifn-γ aa genotype and the higher concentration of ifn-γ. individuals carrying the ifn-γ tt genotype are associated with lower concentrations of ifn-γ when compared to those carrying the ifn-γ at genotype. the ifn-γ + a allele has previously been reported to be associated with some infectious diseases [ ] [ ] [ ] . recently, the ifn-γ + a allele has been shown to be associated with susceptibility to severe acute respiratory syndrome (sars). individuals with ifn-γ + aa and at genotype had a . -fold and . -fold increased risk of developing sars, respectively [ ] . the mechanism by which the ifn-γ + a/t allele influences the sus-ceptibility to sepsis may therefore depend on its role in the regulation of ifn-γ production [ ] . in conclusion, circulating il- β, il- , tnf-α, and ifn-γ were elevated in eos patients; and il- β - c, il- - g, tnf-α - g, and ifn-γ + a alleles were associated with eos in saudi infants. however, due to the small sample size of this study, the cytokine snps were not in the ld. therefore, these results need to be confirmed by a large-scale multicentre prospective study and should be supported by data from other ethnic populations, with the hope that it could be used to develop an early predictor for the prognosis of sepsis in neonates. the relationship between hospital spending and mortality in patients with sepsis million neonatal deaths: when? 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cholesterol-related genetic risk scores are associated with hypometabolism in alzheimer's-affected brain regions il- , il- and cd polymorphisms and sepsis outcome in ventilated very low birth weight infants genetic polymorphisms of il- - and il- - in full term neonates with late onset blood stream infections interleukin- - -genotype, sepsis and cerebral injury in very low birth weight infants genetic variants of tnf-[fc ]a, il- beta, il- receptor [fc ]a-chain, il- and il- genes are not risk factors for sepsis in lowbirth-weight infants genetics and genomics in pediatric septic shock tumor necrosis factor (tnf) and lymphotoxin-alpha (lta) single nucleotide polymorphisms: importance in ards in septic pediatric critically ill patients the role of ifn-gamma in the pathology of experimental endotoxemia variation in the tumor necrosis factor-alpha gene promoter region may be associated with death from meningococcal disease prevalence of two tumor necrosis factor gene polymorphisms in premature infants with early onset sepsis role of interferon-gamma in experimental gram-negative sepsis evidence for ifn-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha cytokine gene polymorphisms in patients infected with hepatitis b virus cytokine gene polymorphisms associated with symptomatic parvovirus b infection association of interferon gamma and interleukin genes with tuberculosis in hong kong chinese the interferon gamma gene polymorphism + a/t is associated with severe acute respiratory syndrome key: cord- -bie veti authors: nan title: ecc- abstracts date: - - journal: int j antimicrob agents doi: . /s - ( ) -x sha: doc_id: cord_uid: bie veti nan f spain introduction: the prevalence of erythromycin resistance (er-r) in group a streptococci (gas) has increased in spain since early s with current rates exceeding % in some regions. this study determined the emm -types associated to erythromycin resistance in spain. material and methods: isolates belonged to the sauce* surveillance collection. rapid sequence analysis of specific pcr products was used to deduce emm -types corresponding to the majority of the known gas m serotypes. pcr primers used: gasm ( ?-tattgcgct-tagaaaattaa- ?) and gasm ( ?-gcaagttctt-cagcttgttt- ?). sequencing was done with the big dye terminator mix and autosequenator (applied biosystems). dna sequences were subjected to homology searches against the bacterial dna database. results: overall, gas isolates ( er-r) were analysed. three m-types (m , st and m ) accounted for . % of the er-r isolates, whereas they just represented a . % of the ery-s. for er-r isolates the strongest association was seen with m (or / ; % ci . Á/ . ), and m was second after m only in the last temporal period of the study ( Á/ ) . no homogeneous distribution of er-r m-types by centres was seen. conclusions: few m-types (leading by m ) are responsible for the er-r in spain. but for m , the remaining er-r m types (st , m and m ) did not show a temporally nor geographically homogeneous distribution. *sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en espana' (susceptibility to the antimicrobials commonly used in the community in spain ) and is the spanish word for the willow tree. significant increase in the prevalence of erythromycin-resistant, clindamycin and miocamycin-susceptible (m-phenotype) streptococcus pyogenes in spain ( ( Á/ purpose: a variety of methods is used for a molecular typing of enterococcus spp. and related gram-positive bacteria. these include dna-based methods such as macrorestriction analysis using pulsedfield gel electrophoresis (pfge), ribotyping, and amplification-based methods such as rapid amplification of polymorphic dna (rapd) and amplified fragment length polymorphism (aflp). we used a homogeneous strain collection of transconjugants resulting from filter-matings with different antibiotic-resistant e. faecium and a recipient isolate from our lab. the influence of transferred antibiotic-resistance determinants on the outcome of different typing methods was investigated. results: fragment patterns resulting from pfge indicated minor differences between the transconjugants and the recipient. in respect to different primers used for rapd, none or only a single fragment shift was detected in the resulting fragment patterns. aflp clusters all transconjugants into a group of major relatedness, but the result was strongly dependent on the mathematical method used for cluster analysis. fragment patterns of digested plasmids showed the possession of different or only widely related plasmids in the transconjugants. conclusions: the results of this study clearly show that under certain situations typing methods commonly used for enterococci and related gram-positive bacteria come to their limits. the sasss network aims to set up a national surveillance study to obtain standardized information on antimicrobial susceptibility to various bacterial pathogens. currently, hospitals are participating in the project from different geographical regions in saudi arabia. during the st year ( ), the sasss focused on setting up this network. overall, high frequencies of resistance to antibiotics to different bacterial pathogens in saudi arabia were seen. geographical variations of resistance were noticed, which could be related to different prescribing practices. approximately, and % of escherichia coli and k. pneumoniae , respectively, were extended spectrum â-lactamases (ebls) producers. resistance of enterobacteriacae group to carbapenem and pipracillin/tazobactam is low. resistance of pseudomonas aeurginosa to various anti-pseudomonal antibiotics including carbapenem is high and alarming. methicillin resistant staphylococcus aureus (mrsa) comprised % of s. aureus isolates. no vancomycin intermediate s. aureus (visa) was detected. high level resistance to gentamicin in enterococcus were seen in % of the isolates and only % of enterococcus facieum were glycopeptide resistant. resistance of streptococcus pneumoniae to penicillin ranged between % to almost %. surveillance of antibiotic resistance on a national level is necessarily to give guidance to practicing physicians on the best agents to use. the world-wide problem of betalactam resistance (r) in streptococcus pneumoniae (sp) has been complicated by increasing r to macrolides and some older fluoroquinolones (fq) (ciprofloxacin cip). aim of our study was to evaluate rate of acquisition of resistance to different fq: cip, sparfloxacin (spx) and levofloxacin (lev) of sp strains with different levels of susceptibility to penicillin (p). fifteen strains were serially and daily passaged in subinhibitory concentrations of these four antibiotics by a gradient plate method until acquisition of resistance. clinical strains isolated from children in day-care centers were used. five strains were susceptible (s) to penicillin (p) (one reference strain, four clinical isolates: p micsb/ . mg/l): five were intermediate (i) to p (one reference strain: p mic . mg/l, four clinical strains p mics: . Á/ mg/l), five were resistant (r) to p (p mics . mg/l). mean of number of passages (n ) necessary to reach i or r level with each fq as selecting agent are in the following table: spx and lev induced resistance but more slowly than cip. our results show that rate of acquisition of resistance to fq is strongly related to alteration of susceptibility to p, probably by modification of cell wall. these results are concordant with clinical results. clinical relevance of phase variation in pneumococcal opacity: nasopharyngeal (np) colonization in children from day care centers (dcc) soa . carsenti h, mancini g, bensoussan m, dunais b, pradier ch, dellamonica p. archet hospital, infectious disease, nice, france streptococcus pneumoniae (sp) adherence to nasopharyngeal (np) epithelium is a prerequisite for induction of otitis transparent sp (t) have been shown to colonize the np of infant rats better than opaque (o) sp. opaque sp has proven more virulent than the t form during systemic infection in a mouse model. aim of this study was to evaluate phase variation in the nasopharynx of children. sp strains were isolated during a winter epidemiology study of np samples in children from family dcc. mics determinations were performed by e -test for penicillin (p), amoxicilline (amx) and ceftriaxone (cro). serotypes were performed using the quellung reaction. upon oil immersion microscopic examination short chains of six to eight cocci were noted as , '/, '/'/, '/'/'/ for absence, , , !/ chains by field, respectively. phase variation was detected on catalase trypticase soja plates, amx and cro mics and bactericidal activity was determined for pairs of o and t variants with different serotypes and susceptibility to penicillin. seventy strains of sp were screened for phase variation. nine out of with chain length , '/ had o variants while out of strains with chain length '/'/ or '/'/'/ showed o variants. proportion of o variants was predominant when chain length increased. serotype f was prevalent. bactericidal activity of o variants showed a four-to eightfold increase of mbc. o variants may be present in np of children while t are predominant form for colonization. these virulent variants with lower level of autolysis showed less susceptibility to killing by antibiotics. they may persist in np and explain the absence of eradication by active molecules. antimicrobial resistance among clinical strains of s. pneumoniae isolated from patients with community-acquired respiratory tract infections (carti) in russia soa . kozlov rs a , bogdanovitch tm a , sivaya ov a , agapova ed b , ahmetova li b , furletova b , gudkova lv b , gugutsidge b , ilyina vn b , marusina b , multich ig b , ortenberg ea b , schetinin ev b , shturmina purpose: to determine the antimicrobial resistance of pneumococci causing carti in different russian cities. methods: a total of non-duplicate strains isolated in russian cities in were studied. antimicrobials tested included penicillin (pen), amoxicillin (amo), erythromycin (ery), azithromycin (azi), clarithromycin (cla), midecamycin (mid), spiramycin (spi), clindamycin (cli), levofloxacin (lev), vancomycin (van), rifampicin (rif), tetracycline (tet) and co-trimoxazole (sxt). susceptibility testing was performed by broth microdilution with interpretation of the results according to nccls guidelines ( ) a map of bacterial resistance in a hungarian region soa . farkas a a , juhasz a b , orosi p a , miszti c c , balogh m d . a kenezy teaching hospital, hygienie, debrecen, hungary , b kenezy teaching hospital, laboratory, debrecen, hungary , c university of debrecen, microbiology lab, debrecen, hungary , d regional hospital berettyóújfalu, laboratory, debrecen, hungary background: regional trends of microbiological resistance pattern constitute basic data and qualifying criteria for effective infection control. purpose: the aim of our study was to establish an internationally compatible regional database in a hungarian county hajdú -bihar. methods: our model is the national nosocomial infections society publications' format from the u.s. published in . it contains data regarding various icu types, ambulatory patients and hospitalised patients. the same format is used for antibiotic utilisation data and device related infections' rates as well. we collected cleaned data of years Á/ from all the microbiological laboratories of our county. results: ciprofloxacin p e. coli . . . Á/above susceptibilities not significantly different from u.s. data were as follows: mr cns, streptococcus pneumoniae /penicillin and rd generation cephalosporin, pseudomonas aeruginosa /piperacillin and enterobacter spp. and escherichia coli /ceftriaxon. conclusions: this database proved to be a very useful tool for choosing primary wards of active surveillance including places for infectious disease physician's visit (icu, rehabilitation unit). additional analysis is needed at an individual institution's level for other heavily used (or useable) antibiotics and bacteria as well (aminoglycosides, beta-lactam Á/beta lactamase inhibitor combinations, nd generation cephalosporins, corynebacteria . to compare epidemiological, clinical, and immunological features of add before and after haart introduction, between the patients (p) diagnosed in Á/ , and the p detected since , in a case-control study. though the mean number of newly diagnosed aids p had a sharp drop in the haart era, from p/year in Á/ to p/year since (p b/ . ), the distribution of add and underlying immunodeficiency showed limited changes. when excluding a greater frequency of tuberculosis (tb) (p b/ . ) and wasting syndrome (p b/ . ), all other add did not show a different frequency before and after . a tendency towards a higher mean cd count at aids disease was noticed: vs cells/ml (p b/ . ), with a significant difference for candida esophagitis , toxoplasmosis, kaposi sarcoma and tb (p b/ . Á/b/ . ). the limited variation of clinical and immunological presentation are attributable to the poor impact of haart before aids recognition: . % of p detected since did not receive haart or had insufficient compliance to antiretrovirals, so that . % of p were aids presenters. during the haart era, an increase of mean age and sexual transmission was found (p b/ . ). notwithstanding the effects of haart on the natural history of hiv disease, the consequences on add distribution and related immunodeficiency were negligible, since most p could not benefit from haart before aids onset. a high clinical suspicion for add should be maintained when facing p with missed or undertreated hiv disease. radata */communication internet platform management of resistance analysis guided haart switch for implementation in clinical practice of hiv-infected individuals soa . paech v, lorenzen t, stoehr a, plettenberg a. ifi, interdisciplinary infectiology and immunology, hamburg, germany purpose: hiv-resistance analyses are indicated to prepare switch of haart in hiv-infected individuals with failure to ongoing haart regimen. specialists at several responsible sites often feel lack of complementary informations if interpretation of resistance analyses is done independent from each other. clinical benefits from resistance analysis assays are sigfnificantly higher for those physicians, who can access external advice from hiv-experts for possible treatment options. the database concept 'radata' (www.radata.de) was developed in germany to generate expert advice for implementation in haart switch. results: fifteen hiv-treatment centres, seven laboratories and high ranked authorities in hiv-medicine contribute to radata database since it is started in january in germany. hiv-infected subjects are eligible to participate at the project after presentation of failure to haart (viral load !/ c/ml). expert advice is generated after all data are evaluated and based on recommendations of Á/ external hiv-experts. observation after therapy switch is scheduled for a period of months. conclusions: radata is a novel database concept with features for evaluation of data and availability of complementary information to participating sites. the project is designed to provide its proficiency to patients and centres from germany and foreign countries. further information will be provided after the number enclosed subjects have enlarged. in vitro effects of hiv infection on abc transporter expression and antiretroviral drug efficacy soa . therefore, we evaluate in primary cultures of human monocytederived macrophages (mdm) and lymphocytes, effects: ( ) of retroviral infection and haart on the expression and activity of p-gp and mrp; and ( ) of specific inhibitors of these host proteins on antiretroviral activities of nrti, non-nrti and ip. results: on the one hand, we evidenced a transitory increase of p-gp mrna expression in lymphocytes and mdm in response to in vitro hiv infection. this was correlated to an increased p-gp cell surface expression and activity, and an increased tnf-alpha production and mrna. in contrast, no significant modulation of mrp was observed. on the other hand, psc and probenecid potentiated in vitro the anti-hiv activity of azt and indinavir. these effects were accentuated when psc and probenecid were combined. conclusion: these results showed that: ( ) hiv infection by increasing abc transporter expression could favorise the efflux of antiretroviral drugs and decrease their pharmacological effects; and ( ) specific inhibitors of these transporters could reverse these deleterious effects. effects of interferon alpha plus ribavirine therapy on frequencies of hcv, hiv and cmv specific cd -t-cell responses in peripheral blood of hiv/hcv coinfected patients after months of treatment soa . methods: two groups of patients with chronic hcv infection were studied: hiv coinfected progressors with antiretroviral therapy and hiv-negative controls. twelve hcv/hiv and hcv patients have already reached months of ifn-alpha'/ribavirine therapy. virusspecific cd -t-cells in peripheral blood were analyzed by ifngamma-elispot-assays using hiv-p , one cmv and three hcv (core, ns , ns ) antigens. results: ( ) at baseline, hcv-specific cd -th -cells frequencies were significantly lower than hiv-and cmv-specific ones; ( ) frequencies of cd -th -cells against hcv as well as against cmv were similar in the two groups; ( ) in hcv'//hiv'/, hcv specific cd -t-cell frequencies did not change between baseline and th month of anti-hcv treatment, decreased in three and increased in only one case. hiv-and cmv-specific frequencies were decreased in seven patients. similar results were observed in hiv-negative group. conclusion: ( ) hcv-specific immune responses might be more prone to tissue compartmentalization than hiv-specific ones; ( ) immune defects induced by hiv infection might not be responsible for the low level of hcv-specific responses observed in hiv-progressors; ( ) ifn-alpha'/ribavirine therapy influence on hcv-and hivspecific cd -t-cell frequencies after months of treatment will be discussed. frequencies of hiv- -p specific th cells (elispot) are correlated with plasma hiv- viral load in a cohort of lt-np and slow progressors soa . martinez v a , alatrakchi n a , costagliola d b , bonduelle o a , agut h c , autran b a , alt study group a . a hopital pitié-salpêtrière, laboratoire d'immunologie cellulaire, paris, france , b faculté de medecine saint-antoine, inserm sc , paris, france , c hopital pitié-salpêtrière, laboratoire de virologie, paris, france background: hiv- -specific t helper- cell responses have been associated with long-term-non-progression (lt-np) in hiv infection but the correlation between frequencies of hiv- -p -specific th cells and viral load has not yet been studied. we prospectively quantified these frequencies by using an ifn-gamma elispot assay in a cohort of lt-np. methods: a cohort of lt-np and slow progressors (infection !/ years and cd counts !/ /mm ) was analysable. hiv- -p specific t cells were analyzed using: proliferation, ifn-gamma eli-spot assays and ifn-gamma production in cell supernatants. results: wide ranges were observed in the frequencies of hiv- -p -specific cd th cells as assessed by elispot ( - sfc/ pbmc) with a median of sfc/ pbmc. these frequencies were negatively correlated with viral load (r /(/ . , p / . ) but not with cd counts and associated with a low level of t cell activation assessed by cd on cd cells (r /(/ . , p / . ). similar results were obtained with t cell proliferation and ifn-gamma production. conclusion: interestingly, the numbers of hiv- -p -specific th cells correlate with plasma viral load, independently of cd counts indicating that: ( ) the defect in hiv- -specific cd th cells does not reflect the global cd depletion; and ( ) these responses are strongly correlated to the control of virus replication. impact of drug Á/drug interactions on therapeutical management of active tuberculosis in hiv infected patients soa . martinez v a , truffot c b , caumes e c , katlama c c , jarlier v b , bricaire f c , jouan m d . a department of infectious and tropical diseases, pitié salpêtrière hospital, institut pasteur, unité de génétique mycobactérienne, paris, france , b department of bacteriology, pitié salpêtrière hospital, paris, france , c department of infectious and tropical diseases, pitié salpêtrière hospital, paris, france , d institut pasteur, unité de génétique mycobactérienne, paris, france since and the use of haart, management of hiv patients with active tuberculosis raised the question of drug Á/drug interactions and therapeutical management of both infections. retrospective cohort study: follow-up of hiv patients with active tuberculosis diagnosed between and . studied data included evolution of tuberculosis and hiv, cd cell counts, plasma hiv viral loads, antituberculosis and antiretroviral regimens. ninety-four percent of patients were treated by quadruple combination antituberculosis drug with rifabutin for five patients. fourteen patients were treated by double antiretroviral therapy of nucleoside reverse transcriptase inhibitors (nrtis) and by triple or more drugs (nrtis and/or nonnucleoside reverse transcriptase inhibitors nnrtis and/or protease inhibitors pis). the median follow-up was months. there was no difference on cd cell counts and viral loads in the two groups and between the patients treated by nnrtis and pis at the diagnosis of tuberculosis, at the time of antituberculosis drug discontinuation and concerning cure rates of tuberculosis. on the other hand, the plasma hiv viral load was significantly better controlled in patients with nnrtis than with pis (p b/ . ). in hiv patients with active tuberculosis receiving haart, antiretroviral combination including nnrtis allows a better control of viral replication than regimen including pis without impact of the use of rifampin or rifabutin. adherence is essential to the effectiveness of antiretroviral therapy. a pharmacy visit would improve the patient advisement. a survey was carried out over a period of months in the u.m.i.t. a self-report was distributed to patients. ninety were evaluated. for %, information's given by the clinician were sufficient and for % the treatment advice cards were useful. however, % of them would like to attend a pharmacy visit. the topics they would prefer to be tackled were drug interactions ( %), side effects ( %) and effect of forgetting ( %). the treatment was well accepted and tolerated for, respectively and % of the patients. the viral load and the cd count were well known by, respectively and %. however, inaccurate pattern of treatment was frequent ( !/ %) and bad adherence was observed: treatment forgotten occasionally ( %), regularly ( %) or inadequate attitude when the treatment was forgotten ( %). number of pills, dose frequency, length of the treatment would be risk factors of nonadherence. for the majority of patients, a pharmacy visit is necessary and beneficial. the first result shows a better understanding of the treatment, an improvement of the adherence and an enhancement of plasma concentration of antiretroviral drugs. in vivo activity of glycopeptides against s. aureus infection in a rabbit endocarditis model: is mic predictive for in vivo efficacy? soa . asseray n, caillon j, lemabecque v, jacqueline c, batard e, potel g, bugnon d. laboratoire antibiologie, faculte de medecine, nantes, france we have studied the in vivo efficacy of vancomycin (v) and teicoplanin (t) against five staphylococcus aureus (sa) strains: two methicillin-susceptible (mssa and ), two methicillin-resistant (mrsa and ) and one glycopeptide-intermediate (gisa ) strain, in a rabbit endocarditis model. mics of v and t (v/t) were . / . , / . , / , . / . , and / , for mssa , mssa , mrsa , mrsa , and gisa , respectively. the animals were randomly infected with one of these strains, then treated for days by v or t. a continuous infusion of v, simulating a mg/kg/ h human dose was used. t was infused as a continuous infusion allowing simulating a mg/kg human dose, following an initial bolus. these regimens achieved clinically relevant serum steady-state concentrations of glycopeptides ( !/ mg/ l). results were as follows: expressed in log cfu/g of vegetations (mean /sd, followed in parenthesis by the number of rabbits used). purpose: to determine the prevalence of the decreased glycopeptides susceptibility among clinical isolates of staphylococcus aureus collected from patients hospitalized in strasbourg university hospital between / / and / / . the susceptibility to glycopeptides of s. aureus isolates collected from hospital environment during approximately the same period was also investigated. methods: the susceptibility to glycopeptides was studied among the mrsa isolates, using: Á/ detection of the decreased susceptibility to glycopeptides using agar plates containing mg/l teicoplanin, Á/ detection of hetero-visa strains using agar plates containing mg/ l vancomycin, Á/ determination of the mics of vancomycin and teicoplanin using the agar dilution and the e -test strips methods. results: thirty-nine percent of s. aureus clinical isolates ( out of strains) are mrsa. no visa or hetero-visa strain was detected. six percent mrsa isolates are teicoplanin intermediate s. aureus strains. in the environment, % s. aureus isolates are methicillinresistant (five out of ). the five strains are all susceptible to glycopeptides. conclusion: the results regarding vancomycin are reassuring. however, the high rate of mrsa and the presence of teicoplanin intermediate s. aureus isolates prove that prevention and control measures need to be improved. comparative investigation of polymerase chain reaction and a conventional methods for detection of methicilin resistant staphylococcus amont clinical isolates soa . kantardjiev tv a , vacheva-dobrevski rs b , panajotov sv a , bachvarova am a , velinov ti a , levterova vs a . a national center of infectious and parasitic diseases, microbiology, sofia, bulgaria , b military medical academy, clinical microbiology, sofia, bulgaria purpose: identification on methicillin resistant staphylococci has a great clinical implication and significant impact of antibiotic therapy. the aim of this study is to compare the disc-diffusion test (ddt), oxacillin agar screen test (oast) and pcr for detection of mec a gene. fifty selective clinical isolates ( staphylococcus aureus and nine s. epidermidis ) determined as methicillin resistant by ddt were enrolled in the study. ddt was performed with oxacillin disk ( mkg/ml) on mueller-hinton agar (mha) without nacl (nccls, ) . oast was performed on mha with % nacl, oxacillin mkg/ ml, t c. these strains was genotypically characterized for the mec a gene presence by pcr method using the mec a - ?-aaa atc cat ggt aaa ggt tgg c- ? and the mec a Á/ ?-agt tct gca gta ccg gat ttg c- ? primers (gibco, brl) . results: in the group of mrs isolates, detected by pcr, positive results were as follow: s. aureus and six s. epidermidis . for six s. aureus isolates ddt and oast were positive; pcr-negative. for two s. aureus and two s. epidermidis isolates pcr was positive; phenotypic methods-negative. conclusions: accurate and rapid detection of mrs is a constant challenge for laboratories. the pcr assay (first time in our country) appears to be more reliable than routine susceptibility testing for the rapid diagnosis of mrsa infections at hospitals, particularly due to the heterogeneous resistance of many strains. . / . ( ) . / . ( ) . / . ( ) . / . ( ) . / . ( ) v . / . * ( ) . / . ( ) . / . * ( ) . / . ( ) . / . ( ) t . / . * ( ) . / . ( ) . / . * ( ) . / . ( ) . / . ( ) distribution and antibiotic susceptibilities of bacteria isolated from suspected urinary tract infections of inpatients in hungary soa . rozgonyi f, csukás z, kamotsay k, szabó d, ostorházi e, berek z, maródi c. institute of medical microbiology, semmelweis university, budapest, hungary between l january and december , a total of , urine samples were cultured % as native urine (nu) and % as uricult-plus (up) (orion diagnostica, finland) . cultivations were negative in % of nu and % of up specimens, while contamination was revealed in % of nu and % of up. in the clinical bacteriologically evaluable positive nu and up cultures, the distribution of the gram-negatives was very similar with the predominance of escherichia coli ( and %) followed by enterobacter spp. the distribution of gram-positives differed significantly according to the types of specimens. nu resulted in % group-d and % group-b streptococcus while up did and . %, respectively. third generation cephalosporins and fluoroquinolons were equally very effective against e. coli strains, while ampicillin inhibited growth of % only. carbapenems, cefepime and the fluoroquinolons were the most active against enterobacter strains. interestingly, trimethoprim'/ sulfarmetoxazole combination could inhibit more than % of enterobacteriaceae strains. piperacillin'/tazobactam ( %), imipenem ( %), and ciprofloxacin ( %) could be the drog of choice against pseudomonas aeruginosa . enterococcus strains were highly sensitive to glycopeptides ( %), nitrofurantoin ( %), imipenem ( %) and amoxicillin'/clavulanic acid ( %). antimicrobial susceptibility levels of escherichia coli isolates cultured from urine at a tertiary care teaching hospital. temporal trend and comparison between community-acquired and nosocomial urinary tract infection soa . nanetti a, manfredi r, valentini r, calza l, chiodo f. uni versity of bologna, infectious diseases, bologna, italy in order to assess the local temporal trend of antibiotic sensitivity of the most common urinary tract bacterial pathogen, all urine-cultured escherichia coli isolates were reviewed as to susceptibility profile, and specimen source (community-versus hospital-acquired infection). when evaluating sensitivity levels of community-acquired pathogens ( Á/ ), a significant resistance rise was limited to cotrimoxazole (p b/ . ) and nalidixic acid (p b/ . ), while a tendency towards increased resistance regarded norfloxacin (p / . ) (fig. ) . when community-acquired e. coli isolates were compared with nosocomial strains (tested in the years Á/ ), a greater susceptibility of community-acquired e. coli isolates was limited to cotrimoxazole versus all other compounds in the year (p b/ . ), while it was extended to amoxicillin, cephalotin, nitrofurantoin and piperacillin in the year (p b/ . ) (fig. ) . on the whole, e. coli showed an elevated sensitivity rate ( !/ % of tested strains) to nitrofurantoin, gentamicin, amikacin, and nd-and rd-generation cephalosporins, while only amoxicillin and piperacillin had a mean resistance rate !/ %, regardless of the community or nosocomial origin. a permanent surveillance of sensitivity levels of the most common pathogens responsible for infectious diseases enables to identify local antimicrobial activity and its temporal variations, and plays a key role in starting empiric therapy, pending bacterial identification and in vitro assays. conclusion: in uti, the antimicrobial agents such as st cg combined with aminoglycosides are recommended as initial treatment as well as the rd cephalosporin generation at monotherapy. in addition, the fluoroquinolones and aminoglycosides are effective in uti. prevalence of resistance mutations to antirretrovirals and relation to virological failure s . garcia f a , suarez s a , alvarez m a , martinez nm a , valera b b , pasquau j b , hernandez quero j a , maroto mc a . a hospital san cecilio, microbiology, granada, spain , b hospital virgen nieves, microbiology, granada, spain purpose: to investigate the prevalence of resistance mutations in the reverse transcriptase (rt) and protease (p) genes of hiv and to relate it with the type of virological failure (vf), we have studied patients ( % naïve or pregnant women, % were first vf, % were second vf, % more than two vf. resistance mutations were investigated using trugene hiv- genotyping kit (visible genetics). results: global prevalence of resistance mutations for rt inhibitors (rti) has been !/ % for m l, d n, k n, m v, l w, t yf, and for l i, m i, l p, a vt, l m for p inhibitors (pi). the prevalence of resistance mutations for the naïve patients studied was very low (a g, v i for rti and l i, m i, m i */all n / */and l p n / for pi). for patients on first vf only k n, m v, t yf (rti) were !/ %, as well as l i, d n, l p (pi); when patients on second vf were studied, then m l, e d, k n, m v, g a, l w, t yf, k qe (rti) and m i, l p, a t (pi) were !/ % prevalence; finally, when patients with more than two vf were studied, the following resistance mutations were !/ %: m l, d n, k r, k n, v i, y c, m v, g a, l w, t yf (rti), and l i, m i, m il, l p, a t, l m (pi). conclusions: the prevalence of primary resistance in the population studied is very low; the prevalence of mutations in the reverse transcriptase and protease genes increase in parallel to the type of virological failure. genotypic resistance in hiv- rna from patient plasma compared with rapid virus isolation and phenotypic resistance in patient pbmcs s . stuermer m, groeschel b, cinatl j, doerr hw. institute for medical virology, university clinic frankfurt, frankfurt, germany objective: to compare hiv- virus isolation in the presence of antiretroviral drugs with plasma hiv- genotyping. materials and methods: hiv- genotyping was performed using the viroseqtm vers. from applied biosystems. interpretation of genotype was done according to international standards. cd -cells were purified from patient plasma and cultivated in microtiter plates coated with anti-cd and anti-cd antibodies in the presence of different concentrations of antiretroviral drugs. virus production was measured using a p antigen assay. phenotypic activity was expressed as % reduction of p concentrations. results: seventeen samples were analyzed. for samples results were obtained from both methods, two samples could not be analysed by phenotyping and four samples not by genotyping. only / samples showed total and / samples partial concordance, / samples showed discordance between the two assays. in discordant samples the genotype gave a definite interpretation. conclusion: hiv- virus isolation and phenotyping from pbmcs may overcome the problem of currently used resistance assays, which analyse only the reverse transcriptase and the protease gene of hiv- . possible mutations in other regions may influence viral fitness and therefore contribute to the growth of the virus population present. the lack of concordance between the two assays is related to the different blood compartments used. the clinical value of resistance tests using pbmcs is under investigation. interleukin- co-operates with a new type i ifn, ifn-tau to inhibit early steps of hiv-i biological cycle s . rogez c a , clayette p a , martin m a , dereuddre-bosquet n a , martal j b , dormont d c . a cea, drm, fontenay-aux-roses, france , b inra, physiologie animale, jouy-en-josas, france , c cea, crssa, ephe, drm, fontenay-aux-roses, france background: type i interferons (ifn) exhibit efficient antiviral activities notably against hiv, but severe side effects restrict their clinical uses. ifn-tau is an ovine or bovine non-cytotoxic type i ifn which displays higher inhibitory effects towards hiv replication than ifn-alpha, particularly in human monocyte-derived macrophages (mdm). the antiretroviral activity of ifn-tau seems to involve antiviral and immunomodulatory mechanisms: il- synthesis is increased in dose-dependent manner in mdm treated with ifn-tau and a specific inhibition of il- biological activity decreases its antiretroviral efficiency. results: after a -h infection, a significant decrease of intracellular hiv rna amount was found in mdm treated with ifn-tau. in parallel, no additive inhibition was observed with ifn-tau during the elongation of proviral dna. these results suggest either an inhibition of hiv nucleocapsid uptake or an immediate hiv rna degradation, and the expression of ?, ?-oas, mxa protein and pkr was then measured. ifn-tau induced the expression of these three host cell factors. the role of il- on these different steps was evaluated and we showed that il- co-operates with ifn-tau during the very early step of hiv biological cycle. conclusion: altogether, these results evidence that ifn-tau uses the same antiretroviral pathway as others type i ifn in mdm, and that il- takes part to its inhibition of early steps of hiv biological cycle. actinomycin-d as a modulator of resistances due to cell-wall active agents like bacitracin (bc) and lysozyme (lz) s . chakrabarty an a , dastidar sg b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india it was observed that development of lzr in the lzr mutants took placed at three different levels and was accompanied with unselected, distinctive and elevated levels of bcr. similarly, bcr in bcr-mutants were also detected at three different levels. although the levels of bcr ( / mg/ml) in the bcr mutants could be raised only by persistent efforts, an increase in the levels of lzr (as cross-resistance) in the same mutants could be easily achieved. a correlation of actinomycin-d resistance with lzr and bcr of the mutant bacteria and the effects of lipase treatment on the same showed a Á/ -fold rise in actinomycin-d resistance of the lzr and bcr mutants of gram-positive bacteria compared with their correspondence wild-types. these findings suggest that lzr and bcr are controlled by several genes accounting for reduced cell-wall and cell-membrane permeability and indirectly, by phenotypic alteration of the lipid content of the cell-wall. thus, the alteration of cell-walls and membranes and a phenotypic extra lipid layer can work in conjunction with the efflux pump mechanisms finally determining the levels of drug-resistance. experimental development of drug resistance to non-antibiotics: a role of alteration of membrane fluidity and efflux systems s . dastidar sg a , mazumdar k a , asok kumar k a , chakrabarty an b . a jadavpur university, pharmaceutical technology, calcutta, india , b department of medical microbiology, calcutta university, calcutta, india drug resistance among clinical strains was studied by selecting mutants resistant to promazine (pr) and methdilazine (md). the results showed that successive step-up mutants of pr and md developed cross-resistance to several unrelated drugs, which in subsequent steps had broader resistance spectra with higher levels of resistance. experiments on the membrane fluidity or permeability of bacterial cells using diphenyl hexatrine (dph), a fluorescent probe on md-mutants showed that three was marked reduction in the membrane fluidity and permeability. when several analogues of the basic phenothiazine structure, e.g. -chlor-methyl-n -methyl-pyrrolidine (cmp), methyl- -methyl- -pyrrolidone- -carboxylate (mmpc), hydroxymethyl-n -methyl-pyrrolidine (hmp) and md with final substitution were tested for antibacterial function on different strains, highest activity was observed with respect to md. with anaerobic bacteria the resistance(s) dependent on efflux pumps showed higher levels of resistance even to md. we have found the non-antibiotic agents triflupromazine, trimeprazine and diclofenac sodium have high degree of activity against vibrios, staphylococci and pseudomonads. the explanation of such a phenomenon in terms of possible efflux pumps will be discussed. csf, plasma and urine pcr in lyme neuroborreliosis s . pícha d a , moravcová l a , lásiková Š a , marešová v a , Ž ïárský e b . a charles university, nd medical school, st clinic for infectious diseases, prague, czech republic , b department of cellular and molecular biology, charles university, rd medical school, st clinic for infectious diseases, prague, czech republic the main reason for high diagnostic value of pcr in neuroborreliosis (nb) is the direct way of spirochete detection. two sets of primers in nested pcr were used: one for plasmide gene encoding ospc protein and second for chromosomal gene s rdna. so far patients with clinically manifested involvement in nb were enrolled into the prospective designed study (being continued). the main including criterion was positive prove of intrathecal specific antibody secretion (in patients) and pcr positivity in csf (in ). all patients were repeatedly examined by neurologist and samples of csf, plasma and urine were taken: ( ) before treatment; ( ) after treatment; ( ) after months. before treatment were patients pcr positive in csf, six in plasma, and in urine. five were parallel positive in csf and plasma and four in all three body fluids. urine after treatment was positive in seven ( %) cases and completely negative after months. the pcr has had relative high sensitivity ( %), but does not rich the sensitivity of antibody index ( %). supported by grant mzcr ; . consumption of imipenem correlates with b-lactam resistance in pseudomonas aeruginosa s . lepper pm a , hö gel j b , trautmann m a , grusa e c . a department of medical microbiology and hygiene, university of ulm, ulm, germany , b department of biostatistics, university of ulm, ulm, germany , c hospital memmingen, central pharmacy, memmingen, germany purpose: in the present study we investigated the monthly consume of three anti-pseudomonas-active antibiotics, namely imipenem, piperacillin/tazobactam (pt) and ceftazidime during a period of years ( Á/ ) . the use of these antibiotics was correlated to the rate of resistance in pseudomonas aeruginosa . results: inspection of the time series for use of imipenem, ceftazidime, and pt, and the corresponding time series for resistance (each available from july to july ) indicates a remarkable coincidence between use of imipenem and resistance against the three antibiotics mentioned. pearsons's coefficient of correlation for the use of imipenem and the resistance against imipenem was . (p b/ . ), between imipenem use and pt resistance was . (p b/ . ), and between imipenem use and ceftazidim resistance . (p b/ . ). we found positive regression coefficients quantifying an association with imipenem use in the same month (p b/ . ) and with the use during the preceding month (p b/ . ). the same was true when checking dependence of ceftadizime resistance (p b/ . ) and pt resistance (p b/ . ) on imipenem use observed during the same month. neither the use of ceftadizime nor of pt could be identified as factors creating resistance to one of the three antibiotics under consideration within a reasonable period of time. conclusion: there might be a strong pressure towards resistance created by carbapenems. this could limit the use of carbapenems for initial empiric therapy. treat hard and fast: short course antibiotic treatment and its relation with patient compliance and effectiveness s . perez-gorricho bpg a , ripoll m b , pechere jc c . a niño jesus hospital, infectious diseases, madrid, spain , b insalud, outpatient consult, madrid, spain , c university of geneve, microbiology, geneve, switzerland 'treat hard and fast ': short course antibiotic treatment and its relation with patient compliance and effectiveness. finding the important implications for the way in which physicians manage patients with mild Á/moderate respiratory tract infections, and the relation of this management with the perception of antibiotic effectiveness, and the compliance with the antibiotic regimen has been the main purpose of the research. in a pan-european market research study of more than patients, designed to determine behaviour to the antibiotic management of mild-moderate respiratory tract infections, patient expectations of antibiotic therapy were identified, particularly those aspects that relate to efficacy and compliance. the study identifies three key drivers of patients perceived antibiotic efficacy: length of antibiotic course, time to onset of symptom relief and time to complete resolution of symptoms. the results demonstrate that once daily treatment for short periods is perceived by patients to be significantly more effective than longer antibiotic courses and thus better meets patient expectations of therapy. in this study, a macrolide, azithromycin, was selected as the drug therapy of shortest course, being the antibiotic with the shortest dosage schedule for common outpatient infections. the perception of efficacy with short course therapy also correlates with overall satisfaction with management by the physician and with patient compliance with antibiotic therapy. purpose of the study: group b streptococci (gbs) remain a major cause of neonatal infections. consensus guidelines have recommended an intrapartum antibioprophylaxis by amoxicillin, which has reduced the incidence of early-onset neonatal gbs infections. however, an increased incidence of beta-lactam-resistant gram-negative neonatal sepsis has been reported. the aim of our study was to analyse the consequences of this antibioprophylaxis on the intestinal microbial colonization of newborns. a study of the fecal flora was carried out on stools samples from days-old newborns divided into groups: group a intrapartum treated mothers (n / ); and group b untreated mothers (n / ). both groups were matched with regards to known factors affecting intestinal microbial colonization: gestational age, type of delivery and feeding. results: colonization by enterobacteria and enterococci was not significantly different and occurrence of amoxicillin-resistant enterobacteria was similar ( / and / in groups a and b, respectively). however, the colonization by clostridia was modified: the number of newborns colonized was significantly less important in group a than in group b (group a: / and group b: / p b/ . ). conclusion: in our study, intrapartum antibioprophylaxis did not affect intestinal colonization by aerobes but reduced significantly colonization by clostridia, potentially anaerobic pathogens. impact of an antibiotic policy restricting the use of b-lactams and macrolides on the incidence of clostridium difficile associated diarrhoea in general medical, renal and elderly patients s . boswell tc a , pacey s b , broomfield s c , westmoreland d c , yates c c . a nottingham city hospital, microbiology, nottingham, uk , b nottingham city hospital, pharmacy, nottingham, uk , c nottingham city hospital, infection control, nottingham, uk the purpose of the study: to investigate the short-term impact of a new antibiotic policy for the treatment of urinary and respiratory infections on the incidence of clostridium difficile associated diarrhoea (cdad) in hospitalised medical, elderly care and renal patients. the results obtained: a policy restricting the use of b-lactams (except parenteral penicillin), and promoting alternative antibiotics including levofloxacin for pneumonia, and doxycycline for non-pneumonic respiratory infections, was launched in july . as a result there was a significant and sustained reduction in use of aminopenicillins, cefuroxime and macrolides, with a corresponding increase in doxycycline and levofloxacin. the incidence of cdad was determined during the st months of the new policy and compared to the last months of the old policy. the incidence of cdad fell from . to . per patients, and from . to . per in-patient days (p b/ . ). in contrast, there was no change in the incidence of cdad in other specialties (surgery, oncology etc.) that had not introduced the new policy. there was no change in the incidence of nosocomial bacteraemia with quinolone-resistant coliforms or mrsa, despite the increased use of levofloxacin. conclusions: hospital-wide reduction of b-lactam and macrolide use in medical patients can result in a significant and immediate reduction in cdad. longer follow-up will determine if this effect is sustained. use of imipenem/cilastatin i.v. (tienam i.v.) for the treatment of low respiratory tract infections in intensive care units s . izzo l a , orsetti r b , boschetto a a , binda b a , della casa u a , caramanico l a , la mazza a a . a department of surgery, universitá degli studi di roma 'la sapienza','p. valdoni', rome, italy , b s. camillo-forlanini, intensive care unit, rome, italy ventilator associated pneumonia (vap) is considered the most frequent infection in the intensive care unit (icu), occurring in Á/ % of patients intubated for longer than h besides nosocomial pneumonia is a common complication in the critically ill surgical or trauma patient. inadequate treatment can lead to the complications of acute respiratory distress syndrome (ards), empyema, and lung abscess. the most important aetiological agents both in vap and in pneumonia which arise as complication in surgical or trauma patients are bacteria, whit a marked predominance of staphylococcus aureus and pseudomonas aeruginosa . the authors present their experience ( cases) on the employment of imipenem/cilastatin i.v. (tienam i. v.) as initial empirical monotherapy at the dose of mg)/ /day or g)/ / day for the treatment of the serious lower respiratory tract infection in an icu. tienam is a well tolerated broad spectrum antibacterial agent that is effective against the majority of gram-positive and gram negative aerobic and anaerobic bacteria including most pseudomonas species. except one patient deceased for causes related to his very poor general conditions and three cases in which has been necessary the addition of an aminoglycoside, in all the other patients the imipenem/ cilastatin (tienam) monotherapy has shown satisfactory clinical and bacteriological responses. clinical auditing of the impact of recommendations on antibiotic treatment s . kinoo j a , david-ouaknine f a , hacquard b a , echard y a , decazes jm b . a centre hospitalier lagny marne la vallée, lagny sur marne, france , b hospital saint louis, paris, france the aim of this study was to assess the impact of curative antibiotic recommendations on suitable prescriptions at lagny-marne la vallée hospital (general hospital, beds). two prospective exhaustive audits were made (all complete hospitalizations, excluding psychiatry, february Á/may and ) of the detailed curative antibiotic prescriptions, before and after distribution of internal recommendations. the same methodology, designed by a multidisciplinary team, was used for both periods. the same antibiotics were available at the pharmacy. the prescriptions were assessed by an infectious diseases specialist and a pharmacist using pre-established criteria: literature recommendations ( audit), internal recommendations ( audit). six hundred and fifty-six prescriptions for patients were collected and analysed in , for patients in . exhaustivity of the recovered prescriptions was over %. patient characteristics, infection sites and microbiological findings were similar for both groups. suitable prescriptions were significantly increased ( Á/ %, p b/ . ). unsuitable prescriptions (economic reasons, too short or too long course, incorrect administration, or underdosage) were significantly reduced. prescriptions for incorrect indications were unchanged and necessary combined treatment not being prescribed, increased. local recommendations improved prescriptions, but efforts have to be done in order to go on the improvement of the practice behaviour. cost-effectiveness analysis of antibiotic therapy in hospitalized patients with copd exacerbations (ae-copd) s . beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy antibiotic costs represent a high burden of total drug costs for hospital administrations. a scientific approach considering also the economic aspects of each therapeutic decision may gain optimal treatment objectives at pondered costs. in our study we retrospectively evaluated the clinical effectiveness and costs of antibiotic therapy in patients with ae-copd. from to , our retrospective study results support previous pharmaco-economic considerations according which in choosing an antibiotic regimen for ae-copd we must take into consideration the expected clinical and microbiological results without forgetting to consider the economic burden of our decisions. significant increase in fungaemia due to non-albicans candida species s . shah pm. klinikum der j.w. goethe universitaet, schwerpunkt infektiologie, frankfurt am main, germany until , predominant candida species in blood cultures was candida albicans . it accounted for . % of all candida species cultured from blood. since then we have observed a gradual increase in number of non-albicans candida species. from , onwards nonalbicans candida species out-number c. albicans . this observation is especially important as non-albicans candida species are generally non-susceptible to azole derivatives and empirical use of azoles in suspected candidaemia should not be recommended. amphotericin b is uniformally active against almost all candida species. echinocandin may be an alternative. see figure below. a search for newer antifungal chemotherapeutics s . chakrabarty an a , dastidar sg b , saha b b , basu l b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india fungal infections due to the mucor-rhizopus (m-z) group present formidable problems due to lack of appropriate and effective drugs against them, as seen in increasing number of clinical situations; death due to mycoromycosis is nearly inevitable. we analysed the biological 'weak-spots' of the mucor-rhizopus group and attempted to devise suitable drugs using their weak-spots. we have noted that like many free-living fungi, the m-z fungi are facultatively chemoautotrophic (can grow on simple sources of carbon and nitrogen and a solution of mineral salts), like the human pathogenic chemoautotrophic nocardioform bacteria. we devised a minimal medium based on that of davis and mingioli, supplemented with simple chemical compounds as sole sources of carbon and nitrogen. the key chemical here was diphenylamine with trypan blue (dpa Á/tb) and other similar sources of c and n. we found that while media free of these chemicals (controls) allowed good growth of different strains of m-z fungi, a mixture of dpa Á/tb completely prevented their growth over a wide concentration range. experiments with immunocompromised mice showed that these drugs at the concentrations used are well tolerated; mice experimentally infected with several clinical isolates of m-z fungi and receiving these chemicals showed that these fungi could not grow in vivo. in vitro activity of newer fluoroquinolones against multi-drug resistant salmonella typhimurium s . nolones resistance is being also reported. we have studied the in vitro activity of b-lactams and fluoroquinolones against multi-drug resistant s. typhimurium from human sources. material and methods: fifty multi-drug resistant s. typhimurium were tested against cefazolin, cefuroxime, cefotaxime, cefepime, ofloxacin, levofloxacin, and moxifloxacin, by the agar dilution method according nccls guidelines. results and conclusions: all the strains were resistant to four or more of the following antibiotics: ampicillin, tetracyclines, chloramphenicol, streptomycin, sulphonamides and nalidixic acid. a high proportion of strains were intermediate or resistant to amoxicillin/clavulanate. we found no resistance to cephalosporins. nevertheless, % were intermediate to first and/or second gen. cephalosporins. cefotaxime and cefepime were the most active cephalosporins (mic : . mg/l). though increasing fluoroquinolones resistance has been described among this kind of strains, no resistance to fluoroquinolones was found here. levofloxacin was the most active fluoroquinolone (mic : . mg/l), followed by ofloxacin (mic : . mg/l) and moxifloxacin (mic : . mg/l). high rates of resistance to antibiotics by salmonellae from diarrhoeic children in zliten-libya s . ghenghesh ks a , ben ali m b , abuhelfaia a b , dufani ma a . a faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya , b faculty of arts and sciences, el-ghomes university, biology, el-ghomes, libyan arab jamahiriya salmonellae are major bacterial cause of diarrhoea in libya particularly in children. included in the present study salmonella species isolated from children with diarrhoea in zliten city-libya. the children aged between a few days to years. the organisms were tested for their susceptibility to antibacterial agents using the disc diffusion method. of the isolates examined, ( %) were resistant to ampicillin, ( . %) to amoxicillin Á/clavulanic acid combination, ( %) to cefoxitin, ( . %) to chloramphenicol, ( . %) to doxycycline, ( . %) to gentamicin, ( . %) to nalidixic acid, ( . %) to trimethoprim Á/sulphamethoxazole and none ( . %) were resistant to norfloxacin. a strong relationship was observed between the availability of antibiotics in the pharmacies of the city and resistance of the isolated salmonellae to these drugs. the misuse of the antibiotics by the community may be an important factor (among others) in the emergence of these high rates of resistance by the salmonellae examined. effect of ceftriaxone along with probiotics administration on intestinal ecosystem and betalactamase activity s . bertazzoni minelli e a , benini a a , zoppi g b . a department of medicine and public health-pharmacology section, university of verona, verona, italy , b department of paediatrics, university of verona, verona, italy oral bacteriotherapy during antibiotic treatment is a much debated topic. aim: to study whether different probiotics can prevent imbalance of the intestinal ecosystem (dysbiosis) in children during therapy with ceftriaxone (cx). methods: fifty-one children (mean age . years) with febrile respiratory tract infections were treated with cx mg/kg/day iv, alone (therapy ) and along with the following preparations: saccharomyces boulardii ( ); enterococcus spp. ( ); lactulose ( ); l. casei gg ( ); l. rhamnosus , l. bifidus and l. acidophilus ( ); b. bifidum and l. acidophilus ( ); and a mixture of various lactobacilli and bifidobacteria at high concentrations ( ). faecal samples, collected before and after treatment, were analysed for microflora composition, cx concentration, and beta-lactamase (bl) activity. results: cx causes intestinal dysbiosis. no c. difficile was found. faecal bl increased after therapy in all treated groups. cx alone increased bl activity in % ( / ) of children (no activity before treatment); a higher incidence ( Á/ %) was found in groups and . after therapies , , , , and , bl activity was found in or more children. cx was detected in % of faecal samples. conclusions: the probiotics administration seems to protect against dysbiosis caused by cx and to contain the increase in faecal bl activity. the effects differ according to the probiotic administered and are peculiar to certain bacterial species. these preliminary data need further studies. comparative study of initial and acquired drug resistance in pulmonary tuberculosis in iran s . mansoori d, arami s, mirabolhasani z. nritld, infectious disease, tehran, islamic republic of iran purpose: resistant to anti-tuberculosis agents particularly multiple drug resistant (mdr) is a major obstacle in treatment tuberculosis in the world. between september and march for smear and culture positive pulmonary tuberculosis patients (old / , new / ) pretreatment susceptibility tests of isolated bacilli to inh, rif, emb and stm were performed by standard proportional method and the results were attributed to three groups: (i) newly diagnosed without any history of treatment; (ii) patients with history of treatment for one course; (iii) patients with history of treatment for two or more courses supposed to be mdr cases. the results were collected for each drug individually and different combinations of two, three and four medications. results: resistance to one, two, three and four drugs was significantly increased in group iii comparing to groups ii and i, also in group ii compared to group i. we observed a high rate of primary resistance to inh and stm in groups i and ii and a high rate of mdr (inh and rif resistance) in groups ii and iii. conclusion: the duration of bacilli exposure to antituberculosis agents in the past is a major factor in developing resistance. in contrast to who's guideline, due to high rate of primary resistance especially to stm in our area, we do not recommend addition of stm for treatment of patients whose initial four-drug regimens have been failed (group ii). donors, to understand host interactions with this bacteria, to develop new methods of diagnosis and define new vaccine candidates. nineteen tb patients and seven healthy donors were enrolled in french hospitals. cellular immune responses were evaluated by lymphoproliferation and ex-vivo quantification of specific th cells by elispot-ifn-gamma assays. four recombinant proteins of m. tuberculosis were tested: esat- , b, erp and tb b . and compared with tuberculin. we confirmed that b (but not esat- in our study) gives higher responses in tb patients compared to donors according to the results in proliferation assay (p / . ). in addition, frequencies of th cd specific cells for esat- and tuberculin were statistical different between the two groups (p / . and . , respectively). . % for b and . % for esat- of the patients tested were responders in elispot-assay versus . % for both in proliferation assay. for the new antigens erp and tb b . , no difference was observed between the two groups. in conclusion, b and esat- are recognised by a large number of our patients. they seem to be promising antigens for the development of new methods of diagnosis or for the development of new vaccines. erp and tb b . are not preferentially recognised by tb patients. other exported antigens will be tested. the incidence of tuberculosis (tb) is increasing worldwide. in recent some years, geographical differences in the incidence of tb in former yugoslavia have been observed. an important rise in tb cases was registered in the bordering region of bosnia. it is likely that poorer living conditions, influenced by war and emotional stress, may promote such rising incidence of tb. renal tuberculosis was diagnosed in patients (female , male ) from district brcko in bosnia, during the period of years, Á/ . at the same time none patient had active pulmonary tb lesions, fibrous lesions were noticed in patients, but we did not diagnose any signs of previous pulmonary tb in seven patients. seven patients developed relapse of renal tb after Á/ years of previous treatment. guided by clinical parameters, precisely done renal echosonography enabled early suspicion and searching for renal tb, by radiological and other methods. bacteriological diagnosis was performed by detection mycobacterium tuberculosis on loewenstein Á/jensen medium in patients. pcr as simple, fast and highly sensitive method enabled diagnosis in incipient stadium of disease, so antituberculous therapy could be instituted some months earlier. prompt diagnosis of renal tuberculosis (using pcr, besides standard methods) is necessary, otherwise delayed diagnosis may be dangerous. a study on resistance to first generation anti-tuberculosis drugs in mycobacterium kansasii s . mirsaeidi sm, farnia p, mohammadi f, mansoori sd, jabbari r, taghizadeh r, masjedi mr, velayati aa. national research inistitute of tuberculosis and lung diseases, infectious diseases and immunology (nritld ), tehran, islamic republic of iran purpose: this research has been performed to determine antibiotic resistance of atypical mycobacteria especially mycobacterium kansasi . results: twenty-three pigmented colonies which indicated atypical agents from nritld's mycobacterium culturebank were selected, they then underwent type identification and antibiogram for inh, rif, etb, stm. nine samples were m. kansasi and were other non-mtb, was m. gordonei , m. xenopi , mac, m. bovis , m. tererra , m. asiatioum , m. marinum and . mean age in m. kansasi cases was '/ . year and in non-kansasi cases '/ . year and in whole society ntm was . '/ . . frequency of resistance in kansasi group were to inh ( %), to rif ( %) to etb ( %), to stm ( %) and prevalence of mdr was ( %) and in non-kansasi group frequency of resistance were ( %) to inh, to rif ( %), to etm ( %) and to stm was ( %), to mdr was ( %). conclusion: a significant difference was seen between the age groups of patients who are affected with m. kansasi and non-kansasi (p b/ . ), also in frequency of resistance to first generation anti-tb drugs. m. kansasi is detected as the most common atypical mycobacterium agent in pulmonary infections and attention to antibiogram is recommended before treatment. buruli ulcer caused by mycobacterium ulcerans , is the third most common mycobacterial after tuberculosis and leprosy in west africa. nowadays, the only effective treatment is surgery. it consists in a large excision of the lesions, often followed by a skin transplant. in this study, the effectiveness of rifampin, amikacin and their combination were estimated in the treatment of mice, which were infected experimentally by m. ulcerans . after weeks of treatment with rifampin, amikacin or their combination, no more viable bacilli were found in infected tissues. the animals were kept for other months. among the mice treated with rifampin alone, two mice out of relapsed. the minimal inhibitory concentration of these isolated strains went from . to mg/ml. the dna sequence, obtained from a -pb of the rpob gene from these strains, showed a missense mutations, which affect a ser- replaced by a phenylalanine. this modification on the gene leads to an important inefficacy of treatment when rifampin was used alone. this study showed that rifampin and amikacin have a bactericidal action on m. ulcerans and that a combination of these antibiotics is necessary to avoid the selection of resistant mutants. histopathologic and electron microscopy studies of a severe isolated hiv enteropathy detected in an aids presenter. favorable response to haart introduction or . manfredi r, calza l, chiodo f. university of bologna, infectious diseases, bologna, italy advanced hiv infection was detected in a heterosexual female with a -year history of chronic diarrhoea and severe wasting, as expressed by a body weight of kg, a cd '/ count of cells/ml, and a plasma viraemia of . million copies/ml. a malabsorption syndrome was confirmed by d-xylose test, but repeated pathogen search tested negative at stool examination and light microscopy, scanning electron microscopy (em), and transmission em study of enteric mucosa. em assays detected an ultrastructural modification of duodenal mucosa never reported to date: an extensive thinning of enterocytic microvilli, disappearance of glycocalix, and large vacuolization of the enterocyte cytoplasm. two weeks after starting an indinavir-based haart, diarrhoea disappeared and our patient significantly gained body weight: kg after months, kg after , and kg after year, paralleling a cd '/ increase to cells/ml, and undetectable hiv viraemia. the subsequent -year follow-up confirmed absence of gut disturbances, a stable body weight, a cd '/ count of Á/ cells/ml, and hiv viraemia persistently b/ copies/ml. repeated endoscopy and related histopathologic and em assays documented a notable improvement of mucosal damage, with complete cure reached after years of haart. a direct intestinal localization of hiv may be responsible for severe diarrhoea, malabsorption, and wasting, though the morphological features of hiv enteropathy are still unclear. haart acts favourably also against isolated hiv-related enteropathy. kaposi's sarcoma in a non-hiv immunocompetent adult: relapsing due to the development of a squamus cell carcinoma or . sioula e a , magira ee a , georgopoulou c a , rontogiani d b , gounaris t a . a evagelismos, internal medicine and infectious diseases, athens, greece , b evagelismos, pathology, athens, greece a -year-old heterosexual hiv negative girl was diagnosed with cutaneous kaposi's sarcoma. the disease was started years earlier with the appearance of lesions on the left feet and on the right knee. absolute number of cd and cd were and cells/dl, respectively with a decreased lymphocyte proliferation. human herpesvirus type had been detected in biopsy specimens and she placed on recombinant interferon alpha- b. follow up few months later the lesions decreased in size. two years after the onset of the disease the patient readmitted because of a mass on the left paratracheal region along with mediastinitis. her body temperature was increased. the patient underwent thoracic ct scan, which demonstrated mediastinal well defined soft tissue infiltration associated with mediastinitis and a well-defined mass in the left paratracheal region. the mass biopsy revealed squamous cell carcinoma. several violaceus lesions were observed on the arms, hands and face. severe bilateral lymphedema of the legs with a reddish papules nodules and tumors from . to cm in diameter on the soles, toes and calves were present. this case illustrates the significant relapsing of the cutaneous kaposi's sarcoma soon after the appearance of and the carcinoma and the mediastinitis. a -year-old male patient with insulin-dependent diabetes underwent cardiac surgery for aortocoronary bypass years ago. two weeks after surgery he developed mediastinitis and sternal osteomyelitis caused by methicillin-resistant staphylococcus aureus (mrsa). twice, revisions and plastic surgery for sternal osteomyelitis were performed. the patient received initially treatment with vancomycin. then the patient received intravenous outpatient treatment with teicoplanin for weeks followed by by treatment with fusidic acid and then trimethoprim/sulfametrol. the fistula was closed. four months later he presented again with substernal pain and purulent discharge. the culture revealed the growth of staphylococci which were first mistaken for coagulase-negative staphylococci. after closer investigation these staphylococci were identified as small variant mrsa. computer tomography (ct) revealed multiple mediastinal abscesses. the patient was treated with intravenous linezolid mg bid for days and then switched to oral linezolid mg bid. the oral therapy was pursued for weeks under close surveillance. the patient improved substantially, the purulent discharge disappeared. the mediastinal abscesses were not detected any longer by ct at the end of treatment. the treatment with linezolid was well tolerated. platelets decreased initially but rose to normal values without treatment modification. nosocomial pneumonia due to stenotrophomonas maltophilia in a profound granulocytopenic patient hospitalized for community-acquired staphylococcus aureus severe sepsis or . radulescu a a , sasca n b , lupse m c , tatulescu d c . a university of medicine and pharmac, epidemiology, cluj-napoca, romania , b the teaching hospital of infectious diseases, laboratory, cluj-napoca, romania , c university of medicine and pharmacy, infectious diseases, cluj-napoca, romania objective: to present the diverse opportunistic infections in immunocompromised patients and treatment difficulties. findings: a -year-old women was admitted to the teaching hospital of infectious diseases cluj with a -day history of fever, myalgia, lumbar pain, hemorrhage syndrome. severe sepsis was diagnosed and the conditions that evolved in it were paronychia in a patient with chronic leukemia having prolonged and profound granulocytopenia due to aggressive treatment with ifn. the condition at admission was critical due to trombocytopenia ( b/ platelets/ml) and hemorrhage syndrome. the evolution was favorable under antimicrobial treatment (imipenem), blood and platelet transfusion, intravenous immunoglobulins, granulocyte colony-stimulating factor, antifungal prophylaxis and supportive care. blood and pus cultures revealed mssa. in the th day of hospitalization she developed bronchopneumonia and respiratory failure. the sputum culture was positive for stenotrophomonas maltophilia susceptible to ceftazidime, fluoroquinolones. treatment was unsatisfactory until introducing ticarcilline Á/clavulanate and ciprofloxacine. she had uneventful recovery despite remaining granulocyto-trombocytopenic. conclusions: treatment of infections with emerging agents in immunocompromised patients is difficult, guidance by results of susceptibility testing being misleading with a poor correlation between the tests and treatment outcome. early disseminated listeriosis in a liver transplant recipient (ltr): a rare case due to an in vitro multiresistant strain or . manfredi r, de ruvo n, vivarelli m, bellusci r, montalti r, la barba g, abtueli aden a, cucchetti a, attard l, calza l, cavallari a. university of bologna, infectious diseases, bologna, italy a ltr receiving cyclosporin, azathioprine and steroids, developed an extraordinary episode of sepsis and pleural effusion due to a multiresistant listeria monocytogenes (lm) isolate. a lm strain serov. showing the same, extensive resistance pattern (all penicillins and stand nd-generation cephalosporins), was isolated from multiple blood cultures and pleural fluid weeks after surgery, while stool exam was negative. our p spent her life in countryside and bred some animals, but denied consumption of uncontrolled food. iv cotrimoxazole administration achieved a complete clinical and microbiological cure in days. underlying immunodeficiency may prompt unusual/severe lm infection, but because of its usual community-acquired origin, lm disease remains infrequent in hospitalized p. only seven anecdotal reports of lm infection were described in ltr, Á/ , all but occurring months Á/years after surgery. an early respiratory and systemic infection caused by a community-acquired lm strain which proved resistant to first-choice antibiotics, but had a favorable response to cotrimoxazole (used only once in a ltr with lm sepsis), characterized our episode. an epidemiological survey retrieved the possible source of this usually community-acquired infection. lm should be regarded as an emerging opportunistic pathogen in ltr, and specific risk factors should be seeked. when immunodeficiency is of concern, the unpredictable sensitivity of lm should prompt in vitro assays to adjust antimicrobial therapy problems for discussion hbv Á/hcv and liver carcinogenesis: where does the viral influence end? or . pappas ga. university hospital, internal medicine, ioannina, greece hepatocellular carcinoma (hcc) is a major clinical problem worldwide, usually evolving over a long-standing liver pathology, in the latter stages in the form of cirrhosis. hbv and hcv chronic infection is a common etiology of cirrhosis, and hence, hcc. a number of studies have attempted to clarify the role of these viruses into the progression towards hcc. does their end in the stage of cirrhosis? is progression towards hcc independent of the etiology of cirrhosis (since alcoholic cirrhosis also proceeds to hcc)? do the trials with interferon alfa for patients with hbv or hcv cirrhosis exhibit a favorable result due to the antiviral properties of interferon, or is interferon exhibiting anti-oncogenic potential?, and is hcc cytokine and hormone sensitive (view ongoing trials with somatostatine analogues versus hcc)? (hence, if we treat alcoholic cirrhosis patients with interferon could we have a favorable response?). which patients with hbv or hcv cirrhosis are eligible for interferon treatment?: interferon treatment is a potential hazard for those with thrombocytopenia. how ethical is it to conduct a randomised trial where one leg of cirrhotic patients is left without antiviral therapy? and on the basis of which classification system should the two legs of such a trial be separated? moreover, do viral proteins with oncogenic potential exist (the controversy over the recently discovered hbv protein is still, unresolved)? a major topic awaiting for a major debate. to assess the role of hiv-associated campylobacteriosis (c) according to haart availability, patients with positive culture were identified since . compared with the Â/ hiv-infected p followed in the last decade, no epidemiological differences were shown, save a greater sexual exposure to hiv (p b/ . ). the introduction of haart caused a drop of frequency of c (from . to . episodes per p-year; p b/ . ), and modified clinical features, with disappearance of dissemination and mortality, reported in and patients before (p b/ . ). hiv-related immunodeficiency and disease stage were significantly related to c features before and after haart availability: p b/ . for cd and neutrophil count, p b/ . for aids diagnosis. most cases ( ) were community-acquired, but alimentary or environmental risk factors were never found. ten patients received cotrimoxazole prophylaxis (nine before ; p b/ . ), while no relationship occurred with steroid or antibiotic use, caused cases out of . a % sensitivity was found to quinolones, followed by cephalosporins ( . %), gentamicin ( . %), macrolides ( . %), and cotrimoxazole ( . %). a Á/ -day antimicrobial therapy cured p , but relapses caused by similar strains occurred in patients within Á/ weeks, all in the pre-haart era (p b/ . ). c still occurs in the haart era, probably due to its varied mode of transmission. the frequency of c is greater in hiv-infected patients, but less frequent visceralization, recurrences, and mortality characterized the haart era. objective: to determine the incidence and risk factors for nosocomial viral respiratory infections (nrvi) and involvement of human coronaviruses (hcov) in a neonatal and pediatric intensive care unit. methods: prospective observational study. nasal samples were obtained by cytological brush at admission and weekly thereafter for all hospitalized infants. nasal samples were taken monthly from staff. virological studies were performed, using immunofluorescence for respiratory syncitial virus (rsv), influenza viruses, paramyxoviruses, and adenoviruses; both immunofluorescence and rt-pcr were used for hcov detection. results: during , hcov related nrvi were detected in nn and six in children. three hcov-related outbreaks were observed (february, august and december), associated with a high prevalence of infection in staff. during august outbreak, hcovinfected nrvi were detected over hospitalized infants. seventy-five of hospitalized preterm nn with gestational age under weeks and . % of staff members were infected. risk factors for nrvi in nn were birth weight, gestational age, ventilation, oxygenation and hospitalization length. ninety-two percent of infected preterm nn were symptomatic, mainly with bradycardia and respiratory worsening. conclusions: these data provide additional evidence for a significant role of hcov in nrvi occurring in hospitalized preterm nn. strain typing and screening of dna targets to assess echinococcus sp transmission in new and old geographic endemic foci or . bart jm a , piarroux r a , dia l b , benchikh-elfegoun mc c , vuitton da a , bardonnet k a . a serf, parasitology, besancon, france , b national centre of veterinary study, parasitology, nouakchott, mauritania , c university of mentouri, parasitology, constantine, algeria purpose: cystic echinococcosis is due to echinococcus granulosus. parasite cycles depending on the main intermediate host species involved in different foci have been described promoting mixed infection in the same definitive host. strain typing is a tool to identify the main intermediate host involved via the dogs in the human infection route and to focus the control measures. many dna targets have been used to compare samples and to access the parasite cycle in different countries. but no study has compared the value of each of these targets. eight targets have been tested in mauritania where echinococcosis is an emergent disease, and in algeria where strain typing has never been done. thirty-five cyst samples from human, ovine, camel and bovine have been tested with six nuclear and two mitochondrial targets. results: the two mitochondrial targets and four out the six nuclear targets have allowed to discriminate the different foci. two strains have been found infectious to human : the 'sheep' strain in algeria and the 'camel' strain in mauritania. conclusion: although overlapping geographically sometimes, this raises the question of the respective genetic evolution of the different strains and of their involving in human infection. alveolar echinococcosis in france: an update or . bardonnet k, bresson-hadni s, bartholomot b, gérard a, watelet j, beytout j, saurin jc, piarroux r, vuitton da, who centre collaborating for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the highest prevalence rate for alveolar echinococcosis (ae) in europe has been found in france. in , a french observatory of human ae was done in order to get data that could be used to evaluate presentation, evolution and management of ae. material-methods: french cases were collected for the period Á/ . registration of every case was performed with the subject's agreement. a questionnaire was filled in by referring to the patients' medical files or to practitioners or to patients themselves. completeness of the collection of cases was ensured by multiplying the sources of information. results: two hundred and sixty nine french patients were registered. sex ratio averaged . mean age at diagnostic was . years. . % of diagnosis was performed in 'echinococcosis free' french areas. symptoms, but not always specific liver symptoms, were present at diagnosis in . % of cases. the liver was the main location of lesions in . % of cases. a wide spectrum of management of the patients was observed, accounting for regional differences. conclusion: this french observatory of human ea will facilitate a better management of the disease at the national level. it shows new epidemiological trends, and especially an extension of the endemic area. can coins and paper currency transmit bacillus anthracis ? or . ghenghesh ks. faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya anthrax is an often fatal bacterial infection caused by bacillus anthracis . recent events that began in september in us has gained the organism worldwide attention and heightened awareness of and concern about anthrax. many cases of anthrax with a number of deaths have been reported as a result of contact with envelopes, sent through postal mail, containing b. anthracis endospores. a number of studies have shown that currency is colonized with bacterial organisms, that include enteropathogens (e.g. shigella sp.), other enteric flora (e.g. escherichia coli ) and potential pathogens (e.g. staphylcoccus sp. , pseudomonas sp. and bacillus sp.). furthermore, methicillin-resistant s. aureus (mrsa) isolates that produced enterotoxin (seb) and toxic shock syndrome toxin- also been reported. all of these studies do agree on that currency may be considered as a method of spreading potentially pathogenic and pathogenic bacteria in the community. therefore, currency could also be a vehicle for spreading other highly pathogenic organisms that include b. anthracis . in addition, the introduction of the 'euro' could also allow such bacteria greater freedom to travel across the euro zone. the threat of using currency, particularly paper notes, in spreading lethal organisms should be investigated and proper measures to prevent the use of such a method by terrorists should be implemented. salvage of temporary femoral catheters for haemodialysis using antibiotics in ambulatory patients or . gerasimovska v, oncevski a, dejanov p. department of nephrology, clinical centre, skopje, the former yugoslav republic, macedonia the stay of femoral catheters (fc) for haemodialysis is typically short-term for several days. we used fc as a temporary vascular access (va) for a longer period of time in outpatients going on regular ambulatory haemodialysis, who had a problem with their permanent access. we analysed patients who were discharged from hosptal with fc. duration time of fc was between and days (average . days) with cummulative total of days. the incidence of bacteriaemia was . episodes/ catheter days. in six patients we had signs of infection, so according to our protocol we took blood cultures from peripheral vein, and from catheter (at same time) and started with antibiotic therapy (ab) systemically and locally (ab was 'locked' in catheter) with different duration of time. dominant microorganism was staphylococcus coagulasa negative, and much less staphylococcus aureus , and enterococcus.ab that were frequently used were: cefotaxim, vancomycin and ciprofloxacin. at one of six patients we removed catheter at once without trying to save the catheter. catheter tip was sent for microbiological analysis too. criteria for catheter-related bacteriemia (crb) was found in only one patient, and for possible crb in five patients after we removed the catheters. in the absence of clinical signs of infection, ab treatment was not provided for positive tip culture alone or for positive blood culture of the catheter with negative blood culture from peripheral vein. advances in meningitis education or . holt de a , tait mi b , cavanna al b , worgan-brown s a , hart b a . a the meningitis trust, stroud, uk , b the computer-aided learning unit, school of health science, university of wales, swansea, uk background: meningitis remains an important cause of death worldwide despite improvements in diagnosis, treatment and prevention. clinical and lay awareness of the disease relies on education, however educational delivery has changed and the introduction of material suitable for computer and internet application is now necessary. we have developed educational material on cdrom and on the internet applicable both at tertiary university and secondary school level. application: a computer-aided learning program on cdrom, covering all aspects of meningitis has been produced. it is suitable for undergraduate teaching of healthcare professionals from student nurse and doctors to pharmacists. in order to reach school children in a form acceptable to both pupils and teachers, we have developed a curriculum-linked website. these applications are simple to use and can be incorporated into existing courses of study, so that issues raised can be discussed with tutors and group peers. comment: the introduction of new methods of teaching and learning mean that compatible educational material must be produced. we believe that these applications, focusing on meningitis, are the first of their kind and that they offer tutors the opportunity to progress their teaching of the disease both in methodology and content. brivudin compared to famciclovir for improved therapy of herpes zoster: effects on acute disease and postherpetic neuralgia or . potentially treatment-related adverse events occurred in . % of the brivudin recipients and in . % of the famciclovir recipients (p / . ). conclusions: in zoster patients ]/ years, brivudin )/ mg and famciclovir )/ mg showed equivalent effects on prevalence and duration of phn. brivudin is as effective as famciclovir in stopping viral replication in acute herpes zoster. brivudin offers the advantage of a once daily dosage regimen while being as well tolerated as famciclovir. activity of complexes of pt(ii) and pd(ii) with pyridine- -carbaldehyde thiosemicarbazone (hfotsc) (acta virol., , , ) with selectivity index (si) . times higher than that of acyclovir (acv). in order to evaluate virus specific response and structure Á/activity relationships we continue our investigations with three pt(ii) and three pd(ii) complexes. the activity was evaluated against sensitive to acv hsv (strain bja) and resistant strains r- (hsv ) and pu (hsv ) and compared to that obtained against strain victoria (hsv ) infection. si was indicative for activity. the virus specific response was demonstrated by the fact that viruses sensitive to acv were also sensitive to pt(hfotsc) ]cl , while acv resistant viruses were sensitive to [ptcl(fotsc) ]. the structure Á/activity relationship was proved by the fact that the less active against hsv infection was [pd(fotsc)]. influenza diagnosis, treatment, and the impact of new antivirals on current treatment behaviours during influenza outbreaks or . schaetz l a , sessa a b , a hoffman-la roche f. basel, switzerland , b italian college of general practitioners, italy introduction: annual influenza epidemics severely affect individuals, families, health care systems and society. the availability of new and specific antivirals provides an opportunity for better management of influenza. methods: during the / and / influenza seasons, physicians ( Â/ /country) and public ( Â/ /country) in the usa and europe were interviewed to determine perceptions of influenza and behaviours for its treatment. results: patients recognise influenza illness as severe and identify it by symptoms of fever, muscle aches/pains and cough. physicians use these symptoms to diagnose influenza clinically ( % fever, % muscle aches/pains, % cough); their main treatment objective being to reduce complications. antibiotics for influenza treatment are broadly recommended/prescribed by about % of european physicians, whereas currently available antivirals are only recommended by %. the recommendation of antivirals by us physicians increased from % (season / ) to % ( / ) and markedly decreased antibiotic use (from to %). experience from the two influenza seasons shows that influenza antivirals are only used while the virus is circulating and that the volume of use is proportional to the size of the outbreaks. conclusions: experiences in the usa show that with prompt outbreak information antivirals can be used appropriately in times of influenza activity. influenza treatment with oseltamivir: costs and benefits for the individual as well as for society or . objective: to evaluate the effects of treatment of influenza with antivirals (oseltamivir) on health outcomes and costs to patients and society. methods: based on clinical trial data and data from the literature a simulation model has been developed. the underlying clinical pathway covers morbidity and mortality due to influenza and its specified complications. health outcome data and costs were attached to events in the model. the model compares various scenarios, which are defined by treatment schemes within defined populations and other parameters. application of the model is shown using uk unit cost data simulating an otherwise healthy adult population comparing oseltamivir with usual care. results: early treatment results in reduced morbidity, which translates into faster recovery and return to normal activities ( . days). lower morbidity and mortality make this a cost-effective intervention from a societal perspective. the analysis covers more than different scenarios and the incremental cost effectiveness ratios will be discussed. conclusion: antiviral treatment appears to be effective in terms of health outcome and cost for otherwise healthy adults from the perspectives of both the individual patient and society. however, this effect is very sensitive to time when treatment is started and the accuracy of the diagnosis of influenza. oseltamivir is well tolerated by all patient groups or . thakar b a , dutkowski r b , froelich e c , gilbride j a , ward p a . a roche global development, welwyn, uk , b f hoffman-la roche, nutley, usa , c f hoffman-la roche, basel, switzerland introduction: oral oseltamivir, the ethyl ester pro-drug of a potent inhibitor of influenza virus neuraminidase, is licensed for the treatment and prophylaxis of influenza in the usa. patients and methods: safety data [adverse events, laboratory safety evaluations] derived from clinical trials involving !/ subjects (including Â/ children and Â/ high-risk adults) and healthy volunteers in a large study investigating ecg parameters. spontaneous event reports from medwatch or yellow-card reports following use by Â/ individuals worldwide. an observational case-control study of !/ subjects with influenza-like illness treated with oseltamivir. results: oseltamivir was well tolerated in clinical trials; drug-related side-effects were limited to transient gi effects occurring in / : exposed individuals. these resolved spontaneously and caused drop out in b/ % of treated subjects. no effects on ecg parameters were noted at doses ]/sixfold above the licensed regimen. oseltamivir had no adverse effects on pulmonary function. no additional effects were identified among high-risk adults or children, or following prolonged dosing for prophylaxis. occasional reports of liver dysfunction have been documented post-marketing but causal association has not been established. conclusions: oral oseltamivir is an effective and safe antiviral suitable for influenza management in all patient groups. the decision to stop the vaccination against smallpox and the loss of specific immunity of a high proportion of the population made apocalyptic the perspective of a natural or provoked re-emergence of smallpox. therefore, it is important to improve the current capacities to prevent or to treat the orthopoxvirus infections. uracil dna glycosylase (udg) is one viral enzyme indispensable to the replication of poxviruses. udg of the copenhagen strain of vaccinia virus (vv) was characterized with the aim of defining specific inhibitors susceptible to be used as a new class of active antiviral substances on the viruses of the orthopoxvirus genus. the activity of this enzyme was analysed in real time, in an original method, on a pcr quantitative instrument by digestion of amplified dna revealed by fluorescent intercaled molecules. this technique was used to screen and select several active antiviral substances on udg. moreover, the antiviral activity was estimated by the cytopathic effect of the vv on infected vero cells. the cytotoxicity was determined by inhibition of trypan blue exclusion. the specificity of action of each tested compound was estimated by the selective index ( % cytotoxic dose/ % effective dose). two antiviral compounds were selected for their inhibitory effect on udg activity and on vv replication in vero cell culture : ('/)- -iodo- ?-deoxyuridine and -chlorouracil. these compounds are candidates for the chemotherapy of poxvirus infections. objective: to study the efficacy and tolerance of russian antiviral drugs produced from dna in a limited resources context. results: the drug derinat was produced from salmons' milt. mm of dna was Á/ kda, hyperchrome effect !/ %, protein content b/ . %. the conjugation of the dna with fe '/ resulted in a new drug named ferrovir which influences dna and rna synthesis during early stages of hiv- replication by blocking the virus's action on cells' metabolism and reduces cytomegalovirus titre in fibroblast cells for . Á/ . ig tcid . a protective effect of ferrovir against fatal herpes encephalitis mice was found. the drugs are not toxic. ic !/ mg/ml. ec of ferrovir against hiv- was mg/ml. in limited clinical trials patients received mg of drugs twice daily ( Á/ days). administration was well tolerated and no side effects were observed. derinat in . % cases of herpesvirus infection ( patients) improved the healing and shortened duration of illness. hiv-infected patients ( ) treated with ferrovir showed sustained, elevated cd '/ counts and a significant reduction in hiv- viral load (median . ig). the apparent remission was found in patients with concomitant hiv and herpes virus infection. conclusions: antivirals show good antiviral potency against rnaand dna-viruses; are well tolerated by patients and are useful in case of mixed infections; low price makes them accessible to populations with low financial resources. ortho total hcv core antigen assay can aid early prediction of response in patients treated with interferon/ribavirin or . lunel f a , veillon p b , payan c b . a ahu angers, laboratoire de bacterio-virologie, angers, france , b chu angers, laboratoire de bacterio-virologie, angers, france aim: evaluate the predictive value of total hcv core antigen assay and viral kinetics in patients with chronic hcv. methods: one hundred and twenty two patients infected by genotype , , or pretreatment viral load (bdna . , chiron) !/ meq/ml, with no previous treatment, received mu interferon (ifn) during months (m). ribavirin was given with ifn after months therapy, for months in patients with detectable rna. viral load was expressed as log (ui/ml) and hcv ag as log (pg/ml)/ ). results: pretreatment ag values were correlated with viral load (r / . ). we observed a rapid decrease of ag ( . log pg/ml) and viral load ( . log ui/ml) after m in sustained responders (sr). in patients who relapsed (rr) after ifn alone, the fall was less important ( . log pg/ml, . log ui/ml) during m . in sr and rr to combination therapy, the decrease of ag and viral load at m was, respectively, (ag: . and . log pg/ml; rna: . and . log ui/ml). we did not observed significant variation of ag and viral load in nonresponders. the negative predictive value of hcv rna and ag after m of treatment were %, and positive predictive values were and %. after month of ifn alone, the hcv ag decrease was highly predictive of sr, correlated with rna negativation and early reduction of hcv rna ( !/ log). conclusion: early measurements of total hcv core antigen are useful to predict long-term response to treatment. lamivudine in the treatment of acute hepatitis b or . vincenti a, meini m, luchi s, de gennaro m, ricciardi l, moneta s, scasso a. infectious diseases department, infectious diseases, lucca, italy acute hepatitis b is a self-limiting infection, but in some cases its course may be particularly severe. we report a case of a -years-old woman affected by acute hepatitis b treated with lamivudine. on admission in the hospital the alanino-aminotransferase was u/l, the aspartate-aminotransferase u/l, bilirubin , mg/dl, hbsag, hbcigm and hbeag were positive, hbv dna was . copies/ml. during the following days, the levels of ast and alt gradually rose; on the th day prothrombine time was %, bilirubin mg/dl and the patient developed signs of encephalopathy. four plasmapheresis were practiced without benefit, so the patient was treated with lamivudine, mg/day. after days of therapy, lamivudine was discontinued because of the appearance of diffuse maculopapular rash. at this time the results of liver function tests were normal; after four months hbsag and hbv dna were no longer detectable. in our patient lamivudine prevented an acute hepatic failure. our experience suggests a promising role of lamivudine in the treatment of acute hepatitis b, but how long such therapy have to be practiced and in which patients? prospective, controlled, clinical studies using lamivudine in patients with acute-hepatitis b are necessary. the cost-effectiveness of amantadine versus symptomatic care in the treatment of influenza or . morris s a , carman wf b , barber j c . a city university, london, uk , b west of scotland specialist virology centre, glasgow, uk , c alliance pharmaceuticals, chippenham, uk aim: to assess the cost-effectiveness of amantadine versus best symptomatic care in the treatment of influenza in the uk. methods: we constructed an economic model populated with parameters from the published literature. the model structure is the same as that used in the economic evaluation of zanamivir published by the national institute for clinical excellence in the uk. we conducted a cost-utility analysis (incremental cost per qaly gained) of amantadine versus best symptomatic care. the analyses are conducted for all adults (average-risk group) and the at-risk population (high-risk group), based on the prevalence of influenza over an average season and when the virus is circulating. the perspective is that of the nhs. results: in the average-risk group the incremental cost per qaly gained of amantadine relative to best symptomatic care is uk£ , during an average influenza season and uk£ , when the virus is circulating. for high-risk individuals the figures are uk£ , and uk£ , , respectively. the results are sensitive to the hospitalisation rate. conclusions : if the threshold for cost-effectiveness is £ , per qaly gained amantadine represents value for money in the treatment of influenza in a variety of scenarios, including the baseline for both average-risk and high-risk groups when the virus is circulating. background: surveillance studies all over the world have revealed an extraordinary increase in the prevalence of penicillin resistant streptococcus pneumoniae . the newer quinolones are believed to have broad activity against s. pneumoniae . methods: a total of penicillin resistant clinical strains isolated from patients at hacettepe children's hospital, ankara, turkey between and were tested for their in vitro susceptibility to various antibiotics that are commonly used in the treatment of respiratory tract infections. the minimum inhibitory concentrations (mics) of the penicillin, amoxicillin/clavulanic acid, doxycycline, azithromycin, clarithromycin, ceftriaxone, ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin were determined using the nccls recommended procedure for e -test. results: the range of mics, mic and mic values for all agents tested against the strains are shown in the table. gemifloxacin and moxifloxacin had the highest in-vitro activity among the quinolones tested. all strains tested were susceptible to b/ . mg/ml gemifloxacin, b/ mg/ml moxifloxacin and mg/ml levofloxacin. conclusions: there is some degree of resistance to all the drugs except the newer quinolones which were active against all isolates studied. purpose: stenotrophomonas maltophilia prevalence is growing, mainly in some hospital areas. s. maltophilia is frequently multidrug resistant. fluoroquinolone (fq) resistance varies from one to another study, but in whole resistance is moderate to high. gyra and parc qrdr partial codes have been recently described. we have studied correlations between fq-resistance and mutations in these sequences in s. maltophilia clinical strains. material and methods: gyra and parc qrdr regions from six fqresistant and two fq-susceptible s. maltophilia clinical strains were amplified and sequenced. mics of ciprofloxacin (cfx), gatifloxacin (gfx) and clinafloxacin (cnfx) were determined by the agar dilution method, according guidelines defined by nccls for p. aeruginosa . results and conclusions: mics ranges of cfx, gfx and cnfx for resistant strains were Á/ , Á/ and . Á/ mg/l. susceptible strains had mics of cfx, gfx and cnfx of , . and . Á/ . mg/l, respectively. most susceptible and resistant strains had no significant mutations in the fragments sequenced. only one resistant strain (mic of cfx mg/l) and one susceptible strain (mic of cfx mg/l) had a significant gyra mutation, the same in both strains (ile Á/val). thus, fq resistance in s. maltophilia shall derive from changes in other areas in the topoisomerases or probably from other mechanisms of resistance, such as efflux pumps. purpose: corynebacterium urealyticum is the cause of encrusted cystitis and other inespecific utis and systemic infections. it is frequently multi-drug resistant, with a high rate of resistance to fluoroquinolones (fq). the mechanisms of resistance to fqs have not been described in c. urealyticum . we describe the c. urealyticum parc gene qrdr region and its relationship with quinolone resistance. materials and methods: the activity of ciprofloxacin (cfx), levofloxacin (lfx), gatifloxacin (gfx), clinafloxacin (cnfx) and moxifloxacin (mfx) against c. urealyticum clinical strains was determined following nccls guidelines for enterococci. we amplified and sequenced their parc qrdr by standard methods. results and conclusions: five strains ( . %) were cfx-susceptible (mic . Á/ . mg/l), had mics Á/ mg/l and ( . %) were highlevel cfx-resistant (mic Á/ mg/l). cnfx was -fold more active than cfx. mfx and gfx had mics of and mg/l. all the strains, including the type strain, showed a c to t change at the position referred to wild type s. aureus parc gene, leading to a ser- -phe change, described as the main parc change in fq-resistant s. aureus . this finding suggest that this mutant sequence, as compared with parc sequences from other grampositives, might be the wild-type for this species, and might explain in part its high resistance rate, and its apparent lightness to develop high-level resistance. purpose: during routine surveillance, we identified ciprofloxacinresistant (mic!/ mg/l) pneumococcal isolates and compared clinical details and resistance patterns. results: they were isolated from sputa ( ) and blood cultures ( ) from adults, most with heart or lung disease. hospital admissions were common; half had been inpatients in the previous months. nineteen patients received quinolones in the preceding months, in part reflecting the local policy (introduced in ) of penicillin and ofloxacin for first line treatment of pneumonia. thirteen patients had radiological signs of pneumonia and were pyrexial with raised inflammatory markers. agar dilution mics for quinolones, including norfloxacin with and without reserpine, penicillin and erythromycin were performed. an increase in norfloxacin mics was noted over the period ( Á/ mg/l) to ( mg/l). fluoroquinolone efflux was suggested in three isolates. resistance to moxifloxacin (mic Á/ mg/l) was noted from onwards. all isolates were serotype v and resistant to penicillin (mic!/ . mg/l). thirty-one were resistant to erythromycin (mic!/ mg/l). conclusion: the policy of using quinolones may have contributed to the development of quinolone resistance and this cluster of isolates. the increasing levels of quinolone resistance observed raise concerns about the future use of newer quinolones for the treatment of respiratory infections. s. maltophilia has emerged in the last years as an important nosocomial pathogen, inherently resistant to most of the antimicrobial agents. new quinolones has been proposed as a treatment of choice because their enhanced activity, but several parameters (t a , atmosphere, method) can affect the results of mics. methods: we have performed mics using two different methods (agar dilution and microdilution) and different conditions: and c of temperature; atmosphere of o and co , and incubation times of , and h. a total of strains were assayed with nine quinolones following standard nccls. comparisons were made between results with Á/ and Á/ h using the x -test (a / . ). results: no differences were found between Á/ and Á/ h results with agar dilution, except with atbs in the case of mics at c co . on the contrary, almost all the atbs showed significant differences in the results with and h using microdilution method, at any condition of ta or atmosphere. comparison of mics (p values, significance level / %) with incubation times of and h at different procedure conditions. the incubation time is a parameter that seems to affect significantly the results of mics of quinolones when microdilution method is used, whereas only few differences can be encountered with the agar dilution method. results: breakpoints were used as proposed by nccls . during the study period the pneumococci resistance was noted as follows: % to pc, % to em and % to sxt. the rank order of activity of the five fqs against multi-drug resistant pneumococci was: cip (mic : mg/l), ofx (mic : mg/l), lvx (mic : mg/l), grx (mic : . mg/l), tvx (mic : . mg/l). conclusions: in romania, fluoroquinolones represent alternative treatment to beta-lactams and macrolides for first-line empirical treatment for respiratory tract infections caused by pneumococci but, continued vigilance for emerging resistance to fqs is further indicated. introduction and material/methods: susceptibility testing (semiautomated broth microdilution method, sensititre, trek diagnostics, usa, following nccls recommendations) was performed with six different quinolones to streptococcus pneumoniae isolates with a ciprofloxacin-cip */mic ]/ mg/l collected in two consecutive sauce$ surveillances in spain ( Á/ / Á/ ). nccls resistance (r) breakpoints were used ( ]/ for ofloxacin-ofl and levofloxacin-lev; ]/ for sparfloxacin-spa; ]/ for gatifloxacin-gat */and moxifloxacin-mox), but for gemifloxacin-gem */where ]/ was used. results were as follows. conclusions: for cip-r isolates gem and mox were the most active agents. gem was the only agent not influenced by cip mic increase regarding prevalence of r , with % resistance for strains with cip mic ]/ mg/l. $sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en españ a' (susceptibility to the antimicrobials commonly used in the community in spain) and is the spanish word for the willow tree. in vitro activity of gatifloxacin and seven other antibiotics against respiratory and urinary tract pathogens from the community. first results of the basic */study ps grimm h, on behalf of a european multicenter study group, institute for med. microbiology, weingarten, germany a total of centers in austria, belgium, france, germany, italy, portugal, spain and switzerland are involved in the basic study (bacterial annual susceptibility information collection). the mics of gatifloxacin (gati), ciprofloxacin (cipro), clarithromycin (clari), benzylpenicillin g (pen), amoxicillin (amox), amoxicillin/clavulanic acid (augm), cefuroxime (cur) and cefixime (cix) were determined using the microdilution method. each center is requested to investigate strains each of the following species: s. pneumoniae (spn), s. pyogenes (spy), s. aureus (sau), e. faecalis (efa), m. catarrhalis (mca), h. influenzae (hin), e. coli (eco), k. pneumoniae (kpn), p. mirabilis (pmi) and p. aeruginosa (pae). so far approximately strains are enrolled. some important mic %/percentage resistance were as follows: from the oral antibiotics tested gatifloxacin has the highest activity and broadest spectrum against all relevant respiratory and urinary tract pathogens. gatifloxacin is a promising alternative for therapy of respiratory tract bacterial infections. in vitro activity of gatifloxacin against bordetella pertussis in comparison with erythromycin, ciprofloxacin and levofloxacin ps bourgeois n, pangon b, ghnassia jc, doucet-populaire f, de versailles ch. microbiologie, le chesnay, france purpose of the study: bordetella pertussis infections are far more common in adults and adolescents than is generally estimated. however, they are often not recognised. infected or colonised adults can act as a reservoir of infection, passing it to children. fluoroquinolones are currently recommended for the treatment of respiratory tract infection in adult patients, which is usually empirical. gatifloxacin is a novel -methoxyquinolone, with a potent activity against both gram-negative and -positive bacteria. the in vitro activity of gatifloxacin was compared with those of erythromycin, the drug of choice for both treatment and prophylaxis of pertussis, ciprofloxacin and levofloxacin, against clinical isolates strains of b. pertussis including erythromycin resistant strains. results: we used the agar dilution method on mueller Á/hinton medium supplemented with % horse blood to determine the mic of each antibiotic. gatifloxacin (mic , . mg/l) was as active as ciprofloxacin and levofloxacin (mic , . mg/l) against both sensitive erythromycin (mic , . mg/l) and resistant erythromycin (mic , !/ mg/l) strains. conclusion: gatifloxacin may be an effective drug in the treatment or prophylaxis of adults with suspected or confirmed pertussis. ex vivo serum activity (killing rates) after gemifloxacin mg versus trovafloxacin mg single doses against ciprofloxacin-susceptible andresistant streptococcus pneumoniae ps calvo a a , giménez mj b , alou l a , gó mez-lus ml a , aguilar l b , prieto j a . a microbiology department, universidad complutense, madrid, spain , b glaxosmithkline, medical department, tres cantos, madrid, spain serum bactericidal activity was measured ex vivo after single dose administration of gemifloxacin (gem) mg and trovafloxacin (tro) mg to healthy volunteers in a randomized, cross-over phase i trial. blood samples were collected h (cmax) after dosing and serum killing rates were determined against a serotype penicillin (pen) Á/ciprofloxacin (cip) susceptible strain (s ) (mics of . , , . and . mg/l for pen, cip, gem and tro) and a serotype pen Á/cip resistant strain (s ) (mics of , , . and . mg/l for pen, cip, gem and tro). tubes with . ml of serum sample and . ml broth ( % todd Á/hewitt'/ % hbss) were incubated over h at c. final inocula was cfu/ml. mean colony counts for samples and controls (k ) are shown in the figure: gem exhibited higher colony counting decrease of the initial inocula, versus tro, for both strains. after h incubation, the initial inocula decrease obtained with tro and the cip susceptible strain was similar to that obtained with gem and the resistant strain, showing a lower influence of cip mic increase in the ex vivo bactericidal activity of gem versus tro. urine bactericidal activity after administration of gemifloxacin and trovafloxacin single doses in a phase i study ps garcía-calvo g a , parra a a , giménez mj b , ponte c a , aguilar l b , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain urine bactericidal activity after o.d. administration of gemifloxacin (gem) mg and trovafloxacin (tro) mg, was assessed in six adult males in a cross-over phase i trial. urine killing rates (ukr) against escherichia coli atcc (mic mg/l of . and . for gem and tro) and s. saprophyticus atcc (mic mg/l . and . for gem and tro) were performed with samples collected at , Á/ , Á/ and Á/ h. a . ml of iso-sensitest broth and . ml of bacterial logarithmic growth were added to ml sample, giving a final inoculum of cfu/ml. colony counting was performed after , , and h incubation. percentages of initial inocula reduction (iir) were calculated. mean urine concentrations measured by bioassay were (mg/l): . , . and . for gem, and . , . and . for tro. against e. coli , an iir of . % was obtained after h incubation with all samples except with tro at Á/ h. against s. saprophyticus an iir of % was obtained after h incubation with all samples except with tro at Á/ h, where bacterial regrowth was found. the maintenance over h of gem urine antibacterial activity suggests its efficacy in the treatment of uncomplicated cystitis. influence of the decreased susceptibility to ciprofloxacin on gemifloxacin versus levofloxacin efficacy in experimental pneumococcal pneumonia in guinea pigs ps garcia-olmos m a , parra a a , gimenez mj b , garcia-calvo g a , ponte c a , aguilar l b , soriano f a . a fundacion jimenez diaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain the efficacy of ciprofloxacin (cip), levofloxacin (lev) and gemifloxacin (gem) in the treatment of pneumococcal pneumonia was assessed in a guinea pig model using three strains (s) with mics (mg/l) of , and . (s ), , and . (s ) and , and (s ) for cip, lev and gem, respectively. intraperitoneal treatments started h after s. pneumoniae intratracheal inoculation, and continued t.i.d up to four doses. ten animals were included in each group. doses (mg/kg) used were , and for cip, lev and gem, respectively, in order to mimic auc - h and cmax obtained in humans after standard doses. animals that survived h after inoculation were sacrificed and colony counts were performed in lungs ( the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of nosocomial pneumonia. our aim was to study the pharmacokinetic Á/pharmacodynamic appropriateness of lfx mg iv bid in the treatment of six inpatients with ventilator-associated pneumonia (vap) ( / years; m Á/ f; / kg). blood and urine samples were collected in steadystate conditions at appropriate intervals. lfx concentrations were analysed by hplc. the aetiological agent was identified in all the cases and its in vitro sensitivity to lfx was always assessed. the results obtained mean values ( /sd) of the major pharmacokinetic parameters were: cmax, . / . mg/ml; vdss, . / . l/kg; t / b, . / . h; cl, . / . ml/min/kg; auc -t, . / . mg/ml h. cumulative urinary excretion was . / . %, confirming that lfx clearance is mainly renal. clinical cure and microbiological eradication were obtained in all the patients after a Á/ day therapy. a suprainfection due to acinetobacter anitratus insensitive to lfx occured in case. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/ ; auc/mic !/ ) in all the cases. the conclusion reached the findings suggest that lfx mg bid iv may be considered effective in the treatment of vap caused by sensitive bacteria. comparative pharmacokinetics of levofloxacin in patients with lower respiratory tract infections (lrti) being treated with sequential therapy ps pea f a , brollo l a , lugatti e b , di qual e a , dolcet f b , talmassons g b , furlanut m a . a institute of clinical pharmacology and toxicology, dpmsc, university of udine, udine, italy , b division of pneumology, sm misericordia hospital, udine, italy the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of lrti. our aim was to study the pharmacokinetic Á/pharmacodynamic appropriateness of a standard switch lfx iv/os regimen ( mg iv od for Á/ days followed by mg os od for Á/ days) in the treatment of seven inpatients with lrti ( / years; m Á/ f; / kg). blood samples were collected in steady-state conditions at appropriate intervals. lfx plasma concentrations were analysed by hplc. the aetiological agent was identified in / cases and its in vitro sensitivity to lfx was assessed. the results obtained absolute oral bioavailability was . / . , with a cmax of . / . vs . / . mg/ml after iv and oral administration, respectively. no significant difference in the main pharmacokinetic parameters was observed between the two routes. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/ ; auc/mic !/ ) in the two assessable cases. all the patients were clinically cured after a Á/ day therapy. the conclusion reached the ad interim findings show that lfx mg od may guarantee per os an exposure similar to that achievable after iv administration, suggesting that sequential therapy may be considered effective in the treatment of lrti. levofloxacine in the exacerbations of copd due to pseudomonas ae ps micheletto c, tognella s, pomari c, dal negro r, ospedale orlandi, div.pneumologia, bussolengo, italy development of antibiotic resistance in bacteria is a problem of great concern. gram-negative bacteria, including multidrug-resistant (mdr) pseudomonas aeruginosa (ps), are responsible for a significant proportion of episodes of copd exacerbations, particularly in elderly ( ). aim was to check the susceptibility to common antimicrobial treatments against ps strains isolated from bronchial secretions in patients with severe exacerbations of copd. methods: microbial investigations were conducted on specimens: spontaneous purulent sputum ( . %), and tracheobronchial aspirates ( . %, collected with a protected specimen brush). results: fifty-seven ps pathogen strains ( cfu) were identified ( . %) and tested over a -month period: ps. aeruginosa . %; ps. putida . %; ps. fluorescens . %, and burkholderia cepacia . %. the assessed susceptibility to most common antibiotics was: levofloxacine ( %), ciprofloxacine ( %), ipenem cil. ( %), ceftazidime ( %); amikacin ( %), and piperacillin'/tazobactam ( %). a much lower susceptibility was found for ticarcillin Á/clavulanic acid ( %), gentamicin ( %), and netilmicin ( %). ( ) at present, levofloxacine proves the most effective antimicrobic option for treating copd exacerbations due to ps infection; ( ) a much more efficient policy of antibiotic prescribing should be promoted in order to prevent the selection of resistant strains in these cases. these results confirm the excellent 'in vitro' activity of levofloxacin against nosocomial gram-negative pathogens, including the esbl producing strains ( % of escherichia coli , e. cloacae and k. pneumoniae were inhibited at . mg/l). levofloxacin was more rapid than ciprofloxacin to determine a bactericidal effect particularly against s. maltophilia . moreover, considering the favourable pk/pd profile, levofloxacin can represent a valid therapeutic option for the treatment of severe gram-negative nosocomial infections. moretti f, quiros-roldan e, casari s, chiodera a, viale p, carosi g. university of brescia, institute of infectious and tropical diseases, brescia, italy a -year-old man, ivdu, hiv positive was attended in aur hospital for fever and toracic pain. a x-chest radiography revealed a round lesion of cm near the lingula with central hyper-diaphan area. lymphocytes cd '/ count was cells/mm and viral load . cp/ ml. hospital stay rhodococcus equi was found in cultures of peripheral blood, faecal and sputum specimens. antibiotic treatment with oral rifampin ( mg/qd) and with intravenous imipenem ( mg tid) was started. due to the persisting fever, immodificated radiography and negativity for p. carinii , mycobacteria and bacteria in bal coltures, imipenem was substituted by parenteral vancomycin ( mg bid). after days, because of persisting fever and increase of the diameter of the lung lesion ( cm) vancomycin was sustituted by oral levofloxacyn ( mg bid), continuing rifampin. after a days course of levofloxacyn therapy the fever remitted. the patient was discharged with levofloxacyn ( mg bid) and rifampin and, after months of follow-up, a radiological control pointed-out a remarkable resolution in the lung lesion. we may suppose that levofloxacyn can be effective for the treatment of r. equi infection, even if more studies (particularly controlled studies) are necessary. liberti a a , izzo b a , loiacono l b , calabria g a , patarino t a , izzo e a . a ii department, naples, d. cotugno hospital, italy , b iii department, naples, d. cotugno hospital, italy the increasing prevalence of salmonella typhi strains with reduced susceptibility of chloramphenicol had prompted the search for other antibiotics with the same efficacy.quinolones are a class of antibiotic with an activity in vitro and in vivo against enteropathogenes. we inwestigated the use of levoxacin in two regimens of treatment of typhoid and paratyphoid infection. patients and methods: thirty-two adult patients were incluted in this study from september to april ; patients had positive culture for s. typhi and six had positive cultures for s. paratyphi . all isolated were fully susceptible to levoxacin. we compared treatment with levoxacin for days, mgr b.i.d. (group , patients), with treatment for days, mgr b.i.d. (group , patients). clinical cure was defined as defervescenze of fever by day of treatment, with an absence of complications and no clinical relapse during the followup. results and conclusion: the clinical cure rate was % ( patients) for group and % ( patients) for group ; the difference in these rates was not statistically significant. the blood cultures of all patients were sterile by day of treatment and remained so until the th month of follow-up, no subjects had clinical or microbiological relapse and all stool cultures remained negative, also. the two regimens of treatment was good tollerated and no adverse event was registered; it was concluded that levoxacin treatment for days in enteric fever is not necessary the mulidrug-resistence of s. typhi led to the use of quinolones as the first-line drug in the treatment of enteric fever. pefloxacin in the treatment of patient with acute infectious diarrhoea ps troselj-vukiae tvb a , strahinja v b , poljak i a , stojanoviae d c , nikoliae n d . a department of infectious disease, university hospital center, rijeka, croatia , b glaxosmithkline, marketing, rijeka, croatia , c dept. rijeka, institute of public health, rijeka, croatia , d dept. rijeka, maritime academy, rijeka, croatia the purpose of the study was to investigate clinical and bacteriological efficiency in and days pefloxacin treatment and to compare it with symptomatic therapy. the results obtained in patients treated with pefloxacin the therapy was clinically effective already in the third day while in the control group this happend in the th day. bacteriological eradication was noted in patients ( %) of the first and patients ( %) of second group in the th days of the treatment. they all had negative cultures and weeks after pefloxacin protocols were completed. only patients ( %) in control group had negative stool cultures in the th day of the treatment and all of them weeks after it ended. there was no statistically significant difference in clinical (p / . ) and bacteriological (p / . ) efficiency between and days pefloxacin treatment protocols. both protocols significantly differed in clinical (p b/ . ) and bacteriological (p / . ) eradication from the control group. the conclusion reached is that the efficiency of pefloxacin (quinolones) in the treatment of acute infectiuos diarrhoea and justifies their use in the more severe forms of the disease. dalhoff a a , ullmann u b , schubert s b . a bayer ag, wuppertal, germany , b university of kiel, institute of med. microbiology, kiel, germany background: the antibacterial activity of moxifloxacin (mxf) was compared to levofloxacin (lev), amoxicillin (amx), clarithromycin (cla) and erythromycin (ery) in an in vitro model. method: pharmacokinetics in bronchial mucosa (bm) and serum (s) following single oral doses of mg mxf or cla and mg lev, amx or ery were simulated using a one compartment model. bacteria tested staphylococcus aureus (nos. , ), streptococcus pneumoniae (nos. , ). aliquots were taken ( Á/ h) and plated on to brain heart infusion agar for enumeration. results: s. pneumoniae was eliminated by all agents studied. significant differences were apparent with s. pneumoniae and the s. aureus strains: objective: to compare the safety and efficacy of once-daily moxifloxacin with once-daily ceftriaxone in the treatment of cap in hiv-infected patients (pts). methods and results: in a retrospective survey, oral moxifloxacin ( mg daily)/ Á/ days) was compared to standard regimen of i.v. ceftriaxone ( g daily)/ Á/ days) for treatment of cap in hiv'/ pts. adults pts with clinical signs and symptoms of cap and consistent chest x-ray findings were included. pts had a median age of years (range Á/ and % were male). demographic characteristics were similar in both treatment groups; pts received mxifloxacin and pts ceftriaxone. clinical success rates were % for moxifloxacin and % for ceftriaxone. at a post-study evaluation approximately weeks later, moxifloxacin-treated pts and ceftriaxone-treated pts had relapsed. the adverse events reported were comparable for both treatment groups. there were four-related adverse events ( gi, headache) for moxifloxacin-treated and ( gi, skin) for ceftriaxonetreated pts. conclusion: the results of this study show that moxifloxacin as oral therapy is as effective and well tolerated as i.v. ceftriaxone in the treatment of hiv'/ pts with cap. therapy with moxifloxacin was not associated with any significant clinical or laboratory abnormalities. these data suggest that once-daily oral administration of moxifloxacin is potentially convenient and cost-effective alternative therapy for cap in pts with hiv infection. moxifloxacin in the treatment of acute maxillary sinusitis after first-line therapy failure or acute sinusitis with high risk of complications ps gehanno p a , berche p b , perrin a b , arvis p c . a ent department, bichat claude bernard hospital, paris, france , b microbiology department, necker enfants malades hospital, paris, france , c bayer pharma, medical affairs department, paris, france the efficacy and safety of moxifloxacin (mxf) mg once daily for days was evaluated in the treatment of acute maxillary sinusitis after first-line therapy failure, or acute sinusitis with high risk of complications. in this prospective, multicenter study, a total of patients with acute bacterial sinusitis confirmed by sinus x-ray were valid for efficacy analysis: one hundred and seventy five patients ( . %) had an acute maxillary sinusitis which failed to respond to a previous antibiotic therapy given for a mean duration of . days, and ( . %) had an acute sinusitis with high risk of complications (frontal: , pan-sinusitis: and sphenoid: ). ninety two patients ( . %) were microbiologically valid. clinical cure and continued clinical cure rates at Á/ and Á/ days post-therapy were . and . %, respectively. clinical cure rates at Á/ days post-therapy were . and . % in sinusitis after first-line therapy failure and in sinusitis with high risk of complications, respectively. bacteriological eradication rate during therapy (day Á/ ) was . %. at Á/ days post-therapy. bacteriological success rates were . % in patients who failed to respond to a previous antibiotic and . % of patients who had sinusitis with high risk of complications. . % of patients experienced drug-related adverse events, abdominal pain ( . %), nausea ( . %) being the most frequently reported events. mxf was rapidly effective and a well-tolerated treatment for this kind of infection. neisseria gonorrhoeae with decreased susceptibility to penicillin and ciprofloxacin: novel mutation patterns in the gyr a and par c genes of ciprofloxacin resistant isolates and plasmid profile of penicillin resistant isolates of n. gonorrhoeae in india (delhi) ps chaudhry u, saluja d. dr. b. r. ambedkar center for biomedical research, university of delhi, delhi, india commercial sex workers (csws) serve as the most important reservoir of gonorrhoea. periodic monitoring of the antimicrobial susceptibility profile of neisseria gonorrhoeae in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. in india, such a surveillance of the in vitro antimicrobial susceptibility of n. gonorrhoeae was established in . significant increasing trend of penicillin and ciprofloxacin resistance with high mic of Á/ and Á/ mg/ml, respectively were found over the years ( Á/ ) . the molecular basis of ciprofloxacin resistance, i.e. mutations in the gyr a and the par c genes of isolates, were analyzed. four isolates (with an mic of mg/ml for ciprofloxacin) harbored triple mutations (ser- to phe, asp- to asn and val- to leu) in the gyr a gene. the third mutation of val- to leu, lies downstream of the quinolone resistance determining region of the gyr a and has not been described before in gonococcus. in addition, these isolates had a phe- to tyr substitution in the par c, a hitherto unknown mutation. the alterations in the par c gene were seen in these isolates only in the presence of changes in the gyr a gene and comprised amino acid changes at codons , , , , and . the presence of b-lactamase plasmid among the penicillinresistant isolates was determined by their plasmid profiles and further confirmation was carried out by a pcr based protocol. our findings suggest that emergence of penicillin and ciprofloxacin resistance in n. gonorrhoeae isolates from a major std center in india, indicates the need for the increased awareness and prudent use of antimicrobials. in vitro activity of newer antibiotics against methicillin-resistant staphylococcus aureus ps gutierrez zufiaurre mn, sanchez hernandez j, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: mrsa are frequently co-resistant to a number of structurally unrelated antibiotics. more than % mrsa are resistant to gentamycin, ciprofloxacin, macrolides and clindamycin. newer antibiotics active against multi-drug resistant grampositives have been developed. we have tested the in vitro activity of newer antibiotics against genetically-characterized, high level ciprofloxacin resistant mrsa. material and methods: thirty-six ciprofloxacin-resistant, gyra/grla mutant mrsa clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), gatifloxacin (gfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method, according nccls guidelines. results and conclusions: all the strains were resistant to cfx, , % were lfx-susceptible and . % were gfx-susceptible. nevertheless, mics of lfx and gfx for all susceptible strains was in the highest extreme of the susceptibility range. mfx was the most active quinolone. almost all the strains were high-level er-resistant with constitutive mlsb phenotype. tl did not improve significantly its behaviour, although it was -fold as active as er against the only er-susceptible strain. va, lin and syn had excellent activity against all the strains. they showed a very homogeneous behaviour against all the strains that were included in a range of Á/ mg/l of lin and va and . Á/ mg/l. sanchez hernandez j, gutierrez zufiaurre mn, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: corynebacterium urealyticum is the etiologic agent of encrusted cystitis and inespecific utis, and can be also involved in systemic infections. c. urealyticum is frequently multi-drug resistant, so only glycopeptide antibiotics and tetracyclines have high susceptibility rates, while fluoroquinolones resistance rates vary significantly. we have tested the in vitro activity of linezolid, telithromycin, synercid and newer fluoroquinolones against multi-drug resistant c. urealyticum clinical strains. material and methods: sixty-four c. urealyticum clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method according guidelines defined by the nccls for enterococci. results and conclusions: results confirm the high resistance rate to older fluoroquinolones and macrolides, with !/ % cfx resistance and % er-resistance. lfx was more active (mic mg/ml). mfx was the most active fluoroquinolones (mic mg/ml). tl improve its behaviour in respect to er (range . Á/ mg/ml). va, lin and syn had excellent antimicrobial activity. no resistant strains were found. mic were , . and mg/ml, respectively. mics were similar for all the strains independently of their resistance to other antibiotics plasma concentrations, urinary excretion and bactericidal activity of linezolid ( mg) versus ciprofloxacin ( mg) in healthy volunteers after a single oral dose ps wagenlehner fme a , wydra s a , onda h a , kinzig-schippers m b , sö rgel f b , naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, (ibmp), nürnberg-heroldsberg, germany purpose of the study: in a randomized cross-over study volunteers received a single oral dose of mg linezolid versus mg ciprofloxacin to assess plasma concentrations (up to h), urinary excretion (by hplc), and urinary bactericidal titers (ubt) up to h. the mean maximum plasma concentration of linezolid was . mg/ ml and of ciprofloxacin . mg/ml. the cumulative renal excretion (mean) of parent drug was %/ % for linezolid/ciprofloxacin. ubts were determined for a reference strain and five gram-positive clinical uropathogens with the following mics (mg/ml) for linezolid/ciprofloxacin: s. aureus atcc ( / . ), s. aureus (mssa) ( / ), s. aureus (mrsa) ( / ), s. saprophyticus (msse) ( / . ), e. faecalis ( / ), e. faecium ( / ). results: median ubts measured within the first h for linezolid were : for enterococcal strains and : to : for the four staphylococal strains. median ubts for ciprofloxacin were : for the two enterococcal strains, : to : for the two ciprofloxacin susceptible, : . for the two resistant staphylococcal strains. areas under the ubt Á/time-curve showed statistically significant differences only for the two ciprofloxacin resistant staphylococcal strains in favour of linezolid. conclusion: linezolid exhibited the same bactericidal activity against ciprofloxacin resistant as susceptible strains. linezolid should be tested for treatment of complicated uti due to gram-positive uropathogens in a clinical trial. whitehouse t a , cepeda ja a , tobin purpose: we performed pharmacokinetics within a double-blind, randomised trial comparing linezolid and teicoplanin in intensive care patients with known or suspected gram-positive infection. they received either mg linezolid intravenously -hourly, or mg teicoplanin -hourly for the first three doses and once daily thereafter (or every rd day if renally impaired). blood samples were collected to create serum pharmacokinetic profiles. linezolid was quantitated by hplc and teicoplanin by fluorescence polarization immunoassay. results: twenty two patients were studied in the linezolid group (m Á/f : , mean age years [range Á/ years]). median treatment duration was . days (range Á/ ). eighteen patients were treated with teicoplanin (m Á/f : , mean age years [range Á/ ]) for median days (range Á/ ). steady state peak concentrations (mean / sd) for linezolid and teicoplanin were . / . and . / . mg/l, respectively. trough concentrations at day were . / . mg/l for linezolid and . / . mg/l for teicoplanin. recommended breakpoints of staphylococcus aureus are mg/l for linezolid and mg/l for teicoplanin. accumulation occurred in one -year-old linezolidtreated patient with impaired renal function. conclusion: current recommended dosing regimens for linezolid and teicoplanin are generally appropriate in the critically ill, though a more detailed analysis is required. stamos g, lebessi e, ioannidou s, paleologou n, kallergi k, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of the study was to investigate the susceptibility of methicillin resistant staphylococcus aureus (mrsa) isolates from a -bed paediatric hospital to quinupristin/dalfopristin (q/d, streptogramin combination) and linezolid (lzd, oxazolidinone). material: we performed a retrospective analysis of mrsa strains, isolated from patients hospitalized in miscellaneous medical departments [neonatal unit ( ) , surgical wards ( ), orthopaedic wards ( ), oncology unit ( ), other wards ( ) and outpatient clinic ( )], during a -year period ( Á/ ) . the sources of isolation were pus ( ), throat ( ), nasal ( ), bronchial ( ), skin ( ), stool ( ) ear ( ) and other ( ) specimens. all isolates were sensitive to glycopeptides, while . % were resistant to gentamicin and . % to erythromycin. methods: the sensitivity testing was performed by a disk diffusion method (bbl sensitivity disks, becton dickinson), according to nccls guidelines. the breakpoint zone diameters for lzd and q/ d were ]/ and ]/ mm for susceptibility and / and / mm for resistance, respectively. results: all isolates were proved to be susceptible to both antibiotics. the mean inhibition zones were . mm for lzd and mm for q/d. conclusions: lzd and q/d are very promising antimicrobial agents, showing excellent activity against mrsa clinical isolates. the prudent therapeutic use is strongly recommended to avoid the emergence of resistance. in vitro activity of streptogramins and oxazolidinones against streptococcus pneumoniae clinical isolates ps stamos g, lebessi e, paleologou n, psatha m, sanida p, zaphiropoulou a, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of this study was to evaluate the in vitro activity of linezolid (lzd), a member of oxazolidinones and the streptogramin combination quinupristin/dalfopristin (q/d) against clinical isolates of streptococcus pneumoniae from a tertiary care paediatric hospital. material: a total of pneumococcal isolates exhibiting reduced susceptibility to common antibiotics were included in the study. the strains were isolated from middle ear fluid ( ), eye ( ), nasal ( ) blood ( ) and other ( ) cultures during the last years. the percentages of the isolates that were resistant to penicillin, erythromycin, cotrimoxazole and clindamycin were . , . , . and . %, respectively. methods: the susceptibility testing was performed by a standard disk diffusion method (bbl sensitivity disks, becton dickinson). in case of marginal results or intermediate sensitivity to quinupristin/ dalfopristin, the mic was determined using the e -test method (ab biodisk). results: all isolates were found to be sensitive to lzd. q/d was very active as well, except for two isolates that exhibited intermediate susceptibility, showing cross-resistance to macrolides and clindamycin, as well. conclusions: the new antimicrobial agents show excellent activity against resistant to common antimicrobials pneumococcal isolates, but the clinical use is not suggested, unless no other therapeutic solutions are available. linezolid is a new oxazolidinone with excellent activity against gram-positive organisms including glycopeptide-resistant strains of staphylococci and enterococci. in icu linezolid has been used for the treatment of severe gram-positive infections in a control trial. the susceptibility pattern of all gram-positive isolates from icu patients has been studied. methods: a total of specimens from icu patients were processed, were from patients enrolled in the antibiotic trial. all methicillin-resistant staphylococcus aureus (mrsa), coagulase-negative staphylococci (cons), enterococcus sp and methicillin-sensitive staphylococcus aureus (mssa) were tested. the break point for linezolid was mg/l and for teicoplanin mg/l. isolates were tested for susceptibility by e -test. results: linezolid ( isolates) mic / (mg/l) were as follows: mrsa (n / ) . / , cons (n / ) . / . , enterococcus sp (n / ) . / . , mssa (n / ) . / . teicoplanin ( isolates) mic / (mg/l) mrsa (n / ) . / . , cons (n / ) / . , enterococcus sp (n / ) . / . , mssa (n / ) . / . . all grampositive isolates were inhibited by concentrations of linezolid below the breakpoint including eight strains of staphylococci resistant to teicoplanin. conclusions: linezolid was highly active against grampositive isolates. resistance to teicoplanin was similar to other reported series. there was no emergence of resistance to linezolid. mrsa colonization is often a limiting factor for discharge from icu. clearance of mrsa is seldom achieved with conventional glycopeptide treatment. the oxazolidinone, linezolid, has excellent soft tissue and respiratory tract penetration and might be expected to eradicate carriage in some patients. we recently performed a doubleblind randomized trial in icu patients with known/suspected gram positive infection. received intravenous linezolid, mg b.d., and patients received teicoplanin mg o.d. mrsa clearance was assessed at end of treatment (eot), at -and days follow-up. results: in the linezolid and teicoplanin groups, and were known to be colonized with mrsa at study entry, respectively, while and were not. detection of clearance of mrsa colonization at eot was % for linezolid vs % for teicoplanin group (x b p b/ . ), at days it was % vs % (x b p b/ . ), and at days % vs % (ns). conclusion: short-term mrsa clearance can be achieved in significantly more patients treated with linezolid however this was not maintained at days, either because of incomplete initial eradication or recolonization. further analysis will follow when molecular typing of the isolates is completed. penetration of linezolid into bone, fat and muscle during hip arthroplasty ps lovering am, bannister gc, zhang j, macgowan ap, southmead hospital, bcare, bristol, uk there are limited data describing the concentrations and penetration of linezolid (lzd) into tissues, such as bone, that can be used to guide therapy for non-vascular infections. here we report the concentrations and penetration of lzd for bone, fat and muscle in comparison with cefamandole (cmd). twelve patients received mg lzd as a min infusion and mg cmd as a bolus injection immediately before hip arthroplasty. bone, fat, muscle and blood samples were collected at timed intervals after the infusion and assayed by a validated hplc method. for bone, peak levels of both agents occurred min after administration with mean levels of lzd . mg/kg versus cmd . mg/kg, decreasing to lzd . mg/kg versus cmd . mg/kg at min. correction for blood concentrations gave penetration of lzd % versus cmd % at min and lzd % versus cmd % at min. for fat and muscle, peak levels occurred min after infusion. mean levels were lzd . mg/kg versus cmd . mg/kg (fat) and lzd . mg/kg versus cmd . mg/kg (muscle). correction for blood concentrations gave penetration of lzd % versus cmd % (fat) and lzd % versus cmd % (muscle). we conclude that linezolid exhibits rapid penetration into bone and associated soft tissues achieving levels in excess of the mic for sensitive organisms; with a similar distribution and penetration profile to agents currently used for treatment of infections in these tissues. bacqué e a , barrière jc a , berthaud n b , desmazeau p b , dutruc-rosset g b , dutka-malen s b , ronan b b . a aventis pharma, chemistry, paris, france , b aventis pharma, disease group, paris, france xrp is a new oral streptogramin composed of semi-synthetic synergistic components in a / (w/w) association: rpr ( d-( -morpholino)methyl- d, g-dehydro pristinamycin i e ) from pi a and rpr [( r )- -deoxy- -fluoro pristinamycin ii b ] from pii b by original synthetic routes. the association has antibacterial activity against staphylococci including methicillin */mls b -resistant strains (mic range: . */ mg/ml), streptococci (mic : . mg/ml), pneumococci including multidrug resistant strains (mic : . mg/ ml), enterococci including vancomycin-resistant strains (mic : mg/ ml), m. catarrhalis and neisseria spp. (mic : . mg/ml), h. influenzae (mic : mg/ml), legionella spp. (mic : . mg/ml) and anaerobes (mic range: . Á/ mg/ml). xrp is generally bactericidal at the concentration of Á/ )/the mic against s. aureus , s. pneumoniae , h. influenzae and demonstrates consequent pae ( . Á/ !/ . h at Á/ )/mic, following an exposure of . Á/ h). mutants of s. aureus to xrp were isolated at low frequencies ( . )/ ( Á/ . )/ ( ) at )/ and )/mic while no mutant could be isolated at )/mic. these results suggest that xrp ( / w/w) is a promising compound for the treatment of community-acquired infections. ex vivo evaluation of rpr /rpr (xrp ), a new oral streptogramin ps berthaud n, diallo n. aventis pharma sa, infectious disease group, paris, france the intra cellular activity of xrp , was assessed in j murine macrophages containing ingested staphylococcus aureus (three strains) or l. pneumophila (one strain). at the concentration of )/ the mic, growth of s. aureus was strongly inhibited after a -h period of incubation (d log cfu/ml vs t controls range: (/ . Á/(/ . , according to the strain tested). at the concentration of )/ the mic, growth of intracellular l. pneumophila was inhibited after a Á/ -h period of incubation (d log cfu/ml vs t controls about (/ . and (/ . at and h, respectively). rpr and rpr alone had also inhibiting effect on bacterial growth (d log cfu/ml vs t controls after h of incubation about (/ . and (/ . , respectively). the bactericidal activity of xrp was also assessed against slowly growing s. aureus , under experimental conditions mimicking those observed in patients with an infected indwelling device (first step of infection: adherence to inert support; declared infection: biofilm model). under the experimental conditions, xrp demonstrated a rapid and potent bactericidal effect against s. aureus adherent to an inert support or included in biofilm. this effect was observed at each of the three concentrations tested ( , , and , and )/ mic, respectively). in vivo evaluation of xrp (rpr /rpr ), a new oral streptogramin ps berthaud n, huet y, aventis pharma sa, infectious disease group, paris, france the oral efficacy of xrp , was assessed in staphylococcus and pneumococcal murine infections. mice were challenged ip ( )/ , and )/ times ld ). abscesses were established by intramuscular injection of about bacteria into the right thigh of mice. pneumonia was established by intranasal injection of about bacteria. mice were treated twice a day for (staphylococcus aureus septicaemia and abscesses), (s. pneunomoniae septicaemia) or days (s. pneumoniae pneumonia). six to days post infection, for septicaemia and abscesses the results were expressed as ed , whereas for pneumonia they were expressed as the dose yielding an average survival time (ast) significantly longer than that of the untreated infected controls. xrp was efficacious in the treatment of experimental infections in mice caused by mls b -sensitive and -constitutively resistant s. aureus (ed range: Á/ and Á/ mg/kg/administration in septicaemia and abscess models, respectively). it was also efficacious in the treatment of infections caused by s. pneumoniae whatever the serotype and the resistance profile of the strains tested (ed range: Á/ mg/ kg/administration in septicaemia, ast: mg/kg/administration). these results suggest that xrp might be effective for the treatment of staphylococcal and pneumococcal community-acquired infections. xrp (rpr /rpr ), a new oral streptogramin: bactericidal activity and pharmacokinetics in a model of streptococcus pneumoniae mouse pneumonia ps berthaud n, huet y, diallo n. aventis pharma sa, infectious disease group, paris, france the bactericidal activity against streptococcus pneumoniae of xrp , a new oral streptogramin composed of two semi-synthetic synergistic components in a / (w/w) association (rpr , pristinamycin i derivative and rpr , pristinamycin ii derivative), was assessed in lungs of mice with pneumonia. mice were inoculated intranasally with about cfu of strain - (mls bresistant). eighteen hours later (t ), animals received xrp ( mg/kg p.o). the administration was repeated , , , and h afterwards. to study the influence of varying the ratio of pi to pii component administered on activity and pk parameters, ratios of rpr to rpr ranging from / to / were also administered under the same conditions. after an oral unitary administration at mg/kg, xrp , as well as ratios of rpr to rpr ranging from / to / , demonstrated strong and quick bactericidal activity in lungs. lung levels of rpr and rpr were generally equal or two times higher than blood levels. resulting rpr /rpr ratios in blood and lung, although not in accordance with the initial ratio administered, were synergistic for Á/ h in blood and for Á/ h in lungs explaining the activity observed. conclusion: based on in vitro data, telithromycin is a good candidate for the treatment of rti. in vitro evaluation of abt- , telithromycin and azithromycin against streptococcus pneumoniae , moraxella catarrhalis , haemophilis influenzae and methicillin-resistant staphylococcus aureus ps steele-moore l, berg d, barnes i, couch k, klein j, holloway w. christiana care, infectious disease, wilmington, us macrolide resistant streptococcus pneumoniae (sp) is a worldwide concern predominantly because these isolates tend to be multiply drug resistant. new agents with increased activity against these pathogens are clinically important. the ketolide class of antimicrobial agents demonstrate excellent in vitro activity against sp, even those that are macrolide resistant. the in vitro activities of the ketolides abt- (a) and telithromycin (t) were compared to azithromycin (az) against clinical isolates of sp, moraxella catarrhalis (m.cat), haemophilis influenzae (h.flu) and mrsa. organisms tested: strains of sp (including az resistant), h.flu, mrsa and m.cat. microdilution mic tests were performed following nccls recommendations using freshly prepared plates containing haemophilis test medium for h.flu, cation adjusted mueller-hinton broth (camhb) with laked horse blood for sp and camhb for m.cat and mrsa. the new ketolides, a and t had superior activity against sp including the az resistant strains (mic s: a / . mcg/ml, t / . , az / ). all compounds had excellent activity against m.cat while none demonstrated activity against mrsa. h.flu activity was comparable among a, t and az. these new ketolides are not currently approved by the fda; however t has been approved in europe. ló pez h, vidal gi, salomó n jm, scaglione m, zitto tr. centro de infectología, infectious diseases, buenos aires, argentina objective: we evaluated the impact of the initial treatment failure rate, hospitalisation and costs in outpatient treatment of adult cap in argentina comparing amoxicillin, clarithromycin and telithromycin. method: a probabilistic model was implemented in outpatient treatment of cap. we estimated an initial treatment with amoxicillin, clarithromycin or telithromycin. we assumed expected clinical cure at . , . and . %, respectively. for those patients with failure treatment we evaluated a second-line of antibiotics (amoxicillin followed by clarithromycin and clarithromycin followed by new fluorquinolone) or hospitalisation. patients with telithromycin and failure treatment must be hospitalised without a second line of outpatient treatment. costs of cap included drug's costs by days, medical visits, chest radiographies, analysis and hospitalisation. results: we estimated treatments in patients and first-line drug failure in , and patients with amoxicillin, clarithromycin and telithromycin, respectively. costs in outpatient treatment were: hospitalisation $ , and $ , . ; second-line drug $ and $ ; second-line hospitalised $ . and $ with amoxicillin and clarithromycin, respectively and hospitalisation with telithromycin $ . conclusions: telithromycin showed lower clinical failure, hospitalisation and costs in cap. some studies suggest shortening cap telithromycin treatment to days helping adherence to treatment and decreasing costs even more. the new macrolides */a good alternative of tetracycline in the treatment of mediterranean spotted fever ps popivanova nip a , petrov aip a , boykinova obb a , kazakova zkk a , baltadjiev agb b . a medical university, infectious disease, plovdiv, bulgaria , b department of anatomy, medical university, plovdiv, bulgaria mediterranean spotted fever (msf) caused by rickettsia conorii appears an endemic disease for some regions in bulgaria. frequently the disease has a severe course with multiple organ lesions. the early and adequate treatment is of extreme importance for the outcome of the disease. searching an alternative antibiotic treatment of this disease we considered macrolides for their good cell and tissue penetration and dose-dependent bacteriostatic and bactericide effect. we treated msf patients with doxycycline )/ mg/day, patients with clarithromycin )/ mg/day, as well as patients with midecamycin )/ mg/day and midecamycin acetate mg/kg/day. as a surrogate marker for treatment evaluation the effect on the febrile syndrome was used. our findings showed that by the th day of treatment the fever normalized in . , . and . % of the patients treated with doxycycline, clarithromycin and midecamycin, respectively. for the same period the patient fever decreased below c in , and . %, respectively. the intoxication symptoms were influenced within the same period equally in all treated patients. conclusions: we suggest that the new macrolides appear a good alternative of tetracycline on patients with msf. erythromycin resistance of gram /'// cocci in bulgaria. benefits of the new macrolides in the treatment of respiratory infections ps popivanova ni a , yovtchev ip b , dobreva md c , argirova ta c . a medical university, infectious disease, plovdiv, bulgaria , b medical university, ear-nose-throat disease, plovdiv, bulgaria , c medical university, microbiology, plovdiv, bulgaria in the recent years we tested erythromycin sensitivity of species of gram /'// cocci isolated from throat and nose secretions, ear and eye effusions, sputa, cerebrospinal fluid and blood cultures, vaginal and urethral secretions, urine and fecal samples from patients with inflammatory diseases of the listed organs and systems. staphylococcus aureus , staphylococcus coagulase /(//, streptococcus â-haemolyticus , enterococcus were isolated. of these microorganisms s. aureus was the most abundant. our resistograms revealed sensitivity of the gram /'// cocci in . and . % for and , respectively, and resistance of . and . %, respectively. in the tests with midecamycin and midecamycin acetat the same microorganisms showed sensitivity of . % and resistance of . %. the clinical findings showed excellent effect of the new macrolides including clarithromycin and azalides */azithromycin. we conclude the resistance of gram /'// cocci and especially of s. aureus to erythromycin increases very quickly and has reached dramatical extent. by now, the new , , and -membered ring macrolides and azalides show high antibacterial activity and good clinical effect. pappas ga, liberopoulos e, tsiara s, elisaf m, tsianos e. university hospital, internal medicine, ioannina, greece quinupristin/dalfopristin (q/d) is a novel injectable streptogramin antibiotic which initiation in therapeutics was hailed as an important step towards treatment of vancomycin-resistant enterococcus faecium (vref) species. initial reports concluded in an excellent response of vref to q/d. reports of q/d-resistant strains of e. faecium have emerged, both in usa and europe. we report two cases of e. faecium bacteremia in which the responsible isolate was not sensitive to q/d. the first patient was a woman with acute leukemia and septicemia. e. faecium was cultured from blood samples: the species was resistant to almost all antibiotics, exhibiting sensitivity only to tetracycline (!), while its sensitivity to q/d was indermediate. the second patient was a man with endocarditis, in whom blood cultures isolated e. faecium sensitive to a number of antibiotics, including ciprofloxacin and vancomycin; still the sensitivity to q/d was indermediate. q/d has not been officially introduced to the antibiotic arsenal of greek medicine. moreover, the drug has never been used in our hospital, not even experimentally. other e. faecium species isolated in our hospital have been sensitive to q/d. how much hope can be put on a drug that seems to be partly useless before its initiation? increasing reports of v/ q-resistant strains of e. faecium from all over europe raises fears that the v/d story might well end before it begins. varadinova t a , diakov t a , karagiozova d a , genova p a , pardon p b , baudry c b , quideau s b . a sofia university, virology, sofia, bulgaria , b et institut du pin, centre de recherche en chimie moleculaire, universite bordeaux , bordeaux, france vegetable tannins posses a wide range of biological activities. the aim of the present study was to evaluate the cytotoxicity of five purified vegetable tannins against mdbk cells. the maximal nontoxic concentrations (mnc) and the concentrations required to inhibit cell growth by % (cc ) were evaluated after , and h periods of action. mnc values after h indicated that compounds stimulated cell surveillance when applied in concentrations lower than . mm. cc values indicated: (i) a decrease in cytotoxicity after h as cc were up to times lower than those observed after the h period, and (ii) a re-increase in cytotoxicity when the period of action was prolonged up to h as cc were times lower than those observed after the h period. these data thus appear to reveal the capability of the investigated natural polyphenolic products to stimulate cell surveillance in a time-dependent manner. antibacterial effect survey of enoxolone on periodontopathogenic and capnophilic bacteria isolated from specimens of patients with periodontitis ps salari mh a , kadkhoda z b , sohrabi n a . a tehran university of medical sciences, pathobiology, tehran, islamic republic of iran , b tehran university of medical sciences, dentistry, tehran, islamic republic of iran objectives: most of the microorganisms associated with periodontitis are capnophilic and anaerobic bacteria. purpose of this study was to detection of antibacterial effect of enoxolone on periodontopathogenic and capnophilic bacteria. methods: in this study periodontopathogenic and capnophilic bacteria were isolated from specimens of patients with periodontitis by culture method. an anti-bacterial activity of enoxolone against these microorganisms was investigated by minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) methods. results: based on our findings the mic, mbc and lethal does (ld ) of enoxolone for actinobacillus actinomycetemcomitans , eikenella corrodens and capnocytophaga were ' , , ', ' , , ', and ' , , ' mg/ml, respectively. conclusion: our results show enoxolone has antibacterial effect on a. actinomycetemcomitans , e. corrodens and capnocytophaga spp. sakalova stn. medical university, microbiology, grodno, belarus we have synthesized diamides of dicarboxylic acids, with components such as -nitrothiazole benzolsulphamides and triazol. all the above compounds exhibited bacteriostatic activity towards some microorganisms. for further studies of bacteriostatic activity of amides and diamides of dicarboxylic acids, as well as for determination of 'structure Á/activity' relationship, we have synthesized a range of monoamides. antibacterial activity of the synthesized compounds was studied in vitro by agar dilution methods. for this purpose, approximately various gram-positive and -negative microorganisms including clinical strains of staphylococcus aureus , bacillus subtilis , serratia marcescens , escherichia coli , proteus morganii , micrococcus lisodeicticus , staphylococcus epidermidis , shigella sonnei , salmonella typhimurium , yersinia enterocolitica . minimum inhibitory concentration was expressed in mg/ml. nitazole was used as a comparison substance. analysis that new derivatives of -nitrotiasole have high antibacterial activity relative towards certain microorganisms included strains obtained from infection department's patients. results will be shown. microbial susceptibility to the essential oil of ziziphora clinopodiodes lam. ps purpose of the study: antimicrobial activities of essential oils vary from oil to oil and from one micro organism to another. the antimicrobial and chemical properties of essential oil from ziziphora clinopodiodes lam. has not been studied and hence the present study was planned to evaluate those properties against a series of micro organisms viz, escherichia coli , staphylococcus aureus , pseudomonas aeroginosa , klebsiella pneumonia , bacillus subtilis , bacillus licheniformis , streptococcus faecalis , candida albicans and saccharomyces cerevisiae . results: z. clinopodiodes lam. essential oil was found to have remarkable antimicrobial property against all the microorganisms but p. aeroginosa . the oil exhibited its best antimicrobial activity within a maximum of min. seventeen components were identified by gas chromatography and mass spectrometry (gc and gc/ms) analysis of the oil, out of which pulegone ( . %), neomenthol ( . %), cyclohexene, -methyl- -( -methenyl)trans ( . %), , -cycloheptadien- -one, , , -trimethyl ( . %), piperitone ( . %), and limonene'/ , -cineole ( . %) constitute major parts of the oil. conclusion: monoterpenes seem to have antimicrobial role. it seems necessary to explore antimicrobial properties of new harmless antimicrobial agents from natural sources as substitutes for common chemical drugs. methanol extract of carpobrotus edulis enhances killing of methicillin resistant staphylococcus aureus phagocytosed by thp- human monocyte derived macrophages and promotes the release of modulators of cellular immunity ps although alkaloids from the family mesembryanthemaceae have anti-cancer activity, species of this family have received little attention. because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, we have studied a crude methanol extract of carpabrotus edulis , a common plant found along the portuguese coast, for properties normally associated with plasma membrane active compounds. the results of this preliminary study show that the extract is non-toxic at concentrations that prime thp- human monocytederived macrophages to kill ingested methicillin resistant staphylococcus aureus and promote the release of lymphokines associated with cellular immune functions. the extract also induces the proliferation of thp- cells within day of exposure and days earlier than that induced by phytohemagglutinin. similar results were obtained with monocyte-derived macrophages isolated from human peripheral blood. the active component or components of the plant extract may be exploited as intracellular active anti-bacterials as well as modulators of cellular immunity. enhancing of erythromycin production by saccharopolyspora erythraea nur with common and uncommon oils ps hamedi j a , malekzadeh f a,b saghafi-nia ae b . a department of biology, faculty of science, university of tehran, tehran, islamic republic of iran , b shafe-e-sari co., antibiotic production co., teheran, iran enhancing effect of various oils on the erythromycin production by saccharopolyspora erythraea nur were evaluated in a complex medium consisting soybean flour and dextrin as the main substrates. the biomass, erythromycin, dextrin and oil concentrations, and ph value were measured on a daily basis. also, the kinds and frequencies of fatty acids of the oils used were determined. saturated fatty acids in the shark oil were higher than that of vegetable oils used. erythromycin concentration in the melon (cucumis melo var. inderus cultivar mashhad ) seed oil containing medium was . times higher than that of the control medium (without oil) and . times higher than that of rapeseed oil containing medium. erythromycin concentration in the other oil containing media, including rapeseed, soybean, shark (carcharhinus dussumieri ), and safflower oils was . , . , . , . time higher than that of control medium, respectively. the melon seed oil had the least enhancing effect on the biomass production, and thus decreasing the cost of the biomass separation. can varicella be eliminated by universal childhood vaccination ? */ epidemiological and economic data from germany ps wutzler p a , banz k b , neiss a c , goertz a d , bisanz h d . a institute for antiviral chemotherapy, university of jena, jena, germany , b outcomes international, basle, switzerland , c institute of medical statistics and epidemiology, technical university, munich, germany , d glaxosmithkline, pharma, munich, germany purpose: universal varicella vaccination in childhood is expected to reduce substantially the number of uncomplicated cases of chickenpox and to decrease the number of complicated cases requiring hospitalization. to generate fundamental data for decisions of the health authorities epidemiological and health-economic data were collected in two large studies. using an age-structured decision analytic model the benefits, costs and cost-effectiveness of a varicella immunization program over a period of years were assessed. results: it could be shown, that the vast majority of varicella cases occur in children aged less than years. in . % of the cases a severe course was assessed. overall incidence of complications was estimated to be . %. a routine varicella vaccination program targeting healthy children could prevent . % of varicella cases and over major complications per year provided the coverage rate would be %. under these conditions the elimination of varicella is predicted to be achievable within Á/ years. a combined measles, mumps, rubella and varicella vaccine is expected to provide the required coverage. conclusions: routine childhood varicella vaccination appears to be a highly efficient strategy to significantly reduce the sizeable burden of varicella and leads to significant savings from both societal and payer's perspective. bulgakova va, balabolkin ii, sentsova tb. scientific center of child health, russian academy of medical sciences, scientific research institute of pediatrics, moscow, russian federation objective: to estimate efficacy of vaccine influvac at children with allergic diseases. methods: twenty children aged Á/ years with allergic diseases received vaccine influvac (solvay pharma). for the control group of children, with allergic pathology did not receive this vaccine because of an intolerance of chicken protein ( children). results: all vaccinated children for the observable season of months did not get influenza. general and aboriginal reactions to a vaccine did not occur. in control group for the observable season two children were ill with influenza and four children with acute respiratory virus infection ( %). among vaccinated children there was an increase in titre to a protective level ( : and above) to all to three strains of influenza days after injection. vaccine influvac can be recommended for an immunisation against influenza of children with an allergic pathology because of efficacy and absence of side effects. ló pez h, zitto tr, vidal gi, cánepa mc, salomó n jm, scaglione m. centro de infectología, infectious diseases, buenos aires, argentina objectives: our study examines the possible economic impact of the influenza on health working adults in argentina, and the intervention cost saving with immunization. methods: this is a theoretical study based on a mathematical model. population data was published in s national statistics. the global incidence of influenza infection was estimated at %. we have estimated the direct cost on influenza infection (outpatient visits, drugs and hospitalization) and indirect cost (work absenteeism and productivity loss) and projected net saving for the Á/ year-old vaccinated group. the vaccine effectiveness was estimated at and %. the price of vaccine was $ each. results conclusion: influenza vaccination is effective in diminishing cases of flu and reducing working-day loss. its a safe and cost-effective vaccine. ló pez h, zitto tr, cánepa mc. centro de infectología, infectious diseases, buenos aires, argentina flu infection is a major cause of illness and one of the most common cause of work absenteeism, increasing institution costs, healthcare provider visits, use of drugs, and decreasing work productivity. vaccine against flu has an effectiveness between and %, in health adults. objective: evaluate the impact of flu-like respiratory tract infections in a health institution staff during year, comparing vaccinated with not-vaccinated groups. methods: we evaluated all causes of absenteeism along year ( ), based on the written note made by the professional who has evaluated ill people, selecting flu-like respiratory infection causes. we evaluated age, working days loss related to illness, and cost on vaccinated and not-vaccinated groups. results: one hundred and sixty eight of the total staff ( people) were vaccinated, of them had flu-like infection, resulting in working days lost. for not-vaccinated group, people had flu and lost days. lost cost for vaccinated group was of $ , and for notvaccinated group, $ . conclusion: we observed a decrease in working days loss and money waste related to flu-like infections on the vaccinated group. because of safety and effectiveness of vaccine against flu, the implementation of vaccination will be cost-effective for all institution staff. we studied functioning of the interferon system in children with atopic bronchial asthma (ba) at the age of Á/ years. the control group included healthy children. we investigated the interferon status (method of grigoryan s.) and serum concentrations of ifngamma (ifng) (elisa). there was a decrease in the ifn-producing ability of leukocytes to the synthesis of ifna at % and ifng at % of children with ba. serum level of ifn of children with ba during all period of illness is compared to the children without predisposition to atopy ( . / . and . / . pg/ml accordingly) was significantly decreased. production of ifna increased after using viferon (recombinant a b ifn and antioxidants). decreased ability of gammainterferonogenesis in the most children was not affected by the action of immunomodulators. there was shown interferon system's dysfunction in the development of atopy and increasing predisposition to respiratory infections and to persistent of atypical infections in children with ba. harxhi a, pilaca a, pano k. university hospital center of tirana, infectious diseases, tirana, albania congo-crimean haemorrhagic fever is viral disease with a high rate of mortality that is caused by a nairovirus, bunyaviride species. this is a zoonotic disease, which affects sporadically humans and is geographically distributed even in eastern europe and balkan. during the months of may and june , in northeast of albania were reported eight cases of haemorrhagic fever. serologic tests performed in the laboratory of reference in thesaloniki, greece confirmed the diagnosis of congo-crimean haemorrhagic fever. in the mean time, who reported the outbreak in southwest kosovo of cases suspected for haemorrhagic fever from which were confirmed laboratory as congo-crimean haemorrhagic fever. we are describing here the clinical history of one of eight cases with cchf in albania. from the epidemiological point of view this case was considered peculiar, as it was the only one hospitaly acquired, and due to the gravity of the haemorrhagic syndrome was admitted at the intensive care unit at infectious diseases service, university hospital center of tirana. results: in the study were included patients ]/ years of age with documented hbsag-carrier ]/ months (average age */ . years, male */ %, female */ %). hbv dna in serum was tested by qualitative and quantitative pcr (commercial test-system ampli-sens hbv). hbeag, hbeab, hbsag were detected by elisa (hoffmann la roche). hbv dna by qualitative pcr was detected in % patients, by qualitative pcr was detected in % patients in the concentration ]/ copies/ml, in . % ]/ copies/ml, in . % ]/ copies/ml, in . % ]/ copies/ml ( fig. ). hbv dna level distribution among the hbsag carriers. elevated ( . the upper limit of normal) alt level was determined in . % of the hbv dna negative and . % of the hbv dna positive patients. hbeag was detected in . % of the hbv dna positive patients and had not been determined in the hbv dna negative patient. eleven percent of patient had the combination of the biochemical, serological and virology criteria, which are typical for active chronic hepatitis b (hbsag-carrier !/ months, hbv dna ]/ )/ copies/ml, elevated alt). conclusion: in smolensk % of the hbsag-carriers have viral replication confirmed by qualitative pcr. eleven percent of them have active chronic hepatitis b. kandemir o a , polat a b , kaya a a . a medicine of faculty, mersin university, clinical microbiology and infectious disease, mersin, turkey , b medicine of faculty, mersin university, pathology, mersin, turkey the exact potential of nitric oxide in the pathogenesis of chronic viral hepatitis is not known. the elevated nitric oxide production is assumed to be responsible for the pathological changes in many inflammatory conditions, mainly via peroxynitrite, a potential oxidant that is produced by the reduction of superoxyde anion with nitric oxide. the intensity and the distribution of the immunohistochemical staining of intrahepatic inducible nitric oxide synthase were studied in the biopsy specimens obtained from patients with viral hepatitis and patients with elevated transaminase levels from other etiology. hepatic inducible nitric oxide synthase staining was significantly more intense in the viral hepatitis group (p / . ). inducible nitric oxide synthase staining levels correlated well with the severity of the viral hepatitis using the knodell's liver histological activity index (r / . , p / . ). among the viral hepatitis group, the pathological distribution of the inducible nitric oxide synthase staining favored the periportal regions whereas less staining was observed in the bile duct and parenchyma regions. as nitric oxide mediated nitration of hepatocellular proteins is found to be elevated in the inflamed hepatic tissues and it well correlated with the severity of the disease, we suggest that inducible nitric oxide synthase can possibly have a critical role in the pathogenesis of chronic viral hepatitis. there were patients under observation who were divided into two groups, the first of patients (eight chronic virus hepatitis; three chronic virus hepatitis'/steatosis; four steatohepatitis; three chronic cryptogenic hepatitis; there were men and four women aged from to . the second group consisted of patients, eight with chronic virus hepatitis; four with steatohepatitis; six with chronic cryptogenic hepatitis. there were men and three women aged from to . diagnosis was confirmed with the help of clinical data, biochemical tests, serological markers, psr-diagnostics and ultrasound examination and computer tomography of the abdomen. in the first group of patients the treatment with ursofalk was administered at the dosage of mg/kg of body mass from month to years with improvement in general condition of the patients: heaviness, pain under the right rib, nausea and skin itch have disappeared. in all the cases, improvement in the biochemical blood analysis took place during treatment. the average index of alt activity was before */ . u/l, after */ . u/l and ast */ . and . u/l; alp */ . and . u/l; ggt */ . and . u/l; chol */ . and . mmol/l; tg */ . and . mmol/l. in the second group of patients the treatment was carried out with various hepatoprotectors during the courses from to months. before the average index of alt activity was . u/l, after */ . u/l; ast */ . and . u/l; alp */ . and . u/l; ggt */ . and . u/l; chol */ . and . mmol/l; tg */ . and . mmol/l. treatment of patients suffering from hepatitis of viral and other aetiologies with ursofalk produces a positive effect on both clinical symptomatic and biochemical indices. remission was more stable during a long period of taking the preparation. the hepatoprotective effect of ursofalk during the years was sustained for the whole of the period of the treatment. after stopping, an acute attack of cytolytic syndrome was observed. with other hepatoprotectors we did not get any improvement in clinical scene of the disease or in biochemical indices. shavlov nm, kletsky sk. minsk, belarus hsv- and - have possibility to damage different organs and systems. sometimes they cause damage of the liver, which resemble viral hepatitis. the etiology of such hepatitis may be confirmed only by results of liver biopsy. we have diagnosed cases of herpetic hepatitis: eight children and four adults. clinical course was different. in five cases the acute beginning took place: high temperature, the jaundice at the Á/ day (the level of bilirubin was Á/ mkmol, especially direct), cholestasis, the pain in upper right part of abdomen. the ascites was found in three patients with acute hepatitis during st week from the beginning of disease. in seven cases the beginning was gradual. the temperature was subfebrile, prolonged; malaise and moderate pain in upper part of abdomen were constant complaint. the jaundice was moderate; bilirubin increased until mkmol. the level of alt was moderately increased ( Á/ times). the blood analysis showed moderate leukocytosis with neutrophilia, and increased sre. the serological markers of hepatitis a, b, c, d were negative in all cases. hsv- and - were found in the blood. the diagnosis was defined by the results of hystochemical investigations, when the viruses were found in liver bioptat, and confirmed with the results of specific treatment. specific damage of liver cells was found: protein dystrophy and specific inclusions in cell nucleus. in all cases the treatment with acyclovir were given. the results we have observed during st week: the temperature became normal, the jaundice decreased and bilirubin was normal during Á/ days. in one case the recidive took place weeks later after treatment. the second course of acyclovir with intron a gave good results. nesic z a , delic d b , prostran m a , vuckovic s a , stojanovic r a . a department of pharmacology, school of medicine, university of belgrade, belgrade, yugoslavia , b clinical center of serbia, institute for infection and tropical disease, belgrade, yugoslavia the large number of unsolved cases of acute and chronic hepatitis has most probably the viral etiology. in mid s, two independent groups of authors reported a new human hepatotropic virus, with flavivirus like genomes, hepatitis g virus (hgv). the aim of this pilot study was to determine the prevalence of hepatitis g viral infection among patients at high risk of exposure to blood and blood products, as well as to evaluate if the risk of hgv infection was higher among them than in the general population. immunoenzyme test on microtitration plate for detection antibodies against hgv e antigen in plasma or sera (r&d systems, minneapolis, usa) was used for evidencing anti-hgv igg antibodies in sera. anti-hgv antibodies were detected in the control group (blood donors) in . % ( / ) patients. prevalence of anti-hgv antibodies among i.v. drug users was evidenced in . % ( // ), in hemophiliacs . % ( / ), in patients acquiring multiple blood transfusion . % ( / ), in hemodialyzed patients . % ( / ) and in patients with transplanted organs . % ( / ). our results suggest that patients exposed to blood or blood products have a higher risk of hgv infection than general population. evaluation of ortho total hcv core antigen assay in assessment and follow-up of patients treated for chronic hcv ps lunel f, veillon p, payan c. chu angers, laboratoire de bactério-virologie, angers, france an assay to quantitate 'total' hvc core antigen (hcv ag) in serum or plasma, may reflect viral load, has been developed by ortho-clinical diagnostics. methods: we evaluated hcv ag with two quantitative assays for hcv rna: bdna . (bayer) and monitor . (roche). we studied samples from untreated patients and from patients with chronic hcv treated with ifn or ifn/ribavirin. results: correlation of ag and quantitative assays was high (r / . for bdna . and . for monitor . ). no difference between the levels of rna and ag among hcv genotypes (r / . Á/ . ) was found. ag values, before treatment, were significantly lower in sustained responders (sr) than in other groups ( . log versus log, p b/ . ). in patients treated with ifn or combination therapy, we found very good correlation between decrease and negativation of ag and viral load: log iu/ml decline after m of interferon was significantly correlated with the negativation of hcv ag and sr. thirty-eight/ of sr had a rna load decrease !/ log iu/ml and / had a negativation of hcv ag after m . conclusion: total hcv core ag appears to be a new tool for monitoring patients with hcv infection. hepatitis c virus rna and hcv core antigen kinetics predict the efficiency of interferon-alfa and ribavirin therapy in naive patients infected by hcv genotype or ps fifty-five patients infected by genotype or were treated with a primary dose of (if hepatitis c virus (hcv) rna b/ meq/ml) or million units of interferon alfa- b (ifn) thrice weekly for months. ribavirin was added at month (m), until m if hcv rna was found positive after m of ifn. the viral kinetic was assessed during the follow up by serial measurements of hcv rna (bdna . and monitor . ) and using a new assay from ortho-clinical diagnostics which is able to quantify total hcv core antigen. sustained virologic response was observed in % of the patients ( / ). after month of ifn treatment, sustained responders had a fall of hcv rna and hcv core antigen higher than non-responders ( . / . log ui/ml versus . / . log ui/ml, p b/ . , for hcv rna) and ( . / . log (pg/ml)/ ) versus . / . log (pg/ml)/ ) p b/ . , for hcv core antigen). after month of ifn, the positive and negative predictive values of sustained response were, respectively, and . % for hcv rna negativation and . and . % for hcv antigenemia negativation. these results suggest that both kinetics of viral load and antigenemia are highly predictive of sustained response. theodorou m, petinelli i, pontikaki d, mela c, blana a, papanastasiou a, toliopoulos a, stavrakaki m, sagkana e. microbiology department, western attika general hospital, greece, egaleo, greece greece has accepted a big number of economic immigrants lately. we investigate the prevalence of hepatitis b/c as well as the epidemiological features that might influence the public health. . economic immigrants from: albania , eastern europe and from asiatic-african countries , visited our hospital to be checked in order to get a health certificate to obtain the green card. they where tested for hepatitis b/c. the serological markers were determined by immunoenzymatic method. all hbsag('/) and anti-hcv were further tested for hbv dna and hcv rna by competitive rt pcr. hcv rna('/) were genotyped by strip hybridization immunoassay. in albanians . % were hbsag('/), . % hbv dna('/), . % anti-hcv('/) and . % hcv rna('/). in east europeans . % were hbsag('/), . % hbv dna('/), . % anti-hcv('/) and . % hcv rna. in asians-africans, . % were hbsag('/) and % hbv dna('/). in pakistanis, . % were anti-hcv('/) and . % hcv rna('/). of the rest of asians-africans, . % were anti-hcv('/) and . % hcv rna('/). albanians: higher prevalence of hbv infection ( . %). greek blood donors: % pakistanis: hcv infection is % (predominance of a type), general greek population: %. public health services in greece and europe must take appropriate measures. el zawawy la a , mohamed on b , ali sm a , eissa me a , allam sr a . a faculty of medicine, parasitology, alexandria, egypt , b high institute of public health, microbiology, alexandria, egypt the purpose of the study was to investigate the influence of schistosomal suppression on the antibody response to hepatitis-b vaccine (hbv) and to study if the vaccine has any protective effect on experimental () infection. the results obtained revealed that infection reduced the serum antibody level against hbv. parasitological and histopathological findings showed significant protection against infection. the conclusion reached was; in order to reduce the incidence of virus-b infection especially in schistosomiasis endemic areas, public health officials should evaluate a policy for regulation of hbv booster vaccination to enhance the population immunity against hepatitis-b infection. cooper e, fisher t, shingadia d. newham general hospital, family clinic, london, uk sustained anti-retroviral combination chemotherapy requires excellent adherence to the regimen so as to suppress viral replication sufficiently to delay the emergence of resistance. if chemotherapy were taken to scale, e.g. in africa, erratic adherence might soon lead to multi-resistant circulating virus. we reviewed our experience in a well established london family clinic with a team including community nurses. we reviewed the records of the african immigrant children, aged Á/ , treated with anti-retrovirals exclusively at our centre throughout . whereas had undetectable hiv rna within the year, only four had undetectable rna throughout the year. four failed therapy through proven resistance mutations, but nine were considered through circumstantial evidence to have rising viral loads primarily because of poor adherence. three were known to have stopped taking drugs for extended periods. the three boys over years were unreliable in adherence, but the one girl in this age-group was fully adherent. our preliminary assessment is that for the children in our families, despite a team approach and home visits, nonadherence to haart may be twice as common as selection of a dominant viral mutant as a primary cause of failure to sustain viral suppression. quiros-roldan e, moretti f, castelli f, el-hamad i, carosi g. the prevalence of hiv related lipodystrophy-syndrome depending on the definition and severity of lipodystrophy ranges from to %. we have retrospectively reviewed the medical records of african patients followed. the characteristics are shown in the . % of africans had triglycerides !/ mg/ml and . % had cholesterol !/ mg/ml, none had both metabolic alterations. glycemia !/ mg/ml was observed in . % patients. it is interesting highlight that in any the africans morphological changes were noted and all of them showed weight stable. although the low prevalence of metabolic alterations may be attributed to the different ethnic alimentary behavior if self-body perception by african is not as accurate as by caucasian on the estimation of the body changes have to be investigated. alabaz d a , alhan e a , yaman a b , evliyaoglu n a , kocabas e a , aksaray n a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey hepatitis a virus (hav) infection is usually asymptomatic in children. however, it may occasionally cause a severe disease with high morbidity and mortality, and loss of school or business days. in a previous study, we have shown that every one of two to three school children from upper social classes living in adana carries high risk of hav infection. it is well known that maternally transmitted anti-hav antibodies interfere with hav vaccination. in an effort to determine the optimal age for hav vaccination, babies ( % girls and . % boys) born in our hospital were prospectively followed up at least months for the presence of maternal antibodies to hepatitis a (anti-hav igg). anti-hav igg titers were measured from the blood specimens obtained at birth from the mothers and from the offsprings at months, , , , , , , , and . the prevalence of positive anti-hav at birth ( %) was similar to those of hav seroprevalance studies carried out in adults in our area. the disappearance of antibodies occurred between the st and st month of life. the prevalence of anti-hav igg among children aged , , , , and months were , , , , and %, respectively. in light of these findings, we suggest that hepatitis a vaccination be given after months of age. earlier vaccination may be ineffective due to interference with maternally transmitted anti-hav antibodies. ghaderi b, alaghebandan r, rastegar lari a. department of microbiology, iran university, tehran, islamic republic of iran diarrhoea is a major public health problem in developing countries. amoebiasis is one of the most common causes of diarrhoea in iran as an endemic area for amoebiasis. little, however, is known about the extent of the condition in our society. the aim of this study is to determine socio-demographic and clinical characteristics of patients with intestinal amoebiasis. during july and august , we collected patients with diarrhoea among patients who visited at a referral hospital in shahriar area (in countryside of tehran), iran. thirty out of patients ( %) had intestinal amoebiasis and were followed up prospectively until the resolution of the illness. nineteen of ( . %) patients were male and the remaining of . % was female. the patients were aged Á/ with mean of . years. most of the patients ( %) were below years of age and the peak of occurrence was between the age of and years. watery diarrhoea with abdominal cramps was the main clinical feature. seventy percent of patients were resident in urban area and the remaining ( %) in rural area. average family income was low and all patients were in low socioeconomic level. water supplying system for all patients was pipeline water. low socioeconomic level associated with poor personal hygiene was the most important factor for highly prevalence of this problem in our society. also it seems that food plays important role in transmission of protozoa then water. the new strategy for allele identification of the genes coding for pertactin and pertussis toxin subunit s in bordetella pertussis ps bordetella pertussis strains demonstrate a significant polymorphism in toxin s subunit and pertactin, which are major protective antigens of the organism. monitoring the changes in prevalence of particular alleles of genes coding for these proteins in local b. pertussis populations is an essential issue in cases of the observed decrease of vaccination effectiveness. we have developed a new method for allele identification of these genes, which eliminates the necessity of dna sequencing. the approach is based on the identification of the number of repeats or the presence of specific nucleotides in the polymorphic regions or residues, respectively, of the genes and utilises products of their full or partial pcr amplification. the nucleotide heterogeneity in each polymorphic site is analysed either by the differential digestion of the amplicons or by the arms (amplification-refractory mutation system) methodology. numbers of repeats in particular regions of the genes are revealed by the size analysis of the adequate pcr products or their restriction fragments. in all cases the presence, size or pattern of dna molecules obtained is visualised by the agarose gel electrophoresis. the preliminary analysis of the recent and archival b. pertussis strains identified in poland was performed using the described approach. the presented strategy provides a much easier, faster and more cost-effective than dna sequencing mean to study the polymorphism of the major b. pertussis antigens. vaccination coverage and history of vaccine preventable infectious diseases among students in second year of medicine and pharmacy of tours university ps borderon jc a , hamed a b , ragot s b . a centre hospitalier universitaire, tours, france , b médecine préventive universitaire, tours, france the purpose of this study was to determine the level of infectious risk in students who will be exposed to patients. information was obtained by a questionnaire for each student, and by checking medical records for immunization coverage and vaccine preventable infectious diseases. answers could be specified for students, of whom females (f) and males (m). the number of non-immunized students was against diphtheria: two, tetanus: three, pertussis: four, poliomyelitis: two, hepatitis b: six, and hepatitis a: , respectively. among the students non-vaccinated against measles, (nine f and five m) had no history of that disease. among ( f and m) non-vaccinated against rubella, ( f and seven m) had no history of that disease, uncertain in seven others (six f and one m). the date of vaccination was often late regarding recommendations. fifteen students had no history of varicella. one student had not received bcg vaccination. fifty-eight students had received two, and three bcg vaccines. post-bcg tuberculin skin testing was missing after first bcg, second and third bcg. the date of the first tuberculin test was often one or several years after bcg vaccination. adverse effects of vaccination were rarely reported: two cases of fever (dt polio, measles); three cases of local reaction (dt polio, dtp polio). one case of contraindication for influenza vaccine: egg allergy. the survey shows failure in immunization coverage actually recommended in health care students. objectives: to present the morbidity of rabies and evaluate the efficiency of our prophylaxis scheme in lasi county. material and method: we made a retrospective study of the rabies cases in the patients admitted in our unit in a th-year period. we have analysed all the clinical, epidemiological and biological aspects. results: in a -year period, cases of rabies were admitted in the clinical infectious diseases hospital lasi. the highest incidence was for Á/ */eight cases ( %); the highest yearly cases were three cases in and . most of the patients were male ( %), came from suburban areas ( patients). eight cases occurred in may Á/june, wild animals were involved in half the cases (fox, wolf). for patients, no prophylaxis was performed and an incomplete course in four cases. the period of time to the appearance of the first symptom was Á/ days. the prophylaxis scheme led to a good protection. conclusions: in lasi county, rabies is a problem with a prevalence of . %/year. trends in the use of antimicrobials in riyadh in were analyzed. data was obtained from a survey of randomly selected families of school children aged Á/ years in a -month period in . one hundred and ninety-nine ( . %) students were on antibiotics in the month preceding the study; ( %) received antibiotics for the diagnosis of pharyngitis; ( %) students antibiotics were prescribed by a physician; and in ( %) the duration of antibiotics was less than week. this study shows a major problem in antibiotics prescription in our community and also the need to establish effective antibiotics policy in general practice to limit the potential emergence of drug resistance bacteria in the community. mir s a , cura a a , erdogan h a , guler s a , sengul gn a , koyu a a , ozinel ma b . a department of pediatrics, ege university medical faculty, izmir, turkey , b department of microbiology, ege university medical faculty, izmir, turkey antibiotic susceptibility spectrum of childhood urinary tract infection agents are geographical variation. the current antibiotic regimens and the selection of antibiotics for prophylaxis should be re-evaluated periodically. the objective of our study was to determine the local resistance rates to antibiotics and to give a direction for the selection of antibiotics in uti treatment. we evaluated urinary culture assays retrospectively, sent from pediatrics and pediatric surgery inpatient and outpatient clinics of our hospital during the last months, and investigated the isolated pathogens and the resistance rates to antibiotics. in addition, the data obtained were compared with of years ago. with respect to the resistance rates to antibiotics of uti pathogens, the resistance rates of e. coli for carbapenems, aminoglycosides and third generation cephalosporins were , and %, respectively as before, but the rates for ampicillin increased from to % and for tmp-smx it increased from to %. we concluded that the resistance profiles to antibiotics should be reviewed every years at least and thus the selection of proper antibiotics would lessen the morbidity as well as the medical expenses. chanturidze tk, tsiklauri r. ministry of labor, health and social affairs, public health, tbilisi, georgia purpose: since infectious diseases (id) are increasing in georgia. this study is aimed to reveal economic barriers of effective id control by assessing financial contribution to id from public and private sources, household's total spending on health and their capacity to pay. sources: ) national household expenditure and revenue survey. ) who fair financial contribution methodology. ) meta-analysis. results: . % of population leaves under the poverty level; % out of total household expenditure (average gel; us$ ) % is spent on food */non-subsistence income covers expenditures on goods and services including health; % of population refuses health services because of inability to pay; public spending on health comprises % of total health expenditures; public spending on id control is below gel per capita; almost all private spending goes to id treatment and equals to . gel per patient. conclusions: insufficient public spending on id control transfers the burden to the population with extremely low capacity to cover health expenses. refusal to utilize health services, and incomplete treatment and increases the threat of id spread and drug resistance. government should increase the allocations to id from public sources for effective id control in georgia. antimicrobial consumption trends in children's university hospital ps ratchina s a , averchenkova l b , jarkova l a . local surveillance of antimicrobial (am) consumption is essential to promote the rational use of this group of drugs. the purpose of this study was to analyze the trends of am use in the children university hospital in and . data on am usage were obtained from the hospital drugstore requests in the -beds multi-ward children university hospital. consumption was expressed as the number of ddd per bed-days (b Á/d). the total am consumption figures were similar in and ( . and . ddds/ b Á/d, respectively) with notable differences in am prescribing patterns. penicillin consumption increased from . to . ddds/ b Á/d mostly due to amoxicillin. the overall aminoglycoside usage remained comparable ( . vs. . ddds/ b Á/d) though amikacin has considerably replaced gentamicin. there was a sevenfold increase of ciprofloxacin ( . vs. . ddds/ b Á/d) along with the evident decrease of tetracycline and co-trimoxazole consumption found ( . vs. . ddds/ b Á/d and . vs. . ddds/ b Á/d, respectively). the tendency to prescribe more effective in respect of the local resistance data and/or more safe am was detected in comparing with that can be explained by the introduction of the local guidelines for infectious diseases management in . clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis */a pilot study ps the aim of this pilot study was to determine the efficacy and tolerability of clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis (ct). fifty-two patients older than years of age with diagnosed chronic chlamydial prostatitis were enrolled. the presence of ct in expressed prostatic secretion or urine specimen voided immediately after prostatic massage was confirmed by isolation on mccoy cells and lugol staining. the majority of patients suffered from suprapubic pain and pain in the groin. twelve patients had no clinical symptoms. according to rectal palpation, prostate gland was normal in patients, tender and soft in and firm in five patients. clarithromycin was administered orally mg twice daily for days. simultaneously the patients' partners received mg orally twice daily for days. clinical efficacy and tolerability of administered clarithromycin were evaluated Á/ days and Á/ weeks after the end of treatment. bactericidal efficacy of administered drug was evaluated Á/ weeks after the end of treatment. the eradication of ct was achieved in out of patients, while patients were clinically cured. two patients had nausea and elevated serum transaminases. in asymptomatic patients, the eradication of ct was achieved in of patients who reported no side effects. this pilot study has shown an excellent efficacy and tolerability of clarithromycin in the treatment of patients with chronic chlamydial prostatitis. women with diagnosis of urinary tract infections (uti) often demonstrate vaginal colonisation with alpha-haemolytic escherichia coli strains. in the present study we decided to evaluate a distribution of virulence genes encoding for cytotoxic necrotizing factor type (cnf ), p-fimbriae, s/f c-fimbriae aerobactin (aer), and afa genes in alpha-haemolytic e. coli strains isolated from gynaecological material in our region and to compare the detected sequences in clinical isolates of other diagnostic groups. of alpha-haemolytic e. coli strains, were isolated from urine, from gynaecological specimen, and were faecal strains. e. coli strains were tested for the production of haemolytic phenotype on blood agar plates. the amplification of virulence factors was performed by pcr according to previously described protocols (le bouguenec et al., ; blanco et al., ; and yamamoto et al., ) . we found that all gynaecological alphahaemolytic strains were positive for cnf , (p b/ . compared to % of urine strains, and p b/ . compared to % of faecal strains). similarly, sfa/foc specific dna sequences were found in % of gynaecological isolates (p / . compared to % of urine strains and p / . compared to % of faecal strains). from this point of view, the female genital tract seems to be a potential reservoir of these uropathogenic e. coli strains. azithromycin in the treatment of pelvic inflammatory disease caused by chlamydia trachomatis ps administered in hospitalized patients with chlamydial pid. the diagnosis was made prior to hospitalization. microbiological analysis of urine, blood and swab specimens collected from endocervix, vagina and urethra confirmed c. trachomatis to be the single suspected causative pathogen of pid. the presence of c. trachomatis in swab specimens from endocervix was examined by dnk/rnk hybridization. azithromycin was administered Á/ days after samples for microbiological analysis were collected in dose of )/ mg iv for days. clinical efficacy and tolerability of therapy were assessed Á/ days after the end of therapy and clinical and microbiological analysis Á/ weeks after completion of therapy. the eradication of c. trachomatis and normalization of gynecological findings were achieved in and disappearance of subjective symptoms in patients. no side effects and deviations from normal values in hematologic and biochemical blood parameters were recorded. this study showed high bactericidal efficacy, rapid clinical effect and good tolerability of once-daily administration of mg azithromycin for days in the treatment of patients with pid caused by c. trachomatis . altindis m a , cevrioglu s b , aktepe oc a , cetinkaya a . a kocatepe university school of medicine, microbiology, afyon, turkey , b kocatepe university school of medicine, obstetrics and gynecology, afyon, turkey diagnosis of the causative organism of the vaginitis is usually based on clinical criteria. a standardized, laboratory based and rapid diagnostic test for the identification of these organisms is desirable. to determine the laboratory method that best predicted the causative organism, we calculated the sensitivity, specificity and predictive value of positive and negative test for clinical criteria, an oligonucleotide probe test (affirm vpiii-bd usa) and compared them with the combination of positive vaginal culture and gram-stained vaginal smear. we evaluated consecutive women aged Á/ years, attending for vaginal discharge. vaginal swab specimens were used for culture of gardnerella vaginalis , trichomonas vaginalis and candida sp, preparation of a vaginal smear for gram-stain interpretation and wet mount evaluation and affirm test. affirm detected g. vaginalis in ( %), candida sp in three ( . %) women and no trichomoniasis case found by any methods. the sensitivity and negative predictive values of affirm test and clinical signs were same ( %) in identifying of bacterial vaginosis. however, affirm test was more specific ( vs %) and also has higher positive predictive value ( vs %) than clinical signs. we did not evaluate the results for patients with candidiasis because of less number. according to these results affirm-microbial identification tests are objective and specific for the rapid diagnosis of the bacterial vaginosis. comparison of efficacy of single dose of tinidazole with standard dose of metronidazole in giardia lamblia infection (preliminary report) ps fallah m a , moshtaghi aa b . a university of medical sciences, parasitology, hamadan, islamic republic of iran , b university of medical sciences, pediatrics, hamadan, islamic republic of iran objectives: giardia lamblia is the most common intestinal protozoa in developing countries. treatment of the infection with metronidazole, the drug of choice, requires a long course of therapy and produced some side effects. the object of this study is to determine efficacy and side effects of tinidazole in g. lamblia infection. this is a preliminary report of an ongoing trial. methods: a randomized controlled clinical trial was carried out and subjects with g. lamblia infection were treated with tinidazole or metronidazole. tinidazole mg/kg single dose and metronidazole mg/kg three times a day for days were given orally to and children, respectively. parasitological cure was documented when there were consecutive negative stool examinations at Á/ weeks after therapy. results: twenty-one of individuals treated with tinidazole and of children treated with metronidazole had parasitological cure. cure rates between two groups were not significant statistically. no major side effects were observed except one case in metronidazole group who had mild headache and abdominal pain for days. conclusions: we concluded, tinidazole at the dose has efficacy equal of metronidazole in the treatment of g. lamblia infection. because of single dose prescription, short course of therapy and good compliance of patients, this preparation is preferred to metronidazole in giardial infection. ebeid fa, seif el-din sh. theodor bilharz research institute, pharmacology, cairo, egypt b-carotein was given in different doses starting from . to mg/kg body weight (b.w.) for different groups of albino mice days before infection with s. mansoni . infection of animals was done by body immersion using egyptian strain of s. mansoni cercariae/mouse. forty-nine days after infection the animals were sacrificed and hepatic and mesenteric worms were extracted and determined. ova count in liver and intestinal tissue and the total number of worms/animals were also determined in experimental groups comparing with infected control animals. the results indicated marked decrease in number of worms and ova count in both liver and intestine comparing with control ones. this reduction increased significantly with increasing dose. it was concluded that b-carotein could be used as a prophylactic agent against s. mansoni infection. barisic z, babic-erceg a, borzic e, zoranic v, carev m, kaliterna v. department of microbiology, public health institute, split, croatia the aim of this study is to determine frequency of pseudomonas aeruginosa urinary tract infection (uti) in outpatient's population in south croatia and to suggest optimal antimicrobial treatment for these patients. during months long observation period, from total number of examined urine specimens, significant bacteriuria was found in specimens. p. aeruginosa was the sixth most common isolate, it was isolated from specimens ( . %). these specimens were taken from different patients. susceptibility testing was performed by disk diffusion method, and the following results were obtained: resistance to cefibuten occurred in . % patients, to norfoxacin in . %, to ciprofloxacin in . %, to gentamicin in . %, to netilmicin in . %, to amikacin in . %, to ceftazidime in . % and to imipenem in . % patients. p. aeruginosa strains showed better susceptibility to tested parenteral antibiotics than to antibiotics for oral use which complicated treatment in outpatients. the best susceptibility was shown to imipenem, but this drug is inappropriate for use in outpatients setting, so the best choice for treatment p. aeruginosa uti in our outpatients is treatment with ceftazidime, and the second choices are aminoglycoside drugs amikacin and netilmicin. silan l a , breza j b , krcmery v jr. c . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school, bratislava, slovakia , c department of pharmacology, st. elisabeth cancer institute, bratislava, slovakia we studied the clinical efficacy of oral treatment with ciprofloxacin/ cpf/ alone and combined with clarithromycin in patients with complicated urinary tract infection/cuti/ with or without an indwelling catherer. patients were randomly allocated to mg cpf/cpf group/ or to mg cpf plus mg cam/combination group/ for days. evaluation was done on day according to the criteria advocated by the japanese urinary tract infection committee. in patients with a urinary catherer, the combination achieved a higher complete bacterial elimination rate / %/ and clinical efficacy rate / %/ than cpf alone / and . %, respectively/. while no significant difference was found in the bacterial elimination rate between the two groups, the clinical effect of the combination / %/ was superior to that of cpf alone / %/ in patients with an indwelling catherer. the better clinical efficacy of the combination may partly be attributed to the antibiofilm effect of cam in the clinical setting. the results also indicated that difficulties still remain in the treatment of cuti in patients with an indwelling catherer. in conclusion, clinical study suggested that cam might have an inhibitory action on biofilm formation in the clinical setting. combination of cam with other appropriate antimicrobial agents may have a favorable effect on the treatment of cuti. vesicoureteral reflux and urinary tract infections */the management of primary vesico-eureteral reflux in children ps the children studied presented with primary vesicoureteral reflux at derer s universitz hospital in bratislava between and . seven hundred and sixty patients, boys and girls, suffering from primary vesicoureteral reflux in age from months to years were tested and systematically analyzed outcomes data for seven treatment alternatives. key outcomes identified were probability of reflux resolution, likelihood of developing pyelonephritis and scarring, and possibility of complications of medical and surgical treatment. available outcomes data on the various treatment alternatives were summarized and the relative probabilities of possible outcomes were compared for each alternative. conclusions: increased of urinary tract infection, vesicoureteral reflux nephropathy includes early diagnosis, appropriate evaluation, effective atb therapy, and surgery indicated. the main determinants of renal damage are bstruction, age, sex, predisposition on renal scarring, reflux grade and laterality, therapeutic delay, individual susceptibility, bacterial virulence and immunogenetic status. ) the one and only absolute indication for surgical management is failure of medical therapy to prevent chronic recurrent pyelonephritis, renal injury or other reflux complications and eliminations of the reflux condition will minimize their likelihood. genetically conditioned immunopathogenic mechanisms are involved in the pathogenesis of the chronic recurrent pyelonephritis in patient suffering from vur. for most children we recommended continuous antibiotic prophylaxis as initial treatment-medical therapy is based on the principle that reflux often resolves with time, and antibiotics maintain urine sterility and prevent infections while the patients awaits spontaneous resolution. ) vur predispose an individual to renal infection, the immunological and inflammatory reaction caused by a pyelonephritic infection may result in renal injury or scarring. silan l a , breza j b . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school of medicine, bratislava, slovakia elderly patients with uti are believed less likely to be cured by antimicrobial therapy than younger patients. the reasons for this poorer outcome have not yet been clarified. we have investigated the efficacy of antimicrobial therapy in elderly patients with complicated uti. five hundred patients, men and women, who had complicated uti/ symptomatic and symptomatic and were Á/ years of age, were treated with one of three different drugs, one was a never quinolone and two were oral cephems. multivariate logistic regression analysis of treatment outcome revealed that the clinical response was significantly related to general underlying diseases and diseases of the urinary tract, but not to age, symptomatic or asymptomatic uti, or infection site such as the kidney or bladder. we concluded that the clinical effectiveness of an antimicrobial agent was not directly related to age, and that urological examination for underlying disease and control of them is quite important for effective treatment and control of complicated utis, especially in elderly patients. the study on the frequency and antimicrobial resistance of escherichia (e ) coli in urine isolates of patients admitted to maribor teaching hospital in and ps rebersek gorisek j a , baklan z a , unuk s a , novak d b . a department for infectious diseases, teaching hospital maribor, maribor, slovenia , b department for microbiology, regional institute of public health maribor, maribor, slovenia purpose: the aim of the study was to determine the frequency and antimicrobial resistance of escherichia coli isolated from urine samples of patients admitted to maribor teaching hospital in and . the frequency and the antimicrobial resistance were compared between years and . methods: in the prospective study going on between and , all urine isolates from patients at maribor teaching hospital were collected and analysed. urine cultures were done using the modified sanford method. the susceptibility testing was performed by disk diffusion method according to nccls. results: in the year , urine isolates and in the year , urine isolates were analysed. e. coli represented . % of urine isolates in and . % of urine isolates in . e. coli resistance rates (%) to amoxycillin was . in the year and . in the year ; to amoxycillin/clavulate was . and . ; to cefalotin was . and . ; to cefaclor . and . ; to trimethoprim sulfamethoxazole was . and . ; to ciprofloxacin was . and . ; to gentamicin was . and . . conclusion: compared to , the frequency of e. coli isolated from urine samples is similar to that in the year . the resistance to amoxycillin, cefaclor and gentamicin is stable. the resistance to trimethoprim sulfamethoxazole and ciprofloxacin is increased and the resistance to amoxycillin/clavulate and cefalotin is decreased. prevalence of the resistance to metronidazole, furazolidone and nitrofurantoin in helicobacter pylori clinical strains ps de la obra sanz p a , roman jl a , lomas e a , villar h b , lopez-brea m a . a hospital de la princesa, microbiology, madrid, spain , b hospital de san agustin, microbiology, aviles, spain the objective of this study was to determine the prevalence of metronidazole, furazolidone and nitrofurantoin resistance in helicobacter pylori clinical isolates. methods: a total of strains of h. pylori were included in this study. all these were tested against metronidazole, and against furazolidone and nitrofurantoin by an agar dilution method. resistance was defined as: metronidazole, mic !/ mg/l; and mic !/ mg/l for furazolidone and nitrofurantoin. results: sixty-eight strains were resistant to metronidazole ( . %). the mic and mic values were and mg/l, respectively. three of strains ( . %) were furazolidone resistant (mic / mg/l), two of these strains were metronidazole resistant (mic / mg/l) and they had mic of mg/l for nitrofurantoin. the mic and the mic were . and . mg/l, respectively for furazolidone. only one of the strains ( . %) was nitrofurantoin resistant (mic mg/l), this strain was metronidazole resistant (mic mg/l) and had a mic / mg/l for furazolidone. the mic and the mic were . and mg/l, respectively for nitrofurantoin. conclusion: the low frequency of furazolidone and nitrofurantoin resistance, compared to metronidazole suggests that the furazolidone and the nitrofurantoin may be good alternatives to metronidazole for treatment of h. pylori infections. antimicrobial resistance of campylobacter isolated from human origins ps zhukhovitsky vg, drabkina iv. department of bacteriology, botkin hospital, moscow, russian federation the purpose of the study was to determine the antimicrobial resistance of thermophilic enteropathogenic campylobacter spp. (tec) isolated from human under acute diarrhoea in in moscow. among tec strains c. jejuni and five c. coli were identified. the antibiotic tested by disk diffusion test on mueller-hinton agar with sheep blood were ampicillin (a), amoxycillin/clavulanate (ac), imipenem (i), meropenem (m), erythromycin (e), clarithromycin (cl), tetracycline (t), doxycycline (d), gentamicin (g), azithromycin (az), chloramphenicol (ch), lincomycin (l), ciprofloxacin (c), nalidixic acid (na). the resistant rate of the tec isolates was highest for na ( %) followed by a ( %), t ( %), d ( %), n ( %) and cl ( %). a moderate resistance rate was obtained for a ( %), ch ( %), az ( %), ac ( %). none of the isolates demonstrated resistance to i, m and g and four of isolates ( %) were sensitive for all the antibiotics tested. mic to na was estimated as mg/l. among na resistant tec strains ( %) were identified as c. jejuni and one ( %) as c. coli . among c. jejuni and c. coli na resistant rate was and %, respectively. one na resistant c. coli and nine na resistant c. jejuni were resistant to ciprofloxacin. ring c, atanassova v. department of food hygiene and microbiology, school of veterinary medicine, hannover, germany aim of the study: poultry meat is known to be often contaminated with salmonella and other foodborne pathogens and thus has to be considered as a possible source for human infections. the aim of the study was to monitor the resistance of salmonella isolates from poultry meat of different european countries to various antibiotics. material and methods: from september to december a total of samples of frozen poultry meat from france, germany, italy, spain, the netherlands and portugal were examined for the prevalence of salmonella using classical cultural detection as well as rflp-pcr. all isolates were tested for their sensitivity towards ampicilline, kanamycine, ciprofloxacine, tetracycline, trimethoprim, sulfamethoxazole, nalidixic acid and erythromycine using standard procedures. results: from . % of all examined samples salmonella spp. were isolated. of these isolates . % were characterized as salmonella , . % as s. hadar and . % as s. typhimurium . nearly % of all isolates were resistant to erythromycin. resistance towards four or more isolates was observed in several cases. discussion: the consumption of poultry meat, if insufficiently prepared, has still to be considered as a major source for human infection with salmonella spp. the question arises whether the resistance of the isolates to various antibiotics is of clinical importance in the treatment of the patients. objective: to provide insight into the epidemiologic situation of salmonellosis for the nis area (the largest area in serbia, with inhabitants */ , ). methods: the material was processed at the institute for public health (epidemiology and microbiology divisions). isolation of microorganisms was performed on apparatus for rapid identification (vitek-biomerieux) and by applying elisa tests and classical microbiological methods. results: in the period Á/ , salmonella laboratory confirmed cases were reported. the greatest number of diseased in the Á/ years group. the most frequent isolated salmonellae were: s. enteritidis ( . %) and s. typhimurium ( . %), s. hadar , s. agona , s. virchow , s. infantis , s. derby , s. enteritidis showed the greatest sensitivity to antibiotics with the infrequent resistance to ampicillin and trimethoprim-sulfamethoxazole. s. typhimurium showed the greater resistance to the wide spectrum of antibiotics and some isolates were resistant to all antibiotics tested. the less common types of salmonella were sensitive to all antibiotics except trimethoprimsulfamethoxazole and ampicillin. conclusion: specific resistance to some antibiotics was related to serotypes. typhoid fever */retrospective study of cases in lebanon ps tohme a, abboud j, ghayad e. hôtel-dieu hospital, internal medicine, beirut, lebanon objectives: to present epidemiological and clinical features of typhoid fever in lebanon. methods: fifty-two patients were seen at hotel-dieu hospital of beirut between and . diagnostic criteria were positive blood culture for s. typhi or paratyphi and/or a somatic o agglutinin titer ]/ / as determined by the widal test with symptoms suggestive of typhoid fever. we also present an epidemiological study of cases registered by the ministry of health during the same period. results: among the cases, % of the patients' ages were between and years and % were less than years. the overall male to female ratio was . and % of cases were seen on january, february and % on august. among the patients, young adults were the most affected. average duration of symptoms before the diagnosis was / days. the main presenting symptoms were: fever ( %), diarrhoea ( %), abdominal pain ( %) and headache ( %). complications were noted in % of cases and digestive complications were the most prevalent. leucopenia was not a helpful diagnostic marker. s. typhi was the most frequent ( %) serotype identified. resistance to ampicilline was %, to cotrimoxazole and chloramphenicol % for each. the mortality rate was %. conclusion: typhoid fever is still an endemic disease in our country and the occurrence of resistant strains of s. typhi will favor ceftriaxone or fluoroquinolones in the treatment. maaloul i a , hammami b a , zambaa f a , elleuch r a , hammami a b , -ben jemaa m a . a chu hedi chaker, service des maladies infectieuses, sfax, tunisia , b chu habib bourguiba, laboratoire de microbiologie, sfax, tunisia although its not very frequent, the psoas abscess is not an exceptional entity. in order to specify its clinical, biological, radiological and evolutionary features, a retrospective study has been led in our service, on a period of years (january Á/december ). on the whole, cases have been listed. they were men and women. the age average was years (extreme Á/ years). the study did not find any underlying diseases, except diabetes mellitus for three patients. the clinical symptoms were dominated by fever with abdomino-lumbar aches ( cases), and psoitis (eight cases). biology showed an inflammatory syndrome in all cases and a hyperleucocytosis in cases. the diagnosis of psoas abscess, evoked on clinical data, has been confirmed by the imagery data: ultra-songraphy ( cases), ct scanning (six cases), magnetic resonance imaging (three cases). the tubercular etiology has been confirmed in six cases, among which two were associated to escherichia coli (one case) and to brucella melitensis (one case). the other etiologic agents were dominated by staphylococcus aureus (eight cases), b. melitensis (two cases), e. coli (one case), bacteroides fragilis (one case), streptococcus anginosus (one case), fusobacterium nucleaticum (one case) and candida glabrata (one case). all patients received an anti-infectious treatment adapted to the micro organism in question. a drainage of the abscess has been realized for patients (percutaneous: nine cases, surgical: six cases). the evolution was favourable for patients. however, two patients had a relapse after stopping the treatment. conclusion: the diagnosis of the psoas abscess, difficult on the clinical data, is based on the imagery techniques (us, ct, rmi). the percutaneous drainage guided by the imagery is recommended (in an etiological and therapeutic aim). associated to an adapted antibiotherapy, it allows to defer a surgical drainage. zezoski mbz, nikolova o, gavriloski pmg. medical center, infectious diseases, prilep, the former yugoslav republic, macedonia purpose: to make a list of the most frequent abdominal changes in patients with human brucellosis. materials and methods: there were new patients with human brucellosis, between and . diagnosis was made using standard clinical, biochemical and serological investigations (bab, wright, coomb's, rvk, -mercaptoethanol, elisa igm and igg), and specially ultrasound examination of the abdomen and retro peritoneum. results: weight loss is the most frequent change, presented in ( . %) patients. follow atypical abdominal pain in ( . %), vomiting in ( . %), diarrhea in ( . %), enlarged liver in ( . %), enlarged spleen in ( . %) and hepatic lesion with increased ast and alt in ( . %). conclusion: although frequent, abdominal changes seldom could be missed in patients with human brucellosis. we recommend routine ultrasound examination with standard biochemical test for liver function, due to avoid unnecessary complications. osteoarticular complications are common in brucellosis. the most common site of involvement is the sacroiliac joint. the osteoarticular complications such as, sacroiliitis and spondylitis are diagnosed with radiologically. in the present study, we aimed to determine the severity (grade) of sacroiliitis by using some laboratory parameters such as esr, crp and tube agg test. seventy-two ( male, female) patients with brucellosis were included in the study. osteoarticular involvement was present in patients. the most common osteoarticular finding was sacroiliitis in the patients ( %). twenty ( ) healthy subjects were formed the control group. there was statistically significant difference between patients and controls regarding esr, crp, and tube agg test (p / . , . , . , respectively). in addition, sacroiliitis has an effect on esr and crp. there was a positive correlation between the grade of sacroiliitis and the value of crp (p / . , r / . ). in conclusion, it has been suggested that, crp may be used as an auxiliary or a secondary parameter in grading sacroiliac joint involvement in brucellosis. magira ee a , papandreoy s a , gounaris t a , spirelis ma a , tasopoulos g a , anagnostopoulou m b , paniara o b , gounari p a , sioulal e a . a evagelismos, internal medicine, athens, greece , b evagelismos, micro- a -year-old greek farmer was admitted to the hospital because of painful scrotal swelling, hepatosplenomegaly, lumbar pain and lowgrade fever accompanied by profuse sweating. his life style included occupational animal exposure ingestion of raw milk and dairy products. the laboratory data were within the normal ranges. focal hypoechoic right testicular lesions, swelling of the concurrent epididimis along with an increase in the vascularity of the right testis were seen on an echo examination. these findings were consisting in unilateral epididimo-orchitis. a ct scan of the lumbar spine area showed a decrease of the signal intensity localized in the anterior aspect of l vertebral body at the diskovertebral junction involving the subchondrial parts of the l and s vertebrae standard tube agglutination test was positive for antibodies to brucella melitensis (titer !/ / ). cultures of blood specimens were positive for b. melitensis . the patient had been given to a combination of antibiotics with doxycycline, streptomycin and rifampin. a remarkable improvement of his clinical condition was showed weeks later. this case illustrates the following point: in areas in which brucellosis is endemic when scrotal abnormalities are seen the possibility of genitourinary tract complications of brucella should be considered. pappas ga a , akritidis nk b , mastora m b , tsianos e a . a university hospital, internal medicine, ioannina, greece , b 'g. hatzikosta ' hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and forms of complications associated with brucella infection. patients and methods: we studied the most recent patients, in all larger series approaching , diagnosed as suffering from brucellosis, and assessed the presence of signs and symptoms of arthritis and spondylitis, or other forms of bone involvement. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures or cultures from other media. results: osteoarticular complications were noted in patients, presenting with arthritis, and presenting with spondylitis. eight patients presented with genitourinary complications, either orcheoepididymitis (four patients), or hematuria resolving with treatment (four patients). meningitis was present in two patients. gastrointestinal complications (vomit and diarrhea) were present in three patients, while one patient presented with ascites. respiratory tract complications, in the form of pneumonia (four patients) or bronchitis (three patients) were noted in seven patients, while one patient with pneumonia exhibited pleural fluid. skin rashes, of macular type, were present in three patients. no patient presented with complications from the heart. hematologic complications were frequent, in the form of severe (one patient) or moderate (two patients) pancytopenia, isolated thrombocytopenia (three patients), or lymphocytosis (eight patients). akritidis nk a , pappas ga b , mastora m a . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and modes of bone and joint involvement in the course of brucellosis. patients and methods: we studied the most recent patients, in all larger series approaching , diagnosed with brucellosis, and assessed the presence of arthritis and spondylitis. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures. results: twenty-three patients exhibited a form of osteoarticular involvement. arthritis was present in patients, most often involving the knees, but also the hips, elbows, even smaller joints as intephalangeal joints of the hand. synovial fluid, when aspirated, was often characterised by an intense mononuclear infiltrate. spondylitis was present in patients, most often involving the lumbar spine, but also the thoracic spine. the characteristic erosion on the upper anterior crest of the vertebral body was visible in plain x-rays in three patients, while mri and bone scan were helpful in other cases. discussion: osteoarticular involvement in the course of brucellosis is the most common focal presentation of the disease. acute brucellosis is often accompanied by bone and joint ache, especially of the lumbar spine, still frank involvement in the form of arthritis and spondylitis is not rare. arthritis usually presents in the acute form of the disease, while spondylitis tends to be characteristic of a chronic form of the disease, often necessitating prolonged use of antibiotics. bosilkovski m, krteva l, caparoska s, grozdanovski k, sajn b. clinic for infectious diseases and febrile conditions, medical faculty, skopje, the former yugoslav republic, macedonia one hundred and twenty-six patients with brucellosis were studied prospectively. seventy-eight ( %) of them had osteoarticular involvement. peripheral arthritis in ( %) patients was the most frequent, followed by spondilitis in ( %), sacroiliitis in ( %), rarely bursitis, tendinitis and osteomyelitis. the overall male to female ratio was : . their average age was (sd ) years. direct contact with animals was the reason for acquisition of the illness in % of patients, in % alimentary or aerogenous route was incriminated, and in % the route of aqisition was unknown. the average duration of the symptoms from the onset to establishing the diagnosis was (sd ) days. the main presenting symptoms were joint pain ( %), sweating ( %), fatigue ( %) and fever ( %). hepatomegaly was present in %. in % of patients, involvement of some other system was evident. comparison with patients, who did not have osteoarticular illness, showed that patients with osteoarticular involvement had significantly more often joint pain, fatigue, weight loss and more prolonged duration of symptoms before the diagnosis was established. doxycycline and chloroquine as combination therapy for chronic q fever endocarditis ps calza l, attard l, manfredi r, chiodo f. division of infectious diseases, university of bologna, s. orsola hospital, bologna, italy introduction: endocarditis is the main clinical manifestation of chronic q fever, occurring in about Á/ % of all reported cases, and it is diagnosed almost exclusively in patients with either cardiovascular abnormalities or an immunocompromised condition. case report: a -year-old caucasian male patient with biological prosthetic aortic valve was first hospitalized because of an interstitial pneumonia. six months later, our patient was re-admitted owing to intermittent fever, chills and weight loss. echocardiographic study showed a small vegetation of mm in diameter on left cusp of aortic valve. serology for coxiella burnetii revealed a complement-fixing igg antibody titer to phase i antigen of more than : , consistent with chronic q fever endocarditis. antimicrobial therapy with i.v. doxycycline and oral chloroquine was started, leading to a clinical and echocardiographical recovery. therapy was continued by oral doxycycline and chloroquine, and the patient remained asymptomatic during a -year follow up. conclusion: the optimal treatment of q fever endocarditis has not been well established: the most effective antimicrobials are fluoroquinolones and rifampin, but chloramphenicol, doxycycline and trimethoprim are also useful. the role of chloroquine in combination with doxycycline seems to be promising, because chloroquine may increase the lysosomal ph, enhancing the doxycycline bactericidal activity. akritidis nk a , pappas ga b , mastora m a , liappis e a , tsianos e b . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scopes: to review the incidence and the forms of lower respiratory tract infection in patients suffering from q fever, and their clinical and radiological characteristics. patients and methods: twenty-seven patients diagnosed as suffering from q fever, were assessed for the presence of lower respiratory tract infection. the diagnosis was confirmed serologically. results: thirteen patients expressed lower respiratory tract pathology, as confirmed by clinical examination and chest x-ray. in of these patients the main cause of admission was respiratory tract symptoms, ranging from dry cough to hemoptysis. chest x-ray was pathological in patients: patients had lobar pneumonia, two of them multiple nodular opacities, and one of them bronchopneumonia. hepatitis was a common finding. all patients were treated with tetracycline. discussion: although coxiella burnetii infection is acquired via the respiratory tract, it is paradoxical that symptoms attributed to the lung are not invariably positive. q fever pneumonia is an atypical pneumonia that usually follows a benign course. diangostic suspicion is usually raised by the epidemiologic pattern and the accompanying mild hepatitis. pleural effusion is not a common finding. the usual radiologic appearance of q fever pneumonia is that of a lobar or segmental pneumonia. one important aspect of q fever pneumonia is its common presentation in the form of multiple nodular opacities often necessitating the exclusion of malignancy. ( ), culture ( ), and serology'/culture ( ). there were Á/ cases per year, mainly in october ( %). a history of exposition to hare was present in / ( %) and to marmot in / ( %). skinning ( / %), animal contact ( / %), bite ( / ) and wound during bait preparation with frozen meat for hunting ( / ) were noted. the initial clinical presentation was ulceroglandular ( %), glandular ( %) and pneumonic ( %). the involved nodes distribution was axillary ( / ), cubital/axillary ( / ) and cubital ( / ). median incubation period was days (range Á/ ); time to consultation days (range Á/ ), and time for effective treatment days (range Á/ ). an initial diagnosis of tularemia was made presumptively in %. effective antibiotic regimen used was aminoglycosides in % ( / ), and tetracyclines in % ( / ). note that intravenous netilmicin was used in cases. complication rate was % ( / ) with one death ( %), and was associated with delay in effective treatment ( !/ days of illness) (p b/ . ). conclusion: in our area tularemia occured mainly in male population, during autumn, with a short incubation period and history of hare contact. delay before appropriate treatment increased the complication rate. bompolaki i, doukakis s, triantafillidou d, polimili g, kastanakis m, nikiforakis k, vittorakis e, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece a severe frontal and/or retroorbital headache represents the most common neurologic manifestation of murine typhus. other neurologic manifestations as confusion, stupor, nuchal rigidity and in severe cases delirium, extreme agitation or coma appear less commonly. eightyfour patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi, were studied from our team. seventy-four patients ( %) presented headache and nine patients ( %) presented confusion. one patient ( . %) presented nuchal rigidity in combination with severe headache and confusion giving us the suspicion of meningitis. in this case a lumbar puncture was performed emergently and the cerebrospinal fluid (csf) was examined. the findings of csf were proteins: mg/dl, wbc: /ml and glucose: mg/dl and its culture was negative. all patients were treated with a specific anti-rickettsial treatment. the outcome of murine typhus was favorable for all patients ( %). no one patient presented neurologic sequelae. conjunctivitis usually accompany rickettsial diseases such as rocky mountain spotted fever, epidemic typhus and murine typhus. eightythree patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january and the first semester of . the clinical examination of these patients revealed the presence of conjunctivitis in / patients ( . %). in the same time these patients referred retroocular pain and mild photophobia. this study showed that in murine typhus the injection of conjunctivae is rather common. almost a quarter of the patients presented conjunctivitis despite the fact, that this ocular manifestation is less severe than in other typhus and spotted fevers. eighty-three patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january and the first semester of . three blood samples were obtained from each patient for the study of their hematological abnormalities. the first sample was obtained on admission, the second sample weeks after the first, the third sample, month after the second. on admission / patients ( %) presented anemia, / patients ( %) presented leukopenia and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. two weeks later anemia was presented in / patients ( %), / patients ( %) presented leukopenia, / patients ( %) presented leucocytosis and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. one month later / patients ( %) had anemia and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. our study showed that early thrombocytopenia and anemia are frequent in murine typhus and that white blood cells count is usually normal. renal function in murine typhus: a study of cases ps doukakis s, polimili g, triantafillidou d, kastanakis m, vittorakis e, palla k, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece the clinical course of murine typhus is usually uncomplicated and the mortality rate is low ( b/ %). advanced age and prolonged interval before administration of a specific anti-rickettsial treatment are correlated with severity of the disease. renal function in murine typhus is usually unaltered except in elderly patients with prolonged hypotension. eighty-three patients with compatible clinical status of murine typhus and high serological titres of antibodies against rickettsia typhi, were studied from our team, during a period of time between january and the first semester of . three blood samples were obtained from each patient for the study of their renal function. the first sample was obtained on admission, the second sample approximately weeks after the first, the third sample, taken from the half of the patients, was obtained one month after the second. on admission / patients ( . %) presented acute renal failure. the outcome of murine typhus was favourable for all patients ( %). the four patients who presented acute renal failure reversed after the administration of anti-rickettsial treatment and careful administration of fluids. in murine typhus the induction of hypovolaemia insufficiently corrected by normal homeostatic mechanisms may lead to prerenal azotaemia. in these cases the immediate onset of an antirickettsial treatment and the correction of hypovolaemia are essential for the rapid clinical improvement of the patient. experimental ocular toxoplasmosis: clinical, histopathological, immunological and therapeutic studies ps el zawawy lae a , hammoda na a , allam sr a , ali sm a , galal as b . a faculty of medicine, parasitology, alexandria, egypt , b faculty of medicine, ophthalmology, alexandria, egypt the purpose of the study was to investigate clinical, histopathological, immunological and therapeutic features of an experimental model of ocular toxoplasmosis in sensitized and non sensitized rabbits and to assess the influence of treatment by interleukin (il- ) on ocular lesions. the results obtained was that 'toxoplasma' retnochoroiditis developed in both groups of rabbits with more pronounced effect in non sensitized animals. administration of il- improved ocular lesions in both groups with more evident effect in sensitized rabbits. immunological findings were consistent with clinical and histopathological observations. the conclusion reached was that; ocular lesions were manifested in non sensitized rabbits more than in sensitized ones. il- revealed a significant impact on improving the host defense against toxoplasm infection in eye. immunotherapy with il- would open the way for a new range of treatment based on immunomodulation. express-diagnostic of streptococcus antigen for the adequate antibiotic therapy in patients with pharyngitis ps pertseva to, konopkina li, kireeva tv. dsma, internal medicine , dniepropetrovsk, ukraine the purpose of the study: evaluation of effectivness of streptococcus express-diagnostic for the adequate antibiotic therapy in patients with pharyngitis. results: we deal with clinical and microbiological comparison in patients with pharyngitis. using of streptococcus antigen express diagnostic in swabs from the backside of pharynx allowed to get positive results in the seven cases ( . %). following cultural study has confirmed these results. positive test was more probable in patients with pronounced fever (more than c), headache, weakness and in cases associated with chronic tonsillitis. isolated streptococcus pyogenes was susceptible to ampicillin, claritromicin, erytromicin, azytromicin, , clindamicin, ceftriaxon, levafloxacin, oxacillin, cefuroxim, roxytromicin. conclusion: using of the express diagnostic of streptococcus antigen allows to restrict groundless prescription of antibiotic therapy in patients with other types pharyngitis (i.e. viral, candidal etc.). alabaz d a , turgut m a , kocabas e a , tumgor g a , yaman a b , alhan e a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey chickenpox is a common viral infection that usually follows a benign, self limited course in healthy children. the most common complication in children with varicella is superimposed cutaneous infections with pyogenic bacteria (streptococcus pyogenes and staphylococcus aureus ). varicella gangrenosum, a necrotising soft tissue infection complicating the vesicular eruption of chickenpox, is rare. here we present three cases with necrotising soft tissue infections following chicken pox. these children were admitted because of common crusted lesions and necrotising soft tissue infection over the neck, the back, and the inguinal area. they all had the contact history and ensuing vesiculopapular rush. these infections were caused by group a streptococci in two cases, and s. aureus in one case. after instituting of appropriate antibiotic therapy, each patient underwent a surgical exploration with fasciotomies and debridement. widespread use of varicella vaccine may decrease invasive infections in children, adolescents, and adults, thus decreasing the burden the disease with its complications impose up on the family and the society . cefprozil is a second generation cephalosporin. the aim of this open, multicentre, non-comparative study was to investigate the efficacy and safety of cefprozil in the treatment of streptococcal tonsillopharyngitis. fifty-eight patients were clinically assessed for signs and symptoms of streptococcal infection. laboratory confirma-tion was sought using three tests; culture, rapid strepto test and estimation of antistreptolysin (aso). one or more tests were done in of the patients. treatment was for days, mg/kg per day in children, mg per day in adolescents. patients were again clinically assessed on the th Á/ th day. the results showed clinical success in patients ( . %) and in of the who had laboratory tests ( . %). two patients had treatment withdrawn because of nausea and abdominal pain ( . %). of the patients with laboratory tests at least one test was ositive. the most helpful was the strepto test giving the quickest positive result in % of those tested ( / ). culture was positive in % of those tested ( / ). the aso test was of limited value. in conclusion, cefprozil showed high clinical efficacy and safety in the treatment of streptococcal tonsillopharyngitis. tsiara s, militadou g, milionis c, elisaf m. internal medicine department, university of ioannina, ioannina, greece streptococcus group b agalactiae (gbs) is a rare pathogen for healthy male adults. we present an old man in whom (gbs) was isolated in blood cultures. case report: a -year-old man was admitted to the hospital in order to investigate osteolytic lesions in the lumbar spine. two weeks before, he experienced severe low back pain radiating to the right leg and fever arising to . c with chills and rigors. a gbs was isolated from blood cultures and treatment with penicillin was initiated. a spine ct scan revealed osteolysis in the t and s vertebrae and the patient transferred to us. he was a previously healthy man. he received only antihypertensive therapy. on admission he was afebrile with arthralgias and myalgias. on clinical examination there was tenderness on the right pleurospondylic angle radiating to the right leg. laboratory data on admission: hb: . g/dl, wbc: . )/ /l, esr: mm/h. biochemical values, serum protein electrophoresis, rectal examination and a prostatic specific antigen (psa) were normal. a bone marrow aspiration and biopsy were negative. a transoesophageal ultrasound revealed vegetation on the right cusp of the aortic valve, with low grade regurgitation. an mri of the lumbar spine revealed infectious myositis with concomitant osteomyelitis involving the l , l vertebrae without any evidence of osteolytic lesions. a thorough investigation did not revealed any underlying immunosuppressive disease. treatment with vancomycin and gentamycin iv was initiated and the patient discharged from the hospital in excellent health after weeks. discussion: gbs infections usually occur in neonates and pregnant, or in adults with underlying disease as diabetes mellitus or immunosuppression. the most common site of infection is soft tissue. we present this case because gbs infections are rare in the elderly in the absence of underlying disease. common sites of involvement are soft tissues, while bone, joints, and heart valves account to Á/ % of the involved organs. although our patient had more than one site of involvement he responded well to medical treatment without surgical debriment or heart valve replacement. objective: to evaluate the safety and efficacy of rhugm-csf in combination with broad-spectrum antibiotics for the treatment of ccnf. methods and results: a retrospective review of all patients (pts) with ccnf treated with antibiotics'/rhugm-csf at our hospital from january to december was performed. five patients were identified with the diagnosis of ccnf. ages ranged from to years; there were three women and two men. dental infection was the most common source of ccnf in %. one patient had acute tonsillitis leading to ccnf. all cases studied experienced infection of the neck with spread into the submandibular, submental, sublingual, retropharyngeal, and parapharyngeal spaces. all infections were polymicrobial. diabetes mellitus was a co-morbidity in one case. all pts were treated with dual antibiotic coverage (vancomycin'/meropenem), rhugm-csf ( mcg/kg/daily given s.c.) and aggressive wound care. rhugm-csf was given for Á/ days. in spite of the severity of the infection all pts recovered and do not experienced local or systemic complications. discussion: ccnf is a severe bacterial infection of the cervical fascia resulting in extensive fascial necrosis with widespread undermining of the surrounding tissues. prompt antibiotic therapy combined with rhugm-csf resulted in a % overall survival in our experience. to our knowledge, this is the first report describing the successful treatment of ccnf with use of broad-spectrum antibiotics combined with rhugm-csf. the following case adds to the clinical manifestations and course of meningococcal disease. a previously healthy -year-old girl presented acutely with high fever purpuric rash including conjuctival haemorrhages and hypotension. the child had also neck stiffness. a presumptive diagnosis of meningococcal septicaemia was made and treatment with penicilline, chloramphenicole, fluids and inotropes was initiated. laboratory investigations showed wbc: /ml, hb: . g/dl, hct: . %, esr: mm in the first hour, pt: s, aptt: s. neisseria meningitidis group b was isolated from the blood cultures. csf obtained after antibiotics were started did not grow n. meningitidis . the patient had an adverse outcome. she died after h of hospitalization. this patient developed a fulminating meningococcal septicaemia. shock is a clinical diagnosis arising from the failure of compensatory mechanisms that maintain perfusion of vital organs at the expense of non vital. septic shock results from loss of circulating plasma volume due to increased vascular permeability and maldistribution of intravascular volume. in young children there is a prevalence of serogroup b meningococcal disease which can be explained by the immaturity of the immune system and by the fact the group b capsule synthesis is known to inhibit alternative complement pathway activation. this case emphasizes the need for further protection against n. meningitidis group b. . results: forty-eight patients were included in this study. the etiological agents were: viral (n / , . %, mean ('x ) age / years), streptococcus pneumoniae (n / , . %,'x age / ), neisseria meningitidis (n / , . %, 'x age / ), s. viridans (n / , %, 'x age / ), p. multocida (n / , age ). the peak incidence of bacterial meningitis was in winter (pneumococcal %, meningococcal %, s. viridans %) .the cerebrospinal fluid (csf) findings in viral meningitis were 'x white cells / /mm , 'x pmn / %, 'x glucose csf/ serum . , 'x protein mg/dl and in bacterial meningitis were 'x white cells / /mm , 'x pmn / %, 'x glucose csf/ serum / . , 'x protein / mg/dl, gram stain was positive in %, culture was positive in %. all pneumococcal and meningococcal strains were susceptible to penicillin. the case fatality rates for pneumococcal and meningococcal meningitis were and . %, respectively. conclusions: the cases of bacterial meningitis were according to typical epidemiological features of age and season. the case fatality rate of pneumococcal meningitis appear to be high regardless of susceptibility to penicillin. none had received pneumococcal vaccine prior to becoming ill. diagnosis and therapy of meningococcal meningitis */trend and particularities of a 'romanian model' ps lintmaer i, moroti r, popescu a, popescu c. institute of infectious diseases matei bals , unit , bucharest, romania background: newer diagnosis methods and antimicrobials are expected to change the management of menigococcal meningitis (mm) and to improve its prognosis. objectives: to determine the changes in the diagnosis methods and therapy of mm patients in a infectious diseases hospital. to compare mm management in bucharest with literature data. methods: retrospective rewiew of mm in adult patients hospitalized over a -year period. our results were compared with other studies made in the s, taken from medline. results: there were episodes of mm during the study period ( episodes in Á/ and episodes in Á/ ) . we noticed a defined diagnosis increase and increased blood culture specificity. the antimicrobial monotherapy was maintained but penicilin was replaced by ceftriaxone. hhc was replaced by dexamethasone in pathogenic therapy. we noticed a shorter length of treatment and a reduced lethality. the most important differences between our results and other studies are: monotherapy regimens are less frequent and therapy lengths are longer; however, prognosis is similar. conclusions: the mm management has been modified in the last Á/ years: prognosis is improved, but the changes do not bring clear cost/ effective benefits. tiouiri th, kilani b, amari l, zouiten f, kanoun kf, ghbontini a, ben chaabene t. rabta hospital, infectious diseases, tunis, tunisia objectives: in order to study epidemiological, clinical and therapeutical characteristics of the infective endocarditis (ie). methods: we reviewed all the cases of ie fulfilling the duke criteria. data were collected during a -year period ( Á/ ) in the unit of infectious diseases. results: one hundred and eight cases were identified. the mean age was . years. sex ratio was . . eighty-five ie ( . %) occured in patients with native valve, and ie ( . %) with prosthetic valve. fever was the most common sign, % had a congestive heart failure, . % had cutaneous signs. the most common primary focus of ie was orthodontic. blood cultures were positive in % of cases. in one case, serological test identified rickettsia conori . streptococci and staphylococci were isolated in . and . %, respectively. echocardiography detected abnormalities in . % of cases. rheumatic heart disease was the most predisposing condition. empirical therapy was based on combination of b lactam with aminoglycoside. recovery was obtained for patients. cardiac surgery was performed in cases. overall mortality rate was . %. conclusion: ie affects young persons. prevention needs eradication of acute rheumatic arthritis. a major outbreak of legionnaires' disease in spain: diagnostics aspects ps guerrero c a , toldos cm a , yagü e g b , ramírez c a , rodríguez t a , -segovia m a . a hospital morales meseguer, servicio de microbiología, murcia, spain , b departamento de microbiología, facultad de medicina, universidad de murcia, murcia, spain objective: to evaluate the value of different methods (serological tests, culture of respiratory secretions, blood cultures and urinary antigen testing) for the diagnosis of legionella pneumophila pneumonia during an outbreak in spain. results: we have studied patients from a recent outbreak of legionellosis in murcia (spain). the diagnosis was achieved in patients. urinary antigens were positive in patients. in the patients with urinary antigen negative the serological response was demonstrated by indirect immunofluorescence (ifa) in patients. all blood cultures processed were negative. sputum samples were obtained from patients, of these l. pneumophila was isolated only in six patients. in all of them direct immunofluorescence test (dfa) was positive. conclusions: although the serological diagnosis was the most sensitive method the urinary antigen testing was of great value in the rapid diagnosis of the legionella's outbreak in murcia. the isolation of l. pneumophila by culture showed a poor sensitivity probably because of the low severity of the illness. purpose: to evaluate chloramphenicol for an initial empiric antibiotic treatment of purulent meningitis in adults. study group: one hundred and twenty patients hospitalized for the diagnosis purulent meningitis in the department in years Á/ , males and females, age range Á/ years, mean age . years. children up to years were not included. method: a retrospective analysis of the study group focused on antibiotic treatment both initial and changes during treatment. results: chloramphenicol was used as an initial antibiotic in ( %), rd generation cephalosporin in ( %), penicillin in ( %), ampicillin in five ( %) and other antibiotic in five ( %), respectively. during treatment chloramphenicol was switched for rd gen cephalosporin in seven of patients with streptococcus pneumoniae meningitis and in five of patients with meningitis of unknown etiology. the reason for the change was non-improving csf formula in three, persisting csf culture positivity in two and persisting coma in seven patients. conclusion: because of repeated necessity to switch chloramphenicol for rd gen cephalosporin during treatment of purulent meningitis of pneumococcal and unknown etiology the initial treatment strategy was changed in . third gen cephalosporin is now used as a first choice antibiotic, what is in consent with international recommendation of treatment. to evaluate and compare groups treated initially with chloramphenicol and with rd gen cephalosporin will need several more years. low prevalence of multi-drug resistant mycobacterium tuberculosis in jerez de la frontera-cadiz (spain) ps alados jc, aller ai, de miguel c, de francisco jl, calbo l. hospital del sas-jerez , microbiologia, jerez de la frontera, cadiz, spain introduction and aim: previous reports indicate that multi-drug resistance mycobacterium tuberculosis (mtb) is an worldwide problem. the aim of this study was to review the resistance of mtb to the first-line antimycobacterial agents in our area. material and methods: over a period of years ( Á/ ), strains of mtb isolated from non-treated patients with tuberculosis ( strain in , in , in and in ) were studied. these isolates were tested for in vitro drugs susceptibility to isoniacid-i, rifampicin-r, streptomycin-s and ethambutol-e using the bact/ alert method (organon teknica) as described by the manufacturer. results: our results showed that . % ( / ) strains were resistant to one or more drugs. single drug resistances were detected on nine strains to i ( . %), one to r ( . %), two to s ( . %), one to e ( . %). three mtb strains were resistant to more than one drug but only one was multi-drug resistant (i'/r).the incidence of i-resistant strains over the period fell from % in to . % in . conclusions: ( ) multi-drug resistance is not an important problem in our area. ( ) isoniacid resistance was declined to an admissible level. to investigate the anti-tuberculosis drug resistance pattern of pulmonary tuberculosis isolates in southern taiwan, an area with higher tuberculosis incidence and mortality than other regions of the island, we performed a hospital-based surveillance at a southern taiwan medical center from to . the combined drug resistance rates to at least one of five first-line agents was . %, and to both isoniazid and rifampin (multi-drug resistance, mdr) was . %, indicating high resistance rates compared with those reported in the who/iuatld global project and in northern taiwan. the resistance rates to two second-line drugs, cycloserine, and kanamycin, were . and . %, respectively. a significant decreasing trend in resistance rates to all drugs except streptomycin was observed during the -year period. though combined drug resistance rate may not be the most accurate tool as it includes previously treated cases which inflates the resistance rate, the observation of trends in the susceptibility of pulmonary tuberculosis in accompany with the increasing percentages of tuberculosis patients receiving complete treatment course and the decreasing percentages of cases lost of follow-up in kaohsiung after the institution of new governmental regulations for case management in suggest the usefulness of intervention programs. lipid profile in patients with multidrug resistant pulmonary tuberculosis ps extrapulmonary tuberculosis may sometimes present with confusing clinical manifestations. a -year-old female patient was admitted with a history of recurrent supra-sternal abscess for year. mri confirmed the presence of sternal osteomyelitis and an anterior mediastinal mass. the diagnosis of tuberculosis was proved by histologic examination and acid-fast stain. the patient was treated with first-line agents, which isoniazid, rifampin, pyrazinamide, and ethambutol. tobacco smoking as a factor of the decrease of chemotherapy effectiveness and of the development of the drug resistance in patients with pulmonary tuberculosis ps shprykov as a , zhadnov vz a , shprykova on b . a medical academy, department of tuberculosis and lung diseases, nyzhny novgorod, russian federation , b medical clinic for infectious # , laboratory of bacteriology, nyzhny novgorod, russian federation studies of the effect of smoking on the results of chemotherapy of patients with tuberculosis of lungs. intensive tobacco smoking slowed down clearance of positive sputum and of lung tissue destruction (in smokers . % and . vs. . % and . % in nonsmokers, p b/ . ). drug-resistant mtb strains have been found to be isolated more often in smokers */ . vs. . % in non-smokers, p b/ . . resistance to streptomycin and isoniazid prevailed, reaching in heavy smokers . and . %, respectively. resistance to rifampicin increased . times. the concentration of rifampicin in the blood serum of heavy smokers decreased in . times. clinical data are in complete correlation with the findings of our experiments: % of experimental cultures developed resistance to streptomycin, isoniazid and less to rifampicin in the study of drug sensitivity under the effect of tobacco smoke condensate. thus, our findings show the development of drug resistance in mtb under the effect of components of tobacco smoke and also showed less effectiveness in therapy. kilani kb, ammari la, tiouiri ht , ben chaabène tbc. rabta hospital, infectious diseases, tunis, tunisia guerrero c a actinomycosis is a chronic disease characterized by abcess formation, tissue fibrosis and draining sinuses. it is caused by anaerobic bacteria belonging to the genus actinomyces. thoracic actinomycosis may involve the lungs, pleura, mediastinum or chest wall. the authors present a case of pulmonary actinomycosis complicating a cervicofacial site. a -year-old man with a history of cervicofacial actinomycosis treated by penicillin g years ago was admitted because of right-sided chest pain for months before presentation, cough and fever. physical examination shows a painless indurated mass in the neck with multiple fistula of the sternum. chest radiograph and ct scan revealed a mass in the upper lobe of the right lung with an infiltrate of the upper lobe of the left one. magnetic resonance imaging confirms the previous lesions, with extending process to the sternum and right collar bone. bronchoscopy was performed while patient was on antimicrobial therapy. culture of bronchoalveolar lavage fluid was negative. transbronchial biopsy was not conclusive. fungal serologies were negative for aspergillosis, histoplasmosis, blastomycosis. bacterial examination of purulent drainage from sternal wound shows inclusion bodies identified as actinomyces. he was treated then with penicillin iv for months, than switched to doxycycline. after months of treatment, he is asymptomatic with radiological improvement. kanellopoulou m a , skarmoutsou n a , iglezos i b , mylona e a , martsoukou m a , apostolopoulou f b , papafrangas e a . a laboratory of clinical microbiology, sismanoglio general district hospital of attica, athens, greece , b nd department of pneumology, sismanoglio general district hospital of attica, athens, greece introduction: achromobacter xylosoxidans is a rare human pathogen. it is an important cause of bacteremia in patients with cardiac diseases, malignancies and immunosuppression. it has been recently recognized as an emerging microorganism in cystic fibrosis (cf), whose its pathogenic role is unknown. aim: to investigate the sensitivity to eleven different antibiotics of a. xylosoxidans strains isolated from adults with cf, during . methods: the susceptibility was tested by kirby bauer and microdilution methods (wider i, fransisco soria melguizo, s.a.), according to nccls recomendations. results: the resistance to antibiotics was as follows : gentamicin, tobramycin, aztreonam %, amikacin %, ceftazidime %, ticarcillin %, carbapenems, cotrimoxazole %, colistin % and piperacillin %. conclusions: ( ) a. xylosoxidans isolated from cf patients appeared resistant to the most usually tested antibacterial agents. ( ) colistin which is used as aerolized antibiotic for cf patients seems to be effective in the half of the isolated strains. ( ) piperacillin was the most active antibiotic against a. xylosoxidans . morris ka, perry jd, jain s, gould fk. microbiology department, freeman hospital, newcastle upon tyne, uk alafosfalin, l-alanyl-l- -aminoethylphosphonic acid, is an antibacterial peptide mimetic which inhibits peptidoglycan biosynthesis. we report the in-vitro activity of this compound in combination with ceftazidime, cefsulodin, fosfomycin, piperacillin/tazobactam, aztreonam, ciprofloxacin and timentin. drug combinations were evaluated against burkholderia cepacia strains, and pseudomonas aeruginosa strains isolated from patients with cystic fibrosis. for this purpose a chequerboard technique was adopted using doubling dilutions of each antibiotic incorporated into a highly defined agar medium free of antagonists. the minimum inhibitory concentrations (mics) and fractional inhibitory concentrations (fics) of all the antibiotic combinations were determined which revealed the antibiotic interaction occurring. alafosfalin in combination with ceftazidime, meropenem, piperacillin/tazobactam and timentin demonstrated the highest percentages of synergy in both b. cepacia and p. aeruginosa . synergy was shown to occur in , , and % of b. cepacia strains respectively, and in , , and % of p. aeruginosa strains. these four combinations were re-tested with all isolates and the results were shown to be reproducible. alafosfalin shows potential as a treatment for cystic fibrosis patients colonised with p. aeruginosa and/or b. cepacia , when applied in combination with these agents. community-acquired pneumonia */does its aetiology matter? ps lintmaer i, popescu a, popescu c. institute of infectious diseases matei bals, unit , bucharest, romania the aetiology of a pneumonia is not one of the criteria used to determine pneumonia's severity. however, it is accepted that identified based Á/based therapy is less expensive (and possibly more effective). objectives: our study aims were to: ( ) to evaluate the role of aetiology identification in pneumonia; ( ) to evaluate the first-line therapy in pneumonia. methods: we conducted a retrospective study in an infectious diseases hospital on patients with pneumonia. we excluded all the cases with nosocomial pneumonia. primary end-point was the day clinical failure (deaths, icu admission), secondary end-points were the average time of fever and length of stay and the antimicrobial regimen changes. results: causative agent identification rate was . %. the evolution was different for patients with identified aetiology compared with other patients in terms of: -day failures, length-of-stay and changes of the antimicrobial regimen. the patients with inadequate first-line therapy had a more severe course of illness with a greater rate of day clinical failure, longer fever and length-of-stay. conclusions: pneumonia's treatment was better for the patients with identified causative agent. that is why we should include aetiology among the pneumonia severity criteria, especially at an 'after -day therapy' re-evaluation. results: pneumomococcal aom was detected in children ( . %) and s.pn. was the only pathogen in . %. the resistance rates of the organism to antibiotics were as follows: penicillin . % (micb/ mg/ml; intermediately resistant . %, mic . mg/ml . %, and mic!/ mg/ml; highly resistant . %), erythromycin . %, clindamycin . %, cotrimoxazole . % and chloramphenicol . %. all isolates were uniformly susceptible to rifambicin and vancomycin. the large majority of pneumococcal isolates ( . %) had the mphenotype and the remaining strains ( . %) the constitutive mls phenotype. a various of serogroups were detected; the serogroup was the most predominant one ( . %), followed by serogroups ( . %), ( . %) and ( . %). the non-typable s.pn. strains compromised the . % of the strains. conclusions: high prevalence of resistance to penicillin, macrolides and cotrimoxazole in pneumococcal aom of childhood was recognized. a strategy for preventing aom caused by drug-resistant pneumococci is mandatory to start. material and methods: a total number of strains were examined. the sensitivity test was performed by kirby bauer, microdilution method (pasco, difco) according to nccls guidelines and by e -test. results: a percentage of . % of s. pneumoniae strains revealed high level resistance to penicillin (mic]/ mg/ml), while the % showed intermediate resistance (mic . Á/ mg/ml). the resistance to erythromycin and cotrimoxazole was . % (mic ]/ mg/ml) and . % (mic]/ / mg/ml), respectively. all strains were sensitive to cefotaxime (mic . mg/ml), vancomycin (mic / . mg/ml), meropenem (mic / . mg/ml) and levofloxacin (mic / mg/ml). conclusions: ( ) the prevalence of high resistance s. pneumoniae to penicillin seems to be low in examined strains ( . %). ( ) intermediate resistance to penicillin of s. pneumoniae isolates was high as expected ( %). ( ) most of the strains were sensitive to erythromycin ( . %) and cotrimozaxole ( . %). ( ) s. pneumoniae isolates were completely ( %) sensitive to levofloxacin, vancomycin and meropenem. beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy aim: of this study was a retrospective ( Á/ ) evaluation of the more effective and practical antibiotic treatment in ae-copd patients (pts) admitted to our unit. methods: before introducing any antimocrobial drug, sputum specimens were collected for microbiological purposes, while blood analysis, to monitor adverse systemic effects, were performed at the beginning and the end of treatment. antibiotic treatment ranged from to days according to four regimens: regimen a ( pts) /oral therapy only: . % with amc g b.i.d.; . % with cip mg b.i.d.; . % with dox mg u.i.d.; . % with lev mg u.i.d.; . % with cla mg b.i.d.; . % miscellaneous. regimen b ( pts) /sequential therapy (e.v. for days /oral): . % with amc g b.i.d.; . % with cla mg b.i.d. regimen c ( pts) /e.v. therapy only: same drugs. regimen d ( pts) /an association of two antibiotics. results: of evaluated pts, only ( . %) required a second regimen of treatment because of failure of the previous one: . % of regimen a; . % of regimen b; . % of regimen c, and % of regimen d. mild adverse effects were detected only in four pts. our results confirm that oral antibiotic treatment is practical, safe, and effective, and can be considered as the first line regimen also in hospitalized patients with ae-copd. becher g a , gillissen a a , rothe m b . a st. george medical center, robert-koch-hospital, leipzig, germany , b filt, lung and chest diagnostics ltd., berlin, germany patients with severe form of chronic obstructive pulmonary disease (copd) are prone by frequent exacerbations. bacterial infections are judged to cause at least half of exacerbations. haemophilus influenzae and streptococcus pneumoniae are the most frequent isolates, gramnegative bacilli account for the severe cases, aggravating the inflammatory process in the airways eventually leading to respiratory insufficiency. the aim of this ongoing placebo controlled, parallel group, mono center study trial is to evaluate beneficial effect of cefixim to reduce bacterial load and pulmonary inflammation in patients (n / ) with acute bacterial exacerbation of severe copd. thus, patients received in randomized fashion either cefixim ( mg/day) or placebo ( days). on days , , and breath condensate is collected using 'ecoscreen' (jaeger germany) for ltb -, il- -, nitrite-and ph-analysis. in parallel sputum gathered for detection of bacterial strains, and for ltb -, and il- quantification purposes. these data are compared to clinical outcome parameters such as lung function tests, radiographic findings, serum inflammatory markers and length of hospital stay. the preliminary data obtained confirm successful antibiotic therapy with oral cefixim in bacterial related acute exacerbations of copd is a useful approach to reduce bacterial load, and concomitantly lower inflammatory indices of the central and peripheral airways leading to clinical improvement of the patients. soriano garcia f a , fenoll a b , fernandez-roblas r a , coronel p c , gimeno m c , rodenas e c , garcia m a , granizo jj d . a fundacion jimenez diaz, microbiology, madrid, spain , b instituto de salud carlos iii, centro nacional microbiologia, majadahonda, spain , c tedec meiji farma, scientific, alcala de henares, spain , d fundacion jimenez diaz, epidemiology, madrid, spain purpose: to describe the susceptibility of streptococcus pneumoniae against cefditoren and other antimicrobials by serotype a multicenter study in south europe was carried out. a total of strains were collected between september and march from adult patients (more than y.o.) with respiratory tract infection (respiratory tract samples and blood cultures). all the isolates were sent to a central laboratory (fundació n jiménez díaz, madrid, spain) where susceptibility test was performed by broth microdilution (sensititre) following nccls recommendations. serotype was determined by quellung reaction and dot assay in carlos iii institute in strains. results: a total of strains ( . %) were not typable. the most prevalent serotypes were ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %). two hundred and sixty-four strains were grouped in different serotypes. the proportion of susceptible strains by serotype to penicillin, erythromycin and levofloxacin were: serotype ( . , . , %); ( . , . , . %); ( . , . , . %); ( . , . , . %); ( . , . , . %); ( . , . , . %). the mic to cefditoren was / . (serotype ); . (serotype , and ) and mg/l (serotype and ). conclusions: the most prevalent serotype was . the susceptibility was higher in serotype than in serotypes and . community acquired pneumonia */a study among closed military community of young people ps martynova av, turkutyukov vb, vostrikova aa, andryukov bg. vladivostok state medical university, epidemiology, vladivostok, russian federation purpose: the etiology of pneumonia is still partly unknown. we should like to clear up an etiological role of respiratory pathogens in community-acquired pneumonia among youth. and we had chosen for it a model of a closed community both investigation of etiology of disease and for further investigation of mechanisms of transmission drug-resistant mechanisms. methods: we studied adults in age of Á/ from closed military collectives who presented to two public hospitals (one urban and one rural) with acute radiologically confirmed pneumonia during winter Á/ . we did blood and lung-aspirate cultures, mycobacterial cultures, serotype-specific pneumococcal antigen detection, and serology for viral and atypical agents. results: streptococcus pneumoniae is recognized as an important cause of community-acquired pneumonia, it probably accounts for % of cases of community-acquired pneumonia among youth. chlamydia pneumoniae and mycoplasma pneumoniae responsible for approximately % of cases. haemophilus influenzae caused . % sever cases of disease, % of all cases were due to moraxella catharralis . conclusion: pneumococcial infection accounted for % of the cases diagnosed. s. pneumoniae was the most common bacterial infective agent, with a low incidence of both m. pneumoniae and s. pneumoniae . other causative pathogens occurred only within groups of individuals with deficiency of immunological status. berezin en a , cardenuto md a , nobuko e b , guerra ml c , brandileone mc d . a santa casa, pediatrics, s. paulo, brazil , b santa casa, microbiology, s. paulo, brazil , c adolfo lutz, microbiology, s. paulo, brazil , d adolfo lutz, bacteriology, s. paulo, brazil to determine antimicrobial susceptibility of sp isolated from the upper respiratory tract, we collected np swab specimens from children, between months and years old. those children attended the outpatient clinic in s. paulo city, with diagnosis of bacterial infection requiring antibiotic therapy between march , to may , . penicillin susceptibility of isolates was determined by screening with oxacillin mcg disk and performing the minimal inhibitory concentration by the e -test. we performed also susceptibility test for amoxicillin and cefaclor. results: sp was recovered from np of children ( . %). the carriage of sp was more prevalent in children attending day care centers, children with young siblings at home, and children with tobacco users at home. the prevalence of penicillin non-susceptible strains was . % all of them with intermediate resistance. all the strains were susceptible to amoxicillin and . % were resistant to cefaclor. serotypes . b, f, n, , a and were the most common. these findings suggest that nasopharyngeal isolates of streptococcus pneumoniae from children with upper respiratory infections can be used to conduct surveillance for antimicrobial resistance in a defined geographic area. we were able to conclude also that penicillin intermediate resistent strains can be susceptible to amoxicillin. hinojosa rm a , saenz a a , collazo m a , echaniz g b . a universidad autonoma de nuevo leon, infectologia, monterrey, mexico , b instituto nacional de salud publica, epidemiologia, cuernavaca, mexico the emergence of penicillin-and multidrug-resistant pneumococcal strains has become a global concern, necessitating the identification of the epidemiological spread of such strains. material: ninety streptococcus pneumoniae clinical isolates were collected from march through march . typing was done by the capsular reaction with pooled, type, or group antisera. susceptibility testing to antimicrobials was done by the e -test and the disk diffusion method. results: only ( %) s. pneumoniae strains were classified by serotyping; the most frequent types were a/b, f, v, f and . the oxacillin screening test detected . % penicillin-resistant s. pneumoniae strains isolated from children and . % from adults. the susceptibility percentage of s. pneumoniae to ceftriaxone was % in both children and adults. s. pneumoniae isolates from children exhibit a susceptibility of % to azithromycin, while in adults % of the isolates were susceptible. for the rest of the antimicrobial agents, the susceptibility ranged from to %. s. pneumoniae had a lower susceptibility to ceftazidime and ciprofloxacin. conclusions: ceftriaxone and azithromycin had a good in-vitro activity against s. pneumoniae strains. but the percentage of penicillinresistant s. pneumoniae detected in this study is alarming, therefore we conclude that a continuous surveillance system is necessary in mexico. vertkine al, prokhorovitch ea, alexanyan la. a retrospective analyses of cases of ambulant pneumonia with fatal outcome was made. among the patients who died from ambulant pneumonia the prevailing age was over ( . %) and the prevailing sex was male ( . %). . % had pneumonia accompanied with some pathology: chronic lung disease ( . %), alcoholism ( . %), diabetes mellitus ( . %). . % of the patients had a big volume of lungs lesion */ . % of the patients suffered from bilobular pneumonia and . % */from trilobate pneumonia. in . % of the cases pneumonia was complicated with abscess formation and/or exudative pleurisy. we studied the antibiotics therapy used for the patients treatment. the change of antibiotics was made only in cases ( . %) whereas in the other cases no change of preparations was made though the signs of the therapy non-effectiveness were obvious. thus, the rational antibiotics therapy with the timely change of non-effective antibacterial drug is significantly important. while choosing the antibiotics, the patient's age, the accompanying diseases, the volume of the lungs lesion and complications which define pneumonia seriousness are to be taken into consideration. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to describe the management of lower respiratory tract infections (lrti) in healthy adults, by general practitioners (gp). methods: a questionnaire was sent to a representative national sample of gps. this questionnaire assessed their perception and management of lrti, the indication for antibiotics (ab) in a case of lrti in a healthy adult with no focal signs and no signs of severity, knowledge of the micro-organisms responsible for acute bronchitis and knowledge of the afssaps (french agency for the safety of health products) recommendations. results: three thousand seven hundred and thirty-eight gps, who reported seeing an average of patients per week, including . / . patients with lrti, returned the questionnaire. the main results are presented in the following for the majority of gps, the main objective of the visit is to determine the indication for antibiotics. according to gps, alp and whooping cough are rare, while atypical pneumonia is frequent. gps also declare that the diagnosis of alp is often easy right from the first visit, in contrast with that of atypical pneumonia. complementary investigations are not often requested. gps consider that they often delay prescription of antibiotics ( %) and declare that they tend to prescribe a macrolide as first-line treatment. finally, gps have a poor knowledge concerning the micro-organisms responsible for acute bronchitis and the majority of gps declare to be familiar with afssaps recommendations. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to study the management of one case of lower respiratory tract infection (lrti) in adults by general practitioners (gps). methods: prospective study conducted on a representative national sample of gps. each gp had to include the first healthy adult patient seen during the -week data collection period, either on a home visit or in the office for recent cough and acute fever !/ . c. clinical data, the diagnostic perception and the therapeutic approach to the patient were collected by means of a standardised questionnaire, distributed by abbott laboratories. results: three thousand seven hundred and thirty-eight general practitioners included patients. conclusions: during lrti in adults, gps observe few focal signs, confirming the marked predominance of bronchitis compared to pneumonia. in view of the frequency of the signs, the diagnosis of pneumonia appears to be overestimated. one-third of clinical situations were diagnosed as 'bacterial superinfection of acute bronchitis', despite it is not a recognised diagnostic entity. antibiotic prescription was immediate in % of cases and delayed in % of cases. this last point shows that clinical practice differs from the gp's perception of their described prescribing practice shown in a simultaneous survey ( % of gps declared that they prescribed antibiotics immediately, while % delayed this prescription). results: thirty-three patients ( men, mean age b years, mean apache ii score . &bdqup;b . ) during the years Á/ were prospectively studied. thirteen patients ( %) had no identifiable risk factor for severe cap. an etiologic factor was revealed in patients ( %). in of them this was achieved with noninvasive methods. psb cultures were taken from eight patients and were positive in only . the offending organisms included: streptococcus pneumoniae in six cases, gnb as the sole pathogen in six cases, haemophilus influenzae (with s. pneumoniae or klebsialla pneumoniae ) in two cases, s. aureus in two and legionella pneumophila in one patient. initial antibiotic regimen a combination of marcodile '/ rd gen cephalosporin /aminoglycoside was successful in patients who all survived and had to be changed empirically or according to culture results in patients who had a mortality of %. the overall mortality rate was %. the identification of the causative factor did not seem to have any impact on survival. conclusion: severe cap in our icu was most often caused by s. pneumoniae and gbn. the high mortality of this entity seems to be influenced by the immediate use of the appropriate antibiotic combination and not by the identification of the causative organism. this underscores the need for knowledge of topical microbiology which helps in designing an effective empirical initial antibiotic regimen. mily mn a , golubev sa b , lugovoy vy a , voronov gg b . a vitebsk emergency hospital, pharmacotherapy unit, vitebsk, belarus , b basic and clinical pharmacology department, vitebsk state medical university, vitebsk, belarus purpose: the study aims were to assess the spectrum and predictors of the antibiotic use during pneumonia management in a regional emergency hospital in belarus. patients, treatment and physicians characteristics of cases ( Á/ ) were collected and possible associations were examined with using defined daily doses methodology (ddd) and american thoracic society (ats) guidelines. results: the mean treatment duration was . / . days, the total antibiotic ddd/ bed-days was . . the ddd/ bed-days of the most used antibiotics were: penicillins . ; aminoglycosides . ; macrolides . ; cephalosporins . ; tetracyclines . . in manova certain ats categories were associated with ddd/day, but not with frequency of definite antibiotic use. ddd/day was higher in case of multilobar infiltrates ( conclusion: our study indicated the low rate of macrolides and cephalosporins using, and the high one of aminoglycosides. antibiotic prescriptions were associated with disease severity and physician personality rather then with empirical choice rules recommended. acute exacerbation of copd: most frequent infecting agents and their suspectability to the different types of penicillins. analysis of medical documentation ps pertseva to, bogatska ke, gashynova ky. dsma, internal medicine , dniepropetrovsk, ukraine number of copd cases has been increased in ukraine. treatment of acute exacerbation (ae) of copd is not always successful because of inadequate antibiotic therapy. the aim of study was to reveal most frequent infecting agents and their susceptibility to the different types of penicillins in patients with ae of copd. medical documentation of patients with ae of copd (type i) was studied. data of sputum analysis and susceptibility of isolated agents to penicillin, ampicillin, oxacillin and amoxicillin/clavulone acid were evaluated. there were patients with haemophilus influencae/parainfluencae ( . %), klebsiella pneumoniae ( %), staphylococcus aureus ( . %), pseudomonas fluorescens , pseudomonas putida , serratia marcescens , serratia liquefaciens ( . % each), streptococcus agalactiae , acinetobacter baumanii ( . % each) in samples of sputum. in . % cases there was mixt infection. . % had no any bacterial agents. only % of agents were susceptible to penicillin, % */to ampicillin, % */to oxacillin. however, % of microorganisms were susceptible to amoxicillin combined with clavulone acid. this study has shown that most frequent infecting agents caused ae of copd were gram-negative microorganisms and s. aureus . according to antibiogram the prescription of amoxicillin/clavulone acid is most expedient in this case. pertseva to, bogatska ke, konopkina li, kireeva tv, gashynova ky. dsma, internal medicine department, dniepropetrovsk, ukraine we examined patients ( men, mean age . / . years) with acute exacerbation of cob (type i). the most frequently isolated agents were haemophilus influenzae and parainfluenzae */eight patients, gram-negative rods in , staphylococcus aureus in two and mixed in six and one patient had no bacterial agents isolated in their sputum. high susceptibility to azithromycin was found in all cases of gram-positive agents and in h. influenzae and parainfluenzae . other gram-negative agents were resistant to this drug in vitro. however, treatment with mg/day during days was clinically effective in . % of cases. only . % of patients had a further acute exacerbation of cob. there were peculiarities of the infecting agents causing acute exacerbation of cob in this study: klebsiella pneumoniae and s. aureus were found more frequently than in other studies. high efficacy of surnamed in the treatment of acute exacerbation of cob was established in . %. . % had partial positive clinical effect after this therapy. there were no patients with adverse events. efficacy and tolerance of amoxicillin mg/kg bid versus amoxicillin mg/kg tid in the treatment of acute otitis media (aom) in children / years ps borek m a , guggenbichler jp b . a biochemie gmbh, international medical department, kundl, austria , b department of pediatrics, university of erlangen, erlangen, germany five hundred and sixteen patients (mean age / . years) with clinical and otoscopic diagnosis of aom were included in a randomized, double blind, multicentre study, and were treated days either with amox mg/kg bid or amox mg/kg tid. assessments were made during therapy (day Á/ ), after end of therapy (eot, day Á/ ) and follow up (fu, day Á/ ). the primary efficacy endpoint was the clinical response at eot defined as success (cure/improvement) or failure. both regimens were well tolerated; one or more drug-related adverse events (aes) were reported in . % ( / ) of bid patients and in . % ( / ) of tid patients. the most frequently reported drugrelated aes in each group were gastrointestinal symptoms (bid . % vs. tid . %), which were mainly of mild or moderate severity. both regimens were clinically equivalent. the higher cure rates in the bid group suggest a possible higher benefit from bid therapy in children / years. children attending family day-care (fdc) should be less exposed to upper respiratory tract infections than those in group day-care (gdc) and therefore to antibiotic treatment; fewer should thus carry resistant bacteria. to test this hypothesis, np carriage of sp and hi with reduced susceptibility to penicillin (pdsp and hi bl'/, respectively) was investigated among children in fdc (maximum three children) and in gdc ( Á/ children) in the alpes maritimes (france) between november and march . a two stage cluster sample of children attending gdc or fdc was selected. np samples were cultured for sp and hi. penicillin susceptibility was tested by disk diffusion and e -test, and b-lactamase production by api-nh † tests (biomerieux, lyon). two hundred and thirty-five children in fdc and in gdc aged Á/ months were sampled. age and sex distribution were similar in both groups. sp was isolated in children in fdc ( %), and in ( . %) children in gdc (p b/ Á/ ). proportions of pdsp were . and . %, respectively (p / . ). hi was present in . % of children in gdc vs. . % in fdc (p b/ . ). proportions of hi bl'/ were . % vs. . %, respectively (p / . ). antibiotic exposure during the previous months concerned . % of children in gdc vs. . % in fdc (p / . ). there was no correlation between antibiotic use and carriage of pdsp or hi b'/ strains. sp and hi carriage rates are significantly lower among children in fdc than in gdc. advising alternative types of daycare for children attending gdc should reduce exposure and thus limit the spread of resistant bacteria. however, the proportion of pdsp and hi bl'/ is similar in both groups and comparable patterns of antibiotic use are observed. continued efforts must concentrate on parental education and enforcement of recommendations for management of pediatric upper respiratory tract infections. during a period between january and august , invasive haemophilus influenzae (hi) isolates had been collected at the children's hospital of tunis. we used haemophilus test medium to test antibiotic susceptibility. the mic of beta-lactams was measured by e -test. beta-lactamase production was determined by using the cefinase test and biotyping by apinh. presence of capsular antigen was determined by using hi typing anti sera. hi strains were isolated from meningitidis ( ), bacteremia ( ) and arthritis ( ). all strains were serotype b and . % of them belonged to biotypes i and ii. amoxicillin resistance with beta-lactamase producing mechanism occured in . %. mic of beta-lactamase producing strains was vs . mg/l in non-producing one. there is no betalactamasenegative amoxicillin resistant among these invasive isolates. antibiotic resistance concerned chloramphenicol: . %, trimethoprim-sulfamethoxazole: . %, tetracycline: . % and kanamycin: . %. invasive hi infections in tunisian children's were always associated with type b strains. introduction of a hib vaccine programme in tunisia is recommended. the aim of this study was to analyse the clinical picture and treatment of neurological manifestations of neuroborreliosis in children. the study included children ( Á/ years) with neuroborreliosis diagnosed on the basis of the clinical and serologic criteria. symptoms of facial palsy occurred in six children symptoms of iii Á/vi cranial nerves palsy in three children, meningitis in four and paresthesias in three. symptoms of v or viii nerve palsy, mental disturbances, radiculoneuritis or cerebellitis were found in singular cases. all children received ceftriaxone intravenously Á/ weeks. total recovery was obtained in children following the first course of therapy. recovery following the second course of therapy (amoxicillin) occurred in one child with mental disorders and one with vi nerve palsy. improvement was achieved after the second course in the patient with radiculitis, however, muscular atrophy persisted. irreversible, unilateral deafness was found in a child with viii nerve palsy in spite of three courses of therapy applied. infection with borrelia burgdorferi in children causes a wide spectrum of neurological manifestations. facial palsy was the most common sign in our study. applying ceftriaxone in the treatment of neuroborreliosis is characterised by a good effectiveness. double infection by c. pneumoniae and m. pneumoniae as a cause of cystic changes in the lungs ps streharova a, moravcikova d. department of infectious diseases, university of trnava, trnava, slovakia chlamydia pneumoniae and mycoplasma pneumoniae are human respiratory pathogens manifested in early childhood. immunological disbalance could trigger autoaggressive diseases. the authors describe the case of a -year-old girl with development of multiorgan failure and septic state, which followed multiple cystic changes in the lungs. the girl did not have a diagnosis of cystic fibrosis. the authors consider that cystic changes are a consequence of double infection by c. and m. pneumoniae. dzarlieva m, momeva l, temelkovska g, balevska p, pejkovska m. medical centre, neonatology, bitola, the former yugoslav republic, macedonia unicef skopje has supported a nationwide safe motherhood needs assessment in representative samples of hospitals. eighteen of maternity wards and facilities renovated and certified as 'babyfriendly'. all mature newborns with successful adaptation to extra uterine life and satisfactory vital parameters are h during the day with their mothers at rooming-in system. aim: with rooming-in system we reached decreasing of incidence of neonatal infections. material and methods: history records of newborns from our department. for the period of months, neonates have been borne. with suspection of infection there */ babies ( . %). newborns borne through meconium stained liquid */ ( . %). results: microbiological findings: from blood culture */staphylococcus coagulaza negative from swabs */staphylococcus aureus , escherichia coli , staphylococcus epidermidis. conclusion: in , from all babies who had risk factors for infection in newborns ( %) we had positive findings and in year (before rooming-in sistem), in ( . %). after that period (with rooming-in) newborns ( . %). with rooming-in system we reached decreasing in incidence of neonatal infections by breaking the chain of infection */only mothers take care of their babies with help of the staff. newborns are in their micro environment, the same they will have at their home. with this practice we have also reduction of nosocomial infections. a study on antibiotic susceptibility and resistance factor transmissibility among antibiotic resistant salmonellae isolated from children affected to diarrhea ps sharifzadeh a. azad university of shahrekord, microbiology, shahrekord, islamic republic of iran in spite of happened drug resistance, antibacterial therapy still is the best route of treatment of salmonellosis in man and animals. in order to detection of dominants serotypes of salmonellae in children and detection of antibiotic susceptibility and r-factor transmissibility among those isolated salmonellae. this study was conducted on diarrheic stool samples were collected from children affected by diarrhea in ayatollah kashany hospital of shahrekord, during spring of to autumn of . after isolation and identification of salmonellae, seven serotypes were detected. one of those was s. typhi and another six serotypes were s. paratyphi b . in order to detection of antibiotic different antibiotic disks were used in disk diffusion method. best results were taken from ceftizoxim, cephtriaxon, cephazolin and chloramphenichol. the r-factor were transferred from isolated salmonellae to escherichia coli k in all of cases of resistance to penicillin and ampicillin. pharyngeal colonization by streptococcus pneumoniae and group a bhs were evaluated in randomly selected school children aged Á/ years in riyadh, saudi arabia. fifty-six children ( . %) had positive culture for either organisms of the isolates from school children, ( %) were s. pneumoniae , of them ( %) were penicillin-sensitive, three ( %) were penicillin-resistant, and two ( . %) were resistant to two antimicrobials. forty isolates of bhs ( %) were group a bhs. all isolates were penicillin and erythromycin sensitive. the carrier rate among school children for penicillin-resistance s. pneumoniae and resistance to two antimicrobials were ( . %) and ( %), respectively. the carrier rate of group a bhs was ( . %). riyadh has a low rate of antibiotic-resistant s. pneumoniae and a similar rate of group a bhs carriers among school children as that seen in temperate areas. boukadida j a , boukadida n b , hannechi n a , said h b , erraii s a , elmhabrech h a . a chu f. hached, laboratoire de microbiologie, sousse, tunisia , b c s base, sousse, tunisia the acute pharyngitis is a very frequent pathology in which group a streptococcus is the most incriminated bacteria. however, other non a b-haemolytic streptococcus (sbna) could be responsible. the aim of this work is to determine the part of each non a b hemolytic streptococci (sbna) in acute pharyngitis and the related antibiotics susceptibility pattern. the study was realized in sousse-tunisia (north africa) during months from may . the origin materials of isolates are throat swab (transystem venturi, copan, bovezzo). the mean age of patients is years with extremes Á/ years. the samples are cultured on blood agar plates in a delay of h maximum. identification was done to samples that have over than b-hemolytic colonies, groupage with pastorex strep. sanofi pasteur france, susceptibility pattern according to nccls norms, mic is determined by e -test. the control strain is s. pneumoniae atcc . twentyone clinical isolates of sbna are distinguished from clinical isolates of b-hemolytic streptococci recovered from patients with acute pharyngitis without symptoms of viruses' infections (tearing, corysa, sneeze). all b-hemolytic streptococcus represents . % of all collected samples. sbna were . % of the isolates. sbna were strains group g streptococci, seven strains group c streptococci and three strains group f streptococci. susceptibility pattern of each sbna to antimicrobial agents is as follow: group g streptococci: peni g / %, amoxicillin / %, pristinamycin %, clindamycin and erythromycin / . %, tetracyclin / . %, telithromycin . % and levofloxacin / . %. group c streptococci: peni g / %, amoxicillin / %, pristinamycin %, clindamycin / . %, erythromycin / . %, tetracyclin / . %, tel-ithromycin / . %, levofloxacin / %. group f streptococci-peni g / %, amoxicillin / %, clindamycin / . %, erythro / . %, tetracyclin / . %, telithromycin / %, levofloxacin / . %. all sbna have mic to penicillin g under . mg/l. according to available data, penicillin g and amoxicillin still the reference treatment of acute bacterial pharyngitis in spite of the new antibiotics introduction. berezin en a , quevedo sg b , nicolla l b , viegas d c , eizenberg b d , pedrosa f b , santos ag a . a santa casa, pediatrics, s. paulo, brazil , b elly lilly, scientific, s. paulo, brazil , c fac. abc, pediatrics, santo andre, brazil , d university hospital, pediatrics, s. paulo, brazil a multicenter, open label, prospective, randomized trial in which patients Á/ years of age with proven gabhs pharyngitis were randomized to receive either -day course of the broad spectrum oral cephalosporin, cefaclor or a -day course of amoxicillin. patients were included if they have signs and symptoms of streptococcal tonsillopharyngitis and a rapid streptococcal rapid test positive . patients were evaluated at days Á/ , Á/ and Á/ posttherapy. pharyngeal cultures were conducted at baseline and at follow-up visit ( Á/ days). we considered for bacteriologic eradication analysis only patients with positive culture to gabhs. there were patients with a rapid streptococcal rapid test. clinical success were achieved in around % of the patients. for evaluate eradication of the initial pathogen we considered patients of the amoxil arm and patients of the cefaclor arm. thirty-four of patients of the amoxil arm ( . %) and of ( %) patients of the cefaclor arm were considered bacteriological cured at the second culture performed at day Á/ . ten days of a penicillin or amoxicillin therapy may not be the best therapeutic choice for all pediatric patients. in developing countries where rheumatic fever is still an important problem to evaluate the bacterial eradication achieved with the different antibiotics may be important. prophylactic affect of saccharomyces boulardii for antibiotic-associated diarrhea in a paediatric age group ps erdeve o, tiras u, camurdan mo, tanyer g, dallar y. ankara education and research hospital, pediatrics, ankara, turkey the process resulting in antibiotic associated diarrhea is the alteration of enteric flora due to bacteria. saccharomyces boulardii is a yeast, isolated from cover of a kind of hazelnut and its usage became widespread recently. there is no enough study about s. boulardii activity at pediatric ages. we aimed to define s. boulardii activity on azithromycin and sulbactam-ampiciline, and associated diarrhea at pediatric age group. the . % of cases only with antibiotic usage developed diarrhea, whereas rate was . % for probiotic using group (p b/ . ). all of the clostridium difficile toxin a defined cases were from sulbactam-ampiciline using group. the rate of diarrhea for sulbactam-ampiciline using group was . %, while it was . % for group used s. boulardii as probiotic beside sulbactam-ampiciline (p b/ . ). it was observed that probiotic usage decreases diarrhea rate four times (p b/ . ). when age groups considered, the rate of sulbactamampiciline associated diarrhea increased at Á/ ages and s. boulardii effect on preventing diarrhea was significant at Á/ ages (p b/ . ). antibiotic associated diarrhea is a common clinical problem at pediatric age group. s. boulardii is a hope giving probiotic especially for sulbactam-ampiciline associated diarrhea. grey e a bilikova e a , hafed bm a , kovacicova g a , chovancova d b , huttova m b , krcmery v a . a school of health, trnava university, trnava, slovakia , b postgraduate academy of medicine, neonatal clinic, bratislava, slovakia the purpose of the study: the aim of the study was to find out, whether artificial abortion of a mother has impact on neonatal infection of her future baby. the results obtained: therefore, we compared neonates with infection, who were born to mothers with past history of abortion ( Á/ artificial abortions within years), with the babies of mothers, who have not experienced abortion before. control group consisted of neonates hospitalized at the same clinic, at the same time period. according to the analysis of risk factors for neonatal infections, it was found, that prematurity ( Á/ weeks of gestation) ( . vs. . %, p . ) and low birth weight- Á/ g ( . vs. . %, p . ) were significantly more frequently observed in babies of mothers with past history of abortion. drug abuse (heroin) ( . vs. . %, p . ) and nicotine use ( . vs. . %, p . ), were more significantly related to neonatal infections in babies of mothers with past history of abortion. etiological analysis showed that only candida albicans ( . vs. %, p . ) was significantly related to neonatal infections in mothers with past history of abortion. the conclusion reached: in conclusion, artificial abortion has not only direct impact to the health of the mother, but also on her next pregnancy. bacteraemia and patient mortality in a peadiatric intensive care unit ps armenian sh, singh j, arrieta ac. children's hospital of orange county, infectious disease, orange, usa purpose: this study will identify the factors that significantly contribute to mortality in patients with bloodstream infections (bsi) at a pediatric intensive care unit (picu). results: medical records of patients who were admitted to the picu and had a documented bsi were reviewed. there were separate episodes of bsi's, with nine patients having multiple bsi's during their hospital stay. a casecontrol model was used. cases were bsi's with eventual mortality (n / ; . %) and controls were those who survived bsi (n / ; . %). patients who died were older ( . vs. . years; pb/ . ), more likely to have a nosocomial bsi ( . vs. . %; pb/ . ), longer hospitalization prior to bsi ( . vs. . days; pb/ . ), and have a polymicrobial bsi ( . vs. . %; pb/ . ). infection related mortality (irm)-defined as death within days of bsi */was significantly higher in those receiving inadequate antibiotic treatment at the time of diagnosis of bsi ( . vs. . %; p b/ . ), as well as in those with gram negative bacteremia and/or fungemia ( vs. %; p b/ . ). logistic regression was used to adjust for potential confounding variables. conclusions: we found that being older, multiple organisms, and a longer hospitalization prior to the bsi were significantly associated with overall patient mortality. irm was significantly higher for those with inadequate initial antibiotic coverage and in those with gram negative bacteremia and/or fungemia. somer a a , gü r d a , diri s a , yalcýn i a , salman n a , ongen b b , gü rler n b . a department of pediatric infectious diseases, istanbul university istanbul medical faculty, istanbul, turkey , b department of microbiology and clinical microbiology, istanbul university istanbul medical faculty, istanbul, turkey teicoplanin is a glycopeptide antibiotic active against a broad range of gram-positive pathogens including methicillin-resistant staphylococci and offers the advantages of once daily administration, choice of administration route, lack of requirement for routine therapeutic drug monitoring and lower propensity to cause nephrotoxiticy and anaphylactoid-like reactions. in this study the efficacy and safety of teicoplanin were evaluated retrospectively in children with serious bacterial infection. sixty-three children ( girls, boys) aged between month and years were treated with teicoplanin (three loading dosages of mg/kg at h intervals, followed by a maintenance dosage of mg/kg/day). the infections treated were pleural empyema (n / ), joint and bone infections (n / ), septicemia (n / ), skin and soft tissue infections (n / ), and lung abscess (n / ). the pathogens isolated were staphylococcus aureus (n / , of which were methicillin resistant), coagulase-negative staphylococci (n / ), s. pneumoniae (n / ), and group a hemolytic streptococci (n / ). the duration of therapy ranged from to days (median days). clinical success (cure plus improvement) was achieved in . % of cases. no side effects attributable to teicoplanin therapy were encountered. teicoplanin appears to be an effective and well-tolerated treatment for serious gram-positive infections in children. the risk of infection is present in all children with acute leukemia. two hundred and sixty episodes of febrile neutropenia were analyzed in children (aged . Á/ years) with aml */ cases and all */ cases over years during cytostatic therapy (protocols: mbfm */ aml and mbfm */ all). a degree of fn occurred in % of cases in aml, in % */in all. the sites of infection were: blood'/ central venous catheter ( %) and respiratory tract ( %). pathogens isolated from blood were: gram-positive */cns */ %, streptococcus spp. */ . %, gram-negative */enterobacteria */ %, pseudomonas aeruginosa */ %, candida spp. */ . %, aspergillus spp. */ . %, other */ . %. for the treatment of fn we used empirical antibiotics regimens. clinical response was noticed: in st line of therapy */ cephalosporins Á/ generation used */ %, carbapenems used */ % as nd Á/ rd lines */vancomycin */ %, amphotericin b */ %. thirtytwo percent of children with aml and . % children with all died because of sepsis. conclusion: carbapenems are more active in the st line of antibiotics therapy both for patients with aml and all. vancomycin is useful in the nd line for patients with all. amphotericin b and vancomycin are useful in combination in the nd line for patients with aml. pavlenishvili ivp. medical academy, pediatrics, tbilisi, georgia our drug of choice in cases of complicated neonatal sepsis i. pavlenishvili, g, iobashvili tbilisi, medical academy georgian society of pediatrics chemotherapy (gspc) with collaborations of drug and therapeutic committee (dtc) of childers hospital 'republic' finished the observation study about nosocomial infections of neonatal icu in above mentioned hospital. study begins from november . during this period we observe cases of neonatal sepsis, most of them were due gram-negative bacteria. thirty-four cases of neonatal sepsis were cause different stains of escherichia coli , proteus spp. and acinetobacter . we notice that during last years the role of acinetobacter in etiology of neonatal sepsis and nosocomial complications of neonatal sepsis in our hospital is gradually increased. as our observation reveals most optimal in the case of complicated neonatal sepsis was (according criteria of dts) use of ceftasidime. almost in all cases we used ceftasidime with success. we have only one case of mortality. since the macrolide resistance (mr) in streptococcus pyogenes has been increasing in europe, we studied the incidence and genetic basis of mr in s. pyogenes in russia. s. pyogenes isolated at baseline and during follow-up visits from children with acute pharyngitis receiving penicillin or midecamycin ( -membered macrolide) were studied. mr was evaluated by agar dilution (nccls), resistance mechanisms */by pcr. a total of s. pyogenes were obtained at the baseline, ( %) of which were erythromycin-resistant: strains ( %) were erm (a)-positive, one */mef (a)-positive, and one was negative for all primers used. all erm (a)-strains were inducibly resistant to clindamycin and represented seven pfge profiles with one profile found in / strains. in three midecamycin treated patients mr was selected during the therapy (one strain had erm (a), one */mef (a), one */ unknown resistance determinant). mef (a)-strain obtained during follow-up visit had different pfge pattern with mef (a) strain isolated at baseline, while both the baseline and follow-up strains with unknown mechanism of resistance had unique pfge pattern. these data showed moderate incidence of erythromycin-resistant s. pyogenes in smolensk. ribosomal methylation (erm (a)) was the most common mechanism and though the polyclonal nature of mr was established, most erm (a)-strains belonged to only a few clones. bacillus cereus infections in traumatology-orthopaedy department: retrospective study and re-evaluation of healthcare practices ps bacillus cereus was cultured for patients from traumatology department who had developed postoperative wound infections between august and march . all patients presented inferior members open fractures, frequently contaminated with telluric material and requiring external fixators. genomic study of clinical isolates by pulsed field gel electrophoresis and random analysis polymorphic dna, allowed us to eliminate an outbreak. furthermore, the reduced delay in which patients developed the infection ( days'//- ) led us to re-evaluate the procotols used in our institution. indeed, all patients had received amoxycillin '/ clavulanate iv g for antibioprophylaxis during anesthetical induction then relayed per os for hours. because of the production of a potent beta-lactamase by the bacteria, this association could not be efficient. furthermore, accordingly to the afnor en norm, we have tested clinical isolates' sensitivity to the principal antiseptics used for antisepsis and disinfection (iodophors, chloride derivated and biguanidines) and observed a major resistance of all strains tested. even if these postoperative wound infections are considered as nosocomial because of the delay in appearance, we actually think that bacillus cereus were initially present in telluric material. this fact led us to propose a systematic screening for bacillus at admission for this type of wound and to administrate quinolones such as ciprofloxacin for prophylaxis. internal thiols and reactive oxygen species in the candidacidal activity exerted by a n-terminal peptide of human lactoferrin ps the purpose of the study: the emergence of candida albicans strains resistant to current antifungals points to the need for new antifungal agents, e. g. antimicrobial peptides. the results obtained : we report that hlf( - ), a synthetic nterminal peptide of human lactoferrin, displays excellent killing effect against fluconazole-resistant c. albicans and that sub-optimal concentrations of this peptide combined with fluconazole act synergistically. previous investigations revealed that hlf( - ) required an energized mitochondrion, atp release by candida , and ligation of atp receptors for its killing effect. we now report that reactive oxygen species (ros) are involved in the killing effect of hlf( - ). since internal thiols protect cells from oxidative damage, our observation that hlf( - ) caused a % reduction of internal thiols in candida is of interest. as expected, n-acetyl-l-cysteine (nac), which is a precursor of glutathione and a ros scavenger, inhibited the killing effect of hlf( - ). diamide, which oxidizes internal thiols, was candidacidal and hlf( - ) and diamide acted synergistically in killing c. albicans and ros production. moreover, the hlf( - )-induced activation of mitochondria was inhibited by nac, indicating that internal thiols/ros affect mitochondrial activity. the conclusion reached : the candidacidal activity of hlf( - ) involves ros production and reduction of internal thiols. objective : an audit was conducted to explore the observation of increasing resistance to ciprofloxacin in enterobacteriacea isolated from specimens from such patients in the hematology unit. results : enterobacteriacea isolates in specimens from such patients processed over the years to were examined. number of specimens per year was */ , , , and respectively and the annual percent ciprofloxacin resistant enterobacteriacea from these was %, %, %, % and %. conclusion : conclusion drawn from the audit was that rapid rise in ciprofloxacin resistance may possibly be attributed to the use of this fluoroquinolone as a single agent in dose of / mg [cut off patient weight kg] twice a day in neutropenic patients till neutrophils exceed /c.mm. subsequent to the audit, prophylaxis protocol was modified to use of oral colistin -mega units/twice a day in combination with ciprofloxacin / mg twice a day. a prospective audit is proposed to test the benefits of this combination. rhinocerebral zygomycosis: diagnostic dilemma for emergency physician: can the associated morbidity and mortality in this rare but deadly disease be reduced? ps samavedam s, guleri as. western infirmary, clinical microbiology, glasgow, united kingdom rhinocerebral zygomycosis [rcz] is a rare, invasive, rapidly progressive opportunistic infection caused by ubiquitous fungi of the order mucorales. it usually occurs in diabetics or immunocompromised patients. the emergency physician will typically see patients with rcz in its earliest stages masquerading as a variety of other less serious diseases. the key markers like necrotic patch on hard palate, nasal septum or turbinate, marked facial pain, and cellulites with marked eye and neurological signs may present late in disease. we report a case of rcz caused by rhizopus arrhizus [oryzae] in an year old woman with poorly controlled diabetes. she presented with a right-sided facial droop of short origin and being generally unwell. ct scan was non-conclusive and delayed presentation of key markers of rcz permitted disease to rapidly progress. despite an intensive antifungal therapy with ambisome and insulin sliding scale, patient rapidly succumbed within days. fine needle aspiration cytology is less invasive, easier and equally effective alternative to pre op biopsy. the key to successful reduction in morbidity and mortality associated with this rapidly fatal disease is -increasing awareness of the disease, an early diagnosis, correction of underlying metabolic derangement, prompt intensive antifungal therapy with amphotericin b and radical surgical debridement of the necrotic tissue. an 'optimal dosage' of ambisome requires discussion. clinical audit in the haematology ward of a tertiary care hospital: study of degree of correlation between bacteraemia and oro-pharyngeal screens in immunocompromised patients over five years and role of antibiotic prophylaxis ps guleri as, butcher i. western infirmary, clinical microbiology, glasgow, united kingdom introduction : a clinical audit was carried out over -years [ ] [ ] [ ] [ ] [ ] in the immunocompromised patients including neutropenic patients and bone marrow [autograft] transplant recipients in hematology ward of gartnaval general hospital, glasgow, a tertiary care center. objective : it was aimed to establish the degree of correlation between bacterial isolates in oro-pharyngeal screen during bacteraemia episodes and role of antibiotic prophylaxis. methods purpose : to assess whether antiretroviral therapy (art) intensification, gm-csf use and remune initiation before stopping art lead to viremia containment, and long periods off art. methods : ten adults with chronic hiv disease, hiv- rna levels (vl) b/ . log copies/ml and median cd '/ -t cell count of /ml were enrolled. after art intensification with ddi ( months) '/ hydroxyurea [hu]( mo.) '/ gm-csf ( mo.) and a remune dose, art was stopped but remune continued. art was resumed if rebound vl did not decrease to b/ . log in months or if cd '/ counts decreased to b/ . results : vl rebounded in all patients after stopping art, and developed an acute retroviral syndrome (ars). cd '/-t cells decreased, and cd '/ '/ increased ( -fold). after a median stoppage of weeks, art was resumed in patients and vl decreased to b/ . log, cd '/ counts were regained, il- and il- levels rose. at the nd interruption, / patients had a rebound, and / had a nd ars, but peak vl and loss of cd '/ were lower (p / . ). after . weeks off art, patients resumed therapy. the breadth and magnitude of hiv-specific activity increased and thymus size grew. the patients were off art for a median of . out of weeks. two of them are off art for and weeks respectively ( vlb/ log). conclusions : this approach led to a high ars incidence, long periods off art, increases in hiv-specific responses, il- and il- levels, and thymus size. urinary tract infection in hospitalized population is a significant problem. this is a study of catherized patients urinary infection in corfu hospital. in Á/ , urine cultures were sent to our bacteriology laboratory. of them were collected from the catheters. clinical data of age and type of disease were analysed. the samples were cultured on mc conkey agar-urotube and vitek cards. the organisms identified with vitek automatic system. the sensitivity was tested with vitek and kirby-bauer method. results: no growth of organisms in . %. positive cultures %. . % of the bacteria were gram((/) rods ( . % e. coli , pseudomonas spp. %), % were gram('/) cocci (enterococcus spp.) and % was candida spp. the resistance of e. coli was: % to ampicillin , % to co-trimoxazol and % to quinolons. e. faecalis was resistant % to vancomycin . conclusions : ( ) the possible interference of acetaminophen in the amoxicillin/ clavulanic acid (a/c) or erythromycin (ery) efficacy in the treatment of acute otitis media (aom), and its possible role in the evolution to otitis media with effusion (ome), were determined in a gerbil model. a f streptococcus pneumoniae strain exhibiting a mic of a/c and ery of / . and . mg/l, respectively, was used. both antibiotics were tested at . and mg/kg. acetaminophen at mg/kg was administered min before each antibiotic dose. antibiotic concentrations in serum and middle ear exudate were determined. both antibiotics significantly reduced the number of culture-positive ears and colony counts, with serum concentrations over the mic of the microorganism for ]/ % of the dosing interval. antibiotic concentrations in middle ear exudate were almost identical in animals receiving and not receiving acetaminophen. clinical and microbiological efficacy was correlated with antibiotic concentrations in middle ear exudate ]/ . times the mic of the microorganism, for both antibiotics. both antibiotics demonstrated efficacy in the treatment of pneumococcal aom, with the same rate of ome. acetaminophen, concomitantly administered, did not interfere the efficacy of the two antibiotics tested and did not prevent the evolution of aom to ome. parra a a , ponte c a , cenjor c b , garcía-olmos m a , giménez mj c , aguilar l c , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b fundación jiménez díaz, otorrinolaryngology, madrid, spain , c glaxosmithkline, medical department, madrid, spain a gerbil model of otitis media with effusion (ome) induced by haemophilus influenzae (amoxicillin/clavulanate-a/c-and erythromycin-ery-mics of / . and mg/l, respectively) was used to evaluate the efficacy of a/c ( / and / mg/kg) and ery ( and mg/kg). antibiotics were administered subcutaneously h post-middle ear inoculation, and continued t.i.d for h, with or without acetaminophen (ap), at mg/kg, administered min before each antibiotic dose. antibiotic concentrations in serum and middle ear (me) were measured by bioassay. me samples for colony counting were collected on day . a/c reduced (p / . ) positive me samples and colony counts versus untreated controls or ery: me positive cultures of % for controls, % for a/c , % for a/c , % for a/c '/ap, % for ery , % for ery and % for ery '/ap. this was due to a/c (but not ery) concentrations in me exceeding . times the mic despite the higher percentage of antibiotic penetration of ery versus a/c ( versus / %). animals receiving ap showed less polymorphonuclear cells and more bacteria in me than those receiving only antibiotics, suggesting that the anti-inflamatory drug diminish the phagocytes and therefore, the efficiency in bacterial clearance. amoxycillin treatment for acute otitis media caused by penicillinresistant streptococcus pneumoniae . a pharmacodynamic analysis pm parra a a , ponte c a , cenjor c b , garcía-calvo g a , giménez mj c , aguilar l c , soriano methods: a serotype f streptococcus pneumoniae strain exhibiting a mic of amoxycillin of mg/l was used in an experimental model performed in gerbils (meriones unguiculatus ) following previously described procedures. amoxycillin was tested at the following doses: . , . , . , . , . and mg/kg. amoxycillin concentrations in serum and middle ear exudate were determined after drug administration. results: doses of ]/ . mg/kg significantly reduced the number of culture-positive ears, colony counts and otorrhoea (p / . ) as compared with untreated controls or animals treated with doses lower than . mg/kg. doses of ]/ . mg/kg achieved antibiotic concentrations in the middle ear . Á/ . times higher than the mic of the infecting strain and serum concentrations over the mic for Á/ % of the dosing interval. conclusions: amoxycillin at doses achieving serum concentrations similar to those obtained in children after standard doses, obtained therapeutic and microbiological efficacy regardless the susceptibility of the infecting strain. better correlation was found between antibiotic efficacy and antibiotic concentrations in middle ear exudate than between efficacy and serum concentrations, which were suboptimal from the pharmacodynamic perspective. increasing prevalence of amoxycillin Á/clavulanate-resistance among e. coli strains in a hungarian university hospital pm veréb i, vígh a, urbán e, hajdú e, nagy e. department of clinical microbiology, university of szeged, szeged, hungary background: amoxicillin Á/clavulanate resistance (acr) is an emerging problem in escherichia coli as reported from different parts of europe. the aims of the present study were to evaluate statistically the prevalence of acr among e. coli isolates and to investigate the genetic background of the resistance. methods: all e. coli strains isolated between and were screened for acr by kirby Á/bauer disc diffusion method. the resistance to other beta-lactam Á/beta-lactamase inhibitor combinations and to different beta-lactam antibiotics were also tested. selected strains underwent determination of beta-lactamase activity. confirmatory tests for suspected extended spectrum beta-lactamase were performed. pcr testing for tem and shv genes were carried out on plasmids isolated from selected strains. results: in out of e. coli strains ( . %) were found to be resistant to amoxicillin clavulanate (amc). most of the resistant strains ( %) were obtained from the genitourinary tract and no acr isolate was found in blood cultures. in out of isolates ( %) proved to be acr and . % were isolated from blood cultures and . % from the genitourinary tract. thirty-five selected strains were further analysed. thirty-two were also resistant to (sam) and six were further resistant to tzp. quantitative beta-lactamase determination showed increased activity in strains which were partially susceptible to amc. the presence of esbl could be proved only in three acr isolates. this open parallel-group study compared the efficacy and tolerability of cef with cfix mg once daily in the treatment of community acquired uncomplicated uti. seventy-eight female patients were randomized to receive either oral cef or cfix for or days. the efficacy of treatment was evaluated by clinical response (by symptoms of uti dysuria frequency urgency suprapubian pain and by clinical signs) by bacteriologic response and health status measures at baseline and posttherapy. results: the clinical cure (complete resolution of symptoms and signs) rate for patients receiving cef was . % of the evaluable patients and . % of the patients receiving cfix. bacteriologic response (based on the results of urine cultures obtained posttherapy) the pathogen was eradicated in . % for cef . % for cfix. no drug related side effects have been reported in cef and side effects were experienced by . % of the patients receiving cfix. improvement in health status comparing visual scale scores baseline and poststudy to have detected a higher change in average score from to in cef, from to in cfix. wilcoxon improvement value was significant on the rd day of therapy in case of cef and on the th day of therapy in cfix group. in conclusion, the results of this study indicate that cef course is more effective than cfix in producing a favourable clinical outcome and achieving higher bacteriologic eradication rate, furthermore cef was better tolerated. cefepime is a fourth generation cephalosporine that has a broader spectrum of antibacterial activity than the third generation cepfalosporines and is more active in vitro against gram-positive aerobic bacteria. the purpose of this study was to measure cefepime concentrations in plasma, bile fluid and gall bladder tissue in patients undergoing cholecystectomy. thirty patients male, female, mean age: years had data acceptable for analysis and were included in this study. all patients received iv g of cefepime. several hours after administration and at different time intervals, during surgery, samples were obtained from plasma, bile fluid and gall bladder tissue concomitantly. antibiotic levels were measured by an agar diffusion method. the mean delta time was / min. the values for plasma, bile fluid and gall bladder tissue, were . / . , . / . and . / . mg/ml, respectively. the plasma/bile fluid ratio was . / . . there was a significant correlation between plasma and gall bladder tissue concentration (r / . , p / b/ . ). a correlation between bile fluid and plasma cefepime concentration was not observed. the minimum inhibitory concentration (mic) data from previous in vitro studies indicate that the cefepime concentration observed in plasma bile and tissue samples of this study would be adequate against typical biliary tract pathogens. furthermore, these cefepime concentrations correlated well with the favorable clinical outcome reported in previous clinical studies in biliary tract infections. there was also good correlation between delta time and plasma and tissue concentrations and if the dose were given closer to the time of surgery, cefepime concentration would be higher reducing the possibility of an infection. objectives: the use of antibiotics may lead to decreased colonization resistance and increased formation of resistant bacteria. present concept was developed to overcome these untoward effects. methods: b-lactamase of bacillus licheniformis was overproduced in bacillus subtilis . this targeted recombinant b-lactamase enzyme (trbl) was released in the small bowel from a controlled-release formulation. beagles (n / ) were treated bid with either mg/kg ampicillin (i.v.)'/placebo (p.o.), mg/kg ampicillin (i.v.)'/trbl (p.o.) or only placebo (i.v.'/p.o.). stool was collected at days and . samples were cultured for total and main groups of aerobic and anaerobic bacteria and yeast. temperature gradient gel electrophoresis (tgge) was used to separate the ribosomal rna genes. results: ampicillin'/placebo group had clearly decreased counts of both aerobic and anaerobic bacteria during the treatment, whereas those receiving trbl had only minor overall changes and some occasional changes by single species. intravenous ampicillin decreased the fecal similarity percentage to %. the similarity percentage during treatment with ampicillin'/trbl did not differ from that of placebo ( vs. %). conclusions: according to our results the trbl can maintain the large intestinal microflora almost unchanged. these results indicate that trbl is a promising novel approach for overcoming the ecological adverse effects on gut flora caused by b-lactam antibiotic agents. adamis since broad-spectrum â-lactams combined with amikacin are often applied for nosocomial infections, their pharmacokinetic interactions might be interesting. one gram of aztreonam and . g of amikacin were administered intravenously single and in combination in six healthy volunteers. blood samples were collected at regular time intervals and concentrations of antimicrobials were determined by a microbiological assay applying a strain developing resistance to single agent after serial passages. mean concentrations of amikacin in serum when administered alone and in combination with aztreonam were . and . , . and . , . and . , . and . , . and . and and . mg/ml immediately after and . , , , and h after infusion of antimicrobials. respective concentrations of aztreonam were . and . , . and . , . and . , . and . , . and . and . and . mg/ml. aucs for amikacin when administered alone and in combination with aztreonam were . / . and . / . mg h/l, respectively. respective auc for aztreonam were . / . and . / . mg h/l. it is concluded that the co-administration of aztreonam and amikacin results in earlier clearance of aztreonam and in higher levels of amikacin compared to the administration of each single antimicrobial. molecular modelling of b-lactams reveals the structural basis for their inhibition of penicillin-binding proteins, susceptibility to b-lactamases and oral bioavailability pm grail bm, gupta s, payne jw. school of biological sciences, university of wales, bangor, uk b-lactam antibiotics are peptide mimetics that act as suicide substrates for transpeptidase enzymes that cross link bacterial cellwall peptides. for the first time, the structural and electronic features needed for their recognition by transpeptidase have been fully described, using innovative molecular modelling techniques to compare the conformational forms adopted by cell-wall peptides and blactams. comparison of features in the backbone and c-terminal regions of conformers of active b-lactam antibiotics and model cellwall peptides, has allowed definition of the molecular recognition template required for substrate recognition by transpeptidase. these shared structural features allow both to act as substrates and to acylate the active-site serine. however, a significant difference in a critical backbone torsion between the two substrates, provides an explanation for the inability of the enzyme Á/antibiotic complex to undergo the deacylation step that causes inhibition of transpeptidase. on the other hand, b-lactamases appear to have evolved molecular mechanisms that facilitate the deacylation reaction through compensating for the altered structural orientations in b-lactams caused by the different backbone torsion. finally, analysis of the conformer repertoires of blactams for structural features required for substrate uptake by peptide transporters, provides insights into how their structures can be tailored for optimal oral absorption. antimicrobial susceptibility of proteus mirabilis clinical isolates producing extended spectrum beta-lactamases (esbls) pm objectives: the aim of the present study was to determine in vitro susceptibility to antimicrobials of proteus mirabilis isolated from urinary tract infections. methods: we studied the susceptibility profile of esbl positive p. mirabilis strains in three adopted children from india with age range from months to years. esbl was identified using the synergic effect of clavulanate with betalactams (ceftazidime and cefotaxime the in vivo efficacy of amoxicillin (amx) sub-therapeutic doses ( . mg/kg, t.i.d for h, achieving serum levels over the mic of only % of the dosing interval) and concomitant specific serotherapy (single intraperitoneal dose of / diluted hyperimmune serum (hs) obtained from mice immunized with the heat-inactivated strain) was assessed in a pneumococcal sepsis balb/c mouse model. mice (five mice/ treatment group) were intraperitoneally infected with . )/ cfu/ ml of a serotype b penicillin-resistant strain (mic of and mg/l for penicillin and amx, respectively). treatments started h after bacterial inoculation. study groups were: control (k; receiving nonimmune serum (nhs)), amx'/nhs, hs, and amx'/hs. survival rates (%) over time were: purpose of the study: the study was performed to determine the consumption of imipenem and resistance of gram-negative pathogens (pseudomonas aeruginosa , acinetobacter sp., klebsiella sp., escherichia coli , proteus mirabilis , serratia marcescens , enterobacter sp. ) to imipenem. gram-negative pathogens were isolated at the sestre milosrdnice university hospital from zagreb, croatia, in and . the imipenem sensitivity testing was performed by disk diffusion and e -test methods. the consumption of imipenem was expressed in ddd/ hospital days in the same periods. results obtained: imipenem resistance of acinetobacter sp. decreased significantly in the year (p / . ), especially in the first months (p / . ) when the lowest consumption of imipenem was recorded. imipenem resistance of other gram-negative pathogens did not decrease significantly. conclusion reached: comsumption of imipenem might lead to changes in resistance to imipenem among acinetobacter strains. vacheva-dobrevski rs, savov ez. military medical academy, clinical microbiology, sofia, bulgaria purpose: acinetobacter baumanii is becoming increasingly frequent nosocomial pathogen at our hospital, and beta-lactam resistant strains are on the increase, especially among icu isolates. to study the susceptibility of a. baumanii clinical isolates to beta-lactams and to determine the esbl-producing strains during , year. a total gram-negative nonfermenters (gnnf) isolates was investigated by semiautomated mini api system (bio merieux, france). eighty-four a. baumanii non-repeated isolates was studied for esbl-producing by double-disk synergy test (ddt) and atb-blse test (bio merieux, france). mics for beta-lactams were determined by e -test (ab biodisk, sweden). results: a. baumanii (n / ) showed a multidrug resistance. the isolates were resistant to cefotaxime ( %), cefoxitin ( %), ceftazidime ( %), amoxicillin/clavulanate ( %), piperacillin ( %), aztreonam ( %), imipenem ( %). the ( %) of investigated a. baumanii expressed esbl activity and originated more frequently from icu ( %). esbls producing strains were isolated from endotracheal aspirate ( %), surgery wounds ( %), blood culture ( . %). conclusions: in general resistance levels were higher in clinical isolates a. baumanii to beta-lactams. the ddt seems to be a practical method for esbl-screening; atb-blse method is more sensitive. our study display to be the first report of esbl-producing a. baumanii strains from our country. carbapenems seems to be the most active agents against a. baumanii . salmonella infantis , strain , was isolated from a newborn baby at wassila bourguiba maternity in tunis. it exhibited high resistance to penicillins, extended-spectrum cephalosporines (cefotaxime, ceftriaxone, ceftazidime, cefpirome) and aztreonam but remained susceptible to cefoxitine and imipenem. involvement and characterization of enzymatic mechanism in b-lactam resistance were investigated in strain . isoelectricfocusing revealed that this strain produced a b-lactamase of pi . this enzyme had a broad-substrate profile, hydrolyzing amoxicillin, ampicillin, ticarcillin, cephaloridine, cefuroxime, cefotaxime, ceftriaxone, cefpirome and ceftazidime. the highest specific activity was observed with ampicillin. cefotaxime was hydrolyzed the most efficiently of the extended-spectum cephalosporines. the pi extended-spectrum b-lactamase (esbl) was inhibited by clavulanic acid and sulbactam. no inhibition of the esbl was observed with mm edta. thus, no metal ion is involved in hydrolysis for this b-lactamase. resistance due to the production of the pi esbl was transferred with dna plasmid into escherichia coli . on the basis of substrate and inhibition profiles and isoelectric point, the pi esbl was not previously described in s. infantis in tunisia. the presence of such a resistance on a plasmid raises concer for rapid dissemination among bacteria and loss of effectiveness of blactams. poizot-martin i a , enel p b , benhaïm s c , vion-dury f c , dinh t c , drogoul mp c , gastaut ja c . a assistance publique hôpitaux de marseille, cisih sud, pr ja gastaut, marseille, france , b assistance publique hôpitaux de marseille, cellule santé publique dmi , marseille, france , c assistance publique hôpitaux de marseille, cisih sud, marseille, france objective: to assess liver biopsy (lb) practices in a cohort of co-infected hcv and hiv patients followed up in an hiv specialized medical unit. method: transversal study with questionnaire among patients in pre-therapeutic's evaluation with pcr'/ and without lb at months. results: among the patients, ( %) are lost of follow up, ( . %) have had lb, ( . %) have no lb. the characteristics of these patients are: median age / . / years; sex ratio / . , cdc-stage a / . % b / . % c / . %, undetectable viral load / . %, median cd / / , anti-retroviral therapy / . %, hcv-genotype / . %; a / . %; / . %. causes of non-made lb are: ( ) refusal from patients because of biopsy's fear / . %; ( ) contraindications because of hiv infection / . % (clinical events / . % which contraindicate anti-hcv treatment, grade iii thrombocytopenia / . % which contraindicate biopsy, non-adherence to previous hiv follow up / . %); ( ) other / . % (alcoholism / . %, psychiatric/depressive disorders / . %, decompensated cirrhosis / . %). drug use or methadone/buprenorphine treatment are not considered as contraindication. conclusion: one-third of patients are afraid of lb. alcoholism and psychiatric/depressive disorders are the principal contraindications to anti-hcv treatment. it seems important to improve information of patients about lb and to focus on alcohol and psychiatric/depressive disorders management in such population. kashiwagi kk a , furusyo nf a , nakashima hn a , kashiwagi sk b , hayashi jh a . a department of environmental medicine and infectious disease, kyushu university, fukuoka, japan , b national kyushu medical center, fukuoka, japan the purpose of the study: the aim of this prospective study was to explore the effect of htlv-i co-infection on the development of hcc among patients with chronic hcv viremia. a total of consecutive patients with chronic hcv viremia were studied and followed-up over a mean period of . years: ( . %) were infected with htlv-i infection and ( . %) were not. the results obtained the annual hcc development rate was . % in patients co-infected with hcv and htlv-i and . % in patients infected with hcv alone. hcc was significantly higher in ( . %) of the patients co-infected patients than in ( . %) of the patients infected with hcv alone (p b/ . , logrank test). in patients under the age of years, hcc development was significantly higher in seven ( . %) of patients co-infected with hcv and htlv-i than in eight ( . %) of patients with hcv alone (pb/ . , logrank test), whereas there was no significant difference in hcc development between patients over age with or without htlv-i infection ( ( . %) of and ( . %) of , respectively). the conclusion reached htlv-i infection accelerates the development of hcc in chronic hcv patients, especially among patients under the age of years. to analyze hbv genotype-related clinical differences among patients with chronic hbv infection, all patients were serially tested for serum alanine aminotransferase (alt) and hepatitis b e antigen (hbeag) and followed up for a mean . ( . ) year period. genotypes b and c were found in ( . %) and ( . %) of the patients, respectively. hbeag positivity and alt abnormality rates at the start of the observation period were significantly higher in genotype c patients ( . and . %) than in genotype b patients ( . and . %). the annual rate of spontaneous hbeag disappearance in genotype b patients was much higher than in genotype c patients ( . versus . %, respectively). patients with genotype c who were continuously hbeag negative from entry had significantly higher alt abnormality ( . %) than those with genotype b ( . %). interestingly, patients with genotype c who became hbeag negative by interferon treatment had high alt abnormality ( . %). all patients with alt abnormality were serum hbv dna positive. these findings indicate that hbv genotype c patients are more severe liver deterioration because of the delay of hbeag disappearance and continued hbv replication after hbeag disappearance. nossik nn a , nebolsin ve b , zheltukhina ga c , yevstigneeva rp c . a the d.i. ivanovsky institute of virology, viral reproduction, moscow, russian federation , b pparminterprisis co., chemistry, mocow, russian federation , c moscow state academy of fine chemical technology, piptide chemistry, moscow, russian federation objective: to study the effects of 'gamma'-l-glutamylhistamine (glu-ha) derivates on non-specific immunity ('alfa'-ifn, 'gamma'-ifn and nk cell activity) and antiviral activity on the experimental influenza and herpes virus infections in mice. the glu-ya and its derivate glu-ii were synthesized by peptide chemistry techniques. the glu-ha and glu-ii was administered i.p. . and . mg/kg before and after influenza virus (type a/aichi) and showed a protective effect even at the high infective dose ( ld ) */the rate of protection / Á/ / % in the positive/control group. they were not very effective in the protection of herpes simplex virus encephalitis in mice. the model of the physico-emotional stress in mice was used to investigate the ifn system and nk cell activity. the production of ifns and nk cell activity of splenocytes decreased in h after the stress and back to normal level in Á/ days. it was shown that glu-ha and glu-ii can protect or substantially prevent the decrease in nk cell activity and ifns synthesis in post-stress period (so normally did not induce the ifns' synthesis). conclusions: the glu-ha and glu-ii showed antiviral effect against influenza virus infection in mice. the immunomodulating activity and ability to normalize the ifn synthesis and nk cell activity depressed the post-stress period and probably play an essential role in the antiviral activity. no dose adjustment of an anti-influenza prodrug oseltamivir is required in patients with hepatic impairment pm oo c a , snell pr b , liu b a , martin d b , simkins t b , small i b , ward p b . a hoffmann-la roche inc., global development, nutley, usa , b roche products ltd., global development, welwyn garden city, uk background: oseltamivir (ose; ro - , tamiflu † ) is an oral ethyl ester prodrug of its active metabolite oseltamivir carboxylate (oc: ro - ), a potent and selective neuraminidase inhibitor of the influenza virus. the purpose of the study is to evaluate the need for ose dosage adjustment in hepatic impaired patients (hi). method: healthy volunteers (hv) versus hi (child-pugh score Á/ ) [matched on the basis of age ( / years), gender and weight ( / %)] were compared. each subject received mg ose. results: based on c max (ng/ml) and auc inf (ng h/ml) analysed using nominal times, ls mean ratios and % ci between hi and hv were similar. ose* values in hi were marginally elevated but not sufficiently to require dose adjustment. the aim of this study is to investigate the influence of molecular structure of macrocyclic pyridinophanes and their analogs on antiinfluenza and antiherpetic activity of these compounds. we used d-qsar approaches on the basis of simple representation of molecular structure. such representation for biologically active substances allows the description of the spatial structure of compounds with the complete stereochemical information. it determines spatial structures either promoting or interfering of the concrete biological activity. it is easy to realize the molecular design of compounds with the given level of activity with the help of the combinations of simplexes. statistic characteristics for qsar of partial least-squares models are satisfactory (r / . Á/ . ; cvr / . Á/ . ). the molecular fragments that increase the antiviral activity were defined and will be demonstrated. this information was used for design and directed synthesis of several novel antiviral agents with predicted high anti-influenza or antiherpetic activities. predicted activities were confirmed experimentally. d-qsar approaches are useful for development of antiviral compounds. this work was partially supported by intas foundation (grant intas - ). lozitsky vp. ukrainian mechnikov research anti-plague institute, chemotherapy, odessa, ukraine the purpose of this study was to research the anti-influenza activity of proteolytic inhibitor e-aca. it prevents the enhancement of proteolysis during the interaction of virions with cell membranes and decreases penetration of virions into cells. e-aca brings down proteolytic cleavage of ha-precursor to ha- and ha-polypeptides and reduces the infectious virus harvest. it shows the prophylactic and therapeutic action during the experimental influenza reducing the enhancement of alkaline proteases activity in lungs after infection. e-aca promotes the intensification of specific antibodies production and cell immunity, prevents vessels' permeability and hemorrhagic phenomena, decreases the destruction of bronchi's epithelium. it reduces the duration of intoxication, catarrhal instances and hyperthermia in sick children. e-aca improves the indexes of immunity, non-specific resistance and decreases the rate of bacterial complications. application of e-aca for treatment influenza and other arvi in children is recommended in ukraine on the base of results of our researches. the higher effects demonstrated as a result of combine usage of e-aca with specific ig, or deitiforin, or unithyol, or ribavirin. in our opinion, the study of effectiveness of e-aca combine application with inhibitors of influenza na is the perspective direction of anti-influenza researches development. we should we treat immediately all varicella patients with acyclovir if the patient is older than years pm during past years in institute for infectious and tropical diseases in belgrade, immunocompetent varicella patients were treated and cured. among them were older than years ( . %). x-rays were performed in all patients. diagnosis of pneumonia was made in patients ( . %), but in ( . %) patients older than years. varicella is a benign, self-limited disease, if it strikes early, i.e. preschool, school children and teenagers. at that time there is no need for specific therapy. but in neonates, immunocompetent adults and in all immunocompromised patients it can be difficult and life-threatening disease. in immunocompetent adult population pneumonia is a very serious, sometimes fatal complication. knowing the pathophysiology of primary varicella Á/zoster infection, specific therapy with acyclovir should be started immediately after making the diagnosis in patients older than years, without waiting for x-ray proof of pneumonia. brivudin compared to acyclovir for the treatment of herpes zoster: effects on acute disease and posttherapeutic pain pm objective: comparison of efficacy and safety of brivudin )/ mg and acyclovir )/ mg, both for days, in the treatment of herpes zoster. methods: randomised, double-blind study on immunocompetent patients ]/ years (brivudin: n / , acyclovir: n / ). a subgroup of patients ]/ years (brivudin: n / , acyclovir: n / ) was examined for the occurrence of posttherapeutic pain in a poststudy survey. posttherapeutic pain was defined as any zoster-associated pain, regardless of intensity, after the end of acute zoster. results: brivudin was superior to acyclovir in reducing time to last occurrence of new vesicles (rr(itt): . [ . Á/ . ], p / . ). the advantage of brivudin was more pronounced in patients ]/ years (rr(itt): . , [ . Á/ . ], p / . ). incidence of posttherapeutic pain was significantly lower with brivudin ( . %) than with acyclovir ( . %, p / . ). duration of pain was comparable in both treatment groups (rr: . , [ . Á/ . ], p / . ). potentially treatment-related adverse events occurred in . % of the brivudin recipients and in % of the acyclovir recipients. conclusions: brivudin mg once daily for days is superior to standard acyclovir in stopping viral replication in acute herpes zoster. in patients ]/ years, brivudin is more effective than acyclovir in reducing the risk of developing posttherapeutic pain. brivudin is as well tolerated as acyclovir. varadinova t a , genova p a , garcia-raso a b , terron a b , fiol j b , badenas f b . a laboratory of virology, sofia university, sofia, bulgaria , b chemistry, universitat de les balears, palma de mallorca, spain we have published that cu(ii) complexes of acyclovir (acv) are active against hsv infection (mbd, ) . here we present data on the activity of acv complexes of ni(ii), cd(ii), co(ii) and ag(i) against resistant to acv hsv strain r- in comparison with the effect against acv sensitive strain victoria. selectivity indexes (si) compared to that of acv were indicative for activity. the following data were obtained: (i) was times less selective inhibitor of strain r- than of strain victoria; (ii) under the action of [cd(acv)cl ], [ni(acv) (h o) ]cl ×/ acv and [ni(acv)(no )] ×/ h o was up to % higher than that in the control; (ii) was times less selective inhibitor than acv; (iv) si of was two times higher for strain r- and five times lower for strain victoria then that of acv. these data show that the selectivity of acv against resistant hsv strains can increase when acv is bond to a proper metal ion. methods: an antiviral activity against herpes simplex virus of the type i (hsv-i/leningrad/ / ) and variant hsv- (vvt/ / r) resistant to acyclovir was determined using commonly accepted method. viruses were grown on a continuous culture. maximal toxic dose was determined by the administration of compounds orally ( mg/kg) or intraabdominally ( mg/kg) to white mice that had mass Á/ g. condition of the animals was controlled during h. mice pneumonia model was used for the testing activity in vivo. results: derivatives tested have activity against hsv- and hsv- resistant to acyclovir. maximum protection of the cells up to % was reached at concentration of compounds Á/ mg/kg. tested compounds have low toxicity and animals did not die after intraabdominal and after per oral administration of the substances. using these compounds led to essential relief of diseases in animals. the number and square of virus specific areas of inflammation in lung was decreased to compare with control untreated group. tested compounds protected animals similar to acyclovir that was used as control. conclusion: derivatives of carboalkoxysulfanilic acids are active against hsv in vitro and in vivo and act on the acyclovir resistant variant viruses. markiewicz r a , szepietowski jc b . a jelfa s.a., medical department, jelenia gora, poland , b department of dermatology, university of medicine, wroclaw, poland background: denotivir is a -benzoamino- ?-chloro- -methyl- isothiazolecarboxanilide anti-inflammatory agent with antiviral and immunomodulatory activities. it possesses also mild antibacterial and antifungal action. the purpose of the study: the aim of this presentation is to give an overview of recent studies demonstrating denotivir efficacy in herpetic infections. the results obtained: in vitro studies revealed that denotivir in the doses below its cytotoxicity (about um) significantly inhibited (by Á/ %). herpes simplex virus (hsv)- and hsv- replication in fibroblast and kidney cell cultures. moreover, it was showed that denotivir in the dose of mg/ml markedly inactivated hsv- after min incubation in c. in giunea pigs research, % denotivir in % dmso appeared to be superior to % dmso alone and untreated groups in the therapy of animal skin infected by hsv- . there was no huge difference in eythema and oedema scorings between studied groups, however in the group treated with denotivir, in contrast to others, no vesicles developed. several clinical studies showed usefulness of denotivir in controlling herpetic infections in dermatology, ophthalmology and otolaryngology. in the majority of studies within few hours after the drug application itch and pain relief was noted and within Á/ days the vesicular lesions were dried up. the conclusion reached: in conclusion, denotivir is an effective antiherpetic agent. this study was conducted on cows affected with teat papillomatosis. in the first step, each cow was located on one of three groups. the first group contained four cows from to years that were treated with fig tree (ficus carica) latex. the second group contained four cows from . to years that were treated with a % solution of salicylic acid, and the third group contained four cows as control. in group one and two following treatment with fig tree latex and salicylic acid, superficial necrosis begun from day and all of the warts disappeared by day . in the control group, after day , there were no changes in number of lesions, but some of them were larger than first observation. on day , one of the marked warts disappeared and on day another wart was disappeared but six were present until day . comparison of effects of salicylic acid and fig latex showed similar effects in treatment of udder papillomatosis in cow. laboratory markers of skeletal muscle toxicity in hiv-infected patients: a cross-sectional case-control survey of frequency, potential correlation with antiretroviral therapy, clinical significance, and outcome pm manfredi r, motta r, patrono d, calza l, chiodo f, boni p. to assess skeletal muscle toxicity among Â/ hiv-infected outpatients (p), the p who had ]/ altered cpk assay ( !/ u/l) between may and november , were compared with p randomly selected among those who had ]/ laboratory exams in this -month interval, in a : case-control study. among the p with altered cpk levels only six were females, and received antiretrovirals. the overall frequency of altered cpk among all p who underwent ]/ laboratory workouts in months was . %. cpk alteration was transient in p, with values ranging from to (mean . / . ) u/l, but was recognized ]/ times in Á/ months in p ( %), of them showing concomitant high aldolase levels ( . Á/ . u/l). a myopathy or a rhabdomyolisis were recognized in four p only; a myositis was confirmed in one p by histopathology. in a multivariate logistic regression analysis, when excluding the unexpected prevalence of the male gender (p b/ . ), no significant difference emerged between p and controls as to age, risk for hiv infection, iv drug use, duration of hiv infection, prior anti-hiv therapy and its length, selected drug combinations, administered nucleoside analogues,hiv disease stage, mean cd '/ count and hiv viremia, signs and duration of lipodystrophy, increased glucose, triglyceride and cholesterol levels, and other therapies. muscle abnormalities, though frequently asymptomatic, are underestimated hiv disease complications, and the role of metabolic (i.e. mitochondrial) alterations, deserves investigation. poor efficacy of non-nucleoside reverse transcriptase inhibitor (nnrti)based salvage haart in hiv-infected patients heavily pre-treated with all other classes of antiretroviral compounds pm manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy poorly comparable literature series show conflicting results of nnrti-based rescue haart: Á/ % rate of virologic success. to assess the response to a Á/ -drug rescue haart including a nnrti, patients (p) treated with nucleoside analogues (na) and protease inhibitors (pi) for !/ and !/ months, respectively but naïve to nnrti, with a viremia !/ copies/ml, were prospectively followed during Á/ years, provided that they ensured a !/ % adherence. efavirenz was used in p, nevirapine in , and delavirdine in two. most p ( . %) had an early laboratory improvement, but mean peak viral load decrease was . log, and a significant reduction vs baseline (p b/ . ) lasted months only. a mean % increase of zenith cd count was obtained (p b/ . ), but only . % of p remained !/ cells/ml year after switch to nnrti. in a multivariate analysis, the concurrent introduction of novel pi(s) ( p) and/or different na(s) ( p) acted favorably until the th month of follow-up (p b/ . ), while genotypic mutations conferring nnrti cross-resistance, usually associated with a broad resistance profile, predicted failure in all p (p b/ . ), and the response did not vary according to duration and type of prior therapy, and selected nnrti. a deep salvage nnrtibased haart has a poor and transient virologic outcome also in nnrti-naïve p, while a more evident and sustained immunologic response is expected. p who can introduce novel pi/na and have no mutations impairing nnrti activity are entitled to a better outcome. fatal lactic acidosis without elevation of liver-enzymes during the treatment with stavudine, didanosine and efavirenz: a case report pm winzer r, langmann p, väth t, zilly m, klinker h. medizinische poliklinik der universität würzburg, schwerpunkt hepatologie/infektiologie, ürzburg, germany nucleoside reverse transcriptase inhibitors (nrtis) cause various side effects, many of which are thought to be due to their effects on mitochondria. a -year-old hiv positive (hiv rna: copies/ml, cd cell count: /ml), obese (body-mass-index: . ), therapy-naïve female patient, who after months of well tolerated and effective antiretroviral therapy (stavudine, didanosine, efavirenz), had slight gastrointestinal discomfort and suddenly developed a lactic acidosis (arterial-ph . [ . Á/ . she died days later despite intensive care (continuous venovenous haemodiafiltration, sodium-bicarbonate infusion, high doses of vitamins, respiration). the pathologic examination showed an enlarged liver ( g) with yellowish appearance and pasty consistency, which microscopically appeared as a massive macro-and microvesicular fatty degeneration, and only slight signs of terminal pancreatitis. this reported case gives evidence that a massive lactate acidosis may develop without previously disarranged laboratory parameters for liver or pancreatic function. a fatal outcome may evolve without further accompanying-illnesses. efficacy and tolerability of atorvastatin in the treatment of hypercholesterolemia in hiv-infected patients receiving haart pm calza l, manfredi r, chiodo f. division of infectious diseases, university of bologna, bologna, italy introduction: significant increases in plasma triglyceride and cholesterol levels have been reported in patients treated with haart, and prolonged metabolic imbalances could significantly act on the longterm prognosis and outcome of hiv-infected persons. patients and methods: fourteen hiv-infected patients on pi-based haart since at least months and presenting hypercholesterolemia ( !/ mg/dl) of at least -month duration and unresponsive to a hypolipidemic diet and physical exercise, have been treated with a single daily dose of atorvastatin ( mg) for months. results: one patient was ecluded from evaluation due to early dropout. ongoing antiretroviral treatment included ritonavir in four cases, indinavir in four, nelfinavir in three, and saquinavir hard-gel in two. at the close of -month follow-up of atorvastatin therapy, a decrease of total cholesterol level of . % versus respective baseline value was observed; eight out of patients reached normal values for cholesterol. mild gastroenteric symptoms were found in only one of the treated patients, while no skeletal muscle and liver toxicity has been observed. discussion: in our study, pharmacological treatment with atorvastatin proved certainly effective in the management of diet-resistant hypercholesterolemia, and was associated with a favourable tolerability and adherence profile. the effect of combination antiretroviral therapy regimens on hiv- proviral dna level in peripheral blood mononuclear cells (pbmcs) was examined in hiv- -positive patients, using endpoint dilution pcr and serially cloning and sequencing of the gag region of hiv- . the major clone was defined as the most numerous of analyzed clones, and observation periods ranged from to months (mean, . / . months). in five patients (one with primary-stage hiv- infection) receiving three antiretroviral drugs, hiv- rna levels reduced to undetectable (i.e. b/ copies/ml). hiv- proviral dna levels and the number of major clones reduced in four of these patients. hiv- rna levels reduced, but remained detectable, in five other patients. in the two remaining patients (both receiving two rather than three antiretroviral drugs) hiv- rna levels increased. these results suggested that the population of the major clones may be affected when hiv- rna levels reduce following combination regimens of antiretroviral therapy. saquinavir hard gel (shg) as a part of a spontaneous Á/ -month deintensification anti-hiv regimen following successful highly active antiretroviral therapy (haart) pm manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy the induction-maintenance concept was poorly studied in hiv'/ patients (p), and shg was never assessed after prolonged response to potent protease inhibitors (pi)-based haart. shg-naïve p who refused indinavir, ritonavir, or nelfinavir-based haart after achieving long-term viral suppression, and resorted to shg'/ nucleoside analogues (na), were followed prospectively. in . % of the p assessed for Á/ months, ]/ na was changed. prior haart was interrupted after . / . months, due to adverse events ( p), or p's request ( p), while a viremia b/ copies/ml was present since . / . months. a viremia of Á/ hiv-rna copies/ml was maintained in p ( . %), while a higher viral load occurred in p after . / . months, and was related to a pre-haart viremia !/ / ml, a more frequent !/ % recovery of cd count, mutations of codons Á/ , and failure to change na (p b/ . Á/ . ). a cd drop !/ %/ cells/ml was found after . / . months in only eight p, who also had virologic failure: immunologic deterioration was earlier and deeper when na were not changed (p b/ . ). all the p who introduced shg'/novel na after a successful !/ -month induction with a potent pi-based haart had a stable Á/ -month outcome. a suboptimal haart including the less effective but better tolerated shg may be effective for !/ year, especially when novel na are introduced, and specific mutations are absent. despite a lower potency, drugs with a good safety and compliance profile may be recovered for simplified regimens. objective: to evaluate efficacy of antiretroviral therapy (art) with two or three drugs in the nervous 'reservoir'. patients: thirteen acute neurological and art naive aids patients underwent a paired and simultaneous sample from plasma and cerebrospinal fluid (csf) for a quantitative detection of hiv- rna (amplicor roche) before art. all patients underwent a ct and/or mr of the brain to perform a diagnosis. all of them had an hiv related neurological acute inflammatory disease. after diagnosis all patients received art: / received two nrti and / received haart including two nrti and one protease inhibitor. all patients underwent a paired and simultaneous follow-up from plasma and csf during the nd month of treatment. results: in all patients baseline levels of hiv-rna were higher (p b/ . ) in the plasma (log . '/ . ) than in the csf (log . '/ . ). the / patients who received dual therapy had undetectable levels (cut-off copies/ml) of viral rna at the follow-up in csf, but not in plasma: three of these seven patients had a detectable plasma hiv- rna. all / patients with haart had undetectable hiv- rna both in plasma and in csf at the follow up. conclusions: dual nrti therapy is rapidly effective in csf (because of an high penetration of drugs through a more permeable blood Á/brain barrier and lower hiv rna baseline levels) but not in plasma. haart is rapidly and equally effective both in csf and plasma. there are no reports of fulminant and fatal hepatic failure after the start of highly active antiretroviral therapy (haart) in an hiv subject without chronic viral hepatitis. case report: a -year-old naive aids woman with clinical symptoms due by a pcp was observed. baseline alt was increased ( . m.n.v.) because of a mild hepatosteatosis and a silent cholelithiasis. serology for hbv, hdv and hcv was negative; igg anti-hav, anti-ebv, anti-cmv and anti-hsv were present. hiv- rna was . log , cd '/ count was /ml. during the pcp treatment with cotrimoxazole alt values increased ( !/ m.n.v.); nevertheless, she completed the treatment. liver enzymes returned to the pre-treatment values over several days. then she started haart with stavudine, lamivudine and efavirenz. after days the patient showed an efavirenz-related skin rush that resolved within days, without treatment discontinuation. fourteen days after the start of haart jaundice appeared. laboratory revealed severe alt increase ( !/ m.n.v.) and hyperbilirubinemia ( mg/dl) and she died because of an acute liver failure syndrome within few days. an haart efavirenzbased regimen can result highly hepatotoxic when given in presence of a hepatosteatosis, of a recent hepatotoxicity caused by a nonantiretroviral treatment and of a previous idiosyncratic reaction to efavirenz. our experience with bulgarian herbal extracts for improving the general condition of hiv-positive patients pm methods: we used a combination of bulgarian herbal extracts and treated six patients, divided in two groups: three with symptomatic and three with asymptomatic hiv-infection. the all three patients with asymptomatic hiv-infection were treated only with herbal extracts, another three patients with symptomatic hiv-infection were treated with combination of herbal extracts and anti-retroviral therapy. the general status of patients has been evaluated by both subjective and objective surveillance. the immunologic monitoring has been performed by absolute count of cd '/ lymphocytes. results: all patients have shown an obvious improvement in their general condition: high spirit and working capacity, good appetite and sleep, a restoration of body weight. the number of cd '/ lymphocytes has been lightly increased or constant. conclusion: the combination of bulgarian herbal extracts has shown significant positive effect on the general condition and improve the quality of life. antifungal activity of in vitro and in vivo combinations of voriconazole with -fluorocytosine and amphotericin b against candida and cryptococcus spp pm hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: the present study was designed to determine whether the activity of voriconazole (vor), a novel triazole, was reduced against candidal and cryptococcal infections by the addition of standard antifungal agents, amphotericin b (amb) and -fluorocytosine ( -fc). vor was tested in combination with standard antifungal agents both in vitro, using a checkerboard mic determination test, and in vivo in immune normal guinea pig models of fungal infections. the results indicate that the efficacy of vor against candida albicans and c. neoformans was not antagonised by amb or -fc in vitro. furthermore, in guinea pig models of systemic candidiasis and intracranial cryptococcosis, no antagonism was observed between the lower doses of vor and either amb or -fc on the basis of reductions in tissue fungal loads. at the highest doses of vor, both amb and -fc showed some antagonism, but the combinations were still effective in significantly reducing fungal tissue loads compared with vehicle-treated control animals. conclusion: these results suggest that vor may be used in combination with standard antifungal agents, and future studies to elucidate the clinical potential of vor combination therapies in the management of candida and cryptococcus infections are warranted. itraconazole in the treatment of pityriasis versicolor pm tiodorovic j, jovanovic d, binic i, nikolic lj. faculty of medicine, clinic of dermatovenerology, nis, serbia, yugoslavia a comparison of two short-term dose schedules with itraconazole was carried out in patients with pityriasis versicolor. the patients were divided in two groups. each group consisted of patients who completed the therapy and controls. the clinical diagnosis was confirmed mycologically, by direct microscopic examination. the first group received mg of itraconazole daily for days. the second group received mg daily for days. the patients were controlled clinically and mycologically and days after the initiation of treatment. erythema, scaling and pruritus was evaluated clinically. clinically and mycologically cured patients accepted as cured. the cure rate were % in the first group and % in the second group at day . the effects of these two groups are similar. none of the patients reported side-effects. fungal urinary infections: emerging species, antifungal susceptibility trends and antibody response pm badawi he a , kamel ai b , fam ns a , el-sayed me a , elian sae c . a theodor bilharz medical research institute (tbmri ), microbiology, giza, egypt , b theodor bilharz medical research institute (tbmri ), urosurgery, giza, egypt , c faculty of medicine, medical microbiology and immunology, cairo university, cairo, egypt objectives: to assess the role of candida species in patients with urinary tract infections (utis) with or without schistosomiasis and/or cancer bladder, to compare chromogenic; chromagar (cma), biggy agar; morphologic (corn meal, rice agar-tween ) media and biochemical candifast test for identification of candida species. susceptibility to antifungal agents using e -test and candifast and the performance of elisa test for detection of anticandida antibodies (igm and igg) in serum were evaluated. results: c. albicans was the most frequent ( . %) species responsible for fungal utis. however, non-albicans species, c. glabrata ( . %), c. tropicalis ( %) and c. krusei ( . %) were also isolated. rice agar-tween was found to be cheap, available and sufficient to make a final identification ( %). cma could not identify c. glabrata . biggy agar could not adequately differentiate candida species. candifast biochemical identification showed low sensitivity of . %. e -test on sabouraud dextrose agar (sda) is simple method for mics determination and could detect s-dd strains in case of azoles. conclusion: the emergence of non-albicans species such as c. glabrata , c. tropicalis and c. krusei have contributed to complicated utis. this necessitates accurate isolation and identification of candida to the species level. morphology on rice agar-tween and antifungal susceptibility using e -test on sda is a simle rapid scheme for routine identification of clinically important yeasts. purpose: classification of allergic fungal rhinosinusitis (afr) is based on the immunologic relationship of the host to the fungus. afr must be differentiated from other fungal rhinosinusitis infections, which include acute invasive, chronic invasive, fungal balls and saprophytic colonization. although many cases of fungal rhinosinusitis is caused by species of aspergillis , dermatiaceous moulds have become an emerging pathogen in immunocompetent individuals. results: our case study involved a year male suffering from facial pain, headache, postnasal drip and loss of smell. he was hiv negative and a nonsmoker. the following laboratory tests were performed: ige- . ( . Á/ . ) iu/ml iga- . ( . Á/ . ) g/l igm- . ( . Á/ . ) g/l allergen specific ige for alternaria */ . ( . Á/ . ) ku/l fbc-normal except eosinophils slightly raised . ( . Á/ . ))/ / l. ct scans indicated fungus proliferation, bone erosion and extension of disease into adjacent anatomic area. sinus tissue following debridement was sent for microscopy and culture. hyphae was microscopically observed and cultures yielded two dermatiaceous fungi, bipolaris spp and alternaria spp . conclusion: it is important to differentiate these two species from curvularia , helminthosporum , drechelria and exserohilum . knowledge of these dermatiaceous fungi is important in directing appropriate antifungal therapy and selecting the correct antigens for postsurgical immunotherapy after initial debridement and irrigation. antifungal activity of in vitro and in vivo combinations of voriconazole with -fluorocytosine and amphotericin b against aspergillus fumigatus pm hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: a key requisite for a new antifungal drug is to demonstrate that it is devoid of significant antagonism in combination with other agents. combinations of the new triazole, voriconazole (vor), and standard antifungal agents ( -fluorocytosine or amphotericin b; -fc or amb) were tested against aspergillus fumigatus in vitro and in guinea pig models of infections to confirm that antifungal activity was not antagonised by using combination therapies. vor was studied in combination with amb or -fc in vitro, using a checkerboard mic determination test, and in vivo, using immune normal and immunocompromised guinea pig models of systemic aspergillosis. results: the results indicate that the potency of vor was not antagonised by amb or -fc in vivo; indeed, at lower concentrations of vor, significant improvements in reducing fungal burden in both in vivo models were achieved by the addition of amb. in vitro, no antagonism was found between vor and amb, although -fc had a significant antagonistic effect on vor activity. conclusion: these results from in vitro and in vivo models of aspergillosis suggest that vor may be used in combination with standard antifungal agents and, therefore, justify further examinations of vor combination therapies in a clinical setting. in vitro activity of caspofungin compared to that of amphotericin b, fluconazole, and itraconazole against candida species pm arikan s, sancak b, hascelik g. department of microbiology and clinical microbiology, hacettepe university medical school, ankara, turkey purpose: to evaluate the in vitro activity of caspofungin against various candida spp. and particularly against isolates with decreased amphotericin b, fluconazole, and itraconazole susceptibilities. methods: susceptibility tests were done by nccls m a microdilution guidelines for clinical candida strains. the mics (mg/ml) were read at and h. results: caspofungin mics at h are shown in the table. mics at h were similar to h readings. expectedly, no evidence of crossresistance was detected between caspofungin and other drugs tested. caspofungin was similarly active against fluconazole-or itraconazolesusceptible and resistant isolates. conclusions: ( ) caspofungin is active in vitro against all candida spp. tested. ( ) caspofungin mics are slightly higher for c. parapsilosis compared to other species. ( ) its activity against fluconazole-and itraconazole-resistant isolates is noteworthy. ( ) validation of these data require clinical investigations. oropharyngeal microbiological samples of bmt patients were evaluated. weekly cultures (days (/ , and '/ ) revealed presence of fungi in patients ( . %): in four ( %) patients before bmt only, in ( %) after bmt only, and in ( . %) both before and after bmt. in one patient candida norvegensis was isolated from the throat, buccal and palatal surfaces. three c. albicans , two c. krusei , and three c. norvegensis from four patients were chosen to comper their antifungal sensitivities and extracellular virulence factors. using fungitest method we determined the sensitivities of these isolates to flucytosine, amphotericin b, miconazole, ketoconazole and fluconazole. the fluconazole sensitivities were also determined by the e -test. on the basis of the fungitest data the three c. norvegensis isolates were sensitive to flucytosine, amphotericin b, and also to ketoconazole. in case of fluconazole and miconazole they proved to be susceptible dependent upon dose. the mic fluconazole values determined by the e -test for the three c. norvegensis isolates were , ]/ and ]/ mg/ml, respectively. the oropharyngeal isolates of c. norvegensis produced high amounts of extracellular aspartic protease and phospholipase similarly to c. albicans strains. these enzymes may contribute to the pathogenesis of this new emerging candida species. in vitro activities of antifungal and antiseptic agents against rhodotorula sp pm preney l, théraud m, guiguen c, gangneux jp. laboratory parasitologie-mycologie, chu de rennes, france purpose of the study: rhodotorula species are common saprophyte yeasts widespread in nature. since the last years, they have been implicated in several severe infections, especially in immunocompromised patients, and various antifungal therapies were used. however, only limited data are available on the susceptibility of rhodotorula sp. to antifungal and antiseptic agents. material and methods: in this work, we evaluated the in vitro activities of eight antifungal agents against strains of rhodotorula ( strains of r. rubra and nine strains of r. glutinis using atb fungus system (biomerieux) and etest strips (ab-biodisk). beside, the effect of eight antiseptic agents was assessed on a suspension of r. rubra . the quantification of yeasts after exposure to antiseptic agents was performed by subculturings using a microtitration method in well plates. results and discussion: all strains tested were susceptible to amphotericin b, fc, and nystatin. twenty-nine out of strains were susceptible to ketoconazole, out of were intermediate to econazole. all strains were resistant to fluconazole (cmi!/ mg/ml) and itraconazole (cmi!/ mg/ml), and out of were resistant to miconazole, suggesting that antifungal therapy must be adapted when rhodotorula yeasts are implicated in invasive infection. beside, min exposure to sodium hypochlorite , chlorhexidine . % or ecodiol (isopropyl alcohol'/alkylamin) showed fungicidal activities. susceptibility testing of aspergillus fumigatus and emerging aspergillus pathogens by a modification of the nccls m -p method pm logotheti m a , kapsanaki-gotsi e b , velegraki a a , zagoura d b . a department of microbiology, mycology reference laboratory, medical school, university of athens, athens, greece , b biology department, section ecology and systematics, university of athens, athens, greece aspergillosis in high risk groups of patients is still associated with high mortality rate ( Á/ %). aspergillus fumigatus is the primary pathogen, while other opportunistic aspergillus species are emerging. amphotericin b (ab), itraconazole (it), voriconazole (vo) and terbinafine (te) minimum inhibitory concentrations (mic) were determined by modifying the nccls m -p microdilution method. stock drug solutions were prepared in rpmi , dimethyl sulfoxide (dmso), and polyethylene glycol (peg ). inocula, of the a. fumigatus group ( ), a. flavus group ( ) ( ) and the m -p quality control strains were prepared according to, and by modifying, the nccls guidelines. plates were incubated at and c and read at and h. peg effectively dissolved it and vo, while either dmso or peg dissolved te. low and c Á/ h ab, it, vo and te mics ( . Á/ . mg/l) were recorded. a. terreus ( ) and a. parasiticus ( ) were resistant to ab. certain clinical isolates demonstrate clinical and in vitro resistance. standardization of susceptibility testing would offer reliable assistance in selecting and monitoring antifungal therapy. otag f, aslan, g, ozturk c. microbiology department, faculty of medicine, mersin university, mersin, turkey rates of opportunistic fungal infections have risen markedly. because some of these species have potential resistance to antifungal agents, rapid presumptive species level identification is crucial in allowing for directed antifungal therapy. in this study, isolated yeasts from the clinical specimens were identified by atb id c (biomerieux, france). the number of identified yeasts were, respectively; ( %) candida albicans , six ( %) c. glabrata , five ( . %) c. tropicalis , three ( . %) c. parapsilosis , two ( . %) c. krusei , two ( . %) c. kefyr , one ( . %) c. guillermondii , one ( . %) c. dubliniensis . twenty-one of strains were investigated for antifungal sensitivities by atb fungus kit (biomerieux, france). the results are as follows: % sensitivity was detected to myconasol, % to flusitozin, nystatin and econasol, % to amphtericin b and ketokonazol. it is important to achieve empirik treatment of the opportunistic candida infections and the following of resistance to antifungals. shakhmatov da, strelchenco, ov. novosibirsk state medical academy, dermatovenerology, novosibirsk, russian federation at the present stage in russia with a background of a high case rate of syphilis, it becomes necessary to exclude biological false positive serological tests. because the serodiagnosis of syphilis has significant limitations, the direct detection of t. pallidum in suspect blood may serve as an alternate diagnostic strategy. polymerase chain reaction (pcr) has been the most widely used amplification method. the study of patients receiving examination related and treatment for syphilis in std clinic and persons directed from other hospitals where routine serologic examination revealed doubtful results. pcr reaction was carried out with nested primer pairs based on the dna sequence of the Á/ and Á/ kda gene of t. pallidum . pcr was utilized with whole blood. a complex of serological tests: fta-abs and tit was used as the &rdqup; gold standard''. as a result the sensitivity of pcr was . % and specificity was . %. selective comparison of pcr results with vdrl, the fta-abs and treponemal immobilisation test (tit) has shown concurrence . %. in conclusion, the preliminary results of pcr in whole blood in syphilis detection revealed its high sensitivity and specificity; possibility to obtain rapid results in unclear cases. chlamydia pneumoniae (cp) is an atypical pathogen whit intracellular location, whose eradication is very difficult. in the past years it has been objects of many studies that lead to the demonstration of a relationship between its presence and the development of widespread multifactorial pathologies such as atherosclerosis and asthma. the lack of its eradication can become an important clinical and social problem. the study objective is the comprehension of pathogen Á/host interaction mechanism, to characterize therapeutics protocols that cold lead to complete eradication of cp from organism. the research had been principally made on the studying the molecular mechanisms that are at the root of pathogen permanence inside host cell. using proliferation and apoptosis tests we underlined a different behaviour of infected cells towards control cells. in presence of p ( mg/ml), i.e. a peptide that can inhibit the proliferation and induce apoptosis in vitro inhibiting nf-kb, uninfected cells proliferation decreased of % in comparison whit the controls, while the one of infected decreased only of about %. moreover, using various apoptosis-inducers, the infected cells showing apoptosis were about % while the uninfected were about %. the caspace iii activity increased significantly in uninfected cells. in conclusion, cp could delay its elimination from the host inhibiting the apoptosis via nf-kb activation. hryniewiecki t a , gzyl a b , rawczynska-englert i a , a department of acquired valvular heart disease, national institute of cardiology, warsaw, poland , b department of sera and vaccines, national institute of hygiene, warsaw, poland infective endocarditis (ie) frequently causes problems in diagnosis, especially where blood cultures are negative and with fungal etiology (also as a fungal superinfection in bacterial ie). the purpose of the study: the purpose of the study was to evaluate the usefulness of broad-range fungal pcr in diagnosis of fungal superinfection of bacterial ie. twenty-five blood samples were taken for analysis from patients with infective endocarditis. ie was diagnosed according to duke criteria including positive blood cultures. suspicion of fungal superinfection was established on serological investigation in five patients, confirmed by blood culture in two patients. control group consisted of patients without infection. dna was isolated using the commercially available s.n.a.p. kit. amplification products were analyzed by gel electrophoresis stained with ethidium bromide. the results obtained: fungal dna was found in two patients with fungal superinfection of bacterial ie confirmed by culture. in the remaining patients with ie and controls no fungal dna was found. the conclusion reached: broad-range fungal pcr is a fast and inexpensive tool for the detection of fungal dna, but it is more prone to contamination than species-specific pcr. the method may be valuable in the identification of fungal superinfection of bacterial ie or diagnosis of fungal ie. rivanera d, lilli d, lozzi ma, piunno m, mancini c. microbiology, science and public health, rome, italy aim: the aim of this study was to evaluate the eia method for detection of antibody to ttv virus (ttv) and to investigate the anti-tt virus prevalence in patients with hepatitis b (hbv) virus, hepatitis c (hcv) virus, in group of 'high risk'subjects to hepatitis and in healthy subjects. the elisa methods (nuclear laser vienna lab) using ttv s and ns antigens: orf ( aa) and orf ( aa) was applied to detect anti-ttv; the serological screening was performed from samples to italian subjects. results: the positive rates of anti-ttv antibodies were . % in patients with hepatitis b Á/c and . % in 'high risk' hepatitis patients. the anti-ttv was also found in . % in healthy people. conclusions: the anti-ttv were detected in all groups studied, however, its positive rate was similar in patients with hepatitis b Á/c and in 'high risk' hepatitis respect to heathly people. our results shown that tt virus is frequent in italy both in patients infected by others transmitted viruses and in general population. the positivity found in healthy adults included in our studies suggests that the virus might be transmitted non-parenterally. the study of pattern of antibody to ttv may be an infectious marker of ttv similar to that of anti-hcv. a stress test on a miniaturized identification system designed for neisseria and haemophilus pm rich m a , bannatyne rm a , memish za b . a king fahad national guard hospital, division of microbiology, riyadh, saudi arabia , b king fahad national guard hospital, infection prevention and control, riyadh, saudi arabia we report an incident that occurred in our laboratory when the bbl crystal identification system for neisseria and haemophilus was used to identify a haemophilus-like-organism. the numerical profile generated was not in the system database. conventional biochemical tests subsequently revealed an identification of brucella melitensis , a common isolate in our area. as a result of this revelation we subjected this system to a mini 'stress-test' with a collection of isolates of b. melitensis . two numerical profiles were obtained, and , neither of which are listed in the system database. brucella species have been misidentified as moraxella species, moraxella phenylpyruvica , and as haemophilus influenzae biotype iv in various identification systems. two cases of laboratory-acquired brucellosis have been attributed to misidentification. to its credit the bbl crystal identification system for neisseria and haemophilus neither generates a profile number with a misidentified organism nor assigns a confidence level. instead it properly directs the user to resort to conventional methods to secure an identification. if further studies on additional brucella isolates and strains from different geographical sources confirm the unique biochemical profiles identified here, it may be worthwhile to incorporate these into the database where they would be of considerable assistance in areas where brucellosis is widespread. cloning and characterization of aflmp in aspergillus flavus pm chong tk, woo pcy, leung asp, yuen ky. the university of hong kong, microbiology, hong kong, hong kong purpose of the study: to clone and characterize an antigenic protein for serodiagnosis of infection caused by aspergillus flavus which is the commonest aspergillus species causing aspergilloma (ao) and invasive aspergillosis (ia) in asia. result obtained: we cloned the aflmp gene, which encodes the first antigenic cell wall protein in a. flavus . aflmp codes for a protein, aflmp p, of amino acid residues, with sequence features that are present in mp p and afmp p, the antigenic cell wall mannoprotein in penicillium marneffei and aspergillus fumigatus that we described previously. it contains a serine-and threonine-rich region for o glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. specific anti-aflmp p antibody was generated with recombinant aflmp p protein purified from escherichia coli to allow further characterization of aflmp p. indirect immunofluorescent staining indicated that aflmp p is present in the cell walls of the hyphae and conidia of a. flavus . furthermore, it was observed that patients with ao and ia due to a. flavus develop a specific antibody response against aflmp p. conclusion reached: this suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with ao or ia, and the protein may represent a good cell surface target for host humoral immunitiy. grape purpose: to investigate the basis for increasing resistance to trimethoprim and sulphamethoxazole. methods: pcr screening for integrons of clinical urinary tract isolates was performed. isolates were tested for resistance to antibiotics. integrons in isolates were sequenced. results: integrons of class were found in isolates and class integrons were found in . eight isolates in the study were resistant to five antibiotics or more and not shown to carry any integron. nineteen of isolates resistant to trimethoprim did not carry integrons. only one of these isolates was shown to carry sul and is thus probably also carrying an integron. none of the isolates were shown to carry dfr , one of five trimethoprim resistance genes known to exist outside integrons. three isolates were resistant to sulphonamides but were not shown to carry neither sul nor sul . only dfr and aad gene cassettes were found in the sequenced integrons. conclusions: resistance to trimethoprim in of trimethoprim resistant isolates is mediated by genes not detectable, as in the case with three sulphonamide resistant isolates. sequenced integrons that contain dfr genes do not carry any gene cassettes mediating resistance to modern antibiotics. unusual diagnosis tool for an unusual presentation of alveolar echinococcosis: report of two cases of local progression after an animal bite pm bardonnet k, bart jm, loiseau j, gérard a, estavoyer jm, heyd b, badet jm, dubiez a, piarroux r, bresson-hadni s. who collaborating centre for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the classical human contamination route for alveolar echinococcosis (ae) is ingestion of eggs. two exceptional human cases are reported with extensive local evolution of ae after a bite. case no. : between and , a patient underwent surgery seven times for a muscle growing tumour which developed after a bite. the diagnosis of muscle ae was assessed on histopathological examination. in , serological tests were in accordance with echinococcus sp infection. case no. : in , a man presented 'cat-scratch fever' with a right supraclavicular tumefaction following a cat bite. between and , five recurrences occurred. different surgical explorations indicated multiple abscesses of the cervical muscles. in , serological tests were in favour of echinococcus sp infection and the pathologist described a parasitic wall suggesting hydatidosis, but specific pcr from histological samples prompted the diagnosis of ae. conclusion: in these exceptional observations, the liver which is the most usual location of ae was lesion-free. the chronic inflammatory ae lesions have developed in the local lymphatic chain area of the bite site. to perform diagnosis in these very unusual forms of ae, it is necessary to add unusual tests such as specific pcr to classical tests. antibiotic resistance in foodborne salmonella is an emerging public health concern. integrons are now recognized as the main genetic vehicles of antibiotic resistance in gram-negative bacteria, including in salmonella . the purpose of the present study was to investigate the presence of class i integrons in resistant isolates of several serotypes of salmonella isolated from poultry products and to determine their association with multidrug-resistance phenotypes. a total of isolates of salmonella belonging to seven different serotypes were tested. the most frequent multiresistant phenotype, found alone or together with other resistances, was to streptomycin and tetracycline. all but seven were resistant to three or more antimicrobial agents, including quinolones and amoxicillin. pcr analysis with the ?cs and ?cs primers detected the presence of class i integrons of . kb in one isolate, with the multiresistant phenotype: amoxicillin, chloramphenicol, streptomycin, trimethoprim-sulphametoxazol and tetracycline. our findings suggest that the uncontrolled use of the antimicrobial agents in food animals may have contributed to the development of the pattern of resistance observed in salmonella isolates. also the presence of integrons in low prevalent human salmonella serotypes but associated with food animals underscores the public health problem of antibiotic resistance acquisition and spread. prevalence and antimicrobial resistance of campylobacter jejuni and c. coli isolated from broilers and pigs in france pm avrain l a , humbert f b , sanders p c , kempf i a . a afssa, umb, ploufragan, france , b afssa, hqpap, ploufragan, france , c afssa, lermvd, fougères, france in , caeca from standard, export or free-range broilers and in , fecal samples from pigs, were collected in french slaughterhouses. prevalence of campylobacter jejuni and c. coli strains was . % in standard, . % in export and % in free-range broilers. in standard and export productions, the most often isolated species was c. jejuni , whereas c. coli was predominant in free-range production. . % samples collected from pigs contained c. coli . the sensitivity of strains to ampicillin, nalidixic acid, enrofloxacin (broilers) or ciprofloxacin (pigs), tetracycline, erythromycin and gentamicin was tested by an agar dilution method. in broilers, the percentages of resistant strains were, respectively , , , , . and % for c. jejuni and , , , , and % for c. coli . in pigs the percentages of resistant c. coli were, respectively , , , , and %. in broiler production, significant differences between distributions of species or percentages of resistant strains were observed according to type of production or administrated antimicrobials. the enzyme dhps (dihydropteroate synthase) participates in the folate synthesis pathway, and is well recognized as the target for sulphonamides. the enzyme preceding dhps in this pathway, pppk (dihydropterin pyrophosphokinase), is another interesting candidate drug target. the metabolic role of pppk is to provide one of the substrates for dhps. earlier studies have suggested that pppk and dhps enzymes need to have physical contact with each other for full enzyme activity. studies of potential interactions between the enzymes have been initiated. so far, indication of a weak interaction has been detected in gelfiltration experiments and the two-hybrid system. to confirm these results, we are currently developing a method to study substrate channeling, as interfering with such interactions could lead to impaired growth and thus be used as inhibitory drugs. we have also cloned and sequenced the operons coding for the enzymes in the folate biosynthesis from different clinical isolates of streptococcus pyogenes . comparisons revealed some isolates with a mosaic structure in the operon, suggesting that horizontal transfer of genetic material has occurred. multi-resistance gene cluster on a plasmid in a clinical isolate of e. faecium pm werner g, hildebrandt b, klare i, witte w. department of nosocomial infections, robert koch institute, wernigerode branch, germany purpose: strain uw was isolated from an urine sample of a patient with a permanent catheter. the purpose of our study was to identify and localize the resistance determinants in this isolate. results: isolate uw was resistant to the following antibiotics: penicillin, ampicillin, gentamicin (high-level), streptomycin (highlevel), erythromycin, clindamycin, vancomycin, teicoplanin, ciprofloxacin, moxifloxacin, nourseothricin, rifampicin, and fusidic acid (lowlevel, mic / mg/l); but showed susceptibilities to oxytetracycline, phosphomycin, chloramphenicol, trimethoprim/sulfamethoxazol, linezolid, and quinupristin/dalfopristin. hybridization, pcr and sequencing experiments localized a cluster consisting of several resistance genes in a composite element on a plasmid. the cluster included genes and transposons tn (vana) Á/tn (ermb) Á/tn (aade Á/ sat Á/apha- ). the plasmid itself was not transferable in filter-matings into a fusidic acid high-level resistant enterococcus faecium recipient while selecting either for erythromycin or vancomycin resistances. however, after transposing a tn -related determinant into uw , determinants became mobilizable with the help of the conjugative transposon. transconjugants were, besides others, high-level resistant to fusidic acid, but susceptible to penicillin and ampicillin. pfge of transconjugants demonstrated a pattern almost identical to the recipient but clearly different from the donor. conclusion: resistance genes in e. faecium could be arranged in a cluster and are mobile via mobilizable/transferable plasmids. lilli d, rivanera d, barbacini ig, lozzi ma, mancini c. department of science and public health, university la sapienza, microbiology, rome, italy aim: hepatitis g virus (hgv), a new rna virus that is parenterally trasmitted has frequentley been found in patients with chronic hepatitis c infection but its role in chronic liver desease is unknown. the aim of this study was to determine the prevalence of hgv infection in patients infected with hcv. ninety-eight patients infected with hcv were evaluated for the presence of hgv rna. the hcv genotypes distribution was genotype b, genotype a, genotype a and four genotype c/ d. hcv rna and hgv rna were detected by rt-nested pcr. results: infection with hepatitis g virus was detected in ( . %) patients and ( . %) were hgv rna negative. none of our patients with genotypes a and c/ d results hgv rna positive. prevalence of hgv infection was % in patients infected with hcv genotype b and . % with genotype a. conclusions: infection with hgv occurred frequently ( . %) in this sample of patients with chronic hepatitis c. we observed a height prevalence of hcv/hgv coinfection in patients infected with hcv genotype a. this association with hcv genotype a was indipendent of the source of infection, infact some of our patients have not history of intravenous drug use. characterization of extended-spectrum beta-lactamase (esbl)mediated resistance in salmonella spp. from durban, south africa pm moodley p a , essack s b , gajee k a , sturm w a . a department of medical microbiology, nelson r. mandela school of medicine, school of infection, university of natal, durban, south africa , b school of pharmacy and pharmacology, university of durban westville, durban, south africa background: gastroenteritis is a common condition among the paediatric population presenting to king edward viii hospital in durban, south africa. from july , we noticed that the susceptibility of the salmonella spp. isolated from stool samples among these children were resistant to multiple antibiotics. aim: to characterize the phenotype of the resistance mechanisms involved. methods: minimum inhibitory concentrations (mics) of ampicillin, azithromycin, ciprofloxacin, cefepime, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, cefoxitin, chloramphenicol, cotrimoxazole and gentamicin were determined by means of the agar dilution method. isolates were subjected to the e -test for extended-spectrum betalactamase (esbl) production. isoelectric focusing was performed as a preliminary step in enzyme characterization. results and conclusion: thirty isolates of multiresistant salmonella spp. were obtained. antibiogram typing revealed six different resistance phenotypes. all isolates depicted ceftazidime/ceftazidime Á/clavulanate ratio of !/ and were considered putative esbl-producers. isolates expressed Á/ beta-lactamases each with pi values ranging between and . indicative of tem-, shv-and/or ctx-m-related esbls. nine isolates expressed two beta-lactamases each and two isolates expressed three beta-lactamases each. there was evidence of the simultaneous expression of both tem-and shv-derived esbls as well as the simultaneous expression of multiple tem-or shv-derived esbls in single isolates, a phenomenon reported in esbl-positive klebsiella pneumoniae isolated at the same hospital. neutrophils exhibit reduced chemiluminescence response to serum opsonized klebsiella pneumoniae producing extended spectrum b-lactamases (esbl) pm objective: to investigate the ability of esbl and non-esblproducing klebsiella pneumoniae isolates treated with human serum to induce a chemiluminescence response in neutrophils. methods: oxidative burst induced by the interaction of esbl (n / ) and non-esbl-producing (n / ) klebsiella pneumoniae isolates with neutrophils from healthy individuals was monitored by measuring the chemiluminescence response (cl). pooled sera from healthy individuals served as source of complement for pretreatment of the bacteria. cl responses triggered by serum treated zymosan served as positive control. the serum opsonized klebsiella strains were arbitrarily graded as high (h) and low (l) inducers of cl when the cl response induced by the bacteria was cl b/ and !/ %, respectively, of that induced by opsonized zymosan. results: out of non-esbl-producing klebsiella isolates, . % induced high cl response in neutrophils whereas only % of esbl-producing klebsiella isolates did so (pb/ . ). conclusions: strains harboring the esbl plasmid were more virulent than non-esbl-producing strains by virtue of their higher tendency to escape serum-dependent recognition by neutrophils. osiris */an automated system for susceptibility testing in agar diffusion technique pm chegrani f, kolbert m, shah pm. universitätsklinik frankfurt, zentrum der inneren medizin med iii schwerpunkt infektiologie, frankfurt am main, germany objective: osiris measured zone sizes were compared to manually measured inhibition zones using round (rp) and mm mueller hinton square agarplates (sqp). variations of / mm in zone size measurements were defined as tolerable. 'very major errors' (vme) were defined as classification of a resistant organism as sensitive by osiris. thirty thousand two hundred and ninety-eight single measurements testing antibiotics on staphylococci and enterobacteriaceae were done according to the din recommendations. results: vancomycin, rifampicin, gentamicin gave the best results on rp with a concordance of , and %. vancomycin, rifampicin, teicoplanin performed best with , , % on sqp testing staphylococci . worst results on rp gave cefuroxim ( . %) and fosfomycin ( . %), on sqp fosfomycin ( . %), ofloxacin ( . %). for enterobacteriaceae amikacin ( %), gentamicin ( %), ciprofloxacin ( %) performed best on rp; worst nalidixinacid ( . %), piperacillin ( . %). concordance on sqp amikacin ( %), cefotaxim ( %), gentamicin ( %), nitrofurantoin ( . %), cotrimoxazol ( %). very major errors were seen in b/ % of all test performed. interpretation: osiris is a rapid and reliable system for susceptibility testing with round and square agarplates and has an excellent expert system. altindis m a , aktepe oc a , kocagoz t b . a kocatepe university school of medicine, microbiology, afyon, turkey , b diomed inc. tr, istanbul, turkey dio-bacit, in a two section plate, that contains % sheep blood agar on one side and sheep blood agar with bacitracin ( mg/ml) was compared for its efficiency in identification of group a beta hemolytic streptococcus (gabs) with other two different growth plates, one containing % sheep blood agar with bacitracin (b) and the other containing b-sxt. we used latex-agglutination for this comparision. throat specimens obtained from cases were inoculated to dio-bacit plates, first to one side with % sheep blood agar and to the other side with b. after an overnight inoculation at c, colonies with beta hemolysis an % sheep blood agar but no growth with b, were inoculated to % sheep blood agar again and antibiogram identification discs containing . u b and . and . mg sxt (oxoid, uk) were placed onto the plate and incubated overnight at c. after that, colonies with beta hemolysis were defined as b-sensitive while colonies resistant to sxt were defined to be gabs. all colonies are serologically classified by latex-agglutination (oxoid, uk). seventy-one ( . %) inoculations revealed growth of gabs at dio-bacit plates. after inoculating these colonies to % sheep blood agar, of them were found to be sensitive to b, while were found to be sensitive to b but resistant to sxt and of them were defined as gabs by latex test. when compared with latex-agglutination test, we found dio-bacit method's sensitivity and spesificity to be and . %, respectively. method: agar diffusion technique as recommended by din was used to determine izs (read using aura and manually) for staphylococci and enterobacteriaceae . variations in automated measured zone sizes of / mm to the manual readings were considered to be within acceptable range. results: six thousand and fifty-two zone sizes were determined for staphylococci and for enterobacteriaceae . mha displayed tendency to smaller zone sizes in automated readings than isa, as well in staphylococci and enterobacteriaceae . on the other side automated readings presented on isa more precise results than mha. overall less major discrepancies ( b/ mm) were found on isa. izs were generally smaller on mha. the tables below show differences in manually and automated measured zone sizes on different media and species. we discovered seven more cases of resistance in the case of metronidazole. we did not have such experience with clarithromycin. conclusion: our results show that e -test is comparable to ad for clarithromycin, but for metronidazole our findings confirm nccls recommendantion. classical ad is time consuming for every day use in the laboratory. the use of screening agar plate with mg/ml of metronidazole to detect possible resistance could be the solution. rokosz a a , sawicka-grzelak a a , meszaros j b , luczak m a . a department of medical microbiology, the university medical school, warsaw, poland , b department of bacteriology, state institute of hygiene, warsaw, poland purpose: to identify esbl-positive strains and to compare two methods applied for the detection of extended-spectrum beta-lactamases (esbls). methods: two hundred and sixty strains of gram-negative rods were cultured from clinical specimens from hospitalized patients. identification of strains was performed in the automatic atb system (biomerieux, france). these strains were identified as esbl-positive on the basis of the double-disc synergy test (ddst according to jarlier et al., ) results. all strains were also determined using a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: cpd (cefpodoxime) and cd (cefpodoxime/clavulanic acid) (oxoid, england). results: consistent results of two methods (ddst and dd) were obtained in the case of from among of examined strains ( . %). consistent results concerned out of strains of enteric rods ( . %) and only five from among other strains (mostly nonfermenting rods). conclusions: the novel method of esbl-producers detection (dd) is more objective and easier for interpretation than the double-disc synergy test (ddst). diagnostic disc test should be used as the basic one or to confirm the results of ddst in difficult cases. assessment of e -test for determining penicillin resistance in pneumococci pm sener b, yeniþehirli g, ercis s, hasçelik g. department of clinical microbiology, hacettepe university medical faculty, ankara, turkey there is a greater need for susceptibility testing methods that distinguish between susceptible and resistant pneumococci. an alternative method could be the e -test, which is compared with the reference agar dilution method in this study. penicillin susceptibility of a total of pneumococci was determined by e -test and agar dilution methods. streptococcus pneumoniae atcc and enterococcus faecalis atcc were used as controls. the results were given in the effect of anoxic conditions on the minimum inhibitory concentration of metronidazole in helicobacter pylori pm de la obra sanz p, lomas e, roman jl, alarcon t, lopez-brea m. the objective of this study was to determine the effect of incubation under anoxic conditions on the metronidazole resistance of helicobacter pylori . methods: a total of clinical isolates were used in this study. mics were determined by an agar dilution method using mueller-hinton agar plus % lysed horse blood. three plates series contained twofold dilutions of metronidazole from to . mg/l were prepared. the first one was incubated under microaerophilic conditions (oxoid) for days; second and third series were incubated anaerobically (anaerobic system, oxoid) for and h, respectively, and were then transferred to the microaerophilic enviroment up to complete days of incubation. results: with microaerophilic incubation, of strains were resistant (mic and mic were and , respectively). with h anaerobic preincubation, four of strains were resistant (mic and mic were . and , respectively). with h anaerobic preincubation, of strains was resistant (mic and mic were . and , respectively). conclusions: anaerobic preincubations causes an increase in sensitivity to metronidazole, the extent of which was dependent on the length of the anaerobic period. methods: the susceptibility to antibiotics was performed by microdilution method according to nccls guidelines. the production of b-lactamase was tested by nitrocefin sticks (oxoid). results: the mics /mics (mg/ml) appeared, respectively: ampicillin / , amoxicillin/clavulanic . / . , cefaclor / , ceftriazone . / . , erythromycin . / . , azithromycin / . / . , clarithromycin . / . , ciprofloxacin . / . , imipenem / . / . , tetracycline / . / / . , trimethoprim/sulfamethoxazole . / . . b-lactamase was detected in . % of the strains. conclusions: ( ) m. catarrhalis isolates were uniformly susceptible to all tested antimicrobials except ampicillin. ( ) the production of blactamase was responsible for ampicillin resistance ( . %). ( ) m. catarrhalis strains had almost the same behavior 'in vitro' to the tested microlides (erythromycin, azithromycin, clarythromycin). rokosz a, sawicka-grzelak a, luczak m. department of medical microbiology, the university medical school, warsaw, poland purpose: to isolate, identify and determine the drug-susceptibility of fungal strains cultured from fecal samples routinely submitted for detection of clostridium difficile and its toxins in cases of antibioticassociated diarrhea (aad). methods: one hundred fecal samples from hospitalized patients were examined (may Á/october ). c. difficile toxins a/b were detected directly in stools with c. difficile tox a/b ii test (techlab † , usa). fecal specimens were inoculated on ccca and candida id (biomerieux, france) media. c. difficile and fungi were identified with standard microbiological procedures. susceptibility of fungal strains to anti-fungal agents was determined (atb fungus, biomerieux, france). results: c. difficile toxins were detected in and c. difficile strains were isolated from of examined specimens. sixty-two fungal strains of genera were cultured from stool samples ( c. albicans isolates). massive fungal growths were observed on primary plates in all cases. fifty-five fungal strains were susceptible to nystatin, -to fluorocytosine, -to amphotericin b, -to ketoconazole, -to miconazole and -to econazole. conclusions: in some cases of antibiotic-associated diarrhea fungal strains are responsible for symptoms of this disease. certain persons having aad should be treated with anti-fungal agents. results: a total of isolates ( . %) were penicillin nonsusceptible (intermediate and resistant) and ( . %) were erythromycin non-susceptible. non-susceptibility to both antibiotics was found in ( . %). conclusions: if penicillin administration eliminates all penicillin susceptible strains, the prevalence of penicillin non-susceptible strains will increase as well as the erythromycin non-susceptible ones. this means that the proportion of erythromycin non-susceptible strains should increase from . to . %. at the same time, if erythromycin eliminate all susceptible strains to this antibiotic, the prevalence of penicillin non-susceptible strains would increase from the initial . to . %. these data can explain the co-selection results observed in different surveillance studies. antimicrobial susceptibility and capsular types/groups of streptococcus pneumoniae isolates causing pneumococcal diseases in bulgaria pm setchanova l a , gergova r a , ioneva m b , sredkova v c . a department of microbiology, medical university, sofia, bulgaria , b department of microbiology, ii city hospital-sofia, sofia, bulgaria , c department of microbiology, faculty of medicine, pleven, bulgaria a prospective study of pneumococcal infections was performed in cooperation with five clinical microbiology laboratories in bulgaria. mics values to antimicrobials and serotype/serogroup distribution were determined for strains of streptococcus pneumoniae . pneumococci were isolated from patients with systemic or respiratory infections. the incidence of penicillin g-intermediate and penicillin gresistant isolate was . and . %, respectively. the rates of resistance to other antimicrobials were: cefotaxime/ceftriaxone */ . %; erythromycin */ . %; clindamycin */ . %; tetracycline */ %; chloramphenicol */ . %; trimethoprim/sulfamothoxazole */ %; ciprofloxacin */ . %; rifampin */ . %. the s. pneumoniae isolates belonged to capsular types/groups. the most common serotypes/serogroups in bulgaria are , , , , , , we aimed to determine the pneumococcal antibiotic resistance rates and the serotypes of those resistant isolates in our hospital. the mic values of isolates (year Á/ ) were determined by agar dilution method. serotyping was performed by using pooled antisera of the pneumo-test. the results were as follows (n / ). objectives: to asses the antimicrobial susceptibility of clinical isolates of pseudomonas aeruginosa obtained from to and to monitor trends in antimicrobial resistance. methods: mics were determined by microdilution testing according to nccls. the antibiotics tested were: ceftazidime (caz), aztreonam (atm), imipenem (imp), gentamicin (cn), tobramycin (tb), amikacin (ak) and ciprofloxacin (cip). results: a total of isolates were included. urine was the most common site of isolation for outpatients isolates ( . %) while for hospitalized patients respiratory samples were the most frequent ( . %). susceptibility to antibiotics was: % caz, . % atm, % imp, . % cn, . % tb, . % ak and . % cip. comparison of susceptibility data through Á/ showed that the increase in the resistance rate was significative for caz ( . vs . %), atm ( . vs %), cn ( vs %), tb ( . vs . %), ak ( . vs . %) and cip ( . vs %). significant differences were found under the following circumstances: isolates from intensive care units and from inpatients were significatively more resistant to caz, atm and imp. isolates from respiratory samples were more resistant to caz and atm and isolates from urine samples were more resistant to cip. conclusions: although the antimicrobial susceptibility level has been decreasing p. aeruginosa isolates still show good susceptibility percentages for all antibiotics tested. antimicrobial susceptibility testing of clinical isolates of bordetella pertussis : report on isolates from rouen, france pm lemee l a , nouvellon m a , caron f b , lemeland jf a . a chu rouen, bacteriologie, rouen, france , b chu rouen, maladies infectieuses et tropicales, rouen, france reports of an increased clinical incidence of pertussis and the development of resistance by bordetella pertussis to erythromycin prompted the collection and antimicrobial susceptibility testing of recent clinical isolates from patients, who were hospitalized in rouen between and . mics of nine antimicrobial agents (erythromycin, josamycin, spiramycin, roxithromycin, ketolide hmr , cotrimoxazole, ciprofloxacin, rifampicin and amoxicillin) were measured by agar dilution method on mueller-hinton agar containing % horse blood. mbcs of erythromycin and rifampicin were also determined against four isolates of b. pertussis . all isolates were fully susceptible to the nine antimicrobial agents tested. mics (mcg/ml) were . for erythromycin, ketolide hmr and ciprofloxacin, . for josamycin, . for spiramycin, roxithromycin and rifampicin, . / . for cotrimoxazole, and for amoxicillin. mbcs (mcg/ml) were . Á/ . for eyrthromycin and . Á/ for rifampicin. in conclusion, our isolates of b. pertussis remain extremely susceptible to all antimicrobial agents tested, especially macrolides. no resistance was detected. finally, if erythromycin remains the molecule of choice, other macrolides (c and c ) also confirm their good in-vitro activity. in addition, the good in-vitro potency of rifampicin, together with its great diffusion within the respiratory tract, suggests that rifampicin has potential clinical efficacy in pertussis too. the emergence of streptococcus pneumoniae (sp) with diminished susceptibility to penicillin g (psdp) suggests the use of other antibiotics such as newer fluoroquinolones (fq). the resistance phenotypes of consecutive pneumococcal strains isolated from patients of four hospitals (observatoire régional des pneumocoques du nord-pas de calais) were studied: strains were susceptible to penicillin g and were psdp. reference strains provided from the centre national de réfeacute;rence des pneumocoques were added to the study. the activity of pefloxacin, ciprofloxacin, norfloxacin, sparfloxacin, levofloxacin and moxifloxacin was studied. reserpine was used to detect the efflux phenotype. methods used were performed according to the recommendations of the comité de l'antibiogramme de la société française de microbiologie. for each strain, the resistance phenotype to fq was deduced by comparison of mics or diameters obtained with those obtained with the reference strains of known phenotypes. fq resistance phenotypes were not correlated to blactam agent susceptibilities. wild type phenotype was observed among . and . % of the susceptible and psdp strains, respectively. a 'wild efflux' mechanism, deduced by addition of reserpine to norfloxacin, represented the predominant phenotype. it was detected among sp susceptible to penicillin g ( . %) as well as among psdp ( . %). the aim of this study was to determine macrolide resistance phenotypes of sp isolated in three french departments (alpes maritimes, doubs, nord) from nasopharyngeal aspirates of children aged months to years attending a dcc. a random sample of children attending randomly selected dccs was obtained during three periods (spring, autumn and winter ) in each department ( children attending dccs and children sampled). analysis of macrolide susceptibility of sp strains was performed using the ca-sfm method. out of strains, . % had decreased susceptibility to penicillin (spdp) and . % were resistant to erythromycin. the triple disk diffusion method (erythromycin (e), clindamycin (cl) and spiramycin) was used to determine resistance phenotypes. macrolide resistance is a well known phenomenon in france and is confirmed by our study. these results show that the constitutive phenotype is predominant as in other parts of europe and the frequency of efflux mechanism is lower than that observed in the usa and canada. developing antibiotic resistance surveillance of helicobacter pylori in england and wales pm elviss nc, owen rj. central public health laboratory, laboratory of enteric pathogens, london, uk purpose: helicobacter pylori antibiotic resistance is a key contributing factor in Â/ % of infected patients failing drug treatment. our aim was to survey rates of primary in-vitro resistance at different locations, and links to disease severity. antral gastric biopsies/cultures were received from phls in chelmsford, mid-essex ( isolates Á/ ); london ( isolates Á/ ); and bangor, north wales ( isolates Á/ ). susceptibilities to metronidazole (mtz), clarithromycin (cla), tetracycline (tet) and amoxicillin (amx) were tested by disc diffusion and also by e -test for cla and mtz. results: overall resistance rates ( isolates) were % for mtz and % for cla. all were susceptible to amx and tet. dual resistance rate was %. breakdown by location showed some marked differences. mtz resistance was highest in london ( %) compared to % in chelmsford and % in bangor. by contrast cla rates were % for london, and about % for bangor and chelmsford. in london, the majority of mtz resistant isolates were from non-uk borne individuals ( % non-uk vs % uk). comparison of duodenal ulcer-associated isolates with those from non-ulcer patients indicated similar rates of mtz resistance ( %). conclusion: resistance rates may vary significantly between locations depending on the local population with non-uk birth being a key risk factor for primary resistance with a mtz resistant strain. local resistance rates should be taken into account in test and treat strategies. potrykus j, benetkiewicz m, wegrzyn g. department of molecular biology, university of gdansk, gdansk, poland purpose of the study : because of their ability to extrude a wide range of compounds, multidrug efflux pumps have recently become an important issue in combating bacterial infections. acrab-tolc is the major efflux system of escherichia coli . we investigated the effect of acra on plasmid-borne and intrinsic chloramphenicol, tetracycline and ampicillin resistance. results and conclusions: recently, we reported a chloramphenicol sensitivity of e. coli mutant expressing cat , the chloramphenicol resistance gene. the strain was shown to bear a nonsense mutation in the acra gene. our studies indicate that this mutation is, at least in part, responsible for the observed chloramphenicol sensitivity phenotype. the mutation seems also to influence the strain's susceptibility to ampicillin and teta (c )-mediated (plasmid-borne) tetracycline resistance. although the teta(c) protein retained its biological function, there was a considerable growth impairment of the mutant strain when cultured in tetracycline containing medium. deletion of the acrab locus prevented any growth in the presence of tetracycline. upon the addition of ampicillin, the mutant underwent lysis more rapidly than the control strain. such was also observed in acrab deletion derivatives of other e. coli strains. we are trying to elucidate the role of the acra gene product in the phenomena described above. existence of efflux pumps in wild type isolates of drug-resistance bacteria pm raja ray rr. medical microbiology and parasitology, calcutta university, kolkata, india efflux pumps possessed by the bacterial cells of different kinds of bacteria had presented as a newer mode of drug resistance in many organisms. the capacity of bacterial cells to cause outward flow of noxious agents was known, however, for a considerable time with respect to tetracycline. recently, interest in the efflux pump system has brought to light some previously ill-understood mechanisms of drug resistance, involving noxious agents, toxins or poisons. we have found high level of resistance in pseudomonads towards cetrimide and other germicides for which no definite chromosomal/plasmid-mediated genes/mechanisms could be identified. likewise, occurrence of nonantibiotic sensitive vibrios, staphylococci and pseudomonads in the background of their high level of resistance to most of the common antibiotics suggest a mechanism of interference with the efflux pump, which accounts for such sensitivity in such cases. involvement of multiple resistance of marine isolates of v. parahaemolyticus to numerous clinically used antibiotics to which they have never been exposed also suggests a possible role of efflux pumps in determining such resistance */that these can simultaneously develop against multiple marine toxins/poisons and other noxious agents. interaction between oxacillin and glycopeptides in a teicoplanin-resistant mutant of staphylococcus epidermidis with reduced susceptibility to vancomycin pm greco aa, ben hassen a. laboratory service of national bone-marrow transplant center, tunis, tunisia we selected a laboratory-generated mutant of staphylococcus epidermidis capable of growing in the presence of mg/l of teicoplanin ( b tm ), from a methicillin-resistant (mic!/ mg/l), teicoplanin-sensitive (mic mg/l) and vancomycin-sensitive (mic mg/l) clinical isolate of s. epidermidis ( b so). in a previous work, we studied the different phenotypic characteristics acquired by the teicoplanin-resistant mutant b tm ( th interdisciplinary meeting on anti-infectious chemotherapy, december , poster sessions, /p ). in this work, we examined the interaction between oxacillin and glycopeptides against this teicoplanin-resistant mutant of s. epidermidis with reduced susceptibility to vancomycin. to study the combined antibiotic activity of oxacillin and glycopeptides, we used different methods: a modified disk diffusion test, the e -test, time-kill assays and population analysis profiles. the synergistic activity of glycopeptides in combination with oxacillin against the teicoplaninresistant mutant b tm was demonstrated with a bactericidal effect. no synergy was seen against the parental strain b so. moreover, the synergy between glycopeptides and oxacillin occurred with suppression of the subpopulation with the highest level of glycopeptides resistance. we concluded that combination of glycopeptides and oxacillin may be a possible alternative in the treatment of infections caused by methicillin-resistant, teicoplanin-resistant s. epidermidis . compositional changes in microcosm biofilms induced by application of minocycline: a preliminary study pm the aim of the study was to observe the effect of application of minocycline upon microcosm dental plaques. the plaques were cultivated in a constant departmenth film fermentor (cdff), which produces biofilms under conditions mimicking those present in vivo. the composition of the biofilms was determined by viable counting on selective and non-selective media. the proportion of antibiotic resistant genera within the biofilm was determined by viable counts utilising media containing minocycline ( mg/ml). before commencing antibiotic pulsing, the biofilms had a total viable anaerobic count of . )/ cfu per biofilm, with negligible ( cfu/biofilm) minocycline-resistant bacteria. however, h after introduction of the antibiotic, the total count had been reduced to . )/ cfu/biofilm whilst the number of minocycline-resistant bacteria had risen to . )/ cfu/biofilm. at the final sampling time point ( h) the total viable anaerobic count was . )/ cfu/biofilm whilst the number of minocycline-resistant bacteria was . )/ cfu/biofilm. hence, there is a very low basal level of inherent resistance to minocycline within microcosm dental plaques, but this increases considerably once the biofilms are exposed to minocycline. mechanism of resistance to aminoglycosides (amg) e. coli isolated from children with community-acquired urinary tract infections (cau-tis) pm methods: during the Á/ years nine centers took part in the study. the mics of antimicrobials were determined by the agar dilution method as described in the nccls guidelines. results: a total of consecutive urine isolates from children aged month to years with cauti were collected. the most frequently isolated species from children with cauti was e. coli ( . %), followed by klebsiella spp. ( . %) and proteus spp. ( . %). results of the in vitro susceptibility testing of e. coli to amg are shown in table below. resistance of the strains was conditioned on production of amg-modifying enzymes. there has been found following phenotypes among resistance strains: gentamicin Á/ tobramycin Á/netilmicin ( . % */aac( )-v and . % */aac( )-iv enzymes) and gentamicin Á/tobramycin ( . %, due to ant( ƒ) enzyme). conclusion: amikacin is most active amg against e. coli . resistance to gentamicin and netilmicin was mainly determined by production of aac( )-v enzyme. effect of b-lactamase inhibitors (b-l-i) on the evolution of resistance (r) to b-lactams (b-l) in gram the b-lactams antimicrobials still are the most frequently used. among the bacteria responsible of high resistance to b-lactams are gramnegative rods; its most frequent mechanism is the production of b-lactamase. the use of b-l-i has reversed partially this mechanism of resistance. we expect changes in the frequencies of r using b-lƒci after more than years. since , the venezuelan group of bacterial resistance, with health institution in the country; analyse and publish data on bacterial resistance of isolates from patients with bacterial infection coming from hospitals. it was used diffusion disk, according nccls. the software program whonet (world health organization net) was used. we follow the trends of r of gram-negative rods to b-l alone and with b-l-i during the decade Á/ . statistical analyses were made by evaluating the differences among percentages of resistance between the two series (p / . ). results and discussion: the difference in r between b-l and b-l/b-l-i are: ( ) piperacillin, piperacillin/tazobactam: between and % of r for most isolated, except for escherichia coli ( %) and serratia spp. ( %); ( ) ampicilin, ampicilin/sulbactam: between and %; ( ) cefoperazone, cefoperazone/sulbactam: between and %. how is expected gramnegative rods resistance to b-lactams with a b-l-i is lower than the b-lactam alone; furthermore the difference between both series, grows higher with time. these results are relevant and they were not expected, since b-l-i have been shown to be b-lactamase inductors. trends in the resistance (r) to b-lactams and others antimicrobials in p. aeruginosa in venezuelan medical centres. nosocomial (nos) and communitarian resistance pm in order to approach the infection produced by resistant bacteria, it is convenient to consider the hospital and the community as two separate ecosystems. the hospital ecosystem has special relevance in the infection and r of gramnegative aerobic bacilli. today, they are the main responsible of nos infection, with special reference to pseudomonas aeruginosa . infection by resistant bacteria is a world wide problem, specially related to nos. since , the venezuelan group of bacterial resistance, with health institution in the country; identify, analyse and publish data on bacterial r to antimicrobials: b-lactams, quinolones and aminoglicosides of isolates from patients with bacterial infection coming from hospitals and the community. it was used diffusion disk, according nccls. the software program whonet (world health organiza-tion net) was used. statistical significance (p / . ) was determined by application of the x technique. we show significant differences in the r of p. aeruginosa nos and communitarian (highest differences: piperacilina / %, tobra / %). we also established significant differences between the r arising in public hospitals and private hospitals (highest differences: ceftazidime / %, amika / %). we show the tendency in decreasing of frequency of r since ; this is more evident in private hospitals (b-lactam and aminoglicosides). new antimicrobials and new mechanism of action, and in the future the new technology will solve today's problem. however, the most important tools we have today are prevention and antimicrobials, and we must make them suitable. susceptibility to antibiotics of enterobacter cloacae and citrobacter freundii from drinking water pm quintera sm, sousa jc, peixe l. department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal the increased use of antimicrobials in farming, together with the practice of raw sewage discharge into receiving waters, has resulted in a significant increase in the number of antibiotic resistant bacteria present in aquatic environment. our objective was to determine the antimicrobial susceptibility, with focus on b-lactam resistance, among enterobacteriaceae strains isolated from raw drinking water samples. several isolates (n / ) of enterobacter cloacae and citrobacter freundii obtained from drinking waters were screened for antibiotic susceptibility patterns, using the agar diffusion technique, according to nccls's procedures. only % of e. cloacae strains, as well as % of c. freundii strains show resistance to amoxicillin and amoxicillin/ clavulanic acid. a reduced incidence of resistance to several others antibiotics was also observed. the obtained results suggest that strains isolated from raw drinking water have greater susceptibility to antimicrobial agents than pathogenic strains from hospital or outpatients infections. the 'natural' antimicrobial resistance phenotypes, usually described for c. freundii and e. cloacae , only seem to apply to strains isolated from human infections. notwithstanding the high susceptibility of the tested isolates to b-lactams, the role of environmental bacteria as a reservoir of resistance genes justify its periodical monitoring as a valid index for resistance spreading. a snapshot of the soil. using bacterial communities for tracing the evolution of metal-resistance pm quintera sm a , sousa jc a , peixe l a , monteiro nm b . a department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal , b department of zoology and anthropology, faculty of sciences, university of oporto, oporto, portugal it is well known that pathogenic bacteria, specially those resistant to antimicrobial agents and heavy metals poses public health risks of great concern, and its detection, namely in soils is generally related to pollution. in this study, the heavy metal resistance patterns of the microflora isolated from polluted (dump area) and unpolluted soil environments were examined. the plate growth covering percentage in the soil samples was determined using mueller-hinton plates supplemented with different heavy metal (al '/, cd '/, cu '/, pb '/, hg '/ and zn '/) concentrations. parallelly, using icp-aes, it was possible to ascertain the real heavy metal concentration for each soil sample. we found that the percentage of plate growth covering from the used samples was closely linked to the level of chemical pollution measured for each location. moreover, using anova, we found significant differences between locations. the dump site showed the highest tolerance to all the tested metals (newman Á/keuls test). this pattern of results was consistent when using the data from the icp-aes. furthermore, it was possible to observe that pseudomonas spp., with a relatively high mic for the studied metals, might become a relevant model for both public health issues and eco-toxicological studies. biochemical characteristics of environmental isolates of listeria monocytogenes pm moshtaghi h a , garg sr b , mandokhot uv b . a shahrekord university, food hygiene, shahrekord, islamic republic of iran , b haryana agricultural university, food hygiene, hisar, india purpose: the investigations were carried out to study the biochemical reactions of listeria monocytogenes isolated from different sources in the environment. results: a total of isolates of l. monocytogenes were obtained from samples of agricultural soil, faecal matter of animals and sewage. all the isolates were gram-positive, small rods, catalase positive, oxidase negative, motile with tumbling motility in hanging drop at Á/ c, aerobic, facultative anaerobic, fermentative and produced acid from glucose. all the isolates of l. monocytogenes were beta haemolytic and positive for camp reaction with staphylococcus aureus . all the isolates were negative for phenyl alanine deaminase, ornithine decarboxylase, lysine decarboxylase, malonate utilization and beta galactosidase tests. these were also negative for acid production from arabinose, d-xylose, mannitol, soluble starch and sucrose but acid was produced in rhamnose, salicin, and trehalose. hydrogen sulfide production was recorded in tripticase soy broth with lead acetate paper strips but negative with triple sugar iron agar. all the isolates were found to hydrolyse aesculin. out of isolates of l. monocytogenes only two produced acid from lactose. in serotyping all the isolates were serotype b. conclusion: we can conclude that l. monocytogenes serotype b at least in fermentation of lactose shows different reactions. methods: the samples were pre-enriched in bhi broth with and without vancomycin ( mg/l) and then plated onto m-enterococcus agar with and without antibiotics: vancomycin ( mg/l), gentamicin ( mg/l), kanamycin ( mg/l), and streptomycin ( mg/l). representative colonies of each morphology were isolated and identified as enterococcus sp as previous described. pcr was used to identify e. faecium and e. faecalis and to characterise vancomycin resistant genotype. api strep was also used in the identification. susceptibility testing to antibiotics was performed by an agar dilution method (nccls). results: three hundred and fifty-three enterococci were isolated from of a total of faecal samples ( %, n / / ). the majority of enterococci were identified as e. faecium , e. faecalis and enterococcus sp. resistance to almost all antibiotics studied was observed: vancomycin */ . %; teicoplanin */ . ; ampicillin */ . %; tetracyclin */ . ; erythromycin */ . %; ciprofloxacin */ . %; chloramphenicol */ . %; gentamicin */ . %; streptomycin */ . %; kanamycin */ . %; linezolid */ %. the vancomycin resistant enterococci presented a vana genotype. conclusion: resistance to several common antibiotics used in therapy was observed among enterococci isolated from healthy human from community. many of these isolates presented multi-resistance. of concern is the presence of vana genotype among these populations that may constitute a reservoir of vancomycin resistant genes. antimicrobial resistance in tetracycline-resistant oral bacteria pm mercury release from dental amalgam may select for mercuryresistant oral bacteria. mercury resistance is often associated with multiple antibiotic resistances. the aims of this study were to determine whether tetracycline-resistant oral bacteria from children with and without amalgam fillings were also resistant to: (a) mercury; and (b) multiple antibiotics. tetracycline-resistant organisms were isolated on iso-sensitest/blood agar containing tetracycline ( mg/ml). the mic of hgcl and several antibiotics were determined using agar dilution (bsac). one hundred and three organisms were isolated from patients without amalgam. ninety-one were streptococcus species, seven neisseria species, three veillonella dispar and two rothia species. fifty-seven percent exhibited resistance to at least one antibiotic, % were mercury-resistant, % were penicillin-resistant, % were ampicillin-resistant and % erythromycin-resistant. fiftytwo organisms were isolated from patients with amalgam. forty-five were streptococcus species, five neisseria species, one v. dispar and one staphylococcus aureus . sixty-three percent exhibited resistance to at least one antibiotic, % were mercury-resistant, % penicillinresistant, % were ampicillin-resistant and % showed erythromycin-resistance. statistically, the results showed that in tetracyclineresistant organisms, the presence of dental amalgam did not affect the level of resistance to mercury or to the antibiotics tested. conway-wallace hl a , mullany p a , bedi r b , wilson m a . a eastman dental institute, university college london, microbiology, london, uk , b eastman dental institute, university college london, transcultural oral health, london, uk the purpose of this study: to determine the prevalence of antibioticresistant oral bacteria in children who had not received antibiotics during the months prior to sampling. plaque samples were obtained from children aged Á/ years and plated onto media containing: penicillin, ampicillin, tetracycline, erythromycin and vancomycin. resistant isolates were enumerated, sub-cultured and frozen for subsequent identification. the process was repeated and months later. the results obtained: bacteria resistant to each of the antibiotics were present in all of the children at each sampling time (except in the case of ampicillin and penicillin at months). the proportion of antibiotic-resistant bacteria in the oral microflora ranged from ]/ . (erythromycin) to / . % (ampicillin). the proportions of bacteria resistant to a particular antibiotic remained reasonably constant over the -month sampling period. in only two cases (penicillin and ampicillin) was there a statistically significant change in the proportions of resistant bacteria at different time periods. the conclusion reached: the results of the study have revealed that bacteria resistant to a wide range of antibiotics may be isolated from children who have not been administered these agents during the months prior to sampling. furthermore, in many cases the proportion of bacteria resistant to a particular antibiotic remains constant over a -month period. antimicrobial use in the intensive care unit: results of a pharmacoepidemiological study in italy pm periti p. e.i.f.t. srl, firenze, italy a retrospective survey of antimicrobial chemotherapy use in intensive care units in italy was carried out in using a computerized questionnaire under the auspices of the journal of chemotherapy. of the icus contacted, . % replied, being mainly general or post-surgical and pediatric units having a mean of beds, nine doctors and nurses. the antimicrobial agents used in these wards were almost always polychemotherapy with prevalent use of beta-lactams, aminoglycosides and glycopeptides or as empirical treatment during the first h after hospital admission. the continual use of medium-high dose combinational antimicrobial chemotherapy was justified by microbiological testing, which revealed that more than one-third of bacterial pathogens were resistant. approximately, % of gram-positive bacteria were methicillin-resistant, whereas about % of gram-negative strains were resistant to at least one of the tested antibiotics. forty percent of the responding icus furnished microbiological testing data, of which three quarters indicated the incidence of chemoresistance of the isolated strains. fungal infections were less frequent than bacterial, the most commonly isolated agent being candida spp. in conclusion, the sample of icus examined showed adequate and reasonable use of antimicrobial agents, with heavy reliance on medium-high dose combination therapy due to the elevated incidence of resistant isolates found. plasma concentrations (p), urinary excretion (u) and bactericidal activity of gatifloxacin (gat) mg versus ciprofloxacin (cip) mg in healthy volunteers after a single oral dose pm boy d a , kinzig-schippers m b , sö rgel f b , well naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, ibmp, nürnberg-heroldsberg, germany twelve volunteers received a single oral dose of mg gat versus mg cip to assess p up to h, u (by hplc), and urinary bactericidal titers (ubt) in eight intervals up to h. the mean pmax of gat/cip was . / . mg. the ucum (mean) for gat/cip was . / . %. the ubts, i.e. the highest twofold dilution of urine still bactericidal, were determined for nine uropathogens and one reference strain */mics (mg/ml) (microdilution) for gat/cip: escherichia coli atcc ( . / . ); e. coli ( . / . ); klebsiella pneumoniae ( . / . ); proteus mirabilis ( . / . ); pseudomonas aeruginosa ( / . ); s. saprophyticus ( . / . ); two strains of s. aureus ( . / . ); two strains of e. faecalis ( . / and / ). the median ubts measured within the first h for gatifloxacin were between : and : for the five gram-negative strains (incl. p. aeruginosa ) and between : and : for the five gram-positive strains. the median ubts for ciprofloxacin were between : and : for the gramnegative strains (incl. p. aeruginosa ) and between : . and : for the five gram-positive strains. for the ubts up to h, gat was significantly superior to ciprofloxacin in all gram-positive strains, not different in the two e. coli strains, and inferior in the klebsiella , proteus and pseudomonas strains. for the ubts at Á/ h, gat was generally superior to cip, but showed no difference in the proteus and pseudomonas strains. gat showed overall comparable urinary bactericidal activity as cip. this is in agreement with a clinical study performed previously. malaria is one of the most prevalent endemic infectious disease affecting humans. in bichat hospital cases of malaria acute illness were reported during . among them, patients were hospitalised and intravenously treated by quinine. this retrospective study consisted of comparing the therapeutic drug monitoring (tdm) of quinine distinguishing, respectively and patients cured in infectious medical department (imd) and intensive care unit (icu) where a standardised quinine regimen was established ( and % malaria attacks, respectively). in icu, the treatment consisted of an infused loading dose mg/kg/ h of quinine diluted in % glucose followed by mg/kg/day. plasma quinine maximal concentrations were assessed after selective liquid Á/liquid extraction and spectrofluorometry detection. statistical analysis was performed using t -test. results showed that patients had comparable weight ( . / . and . / . kg) but quinine doses and plasma concentrations were significantly different in icu and imd, respectively ( . / . versus . / . mg/kg/day, pb/ . and . / . versus . / . mg/l, p b/ . ). in icu and imd, respectively: and % were in the therapeutic range ( Á/ mg/l) with and % below the requested therapeutic concentration ( mg/l) and and % above the limit of toxicity ( mg/l) conveying the importance of tdm in intravenous quinine treatment to avoid infra-therapeutic or toxic concentrations. simultaneous central nervous system distribution using microdialysis and pharmacokinetic Á/pharmacodynamic modelling of the electroencephalogram effect of norfloxacin in rats pm chenel m, marchand s, dupuis a, bouquet s, couet w. university of pharmacy, pharmacology, poitiers, france purpose: to investigate the epileptogenic activity of norfloxacin by a pharmacokinetic Á/pharmacodynamic (pk Á/pd) modelling approach and to assess the contribution of distributional processes across the blood Á/brain barrier (bbb) to the delayed effect. methods: rats (n / ) received an iv bolus dose of norfloxacin ( mg/kg). convulsant effect was quantified by electroencephalogram (eeg) recording during h post-dose. arterial blood samples were collected for drug assays in plasma. unbound norfloxacin concentrations were monitored in brain extracellular fluid (ecf) using microdialysis with in vivo calibration of the probes by retrodialysis with ciprofloxacin. results: the eeg effect reached its maximum between and min post-dose. a pk/pd effect compartment model was successfully fitted to these data. the relationship between effect and concentration at the effect site was best described by a spline function. norfloxacin concentrations in brain ecf were relatively low compared to plasma levels (ecf/plasma areas under curve (auc) ratio equal to . / . %), but central distribution was rapid. therefore, the effect versus brain ecf concentrations curves still exhibited a marked hysterisis. conclusion: the delay observed between plasma concentrations and norfloxacin convulsant effect cannot be explained by a slow distribution of norfloxacin across the bbb. pagoulatou a a , kanellakopoulou k b , vafiadou m a , kostakopoulos th c , kastriotis i b , giamarellou h c . a department of anesthesia, sismanoglio general hospital, greece , b th department of internal medicine, athens medical school, athens, greece , c department of urology, athens medical school, athens, greece csf kinetics of van and fu were studied in patients who underwent short urological surgery under spinal anesthesia. patients were excluded if they were already receiving an antibiotic or were suffering from renal and hepatic dysfunction. van was administered at g over h infusion. serum and csf samples were collected post-dose and the mean serum levels were as follows: min Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (six patients) and Á/ h: . mg/ml (seven patients). fu was administered at mg dose over h infusion. serum and csf samples were taken post-dose and the mean serum concentrations were found as follows: Á/ min: . mg/ml (six patients), min Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (five patients). in csf, both van and fu were undetectable. it is concluded that in the absence of meningeal inflammation van and fu do not penetrate (with the applied microbiological assay) the csf barrier. comparison of the pharmacology of intravenous and orally given moxifloxacin in an in-vitro model pm wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: the intravenous form (iv) of mg moxifloxacin (mox), one of the newer fluoroquinolones, has been recently approved by the fda. during the iv treatment higher peak serum concentrations are achieved in comparison to the oral administration (po) of the same dose. the antibacterial activity of fluoroquinolones is concentration dependent. we therefore simulated human pharmacokinetics of single po and iv dosages of mg mox in an in-vitro model using six different gram-negative and -positive pathogens to elucidate the different effect of these two dosing schedules. results: the comparison of the pharmacological parameter auc/ mic shows an increase ( table ) that could predict an enhanced antibacterial effect. however, the analysis of the killing curves with the following parameters, ka.max (maximal killing activity) and aac (area above the killing curve between and h), reveals no major difference between the po and iv dosage. conclusion: the serum concentration after oral administration is already sufficiently high to show the optimal bactericidal effect of mox that can only be slightly increased by higher peak concentrations and higher auc/mic ratios. thus the concentration dependence is not linear but ends already at concentrations achievable by oral dosing and documents that auc/mic calculations cannot easily be translated into dosing schedules. background : bacteria growing in vivo multiply much more slowly than in vitro. whether the bactericidal activity of quinolones may be affected by an increase in generation time (g) was studied in batch cultures. methods: by limiting the nutrient supply, generation times were lengthened from approximately . to . h up to . h. alternatively, the quinolones were added to the bacterial cultures during the lag-, exponential-and stationary phase. recent clinical isolates of escherichia coli were exposed to multiples of the mics of ciprofloxacin or norfloxacin. the 'killing rates' were calculated in analogy to the growth rate. results: the bactericidal activity of the quinolones tested against e. coli was minimally influenced by the reduced generation time. ciprofloxacin concentrations of ]/ )/mic eliminated the test strains within / h from the test system if added during the lag or exponential growth phase; four times higher concentrations were needed to reduce cfus by % within h, if added during the stationary phase. norfloxacin was significantly less active. conclusion: in contrast to norfloxacin, the bactericidal activity of ciprofloxacin is minimally affected by the generation time or growth phase of the bacteria. wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: moxifloxacin (mox) is one of the newer fluoroquinolones, now available for parenteral application. the pharmacology of an intravenous once-daily dose (od) of mg mox was determined with five gram-negative and -positive pathogens (streptococcus pyogenes , streptococcus pneumoniae , moraxella catarrhalis , escherichia coli , and klebsiella pneumoniae ). a twice-daily dose (bid) of mg mox was simulated with the gram-positive species in order to increase the bactericidal effect. results: to determine the efficacy, killing curves were analyzed, and following parameters were calculated: ka.max: maximal killing activity [log cfu]; ka. conclusion: an intravenous once-daily dose of mox is active against all tested pathogens. the gram-negative species are rapidly killed (ka. h similar to ka.max). there is no pronounced initial effect on the two gram-positive species but a general slow reduction in the viable cell count (ka.max is reached after h). the efficacy of mox (measured as aac and ka.max) on s. pyogenes and s. pneumoniae is to some extent increased after the second dose. however, the analysis of the killing curves reveals no major difference between od and bid. even the od nearly gives the maximal bacterical activity of mox against gram-positive pathogens. objectives: to evaluate the dose proportionality of amoxicillin and to compare the respective pk/pd parameters of two dosage regimens. methods: the dose proportionality of amoxicillin was evaluated using linear regression of mean auc -inf and c max data of different bioequivalence studies (n / volunteers) performed with formulations containing various amounts of amoxicillin alone or in the combination with clavulanic acid. the volunteers received a single oral dose in the range of Á/ mg. amoxicillin plasma concentrations were determined by hplc/uv or lc/ms/ms methods. time above mic (tmic) expressed in% of dosing interval was calculated with three target mic values ( . , . and . mg/l) for mg hourly and g -hourly dosage regimens. results: the absorption of amoxicillin (auc -inf ) showed a linear dependence with a correlation coefficient of . . the correlation coefficient of the linear regression for the cmax dependence on the actual dose was . . the respective tmic for both dosage regimens were very similar, with largely overlapping confidence intervals, supporting a pd breakpoint of mg/l for the g -hourly regimen (tmic ]/ mg/l: . %, % ci . , . %). conclusion: this analysis shows the dose proportionality of amoxicillin over the dosage range of Á/ mg and supports the pharmacodynamic rationale for a g bid dosage regimen. piperacillin/tazobactam concentration profile after high dose administration pattern in nosocomial pneumonias due to mecanical ventilation pm pedeboscq s a , gruson d b , bassoua v a , hilbert g b , pometan jp a . a st. andré hospital, pharmacy, bordeaux, france , b pellegrin hospital, reanimation, bordeaux, france the piperacillin (p)/tazobactam (t) antibacterial spectrum covers the largest part of bacteria responsible for pneumonias due to mechanical ventilation. but, due to important bacterial inoculum and pharmacokinetic parameter modifications in intensive care patients, high doses of beta-lactamines seem to be necessary to obtain antibiotic concentrations above suspected bacteria's mic (minimal inhibitory concentration) . this led us to compare, in patients with pneumonia due to mechanical ventilation, two intermittent administration patterns: g three times a day (usual pattern) versus g four times a day (high dose pattern). this study is carried out in collaboration with intensive care unit, bacteriological department and pharmacy where antibiotic concentrations are determined. twenty-three takings of blood are executed within a h period, in addition to two bronchial secretion samples. concerning p seric concentrations, the high dose pattern seems to be more adapted because of relatively high residual concentrations ( !/ mg/ml). three hours after each injection, t seric concentrations are lower than the mg/ml activity threshold. first and second day residual bronchial concentrations of p seem to be sufficient although t concentrations are below activity threshold. these results are to be correlated with the mic determined by the bacteriological department, and only this correlation will make us able to conclude the better efficacy of the high dose pattern in intensive care patients. anti-inflammatory drugs interference in absorption and tissue penetration of amoxycillin pm del fiol fs a , menon sz b , caramez th b , celotto tf b , lopes ras b . a university of sorocaba, pharmacology, sorocaba, brazil , b school of pharmacy, university of sorocaba, sorocaba, brazil antibiotics and anti-inflammatories are frequently associated in clinical practice. there is some concern about the quantity of antibiotic that reaches the infection sites, which may be reduced in the presence of an anti-inflammatory drug. the purpose of the present study was to analyse how steroids (dexamethasone (dexa)) and aines (celecoxib (cele)) influence on the penetration of amoxicillin to inflamed tissues. thirty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs to form granulomatous tissue. one week later the animals were divided into three groups. one group received only amox ( mg/kg), another received amox ( mg/kg) plus cele ( . mg/kg) and the last received amox ( mg/kg) plus dexa ( . mg/kg). one hour later the animals were sacrificed and the concentration of amoxicillin in the serum and tissue investigated. there was no difference among the groups in the quantity absorbed (amox / . / . mg/ml; amox'/cele / . / . mg/ml and amox'/dexa / . / . mg/ml). there was a reduction in the tissue concentration of amoxicillin (p b/ . tukey-kramer) for the group that received the drug with dexamethasone. for the other groups, there was no difference in the tissue concentration of amoxicillin. the results indicated that in inflamed tissue, a significant reduction of antibiotic penetration was induced by sinultaneous dexamethasone therapy. prediction of the optimal amoxicillin dose regimen based on coupling of pharmacokinetic data and bactericidal activity pm background: given its short half-life, amoxicillin (amx) should be administered at least three times a day to patients with acute exacerbations of chronic bronchitis, in order to achieve serum concentrations well above the mic of the responsible pathogen. however, several authors have recommended twice-daily administration of a higher dose for a shorter period. we assessed the relationship between amx sputum concentrations and antibacterial activity following two treatment schedules in healthy volunteers. subjects and methods: twelve healthy volunteers were randomized to receive amx for days at a dose of either g bd or mg bd. serum and sputum were collected every day, h after the morning administration, and again days after the last dose. amx concentrations were determined by hplc with fluorometric detection. sputum killing activity was determined against haemophilus influenzae , streptococcus pneumoniae and moraxella catarrhalis . results: mean serum concentrations measured h after the morning administration were . ( mg bd) and . mg/l ( g bd), and were above the mics of the three microrganisms. in contrast, sputum concentrations were always below . mg/l. in terms of sputum killing activity, g bd was more effective than mg bd against s. pneumoniae and m. catarrhalis , whereas no sputum samples were active on h. influenzae . conclusion: the optimal amoxicillin treatment schedule cannot be established on the basis of serum pharmacokinetics only. galmiche h, louchahi k, tod m, drugeon h, giroud jp, rouveix b. service de pharmacologie clinique, hopital cochin, paris, crepit , hopital avicenne, bobigny, service de microbiologie, hopital laennec, nantes, france background: cysteine-based mucolytics are commonly used in combination with antibiotics to treat patients with acute exacerbations of chronic bronchitis (aecb). they are also used to allow in vitro mic determination in sputum specimens. we conducted an in vitro and ex-vivo compatibility study designed to detect a possible interaction between mucolytics and antibiotics. methods: serial samples of bronchial secretions were collected from aecb patients and from healthy volunteers who received g of amoxicillin twice a day for days. two mucolytics were used to fluidify sputum specimens: , -dihydroxy- , -dithiolbutan (digest-eur † ) and acetylcysteine ( % solution). amoxicillin was assayed using a chromatographic system with fluorometric detection. each sample was also tested in a microbiological assay. results: amoxicillin could not be detected in the presence of the mucolytic agents. conclusions: this mucolytic Á/amoxicillin interaction may be explained by amoxicillin fixation to fluidified mucoproteins, and should be taken into account when assessing antibiotic efficacy in vivo. del fiol fs a , ferro c b , albuquerques et b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil physicians frequently recommend that macrolides should be administered with milk to decrease the discomfort they cause. thus the objective of this study was to verify the interference of milk in the absorption and distribution of erythromycin (eryt); clarithromycin (clar); roxithromycin (roxi) and azithromycin (azit). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs eryt, clar, roxi and azit with and without milk ( . ml/kg [ca'/'/] / . mg/ml). the animals were sacrificed and the serum and tissue concentration of the drugs was investigated. there was no reduction (pb/ . tukey-kramer) in the serum and tissue concentration in the presence of milk for azit and clar. there was a % reduction for roxi in the serum concentration in the presence of milk ( . / . and . / . mg/ml), but no alteration in the tissue concentration. there was a % reduction for eryt (p b/ . ), in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and a % reduction in the tissue concentration. the milk decreased the effectiveness of treatments with erythromycin and roxithromycin and the bioavailabilities of this macrolides were affected by co-administration with milk. del fiol fs a , souza gp b , duzzi mr b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil the degree to which tetracyclines are absorbed differs greatly. this absorption is impaired by the concurrent ingestion of divalent and trivalent cations. thus the objective of this study was to investigate the interference of milk in the absorption and distribution of tetracycline (tetr), oxytetracycline (oxyt), minocycline (mino) and doxycycline (doxy). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their back for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs: tetr; oxyt; mino; and doxy with and without milk ( . ml/kg [ca'/'/] / . mg/ml). the animals were sacrificed and the drug concentrations in the serum and tissue were determined. there was no reduction (pb/ . tukey-kramer) in the serum and tissue concentrations in the presence of milk for mino. there was a % reduction (p b/ . ) for doxy in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. for oxyt, there was a reduction of % (pb/ . ) in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. the tetr results show a . % reduction (p b/ . ) in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. milk decreased tetracycline bioavailability and effectiveness. isotopic studies with oxine labelled platelet. platelet kinetics in thrombocytopenic malaria patients pm introduction : thrombocytopenia is a common feature in human malaria ( ). excessive splenic platelet pooling has been suggested to play a role in uncomplicated cases of malaria, but a moderately shortened platelet life span during the period with decreasing parasitaemia seems the most plausible cause of the frequently observed thrombocytopenia ( , ). consumption coagulopathy, eventually manifested as disseminated intravascular coagulation, has been described in malaria ( ). in uncomplicated malaria, however disseminated intravascular coagulation is rarely found ( ). results: in malaria patients the sequestration was not different to normal. platelet half-life was reduced in patients with p. falciparum malaria to Á/ h (normal Â/ days). in one patient with p. vivax malaria platelet half live was . h. conclusion: no significant differences in the sequestration of platelets when compared to healthy individuals could be detected by in-labelled platelet scintigraphy. especially, no enhanced splenic sequestration, as previously expected, was the cause of the thrombocytopenia. therefore, other mechanisms than sequestration are responsible for the dramatically reduced life span of the platelets during acute malaria. zaharanka ag, rozhdestvensky da. vitebsk state medical university, vitebsk, belarus aim: the studying of chromatingeterogenic test (ct) results in sperm of subjects taking doxycycline (d) and some macrolides (erythromycine (e), jozamycine (j), and azytromycine (a)) in moderate therapeutic doses. methods: forty healthy volunteers ( Á/ years) were studied. daily dose of d was . ; e was administered in dose . four times per day days; j */ . before meals twice daily days; a */ . before meals once daily days. ct for evaluation of dna condition in human spermatozoids was performed before treatment (twice), on the th and th days of treatment, as well as after and months after treatment course completing. results: ct data analysis revealed that the mean amount of defective spermato-zoids before treatment was . '/ . %. by the th day of d treatment the index of de-natured dna was . '/ . % (pb/ . ), by the th day */ . '/ . % (pb/ . ). one and months after the d treatment course the amount of generative cells with denatured dna was . '/ . and . '/ . %, respectively (p b/ . in both case). under e treatment the amount of defective spermatozoids changed as . '/ . ( th day), . '/ . ( th day), . '/ . (after month), and . '/ . % (after months) (pb/ . in any case). under a using the ct results at the same control points were . '/ . , . '/ . , . '/ . , . '/ . % (pb/ . in any case); and under j treatment */ . '/ . , . '/ . , . '/ . , . '/ . %, respectively (p !/ . in any case). conclusions: the data obtained permit to conclude that d demonstrates the high level of toxicity to male generative cells. this effect preserves during months after the course of d treatment. objective: to study the effect of aggressive isolation and decontamination measures to control an outbreak of multi-resistant acinetobacter baumanii (mr-ab) in an icu. the outbreak: the index case was transferred from a mediterranean hospital, directly into an open-plan -bedded icu, with severe injuries to his head and thorax. he died shortly after admission. sputum, bronchoalveolar lavage fluid, blood cultures and a chest drain swab grew mr-ab, resistant to ampicillin, co-amoxiclav, aztreonam, amikacin, ceftazidime, cefotaxime, cefuroxime, ciprofloxacin, gentamicin, meropenem, piptazobactam, tobramycin and sensitive only to colistin. within days, mr-ab was isolated from two further icu patients. all isolates demonstrated identical antimicrobial susceptibility profiles. the icu was closed to admissions and thoroughly cleaned. all patients were isolated and their contacts screened. the icu was reopened, however, mr-ab was isolated from a fourth patient. this patient was isolated, the icu closed, for a second time, thoroughly cleaned, and all contacts isolated until discharge. all subsequent patients screened were negative for mr-ab conclusion: this illustrates the importance of aggressive isolation measures and thorough supervised cleaning in control of an outbreak, and the need to screen patients for resistant bacteria before admission to the intensive care unit in a general hospital. extended spectrum beta-lactamase-positive bacteria isolated in neonatal intensive care unit pm sandorcinova z a , siegfried l a , kmetova m a , viragova s b . a institute of medical microbiology, faculty of medicine, university of p.j. safarik, kosice, slovakia , b hospital, kosice-saca, neonatal intensive care unit, kosice, slovakia extended-spectrum beta-lactamases hydrolyse all penicilins, cephalosporins, including third-generation cephalosporins and aztreonam. esbl are predominantly produced by klebsiella spp. but may be presented in other enterobacteriaceae, too. the aim of present study was to investigate the occurrence of esbl-producing bacteria isolated from patients hospitalized at the neonatal intensive care units (icu). fifty escherichia coli and klebsiella spp. were isolated from rectum of patients hospitalized at the neonatal icu. the mics of antimicrobial agents were determined by the standard agar plate dilution method according to the nccls guidelines. for screening of esbl production we investigated strains showing reduced susceptibility (mic equal and/or more than ml/l) to at least one of third generation cephalosporins. esbl production was detected by double disk synergy test (ddst), e -test for esbl, and pcr employing specific primers for the presence of blashv and blatem genes. by using ddst and e -test, among e. coli isolates, an expression of esbl was detected in the three by the former method, while among klebsiella spp. isolates, a production of esbl was found in the two by the latter method. in esbl-positive e. coli strains the presence of blatem genes fragments was detected while in esbl-positive klebsiella spp. genes encoding for shv-type beta-lactamases were found. isolation of staphylococci from wound swabs and their susceptibility to antibiotics pm markov mij, shopovski e, despotovski v, angelevski a, nikolovski b. military hospital, microbiology, skopje, the former yugoslav republic, macedonia purpose: to determine percent of staphylococci from wound swabs and to establish their susceptibility to antibiotics. material and method: the wound swabs have been evaluated with standards microbiological techniques. bacteria have been identified with strips from the 'atb expression' system. the susceptibility testing has been performed with strips with dilution technique, read by the same system. results: during the last years ( Á/ ), a total of wound swabs have been evaluated in the military hospital in skopje. positive bacterial finding have been determined in ( %) swabs with isolated bacterial species, from which ( . ) were staphylococci: staphylococcus aureus ( . %) isolations ( methycillin resistant s. aureus ); s. epidermidis ( . %); s. haemolyticus ( . %); s. hominis ( . %); s. chromogenes and s. lugdunensis nine ( . %); s. intermedius , s. lentus , s. sciuri and s. warneri all with seven ( . %) isolations. the susceptibility of s. aureus was to: penicillin %, ampicillin %, amoksicillin/clavulonic acid %, ceftazidime %, gentamicin %, tetracycline's %, erythromycin %, lincomycin %, ciprofloksacin %, cotrimoxazole %, vancomycin %, fusidic acid %, cefixime %, and azitromicin %. conclusion: in our study most frequently isolated bacteria from the wound swabs were staphylococci, especially s. aureus . susceptibility, except for the penicillin ( %), was high to other antibiotics. the study went on from january to december . at maribor teaching hospital, staphylococcus aureus isolates were collected. in , ( %) and in , ( . %) were mrsa. mrsa were recovered from routine clinical material and from surveillance swabs (nose, throat, skin). for isolation, conventional culture media were used and for surveillance swabs mrsa-screening plate (manitol salt agar with % oxacillin) and trypticase soy broth with . % nacl were added. s. aureus was identified by catalase, dna-se and tube-coagulase test. antibiotic susceptibility was determined by the disk-diffusion method according to ncci guidelines. all mrsa isolates were tested for sensitivity to the following antimicrobials: gentamicin, netilmicin, ciprofloxacin, erythromycin, chloramphenicol, tetracycline, cotrimoxazol, vancomycin and clindamycin. it was found that all mrsa isolates were sensitive to vancomycin and partially or totally resistant to the rest. there were no important differences between the years and . our mrsa isolates were completely ( %) susceptible to vancomycin, but resistant to the other antimicrobials in use to some extent. although the monitoring of mrsa susceptibility to antimicrobials once a year did not show any important change in antimicrobial resistance, the periodical monitoring of mrsa susceptibility to antimicrobials and revaluation of current treatment regimens of mrsa infections is necessary. staphylococcus aureus strains with reduced susceptibility to vancomycin among clinical isolates in university hospital in warsaw pm the visa and especially h-visa are very difficult to be found in the routine laboratory. in our investigations we examined of s. aureus strains isolated and stored in our laboratory for several last years ( Á/ ) . most strains were isolated in , and some in . for all staphylococcal strains mrsa as well as mssa the mics of vancomycin were performed by the standard dilution method. among strains isolated in the last year three strains were recognized as visa (mic values were mg/l). the frequency of visa was . %. in the aim of founding the h-visa strains the population analysis was used. for this analysis all strains growing on the concentration mg/l of vancomycin from the inoculum were chosen. it was strains, but only of them were recognized as h-visa. the frequency of h-visa among investigated strains was about %. most but not all of the h-visa and all visa strains were methicillin resistant. in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci isolated from patients of surgical intensive care unit pm kucukates e, karayel n, kansiz e. institute of cardiology, university of istanbul, istanbul, turkey objectives: oxacillin-resistant staphylococci have emerged as a major infection control problem in our hospital. the aim of this study was to evaluate the in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci. material and methods: this study was performed between january and december , at university of istanbul, institute of cardiology. the antimicrobial susceptibilities of staphylococci isolates for vancomycin, teicoplanin and oxacillin have been investigated by e -test according to nccls guidelines. results: fifty-five ( . %) of clinical isolates were staphylococcus aureus . one hundred and twenty-four ( . %) of clinical isolates were coagulase negative staphylococci (cns). none of staphylococci isolates were resistant to vancomycin. but three of cns isolates were intermediate and six of cns isolates were resistant to teicoplanin. twenty-eight ( . %) of s. aureus were resistant to oxacillin. ninety ( . %) of cns isolates were also resistant to methicillin. conclusions: nosocomial staphylococcal infections, especially in intensive care units increase day by day. staphylococcal infections are a major problem in many hospitals. according to our experiences the rate of oxacillin resistant staphylococci isolates in our hospital has also increased. methicillin-resistant staphylococcus aureus (mrsa) is a clinically significant pathogen because mrsa is resistant to many kinds of antibiotics and causes nosocomial infections around the world. the antiseptics are used for prevention of infections by mrsa. antisepticresistant mrsa strains have been isolated from clinical specimens. antiseptic resistance genes confer resistance to many kinds of drugs structurally and the resistance mechanism is the energy-dependent drug efflux system. in addition, the fluoroquinolone (fq)-resistance gene, nora , confers also resistance to many kinds of antiseptics. we studied the relation of the susceptibility to antiseptics and fqs of mrsa strains isolated in japan. a total of strains of mrsa were isolated from hospitals in japan from to . acriflavin (af), acrinol, benzethonium chloride, benzalkonium chloride and chlorhexidine digluconate were used as the antiseptics. norfloxacin and sparfloxacin were also used in this experiment. the mic was determined by agar double-dilution method as recommended by the nccls. about % of mrsa showed resistance to af (mic: !/ ug/ml). no strain was resistant to a specific antiseptic. fq-susceptible strains were susceptible to all antiseptics. this study showed that antiseptic-resistant mrsa are widely spread at hospitals in japan. the drug of choice in treatment of serious infections caused by mrsa was still vancomycin, however sometimes failures were observed, especially in monotherapy. some conflicting are present in literature about an effect of combined action of vancomycin and betalactams. in the present work, the common effect of vancomycin and methicillin against chosen staphylococcus aureus strains was examined. the investigated strains were characterized as visa, h-visa and clones obtained from h-visa in population analysis. two methods were performed: e -tests with methicillin and vancomycin placed on the media supplemented with the second antibiotic and the chessboard micro-analysis with increasing concentrations of both antibiotics. the fic indexes were calculated for different combinations of concentrations. on the basis of the fic indexes it was shown that the simultaneous action of vancomycin and methicillin was synergistic in all examined strains visa and h-visa, but only in appropriate concentrations. in different combinations the observed effect was addition or indifference. antagonism was never observed. the synergistic effect was not observed in the case of standard s. aureus strain sensitive to methicillin. supplementation of media with % of nacl substantially decreased the observed effect. incidence of antibiotic resistance in staphylococcus aureus strains in hungary with special reference to mrsa pm ghidán Á , maró di c, csukás z, kamotsay k, szabó d, ostorházi e, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary between january and december , a total of staphylococcus aureus strains isolated from patients admitted to the clinics of the semmelweis university were examined for antibiotic sensitivity with the disc diffusion test. resistance to individual antimicrobials were as follows: penicillin %, oxacillin %, erythromycin %, ciprofloxacin %, amikacin %, netilmicin %, tobramycin %, gentamicin %, clindamycin %, mupirocin %, tetracyclines %, chloramphenicol %, teicoplanin % and vancomycin %. all mrsa were b-lactamase producer. they showed coresistance to erythromycin ( %), ciproflxacin ( %), amikacin ( %), netilmicin ( %) and mupirocin ( %). multiple resistant mrsa strains to mupirocin'/tetracyclines'/chloramphenicol amounted to . %. triple resistance to oxacillin'/ciprofloxacin'/netilmicin was %. the detection of meca gene by pcr in randomly chosen mrsa qualified with mg oxacillin disc resulted in only % meca positivity indicating that the traditional disc diffusion test overestimates the frequency of mrsa strains particularly in such an environment where the usage of penicillins and cephalosporins is so liberal as in hungary. consequently, the selective pressure for blactam-resistance and b-lactamase induction exists everywhere. this conclusion is coherent with the relatively low frequency of multiple resistant mrsa strains and urge the need of a routinely available genetic method to apply for mrsa detection. objectives: the main objectives of this study were to monitor antibiotic resistance, identify new/emerging resistance mechanisms at an early stage, prevent their dissemination, early detection and prevent the outbreaks. methods: our laboratory used antibiotic disc sensitivity testing methodology (nccls ) . zone sizes were measured objectively using a biomic automated radius zone reader. results: throughout years (january till december ) we have surveyed organisms collected from outpatient departments ( , . %), radio-oncology department ( , . %), medical department ( , . %), obg department ( , . %), surgical-oncology non icu department ( , . %), icu department ( , . %). from ( %) strains of enterobacteriaceae ( . %) were resistant to th generation fluoroquinolone and ( . %) strains were esbl positive. from ( %) strains of staphylococcus aureus were only five ( . %) strains resistant to methicilin (mrsa). we collected ( %) strains of enterococci, whereabout only two ( . %) were resistant to glycopeptides (vre). from ( %) strains of pesudomonas aeruginosa , ( . %) were resistant to aminoglycosides. conclusions: national restrictive antibiotic policy hand in hand with local hospital antibiotic policy and regular rotation of antibiotics used in prevention and treatment on all departments is leading in our case in positive situation in antibiotic resistance in comparing with other slovakian and european centers. antimicrobial resistance of nosocomial strains of staphylococcus aureus pm dekhnich av, stratchounski ls, edelstain ia, narezkina ad. institute of antimicrobial chemotherapy, smolensk, russian federation purpose: to determine the antimicrobial resistance of staphylococcus aureus causing nosocomial infections in smolensk regional hospital. results: a total of s. aureus strains isolated during Á/ were studied. antimicrobials tested included oxacillin (oxa), erythromycin (ery), clindamycin (cli), gentamicin (gen), vancomycin (van), linezolid (lnz), tetracycline (tet), chloramphenicol (chl), rifampicin (rif), fusidic acid (fus), trimethoprim/sulfamethoxazole (ts), ciprofloxacin (cip), mupirocin (mup), quinupristin/dalfopristin (qd). susceptibility testing and its interpretation were performed by agar dilution according to nccls guidelines where applicable. results are presented in the conclusions: the most active antimicrobials were vancomycin, linezolid, quinupristin/dalfopristin, fusidic acid, mupirocin, followed by co-trimoxazole, rifampicin. beta-lactams, macrolides, lincosamydes, tetracyclines and chloramphenicol should not be used for the treatment of nosocomial s. aureus infections. we investigated all staphylococcal infections within years among neonates hospitalized for infection in the neonatal icu in a tertiary neonatal referral center. univariate analysis, to assess risk factors for neonates infected with staphylococcus aureus ( ) vs. without s. aureus ( ) was performed. from the total number of cases, in cases s. aureus was isolated from various samples; in cases from blood cultures, in cases from urine, in cases from eye swabs and in cases from gastric content (no significant differences in comparison with control group). colonization with s. aureus , was a predictor of infection: nasal swabs, throat swabs, ear swabs, skin swabs and umbilical swabs were significantly more commonly observed in neonates infected with s. aureus , than with other infections. etiological analysis showed that co-pathogens escherichia coli and viridans streptococci were significantly more frequently associated with neonatal infection caused by s. aureus , in comparison to other organisms. according to localization of infection site, conjunctivitis and thrush stomatitis was the commonest s. aureus neonatal infections. outcome was similar to other infections and without any significant differences between both groups. mortality was similar to other infections, probably because: initial therapy in our centre contains an antistaphylococcaly active agent (cefuroxime or cefotaxime plus aminoglycosides). morozova ot, semina na. laboratory of hospital infections, central research institute of epidemiology, moscow, russian federation purpose is to study the role of enterococcus spp. in the aetiology of nosocomial infections among the patients of the childrens clinical hospital and susceptibility of these strains to antibiotics. methods: the strains of enterococci were isolated from patients with hospital infections in Á/ . results: the aetiological structure of enterococcus -infections showed the predominance of skin and soft tissue infections ( . %), urinary tract infections ( . %), bloodstream infection ( . %), pneumoniae ( . %), infection of central nervous system, gastrointestinal tract, eye, surgical wound infections were of rare incidence ( Á/ . %). various nosological forms of infections were caused more often by e. faecalis than e. faecium ( . , . %). the antibiotic resistance to ampicillin and other beta-lactams occured in % of e. faecium isolates, but all strains of e. faecalis were susceptible to these drugs. high-level gentamicin resistance demonstrated e. faecalis isolates */ . %, e. faecium */ %; and high-level streptomycin resistance showed e. faecalis */ . %, e. faecium */ . %. all the e. faecalis were active against fluoroquinolones, but e. faecium were resistant in . %. there were no vancomycin resistant enterococci. conclusion: e. faecalis predominated in the aetiological structure of nosocomial infections due to enterococcus spp. antibiotic resistance patterns for two species of enterococci were different, all the strans were susceptible to vancomycin. evaluation of antimicrobial resistance of enterococcus spp. experience of years ( Á/ ) pm lopez-barba j, jesus de la calle i, solino-ocañ a i, rodríguez-iglesias m, perez-ramos s. microbiology laboratory, puerto real university hospital, cádiz, spain objective: determination of quantitative changes in antimicrobial resistance of enterococcus isolated from clinically significant not urinary samples of patients remitted to the laboratory of microbiology during a years period ( Á/ ) . material: the period of the study was comprised between and . the samples has been processed for the isolation of enterococcus following conventional methods. were isolated enterococci strains. the identification and susceptibility to antibiotics have been performed in automated system microscan(c) dade-behring(c) through panels combo cgp. the data were processed by the statistical system statgraphics plus . . results: of the enterococcus , have been identified e. faecalis and e. faecium . the resistance is shown in table . conclusions: the resistance to va and tei of e. faecalis remains through last years ( Á/ %). the high resistance to erythromycin and tetracilin ( !/ %) and the resistance ( Á/ %) to quinolones, antibiotics all of them used in community-acquired infections justify the susceptibility testing to the clinical strains isolated of this group of microorganism. in e. faecium the antimicrobial resistances was high and increasingly to imipenem, meropenem, erythromycin and quinolones. characteristics of strains e. faecium colonizing the neutropenic patients pm abbassi ms, achour w, gréco a, ben hassen a. laboratory of bone marrow transplant center, tunis, tunisia digestive colonization by enterococcus faecium in the neutropenic patients under gut decontamination is important. seventeen multiresistant strains of e. faecium isolated from stools of seven neutropenic patients were the target of an epidemiological analysis through the determination of the mics of amoxicillin, gentamicin, vancomycin, the transferability gentamicin resistance to the recipient strain e. faecalis jh - by filter-mating assay, analysis of plasmid profiles of e. faecium -strain and of transconjugants and the amplification by pcr of the gene aac( ?)-aph( ??) coding for the bifunctional enzyme by using primer m who gives a fragment of kb. among the seventeen strains, eleven had the same antibiotype a , had a gentamicin mic!/ mg/l. the mic of the amoxicillin was of mg/l. all the strains were sensitive to vancomycin. ten strains harbored a plasmid of kb transfered at a frequency of . Á/ , also found in gentamicin-resistant transconjugants. however, strains belong to nine distinguished plasmids profiles. all high-level gentamicin resistant-strains had a positive pcr amplification of the aac ( ?)-aph ( ƒ) gene. the features of the studied strains establish their endogen origin, specific for every patient, sharing only high-level resistance to gentamicin. gut decontamination treatment with gentamicin enhance either the spread and the preservation of easy-transferable plasmid carrying genetic transposable element. frequency and antibiotic resistance of bacteria isolated from patients suffering infectious complications following the implantation of prosthetic devices pm kristó f k, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary for patients with indwelling joint prosthesis, early recognition and prompt therapy for infection in any location may be critical to reduce the risk of seeding the joint implant heamatogenously. a year period ( ) a total of swabs of aspiration from patients with infectious complications following the implantation of prosthetic devices were cultured. cultivation and identification of the strains were performed by conventional methods and by vitek system (biomȇrieux) and susceptibility testing by disc diffusion. potentional pathogens were recovered in cases ( . %). gram positive cocci, in particular staphylococcus spp. proved to be the most commonly isolated bacteria. coagulase-negative staphylococcus (cns) was isolated more frequently ( %), followed by s. aureus ( %), enterococcus faecalis and e. faecium ( %), gram-negatives ( %), anaerob isolates ( . %). resistance to individual antimicrobials of s. aureus and cns were as follows: methicillin and %, clindamycin . and . %, fluoroquinolones and %. mupirocin resistant strains of s. aureus were not found, while . % were among the cns strains. our results could be essential for the rational selection of treatment at our orthopedic wards. results: in the analysed period the percentage of isolated enterococcus sp. strains among all non-repetitive clinical isolates in , and was . , . and %, respectively. cultured strains were identified as e. faecalis, e. faecium, e. gallinarum and e. avium . the most prevalent was e. faecium strains, isolated with a frequency of , and %, respectively. vancomycin-resistant strains were all identified as e. faecium and in they comprised % of all isolates of this species. conclusions: ( ) the frequency of isolation of enterococcus sp. in blood cultures of haematological patients remained relatively stable in Á/ . ( ) the predominant enterococcal species isolated from these patients was e. faecium . ( ) in we recorded for the first time an emergence of vancomycin-resistant e. faecium , which comprised % of all isolates of this species. kalai s, ben hassen a, achour w, greco a. laboratory of microbiology, national bone marrow transplantation center, tunis, tunisia from april to june , non-repeated strains of pseudomonas aeruginosa were isolated from immunocompromised patients. thirty-six percent of strains were isolated from abscess, % from blood culture and % from urine. susceptibility to antibiotic was studied by the routine disk diffusion method (ca-sfm). mics were determined using agar dilution to antibiotics ( b-lactams, four aminosides and two fluoroquinolones). serotyping of the different trains was performed using antisera to the international antigenic typing systems serotypes. the study showed % of resistance to c cochin port royal hospital, service de gynecologie-obstetrique site st vincent-de-paul, paris, france , d cochin port royal hospital, cclin, paris, france aims: to determinate risk markers of an outbreak of postpartum endometritis due to group a streptococcus . design: a case-control study using data collected with a structured form. setting: the cases of postpartum endometritis were diagnosed in the department of obstetric of the paris hospital network during days (december Á/january ). the group of controls consisted of women delivered in the same department during the same period. participants: cases (n / ) and controls (n / ). findings: cases had smoked more often during pregnancy ( vs. %; p / . ) and received more often immunosuppressive treatment than controls ( vs. %; p / . ). instrumental delivery has been needed more often for cases than controls ( vs. %; p / . ). cases had been hospitalized after delivery in a ward z of the department more often than controls ( vs. %; p / . ). they had been examined after delivery more often by a midwife x ( vs. %; p / . ) and a nurse y has provided care to cases and not to controls ( vs. %; p / . ). conclusion: smoking, receiving immunosuppressive treatment during pregnancy, and instrumental delivery were significantly associated with postpartum endometritis (pb/ %). a midwife and a nurse might be involved in the transmission of the infection. petoukhova i a , sokolova e a , dmitrieva n a , nummaev b b . a laboratory microbiology, cancer research center of russia, moscow, russian federation , b department of oncogynecology, cancer research centre, moscow, russian federation the aim of the study was to assess efficacy of perioperative ap. total pts were included. two hundred and one pts with cervical cancer (cc) undergone extensive hysterectomy, pts with cancer of vulva (cv)-extensive vulvoectomy, pts with ovarian cancer (oc) Á/ extensive/combined operations. fifty-one pts (group ) received ap with amoxicillin/clavulanate (am/cl) . g iv min prior to operation, then . g iv thrice per day for Á/ days. fifty pts (group ) received cefotaxime (ctx) g iv min prior to operation, then g four times per day for Á/ days'/metronidazole (mtz) mg two times per day for the same period. one hundred and forty pts were retrospective control (they received ii Á/iii generation cephalosporins or linkosamides only after operation). the rate of swi in pts with cc, cv and oc was . , , %, respectively; dwi */ . , and %, respectively; uti */ , , . %, respectively. the ap with am/cl was more effective compared to ctx'/mtz (swi */ vs %, respectively, pb/ . , dwi */ . vs %, respectively, p b/ . ). the rate of postoperative uti was equivalent in two groups ( vs %, p /n.s.). thus, ap with am/cl is preferrable option in extensive operations in og pts. fifty-eight patients were randomized into two groups. group , a treatment consisting of patients and group , consisting of patients as a control. the patients were at the age of / . and had had different surgical interventions with general anaesthesia from to h and accompanying copd ( %). artificial pulmonary ventilation was used in cases. in the early post-surgery period the patients of group were administered inhalation therapy, including ipratopium of bromide in the combination with fenoterol (atrovent, berodual) and ambrocsol (lazolvan) through a nebulizer. the inhalation therapy was not administered to the patients of group . under the influence of the inhalation therapy pulmonary ventilation and respiratory metabolism was restored faster in all the resuscitation patients (in group */by the end of the first h, in group */on the rd Á/ th day). the percent rate of pef was . '/ . and . '/ . , respectively. artificial pulmonary ventilation ended in . and . h, respectively. the time of the patients' stay in the resuscitation department was . days in group and . days in group . by the end of the st week pneumonia developed in one patient from group and in eight patients in group . aerosol therapy application accelerates medication delivery to the respiratory tracts, increases the local activity and effects good prophylaxis for surgical hospital pneumonia. variation in ethiology of early and late onset ventilator associated pneumonia pm nikolopoulos j, daganou m, michailidou m, karabela e, kavada k, retsou s, antoniadou a, rasidakis a. department of respiratory abstracts s failure and icu, sotiria general and chest disease hospital, athens, greece purpose: to compare the distribution of causative microorganisms, their susceptibility to antibiotics and outcome of 'early' and 'late' vap in a greek icu. methods and results: retrospective study of mechanical ventilated (mv) patients (pts) with early and late vaps during a -month period. diagnosis of vap was made by clinical, radiographic criteria and quantitative cultures of bronchial secretions. vap was diagnosed in pts ( %) out of consecutive admissions in icu. all pts before the development of vap received antibiotics. three episodes of vap ( . %) were developed before the th day of mv (early vap) and were caused: ( ) by multi-resistant acinetobacter; and ( ) by antibiotic-susceptible pseudomonas aeroginosa . in this group one pt died from septic shock related to vap and two pts survived. fourteen pts ( . %) developed vap after the th day of mv (late vap). five cases were caused by multi-resistant p. aeroginosa , two cases by mrsa, two cases by multi-resistant acinetobacter, two cases by susceptible to antibiotics klebsiella pneumoniae , and three were polymicrobial and caused by multi-resistant microorganisms (mrsa and gnb). four pts died ( . %) from septic shock related to vap, five pts ( %) died because of another cause and five pts ( %) survived. conclusions: early and late onset episodes of vap were caused by 'potentially drug-resistant bacteria'. p. aeroginosa as a cause of early vap was susceptible. mortality attributed to early and late vap was similar. antibiotic prophylaxis in oncological and major reconstructive orthopaedic surgery pm de biase p, ciampalini l, astone a, capanna r. azienda ospedaliera careggi, oncological and reconstructive centre, aoc, florence, italy during last year patients scheduled for oncological surgery or major reconstructive procedures were randomised to either vancomycin or teicoplanin prophylaxis. prophylaxis was performed with either vancomycin g i.v. twice daily or teicoplanin once daily i.v. two hundred patients were included. four patients did not agree the study protocol and were excluded. we treated patients with teicoplanin and patients with vancomycin. out of the patients were operated for oncological disease, while the remaining underwent major orthopaedic procedures. we experienced cases of red man syndrome, and five cases of moderate hypotension. five patients had postoperative complications: two deep venous thrombosis, one pulmonary embolism, two postoperative haematoma. in five patients we observed a wound dehiscence; two of these patients showed clinical sign of ssi and microbiological examinations were positive for mrsa. one patient recovered from infection with medical therapy, while the other patient showed a local tumour recurrence and was amputated at the thigh. at last surgery infection was still present clinical and at microbiological examination. in conclusion we had an infection rate of . % which is comparable to the infection rate of a 'clean' surgery in patients with normal risk of infection. teicoplanin showed lower toxicity, has a longer half-life and has a simpler way of infusion and it is our current choice in high risk surgery. injuries with contaminated sharp articles in health care workers in general hospital celje, slovenia pm lesnicar g, sibanc b. department of infectious diseases, medical center celje, celje, slovenia in a prospective study carried out from january till june , we registered subcutaneous injuries with sharp objects, mostly in nurses and cleaning service workers. in % of cases the incident occurred outside the hospital, in persons who were not medical workers. in cases the injury causing object was a needle that had been used in known patients, of which were hepatitis b positive. fifty-five ( . %) of the injured health workers had been previously vaccinated against hepatitis b; the protective antibodies to hepatitis b in the blood were found in / ( . %) health workers only, while the tests for antibodies to hepatitis c and hiv were negative in all cases. following the incident, the majority of the injured persons, i.e. , were vaccinated against hepatitis b, while persons ( . %) also received passive prophylaxis with human immunoglobulin against hepatitis b. none of the injured persons have developed the disease or showed evidence of sero-conversion. in the year the general as well as specific preventive measures practised in our hospital became more rigorous. thus, approximately % of our health workers at risk have already been vaccinated against hepatitis b. to improve measures preventing dissemination of multidrug-resistant bacteria (mrb), a cross-sectional survey ( ) was conducted to analyse healthcare workers' (hcws) isolation precaution knowledge for mrb infection at investigation and outpatient departments excluding four declaring not to be involved in care to mrb patients (emergency, obstetrics, nuclear medicine, and bacteriology). two hundred and eight hcws answered ( % of the paramedical staff, % of the physicians). thirty-three percent of them reported they do not know frequently or always the patient mrb status. they ( %) wish mrb status to be mentioned on the test form or on the advice request letter. mrb patient visit or test was appropriately timed in % answers. gowns ( %) or masks ( %) use were not systematically reported. other hcws ( %) reported better isolation precaution knowledge than physicians ( %) and nurses ( %) than investigation assistants ( %). physicians declared lower compliance with use of gowns, gloves or draw-sheets than other hcws. they had also lower education in isolation precautions and were less interested in education programs. this study suggests the necessity to improve mbr infection information. physicians and investigation assistants seem to be insufficiently aware of hospital infection control. therefore, education strategies targeted at physicians and investigation assistants working at outpatient and investigation departments should be developed. outbreak of clostridium difficile -associated diarrhoea in infectious disease department: risk factors and hygiene measures assessment pm henoun loukili n, martin m, remy v, hansmann y, christmann d. hôpitaux universitaires de strasbourg, maladies infectieuses et tropicales, strasbourg, france (shock)'/ * (bedridden status)'/ * (age !/ years)'/ * (previous antibiotic treatment) and points (women) / * (shock)'/ * (bedridden status)'/ * (age !/ years)'/ * (immunosuppression). the vast majority of patients ( and % of males and females, respectively) could be classified in the subgroups with lower scores (six points or less) which had a very limited risk of death ( Á/ . and Á/ . % for men and women, respectively), whereas for patients in the highest score subgroup ( points or more), the risk was % for men and % for women. conclusion: risk stratification of patients with ap is possible from simple clinical variables available on admission. procalcitonin (pct) and c-reactive protein (crp) for differentiation of systemic inflammatory response syndrome (sirs), sepsis and severe sepsis pm lupse m a , ursu l b , slavcovici a a , carstina d a . a clinical department, teaching hospital of infectious diseases, cluj-napoca, romania , b laboratory department, teaching hospital of infectious diseases, cluj-napoca, romania objectives: to evaluate the value of pct in the differentiation of patients with sirs, sepsis, severe sepsis and bacteremia in comparison to crp. design: prospective study including patients who meet criteria for sirs, sepsis or severe sepsis (consensus conference of the accp/ sccm) admitted over -month period. patients and method: a total of patients were included: eight with sirs, with sepsis and with severe sepsis. sixteen from patients had bacteremia. pct and crp were evaluated in the first h after admission: pct by brahms pct-q test and crp by turbidimetric assay. the sensitivity, specificity, predictive value of different cutoff points for crp and pct were determined. results: with a cut off point of . ng/ml for pct and . mg/dl for crp sensitivity and specificity for sepsis were %, respectively % (ppv . , npv . ) and %, respectively % (ppv . , npv ). a cutoff point of ng/ml for pct accurately predict severe sepsis (sensitivity %, specificity %, ppv ). a pct level of at least ng/ml was a good predictor for bacteremia (sensitivity %, specificity %, ppv . and npv ). conclusion: pct is a good discriminating marker to characterize the level of inflammation caused by infection and can predict bacteremia . giamarellou h a , aoun a b , klastersky j b , anagnostopoulos n c , galani l c , grecka p c , panaretou e c , papageorgiou e c , repoussis p c , syrseloudis pct has been considered as a useful diagnostic marker in neutropenic patients with bacteremia and/or severe sepsis (giamarellos-bourboulis ej et al. clin infect dis ; : ) . in an attempt to define its value in the diagnosis of localized infections in neutropenic hosts, daily determinations of pct and of c-reactive protein (crp) were performed before and after the onset of fever in subjects male and female aged . / . years with various haematologic malignancies (aml , nhl , mds , all ) developing neutropenia ( b/ pmns/mm ) after chemotherapy. thirty-three patients were presented with fever of unknown origin (fuo) and with localized bacterial infections (lbi; pneumonia , acute pyelonephritis , soft tissue infections , acute pharyngitis ). pct was determined by an immunoluminometric assay and crp by nephelometry. it is concluded that febrile neutropenia followed by a localized bacterial infections is accompanied by significantly higher levels of pct than in case of fuo ( . / . vs . / . ng/ml). similar differences are not observed with crp, which lacks the appropriate specificity. our study included patients who were categorized as having proven ( ), probable ( ), or possible ( ) systemic fungosis according to eortc criteria; showed no sign of infection, and were used as controls. blood samples were received on the st, rd, and th day from the onset of signs of a fungal infection, and then twice a week. pct levels were determined by an immunochemioluminent assay, and candida and aspergillus antigen levels by elisa. in only five patients pct indicated early signs of infection, albeit at barely detectable limits. six patients, however, showed significantly increasing titres preceding time of death. positive antigens titres were observed only in patients who had proven or probable systemic fungosis. only half of the control group had negative antigen titres; a high rate of false negatives was also observed. both pct and antigens titres increased in parallel in / patients with unfavorable outcome. pct and antigens titres cannot reliably indicate early diagnosis of systemic fungal infections although may be used as a prognostic tool of severity. lactulose, a factor that decreases endotoxaemia, in obstructive jaundice? pm koutelidakis im a , papaziogas v a , makris i a , giamarellos-bourboulis ej b , giamarellou h b , papaziogas t a . a thessaloniki med school, nd surgical clinic, thessaloniki, greece , b th department internal medicine, athens medical school, athens, greece bacterial translocation is a process implicated in the pathogenesis of spontaneous peritonitis. in order to evaluate the impact of lactulose administration on systemic endotoxaemia, obstructive jaundice was induced in rabbits by common bile duct ligation. animals were divided into two groups, group a of five rabbits not receiving lactulose and group b of six rabbits, which received . ml/kg of lactulose orally by an oral catheter. blood was collected daily, before and after operation for a total duration of four days. samples were applied for culture and for determination of endotoxins (lps) by the lal qcl- assay. concentrations of lps (mean /sd) of group a were . / . , . / . , . / . and . / . eu/ml on the st, nd, rd and th day, respectively. respective concentrations of lps (mean /sd) of group b were . / . , . / . , . / . and . / . . all blood cultures were sterile in both groups. differences activity of linezolid against nosocomial strains of staphylococcus aureus in russia: results of multicentre study pm were included in the study. antimicrobial susceptibility testing was performed by agar dilution method in accordance with the nccls recommendations. all tested strains including mrsa strains ( . % of all strains) were found to be susceptible to linezolid with the mic ranged from . to mg/l. both mic and mic were mg/l. conclusions: linezolid had excellent in vitro activity that was not affected by resistance to other classes of antimicrobials susceptibility to antiseptics of mrsa isolated in japan during Á % to piperacillin, % to ceftazidime, % to cefepime, % to imipenem, % to amikacin and % to ciprofloxacin. fiftythree percent of p. aeruginosa strains were multiresistant ( strains) and were isolated in patients. wild phenotype to b-lactams was observed in % of strains. the most frequent b-lactams resistance phenotypes were: cephalosporinase over production ( %) and penicillinase ( %). imipenem, ceftazidime and piperacillin-tazobactam were the most active b-lactams (mic of . and mg/l, respectively) these results showed high rates of antibiotic resistance and predominance of o serotype in multiresistant strains compared to the o serotype in europe. infectious complications sustained by stenotrophomonas (xanthomonas ) maltophilia in hiv at present, very limited informations are available about s. maltophilia infections in the setting of hiv disease. patients and methods: a retrospective survey of clinical and microbiological records of hiv-infected patients referring to out tertiary care centre between and was performed, in order to identify all episodes of s. maltophilia infections, and analyze its epidemiological, clinical, and microbiological variables. results: sixty-one episodes of s. maltophilia infection were observed in patients: sepsis/bacteraemia in cases ( . %), lower airways infection in five, urinary tract infection in four, pharyngitis in two, lymphadenitis and liver abscess in one case each. forty-seven out of episodes of s. maltophilia infections ( %) occurred as nosocomial disease, generally in association with advanced immunodeficiency, neutropenia, instrumentation, and prior antimicrobial therapy. bacterial isolates showed an elevated resistance profile against many betalactam compounds, aztreonam, imipenem, and aminoglycosides. conclusion: s. maltophilia represents an emerging opportunistic pathogen in hiv-infected patients extended spectrum beta-lactamases producing germs in intensive care units pm university of medicine and pharmacy 'victor babes ', microbiology, timisoara, romania injury and complications lasted months, demanded for surgical procedures and total cost was comparison of different methods for detection of extended spectrum beta lactamases (esbls) and their genetic relatedness among enterobacteriaceae clinical isolates in a research medical institute pm methods: one hundred out of isolates that were screened positive for esbls were tested with double disk synergy test (ddst), three dimensional test (tdt), e -test-esbl and vitek-esbl test. pulsed field gel electrophoresis (pfge) analysis was applied to esbls; five klebsiella pneumoniae and eight escherichia coli . results: revealed the prevalence of esbls in . % of clinical isolates. the sensitivities of the ddst, tdt, e -test and vitek were , , . and . %, respectively. in the ddst, aztreonam was the most sensitive indicator ( . %). pfge demonstrated that % of k. pneumoniae were derived from a single clone whereas . % of e. coli isolates were derived from two different clones. non-clonal origin was demonstrated in % of k. pneumoniae and . % of e. coli . conclusion: there is an increased prevalence of esbls. the ddst is the most sensitive, practical and cost effective diagnostic method reliable for routine use in our laboratory. both clonal spread and plasmid dissemination contributed to the concurrent nosocomial outbreaks caused by esbl-producing k severe nosocomial infections due to stenotrophomonas maltophilia pm the teaching hospital of infectious diseases total og pts after extensive hysterectomy ( pts), extensive vulvoectomy ( pts) and extensive/combined operations for ovarian cancer ( pts) were analysed. ic developed in pts. one hundred and sixtyseven pts had no ic. twenty-eight of rf analysed were independent rf of ic. most important included: age !/ years (p / . ), grade Á/ obesity (p / . ), diabetes mellitus (p / . ), diagnosis of cervical cancer (p / . ), history of pre-cancer of vulva postpartum endometritis due to group a streptococcus : a case-control study pm unite operationnelle d 'hygiene all isolates were sensitive to vancomycin. among gram-negative bacteria klebsiella spp. was isolated in . % of cases, acinetobacter baumanii in . %, enterobacter spp. in . %, providencia spp. in . %. of the klebsiella spp. isolates % were resistant to amikacin, % to cephalosporins, % to piperacillin/ tazobactam. all were sensitive to imipenem. of the a. baumanii isolates % were resistant to amikacin, aztreonam, cefoperazone, cefotaxime, ceftriaxone, piperacillin; % to ampicillin-sulbactam, % to ceftazidime, and % sensitivity to imipenem antimicrobial activity of selected pharmacopoeial antiseptics analysed according to european standards pm european committee for standardisation approved several european standards (en), describing test methods establishing, whether an antiseptic has or does not have a bactericidal or fungicidal activity under the laboratory conditions defined by en. the aim of the study was to investigate, if some chemical compounds in concentrations recommended by polish pharmacopoeia for skin disinfection, comply european standards requirements. methods: basic bactericidal (en ) and fungicidal (en ) activity were investigated as well as bactericidal activity of products for hygienic and surgical handrub and hand wash used in human medicine (pren ). all methods and used neutralizers were validated. standard strains: staphylococcus aureus , pseudomonas aeruginosa , escherichia coli , e. hirae , candida albicans and a. niger were used, when en standards were evaluated. results: ethanol, izopropanol and n -propanol caused viable microbial count reduction required by ens in pharmacopoeial concentrations the purpose of the study: we collected bacteriological samples from adult and neonate patients who were admitted in intensive care units (icu) . the aim was to observe the colonization status with microbes that may have a nosocomial potential and to establish circulating phenotypes in icus. the results obtained from a total of samples strains of gram negative bacteria (enterobacteriaceae family) were isolated. fourteen strains showed extended spectrum beta-lactamases (esbl) phenotype (eight strains of klebsiella pneumoniae , three of escherichia coli , two of klebsiella ornithynolitica , one of klebsiella oxytoca ). we used both disc diffusion test (extended antibiotic susceptibility test and synergy test to visualize 'champagne stopper' pattern) and mini api † system.the conclusion reached: we put in evidence a massive colonization with germs that may have a nosocomial potential especially microbes that produce esbl ( . % from all enterobacteriaceae isolated) which implies a rational policy in prescribing antibiotics in hospitals from western part of romania.carbapenem activity against nosocomial gram-negative rods pm sawicka-grzelak a a , rokosz a a , meszaros j b , luczak m a . a department of medical microbiology, university medical school, warsaw, poland , b department of general and transplantation surgery, university medical school, warsaw, poland purpose: to determine a susceptibility of nosocomial gramnegative rods to carbapenems.methods: two hundred strains of gram-negative rods were cultured from clinical specimens from hospitalized patients (july Á/november ). identification of strains was performed in the automatic atb system (biomerieux, france). susceptibility of strains to carbapenems: imipenem and meropenem was determined with disc diffusion method according to nccls recommendations. esbl-producing strains were detected with double-disc synergy test (ddst according to jarlier et al., ) or a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd ) (oxoid, england) .results: one hundred and ten strains of enteric rods and strains of non-fermenting rods were cultured. twenty eight ( %) esblpositive strains were detected. carbapenems were active against % of enteric rods. the percentage of non-fermenting rods susceptible to imipenem was and to meropenem */ .conclusions: carbapenems: imipenem and meropenem demonstrated high activity against clinical strains of enteric rods. however, the antibiotics were less active against nosocomial strains of nonfermenting rods.inhaled antibiotics against multiresistant bacteria in bronchial secretions of icu patients: a preliminary report pm horianopoulou m a , kanellopoulou m b , paraskevopoulos i a , valakis k a , kyriakidis a a , lambropoulos s a . a intensive care unit, sismanoglio general hospital, athens, greece , b department of microbiology, sismanoglio general hospital, athens, greece purpose: the aim of this study was to assess the effectiveness of aerosolized ampicillin/sulbactam, ceftazidime and colistin, in icu patients with multiresistant acinetobacter baumannii or pseudomonas aeruginosa colonization of the respiratory tract.methods: fifty-three intubated, mechanically ventilated patients participated in the study. multiresistant a. baumannii , sensitive only to ampicillin/sulbactam, or p. aeruginosa , sensitive to ceftazidime or colistin, were isolated from the bronchial secretions ( Á/ cfu/ ml). all patients were subsequently treated with intravenous ampicillin/sulbactam, ceftazidime or colistin, whereas of them were also given the same antibiotic in aerosolized form.results: a decrease in the number of colonies by Á/ cfu/ml was observed, following Á/ days of combined treatment with both intravenous and inhaled antibiotic. none of the patients developed vap. in the patients who only received the antibiotic intravenously, the decrease ranged from zero to cfu/ml, after days of treatment. two of patients developed vap.conclusions: our results suggest that the administration of aerosolized antibiotics represents an effective means of preventing ventilator-associated pneumonia caused by a. baumanni and p. aeruginosa . introduction: pseudomonas aeruginosa is an important nosocomial pathogen. resistance to certain beta-lactam antimicrobial agents among p. aeruginosa is increasing. despite the development of new antibiotics multiresistant strains of p. aeruginosa represent an important therapeutic problem. the aim of this study was to investigate the activity of imipenem, amikacin, piperacillin, ciprofloxacin, ceftazidime, against clinical isolates of p. aeruginosa . methods: a total of isolates by tracheal aspiration from hospitalized patients, admitted to intensive care units were identified as p. aeruginosa using an algorithm that included: gram stain, pigment, oxidaze ('/ ,) and gram negative identifications microscan walkaway- (dade behring) were used according to the manufactures instructions. minium inhibitory concentrations were determined using walkaway, interpretation based on ncclsm -s , january ' .results: the respiratory tract was the single site of isolation for this study. the best activity was showed by imipenem %, followed by amikacin %, piperacillin, pip/tazobactam, ciprofloxacin had the same sensitivity %.conclusion: a high level resistance to antibiotics was observed to p. aeruginosa isolated from tracheal aspiration. carbapenems seem to be the most active against p. aeruginosa in this study. materials and methods: clinical samples were collected from patients admitted to this hospital. only one isolate per patient was included. antimicrobial susceptibility testing was performed as recommended by the nccls. all bacterial isolates were tested by dd and ad to provide a comparison of both test results. very major error was considered when the strains were resistant (r) by ad and susceptible (s) by dd and major error when s by ad and r by dd. categories of s and r were stablised using the breakpoints suggested by mensura ( ) . colistin r strains was typed by rep-pcr.results: among the strains included in this study ( . %) were s to colistin and five ( . %) were colstin r by ad, of this five colistin r strains four ( . %) were s to colistin by dd and one was r by both methods. all r isolates were similar by rep-pcr.conclusions: most of the ad colistin resistant strains were s when tested by dd indicating that this method is not useful to determine the resistance to colistin. rep-pcr patterns show that the spread of a colistin r clone seems to be involved. methods: gnb were isolated from per-operative biopsies (pob) and/ or from articular punction (ap). patients (pts) received cfp, g bid'/ ofl, mg tid or cip, mg bid intravenously for days, followed by a prolonged oral fq monotherapy. cure was defined as: resolution of all clinical signs of infection, normalization of the biological inflammatory profile at the end of treatment (eot) and absence of infection at the same site during the post-treatment followup period (ptfu).results: all of the studied patients [mean age / years] had hospital acquired bji. seventeen/ had an infected orthopedic device (prosthetic joints / , other orthopedic prosthetic devices / ). culture of pob and ap yielded to pseudomonas sp. ( ), enterobacter cloacae ( ), others ( ). vancomycin was added for six pts co-infected by gnb-mrsa. nineteen/ pts underwent a surgical intervention (debridement / , removal-replacement / , amputation / ). after ptfu period of months (range Á/ ), the overall success rate was / ( . %) without serious adverse events.conclusion: cfp Á/fq combination was safe and efficient in the treatment of hypercase gnb, bji.treatment of posttraumatic mrsa osteomyelitis of the femur with longterm cotrimoxazole */a case report pm the authors report a case of posttraumatic osteomyelitis of the femur caused by methicillin-resistant staphylococcus aureus (mrsa), following the shot injury. relapses of the infection occured in months interval and were treated by revision, debridement, lavage and vancomycin. because of laboratory signs of renal insufficiency vancomycin became contraindicated for treatment of the third relapse of infection and the different approach was employed: classic open treatment of bone infection sec. orr was combined with a long-term administration of high-dose cotrimoxazole. the patient was given cotrimoxazole mg daily divided in four doses ( mg/kg/ h) for months, then for gastrointestinal complaints with lowered dose of g daily for next months. the wound completely healed. during months after the final surgery there was no relaps of infection, but the atrophic pseudoarthrosis of the femur resulted. the patient can walk with a rigid orthesis and two crutches. the whole treatment of the objective: to present a variety of severe nosocomial infections due to stenotrophomonas maltophilia in patients hospitalized in tertiary medical units from cluj.results: during the last year nine strains of s. maltophilia obtained from patients with severe infections and hospitalized in different wards were isolated. all but one were considered nosocomial infections: four cases of pneumonia, one urinary tract infection, three cases of surgical wound infections and one case of endocarditis under surgical treatment. the cases of pneumonia were either primary occurring in a granulocytopenic patient with leukemia or secondary in patients that underwent surgical treatment. in the case of endocarditis the ethiology was established after surgery from the damaged valve in a negative hemoculture patient with a poor outcome under medical treatment. in all cases of surgical wound infection bacteremia occurred diagnosed on clinical basis in the presence of severe sepsis or hematogenous dissemination in the lung. the urinary tract infection occurred in a patient after urinary surgery and having a catheter in place. the immediate evolution was favorable in all cases but treatment was difficult due to the highly resistant strains and to underling diseases.conclusions: s. maltophilia should be considered in nosocomial severe infections and prophylaxis by interrupting environmental transmission has to be promoted. sawicka-grzelak a, rokosz a, luczak m. department of medical microbiology, university medical school, warsaw, poland purpose: to identify and determine the drug-susceptibility of esblpositive strains isolated from urine samples.methods: seven hundred and twelve strains of gram-negative rods were cultured from urine samples from hospitalized patients during months (july Á/november ). identification and susceptibility were performed in the automatic atb system (biomerieux, france) using id e, id gn and atb ur strips. esbl-activity was detected with double-disc synergy test (ddst according to jarlier et al., ) or using a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd ) (oxoid, england).results: five hundred and ninety-five strains ( . %) belonging to enterobacteriaceae family, strains ( . %) of non-fermenting rods and two strains ( . %) of other gram-negative rods were isolated. eighty-two esbl-producing strains ( . % of all strains) were detected. fifty-nine esbl-positive strains were susceptible to nitrofurantoin, -to norfloxacin and ciprofloxacin and -to fosfomycin.conclusions: esbl-positive strains were detected most frequently among enteric rods ( strains). nitrofurantoin and quinolones were the most active in vitro antibacterial agents against examined esblpositive uropathogens. results: the mechanisms of resistance were evaluated phenotypically using different aminoglycosides. a total of aminoglycoside resistant gram-negative strains were studied. one hundred fifty-eight strains were collected in Á/ , in and in . the resistant profiles were determined: enterobacteriaceae */ ; pseudomonas aeruginosa */ ; acinetobacter spp. */ . the most frequently phenotypes were gt (gentamicin, tobramycin) */ % and gtnet (gentamicin, tobramycin, netilmicin) */ %. the gt phenotype due to production ant( ƒ)-i enzyme, the gtnet Á/aac( )-v ( strains), aac( )-iv */one strain and aac( ƒ)-i */one strain. the resistance to amikacin in % strains was due to production aac( ?)-i ( %) and aph( ?)-vi ( %). the most of examined strains were simultaneously resistant to kanamycin and neomycin caused by production of aph( ?)-i ( %). only seven strains were resistant to all aminoglycosides due to impermeability of outer membrane. no substantial differences were observed between years.conclusions: the main mechanism of aminoglycoside resistance is fermentative modification. the high rate to gentamicin and tobramycin was due to production of ant( ƒ)-i and aac( )-v. amikacin and isepamicin were the most active aminoglycosides against gramnegative nosocomial isolates.the incidence of clostridium difficile associated diarrhoea (cdad) increased in our department from january to june .objective: to confirm the out break of cdad, to identify the risk factors and assess the effectiveness of the measures implemented for controlling this outbreak.methods: cdc definitions were used to identify the cases. the scope of the outbreak was defined. cdad incidences during the outbreak period and during the same period in were compared. risk factors (reduced mobility, antibiotic treatments . . .) were studied for patients whom length of hospital stay (lhs) was more than days. contact precautions and environmental cleaning with clona implemented were assessed.results: seventeen episodes of cdad were identified. sex ratio: . , mean age / . , mean lhs / days, mean delay for cdad occurring / days. one hundred and fifty-two patients involved in the study of risk factors. relative risk (r.r.) evaluated were: blactams (r.r. / . , ic %: . Á/ . ), reduced mobility (r.r. / . , ic %: . Á/ . ). incidence of cdad was less than two cases per days of hospitalisation after june .conclusion: we confirmed the outbreak of cdad in our department b-lactams and reduced mobility were identified as risk factors for cdad. measures implemented to control the outbreak were effectiveness.positive heart transport fluid cultures associated with severe infections in heart transplant recipients pm at the mount sinai hospital in new york city heart transplants were performed between and june . cultures were routinely performed on all heart transplant transport fluids. culture data was available for of these patients. in total / ( . %) were positive for bacteria, fungi or both. the organisms isolated included coagulase negative staphylococci ( ), pseudomonas aeruginosa ( ), staphylococcus aureus ( ), acinetobacter baumanii ( ), serratia mercescens ( ), enterobacter cloacae ( ), escherichia coli ( ), proteus mirabilus ( ), enterococcus faecalis ( ), viridans streptococci ( ), and fungi (aspergillus fumigatus ( ), penicillium species ( ), and rhodotorula rubra ( ). two heart transplant recipients had two organisms isolated from the transport fluid. isolation of resistant gram-negative bacilli in the transport fluid was associated with significant infection in / patients ( %) with the same organism. the observed infections were pneumonia secondary to e. cloacae , sternal wound infection secondary to p. aeruginosa , and bacteremia secondary to p. aeruginosa . it appears prudent to provide prophylaxis against resistant gram negative bacilli to prevent infections. zacharof ak, flevaris c, petrogianopoulos c, karachalios g, vroulis j, chartzoulakis g, drakogiorgos g, loizidou a, svoukas g. nd department of internal medicine, hellenic red cross hospital, athens, greece objective: we studied the trends of nosocomial bloodstream infection and calculated the population-attributable risk for death among hospitalized patients. methods: we perform a -year retrospective study for all patients (n / ), admitted to our department between and .results: between and , a total of patients developed episodes of nosocomial bloodstream infection. the crude infection rates increased linearly from . to . per discharges ( . Á/ . episodes per patient-days) during the -year study period. increases in the infection rates were due to gram-positive cocci, yeasts and essentially explained by infections caused by coagulasenegative staphylococci, staphylococcus aureus , enterococci, and candida species, respectively. although the crude mortality in patients with nosocomial bloodstream infections decreased from % in to % in , the in-hospital population-attributable mortality among infected patients increased from . deaths per discharges in to . per discharges in . the etiologic fraction or the proportion of deaths in patients with bloodstream infection to all deaths occurring in the hospital increased from . % in to . % in .conclusions: the incidence and the population-attributable risk for death among patients experiencing nosocomial bloodstream infections increased progressively during the last years in our department.ventilator-associated pneumonia before and after intensive care unit temporary closure pm results: we compared the incidence, causative organisms and mortality of vap in two different time periods. period june to december . period june to december . between those two periods the icu remained closed for months because of reconstruction works.period : sixty-seven consecutive patients (pts) were studied with bronchial secretions cultures at least days after mechanical ventilation (mv) initiation. the vap was diagnosed by clinical, radiological and microbiological criteria in pts ( %). causative organisms included: pseudomonas aeruginosa , acinetobacter , staphylococcus aureus , klebsiella pneum. , enterobacter . in three cases vap was proved polymicrobial. fourteen ( ) episodes of vap ( %) were developed after days mv (late vap) and were attributed to multiresistant microorganisms. mortality of vap was %.period : among consecutive pts, vap was diagnosed in ( %). causative organisms included: acinetobacter , p. aeruginosa , s. aureus , k. pneumoniae , escherichia coli . five ( ) cases were polymicrobial and cases were 'late vap' ( %). causative microorganisms had similar patterns of sensitivity to antibiotics (compared to period one). mortality of vap was %.conclusion: temporary icu closure had no significant influence on the incidence, distribution of causative organisms, their sensitivities to antibiotics and mortality of the vap. efstathiou sp a , pefanis av a , tsioulos di a , tsiakou ag a , zacharos id a , kanavaki s b , mountokalakis td a . a third university department of medicine, sotiria general hospital, athens, greece , b microbiology laboratory, sotiria general hospital, athens, greecepurpose: the aim of this study was to derive a scoring system for the prediction of outcome in adult patients with acute pyelonephritis (ap) severe enough to need hospitalization. therefore, the charts of patients ( men, median age years) were reviewed.results: logistic regression analysis identified in both sexes four independent correlates of in-hospital mortality, the coefficients of which divided by . and rounded to the nearest interval, resulted in the following integer-based scoring system: points (men) / * between concentrations of lps of the two groups were statistically significant on the nd and the th day (p b/ . ). it is concluded that the administration of lactulose may decrease systemic endotoxaemia in the field of obstructive jaundice.nosocomial infections: a prevalence study in the island of crete pm doukakis s a , tzimis l b , perogambrakis g a , kalloniatou m a , christodoulakis n a , evaggelopoulos a a , koutsoumba d a , kastanakis s a . a first medical department, 'saint george ' general hospital, chania, greece , b pharmacy department, 'saint george ' general hospital, chania, greeceprevalence surveillance is a rapid and inexpensive mode to estimate the problem of hospital-acquired infections (hais). to study the problem of nosocomial infections in our hospital, a prevalence study was made from our team in . the study included patients (the total number of hospitalized patients at the time of the study). from these patients were males ( . %) and females ( . %). one hundred and ninety-one patients ( . %) belonged in the groups of age between and years. fifty-seven patients had a urine catheter ( . %). one hundred and fifty-five/ patients ( %) received antibiotics and from these patients received one antibiotic and the remaining patients two or more. a nosocomial infection was found in patients and consequently the prevalence of hais was . %. among these, urinary tract infections were six ( . %), lower respiratory tract infections were three ( . %), surgical site infections were three ( . %), and bloodstream infections was one ( . %). the incidence of multiresistant bacteria was primarily enterococcus spp and secondary, pseudomonas aeruginosa , enterobacter spp, klebsiella pneumoniae , escherirchia coli , staphylococcus aureus , enterobacter cloacae . unfortunately prophylactic chemotherapy of long duration was found despite the suggestions of the infection control committee. regarding age the highest incidence of hais occurred in the third age group. bagirova ns, dmitrieva n. laboratory of microbiology, cancer research center of russia, moscow, russian federation objectives: to determine the pathogens and susceptibility to antimicrobials.methods: blood samples were collected from adult pts ( Á/ ). the bacteraemic episodes were classified according to the definitions of the cdc. laboratory detection of bacteraemia and fungaemia was performed according to cumitech b (blood cultures iii, ). susceptibility testing was performed by disk diffusion method (nccls).results: the total number of blood samples */ , -positive ( . % episodes of significant bacteraemias). bsi was confirmed microbiologically in of febrile pts ( . %). the most frequent pathogens were gram('/) cocci ( . %) (p b/ . ), gram((/) bacilli */ . %, fungi */ . %. coagulase-negative staphylococci (cns) represented . %, staphylococcus aureus . %, streptococcus spp. . %, enterococcus spp. . %, enterobacteriaceae . %, pseudomonas aeruginosa . %, other non-fermenting */ . %, yeast . %, mould . %, anaerobes . %. one hundred percent cns were resistant to penicillin, . % to oxacillin, . % to clindamycin, . % to cefazolin, . % to ceftazidime, . % to ciprofloxacin, . % to gentamicin, and no isolate was resistant to vancomycin. the predominant pathogens in all types of hm were gram('/) cocci (mainly cns). all gram('/) microorganisms were sensitive to vancomycin. katashinsky o a , opriatova t b , tchuev p c . a state clinical hospital, anaesteziology, odessa, ukraine , b state clinical hospital, bacteriology, odessa, ukraine , c medical university, anaesteziology, odessa, ukrainethis investigation was carried out in odessa state clinical hospital during Á/ . of the patients with post-operation complications, % of gram-negative cultures were sensitive to ceftazidime and % to amikacin. sixty-nine percent of gram-positive cultures were resistant to penicillin g, but they were sensitive to vancomycin and nitrofurantoin in and % of cases, respectively. sixty percent of isolates of pseudomonas aeruginosa were sensitive to ceftazidime and % to amikacin. rates of resistance to carbenicillin and gentamicin were and %, respectively. one hundred and fortythree isolates of escherichia coli were studied and % of them were resistant to ampicillin % to cephalothin and % to tetracycline. of the isolates tested against ciprofloxacin, all were sensitive. one hundred and eight isolates of staphylococcus aureus were studied and they were resistant only to penicillin g ( %). staphylococcus aureus was sensitive to erythromycin ( %), tetracycline ( %), oxacillin ( %) and vancomycin ( %). all isolates enterococcus faecalis were sensitive to nitrofurantoin and % to ciprofloxacin. the majority of s. aureus and e. faecalis isolates were susceptible to most other antibiotics, but the majority of e. coli isolates were resistant to the studied antibiotics expect ciprofloxacin.campylobacter foetus bacteraemia in an immunocompromised patient: a case report pm monno r a , ierardi e b , rendina m b , ceci g a , luzzi i c , de vito d a , rizzo g a , francavilla a b . a department of internal medicine and public health, university of bari, bari, italy , b department of emergency and organ transplantation, university of bari, bari, italy , c istituto superiore di sanità, rome, italy a -year-old woman was admitted for recurrent fever. the patient underwent a liver transplantation and splenectomy in . she had followed immunosuppressive therapy until when tacrolimus was added for chronic rejection. in non-hodgkin lymphoma was diagnosed and chemotherapy was started. six months later because of the presence of two metastatic encephalic foci affecting the optic chiasm, a new chemotherapy course was started with the regression of lesions. in january she was treated with steroid recycle and cyclosporine Á/azathioprine Á/prednisone reintroduction. fever occurred after months and cytomegalovirus (cmv) infection was diagnosed. treatment with ganciclovir was started with clinical remission. in november cmv infection recurred and blood cultures were positive for a bacterium that was identified as campylobacter fetus . the patient was successfully treated with intravenous ciprofloxacin. bacteremia frequently occurs in cancer patients. bacteremia due to c. fetus are rare, occurring mainly in immunocompromised patients. c. fetus expresses a proteinaceous surface layer that confers serum resistance. in our patients steroid and immunosuppression may have contributed to the development of lymphoma. all of these factors and chemotherapy have contributed to cmv infection and all have made the patient susceptible to bacteremia with this infrequently found bacterium. the clinical microbiologist should be aware of this infection in immunocompromised host. minenko sv, dmitrieva nv, sokolova en, ptushkin vv. bone marrow transplantation department, n.n. blokhin cancer research center, moscow, russian federation c'/a is the standard regimen as empirical therapy for febrile neutropenia (fn). activity of c against g'/ bacteria and g(/ bacteria, producing chromosomally-mediated b-lactamases (e.g. ampc) is suboptimal. cep is active against a broad range of g'/ and g(/, including ampc producing bacteria. the purpose of the study was to compare the efficacy of two regimens in the treatment of fn.methods: patients with fn received either cep ( g/ h) or c ( g/ h) plus amikacin ( mg/kg/day) or netilmicin ( mg/kg/day). data were collected prospectively.results: a total of pts with episodes ( / ) of fn were included. fifteen/ in cep group and / in c'/a group. the median duration of neutropenia grade , distribution of age, sex and underlying disease were comparable in both arms. mdi was in and %, cdi in and % fuo in and % in fep and c'/a groups correspondingly. response to the initial empirical regimen according to ihs criteria was in % of cep and . % of c'/a groups (p / . ). modification of therapy with a change of cep or c to carbapenem took place in and % of cep and c'/a groups (p / . ). no patient in either treatment group died due to the presenting infection. tolerability of cep was good and no laboratory abnormalities took place. transient elevation of serum creatinin level was observed in two patients c'/a group.conclusions: cep monotherapy is as effective as c'/a combination in the treatment of patients with fever and granulocytopenia purpose of the study: retrospectively, we analysed patients with intraabdominal infection for aetiology, risk factors, and outcome from hospitals in slovak republic within year (march Á/march ). results: in this group significantly more frequent were older patients ( !/ years) with cancer ( vs. %, p b/ . ). acinetobacter spp. ( vs. %, p b/ . ) and enterobacteriaceae ( vs. %, pb/ . ) were predictive for monoinfection. pre-treated patients with other antibiotics had inferior prognosis and more risk factors: permanent urinary catheter ( vs. %, pb/ . ), ventilation or intubation ( vs. %, pb/ . ) and polymicrobial infection ( vs. %, pb/ . ). the risk factors with poor prognosis were enterobacteriaceae ( vs. %, pb/ . ), diabetes mellitus as underlying disease ( vs. %, p b/ . ) and uraemia ( vs. %, p b/ . ). surprisingly, negative prognostic factor was also non-effective previous antibiotic therapy. failed patients died significantly more frequently patients due to underlying disease. cefoperazone/sulbactam was shown to be useful, effective and well tolerated also in one group of patients with % efficacy of treatment, and it belongs to a group of antibiotics suitable for treatment of nosocomial infections. key: cord- -e rhioty authors: rowland, raymond r.r. title: the interaction between prrsv and the late gestation pig fetus date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: e rhioty porcine reproductive and respiratory syndrome virus (prrsv) crosses the placenta during late gestation and productively infects the fetus. virus replication and cytokine responses were measured in tissues of fetuses recovered at – days of gestation, just prior to parturition. at the time of recovery, gross anatomical abnormalities were evident in both infected and non-infected fetuses from the infected dams. virus isolation and immunohistochemistry identified the thymus as the primary site of virus replication. steady state rt-pcr amplification of inflammatory, th and th cytokines, showed elevated ifn-γ and tnf-α mrnas in tissues from infected fetuses, which corresponded to elevated cytokine proteins in serum but not amniotic fluid. further evidence for induction of immunity was found in the hyperplastic response of lymph nodes, which included the development of germinal centers occupied cdw + b cells. collectively, these data support the notion that the immunocompetent fetus is capable of initiating an antiviral response, which is compartmentalized within the infected fetus. furthermore, fetal pathology may not be a direct result of virus replication in the fetus. porcine reproductive and respiratory syndrome (prrs) is caused by an enveloped positive-stranded rna virus, prrsv, belonging to the family arteriviridae cavanagh, ; nelsen et al., ; wensvoort et al., ) . other members of the arterivirus group include lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv; for review see plagemann, ) . the arteriviruses, toroviruses, roniviruses and coronaviruses form a single order, nidovirales. arteriviruses structurally resemble togaviruses, but similar to coronaviruses, replicate via a nested -co-terminal set of subgenomic mrnas that possesses a common leader and a poly-a tail (reviewed in snijder and mulenberg, ) . the arteriviruses exhibit several important properties relevant to the study of viral pathogenesis, including cytopathic replication in macrophages, the capacity to establish and maintain an asymptomatic infec-tion, as well as cause severe and fatal disease (plagemann, ) . infection of adult pigs with prrsv usually produces a non-fatal disease, characterized by flu-like symptoms, a transient elevation in temperature and inappetance (reviewed in benfield et al., ; christianson et al., ) . the reproductive form of prrs occurs following the infection of late gestation pregnant gilts or sows. natural infection of the fetus with prrsv is initiated with the infection of gilts and sows at days gestation. after productive replication on the maternal side, the virus crosses the placenta and productively infects the fetus. the mechanism of transplacental infection is unknown, but could be similar to the infected "trojan horse" macrophage, described for ldv (cafruny and bradley, ) . since the pig fetus becomes immunocompetent at about days of gestation, prrsv infection occurs in an immune environment containing functional b and t cells. accordingly, virus-induced reproductive failure can present clinically as delayed returns to estrus, as well as abortions, mummified fetuses, stillborn and weak-born pigs christianson et al., ; collins et al., ; mengeling et al., ; rossow et al., ; rowland et al., ) . surviving neonates can exhibit the severest form of respiratory disease with mortality sometimes reaching % within three weeks after birth (feng et al., ; rossow et al., ; rossow, ) . the complex pathology following exposure to prrsv in utero represents a unique form of the disease referred to as congenital prrs (rowland et al., ) . the purpose of this study was to characterize the interaction between prrsv and the pig fetus by ( ) identifying sites of virus replication, ( ) measuring immune and inflammatory cytokines in different compartments, and ( ) evaluating the response of lymph nodes. experiments involving animals were approved by the kansas state university iacu committee. pregnant sows, obtained from a closely monitored prrsv-negative herd, were challenged at days gestation with a sixth passage isolate of sd- , a typical north american field isolate (rowland et al., ) . the methods for the preparation of the prrsv inoculum on marc- cells and infection of pigs are described in rowland et al. ( ) . virus was cultivated on marc- cells in mem supplemented with antibiotics (pen/step) and % fbs. dams, at days gestation were challenged with approximately tcid of virus diluted in ml of culture medium. one half of the inoculum was administered by intramuscular injection in the neck. the remaining dose was administered intranasally. mock-infected sows were challenged with medium recovered from marc- cells. dams were monitored for clinical signs and blood collected weekly. at between and days of an approximate days gestation period, the dams were euthanized. the uterine horns were immediately removed and the individual fetuses with intact placenta were carefully removed and immediately necropsied. a sample of amniotic fluid was collected prior to removal. the brachial artery of each fetus was severed and blood collected using a disposable syringe and serum stored at − • c. maternal, accessory and fetal tissues were collected and stored in formalin for histological staining and immunohistochemistry (ihc), or storage in rnalater (ambion) for rt-pcr of cytokine mrnas. prrsv-specific antibody was measured in sera using the herdcheck ® prrs elisa (idexx) and performed by personnel at kansas state university veterinary diagnostic laboratory. serology results were reported as a sample/positive (s/p) ratio. an s/p ratio greater than . was considered positive for prrsv antibody. virus isolation (vi) in serum and tissues was performed as described in rowland et al. ( ) . briefly, serum was serially diluted in mem supplemented with pen/step antibiotics and % fbs and placed on well plates of confluent marc- cells. after three days, plates were fixed in % acetone and stained with fitc-sdow- anti-nucleocapsid antibody, diluted in pbs with % fbs (nelson et al., ) . the results were reported as the log of the inverse dilution of the last positive well. virus isolation from tissues was the same except that tissues were weighed and homogenized in hanks balanced salt solution and then centrifuged at × g for min to remove debris. the sequencing of the hypervariable region of orf is described in rowland et al. ( ) . total rna was prepared from serum or infected marc- cells using an rneasy kit (qiagen) according manufacturer's instructions. for pcr, cdna was prepared using mlv reverse transcriptase (promega) and msb as the primer. the sense and antisense primers for the outer amplification were msa, -cttcgtcccttcttttcctcgtgg, and msb, -ccgctctagagccaacgatagagtctgc, respectively. the product was re-amplified with a nested set of sense and antisense primers, a, -accgtgtatgttaccatcacagcc and b, acgggaaagatgacaaaactctcc. thirty-two cycles of amplification were performed for each primer pair. the conditions for both amplifications included a • c denaturing step ( s), a • c annealing step ( s), and a • c ( s) polymerization step. the final pcr product, which contained the last nucleotides of orf , the nucleotide untranslated region (utr), and the first nucleotides of orf was sequenced directly by automated dna sequencing. pcr products were cloned into a pcr . ta cloning vector (invitrogen), propagated in escherichia coli and individual plasmids sequenced using m forward and reverse primers. sequences were analyzed using gene jockey ii software. tissue samples for rt-pcr were immediately placed in rna-later (ambion) and stored at − • c. total rna was extracted from approximately g of tissue using rneasy kit (qiagen) according to manufacturer's instructions. the design of cytokinespecific primers and rt-pcr procedures were performed according to reddy and wilkie ( ) . primer sequences are listed in table . rna was diluted to a final volume of l in nuclease free water. cdna was prepared from l of total rna by reverse transcription using molony murine leukemia virus reverse transcriptase (promega) and random hexamers as primers. the amplification of ␤ m mrna was used as an internal control. pcr amplification of cytokine and control cdnas consisted of cycles ( s at • c, s at • c, and s at • c) and dna products electrophoresed on a . % agarose gel and visualized using ethidium bromide. the identity of the dna products was confirmed by dna sequencing. tissue samples were collected and immediately placed in % buffered formalin. paraffin-embedded thin sections were mounted on slides, deparaffinized and stained with hematoxylin for the detection of prrsv antigen, slides were incubated for min with a : dilution of mab sr- anti-nucleocapsid antibody (rural technologies). other antibodies included a polyclonal anti-human cd and b cell antibodies, anti-cdw and anti-cd ␣. bound antibody was detected with biotinylated goat anti-mouse or anti-rabbit ig followed by avidin-hrpo and dab chromagen (ventana medical). slides were counterstained with hematoxylin. the experiment incorporated four prrsv-infected and twomock-infected dams. all maternal serum samples were vi-negative and seronegative for prrsv prior to infection. between one and two weeks after virus challenge, all infected sows were vi-positive in serum, confirming the presence of an active infection. by the time of necropsy, the concentration of circulating virus in the dams had dipped to below detectable levels by vi. a total of viable fetuses were recovered from the four infected dams (see table for summary). two fetuses were dead and partially autolysed and not subjected to further study. ( %) of the viable fetuses were positive for prrsv by vi. since dams were vi-negative in blood at the time of necropsy, it was concluded that the presence of virus serology results, shown in parentheses, are presented as the s/p ratio. s/p ratios greater than . were considered positive for prrsv antibody. dead fetuses were partially autolysed and not tested. gross pathology key: * , partially mummified; * , non-viable fetus or relatively low quantity of amniotic fluid; * , necrotic placenta; * , merconium-and/or blood-stained amniotic fluid; * , small, underdeveloped fetus. in the fetal circulation was the result of virus replication in the fetus and not from contamination with maternal blood. (the virus isolation technique is not subject to false positives from minute amounts of cross-contamination with viral protein or rna.) the number of infected fetuses in each litter varied from no infected fetuses (dam no. ) to five of ( %) infected fetuses for dam no. . fetuses that were vi-negative in serum were confirmed as prrsv-negative by vi-negative results in placenta, lung, lymph nodes and thymus (data not shown). one fetus, - , was seropositive for prrsv (s/p ratio = . ). since there is no maternal transfer of antibody from mother to fetus (tizard, ) , it was concluded that this fetus generated an antibody response to prrsv in utero. of the infected fetuses showed some form of gross pathology, including growth retardation (two fetuses) or reduced amounts and/or merconium-stained amniotic fluid (five fetuses). ongoing virus replication in the fetus as the source of these gross pathological changes is questionable, since non-infected fetuses from infected dams exhibited similar changes. for example, fetuses from dam no. (see fig. ) were either merconium stained (two fetuses), non-viable, possessed reduced amniotic fluid levels (four fetuses) or small (one fetus). except for one autolysed fetus, the fetuses from the two control dams showed no evidence of gross pathology (data not shown). rna viruses frequently exist as a heterogeneous population, frequently referred to as a quasispecies. the appearance or disappearance of individual viral sequences within a quasispecies population is often used as evidence to support the existence of positive or negative selection during infection (elena et al., ; forns et al., ; tsibris et al., ) . mutations that appear in the prrsv genome are useful as markers to identify and follow the appearance and disappearance of viruses within the population (allende et al., ; rowland et al., ) . dna sequencing of orf pcr products, amplified directly from the sera of infected dams, table frequency of t at nucleotide position of orf . consensus nucleotide at position of orf t:c ratio in cloned pcr products (percent) t / ( ) fetus - t / ( ) fetus - t / ( ) fetus - c / ( ) fetus - t / ( ) fetus - t / ( ) identified one dam, no. , which possessed a virus with a mutation within the hypervariable region of orf . the change detected by sequencing the pcr product was a c to t (u for rna) nucleotide transition at position that resulted in a non-conserved amino acid change from threonine to isoleucine in gp . the t- mutation was not detected after sequencing the orf pcr products from the other dams (see table ). pcr products were cloned into a ta plasmid and the individual plasmids sequenced. even though t- was detected in the pcr product from dam no. , the sequence of individual clones showed that two of the five sequences possessed a c at position mutation, which indicated that viruses with the wild-type orf sequence were still present in the population. whole pcr and plasmid-cloned pcr products were sequenced for the five infected fetuses from dam no. . the frequency of the t- mutation ranged from % (fetus - ) to % (fetuses - and - ). these results indicate that the fetus is capable of selecting for a particular virus population, which either arises in the dam or fetus. therefore, fetal infection is a potential source of prrsv diversity. during acute infection of the postnatal pig, the largest quantity of virus and greatest number of cells supporting virus replication are found in the lung, a consequence of targeting alveolar macrophages. during the later stages of prrsv infection, secondary lymphoid organs, including tonsil and lymph nodes, become sources of virus replication (allende et al., ; rossow, ; rowland et al., ; . virus replication in fetal tissues was assessed using a combination of virus isolation and ihc detection of nucleocapsid antigen in formalin-fixed tissues. as summarized in table , virus was isolated from all tissues from infected fetuses, including placenta, umbilical cord, heart, lung, spleen, lymph nodes and thymus. overall, the thymus contained the largest quantity of virus. nine of ten fetuses yielded measurable amounts of virus with five of the ten fetal thymuses producing titers greater than . . for lung, of fetuses were vi-positive and only one fetal lung yielded a virus titer greater than . . the recovery of virus from a tissue can represent virus in circulating blood. therefore, to determine if cells in the thymus and other tissues were a source of prrsv, tissue thin sections were stained with prrsv anti-nucleocapsid antibody. the ihc staining procedure included two sets of negative controls: tissue thin sections from non-infected fetuses and from infected fetuses stained with only secondary antibody. both controls were negative for staining (data not shown). the results in table showed the largest number of positive tissues for the thymus ( of positive), followed by spleen ( of positive) and lymph node ( of positive). within the thymus, antigen-positive cells were located in both medullar and cortical regions (data not shown). prrsv antigen-positive cells were not detected in lung or tonsil. taken together, the virus titration and ihc results identify the thymus as a principal source of virus replication in the prrsvinfected fetus. in the post-natal pig, prrsv infection results in distinct pathology in the lung, including the appearance of interstitial pneumonia. representative lymph node and lung tissues from non-infected and infected fetuses are shown in fig. . there was no discernable difference between lungs from infected and non-infected fetuses (compare fig. panels c and d) . lymph nodes from prrsv-negative fetuses appeared largely undeveloped and devoid of well-defined germinal centers. in contrast, the lymph nodes from prrsv-infected fetuses appeared much more pronounced and enlarged. at the microscopic level, the increased lymph node volume was associated with increased numbers of cells and the formation of distinct germinal centers (see fig. panels a and b) . the overall appearance is consistent with an antigen-activated lymph node. to determine the source of the increased cell volume, formalin-fixed thin sections of lymph nodes were stained with t cell-specific (cd ) and b cell-specific (cdw and cd ␣) antibodies. representative results for mandibular lymph nodes from infected and non-infected fetuses are shown in fig. . the lymph nodes from both infected and non-infected fetuses showed exten- sive areas of staining with anti-cd , indicating the presence of t cells. lymph nodes from non-infected fetuses were negative for cwd staining, but possessed some regions that were positive for cd ␣. the b cell receptor for antigen (bcr) signal transduction complex is composed of a heterodimer of ig␣ and ig␤ chains, which are also known as cd ␣ and cd ␤, respectively. cd ␣ staining in the lymph node from non-infected fetuses is consistent with the presence of pro-and pre-b cells (lee et al., ) . the principle difference between infected and non-infected fetuses was found in an overall increase in cd ␣+ cells as well as the appearance of cdw + cells, which were associated with germinal centers. cdw is beta-galactoside alpha- , -sialyltransferase, which is up-regulated in activated b cells (erikstein et al., ) . the absence of cdw staining in non-infected fetuses is consistent with the overall quiescent nature of the non-stimulated fetal immune system. the up-regulation of cdw after prrsv infection is consistent with b cell activation and the formation of mature germinal centers in response to infection. it should be noted that infected fetuses showed different degrees of staining, a likely consequence of the different stages of fetal infection; i.e. less staining was the result of early infection. together, the overall morphology and lymphocyte marker expression results indicate that the increased volume in the lymph nodes of infected fetuses is largely the result of increased numbers of mature activated b cells, which occupy germinal centers. immune cytokines are important factors in antiviral immunity and can influence the outcome of pregnancy (arck et al., ; basurko et al., ). the detection of cytokine gene expression in tissues was performed using a steady state rt-pcr procedure. rt-pcr was performed on rna isolated from four fetuses randomly chosen from the two mock-infected dams and four randomly selected prrsv-infected fetuses. the tissues selected for rt-pcr amplification were lung, lymph node and placenta. lung and lymph node represent sites cytokine alterations in the post-natal pig. placenta was selected as an accessory tissue located at the fetal maternal interface. amplification of mrnas included cytokines associated with inflammatory (il- , il- ), th (il- , ifn-␥, il- ) and th /regulatory (il- , il- ) responses. the amplification of ␤ m mrna was included as an internal control. the determination of a cytokine response was based on the presence or absence of a pcr product. il- , il- , il- and il- products were detected in tissues from both control and infected fetuses. because of the qualitative nature of the pcr method, it was not possible to accurately determine quantitative differences between control and infected fetuses; and therefore, these cytokines were not subjected to further study (data not shown). il- mrna was not detected in any of the selected tissues for control and infected fetuses. marked differences in expression were observed for tnf-␣ and ifn-␥ mrnas. the results for ifn-␥ and tnf-␣ from lung, mandibular lymph node and placenta are presented in fig. . the results for il- are also shown. the internal control mrna, ␤ m, was amplified from all tissues, indicating that the rna was intact. il- was amplified from lung and mandibular lymph nodes from two of the four control fetuses and from all infected fetuses. the presence of il- was not unexpected, since increased il- production is associated with fetal t cell responses (lin et al., ; rainsford and reen, ) . as shown in fig. a , ifn-␥ and tnf-␣ pcr products were not detected in lung, lymph node or placenta from the non-infected fetuses. however, ifn-␥ pcr products were obtained for lung and lymph nodes from infected fetuses. one infected fetus, - , yielded a faint ifn-␥ product for placenta. for infected fetuses, tnf-␣ mrna was amplified from lung, but not lymph node or placenta (fig. b) . to determine if cytokine gene expression was the direct result of prrsv infection, rt-pcr for ifn-␥ and tnf-␣ was performed on the same tissues from fetuses of infected dam no. , which produced only prrsv vi-negative fetuses (see fig. ). the analysis of mrna expression in lungs and lymph nodes from six fetuses from dam no. fig. . cytokine gene expression in fetal tissues. rt-pcr for cytokine mrnas was performed on lung (l), mandibular lymph node (n) and placenta (p) for control (panel a) and infected (panel b) fetuses as described in the text using the primers in table . amplification of ␤ -microglobulin mrna was used as an internal control. pcr products were electrophoresed on a . % agarose gel and stained with ethidium bromide. showed only the amplification of the ␤ m, but not tnf-␣ or ifn-␥ mrnas (data not shown). similar results were obtained from virusnegative fetuses located immediately adjacent to infected fetuses (data not shown). in order to confirm that tnf-␣ and ifn-␥ were produced during infection, cytokine protein levels were measured in fetal sera and amniotic fluid from infected fetuses and fetuses from mock-infected dams. as shown in fig. , the concentration of tnf-␣ in serum for prrsv-infected fetuses ranged between and pg/ml compared to less than pg/ml for control fetuses. even though the maximum quantities obtained for infected fetuses were near the lower limit of detection for the elisa test, it was clear that tnf-␣ was elevated during infection. detectable concentrations of tnf-␣ (> pg/ml) were found in amniotic fluid from only two control and two infected fetuses. compared to fetuses from mockinfected dams, ifn-␥ concentrations were detected in sera, with values ranging from a low of to more than pg/ml. a maximum level of pg/ml was obtained for a single non-infected fetus. ifn-␥ was not detected in amniotic fluid from either the infected or control fetuses. the presence of ifn-␥ and tnf-␣ in serum, but not amniotic fluid, further supports the notion that the ifn-␥ and tnf-␣ responses are primarily restricted to the prrsv-infected fetus and do not extend to the accessory tissue compartments. this study characterizes the unique biology associated with the interaction between prrsv and the late gestation fetus. consistent with the body of published literature, the fetuses from the infected dams obtained in this study exhibited several anatomic pathological features typical of prrsv infection of the fetus, including lesions associated with the accessory organs, such as umbilical cord and amniotic sac (lager and halbur, ; mengeling et al., ) . one interesting observation from this study was the apparent absence of a correlation between the presence of gross abnormalities and productive fetal infection. for example, several fetuses from dam no. exhibited several types of gross pathology, including death, growth retardation, or merconium/blood stained amniotic fluid. there were also examples of productively infected fetuses that showed no evidence of gross pathology (see fetuses - , - , - , - , - in fig. ). the mechanism for fetal pathology remains unclear, but the results suggest that the source pathology is likely result of the infection of tissues on the maternal side and damage to maternal tissues or production of maternal factors that affect the fetus. in this study we did not observe lesions in the myometrium or placenta; however, stockhofe-zurwieden et al. ( ) reported prrs virions budding from maternal vascular endothelial cells at the maternal-fetal interface. lager and halbur ( ) reported damage to the myometrium during prrsv infection. virus-associated lesions in the myometrium are observed for horses infected with eav (coignoul and cheville, ) . the appar-ent discrepancy between pathology and infection helps to explain the stealthy nature of the prrsv. a significant number of apparently healthy, but infected fetuses, are likely go on to become healthy growing pigs with the capacity to shed virus. rna viruses often exist as a population of closely related sequences, frequently referred to as a quasispecies. the appearance or disappearance of individual gene sequences in the population over the course of infection is used as evidence for changes in fitness that result from selection. prrsv variants with mutations in orf typically appear during the serial passage of virus in culture and during infection of pigs allende et al., ) . the significance of mutations in orf as a source for increased fitness during infection is not completely understood; but is useful for following the fate of individual prrsv subpopulations over the course of a long-term infection . in this study, one dam, no. , showed evidence of a mixed prrsv infection as indicated by the presence of two different orf sequences, which were distinguished from each other by a nucleotide substitution at position in orf . the c to t change was observed in approximately % of the cloned plasmid products obtained from dam no. (see table ). however, the same frequency was not transferred to the individual fetuses, which included two fetuses that possessed only viruses with the t- mutation and a single fetus with a virus population dominated by c at position . these results suggest that fetal infection can alter the selection of prrsv variants and may represent a source of prrsv genetic diversity. to identify the targets of virus replication in the fetus, a variety of tissues were assessed for the presence of virus and virus-infected cells. vi, as opposed to more sensitive approaches, such as pcr, was selected as the means for measuring virus, because the relative insensitivity of vi avoids the possibility of false positive pcr results that might result from small amounts of contaminating maternal material. within the group of selected tissues, virus could be isolated from all tissues. however, the most consistent source and largest quantity of virus were obtained from the thymus. collectively, these data identify the thymus as a primary site of virus replication in the prrsv-infected fetus and confirm an earlier observation for prrsv by benson et al. ( ) . the specific cell population in the thymus targeted prrsv replication remains unknown. the natural predisposition of maternal immunity towards th like responses aids in protecting the fetus by blocking cell-mediated th -related allorejection responses (arck et al., ; entrican, ; raghupathy, ; recently reviewed in challis et al., ) . the negative influence of th cytokines on fetal development can be demonstrated experimentally in mice, which show that a single injection of il- or ifn-␥ into certain strains induces fetal resorption (lala, ; chaouat et al., ) . tnf-␣, when infected into mice is abortifacient (clark et al., ) . th cytokines can also have important long-term impacts. for example, newborn mice that survive maternal infection with influenza virus exhibit behav-ior similar to hyperanxiety and autism. the behavioral changes are a consequence of the altered distribution of dopamine and glutamine receptors during fetal brain development. the effect of influenza virus infection on the fetal brain can be mimicked by the administration of poly i:c, an inducer of ifn (shi et al., ) . furthermore, the addition of ifn-␥ to hippocampal neuron cultures alters the distribution of glutamate receptors, sufficient to affect synaptic activity between neurons (vikman et al., ) . virus infections during pregnancy present an interesting paradox: those cytokines that protect the fetus from viral infection, tend to inhibit fetal development or potentiate rejection of the fetus; while those cytokines that maintain and promote fetal development are associated with the inhibition of antiviral immune responses. because of the potential negative impact of th cytokines on fetal outcome, there is a natural predisposition for the fetus to block the induction of th -associated cytokines. for example, the ifn-␥ response in the developing fetus can be blocked by a combination of factors, including ( ) defects in the capacity of fetal dendritic cells to express mhc class ii and synthesize il- , ( ) hypermethylation of the ifn-␥ gene in fetal t cells, ( ) the absence of target macrophages, and ( ) the presence of an immune environment dominated by th /regulatory cytokines (goriely et al., ; langrish et al., ; melvin et al., ; marodi et al., ; murphy et al., ; prescott et al., ; white et al., ,) . with this in mind, there are examples of viruses, such as epstein-barr virus (ebv), which are capable of stimulating antigen-specific ifn-␥ responses in human umbilical cord blood lymphocytes (ito et al., ; wilson and morgan, ) . these and other data provide a description of the fetus as capable of initiating a robust antiviral th immune response (chaouat et al., ; chipeta et al., ; murphy et al., ) . in this study ifn-␥ and tnf-␣ mrnas were identified in tissues from infected fetuses. rna message was not identified in tissues of fetuses from mock-infected dams or non-infected fetuses from infected dams. this indicates that altered gene expression is the direct result of fetal infection. up-regulated expression was confirmed by the presence of detectable levels of ifn-␥ and tnf-␣ proteins in serum. furthermore, the results indicate that inf-␣ and ifn-␥ cytokines response are compartmentalized within the fetus and do not extend to other compartments, such as amniotic fluid or placenta, thus lessening the probability of allorejection by the dam. from these data, it is apparent that the fetus is capable of initiating a th -like response; however, the capacity of ifn-␥ and tnf-␣ to control prrsv infection in the fetus is not known. additional evidence for induction of virus-specific immunity was found in the development of germinal centers containing activated (cdw +) b cells and seroconversion in at least one fetus. pro-inflammatory cytokines, such as tnf-␣ and ifn-␥ can contribute to pulmonary distress through the activation of alveolar macrophages and other cell populations. the increased quantities of ifn-␥ and tnf-␣ found in the lungs of prrsv-infected fetuses may not be sufficient to cause pulmonary damage to the fetal lung, primarily because fetal macrophages have a reduced capacity to respond to inflammatory stimuli. in addition, il- , up-regulated in response to prrsv infection, is a potent antagonist of ifn-␥ and tnf-␣ activation of macrophages (burchett et al., ; marodi et al., ; johnsen et al., ; thanawongnuwech et al., ) . however, within days after birth, adult macrophages, including mature alveolar and intravascular macrophages emerge into an environment already enriched in ifn-␥ and tnf-␣. the outcome is the rapid recruitment, activation, and cytopathic killing of large numbers of virus-permissive macrophages (choi and chae, ) . this scenario as a cause of severe interstitial pnuemonia in the prrsv-infected newborn requires further investigation, but has obvious implications in the etiology of postnatal pulmonary complications following virus infections of the fetus. porcine reproductive and respiratory 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introduction, chapter quantitative deep sequencing reveals dynamic hiv- escape and large population shifts during ccr antagonist therapy in vivo interferongamma-induced changes in synaptic activity and ampa receptor clustering in hippocampal cultures differential patterns of methylation of the ifn-gamma promoter at cpg and non-cpg sites underlie differences in ifn-gamma gene expression between human neonatal and adult cd ro-t cells primary immune responses by cord blood cd (+) t cells and nk cells inhibit epstein-barr virus b-cell transformation in vitro this work was partially supported by the usda national research initiative for competitive grants program grants # - - . key: cord- - me ugkg authors: wang, xiaona; li, fengsai; han, meijing; jia, shuo; wang, li; qiao, xinyuan; jiang, yanping; cui, wen; tang, lijie; li, yijing; xu, yi-gang title: cloning, prokaryotic soluble expression, and analysis of antiviral activity of two novel feline ifn-ω proteins date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: me ugkg cats are becoming more popular as household companions and pets, forming close relationships with humans. although feline viral diseases can pose serious health hazards to pet cats, commercialized preventative vaccines are lacking. interferons (ifns), especially type i ifns (ifn-α, ifn-β, and interferon omega (ifn-ω)), have been explored as effective therapeutic drugs against viral diseases in cats. nevertheless, there is limited knowledge regarding feline ifn-ω (feifn-ω), compared to ifn-α and ifn-β. in this study, we cloned the genes encoding feifn-ωa and feifn-ωb from cat spleen lymphocytes. homology and phylogenetic tree analysis revealed that these two genes belonged to new subtypes of feifn-ω. the recombinant feifn-ωa and feifn-ωb proteins were expressed in their soluble forms in escherichia coli, followed by purification. both proteins exhibited effective anti-vesicular stomatitis virus (vsv) activity in vero, f (feline kidney cell), madin–darby bovine kidney (mdbk), madin–darby canine kidney (mdck), and porcine kidney (pk- ) cells, showing broader cross-species antiviral activity than the intercat ifn antiviral drug. furthermore, the recombinant feifn-ωa and feifn-ωb proteins demonstrated antiviral activity against vsv, feline coronavirus (fcov), canine parvovirus (cpv), bovine viral diarrhea virus (bvdv), and porcine epidemic diarrhea virus (pedv), indicating better broad-spectrum antiviral activity than the intercat ifn. the two novel feifn-ω proteins (feifn-ωa and feifn-ωb) described in this study show promising potential to serve as effective therapeutic agents for treating viral infections in pet cats. as cats continue to become more popular as household companions and pets [ ] , a variety of viral infections pose a serious threat to felines, including a high incidence of feline leukemia virus (felv) [ ] , feline coronavirus (fcov) [ ] , feline immunodeficiency virus (fiv) [ ] , and feline panleukopenia virus (fpv) [ ] . currently, the only preventative vaccines and therapeutic drugs available for use in pet cats have limited effectiveness. nevertheless, interferons (ifns) play an increasingly complementary role in homology and phylogenetic tree analysis of the feline ifn-ω genes isolated in this study were analyzed using dnastar and mega software. in addition, the characteristics of the feifn-ω genes and proteins were analyzed by several online bioinformatics software programs: signal peptide cleavage sites were analyzed by the signalp . server at http://www.cbs.dtu.dk/services/signalp- . /; phosphorylation sites were analyzed by the netphos . server at http://www.cbs.dtu.dk/services/netphos/; n-glycosylation sites were analyzed by the netnglyc . server at http://www.cbs.dtu.dk/services/netnglyc/; o-glycosylation sites were analyzed by the yinoyang . server at http://www.cbs.dtu.dk/services/yinoyang/; subcellular localization was analyzed by the targetp . server at http://www.cbs.dtu.dk/services/targetp; transmembrane regions were analyzed by the tmhmm . server at http://www.cbs.dtu.dk/services/tmhmm/; antigen epitopes and hydrophobicity were analyzed by the bepipred . server at http://www.cbs.dtu. dk/services/bepipred- . /; and secondary and three-dimensional structures were predicted by sopma at https://npsa-prabi.ibcp.fr/cgi-bin/npsa. in this study, the genes encoding feifn-ω that were isolated by rt-pcr were subcloned as a kpni and bamhi-generated (new england biolabs, ma, usa) gene fragment into the prokaryotic soluble expression plasmid pcold-tf, giving rise to recombinant pcold-feifn-ω. after that, the recombinant plasmid was transformed into e. coli bl (de ) competent cells and validated by pcr and sequencing analyses, thus generating the recombinant e. coli strain pcold-feifn-ω/bl . we then used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) analysis to determine the optimal conditions for expression of the recombinant feifn-ω in pcold-feifn-ω/bl cells via induction by isopropyl β-d-thiogalactoside (iptg, sigma, st. louis, mo, usa). briefly, for optimization of the iptg concentration, the recombinant e. coli strain was grown in luria-bertani broth (sigma, st. louis, mo, usa) supplemented with µg/ml ampicillin at • c until the optical density at nm was approximately . . then, iptg was added at final concentrations of . mmol/l, . mmol/l, . mmol/l, . mmol/l, or . mmol/l, and the cultures were continually cultivated for viruses , , of another h. after centrifugation at , × g for min, the cell pellets were lysed and analyzed by % sds-page. to determine the optimal induction time, the recombinant strain was induced by the optimized final concentration of iptg for h, h, h, h, and h. following centrifugation and cells lysis, the proteins were again analyzed by % sds-page. the fusion protein expressed by the pcold-feifn-ω/bl bacteria cells was subjected to cleavage by the c protease (sigma, st. louis, mo, usa). the target recombinant feifn-ω protein was then purified using ni + affinity chromatography columns according to the manufacturer's instructions, followed by confirmation analysis using sds-page. the purified feifn-ω protein was stored at − • c until use. vsv was used as a virus model to evaluate the antiviral activity of the recombinant feifn-ω via the in vitro microdose cytopathic effect inhibition assay (mcia) according to the method described previously [ , ] with slight modifications. briefly, µl of the purified feifn-ω sample was serially diluted ten-fold in dmem containing % fbs and transferred to confluent f cell monolayers in -well cell culture plates, and then incubated at • c in % co for h. f cells without ifn treatment were used as a control group. after incubation, vsv (moi = ) was prepared as described above, added into the -well plate, and incubated for - h until the cpe of the cells in the viral control group reached %. next, the culture medium was removed, and the cells were stained with . % crystal violet in % ethanol at • c for min. the cells were then destained with . % acetic acid in % ethanol at • c for min before determining the absorbance of each well at nm. each sample was performed with eight biological replicates and three technical replicates. the antiviral activity of the ifn was calculated as the method previously described, which is expressed as units per milligram according to the ratio of ifn titer and protein concentration [ ] . in parallel, the intercat ifn antiviral drug (toray industries, tokyo, japan) was used as an ifn treatment control. in addition, we determined the species-specific antiviral activity of the recombinant feifn-ω by conducting mcias using vsv in f , vero, mdck, mdbk, and pk- cells. broad-spectrum antiviral activity of the recombinant feifn-ω was determined by conducting mcias using vsv, fcov, cpv, bvdv, and pedv. the results are shown as mean ± sem (n = ) for three independent experiments. statistical analyses were performed using graphpad prism v . software. tukey's multiple comparison tests and one-way analysis of variance (anova) tests were used to analyze the significance of the differences between groups: * p < . ; ** p < . ; and *** p < . . we first collected splenic lymphocytes from cats infected with vsv and treated with poly(i:c) simultaneously. we then performed rt-pcr assays using a pair of degenerate primers to identify two genes encoding feifn-ω, referred to as feifn-ωa and feifn-ωb ( figure a) . these two novel genes were deposited in the genbank with accession numbers mk and mk . following nucleotide sequence homology analysis, we found that the feifn-ωa gene (mk ), with a size of bp, shared a maximum nucleotide sequence homology of . % ( . % amino acid homology) with the known subtypes of feifn-ω (feifn-ω to feifn-ω ) genes published in the genbank (figure c ). the feifn-ωb gene (mk ), with a size of bp, shared a maximum sequence homology of . % ( . % amino acid homology) with the known subtypes of feifn-ω ( figure d ). the sequence homology shared between the feifn-ωa and feifn-ωb genes was only . % (figure b) , indicating that the two genes identified in this study were novel feifn-ω genes. we constructed a phylogenetic tree comparing the feifn-ωa/ωb genes identified in this study with other ifn gene sequences published in genbank from different animals using the software dnastar (megalign) and mega . the results showed that the feifn-ωa/ωb genes belonged in the type i ifn family, but that their evolutionary relationship to the known feline ifn-ω genes already published in the genbank (figure ) was distant, indicating that the feifn-ωa and feifn-ωb genes, identified for the first time here, are new subtypes of feline ifn-ω. viruses , , x for peer review of we constructed a phylogenetic tree comparing the feifn-ωa/ωb genes identified in this study with other ifn gene sequences published in genbank from different animals using the software dnastar (megalign) and mega . the results showed that the feifn-ωa/ωb genes belonged in the type i ifn family, but that their evolutionary relationship to the known feline ifn-ω genes already published in the genbank (figure ) was distant, indicating that the feifn-ωa and feifn-ωb genes, identified for the first time here, are new subtypes of feline ifn-ω. the characteristics of the novel feline ifn-ω proteins (feifn-ωa and feifn-ωb) were analyzed using several online bioinformatics software programs, including the identification of potential signal peptide cleavage sites, n-glycosylation sites, o-glycosylation sites, phosphorylation sites, subcellular localization, and transmembrane regions. the results from these detailed analyses are displayed in table . in addition, we also used online software algorithms to predict antigen epitopes, hydrophobicity, and the secondary and three-dimensional structures of the feifn-ωa and feifn-ωb proteins. as shown in figure figure b ). the maximum hydrophobicity of feifn-ωa was . , and the minimum hydrophobicity was − . . the maximum hydrophobicity of feifn-ωb was . , and the minimum hydrophobicity was − . . the secondary structure of the two feifn-ω proteins was predicted using sopma software and revealed that feifn-ωa contained . % alpha helix, . % beta sheet, and . % irregular curl structures (figure c ), whereas feifn-ωb contained . % alpha helix, . % beta sheet, and . % irregular curl structures ( figure d ). the three-dimensional structures of feifn-ωa and feifn-ωb were predicted with swiss-model software and are shown in figure e ,f, respectively. ( ) . % extracellular . % intracellular % mitochondrion intracellular a signal peptide cleavage sites were analyzed by signalp . server at http://www.cbs.dtu.dk/services/ signalp- . /; b phosphorylation sites were analyzed by netphos . server at http://www.cbs.dtu.dk/services/ netphos/; c n-glycosylation sites were analyzed by netnglyc . at http://www.cbs.dtu.dk/services/netnglyc/ and o-glycosylation sites were analyzed by yino yang . at http://www.cbs.dtu.dk/services/yinoyang/; d subcellular localization was analyzed by targetp . server at http://www.cbs.dtu.dk/services/targetp; e transmembrane region was analyzed by tmhmm server v. . at http://www.cbs.dtu.dk/services/tmhmm/. the genes encoding feifn-ωa and feifn-ωb were subcloned into the prokaryotic soluble expression vector pcold-tf and then transformed into e. coli bl competent cells, thus generating the recombinant protein expressing e. coli strains pcold-feifn-ωa/bl and pcold-feifn-ωb/bl . after inducing their expression with iptg, the rfeifn-ωa ( figure a ) and rfeifn-ωb (figure b ) proteins fused with the trigger factor (tf) of approximately kda and were primarily expressed in their soluble forms. subsequently, we optimized the expression conditions of the rfeifn-ω proteins from the pcold-feifn-ωa/bl and pcold-feifn-ωb/bl e. coli strains. our observations indicated that the optimal induction conditions for rfeifn-ωa protein expression were an iptg concentration of . mmol/l ( figure c ) and an induction time of h (figure d) , and the optimal induction conditions for rfeifn-ωb protein expression were an iptg concentration of . mmol/l ( figure e ) and an induction time of h (figure f ). after that, the fused proteins (rfeifn-ωa/ωb+tf) were purified by his-tag ni + affinity column chromatography and subjected to cleavage using the c protease. the cleaved proteins were then purified by his-tag ni + affinity column chromatography once again, after which we collected the purified recombinant rfeifn-ωa and rfeifn-ωb proteins with molecular weights of approximately kda and kda, respectively ( figure g ). viruses , , x for peer review of the genes encoding feifn-ωa and feifn-ωb were subcloned into the prokaryotic soluble expression vector pcold-tf and then transformed into e. coli bl competent cells, thus generating the recombinant protein expressing e. coli strains pcold-feifn-ωa/bl and pcold-feifn-ωb/bl . after inducing their expression with iptg, the rfeifn-ωa ( figure a ) and rfeifn-ωb (figure b ) proteins fused with the trigger factor (tf) of approximately kda and were primarily expressed in their soluble forms. subsequently, we optimized the expression conditions of the rfeifn-ω proteins from the pcold-feifn-ωa/bl and pcold-feifn-ωb/bl e. coli strains. our observations indicated that the optimal induction conditions for rfeifn-ωa protein expression were an iptg concentration of . mmol/l (figure c ) and an induction time of h (figure d) , and the optimal induction conditions for rfeifn-ωb protein expression were an iptg concentration of . mmol/l (figure e ) and an induction time of h (figure f ). after that, the fused proteins (rfeifn-ωa/ωb+tf) were purified by his-tag ni + affinity column chromatography and subjected to cleavage using the c protease. the cleaved proteins were then purified by his-tag ni + affinity column chromatography once again, after which we collected the purified recombinant rfeifn-ωa and rfeifn-ωb proteins with molecular weights of approximately kda and kda, respectively (figure g ). we determined the antiviral activity of rfeifn-ωa and rfeifn-ωb using microdose cytopathic effect inhibition assays (mcias) and vsv as the viral model that was propagated on f cells. as shown in figure , both rfeifn-ωa and rfeifn-ωb demonstrated good antiviral activity similar to the intercat ifn (feline ifn antiviral drug) positive control. however, no antiviral effect in negative control groups (cells untreated with the ifns) was detected. viruses , , x for peer review of the black arrow represents the target fusion protein and red arrowhead represents the target fusion protein expressed under the optimized condition. we determined the antiviral activity of rfeifn-ωa and rfeifn-ωb using microdose cytopathic effect inhibition assays (mcias) and vsv as the viral model that was propagated on f cells. as shown in figure , both rfeifn-ωa and rfeifn-ωb demonstrated good antiviral activity similar to the intercat ifn (feline ifn antiviral drug) positive control. however, no antiviral effect in negative control groups (cells untreated with the ifns) was detected. we then tested whether the antiviral activities of rfeifn-ωa and rfeifn-ωb were species-specific. to address this question, we performed mcias with vsv propagated in several different cell lines from different animal species, including f cells (cat), vero cells (monkey), mdck cells (dog), mdbk cells (cattle), and pk- cells (pig). as shown in figure , the purified recombinant feifn-ωa and feifn-ωb proteins showed antiviral activity in both homologous animal cells (f cells) and heterologous animal cells (vero, mdck, mdbk, and pk- cells) in vitro. the antiviral activity of the rfeifn-ω proteins in heterologous animal cells was significantly stronger than that of the intercat ifn control. although the intercat ifn displayed high antiviral activity in f and vero cells, it had significantly weak antiviral activity in mdck and mdbk cells, and no antiviral activity in pk- cells. furthermore, the antiviral activity of rfeifn-ωb was better than that of rfeifn-ωa in homologous and heterologous animal cells, but especially in homologous animal cells that originated from cats. however, no antiviral activity was detected in cells' control groups (cells untreated with the ifns). we then tested whether the antiviral activities of rfeifn-ωa and rfeifn-ωb were species-specific. to address this question, we performed mcias with vsv propagated in several different cell lines from different animal species, including f cells (cat), vero cells (monkey), mdck cells (dog), mdbk cells (cattle), and pk- cells (pig). as shown in figure , the purified recombinant feifn-ωa and feifn-ωb proteins showed antiviral activity in both homologous animal cells (f cells) and heterologous animal cells (vero, mdck, mdbk, and pk- cells) in vitro. the antiviral activity of the rfeifn-ω proteins in heterologous animal cells was significantly stronger than that of the intercat ifn control. although the intercat ifn displayed high antiviral activity in f and vero cells, it had significantly weak antiviral activity in mdck and mdbk cells, and no antiviral activity in pk- cells. furthermore, the antiviral activity of rfeifn-ωb was better than that of rfeifn-ωa in homologous and heterologous animal cells, but especially in homologous animal cells that originated from cats. however, no antiviral activity was detected in cells' control groups (cells untreated with the ifns). next, we wanted to investigate whether the antiviral activities of the recombinant feifn-ωa and feifn-ωb proteins were also broad-spectrum. we performed mcias using a wide range of different viruses, including vsv, fcov, cpv, bvdv, and pedv. as shown in figure , rfeifn-ωa and rfeifn-ωb both displayed in vitro antiviral activity against vsv, fcov, cpv, bvdv, and pedv. their antiviral activities were especially strong against fcov and vsv. in contrast, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of rfeifn-ωa. no notable differences were observed between rfeifn-ωa and rfeifn-ωb with cpv, bvdv, or pedv. in addition, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of intercat ifn, and the antiviral activities of both rfeifn-ωa and rfeifn-ωb against cpv, bvdv, and pedv were significantly higher than that of intercat ifn. in fact, we observed no antiviral activity against bvdv or pedv by intercat ifn. unsurprisingly, there was no antiviral activity detected in cells' control groups (cells untreated with the ifns). figure . the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; ** p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the figure . the species-specific antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the intercat ifn group. comparison of the rfeifn-ωb group and rfeifn-ωa group also indicated significant differences (# p < . ). next, we wanted to investigate whether the antiviral activities of the recombinant feifn-ωa and feifn-ωb proteins were also broad-spectrum. we performed mcias using a wide range of different viruses, including vsv, fcov, cpv, bvdv, and pedv. as shown in figure , rfeifn-ωa and rfeifn-ωb both displayed in vitro antiviral activity against vsv, fcov, cpv, bvdv, and pedv. their antiviral activities were especially strong against fcov and vsv. in contrast, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of rfeifn-ωa. no notable differences were observed between rfeifn-ωa and rfeifn-ωb with cpv, bvdv, or pedv. in addition, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of intercat ifn, and the antiviral activities of both rfeifn-ωa and rfeifn-ωb against cpv, bvdv, and pedv were significantly higher than that of intercat ifn. in fact, we observed no antiviral activity against bvdv or pedv by intercat ifn. unsurprisingly, there was no antiviral activity detected in cells' control groups (cells untreated with the ifns). viruses , , x for peer review of figure . the species-specific antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the intercat ifn group. comparison of the rfeifn-ωb group and rfeifn-ωa group also indicated significant differences (# p < . ). next, we wanted to investigate whether the antiviral activities of the recombinant feifn-ωa and feifn-ωb proteins were also broad-spectrum. we performed mcias using a wide range of different viruses, including vsv, fcov, cpv, bvdv, and pedv. as shown in figure , rfeifn-ωa and rfeifn-ωb both displayed in vitro antiviral activity against vsv, fcov, cpv, bvdv, and pedv. their antiviral activities were especially strong against fcov and vsv. in contrast, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of rfeifn-ωa. no notable differences were observed between rfeifn-ωa and rfeifn-ωb with cpv, bvdv, or pedv. in addition, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of intercat ifn, and the antiviral activities of both rfeifn-ωa and rfeifn-ωb against cpv, bvdv, and pedv were significantly higher than that of intercat ifn. in fact, we observed no antiviral activity against bvdv or pedv by intercat ifn. unsurprisingly, there was no antiviral activity detected in cells' control groups (cells untreated with the ifns). figure . the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; ** p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the figure . the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < . ; ** p < . ; *** p < . ) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the intercat ifn group. a comparison of the rfeifn-ωb group and rfeifn-ωa group also indicated significant differences (# p < . ). currently, cats are continuing to become more and more common as household pets. we know of a wide variety of viral infections that can seriously endanger the health of pet cats, such as felv, fiv, fipv, fcov, and feline calicivirus (fcv). interferon represents a promising potential therapeutic agent that can effectively treat pet cats infected with these viruses. in this study, we identified two new genes encoding feline ifn-ω (feifn-ωa and feifn-ωb) in the spleen lymphocytes of cats. following sequence homology analysis, we found that feifn-ωa and feifn-ωb shared maximum nucleotide sequence homologies of . % and . %, and maximum amino acid homologies of . % and . % with the previously published subtypes of feline ifn-ω, respectively. in addition, phylogenetic tree analysis of ifns in cats and other species constructed using neighbor-joining analysis revealed that feifn-ωa and feifn-ωb do belong to the type i ifn family, but their evolutionary relationship to known feline ifn-ω genes was distant, indicating that feifn-ωa and feifn-ωb were indeed new subtypes of feline ifn-ω. we deposited these two genes into the genbank with the accession numbers mk (feifn-ωa) and mk (feifn-ωb), thus enriching the ifn-ω data submitted to the genbank [ ] . in this study, the characteristics of the feifn-ωa and feifn-ωb proteins, such as signal peptide sequences, signal peptide cleavage sites, phosphorylation sites, glycosylation sites, antigen epitopes, hydrophobicity, and transmembrane regions were analyzed using bioinformatics to provide better theoretical guidance for the functional study of these proteins. the mature proteins, with normal biological activity, were formed only after the signal peptide sequence was removed from the precursor protein, thus allowing them to be secreted outside the cell membrane [ , ] . we used online software to predict that the signal peptide sequence of the feifn-ωa/ωb proteins consists of amino acid residues, and that the signal peptide cleavage site is located between residues gly and cys . the results indicated that the recombinant feifn-ωa and feifn-ωb proteins could be expressed in vitro in their soluble forms. glycosylation is an important post-translational modification process that can affect the antigenic determinants, charge properties, enzymatic properties, and thermal stability of proteins. similar to previous reports for the known feifn-ω subtypes [ ] , we observed no n-glycosylation sites present in feifn-ωa or feifn-ωb. however, our analyses predicted nine potential o-glycosylation sites in feifn-ωa and six potential o-glycosylation sites in feifn-ωb, which is different from previous reports on the other known ifn-ω subtypes [ ] . studies have shown that glycosylation sites can play an important role in determining the activity of ifns [ , ] . for example, glycosylated ifn-ω has been observed to be markedly more potent than non-glycosylated ifn-ω against hepatitis c virus, bvdv, yellow fever virus, and west nile virus, with even more superior effects than ifn-α, ifn-β, and ifn-γ [ , ] . insect/baculovirus expression systems are one of the most effective eukaryotic expression systems for preparing feline ifns. these expression systems are capable of making post-translational modifications, such as glycosylation, which allows proteins to fold correctly, thus producing highly active and stable ifns [ ] [ ] [ ] [ ] [ ] . however, the application of these systems is limited by drawbacks, such as low yield of ifn protein and complicated operation requirements [ ] . therefore, e. coli expression systems are still the most widely used prokaryotic expression system for protein production with their characteristics of simple procedures, large-scale production, and low cost [ ] [ ] [ ] . generally, recombinant ifn is produced by e. coli expression systems in an insoluble inclusion body form that has not been modified or folded correctly, resulting in the loss of its biological activity [ , ] . to obtain a soluble and biologically active ifn protein, the inclusion bodies need to be denatured and then renatured, which is a time-consuming, laborious process [ ] . in this study, we selected the prokaryotic soluble expression system pcold-tf to prepare recombinant feifn-ωa and feifn-ωb. the pcold-tf system is a highly efficient soluble expression system with a his-tagged tf that allows the expressed protein to be effectively modified, folded, and secreted into the cytoplasm [ , ] . following the construction of recombinant e. coli strains and induction by iptg, the rfeifn-ωa/ωb proteins were expressed in their soluble forms and analyzed via sds-page. we optimized expression conditions for the rfeifn-ωa and rfeifn-ωb proteins and purified them using his-tag ni + affinity column chromatography. both proteins showed antiviral activity against vsv in microdose cytopathic effect inhibition assays using f cells. our results provide a basis for further studies into the development of rfeifn-ωa and rfeifn-ωb as therapeutic agents for viral infections. meanwhile, our data provide evidence that the pcold-tf expression system can be used to produce ifn with full bioactivity. recombinant feline interferon-ω (rfeifn-ω) was the first licensed immunomodulator for use in treating viral infections in cats, especially fiv and felv infections [ , , ] . furthermore, rfeifn-ω also exhibits therapeutic effects against other feline viral infections, such as fcv, feline parvovirus, and feline herpesvirus- [ ] , as well as viruses that originate in other animals, such as foot-and-mouth disease virus, influenza virus, bvdv, vsv, prv, and rotavirus [ , , ] . in this study, our results demonstrated that both rfeifn-ωa and rfeifn-ωb had antiviral activity in homologous animal cells (f cells, cat) and heterologous animal cells (vero cells, monkey; mdbk cells, cattle; mdck cells, dog; pk- cells, pig), indicating that rfeifn-ωa and rfeifn-ωb have broad cross-species antiviral activity in vitro, similar to previously published findings in mdbk and mdck cells [ ] . in contrast, the antiviral activities of rfeifn-ωb in homologous and heterologous animal cells were better than that of rfeifn-ωa. intriguingly, the rfeifn-ωa and rfeifn-ωb proteins exhibited antiviral activity in mdck cells, despite the absence of ifn-ω in canines [ , ] . we speculate that different ifns with different physiological functions are likely responsible for the difference of results obtained in our study compared to other published studies. furthermore, analysis of the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb revealed that they were effective against vsv, fcov, pedv, bvdv, and cpv. out of those five viruses, antiviral activity was strongest against vsv and fcov. no significant differences in antiviral activity against cpv, bvdv, or pedv were observed between rfeifn-ωa and rfeifn-ωb. however, the overall broad-spectrum antiviral activity of rfeifn-ωb was significantly stronger than that of rfeifn-ωa. recently, combination antiviral therapy has become a common practice in treating feline viral infections due to pharmacokinetics and the short half-life of ifn alone [ , ] . there are many published studies focused on the combination of ifn-ω with other therapeutic agents, such as chemotherapeutic agents [ ] , ifn-α [ ] , and ribavirin [ ] , suggesting this is an attractive strategy to use against viral infections. we further evaluated the antiviral effects of rfeifn-ωa and rfeifn-ωb combined with feline il- against vsv and fcov in f cells. our results revealed that the in vitro antiviral activity of combination therapy was significantly increased compared to that of rfeifn-ωa or rfeifn-ωb alone, indicating that rfeifn-ω combination therapy may represent a more potent option than monotherapy for treating viral infections in cats; however, this hypothesis requires further exploration in vivo. in summary, the rfeifn-ωa and rfeifn-ωb obtained in this study exhibit significant broad-spectrum antiviral activity in both homologous and heterologous cells in vitro, particularly rfeifn-ωb, suggesting a promising candidate for the development of an effective therapeutic agent against viral infections in cats and other animals. in this study, two new subtypes of feline ifn-ω (ωa and ωb) were identified and characterized. they shared a maximum nucleotide sequence homology of . % and . % and a maximum amino acid homology of . % and . % with the previously known subtypes of feifn-ω, respectively. we analyzed the characteristics of feifn-ωa and feifn-ωb in detail using bioinformatics followed by soluble expression and optimization of induction conditions in e. coli. our data showed that purified recombinant feifn-ωa and feifn-ωb had broad-spectrum antiviral activity in homologous and heterologous animal cells, suggesting they are candidates for the development of effective therapeutic agents to be used against viral infections in pet cats. and, our research is underway to systematically evaluate the effectiveness of the two novel feifns as therapeutics agent for cat viral infections in vivo. in addition, the reported feifn-ω sequences will enrich the ifn data submitted to genbank. epidemiology of naturally occurring feline urologic syndrome feline leukemia virus infection: importance and current situation in switzerland effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis the protective rate of the feline immunodeficiency virus vaccine: an australian field study cell cycle s phase markers are expressed in cerebral neuron nuclei of cats infected by the feline panleukopenia virus interferon-omega: current status in clinical applications cloning, expression and antiviral bioactivity of 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interferon-omega for treatment of cats with acute upper respiratory viral disease constitutive and trophoblast-specific expression of a class of bovine interferon genes characterization and antivirus activities of a novel bovine ifn-omega safety pharmacology, toxicology and pharmacokinetic assessment of recombinant human omega-interferon produced from cho-ss cells a comparison of the antiproliferative properties of recombinant human ifn-alpha and ifn-omega in human bone marrow culture effect of recombinant feline interferon-ω alone and in combination with chemotherapeutic agents on putative tumour-initiating cells and daughter cells derived from canine and feline mammary tumours liposomal plasmid dna encoding human thymosin α and interferon ω potently inhibits liver tumor growth in icr mice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- - qemb x authors: li, li; qiao, dan; fu, xiaoying; lao, suihua; zhang, xianlan; wu, changyou title: identification of m.tuberculosis-specific th cells expressing cd generated in vivo in pleural fluid cells from patients with tuberculous pleurisy date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: qemb x th cell-mediated immune responses at the site of active infection are important to restrict the growth of m.tuberculosis (mtb) and for the spontaneous resolution of patients with tuberculous pleurisy (tbp). in the present study, we found that without any stimulation, cd (+) t cells in pleural fluid cells (pfcs) from patients with tbp expressed significantly higher levels of cd than pbmcs from patients with tuberculosis (tb) or healthy donors. cd (+)cd (+) t cells expressed t-bet and il- rβ . after stimulation with mtb-specific antigens, cd (+)cd (+) t cells expressed significantly higher levels of ifn-γ, il- and tnf-α than cd (+)cd (−) t cells, demonstrating that cd (+)cd (+) t cells were mtb-specific th cells. in addition, cd (+)cd (+) t cells were mostly polyfunctional th cells that simultaneously produced ifn-γ, il- , tnf-α and displayed an effector or effector memory phenotype (cd ra(−)ccr (−)cd l(−)cd (−)). moreover, the percentages of cd (+)cd (+) t cells were significantly and positively correlated with polyfunctional t cells. interestingly, sorted cd (+)cd (+) but not cd (+)cd (−) fractions by flow cytometry produced ifn-γ, il- and tnf-α that were significantly regulated by cd (+)cd (+) treg cells. taken together, based on the expression of cd , we found a direct quantitative and qualitative method to detect and evaluate the in vivo generated mtb-specific polyfunctional cd (+) t cells in pfcs from patients with tbp. this method can be used for the potential diagnosis and enrichment or isolation of mtb-specific th cells in the investigations. tuberculous pleurisy (tbp) is characterized by an intense chronic accumulation of inflammatory cells at the disease site, including the activation of th cells and their preferential recruitment to the affected areas [ , ] . however, tbp remains difficult to identify despite numerous diagnostic tools [ ] . currently, the definite diagnosis of tuberculosis (tb) pleural effusions depends on the demonstration of acid-fast bacilli in the sputum, pleural fluid, or pleural biopsy specimens [ ] . moreover, high levels of ifn-c are detected in tuberculosis pleural fluid [ , ] . therefore, measurement of ifn-c in the pleural fluid has also gained wide acceptance in the diagnosis of tb pleural effusions [ ] . however, these assays did not provide information regarding antigen-responsive cells. an abundance of immunocompetent cells in the pleural effusion provides investigators with a good model to study the correlates of a protective immune response at the site of infection. the enrichment of th cells in pleural fluid has been previously documented [ ] . however, detailed phenotypic and functional characterizations for these cells are still unknown. the bcg vaccine is an attenuated strain of mycobacterium bovis and includes loss of the esx locus, which encodes two family members, culture filtrate protein of kda (cfp- ) and early secretory antigenic target (esat- ). these antigens have an important diagnostic role in distinguishing bcg vaccination from mtb infection [ ] [ ] [ ] [ ] [ ] . although esat- and cfp- contain numerous potential t cell epitopes, the immune response during infection is often focused toward a few immunodominant epitopes. in the present study, we selected the six most dominant peptides identified in esat- and cfp- , as reported to be frequently recognized by subjects in indian, european, cambodian, and middle eastern cohorts with both latent and active tb [ ] [ ] [ ] [ ] [ ] [ ] . it has already been demonstrated that t-lymphocytes in the lungs, both in normal individuals and in those with granulomatous disease, are almost entirely cd ro + memory t cells and express both early-and late-activation markers, such as cd and cd respectively [ ] . previous data have demonstrated that almost all of the ccr + and ccr + cd + t cells recruited to the lungs of patients with active tb express the memory t-cell phenotype and that the majority may have been recently activated [ ] . cd is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates probably suggests that it plays a role in the pathogenesis of inflammatory diseases [ ] . previous studies have also demonstrated the usefulness of the determination of cd on cd + t cells after in vitro stimulation with tuberculin as a rapid indicator of immune sensitization against mtb [ , ] . however, these studies did not directly assess the function of cd + cells. the present study was initially designed to analyze the discrepancy of phenotypes among pleural fluid cells (pfcs) from patients with tbp, pbmcs from tb patients and normal donors. we found a significant increase in cd expression on cd + t cells in pfcs. importantly, cd + cd + cells were mostly th cells specific for esat- and cfp- peptides. the phenotypic and functional analysis of cd -expressing cells strongly suggested that cd could be a useful marker for the identification or enrichment of antigen specific th cells at local sites following mtb infection. cd may be useful for diagnostic procedures, including specific immune monitoring during tb infection or after vaccination, as well as for therapeutic applications such as targeted adoptive th cell therapies. significantly higher expression of cd on cd + t cells in pfcs from patients with tbp than in pbmcs from tb patients and healthy donors without any stimulation our initial experiments assessed the surface expression of cd on cd + t cells from pfcs of patients with tbp and pbmcs from tb patients (ptb) and healthy donors (hd). as expected, without any stimulation, a significantly higher level of cd expression was observed on cd + t cells from pfcs of tbp patients than on pbmcs from either ptb or hd ( fig. a and b, p, . ). moreover, higher levels of cd expression ( . % . %, mean sd) on cd + t cells were observed in % of the pfcs from individuals with tbp than on the pbmcs of ptb or hd ( . % . % for healthy donors and . % . % for tb patients, mean sd). no significant difference in cd expression was observed on pbmcs from ptb and from hd ( fig b, p = ns) . in addition, we also demonstrated that cd + cd + t cells expressed significantly higher levels of hla-dr than cd + cd t cells, a molecule which is usually induced on cd + t cells after activation (fig c and d , p, . ). unexpectedly, no significant difference was observed in the expression of cd , another activation marker, which is usually temporally expressed on activated t cells or constitutively expressed on regulatory t cells (fig c and d , p = ns). significantly higher expression of t-bet and il- rb by cd + cd + t cells than by cd + cd t cells in order to confirm whether cd + cd + t cells in pfcs were th cells, we further evaluated the expression of t-bet, an important transcription factor specific for th cells. the results showed that more than % of cd + cd + t cells and about . % of cd + cd t cells expressed t-bet (fig a) . the expression of t-bet on cd + cd + t cells was significantly higher than that on cd + cd t cells (fig b, p, . ). we next examined the expression of il- rb , another marker highly specific for differentiated th cells. similar to the results obtained for t-bet expression, the majority of cd + cd + t cells constitutively expressed il- rb ( fig. a) , and the expression was significantly higher than that observed on cd + cd t cells (fig c, p, . ), further confirming that the cd + cd + t cell subset was highly enriched for th cells. esat- and cfp- peptides-specific th cells were preferentially enriched within cd + cd + t cells because cd + cd + t cells were predominantly th cells, we aimed to examine whether direct ex vivo evaluation of surface expression of cd without in vitro upregulation permits direct assessment of mtb-specific th cells. our initial experiments indicated that stimulation of pfcs with esat- and cfp- peptides resulted in increased surface cd expression that is difficult to elucidate (data not shown). short-term stimulation of these cells includes incubation with secretion inhibitory brefeldin a (bfa), which can block the transport of newly synthesized cd molecules to the cell surface and thus allows for the direct assessment of the relationship between in vivo cd expression and cytokine production. after culturing pfcs with esat- / cfp- peptides and bfa, we analyzed the production of th cytokines by intracellular cytokine staining and polychromatic flow cytometry. as shown in fig a and b , cd + cd + t cells produced greater amounts of ifn-c, il- and tnf-a than did cd + cd t cells. statistical results demonstrated that the percentages of ifn-c, il- or tnf-a-producing cd + cd + t cells were significantly higher than those for cd + cd t cells (p, . , fig c) . in order to further confirm that the responses of t cells were specific for mtb, we also stimulated pfcs from tuberculous pleurisy patients with other irrelevant antigens such as sars-cov s peptides (stffstfkcygvsatkl) and (nfsqilpdplkptk rsfi) as well as hepatitis b virus surface antigen (hbsag). we found that these irrelevant antigens could not induce the production of ifn-c, il- and tnf-a by neither cd + cd + nor cd + cd t cells. however, following stimulation with mtb-specific peptides of esat- /cfp- , cd + cd + t cells from the same patients expressed high levels of ifn-c, il- and tnf-a (data not shown). it has been suggested that t cells simultaneously producing ifn-c, il- and tnf-a are associated with protective immunity and concomitant beneficial outcomes, at least in chronic viral infections such as hiv. we therefore compared the expression of ifn-c, il- and tnf-a in cd + cd + , cd + cd and total cd + t cells after short-term in vitro stimulation with esat- / cfp- peptides. we showed that cd + cd + and cd + cd t cells concurrently and individually produced ifn-c, il- and tnf-a (fig a, b ). this assay also identified cells that expressed cd but lacked expression of any cytokine measured (as evidenced by a subset of cd + cd + ifn-c il- tnf-a cells). these cells probably contained other effector functions, for example, the secretion of mip- b or other cytokines. the total antigen-specific responses were defined as the percentages of t cells expressing any combination of ifn-c, il- or tnf-a, and thus, seven distinct functional populations could be delineated (ifn-c/il- /tnf-a triple expressers; ifn-c/il- , ifn-c/tnf-a or tnf-a/il- double expressers; or ifn-c, il- or tnf-a single expressers). interestingly, polyfunctional t cells (ifn-c/il- / tnf-a triple expressers) dominated the cd + cd + t cell response. however, frequencies of t cells producing only tnf-a were the highest when analyzing the distribution of the seven subsets within either the cd + cd population or all cd + t cells (fig c, d) . the percentages of double expressers were similar within cd + cd + , cd + cd and total cd + t cells. our results demonstrated that total mtb-specific cd + t cells were not dominated by polyfunctional t cells. however, polyfunctional t cells were primarily enriched within cd + cd + t cells. we further analyzed the correlation between the percentages of cd -expressing cd + t cells and polyfunctional t cells. interestingly, significant positive correlation between cd + cd + t cells and polyfunctional t cells was observed ( fig e, cd + cd + t cells were mostly effector memory t cells we next investigated the phenotype of cd + cd + t cells relating to naïve/memory markers. as shown in fig a, cd + cd + t cells were predominantly cd ro + cd ra , thereby exhibiting a memory cell phenotype. in contrast, cd + cd t cells contained significantly higher percentages of cd ra + and lower percentages of cd ro + cells than did cd + cd + t cells. moreover, we also examined the expression of cd and cd and found that cd + cd + t cells had significantly lower expression of both markers ( fig b) . to further validate whether mtb-specific cd + cd + t cells were memory cells, we tested the phenotype of ifn-c-secreting cd + cd + t cells by assessing typical memory t cell markers. as expected, the majority of cd + cd + ifn-c + t cells displayed a cd ra ccr cd l cd effector memory cell phenotype (fig c) , suggesting that these cells were capable of producing multiple cytokines quickly at local sites. we further investigated whether purified cd + cd + t cells, stained by fluorescently labeled monoclonal antibody and sorted by flow cytometry, would be compatible with assays that required live cells. we first purified cd + t cells and cd + cells by positive selection with microbeads. thereafter, cd + cd + and cd + cd t cell fractions were obtained by flow cytometrybased cell sorting (fig a) . we then cocultured cd + cd + or cd + cd t cells with cd + cells (as antigen-presenting cells) in the presence of esat- /cfp- peptides (p -p ) and measured the production of th cytokines. notably, considerable levels of ifn-c and il- were produced by cd + cd + t cells, whereas little was observed from the cd + cd fractions. however, tnf-a production was readily detected in unstimulated cells, and significantly higher tnf-a was measured following stimulation of the cd + cd + fractions ( fig b) . moreover, significantly higher levels of ifn-c, il- and tnf-a were produced by cd + cd + cells than by cd + cd t cells (p, . , fig b) . we also assessed percentages of th cytokineproducing cells by flow cytometry. as expected, cd + cd + t cells were highly enriched for ifn-c, il- and tnf-a production compared with cd + cd cells (fig c-e) . thus, cd expression provides a means to identify the vast majority of antigen-specific cd + t cells, regardless of the type of cytokine measured. importantly, our assay conditions based on the cd coculture assay retained the ability of cells to secrete multiple cytokines. cd + cd + t cells inhibit production of th cytokines by cd + cd + t cells in order to examine whether cd + cd + treg cells could directly inhibit cytokine production by cd + cd + t cells, we sorted cd + cd + and cd + cd + t cells by flow cytometry (fig a) . in order to further confirm that cd + cd + t cells were regulatory t cells, we found that the majority of cd + cd + t cells (. %) expressed foxp (data not shown), the specific marker for this subset. we further confirmed this observation by incubation of sorted cd + cd + or cd + cd + t cells with cd + t cells, respectively. the result demonstrated that neither ifn-c nor tnf-a was produced by the cd + cd + t cells, whereas cd + cd + t cells produced large amounts of ifn-c and tnf-a upon coculture (fig b) , confirming that cd + cd + t cells were hyporesponsive and represented regulatory t cells. we then cocultured cd + cd + with cd + cd + t cells at different ratios in the presence of cd + cells. cd + cd + cells stimulated with esat- /cfp- peptides (p -p ) produced considerably greater amounts of th cytokines compared with unstimulated cells. the addition of cd + cd + t cells into cultures resulted in the inhibition of ifn-c, il- and tnf-a production in a dose-dependent manner (fig c-e) . were gated on cd + cd + ifn-c + and cd + cd + ifn-c cells and analyzed for the expression of memory markers cd ra, ccr , cd l and cd , as indicated. upper panel represents the control. lower panel represents the expression of each marker. cd + cd + ifn-c + t cells are shown in black, whereas cd + cd + ifn-c t cells are depicted in gray. the expression of each marker within cd + cd + ifn-c + t cells was overlaid with cd + cd + ifn-c t cells. the numbers in the quadrants indicate the percentage of cells among cd + cd + ifn-c + t cells. one representative result from ten independent experiments with similar results is shown. doi: . /journal.pone. .g overall, our assay conditions showed that cd + cd + cells could inhibit th cytokine production by cd + cd + t cells. without treatment, tuberculous pleurisy (tbp) usually resolves spontaneously [ ] and thus is thought to be a useful model to study the correlates of protective immune responses at the site of infection [ , ] . meanwhile, the diagnosis of tbp is difficult and continues to pose clinical challenges. currently, the two major advances in the diagnosis of tbp have been pleural biopsies [ ] and adenosine deaminase (ada) assessment in pleural fluid [ , ] . the free (unstimulated) pleural fluid ifn-c assay is another method established for the diagnosis of tbp [ ] . however, both ada and ifn-c are non-specific markers of inflammation [ ] . the detection of t cell responses specific for defined antigens is essential for the evaluation of pathogenic or protective immunity induced by infectious pathogens or vaccines. cd + t cell responses are commonly determined by the expression of various cytokines, such as ifn-c, il- and tnf-a for th cells, after short-term stimulation with specific antigens. the development of ifn-c release assays (igras) has emerged as an attractive alternative to the tuberculin skin test for detection of latent tb infections and might also contribute to assisting in the diagnosis of tbp [ ] . igras are based on the principle that t cells of individuals sensitized with tb antigens produce ifn-c when they re-encounter highly mtb-specific antigens such as esat- and cfp- . however, this assay precludes further analysis because it is lethal to cells. moreover, the igras are not recommended on either the blood or the pleural fluid for the diagnosis of tbp [ ] . consistent with other studies, we found that the expression of cd was significantly increased on cd + t cells in pfcs from patients with tbp [ ] . we further demonstrated that the majority of cd + cd + cells were th cells by analyzing expression of the th markers t-bet and il- rb . in order to figure . cd + cd + cells inhibit the production of th cytokines by sorted cd + cd + t cells. (a) cd + cd + and cd + cd + cells were sorted from purified cd + t cells. (b) production of ifn-c and tnf-a by sorted cd + cd + and cd + cd + cells in the presence of purified cd + t cells following stimulation with p -p . (c-e) sorted cd + cd + and cd + cd + cells were cultured at different ratios and cocultured with purified cd + cells in the presence or absence of p -p . the supernatants were collected, and cytokine production was assessed by elisa. one representative result from three independent experiments with similar results is shown. significant differences compared with incubation with p -p in the absence of tregs are indicated (*, not significant;**, p, . ;***, p, . ). doi: . /journal.pone. .g figure . isolation of viable cd + cd + and cd + cd t cells. (a) cd + cd + and cd + cd cells were sorted from magnetic beadpurified cd + t cells. (b) sorted cd + cd + and cd + cd cells were cocultured with purified cd + cells in the presence or absence of bcg or p -p . supernatants were collected and cytokine production was assessed by elisa. significant differences compared with cd + cd fractions are indicated (***, p, . ). (c, d) after stimulation for hours, cd + t cells were gated and the expression of th cytokines by cd + cd + and cd + cd cells was analyzed. numbers in the quadrants indicate percentages of cells among the cd + cd + or cd + cd populations. (e) the frequencies of ifn-c, il- or tnf-a-producing cd + cd + (closed circles) or cd + cd t cells (open circles, n = - ) are demonstrated. horizontal lines represent mean value. doi: . /journal.pone. .g further confirm that cd + cd + cells were mostly mtb-specific t cells, we performed intracellular cytokine staining. we noted that stimulation of pfcs resulted in increased surface cd expression. we overcame this limitation by incubation with bfa during short-term stimulation. using this method, we were able to directly assess the relationship between in vivo cd expression and cytokine production. using polychromatic flow cytometry, we demonstrated for the first time that the majority of mtb specific th cytokine-producing cells were enriched within the cd + cd + subset, whereas cd + cd cells scarcely produced cytokines. in addition, pfcs from tuberculous pleurisy patients were stimulated with sars-cov s peptides (stffstfkcygvsatkl) and (nfsqilpdplkptkrsfi) as well as hepatitis b virus surface antigen (hbsag). these irrelevant antigens could not induce the production of ifn-c, il- and tnfa by neither cd + cd + nor cd + cd t cells. however, following stimulation with mtb-specific peptides of esat- / cfp- , cd + cd + t cells from the same patients with tuberculous pleurisy expressed high levels of ifn-c, il- and tnf-a, indicating that the response was mtb-specific. most importantly, the detection of cd is useful for the direct identification of mtb-specific th cells in vivo without further stimulation. t cells that produce multiple factors simultaneously are termed polyfunctional t cells and have been shown to provide vaccineinduced immunity and protection against disease progression in hiv- infection [ , ] . in the present study, analysis of total cd + th subpopulations indicated that the majority of th cells produced only one cytokine. however, polyfunctional cd + t cells simultaneously producing ifn-c, il- and tnf-a dominated the cd + cd + t cell population, but the percentage of tnf-a single expressers was higher in both the cd + cd and the total cd + t cell subsets compared with cd + cd cells. our results indicated that detection or isolation of cd + t cells is a good method for the identification or enrichment of polyfunctional t cells. interestingly, significant positive correlation was observed between the percentages of cd -expressing cd + t cells and polyfunctional t cells. our results demonstrated for the first time that the expression of cd may be a useful marker for the definition of mtb-specific polyfunctional t cells in vivo. a number of studies in humans suggested that polyfunctional t cells may indeed be involved in mediating protection in tb [ ] [ ] [ ] [ ] [ ] [ ] . however, it has also been indicated in the recent studies that polyfunctional cd + t cells may be a useful biomarker of active tb and that polyfunctional responses are not associated with protection against tb [ ] . in the present study, we hypothesized that the highly increased frequencies of cd -expressing cd + t cells (polyfunctional t cells) in pleural fluid may be associated with protection against tb and thus lead to the self-resolution tendency of tbp. a previous study has indicated that cd + cd + cd cells represented a new subset of regulatory t cells in the tumor model [ ] . these cd + cd + cd cells are hyporesponsive and can suppress proliferation of cd + t cells in a cell-to-cell contact manner. in our study, however, cd + cd + t cells represented effector or effector memory cells and quickly produced large amounts of cytokines following short-term stimulation. moreover, we also assessed other markers (foxp and ctla- ) essential for the suppressive function of regulatory t cells and found that these molecules were not differentially expressed by cd + cd + and cd + cd t cells, further confirming that cd + cd + t cells were not regulatory t cells (data not shown). these differing results were probably associated with discrepancies in local circumstances, including cytokines, chemokines and antigen presenting cells that differ between tumor and inflammation contexts. moreover, it remains to be determined if cd is induced by the pleural fluid environment or if cd + cd + t cells are selectively recruited from the circulation. previous studies have indicated that cd + cd + t cells can inhibit antigen-specific t cell responses in tb patients [ ] [ ] [ ] [ ] [ ] [ ] . however, none of these studies has isolated mtb-specific cd + t cells directly. in the present study, we purified viable cd + cd + and cd + cd + treg cells directly and found that cd + cd + treg cells could inhibit cytokine production by cd + cd + t cells. in addition, our results clearly demonstrated that cd + cd + t cells were hyporesponsive and scarcely produced cytokines following short-term stimulation, further confirming that these cells were regulatory cells. in our previous studies, we have demonstrated that the suppressive effect of treg cells on ifn-c production by cd + cd + t cells [ ] and cd t cells [ ] was mainly mediated via the release of il- but not tgf-b. in summary, we demonstrated that cd as a useful marker for mtb-specific th cells in pfcs from patients with tbp enabled a direct ex vivo estimation of the quantity, as well as the quality, of mtb-specific th responses. the majority of cd + cd + t cells but not total cd + t cells, were dominated by polyfunctional t cells. importantly, significantly positive correlation was observed between cd -expressing cd + t cells and polyfunctional t cells, suggesting that cd is a good marker for the identification or enrichment of polyfunctional t cells in pfcs. moreover, isolation of live mtb-specific th cells according to surface cd expression without any stimulation offers an opportunity for further investigation. written informed consent was obtained from all patients and healthy donors. ethics approval for the present study was obtained from the ethics committee of the zhongshan school of medicine, sun yat-sen university (guangzhou, china) and the chest hospital of guangzhou (guangzhou, china). a total of fifty patients with tuberculous pleurisy ( females and males, range - years of age) were recruited from the chest hospital of guangzhou, china. diagnosis of pleural effusion from tb etiology was based on one of the following criteria: (i) demonstration of mtb on pleural fluid smear (by the ziehl-neelsen method); (ii) pleural fluid or pleural biopsy specimens growing m. tuberculosis on lowenstein-jensen medium; or (iii) histological evidence of caseating granuloma on biopsy specimens of pleural tissue with positive staining for m. tuberculosis. twentyone patients ( females and males, range to years of age) with active pulmonary tuberculosis (culture-confirmed pulmonary tb) were also included in this study. patients who had been previously diagnosed with hiv, hbv, or hcv or with a history of autoimmune diseases were excluded from the study. none of the patients was receiving mtb-related treatments at the time of the study. twenty-two healthy volunteers between the ages of and were recruited from sun yat-sen university. for the detection of mtb-specific immune responses, we selected six highly immunogenic and largely hla-dr-restricted peptides. four peptides were derived from esat- , and two were derived from the cfp- protein of mtb. synthetic peptides of amino acids (aa) in length were obtained from shenzhen hanyu pfcs were isolated by lysing erythrocytes using ammonium chloride solution and resuspended to a final concentration of cells/ml in complete rpmi medium (invitrogen, grand island, ny) supplemented with % heat-inactivated fetal calf serum (fcs; hyclone, logan, ut), u/ml penicillin, mg/ml streptomycin, mm l-glutamine, and mm mercaptoethanol. peripheral blood mononuclear cells (pbmcs) were isolated by ficoll-hypaque gradient centrifugation of heparinized venous blood obtained from healthy individuals or tb patients. for the detection of intracellular cytokines, cells were incubated at a concentration of cells/ml with mg/ml peptides plus mg/ml anti-cd and mg/ml anti-cd d for h in the presence of brefeldin a (bfa, mg/ml; sigma-aldrich, st louis, mo). for the detection of t-bet, surface markers were first stained and cells were then fixed in % formaldehyde, permeabilized in % methanol and labeled with anti t-bet mab. the expression of il- rb was detected by indirect immunofluorescence analysis. briefly, cells were incubated with mab for il- rb , followed by biotin-sp-conjugated anti-rat igg f(ab') fragments, and were finally incubated with streptavidin-pe. all incubations were performed at uc and cells were washed twice between incubations. flow cytometry was performed using bd facscalibur (bd biosciences) or facsaria ii (bd biosciences) and the data were analyzed using flowjo software (treestar, san carlos, ca). cd + and cd + cells were isolated from pfcs by positive selection with anti-cd or anti-cd microbeads (miltenyi biotec, bergisch gladbach, germany). for isolation of cd + cd + , cd + cd and cd + cd + cells, purified cd + t cells were stained with pe-conjugated cd and fitc-conjugated anti-cd and then sorted on a facsaria ii high-speed cell sorter. the purity of cells, as assessed by flow cytometry, was - % for each cell subset. purified cd + cd + , cd + cd or cd + t cells were cocultured with purified cd + cells (at ratio of : ) in the presence or absence of bcg, esat- and cfp- peptides. after hours, cell-free supernatants were collected and assessed for ifn-c, il- and tnf-a production by elisa kits (bd bioscience pharmingen). for the detection of intracellular cytokines, cells were cultured for hours and then subjected to flow cytometry analysis. to examine the role of cd + cd + cells in the regulation of cytokine production by sorted cd + cd + t cells, purified cd + cd + (teff), cd + cells and cd + cd + cells (treg) were co-cultured in various ratios (teff:treg) in the presence of esat- and cfp- peptides. cell-free supernatants were collected and assessed for ifn-c, il- and tnf-a by elisa. the wilcoxon matched pairs test (two-tailed) was performed to determine statistical differences between the groups using graphpad prism software version . inhibition of cytokine production by cd + cd + t cells was analyzed using the paired student's t test. a value of p, . was considered statistically significant. conceived and designed the experiments: cyw. performed the experiments: ll. analyzed the data: ll. contributed reagents/materials/ analysis tools: dq xyf shl xlz. wrote the paper: ll. some problems concerning local cellular immunity in tuberculosis production of the rantes chemokine in delayed-type hypersensitivity reactions: involvement of macrophages and endothelial cells can pleural tuberculosis be diagnosed using interferongamma release assays? diagnosis and treatment of tuberculous pleural effusion in compartmentalization of pro-inflammatory cytokines in tuberculous pleurisy cytokines in pleural liquid for diagnosis of tuberculous pleurisy update on tuberculous pleural effusion cytokine polarization in miliary and pleural tuberculosis mycobacterium tuberculosis and the host response development of new vaccines and diagnostic reagents against tuberculosis effect of bcg vaccination on risk of mycobacterium tuberculosis infection in children with household tuberculosis contact: a prospective community-based study the development and impact of tuberculosis vaccines recent findings in immunology give tuberculosis vaccines a new boost direct ex vivo analysis of antigen-specific ifn-gamma-secreting cd t cells in mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment enumeration of t cells specific for rd -encoded antigens suggests a high prevalence of latent mycobacterium tuberculosis infection in healthy urban indians characterization of a mycobacterium tuberculosis peptide that is recognized by human cd + and cd + t cells in the context of multiple hla alleles differential t cell responses to mycobacterium tuberculosis esat in tuberculosis patients and healthy donors aspartic acid homozygosity at codon of hla-dq beta is associated with susceptibility to pulmonary tuberculosis in cambodia human th cell lines recognize the mycobacterium tuberculosis esat- antigen and its peptides in association with frequently expressed hla class ii molecules in situ activation of helper t cells in the lung expansion of ccr + cd + t-lymphocytes in the course of active pulmonary tuberculosis cd controls the pathogenesis of allergic airway inflammation cd expression on cd + t lymphocytes after in vitro stimulation with tuberculin is an indicator of immune sensitization against mycobacterium tuberculosis antigens restoration of cellular immunity against tuberculosis in patients coinfected with hiv- and tuberculosis with effective antiretroviral therapy: assessment by determination of cd expression on t cells after tuberculin stimulation pleural tuberculosis: an update cytokine production at the site of disease in human tuberculosis correlates of protective immune response in tuberculous pleuritis diagnostic accuracy of adenosine deaminase in tuberculous pleurisy: a meta-analysis adenosine deaminase activity is a sensitive marker for the diagnosis of tuberculous pleuritis in patients with very low cd counts novel tests for diagnosing tuberculous pleural effusion: what works and what does not? interferon-gamma release assays for the diagnosis of tb pleural effusions: hype or real hope? increased susceptibility to apoptosis of cd dimcd + nk cells induces the enrichment of ifn-gamma-producing cd bright cells in tuberculous pleurisy hiv nonprogressors preferentially maintain highly functional hiv-specific cd + t cells the novel tuberculosis vaccine, aeras- , induces robust and polyfunctional cd + and cd + t cells in adults immunisation with bcg and recombinant mva a induces long-lasting, polyfunctional mycobacterium tuberculosis-specific cd + memory t lymphocyte populations modified vaccinia ankara-expressing ag a, a novel tuberculosis vaccine, is safe in adolescents and children, and induces polyfunctional cd + t cells bacillus calmette-guerin vaccination of human newborns induces t cells with complex cytokine and phenotypic profiles detection of polyfunctional mycobacterium tuberculosis-specific t cells and association with viral load in hiv- -infected persons dynamic relationship between ifngamma and il- profile of mycobacterium tuberculosis-specific t cells and antigen load multifunctional cd + t cells correllate with active mycobacterium tuberculosis infection cd + cd + cd -t cells, a new subset of regulatory t cells, suppress t cell proliferation through membrane-bound tgf-beta regulatory t cells are expanded in blood and disease sites in patients with tuberculosis increased frequency of cd (+)cd (high) treg cells inhibit bcg-specific induction of ifn-gamma by cd (+) t cells from tb patients cd (+)cd (+)foxp (+) regulatory t cells suppress mycobacterium tuberculosis immunity in patients with active disease regulatory t cells depress immune responses to protective antigens in active tuberculosis foxp + regulatory t cells suppress effector t-cell function at pathologic site in miliary tuberculosis a role for cd +cd + t cells in regulation of the immune response during human tuberculosis cd + cd + treg cells inhibit human memory gammadelta t cells to produce ifn-gamma in response to m tuberculosis antigen esat- key: cord- -j daoiq authors: resende, lucilene aparecida; roatt, bruno mendes; aguiar-soares, rodrigo dian de oliveira; viana, kelvinson fernandes; mendonça, ludmila zanandreis; lanna, mariana ferreira; silveira-lemos, denise; corrêa-oliveira, rodrigo; martins-filho, olindo assis; fujiwara, ricardo toshio; carneiro, cláudia martins; reis, alexandre barbosa; giunchetti, rodolfo cordeiro title: cytokine and nitric oxide patterns in dogs immunized with lbsap vaccine, before and after experimental challenge with leishmania chagasi plus saliva of lutzomyia longipalpis date: - - journal: vet parasitol doi: . /j.vetpar. . . sha: doc_id: cord_uid: j daoiq in the studies presented here, dogs were vaccinated against leishmania (leishmania) chagasi challenge infection using a preparation of leishmania braziliensis promastigote proteins and saponin as adjuvant (lbsap). vaccination with lbsap induced a prominent type immune response that was characterized by increased levels of interleukin (il-) and interferon gamma (ifn-γ) production by peripheral blood mononuclear cells (pbmc) upon stimulation with soluble vaccine antigen. importantly, results showed that this type of responsiveness was sustained after challenge infection; at day and after l. chagasi challenge infection, pbmcs from lbsap vaccinated dogs produced more il- , ifn-γ and concomitant nitric oxide (no) when stimulated with leishmania antigens as compared to pbmcs from respective control groups (saponin, lb- treated, or non-treated control dogs). moreover, transforming growth factor (tgf)-β decreased in the supernatant of slca-stimulated pbmcs in the lbsap group at days. bone marrow parasitological analysis revealed decreased frequency of parasitism in the presence of vaccine antigen. it is concluded that vaccination of dogs with lbsap vaccine induced a long-lasting type immune response against l. chagasi challenge infection. in the studies presented here, dogs were vaccinated against leishmania (leishmania) chagasi challenge infection using a preparation of leishmania braziliensis promastigote proteins and saponin as adjuvant (lbsap). vaccination with lbsap induced a prominent type immune response that was characterized by increased levels of interleukin (il-) and interferon gamma (ifn-␥) production by peripheral blood mononuclear cells (pbmc) upon stimulation with soluble vaccine antigen. importantly, results showed that this type of responsiveness was sustained after challenge infection; at day and after l. chagasi challenge infection, pbmcs from lbsap vaccinated dogs produced more il- , ifn-␥ and concomitant nitric oxide (no) when stimulated with leishmania antigens as compared to pbmcs from respective control groups (saponin, lb-treated, or non-treated control dogs). moreover, transforming growth factor (tgf)-␤ decreased in the supernatant of slcastimulated pbmcs in the lbsap group at days. bone marrow parasitological analysis revealed decreased frequency of parasitism in the presence of vaccine antigen. it is concluded that vaccination of dogs with lbsap vaccine induced a long-lasting type immune response against l. chagasi challenge infection. crown copyright © published by elsevier b.v. all rights reserved. visceral leishmaniasis with a zoonotic feature is caused by protozoan species belonging to the complex leishmania donovani (leishmania infantum syn. leishmania chagasi, in latin america) and is widely distributed in the mediterranean basin, middle east, and south america (desjeux, ) . canines are the main reservoir for the parasite in different geographical regions of the globe and play a relevant role in transmission to humans (deane, ; dantas-torres, ) . thus, the current strategy for control of the disease includes the detection and elimination of seropositive dogs alongside vector control and therapy for human infection (tesh, ) . chemotherapy in dogs still does not provide parasitological cure (noli and auxilia, ) , and for this reason a vaccine against visceral leishmaniasis (vl) would be an important tool in the control of canine visceral leishmaniasis (cvl) and would also dramatically decrease the infection pressure of l. chagasi for humans (hommel et al., ; dye, ) . toward this purpose, establishing biomarkers of immunogenicity is considered critical in analyzing candidate vaccines against cvl (reis et al., ; maia and campino, ) , and this strategy is being used to identify the pattern of immune response in dogs and to further the search for vaccine candidates against cvl (reis et al., ) . several studies have reported the potential of different cvl vaccines to trigger immunoprotective mechanisms against leishmania infection (borja-cabrera et al., ; rafati et al., ; holzmuller et al., ; giunchetti et al., ; lemesre et al., ; araújo et al., araújo et al., , fernandes et al., ; giunchetti et al., a,b) . the polarized immune response described in a mouse model during leishmania infection (mosman et al., ; barral et al., ; kane and mosser, ; murray et al., ; trinchieri, ) does not occur in dogs, with different studies demonstrating the simultaneous presence of interferon (ifn)-␥ and interleukin (il)- (chamizo et al., ; lage et al., ; menezes-souza et al., ) . in addition, a mixed profile of cytokines has been described during cvl, with high levels of ifn-␥, il- , and transforming growth factor (tgf)-␤, concomitant with reduced expression of il- according to skin parasite load (menezes-souza et al., ) . studies evaluating other biomarkers of immunogenicity induced by the lbsap vaccine (composed of l. braziliensis promastigote proteins plus saponin as the adjuvant) have demonstrated higher levels of circulating t lymphocytes (cd + , cd + , and cd + ) and b lymphocytes (cd + ) and increased levels of leishmania-specific cd + and cd + t cells (giunchetti et al., , a . lbsap vaccine is considered safe for administration, without induction of ulcerative lesions at the site of inoculation vitoriano-souza et al., ) . moreover, lbsap vaccinated dogs presented high ifn-␥ and low il- and tgf-␤ expression in the spleen, with significant reduction of parasite load in this organ (roatt et al., ) . additionally, lbsap displayed a strong and sustained induction of humoral immune response, with increased levels of anti-leishmania total igg as well as both igg and igg , after experimental challenge (roatt et al., ) . considering the promising results of the lbsap vaccine, we aimed to further evaluate the immunogenicity biomarkers before and after experimental l. chagasi challenge . thus, the profile of different cytokines (il- , il- , tgf-␤, il- , ifn-␥, and tumor necrosis factor [tnf]-␣) and nitric oxide (no) in supernatants of peripheral blood mononuclear cell (pbmc) cultures were evaluated before the first immunization (t ), days after completion of the vaccine protocol (t ), and at time points (t ) and (t ) days after experimental l. chagasi challenge. the frequency of parasitism in the bone marrow was also evaluated until t . . . animals, vaccination and experimental challenge with l. chagasi plus saliva of lutzomyia longipalpis twenty male and female mongrel dogs that had been born and reared in the kennels of the instituto de ciências exatas e biológicas, universidade federal de ouro preto, ouro preto, minas gerais, brazil, were treated at months with an anthelmintic and vaccinated against rabies (tecpar, curitiba-pr, brazil), canine distemper, type adenovirus, coronavirus, parainfluenza, parvovirus, and leptospira (vanguard ® htlp /cv-l; pfizer animal health, new york, ny, usa). the absence of specific anti-leishmania antibodies was confirmed by indirect fluorescence immunoassay. experimental dogs were divided into four experimental groups: (i) control (c) group (n = ) received ml of sterile . % saline; (ii) lb group (n = ) received g of l. braziliensis promastigote protein in ml of sterile . % saline; (iii) sap group (n = ) received mg of saponin (sigma chemical co., st. louis, mo, usa) in ml of sterile . % saline; and (iv) lbsap group (n = ) received g of l. braziliensis promastigote protein and mg of saponin in ml of sterile . % saline. all animals received subcutaneous injections in the right flank at intervals of weeks for a total of three injections. the challenge of experimental animals was performed after days of vaccination protocol. in this sense, all dogs received intradermally . × promastigotes of l. chagasi stationary phase of cultivation, in the inner side of the left ear, in addition to acini of the salivary gland of l. longipalpis. this preliminary stage of the study was performed from to . promastigotes of l. braziliensis (mhom/br/ /m ) were maintained in in vitro culture in nnn/lit media as previously described . briefly, parasites were harvested by centrifugation ( × g, min, • c) from -day-old cultures, washed three times in saline buffer, fully disrupted by ultrasound treatment ( w, min, • c), separated into aliquots, and stored at − • c until required for use. protein concentration was determined according to the method of lowry (lowry et al., ) . the lbsap vaccine was previously described by giunchetti et al., and registered at the instituto nacional da propriedade industrial (brazil) under patent number pi - ( february ). peripheral blood samples were collected before the first immunization (t ), days after completion of the vaccine protocol (t ) and at time points of (t ) and (t ) days after experimental l. chagasi challenge by puncture of the jugular vein in sterile heparinized ml syringes. to obtain pbmcs for the in vitro analysis, the blood collected was added over ml of ficoll-hypaque (histopaque ® , sigma) and subjected to centrifugation at × g for min at room temperature. the separated pbmcs were resuspended in gibco rpmi medium, washed twice with rpmi , centrifuged at × g for min at room temperature, homogenized, and finally resuspended in rpmi at cells/ml as previously described . the in vitro assays were performed in -well flatbottomed tissue culture plates (costar, cambridge, ma, usa), with each well containing l of culture medium ( % fetal bovine serum/ % streptavidin/penicillin, mm lglutamine, and . % ␤-mercaptoethanol in rpmi ) and l of pbmcs ( . × cells/well) with l of vaccine soluble antigen (vsa; l. braziliensis, g/ml) or l of soluble l. chagasi antigen (slca, g/ml) obtained according to reis et al. (reis et al., a,b) . one-hundred l of rpmi was added in place of the antigenic stimulus in the non-stimulated control cultures. incubation was carried out in a humidified incubator with % co , at • c for days, after which the supernatants were collected and stored in a freezer at − • c for detection of cytokine and no. the in vitro evaluation was performed with the supernatant of pbmcs collected at t , t , t and t , which were stored as described above. cytokine levels were determined by enzyme-linked immunosorbent assay (elisa), purchased from r&d systems (minneapolis, mn, usa), according to the manufacturer's instructions. duoset elisa was used for analysis of tnf-␣ (anti-canine tnf-␣/tnfsf a immunoassay; catalog number: dy ); il- (anti-canine il- , catalog number: dy ); il- (anti-canine il- /il- p , catalog number: dy ); and ifn-␥ (anti-canine ifn-␥, catalog number: dy b) cytokines. the level of tgf-␤ was quantified by elisa using the quantikine ® kit (mouse/rat/porcine/canine tgf-␤ immunoassay, catalog number mb b). il- cytokine was evaluated using monoclonal anti-canine il- antibody (catalog number: mab ) as capture antibody; recombinant canine il- (catalog number: cl) for obtaining the standard curve; and biotinylated anti-canine il- antibody (catalog number: baf ), streptavidin (r&d systems, dy ), and substrate solution ( : mixture of h o and tetramethylbenzidine, product code - - , lot. no. rb ). minimum sensitivity was pg/ml for tnf-␣, pg/ml for il- , pg/ml for il- , pg/ml for ifn-␥, pg/ml for tgf-␤, and pg/ml for il- . all experiments were performed using -well plates (costar ® , washington, dc), according to r&d systems instructions. the reading was performed using the microplate automatic reader (el , biotek, winosski, vt) at a wavelength of nm. quantification of levels of no was performed indirectly by measuring nitrite in supernatants of pbmc cultures by griess reaction (green et al., ; gutman and hollywood, ) . duplicate samples were grown in -flat bottom wells (nunc, naperville, il). briefly, a -l aliquot of cell-free culture supernatant was mixed with l of griess reagent ( % sulfanylamide, . % naphthylethylenediamide-dihydrochloride, and . % phosphoric acid, all from sigma). following min of incubation at room temperature in the dark, the absorbance was measured at nm by using a microplate reader (biotek, el ). the concentration of nitrite was determined by interpolation from a standard curve constructed by using sodium nitrite solutions of known concentration in the range - m. to discount the interference of nitrites already present in the culture medium, data were calculated taking into account the blank for each experiment, assayed by using the medium employed for the in vitro pbmc cultures. the results were first expressed as nitrite concentration (m). bone marrow was obtained to evaluate the frequency of tissue parasitism in the different groups. dogs were anesthetized with an intravenous dose ( mg/kg body weight) of sodium thiopental (thionembutal ® ; abbott laboratories, são paulo, brazil), and bone marrow fluid was removed from the iliac crest under aseptic conditions. the bone marrow aspirates were used to study the presence of l. chagasi parasites by pcr. dna of bone marrow samples was extracted by wizard tm genomic dna purification kit (promega, madison, wi, usa) according to the manufacturer's instructions. pcr was performed as previously described (degrave et al., ) using the primers forward: [ -ggg(g/t)aggggcgttct(g/c)cgaa- ] and reverse: that amplified a dna fragment of base pairs (bp) from the conserved region of leishmania minicircle kdna. briefly, the pcr assay reaction mixture contained . l of dna preparation, . mm dntps, mm tris-hcl (ph . ), mm kcl, . mm mgcl , pmol of each primer, and u taq polymerase (invitrogen) to a final volume of l. pcr amplification was performed in a veriti thermal cycler well thermocycler (applied biosystems ® , irvine, ca, usa), over cycles consisting of min at • c (denaturation), min at • c (annealing), min at • c (extension), and min at • c (final extension). positive [genomic dna of l. chagasi (mhom/br/ /bh )] and negative (without dna) controls were included in each test. amplified fragments were analyzed by electrophoresis on % table levels of tgf-␤ in the pbmcs from dogs before the first vaccine dose (t ), following completion of the vaccine protocol (t ), and after early (t ) and late (t ) time points following l. chagasi challenge. the results are presented with regards to stimulation with soluble l. chagasi antigen (slca) in the following groups: c (control) and lbsap (killed l. braziliensis vaccine plus saponin). polyacrylamide gel and ethidium bromide-stained for the pcr product identification. the parasitological investigation was performed until days after l. chagasi challenge. statistical analyses were performed using prism . software package (prism software, irvine, ca, usa). normality of the data was demonstrated using a kolmogorov-smirnoff test. paired t-tests were used to evaluate differences in mean values of cytokines levels, considering the comparative analysis of t and t (fig. ) or t (fig. ) or t (fig. ) , in each group evaluated. unpaired ttests were used to evaluate differences in mean of values of tgf-␤ (table ) . analysis of variance (anova) test followed by tukey's multiple comparisons were used in the evaluation between the different treatment groups for cytokines (figs. - ) and nitric oxide (fig. ) analysis. differences were considered significant when p values were < . . to determine the impact of lbsap vaccination on the immune response, we evaluated the cytokine profile (tnf-␣, il- , ifn-␥, il- , and il- ) in the supernatant of pbmc stimulated with vsa ( fig. a) or slca (fig. b) . in this context, we performed a comparative analysis between t and t , in addition to the comparisons between experimental groups at each time point. in the comparison between t and t , the sap group showed increased levels (p < . ) of tnf-␣ and ifn-␥ production at t with vsa stimulation. additionally, the lb group presented higher levels (p < . ) of il- in vsa-stimulated pbmcs at t , as compared to t . in contrast, in slca-stimulated cultures, the lb group displayed lower levels of tnf-␣ at t as compared to t in slca-stimulated cultures (p < . ). interestingly, the lbsap vaccine induced higher levels of both il- and ifn-␥ at t in vsa-stimulated pbmcs. similarly, in the presence of slca, increased levels (p < . ) of ifn-␥ were observed in the lbsap group at t . the comparison between the experimental groups, in different time points, revealed increased levels (p < . ) of ifn-␥ in vsa-stimulated cultures from the lb group, as compared to c group in t . interestingly, higher (p < . ) levels of this cytokine were observed in the vsa-stimulated culture of lbsap group when compared to c and sap groups, at t . similarly, in slca-stimulated cultures, lbsap group displayed increased (p < . ) levels of ifn-␥ in relation to c, sap and lb groups at t . in addition, at t , lbsap group showed increased (p < . ) levels of il- in relation to c and sap groups, in addition to reduced (p < . ) levels of il- when compared to lb group, in vsa-stimulated cultures. the early immune response after l. chagasi challenge was analyzed in different groups. we determined the cytokine patterns in the supernatant of pbmcs comparing the different treatments (vsa - fig. a and slca - fig. b) , different time points (t and t ), and different experimental groups, at each time point. comparison between t and t showed that the c group had increased levels of tnf-␣ production (p < . ) and lower levels of il- production (p < . ) at t upon vsa and slca stimulation. additionally, c group had higher levels of il- in slca-stimulated pbmcs (p < . ) and higher levels of ifn-␥ production in vsa-stimulated pbmcs (p < . ) at t . the sap group showed increased levels (p < . ) of tnf-␣ and il- production and reduction of il- levels at t . in slca-stimulated cultures, the sap group presented higher levels (p < . ) of tnf-␣ and ifn-␥. the lb group showed increased levels (p < . ) of tnf-␣, il- , and il- production and reduction of il- in vsastimulated pbmcs at t . in cultures stimulated with slca, the lb group shown increased levels (p < . ) of ifn-␥. interestingly, the lbsap vaccine induced higher levels of il- at t in pbmcs stimulated with vsa. furthermore, in the presence of slca, lbsap vaccine induced higher levels of ifn-␥ (p < . ). the reduced levels of il- , which occurred in the other groups, were retained (p < . ) in the lbsap group at t for both stimuli (vsa and slca). the comparative analysis between the experimental groups showed, at t , increased levels (p < . ) of il- , in slca-stimulated cultures in the lb group and vsastimulated cultures in the lbsap group, in relation to c group. interestingly, the slca-stimulated pbmcs from lbsap group showed increased levels (p < . ) of il- compared to lb and sap groups at t . furthermore, increased levels (p < . ) of ifn-␥ in the lbsap when compared to c, sap and lb groups were observed. . . lbsap vaccine elicited a long-lasting type immune response at t , displaying higher levels of ifn- the late immune response after l. chagasi challenge was studied in different groups with regard to the cytokine t t t t t t t t t t t t t t t t t t t t . the x-axis displays the cytokines evaluated (tnf-␣, il- , ifn-␥, il- , and il- ). the y-axis represents the mean values (pg/ml) ± sd from groups of five animals/evaluation time; the left y-axes depict the tnf-␣, il- , and il- levels, while in the right y-axes represent the il- and ifn-␥ cytokine levels. significant differences (p < . ) between values measured at t (before the first dose) and t ( days after the third dose) are indicated by connecting lines, whereas the symbols c, sap, and lb indicate significant differences in relation to c, sap, and lb, groups, respectively, at the same stimulus and time of evaluation. levels in the supernatant of pbmcs treated with vsa ( fig. a) or slca (fig. b) , and the t and t data were compared, besides the comparisons between experimental groups, at each time. in the comparison between t and t , the results showed that the c group had increased levels of tnf-␣ in vsa-stimulated pbmcs (p < . ) and decreased levels of il- production (p < . ) in the presence of slca at t . the sap and lb groups presented increased levels of il- (p < . ) at t in the presence of the vsa stimulus, as compared to t . in the presence of the slca stimulus, the lb group had decreased levels of il- (p < . ) at t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t . the x-axis depicts the cytokines evaluated (tnf-␣, il- , ifn-␥, il- , and il- ). the y-axis represents the mean values (pg/ml) ± sd from groups of five animals/evaluation time; the left y-axes illustrate the tnf-␣, il- , and il- levels, while the right y-axes represent the il- and ifn-␥ cytokine levels. significant differences (p < . ) between values measured at t (before the first dose) and t ( days after the l. chagasi challenge) are indicated by connecting lines, whereas the symbols c, sap, and lb indicate significant differences in relation to c, sap, and lb, groups, respectively, at the same stimulus and time of evaluation. t , as compared to t . similarly, the group lbsap had decreased levels of il- (p < . ) at t , as compared to t , but this difference was only observed in the presence of the vsa stimulus. interestingly, in this group, levels of il- (in the presence of vsa) and ifn-␥ (in the presence of slca) were higher compared to t (p < . ). whereas this was a time-delayed response post-challenge with l. chagasi (t ), this result indicates an immune response predominantly of the type , induced by vaccination with lbsap. comparative analysis between the experimental groups showed increased (p < . ) levels of tnf-␣ in vsastimulated cultures of lb group in relation to c group, at t . interestingly, at t , increased (p < . ) levels of t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t . the x-axis displays the cytokines evaluated (tnf-␣, il- , ifn-␥, il- , and il- ). the y-axis represents the mean values (pg/ml) ± sd from groups of five animals/evaluation time; the left y-axes illustrate the tnf-␣, il- , and il- levels, while in the right y-axes represent the il- and ifn-␥ cytokine levels. significant differences (p < . ) between values measured at t (before the first dose) and t ( days after the l. chagasi challenge) are indicated by connecting lines, whereas the symbols c, sap, and lb indicate significant differences in relation to c, sap, and lb, groups, respectively, at the same stimulus and time of evaluation. ifn-␥ in lbsap group was observed in relation to c, sap and lb groups, in slca-stimulated pbmcs. the levels of tgf-␤ are shown in table , which focuses on the analysis using supernatant of pbmcs simulated with slca. we evaluated the data using a comparative analysis between the control and lbsap groups at t and t as well as at t and t for the l. chagasi challenge. interestingly, there was a decrease in tgf-␤ in the group immunized with lbsap compared to group c at t . since the production of no is considered to be a key element in mechanisms that mediate the elimination of intracellular pathogens, the levels of antimicrobial oxidant produced by in vitro antigen-stimulated pbmcs derived from dogs vaccinated with lbsap were determined (fig. ) . at t a reduction (p < . ) was observed in the levels of the reactive no in vsa-stimulated cultures compared to the respective control cultures of the groups c, sap, lb, and lbsap (fig. a) . at t , significantly increased nitrite levels (p < . ) in the vsa-and slca-stimulated cultures were observed in the sap group compared with cultures receiving the same stimuli in the c and lb groups. slca-stimulated cultures in the c and lb groups showed a significant reduction of no levels when compared to the respective control cultures (fig. b ). in addition, the c group presented higher levels of no in control cultures in relation to vsa-stimulated cultures (fig. b) . interestingly, in the lbsap group, higher (p < . ) levels of no levels were recorded in the supernatant of slca-and vsa-stimulated cultures at t when compared with cultures receiving the same stimuli in groups c and lb. the parasitological investigation was performed until days after l. chagasi challenge. by t two dogs from group c, four dogs from group sap, and one dog each from the lb and lbsap groups were diagnosed as positive. it is interesting to note also, that until the period in which they were accompanied (t ) all experimental groups remained asymptomatic. increased vl incidence in the world and especially in brazil have motivated studies and evaluations of anti-cvl vaccines because of the epidemiological importance of dogs in the biological cycle of the parasite (palatnik-de-sousa, ) . aiming to guide the rationale for developing anti-cvl vaccines, studies have been performed to identify biomarkers of immunogenicity before and after l. chagasi challenge (gutman and hollywood, ; reis et al., ) . type and type immune responses and immunomodulatory cytokines are considered the main targets for identifying resistance biomarkers following vaccination against cvl (reis et al., ; fernandes et al., ; carrillo et al., ; de lima et al., ) . results from previous studies using lbsap, the anti-cvl vaccine, showed high immunogenic potential, with induction of increased levels of circulating t lymphocytes (cd + , cd + , and cd + ) and b lymphocytes (cd + ), and higher levels of cd + and cd + t cells that were leishmania specific roatt et al., ) . in these studies, lbsap vaccine elicited strong antigenicity related to the increased levels of anti-leishmania igg isotypes after vaccination , and a strong and sustained induction of humoral immune response after experimental challenge, with increased levels of anti-leishmania total igg, igg and igg (roatt et al., ) . furthermore, lbsap vaccinated dogs presented high ifn-␥ and low il- and tgf-␤ expression in spleen with significant reduction of parasite load in this organ (roatt et al., ) . in addition, lbsap vaccine displayed safety and security for the administration vitoriano-souza et al., ; moreira et al., ) . however, there are few studies evaluating the cytokine profiles associated with cvl and in anti-cvl vaccines, which might serve as biomarkers to identify resistance and susceptibility. thus, this study aimed to evaluate the cytokine profile and no induced by immunization before and after experimental challenge with l. chagasi and sand fly saliva. in addition, the frequency of bone marrow parasitism was included in the evaluation. we thus performed a comparative analysis of the cytokine profile before immunization (t ), after completion of the vaccine protocol (t ), and at early (t ) and late (t ) time points after experimental challenge with l. chagasi. the production of distinct cytokines was evaluated during the vaccination protocol and after l. chagasi and sand fly saliva experimental challenge. the analysis of il- levels has been considered a morbidity marker during ongoing cvl (quinnel et al., ; brachelente et al., ; chamizo et al., ) , as well as in a murine models of vl (miralles et al., ) . we observed that the group vaccinated with lbsap showed increased levels of il- as compared to the c group. however, increased levels of ifn-␥ in the lbsap group were also observed. according to manna et al. ( ) , it is possible to maintain a standard of resistance in cvl even in the presence of il- , as long as there are elevated levels of ifn-␥. nevertheless, our results do not suggest a typical profile linking this cytokine with a resistance or susceptibility pattern in cvl. similar to our study, a previous study (manna et al., ) did not associate il- with resistance or susceptibility to natural l. chagasi infection in cvl. in contrast, levels of il- in splenocytes from dogs naturally infected with l. chagasi and presenting different clinical signs, indicated that this cytokine could be a biomarker present during the course of infection in cvl (lage et al., ) . similarly, il- has also been associated with susceptibility to cvl (pinelli et al., ; lage et al., ; alves et al., ; boggiatto et al., ) and human vl (nylen and sacks, ) . our data showed increased levels of il- at t and t in the lb group and at t in the sap group. in contrast, we observed decreased levels of il- in lbsap in relation to the lb group at t in vsa-stimulated pbmcs. we hypothesize that lower levels of il- during the immunization protocol and the lack of significance in il- levels after experimental challenge with l. chagasi in the lbsap contributes to the establishment of a more efficient immune response in these vaccinated dogs. in addition, the cytokine tgf-␤ has been associated with progression of leishmania infection in a murine model (barral et al., ; virmondes-rodrigues et al., ; gantt et al., ) . few studies have been performed in cvl; however, existing studies show increased levels of tgf-␤ in both asymptomatic and symptomatic dogs naturally infected with l. chagasi . our results displayed decreased levels of tgf-␤ in slca-stimulated cultures of lbsap group at t . these results suggest that vaccination with lbsap may trigger reduced tgf-␤ production after experimental challenge. in fact, a previous work (alves et al., ) reported high levels of tgf-␤ associated with increased parasite load in lymph nodes from symptomatic dogs naturally infected with l. chagasi and an association between this cytokine and cvl morbidity. therefore, it is possible that the reduced levels of tgf-␤, associated with higher levels of il- and ifn-␥, after l. chagasi and sand fly saliva challenge, would contribute to establishing immunoprotective mechanisms induced by lbsap vaccination. type cytokines have also been considered as a prerequisite for evaluating immunogenicity before and after l. chagasi experimental challenge in anti-cvl vaccine clinical trials (reis et al., ) . thus, we analyzed tnf-␣, il- , and ifn-␥ levels. some studies have established that tnf-␣ together with ifn-␥ are associated with a resistance profile against cvl (pinelli et al., (pinelli et al., , chamizo et al., ; carrillo et al., ; alves et al., ) . however, it is not a consensus that tnf-␣ profile would be a good indicator of resistance or susceptibility after l. chagasi infection, considering the similar levels of tnf-␣ showed in dogs presenting distinct clinical signs lage et al., ) . moreover, lbsap group did not present any differences in tnf-␣ levels when compared to other experimental groups. in fact, our data were similar to leishmune ® results, that did not present differences in the expression of this molecule (araújo et al., ; de lima et al., ) . in addition, assessment of il- levels in the group immunized with lbsap revealed increased levels of this cytokine at t , t , and t in the presence of vsa stimulation, compared to t . interestingly, higher levels of il- after vaccine protocol in relation to c and lb group (t , in vsa-stimulated cultures), and in the early period post challenge in relation to sap and lb groups (t , in slca-stimulated cultures) was the hallmark of lbsap group. since this cytokine has been associated with protection in cvl (strauss-ayali et al., ; menezes-souza et al., ) , high levels of il- and impaired tgf-␤ production would indicate the establishment of immunoprotective mechanisms induced by lbsap vaccination. ifn-␥ is considered an important pro-inflammatory cytokine for establishing protective immunity against the leishmania parasite, inducing no synthesis, and activating microbicidal function in macrophages (trinchieri et al., ; reiner and locksley, ) . thus, no is considered one of the most important molecules responsible for killing intracellular parasites such as those of the leishmania genus (heinzel et al., ; bogdan, ; sisto et al., ; gradoni and ascenzi, ) . in this context, we found that the lbsap group had increased levels of ifn-␥ after the vaccine protocol (t ), presenting sustained improvement at the early (t ) and late (t ) time points after l. chagasi experimental challenge in the presence of the slca stimulus, compared to t . interestingly, after the vaccination protocol (t ), the lbsap group showed increased levels in ifn-␥ in vsa or slca-stimulated cultures compared to other groups. moreover, in both early (t ) and late (t ) period post challenge, the lbsap group remained producing increased levels of leishmania-specific ifn-␥, as compared to the respective stimulated cultures (vsa or slca) from the other groups. furthermore, the increased ifn-␥ levels at t was concomitant with higher no amounts in cultures stimulated with slca and vsa. since ifn-␥ is associated with a resistance profile to leishmania infection in different experimental models (squires et al., ; andrade et al., ; murray et al., ; carrillo et al., ; fernandes et al., ) , our data revealed an intense leishmania-specific induction of ifn-␥ after immunization with lbsap. considering the lack of a sufficient amount of biological material, we performed pcr analysis to assess the parasite burden. however, only the lbsap and lb groups showed one dog each with positive parasitological results, which may indicate that the antigen of l. braziliensis can induce protection after experimental l. chagasi challenge. further investigations will focus on the efficacy of the lbsap vaccination in protecting against an experimental challenge with l. chagasi, using quantitative pcr. in conclusion, our data point to a prominent type immune response is elicited by higher levels of il- and ifn-␥ following complete vaccination and after l. chagasi challenge. additionally, the levels of tgf-␤ are reduced in the early immune response after l. chagasi challenge, while no production is enhanced at a late time point following l. chagasi challenge. furthermore, based on bone marrow parasitological analysis, the frequency of parasitism is decreased in the presence of the vaccine antigen. thus, lbsap vaccine appears to elicit prominent, long-lasting type immunogenicity. expression of ifn-gamma, tnf-alpha il- and tgfbeta in lymph nodes associates with parasite load and clinical form of disease in dogs naturally infected with leishmania (leishmania) chagasi evaluation of the immune response and production of interferon in canine visceral 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tissue granulomatous response interleukin- augments a th -type immune response manifested as lymphocyte proliferation and interferon gamma production in leishmania infantum-infected dogs control of zoonotic visceral leishmaniasis: is it time to change strategies? interleukin- production by effector t cells: th cells show self control producer cells of interleukin transforming growth factor beta and immunosuppression in experimental visceral leishmaniasis kinetics of cell migration to the dermis and hypodermis in dogs vaccinated with antigenic compounds of leishmania braziliensis plus saponin the authors are grateful for the use of the facilities at cebio, universidade federal de minas gerais and rede mineira de bioterismo (fapemig). this work was supported by fundaç ão de amparo a pesquisa do estado de minas gerais, brazil (grant: cbb-apq- - ; cbb-apq- - -ppsus; cbb-apq- - ), conselho nacional de desenvolvimento científico e tecnológico- cnpq, brazil (grant: / - -papes v/fiocruz; / - ; / - ) and capes. rco, oamf, rtf, cmc, abr and rcg are grateful to cnpq for fellowships. the authors also thank the boldface editors for the critical reading of the manuscript, editorial suggestions and changes. key: cord- -jgbjxgh authors: graham, simon p.; mclean, rebecca k.; spencer, alexandra j.; belij-rammerstorfer, sandra; wright, daniel; ulaszewska, marta; edwards, jane c.; hayes, jack w. p.; martini, veronica; thakur, nazia; conceicao, carina; dietrich, isabelle; shelton, holly; waters, ryan; ludi, anna; wilsden, ginette; browning, clare; bialy, dagmara; bhat, sushant; stevenson-leggett, phoebe; hollinghurst, philippa; gilbride, ciaran; pulido, david; moffat, katy; sharpe, hannah; allen, elizabeth; mioulet, valerie; chiu, chris; newman, joseph; asfor, amin s.; burman, alison; crossley, sylvia; huo, jiandong; owens, raymond j.; carroll, miles; hammond, john a.; tchilian, elma; bailey, dalan; charleston, bryan; gilbert, sarah c.; tuthill, tobias j.; lambe, teresa title: evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored covid- vaccine candidate chadox ncov- date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: jgbjxgh clinical development of the covid- vaccine candidate chadox ncov- , a replication-deficient simian adenoviral vector expressing the full-length sars-cov- spike (s) protein was initiated in april following non-human primate studies using a single immunisation. here, we compared the immunogenicity of one or two doses of chadox ncov- in both mice and pigs. whilst a single dose induced antigen-specific antibody and t cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in sars-cov- neutralising titres. as sars-cov- began to spread around the world at the beginning of several vaccine platform technologies were employed to generate candidate vaccines. several use replicationdeficient adenoviral (ad) vector technology and express the sars-cov- spike (s) protein. the first phase i clinical study of an ad -vectored vaccine has been reported , chadox ncov- (azd ) phase i trials (nct ) began in april with phase ii and iii trials (nct ) started soon thereafter, and an ad -vectored vaccine is expected to enter phase i shortly. typically, only one dose of ad-vectored vaccines has been administered in early preclinical challenge studies or clinical studies against emerging or outbreak pathogens - . rhesus macaques immunised with a single dose of chadox ncov- were protected against pneumonia but there was no impact on nasal virus titers after high dose challenge to both the upper and lower respiratory tract . to increase antibody titres and longevity of immune responses, a booster vaccination may be administered. homologous prime-boost immunisation resulted in higher antibody titres including neutralising antibodies and a trend towards a lower clinical score in a mers-cov challenge study . here, we set out to test the immunogenicity of either one or two doses of chadox ncov- in mice and pigs, to further inform clinical development. 'prime-boost' vaccinated inbred (balb/c) and outbred (cd ) mice were immunised on and days post-vaccination (dpv), whereas, 'prime-only' mice received a single dose of chadox ncov- on day . spleens and serum were harvested from all mice on day ( weeks after boost or prime vaccination). analysis of sars-cov- s protein-specific murine splenocyte responses by ifnγ elispot assay showed no statistically significant difference between the prime-only and primeboost vaccination regimens, in either strain of mouse ( figure a ). intracellular cytokine staining (ics) of splenocytes ( figure b) showed, in both mouse strains, that the response was principally driven by cd + t cells. the predominant cytokine response of both cd + and cd + t cells was expression of ifn-γ and tnf-α, with negligible frequencies of il- + and il- + cells, consistent with previous data suggesting adenoviral vaccination does not induce a dominant th response , . there were no signficant differences in cd + and cd + t cell cytokine responses between prime-only and primeboost mice. prime-only and prime-boost pigs were immunised on dpv and prime-boost pigs received a second immunisation on dpv. blood samples were collected weekly until dpv to analyse immune responses. ifn-γ elispot analysis of porcine peripheral blood mononuclear cells (pbmc) showed responses on dpv ( weeks after boost) that were significantly greater in the prime-boost pigs compared to prime-only animals (p < . ; figure c ). the prime-boost dpv responses were greater than responses observed in either group on dpv, but inter-animal variation meant this did not achieve statistical significance. ics analysis of porcine t cell reponses showed a dominance of th -type cytokines (similar to the murine response) but with a higher frequency of s-specific cd + t cells compared to cd + t cells ( figure d ). however, cd + and cd + t cell cytokine responses did not differ significantly between vaccine groups or timepoints ( vs. dpv). sars-cov- s protein-specific antibody titres in serum were determined by elisa using recombinant soluble trimeric s (fl-s) and receptor binding domain (rbd) proteins. a significant increase in fl-s binding antibody titres was observed in prime-boost balb/c mice compared to their prime-only counterparts (p < . ), however, the difference between vaccine groups for cd mice was not significant (figure a ). antibody responses were evaluated longitudinally in pig sera by fl-s and rbd elisa. compared to pre-vaccination sera, significant fl-s specific antibody titres were detected in both prime-only and prime-boost groups from and dpv, respectively (p < . ; figure b ). fl-s antibody titres did not differ signifcantly between groups until after the boost, when titres in the prime-boost pigs became significantly greater with an average increase in titres of > log (p < . ). rbd-specific antibody titres showed a similar profile with significant titres in both groups from dpv (p < . ) and a further significant increase in the prime-boost pigs from dpv onwards which was greater than the prime-only pigs (p < . ; figure c ). sars-cov- neutralising antibody responses were assessed using a virus neutralisation test (vnt; figure d ) and pseudovirus-based neutralisation test (pvnt; figure e ). after the prime immunisation, sars-cov- neutralising antibody titres were detected by vnt in and dpv sera from / prime-boost and / prime-only pigs. two weeks after the boost ( dpv), neutralising antibody titres were detected and had increased in all prime-boost pigs, which were significantly greater than the earlier timepoints and the titres measured in the prime-only group (p < . ). in agreement with this analysis, serum assayed for neutralising antibodies using the pvnt revealed that antibody titres in dpv prime-boost pig sera were significantly greater than earlier timepoints and the prime-only group (p < . ). statistical analysis showed a highly significant correlation between pvnt and vnt titres (spearman's rank correlation r = . ; p < . ). in this study, we utilised both a small and a large animal model to evaluate the immunogenicity of either one or two doses of a covid- vaccine candidate, chadox ncov- (now known as azd ). small animal models have variable success in predicting vaccine efficacy in larger animals but are an important stepping stone to facilitate prioritisation of vaccine targets. in contrast, larger animal models, such as the pig and non-human primates, have been shown to more accurately predict vaccine outcome in humans [ ] [ ] [ ] . the mouse data generated in this study suggested that the immunogenicity profile was at the upper end of a dose response curve, which may have saturated the immune response and largely obscured our ability to determine differences between prime-only or prime-boost regimens. we have developed the pig as a model for generating and understanding immune responses to vaccination against human influenza [ ] [ ] [ ] and nipah virus , .the inherent heterogeneity of an outbred large animal model is more representative of immune responses in humans. extensive development of reagents to study immune responses in pigs in recent years has extended the usefulness and applicability of the pig as a model to study infectious disease. these data demonstrate the utility of the pig as a model for further evaluation of the immunogenicity of chadox ncov- and other covid- vaccines. we show here that t cell responses are higher in pigs that received a prime-boost vaccination when compared to prime only at day , whilst comparing responses days after last immunisation demonstrates the prime-boost regimen trended toward a higher response. in addition, chadox ncov- immunisation induced robust th -like cd + and cd + t cell responses in both pigs and mice. this has important implications for covid- vaccine development as virus-specific t cells are thought to play an important role in sars-cov- infection [ ] [ ] [ ] [ ] [ ] . while no correlate of protection has been defined for covid- , recent publications suggest that neutralising antibody titres may be correlated with protection in animal challenge models , . a single dose of chadox ncov- induces antibody responses, but we demonstrate here that antibody responses are significantly enhanced after homologous boost in one mouse strain and to a greater extent in pigs. however, it is likely that a combination of neutralising antibodies and antigen-specific t cells would act in synergy to prevent and control infection, as we have recently shown in the context of influenza vaccination , . whilst human immunogenicity and clinical read-outs are a critically meaningful endpoint, studies in small animals and pigs will help prioritise candidates to be tested in humans. further clinical studies are needed to assess immunogenicity after prime-boost vaccination and the impact on clinical efficacy and durability of the immune response. mouse and pig studies were performed in accordance with the uk animals (scientific procedures) act and with approval from the relevant local animal welfare and ethical review body (mice -project license p b f , and pigs -project license pp ). the principles of the r's were applied for the duration of the study to ensure animal welfare was not unnecessarily compromised. vero e cells were grown in dmem containing sodium pyruvate and l-glutamine (sigma-aldrich, poole, uk), % fbs (gibco, thermo fisher, loughborough, uk), . % penicillin/streptomycin ( , u/ml; gibco) (maintenance media) at °c and % co . sars-cov- isolate england- stocks were grown in vero e cells using a multiplicity of infection (moi) of . for days at °c in propagation media (maintenance media containing % fbs). sars-cov- stocks were titrated on vero e cells using mem (gibco), % fcs (labtech, heathfield, uk), . % avicel (fmc biopolymer, girvan, uk) as overlay. plaque assays were fixed using formaldehyde (vwr, leighton buzzard, uk) and stained using . % toluidine blue (sigma-aldrich). all work with live sars-cov- virus was performed in acdp hg laboratories by trained personnel. the propagation, purification and assessment of chadox ncov- titres were as described previously . a synthetic dna, encoding the spike (s) protein receptor binding domain (rbd; amino acids - ) of sars-cov- (genbank mn ), codon optimised for expression in mammalian cells (idt technology) was inserted into the vector popinttgneo incorporating a c-terminal his tag. recombinant rbd was transiently expressed in expi ™ (thermo fisher scientific, uk) and protein purified from culture supernatants by immobilised metal affinity followed by a gel filtration in phosphate-buffered saline (pbs) ph . buffer. a soluble trimeric s (fl-s) protein construct encoding residues - with two sets of mutations that stabilise the protein in a pre-fusion conformation (removal of a furin cleavage site and the introduction of two proline residues; k p, v p) was expressed as described . the endogenous viral signal peptide was retained at the nterminus (residues - ), a c-terminal t -foldon domain incorporated to promote association of monomers into trimers to reflect the native transmembrane viral protein, and a c-terminal his tag included for nickel-based affinity purification. similar to recombinant rbd, fl-s was transiently expressed in expi ™ (thermo fisher scientific) and protein purified from culture supernatants by immobilised metal affinity followed by gel filtration in tris-buffered saline (tbs) ph . buffer. for analysis of t cell responses in pigs, overlapping mer peptides offset by residues based on the predicted amino acid sequence of the entire s protein from sars-cov- wuhan-hu- isolate (ncbi reference sequence: nc_ . ) were designed and synthesised (mimotopes, melbourne, australia) and reconstituted in sterile % acetonitrile (sigma-aldrich) at a concentration of mg/ml. three pools of synthetic peptides representing residues - (pool ), - (pool ) and - (pool ) were prepared for use to stimulate t cells in ifn-γ elispot and intracellular cytokine staining (ics) assays. for analysis of t cell responses in mice, overlapping mer peptides offset by residues were designed and synthesised (mimotopes) and reconstituted in sterile % dmso (sigma-aldrich) at a concentration of mg/ml. two peptide pools spanning s region (pool : to and - , pool : - ) and peptide pools spanning s region (pool : to , pool : to ) were used for stimulating splenocytes for ifn-γ elispot analysis, and single pools of s (pool and pool ) and s (pool and pool ) were used to stimulate splenocytes for ics. mice: inbred female balb/colahsd (balb/c) (envigo) and outbred crl:cd (cd ) (charles river) of at least weeks of age were randomly allocated into 'prime-only' or 'prime-boost' vaccination groups (balb/c n= and cd n= ). prime-boost mice were immunised intramuscularly with infectious units (iu) ( . x virus particles; vp) chadox ncov- and boosted intramuscularly four weeks later with × iu chadox ncov- . prime-only mice received a single dose of iu chadox ncov- at the same time prime-boost mice were boosted. spleens and serum were harvested from all animals a further weeks later. pigs: six - -week-old, weaned, female, large white-landrace-hampshire cross-bred pigs from a commercial rearing unit were randomly allocated to two treatment groups (n = ): 'prime-only' and 'prime-boost'. both groups were immunised on day with × iu ( . × vp) chadox ncov- in ml pbs by intramuscular injection (brachiocephalic muscle). 'prime-boost' pigs received an identical booster immunisation on day . blood samples were taken from all pigs on a weekly basis at , , , , , and dpv by venepuncture of the external jugular vein: ml/pig in bd sst vacutainer tubes (fisher scientific) for serum collection and ml/pig in bd heparin vacutainer tubes (fisher scientific) for peripheral blood mononuclear cell (pbmc) isolation. mice: antibodies to sars-cov- fl-s protein were determined by performing a standardised elisa on serum collected -weeks after prime or prime-boost vaccination. maxisorp plates (nunc) were coated with ng/well fl-s protein overnight at °c, prior to washing in pbs/tween ( . % v/v) and blocking with blocker casein in pbs (thermo fisher scientific) for hour at room temperature (rt). standard positive serum (pool of mouse serum with high endpoint titre against fl-s protein), individual mouse serum samples, negative and an internal control (diluted in casein) were incubated for hours at rt. following washing, bound antibodies were detected by addition of alkaline phosphatase-conjugated goat anti-mouse igg (sigma-aldrich), diluted / in casein, for hour at rt and detection of anti-mouse igg by the addition of pnpp substrate (sigma-aldrich). an arbitrary number of elisa units were assigned to the reference pool and od values of each dilution were fitted to a -parameter logistic curve using softmax pro software. elisa units were calculated for each sample using the od values of the sample and the parameters of the standard curve. pigs: serum was isolated by centrifugation of sst tubes at × g for minutes at rt and stored at - °c. sars-cov- rbd and fl-s specific antibodies in serum were assessed as detailed previously with the exception of the following two steps. the conjugated secondary antibody was replaced with goat anti-porcine igg hrp (abcam, cambridge, uk) at / , dilution in pbs with . % tween and % non-fat milk. in addition, after the last wash, a µl of tmb (one component horse radish peroxidase microwell substrate, biofx, cambridge bioscience, cambridge, uk) was added to each well and the plates were incubated for minutes at rt. a µl of biofx nmstop reagent (cambridge bioscience) was then added to stop the reaction and microplates were read at nm. end-point antibody titres (mean of duplicates) were calculated as follows: the log od was plotted against the log sample dilution and a regression analysis of the linear part of this curve allowed calculation of the endpoint titre with an od of twice the average od values of dpv sera. the ability of pig sera to neutralise sars-cov- was evaluated using virus and pseudovirus neutralisation assays. for both assays, sera were first heat-inactivated (hi) by incubation at °c for hours. virus neutralization test (vnt): starting at a in dilution, two-fold serial dilutions of sera were prepared in well round-bottom plates using dmem containing % fbs and % antibiotic-antimycotic (gibco) (dilution media). μl of diluted pig serum was mixed with μl dilution media containing approximately plaque-forming units (pfu) sars-cov- for hour at °c. vero e cells were seeded in -well flat-bottom plates at a density of × cells/ml in maintenance media one day prior to experimentation. culture supernatants were replaced by µl of dmem containing % fcs and % antibiotic-antimycotic, before µl of the virus-sera mixture was added to the vero e cells and incubated for six days at °c. cytopathic effect (cpe) was investigated by brightfield microscopy. cells were further fixed and stained as described above, and cpe scored. each individual pig serum dilution was tested in quadruplet on the same plate and no sera/sars-cov- virus and no sera/no virus controls were run concurrently on each plate in quadruplet. wells were scored for cytopathic effect and neutralisation titres expressed as the reciprocal of the serum dilution that completely blocked cpe in % of the wells (nd ). researchers performing the vnts were blinded to the identity of the samples. pseudovirus neutralisation test (pvnt): lentiviral-based sars-cov- pseudoviruses were generated in hek t cells incubated at °c, % co . cells were seeded at a density of . x in well dishes, before being transfected with plasmids as follows: ng of sars-cov- spike, ng p . (encoding for hiv- gag-pol), ng csflw (lentivirus backbone expressing a firefly luciferase reporter gene), in opti-mem (gibco) along with µl pei ( µg/ml) transfection reagent. a 'no glycoprotein' control was also set up using carrier dna (pcdna . ) instead of the sars-cov- s expression plasmid. the following day, the transfection mix was replaced with ml dmem with % fbs (dmem- %) and incubated at °c. at both and hours post transfection, supernatants containing pseudotyped sars-cov- (sars-cov- pps) were harvested, pooled and centrifuged at , x g for minutes at °c to remove cellular debris. target hek t cells, previously transfected with ng of a human ace expression plasmid (addgene, cambridge, ma, usa) were seeded at a density of × in µl dmem- % in a white flat-bottomed -well plate one day prior to harvesting of sars-cov- pps. the following day, sars-cov- pps were titrated -fold on target cells, with the remainder stored at - °c. for pvnts, pig sera were diluted : in serum-free media and µl was added to a -well plate in quadruplicate and titrated fold. a fixed titred volume of sars-cov- pps was added at a dilution equivalent to signal luciferase units in µl dmem- % and incubated with sera for hour at °c, % co . target cells expressing human ace were then added at a density of x in µl and incubated at °c, % co for hours. firefly luciferase activity was then measured with brightglo luciferase reagent and a glomax-multi + detection system (promega, southampton, uk). pseudovirus neutralization titres were expressed as the reciprocal of the serum dilution that inhibited luciferase expression by % (ic ). mice: single cell suspension of mouse spleens were prepared by passing cells through μm cell strainers and ack lysis (thermo fisher) prior to resuspension in complete media (mem supplemented with % fcs, pen-step, l-glut and -mercaptoethanol). for analysis of ifn-γ production by elispot assay, splenocytes were stimulated with s peptide pools at a final concentration of g/ml on ipvh-membrane plates (millipore) coated with g/ml anti-mouse ifn-γ (clone an ; mabtech). after - hours of stimulation, ifn-γ spot forming cells (sfc) were detected by staining membranes with anti-mouse ifn-γ biotin mab ( µg/ml; clone r a , mabtech) followed by streptavidin-alkaline phosphatase ( µg/ml) and development with ap conjugate substrate kit (bio-rad). for analysis of intracellular cytokine production, cells were stimulated with μg/ml s peptide pools, media or cell stimulation cocktail (containing pma-ionomycin, biolegend), together with μg/ml golgiplug (bd biosciences) and μl/ml cd a-alexa for hours in a -well u-bottom plate, prior to placing at o c overnight. following surface staining with cd -buv , cd -percp-cy . , cd l-bv , cd -bv , cd -apc-cy and live/dead aqua (thermo fisher), cells were fixed with % neutral buffered formalin (containing % paraformaldehyde) and stained intracellularly with tnf--af , il- -pe-cy , il- -bv , il- -pe and ifn-γ-e diluted in perm-wash buffer (bd biosciences). sample acquisition was performed on a fortessa (bd) and data analysed in flowjo v (treestar). an acquisition threshold was set at a minimum of events in the live cd + gate. antigen-specific t cells were identified by gating on live/dead negative, doublet negative (fsc-h vs fsc-a), size (fsc-h vs ssc), cd + , cd + or cd + cells and cytokine positive. total sars-cov- s specific cytokine responses are presented after subtraction of the background response detected in the media stimulated control spleen sample of each mouse, prior to summing together the frequency of s and s specific cells. pigs: pbmcs were isolated from heparinised blood by density gradient centrifugation and cryopreserved in cold % dmso (sigma-aldrich) in hi fbs . resuscitated pbmc were suspended in rpmi medium, glutamax supplement, hepes (gibco) supplemented with % hi fbs (new zealand origin, life science production, bedford, uk), % penicillin-streptomycin and . % -mercaptoethanol ( mm; gibco) (crpmi). to determine the frequency of sars-cov- s specific ifn-γ producing cells, an elispot assay was performed on pbmc from , , and dpv. multiscreen -well plates (mahas ; millipore, fisher scientific) were pre-coated with µg/ml anti-porcine ifn-γ mab (clone p g , bd biosciences) and incubated overnight at °c. after washing and blocking with crpmi, pbmcs were plated at × cells/well in crpmi in a volume of µl/well. pbmcs were stimulated in triplicate wells with the sars-cov- s peptide pools at a final concentration of µg/ml/peptide. crpmi alone was used in triplicate wells as a negative control. after hours incubation at °c with % co , plates were developed as described previously . the numbers of specific ifn-γ secreting cells were determined using an immunospot ® s analyzer (cellular technology, cleveland, usa). for each animal, the mean 'crpmi only' data was subtracted from the s peptide pool , and data which were then summed and expressed as the mediumcorrected number of antigen-specific ifn-γ secreting cells per x pbmc. to assess intracellular cytokine expression pbmc from and dpv were suspended in crpmi at a density of × cells/ml and added to µl/well to -well round bottom plates. pbmcs were stimulated in triplicate wells with the sars-cov- s peptide pools ( µg/ml/peptide). unstimulated cells in triplicate wells were used as a negative control. after hours incubation at °c, % co , cytokine secretion was blocked by addition : , bd golgiplug (bd biosciences) and cells were further incubated for hours. pbmc were washed in pbs and surface labelled with zombie nir fixable viability stain (biolegend), cd -percp-cy . mab (clone - - , bd bioscience) and cd β-fitc mab (clone ppt , bio-rad antibodies). following fixation (fixation buffer, biolegend) and permeabilization (permeabilization wash buffer, biolegend), cells were stained with: ifn-γ-af mab (clone cc , bio-rad antibodies, kidlington, uk), tnf-α-bv mab (clone mab , biolegend), il- mab (clone a d f h , invitrogen, thermo fisher scientific) and il- bv mab (clone mp - d , biolegend) followed by staining with anti-mouse igg a-pe-cy (clone rmg a- , biolegend). cells were analysed using a bd lsrfortessa flow cytometer and flowjo x software. total sars-cov- s specific cytokine positive responses are presented after subtraction of the background response detected in the media stimulated control pbmc sample of each pig, prior to summing together the frequency of s-peptide pools - specific cells. graphpad prism . . (graphpad software, san diego, usa) was used for graphical and statistical analysis of data sets. anova or a mixed-effects model were conducted to compare responses over time and between vaccine groups at different time points post-vaccination as detailed in the results. neutralising antibody titre data were log transformed before analysis. neutralising antibody titre data generated by the vnt and pvnt assays were compared using spearman nonparametric correlation. p-values < . were considered statistically significant. were immunised on day and with chadox ncov (prime-boost) or chadox ncov on day (prime-only); pigs (n= ) were immunised with chadox ncov- on days and (primeboost), or only on day (prime-only). to analyse sars-cov- s-specific t cell responses, all mice were sacrificed on day for isolation of splenocytes and pigs were blood sampled longitudinally to isolate pbmc. following stimulation with sars-cov- s-peptides, responses of murine splenocytes (a) and porcine pbmc (c) were assessed by ifn-γ elispot assays. using flow cytometry, cd + and cd + t cell responses were characterised by assessing expression of ifn-γ, tnf-α, il- , il- and il- (mice; b) and ifn-γ, tnf-α, il- and il- (pigs; d). each data point represents an individual mouse/pig with bars denoting the median response per group/timepoint. : sars-cov- s protein-specific antibody responses following chadox ncov- primeonly and prime-boost vaccination regimens in mice and pigs. inbred balb/c (n= ) and outbred cd (n= ) were immunised on day and with chadox ncov (prime-boost) or chadox ncov on day (prime-only), whereas, pigs were immunised with chadox ncov- on days and (prime-boost), or only on day (prime-only). to analyse sars-cov- s protein-specific antibodies in serum, all mice were sacrificed on day and pigs were blood sampled weekly until day . antibody units or end-point titres (ept) were assessed by elisa using recombinant sars-cov- fl-s for both mice (a) and pigs (b), and recombinant s protein rbd for pigs (c). sars-cov- neutralising antibody titres in pig sera were determined by vnt, expressed as the reciprocal of the serum dilution that neutralised virus infectivity in % of the wells (nd ; d) , and pvnt, expressed as reciprocal serum dilution to inhibit pseudovirus entry by % (ic ; e). each data point represents an individual mouse/pig sera with bars denoting the median titre per group. a single dose of chadox mers provides broad protective immunity against a variety of mers-cov strains. biorxiv chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques. biorxiv a single dose of chadox mers provides protective immunity in rhesus macaques antigen encoded by vaccine vectors derived from human adenovirus serotype is preferentially presented to cd + t lymphocytes by the cd α+ dendritic cell subset immunization with an adenovirus-vectored tb vaccine containing ag a-mtb effectively alleviates allergic asthma the pig: a model for human infectious diseases large animal models for vaccine development and testing the contribution of non-human primate models to the development of human vaccines comparison of heterosubtypic protection in ferrets and pigs induced by a single-cycle influenza vaccine immunogenicity and protective efficacy of seasonal human live attenuated cold-adapted influenza virus vaccine in pigs aerosol delivery of a candidate universal influenza vaccine reduces viral load in pigs challenged with pandemic h n virus vaccine development for nipah virus infection in pigs bovine herpesvirus- -vectored delivery of nipah virus glycoproteins enhances t cell immunogenicity in pigs. vaccines (basel) targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid- patients clinical and immunological features of severe and moderate coronavirus disease transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients presence of sars-cov- reactive t cells in covid- patients and healthy donors. medrxiv sars-cov- infection protects against rechallenge in rhesus macaques dna vaccine protection against sars-cov- in rhesus macaques vaccination with viral vectors expressing np, m and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice a serological assay to detect sars-cov- seroconversion in humans this study was supported by engineering sarah gilbert and teresa lambe are named on a patent application covering chadox ncov- . the remaining authors declare no competing interests. the funders played no role in the conceptualisation, design, data collection, analysis, decision to publish, or preparation of the manuscript. correspondence and material requests to professor simon p. graham (simon.graham@pirbright.ac.uk) and professor teresa lambe (teresa.lambe@ndm.ox.ac.uk). key: cord- -u gt fh authors: teijaro, j.r. title: pleiotropic roles of type interferons in antiviral immune responses date: - - journal: adv immunol doi: . /bs.ai. . . sha: doc_id: cord_uid: u gt fh since isaac's and lindenmann's seminal experiments over years ago demonstrating a soluble factor generated from heat killed virus-stimulated chicken embryos could inhibit live influenza virus replication, the term interferon has been synonymous with inhibition of virus replication. while the antiviral properties of type interferon (ifn-i) are undeniable, recent studies have reported expanding and somewhat unexpected roles of ifn-i signaling during both acute and persistent viral infections. ifn-i signaling can promote morbidity and mortality through induction of aberrant inflammatory responses and recruitment of inflammatory innate immune cell populations during acute respiratory viral infections. during persistent viral infection, ifn-i signaling promotes containment of early viral replication/dissemination, however, also initiates and maintains immune suppression, lymphoid tissue disorganization, and cd t cell dysfunction through modulation of multiple immune cell populations. finally, new data are emerging illuminating how specific ifn-i species regulate immune pathology and suppression during acute and persistent viral infections, respectively. systematic characterization of the cellular populations that produce ifn-i, how the timing of ifn-i induction and intricacies of subtype specific ifn-i signaling promote pathology or immune suppression during acute and persistent viral infections should inform the development of treatments and modalities to control viral associated pathologies. many viruses harbor viral proteins with specific functions geared toward preventing ifn-i production and/or signaling, highlighting the evolutionary selective pressure exerted by ifn-i during viral replication (devasthanam, ) . the absence of ifn-i signaling during acute virus infection in vivo increases virus replication, dissemination, and lethality during multiple viral infections in animal models. global deletion of ifnar results in enhanced mortality during vesicular stomatitis virus (vsv), vaccinia virus (vv), west nile virus (wnv), and lymphocytic choriomeningitis virus (lcmv) infections (muller et al., ) . moreover, infection of ifnar ko mice with acute lcmv armstrong (arm) (nakayama et al., ; zhou, cerny, fitzgerald, kurt-jones, & finberg, ) and treatment of arm-infected mice with an ifnar neutralizing antibody elevated viral loads and promoted virus persistence (teijaro et al., ; wilson et al., ) . dendritic cell-specific deletion of ifnar results in elevated virus replication and systemic persistence of the cw strain of murine norovirus (mnov) despite increased cell-mediated and humoral adaptive immune responses (nice et al., ) . ifn-i signaling has been shown to be essential for controlling wnv infection and restricting viral pathogenesis (sheehan, lazear, diamond, & schreiber, ) . mice deficient in ifnar signaling display increased susceptibility to wnv infection (pinto et al., ; samuel & diamond, ) . during infection with the coronavirus, mouse hepatitis virus (mhv-a ), the magnitude of the ifn-i and -ii responses directly correlated with viral loads (raaben, koerkamp, rottier, & de haan, ). moreover, ifn-i produced by plasmacytoid dendritic cells (pdcs) was essential to control virus replication and prevent mortality following mhv-a infection in mice (cervantes-barragan et al., ) . during experimental infection of mice and nonhuman primates with the lassa hemorrhagic fever virus, delayed or reduced induction of ifn-i and downstream gene signatures correlated with high viral loads and fatal outcome (baize et al., ; yun et al., ) . deletion of ifn-i related signaling pathways during respiratory virus infections in animal models results in diverse effects depending on the virus strain and genetic background (durbin et al., ; price, gaszewska-mastarlarz, & moskophidis, ) . in the context of respiratory viral infection, genetic deletion of stat reduced virus control, enhanced pathology, and mortality during sars-cov and influenza virus infection (durbin et al., ; frieman et al., ) . interestingly, stat -deficient animals were highly susceptible to influenza virus infection, displaying elevated viral titers and increased pathology compared to stat -sufficient mice. studies in mouse models of influenza virus have revealed conflicting evidence for the role of ifnar in controlling influenza virus replication, morbidity, and mortality. infection of ifnar À/À mice with the pr strain of influenza virus resulted in altered recruitment of ly c hi vs ly c int monocytes in the lung, translating into increased production of the neutrophil chemoattractant, kc (cxcl ), elevated numbers of neutrophils in the lung and increased morbidity and mortality (seo et al., ) . therefore, modulation of type interferon signaling and production needs to be balanced to have enough to control virus infection but not promote excessive inflammation. the discrepancy between influenza pathogenicity in ifnar and stat -deficient mice was later clarified when animals lacking both ifnar /ifn-λ were unable to control influenza virus replication. this is further supported in humans where null mutations in the human interferon regulatory factor- gene results in reduced ifn-i and -iii production from myeloid dcs and pdcs and life-threatening seasonal influenza virus infection (ciancanelli et al., ) . exposure of bone marrow cells to ifn-i prior to their recruitment to lung endows these cells with an antiviral program that protects from virus infection after entry into the infected lung (hermesh, moltedo, moran, & lopez, ) . deletion of the ifn-β or ifnar genes in mice with a functional mx gene increased virus replication and reduced the ld -fold (koerner, kochs, kalinke, weiss, & staeheli, ) . infection of ifnar -deficient mice with low dose mouse adapted h n influenza viruses resulted in mortality, elevated viral loads, exacerbated lung pathology, and reduced numbers of il- -producing cells as compared to ifnar -sufficient controls (arimori et al., ) . moreover, exogenous administration of il- to ifnar -deficient animals following influenza virus infection partially restored survival and ameliorated lung pathology. thus, ifn-i can be protective during influenza virus infection either through suppressing virus spread or prompting induction of immune-suppressive cytokines to reign in excessive inflammation. in addition to directly inhibiting virus propagation, ifn-i also has potent immune stimulatory functions which support the resolution of virus infection. ifn-i promotes upregulation of mhc-i expression in multiple cell lineages (lindahl, gresser, leary, & tovey, a , b , which is required for optimal t cell stimulation, differentiation, expansion, and killing of virus-infected cells. autocrine signaling of ifn-i on dendritic cells promotes their activation and t cell stimulatory capacity (montoya et al., ) . ifn-i signaling during virus infection promotes conversion of pdcs into myeloid derived dcs and impairs hematopoietic differentiation of bone marrow progenitors into dcs (sevilla, mcgavern, teng, kunz, & oldstone, ; zuniga, mcgavern, pruneda-paz, teng, & oldstone, ) . following exposure to ifn-i, metallophilic macrophages induce expression of the usp protein which prevents jak phosphorylation and inhibits ifn-i signaling in these cells. in turn, repression of ifn-i signaling allows for restricted virus replication in these macrophages, promoting the production of viral antigens which are recognized by b cells, the final result is the facilitation of antiviral antibody generation and enhanced virus control (honke et al., ) . ifn-i also exerts potent costimulatory effects directly on cd t cells, enhancing cd t cell proliferation upon ifnar signaling (curtsinger, valenzuela, agarwal, lins, & mescher, ; kolumam, thomas, thompson, sprent, & murali-krishna, ) . the timing of cd t cell exposure to ifn-i significantly influences the differentiation and magnitude of the response (welsh, bahl, marshall, & urban, ) . exposure of naïve cd t cells to apc and ifn-i prior to antigenic stimulation promotes the maintenance of a naïve phenotype with reduced proliferation despite production of effector cytokines. direct ifn-i signaling on naïve and memory t cells promotes rapid apoptosis, inhibits proliferation, and promotes early effector differentiation of memory cells upon exposure. blockade of ifn-i signaling during wnv infection has significant effects on t cell expansion, cytokine production, and differentiation when administered during the maturation phase of the t cell response, however, had no effect when given prior to infection (pinto et al., ) . moreover, low dose priming with the vv ankara strain had little effect on effector or memory t cell recall in ifnar À/À mice (volz, langenmayer, jany, kalinke, & sutter, ) . in addition to t cells, ifn-i signaling is known to be important for nk cell function. ifn-i signaling promotes nk cell cytolytic capacity and survival during acute viral infection (hwang et al., ; martinez, huang, & yang, ; nguyen et al., ) and was recently reported to protect antiviral cd t cells from nk cell lytic effects (crouse et al., ; xu et al., ) . reconstitution of ifnar À/À mice with ifnar +/+ nk cells restored early control of vv infection in vivo (martinez et al., ) , suggesting that nk cell intrinsic ifnar signaling is important for early control of vv replication. moreover, direct ifn-i signaling on nk cells was required to induce nk cell ifn-γ production during acute lcmv infection. early ifn-γr signaling was required for promoting initial virus control in the peritoneum (mack, kallal, demers, & biron, ) , suggesting that ifn-i signaling directly on nk cells promotes virus control during acute lcmv infection. ifn-i signaling during viral infection can also signal to regulatory t cells and subsequently alter their suppressive functions. it was recently demonstrated that ifnar signaling on foxp + tregs limits their suppressive function during acute lcmv infection, thus promoting virus control (srivastava, koch, pepper, & campbell, ) . deletion of ifnar on foxp + cells blunted virus-specific t cell responses and elevated virus loads. thus, ifn-i signaling on suppressive t cell populations temporarily suspends suppressive function and allows for optimal antiviral t cell responses during an ongoing viral infection. similar to effects on t cells, ifn-i signaling has both positive (le bon et al., ) and negative effects on antiviral b cell responses. the survival and maturation of immature b cells can be inhibited by ifn-i signaling (lin, dong, & cooper, ) . in contrast to immature b cells, ifn-i signaling promotes b cell activation, antibody production, and isotype switch following influenza, vsv, and wnv infection (coro, chang, & baumgarth, ; fink et al., ; purtha, chachu, virgin, & diamond, ; rau, dieter, luo, priest, & baumgarth, ). however, it was also reported that influenza virus-specific antibody levels were elevated at later time points following influenza virus challenge in ifnar -deficient mice compared to ifnar -sufficient controls (price et al., ) . during acute lcmv infection, blockade of ifn-i signaling in both wild-type and stat -deficient mice enhanced t follicular helper cell (t fh ), germinal center b cell differentiation, and anti-lcmv antibody responses (ray et al., ) . elevated antibody responses during acute viral infections following ifnar blockade suggest that, in certain circumstances, ifn-i signaling can restrain optimal antiviral antibody responses. the correlation of an aggressive immune response and severe disease following influenza virus infection in humans and animal models has been discussed previously (la gruta, kedzierska, stambas, & doherty, ). an aggressive innate response, with elevated recruitment of inflammatory leukocytes to lung, likely contributed to the morbidity of the influenza infection (ahmed, oldstone, & palese, ; kobasa et al., ) . in fact, lung injury during infection of macaques with the h n influenza virus strain directly correlated with early dysregulated inflammatory gene expression, including elevated ifn-i signatures (cilloniz et al., ; kobasa et al., ) . more recently, clinical studies on avian h n -infected humans documented a significant association between excessive early cytokine responses and immune cell recruitment as predictive of poor outcome (de jong et al., ). an aberrant cytokine/chemokine response was observed in patients with severe disease during the most recent h n pandemic in (arankalle et al., ) . type i interferon signaling is well known to inhibit influenza virus replication and spread (garcia-sastre & biron, ) . the production of the ns protein, one of viral proteins, acts to inhibit type interferon production and signaling (hale, randall, ortin, & jackson, ) , suggesting that ifn-i signaling exerts substantial selection pressure on virus fitness. deletion or mutation of the ns gene results in significant increases in the levels of type interferon in infected cells and significantly lower virus titers both in vitro and in vivo (garcia-sastre, egorov, et al., ; jiao et al., ; kochs, garcia-sastre, & martinez-sobrido, ) . despite strong evidence demonstrating extensive antiviral properties of ifn-i, several studies also suggest pathogenic roles for ifn-α during influenza virus infection. the production of several proinflammatory cytokines and chemokines is known to be amplified by ifn-i receptor signaling. in addition to protective effects of ifn-i signaling, pathogenic roles for ifn-i have been reported during influenza virus infection ( fig. a) . appearance of ifn-α in lavage fluid directly coincides with symptom onset during human experimental influenza virus infection (hayden et al., ) , suggesting that ifn-i signaling and pathological responses in humans temporally coincide. recently, it was paradoxically reported that deletion of ifnar or depletion of pdcs in svev mice inhibited pulmonary pathology and improved survival following lethal influenza virus challenge (davidson, crotta, mccabe, & wack, ) . reduced immune pathology and enhanced survival in mice deficient in ifn-i signaling transpired without significant increases in viral loads or impediment of eventual viral clearance (fig. b) . in contrast to deletion of ifn-i signaling, treatment of influenza virus-infected mice with ifnα resulted in enhanced morbidity and mortality; thus, ifn-i can promote pathological consequences during acute influenza virus infection. over the past years, we identified that therapeutic administration of sphingosine phosphate (s p) analogs early during influenza virus infection in mice resulted in reduced morbidity and mortality . s p is a lipid metabolite converted from ceramide precursors to sphingosine. fig. ifn-i signaling enhances cytokine/chemokine amplification, innate immune cell recruitment, and immune pathology during respiratory viral infections. (a) viral infection in the lung with influenza or sars-cov promotes the induction of delayed ifn-i production which enhances cytokine/chemokine production, recruitment of nk cells, and neutrophils and inflammatory macrophage/monocytes all which contribute to lung immune-mediated pathology. (b) blockade or genetic deletion of ifnar blunts cytokine/chemokine amplification, inhibits recruitment of nk cells, neutrophils, and inflammatory macrophages/monocytes resulting in reduced immunopathology, and improved survival. treatment of mice with s p r agonists early during influenza virus infection suppresses ifn-i amplification from plasmacytoid dendritic cells which lowers ifn-i levels. the end result is blunting of cytokine/chemokine amplification, inhibition of nk cell, neutrophil, and inflammatory macrophage/monocyte recruitment into the lung, reduced immunopathology, and improved survival. the subsequent phosphorylation by sphingosine kinase and produces bioactive s p in vivo where it acts on s p-specific g-protein couples receptors (gpcrs) (chalfant & spiegel, ) . the levels of bioactive s p are regulated through the actions of s p phosphatases and lyases which dephosphorylate and degrade s p, respectively. highest levels of s p are found in the blood and lymph with significantly lower levels maintained in peripheral tissues (cyster, ) . s p binds and signals through five gpcrs denoted as s pr - which couple to various g-protein signaling effectors. the expression of s p receptors is heterogeneous, being found on both hematopoietic and nonhematopoietic lineages (im, ) . the functional coupling to multiple heterotrimeric g-proteins promote the diverse cellular functions associated with s p receptor signaling. signaling through these five receptors is known to modulate multiple cellular processes including: cell adhesion, migration, survival, proliferation, endocytosis, barrier function, and cytokine production (rivera, proia, & olivera, ) . recently, we identified a novel regulatory function of s pr signaling in blunting early cytokine amplification and innate immune cell recruitment following influenza virus infection (fig. b) . early administration of a promiscuous s pr agonist, aal-r, or an s p r-selective agonist (cym- ) significantly blunted production of multiple pro-inflammatory cytokines and chemokines following infection with either wsn or human pandemic h n influenza virus walsh et al., ) . further, both aal-r-and cym- -mediated reduction of early innate immune cell recruitment and cytokine/chemokine production correlated directly with reduced lung pathology and improved survival during h n influenza virus infection. while these s pr agonists clearly inhibited innate immune responses, significant inhibition of activated t cell recruitment into the lung at various times post infection occurred in mouse adapted (marsolais et al., ) and human pathogenic strains of influenza virus . the above findings were extended using genetic and chemical tools to probe functions of the s p receptor (s p gfp knockin transgenic mice, s p receptor agonists and antagonists), revealing that pulmonary endothelial cells modulate innate immune cell recruitment and cytokine/chemokine responses early following influenza virus infection . importantly, s p r agonist treatment blunted cytokine/chemokine production and innate immune cell recruitment in the lung independently of endosomal and cytosolic innate sensing pathways (teijaro, walsh, rice, rosen, & oldstone, ) . further, s p r signaling suppression of cytokine amplification was independent of multiple innate signaling adaptor pathways but required the myd adaptor for cytokine amplification following influenza virus challenge. immune cell infiltration and cytokine production were found to be distinct events, both orchestrated by signaling through the s p r. suppression of early innate immune responses through s p r signaling also reduced mortality during infection with human pathogenic strains (h n / swine) of influenza virus in a ferret model, demonstrating that s pr -mediated blunting of influenza virus pathogenesis in mice could be extended to a model more closely resembling human disease. the link between s pr and ifn-α amplification following influenza virus infection was striking. in fact, the absence of ifnar abolished cytokine amplification and the capacity of s p r agonists to further blunt cytokine/chemokine responses (teijaro et al., . to understand how s pr signaling regulates ifn-α and cytokine amplification, we assessed the pulmonary cell subsets that produce ifn-α and cytokines/chemokines following influenza virus challenge. expression of s p r was quickly observed in purified pdcs; moreover, s p r agonists suppressed ifn-i induction/amplification from both mouse and human pdcs following influenza virus simulation (teijaro et al., ) . further mechanistic studies revealed that s p r agonist-mediated suppression was independent of gi/o signaling and required signaling through the s p r c-terminus. biochemically, s p r agonists accelerated the turnover of ifnar and promoted trafficking to lysosomes for degradation, abrogating stat phosphorylation, blunting the ifn-i autoamplification loop. the fact that ifn-i production/signaling can down modulate s pr expression/activity indirectly through upregulation of cd which promotes internalization of s pr in t cells is significant (shiow, ) and suggests that s p r and ifn-i signaling are closely linked and capable of counter regulating one another. an additional study also reported ifn-i modulation in pdcs via other s prs (dillmann et al., ) , suggesting that this phenomenon could be more promiscuous than originally thought. similar to influenza virus infection, aberrant innate cytokine/chemokine responses and immune cell recruitment into lungs correlate with disease severity in human patients (huang et al., ) . ifn-i signaling during murine sars-cov infection appears to be dispensable for virus control while also potentiating immune pathology. however, the role ifn-i signaling plays in this pathology has only recently been systematically addressed. deletion of ifnar in mice does not mirror the enhanced viral loads or pathological consequences observed in stat À/À mice in sars-cov infection, suggesting an ifnar -independent stat- -dependent pathway is necessary for controlling sars-cov (frieman et al., ) . this study provocatively suggests that ifn-i signaling is dispensable for controlling sars-cov replication in vivo. recently, an important study was published where the authors further highlighted the importance of ifn-i signaling in respiratory virus pathology by reporting that delayed ifn-i induction and signaling during sars-cov infection in mice promoted the development and infiltration of inflammatory monocyte-macrophages into the lung, resulting in exacerbated lung pathology and lethal pneumonia (channappanavar et al., ) . attenuation of ifn-i signaling either through genetic deletion or through antibody neutralization of ifnar prevented inflammatory monocyte-macrophage infiltration into the lung, abrogated lung immune pathology, and resulted in mild clinical disease. importantly, genetic deletion or blockade of ifn-i signaling resulted in control of viral loads similar to control animals, reinforcing that ifn-i signaling is dispensable for control of sars-cov infection in vivo. one possibility is that in the absence of ifn-i signaling, induction of an ifn-iii (ifn-λ) antiviral program may effectively limit viral replication. the results found in this study were strikingly similar to those found in influenza virus-infected svev mice and suggest that strategic modulation of ifn-i signaling could ameliorate pathologies associated with severe respiratory virus infection. collectively, the studies above suggest that ifn-i signaling is essential to cytokine and chemokine amplification and innate immune cell recruitment and can promote excessive immunopathology during acute respiratory viral infections (fig. ) . importantly, that ifn-i production and signaling can be blunted without enhancing virus propagation following acute respiratory viral infection suggests that this pathway can be modulated without compromising host antiviral responses. the correlation between blunting ifn-i signaling, lessened immune pathology, and improved survival during multiple respiratory viral infections highlight the need to mechanistically dissect how ifn-i promotes immune pathology during these infections. the role of ifn-i signaling in restraining chronic/persistent viral infection is well documented. inhibition of ifn-i signaling by antibody blockade of ifnar results in elevated virus replication early following lcmv cl infection and treatment of mice with ifn-i during the early stages of persistent lcmv infection promotes rapid virus control (wang et al., ) . mechanistically, ifn-i therapy increased expansion of virus-specific cd t cells and prevented t cell exhaustion; however, whether this was due to ifn-i-mediated immune stimulatory effects, lowering of antigen levels, or both was not systematically addressed. an additional study reported that deletion of the - oligoadenylate synthetase-like gene prior to lcmv cl infection facilitated sustained ifn-i production/signaling, promoted t cell expansion, reduced t cell exhaustion, and promoted rapid virus control (lee, park, jeong, kim, & ha, ) . similar to persistent lcmv infection, ifn-i administration can exert protective effects through slowing siv replication and disease progression if administered early following infection (sandler et al., ) and has shown some efficacy in patients with persistent hiv infection (asmuth et al., ; azzoni et al., ) . moreover, treatment with pegylated ifn-α in conjunction with the antiviral drug ribavirin was the standard of care for treating patients with chronic hepatitis c virus (hcv) infection until recently (heim, ; moreno-otero, ) . however, despite success in hcv therapy, the modest efficacy observed following ifn-α administration requires ribavirin and, even in combination, only a slim majority of patients respond. moreover, patients who fail to control hcv following ifn-i therapy were reported to express a higher ifn-i gene signature prior to treatment (sarasin-filipowicz et al., ) . similar trends were observed following ifn-i administration during hiv and siv infections, where ifn-i administration had only the modest effects if given during established persistent infection (asmuth et al., ; hubbard et al., ) . the reasons for the discrepancies observed in human persistent viral infections, where ifn-i therapy can promote control ( - % of hcv patients) while in others (during established hiv infection) minimal benefit is observed, remain unknown. one could imagine a scenario where in some persistently infected hcv patients, elevated ifn-i signatures persist, and addition of pegylated ifn-α provides minimal benefit while patients with lower ifn-i signatures respond to the therapy. whether treatment with pegylated ifn-α earlier during infection (prior to sustained ifn-i signatures) would be beneficial would be interesting to discern. a similar profile appears to exist in persistent siv infection, where early administration of ifn-i promotes control of viral loads and pathogenesis, while later administration has modest effects on viral titers and disease outcome. during infection with a model gamma herpesvirus, mhv , the lack of ifn-i signaling exacerbated virus replication, increased reactivation from latency, and resulted in enhanced morbidity and mortality (barton, lutzke, rochford, & virgin, ; dutia, allen, dyson, & nash, ) . taken together, ifn-i therapy may be beneficial during the early stages of persistent, latent chronic viral infection, or infections with lower ifn-i signatures; however, blocking ifn-i signaling either alone or in conjunction with antiviral or immune checkpoint therapies may prove more effective once virus persistence and elevated ifn-i signatures are established. however, the ultimate outcome will likely depend on the persistent virus studied, genetic susceptibilities of individuals, and subtype and timing of ifn-i species produced; all which require further investigation. moreover, given the undesirable side effects of ifn-i administration, ifn therapy can do as much harm as good during viral infection, highlighting the need for developing alternative approaches to treat persistent viral infections. during persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. elevated ifn-i signatures have been observed during lcmv infection in mice (hahm, trifilo, zuniga, & oldstone, ) and hiv and hcv infections in humans and nonhuman primates (bosinger et al., ; jacquelin et al., ; wieland et al., ) . chronic immune activation following hiv infection has been reported, and suppression of this hyperactivated state has been proposed as a potential strategy to alleviate hiv-associated pathologies (boasso, hardy, anderson, dolan, & shearer, ; d'ettorre, paiardini, ceccarelli, silvestri, & vullo, ) . disease following experimental siv infection in rhesus macaques correlates with elevated ifn-i production and inflammatory signatures (jacquelin et al., ; manches & bhardwaj, ). in contrast, siv infection in sooty mangabeys and african green monkeys, which develop modest pathology despite equivalent viral loads as macaques, correlate with reduced ifn-i and inflammatory gene signatures (bosinger et al., ) . similar correlations with respect to reduced immune activation exist in hiv-infected elite controllers, although whether reduced immune activation follows virus control is uncertain (deeks & walker, ; saez-cirion et al., ) . blockade of pd- signaling during chronic siv infection reduces hyperimmune activation and microbial translocation in rhesus macaques and lowers ifn-i signatures in the blood and colon (dyavar shetty et al., ) . moreover, an elevated interferon signature is observed in hcv-infected patients despite limited control of virus replication and development of liver pathology (guidotti & chisari, ; su et al., ; wieland et al., ) . in fact, hcv infection in culture blocks isg protein expression through activation of rna-dependent protein kinase (garaigorta & chisari, ) , creating a paradoxical ifn-i-dependent viral advantage. thus, ifn-i signaling pathways have the potential to aid viral fitness and promote pathology during persistent viral infection. these studies further highlight the viability of the ifn-i signaling system as a target to promote control of persistent viral infection. while the literature suggests a causative role for ifn-i in contributing to pathogenesis of persistent virus infections, definitive studies assessing how ifn-i neutralization affects the outcome of virus persistence were lacking until recently. two laboratories assessed the role ifn-i signaling plays during persistent infection using the lcmv clone- (cl ) strain of virus. during their investigation, they found that blockade of ifn-i signaling using an ifnar neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules il- and pd-l and restored lymphoid architecture in mice persistently infected with lcmv (fig. ) . importantly, blockade of ifnar both prior to and following established persistent lcmv infection promoted faster virus clearance and required an intact cd t cell compartment (teijaro et al., ; wilson et al., ) . blockade of ifn-i signaling significantly enhanced cd t cell differentiation into th effectors as well as increased t fh cell differentiation (osokine et al., ) . the above studies demonstrate for the first time a direct causal link between ifn-i signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and long-term virus persistence. more recently, it was reported that during cl infection, both type i and ii interferon promoted the induction and suppressive capacity of cd + cd + immune regulatory dcs (iregdcs), respectively (cunningham et al., ) . while ifn-γ promoted the differentiation of iregdcs from monocytes, ifn-i promoted the suppressive functions of iregdcs. genetic deletion of ifnar prevented the expression of pd-l and production of il- from iregdcs, relieving their suppressive capabilities. in addition to modulating the suppressive capacity of iregdcs, ifn-i signaling also limited their generation/expansion. during mnov infection, selective genetic deletion of ifnar in dcs increased expression of the cellular activation markers cd , cd , and mhcii, suggesting that direct ifn-i signaling on dcs may be responsible for restraining dc function in vivo (nice et al., ) . generation of elevated numbers of iregdcs was also observed during hiv and mycobacterium tuberculosis infections as well as cancer, suggesting that iregdc generation is common in immunosuppressive environments. the ifn-i-driven immune-suppressive state during persistent lcmv infection also inhibits macrophage function. a recent study found that mice infected with the persistent docile strain of lcmv have impaired humoral immune responses to a superinfecting vsv infection (honke et al., ) . the absence of virus replication in cd + macrophages was not due to antiviral cd t cell-mediated killing of cd + macrophages but instead the result of sustained ifn-i responses and an elevated ifn-i antiviral gene program. in turn, reduction in vsv replication and antigen production in cd + macrophages reduced antigen production in these cells which was essential for antiviral antibody generation. the existence of multiple ifn-i subspecies ( ifn-α species in mice and in humans in addition to ifn-β) suggests that either the ifn-i system requires redundancy to be effective or that individual ifn-i species evolved to execute specific functions. certainly, different ifn-α species and β display varying degrees of affinity for the ifnar / receptor complex (ng, mendoza, garcia, & oldstone, ; thomas et al., ) , with ifn-β displaying the highest binding affinity. lcmv persistence was influenced more by ifn-β than ifn-α signaling as treatment of mice infected with lcmv cl with an ifn-β neutralizing antibody displayed accelerated virus clearance compared to a polyclonal ifn-α antibody which had minimal effects on virus control (ng et al., ) . ifn-β neutralization did not exacerbate early virus replication, improved lymphoid architecture, and enhanced virus-specific cd and cd t cell responses. however, while ifn-β neutralization clearly promoted faster virus clearance as compared to neutralization with a polyclonal ifn-α antibody, the contribution of ifn-α species not neutralized by the polyclonal antibody used was not investigated. nevertheless, neutralizing ifn-β may promote adaptive immune control of virus without significantly affecting virus replication and thus may represent a safer approach to promoting control of persistent virus infection in vivo. the dichotomy between ifn-α and β was further highlighted upon infection of new zealand black (nzb) mice with lcmv cl . infection of nzb mice with cl resulted in early lethality that was found to be due to cd t cell-dependent thrombocytopenia and pulmonary endothelial cells loss (baccala et al., ) . interestingly, despite upregulation of pd- /pd-l expression and il- production, t cell function remained intact. moreover, this enhanced pathology correlated with elevated ifn-i protein levels and gene signatures; however, unlike infection in c bl/ j mice, the pathology required ifn-α signaling and was ifn-β independent. it was recently reported that ifn-β signaling required binding to ifnar but was independent of ifnar . deletion of ifnar ameliorated lps-induced sepsis induction, while ifnar À/À mice were unaffected (de weerd et al., ) ; thus, it would be interesting to test how ifnar À/À nzb mice respond to cl infection. the above studies demonstrate that ifn-α and -β species can differentially modulate immune responses in various viral infections, highlighting the importance of future investigation into how different ifn-i subtypes modulate viral control and disease pathogenesis. several important questions still remain that provide exciting avenues for investigating the roles of ifn-i signaling during viral infection in the future. although ifn-i signaling can trigger various downstream effector pathways, how signaling via select ifn-i species dictate specific outcomes following viral infections remain incompletely understood. specifically, there is a great need to understand the roles individual ifn-i-α and -β subsets play in restraining viral replication or promoting immune inflammatory/suppressive programs in vivo. further, how ifn-i signaling in specific cellular subsets in vivo regulates immune pathological and immunesuppressive responses will be interesting to dissect. the ifnar -floxed mouse strain which was generated recently will be instrumental in future studies to investigate this question. illuminating what cell types require ifn-i signaling in vivo should pave the way for generating a detailed understanding of the cellular and molecular mechanisms by which ifn-i signaling acts to promote immune pathology and suppression in acute and persistent viral infections. the capacity of ifn-i signaling to promote immune pathology during acute respiratory viral infection appears in animal models of both influenza and sars-cov infection. the necessity of ifn-i signaling to restrain viral spread during acute viral infection suggest that targeting the ifn-i signaling pathway may be ill advised. however, one wonders whether targeting specific ifn-i species to suppress detrimental inflammation can be achieved without compromising virus clearance during acute respiratory viral infections. moreover, the production of ifn-λ during respiratory viral infection may be sufficient to control viral loads while ifn-i signaling is inhibited. recent results in mouse models suggest this may be possible; however, further studies are needed. moreover, whether the effects observed in mice will translate to human respiratory viral infections is unknown and should be investigated with caution. in the context of the immune-suppressive programs elicited by ifn-i signaling during persistent virus infection, the recent demonstration that blockade of ifn-β enhanced virus control by inducing improved lymphoid architecture and enhanced virus-specific cd and cd t cell responses, suggest that targeting selective ifn-i species can redirect immune responses sufficiently to promote immune-mediated virus control. importantly, relief of the immune-suppressive environment in this case was not accompanied by elevated viral loads following treatment with ifn-β-neutralizing antibody, suggesting that more selective modulation of specific ifn-i species can allow for preservation of some antiviral functions. the mechanisms by which the different ifn-i species interact with the ifnar and ifnar receptors to induce differential downstream signaling suggests this pathway could be manipulated pharmacologically. it is interesting to postulate whether small molecules or biologics could be developed to block binding/signaling of specific ifn-i species (i.e., ifn-β or specific α-species). for example, could ifn-β signaling be selectively inhibited without altering ifn-α species engagement with the ifnar / receptor complex during ongoing viral infection using a small molecule or antibody therapeutic? could a small molecule be designed to reverse aspects of the immune-suppressive environment and promote virus control without compromising virus replication? on the contrary, could selective ifn-i agonists be developed to increase ifn-i signaling in a productive way to lower viral loads and bring persistent/chronic viral infection under control? a similar question could be posited during acute viral infections where ifn-i signaling promotes aberrant inflammation and immune pathology. moreover, it would be interesting to investigate whether selective biological or pharmacological modulation of ifn-i signaling may translate to treat autoimmune disease states associated with elevated and sustained ifn-i signaling. however, any therapy that enhances or blocks ifn-i signaling will need to be approached carefully, given the delicate balancing act required for controlling virus replication while safely modulating immune responses. protective immunity and susceptibility to infectious diseases: lessons from the influenza pandemic role of host immune response and viral load in the differential outcome of pandemic h n ( ) influenza virus infection in indian patients type i interferon limits influenza virus-induced acute lung injury by regulation of excessive inflammation in mice pegylated interferon-alpha a treatment of chronic siv-infected macaques safety, tolerability, and mechanisms of antiretroviral activity of pegylated interferon alfa- a in hiv- -monoinfected 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endothelial cells are central orchestrators of cytokine amplification during influenza virus infection mapping the innate signaling cascade essential for cytokine storm during influenza virus infection. proceedings of the national academy of sciences of the united states of america structural linkage between ligand discrimination and receptor activation by type i interferons rapid expansion of cd + t cells in wild-type and type i interferon receptor-deficient mice correlates with protection after low-dose emergency immunization with modified vaccinia virus ankara suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus timing and magnitude of type i interferon responses by distinct sensors impact cd t cell exhaustion and chronic viral infection type interferons and antiviral cd t-cell responses simultaneous detection of hepatitis c virus and interferon stimulated gene expression in infected human liver blockade of chronic type i interferon signaling to control persistent lcmv infection type i interferon protects antiviral cd + t cells from nk cell cytotoxicity functional interferon system is required for clearance of lassa virus role of interferon regulatory factor in t cell responses during acute lymphocytic choriomeningitis virus infection bone marrow plasmacytoid dendritic cells can differentiate into myeloid dendritic cells upon virus infection key: cord- -gnno elo authors: wang, ziran; hao, zhuang; yu, shifeng; huang, cong; pan, yunlu; zhao, xuezeng title: a wearable and deformable graphene-based affinity nanosensor for monitoring of cytokines in biofluids date: - - journal: nanomaterials (basel) doi: . /nano sha: doc_id: cord_uid: gnno elo a wearable and deformable graphene-based field-effect transistor biosensor is presented that uses aptamer-modified graphene as the conducting channel, which is capable of the sensitive, consistent and time-resolved detection of cytokines in human biofluids. based on an ultrathin substrate, the biosensor offers a high level of mechanical durability and consistent sensing responses, while conforming to non-planar surfaces such as the human body and withstanding large deformations (e.g., bending and stretching). moreover, a nonionic surfactant is employed to minimize the nonspecific adsorption of the biosensor, hence enabling cytokine detection (tnf-α and ifn-γ, significant inflammatory cytokines, are used as representatives) in artificial tears (used as a biofluid representative). the experimental results demonstrate that the biosensor very consistently and sensitively detects tnf-α and ifn-γ, with limits of detection down to . and . pm, respectively. the biosensor, which undergoes large deformations, can thus potentially provide a consistent and sensitive detection of cytokines in the human body. human biofluids (such as saliva, tears and sweat) are attractive clinical diagnostic bio-media containing numerous cytokines (with a molecular weight lower than kda) [ ] [ ] [ ] . they can be easily collected without skin-piercing. abnormally elevated levels of cytokines in human biofluids are considered to be closely related to the attack of some severe diseases, such as coronavirus disease (covid- ) , and chronic diseases [ ] [ ] [ ] . hence, the capacity for the continuous detection of cytokine levels in human biofluids for high risk populations, daily, is of great significance for offering information on health conditions and thereby gaining valuable time, which can then be used to take effective preventative measures before the attack from such diseases [ , ] . wearable sensors, which could be attached to the non-planar human body surface and complete on-site signal transduction, seem to be capable of performing such work. graphene, an attractive two-dimensional nanomaterial, is extremely sensitive to its surface charge distribution, and widely used as the transducer for sensors due to its outstanding electrical properties [ ] [ ] [ ] . especially, with the aid of aptamers, the graphene field-effect transistor (gfet) can enable the sensitive, rapid and label-free detection of cytokines [ ] [ ] [ ] . to date, efforts have been made to use gfet biosensors in wearable applications due to the high mechanical flexibility of graphene [ ] [ ] [ ] . such sensors are fabricated on sheets of polymers, such as polydimethylsiloxane (pdms), polyester (pet) and polyethylene naphthalate (pen) [ ] [ ] [ ] . however, these polymer sheets, whose overall thicknesses are µm or more, would hardly be subject to large deformations and curvature (with radii ranging from to mm). as such, gfet biosensors with an extremely thin substrate that can sustain the large deformations involved in physiologically and biochemically relevant measurements on non-planar human body surfaces are still highly desirable. in this paper, we present a wearable and deformable aptameric gfet biosensor that is designed to enable the sensitive, consistent and time-resolved monitoring of cytokines in human biofluids. the biosensor is fabricated on a biocompatible and ultrathin, polymer-supporting substrate (figure a ). due to the employment of this substrate, whose thickness is only . µm, the biosensor, with good mechanical durability, is capable of conforming to non-planar surfaces such as the human skin or eyeball and withstanding large deformations, including bending and stretching, whilst maintaining consistent and sensitive responses. moreover, tween is used to modify the graphene surface to effectively suppress nonspecific adsorption, thus enabling the biosensor to detect cytokines (tnf-α and ifn-γ, significant inflammatory cytokines, were used as representatives) in artificial tears (used as a biofluid representative). the experimental results indicate that our aptameric gfet biosensor can realize the highly sensitive detection of tnf-α and ifn-γ, with limits of detection down to . and . pm, respectively. furthermore, the time-resolved monitoring of tnf-α in artificial tears under different tensile strains with consistent sensing responses is enabled. as a result, our biosensor can be potentially used in wearable applications for monitoring an individual's health conditions and predicting the attack of chronic diseases. nanomaterials , , x for peer review of enable the sensitive, rapid and label-free detection of cytokines [ ] [ ] [ ] . to date, efforts have been made to use gfet biosensors in wearable applications due to the high mechanical flexibility of graphene [ ] [ ] [ ] . such sensors are fabricated on sheets of polymers, such as polydimethylsiloxane (pdms), polyester (pet) and polyethylene naphthalate (pen) [ ] [ ] [ ] . however, these polymer sheets, whose overall thicknesses are μm or more, would hardly be subject to large deformations and curvature (with radii ranging from to mm). as such, gfet biosensors with an extremely thin substrate that can sustain the large deformations involved in physiologically and biochemically relevant measurements on non-planar human body surfaces are still highly desirable. in this paper, we present a wearable and deformable aptameric gfet biosensor that is designed to enable the sensitive, consistent and time-resolved monitoring of cytokines in human biofluids. the biosensor is fabricated on a biocompatible and ultrathin, polymer-supporting substrate (figure a ). due to the employment of this substrate, whose thickness is only . μm, the biosensor, with good mechanical durability, is capable of conforming to non-planar surfaces such as the human skin or eyeball and withstanding large deformations, including bending and stretching, whilst maintaining consistent and sensitive responses. moreover, tween is used to modify the graphene surface to effectively suppress nonspecific adsorption, thus enabling the biosensor to detect cytokines (tnf-α and ifn-γ, significant inflammatory cytokines, were used as representatives) in artificial tears (used as a biofluid representative). the experimental results indicate that our aptameric gfet biosensor can realize the highly sensitive detection of tnf-α and ifn-γ, with limits of detection down to . and . pm, respectively. furthermore, the time-resolved monitoring of tnf-α in artificial tears under different tensile strains with consistent sensing responses is enabled. as a result, our biosensor can be potentially used in wearable applications for monitoring an individual's health conditions and predicting the attack of chronic diseases. the monolayer graphene sheet was ordered from graphenea inc. (cambridge, ma, usa). -pyrenebutyric acid n-hydroxysuccinimide ester (pase), ethanolamine, tween , human interleukin- (il- ), tnf-alpha and ifn-gamma were purchased from sigma-aldrich (st. louis, mo, usa). human growth hormone (gh) and human epidermal growth factor (egf) were ordered from acro biosystems (newark, de, usa). ultrathin mylar films ( . µm thickness of polyethylene terephthalate membranes) were ordered from chemplex industries (palm city, fl, usa). artificial tears were ordered from alcon laboratories. the aptamer specific to tnf-alpha ( -nh -tgg tgg atg gcg cag tcg gcg aca a- ) and ifn-gamma ( -nh -ggg gtt gtt tgt gtt ggg tgt tgt gt- ) was synthesized and purified by sangong biotech (shanghai, china). the gfet biosensor was fabricated following our previous nanofabrication process [ , , ] . briefly, an ultrathin mylar film ( . µm) was placed on a glass slide as the biosensor's substrate. subsequently, drain, source and gate electrodes ( / nm of cr/au) were patterned onto the film using a lithography process, including e-beam evaporation and lift-off. the biosensor was then exposed to the oxygen plasma to remove the residue on the surface. a monolayer graphene sheet was transferred onto electrodes as the conducting channel, using a polymethyl methacrylate (pmma) carrier layer. after dissolving the pmma layer with acetone, the graphene was biochemically functionalized to enable the biomarker detection. the fabricated biosensor was ultra-flexible and capable of conforming to the underlying surface, such as a human wrist or eyeball (figure b,c) . finally, the biosensor was mounted onto a pre-stretched elastomer to obtain the necessary stretchability, which allowed the biosensor to be stretched from a % to % extension ( figure d ). to achieve the biochemical functionalization, the biosensor was first immersed in mm -pyrenebutanoic acid succinimidyl ester (pase) solution for h at room temperature. pase was modified on the graphene surface through π-π stacking, which was used to link the aptamer. after incubating in the µm aptamer solution for h, the device was washed with phosphate buffer (pbs) to remove free aptamer. ethanolamine was then used to quench the unreacted pase on the graphene by soaking in mm ethanolamine solution for h. finally, the biosensor was immersed in . % tween solution to passivate the uncoated graphene area. during the operation, a volume of µl of analytes (tnf-α, ifn-γ and control proteins) at a given concentration was added to a polydimethylsiloxane (pdms) open well, which was mounted on the graphene conducting channel to hold the analyte solution. in the experiments, µl of artificial tears was added to a ml centrifugal tube with µl of × pbs. then, the mixture solution was stored at • c before protein solution configurations. a microscope image of the gfet biosensor fabricated on the ultrathin film is shown in figure a . the surface functionalization of the graphene with pase was confirmed using raman spectra (figure b ). the modification of the pase split the g band, illustrating the coupling of graphene with the pyrene group on pase. furthermore, the dirac point shift ∆v dirac ,where ∆v dirac = v dirac -v dirac, with v dirac, the dirac point obtained in the solution without biomarker, before and after pase, aptamer and tween functionalization was measured with the gate voltage v g increasing from − . to . v at a fixed drain-source voltage v ds of . v (figure c,d) . ∆v dirac was observed to be monotonically increased after pase functionalization, indicating that the p-type doping of the graphene had been induced. upon the attachment of the aptamer specific to tnf-α and ifn-γ, ∆v dirac decreased by . and . v, respectively. tween , a chemically stable nonionic surfactant, was used to block the uncoated graphene area to suppress the nonspecific adsorption in artificial tears due to its low binding affinity to the abundant non-target molecules present in the tears [ , ] . after the modification with tween , the dirac point v dirac shifted towards the direction of the negative gate voltage, illustrating that tween induced the n-type doping of the graphene. thus, it was concluded that the biosensor was successfully functionalized using different aptamers. (figure c,d) . Δvdirac was observed to be monotonically increased after pase functionalization, indicating that the p-type doping of the graphene had been induced. upon the attachment of the aptamer specific to tnf-α and ifn-γ, Δvdirac decreased by . and . v, respectively. tween , a chemically stable nonionic surfactant, was used to block the uncoated graphene area to suppress the nonspecific adsorption in artificial tears due to its low binding affinity to the abundant non-target molecules present in the tears [ , ] . after the modification with tween , the dirac point vdirac shifted towards the direction of the negative gate voltage, illustrating that tween induced the n-type doping of the graphene. thus, it was concluded that the biosensor was successfully functionalized using different aptamers. the capability of the biosensor for cytokine detection was assessed using tnf-α and ifn-γ ( figure )-inflammatory cytokines related to inflammation, covid- and cancers [ , ] . as the tnf-α concentration increased from . to nm (figure a) , vdirac decreased by . v, from . to . v. it could be observed that vdirac monotonically decreased with increasing tnf-α concentrations, suggesting the successful detection of tnf-α in artificial tears using the fabricated biosensor. the equilibrium dissociation constant, defined kd, was investigated to study the binding affinity between the aptamer and tnf-α (figure c ). the normalized Δvdirac was employed to address the effect of device-to-device variations, defined as Δvdirac/Δvdirac,max (vdirac,max is the dirac point corresponding to the maximum tnf-α concentration tested). Δvdirac/Δvdirac,max was determined using the capability of the biosensor for cytokine detection was assessed using tnf-α and ifn-γ ( figure )-inflammatory cytokines related to inflammation, covid- and cancers [ , ] . as the tnf-α concentration increased from . to nm (figure a) , v dirac decreased by . v, from . to . v. it could be observed that v dirac monotonically decreased with increasing tnf-α concentrations, suggesting the successful detection of tnf-α in artificial tears using the fabricated biosensor. the hill-langmuir equation [ ] . based on the fitted curve, the kd was calculated to be . ± . nm. the limit of detection (lod) was estimated based on the three-sigma rule, and the sigma was obtained from the standard deviation of the experimental and fitted data. the lod was calculated to be . pm, which is many times lower than that for existing biosensors for tnf-α detection. subsequently, the biosensor was employed to test the detection of ifn-γ in artificial tears ( figure b,d) . as the ifn-γ concentration increased from . to nm, the maximum Δvdirac was . v, decreasing from . to v. the sensing signal was also consistently shifted in the direction of the negative gate voltage with the increasing ifn-γ concentrations. this was expected from the electrostatic mechanism [ ] . after binding with the cytokines, the aptamer together with the negatively charged cytokines was brought closer to the graphene surface, which enabled the charge redistribution in the graphene, due to electrostatic induction [ , ] . hence, a detectable change in the drain-source current was measured. the equilibrium dissociation constant kd was estimated to be . ± . nm (figure d) , and the lod was calculated to be . pm. thus, the biosensor shows a consistent and sensitive response, able to detect cytokines in artificial tears with a lower lod than most other existing methods (table ) . the equilibrium dissociation constant, defined k d , was investigated to study the binding affinity between the aptamer and tnf-α (figure c ). the normalized ∆v dirac was employed to address the effect of device-to-device variations, defined as ∆v dirac /∆v dirac,max (v dirac,max is the dirac point corresponding to the maximum tnf-α concentration tested). ∆v dirac /∆v dirac,max was determined using the hill-langmuir equation [ ] . based on the fitted curve, the k d was calculated to be . ± . nm. the limit of detection (lod) was estimated based on the three-sigma rule, and the sigma was obtained from the standard deviation of the experimental and fitted data. the lod was calculated to be . pm, which is many times lower than that for existing biosensors for tnf-α detection. subsequently, the biosensor was employed to test the detection of ifn-γ in artificial tears (figure b,d) . as the ifn-γ concentration increased from . to nm, the maximum ∆v dirac was . v, decreasing from . to v. the sensing signal was also consistently shifted in the direction of the negative gate voltage with the increasing ifn-γ concentrations. this was expected from the electrostatic mechanism [ ] . after binding with the cytokines, the aptamer together with the negatively charged cytokines was brought closer to the graphene surface, which enabled the charge redistribution in the graphene, due to electrostatic induction [ , ] . hence, a detectable change in the drain-source current was measured. the equilibrium dissociation constant k d was estimated to be . ± . nm (figure d) , and the lod was calculated to be . pm. thus, the biosensor shows a consistent and sensitive response, able to detect cytokines in artificial tears with a lower lod than most other existing methods (table ) . to investigate the specificity of the biosensor, egf and gh, two related proteins, were chosen as control proteins. the biosensor, modified with the aptamer specific to tnf-α, was firstly exposed to control proteins in artificial tears (figure a ). the sensing signal for tnf-α significantly increased with the adding of tnf-α concentrations. the normalized ∆v dirac /∆v dirac,max for tnf-α at nm was over six times larger than that for control proteins ( . %) at the same concentration. additionally, the dissociation constant k d for the control proteins was investigated. k d was estimated to be . × and . × nm for egh and gh, respectively, which is much larger than the k d for tnf-α ( . nm). subsequently, the biosensor modified with the aptamer specific to ifn-γ was tested using these control proteins (figure b) . the ∆v dirac /∆v dirac,max for ifn-γ is also six times larger than those for the control proteins ( . %) at the same concentrations. furthermore, the k d was calculated to be . × and . × nm for egh and gh, respectively, which is tens of thousands of times larger than the k d for ifn-γ ( . nm). hence, the biosensor has a high level of specificity for the target cytokine in artificial tears. nanomaterials , , x for peer review of graphene-based sensor aptamer artificial tears . pm (this work) to investigate the specificity of the biosensor, egf and gh, two related proteins, were chosen as control proteins. the biosensor, modified with the aptamer specific to tnf-α, was firstly exposed to control proteins in artificial tears (figure a ). the sensing signal for tnf-α significantly increased with the adding of tnf-α concentrations. the normalized Δvdirac/Δvdirac,max for tnf-α at nm was over six times larger than that for control proteins ( . %) at the same concentration. additionally, the dissociation constant kd for the control proteins was investigated. kd was estimated to be . × and . × nm for egh and gh, respectively, which is much larger than the kd for tnf-α ( . nm). subsequently, the biosensor modified with the aptamer specific to ifn-γ was tested using these control proteins (figure b) . the Δvdirac/Δvdirac,max for ifn-γ is also six times larger than those for the control proteins ( . %) at the same concentrations. furthermore, the kd was calculated to be . × and . × nm for egh and gh, respectively, which is tens of thousands of times larger than the kd for ifn-γ ( . nm). hence, the biosensor has a high level of specificity for the target cytokine in artificial tears. to verify the feasibility of the biosensor for wearable applications, time-resolved measurements were employed, using the biosensor modified with an aptamer specific to tnf-α as a representative. as shown in figure a , the device was first exposed to different concentrations of tnf-α in artificial tears. the characterization started with the injection of the artificial tears in the pdms well, then the artificial tears spiked with various tnf-α concentrations were injected after each binding equilibrium at corresponding concentrations. the changes in the drain-source current are denoted as Δids, and the maximum change is denoted as Δids, max. the sensing signal was observed to increase stepwise and significantly from to . with increasing tnf-α concentrations from to nm. moreover, it was found that equilibrium was reached after the binding of aptamer with tnf-α within min at different concentrations. by contrast, the Δids/Δids, max-generated by related cytokines (ifn-γ and il- )was less than % compared with that for tnf-α at the same concentration. to verify the feasibility of the biosensor for wearable applications, time-resolved measurements were employed, using the biosensor modified with an aptamer specific to tnf-α as a representative. as shown in figure a , the device was first exposed to different concentrations of tnf-α in artificial tears. the characterization started with the injection of the artificial tears in the pdms well, then the artificial tears spiked with various tnf-α concentrations were injected after each binding equilibrium at corresponding concentrations. the changes in the drain-source current are denoted as ∆i ds , and the maximum change is denoted as ∆i ds, max . the sensing signal was observed to increase stepwise and significantly from to . with increasing tnf-α concentrations from to nm. moreover, it was found that equilibrium was reached after the binding of aptamer with tnf-α within min at different concentrations. by contrast, the ∆i ds/ ∆i ds, max -generated by related cytokines (ifn-γ and il- )-was less than % compared with that for tnf-α at the same concentration. nanomaterials , , x for peer review of until it achieved the shape of the human eyeball (which was accompanied by the biosensor, mounted on top). the diaphragm deflection induced a large-magnitude stretching of the biosensor ( % tensile strain), serving as an example mimicking the large deformation of the wearable device for cytokine detection on the human body (figure d ). the sensing response to tnf-α was sensitive and consistent with the test on a flat surface (figure a) , and the degradation of the signal was less than % at the same concentration for the biosensor under two tensile strains. overall, the results demonstrate that the fabricated biosensor has promising prospects for being embedded with wearable devices for the monitoring of cytokines in body fluids. we presented a wearable gfet biosensor for the time-resolved monitoring of cytokines in artificial tears. the modification of the graphene with the aptamer enabled the highly specific detection of the target cytokine. tween , which occupies the uncoated area of the graphene, offers a capability of suppressing nonspecific adsorption; the biosensor hence affords a high level of sensitivity for cytokine detection in artificial tears, with a lod down to . and . pm for tnf-α and ifn-γ, respectively. in addition, the time-resolved monitoring of tnf-α in artificial tears using the biosensor under different tensile strains has been demonstrated. the sensing response for the target cytokines is consistent with that found on the flat surface. these results demonstrate that this wearable gfet biosensor is a critical step toward the general application of sensors for the monitoring of disease biomarkers in the human body. additionally, we present a potential capability for wearable applications using the biosensor mounted on a homemade diaphragm chamber for cytokine detection (figure b ). the diaphragm is a stretchable membrane, which can be made to swell or shrink by pumping air into the chamber to mimic the human eyeball. the biosensor was first mounted on the planar diaphragm ( % tensile strain) to test cytokine detection (figure c ). then, the diaphragm was inflated with compressed air until it achieved the shape of the human eyeball (which was accompanied by the biosensor, mounted on top). the diaphragm deflection induced a large-magnitude stretching of the biosensor ( % tensile strain), serving as an example mimicking the large deformation of the wearable device for cytokine detection on the human body (figure d ). the sensing response to tnf-α was sensitive and consistent with the test on a flat surface (figure a) , and the degradation of the signal was less than % at the same concentration for the biosensor under two tensile strains. overall, the results demonstrate that the fabricated biosensor has promising prospects for being embedded with wearable devices for the monitoring of cytokines in body fluids. we presented a wearable gfet biosensor for the time-resolved monitoring of cytokines in artificial tears. the modification of the graphene with the aptamer enabled the highly specific detection of the target cytokine. tween , which occupies the uncoated area of the graphene, offers a capability of suppressing nonspecific adsorption; the biosensor hence affords a high level of sensitivity for cytokine detection in artificial tears, with a lod down to . and . pm for tnf-α and ifn-γ, respectively. in addition, the time-resolved monitoring of tnf-α in artificial tears using the biosensor under different tensile strains has been demonstrated. the sensing response for the target cytokines is consistent with that found on the flat surface. these results demonstrate that this wearable gfet biosensor is a critical step toward the general application of sensors for the monitoring of disease biomarkers in the human body. simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry: allergics vs. non-allergics measurement of cytokines in sweat patches and plasma in healthy women: validation in a controlled study acute phase protein and cytokine levels in serum and saliva: a comparison of detectable levels and correlations in a depressed and healthy adolescent sample wearable sensors for biochemical sweat analysis methylxanthine drug monitoring with wearable sweat sensors flexible electronics toward wearable sensing recent advances in cytokine detection by immunosensing ultrasensitive detection of cytokines enabled by nanoscale zno arrays high-performance flexible graphene field effect transistors with ion gel gate dielectrics the rise of graphene fully rollable transparent nanogenerators based on graphene electrodes graphene-based fully integrated portable nanosensing system for on-line detection of cytokine biomarkers in saliva an ultraflexible and stretchable aptameric graphene nanosensor for biomarker detection and monitoring modulating the linker immobilization density on aptameric graphene field effect transistors using an electric field large-area ultrathin films of reduced graphene oxide as a transparent and flexible electronic material graphene-based wireless bacteria detection on tooth enamel ultrafine graphene nanomesh with large on/off ratio for high-performance flexible biosensors measurement of cytokine biomarkers using an aptamer-based affinity graphene nanosensor on a flexible substrate toward wearable applications high-performance, flexible enzymatic glucose biosensor based on zno nanowires supported on a gold-coated polyester substrate a wearable and highly sensitive pressure sensor with ultrathin gold nanowires real-time monitoring of insulin using a graphene field-effect transistor aptameric nanosensor an integrated flexible and reusable graphene field effect transistor nanosensor for monitoring glucose rapid, label-free, electrical whole blood bioassay based on nanobiosensor systems graphene-based biosensors for detection of bacteria and their metabolic activities angiopoietin- sensitizes endothelial cells to tnf-α and has a crucial role in the induction of inflammation elevations of serum cancer biomarkers correlate with severity of covid- label-free biosensors based on aptamer-modified graphene field-effect transistors detecting multiple cell-secreted cytokines from the same aptamer-functionalized electrode detection of interferon gamma using graphene and aptamer based fet-like electrochemical biosensor the authors declare no conflict of interest. key: cord- - xf dw authors: parra, beatriz; hinton, david r.; lin, mark t.; cua, daniel j.; stohlman, stephen a. title: kinetics of cytokine mrna expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: xf dw abstract the potential role(s) of cytokines in the reduction of infectious virus and persistent viral infection in the central nervous system was examined by determining the kinetics of cytokine mrna expression following infection with the neurotropic jhm strain of mouse hepatitis virus. mice were infected with an antibody escape variant which produces a nonlethal encephalomyelitis and compared to a clonal virus population which produces a fulminant fatal encephalomyelitis. infection with both viruses induced the accumulation of mrnas associated with th - and th -type cytokines, including ifn-γ, il- , and il- . peak mrna accumulations were coincident with the clearance of virus and there was no obvious differences between lethally and nonlethally infected mice. tnf-α mrna was induced more rapidly in lethally infected mice compared to mice undergoing a nonfatal encephalomyelitis. rapid transient increases in the mrnas encoding il- , inos, il- α, il- β, and il- occurred following infection. nonlethal infections were associated with increased il- , il- β, and earlier expression of il- , while lethal infections were associated with increased inos and il- α mrna. these data suggest a rapid but differential response within the central nervous system cells to infection by different jhmv variants. however, neither the accumulation nor kinetics of induction provide evidence to distinguish lethal infections from nonlethal infections leading to a persistent infection. accumulation of both th and th cytokines in the central nervous system of jhmv-infected mice is consistent with the participation of both cytokines and cell immune effectors during resolution of acute viral-induced encephalomyelitis. introduction (wesselingh et al., ) . variations between viral infections resulting in cns the goal of the immune response during viral infection inflammation prompted an examination of the temporal is to limit replication via induction of both nonspecific and induction of cns cytokines during fatal and nonfatal cns specific antiviral effectors. acute viral infections of the ceninfections by variants of the jhm strain (jhmv) of mouse tral nervous system (cns) result in vigorous, but in some hepatitis virus (mhv). in immunocompromised hosts instances limited, host immune responses (sedgwick and jhmv replicates unchecked in the cns demonstrating dorries, ) . in contrast to responses in the periphery the importance of immune effectors in limiting cns virus where limiting virus replication can generally be carried replication ; houtman and out with minimal regard to tissue damage, within the cns fleming, b; . effector checks and balances minimize inflammatory-mediated mechanisms implicated in protection and clearance of damage while limiting viral-induced cytopathology. al-jhmv from the cns include cell-mediated immunity and though a wide range of immune effectors are often induced, both neutralizing and nonneutralizing antibodies. jhmv predominant anti-viral mechanisms appear related to the provides an interesting paradigm of acute viral encephapathogenesis strategy of the individual agent. for example, litis not only because of its associated demyelination infection of mice with lymphocytic choriomeningitis virus (weiner, ; lampert et al., ) but also because induces a predominant cd / cytotoxic t lymphocyte (ctl) some immune effector mechanisms prevent death via response (lehmann et al., ) . by contrast, resolution of directly reducing cns virus replication while other immeasles virus-encephalitis in mice is mediated by cd / t mune effectors prevent death without significantly altercells and correlates with the local production of ifn-g ing virus replication hout-(finke et al., ) . finally, resolution of sindbis virus-inman and fleming, b; . a duced encephalitis is related to induction of neutralizing common theme appears to be prevention of neuronal infection by reducing viral load or preventing neuronal infection, most likely via cytokines. tion and clearance of jhmv from the cns are not yet and rarely neurons ( . v- ). these viruses contrast to the predominantly neuronotropic oblv- variant previously clear. the antiviral effects of cd / t cells appear to be due to direct lysis of infected cells; however, cd / and examined (pearce et al., ; . cd / t cells may also exert antiviral activity indirectly material and methods via cytokine secretion (biron, ) . neither infected neurons nor oligodendrocytes appear susceptible to major mice and viruses histocompatibility (mhc) class i-mediated killing in vivo, c bl/ mice were purchased from the jackson laboconsistent with the inability of jhmv-specific ctl to clear ratory (bar harbor, me) at weeks and maintained in virus from infected oligodendroglia (stohlman et al., the university of southern california vivarium. all mice b). furthermore, clearance of jhmv from the cns were used at weeks of age. to produce a lethal infecis inhibited, but not abolished, in mice genetically defition, mice were infected by intracerebral inoculation (i.c.) cient in perforin-mediated cytolysis (lin et al., ) . with pfu of the plaque-purified dm isolate of jhmv these data suggest the possibility that cytokines contrib- (stohlman et al., ) in a volume of ml. this virus has ute to either clearance or protection from jhmv infection. the plaque size and pathogenesis similar to the parental during jhmv infection of the cns there is an abrupt suckling mouse brain pool of jhmv originally described increase in mrna encoding interleukin- (a and b), ilby weiner ( ) and produces a lethal encephalomyeli- , tumor necrosis factor (tnf)-a, and interferon (ifn)-g, tis with minimal demyelination apparent at the time of at the time of maximal decrease in virus replication and death. to produce a sublethal infection, mice were inmononuclear cell infiltration (pearce et al., ) . no ifnfected with pfu of the . v- monoclonal antibodyg mrna was detected in immunodeficient mice, sugderived neutralization-resistant variant of jhmv (fleming gesting this cytokine may be important during viral clearet al., ) . this variant replicates predominantly in oliance (pearce et al., ) . consistent with this concept, godendroglia producing a flaccid paralysis. although vimice treated with anti-ifn-g are more susceptible to ral antigen is cleared from survivors by days postinfec-jhmv, while administration of ifn-g provides protection tion (p.i.), viral rna persists for at least months (adami (smith et al., ) . il- , tnf-a, and type nitric oxide et al., ) . groups of at least mice were sacrificed synthase (inos) have also been detected in the cns at various times p.i. immunosuppression was induced during acute jhmv infection (sun et al., ; by lethal irradiation ( r) hr prior to infection. shamet al., a; while il- b, il- , tnf-a, infected mice were injected i.c. with ml of sterile endoand inos were detected in the cns of chronically intoxin-free phosphate-buffered saline (pbs). fected mice (sun et al., ) . the complex interactions of multiple immune effector mechanisms during jhmv virus titration infection may reflect both the relative immune privilege virus titers were determined by plaque assay using of the cns (sedgwick and dorries, ) as well as the monolayers of dbt cells as previously described (stohlspecific tropism of the virus for cns cell types. neuroman et al., ) . one-half of the brain was homogenized tropic mhv isolates differ in tropism and include viruses using tenbrock tissue homogenizers in . ml of dulbecwith predominant tropisms for astrocytes, microglia, and co's pbs, ph . . the remaining half was taken for histooligodendroglia as well as neurons (fleming et al., ; pathology or rna extraction (see below). following cen- perlman and reis, ; kyuwa and stohlman, ; trifugation at g for min at Њ, supernatants were pearce et al., ; stohlman et al., a) . the balance assayed immediately or frozen at Њ. data presented between limiting viral replication and preserving cns are the average titer of groups of three or more mice. function occasionally results in incomplete viral clearance and a persistent cns infection which may or may antibody titration not involve the continued presence of infectious virus jhmv-specific igm, igg , and igg a antibodies were quantitated by elisa as previously described (lin et al., b) . persistence of infectious virus correlates with the ) using rabbit anti-mouse igm, igg , or igg a antipresence of ctl escape variants (pewe et al., ) . bodies (cappel, costa mesa, ca). concentrations of se-to understand the complex interrelationships between rum antibodies were expressed as the highest dilution encephalitis, protection, and viral clearance leading to a with o.d. values three times above background level. persistent infection of the cns, the expression of pro-neutralizing antibodies were tested in serum as preand anti-inflammatory cytokine mrnas in the cns of viously described (lin et al., ) . mice undergoing either lethal or sublethal jhmv infection were compared. the two jhmv chosen for study infect histology either primarily microglia and astrocytes, less frequently oligodendroglia and neurons (dm) or primarily oligoden-histopathologic analysis was performed as previously described (stohlman et al., a) . briefly, tissues were droglia, much less frequently microglia and astrocytes, fixed for hr in clark's solution ( % ethanol, % glacial with p-atp-labeled internal oligonucleotide probes. membranes were washed (three times; ssc, . % acidic acid) and embedded in paraffin. sections were stained with hematoxylin and eosin or luxol fast blue. distri-sds; room temperature), exposed to storage phosphor screens (molecular dynamics, sunnyvale, ca), and bution of jhmv antigen was examined by immunoperoxidase staining (vectastain-abc kit; vector laboratory, burlin-scanned using a phosphorimaging scanner (molecular dynamics). game, ca) using the anti-jhmv mab j . specific for the viral nucleocapsid protein (fleming et al., ) . radioactive signals of cytokine cdna were quantified and normalized to the house-keeping enzyme hypoxanthine phosphoriboxyltransferase (hprt) values to adjust cytokine mrna expression for differences in cdna as previously described (cua et al., (cua et al., , . the sample with the highest specific brains were processed individually to prevent contamination. rna was isolated from half brains by homogeni-activity was designated the % maximal response and values for the remainder were derived as percentage of zation at room temperature in guanidinium isothiocyanate using tenbrock tissue homogenizers as previously the highest value. data shown are mean values for - mice at each time point { standard deviation. described (cua et al., ) . samples were sheared prior to centrifugation through . m cesium chloride at , g for hr to isolate rna. the cdna were pre-results pared using avian myeloblastosis reverse transcriptase acute and subacute jhmv-induced encephalitis (promega, madison, wi) and oligo dt primers (promega) for min at Њ. expression of cytokine mrna was fatal encephalomyelitis induced by jhmv is associated with minimal demyelination (kyuwa and stohlman, determined by semiquantitative pcr analysis, following procedures previously described (cua et al., (cua et al., , (cua et al., ). houtman and fleming, b) . this contrasts with infection by . v- which produces an acute nonfatal en-pcr was performed using amplitaq dna polymerase (perkin-elmer, branchburg, nj) and specific cytokine cephalomyelitis with extensive demyelination (fleming et al., ; wang et al., ) . although both viruses primers for ifn-g, il- a, il- b, il- , il- , il- , tnf-a (murphy et al., ; cua et al., ) , and il- p . the replicated rapidly to high titer in the cns (fig. a) , jhmvinfected mice succumbed within days while . v- -sequences of the il- p oligonucleotides primers and probe used are as follows: primer, gac cct gcc infected mice underwent a subacute disease with little or no mortality (fig. b) . peak . v- replication was at cat tga act ggc; primer, caa cgt tgc atc cta gga tcg; oligoprobe, tgt ctg cgt gca agc tca day while the peak of jhmv replication was delayed until day . . v- clearance began at day p.i. and by gga. amplification was carried out using cycles of one denaturation step at Њ ( sec), primer annealing day virus was nearly undetectable. by contrast, titers in jhmv-infected mice initially decreased at day p.i. at Њ ( sec), extension step at Њ ( . min), followed by a final extension step for min. for il- and inos a and detectable virus was still present in the cns of moribound mice at day p.i. (fig. a ). during lethal jhmv nested pcr was performed by using internal primers in a second round of pcr ( cycles) under the conditions infection, virus replication within the cns is not reduced as rapidly as in mice which survive infection ( fig. a ) described above. the oligonucleotide primers used in the second pcr for il- were the corresponding se-consistent with the notion that rapid clearance correlates positively with protection. consistent with these findings, quences for the -primer and the probe described by cua et al. ( ) . the nucleotide sequence for the il- immunohistologic examination of the brains of jhmvinfected mice at day showed abundant viral antigen oligonucleotide probe was ttg aag gag gtc aca gga gaa ggga (sideras et al., ) . the and outer in regions of encephalitis while only focal residual viral antigen was found in . v- -infected animals (fig. ) . en-primer sequences for inos were gcc ttc cgc agc tgg gct gt and atg tgg tag cca cat ccc gag cephalitis was prominent in mice infected with either . v- or jhmv and no differences in the amount or distri-cc, respectively (lyons et al., ) . internal and inos primers were agc tac tgg gtc aaa gac aag bution of mononuclear cell infiltrates were found at day (fig. ) . no serum neutralizing antibodies were de-agg ct and the outer primer, respectively. the oligonucleotide probe consisted of the sequence ctc cct tected in either group by day postinfection, even though the virus titer in the cns had declined over log (lin tcc gaa gtt tct ggc agc a. for quantification, pcr products were diluted in dena-et al., ; data not shown). in contrast to neutralizing antibodies, igm was first detected at day post- . v- turing solution ( . n naoh, mm edta), neutralized with tris-hci ( . m; ph . ), and transferred to . infection (data not shown) and both igg and igg a were detected as early as days p.i. (fig. c) . the igg and mm nytran membranes (schleicher & schuell, keene, nh) using a minifold i dot blot apparatus (schleicher & igg a response suggest the absence of a shift toward either a th -or th -type response reported to be in-schuell). membranes were hybridized ( Њ; overnight) volved in the response to sindbis virus-induced encepha-deficient mice, which showed no evidence of ifn-g mrna, suggests tnf-a may also contribute to inos litis (wesselingh et al., ) . mrna induction (colasanti et al., ; gazzinelli et al., ) . consistent with this notion, tnf-a mrna was first proinflammatory cytokines detected at day in mice undergoing a lethal infection the mrna encoding ifn-g increased in both groups and at day in mice sublethally infected (fig. c ). similar of mice through day postinfection, consistent with the to the kinetics of ifn-g, tnf-a mrna increased until rapid accumulation of both nk and t cells in the cns of death of lethally infected mice. in mice undergoing a infected mice (williamson et al., ; williamson, ) sublethal infection, tnf-a mrna declined following the (fig. a) . no ifn-g mrna was detected in either shampeak of virus replication and approached baseline levels infected mice or in infected immunodeficient mice. durby days p.i. ing the lethal jhmv infection ifn-g mrna did not in-similar to both tnf-a and inos, il- is secreted from crease between day and day . however, in mice macrophages during the induction of cell-mediated imundergoing a sublethal infection the level of ifn-g mrna munity and protects from a number of viral infections continued to increase to day and remained elevated, via a ifn-g-dependent mechanism (ozmen et al., ; suggesting the possibility that ifn-g is important follow- orange and biron, ) . no il- mrna was found foling infection with a jhmv variant tropic for oligodendroglowing sham infection; however, il- mrna increased lia. even though ifn-g mrna increased during the early rapidly and peaked at days following both infections phase of infection, a sharp transient increase in inos (fig. a ). increased il- mrna also occurred in immu-mrna was detected at day p.i. in mice with a lethal nodeficient mice at days p.i., suggesting a direct reencephalomyelitis (fig. b) . only a slight increase was sponse to infection which may be related to the recently detected in mice undergoing subacute encephalomyelidescribed ifn-g-independent induction of il- (heinzel tis. interestingly, infection of immunodeficient mice with et al., ) . il- mrna levels decreased after day jhmv induced the accumulation of inos mrna to apand nearly approached base line levels found in uninproximately % the level found in infected immunocomfected mice by days p.i. petent mice, suggesting a direct response to viral infec-the il- b mrna level found at day p.i. declined by days p.i., consistent with induction of an early transient tion. the increase in inos and tnf-a mrna in immuno- increase in il- b mrna in sham-infected mice (fig. b ). subacute infection (fig. c ) and then declined but never returned to baseline. in lethally infected mice the peak il- b mrna peaked at day following sublethal infection and subsequently declined as virus was cleared of il- a mrna was delayed (day p.i.) and then declined as the animals succumbed to infection (fig. c ). il- from the cns. following a lethal infection, the quantity of il- b mrna increased from day p.i. until death. il-mrna peaked at day postinfection in lethally infected mice and declined by day as virus was cleared from a mrna peaked at day p.i. in the mice undergoing a the cns (fig. d) . in contrast to the lethal infection, the of il- mrna expression following acute and subacute infections showed that the levels increased in parallel levels of il- mrna increased rapidly and peaked at day p.i. following subacute infection. the level then through day p.i. (fig. a ). in . v- -infected mice, the level of il- mrna continued to increase until day p.i. declined rapidly by day and had reached baseline levels by day p.i. no il- mrna was detected in sham-and then declined slightly by day . infected mice, suggesting a rapid response to virus infection. very low levels of il- mrna were detected in im-discussion munodeficient mice infected with either virus. jhmv produces an acute cns infection associated with several immune effector mechanisms, including th -related cytokines both cd / and cd / t cells houtman and fleming, b) . kinetic analysis of cellular igg and igg a virus-specific antibodies were detected in survivors of jhmv infection; however, there appeared cns infiltrations during jhmv infection of mice shows that nk cells accumulate prior to cd / t cells, which in to be little relationship between induction of antibody and control of jhmv infection within the cns. induction of both turn precede accumulation of cd / t cells and macrophages (williamson et al., ; williamson, ) . there isotypes suggest that th and th cytokines are induced by jhmv infection. the kinetics of il- mrna induction is no direct evidence for a role of nk cells in suppressing jhmv replication (houtman and fleming, a) ; how-was of interest due to the association of il- with reduced th activity in vitro and with remission during exper-ever, cd / ctl appear to be critical immune effectors (williamson and stohlman, ; stohlman et al., b) . imental allergic encephalomyelitis (kennedy et al., ) . il- mrna was first detected at day p.i. in lethally recent analysis of jhmv pathogenesis in mice deficient in perforin suggests that in addition to cytolytic effectors infected mice, but not until day postinfection in the cns of the mice undergoing a subacute encephalitis. however, other immune components also contribute to sterilizing immunity (lin et al., ) . similarly, the adoptive transfer at the time most lethally infected mice were about to succumb to infection (day ), there was no difference in the of virus-specific cd / t cells to jhmv-infected mice demonstrates that some clones protect via reducing viral peak levels of il- mrna between the two groups. the kinetics of il- mrna accumulation differed between the replication (yamaguchi et al., ) , while others protect without reducing virus replication , groups; il- mrna accumulation in mice undergoing a sublethal infection was slower and remained at peak lev-suggesting that cytokines may play an important role in providing sterile immunity. els until day p.i., prior to declining to near basal levels by day . no il mrna was detected in the cns of in general the kinetics of cytokine mrna expression correlated with the temporal presence of cns infiltrating sham-infected or infected immunodeficient mice. no il- mrna was detected following a single amplification dur-mononuclear cells. many cytokine transcripts, with the exceptions of il- , il- a, and il- , were maximally ex-ing lethal or sublethal jhmv infections. however, after a second amplification, low abundant mrnas were de-pressed by day p.i., near the peak inflammatory cell infiltration and during the elimination of virus from the tected (fig. a) . no il- mrna was detected in either sham-infected or infected immunosuppressed mice fol-cns (williamson et al., ; williamson, ) . previous data using the oblv- jhmv variant which has a selec-lowing two amplifications (data not shown). the kinetics tive tropism for neurons suggested a correlation between increasing time following subacute infection, consistent with the resolution of encephalitis. it is interesting that ifn-g induction, t cell accumulation, and reduction of virus replication (pearce et al., ) . the semiquantita-the cns of mice with active macrophage-mediated demyelination (day p.i.) showed little evidence of tnf-tive kinetic analysis of ifn-g mrna in the cns of mice undergoing both lethal and sublethal jhmv infections a mrna, consistent with the inability of anti-tnf-a to prevent jhmv-mediated demyelination (stohlman et al., supports the positive correlation between ifn-g and viral clearance. however, the oblv- jhmv variant is a). a surprising number of mrnas peaked relatively early cleared from the cns of ifn-g-deficient mice , consistent with ifn-g exhibiting poor in vitro following jhmv infection. the mrnas encoding inos, il- , il- a, il- b, and il- peaked either prior to or anti-jhmv activity (zhang et al., ) and inability of rifn-g to inhibit cns virus replication (smith et al., ) . coincident with initiation of viral clearance. in most cases (except inos mrna) the levels were either higher or these data contrast with other viral-induced encephalopathies in which ifn-g plays a significant role (kundig et increased more rapidly in the mice undergoing subacute infections. accumulation of inos mrna was first de-al., ; finke et al., ) , including some (yu et al., ) , but not all (wesselingh et al., ) , neuronotropic tected in mice undergoing a lethal infection coincident with the initial detection of ifn-g mrna. however, the viruses. the kinetics of ifn-g mrna induction suggests that it may play a more prominent role in the pathogene-mrna levels declined as virus replication declined, suggesting a direct effect of virus on inos induction. in sis of jhmv variants with predominant tropisms for microglia, astrocytes (jhmv), or oligodendroglia ( . v- ). contrast to lethal infections, inos mrna lagged detection of ifn-g in mice undergoing subacute infections and the isotype diversity of the anti-jhmv antibody response suggests that both th and th subsets of cd / increased to less than % the level detected in mice undergoing a lethal infection. similar to the recent data t cells are activated during infection. il- mrna accumulation in the cns corresponds to infiltration of th cells demonstrating low levels of inos in the cns of both nude mice and mice deficient in ifn-g , (cua et al., ) and kinetic analysis suggests that t cells expressing th cytokine profiles are recruited into inos mrna in immunodeficient mice was approximately % the levels detected in the cns of intact mice at day the cns with nearly equal kinetics in both lethally and sublethally infected mice (fig. a) . il- increases the p.i. although jhmv is susceptible to inhibition by inos in vitro, inos is not associated with in vivo protection severity of encephalitis (ikemoto et al., ) and could potentially play a role in jhmv persistence via inhibition . il- , predominantly produced by cells of the myelo-of viral clearance (moran et al., ) . in support of the recruitment of th cells, il- mrna also increased with monocytic lineage, is associated with the induction of th cd / t cells (brunda, ) . il- mrna peaked kinetics similar to those of ifn-g and il- . whether this difference in detection of th cytokines is due to differ-early (day ) in mice undergoing both lethal and sublethal jhmv infections. however, no significant differences ences in mouse strains or the selective tropism of the virus is not known. it is interesting that although il- is were found comparing mrna levels in immunodeficient mice to intact mice. this may suggest that jhmv infection secreted by activated microglia in vitro (lodge and sriram, ) , no il- mrna was detected in sham-induces transcription of il- mrna in cns cells. in addition, . v- infects predominantly, but not exclu-infected or immunodeficient mice. this contrasts with other cytokine mrna detected in either sham-infected sively, oligodendroglia, while jhmv infects predominantly microglia and astrocytes. the relatively higher or virus-infected immunodeficient hosts (see below). tnf-a mrna is induced following jhmv infection level of il- mrna in . v- -infected mice suggests the possibility that oligodendroglia transcribe il- mrna in (pearce et al., ; stohlman et al., a; sun et al., ) and tnf-a is present during both the acute and response to jhmv infection, similar to the induction of il- mrna following measles virus infection of oligo-persistent jhmv infections. tnf-a mrna is not translated in jhmv-infected cells (stohlman et al., a ), al-dendroglia (yamabe et al., . during both the lethal and sublethal infections the il-though it may be secreted by adjacent but not infected cells. in addition, inhibition of tnf-a, which prevents a mrna peaks appear to coincide with replication and not clearance, suggesting that infection induces a rapid experimental autoimmune encephalitis (ruddle et al., ) , has no effect on either jhmv-induced encephalitis induction of il- a mrna. these data contrast to the association of il- a mrna and the clearance of the oblv-or demyelination (stohlman et al., a) . as anticipated, based on the relative tropism of the two viruses analyzed, variant of jhmv (pearce et al., ) , suggesting an additional difference in cytokine responses depending tnf-a mrna accumulated initially in the cns of mice infected with jhmv. however, by day p.i. there was on the tropism of the virus analyzed. il- b mrna, previously detected in the cns of jhmv-infected mice little difference in the levels of tnf-a mrna in the two groups. finally, the level of tnf-a mrna decreased with (pearce et al., ) , increased directly after infection at day p.i. however, the level was approximately the same suggests it may play a positive role in reducing the extent of cns inflammation thereby inadvertently contributing as the level detected in sham-infected mice, suggesting it was induced by trauma. in all mice the levels subse-to persistent infection. some aspects of our data, i.e., the rapid induction of il- mrna in mice infected with . v-quently dropped by day p.i. the levels of il- b mrna peaked at day following . v- infection and at day , suggest that infection of specific cell types may influence the induction of cytokine mrna (yamabe et al., following jhmv infection, suggesting il- b mrna was also induced by infection. analysis of the levels in immu- ) . this supports the notion that the cytokine mrna patterns more closely reflect diversity of the immune re-nodeficient mice were consistent with the notion that infection, and not immune infiltrates, contributed the ma-sponse to an individual agent, although differential secretion of cytokines following infection of unique cns cell jority of the il- b mrna levels. il- , another pleiotropic cytokine with numerous ef-types cannot be ruled out (benveniste, ; sun et al., ) . while the kinetics of ifn-g, il- , and il- showed fects on immune responses (van snick, ) , was also detected early following both lethal and sublethal infec-little difference between the groups undergoing lethal or sublethal infections, mrnas encoding il- and il- b tions. by contrast, il- mrna was also only detected at days p.i. with the neuronotropic oblv- jhmv variant either appeared more rapidly (il- ) or accumulated to higher levels (il- b) following infection with . v- virus. (pearce et al., ) . kinetic analysis shows that the levels of il- mrna peaked at day post- . v- infection by contrast the induction of inos and il- a mrnas were increased in mice undergoing a lethal infection. these and at day post-jhmv infection. interestingly, analysis of the mrna levels in the immunodeficient mice showed data suggest that an early induction of il- , and possibly il- b, are associated with sublethal infection or the dif-virtually no induction of il- mrna, suggesting that in contrast to il- a and il- b an intact immune response ferent tropisms exhibited by these two jhmv variants. however, during both infections the mrna levels de-was required for il- mrna induction. rapid induction of il- mrna following jhmv infection is consistent with creased as virus was cleared. similarly, there appears to be an inverse correlation between a rapid induction other models of viral-induced encephalitis in which it also precedes ifn-g (moskophidis et al., ) . although of inos mrna and sublethal disease, consistent with the recent demonstration that although inos is protective in both il- and il- are cofactors for ctl induction (chen and zlotnik, ; takai et al., ) , kinetic analysis is vitro, inhibition of inos activity in vivo appears to have no effect on jhmv pathogenesis . taken consistent with the notion that il- , and not il- , may be involved in the induction or recruitment of jhmv-specific together, kinetic analyses of the induction of cytokine mrna during the lethal and sublethal jhmv infections ctl. jhmv infection induces il- secretion from both brain endothelial cells and astrocytes following in vitro are consistent with the accumulation of both th -and th -associated cytokines and support the interaction of infection with jhmv (joseph et al., ) , consistent with data showing that it is produced by resident cns cells multiple cellular and soluble effector mechanisms whose balance may be critical in providing protection and steri-following infection with lymphocytic choriomeningitis virus (frei et al., ) . it is interesting that il- mrna lizing immunity. peaks first in mice infected with the . v- variant compared to jhmv, which infects a significantly larger num-acknowledgments ber of astrocytes. the rapid induction of il- and il-we thank wen-qiang for excellent technical assistance. this work b following infection with . v- is consistent with the was supported by grant ns from the national institutes of health. induction of these mrna in oligodendroglia infected by escherichia coli lipopolysaccharide and tumor necrosis factor alpha a paradigm for virus-induced demyelinating disease. trends microbiol. , - . stimulation recovery from acute virus infection. role of cytotoxic t lymphocytes in the eliminatiation factor self-antigen-intion of lymphocytic choriomeningitis virus from spleens of mice duced th responses in experimental allergic encephalomyelitis (eae)-resistant mice exposure to t helper cytokines in vivo before encounter with antigen selects for perforin-mediated cytolysis regulation of microglial activation t helper subsets via alterations in antigen-presenting cell function gamma interferon is a major mediator of antiviral defense in experiing and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv- ) rus-infected mice production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of in-weiner on the cellular source and function of interleukin produced in the central nervous system in viral disease. ase chain reaction method in schistosoma mansoni infected mice an absolute and restricted requirement for il- in natural killer cell ifn-g production and antiviral bral toxoplasmosis is induced by in vivo neutralization of tnf-a and correlates with the down-regulated expression of inducible nitric defense the in vivo oxide synthase and other markers of macrophage activation ifng independent production of il- during murine endotoxemia cytokine induction during t-cell mediated clearance of mouse hepa-immunol dissociation of demyelination titis virus from neurons in vivo the astrocyte is a target cell in mice and viral clearance in congenitally immunodeficient mice infected with murine coronavirus jhm pathogenesis of mouse hepatitis virus-induced demyelination cytotoxic t cell-resistant variants are selected in a virus-induced small amounts of exogenous il- increase the severity of demyelinating disease grunencephalitis induced in mice by the intranasal infection of herpes simplex virus type interleukin- induction in vitro in mouse brain endothelial cells and astrocytes allergic encephalomyelitis the immune system response by exposure to mouse hepatitis virus (mhv- , jhm) igg induction factor: a single molecular entity with multiple of mice with experimental autoimmune encephalomyelitis reveals that il- mrna expression correlates with recovery - . dependent ifn-g exerts an antiviral effect in the central nervous system but not in peripheral solid organs pathogenesis of a neurotropic of two plaque morphology variants of the jhm neurotropic strain murine coronavirus strain jhm in the central nervous system. seminar virol mechanisms of demyelination in jhm virus encephalomyelitis. electron microscopic cells prevent a lethal infection but do not inhibit virus replication tumor necrosis between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus mouse hepatitis virus-specific cytotoxic t lymphocytes protect from lethal infection without eliminating virus from the central williamson hepatitis virus strain jhm virus-specific t cells in the central nervous activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus b cell stimulatory factor- is involved in the differentiation of yamabe , gene expression in measles-infected adult human glial cells protection of mice from a lethal coronavirus infection in the central induced by murine hepatitis virus, jhm strain (mhv- ) is immunologically mediated pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) role of interferon-g in immunity to herpes simplex virus expression of gamma interferon by a coronavirus defectivealphavirus encephalitis suggests a predominant type t cell response effective clearance of key: cord- - xpma authors: onal, merih; onal, ozkan; turan, alparslan title: can secondary lymphoid organs exert a favorable effect on the mild course of covid- in children? date: - - journal: acta oto-laryngologica doi: . / . . sha: doc_id: cord_uid: xpma nan coronavirus disease is characterized by severe acute respiratory syndrome which is related to a novel virus named coronavirus (sars-cov- ) [ ] . according to a report presented by world health organization; patients under the age of accounts for only . % of patients found to be covid- positive [ ] . when the reports in the literature on children who are covid- positive are reviewed, it can be established that children without underlying disorders such as respiratory dysfunction and immunosuppression have a milder course of disease without evaluating their age groups and epidemiological characteristics [ ] . it is not yet known why a large majority of covid- cases in children have a milder course than adults. but, various theories have been put forward on this issue. for example, it has been claimed that the binding capacity of angiotensin converting enzyme ii (ace-ii), which is thought to be coronavirus cell receptor, is lower in children, which decreases viral load, leading to a milder disease [ ] . it has even been stated that children may still remain asymptomatic for this reason even after contracting the covid- virus [ ] . another theory proposes that children remain usually indoors and hence are less exposed to pathogens, but examples of children living in rural areas suffice to refute this theory [ ] . although the immune system developed via vaccination is effective against pandemic diseases, the lack of an effective vaccine against covid- so far, suggests the presence of other defense mechanisms against covid- in children [ ] . in this editorial, we want to discuss a theory, which has not been mentioned before. it is known that the immune system of children and adults is functionally and structurally different. palatine and pharyngeal tonsils are important organs of the immune system, and they protect the body from pathogens invading the upper respiratory tract, especially in young children [ ] . palatine and pharyngeal tonsil tissues are secondary lymphoid organs that prepare a continuous immune response and exhibit immune activity in childhood, which are located at the entry point of the respiratory and digestive system. they make up the first defense mechanism and carry out cellular and humoral immune functions against antigens that enter the body from the respiratory and digestive system mainly up to puberty [ ] . recent studies have demonstrated that localized inflammatory changes in the lower and upper respiratory tract may give rise to systemic responses [ ] . adenoids are lymphoepithelial tissues predominantly composed of t lymphocytes, macrophages, and dendritic cells. leukocytes on the surface secretion of adenoids may release iga, igg, and igm, required in antigen phagocytosis. adenoid surface secretion also contains many active t cells participating in cellular immunity. t lymphocytes in adenoid tissue are extremely important for mounting an effective immunity reaction. particularly cd t lymphocytes play an efficient role in immune response both via cytolysis and production of cytokine, chemokines, and microbicidal molecules. when ifn-a production, released by t lymphocytes, decreases for any reason, children become vulnerable to contagious viral diseases. furthermore, pathogenic bacteria grow rapidly in adenoid tissue [ ] . interferon (ifn), is a glycoprotein synthesized by host cells exposed to the virus as an antiviral response and forms the first line of defense against viral infections [ ] . ifn, inhibits virus replication by stimulating the production of antiviral proteins and protects normal cells from viruses. in addition, it activates cytotoxic t lymphocytes, nk natural killer cells, and macrophages, eliminating the virus from cells [ ] . if there is a deficiency of endogenous ifn owing to immaturity of the immune system, the antiviral response decreases and viral infections arise [ ] . ifn production in the early periods in children may give them an advantage against viral diseases. besides, it has also been recognized that secretory iga, which is the main antibody class released from adenoid tissue, has a significant role in mucosal immunity, bonding of bacteria and viruses, and invasion of epithelium by pathogens [ ] . in a study on patients with hypertrophic adenoid tissue, recurrent upper and lower respiratory tract infections were demonstrated along with high serum myeloperoxidase levels (indicating neutrophil activation), increased serum eosinophilic cationic protein levels (indicating eosinophil activation) and high cd glycoprotein levels (indicating monocyte/macrophage activation) [ ] . actually, it was stated that one of the probable causes of the milder course of covid- in children may be that the amount of myeloperoxidase is lower in the lungs of children [ ] . it is known that human tonsils are immunologically reactive lymphoid organs carrying out humoral and cellular immunity functions as a response to various antigens and displaying b and t cell activity [ ] . it is known that tonsils in children harbor a higher concentration of helper and cytotoxic lymphocytes than adults [ ] . however, in a study, in the nasopharynx of children with tonsillar hypertrophy (mean age ), adenovirus, bocavirus- , coronavirus and rhinovirus were detected at higher rates compared to the recurrent tonsillitis group (mean age ) [ ] . in the same study, although a difference was expected in tonsillar hypertrophy and recurrent tonsillitis groups in terms of the levels of interferons (ifn-a, ifn-b, ifn-c, il- , il- ) , which are cytokines with antiviral activity and whose expression is induced by viral infection, not detected. it is thought that this may be due to the fact that tonsil hypertrophy is a result of chronic inflammation in the tonsils and the same interferon pathways are activated equally in both tonsillar hypertrophy and recurrent tonsillitis [ ] . in addition, il- levels, which has anti-inflammatory properties, was found to be higher in tonsillar hypertrophy group than recurrent tonsillitis group. but, it should be added that there was no difference in virus detection between groups at the intratonsillar level in the same study [ ] . in a recent crosssectional study in which pediatric and adult patients undergoing tonsillectomy were included (mean age ), it was shown that no virus was shown to be associated with tonsil diseases and respiratory diseases [ ] . the wide age range of the study may be associated with this result. even though high serum ig g and ig a levels have been demonstrated in various studies in patients with chronic tonsillitis there are also other studies, indicating that hypofunction of local and systemic immunity brings about inflammation and/or hypertrophy of adenoids and tonsils [ ] . in fact, it is believed that when there is chronic inflammation in these organs, they do not play part in protection against upper respiratory tract infection and even inhibit immune response, enhancing the severity of the infection [ ] . this may be one of the presumable mechanisms of immune deficiency in children who have a severe clinical picture in covid- similar to adults. it is our suggestion that, with epidemiological studies both in children and adults, the structure of secondary lymphoid organs, forming the first line of defense for the body, is an issue that should be comprehensively addressed. no potential conflict of interest was reported by the author(s). https://orcid.org/ - - - ozkan onal http://orcid.org/ - - - alparslan turan http://orcid.org/ - - - acute myocardial injury: a novel clinical pattern in children with covid- world health organization. world health organization-china joint mission on coronavirus disease why is covid- so mild in children? a pneumonia outbreak associated with a new coronavirus of probable bat origin a covid- transmission within a family cluster by presymptomatic infectors in china epidemiology of covid- among children in china seroepidemiological analysis of influenza pandemics in shizuoka prefecture and all japan short-and long-term impacts of adenoidectomy with/without tonsillectomy on immune function of young children < years of age: a cohort study immunology of tonsils and adenoids: everything the ent surgeon needs to know nasal allergen provocation induces adhesion molecule expression and tissue eosinophilia in upper and lower airways children with recurrent otitis show defective ifn-gamma producing cells in adenoids current scenario of covid- in pediatric age group and physiology of immune and thymus response diagnosis and treatment of novel coronavirus infection in children: a pressing issue differential expression of immunoglobulin a in the adenoids of children with and without exudative otitis media adenoid hypetrophy: definition of some risk factors serum immunoglobulins in patients with chronic tonsillitis early stage impacts of tonsillectomy on immune functions of children tonsillar cytokine expression between patients with tonsillar hypertrophy and recurrent tonsillitis intratonsillar detection of distinct viruses: a cross-sectional study profile of immunoglobulin production in adenoid and tonsil lymphocytes key: cord- -lcl jn authors: acharya, dhiraj; liu, guanqun; gack, michaela u. title: dysregulation of type i interferon responses in covid- date: - - journal: nat rev immunol doi: . /s - - -x sha: doc_id: cord_uid: lcl jn infection with sars-cov- can lead to excessive production of pro-inflammatory cytokines, but the production of type i interferons, which are key antiviral mediators, is reportedly blunted. here, we discuss how imbalanced interferon responses may contribute to the pathology of covid- . covid- is characterized by a mild to severe respiratory illness that appears to be influenced by age and comorbidities. critically ill patients often develop acute respiratory distress syndrome (ards) or multi-organ injuries as a result of secondary haemophagocytic lymphohistiocytosis (shlh). both ards and shlh are characterized by overzealous cytokine production and excessive inflammation, the hallmarks of cytokine release syndrome (crs). the crs elicited by sars-cov- is reminiscent of the immune dysregulation caused by other highly pathogenic respiratory viruses, including the related betacoronaviruses sars-cov and mers-cov. markedly elevated plasma levels of pro-inflammatory cytokines, including il- and tumour necrosis factor (tnf), as well as of several chemokines have been detected in patients with covid- (ref. ). airborne sars-cov- infections in humans initiate from the virus entering nasal and airway epithelial cells through binding to angiotensin-converting enzyme (ace ). tmprss , a cellular protease that activates the sars-cov- spike protein, colocalizes with ace and can prime sars-cov- fusion directly at the plasma membrane. in the lungs, sars-cov- infects type i and type ii alveolar epithelial cells, as well as alveolar macrophages that are among the first producers of pro-inflammatory cytokines. as key components of the immediate antiviral response, type i interferons (hereafter referred to as ifns) are crucial for restricting viral replication and spread, through autocrine and paracrine type i ifn receptor (ifnar) signalling. however, minimal amounts of ifns have been detected in the peripheral blood or lungs of patients with severe covid- (refs , ), which is in contrast to what is seen in patients infected with highly pathogenic influenza viruses. interestingly, although low levels of systemic ifn production appear to correlate with severe covid- (ref. ), the local induction of ifns and ifn-stimulated genes (isgs) has been noticeable in the bronchoalveolar lavage (bal) of some critically ill patients . this was attributed to the activation of specialized immune cells such as lung-resident dendritic cells (dcs). in particular, plasmacytoid dcs were shown to produce ifnα in response to sars-cov. in patients with sars who did not receive corticosteroids, ifnα was detected in plasma during the 'pre-crisis' phase but subsided during the 'crisis' phase . in a mouse model of sars-cov infection, local ifn responses in the lungs were delayed relative to peak viral replication, which impeded virus clearance and was associated with the development of crs . the kinetics of the systemic and the local ifn responses that occur during covid- remain to be fully elucidated, as well as their respective contributions to covid- pathogenesis and disease severity. the dysregulated ifn responses are indicative of the effective immunomodulatory strategies used by betacoronaviruses. during the incubation phase, sars-cov- replicates stealthily in host cells without detectably triggering ifns, leading to high viral loads . coronaviruses are known to induce the formation of membranous compartments dedicated to viral rna synthesis and thereby conceal viral pathogen-associated molecular patterns (pamps; for example, viral rnas) from detection by host pattern recognition receptors (prrs), such as rig-i and mda . furthermore, several conserved betacoronavirus proteins, predominantly non-structural proteins (nsps), are known to exert direct ifn-antagonistic activities. some modify specific features of the viral rna (by catalysing guanosine-n and ribose- ʹ-o methylation) to avoid recognition by specific prrs (for example, nsp and nsp ), while others, such as nsp and nsp , inhibit the signal transduction mediated by prrs and by ifnar, respectively . by contrast, the nucleocapsid protein of sars-cov has been shown to directly activate nf-κb. the robust production of pro-inflammatory cytokines and chemokines, with a limited production of ifns, during sars-cov- infection suggests effective activation of nf-κb but not that of ifn-regulatory factor (irf ) and irf (ref. ). it will be important to determine exactly how sars-cov- antagonizes ifn induction and ifnar signalling. as a central liaison between the innate and adaptive immune systems, ifns are imperative to regulating the activation and functions of various immune cell populations. importantly, during sars-cov or www.nature.com/nri mers-cov infection in mice, ifns directly regulate the pulmonary infiltration of monocyte-derived macro phages. whereas blocking ifnar signalling markedly reduced macrophage infiltration, delayed ifn induction by sars-cov led to the accumulation of highly activated macrophages in the lungs that induced immunopathology . by contrast, ifnar inhibition enhanced the recruitment of neutrophils to the lungs in mers-cov-infected mice, leading to elevated production of pro-inflammatory cytokines . impaired ifn production during severe covid- may also lead to an imbalance in the pro-inflammatory versus pro-repair functions of airway macrophages. patients who died from sars-cov showed an accumulation of pro-inflammatory macrophages but a deficiency in wound-healing macrophages in the lungs; this was associated with higher serum levels of neutralizing antibodies against the spike protein of sars-cov . other innate immune cells such as natural killer (nk) cells are also regulated by ifns during coronavirus infection. inhibition of ifnar signalling suppressed the accumulation of nk cells in the lungs of mers-cov-infected mice , which may dampen the early clearance of virusinfected cells. while patients with severe covid- showed profound depletion and functional exhaustion of nk cells , it is unclear whether this nk cell dysfunction is due to dysregulation of ifn responses. severe covid- is associated with impaired t cell responses that manifest as lymphopenia and functional exhaustion of cd + and cd + t cells . impaired t cell responses can result from deficient ifn production, as ifns promote the survival and effector functions of t cells. blocking ifnar signalling during mers-cov infection attenuated the development of virus-specific cd + and cd + t cells in mice . although the early production of ifns is crucial for an efficient t cell response, a delayed ifn response can inhibit t cell proliferation or t cell egress from lymphoid organs, or it can cause functional exhaustion and cell death of t cells. the lung injury associated with crs in patients with severe covid- indicates a possible failure to activate immunosuppressive mechanisms in a timely manner. indeed, regulatory t (t reg ) cell counts in patients with covid- have been shown to inversely correlate with disease severity . ifns are known to be crucial regulators of the development of t reg cells. it is thus tempting to speculate that the deficient or dysregulated ifn responses elicited by sars-cov- infection may influence the generation of t reg cells during the recovery phase of covid- . future studies should explore how ifn dysregulation during covid- might shape t cell responses and, given that cd + t cell activation is crucial for the development of b cell immunity, how this may in turn affect antibody responses. that ifn dysregulation represents a key determinant of covid- pathogenesis highlights its potential for therapeutic intervention. prophylactic administration of ifns, which elicits a pre-existing antiviral state in target cells, may block viral infection at the very early stage. daily ifnα nasal drops along with standard personal protective equipment (ppe) were shown to protect at-risk health-care workers from covid- over days without noticeable adverse effects (nct ). the use of ifns as a treatment for covid- remains controversial, particularly regarding the timing of administration. early ifn treatment before peak viral replication protected mice from lethal sars-cov or mers-cov challenge, whereas late ifn administration impeded viral clearance and aggravated immunopathology , . clinical studies on sars-cov and mers-cov have also shown inconclusive effects of ifn in combination with antivirals on disease outcomes, which is likely due to varied timing of administration and also comorbidities. importantly, ace has been recently identified as an isg in human airway epithelial cells . whether prophylactic or therapeutic ifn administration may enhance the entry and replication of sars-cov- in target cells during disease progression is a potential safety concern. further studies should also determine the contributions of host genetics, age and comorbidities to the therapeutic effectiveness of ifns. while several ongoing clinical trials are evaluating the efficacy of ifn treatment for covid- , a deeper understanding of the spatiotemporal kinetics of ifn responses during clinical sars-cov- infections is warranted to inform ifn-related therapeutics and vaccine design. imbalanced host response to sars-cov- drives development of covid- impaired type i interferon activity and exacerbated inflammatory responses in severe covid- patients heightened innate immune responses in the respiratory tract of covid- patients interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection functional exhaustion of antiviral lymphocytes in covid- patients dysregulation of immune response in patients with coronavirus (covid- sars-cov- receptor ace is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues the authors contributed equally to all aspects of the article. the authors declare no competing interests. clinicaltrials.gov: https://clinicaltrials.gov/ key: cord- -ywb cq authors: sarkar, indranil; garg, ravendra; van drunen littel-van den hurk, sylvia title: selection of adjuvants for vaccines targeting specific pathogens date: - - journal: expert rev vaccines doi: . / . . sha: doc_id: cord_uid: ywb cq introduction: adjuvants form an integral component in most of the inactivated and subunit vaccine formulations. careful and proper selection of adjuvants helps in promoting appropriate immune responses against target pathogens at both innate and adaptive levels such that protective immunity can be elicited. areas covered: herein, we describe the recent progress in our understanding of the mode of action of adjuvants that are licensed for use in human vaccines or in clinical or pre-clinical stages at both innate and adaptive levels. different pathogens have distinct characteristics, which require the host to mount an appropriate immune response against them. adjuvants can be selected to elicit a tailor-made immune response to specific pathogens based on their unique properties. identification of biomarkers of adjuvanticity for several candidate vaccines using omics-based technologies can unravel the mechanism of action of modern and experimental adjuvants. expert opinion: adjuvant technology has been revolutionized over the last two decades. in-depth understanding of the role of adjuvants in activating the innate immune system, combined with systems vaccinology approaches, have led to the development of next-generation, novel adjuvants that can be used in vaccines against challenging pathogens and in specific target populations. development of vaccines against infectious diseases is one of the most remarkable accomplishments in the history of mankind [ ] . smallpox has been eradicated from the world, and other diseases like diphtheria, poliomyelitis, pertussis, measles, and neonatal tetanus are significantly controlled by vaccination [ , ] . while live attenuated vaccines are immunogenic, there is a chance of live virus-induced disease progression in populations with underdeveloped or compromised immune systems [ ] . in contrast, inactivated virus vaccines are safe, but unsuitable when natural infection by the pathogen itself fails to induce any long-term immunity. recombinant subunit vaccines are considered as one of the most attractive modern vaccine types due to their high safety profiles. however, subunit vaccines are not inherently immunogenic [ , ] . to overcome this limitation, adjuvants are incorporated in subunit vaccines to enhance immunogenicity of the vaccine antigen. adjuvants facilitate the development of vaccines targeting pathogens against which live attenuated or inactivated vaccines are ineffective or undesirable. identification and selection of new adjuvants is thus critical, but also challenging, for successful subunit vaccine development. the fact that only a few adjuvants were approved for use in human vaccines till a few years ago can be at least partially attributed to the dearth of knowledge of the mechanism of action of adjuvants. however, presently six adjuvants (alum, as , mf , as , as and cpg odn) are approved for use in human vaccines. this was possible because structural characterization of several adjuvants and identification of various pattern recognition receptors (prrs) and co-stimulatory ligand receptors have enabled us to better understand the mode of action of adjuvants at a molecular level. understanding the mode of action of adjuvants is critical in designing vaccines that elicit pathogen-specific effector and long-term memory responses and in assessing the adjuvant safety at developmental and regulatory stages. the possible mechanisms of action by which adjuvants exert their adjuvanticity are discussed in the subsequent sections and represented schematically in figure . adjuvants as a delivery system in subunit vaccines [such as liposomes, immune stimulating complexes (iscoms) and nanoparticles] are considered effective in stimulating protective immunity [ ] . such adjuvants prevent rapid degradation of proteins and peptides in vivo, thereby enhancing the dose effectiveness of the vaccine antigen. liposomes are used in vaccine formulations against influenza, chlamydia, malaria, and tuberculosis (tb). coadministration of antigen with cationic liposomes leads to the induction of stronger antigen-specific immune responses than neutral or anionic liposomes [ ] . liposomes are effective vaccine delivery systems and act as carriers in adjuvants such as as , a liposome-based formulation consisting of monophosphoryl lipid a (mpla) and qs- [ ] . improved saponin-based tensoactive adjuvants (iscom, iscomatrix, and matrix-m tm ) are particulate antigen delivery systems with powerful immunostimulating activity [ ] . in iscoms, saponin, cholesterol and phospholipid form cage-like structures ( - nm in diameter). the adjuvant iscomatrix has a structurally similar structure but without the incorporated antigen (the antigen can be formulated with iscomatrix to prepare an iscomatrix vaccine) [ ] . both antigen delivery and immunostimulatory properties are present in iscomatrix [ ] . within the first few hours of injection, the antigen-iscomatrix complex traffics into draining lymph nodes (dlns) where antigen delivery takes place for uptake by the resident dendritic cells (dcs) and other antigen presenting cells (apcs) [ ] . iscoms are currently used in the development of influenza vaccines and iscomatrix in hepatitis c virus (hcv), influenza and candidate cancer vaccines in humans. the matrix-m tm adjuvant is being evaluated in vaccines against influenza, herpes simplex virus (hsv) type and malaria [ ] . nanoparticles are polymeric colloidal carriers of antigens, which enable site-directed delivery and prolonged release of antigen and facilitate alternative modes of vaccine administration (such as inhalation, optical or topical delivery). examples of polymeric nanoparticles are poly(lactic-co-glycolic acid) (plga), poly(lactic acid) (pla), poly(glycolic acid) (pga), poly(hydroxybutyrate) (phb) and chitosan [ ] aluminum salts are also used as delivery systems. crystals of alum bind to and alter the lipids of the dc plasma membrane to trigger cell activation that facilitates delivery of antigen, without alum itself being internalized by the dcs [ ] . aluminum salts are used as adjuvants in human vaccines against diphtheria, tetanus, pertussis, rabies, anthrax, and hepatitis a and b [ ] . in vitro studies revealed that the oil-inwater emulsion adjuvant, mf increases both phagocytosis and pinocytosis indirectly to promote better antigen uptake by apcs. instead of directly targeting dcs for antigen uptake, mf acts upstream by promoting influx of dc precursor cells and augmenting their differentiation into dcs [ ] . the safety of mf was demonstrated in various clinical studies with antigens from hepatitis b virus (hbv), hcv, cytomegalovirus (cmv), hsv and human immunodeficiency virus (hiv) [ ] . similar to mf , as does not directly activate dcs in vitro. intramuscular injection of as in mice promotes influx of monocytes, dcs, and granulocytes into the dlns. as is article highlights • identification of new prrs and their agonists are expected to lead to the identification of more adjuvants; in particular, prr agonists in combination adjuvants hold great promise. • systems vaccinology will provide a better understanding of the mode of action of adjuvants and allow identification of unique biomarkers of adjuvanticity. • there are many pathogens for which host-pathogen interactions have not been characterized in detail. such knowledge on hostpathogen interaction combined with the mechanism of action of adjuvants will lead to the use of specific adjuvants in vaccines against distinct pathogens. figure . schematic representation to highlight the possible mechanism of action by which adjuvants exert their adjuvanticity. adjuvants can serve as a depot that mediates recruitment of apcs or act as a delivery system to facilitate uptake of antigen by the apcs. adjuvants may activate innate immune responses by signaling through cell surface clrs (such as dectin- , dectin- , mincle), cytosolic nlrs, cell surface tlrs, endosomal tlrs or cytosolic rig-i and mda . signaling via prrs may lead to the activation of several transcription factors, which results in the production of pro-inflammatory cytokines, chemokines and type i ifns. secretion of chemokines due to adjuvants may also result in the recruitment and infiltration of more immune cells. adjuvants can activate c-gas that participates in the stingmediated irf -type i ifn pathway. adjuvants can enhance the expression of mhc and co-stimulatory molecules to mediate efficient presentation of antigen to naïve cd + t cells. depending upon the class of adjuvant, cellular (th ) and/or humoral (th ) immune responses may be induced. adjuvants also play important roles in gc reaction, affinity maturation and long-lived memory responses as a part of humoral immunity. also responsible for enhanced antigen uptake, by monocytes in particular, and antigen presentation in the dlns; this process is mediated by the presence of α-tocopherol in as [ , ] . the safety of squalene-based adjuvants (such as mf and as ) has been demonstrated by toxicological studies in animal models as well as in phase i-iii studies in humans [ ] [ ] [ ] depot effect refers to slow and prolonged antigen release at the site of injection providing continuous stimulation of the immune system. this facilitates enhanced antigen uptake by the apcs, which correlates to induction of high antibody titers. adjuvanticity of alum was originally thought to be due to depot effect; however, according to recent evidence, a depot effect is not the only mechanism by which it exerts its adjuvant activity [ ] . in a mouse model, alum was found to rapidly induce an inflammatory environment (via induction of inflammatory chemokines) that in turn triggers neutrophil recruitment and swarming at the injection site. furthermore, alum induces neutrophil death, resulting in the release of extracellular dna strands (neutrophil extracellular traps or nets) that play a significant role in the adjuvant action of alum [ ] . oil-in -water emulsions such as emulsigen®, water-in-oil emulsions such as cationic adjuvant formulation (caf) (a cationic liposome consisting of a combination of dimethyldioctadecylammonium/α,α'-trehalose , '-dibehenate or dda/tdb), as well as biodegradable micro-and nano-particles exhibit adjuvant activity via a depot effect in mice [ , ] . cationic liposomes exhibit long depot effects at the site of injection and strong electrostatic interactions with apcs. in contrast, adjuvants such as mf or iscoms do not require depot formation to exert their adjuvant activities; rather, antigen and adjuvant are cleared rapidly from the site of administration. a biodistribution study of as in mice conducted by radiolabelling each component of as revealed that all constituents of as infiltrated into the dlns within min of injection [ ] and that - % of each constituent of as was cleared from the injection site days post injection, suggesting dissociation of as [ ] . similarly, intramuscular injection of an as -adjuvanted vaccine in mice indicated rapid clearance of antigen and qs- from the injection site and into the dlns. differential biodistribution dynamics and pharmacokinetics of antigen and qs- suggested that the as and the vaccine antigen were not physically associated with each other [ ] . similarly, in matrix adjuvant formulations (consisting of nanoparticles comprised of saponin, cholesterol, and phospholipid), a physical association between matrix and antigen is not required for potent immune stimulation [ ] . in contrast, electrostatic interaction of antigen with adjuvant is required for optimal adjuvant activity of caf . similarly, optimal adjuvanticity of virosomes is achieved when the antigen of interest is associated with the virosome either through encapsulation or attachment to the bilayer via hydrophobic interaction [ ] . the success of the yellow fever vaccine yf- d, a live attenuated virus vaccine, can be attributed to its ability to activate multiple tlrs including tlr , , and , on or in dcs in mice [ ] . yf- d also activates human monocyte-derived dcs and plasmacytoid dcs (pdcs) [ ] . tlr -tlr complexes are activated by the lipopeptide analog pam csk , while tlr -tlr complexes are activated by the macrophage activating lipopeptide- (malp- ) from mycoplasma [ ] . poly(i:c) is a ligand for endosomal tlr , cytosolic retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated protein (mda ). poly(i:c) and its two derivatives, polyi:c u (ampligen) and poly(ic:lc) (hiltonol) are used in clinical trials against both tumors and infectious diseases such as hiv [ ] . tlr is targeted by monophosphoryl lipid (mpl)a. as (containing mpl) is approved for use in hbv (fendrix) and human papilloma virus (hpv) vaccines (cervarix) [ , ] . as (containing mpl) is also used in a malaria vaccine (rts,s) and the varicella zoster virus vaccine (hz/su) that have been found to be efficacious in phase iii trials [ ] . tlr recognizes bacterial flagellin. tlr signaling in cd + cd b + dcs plays an important role in intestinal iga production and th differentiation, and leads to myd -dependent, strong nuclear factor (nf)-κb activation and th- type immune responses in mice [ , ] . in contrast, flagellin acts as a th -polarizing factor for cd + t cells from human neonates and adults [ ] . tlr and tlr recognizing single-stranded rna (ssrna) molecules are targeted by small-molecule immune potentiator (smip)-based adjuvants such as imiquimod and resiquimod used in hpv virus-like particle (vlp) vaccines [ ] . tlr signaling induces b cell-mediated production of immunoglobulins (ig), interleukin (il)- and tnf-α, and natural killer (nk) cellmediated production of ifn-γ, while tlr signaling induces t cell proliferation, production of ifn-γ, il- , and il- , and memory t cell activation [ ] . tlr recognizes unmethylated cpg motif-containing microbial dna or immunostimulatory sequences (iss). tlr agonists are used in hbv vaccines to promote higher levels of protective antibodies. consequently, fewer immunizations and lower antigen doses are needed. in mice, tlr signaling leads to th type pro-inflammatory responses (il- , il- , il- , il- , tnf-α and ifn-γ), upregulation of major histocompatibility complex (mhc) and costimulatory molecules, and increased cd + t cell responses, while tlr -mediated b cell activation is responsible for induction of humoral immunity and antibody class switching [ ] . intracellular nlrs such as nod and nod receptors recognize diaminopimelatic acid (dap)-containing muropeptide from gram-negative bacteria, while nod detects the muramyl dipeptide (mdp) component present in all bacterial peptidoglycans. the adjuvanticity of the mucosal adjuvant cholera toxin (ct) is mediated through the nod receptor [ ] . adjuvants that are inducers of damage-associated molecular patterns (damps) trigger innate immune responses in vivo by damaging the host cells, thereby resulting in the release of damp factors (ex. rna, dna) for subsequent activation of the innate immune receptors [ ] . the cytosolic receptor nlrp is recognized by adjuvants such as quil-a and chitosan, as well as atp, mdp, uric acid crystals and silica. these compounds generate damp signals, such as reactive oxygen species (ros) or induce potassium efflux to activate nlrp . alum's adjuvanticity in mice is also attributed to the activation of nlrp / nacht (via swelling and rupture of the phagolysosome, release of cathepsin b into cytosol, subsequent activation of caspase and release of il- β), leucine-rich repeat (lrr), and pyrin-paad-dapin domain (pyd) domains-containing protein (nalp ) inflammasome, release of uric acid or activation of the stimulator of interferon (ifn) genes (sting)-ifn regulatory factor (irf) pathway due to the release of dna [ , ] . however, validation of these hypotheses for humans is warranted (ex. a direct role of il- β in adjuvanticity of alum in humans is debatable) [ ] . for instance, it is generally concluded that nlrp inflammasome and caspase are sometimes, but not always, required for induction of th cellassociated antibody responses in response to aluminum salts in vivo [ ] activation of caspase- is nlrp -dependent in vitro. however, no such role of nlrp is observed for qs- in vivo. qs- when formulated in liposomes activates human dcs by promoting cholesterol-dependent endocytosis with subsequent lysosomal destabilization and syk activation similar to alum [ ] . in mice, endocine, a lipid adjuvant, causes cellular damage to generate damps such as lactate dehydrogenase (ldh), dna and rna [ ] . chitosan induces release of mitochondrial dna into the cytoplasm to activate the nlrp inflammasome in mice [ ] . mf (but not aluminum hydroxide or calcium phosphate adjuvants) induces release of extracellular atp from the muscle in mice that acts as a danger signal [ ] . other damp adjuvants such as hydroxypropyl-βcyclodextrin (bcd) induce local cellular stress and death resulting in the release of the host cellular dna that serves as a damp to induce th -type immune responses [ ] . cytosolic dsdnas are sensed by several other prrs such as absent in melanoma (aim ), as well as by the protein cyclic guanosine monophosphate-adenosine monophosphate (cgamp) synthase (cgas), which simultaneously leads to the activation of sting-dependent tbk -irf -ifn- pathways and rela-tnf-α pathways [ ] . sting can bind cyclic dinucleotides (cdn), cyclic di-gmp (cdg) and cyclic di-amp (cda). in mice, cdg appears to be a safer mucosal adjuvant than cholera toxin [ ] and to promote protective immunity against h n influenza, staphylococcus, streptococcus and klebsiella infections. in mice, chitosan triggers release of intracellular dna that results in the engagement of the cgas-sting pathway in dcs to induce type ifn production and isgs, thereby promoting robust th immunity. this leads to the upregulation of costimulatory immune markers and the subsequent activation of dcs, as well as induction of igg c and cell-mediated immunity (cmi) [ ] . carbohydrate-based adjuvants include glucans, fructans, mannans, chitin/chitosan and other carbohydrate compounds derived from mycobacterium spp. (including lipoarabinomannan, muramyldipeptide/mdp, trehalose- - -dimycolate/tdm), as well as lps and saponin compounds (including qs- , a saponin in an oil-in-water emulsion) [ ] . in human monocyte-derived dcs (modc), qs- (also a component of as ) is endocytosed via the action of membrane cholesterol, with subsequent lysosomal destabilization and syk activation to promote a pro-inflammatory transcription program. in addition, cathepsin b (a lysosomal cysteine protease) is required for activation of modcs in vitro and also required for adjuvant activity of qs- in vivo [ ] . in another study, ln-resident cd b + cd + macrophages played a key role in the adjuvant activity of qs- . upon intramuscular injection in mice, qs- leads to rapid induction of local innate immune responses in the dlns and co-localises with ln-resident macrophages that are crucial for innate and effector responses to antigens formulated with qs- (via caspase / and inflammasome activation) [ ] . the primary mechanism of action of carbohydrate-based adjuvants involves interaction with prrs such as tlrs, nod and c-type lectin receptors (clrs) dectin- , dectin- and mincle on monocytes and apcs. this interaction activates nf-κb to induce inflammatory chemokine and cytokine responses [ ] . carbohydrate adjuvants also activate complement pathways to generate complement components acting as opsonins and chemokines. other important mechanisms of action of carbohydrate-based adjuvants include chemotaxis of lymphocytes, inflammasome activation (e.g. zymosan and mannans), and pore-formation, facilitating antigen entry into apcs (via interaction with cholesterol in the plasma membrane, e.g. qs- ) [ ]. adjuvants induce a series of signal transduction pathways to exert their action at both innate and adaptive levels. intramuscular injection of mpl or aso in mice is responsible for nf-κb activation in the muscles and local dlns [ ] . synthetic derivatives of mpl induce activation of tlr and selectively activate the p mapk pathway, which is strongly associated with optimal induction of ifn-γ-induced protein (ip- ), tnf-α and il- in mice [ ] . injection of as adjuvanted vaccine promotes release of ifn-γ by ln-resident nk cells and cd + t cells. pathway enrichment analysis of differentially expressed genes in injection site-dlns revealed that the ifn-signaling pathway was most enriched at and h, while the il- -driven anti-inflammatory pathway was also triggered by as at h post-injection. increased levels of ifnγ were detected early in the serum and dlns of humans and macaques vaccinated with as -adjuvanted vaccine, respectively [ ] . cellular and molecular synergy between mpl and qs- used in as in combination was responsible for this early ifn-γ response, which in turn, enhances the immunogenicity of as -adjuvanted vaccines [ ] . il- as an adjuvant activates janus kinase (jak)-signal transducer and activator of transcription (stat), phosphoinositide -kinase (pi k) and mapk pathways, thereby promoting b-cell and t-cell differentiation via sustained activation of stat and th differentiation through irf [ ] . subtle chemical alterations to mpla were made to develop a designer smip-based tlr -agonist known as sla, which induces trif signaling to produce th biased cytokines and chemokines like ifn and ip- , and less il- β. sla in oil-in-water emulsion (sla-se) was produced capitalizing on the knowledge that a combination of ifn and caspase-dependent inflammasome signaling leads to powerful adjuvant action [ ] . activation of the nf-κb pathway, as well as the p and jnk mapk pathways, programs dcs to produce il- p and to induce th responses. the extracellular signal-regulated kinase (erk)-c-fos mapk pathways favor th -type responses, while erk-retinaldehyde dehydrogenase (raldh) enzymes or β-catenin program dcs to induce t regulatory (treg) responses. similarly, complete freund's adjuvant (cfa) induces transcription of mhc-ii and b cell activation markers via the lyn-syk-pi k, the calcineurin-nuclear factor of activated t-cells (nfat) and the ras-mek-erk signaling pathways [ ] . saponin adjuvanticity relies on myd -mediated and il- receptor-signaling pathways [ ] . chitosan engages cgas-sting pathways to induce igg c and th responses in mice [ ] . intact myd signaling in each of the three types of apcs (dcs, macrophages and b cells) is essential for robust activity of tlr ligand-based vaccine adjuvants (porb, a tlr ligand and cpg, a tlr ligand) such as induction of in vivo cytokine responses, germinal center (gc) formation and antibody production [ ] . based on microarray analysis, mosca et al. demonstrated that three potent human vaccine adjuvants, mf , cpg odn, and alum, modulate a common set of genes ['adjuvant core response genes'] encoding cytokines, chemokines, innate immune receptors, ifn-induced proteins and adhesion molecules in mouse muscles [ ] . the establishment of a local immunocompetent environment due to such nonpathogenic inflammatory responses is associated with vaccine adjuvanticity. when compared to cpg odn and alum, mf was found to be the stronger inducer of adjuvant core response genes, which was reflected in enhanced and more rapid influx of mhc-ii + and cd b + cells into the injection site and more efficient transport of antigen to the dlns [ ] . both alum and mf induced chemokines involved in cellular influx such as ccl , ccl , ccl , and cxcl- [ ] . the presence of α-tocopherol in as promotes induction of leukocyte-recruiting chemokines (ccl , ccl and ccl ), neutrophil-mobilising cytokine (granulocyte colonystimulating factor (csf )) and pro-inflammatory cytokine/ chemokines (il- and cxcl- ), in mice which is in agreement with increased recruitment of granulocytes and antigenloaded monocytes into the dlns [ ] . aluminum adjuvants facilitate recruitment and differentiation of inflammatory monocytes (f / int cd b + lyg − ly c + ) into inflammatory dcs, thereby enhancing both humoral and cellular immunity [ ] . many of the above results with alum and mf have been confirmed in non-human primates [ ] . for example, vaccination with hiv vaccine adjuvanted with alum or mf triggered recruitment of monocytes, dcs, and neutrophils in the muscle [ ] . subcutaneous administration of iscomatrix in sheep induces a rapid and transient production of cytokines (il- , il- , ifn-γ) and influx of innate cells such as nk cells, nkt cells, neutrophils, migratory dcs (cd + cd − ) and cd α + dcs to the dlns [ ] . a combination adjuvant consisting of poly(i:c), a host defense peptide and pcep when delivered intranasally transiently induces production of chemokines and cytokines in murine respiratory tissues, which promotes infiltration and activation of dcs, macrophages, and neutrophils to generate improved mucosal and systemic immune responses [ ] . (a) improvement of the quality of antibody responses: innate immune responses play a profound role in regulating the magnitude, quality and persistence of antibody responses. the magnitude of the antibody response is critical in conferring protection against diphtheria, hepatitis a, lyme disease, tetanus, yellow fever, polio, rabies, and pneumococcal infections [ ] , while for rsv and meningococcal infections, the magnitude and quality of the antibody and cell-mediated response are important. adjuvant systems such as as are used in malaria (rts,s), herpes zoster (hz/su), tb and hiv vaccines, while as is used in several influenza vaccines such as trivalent inactivated h n influenza, h n prepandemic influenza, and candidate h n and h n pandemic influenza vaccines. as is used in licensed hpv- / and hbv vaccines. such adjuvant systems are known to augment antigen-specific t cell and antibody responses [ ] . in a murine study, the er stress-related pathway was found to potentially contribute to the adjuvanticity of as in vivo [ ] . furthermore, the expression of the er stress sensor kinase ire α by myeloid cells was involved in adjuvant activity of as . the er stress-related pathway was required for as mediated induction of il- , robust tfh responses, and antigenspecific antibody affinity maturation. il- plays a crucial role in differentiation of tfh cells as well as in the expression of il- by tfh cells and in production of antibodies [ ] . alum-adjuvanted vaccines act on il- producing gr- + cells to facilitate optimal priming, clonal expansion and antibody production by antigen-specific b cells in mice [ ] . in b cells, tlr ligands as adjuvants induce upregulation of surface markers involved in antigen uptake (mhc-i and mhc-ii) and surface markers involved in cross-talk with the t cells (cd , cd , and cd ), which ultimately leads to increased antigenspecific antibody production [ ] . however, when as is used as an adjuvant in the same hbv and hpv vaccine, instead of aluminum salts, higher levels of antibodies are induced in humans, indicating the added benefit of mpl (a tlr agonist) in humans [ ] . emulsigen®, an oil-in-water adjuvant, similar to mf and as , boosts innate responses and increases the number of cd + t cells required for robust antibody responses [ ] . mf supports induction of t follicular helper (tfh) cells and gc responses to vaccination by an unknown mechanism [ ] . in humans, mf and as promote early inflammation similar to results from animal studies [ ] . type ifns responses are induced in children as early as day postimmunization with mf -adjuvanted influenza vaccine, but in children immunized with non-adjuvanted vaccine, type i ifn responses are weaker and delayed. such early innate immune responses are reflected in induction of antigenspecific tfh cells days post-vaccination, and enhanced antibody responses in humans immunized with mf -adjuvanted influenza vaccine [ ] . (b) induction of gc reactions to promote memory b cell development: immunological memory is a distinctive hallmark of the adaptive immune system that contributes to protective immunity against infectious diseases. the gc reaction is central to memory development. induction of certain key molecules such as cd , inducible t-cell costimulator (icos), il- , programmed death-ligand (pd- ), cd , irf , and b-cell lymphoma protein (bcl- ) play a critical role in regulation of gc differentiation, affinity maturation and long-lived memory responses [ ] . tlrs expressed on gc b cells, follicular dcs (fdcs) and t cells have a profound effect on induction of antibody responses. nanoparticles resembling virions in size and containing tlr ligands, mpl and r , in combination with h n hemagglutinin mediate increased persistence of gcs, which significantly influence the differentiation of memory b cells critical for long-lived antibody responses in mice [ ] . a subset of cd + t cells, icos + cxcr + cxcr + t cells, was associated with protective antibody responses conferred by a trivalent split-virus influenza vaccine and efficiently induced memory b cells to differentiate into plasma cells [ ] . novel adjuvants may enhance b-cell activation in gcs and bonemarrow plasma cell survival. for example, the heat-labile enterotoxin (lt) of escherichia coli, ltk , when administered parenterally to neonatal mice, facilitates maturation of follicular dcs and generation of gcs [ ] . . . induction of cellular immunity: effector th /th / th and memory t cell responses signaling via tlr , tlr , tlr , tlr , and tlr promotes th biased immunity, while signaling via tlr /tlr , tlr /tlr and tlr promotes th -biased immunity. cd c + cd b − cd α + dcs localized in the marginal zones of lns induce th responses, as well as exhibit cross-presentation of antigens in vivo and ex vivo in mice [ ] . in humans, bdca + (cd c + ) and bdca + (cd + ) dcs (equivalent to murine cd α − and cd α + , respectively) are involved in cross-presentation of extracellular antigens [ ] . as a result of tlr -mediated enhanced mhc-i expression and type i ifn production, poly(i:c) promotes antigen cross-presentation to cd + t cells and antigen-specific ctls. in contrast, alum promotes th responses (strong antigen-specific igg and ige) and does not induce cd + t cell immunity, and even inhibits th immune responses in mice [ ] . however, when alum is present in combination with mpla, th responses can be generated as is found for aso [ ] . it is unclear whether such an aluminum salt-induced th bias exists in humans [ ] . rather, poor t cell responses are induced by aluminum salts in humans, possibly due to poor stimulation of the innate immune system [ ] . squalene-based oil emulsion is a potent inducer of both th -and th -mediated immunity and is well tolerated [ ] . adjuvants such as qs- , mf or cfa preferentially induce th -biased or a mixed th / th and th /th immune response. experimental cafs combined with immunostimulators such as tdb in tb vaccines stimulate both cellular and humoral immune responses, as well as promote efficient polyfunctional memory t cells, and th -and th -biased immune responses in mice [ ] . a sting-activating adjuvant, cdn, when formulated in a subunit vaccine and delivered intranasally, promoted a robust th immune response that correlated with long-lasting enhanced protection against mycobacterium tuberculosis in mice. adjuvanted vaccines promoting th responses may protect against intracellular pathogens by recruiting protective t cells earlier during infection [ ] . in neonates, cd + t cells are polarized towards th responses and reduced th responses. however, novel adjuvants such as ic and caf can induce adult-like th responses in newborn mice [ ] . cdg as a mucosal adjuvant induces th and th immune responses in mice [ ] . caf predominantly induces cd + t cell responses, while caf (consisting of dda, tdb and poly(i:c)) induces both cd + and cd + t cell responses [ ] . replacing as in an rts,s/ as candidate malaria vaccine with as improved antibody and cd + t cell responses, as well as protective efficacy as demonstrated by a randomized, double-blind, phase a trial [ ] . activated murine sub-capsular macrophages produce il- , which promotes generation of cd + t cells, while activated apcs also produce il- or il- , promoting generation of tfh cells, which in turn favors production of high-avidity antibodies by b cells in both mice and humans [ ] . intramuscular injection of mice with as induces early ifn-γ production by ln-resident nk cells, which is mediated by synergistic action of il- and il- in promoting ifn-γ responses. early ifn-γ production by nk cells is a prerequisite for optimal activation of dcs and induction of antigen-specific cd + t responses to as -adjuvanted antigens [ , ] . such ifn-γ responses were also observed in lns of macaques when they were injected with as [ ] . elevated levels of ifn-γ in the serum at day post-immunization and an increase in the number of cytokineproducing antigen-specific cd + t cells was also observed in humans immunized with rts,s malaria vaccine [ ] . the mechanisms of action of different adjuvants are summarized in table . the adjuvants that are licensed for use in human vaccines are listed in table , while the adjuvants in clinical trials are listed in table . . selection of adjuvants based on their mechanism of action against distinct types of pathogens mucosal surfaces are an attractive target for pathogens whose port of entry are gastrointestinal (e.g. polio virus, escherichia, salmonella, shigella, vibrio and helicobacter), respiratory (influenza virus, m. tuberculosis or mtb, adenovirus, coronavirus, rhinovirus and rsv) or urogenital tract (hsv, hpv, hiv- , chlamydia and neisseria) [ ] . mucosal adjuvants can be categorized as toxin-based (lt and ct), immunostimulatory (mpl, cpg, and qs ) and delivery system (emulsigen® and iscoms). two commonly used oral toxin-based adjuvants are [ ] . both are potent, but also toxic, when used as mucosal adjuvants. protective efficacy was attained when intranasal vaccines containing mutant lt adjuvants were used against hsv, bordetella pertussis and streptococcus pneumoniae [ ] . natural infection with rsv induces poor antibody responses with impaired effector functions, and perturbs localization and persistence of effector and memory t cells [ ] . induction of a potent, local mucosal immune response is required to prevent infection and a high systemic antibody response is also required to interrupt disease progression. nanoemulsions are safe for intranasal delivery and induce th and th responses in mice with no significant inflammation [ ] . sastry et al. tested multiple adjuvants such as sigma adjuvant system (sas, an oil-in-water adjuvant), carbopol, alum, adjuplex, poly(i:c), poly(ic:lc), mpla, addavax and montanide isa, and found that the adjuvantmediated increase in rsv-specific neutralizing antibody responses was context-dependent (i.e. whether pre-existing immunity was present or not) and species-specific (i.e. mice vs. calves) [ ] . formulation of the rsv fusion (f) protein with a combination adjuvant, triadj, elicited protective, mucosal and systemic, immune responses against rsv in mice and cotton rats [ ] . the mucosal epithelial barrier limits the bioavailability of vaccine antigens for sampling by apcs. adjuvants such as polyethyleneimine and chitosan are used as penetration enhancers and immunostimulants to augment binding to the mucosal surfaces and activate innate immunity in a mouse model [ ] . chitosan polymeric nanoparticles stimulate the nasal-associated lymphoid tissues (nalt) to produce mucosal secretory iga, igg, tnf-α, il- , and ifn-γ. pro-inflammatory cytokines and chemokines also act as mucosal adjuvants. proinflammatory cytokines such as il- , il- , il- , il- , and il- induce mucosal cd + ctls and antigen-specific iga. similarly, chemokines such as ccl (or mcp- ) enhance mucosal iga and ctl responses [ ] . neutralizing antibodies may protect against some acute self-limiting mucosal pathogens, but for highly invasive pathogens causing chronic infections (such as hiv, hcv, herpesviruses, and mycobacteria), mucosal innate and adaptive immune responses including cd + , th , and cd + ctls, as well as secretory iga and igg neutralizing antibodies at the port of pathogen entry, are required for effective and optimal protection [ ] . mucosal adjuvant-containing vaccines elicit both local and systemic immune responses, effective at local as well as distant sites [ ] . to control enteropathogens, orally administered vaccines must overcome challenges of antigen degradation and immune tolerance [ ] . biodegradable micro-or nanoparticles are required that are resistant to low ph and can target antigen to m cells. u-omp , a bacterial protease inhibitor from brucella abortus, is an oral adjuvant suitable for subunit vaccine formulation, which can inhibit stomach and gut proteases and delay antigen digestion at the lysosome to enhance antigen presentation and recruitment of immune cells to gastrointestinal mucosa [ ] . intranasal immunization of mice with poly-i:c u (ampligen) in an h n influenza vaccine promoted increased levels of protective, mucosal iga and systemic igg [ ] . however, only a few mucosal vaccines are licensed for humans, primarily due to a dearth of safe and effective mucosal adjuvants [ ] . although during the last few decades there has been a constant development of new and effective mucosal adjuvants, most of them are toxic. for example, ltk when intranasally injected can trigger transient facial nerve paralysis or bell's palsy [ ] . thus, there is an urgent need to address safety issues of mucosal adjuvants. in a phase iii trial, poly-i:c u (ampligen) was demonstrated to be safe [ ] . a randomized phase i/ii trial was conducted to determine the safety and efficacy of ampligen in patients with stage ii-iv human epidermal growth factor receptor (her )-positive breast cancer. the result from this trial will give important insight into the application of ampligen in therapeutic cancer vaccines [ ] . pathogenic fungi and protozoan parasites have complex life cycles and switch among several different forms during their life. histoplasma capsulatum grows as a mold in the soil at low temperature, but upon inhalation into the lungs, it switches to yeast form and causes histoplasmosis. interaction of infected macrophages with cd + and cd + t cells leads to increased production of th cytokines, il- , ifn-γ, and tnf-α, that are critically important in generating protective immunity against h. capsulatum infection in mice. leukotrienes, lipid mediators derived from arachidonic acid metabolism, are found to be potent adjuvants against such fungal infections [ ] . malaria vaccine development is impeded by the complex life cycle of plasmodium spp., the intracellular stage in its life cycle, large physical size, surface antigenic diversity and enormous genetic and genomic plasticity [ ] . the parasite replicates intracellularly (and thus is partially protected from immune recognition) and also sequesters any innate immune ligand away from prrs in the sporozoite and gametocyte stages of their life cycle. a malaria vaccine needs to establish humoral immunity to prevent merozoites from entering the erythrocytes and the liver or destroy the merozoites through opsonization and cmi. rts,s/as (mosquirix) is a malaria candidate vaccine targeted against the infectious sporozoite stage and designed to enhance both antigen-specific humoral and cellular immunity. th effector cells are essential to target asexual blood stages, while eventual control and/or clearance of the parasites requires antibody-mediated responses [ ] . mpl and qs- , the two components used in as have important functions. mpl is a tlr agonist that induces production of ifn-γ by t cells and antibody isotype switching to igg a/c in mice, while qs- induces neutralizing antibodies and cytotoxic t cell responses [ ] . as requires synergistic activities of both mpl and qs- for optimal adjuvant activity. as in combination with plasmodium antigens induces rapid and transient innate immune responses in the injection site and dlns, activates immune cells (including apcs), as well as generates -fold higher antibody titers when compared to natural exposure [ ] . however, in a large phase iii trial in children and young infants in seven sub-saharan african countries, although rts,s/as prevented a considerable number of cases of clinical malaria in infants and young children over - years, the vaccine efficacy declined with subsequent follow-ups in the infants, and did not provide significant protection against severe malaria [ ] . nonetheless, rts,s/as plays a significant role in the control of malaria in areas of high transmission when used in conjunction with other effective preventive measures (rts,s clinical trials partnership). poly(i:c) and its derivatives are of great importance for vaccines that need to induce a th /ctl immune response against various viruses and pathogens including p. falciparum [ ] . pam csk was used in a malaria vaccine containing p. falciparum circumsporozoite protein b cell epitopes and universal t cell epitopes, which resulted in the induction of high titers of antigenspecific igg , igg and igg in immunized volunteers [ ] . herpesviruses are large viruses with a complex genome. primary infection with varicella zoster virus (vzv) causes varicella (chickenpox) and may go into latent phase in human cranial and dorsal root ganglia. aging or immune dampening results in decline of vzv-specific cmi, which may induce reactivation of the virus and cause shingles. hence, cmi is necessary to prevent reactivation of the latent virus. the vzv vaccine hz/su (shingrix) composed of the vzv glycoprotein e subunit (ge) antigen and as b was recently approved for the prevention of herpes zoster in adults aged years or older [ ] . as was selected as the adjuvant for the vzv vaccine, because compared to other adjuvant systems, as induced higher numbers of ifn-γ secreting cd + t cells, and thus improved t cell as well as antibody responses, with acceptable clinical safety profiles [ ] . hpv effectively evades innate immunity by inhibiting the ifn receptor signaling pathways and activation of isgs via the e and e proteins. hpv also downregulates tlr and does not induce any danger signal to alert the immune system [ ] . this prolongs the duration of infection and delays the onset of adaptive immunity. thus, an effective cmi is required to clear and control hpv infection. effective vaccine-induced immunity against hpv should consist of cmi to the early proteins, e and e , and neutralizing antibodies against the virus coat protein l . two currently approved hpv vaccines, cervarix (a bivalent hpv / vaccine, gsk) and gardasil (a quadrivalent hpv / / / vaccine, merck) are highly protective against hpv , , and [ ] . both are li vlps; however, cervarix is as adjuvanted, while gardasil is aahs (amorphous aluminum hydroxyphosphate sulfate)-adjuvanted. vlps strongly activate the stromal dcs in the injection site that migrate to the dlns, or may directly bind to the surface of apcs or other immune cells and migrate to the lns, where they prime naïve b cells [ ] . according to a recent study in girls aged - years, two doses of cervarix elicited superior hpv- / antibody responses compared to two or three doses of gardasil. the differences in immunogenicity between the two vaccines may be due to the different types of adjuvants used. as enhances humoral immune responses and cmi by triggering local and transient cytokine responses that promote enhanced activation and presentation ability of apcs [ ] . significantly higher antibody titers are induced in mice immunized with hpv- l vlps adsorbed onto aahs as compared to vlps adsorbed onto aluminum hydroxide along with induction of an improved l -specific ifn-γ secreting t cell response [ ] . mtb causing tb is an intracellular pathogen that has the ability to survive within the hostile environment of the alveolar macrophages after being phagocytosed and to multiply unchecked. cd + t cells, cd + t cells, ctls, th cells, nk cells, and activated macrophages are critical in controlling mtb infections. bacillus calmette-guérin (bcg) vaccine fails to protect adults from pulmonary tb and prevent transmission of mtb in adolescents and adults [ ] . thus, there is an urgent need for improved vaccines against tb. one of the potential vaccine strategies against mtb is to eliminate or control latent infection and prevent reactivation or progression to clinical tb in latently infected patients. this may be accomplished by incorporating adjuvants that are capable of inducing both cd + and cd + t cell responses in both immunocompetent and immunocompromised individuals. mechanisms of antibody-mediated protection against tb include opsonization, complement activation, and fc receptor engagement. current research is focused on adjuvants that act on innate lymphoid cells (ilcs), nk cells and non-classical t cells such as cd , mr , hla-e and γδ t cells present in large numbers in the circulation and mucosa [ ] . although the immune correlates of protection from tb disease are not validated yet, vaccines currently in clinical development predominantly focus on generating cd + and cd + th -type immune responses. adjuvants such as mineral salts, saponin, emulsigen®, micro-or nanoparticles, toxin derivatives, cationic lipids, cpg dna, adjuvant systems and cytokines have been tested in subunit vaccine preparations, either alone or in combination with bcg in a prime-boost strategy [ ] . the strongest th -inducing adjuvants for tb are unmethylated mycobacterial dna and cpg odn, which promote ctl activation and ifn-γ production [ ] . tlr / and tlr / ligands are presented on the surface of mtb (triacylated and diacylated forms of mycobacterial p lipoprotein) or secreted by the bacterium, while nlrs such as nod are responsible for intracellular recognition of mycobacteria [ ] . novel adjuvants, including dda, tdb, ic , poly(i:c), gelatin, cpg odn, mpla, glycopyranosyl lipid adjuvant (gla) in combination with squalene (se) known as gla-se, mf , caf , and as b are also being clinically tested. dda promotes generating humoral, cell-mediated and ifn-γ responses against mtb, while as and mf induce strong th immunity against mtb. all these adjuvanted subunit vaccines induce protective immunity and enhance bcg-primed immunity in animal models [ ] . in a randomized, double-blind, phase b trial, a candidate tuberculosis vaccine, m /as e , demonstrated a clinically acceptable safety profile and conferred % protection against active pulmonary tuberculosis in adults with latent mtb infection [ ] . nanoparticle-based vaccines are critical for the induction of protective th -type immune responses to intracellular pathogens. the liposomal caf adjuvant promoted th and long-lasting memory t cell response in human tb vaccination trials. caf -adjuvanted tb vaccine stimulates the clr, and mincle, and triggers the syk/card signaling cascade to activate the th signaling pathway [ ] . one of the biggest challenges in vaccine development is the fact that the immunological mechanisms that govern vaccine safety and efficacy are still largely unknown. in recent years, systems vaccinology has emerged as an interdisciplinary approach that relies on high-throughput omics-based techniques to study vaccine-induced changes in the entire genome, set of transcripts, proteins, and metabolites in various tissues. a systems vaccinology approach has been used to elucidate immune responses to vaccines against yellow fever [ ] , influenza [ ] , malaria [ ] , smallpox [ ] and hiv [ ] . in addition, a systems vaccinology approach identified molecular and cellular immune signatures of a vaccine against bordetella pertussis [ ] . ip- was identified as an early innate immune signature that correlated with antibody responses to an ebola vaccine (rvsv-zebov) [ ] . computational analysis of the transcriptomic profile in human peripheral blood mononuclear cells (pbmcs) induced by yellow fever vaccine yf- d identified two molecular signatures: eukaryotic translation initiation factor alpha kinase (eif ak ) and tnfrsf , encoding the receptor for the b-cell growth factor blys-baff [ ] . eif ak correlated with the magnitude of the cd + t cell responses, while tnfrsf correlated with the magnitude of neutralizing antibody responses. other genes such as calreticulin, c-jun, and glucocorticoid receptor were also induced by yf- d, and this induction correlated with cd + t cell responses [ ] . in another study with young healthy adults, intramuscularly administered tiv induced higher antibody levels and plasmablasts when compared to intranasally delivered live attenuated influenza vaccine (laiv) with induction of distinct transcriptional signatures such as enhanced expression of type ifn genes in laiv recipients, but not in tiv recipients [ ] . based on a systems vaccinology approach, tlr agonists as adjuvants were found to potently enhance the immunogenicity of influenza vaccine, resulting in an improved antibody response in humans [ ] . the longevity of the immunoglobulin response post vaccination could be predicted from the ability of the adjuvanted vaccine to induce proliferation of antigen-specific il- + icos + cxcr − cd + t cells in the peripheral blood. systems vaccinology also identified two biomarkers (junb and ptx ) of mf and the skeletal muscle tissue cells (in addition to apcs) as direct target of mf for its adjuvant action in mice [ ] . caproni et al. investigated molecular signatures induced by different tlr-dependent (cpg odn, resiquimod and pam csk ) and tlr-independent (mf and alum) adjuvants in influenza subunit vaccines to establish the innate immune correlates of adjuvanticity by using dna microarrays in a mouse model [ ] . two adjuvants, mf and pam csk increased overall antibody and hai titers, and induced active infiltration of cd b + cells, especially neutrophils, to the injection site. this suggests early induction of cd b + cells due to an emulsion-based adjuvant to be predictive of subsequent robust humoral immunity. systems vaccinology has also been applied to identify novel mechanisms of induction of th responses by an adjuvant. for instance, the th -promoting adjuvant activity of cysteine protease allergen is dependent on the production of ros by dcs. as a result of induction of ros, oxidized lipids are induced that in turn promote epithelial cell-mediated production of thymic stromal lymphopoietin (tslp), resulting in the recruitment of il- + basophils to the lns for induction of th -type immune responses in mice [ ] . genes associated with memory b cell formation and productive antibody responses such as bcl , bcl a, tank, plcg , and cd are induced when mice are immunized with ovalbumin (ova) adjuvanted with tlr and tlr agonists [ ] . in a study with the candidate malaria vaccine rts,s/as b in human subjects, enhanced expression of genes involved in immunoproteasome formation, psme in particular, was found to be responsible for conferring protection from parasitemia. induction of the immunoproteasome enhances mhc antigen presentation, which in turn, indirectly enhances antibody responses and directly augments cd + t cell development and production of ifn-γ, tnf-α, il- , and cd l. the above immune signatures may contribute to the protective efficacy of the candidate malaria vaccine [ , ] . a comparative systems analysis of four vaccine adjuvants, gla-se, ic , caf , and alum, in mice revealed distinct molecular signatures. gla-se induced massive changes in the transcriptomic profile in the whole blood and dlns that correlated with increased cellular influx (such as cd c + gr + mdcs) in the dlns, in contrast to limited transcriptomic changes induced by other adjuvants. co-expression analysis of differentially expressed genes in whole blood revealed that caf and gla-se (but not ic ) induced transcriptional signatures related to innate immune responses. the analysis also revealed modules enriched for genes associated with tfh and gc-mediated b cell responses; for example, gla-se induced nfatc , nfatc , and il r; caf induced batf and ic induced pou af . a systemic analysis of protective immune responses to three rts,s vaccinations with a subsequent controlled human malaria challenge of the vaccine recipients with plasmodium-infected mosquitoes was carried out. molecular signatures of b cell and plasma cells in human pbmcs were found to be positively correlated to protection, while the nk cell signatures correlated negatively with protection, indicating multiple mechanisms of protective immunity against p. falciparum [ ] . in a study by burny et al., different adjuvants [as b , as e , as , as , and alum/al(oh) ] induced common innate pathways and were responsible for improved adaptive responses when used with a model antigen (hbv surface antigen or hbsag) in humans. as b , as e , and as induced comparable innate profiles and so did as and alum. furthermore, the ability to activate innate immunity (ifn-signaling pathway, in particular) was linked to enhanced adaptive responses elicited by as -and as -adjuvanted vaccine. early changes in immune markers, such as crp, il- , ifn-γ, and ip- , correlated with the magnitude of the adaptive responses [ ] . systems vaccinology also identifies signatures of vaccine safety and efficacy. non-specific adverse side effects observed for vaccines that fail in human clinical trials are frequently associated with over-stimulation of certain components of the innate immune system. systems vaccinology can be applied to screen adjuvants to help design protective and safe vaccines [ ] . correlates of protection have been established for a number of licensed vaccines as reviewed by tomaras et al. [ ] . however, attempts to identify correlates of protection are still ongoing for tb, while the commonly assumed immune correlates often fail to correctly predict an individual's risk of developing malaria [ ] . for hiv, complex immune correlates of protection characterized by multiple types of immune responses are found to be involved in controlling hiv- transmission [ ] . for vaccines for which the immune correlates of protection are unknown, systems vaccinology approaches can be used to identify signatures induced rapidly after vaccination that will help to predict the later immune outcome. a systems vaccinology approach can also help in identifying vaccine non-responders as well as vaccine high-and low-responders [ ] . innate and adaptive immune responses are profoundly influenced by any significant changes in metabolic activity. inflammation triggered by vaccine adjuvants results in a shift in energy supply leading to metabolic acidosis and impaired oxygen supply, which in turn results in phenotypic shifts. these phenotypic shifts heavily affect the metabolic state of an individual. lipid metabolism plays an important role in inflammation. liquid chromatography-mass spectrometry (lc-ms) is employed to identify and quantify cell-or tissue-specific metabolites [ ] . metabolite immune-correlates such as nucleotides, amino acids, lipids, fatty acids, and anti-oxidants may represent inflammatory mediators and/or biomarkers that profoundly influence several inflammatory processes such as cellular infiltration, activation of signaling pathways and oxidative stress [ ] . thus, a comprehensive understanding of the molecular signatures induced by adjuvants early after vaccination will help to predict the later adaptive immune responses in humans. furthermore, such knowledge will also improve or help in (re-)designing next-generation adjuvants and drive the development of next-generation vaccines with the concerted effort of vaccinologists, clinicians, systems biologists, statisticians, as well as industrial and regulatory authorities. the relationship between adjuvants, innate pathways/ receptors activated, immune responses triggered, and the type of pathogens ideal for such adjuvant-mediated immune responses are summarized in table . in this review article, we have summarized the mechanism of action of different classes of adjuvants. we also discussed why this knowledge is important in context of distinct disease targets and how this knowledge can be utilized to improve the development of adjuvanted vaccines against challenging pathogens. we also briefly highlighted the important role of the new-age systems vaccinology approaches in better understanding an adjuvant's mode of action, and identification of unique cellular and molecular biomarkers of adjuvanticity. it is important to note here that mouse models offer flexibility and accessibility to study intricate facets of the mechanism of action of adjuvants and responses to immunization with adjuvanted vaccines. while the results from animal studies often overlap with the results from human studies, there are several dissimilarities as well. for instance, mf -adjuvanted vaccine in mice induces both cell-mediated and humoral immune responses, which is not observed in humans at any age, rather they tend to develop a th -th response. even in humans, immune responses need to be investigated not only in the serum but also in the dlns and other lymphoid organs; not only at the priming site but also in the distant effector sites such that a holistic and reliable assessment of mechanism of action of adjuvant and/or vaccine can be made encompassing all possible immune parameters. even the immunological correlates that have recently been identified by gene profiling or systems vaccinology for different adjuvants/vaccines are defined more at the population level and much less at the individual level [ ] . all these considerations must be taken into account while designing effective and safe vaccines/adjuvants. recent advancements have allowed researchers to conclude that clinical-grade adjuvants have distinct immunological profiles and signatures, which can be used to target different pathogens. based on pathogen-specific immune response requirements (i.e. th , th or th responses, or mixed th / th or th /th responses, etc.), next-generation adjuvants can be rationally developed and incorporated into human vaccines. currently, all approved human adjuvants mostly induce only antibody responses. however, recent adjuvant research has led to the development of novel adjuvants capable of inducing cmi (especially required for malaria, tb and hiv), as well as antibody responses. new immunostimulatory adjuvants or immunomodulatory compounds are under investigation to induce cmi and high antibody titers. novel combination adjuvants are being tested in candidate human vaccines with promising results that have strong implications for use in vaccines against challenging infectious pathogens and different target populations. this is potentially due to activation of multiple innate immune sensing signal transduction pathways by combination adjuvants. novel adjuvants are required that can target emerging new pathogens or reemerging old pathogens. such pathogens often have a more complex host-pathogen interaction, which needs better understanding and further characterization. among these new-generation adjuvants, several are proprietary, which may make it difficult to purchase them and conduct independent parallel trials. factors such as genetic background, preexposure to pathogens or vaccine antigens, age, nutritional and immunological status of vaccine recipients, all dictate the final effectiveness of adjuvanted vaccines. nevertheless, with the aid of structural, systems and reverse vaccinology, epitope prediction and other technological advancements, adjuvant technology is now gradually progressing towards a more personalized approach. there has been an exponential growth in the field of adjuvant research. while alum was historically used as the only licensed adjuvant for more than years, six new adjuvants were approved in the last years. the next five years will see substantial progress in obtaining licensure for more varied types of adjuvants. understanding innate immunity and the role of prr agonists as adjuvants in stimulating the innate immune system has revolutionized adjuvant technology. systems biology has immense contribution in the development of effective and potent adjuvants, and will continue to do so in the coming years. correlates of adjuvanticity or immune signatures, and biomarkers of adjuvant safety and protective efficacy will further streamline adjuvant research. tailor-made adjuvants will find their use against distinct pathogens and in specific target populations. better characterization of adjuvants by new omics-based technologies will facilitate licensing of new adjuvants. since there is a pressing need for developing vaccines against a multitude of very virulent/emerging pathogens, we need to develop subunit vaccines and not live vaccines, and hence adjuvant selection is critical. the mucosal surface is the preferred route of entry for most pathogens. therefore, mucosal immunization is considered to be most effective in preventing mucosally transmitted infections. however, the major hurdle in the development of mucosal vaccines is the lack of safe and effective mucosal adjuvants due to toxicity issues. there is specific need for standardized, more comprehensive and pertinent methodologies for safety evaluation to enable development of safe mucosal adjuvants [ ] . mucosal adjuvants are also required to promote bioavailability of vaccine antigen. another requirement is the development of mucosal adjuvants with an optimal targeting ability so as to reduce undesirable adverse side effects. since efficacy and toxicity of most mucosal adjuvants appear to be intrinsically linked, a risk-benefit ratio needs to be ascertained for these adjuvants. attention must also be directed to studying antigen-adjuvant interactions instead of irrational mixing of an adjuvant in a vaccine formulation [ ] . oil-in-water emulsions are very promising adjuvants and characterization/analysis of components added in emulsion preparations in more detail will facilitate improvement of such adjuvants [ ] . papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers vaccine-preventable disease table working group. historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states modes of action for mucosal vaccine adjuvants vaccine adjuvants: from to and beyond. vaccines (basel) an overview of adjuvant formulations and delivery systems liposomal vaccine delivery systems liposomes as vaccine delivery systems: a review of the recent advances iscom-based vaccines: the second decade 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receptor ligand-based vaccine adjuvants require intact myd signaling in antigen-presenting cells for germinal center formation and antibody production molecular and cellular signatures of human vaccine adjuvants identification of molecular and cellular biomarkers induced in mouse muscle upon injection with mf oil-in-water emulsion the adjuvants aluminum hydroxide and mf induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells adjuvant system as containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells induction of lymphocyte recruitment in the absence of a detectable immune response littel-van den hurk s. formulation of the respiratory syncytial virus fusion protein with a polymer-based combination adjuvant promotes transient and local innate immune responses and leads to improved adaptive 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proteases requires dendritic cell-basophil cooperation via ros-mediated signaling key roles of adjuvants in modern vaccines systems analysis of protective immune responses to rts,s malaria vaccination in humans complex immune correlates of protection in hiv- vaccine efficacy trials identification of immune signatures predictive of clinical protection from malaria systems vaccinology: enabling rational vaccine design with systems biological approaches new approaches to understanding the immune response to vaccination and infection vaccine adjuvants: putting innate immunity to work the importance of adjuvant formulation in the development of a tuberculosis vaccine recent advances of vaccine adjuvants for infectious diseases the authors like to thank all the current and previous members from the laboratory as well as the animal care facility at vido-intervac, university of saskatchewan, canada for their contribution. this is vido-intervac manuscript no . publication of this manuscript is supported by grant mop from the canadian institutes of health research (cihr). the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. key: cord- - nrzfon authors: stanifer, megan l.; kee, carmon; cortese, mirko; triana, sergio; mukenhirn, markus; kraeusslich, hans-georg; alexandrov, theodore; bartenschlager, ralf; boulant, steeve title: critical role of type iii interferon in controlling sars-cov- infection, replication and spread in primary human intestinal epithelial cells date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: nrzfon sars-cov- is an unprecedented worldwide health problem that requires concerted and global approaches to better understand the virus in order to develop novel therapeutic approaches to stop the covid- pandemic and to better prepare against potential future emergence of novel pandemic viruses. although sars-cov- primarily targets cells of the lung epithelium causing respiratory infection and pathologies, there is growing evidence that the intestinal epithelium is also infected. however, the importance of the enteric phase of sars-cov- for virus-induced pathologies, spreading and prognosis remains unknown. here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of sars-cov- lifecycle in human intestinal epithelial cells. our results demonstrate that human intestinal epithelial cells fully support sars-cov- infection, replication and production of infectious de-novo virus particles. importantly, we identified intestinal epithelial cells as the best culture model to propagate sars-cov- . we found that viral infection elicited an extremely robust intrinsic immune response where, interestingly, type iii interferon mediated response was significantly more efficient at controlling sars-cov- replication and spread compared to type i interferon. taken together, our data demonstrate that human intestinal epithelial cells are a productive site of sars-cov- replication and suggest that the enteric phase of sars-cov- may participate in the pathologies observed in covid- patients by contributing in increasing patient viremia and by fueling an exacerbated cytokine response. coronaviridae is a large family of single-stranded positive-sense enveloped rna viruses that can infect most animal species (human as well as domestic and wild animals). they are known to have the largest viral rna genome and are composed of four genera (cui et al., ) . generally, infection by coronaviruses results in mild respiratory tract symptoms and they are known to be one of the leading causes of the common cold (moriyama et al., ; paules et al., ) . however, in the last years, we have witnessed the emergence of highly pathogenic human coronaviruses: the severe acute respiratory syndrome-related coronavirus (sars-cov- ), the middle east respiratory syndrome-related coronavirus (mers-cov) and, at the end of , the severe acute respiratory syndrome-related coronavirus- (sars-cov- ) . sars-cov- is responsible for the coronavirusassociated acute respiratory disease or coronavirus disease (covid- ) and represents a major global health threat and coordinated efforts are urgently needed to treat the viral infection and stop the pandemic. although sars-cov- primarily targets cells of the lung epithelium causing respiratory infection, there is growing evidence that the intestinal epithelium can also be infected. multiple studies have reported gastro-intestinal symptoms such as diarrhea at the onset of the disease and have detected the prolonged shedding of large amounts of coronavirus genomes in the feces even after the virus was not detectable in oropharyngeal swabs (wu et al., b; xiao et al., ; xing et al., ; xu et al., b) (wölfel et al., ) . although one study revealed the isolation of infectious virus particles from stool samples , to date, it remains unclear how many people shed infectious viruses in feces. most critically, it remains unknown whether or not there is a possibility for fecal transmission of sars-cov- but multiple health agencies worldwide have highlighted this possibility. the presence of such a large amount of coronavirus genomes in feces is hardly explainable by a swallowing virus replicating in the throat or by a loss of barrier function of the intestinal epithelium which will allow the release of viruses or genomes from the inside of the body (circulation or lamina propria) to the lumen of the gut. instead, it is likely due to an active replication in the intestinal epithelium. recently, intestinal biopsies of sars-cov- infected patients clearly show the presence of replicating viruses in epithelial cells of the small and large intestine (xiao et al., ) . sars-cov- infection of the gastrointestinal tract is supported by the fact that ace , the virus receptor (hoffmann et al., ) , is expressed in intestinal epithelial cells (zhao et al., ) (lukassen et al., ) (wu et al., a) (venkatakrishnan et al., ) and single cell sequencing analysis suggest that its expression is even higher on intestinal cells compared to lung cells (xu et al., a) .this highlights that sars-cov- is not restricted to the lung but also infects the gastrointestinal tract. importantly, many animal coronaviruses are well known to be enteric and are transmitted via the fecal-oral route (wang and zhang, ) . additionally, the presence of human pathogenic coronaviruses in the gastrointestinal tract was previously reported for sars-cov- and mers-cov but remained seriously understudied (leung et al., ; wong et al.; zhou et al., ) . although it is now clear that human coronaviruses, particularly sars-cov- , are found in feces and can infect the gastrointestinal tract, the importance of its enteric phase for viremia, pathogenesis and patient prognosis remains unknown. to combat the current pandemic of covid- and to prepare for potential future emerging zoonotic coronaviruses, we need to gain a better understanding of the molecular basis of sars-cov- infection, replication and spread in a tissue-specific manner. here, we engaged in studying sars-cov- infection of human intestinal cells. for this, we exploited both human intestinal epithelial cell lines and human organoid culture models to characterize how these cells support sars-cov- replication and spread and how they respond to viral infection. direct comparison of both primary and transformed cells show that human intestinal epithelial cells fully support sars-cov- infection and de-novo production of infectious virus particles. interestingly, viral infection elicited a robust intrinsic immune response where type iii interferon mediated response was shown to be significantly more efficient at controlling sars-cov- replication and spread as compared to type i interferon. importantly, human primary intestinal epithelial cells responded to sars-cov- infection by producing only type iii interferon. taken together, our data clearly highlight the importance of the enteric phase of sars-cov- and this should be taken into consideration when developing hygienic/containment measures, and antiviral strategies, and when determining patient prognosis. as there is growing evidence that the gastro-intestinal tract is infected by sars-cov- , we engaged in studying virus infection, replication and spread in human intestinal epithelial cells (iecs). first, sars-cov- (strain bavpat ) was propagated in the green monkey cell line vero (see methods section). to detect viral infection, we used an antibody directed against a region of the nucleoprotein (np) which is conserved between of sars-cov- and sars-cov- . additionally, we used the j antibody which detects double-stranded rna (dsrna) which is a hallmark of rna virus replication (targett-adams et al., ) . cells positive for np were found to be always positive for dsrna; the np signal was found to be dispersed within the cytosolic area whereas dsrna were found in discrete foci likely corresponding to replication compartments (harak and lohmann, ) (fig. s a ). supernatants of infected vero cells were collected at hours post-infection (hpi) and the amount of infectious virus particles present was measured using a tcid approach on vero cells (fig. s b ). the colon carcinoma derived lines t and caco- cells were then infected with sars-cov- at a moi of . (as determined in vero cells) and, at different time post-infection, cells were fixed and immunostained using the anti-np and anti-dsrna antibodies (fig. a) . results show that sars-cov- infected caco- cells were readily detected as early as hpi and, by hpi, most of the cells were infected (fig. b) . similar results were observed in the t cells, but detection of infection was slightly delayed compared to caco- cells and, although the same amount of sars-cov- was used to infect these cells, only around % of the cells were found infected at hpi (fig. b) . these observations were in agreement with the increase in viral genome copy numbers over time (fig. c ) and the release of infectious virus particles in the supernatant of infected t and caco- cells (fig. d) . interestingly, infection of iecs by sars-cov- was associated with the generation of an interferon (ifn)-mediated intrinsic immune response. concomitant with the differences observed in virus replication and denovo virus production observed between t and caco- cells, t cells mounted a much stronger immune response compared to caco- cells (fig. e ) although much less t cells were infected (fig. b) . all together these results show that iecs are readily infected by sars-cov- and that infection of caco- cells lead to a weaker intrinsic immune response which is associated with more de-novo infectious virus production compared to t cells. this observation suggests that the ifn-mediated immune response controls sars-cov- infection in iecs. to directly test the function of ifns in controlling/restricting sars-cov- replication and spread in human iecs, t and caco- cells were mock-treated or pre-treated with type i (ifnb ) or type iii (ifnl) ifns. hrs post-treatment, cells were infected with sars-cov- at a moi of . (as determined in vero cells), in the presence or absence of ifns ( fig. a) . hpi, cells were immunostained using both the anti-np and anti-dsrna antibodies. results to address whether the endogenous levels of ifns generated by iecs controls sars-cov- replication and spread, we exploited our previously reported iecs depleted of either the type i ifn receptor (ifnar ) (ar-/-), the type iii ifn receptor (ifnlr ) (lr -/-) or depleted of both ifn receptors (dko). to control that our cells were functionally knocked-out for the type i ifn (ar-/-) and/or the type iii ifn receptor (lr-/-), t cells were treated with either ifn and the production of the ifn stimulated gene ifit was evaluated. as expected, type i ifn receptor knock-out cells (ar-/-) only responded to ifnl, whereas type iii ifn receptor knock-out cells (lr-/-) only responded to ifnb (fig. s ). the ifn receptor double knock-out cells (dko) did not respond to either ifn (fig. s ). wt or ifn receptor knock-out t cells were infected with sars-cov- at a moi of . (as determined in t cells). hpi, cells were immunostained using the anti-np antibody and the number of infected cells was quantified using fluorescent microscopy (fig. a) . results showed that depletion of the type i ifn receptor (ar-/-) resulted in a slight increase in the number of infected cells. importantly, depletion of the type iii ifn receptor (lr-/-) resulted in a massive increase of cell infectivity by a factor of around seven. similar results were obtained when both the type i and the type iii ifn receptors were depleted (dko) (fig. b) . interestingly, this increase in the number of infected cells upon depletion of the type iii ifn receptor (lr-/-) was associated with a significant increase in viral genome copy numbers (fig. c ) and with a three orders of magnitude increase in de-novo infectious virus production (fig. d ). together these results suggest that the type iii ifn-mediated immune response actively participates in controlling sars-cov- infection in human iecs. to unambiguously address the importance of the ifn-mediated antiviral response, we used the pan-jak inhibitor (pyridone- ) to inhibit the stat phosphorylation activation and block the production of interferon stimulated genes (isgs). as we previously reported, treatment of t cells with the pan-jak inhibitor fully inhibits signal transduction downstream both the type i and type iii ifn receptors ( (pervolaraki et al., ) and data not shown). mock and pyridone- pre-treated t cells were infected with sars-cov- for hrs and analyzed using fluorescence microscopy following immunostaining using the anti-np antibody. results show both a significant increase in the number of infected cells (fig. e ) and an increase of viral genome copy number in cells treated with the pan-jak inhibitor (fig. f ). importantly, and in agreement with the results observed in cells depleted of the type iii ifn receptor, this increase in infectivity was also associated with an increase in infectious denovo virus particle production ( fig. g ). all together, these results strongly support a model where the type iii ifn mediated signaling controls sars-cov- infection in human intestinal epithelial cells. to address whether primary human iecs can be infected by sars-cov- and support denovo virus production, we used human colon derived organoids from two distinct donors. intact ultrastructural organization and differentiation to all cell types (e.g. enterocytes, goblet cells, enteroendocrine cells, and stem cells) was confirmed using confocal fluorescent microscopy ( fig. a ) and quantitative-rt-pcr against cell type specific transcripts (fig. b ). as quantification of the number of infected cells in d organoids is very challenging, we exploited previously established protocols to differentiate and infect human intestinal organoids in two dimensions with viruses (ettayebi et al., ; stanifer et al., ) . non-differentiated organoids were seeded on human collagen-coated ibidi chambers. at hrs post-seeding, differentiation was induced by removal of wnt a and reducing the amounts of r-spondin and noggin for four days. upon full differentiation, organoids were infected with sars-cov- . at hpi, the infection was analyzed by immunostaining using the anti-np and anti-dsrna antibodies and by quantitative rt-pcr ( fig. c ). results show that independent of the donor, colon organoids were readily infected by sars-cov- as noted by the presence of cells positive for both np and dsrna (fig. d ). quantification revealed that around - % of cells were infected in each donor (fig. e ). this infection was associated with an increase of viral genome copy number (fig. f ). interestingly, infection of organoids led to no type i interferon (ifnb ) production but an extremely large up regulation of type iii ifn (ifnl) ( fig. g and fig. s ). to determine if exogenously added ifns could prevent sars-cov- infection, colon organoids were mock or pre-treated with ifnb and ifnl and then subsequently infected with sars-cov- for hrs. results show that pre-treatment of colon organoids with both ifnb and ifnl significantly impaired infection (fig. h ). this was associated with a reduction of sars-cov- genome copy numbers (fig. i ) and a decrease in infectious de-novo virus particle production (fig. j ). all together these results show that human colon organoids can support sars-cov- infection, replication and spread and that the type iii ifn response plays a critical role in controlling virus replication. human primary intestinal epithelial cells support robust replication of sars-cov- and secretion of infectious de-novo virus particles. we found that around % of the cells are infected in a human colon derived organoid leading to a modest but significant increase in viral genome copy number (fig. f ) and de-novo infection virus particles (fig. j ) over time. this modest replication of sars-cov- is explained by the fact that (i) only a small fraction of the cells is infected by sars-cov- , and (ii) as shown in this work, ifns are potent inhibitors of sars-cov- replication ( fig. and ) and organoids are highly immunoresponsive upon viral infection (stanifer et al., ) (fig. g) . it is well known that in vivo intestinal epithelium cells are less immunoresponsive because of the gut microenvironment (e.g. microbiota and tissue specific immune cells) and as such they will very likely show a severely dampened immune response allowing for an even greater sars-cov- replication. importantly, the intestinal epithelium is the largest organ in the body and even if only a few percent of the cells are infected it will result in the generation of an extremely large amount of de-novo viruses. analysis of the single-cell rna sequencing data from the colon atlas revealed that only . % of human colon epithelial cells express very low levels of the sars-cov- receptor ace- ( fig. s a-e) . this is very different to the small intestine where ace- appears to be more expressed (qi et al., ) . this low ace- level could explain why we have a small percentage of infected cells in our colon organoids (fig. e) . on the contrary, tmprss seems to be not a limiting factor in the colon (fig. s c-e) . interestingly, q-rt-pcr and western blot analysis do not support single cell analysis and clearly show that ace- is expressed in both our carcinoma derived lines and our colon organoids (fig. s f-g) . the discrepancy between the single-cell rna sequencing and classical molecular and biochemical approaches is likely the results of (i) the sequencing being not deep enough to detect ace- in individual cells and (ii) rna expression not necessarily matching the protein expression levels. these observations highlight that although analyzing data from single-cell rna sequencing atlases could be very informative, their findings should be validated in tissues as they may be mis-leading or miss important sites of virus replication. the human colon carcinoma caco- cells produce very large amounts of infectious viral particles (between and infectious particles per ml). this is higher than the titers obtained in vero cells which are commonly used to isolate and propagate sars-cov- (harcourt et al., ) and where we routinely obtained titers of and infectious particles per ml in vero cells (fig. s b) . interestingly, in a study comparing different human cell lines, caco- cells were the only cell type found to support sars-cov- replication and, compared to green monkey cells, caco- cells were very efficient in producing infectious de-novo virus particles and did not show cytopathic effects (mossel et al., ) . these observations that caco- are excellent culture models supporting both sars-cov- and sars-cov- further highlight the potentially central role of intestinal epithelial cells in covid- patients. intriguingly, t cells, which are also colon-carcinoma derived cells supported sars-cov- infection, replication and spread but to a much lesser extent compared to caco- cells. analysis of the intrinsic immune response generated upon infection of both cell lines revealed that t cells are more immunoresponsive compared to caco- cells. in light of the results obtained by pretreating cells with exogenous ifns, we propose that t cells, by being able to mount a stronger and faster immune response compared to caco- cells can better restrict sars-cov- infection. this model is fully supported by our ifn receptor knock-out t cells, in which virus replication and de-novo virus production are drastically increased to levels similar to the ones observed in caco- cells. exogenously added ifns (both type i and type iii ifns) induce an antiviral state in our human intestinal epithelial cells, thereby restricting sars-cov- replication in these cells. similar observations were made with type i ifn in vero cells (mantlo et al., ) . interestingly, infection of the calu- human lung epithelial cells by sars-cov- seem to also mount an immune response (lokugamage et al., ) importantly, our work shows that, compared to the airway epithelium, the intestinal epithelium produces a typical antiviral response. this highlights that host/pathogen interaction should be considered in a tissue specific manner as different cellular responses and viral countermeasures might be established between the lung, the gut and other organs. interestingly, in human colon organoids we observed that only type iii ifn is made upon sars-cov- infection, although human intestinal organoids are capable of making both type i and iii ifn upon enteric virus infection (pervolaraki et al., ; stanifer et al., ) . the lack of type i induction appears to be specific to sars-cov- and it is likely that this virus encodes a specific antagonist which counteracts the production of type i ifn only. however, further studies are necessary to prove this novel concept. we propose that the gut is an active site of replication for sars-cov- and this could account for the viremia observed in covid- patients and for the presence of large amounts of sars-cov- genomes in the feces. the origin of the replicating sars-cov- in the intestinal epithelium is still not clear. to date only one paper reported the isolation of infectious virus from stool samples . further characterization of the sars-cov- enteric lifecycle is necessary to determine whether the viral infection observed in the gut is due to fecal/oral transmission or is a manifestation of virus spreading from the lung to the gut. in the context of the gut we foresee that at the onset of sars-cov- infection, human intestinal epithelial cells will mount an antiviral response through the type iii ifn signaling pathway. as immune cells participate in mounting an innate immune response, type i ifn will be secreted from these cells and will be able to act on intestinal epithelial cells further reinforcing their antiviral state against sars-cov- . in respect to the severe pathologies observed in the lung, which are believed to be caused by a cytokine storm, the findings that lung epithelial cells mount a muted immune response upon sars-cov- infection suggests that the cytokines are coming from an alternative source. this source could be from local immune cells but also from the gastro-intestinal mucosa given the large immune response generated by primary human intestinal epithelial cells. as a matter of fact, many cytokines are released from epithelial cells towards the lamina propria (tissue side) (stanifer et al., ) , and will quickly enter the circulation to potentially fuel and promote the inflammation and pathology observed in the lung. in conclusion, the gastro-intestinal tract is an active site of sars-cov- replication and this should be considered when developing antiviral strategies as it may participate in the viremia and potentially reinfection. from a patient prognosis point of view, the severity of the disease should be correlated with the extent of the enteric replication. ifnl (il a) (# - k) and ifnl (il- b) (# - k) were purchased from peprotech and were used at a concentration of ng/ml each to make a final concentration of ng/ml, pyridone (calbiochem # - )was used at a final concentration of um. all sars-cov- infections were performed the moi indicated in the text. media was removed from cells and virus was added to cells for hour at °c. virus was removed, cells were wash x with pbs and media or media containing inhibitors/cytokines was added back to the cells. d organoid seeding. -well ibidi glass bottom chambers were coated with . % human collagen in water for h prior to organoids seeding. organoids were collected at a ratio of organoids/transwell. collected organoids were spun at g for mins and the supernatant was removed. organoids were washed x with cold pbs and spun at g for mins. pbs was removed and organoids were digested with . % trypsin-edta (life technologies) for mins at °c. digestion was stopped by addition of serum containing medium. organoids were spun at g for mins and the supernatant was removed and organoids were re-suspended in normal growth media at a ratio of µl media/well. the collagen mixture was removed from the ibidi chambers and µl of organoids were added to each well. rna isolation, cdna, and qpcr. rna was harvested from cells using rnaeasy rna extraction kit (qiagen) as per manufactures instructions. cdna was made using iscript reverse transcriptase (biorad) from ng of total rna as per manufactures instructions. q-rt-pcr was performed using itaq sybr green (biorad) as per manufacturer's instructions, tbp or hprt were used as normalizing genes. primer used: performed, and cells having fewer than genes and more than % of umi count mapped to mitochondrial genes were discarded. consecutively, the resulting datasets were normalized, scaled and high-variance genes genes were selected. reciprocal pca-based data integration was done to merge the samples. afterwards, the resulting batch-corrected counts were used for calculating pca-based dimensionality reduction and unsupervised louvain clustering. furthermore, umap visualization was calculated using neighboring points for the local approximation of the manifold structure. cell type annotation was based on the unsupervised clustering and the metadata provided by the colon atlas (smillie et al., ) . statistics. unless otherwise stated, statistical analysis was performed by a two-tailed unpaired t test using the graphpad prism software package. all samples were analyzed without blinding or exclusion of samples. zhou, j., li, c., zhao, g., chu, h., wang, d., yan, h.h.-n., poon, v.k.-m., wen, l., wong, b.h.-y., zhao, x., et al. ( ) . human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus. sci. adv. , eaao . sars-cov- launches a unique transcriptional signature from in vitro, ex vivo comparative replication and immune activation profiles of sars-cov- and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid- type i and type iii interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia origin and evolution of pathogenic coronaviruses replication of human noroviruses in stem cell-derived human enteroids ultrastructure of the replication sites of positive-strand rna viruses isolation and characterization of sars-cov- from the first us covid- patient (microbiology) sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor enteric involvement of severe acute respiratory syndrome-associated coronavirus infection sars-cov- sensitive to type i interferon pretreatment outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle sars-cov- receptor ace and tmprss are primarily expressed in bronchial transient secretory cells potent antiviral activities of type i interferons to sars-cov- infection seasonality of respiratory viral infections exogenous ace expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication coronavirus infections-more than just the common cold type i and type iii interferons display different dependency on mitogen-activated protein kinases to mount an antiviral state in the differential induction of interferon stimulated genes between type i and type iii interferons is independent of interferon receptor abundance ifn-λ determines the intestinal epithelial antiviral host defense single cell rna sequencing of human tissues identify cell types and receptors of human coronaviruses intra-and inter-cellular rewiring of the human colon during ulcerative colitis reovirus intermediate subviral particles constitute a strategy to infect intestinal epithelial cells by exploiting tgf-β dependent pro-survival signaling asymmetric distribution of tlr leads to a polarized immune response in human intestinal epithelial cells visualization of double-stranded rna in cells supporting hepatitis c virus rna replication knowledge synthesis from million biomedical documents augments the deep expression profiling of coronavirus receptors animal coronaviruses: a brief introduction emerging and re-emerging coronaviruses in pigs detection of sars-cov- in different types of clinical specimens virological assessment of hospitalized patients with covid- covid- and the digestive system single-cell rna expression profiling of ace , the putative receptor of wuhan -ncov prolonged presence of sars-cov- viral rna in faecal samples evidence for gastrointestinal infection of sars-cov- prolonged viral shedding in feces of pediatric patients with coronavirus disease high expression of ace receptor of -ncov on the epithelial cells of oral mucosa characteristics of pediatric sars-cov- infection and potential evidence for persistent fecal viral shedding interferon-λ enhances adaptive mucosal immunity by boosting release of thymic stromal lymphopoietin single-cell rna expression profiling of ace , the receptor of sars-cov- (bioinformatics) we would like to acknowledge vibor laketa and the infectious diseases imaging platform (idip) at the center for integrative infectious disease research, heidelberg, germany, for support with image acquisition and analysis. we also thank christian drosten at the charité, berlin and the european virus archive (evag) for the provision of the sars-cov- strain bavpat . the authors declare no competing interests. key: cord- -reez md authors: wang, zhenling; pan, hailong; jiang, boguang title: type i ifn deficiency: an immunological characteristic of severe covid- patients date: - - journal: signal transduct target ther doi: . /s - - - sha: doc_id: cord_uid: reez md nan recently, a paper published in science by hadjadj et al. reported that type i interferon (ifn) deficiency, could be a hallmark of severe coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ). severe covid- was also associated with a lymphocytopenia, persistent blood viral load, and an exacerbated inflammatory response (fig. ) . these findings provide insights into the treatment of severe covid- patients with type i ifn. severe covid- is clinically characterized by two-phase disease progression. a secondary respiratory worsening - days after the first onset of symptoms can occur early in the disease. among the respiratory failure patients, young adults (aged years and lower) with previously mild comorbidities have a relatively high rates. the respiratory failure is concomitant with characteristic ct scan, lymphocytopenia, high prothrombin time, and d-dimer levels. this biphasic evolution suggests a dysregulated inflammatory host response driven by virus resulting in an imbalance between proand anti-inflammatory mediators. however, the immunological features and mechanisms involved in covid- severity are unclear. in order to test whether the severity disease can be caused by sars-cov- viral infection and hyperinflammation, hadjadj et al. conducted a comprehensive immune analysis of grouped covid- patients with different disease severity. first, to identify whether the severe disease induced lymphocytopenia, hadjadj et al. compared the peripheral blood leukocytes density of variously severe patients by combining mass cytometry with visualization of high-dimensional single-cell data based on t-distributed stochastic neighbor embedding. there is a significantly decreased density of nk cells and cd + t cells in severe and critical patients, while the density of b cells and monocytes was increased. the authors determined the functional status of specific t-cell subsets (cd + /cd + ) and nk cells based on the expression of activation (cd , hla-dr) and exhaustion (pd- , tim- ) markers. they observed that the activated nk and cd + /cd + t cells were increased in all infected patients, while the exhausted cd + /cd + t cells and nk cells were increased in only severity patients. this result supported lymphocytopenia correlates with disease severity. subsequently, hadjadj et al. characterized the disease severity by studying immunological transcriptional signatures, the expression of immune-related genes in peripheral white blood cells was quantified by direct probe hybridization. and the differentially expressed genes evaluating severity grades were identified. patients with various disease severity were separated by principal component analysis and the severity-related genes were enriched by gsea (q value < . ). a severity-dependent manner was found in activation level of the innate and inflammatory pathways. ifn responses were crucial in anti-viral immune, but were tested relatively lower in severe cases of covid- . here, in critical covid- patients, the genes involved in type i ifn signaling (such as ifnar , jak , tyk ) were upregulated, whereas the ifnstimulated genes (isgs) (such as mx , ifitm , ifit ) were dramatically downregulated. compared with patients that had mild-to-moderate infection, the isg score (based on the mean expression value of six isgs defining a type i ifn signature) was significantly reduced in critical patients. being consistent with isg scores, ifn-α protein in plasma was significantly lower in critical than in mild-to-moderate patients by simoa digital elisa. isg score and plasma ifn-α from blood collected prior to respiratory failure revealed that the low type i ifn response preceded clinical deterioration to critical status. then, the global type i ifn activity was measured by an in vitro cytopathic assay (protection of madin-darby bovine kidney cells against cell death after infection with vesicular stomatitis virus). there is a lower ifn activity in serum of severe or critical patients than that of mild-to-moderate patients. they next stimulated whole blood cells with ifn-α and observed a comparable increase in isg score in any groups. among these patients, there is a higher blood viral load in severe and critical patients evaluated by digital pcr. by correlated analysis of viral loading with ifn-α production either on protein or on gene level, the authors suggest that the most severe cases of covid- are featured by impaired ifn-α production. hypercytokinaemia observed in severe covid- cases is characterized by increased plasma levels of pro-inflammatory cytokines. thus, interleukin (il)- , tumor necrosis factor (tnf)-α, il- β, and il- proteins in the plasma of patients were quantified using simoa elisa assay. il- and tnf-α were detected with high levels in severe and critical patients compared to mild-tomoderate patients. while il- β and il- α were not detected. this study suggests that severity of covid- was characterized by increased tnf-α and il- production. to further explore the transcription factors that may cause the excessive inflammatory response of covid- , the authors observed that upregulated genes in severe or critical patients mainly belonged to the nf-κb pathway by a kinetic analysis. aberrant nf-κb pathway activation resulted excessive innate immune sensor activation by pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (dapms). two markers of cellular necrosis and damage, namely ldh and receptor-interacting protein kinase- , were found to be involved in disease severity and significantly elevated in patients with severe covid- . while mounting specific immune response, target organ infiltrations of neutrophils and monocytes could be facilitated by overproduction of chemokines thus contribute to development of acute respiratory distress syndrome. therefore, the expression of chemokines (cxcr , cxcl ) and chemokine receptors (ccr , ccl ) of neutrophils and monocytes was detected. neutrophil chemokine receptor cxcr , monocyte chemotactic factor ccl , and receptor ccr were significantly upregulated in severe and critical patients. overall, these results provide a framework of ongoing inflammatory cascade that impaired type i inf production and high viral load perhaps fueled by both pamps and damps. overall, hadjadj et al. identified an impaired type i ifn response, characterized by no ifn-β and low ifn-α production and activity, should be a hallmark of severe covid- . clinically, type i ifn deficiency is associated with hyperinflammation driven by nf-κb and lower viral clearance. the authors indicated that ifn response is possible to incorporate as an indication to assess early severe covid- . the application of ifn administration and targeted antiinflammatory therapies may aid in the development of improved treatments to overcome sars-cov- infection. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. © the author(s) fig. immunological characteristics of severe covid- patients. impaired type i ifn response featured immunological characteristics of severe covid- patients, accompanied by lymphocytopenia, hypercytokinaemia, and high blood viral load. impaired type i interferon activity and inflammatory responses in severe covid- patients early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia clinical and immunological features of severe and moderate coronavirus disease clinical features of patients infected with novel coronavirus in wuhan covid- : consider cytokine storm syndromes and immunosuppression this work is supported by the national major scientific and technological special project for "significant new drugs development" ( zx - - ). competing interests: the authors declare no competing interests. key: cord- - qiwui u authors: torrecilhas, a c t; faquim-mauro, e; da silva, a v; abrahamsohn, i a title: interference of natural mouse hepatitis virus infection with cytokine production and susceptibility to trypanosoma cruzi date: - - journal: immunology doi: . /j. - . . .x sha: doc_id: cord_uid: qiwui u mouse hepatitis virus (mhv) infection can have a pronounced impact on several investigation areas. reports on natural mhv outbreaks are rare and most studies have been conducted by deliberately infecting mice with mhv laboratory strains that cause moderate to severe disturbances to the immune system. we have investigated the effects of a natural acute outbreak of mhv in our otherwise specific-pathogen-free (spf) inbred mouse colonies, and of enzootic chronic mhv infection on cytokine production and resistance to the intracellular pathogen trypanosoma cruzi. we found that balb/c and/or c bl/ spf mice that had been injected with t. cruzi blood trypomastigotes from recently mhv-contaminated (mhv(+)) mice developed significantly higher parasite blood counts, accelerated death, and showed higher il- production by spleen cells than their counterparts whose t. cruzi inoculum was derived from mhv-negative (mhv(−)) donors. interferon-γ (ifn-γ) production by mhv(+) and mhv(−) mice was not significantly different. in contrast, t. cruzi infection of chronically mhv-infected mice did not result in major changes in the course of infection when compared with that observed in mice from mhv(−) colonies, although a trend to higher parasitaemia levels was observed in balb/c mhv(+) mice. nevertheless, both balb/c and c bl/ t. cruzi-infected mhv(+) mice had diminished ifn-γ production to parasite-antigen stimulation in comparison with similarly infected mhv(−) mice. interleukin- (il- ) production levels by spleen cells did not differ between chronic mhv(+) and mhv(−) mice, but ifn-γ neutralization by monoclonal antibody treatment of anti-cd -stimulated spleen cell cultures showed higher levels of il- synthesis in mhv(+) balb/c mice. immune responses and on their interference with experimental models of infection. most reports deal with virulent laboratory mouse hepatitis virus (mhv ) collectively designates corona strains, although more recently, attenuated mhv laboratory viruses of a wide range of virulence. the strains that are strains have been used in an attempt to mimic the prevalent endemic in most mouse colonies over the world show relatively strains. the objective of this study was to investigate the low virulence and animals from infected colonies do not effects of a natural acute outbreak of mhv due to accidental present overt signs of illness. nevertheless, although tolerated exposure, in our otherwise specific-pathogen-free (spf ) inbred by many researchers, evidence has accumulated over the years mouse colonies, and of enzootic chronic mhv infection on that results from several investigation areas can be comprocytokine production and resistance to the intracellular pathomised by concomitant mhv infections. in particular, the gen trypanosoma cruzi. study of immunological parameters that determine resistance trypanosoma cruzi is a dygenetic protozoan that infects to infections can be seriously affected by mhv infection several kinds of mammals and is the aetiological agent of (reviewed in refs , ) . there are few studies on the effects of chagas' disease in man. the parasite replicates in the cytoinfection by natural-low-virulence enzootic mhv strains on plasm of virtually any nucleated cell type including macrophages; non-dividing forms of the parasite are found free in more resistant. susceptible mice have higher parasite numbers ), pneumonia virus of mice (pvm ), minute virus of mice (mvm ), lymphocytic chorio-meningitis virus (lcmv ), in the blood and tissues and most animals die within weeks of inoculation of as few as parasites; in contrast, resistant sendai virus, ectromelia virus, mouse polio virus (gd ), and on elisa for the bacteria mycoplasma pulmonis. all pro-mice have lower parasite loads and survive infection with parasites. both t. cruzi-resistant and -susceptible cedures with the animals were in accordance with the principles of the 'brazilian code of laboratory animals use'. mouse strains show elevated production of interferon-c (ifn-c) during infection and very low interleukin- (il- ) and il- production. - ifn-c and tumour necrosis factor-a trypanosoma cruzi infection, parasitaemia counting and experimental design (tnf-a), triggered by il- , are essential to control of parasitism by innate and acquired cellular immune systems in infective blood trypomastigotes were obtained from y strain t. cruzi-infected anaesthetized mice by drawing cardiac blood; mice. - il- exerts an important in vivo regulatory role on ifn-c and nitric oxide production , and its absence in motile blood forms were counted and the desired number of parasites was injected intraperitoneally (i.p.). infection was il- -gene-deprived mice , or neutralization by monoclonal antibody (mab) treatment results in lower parasitism, maintained by weekly i.p. infection of balb/c mice. in the experiments designated 'acute' mhv infection, parasites were whereas increased parasitism occurs when mice are treated with recombinant il- (ril- ) or receive il- -and il-maintained in mice coming from either recently infected mhv+ or from mhv− colonies and or forms were -producing t cells. il- , depending on the parasite strain, is also involved in negative regulation of parasitism. inoculated in recipient balb/c or c bl/ mice from mhv− colonies. in later experiments, designated 'chronic' mhv we found that balb/c and c bl/ mice that had been injected with blood trypomastigotes from recently mhv-infection, the t. cruzi strain was started anew from tissueculture-grown trypomastigotes and maintained in mhv− contaminated mice became much more susceptible to t. cruzi infection and produced higher il- levels than their counter-mice, whose blood was used as a source of t. cruzi to infect recipients derived from mhv+ colonies that had been infected parts whose t. cruzi inoculum was derived from mhvnegative (mhv−) donors. comparison between mice coming for more than months or from mhv− colonies. in these experiments the infective t. cruzi dose was , , or from chronically mhv-infected and from mhv− colonies, showed higher parasitaemia levels in balb/c mhv-positive blood forms in balb/c mice and , (not shown), , or blood forms in c bl/ mice. as c bl/ (mhv+) mice but otherwise no major significant differences in susceptibility to t. cruzi. nevertheless, quantitative differ-mice are much more resistant to t. cruzi infection, the high inocula would allow comparison between mhv− and mhv+ ences in ifn-c, il- and nitric oxide production were found between mhv-infected and uninfected mice. c bl/ colonies submitted to moderate to severe t. cruzi infection. parasitaemia determination was performed by direct microscope (× ) counting of motile parasites in a ml fresh materials and methods blood sample, obtained from the lateral tail veins. mice and mhv infection c bl/ and balb/c female mice ( - -week-old), acutely spleen cell cultures spleen cell suspensions were prepared from t. cruzi-infected or chronically infected with mhv were obtained from biotério de camundongos isogênicos do departamento de imunologia and uninfected mice, mhv+ or mhv−, depleted of erythrocytes by hypotonic lysis with distilled water and resuspended do icb/usp (são paulo, sp, brazil ). mice with negative serological tests for mhv, from both these strains, were in rpmi- (cultilab, campinas, sp, brazil ) complete medium containing % fetal calf serum (fcs; cultilab) and obtained from biotério de camundongos isogênicos da universidade estadual de campinas (campinas, sp, brazil ). supplemented with glutamine, -mercaptoethanol ( -me ) and antibiotics as described. spleen cell suspensions were pooled mhv− mice were housed and handled separate from and before those coming from mhv-contaminated animal facili-from three mice and were cultured in duplicate or triplicate in -well flat-bottomed plates at /ml or at × /well ties. the mice were fed autoclaved food and water, and were handled using disposable gloves. mhv infection was diagnosed and stimulated with t-ag ( × frozen-thawed tissue culture trypomastigotes parasites) prepared as described. by antibody testing of the sera. the mhv enzyme-linked immunosorbent assay ( elisa) diagnostic kits sold by charles concanavalin a (con a at · m/ml ) or plate-bound anti-cd [mab - c , americal type culture collection (atcc ) river laboratories ( wilmington, ma) were used according to the manufacturer's instructions. when the outbreak of crl ], coated at mg/ml, ml/well ) were also used as t-cell stimulants. . supernatants from the cultures were mhv was detected, levels of anti-mhv antibodies were very high with corrected optical density (od) values for sera harvested after hr from the higher cell-density cultures and after hr from the lower-density cultures. the following ranging from · to · (positive test values > ). these were calculated (as indicated by the manufacturer) by the following neutralizing rat mab anti-mouse cytokines were incorporated to the cultures in some experiments: jes - a anti-il- , and formula: [(od obtained for the test serum diluted at / incubated with the cells containing the virus) -(od obtained xmg . anti-ifn-c; both were used at final concentrations of mg/ml. the anti-cd mab gk . was added to cultures for the same serum and dilution incubated with uninfected cells)]/ · . fluorescence antibody testing was also performed at mg/ml. as an additional control and ranked + + + + for serum incubated with mhv-infected cells. the colonies were serologi-cytokine assays cytokine levels in the culture supernatants were measured by cally negative on elisa and immunofluorescence testing for the following viruses: respiratory-enteric orphan virus (reo two-site sandwich elisa using the following mab pairs of which the second cited was biotinylated. ifn-c, xmg . and when the inoculum came from mhv− donor mice. the an ; il- , jes- a and sxc- ; il- , b and differences in parasitaemia could be observed with inocula of bvd g . minimum levels of detection for the assays or blood trypomastigotes (fig. a,b) . moreover, for were:. ifn-c, · ng/ml; il- , · units/ml; and il- , balb/c mice injected with mhv+ inocula, mortality reached · ng/ml. the rat anti-mouse cytokines producing % by day ( t. cruzi forms) and % by day hybridomas were a generous gift from dr r. l. coffman, ( t. cruzi forms) whereas all recipients of mhv− inocula dnax research institute of molecular and cellular biology, survived to days after infection. all c bl/ mice palo alto, ca. standard curves were obtained with recombisurvived after infection whether inoculated with t. cruzi from nant mouse cytokines. the supernatants were tested in serial mhv+ or mhv− donor mice. twofold dilutions and the results were expressed as the arith-serological tests for mhv became positive in t. cruzimetic mean of duplicate determinations. the sd did not infected (from mhv+ donors) c bl/ mice by day after exceed % of the mean. infection and in % of a group of balb/c mice that survived to days after being infected with t. cruzi statistical analysis forms. recipients of inocula from mhv− mice remained the significance of differences in parasitaemia between distinct serologically negative for mhv. looking into possible causes experimental groups was examined by analysis of variance with of the observed increase in susceptibility to t. cruzi of mice repeated measurements followed by tukey's test for multiple infected with mhv+-derived inocula. we investigated whether comparisons. differences of · log or over were significant cytokine production was altered concomitant to the viral at least at p< · . differences in cytokine production levels infection. spleen cells (stimulated with con a or t-ag) from were tested by bonferroni's multiple comparison test. mice that received an mhv+ inoculum of t. cruzi produced detectable and much higher levels of il- during the first weeks of infection than spleen cells from mice that results received an identical inoculum of t. cruzi derived from t. cruzi infection and cytokine production in mhv− mice mhv− mice. in fact, il- production in this last situation inoculated with t. cruzi blood forms obtained from donor-mice was very low and often below · units/ml (table ). il- that were serologically mhv+ or mhv− production, on day after infection, in balb/c (but not in c bl/ ) mice was cd -activation dependent as treatment our preliminary observation, suggestive of an infection with gk . mab suppressed most of its synthesis outbreak in the mouse colonies, was increased susceptibility ( units/ml in untreated versus units/ml in gk . treated to t. cruzi infection first detected in balb/c mice. as the cultures in con a-stimulated and units/ml versus diagnosis of mhv infection was confirmed, we first investi- units/ml, respectively, in t-ag-stimulated balb/c spleen gated how a t. cruzi inoculum derived from donor mice that cell cultures). in c bl/ mice, the values were units/ml had concomitant mhv infection would compare with a in untreated versus units/ml in gk . -treated con similar inoculum originated in mhv− mice in regard to their a-stimulated cultures and versus units/ml, respectively, course of infection in a t. cruzi-susceptible (balb/c) and in after t-ag stimulation. in spite of the higher il- proa resistant (c bl/ ) mouse strain. as shown in fig. , duction by spleen cells from mice that had received the parasitaemia levels during the first days of infection were mhv+ inoculum, ifn-c levels were not significantly different significantly higher for both strains of mice when infected with t. cruzi blood forms derived from mhv+ donors than from those secreted by spleen cells from mhv− t. cruzi recipients and il- production was below detection levels in resulted in significant increases in ifn-c production of the order of - % (fig. ) . we next investigated whether all groups (data not shown). infecting mhv− or mice that were chronically infected mhv with t. cruzi would affect cytokine production and/or alter cytokine production by spleen cells from mice derived from the course of infection. spleen cell cultures from balb/c and c bl/ mice coming in contrast with the marked increase in parasitaemia and (data not shown). production of ifn-c by spleen cells from susceptibility to t. cruzi determined by the situation of acute mhv+ c bl/ and balb/c mice cells to con a stimulation simultaneous infection with blood parasites derived from was not significantly higher than in spleen cell cultures from acutely infected mhv+ donors as described above, no statis-mhv− animals (fig. ) . production of ifn-c to con a was tically significant differences of parasitaemia levels or mortunder control by il- both in mhv− and in mhv+ mice ality were observed between chronically infected mhv+ and because the addition of anti-il- mab a to cultures mhv− balb/c or c bl/ mice infected with t. cruzi. however, peak t. cruzi parasitaemia levels were attained earlier and were - % higher in balb/c mhv+ mice as compared to mhv-free mice (data not shown). differences in cytokine production between t. cruziinfected mhv+ and mhv-free groups of mice were only observed in the first week of infection. balb/c mice spleen cell cultures from mhv+ mice infected with t. cruzi produced lower ifn-c levels to con a and t-ag stimulation than cultures from mhv− mice (fig. ) . although the potential to produce ifn-c to polyclonal con a stimulation was preserved in mhv+c bl/ mice, ifn-c production was markedly suppressed on parasite-specific stimulation (fig. ) . the comparison of il- production levels among balb/c and c bl/ mice, mhv− or mhv+, infected with t. cruzi yielded no significant differences (data not shown). nevertheless, when balb/c (but not c bl/ ) spleen cell uninfected mhv+ mice (fig. , normal group) , suggesting mice, suppression of ifn-c responses is limited to the antigenspecific compartment whereas mhv infection affects more that the potential to increased il- production stimulated by mhv infection was, in these mice, maintained in check by severely balb/c mice by suppression of both polyclonal and specific ifn-c responses. ifn-c. taken together, these results indicate that in c bl/ immune response have been performed in situations of deliberate infection of mice with mhv laboratory strains of varying enzootic mhv strains are of low virulence, mostly entervirulence that cause moderate to severe disturbances to the otropic and notoriously difficult to isolate and to grow in immune system. mice infected with the pantropic strain of vitro. when the outbreak was detected in the mouse colony, medium-virulence, jhm, show suppression of con a-induced we unsuccessfully tried to isolate infective viral particles from proliferation, decreased il- and il- synthesis and a delay the plasma and liver of seropositive mice. immunosuppressing in ifn-c production in the first week of infection, whereas, the animals with cyclophosphamide also failed to promote later in infection, large amounts of ifn-c are produced by viral isolation. the difficulty in isolating low virulence enzootic balb/c mice. - macrophage function was impaired in mice enterotropic mhv, as opposed to polytropic laboratory mhv infected with this strain or with naturally occurring mhv strains, has been frequently reported. , , we had (as many strains. , increased message levels for ifn-c, il- , il- , other authors) to rely on serum antibody screening and tnf-a and inducible nitric-oxide synthase (inos ) were found serological conversion as a criterion to identify occurrence of in the brain of mice infected with a neurotropic jhm variant mhv infection. serological testing, although highly specific, strain. mhv a- is yet another laboratory strain, but does not distinguish between acute and chronic mhv infection of relatively low virulence that, although subclinical, is and thus we will discuss the immunological alterations found accompanied by increased production of ifn-c and suppresin the group of mice that had recently become seropositive for sion of con a spleen responses. , mhv (recent mhv-testers) in comparison with those found there have been few reports on immunological alterations in mice originating from colonies with longstanding record of resulting from natural mhv outbreaks. however, established mhv seropositivity. both groups of mice were serologically 'chronic' natural infection with mhv was reported to affect negative on testing for several other mouse pathogenic viruses mostly splenic t lymphocytes, with a - % decrease in and for mycoplasma pulmonis (see the materials and methods). proliferative responses to con a and resistance to the effects we have found a much more severe course of t. cruzi of nor-adrenalin or dibutyryl-camp. we found that lymphoinfection in mhv− mice infected with blood forms obtained proliferative responses of spleen cells from balb/c and from mouse colonies that had recently become seropositive c bl/ mice, mhv+ or mhv− recipient mice to con a, for mhv, as compared with mice inoculated with parasites t-ag, or anti-cd stimulation were not significantly different. derived from mhv− donors; increased susceptibility to infec-suppressed lymphoproliferative responses to these stimuli, tion was accompanied by increased synthesis of il- by commonly seen in the course of t. cruzi infection, were spleen cell cultures. interleukin- down-regulates il- and observed from day of infection, with minimal levels of ifn-c production, besides antagonizing ifn-c-and tnf-asuppression seen on day and recovery by day (data not dependent macrophage activation and intracellular t. cruzi shown). thus, in the course of t. cruzi infection, we did not killing. , , , - in spite of the enhanced il- secretion by observe, neither in recently seropositive recipient mice nor in spleen cells from mhv+ recipients, no decrease of ifn-c mice from chronically mhv-infected colonies, suppression of levels, in comparison to recipients of mhv− donors could be lymphoproliferation to con a, to parasite antigens or to antidetected in these same cultures. however, the data on enhanced cd stimulation that could be ascribed to the viral infection. ifn-c synthesis by anti-il- mab treatment, showed that however, its occurrence could have been masked by the intense endogenous il- was down-regulating ifn-c production in suppression characteristic of acute t. cruzi infection. this situation of probable acute concomitant infection with our results on the aggravation of t. cruzi infection in the parasite and the virus. the maintenance of in vitro ifn-c balb/c and c bl/ mice by using parasite inocula mainsecretion rates in the presence of increased il- concentained in mice that had positive serology for mhv are in trations has been described upon treatment of t. cruzi-infected agreement with a previous report on cba/j mice infected with mice with high doses of ril- that aggravate infection. t. cruzi derived from corona virus-positive mice. , these both mouse strains, balb/c and c bl/ , respectively, authors were able to demonstrate, in the plasma, infective susceptible and resistant to t. cruzi, showed increased parasitavirus particles that could be neutralized by anti-mhv antiemia and augmented il- production, but increased mortality serum. nevertheless, the underlying mechanisms leading to as a consequence of mhv infection was not observed for increased susceptibility to the parasite were not explored. c bl/ mice and they maintained their resistant phenotype we further investigated the influence of a longstanding to t. cruzi infection. although mouse strain resistance to ( year) established endemic mhv infection on the course of mhv infection is highly dependent on the mhv strain, studies t. cruzi infection and immune response. in contrast with the with laboratory mhv-strains have concluded that indifferently marked effects on the immune response observed in mhv− to the degree of mhv-strain virulence, replication in macrohosts infected with t. cruzi derived from recently mhv+ mice, phages does always occur. viral replication inside macrothe disturbances of immune response found in mice from phages may directly interfere with t. cruzi killing ability by chronically mhv-infected colonies were milder. production these cells and stimulate a number of macrophage functions of il- was similar between mhv+ and mhv− mice, while including il- synthesis. in this regard, c bl/ mice are parasite-antigen-stimulated ifn-c production was lower in t. semisusceptible to most mhv strains; , as production of cruzi-infected balb/c and c bl/ mice. however, when il- by mhv-infected c bl/ mice (but not by balb/c neutralization of ifn-c in the cultures unmasked the full mice) was cd +-activation independent, virus-infected or potential of il- secretion, mhv+ balb/c mice (but not virus-stimulated macrophages could be the main source of c bl/ ), showed higher il- production levels. this result this cytokine. is duration of mouse hepatitis virus infection: studies in immunocompetent and chemically enterotropic mouse hepatitis virus. lab immunosuppressed mice the cellular synergism between tumor necrosis factor-alpha and interferonand molecular pathogenesis of coronaviruses. lab animal sci gamma on macrophage activation for the killing of intracellular , . trypanosoma cruzi through a nitric oxide-dependent mechanism the microbicidal activity of interferon-gamma-treated san diego cytokines in innate and acquired dependent, nitrogen oxide-mediated mechanism inhibitable by immunity to trypanosoma cruzi infection tumor necrosis factor alpha mediates resistance to trypanosoma cruzi: early parasite pro-infect immun , . liferation and host resistance in inbred strains of mice trypanosoma cruzi: serum sci , . antibody reactivity to the parasite antigens in susceptible and ifn-gamma and il- production during trypanosoma cruzi infec- l failure of immune cell proliferation and interleukin production trypanosoma cruzi: mainten-adv exp med biol , . ance of parasite-specific t cell responses in lymph nodes during trypanosoma cruzi infection alters in vitro splenic t cell proliferation and cytokine production. suppresses nuclear factors that bind to specific sites on the lab animal sci , . interleukin- enhancer characterization of accessory cell function during acute infection of balb/cbyj mice with lactate dehydrogenase-elevating virus-permissive macrophages and t lymphocyte alterations. virus res , . mouse hepatitis virus (mhv ), strain jhm effect of d.a. ( ) mouse hepatitis virus suppresses modulation of inapparent murine hepatitis virus infections on macrophages and mouse spleen t cell activation murine virus contaminant of trypanosoma ( ) kinetics of cytokine mrna expression in the central cruzi experimental infection. rev inst med trop são paulo , nervous system following lethal and non-lethal coronavirus- . induced acute encephalomyelitis trypanosoma cruzi: murine virus contaminant of the experimental infection key: cord- -g a xf authors: shin, hye jin; kim, chonsaeng; cho, sungchan title: gemcitabine and nucleos(t)ide synthesis inhibitors are broad-spectrum antiviral drugs that activate innate immunity date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: g a xf nucleoside analogs have been frequently identified as antiviral agents. in recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)ntp pools. intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. the precise crosstalk between these two independent processes remains to be determined. nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular dna/rna polymerases [ ] . more recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. some nucleoside analog drugs targeting specific viral polymerases (acyclovir for herpesviruses, zidovudine for human immunodeficiency virus (hiv), and sofosbuvir for hepatitis c virus (hcv)) have been successful in clinical trials [ ] [ ] [ ] [ ] and are currently in use for the treatment of virus-infected patients. another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with ifn-α [ ] . importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. the primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( figure ). alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. the other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. there have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [ ] [ ] [ ] [ ] . by targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)ntp pools. as viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. a more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes (isgs). importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. however, the precise mechanism of this crosstalk remains to be elucidated. there is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. they have been well-summarized in a previous report [ ] . in the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. more importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( figure ). alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. the other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. there have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [ ] [ ] [ ] [ ] . by targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)ntp pools. as viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. a more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes (isgs). importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. however, the precise mechanism of this crosstalk remains to be elucidated. there is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. they have been well-summarized in a previous report [ ] . in the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. more importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. figure . the mechanism of antiviral effect of nucleos(t)ide analogs. nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [ , ] . however, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of rna viruses, including middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov), zika virus (zikv), hcv, poliovirus (pv), influenza a virus (iav), hiv, and enteroviruses (ev) [ ] [ ] [ ] [ ] [ ] [ ] . the antiviral activities of gemcitabine against the abovementioned viruses are summarized in table . mers-cov and sars-cov belong to the family of coronaviridae and are causative agents of severe viral respiratory illness in humans. to efficiently select appropriate antiviral drug figure . the mechanism of antiviral effect of nucleos(t)ide analogs. nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [ , ] . however, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of rna viruses, including middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov), zika virus (zikv), hcv, poliovirus (pv), influenza a virus (iav), hiv, and enteroviruses (ev) [ ] [ ] [ ] [ ] [ ] [ ] . the antiviral activities of gemcitabine against the abovementioned viruses are summarized in table . mers-cov and sars-cov belong to the family of coronaviridae and are causative agents of severe viral respiratory illness in humans. to efficiently select appropriate antiviral drug candidates, dyall et al. screened fda-approved drugs in virus-infected vero e cells and identified gemcitabine as one of drugs with antiviral activity against both mers-cov and sars-cov (ec of . µm and . µm, respectively) [ ] . more recently, gemcitabine was shown to effectively suppress zikv infection and replication in human retinal pigment epithelium (rpe) cells, particularly at non-cytotoxic concentrations (ec of . µm vs. cc of > µm) [ ] . zikv, a member of the flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. effective antiviral activities of gemcitabine were also found for the replication of hcv in huh- cells and the infection of hiv in u -magi-cxcr cem cells, with estimated ec s of nm and . nm, respectively [ , ] , which were lower concentrations than those used in cancer therapy [ ] . in the case of hiv, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced hiv infectivity by increasing the viral mutation frequency [ ] . in a follow up study, clouser et al. further reported the antiviral effect of gemcitabine against hiv-related retrovirus, murine leukemia virus (mulv), in vitro (ec of . nm) and even in murine aids model [ ] . a significant antiviral effect of gemcitabine on iavs was also reported for rpe cells by denisova et al. (ec of . µm) [ ] . they also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on sindbis virus and herpes simplex virus- (hsv- ) (> log reduction in virus titer) but relatively weak effects on semliki forest virus and human echovirus , and minimal effects on bunyamwera virus, measles virus (mev), and vaccinia virus [ ] . the antiviral effect of gemcitabine on evs, initially performed on coxsackievirus b (cvb ), was found from screening fda-approved drugs in cvb replicon-harboring vero cells by our group (ec of . µm) [ ] . its broad-spectrum antiviral activity on evs was further identified by observing a similar inhibitory effect on enterovirus (ev ) and human rhinoviruses (hrvs) (ec s of and - µm, respectively). in the case of hrv, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model [ ] . in this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including tnf-α and il- β, and the number of lung infiltrating lymphocytes. more recently, zhang et al. also identified gemcitabine as the best anti-pv inhibitor from a screen of fda-approved drugs in pv replicon-harboring hela cells (ec of . µm) [ ] . as previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of rna viruses and has a therapeutic potential for the treatment of various virus-associated diseases. moreover, it is possible that gemcitabine is effective for other untested rna viruses. because gemcitabine is a deoxycytidine analog that interferes with dna as well as rna synthesis, dna viruses may not be the exception. consistent with this possibility, there has been a report that the infection of hsv- , which is a representative dna virus classified into the herpesviridae family, was strongly affected by gemcitabine [ ] . most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. this is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as mers-cov, sars-cov, and zikv, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. in this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. importantly, it is noteworthy that zikv was the most strongly affected by gemcitabine, with a low nanomolar ec , which was lower than that used in cancer therapy [ , ] . even for other viruses with a relatively high ec , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. in this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. as an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few rna viruses, was reported against evs such as cvb and ev [ ] . as previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed hiv infectivity both in vitro and in vivo [ , ] . however, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models [ , ] . more extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. however, our group has recently reported that gemcitabine had an anti-ev effect by targeting the salvage pathway of pyrimidine biosynthesis [ ] . moreover, gemcitabine strongly induced the expression of several isgs including cxcl , irf , irf , ifit , and ddx , which were the major effectors in the innate immunity that defended the host against the virus infection. these results were consistent with a previous report that gemcitabine stimulated the production of ifn-β and ifn-γ in iav-infected rpe cells [ ] . importantly, the activation of isgs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. similar phenomena in terms of isg activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in table [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid (mpa) are inhibitors of inosine- -monophosphate (imp) dehydrogenase (impdh), which is a key enzyme of the purine biosynthesis pathway. these inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. both have antiviral activities against viruses such as hcv, hepatitis e virus (hev), mers-cov, dengue virus, yellow fever, hepatitis b virus, west nile virus (wnv), chikungunya virus (chikv), and iav [ ] [ ] [ ] [ ] [ ] [ ] [ ] , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against hcv and hev shown to involve the stimulation of isgs [ , ] . for the antiviral activity of ribavirin against hcv, ribavirin specifically induced the expression of irf , irf , and isg mrnas, which are known to be important for anti-hcv immune responses [ ] . isg activation occurred through an undefined mechanism that was different from the classical ifn signaling, intracellular dsrna sensing pathway, toll-like receptor and nuclear factor b pathways. more importantly, ribavirin-induced isg activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting impdh inhibition-mediated isg activation as an alternative innate immunity pathway. like ribavirin, mpa remarkably induced the expression of several isgs, including irf , irf , isg , ifi , irf , cxcl , ifit , and ifitm mrnas in naïve or hev-infected huh- cells, and the induction of isgs was at least partially abrogated by the use of supplemented guanosine [ ] . mechanistically, the induction of isgs by mpa was independent of the classical jak/stat system, which is similar to that observed with ribavirin [ ] . similar results were obtained with several impdh or impdh inhibitors, with various affinities, that were custom-designed and synthesized [ ] . as shown in table , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase (dhodh), an essential enzyme in de novo pyrimidine synthesis. lucas-hourani et al. identified dd as an interferon-sensitive response element (isre)-stimulating compound from high-throughput screening, and further analyses suggested that it was a dhodh inhibitor with a strong antiviral activity against various viruses including mev, chikv, and wnv [ ] . dd enhanced the expression of several isgs, which were almost completely suppressed by the addition of supplemented uridine, indicating dhodh inhibition-mediated isg activation. moreover, the antiviral activity of and isg activation by dd required the interferon regulatory factor (irf ) transcription factor, a master regulator of antiviral gene expression [ ] , which was consistent with the observation that the anti-hcv activity of mpa was partially mediated by irf [ ] . in this study, similar results were shown with brequinar, another well-known dhodh inhibitor. fa- is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting dhodh and inducing the expression of isgs such as ifnb , cxcl , isg , and ccl [ ] . however, whether isg activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. the mechanism of nucleotide synthesis inhibitor-induced isg activation is still presently unclear. nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced isg activation is independent of the classical jak/stat-mediated ifn signal [ , , ] . first, wang et al. clearly showed that isg activation and anti-hev activity induced by mpa or brequinar was not mediated by jak [ ] . second, irf induction by ribavirin was not affected by knockdown of stat , while that of ifn-α was strongly affected under the same conditions [ ] . third, our recent study with gemcitabine further confirmed ifn signal-independent isg activation by parallel studies comparing the effects of gemcitabine and ifn-α. in our study, the phosphorylation of stat at tyr , which was dramatically triggered by ifn-α, did not occur when treated with gemcitabine [ ] . moreover, the upregulation of ddx mrnas induced by gemcitabine was not affected by irf knockdown, which was contrary to the result that ifn-α-induced upregulation of ddx mrnas was significantly suppressed under the same conditions. consistent with above observations, there have been some reports that isgs was induced in the absence of jak or stat activation [ , ] . despite limited data, we speculate the scenario of isg activation that is independent of jak/stat-mediated ifn signal. purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as impdh and dhodh, leading to the depletion or imbalance of the (d)ntp pool. inactivation of metabolic enzyme(s) itself or consequently altered nucleos(t)ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of isgs, possibly through the relay of kinases and transcription factors. based on the previously mentioned reports, this signal is less likely to be dependent on stat / -irf (ifn-stimulated gene factor ; isgf ), at least for gemcitabine, which is the major transcriptional complex in the ifn-induced jak/stat pathway. it should also be considered that thomas et al. excluded the involvement of an intracellular double-stranded rna sensing pathway, toll-like receptor and nuclear factor κb pathways, as well as a classical ifn signal in the activation of isgs induced by ribavirin [ ] . despite the consensus of isg activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of isgs, at least with different patterns [ ] . targeting an enzyme in which pathways (purine or pyrimidine synthesis) or steps (early/late and de novo/salvage) produce different levels of intermediates and nucleos(t)ides will consequently result in diverse outcomes of isg activations. there might be more than one signaling pathway involved. the synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward isg activation. systematic analyses of signaling kinases, irfs, and stats using sirna knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos(t)ides should therefore clarify the underlying molecular mechanisms of isg activation by purine/pyrimidine biosynthesis inhibitors. as newly emerging or re-emerged viruses such as sars-cov, mers-cov, and zikv have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. in this regard, nucleoside analogs that directly target viral rna-dependent rna polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. nucleoside analogs probably induce different subsets of isgs, at least with a different pattern, leading to various combinations of isgs and resulting antiviral outcomes. moreover, according to schoggins et al., different viruses are affected by distinct subsets of isgs and some isgs such as irf , mb d , hpse, ddx , mda, and ifitm act broadly on various viruses [ ] . thus, more systematic analyses on the subsets of isgs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. given the clinical side effects of ifn treatment, nucleotide analogs that differ from ifn in the activation of subsets of isgs need to be considered as alternatives. nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered. advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases the purine path to chemotherapy vidarabine versus acyclovir therapy in herpes simplex encephalitis a controlled trial comparing continued zidovudine with didanosine in human immunodeficiency virus infection. the niaid aids clinical trials group sofosbuvir for previously untreated chronic hepatitis c infection ribavirin potentiates interferon action by augmenting interferon-stimulated gene induction in hepatitis c virus cell culture models the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase ribavirin inhibits in vitro hepatitis e virus replication through depletion of cellular gtp pools and is moderately synergistic with α interferon a novel potent inhibitor of inosinate dehydrogenase activity and guanylate biosynthesis cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis e virus replication evaluation of the antitumor activity of gemcitabine ( , -difluoro- -deoxycytidine) understanding ribonucleotide reductase inactivation by gemcitabine repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism cell-based high-throughput screening assay identifies , -difluoro- -deoxycytidine gemcitabine as a potential antipoliovirus agent obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection activity of a novel combined antiretroviral therapy of gemcitabine and decitabine in a mouse model for hiv- synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses cellular growth kinetics distinguish a cyclophilin inhibitor from an hsp inhibitor as a selective inhibitor of hepatitis c virus gemcitabine in leukemia: a phase i clinical, plasma, and cellular pharmacology study exploiting drug repositioning for discovery of a novel hiv combination therapy antiviral activity of gemcitabine against human rhinovirus in vitro and in vivo gemcitabine, a broad-spectrum antiviral drug, suppresses enterovirus infections through innate immunity induced by the inhibition of pyrimidine biosynthesis and nucleotide depletion mycophenolic acid, an immunomodulator, has potent and broad-spectrum in vitro antiviral activity against pandemic, seasonal and avian influenza viruses affecting humans identification of active antiviral compounds against a new york isolate of west nile virus cellular impdh enzyme activity is a potential target for the inhibition of chikungunya virus replication and virus induced apoptosis in cultured mammalian cells inhibition of dengue virus replication by mycophenolic acid and ribavirin ribavirin and mycophenolic acid markedly potentiate the anti-hepatitis b virus activity of entecavir calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis e virus mycophenolic acid augments interferon-stimulated gene expression and inhibits hepatitis c virus infection in vitro and in vivo antiviral activity of tiazofurin and mycophenolic acid against grapevine leafroll-associated virus in vitis vinifera explants effects of cyclosporine, azathioprine, mizoribine, and prednisolone on replication of human cytomegalovirus in vitro and in vivo activities of anti-influenza virus compound t- characterization of dengue virus resistance to brequinar in cell culture anti-hiv- activity of leflunomide: a comparison with mycophenolic acid and hydroxyurea effect of leflunomide and cidofovir on replication of bk virus in an in vitro culture system inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity broad-spectrum inhibition of common respiratory rna viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (rsv) inhibition of dengue virus through suppression of host pyrimidine biosynthesis d , a non-nucleoside inhibitor of influenza virus infection that interferes with de novo pyrimidine biosynthesis roles of jaks in activation of stats and stimulation of c-fos gene expression by epidermal growth factor critical roles for both stat -dependent and stat -independent pathways in the control of primary dengue virus infection in mice interferon-stimulated genes: roles in viral pathogenesis author contributions: hye jin shin, chonsaeng kim, and sungchan cho wrote the manuscript. the authors declare no conflict of interest. key: cord- -fzsczb m authors: rubio, nazario; de felipe, carmen title: demonstration of the presence of a specific interferon-γ receptor on murine astrocyte cell surface date: - - journal: journal of neuroimmunology doi: . / - ( ) - sha: doc_id: cord_uid: fzsczb m abstract interferon gamma (ifn-γ) is a pleiotropic lymphokine produced by t-lymphocytes which acts as a soluble mediator in immunological reactions. in addition to several immune target cells, such as monocytes and macrophages, it acts on the principal glial population, the astrocytes, inducing ia antigen expression. we have developed a binding assay for i-labeled recombinant murine ifn-γ, and show that, using this assay, ifn-γ interacts with a single specific receptor on the murine astrocyte cell membrane. the binding is specific and saturable and it takes place with a k d = . × − m, with , receptor molecules per astrocytic cell. the binding shows, as for macrophages, species specificity. using an immune assay including rabbit antibodies to ifn-γ and i-labeled protein a, we have demonstrated an internalization of the ligand. this is an energy-dependent process, as around % of the bound ifn-γ is endocytosed after h at °c when cultures are maintained in complete culture medium. a paradox exists in neuroimmunology as the central nervous system (cns) does not express class ii major histocompatibility complex (mhc) antigens (williams et al., ) and pathological immune reactions take place within the cns. this paradox can be resolved by the finding that interferon gamma (ifn- ) and some viral infections, such as those induced by murine coronavirus (suzumura et al., ) , theiler's murine encephalomyelitis virus (rodriguez et al., ) or neurotropic routine hepatitis virus (massa et al., ) can induce the expression of new antigens in astrocytes. several studies have shown that ifn-y, also known as immune interferon, induces the expression of class ii mhc antigens in macrophages (steeg et al., ) and astrocytes (hirsch et al., ; wong et al., ) . the fact that a t-lymphocyte lymphokine produces such a striking effect on glial cells may have high functional significance. it has been demonstrated that ifn-y induces la antigen expression on astrocytes (fierz et al., ) and that astrocytes activated in this manner can present antigens such as myelin basic protein to tlymphocytes (fontana et al., ) . these autoaggressive t-cells can lysc presenting astrocytes (sun and wekerle, ) . conversely, astrocytes do not produce ifn- , even when subjected to a superinduction regimen (tedeschi et al., ) . we have undertaken the study of the interaction between recombinant ifn-t and its receptor on the murine astrocyte cellular membrane from a physicochemical point of view. here we describe in detail the binding characteristics of this important neuroimmunological reaction. recombinant mouse gamma interferon, free of protein stabilizers was purchased from holland biotechnology, leiden, the netherlands and was labeled with j i (amersham international, u.k.) utilizing the enzymobead iodination lactoperoxidase reagent (bio-rad, richmond, ca, u.s.a.) (marchalonis, ) . the ~ i-rifn- and free iodine were separated on a disposable pd- sephadex g- m column (pharmacia, uppsala, sweden). the specific activity obtained was ci/mmol and was % trichloroacetic acid (tca) precipitable. the iodinated rifn-t migrated mostly as a single peak with an apparent molecular weight of , when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis slabs. protein a from staphylococcus aureus (sigma chemical co., st. louis, mo, u.s.a.) was labeled by the chloramine t method (greenwood et al., ) . astrocyte cultures were prepared by mechanical dissociation of the cerebral cortex from newborn wistar rats or sjl/j mice. the cortex was isolated under a dissecting microscope and carefully cleaned of choroid plexus and meninges. cell suspensions were filtered through /zm pore size mesh into dulbecco's modified eagle medium (dmem) containing % fetal calf serum (fcs) and gentamicin (flow laboratories, u.k.). after centrifugation, cells were filtered through a #m mesh sieve, plated in cm -~ tissue culture flasks (costar, cambridge, ma, u.s.a.) and cultured at ° c. the medium was changed after days of culture and, subsequently, times a week for the entire culture period. cultures were enriched in astrocytes by removal of less adherent oligodendrocytes by shaking for h at ° c and rpm in an orbital shaker. cellular confluence was observed days after plating with a polygonal fiat cell morphology. the content of a mean of . % astrocytes was confirmed by indirect immunofluorescence staining of methanol-fixed cultures using rabbit anti-gliai fibrillary acidic protein (gfap) antiserum (dakopatts, denmark) and fluorescein-labeled goat anti-rabbit lgg (miles laboratories, elkhart, in, u.s.a.). the lack of noticeable oligodendrocytcs and microglial/macrophage cells was determined by using a guinea pig anti-myelin basic protein (mbp) antiserum prepared by ourselves (rubio and cuesta, ) and a monoclonal anti-mac- antibody (serotec, oxford, u.k.). secondary fluorescein-labeled antibody against guinea pig and rat igg were purchased from sigma. the rat astrocytoma c- was obtained from the american type culture collection, u.s.a., and cultured in dmem supplemented with % fcs and gentamicin. confluent astrocyte monolayers in mm diameter -well plates (costar) were used in the binding experiments. ceils were incubated at different temperatures, times and with or without unlabeled ifn- , as stated in the text. the buffer used was phosphate-buffered saline (pbs) containing . % bovine serum albumin (bsa) from sigma. after washing times with the above buffer, cells were detached from the plastic surface with n naoh at °c and counted in an lkb-wallac compugamma counter. monolayers of astrocytes in mm diameter -well plates were incubated with a saturating amount of unlabeled ifn- . binding and endocy-tosis were allowed to take place at °c or °c for different periods of time. after two washes with pbs-bsa to remove free ifn- , cells were fixed for rain at room temperature in freshly prepared % formaldehyde in pbs. after cells were washed, they were incubated with a rabbit anti-murine ifn-y antiserum (kindly provided by dr. robert c. curry, bristol-myers, wallingford, ct, u.s.a.) diluted : , at °c for min. after two further washes, the antibody bound to ifn-y available on the astrocytes surface was detected by incubation with radioiodinated staphylococcal protein a ( , cpm, min, ° c). after washing, cells were detached with n naoh and counted. to assess total internalized ligand, we removed surface-bound i-ifn-y from twice-washed cells with ml of . m nac - mm glycine, ph . at °c for min (costlow and hample, ) . after two more washes with pbs, cells were detached and counted in the gamma counter. scatchard and hill plot analysis of the data from binding assays was performed employing the ligand program (munson and rodbard, ) . the data shown represent mean results of three reproducible experiments + standard deviations. fig. shows the kinetics of i-labeled ifn- binding to pure astrocyte cultures. care has been exercised to use only cultures containing around % antrocytes, free of oligodendrocytes or microglial/macrophage cells according to criteria explained under materials and methods. saturation of the binding capacity of × astrocytic cells takes place in min at ° c. at ° c, a min incubation period is required to reach maximal binding. maximal binding is found to be around % of the input, which is kept constant in , cpm per well in this work. to demonstrate that the binding shown in fig. is specific, labeled ifn- was allowed to bind cells in the presence of a -fold excess of the unlabeled recombinant molecule. the saturation of specific receptor sites is depicted in fig. , and shows that the binding of ifn-t to cells is inhibited by the presence of a constant amount of unlabeled ligand. if specific binding is defined as that binding which is inhibited in the presence of an excess of 'cold' ifn-y, it is approximately % in this system. when these data are analyzed by the li-gand program, a scatchard plot (scatchard, ) is obtained showing a k d of . × - m and a bma x of . × - m (fig. , inset) . the number of receptor molecules per cell is calculated to be , . the hill plot shows that the astrocyte surface receptor binds ifn-y in a homogenous, noncooperative manner. halfsaturation is achieved at a concentration of free ligand of about pmol ( . × - m) that is in the range of the k d reported. different numbers of mouse astrocytes were allowed to attach to mm diameter wells and incubated with , cpm of ifn-y. as shown in fig. , the binding reaches a plateau when - x cells are plated per well. this indicates that the binding is receptor-specific and cell number-dependent. as described by other authors in studies about ifn-y receptor in macrophages, rat cells do not recognize murine ifn-y (celada et al., ) . wistar rat astrocytes do not bind any ligand during the time of incubation used by us (fig. ) . c- cells, an astrocytoma cell line of rat origin, do not either show any binding (not shown). these data confirm the species specificity of the receptor molecule, present also in astrocytes. the above results provide direct evidence for high affinity binding of 'immune' interferon to a specific cell surface receptor on murine astro- the use of a rabbit antibody against murine ifn-t and i-labeled protein a allows us to study the availability of the leukin on the astrocyte surface. when cell receptor-bound unlabeled ifn-t remains at °c in complete culture medium (dmem + % fcs) it begins to be internalized after min. after h only % of the maximal amount of bound ifn-t remains available to the specific antibody plus protein a complex. if the binding and further incubation are performed at °c, internalization does not occur (fig. ) . no endocytosis takes place when the complete culture medium is substituted by pbs. we assume that the detection of lower antibody binding is due to an energy-dependent in- ternalization process rather than to a release from the cell receptor, because of the high affinity of the binding (k a = . × - m) and be- causc the hypothetic release does not take place either at °c or in pbs at °c. these data demonstrated an energy-dependent internalization of ifn-,/ by astrocytes, as described by others for macrophages (anderson eta[., ) . to further demonstrate internalization of the ligand, we assessed the amount of intracellular radioactivity after removing surface-bound inf-t at low ph (glycine buffer, ph . ). table shows that at °c no internalization takes place in a significant manner when the time of incubation is increased. conversely, at °c the ph . -resistant label (internalized) increases, reaching _+ . % of maximal binding after min of incubation. the amount of cpm bound after min at °c is defined as % of binding. interferon gamma is a lymphokine with a wide array of biological effects on immune and nonimmune cells (trinchieri and perussia, ) . these effects are thought to be mediated by a cell surface receptor (aguet, ) which does not bind ifn-a or ifn-/ (branca and baglioni, ) . the human ifn-y receptor has been purified and characterized from a biochemical point of view showing a molecular weight of around (i, (novick et al., (novick et al., , . monoclonal antibodies with the ability to block the binding of i i-labeled ifn-,/to its receptor have been raised against it (novick et al., ; depla and de ley, ) . finally, the gene encoding the human ifnreceptor has recently been cloned (aguet et al., ) . in addition to the macrophages, its main target cell (schreiber et al., ) , glial astrocytes have been reported to be committed to an important immune role when in contact with ifn-y (hirsch et al., ; wong et al., ) . our results clearly demonstrate the presence of a specific receptor for ifn--/ on murine astrocytes. the binding takes place in a bimolecular reversible manner with high affinity, k~ = . × . m. this affinity constant is higher than that found for rlfn-y on human fibroblasts ( - × -s m) (anderson et al., ) and mouse macrophages ( . × -s m) (celada et al., ) and lower than that rcportcd for human monocytes ( - × ' m) (finbloom et al., ) . the number of binding sites per cell reported here ( , sites/cell) is similar to the number found for murine macrophages (-~ , ) (celada et al., ) and human fibroblasts (anderson et al., ) and somewhat higher than that observed on human monocytes ( sites/cell) (finbloom et al., ) . the binding is specific and saturable (fig. ) . it is also species-specific, as wistar rat astrocytes and the rat astrocytoma cell line c- do not bind murine ifn-y, maintaining the species specificity found in macrophages. there is a somewhat paradoxical situation concerning the apparent lack of specific binding of mouse ifn-y to rat cells and the biological effects detected in rat astrocyte cultures (fiertz et al., ) . this could be explained by the presence of a low affinity receptor only noticeable after very long periods of incubation (celada et al., ) . the human fibroblast receptor for ifn-y has been reported to be internalized at °c (anderson et al., ) , we have observed the same phenomenon in an immune assay set up with specific antibodies and protein a labeled with ~-~-si. the endocytosis of ifn-y bound to the cell surface receptor takes place at °c but not at °c and a rich culture medium (dmem + % fcs) is required, probably to maintain an active metabolic state in the cultured astrocytes. further demonstration of internalization by an energy-dependent process is provided for the direct measure of the intracellular labeled ligand which takes place at °c but not at °c. high affinity binding of l-' l-labelled mouse interferon to a specific cell surface receptor molecular cloning and expression of the human interferon-y receptor specific binding of -human interferon-y to high affinity receptors on human fibroblasts human interferon-y is internalized and degraded by cultured fibroblasts evidence that type i and ii interferons have different receptors evidence for a gamma interferon receptor that regulates macrophage tumoricidal activity prolactin receptors in cultured rat mammary tumor cells. energy-dependent uptake and degradation of hormone receptors monoclonal antibodies against the human interferon-y receptor(s). mol. immunol astrocytes as antigen-presenting cells. i. induction of la antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation the characteristics of binding of human recombinant interferon-y to its receptor on human monocytes and human monocyte-like cell lines astrocytes present myelin basic protein to encephalitogenic t-cell lines the preparation of i-labeled human growth hormone of high specific radioactivity expression of la antigens by cultured astrocytes treated with gamma-interferon an enzymic method for the trace iodination of immunoglobulins and other proteins viral particles induce la antigen expression on astrocytes ligand: a versatile computerized approach for characterization of ligandbinding systems the human interferon-y receptor. purification, characterization and preparation of antibodies monoclonal antibodies to the human interferon-y receptor: blocking of the biological activities of interferon-y and purification of the receptor immune response gene products (ia antigens) on glial and endothelial cells in virus induced demyelination lack of cross-reaction between myelin basic proteins and putative demyelinating virus envelope proteins the attraction of proteins for small molecules and ions monoclonal antibodies to murine y-interferon which differentially modulate activation and antiviral activity regulation of murine macrophage la antigen expression by lymphokine with immune interferon activity ia-restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes astrocytes produce interferon that enhances the expression of h- antigens on a subpopulation of brain cells immune interferon: a pleiotropic lymphokine with multiple effects distribution and quantitation of hla-abc and dr (ia) antigens on human kidney and other tissues interferongamma induces the expression of h- and la antigens on brain cells key: cord- -lbikoyo authors: beidas, meshal; chehadeh, wassim title: effect of human coronavirus oc structural and accessory proteins on the transcriptional activation of antiviral response elements date: - - journal: intervirology doi: . / sha: doc_id: cord_uid: lbikoyo objectives: the molecular mechanisms underlying the pathogenesis of human coronavirus oc (hcov-oc ) infection are poorly understood. in this study, we investigated the ability of hcov-oc to antagonize the transcriptional activation of antiviral response elements. methods: hcov-oc structural (membrane m and nucleocapsid n) and accessory proteins (ns a and ns a) were expressed individually in human embryonic kidney (hek- ) cells. the transcriptional activation of antiviral response elements was assessed by measuring the levels of firefly luciferase expressed under the control of interferon (ifn)-stimulated response element (isre), ifn-β promoter, or nuclear factor kappa b response element (nf-κb-re). the antiviral gene expression profile in hek- cells was determined by pcr array. results: the transcriptional activity of isre, ifn-β promoter, and nf-κb-re was significantly reduced in the presence of hcov-oc ns a, ns a, m, or n protein, following the challenge of cells with sendai virus, ifn-α or tumor necrosis factor-α. the expression of antiviral genes involved in the type i ifn and nf-κb signaling pathways was also downregulated in the presence of hcov-oc structural or accessory proteins. conclusion: both structural and accessory hcov-oc proteins are able to inhibit antiviral response elements in hek- cells, and to block the activation of different antiviral signaling pathways. human coronavirus oc (hcov-oc ) is an enveloped, positive-sense rna virus classified as a betacoronavirus, the same genus as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses. hcov-oc infection has been associated mainly with upper respiratory tract symptoms and exacerbation of asthma and pneumonia in some groups and institutional settings [ ] [ ] [ ] [ ] . the virus has also been associated with severe neurological disorders like acute disseminated encephalomyelitis in children [ , ] . while most studies have focused their attention on the immunopathology of sars-cov and mers-cov, there has not been the same interest for hcov-oc . indeed, there are few studies concerned with the molecular mech-intervirology ; : - doi: . / anisms governing hcov-oc infection and its effect on the intracellular host defenses. hcov-oc is over kb in length, and similarly to the other coronaviruses, it is composed of the structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n) [ ] . uniquely, there are two accessory proteins interspaced between these structural proteins called ns a and ns a [ ] . these proteins are not essential for replication; however, they might play a role in the pathogenesis of coronavirus infection [ ] . the interferon (ifn) induction and signaling pathway is the major branch in the innate immune response against viruses. the induction of type i ifn is initiated by molecular sensing of viral rna by pattern recognition receptors such as tlr, rig-i, and mda [ ] . the adaptor protein mavs mediates the signals from rig-i and mda to activate the kinases tbk and ikkε, which in turn phosphorylate irf and irf transcription factors [ ] . irf and irf dimerize to undergo nuclear translocation and initiate the transcription of type i ifn. the adaptor proteins myd and trif mediate signals from tlrs and activate irak , traf , and the aforementioned kinases [ ] . the irfs along with nuclear factor kappa b (nf-κb) can then be phosphorylated to translocate into the nucleus and establish the transcription of type i ifn by binding cognate sites on the ifn-β promoter [ ] . nf-κb specifically binds to the nf-κb response element (nf-κb-re) to regulate the expression of pro-inflammatory and cell survival genes [ ] . ifn signaling via the jak/stat pathway is established when type i ifn binds to the ifn receptor. this leads to activation of the kinases jak and tyk . these kinases in turn phosphorylate the transcription factors stat and stat [ , ] . phosphorylated stat and stat then dimerize and undergo nuclear translocation, where they form a complex with irf called the isgf . this complex will bind the ifn-stimulated response element (isre) and initiate the transcription of isgs. these isgs assist the cell in counteracting the viral infection by establishing an antiviral state [ ] . sars-cov and mers-cov were shown to inhibit the transcriptional activity of isre, ifn-β promoter or nf-κb-re [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the effect of different hcov-oc proteins on the transcriptional activation of antiviral response elements has not yet been investigated. in this study, the ability of hcov-oc structural (m and n) and accessory proteins (ns a and ns a) to antagonize the transcriptional activation of isre, ifn-β promoter, and nf-κb-re was investigated. human embryonic kidney (hek- ) cells (american type culture collection atcc, manassas, va, usa) were cultured in cm tissue culture flasks in dulbecco's minimal essential medium containing glutamax tm (life technologies corporation tm , grand island, ny, usa). the medium was supplemented with % fetal bovine serum, fungizone ® ( µg/ml), penicillin g ( , u/ml), and streptomycin sulfate ( , pg/ ml) (life technologies tm ). monolayers of hek- cell culture flasks were incubated at ° c in the presence of % carbon dioxide (co ) and % humidity. the qiaamp ® viral rna minikit (qiagen tm gmbh, hilden, germany) was used to isolate rna from hcov-oc (atcc vr- ) according to the manufacturer's instructions. the hcov-oc ns a, ns a, m, and n genes were amplified by a twostep reverse transcription-polymerase chain reaction (rt-pcr) using the geneamp ® rna core kit (applied biosystems tm , foster city, ca, usa), and pmol of previously described primers [ ] on geneamp ® pcr system (applied biosystems tm ). the rt reaction conditions were: annealing at ° c for min, denaturation at ° c for min, and cooling at ° c. thereafter, the cdna was amplified by pcr using the following conditions: denaturation at ° c for min, then cycles of denaturation at ° c for s, annealing at ° c for s, and extension at ° c for s, followed by a final extension step at ° c for min, and cooling at ° c. pcr products were run on agarose gel and the bands that were size-specific for the amplified gene of interest, were cut and purified by wizard ® sv gel and pcr clean up system (promega tm , madison, wi, usa) according to the manufacturer's instructions. rna of influenza a virus (h n , a/hong kong/ / strain; atcc vr- ) was also isolated and used to amplify ns gene by rt-pcr using ns -specific primers [ ] . influenza a ns protein was used in the following experiments as inhibitor of the innate immune host response [ , ] . rt-pcr products were cloned using the pacgfp -n in-fusion ® ready vector (clontech tm , takara bio company, mountain view, ca, usa) that encodes a green fluorescent protein (gfp) from aequorea coerulescens allowing the expression of the fusion protein to the n-terminus of acgfp . the ligation reaction was set up by utilizing the in-fusion ® hd cloning kit (clontech tm ). competent top escherichia coli cells (invitrogen tm , carlsbad, ca, usa) were used for transformation. the pureyield tm plasmid miniprep kit (promega tm ) was used to isolate the vector according to the manufacturer's instructions. confirmation of successful cloning was achieved using restriction digestion and direct sequencing (data not shown). hek- cells were seeded at × per well of -well plates, and transfected with ns a-pacgfp , ns a-pacgfp , m-pacgfp , n-pacgfp , or ns -pacgfp vector using lipofectamine ® (promega tm ) according to the manufacturer's instructions. expression of ns a, ns a, m, n and ns proteins was confirmed by indirect immunofluorescence assay using anti-gfp monoclonal antibody ( μg/ml) (clone jl- , clontech tm ) (data not shown). expression of n and ns proteins was further confirmed by immunofluorescence assay using monoclonal antibody against hcov-oc n (emd millipore tm , billerica, ma, usa) and polyclonal antibody against influenza a ns protein (emd millipore tm ), respectively (data not shown). the ifn-β promoter was amplified using previously described primers [ ] , and inserted into the reporter pgl . (luc cp/ne) vector (promega tm ) between the restriction sites bglii and hindiii according to the manufacturer's instructions, creating the pifnβluc plasmid. the reporter vectors coding for firefly luciferase under the control of isre, pisre-luc (pgl . [luc p/isre/hygro]) or nf-κb-re, pnfκb-re-luc (pgl . [luc p/nf-κb-re/hygro]) were purchased from promega tm . following h of transfection with one of the expression vectors cited above, hek- cells were co-transfected with pifnβ-luc, pisre-luc or pnfκb-re-luc vector at . μg/well using lipofectamine ® (promega tm ). renilla luciferase vector (prl-tk) (promega tm ) was used at ng/ well as an internal control for transfection efficiency. hcov-oc ns a, ns a, m, and n protein total rna from transfected and mock-transfected hek- cells was extracted using rneasy ® kit (qiagen tm ) with on-column dnase digestion according to the manufacturer's instructions. the rt first strand kit (qiagen tm ) was used for cdna synthesis. the rt reaction was then added to rt sybr green rox qpcr mastermix (qiagen tm ), and the human antiviral response pcr array (qiagen tm ) was used to profile the antiviral gene expression in hek- cells on abi fast real-time pcr system (applied biosystems tm ). the pcr array monitored the transcriptional activity of different genes involved in the activation of antiviral and pro-inflammatory proteins following amplification of rna transcripts by real-time rt-pcr. however, we only profiled the expression of genes relevant to the antiviral response elements. cycle threshold (c t ) values were exported to an excel file to create a table of c t values. this table was then uploaded on to the qiagen data analysis web portal (http://www.qiagen.com/geneglobe). c t values were normalized to glyceraldehyde -phosphate dehydrogenase. fold regulation comparison was used to profile antiviral gene expression. fold change/regulation was calculated using the delta-delta c t method (∆∆c t ). the fold regulation threshold was ≥ for upregulation and ≤- for downregulation. fold change in firefly luciferase from three different experiments was summarized as mean ± standard deviation. the difference in fold change mean between two groups was determined by independent-samples t test. p values < . were considered sig-nificant. all statistical analyses were performed using spss statistics software version . for windows (ibm corporation, armonk, ny, usa). graphpad prism software (graphpad software inc., la jolla, ca, usa) was used to generate all charts. effect of hcov-oc proteins on the transcriptional activation of antiviral response elements. the transcriptional activity of isre, ifn-β promoter, and nf-κb-re was assessed by measuring the firefly luciferase levels in hek- cells transfected with ns a-pacgfp , ns a-pacgfp , m-pacgfp , n-pacgfp , or ns -pacgfp vector. in control cells nonexpressing viral protein, the firefly luciferase levels were highest following h of stimulation with one of the inducers (data not shown). in the absence of an inducer, the firefly luciferase levels in hek- cells expressing one hcov-oc protein were similar to the background levels ( fig. a, b) . in ifn-α-treated hek- cells, the expression of firefly luciferase under the control of isre or ifn-β promoter was inhibited in the presence of one of the tested hcov-oc proteins or in the presence of influenza a ns protein (fig. a, ) . the transcriptional activity of isre was also inhibited in sev-stimulated hek- cells in the presence of one hcov-oc protein (fig. b) . in mock-transfected hek- cells, sev induced only low levels of firefly luciferase under the control of ifn-β promoter (data not shown), and therefore the effect of hcov-oc proteins on the transcriptional activity of ifn-β promoter in sev-stimulated hek- cells could not be assessed. following the challenge with tnf-α, the transcriptional activity of nf-κb-re was inhibited in the presence of one of the tested hcov-oc proteins or in the presence of influenza a ns protein (fig. ) . to investigate whether the inhibition of the transcriptional activity of antiviral response elements in the presence of hcov-oc proteins can be associated with a downregulation of the expression of antiviral genes, pcr array profiling of antiviral genes was carried out in trans-fected and mock-transfected hek- cells. following sev challenge of hek- cells, the expression of genes involved in the type i ifn and nf-κb signaling pathways was downregulated in the presence of hcov-oc structural or accessory proteins (fig. ) . similar results were obtained in the presence of the influenza a ns protein. the mean negative fold change for tradd gene in the presence of accessory protein ns a was greater than in the presence of other hcov-oc proteins (p < . ). the expression of irf gene was more downregulated in the presence of ns a or n protein than in the presence of other hcov-oc proteins (p < . ), whereas the expression of tlr gene was more downregulated in the presence of ns a protein than in the presence of other hcov-oc proteins (p < . ). remarkably, irf and ifna gene expression was more downregulated in the presence of ns a, ns a or n protein than in the presence of influenza a ns protein. similar to influenza a ns protein, hcov-oc structural (m and n) and accessory (ns a and ns a) proteins were able to inhibit the transcriptional activity of antiviral response elements, isre, ifn-β promoter, and nf-κb-re, and to downregulate the expression of several genes involved in the activation of an antiviral response. the inhibition of the isre, ifn-β promoter, and nf-κb-re activity, and the downregulation of the expression of ifna , irf , tlr , myd , tradd, and mavs in the presence of each hcov-oc accessory protein suggest that hcov-oc ns a and ns a have the potential to block the type i ifn and nf-κb pathways. previous studies have shown that sars-cov and mers-cov accessory proteins have a role in innate immune evasion [ , , [ ] [ ] [ ] . however, hcov-oc accessory proteins are not homologous to sars-cov and mers-cov accessory proteins. also of interest, the murine coronavirus accessory protein a, which is homologous to hcov-oc ns a protein, was shown to antagonize ifn induction [ ] . sars-cov and mers-cov m proteins can suppress the activity of ifn-β promoter and isre [ , , ] . nf-κb activation is also inhibited when sars-cov m protein is expressed in vero or hela cells [ ] . our data showed that the transcriptional activity of isre, ifn-β promoter, and nf-κb-re was inhibited in the presence of hcov-oc m protein. in addition, like the accessory proteins, the hcov-oc m protein was able to downregulate the expression of ifna , irf , tlr , myd , tradd, and mavs. these findings suggest that in addition to its function in the assembly of virions [ ] , hcov-oc m protein is an antagonist of the host antiviral defenses. the coronavirus n protein binds genomic rna to form the helical capsid, and has a critical role in coronavirus replication [ , ] . the sars-cov n protein can activate nf-κb-re in vero e cells [ ] . however, sars-cov n protein inhibits the promoter activity of isre, ifn-β promoter, and nf-κb-re in t cells [ ] . our study showed that the transcriptional activity of isre, ifn-β promoter, and nf-κb-re was inhibited in the presence of hcov-oc n protein. lai et al. [ ] showed that after h of tnf-α treatment, hcov-oc n protein potentiates nf-κb expression in t cells by binding its inhibitor, mirna . mirna expression is induced by stimuli which activate nf-κb, such as tnf-α and lipopolysaccharide [ ] . unfortunately, the authors did not show results for nf-κb-re activation after h of tnf-α treatment, the optimal time for nf-κb-re activation in our experiments. the influenza a virus that is known to inhibit irf , ap- , and nf-κb activation [ , ] was shown to upregulate the mirna expression [ ] . we speculate that the expression of n protein results in transient downregulation of nf-κb expression through downregulation of its mediators (e.g., tlr , myd , tradd), leading to early inhibition of nf-Κb-re activation, and that the accumulation of mirna in cells with time will lead to the formation of n protein/mirna complex and the removal of inhibition on nf-kb and nf-kb-re. in conclusion, hcov-oc has the ability to inhibit the transcriptional activation 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replication by microrna- through targeting mcpip this work was in part supported by kuwait university research administration grant no. ym / . the authors report no conflicts of interest. key: cord- -erzpz c authors: downey, jeffrey; pernet, erwan; coulombe, françois; divangahi, maziar title: dissecting host cell death programs in the pathogenesis of influenza date: - - journal: microbes infect doi: . /j.micinf. . . sha: doc_id: cord_uid: erzpz c influenza a virus (iav) is a pulmonary pathogen, responsible for significant yearly morbidity and mortality. due to the absence of highly effective antiviral therapies and vaccine, as well as the constant threat of an emerging pandemic strain, there is considerable need to better understand the host–pathogen interactions and the factors that dictate a protective versus detrimental immune response to iav. even though evidence of iav-induced cell death in human pulmonary epithelial and immune cells has been observed for almost a century, very little is known about the consequences of cell death on viral pathogenesis. recent study indicates that both the type of cell death program and its kinetics have major implications on host defense and survival. in this review, we discuss advances in our understanding of cell death programs during influenza virus infection, in hopes of fostering new areas of investigation for targeted clinical intervention. influenza viruses cause the most recurring respiratory disease in humans, with outbreaks likely occurring since at least the middle ages [ ] . present estimates indicate that each year, seasonal influenza affects e % of the world's population, resulting in e million cases of severe illness and between to deaths [ ] . in addition to seasonal outbreaks, influenza pandemics sporadically emerge, causing markedly higher mortality. in , the worst pandemic in recorded history caused more than million deaths worldwide [ ] . since then, three other pandemics have occurred: first in , then again in and finally in [ ] . currently, there are major concerns surrounding the emergence of the new highly pathogenic avian influenza (hpai) viruses of h n , h n , h n , or the more recent h n dthe only hpai strain to cross from eurasia to america [ e ]. one of the key characteristics of highly infectious iav and hpai viruses is that they are poorly glycosylated and, thus, able to bypass the upper airway defense mechanisms to reach the lower respiratory tract, leading to pneumonia [ ] . histopathologic studies of influenza autopsies from outline the key changes characteristic of severe iav viral pneumonia. these include "… varying degrees of acute intra-alveolar edema and/or hemorrhage, and diffuse alveolar damage in addition to necrotizing bronchitis and bronchiolitis" [ ] . similarly, in the recent h n outbreak, viral antigen could be found in the alveolar space, suggesting pandemic strains of iav have the capacity to infect distal areas of the lung and directly cause alveolar damage. these occurrences collectively predispose to a clinical condition termed acute respiratory distress syndrome (ards), which may occur as a complication of primary viral pneumonia [ ] . ards is precipitated by cell death among the epithelialeendothelial barrier (eeb) of the distal lung, barrier rupture, fluid leakage into the alveolar lumen and respiratory insufficiency. hence, understanding and disrupting the strategies employed by the virus to damage the lower airways is critical in preserving pulmonary architecture. the most distal structure of the lung, the alveolus, is constantly patrolled by alveolar macrophages (am ) and is delimited by the eebda bi-cellular layer composed of type i and type ii alveolar epithelial cells (aec-i and aec-ii), facing the air space, and endothelial cells that form the pulmonary capillaries. the identification of the infective target cells of severe pulmonary viruses within the alveolus is critical in evaluating the potential effects of cell damage on lung function. for example, ex vivo infection of human lungs with middle east respiratory syndrome coronavirus (mers-cov)d a recent zoonotic virus with a fatality rate of e % in humansdshowed that aec-i, aec-ii and endothelial cells can all be infected and killed [ e ] . in addition, while mers-cov productively replicates in human macrophages and t lymphocytes, it is also cytotoxic in these cells [ , ] . interestingly, the tropism of the virus appears to have a significant impact on severity of disease. for instance, in comparison to mers-cov that infects both structural cells and leukocytes and causes high mortality, severe acute respiratory syndrome (sars)-cov only infects structural cells, causing less mortality [ ] . in iav infection, a number of reports identify aec-ii as the primary replicative niche in the human lung for highly pathogenic strains, while low-pathogenicity strains fail to penetrate the lower airways [ e ] . hpai also infects human endothelial cells in vitro and some evidence suggests that infection of the endothelium may occur in vivo [ , ] . in addition, iav antigens are found in human am and the virus can productively infect and kill human macrophages [ , , , , , e ] . thus, similar to the highly pathogenic mers-cov, highly virulent iav shows tropism for both human alveolar structural cells and leukocytes, highlighting the severity of pandemic strains. am within the airways are the first leukocytes to encounter the majority of pulmonary pathogens and their initial response to infection is critical in "setting the tone" and orchestrating an effective immune response. studies examining how am are altered following pulmonary viral infection suggest that the loss of eeb integrity, reduction in regulatory mediators and activation of multiple pattern-recognition receptors (prr) lead to these cells becoming pro-inflammatory, rather than regulatory [ ] . upon infection with iav, am initiate the immune response by producing chemokines and cytokines, including type i interferons (ifn-i; primarily composed of ifn-a/b), as well as instructing adaptive immune responses [ , ] . if adequately regulated, these responses lead to effective immunity, clearance of the virus with minimal immunopathology and return to pulmonary homeostasis. if dysregulated, however, these responses can have dramatically deleterious effects, marked by impaired viral clearance, increased immunopathology and enhanced susceptibility to infection. thus, pulmonary immune responses must be tightly regulated to effectively eliminate viruses that reach the distal airways, while minimizing immunopathology in order to maintain optimal gas exchange [ ] . the death of host cells as a result of infection has been recognized for over a century and it is now understood that pathogeninduced cell death may occur through a variety of complex mechanisms. initially, cell death was discussed in dichotomy as either: ( ) apoptosis, considered an activedor programmeddprocess that preserves the plasma membrane and leads to the deletion of cells with little tissue disruption and no inflammation; or ( ) necrosis, a passive form of cell death leading to complete disruption of the plasma membrane and spillage of cellular contents, causing inflammation and tissue damage [ ] . however, this simplification of the cell death program has altered considerably over the last couple of decades. there are now several distinct forms of programmed cell death that have been described in addition to apoptosis, including pyroptosis, paraptosis and necroptosis [ ] . interestingly, some of these cell death modalities have been observed in autopsies of iav-infected humans, particularly hpai cases [ , ] ; yet, the consequences of these cell death programs on host immunity and the pathogenesis of iav are not fully understood. therefore, a more complete characterization of the forms of cell death occurring in iav infected lungs may define why certain cell types are more, or less, susceptible to iav-induced cell death and how this impacts viral propagation. furthermore, different forms of cell death may have distinct consequences on the virulence of iav strains by differentially modulating the immune response. this suggests that regulated modulation of cell death programs during iav infection may be an appealing target for anti-iav therapies. in this review, we focus on the current state of knowledge of cell death during influenza infection and explore research avenues that aim to dissect cell death programs, as well as their ensuing consequences in the pathogenesis of this threatening virus. considering the mechanisms of cell death have been extensively reviewed elsewhere [ , ] , herein we focus on severe influenza infection and the immunological effects of influenza-induced cell death in the lung structural cells (e.g. epithelial cells) as well as immune cells (e.g. macrophages). finally, we suggest that manipulation of specific cell death programs at different stages of infection may offer avenues for novel therapy to iav by both sequestering viral replication and promoting host resistance mechanisms through immunomodulation. iav utilizes epithelial cells as its primary replicative site and all pulmonary epithelial cells are permissive to infection and subsequent replication. however, proximity of the replication to distal portions of the lung is strongly associated with an increase in virulence capacity of iav strains. for example, seasonal influenza strains, or those of low pathogenicity, replicate primarily in the upper airways and are rapidly killed in the pharynx and proximal lung by host resistance mechanisms, causing minor acute disease. on the contrary, hpai and highly pathogenic h n strains bypass immune mechanisms of the upper airways to replicate in the distal portions of the lung and this distal tropism is highly correlated with primary viral pneumonia and mortality [ ] . altered tropism of iav to the lower airways is thought to be mediated by two mechanisms. first, iav infects cells by its hemagglutinin (ha) binding to terminal sialic acids on the plasma membrane of cells, attached either via an a , or a , linkage. both a , and a , linkages are present throughout the human lung; however, preferential binding of a , linked residues by iav hemagglutinin favors more distal and severe infections. second, glycosylation of iav hemagglutinin is inversely correlated with pathogenicity, as highly glycosylated hemagglutinins are more efficiently trapped and cleared from the upper airways by ciliary motion [ ] , while poorly glycosylated iav bypass this host defense mechanism and access the lower airways [ ] . for instance, the pandemic h n strain exhibited only glycosylation site on its hemagglutinin protein and hpai strains are known to preferably bind a , residues, due to their enhanced expression on avian epithelial cells. in the lower airways, it was shown in a human lung explants model that approximately % of influenza positive cells are aec-ii cells, with a minor contribution of am and other cell types [ ] . interestingly, iav replication is enhanced in aec-ii compared to bronchial epithelial cells [ ] . therefore, highly pathogenic strains of iav have mechanisms to reach their preferred replicative niche in aec-ii in the distal lung, suggesting that maintenance of pulmonary epithelial cells is beneficial to the virus in promoting its replication. however, considering that iav is also lytic in epithelial cells of humans [ ] and mice [ ] hints that at certain time points after infection, cell death may be involved in preventing or facilitating viral propagation, escape and reinfection. therefore, the consequences of cell death in structural cells, and in particular aec-ii, on host immunity to iav must diverge depending on the kinetics of the infection. apoptosis comes from the ancient greek word meaning "falling off" and is the most characterized form of programmed cell death. proceeding in a non-inflammatory manner, apoptosis likely evolved to meet the growing demand to remove aging or damaged cells from the body as more complex and multicellular organisms appeared and, as a result, the apoptotic machinery remains evolutionarily conserved. indeed, at homeostasis in an adult human, it is estimated that between and billion cells are being cleared daily via apoptosis [ ] . this number can then augment dramatically in developmental stages and aging. thus, the physiological demand for cellular turnover in a non-inflammatory manner is essential to maintain homeostasis. the signature of early apoptosis involves condensing of chromatin within the nucleus, compartmentalization of cellular organelles and the shrinking of the cell membrane. ultimately, these compartments bleb off in the form of apoptotic bodies, which are phagocytosed and degraded by macrophages and other phagocytic cells in a process known as efferocytosis [ ] . importantly, the cell membrane of apoptotic cells remains intact throughout and the cell shrinks rather than ruptures, as appearing in necrotic forms of cell death. thus in apoptotic cells, there is no leakage of cytosolic contents to initiate an inflammatory response [ ] . there are two major pathways that lead to apoptosis: intrinsic and extrinsic, both of which primarily converge on a family of proteins called caspases that cleave cellular substrates and lead to the morphological signature of apoptosis [ ] . intrinsic apoptosis is initiated by the mitochondrion in response to internal cellular stress and involves activation of caspase by cytochrome c. extrinsic apoptosis, on the other hand, is dependent upon death ligands of the tnf superfamily like trail or fasl binding their cognate cell surface receptors and the subsequent activation of caspase [ , ] . in addition to its physiological role, apoptosis is involved in promoting resistance or susceptibility to intracellular pathogens. for example, virulent mycobacterium tuberculosis (mtb) contains an anti-apoptotic gene (nuog) that diminishes both innate and adaptive immunity to promote the bacterial growth [ ] . in contrast to mtb, apoptosis induced by listeria monocytogenes is an immune evasion strategy, allowing the bacteria to disseminate [ ] . thus, it appears that apoptosis can be both protective and detrimental to the host depending on the pathogen. interestingly, both extrinsic and intrinsic pathways of apoptosis were shown to be activated in influenza-infected cells [ ] . this observation is well established, being described in human autopsies for almost a century, beginning with the pandemic, where pronounced epithelial desquamation, hyalination and sloughing were noted [ ] . experimentally, apoptosis of iav-infected epithelial cells was shown to be dependent upon viral replication, as an inactivated virus failed to induce apoptosis in mice [ ] and human cells [ ] . moreover, the magnitude of epithelial cell apoptosis was positively associated with iav strain pathogenicity in vivo [ , , ] and in vitro [ , ] in both mice and humans. however, whether apoptosis promotes host resistance or iav dissemination remains to be determined. logically, apoptosis of epithelial cells could represent a resistance strategy to iav, as it eliminates the intracellular niche required for viral replication. support for the protective capacity of apoptosis is that critical host antiviral factors actively induce epithelial cell apoptosis. upon viral infection, ifn-i (ifn-b) is rapidly produced, following recognition of the viral genome by pattern-recognition receptors (prr). in iav infection, rna sensing is predominantly mediated by the cytosolic retinoic acid-inducible gene (rig-i), which interacts with the mitochondrial antiviral signaling protein (mavs) at the mitochondrial outer-membrane to initiate ifn-i production mainly via the transcription factor interferon regulatory factor (irf ) (reviewed in ref. [ ] ). ifn-i then signals through a heterodimeric receptor complex of ifnar and , leading to sustained ifn-i (largely ifn-a) production and an upregulation of a large subset of genes, together termed interferon stimulated genes (isgs). isgs then serve to halt viral replication through both direct or indirect mechanisms. interestingly, both ifn-i and isgs have been shown to induce apoptosis. following iav infection, ifn-i signaling activates caspase-dependent apoptosis in aec-ii cells [ ] , and the pharmacological inhibition of these caspases blocks apoptosis and enhances viral replication. additionally, several classical apoptosis proteins, including caspase and trail, are isgs upregulated by ifn-i signaling. thus, iav sensing causes the release of ifn-i that induces epithelial cell apoptosis directly through caspase activation downstream of the ifnar / complex, and indirectly by promoting the transcription of pro-apoptotic isgs. other isgs have been equally implicated in promoting apoptosis in iav-infected cells as an antiviral mechanism. for example, protein kinase r (pkr) is an isg known to inhibit iav replication through a variety of mechanisms. pkr can directly sense dsrna generated during viral replication to induce fas expression and fadd-dependent apoptosis [ ] , as well as inhibit host and viral protein translation through the phosphorylation of eif a [ ] in iav-infected cells [ ] . moreover, pkr has been shown to increase ifn-i production in fibroblasts directly in response to another rna virus: semliki forest virus [ ] . thus, it appears pkr and other isgs contribute to antiviral immunity by promoting ifn-i responses, restricting viral protein translation and inducing apoptosis. in addition to being directly antiviral, apoptosis also protects against iav infection through the resolution of the immune response. in response to hpai, bronchiolar epithelial cell apoptosis limits excess pro-inflammatory cytokine (ie. tnf) release to dampen immunopathology and avoid the onset of ards [ ] . furthermore, a recent study has implicated club cells, a subset of bronchiolar epithelial cells, in augmenting immunopathology to an h n iav virus, by failing to undergo apoptosis following viral clearance [ ] . therefore, the protective capacity of apoptosis in structural cells is both antiviral and immunomodulatory. given the importance of apoptosis in antiviral immunity, it is not surprising that iav has encoded factors to block its induction. the principal iav virulence factor, non-structural protein (ns ), is rapidly transcribed upon infection and is the first protein expressed in infected cells. ns blocks early apoptosis in epithelial cells and fibroblasts by inhibiting ifn-i induction [ ] and activating the host pro-survival pi k/akt pathway to facilitate viral replication [ ] . importantly, these functions are critical in viral fitness, as strains lacking a functional ns are severely impaired in their virulence. taken together, all the above studies highlight the importance of apoptosis of structural cells in promoting immunity to influenza, by limiting the intracellular niche necessary for viral replication. however, the notion that apoptosis solely represents a hostdriven antiviral strategy is confounded by the fact that iav encodes both anti-and pro-apoptotic factors. for example, h n ns facilitates airway epithelial apoptosis [ , ] , suggesting a strainor expression level-specific role for this protein in iav pathogenesis. furthermore, recent study has characterized h n iav nucleoprotein (np) as a pro-apoptotic viral protein in human airway epithelial cells [ ] that targets the host's anti-apoptotic protein ap [ ] . in fact, host apoptotic machinery has been shown to be essential in promoting new virion production in epithelial cells in vitro [ e ], in a caspase- - [ ] and - dependent manner [ ] , and in vivo by iav-manipulation of annexin-a [ ] . these findings outline iav as an effective regulator of the host's apoptotic machinery in structural cells, capable of both inducing and blocking apoptosis to further its pathogenesis. the paradoxical role of apoptosis in immunity to iav, which appears to both prevent and permit viral dissemination, can perhaps be explained by the kinetics of the apoptotic response in epithelial cells (fig. ) . immediately upon infection, it is beneficial for iav to block epithelial cell apoptosis to avoid destroying its replicative niche and this is primarily mediated by viral ns . early blockage of apoptosis by iav is counteracted by host mechanisms, such as ifn-i signaling, to induce apoptosis and resist viral replication [ ] . yet, following initial replication cycles, at later time points, iav must activate apoptotic pathways to generate new infectious virions, promote budding at the cell surface and facilitate subsequent rounds of infection in neighboring cells. thus, pharmacological inhibition of apoptosis in humans during the later stages of infection may offer appealing therapeutic avenues, either by blocking pro-apoptotic pathways [ ] or enhancing antiapoptotic proteins [ ] . interestingly, neutralization of proapoptotic trail or fas signaling post-iav infection in aec-ii cells decreased iav load [ ] . similarly, mice treated with decoy fas in vivo to block fasl signaling were protected from lethal iav infection, when compared to untreated mice [ ] . our understanding of the interplay between influenza, host apoptotic machinery and resistance mechanisms has increased exponentially recently. however, much of our knowledge still derives from study in vitro using human or mouse cells and, thus, the exact effects of these pathways on disease outcome remain to be determined. like apoptosis, the observation that iav causes necrosis in epithelial cells has long been established. yet, the impact of iavinduced epithelial cell necrosis on the host immune response, and the factorsdviral or host-deriveddinvolved in this process are just beginning to become known. canonically, necrosis was considered a passive form of cell death associated with increased injury or disease severity. however, it is now well established that many forms of necrosis are, in fact, programmed. necrotic cell death is delineated from apoptosis by a disruption of the cell membrane and spilling out of cytosolic contents that act as dangerassociated molecular patterns (damps). these damps are sensed by neighboring cells, resulting in a robust inflammatory response [ , ] , thus, generating a potential positive feedback loop of tissue damage and immunopathology. the observation that hpai [ ] and pandemic h n often induce necrosis in epithelial cells, while seasonal h n appears only to cause it in immune compromised hosts [ ] , may at least partly explain the association between severity of disease and necrosis of the epithelium. moreover, other strains of highly pathogenic viruses such as dengue virus [ ] or mers-cov [ ] appear to induce considerable necrosis in epithelial cells to promote viral replication and propagation, while less virulent viruses like rhinovirus, or sars-cov induce limited or no necrosis [ , ] . in vitro, h n iav induces necrosis in human lung bronchiolar epithelial cells, coupled with massive release of proinflammatory cytokines and neutrophil chemoattractant cxcl , which is highly suggestive of a link between necrosis of the epithelium and ards [ ] . collectively, these observations promote the notion that epithelial cell necrosis is detrimental to the host and is consequential of severe infection. the most characterized form of programmed necrosis is ripk dependent programmed necrosis (termed necroptosis). necroptosis is dependent upon the formation of the necrosome, which consists of ripk and ripk , interacting through their rip homotypic interaction motifs (rhim). typically, ripk and ripk are cleaved by caspase- , causing their inactivation and leading to extrinsic apoptosis. however, when caspase- activity is compromised, the necrosome is activated to induce necroptosis. known mediators downstream of the necrosome include mixed lineage kinase domain like pseudokinase (mlkl). mlkl is a substrate for ripk and induces necroptosis by a combination of the assembly of a pore-forming complex at the plasma membrane, or as a platform for ca þ -mediated necrosis [ ] . although the exact executioner mechanisms of necroptosis remain unknown, recent study has implicated the plasma membrane repair machinery escrt iii as a potential mediator [ ] . necroptosis regulates a variety of inflammatory processes, including ischemic heart disease, bacterial infection and embryogenesis [ ] . interestingly, there is accumulating evidence for a role of necroptosis in promoting immunity to viral infection. seminal work showed necroptosis to be essential in antiviral defense, as ripk À/À mice succumbed to vaccinia virus infection [ ] . moreover, herpes simplex viruses (hsv- / ) and murine cytomegalovirus (mcmv) encode inhibitors of necroptosis, ul and m respectively, suggesting that viruses may block necroptosis as part of their pathogenesis [ , ] . although no iav inhibitors of necroptosis have yet been identified; most recently, ripk -dependent necroptosis of murine fibroblasts and alveolar epithelial cells was demonstrated to be critical in promoting iav clearance from the lungs and, as a result, ripk -deficient mice were highly susceptible to iav infection coupled with enhanced viral loads [ ] . thus, this study hints at a protective role for necroptosis in immunity to iav. however, human studies are required to investigate the potential clinical targeting of the necroptotic pathway in protection against iav infection. certainly, apoptosis and, more recently, necrosis have dominated study of iav-mediated cell death. however, the role of other forms of cell death in immunity to infectious diseases, such as iav, is beginning to be recognized. pyroptosis is a form of rapid inflammatory cell death with cell membrane rupture and dna damage, which is dependent upon caspase- and certain nod-like receptors. pyroptosis is coupled with proinflammatory il- b and il- release from pyroptotic cells, which contributes in part to its inflammatory nature [ ] . caspase- activation and subsequent cytokine release induced by iav in murine lung fibroblasts has been shown to inhibit viral pathogenesis [ , ] . yet, whether caspase- activation in these models led to pyroptosis, or rather defined a pyroptosis-independent role for caspase- was not carefully elucidated. thus, more study of the role of caspase- activation and pyroptosis in immunity to iav is needed. although epithelial cells constitute the major replicative niche for iav, the lung is a complex organ and thus other structural cells can be infected with iav. pulmonary fibroblasts are the most substantial subset of structural cells in the lung interstitium, playing a significant role in the repair process to injurious exposure and pulmonary inflammatory disorders, such as asthma and copd [ ] . fibroblasts are used extensively in iav studies as a model of lung structural cells. as in epithelial cells, fibroblasts are sensitive to both iav-induced apoptosis [ ] and necroptosis [ ] in vitro. however, the contribution of fibroblasts in immunity to iav infection is not well understood. whether fibroblasts are a target for iav infection in vivo and/or whether their response is analogous to, or completely independent of, epithelial cells, requires further investigation. understanding the role of fibroblasts in the pathogenesis of iav may provide additional targeted therapy against iav infection. similarly, endothelial cells represent a substantial compartment of the lung and in vitro infection of human endothelial cells led to productive iav replication and increased vascular leak, mediated by apoptosis [ ] , particularly upon infection with hpai (e.g. h n ) [ ] . hpai has been shown to disseminate from the lung systemically upon infection in mice [ ] . this has equally been observed in humans, where viral rna was found in multiple organs, including the brain and placenta, of fatal cases of h n [ , ] . the ability of hpai viruses to disseminate to other tissues is predominately mediated by changes to the cleavage sites of the viral ha protein, which is essential for viral entry into cells. in the case of low virulence seasonal strains of iav, ha cleavage occurs at a single basic arginine by trypsin-like proteases found exclusively in cells of the respiratory tree and gastrointestinal tract. on the other hand, hpai ha proteins contain polybasic cleavage sites that permit cleavage by furin proteases that are ubiquitously expressed. thus, this allows hpai strains to disseminate and cause systemic infection [ ] . additionally, pandemic and highly virulent strains of the virus, including hpai and the h n strain, are known to completely exhaust the replicative niche of epithelial cells of the lung over the course of infection as a by-product of overly exuberant replication and failure of immune response to control viral propagation. thus, it is appealing to associate the infection of endothelial cells by hpai viruses with subsequent systemic dissemination, made possible by the polybasic cleavage sites. however, this is complicated by the fact that no virus was found in endothelial cells of fatal cases of h n infection [ ] , yet virus was found in hyaline membranes and endothelial cells of fatal cases of the h n pandemic, where dissemination to other organs was not noted [ ] . as there is no known evolutionary advantage for iav to disseminate to other organs, a greater level of study is required to confirm what role infection of endothelial cells in humans plays and whether endothelial cell death is a pro-host or pro-pathogen strategy. during influenza virus infection, the pulmonary inflammatory response is characterized by early infiltration of leukocytes, including neutrophils and mononuclear cells and later by adaptive immune lymphocytes [ , ] . autopsy reports of humans infected with hpai and pathogenic strains of h n exhibited severe pulmonary leukopenia, which together constituted important clinical features of severe infection and fatal cases [ , ] . importantly, this leukopenia was mainly contributed to iav-induced death of these cells at the site of infection, rather than failed recruitment or effector function in the lung [ e ]. interestingly, pulmonary iav infection has also been shown to have profound effects on bone marrow hematopoiesis in an ifn-i-dependent manner [ ] . thus, the observed leukopenia could alternatively be explained by other mechanisms, such as exhaustion of the bone marrow hematopoietic stem cells due to severe infection. hematopoietic stem cell exhaustion is a phenomenon often observed during chronic bacterial or viral infections [ , ] , but remains poorly characterized in iav infections. nevertheless, these findings illustrate the importance of preserved leukocyte viability and function in host defense to highly pathogenic strains of iav and activation of the cell death program in these cells may represent a strategy of immune evasion by iav. although iav has been shown to contribute to cell death in the majority of leukocytesdincluding neutrophils, dendritic cells and lymphocytesdthis review will focus on cell death in pulmonary monocyte/macrophages, given their critical role in the initial response to the infection. pulmonary macrophage populations change considerably during iav infection. at homeostasis, tissue resident am constitute the vast majority of cells in the airways and are the first leukocytes to encounter the virus, following replication in epithelial cells [ ] . am can then be readily infected [ , ] and activated to secrete a spectrum of cytokines and chemokines that orchestrate both innate and adaptive immune responses to iav and other pulmonary viral infections [ , , ] . over the course of infection, am populations are diminished, while inflammatory monocytederived macrophages (md-m , also called exudate macrophages) expand and then contract during the resolution phase [ ] . both am and md-m have been linked to host protection and disease pathogenesis depending on the magnitude of activation of the immune response, which is directly associated with disease tolerance [ ] . invariably, loss of am causes increased susceptibility to infection, due to reduced production of antiviral ifn-i (mainly ifn-b) leading to uncontrolled viral replication and increased immunopathology [ , , , ] . thus, am represent a logical target for iav to induce cell death and, indeed, several studies have reported early apoptosis post-infection in am [ e ]. similarly, recruited m are highly permissive to iav infection and susceptible to iav-induced cell death [ , ] . collectively, these findings suggest that in contrast to the biphasic role of epithelial cell apoptosis in preventing or promoting pathogenesis, highly virulent iav rapidly infects and induces early death in pulmonary m to suppress antiviral responses. apoptosis occurs in m during iav infection and can be either triggered intrinsically by viral proteins or activated extrinsically by host factors in the pulmonary microenvironment. while the induction of apoptosis by viral proteins has been studied extensively in structural cells, fewer studies have investigated it in m [ , , , , ] . interestingly, influenza virus contains an essential gene, polymerase basic protein -frame (pb -f , a product of an alternate reading frame of the pb gene), which increases apoptosis-mediated pathogenicity in iav [ ] . functionally, pb -f has been shown to be the main inducer of apoptosis in monocytes/m , but not epithelial cells [ , ] . the pro-apoptotic effect of pb -f is elicited by its translocation to the mitochondrion [ ] . in mitochondria, pb -f interacts with components of the permeability transition pore complex (ptpc) ant and vdac [ ] to disrupt the mitochondrial inner membrane potential, followed by cytochrome c release and the induction of apoptosis, which subdues the m antiviral capacity and promotes host susceptibility [ ] . iav-induced apoptosis in m is not solely caused by viral protein expression and may result from the activation of the extrinsic pathway by host factors [ , , ] . interestingly, iav-infected m are the main source of the key antiviral cytokine: ifn-i [ , , ] and can induce m cell death via the upregulation of isgs. moreover, infection of human m with hpai strains induces trail release to trigger the apoptotic cascade [ , ] . equally, upregulation of fas and secretion of fasl at later time points postinfection have been shown to induce apoptosis selectively in recruited m [ , ] , a process which is thought to contribute to immune contraction and the return to homeostasis. of note, although tnf has been reported to induce apoptosis in iav-infected epithelial cells, it does not appear to mediate m apoptosis [ ] . thus, during iav infection, activation or inhibition of apoptotic pathways are cell-type specific and can either promote or prevent iav pathogenesis. therefore, this complex network certainly needs to be interrogated more intensely. the outcome of iav-induced m apoptosis is still controversial, as it may favor both the host and the virus. nonetheless, much of the literature suggests a host-detrimental effect of m apoptosis. for instance, hpai and pandemic h n influenza viruses hijack innate antiviral responses by inducing apoptosis in m to decrease m -antiviral cytokines (primarily ifn-i) and promote viral replication [ , , ] . considering residential am are the first immune cells encountered by the influenza virus and are critical in mounting an immune response, it is not surprising that iav efficiently infects and kills am to suppress their antiviral responses. thus, shortly after severe iav infection, the original pool of resident am decreases, resulting in enhanced viral replication [ , ] . similarly, limiting am cell death through the exogenous administration or overexpression of gm-csfdthe critical growth and maintenance factor for am dis protective, leading to lowered pulmonary viral load, decreased immunopathology and increased survival of mice after iav infection [ , ] . in addition to inhibiting the early antiviral responses, iav-induced m apoptosis might skew the balance from a protective to pathological immune response. during iav infection, it has been shown that mice lacking type i ifn signaling die from an uncontrolled inflammatory response, as ifn-i signaling was essential to induce anti-inflammatory il- production by "regulatory" m [ ] da subset of m known to be more susceptible to iav-induced apoptosis [ ] . mechanistically, iav-induced m apoptosis is mainly mediated by the viral protein pb -f and its expression has been linked to delayed viral clearance [ ] . expression of the pb -f protein in a mouse-adapted h n strain resulted in early increased viral titers and death of infected m [ ] . to prevent pb -f -induced early m apoptosis, we have recently shown that the host mitochondrial nlr-member nlrx directly interacts with pb -f within mitochondria to disarm its apoptotic function. in iavinfected nlrx À/À mice, or m in vitro, higher levels of apoptosis were observed compared to wt m . this was associated with reduced production of ifn-i and increased viral loads in vitro and in vivo [ ] . thus, by inducing m cell death, iav hijacks the host's key antiviral responses, resulting in both enhanced iav replication/ dissemination and impairment of the anti-inflammatory response, which drives immunopathology and potentially ards (fig. ) . however, recent findings indicate that in some cases, apoptosis of pulmonary m may be beneficial for the host. indeed, early apoptosis of hpai-infected porcine am correlated to decreased viral replication and magnitude of the inflammatory response [ ] . aside from viral proteins, our laboratory recently demonstrated that iav induces production of the host eicosanoid prostaglandin e (pge ) to directly inhibit early production of ifn-i, which increased viral loads through reduced m apoptosis. thus, whether apoptosis is protective or pathological seems, again, to be connected to the kinetics of its induction. early post-infection, m viability is essential to promote antiviral immunity and this is countered by iav proteins such as pb -f . later, however, m apoptosis becomes protective, as the apoptotic vesicles stimulate adaptive immunity by promoting cross-presentation [ ] . to conclude, apoptotic cell death of macrophages appears to be a double-edged sworddboth beneficial and detrimental to the host, depending on iav virulence factors and host mediators. further work is needed with closer inspection of the kinetics of expression of viral proteins (e.g. pb -f , ns ) as well as host factors (eg. nlrx , ifn-i, eicosanoids) and their link with kinetics of m cell death (early versus late induction) to fully understand whether timing or, rather, specific pathways or types of apoptosis dictate this complex phenomenon. despite apoptosis being the main m death modality observed in iav infections, there is also evidence of necrosis [ , ] and, specifically, of the more recently characterized ripk -mediated necroptosis. however, iav-induced macrophage necrosis has been poorly studied in vivo and most of our knowledge is confined to in vitro observation. at low multiplicities of infection (moi), necrosis is not substantially induced in m [ , , ] . in addition, we and others have recently shown that iav does not induce ripk mediated necroptosis in m , both in vivo and in vitro, but, rather, ripk plays a crucial role in the production of il- b [ ] and ifn-i [ ] and, as a result, mice deficient in ripk are extremely susceptible to iav infection. however, following severe infection with a high viral titer, iav induces necroptosis through the ripk eripk ecaspase complex in vitro [ ] , suggesting a potential role for ripk -mediated necroptosis in depletion of m during infection with highly virulent strains. synergy between these two observations may come from the pleiotropic function of ifn-i, which has been also shown to induce m necroptosis in vitro or in vivo during bacterial infections [ e ]. thus, it can be envisioned that dysregulated ripk -mediated ifn-i production in severe infection may, in fact, become detrimental to the host by inducing necroptosis of pulmonary m , suggesting a threshold for protective ifn-i production and deleterious immunopathology induced by m necroptosis. of note, pathways known to induce ifn-i in response to iav and other viruses, such as tlr [ ] , pkr [ ] and dna-dependent activator of ifn-regulatory factors (dai) [ ] have also been shown to trigger necroptosis, advocating a potential link between type i ifn production and necroptosis induction. finally, iav-infected macrophages produce il- b through inflammasome activation and caspase- cleavage, which are characteristics of pyroptotic cell death [ , ] . however, there was no difference in cell death in iav-infected nlrp À/À (lacking the nlrp inflammasome) and wt m , in vitro [ ] , suggesting that pyroptosis does not occur in m during iav infection. certainly, further investigation is required to fully understand the precise mechanisms involved in this process. the lung represents a diverse microenvironment filled with numerous cellecell interactions with high turnover under homeostatic and pathological conditions. in particular, epithelial cells of the ebb and am exist in close proximity and are known to regulate each other's functions at homeostasis and upon infection. over the last ten years, we have gained appreciable knowledge of how pulmonary macrophages induce apoptosis in infected epithelial cells. in , herold et al. showed for the first time that secretion of trail by m induced apoptosis of aec and contributed to lung injury during iav infection [ ] . further investigation also revealed that secretion of ifn-i, and specifically ifn-b, by infected am was responsible for autocrine trail expression and epithelium injury [ ] . most importantly, an upregulation of trail expression and secretion was observed in am from pandemic h n -infected individuals [ ] . in addition to potentiating lung injury, this could potentially lead to viral dissemination by promoting efferocytosis. efferocytosis is the phagocytosis of apoptotic bodies released from dying cells by neighboring m [ ] . recently, annexin a , a key protein involved in efferocytosis [ , , ] , was shown to facilitate iav replication and dissemination in vivo [ ] . interestingly, tcherniuk et al. showed that newly formed viral particles contained annexin a on their membranes, increasing their infectivity and viral replication through enhanced uptake [ ] . in addition, activation of formyl peptide receptor /lipoxin a receptor (fpr /alx) via annexin a , increased pulmonary viral load and susceptibility to infection [ ] . hence, one can envision a model wherein the induction of epithelial cell apoptosis by am derived ifn-i leads to the presence of annexin a -expressing viral particles on the surface of infected cells. these new viral particles are, in turn, recognized and taken up by bystander m , infecting them and preventing their antiviral responses. this leads to increased viral replication that subsequently enhances the magnitude of the immune responsedthus, continuing this vicious cycle of lung injury/reinfection. this may potentially explain how hpai and pandemic h n viruses strip the pulmonary epithelium and leave the host leukopenic, as seen in fatal cases. therefore, discovery of factors that can mitigate this pathological cycle would be of clinical significance in combatting highly pathogenic strains of iav. despite influenza virus infections likely dating back to the middle ages and considerable burden on global health, there is no effective therapy against iav. to prevent infection, vaccination strategies are deployed worldwide. however, the protection conferred by vaccination is highly variable, due to the ability of the virus to shift antigens, leading to vaccine mismatch and a decrease in efficacy. antiviral therapies, like neuraminidase inhibitors (i.e. oseltamivir) that block viral egress to potentially ameliorate disease are currently in use. despite considerable efficacy in murine models in lowering viral titers and improving disease outcome [ , ] , its protective effect in humans is controversial and limited benefit has been shown in the majority of patients [ , ] . additionally, highly resistant strains of iav to oseltamivir have recently emerged that raises growing concern [ ] . thus, these factors collectively suggest that the dogma of only targeting the virus and not the host's immune response in the generation of anti-iav drugs needs to be revisited. indeed, there are several "proof-of-concept" studies highlighting the promising therapeutic potential of targeting host cell death programs during iav-infection. first, anti-trail treatments selectively attenuated epithelial cells apoptosis, lung injury and increased survival of mice after iav infection. secondly, we recently demonstrated that targeted inhibition of mpges- , the key enzyme in pge production, selectively regulated pulmonary m apoptosis to boost antiviral and immunoregulatory properties. importantly, mpges- inhibition was effective even after three days of iavinfection, coinciding with the onset of symptoms in humans. in line with this, a recent study by hung et al. showed a reduction in viral titers, hospital stay and mortality when individuals infected with h n iav were given a combinatory therapy of clarithromycin (antibiotic) enaproxen (cox inhibitor) e oseltamivir (neuraminidase inhibitor) [ ] , compared to oseltamivir treatment alone, highlighting the importance of combining immunomodulatory therapies and those directly targeting the pathogen. additionally, we suggest ripk as an attractive target for immunomodulatory therapy. recently, the antiviral capabilities of ripk have been shown to be at least twofold, by both inducing epithelial cell necroptosis and promoting ifn-i responses from pulmonary m to potentiate survival to lethal iav infection. therefore, agonists of ripk may promote viral clearance via both epithelial cells and macrophages and ultimately confer survival to severe disease. this shows that combining treatments that differentially target structural cell and pulmonary m can significantly improve the outcome of influenza virus infection. the constant exposure of humans to potentially life-threatening respiratory pathogens presents ongoing challenges to clinicians. the knowledge gained from the studies of cell death programs in epithelial cells and macrophages during iav infection provide significantly better understanding of host mechanisms involved in protective or pathological immunity to iav infection. however, further investigation is still required to identify the kinetics of cell death during influenza infection, which appears to be critical in regulating antiviral and anti-inflammatory responses. in addition, most of our knowledge comes from in vitro experiments and thus a need for greater tools to investigate cell death in vivo and discovery of new cellular markers of the various cell death programs will be required for developing a novel therapy in influenza virus infection. . all authors declare that no conflicts of interest exist. handbook of geographical and historical pathology influenza (seasonal) fact sheet. who influenza: the mother of all pandemics reviewing the history of pandemic influenza: understanding patterns of emergence and transmission clinical and epidemiological characteristics of a fatal case of avian influenza a h n virus infection: a descriptive study human influenza a h n virus related to a 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induces necroptosis in macrophages during infection with salmonella enterica serovar typhimurium tolllike receptor -mediated necrosis via trif, rip , and mlkl dlm- / zbp ) is a cytosolic dna sensor and an activator of innate immune response inflammasomes as mediators of immunity against influenza virus alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules macrophage-expressed ifn-beta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice annexin i is an endogenous ligand that mediates apoptotic cell engulfment impaired phagocytic mechanism in annexin null macrophages formyl peptide receptor plays a deleterious role during influenza a virus infections virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic a/vietnam/ / influenza virus in mice aerosol administration increases the efficacy of oseltamivir for the treatment of mice infected with influenza viruses neuraminidase inhibitors for preventing and treating influenza in healthy adults and children oseltamivir treatment for influenza in adults: a meta-analysis of randomised controlled trials efficacy of clarithromycinenaproxeneoseltamivir combination in the treatment of patients hospitalized for influenza a (h n ) infection: an open-label randomized, controlled, phase iib/iii trial annexin regulates dc efferocytosis and cross-presentation during mycobacterium tuberculosis infection key: cord- -afce g authors: perales-linares, renzo; navas-martin, sonia title: toll-like receptor in viral pathogenesis: friend or foe? date: - - journal: immunology doi: . /imm. sha: doc_id: cord_uid: afce g viral infections frequently induce acute and chronic inflammatory diseases, yet the contribution of the innate immune response to a detrimental host response remains poorly understood. in virus-infected cells, double-stranded rna (dsrna) is generated as an intermediate during viral replication. cell necrosis (and the release of endogenous dsrna) is a common event during both sterile and infectious inflammatory processes. the discovery of toll-like receptor (tlr ) as an interferon-inducing dsrna sensor led to the assumption that tlr was the master sentinel against viral infections. this simplistic view has been challenged by the discovery of at least three members of the dexd/h-box helicase cytosolic sensors of dsrna that share with tlr the toll–interleukin- receptor (tir) -adapter molecule tir domain-containing adaptor protein interferon-β (trif) for downstream type i interferon signalling. data are conflicting on the role of tlr in protective immunity against viruses in the mouse model. varying susceptibility to infection and disease outcomes have been reported in tlr -immunodeficient mice. surprisingly, the susceptibility to develop herpes simplex virus- encephalitis in humans with inborn defects of the tlr pathway varies, and tlr -deficient humans do not show increased susceptibility to other viral infections. therefore, a current challenge is to understand the protective versus pathogenic contribution of tlr in viral infections. we review recent advances in the identification of tlr -signalling pathways, endogenous and virus-induced negative regulators of the tlr cascade, and discuss the protective versus pathogenic role of tlr in viral pathogenesis. viral infections frequently induce acute and chronic inflammatory diseases, yet the contribution of the innate immune response to a detrimental host response remains poorly understood. in virus-infected cells, double-stranded rna (dsrna) is generated as an intermediate during viral replication. cell necrosis (and the release of endogenous dsrna) is a common event during both sterile and infectious inflammatory processes. the discovery of toll-like receptor (tlr ) as an interferon-inducing dsrna sensor led to the assumption that tlr was the master sentinel against viral infections. this simplistic view has been challenged by the discovery of at least three members of the dexd/h-box helicase cytosolic sensors of dsrna that share with tlr the toll-interleukin- receptor (tir) -adapter molecule tir domaincontaining adaptor protein interferon-b (trif) for downstream type i interferon signalling. data are conflicting on the role of tlr in protective immunity against viruses in the mouse model. varying susceptibility to infection and disease outcomes have been reported in tlr -immunodeficient mice. surprisingly, the susceptibility to develop herpes simplex virus- encephalitis in humans with inborn defects of the tlr pathway varies, and tlr -deficient humans do not show increased susceptibility to other viral infections. therefore, a current challenge is to understand the protective versus pathogenic contribution of tlr in viral infections. we review recent advances in the identification of tlr -signalling pathways, endogenous and virus-induced negative regulators of the tlr cascade, and discuss the protective versus pathogenic role of tlr in viral pathogenesis. mammalian cells are able to detect viruses at multiple steps. therefore, it is likely that cells benefit from redundant mechanisms to detect and limit viral infections. recognition of viral nucleic acids by intracellular toll-like receptors (tlrs) entails the early steps of the immune response elicited during viral infections. for the past years, efforts have been made to define the role of tlr in the antiviral response and to elucidate at the molecular level the tlr pathway as a 'major sentinel' against viruses. in contrast to tlr , which recognizes single-strand (ss) rna (therefore its role could be restricted to certain types of rna viruses), tlr has, at least in principle, the ability to play a crucial role in the antiviral response against most viruses by its ability to sense double-stranded (ds) rna, a common intermediate of replication among many viruses. intriguingly, dsrna of non-viral origin and, in particular, endogenous mrna and rna released from necrotic cells have been shown to trigger the tlr pathway. , toll-like receptor is broadly expressed and well conserved among vertebrates and its expression has been confirmed in immune and non-immune cells. a functional antiviral role of tlr against certain viruses has been demonstrated in various human and murine cells as well as in the mouse model (recently reviewed in ref. ) . interestingly, tlr has been implicated in licensing myeloid dendritic cells (dcs) for cross-priming of cd t cells in the mouse, and a certain subset of human dcs that highly express tlr exhibit a better capacity for cross-presenting apoptotic and necrotic cell antigens after tlr stimulation. indeed, at least in part, the strategy of using tlr ligands as adjuvants in vaccine therapy is based on the selective high expression of tlr in human cd + dcs and mouse cd a + dc subsets. in contrast to the beneficial roles of tlr in antiviral response and cellular immunity, emerging evidence suggests that tlr also mediates pathogenesis, which may or may not be directly related to the exacerbation of the antiviral response. this review provides background on the tlr pathway, highlights the negative regulation of tlr signalling by endogenous and virus-encoded proteins, and discusses emerging evidence on the detrimental contribution of tlr in viral pathogenesis. the tlr is located in the endoplasmic reticulum of unstimulated cells, and upon dsrna stimulation, it traffics to endosomes to encounter its ligand through a phdependent mechanism that also requires tlr dimerization. the interaction between the endoplasmic reticulum membrane protein unc- b and tlr is crucial for tlr trafficking and signalling. in addition, recent structural studies have demonstrated that the length (at least bp) and structure of dsrnas play critical roles in tlr recognition and signalling. emerging evidence suggests that tlr proteolysis by asparagine endopeptidase and cathepsins in endolysosomes may represent a general regulatory strategy for all tlrs involved in nucleic acid recognition. the tlr proteolytic processing regulates stability and endosomal localization, but its role in tlr function remains poorly understood. furthermore, tlr tyrosine phosphorylation in the endosome is essential for recruiting the adaptor protein toll-interleukin- (il- ) receptor (tir) domain-containing adaptor protein interferon-b (ifn-b) (trif) to the tir domain of tlr . this is in contrast to other endosomal tlrs (tlr - ) that do not require tyrosine phosphorylation, possibly owing to their myd -dependent, trif-independent signalling. to recruit trif upon recognition and dimerization, tyr and tyr residues in the cytoplasmic domain of tlr are sufficient for downstream signalling when phosphorylated. two studies have reported a differential role for the tyrosine kinases c-src and epidermal growth factor receptor in mediating tlr phosphorylation. these studies support the current model that after dsrna stimulation, tlr binds epidermal growth factor receptor before c-src does. interestingly, epidermal growth factor receptor is required for tyr phosphorylation, and src is required for tyr phosphorylation. in addition, phosphoinositide -kinase and bruton's tyrosine kinase have been implicated in tlr phosphorylation. phosphoinositide -kinase is required for the full activation of ifn regulatory transcription factor (irf ) and nuclear factor-jb (nf-jb), but its role in tlr signalling remains to be determined. bruton's tyrosine kinase directly phosphorylates the tyr residue of tlr . in bruton's tyrosine kinase-deficient macrophages, tlr -induced phosphoinositide -kinase, akt and mitogen-activated protein kinase (mapk) signalling and activation of nf-jb, irf , and activator protein (ap- ) transcription factors are all defective. triggering trif recruitment by tlr dimerization and tyr phosphorylation results in the stimulation of the transcription factors irf , nf-jb and ap- through two branches. nf-jb activating kinase (nak) -associated protein mediates trif association with the tumour necrosis factor (tnf) receptor-associated factor (traf ) together with traf family member-associated nf-jb activator (tank)-binding kinase (tbk ) and ijb kinase e (ikke) complex, which subsequently promotes the phosphorylation, dimerization and translocation of irf into the nucleus. the second branch drives the activation of nf-jb and ap- and is mediated by the protein kinase receptor-interacting protein (rip ) and the e ubiquitin protein ligase traf , which interact with trif and recruit tab (tak binding protein ), tab (tak binding protein ), and tak (transforming growth factor-b-activated kinase ). this branch results in the activation of the ikk complex (nuclear factor-jb essential modulator, ikka, ikkb), the mapk pathway, and further activation of nf-jb and ap- , respectively. the rip and traf branches result in the activation of the antiviral and pro-inflammatory response by the induction of type i ifn and other cytokines. intriguingly, rip interaction with trif leads to cell death through a fas-associated protein with death domain/caspase- -dependent and mitochondrion-independent pathway (rip /fas-associated protein with death domain). indeed, the tlr -trif axis has a cytopathic potential that might be developed during infection. taken together, these data suggest that upon tlr -trif stimulation and activation, three major outcomes can be predicted: (i) the development of the antiviral response mediated by irf activation and further type i ifn production; (ii) a cytopathic effect or cell death through caspase- activation by rip ; and (iii) the generation of a pro-inflammatory environment by the activation of nf-jb and ap- . many of the adaptors and molecules involved in tlr signalling are shared by other tlr pathways. for example, trif is also an adaptor in a branch of the tlr and perhaps the tlr pathways and modulates the tlr pathway by inducing proteolytic degradation of tlr . unexpectedly, evidence is emerging on the role of trif in cytosolic sensing of dsrna. in particular, trif is an adaptor of a novel dsrna sensor complex consisting of the rna helicases ddx -ddx -dhx that has been identified in the cytosol of myeloid dcs. hence, the challenge is to understand specificity and redundancy among tlrs and other known and yet to be discovered pathways. moreover, several positive and several negative , [ ] [ ] [ ] [ ] [ ] regulators of the tlr pathway have been identified. here, we focus on the negative regulation of the tlr pathway and discuss endogenous and virus-encoded negative regulators of the tlr pathway. the signalling balance must be maintained during the development of any immune response to prevent tissue damage and, potentially, autoimmunity. emerging evidence demonstrates that endogenous negative regulators play a key role in the fine tuning of innate signalling pathways. several adaptors and downstream molecules of the tlr pathway have been shown to be negatively modulated by various mechanisms and regulators. among these, we discuss recent advances in the regulation of trif, traf , rip and traf (table ) . a number of molecules have been demonstrated to affect trif under sterile and infectious conditions in various studies using in vitro cell approaches. sterile aand armadillo-motif-containing protein (sarm) is a tir domain-containing adaptor that has been shown to inhibit trif, resulting in down-regulation of trif-dependent cytokine and chemokine induction and ap- activation through the mapk pathway. trif is also involved in the tlr , myd -dependent signalling pathway, and the adaptor translocating chain-associated factor (tram) mediates trif recruitment. tag (tram adaptor with gold domain), a splice variant of tram, is an adaptor that negatively modulates tlr -tram signalling from endosomes by displacing trif, so inhibiting the signal transduction. interestingly, integrins may play a role in the regulation of some tlrs. the integrin cd b activates syk and promotes degradation of trif via the e ubiquitin ligase cbl-b. triad a is an e ubiquitin ligase that modulates tlr signalling by degradation of the tir domains of trif and rip . tripartite motif (trim) proteins and, in particular, trim have been identified as novel negative regulators of the innate immune response. trim expression is enhanced after tlr stimulation and negatively regulates tlr -mediated type i interferon signalling by targeting trif for proteasomal degradation. importantly, trim negatively regulates tlr / -and rig-i-mediated ifn-b production and antiviral response by targeting nf-jb-activating kinaseassociated protein (nap ) and traf . finally, a novel role for adam (a disintegrin and metalloprotease) as a negative regulator of tlr and tlr signalling by a mechanism involving trif degradation has recently been identified. traf assembles lys linked polyubiquitin chains and forms a protein complex with tbk and ikke, then binds to trif and acts as a mediator of the irf activation, which results in the phosphorylation of irf . deubiquitinating enzymes (dubs) are proteases that specifically cleave ubiquitin linkages, negating the action of ubiquitin ligases. de-ubiquitinating enzyme a (duba) selectively cleaves the polyubiquitin chains on traf , resulting in its dissociation from the downstream signalling complex and the reduction of the type i ifn response. interestingly, mip-t has been suggested to function in the cilia to facilitate infection with viruses that preferentially infect the apical ciliated side of the respiratory and gastrointestinal epithelia by compromising innate immunity, although this hypothesis requires further investigation. the negative role of mip-t in the production of type i ifn by inhibition of traf complex formation is unique as mip-t appears to affect traf ubiquitination only slightly but is capable of preventing traf from engaging downstream transducers. the rip kinase is negatively regulated by rip , triad a and a . meylan and colleagues have shown that rip inhibits rip by competition, down-regulating the trif-rip -induced nf-jb pathway. similarly to duba, a is a de-ubiquitinating enzyme that has been found to act in the cytoplasm as a negative regulator of tlr responses, affecting rip and traf and thereby terminating tlr-induced nf-jb signalling. in addition, a interacts with trif and inhibits tlr -and sendai virus-induced activation of ifn-sensitive response element and ifn-b promoter, suggesting that a is a virus-inducible protein that blocks both inflammatory and cellular antiviral responses. a variety of negative regulators affect traf , a signal transducer shared by the trif and myd axis. tlrs other than tlr depend on the adaptor protein myd , and its activation from different sources converges in the recruitment of traf . therefore, it is not surprising to find several molecules that interact with and tightly regulate traf . in addition to a , traf is negatively regulated by the dub tumour suppressor cylindromatosis, which negatively regulates the tnf receptor-induced activation of nf-jb and c-jun n-terminal kinase by reversing ubiquitination of its target and possibly by a transient interaction with nuclear factor-jb essential modulator. , activation of nf-jb and ap- leads to production of pro-inflammatory signals as well as cylindromatosis. therefore, it generates an autoregulatory loop that controls the immune response through traf . tank (also known as i-traf) has been identified as a traf-binding protein. the presence of tank in unstimulated cells mediates the inhibition of traf by blocking its ubiquitination. tank seems to negatively regulate pro-inflammatory cytokine production, rather than the ifn response induced by tlr signalling. traf is also regulated by pellino- , a family of proteins that have been shown to be subject to various forms of post-translational modifications. it was demonstrated that trif signalling induces its expression, and through its e ubiquitin ligase capability, pellino- inhibits traf and further activation of irf , resulting in down-regulation of type i ifn expression. a potent negative regulator of traf is the ubiquitin-specific protease (usp ), which plays an essential role in modulation of the tlr/il- r signallingmediated innate immune response by cleaving ubiquitin linkages in a dub activity-dependent manner. similarly, the orphan nuclear receptor small heterodimer partner is a transcriptional co-repressor that regulates hepatic metabolic pathways and has been found to inhibit tlr signalling by regulating the polyubiquitination of traf . the nucleotide-binding domain leucine-rich repeat containing (nlr) proteins serve as regulators of inflammatory signalling pathways. the mitochondrial nlrx protein interacts with traf and inhibits nf-jb activation. upon tlr stimulation, nlrx dissociates from traf to allow its function. nlrx is an important regulator of the antiviral response against rna viruses,because it also counteracts the rig-i-mavs (mitochondrial antiviral signalling protein) cytoplasmic pathway. in the previous section, we described a number of endogenous negative regulators that target trif, traf , rip and traf , thereby controlling the signalling cascades mediated by tlr stimulation to protect the host from potentially harmful, excessive immune activation. during co-evolution with the host, viruses have evolved mechanisms to counteract cell responses against infection for their own benefit and diminish the immune response at multiple levels. here we discuss the various strategies that some viruses have developed to negatively regulate the tlr pathway by targeting trif, trim , traf , traf and rip (table ) . one viral strategy to interfere with the tlr signalling pathway is to down-regulate the expression of the adaptor trif. some viral non-structural proteins with serine protease activity have been identified to target trif for proteolysis. the ns / a of hepatitis c virus (hcv), the hepatitis a virus (hav) protease-polymerase processing intermediate ( cd), the c(pro) cysteine protease of coxsackievirus b (cvb ), and the enterovirus (ev ) c protease cleave trif and disrupt the tlr signalling. additionally, the replication and transcription activator that is necessary and sufficient for the switch from latent to lytic infection with kaposi's sarcoma-associated herpesvirus, mediates the degradation of trif through the ubiquitin-mediated proteasome pathway. little is known about how viruses may regulate the activity of the endogenous negative regulator trim . as discussed above, the expression of this e ligase is up-regulated after tlr stimulation. trim promotes k linked polyubiquitination and proteasomal degradation of nap and trif and negatively regulates tlr -mediated type i ifn signalling. the picornavirus enterovirus induces trim degradation, suggesting that reduced expression of trim may be beneficial for the virus by targeting molecules yet to be discovered. the precise role of trim suppression in picornavirus infection remains unknown. in contrast, trim expression was induced upon vesicular stomatitis virus infection, an ssrna virus recognized by rig-i. over-expression of trim decreased ifn-b production and increased vesicular stomatitis virus replication in the presence or absence of polyriboinosinic polyribocytidylic acid [poly(i:c)], suggesting that virus-induced trim negatively regulates antiviral immune responses. some viral proteins have the ability to interfere with the antiviral pathways by sequestration of cellular proteins such as traf and traf that play key roles in the antiviral response. for instance, the m protein of the highly pathogenic severe acute respiratory syndrome coronavirus prevents the formation of the traf -tank-tbk /ikke complex and thereby inhibits tbk /ikke-dependent activation of irf /irf transcription factors. vaccinia virus inhibits tlr -induced nf-jb activation by sequestering traf and il- receptor-associated kinase-like through interaction with the poxvirus protein a r. taken together, these findings provide evidence of the complex regulatory networks that viruses exploit to counteract the tlr signalling pathway. the question that arises is how crucial is tlr in antiviral immunity in vivo. surprisingly, data from the literature suggest that tlr may be a double-edged sword that functions in both defence and offence in host immunity to viruses. because tlr recognizes dsrna, which is produced as a replicative intermediate by rna and dna viruses, and some viruses contain dsrna genomes, the tlr pathway has the potential to control immunity to most of the clinically relevant viral infections in humans, caused by flaviviruses, hepatitis b and c viruses, herpesvirus, rotavirus, retroviruses, orthomyxoviruses and poxviruses, among others. however, the role of tlr in human immunity is still largely unknown, and our understanding is based mostly on studies using human primary or immortalized cells (table , fig. ). a remarkable exception is the knowledge gained from human patients with inborn errors in the tlr pathway who do not display susceptibility to viral infections other than herpes encephalitis. herpes simplex virus (hsv- ) is a highly prevalent neurotropic virus that infects the central nervous system (cns) and can generate herpes simplex encephalitis in children with inborn errors of tlr immunity. it has been shown that impaired tlr and unc- b-dependent ifn-a/b intrinsic immunity to hsv- in the cns, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of herpes simplex encephalitis in children with tlr -pathway deficiencies. , moreover, li and colleagues have demonstrated that among ifns, endogenous ifn-k also participates in tlr -mediated antiviral activity in primary human astrocytes. to date, tlr is the only tlr that has been shown to play a non-redundant role for protection against hsv- infection in the cns, although tlr may have a redundant role against other viruses in humans. taken together, these findings suggest that the role of human tlr in protection against herpes simplex encephalitis is unique. moreover, hidaka and colleagues performed a genetic analysis including tlr genes that could be involved in the recognition of influenza a virus (iav) on patients with influenza-associated encephalopathy and found a missense mutation (f ) in the tlr gene. this finding correlated the deficiency of tlr with the encephalopathy induced by iav. in contrast, a genetic evaluation of tlr identified the wild-type tlr rs allele to be a risk factor in tick-borne encephalitis virus infection, a pathogen that can be asymptomatic or can cause severe symptoms in the cns. this study suggests that a functional tlr is a risk factor for tickborne encephalitis virus infection. in contrast, emerging evidence suggests that tlr may exhibit protective as well as detrimental functions in the context of other human viral infections. hepatitis c virus is an ssrna virus that induces chronic hepatitis and may lead to hepatocellular carcinoma. tlr senses dsrna of hcv upon infection in cultured hepatoma cells as well as in primary human hepatocytes, leading to nf-jb activation and to secretion of pro-inflammatory cytokines and chemokines. these data suggest that tlr may contribute to the intrahepatic and unbalanced pro-inflammatory response that characterizes the pathogenesis of hcv-associated liver diseases. interestingly, a link has been reported between the tlr pathway and induction of glomerulonephritis in patients with chronic hcv infection. this disease is characterized by inflammation of the blood vessels in the kidneys. in hcv-associated glomerulonephritis, tlr mrna and protein expression are up-regulated in mesangial cells, and upon stimulation with tlr ligand, an increment of selected cytokines and chemokines that are characteristic of the disease is observed. in addition, some tlr polymorphisms have been associated with chronic hepatitis b and hepatitis b-related acute-on-chronic liver failure and hepatocellular carcinoma. furthermore, there is a close relationship between the presence of tlr rs /ct polymorphism and an increased risk of pneumonia in children infected by the pandemic a/h n / influenza virus. although animal models are being evaluated (discussed below), the precise role of tlr against influenza in humans remains to be determined. in the context of rotavirus infection, up-regulation of epithelial tlr expression during the contribution of tlr in hiv- infection is poorly understood. activation of the tlr pathway with poly(i: c) induces an antiviral state that limits hiv- infection in primary human macrophages and human fetal astrocytes by different mechanisms. in addition, when balb/ c mice were immunized with purified recombinant hiv- envelope gp or influenza haemagglutinin protein together with poly(i:c), epitope-specific cd + mhc class i molecule-restricted cytotoxic t lymphocytes were primed from naive cd + t cells in vivo, suggesting that activation of the tlr pathway enhances class i processing of exogenous protein and induction of hiv-specific cd + cytotoxic t lymphocytes. interestingly, a common tlr allele confers immunologically mediated protection from hiv- . taken together, these findings suggest that triggering of tlr with specific ligands could have therapeutic potential against hiv- infection in humans. however, extreme caution is advised because tlr may also make a detrimental contribution of tlr in hiv- -induced pathogenesis. for example, tlr is elevated and it is expressed in proximity to infiltrating mononuclear cells in biopsy specimens from patients with hiv myopathies. immunity to viral infection in tlr -deficient mice viral infections in mice with tlr deficiency are useful models to better understand the complexity of tlr mediated immune responses in vivo. the role of tlr has been tested in at least different viral infections in mice (table , fig. ). most of the viruses studied so far have neurotropic, respiratory, liver or cardiac tropisms. the role of tlr has been studied in tlr -deficient mice with various genetic backgrounds with the following neurotropic viruses: west nile virus (wnv) (flaviviridae, positive-sense ssrna), poliovirus and theiler's murine encephalomyelitis virus (tmev) (picornaviridae, positivesense ssrna), hsv- and hsv- (herpesviridae, dsdna) and t reovirus (reoviridae, segmented dsrna). the cns is separated from the rest of the body by the bloodbrain barrier; the cerebrospinal fluid is also separated from the periphery by the blood-cerebrospinal fluid barrier of the choroid-plexus epithelium. this structure makes the brain an immune-privileged tissue where, during viral infection, the tight balance between a controlled antiviral response and excessive immune activation determines the pathological outcome. west nile virus infection induces encephalitis in humans and mice. the role of tlr in wnv encephalitis remains controversial, and both protective and pathogenic roles have been reported in the mouse model. perhaps these conflicting data reflect the use of different routes of virus inoculation and strains. for instance, tlr deficiency protected mice that were infected intraperitoneally from wnv lethal infection, reduced blood-brain barrier permeability, and decreased systemic tnf-a and il- production suggesting a detrimental role of tlr . in contrast, increased mortality was observed in tlr -deficient mice in association with elevated virus burden in neurons in the brain, suggesting that tlr has a protective role against wnv in the cns. however, the same group found that serum ifn-a levels, viral load in non-neural tissues, and mortality rates did not differ significantly between mda -deficient mice and wild-type mice. in contrast, serum ifn production was abrogated and the viral load in non-neural tissues and mortality rates were both markedly higher in trif-deficient and tlr -deficient mice than in wild-type mice. these results suggest that multiple pathways are involved in the antiviral response in mice and that the tlr -trif-mediated signalling pathway plays an essential role in the antiviral response against poliovirus infection in the mouse model. the role of tlr -mediated signalling in the protection and pathogenesis of tmev-induced demyelinating disease is intriguing and warrants further investigation. in this case, activation of tlr signal transduction before tmev infection leads to pathogenesis via over-activation of the pathogenic immune response. in contrast, tlr -mediated signalling during viral infection protects against tmev demyelinating disease by reducing the viral load and modulating immune responses. herpes simplex virus is a major cause of encephalitis, whereas hsv- can give rise to meningitis as well as encephalitis, sacral radiculitis and myelitis in humans. the role of tlr in herpes simplex encephalitis has been recently studied in the mouse model using the hsv- strain. interestingly, tlr -deficient mice were hypersusceptible to hsv- infection in the cns after vaginal inoculation. although tlr -deficient mice did not exhibit a global defect in innate immune responses to hsv, astrocytes were defective in hsv-induced type i ifn production. overall, these data suggest that tlr acts in astrocytes to sense hsv- infection immediately after entry into the cns, possibly preventing hsv from spreading beyond the neurons mediating entry into the cns. although hsv- strain has been studied in tlr -deficient mice, the role of tlr in hsv- infection of the cns has not been fully investigated. however, the study above has revealed a key function of tlr in cd t-cell priming to hsv- . finally, the role of tlr in reovirusinduced neuropathogenesis has been addressed using the t reovirus strain dearing (t d). no differences were observed in cns injury, viral loads, and mortality in tlr -deficient compared with wild-type mice, suggesting that at least for the t d strain, tlr does not play a significant biological role in reovirus neuropathogenesis. the contribution of tlr in respiratory viral infections has been tested for rhinovirus, influenza a virus, vaccinia, and respiratory syncitial virus in the mouse. rhinovirus is the most frequent cause of asthma exacerbations and can be studied in vivo by using the rhinovirus type rv b strain, a minor group virus that replicates in mouse lungs. by using tlr -deficient mice, wang and colleagues showed that lack of tlr was beneficial for the infected mice. tlr -deficient mice showed reduced lung inflammatory responses and reduced airway responsiveness, suggesting that in the context of rhinovirus infection, binding of viral dsrna to tlr initiates proinflammatory signalling pathways leading to airway inflammation and hyper-responsiveness. similarly, in the absence of tlr , iav-infected mice had increased survival rates even though the viral burden in lungs was elevated, and it correlated with significantly reduced proinflammatory mediators as well as a lower number of cd + t lymphocytes in the bronchoalveolar airspace, suggesting a detrimental pro-inflammatory role of tlr in the lungs of iav-infected mice. furthermore, respiratory syncytial virus preferentially infects airway epithelial cells, causing upper respiratory infections, asthma exacerbations and pneumonia. respiratory syncytial virus induces tlr up-regulation, leading to a priming of lung epithelial cells for subsequent exposure to extracellular dsrna. upon tlr stimulation, activation of nf-jb and increased levels of il- amplify epithelial cell responsiveness, resulting in an altered inflammatory response. additionally, aeffner and colleagues reported that dsrna induces similar pulmonary dysfunctions to respiratory syncytial virus infection in mice. respiratory syncytial virus induces nucleotide/p y purinergic receptormediated impairment of alveolar fluid clearance, which results in the formation of lung oedema. the role of tlr in the pathogenesis of vaccinia virus, a prototype poxvirus, has been investigated using a recombinant strain western reserve vaccinia virus that expresses firefly luciferase in the mouse. tlr -deficient mice had substantially lower viral replication in the respiratory tract and diminished dissemination of virus to abdominal organs. mice lacking tlr had reduced disease morbidity and lung inflammation that correlated with reduced recruitment of leucocytes to the lung. interestingly, mice lacking tlr also had lower levels of inflammatory cytokines, including il- , monocyte chemoattractant protein- and tnf-a in serum and/or bronchoalveolar lavage fluid, but levels of ifn-b did not differ between genotypes of mice. taken together, these results show that, in the respiratory tract, tlr might have a detrimental role and its stimulation leads to potential over-activation of the pro-inflammatory response in the airways that is detrimental to the host. mice that are tlr -deficient have been shown to be highly susceptible to encephalomyocarditis virus (emcv) , and coxsackieviruses b and b - infections, resulting in significant mortality. these viruses induce cardiac injury or diabetes depending on the virus strain, and are considered a model of virus-induced autoimmune inflammation in the heart and pancreas. lack of tlr leads to an impaired expression of pro-inflammatory response in the heart and the liver of mice infected with emcv, and in the pancreas of mice infected with the emcv variant of pancreas b-cell tropism, facilitating the increment of viral burden in these tissues. , greater cardiac damage and increased viral load in heart, serum and liver are also observed in tlr -deficient mice compared with immunocompetent mice infected with coxsackieviruses b and b . [ ] [ ] [ ] remarkably, tlr and its adaptor trif may have differential roles in viral myocarditis resulting in different t helper type (th ) responses that uniquely influence the progression to chronic myocarditis. in particular, tlr -deficient mice developed an il- -dominant th response during acute coxsackievirus b myocarditis with elevated markers of alternative activation, whereas trif-deficient mice had elevated the th -associated cytokine il- . although tlr -or trif-deficient mice developed similarly worse acute coxsackievirus b myocarditis and viral replication compared with control mice, disease was significantly worse in trif-than in tlr -deficient mice. this is the first report in the literature to demonstrate a differential role of trif and tlr in a mouse model of viral pathogenesis. these findings, although intriguing, suggest that tlr may also signal independently of trif under at least some circumstances. indeed, emerging evidence demonstrates the existence of a new branch of tlr signalling that does not lead to gene induction but affects many cellular properties, such as cell migration, adhesion and proliferation. surprisingly, for this effect of tlr signalling, the adaptor proteins trif and myd were not required. the effects of the new pathway were mediated by the proto-oncoprotein c-src, which bound to tlr after dsrna stimulation of cells. this is the first example in the literature of tlr-mediated cellular effects of an adaptor-independent effect of any tlr. finally, the role of tlr in viral hepatitis using tlr deficient mice remains poorly understood. a caveat in the literature is that the viruses studied so far (emcv, coxsackievirus b , poliovirus and the phlevovirus punta toro virus) are not strictly hepatotropic and induce multiorgan pathogenesis. nevertheless, mice lacking tlr expression developed higher viral load in the liver and increased mortality compared with their controls after intraperitoneal infections with emcv and coxsackievirus b and intravenous infection with poliovirus. in contrast, tlr -deficient mice demonstrate increased resistance to lethal infection and have reduced liver disease associated with hepatotropic punta toro virus infection. although infectious challenge produced comparable liver and serum viral loads, mice lacking tlr were able to clear systemic virus at a slightly faster rate, and exhibited fewer inflammatory mediators. in particular, tlr -deficient mice had lower levels of il- , monocyte chemoattractant protein- , ifn-c, and ran-tes than wild-type animals. therefore, the tlr -mediated response to punta toro virus infection is detrimental to disease outcome. although il- is critical to establish antiviral defence in this model, it also contributes to pathogenesis when released in excess through a tlr -mediated mechanism. in addition, non-specific activation of innate immunity can drastically enhance susceptibility to immune destruction of the liver through activation of tlr . immune destruction of the liver requires first the priming of liver-specific t cells. these activated t cells then have to migrate into the target organ, where autoimmunity finally occurs. usually, the liver does not attract liver-specific t cells because chemokines are expressed at low levels in the liver. however, pro-inflammatory signals derived from ligation of tlr can lead to homing of cd + t cells to the liver with subsequent enhancement of liver disease by a mechanism mediated by ifn-a-and tnf-a-dependent up-regulation of genes and their products, such as the chemokine cxcl , involved in t-cell homing and migration. this pathogenic mechanism may explain observations suggesting that autoimmunity, including hepatitis, can be promoted by viral infections. overall, data from the mouse model suggest that the protective versus pathogenic contribution of tlr in viral infections is multifactorial. however, some conclusions can be drawn. first, there are differences among type of viruses, but the role of tlr in viral pathogenesis does not exhibit a differential trend among ssrna, dsrna and dna viruses. second, tlr may exhibit differential roles that are tissue specific. in this regard, the detrimental contribution of the tlr pathway in autoimmune hepatitis through activation of certain cytokines and chemokines involved in t-cell migration to the liver is remarkable. however, tlr is protective in three and detrimental in only one of the four hepatotropic viruses tested to date in the mouse (the phlebovirus punta toro virus). perhaps among these viruses, the pathogenesis of punta toro virus represents a better example of virusinduced hepatitis in the absence of other major organ involvement. in the cns, tlr seems to be protective rather than detrimental against infection with certain viruses, and at least in the case of the flavivirus wnv, controversial results remain to be further evaluated. in contrast, studies suggest that tlr may have a pathogenic role in the lung at least in the context of the four respiratory viruses that have been evaluated. third, experimental factors (e.g. route of virus inoculation and mouse strain) may affect the interpretation of the results obtained in certain studies. although the role of tlr in protection against viral infections may vary among viruses, a detrimental contribution of tlr in viral pathogenesis is emerging from studies in the mouse and in certain viral infections in humans (fig. ) . tlr participates in both defence and offence in host immunity to viruses. a working model of the role of tlr in antiviral (protective) and immunoregulatory (detrimental) responses based on data from the literature is shown in fig. . we have much to learn about the mechanisms that drive tlr -mediated disease in the context of viral infections and, importantly, in autoimmunity and in cancers that occur after certain infections. the role of tlr in the fine-tune regulation of t-cell polarization may have a major pathogenic impact in the context of some viral infections. virally infected cells generate signals that act on dcs to favour cross-priming. upon dsrna recognition, tlr induces type-i ifn secretion known to enhance cross-presentation by dcs and to mediate an antiviral immunity against a number of viral infections. this event ultimately results in th responses and cytotoxic t lymphocyte activation. the beneficial or detrimental role of tlr balancing the response towards th immunity upon tissue-specific viral infections would greatly diverge depending on the ability of the cellular environment to modulate and balance it by generating a th counterpart response. moreover, a single viral pathogen conventionally defined as th -inducing bears a variety of ligands capable of engaging multiple tlrs that can also stimulate a th response, although to a different degree. therefore, a better understanding of the th /th response modulation through tlr stimulation in virus-driven tissue-specific pathogenesis is essential. in the context of chronic rna viral infections that result in sustained ifn-a/b signalling, without viral control, the tlr -trif axis may play important roles in determining how the balance between antiviral and immunoregulatory pathways affect protective versus detrimental responses. it will be interesting to determine whether tlr has a non-redundant, protective role against viral infections other than in herpes simplex encephalitis in humans. a current challenge is to fully understand this remarkable and perhaps unique role. another intriguing challenge is to identify tlr -dependent, trif-independent pathways, and their contributions not only during infections but also in autoimmunity and cancer. our understanding of the potential differential roles of tlr in certain organs is in its infancy, and at least in liver and kidney, activation of tlr may induce autoimmune hepatitis and glomerulonephritis, respectively. triggering of tlr with specific ligands such as poly(i:c) is being evaluated to enhance the antiviral response. , , based on the emerging detrimental role of tlr in certain diseases, the question remains how therapeutic activation of tlr for antiviral purposes may aggravate pathogenesis in some individuals. we can probably expect the development of specific therapeutic approaches to diminish tlr -driven disease in the future. , and induce the activation of the transcription factors interferon (ifn) regulatory transcription factor (irf ), nuclear factor-jb (nf-jb) and activator protein- (ap- ) through two branches. tlr activation leads to (i) the development of an antiviral response mediated by irf activation and further type i ifn production; (ii) cell death through a fas-associated protein with death domain /caspase- -dependent and mitochondrion-independent pathway (rip / fas-associated protein with death domain); and (iii) the generation of a pro-inflammatory environment by the activation of nf-jb and ap- , and the mitogenactivated protein kinases (mapk) the extracellular signal-regulated protein kinase / (erk), the p map kinases (p ), and the c-jun nterminal kinase (jnk), which differentially regulate many cellular functions including inflammation-mediated inflammatory processes. despite the detrimental role of tlr in viral pathogenesis being poorly understood, three major mechanisms have been identified: (i) over-expression of tlr (only observed in some viral infections but not others); (ii) over-production of cytokines [tumour necrosis factor-a (tnf-a), interleukin- (il- ), il- p /p , and ifn-c], chemokines [regulated on activation, normal t-celll expressed and secreted (rantes), il- , and monocyte chemoattractant protein- (mcp- )], and the immune suppressive molecule programmed death ligand- (pdl- ); (iii) dysregulation of t helper type and (th /th ) polarization. however, the precise molecular mechanisms that lead to tlr hyper-responsiveness are unknown. overproduction of inflammatory mediators leads to dysregulation of leucocyte trafficking, blood-brain barrier (bbb) permeability, and mechanisms to be identified. a detrimental role of tlr has been identified in mice infected with west nile virus (wnv) and theiler's murine encephalomyelitis (tmev) (encephalitis), punta toro virus (ptv) (hepatitis), and in respiratory infections caused by respiratory syncytial virus (rsv), vaccinia virus, influenza a virus and rhinovirus rv b (pneumonia). in humans, tlr may contribute to glomerulonephritis in patients with chronic hepatitis c virus infection. finally, tlr hyper-responsiveness may play a detrimental role in virus-triggered autoimmunity. pattern recognition receptors and the innate immune response to viral infection mrna is an endogenous ligand for toll-like receptor tlr is an endogenous sensor of tissue necrosis during acute inflammatory events molecular evolution of vertebrate toll-like receptors: evolutionary rate difference between their leucine-rich repeats and their tir domains antiviral responses induced by the tlr pathway human cd + (bdca- ) + dendritic cells (dcs) represent a unique myeloid dc subset that cross-presents necrotic cell antigens dimerization of toll-like receptor (tlr ) is required for ligand binding unc b delivers nucleotidesensing toll-like receptors to endolysosomes structural basis of toll-like receptor signaling with double-stranded rna nucleic acid recognition by toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase cleavage of toll-like receptor by cathepsins b and h is essential for signaling two tyrosine residues of tolllike receptor trigger different steps of nf-jb activation toll-like receptor associates with c-src tyrosine kinase on endosomes to initiate antiviral signaling epidermal growth factor receptor is essential for toll-like receptor signaling tipe controls innate immunity to rna by targeting the phosphatidylinositol -kinase-rac pathway bruton's tyrosine kinase phosphorylates toll-like receptor to initiate antiviral response toll/ il- receptor domain-containing adaptor inducing ifn-b (trif) associates with tnf receptor-associated factor and tank-binding kinase , and activates two distinct transcription factors, nf-jb and ifn-regulatory factor- , in the toll-like receptor signaling recognition of double-stranded rna and activation of nf-jb by toll-like receptor mechanisms of the trif-induced interferon-stimulated response element and nf-jb activation and apoptosis pathways signaling of apoptosis through tlrs critically involves toll/il- receptor domain-containing adapter inducing ifn-b, but not myd , in bacteria-infected murine macrophages role of adaptor trif in the myd -independent toll-like receptor signaling pathway trif mediates toll-like receptor -dependent inflammatory responses to borrelia burgdorferi trif modulates tlr -dependent responses by inducing proteolytic degradation of tlr ddx , ddx , and dhx helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells new insights into the regulation of signalling by toll-like receptors and nod-like receptors shp- phosphatase negatively regulates the trif adaptor protein-dependent type i interferon and proinflammatory cytokine production the ubiquitin-modifying enzyme a is required for termination of toll-like receptor responses the human adaptor sarm negatively regulates adaptor protein trif-dependent toll-like receptor signaling traf-interacting protein (trip) negatively regulates ifn-b production and antiviral response by promoting proteasomal degradation of tank-binding kinase duba: a deubiquitinase that regulates type i interferon production sarm inhibits both trif-and myd -mediated ap- activation tag, a splice variant of the adaptor tram, negatively regulates the adaptor myd -independent tlr pathway integrin cd b negatively regulates tlr-triggered inflammatory responses by activating syk and promoting degradation of myd and trif via cbl-b triad a regulates ubiquitination and proteasomal degradation of rip following disruption of hsp binding trim negatively regulates tlr -mediated ifn-b signaling by targeting trif for degradation tripartite motif-containing protein negatively regulates tlr / -and rig-i-mediated ifn-b production and antiviral response by targeting nap e ubiquitin ligase tripartite motif negatively regulates tlr-mediated immune responses by proteasomal degradation of tnf receptor-associated factor in macrophages trif-mediated tlr and tlr signaling is negatively regulated by adam age-dependent tlr expression of the intestinal epithelium contributes to rotavirus susceptibility rip is an essential mediator of toll-like receptor -induced nf-jb activation de-ubiquitination and ubiquitin ligase domains of a downregulate nf-jb signalling a is a potent inhibitor of tlr -and sendai virus-induced activation of nf-jb and isre and ifn-b promoter the tumor suppressor cylindromatosis (cyld) acts as a negative regulator for toll-like receptor signaling via negative cross-talk with traf and traf cyld is a deubiquitinating enzyme that negatively regulates nf-jb activation by tnfr family members tank is a negative regulator of toll-like receptor signaling and is critical for the prevention of autoimmune nephritis pellino targets the irf pathway and facilitates autoregulation of tlr -and viral-induced expression of type i interferons ubiquitin-specific protease mitigates toll-like/interleukin- receptor signaling and regulates innate immune activation the orphan nuclear receptor shp acts as a negative regulator in inflammatory signaling triggered by toll-like receptors nlrx protein attenuates inflammatory responses to infection by interfering with the rig-i-mavs and traf -nf-jb signaling pathways immune evasion by hepatitis c virus ns / a proteasemediated cleavage of the toll-like receptor adaptor protein trif disruption of tlr signaling due to cleavage of trif by the hepatitis a virus protease-polymerase processing intermediate, cd the coxsackievirus b c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling cleavage of the adaptor protein trif by enterovirus c inhibits antiviral responses mediated by toll-like receptor kaposi sarcoma-associated herpesvirus degrades cellular toll-interleukin- receptor domain-containing adaptor-inducing b-interferon (trif) enterovirus induces degradation of trim , a potential e ubiquitin ligase severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk /ikke complex the poxvirus protein a r targets toll-like receptor signaling complexes to suppress host defense tlr deficiency in patients with herpes simplex encephalitis impaired intrinsic immunity to hsv- in human ipsc-derived tlr -deficient cns cells herpes simplex virus encephalitis in a patient with complete tlr deficiency: tlr is otherwise redundant in protective immunity induction of interferon-c contributes to toll-like receptor -mediated herpes simplex virus type inhibition in astrocytes inborn errors of anti-viral interferon immunity in humans a missense mutation of the toll-like receptor gene in a patient with influenza-associated encephalopathy a functional toll-like receptor gene (tlr ) may be a risk factor for tick-borne encephalitis virus (tbev) infection activation of chemokine and inflammatory cytokine response in hepatitis c virus-infected hepatocytes depends on toll-like receptor sensing of hepatitis c virus double-stranded rna intermediates novel role of toll-like receptor in hepatitis cassociated glomerulonephritis association of toll-like receptor polymorphisms with chronic hepatitis b and hepatitis b-related acute-on-chronic liver failure toll-like receptor genetic variants and susceptibility to hepatocellular carcinoma and hbv-related hepatocellular carcinoma toll-like receptor gene polymorphisms and severity of pandemic a/h n / influenza in otherwise healthy children a role for microrna- modulation in the anti-hiv- effects of toll-like receptor stimulation in macrophages tlr ligation activates an antiviral response in human fetal astrocytes: a role for viperin/cig polyriboinosinic polyribocytidylic acid [poly(i:c)]/tlr signaling allows class i processing of exogenous protein and induction of hiv-specific cd + cytotoxic t lymphocytes a common polymorphism in tlr confers natural resistance to hiv- infection expression of toll-like receptors by human muscle cells in vitro and in vivo: tlr is highly expressed in inflammatory and hiv myopathies, mediates il- release and up-regulation of nkg d-ligands inflammatory events at blood-brain barrier in neuroinflammatory and neurodegenerative disorders: implications for clinical disease nile virus infection and immunity toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis toll-like receptor has a protective role against west nile virus infection the toll-like receptor -mediated antiviral response is important for protection against poliovirus infection in poliovirus receptor transgenic mice tlr signaling is either protective or pathogenic for the development of theiler's virus-induced demyelinating disease depending on the time of viral infection herpes simplex virus infections of the central nervous system: encephalitis and meningitis, including mollaret's tlr deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of cns infection in mice cutting edge: priming of cd t cell immunity to herpes simplex virus type requires cognate tlr expression in vivo does toll-like receptor play a biological role in virus infections? mda and tlr initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness detrimental contribution of the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia respiratory syncytial virus induces tlr protein and protein kinase r, leading to increased double-stranded rna responsiveness in airway epithelial cells double-stranded rna induces similar pulmonary dysfunction to respiratory syncytial virus in balb/c mice tlr increases disease morbidity and mortality from vaccinia infection toll-like receptor is an essential component of the innate stress response in virus-induced cardiac injury rna sensor-induced type i ifn prevents diabetes caused by a b cell-tropic virus in mice a critical link between toll-like receptor and type ii interferon signaling pathways in antiviral innate immunity th regulation of viral myocarditis in mice: different roles for tlr versus trif in progression to chronic disease toll-like receptor signaling on macrophages is required for survival following coxsackievirus b infection a trif-independent branch of tlr signaling tlr deletion limits mortality and disease severity due to phlebovirus infection immunoprivileged status of the liver is controlled by toll-like receptor signaling toll-like receptor promotes cross-priming to virus-infected cells antiviral innate immunity disturbs podocyte cell function protective role of toll-like receptor -induced type i interferon in murine coronavirus infection of macrophages nlrx negatively regulates tlr-induced nf-jb signaling by targeting traf and ikk rpl and snm wrote the manuscript. figs and were created by rpl and snm respectively. we apologize to any investigators whose work we have omitted due to space limitations. we acknowledge funding to the authors' laboratory by public health service grants to snm (ns , ai and dk from the national institute of neurological disorders and stroke, the national institute of allergy and infectious disease, and the national institute of diabetes and digestive and kidney diseases, respectively). the founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. diana winters (drexel university college of medicine academic publishing services) is acknowledged for manuscript editing. none. key: cord- -osstpum authors: nan title: abstracts oral date: - - journal: am j transplant doi: . /j. - . . .x sha: doc_id: cord_uid: osstpum nan abstracts evl (target - ng/ml), aza or mmf with standard (sd) or reduced (rd) csa. data at months post-tx is presented here. results. the proportion of patients with cmv events, including serious adverse events and laboratory evidence of cmv infection, has remained low and broadly similar following introduction of cc administration of evl and use of concomitant rd-csa. cmv syndrome and cmv organ involvement occurred in < % of recipients receiving evl in each of the studies. the highest rates of all cmv parameters occurred with mmf + sd-csa or aza + sd-csa. conclusion. the low incidence of cmv infection and cmv-related events observed in heart tx patients receiving fd-evl and sd-csa as de novo immunosuppression has been maintained in newer regimens of cc-evl with rd-csa. cmv syndrome and cmv organ involvement are rare with evl-based immunosuppression after cardiac tx. the low rate of cmv infections in evl-treated patients may contribute to improved long-term outcome and a lower incidence of cav; confirmatory data are awaited. fibronectin-α β interactions enhance p mapk phosphorylation and metalloproteinase- expression in cold liver ischemia/reperfusion injury. sergio duarte, xiu-da shen, takashi hamada, constantino fondevila, ronald busuttil, ana j. coito. the dumont ucla transplant center. expression of endothelial fibronectin (fn) is an early event in liver ischemia/reperfusion (i/r) injury. we have recently shown that cs peptide facilitated blockade of fn-α β leukocyte interactions regulates metalloproteinase- (mmp- ) expression in steatotic orthotopic liver transplants (olt). this study tests the function of the cs peptide therapy upon mmp- , and further dissects putative mechanisms, in an alternate model of cold ischemia/reperfusion (i/r) injury. methods and results: cs peptides were administrated through the portal vein of sprage-dawley (sd) rat livers before and after h cold storage ( µg/rat). sd recipients of olts received an additional dose of cs peptides h post-olt. cs therapy significantly increased the d olt survival rate ( % vs. %, n= /gr, p< . ). cs peptides reduced sgot levels (u/l) at h ( ± vs. ± p< . ) and h ( ± vs. ± , p< . ) post-olt. cs treated olts showed good preservation of lobular architecture, contrasting with severe necrosis and sinusoidal congestion in controls. moreover, cs treated livers were characterized by a profound decrease in t ( ± vs. ± , p< . ), nk ( ± vs. ± , p< . ) and ed ( ± vs. ± , p< . ) cells as early as h after i/r. neutrophils, as indicated by mpo activity ( . ± . vs. . ± . , p< . ) were depressed by cs therapy. this correlated with decreased mrna expression of tnf-α ( . ± . vs. . ± . , p< . ) and cycloxygenase- ( . ± . vs. . ± . , p< . ) . leukocyte transmigration is dependent upon adhesive and focal matrix degradation mechanisms. mmp- , which is inducible and expressed by infiltrating leukocytes, was profoundly depressed in h cs- peptide treated olts, at both mrna ( . ± . vs. . ± . , p< . ) and protein ( . ± . vs. . ± . , p< . ) levels. moreover, mmp- activity evaluated by zymography was reduced by ∼ -fold in the cs- group. interestingly, phosphorylation of mitogen-activated protein (map) kinase p ( . ± . vs . ± . , p< . ) was selectively downregulated in the h cs- treated olts. in conclusion, this work supports a broad regulatory role for fn-α β interactions on mmp- expression by leukocytes, likely mediated through activation of p mapkinase, and matrix pathological breakdown associated with leukocyte infiltration. this data provides the rationale for the development of novel therapeutic approaches in cold liver i/r injury. ischemia reperfusion injury is the most common cause of acute kidney injury in both native and allograft kidneys. the pathogenic mechanisms of renal ischemia reperfusion injury include changes at the level of the microvasculature as well as the tubules. within the microvasculature, leukocyte-endothelial interactions likely play a role and contribute to the well-established microvasculature dysfunction (e.g., endothelial permeability) and alterations in endothelial-leukocyte interaction occur during ischemic acute kidney injury. our previous studies have demonstrated that t cells modulate renal ischemia reperfusion injury in a murine model, we hypothesized that t cells could mediate changes in renal vascular permeability during ischemia reperfusion injury. we performed a min bilateral renal ischemia followed by reperfusion in c bl wild type mice and in t cell deficient (nu/nu) mice with or without t-cell adoptive transfer from their wild type littermates and evaluated rvp by evans blue dye extravasations (ebde). the time course studies of rvp showed marked increases in renal ebde within the early - hrs after ischemia. cd positive pan t-cells but neither cd nor cd t cells were found to infiltrate into post ischemic kidney within hrs, and comparison was made with other leukocytes using immunohistochemistry technique. gene microarray analysis demonstrated that the gene for tnf-α (tnfaip ), a potent mediator of microvascular permeability as well as a t cell product, was increased in the kidney early after ischemia. tnf-α, ifn-γ and il- protein by an intracellular cytokine staining technique was found increased early in peripheral circulating t cells and later in renal t cells after renal ischemia. the rise in rvp was significantly attenuated in t cell deficient mice nu/nu at hrs compared to the wild type littermates and this attenuated rvp in t cell deficient mice was restored after adoptive transfer of splenic t cells from wild type littermates into these nude mice. these data demonstrate that t cells traffic early into post ischemic kidney, produce tnf-α and other cytokines that can increase rvp, and directly participate in the increased rvp during acute ischemia reperfusion injury. t cell-endothelial interactions are a likely mechanisms underlying the pathophysiologic role of t cells in renal ischemia reperfusion injury . background: ischemia/reperfusion injury (iri) is a major cause of organ dysfunction after intestinal injury and transplantation. the interaction between the innate and adaptive immune systems has become a major area of focus in this field. our preliminary work showed that mice undergoing intestinal iri experienced worse survival and tissue injury in conjunction with increased infiltration of pmn and cd + cells as compared to sham mice. the purpose of this study was to investigate the role of the t cell in intestinal iri through the use of genetically deficient mice. methods: under anesthesia, male c bl wild-type mice (wt) and cd knockout mice (cd ko) underwent min of warm intestinal iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at h and h. tissue was analyzed by histology, cd immunostaining, myeloperoxidase activity (mpo), and semi-quantitative pcr for several cytokines/chemokines. results: wt had significantly worse survival compared to cd ko ( % vs. %, p= . ), and worse histopathological injury mostly involving mucosal sloughing. wt had higher mpo activity than cd ko ( . ± . vs. . ± . at h, p= . , and . ± . vs. . ± . at h, p= . ) and increased cd + cell infiltration. there was also increased mrna production of cytokines/chemokines in wt vs. cd ko at both timepoints; specifically, the data with respect to b-actin at h was: il- ( . conclusion: this study demonstrates for the first time in the intestine that cd + t cells are important mediators of iri. in the absence of cd + t cells, better outcomes were associated with less pmn infiltration implying a link between the innate and adaptive immune systems. an alteration in chemotactic signaling potentially effected by cd + t cells may play a role in this process. these results confirm the important function of cd + t cells in intestinal iri and justify further investigation into their complex role in this process. messenger rna assessment in urine sediments from renal transplant patients is rapidly evolving as a non-invasive diagnostic tool. t cells, macrophages, and often b cells are present in rejecting kidney grafts. we questioned whether urinary mrna levels of markers representing these cell types ((regulatory) t cells: cd ε, foxp , cd , tgf-ß; macrophages: cd , s a ; b cells: cd ) are associated with rejection. materials in our institute urine samples a year from over renal transplant patients are being collected for mrna quantitation purposes. urine sediments are pelleted and stored in rna-preserving solution. we initially investigated the effect of incubation time of urine on mrna integrity. next, in a case-control study urine samples taken at time of biopsy-proven rejection were compared to samples taken during stable graft function. median time of sampling ( days versus days posttansplant) did not significantly differ between groups. rna ( . ± . µg) was extracted using rneasy spin columns. message of the markers mentioned above was quantified with q-pcr and normalized. results incubation of urine for h or longer at room temperature after acquisition resulted in a % decrease in s rrna signals (p< . ). however, storage of urine for up to h at ºc did not result in decreased mrna expression. in the case-control study, all markers tested showed the highest expression levels in urine rejection samples. when corrected for multiple comparisons, only tgf-ß ( -fold, p= . ) and foxp ( -fold, p= . ) expression was significantly increased in rejection samples compared to non-rejection samples. tgf-ß levels highly correlated with foxp levels (r = . , p< . ), suggesting a mechanistic relationship in vivo between the two molecules. conclusion rna integrity in urine samples stored at ºc is maintained for at least h. detection of increased tgf-ß and foxp message in urine from patients with a kidney transplant represents a means for indicating occurrence of graft rejection. this implies that mrna urinalysis renders a suitable molecular tool for non-invasive patient monitoring in clinical practice. the data furthermore suggest that rejection is associated with tgf-ß-mediated immune mechanisms in which regulatory t cells play a role. the significance of b cell and plasma cell infiltration in renal allografts remains controversial. we previously established that transcript sets associated with ifng effects or t cells reflect the inflammatory burden in renal allografts. in the present study, we identified b cell associated transcripts (bats) and immunoglobulin transcripts (igts), reflecting b cell and plasma cell infiltration, respectively. using microarrays, we analyzed bat and igt expression in relationship to histologic lesions, diagnosis, and renal function in renal allograft biopsies. immunostaining confirmed that bat and igt expression was associated with b cells and plasma cells in the graft. expression of bats and igts was increased in biopsies with rejection ( . ± . , . ± . ) compared to non-rejection ( . ± . , . ± . ), but was not different between t cell ( . ± . , . ± . ) and antibody mediated rejection ( . ± . , . ± . ) and also occurred in some non-rejecting biopsies (recurrent gn). bat and igt scores correlated strongly with time post transplant (fig ), which was best modeled as a dichotomous relationship: biopsies ≤ months did not express bats or igts above the level of control kidneys. other inflammatory markers were not time dependent. in biopsies ≥ months, bat and igt expression correlated with interstitial inflammation, tubular atrophy, and interstitial fibrosis. in a multiple regression analysis, only time post transplant and interstitial inflammation were independently related to bat and igt scores. when correcting for time post transplant, bat and igt scores did not correlate with renal function at the time of biopsy or future function months post biopsy. bats and particularly igts are a time dependent feature of injured and inflamed renal allografts. corrected for the effect of time, bats and igts do not correlate with outcomes, indicating that b cell and plasma cell infiltrates have no specific role for the mechanism of injury independent of the inflammatory burden. their accumulation in late allografts may indicate emergence of specialized lymphoid compartments in tissues with long standing low level inflammation. pearson correlation coefficient between pbts expression and renal function pbts egfr at %change in egfr from biopsy months after biopsy baseline to biopsy biopsy to months after qcats - . * - . * - . * - . grits - . ** - . ** - . ** - . irit_d - . - . - . . irit_d - . ** - . ** - . ** . * irit_d - . ** - . ** - . * - . kt . ** . . ** - . * p< . , ** p< . thus the pbts have prognostic value. surprisingly, the transcripts in the biopsy with greatest prognostic value for future gfr or recovery of gfr were not related to rejection (cytotoxic t cell or ifng associated) but to the degree of injury response. we propose that the common pathway linking various injuries (immune and non immune) to gfr is through the degree of injury response these events induce in the epithelium, particularly those in the irit_d set. early rejection from anamnestic response in offspring-to-mother or husband-to-wife kidney transplant. kwan tae park, song chul kim, duck jong han. surgery, asan medical center, seoul, korea. accelerated rejection can be developed in the immediate post-transplant period resulting from anamnestic response due to the exposure to fetal hla antigen during the previous pregnancy in case of offspring-to-mother or husband-to-wife. however, accelerated rejections in these groups have been rarely reported. cases of offspring-to-mother (offspring group) and cases of husband-to-wife (spouse group), who has been sensitized to spouse via their children, underwent kidney transplants from january of to august of at our institution and retrospectively reviewed. control group was female kidney recipients transplanted at the same period from living related donors other than offspring and from living unrelated donors other than husband. acute rejection (ar) rate within months after transplant were . % ( / ) in offspring group and . % ( / ) in spouse group. the ar rates were not different between the two groups, however they were significantly higher than control group in both groups, namely . % ( / ) for offspring control and . % ( / ) for spouse control. the mean onset of ar was . day ( - ) and it was significantly later than that of control groups ( . day, p< . ) and % of ar were accelerated rejections within days after transplant. proportion of acute humoral rejection was significantly higher in both groups than control group ( . % vs . % in offspring group, % vs % in spouse group). most of the ar was successfully reversed by steroid pulse and/ or plasmapheresis, ivig, rituximab. any risk factors for the ar such as number of pregnancy, preoperative cdc cross matching, pra, immunosuppressant and antibody induction couldn't be identified. serum creatinine level in ar patients were higher than ar free patients by postoperative month, but last follow up creatinine level didn't show any statistical difference ( . mg/dl in ar vs . mg/dl in ar free). year graft/patient survival rate were not different between ar and ar free groups and study and control groups. risk of higher rejection rate accompanied with higher accelerated and humoral rejection was identified in female kidney recipients from offspring or spouse donor in comparison with control group. considering the anamnestic response of this cohort of female recipients, more prudent preoperative screening and careful immediate postoperative immune monitoring are required for the avoidance of rejection and impaired graft survival. kaplan-meier survival curves showed that ∆upc > . were associated with decreased patient and allograft survival. these data show for the first time that regardless of the histopathology, ∆upc > . one month after rejection is associated with poor patient/allograft outcomes. from histology to microarrays the histopathological hallmark of t cell mediated rejection (tcmr), is interstitial infiltration. assessing infiltration by histology is arbitrary, limited in reproducibility, and has never been assessed against independent standards. we recently reported that a set of cytotoxic t cell-associated transcripts (qcats) could quantitatively assess the t cell burden in tissue. objective of the present study was to re-examine the current diagnostic criteria in relationship to the qcats. in renal allograft biopsies, we assessed how histology predicts the qcat burden. an independent diagnostic threshold for qcats was established in control samples. applying this qcat threshold revealed current diagnostic criteria for histology to be flawed; the threshold for interstitial infiltration is too high ( % specificity, % sensitivity), the types of infiltration to be considered (i.e. i-banff) are wrongly defined, and tubulitis is not increasing diagnostic accuracy. changing the criteria by lowering the histological threshold for cortical infiltration from % to %, taking into account all interstitial cellular infiltration (= i-total) and ignoring tubulitis increased sensitivity to % with a decreased specificity of %. but, this includes biopsies having interstitial infiltration without qcats, indicating that histology might not discriminate between 'active' and 'inactive' infiltration. both the refined histological and the qcat threshold had prognostic value in terms of future renal allograft function. we propose a refined histological scoring system that better predicts the active t cell burden and outcome than the current banff criteria for renal allograft rejection. there has been an increasing and well justified interest regarding the long term renal consequences of kidney donation. the collective evidence suggests, however, that kidney donors enjoy a normal life span and their lifetime risk of esrd may not be different from non-kidney donors. assessing kidney function utilizing serum creatinine is not without limitations. therefore, to better assess kidney function, yeas ago, we began a large effort to measure gfr using the plasma disappearance of iohexol and first void urinary albumin/creatinine ratio (acr) in randomly selected kidney donors who donated at our institution. results: we have performed over donor uninephrectomies since the inception of our program in . of these, donors underwent iohexol gfr at our general clinical research center. microalbuminuria is defined as acr between - mg/g and macroalbuminuria as acr> mg/g. the mean age was . ± . years, . ± . years have elapsed since donation, hemoglobin was . ± . g/l, systolic blood pressure (sbp) was . ± . mmhg and diastolic blood pressure (dbp) was . ± . mmhg. . % of donors had a gfr greater than ml/min/ . m and . % had a gfr between - ml/min/ . m . the detailed renal profile of these donors is shown in the table below. multivariate analysis that adjusted for age, gender, ethnicity, time from donation, sbp, dbp and body mass index (bmi) identified age at donation, female gender and bmi as independent predictors for gfr< ml/min/ . m and time from donation and systolic blood pressure as independent predictors for micro and macroalbuminuria. conclusion: this is the largest effort describing measured gfr in previous kidney donors. it is reassuring to find out that the majorities have a preserved gfr and only a minority has albuminuria. the risk factors for reduced gfr and albuminuria are analogous to what has been described in the general population. cystatin c levels and long-term donor health markers cystatin-c < (n= ) psychosocial and physical health of lkds following donation is of utmost importance. unfortunately, this issue has been neglected to a large extent. we sought to examine the current practices regarding psychosocial follow-up of lkds after surgery across transplant (tx) centers. we conducted a -question online survey regarding this practice and issues related to lkd. the survey was e-mailed via listservs of professional tx societies. several questions allowed for more than one response. characteristics of the tx centers that participated in the survey are found in table . only . % actively follow donors regarding mental health, substance abuse, or quality of life issues after donation. the professionals most often involved are social workers ( %) and nurse coordinators ( %). the majority of follow-up is conducted via phone ( . %); though appointments ( . %) and questionnaires ( . %) are also utilized. % initiate follow-up with donors - weeks post-operatively, whereas only % of programs maintain contact at both - months and months. . % offer post-operative psychosocial support; % only under certain circumstances. this support is provided by social workers ( %), psychologists ( %), and/or psychiatrists ( %). support is available indefinitely in % of programs. . % assume the costs of post-operative medical and psychosocial care indefinitely; . % for a specified period of time. . % bill the recipient's insurance and . % bill the donor's insurance. . % of centers accept donors without health insurance and only . % purchase insurance on behalf of donors to cover post-operative health care needs. the results of this survey clearly demonstrate that post-operative psychosocial follow-up of lkds is uncommon and that current practices are widely variable. without routine follow-up of donors, tx centers are less likely to capture post-operative psychosocial issues that may result from organ donation. standardized post-operative follow-up of lkds should become a mandatory part of their care, which will require increased support from health care policy makers. the role of hepatitis c and race in patient and graft survival in combined kidney and liver transplantation. dilip moonka, ravi k. parasuraman, kim a. brown, alissa kapke, dean y. kim. gastroenterology, henry ford health systems, detroit, mi; nephrology, henry ford health systems, detroit, mi; biostatistics, henry ford health systems, detroit, mi; transplant institute, henry ford health systems, detroit, mi. the influence of hepatitis c (hcv) and race are not well understood in combined kidney-liver transplant (klt). hcv has a negative impact on patient and graft survival in liver recipients whereas african-american (aa) race is a negative prognostic factor in kidney recipients. aim: to determine the influence of hcv and aa race on patient and graft survival in klt. methods: the unos public use database was used to identify patients undergoing klt who had known hcv ab status. % were aa and % were non-hispanic white (nhw). . % were hcv ab positive. groups were assessed for patient and graft survival. results: there is a significant gradient in patient survival and both kidney and liver survival from nhw patients without hcv to aa patients with hcv (table) . the patient survival at five years drops from % to % along this gradient. there is also a % drop in liver and an % drop in kidney graft survival. on pairwise testing, the difference in patient survival at yr between all patients without hcv and those hcv ab positive drops from % to % (p= . ). the difference in yr survival between all nhw and aa patients (regardless of hcv status) drops from % to % (p= . ). on multivariate analysis, hcv and the combination of hcv and aa race remains associated with diminished survival but race alone does not. conclusions: patients with hcv who undergo a klt transplant are at increased risk for poor overall survival and poor survival of kidney and liver grafts. this effect appears even more pronounced in aa patients. this knowledge is critical to individual centers in assessing risk and benefit from klt in these groups and these groups represent an opportunity for improved interventions. kidney: pediatrics background: pediatric renal transplant recipients have excellent short-term outcomes but long-term success is compromised by complications of chronic immunosuppressive medications and chronic allograft nephropathy. studies show that calcineurin inhibitors and steroids can be individually avoided in pediatric renal transplantation. building on that experience we designed this study to optimize short and long-term renal allograft function with minimal chronic immunosuppression using a steroid-free, calcineurininhibitor withdrawal protocol in low risk pediatric renal transplant recipients. methods: unsensitized pediatric recipients of a first living donor kidney transplant received doses of campath- h ® ( . mg/kg), day pre-and post-transplant. subjects received tacrolimus and mmf immediately post-transplant until week - when they underwent protocol renal biopsy and were changed to sirolimus and mmf if rejection free. the planned subjects have been enrolled; this report describes the clinical outcomes of the with year of follow up. results: the mean subject age is . yrs; . % are female and . % caucasian. at transplant, / were cmv seronegative and / were ebv seronegative. protocol therapy was discontinued in eight subjects due to: rejection ( ), mouth ulcers ( ), leukopenia ( ) , unrelated ( ). clinical acute rejection (ar) occurred in subjects ( %) and had subclinical ar; ar was cellular rejection in subjects, and humoral in at days post-transplant who had an undetected positive crossmatch to class ii hla. there were two graft losses, one due to recurrent fsgs and one due to medication non-adherence. there were no cases of ptld and no deaths. leukopenia occurred in subjects ( . %). there were infections of which were urinary tract infections ( . %) and were pneumonia ( . %). conclusions: minimization of immunosuppression using a steroid-free, calcineurinwithdrawal protocol in low risk pediatric renal transplant recipients appears to be well tolerated with acceptable rates of clinical ar and no serious infections months after transplantation. male; % white). substantial increases in bmiz were observed within the first mo.; no changes were seen from to mo. baseline bmiz category (<- . , - . to + . , >+ . ) influenced the pattern of change. subjects with low bmiz (<- . ) at baseline experienced the greatest increases in bmiz, but overweight was rare; increases tended to result in a bmiz in the healthy range. those with high bmiz (>+ . ) at baseline demonstrated no significant change in bmiz post-tx. younger age at tx (highest risk for those to y. at tx) more remote date of tx, and baseline bmi between the th and th percentiles were significant independent risk factors for unhealthy weight gain both at mo. and persisting at mo. post-tx. weight gains occur early after tx and tend to persist. counseling focused on prevention of weight gain should be a routine part of post-tx care, with the most intense efforts concentrated on the highest risk patients: young, healthy weight children. avoidance of early weight gains may have an important impact on bmi over the long term. referral. lindsey a. pote, jennifer trofe, erin h. wade, jorge baluarte, alden doyle, simin goral, karen warburton, robert grossman, jo ann palmer, roy d. bloom. pharmacy, hosp of the univ of penn; nephrology, univ of penn; transplant surgery, children's hospital of philadelphia. intro: few data exist on outcomes in pediatric kidney recipients who transition to an adult transplant program. purpose: to examine outcomes in pediatric kidney recipients who transition to an adult transplant program and to identify characteristics associated with non-adherence related graft loss. methods: retrospective, single center analysis of pediatric kidney recipients who transitioned to an adult program. results: for the cohort overall, transition to the adult program occurred a mean of ± . months following transplantation. mean serum creatinine at transition was . ± . mg/dl and mean patient age . ± . years. % of patients received living donor kidneys. within ± . months of transition, ( . %) of patients experienced graft loss. causes of graft loss included admitted non-adherence (n= ), recurrent disease (n= ), chronic progressive graft dysfunction (n= ) and bk nephropathy (n= ). graft loss occurred a mean of ± months post transition for non-adherent patients and . ± months for adherent patients (p=ns). the characteristics of the cohort is shown in the table according to whether or not patients had documented non-adherence related graft loss. conclusions: ) graft loss commonly occurs within years following transition and is attributable to both patient non-adherence and late referral by the pediatric transplant program, ) non-adherence related graft loss was more common in males, ) factors unassociated with non-adherence include ethnicity, prior transplantation, age at transplant, & duration of dialysis, ) the development of collaborative pediatric-toadult transition clinics may enhance adherence and lead to improved graft outcomes in this population. this -year, prospective randomized pilot study compares the effect of conversion to sirolimus (srl) vs. continued mycophenolate meofetil (mmf) in patients with chronic allograft nephropathy (can), on histological progression. we present -year data on safety and renal function. participants > year post-transplant with can (banff ≥ci , ct ) on tacrolimus, mmf and prednisone were randomized to continue mmf or convert to srl (target - ng/ ml). tac dose was minimized (target - g/l), and renal biopsy performed at baseline, , years. -month interval monitoring included adverse event (ae) reporting, egfr (schwartz) and immunosuppressant levels. / (mmf= , srl= ) have completed -year follow-up. baseline gender, ethnicity, previous acute rejection episodes, can grade, proteinuria (upcr) were similar. srl had lower baseline egfr ( ± vs. ± ml/min/ . m , p= . ). aes were reported (mmf= , srl= ), serious ae (sae: mmf= , srl= ). common saes were similar: dehydration & elevated creatinine ( ), gastroenteritis ( ) and rejection (ar). episodes ( patient) of ar occurred in mmf group and ( patients) in srl group, all attributable to non-adherence. there were no sustained change in electrolytes, hg and total wbc counts in the st year, except neutrophil counts were lower in the srl group ( mo: mean . vs. . , p< . ). platelets were significantly lower at months (srl) but not thereafter ( month: mean ± vs. ± x e ). cholesterol and tg increased early (p< . ), but only cholesterol persisted after years (mean . vs. . mmol/l, p< . ). in srl group only, upcr increased over years (∆upcr ± mg/mmol, p< . ). this was not associated with hypoalbuminemia at years. egfr was lower in srl vs. mmf after months (p< . ), but the rate of change in egfr from baseline was similar between groups (- ± vs. - ± ml/min/ . m , p=ns). serious adverse events did not differ significantly between the srl and mmf groups. sustained srl treatment was associated with mild neutropenia, hypercholesterolemia and proteinuria after years. egfr was reduced at baseline compared with mmf, and both groups had similar rates of decline in gfr over time. allograft recipients. maarten naesens, oscar salvatierra, li li, minnie sarwal. department of pediatrics, stanford university school of medicine, stanford, ca. background: in contrast to adult kidney recipients, little is known about the long-term evolution of tacrolimus pharmacokinetics in pediatric kidney transplant recipients. methods: one-hundred five pediatric recipients of a kidney allograft, all treated with a corticosteroid-free immunosuppressive protocol, were included. the evolution of tacrolimus doses and exposure was recorded at , , , , and months after transplantation, as well as all pre-dose trough levels (c ; n= ) obtained in the first years after transplantation. results: dose-corrected tacrolimus exposure (c /dose/kg) increased in the first years after kidney transplantation in pediatric recipients (table , figure ). this decrease in dose requirement by time was only significant in children older than years at the time of transplantation ( figure ). in addition, the younger patients had significantly higher dose requirements compared to older recipients, which translated in marked underexposure in - % of patients < years of age in the first days after transplantation. conclusion: pediatric kidney transplant recipients exhibit maturation of tacrolimus pharmacokinetics with time after transplantation. this can not be explained by differences in corticosteroid use, as all patients were treated with a corticosteroid-free protocol. the higher dose requirements for younger recipients and the absence of tacrolimus maturation in the youngest recipients suggest that age-dependent changes in tacrolimus intestinal first-pass effect, metabolism or distribution play a role. whether age-specific tacrolimus dosing algorithms will improve outcome needs further study. determinants of dose-corrected pre-dose trough levels (c /dose/kg) aih may present as acute hepatitis in % of patients (pts) and result in alf. early administration of corticosteroids may obviate the need for liver transplantation (lt), but features which identify aih in pts with alf have not been determined. aim: to identify clinical and histological features which distinguish alf due to aih. methods: / pts in the alf study group registry had no evidence of viral, metabolic, vascular, and drug/toxic liver injury. all had alf defined by acute disease, encephalopathy, and coagulopathy. based upon admission clinical features, / had probable aih, and were considered "indeterminate." liver biopsies (lbx) available from pts were reviewed by a blinded expert hepatopathologist. clinicopathologic correlations were analyzed retrospectively. results: all lbx available from pts with suspected aih had classical histologic features of aih. moreover, / ( %) lbx from pts with indeterminate alf also had aih features: extensive necrosis ( %), fibrosis ( %), cirrhosis ( %), interface hepatitis ( %), and plasma cell-rich inflammation ( %). of all lbx with aih features, centrilobular lesions associated with acute, severe aih (am j surg path ; : ) were frequent: plasma cellrich central venulitis ( %), exclusive centrilobular necroinflammation ( %), and pericentral dilatation/congestion ( %). clinical characteristics and outcomes of the pts with aih based upon laboratory and histologic findings differed significantly from pts whose alf remained indeterminate: aih pts were older ( v y), predominantly female ( v %), and had longer jaundice-encephalopathy interval ( v d) , lower alt ( v u/l), higher globulins ( . v . g/dl), lower creatinine ( . v . mg/ dl), and a higher prevalence of ana ( v %) and asma ( v %) (all p<. ). although spontaneous survival did not differ, more aih pts underwent lt ( v %), and more survived month after enrollment ( v %; p<. ). conclusions: using histologic criteria to classify pts with indeterminate alf, aih accounted for at least % of the alf study group registry, and represented % of indeterminate alf. lbx should be performed in all patients with alf of obscure etiology, in particular to identify centrilobular necroinflammatory lesions, which appear to be specific indicators of acute and severe aih. (u- from niddk). ( ) ( ) ( ) ( ) ( ) . mikel gastaca, miguel montejo, lluis castells, antonio rafecas, antonio rimola, ramon barcena, federico pulido, magdalena salcedo, martin prieto, manuel de la mata, jose r. fernandez, jose m. miro, the spanish lt in hiv-infected patients working group. hospital de cruces, bilbao, spain; hospital vall d´hebron, barcelona, spain; barcelona, spain; univ. of barcelona, barcelona, spain; hospital ramon y cajal, madrid, spain; hospital de octubre, madrid, spain; hospital gregorio marañon, madrid, spain; hospital la fe, valencia, spain; hospital univ. reina sofia, cordoba, spain. background and aim: we report on the preliminary results of the prospective multicenter spanish study in hiv- infected patients who underwent olt. methods: the prospective multicenter spanish study fipse-olt-hiv -gesida - was initiated in january . inclusion criteria follows the rules previously described in the spanish consensus document. hiv-infected patients transplanted between january and december were included in this study. results: olt were consecutively performed in hiv- infected patients in the period of the study. median (iqr) follow-up is ( - ) months. median (iqr) age was years ( - ), % of the recipients were male and former drug abuse was the most common hiv- risk factor ( %). % of the patients were transplanted due to hcv-related cirrhosis and % due to hbv cirrhosis. median (iqr) meld score was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . pre-olt median (iqr) cd cell count was ( - ) cells/mm and %patients had undetectable plasma hiv viral load. immunosuppression was based on tacrolimus in % of the patients. haart was re-started in a median (iqr) time of ( - ) days after olt and was based on efavirenz in % of the cases. acute rejection occurred in patients ( %). twenty patients died ( . %) mainly due to hcv recurrence ( %). antiviral therapy with peg-interferon plus ribavirin was initiated in patients obtaining a sustained viral response in of them ( %). patient survival ( % confidence intervals) at , , and years was % ( - %), % ( - %), % ( - %) and % ( - %), respectively. conclusion: for selected hiv- infected patients under haart therapy, olt is a safe and effective procedure at mid-term. as in the hiv-negative population, hcv recurrence is the mayor cause of concern and response to antiviral therapy is still disappointing. hepatitis c virus and objectives: describe the -year experience of a university hospital treating chronic hepatitis c relapse, histologically diagnosed after liver transplant. material & methods: from september through july , all the patients subject to liver transplant with histological diagnostics of chronic hepatitis relapse were submitted to antiviral treatment. treatment was suspended if there was a severe adverse reaction to the drugs used, non-adherence, severe rejection or no response after at least months. hcv positive serology patients, alt increase ( , x nl) and hepatic biopsy with metavir rating showing structural and/or periseptal or parenchymatous portal inflammatory activity /= were included. from to , all patients were treated with conventional interferon and ribavirine, regardless of the genotype. from october on, patients with genotype were treated with ribavirine and pegylated interferon and genotype were treated with conventional interferon and ribavirine. results: patients were treated during this time period, thirty-seven male ( . %). their average age was . . % were genotype , . % were genotype . average time between transplant and beginning of treatment was months. average treatment was ( - ) months. . % received conventional interferon, . %, pegylated alpha a and . %, pegylated alpha b. . % had one or more cellular rejection episodes before vhc relapse diagnostics and were treated with corticosteroids. sustained virological response (svr) occurred in . %. three patients had a virological response at the end of treatment (evr) and have not completed six months post treatment. out of patients with svr, . % were genotype , . % genotype and . % were not genotyped. considering genotypes, svr was detected in . % with genotype , in % with genotype . srv was detected when we examined hepatic biopsy, in . % of the f /f patients ( out of ), . % of the f patients ( ), . % of the f patients ( ) . survival period of years for svr patients was % ( out of ) and . % for patient without svr (p = . -kaplan-meier). five-year survival was . % for patients with genotype and % for patients with genotype . conclusions: our results show it is possible to get good five-year survival rates for treated patients. however, handling adverse reactions and long term treatment still pose difficulties in pursuing better svr rates. the impact of alcoholic liver disease (ald) and background: although liver transplant survival benefit has been shown to be associated with meld score, disease-specific analyses have not been reported. we evaluated, using srtr data, the effect of ald and/or hcv infection on transplant waitlist and post-transplant mortality, and survival benefit of deceased donor liver transplants. methods: , patients age ≥ , who were listed between sept and dec , and followed until dec , , were classified into cells according to hcv or ald status: hcv+, ald-: , ; hcv+, ald+: , ; hcv-, ald+: , ; hcv-, ald-: , ). cox regression was used to estimate waiting list mortality and post-transplant mortality separately. survival benefit, which encompasses both pre-and post-transplant events, was assessed using sequential stratification; an extension of cox regression which matches transplant recipients by meld and organ procurement organization to patients on active on the waitlist at the time of transplant. results: hcv significantly (p= . ) increased waitlist mortality, with a covariate-adjusted hazard ratio (hr) for hcv+ vs. hcv-of hr= . (p= . ). the impact of hcv+ was greater among ald+ candidates (hr= . ; p< . ), but was also significant among ald-candidates (hr= . ; p= . ). the contrast between ald+ and ald-waitlist mortality was only significant among hcv+ candidates (hr= . ; p= . ). post-transplant mortality was significantly higher among hcv+ vs. hcv-recipients (hr= . ; p=. ); there was no difference between ald+ vs. ald-recipients. survival benefit of liver transplantation was significantly lower among hcv+ compared to hcv-recipients with meld - , but significantly higher for hcv+ recipients with meld scores of ≥ . a diagnosis of ald did not influence the survival benefit of transplantation at any meld score. conclusion: despite higher waitlist mortality, hcv+ recipients had significantly lower liver transplant survival benefit than hcv-recipients, within categories defined by meld score. in contrast, transplant survival benefit was not influenced by ald. transplanted for hbv-related cirrhosis. giuseppe tisone, daniele di paolo, ilaria lenci, laura tariciotti, andrea monaco, manuele berlanda, linda de luca, giuseppe iaria, alessandro anselmo, irene bellini, mario angelico. hepatic surgery and transplantation, university of rome tor vergata, rome, italy. background and aim: post-transplant active immunization with hbsag vaccine is a potential prophylaxis strategy against hbv-recurrence after liver tranplantation due to hbv-related disease. previous studies showed conflicting results using standard vaccines, whereas the use of the new adjuvant -deacylated monophosphoryl-lipid-a (mpl) significantly increased patient's immunization rate. we investigated the efficacy of a long-term ( months) accelerated (monthly doses) vaccination schedule using the mpl-adjuvanted vaccine administered with and without concomitant hbig. methods: patients (m/f: / ) transplanted for hbv-related cirrhosis ± months earlier were recruited. all were hbsag and hbv dna negative in serum and cccdna negative in liver tissue; ( . %) were co-infected with hcv and ( . %) with hdv. study protocol consisted of consecutive monthly intramuscular vaccine doses (hbsag mg plus mpl mg) given together with lamivudine ( mg/daily). each of the initial doses (first cycle) was administered within days after iu hbig i.v. infusion, while the last doses were given after complete hbig withdrawal (second cycle). hbsab titre was determined before each vaccine dose and during the follow-up. all patients were maintaiened on low-level immunosuppression. preliminary results: all patients completed first vaccination cycle; ( . %) patients received adjuvanted vaccine doses (first and second cycles) and were monitored during months follow-up after vaccination end. no side effects occurred, nor evidence of hbv recurrence. at the end of first cycle all patients achieved an anti-hbs titre > iu/l (mean ± iu/l) and ( . %) a titre greater than iu/l. at the end of follow-up / ( . %) and / ( . %) had an anti-hbs titre greater than (mean ± ui/l) and iu/l, respectively. conclusions: nine months after hbig withdrawal more than half of the patients reached and maintained a protective anti-hbs titre (> iu/l). this intensive schedule using the mpl-adjuvanted vaccine, given in combination with hbig and lamivudine, seems to be more effective than previous hbv vaccination protocols, although a longer follow-up is needed to assess its final effectiveness. heterologous immunity via alloimmune responses to hepatitis c virus replication after liver transplantation. hideki ohdan, masahiro ohira, yuka tanaka, kohei ishiyama, nobuhiko hiraga, michio imamura, kazuaki chayama, toshimasa asahara. surgery, hiroshima university, hiroshima, japan; internal medicine, hiroshima university, hiroshima, japan. the immunosuppressive environment in liver transplantation (lt) recipients infected with hcv is believed to be associated with the progression of hcv reinfection. in the present study, we simultaneously monitored hcv levels and alloimmune status in hcv-infected lt recipients. for evaluating the immune status of hcv-infected lt recipients, we employed a mixed lymphocyte reaction (mlr) assay using a cfselabeling technique. the kinetics of the stimulation index of anti-donor reactive cd + t cells clearly mirrored that of the hcv rna titer, and a significant reverse correlation was observed between the parameters (r = . ). we did not observe a similar relationship between the stimulation index of an anti-third party cd + t cells and the hcv rna titer (r = . ). one possible explanation for this phenomenon might be that cytokines outputted by t cells responding to allostimulation display the anti-hcv activity either directly or indirectly through the activation of bystander immunocytes. to investigate such possibilities, we performed a transwell culture assay comprising a mlr culture in the upper chamber and genomic hcv replicon cell culture in the lower chamber to mimic the anatomical features of the interaction between hcvinfected hepatocytes and alloreactive t cells that infiltrate the portal area in the liver. when one-way mlr was performed using one-halpoidentical combination in the upper chamber, hcv replication was significantly suppressed in the lower chamber containing hcv replicon cells. hcv replication was further suppressed when mlr was carried out with a complete allogeneic combination. this inhibiting effect was dependent on their ifn-γ secreting activity. in addition, a similar result was obtained when ifn-γ-secreting nk/nkt cells stimulated with il- were cultured in the upper chamber. in conclusion, there was a close relationship between the anti-donor and the anti-hcv immune status in the lt recipients infected with hcv. cytokines such as il- and ifn-γ may be produced in response to allostimulation, and even these cytokines do not cause graft rejection, display anti-hcv activity. the elucidation of such a possible heterologous immunity via alloimmune responses to hcv replication might lead to the establishment of a novel method to prevent the progression of hcv reinfection in hcv-infected lt recipients. hepatitis c e region diversity after liver transplantation: a report from the hepatitis c iii trial. juan f. gallegos-orozco, hugo e. vargas, georges netto, gary l. davis, , and . % are morbidly obese . the goal of this study was to quantify the effect of bmi on access to transplantation (att), likelihood of receiving a transplantation (lrt), turndown rates (tr), and survival benefit (sb). methods: att was defined as either registering for the deceased donor waiting list or receiving a live donor transplant, and was analyzed based on the usrds registry using logistic regression. lrt was based on time spent in active status on the waiting list before receiving a transplant, and was analyzed based on the unos waiting list dataset using cox proportional hazards time-to-event analysis. tr was defined as the relative likelihood of being turned down for an organ offer by a provider other than the patient, and was analyzed based on the unos organ turndown dataset using negative binomial regression. sb was defined as survival after kidney transplantation versus survival on the deceased donor waiting list, and was analyzed based on the unos waiting list dataset using a time-dependent cox survival benefit model. all models were adjusted for known factors influencing those outcomes. lgbp lsg lsg mean follow-up (months) . (range - ) (range - ) . ( , ) patients > months since operation (n) / / / mean % ewl at > months (range - ) (range - ) ( , ) transplant candidate at > months / / / underwent transplant / / / complications developed in two patients (both with cirrhosis) and there was no mortality. mean follow-up was . months, and mean ewl at months or later was % (esrd), % (cirrhosis) and . % (esld). obesity associated comorbidities were improved or resolved in all patients. serum albumin and other nutritional parameters months or later after surgery were similar to preoperative levels in all groups. at most recent follow-up, out of patients ( %) have reached our institution's body mass index (bmi) limit for transplantation and are awaiting transplant; one patient with esld has undergone a successful lung transplant and one patient with cirrhosis has undergone a successful liver transplant. factors that contribute to inequitable access to the transplantation network (unos/ optn) include socioeconomic status, geographic location and delayed referral. the goal of the meld allocation system was to assign priority to the sickest candidate and assure equitable access to liver transplantation (lt). the meld has been validated as a reliable marker for liver disease severity. at the time of referral to the transplant center or listing a high meld (hm) is an indirect measure of delayed referral. therefore, the aim of this study is to identify factors associated with a hm at the time of listing for lt. method: using the unos database, we identified all adult candidates listed for lt from to . patients who received meld upgrade (i.e. hcc, fhf) and those listed for multi-organ transplant were excluded. the data collected included demographics, insurance payor, diagnosis, and meld score. meld score at time of listing was categorized as low (< ) and high (> ) and insurance type as private, medicaid and government (excluding medicaid) . results: during the study period , candidates were added to the lt waiting list. of these, there were , ( . %) males with a median age of (range - ) . caucasians were , ( . %), hispanic ( . %), african americans ( . %) and the rest ( . %). the underlying liver disease was hepatitis c ( . %), alcohol ( . %), alcohol/hepatitis c ( . %), pbc/ psc ( . %), cryptogenic ( . %) and others ( . %). age, gender and liver disease etiology were not associated with a hm at listing. a hm was associated with ethnicity: aa ( . %), hispanic ( . %), caucasian ( . %)[p< . ] and insurance type: medicaid ( . %), government ( . %) and private ( . %)[p< . ].there was no strong interaction between ethnicity/race and insurance in combination as predictors of hm i.e. both were independent. conclusion: ( ) aa and lt candidates with medicaid insurance are more likely to have a hm score at initial listing ( ) hispanics had the lowest rate of private insurance compared to caucasians with the highest rate. the above results suggest that type of insurance and ethnicity are independently associated with a hm (i.e. sicker patients) at listing. since a hm score is associated with increased mortality, implementation of strategies that result in timely and equitable access to the transplantation network regardless of the insurance type or ethnicity of the candidate(s) should be a priority. marked increase in the use of inactive status on the kidney transplant waiting list. kim nguyen, valarie ashby, , further wait time accrued only after active status was restored. on / / , the optn implemented a change in policy that provides for the accrual of waiting time during the entire interval that wl candidates are designated as s . methods: we investigated the impact of this policy change on the patterns of s designation, using the srtr/optn database. initial and subsequent status on the wl were determined for , candidates placed on the ki tx wl from / / - / / . the probability of becoming active (including receiving a living donor tx) on the wl was calculated for those who were s at listing before and after the policy change. results: table shows trends in the use of s before and after the policy change. of the , candidates listed as s before the policy change, % became active within months and % within year of wl. for the , candidates listed as s after the policy change, the corresponding figures are % and % respectively. figure demonstrates that, since the implementation of the new policy, the % of patients initially wl in s at most us tx centers has increased. conclusion: since the implementation of this policy, the number and % of pts wl as s at most ki tx centers, and the duration of s for those initially wl as s has increased dramatically. the implications of this practice on access to ki tx and survival warrants further investigation. can abstracts ( %) patients were retransplanted, and ( %) patients had panel reactive antibody > . the mean cumulative thymoglobulin dose administered was . mg/kg, and the primary maintenance immunosuppression started prior to discharge was a sirolimuscontaining regimen ( %). at the end of the follow-up period, ( %) patients had functioning grafts and ( %) patients experienced graft loss. of the patients with graft loss, ( %) patients experienced graft loss secondary to death. no pertinent differences were identified between groups. the graft survivals for african american recipients with african american or caucasian donors were % vs. % at year and % vs. % at year . conclusions: our study results suggest that donor race does not adversely affect graft outcomes in african american cadaveric kidney recipients with modern immunosuppression. donor racial differences may not play a primary role in the inferior graft outcomes of african american cadaveric kidney recipients. cardiac evaluation before kidney transplantation: are we screening too often or not enough? krista l. lentine, m. a. schnitzler, j. j. snyder, d. c. brennan, p. hauptman, p. r. salvalaggio, b. kasiske. evaluation for ischemic heart disease (ihd) is a common but non-standardized practice before kidney transplant. we retrospectively studied pre-transplant cardiac evaluation (ce) practices among a national sample of renal allograft recipients. methods: we examined usrds data for medicare beneficiaries transplanted in - with part a and b benefits from dialysis initiation through transplant. clinical traits defining "high" expected ihd risk were defined as diabetes, prior ihd, or > other coronary disease risk factors. pre-transplant ce were identified by billing claims for noninvasive stress tests and angiography. we quantified individuals with claims for coronary revascularization procedures between ce and transplant, and abstracted post-transplant acute myocardial infarction (ami) events from claims and death records. results: among , eligible patients, . % ( . % of high-risk and . % of lower-risk) underwent ce before transplant. overall, . % of patients who received ce also received pre-transplant revascularization, including only . % of lower-risk patients studied by ce ( table a) . the adjusted odds of transplant without ce (higher or for no ce, table b ) increased sharply with younger age and shorter dialysis duration. increased likelihood of transplant without ce also correlated with black race, female sex, and certain geographic regions. post-transplant ami rates in patients transplanted without ce allow assessment of whether ce was appropriately deferred in those who indeed face low ihd-risk after transplant. among patients transplanted without ce, the -yr incidence of post-transplant ami was % and % in lower and high-clinical risk groups, respectively, but varied by clinical traits within these groups. in lower-risk patients transplanted without ce, blacks patients faced increased ami-risk compared to whites (adjusted hr . , % ci . - . ). conclusions: observed ce practices demonstrate a low yield of pre-transplant revascularization but also raise concern for socio-demographic barriers to evaluation access. women who become pregnant in the first two years after kidney transplantation have a higher risk of graft loss. nadia zalunardo, olwyn johnston, caren l. rose, john s. gill. division of nephrology, university of british columbia, vancouver, bc, canada. existing information regarding the risks of pregnancy is derived from voluntary data sources. using medicare claims files from the usrds ( usrds ( - , we examined pregnancies (defined by presence of an inpatient icd- billing code for pregnancy or pregnancy-related complication) in the first years after kidney transplantation (ktx) among women aged - years whose primary insurance payor was medicare (n= , ) . patients were followed until graft failure, death or december , . there were pregnancies identified in women during the first post-transplant years. women who became pregnant during the st or nd post-transplant year had a shorter time to graft loss than women who became pregnant during the rd year (figure). to minimize the survivor bias among women who became pregnant during the rd posttransplant year, we determined the association between the timing of pregnancy and graft survival among the subset of women (n= ) who had graft survival of at least two years using a cox multivariate regression adjusted for age, race, cause of esrd, donor source, calendar year of transplant, dialysis vintage, maintenance immunosuppression, and gfr at months after ktx. pregnancy in the st year (hr . , % ci . - . ), and second year (hr . , % ci . to . ) was associated with an increased risk of graft loss compared to pregnancy during the third post-transplant year. we concluded that women who are able to wait should be counseled to become pregnant after the second post transplant year. the aging donor and recipient populations have led to new challenges in simultaneous kidney-pancreas transplantation (skpt). the purpose of this study was to retrospectively review our single center experience in skpt with respect to extended (ex) donor (d) and recipient (r) criteria. methods: over a month period, we performed skpts with enteric drainage ( portal venous drainage). ex ds were defined as age < (n= ), > (n= , mean age . yrs), or donation after cardiac death (dcd, n= ). all dcd donors were managed with extracorporeal support. ex rs were defined as age > (n= , mean age . yrs) or those with a pretransplant serum c-peptide level > . ng/ml (n= , mean . ng/ml). all rs received depleting antibody induction ( ratg, alemtuzumab) with tacrolimus, mmf, and tapered steroids ( steroid-free). results: a total of ds ( %) and rs ( %) met the above ex criteria. median waiting time was months, mean pancreas preservation was hours, and median length of stay was days. with a mean follow-up (f/u) of months, patient (pt) ( % ex d vs % non-ex d), kidney ( % ex d vs % non-ex d) and pancreas graft survival ( % ex d vs % non-ex d) rates were similar between d groups (all p=ns). the incidences of delayed kidney graft function ( % in each group) and early pancreas graft loss due to thrombosis ( % ex d vs % non-ex d) were also comparable between d groups. with regard to r groups, pt ( % vs %, p= . ) and kidney ( % vs %, p=ns) graft survival (gs) rates were slightly lower in the ex r group compared to the non-ex r group, respectively. however, death-censored kidney gs rates ( % ex r vs % non-ex r) were comparable between groups. uncensored pancreas gs rates ( % ex r vs % non-ex r) were similar. the incidences of acute rejection, surgical complications, infection, and other morbidity were comparable regardless of d or r group. at year (or latest) follow-up, renal and pancreas functional parameters were similar between d groups. however, the ex r group demonstrated slightly compromised renal and pancreas allograft function and a greater need for oral hypoglycemic agents. conclusion: intermediate-term outcomes in skpt from selected ex ds or rs have comparable outcomes, although ex r criteria may represent a risk factor for pt survival and functional outcomes. conclusion: spk recipients with functioning pancreas grafts have significantly improved kidney and patient survival compared to ld ka and dd ka. however, early pancreas graft failure results in kidney and patient survival rates similar to ka recipients. spk provides optimal outcomes for patients with dm , but long-term risk associated with early pancreas loss may be a consideration when selecting spk vs ka transplantation. the impact of long-term metabolic control on renal allograft and patient survival in type diabetes. christian morath, martin zeier, bernd dohler, jan schmidt, peter p. nawroth, gerhard opelz. nephrology, university of heidelberg; transplantation immunology, university of heidelberg; transplantation surgery, university of heidelberg; endocrinology, university of heidelberg. it is a matter of debate whether pancreas allografts independently contribute to renal transplant and patient survival in type diabetics who received a simultaneous pancreas kidney transplant (spk). using the data of the collaborative transplant study (cts), we studied type diabetic recipients of deceased donor kidneys (ddk), living donor kidneys (ldk), or spk performed during two time periods: to and to . we analyzed graft and patient survival rates for a maximum of years. ddk recipients showed inferior graft and patient survival compared to ldk and spk recipients in both time periods. ldk recipients had a superior graft survival rate initially, but the survival rate of kidneys in spk recipients reached the level of ldk toward the end of the follow up period. the results of patient survival paralleled those of kidney graft survival: an early advantage of ldk as compared to spk faded away during follow up. multivariate analysis, in which pretransplant cardiovascular risk assessment was appropriately considered, showed that patient survival of spk recipients was superior to that of ldk recipients beyond the tenth year after transplantation (hazard ratio hr = . , p = . ). this was reflected by a lower cumulative cardiovascular death rate in recipients of spk ( . %) compared to recipients of ddk ( . %) or ldk ( . %) . the early survival benefit of ldk compared to spk is lost during long-term follow up, probably related to improved glycemic control in spk recipients. proposal for a grading rejection schema in pancreas allograft biopsies from a multidisciplinary panel of pathologists, surgeons and nephrologists. .normal .indeterminate septal inflammation that appears active but the overall features do not fulfill the criteria for mild cell mediated acute rejection. active septal inflammation involving septal structures and/or focal acinar inflammation. -grade ii / moderate acute cell mediated rejection multifocal (but not confluent or diffuse) acinar inflammation with spotty acinar cell injury and drop-out and/or minimal intimal arteritis -grade iii / severe acute cell mediated rejection diffuse, (widespread, extensive) acinar inflammation with focal or diffuse multicellular /confluent acinar cell necrosis.and/or moderate or severe intimal arteritis and/or transmural inflammation -necrotizing arteritis chronic active cell-mediated rejection. chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell infiltration in fibrosis, formation of neo-intima) .antibody mediated rejection =c d positivity + confirmed donor specific antibodies + graft dysfunction -hyperacute rejection -accelerated antibody mediated rejection severe, fulminant form of antibody mediated rejection with morphological similarities to hyperacute rejection but occurring later (within hours or days of transplantation). -acute antibody mediated rejection specify percentage of biopsy surface with interacinar capillaries positive for c d. .chronic allograft rejection/graft sclerosis stage i (mild graft sclerosis) < % of the core surface stage ii (moderate graft sclerosis) - % of the core surface. stage iii (severe graft sclerosis) > % of the core surface . other histological diagnosis e.g. cmv pancreatitis, ptld, etc. a simple, reproducible, clinically relevant and internationally accepted schema for grading rejection should improve the level of diagnostic accuracy and positively affect patient care. rank test). similar results were obtained when death was included as a cause of graft loss (data not shown). conclusion: par is associated with worse long term kidney graft survival than kar. it is likely that patients that experience par within the first year have also experienced undiagnosed sub-clinical kar. this adds associative data to the argument that the likelihood of concordance between par and kar is high and therefore, the need for increased monitoring of kidney function after par is warranted. the most efficacious immunosuppressive (immuno) regimen for spkt is debated, and information on the best regimen to prevent amr is particularly scarce. we performed a retrospective comparative cohort study that included spkt patients (pts) transplanted in - , who received maintenance immuno with tac, mmf and steroids. two groups were compared: pts who received induction with alemtuzumab (alem) (n= ) and pts who were induced with basiliximab (basilmab) (n= ). donor and recipient characteristics were similar. kt acute cellular rejection (acr) was more frequent with basilmab ( -yr . % vs . %, p= . ), but the incidence of biopsy-proven kt amr was similar ( -yr % with basilmab vs . % with alem, p=ns). no differences in prevalence were detected considering early amr (< th day) or late amr (> th day) separately. multivariate analyses showed that the only significant risk factor for amr was female gender (adjusted hr . , p= . ). the biopsy-proven kt rejection was associated with clinical pt rejection in out of the amr cases ( %), without differences between the groups. post-rejection kt graft survival was similar in both groups ( -yr basilmab/alem . / . %), but deathcensored kt survival was lower with alem ( / . %, p= . ). the predominant cause of kt loss in pts induced with basilmab was death-with-function (n= ), while in pts induced with alem acute or chronic amr was the most common cause of kt attrition (n= ). pancreas survival was similar between both groups. more pt losses due to ar occurred in alem-treated group than in the basilmab group ( vs ) . nearly all early episodes resolved with treatment and were not associated with worse graft survival. conversely, late amr episodes carried a worse prognosis, with decreased -yr kt ( . % vs . %, p< . ) and pt graft survival ( . vs . %, p< . ). late amr was associated with graft loss in multivariate cox models (kidney loss hr . , p= . and pancreas hr= . , p= . ) . amr is common in spkt recipients. acr is better prevented with alem than with basilmab, but no relevant difference is found between alem and basilmab in amr. late (as opposed to early) amr episodes are associated with significant reduction in spkt survival rates despite treatment. preventive and/or different treatment strategies are required to address late amr in spkt. intraoperative fluorescence imaging (ifi) in pancreas transplantation (pt) to determine vascular patency and allograft perfusion. edmund q. sanchez, srinath chinnakotla, marlon f. levy, robert m. goldstein, goran b. klintmalm. baylor regional transplant institute, dallas/ft. worth, tx. thrombotic complications of pt are well known. we report ifi using the spy device to assess immediate vascular patency and allograft perfusion. our controlled experience is presented here. methods: pts were imaged intraoperatively using the spy device under our irb approved study. indocyanine green solution ( . mg/ml) was injected into central venous catheters after completion of the vascular anastomoses of whole pancreaticoduodenal allografts. the pancreas transplants were performed in the retroperitoneal portal and enteric drained technique described by boggi, et al. imaging of the allograft vasculature, perfusion of the pancreas, and perfusion of the duodenal segments was performed and recorded intraoperatively. all video sequences were archived for later review. results: ifi on pancreas transplants ( simultaneous pancreas-kidney and pancreas after kidney) was performed and video sequences were recorded. all pancreas allografts demonstrated intraoperative vascular patency and complete pancreatic and duodenal perfusion. there were no side effects seen. all pancreas transplants had immediate graft function. one patient was re-explored on postop day due to persistent acidosis and hypotension. repeat ifi demonstrated vascular patency and perfusion, despite an ischemic external physical appearance. there were no vascular complications that required reoperation, nor were there any graft thromboses. characteristic slow venous outflow was seen in each case. conclusion: ifi with the spy device is a simple and effective method to determine immediate patency and perfusion of the whole pancreaticoduodenal allograft. its use is beneficial in re-exploration to rule out infarcted pancreas allografts. further development in quantification of vascular flow rates and allograft perfusion indices by this device is abstracts in progress. once these are established, this information will be studied in a controlled fashion, and will be compared with clinical outcomes. we have demonstrated safety and developed a protocol for using the spy device in ifi of pancreas transplants. tolerance/immune deviation i tolerance without immunosuppression: exploiting epigenetic regulation as a new approach to achieving donor-specific allograft tolerance. liqing wang, ran tao, joel a. friedlander, wayne w. hancock. pathology & immunology, children's hospital of philadelphia & upenn, philadelphia, pa. given toxicities of all current immunosuppressive agents, plus complications of malignancy, infection and chronic rejection, maintaining the search for new approaches to tolerance induction is essential. while weaning of immunosuppression is fraught with risks and costimulation blockade has not yielded the expected boon, use of a broad histone deacetylase inhibitor (hdaci), such as trichostatin a (tsa) or suberoylanilide hydroxamic acid (saha), promotes foxp acetylation of foxp and binding to target genes, leading to enhanced treg suppressive function, and wks of therapy with hdaci and low-dose rapamycin can induce allograft tolerance. we now show that in addition to acetylation, modulation of a second major epigenetic mechanism, dna methylation, has salutary effects, such that combined use of an hdaci and a dna methyltransferase inhibitor (dnmti, e.g. -aza- deoxycytidine) has potent effects. microarray analysis of the effects of hdaci on tregs showed down-regulation of multiple genes associated with dna methylation, including dnmt, methyl-cpgbinding domain and associated proteins, leading us to test the effects of dnmti use on treg function. dnmti administration decreased foxp gene methylation, enhanced treg gene expression, and increased treg suppression in vitro, and led to a dose-dependent prolongation of cardiac allograft survival (balb/c->c bl/ ) in vivo (p< . ). the combination of hdaci and lower doses of dnmti, administered for just wks, led to permanent engraftment (> d, p< . ), and the permanent acceptance of second donor allografts but acute rejection of third-party (cba) cardiac allografts indicated induction of donor-specific tolerance post-epigenetic therapy. analysis of long-surviving cardiac allografts showed a minor infiltrate consisting primarily of foxp + tregs and an absence of chronic rejection (transplant arteriosclerosis, myocardial fibrosis), whereas combined epigenetic therapy led to only minor prolongation of allograft survival in treg-depleted recipients. in summary, brief use of clinically approved agents (an hdaci plus an dnmti), can synergistically enhance treg function and induce tregdependent donor-specific allograft tolerance without use of any immunosuppression. we conclude that insights gained through epigenetic targeting may provide completely new approaches to the taming and regulation of otherwise powerful immune responses post-transplantation. a novel epigenetic approach to generate mouse and human cd + cd + foxp + regulatory t cells. girdhari lal, nan zhang, william van der touw, yaozhong ding, jonathan s. bromberg. gene and cell medicine, mount sinai school of medicine, new york city, ny. background: constitutive foxp expression is required for stable and suppressive cd + cd + regulatory t cells (treg) . we previously showed that demethylation of an upstream cpg island of the foxp promoter is characteristic of stable and suppressive treg. using dna methyltransferase inhibitors, we present a novel method to generate antigen-specific treg from mouse and human cd + cd -t cells. methods: naïve cd + cd -t cells and natural treg (ntreg) were purified from wild type or foxp -gfp transgenic mice. human naïve cd + cd -t cells were purified from pbmc. t cells were cultured with irradiated antigen presenting cells in the presence of il- , anti-cd ε mab, tgfβ or the dna methyltransferase inhibitor -aza- '-deoxycytidine (zdcyd), which demethylates the upstream promoter. foxp expression was determined by flow cytometry and qrt-pcr. results: naïve t cells cultured under stimulatory conditions with zdcyd express increased foxp mrna ( fold) and intracellular foxp protein ( % of cells vs % without zdcyd). foxp expression is synergistically enhanced with tgfβ ( ± . % vs . ± . % zdcyd alone or . ± . % tgfβ alone), similar to ntreg ( . ± . %). tgfβ in combination with other dna methyltransferase inhibitors (procainamide, hydralazine, rg ) also induces foxp . zdcyd plus tgfβ induced treg stably express foxp and similar surface markers and cytokine mrna as ntreg. zdcyd plus tgfβ induced treg suppress proliferation of cd + cd -t cells, prevent cd + cd -cd rb hi induced colitis in scid mice, and enhance islet allograft survival. zdcyd plus tgfβ induce foxp expression in antigen-specific wild type or t cell receptor transgenic cd + t cells stimulated with alloantigen or peptides, and in naïve cd + cd -t cells. in vivo administration of zdcyd preferentially preserves thymic cd + cd + foxp + treg generation. zdcyd alone or zdcyd plus tgfb induce strong foxp expression in human cd + cd -t cells compared to tgfβ alone. zdcyd plus tgfβ induced human treg suppress the proliferation of naïve cd + cd -t cells, whereas tgfβ-induced treg do not. conclusion: dna methyltransferase inhibitors induce stable and suppressive foxp + treg from peripheral cd + cd -t cells. these finding have important implications for understanding t cell development and differentiation, and provide a clinically applicable technique for manipulating epigenetic regulation for the generation of treg for tolerance. macrophages driven to a novel state of activation can promote tolerance. katharina kronenberg, seiichiro inoue, james a. hutchinson, beate g. brem-exner, gudrun e. koehl, hans j. schlitt, fred fandrich, edward k. geissler. surgery, university of regensburg, regensburg, germany; surgery, university hospital schleswig holstein, kiel, germany. background: the use of immunomodulatory cells in organ transplantation (tx) is a promising tolerance-induction strategy. here we describe a novel macrophage population capable of enriching t regulatory cells (treg) and promoting tolerance. methods: balb/c bone marrow, spleen and blood mononuclear cells were cultured with m-csf for days, with a hr pulse of ifn-γ on day ; the resultant adherent cells are referred to as ifnγ-induced monocyte-derived cells (ifnγ-mdc) . residual lymphocytes from these cultures were recultured with the adherent ifnγ-mdc for an additional days. ifnγ-mdc were phenotyped by facs and immunomodulation of lymphocytes was determined by cell counting and facs analysis for tregs. mouse heterotopic heart tx was used for in vivo testing. results: ifnγ-mdc highly express cd b/c, f / and pd-l . compared to classically-activated (m ) macrophages, ifnγ-mdc express less cd /cd , but higher cd c. ifnγ-mdc profoundly delete activated, but not resting, lymphocytes (> %), with cell contact (via transwell system) and caspases (via inhibitors) being essential. most interestingly, ifnγ-mdc highly enrich lymphocytes for cd + cd high foxp + treg ( ± %; n= ). the same, or higher, proportions of treg developed when ifnγ-mdc were produced from macs-purified cd b + and cd + cells ( : ) . ifnγ-mdc culture supernatant showed increased il- (elisa) vs. monocyte control cultures ( ± pg/ml vs. < pg/ml, respectively; n= ), suggesting il- as one potential mediator. ifnγ receptor signaling and cd interactions are required for treg enrichment, as confirmed using ifnγ receptor and cd knock-out mice (c bl/ mice). ifnγ-mdc derived from cd b + cells and lymphocytes of ova-transgenic otii mice showed enrichment of treg by -fold with high-affinity ova peptide ( - ) vs. treg levels using a low-affinity ova peptide (ova - ), suggesting ag-specific treg cells can be enriched with ifnγ-mdc. finally, a single post-heart tx (d+ ) i.v. injection of x donor (c h)-derived ifnγ-mdc prolonged allograft survival in balb/c recipients from . ± . d in controls (n= ) to . ± . d (n= ; p= . ). conclusions: ifnγ-mdc are a novel macrophage population capable of deleting activated t cells and highly enriching remaining lymphocytes for tregs. therefore, ifnγ-mdc could be useful in a clinical setting for promoting transplant tolerance. plasmacytoid dc therapeutic potential is independent of ido induction due to elevated dap expression. tina l. sumpter, bridget l. colvin, zhiliang wang, andrew l. mellor, angus w. thomson. , starzl transplantation institute, dept. of surgery, university of pittsburgh, pittsburgh, pa; immunology, university of pittsburgh, pittsburgh, pa; immunotherapy center, medical college of georgia, augusta, ga. optimizing the mechanisms of pdc tolerogenicity may facilitate their use in the development of cell-based tolerance induction strategies. pdc may undermine t cell function via inducible expression of ido, a tryptophan catabolizer that enhances t cell apoptosis. ido is negatively regulated by dap . in some systems, loss of dap correlates with immunostimulatory activation of pdc, while in others, its absence correlates with enhancement of pdc tolerogenicity through induction of ido. in these studies, we characterized the ability of wt and ido-deficient pdc to attenuate murine heart allograft rejection in order to define the contribution of ido and its regulation by dap relative to the immunoregulatory potential of pdc. methods: pdc and control myeloid (m) dc were generated from bm cultures of wt or ido ko c bl/ (b ; h b ) mice. wt mdc, pdc or ido ko pdc were pulsed with alloag (balb/c; h d ), stimulated with cpg, and then injected i.v. into syngeneic (b ) recipients d before heart transplant. donor ag-specific activation of t cells was analyzed by mlr and elisa. dap expression was evaluated by rt-pcr and abrogated with sirna. results: administration of ido ko pdc d before transplant prolonged graft survival beyond that seen with control mdc, but not to the extent seen with wt pdc, suggesting involvement of ido. pdc cultures from ido ko mice exhibited a more mature phenotype than their wt counterparts with reduced expression of co-regulatory molecules (e.g., icosl). although ido ko pdc induced enhanced t cell alloactivation compared to wt pdc, use of the ido inhibitor -mt indicated that wt pdc do not produce functional ido. further analyses showed wt pdc exhibited high levels of dap mrna expression. silencing dap had no effect on the ability of wt pdc to activate allogeneic t cell proliferation, but significantly enhanced ifnγ secretion. addition of -mt to mlr with dap -silenced pdc increased t cell proliferative responses to alloag, verifying ido induction in dap -silenced pdc. conclusions: these data underscore the prophylactic potential of pdc for cell-based therapy that may be independent of ido, due, in part, to elevated dap expression. additionally, our data support a role for dap in both immunostimulation and immunoregulation by pdc. rapamycin preferentially blocks the expansion of potentially tolerogenic plasmacytoid dendritic cells in vivo. heth r. turnquist, angus w. thomson. , starzl transpl inst and dept of surgery; dept of immunol, univ of pittsburgh sch of med, pittsburgh, pa. rapamycin (rapa), a 'tolerance-sparing' immunosuppressant with anti-proliferative properties, inhibits myeloid (m) dendritic cell (dc) differentiation/maturation in vitro. rapa decreases cd c + dcs in the mouse spleen and suppresses the expansion of total cd c + dc following administration of the dc growth factor, fms-like tyrosine kinase ligand (flt l). however, the influence of rapa on plasmacytoid (p) dc,the principal type- ifn producers in the body, known to regulate innate and adaptive immune responses, and reported to promote experimental transplant tolerance, has not been evaluated. methods: dc were propagated from bone marrow for days (d) in flt l, in the absence or presence of a clinically-relevant dose of rapa ( ng/ml). in addition, dcs were mobilized in c bl/ mice by i.p. flt l ( mg/d for d; d - ). untreated and flt l-treated groups also received rapa ( . mg/kg/d i.p.; d - ). on d , dc were isolated by density gradient centrifugation and/or cd c + positive selection. mdc (cd c + cd b + ) and pdc (cd c lo cd b -b + ) were identified by flow cytometry. results: rapa suppressed pdc and mdc generation in vitro; rapa-dc-treated cultures had only % of the pdc and % of the mdc found in control conditions. in normal mice, both mdc ( % of control) and pdc ( . %) numbers were reduced significantly by rapa administration. rapa, when given concurrently with flt l, blunted the typical profound expansion of mdc. specifically, flt l and rapa-treated mice displayed a ± x increase over control (steady state) numbers compared to a ± x increase in mice treated with flt l alone. flt l-induced expansion of pdc was to a much greater extent impacted by rapa, as absolute numbers of pdc increased only . ± . x over control numbers compared to ± x increase in absolute splenic pdc in flt l-treated mice. conclusion: these data identify rapa as a selective suppressor of pdc generation, as described for corticosteroids. this has significant implications, given the use of rapa following organ transplantation, and the suggested importance of secondary lymphoid tissue pdc for promotion of transplant tolerance mediated by treg. due attention to these disparate effects of rapa on regulating cell populations will be necessary to optimize therapeutic regimens for safe promotion of tolerance. the liver is tolerated better than other transplanted organs. this may reflect the inherent tolerogenicity of liver dendritic cells (dc) and interactions with foxp + regulatory t cells (treg). recent studies have shown that cd expression on dc correlates with induction of foxp + treg, and can be enhanced in the presence of transforming growth factor-β (tgf-β) and retinoic acid (ra), both of which are produced in the liver. this study evaluates cd expression on liver plasmacytoid (p) and myeloid (m) dc and the potential of liver dc subsets to induce tregs. methods: pdc and mdc were magnetically isolated from livers and spleens of c bl/ mice and co-cultured with cfse-labeled allogeneic (balb/c) cd + cd -t cells. after or d of co-culture, t cells were assessed for cfse dilution and intracellular foxp expression. rhtgf-β and either all trans or cis-ra were added to co-cultures. cd expression was evaluated by flow cytometry. results: cd expression was elevated on liver pdc and mdc compared to spleen pdc or mdc, with higher expression on liver mdc. pdc from the liver and the spleen were poor inducers of foxp in cd + cd -t cells compared to mdc. foxp induction was enhanced with tgf-β when splenic pdc were used as stimulators. however, when liver pdc were used as stimulators, tgf-β had no effect on foxp induction. ra (either all trans or cis) enhanced induction of foxp + cells in cd + cd -t cells in the presence of tgf-β when splenic pdc but not liver pdc were used as stimulators. tgf-β and tgf-β with ra also enhanced foxp induction in cd + cd -t cells when either spleen or liver mdc were used as stimulators, though splenic mdc were superior inducers of foxp . conclusions: many studies have focused on naturally-occurring foxp + tregs in systemic tolerance. our data show that liver mdc and pdc are poor inducers of foxp in t cells, even in the presence of tgf-β and ra. these data suggest that the inherent tolerogenicity of liver dc subsets may be independent of treg induction or that liver dc interact with treg through alternative mechanisms. background: t cell-mediated immune rejection occurs in organ transplantation. in addition to mhc-tcr signaling, t cell activation requires costimulation from antigen presenting cells. b molecules (cd /cd ) and cd are critical costimulatory molecules in t cell activation. insufficient or lack of costimulation results in inactivation or tolerance. we hypothesized that blocking the costimulation pathway using small interfering rna (sirna) expression vector can prolong allogeneic heart graft survival. method: vectors that express hairpin sirnas specifically targeting cd and cd were prepared. recipients (balb/c mice) were treated with cd and/or cd sirna vectors, and days prior to heart transplantation. control groups were injected with a blank vector and sham treatment (pbs). after sirna treatment, a fully mhcmismatched (balb/c to c /bl ) heart transplantation was performed. result: allogeneic heart graft survival (> days) was approximately % in the mice treated simultaneously with cd and cd sirna vectors. in contrast, allogenic hearts transplanted into recipients treated with blank vector and pbs stopped beating within days. hearts transplanted into cd or cd sirna vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than days. real time pcr and flow cytometric analysis showed an upregulation of foxp expression in spleen lymphocytes and a concurrent downregulation of cd and cd expression in splenic dendritic cells of sirnatreated mice. an mlr, using splenic dendritic cells (dcs) isolated from tolerant recipients, showed a significantly lower t cell proliferation capacity in cd -and cd -sirna vector-treated mice, compared to control groups. tolerant dcs from cd -and cd -treated recipients promoted cd +cd +foxp + regulatory t cell differentiation. finally, tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema in mice treated with cd and cd sirna vectors. conclusion: this study demonstrates that the simultaneous silencing of cd and cd genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation. costimulation blockade (cob) of the cd /b pathway has long been suggested as a means of attaining indefinite, well-tolerated, antigen-specific prophylaxis from allograft rejection. although effective in rodent models, cd /b blockade is not alone sufficient to prevent rejection in more robust primate models. the relative presence of allocrossreactive memory t cells is one mechanism of cob resistant rejection. lfa -ig is an approved agent for the treatment of psoriasis, shown clinically to deplete tem cells in psoriatic lesions. we hypothesize that lfa -ig specifically targets cob resistance cells, including heterologous alloreactive t cell memory, while avoiding interference of peripheral mechanisms that foster cob-mediated allograft acceptance. rhesus monkeys underwent mhc mismatched renal allotransplantation. ctla -ig was given mg/ kg iv on days - , , , , , , and mg/kg iv on days , , , . lfa -ig, ( . mg/kg iv) was given on day - , , , then weekly ( wks). sirolimus was given orally ( mg/kg) days - and donor specific transfusion ( ml/kg iv) on day - . animals were followed by polychromatic flow-cytometry to quantify t cell subsets. lfa -ig synergized with ctla -ig successfully preventing renal allograft rejection in this model and leading to indefinite survival in / animals (table) . this enhanced survival was associated with a significant reduction in tem cells in animals treated with lfa -ig compared to animals without lfa -ig treatment (figure). lfa -ig withdrawal led to tem re-population and rejection. this therapy regimen, all clinically available agents, promotes cob-mediated graft acceptance without the use of calcineurin inhibitors, steroids, or gross t cell depletion. the aim of this study was to investigate the immunological pathways that regulate t cell dependant allograft responses in the hope of finding novel immunomodulatory compounds. islet (and skin) allografts were transplanted into full mhc-mismatched wild type (wt) and baff-transgenic (baff-tg) recipient mice. allograft function/ rejection was monitored by blood glucose levels and/or confirmed by nephrectomy. histology, splenocyte populations & t cell functions were analyzed. the b cell activation factor from the tnf family (baff) is critical for b cell survival. t cells express baff receptors suggesting that baff may also play a role in t cell responses. contrary to expectations, we found that baff-tg mice accepted a full mhc-mismatched islet allograft for > days. in addition, baff-tg mice also showed delayed skin graft rejection. this was due to a t cell intrinsic change in allo-responsiveness as shown by a failure of purified baff-tg t cells to reject an allograft in adoptive transfer experiments. however, baff-tg t cells were not anergic per se as they proliferated normally to mitogens in vitro and to antigenic challenge in vivo. intriguingly, baff-tg mice harbored an increased frequency of t regulatory cells in the periphery as compared to wt mice. elimination of cd + t cells restored normal allograft rejection to baff-tg mice, demonstrating that the increased number of treg cells were responsible for their altered allo-immunity. in a second approach, baff-tg t cells depleted of cd + cd + t cells were adoptively transferred to transplanted rag recipients, and in this case the baff-tg cd + cd -t cells rejected their allograft. proliferation assays showed that excessive baff was not promoting treg expansion by driving proliferation in the periphery. adoptive transfer of gfp expressing syngeneic splenocytes did not exhibit enhanced survival in the presence of excessive baff. however, analysis of treg cells in the thymus of baff-tg mice showed an increased intrathymic frequency. together these results demonstrate that baff-tg mice harbor an expanded number of treg cells that prevent th -type immune responses including allograft rejection. furthermore, we demonstrate that manipulating baff levels may provide a means to generate immunosuppression free dominant allograft tolerance. we have previously reported the outcome of pig-to-baboon thymokidney transplants from galt-ko miniature swine using an immunosuppressive regimen designed to facilitate the induction of tolerance. although the results were superior to results using hdaf thymokidneys, there was a high rate of early post-operative complications. we investigated whether the elimination of steroids and whole body irritation (wbi) from the treatment protocol would decrease the complication rate in the perioperative period. methods: baboons received thymokidney transplants from galt-ko miniature swine. the immunosuppressive regimen was based on the previously published protocol, but eliminated steroids and substituted rituximab for wbi. it consisted of thymectomy day - , splenectomy day , atg, rituximab, mmf and anti-cd mab. renal function was assessed by serum creatinine levels. evidence for baboon thymopoiesis in the pig thymus was assessed by facs and immunohistochemistry. results: one animal died due to a drug reaction at pod with normal renal function and no evidence of infection. the remaining baboons showed no signs of rejection although igm deposition was observed, likely due to preformed non-gal nab. there were no deaths due to rejection. although early infectious complications were not seen, late complications leading to mortality were still observed, including systemic cmv infection (pod ), pleural effusions (pod , ) , ards with pulmonary hemorrhage (pod , ) and acute myocardial infarction (pod ). two animals showed evidence for early thymopoiesis in the pig thymus by facs and immunohistochemistry. cd +/cd + thymocytes were seen in the thymic grafts, indicating t cell development was supported by the pig thymus. the average survival of the steroid-free group was days including the animal with the drug reaction and days excluding it, compared to . days in the regimen that included steroids/wbi. conclusions: a steroid-free and wbi free regimen designed for the induction of tolerance across the pig-to-baboon xenogeneic barrier had fewer early post-operative complications than previous regimens. greater than -day average survival of recipients of life-supporting xenogeneic thymokidneys was observed without rejection. this regimen also appears to permit baboon thymopoiesis in the pig thymus. purpose: the effects of human decay accelerating factor (hdaf) addition to the galactosyl transferase knock-out (galt-ko) background in transgenic pig kidney xenotransplantation in baboons has not been previously studied. methods: baboon recipients of galt-ko (n= ) or galt-ko/hdaf (n= ) pig kidneys received flolan, heparin and/or aspirin. steroids (s), cobra venom factor (v), atg (a), mmf (m), and/or a low-dose cd blockade (c) regimen were given. results: pre-transplant (tx) anti-non-gal antibody (ab) titers were inconsistently associated with early galt-ko kidney xenograft failure. high d-dimer levels hours post-tx were closely associated with early galt-ko graft loss but not when assessing levels of c a, btg, or increased cd p expression on circulating platelets. regimens lacking mmf and/or cd blockade elicited by day - large anti-donor non-gal ab responses . post-operative creatinine levels for the galt-ko/hdaf recipients were lower along with superior graft survival compared to galt-ko; d-dimer, anti-donor non-gal ab, c a, btg, and f + measurements are in progress for the galt-ko/hdaf recipients. galt-ko/hdaf v,a,m,s,c > * tbp . ; . * graft survival at time of abstract submission as graft still functioning at time of abstract submission; tbp --to be processed ! --anuric; non-life supporting, but appeared viable, pink, and well-perfused until day conclusion: early failure of vascularized galt-ko organs is closely associated with activation of coagulation pathways; whether this is triggered primarily by anti-non-gal ab or by other mechanisms is not addressed by our study. preliminary results using galt-ko/hdaf pig kidneys show promise in attenuating or possibly preventing these and/or other such mechanisms. the established efficacy of cd -based costimulation blockade with mmf to prevent induced anti-non-gal antibody elaboration is confirmed by our preliminary findings. recovery of cardiac function after pig-to-primate orthotopic heart transplant. christopher g. a. mcgregor, william r. davies, keiji oi, henry d. tazelaar, randall c. walker, krishnaswamy chandrasekaran, guerard w. byrne. mayo clinic, rochester, mn. xenotransplantation has been proposed as a method to alleviate the shortage of donor organs. we report survival of up to weeks of three orthotopic transplants after pig-tobaboon cardiac xenotransplantation. pig-to-baboon orthotopic transplantation was performed using an adult baboon recipient and cd transgenic pig. the recipients were treated with an α-gal polymer to block the effects of anti-gal antibody and with atg induction and a tacrolimus and sirolimus based maintenance immunosuppression. heart function was continuously monitored by intramyocardial electrocardiography and every - days by serial echocardiography. standard hematological, serum chemistry and cardiac troponin levels were monitored every - days. the animals survived , , and days in a healthy condition. mortality was a result of pneumonitis, respiratory failure and bowel infarction respectively. one recipient (survival days) showed impaired perioperative left ventricular function with an ejection fraction of % on echo. over the next two weeks, the ejection fraction in this animal returned to normal ( %) and troponin levels normalized. this study shows the longest survival of orthotopic cardiac xenografts to date with recovery of impaired heart function after early ischemia reperfusion injury. this significant improvement in ventricular function suggests that the normal reparative processes appear to function across the xenotransplantation barrier, supporting the potential clinical potential of cardiac xenotransplantation. purpose: to determine whether the galactosyl transferase gene knock-out (galt-ko) protects pig lungs from hyperacute lung rejection (halr) by human blood. the effects of adding human decay accelerating factor (hdaf) to the galt-ko background will also be examined for the first time. methods: heparinized fresh human blood was used to perfuse galt-ko pig lungs (n= ). lung function was assessed by flow, pulmonary vascular resistance (pvr), oxygen transfer, and tracheal edema using pre-defined survival endpoints. historical wild-type (wt) pig lungs (n= ) provide context for analysis. additionally, two pig lungs expressing galt-ko/hdaf were recently examined, one of which received treatment with xigris. results: median lung survival in galt-ko lungs was minutes (range min to > min) vs. minutes in wt lungs (range min to min, p= . ); / galt-ko/hdaf pig lungs survived > min. pvr at minutes for galt-ko lungs was ± mmhg-min/l vs.wt lungs ( ± mmhg-min/l at minutes) vs. mmhgmin/l for each galt-ko/hdaf lung. complement activation (∆c a) at minutes was significantly lower with galt-ko (∆c a ± ng/ml) than wt lungs (vs. ± ng/ml, p= . ). galt-ko was also associated with reduced platelet activation: only ± % of circulating platelets express cd p at min. vs. ± % in the wt group (p= . ); ∆βtg at min [ ± iu/ml (galt-ko) vs. ± iu/ ml (wt)], and less thrombin formation (∆ f + ) at minutes (galt-ko: ± . nm vs. wt: ± nm). ∆c a, % of circulating platelets expressing cd p, ∆βtg, and ∆ f + measurements are in progress for galt-ko/hdaf. platelet sequestration was delayed as mean % of the initial platelets remaining in the galt-ko lung perfusate was ± % at min vs. ± % in the wt lung group (p= . ). neutrophil sequestration was also diminished in galt-ko ( ± % residual) vs. wt lung ( ± % residual, p= . ). conclusion: galt-ko pig lungs are significantly protected from several facets of halr: complement and coagulation cascade activation, neutrophil sequestration, and platelet activation are all partially attenuated by this modification alone. preliminary results suggest that galt-ko/hdaf lungs offer even further protection. disseminated intravascular coagulation is associated with tissue factor expression on recipient platelets and monocytes. chih che lin, , , mohamed ezzelarab, corin torres, david ayares, anthony dorling, david k. c. cooper. thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; revivicor inc., blacksburg, va; department of immunology, imperial college london, hammersmith hospital, london, united kingdom; department of surgery, chang gung memorial hospital, kaohsiung, taiwan. purpose acute humoral xenograft rejection (ahxr), frequently associated with disseminated intravascular coagulation (dic), remains a challenge in pig-to-primate xenotransplantation (tx). a previous in vitro study showed that recipient platelets and monocytes were induced to express tissue factor (tf) after incubation with porcine endothelial cells, and we speculated this may contribute to thrombosis. the present study investigated whether circulating (extragraft) monocytes and platelets express tf and whether this relates to the development of dic after pig tx. methods baboons (n= ) received an aortic patch or kidney from either a wild-type (wt) or α , galactosyltransferase gene-knockout (gtko) pig, with or without immunosuppressive therapy. baboon monocytes and platelets were isolated from blood before and after tx. surface tf phenotype was determined by flow cytometry. functional tf activity was determined by clotting time assays after mixing monocytes and platelets with recalcified factor vii (fvii)-deficient plasma with or without added fvii. before tx, monocytes and platelets did not express tf or display tf-dependent procoagulant activity. after gtko aortic patch tx, the baboons were euthanized by day without developing dic. however, by day , circulating platelets (but not monocytes) expressed tf and promoted tf-dependent clotting in recalcified plasma. in the absence of immunosuppression, wt kidneys survived < day before the onset of dic, at which time tf activity was detected on both platelets and monocytes. after gtko kidney tx in immunosuppressed baboons, platelets expressed tf as early as day . in contrast, monocytes began to express tf only at the onset of dic. this study links expression of tf on recipient monocytes and platelets with the development of dic. expression on platelets appeared to predict the subsequent development of dic, whereas that on monocytes was associated with the onset of dic. whilst our observations do not establish a causal relationship, they provide the basis for further study. international human xenotransplantation inventory. antonino sgroi, leo buhler, megan sykes, luc noel. surgical research unit, department of surgery, geneva university hospital, geneva, switzerland; transplantation biology research center, massachusetts general hospital, boston, ma; world health organization, geneva, switzerland. background: xenotransplantation carries inherent risks of infectious disease transmission to the recipient and even to society at large, and should only be carried out with tight regulation and oversight. a collaboration between the international xenotransplantation association, the university hospital geneva and the world health organization has established an international inventory (www.humanxenotransplant. org) aiming to collect basic data on all types of xenotransplantation practices on humans that are currently ongoing or have been recently performed. methods: we collected information using publications in scientific journals, presentations at international congresses, internet-based information, and declarations of ixa members. an electronic questionnaire is available on the website www. humanxenotransplant.org, which can be filled out and sent to the office in geneva. results: we identified a total of recent or current human applications of xenotransplantation, eight were currently ongoing and one will start soon. the source animal was: pig (n= ), sheep (n= ), calf (n= ), rabbit (n= ), blue shark (n= ), hamster (n= ), and unknown (n= ). all trials transplanted xenogeneic cells, i.e. islets of langerhans (n= ), hepatocytes (n= ), kidney cells (n= ), chromaffin cells (n= ), embryonic stem cells (n= ), fetal (n= ) and adult cells (n= ) of various organs. the treatments were performed in different countries, in europe, in russia, in asia, mexico, in usa and in africa. six countries had no national regulation on xenotransplantation. conclusion: several clinical applications of cell xenotransplantation are ongoing around the world, often without any clear governmental regulation. this information should be used to inform national health authorities, health care staff and the public, with the objective of encouraging good practices, with internationally harmonized guidelines and regulation of xenotransplantation. cells from α , -galactosyltransferase gene-knockout pigs additionally transgenic for human membrane cofactor protein demonstrate resistance to human complement-mediated cytotoxicity. hidetaka hara, cassandra long, mohamed ezzelarab, peter yeh, carol phelps, david ayares, david k. c. cooper. surgery, thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; revivicor, inc., blacksburg, va. purpose: ( ) to compare the antibody binding and complement-mediated cytotoxicity (cdc) of human sera to pig peripheral blood mononuclear cells (pbmc) and porcine aortic endothelial cells (paec) from (i) wild-type (wt), (ii) α , -galactosyltransferase gene-knockout (gtko), (iii) human membrane cofactor protein transgenic (mcp) and (iv) gtko/mcp pigs. ( ) to investigate the effect on binding and cdc of human sera following activation of paec. methods: pooled human serum was tested by flow cytometry for binding of igm and igg ( % serum concentration) and cdc ( % serum concentration) to pbmc and paec from wt, gtko, mcp, and gtko/mcp pigs. paec from all pig types were activated by ifn-γ, and again tested for antibody binding and cdc. results: there was higher binding of igm and igg to wt and mcp than to gtko and gtko/mcp pbmc and paec, but there were no differences in binding (i) between wt and mcp cells or (ii) between gtko and gtko/mcp cells. cdc of wt pbmc and paec was significantly greater than of gtko, mcp, and gtko/mcp pbmc (wt %; gtko %; mcp %; gtko/mcp %) and paec (wt %; gtko %; mcp %; gtko/mcp %) (both p< . ). importantly, there was no lysis of gtko/ mcp paec. after activation of paec, although cdc was increased against wt and gtko paec, mcp and gtko/mcp paec demonstrated significant resistance to lysis (wt %; gtko %; mcp %; gtko/mcp %). conclusions: ( ) cdc of all types of genetically-engineered pbmc and paec was significantly lower than of wt pbmc and paec, with lysis of gtko/mcp paec being significantly lower than to gtko or mcp cells. ( ) paec from both mcp and gtko/mcp showed resistance to lysis even after activation of the cells. ( ) organs from gtko/mcp pigs should provide considerable protection against cdc. role for cd -sirpα signaling in human t cell proliferation in response to stimulation with porcine antigen presenting cells. hiroyuki tahara, hideki ohdan, kentaro ide, toshimasa asahara. department of surgery, hiroshima university, hiroshima, japan. we have previously proven that genetic induction of human cd on porcine cells provides inhibitory signaling to signal regulatory protein(sirp) α on human macrophages; this provides a novel approach to preventing macrophage-mediated xenograft rejection. a recent report indicated that the similar cd -sirp system negatively regulates the functions of both t cells and antigen presenting cells(apcs) in humans. we hypothesize that the interspecies incompatibility of cd may also act as an additional barrier of t cell-mediated xenograft rejection. we have analyzed the frequency and proliferative activity of human t cells responding to either porcine or allogenic apcs by using in vitro mixed lymphocyte reaction (mlr) assay with a cfse-labeling technique. irradiated stimulator porcine or human peripheral blood mononuclear cells(pbmcs) were cultured with cfse-labeled responder human pbmcs. by fcm analysis, the number of division precursors was extrapolated from the number of daughter cells of each division and from the mitotic events, and precursor frequencies in cd + and cd + t cell subsets. using these values, stimulation index(si) was calculated. the frequencies of alloreactive and xenoreactive cd + t cell precursors were almost identical, . ± . % and . ± . %, respectively (n= - , for each type of precursor). however, the si of xenoreactive cd + t cells was significantly higher than that of alloreactive cd + t cells, indicating a stronger reaction by a single xenoreactive cd + t cell. in the presence of human cd -fc(containing the extracellular domain of human cd fused to the fc portion of human ig, µg/ml), the si of xenoreactive cd + t cells was significantly reduced to a level similar to that of alloreactive cd + t cells (n= , p< . ), although the frequencies of their precursors were absolutely uninfluenced. the frequencies of alloreactive and xenoreactive cd + t cell precursors were also identical, i.e. . ± . %, . ± . %, respectively (n= - ). the si of both alloreactive and xenoreactive cd + t cells did not differ: however, that of xenoreactive cd + t cells was significantly suppressed in the presence of human cd -fc (n= , p< . ). these results suggest that the t cell responses to porcine cells are stronger than those to allogeneic cells because of the interspecies incompatibility of cd . moreover, genetic manipulation of porcine apcs to induce human cd expression might attenuate human t cell-mediated xenograft rejection. reactivation of human cytomegalovirus (hcmv) is a potential risk following the clinical application of pig-to-human xenotransplantation. since little is known about undesirable side-effects arising from hcmv infection of porcine organs, we investigated the capabilities of various hcmv strains to infect porcine endothelial cells (pec) and putative immunological consequences thereof. pec from different anatomical origins were incubated with the hcmv laboratory strains ad and tb /e or a clinical isolate at a multiplicity of infection (moi) ranging from . to . viral replication kinetics, evolution of cytopathology, and lytic end points were analyzed. consequences of pec infection on human nk cell activation were evaluated using xenostimulation assay assessing nk cell ifng secretion and cytotoxicity. infection was evident and a maximum percentage of infected cells was reached at a moi of . hcmv replicated in all tested pec types, with a fraction of infected cells ranging from % to %. ad infection of a (microvascular ec) resulted in cytopathic effect (cpe) development by dpi and in lysis of % of the cells at dpi. contrary, no cpe was observed in ped (aortic ec) up to dpi. infection of a with tb /e was non-lytic and resulted in accumulation of both extra-and intracellular virus. virus titers reached a maximum at dpi, peaking at levels -fold higher than residual input virus. we then determined whether infected pec supported a complete replication cycle and produced viral progeny. after dpi pec supernatants and lysates revealed the production of significant amounts of virus. preliminary results showed that coculture of human nk cells and infected pec resulted in increased nk killing and ifng production. altogether, these findings provide evidence that pec are permissive and support the complete productive replication cycle of hcmv. however, cpe are cell-type and strain dependent. moreover, pec infection leads to modification of the xenogeneic cytotoxicity mediated by human nk cells. our findings allow a better estimation of the potential role of hcmv cross-species infection following xenotransplantation and may be crucial to guide future clinical trials. chronic rejection/injury: innate and adaptive immunity i testing for donor-specificity of antibodies post-transplantation increases the predictability of chronic rejection. mikki ozawa, arundhati panigrahi, paul i. terasaki, narindar mehra. one lambda, inc., canoga park, ca; new delhi, india; terasaki foundation laboratory, los angeles, ca. purpose: post-transplant hla antibodies have been shown to be linked to chronic graft failure, yet some recipients do well in the presence of antibodies. we investigated whether testing for donor-specificity of antibodies improves the predictive value of antibodies in regards to chronic rejection. methods: in a prospective study of renal transplant recipients, post-transplant sera were tested for hla and mica antibodies, and positive sera were tested by single antigen beads to determine whether the antibodies were donor-specific. one year after testing, biopsy and clinical data on the graft status were collected. patients who did not have follow-up or died with function were excluded from analysis. results: of the patients with follow-up and abdr typing info, ( %) were found to have antibodies. sixteen of those had donor-specific a, b, or dr antibodies (dsa), while had non-donor specific ab's (ndsa). sixty patients had only dp, dq, cw, or mica antibodies, and were grouped into "donor-specificity unknown", as these typing were not available at the time of this report. table summarizes the antibody groups and transplant outcome one year later. interestingly, the mean serum creatinine values at the time of antibody testing were very similar among the groups, regardless of the presence of antibody or the type (ranged . to . mg/dl). one year later, however, a striking % of dsa patients had either returned to hemodialysis, were regrafted, had died, or had biopsy-proven chronic rejection, compared to only % in ndsa patients and % in those who were negative (dsa vs. neg, p= . ). mica antibodies also showed significant association with graft failure or can (p= . ), and donor mica typing is underway. conclusion: in this prospective study of renal patients, dsa detected posttransplantation were highly correlated with chronic rejection. inclusion of specificity analysis in post-transplant antibody monitoring would significantly improve the predictability of chronic rejection. donor cd t cells within solid organ transplants contribute to graft rejection. thet su win, sylvia rehakova, margaret negus, kourosh saeb-parsy, martin goddard, eleanor bolton, andrew bradley, gavin pettigrew. university of cambridge; papworth hospital, united kingdom. this study examines the contribution of donor cd t cells within heart allografts to the development of graft vasculopathy, by providing help to recipient b cells for generating effector autoantibody responses. b mice were transplanted with mhc class ii-disparate bm hearts. the allo-and auto-antibody responses were quantified, and their contribution to allograft vasculopathy assessed by incorporating b cell deficient mice as recipients. the role of donor cd t cells in providing help for autoantibody was examined by removing cd t cells from donor hearts before transplantation by three approaches: treating with anti-cd mab, administering gy lethal irradiation, and using bm donors that are genetically deficient in t cells. the contribution of donor cd t cells to autoanitbody development was further assessed by challenge with bm cd t cells two weeks prior to transplantation. bm heart grafts developed progressive vasculopathy and were rejected slowly (mst= days, n= ). the contribution of antibody-mediated effector mechanisms was confirmed by histopathological evidence (fibrinoid necrosis and vascular proliferation), by complement c d staining, and by a reduction in the severity of vasculopathy and long-term graft survival in b cell deficient recipients. surprisingly, no alloantibody was detected, but recipients instead developed autoantibody. the autoantibody response was completely dependent upon the provision of help from donor cd t cells; it was abrogated by transplanting cd t cell deficient hearts. to further confirm an effector role for autoantibody in vasculopathy development, b mice were primed for humoral autoimmunity, but not for alloimmunity, by injecting with highly purified bm cd t cells prior to transplantation. heart grafts were rejected more rapidly (mst= days, n= , p< . ), and demonstrated severe vasculopathy. finally, bm-chimeric recipients that lacked mhc class ii expression only on b cells did not develop autoantibody, suggesting that cognate interaction between the donor cd t cells and mhc class ii of recipient b cells is crucial for post-transplant humoral autoimmunity. our results demonstrate the novel finding that help for autoantibody production is provided by graft-versus-host cognate recognition of recipient b cell mhc class ii by donor cd t cells. this autoantibody contributes to the development of vasculopathy independently of alloantibody. allograft rejection. gang chen, huiling wu, jainlin yin, josette m. eris, steven j. chadban. collaborative transplantation research group/renal medicine, university of sydney/rpah, sydney, nsw, australia. toll like receptors (tlrs) are innate immune receptors that play an important role in innate immunity but also provide a link between innate and adaptive immunity. myd is a key tlr signal adaptor. a recent clinical study reported that activation of innate immunity in heart-transplantation recipients through tlr contributes to the development of chronic rejection after cardiac transplantation. in this study, we aimed to determine whether tlr -myd signaling is required for the development of chronic allograft damage using tlr -/and myd -/mice in a murine model of chronic cardiac allograft rejection. methods: cardiac transplants were performed: b .c-h- bm (b .h- bm ) hearts to wt, myd -/and tlr -/mice (all c bl/ -h- b -single mhc class ii mismatch) as allografts (n = - /group) and c bl/ to c bl/ isografts (n = ). blood and tissue were harvested at day after transplantation. cell infiltration, fibrosis and vasculopathy were assessed histologically (grade - ). cd + foxp + cells in the blood and spleen were measured by flow cytometry analysis. results: all hearts remained pulsatile until sacrifice. histology of tlr -/and myd -/recipients showed protection from leukocyte infiltration ( . ± . & . ± . vs . ± . , p < . ), fibrosis ( . ± . & . ± . vs . ± . , p < . ) and vasculopathy ( . ± . & . ± . vs . ± . ) at day post transplantation compared to wt recipients. by facs, the ratio of cd + foxp + regulatory t (treg) cells to total cd + cells at day post transplantation in spleen was significantly increased in tlr -/and myd -/recipients versus wt allograft and isograft mice ( . ± . & ± . % vs . ± . & . ± . %, p < . ). conclusion: absence of tlr and myd signaling reduces chronic allograft damage in a murine model of chronic cardiac rejection. one mechanism of protection may be enhancement of regulatory t cell function. promotion of treg generation via the tlr / myd pathway may be one important consequence of cross-talk between innate and adaptive immunity. pathway: implication in transplant arteriosclerosis. thibaut quillard, stéphanie coupel, flora coulon, juliette fitau, maria-cristina cuturi, elise chiffoleau, béatrice charreau. inserm u , itert, nantes, france. endothelial cell (ec) activation, injury and apoptosis are the key events associated with transplant arteriosclerosis (ta) and chronic allograft rejection (cr). notch is a major signalling pathway controlling vascular function and injury. nonetheless, the involvement of notch, at endothelial level, in both ta initiation and progression remains unknown. using a fully mhc mismatched rat cardiac allograft model of cr, we found that ta at day correlates with a strong decrease in both expression and activity of the notch pathway in transplant as compared to tolerant and syngeneic controls. the present study investigates the contribution of notch activity to ec survival. in this purpose, a recombinant adenovirus, encoding the notch intracellular domain (nicd) and the reporter gfp cdnas was constructed and used to maintain notch activity in cultured primary human arterial ec. our results demonstrate that nicd protects ec from cell death induced by tnfα in the presence of chx or by anoïkis as measured by annexinv and dna content staining. nicd mediates cytoprotection through the regulation of key-apoptosis molecules. using an apoptosis-dedicated qpcr array, we found that out of a panel of apoptosis-related genes were significantly regulated. nicd upregulated bcl , bcl a and bcl-xl ( . -, . -and . -fold compared to noninfected cells, respectively). in addition, pro-apoptotic genes bim, drp , bok and cd were markedly downregulated in transduced cells ( . -, . -, . -and . -fold decrease, respectively). western blotting analysis confirmed the induction of protective molecules (bcl and bcl-xl) and the inhibition of pro-apoptotic proteins (bad, bak and bax). consistent with these data, nicd conducted phosphorylation of akt as well as a reduction of pten expression, suggesting that the cytoprotective activity of nicd may be mediated by recruitment of the pi k survival pathway. to conclude, our findings indicate that ta correlates with a decreased notch signaling in transplant and that activation of the notch pathway in vascular ec prevents apoptosis by promoting protective gene expression and survival pathway. these data suggest that controlling notch activity may prevent ec dysfunction associated with ta and cr. donor age intensifies the early immune response. christian denecke, xupeng ge, irene kim, daman bedi, anne weiland, anke jurisch, steven mcguire, johann pratschke, stefan g. tullius. div. of transplant surg, bwh, transplant surg res lab, boston; dept. of surgery, charite berlin, germany. increasing numbers of organs from elderly donors are currently utilized for transplantation. advanced donor age may not only be associated with physiological impairments but also with a modified immune response of the recipient. we hypothesized a more potent early immune response following the transplantation of elderly donor organs and we analyzed the immune response in a mouse heart transplant model. young b mice received heart allografts from , and mths old bm donors. the recipients immune response and intragraft changes were analyzed. elderly, non-manipulated hearts contained overall significantly elevated frequencies of cd + and cd + t-cells and dcs (cd c + ) ( mths vs. mths: cd + : . % vs. . %, cd + : . % vs . %, cd c + : . % vs. . %, p< . ). following engraftment of mths old heart grafts numbers of activated dc's (cd c + i-a b+ and cd c + cd + ) had significantly increased in recipient spleens (day :p< . ). in parallel, frequencies of effector/memory phenotype t-cells (cd + cd high cd l low and cd + cd high cd l low ) were significantly elevated with increasing age ( vs. vs, mths: cd + cd high cd l low : . % vs. . % vs. . %, respectively, p< . ). in addition, tregs (cd + cd + foxp + ) were also elevated ( vs. vs, mths: . %vs. . %vs. %,p< . ) t-cell alloreactivity, as measured by ifnγ-production, increased with donor age ( mths vs. mths vs. mths: . ± . vs . ± . vs. . ± . ifnγ-producing spots/ x cells, p< . ). mixed lymphocyte reaction (mlr) at day revealed a gradual increase in splenocyte proliferation with advancing donor age (p= . ) indicating an enhanced immunogenicity of older organs. immunohistochemical staining confirmed augmented cd + and cd + t-cell infiltrates and an intense ki positivity of gics in mths old heart grafts, emphasizing an intensified immune response towards organs from old donors. in summary, old native heart transplants contain an overall higher number of passenger leukocytes contributing to an increased immunogenicity of these organs. after transplantation, a more potent dc and t-cell activation and intragraft t-cell infiltration was observed in elderly organs. nox and chronic kidney allograft interstitial fibrosis. shannon reese, madhu adulla, surmeet bedi, jose torrealba, deb hullett, arjang djamali. nephrology, uw madison, madison, wi. to determine the role of oxidative stress (os) in kidney allograft fibrosis, we are developing strategies that would decrease the generation of reactive oxygen species (ros) instead of using ros scavengers, the standard approach to antioxidant therapy so far. we therefore started to examine the expression of nadph oxidase (nox) subunits (nox , nox , p and p phox) and their distribution in human and rat kidney allografts with chronic interstitial fibrosis (iftanos). using double-staining immunofluorescent studies in human allografts (n= ) we showed that nox was present in injured tubules costained with αsma ( figure . ) similarly, interstitial macrophages (cd + -nox + ) and myofibroblasts (αsma + -nox + ) but not cd + t cells or s a + fibroblasts expressed high nox levels, suggesting that nox is involved in the pathogenesis of allograft fibrosis via epithelial-tomesenchymal transition (emt), macrophage and myofibroblasts activation. we then examined the coexpression of nox , nox and p phox in normal and transplant kidneys with iftanos and showed that these molecules were upregulated in the latter group and that nox and nox were associated with p phox expression. these results were confirmed in the fisher to lewis rat kidney transplant model (figure . ). immunoblot analyses at weeks and months showed that nox and nox levels were increased in the allogeneic (n= ) compared to syngeneic transplants (n= ). greater nox levels were composite tissue allotransplantation (cta) is a recently introduced option for limb replacement and reconstruction of tissue defects. as other allografts, a cta grafts can undergo immune-mediated rejection, and standardized criteria are required for characterizing and reporting severity and types of rejection. this manuscript documents the conclusions of a symposium on cta rejection held at the ninth banff conference on allograft pathology in la coruna, spain on june , , and proposes a working classification scheme, the banff cta- , for the categorization of cta rejection. this classification was derived from public international consensus discussions regarding all published scoring systems for cta rejection. given the current limited clinical experience in cta, a formal histological classification was established for acute skin rejection with the understanding that other types of rejection involving other tissues will be developed with periodic review of this emerging field. it was agreed that the defining features to diagnose acute skin rejection would include inflammatory cell infiltration with epidermal and/or adnexal structure involvement, epithelial apoptosis, dyskeratosis, and necrosis, and that severity of rejection will be graded under five categories. this classification refines proposed schemas, represents international consensus on this topic, and establishes a working collective classification system for cta reporting. the role of skin biopsies in diagnosing clinical rejection in hand composite tissue allotransplantation (cta). christina l. kaufman, , ruben n. gonzales, kadiyala v. ravindra, , brenda w. blair, joesph f. buell, , warren c. breidenbach. , , christine m. kleinert institute, louisville, ky; kleinert kutz and associates, louisville, ky; jewish hospital transplant center, louisville, ky; surgery, university of louisville, louisville, ky. aim: traditionally allograft biopsy has dictated treatment of allograft rejection. we hypothesize that histological grade of the biopsy may over diagnose rejection in cta hand cta is unique in that the organ is external and early signs of rejection can be viewed directly. skin is the first tissue in the cta graft to show rejection. methods: for this study, rejection by defined by requirement of treatment. these episodes were compared with histological grade, and hand appearance. we reviewed skin biopsies taken during , and years of follow up. results: three observations surfaced. first, a rejection grading scale for cta is still evolving. a review of the literature showed at least four different criteria are in use. the criteria used to grade biopsies at our center changed over the year follow up. bias towards calling higher grades of rejection, and more aggressive treatment occurred in the first two patients. a re-read of random biopsies taken from the first two patients showed a down-grade of rejection to grade ii or i in five cases. secondly, concomitant cmv infection in the last patient prevented the aggressive treatment of rejection. despite high grade histology, the swelling, rash and redness responded to topical immunosuppression. systemic treatment was not necessary. analysis indicated that swelling and/or rash, and the percentage of skin involvement seemed to be more informative markers of existing (or resolving) alloreactivity than was histology. the biopsies taken from the graft shown below showed a grade iii histology. thirdly, changes in hand function were not associated with changes in biopsy grade. conclusion: the histologic grade of skin biopsies from hand allografts appears to over estimate rejection. alterations in immunosuppression shoud be based on appearance of the allograft, and clinical course as much as on the histologic grade of the biopsy. expression of molecular mechanisms of lymphozyte trafficking correlates closely with skin rejection in human hand transplantation. theresa hautz, bettina zelger, gerald brandacher, hans g. mueller, andrew w. p. lee, raimund margreiter, stefan schneeberger. dept. of general and transplant surgery, innsbruck medical university, austria; dept. of pathology, innsbruck medical university, austria; dept. of dermatology, innsbruck medical university, austria; div. of plastic surgery, university of pittsburgh. introduction: to understand in greater depth the molecular mechanisms involved in skin rejection in hand transplantation, we investigated key molecular markers of lymphocyte trafficking, cellular rejection and antibody mediated rejection in human hand transplantation. methods: a total of skin biopsies taken from three bilateral hand transplants were assessed by h&e histology (grades as per previously a published classification - b) as well as immunohistochemistry using antibodies for following markers: lymphocyte function-associated antigen (lfa)- = cd a, intercellular adhesion molecule (icam)- = cd , selectin e = cd e, selectin p = cd p, ve-cadherin = cd , human leukocyte antigen (hla) ii (dp, dq, dr), psoriasin = s a and c d. levels of expression were assessed ( , +, ++, +++) and read in the light correlated with the rejection as well as time after transplantation. results: rrejection ranged between grade and a with an average score of . . in healthy skin, none of the markers investigated was consistently up-regulated. upon rejection, cd , cd e and cd p staining in endothelial cells was significantly increased. expression of cd e and cd p correlated well with severity of rejection. the majority of infiltrating lymphocytes stained positive for cd a. interestingly, also kerationcytes were highly positive for cd a at the onset of rejection. cd was detected on endothelial cells, but its occurrence did not correlate with rejection. psoriasin expression was observed in keratinocytes in a basal and focal pattern and correlated well with rejection. for c d, no consistent staining pattern was observed indicating that antibody mediated rejection did not play a role in these patients. conclusion: molecular markers involved in lymphocyte trafficking are up-regulated upon skin rejection after hand transplantation and represent promising target for prophylaxis and treatment of rejection in composite tissue allotransplantation. investigation of the immunomodulatory phenotype of infiltrating lymphozytes in skin rejection of human hand allografts. theresa hautz, gerald brandacher, bettina zelger, hans g. mueller, andrew w. p. lee, raimund margreiter, stefan schneeberger. dept. of general and transplant surgery, innsbruck medical university, innsbruck, austria; dept. of pathology, innsbruck medical university, austria; dept. of dermatology, innsbruck medical university, austria; div. of plastic surgery, university of pittsburgh. introduction: skin rejection has complicated the postoperative course in human hand transplantation. to better define the characteristics of the lymphozytic infiltrate human hand transplant biopsies have been investigated for expression of foxp and indoleamine , -dioxygenase (ido), a key regulatory enzyme to induce t-lymphocyte unresponsiveness. methods: a total of skin biopsies taken from three bilateral hand transplant recipients during the first years after transplantation were assessed by h&e histology (graded as per previously published classification - b) as well as immunohistochemistry for ido and foxp . levels of expression were assessed ( , +, ++, +++) and interpreted in the light of clinical courses, time after transplantation, severity of rejection as well as markers for lymphozyte migration. results: overall, rejection ranged between grade and a with an average score of . . ido was found constitutively expressed in the endothelium independent of rejection. ido expression in the cellular infiltrate was significantly increased upon and correlated well with severity of rejection (rejection grade , . +/- . : rejection grade , . +/- . p< . ). foxp positive t-cells were mainly found in severe rejection (rejection grade , . +/- . : rejection grade , . +/- . p= . ). ido expression correlated well with foxp expression, although the overall staining intensity for foxp was lower. a strong tendency towards higher expression of ido as well as foxp towards later time-points after transplantation was observed (year -foxp . +/- . , ido . + /- . [n= ] , year -foxp . +/- . , , year -fox . +/- . , ). expression of ido correlated closely with expression of e-selectin, p-selectin, icam and lfa- . conclusion: characteristics of the cellular infiltrate indicate a strong tendency towards self limitation of the alloimmune response towards the skin with both time after transplantation as well as severity of rejection in human hand transplantation. further studies are warranted to clarify the clinical relevance of these findings. prospective analysis of the immunologic profile of a hand transplant recipient in the first year. kadiyala v. ravindra, warren c. breidenbach, joseph buell, suzanne t. ildstad. department of surgery, university of louisville, louisville, ky; kleinert, kutz, and associates, louisville, ky; institute for cellular therapeutics, university of louisville, louisville, ky. introduction: a major obstacle to the wider application of hand transplantation is the long term complications associated with immunosuppression. minimization of immunosuppression is an important goal in all transplant recipients. currently there are no accurate tools to evaluate the immunological responsiveness which might help tailor the level of immunosuppression for an individual patient. the response of recipient lymphocytes to pha, candida, and alloantigen may represent laboratory tool towards this end. it has been reported that these responses are hierarchical with response to alloantigen being the first to be lost, followed by candida and finally pha. a year old male received a proximal forearm transplant in november . immunosuppression included induction with a single mg dose of alemtuzumab and maintenance with tacrolimus and mycophenolate mofetil. the patient developed an episode of cytomegalovirus infection followed by acute rejection after reduction of his immunosuppression during the rd post-operative month. these were successfully treated with ganciclovir and topical tacrolimus & steroids respectively. blood samples were drawn at selected time points, and subjected to phenotyping of lymphocyte subsets and immune monitoring for circulating peripheral blood regulatory t cells (t reg ) and proliferative responses to phytohemagglutinin (pha), candida, and alloantigen. results: alemtuzumab induction resulted in profound lymphopenia. at week and month, the response to pha was intact (stimulation index and respectively), but response to alloantigen and candida suppressed (si < ). a similar immunologic profile persisted up through months. at year, the pha and candida responses are robust (si and respectively), but alloresponses have not returned. current immunosuppression consists of tacrolimus ( - ng/ml) and mycophenolate mofetil ( mg b.i.d.). there is no gross evidence of acute or chronic rejection. conclusions: induction with alemtuzumab alters the recovery of immune response: recovery to candida was delayed beyond months and to alloantigens beyond a year. in light of this, further reduction of immunosuppression may be contemplated in future hand transplants without the risk of rejection. composite tissue allotransplantation has achieved significant clinical advances despite an adequate preclinical model to study technical and immunosuppressive strategies. we have developed a non-human primate model of facial composite tissue allografts (cta). unilateral lower hemi-facial cta (bone, muscle, skin) transplants were performed between mismatched cynomolgus monkeys. immunosuppression consisted of days of continuous iv tacrolimus monotherapy followed by tapered daily im doses. six animals received prophylactic gancyclovir. all animals had serial transplant biopsies. ten transplants have been performed, with one loss secondary to line infection on day without evidence of rejection. two ctas had evidence of chronic rejection (day , ); with development of alloantibody after days. five ctas had prolonged survival (day , , , , ) , but developed ptlds resulting in experimental endpoints. all animals had clinically normal grafts, but animals showed histological evidence of mild rejection not treated with any additional immunosuppressive therapy. ptld tumors were analyzed using short tandem repeats (str) to define donor or recipient origin. str analysis demonstrated donor origin of ptld tumors and recipient origin in animal. none of the ptld animals had clinical evidence of rejection of skin, bone, or muscle. ebv was not detected in the serum of tested animals, and ganciclovir therapy had no effect on the development of tumors. tacrolimus levels of ptld animals were higher than animals with rejected grafts ( vs. ng/ml; p= . ). two additional animals have healthy grafts (day +, +) without evidence of rejection or ptld, and have been converted to rapamycin after day . we have developed a preclinical model for facial cta transplantation that achieves prolonged graft survivals with tacrolimus monotherapy. the high incidence of ptld tumors of donor origin represents an outcome similar to bone marrow transplantation in contrast to its rarity in solid organ transplantation. our findings are a cautionary note regarding ctas that include vascularized bone elements. immunosuppressive protocol modifications have been made in an effort to decrease the incidence of these donor-derived ptlds. heterotopic heart transplantation: the united states experience. jama jahanyar, tarek a. sibai, matthias loebe, michael m. koerner, guillermo torre-amione, george p. noon. dept. of surgery, baylor college of medicine, houston, tx. heterotopic heart transplantation (hht) is utilized in patients (pts) who do not qualify for standard orthotopic heart transplantation (oht). specific indications include refractory pulmonary hypertension and a donor-recipient size mismatch. the objective of this study was to analyze the unos database and compare outcomes of hht to oht. the unos database with more than pts undergoing thoracic organ transplantation in the u.s. between and was reviewed (based on optn data as of may , ) . primary endpoint of this study was overall survival and subgroup survival [pts with transpulmonary gradient (tpg)> , ischemic (icm) and dilated cardiomyopathy (dcm)]. secondary endpoint was assessment of pretransplant criteria. exclusion criteria were retransplantation and missing transplant dates. of who underwent oht and who underwent hht, and respectively, were enrolled in this study. [ ] [ ] [ ] [ ] [ ] [ ] survival after oht is superior to hht. this survival benefit however, disappears in pts with a tpg> . overall the survival after hht is superior to the reported survival in pts who undergo lvad implantation as destination treatment (rematch-trial/ year survival of %). thus in selected pts, especially those with elevated tpgs, hht should be considered a viable option with overall good results. ventricular assist device as a bridge to heart transplantation in children: can we afford it? william t. mahle, glen ianucci, robert n. vincent, kirk r. kanter. sibley heart center cardiology, emory university school of medicine, atlanta, ga. ventricular assist devices (vads) allow children with severe heart failure to be bridged to successful heart transplantation (ht). vads are being used with increasing frequency in the pediatric population and newer devices allow even young infants to be supported. vad implantation and maintenance, however, is quite expensive and the cost-effectiveness of vad use in adults has been questioned. to date, an economic analysis of vad support in children has not been undertaken. methods: we used pediatric health information system (phis), an administrative database of the child health corporation of america (a consortium of children's hospitals in north america), to determine the costs related to vad use in children. data on subjects < yrs of age from - were reviewed. hospital charges were converted to costs based on hospital-specific cost-to-charge ratios. projected survival for subjects who were successfully bridged to ht was derived from published data. costutility was expressed as cost per quality-adjusted life years (qalys) saved, expressed in us dollars. all future costs and benefits were discounted at %. results: the median age at implantation from the phis database was . years, range days to yrs. the mean hospital cost per patient was $ , . estimated survival to heart transplantation was % and estimated successful explantation without transplantation was %. the calculated cost-utility for vad as a bridge to transplantation was $ , /qaly saved. if one assumes that the children who survived to vad explantation would otherwise have a high risk of hospital death ( %) without vad support, then the calculated cost-utility would be $ , / qaly saved. even if abstracts survival to transplantation exceeds %, vad implantation does not achieve a favorable cost-utility ratio. vad support in pediatric heart only becomes cost-effective if recovery and explantation can be achieved in over % of subjects. conclusions: vad supports serves as an effective bridge to heart transplantation in children. however, the cost-utility of this strategy is above the generally accepted threshold for cost-effectiveness ($ , /qaly). in a setting of limited healthcare resources vad as a bridge to transplantation may not be justified. purpose: heart transplantation (tx) in patients with hla sensitization presents challenges in organ allocation. virtual crossmatch (vxm), in which recipient hla antibodies, identified by labscreen pra beads, are compared to the prospective donor hla-type, could increase the use of allografts from distant donors (dd). accuracy of vxm and outcomes of this approach in heart tx are not known. methods and materials: to increase time-efficiency at the time of organ allocation, crossmatch testing is frequently initiated on pre-set trays which contain sera of multiple prospective sensitized recipients. we used results from these studies to determine expected accuracy of vxm. we assessed outcomes of allocation algorithm implemented in in sensitized patients. conventional prospective crossmatch was done when allografts were procured from local donors (ld), while vxm was utilized when allografts were offered from dd. there were direct t-cell ahg crossmatch tests done with sera of potential allograft recipients who had preformed hla-antibodies of known class i hla specificities. as shown in table , the positive and negative predictive values (ppv, npv) of vxm were % and %. table shows outcomes in sensitized patients who were eligible for vxm approach. received allografts from ld with negative prospective crossmatch while ( %) received allografts from dd with negative vxm. three dd patients had a positive retrospective crossmatch -npv of vxm was % in this cohort. vxm has high negative and positive predictive values, accurately predicting results of standard direct crossmatch in most patients. vxm allows use of allografts from dd and is likely to improve organ allocation in the disadvantaged group of sensitized patients. single center experience with new heart allocation system implemented by united network of organ sharing. biljana pavlovic-surjancev, nilamkumar patel, linda dusek, james sinacore, jennifer johnson, cassie bessert, alain heroux. heart failure/heart transplant program, loyola university medical center, maywood, il. purpose: in july , united network of organ sharing (unos) implemented a new allocation system for adult heart transplant (tx) candidates with the following sequence: local status a→local status b→zone a status a→zone a status b→local status . the purpose of this study is to evaluate the impact of new system on heart tx patients at a single center in region . methods: patients transplanted during year (y) prior to new system ( - - to - - , group , n= ) and y following new system ( - - to - - ,group , n= ) were compared for unos status at the time of transplant, waiting time, ischemia time, length of the hospital stay (los) before and after transplant, donor age, procurement-team travel distance and cost. results: new system significantly decreased median waiting time, but increased median ischemia time without affecting short-term survival: patient died in each group. number of transplants increased % in group mostly due to increased number of status a patients supported by iabp without change in number of status b and patients. median pre-transplant los increased -fold in group (p= . ), whereas mean pre-transplant los increased by days. in group , thirteen patients had donor heart procured in zone a and thirteen patients had donor heart procured locally, whereas in group , all donor hearts were procured locally. median procurement-team travel distance increased -fold and median travel cost -fold in group (p< . ). clinical outcomes associated with simultaneous heart-kidney transplantation. tariq shah, , , , suphamai bunnapradist, jagbir gill, steven k. takemoto. the figure below indicates recipients of shk transplants (open symbols) had lower rates of rejection when compared to heart (square) or kidney (diamond) recipients. survival rates were initially higher for kidney recipients with rates for shk similar to heart recipients. the hazard ratio (hr) for graft loss was higher for shk recipients compared to kidney, but lower when compared to heart recipients. the lower hazard for shk in heart allografts might be attributed to risk associated with pretransplant dialysis (hr= . , . - . , p< . ) . approximately % ( , ) of cardiac transplant recipients initiated dialysis prior to transplantation. the differing rates of shk rejection observed in the heart and kidney analytic files might be attributed to susceptibility or treatment of heart and kidney allografts, rejection monitoring or reporting. conclusion: retrospective examination of data provided to the optn indicates simultaneous heart-kidney transplantation seems to be effective for cardiac transplant candidates who require dialysis. abstract# objective: krp , a novel structural analog of fty , has been documented to display -fold greater selectivity of agonism for sphingosine- -phosphate (s p) type (s p ) receptor compared with s p receptor. clinical trial has shown that fty produced dose-limiting toxicity-bradycardia, due to its effect on s p receptor. we have tested the effect of krp on the survival of islet allografts either alone or combined with local delivery of cd + cd + foxp + t regulatory (treg) cells. previous studies using knockout mice have documented a key role for the integrin cd in promoting allograft rejection. these data are consistent with a critical role for cd expressing cells in this process. however, a direct test of this hypothesis has proven problematic due to the lack of mabs that efficiently deplete cd + cells in wild type hosts. to circumvent this problem, we conjugated the non-depleting anti-cd mab, m , to the toxin, saporin (sap), to produce an immunotoxin (m -sap) that selectively depletes cd -expressing cells in vivo. treatment of naive mice with m -sap selectively depleted cd + cd + cells and dramatically reduced the overall frequency of cd + lymphocytes in diverse compartment including intestinal intraepithelial lymphcyte,spleen and mesenteric lymph node. m -sap also depleted cd + dendritic cells (cd c + ) and t regulatory cells (tregs, cd + cd + ) in the above compartments. in the thymus, m -sap depleted cd -expressing cells in both cd -cd and cd -cd + subpopulations, both of which express cd , leading to a dramatic reduction in the number of thymocytes( . ± . × vs. . ± . × , p< . , in control vs. treated respectively). we next assessed the effect of m -sap in a fully allogeneic islet transplantation model (balb/c→c bl/ ). m -sap produced long-term (lt) graft survival (> d, n= ,vs. untreated median survival time d, n= ). unconjugated m or isotype control (igg-sap) did not significantly prolong islet allograft survival. graft histology showed little, if any, lymphocyte infiltration surrouding islet transplants in lt mice. in contrast, intense lymphocyte infiltration with disruption of islet morphology was observed in untreated mice. pretreatment of donor islets with m -sap did not significantly prolong allograft survival indicating that the immunosuppressive effect of m -sap resides at the level of host. interestingly, we found a - fold increase in both percentage and number of foxp + cd + cd + cells in the spleen and draining lymph node from lt mice, though it remains to be determined whether such cells account for graft acceptance in m -sap treated recipients. in summary, these data document that depletion of cd expressing cells promotes long-term islet allograft survival. these findings point to a novel strategy for therapeutic intervention in islet allograft rejection. pancreatic objective: to engineer pancreatic islets in a rapid and efficient manner with a novel form of fasl protein chimeric with core streptavidin and test the efficacy of engineered islets for long-term survival in allogeneic hosts. methods: balb/c pancreatic islets were engineered first by cell surface modification with biotin followed by the display of a chimeric form of fasl protein that consists of extracellular domain of fasl fused c-terminus with core streptavidin (sa-fasl). sa-fasl-engineered islets were transplanted into streptozotocin diabetic c bl/ mice under transient cover of rapamycin. unmodified islets or those engineered with streptavidin protein (sa) served as controls. results: all the islets showed effective engineering with sa-fasl, which persisted on the surface of islets for weeks in vitro as assessed by confocal microscopy. all the islets (n= ) engineered with sa-fasl survived over the observation period of - days without detectable signs of rejection. in marked contrast, all the unmodified (n= ) and sa-engineered (n= ) islets underwent acute rejection within days. the observed tolerance was localized to the engineered islets as unmodified second set of islets transplanted under contralateral kidney of long-term (> days) graft recipients were rejected in a normal tempo (mst= ± days) without any effect on the survival of primary islets. conclusions: engineering pancreatic islets with exogenous immunomodulatory molecules, such as sa-fasl, in a rapid (∼ hrs) and efficient ( % of targeted islets) manner represents a novel means of immunomodulation with considerable therapeutic potential for the treatment of type diabetes. supported in parts by nih (r dk , r ai , r ai , r hl ), jdrf ( - - ) the ability of embryonic stem (es) cells to form cells and tissues from all three germ layers can be exploited for the generation of cells that can be used to treat diseases. in particular, successful generation of hematopoietic cells from es cells could provide safer and less immunogenic cells than bone marrow cells, that require severe host preconditioning, when transplanted across mhc barriers. in the past, it has been difficult to derive hematopoietic cells from es cells. it has now become clear that this was due to the lack of self-renewal properties by these newly developed progenitor cells. here, we exploited the self-renewal properties of ectopically expressed hoxb , a homeobox transcription factor, to generate hematopoietic progenitor cells (hpcs) that successfully induce high level mixed chimerism and long-term engraftment in recipient mice. hoxb -transduced svj es cells (h b ) were allowed to form embryoid bodies. these were dismantled after days and the cells treated with a cocktail of hematopoietic cytokines. by day , es cells had formed hpcs. these newly generated hpcs were cd +, cd +, cd + but poorly expressed mhc class i molecules and no class ii. the hpcs fully restored splenic architecture in rag -/-γ c -/immunodeficient mice, abstracts comparable to bone marrow. additionally, hpc-derived newly generated t cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus (lcmv) and specifically secreted il- and ifn-γ upon cd stimulation. further, hpc-derived antigen presenting cells (apcs) in chimeric mice efficiently presented viral antigen to wild type (wt) t cells. in syngeneic recipient mice, hpcs engrafted and formed more robust t and b cell populations. the majority of the hpc-derived cells were however, gr- + , suggesting a bias towards myeloid cells by the hoxb . interestingly, these cells successfully engrafted in allogenic mrl (h k ) and balb/c (h d ) recipients without the need for immunosuyprresion. this ability to form mixed chimerism across mhc barriers is a consequence of their lack of mhc, cd and cd expression. our results demonstrate for the first time that leukocytes derived from es cells ectopically expressing hoxb are immunologically functional and escape immunological rejection when transplanted across mhc barriers allowing the induction of mixed chimerism. normalization the de is derived from the anterior segment of the primitive streak which corresponds to the early and mid-gastrula organizer during early embryo development, from which many of the major visceral organs, including the liver, pancreas, lung, thyroid and intestines are derived. es cells were cultured in a serum-free medium containing activin a and bfgf for - days, the differentiated cells developed into an epithelial monolayer yielding more than % of cxcr expressing cells. cxcr has been reported as a cell surface marker of the definitive endoderm. molecularly, the differentiated es cells express typical definitive endodermal genes in particular, foxa , sox- , gsc, and hnf α. the cxcr + definitive endodermal cells were further purified using immunomagnetic bead separation to more than % purity in order to eliminate teratoma-forming cells. to study their engraftment and regenerative capacity, these newly differentiated cells were intravenously infused into a mouse with carbon tetrachloride-induced liver injury. harvested livers from these animals showed large de-derived cells positive for albumin suggesting that they were de novo generated hepatocytes. a second cell type was ck- expressing, suggesting that the engrafted cells also differentiated into cholangiocytes. therefore, we further transplanted these de cells into a factor viii null mouse and asked whether these cells could correct factor viii activity. plasma factor viii activity (coamatic assay) fully normalized to that of wild type mice and has remained stable over days. these data suggest that es cell-derived de progenitor cells can restore factor viii activity in hemophilia a mouse model, presumably through protein production in de novo generated hepatocytes. more importantly, none of these animals developed teratomas. thus, es-cell derived cells can potentially be coaxed to form cellular transplants with curative capabilities. objective: we have established a novel approach, protex, to rapidly and efficiently engineer primary cells, tissues, or organs to display on their surface exogenous proteins of interest for immunomodulation. this approach involves generation of chimeric proteins with core streptavidin, biotinylation of cells, and the transient display of chimeric proteins on the cell surface. in this study, we displayed a chimeric form of fasl (sa-fasl) on the surface of bone marrow cells and tested the efficacy of these cells to establish mixed chimerism in allogeneic hosts under nonmyeloablative conditions. methods: balb/c bone marrow cells were engineered with sa-fasl and million of these cells were transplanted into c bl/ mice subjected to various doses of total body irradiation days earlier. a short course of rapamycin was used to enhance the tolerogenic effect of sa-fasl. bone marrow cell recipients were typed for multilineage chimerism at various times post-transplantation and tested for donor-specific tolerance using skin grafts. results: all the animals (n= ) treated with cgy total body irradiation and transplanted with sa-fasl-engineered donor cells showed significant levels of chimerism ( - %) on day post-transplantation that showed a steady increase overtime and reached to - % on day post-transplantation. in marked contrast, none of the control animals (n= ) receiving bone marrow cells and a short course of rapamycin showed detectable chimerism. chimerism was multilineage and associated with donor specific tolerance since chimeric animals accepted donor, but rejected third party skin grafts. conclusions: engineering bone marrow cells in a rapid (∼ hrs) and efficient ( % targeted cells) manner with exogenous proteins having immunoregulatory functions provides a new and effective means of immunomodulation to establish mixed allogeneic chimerism under nonmyeloablative conditions with significant potential in clinical bone marrow transplantation. funded in parts by nih (r dk , r ai , r ai , r hl ), jdrf ( - - ) , ada , and aha fellowship b. chronic allograft dysfunction is still a major clinical problem in organ transplantation. morphologically it is characterized by changes suggestive of an alloantibody mediated mechanism such as glomerulopathy and vasculopathy or by non-specific changes such as interstitial fibrosis and tubular atrophy. alloantigen dependent as well as -independent factors contribute to the pathogenesis of these changes. in this study we analysed allospecific t cells from the peripheral blood of kidney transplant patients under different immunosuppressive protocols with or without chronic allograft dysfunction. renal allograft recipients of our renal transplant clinic were screened at least six months after transplantation. all patients were on a calcineurin-based immunosuppressive protocol consisting of cyclosporine (csa)/mycophenolate mofetil (mmf)/steroid, tacrolimus (tac)/mmf/steroid, or csa/steroid. patients had to be mismatched for one or more of the five candidate hla-dr antigens for which synthetic peptides were available (dr , dr , dr , dr , and dr ). patients with biopsy proven chronic allograft nephropathy (can)with an elevated serum creatinine level of ≥ . mg/dl were compared with patients with stable allograft function (serum creatinine < . mg/dl). t cell lines were generated from peripheral blood lymphocytes of renal transplant recipients against donor-derived hla-dr peptides presented by self apc. t cell lines generated from patients with can produced significantly more ifn-γ, while those generated from stable patients produced il- associated with a low proliferation index in response to the donor-derived mismatched hla-dr allopeptide in vitro. moreover, significantly more cd +cd +foxp + t cells were found in stable patients on tac and mmf as compared to patients on csa and mmf. interestingly, a higher gene expression of cd , cd , ctla- , foxp , and il- was observed in those patients. taken together a th (ifn-γ) alloimmune response is deleterious and promotes chronic graft damage, while a th (il- ) response seems to be associated with a lower incidence of chronic allograft nephropathy. an immunosuppression based on tac and mmf seems to favour cd +cd +fosp + t cells and to allow long-term engraftment with stable renal function. cyclosporin induces epithelial to mesenchymal transition in renal grafts. the expression of epithelial to mesenchymal transition (emt) markers is a reliable predictor of the progression towards interstitial fibrosis and tubular atrophy of the renal grafts. in vitro experiments suggest that calcineurin inhibitors (cni) can induce emt of tubular epithelial cells. although no evidence was ever provided in vivo, this suggests that emt could be involved in the pathogenesis of renal fibrosis induced by cni. we have previously reported the results of a prospective randomized trial comparing the elimination at month of either cyclosporine (csa, n= ) or mycophenolate (mmf, n= ) from a triple drug regimen in de novo renal transplant patients. all of them had systematic graft biopsies at months and post engraftment. in the leftover material, we retrospectively detected in tubular cells and by immuno-histochemistry the expression of two validated markers of emt: the de novo expression of vimentin (vim) and the cytoplasmic translocation of b-catenin (cat). we were able to measure the emt score at both months and in a total of patients ( in each group). in the csa group, the vim and cat scores had progressed between and months from . calcineurin inhibitors (cni) are efficacious but nephrotoxic immunosuppressives. arteriolar hyalinosis is one of the characteristic histological correlates of this toxicity. yet, time course of this lesion, its reversibility, dose-dependency and the discrimination between drug-related effects, diabetic and hypertensive vasculopathy remain unclear. aim of this study was to evaluate the prevalence and time course of cni-related vascular changes after renal transplantation (tx) in protocol (pbx) as well as in indication biopsies (ibx) and to correlate this to the cni blood levels, blood pressure and diabetes. from patients, a total number of pbx, taken at weeks (n= ), (n= ) and months (n= ) after tx and ibx were classified according to banff-criteria. assessement of cni toxicity included: isometric vacuolisation of tubules (isovac), intimal arteriolar hyalinosis (iah) and nodular arteriolar hyalinosis (nah) and vacuolisation of small vessel smooth muscle cells (vsm). % of patients received either cyclosporine or tacrolimus. in pbx, isovac was present in %, % and % of patients at weeks, and months post-tx, respectively; iah in %, % and %; nah in %, % and %; vsm in %, % and %. in late ibx (> years post-tx) the prevalence of the analyzed parameters apart from isovac ( %) was markedly higher: % for iah, % for nah and % for vsm. in pbx, vsm, iah and nah were associated with each other but not significantly dependenct on blood pressure, rejection episodes or diabetes. through levels of cnis were not different between patients with and without vascular hyalinosis. in patients with vsm and iah in late ibx one third had these lesions already present in earlier biopsies. conclusion: the prevalence of presumed morphological signs for vascular cni-toxicity in pbx is low and constant and apparently, not associated with cni blood levels, hypertension or diabetes. in contrast, in ibx later than two years after transplantation, prevalence of vascular cni-toxicity signs is much higher. this emphasises that cni reduction protocols should be regarded within the first six months after transplantation, when vascular changes are still marginal. further elucidation of precursor lesions in pbx would help to find out patients at risk for cni-induced vascular changes. donor introduction: achieving donor specific tolerance has been the goal of the transplant community. success has been reported with donor stem cell transfusion in animal studies and prevention of chronic rejection in cardiac allograft recipients in the clinic. this report details the results of the initial phase of a study in humans. methods: a prospective phase i/ii fda approved pilot protocol was initiated to evaluate the effects of donor graft facilitating cell (fc)/stem cell infusion in kidney transplant recipients. conditioning was performed with cgy of total body irradiation. bone marrow processed to remove gvhd-producing cells but retain cd + /tcr -fc and stem cells was infused hours post-operatively. the dosage of stem cells was limited by the t cell dosage. the starting dose was x t cells. this was increased in steps of x per patient. as the study spans a -year period, the immunosuppression changed: patients received cyclosporine (cya), mmf and prednisone; were induced with basiliximab and maintained on cya( )/fk( ), cellcept and prednisone; and received alemtuzumab induction and maintenance with fk and mmf. all patients underwent tolerance testing and immunoprofiling studies. results: of the patients, received live donor kidney transplants. one graft was lost from arterial thrombosis on day . delayed graft function was seen in patients. good long-term graft function was seen in the other patients. acute rejection was noted only in and infectious complications (cmv- , histoplasma- ) in patients. two patients died with functioning grafts -one at years from lung cancer and another from complications from diabetes at years. six patients are alive with functioning grafts at mean follow-up of . years with a mean creatinine of . mg/dl. no gvhd was detected in any of the patients. macrochimerism was not detected in any of the patients at any point. notably, in spite of the fact that durable engraftment was not yet achieved, none of the patients were sensitized as a result nor did they experience immunologic sequelae. conclusions: in our patients there were no untoward sequelae related to either the conditioning regimen or marrow infusion. the incidence of acute rejection even on longterm follow up has been low. we currently propose to reduce the immunosuppression further in these patients. a pilot study was performed to evaluate whether immune cell depletion with alemtuzumab would permit post-transplant weaning of maintenance immunosuppression in well-matched renal transplant recipients. patients received alemtuzumab mg intravenously on the day of the transplant and the subsequent days while sirolimus and tacrolimus were started on day . tacrolimus was discontinued at day in all patients. extensive immune monitoring was performed at year. at current follow-up ( to months), all patients are alive with a functioning graft (median mdrd gfr= ml/ min). one patient experienced clinical and biopsy-proven rejection at months. all other patients remain on sirolimus monotherapy. four patients have been weaned to mg of sirolimus daily as their sole immunosuppressive agent, with resulting blood levels of - ng/ml. these patients have no evidence of donor-specific alloantibody, are unresponsive or hyporesponsive to donor cells by the cytokine kinetics test, and have a regulator phenotype to soluble donor antigens by trans-vivo dth. flow cytometry of peripheral blood demonstrated increased foxp expression in the cd +cd + population (p= . ). naive b cells (cd /cd neg) cells increased in of patients (p= . ) and memory b cells increased in all recipients (p= . ) when comparing pretransplant to year timepoints. other than the patient with rejection, -month protocol biopsies did not show any evidence of rejection, although of showed focal c d positivity and diffuse positivity. these patients also had evidence of alloantibody by luminex xmap testing. in conclusion, the cytokine kinetics test, alloantibody testing, and trans-vivo dth assay abstracts results correlated with clinical evolution of patients who successfully weaned both tacrolimus and sirolimus without rejection or alloantibody. the flow cytometry findings described occurred regardless of clinical evolution and may represent alterations of the immune system inherent in the treatment protocol independent of individual patient responses to the graft. however, the functional assays of cytokine kinetics assay and trans-vivo dth may be of potential use to correlate with the clinical immune status of the kidney transplant recipient. we have previously reported the short-term results of alemtuzumab (campath- h) pre-conditioning with tacrolimus monotherapy and subsequent spaced weaning in living donor kidney transplantation (ldkt). we report here our year experience. methods: we performed consecutive unselected ldkt (donor kidneys were removed laparoscopically) from / / to / / using mg ( . mg/kg) alemtuzumab and tacrolimus monotherapy. at months post-transplant and every to months interval, we used clinical data (including elisa antibody titers, cylex t-cell activation assay, and identification of donor specific antibodies) to wean tacrolimus when possible (bid-->qd-->qod-->tiw-->biw-->qwk). the recipients included hiv+, pediatric recipients, and re-transplants. the mean follow up was . + . days. results: actuarial recipient survivals at -, -, -years were . %, . %, and . %, respectively. graft survivals at -, -, -years were . %, . %, and . %, respectively. the mean creatinine (mg/dl) at -, -, -years were . + . , . + . , and . + . , respectively. the mean gfr (ml/min/ . m ) at -, -, -years were . + . , . + . , and . + . , respectively. the cumulative incidence of acute cellular rejection (acr) at -, -, -, -, -, -, -, and > conclusions: in the current era low risk patients infrequently have ar, and have excellent short and long term graft survival without the use of depleting antibodies. given the increased costs of these drugs, the indications for using depleting antibodies in low risk ktrs of scd kidneys should be further clarified. kidney transplantation prolongs survival in hepatitis c virus-positive (hcv+) patients with end-stage renal disease (esrd). however, the effects of induction therapy and chronic immunosuppression are unknown on the course of hcv infection and potential for cirrhosis in renal transplantation (rtx) recipients. we have retrospectively assessed parameters of liver function, child-pugh (cp) and meld scores in hcv+ esrd patients who received induction therapy with t-cell depletion (group : thymoglobulin, n= ) or an il- inhibitor (group : basiliximab, n= ). pre-rtx liver biopsies were similar in group and . patients were followed for a mean of days (range to days) following rtx and received tacrolimus, mycophenolate mofetil, and sometimes steroids post-transplant. overall graft survival was % in group and % in group (p > . ). data were analyzed pre-rtx, at and days and at time of last follow-up. serum ast, alt, platelets, inr, albumin and bilirubin did not change following rtx in either group. cp scores in group were not significantly changed after rtx ( . ± . to . ± . at last follow-up, p= . ). group patients on steroid-free protocols (n= ) demonstrated declining cp scores from . ± . to . ± . at last follow-up that were not statistically significant (p= . ). group patients on steroids showed opposite trends of cp: . ± . pre-rtx vs . ± . at last follow-up that similarly did not reach statistical significance (p= . ). group cp scores declined from . ± . to . ± . at last follow-up (p= . ). there was no difference between cp or meld scores at any point between the groups. as expected, meld scores improved significantly following rtx and remained low up until the final visit (p= . ); this was attributed to the drop in serum creatinine post-rtx. neither the use of thymoglobulin or basiliximab resulted in acute hepatitis resurgence or the development of cirrhosis post-transplant. we have not identified any association between choice of induction agent or maintenance immunosuppression regimens, including steroid withdrawal, with impaired hepatic function or progression to liver cirrhosis in hcv+ rtx patients. t cell depletion was well-tolerated by hcv+ rtx patients and resulted in good graft outcomes. interestingly, cp scores declined after renal transplantation in the basiliximab induction group. kidney: complications i prevalence background: a few years ago we observed an expansion of blood gd t cells following cytomegalovirus (cmv) infection in kidney transplant recipient (ktr). we recently demonstrated that these cells share a strong reactivity against cmv infected cells and tumor epithelial cells in vitro. an implication of gd t cells in the immune surveillance against cancer has been demonstrated in mouse and strongly suggested in human. we tested here the hypothesis of a protective role of cmv-induced gd t cells against neoplasia in ktr through: / a longitudinal case / control (ktr with cancer / ktr without cancer) study where gd t cell percentages were determined before and after cancer diagnostic (n= ), / a retrospective follow-up of ktr for . years looking for risk factors for malignancy. results:the median of gd t cell percentage in patients with malignancies was significantly lower when compared to control patients , and months before the diagnostic of the cancer (p< . ). using a conditional logistic model, we determined that patients with a gd t cell percentage above % were protected from cancer (p< . ). a significant association between increase of the vd neg gd t cell subset and lower cancer occurrence was only retrieved in the ktr who experienced pre-or post-graft cmv infection. finally, using univariate and multivariable analysis, absence of pre-or post-graft cmv infection in ktr was associated with a risk of cancer . times more elevated (p= . ). this study reveals an unexpected protective role of cmv against cancer in ktr most probably via the expansion of gd t cells cross-reactive against cmvinfected and tumor cells. background: viral infection (vi) is a morbidity factor in transplant recipients (tx pts). induction therapy (ind-rx) is a known risk factor for vi. although cam is thought to be a more potent ind-rx than zen, we have previously shown similar cmv infection rates in each. we have also shown that cmv-tc analyzed by cytokine flow cytometery (cfc) are consistently detectable in cmv sero(+), but not sero(-) individuals. cmv-tc(-) was associated with persistence of cmv infection in tx pts. here, we report on the effect of ind-rx on cmv-tc in kidney tx pts. methods: pre-tx samples from cmv-sero(+) pts and post-tx samples from pts were submitted for cmv tc-cfc. whole blood was incubated with a pooled overlapping peptide mixture consisting of peptides from cmv pp and brefeldin a at degrees for hours and room temperature overnight. ifnγ+cd + cells were enumerated by cfc and results were expressed as ifnγ+cd + cell%. results > . % were considered as (+ infection associated graft loss during the entire study period is shown in figure . infections contributing to renal allograft loss increased significantly from to . this may be due to increase use of both induction agents and potent maintenance regimens. this is an important cause for poor long-term graft outcome despite decreasing rejection rates and a balance has to be maintained between prevention of rejection and avoidence of infection. serum creatinine (scr) at procurement was ± µmol/l. the incidence of donor hypertension, diabetes, and death from cerebrovascular origin was %, %, and % respectively. multivariate analysis showed that the only clinical parameters associated with a low egfr were donor scr and donor hypertension. nyberg or pessione scores were not significantly associated with a low egfr. regarding d biopsies, univariate analysis showed that % of sclerotic glomeruli (sg, p= . ), arteriolar hyalinosis (p= . ), mean remuzzi score (p= . ) and mean cadi score (p= . ) were all significantly associated with a low egfr. a logistic regression showed that an integrated score including: i) donor scr (± µmol/l), ii) hypertension, and iii) sg (± %) had the highest performance in predicting a low egfr at yr compared to clinical or histological parameters alone. using this composite score, the adjusted or for the prediction of a low -yr egfr ranged from if none of the factors were present, to . (if sg > % was associated with one of the clinical factors), and to . (if the factors were present, p= . ). conclusion: this study highlights that d biopsies are useful to predict graft outcome particularly in md population, and may perform better than clinical scores alone. in this population, a simple and routinely applicable integrated scoring strongly predicts a poor graft outcome, which may allow an optimized allocation of marginal donors. prospective kidney transplantation from small pediatric donors is increasingly being utilized as a means to optimize the organ supply, however the single most common specified reason for the discard of pediatric kidneys is vascular damage, such as shortening of the suprarenal aorta or injury to the renal artery orifices, which often precludes en bloc transplantation (ebk). at our center, damaged kidneys were salvaged by transplantation as singles (sk background: in an effort to maximize the number of recipients transplanted per donor, transplant centers in our donor service area (dsa) voted to preferentially allocate local and imported en-bloc pediatric donor renal allografts to centers willing to transplant two individuals with single allografts. after year of implementing this policy into action, we report on our initial experience. methods: from july to june we reviewed our experience with adult single allograft recipients of pediatric donors less than months of age. there were no exclusions based on age or size with exclusion criteria consisting of donor age < months and single allograft size < cm. all but recipient received rabbit anti-thymocyte globulin induction, tacrolimus, mycophenolate mofetil, and rapid steroid withdrawal. results from this cohort were compared to consecutive recipients of adult single allografts from standard criteria donors with the same immunosuppression protocol used as historical controls. results: pediatric single allografts with median donor age of months (range - ) were transplanted into adults with median age years (range - ). showed that non-white race was associated with increased risk of death (p< . ). this effect of race was attenuated when ltx center was taken into account [b] . finally, with the addition of meld in the model, the effect of race on waitlist mortality all but disappeared [c] . conclusions: on the surface, minority patients may appear to have higher mortality on ltx waitlist compared to caucasian counterparts. however, this association is predominantly a result of minority patients having a higher meld score, although ltx center-specific mortality may contribute. these data suggest that waitlist outcome may be improved by optimizing referral of minority patients. background: in cirrhotic patients awaiting liver transplantation (lt), low serum sodium (na) predicts short term pre-lt mortality, independently of meld. incorporation of na into meld has been recommended to improve prognostic accuracy (meld-na; gastroenterology : gastroenterology : , ). however, short term interventions such as water restriction that improve na may have little effect on prognosis. hypothesis: the lowest level of serum sodium in the preceding (na ), (na ) or days (na ) may be better than the current serum sodium (nac) for predicting pre-lt cirrhotic mortality. methods: we reviewed electronic records of cirrhotic veterans referred for consideration of lt, / / - / / . date of most recent na at referral was chosen as time zero for determining nac, na , na , and na and for assessing subsequent survival. findings: within days, patients died pre-lt ( %) and underwent lt ( %). na at all time points was associated strongly (p<. ) with prelt death (censored at lt). areas under receiver operating characteristic curves (aurocs) for na , na and na as predictors of d prelt mortality (mean±se) were . ±. , . ±. and . ±. , respectively, compared to . ±. for nac (all p<. vs. nac). on multivariable logistic regression analysis, meld and na were independent predictors of d prelt mortality, with best discrimination given by the following model: meld-na = meld + ( -na )* . with value of na capped at . aurocs for meld-na , meld-na, and meld were . ±. , . ±. and . ±. , respectively. findings were similar when patients with hcc at referral (n= ) were excluded (aurocs . ±. , . ±. and . ±. , respectively), and when day survival endpoint was changed from "death censored at lt" to "death or lt" (auroc's . ±. , . ±. , and . ±. , respectively). conclusion: short term improvement in na may mask true prelt mortality risk. the lowest na in the preceding days is a better prognostic indicator than current sodium. substitution of meld-na for meld would permit more accurate "sickest first" organ allocation, while at the same time allowing prelt correction of na without loss of priority. prospective validation of meld-na , in comparison to meld-na and meld, is warranted. hyponatremia does not affect survival following liver transplantation. byung cheol yun, w. ray kim, y. s. lim, joanne t. benson, walter k. kremers, terry m. therneau. gastroenterology and hepatology, mayo clinic college of medicine, rochester, mn. background: hyponatremia is a common yet important complication of cirrhosis. serum sodium (na) has been found to be an important predictor of survival in patients with cirrhosis. models incorporating na have been proposed for liver allocation. concerns have been raised, however, that liver transplantation (ltx) in hyponatremic patients will adversely affect the outcome. in this work, we assessed the effect of pre-ltx na on the short term survival following ltx. methods: patient-level data on all waitlist registrants in the us for and were obtained from the organ procurement and transplantation network. demographic, clinical and laboratory data at the time of ltx and outcomes following ltx were extracted. the relationship between na pre-ltx and survival post-ltx was analyzed using multivariable regression analyses. results: there were primary transplants that met the inclusion criteria between and . the median na in meq/l at the time of ltx was (interquartile range, iqr: - ). there were patients who had a na ≤ meq/l. the mean meld score was . (sd . ). median follow up was (iqr: - ) days. the overall -and -day survival post-olt was % and %, respectively. in a multivariable logistic regression model, meld was associated with . -fold increase in -day mortality ( confidence interval: . - . ), while na did not have impact on survival (hr= . , % ci: . - . ). the figure represents the risk of -day mortality according to na after adjustment for meld, which clearly shows absence of mortality increase over a wide range of na. conclusions: hyponatremia at the time of ltx has no detrimental impact on short term patient survival following ltx. although these data do not address morbidity (e.g., central pontine myelinolysis), there is no evidence that incorporation of na in organ allocation will lead to diminished survival. objective. this study examined the relationship between meld at liver transplantation (lt) and post-lt quality of life (qol). methods. adult lt recipients (n = ) at two centers completed the sf- and transplant symptom frequency questionnaire (tsfq) -year post-lt. high sf- scores indicate better qol; high tsfq scores indicate more symptomatology. clinical (lab) meld at lt, demographic characteristics, presence of ascites, encephalopathy, and variceal bleeding pre-lt, current employment status, presence of co-morbid medical conditions, and bmi were collected from medical records. results. primary lt indication was viral hepatitis ( %), cholestatic liver disease ( %), or hepatocellular disease ( %), and % had ascites, % encephalopathy, and % gastroesophageal bleeding. mean meld at lt was ± . there was almost no correlation between meld and sf- physical (r = . ) and mental (r = . ) functioning. statistically significant yet weak correlations were found between meld and physical functioning (r = - . ) and role functioning -physical (r = - . ). meld was not significantly correlated with any other sf- scales (r's - . to - . ). meld was not significantly correlated with any tsfq domains: affective distress (r = . ), neurocognitive symptoms (r = . ), gastrointestinal distress (r = - . ), physical appearance changes (r = . ), appetite and weight changes (r = . ), and miscellaneous symptoms (r = . ). older age (ß = - . ), female sex (ß = - . ), viral hepatitis (ß = . ) or cholestatic disease (ß = . ), higher bmi (ß = - . ), and > medical co-morbidity (ß = . ) were significant predictors of lower qol as measured by the sf- (adj r = . , f = . , p < . ). older age (ß = . ), female sex (ß = . ), higher bmi (ß = . ), history of variceal bleeding (ß = . ), and > medical co-morbidity (ß = . ) were predictive of more symptoms on tsfq (adj r = . , f = . , p < . ). meld was not predictive of qol. conclusions. higher disease severity, as measured by meld, at lt does not portend a worse qol outcome for patients -yr after transplantation. other pre-lt indicators of decompensation also do not predict post-lt qol. post-lt qol is affected more by other variables, including age, sex, bmi, and medical co-morbidities. introduction: racial disparities in access to cadaveric renal allografts have been well described for renal transplantation. however, little is known about differences in orthotopic liver transplantation (olt) rates for patients of minority racial groups following listing. the purpose of the current study was to determine if there is difference in rate of transplantation among racial groups and to examine the potential reasons for the disparity. methods: the united network for organ sharing (unos) database was obtained. data was extracted for adult olts greater than years of age performed from / - / . transplants for which recipient race or model for end-stage liver disease (meld) score at listing were unknown and patients active on the list were excluded. rates of transplantation as well as differences in reasons for de-listing (transplantation, death/deterioration, and improvement) were examined. in an effort to examine only patients with chronic liver disease, further analysis was performed excluding patients with acute fulminant liver failure and retransplants. results: the database contained complete meld and race information on , olts. seventy-four percent of patients were caucasian, % hispanic, % african-american, and % were asian. as seen in table , laboratory meld score at removal differed between racial groups. examining pair-wise comparisons of the three minority groups to caucasians, only hispanics differed in reason for delisting (table ) . subgroup analysis excluding acute hepatic failure patients and retransplants showed similar results with hispanic patients being more likely to die/deteriorate as compared to other racial groups ( % deaths vs. % deaths for caucasians), and being less likely to receive a transplant ( % of hispanics vs. % of caucasians, p< . ). conclusion: hispanic patients, although listed with higher meld scores, are transplanted less often than caucasian patients and are more likely to die/deteriorate while awaiting olt. reasons for this discrepancy are unclear and merit further attention. background: since the implementation of the model for end-stage liver disease (meld) for liver allocation, an increasing number of candidates with renal insufficiency have undergone orthotopic liver transplantation (olt). since candidates with renal insufficiency have higher post-transplant morbidity and mortality, meld-based allocation may be shifting some waiting list mortality to the post-transplant period in these candidates. the objective of this study was to evaluate the survival benefit among candidates with renal insufficiency who underwent olt. methods: scientific registry of transplant recipients data for adult candidates age ≥ initially listed for olt between / / and / / (n= , ) were analyzed. the effect of serum creatinine on the survival benefit (contrast between waiting list and post-transplant mortality) was assessed by sequential stratification, an extension of cox regression. each recipient was matched with candidates active on the waiting list in the same organ procurement organization with the same meld score. results: for meld scores - , the survival benefit of olt significantly decreased as serum creatinine increased. among candidates transplanted at meld - , the % with serum creatinine > . mg/dl ( %) experienced no significant survival benefit ( figure) . candidates transplanted at meld ≥ experienced significant olt benefit irrespective of serum creatinine level. conclusions: comparing two patients with meld ≥ , the patient with higher creatinine experiences significantly less survival benefit from liver transplantation. almost onequarter of patients transplanted at meld - experienced no survival benefit from olt based on years of follow-up. therefore, more careful assessment of candidates is required in order to maximize the survival benefit gained by the wait-listed end-stage liver disease population as a whole. liver: living donors and parial grafts i assessment introduction: consideration of the risks and benefits of a procedure are critical in medical decision making. however, relatively little is known about risk tolerance amongst donors and transplant professionals in live-donor liver transplantation (ldlt). we conducted confidential semi-structured interviews in a convenience sample of donors, non-donors (individuals who had been assessed for donation but did not donate) and transplant team members. in addition to examining issues surrounding decision making for ldlt donation, we sought to assess the tolerance of participants, above which they would no longer contemplate donation, for a number of potential outcomes following ldlt. the outcomes that participants were asked to consider included their tolerance for risk of donor death, risk of serious donor complication, as well as risk of recipient death following transplantation. the interviews were conducted sequentially, data was coded quantitatively, and the study terminated once saturation was reached. (pre, weeks , , , and post-donation) . ambivalence detected by staff or described by donor was recorded. donor and recipient characteristics were examined and compared between ambivalent and non-ambivalent groups. results: staff identified and self identifed ambivalent donors were not equivalent. staff assessments indicated ambivalent donors ( male, female). donors self-identified as ambivalent ( male, female); donors were on both lists ( male, female). the combinations of brother to brothers and sons to fathers were the most common pairs among ambivalent donors and more common than in total donor cohort. recipient diagnosis of alcohol or hepatitis c related liver disease was more common in ambivalent donors. ambivalent donors were more likely to be college educated and to express significant religious affiliations than the total rhl donor group. all but ambivalent donor indicated that they would donate again on the year qol survey. conclusions: ambivalence about rhl donation is present in approximately % of candidates who complete donation. staff-identified and self-identified groups showed only % overlap; however, both groups showed similar characteristics. brother-tobrother and son-to-father pairings and recipients with perceived self-induced liver failure were more common in both groups compared to total donor cohort. ambivalent donors had more education and stronger religious or spiritual identification than the entire cohort. only donor indicated persistent doubt about donation. these results suggest that expressed or perceived donor candidate ambivalence may represent a process of careful consideration and should not be used sole basis for donor disqualification. the impact of donor age on recipient outcome for adult right-lobe living donor liver transplantation (rldlt) is unclear. aim: to analyze the effect of donor age on recipient outcome following rdldt. methods: since we have performed rldlt (mean donor age years, range - years), including donors age years or older. we analyzed the effects of donor age, as a continuous or categorical (< vs > years) variable, on recipient outcome. recipient outcome measures included biochemical markers of hepatocytes injury (ast, alt) and graft function (inr, bilirubin), postoperative infections, bleeding, biliary complications, acute cellular rejection, as well as patient and graft survival. analyses were carried out stratified for higher recipient meld scores (< vs. > ), recipient age (< vs. > years), and hepatitis c virus (hcv) infection (presence vs. absence). results: -year patient and graft survival after rldlt was % and %, respectively. donor age as a continuous variable was associated with increased ast (p= . ) and alt (p= . ) release after transplantation, while no effect was observed on inr or bilirubin. rldlt using donors above years of age resulted in an increased incidence of biliary strictures ( % vs. %, p= . ), postoperative cholangitis ( % vs. %, p= . ). no effect of donor age was found for the following recipient outcome measures: the number of bile ducts supplying the graft, type of biliary reconstruction required; rejection, hemorrhage, pulmonary or urinary tract infections, renal failure, or length of hospital stay. -year patient survival was identical for patients receiving grafts from donors below or above years of age ( % vs %, p= . ). similarly, -year graft survival was comparable for young and old grafts ( % vs %, p= . ). recipient age (< vs > years), recipient meld score (< vs > ), or hepatits c status of the recipient did not impact on the effect of age on patient or graft survival. conclusion: in this single center series of rldlt, the use of selected older donors did not impair graft and patient survival, but was associated with an increased rate of biliary strictures. background: biliary stricture rate after living donor liver transplant (ldlt) in adults remains relatively high in comparison to the stricture rate after adult cadaveric liver transplant or ldlt in pediatric patients. the etiology or risk factors for biliary stricture development at present time are uncertain. purpose: to determine the risk factors for biliary stricture after right lobe (rl) ldlt. methods: from / to / , ldlt procedures were performed in adult recipients. eleven patients were excluded from analysis due to < days follow up or need for retransplant. the following data was prospectively collected: . demographics, . acuity of illness, . number of bile ducts, . type of biliary reconstruction, . graft to recipient weight ratio, . hemodynamic parameters, . outcomes. these parameters were compared in patients with and without strictures. results: mean follow-up for patients is days (range: - ). of patients died during the follow-up range and required whole liver re-transplants. patients ( %) developed a biliary strictures during the follow-up period. comparison of risk factors in patients with and without strictures revealed the following results: mean meld > bile duct grwr* < . neither meld score, number of bile ducts or type of biliary reconstruction appear to be contributing factors to the development of bile duct stricture following rl ldlt. the biliary stricture rate was related to the volume of transplanted liver and post transplant graft recovery. therefore, the development of biliary strictures in some patients may represent yet another feature of small-for-size syndrome. background: ox and cd can be expressed by both foxp + tregs and activated t effector cells. however, the question as to how ox and cd function, individually or collectively, in regulating such functionally different t cell subsets in transplant models remains poorly understood. in some models, blocking cd costimulation is remarkably effective in prolonging graft survival, but targeting cd alone rarely creates tolerance. but the role of ox in regulating the cd blockade induced tolerance is completely unknown. in the present study we critically examined the role of ox in the activation of cd deficient t effector cells as well as in the regulatory function of foxp + tregs. we also examined the effect of ox on the induction of new foxp + tregs/th cells from activated cd deficient t effector cells. the impact of ox in the induction of allograft tolerance was examined using an islet transplant model. we found that cd deficient foxp + tregs constitutively expressed ox on the cell surface, but the cd deficient t effector cells did not. however, when the t effector cells were sorted and stimulated in vitro, ox expression could be abstracts readily induced on the t effector cells. to further examine how ox regulates such functionally different t cell subsets, we found that ox delivers potent costimulatory signals to t effector cells, which prevent the induction of new foxp + tregs from activated t effector cells but promote their differentiation to th cells but not th cells. surprisingly, ox costimulation to cd deficient foxp + tregs completely inhibited their regulatory functions. in an islet transplant model, we showed that cd deficient mice can reject the dba/ islet allografts, but blocking ox costimulation readily induced donor specific tolerance (mst> days), and this tolerant status was critically dependent on the induction of foxp + tregs. in contrast, treatment of cd deficient recipients with a agonist anti-ox mab precipitate rapid islet allograft rejection, suggesting that ox costimulation is critically important in the induction of transplant tolerance. conclusions: our data suggest that ox is a costimulatory molecule to t effector cells but a powerful negative regulator for foxp + tregs. thus, a key role for ox in the induction of transplant tolerance is the control of t cell mediated regulation. background: foxp is a winged-helix family transcription factor that is the master regulator for the development and function of regulatory t cells (treg). we investigated the molecular mechanisms important for regulation of foxp expression, and defined the structure of the active foxp promoter in cd + t cell lineages. methods: purified cd + cd -foxp -gfp -t cells (naïve) and cd + cd + foxp + gfp + treg were cultured with antigen presenting cells in the presence of il- , anti-cd ε mab, tgfβ or the dna methyltransferase inhibitor -aza- '-deoxycytidine (zdcyd). foxp promoter structure and activity were monitored with methylation-specific pcr, disulfite-sequencing, chromatin immunoprecipitation (chip) assays, electrophoretic mobility shift assay (emsa) and luciferase promoter assay. the foxp promoter has an upstream cpg island ∼ kb from the transcriptional start site. disulfite-sequencing and methylation-specific pcr analysis showed that this region is heavily methylated in naïve cd + t cells and tgfβ induced peripheral treg, but demethylated in thymic derived natural treg (ntreg). chip analysis showed that the methylated cpg island is bound specifically by the dna methyltransferases and b. zdcyd causes demethylation of the cpg island, and in combination with tgfβ, synergistically induces foxp expression. chip assays for acetylated histone and sp , both markers of gene activation, showed that the cpg island is acetylated and bound by sp in ntreg and zdcyd plus tgfβ induced treg, but not in activated cd + t cells or tgfβ induced treg. emsa likewise shows the cpg island binds sp . in contrast to the upstream promoter, the structure of the first intronic promoter differs markedly between ntreg and tgfβ induced treg, but is not affected by zdcyd. the upstream cpg island also possesses enhancer activity that is repressed by dna methyltransferases. zdcyd plus tgfβ induced treg have stable foxp expression and enhanced suppressive functions in vitro and in vivo. conclusion: these results demonstrate that ntreg and tgfβ induced treg are genetically distinguished from each other by the epigenetic structure of a unique upstream cpg island of the foxp promoter. the function of this region is regulated by dna methylation and histone acetylation. zdcyd demethylates the promoter, leading to enhanced and stable expression of foxp and suppressor activity, similar to ntreg. this has important implications for biology, and generating treg for tolerance. chemokine background: trafficking of lymphocytes through lymphatics to secondary lymphoid organs is crucial for immune responses. we previously showed that regulatory t cell (treg) function required trafficking from the inflammatory graft site to the local draining lymph node (dln). since the mechanisms that regulate migration through afferent lymphatics are poorly understood, we explored the role of chemokine receptors on treg for afferent lymphatic migration in an islet transplantation model. methods: islets were transplanted from balb/c mice into foxp gfp c bl/ mice. treg from wild type, ccr -/-, ccr -/-, ccr -/-, or ccr -/-c bl/ mice were isolated, labeled with red dye pkh , and transferred intravenously, or locally into the islet allograft. treg migration to islet grafts and dln was determined by flow cytometry and immunohistochemistry. endogenous foxp gfp+ treg and transferred pkh labeled treg were sorted from the islet grafts and the dln, and chemokine receptor and sphingosine -phosphate receptor (s p ) expression were determined by rt-pcr. islet allograft survival was determined by measurement of blood glucose. results: freshly isolated treg expressed s p and the chemokine receptors ccr , ccr , ccr , and ccr . endogenous treg, and both intravenously and locally transferred treg, that were recovered from islet allografts and dln expressed similar levels of ccr and ccr . ccr was expressed preferentially on islet migrating treg, while s p and ccr were expressed preferentially in dln migrating treg. locally transferred treg migrated to the dln, but ccr -/-treg were not able to migrate to the dln. ccr -/and ccr -/-treg were impaired in their ability to migrate to the dln. this suggested that these three chemokine receptors all regulated treg entry into afferent lymphatics and migration from the graft to the dln. in contrast, ccr -/-treg migrated normally from the islet to the dln. importantly, ccr -/-, ccr -/and ccr -/-, but not ccr -/-treg, were impaired in their ability to prolong islet allograft survival when transferred locally in the islet allograft. conclusion: treg migrate from the inflammatory site of the allograft to draining secondary lymphoid tissue through afferent lymphatics. this process depends on ccr , ccr , and ccr ; and is crucial for full treg function in vivo. these results demonstrate a novel role for sequential migration from the graft to the dln in treg function and suppression. epigenetic regulation of gene expression provides a major, and especially beyond oncology, largely unexplored means to regulate host immune cell functions. our ongoing analysis of histone deacetylase (hdac) expression by foxp + naturally occurring murine regulatory t (treg) cells showed tcr-activated tregs had - fold more hdac mrna than corresponding resting treg or non-treg cells. in various cell types, hdac deacetylates alpha-tubulin, cortactin, and hsp , abrogates formation of the aggresome, and blocks the unfolded protein response, though nothing is known regarding these pathways in tregs. we found that an hdac -specific inhibitor, tubacin (but not the control compound, niltubacin), increased treg suppressive function in vitro (p< . ), in association with increased expression of ctla, il- , gitr, pd- and other treg-associated genes (p< . ), and increased treg foxp protein (though not mrna) expression. tubacin enhanced the conversion of cd +cd -cells into cd + foxp + treg in vitro, and globally decreased cytokine production, with the exception of il- and il- mrna. comparable and dose-dependent effects were seen using the hsp inhibitor, geldanamycin, suggesting that the effects of hdac inhibition were mediated, at least in part, by blocking the chaperone effect of hsp . use of tubacin in vivo significantly decreased the severity of colitis in two murine inflammatory bowel disease models (p< . ), dextran sodium sulfate-induced colitis and the cd +cd lhigh adoptive transfer model of colitis, as assessed by standard clinical and histologic criteria. in addition, days combined use of tubacin and a subtherapeutic dosage of rapamycin led to significantly prolonged cardiac allograft survival (balb/c->c bl/ ) compared to use of either agent alone (p< . ). our data show that use of the first known small molecule inhibitor of one specific hdac has important therapeutic effects, including enhancing the production and suppressive function of tregs. while ongoing studies are directed towards unraveling the interactions of hdac -dependent pathways and treg functions, the current data indicate the importance of understanding the functions of hdacs to the development of entirely new ways to regulate host immune responses. dendritic cells supply paracrine il- for treg cell functional activity. regulatory cd +cd + t cells (tregs) are important for the maintenance of immune tolerance, and immunotherapy with tregs is being explored for organ and cell transplantation. treg development, expansion and function depend on il- . because tregs do not make il- , they must obtain il- from another cell. although cd + teffectors are a logical candidate, the identity of the paracrine source of il- for tregs is not substantiated. we explored whether dendritic cells (dcs) could serve as the paracrine source of il- for treg and rd , a cd +cd + regulatory hybridoma. using four dimensional live cell imaging we demonstrate that treg and rd cells establish tight contact with dcs, and cd is localized at these contacts. using the il- elispot and real-time rt-pcr we found that splenic dcs and the jawsii dc cell line constitutively make il- . lps and cpg increases dc production of il- . co-culture with jawsii dc cell line significantly upregulates cd expression on alloreactive do . tregs and rd cells, but not on do . cd + teffector cells. tregs and rd cells are functionally suppressive after activation by wild type but not il- knock-out allogeneic dcs, and anti-cd inhibits the function of treg and rd cells in a dose response fashion. in contrast, wild type and il- knock-out dcs are equally able to activate alloreactive cd +cd -cells. supplemental il- at high ( u/ml) but not low doses ( u/ml) restores the function of alloreactive tregs and rd that were activated by il- ko dcs. these data indicate that treg cells acquire il- from dendritic cells for their gain of function and validate dendritic cells as a paracrine source of il- for treg. introduction: previously, it has been demonstrated that foxp , a gene required for the development and function of regulatory t cells, was highly expressed in the graft during cardiac rejection, suggesting infiltration of regulatory t cells in the transplanted organ during an allogeneic response. in this study, we investigated whether graftinfiltrating t cells expanded from rejecting human cardiac allografts exhibit immune regulatory activities. methods: graft-infiltrating lymphocytes (gils) cultured from endomyocardial biopsies (emb; n= ) with histological signs of acute cellular rejection were expanded in the presence of donor-antigens in il- /il- -enriched medium for - weeks. flow cytometry was used to analyze the expression of cd , cd , cd and foxp . to analyze the immune regulatory function, we performed mlrs with peripheral blood mononuclear cells (pbmc) of the patients and irradiated donor or third party spleen cells in the absence and presence of gils (ratio : ). results: of the cd + gils, % (median; range: - %) stained positive for foxp . this foxp expression was detected in both cd + and cd + t-cell population (median: % and %, respectively). functional analysis demonstrated that gils suppressed the antidonor proliferation of responder t cells (range % inhibition: - %). interestingly, this suppression was predominantly achieved by cd + gils: depletion of cd + cells from the gils population diminished the inhibitory effect, whereas addition of solely cd + gils to the mlr abundantly suppressed the anti-donor response (range % inhibition: - %). in contrast, gils did not inhibit the proliferation of t cells stimulated with third-party antigens. the figure below depicts a representative example. graft-infiltrating lymphocytes expanded from rejecting cardiac allograft exhibit donor-specific immune suppressive activities. these results suggest that during acute cellular rejection, graft-infiltrating lymphocytes not only consist of graft-destructing effector t cells, but may also comprise immune regulatory cells of the cd + phenotype. the context: it has been previously suggested that a liver allograft is immunoprotective and able to decrease the rate of rejection of a donor-specific allograft of another organ. it has been recently proposed that allografts other than the liver may also be immunoprotective. objective: the aim of this analysis was to examine one year rejection rate and the incidence of rejection free survival of all combined transplants in the collective us experience to gain insight to any possible protective effect of one organ for another. methods: the united network of organ sharing (unos) provided de-identified patientlevel data. analysis included all recipients transplanted between january , and october , who were years or older (except intestinal transplants). rejection at one year was defined as treatment for one or more episodes of rejection. results: analysis included a total of , patients who received either one, or combined, simultaneous or sequential, organ transplants in all possible combinations. results are summarized in figure (one-year organ allograft rejection rate). the collected data demonstrate that the rejection rate of donor-specific organ allografts which accompanied primary liver, kidney, and heart transplants was significantly lower in combined transplants as compared to that of the primary allograft transplanted alone. this was not true, however, for intestinal and pancreatic allografts where protection for the accompanying organ was not observed. we further demonstrate that transplantation of two organs of the same type (double kidneys or double lungs), i.e. increase in antigen load, also leads to decreased rates of rejection of the allografted organs. conclusions: in combined simultaneous transplants, the heart, liver, and kidney allografts appear themselves to be protected, and to protect the other organ from rejection. increased antigenic load of identical antigens in case of double lung and double kidney transplants appears to also offer immunologic protection against rejection, perhaps by different mechanisms. background: a all ( -center adult-to-adult living donor liver transplantation cohort study) has identified risk factors for mortality after aaldlt, including center experience. the aim of this study was to determine if a all findings are reflected in the national experience. methods: aaldlt at a all (n= ) and non-a all centers (n= ) from / / to / / in the scientific registry of transplant recipients database were analyzed. cox regression models adjusted for recipient and donor characteristics were fitted to test associations with mortality risk after aaldlt, including center type (a all vs. non-a all) and case number (for each aaldlt at each center). results: aaldlt were performed at a all and non-a all centers. there was no significant difference in overall mortality risk between a all and non-a all centers. significant predictors of death (both groups combined) included donor age (hazard ratio (hr)= . per years, p= . ), recipient age (hr= . per years, p< . ), diagnosis of hcv (hr= . , p= . ) or hcc (hr= . , p< . ), and earlier center experience (aaldlt case number ≤ , hr= . , p< . ). there was no significant effect of transplant year after adjusting for experience. cold ischemia time > . hours was associated with higher mortality (hr= . , p= . ); this effect was similar in a all and non-a all centers. there were no significant interactions between center type and any predictor except center experience ( figure) . compared to later experience, earlier center experience was associated with significantly higher mortality risk in both a all (hr= . , p< . ) and non-a all centers (hr= . , p< . ). survival during early experience was significantly worse at a all vs. non-a all centers (hr= . , p= . ), but survival in later experience was similar. conclusions: after the first cases, aaldlt survival was similar at a all and non-a all centers, and similar significant mortality risk factors were identified, including center experience. these analyses support the generalization of findings from a all centers to others performing aaldlt. abstract# rejection with hemodynamic compromise (hc) and chronic allograft vasculopathy (cav) impact survival in pediatric heart transplantation (phtx). we showed that high pro-inflammatory / lower regulatory cytokine gene polymorphism (gp) profile increased the risk for acute rejection. in this analysis, we assessed the effect of genetic factors on hc and cav. methods: phtx with clinical and gp data for cytokines (tnf-α a- g; inf-γ t+ a; il- g- a, c- t, c- a ; il- c- t; il- t- c; il- g- c), growth factors (tgfβ- t+ c, c+ g; vegf a- c, c- t, g+ c), effector molecules (fas a- g; fasl c- t) and pharmacogenomics (abcb c t, g t/a) were analyzed regarding hc and cav. results: adjusting for recipient black race and age with cox regression models, we identified the following risk factors: il- high was associated with lower rates of hc. low th (inf-γ, tnf-α) with high th (il- , il- ) cytokine gp profiles were protective for hc in combination with il- high. carriers of fas high experienced higher rates for hc and cav and high fas-fasl combination doubled the relative risk for cav. abcb cc/ gg genotypes were also associated with lower rates of hc (table ) . conclusion: in this large multi-center study gps with higher regulatory profiles and increased drug transport were associated with a lower incidence of hc. a genetic proapoptotic profile might contribute to the pathogenesis of cav. sponsorship: this work was supported by p hl - from the national heart lung and blood institute, national institutes of health. it has recently been reported that cd d-restricted nkt cells that express invariant tcr (inkt cells) play an important role in the production of autoantibodies through the interaction with b- cells. this observation prompted us to investigate the possible role of inkt cells in the production of antibodies (abs) against transplant-related antigens, such as abo blood group carbohydrates and histocompatibility complex allopeptides, in a mouse model. we have previously demonstrated that b cells with receptors for blood group a carbohydrates were found exclusively in a cd b + cd + b- subpopulation of mice, resembling humans with blood group o or b. immunization with human blood group a red blood cells (a-rbcs) elicited the extensive production of anti-a igm and igg. furthermore, the number of b- cells with receptors for a carbohydrates increased in the peritoneal cavity. in cd d -/and vα -/-balb/c mice, which lack inkt cells, such elicited production of anti-a igm was not observed, even after immunization with human a-rbcs. however, class ii -/-balb/c mice, which lack cd + t cells but maintain normal levels of inkt cells, exhibited levels of anti-a igm production comparable to those in wild-type (wt) balb/c mice. moreover, anti-a igg production was absent in cd d -/-balb/c mice even after the immunization, indicating that although inkt cells crucially contribute to anti-a igm production and igg class switching, helper t cells do not. notably, the proportion of b- cells in the livers of cd d -/-balb/c mice was significantly reduced ( . ± . %, n = ) when compared to that in wt mice ( . ± . %, n = ). we next immunized cd d -/and wt balb/c mice twice with × allogeneic b mouse thymocytes, and thereafter detected the anti-b (allopeptides) abs by flow cytometry. in the cd d -/mice, anti-b igm production was comparable to that of wt mice, and igg class switching also occurred normally. these findings indicated that inkt cells play a pivotal role in the production of abs specific for blood group carbohydrate determinants that are believed to be t cell independent, but are not required in the production of abs for allopeptides that are believed to be t cell dependent. the depletion of inkt cells or the suppression of their function might constitute a novel approach for preventing antibody-mediated rejection in abo-incompatible transplantation, or in xenotransplantation, which involves similar carbohydrate antigens. background static cold storage (cs) is the most widely used organ preservation method for deceased donor kidney grafts. retrospective analyses have indicated that preservation by hypothermic machine perfusion (mp) may lead to improved outcome after renal transplantation. however, there is a lack of sufficiently powered prospective studies to test the presumed superiority of mp. in an international prospective randomized controlled trial we enrolled kidney pairs of consecutive deceased donors and randomly assigned one organ to mp and the contralateral kidney to cs preservation. follow-up was directed at all recipients of these grafts. the primary endpoint was delayed graft function (dgf). mp significantly reduced the risk of dgf (or . ; p= . ) and more than halved the incidence of primary non-function after transplantation, when compared to cs ( . vs. . %; p= . ). furthermore, mp significantly reduced the risk of graft failure in the first months post-transplant (hr . ; p= . ). in recipients who developed dgf, -month graft survival was better if their transplanted kidney was machine perfused ( vs. %; p= . ). hypothermic machine perfusion reduces the risk of delayed graft function, primary non-function, and graft failure in deceased donor kidney transplantation when compared to static cold storage. furthermore, mp alleviates the deleterious effect of dgf on graft survival. we investigated the trafficking of cells after skin and heart transplantation in a dynamic fashion through the use of in vivo microscopy. antigen presenting cells were followed using mhc-cl-ii-gfp and cd c-gfp transgenic mice. vascularized and non vascularized skin grafts as well as heart transplants were used in syngeneic as well as allogeneic settings. after syngeneic non-vascularized skin transplantation, we observed an early and massive cellular infiltration of host cells into the graft as early as hours post-transplant with a gradual accumulation in the dermis. the accumulation of host-derived cells was accelerated after graft vascularization at day / post transplantation. this graft infiltration by recipient cells was more pronounced with vascularized skin grafts, and to a higher degree in heart transplants. recipient cells similarly infiltrated allogeneic grafts early on and in larger numbers than for syngeneic grafts by day / post-transplantation. when visualizing mhc-cl-ii-gfp recipient cells in a syngeneic skin transplant, recipient dcs invaded the graft early on and, by weeks post transplant gradually replaced graft dcs in the dermis (dermal dcs) and the epidermis (langerhans cells). donor dcs could still be seen in the graft up to days post transplant. however, virtually all donor langerhans cells were eventually replaced by recipient ones in a concentric fashion suggesting that the new langerhans cells originate from the recipient skin adjacent to the graft and not from centrally-derived precursor cells. the vascular endothelium of a syngeneic transplant was partially replaced by recipient vascular endothelial cells in a centripetal fashion with more recipient-derived vascular endothelium present at the periphery of the graft and more donor-derived endothelium remaining in the center of the graft. therefore, the graft can be seen as a "chimera" of cells from donor and recipient origin. the presence of recipient endothelial vascular cells and dcs within the graft may be important for maintaining the indirect response thought to be responsible for chronic rejection. objective: maturation resistance and tolerogenicity can be conferred on dendritic cells (dc), -crucial regulators of t cells, by exposure to rapamycin (rapa), a tolerance-sparing immunosuppressant. the mechanisms underlying this acquired unresponsiveness, typified by diminished responses to toll-like receptor (tlr) or cd ligation, have not been identified. thus, our objective was to elucidate a molecular basis for rapa-induced dc maturation resistance. methods: rapa administration was used to condition splenic dc in vivo and bone-marrow derived dc in vitro. dc maturation was monitored by assessment of co-stimulatory molecule expression, cytokine production, and t cell allostimulatory capacity. to identify negative regulators of maturation, microarray analysis and quantitative rt-pcr was completed, and findings confirmed via western and flow cytometric analyses. results: in vitro or in vivo exposure of myeloid dc to rapa elicited de novo production of il- β by otherwise immature dc (cd lo ). interestingly, dc il- β production, acting in an autocrine/paracrine fashion, promoted dc overexpression of the il- receptor(r) family member, st l, and enhanced its surface expression. st l is the receptor for il- , an il- family member, and has also been implicated as a negative regulator of tlr signaling. consistent with this regulatory function, il- β-induced st l expression suppressed the responsiveness of rapa-conditioned dc to tlr or cd ligation. conclusion: rapa causes de novo production of il- β by immature dc, upregulating st l, and establishing a barrier to dc maturation following exposure to tlr or cd ligation. as such this work identifies a novel mechanism by which a clinically-important immunosuppressant impedes the capacity of dc to mature and consequently stimulate effector/adaptive t cell responses. these findings are particularly relevant to the potential use of rapa-conditioned dc as "negative" cellular vaccines to block alloag-specific responses, as exposure to endogenous and exogenous inflammatory stimuli can induce dc maturation and negate the tolerogenic properties of immature dc. exosomes are nanovesicles ( - nm) released to the extracellular milieu by different cell types. exosomes secreted by dendritic cells (dcs) and other apcs express mhc ag, adhesion molecules and costimulatory molecules oriented on the membrane surface with their binding domains facing outwards. thus, exosomes released by graft-infiltrating leukocytes (gils) could function as "ag-presenting vesicles" or as vehicles to transfer alloag between recipient's apcs during elicitation of t-cell allo-immunity. aims: to test if (i) gils activate anti-donor t-cells in secondary lymphoid organs by releasing exosomes with alloag into systemic circulation; or (ii) gils that traffic to the spleen as passenger leukocytes use exosomes as a local mechanism to transfer alloag to recipient's dcs. methods: exosomes were isolated from supernatants of bm-derived [c bl/ (b ), ia b ] dcs pulsed with the balb/c iea - allopeptide and purified by ultra-filtration and ultra-centrifugation on a %sucrose/d o gradient. we used pkh + exosomes and cd . congenic b mice for traffic studies, heart (heterotopic) and skin transplantation models (balb/c→b , thy . + ), and cfse-labeled h . tcrtg cd t-cells (thy . + ) specific for ia b (b ) loaded with iea - (balb/c). dcs were genetically engineered to release exosomes expressing green fluorescent protein (gfp). we have previously shown that blood-borne exosomes carrying balb/c alloag are reprocessed by different subsets of splenic dcs for presentation to indirect pathway h . cd t-cells. here, we demonstrated that although gils of cardiac and skin allografts release exosomes ex vivo, they did not secrete enough concentrations of exosomes with alloag into circulation to stimulate donor-reactive t-cells in lymphoid organs. instead, our findings indicate that migrating dcs (generated in vitro or isolated from gils), once homed in the spleen, they transfer exosomes expressing gfp and carrying allopeptides to spleen-resident dcs of the recipient, identified by the congenic marker cd . . conclusion: exchange of exosomes between dcs in lymphoid organs might be a mechanism by which passenger leukocytes transfer alloag to recipient's apcs in secondary lymphoid organs. t cell activation is critical in initiating adaptive immunity, and pkcθ, a novel member of the pkc family, mediates non-redundant functions in the t cell receptor; however, its role in the mediation of allograft rejection remains unclear. this study is aimed at investigating whether alloimmune response can be alleviated by a deficiency of the pkcθ molecule, and whether transgenic expression of anti-apoptotic bcl- methods. wild-type (wt) cardiac allografts were transplanted into pkcθ -/mice, with or without sub-therapeutic anti-cd mab. purified pkcθ -/or pkcθ -/-/ bcl-x l t cells were adoptively transferred into rag -/mice engrafted with cardiac allografts. lymphocyte proliferation assays were performed (cfse). nf-kb activation was assessed by bioluminescence imaging (bli) using luciferase transgenic mice under the control of a nf-kb promoter. results. the cardiac allografts were rejected in a delayed fashion in pkcθ -/mice with increased nf-kb activation; however, sub-therapeutic anti-cd mab (that normally delays rejection of cardiac allograft) induced long-term survival of cardiac allografts. the cardiac allografts were permanently accepted in rag -/mice with adoptive transfer of pkcθ -/-t cells, and the rejection can be elicited by transfer of pkcθ -/-/ bcl-x l t cells. in a lymphocyte proliferation assay, pkcθ -/-t cells displayed greatly reduced proliferation. in response to cd and cd stimulation, pkcθ -/-t cells underwent accelerated apoptosis and reduced th , th , and treg subsets compared to the wt t cells. bcl-x l restored the survival of the pkcθ -/-t cells. conclusions. the results suggest that pkcθ mediates the alloimmune response. bcl-x l transgene prevents pkcθ -/-t cell apoptosis and re-elicits allograft rejection. tolerogenic dendritic cells (dc) are immature, maturation-resistant(mr) or alternatively-activated dc that express mhc molecules and low levels or absent costimulatory signals. although mrdc administration has successfully prolonged allograft survival in murine models, the mechanism of action in vivo remains unknown. aim: to test in vivo if the down-regulation of the anti-donor response induced by donor-derived tolerogenic dc is due to: (i) direct interaction of the tolerogenic dc with donor-reactive t cells or (ii) by reprocessing of the tolerogenic dc into alloantigen (alloag) by recipient apc for interaction with indirect pathway t cells. methods: dc were generated in vitro by culturing balb/c bone marrow cells for - days in medium with gm-csf + il- supplemented with nm α, -( h) vitamin d (vd ). we used a model of heterotopic vascularized allogeneic heart transplantation [balb/c into c bl/ (b )] and cd t cells from h . tcrtg mice that recognize b ia b loaded with the balb/c allopeptide ieα - (indirect pathway) . results: we demonstrated that vd renders dc maturation resistant (vd -mrdc) as vd -mrdc fail to up-regulate co-stimulatory molecule expression, release il- p , or stimulate allo-responsive t cells after challenge with potent dc-maturation stimuli. adoptive transfer (i.v.) of balb/c vd -mrdc (day - ) significantly prolonged survival of balb/c heart grafts in b mice in the absence of immunosuppressive therapy. interestingly, we found that in vivo, balb/c vd -mrdc induced proliferation of indirect pathway h . cd t cells in the spleens of b recipient mice, indicating that reprocessing of the balb/c dc by host (b ) apc does occur. proliferation of h . cd t cells in response to balb/c vd -mrdc resulted in defective activation (cd l high , cd low ) of h . t cells, leading to their peripheral deletion and outgrowth of cd + foxp + treg cells. reprocessing of balb/c vd -mrdc was performed by recipient splenic cd c high cd α neg dc, and donor alloag continued to be presented through the indirect pathway for days after donor dc administration. conclusion: these results suggest that dc-based therapies downregulate t cell allo-immunity and prolong allograft survival, at least in part, through reprocessing of the tolerogenic dc into alloag by recipient apc. early introduction: alloreactive memory t cells are present in all transplant recipients due to prior direct sensitization or heterologous immunity. these cells are known to circumvent tolerance induction and/or prevent indefinite graft survival in several models, but mechanistic details of their function are unknown. the goal of this study was to test the hypothesis that cd memory t cells initiate alloreocognition and express effector functions within hours of reperfusion. methods: syngeneic or a/j (h- a ) hearts were transplanted into wt c bl/ (h- b ), cd -/-, cd -/-, or rag -/-recipients. rna and protein were prepared from total graft homogenates and analyzed by qrt-pcr and elisa. rag -/-mice received x wt or ifng-/- c cells and were used as recipients weeks after reconstitution. donor-specific cd memory cells were purified from wt spleens weeks after a/j skin grafting, and donor-specific effector cd cells were purified from spleens of cd . mice days after a/j heart transplantation. flow cytometry was used to quantify graft infiltrates. results: allografts contained elevated levels of ifng and cxcl mrna at , and hrs post-transplant vs. isografts. detectable cxcl protein was produced in allografts from wt and cd -/-recipients but not in isografts or allografts from cd -/-or rag -/recipients. treatment with ctla -ig and mr failed to reduce cxcl production. reconstitution of rag -/-mice with ifng sufficient or deficient c tcr transgenic cd cells indicated that early allospecific cxcl production absolutely requires ifng made by recipient cd cells. although donor-specific ifng production was undetectable in splenocytes until day - post transplant, graft-infiltrating cd hi cd l lo cd t cells were present as early as hrs post-transplant. in adoptive transfer studies, effector-memory cd t cells reconstituted early allospecific cxcl production in cd -/-mice. lastly, primed cd t cells adoptively transferred at day post-transplant readily infiltrated allografts in control but not cd depleted recipients. conclusions: cd memory t cells infiltrate allografts rapidly post-transplant, produce ifng, and propogate an inflammatory environment which optimizes recruitment of primed effector t cells. successful neutralization of this early allorecognition pathway should provide valuable adjunctive therapy to improve graft function and survival. background: allogeneic t cell stimulation requires not only antigen-specific signals but also costimulatory signals, most importantly between cd / on the antigen presenting cell (apc) and cd and ctla on the t cell. engagement of the t cell receptor without costimulation can lead to anergy and the induction of regulatory t cells (tregs). t cell activation is also controlled by expression of the tryptophan-catabolising enzyme indoleamine , -dioxygenase (ido). depletion of this essential amino acid, and/or the production of tryptophan metabolites inhibits t cell proliferation. methods: a genetic approach to confer tolerogenic properties on murine dendritic cells (dcs) has been explored using lentiviral vectors, based on the equine infectious anaemia virus. firstly, an intracellular method that prevents costimulation has been developed: a fusion protein consisting of ctla and kdel [an endoplasmic reticulum (er) retention signal] is expressed in dcs. the ctla -kdel binds to cd / in the er and prevents expression of these proteins on the dc surface. a second approach uses an elevated expression of the ido enzyme by transduced dcs. results: ctla -kdel-or ido-transduced dcs were unable to induce allogeneic t cell proliferation. however, using two-stage dc:t cell co-culture assays, it was shown that ctla -kdel-, but not ido-transduced dcs, can induce donor-specific t cell anergy in vitro and in vivo. tolerance to both the direct and indirect pathways was shown using ctla -kdel-transduced dcs. linked suppression was mediated by the generation of donor-specific tregs. ido-transduced dcs did not generate tregs. furthermore, it was shown separately that dcs expressing ido whilst lacking cd / expression for potential ligation by ctla (although ctla -cd / ligation upregulates ido, it downregulates t cell activation) failed to generate or even sustain foxp + treg populations. the ability of the transduced dcs to induce tolerance to allografts was assessed in a complete mismatch and cbk→cba (indirect pathway) corneal graft model. these results support a clinical strategy to induce treg-mediated, donorspecific transplantation tolerance using ctla -kdel-, rather than ido-expressing dcs. indirect cd t cells that recognise processed alloantigen on recipient apc can provide help to alloreactive cytotoxic cd t cells that recognise intact mhc i alloantigen on donor apc, but exactly how such 'un-linked' help is provided is not clear. the respective abilities of direct and indirect pathway cd t cells to provide help for cytotoxic cd alloimmunity were examined in a mouse model of heart graft rejection in which the recipients contain only monoclonal helper cd t cells, specific for self-restricted h-y antigen (female b mar/rag -/mice). mice were additionally reconstituted with b cd t cells, and then challenged with female balb/c (no cd t cell help), or male balb/c (indirect pathway help), or male b xbalb/c f hearts (direct pathway help) . un-reconstituted mar/rag -/mice lack effector b and cd t lymphocytes, and consequently all heart grafts survived indefinitely. in contrast, reconstituted mar/ rag -/mice rejected male f grafts rapidly (mst d), whereas female balb/c grafts survived indefinitely, confirming a cd -dependent effector role for the transferred cd t cells. cd t cell help through the indirect pathway, although sufficient to elicit graft rejection, was less efficient than direct pathway help, because male balb/c grafts were rejected more slowly than the f grafts (mst d, p< . ). we next considered whether indirect pathway cd t cells provide help through recognition of mhc ii complexes on the surface of alloreactive cd t cells, in analogous fashion to the cognate interaction between b and t lymphocytes. in support, reconstitution of mar/rag -/recipients with instead, mhc ii-deficient cd t cells, resulted in slower rejection of male balb/c hearts (mst d, p< . ), whereas male f grafts, that still permit provision of linked help, were rejected at the same tempo. most tellingly, mar/rag -/mice that received simultaneously a female balb/c heart and male b apc (to activate mar cd t cells) rejected their grafts rapidly when reconstituted with male cd t cells (mst d). in contrast, grafts survived indefinitely when female cd t cells were transferred. flow cytometric analysis of mitogenstimulated cd t cells revealed surface mhc ii expression. indirect allorecognition can provide help for generating cytotoxic alloimmunity, but not as effectively as through the direct pathway. indirect pathway help is potentiated by linkage through recognition of cd mhc ii. purpose: tolerogenic properties of dendritic cells (dc) are supported and preserved by conditioning with the immunosuppressant rapamycin (rapa). the ability of rapaconditioned, recipient-derived dc pulsed with alloantigen (alloag) to suppress both direct and indirect alloag-specific t cells in the absence of immunosuppression has been demonstrated in a murine allograft model. dc can acquire intact mhc from cells or cell lysates. however, the ability of alloag-pulsed rapa-dc to immunomodulate directly-reactive alloag-specific t cells has not been formally demonstrated. methods: dc were generated from c bl/ (b ; h b ) bone marrow cells in gm-csf and il- . rapa was added to indicated cultures (rapa-dc) beginning on day (d) . on d , cd c + bead-purified rapa-dc or non-treated control dc (ctr-dc) were incubated with balb/c (h d ) splenocyte lysates ("alloag pulsing"). following incubation, the dc were harvested and the level of donor and recipient mhc molecules on cd c + cells determined by flow cytometry and immunofluorescent imaging. surface levels of cd , b -h (programmed death ligand- ; pd-l ), and fas-l were compared. pulsed-dc were also incubated with cd + t cells from rag -/- c mice for d. c cd + cells express t cell receptors specific for h -l d , a mhc class i molecule of balb/c. following incubation, c cell proliferation and apoptosis were both assessed. results: ctr-and rapa-dc presented detectable levels of directly-transferred mhc class i and ii on their surface after incubation with allogeneic balb/c cell lysate. donor mhc presented by "pulsed" recipient dc stimulated directly-reactive, alloag-specific c t cell proliferation. however, only rapa-dc induced apoptosis in the overwhelming majority of these cells responding via the direct pathway. induction of apoptosis correlated with an increased level of surface fas-l on rapa-dc and their comparatively low level of cd relative to pd-l . conclusions: rapa-conditioned dc can present intact mhc molecules acquired from lysates of allogeneic splenocytes and concurrently induce apoptosis of directlyreactive alloag-specific cd + t cells. as such, we provide mechanistic insight into a mechanisms by which alloag-pulsed, recipient-derived rapa-dc may facilitate allograft tolerance. background: t regs actively regulate alloimmune responses and promote transplant tolerance. atg, a widely used induction therapy in organ transplantation, depletes peripheral t cells but may preferentially spare t regs . sirolimus is thought to expand natural t regs . b t cell costimulatory blockade inhibits effector t cell (t eff ) expansion and may promote regulation. we investigated the effect of combining mouse atg (matg), ctla ig and sirolimus on stringent skin allograft survival, and studied the mechanisms by determining t reg /t eff balance in vivo using a unique model (abm-tcrtg-foxp /gfp reporter mouse conclusion:this is the first report to establish that t cell depletion with matg combined with ctla ig and sirolimus synergize to prolong stringent fully allogeneic skin allograft survival by promoting regulation and tipping the t reg /t eff balance by both preserving t regs and facilitating generation of new t regs by a conversion mechanism. these results provide the rationale for translating such a novel therapeutic combination to promote regulation and tolerance in primates and human organ transplantation. expansion of cynomolgus cd +cd +foxp + regulatory t cells using low dose anti-thymocyte globulin. to test low dose atg in vivo, mg/kg ( % of depleting dose) was administered thrice (day , and ) to a naïve monkey and to a monkey that was treated concurrently with sirolimus (trough - ng/ml) after heart transplantation. in the naive monkey, lowdose atg led to expansion of cd +cd +foxp + tregs in peripheral blood (baseline . %, day = . %, day = . %) and in lymph nodes (baseline . %, day = . %, day = . %) without causing t cell depletion. similarly, in the transplanted monkey peripheral blood tregs expanded from . % at baseline to . % on day . low dose atg is not only able to expand tregs ex vivo by proliferation of natural cd +cd + cells, but can equally induce tregs in vivo without lymphodepletion. these findings provide the rationale for development of tolerance inducing strategies based on enhancing regulatory mechanisms in human transplant recipients. immunological background⁄aim: previously, we have shown that combination of human anti-cd mab, d and tactolimus exerts additive immunosuppressive effect and markedly prolongs renal allograft survival in cynomolgus monkeys. in this study, we further evaluated the immunological aspects among these transplant recipients. method: kidney transplantations were performed across mhc mismatched cynomolgus monkeys. transplant recipient was given either no-treatment, tacrolimus ( mg⁄kg⁄day, po), d ( mg⁄kg, iv) or tacrolimus+ d (n= ⁄group). peripheral lymphocyte population, mlr and serum anti-donor antibody levels and graft histology were assessed. results: mean graft survival for no-treatment, tacrolimus, d and tacrolimus+ d treatment groups was . ± . , . ± . , . ± . and . ± . days, respectively. peripheral cd + cells partially declined in both d alone and d + tacrolimus given animals at the early post-operation period, although the numbers recovered thereafter. cd + and cd + cells were unaffected. cd + effector memory population was reduced by addition of tacrolimus to d (fig. a ). mlr against donor and rd party antigens were suppressed in both d and tacrolimus+ d groups (fig. b) . addition of tacrolimus further reduced graft cd + , cd + and cd + cellular infiltration (fig. c ). anti-donor antibodies were detected in sera during the treatment course of d ; however, they did not develop under the tacrolimus+ d treatment. graft c d deposition correlated with serum anti-donor antibody levels. the d inhibits both cellular and humoral responses against donor antigens. addition of tacrolimus strengthens these immunosuppressive effects of d , leading to further prolongation of graft survival. objectives: allogeneic islet transplantation offers the potential for cure from diabetes. application of this therapy, however, is limited by immunologic mechanisms requiring medical therapy to prevent rejection of the islets. costimulatory blockade of the cd / cd /cd and the cd /cd pathways has shown promise in ameliorating the immune response to allow engraftment and function of islets. we have evaluated a new drug regimen consisting of induction therapy with a , a murine anti-cd antibody, and basiliximab and maintenance treatment with ctla ig and sirolimus in diabetic rhesus macaques which received allogeneic islets. methods: allogeneic rhesus macaque islets ( , ie/kg ± , ) were transplanted intraportally into diabetic rhesus macaques (n= ) under the following immunosuppressive regimen: short term administration of anti-il- receptor (basiliximab) and anti-cd ( a ), with maintenance immunosuppression using sirolimus for days and abatacept (ctla ig) for long term therapy. weekly peripheral blood flow cytometric and cmv viral load monitoring was performed. results: recipients treated with this immunosuppressive regimen had immediate return to normoglycemia following islet transplant. the graft survival in the first three animals was , and days. the fourth animal continues to exhibit good glycemic control at his current post-operative day . each of these animals had monthly intravenous glucose tolerance tests with monitoring of blood glucoses and c-peptides with further evidence of glycemic response and c-peptide generation. flow cytometry confirms cd blockade during the administration of a and return of cd after cessation of therapy. the treatment was well tolerated with minimal evidence of cmv reactivation and no evidence of thrombocytopenia or thromboembolism. conclusions: these preliminary results indicate that cd /cd costimulatory-based immunosuppressive regimens can protect allogeneic islets from rejection. furthermore, a appears to adequately block cd to facilitate this engraftment and function as demonstrated by flow cytometry. iwami, , qi zhang, osamu aramaki, nozomu shirasugi, katsuya nonomura, masanori niimi. surgery, teikyo university, tokyo, japan; renal and genitourinary surgery, hokkaido university, sapporo, japan. many studies have shown immunosuppressive effects of dietary intake of fish oil containing eicosapentaenoic acid (epa) in various models such as autoimmune diseases and transplantation. however, its mechanisms remain uncertain. furthermore, there have been no studies examining the effect of purified epa. here we determined the ability of purified epa to inhibit alloimmune response in mouse cardiac transplantation model. methods: cba recipients (h- k ) were given single injection of purified epa intraperitoneally on the same day as transplantation of a heart from c bl/ donors (h- b ). mixed leukocyte reaction (mlr) assay and enzyme linked immunosorbent assay (elisa) were also performed to evaluate the effect of purified epa on cell proliferation and cytokine production. to determine the presence of regulatory cells, adoptive transfer study was conducted. results: untreated cba recipients rejected c bl/ cardiac allografts with median survival time (mst), days. in contrast, cba recipients treated with purified epa ( . g/kg) had significant prolongation of allograft survival (mst, > days). cba recipients treated with . g/kg purified epa eventually rejected allografts (mst, days). in mlr assay, treatment with . g/kg purified epa suppressed alloproliferation of splenocytes in the recipients. the treatment also inhibited production of il- , il- and ifng by the splenocytes in the recipients. when splenocytes were harvested from the recipients treated with . g/kg purified epa days after cardiac allografting and were adoptively transferred into naïve secondary recipients, the adoptive transfer induced significant prolongation of cardiac allograft in nave secondary recipients (mst > days, compared to that in the recipients with adoptive transfer of naïve splenocytes, mst, days). conclusions: purified epa induced significantly prolonged survival of fully mismatched cardiac allografts, and generated regulatory cells. background: chronic allograft nephropathy (can), the most common cause of late kidney allograft failure, is not effectively prevented by the current regimens. activation of extracellular signal-regulated kinases / (erk / ) mediating intracellular signal transduction from various growth factor stimuli is required for tgf-β production, which plays a key role in the development of can. hence, the therapeutic potential of disruption of erk / signaling to prevent can was examined in an experimental model. methods: kidney donors from c bl/ j mice (h- b ) were transplanted to bilaterally nephrectomized balb/c recipient mice (h- d ). the recipients were treated with ci (mek-erk / inhibitor) or vehicle after days post-transplantation for days. can was evaluated with the banff working classification. results: all six allografts receiving ci treatment were survived, while two out of seven grafts were lost in vehicle-treated group. at the end of experiment, the function of grafts in ci treated recipients had been maintained, indicated by lower levels of serum creatinine and bun ( ± µm and ± mm, n= ) as compared to those ( ± µm and ± mm, n= ) in vehicle group (creatinine, p= . ; bun, p= . ). pathological evaluation indicated that ci reduced can, reflected by a lower can score in ci -treated group ( . , n= ) as compared to that ( . , n= ) in vehicle controls (p= . ). further examinations showed that ci treatment resulted in inhibition of phosphorylation of erk / and reduction of tgf-β levels in grafts. in vitro ci potently suppressed not only growth factors-stimulated erk / activation and tgf-β biosynthesis in renal tubular epithelial cells, but also attenuated alloantigenstimulated t cell proliferation. conclusion: our data suggest that interference of erk / signaling with pharmacological agent (i.e. ci ) has therapeutic potential to prevent can in kidney transplantation. objective: this is the first study to investigate the role of a novel jak and sykinhibitor, r , in the prevention of obliterative airway disease (oad), the major obstacle after lung transplantation. methods: trachea from brown-norway (bn) donors were heterotopically transplanted in the greater omentum of lewis (lew) rats. recipients were treated for days with r ( , , , or mg/kg), rapamycin ( . or mg/kg), or left untreated. allografts were recovered and processed for histological evaluation determining degree of luminal obliteration, percentage of respiratory epithelial coverage, and mononuclear cell infiltration. donor reactive (igg) antibodies from the recipient's serum were determined using flow cytometry. results: r at , , and mg/kg significantly inhibited luminal obliteration in a dose dependent manner ( ± %, ± %, ± %; p= . vs. no medication). rapamycin in both concentrations significantly inhibited luminal obliteration ( ± %, ± %; p< . vs. no medication) similarly to r at and mg/kg. r at and mg/kg significantly preserved respiratory epithelium compared to r at and mg/kg ( ± %, ± % vs. ± , ± %; p= . ) and was superior to rapamycin in epithelial preservation ( ± %, ± % vs. ± %, ± %; p= . ). all r and rapamycin-treated recipients expressed decreased numbers of peritracheal mononuclear cells in a dose dependent manner (p< . ). r , , and mg/ kg treated recipients had significantly reduced igg levels versus untreated recipients ( ± , ± , ± vs. ± ; p< . ). all r treated recipient thymus and spleen weights were significantly lower compared to the untreated group (p= . ). bun, cr, and cholesterol levels were unaffected in r treated recipients. conclusion: r potentially exhibits its inhibitory effect by preserving the respiratory epithelium, rather than by rapamycin's mechanism of reduced smooth muscle cell (smc) proliferation. r occupies a beneficial pharmacokinetic profile, lacks nephrotoxic and atherogenic properties, and provides a favorable alternative to rapamycin in the treatment of chronic rejection in lung transplant recipients. genz- is a novel, oral immune-modulatory agent identified in a high-throughput screen designed to find inhibitors of tnfα-induced apoptosis. the molecular target of the compound remains under investigation but is likely downstream of the tnfα cell surface receptor. in vitro studies have shown genz- to be an effective inhibitor of the tnfα-triggered caspase cascade but not anti-cd or fas-mediated apoptosis, and thus may act by inducing allograft resistance to immune attack rather than suppressing the alloimmune response per se. it has been shown to synergize with sirolimus in murine heterotopic cardiac allotransplant models. in order to test this promising new agent in a more clinically relevant model of solid organ transplantation, we studied genz- in a mismatched nhp (rhesus macaque) renal transplant model. genz- (n= ) was administered ( mg/kg, iv, days - ) with sirolimus ( mg/kg, po, days - ). five control animals received only sirolimus and vehicle ( mg/kg, po, days - ). all animals were followed serially by polychromatic flow cytometry to determine the relative and absolute number of cd + and cd + t cell subsets. time to allograft rejection, the primary end point, was determined by a significant rise in serum creatinine and bun, as identified with biweekly monitoring. after diagnosis of rejection, allografts were removed for histological and transcriptional studies, along with splenocytes for immune function assays. in this pilot study, prolongation of rejection-free survival was significantly improved with genz- and sirolimus combined vs. sirolimus alone ( . days vs. . days, respectively, p = . ). given these initial results, we have initiated a larger study (n= ) to optimize the dose and duration of genz- . five animals, transplanted within the past month remain alive and well in this study. further investigation of this agent will allow us to better understand the benefit of inhibiting tnfα-mediated apoptotic effects in both cellular alloimmune response and allograft injury in solid organ transplantation. targeting purpose: allospecific t memory cell responses are present in transplant recipients from exposure to cross-reacting antigens. we have previously reported that lfa- inhibition suppresses primary cd -dependent rejection responses which are not controlled by any conventional immunosuppressive strategy. these studies were conducted to analyze the efficacy of this anti-lfa- ab for control of cd -dependent responses in sensitized hosts. methods: fvb/n (h- q ) donor hepatocytes were transplanted into c bl/ (h- b ) or cd ko (h- b ) recipients. memory responses were analyzed by retransplantation with a second fvb/n allogeneic hepatocyte transplant. cohorts of mice were treated with anti-lfa- mab and observed for hepatocyte survival or magnitude of cd + t cell mediated allospecific cytolytic activity. results: the untreated secondary cd ko and c bl/ recipients rejected hepatocyte allografts with enhanced kinetics in comparison to the primary graft (mst= day vs day , and mst= day vs. day , respectively; p < . ). anti-lfa- mab treated cd ko recipients demonstrated delayed rejection (mst= day vs day ; p= . ) compared to secondary rejection in untreated cd ko hosts. anti-lfa- mab treatment did not delay rejection in sensitized c bl/ recipients (mst= day ) but did significantly reduce the in vivo allospecific cytotoxic effector function in c bl/ secondary recipients ( . ± . %; p= . ) as compared to untreated controls ( ± . %). the residual cytotoxicity observed in anti-lfa- mab treated c bl/ recipients is comparable to the in vivo cytotoxicity of cd -depleted c bl/ secondary recipients ( . ± . %) and is likely mediated by alloantibody. in fact, the level of allospecific cytotoxicity in anti-lfa- mab treated sensitized c bl/ recipients correlated with the amount of alloantibody present in recipient serum. conclusion: in conclusion, treatment with anti-lfa- mab delayed (cd -independent) cd -dependent rejection in sensitized recipients but did not delay rejection in sensitized cd -sufficient c bl/ recipients. despite the efficacy of treatment with anti-lfa- mab to significantly reduce the in vivo allospecific cytotoxic effector function in sensitized c bl/ mice this strategy did not delay rejection. this is likely due to alloantibody mediated rejection in sensitized c bl/ (but not cd ko recipients) which is not suppressed by treatment with anti-lfa- mab. abstract# cytomegalovirus (cmv) represents a major cause of infectious complications after transplantation. recently, chronic infections with lcmv, hiv or hcv were shown to be associated with functionally anergic t-cells characterized by high expression of the programmed death (pd)- molecule. this study was carried out to characterize functional exhaustion of cmv-specific cd t-cells as determinant of impaired cmv-control and to elucidate whether the pd- pathway may be operative in active cmv-infection after renal transplantation. cmv specific cd t cells from controls, hemodialysis patients, and renal transplant patients were quantified using flow cytometry and analysed for their expression of pd- and cytokines ifnγ and il . cmv specific proliferation was analysed by cfda-se dilution. in viremic transplant-recipients, a significantly higher proportion of cmv-specific cd t-cells were pd- positive (median . %) as compared to non-viremic transplant patients ( . %), dialysis-patients ( . %) or controls ( . %, p< . ). in line with functional impairment, pd- positive t-cells produced significantly less ifnγ per single cell as compared to pd- negative t-cells (mean fluorescence intensity . ± . versus . ± . , p< . ). moreover, unlike controls or non-viremic patients, the majority of cmv-specific t-cells from viremic patients showed a long-term loss of il- production. interestingly, functional anergy of pd- positive cmv-specific cd t-cells was reversible in that antibody-mediated blockade of pd- signaling with its ligands pd-l /-l led to a fold increase in cmvspecific proliferation. in conclusion, expression of pd- defines a reversible defect of cmv-specific cd t-cells, and blocking pd- signaling may provide a potential target for enhancing the function of exhausted t-cells in chronic cmv-infection. differential background: some patients with cmv disease may be simultaneously infected with multiple viral strains. it is unknown if different strains clear differently with the commencement of antiviral therapy. we assessed response to antiviral therapy in patients with simultaneous co-infection with multiple strains of cmv. methods: pcr-based strain typing of cmv was performed using the glycoprotein b gene of cmv (gb - ) in a cohort of organ transplant recipients with cmv disease. from this, patients were identified that had simultaneous infection with ≥ cmv strains. quantitative assessment of each of the strain types was performed at regular intervals after starting antiviral therapy. results: the different types of multi-strain infections were gb +gb ( / , %), gb +gb ( / , %), gb + gb ( / , %), gb +gb ( / , %), gb +gb ( / , %) and gb +gb ( / , %). / ( %) were simultaneously infected with or different genotypes. within individual patients, there was trend for gb cmv load ( . log genomes) to be lower than the other genotypes (p= . - . ) at the onset of disease. decay kinetics for all genotypes showed a bisphasic response with a st phase decline of ∼ . days and a nd phase of ∼ days. st phase delines were fastest for gb (p= . vs gb and ) while gb decline was slower than gb during the st phase. nd phase declines were similar between gb and ( days and . days) but were slower for gb and ( . days; p= . ). there was a significant correlation between st phase decline and log decline from baseline by day (r= . ; p= . ). relative fitness calculations revealed complex fitness dynamics between genotypes although gb was always less fit than gb , and , and gb and were always less fit than gb . conclusion: in patients with cmv disease who have simultaneous coinfection with multiple strains, the st and nd phase declines in gb are significantly slower than either gb or and have a lower log decline from baseline by day . these data indicate that either a significant fitness difference exists between cmv strains or that antiviral control of replication may be linked to cmv gb genotype and should aid our understanding of treatment success and failure. one introduction: parvovirus b (pvb ) is a single-stranded dna virus that was first reported to affect transplant (tx) recipients around years ago. in the kidney tx setting pvb has been reported to cause anemia and proteinuria. reported incidence in a general kidney transplant cohort has been reported to be between %- % and as high as % in an anemic kidney tx population. here we report our incidence of pvb infection over a year span. patients and methods: all records of kidney tx recipients from until were reviewed for the presence of pvb infection. there were kidney tx performed during this period. diagnosis of pvb infection was made either by detection of pvb via pcr in a blood/tissue sample or by detection of virus on renal tissue by immunostain. in patients found to have pvb infection; presence of anemia, proteinuria, concurrent infection and acute rejection rates were examined. response to treatment with ivig was also evaluated. results: incidence of infection was . % as patients were found to have evidence of infection. average time from tx to diagnosis of infection was . months (range days- months). average creatinine at diagnosis was . mg/dl. anemia was present in % of patients with an average hematocrit of . %. proteinuria was present in % of patients with evidence of pvb infection. co-infection was noted in patients ( cmv, ebv) and acute rejection was noted in % of individuals within months of diagnosis. collapsing glomerulopathy (cg) was present in patients and they all had subsequent graft loss at an average of months after diagnosis. of the patients with cg had proteinuria along with anemia and were caucasian. % ( / ) of all patients with evidence of pvb infection received ivig and cleared their infection. one of the remaining pts without ivig spontaneously cleared their virus. conclusion: although the incidence of pvb infection in our kidney tx cohort was very low, its presence portends an unfavorable outcome. the presence of cg associated with pvb is an especially devastating lesion with very poor outcomes. response to treatment with ivig and reduction of immunosuppression is variable. based on our data it seems reasonable to screen all tx patients with unexplained anemia and concurrent proteinuria as early detection of pvb may be crucial. background: prior to transplant, screening for latent tuberculosis (ltbi) by tuberculin skin test (tst) is recommended. the accuracy of tst in end stage renal disease however may be limited. the quantiferon®-tb gold assay (qft) detects interferon-δ produced by peripheral blood t-cells in response to tb specific antigens and may be more accurate for diagnosis of ltbi. methods: this prospective single center study compared the tst to qft for the diagnosis of ltbi in a cohort of adult patients listed or undergoing workup for renal transplantation. all patients had both tst and qft performed. additional data collected included demographics, tb risk history and chest x-ray results. based on demographic and radiographic findings, patients were classified as high or low risk for ltbi. a positive tst was defined as ≥ mm and positive qft as ≥ . iu/ml. results: a total of patients were enrolled. complete data was available for subjects ( did not return to have tst read). the mean age was . +/- . years with ( . %) males and ( . %) females. the most common etiologies of renal diseases were diabetes ( . %) and glomerulonephritis ( . %). most subjects ( of ) were on renal replacement therapy (hemodialysis in . % and peritoneal dialysis in . %). twenty ( . %) subjects had received bcg and ( . %) were born in or lived in a country in with tb prevalence rate > / population. fifteen ( . %) subjects were considered to be at high-risk for ltbi. overall ( . %) had a positive tst and ( . %) had a positive qft. the qft was indeterminate in subject due to a low mitogen response. agreement between the tests was % (k= . , p< . ). in low-risk subjects (n= ) the tst was negative in all and the qft was negative in and indeterminate in . in clinically high-risk subjects, ( %) had a positive tst and ( %) had a positive qft. the subjects with discordant results, both from tb endemic countries, had both completed treatment for ltbi years prior and remained tst positive, but were qft negative. in renal transplant candidates, the tst and qft are comparable for the diagnosis of ltbi. the qft has the advantage of being completed in a single visit and in our cohort indeterminate results were uncommon. optimal utilization of htlv i/ii positive organs -a nationwide survey. objective: we recently presented data from the unos database that demonstrated no significant difference in graft or patient survival between htlv i /ii (+) and (-) liver recipients. several organ procurement organizations (opo) including our own, do not offer htlv i /ii positive organs while many others find it difficult to place them. despite this, the number of htlv (+) organs is increasing with utilized in alone. this prompted us to evaluate the practical difficulties in placing these organs so as to improve utilization of these "high risk" life saving organs. medthod: a telephone/email survey of all the opos in usa was done over a month period from october to november . results: of the opos, responded. all screen patients for htlv i/ii with elisa. centers confirm with repeat elisa, confirm with western blot and centers do not pursue further. of the centers offer the htlv i/ii positive organs. there were a total of positive donors in the past years of which organs from donors ( . %) were placed. centers offer all the organs while offer one or more organs selectively based on accepting centers. none have been able to place the pancreas. only liver and kidney were commonly accepted. several centers noted a high false positive rate. based on the unos regional analysis data, % of the organs are utilized in ny state alone. many opos did not know which particular centers accept these organs and consequently spend a lot of time and effort in order to place them. a majority wanted to have a list of transplant centers that accept these organs. conclusions: htlv i/ii organs are being underutilized. moreover, our prior analysis of unos data shows that these life saving organs are shared more nationally than loco-regionally which is associated with a poorer outcome. increased knowledge of successful htlv (+) donation and the centers that are willing to utilize these organs in the appropriate setting will help expand the donor pool and decrease mortality on the waiting list. increasing traditional two-drug chronic immunosuppression (is) used in organ transplantation (tx) is associated with development of ebv-driven complications because of impairment of anti-viral cd + t cell surveillance. since the long-term impact of alemtuzumab preconditioning combined with tacrolimus monotherapy on ebv immunity after tx has not been studied, here we aim to analyze the frequency and function of peripheral blood ebv-specific cd + t cells. thirteen ebv + stable kidney transplant (ktx) recipients and ebv + healthy controls were recruited to this cross-sectional study. all patients received alemtuzumab preconditioning, followed by tacrolimus monotherapy. blood samples were collected at least year post-tx to allow immune reconstitution. the ebv-specific cd + t cell phenotype and function were screened by flow cytometry and ifng elispot assay. hla-a restricted ebv-lytic (bmlf ) and latent (lmp a) peptides were used to generate tetramer (tmr) probes, and for functional screening in elispot. circulating cd + t cells from ktx patients had recovered by year, and were comparable to those of healthy controls ( . %± . vs % ± . , p= . ). moreover, the memory distribution and the frequency of ebv-specific cd + t cells detected in patients and controls (bmlf -specific: . %± . vs . %± . , p= . , and lmp specific: . %± . vs . %± . p= . ) were similar. in contrast, the frequency of functional type- (ifn-g producing) ebv-specific cd + t cells was significantly lower in ktx patients than in healthy controls (bmlf : ± spots/ cd t cells vs ± p= . , and lmp : ± vs ± p= . ). accordingly, on average, only - % of circulating ebv-specific cd + t cells from ktx patients produced ifn-g, while - % of effector cells were functional in healthy controls. addition of il- ( iu/ml) during the elispot assay reversed the hypo-responsiveness of type- (ifn-g) ebv-specific cd + t in patients (range - fold increase), suggesting that these effector cells were anergic. these results support the notion that alemtuzumab-induced lymphocyte depletion followed by tacrolimus monotherapy renders ebv-specific cd + t cells anergic in vivo, a state that can be readily reversed by cytokines such as il- , which are commonly released during immune activation. background: the epidemiology of the transmission of cytomegalovirus (cmv) from organ donors to recipients is not completely understood. we studied donor to recipient transmission patterns by analyzing viral genomic variants through the use of cmv glycoprotein b (gb) genotyping by real-time pcr. polymorphisms in gb ul allow discrimination of distinct genomic variants (gb - ). methods: organ transplant recipient pairs or triplets were included in the study if: a) they had cmv infection, b) they received an organ from a cmv seropositive donor, and c) there was at least one other recipient from the same donor that also developed cmv infection. genotyping (gb - ) was performed by quantitative real-time pcr on stored blood samples. clinical charts were reviewed to evaluate the clinical characteristics and outcome of cmv infection. results: of the cmv seropositive donors screened, were multiple organ donors for which or more of their recipients developed cmv infection. the total number of recipients from these donors was (median of recipients per donor). of these recipients, ( %) had cmv infection ( recipient pairs and recipient triplets). the prevalence of genotypes was gb (n= ; %), gb (n= ; %), gb (n= ; %), gb (n= ). mixed infection with two concurrent genotypes was present in patients ( %). overall concordance between cmv gb genotype in recipient pairs was . % ( / ). if both recipients were cmv seronegative (d+/r-) the gb concordance in recipients was % ( / pairs). gb concordance was % ( / pairs) if one of the recipients was seronegative and the other seropositive. concordance was % ( / pair) if both recipients were seropositive. concordance between genotypes was seen in / ( %) recipients triplets. in seropositive recipients with cmv viremia, the origin of the cmv strain was thought to be donor derived in / ( %) and of reactivation of the recipients own virus in / ( %) of the cases. no difference in clinical outcome or organ tropism was seen between genotypes. based on an analysis of strain concordance within recipients from common donors, transmission patterns of cmv can be assessed. in d+/r+ transplant patients, donor strain superinfection accounts for the approximately two-thirds of cmv infection. backgroud: although map kinases have been implicated in the pathophysiology of liver iri, their functional significance in the mechanism of tlr mediated pro-inflammatory immune regulation, remains to be elucidated. methods: map kinase activation in a murine model of liver warm iri ( min. ischemia, h reperfusion) was determined by western blots. chemical inhibitors of erk (u , µm in vitro or mg/kg in vivo), jnk (sp , µm or mg/kg), and p (sb , µm, mg/ kg) map kinases were utilized in vitro in primary bm-derived macrophage cultures stimulated with lps ( ng/ml); or in vivo in liver pro-inflammatory immune responses induced by lps ( µg/mouse, i.p.) or iri. results: erk and jnk, but not p , map kinase activation were readily detected in liver iri. in primary macrophage cultures, lps induced pro-and anti-inflammatory genes, including tnf-α, il- β, il- , il- , inos and cxcl . erk inhibitor mainly suppressed il- β and il- ( % and % resp), whereas jnk inhibitor suppressed the majority of genes. in lps-induced liver inflammation, erk inhibitor suppressed il- , il- β, inos and il- by > %, but failed to affect tnf-α/cxcl . jnk inhibitor, on the other hand, preferentially inhibited pro-inflammatory genes, but marginaly affected il- (< %). and produced comparable suppression of pro-/anti-inflammatory genes (figure ). interestingly, tnf-α was the least responsive gene subjected to map kinase regulation. conclusion: erk and jnk map kinase activation: / are required for tlr activationinduced pro-inflammatory gene induction; / play critical role in the development of ir-mediated liver immune response/tissue injury. background: the jak/stat signaling is one of the major pathways for cytokine signal transduction. the signal transducer and activator of transcription (stat ) is mainly activated by ifn-α/ß/ifn-γ. the activation of stat by ifn-γ has been implicated in hepatic inflammation. we have shown that activation of toll-like receptor (tlr) complex initiates pro-inflammatory response leading to liver ischemia/reperfusion abstracts injury (iri). indeed, tlr signaling in vitro activates stat , which in turn triggers production of type- ifn-dependent cxcl (ip- ). this study was designed to analyze the cross-talk between stat and the map kinase (erk) downstream of jak/ stat signaling pathways. methods & results: we used a mouse liver model of partial warm ischemia ( min), followed by reperfusion ( h) . first, we employed stat ko (n= ) and control wt (n= ) mice. the hepatocellular damage, as measured by salt levels (iu/l), was significantly decreased in / stat ko mice (p< . ); the remaining / of stat ko showed salt levels comparable with wt. hence, we distinguished two groups of stat "protected" vs. "nonprotected" ko recipients. histology revealed minimal sinusoidal congestion without edema/vacuolization or necrosis in stat ko "protected" group. the induction of mrna coding for tnfα/ il- was higher in stat ko "nonprotected" livers. the expression of cxcl , the product of stat activation downstream of tlr in type i ifn pathway, was profoundly and selectively depressed in livers from stat ko "protected" mice, as compared to iri susceptible livers. similarly, western blot-assisted phospho-erk expression was up regulated selectively in the stat ko "protected" group. in the second series, c bl/ mice were treated h prior to liver ischemic insult with jak- inhibitor (tyrphostin ag ; mg/kg, i.p.; n= ), or vehicle (n= ). the hepatocellular damage, as measured by salt levels (iu/l), and histology was significantly decreased in ag group, as compared with controls (mean = vs. ; p< . ). the disruption of jak/stat signaling by inhibiting jak uniformly ameliorates the inflammatory immune response in liver iri. however, the blockage of stat alone is insufficient to reproducibly exert cytoprotection. as jak is upstream of stat as well as upstream of map kinase (erk), this study highlights the role of both signaling pathways in hepatic iri. purpose: tlr is required for maximal ischemic injury of the heart, liver, lung, and kidney. to better understand the mechanisms of tlr action, we investigated a murine model of ischemic kidney disease and examined endothelial tlr expression. methods: . animal ischemia reperfusion injury(iri): the right kidney of wildtype(wt) c bl/ , or tlr -deficient(ko) c bl/ scn mice was removed. the left pedicle was clamped for min, followed by hr reperfusion. sham animals were controls. . bone-marrow chimera: four groups of bm chimeras were created: wt→wt; ko→wt; wt→ko; ko→ko ( recipients/grp). wks later, chimerism was confirmed by tail and blood genotyping, and mice subjected to renal iri. . tlr mrna detected by dig-labeled antisense; tlr on endothelium by anti-tlr and anti-cd . . total genome mrna expression on ischemic vs. sham wt mice, ischemic vs. sham ko mice( mice/grp) determined using affymetrix mouse genome . genechips followed by genesifter analysis. quantitative real-time rt-pcr confirmed candidate genes. . ms endothelial cells were treated with h o ( um) for min, cultured for hr and then expression of tlr and endothelial genes determined. results: tlr -deficient mice had less renal injury as assessed by pathology and function. radiation chimeras showed that radioresistant parenchymal cells and radiosensitive leukocytes were both required for maximal injury. immunohistology and in situ hybridization identified endothelia in the outer medulla as a major tlr expressing cell type at hr post-reperfusion. genechip analysis revealed a panel of cytokine, chemokines, proinflammatory and cell-cycle related genes that were differentially expressed on iri versus sham kidneys. real-time rt-pcr confirmed that the following pro-inflammatory endothelial genes increased after ischemia in wildtype but not tlr ko mice: tlr , pentraxin related gene(ptx ), and endothelial cell-specific molecule (esm ). real-time rt-pcr showed that in vitro h o treatment of endothelial cells induced expression of tlr ( . -fold), ptx ( . -fold), and esm ( -fold). we found that endothelia in the outer medulla increase their expression of tlr after ischemia, and that the endothelial genes ptx and esm increase only in wt ischemic kidneys. we also found that h o , which mimics reactive oxygen species generated during iri, directly increases these same endothelial genes in vitro. nkg d is an activating receptor expressed on nk cells and cd + t cells. ligands for nkg d including rae- , mult- and h in mouse, may be upregulated by tissues in response to stress. a study in mouse macrophages described upregulation of rae- in response to stimulation through tlr by lps. we have shown that tlr mediates kidney ischemia reperfusion injury (iri) and also observed rae- upregulation in iri kidneys. we now determined whether: ) kidney iri could induce the expression of nkg d ligands: ) expression of nkg d ligands is tlr dependent; ). bone marrow (bm) derived cells or parenchymal kidney cells express nkg d ligands. methods. kidney-ischemia was induced in tlr -/-, myd -/and wt mice for min. blood and tissue were harvested at days , and . primary cultures of mouse tubular cells (tecs) were also subjected to ischaemia (mineral oil overlay for hr) or tlr activation (lps stimulation). bm chimeric mice were generated by transplanting bm into irradiated recipient mice before iri was induced. results. rae- mrna level in iri kidney was increased from day to day , peaking at day compared to sham operated kidney ( - fold increase, p < . ) measured by real time pcr. rae- protein was detected in renal tubular cells from ischemic kidney but not from shamoperated control by flow cytometry. mult- mrna expression was also increased from day to day ( - fold increase, p < . ). both rae- and mult- mrna levels were reduced in tlr -/and myd -/-iri kidneys ( - fold reduction) versus wt controls (p < . ). tlr -/and myd -/primary cultured tecs submitted to iri in vitro also showed less rae and mult- mrna expression than wt controls (p < . ). lps stimulated rae- and mult- expression in wt but not in tlr -/-tecs. tlr -/mice bearing wt hemopoietic cells had significantly lower kidney rae- and mult- mrna expression after iri versus wt mice with tlr -/-bm (p < . ). conclusion. kidney iri causes rae- and mult- expression and kidney parenchymal cells are the dominant source. tecs can be stimulated to express rae- and mult- by ischemia or lps via the tlr pathway in vitro and deficiencies in this pathway provide protection against iri and rae- and mult- expression in vitro and in vivo. thus, kidney iri causes upregulation of nkg d ligands by parenchymal kidney cells via tlr . background: intestinal ischemia/reperfusion injury (iri) is a major clinical problem. although toll-like receptor (tlr ) has been implicated as a potential link between the innate and adaptive immunity, little is known on its role in intestinal iri. our preliminary research in intestinal iri has shown that, compared to sham controls, wt mice had decreased survival, worse tissue injury/apoptosis, increased pmn infiltration, increased cd + cell infiltration, and increased production of tlr , chemokines, and adhesion molecules. here we used tlr ko mice to further investigate the role of tlr in intestinal iri and its effects on cytokine/chemokine programs and apoptotic signaling. methods: c bl wt and tlr ko mice underwent min of total jejunoileal warm iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at h and h. tissue analysis included histopathology, cd immunostaining, myeloperoxidase (mpo) activity, rt-pcr for chemokines/ cytokines, and western blots for apoptotic and ho- protein expression. results: tlr ko had superior survival compared to wt ( % vs. %, p< . ). on histopathology tlr ko had near normal-appearing villous architecture, while in contrast, wt showed mucosal erosions and villous congestion/hemorrhage. tlr ko had reduced cd + cell infiltration as compared to wt ( . ± . vs. . ± . per hpf at h, p< . ; and . ± . vs. . ± . per hpf at h, p< . ). early mpo activity was also reduced in tlr ko ( . ± . vs. . ± . u/g at h, p< . ). rt-pcr analysis demonstrated decreased production of mrna for ip- , mcp- , rantes, and ifn-γ and increased production of il- in tlr ko. there was decreased protein expression of caspase- and increased expression of bcl- and ho- in tlr ko mice. conclusion: the genetic absence of tlr exerts protection against intestinal iri, demonstrating for the first time that tlr is required for intestinal iri. the absence of tlr signaling reduces iri through reduced neutrophil and t cell chemotaxis, and up-regulation of protective molecules. these results support data that tlr is a mechanistic link between the innate and adaptive immunity, implicating tlr as a potential therapeutic target for the prevention of intestinal iri. it is well known that liver steatosis increases hepatic vulnerability to ischemia/ reperfusion (i/r) injury as part of the transplantation process. endotoxin (lps) is thought to be a major contributing factor to the pathogensis of i/r. during portal occlusion, lps is translocated across the mesenteric tissue barrier into the portal circulation, and is delivered as a large bolus to the liver at the point of reperfusion. at this time, lps is mainly recognized by toll-like receptor (tlr ). this leads to downstream signaling and the production of proinflammatory products that ultimately lead to cellular inflammation, necrosis, and apoptosis. it is well known that steatotic livers are highly sensitive to endotoxin as compared to their lean counterparts post-i/r, and we have previously seen that monoclonal antibody blockade of endotoxin dramatically improves animal survival after i/r. therefore, we propose the novel hypothesis that tlr signaling is a major contributor to cellular damage after steatotic hepatic i/r. to test this hypothesis, we subjected male -week-old c bl/ j (control) or c bl/ scn (tlr deficient, tlr ko) mice to a high-fat diet (hfd) for four weeks. then, we subjected the animals to minutes of total hepatic ischemia and or hours of reperfusion. there was a dramatic improvement in animal survival in the hfd tlr ko animals versus control hfd animals at hours ( % vs. % in control hfd animals, p< . ). there was significantly more liver necrosis (as measured by a grading scale from - ) in the control hfd animals as compared to the tlr ko hfd animals ( . ± . in control vs. . ± . in tlr ko, p< . ). in addition, we see significant increases in the message level of the proinflammatory cytokines il- , il- , and ifn-γ at one hour in the control hfd animals that is abrogated dramatically in the tlr ko hfd animals. we do not see these dramatic changes in the control animals fed a normal diet. despite the significant increases in inflammation in the control hfd animals versus the tlr ko hfd and normal diet control animals, we do not see changes in the tlr message level or endotoxin boluses, implying an increased sensitivity in the absence of an increased number of receptors. tlr is a critical molecule in the pathogenesis of steatotic liver ischemia/reperfusion, and represents a potential therapeutic target for expansion of the donor pool. the background: neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia and reperfusion injury (iri). particularly, neutrophil elastase (ne) accounts for a substantial portion of the neutrophil function. this study was designed to explore the role of, and mechanism by which ne exerts its function in a mouse model of liver warm iri. methods: partial warm ischemia was produced in the left and middle hepatic lobes of c bl/ mice for min, followed by - h of reperfusion. mice were treated with ne inhibitor (nei; mg/kg p.o.; gw a; n= ) or control (n= ) at min prior to the ischemia insult. after h or h of reperfusion, sast/salt levels and intrahepatic neutrophil accumulation (myeloperoxidase [mpo] activity) were assessed. the pro-inflammatory cytokine (tnf-α, il- ), chemokine (cxcl- , cxcl- , ip- ) and toll-like receptor (tlr) gene expression profiles were screened by rt-pcr. liver samples were collected for histological grading, and detection of neutrophil infiltration by the naphtol as-d chloroacetate esterase stains. results: nei treatment significantly reduced sast/salt levels, as compared with controls ( ± vs ± ; p< . / ± vs ± ; p< . at h, and ± / ± vs ± / ± ; p< . at h). the expression of pro-inflammatory cytokines, and chemokines was significantly reduced in the nei treatment group (tnf-α in h; p< . , il- in h and h; p< . and p< . , cxcl- in h; p< . , cxcl- in h and h; p< . and p< . , ip- in h; p< . ). the mpo activity (u/g) was also significantly reduced following nei treatment ( . ± . vs . ± . ; p< . ). tlr expression was selectively diminished in nei pretreated livers ( h; p< . ). histological examination of liver sections has revealed that unlike in controls, nei treatment markedly reduced edema, diminished centrilobular ballooning/sinusoidal congestion, ameliorated hepatocellular necrosis, and decreased local neutrophil infiltration. conclusion: the inhibition of ne ameliorated hepatocellular damage, reduced local inflammatory responses, and neutrophil activity/infiltration in a stringent mouse liver model of warm iri. interestingly, it also downregulated the innate tlr signaling. this study documents the previously unrecognized ne -tlr cross talk, and implies neutrophil elastase in the signal transduction pathway instrumental for liver iri. lymphocyte lymphocytes are involved in the early pathogenesis of ischemia-reperfusion injury (iri) in kidney; however, their role during healing is unknown. this has direct clinical consequence since lymphocyte-targeting agents are currently administered to prevent rejection during recovery from iri in renal transplants. c bl/ mice underwent unilateral clamping of renal pedicle for min, followed by reperfusion, and were sacrificed at day . mice were treated with saline (c), methylprednisolone (pred) or mycophenolate mofetil (mmf) i.p. daily from day until sacrifice (n= /group). lymphocytes were isolated from the kidneys, counted and stained with monoclonal antibodies. kidney damage (% damaged tubules) and proliferation (ki assay) were assessed. flow cytometry analysis demonstrated increased numbers of tcrβ + cd + and tcrβ + cd + t and tcrβ -nk . + nk, but not cd + b cells at day in the ischemic (ir) kidneys compared to contralateral. regulatory t cells, tcrβ + cd + cd + foxp + , and t cell subsets tcrβ + cd + cd + and tcrβ + nk . + also increased. moderate tubular damage in cortex, severe injury in outer medulla and increased proliferation in both compartments characterized the repair phase. pred improved histological damage in ir kidneys, while mmf worsened it. proliferative index correlated with histology in outer medulla. pred reduced the total counts and activation of tcrβ + cd + and tcrβ + cd + t cells in ir kidneys, and increased the percentage of tcrβ + cd + cd + among total tcrβ + cd + t cells. mmf reduced all lymphocyte subsets, decreased the percentage of tcrβ + cd + cd + foxp + among total tcrβ + cd + t cells, and lowered il- tissue levels. il- and platelet derived growth factor-bb protein levels were also decreased in ir kidneys from mmf-treated mice. in conclusion, specific trafficking and phenotypic changes of kidney-infiltrating lymphocytes occur during recovery from renal iri, and lymphocyte-targeting agents, pred and mmf, alter tubular cell structure, proliferation, and inflammatory response in the repair phase. background: ho- plays an important cytoprotective role in a variety of organ injury models. we have shown that ho- exhibits potent cytoprotective effects against liver i/r injury. this study explores the function and mechanism of ho- in liver i/r injury by using sirna that suppress ho- expression both in vitro and in vivo. methods: using a partial liver warm ischemia model, c bl/ wide-type (wt) mice (n= /gr) were injected with ho- sirna/nonspecific control sirna ( mg/kg, i.v. at day - ) or ad-ho- /ad-β-gal ( . x pfu, i.v. at day - ). sham control wt underwent the same procedures, but without vascular occlusion. mice were sacrificed at h of reperfusion; liver tissue and blood samples were collected for future analysis. in in vitro studies, ypen- endothelium cells were transfected with ho- sirna ( nm) or ad-ho- / ad-β-gal. results: ho- sirna treated mice showed significantly increased sgot levels (iu/l), as compared with nonspecific control sirna or ad-ho- ( ± vs. ± and . ± , respectively; p< . ). these correlated with histologic suzuki's grading of liver i/r injury, with ho- sirna showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis; nonspecific control sirna showed moderate edema, sinusoidal congestion/cytoplasmic vacuolization. in contrast, ad-ho- revealed only minimal sinusoidal congestion without edema or necrosis. ho- sirna significantly increased local neutrophil accumulation and caspase- activity, and increased the frequency of apoptotic cells ( . ± . vs. . ± . and . ± . , respectively; p< . ), as compared with nonspecific control sirna or ad-ho- . both ypen- endothelium cells and wt mice treated with ho- sirna revealed markedly increased caspase- activity and reduced ho- expression. in contrast, ad-ho- significantly decreased caspase- activity and increased ho- and anti-apoptotic bcl- /bcl-xl expression. conclusion: this study provides evidence that ho- exerts cytoprotection against i/r injury by regulating liver apoptosis and inhibiting caspase- activation pathway. organ specific sirna is not only a powerful tool to study local gene function, but it may also provide novel therapeutic application in transplant recipients. islet cell transplantation has recently emerged as one the most promising therapeutic approaches for diabetic patients to improve glycometabolic control. one major problem of the procedure is the requirement of an immunosuppression protocol capable of counteract both auto and allo-immune response. recent data suggest that anti-thymoglobulin (atg) can halt efficiently the mounting of an alloresponse and the recurrence of autoimmunity in nod mice by expanding antigen specific t-regulatory cells. we retrospectively reviewed our casuistry type diabetic kidney-transplanted patients who underwent islet transplantation using an immunosuppressive protocol based on atg or daclizumab (as induction treatment) plus cyclosporine and mmf as maintenance therapy. patients underwent islet after kidney transplantation in our center. thirty-four patients received a time course of atg as induction ( mg per day for - days), (number of islet infused= , ± , ), and patients received daclizumab at induction ( mg/kg every weeks for weeks); (number of islets= , ± , ). no major adverse events were recorded in our center; no malignancies or infections outbreaks were evident. patients in the atg induction group showed a better islet survival rate compared to daclizumab (p= . ), according to c-peptide> ng/ml. a sustained and prolonged c-peptide secretion was evident in the atg group; while in the daclizumab group only patient was functioning at year. interestingly, in the atg, which reached a longer follow-up, we cannot observe a loss of beta cell mass according to our metabolic test, suggesting the preservation of islet mass. in conclusion, atg can provide a good protection towards both allo and auto immune response. the next step will be to use atg in a calcineurin free protocol, allowing a better expansion of t-regs. introduction: despite consistent achievement of insulin-independence, recent data indicate that the long term success of islets transplants using the edmonton protocol is < % at years. the cause of the nearly universal late islet allograft failure remains unknown but hypotheses include: allo or autoimmune injury, marginal mass exhaustion, hepatic site related dysfunction, and immunosuppression toxicity. we examined the series of islet transplants performed at our institution and noted marked differences in the outcome and complications in ia and iak groups. our results may provide insight as to the cause of chronic islet loss. methods: thirty-one islet infusions were administered to ia (n= ) and iak (n= ) type- diabetics between / - / . ia and iak had similar demographics and transplanted islet mass ( , vs , ieq/kg). ia received edmonton like immunosuppression with zenapax induction and cni/srl, whereas iak patients received zenapax and: cni/mmf/pred ( ), cni/mmf ( ), cni/srl ( )). results: insulin-independence was achieved in all but patients who completed therapy ( others withdrew). compared with ia, iak exhibited better glycemic control ( -month mean hba c . vs . , stimulated c-peptide . vs . ), and improved islet survival; all ia eventually failed and only / were insulin free for > -years, whereas only / iak grafts have failed with exhibiting continued robust function at > ,> ,> , and > months with / fully insulin independent. in addition, all ia patients demonstrated immune sensitization post graft failure versus / iak. all ia developed mouth ulcers versus only / iak. conclusions: ia and iak exhibit striking differences in outcome and complications. the absence of mouth ulcers and lack of sensitization in iak may relate to steroid use and continued immunosuppression for the kidney graft, respectively. the superior outcome of the iak cohort may be a result of differences between the two groups including the use of maintenance steroids, prior exposure to thymo or the chronically immunosuppressed state of the iak recipient. perhaps the most interesting correlation with outcome is the absence of the anti-proliferative agent sirolimus in the iak group. our results provide clues to the cause of chronic islet transplant failure and may lead to novel approaches to avoid it. islet background: although islet transplantation has become an option for treatment of type diabetes, all currently used immunosuppressive protocols have significant renal and islet toxicity. we describe a novel immunosuppressive protocol using sirolimus and the anti-lfa antibody efalizumab that permits prolonged islet allograft survival without the need for steroids or calcineurin inhibitors (ci). methods: between february and august , consecutive type diabetic patients with hypoglycemic unawareness and normal renal function received allogeneic pancreatic islet transplants. induction immunosuppression consisted of doses of thymoglobulin given on pre-transplant days - and - , efalizumab ( mg/kg sq/week starting on d - ), and sirolimus. maintenance immunosuppression consisted of sirolimus and efalizumab. results: all patients achieved insulin independence after single islet infusions (mean ieq/kg= , ). three of remain insulin independent or more months after transplant (table ) . patient resumed low dose insulin (approximately % of original dose) weeks after transplant and is awaiting a second islet infusion. her blood glucose control is markedly improved and she has not experienced any hypoglycemic episodes. all patients show persistent c-peptide secretion and have stable renal function. side effects due to efalizumab were limited to transient irritation at the injection site. conclusions: thymoglobulin induction followed by sirolimus and efalizumab maintenance is well tolerated and allows prolonged islet allograft survival. this protocol is the first ci/steroid free islet regimen resulting in insulin independence with a single donor islet infusion. by eliminating ci, this protocol minimizes renal and islet toxicity and may thus improve long-term islet survival and function. long . clinical and metabolic profiles were assessed every months for months. results: si-exn group was on this drug for median time of days pre-si. both groups were similar except for duration from post-completion to graft dysfunction ( ± vs ± in si-exn and si-c group respectively, p< . ) and duration of graft dysfunction before si ( ± vs ± , p= . ). si-c and si-exn groups received mean of ± and ± ieq/kg, respectively (ns). only / of si-c patients achieved insulin independence for , and > days after si. all subjects in si-exn group achieved insulin independence for more than , , , days. at months insulin independence was % in si-c and % in si-exn group. comparing pre and post-si, si-exn group had significantly lower a c at - months and lower auc glucagon at and months (p< . ). auc c-peptide in si-exn group was significantly higher than si-c at and months. intravenous glucose tolerance test showed significantly increased acute insulin responses to glucose at - months in si-exn and at and months in si-c group. si-exn group had more acute c-peptide response to glucose than si-c at and month (p< . ). acute exenatide administration during intravenous glucose tolerance test at month in si-exn revealed significantly increased acute insulin and c-peptide response to glucose which indicates improved first phase insulin release. conclusion: supplemental islet infusions under exenatide lead to insulin independence, restore first phase insulin secretion and result in long term insulin independence. exenatide this prospective phase / trial aimed to demonstrate safety and reproducibility of allogeneic islet transplantation (tx) in type diabetic (t dm) patients and implement a strategy to achieve and maintain insulin-independence with minimal islets. ten c-peptide negative t dm subjects with hypoglycemic unawareness received - intraportal allogeneic islet tx. four subjects (group ) received the edmonton immunosuppression regimen (daclizumab, sirolimus, tacrolimus). the next subjects (group ) received etanercept, exenatide and the edmonton regimen. we followed all subjects for months after the first tx. the primary efficacy end point was insulin independence. secondary endpoints were hba c, fructosamine, ogtt, mixed meal test, glucagon stimulation test, ivgtt and hypoglycemia. to study the effect of exenatide, we compared frequently sampled ivgtt, c-peptide, proinsulin, amylin and glucagon with and without exenatide. two self-limiting bleeds occurred in infusions. all subjects became insulin independent. group received a mean total number of islets (ein) of , , ± , in (n= ) or (n= ) tx, whereas group became insulin independent after tx ( , ± , ein, p= . ). all group subjects remained insulin free through the -month follow-up. two group subjects resumed insulin: one after immunosuppression reduction during an infectious complication, the other with severe gastroparesis and exenatide intolerance. hba c reached normal range in both groups ( . ± . at baseline to . ± . after - tx in group vs. . ± . to . ± . after tx in group ). baseline hba c was significantly higher in group than group (p= . ). pre-and post-tx hypo scores were . ± . and in group vs . ± . and . ± . in group . glucagon levels decreased significantly in all subjects. in group , the decrease in glucagon levels after challenge tests with and without exenatide was . fold more significant after exenatide in all subjects ( background istx has been investigated as treatment for type dm. however, the hope this approach would result in long-term freedom from exogenous insulin has failed in practice. techniques for isolating islets have advanced and with availability of new is agents, strategies can now be developed specifically for istx that will provide greater immunologic protection w/o diabetogenic side effects. rejection/ vascularization still remain major limitations for success of istx. we hypothesize that bm is an accessible, immunologic privileged space with natural well-developed vasculature and may be a suitable site for istx. method wistar rats were used as donors/ recipients. dm was induced by iv-streptozocin. rats who had morning glc levels > mg/dl on two separate occasions were used as recipients. ptx was performed as normal rodent standard. islets were isolated from pancreas by distending pancreatic duct with liberase. islets were separated on a discontinuous histopaque density gradient, further purified, counted, divided into aliquots transplanted into different sites (liver, bm). bw, glc and c-pep were measured in all groups before/after tx. background: in february , the islet community was notified of a possible bovine product contamination in the collagenase enzyme (liberase hi) used for human islet isolations. to eliminate the potential hazard of bovine spongiform encephalopathy, we successfully adapted our human islet processing procedure to utilize a different gmp collagenase and neutral protease (nordmark/serva). here we describe what we consider the most important factors for achieving reproducible and clinically useable islet isolations. methods/results: a standard isolation protocol involving controlled enzymatic digestion followed by density gradient purification was used. seventeen donor pancreata were processed and ten were ultimately used for clinical transplantation. eight of these successful isolations were performed using the nordmark/serva enzymes (table ) . the following factors were identified as being important for ensuring successful islet yields: ) donor age and size: male donors - years old who were tall (> cm) and heavy (> kg) conclusions: incorporation of several important modifications into our existing islet isolation protocol has allowed us to routinely obtain high quality islet isolations using an alternative, bovine product-free gmp enzyme. (n= )], and in pts immuno was cni-free. tac-treated pts were younger, more sensitized and more frequently re-kt. dgf was more frequent in pts receiving cni-free immuno. as a group, these pts were older, more frequently received a kt from a donor after cardiac death and less frequently from a living donor. acute cellular rejection (acr) was diagnosed in pts ( . %), occurred before day th (e-acr), and after this date (l-acr). c d-positive amr was diagnosed in pts ( . %), of which were early (e-amr) and were late episodes (l-amr rates of acute rejection (ar) and background: an increasing number of hs patients are being transplanted using desensitization protocols. these patients are considered high risk for ar, particularly antibody mediated rejection (amr). here we examined our ar rates and treatment outcomes for our hs kidney transplant (kt) recipients using our most current desensitization strategies. methods: between june (when rituximab was introduced to our desensitization and treatment protocols) to july , hs kt patients were transplanted using combinations of high-dose ivig, rituximab, and plasmapheresis (pp) for desensitization. we examined the overall ar, c d-cell-mediated (cmr), and c d+amr rates. amr episodes were treated with steroids, high-dose ivig, and rituximab. refractory and rapidly progressive amr was treated with pp. treatment outcomes and differences in ar rates between deceased (dd) and living donors (ld) was examined. recent reports suggest that treatment with the monoclonal anti-cd antibody, rituximab (rtx) may improve renal graft survival in antibody mediated rejection (amr); however optimal dosing for this indication is unknown. we examined the efficacy of a single low dose ( mg) of rtx for treatment of refractory amr in order to limit significant infective complications associated with conventional dosing regimens ( mg/m ). rtx was used in seven consecutive patients who had refractory amr as judged by ongoing biopsy evidence of amr after weeks of standard therapy consisting of pulse methylprednisolone and plasma exchange with low dose ivig ( mg/kg) (pe/ivig). all patients received tacrolimus and mycophenolate mofetil. amr was defined as ) characteristic histology on biopsy (banff criteria), ) graft dysfunction and ) presence of a donor specific anti-hla antibody (dsab). b-cell counts (cd ) and serum creatinine (scr) were monitored. pe/ivig was ceased in all cases after rtx dosing. the average follow-up since rtx dosing is . months (range . - . mths). all patients still have functioning grafts ( % month graft and patient survival), with current mean scr levels ( ± µmol/l) significantly lower than mean peak rejection levels ( ± µmol/l) p= . . cd counts fell to zero and remained < x /l for > months in all patients. dsabs remain detectable by luminex flow beads in all patients despite stabilization of scr. two of patients developed an infective complication post rtx dosing. one patient developed bacterial pneumonia requiring hospital admission whilst a second patient developed cmv viraemia and later bk nephropathy which has not led to significant graft dysfunction. hence, infectious complications are far less than those reported for multiple standard dose regimens in similar patient groups. large clinical trials are required to confirm the efficacy of rtx in treating amr as well as optimal dosing. standard dosing is based on oncology treatment regimens which are likely to be excessive for amr in patients already markedly immunosuppressed. our data suggests single low-dose rtx for refractory amr results in excellent patient and graft survival and leads to low rates of serious infective complications in the short-term. renal the demographic and clinical data were similar between +cxm and negative cxm groups. the rejection rate was significantly higher and the length of stay was significantly longer in +cxm group as shown in the table. the ra survival rates were %, % and % lower at , and years post transplant respectively among + cxm recipients. however, this did not reach significance mostly due to small sample size (p= . ). the inferior ra outcome is seen during the initial few years after transplantation that disappears after years. in clkt, pre-transplant +cxm can result in higher ra rejection rate and longer length of stay in hospital. la may not always confer immunological protection to ra in + cxm clkt and its true burden may be under recognized since cxm is not routinely performed in all clkt. study of antibody specificities or la volume to determine the effect of +cxm on ra outcome is essential. skpt in patients with positive cdc b-cell and/or flow cytometry crossmatch is associated with high amr rate despite low dose ivig and r-atg/alemtuzumab induction. pancreas graft survival is inferior in patients with positive crossmatch while kidney graft and patient survival are similar to that of negative crossmatch recipients. majority of amr can be reversed with treatment but further long-term follow-up is needed to determine the impact on graft survival. ten the first european phase iii trial with tacrolimus in the early ´s clearly showed advantages for tacrolimus in terms of acute rejection (ar) vs the original cyclosporin formulation. however, no advantages were seen in survival rates. the present investigator-initiated, observational follow-up study collected data on patients included in the original cohort of patients who participated in this large study and who were randomised to a triple immunosuppressive regimen consisting of tacrolimus, azathioprine and steroids (tac/aza/ster, n= ) or cyclosporine, aza and steroids (csa/aza/ster, n= ). efficacy and safety parameters assessed at follow-up included: acute rejection; patient and graft survival; renal function, vital signs, basic lab results and immunosuppressive regimen for the patients years after completion of the original study. results: currently, data are available from % of the patients. all assessments were conducted following the intent-to-treat principles. more patients in the tac than in the csa group remained on the randomised treatment. while kaplan-meier (k-m) estimates for patient survival at year show comparable ( % tac vs %csa) results, k-m estimates for graft survival demonstrate an advantage for the tacrolimus cohort ( % tac vs % csa). graft half-life estimates (gjertson and terasaki method) yielded overall results of . years (tac) and . years (cs). in both treatment groups, ar was associated with inferior long-term results. for patients with ar, half-life estimates were . and . years for tac and csa, respectively, while in patients without ar half-lives were . (tac) and . (cs) years. mean serum creatinine after years was significantly lower in the tac cohort vs the csa group ( . mg/dl tac vs . mg/ dl csa, p< . ). mean glomerular filtration rates (mdrd estimate) were . ml/ min in tac and . ml/min in cs. after ten years of follow-up, the mean (c- ) levels were . mg/ml (tac) and mg/ml (cs). conclusion: analysis of this long term follow-up of a large study confirms the benefits of a tacrolimus-based therapy. it also confirms the concept that freedom from early acute rejection is associated with superior long term results in renal fuction. therefore, long term renal allograft function is remarkably good in tac patients. cyclophosphamide a novel therapy for ivig/plex-resistant antibody-mediated rejection. (c)). moreover subgroup analyses within the different groups were made to identify potential differences. groups were analyzed for survival, graft rejection and graftvasculopathy (cad). kaplan-meier analysis was used and log-rank test was performed to detect differences. results: a total of transplants ( . %) were blood group compatible. the majority (n= ) were a transplants ( %) followed by b (n= ; %), aab (n= ; %), bab (n= ; %) and ab (n= ; %). overall survival comparison showed no significant difference in long-term survival ( -year) recent data has shown the utility of urinary biomarkers to predict delayed graft function early following renal transplantation, but their ability to predict longer-term allograft function is less clear. we evaluated whether urinary biomarkers measured in the early post-transplant period would predict allograft function at and months after renal transplantation. urinary biomarkers including n-acetyl-β-d-glucosaminidase (nag), kidney injury molecule- (kim- ) and matrix metalloproteinase- (mmp- ) were measured in patients at hours , , , and days and following renal transplantation. glomerular filtration rate (gfr) at and months were calculated using the mdrd equation. all patients had functional allografts at months and patients lost allografts after months. levels of individual biomarkers at different post-transplant time points were correlated with and month gfr using spearman's correlation (rho) and are shown in the urinary nag levels at post-transplant hours , , and days and showed significant negative correlation with -month gfr. nag levels at hour and days and maintained the significant negative correlation with -month gfr. urinary kim- levels at the hour point showed significant negative correlation with -month gfr and kim- levels at hour and day displayed significant negative correlation with -month gfr. urinary mmp- levels at no time points had significant correlation with either -month or -month gfr. urinary biomarkers particularly nag shows promise as a tool in the early prediction of longer-term allograft function following renal transplantation. in kidney disease chemokines are involved in the recruitment of leukocytes causing kidney damage and progression to endstage renal failure. similar mechanisms are proposed for chronic allograft failure in renal transplant recipients. the chemokine attracted leukocytes produce proinflammatory and profibrotic cytokines contributing to fibroblast proliferation, matrix production and tubular atrophy. the role of chemokines in the acute post-ischemic tubular necrosis early after renal transplantation and their impact on long term allograft outcome has not been investigated yet. methods patients ( m, f, mean age . yrs) with dgf (with a median number of . dialysis sessions) developed biopsy proven acute tubular necrosis during the first three weeks after transplantation. the biopsies were studied by immunostaining for ki , ccr , ccr , rantes, mcp- , cd and cd , respectively. the patients were followed for a mean time of . yrs. none of them developed a rejection episode in the follow up time. after follow up the mean serum creatinine was . mg/dl ( . - . the goal of a tx is for the recip to achieve long-term survival (surv.), with continued graft function, equivalent to the gen. population. we studied subsequent outcome in , -yr kidney tx survivors (tx - ) ; % living donor (ld); % st tx; % female; % type diabetes; mean age at tx (±se), ± yrs. actuarial -yr surv. is shown in table ; ld patient, graft, and death-censored graft surv. is signif. ↑vs. deceased donor (p<. ). mean creatinine (± se) at yrs was . ±. ; yrs, . ±. ; yrs, . ± ; yrs, . ± . the major causes of late graft loss (gl) were death with function (dwf) and chronic rejection (can). by multivariate analysis, risk factors for gl after yrs were age ≥ yrs at tx; type diabetes, retx, hla mm ≥ ; ≥ acute rejection episode, and pretx cardiac, peripheral vascular, or liver disease (p<. ). the most common cause of dwf was cardiovascular disease (cvd); however, > % deaths were due to malignancy. the use of kidneys from donors with a positive serology for hcv into hcv(+) recipients still remains controversial. in , our units adopted this policy, subsequently modified in , so these kidneys were limited to recipients with a positive rna hcv before transplantation. the aim of the present analysis was to review the long-term safety of our policy. since january to february , hcv(+) patients with a negative hbsag and not treated with interferon received a kidney transplant in our units. patients were transplanted from an hcv(-) donor(group ) and from an hcv(+) donor(group ). median follow-up time was (ir - ) months. remarkably, group showed a significantly higher donor age ( . ± . versus . ± . years;p< . ) and recipient age ( . ± . versus . ± . years;p< . ), as well as a worse hla compatibiliy than group . immunesuppressive therapy did not significantly differ between the two groups. group notably, only patients died because of a liver disease ( in group versus in group ) and only patients developed a hepatocarcinoma (both in group ). in summary, no significant differences were observed in hcv(+) recipients according to hcv serology of the donor, in terms of death censored graf survival, patient survival and evolution of liver disease. therefore, our experience clearly demonstrates that the use of kidneys from hcv(+) donors into hcv(+) recipients is a safe strategy in the long-term and a wise way of using these kidneys, that otherwise would be lost at a moment of shortage. introduction: deceased donor kidneys are allocated to adult candidates in the u.s. primarily by waiting time and hla similarity. we investigated two alternative allocation systems, lyft-scd and lyft-dy, that prioritize offers of kidneys to adults based in part on lyft. methods: based on characteristics of each candidate and donor, lyft was calculated as the difference between expected median years of life for the candidate with that kidney donor transplant (tx) versus without a kidney. years on dialysis were weighted at . of years with a functioning graft. the relative risk of graft failure for each donor was calculated from donor factors using a cox model and the donor profile index (dpi), the percentile score of that risk among kidneys used in ( %=greatest risk of graft failure). using actual candidates and deceased donors and acceptance patterns from , we modeled allocation with the kidney-pancreas simulated allocation model (kpsam). kpsam sequentially offers kidneys to candidates according to user-specified allocation rules, and models the probability for kidney placement and post-tx survival. the lyft-scd model allocated scd kidneys by lyft and ecd kidneys by dialysis years (dy). the lyft-dy model allocated organs according to the formula [. lyft( -dpi)]+[dy(. dpi+. )]+[ pra/ ] to adult kidney tx candidates. results: the table compares a year's worth of kidney-alone recipient characteristics, lifespan after tx and additional lifespan (v. dialysis) due to tx among all kidney (including spk) recipients, and the donor/recipient age correlation (r) under current national kidney allocation, allocation using lyft for scd, and allocation using lyft-dy within each donation service area. conclusions: allocation systems incorporating lyft have the potential to increase the number of years of life attainable from tx and are being considered for the allocation of kidneys within the u.s. introduction : organ shortage for transplantation has led to the use of marginal kidneys, which has been associated with poorer but still acceptable results. in our center, the transplantation of two very marginal kidneys into a single recipient was used as a strategy to increase the number of organs available. the present study reports renal function and survival of dual-kidney transplants (dkt) performed at our institution. methods : from october to june , transplants were performed at our center, from which were dkt. kidney selection for a dkt was based on refusal of the kidneys for single kidney transplantation (skt), donor's age ≥ years and ≥ % of glomerulosclerosis on pre-implantation biopsy. the calculated creatinine clearance (crcl, ml/min), graft and recipient survival from dkt were compared to those of skt from ecd (based on unos criteria, n= ) and ideal donors (id, aged to y, n= ) over a seven-year period. results : dkt donors were significantly older than the two other groups (dkt: year old, ecd: , id: ). dual transplants were offered to older recipients by design ( , , ). delayed graft function was more prevalent for dkt and ecd than with id ( %, %, %). twelve, and -month crcl were similar for dkt and ecd but lower than id (twelve months: , , ; months: , , ; months: , , , respectively). patient survival, actuarial graft survival and actuarial graft survival censored for death were similar between the groups. conclusion : to our knowledge, this study reports short and long-term outcome of the largest cohort of dkt performed at a single institution. it shows that dual-kidney transplantation is a more complex intervention with more risks of surgical and post-op complications (data not shown). however, with adequate surgical expertise, dkt patients can expect long-term results comparable or even better than ecd. in our center, the use of dkt has increased the number of transplantations by % for the period of study. it allowed older patients to receive a graft within shorter delays. moreover, it permitted a more judicious allocation of a resource that is still limited. an results: very few differences resulting from donornet were seen. for regionally exported kidneys, cold ischemia time (cit) was . h before donornet (bd) and . h after donornet (ad); . % of regionally exported kidneys had cit> h bd, as compared with . % ad. for nationally exported kidneys, cit was . h bd and . h ad; . % of nationally exported kidneys had cit> h bd, as compared with . % ad. the same hucs of exported kidneys bd were also hucs ad. of the top centers utilizing exported kidneys bd, only one was not within the top centers utilizing exported kidneys ad. when we looked at cit for kidneys exported to hucs, again there was little difference before or after donornet, with a trend to possibly longer cit for hucs. for kidneys regionally exported to hucs, cold ischemia time (cit) was . h before donornet (bd) and . h after donornet (ad); % of regionally exported kidneys had cit> h bd, as compared with . % ad. for kidneys nationally exported to hucs, cit was . h bd and . h ad; . % of nationally exported kidneys had cit> h bd, as compared with . % ad. conclusion: the first months of national implementation of donornet were not successful in decreasing cit for regionally or nationally exported kidneys. furthermore, the centers to which a high proportion of these kidneys were exported did not change as a result of donornet, and for these centers allocation efficiency is no better and might even be worse than before. machine cold machine preservation has been shown to improve outcome following transplantation of deceased heart-beating donor kidneys. to determine whether cold machine preservation also improves outcome of kidneys donated after cardiac death (dcd) we undertook a multicentre randomized controlled trial in the uk comparing machine perfusion with simple cold storage. one kidney from each dcd donor was randomized to machine perfusion (organ recovery systems lifeport device with kps- solution), the other to perfusion with uw (viaspan) solution followed by simple cold storage. the primary outcome measure was the need for dialysis in the first days (delayed graft function, dgf); secondary outcome measures included gfr at week (mdrd technique); and month graft and patient survival. at the time of writing, one week outcome data are available for all patients. a sequential design was used with the data inspected after transplants and then every . all recipients received basiliximab, mycophenolate sodium, tacrolimus and prednisolone. the significance of the initial results has been tested using paired t tests and mcnemar's exact tests. recruitment was stopped after donors when the unadjusted sequential analysis concluded that there was no difference in the incidence of dgf. of the kidneys were transplanted; one was not used for anatomical reasons. one kidney suffered primary non function; it had been machine preserved. the results shown below represent intention to treat. introduction: split liver transplantation has been adopted as an alternative to expand the organ donor pool. while the adult/child split liver (a/csl) in which an extended right graft (erg) is transplanted into an adult and a left lateral segment (lls), transplanted into a child, has gained a wide acceptance within the transplantation community, adult/ adult split liver (a/asl) in which the liver is split into a full right (fr) and full left (fl) is still performed very rarely. we analyzed the experience at our center with split liver grafts over a period of years. material and methods: between october and october we performed a total of liver transplants in recipients ( / children and / adults). among the ( children and adults) recipients of a primary isolated liver transplant, ( children and adult) were transplanted with a a/c or a/a sl. the recipients of a whole size graft ( adults and children) were used as a control. results: adults: patients received a split liver graft ( erg graft and fl/fr grafts) and a whole liver graft. overall the incidence of biliary complications using a split liver graft was % and with a whole liver graft % (p= , ).for the recipients of a split liver graft and years patient and graft survival was %/ % and %/ %. recipients of a whole liver graft had a and years patient / graft survival of %/ % and %/ % respectively. children: children received a split graft ( lls, erg and fl) and a whole size graft. biliary complications occurred in % of the recipients of a split liver graft and % of the recipients of a whole size graft (p < , ). among the recipients of a split graft and years patient / graft survival was %/ % and %/ % respectively. the recipient of a whole size graft had a and years patient / graft survival of %/ % and %/ %. conclusion: incidence of biliary complications is significantly higher using a split graft. at a high volume center, use of split liver grafts reached comparable and even better results of a whole liver graft in terms of patient and graft survival. thus, the use of split grafts should be strongly encouraged to expand the donor pool. a groups of primary ldlt and ddlt recipients were identified by matching diagnosis, meld score, and recipient age. cost data, acquired from the hospital financial database and inflation adjusted and clinical outcomes were compared between the groups. background: liver transplant recipients with (gw/rw) < . % are thought to have a higher incidence of post-operative complications, including small for size syndrome (sfss), and overall inferior outcomes. we analyzed a cohort of partial liver graft recipients and compared those with gw/rw < . % to those with gw/rw > . %. results: between and , adult patients underwent partial graft liver transplant. seventy-six grafts were from live donors (ldlt) and the remaining were from deceased donor split-liver transplants (slt). of these, patients had gw/ rw < . % ( ldlt, slt), and had gw/rw > . % ( ldlt, slt). median follow-up was . months and did not differ between the two groups. baseline demographics including donor and recipient age, and meld at the time of transplant also did not differ between groups (table) . three-month and -year graft survival for the two groups was comparable. three recipients with gw/rw < . % developed sfss ( . %); all three had inflow modification with splenic artery ligation (sal), and of the grafts was lost at one-year. eight recipients with gw/rw > . % developed sfss ( . %) (p = ns); six underwent sal and none had graft loss at one-year. there was a trend toward increased hepatic artery thrombosis with the smaller grafts that did not reach statistical significance (p = . ). the incidence of other surgical complications was similar between the two groups (table) . the diagnosis of rejection in post-transplant kidney disease depends largely on the assessment of biopsy pathology, which suffers from arbitrary scoring, subjectivity, and sampling variance. with microarray gene expression data from biopsies for cause, we used predictive analysis of microarrays (pam) to build a rejection classifier. because of the imperfect gold standard, the classifier cannot and should not have extremely high predictive accuracy. the goal was to see if microarrays could detect a robust rejection signature despite this uncertainty, and to identify the top genes distinguishing rejecting from non-rejecting biopsies. by examining discrepancies between pathology diagnoses and pam predictions, and using clinical follow-up information, we combine the strengths of each method to produce a more accurate diagnostic system. biopsies with rejection plus clinical episodes (fig. ) have higher positive predictive value than those lacking episodes. the majority of episodes occur in the top part of fig. , indicating that a valid rejection signature is being detected. of the exceptions, most had been treated with steroids before the biopsy, suppressing their gene expression patterns. samples called borderline rejection separated into two distinct high/low probability groups using pam, with all the cases classified as clinical rejection episodes occurring in the high probability group. of the genes chosen by the classifier, had previously been annotated in our studies of mouse kidney graft rejection. gzma, gzmb, and prf ranked th, nd, and th respectively. the top five genes were: cxcl , gbp , cxcl , indo, and cxcl , all previously annotated in rejecting mouse kidneys as interferon-γ induced genes. in summary, gene expression-based classifiers, combined with histopathology, promise more accurate diagnostic assessment of the state of kidney transplants at the time of biopsy. moreover our data driven classifier independently identifies the genes previously annotated in experimental studies. influence of donor background: renal-cell associated tlr activation was found to be important in mediating ischemia reperfusion injury to murine kidneys. we hypothesized that genetic variations within the tlr gene of the kidney donor affects the rate of delayed graft function (dgf). methods: we genotyped the functional tlr polymorphisms (snps) d g (rs ) and t i (rs ) in kidney donors from centers and correlated them with the occurrence of dgf. dgf was defined as the need of dialysis within days post-transplant or less than % drop in creatinine within the first hours after transplant. in a sample of patients from one center, we analysed intragraft hmgb- (high mobility group box protein- ) gene expression in pre-and post-implantation biopsies. statistical analyses were performed using the spss statistical package. results: both tlr snps were in hardy-weinberg equilibrium, and were combined for further analysis. recipients of a tlr mutated kidney showed a significant lower rate of dgf (p= . ), which was persistent after correction for known donor-derived risk factors (donor age, ecd vs. dcd-kidney, cold ischemia time, p= . , hr . , cl . - . ). additionally, we confirmed the presence of a possible tlr ligand, hmgb- , in pre-and post-implantation biopsies. we also confirmed the presence of a functional tlr receptor in human proximal tubular cells which expressed mcp- , il -β, il- and tnf-α after specific tlr agonist treatment. conclusion: human renal tubular epithelial cells express tlr and a functional tlr snp in donor kidney is associated with lower rate of delayed graft function. background: methylation of promoter cpg islands has been associated with gene silencing and demonstrated to lead to chromosomal instability. we postulate that differences in methylation patterns observed in tissues ranging from normal to cirrhosis to hcc may lead to the discovery of direct precipitating events that contribute to tumorigenesis in hcv-hcc patients. methods: dna from hcv-hcc tumors and corresponding non-tumor hcv cirrhotic tissues as well as independent hcv cirrhotic tissues and normal liver tissues were bisulfite treated and hybridized to the illumina goldengate methylation beadarray cancer panel i for interrogating cpg sites in the promoter regions of distinct genes. for each cpg site, a summary statistic representing "percent methylated" was estimated. for each cpg site, a jonckheere-terpstra test was applied to identify whether there was a significant monotonic trend in percent methylated across the independent normal, cirrhotic, and hcc tissues. in addition, the paired hcc and non-tumor cirrhotic tissues were analyzed using a paired t-test. the q-value method for estimating gene-wise false discovery rates (fdr) was used to adjust for multiple comparisons; cpg sites with an fdr< . were considered significant. to identify if serological responses to allogenic non-hla renal compartmentspecific antigens can be detected after renal transplantation (txp). methods: paired pre-and post-transplant (mean time months) serum samples from pediatric kidney allograft recipients were used for identification of de novo non-hla antibody formation assessed using invitrogen protoarray (v ), measuring signal post-pre. probes from the protoarray and cdna microarray platforms were re-annotated to current ncbi entrez gene identifiers using ailun. cdna arrays (gse: ) from normal kidney regions (inner and outer cortex, inner and outer medulla, papillary tips, renal pelvis and glomeruli; see table) were analyzed for compartment-specific genes by sam, and the highest-ranked genes in each compartment (ks two-sample test p < . ) and were ranked against each individual patient's numerical antibody response across the proteins on the protoarray. results: in % of patients ( / ), kidney-specific serological responses against the renal pelvis were the first to be detected, followed by responses against renal cortex. control gene expression data sets from other solid organs (gse: ; lung, heart, pancreas) did not demonstrate immune-response enrichment. the presence of these antibodies was not associated with donor specific hla class i or ii antibody levels. conclusion: we demonstrated de novo formation of circulating non-hla non-abo antibodies after txp, irrespective of hla antibodies, are detected against kidneyspecific protein targets. as these antibodies are not seen against other solid organs, the response is likely allogeneic. the renal pelvis and renal cortex have the highest immunogenic potential. we conclude that urinary cell mrna profiles that include levels of mrna encoding tubular proteins and mrna encoding proteins implicated in emt offers a noninvasive means ascertaining renal allograft status with respect to presence or absence of can. functional functional annotation of these genes identified cell cycle pathway and carboxylic acid metabolism pathway as potentially relevant for both datasets. altogether, these data suggest that as compared with recipients requiring on-going immunosuppression, operationally tolerant liver recipients exhibit traits of immunological quiescence and activation of natural killer related pathways. liver and kidney tolerant states appear to be fundamentally different biological processes, although some common pathways could be relevant to both settings. genetic introduction: both normal and fibrotic renal allografts develop a large number of persistent changes in pro-inflammatory gene transcripts early after transplantation ( ) . the aim of the current study was to determine if this "transplant effect" is attenuated by immunosuppression, fibrosis or hla-match. methods: we studied histologically normal implantation biopsies (t ) and m protocol biopsies (t ) from living donor kidney transplant recipients who had no identifiable post-transplant complication (no acute rejection, polyoma virus, etc). patients were stratified into distinct clinicopathologic groups. the first three groups had normal histology at both t and t and included: ) hla identical-tac treated (n= ); ) non-hla identical-tac (n= ); ) non-hla identical srl (tac-free, n= ). a fourth group was histologically normal at t but developed mild fibrosis by t (n= ). mrna expression from these biopsies was measured using the u plus . microarray and custom taqman low density arrays (tlda). data for each group was tested using a generalized linear model and changes common to each dataset identified (p< . ). results: in addition to transcript changes specific for each clinicopathologic condition, a large number of transcripts (n= ) were altered in all groups. % (n= ) had higher expression in all comparisons at t versus t , including fibronectin and collagen iv. % (n= ) of transcripts were considered down-regulated, including interleukin and β. gene ontology and signaling pathway analyses showed many of the transcripts to be involved in pro-inflammatory and immune response signaling pathways (ex. tlr, il- /- , etc). custom tldas were used to study several genes considered not detected by microarray. this included tgf-β , cd- e/- /- and vegf, all of which were significantly up-regulated in multiple comparisons. conclusions: persistent changes in pro-inflammatory transcripts occur in all renal transplants. this "transplant effect" cannot be explained by calcineurin-inhibitors (occurs in tac-free immunosuppression), increased alloreactivity (similar in hla identical/ non-identical) or fibrosis. a likely cause is persistent changes from ir injury. the role of the "transplant effect" in long-term allograft survival is unclear, but it likely interacts with other forms of graft injury. background: a critical gene-set for acute renal rejection (ar) diagnosis in peripheral blood has been previously identified by our group using cross-platform hybridization of unique peripherla blood samples with matched biopsy diagnosis, with ar (n= ) and without ar (sta; n= ), with cross-hybridization across human microarray platforms: k cdna lymphochip, k agilent and k affymetrix hu plus. in this study we proposed to verify and validate this gene-set for ar diagnosis and prediction by peripheral blood pcr-based analysis. method: / peripheral blood samples from the microarray gene-set were selected for -well format multiplex abi-taqman qpcrv validation of ar diagnosis across the gene-set. an independant set of new, time and immunosuppression matched, peripheral blood samples ( sta, ar) were used as a test-set for blinded prediction of ar diagnosis using qpcr across the most informative genes. sequential samples from ar patients (at ar, - months prior to, and after ar) were also examined by qpcr for these genes for blinded ar prediction, prior to biopsy proven ar. prediction models were generated using logistic regression analysis and p< . considered significant. result: in the validation set of samples, / genes were highly significant for confirming peripheral blood-ar diagnosis (p< . ). in the test group of samples, ar was predicted with a very high level of sensitivity and specificity (ppv and npv> %). in the group of ar patients with sequential samples pre-and post-ar, expression for these genes were significantly elevated in the pre-ar samples (vs. sta, p< . ) but were not different between pre-ar and ar values. conclusion: a highly specific and sensitive biomarker panel of genes has been derived and tested by cross-platform microarray analysis. thsi gene-set has now been validated and further verified by multiplex pcr, for ar diagnosis and prediction, even months prior to the rejection event. utility of this gene-set should be further validated in real-time by serial longitudinal peripheral blood testing, for more senstive and specific minimally invasive monitoring for graft rejection. tolerance/immune deviation ii background: our previous studies showed that regulatory t cells (treg) execute suppressive function in both the inflammatory site of the allograft and the draining lymph node (dln) to suppress allograft rejection. we explored how treg influenced the migration and function of antigen specific effector t cells and dendritic cells (dc) at these two sites in an islet transplant model. methods: treg were sorted from wild type or ccr -/-c bl/ mice, labeled with pkh , and transferred directly to islet allografts (balb/c into c bl/ ). effector cd t cells (te) from alloantigen specific t cell receptor transgenic mice were labeled with csfe and transferred intravenously. treg and te migration to islet grafts and dln were analyzed with flow cytometry and immunohistochemistry. chemokine expression was determined by rt-pcr. cx cr gfp mice, in which dc are internally labeled with gfp, served as islet donors, and donor dc migration was tracked with fluorescence microscopy. results: during priming and rejection, islet allografts expressed a panel of inflammatory chemokines shortly after transplantation that recruited te to the islets. te accumulated and proliferated in both the islet allograft and the dln. concomitantly, donor derived dc migrated from the islet to the dln as early as day after transplantation. local transfer of treg into the islet allograft inhibited islet chemokine expression, inhibited donor dc migration in an il- and tgfb dependent fashion, and reduced te migration to and proliferation in the graft. the transferred treg also migrated from the islet allograft to the dln, and directly inhibited te accumulation and proliferation in the dln, and migration of te to the islet. ccr -/-treg, which could not migrate to the dln but were retained within the graft, and were far less effective than wild type treg in inhibiting te migration and proliferation into both the islets and dln. conclusions: treg migrate to the islet and suppress parenchymal cell chemokine expression, dc migration, and te responses in the islet allograft. treg also migrate to the dln to suppress te proliferation and migration. treg trafficking from the allograft to the dln is crucial for suppressive function in order to target the separate effector mechanisms. these results demonstrate novel and important functions for migration in treg induced suppression and graft survival. tregs cd +foxp + regulatory t cells (tregs) play an important role in transplant tolerance, yet basic aspects of treg biology including the mechanisms involved in their induction remain unclear. α-cd rb is a potent tolerogenic agent that induces a x increase in number of tregs in wt mice, even in the absence of allo-antigen. here we use foxp red fluorescent protein reporter knock-in mice to study treg homeostasis and identify the mechanisms by which α-cd rb induces tregs. cfse-stained highly sort-abstracts purified foxp + or foxp -cells were adoptively transferred into fully replete naive wt congenic mice. whereas only - % of transferred foxp -cells undergo homeostatic proliferation (hp) over d, - % of foxp + cells proliferate -a surprisingly high rate of hp. moreover, treatment with α-cd rb markedly enhanced hp by transferred foxp + cells, resulting in a x increase in cell number. α-cd rb induced de novo foxp expression in - % of transferred foxp -cells (many of which subsequently underwent hp). thus, α-cd rb induces both conversion of foxp -to foxp + cells and promotes hp in foxp + cells in the absence of exogenous antigen. we then addressed the signals controlling basal hp of tregs and both hp and conversion mediated by α-cd rb. cfse-stained congenic cd + cells adoptively transferred into naive wt mice were untreated, or received csa, α-cd rb, or both and foxp + and foxp -cd cells were assessed on d . calcineurin inhibition greatly reduced basal hp of transferred foxp + cells compared to untreated mice. moreover, csa completely abrogated increased treg hp induced by α-cd rb, although conversion still occurred. next we assessed the role of tgfβ. we found that blocking tgfβ signaling with α-tgfβ shortened allograft survival, but had no effect on basal treg hp, or on treg hp or conversion by α-cd rb. finally, although exogenous antigen is not required, basal and α-cd rb-induced hp may still require recognition of self-ag bytregs. indeed, preliminary results reveal that when cfse-labeled cd + cells are transferred into mhcii ko mice, basal hp by tregs is reduced and not restored by α-cd rb. thus, α-cd rb alters normal controls regulating hp by treg. moreover, both basal and α-cd rb-mediated hp requires intact calcineurin activity and tcr signaling, but not tgfβ. understanding the signals controlling hp in treg may reveal new therapeutic targets for tolerance induction. we report the first demonstration of a tolerance-inducing strategy based on posttransplant administration of allo-ag-specific treg (aastreg) and rapamycin (rapa), in a fully allogeneic, unmanipulated mouse heart transplant model. enrichment of naturally-occurring aastreg represented the first step to counterbalance the high frequency of alloreactive t cells. selection was achieved by co-incubation of freshly-isolated cd + cd + t cells with donor bone marrow-derived dendritic cells (dc). the source of stimulatory factors necessary to sustain treg proliferation was the supernatant of cd + cd -t cells co-cultured with allogeneic dc (mlrsup). use of mature (cd high ) dc favored extensive expansion of foxp cells capable of il- production (t h ). co-culture of treg with immature dc in the presence of mlrsup rendered a t cell population with regulatory phenotype: foxp + , gitr + , ctla- + , ccr + , cd l -. these cells inhibited in vitro effector t cell proliferation at : and : treg:t cell ratios. the suppressive activity was ag-specific; no significant inhibition was evident when effector t cells were stimulated by third party dc. aastreg were then tested for their ability to induce transplant tolerance in a mouse heterotopic heart allograft model (balb/c to c bl/ ; unmanipulated). rapa was used: i) to inhibit effector t cell proliferation, while sparing treg activity and thus enhancing the in vivo treg:effector t cell ratio; ii) to promote resolution of the inflammatory state and preserve the susceptibility of conventional t cells to treg suppression. graft recipients received rapa ( mg/kg/d; d - ) and x aastreg i.v. on d . in comparison to the untreated group (median survival time, mst= d; n= ), rapa alone extended mst to d (n= ), with no long-term graft survival. under cover of rapa, aastreg exerted a profound tolerogenic effect: > % recipients exhibited longterm graft survival (mst> d; n= ). this effect was stronger than polyclonal treg administration: % long-term survivors (mst> d; n= ). moreover, the tolerogenic effect was ag-specific, as aastreg selected against third party dc (c h/hej) did not prolong graft survival in comparison to the rapa-only control group. these results indicate the feasibility and therapeutic potential of ag-specific tolerogenic cell therapy based on post-transplant administration of selected aastreg. direct intravenous immunoglobulins (ivig) is an effective treatment for t-cell mediated graft rejection and autoimmune diseases. ivig treatment is associated with rapid clinical improvements without the side effects of global immunosuppression. this prompted us to ask whether ivig might enhance directly the suppressive function cd +cd +foxp + regulatory t cells in vitro and in vivo. in vitro, mouse cba/ca (h k ) total cd + or cd + cd responder cells were stimulated with c bl/ (h b ) splenocytes, and human cd + or cd + cd cells were stimulated with allogeneic antigen presenting cells. in vivo, * total cd + or cd + cd -t cells of cba/ca mice were adoptively transferred into cba/rag -/mice, and one day later transplanted with a tail skingraft of c bl/ mice. ivig was administered i.v. on day , , , and . human serum albumin (hsa) was used as a control. binding of ivig and activation status of foxp + cd + t cells were determined. in vivo, only when total cd + t cells, but not cd -t cells, were adoptively transferred, ivig protected against t-cell mediated rejection of the fully mismatched skingraft (mst: ivig > vs hsa days, p< . ). ivig binds to ± % of foxp + t cells, while binding to foxp -t cells was minimal. this binding was partially fcγ receptor mediated. furthermore, ivig treatment resulted in activation of cd + t cells as detected by increased expression of phosphorylated zap /syk, which could be abrogated by specific tyrosine kinase inhibition. in vitro, ivig inhibited the alloproliferative response of murine cd + t cells by ± %, but after depletion of cd + t cells, this inhibition decreased to ± % (n= , p< . ). similar results were found in human mlr, where depletion of cd + t cells resulted in twofold reduction in ivig mediated suppression. significantly, incubation of human cd + cd + with ivig enhanced their ability to suppress allogeneic t-cell proliferation (ivig ± % vs hsa ± % of inhibition, n= , p< . ) . our results identify a novel pathway through which ivig treatment induces direct functional activation of both mouse and human regulatory t cells. immediate binding and activation of regulatory t cells is one of the mechanisms in the immunomodulatory repertoire of ivig, which allows rapid inhibition of allogeneic responses, and therefore can be a valuable tool after organ transplantation. generation foxp is a dna-binding protein that is necessary for regulatory t cell (treg) function, though recent data indicate that detection of foxp mrna or protein alone does not necessarily indicate whether the associated foxp + cell is functional or not. to that end, our analysis of foxp sequence showed the presence of an rxxr motif, conserved between mice and humans, that is located amino acids (aa) from the carboxy terminus of foxp and is a potential recognition sequence for cleavage by proprotein convertase enzymes. we found several proprotein convertases are expressed by naturally occurring tregs, with further upregulation upon tcr activation. we generated an antibody against the aa carboxyl peptide of foxp , and by western blotting detected a peptide of about . -kda, consistent with a -aa long carboxy-terminal foxp cleavage product. both cleaved and uncleaved forms of foxp were resolved by sds-page, and the cleaved form was found only in the dna-bound fraction. the requirement of proteolytic cleavage, at the intact tetrabasic rxxr motif ( rkkr ), for full treg function was shown by abolishing the motif through mutagenesis, followed by retroviral expression of mutants, and analysis of proteolytic cleavage by western blotting. assays of treg function by cells expressing either long-or short-foxp mutants showed short-foxp (missing the carboxy-terminal -aa) suppressed teff cell proliferation significantly more effectively than wt foxp or an engineered and cleavage-resistant mutant, termed long-foxp (p< . ). moreover, adoptive transfer of cells expressing short-foxp prevented experimental colitis more effectively than wt-foxp (p< . ), and both were more effective than cells expressing long-foxp . animals that received cells expressing short-foxp gained weight and were protected from disease. thus, function of foxp is regulated at a post-translational level by proteolytic cleavage and the short form of foxp represents the active form. our data shows proteolytic cleavage of foxp is key to the generation of functional tregs, with enzymes(s) of the proprotein convertase family likely playing an important role in this process and providing an extra and hitherto unrecognized important level of regulation for foxp . blocking since tgf-β is secreted in a latent form and active tgf-β is rapidly cleared from circulation. in order to make long-lasting active form of tgf-β, the mutant tgf-β was fused to a human igg fc component. the secreted human mutant tgf-β/fc (hmtgf β /fc) fusion protein stained with both anti-human tgf-β and anti-human igg antibodies, confirming the cytokine and isotype specificity of tgf-β moiety and fc domain. moreover, in vitro bioassay, hmtgf-β/fc inhibited il- dependent ht- cell proliferation in a dose dependent manner and had a circulating half-life of hours in mice. in vitro, anti-cd and anti-cd triggered cd + or cd + t cell proliferation, measured by h-thymidine uptake, could be synergistically inhibited by tgf-β and rapamycin. at the same time, the two reagents when added together could synergistically promote the induction of cd + foxp + t cells from peripheral naïve cd + foxp -t cells. in a pancreas islet transplantation model in which the fully mhc mismatched dba/ islets were transplanted into c bl/ mice rendered diabetic by streptozotocin, mean survival time of islet was days in control recipients (n= ). administration of muttgf-β/fc resulted in a delayed islet allograft rejection (mst; days, n= ). treatment with rapamycin prolonged islet grafts survival in % of recipients. in contrast, combined treatment with tgf-β/fc and rapamycin by four doses produced indefinite islet allograft survival in % cases. we have produced the long-lasting active hmtgf-β/fc fusion protein. the tgf-β/fc is synergistic with rapamycin to convert naïve cd +foxp -t cells into cd +foxp + tregs and promote long-term islet allograft engraftment. the the recognition of microbial motifs by the innate immune system leading to the stimulation of adaptive immune responses may explain the relationship between infections and susceptibility to graft rejection. a number of infectious agents have been reported to prevent the induction of allograft tolerance, however, none of these can reverse established tolerance, a situation that is highly relevant to clinical transpalntation. we here report that established allograft tolerance (induced with anti-cd + donorspecific transfusion) can be reversed in a cardiac transplantation mouse model following infection with listeria monocytogenes. this reversal of tolerance was dependent on the presence of both cd + and cd + cells, as well as of the tlr/il- /il- adaptor molecule, myd . we hypothesized that the reversal of tolerance requires that alloreactive conventional t cells (tconv) escape dominant regulation by pre-existing tregs. these alloreactive tconv can then proliferate resulting in an increased ratio of tconv:tregs favoring the reversal of established tolerance. indeed, we observed that tolerant grafts harbored low numbers of infiltrating cd + and cd + t cells ( - . x /heart), with - % of the infiltrating cd + cells co-expressing foxp (n= ). following the reversal of tolerance and allograft rejection, a - fold increase in cd + foxp and cd + foxp -tconv was observed in the graft, but no increase in the foxp + subset. in contrast, we observed no significant changes in the splenic t cell subsets, raising the possibility that the escape from tregs occurs locally within the graft. infection of tolerant il- -or ifnar -deficient recipients with listeria monocytogenes failed to reverse tolerance, consistent with a hypothesis that the initial escape of tconv from regulation may depend on il- , while their proliferation may be type i ifn-dependent. in summary, the conditions for the reversal of tolerance is more stringent than the prevention of tolerance, and requires myd -signalling and the presence of both cd + cells and cd + cells, as well as il- and type i ifn signaling. these studies point to the potential impact of bacterial infections on established tolerance, and to novel strategies to monitor and facilitate the maintenance of tolerance in the clinic. th our laboratory has identified kα tubulin, as an epithelial autoantigen to which immune response occurs following human ltx. further, we have shown a strong correlation between the presence of antibodies (abs) to kα tubulin and development of chronic rejection ie bronchiolitis obliterans syndrome (bos). goal of our study is to test the hypothesis that epithelial damage due to allo immunity results in remodeling exposes otherwise cryptic self antigens including kα tubulin and collagen type v (coll v) leading to cellular immune reactivity and ab production against these auto-antigens which may play a role in the pathogenesis of bos. patients who developed anti-hla ab and patients who had no detectable anti-donor hla abs post-ltx by luminex assay were analyzed for development of abs to kα tubulin and coll v using elisa method developed in our laboratory. the elisa for kα tubulin used recombinant protein ( µg/ml) purified on affinity resin. the elisa for collagen v uses commercially available human collagen v ( µg/ml). elispot assay for ifnγ and il- were performed for bos-and bos+ patient pbls (at the time of bos diagnosis), after stimulations with kα tubulin protein. the levels of anti-tubulin abs and anti-coll v abs were increased significantly in bos+ ltx recipients with anti-hla compared to those with no anti-hla, table (p< . ). . surprisingly, both cd and cd subsets induced long-term survival of secondary test grafts (> days). however, only the cd + t-reg prevented tvs (ni= + , + % of vessels), as compared to cd + t-reg (ni= + in + % of vessels). the cytokine profile indicated a dominant il- response. allo-antibody analysis showed up-regulation il- /il- dependent igg and igg c. conclusion: allochimeric protein-therapy and csa treatment generate t-reg that prolong graft survival, but only cd + t-reg generated from allochimeric protein-therapy prevent tvs. this cd + t-reg may be responsible for long-term graft maintenance through its unique ability to control anti-inflammatory and alloantibody responses. immunodominant h background: minor histocompatibility ags (mihas) are self peptides, derived from proteolytic processing of normal cellular proteins. miha incompatibility can induce t cell response to dominant mihas and facilitates expansion of ctls and graft rejection. however, the contribution of ctls recognizing these mihas in solid organ transplantation has not been fully evaluated. methods: balb.b (h- b ) donor hearts were transplanted heterotopically to c bl/ (h- b ) recipients. in vivo alloreactive cd t cells were monitored with peptide/mhc multimers. α-lfa- mab was given to recipients to prevent rejection. various numbers of h -specific cd t cells or cd t cells lacking h specificity were adoptively transferred into balb.b graft bearing b .scid recipients. results: % of transplantation recipients developed acute rejection and showed markedly increased numbers of h -specific cd t cells at day - in spleen. abundant cd infiltration was found and selective infiltration of h -specific cd t cells in the graft was confirmed with flow cytometry and in situ tetramer staining. α-lfa- mab treatment prevented acute rejection (mst> ) and allogeneic t cell expansion. it also profoundly attenuated neointimal hyperplasia ( graft-bearing b .scid mice. we found that the degree of acute rejection positively correlated with the number of h -specific cd t cells transferred. however, transferred h -specific cd t cells did not cause chronic rejection. interestingly, greater numbers of cd t cells with h immunodominance were found in spleen, blood and graft at days after adoptive transfer of cd lacking h specificity compared to h -specific cd t cell transfer. conclusion: h responses dominate other miha immune responses during acute rejection after balb.b to b cardiac allograft. we confirmed a role of immunodominant h specific cd t cells as pathological effector t cells in acute rejection but not in chronic rejection. maintaining a h response may be beneficial in allotolerance to suppress miha responses that could induce chronic rejection in long-term grafts. chronic lung allograft rejection, bronchiolitis obliterans syndrome (bos), affects up to % of transplant survivors years post-ltx. human neutrophil peptides (hnp - / α defensins), have been identified in the lavage fluids from patients with chronic rejection by proteomics. goals of our study were to determine the role of anti-hla abs and defensins in bos and to define the interactions between α- antitrypsin (aat), and defensins in regulating inflammation leading to epithelial cell proliferation and bos pathogenesis. bal and serum samples (post-ltx and pre bos) from bos+, bos-patients and normals were analyzed by elisa for α defensins (hnp - ), human β defensin (hbd ) and aat. small airway epithelial cells (saec) were treated with hnp or , with or without equimolar aat or with anti-hla abs and analyzed for levels of hbd production (elisa), cytokine and chemokine (luminex), and cell surface adhesion molecules (icam, vcam) by facs. bal and serum from bos+ patients had high levels of hnp - and hbd compared to bos-recipients or normal sera (p< . ) ( table ). there was also a significant decrease in aat levels in bos+ compared to bos-or normal serum (p= . ) ( table ) . saec produced human β defensins following anti-hla ab stimulation or by hnp treatment (table ) . there was increase in adhesion molecules (icam, vcam, folds) cytokines {il- , il- r α ( folds), il- β, il- ( folds)}, il- ( folds decrease), chemokines (il- , mcp , - folds increase) and growth factors (egf and vegf, . folds increase) in response to treatment with hnp or hnp compared to untreated saec, that was inhibited by aat. increased defensins and decreased aat levels were seen in lavage and serum of ltx recipients with bos. anti-hla abs further stimulate defensin production by saec. we conclude that chronic stimulation of epithelial cells both by defensins and anti-hla abs can lead to increased growth factor production contributing to the pathogenesis of bos. under tubular atrophy/interstitial fibrosis (ta/if) remains a major cause of late kidney allograft loss and epithelial mesenchymal transformation (emt) is now appreciated as a key feature in this process. we hypothesize that macrophages play a direct role in the development of ta/if by creating a profibrotic microenvironment and stimulating emt within the allograft. in human kidney allografts with ta/if (n= ), macrophage infiltration detected by immunostaining was significantly upregulated (mean score . ± . ) compared to biopsies without corresponding pathological changes from recipients with stable function (n= ; . ± . ; p< . ) and the extent of this staining also correlated to the magnitude of graft dysfunction (p< . ). rt-pcr in ta/if grafts also showed marked upregulation for emt markers αsma ( . ± . -fold; p< . ), s a ( . ± . -fold; p< . ), and vimentin ( . ± . -fold; p< . ), compared to stable function allografts. to explore the macrophage-emt relationship, we co-cultured freshly isolated human monocytes over a primary culture of human proximal tubular epithelial cells (ptecs) using culture inserts allowing media exchange but forbidding contact between the two cell populations. by rt-pcr, co-cultured epithelial cells showed significant downregulation of bmp ( . ± . -fold; p< . ), a negative regulator of emt, and epithelial marker e-cadherin ( . ± . -fold; p< . ), with marked upregulation of emt genes s a ( . ± . -fold; p< . ) and αsma ( . ± . -fold; p< . ) compared to ptecs cultured in media alone. investigating the mechanism of monocyte driven emt, we evaluated the effects of am , a synthetic retinoid that also inhibits il- and vegf signaling. am markedly reversed the transcriptional profile with reduction of emt markers s a ( . ± . -fold; p< . ), αsma ( . ± . -fold; p< . ) and vimentin ( . ± . -fold; p< . ), and a simultaneous increase in expression of bmp ( . ± . -fold; p< . ), thus reversing the pro-emt milieu. these results indicate that human monocytes induce transcription of emt related genes in ptecs, even in the absence of physical contact. moreover, this phenomenon appears to be mediated in part by il- , vegf, and other pathways mediated by the retinoic acid receptor. thus, in addition to their typical immune functions, macrophages support a milieu within allografts that promotes ta/ if in humans. further investigation into this novel pro-ta/if pathway could ameliorate chronic injury and improve graft survival. old donor kidneys are more likely to develop long-term graft failure, especially if they are exposed to stresses (e.g. rejection). this limited ability of old tissue to withstand stress may be due to its reduced capacity of replication and regeneration. telomere shortening determines lifespan and regenerative capacity. we showed that telomere shortening occurs in old human kidneys. late-generation terc ko mice with critically short telomeres have a reduced lifespan, show an accelerated aging phenotype and are an ideal model to resemble the situation in old human donors. this study addressed the question whether iri causes greater damage in kidneys from late-generation terc ko mice. we studied terc wildtype (wt), early-(g ) and late-generation (g ) terc ko mice day (wt:n= ; g :n= ; g :n= ), (wt:n= ; g :n= ; g :n= ), (wt:n= ; g :n= ; g :n= ) and days (wt:n= ; g :n= ; g :n= ) after iri. acute tubular necrosis was found mainly on days and and was significantly higher in terc ko g kidneys. tubular atrophy (ta) and intersitial fibrosis (if), reflecting chronic damage, were first detected at day and increased in all mice with a significantly higher extent of ta/if in terc ko g kidneys at day ( fig.) . the cell cycle inhibitor p , a downstream mediator of senescence induced by telomere shortening, was significantly upregulated in all groups after iri. p levels were highest in terc ko g kidneys at day ( fig.) . proliferative capacity, as measured by ki- immunostainings at day , was significantly lower in tubular, glomerular and interstitial cells of kidneys from terc ko g mice. we show that late-generation terc ko mice with critically short telomeres have a greater susceptibility towards acute injury and develop more chronic renal damage. this is likely due to the reduced capacity of these kidney to proliferate and thereby to regenerate. our data strongly suggest a pathogenetic role of telomere shortening, an important senescence mechanism, for the development of long-term graft failure. results: belatacept treatment had no effect on the number of peripheral blood treg cells and t cell suppression assays. the percentage of foxp cells was significantly elevated in rejecting kidney allografts in belatacept-treated patients compared to cnitreated patients. conclusions: following chronic belatacept therapy, the number and function of peripheral blood treg cells are maintained in kidney transplant patients. belatacept enhances the treg population in the allograft in patients with acute rejection. therefore, our data suggest that co-stimulation blockade with belatacept does not affect treg homeostasis. the increased number of treg cells in rejecting allografts in belatacepttreated patients may provide a novel mechanism whereby belatacept can mitigate the severity of acute rejection and improve graft survival. the steady-state auc of n-desmethyl-aeb was minor in comparison to aeb and similar between the patient groups: ± ng.h/ml in transplantation vs ± ng.h/ml in psoriasis (p= . ). demographic covariates: in the first week posttransplant with patients receiving fixed-dose aeb , intersubject variability for c was % and for auc was %. aucs were similar in men vs women ( ± vs ± , p= . ). age, which ranged from - years, did not influence auc based on regression analysis (r = . , p= . ). there was a borderline-significant negative correlation between weight (range, - kg) and auc (p = . ); however, its clinical relevance was low in that it could explain < % of the variability in auc (r = . ). there was a significant positive correlation between aeb c and auc (r = . , p< . ). conclusions: ( ) in the first week posttransplant, patients achieved aeb blood levels anticipated for this regimen. ( ) there was notable intersubject pharmacokinetic variability at this time but it was not attributable to standard demographic factors such as sex, age, or weight. ( ) a good correlation was noted between c and auc suggesting that c might serve as a marker for total drug exposure. a we studied which is protocol would be best after rapid discontinuation of p. between / and / , st and nd kidney tx recips were randomized: csa-mmf (n= ) vs. high tac-low srl (n= ) vs. low tac-hi srl (n= ) (for tac and srl levels, high = - , low = - ). all received thymoglobulin (tmg) doses, and p for days; tmg was continued in dgf. min f/u = yr; mean = ± mos. there was no diff between groups in recip age, gender, ethnicity, prim dis, donor source, % retx, pra; or in donor age, gender, ethnicity. there was no signif diff. between groups (intention to treat) in actuarial patient, graft, death-censored (dc) graft, acute rejection (ar), or biopsy-proven chronic rejection rates (cr), serum cr level or calculated (mdrd) gfr or in studied side effects. cr(sd) . (. ); . (. ); . ( . ); . ( ); . (. ) . (. ); . ( ); ( . ); . ( . ); . (. ) . (. ); . (. ); . (. ); . (. ); . (. ) mdrd gfr(sd) ( ), ( ), ( ), ( ), ( ) ( ), ( ), ( ), ( ), ( ) ( ) the most common cause of graft loss was death with function (csa %, high tac %, low tac %). the majority of recips in each group remained p-free (csa %, high tac %, low tac %) but a large % were not on the medications which they were randomized to (csa %, high tac %, low tac %). we found a signif ↑ of new onset diabetes (nodm) (p=. ) in the tac-srl groups: csa %, high tac %, low tac %. there was a trend towards more use of lipd lowering medications in the tac/srl groups. in summary, all is protocols were effective; with min f/u yr for each group, patient and graft survival and ar rates are similar. we found an increased nodm and possibly hyperlipidemia in the tac/srl groups. purpose: we present preliminary -year results from a worldwide trial comparing srl regimens with tac+mmf. methods: renal transplant recipients (n= ) were randomly assigned to the following treatment regimens: group : srl ( - ng/ml, then - ng/ml after week ) + tac ( - ng/ml) with elimination at weeks (n= ); group : srl ( - ng/ ml through week , - ng/ml thereafter) + mmf (up to gm/day) (n= ); or group : tac ( - ng/ml through week , - ng/ml thereafter) + mmf (up to gm/day) (n= ). all patients received corticosteroids and daclizumab. in june , group was terminated (after all patients were accrued) because of increased acute rejection (ar) rates. results: demographic characteristics were similar between groups except for more females in group . patient and graft survival were also similar among groups (see table) . biopsy-confirmed ar (bcar) was significantly greater in group compared with groups and , p< . , and most occurred in the first months posttransplant with preponderance during the first months. subtherapeutic srl trough concentrations were reported in a large number of rejectors in group . most of the rejections in group occurred within the first months before tac was eliminated. all ars were mild to moderate; grade ii ars were proportionally greater in group . mean nankivell gfr was numerically higher in group . preliminary results at years show excellent patient and graft survival and similar renal function among treatment groups, despite higher ar rates in group . early adequate exposure to sirolimus is mandatory to achieve desired (low bcar rates) results. the goal of rapid discontinuation of prednisone (rdp) after kidney transplantation is to minimize prednisone-related side effects without increasing acute rejection (ar) rates or decreasing long-term graft survival. to date, studies have shown that rdp is associated with decreased prednisone (p)-related sided effects, and randomized trials of rdp (vs. long-term p) have shown little or no ↑ in ar rates. however, concern remains that long-term graft survival will be worse with rdp protocols. we studied t½ (the time it takes for ½ of the grafts surviving at year to subsequently fail) for st tx recipients treated with rpd ( - ) (n = ) (antibody, cni, antimetabolite [mmf or srl] , and rdp) vs. st tx historical controls ( ) ( ) ( ) ( ) ( ) ( ) (n = ) treated with a protocol of antibody, cni, antimetabolite [mmf] , and long-term p. table shows characteristiscs of the groups. pra; rdp were more likely to get a living unrelated donor transplant. t ½ is shown separately for living (ld) and deceased (dd) donor transplants in table . there was no significant difference in t½ between groups. we conclude that, compared to historical controls, rapid discontinuation of prednisone can be done without any detrimental impact on long-term outcome. a prospective, randomized study to confirm this observation is necessary. successful a) . we enrolled patients (table ) with an average follow-up of . + . months. subjects - years old included primary and re-transplants, with primary endpoints of renal function and can. follow-up averaged . + . months in patients withdrawn from ci ( from group , from group ). we found no increased rejection after ci discontinuation and that both ratg induction dosing regimen and ci discontinuation significantly impacted renal function and the development of can. we successfully discontinued both calcineurin inhibitors and steroids with either single or divided-dose ratg induction, and single-dose ratg induction independently associated with improved renal function and reduced can. study demographics single-dose ratg ( mg/kg x ) divided-dose ratg ( . mg/kg x ) group (n = ) group (n = ) group (n = ) group (n = ) age . ± . . in subset analysis, we also find an enhanced negative impact of srl when combined with steroids (str) in patients without dgf. conclusion: this data supports previous findings that srl may be associated with a sustained survival disadvantage apparent early post transplant, and that this effect appears exacerbated when combined with dgf or str. several potential explanations for this effect may include srl-associated prolonged early graft dysfunction, hyperlipidemia or proteinuria. however, patients initiating srl after discharge may not evidence this survival disadvantage, and this question was not addressed here.these findings may be of particular importance to centers employing combined srl and str therapy, as well as to define the best mode of support during recovery from dgf. prospective randomized studies would be necessary to evaluate these. table shows cai in both groups from to years. in slr group the doses of cin were significantly lower from one through years (p= . ). cai due to interstitial fibrosis/ tubular atrophy was significantly lower in slr group at years. our data shows that year patient and graft survival, graft function, bpar and scar were comparable between mmf and slr groups despite lower prevelance of interstitial fibrosis/tubular atrophy in slr group. registry analyses suggest that tac/mpa immunosuppression is associated with superior kidney graft survival vs. tac/srl. large single-center experience may assist in clarifying these findings, by examining outcomes related to specific utilization practice. we retrospectively examined the outcomes of consecutive first renal transplants ( % deceased donor, % living donor) at a single center, treated with tac/srl or tac/mpa. graft and patient survival, acute rejection rates, and yr egfr were analyzed by era of transplant ( - vs. - ) . changes in tac/srl utilization between eras included elimination of the srl loading dose and a reduction in tac target trough concentrations. summary. compared to tac, srl+cya was associated with a % decreased risk of skin ca that was statistically significant. although srl+cya was associated with a % decreased risk of de novo solid ca, it did not reach statistical significance. this reduced risk may not reflect the unique aspects of srl itself, but may be a reflection of practice pattern, patient selection or center effect. the backgrounds: a number of studies have observed increase of malignancies following renal transplantation. however, the incidence and the site of malignancies were quite different by the follow-up times, the era and the region. methods: we reviewed the records of renal transplant recipients in our institute between and and recorded the incidence and types of de novo malignancies. they were divided into two groups by immunosuppressive era; azathioprine (aza) era ( . - . : n= ) and calcineurin inhibitor (cni) era ( . -: n= ) . results: a total of ( in aza era and in cni era) kidney recipients out of developed malignancies. the tumors included gi-tract cancers, liver cancers, skin cancers, tongue cancers, breast cancers, renal cell carcinomas, thyroid cancers, leukemia, lymphoma, one lung cancer, one uterus cancer and one kaposi's sarcoma (ks). the average interval between transplantation and development of malignancy was ± ( - ) months. mortality was high in liver cancer ( %) and leukemia ( %). cumulative incidence of malignancies of all recipients in , , , years were . %, . %, . % and . %, respectively. graft-loss censored cumulative incidence, which was calculated to see the incidence among graft survivors under continuing immunosuppression, of all recipients in , , , years were . %, . %, . % and . %. that of , and years in cni era was . %, . % and . %, while that in aza era was . %, . % and . %, showing early higher incidence in cni era outstripped by aza era by years. site of malignancy in cni era occurring within years, which was never observed in aza era, was focused on liver, leukemia (including atl), ks and ptld. discussions:our results demonstrated that recent potent immunosuppressive regimen shortened the interval between transplantation and viral-related malignancies. however, long-term incidence of whole malignancies has been decreasing by minimizing chronic immunosuppression in our institute. the impact of transplant center practice on the association between pulsatile perfusion and delayed graft function. jagbir gill, david gjertson, suphamai bunnapradist, michael cecka. ucla, la, ca. the use of pulsatile perfusion (pp) is increasing in the us, but practice varies widely among transplant (tx) centers. we describe the variability of pp use and its impact on post transplant outcomes. methods: we identified all cadaveric kidney tx from - using optn/unos data. the cohort was stratified by tx center pp use as follows: low pp centers ( - % pp use), med pp centers ( - % pp use), and high pp centers (> % pp use). donor characteristics and the incidence of dgf were compared between and within each strata. results: pp was used by % of centers, however most centers used pp < % of the time ( . %). compared to low and med pp use centers, kidneys pumped in high pp centers were from donors that were younger, had a lower mean terminal serum creatinine, and had a lower incidence of cva and hypertension. the overall incidence of dgf was lowest in the high pp centers ( . %), compared to the med ( . %) and low pp ( . %) centers. the rates of dgf within each strata (high, med, and low pp centers) for tx performed using pp versus cold storage (cs) are outlined in the table below . within each strata, the rate of dgf did not differ between tx performed with and without pp. in ecd transplants, pp was associated with lower rates of dgf across all center groups. however, the impact of pp on scd and dcd transplants was less significant and varied across centers by pp use. hla sensitized patients ( - %) in our dsa are now transplanted at a rate of %, which is significantly higher (p = . ) than the % rate when ua weren't entered into unet. furthermore, of kidneys imported into our dsa and allocated to the dsawide renal candidate list (since ua entry started), % ( / ) were transplanted into sensitized candidates ( % to %), each of whom had a negative flow or ahg t cell igg crossmatch. conclusion. the higher transplantation rate for hla sensitized patients in our dsa shows that virtual a, b, & c crossmatching yields a dsa-wide ranked list of sensitized candidates likely to have a negative final class i (t cell) crossmatch and be transplanted. the data also lend support to the notion that sharing kidneys across dsa boundaries for hla sensitized candidates, based on a negative virtual hla class i crossmatch, has merit. predicting introduction: predicting graft outcome after renal transplantation based on donor histological features has remained elusive and is subject to institutional variability. we propose a pre-transplant donor path scoring system that reliably predicts graft outcome regardless of recipient ® characteristics. methods: we retrospective analyzed imported cadaveric renal transplants which were initially rejected by other centers due to donor parameters between / - / . all kidneys were re-biopsied at our center prior to implantation. morphometric analysis performed consisted of measuring glomerular-size arterioles, interlobular, and arcuate/ interlobar arteries and wall to lumen ratio (wlr) was calculated; calculating % glomerulosclerosis (gs); the presence of arteriolar hyalinosis (ah), scar, periglomerular fibrosis (pfg), and acute tubular necrosis in the biopsies. the patients were followed for a mean of months. multivariate cox analysis was done to evaluate the predictive value of these path variables to graft outcome.results: ah, gs> %, wlr> . , pgf, and scar were found to independently predict graft outcome. the unos board of directors has approved the change from using panel reactive antibodies (pra) to calculated pra (cpra). the cpra is a formulated pra based upon the frequency of the specificities of hla antigens found in the donor pool and is expected to standardize the degree of patient sensitization. donor organs expressing unacceptable antigens will not be offered to a recipient with donor (hla) antigen specific antibodies (dsa). highly sensitive, single antigen bead and solid phase assays (flow pra and luminex) are used to identify these hla antibodies (abs). each transplant center can determine the criteria used for identifying an unacceptable antigen, for example, based upon dsa titer or the fluorescence intensity (fi) coming from the donor-specific single antigen bead. it is unclear whether abs identified by these techniques are clinically relevant for organ allocation. we retrospectively evaluated flow-pra, flow cytometry crossmatching (fcxm), hla ab specificities and titers of pre-transplant (tx) sera from transplant recipients of deceased renal allograft donors transplanted following a negative cytotoxic-anti-human globulin crossmatch. the two year graft survival of % for the recipients ( / , %) with low-titer (≤ : ) donor specific hla ab and a negative (-) fcxm was significantly better when compared to the % two year graft survival of the % ( / ) of recipients presenting with (+) dsa but a (+) fcxm (p< . ). recipients ( / , %) with non-donor abstracts specific hla abs (high or low titer) and a (-) fcxm also experienced a better two year graft survival of % compared to the % for the other % of recipients with (+) fcxms (p < . ). these data suggest that in the presence of donor-specific or non-donor-specific hla abs you can not predict the crossmatch outcome without actually performing the crossmatch which will then influence donor organ allocation and graft survival outcome. in the face of low-titer dsa and a (-) fcxm recipients experienced excellent graft outcome when compared to recipients with (+) fcxms. therefore, donor organ allocation based on cpra (ab specificity and unacceptable ags) utilizing highly sensitive single antigen bead and solid phase luminex assays may disadvantage recipients (no donor crossmatch) who could otherwise be successfully transplanted. aim: to assess vegfr expression in hcc and the adjacent benign cirrhotic parenchyma and its correlation with tumor differentiation, vascular invasion, and tumor morphologic parameters. background: hcc is a highly vascular tumor in which angiogenesis is mediated in part by vegf. vegf is highly expressed in hcc and mediates its effects through multiple receptors including vegfr . the tyrosine kinase inhibitor sorafenib inactivates the vegfr receptor and exhibits anti-tumor effects in hcc. clinical significance of vegfr expression with respect to tumor parameters has not been evaluated. patients and methods: immunohistochemical staining for vegfr was performed in hcc and corresponding adjacent cirrhotic liver from patients undergoing liver transplant. patients had hcc within mc. stains were scored by estimating the % of positive surface area in veins, arteries, and sinusoidal lining cells. data are presented as median [p , p ]; wilcoxon signed rank and wilcoxon rank sum tests were used. results: vegfr levels in hcc were significantly correlated to levels in adjacent non-tumorous liver. higher levels of vegfr in non-tumorous liver were associated with higher levels in hcc from the same patient. vegfr levels were significantly higher in hcc compared to adjacent areas (p< . ). vegfr levels in hcc were not significantly different between patients who fell within mc and those beyond mc. however, vegfr levels were significantly higher in the adjacent arteries of nontumorous liver ( . [ , ] vs. [ , ]; p= . ) in those patients with hcc beyond mc. subjects with moderate or poor differentiation had significantly higher levels of vegfr in sinusoids and veins of hcc and in the sinusoids of adjacent non-tumorous liver. there was no correlation with vascular invasion. conclusions: elevated vegfr in hcc correlates with elevated vegfr in adjacent cirrhosis, suggesting that high expressing hcc arise in a high vegfr expression, pro-angiogenic environment. moreover, higher vegfr expression in background cirrhosis correlates with advanced hcc (beyond mc), suggesting that anti-angiogenic agents may prevent tumor formation or progression in cirrhotic patients. this novel concept warrants further study. . we further attempted to find a subgroup of patients combining tumor size, number of nodules and various levels of afp which together would correlate strongly with pdiff tumors. however, the distribution was erratic and even in extreme outliers (> nodules, > cm in maximum size, with or without high afp), where transplantation is currently not indicated according to any current expanded tumor inclusion criteria, the incidence of pdiff did not exceed %. conclusions: pdiff is most highly associated with tumors that exceed the milan criteria and the expanded criteria currently used for liver transplantation. thus, tumor biopsy would not appear to be of benefit except perhaps in those patients with a high afp level. however, if tumor criteria are further extended in the future, tumor biopsy may be justified in those with higher tumor burdens. < cm) ), , ( . %) at ls=t ( tumor < cm or tumors ≤ cm), and ( . %) with ls> . results: overall survival at months was . %, with significant differences seen for listing stage (ls = . %, ls = . % ls> = . %, p= . ) and by histologic stage as determined by pathology (p<. ). survival for patients with histologic stage b hcc was only % at months, versus % for stage . patients with tumors greater than cm both by listing stage and by pathology fared poorly compared to those with smaller tumors (p= . . ). patients listed who received ablation treatment (at) preoperatively and who had their tumors "down-staged" had better results (p= . ). we observed no difference in at types (tace vs rfa). those with micro or macrovascular invasion had lower survival rates, at . % and . %, respectively (p <. ). afp > continues to be a significant predictor of lower post-transplant survival, with a survival rate of . % at -months (p<. ). conclusions: we conclude that listing and histologic tumor size, presence of micro/ macrovascular invasion, and high afp are associated with poorer lt results. at is emerging as potentially effective treatment for improving lt outcomes for hcc recipients. introduction: so far, milan criteria are used to select patients with hepatocellular carcinoma (hcc) for liver transplantation (lt). herein we compare prognostic markers in patients with pretreatment by transarterial chemoembolization (tace) to a second cohort transplanted without tace pretreatment. patients and methods: between september and october , patients with hcc underwent lt at our institution. eighty-two patients were pretreated by repeatedly performed tace whereas in patients none or other forms of pretreatment had been used (non-tace group). tace was performed using lipiodol and mitomycin. every weeks tace was repeated until transplantation. tumor response was assessed by ct scans ( -week intervals). results: sixty-seven percent of the patients transplanted after tace pretreatment exceeded the milan criteria compared to % in non-tace patients. the proportion of recurrence-free patients was comparable in the tace and non-tace group ( . % and . %, respectively). in the univariate analysis grading, angioinvasion and progress-free tace were significant predictors for recurrence after lt in patients with tace pretreatment. progress-free tace was the only significant predictor of recurrence in the multivariate analysis. in the non-tace group t classification, number of nodules, grading, angioinvasion, milan criteria and underlying disease (hcv versus all other diseases) were significant. after tace pretreatment freedom from recurrence was . % in patients with stable disease or regress but only . % in patients with progress during tace. conclusions: tace pretreatment is capable of selecting a biological entity of tumors which differs significantly from untreated tumors. this statement is deduced from the remarkable differences of predictors for tumor recurrence in patients with and without tace pretreatment. moreover, tace patients can be separated into two groups: those who experienced tumor progress during repeatedly performed tace and those who did not. stable disease during the continued tace before transplantation resulted in remarkably low tumor recurrence. liver with a median follow-up of months, there was no statistical difference in the year overall survival in the m ( %) and m+ ( %) groups (p= . ). the -year disease free survival was significantly higher in the m ( %) vs. m+ ( %) groups (p= . ). when stratifying for ucsf criteria tumors, the -year disease free survival was milan ( %) vs. ucsf ( %) vs. beyond ucsf ( %) groups (p= . ). univariate analysis demonstrated the following factors to be associated with disease recurrence: intermediate waiting time - months (p= . ), preoperative tace (p= . ) or resection (p= . ), more than tumors (p= . ), and max tumor size over cm (p= . ). multivariate analysis controlling for age, gender, and waiting time demonstrated that preoperative resection hr . ( %ci . - . ), more than tumors hr . ( %ci . - . ) and max tumor size greater than cm hr . ( %ci . - . ) were independently associated with disease recurrence. conclusions: the overall survival after liver transplantation is excellent in the m and m+ groups, far exceeding survival rates that can be obtained via any other modality. the current unos hcc algorithm should be reconsidered, to allow extra listing points for selected patients with hcc that exceed both the milan and ucsf criteria. postoperative use of intense insulin therapy in liver transplant recipients. lama m. hsaiky, iman e. bajjoka, dhaval patel, marwan s. abouljoud. transplant institute, henry ford hospital, detroit, mi. hyperglycemia and insulin resistance are common post liver transplant, even in patients with no history of diabetes. in the general surgical intensive care unit (sicu) population, the use of intensive control of blood glucose has recently been shown to reduce both morbidity and mortality. thus far, limited data exist in the liver transplant population. purpose: assess the impact of intensive insulin therapy to maintain blood glucose at or below mg/dl immediately post liver transplantation in the surgical intensive care unit (sicu). methods: a retrospective evaluation of liver transplant recipients who received two different insulin protocols in the sicu was performed. prior to january , patients were assigned to receive sliding scale insulin therapy (ssit) to maintain blood glucose (bg) less than mg/dl. the intensive insulin therapy (iit) was implemented in august with a goal bg level between - mg/dl. the following data was analyzed: bg ranges, need for mechanical ventilation, blood transfusions, infection rate in the sicu, rejection episodes and patient survival. results: a total of liver transplant patients were evaluated; of which patients were in the ssit group and the other in the iit group. demographic characteristics were comparable between the two groups. in the iit, % of the bg readings were maintained at < mg/dl versus % in the ssit. the incidence of hypoglycemia (bg < mg/dl) was less than % in both groups. the need for mechanical ventilation was . days vs. . days and the overall number of blood transfusion was on an average of . vs. units (p< . ) in the ssit vs. iit group, respectively. iit also reduced overall sicu infection rate by % (p= . ). the rate of acute cellular rejection at months post transplant was less in the iit, % vs. % in the ssit group (p= . ). moreover, mortality during hospital stay was reduced from % in the ssit to % in the iit group. conclusions: the use of intense insulin therapy immediately post liver transplantation has resulted in reducing infection rate and rejection episodes and a trend for reduced morbidity and mortality among post surgical liver transplant recipients without the adverse effects of hypoglycemia. medical epidemiology of patients surviving ten years after liver transplantation. kerri a. simo, stephanie e. sereika, david a. gerber. abdominal transplantation division, department of general surgery, university of north carolina, chapel hill, nc. background: as the population of long term survivors of liver transplantation (olt) grows, their medical epidemiology has become increasingly important. the goals of this study were to define a collective profile of liver transplant recipients ≥ years post olt and to compare their co-morbidities with those of the general population. in , the national health survey reported that % of the us population had hypertension, . % had diabetes/impaired fasting glucose, and . % had chronic kidney disease. methods: a retrospective review of a prospectively collected database of adult patients who underwent olt at a single transplant center from september , to october , was performed. inclusion criteria consisted of survival ≥ years post olt with > year follow up. results: seventy-one patients met inclusion criteria. ninety percent of patients had ≥ years follow up. the mean age at transplant was (range - ). the mean calculated meld score was (range - , median= ). indications for olt were hcv( %), alcohol( %), cryptogenic( %), autoimmune( %), hbv( %), psc( %), pbc( %), and other( %). seven patients required retransplant during the first years. an additional patients underwent other operations: arterial or biliary revisions, hernia repairs and non-transplant related ( abdominal). during analysis, the following medical co-morbidities were found: patients( %) had hypertension ( new onset), ( %) diabetes ( new onset), ( %) renal insufficiency and renal failure ( new onset), ( %) cardiovascular disease (all new onset). nine patients ( %) were diagnosed with de novo cancer. medications for chronic health problems included patients on diabetic medications, on antihypertensives and on lipid lowering agents. initial immunosuppression consisted of % on steroids, % on cyclosporine, % on tacrolimus, % on mycophenolate mofetil (mmf) and % on azathioprine. immunosuppression at years consisted of % on cyclosporine, % on tacrolimus, % on steroids, % on mmf, % on sirolimus, % on mycophenolate sodium, % on azathioprine. no patients were on triple therapy, were on dual therapy, and were on monotherapy. summary: patients alive years post olt have a significantly higher incidence of hypertension, diabetes, and renal disease than the general population. this study supports conscientious medical follow up to ensure continued meaningful survival. liver transplantation in the morbidly obese (bmi> ). c. quintini, l. kauzman, k. hashimoto, p. ding, t. doago uso', n. sopko, j. rosenblum, f. aucejo, c. winans, d. kelly, b. eghtesad, d. vogt, j. j. fung, c. miller. general surgery -liver transplant service, cleveland clinic oh. morbid obesity (mo) is a problem seen with increasing frequency among candidates for olt. mo is considered a contraindication for olt in some centers without clear evidence to support such a practice. our aim is to describe outcomes for olt in patients with a bmi> kg/m in a single center. methods. between / and / , olts were performed in patients. / patients with a bmi> were compared to all other olt patients with a bmi< . we analyzed patient and graft survival, operative time, blood transfusion requirements, and post operative events (icu and overall length of stay los, surgical complications and infections). we also analyzed the post transplant weight records of our study group at - and months. results. results are summarized in table . outcomes of olt in the morbidly obese are no worse than those of other patients undergoing olt. bmi is often artificially elevated in end-stage cirrhotic due to severe fluid retention. these patients exhibit rapid weight loss following transplantation that is likely due to extra-cellular fluid loss from the improved homeostatic milieu provided by the new liver and possible improvement in renal function. the presence of obstructive cad was not associated with increased peri-operative morbidity or mortality. these patients experienced similar patient and graft survival irrespective of the degree of cad. significant unmodified cad should not represent an absolute contraindication to liver transplantation. olt was also examined. we found that using a trj of . m/s as a cut-off created two age-matched groups with significantly different survival curves at one year (p = . ) with a relative risk of mortality of . for trj ≥ . m/s. this study underscores the importance of screening tte for the presence of pph in the evaluation of patients for olt, suggesting that clinically significant pph may be present at lower trj than typically prompt right heart catheterization. some of the limitations of the study are the variability of the time from pre-olt echo to transplant, and the assumption of rap = without assessment of ivc size. we are using these data as a framework for further prospective trials to better assess the clinical applications of the findings. hyperlipidemia has been shown to predict faster chronic kidney disease (ckd) progression over the long term in lung allograft recipients. it is unknown whether disordered lipid metabolism may also aggravate the early loss of renal function often seen in this patient population in the immediate post-operative period. we studied lung allograft recipients transplanted between january and december . pertinent demographic and clinical variables were recorded at baseline and one month post-transplant, including creatinine levels and fasting lipid panels. logistic regression models were created to investigate an independent association between lipid levels and change in renal function by one month post-transplant. mean +/-sd baseline creatinine was . +/- . mg/dl and low density lipoprotein (ldl) was +/- mg/dl, the latter remaining unchanged at one month. in contrast, by one month post-transplant the mean creatinine level of survivors increased significantly to . +/- . mg/dl (p < . ), with a overall mean increase of % above baseline. the highest quartile of patients that fared the worst experienced a rise in creatinine > % above baseline. on univariate analysis, there was a strong trend toward those with one month ldl values in the highest quartile (i.e., > mg/dl) having an increased odds of experiencing a rise in creatinine by one month > % above baseline (or . , p= . ). after controlling for age, gender, pre-transplant creatinine, bmi, and the presence of diabetes prior to transplant an ldl value in the highest quartile by one month post-transplant was the only variable independently predictive of a rise in creatinine > % above baseline at one month (or . , p= . ). in summary, hyperlipidemia occurring early post-lung transplant predicts faster loss of renal function soon after surgery. though speculative, given the known beneficial effects of lipid lowering agents such as statins on the rate of ckd progression, perhaps timely initiation of these medications after surgery may also attenuate the early decline in renal function often observed in this patient population. the risk for acute cellular rejection (acr) following lung transplantation (ltx) is still a problem despite heavy multidrug immunosuppression. induction therapy with potent t-cell depleting agents have facilitated the implementation of minimal post-transplant immunosuppression.the impact of this protocol on the activation of proinflammatory cytokines and effector molecules that affect the cellular rejection process is not well determined. in this study, we evaluated the relationship between upregulation of t-cell and macrophage-dependent inflammatory cytokines detected by molecular methods and the clinical status of ltx patients. we studied ltx patients who received anti-lymphocytic induction therapy(thymoglobulin n= or campath- h n= ) followed by maintenance immunosuppression with tacrolimus and low-dose steroids. we analyzed bronchoalveolar lavage (bal) mrna samples ( - per ltx patient) by real-time pcr for gzmb, ifn-γ, il- , mcp- , rantes, tnf-α, and gapdh (control). the abi prism sds and fast real-time pcr systems were used and data were analyzed by the dct and -ddct methods. we determined the relationship of categorical outcomes (rejection-no rejection) with continuous variables (number of cycles) by anova. early acr that occurred within the first days post-ltx was associated with an increase (> - fold) of macrophage-specific mrna (mcp- , rantes, tnf-α). / thymo-treated ltx patients experienced early acr while only / campath- h-treated patients had early acr (p< . ). in contrast, late acr that occurred greater than days post-ltx was associated with a significant upregulation ( - fold) of t-cell dependent mrna (gzmb and ifn-γ) in addition to increases of rantes and mcp- mrna. overall, ltx patients who experienced early or late acr (n= ) exhibited higher mrna levels for the above mediators compared to stable patients (n= ). our data indicated that in early post-t-cell depletion, the acr phenotype was characterized mainly by macrophage activation. in contrast, greater than months post-ltx with the recovery of cd + t-cells, the acr phenotype was associated with cytotoxic t-cell activation. furthermore, sensitive molecular methods may detect the activation of pro-inflammatory mediators within the allograft prior to the diagnosis of rejection by transbronchial biopsies and may impact optimal patient management. antibody-mediated rejection (amr) is defined by the presence of donor-specific alloantibodies, markers of complement activation and the clinical phenotype of organ dysfunction. compared to renal and cardiac allografts, very few amr cases are documented in lung transplantation (ltx). methods. we report here on seventeen ltx patients exhibiting: ) donor-specific hla antibodies (dsa); ) linear, continuous subendothelial c d deposition in lung allograft; ) lung allograft dysfunction. the presence and specificity of dsa were determined by elisa and/or luminex. c d deposition was assessed by immunohistochemistry in transbronchial biopsy paraffin blocks. allograft dysfunction was considered when either biopsy-proven acute cellular rejection (acr, ≥ ishlt a ) or bronchiolitis obliterans (bos) were diagnosed. the average detection of dsa occurred in the first year post-ltx, on ± postoperative day (pod), range to days. thirteen ( %) of amr patients were females. an anamnestic humoral response was encountered in cases (one pregnancyrelated and three re-transplant patients), while de novo dsa were detected in cases ( %). the percent-reactive antibody (pra) was lower in de novo cases ( %) when compared to memory response ( %, p< . ). anti-class i dsa were found in cases, anti-class ii in cases, while patients exhibited both class i and class ii dsa. specific vascular c d deposition was detected in ( %) patients. lung allograft dysfunction was considered in ( %) cases, while three patients with dsa and specific c d deposition fulfilled the criteria of sub-clinical amr. in patients where plasma-exchange/ ivig were applied, antibody titers dropped from : to : or : in two cases; in a third case, antibody titer remained high post-pheresis ( : ) and the graft failed. conclusions: both anti-class i and anti-class ii, low pra or high pra, pre-formed or de novo dsa can be detrimental for lung allograft. the presence of donor-specific alloantibodies, vascular c d deposition and allograft dysfunction shows that amr criteria can also be met in ltx. rationale: recently, inhaled cyclosporine has been shown to reduce mortality and bos. previously, we demonstrated that apically-dosed cyclosporine poorly transmigrated differentiated human airway epithelial cells in vitro (using a transwell system). only ∼ % of the apically deposited cyclosporine passed through the epithelial layer whereas most of the drug accumulated within the epithelium. thus inhaled cyclosporine may not be capable of inactivating airway allogeneic t cells since it may not reach the airway wall in high concentration. in this study, we hypothesized that inhaled cyclosporine alters airway epithelial signaling cascades that may ultimately result in reduced inflammatory cell recruitment to the lung. methods: human tracheobronchial epithelial (htbe) cells from healthy donors were grown in ali media to confluence at an air-liquid interface in millicells. differentiated htbes were treated on their apical surface with cyclosporine concentrations of and , ng/ml or vehicle for hr to mimic the effects of inhaled cycolsporine. the basilar and apical compartments (n= - for each) were then assayed for cytokine secretion (il , il , il , il- p , tnf, eotaxin, mcp- , gm-csf, rantes and egf) by luminex assays in pg/ml. results: , ng /ml of cyclosporine markedly blunted the basilar secretion of il- ( ± vs ± , p= . ), rantes ( ± vs ± , p= . ), gm-csf ( ± vs ± , p= . ) and mcp- ( ± vs ± , p= . ). il and eotaxin were unaffected; tnfα, il β and il p were undetectable. at ng/ml cyclosporine, cytokine secretion was decreased to a lesser extent. a similar pattern of diminished cytokine secretion was seen in the apical compartment of this system. last, apical, but not basal, egf secretion was augmented by cyclosporine ( ± vs ± pg/ml, p= . ). conclusion: cyclosporine decreased the secretion of critical cytokines and chemokines from human airway epithelial cells. these mediators are known to enhance mononuclear and t cell recruitment in a large variety of animal models of disease as well as in clinical studies. inhaled cyclosporine may work by reducing cell recruitment. this hypothesis will require in vivo testing. funded by: cf foundation. background: cytomegalovirus (cmv), human herpes virus - and - (hhv- and - ) are β-herpesviruses that commonly reactivate and have been proposed to trigger acute rejection and chronic allograft injury. the role of these viruses in the development of bos after lung transplantation remains unclear. we assessed the contribution of β-herpesvirus infection in the allograft by a prospective molecular assessment of serial broncho-alveolar lavage (bal) samples in lung transplant recipients. methods: quantitative real-time pcr of bal samples were performed for cmv, hhv- and hhv- in a prospective cohort of lung transplant recipients. a time-dependent cox regression analysis was used to correlate the risk of bos and acute rejection in patients with and without β-herpesviruses infection. results: patients were included in the study over a period of years. a total of samples from bal were obtained (median per patient). / patients ( %) had at least one positive result for one of the β-herpesviruses: patients ( %) for cmv, patients ( %) for hhv- , and patients ( %) for hhv- . median time to detection was days (range - ) for cmv, days (range - ) for hhv- , and days (range - ) for hhv- . median peak viral load was , copies/ml (range - , , ) for cmv, copies/ml (range - , ) for hhv- , and copies/ml (range - , ) for hhv- . acute rejection (≥ grade ) occurred in % and bos (≥ stage ) in %. in the time dependent cox regression model, the relative risk of bos or acute rejection was not increased in patients with cmv, hhv- , or hhv- reactivation. for example, the hazard ratio of cmv and bos was . ( % ci . - . , p= . ) and for cmv and acute rejection was . ( % ci . - . , p= . ). in many of the patients, β-herpesvirus reactivation occurred after the acute rejection episode likely reflecting augmented immunosuppression. abstract# mannose binding lectin in this large cohort of lung transplant recipients, local reactivation of cmv, hhv- and hhv- in the allograft was very common. however, despite high viral loads in many patients, infection was not significantly associated with the development of acute rejection or bos. introduction: the role of chemokine receptors in regulating donor-specific responses to allografts is poorly understood. cd + cd + t cells regulate alloreactive cd t cell responses and acute rejection of single class ii mhc-disparate cardiac allografts in c bl/ mice. ccr is expressed by a small proportion of cd + cd + t cells but the requirement for these cells in regulating alloreactive t cell responses remains poorly understood. the goal of this study was to investigate the role of ccr + t regulatory cells in acute rejection of single class ii mhc-disparate cardiac allografts. methods: wild-type c bl/ (h- b ) and b .ccr -/received heterotopically transplanted b .h- bm mice heart grafts. the presence of cd + foxp + t cells in the recipient spleen and in heart allografts was determined by flow cytometry. foxp mrna expression in the heart grafts was analyzed by qrt-pcr. donor-specific cd + t cells producing ifn-g or il- in allograft recipient spleens were enumerated by elispot assay. cell sorted naïve wild-type cd + cd + t cells and cd + cd -t cells were adoptively transferred to wild-type and ccr -/mice before the cardiac transplant. results: in wild-type recipients > % b .h- bm cardiac grafts survived more than days whereas ccr -/recipients rejected the allografts within days ( days mean survival) with intense cd + t cell infiltration in the graft. donor-reactive ifn-g and il- producing cd + t cell numbers were increased -fold in the spleens of ccr -/vs. wild-type recipients at day post-transplant and in contrast to wild-type recipients these numbers were sustained for at least more days. allograft infiltrating cd + cd + foxp + cells and intra-graft foxp mrna expression were clearly present in allografts from wild-type recipients and were virtually absent in allografts from ccr -/recipients. transfer of purified wild-type cd + cd + t cells to ccr -deficient mice resulted in the long-term survival of % of b .h- bm cardiac allografts. conclusion: ccr + regulatory t cells control the magnitude and function of the alloreactive t cell immune response to single class ii mhc-disparate cardiac allografts. profile that were distinct from those of cd +cd + tregs, naïve cd +cd -t-cells, and activated cd + t-cells. furthermore, the cd + converted dn t-cells were highly potent in suppressing antigen specific alloimmune responses in vitro. in this study, we further characterized and test the functional potential of the converted dn t-cells in vivo. we showed that the converted dn t-cells retained a stable phenotype after re-stimulation in vitro and in vivo. il- was capable of breaking the anergic status and reserving the suppressive function of dn t-cells. in an immunocompetent mhc completely mismatched islet transplant model, the transfer of x dn t-cells (converted from cd +cd -t-cells of naïve c bl/ mice by co-culture with mature dba/ dc plus ril- in mlr for days) resulted in a statistically significant prolongation of alloantigen specific dba/ strain, but not third party c h strain, islet allograft survival in c bl/ recipients in comparison with that of untreated control group. as il- was capable of breaking the anergic status and reserving the suppressive function of dn t-cells, we added il- /fc, a long-lasting form of il- , and low dose rapamycin with dn t-cells in a mhc mismatched skin allograft model. the single transfer of x dn t-cells plus days il- /fc and low dose rapamycin treatment significantly prolong dba/ skin allograft survival in c bl/ recipients in comparison with untreated group (mst days vs. days, p= . ) and il- /fc plus rapamycin treated group mst ( days vs. days, p= . ). the results of using ex vivo cd + t-cells converted dn t-cells in skin and islet transplantation models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the prevention of allograft rejection. jessamyn bagley, jonathan g. godwin, joren madsen, john iacomini. introduction: it has been suggested that natural killer (nk) cells are critical mediators that connect the innate and adaptive immune response. cytokine production by nk cells contributes to the polarization of immune responses to t helper , and nk cells express co-stimulatory molecules that may affect t cell proliferation. recent work has shown that nk cells are involved in the chronic rejection of parental cardiac grafts by f recipients. we hypothesized that given the role of nk cells in t cell activation and proliferation, nk cells may play a role in the development and function of t regulatory cells (treg) which control alloreactive responses. methods: nk, cd + t cells and cd +cd + treg were purified from the spleens of c bl/ j mice using macs bead separation followed by fluorescence activated cell sorting. naïve cd +cd -t cells from c bl/ j mice (h- b) were placed in culture with tgf-beta in the presence or absence of nk cells, and the development of treg was monitored by assessing foxp expression by intracellular cytokine staining and flow cytometry. in addition, the effect of nk cells on the function of treg was measured by elispot assay. results: following days of culture with tgf-β and anti-cd antibody, cd +cd -t cells acquire foxp expression. the addition of activated nk cells to cultures with cd +cd -t cells and tgf-β prevented the acquisition of foxp expression by cd cells. tregs induced by stimulation of cd +cd -t cells with anti-cd in the presence of tgf-β are capable of suppressing the production of il- , ifn-γ and il- by cd t cells in response to fully allogeneic balb/c stimulators. however, in the presence of activated nk cells, induced tregs fail to function, and production of il- , ifn-γ and il- by cd t cells is restored. these experiments were repeated with natural cd +cd + tregs isolated directly from mice without induction, and found that the ability of natural treg to suppress a cd t cell response to alloantigen was impaired in the presence of activated nk cells. conclusions: the presence of activated nk cells in culture can prevent the development of induced treg. furthermore, the presence of activated nk cells interferes with the function of mature treg in culture and allows a productive cytokine response by cd effector cells in response to alloantigen. results: both the syngeneic b cells and allogeneic dba/ cells survived nicely in the rag-/-il- rg-/-mice. however, the allogeneic dba/ cells, but not the syngeneic b cells, were readily killed by the nk cells in the rag-/-mice. however, both rag-/-and rag-/-il- rg-/-mice accepted the dba/ skin allograft long term without any sign of rejection (> days). thus, nk cells by themselves, though cytolytic to dba/ cells, fail to reject the dba/ skin allografts. to test the hypothesis that the activation status of nk cells may dictate their alloreactive potential, we treated the rag-/-mice with il- /il- ra complex to maximally stimulate the nk cells in vivo. we found that il- is remarkably potent in stimulating nk cells in vivo; and nk cells stimulated by il- express an activated phenotype and are surprisingly potent in mediating acute skin allograft rejection in the absence of any adaptive immune cells, as il- treated rag-/-, but not the rag-/-il- rg-/-mice, readily rejected the dba/ skin allografts (mst= days). nk cell-mediated graft rejection doesn't show features of memory responses. and suggests that the fate of the allografts may depend on the activation status of nk cells and the availability of nk stimulating cytokines. background: t-bet is a transcription factor that promotes th development. both t-bet and the cytokines ifng and il- have been implicated as negative regulators of th . in contrast, il- promotes th development. il- production is associated with granulocytic pathologies in several disease states. hence, this study assessed the relationship between t-bet, ifng, il- and il- in th induction and granulocytic infiltration of cardiac allografts. (ifng-/-) mice were transplanted with balb/c cardiac allografts. recipients were left untreated, depleted of cd + or cd + cells, treated with anti-cd l mab, and/or treated with neutralizing anti-il- or anti-il- mab. graft histology was assessed and primed donor-reactive th responses were quantified by elispot. intragraft expression of il- and the th transcription factor rorgt were quantified by real-time rt-pcr. results: wt and t-bet-/-mice rejected their allografts at a similar tempo but with distinct pathologies: allografts in t-bet-/-recipients were heavily infiltrated with granulocytes while graft infiltrating cells in wt recipients were primarily mononuclear. while th and th responses were readily detectable in t-bet-/-recipients, th dominated the response in wt mice and th were not detectable. depletion of cd + cells prolonged graft survival in wt, but not in t-bet-/-recipients suggesting that cd + cells mediated rejection independent of cd + help in t-bet-/-mice. cd + cells were the source of il- in t-bet-/-recipients. anti-cd l therapy promoted long-term allograft survival in wt recipients, but not t-bet-/-mice unless cd + cells were depleted. additionally, anti-cd l therapy inhibited th responses in both wt and t-bet-/-recipients, but not the cd + th response in t-bet-/-mice. eliminating ifng and il- failed to induce il- production, while neutralizing il- reduced the th response in t-bet-/-mice. conclusions: while cd + th have been described in detail, cd + th have received less attention. in t-bet-/-allograft recipients, cd + th emerge independent of cd + help. cd + th are resistant to anti-cd l therapy and are associated with granulocyte infiltration of the graft. these data implicate t-bet, as opposed to ifng and il- , as a negative regulator of the th response while il- is required for cd + th induction. nan zhang, bernd schroppel, girdari lal, jordi c. ochando, jonathan s. bromberg. background: cd + cd + foxp + regulatory t cells (treg) are important in suppressing immunity to prolong allograft survival. treg migration and its effects on suppressive activity are poorly understood. we determined treg migration patterns and effector function in an islet allograft model. ccr -/-, ccr -/-, ccr -/-, ccr -/-, l-selectin (cd l) -/-, and fucosyltransferase (fuct) iv-vii -/-c bl/ mice were used to generate treg. treg were transferred intravenously, or locally to islet allografts, following islet transplantation (balb/c into c bl/ ). transferred treg were labeled with red dye pkh and migration to islet grafts, draining lns (dln), peripheral lns and spleen were tracked with fluorescence microscopy and flow cytometry. islet allograft survival was determined by measurement of blood glucose. results: treg expressed p-selectin ligand, cd l, and a panel of chemokine receptors similar to other t cell subsets. intravenously transferred wild type treg migrated to both islet grafts and dln, and prolonged allograft survival. cd l -/or ccr -/-treg, which migrated to islets but not dlns, prolonged allograft survival as potently as wild type treg. fuct iv-vii -/-treg, which lack e-selectin and p-selectin ligands, migrated to dln, but not islets, and did not prolong graft survival. similarly, ccr -/-, ccr -/-, and ccr -/-treg migrated to dln, but not islets, and did not prolong graft survival. when locally transferred to the islet graft, treg also migrated from the allograft to the dln, and prolonged graft survival even longer than after intravenous transfer. locally transferred ccr -/-, ccr -/-, or ccr -/-treg were not able to migrate from the islet to the dln, and were impaired in their ability to prolong islet survival. conclusion: treg migration to allografts is essential for their suppressive function; migration to lymphoid tissues alone is not sufficient to prolong graft survival. treg migration from the islet allografts to the dlns, via afferent lymphatics, is also required for optimal suppressive function and graft survival. the sequential migration from the site of inflammation and then to dlns is necessary for treg to execute fully their suppressive program. these results demonstrate a novel and important aspect of migration in treg suppression and tolerance. abstracts failure is unknown. methods: patients with iothalamate gfr < ml/min (n= ) or on dialysis (n= ) at the time of liver transplant evaluation had undergone a percutanous ct guided renal biopsy. prior to the biopsy an inr ≤ . and platelet count ≥ , / ml were achieved in the majority of cases. all patients were monitored overnight for complications. candidates were listed for slk if pathology showed ≥ % glomerulosclerosis (gs) or ≥ % interstitial fibrosis (if). results: patients were eligible for slk and for lta.creatinine was higher in slk candidates but not clinically different. background -it is well known that delayed graft function (dgf) is costly for those who received a renal transplant. however, the true cost of dgf is unknown. methods -we estimated the cost of dgf for adult cadaveric renal recipients in the usrds - who had medicare as their primary payer. those included were restricted to single organ recipients as well as those who had no previous transplants. cost was defined as the accumulated average cost per day for everyone with a functioning graft on that day. we examined the total cost of dgf, cost associated with dialysis, and non-dialysis cost for all patients combined and separately by donor type; standard criteria donors (scd), expanded criteria donors (ecd), and non-heart beating donors (dcd) as well as time to graft failure or no graft failure. background: based on adverse outcomes during the first year post-transplant, the optn membership and professional standards committee (mpsc) peer-review process flags transplant programs for further review using one of two methods. programs performing at least transplants during at . year cohort are flagged based on the comparison of observed to expected event counts (death or graft failure) and the corresponding p-value (< . ). programs performing or fewer transplants are flagged if they have any adverse events. this leads to different flagging rates for programs of different sizes. the p-value has low sensitivity to identify poorly performing programs with a "moderate" number of transplants ( to ) whereas flagging every event results in a high false positive rate for "small" centers with < transplants. during the july review, % of "small" centers and % of centers with + transplants were flagged for review (all organs). the scientific registry of transplant recipients (srtr) has developed alternative approaches for flagging centers with more consistent flagging rates. methods: the new method would allow for different choices of sensitivity (rr) and specificity (p-value) for different purposes and would flag program if either {p-value < . (a different value could be chosen)} or {observed / expected > rr}. the resulting false negative rate is less than % for centers with the given rr. this approach avoids an arbitrary definition of small versus large programs, and has sensitivity (or power) > % to flag programs with the selected rr, regardless of size. results: the following discussion: this approach gives a more balanced distribution of flagging across programs of different sizes and has sensitivity > % for transplant programs of all sizes. with the choice of rr= . , the overall number of centers flagged would be nearly unchanged from current methods. center performance ratings are of increasing importance to the transplant community with the introduction of the cms final rule. ongoing debate exists regarding how much center ratings are directly a reflection of quality of care or whether ratings can be substantially influenced by exogenous factors. the study examined data from the national srtr database from - . centers' semi-annual graft and patient survival were calculated along with a comparison with expected outcomes adjusted for covariates used by the srtr. centers meeting three criteria for poor performance were categorized within each cohort. patient characteristics at each center were compared with performance evaluations. overall, half of transplant centers met criteria for low performance in at least one of the semi-annual intervals. several center factors investigated were not significantly associated with the likelihood to meet low performance criteria including the proportion of older, obese and privately insured patients. in contrast, centers with higher levels of ecd transplants (most ecds= % vs least ecds= %), african american recipients (most aas= % vs least aas= %) and patients with low albumin level (lowest albumin = % vs highest albumin = %) were more likely to meet low performance criteria. approximately half the centers that initially met criteria for low performance no longer met criteria when excluding ecd transplants and african american recipients from the performance assessment. conclusions given the extreme implications of performance ratings for transplant centers including possible loss of funding to centers with low performance, it is critical that we recognize potential weaknesses and biases of performance ratings. our results are important towards understanding factors related to performance ratings and raising questions as to whether risk adjustment techniques are adequate for fair transplant center performance evaluations. rather than only risk adjustment, stratified evaluations may be a partial solution to remove disincentives to performing higher risk transplants. it is also important to recognize factors not associated with low performance such that centers do not unnecessarily limit access to groups based on perceived deleterious impact on ratings. model. olaf boenisch, takaya muramatsu, francesca d'addio, robert padera, hideo yagita, nader najafian. transplantation research center, brigham and woman's hospital and children's hospital, boston, ma; immunology, juntendo university of medicine, tokyo, japan. t cell immunoglobulin and mucin domain (tim)- , a molecule expressed on terminally differentiated th cells, is an important regulator of th autoimmunity and in induction of transplantation tolerance. its functions in alloimmune responses during acute and chronic rejection are unknown. tim - , a novel blocking antibody of tim- , was administered to recipients in various donor-recipient strain combinations on days ( ug), , , , and ( ug) after transplantation. the frequency of ifng-, il- -, and granzyme b-producing splenocytes was measured by elispot. these data establish the regulatory functions of tim- in alloimmune responses in solid organ transplantation models. the inhibitory actions may be secondary to modulation of effector or regulatory t cells and appear to dominate in conditions of low levels of t cell activation, due to a restricted degree of allogeneic mismatch or absence of cd costimulation. the i-r injury in transplanted kidney is a major cause of dgf, an event associated with an increased risk of acute rejection. adaptative immunity was suggested to play a role in the pathogenesis of renal i-r injury, although the influence of the th /th bias in this scenario is still debated. thus, the aim of the present study was to evaluate the features of t cell response during i-r injury at the peripheral and tissue level in renal graft recipients with dgf. the mrna levels of specific th (t-bet) and th (gata- ) transcription factors were evaluated in circulating lymphomonocyte of kidney transplant recipients with early graft function (egf) (n= ) and dgf (n= ), before (t ) and hours after transplantation (t ) by real time pcr. infiltrating lymphocytes were characterized in graft biopsies of patients with dgf (n= ) and in a control group of patients with tubular damage by acute cni toxicity (n= ) by immunohistochemistry. in addition, we evaluated the th /th bias at the renal level in a pig model of i-r injury. t-bet/gata- mrna ratio was similar in the groups of patients at t . at t the dgf group presented a significantly higher increase of t-bet/gata- ratio compared with the egf group ( ± vs ± % of t , p< . ). moving to the tissue level, dgf patients presented a number of interstitial cd + ( . ± . vs . ± . , p= . ) and cd + ( . ± . vs ± , p= . ) t cells significantly higher compared to the control group, while no significant differences were observed in cd + cells number between the two groups. also at the tissue level the ratio between t-bet + and gata- + cells was significantly higher in the dgf compared with the control group ( . ± . vs . ± . , respectively, p= . ). to confirm that these changes were due to i-r, we investigated the presence of t-bet + and gata- + cells in a pig model of i-r injury. interestingly, the ratio was significantly increased after hours of reperfusion (basal . ± . vs hours . ± . ; p= . ). in conclusion, our results suggest that kidney transplant recipients with dgf present a bias toward a th -driven immune response both at the peripheral and at the tissue level. this event, due to the i-r process, as suggested by the animal model, may represent a link between dgf and acute graft rejection. as expected a strong negative association between duration of dialysis and patient survival was seen in caucasian recipients. in contrast the relationship between patient survival and duration of dialysis was u shaped in minorities with the worst patient survivals seen among preemptive transplants and those patients with over months of dialysis. the difference in outcomes between caucasians and minorities could be related to the biologic differences in the effect of dialysis on subsequent transplantation or to a differential selection bias introduced by duration of dialysis in the two populations. anti-carbohydrate natural antibodies. lorenzo benatuil, jonathan g. godwin, shamik gosh, john iacomini. transplantation research center, boston, ma. background. we constructed immunoglobulin gene knock-in mice lacking expression of the αgal epitope that carry rearranged vh and vl genes encoding antibodies specific for the carbohydrate antigen galα - galβ - glcnac-r (αgal). here, we describe two novel populations of b cells in the spleen of these m vhvlgt mice and their role in the production of αgal specific antibodies. methods. m vhvlgt mice were sacrificed and single cell suspensions from spleen, bone marrow, peripheral lymph nodes and peritoneal cavity were prepared and stained with different antibodies and with fluorescently labeled αgal-bsa for flow cytometric analysis. using multiparameter cell sorting, we purified marginal (mz) and follicular (fo) zone b cells, in addition to two novel splenic b cell populations. cells were adoptively transferred into b cell deficient µmt/gt double knock-out mice. serum samples were collected, and production of αgal specific igm antibodies was assessed by elisa. to investigate these b cell tolerance mechanisms, we employed mice with naturally occurring antibodies (abs) against human blood group a carbohydrates in their sera and possessing b cells with receptors for blood group a determinants. b cells with receptors for a carbohydrates in mice belong to the cd + b- subset, with phenotypic properties similar to those of human b cells. when these cells were temporarily eliminated by injecting synthetic a carbohydrates, subsequent treatment with cyclosporin a or tacrolimus, which blocks b- cell differentiation, completely inhibited the reappearance of b cells with receptors for a carbohydrates in mice. it is probable that calcineurin inhibitors used for preventing t cell-mediated rejection simultaneously suppress b- cell differentiation. however, despite a very limited dose of calcineurin inhibitors, the b cell tolerance toward blood group a antigens was persistently maintained in the blood group a-to-o liver transplant recipient. b cell tolerance after abo-incompatible transplantation might be a consequence of presentation of blood group carbohydrate antigens by cells in the engrafted liver. immune fluorescence staining of the human liver reveals that blood group antigens are predominantly expressed on the liver sinusoidal endothelial cells (lsecs). we have previously proven that lsecs, which constitutively express fas-l and pdl- have the capacity to tolerize alloreactive t cells. taken together, we hypothesize that blood group antigen-reactive b cells are also tolerized through the interaction with the lsecs. in order to address this possibility, we used α- , galactosyltransferase-deficient mice. when the α-gal-expressing lsecs isolated from wild-type mice were adoptively transferred via the portal vein into the splenectomized congeneic α-gal-deficient mice, these mice lost the ability to produce anti-α-gal abs (n = ). this finding suggests that the lsecs expressing blood group carbohydrates play a pivotal role in the tolerization of newly developed b cells specific for the corresponding carbohydrate antigens after abo-incompatible liver transplantation. success in kidney transplantation has resulted from control of t-cell-mediated acute rejection. however, little has been done to improve the fate of patients who possess pretransplant donor-specific antibody (dsa), and no proven therapies exist to specifically prevent dsa formation post-transplant. while methods to detect and characterize dsas are clearly useful, the b-cell subsets that produce dsas or more importantly sustain their production are poorly characterized. this study set out to investigate the fundamental mechanisms determining the formation of donor-specific memory b cells and plasma cells in novel mouse models of dsa formation. we have developed two complementary and novel systems to track the phenotypic and functional properties of polyclonal donor-specific b-cells. the first system involves allosensitization between normal c bl/ and balb/c mice. we used advanced flow cytometric methods to track donor-specific b cells with h- k b or h- k d tetramers. tetramers are comprised of four identical h- molecules bound to a fluorescently labeled steptavidin molecule that is able to bind the donor-specific b-cell antigen receptor (surface immunoglobulin). in our second system, we use donor mice that constitutively express full-length membranebound chicken ovalbumin (mova) protein in all tissues. analogously, ova specific-b cells can be analyzed flow cytometrically with the use fluorescently-labeled ovalbumin. both systems allow us to identify donor-specific memory b cells ( aad -cd -cd -ova/ tetramer + igd -b + cd -) and plasma cells ( aad -cd -cd -ova/tetramer + igd -fb lo cd + ) during skin graft rejection (day post transplant). we were also able to track the development of a stable memory cell population in the bone marrow and spleen for > days cells.for the ova system quantitative elisa and elisot measurements for serum dsa and dsa-secreting cells, respectively, correlated with the development of donor-specific memory b cells. using these assays we will be evaluate polyclonal donor-specific b memory subsets under multiple conditions of immunity and tolerance and for the first time, characterize their functional properties and migration patterns. these experiments will provide new information on the basic biology of the memory b-cell response to allografts in mice and facilitate the development of these methods in sensitized patients that may lead to critical therapeutic opportunities for dsa production. in the tnf-related b-lymphocyte survival factor, blys/baff, is critical for primary follicular (fo) and marginal zone (mz) b-cell survival. in vivo neutralization of blys/ baff, using a newly developed monoclonal antibody, depletes the fo and mz b-cell compartments. here, we hypothesized that targeting b-lymphocytes, via the blys/baff pathway, could promote humoral tolerance to islet allografts. cohorts of stz-diabetic c bl/ mice were transplanted with isolated islets from balb/c donors. treatment with anti-blys/baff alone ( mcg/mse x doses+ mcg/wk/mse) did not protect islet allografts from acute rejection (mst= d; n= ) . on the other hand, a -day course of rapamycin ( mg/kg) prevented acute allograft rejection (mst= d; n= ). when rapamycin was combined with the anti-blys regimen, islet allograft survival was markedly prolonged (mst> d; n= ). importantly, islet allograft survival was coincident with the absence of detectable serum alloantibodies and blunted donor specific t-cell responses. following treatment with anti-blys, b-lymphocyte compartment reconstitution was detectable at days following treatment and was characterized by stringent selection at the transitional→fo b-cell tolerance checkpoint. overall, these data indicate that the blys/baff pathway may be a logical target of immunomotherapy for the achievement of humoral transplantation tolerance. reza tavana background: highly sensitized patients' ability to be transplanted is severely compromised because of high level of antibodies against various hla antigens. interleukin- is a type i cytokine that signals through a receptor composed of the il- r and the common cytokine receptor -chain ( c ). it is produced by t-cells and has been shown to be the contributing factor in terminal differentiation of memory b-cell to anti-body producing b-cell and plasma cells. objective: we evaluated the effect of il- co-stimulation on antibody production capacity of b-cells and also measured the expression of il- r on b lymphocytes of highly sensitized patients compared to non-sensitized patients on the kidney transplant list. methods: patients with a pra level > % (sensitized) were compared to non-sensitized patients from the transplant waiting list. after consent was obtained, peripheral blood was taken from patients before initiating hemodialysis. leukocytes were labelled with anti-cd , anti-il- antibodies and paraffin fixed after rbc lysis. il- r expression was measured by flow-cytometry over facscan machine on the same day. the expression of the il- r is significantly higher on b-lymphocytes of highly sensitized patients (hsp) compared with non sensitized patients (nsp) (fig .p< . ). in-vitro co-stimulation of isolated peripheral b-cell with il- and anti-cd results in higher igg production compared with anti-cd- or il- stimulation alone (fig ) . conclusions: il- is an important cytokine in b-lymphocyte stimulation and increases igg production. il- receptor on b-lymphocytes is up-regulated in sensitized kidney transplant recipients. il- to il- r interaction between t and b-lymphocytes may be an important pathway in antibody production in highly sensitized renal transplant recipients. we created mixed bone marrow chimeras in irradiated µmt (b cell-deficient) recipients by transplantation of syngeneic bone marrow from µmt, wildtype (wt) and mhcko (lack mhc i & ii expression on all cells) mice. µmt+mhcko chimeras lack expression of mhc i & ii on b cells but not other professional apcs such as dcs hence antigen presentation specifically by b cells is disrupted. allograft rejection and development of alloreactive memory t cells in µmt+mhcko chimeras was compared to µmt+wt chimeras that had intact antigen presentation by both b cells and apcs. skin allograft rejection was comparable between µmt+mhcko and µmt+ wt chimeras (mst = and days, respectively, p = . , n = /grp). however, heart allograft rejection was significantly delayed in the µmt+mhcko chimeras compared to µmt+ wt chimeras (mst = and days, respectively, p= . , n = / grp). development of alloreactive memory t cells was assessed in chimeras at weeks after skin allograft rejection by quantitation of antigen-specific ifnγ producing t cells. alloreactive cd and cd memory t cells were significantly fewer in µmt+ mhcko chimeras ( -fold fewer cd , p = . , and -fold fewer cd , p = . , n = /grp) than in µmt+ wt chimeras. these results show that the disruption of antigen presentation by b cells significantly delays heart but not skin allograft rejection. development of alloreactive cd and cd memory t cells is significantly impaired in the absence of antigen presentation by b cells. conclusions: antigen presentation by b cells accelerates heart allograft rejection and leads to development of alloreactive memory t cells. these findings emphasize antibodyindependent functions of b cells in promoting alloimmune responses and highlight the need for b-cell targeted therapies to improve long-term allograft survival. purpose: to test whether depletion of cd + b cells at the time of engraftment alters the prevalence of anti-donor alloantibody (ab)or severity of cav in the context of therapeutic immunosuppression with (csa) or high dose cd inhibition. methods: forty-five mlr-mismatched heterotopic cardiac cynomolgus allograft recipients were treated with high dose anti-cd monotherapy (αcd ; n= , with atg induction) or αcd with additional anti-cd therapy (rituximab mg/ kg q wk for weeks: αcd + αcd ; n= , with atg). thirteen other animals received therapeutic csa (target trough > ng/ml), five of which received additional αcd . graft survival was censored at days. ab was deemed (+) if present (by flow cytometry) around time of explant. results: animals died with beating grafts, mainly with atg-associated lung pathology or infectious etiologies and are excluded from this analysis. animals that had sustained αcd levels < µg/ml after protocol day were considered "subtherapeutic" for this reagent and also excluded from further consideration. graft survival with αcd + αcd (median > d) and proportion of grafts surviving to days ( / ) was significantly increased relative to αcd alone (median d, p= . ; / > d). graft survival with csa + αcd (median > d) and proportion of grafts surviving to days ( / ) was increased relative to csa alone (median d, / > d). with therapeutic αcd (trough level > µg/ml until graft explant), / ( %) developed ab vs. / with αcd + αcd ( %) (p= . ). average cav score for the αcd group was . ± . vs. . ± . in the αcd + αcd group (p= . using unpaired t test). preliminary cav scoring suggests that added αcd inhibited cav (scores ranging . - . ) relative to csa alone (median . ; range . - . ); ab analysis is in progress for these groups. conclusions: using αcd is associated with significant attenuation of cav when used with either "therapeutic" αcd or csa. our findings demonstrate for the first time that αcd reduces the severity of cav in conjunction with both conventional immunosuppression and costimulation pathway blockade. mechanisms include inhibition of ab production, and perhaps others that remain to be defined. subsaturating concentrations of anti-hla antibody ( sensitized recipients having donor specific anti-hla abs have been successfully transplanted following a conditioning regimen employing plasmapheresis with or without pooled human immunoglobulin. in vitro studies have shown that exposure of ecs to subsaturating concentrations (ssc) of anti-hla ab (priming) followed by subsequent exposure to high concentrations (hc) of anti-hla induces the expression of protective genes, bcl and ho- and confers protection to complement mediated lysis of ecs. however, the molecular events following priming with exposure to ssc of anti-hla ab still remains undefined. to determine the priming events and to define the kinetics of protective gene expression, we exposed human aortic ecs to ssc of anti-hla ab for hrs and re-exposed them to saturating concentrations of anti-hla abs. ecs were collected at , , and hrs after exposure to ssc as well as hrs after exposure to hc. expression profile of signaling intermediates (mapk, wnt, nf-kb, hedgehog, pi kinase, stress pathway, tnf family) in the ecs were analyzed by gene array. analysis of the bcl and ho- expression showed no significant increase in expression following exposure to ssc of w / (priming) or control ab alone at any of the time points ( background/aims: microcirculation disturbance, endothelial injury and cytokine overproduction are implicated in the pathophysiology of hepatic ischemia reperfusion injury (iri). thrombomodulin (tm) is a membrane-bound endothelial thrombin receptor that accelerates thrombin-catalyzed protein c activation, inhibits thrombin-induced fibrin formation, and also regulates inflammation. in the present study, we investigated the effects of recombinant human soluble tm (rhstm) on hepatic iri in the rat. methods: wister hannover rat was used for preparing ischemia/reperfusion model. hepatic iri was induced by subjecting % area of the rat liver to minutes of ischemia followed by h of reperfusion. the rats were randomly assigned to a group receiving an intravenous injection of rhstm ( mg/kg body weight) and to a group treated with saline minutes prior to the beginning of reperfusion. sinusoidal endothelial cells (secs) and kuppfer cells (kcs) were isolated using centrifugal elutriation. the plasma levels and the concentration in cultured cell supernatant of il- and tnfα were measured by specific enzyme immunoassays. results: the plasma alt, ast and hyaluronic acid levels were significantly decreased, and the histological damage of the liver was attenuated in rhstm-treated rats as compared to control rats. using laser doppler flow-meter we found that rhstm treatment improved hepatic microcirculation. the intrasinusoidal fibrin deposition, injury of secs and liver dysfunction during hepatic iri were weaker in rhstm-treated rats than in control rats. tm activity in secs was significantly recovered, and plasma il- and tnfα were significant decreased in rhstm-treated rats as compared to control rats. further, il- and tnfα production in isolated kcs was also significant decreased in rhstm-treated rats as compared to control rat . conclusion: the present results suggest that rhstm is useful for the prevention of secs damage and kcs activation induced by iri. our present study also suggests that disturbance of hepatic microcirculation is induced in part by intrasinusoidal microthrombus formation and by locally released inflammatory cytokines from kcs. protective effects of preservation solution including activated protein c in small-for-size liver transplantation in rats. background: small-for-size liver graft is a serious obstacle of partial orthotopic liver transplantation (olt). however, various therapeutic strategies including surgical innovations, pharmacological agents and gene therapies to protect small-for-size liver graft have not yet been developed. aims: activated protein c (apc) is known to have cell protective properties via its anti-inflammatory and anti-apoptotic activities. this study aimed to examine the cytoprotective effects of preservation solution containing apc on olt using smallfor-size rat liver graft ( % partial liver). methods: liver grafts were assigned to two groups: in the control group, the grafts were flushed and stored in histidine-tryptophan-ketoglutarare (htk) solution alone for h; in the apc group, in htk solution containing apc for h. results: the apc group significantly increased -day graft survival from % to %, decreased levels of transaminase, and improved histological features of hepatic iri compared to the control group. myeloperoxidase activity demonstrated that the apc group markedly suppressed the infiltrations of neutrophil. hepatic expressions of tumor necrosis factor-α and il- of the apc group were remarkably decreased. the apc group significantly reduced serum hyaluronic acid levels, indicating attenuated sinusoidal endothelial cell injury. moreover, the apc group markedly increased hepatic levels of nitric oxide caused by upregulated endothelial nitric oxide synthesis (nos) together with downregulated inducible nos, and decreased hepatic levels of endothelin- . finally, hepatocellular apoptosis of the apc group was remarkably suppressed by downregulated hepatic caspase- and caspase- activities. conclusions: preservation solution containing apc inhibited pro-inflammatory cytokine synthesis, which leads to hepatocellular apoptosis and liver injury. one of the cytoprotective effects of the apc treatment was to upregulate hepatic enos, followed by increased expression of hepatic no, and to decrease expression of hepatic et- , resulting in the prevention of microcirculatory disturbance. preservation solution containing apc is a potential novel and safe product for small-for-size liver transplantation to improve liver graft function and animal survival. deletion of cd on natural killer cells attenuates hepatic ischemia/ reperfusion injury. living donor liver transplantation (ldlt) has emerged as a solution to ease organ shortage in orthotopic liver transplantation. however, ldlt is often complicated by small-for-size liver graft that is highly susceptible to injury and shows decreased liver regeneration. suppression of liver regeneration in small-for-size grafts correlates with impaired priming as a result of limited nf-κb activation and decreased production of the priming cytokines tnf and il- . we have shown that the hepatoprotective protein a promotes liver regeneration, partly through blockade of the cyclin dependent kinase inhibitor p . however the impact of a expression in livers on il- production and/ or signaling were still unknown. in this study we demonstrate that secretion of il- (elisa) following treatment with lps or tnf was moderately lower in hepatocytes transduced with a recombinant a adenovirus (rad) as compared to non-transduced or rad.β-galactosidase transduced cells. this indicates that il- production in hepatocytes is not solely nf-κb dependent (not totally blocked by the nf-κb inhibitor a ). despite similar or lower il- levels, il- signaling as evaluated by phosphorylation of stat (western blot; wb) was enhanced in a expressing hepatocytes. this was confirmed in experiments showing that a increases stat- phosphorylation in response to exogenous human il- . accordingly, hepatocyte proliferation was significantly higher in rad.a transduced hepatocytes as opposed to controls. the pro-proliferative function of a mapped to the zn domain. since the balance of il- signaling in hepatocytes is finely regulated through a negative feed-back loop provided by the il- /stat- dependent induction of suppressor of cytokine signal- (socs ), we investigated whether a affects il- signaling by modulating socs expression. our results indicate that a indeed decreased il- mediated upregulation of socs (wb). this later result was confirmed in vivo. improved regeneration and survival in a treated livers correlated with a substantial decrease in socs levels before and hours following extended ( %) liver resection in mice. these results suggest that a enhances priming of hepatocytes by il- likely through down-regulation of socs . this added to the effect of a on p would further enhance its pro-proliferative function in hepatocytes to benefit survival and function of small-for-size liver grafts. background: we have previously demonstrated that silencing inflammatory, apoptosis and complement genes can prevent ischemia-reperfusion (i/r) injury occurring in heart transplantation. however, the method for efficiently delivering sirna into donor organ has not been established. this study was designed to develop a new method to induce gene silencing by coronal artery infusing with sirna solution for prevention i/r injury in heart transplantation. methods: multiple sirnas that specifically target tnfa, caspase , and c a receptor (c ar) genes were generated and selected. sirna protection of donor organs was evaluated in a rat heart transplantation model. heart grafts from lawis rats were infused with sirna solution via coronal artery and preserved in hkt solution at ° c for hrs, and subsequently transplanted into syngeneic lawis rats. cardiac functions were assessed by heart beating rate. gene expression at mrna level was determined by qpcr. the i/r injury was assessed by immunohistochemistry. results: after donor heart perfusion with sirna solution, sirna was found to enter the myocardial cells, indicated by the fluorescence emitted from the dye labeled sirna. the levels of tnfa, caspase and c ar genes were significantly up-regulated in the grafts after ex vivo preservation for hrs. these up-regulated tnfa, caspase , and c ar genes were significantly knocked down by sirna infusion. using a sirna infused organ as a donor, the graft survival was significantly prolonged in heart transplantation. while sirna solution-treated heart grafts retained strong heartbeat up to the end point of observation (> days), the control grafts lost function within days. in addition, an improved cardiac function was observed in the graft preserved in sirna solution. the protection of graft by sirna solution is associated with prevention of i/r injury. sirna solution-treated organs exhibited almost normal histological structures as well as less neutrophil and lymphocyte infiltration, compared with control solution-treated organs. conclusions: this study developed a novel ex vivo sirna delivery system using coronal artery infusion, which can effectively silencing genes in donor hearts and prevent cardiac i/r injury. background: ischemia/reperfusion (i/r) insult is a prime factor leading to liver dysfunction. apoptosis plays key role in the early graft loss following orthotopic liver transplantation (olt). bcl-xl has been showed to exert an anti-apoptotic function both in vitro and in vivo. this study was designed to evaluate potential cytoprotective effects and mechanisms for bcl-xl in liver i/r injury by ad-bcl-xl gene transfer. methods: a mouse model of partial min warm hepatic ischemia followed by h of reperfusion was used. balb/c wide-type (wt) mice (n= /gr) were injected with ad-bcl-xl or adβ-gal reporter gene ( . x pfu, i.p. at day - ). sham control wt mice underwent the same procedure, but without vascular occlusion. mice were sacrificed after h of reperfusion; liver tissue and blood samples were collected for future analysis. results: ad-bcl-xl treated mice showed significantly lower sgot levels (iu/l), as compared with ad-β-gal or wt controls ( ± vs. ± , and ± , respectively; p< . ). these correlated with histologic suzukis grading of hepatic i/r injury, with wt/ad-β-gal controls showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis. in contrast, wt mice treated with ad-bcl-xl revealed minimal sinusoidal congestion without edema/vacuolization or necrosis. ad-bcl-xl gene transfer significantly reduced local neutrophil accumulation and apoptosis ( . ± . of tunel+ cells in ad-bcl-xl vs. . ± . and . ± . of tunel+ cells in wt or ad-β-gal treated mice; p< . ). unlike in controls, intragraft expression of mrna coding for tnf-α, e-selectin/icam- , and ip- /mcp- remained depressed in the ad-bcl-xl group. ad-bcl-xl gene transfer markedly depressed the activation of nf-κb, caspase- , and increased ho- , a , and bcl- /bcl-xl expression, as compared with wt/ad-β-gal controls. conclusion: this study demonstrates that inhibition of nf-κb activation contributes to the cytoprtective effects after bcl-xl gene transfer in hepatic i/r injury. the induction of anti-oxidant ho- and anti-apoptotic a , bcl- by bcl-xl gene transfer exerts synergistic cytoprotective effect against antigen-independent hepatic inflammatory injury induced by i/r. hepatocyte background: primary graft non-function (pnf) affects survival and function of renal allografts. pnf, secondary to the ischemic and inflammatory injury in the peri-transplant period, leads to acute tubular necrosis and predisposes to acute rejection. defining new preconditioning regimens to reduce pnf are desirable. a is part of a negative antiinflammatory loop aimed at inhibiting nf-κb in renal proximal tubular epithelial cells (rptec). hepatocyte growth factor (hgf) is a pleiotropic growth factor upregulated in acute kidney injury and acute rejection likely to modulate inflammation and promote repair. in the present study we evaluated the effect of hgf on rptec and hypothesized that some of its protective functions may relate to the upregulation of a . methods and results: treatment of rptec with hgf ( ng/ml) led to a . ± . (n= ; p= . ) fold increase in a mrna (real time-pcr), which translated into a significant . ± . fold increase (n= ; p= . ) in a protein by h, as shown by western blot (wb). two lines of evidence suggested that upregulation of a by hgf was nf-κbindependent. hgf did not degrade iκbα in rptec (wb) nor upregulated the nf-κb dependent molecule icam- , as shown by flow cytometry analysis (facs). further, a was still upregulated in rptec expressing the nf-κb inhibitor iκbα, both at the mrna and protein levels. upregulation of a by hgf protected rptec from a subsequent inflammatory insult, here mimicked by the addition of tnf. pretreatment of rptec with hgf for hours blunted tnf-induced ( u and u/ml) upregulation of icam- , as analyzed by facs. conclusion: to our knowledge this is the first demonstration that a could be upregulated in rptec, in a non-nf-κb dependent manner. further studies are carried out to elucidate the transcription factors involved in hgf-induced upregulation of a . from a clinical standpoint, these results highlight the unique ability of hgf to protect rptec from inflammation by inducing the anti-inflammatory protein a , remarkably without triggering other pro-inflammatory signals. we propose that hgf-based therapies could serve in preconditioning regimens to prevent ischemia/reperfusion injury and reduce pnf and acute rejection in renal transplantation. background. liver ischemia reperfusion injury (iri) is one of the main causes of graft dysfunction and rejection in liver transplantation. it has been documented that iri is associated with inflammatory and complement pathway activation. this study was designed to investigate the efficacy of small interfering rna expression vector (shrna) targeting tnf-α and complement (c ) genes in the protection of mouse liver iri. methods. shrna expression vectors were constructed for tnf-α and c genes. mice received shrna by hydrodynamic injection prior to iri, which consisted of interrupting blood supply to the left lateral and median lobes of the liver for minutes followed by reperfusion. iri was evaluated using liver histopathology, as well as levels of serum alanine transferase (alt) and aspartate transaminase (ast). neutrophil accumulation was determined by a myeloperoxidase (mpo) assay. lipid peroxidation was assessed by malondialdehyde (mda) levels. realtime pcr was used to test gene silencing efficacy in vitro and in vivo. result. we demonstrated that iri is associated with an increase in tnf-α and c mrna levels in liver tissue hours after reperfusion. shrna-treatment effectively down-regulated tnf-α and c expression in iri livers. in comparison with vehicle control, the serum levels of alt ( . ± . u/l vs . ± . u/l) and ast ( . ± . u/l vs . ± . u/l), were significantly reduced in mice treated with tnf-α and c shrna. additionally, the neutrophil accumulation and lipid peroxidase-mediated tissue injury, detected by mpo and mda respectively, were improved after shrna treatment. tissue histopathology showed an overall reduction of injury area in shrna-treated mice. conclusion. this is the first demonstration that liver iri can be prevented through gene silencing of inflammatory genes and complement genes, showing potential for shrna-based clinical therapy. kidney -acute rejection: antibody-mediated rejection the ( ) in the first three days after transplantation, a temporary decrease in dsa was observed in all amr cases, and all of them quickly rebounded thereafter; ( ) c d can be detected very early (can be seen on day , % in day protocol biopsies, frequently in the absence graft dysfunction, and % in index biopsies); ( ) the pathologic changes observed in sequential biopsies were c d deposition followed by acute tubular injury, then interstitial inflammation and peritubular capillary margination seen in index biopsies; ( ) pure amr occurred early ( % at day ), usually evolving into mixed amr with accompanying cellular rejection ( % in index biopsies); ( ) most recipients ( %) had initial graft function before developing amr; ( ) background: antibody-mediated rejection (amr) has been recognized as a major problem in abo-incompatible (abo-i) renal transplantation (rtx). however, little is known about the long-term impact of amr in the abo-i renal transplant setting, especially after the introduction of a tacrolimus (fk)-based immunosuppressive regimen. the aim of this study was to assess the long-term impact of amr on the clinical and pathological outcomes in abo-i rtx. methods: fifty-eight patients who underwent abo-i rtx at our institution between march and december under an fk-based immunosuppressive regimen were enrolled in this study. protocol biopsies were performed regardless of renal function at one month and one year after rtx. fifty-six of the patients received the biopsy at one month and of patients underwent biopsy at one year posttransplant. amr was diagnosed by morphological features based on the banff ' update and other characteristic findings for amr previously reported, such as mesangiolysis, interstitial hemorrhage, and cortical infarction. we evaluated graft survival, incidence of chronic rejection characterized by transplant glomerulopathy (tgp) at one year posttransplant, and renal function using serum creatinine at three years posttransplant according to the incidence of amr at one month posttransplant. the overall graft survival rate at , , and years after rtx was %, %, and %, respectively. the incidence of amr at one month was % ( / ). the graft survival rate of the patients with amr was significantly lower than that of the patients without amr (p< . , years: % vs %, years: % vs %). the incidence of tgp in the patients with amr was significantly higher than that of the patients without amr (p< . , % vs %). the serum creatinine concentration at three years after rtx was significantly higher in the patients with amr than in those without amr (p< . , . mg/dl vs . mg/dl). in this study, we revealed that amr in abo-i rtx is associated with not only graft loss but also the progression of chronic renal impairment, functionally as well as pathologically, even after the introduction of an fk-based immunosuppressive regimen. further studies are needed to establish a more effective immunosuppressive regimen, such as rituximab induction therapy; against amr in abo-i rtx. conclusions: de novo dsa in ar is an independent predictor of graft loss and its degree of influence is comparable to other established risk factors (aa race, dgf, increased baseline creatinine). additional studies are warranted to: ) confirm the predictive ability of dsa and ) determine whether reduction/eliminattion of dsa will allow improvements in graft survival. was performed in all the recipients before and six months after lrkt. graft biopsies were performed as well within and after six months of the transplantation (tx). all the data of recipients were collected prospectively during the period of follow-up. humoral rejection rate, donor specificity, and time of appearance of the de novo abs were retrospectively studied. results among the lrkt recipients, ( %) showed negative/negative results, ( %) showed positive/positive results, ( %) showed positive/negative results, and ( %) showed negative/positive results (de novo abs) in the pre-/post-transplant flow-pra analysis. among the cases with de novo abs, ( %) had donorspecific abs (dsa) and the remaining ( %) had non-donor specific abs (ndsa) as determined by lab single antigen analysis. four of the five recipients ( %) with dsa showed evidence of both vascular and humoral rejection in the graft biopsies performed within months of the transplantation, while one of the eight recipients ( %) with ndsa showed evidence of cellular rejection during the same period. a -year graft survival rate of the recipients with de novo abs was %, compared with %, % and % in other groups without de novo abs (p= . ). conclusions lrkt recipients with developing de novo abs has much higher incidence of humoral rejection and worse prognosis, especially those with donor-specific de novo abs. cautious monitoring for the appearance of anti-hla antibodies should be adopted after transplantation, even in patients without anti-hla ab prior to the transplantation. despite significantly higher response than the males w/o amr (p< . ), the other females did not experience amr. conclusions: ) cfc is a novel assay to measure allo/ do cd -cell responses, assess the degree of sensitization, and predict amr in hs, ) allo/do cd -cell numbers are elevated in many hs, but not nc, ) hs w/ high(+) cfc are at increased risk for amr and may need additional pre-tx desensitization, ) allo/do reactivity are higher in hs females, which may explain their higher rate of amr, ) cfc cut off levels for amr prediction may be higher in females than males, ) monitoring hs using the cfc pre-and post-desensitization may help determine the efficacy of desensitization and risk for amr. were treated with plasmapheresis, ivig and rituximab, and pts with l-amr received ivig and rituximab. the -year gs post-amr in pts with e-amr and focal c d staining was % vs. % in pts with diffuse staining; while cases of l-amr with focal c d deposition had a gs of % vs. % in cases with l-amr and diffuse staining. the number of cases with focal staining was low, and the numerically evident differences were not statistically significant (log-rank p=ns). notably, when losses due to death with a functional graft were censored, post-amr gs was significantly lower in pts with e-amr and focal staining than in their counterparts with diffuse c d deposition ( % vs. %, log-rank p= . ). in this retrospective single center study, focally positive c d amr carries a worse prognosis than previously thought, and causes a significant reduction of gs. whether any degree of c d staining in the context of kt dysfunction should be treated as amr remains a pending question. association time of biopsy was . ± . mo after kt. however, cases were biopsied in the st year posttransplant. the extent of c d staining was graded as < % ( ), - % ( ), and ≥ % ( ) of ptc, and the intensity was graded as none ( ), light ( ), and strong ( ) staining. these findings demonstrate the significant discordance between detection of dsa and c d, which is a relatively specific histological evidence of ab-mediated injury. this factor should be taken into account when clinical decisions for treatment of patients with either c d or dsa positivity are made. the observed discrepancy could be partially due to the technique used for staining of the biopsy specimens, inability to detect anti-hla dsa with the available technology, or non-hla dsa. long term follow up data are needed to evaluate the impact of these markers on graft outcomes. introduction epithelial to mesenchymal transition (emt) is a potential mechanism of tissue fibrogenesis. in a previous study, we had reported that the early expression of emt markers was associated with the progression of renal grafts towards interstitial fibrosis and tubular atrophy (if/ta). here, we report the long-term follow-up of this cohort, paying a special attention to the evolution of graft function. patients and methods patients engrafted with a kidney from a cadaveric (n= ) or a living (n= ) donor, and in whom sequential protocol biopsies had been performed at and months, were included. the phenotype of epithelial cells was studied at three months according to the expression of vimentin (an intermediate filament normally expressed by fibroblast-like cells) and to the cellular localization of β-catenin. grafts in which these two markers were abnormally expressed by more than % of tubular cells were considered as emt+ grafts. serum creatinine and creatinine clearance (estimated abstracts by gault and cockcroft index) were collected from to months post-transplant and compared according to the emt status of the graft. results multivariate analysis demonstrated that the early expression of emt markers was an independent risk factor of the progression of graft fibrosis between and months. more importantly, these early phenotypic changes were associated with a progressive and sustained deterioration of the graft function : emt+ patients had a statistically higher serum creatinine from twelve months after transplantation, and a significantly lower creatinine clearance from months after transplantation (emt+ . ± . ml/min vs emt- . ± . ml/min, p= . ). the difference was persistent at months. conclusion the expression of emt markers by tubular epithelial cells at an early time point post-transplant (three months) is highly suggestive of an ongoing fibrogenic process, and has repercussions on the long-term graft function. therefore, these epithelial phenotypic changes are relevant and promising biomarkers for an early detection of if/ta. we recently reported that % of transplant glomerulopathy (tg) has evidence of alloantibody-mediated injury in biopsies for cause. we found that / of tg is c d+ab+ and / is c d-ab+ suggesting that c d staining is not sensitive enough to detect all biopsies with antibody-mediated injury. we aimed to develop a new laboratory test to detect biopsies with antibody-mediated rejection (abmr) which are missed by c d. using affymetrix microarrays, we analyzed gene expression in renal allograft biopsies for cause. we previously reported that both abmr and t cell-mediated rejection (tcmr) biopsies show increased expression of transcript sets associated with cytotoxic-t cells (cats) and gamma-interferon effects (grits) compared to biopsies without rejection (p< . ). however, abmr biopsies were discriminated by a selective increased expression of "endothelial cell-associated transcripts" (endats). these genes included established endothelial markers such as vwf, pecam , sele, cd , and cadherin , which are involved in endothelial-cell activation. hierarchical clustering of biopsies with ab+ using endats identified a group of c d-ab+ biopsies (n= ) clustered with c d+ ab+ biopsies (n= ). thus % of biopsies with antibody ( of ) had increased endat-scores despite being negative for c d. these c d-ab+ biopsies with high endats, had higher scores for cats and grits, increased incidence/severity of tg, tubular atrophy/interstitial fibrosis, and worse future graft function (p< . ), but similar incidence of tcmr or borderline lesions, in comparison to c d-ab+ biopsies with no increase in endats. the c d-ab+ cases with endothelial activation show extensive inflammation in the allograft, as measured by the gene sets, which is similar to c d+ abmr. there are a significant number of cases with alloantibody and no c d that show increased expression of endothelial genes. thus the transcriptomics detects deteriorating c d-allografts with ongoing alloantibody mediated injury. we conclude that increased expression of endothelial genes provides a new feature of abmr, and can be used as a new diagnostic test to detect and treat c d-abmr. probabilistic ( ) . the bayesian network model was analyzed and, interestingly, cd was critically related to tg, suggesting a b-cell mediated process. tg was also predicted by upregulation of ccl , ccl , ccl , cxcl , il- , il- , and icam gene expression. ten percent of the samples were excluded randomly from the initial model, and subsequently used for cross validation. in the validation analysis, the model effectively predicted tg (auc of . , % ppv) and sf (auc of . , % ppv). this study provides a compelling and clinically relevant example of the combination of quantitative gene expression with probabilistic bayesian modeling to predict renal allograft pathology. potentially important molecular pathways associated with transplant glomerulopathy were also identified. the application of this integrated approach has broad implications in the field of transplant diagnostics and interpretation of large data sets. , , , , , and months respectively. the daily dose and blood levels of tac were significantly lower in tac/slr group compared to tac/mmf group. renal function is shown. despite lower prevelance of cai in tac/slr group long-term graft function and patient and graft survival are comparable between tac/mmf and tac/slr groups. objective: the aim of this study was to determine if ethinicity impacts graft outcomes in kidney transplant patients converted to sirolimus (srl) and either maintained on calcineurin inhibitors (ci) or mycophenolate (mmf) with steroids. methods: this was a retrospective analysis of all kidney transplants converted to srl and transplanted from / to / . patients were divided into groups: group : aas converted to srl + continued on ci; group : non-aas converted to srl + continued on ci; group : aas converted to srl + continued on mmf; group : non-aas converted to srl + continued on mmf. pediatrics and multiorgan transplants were excluded. results: a total of patients were included ( % aa). demographics, baseline immunosuppression, and reason for srl conversion were similar between groups. table displays characteristics and outcomes. patients converted to srl+ci regimens had higher rates of acute rejection before srl conversion (p< . ), but equal rates after conversion. development of proteinuria was similar across groups. figure displays the graft survival rates for each group. aa patients converted to srl+mmf tended to have poorer outcomes compared to aa patients converted to srl+ci. non-aa patients converted to srl+mmf tended to have better graft outcomes compared to non-aa patients coverted to srl+ci, although this did not reach statistical significance(p= . ). conclusion: aas converted to srl may benefit from continued ci, while non-aas converted to srl appear to have better outcomes with mmf. further prospective studies are warranted to confirm these findings. aa srl+ci (n= ) there are no large registry studies evaluating the correlation of allograft failure for recipients of kidneys from the same deceased donor. we examined outcomes in such recipient pairs using data from the united states renal data system. methods: we studied the correlation of graft failure events within , pairs of same-donor recipients transplanted during through . analyses were limited to patients with functioning grafts months post-transplant (tx) and adjusted for known donor, recipient, and tx management factors. we estimated odds ratios to measure the increased risk for , , and -year graft failure and death-censored graft failure when the contralateral kidney had such an event. we also evaluated the effect of recipient pairs transplanted at the same center vs different centers. results: there is a strong correlation in outcomes for recipients with the same donor (table) . the correlation was stronger within pairs transplanted at the same center than for those transplanted at different centers. differences in the correlation of graft failures within pairs transplanted at the same versus separate centers diminished over and were absent by years post-tx. results for death-censored allograft failure were similar. conclusion: unmeasured donor factors contribute significantly to the correlated graft failure outcomes in paired recipients of deceased donor kidneys and need further study. kidney tx outcomes in the first year may be affected by differences in management between transplant centers, more so than in subsequent years post-tx. odds ratio of death-censored graft failure and graft failure in recipients of a donor pair, given that the outcome occurred in the recipient of the contralateral kidney, with both recipients being transplanted at the same (s) center or at different (d) centers. all odds ratios significant with p< . . the deterioration of kidney allograft function (dekaf) study is a nih-funded multicenter observational study of late allograft (ktx) loss. the study examines two cohorts: a "long-term cohort" (ltc) of prevalent ktx with scr < . mg/dl with deterioration of function ( % increase in scr or proteinuria) and a "prospective cohort" (pc) of incident ktx developing a persistent > % increase in scr. we examined the pathologic features of the first renal biopsy (bx) obtained for new onset deterioration in each cohort. all bx were read and scored centrally using a modified banff schema. on average, bx were obtained at (pc) and (ltc) months post-tx. mean scr was similar, but more patients (pts) in ltc had proteinuria. moderate to severe interstitial fibrosis and tubular atrophy (ta) were more prevalent in ltc than pc ( vs % ci score ≥ ; vs % for ta). the rate of vascular sclerosis > % was similar ( . % pc vs . % ltc); however, hyaline arteriolar sclerosis > % was more common in the ltc ( vs . %). interstitial inflammation (i) and tubulitis (t) scores were similar in both cohorts. however, more pts in ltc had peritubular capillary infiltrates (> cells) and evidence of tx glomerulopathy. while rates of interstitial inflammation and tubulitis sufficient to warrant diagnosis of acute rejection are similar in the prospective and long-term cohorts, long term cohort pts had more proteinuria, interstitial fibrosis, tubular atrophy, hyaline arteriolar sclerosis, and transplant glomerulopathy. analyses of histologic findings and renal outcome are ongoing. the term chronic allograft nephropathy (can) has been abolished by the last banff meeting report (am j transplant, ) and categories have been introduced for chronic changes: chronic active t cell-mediated rejection and chronic active humoral rejection (cahr). aim of the study was to review all cases of can diagnosed in the last years and to identify immunohistochemical markers of chronic rejection. a cohort of cad pts with biopsy-proven can was analyzed. each case was reviewed and assigned into groups according to banff criteria: chronic rejection (cr), chronic calcineurin toxicity (cnit) or chronic lesions not otherwise specified (nos). cd +, cd +, cd +, cd + cells and c d deposits were assessed by immunohistochemistry. twenty-eight pts were classified as cnit, as cr, of which were cahr, and as nos. serum creatinine and h proteinuria at renal biopsy, extent of interstitial fibrosis and glomerulosclerosis were not significantly different among groups (table ).the number of cd + cells was higher at ti level in cr compared to cnit (table ;*p=. ). cd + cells were higher at ti and g level in cr compared to cnit (table ;*p=. ). ti and g cd + cells were not different among the groups (table ). the number of g cd + cells was increased in cr compared to cni and nos (table ;*p=. ). no significant difference in cd , cd , cd , cd expression was found at ti and g level between c d + and c dcases of cr. cd , cd , cd but not cd expression at ti level correlated with ti fibrosis (r =. , . , . , respectively, p<. ) at the univariate analysis. only ti cd + cells independently correlated with fibrosis at multiple regression analysis. in conclusion, our data suggest that: morbid obesity limits access to kidney transplantation and predicts adverse transplant outcomes. there are limited data on the safety and efficacy of gastric bypass (gb) as a weight reduction therapy among transplant candidates and recipients. methods: we examined usrds registry data to identify medicare-insured kidney transplant candidates and recipients with billing claims for gb procedures. gb were categorized according to occurrence before listing, on the waitlist, or after transplant. we studied the clinical characteristics of gb-treated patients, and subsequent outcomes including progression from listing to transplant and -day mortality. usrds surveys bmi data at dialysis start, waitlist entry, transplant, and transplant anniversaries.we computed changes between most recently reported body mass index (bmi) values preceding and following gb, when available. results: we identified transplant candidates treated with gb before listing, who underwent gb on the waitlist, and gb cases after transplant. patients treated with gb were most commonly female, white race, and without diabetic or hypertensive renal failure (table) . -day mortality after gb, calculable for listed and transplanted patients, was . % and . %, respectively. transplant recipient experienced graft failure within days of gb. of patients treated with gb on the waitlist, proceeded to transplant. post-gb weight loss was detected for % with gb pre-listing, % with gb on the waitlist, and . % with gb after transplant. among patients listed for transplant in the same era and bmi > at first dialysis who were not treated with gb, % had lost weight between dialysis start and listing. conclusions: gb has been performed in small numbers of kidney transplant candidates and recipients, and is followed by weight-loss in the majority of cases. peri-operative mortality is comparable to reports in patients without kidney disease. gb warrants prospective study as a strategy for reducing complications of obesity in esrd. introduction: the present study investigated the incidence of posttransplant diabetes mellitus (ptdm) and calculated the risk of developing ptdm under a tacrolimus and mycophenolate mofetil (mmf)-based immunosuppression based on clinical characteristics, tacrolimus-pharmacokinetics, and genetic polymorphisms related to tacrolimus-pharmacokinetics, cytokines and diabetes mellitus. methods: seventy-one non-diabetic adult kidney recipients (male , female ) were studied. patients with continuous high plasma glucose levels, over . mg/dl of hemoglobin a c, or requiring insulin and/or oral anti-diabetic agents for more than months after transplantation at -year after transplantation were diagnosed as having ptdm. fifteen genomic polymorphisms were assessed. results: one year after transplantation, recipients ( . %) developed ptdm. positive risk factors were age (p= . ) and body mass index (p= . ). there were no significant differences in acute rejection rate, total steroid doses, tacrolimuspharmacokinetics or its related to genetic polymorphisms between the two groups. the frequencies of ptdm were significantly higher in patients with adiponectin t g tt genotype than in those with the g allele (p= . ), and in patients with glucocorticoid receptor (nr c ) bcl i cc genotype than in those with the g allele (p= . ). conclusions: the incidence of ptdm at -yr after transplantation was . % in our cohort. elder or obese patients were risky for the development of ptdm. the presence of the adiponectin t g tt or nr c bcl i cc genotype may also be risk factors for ptdm, suggesting that insulin and glucocorticoid sensitivity-related genes are associated with the development of ptdm. analysis of these genotypes is a possible method of predicting a patient's risk for developing ptdm and would be a valuable asset in selecting appropriate immunosuppressive regimens for individuals. pharmacokinetics persistent hyperparathyroidism (hpt) with hypercalcemia is common after renal transplantation. studies have shown that treatment with cinacalcet corrects hypercalcemia and lowers pth levels in these patients. so far cinacalcet's steadystate pharmacokinetics and their correlation with pharmacodynamics (pk/pd) have only been studied in hemodialysis patients, but not in renal transplant recipients with persistent hpt. to gain further insight into cinacalcet's effects on calcium-phosphate homeostasis, we determined its steady-state pharmacokinetics and pharmacodynamic effects in these patients. in a prospective, single center, open label study we examined the effect of a -week treatment with mg and subsequent -week treatment with mg cinacalcet daily on calcium-phosphate homeostasis over hours and determined the steady-state pharmacokinetics of cinacalcet in stable renal allograft recipients. the urinary calcium excretion was determined in timed urine samples. median auc - was . ng*h/ml and c max was . ng/ml for mg cinacalcet which is higher, and oral clearance (cl/f) was . l/h which is lower in renal transplant recipients compared to previously published data of hemodialysis patients ( mg cinacalcet auc - , c max . , cl/f ). we also observed a non-proportional increase of auc - after doubling of the cinacalcet dose. the once daily administration of cinacalcet dose-dependently reduced ipth and serum calcium. cinacalcet and parathyroid hormone (pth) concentrations showed an inverse correlation and were fitted to a simple emax model (e max = % reduction vs. baseline, ec = ng/ml). the -hour fractional urinary excretion of calcium was increased after mg cinacalcet (baseline . ± . %, mg . ± . %, mg . ± . %). renal function remained stable. cinacalcet's higher and non-proportional increase of auc - in transplant recipients compared to hemodialysis patients evokes the possibility of a pharmacokinetic interaction with concomitant cyclosporine treatment. cinacalcet effectively corrected the biochemical abnormalities of persistent hpt. the transient calciuria could potentially favor nephrocalcinosis and reduce bone mineral density, suggesting that higher doses of cinacalcet need to be used with caution in renal transplant recipients with severe persistent hyperparathyroidism. screening for proteinuria in the kidney transplant clinic. bryce a. kiberd, romuald panek. dalhousie university, halifax, ns, canada. proteinuria is a predictor of progression in kidney disease. it is not clear whether measuring albuminuria will have greater clinical utility over measurement by dipstick or total proteinuria in kidney transplant recipients. there has also been a trend away from using hour collections to using spot urine albumin/creatinine (ac) and protein/creatinine (pc) ratios. we compare the prevalence of proteinuria estimated by dipstick, ac and pc in prevalent patients (> months post transplant) in the kidney transplant clinic. significant albuminuria defined as ≥ mg/g was present in % ( / ). albuminuria was seen in % ( / ) with negative and % ( / ) with trace dipstick proteinuria. significant predictors of albuminuria in a mulitvariate logistic analysis were egfr (or . per ml/min/ . m , % ci . - . p= . ), diastolic bp (or . per mmhg, % ci . - . p= . ), and mmf use (or . , % ci . - . p= . ). macroalbuminuria (ac> mg/g) was seen in . % ( / ) and significant predictors in a mulivariate logistic analysis were lower egfr and higher systolic bp. sirolimus use was associated with more macroalbuminuria and mmf use with less macroalbuminuria. in a subset of patients followed for > years prior gfr loss was considerably greater (p= . ) in patients with albuminuria (- . ml/min/ . m /year) compared to those without (- . ml/min/ . m /year). however other measures of proteinuria were also significantly (p for trend) associated with prior gfr loss (ml/min/ . m /year) as shown in the < . (n= ) . - . (n= ) > . (n= ) ∆ egfr/year - . - . - . . * a pc cut point of . g/g had a sensitivity and specificity for albuminuria > mg/g of % and % respectively (c= . , % ci . - . ), and a pc cut point of . g/g had a sensitivity and specificity for macroalbuminuria > mg/g of % and % respectively (c= . , % ci . - . ). ac may be more sensitive and therefore have more clinical utility than other measures of proteinuria for progression. however prospective follow up of renal function change and cv outcomes is required. serum creatinine is a crude marker of gfr in renal transplant recipients and changes in gfr are frequently not accompanied by commensurate changes in serum creatinine concentration. serum cystatin c and estimates of gfr (egfr) based on cystatin c have been shown to be more accurate than serum creatinine and creatinine-based egfr in renal transplant recipients. the purpose of this study was to determine whether the filler, lebricon and rule cystatin c-based egfr equations were better able to detect changes in true gfr than the mdrd and cockcroft gault creatinine-based egfr equations. we performed two measures of m tc-dtpa gfr, serum creatinine and serum cystatin c on each of stable renal transplant recipients at least months apart. we calculated and compared the percent annual change in the measured gfr and the estimated gfr using the various gfr estimation equations. we also determined the sensitivity, specificity, positive predictive value and negative predictive value of each prediction equation for the detection of decline in measured gfr. results are presented below: the cystatin c and creatinine-based egfr equations all demonstrated poor sensitivity and diagnostic performance to detect a decline in gfr. novel equations derived and validated in the transplant population are needed to accurately assess kidney function over time. background: hypercalcemia, hypophosphatemia and renal phosphate wasting are common after kidney transplantation and are related to persistent hyperparathyroidism and hyperphosphatoninism. animal data suggest that these alterations in mineral metabolism may contribute to nephrocalcinosis and progressive graft dysfunction. supporting clinical data are limited. aim: to test the hypothesis that nephrocalcinosis is highly prevalent in the early posttransplant period and is related to a disturbed mineral metabolism. methods: biomarkers of mineral metabolism (including albumin-corrected serum calcium [ca c ], serum phosphorus [p], biointact pth, calcidiol, calcitriol and alkaline phosphatase) and renal calcium and phosphorus excretion parameters were prospectively assessed in renal transplant recipients ( % male, mean age ± yrs) at the time of their -month protocol biopsy. these protocol biopsies were screened for the presence of microcalcifications. intratubular, interstitial and/or cytoplasmatic microcalcifications were observed in . % of biopsies. calcifications were more prevalent in recipients of a living related donor as compared to cadaveric donor. high serum ca c levels, high serum pth levels, a high urinary ca×p product and high fractional excretion of p and low serum p levels were significantly associated with renal microcalcifications (see figure below). microcalcifications were not related to the fractional excretion of ca, use of diuretics, immunosuppressive regimen, serum alkaline phosphatase level and history of delayed graft function. the extent of microcalcifications correlated significantly with the severity of mineral metabolism disturbances. conclusion: our data demonstrate that nephrocalcinosis is highly prevalent in the early posttransplant period and suggest that a disordered mineral metabolism is implicated in its pathogenesis. polymorphism in abcb , the gene encoding for p-glycoprotein, predicts recovery of graft function early after kidney transplantation. the pharmacokinetics of cyclosporine (csa) is characterized by wide inter-individual variability. this might be particularly relevant in the early post-transplant period, due to the detrimental effects of the drug on the kidney. p-glycoprotein (p-gp), the product of the abcb gene, plays a key role in the distribution of csa at cellular level. single nucleotide polymorphisms (snps) of abcb might potentially influence the response of patients to csa. in particular, the snp in position of the exon , despite its silent nature, has been recently associated with altered specificity for its ligand, such as csa (kimchi-sarfaty et al, science , : ) . whether p-gp pharmacogenetics would help to guide csa treatment early post-transplant remains ill defined. we sought to evaluate the effects of the snps in the exon on the rate of recovery of graft function, as estimated gfr early postoperatively, in kidney transplant patients given csa as part of their immunosuppressive regimen. the frequency of dgf (as need for post-operative dialysis) among the different abcb genotypes was also estimated. of the kidney transplant recipients, % had the abcb wild type (c/c) genotype in exon , % were heterozygous (c/t) and % were homozygous (t/t) for the polymorphic variant in position . gfr values were significantly lower in patients carrying one of the two mutant alleles than in the wild type ( figure) . the frequency of dgf was %, % and % in patients with the cc, ct and tt genotypes, respectively. these findings demonstrate that in patients carrying the ct or tt mutant alleles in exon of the abcb gene and given csa, the recovery of graft function is less prompt, and the risk to develop dgf higher than in wild-type cc genotype. pre-transplant screening for abcb polymorphism would help to identify patients who may safely receive csa early post kidney transplantation. cigarette cigarette smoking has shown to reduce graft and patient survival in renal transplant patients. however, whether it could directly produce allograft disfunction has not been investigated. the aim of this study was to assess the smoking influence on renal graft function. we studied a cohort of adult renal transplant patients, transplanted from jan/ to dec/ and followed until dec/ . smoking habits were recordered at the time of transplant (never, former, current smoker). during summer of , a telephonical survey allowed us to obtain complete information about smoking habits in patients (aged . ± , % male): status (never, former, current), years of habit, years of quit, and number of cigarette smoked per day. number of "pack-years" was calculated. renal function was measure by inverse serum creatinine at rd month and then annually. time to decline a thirty percent in inverse serum creatinine was registered. patients were divided in two groups: those who always smoked during all transplant period (smokers, n= ) and those who did not (n= renal insufficiency occurs frequently after extrarenal transplantation as a result of acute tubular necrosis at transplantation, high blood pressure, and cni toxicity. we performed renal biopsies in patients after heart ( ), lung ( ) , liver ( ), bone marrow ( ), and cornea ( ) transplantation since . the time from transplantation to biopsy was ± months in general and was longest after liver ( ± months) and shortest after bone marrow transplantation ( ± months). the histologic changes were: tubular atrophy/interstitial fibrosis of ≥ % in % of biopsies (heart biopsies %, lung %, liver %, bone marrow %); acute tubular changes in % (heart %, lung %, liver %, bone marrow %); arteriolar hyalinosis in % (heart %, lung %, liver %, bone marrow %); arterionephrosclerosis in % (heart %, lung %, liver %, bone marrow %); glomerular sclerosis of ≥ % in % (heart %, lung %, liver %, bone marrow %); glomerulonephritis in % (heart %, liver %, bone marrow %; that means iga-nephropathy after heart and liver, immune complex nephritis twice after liver, mpgn after liver, and membranous glomerulonephritis and minimal changes after bone marrow transplantation); thrombotic microangiopathy in % (lung %, liver %, bone marrow %); finally one case of polyoma nephritis after lung transplantation. among heart and lung transplantations, patients needed kidney transplantation ( . %) after ± months; and among liver transplantations, needed kidney transplantation ( . %) after ± months (sign. later, p= ). conclusion: as expected, most histologic changes were those of cni toxicity and hypertension. surprising is the high number of glomerulonephritis under immunosuppression. thrombotic microangiopathy without the typical clinical signs were interpreted as cni-related toxicity and seems to occur more often after extrarenal than after renal transplantation. patients with heart and lung transplantation reach end-stage renal failure more often and earlier than patients with liver transplantation. the context: living kidney transplantation, a superior therapy to deceased donor kidney transplantation, is underutilized. states have enacted legislation and the federal government has launched initiatives to compensate living organ donors, but the effect of policy on improving living kidney donation rates in the united states is unknown. objective: to determine whether public policies are associated with changes in living kidney donation rates in the continental u.s. design, setting, and study subjects: series of cross-sectional analyses using records of state legislatures in continental states and living kidney donation rates from the united network for organ sharing. main outcome measures: living kidney donation rate during each year from - and change in donation rates before and after legislation enactment in each state and launch of federal initiatives. results: from january through december , states enacted legislation for living donors ( mandating paid leave, tax deductions, unpaid leave, encouraging paid leave). few states (n= ) enacted legislation prior to . there was a steady increase in the mean living kidney donation rate in the continental u.s during the study period (mean (standard deviation) annual increase in donations . ( . ) donations per , , population). in analyses accounting for length of time state legislation had been enacted, the types of legislation enacted, and the incidence and prevalence of esrd in each state, there was a slightly (but not statistically significantly) greater average annual increase in donations after compared to before state legislation enactment (annual increase in donations per , , population [ % confidence interval ( introduction: accurate and precise renal function assessment is essential in the evaluation of prospective kidney donors. while direct measurement of gfr is the "gold standard", it is not widely available. moreover, creatinine (scr)-based estimation equations are suboptimal to assess kidney function in this setting. ct scans are increasingly being used to study renovascular anatomy in donors and has replaced angiographic exams in many institutions. d imaging reconstruction allows for kidney volumes (kv) measurements which have been shown to highly correlate with measured gfr in this population. thus, the purpose of this study was to develop a model to estimate measured gfr that not only incorporates scr and demographic data but also kv as measured by d ct scans. methods: individuals who underwent donor evaluation were identified. an automated segmentation algorithm was used to measure renal parenchymal volume from preoperative abdominal cts. patient demographics and scr values were obtained from the medical records. gfr (normalized for bsa) was measured by i iothalamate renal clearances (igfr). an analysis of covariance model was created to correlate measured igfr with kv, patient age, sex, race, weight, height and scr. pearson's correlation coefficient was calculated for each variable. results: kv (p< . ), age (p< . ), scr (p< . ) and weight (p< . ) significantly correlated with igfr. sex ( . ), race ( . ) and height ( . ) were not statistically significant. the new fitted regression model is: kv-egfr (ml/min/ . m ) = . -( . *age) + ( . *weight) + ( . *volume) -( . *scr). we then compared the performance of the kv-egfr model to the re-expressed mdrd equation using calibrated scr assay. the r was . vs . , respectively; signed median % difference was + . % vs - . %, respectively (% difference b/w estimated gfr and igfr); and accuracy within % (% of estimated gfr values that fall within % of igfr) of . % vs . %, respectively. finally, the kv-egfr model was closer to igfr (in absolute values) than the mdrd eq. in / ( . %) cases vs / ( . %) cases, respectively. conclusions: kidney volumes highly correlate with igfr and the proposed gfr estimation model outperforms the mdrd equation in potential living kidney donors. the kv-egfr model could be used to estimate donor gfr in lieu of i iothalamate gfr which is less clinically available. for donor selection, current reports identify unsuspected renal pathology by time -biopsy. aim: to explore whether the findings at time -renal biopsy (bx) correlates with pre-donation clinical data including renal function. methods: kt databases from institutions were reviewed. time -renal bx are routinely performed from the upper pole during back-table and evaluated by nephropathologist for interstitial fibrosis (if), tubular atrophy (ta), arteriolar hyalinosis (ah), mesangial increase (mi), and glomerulosclerosis (gs). pre-donation data gathered from the donors were demography, body weight, bmi, systolic/diastolic bp, scr, proteinuria, and egfr by levey equation clinical data is summarized in the table. and gs showed no correlation. multivariate analysis failed to sustain the significant associations found on bivariate analysis, most likely due to a low event/parameter relation. conclusions: a significant correlation was observed between time -bx findings and clinical pre-donation parameters. whether these histological findings at the time of kidney donation represent a higher burden/risk for the remaining kidney ought to be evaluated during follow-up. in an era where living donation is increasing, we should advise a closer surveillance of these donors in order to modify risk factors that participate in kidney damage progression. predictors of poor early graft function following laparoscopic donor nephrectomy (ldn). matthew cooper, abdolreza haririan, stephen jacobs, michael phelan, benjamin philosophe, stephen bartlett, joseph nogueira. dept of surgery, urology, and medicine, university of maryland, baltimore, md. ldn has become the standard of care in many transplant centers. poor early graft function remains an important complication. we conducted a retrospective study to evaluate the risk factors for slow or delayed graft function following ldn methods: donor and recipient records from the first ldn were reviewed ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: slow graft function (sgf) was defined as cr> . mg/dl at pod and dgf as the need for dialysis within the first week following transplantation. donor variables examined included age, sex, race, bmi, egfr, and number of renal arteries. recipient variables included age, sex, race, bmi, prior tx, pre-tx dm, history of smoking and drug usage. additional variables evaluated included degree of relationship (lurt), hla mismatch, antilymphocyte induction, de novo cni usage, r v. l nephrectomy, wit, total or time, and performance of simultaneous deceased donor pancreas tx (splk). univariate analysis was performed with significance defined as p< . . significant variables were then included in the multivariate analysis. background: a kidney exchange program is the logistic solution for patients with positive cross match (x + ) or abo incompatible donors. a major problem in all kidney transplantation programs is the sensitized recipient. we analyzed the success rate for immunized recipients in the dutch kidney exchange program. methods: from january till december donor-recipient pairs were registered. there were couples in the x + group while pairs were abo incompatible. in the x + group the median pra was % ( - %). to create new combinations a match program was run times (every months) with a median of ( - ) participating couples. allocation criteria included bloodtype (first identical, then compatible), hla match probability within the actual exchange donor pool to ensure that highly sensitized recipients have the best chance to receive a kidney, and the waittime on dialysis. cross matches between new donor and recipient were performed centrally in our reference laboratory with cdc-tests. results: after match runs, we found matching couples for / ( %) x + pairs, for / ( %) abo incompatible pairs with o recipients and for / ( %) abo incompatible pairs with non-o recipients. median pra of the recipients in the x + group was % ( - %). after match runs chances for success became small. the overall success rate for abo incompatible and x + pairs in the dutch kidney exchange program after years is %. however, the success rate for immunized patients in the x + group is significantly higher ( %) as our match program gives priority to those recipients with the smallest chance of finding a compatible donor in each match run. thus our kidney exchange program is especially suited for immunized patients. although paired kidney donation (pkd) program is an established method to overcome incompatibilities between kidney donor-recipient pairs (drp), significant proportion of the incompatible drp participating in such program could remain unmatched. domino-kidney transplantation (kt) in which altruistic living non-directed donor kidney (lndk) is offered to a pool of incompatible drp, and is used to initiate a chain of pkd transplants, could provide more opportunities of kidney transplantation to drp in pkd program. we introduce our experience of multicenter domino kt for the last years sixteen hospitals participated in the domino-kidney transplantation between february, and july, . domino-kidney transplants were performed with domino-kt chains initiated by altruistic lndk. -pair chains were , -pair chains , -pair chains , -pair chains , and -pair chains . the development of a multi-regional kidney paired donation program. using an optimization matching algorithm the new england program for kidney exchange (nepke) efficiently matches incompatible donor/recipient pairs. in , the mid-atlantic paired exchange program (mapep) and other individual centers began sharing data with nepke. this is a report of the success of two regional programs working together to increase the probability of participants in both programs finding a compatible match. methods: incompatible pairs and non-directed donors (ndd) are referred to nepke through transplant centers. donor and recipient abo, hla and recipient hla antibody screening are entered into the computer database. utilizing the optimization program, searches for compatible matches are conducted every to days. the program identifies potential and -way matches, ndd chains, and list exchange chains. following the determination of compatibility nepke notifies transplant centers involved of the potential match and centers accept or decline the offer. transplant centers notify their pairs and preliminary crossmatches are performed. a conference call is scheduled to coordinate simultaneous donor nephrectomies and recipient transplants. surgeons speak prior to incision to ensure simultaneous donation. results: from july to december , pairs and ndds have entered nepke. during this time frame over one thousand possible matches were identified, with the majority of these matches involving the same pairs in multiple matches. after "optimizing" and eliminating multiple matches two-way exchanges; three-way exchanges; three-way list chain exchanges; and ndd chain matches were offered to transplant centers as possible matches. most common reason offers were declined include: positive crossmatch ( . %); donor factors ( %), and recipient inactive or transplanted ( %). four offers occurred in mapep alone pairs, in nepke, and offers were cross-regional. three matches are pending for additional transplants. one previous and one pending transplant are the result of cross-regional exchanges. five transplants performed and pending involve ndd chains. one pending match involves a list exchange chain. conclusion: using a computerized optimization algorithm to match and way exchanges, ndd and list exchange chains has lead to a substantial increase in the number of kpd matches and transplants performed. cross-regional coordination is feasible and expands the number of transplants performed beyond the ability of individual exchange programs. as living donors become an increasingly important source of life-saving organs, there is growing concern about the lack of comprehensive research on donor outcomes. in addition to possible long term consequences, there is a risk that donors will experience complications during or following surgery. of the , living kidney donors in - , none died during surgery, and . % (n= ) needed blood transfusions during surgery. in the six weeks following donation, . % (n= ) had at least one serious adverse event (sae): . % (n= ) needed readmission following initial discharge, . % (n= ) needed an interventional procedure, . % (n= ) needed re-operation, . % (n= ) had vascular complications, and . % (n= ) had other complications. when all saes were considered in combination, the rate was . %. because over % of ldr forms were submitted by transplant centers fewer than weeks post-donation, all complication rates should be considered minimum estimates. one donor was reported to have died from donation-related causes within weeks of donation. the number of living donor kidney transplants performed by a transplant center in - ranged from to transplants. additionally, the risk of donor complications is not equal across transplant centers. for example, there was a significant correlation between the number of living donor kidney transplants performed at a transplant center and the percentage of that center's patients who were readmitted within weeks of donation, with greater donor volume associated with a lower rate of readmission. living kidney donation is relatively safe, but prospective donors should be made aware that there is a non-trivial risk ( . %) of short-term complications post-donation. as with many other major surgical procedures, complication rates are lower, on average, at institutions that perform a larger number of these procedures. we sought to determine if intensive screening improves detection of polyomaviral reactivation in asymptomatic patients and pre-emptive stepwise modification can improve outcome of polyomaviral nephropathy (pvn). methods:this is a prospective single center study. we randomly assigned de novo kt (cluster randomization) to intensive screening (is:n= ) and routine care (rc: n= ) for the detection of decoy cells. is was initiated at week- of kt and rc at the time of increase in serum creatinine. this was complemented with urine and blood nucleic acid testing. all patients had biopsies performed for detection of polomya nephritis (pvn). both groups were treated with pre-specified stepwise modification of it based on cell cytology and viremia (step : decrease dose of cellcept by %, step : decrease dose of tacrolimus by %, or switch to sirolimus therapy, step : discontinue cellcept). primary outcome included persistence of decoy cells/viremia following each step in modification of it every three months and secondary outcomes included acute rejection, graft function and graft loss. results: polyomaviral reactivation developed in . % in is group and % had pvn without changes in serum creatinine. the estimated cumulative rate of primary outcome in the is versus rc groups, -months ( % vs. %) relative risk (rr), . ; %ci, . - . ;p= . ); -months ( %vs. %) (rr= . ; % ci, . - . ; p= . ); and -months ( %vs. %) (rr= . ; % ci, . - . ; p= . ). secondary outcomes: despite similar degrees of step-wise is modification, rate of acute rejection non-significant (p= . ), but % of patients in rc loss the graft vs no graft loss in is group. patients who continue to remain on tacrolimus vs. those who were switched to sirolimus therapy had persistent viremia (or= . ; %ci, . - . ; p= . ). conclusion: is for poloymaviral reactivation allows early detection of pvn in the presence of stable graft function. stepwise modification in it resulted in early resolution of decoy cells and viremia in both groups, albeit slowly in rc group, and it did not prevent the graft loss in rc group. background: dna sequencing of the bk viral (bkv) genome non-coding control region (nccr) from individual patient isolates demonstrate divergent sequence and alterations in the arrangement of modularly conserved sequence blocks (p-q-r-s) ranging in size from to base pairs. aim: primary aim to molecularly clone and analyze patient-derived bk virus nccr sequence variants. our secondary aim is to determine if reporter gene constructs of patient-derived nccr variants differ in their promoter activity upon transfection into a mammalian cell line (vero) and a human primary tubular epithelial cell line. methods: bkv dna was amplified and sequenced from blood and urine samples of renal transplant recipients. via sequence alignment, unique nccrs were determined. these sequences were pcr amplified and cloned into reporter plasmids containing the renilla luciferase gene. promoter activity was measured via luminometer hours after transfection into vero cells and human tubular epithelial cells. results: variation in naturally occurring bkv nccr promoter regions exist as single basepair insertions and deletions, and insertions or deletions of partial sequence blocks (p-q-r-s). single basepair substitutions were most commonly seen ( % of analyzed samples). promoter activity within vero cells ranged from % to % as compared to nccr activity of an archetypal strain (wwb). low promoter activity (< %) was seen in isolates with duplications of the p block and large deletions of the r block. conclusion: these sequence blocks are rich in regulatory elements and control the expression of both bkv structural and regulatory genes. variation in these cisacting eukaryotic transcriptional promoter binding sites corresponds to differential promoter activity in naturally occurring bkv isolates. current plans include repeating the promoter activity studies in human primary tubular epithelial cells. humoral and cellular immunity to polyomavirus bk large t and vp antigens after pediatric kidney transplantation. polyomavirus bk-associated nephropathy (bkvn) has emerged as a cause of graft failure after kidney transplantation (ktx). in a cohort of pediatric renal recipients undergoing prospective three-monthly monitoring for bk by blood and urine q-pcr, we evaluated antibody response, measured by enzyme immunoassay using bk vlp, and cellular immune response, reported as frequency of ifnγ-secreting cells in a elispot assay after -day stimulation with bkv large t (lt) and vp peptides. we could not observe any influence of recipient pre-transplant bkv-specific igg or t-cell levels, which were generally low, on bkv infection after allografting. after transplantation, both specific igg levels, and frequency of bkv-specific t cells increased according to the degree of viral exposure. in detail, bkv-seropositive patients who never reactivate the virus (group , n= ) did not show significant increase in igg levels (from a median od of . at month + to . at the end of follow-up), while patients with urinary shedding alone (group , n= ) or with viremia (group , n= ) increased from a median od of . to . (p= . ), and . to . (p> . ). in the case of cellular immunity, vp -specific t-cells increased in the three groups. conversely, lt-specific t-cells, which were high (median sfu/ cells) and remained unchanged throughout the follow-up period in group patients, had a significant increase in recipients belonging to both group and . interestingly, patients with urinary shedding who do not progress to viremia show a median -fold increase in lt-specific t-cell levels at peak viruria, compared to no increase observed in patients who develop viremia. the latter group mount a significant response to lt only after therapeutic reduction of immunosuppression. at peak viruria, viremic patients already show a -fold rise in specific igg compared to the . increase observed in group recipients. our data suggest that inability to reach protective levels of bkv lt-directed t cells, rather than specific igg, predispose ktx recipients to bkv replication. introduction:the antibody response to human bkv virus (bkv) is incompletely characterized. antibody responses to the vp- protein have been detected in kidney transplant patients, but it is not known if these have virus neutralizing activity. methods:recombinant bk, jc, and sv virus like particles were used to produce a panel of monoclonal antibodies. these antibodies were characterized for isotype and for ability to bind the respective antigens in elisa assays. to test neutralizing activity, bkv gardner strain (atcc# vr ) viral particles were incubated with the corresponding antibodies for hours at degrees c, and used to infect wi cells. bkv infection was monitored by quantitative real time pcr using primers directed against the vp- gene. neutralizing activity was defined as greater than % inhibition of viral yield. results:the monoclonal antibodies were of the igg a or igg b class with the exception of one igg and one igm antibody. all anti-bkv monoclonal antibodies bound to bkv capsids in-vitro in elisa assays. this binding affinity was species specific, as only antibody showed weak binding activity to jcv and sv capsids. neutralization of infectious bk virus was shown for / antibodies. denaturation of capsid proteins indicated that the monoclonal antibodies recognized primarily conformational epitopes, with only monoclonal antibody appearing to have a linear component. paucity of linear epitopes was further suggested by lack of reactivity of sera from bkv seropositive subjects in elisa assays based on genotype-specific short peptide sequences derived from the bkv vp- loop region. four monoclonal antibodies each generated from jcv capsids and sv capsids did not show any bkv neutralizing activity. conclusions: bkv vp- protein capsids contain species specific conformational epitopes which can elicit virus neutralizing and non-neutralizing antibody responses. measurement of these antibodies in renal transplant recipients may have diagnostic and prognostic applications. bkv specific monoclonal antibodies deserve further study as potential therapy of acute infections in the viremic phase. impact of immunosuppression reduction in bk viremic patients: year follow-up. s. kuppachi, a. guasch, c. p. larsen, k. e. kokko. transplant center, emory university, atlanta, ga. background: development of bk nephropathy is a risk factor for allograft loss. bk viremia (bkv) precedes the development of bk nephropathy. it has been reported with -year follow-up that reduction of immunosuppression leads to control of bkv. here, we report our -year follow-up experience of bkv patients that were identified by a prospective screening protocol and managed by sequential reduction of immunosuppression. methods: all kidney or kidney-pancreas transplant recipients at emory university between / - / were screened prospectively for bkv by real time pcr during follow-up visits ( - , , , , ) . patients with a bk viral load of greater than , copies/ml blood received a kidney biopsy to screen for bk virus nephropathy by immunohistochemistry. bkv without nephropathy resulted in reduced immunosuppression by a % reduction in mycophenolate dose. bkv with nephropathy resulted in discontinuation of mycophenolate. all identified bk patients were monitored every - weeks until viral load was below , copies/ml. immunosuppression was further reduced if viral loads failed to decrease. results: recipients were followed over a -month period. patients had received simultaneous kidney and pancreas, a liver and kidney and the rest kidney alone. of ( %) patients developed bkv within a year from time of transplantation. average time to diagnosis of bkv was . months. average dose of mycophenolate at years was . g/d in the bkv negative population as compared to . g/d in the bkv positive population. average hour trough blood level of tacrolimus at years was . ng/ml in the bkv negative population as compared to . ng/ml in the bkv positive population. survival rates for both patient and organ were comparable at years in bk viremic vs bk negative patients ( % vs %) and ( % vs %) respectively. while bk viremia is a historic risk factor for organ loss, prospective monitoring and reduction of immunosuppression is associated with comparable year patient and organ survival to patients that never develop bk viremia. background: there are currently no bk virus (bkv) specific therapies available for clinical use. this study evaluates viral large t antigen as a potential target for drug development, since (a) this is a key molecule that participates in several different stages of viral replication, (b) has no homologous human protein, and (c) offers multiple functional domains for chemical binding, particularly the atp binding site, the dna binding site, the hexamerization surfaces, and the hinge region. methods: virtual screening and protein modeling techniques were applied to bkv large t antigen using a model developed from the known crystal structure of sv large t antigen. two different structural states of large t antigen (monomer and dimer structure) in three different states (with the nucleotide pocket empty, with bound adp and bound atp), were evaluated for a total of large t antigen receptor conformations. results: a computational solvent mapping analysis of small molecular probes allowed identification of multiple functional sites, which represent potential drug binding pockets on the large t antigen molecule. it was possible to classify molecular conformations centered on the atp binding site, hexamerization surface and hinge region of the viral protein. we docked medium sized fragments (< da) to confirm the results obtained by computational solvent mapping, and further characterize chemical properties of the atp binding site. in another approach, known chemical structures of hsp and rho-kinase inhibitors were used to search compound databases and obtain a subset of compounds (mean size of da) capable of docking large t antigen. cross-referencing the top solutions obtained by energy ranking, we were able to identify compounds that bind large t antigen in all conformational states. a subset of compounds simultaneously binds the atp binding site, hexamerization surface and hinge region of bkv large t antigen. conclusions: virtual screening and three dimensional homology modeling technology has allowed us to identify compounds that can bind multiple sites on the large t antigen. these compounds are predicted to preferentially inhibit viral replication without the toxicity expected from simultaneous inhibition of host cell kinases. bk virus (bkv), a human polyomavirus, causes bkv nephritis, which often leads to graft loss after renal transplantation. currently, the only efficient therapy against bkv nephritis appears to be a reduction/change of immunosuppressive agents, and this may increase the inherent risk of rejection. since human renal proximal tubular epithelial cells (hrptec) represent a main natural target of bkv nephropathy, the analysis of bkv infection of hrptec is likely to provide necessary additional insight into bkv biology and contribute to the development of strategies for treatment of bkv nephritis. here we report the ability of -hydroxy- -methyl-glutaryl coenzyme a (hmg-coa) reductase inhibitor pravastatin, which is routinely used to treat hypercholesterolemia, to repress bk virus entry pathways in hrptec and, correspondently, prevent bkv infection. the percentage of hrptec infected with bkv was assessed by immunofluorescent analysis in the absence and presence of pravastatin. both, the percentage of bkv infected cells and the intensity of bkv infection, assessed by western blotting using antibodies against large t antigen, were significantly decreased in hrptec treated with pravastatin. it is likely, that pravastatin's inhibitory effect is explained by depletion of caveolin- , a critical element of caveolae. we demonstrate that bkv enters hrptec by caveolarmediated endocytosis and disruption of caveolin mrna and protein inhibits bkv infection of hrptec. we provide evidence that pravastatin dramatically decreased caveolin- expression in hrptec and interfered with internalization of labeled bkv particles. our data suggest that pravastatin, acting via depletion of caveolin- , prevented caveolar-dependent bkv internalization and repressed bkv infection of hrptec. our data represent the first report of inhibitory action of statins upon bkv infection. results: . % of patients were caucasian, . % hispanic, . % african-american and . % asian. overall -and -yr patient survivals were % and %. stratified by race, only african-american survivals differed from caucasian ( -year survivals of % vs. %; figure ). compared to caucasians, african-american patients were younger ( yrs ± . vs. yrs ± . ), were more likely to be status ( . % vs. . %), to have a serum creatinine ≥ . mg/dl ( % vs. %), to receive an multi-organ transplant ( % vs. %), to have fulminant hepatic failure ( % vs. %), to have a higher meld score ( . ± . vs. . ± . ) , to be in the icu ( % vs. %), and to be ventilated pre-transplant ( % vs. %); all p-values < . . after adjustment for each of these variables, race was still an independent predictor of mortality (p= . , hr: . , ci: . - . ). multivariate analysis of the african-american group (n= ) alone revealed that a bmi greater than (p= . , hr: . , ci: . - . ), a creatinine greater than . mg/dl (p= . , hr: . , ci: . - . ), and icu admission pretransplant (p= . , hr: . , ci: . - . ) are independent predictors of mortality in this subset of patients. conclusion: in the current meld era, race is still a predictor of worse outcomes, even after adjustment for multiple clinical variables. further work is necessary to elucidate why this disparity exists. the purpose of this study was to analyze the effects of successive pregnancies in female liver transplant recipients on newborn and maternal outcomes. data were collected from the national transplantation pregnancy registry via questionnaires, phone interviews and hospital records. analyses for linear trends (proportions and continuous variables) were done by chi square and least squares regression. there were outcomes of pregnancies, including twins. of the liver recipients who had a first pregnancy, had between one and four subsequent pregnancies. there were no significant differences in the variables analyzed as noted in the conclusions: successive pregnancies in liver transplant recipients are not associated with adverse fetal outcomes and/or increased maternal graft loss. female liver recipients with excellent allograft function without significant recurrent disease or chronic rejection who wish to have more than one pregnancy should not be discouraged to conceive. recorded and categorized in a blinded fashion. univariate, multivariate and survival analyses were performed. over a . -year period ( ) ( ) ( ) ( ) ( ) ( ) , olt recipients were randomized to receive a cca with biliary stent (n= ) or cca alone (n= ). patients with hepatic artery thrombosis (n= ; . %) were excluded. there was no significant difference in demographic or graft-related variables. the mean age at transplant was years, % were male, the mean meld was and % had hepatitis c. the mean donor age was years, % of donors were male, and the mean cold ischemia time was . hrs. the only complication related to the biliary stent was one occlusion. the rate of overall bc in the stented patients was . % vs. . % in non-stented patients, (p=ns). however, stented patients had significantly less bc in the first -days post-olt ( . % vs. . %, p< . ) and significantly less anastomotic leaks ( . % vs. . %, p< . ). over the year following olt, stented patients also required less biliary therapeutic interventions (mean . vs. . interventions/patient, p< . ) and fewer readmissions (mean . vs. . readmissions/patient, p< . ). we also observed improved late graft survival (> mo) in the stented group. intraoperative stenting of the cca at olt does not appear to reduce the long-term rate of bc but it decreases the incidence of biliary leaks and significantly improves many facets of early patient management. is background: long-term outcomes after retransplantation of the liver (re-olt) is inferior compared to primary olt. however, the survival benefit of re-olt based on model for end stage liver disease (meld) is not known. a single-center analysis of adult patients who underwent re-olt between february to february was performed. survival benefits, at a given meld score, were calculated by comparing re-olt survival at months to expected -month survival without retransplantation results: of pts, underwent re-olt, and received transplants. the figure shows re-olt survival benefit at any meld score with increased significance in patients with meld scores > . although meld scores - predicted the highest mortality after re-olt, they also demonstrated survival benefit. multivariate cox regression identified cold ischemia time > hrs (rr . , p . ), meld - (rr . , p . ), time from first olt ( days - year, rr . , p . ) and third transplant (rr . , p . ) as independent predictors for mortality following re-olt. conclusions: re-olt should be considered in all patients even with low meld scores. although patients with high meld scores ( - ) exhibit poor survival outcomes, a survival benefit is achieved. survival benefit that considers both probability of death without re-olt and expected survival with re-olt, should be used for selection of retransplantation candidates. this study analyzed posttransplant complications, meld score, and donor ages and their effect on length of stay (los). methods: this irb approved retrospective review of our prospectively maintained database included liver transplant recipients transplanted between - . los < days, - d, and > d were analyzed. primary analysis to look at los, meld, and donor age was performed using wilcoxon two-sample test. complication groups were analyzed using logistic regression and odds ratio estimates were determined. kaplan-meier for patient survival for the los groups was performed. univariate analysis: allograft dysfunction, vascular complication of liver allograft, intra-abdominal (other than liver), biliary, cardiac, pulmonary, neurologic, sepsis, renal, and endocrine were statistically significant (p< . ) using fisher exact test. multivariate analysis: using logistic regression, significant complications were analyzed to determine the complications linked with los > d and > d. analysis of liver transplants demonstrated increasing los with increasing meld in each donor age group ( - , - ) p< . . for donor age > , this relationship was not significant (p= . ). multivariate analysis using logistic regression determined odds ratio estimates for each complication resulting in los > days and > days. los > days and los > days were associated with different complications (as shown above). allograft dysfunction (including pnf) and renal complications were not significant factors in multivariate analysis. patient survival significantly decreased (p< . ) with increased los > days. introduction: some patients with primary biliary cirrhosis (pbc) may require longterm corticosteroid (cs) therapy following liver transplantation (olt) due to recurrent inflammation in the graft. our center has attempted to minimize cs use in all of our olt recipients. we reviewed our experience in this cohort of patients to determine ) patient outcome including recurrent disease and ) long-term requirement for cs use in pbc patients. methods: from to , , olts were performed in , adults at the university of colorado of which patients ( . %) with pbc received allografts. recurrence was defined by characteristic histologic changes on biopsy. bivariate and multivariate analyses were used to evaluate predictors of cs withdrawal. potential predictors of cs discontinuation were considered: age, gender, bmi, race, presence of inflammatory bowel disease (ibd), type of graft (cadaver or living donor (ld), recurrence of aih, warm ischemia time, follow up time (time since transplant), and immunosuppressant (is). results: overall survival at years was %. the , and year recurrence-free survival was , , and %, respectively. disease recurred in patients ( . %). of these patients, none received a second transplant because of recurrent disease. cs was withdrawn in % of patients at time of review. independent predictors of cs discontinuation are age (>median) (p = . ) and ld graft type (p= . ). conversely, cyclosporine (csa) (p= . ), female gender (p= . ), and bmi > (p= . ) were negatively associated with cs withdraw. interestingly, cs withdrawal did not influence pbc recurrence. conclusions: ) long-term outcomes in pbc patients are favorable and disease recurrence can be managed medically without abstracts re-transplantation. ) using an aggressive cs minimization approach, almost / of the patients were cs-free at the time of last follow-up. ) increasing age and ld grafts were associated with successful cs withdraw; while csa, female gender, and increasing bmi were associated with unsuccessful cs withdraw. incidence cholestatic disease (cd), either chronic rejection or recurrent primary sclerosing cholangitis (psc), post liver transplantation (lt) occurs in - % of psc grafts. the study objectives were to evaluate the incidence and long-term outcome of cd. from to , grafts in consecutive psc patients and grafts in concurrent alcoholic liver disease (ald) patients were compared. cd was diagnosed by biliary imaging and/or histology. median follow-up was months. the groups were similar including meld score and cold ischemic time; however, roux-en-y biliary anastomosis was more common in the psc group ( % vs. %, p< . ). the psc group had more cmv hepatitis ( % vs. %, p< . ) and acute rejection ( % vs. %, p< . ), and fewer biliary anastomotic strictures ( % vs. %, p< . ). cd occurred in psc grafts and ald grafts (p< . ). the incidence of cd was greater in the psc group (p= . , fig. ). no significant risk factors for cd were identified. in the psc group the graft survival was lower in the cd group (p= . , fig. ) ; however, patient survival at and years was not effected by cd: % and % vs % and % because the re-lt rate was greater in this group ( % vs. %, p< . ). at years, patient survival in the psc group was better than for ald ( % vs %, p< . ). long-term outcome post lt for psc is good and better than for ald; however, cd continues to develop beyond the first years with a prevalence of % at years. graft loss in patients with cd is high, but with re-tx, patient survival is similar to non-cd patients. further studies to distinguish chronic rejection vs. recurrent psc may provide insight into the prevention of cd following lt for psc. discussion: our analysis showed that patients with aih have a worse long term survival compared to pbc and psc after ddlt. this may be explained by possibly more advanced disease at the time of presentation or more septic complications due to pretransplant salvage therapy with immunosuppressants. also,pbc had the worst relative outcome after ldlt in this group-possibly explained by the older age of the recipients. overall, ldlt offered better outcomes than ddlt in terms of survival in patients with aih,pbc and psc. this study highlights an important and previously unvisited aspect of transplantation for autoimmune and cholestatic liver diseases. inflammatory bowel disease course in patients transplanted for primary sclerosing cholangitis. ariana wallack, joel s. levine, lisa forman. internal medicine, univ. of colorado hsc, aurora, co; gastroenterology and hepatology, univ. of colorado hsc, aurora, co. the natural history of ibd following liver transplant (lt) for psc is unknown. prior studies have not shown factors consistently associated with disease activity, but were limited by small sample sizes. the aim of the study is to describe our experience with ibd post-lt in patients with psc and determine factors predictive of disease activity. a survey was mailed to liver recipients transplanted for psc between and asking about medications, ibd activity and quality of life after lt. responses were linked to our lt database. results ( %) recipients responded. % were male with a median of ( - ) months since transplant. % were caucasian with a median transplant age of ( - ) years. % developed recurrent psc post-lt. % had a diagnosis of ibd ( % uc). immunosuppression included tacrolimus in % and cyclosporine in %. % experienced at least one episode of acute rejection. % rated their quality of health - on a scale of - . there was no significant difference in demographic variables between ibd and non-ibd cohorts. % of respondents had ibd pre-lt. of the patients without pre-existing ibd, developed ibd post-lt. of the de novo ibd cohort, % were male, median transplant age was years ( - ), and % were caucasian. ibd developed in % at more than years post-lt. there was no statistical difference between the de novo ibd cohort and those with pre-existing ibd except for ibd type ( % uc vs %, p= . ). significantly more patients were not on any ibd medications post-lt compared to pre-lt ( % vs %, p= . ). fewer post-lt patients were on aminosalicylates ( % vs %, p= . ) and prednisone ( % vs %, p= . ) compared to pre-lt. post-lt recipients developed fewer ibd flares requiring hospitalization ( % vs %, p= . ). % reported improvement in ibd activity post-l. % and % reported no change and worsening activity, respectively. there was no significant difference between reported disease activity and immunosuppression, cmv status, rejection rate, recurrent psc, transplant age, gender or race. background: symptomatic cmv continues to be a significant problem. the costs associated with its development remain high without an optimal method for prevention. the aim of this study was to measure the pharmacoeconomic impact of implementing an abbreviated pre-emptive monitoring strategy versus valganciclovir (vgc) prophylaxis in a large teaching hospital. methods: costs for this analysis were based on a societal perspective, including drug, personnel, hospital, outpatient infusion and monitoring costs related to resources needed to perform pre-emptive monitoring and treatment of cmv related events. time per hr costs were nurse/data coordinator $ , physician $ , and pharmd $ . cmv pcr cost was $ . vgc cost per mg was calculated for prophylaxis, treatment and days of consolidation as needed based on an awp ($ . per mg). estimated cost of cmv syndrome was based on cost of picc line placement, drug, personnel and supply costs based on days of therapy and was estimated to be $ , . . inpatient admission cost per day were $ . results: a total of patients were included in this analysis. baseline and transplant demographics were well matched. table displays the total direct and indirect costs accumulated for each group. cmv syndrome occurred in three patients for each group. there was no cmv disease in either group. % of patients in the pre-emptive group had dnaemia, but only patients required oral anti-viral therapy. provider time and lab monitoring costs were significantly higher in the preemptive group, while direct medication cost was significantly higher in the prophylactic group. conclusions: frequency of disease severity and outcomes were equal in each group. although the overall costs between strategies is equivocal, allocation of resources to provide pre-emptive monitoring places the burden of disease prevention on the health care system versus the patient necessitating further abbreviation of these strategies. we propose a non-simultaneous form of kidney paired donation that starts with a living, non-directed donor (lnd). a paired donation matching algorithm was developed to allow for lnds to start potentially never-ending altruistic donor (nead) chains in addition to closed loops of -, -and -way exchanges. results: in july , a lnd from michigan traveled miles to donate a kidney to a woman whom he had never met in arizona. the following week, the arizona recipient's husband donated one of his kidneys to a -year-old woman in ohio. the following month, the mother of this recipient traveled to a city hours away to donate her kidney to a patient whose incompatible donor simultaneosly gave a kidney to the fourth patient in the chain. the incompatible donor for this fourth recipient is now slated to give her kidney to a patient in maryland. over the past months, transplant programs have partnered to perform paired donation transplants. demonstrating the advantage of nead chains over classic paired donation, of transplants resulted from altruistic donor chains. conclusion: in order to fully realize the potential of the above approach, one must be willing to supplant two prevalent ideas: ) that kidneys from altruistic donors should be given to the top candidate on the deceased donor waiting list, and ) that paired exchanges must be done simultaneously. while there are certain pitfalls to using chains of donors as opposed to traditional "swaps" (i.e. the possibility that a donor could renege, or the accumulation of type ab donors who are not likely to be able to begin another chain), this proposed paradigm shift could result in a very significant increase in both the number and quality of paired donation kidney transplants. purpose: medical literature and national best practices correlate families' understanding of brain death with organ donation rates. analysis of hospital data demonstrated variability in family communication. although surgical residents frequently interact with family members of potential donors and play a critical role in the donation process, they receive no formal cst. we sought to determine if pre-training residents improved performances in cst involving explanation and notification of brain death to family members and aided efforts to achieve the national donation rate goal of %. methods: in collaboration with a regional organ procurement organization (opo), an educational model for end of life cst was developed. surgical residents were divided into groups. the first group (n= ) attended a -hour didactic session at the opo that included role-playing exercises explaining brain death to families. opo staff, trained to serve as family role-players and skills station coaches, debriefed residents after each simulation. the second group (n= ) received no specialized training. six weeks later both resident groups participated in formal videotaped family communication simulations. independent observers (hospital faculty and senior opo staff), blinded to training, evaluated residents' communication skills using a -parameter assessment tool. all residents reviewed their videotaped performances and evaluations, then repeated the simulations after six months. results: during the study period, the pre-trained resident group assessment scores increased by % (p= . ), while the untrained group increased by % (p<. ). while evidence of improvement existed in both groups, pre-trained residents consistently scored higher when compared to untrained ( % vs. %, p= . ). during this same time period, donation rates in the surgical intensive care unit (sicu) increased by %. conclusion: our educational model demonstrated effective training in communication skills during end of life discussions. although a direct relationship cannot be established, donation rates in the sicu increased after resident training. incorporation of this training program into resident education has the potential to improve donation rates and increase the number of organs available for transplantation. program. saverio mirarchi, graeme n. forrest, benjamin philosophe. medicine, university of maryland, baltimore, md; surgery, university of maryland, baltimore, md. objective: to improve the efficiency of care for solid organ transplant patients by having internists work in conjunction with transplant surgeons to manage transplant patients admitted to the hospital more than days post organ transplant. this includes patients who have undergone kidney, pancreas, and liver transplants which our center transplants over of these organs/year. in , the organ transplant program was divided into a surgical transplant service (sts) and medical transplant hospitalist service (mths). the mths consists of four full time physicians, one part time physician, and one nurse practictioner with daily rounds from a transplant surgeon on a weekly rotating schedule. methods: we analyzed our data from the past five years since the inception of the mths at a large university medical center. the length of stay (los) for the mths and sts were compared to data from the previous combined program as well as data from the university health consortium (uhc). in addition, we looked at the cost of care to determine if there were any savings related to reduced los and adjusted for the combined salary of the mths. results: in the review period, the total admissions for both the mths and sts averaged admissions/year. over the past five years the mths showed a major decrease in the los index, defined as the ratio between the observed los and the expected los based on uhc data. this index dropped from . to . for patients on the mths. there was also a parallel drop in the sts los index from . to . . this was associated with a significant cost savings for the hospital. on average, the program has realized an average savings of approximately $ , , per year. when accounting for the combined salary for the mths group which is , dollars/year, the total savings over the year period this amounts to $ . million. conclusion: the creation of a mths working within an organ transplant program at a large university center has been able to demonstrate a major decrease in los for transplant patients which has resulted in decreased costs and improved efficiency. this has also allowed more focused care for a complex medical population. we believe that our program can serve as a model for similar programs at other busy transplant sites. with increasing demand for kidney transplants(tx), more patients are opting to travel outside the us to obtain transplantation. we describe the characteristics and outcomes of kidney tx recipients followed at our center who traveled abroad for a kidney tx between and . methods: data were obtained via chart review. we compared demographics to all tx recipients at our center during the same period and compared post-tx outcomes to a cohort of patients transplanted at our center matched for age, race, tx year, dialysis time, prior tx, and donor type. median follow-up time was days (range - ). results: demographics are outlined in table. patients transplanted abroad were more likely to be asian and had shorter dialysis times. most patients were transplanted in china ( %) followed by iran ( %), the philippines ( %), and india ( %). living unrelated tx were most common. all patients were discharged on a calcineurin inhibitor, pred, and either mmf ( %), aza ( %), or rapamycin ( %). patients received induction. only patients received cmv prophylaxis. the median duration of hospitalization was days. the median time post-tx to initial visit at our center was days. patients required urgent admission to hospital, of whom lost their grafts. patients ( graft survival at -year was % for both "tourists" and matched recipients at our center. median time to graft loss was ( - ) days. mean scr and rejection -year post tx was not significantly different between recipients transplanted abroad and at our center. conclusion: compared to patients transplanted locally, patients transplanted abroad had similar graft survival and renal function post tx, but had a high incidence of infectious complications. [background] the primary benefits anticipated following successful induction of allograft tolerance are avoidance of the complications of long-term immunosuppression and prevention of chronic rejection. we have previously reported successful induction of renal allograft tolerance in recipients of hla mismatched combined kidney and bone marrow transplantation (ckbmt). no evidence of chronic rejection has been observed in these recipients after immunosuppression-free periods of to years. in the current study, we have evaluated the longer-term economic impact of this approach. [method] the conditioning regimen for ckbmt included cyclophosphamide, thymic irradiation, anti-cd mab, and a calcineurin inhibitor, which was discontinued after - months. stable renal transplant recipients receiving ongoing triple or double drug immunosuppressive therapy with compatible follow up times were compared with the four tolerant recipients. tolerant patients and stable patients on maintenance immunosuppression were also comparable with respect to their age, their original disease and donor-recipient histocompatibility. [results] the perioperative charges for ckbmt were approximately $ , higher than those for conventional living donor kidney transplantation. after - months, continuing medications in ckbmt recipients included only occasional over-thecounter analgesics. in contrast, the stable conventionally treated recipients were taking an average of pills daily. these included treatments required for de novo diabetes ( %), hypertension ( %), hyper lipidemia ( %) and gastro-intestinal symptoms/ prophylaxis ( %) in addition to their maintenance immunosuppression. these needs resulted in annual maintenance charges of over $ , /year/allograft recipient and do not include unmeasured costs related to issues such as quality of life or absences from employment. [conclusion] even with this admittedly expensive approach to tolerance induction, the overall medical costs for conventional kidney transplantation with ongoing immunosuppression and treatment for complications will exceed the cost of tolerance after approximately years. increasing african american donation rates in a midwest metropolitan community. susan gunderson, susan mau larson, david m. radosevich, clarence jones, bill tendle, tiffany scott. lifesource, st. paul, mn; transplant information services, university of minnesota, minneapolis, mn; southside community health services, minneapolis, mn. purpose: african americans are underrepresented in deceased organ donation in this + million community. historically minimal educational outreach had occurred and this study was designed to understand the community's disposition toward donation and to increase support for donation. methods: television, newspaper, and radio advertising aired over a month period beginning in using the nationally produced donate life-african american campaign as the primary intervention. other components included related faith-based and community outreach. the opo partnered with a minority focused community health clinic to survey african americans pre-and post-intervention. a mailed, selfadministered survey was sent to a sample drawn from organizational lists with a high likelihood of including african american households. for the evaluation, the sample was separated into ) community members and ) church members exposed to faithbased campaigns. results: african americans in this community have a sophisticated understanding of the importance of donation (average of knowledge questions scored correctly) in pre-intervention survey. in both community and church groups media exposure and donation knowledge increased after the media campaign (p< . and p= . respectively). similarly, donor designation rates for the entire population on the drivers licenses increased ( . % versus . %, p= . ). among all african americans the propensity to donate was, however, unchanged following the campaign. propensity to donate increased (p= . ) in the community sample whereas there was a reduced propensity to donation (p= . ) in the church sample. during the study time period the opo also experienced significant increases in donation authorization rates among african-americans, increasing from % to %. summary: a media based campaign combined with grassroots outreach is an effective tool to increase knowledge and awareness. partnership between the opo and community and faith based leadership was a significant positive byproduct of the project and should be included in future outreach efforts. background: it has been hypothesized that the clinical benefits of anti-thymocyte globulin (atg) induction therapy do not completely result from immunodepletion, but may also result from induction of immunoregulatory t-cells (treg). in this prospective, controlled study we investigated the effect of atg-induction therapy on the frequency and phenotype of peripheral cd + foxp + cd -/low t-cells in kidney transplant patients. methods: after transplantation, patients received atg-induction therapy (thymoglobulin ® ) and triple therapy consisting of tacrolimus, mmf and steroids. the control group (n= ) received triple therapy only. by flow cytometry, t-cells were analyzed for markers associated with immune regulation: cd , foxp and cd . within the foxp + t-cell population, the cd ro (memory) and ccr (homing receptor) markers were characterized. results: pre-transplant levels of cd + foxp + cd -/low t-cells in all patients were - % (median %) of cd + t-cells. one wk post atg induction therapy, no measurable numbers of treg were present. at wks post atg-induction therapy, a higher proportion of the first detectable t-cells expressed foxp compared to the control-group (atg vs. control-group; vs. %, median, respectively, p= . ) and then returned to pre-transplant levels at wks. this increased proportion of cd + foxp + cd -/ low t-cells resulted in a significantly higher foxp + /foxp neg ratio than in the controlgroup at wks; . vs. . , median, p= . ) . at wks, we found a decline in the proportion of naive foxp + treg (cd ro neg ccr + pre-transplant vs. post-transplant; vs. %, median, p= . ) which was associated with a rise in the proportion of memory foxp + treg (cd ro + pre-transplant vs. wks post-transplant; vs. %, p= . ). moreover, the proportion of memory t-cells exceeded that in the control-group at wks (atg vs. control-group; % vs. %, median, p= . ) which was mainly due to an increase in the proportion of effector memory foxp + treg (cd ro + ccr neg ). conclusion: after atg-induced immune cell depletion, a shift towards cd + foxp + cd -/low peripheral regulatory t-cells with the memory phenotype was measured in kidney transplant patients. this finding suggests that atg treatment triggers the generation of de novo peripheral regulatory t-cells by homeostatic proliferation. introduction in a prospective study, we investigated whether donor-specific regulatory cd + cd bright+ foxp + t cells develop in kidney transplant patients. methods we analyzed the percentage and function of peripheral regulatory cd + cd bright+ foxp + t cells of patients before, , and months after kidney transplantation. the immune regulatory capacities of cd + cd bright+ foxp + t cells were assessed by their depletion from pbmc and reconstitution to cd neg/dim responder t cells at a : ratio in the mlr. in the first year after transplantation, peripheral cd + cd bright+ foxp + t cells decreased from , % ± , pre-transplantation to , % ± , at months (p< . ). while mlr reactivity to rd party-ag ( rd p) significantly improved (p< . ), the reactivity against donor antigens remained low. functional analysis demonstrated potent donor-specific regulatory activities by cd + cd bright+ foxp + t cells after transplantation. depletion of cd + cd bright+ foxp + t cells from pbmc resulted into increased proliferation upon stimulation by donor antigens (p< . ). upon reconstitution, the capacity of cd + cd bright+ foxp + t cells to control the proliferation of anti-donor reactive cd neg/dim t cells increased over time: from % (median) pre-transplant to % post-transplant at month (p< . ). moreover, the anti-donor regulatory activities by the cd + cd bright+ foxp + t cells were significantly more vigorous than those controlling rd p-ag stimulated responder t cells ( %, p< . ). the generation of potent donor-specific regulatory cd + cd bright+ foxp + t cells in the periphery of kidney transplant patients prevents the development of adequate alloreactivity. conclusions: these results indicated that anti-donor responses could be detected even in a significant proportion of "stable" long-term hla identical kidney transplant recipients. speculatively this may be the cause of late graft failures in this group of patients. expression klotho is a gene almost exclusively expressed in renal distal tubules and loss of expression is associated with accelerated aging. in various models of acute and chronic renal injury, and with normal aging, renal klotho expression has been found to decrease. increased donor age is associated with poorer long term allograft function. we hypothesized that klotho mrna expression in renal implant biopsies would correlate with donor kidney quality, as determined by donor chronologic age and peri-transplant renal injury. our recent unsupervised microarray analysis of renal implant biopsies revealed a continuum of organ quality across all samples ranging from the best living donor (ld) kidneys at one end to the worst performing deceased donor (dd) kidneys at the other (am j transpl , nov ,epub). three predominant groups were identified of ld, dd kidneys with low risk ( . %) of delayed graft function (dgf) and dd kidneys with high risk ( %) of dgf (p < . ). analysis of klotho gene expression among these groups also revealed a spectrum of expression from highest levels in ld to lowest in dd kidneys, especially those who developed dgf. differences were highly significant among ld vs dd kidneys (p < . ), although donor age was not different between these groups. klotho transcript levels were significantly different among kidneys that developed dgf compared to those with immediate graft function (igf) (p < . ). in conclusion, reduced klotho gene expression is associated with grafts at risk of poorer function and appears to be independent of donor age. decreases in klotho expression likely reflect other factors impacting the renal tissue, which may impact potential for repair and ultimately allograft function. anti renal allograft rejection episodes produce a stereotypical response in the allograft characterized by infiltration by cytotoxic t lymphocytes (ctl), potent ifng response by donor and recipient cells, and decreased transcripts associated with the epithelium. we previously identified pathogenesis based transcript sets (pbts) that reflect the disturbance in rejecting allografts (qcats -ctl, grits -ifng response, kts -decreased function, ajt : , . we hypothesized that anti-rejection treatment would reverse the transcriptome changes of rejection more than histopathologic lesions. using microarray analysis we measured expression of these pbts in antibody mediated rejection (abmr)( untreated, treated) and t-cell mediated rejection (tcmr) ( untreated, treated) biopsies, normalized to nephrectomy samples. histopathology scoring of the primary rejection lesions were analyzed; interstitial inflammation(i), tubulitis(t), intimal arteritis(v), and glomerulitis(g) (fig a) . of these, only i and t differentiated between abmr and treated abmr (p< . ) yet none differentiated between tcmr and treated tcmr. pbts on the other hand differentiated treated from untreated for both abmr (p< . ) and tcmr (p< . ) with all pbts (fig b/c) . the changes in transcript expression in treated cases were dramatic. particularly impressive was the consistent correction of the disturbances in pbt expression despite the heterogeneous treatment following abmr episodes. unlike abmr treatment, tcmr treatment always included steroids which may contribute to the more pronounced changes compared to abmr. the time of treatment before the biopsy, which also varied within the groups, did not affect the changes in pbt expression since values in treated cases approached those of nephrectomy samples. thus, histologic rejection lesions persist following anti-rejection treatment whereas transcript disturbances of rejection are greatly reduced compared to untreated cases. this study suggests that assessment of anti-rejection treatment is more sensitively monitored by transcript expression than histopathology. blood deciphering the mechanisms of tolerance and chronic immune-mediated rejection remains a major goal in transplantation. data in rodents suggests that toll-like-receptors (tlr), regulators of innate immune responses, play a role in determining graft outcome. however, few studies have focused on tlr in human kidney transplant recipients. we addressed this issue by analyzing the peripheral blood (n = ) and graft biopsies (n = ) of renal transplant patients and healthy volunteers. we analyzed, for the first time, the expression of tlr in pbmc from kidney recipients with contrasted situations: operational tolerance and chronic immune-mediated rejection (banff ), compared to patients with normal histology and stable graft function, non transplant patients with renal failure and healthy volunteers. we found that myd and tlr were significantly contrasted in the pbmc, and in particular in monocytes, of patients with chronic immune-mediated rejection vs. operational tolerance. chronic rejection patients had significantly increased tlr and myd compared to operationally tolerant patients, who resembled healthy volunteers and non transplant patients with renal failure. interestingly, analysis of tlr transcripts in graft biopsies from patients with normal histology or chronic immune-mediated rejection reflected the blood findings, with a significant increase of tlr in chronic immune-mediated rejection. thus, we provide data to support a link between tlr expression and long-term graft outcome. the role of tlr and their endogenous ligands in mediating allograft rejection or acceptance therefore warrants further investigation and could give rise to new strategies of therapeutic intervention. our results suggest that peripheral blood tlr shows potential as a biomarker of chronic immune-mediated rejection and that measuring blood tlr levels may help to identify patients experiencing chronic rejection who require a biopsy. moreover, our data suggest that absence of tlr signaling may be a feature of operational tolerance to kidney grafts. identification of immune identification of immunological tolerance is an important prerequisite in order to establish an individually-tailored approach to the post-transplant management of allograft recipients. it will also provide new insight into the mechanism underlying the balance between tolerance and rejection. here we present data from a multi-centre study aimed at identifying tolerance to renal allografts. we have collected samples from five selected groups of renal transplant recipients: drug-free tolerant patients that were functionally stable despite remaining immunosuppression-free for more than one year; functionally stable patients on minimal immunosuppression (< mg/ day prednisone); stable patients maintained with calcineurin inhibitors (cni); stable patients maintained on cni-free immunosuppression regimen; and patients showing signs of chronic rejection. a group of age and sex matched healthy volunteers was also included as control. several biomarkers and bioassays, were combined to provide an immunological 'fingerprint' of the tolerant state. immunophenotype showed a selective expansion of peripheral blood b and nk lymphocytes in drug-free tolerant patients. this group of patients was also characterized by the absence of anti-donor specific antibodies. the differential expression of several immune relevant genes and a high ratio of foxp / α- , -mannosidase expression in these patients was observed. tcr landscape analysis highlighted differences between the vβ repertoires of drug-free tolerant recipients and chronic rejection patients. additionally, direct pathway donor-specific hyporesponsiveness by ifnγ elispot and lack of indirect pathway anti-donor responses assessed by trans-vivo dth were detected in drug-free patients. the diagnostic capabilities of the combined results of several of the above mentioned biomarkers and bioassays are as follows: specificity . , sensitivity of . and a positive predictive value of . %. these biomarkers could be used to inform drug weaning protocols of kidney transplant recipients. bile acid aspiration stimulates lung allograft immunity. bile acids detected in the broncho alveolar lavage (bal) as a marker of aspiration has been associated in a dose dependent fashion to earlier development of bronchiolitis obliterans syndrome. we sought to study the relationship between bile acids and active immune molecules as detected in the bal. methods: bal collected prospectively from lung transplant recipients at routine surveillance bronchoscopies were assayed for bile acids. samples were then assayed by luminex for cytokines , and by elisa for pulmonary collectins (sp-a, sp-d). results were analyzed according to levels of bile acids as per roc testing for accuracy for bronchiolitis obliterans syndrome diagnosis (high levels ≥ . µmol/l). results: we prospectively examined lung transplant recipients and a total of bal samples were collected. in no bile acids were detected, low levels were present in , and high levels were detected in samples. samples with high bile acids had significantly greater innate (tnf-a, il- b, il- , il- , il- ) and adaptive (ifn-g, il- ) cytokines as well as greater chemokines (mcp- , il- ) compared to the other samples. in contrast pulmonary collectins sp-a and sp-d were significantly reduced in samples with high bile acids. the figures shows the median and interquartile range for each molecule according to bile acid levels. conclusion: bile acids detected in the bal as markers of aspiration stimulate the lung allograft immunity in a dose dependent fashion. in particular high levels of bile acids are associated with an impaired lung specific innate defense system provided by the pulmonary collectins and with a broncho-alveolar district "cytokine storm". tolerance/immune deviation iii the results of using ex vivo cd + t-cells converted dn t-cells in nod mouse models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the treatment of autoimmune type i diabetes. horng-ren yang, gouping jiang, john j. fung, shiguang qian, lina lu. immunology and general surgery, cleveland clinic, cleveland, oh. liver transplant tolerance was recognized by spontaneous acceptance of liver allograft in many species. the underlying mechanism remains unclear. interestingly, although liver allografts are accepted, hepatocyte transplants in the same combination are promptly rejected, indicating a crucial role of liver tissue cells in immune suppression. we have demonstrated a profound t cell inhibitory activity of hepatic stellate cells (hpsc), which are known to participating in repairing and fibrosis during liver injury. addition of activated (a) hpsc, but not quiescent hpsc, significantly inhibited allo-dc induced-t cell proliferative responses (mlr) in a dose dependent manner, which was associated with enhanced t cell apoptosis (tunel). neutralization of b -h by anti-b -h mab significantly reduced the hpsc-induced t cell apoptosis and reversed the inhibition of t cell proliferation, suggesting a key role of b -h . to evaluate this in vivo, balb/c islets ( ) were co-transplanted with x activated hpsc (b ) into stz-induced diabetic b recipients. co-transplant with hpsc effectively protects islet allografts from rejection. this was associated with reduction of graft infiltrating t cells and enhancement of apoptotic activity. co-transplant with hpsc from b -h -/livers markedly lost their islet graft protective capacity, associated with less apoptosis of infiltrating cells. to determine the subsets of apoptotic t cells, t cells were isolated from spleen, draining lymph nodes, and grafts for phenotype and function analyses. on pod , a marked reduction of graft infiltrating cd + ( . %) and cd + t cells ( . %) was seen in hpsc co-transplant group, as compared to islets alone, which was further progressed thereafter. cd t cells dropped ∼ folds on pod . cd + / cd + ratio was increased from . at the early pod to . in the long-term survival grafts. adoptive transfer of cfse-labeled des transgenic t cells was used to track the response and fate of antigen-specific cd + t cells. the results showed active division of des + cells within the allograft in both the islet only and hpsc co-transplantation groups. however, accumulation of these des + cells was significantly lesser in the hpsc co-transplantation group as compared to that in the islet only group ( . × vs. × cells per graft). these findings suggest that hpsc induce antigen-specific cd + t cell death, and may not inhibit their activation. donor-specific memory t cells are potent mediators of allograft rejection due to their ability to proliferate and give rise to cytotoxic and inflammatory cytokine-secreting effectors within hours of stimulation. furthermore, memory t cells have been shown to be relatively resistant to the effects of many tolerance-induction protocols, including blockade of the cd and cd pathways. while seminal studies have shown that donor-reactive memory cells, generated through pre-sensitization with donor tissue or infection with pathogens with cross-reactive epitopes, contribute to costimulation blockade-resistant rejection of fully mhc disparate allografts, the role of memory t cells specific for minor antigens in costimulation blockade-resistant rejection has not been well studied. we addressed the ability of memory t cells specific for a single donorderived class i epitope to mediate this process. tcr transgenic t cells (ot-i) specific for siinfekl/kb were adoptively transferred into naive b recipients, which were then infected with ovalbumin-(siinfekl) expressing listeria monocytogenes (lm-ova). at memory, mice received a skin graft expressing ovalbumin (mova), and therefore the siinfekl epitope. results showed that the transferred ot-i t cells proliferated in response to lm-ova, but not in response to a control infection with wild-type listeria (lm). at day , ot-i t cells comprised ∼ % of the total cd + t cell compartment. during memory, the siinfekl-specific cells comprised ∼ - % of the total cd + t cell compartment. engraftment of mova skin on lm-ova memory recipients resulted in rejection with accelerated kinetics relative to lm infected controls (mst= d vs d). following treatment with ctla- ig and anti-cd , / lm-ova infected recipients experienced costimulation blockade-resistant rejection, while lm-infected controls went onto long-term graft survival (p< . ). lm-ova-infected recipients also resisted the engraftment of mova-expressing donor bone marrow following a tolerance induction protocol containing busulfan, ctla- ig, and anti-cd . these results suggest that memory t cells specific for a single surrogate minor antigen are sufficient to induce costimulation blockade-resistant rejection, and support the feasibility of using this model to study the specific requirements for donor-reactive memory t cell activation and tolerance induction during transplantation. the during an immune response, cd helper t cells can be instructed by non-antigenspecific signals to differentiate into functionally distinct subsets with mutually exclusive patterns of cytokine production. we find that ikaros, a zinc finger transcription factor required for lymphocyte development, is crucial for the development of polarized t helper subsets. in the absence of ikaros dna binding activity, cd t cells induced to undergo th differentiation in vitro or in vivo produce high levels of il- , but fail to silence expression of ifn-gamma and il- , cytokines that contribute to inflammatory disease processes such as autoimmunity and organ transplant rejection. similarly, ikaros is required for repression of il- and il- expression by th cells, and inhibition of ifn-gamma and il- production by th cells. our results show that ikaros controls the expression of transcription factors such as gata- , c-maf, stat- , and runx , and in polarized th cells, ikaros inhibits ifn-gamma gene expression through direct repression of the t-bet locus. these studies place ikaros, a dna binding protein previously recognized only as a regulator of lymphocyte development, as a master regulator of peripheral t cell differentiation and function. introduction as the fastest growing subpopulation seeking organ transplantation is > yrs of age, it will be imperative to discern how aging impacts the acquisition of transplantation tolerance. prior work has demonstrated that viral infections induce the development of alloreactive t cells, which impede the induction of transplantation tolerance. in this study, we tested the hypothesis that aging alters host defense against viruses leading to the development of cross-reactive t cells, which impair transplantation tolerance induction. we first examined how aging modifies the function of plasmacytoid dcs (pdcs), as ifnα production by pdcs is essential for control of viral infections. using both in vitro and in vivo murine systems, we found that aged pdcs produced lower levels of ifnα in response to tlr activation with cpg sequences or herpes simplex (hsv)- virus (elisa). aged mice ( - mths) failed to the clear this virus as effectively as young ( - mths) mice. this was associated with increased liver inflammation, the release of systemic th -skewing cytokines, il- and il- , and augmented splenic il- levels in aged mice (elisa). prior to transplantation, unmanipulated aged mice (b or cba background) produced more donor-specific il- effector-memory t cells as compared to unmanipulated young mice (elispot). furthermore, mlr assays demonstrated that aged memory cd + t cells from unmanipulated mice produced significantly more il- in response to donor antigen compared to young t cells (elisa). to determine if aged recipients manifest an altered response to therapies that prolong allograft survival, aged and young b mice received balb/c skin allografts and perioperative anti-cd and anti-cd . we found that aged mice rejected their allografts significantly faster (median survival days) than young mice (mst, days, p < . ). similar results were noted when we employed perioperative treatment with anti-cd + dst and when we altered the donor-recipient (b to cba) strain combination. pre-treating aged mice with an anti-il- mab improved the efficacy of the graft-prolonging therapy compared to aged mice that received control mab (p = . ). conclusion our results suggest that impaired control of viral infections with aging leads to the generation of alloreactive il- producing t cells that impair therapies that may induce transplantation tolerance. prevention of type diabetes by thymus genetic modification with protective mhc class ii molecules. jesus r. paez-cortez, michela donnarumma, chaorui tian, john iacomini. transplantation research center, boston, ma. introduction. susceptibility to type diabetes is determined by multiple genetic factors, among the strongest of which is the inheritance of at-risk genes that lead to disease development. here we examined whether diabetes can be prevented by providing protective mhc class ii genes through directly infecting the thymus of diabetes prone nod mice. methods. after direct exposition of the thymus, lentiviruses encoding control (phage-cmv-dsred-ires-zsgreen-w) or protective mhc class ii iaβ d (phage-cmv-iaβ d -ires-zsgreen-w) genes were injected in a single thymic lobe of to week old female euglycemic nod mice. gene expression was determined by microscopic examination at different time points after injection and blood glucose levels were monitored weekly to examine whether this approach prevents the development of diabetes. the presence of diabetogenic cd + t cells were detected by flow cytometry via tetramer staining of splenocytes. pancreatic islet integrity and insulin production was assessed by immunohistochemistry. results. viral gene expression was observed exclusively in thymic epithelial cells beginning at days post-injection in both groups. nod mice injected with control lentivirus developed diabetes by weeks post injection, similar to non-treated animals ( - weeks). in contrast, phage-cmv-iaβ d -ires-zsgreen-w injected animals remained normoglycemic at months post-injection. diabetogenic cd + t cell population were not detected in splenocytes of iaβ d injected animals using mhc class i tetramers. islet integrity and insulin production was preserved in the treated group, in marked contrast to controls, which exhibited characteristic lymphocytic infiltration of islet cells and low insulin storage. conclusions. thymic genetic modification with protective a mhc class ii iaβ d molecule can be used to prevent diabetes in nod mice. central deletion of diabetogenic t cell populations may be involved in prevention of autoimmunity in these animals. role of invariant nkt cells in liver sinusoidal endothelial cell-induced immunosuppression of t cells with indirect allospecificity. masayuki shishida, hideki ohdan, yuka tanaka, masataka banshodani, yuka igarashi, toshimasa asahara. surgery, hiroshima university, hiroshima, japan. we have reported that liver sinusoidal endothelial cells (lsecs) endocytose portally injected allogeneic splenocytes and can negatively regulate t cells with indirect allospecificity via the fas/fasl pathway. as a result of in vitro transmigration across the lsecs from balb/c mice treated with a portal injection (pi) of b mhc class iideficient (c d) splenocytes, the naive balb/c cd + t cells lost their responsiveness to the stimulus of balb/c splenic antigen presenting cells (apcs) that endocytosed the donor-type alloantigens. however, they maintained a normal response to the stimulus of balb/c apcs that endocytosed third-party c h alloantigens. in the present study, we examined whether invariant nkt (inkt) cells influence the ability of lsecs to endocytose irradiated allogeneic cells. balb/c wild-type (wt) mice or balb/c cd d-deficient (cd d -/-) mice that lacked inkt cells were portally injected with × irradiated b c d splenocytes labeled with pkh- . only . ± . % lsecs endocytosed the labeled splenocytes in the balb/c cd d -/mice at h after pi, whereas . ± . % lsecs endocytosed the labeled splenocytes in the wt control mice (p < . , n = each). when balb/c wt mice intraperitoneally received µg α-galcer, before pi of b c d splenocytes, the expression of mhc class ii on lsecs and endocytic activity of lsecs were enhanced. thus, we found that the endocytic activity of lsecs was regulated by the inkt cells. intraportal adoptive transfer of lsecs isolated from balb/c wt mice, treated with a pi of b c d splenocytes, into balb/c mice significantly prolonged the survival of subsequently transplanted heart allografts (n = ) as compared to the adoptive transfer of lsecs isolated from balb/c cd d -/mice, treated similarly, into the balb/c mice (n = ). however, intraportal adoptive transfer of lsecs isolated from balb/c wt mice, which received µg α-galcer intraperitoneally prior to pi of b c d splenocytes, into balb/c mice did not result in a further prolonging of the effect (n = ). these findings indicate that inkt cells are required for such lsec-induced immunosuppression of t cells with indirect allospecificity; however, α-galcer-induced activation of inkt cells does not promote such suppressive effects on these t cells. in conclusion, naive inkt cells play a pivotal role in the lsec-induced immunosuppression of t cells with indirect allospecificity. notwithstanding the considerable amounts of in-vitro data supporting the nonimmunogenicity and immunomodulatory effects of mesenchymal stem cells (msc), scanty and conflicting data are available on their in-vivo immunomodulatory capacities. in this study we formally investigated whether msc had immunomodulatory properties in solid organ transplantation, using a semi-allogeneic heterotopic heart transplant mouse model, and studied the underlying mechanism(s). msc, isolated from bone marrow by adherence, were depleted from cd +cd b+ cells before injection. bone marrow-induced hematopoietic mixed chimerism is the most robust mechanism for the induction of transplantation tolerance. however, immunogenicity of bone marrow cells requires harsh immunosuppressive regimens that can lead to severe sideeffects including death. here, we examined whether embryonic stem (es) cells can be successfully coaxed to form hematopoietic progenitor cells (hpc) which potentially could be less immunogenic than bone marrow cells. here, we transduced es cells with hoxb , a hematopoietic transcription factor that confers self-renewal properties to hematopoietic cells and differentiated them into hematopoietic cells. transduced cells had a - fold greater proliferation capacity than controls. at the end of the differentiation procedure, most cultures were > % cd + . hpcs were purified using immunomagnetic bead separation. the separated cells were further characterized for leukocyte markers and showed a high percentage of cd , cd , cd and low class i, but no class ii expression. further, they poorly express co-stimulatory molecules such as cd and cd . when transplanted in rag -/γ c _/_ mice, hpcs fully reconstituted bone marrow, forming multi-lineage hematopoietic cells. to now determine whether these cells engraft in allogenic recipients, the cells were transplanted in syngeneic and allogenic mrl mice. all transplanted animals became chimeric (n> ), reaching - % after days. thereafter donor cells declined as a result of out-competition by resident bone marrow cells. this pattern was identical in both syngeneic and allogenic recipients. unexpectedly, allogenic chimeric mice became tolerant to donor-type cardiac allografts as monitored over days. grafts showed no mononuclear cell infiltration or signs of chronic rejection. interestingly, the t cells in tolerant animals showed responses to mrl alloantigen similar to that of controls, suggesting that our protocol was likely non-deletional, but could involve regulatory t cells. indeed, when stained for cd + foxp + cells, the allografts showed a high percentage of these cells, but not in controls, confirming our hypothesis. thus, these data show for the first time the potential of es-derived hpcs to regulate engraftment of allografts, providing an alternative approach for the induction of transplantation tolerance. we had previously shown that a is part of the regulatory atheroprotective response of endothelial (ec) and smooth muscle (smc) cells to injury. a is a nf-κb dependent gene with potent anti-inflammatory effects in ec and smc, through blockade of nf-κb. a also serves an anti-proliferative function in smc and opposite anti-apoptotic or pro-apoptotic functions in ec and neointimal smc. based on these functions, a would be a good candidate to prevent transplant arteriosclerosis (ta) and chronic rejection in vascularized organ grafts. this is supported by a expression in ec and smc correlating with the absence of ta in rat kidney allografts and long-term functioning human kidney allografts. fully mismatched c bl/ (h b ) and balb/c (h d ), were used as donors and recipients of an aortic to carotid allograft. in this combination, ta lesions start at weeks and become occlusive by weeks when left without immunosuppression. a expression in the graft was achieved by recombinant adenoviral (rad) mediated gene transfer prior to retrieval. control mice were infused with saline or control rad beta-galactosidase. the grafts were harvested at weeks and analyzed for ta lesions by measuring intima to media ratios (i/m) and for markers of inflammation and of the immune response by immunohistochemistry. a expressing vessels were significantly protected from intimal hyperplasia with i/m reaching . ± . as compared to saline ( . ± . ) and beta-gal ( . ± . ) treated vessels. this effect of a did not associate with a decrease in infiltrating cd , cd or cd t cells in a vessels as compared to controls. a possible modification of the phenotype of these t cells (t-regs vs. effector t cells) is being explored. rather, protection from ta correlated with increased expression of endothelial and inducible nitric oxide synthases (nos) in ec and smc of a expressing vessels, suggesting that this effect was, at least in part, related to increased in situ production of nitric oxide. in conclusion, we present the first direct evidence that expression in the vessel wall of the anti-inflammatory and atheroprotective protein a prevents transplant arteriosclerosis through a mechanism implicating increased expression of nos. carbon monoxide inhalation reverses established chronic allograft nephropathy through the no pathway. g. faleo, a. nakao, j. kohmoto, r. sugimoto, k. tomiyama, a. ikeda, m. a. nalesnik, d. b. stolz, n. murase. thomas e starzl transplantation institute, university of pittsburgh, pittsburgh, pa. chronic allograft nephropathy (can) is the most common cause of graft loss; however an established therapeutic strategy in preventing/treating can is not yet available. we have previously shown that carbon monoxide (co) effectively inhibits can development. here, we examine the mechanisms of co in overturning can through vascular endothelial cell protection. methods: orthotopic kidney transplantation (ktx) was performed in lewis to binephrectomized bn rats under brief tacrolimus ( . mg/kg, d - , im). by d after ktx, bn developed can with decreased creatinine clearance (ccr, . ± . ml/min), significant proteinuria ( . ± . mg/ h), and increased banff scores for intimal arteritis, interstitial fibrosis, and tubular atrophy. recipients were then treated with inhaled co ppm from d to d . results: inhaled co effectively reversed the severity of can and markedly improved renal function at d (table) and recipient survival (> d vs. d air control). co treatment resulted in reduced cytokine mrna levels (tnf-α, ifn-γ) and improved banff scores compared to untreated controls. in untreated allografts, cd expression on peritubular capillaries (ptc) was markedly diminished, while co-treated grafts showed normal cd expression, suggesting significant improvement in maintaining ptc integrity with co. interestingly, enos and inos protein expression was significantly upregulated in untreated grafts, while it was maintained at steady levels in co-treated grafts. immunohistochemistry revealed enos expression on vascular endothelial cells while inos on infiltrates. further, serum nitrate/nitrite levels were significantly higher in untreated than in co-treated recipients. elevated levels of mda, a marker for oxidative stress, in air control group were accordingly reduced in co-treated grafts. renal cortical blood flow data showed a better perfusion in co-treated group at d ( . chronic allograft vasculopathy (cav) is a component of chronic rejection and a major cause of graft loss. non-immunologic factors and indirect allorecognition participate in the pathogenesis of cav. new therapies with tolerogenic dendritic cells (dcs) are based on in situ-delivery of alloag to "quiescent" dcs of the recipient's lymphoid organs via apoptotic cells, vesicles or particles. we have shown that the ability of apoptotic cells to deliver alloag and an inhibitory signal to dcs down-regulates the indirect alloresponse and prolongs allograft survival in mice. aims: to test if targeting of recipient's dcs in situ with donor apoptotic cells ameliorates cav by down-regulating indirect pathway allo-immunity. methods: we performed functional aortic (abdominal) transplantation in mice [balb/c→c bl/ (b )]. b mice were injected i.v. with balb/c uvb-induced early apoptotic splenocytes (d- ). sixty days later, grafts were evaluated in sections with h&e, vangieson's (elastic fibers) and masson's (collagen) techniques. cfse-labeled h . tcrtg cd t-cells specific for ia b (b ) loaded with ieα - (balb/c) were used to evaluate the indirect pathway t-cell response. results: pkh + balb/c apoptotic cells injected (i.v.) in b mice were captured by splenic cd and cd α + dcs, but not plasmacytoid dcs. splenic dcs with apoptotic cells remained quiescent in vivo (mhc-i/ii lo , cd / lo , icosl + , pdl- / + ) and were unable to up-regulate mhc-ii and cd upon culture with gm-csf. injection of donor apoptotic cells induced defective activation and deletion of indirect pathway h . cd t-cells. therapy with donor apoptotic splenocytes reduced intimal thickness in aortic allografts ( ± vs. ± mm in controls; p< . ) and proliferation of α-smooth muscle cells and collagen deposition. the effect of apoptotic cells was allospecific, superior than that of cells alive, and depended on the physical properties of apoptotic cells, since necrotic cells did not achieve the effect. treatment with donor apoptotic cells decreased significantly the indirect pathway t-cell response (assessed by elispot for ifn-γ) and reduced the level of circulating alloab. conclusion: in situ-targeting of recipient's dcs with (early) apoptotic cells carrying donor alloag is a novel approach to prevent cav by down-regulating the indirect pathway alloresponse. the antifibrotic agent, pirfenidone, has direct inhibitory effects on t cell activation, proliferation, and cytokine and chemokine production, leading to suppression of host alloresponses. gary a. visner, fengzhi liu, hanzhong liu, liqing wang, wayne w. hancock. medicine, children's hospital boston, boston, ma; pathology, children's hospital of philadelphia, philadelphia, pa. there is an urgent need to develop new therapies effective against the fibrotic and other complications of chronic allograft rejection. while pirfenidone (pfd) is an established anti-fibrotic agent, we previously showed that pfd treatment reduced acute rejection in a rat lung transplant model suggesting that it might have direct immune modulating properties. accordingly, in this study, we tested the effects of pfd on t cell responses. we first evaluated whether pfd alters t cell proliferation and cytokine release in response to t cell receptor (tcr) activation in vitro. since pfd can inhibit tgf-β by mononuclear cell fractions, we also examined whether pfd affects the suppressive effects of regulatory t cells (cd +cd +). the effects of pfd on alloantigen-induced t cell proliferation in vivo were then assessed by adoptive transfer of cfse-labeled t cells across a parent->f mhc mismatch, as well as by using a murine heterotopic cardiac allograft model (balb/c->c bl/ ). pfd was found to significantly inhibit tcr-stimulated cd + t proliferation in vitro (p< . ), whereas cd + t cell proliferation was not significantly affected. while the beneficial effects of pfd were not associated with increased cd + t cell apoptosis, pfd use inhibited tcr-induced production of multiple cytokines and chemokines, including . interestingly, there was no change on tgf-β production by purified t cells, and pfd also had no effect on the suppressive properties of naturally occurring regulatory t cells. similar to the in vitro studies, pfd inhibited allo-antigen-induced t cell proliferation in vivo (parent->f model), and showed synergistic effects with low dose rapamycin in this model. lastly, though pfd alone did not affect the tempo of acute cardiac allograft rejection across a full mhc mismatch, use of pfd plus a subtherapeutic regimen of rapamycin significantly prolonged allograft survival (p< . ), decreased mononuclear cell infiltration and prevented development to chronic rejection, including arteriosclerosis and myocardial fibrosis. we conclude that pfd may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses. we have previously reported studies in miniature swine showing that transplantation (tx) of prevascularized donor islets as part of composite islet-kidney (i-k) reversed diabetic hyperglycemia across fully allogeneic barriers, while free islets did not. in order to test the potential clinical applicability of this strategy, we have extended it to a fully allogeneic nonhuman primate model. methods: two diabetic baboons received composite iks and one diabetic baboon received free islets across fully allogeneic barriers. ( ) i-k preparation in donors: two i-ks were prepared by isolating islets from % partial pancreatectomies and injecting them under the autologous renal capsule, allowing for vascularization before allogeneic tx. these i-ks were harvested at days and for allogeneic ik tx. ( ) induction of insulin-dependent diabetes (iddm) and allogeneic i-k or free islet tx: all recipients received streptozotocin at mg/m x (body surface area). iddm was induced successfully in two animals, while one animal required total pancreatectomy to induce iddm. after confirming iddm by fasting blood sugar (fbs) > mg/dl for consecutive days, either i-ks or free islets were transplanted (single donor to each recipient) with atg (day - ) followed by mmf and low-dose tacrolimus. free islets were injected into the liver through the ileocolic vein. islet function was assessed by fbs and renal function was assessed by serum creatinine. immunologic status was examined by cml/mlr assays. results: all three recipients had strong ctl/mlr responses to donors pretx, indicative of a fully allogeneic combination. fbs decreased immediately after i-k tx and no insulin therapy was required throughout the experimental period (days and ). bs levels averaged . +/- . mg/dl in the first baboon and . +/- . mg/dl in the second. normal creatinine levels (< . mg/dl) were maintained by these life-supporting ik grafts. in contrast, the recipient of allogeneic free islets had unstable bs levels and required insulin from day (bs at day ) conclusions: life-supporting i-ks from single donors achieved glucose regulation without insulin therapy and maintained normal renal function. these results demonstrate the feasibility of composite i-k tx in a non-human primate allogeneic model, with possible clinical applicability for the cure of diabetic nephropathy. donor cell infusion without immunosuppression as a novel therapy for induction of donor-specific tolerance in islet cell transplantation. xunrong luo, kathryn pothoven, derrick mccarthy, matthew degutes, aaron martin, xiaomin zhang, guliang xia, dixon kaufman, stephen miller. medicine, northwestern university; microbiology and immunology, northwestern university; surgery, northwestern university, chicago, il. background in autoimmune models, peptide-pulsed splenic antigen presenting cells that are chemically fixed with ethylcarbodiimide (ecdi) have been used as a powerful and safe method to induce antigen specific t cell tolerance. ecdi fixed donor cells for allo-antigen specific transplant tolerance has not been well studied. material and methods c bl/ mice were rendered diabetic by stz. kidney subcapsular allogeneic islet transplant was performed days after diabetes stabilization (day ). x ecditreated donor splenocytes were injected i.v. either once (day - ) or twice (day - and day + ). animals were analyzed for graft outcome. results control mice receiving islet graft alone rejected the graft between day to day (mst = days, n= ). mice receiving one dose of ecdi-treated donor splenocytes (on day - ) showed similar graft survival as controls (mst = days, n= ). in contrast, mice receiving doses of ecdi-treated donor splenocytes (on day - and day + ) showed significant prolongation of graft survival with . % functional grafts at day (n= ), and some remained functional > days. this protection is donor-specific as mice receiving ecdi-treated sjl cells rejected balb/c islet grafts as controls (mst = days, n= ). immunohistochemistry of protected grafts showed positive insulin staining within well-defined islet architecture. peri-islet infiltrates were composed of cd +, cd +, and cd c+ cells, with occasional foxp + cells. anti-donor antibody production (igg ,g a,b, g ) was completely abolished in long-term graft survivers. this tolerance was undisturbed by anti-cd antibody treatment during maintenance stage, but could not be established if treatment was given around the time of the first donor cell infusion. in addition, lack of pd-l also impaired tolerance induction evidenced by using pd-l -/as recpients. conclusion . multiple infusions of ecdi-treated donor splenocytes significantly prolonged allo-graft survival in the islet transplant model. . the protective effect is donor-specific and is dependent on regulatory t cells as well as the pd-l signaling pathway. therapy with ecdi-treated donor cells may emerge to be a novel and potent agent for induction of donor-specific transplant tolerance. mice. rebecca stokes, k. cheng, c. scott, w. hawthorne, p. o'connell, j. e. gunton. garvan institute, darlinghurst, nsw, australia; nptu, westmead, nsw, australia. the aim was to investigate the effects of increasing hif- α protein in human islets upon islet-transplant outcomes. hif- α is a transcription factor which co-ordinates a program of cellular responses to stressors including hypoxia. in other cell-types hif- α improves survival following hypoxic-challenge. hif- α functions as a heterodimer with arnt which we have shown to be important for normal β-cell function ( ) . islet transplantation subjects islets to hypoxia. it is thought up to % of islets die within week of transplantation and this is at least partly due to hypoxia. the role of hif- α in β-cell function is unknown. we hypothesized that increasing levels of the protective factor hif- α in islets before transplantation would improve survival and engraftment and thus improve islet transplant outcomes. isolated human pancreatic islets from separate donors were cultured overnight in control media, or media supplemented with desferrioxamine (dfo), a small molecular stimulator of hif- α protein. islets were transplanted into diabetic scid mice. there were transplant groups, with mice receiving: .supra-physiological-mass transplant of control-cultured ieq (islet equivalents) .minimal-mass-transplant of control-cultured ieq, or .minimal-mass-transplant of ieq cultured with dfo. for each human donor, at least of each of the transplant groups was performed to avoid the confounder of inter-donor variability. recipients of control ieq cured in % of cases. minimal-mass-transplantation was ineffective: ieq cured % mice at -days. however, minimal-mass-transplant of dfo treated islets had % success (p< . vs group and p=ns vs -control-ieq). blood glucose levels were markedly improved in the dfo treated group compared to ieq control group (p< . ) and equivalent to control ieq transplants (p=ns). this data demonstrates increasing hif- α in human islets prior to transplantation markedly improves islet transplant outcomes. hif- α and dfo may have a therapeutic role in human islet transplantation. long-term disappearance of neovascularization of transplanted islets. eba hathout, nathaniel chan, annie tan, john chrisler, john hough, naoaki sakata, john mace, ricardo peverini, richard chinnock, lawrence sowers, andre obenaus. loma linda university, loma linda, ca. we recently reported an in vivo time-line for neovascularization of transplanted islets using dynamic contrast enhanced (dce) magnetic resonance imaging (mri) over a -day period. however, vascularization of transplanted islets must be maintained for extended periods to provide long-term function. in this dataset, we investigated whether vascularization was maintained in transplanted feridex-labeled syngeneic murine subcapsular islets ( ieq per kidney) using dce imaging on an . t mr scanner and subsequent immunohistochemistry over days. sub-capsular transplants could be visualized at post-transplant days and using t weighted imaging. however, the islets could not be seen on mri at post-transplant day . injection of the contrast agent gadolinium (gd)-dtpa for dce at , and days showed increased signal in the transplant area. at days, there was no change in signal intensity after contrast injection during dce. immunohistochemistry confirmed mri and dce findings. these results suggest that islet neovascularization occurs early after transplantation but is likely not maintained for the -day duration of our experiments. this work was supported by nih/niddk grant # r dk . a) t imaging at day clearly identifies iron-labeled islets (arrows) in the subcapsular region. no iron-labeled islets are observed at day (arrows). b) dce imaging for neovascularization of transplanted islets in the subcapsular region demonstrates a temporal decline in signal intensity. oleanolic acid, a natural triterpenoid, significantly improves islet survival and function following transplantation. n. angaswamy, d. saini, s. ramachandran, n. benshoff, w. liu, n. desai, w. chapman, t. mohanakumar. , surg, wusm; path & immunol, washington univ sch med, st. louis, mo. oleanolic acid (oa), a triterpenoid in medicinal herbs, is an integral part of normal human diet. oa has anti-oxidant, anti-inflammatory properties (inhibits inos & cox ) & lowers plasma glucose levels. we hypothesis that these properties of oa will prevent early islet cell loss following transplantation & also benefit long term function of allograft. c bl/ mice, made diabetic by streptozotocin ( mg/kg) were transplanted with balb/c islets (isolated by collagenase digestion) under kidney capsule. oa ( . mg/day) was administered i.p. in µl of pbs (with m dmso) or pbs-dmso as vehicle control daily from day - onwards. blood glucose was monitored daily. immunohistochemical analyses of grafts were performed for cd & cd markers. cellular immune responses to donor antigens & cytokines produced by cells and in sera were measured using elispot & luminex assays. effect of oa on function of transplant with suboptimal dose of islets ( - ) was also analyzed. optimal dose of islets ( ) transplanted into diabetic bl/ mice administered with oa significantly reduced time taken to reverse diabetes following transplantation (< ± vs ± days, p= . ). further, oa treatment reversed diabetes even with suboptimal dose ( ) of islets while untreated animals did not achieve normoglycemia. as expected, control diabetic mice rejected on ± days whereas, oa administration alone prolonged islet allograft survival to ± days (p< ). oa treatment resulted in > fold increase in serum kc, il- & vegf (p< . ) & fold decrease in mcp- , ip- & il- (p< . ) in luminex assay. stimulation of splenocytes from oa treated mice with donor balb/c cells resulted in significantly reduced ifng ( . fold), il- ( . ), il- ( . ) & il- ( ). in addition, proliferation in mlr was also reduced . fold. immunohistochemical analysis of grafts showed significant reduction in cellular infiltration in oa treated animals with reduction in both cd and cd t cells. daily administration of oa markedly improved islet engraftment & function with reversal of diabetes even when suboptimal dose of islet were transplanted. further, oa treatment allowed significant long term survival of allograft with no other immunosuppression. we demonstrate that prevention of inflammatory signaling cascades by oa resulted in marked reduction of cellular infiltration into graft allowing long term function of allograft. endoplasmic reticulum stress may be an important cause of cell loss after human islet isolation. soon hyang park, michel tremblay, steven paraskevas. surgery, mcgill university health center, montreal, qc, canada; mcgill cancer center, mcgill university, montreal, qc, canada. purpose: to evaluate the presence of endoplasmic reticulum (er) stress, induced by conditions to which islets are subjected during isolation (ischemia, nutrient deprivation, thermal stress and cytokine release) in human islets and to determine if this leads to the unfolded protein response (upr), which could alter cell survival. methods: human islets were purified from cadaveric pancreata by collagenase dissociation and continuous density gradient purification. islet preparations were cultured in serum-free medium and sampled at the end of isolation and daily thereafter. total mrna was purified and gene expression evaluated by rt-pcr. activity in upr signaling pathways was evaluated by immunoblot. apoptosis was measured by a caspase- activity assay. representative trends observed in > isolations are described. results: following isolation, a rapid increase in upr signaling was observed in the perk and ire- modules of the upr. these include the phosphorylation of perk target eif ? and splicing of mrna for the transcription factor xbp- . these changes occurred concurrently with a rapid spike in jnk activity and a rise in expression of the upr target gene chop. after these signals peaked, caspase- activity increased with time (apoptotic cells), as did expression of er chaperone bip (surviving cells). conclusion: we consistently observed upr activation in human islets. er stress and the upr may be one important and unrecognized cause of apoptosis in this context. current investigations focus on upr modification and determination of a causal relationship with apoptotic cell death. immunosuppression and the risk of renal transplant failure due to recurrent glomerulonephritis. atul mulay, carl van walraven, greg knoll. the ottawa hospital, ottawa, on, canada. glomerulonephritis (gn) is the most common cause of end-stage renal disease among those who undergo kidney transplantation. recurrent gn is a major cause of kidney transplant failure. immunosuppressive medication is used to treat gn in the native kidney prior to the development of end-stage renal disease but the impact of different immunosuppression on recurrent gn post-transplantation is unknown. we used the united states renal data system to determine the association of routine post-transplantation immunosuppressant use with time to renal allograft failure due to recurrent gn. immunosuppressants were treated as time-varying covariates. the study-cohort included patients with kidney failure due to gn who received first kidney transplant between and . the study cohort included , patients with a median follow-up of months. ten-year overall graft survival (including death as graft loss) and death-censored graft survival was . % and . % respectively. use of cyclosporine (hazard ratio . ; % ci . - . ), tacrolimus (hazard ratio . ; % ci . - . ), azathioprine (hazard ratio . ; % ci . - . ) or mycophenolate mofetil (hazard ratio . ; % ci . - . ) was not associated with risk of graft failure due to recurrent gn after adjusting for important covariates. there was no difference of recurrent gn causing graft failure between cyclosporine and tacrolimus (p= . ) or between azathioprine and mycophenolate mofetil (p= . ). however, change in any immunosuppressant during follow-up was independently associated with graft loss due to recurrence (hr . , % ci . - . , p= . ).when we restricted the analysis to patients who had no change in immunosuppression during follow-up we again found no association between any of the immunosuppressive medications and the risk of graft loss due to recurrent gn. despite the increased use of tacrolimus, cyclosporine and mycophenolate mofetil to treat gn in native kidney disease, the use of these medications following kidney transplantation had no impact on the risk of graft loss due to recurrent gn. glomerulosclerosis. junichiro sageshima, gaetano ciancio, alessia fornoni, linda chen, carolyn abitbol, jayanthi chandar, warren kupin, giselle guerra, david roth, sherry shariatmadar, gaston zilleruelo, george w. burke iii. university of miami miller school of medicine, miami, fl. background: disease recurrence is a major obstacle of kidney transplant for focal segmental glomerulosclerosis (fsgs). anti-cd antibody (rituximab) has been used for nephrotic syndrome of native kidney. the significant reduction of proteinuria in transplant recipients with fsgs recurrence was also reported after rituximab use for posttransplant lymphoma. we hypothesized that rituximab induction could alter the posttransplant course of fsgs recipients, particularly in those patients with rapid progression to end-stage renal disease who are higher risk of recurrence. methods: we compared the outcome of transplants for primary fsgs treated with and without rituximab. from jan. to dec. received renal allografts along with our "standard" immunosuppressive protocol, consisting of tacrolimus, mycophenolate, corticosteroids, antithymocyte globulin and/or daclizubab. from jan. to dec. received rituximab in addition to the "standard" immunosuppression. posttransplant proteinuria was treated with plasmapheresis (pp) and maintenance angiotensin blockade (ab). results: there was no adverse event related to rituximab infusion. the overall incidence of posttransplant proteinuria was significantly lower in recipients with rituximab induction (p < . ). four recipients treated with "standard" immunosuppression developed massive proteinuria (u-protein/creat. > ) immediately following transplantation; they responded poorly to pp and ab. four other recipients had moderate proteinuria. in contrast to this, of the patients induced with rituximab, only had massive proteinuria and had mild to moderate proteinuria which responded well to pp and ab. with a median follow-up of months, there was no significant difference of graft survival between groups ( -year survival: % without rituximab vs. % with rituximab). a half of the graft loss was related to non-compliance. conclusion: while the mechanism of action is unclear, our observation indicates that rituximab induction may decrease the incidence and severity of recurrence of fsgs following kidney transplantation. a larger-scale study is desirable to confirm this observation. mesangial chimerism in recurrent iga nephropathy. geoffrey talmon, dylan miller. department of pathology and laboratory medicine, mayo clinic, rochester, mn. background iga nephropathy (in) is the most common primary glomerulonephritis and nearly % of patients who undergo a renal transplant for in recur. data support that bone abstracts marrow-derived cells are capable differentiating into various mesenchymal cells within the kidney. the extent to which this phenomenon versus proliferation of resident mesenchymal cells is involved in populating mesangium is not well understood. the mesangial injury and/or hypercellularity seen in in provides a robust in vivo model for determining if this phenomenon is prevalent in human kidneys. design follow-up biopsies from male patients receiving female renal allografts for in that showed recurrent disease were selected. fluorescent in-situ hybridization and immunofluorescent staining was performed on unstained slides from the paraffinembedded tissue for smooth muscle actin, x, and y chromosome centromeres. cells within nonsclerotic glomeruli with triple positivity (y+) were assumed to be mesangial cells derived from the recipient. results four cases of recurrent in with nonsclerotic glomeruli were obtained, each displaying at least minimal mesangial proliferation by light microscopy (one "minimal", two "mild", one "moderate"). mesangial cells with y chromosome centromeric material were observed in each case ( %). between and mesangial cells were present in each glomerulus (mean . ) with no to three y+ cells seen (mean . ). these accounted for between % and % of mesangial cells in individual glomeruli. the ratio of y+ to total mesangial cells in each case ranged from : . . to : . (mean : . ). the case exhibiting minimal mesangial hypercellularity had a ratio of : . , those with mild had ratios of : . and . . , and that with moderate : . . conclusions recipient-derived mesangial cells make up a fraction of the population of glomerular cells in renal allografts affected by recurrent in. although the number of cases is small, the number of recipient-derived cells does not seem to be directly related to the degree of mesangial hypercellularity seen by light microscopy. the consistent presence of these "colonizing" cells in patients with recurrent in does, however, suggest that there may be a role for targeted therapy directed against circulating recipient cells. in the mid 's, patients developing esrd secondary to systemic lupus (sle) were deemed to be poor transplant candidates because of concern for early recurrent lupus nephritis (rln) leading to allograft loss. subsequently, rln was considered an unusual complication of kidney transplantation, occurring in < % of allografts. however, over the last decade, several reports have shown the frequency of rln to range from - %. we sought to determine the frequency of rln at our center and to identify any clinical variables associated with rln. between / between / - / allografts in patients with esrd due to sle functioned for more than days after engraftment. immunosuppression consisted of azathioprine (aza), or cyclosporine (csa) and aza, or mycophenolate (mmf) and csa, or tacrolimus and mmf depending on the date of transplant. all received steroids. proteinuria was defined as + on dipstick or urine protein/creatinine ratio > . . medical charts were reviewed. we found pathologic evidence of rln in ( %) of patients who underwent biopsy due to allograft dysfunction or proteinuria, comprising % of all patients transplanted for sle. characteristics of these patients are shown below: randomized studies have shown little or no increase in ar in kidney tx recips on p-free is; but concern remains about long-term outcome. we present -yr f/u of a protocol incorporating rapid (< days) discontinuation of p, with now over patients transplanted using this protocol. between / between / and / between / , adult tx recips were treated with thymoglobulin ( doses)(extended in dgf), p ( days), a cni, and either mmf or srl. of these, were ld ( lurd); dd. of the , % were female; % white; mean recipient age was ± years and mean donor age was . ± . years. diabetes was present in . % of the recipients and . % of the transplants were retransplants. the peak pra was > % in % of recipients; % had tx pra > %. table shows actuarial survival rates. graft survival rates were significantly better in ld vs dd transplants (p= . ) and and acute rejection rates lower (p= . ). compared to national data from srtr, overall outcomes were not significantly different. with mean follow-up of . years, a total of ( %) recipients have died -the most common cause being cerebrovascular accident ( %) followed by malignancy ( %). there were only ( . %) patient deaths due to cardiac causes. of ( . %) graft losses, ( %) were from dwf (death with function); ( %) from cr/can. renal function has been stable with mean serum cr of . mg/dl at and years posttransplant with cretinine clearance of and respectively. at years posttx, compared to ppretransplant values, recipients showed a . % increase in weight, a % decrease in serum cholesterol, and a . % decrease in serum lipid values. % of the kidney recips remain p-free; the most common reason for restarting p was acute rejection (ar). conclusion: short-term data suggests kidney tx recips do well with rapid discontinuation of p. our intermediate-term data suggests that patient and graft survival rates remain good and renal function remains stable. ongoing long-term follow-up is necessary. background. since / our program has employed a steroid-free, rapamycin and neoral maintenance immunosuppression regimen for kidney transplant recipients. prior to that time recipients were treated with prednisone, mmf and neoral. we noted a significant reduction in acute cellular rejection (acr) after implementing this regimen. this retrospective analysis was performed to examine the impact, if any, on the incidence, character, and outcomes of early ahr. results. this study includes consecutive kidney recipients transplanted between / and / . there were recipients in the prednisone, mmf, and neoral era (grp ) and in the steroid-free, rapamycin and neoral era (grp ). recipient age, gender, african-american race, ab and dr mismatch, and frequency of pra> % was not statistically significantly different between the groups. however, % of grp pts vs % of grp pts received a living donor kidney due to a more recent volume increase in this procedure (p< . ). there were a total kidney biopsies in pts performed in the first months post-transplant; in pts when excluding pre-perfusion biopsies. nineteen percent ( / ) of these showed > % peritubular capillary (ptc) c d deposition. comparison of grp to grp demonstrated the following: ) the incidence of clinical acute rejection in the first months was . % ( / ) in grp and . % ( / ) in grp (p< . ), ) the overall incidence of a c d+ biopsy was similar in the groups ( conclusions. despite a significant reduction in the incidence of acute rejection using our newer, steroid-free immunosuppression protocol, there has been no reduction in the incidence of early ( - months) ahr evidenced by c d+ kidney biopsy. however, the percentage of ahr unassociated with acr has significantly increased. the poor graft survival in pts with early ahr has not improved with our newer immunosuppression regimen. conclusions: four risk factors for ar were identified in the rdp study population: retx, aa race, age - (vs. > ) and pra > (vs. < ). four risk factors for gl were identified: pre-tx t dm, ar, dgf and dd tx (when ar and dgf omitted). these risk factors for ar and gl are the same as we observed in prednisone-containing protocols. additionally, many of these factors are not modifiable. identification of high risk groups allows for individualization of is. increasing lds and utilization of is protocols to decrease or minimize dgf and ar are goals for improving graft outcome. effect with the advent of more potent immunosuppression hla matching has been deemphasized in the allocation of deceased donor kidneys due to the limited impact on acute rejection and graft survival. an unforeseen consequence of poorer matching could be an increase in sensitization of patients in need for a repeat transplant. our study examined candidates listed in the us from - from the srtr database that were re-listed following loss of a primary kidney transplant (n= , ). the primary outcome of the analysis was change in pra from the listing prior to recipient's initial transplant to the subsequent listing. absolute change in peak and current pra levels were examined in general linear models as well as the proportion of patients with a rise in pra level in a logistic model. results hla(a,b,dr)-matching in the primary transplant was strongly associated with change in pra level (p<. , figure ). among recipients with -hla mm, over % had a rise in pra at re-listing as compared to % of -hla mm recipients. younger recipient and donor age, males, deceased donor transplants and african american recipients were also significantly associated with elevation in pra. in addition, the effect was apparent stratified by primary donor type. while there might be a limited impact of hla matching on graft survival, many patients might be negatively impacted from poor hla matching from their first transplant when needing a second transplant. as high pra is one of the strongest risk factors for not getting transplanted, this should be taken into account when evaluating the impact of hla matching in kidney transplantation. this might be particularly important in younger patients and in patients with a long life expectancy in general because of the high likelihood of needing a second transplant during their lifetime. the were about % less likely to die or lose an ecd kidney from a donor aged - and % more likely with kidneys from donors older than . a similar trend was seen with older recipients, but the risks seemed to be lower. logistic regression indicates recipients older than age were times more likely to have a donor older than age than recipients younger than , hypertension and creatinine > . were less likely in older donor kidneys. interestingly, the use of these kidneys relative to the total number of ecd kidneys has decreased since (odds ratio= . , . - . , p< . ). conclusion: an increase in the supply of kidneys might be achieved with increased utilization from deceased donors older than age . outcomes were similar to those from donors age - in recipients that were older than age . outcomes , but approximates non-dcd survival thereafter. dcd listing for retransplantation and graft failure progressed continuously over days versus days in non-dcd. when retransplanted, dcd recipients waited longer and received higher risk allografts (p= . ) more often from another region. more dcd recipients remain waiting for retransplantation with fewer removed for death, clinical deterioration, or improvement. conclusions: dcd utilization is impeded by early outcomes and a temporally different failure pattern that limits access to retransplantation. allocation policy that recognizes these limitations and increases access to retransplantaton is necessary for expansion of this donor population. orthotopic liver transplantation with allografts from dcd donors. roberto c. lopez-solis, background: in the current era of liver transplantation, organ shortage continues to be a significant problem. the use of extended criteria allografts from donation after cardiac death (dcd) donors to increase transplantation rates is widely practiced. this study is a review of one of the largest single center experiences utilizing dcd donors in the world with a follow-up of almost years. methods: from / / to / / , , liver transplants were performed at our institution, ( . %) of which were liver allografts from dcd donors. patient and donor demographics, recipient and graft survival, and the incidence of primary non function, hepatic artery thrombosis, retransplantation, and bile duct complications were analyzed for this subset of recipients. results: kaplan meier analysis showed a -and -year patient and graft survival of % and %, and % and %, respectively. the mean age of recipients was ± years with an average meld score of . ± . (range, to ), and there were male patients ( %). donor mean age was ± years and cold ischemia time was ± minutes. one hundred and three patients ( . %) are alive and ( . %) underwent retransplantation. the incidence of primary non-function was . % ( patients) and hepatic artery thrombosis was . % ( subjects the fda warns against using sirolimus (srl) in liver transplants, reporting increased hepatic artery thrombosis (hat), excess mortality and graft loss when srl is used as initial immunosuppression (is) with calcineurin inhibitors. we report the largest experience to date of patients with srl used as initial is, assessing hepatic artery complications and survival outcomes. materials and method all olt pts from - were reviewed. those using srl as initial is were identified, and the remaining olt pts from that time period were used as controls. ultrasound assessed graft vascular status and any issues were verified by angiogram. . there were no significant difference in demographics variables, lt indication or pre-lt meld score between the two gr. mean follow-up was . ± . months. all the enrolled pts were treated with abstracts an initial dose of cs of mg/kg/day, to target ng/ml for the first days. pts were randomized on day into one of the two following gr on a : basis. ev gr: initial dose of ev was mg/day, to reach blood level of ng/ml. the dose was increased on day , when cs was discontinued, in order to reach an ev blood level between and ng/ml. cs gr: after the th post-operative day the dose of cs was adjusted to a target level of ng/ml until day , then to ng/ml until the end of month . all pts received basiliximab induction on day and after lt. pts were weaned off prednisone by weeks. pt survival at and months was similar in the ev and cs gr ( . % and . % vs . % and . % respectively; p= ns). causes of death were sepsis ( ), hcv recurrence ( ), pulmonary embolism ( ) in the ev gr, and sepsis ( ), rupture of splenic artery aneurysm ( ) the overall incidence of infection episodes was comparable between two gr ( . % ev gr vs . % cs gr; p=ns). cholesterol but not triglycerides increased in the ev gr compared with the cs gr (p<. ); ev dose reduction decreased such parameters without the need for statin implementation. conclusion ev monotherapy in de novo lt showed similar patient survival and incidence of morbidity compared to a cs immunosuppressive protocol. the primary endpoint was achieved inasmuch as renal function was statistically better in the ev gr. background: sir is a potent immunosuppressive agent that inhibits t-cell activation and proliferation. in lt recipients, sir has primarily been used as a renal-sparing agent, but its toxicity and tolerability in this population has not been well defined. aims: to identify the adverse effects and predictors of discontinuation of sir in lt recipients. methods: records from adult lt recipients transplanted between / and / were reviewed. reasons for starting and discontinuing sir were captured, as were all significant adverse effects and laboratory abnormalities. factors predicting sir discontinuation in univariate analysis were further analyzed by multivariable logistic regression (mlr). results: mean age of the study group was ± years, and % were male. underlying liver disease was hcv ± alcohol in %, % had hepatocellular carcinoma, and % received living donor grafts. calcineurin inhibitors (cni) were started post-operatively in % ( % tacrolimus/ % cyclosporine), with or without mycophenolate and prednisone. patients ( %) started sir a median of days (iqr: - ) post-lt primarily for renal insufficiency ( %) or cni neurotoxicity ( %). sir was overlapped with tacrolimus and cyclosporine in % and %, respectively. prior to starting sir, total cholesterol was ± mg/dl, ldl-cholesterol ± , triglycerides ± . peak lipids after sir were ± , ± , and ± mg/dl, respectively, despite lipid-lowering therapy. serum creatinine was . ± . and . ± . mg/dl before and after sir, respectively. before sir, % of patients had no proteinuria, but only % had no proteinuria after sir. high range proteinuria (> mg/dl) was noted in % before and % after sir. finally, sir was discontinued in total of ( %) patients, for indications of cytopenias ( %), hyperlipidemia ( %), mouth ulcers ( %), sepsis ( . %), skin reactions ( . %), nephrotic syndrome ( %), gi intolerance ( %), pneumonitis/boop ( . %), myopathy ( . %), and combinations of above ( %). mlr failed to identify any pretreatment predictors of discontinuation. conclusions: immunosuppression with sir improves azotemia at the expense of considerable hematologic, metabolic, dermatologic, renal, pulmonary and muscle toxicity. considering the high incidence of proteinuria after sir treatment, the use of sir as a less nephrotoxic agent must be re-considered. and to identify the most effective protocol. peripheral blood was obtained from ebvseronegative and ebv-seropositive pediatric heart (h) tx patients. lcl vs dc-based methods were compared as follows: (i) lcl (ii) lcl + il- (iii) type- polarized dc (treated with il -b, tnf-a, il- and ifn-g) loaded with mhc class i-restricted ebv-peptide pool (dc/pep.) and (iv) dc/pep. + il- . the ebv-specific cd + t cell phenotype and function were screened using flow cytometry, ifng elispot and cytotoxicity assays. the yields and the functional activities of in vitro co-cultures differed based on the induction method employed, and on the ebv status of the patients tested. for the ebv-seropositive pediatric htx patients, all four methods resulted in the successful expansion of functional type- ebv-specific cd + t cells, suggesting that memory cd + t cell are readily reactivated in vitro. for the ebv-seronegative pediatric tx patients however, only the lcl + il- approach resulted in significant augmentation of type- ebv-specific ctls that were competent to secrete ifn-g ( ± / cells) and to kill ( ± lu/ cells) ebv + targets. we found that il- secreted by lcl (and not by dc) was critical in triggering expression of il- rb on naive cd + t cells, and rendering these cells responsive to il- p . further addition of exogenous il- p (which is generally not produced by lcl) proved to be essential for effective type- priming. however, blocking il- during ebv-priming has abolished il- rb expression and subsequent ifng production. these results demonstrate that the inducible expression of il- rb on naïve cd + t cells was dependent on il- , and support the critical early role of ebv infected b cells in the in vivo priming of naïve precursors into potent ebv type- cd + t cell in children. serial ebv load monitoring in pediatric heart transplant patients (phtx) has identified a group of asymptomatic children that exhibit persistently high ebv loads in peripheral blood (≥ , copies/ml on at least % samples over a period of at least months). these patients have a high rate of progression to late ptld. our goal is to characterize the deficiency of ebv-specific cd + t-cell immunity that allows this state to occur and be maintained. twenty-one stable ebv + phtx patients were categorized as follows: group (n= ) no detectable viral load; group (n= ) low viral load (≤ , copies/ ml); group (n= ) high viral load (≥ , copies/ml). twelve healthy subjects were recruited as controls. flow cytometric analysis with hla-a ebv-tetramer (tmr) probes in conjunction with mabs against memory/activation markers was performed on peripheral blood cd + t cells, and their ebv-specific ifn-γ production was measured by elispot. ebv-"latent" specific cd + t cells in g patients were mostly cd l + / cd ro + (central memory) and expressed heterogeneous levels of pd- and high cd (il- receptor α), the ebv-lytic-specific cd + t cells were more frequent, and biased toward cd l -/cd ro + (effector memory) and cd l -/cd ra + (stable effector memory), corresponding to terminally differentiated memory compartments. this cell population also expressed heterogeneous levels of pd- and down-regulated cd . in contrast, both ebv-lytic and -latent specific tmr + cd + t cells from g patients were homogeneously cd l + /cd ro + (effector memory), cd + and cd -, suggestive of "recently activated" phenotype. interestingly, although patients in groups g and g had high frequencies of ebv-specific tmr + cd + t cells (g . %± . , g . %± . , p= . ), only g patients exhibited a direct correlation between tmr + cd + t cells and ebv-specific ifn-γ production. these results demonstrate that different levels of chronic ebv-antigenic pressure trigger significant differences in the phenotypic and functional features of ebv-specific cd + t cells from phtx, suggesting that the immunologic characterization of high ebv load carrier state is a combined "activated" phenotype with "exhausted" function of ebv-specific cd + t cells. ebv-encoded lmp indirectly activates the jak/stat pathway through induction of ifnγ. abstracts evidence of ptld. children were enrolled at sites. mean age at transplant . years, mean time to ptld months . organs transplanted were lung , heart , kidney . all ptld were of b cell origin and expressed cd and all were ebv positive. histology was: polymorphic , monomorphic , hodgkins-like . patients received doses, patients doses and patient doses. treatment was associated with minimal side effects in , mild-moderate infusion related reactions in and moderate reaction in . no patient had treatment discontinued because of side effects. twelve patients ( %) showed complete response after - doses, had progressive disease and one had stable disease. at months, ( %) were alive with one graft loss (kidney) and none with residual disease. at latest follow-up (mean . months, - ), remain alive with further graft loss (lung) and no ptld. the deaths occurred between . and months and were associated with progressive disease ( ), chronic rejection ( ), and complications of elective surgery ( ) . conclusions: these findings support prior registry data and suggest that rituximab without chemotherapy is a successful second line treatment in approximately two-thirds of children with refractory ptld. cancer after organ transplantation in france. jean michel rebibou, fabienne pessione, francois aubin, bernard loty. agence de la biomedecine, france. cancer prevention appears as a major challenge in transplantation. estimating cancer frequency is a major step toward designing prevention policy. we report on patterns of cancer incidence among transplantations registered in the french data base. ). the risk of lung cancer is higher for recipients of a thoracic organ and the risk of kidney cancer appeared higher for kidney recipients. ten year cumulative incidence was . % for all cancer and transplantation types, . % for nhl. multivariate analysis demonstrated that cancer risk increased with recipient age (p< - ), year cumulative incidence was % for recipients older than year. it was higher in male (p< - ) and in thoracic organ recipients when compared with kidney recipients (p< - ). cancer incidence did not vary according to the transplantation period ( ( - ( vs ( - . p= . ) and nhl risk was significantly lower during the period - (rr= . , p= . ). this work does not report any increase in cancer incidence among transplant recipients while cancer incidence increased in the general population. the observed decrease of nhl risk is of particular interest. the both costimulatory and co-inhibitory signals are delivered by b ligands through the cd family of receptors on t lymphocytes determining the ultimate immune responses. although b -h , a recently discovered member of the b family, is known to negatively regulate t cell immunity in autoimmunity and cancer, its role in transplantation rejection and tolerance has not been established. to study its role in physiologic rejection processes, we first treated b wt recipients of balb/c hearts with a blocking mab against b -h or isotype igg control and found no difference in graft survival (mst days, n= vs. , n= ). however, b -h blockade resulted in accelerated allograft rejection in cd deficient b recipients (mst . vs. , n= , p= . ) , indicating that b -h signaling can mediate negative regulation in the absence of cd costimulation. we next studied b - /b - double deficient (dko) b recipients of balb/c heart allografts, as these mice are truly independent of cd /ctla- :b signals. while cardiac allografts were accepted in control dko recipients (mst> , n= ), blocking b -h precipitated rejection (mst , n= , p= . ) demonstrating non-redundant functions of these two negative pathways. based on these results, we next evaluated the role of b -h in acquired transplantation tolerance by blocking cd :b using ctla -ig. b -h blockade abrogated prolongation of allograft survival by ctla -ig ( µg on day ) in the fully mhc-mismatched cardiac allograft model (mst vs. . , n= , p= . ) . we conclude that the novel b -h molecule can regulate alloimmune responses independent of an intact cd /ctla- :b costimulatory pathway. the interplay between positive (stimulatory) and negative (regulatory) costimulatory signals is an important determinant of the outcome of the alloimmune response and could be exploited to induce tolerance. specific in a model of mhc-mismatched kidney allograft in the rat, treatment with anti-cd antibodies induced a form of tolerance independent of treg cells but associated with a two-fold accumulation of mdsc in the blood. to further characterize these cells, we analyzed their phenotype and mechanism of action by flow cytometry, western blotting and suppression assays. mdsc expressed cd and cd , nkrp- , cd a (sirpa), cd a, cd b, his and for a fraction of them cd but did not express mhc class ii molecules. mdsc dose-dependently suppressed the proliferation of t cells in mlr and after stimulation with anti-cd + anti-cd antibodies. although detected in blood, bone marrow, spleen and lymph nodes, mdsc were only suppressive in blood and bone marrow. this suppression was lost after physical separation from the responding t cells by semi-permeable membranes in transwell assays, as well as after addition of l-nmma, a selective inhibitor of inducible nitric oxide synthase (inos), suggesting a role for no in the suppression. western blot analyses revealed that inos was expressed only after contact between mdsc and activated cd + cd effector t cells and to a much lesser extent after contact with activated cd + cd high t reg cells. mdsc affected the viability of stimulated cfse-labeled effector t cells by blocking their proliferation but not their activation. in contrast, mdsc did not block the response of t reg cells stimulated by anti-cd + anti-cd antibodies. this selective suppression of effector but not of regulatory t cells was confirmed by cytokine profile analyses. in vivo, the expression of inos was higher in the blood of tolerant recipients, as well as in the graft, as compared with isografted recipients. in addition, the injection in stable tolerant animals of aminoguanidine, which inhibits inos, induced graft rejection within weeks. in conclusion, these results suggest that mdsc, accumulated in the blood of tolerant recipients of kidney allografts, release high levels of no after contact with activated effector t cells and specifically control their proliferative response. liver non-parenchymal components inhibit dendritic cell differentiation and maturation. ching-chun hsieh, horng-ren yang, guoping jiang, john j. fung, shigunag qian, lina lu. immunology and general surgery, cleveland clinic, cleveland, oh. the inherent tolerogenicity of liver allografts could be due to comparatively large numbers of potentially tolerogeneic antigen presenting cells, in particular dendritic cells (dc). it is not clear whether the unique antigen presenting function of liver dc is intrinsic, or is altered by the microenvironmental factors within the liver. in the present study, we investigated the effect of hepatic stallet cells (hpsc), a unique tissue cells in the liver which were actively expanded in the liver allografts, on generation and function of bone marrow derived dc (bm dc). we hypothesize that liver hpsc may modulate immune response via inhibition of liver dendritic cells (dc) which are known of bone marrow (bm) origin. in this study, dc were propagated from b bm cells with gm-csf for days. irradiated hpsc isolated from b mouse liver were added at the beginning into the culture at hpsc : bmdc progenitor ratio of : . the differentiation, maturation and function of propagated dc were determined by characterizing their surface molecule expression and functions of instructing t cell activation / differentiation. the results showed that addition of hpsc markedly blocked the differentiation of dc from bm precursors (most cells remained in cd b + cd cprecursors stages). the incidence of cd c + cells was % vs. . % in normal bm dc culture (without hpsc). the presence of hpsc also prevented maturation of cd c + dc, as evidenced by low expression of cd , cd and cd , but high of b -h . the inhibitory effect appeared to be mediated by soluble factor (s) produced by hpsc, since addition of the hpsc culture supernatant or transwell culture provided comparable inhibitory activity. culture of allogeneic cd + t cells with hpsc-dc elicited poor proliferative response in a d mlr assay, with low il- and ifn-γ production. three color staining of t cells stimulated by hpsc-dc showed that cd + foxp + t cells were preferentially expanded, suggesting that hpsc-dc were capable of inducting treg. in contrast, bm dc induced vigorous cd + t cells proliferation, most of activated cd + t cells were cd + foxp associated with high levels of ifnγ and il- in the culture, indicating induction of th cells. in conclusion, liver tissue cells, such as hpsc markedly inhibit dc differentiation and maturation, suggesting that the tolerogenic property of liver dc may not be intrinsic, but is altered by the microenvironmental factors in the liver. . allograft rejection was also significantly accelerated when bm hearts were transplanted into cd ko recipient (mst days). to investigate the mechanisms of these findings, lymphocytes harvested at day post-tx from spleens and regional lymph nodes were assessed by flow cytometry for phenotypic differentiation of the activation status, regulatory t cell markers and effector cell generation. the ratio of effector to regulatory cells was . : . : . in the wt, cd ko and b dko recipients, respectively. cytokine production was evaluated by elispot using donor specific antigen stimulation of recipient splenocytes. the mean ifn-γ production was spots per , splenocytes in wt, in cd ko and in b dko. this study for the first time demonstrates the paradoxical role of b -cd co-stimulation in fully allogeneic and class ii mismatched grafts and proposes a possible mechanism through the re-alignment of the effector to regulatory t cell ratio in favor of a highly alloreactive immune response. the major concern regarding kidney donation has been whether the occurrence of hyperfiltration, on the background of increasing prevalence of hypertension with aging and the decline in glomerular filtration rate (gfr) noted in some people as they get older, may put donors at a higher risk for progressive kidney disease. we have previously reported on donors > years after donation and found them to incur no excessive risk of hypertension or kidney disease. herein, we report on renal and non-renal outcomes on the world's largest experience of kidney donors to date (n= ) who donated more than years ago. methods: kidney donors were asked to fill out a survey detailing their medical history since donation and obtain a physical exam and laboratory testing by their local physician. results are expressed as mean±standard deviation (sd the graph below depicts the cross-sectional distribution of last serum creatinine available years or more after donation regardless whether the donor is presently alive or not though the majority is from currently alive donors. conclusion: in the longest follow-up of kidney donors to date, this data indicates that - decades of living with one kidney has no serious adverse renal effects and a prevalence of hypertension that is probably similar to the general population considering the age of these donors. centers ) . before and after adjustment for comorbidity, ( . %) and ( . %) centers, respectively, met all criteria for review, but only met criteria for review both before and after comorbidity adjustment. we conclude that failure to adjust for pre-existing recipient comorbidity results in grossly inaccurate estimation of expected gf per ktx center, more often resulting in expected gf that is too low. using data that are not adjusted for comorbidity to judge the quality of tx programs could encourage denial of access to high-risk patients. introduction: the purpose of our study was to examine temporal trends in the regionalization patterns and center volume-outcomes relationship for lung transplantation in the united states over the past decade. a retrospective analysis of all adult single-organ lung transplants included in the scientific registry of transplant recipients for three consecutive time periods between and was performed. for each time period, lung transplant centers were divided into three groups based on each center's annual volume of the procedure (low-volume group = - procedures per year, medium-volume group = - procedures per year, high-volume group = greater than procedures per year). oneyear observed-to-expected patient death ratios were then calculated and compared for each group in each time period. a temporal analysis of the percentage of transplants being performed relative to center volume was also performed. statistical comparisons were made using chi square testing. results: a total of , lung transplant procedures were included in the analysis. in period , there was not a significant difference in the one-year observed-to-expected patient death ratio of low-volume lung transplant centers when compared to high-volume centers (ratio . for low-volume centers vs. . for high-volume centers, p = . ). by period , however, a significant relationship between center volume and outcomes had emerged (ratio . for low-volume centers vs. . for high-volume centers, p = . ). over this same time period, the percentage of lung transplants within the united states that are performed at low-volume centers has decreased significantly (from . % of all lung transplants in period to . % in period , p < . ), while the percentage being performed at high-volume centers has increased significantly (from . % of all lung transplants in period to . % in period , p< . ). conclusions: a significant relationship between center volume and patient outcomes has emerged for lung transplantation over the past decade. at the same time, the percentage of these procedures being performed at high-volume centers has increased. these findings suggest that regionalization patterns for a given procedure may be influenced by the presence or absence of a volume-outcomes relationship for that procedure. liver transplantation (lt) has emerged as one of the few curative treatment modalities for patients with hepatocellular carcinoma (hcc). however, the increase in the incidence of hcc recurrence due to immunosuppressants administered after lt is a serious issue. we have recently proposed a novel strategy of adjuvant immunotherapy for preventing the recurrence of hcc after lt: intravenous administration of il- -stimulated natural killer (nk) cells extracted from donor liver graft to liver transplant recipients. since the immunosuppressive regimen currently used after lt reduces the adaptive immune components but well maintains the innate components of cellular immunity, the augmentation of nk cells response might be a promising immunotherapeutic approach. we confirmed that the il- -stimulated donor liver nk cells exhibited a significantly high level of trail and showed vigorous cytotoxicity against an hcc cell line without cytotoxicity against normal cells. after obtaining approval from the ethical committee of our institute, we successfully applied this therapy to cirrhotic patients with hcc from january . the average number of nk cells that had been administered to lt recipients at days after lt was . ± . × cells/body. the lt recipients were categorized as follows: ( ) based on the milan criteria, recipients met the criteria while did not, and ( ) based on the tnm stage, recipients were categorized as pathological tnm stage i; , stage ii; , stage iii; and , stage iv. in our institute, the -year recurrence-free survival rates of the lt recipients treated with and without this therapy were % and . %, respectively. kinetic studies revealed that in the early postoperative period, the peripheral blood obtained from the treated lt recipients exhibited a significant improvement in cytotoxicity against hcc cell line as compared to the untreated lt recipients (p < . ). furthermore, flow cytometric analyses revealed that the frequency of trail + nk cells increased remarkably in the peripheral blood of the treated lt recipients (p < . ). in conclusion, the administration of il- -stimulated donor liver nk cells contributes to the promotion of host anticancer activity and has the potential to regulate hcc recurrence after lt. abstract# vegf, a well-established angiogenesis factor, is expressed within allografts at high levels in association with acute and chronic rejection. in previous studies, we have reported that vegf possesses potent proinflammatory properties in part via its ability to mediate leukocyte trafficking into allografts. recently, we discovered that vegf mediates cd + and cd + t cell migration via interaction with its receptor kdr (also called vegfr ). blockade of t cell kdr significantly inhibits transendothelial migration. these observations suggest that kdr may be a novel t cell receptor for allogeneic lymphocyte recruitment. here, we first examined the expression of kdr on peripheral human cd + and cd + t cells by facs analysis. we found that ≤ % of circulating t cells express kdr, and its expression was at low levels on individual unactivated t cells. further, we found that the expression of kdr on t cells increased markedly following activation with mitogen and following interactions with activated allogeneic endothelial cells. induced expression of kdr on cd + and cd + t cells was at a similar level as that observed on endothelial cells. therefore, kdr appears to be selectively expressed on t cells, that traffick into allografts. to test the pathophysiological significance of these observations, we analyzed the expression of kdr in a total of cardiac, and renal, human allograft biopsies. we correlated the expression of kdr with cd + t cell infiltrates; and by double immunofluorescence staining, we determined co-expression of kdr on individual cd + t cell infiltrates. in cardiac allografts we found that kdr was expressed throughout the endomyocardium, and was most notable on endothelial cells in all biopsies examined. by grid counting of - areas of each biopsy, we found that the mean number of cd + t cell infiltrates ranged from to cells/hpf (x mag.). by double staining, we noted that kdr was expressed on ± % (mean±sem) of these cd + t cell infiltrates. similarly, we found that kdr was co-expressed on cd + t cells within renal allografts. while infiltrates were more focal, again ± % (mean±sem) of graft infiltrating t cells expressed kdr. collectively, these observations for the first time identify kdr as a novel receptor on allogeneic t cells. we suggest that intragraft vegf may interact with t cell kdr to facilitate homing and recruitment of allospecific lymphocytes into allografts. interaction of infiltrating cd + t cells and tissue cells in tolerant liver allografts: using tcr transgene approach. guoping jiang, qiwei zhang, horng-ren yang, kathleen brown, john j. fung, lina lu, shiguang qian. immunology and general surgery, cleveland clinic, cleveland, oh. the liver allografts are accepted without requirement of immunosuppression in mice. the underlying mechanisms are not completely understood. we hypothesized that it resulted from an abortive t cell response within the liver due to hyporesponsiveness or apoptosis. to test this, we examined the activation and fate of allo-ag specific cd + t cells following liver transplants (ltx) compared with heart transplants (htx) that were acutely rejected. following transplantation [b (h b )→c h (h k )], cfse labeled cd + t cells ( x ) from des tcr tg mice (h k b specific tcr) were adoptively transferred into recipients. animals were sacrificed two days following des t cell administration for analyses of t cells in the grafts or draining lymph nodes (d-ln). host cd + leukocytes were quickly infiltrated following ltx. among cd population ∼ % were cd + and ∼ % cd + . cd + t cells were further increased thereafter in grafts and d-ln, associated with high inf-g production. cd + t cells in the liver grafts rapidly reduced to % by pod , and to % by pod . however, the incidence of cd + t cells remained high. cfse dilution assay and elispot showed an active division of des + t cells in liver allografts either on pod or . these ag-specific cd + t cells functioned well evidenced by ifn-g production in response to allo-ag. however, compared to htx, the accumulation of des + cells in grafts was significantly lower in ltx [ . % ( x /heart), and . % ( . × /liver)] on pod , and further dropped to . % ( . × /liver) on pod . the expended cohorts of adoptively transferred cells followed by their elimination suggested elimination of ag-specific cd + cells. to examine the role of liver environment, graft cd non-parenchymal cells (npc) were isolated tested fro regulatory effect on des + t cell response. liver allografts showed significantly expansion of cd -npc, which were donor mhc class i + (h b + ), b -h + , trail + and low for cd and cd . these cells did not inhibit des + t cell proliferation in response to b spleen stimulation, but significantly enhanced their death, which was dependent on b -h /trail. this was confirmed by using cd -npc from b h -/or trail -/mice. in conclusion, activated t cells in liver grafts may stimulate tissue cells to express inhibitory and death inducing molecules, resulting in t cell death and graft acceptance. foxp + graft-infiltrating lymphocytes (gil) have been detected in the rejecting allografts of transplant patients. foxp is a marker for regulatory t cells (t reg ). published reports suggest that human foxp can be upregulated following tcr stimulation without induction of regulatory function. to investigate whether foxp + gil during acute rejection are t reg , we analyzed the phenotype of gil harvested from acutely-rejected non-human primate kidney allografts. methods: renal allografts with histologically-confirmed acute rejection were harvested at the time of necropsy from macaques that had undergone experimental transplantation. following digestion of kidney fragments using collagenase, gil were isolated using lymphocyte-separation media and cryopreserved. axillary lymph nodes (ax ln) were also isolated either at the time of necropsy or by biopsy. for seb-stimulated lymphocytes, ax ln cells were stimulated for days in the presence of ng/ml seb for - days. to perform intracellular interferon-gamma (ifng) analysis, cells were stimulated with pma and ionomycin in the presence of brefeldin a for - hours at °c. samples were prepared for flow cytometry by first staining for extracellular antigens. cells were then fixed and permablized (kit from ebiosciences) prior to intracellular staining for foxp , ki and ifng. results: the percentage of foxp + in cd + gil was similar to that found in ax ln ( table ). the majority of these cells were cd + . while % of the cd + /cd + /foxp + population of both ax ln and gil were cd + , a significantly higher frequency of cd + and ki + cells were found in gil (p < . ; student's t-test). simlar levels of cd and ki expression were found in seb stimulated lymphocytes. unlike the seb-stimulated lymphocytes, few ifng -producing cells were demonstrated following pma/ionomycin stimulation of gil. conclusion: our current data indicates that the majority of foxp + gil from acutely rejecting renal allografts are recently activated cd t cells that lack effector function. this suggests that foxp t cells within rejecting allografts may indeed be t reg . findings in mouse models of transplantation often fail to translate well in humans. three variables may account for the discrepancy: ( ) evolutionary divergence between mice and humans, ( ) influence of infection history on alloimmunity, and ( ) use of highly inbred strains of laboratory mice. here, we investigated whether the use of inbred mouse strains skews the rejection phenotypes and their response to treatment due to decreased genetic diversity and/or fixation of undesirable genetic loci, known as inbreeding depression. we examined heterotopic cardiac allograft survival in outbred and inbred mouse populations in the presence or absence of immunosuppression. in the absence of immunosuppression, heart transplantation within or between outbred stocks of mice (n = ) resulted in three distinct rejection phenotypes that resemble accelerated ( - days), acute ( - days), and chronic rejection (> days), respectively. in contrast, all fully allogeneic grafts transplanted between inbred mice (n = ) were rejected acutely ( - days) as were historical controls (n > ). the accelerated phenotype, present in % of outbred to outbred transplantations, was characterized by extensive hemorrhagic necrosis of the heart with thrombosis, neutrophil margination and neutrophilic arteritis, and did not correlate with donor:recipient mhc ii disparity. immunosuppression with t cell costimulation blockade did not prevent accelerated rejection (incidence = % in the treated group, n = ) but did convert the acute rejection phenotype into longterm allograft survival in all groups studied. the same accelerated phenotype was observed if transplantation was performed from outbred to inbred mice (incidence = %; n = ) but could not be duplicated if inbred to outbred transplantation was performed (n = ). finally, c depletion with cobra venom factor abrogated the accelerated rejection phenotype in outbred to outbred transplantations (n = ), suggesting a role for complement in the pathogenesis of this phenotype. in summary, our data ( ) indicate that the use of outbred mouse stocks may uncover clinically-relevant rejection phenotypes not observed in inbred mouse strains, and ( ) underscore the importance of the donor background in determining the phenotype of rejection. outbred mouse stocks may provide a platform to uncover mhc-unlinked genetic loci that play an important role in the outcome of solid organ transplantation. th are limited in their ability to reject allografts. elderly recipients represent the most rapidly growing segment of patients on the waiting list. however, little is known about age-dependent alterations of the immune response in organ transplantation. we examined age dependent t-cell functions in a transgenic mouse transplant model. effector t-cell phenotype, -function, cytokine production and regulatory t-cell function were analyzed in and mths old b mice. in an in vivo transplant model, bl/ nude mice were reconstituted with x young or old transgenic alloantigen-specific cd + tcells and engrafted with bm skin grafts. t-cell phenotype and cytokine secretion were sequentially analyzed in all lymphatic compartments. splenocytes of naïve old b mice contained significantly higher frequencies of t-cells with an effector/memory phenotype (cd + cd high cd l low and cd + cd h igh cd l low; p< . ). in vitro proliferation and ifnγ-production were significantly reduced in aged mice indicating an impaired t-cell response with increasing age as assessed by mlr (p< . ) and elispot (p< . ). in parallel, regulatory functions remained age-independent as alloantigen-specific cd + cd + foxp + t-cells isolated from sensitized old mice demonstrated a dose-dependent well preserved suppressor function. next, we tested the age-dependent alloantigen -specific cd + t-cell function in a transgenic skin transplant model: age did not significantly impact rejection kinetics (young vs. old: . vs. . days, n.s.) however, t-cell migration and activation were significantly different: fewer numbers of activated cd + cd + and effector/memory phenotype t-cells (cd + cd high cd l low ) were found in recipient spleens (p< . ) and draining lymph nodes (dln) (p< . ) after transfer of old t-cells. chemokine receptor staining revealed less cxcr + and ccr + t-cells in dln following the transfer of old t-cells (total cell numbers x : cxcr + : . ± . vs. . ± . , ccr + : . ± . vs. . ± . , p< . ). this was paralleled by reduced intragraft t-cell infiltration as observed by immunohistochemistry. in summary, native elderly mice showed an increased frequency of effector memory t-cells but an overall impaired t-cell response. regulatory -t-cell function remained preserved. in vivo allospecific cd + t-cell activation and migration was impaired in elderly transplant recipients. the background: the sensitized transplant recipients may undergo an "accelerated" form of rejection, which is mediated by t cell-dependent mechanisms. these patients often experience increased rate of early rejection episodes, which are difficult to control with currently used immunosuppressive agents. methods: in our model of cardiac graft rejection in sensitized recipients, b mice are first challenged with b/c skin, followed - days later by b/c heart transplant (htx). unlike in naive hosts, htx rejection and alloreactive cd activation in this model are cd blockade-resistant. we first performed systemic analysis at the intragraft transcriptional level by microarray to identify disparities in local immune responses in naive vs. sensitized hosts. aiming to improve the efficacy of costimulation blockade in the sensitization settings, we then determined the role of cd t cells in costimulation blockade-resistant alloimmune response by using cd depleting (gk . ) vs. cd blocking (yts ) ab, in conjunction with cd blockade (mr ). results: htx harvested from groups of naïve (day - ), sensitized (day - ), control ig or mr ab treated mice (n= /gr) were subjected to microarray analysis. mr treatment suppressed htx expression of proinflammatory genes (il- β, il- , tnf-α), and t cell-targeted chemokines (rantes, mig, cxcl ) early after htx in naïve, but not sensitized recipients. five groups of sensitized mice treated at the time of htx with: ( ) ). ctl activation was determined by facs phenotyping at day and . the simultaneous blockade of cd costimulation and cd help, but not a single blockade with mr ab or anti-cd ab, was required to inhibit peripheral alloreactive cd activation in sensitized mice. additionally, cd activation in the absence of cd help showed defective cytotoxic molecule profile, with suppressed perforin but upregulated granzyme b expression at the graft site. conclusion: cd blockade-resistant cd activation is critically dependent on cd t cells. this study provides novel immunological basis to study the potential synergy between adjunctive cd and cd targeted therapies to control accelerated graft rejection in sensitized hosts. mediators. g. einecke, l. g. hidalgo, p. f. halloran. department of medicine, university of alberta, edmonton, canada. the hallmark of t cell mediated rejection (tcmr) are interstitial inflammation and tubulitis. the mechanisms of tubulitis and epithelial deterioration during tcmr are unknown. we previously showed that tubulitis in mouse allografts is independent of cytotoxic molecules (gzma/b, prf) and is preceded by molecular changes with loss of epithelial genes, reflecting epithelial dedifferentiation. human tcmr is associated with loss of the same epithelial genes and re-expression of embryonic pathways (wnt, notch). we hypothesized that tcmr is mediated through soluble factors released by effector t cells or macrophages in the interstitium, and that supernatants of effector t cells would simulate these changes in epithelial cultures. we established an in vitro model in which cultured primary human renal epithelial cells are incubated with supernatants from effector t cell/monocyte co-cultures. the transcript changes in this model, analyzed by microarrays, closely simulated those in human and mouse tcmr (fig ), with loss of epithelial transcripts, activation of wnt/ notch pathways, and increased expression of ifng-inducible and injury-related transcripts previously defined in our mouse model. some of these changes were reproduced by incubation of epithelial cells with ifng or tgfb. the in vitro model identified additional epithelial transcript changes not previously identified in vivo (not affected by ifng or ischemic injury, not expressed in t cells, macrophages, b cells, or nk cells). expression of these transcripts (n = ) was highly altered in human tcmr compared to nonrejecting biopsies of human renal allograft biopsies and distinguished tcmr from antibody-mediated rejection in a hierarchical cluster analysis. thus we have established an in vitro model that closely simulates the epithelial events during human tcmr and confirms that these changes are independent of direct contact with inflammatory cells, supporting the hypothesis that interstitial effector t cells mediate allograft deterioration by soluble mediators. together with previous mouse and human data these results provide the first in vitro model of the epithelial consequences of tcmr. objective. c split product deposition to hla antigen-coated microparticles ([c d] flowpra) was previously shown to be a specific marker of c d-positive antibodymediated rejection (amr). the objective of this study was to assess the predictive value of [c d]flowpra reactivity in a cohort of non-biopsied patients with stable graft function during the first year. methods. a total of kidney transplant recipients were enrolled (inclusion criteria: functioning graft at months; prospective collection of sera taken before and at - , , and months after transplantation). included patients were serially screened for humoral panel reactivity applying [igg] and [c d]flowpra screening. results. fifty-four of the included recipients had stable graft function within the first year and were not subjected to diagnostic renal biopsy. in this particular patient group, detection of complement-fixing hla reactivity tended to be less frequent than in the patients with biopsied graft dysfunction (≥ % [c d]flowpra before transplantation: % vs. % of recipients, p= . ; ≥ % [c d]flowpra after transplantation: % vs %, p= . ). in line with our previous results, within the group of biopsied patients, pre-and/or post-tx [c d]flowpra reactivity was tightly associated with the immunohistochemical detection of peritubular capillary c d deposition (p= . ) reflecting ongoing amr. remarkably, in initially stable patients, detectable [c d] flowpra reactivity was not associated with inferior long-term outcomes. within this patient group, recipients with and without (pre-and/or post-transplant) c d-fixing anti-hla reactivity did not differ with respect to yr allograft survival (p= . ), yr serum creatinine levels ( . vs. . mg/dl; p= . ), and proteinuria at yrs ( . vs. . g/ h; p= . ). similar results were obtained for a comparison of [igg]flowpra positive vs. negative subjects. conclusion. our data suggest that a considerable number of patients with initially stable graft function may have excellent long-term graft function despite serologically detectable levels of (complement-fixing) alloreactivity. for these antibody-positive recipients, a potential role of graft accommodation can be speculated. the immunoproteasome subunit beta as a novel peripheral blood and intragraft biomarker of chronic antibody mediated allograft rejection in clinical transplantation. joanna ashton-chess, in an attempt to identify non-invasive biomarkers of specific histological scarring, we compared publicly available gene sets derived from microarray studies of human renal transplant biopsies published in the literature with our own microarray data derived from studies of rat heart allografts. in this way we identified an immunoproteasome subunit (proteasome subunit beta -psmb ) as a potentially interesting candidate. psmb is one of three members of the immunoproteasome that are induced by abstracts interferon gamma. messenger rna profiling in renal transplant biopsies (n = ) with normal histology, interstitial fibrosis and tubular atrophy, calcineurin inhibitor toxicity, transplant glomerulopathy or chronic antibody-mediated rejection (banff ) revealed psmb to be strongly and significantly increased in chronic antibody mediated rejection vs. the other three histological diagnoses. receiver operator characteristic (roc) curve analysis showed that psmb mrna could diagnose chronic antibody-mediated rejection with an auc of . , a sensitivity of . and a specificity of . . moreover, psmb mrna was significantly increased in the pbmc (n = ) of patients with chronic antibody-mediated rejection compared to those with normal histology. roc analyses revealed an impressive auc of . with all patients being correctly classified. similar results were also observed in a rat allograft model where psmb was significantly increased at day post transplantation in both the heart allograft and the pbmc of animals presenting chronic transplant vasculopathy vs. syngeneic grafts. moreover, inhibition of the proteasome by administration of velcade® at . mg/kg every other day for the first days post transplantation significantly and dose-dependently prolonged allograft survival (mst . days in velcade-treated vs. . days in untreated animals).together our data point towards psmb as a blood and intragraft biomarker of chronic antibody-mediated rejection as well as a potential therapeutic target. furthermore, our results suggest that using a threshold of psmb in the blood could help in guiding the decision to biopsy in the clinic. baff monitoring after b-cell depletion therapy for acute renal transplant rejection. valeriya zarkhin, snehal mohile, li li, jonathan martin, minnie sarwal. pediatrics, stanford university, stanford, ca. introduction: the objective of this study was to investigate the interaction between b-cell activation factor of the tnf family (baff) level and circulating b-cell repopulation in pediatric patients with acute kidney transplant rejection treated with the b-cell-depleting agent rituximab. methods: pediatric patients ( - yrs) with biopsy proven b-cell positive ar were treated with steroids and rituximab ( x mg/m /dose/week). all patients were followed up for months. peripheral blood cd cells and donor specific antibodies (dsa) were monitored monthly. serum level of baff was measured by elisa at ar, , , , and months post-ar treatment and correlated with clinical outcomes. results: complete depletion of circulating and intragraft b-cells was observed with rituximab, with improvement in ar grade in all patients. the median time of peripheral b-cells repopulation was months (range - months, fig. a) . no correlation was found between pre-treatment peripheral b-cell number and the b-cell repopulation time (r= . , p= . ). baff levels rose significantly with b-cell depletion with maximum values at months post-treatment ( . fold increase, p= . ) and returned to pre-treatment levels, with b-cell recovery, at months (fig. b) . serum baff levels correlated positively with b-cell depletion > months (r= . , p= . , fig b). a lack of depletion of dsa i, but not dsa ii correlated with higher baff levels (r= . , p= . ). the timing of b-cell repopulation and depletion of dsa i may be dependant on serum baff level. anti-baff treatment may be considered in addition to rituximab or standard immunosuppressive treatment protocols in patients with persistent and/or antibody mediated rejection. the background: the development of donor specific hla antibodies (dsa) post-transplant has been associated with graft failure. we have shown in a longitudinal study that increases in dsa may precede rejection by months. this retrospective analysis evaluates changes in maintenance immunosuppression (mi) and appearance of dsa in stable transplant recipients. methods: sera from stable renal transplant recipients were collected at - month intervals and tested for the presence of dsa. the types and doses of immunosuppression were correlated with the appearance of dsa. two hundred eighty stable renal transplant recipients who received either a deceased or a living donor kidney were monitored post-transplantation for the development of dsa. patients have been followed for to years and had a minimum of serum samples analyzed. all recipients received anti-lymphocyte induction therapy. maintenance immunosuppression (mi) consisted of a calcineurin inhibitor, prednisolone, and mycophenolic acid. hla single antigen beads analyzed in the luminex instrument were used to establish donor specificity of the antibodies. a chart review was undertaken to determine the doses of the mi posttransplantation. all mi was managed by the transplant team and changed according to clinical indications without regard to dsa. results: of the patients monitored developed dsa post-transplantation with a functioning graft. dsa was against hla-class ii antigens in of ( %); class i antigens in of ( %); and against both class i and ii antigens in of ( %). dsa against class ii was against dq in all except one case. in the majority of the recipients the appearance of dsa was preceded with dose reduction of the mi, either calcineurin inhibitor or mycophenolic acid or both. conclusions: our data show that dsa developed predominantly against hla-class ii antigens and that the appearance of dsa was often preceded with reduction of one or more of the mi. this data shows the importance of monitoring dsa with mi decreases in a stable allograft recipient. antibody production and antigen presentation are directly inhibited by mycophenolate mofetil. anat r. tambur, joe leventhal, nancy d. herrera, joshua miller. division of organ transplantation, northwestern university, chicago, il. immunosuppressive medications are primarily designed to target t cell proliferation. mycophenolate mofetil (mmf) exerts its effect by inhibiting de-novo synthesis of guanine, a dna building block. we, and others, have previously shown that mpa (active metabolite of mmf) affects the differentiation of monocytes into dendritic cells (dc). we further demonstrated that cell-surface receptors associated with antigen up-take and antigen-processing and presentation (cd and cd ) are down regulated when cells are matured in the presence of mpa. this phenotype translated into a decreased uptake of alloantigens and reduced stimulation of t cells. we concluded that mmf inhibits also cell functions requiring mrna synthesis. we now present data regarding the role of mpa in maturation and function of b-lineage cells. pbmcs from subjects were cultured in the presence of cpg, il- , il- and cd l for days to induce memory b and plasma cell maturation in-vitro. cultures were performed in the presence or absence of mpa ( ugr/ul) for the length of the incubation period. in-vitro stimulation of b cells increased the memory population (cd + cd +) from . +/- . % to +/- %. similarly, plasma cells were increased from . +/- . % to . +/- . %. the addition of mpa to the culture inhibited stimulation for both memory and plasma cells ( . +/- % p= . ; and . +/- . % p=ns, respectively). we have further analyzed the effects of mpa on antibody secretion using an elisa (measuring soluble antibodies) as well as a b-cell elispot assay (assessing the number of b cells that produce antibodies). while an expected increase in od values was observed for stimulated samples compared with non-stimulated samples, a significant decrease was observed when stimulation occurred in the presence of mpa. nonstimulated cells: . +/- . ; stimulated cells: . +/- . ; mpa treated stimulated cells: . +/- . ; p< . ). the number of antibody producing cells was also significantly lowered when cultures were done in the presence of mpa (a mean of cells were counted for the stimulated cells compared with - cells for non-stimulated and mpa-treated-stimulated cells). to our knowledge this is the first time where in-vitro experimental data document the inhibitory effect of mpa on b lineage cells and antibody secretion, although clinically known for some time. these results confirm our previous observations regarding the effects of mmf on non-proliferating immune cells. a non-allogeneic stimulus triggers the production of de novo hla and mica antibodies. luis e. morales-buenrostro, , lluvia a. marino-vazquez, anh nguyen, paul i. terasaki, josefina alberu. nephrology and transplantation., instituto nacional de ciencias medicas y nutricion salvador zubiran, mexico city, df, mexico; terasaki foundation laboratory, los angeles, ca; one lambda inc., canoga park, ca. background: in a previous study, we found that healthy people developed hla abs after immunization against hepatitis b virus. the aim of this prospective study was to establish if stimulation with influenza vaccine is capable of triggering the production of hla and mica abs. methods: we determined the presence of hla and mica abs (de novo and preformed abs) in groups of patients vaccinated against influenza: a) healthy adults, b) esrd patients, and c) tr. additionally, we followed healthy unvaccinated people without exposure to sensitizing factor: d) control group. sera samples were collected at baseline (pre influenza shot), at week, and monthly up months after immunization. hla abs were assessed with labscreen single antigen beads for luminex. all samples of each patient were tested simultaneously. a luminescence value higher than was considered positive only if it was times the baseline value. we analyzed the data using chi square, one way anova test, and logistic regression. results: the table shows the types of abs in each group. interestingly, we found preformed abs across all four groups, including the control group (which is free of any known sensitizing factors). the proportion of de novo abs was higher in the group b and c. multivariate analysis shows that the only independent factor associated with development of de novo abs was the presence of preformed abs. we observed a nonspecific immunologic response triggered by external stimulus and was not necessarily associated to the vaccine in people previously sensitized. a introduction. decisions about the minimization and ultimate withdrawal of immunosuppression (is) would be facilitated by the identification of biomarkers associated with operational tolerance (ot). methods. as part of an itn/nih supported study tolerant kidney transplant recipients (off all is for > yr with stable function, n= ) were compared to recipients with stable function on is (sis, n= ), recipients with chronic allograft nephropathy (can, n= ), and healthy volunteers (hv, n= ). pbmc, whole blood total rna, and urine samples from each group were examined using flow cytometry, microarrays, and rt-pcr respectively. results. analysis of microarrays revealed significantly higher expression of b cell differentiation genes in tolerant recipients compared to the sis and can groups. consistent with this finding, tolerant recipients also displayed higher numbers of naïve b cells in peripheral blood and increased expression of cd in urine relative to the sis and can groups. no differences in treg or genes associated with regulatory cells were observed in tolerant recipients relative to other groups. these analyses failed to demonstrate significant differences between tolerant recipients and hvs although support vector machine learning methods suggested potential differences in a number of genes including nfat and calcineurin. finally, relative to tolerant patients, those with can showed decreased numbers of t and nk cells and expressed lower levels of genes associated with immune cell activation in peripheral blood. conclusions. differences in b cell numbers may be useful in identifying tolerant renal transplant recipients or those predisposed to developing tolerance and could potentially provide insights into the mechanisms of tolerance. erythrocyte development of antibodies (abs) to mismatched donor hla antigens has been associated with acute and chronic rejection. complement activation, and c d deposition, has been correlated with humoral rejection of allografts. however, utility of c d staining in ltx has been controversial. a recent study (arthritis rheum. nov; ( ) : - ) shows a strong correlation between erythrocyte bound c d (e-c d) in diagnosis and monitoring of sle. goal of our study is to determine the utility of measuring e-c d in the diagnosis of humoral rejection following human ltx. ltx recipients were analyzed post-ltx for e-c d using facs of rbcs incubated with anti-c d (quidel) followed by fitc-goat anti-mouse. normals were also analyzed. the serum was analyzed for development of anti-hla abs by solid phase assays and for the presence of autoabs to kα tubulin and collagenv (elisa). biopsies from patients were stained immunohistochemically for c d deposition. summary of results are presented in table . infection= / ar= / % e-c d in normals- . %; mfi in normals- . ; mfi=mean fluorescence intensity; ar=acute rejection; dsa=donor specific abs out of patients show significant increase in the % bound e-c d (p< . ) as compared to controls. / had anti-hla and / had autoabs which were significantly different from those with low e-c d (p= . ). staining of the biopsies showed c d deposition in recipients with increased e-c d. all patients with acute humoral rejection had elevated e-c d. we conclude that there is a significant correlation between increase in % e-c d in ltx recipients and development of abs to either hla antigens or auto-antigens during the post-ltx period. biopsies from patients with increased e-c d showed deposition of c d in the allografts. preliminary data suggest that measurement of e-c d using a non-invasive method of flow cytometry may be of value in monitoring ltx patients for humoral rejection. innate immunity: chemokines, cytokines innate immunity is emerging as an important initiator and modulator of the adaptive immune response. in the setting of transplantation, ischemia-reperfusion injury and tissue trauma appear to potentiate the alloimmune response. one of the mechanisms through which the innate immune system modulates an adaptive immune response is dendritic cells (dc). in this study, we examined dc activation in a mouse model of skin transplantation by monitoring the expression of mhc ii and p chain of the proinflammatory cytokine il- . the p expression was detected in live cells using the yet reporter mouse, in which a transgene for yellow fluorescent protein (yfp) was placed downstream of the endogenous il- p gene, thus faithfully "reporting" p expression. skins from c bl/ or balb/c donors were grafted on the dorsal thorax of c bl/ .yet mice. draining and non-draining lymph nodes (ln) were harvested at hours and examined for yfp expression by fluorescent microscopy. a marked increase in the numbers of yfp-expressing dc was observed in draining ln in both syngeneic and allogeneic graft recipients. surprisingly, yfp-expressing dc also increased in nondraining ln when compared to non-transplanted controls. similarly, dc in draining and non-draining ln showed higher mhc ii expression than those in non-transplanted controls. upregulation of mhc ii was highest at hours and decreased significantly by - hours. to assess whether dc activation in non-draining ln was functionally significant, we monitored the activation of adoptively transferred ovalbumin-specific ot-ii tcr transgenic t cells in response to footpad antigen challenge in mice with or without a syngeneic skin transplant on the contralateral upper thorax. otii t cells in transplanted animals proliferated approximately -to -fold better and a higher percentage of the otii cells produced il- than those from non-transplanted animals. thus, local surgical trauma results in a widespread, time-limited, functional activation of dc that appears to act as a partial adjuvant for t cell responses. together, these data suggest that surgical trauma may incite a systemic barrier to transplantation via the activation of dc and therapeutic interventions that reduce the surgical trauma and dc activation may help to improve survival of transplanted grafts. tolerance of cardiac allografts: studies with cx cr -deficient mice. takaya murayama, katsunori tanaka, takuya ueno, mollie jurewics, guleria indira, fiorina paolo, paez jesus, smith n. rex, sayegh mohamed, reza abdi. transplantation research center, renal division, brigham and women's hospital, harvard medical school, boston, ma; department of pathology, massachusetts general hospital, harvard medical school, boston, ma. although donor/tissue dendritic cells (ddc) have long been known to play a key role in mounting alloimmune responses, however, their generation, trafficking and role in tolerance have not rigorously examined. we have used b .fvb-tg (itgax-dtr/ egfp) mice which has a gfp linked to the cd c promoter. using these mice as the donors of heart allograft transplantation provided us a unique model to study ddc posttransplantation. our trafficking data indicate there is a rapid migration of ddc into spleen ( hours post transplantation) but not lymph nodes and that ddc were unexpectedly detected in the spleen of the recipients long after rejection of heart allografts suggesting ddc could escape from the immunosurveillance of host immune system. our data also show that ddc proliferate in the lymphoid tissue of the recipients and co-express class ii molecule of the recipients. we then show that cx cr pathway regulates generation abstracts of heart tissue dc constitutively. as compared to wt hearts, cx cr -/hearts contain lower number of dc and transplanting cx cr -/donor hearts into wt balb/c mice led to significant prolongation of allograft survival without immunosuppression (mst of vs. days, respectively). increasing cx cr -/heart dc by implanting donors with flt -producing hybridoma cells has restored the time of rejection. unexpectedly, induction of long-term survival with anti-cd blockade (mr ) and ctla- ig (but not low dose rapamycine) was abrogated when cx cr -/hearts were used as donors, with concomitant lesser tregs in the cx cr -/heart allografts as compared to wt. furthermore, co-transplanting hearts from wt and cx cr -/into the same recipient treated with mr resulted in significant prolongation of cx cr -/heart allograft survival. depleting the ddc of heart donors prior to transplantation with diphtheria toxin also worsened markedly chronic rejection in the recipients at day post-transplantation. our data indicate that, in contrast to the widely accepted dogma, the presence of donor dc in graft tissue is not only central to allograft rejection but also is necessary for the induction and maintenance of peripheral tolerance. local c a interaction with c ar on dcs modulates dc function, subsequently up-regulating allospecific t cell responses. qi peng, ke li, steven h. sacks, wuding zhou. mrc centre for transplantation, department of nephrology and transplantation, king's college london, guy's campus, london, united kingdom. the innate system of immunity plays an important role in ischemia-reperfusion injury and allograft rejection. the early stages of inflammatory processes are accompanied by complement activation. one biological consequence of this activation is the release of potent inflammatory anaphylatoxins, c a and c a, which have been reported to regulate a range of inflammatory responses. we previously reported that dcs express c ar and c ar, and c a-c ar interaction has a positive impact on murine bm dcs, in terms of activation phenotype and capacity for ag uptake and allostimulation. however, the role of c a in modulating dc function remains unclear. the aim of this study is to investigate the role of local c ar signalling in modulating murine bm dc function and subsequent regulation of the allospecific t cell response. we first evaluated if c a-c ar interaction could result from local expression of factors. our results showed that c ar mrna was detected in wt dcs at different stage of dc culture by rt-pcr, and c a was detected by elisa in the culture supernatants from different stages of dc culture. we next determined if c a-c ar interaction modulates dc function in allospecific t cell stimulation in vitro and in vivo. we found that bm dcs cultured from c ar-/-mice or treated with c ar antagonist (c ara, w ) exhibited a less activated phenotype (producing significantly less il- and more il- , in response to lps stimulation); both c ar-/-and antagonist-treated dcs (lps stimulated) showed reduced capacity to stimulate naïve alloreactive t cells, as measured by ifn-γ production and thymidine uptake. as regards interaction in vivo, following i.p. administration of the c ara-treated dcs into allogeneic mice for days, ex vivo mixed lymphocyte reaction showed that cd + t cells from those recipients have reduced thymidine uptake, but increased il- production compared to that with untreated dcs. conversely, dcs treated with c ar agonist (c a) exhibited a more activated phenotype (producing more il- and less il- ) and were more potent in allospecific t cell stimulation. our findings demonstrate that murine bm dcs can express c ar and c a can be generated locally; c a-c ar interaction up-regulates murine bmdc activation and their allostimulatory capacity. thus, targeting c a-mediated signal may be able to prevent allograft injury. role of tnfα in early chemokine production and leukocyte infiltration into heart allografts. daisuke ishii, austin d. schenck, robert l. fairchild. immunology, glickman urological and kidney institute., cleveland clinic, cleveland, oh. objectives: the acute phase cytokines il- and tnfα are produced early during inflammatory processes, including wound healing and ischemia/reperfusion. the goal of this study was to investigate the role of these cytokines in the induction of early chemokine production and leukocyte infiltration into heart allografts. methods: c bl/ (h- b), balb/c (h- d), and balb/c.il- -/-mice received vascularized syngeneic or complete mhc mismatched, a/j (h- a), cardiac grafts. grafts were retrieved at different time points and total rna and tissue protein were prepared and analyzed by quantitative rt/pcr and elisa to test expression levels of tnf-α, il- , cxcl /kc, cxcl /mip- , and ccl /mcp- in the grafts. anti-tnfα mab ( ug) was given at the time of transplantation with or without anti-cd mab ( ug on days and ). infiltration of cd +, cd + t cells, neutrophils and macrophages was assessed by flow cytometry and immunohistochemistry. donor-reactive t cell priming to ifn-g producing cells in the recipient spleen was measured by elispot. results: expression of tnfα and il- mrna reached an initial peak at hrs post-transplant and a second peak at - hrs with equivalent levels in both iso-and allo-grafts. the neutrophil and macrophage chemoattractants cxcl /kc, cxcl / mip- and ccl /mcp- reached peak levels at hrs post-transplant in both sets of grafts and then declined to background levels. il- deficiency in the recipient or the cardiac allograft did not prolong allograft survival. in untreated mice, heart allografts were rejected at . ± . days after transplantation. anti-tnfα mab decreased neutrophil and macrophage chemoattractant levels % at hrs post-transplant and subsequent neutrophil, macrophage and cd + cell infiltration into the allografts as well as extended graft survival to . ± . days. anti-tnfα mab also decreased the number of donor-reactive ifn-g producing cd t cells almost % on day post-transplant. whereas anti-cd mab prolonged survival to day , administration of anti-tnfα and anti-cd mab delayed rejection to day and resulted in the long-term (> days) survival of % of the heart allografts. conclusions; these data indicate that anti-tnfα antibodies can delay donorreactive cd t cell priming and leukocyte infiltration into heart allografts rejection. as a conjunctive therapy, tnfa antibodies can promote long-term survival of the allografts. introduction recent evidence indicates that inflammation impairs immune regulation, yet the mechanisms behind this effect are not clear, in particular in regards to transplantation tolerance. in this study, we investigated the role of inflammatory cytokines, il- and tnfα, in transplantation tolerance induction. we first examined the impact of il- + tnfα on in vitro t cell alloimmune responses. t cells that were stimulated by allogeneic apcs in conditioned media harvested from lps-activated dcs proliferated more than apc-stimulated t cells that were cultured in conditioned media derived from non-lps-activated dcs. the ability of cd and cd t cells to respond to allogeneic apcs in the lps-activated conditioned media was significantly impaired by the addition of either anti-il- or anti-tnfα mabs. the addition of both mabs further diminshed t cell proliferation, indicating that il- and tnfα synergize to augment in vitro t cell allostimulation. in support of these findings, we noted reduced t cell proliferation during the mlr when t cells were cultured in conditioned media derived from either il- -/-or tnfα-/-lpsactivated dcs. furthermore, these diminished responses was restored by the addition of recombinant il- or tnfα, respectively. to examine the in vivo implications of these findings, we employed a murine skin allograft model, with recipients that were either b wild type, il- -/-or tnfα-/-. these groups were transplanted with a balb/c skin graft and treated with (or without) perioperative costimulatory blockade (ctla ig and anti-cd ). in the absence of immune modulation, all groups rejected balb/c skin allografts at a similar tempo (< days). in the presence of costimulatory blockade, il- -/-recipients (median survival time, mst = days, p = . ) rejected their allografts at a slower tempo compared to wt (mst = days) or tnfα-/-recipients (mst = days). however, this response in il- -/-recipients was further delayed by administering a tnfα inhibiting mab (mst = days), indicating that synergy between il- and tnfα occurs in vivo and prevents the ability of costimulatory blockade to delay the onset of allograft rejection. conclusions we conclude that synergy between il- and tnfα augments t cell alloimmune responses and impairs the effects of costimulatory blockade to delay allograft rejection. abstract# background: calcineurin inhibitors (cni) are involved in the development of post transplant diabetes mellitus (ptdm). changes in insulin secretion and sensitivity are central mechanisms involved in the development of ptdm. in addition alterations in endothelial function seem to be involved. the present study investigated the effect of cni's on these factors. methods: in a predefined sub-study of a previously published randomized trial it was aimed to compare the effect of cni treatment (n= ) with complete cni-avoidance (n= ) on insulin secretion and sensitivity as well as endothelial function. an oral glucose tolerance test and endothelial function investigation with laser doppler flowmetry was performed in patients, weeks and months following transplantation. results: insulin sensitivity differed already weeks posttransplant and was significantly better after months in patients never treated with cni drugs (p= . ). endothelial function was significantly correlated with insulin sensitivity (n= , r = . , p= . ) at weeks posttransplant, but not after months (p= . ). insulin secretion tended to be higher in cni treated patients both at week and month (p= . ). conclusions: findings in the present study indicate that long-term cni treatment reduce insulin sensitivity which was associated with impaired endothelial function. in response to this peripheral insulin resistance a tendency towards a compensatory increase in insulin secretion was seen. these effects combined may indicate a future risk for premature cardiovascular disease in cni treated renal transplant recipients, but this hypothesis needs further study. group(n) ktx ptx(kptx) ecd re-tx pra(> %) race(aa) low immunologic risk alem ( ) overall pt, ktx, and ptx survival are , , and % at months median follow-up. actuarial survival rates, initial length of stay, delayed graft function, steroid free rates, major infection, and incidence of ptld ( ratg pt) were similar for alem and ratg groups, but treated acute rejection (ar) occurred in ( %) alem pts compared to ( %) ratg pts (p= . ) and biopsy proven rejection (bpar) in ( %) alem pts compared to ( %) ratg pts (p= . ). only alem bpar has occurred after months. total daily mmf doses were similar for alem and ratg groups at months ( ± mg vs ± mg). neupogen use was greater in the alem group, ( %), than in the ratg group ( ( %), p= . ). excluding pak, chronic allograft nephropathy (can) was observed in ( %) alem pts and ( %) ratg pts. (p= . ). conclusions: alem and ratg induction both provide excellent and yr pt, ktx, and ptx survival. alem is associated with lower acute rejection rates and perhaps less can, but requires increased neupogen administration to help maintain mmf dosing. thymoglobulin dosing intensity and density: effects on induction efficacy ruth-ann m. lee, adele h. rike, background: alemtuzumab (campath h) has been used as induction therapy for kidney transplant recipients with acute rejection rates reported by us and others of to % at one year. the histologic type of rejection and the time frame for occurrence after treatment with alemtuzumab have not been well established. this study is a retrospective single center review of acute rejection episodes of kidney transplant recipients treated with alemtuzumab induction with respect to the kinetics and histologic patterns of acute allograft rejection. methods: from / / to / / , kidney transplants were done meeting the inclusion criteria for this review. all patients had negative t and b cell flow cytometric crossmatches and received induction therapy with alemtuzumab mg iv intra-operatively, methylprednisolone - mg during the first hours, and were then maintained on tacrolimus (target level - ) and mycophenolate mofetil without steroids. all episodes of biopsy-proven acute rejection (ar) were reviewed. patients in pre-transplant desensitization protocols or with documented non-adherence with medications were excluded from the analysis. results: a total of of patients ( . %) experienced ar during the study period, with a mean follow up of months (range - months). of the ar episodes, ( %) occurred within the first months post-transplant, ( %) occurred between - months, ( %) occurred between - months, ( %) occurred between - months, and ( %) occurred more than one year post-transplant. of the rejection episodes within the first months post-transplant, / occurred within the first days. histologic analysis showed that / rejection episodes ( %) within the first months included an antibody mediated component ( / within the first days.) in contrast, / rejection episodes ( %) which occurred greater than months post-transplant were antibody mediated. of the patients with antibody mediated rejection, only patients had panel reactive antibody (pra) levels > % at the time of transplant. conclusion: this large experience with alemtuzumab induction therapy with a steroidfree maintenance protocol, demonstrates that the majority of rejection episodes occur within the first months post-transplant, with the largest fraction in the first months. a significant number of early rejection episodes are antibody mediated and occur in unsensitized recipients. in a randomized, international, multicenter study, comparing the use of thymoglobulin (tmg) and basiliximab (bas) in recipients at high risk for delayed graft function (dgf) or rejection, tmg was associated with less acute rejection ( . % vs . %, p= . ) and a lower triple endpoint (rejection, death or graft loss, . % tmg vs . % bas, p= . ) but not a significantly lower quadruple endpoint including dgf. the purpose of this study was to compare the efficacy of tmg and bas for induction stratified by donor source: standard criteria donor (scd), extended criteria donor (ecd) or donor with hypertension (htn). methods: retrospective review of data collected in the original randomized trial. data-capture limitations necessitated defining ecd as donor age > or donor age between and with both a donor history of htn and donor renal insufficiency (history of atn or creatinine above . mg/dl during the hours prior to organ recovery/start of cold ischemia time). results: recipients received ecd kidneys [tmg n= ( . %), bas n= ( . %), p=ns]. outcomes are presented below. there were no differences in the rates of dgf between the groups examined. conclusion: standard and non-htn donor recipients had a tremendous benefit of tmg compared to bas with less acute rejection and death. contrary to the perceived niche of tmg in ecd recipients, tmg has its most beneficial effect in scd recipients and recipients of donors without htn at risk for acute rejection or dgf. evaluation we have changed our immnosuppressive protocol in abo-incompatible kidney transplantations and attempted to determine whether the changes in agents have resulted in better outcomes. we used tacrolimus(fk), mycophenolate mofetil(mmf) and methylprednisolone(mp) in immunologically high risk patients between and . moreover, we performed splenectomy at the time of the transplant surgery in patients (group ) with aboincompatibilities between ansd , and administered rituximab as an alternative to splenectomy in patients(group ) with abo-incompatibilities between and . in this study, we compared the graft survival rates as well as the incidence of acute rejection in these two treatment eras. the graft survival rate at one year was % in group and % in group (p=ns). the graft was lost in one case of the cases of group due to insufficient doses of the immunosuppressive drugs. the incidence rate of acute rejection was % ( / ) in group , and % ( / ) in group (p< . ). there were no significant differences in serum creatinine level one year after transplanatation between two groups( . ± . in group vs. . ± . mg/dl in group ). no serious adverse events associated with rituximab or splenectomty were encountered in either groups. in abo-incomaptible kidney transplantation, rituximab under fk/mmf combination as an alternative to splenectomy seems to yield an excellent result in terms of the incidence rate of acute rejection. introduction: the choice of immunosuppression in the elderly kidney transplant recipient remains unclear. the objective of this study was to compare outcomes with different t cell-depleting induction agents in the elderly. method: all solitary kidney transplant recipients over the age of years that received induction therapy with either alemtuzumab or thymoglobulin from to were included in this unos analysis. overall graft survival, the risk of graft loss, and the risk of rejection were compared using kaplan meier, cox proportional hazards, and logistic regression, respectively. results: patients receiving alemtuzumab had a significantly lower -year graft survival ( . %) than patients receiving thymoglobulin ( . %), p= . ) (figure) after adjusting for other risk factors, alemtuzumab had a higher risk of graft loss compared to patients given purpose. the aim of this prospective randomized study was the comparison of efficacy and incidence of adverse events in two induction therapy regimens (atg versus basiliximab) in patients receiving a dual immunosuppression. methods. recipients of first or second deceased donor kidney transplants were prospectively randomized to receive either atg (fresenius) or basiliximab (novartis) as induction therapy. dual immnosuppression consisted of tacrolimus (astellas) and methylprednisolone. cmv prophylaxis was not applied on a regular basis. statistical analysis was performed with fisher's exact or chi-squared test, anova or mann-whitney u test, kaplan meier curves and log-rank test. results. patient characteristics of populations treated with atg versus basiliximab were similar concerning average age ( years), gender and dialysis time prior to transplantation ( vs. months). average donor age and cold ischemia were also comparable ( vs. years and vs. minutes). the actuarial -year patient survival for the atg subpopulation is , % in comparison to % in the basiliximab group (n.s.). analyzing graft survival after years, rates of , % in atg patients compared to % in the basiliximab group can be observed (n.s.). the incidence of acute rejection episodes was similar in both groups (atg: n= vs. basiliximab: n= ). ( %) patients in the atg group and ( , %) in the basiliximab group showed a delayed graft function. serum creatinine was not significantly different at and years (atg: , ± , mg/dl and , ± , mg/dl vs. basiliximab: , ± , mg/dl and , ± , mg/dl). patients in the atg group had a higher rate of cmv infections (n= vs. n= ; p= , ), whereas patients treated with basiliximab had significantly more hematological complications like anaemia, leukopenia and thrombocytopenia. conclusion. comparing induction therapy with atg and basiliximab, our data shows similar patient and graft survival rates with slightly better results in the atg group. patients treated with atg had a higher rate of cmv infections but less hematological complications. predicting cardiovascular events (cvd) and the varied effects of immunosuppressive medication on cvd risk factors requires an understanding of how traditional and nontraditional risk factors impact cvd after kidney transplantation (ktx). single-center studies have generally lacked statistical power and generalizability. registry studies have lacked sufficient data on cvd risk factors. the port project is creating a multicenter international database of ktx recipients with the primary objective of developing risk prediction models for post-transplant cvd. as a preliminary data assessment, an analysis was done on , ktx from - from transplant centers representing european centers, north american centers, and centers from asia/oceania. all data were extracted from preexisting databases at each individual transplant center and processed into the consolidated port database. , major adverse cardiac events (mace) were identified, defined as non-fatal or fatal myocardial infarction, cardiac abstracts arrest, and sudden death. the mace-free survival curves by participating center are shown in the figure. in this preliminary analysis, the overall one-year cumulative incidence of mace was . %, ranging from . % to . % across centers; and the five-year cumulative incidence was . %, ranging from . % to . %. the prevalence of diabetes pre-transplant varied from % to % across centers. in cox proportional hazards models adjusted for age, gender, race, donor type, transplant center, and reported history of diabetes, hypertension (htn), and ami, patients with a reported history of ami had a % increased risk ( %- %, p< . ) for mace. for the final analysis, the definition of mace will be expanded to include major revascularization events. the final port database will be used to develop and validate an equation to predict mace and other health outcomes of interest, after accounting for differential cvd risk factors internationally. abstracts to center. the most commonly used bp medications were beta-blockers, followed by ccbs, and both were used with the same frequency at and months. in contrast, the percentage of patients on an acei or arb more than doubled between and months, suggesting reluctance to use these agents early after tx (a practice not necessarily evidence-based); however, more than % of subjects did not receive acei/arb therapy even at months. fewer than half of all patients received aspirin, including only % with dm and/or cvd. similarly, only half with dm and/or cvd received a statin at and months. these data indicate current management of ktx recipients fails to utilize optimal cvd risk reduction measures in a timely fashion, perhaps missing an opportunity to reduce long-term morbidity and mortality from cvd in this at-risk population. background: cardiovascular (cv) risk reduction has been a primary reason for pursuing early corticosteroid withdrawal (ecswd). to date, actual cardiovascular event data (cve) (rather than cv risk) has not been reported for ecswd. therefore, we analyzed and compared actual cv events (cve) and cv-related survival in ecswd (≤ days) and chronic corticosteroid (ccs) pts. methods: cve and heart failure (hf) data were prospectively collected. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/ transient ischemic attack. hf events were defined as pulmonary edema or hf diagnosis. conclusions: rtx recipients receiving ecswd experienced: ) fewer cve and ) a trend toward overall better pt survival. these differences in cve and pt survival do not present until at least yrs ptx. and therefore require long term followup to become evident. abstracts immunohistochemistry was performed for cd + and cd + cells. results were compared with those of the patients on maintenance is (gr-is n= ) and the liver tissue from normal subjects (gr-normal n= ). results: the follow-up time in gr-tol was longer than that in gr-is.(gr-tol and gr-is: m and m, p< . ) in gr-tol, typical features of neither acute nor chronic rejection were observed following banff criteria. the extent of graft fibrosis in gr-tol, however, was greater, than those in gr-is and gr-normal (gr-tol, gr-is and gr-normal ; . , . and (ishak's modified staging )(gr-tol vs. gr-is, gr-is vs.gr-normal p< . ). each number of cd + and cd + cells in graft infiltrates was increased in gr-tol, compared with that in gr-normal, but equivalent with that in gr-is (cd + gr-tol, gr-is and gr-normal ; . , . and . cells/field, gr-tol vs.gr-normal p< . , gr-tol vs.gr-is ns / cd + gr-tol, gr-is and gr-normal; . , . and . cells/field gr-tol vs.gr-normal p< . , gr-tol vs.gr-is ns). conclusions+discussion : in tolerant graft after pediatric living-donor ltx, neither acute nor chronic rejection was observed, but fibrosis developed. because of the similar extents of cd + and cd + cells infiltrates and different follow up time between tolerant and immunosuppressed patients, it remains questionable whether fibrosis in tolerant graft is antigen-dependent. serial protocol biopsy before and after starting weaning is will detect fibrosis early, and observing whether reintroduction of maintenance is reverses fibrosis in that case will answer this question. operational tolerance may not always guarantee intact graft morphology. development of "operational tolerance" after pediatric liver background : in the setting of our pediatric living-donor liver transplantation (ltx), % of all the patients (significantly higher proportion, compared with those of other transplant centers) achieved complete withdrawal of immunosuppression (is), which is reffered to as "operational tolerance". nonetheless, some patients encountered rejection while they were undergoing weaning from is. it is,therefore,essential to identify and characterize the differences that will enable patients in these two distinct populations to be distinguished reliably. methods: the study groups consisted of group tolerance(gr-tol) in which patients are successfully weaned off from is, and group rejection (gr-rej) in which patients experienced clinically evident rejection during or after weaning process. the correlation between the clinical outcome (success or failure of weaning is) and following parameters was assessed ; donor/recipient age, donor/recipient gender, abo compatibility, hla mismatch, graft size, early (< month) rejection episode and initial immunosuppression.results: there was no difference between gr-tol and gr-rej with respect to donor/recipient age (gr-tol and gr-rej; y and y ns/ m and m ns), or donor/recipient gender (gr-tol and gr-rej (female); % and % ns/ % and % ns). abo compatibility did not differ between the two groups(gr-tol and gr-rej (i dentical:compatible:incompatible); %: %: %and %: %: % ns).the presence of hla-b mismatch was more frequent in gr-tol than that in gr-rej (gr-tol and gr-rej; % and % p< . ), while the presence of hla-a or dr mismatch did not affect success or failure of weaning is(hla-a gr-tol and gr-rej; % and % ns, hla-dr ; % and % ns). graft size did not differ between the two groups(gbwr gr-tol and gr-rej; . % and . % ns). the patients in gr-rej experienced early rejection more frequently than those in gr-tol(gr-tol and gr-rej; % and % p< . ). mean trough level of tacrolimus within days after ltx was compatible between the two groups (gr-tol and gr-rej; ng/ml and ng/ml ns). conclusions: development of operational tolerance after pediatric ltx was associated with the absence of early rejection and the presence of hla-b mismatch between donors and recipients. auxiliary partial orthotopic liver transplantation (apolt) in children with fulminant hepatic failure. patients with fulminant liver failure (fhf) who undergo auxiliary partial orthotopic liver transplantation (apolt) have a chance to come off immunosuprresion (isp) when the native liver regenerates. it may be most beneficial for children with fhf; however, the literature regarding its use in children has been limited. from the beginning of the pediatric liver transplant program at our institution, patients underwent liver transplantation for fhf. of those, received apolt and the remaining standard liver transplantation (olt). seven children (age months to years) who received apot (apolt group) were compared to matched control group of patients (olt group). since apolt was offered routinely at out instituting since , of apolt cases were done since . in apolt group, either left lateral segment or left lobe graft was used. recipients left lobe was removed in all cases. in olt group, received whole liver graft and received partial liver graft. all native livers showed submassive to massive necrosis at the time of transplant in pathology. all children ( %) in apolt group are currently alive with a median follow up of days (range - days) where ( %) patients are alive in olt group (median follow up days). six of children in apolt group ( %) showed native liver regeneration. first four apolt recipients ( %) are currently off isp with fully regenerated native liver. two of those patients developed complete atrophy of the graft liver, one underwent graft removal due to sepsis caused by severe rejection. one remaining patient who is off isp is displaying progressive atrophy of the transplant liver. incidence of acute rejection was % ( / ) in apolt group vs % ( / ) in olt group. other postoperative complications included hepatic artery thrombosis (hat) (n= ), bile leak (n= ), bilary structure (n= ) and bowel obstruction (n= ) in apolt group, hat (n= ), bile leak (n= ), bowel perforation (n= ), chylothorax (n= ) and aplastic anemia (n= ). median posttransplant length of stay was days in apolt and days in olt group. conclusions: apolt was safely performed in children with fhf. significant proportion of recipients displayed native liver regeneration and came off immunosuppression. natural killer cell dysfunction in pediatric acute liver failure. nada yazigi, greg tiao, alexandra filipovich, john bucuvalas. in pediatric patients, indeterminate acute liver failure (alf) accounts for ∼ % of all cases, and carries a particularly poor prognosis without transplantation. evidence exists to suggest that acute liver failure may reflect a disproportionate immune response to a common stimulus. nk cells comprise a central component of the innate immune system. we hypothesized that nk cell dysfunction (innate or secondary to an antigenic insult) plays a pathologic role in indeterminate alf. we reviewed peripheral nk cell function in a series of consecutive children cared for at cincinnati children's hospital, who met criteria for indeterminate alf as defined by the pediatric alf study group. peripheral blood testing was carried for nk cell number, cytolytic function and perforin and granzyme activity as part of our clinical alf protocol. seven of fifteen patients had nk cell dysfunction. only the severity of cholestasis was statistically higher in the nk cell dysfunction group. there was no statistical difference between the groups with respect to age, inr, or peripheral blood cell counts. of seven patients with nk cell dysfunction died in contrast to none of eight in the normal nk cell group. of the patients with nk cell dysfunction who eventually received a liver transplant, had severe early recurrence of chronic hepatitis in the graft at a year follow up. those outcomes are in sharp contrast to the group with no nk cell dysfunction where patients needed transplantation, but all had no complications both on short or long term follow up. we documented nk cell dysfunction at the time of alf diagnosis in / pediatric patients with indeterminate alf. this subgroup of patients was found to have higher: mortality, risk of infections, as well as recurrent disease in the graft. our findings suggest that nk cell dysfunction is involved in the pathogenesis of indeterminate alf. as such, it could therefore be a prime target for therapeutic intervention, with goals to rescue the patient from liver failure and /or to improve post-transplantation outcomes. background hrs is a reversible renal failure which occurs in pts with advanced liver disease and portal hypertension and is characterized by a marked decrease in gfr and rpf in absence of other identifiable causes. vasodilation theory is currently the most accepted hypothesis to explain the pathogenesis. in decompensated cirrhotics, probability of developing hrs is - %/yr and increases to % at yrs. ideal treatment is ltx. however, there is an urgent need for effective alternative tx to increase surv chances for pts until ltx can be performed. interventions that have shown some promise are vasoconstrictors in splanchnic circulation and tips. main objective was to compare efficacy of two different regimens (albumin/terlipressin resp hes/terlipressin both w/ wo midotrine) against tips whereas grf was considered as primary efficacy endpoint. pts/tx dx of hrs was based on criteria, as proposed by international ascites club. only pts with esld on the waiting list for ltx were eligible to be enrolled. pts were assigned to tx arms and randomized w/wo midotrine. volume/vasoconstrictor tx lasted for d, mitodrine was continued; follow up for d. results iia (albumin/terlipressin); iib (hes/terlipressin); iic (tips) discussion combination of volume expansion/vasoconstriction improved effectively gfr in pts with hrs. use of albumin shows no advantage compared to (cost effective) hes. although a marked improvement was observed during iv-treatment, renal fct deteriorated upon treatment withdrawal whereas pts with continued mitodrine showed superior long term outcome. we analyze our single institution experience to quantify the long-term incidence of renal failure based on month gfr compared to subsequent determinations. methods: this is an irb approved retrospective review of the prospectively maintained database of lt recipients. exclusion: patients on renal replacement therapy (rrt) at time of transplant, combined liver kidney, fulminant hepatic failure, and < -year follow-up. gfrs (i iothalamate glofil method) were measured at initial evaluation (ie), month (m ), year (y ), (y ), (y ), (y ), and (y ). patients were grouped by gfr > (g ), - (g ), and < (g ). ie and m gfr were used as starting points for longitudinal analyses. paired data analysis for ie, m , y and y was also performed. renal failure was defined as gfr < , received kidney transplant, on dialysis, or on kidney transplant list. results: liver transplant patients were reviewed between and . paired glofil data was available for the y and y analysis in patients. m gfr correlated more with long-term renal function (p< . ). g demonstrated largest reductions in gfr over time. g and g , when corrected for patients that got kidney transplantation and rrt, demonstrated progressive reduction in gfr. g , g , and g were statistically significant (p< . wilcoxon two-sample test). this study clearly demonstrates progressive decline in gfr continuing out to years after liver transplantation. m gfr correlates better with long-term renal function compared to ie gfr (not truly reflective of renal function at time of lt). if m gfr < , data showed a high rate of renal failure in our paired data analysis by y (p< . ). by correcting for patients with renal failure, the previously reported stability in gfr between y and y is not seen. analysis of grouping demonstrates that g patients at m have lower incidence of renal failure > years after lt. g and g patients will be at higher risk for renal failure each year after transplantation. introduction: calcineurin inhibitors have demonstrated efficacy in liver transplantation. however, they have a potential to impair renal function. delayed tacrolimus (tac) administration may reduce the risk of renal dysfunction. methods: a prospective study included liver transplant pts randomised to delayed introduction of tac (day ) + daclizumab (dac) (group a) or to immediate tac administration (group b). in both groups tac t was - ng/ml until week and - ng/ml thereafter. mmf was given at g/d for months, and corticosteroids (cs) at standard doses. pts with a serum creatinine (scr) > µmol/l at hours (h ) were excluded. the primary endpoint was the rate of pts with a mean scr > mmol/l at month . month results are presented. results: pts were randomised. baseline characteristics were similar. at month , mean tac t was . (group a) and . ng/ml (group b median follow-up post-tx was years ( - ). most frequent tx indications were alcoholic ( %) and hcv ( %) cirrhosis. amdrd glomerular filtration rate (gfr) was < ml/min/ , m in % (bt), % ( m), % ( y) and % ( y) of the patients. changes in gfr were then compared according to the immunosuppressive protocol: -group "cni+mmf" = a calcineurin inhibitor (cni) + mycophenolate mofetil (mmf). -group "cni" = a cni without mmf. in this group, some patients received only cni and some cni + azathioprine. there was no difference between those sub-groups, neither on rf nor on cni doses. all those patients were thus pooled. in both groups, gfr decreased from bt: - % in "cni+mmf" vs - % in "cni" at m (p= . ), - % vs - % at y (p= . ), and - % vs - % at y (p= . ). although their mean gfr bt was lower ( vs ml/min/ . m , p= . ), the decrease in rf in "cni+mmf" patients was less severe. nearly % of the patients had renal insufficiency in the years following liver tx. the reduction in the gfr is less pronounced in patients treated with mmf even if they were significantly more at risk bt. except at m, there was no difference in cni doses between the groups, suggesting that the sustained lower decrease in rf observed in "cni+mmf" may not be only explained by a cni dose reduction. acute rejection is a complex biologic process involving multiple cell types, cytokines and chemokines/chemokine receptors. we hypothesized that an mrna panel that included genes implicated in the anti-allograft response would distinguish allografts undergoing acute rejection from normal allografts with a high degree of accuracy. we tested this hypothesis by measuring levels of urinary cell mrna and peripheral blood cell mrna for cell surface proteins cd , cd , cd , cd , and ctla ; chemokines/ chemokine receptors ip , mig, cxcr ; cytotoxic attack molecules granzyme b(gb) and perforin, and immunoregulators foxp , tgf-beta and il- . gene specific primer pairs and probes were used in pre-amplification enhanced real time quantitative pcr assays to measure mrna and transcripts for s rrna. for each cell source, we used logistic regression to identify a linear function of up to log-transformed measures that would distinguish biopsies of ar patients from those of stable transplant patients. our study demonstrates that molecular signatures developed using urinary cell levels of just genes (signature , urinary cell levels of mrna for ctla , foxp , gb, cd , and mig), or a combination of urinary and blood cell levels (signature , urinary cell level of ctla mrna and peripheral blood cell levels of cd and ctla ) differentiate ar from stable biopsies with % sensitivity and % specificity. blood cell levels alone are also informative, but less so. we conclude that molecular signatures, developed from noninvasively ascertained mrna profiles of urinary cells/ peripheral blood cells, predict acute rejection with extraordinary accuracy. clinical trials to validate the predictive value of these signatures are worthy of pursuit. we have described the association of cd + b cell infiltrates in renal transplant (tx) biopsies (bxs) with acute cellular rejection (acr) and tx dysfunction (dysfx). we have also found metabolically active plasma cells (pcs) staining for s ribosomal protein (s rp) within these txs. herein, we report the significance of cd + pcs in rejection (rj) and evaluate the impact of cd , cd , and s rp on long term tx fx by calculated creatinine clearance (crcl). we studied tx bxs from pediatric (ped) patients (pts) who were bxed for suspicion of rj from nov to nov . pts were given daclizumab and maintained on prednisone, mycophenolate mofetil, tacrolimus or cyclosporine. immunohistochemical staining and quantification for cd , cd , s rp, and c d were performed under x light microscopy. bxs were classified by modified banff criteria. crcl was followed yr post-bx. cd + pcs were associated with c d-negative acr (p= . ) but not antibody mediated rj (amr, p= . ). roc analysis confirmed > cd + cells/hpf strongly associated with acr, yielding % sensitivity, % specificity, correctly classifying % and comprising total roc area . ( % ci . , ). higher cd counts at bx correlated with worse tx fx (fig ) . a univariate regression model showed that cd , cd , s rp and time were associated with a decline in tx fx at bx. multivariate model showed that cd , cd , and time had the main effects on crcl decline, with s rp dropping out. all patients regardless of rj status had a ml/min/ . m crcl decline exerted by time (p= . ). pts with cd had an additional sustained ml/min/ . m crcl decline seen yrs post-bx (p= . ). pts with cd also had ml/min/ . m crcl decline at bx (p= . ), but there was an interaction between time and cd that negated a sustained effect (p= . ). this study identifies a numerical threshold of > cd cells/hpf that is associated with acr and tx dysfx. infiltrating cd cells had the greatest, sustained effect on tx dysfx. we conclude that cells of the b lineage, particularly cd , play a key, but undefined, role in acr. intragraft there is now evidence that foxp + cells are not indicators of tolerance, since foxp is also increased during acute rejection. however, it is unknown whether foxp + cells are present during chronic antibody mediated rejection. moreover, the relative balance of regulatory, effector and cytotoxic pathways in chronic vs. acute injury has yet to be explored. here we addressed this issue. intragraft regulatory, effector and cytotoxic transcriptional profiles were analysed within renal transplant biopsies (n = ) classified (banff ) as displaying normal histology, chronic calcineurin inhibitor toxicity (cnitox), chronic antibody mediated rejection (camr) and acute cellular rejection (acr). granzyme b, tbet and foxp mrna were measured by quantitative pcr and foxp -positive cells were additionally quantified in graft biopsies by immunohistochemistry. distinguishing mrna profiles were analyzed in the peripheral blood (n = ). our data show that foxp mrna is increased not only in acr (p< . ) but also in camr (p< . ). expression of foxp mrna correlated tightly with the density of foxp protein-positive cells by immunohistochemistry (spearman r = . ; p < . ); foxp + cells were found in aggregates and within tubules. moreover, graft cytotoxic, effector and regulatory pathways were all found to be active in chronic as well as acute graft injury. significant increases in granzymze b, tbet and foxp mrna were observed in camr, cni-tox and acr compared to normal histology (p< . , p< . or p< . ). however, differences in the relative contribution of each pathway were evident, with significant accumulation of foxp mrna predominating in acr and granzyme b predominating in camr. thus, camr can be distinguished from both acr and cni-tox by an unfavorable intragraft granzyme b/foxp mrna ratio (p< . ). interestingly, this ratio was reversed in the blood, suggesting different migratory patterns for regulatory and cytotoxic cells between the blood and the graft.our data thus confirm that intragraft and peripheral blood foxp accumulation is also a feature of camr of kidney grafts. moreover, camr can be distinguished from other graft injury types based on its intragraft or blood cytoxicity/regulatory profile. survival of solid organ grafts depends on life long immunosuppression which results in increased rates of infection and malignancy. induction of tolerance to allograft would represent the optimal solution for controlling both chronic rejection and side effects of immunosuppression. we previously showed that operational tolerance after kidney transplantation could occur in some patient. here, the potential of high throughput microarray technology allowed us to study the peripheral blood gene expression profile associated to operational tolerance and chronic rejection in a cohort of human kidney graft recipients (n= ). microarrays were used to compare the gene expression profile of pbmc from patients with chronic rejection and drug-free operationally tolerant recipients. results have been treated using a classical statistical and a non-statistical analysis based on the identification of key leader genes associated respectively to chronic rejection and operational tolerance, either as those mostly changing their expression or having the strongest interconnections. differentially expressed genes were identified between operational tolerant patients and patients with chronic rejection. abstracts defined as missing > % of prescribed doses on mam and mems, and > sd among consecutive blood serum levels. results: participants were transplant patients (m = . + . years old, % male, . % caucasian). on the mam, . % of the patients acknowledged some non-adherence but minimized how many doses they missed. using mems technology, % had some non-adherence and specifically, . % of the participants missed doses and only % of their doses were taken within the allowable time frame. using > sd criteria for blood serum levels, % of the participants were considered non-adherent. non-adherence worsened with years since transplant. more missed (r = . , p = . ) and late doses (r = . , p = . ) on the mam and > sd among blood serum levels (r = . , p = . ) was associated with higher incidence of acute rejections. adherence data was examined for patients with documented acute rejections (n = ). sensitivity and specificity of each detection method was also examined. the mam and sd detection methods each identified non-adherence in % of the patients with acute rejections; mems did not identify any additional non-adherent patients. only % of the patients with acute rejections were identified consistently by all three adherence detection methods; all patients with acute rejections were identified by at least one method. discussion: non-adherence worsening with time since transplant and was associated with acute rejections. since no single method of detecting adherence identified all the patients with acute rejections, multi-method adherence assessments should be used to accurately capture patients who are non-adherent. non na is a leading cause of allograft loss and results from multiple factors. locus of control (loc) and beliefs regarding health have been associated with adherence in other populations. randomly chosen ktr's were interviewed using a confidential questionnaire administered by an outside investigator that included questions regarding loc, health beliefs and self-efficacy. the population was % female, % black, % hispanic, % deceased donor kidney, % diabetic, % greater than high school education, % employed, % married or cohabiting, % income < k per year, % insured by medicaid. mean age . ± . yrs, time on dialysis . ± mos, months since transplant . ± . , total meds . ± . . by pearson correlation, non-adherence (na), defined as "having missed doses of immunosuppression over the preceding months", was not correlated with race, gender, income, type of insurance, age, marital status, type of txp, mos on dialysis, time since transplant, or number of medications. na was correlated with higher education level (r= . , p= . ), current employment (r= . , p= . ), and knowledge of most recent creatinine value (r= . , p= . ). na was associated with concerns regarding prednisone (long term effects, dependency) r= . , p= . , feelings of greater personal control over illness (r= . , p= . ), and inversely correlated with powerful others loc (feeling that one's health is dependent on other people), r=- . , p= . and belief in the necessity of medication for maintenance of transplant health, r=- . , p= . . we conclude, in our population of inner-city patients: . na is not associated with standard demographic factors including income, race and gender. . contrary to findings in other populations, na is associated with higher education and current employment. . na is associated with knowledge about creatinine value, concern regarding long-term effects of prednisone and disbelief in the importance of transplant medications. . na is associated with feelings of personal control and feeling that powerful others (e.g. health care providers) are not of high importance in the outcome of illness. . education programs designed to address na in this population should be targeted towards altering negative beliefs regarding medications and stress the importance of partnering with the transplant team for optimal long-term outcome. purpose: the present study aimed to prospectively examine the relationships among nonadherence, health-related quality of life (hrqol), and family factors in adolescent kidney, liver, and heart transplant recipients. method: adolescent transplant recipients aged to years (m = . , sd = . ; % female; % kidney, % liver, % heart) and their parents participated. at baseline and -month follow-up assessments, adolescents and their parents independently completed phone interviews assessing self-/proxy-reported medication adherence, hrqol, and family cohesion and conflict. medical record reviews were conducted to obtain current medications, immunosuppressant drug assays, and clinical outcomes in the past year (i.e., rejection episodes, hospitalizations, graft loss). results: at baseline, adolescents classified as nonadherent based on self-report and tacrolimus standard deviation (sd) reported significantly lower general health perceptions (f( , ) = . , p < . ), self-esteem (f = . , p < . ), mental health (f = . , p < . ), and behavior hrqol (f = . , p < . ) compared to adolescents classified as adherent. similarly, parents of adolescents classified as nonadherent reported significantly lower physical functioning (f = . , p < . ), self-esteem (f = . , p < . ), and behavior hrqol (f = . , p < . ) for their adolescents. family conflict was correlated with adolescent report of behavior (r = -. , p < . ), physical functioning (r = -. , p < . ), self-esteem (r = -. , p < . ), and mental health hrqol (r = -. , p < . ). family conflict was correlated with parent report of behavior (r = -. , p < . ) and physical functioning (r = . , p < . ). improvement and deterioration in hrqol from baseline to -month follow-up is currently being examined. it is expected that increased family conflict and decreased medication adherence will be associated with deteriorations in hrqol. the interrelationships between medication adherence, family conflict, and hrqol domains such as self-esteem and mental health suggest that interventions targeting these domains may result in improvements in medication adherence behavior. the use of cam in general is associated with non-disclosure by patients to physicians. randomly chosen ktrs were interviewed using a confidential questionnaire administered by an outside investigator, including questions on cam usage, whether it was doctor-recommended, and whether the patient disclosed use. cam was defined as ingestion of herbal or other preparations, use of mind-body techniques or manipulation of the body for healing by someone not an allopathic medical provider. use of vitamins and spirituality were excluded. the population was % female, % black, % hispanic, % deceased donor kidney, % diabetic, % > high school education, % employed, % married or cohabiting, % income < k per year, % insured by medicaid. mean age . ± . yrs, time on dialysis . ± mos, months since transplant . ± . , total meds . ± . . % of patients (n= ) used cam. by pearson r, cam use was correlated with na to immunosuppressants, p= . , r= . , blood sugar-lowering medications, p= . , r= . , and cholesterol lowering medications, p= . , r= . , worries about long-term effects of medicines, p= . , r= . , belief that doctors place too much trust in medication, p= . , r= . , and that natural remedies are safer than medicines, p= . , r= . . cam use was inversely related to belief that health depends on allopathic medicines, p= . , r= - . , medicines protect from worsening disease, p= . , r= - . , and that following doctors orders is the best way to stay healthy, p= . , r= - . , and that having a kidney transplant makes them feel happy, p= . , r= - . . we conclude, in our population of inner-city patients: . use of cam is correlated with medication non-adherence. . patients who use cam are more worried about long term effects of medication, believe that natural remedies are safer than medications and that doctors place too much trust in medication. . patients who use cam do not believe that their health depends on allopathic medication, that medicines protect from worsening disease, or that following a doctor's orders is the best way to maintain optimal health. . patients who use cam are less happy with their kidney transplant. . disussing cam use and motivation for use is important in the transplant clinic and may alert the provider to possible risk for non-adherence. by multivariate cox analysis the risk of tg related to the presence of hla-iiab death censored graft loss occurred in . % of patients without tg and in . % of patients with tg (p< . ) hla-iiab are associated with higher risk of tg and reduced graft survival. furthermore, the risk of tg and its prognosis relate to the level of hla-iiab quantitated in a solid phase assay term survival of cardiac allografts in wild-type mice by alloantigen (alloag)-specific foxp + cd + cd + natural regulatory t (nt reg ) cells. guliang xia, jie he methods: fresh naive cd + cd + nt reg were isolated from congeneic b .pl mice via automacs and enriched for alloag specificity by in vitro culture with either anti-cd / cd -coated dynabeads (d - ), then donor bone marrow-derived dendritic cells % (d+ ) for dc/beads-expanded nt reg , while total fold of expansion of nt reg remained similar ( . ∼ . for beads/dc-or . ∼ . for dc/beads-expansion) regardless of the presence or absence of tgf-β. introducing ra ( nm) into bead/dc-based, tgf-β/ il- -conditioned culture resulted in marginal improvement with . % (d+ ) nt reg being foxp + . in mlr assays, nt reg expanded with tgf-β/il- exerted more potent suppression than cells conditioned with il- alone. in vivo, beads/dc-expanded, tgf-β/ il- -conditioned nt reg synergized with transient host t cell-depletion (anti-thy . mab i.p. µg at d- & µg on d+ ) in c bl/ mice to suppress balb/c heart allograft rejection with . % (n= ) and % (n= ) allografts surviving over days when x or x cells/mouse were injected immediately post-transplant, respectively. anti-thy . treatment alone led to only . % long-term survival. infused nt reg survived long-term ( . % circulating t cells ( x cell dose) or . % ( x cell dose) at d+ post-transplant) and expressed high level foxp ( ∼ %) in vivo. long-term surviving allografts showed characteristics of 'acquired immune privilege' with cellular infiltrates that were foxp + , tgf-β + , il- + and indoleamine , -dioxygenase (ido) + , although signs of mild to moderate chronic rejection were still evident conclusion: t-bet deficiency results in up-regulation of il- expression in addition to th associated cytokines resulting in acceleration of chronic rejection despite profound deficiency of ifn-γ. t-bet deficiency may contribute to the alloimmune responses independent of ifn-γ by in situ hybridization, tir mrna level was higher (p< . ) in cortical tubuli, glomeruli, perivascular and peritubular areas of kidney grafts at , and - d post-tx, than in naive kidneys. to assess how local expression of tir affects the outcome of kidney grafts, we transplanted tir -/-b x kidneys into dba/ mice. most ( %) recipients of tir -/-kidneys rejected their grafts with a median survival of . d (n= , p< . vs wt) and had more severe graft dysfunction (bun levels) at day , and - days post-tx, than recipients of a wt allograft (p< . ) opticept trial: efficacy and safety of monitored mmf in combination with cni in renal transplantation at months. r trough-based dose adjustments were made in the mmf cc arms. antibody induction and/or corticosteroids were administered according to center practice. primary endpoints were the proportion of patients with treatment failure (biopsy-proven acute rejection [bpar], graft loss, death), and mean percent change in calculated glomerular filtration rate (gfr; nankivell equation) at months. safety endpoints were incidences of adverse events (aes) and serious aes baseline characteristics did not differ among treatment groups with living donors accounting for approximately % of grafts. % received tacrolimus (tac) and % cyclosporine (cya): cni doses and levels were significantly lower in group a. mmf doses were greater in cya-treated subjects in all groups cya treated patients and in group a (p= . ); stability of renal function over time was greatest in group a. despite higher mmf doses in group a (p< . ) at most time points, significantly fewer mmf withdrawals occurred in group a vs. groups b and c. conclusions: a concentration-controlled mmf and reduced level cni regimen is not inferior to that of fixed-dose mmf and standard-dose cni as regards bpar and other end points. this regimen facilitated higher mmf dosing without an overall increase in adverse effects, and with a trend toward preservation of kidney function versus standard-dose cni regimens comparison at one year of interstitial fibrosis (if) by automatic quantification in renal transplant recipients with cyclosporine (csa) discontinuation and sirolimus (srl) introduction introduction: we previsouly reported the clinical results of a multicentric study showing that csa conversion to srl at week (w) is associated with a significant improvement in renal function. using routine renal biopsy (rb) performed at w during this study routine rb was performed at w . for each rb, a section was imaged using a colour video camera and analyzed by a program of colour segmentation which automatically extracts green colour areas characteristic of if. results were expressed as percentage of if and grade according to banff classification. results: male donor gender was associated with higher if ( ± % vs. ± %, p = . ). if was numericaly higher in patients who had experienced acute rejection ( ± %, n = vs. ± %, n= , p= . ) there was a positive correlation between renal function and the percentage of if on rb (p= . ). despite significant improvement of renal function at w in the srl group intent to treat (n= ) mean if (%) grade i (%) grade ii (%) grade iii (%) sirolimus (n= ) conclusion: despite significant improvement in renal function after csa to srl conversion at months, we found no difference of if on rb at w . the observed improvement of renal function may be due to a hemodynamic effect. a longer delay may be necessary to observe histological improvement. the higher if score than the one previously reported by others may be explained by the use of expanded criteria donors abstract# effects of cni or mmf withdrawal on carotid intima media thickness in renal transplant recipients methods: we included stable renal transplant patients on cni-based immunosuppression, including steroids ( mg/d) and mmf ( g/d), who were randomized to mmf-withdrawal (group a: csa-auc ng*h/ml) or cni-withdrawal (group b: auc-mpa µg*h/ml). patients were treated for traditional risk factors according to stringent predefined targets. ambulatory bloodpressure (abpm), lipids, estimated creatinine clearance (mdrd) and imt were measured at baseline and after months. results: groups were comparable with respect to demographic characteristics, immunological profile, renal function, systolic and diastolic bloodpressure and lipids. mean duration of follow-up was . ± . months. only patient ( . %) in group b and patients ( . %) in group c experienced acute rejection despite adequate exposure (p= . ). imt did not change final renal function outcomes from the spare-the-nephron (stn) trial: mycophenolate mofetil (mmf)/sirolimus (srl) maintenance therapy and cni withdrawal in renal transplant recipients purpose: to compare the effect on renal function of maintenance immunosuppression with mmf and srl to that of mmf and a cni in renal allograft recipients. methods: in a -year open-label, prospective, randomized, controlled, multicenter study, subjects maintained on mmf and a cni were randomized - days posttransplantation to either mmf ( - . g bid) plus srl ( - mg followed by ≥ mg/ day results: outcomes of the first subjects receiving mmf/srl and receiving mmf/cni (tac, n= ; csa, n= ) completing year of follow-up will be reported here. final outcomes of all subjects will be presented at the congress. mean time from transplant to randomization in both groups was days. groups were similar at baseline for all reported renal function endpoints after months of therapy, maintenance immunosuppression with mmf/ srl after cni withdrawal appears to preserve renal function when compared with a mmf/cni-containing regimen improved outcomes after de novo renal transplantation: -year results from the symphony study. h. ekberg, h. tedesco-silva frei, y. vanrenterghem, p. daloze, p. halloran at years, the rate of uncensored graft loss was lowest in patients receiving tacrolimus ( % vs - % in other groups; kaplan-meier estimates). gfr at the end of the core study was slightly better in the follow-up itt patients ( - ml/ min) than in the core study itt patients ( - ml/min), suggesting inclusion of betterperforming patients in the follow-up. renal function was generally stable over year . a slight improvement in gfr in the sirolimus group (+ . ml/min) was observed, whereas the tacrolimus group still had superior gfr ( vs - ml/min in other groups). conclusions: in follow-up patients, renal function was stable during the second year and gfr differences were less marked than at year a prospective randomized study of alemtuzumab vs rabbit anti-thymocyte globulin induction in kidney and pancreas transplantation gautreaux, s. iskandar, p. adams, r. stratta. surgery; medicine; pharmacy alemtuzumab (alem) and rabbit anti-thymocyte globulin (ratg) are the most commonly used t-cell depleting induction agents in kidney (k) and pancreas (p) transplantation (tx) expanded criteria donors (ecd) were included. results: between / / and / / pts enrolled and pts were transplanted. of pts, ( %) had ktx alone, ( %) kptx, and ( %) paktx. of ktx alone, ( %) were deceased donor, and ( %) were ecds. recipient age, race, re-tx abstract# purpose: to determine the impact of alginduction on long-term outcomes post-renal tx. methods: between / and / , consecutive adult pts received a deceased donor renal tx at a single institution results: the incidence of acute rejection was lower in gr. ( % vs. %, p< . ). the incidence of cmv infection was % in gr. and % in gr. (p=ns). the overall incidence of cancer was abstract# single-dose induction with rabbit anti-thymocyte globulin (ratg) safely improves renal allograft function and reduces chronic allograft nephropathy clifford miles, gerald groggel, lucile wrenshall. divisions of transplantation and nephrology we conducted a prospective, randomized trial in renal transplant recipients comparing two dosing protocols [single dose ( mg/kg) vs. divided doses ( . mg/kg for doses)] of rabbit anti-thymocyte globulin (ratg; thymoglobulin®). we present herein the results of the first patients throughout the first months post-transplantation, recipients of kidneys from non-marginal deceased donors derived the greatest benefit in renal function (egfr) from the single-dose regimen (p = . ). the incidence of chronic allograft nephropathy (can) was also lower in the single-dose group, in both clinically-indicated and protocol biopsies combined (p = . ) and in -month protocol biopsies alone high risk (race, pra) ( %) in multivariable regression, allograft failure strongly predicted increased risk of subsequent cve. among listed candidates, receipt of a transplant was associated with significant time adjusted for baseline factors, cve after transplant predicted increased risk of subsequent mortality: hr . (ci . - . ) after is microalbuminuria post-renal transplantation is related to inflammation and cardiovascular risk our objective was to define the relationship between microalbuminuria and these risk factors in stable rtr. methods: over one year, we identified stable rtr who were at least months post-transplant and provided successive urine albumin-to-creatinine ratio (acr) measurements, excluding those with recent illness and overt proteinuria. microalbuminuria was defined as averaged acr ≥ . in men and . in women (cda ). framingham-based traditional as well as novel cardiovascular risk factors associated with microalbuminuria were determined by univariate (p < . ), followed by stepwise backwards elimination (p > . ) multivariate logistic regression analysis microalbuminuria did not correlate with prior acute rejection, delayed graft function, or any specific antihypertensive or immunosuppressive agents. conclusions: post-transplant microalbuminuria is highly prevalent and is associated with elevated crp, elevated bp, and smoking. its relationship to these other factors suggests that it reflects an inflammatory state in otherwise stable patients and thus may indicate graft and patient health the first year after kidney transplantation (tx) is associated with increased mortality relative to dialysis. early post-tx deaths are often cardiovascular (cv) and frequently occur after the first week post-tx. ctnt is a sensitive and specific maker of myocardial injury. in this study we investigated whether ctnt relates to early post-tx survival. methods: patients received kidney tx from / to / , % from living donors. ctnt was measured during the pre-tx workup and periodically while on the tx waiting list. patients ( %) had a dobutamine stress echo (dse) and ( %) had a coronary angiogram. the combined end point of the study was death or major cardiac events. survival was censored for graft loss. results: mean age was + , % males. pre-tx ctnt level was elevated (> . ng/ ml) in % of patients other dse derived parameters did not relate significantly to survival. ctnt further stratified the risk associated with other variables. thus, among patients with ef< %, year survival was %, % and % (p= . ) in patients with ctnt < . , . - . and > . , respectively. similarly, these ctnt ranges stratified risk in patients with low albumin conclusion: an elevated pre-tx ctnt is a strong and independent predictor of reduced early post-tx survival. ctnt allows stratification of risk in patients who have other risk factors such as low ef, low serum albumin and dialysis> years. in all patients, independent of any other variables, a normal ctnt was an excellent predictor ( %) of survival abstract# validation of framingham risk assessment by actual cardiovascular event data in renal transplant recipients alloway, michael cardi, gautham mogilishetty, shazad safdar excellent outcome after liver transplantation in children with cystic fibrosis some studies have reported benefits of liver transplantation (lt) in cf patients, but large outcome studies are not available. we report the outcomes of a large cohort of cf patients undergoing lt. methods: pre and post-lt patient characteristics, post-lt morbidity and mortality, and patient and graft survival were patients age < yr) received a st isolated lt. cf patients were listed for st lt, neither waitlist deaths nor the probability of death from time of listing was different from non-cf. ( . %) cf patients underwent lt with an average followup of yrs ( - yrs) average peld: . ( . % had a peld < ), median age: . yrs ( . - . ) graft survival in cf patients was . %, . %, and . % at , , and yrs compared to . %, . %, and . %. rejection rates were not different ( . % cf vs . % non-cf @ yrs with % of these patients requiring dialysis. standardized height and weight scores showed no improvement over years followup in the cf patients (height z - . at tx to - . at yrs., weight z - . to - . ), but tended to improve in the non-cf group in addition, death rates from time of listing are not increased compared to non-cf patients. these data support lt as a treatment for cf liver disease, but studies investigating the lack of growth improvement and increased renal complications in these patients may further improve outcomes. abstracts full cni group. conclusion: compared to full cni, low cni/mmf a) allows renal function to recover in patients with impaired renal function at the time of ltx and b) preserves long term renal function. cni sparing in combination with mmf may become cellular islet autoimmunity influences clinical outcome of islet cell transplantation methods: twenty-one t d patients received cultured islet cell grafts prepared from multiple donors and transplanted under anti-thymocyte globulin (atg) induction and tacrolimus plus mycophenolate mofetil (mmf) maintenance immunosuppression. immunity against auto-and alloantigens was measured before and during one year after transplantation. cellular auto-and alloreactivity was assessed by lymphocyte stimulation tests against autoantigens and cytotoxic t lymphocyte precursor assays, respectively. humoral reactivity was measured by auto-and alloantibodies. clinical outcome parameters remained blinded until their correlation with immunological parameters. results: all patients showed significant improvement of metabolic control and out of became insulin-independent. multivariate analyses showed that presence of cellular autoimmunity before and after transplantation was associated with delayed insulinindependence (p= . and p= . , respectively) and lower circulating c-peptide levels during the first year after transplantation (p= . and p= . , respectively). / patients without pre-existent t-cell autoreactivity became insulin-independent, versus / patients reactive to both islet autoantigens gad and ia- before transplantation. autoantibody levels and cellular alloreactivity were not associated with outcome. conclusions: cellular islet-specific autoimmunity affects clinical outcome of islet cell transplantation under atg-tacrolimus-mmf immunosuppression bmp- is downregulated & tgfβ to bmp- ratio favors emt during acute rejection of human renal allografts allospecific cd + t-cells predict rejection risk and measure immunosuppressive effect after abdominal organ transplantation in recipients methods: allospecific cd +t-cells were measured in < hours with polychromatic flow cytometry to identify rejectors (who had experienced acute cellular rejection within days post-transplantation) in single mixed leukocyte responses (mlr) from cross-sectional recipients- children with liver or intestine allografts, and adults with renal allografts. where possible, results were correlated with proliferative alloresponses measured by cfse-dye dilution (n= ), allograft biopsies (n= ), and expression of ctla , a negative t-cell costimulator, which antagonizes cd -mediated effects (n= ). results: in the first children, logistic regression identified donor-specific, memory cd + t-cytotoxic cells (tc) as enhanced among rejectors, compared with non-rejectors ( ± vs ± per , cells, p= . ), relatively drug-resistant (r with drug levels =- . , p=ns), with greatest sensitivity/specificity (> %) for rejectors noninvasively developed molecular signatures accurately predict acute rejection of human renal allografts greater emotional well-being (sf- ) and felt that their transplant interfered significantly less with various aspects of their life (iirs). conclusions: findings highlight the potential utility of assessing attachment style in transplant populations cni sparing in de novo renal transplantation: -year results from the symphony study one background: single center non-randomized results with steroid avoidance have shown patient and graft benefits. methods: unsensitized, primary kidney recipients, - yrs of age, were enrolled from us transplant programs ( )( )( ), in a prospective : randomized multicenter study of steroid-free (sf) vs. steroid-based (sb) immunosuppression with matched demographics. . % of sf and . % of sb were african americans and . % of sf vs. . % of sb had esrd from fsgs. sf patients received extended ( mo) vs. standard ( mo) daclizumab induction in the sb group. patients in both arms received tacrolimus and mmf maintenance. protocol biopsies were performed at , , and mo, and for renal dysfunction. primary end-points were differences for standardized height scores and biopsy proven acute rejection (bpar) at year. results at year: sf and sb patients were enrolled; sf and sb were - yrs of age. patient survival was % in both arms. graft survival was similar ( . % in sf vs. . % in sb). intent to treat median delta height sds scores from baseline for different age groups were: . for sf and . for sb in the - yr old (p= . ); . for sf and . for sb in the - yr old (p= . protection of liver ischemia reperfusion injury by silencing of tnf-α and complement genes. roberto hernandez-alejandro, xusheng zhang, dong chen, xiufen zheng, hongtao sun, weihua liu, marianne beduhn, aminah shunnar, motohiko suzuki, norihiko kubo, bertha garcia, anthony jevnikar, , living kidney donation is rapidly increasing worldwide to offer a partial (?) solution for the numerous esrd wait-listed pts. in spite of properly followed guideline criteria conclusion: dcd donors are a viable source of liver allografts for transplantation. patients who receive dcd livers have outcomes comparable to subjects who receive grafts from brain dead donors. use of dcd livers from donors over years of age is accompanied by a higher incidence of retransplantation and biliary complications. background: hypothermic machine perfusion (hmp) is in its infancy in liver transplantation (ltx). potential benefits include diminished reperfusion injury and improved early function. methods: the study was designed as a phase trial of liver hmp. exclusion criteria included: multiple organ recipients, meld> , icu patients, and patients > years of age. donor livers > years, biopsy with > % macrosteatosis and dcd were also ineligible for hmp. seventeen patients were enrolled transplanted with livers that underwent hmp for - hours using dual centrifugal perfusion with vasosol solution at - °c. patient, operative and early outcome variables were recorded. we compared outcomes to matched cold stored (cs) controls from the same era. results: all hmp grafts functioned immediately by usual clinical criteria with intraoperative bile production. results are summarized in table . synergy between il- and tnfα promotes t cell alloreactivty and impairs the graft-prolonging effects of costimulatory blockade. hua shen, bethany m. tesar, wendy e. walker, daniel r. goldstein. internal medicine, yale university, new haven, ct. a novel role of th cells in allograft rejection and vasculopathy. francesca d'addio, jesus paez-cortez, m. javeed ansari, laurie glimcher, john iacomini, mohamed sayegh, xueli yuan. transplantation research center, renal division, brigham and women's hospital, boston, ma; harvard school of public health, boston, ma. introduction: transcription factor t-bet plays a crucial role in th /th development. here, we investigated the role of t-bet in th differentiation and function of th cytokines in allograft rejection using an mhc class ii mismatched model of cardiac allograft vasculopathy. methods/results: cardiac allografts from bm mice were transplanted into wild-type as well as t-bet and ifn-γ deficient c bl/ recipients. t-bet-/-mice showed significantly accelerated allograft rejection (mst= . ± . days). however, as previously reported, all ifn-γ-/-and majority of the c bl/ mice accepted grafts for greater than days. upon in vitro stimulation of recipient splenocytes by irradiated donor cells, t-bet-/-and inf-γ-/-lymphocytes produced significantly less inf-γ and more th cytokines. interestingly, production of the proinflammatory cytokines il- and il- was significantly higher in t-bet-/-( ± . and ± . pg/ml) than c bl/ ( . ± . , . ± . pg/ml, p= . and . compared to t-bet-/-) and inf-γ-/-( . ± . , . pg/ml, p= . and . compared to t-bet-/-) mice. in vivo administration of il- neutralizing antibody (mab ) significantly prolonged survival of bm hearts (mst> days, p< . compared to the . ± . days of the control igg group) in t-bet-/-mice. immunofluorescence staining of bm hearts harvested from t-bet-/-recipients indicated that both cd and cd infiltrating lymphocytes produced il- . however, t-bet-cd double knockout mice did not reject bm heart grafts, nor did the grafts exhibit chronic vasculopathy. in contrast, t-bet-cd double knockout mice rejected (mst: . ± . days). splenocytes from t-bet-cd knockouts produced significant lower il- ( . ± . ) and il- ( . ± . pg/ml) than observed in t-bet knockouts ( ± . and . ± . pg/ml) and t-bet-cd knockouts ( . ± . and . ± . pg/ml respectively) recipients when re-stimulated with donor cells, while there was no significant difference in inf-γ production. induction in the elderly transplant recipient: an analysis of the optn/ unos database. suphamai bunnapradist, steven takemoto, jagbir gill, tariq shah. medicine-nephrology, ucla, la, ca; medicine-nephrology, national institute of transplantation, la, ca.we examined the incidence and mortality implications of cerebrovascular events (cve) after kidney transplant. we also compared variations in risk on the transplant waitlist and after allograft failure. methods: we used registry data from the us renal data system to retrospectively investigate ischemic stroke (is), hemorrhagic stroke (hs) and transient ischemic attacks (tia) among , adults who received kidney transplants in - with medicare as primary payer. patients with prior indications of cve in the registry were excluded. we ascertained events from billing claims, and estimated incidence of first events by the product-limit method. at-risk time was censored at: loss of medicare, yr transplant anniversary, non-cve death or end of study ( / / ). cox regression was used to identify independent correlates of cve, and to examine cve events as time-dependent mortality predictors. we estimated cve incidence after graft failure among patients without cve diagnoses prior to graft loss (n= , ), and amongthe association between hyperuricemia at six months after kidney transplantation and the development of new cardiovascular disease, many studies have previously reported safe withdrawal of prednisone (pw) late after kidney transplantation (ktx). to determine the best immunosuppression regimen during the pw, we performed a prospective trial with stable ktx patients randomized to either csa or 's' based regimen. methods: all patients received antibody induction therapy at the time of rtx and maintained on csa, p and cellcept®. patients excluded if they had > acute rejection, > gm/d proteinuria or serum creatinine > . mg/dl. patients were enrolled and data presented for patients with > weeks follow-up (f/u) with mean f/u of . ± . weeks. no differences observed in baseline characteristics in both groups. all patients then randomized to either csa (n= ) or 's' (n= ) and cellcept® converted to equivalent dose of ms. csa dosed by c level ( -hour) with goal level of ng/ml. sirolimus target level was ng/ml. results: patients withdrew from study, patients on s returned to csa regimen because of side effects. patients in the csa group and patient in 's' group had ar ( of them due to drug non-compliance). death censored graft survival was %. mean csa drug level acheived was ± ng/ml and 's' drug level was . ± ng/ml. no significant differences noted in hematological values or bp measurements. csa ( purpose: the clinical significance of c d positiviity in patients with acute rejection is well defined but its significance in stable graft function is undetermined. this study was performed to evaluate the clinical outcome of protocol biopsy-proven c d positive renal transplants with stable graft function in the early posttransplantation period. methods: renal allograft biopsies were included. protocol biopsies (n= ) were performed from stable allografts on day posttransplantation, and indication biopsies (n= ) were performed from dysfunctioning allografts. incidence of c d positivity was compared between protocol and indication biopsies. clinical characteristics, biopsy findings, graft function, acute rejection episodes, and graft survival rates were compared between the c d-positive and c d-negative grafts in each group. results: c d deposition in protocol biopsies was detected in of biopsies ( . %), whereas . % ( of biopsies) in indication biopsies. the histological findings of c d-positive protocol biopsies were minimal inflammation of tubulointerstitium. on the other hand, those of c d-positive indication biopsies were various including acute humoral rejection, acute cellular rejection, acute tubular necrosis and calcineurin inhibitor toxicity. in the protocol biopsy group, graft function during year after biopsy, acute rejection rate, and cumulative graft survival did not differ between the c d-positive and c d-negative grafts. all c d-positive allografts maintained stable graft function without any antirejection therapy. in the indication biopsy group, graft function during year after biopsy and acute rejection rate did not differ between the c d-positive and c d-negative grafts. however the cumulative graft survival rate was worse in the c d-positive grafts than the c d-negative ones (p= . ). conclusion: c d positivity associated with allograft dysfunction indicates a poor graft outcome. however, c d-positive allografts with stable graft function in the early posttransplantation period take an indolent course. are methods: this was a retrospective single centre study reviewing all the adult patients who had a kidney transplant biopsy between april and october at guy's hospital. results: patients had diffuse (> %) c d staining out of who had kidney transplant biopsies in this time and had been followed up within the centre. of these patients, also had dsa prior or at the time of biopsy. the fall in egfr in this group a year post biopsy was greater than those with diffuse staining for c d but no dsa. the mean change in egfr from the day of biopsy at a year was - . ml/min/ . m (+/- . ) in those with dsa compared with + . ml/min/ . m (+/- . ) for those with c d but without dsa. the changes in egfr from the pre-biopsy baseline at one year showed a fall in egfr in both groups but this was greater in those with dsa (- . compared with - . ml/min/ . m ).of patients who never had diffuse or focal c d staining on biopsy, only had had dsa tested. of these only ( %) had a positive dsa result. from these eight, four had features of rejection and four did not. one person in each of these groups is dialysis dependant and one person in the rejection group has egfr < ml/min/ . m . although small numbers, this outcome appears to be worse than that of c d negative patients with no dsa but features of rejection who in fact showed an improvement in egfr from the day of biopsy by . m l/min/ . m or an improvement from their pre-biopsy baseline of . m l/min/ . m at one year. conclusion: dsa is of additional value in evaluating risk of graft failure. this appears to be of value in those with and without diffuse c d staining on biopsy. utility of post-transplantation flow cytometry crossmatching in predicting graft outcomes. michelle willicombe, graham shirling, ray fernando, henry stephens, paul sweny, peter j. dupont. department of renal medicine, royal free hospital, london, united kingdom; histocompatibility laboratories, anthony nolan trust, london, united kingdom. de novo development of donor-specific anti-hla antibodies after renal transplantation may be associated with increased rejection and decreased graft survival. flow-cytometry crossmatches (fcxm) have been suggested as method of screening for development of donor-specific hla antibodies post-transplantation, but interpretation of crossmatch results can be confounded by antibodies directed against antigens other than hla. we assessed the impact of developing a positive fcxm post-transplantation on clinical outcomes in a cohort of live donor renal allograft recipients. methods: patients were studied. / ( %) received tacrolimus-based and / ( %) ciclosporin-based immunosuppression. median follow-up was months. all patients had negative complement-dependent cytotoxic (cdc) t cell crossmatches pretransplantation. / ( %) in the group with a positive fcxm had an acute rejection episode in the first months compared with / ( %) in the group with a negative fcxm (p=ns). graft function at months was not different between the groups (positive fcxm -median creatinine mmol/l; negative fcxm -median creatinine mmol/l; p=ns). / grafts ( %) were lost within the first year in the positive fcxm group compared with / ( %) in the group with negative post-transplant fcxm (p=ns). the development of a positive fcxm post-transplantation alone is not predictive of adverse clinical outcomes. this may be explained by the poor correlation between a positive fcxm and the presence of antibody directed against mismatched donor hla antigens. surveillance for development of donor-specific anti-hla antibodies after transplantation may be best performed using high-resolution bead technologies rather than fcxm. use of the fcxm alone, without establishing antibody profiles, is of limited predictive value. based upon the amount of antibody (ab) measured by the titer of donor specific hla antibodies, dsa, or the fluorescence intensity (fi) of the donor specific single antigen bead. it is unclear whether abs indentified by sensitive single antigen bead and solid phase assays (flow pra and luminex) correlate with and are predictive of a clinically relevant end-point (a + fcxm). we evaluated the pra, dsa, bead specific ag fi and fcxm reactivity of pre-transplant (pre-tx) sera from recipients of a deceased donor renal allograft to determine whether amount of ab (measured by fi) predicts a (+) fcxm. patients with a (+) dsa, a (+) fcxm and pre-tx class i pra ≥ % (n = , mean pra of ± %) when compared to patients with pre-tx pra < % (n = , mean pra ± %) had comparable mean fis ( , ± , vs , ± , ) , fi ranges ( , - , vs , - , ) and median fis ( , vs , ). the class ii comparisons were of the same pattern. surprisingly, patients with a (+) dsa, a (-) fcxm and pre-tx class i pra ≥ % (n = , mean ± %) compared to patients with pre-tx pra < % (n = , mean pra ± %) had comparable mean fis ( , ± , vs , ± , ) , fi ranges ( , - , vs , - , ) we have recently showed that pre-treatment of the donor with epo causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. -aminoisoquinolinone ( -aiq) a potent water soluble parp inhibitor has proven to reduce renal ischemia-reperfusion (i/r) injury. the aim of our study was to determine the effects in the graft and in the receptor of the pre-treatment of the donor with epo and treatment of the recipient with -aiq, in a porcine model of dcd kidney transplantation. material/methods: landrace pigs were killed by lethal injection; their kidneys were subjected to min of warm ischemic time (wit) and then transplanted after h of cold storage in celsior. in the pre-treated group, donors received a single dose of epo ( iu/kg) min before cardiac arrest. in the treated group, recipients received a continuous dose of -aiq ( mg/kg/h) minutes before reperfusion and maintained during minutes. blood, urine and renal tissue samples were collected at the end of the experiment for biochemical, histological and immunohistochemistry (pars, inos and cox- ) evaluation. data analysis performed with graph pad prism statistical package; p< . considered statistically significant. results:transplantation of kidneys from dcd resulted in: a significant rise of the levels of creatinine, n-acetil-b-d-glucosaminidase, glutathione-s-transferase, ast, ldh, alt, fractional excretion of na+, interleucin and , malondialdehyde levels and myeloperoxidase activity (p< . ); a significant reduction in urine flow and creatinine clearance, disturbances in the histological and imunohistochemistry pattern. administration of epo before ischemia and -aiq before reperfusion reduced significantly the biochemical (p< . ), histological and imunohistochemical evidence of glomerular dysfunction and tubular injury. they also reduced systemic injury, inflammatory response and oxidative stress. conclusions:pre-treatment of the donor with epo and treatment of the recipient with -aiq causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. in the hmp group perfusate ast levels strongly correlated with recipient peak ast by linear regression (p< . ). conclusions: hmp of liver grafts provides safe and reliable preservation in our pilot series. perfusate ast may allow pretransplant prediction of reperfusion injury. a larger randomized trial will be necessary to demonstrate the magnitude of benefits of hmp over cs in ltx.purpose: liver discard rates have increased in a large, urban organ procurement organization from % to %. the reason is likely the result of increased transplant surgeon willingness to consider organs close to the margin of clinical acceptability. however, the costs associated with recovering these organs are high if the liver is discarded. we endeavored to determine whether a model based on pre-recovery data could predict liver discard introduction: we report our years experience with the use of campath- h (c h) in adult liver transplantation. from december until july we administered c h induction with low dose maintenance tacrolimus immunosuppression to adult recipients of a liver allograft. most common primary diseases were laennec (n= ), cryptogenic cirrhosis (n= ) and autoimmune: psc (n= ), pbc (n= ) and aih (n= ). the first dose of c h was administered immediately before (n= ) or after (n= ) the transplant procedure. follow up was until september, . results: five year patient and graft survival was % and % respectively. there were deaths due to stroke (n= ), chronic rejection (n= ), failure to thrive/pneumonia (n= ), sepsis (n= ), hepatic artery thrombosis, hcc, prostate cancer, graft lymphoma and non-compliance (one each). seven patients were retransplanted, for primary non function (n= ), portal vein thrombosis (n= ), hepatic artery thrombosis (n= ), hepatitis b (n= ) and chronic rejection (n= ). thirty six patients had biopsy proven rejection episodes: mild (n= ), moderate (n= ) or severe (n= ). the average tacrolimus hour trough levels were . , . ng/ml and . ng/ml for the rst, nd and th year post-transplantation, respectively. there was no significant difference in the outcome of the transplant so far, between patients that received c h before or after the transplant procedure. immunosuppression-related complications included a). opportunistic infections: most common were herpes zoster (n= ), cmv (n= ), and herpes simplex (n= ), b). neoplasms: skin cancer (n= ), kaposi sarcoma (n= ), lymphoma (n= ) and c). nephrotoxicity: five patients received a kidney graft for diabetic nephropathy (n= ), nephrotic syndrome (n= ) and calcineurin nephrotoxicity (n= ). conclusion: the use of c h induction with half the usual dose of tacrolimus is an effective regimen in adult liver transplantation. the timing of c h administration does not seem to affect the clinical outcome so far. a. david mayer, james m. neuberger. the liver unit, queen elizabeth hospital, birmingham, united kingdom. introduction: in the prospective respect study, primary liver transplant patients were randomised to of groups: a) standard-dose tacrolimus (target trough level > ng/ml) for the st month; b) g mycophenolate mofetil (mmf) iv until at least day , g po thereafter + reduced-dose tacrolimus (target trough level ≤ ng/ml); and c) mmf as in group b + reduced-dose tacrolimus introduced on day (target trough level ≤ ng/ml) + daclizumab on days and . steroids were given in all groups according to local centre protocol. results at year showed that g mmf + delayed and reduced tacrolimus + daclizumab is associated with significantly less impairment of renal function compared with standard treatment. here we present the results of the per protocol (pp) population. methods: the pp population, which was defined prior to the sub-group analysis, consisted of patients from the full analysis set who had no inclusion/exclusion criteria violation, had at least one creatinine clearance (crcl) value beyond months, were treated according to the protocol and did not receive any prohibited medication during the first days and for less than week at any time during the study. a composite endpoint comprising freedom from renal dysfunction (≥ % decrease from baseline in calculated crcl), acute rejection, graft loss or death was also investigated. results: the full analysis set included , and patients, whereas the pp population only included , and patients in groups a, b and c, respectively. the mean difference in calculated crcl from baseline to year was significantly smaller in group c compared with group a (- . ml/min vs - . ml/min, p = . ), but was not significantly different between groups a and b (- . ml/min). the incidences of death (n = , , ) and graft loss (n = , , ) were similar in all groups. the incidence of the composite endpoint at year was in both the full analysis set and the pp population significantly lower in group c compared with group a (pp population: % vs %, p < . ), but was not significantly different between groups a and b ( %). the pp analysis confirms the results from the full analysis set that g mmf + delayed and reduced tacrolimus + daclizumab is associated with less impairment of renal function compared with standard treatment with no negative effect on death and graft loss. aims: post transplant lymphoproliferative disorder (ptld) is a serious complication of solid organ transplantation that is closely associated with epstein barr virus (ebv) infection. ebv + ptld lymphomas express several latent viral genes including latent membrane protein (lmp ), a proven oncogene that is essential for human b cell transformation. lmp is able to activate erk, jnk, p , nfκb and pi k. the aim of this study is to determine whether lmp isolated from ptld tumors differs in signaling ability from lmp derived from the b. strain of ebv, originally isolated from a patient with infectious mononucleosis. methods: lmp variants isolated from a panel of ebv + ptld-associated b cell lines were cloned and sequenced. inducible chimeric constructs containing the lmp c-terminus and ngfr transmembrane domain were created for each tumor variant and expressed in the burkitts b lymphoma cell line bl . lmp signaling in bl clones was induced by crosslinking of ngfr. activation of p , erk, akt and jnk was assayed by western blotting (wb) with phospho-specific antibodies. nfκb activation was assayed by wb for iκb and cfos induction was analyzed by wb and the transam cfos binding assay. results: all three tumor variants of lmp , as well as the b. lmp isoform, were able to induce p activation within min of ngfr crosslinking while akt and jnk were activated within min. all variants showed similar ability to activate nfκb. however, tumor lmp variants induced prolonged erk activation (up to hrs) while the b. lmp variant induced a transient response. cfos is induced only during the sustained phase of erk activation. indeed, the tumor variants of lmp , but not b. lmp , were able to induce cfos protein. similarly, cfos binding to the ap consensus site was only observed in tumor lmp -induced nuclear lysates. two mutations in the c-terminus-aa (s vs g) and aa (t vs s) -are conserved in the tumor variants lmp compared to b. lmp . point mutation of either of these amino acids from the b. to tumor variant version allowed for sustained activation of erk and subsequent cfos induction and binding to the ap site. conclusion: tumor-derived lmp has enhanced ability to induce the cfos oncogene and this property can be localized to two amino acids in the c terminus. these findings suggest that these specific amino acid residues of lmp are important in determining whether ebv infection is benign or results in ptld. the absence of interferon regulatory factor- (irf- ) confers protection against the liver ischemia and reperfusion injury through an il- independent pathway. elizabeth r. benjamin, xiu-da shen, feng gao, yuan zhai, genhong cheng, ronald w. busuttil, jerzy w. kupiec-weglinski. surgery, dumont-ucla transplant center, los angeles, ca. toll-like receptor (tlr ) mediated liver reperfusion damage after warm ischemia requires signaling through the myd -independent, irf -dependent pathway with cxcl- (ip- ) playing a central role in the injury development. studies using cxcl- ko mice have shown that these mice are protected through an il- dependent mechanism. we chose to investigate irf , upstream of cxcl- , to further characterize its role in the injury progression, and to better understand the involvement of il- in this pathway. methods: we used irf ko mice and their wt counterparts in a model of partial hepatic warm ischemia with , , and h of tissue reperfusion (n= ko, wt at and h; n= ko, wt at h). wt bone marrow derived macrophages were generated and stimulated with lps to determine the kinetics of il- production. tissue was analyzed for histology and mrna levels were measured by qpcr. results: kinetic studies showed peak il- production at and h post-reperfusion (pr). on pathology, irf ko mouse livers were protected from ir injury both early pr, at and h, and at hrs pr when compared to wt. consistent with these data, il- mrna induction was decreased in irf ko, as compared with wt at , , and h. although il- induction was maintained in the cxcl- ko mice, the irf ko mice showed decreased levels of il- at h pr. by h, il- levels were normalized to wt. conclusion: irf ko mice are protected from liver ir injury with evidence of this protection as early as h pr. although cxcl- ko mice are protected from ir injury with maintained il- expression, the absence of the upstream molecule, irf , confers protection in an il- independent manner. these data suggest a novel mechanism of ir injury mediated by irf in the liver. background: bone marrow (bm) transplantation may induce donor-specific tolerance to prevent rejection of allogeneic solid organs while maintaining immunity against infections and tumors. currently allogeneic bm transplantation is limited by donor t cell mediated graft-versus-host disease (gvhd), as well as a variable requirement for recipient marrow ablation and high numbers of donor bm cells. furthermore, sustained macro-chimerism has not yet been easily or predictably achieved in partially ablated patients or large animals. while rejection of allografts is mediated primarily by recipient t cells, recent studies have demonstrated the capacity of nk cells to reject allogeneic bm and to prevent long-term mixed chimerism. thus, nk cells represent a barrier to long term bm engraftment even with t cell tolerance. we have previously identified a novel type of regulatory t (treg) cell with a "double negative" (dn) phenotype (tcrab + cd + cd -cd -). dn-treg cells can effectively suppress anti-donor t and b cell responses and prolong graft survival in allo-and xenotransplantation models. we therefore tested the capacity of dn-treg to alter nk cell function. methods: c bl/ bm cells were i.v. injected into sub-lethally-irradiated ( . gy) cb f (h- b/d) in a "parent to f " model, or into allo-disparate balb/c mice. bm cells were co-transplanted with various numbers of c bl/ dn-treg cells or cd + or cd + t cells as controls. recipient spleen cells were collected days after to detect donor progenitors in a colony-forming-unit (cfu) assay. mice then received cardiac (n= ) or skin transplants (n= ) to confirm tolerance. we found that donor-derived dn-treg cells suppress nk cell-mediated allogeneic bm graft rejection in both "parent-to-f " and fully mhcmismatched bm transplantation models. adoptive transfer of dn-treg cells with donor bm cells promoted the establishment of stable mixed chimerism and donor specific tolerance to bm donor cardiac and skin grafts (mst> days), without inducing gvhd in sub-lethally irradiated mice. perforin deficient dn-treg cells were unable to efficiently inhibit nk cell function, and donor bm did not engraft. these results demonstrate a potential approach to control innate immune responses and promote allogeneic bm engraftment and donor specific tolerance through the use of dn-treg cells.framingham risk score (frs) predicts cardiovascular (cv) risk in the general population, but may underestimate cv risk in kidney transplant (txp) patients (pts). frs has not previously been validated by prospective cardiovascular event (cve) data collection in kidney txp pts. the purpose of this study was to validate frs with actual observed cve data in kidney txp pts. methods: cve data was collected at routine intervals in our kidney txp pts and entered in a cardiovascular risk database. frs was calculated from baseline to yrs posttransplant (ptx) individual frs factors of age, sex, smoking, diabetes mellitus (dm), high-density lipoprotein (hdl), total cholesterol (tc), and blood pressure (bp) were evaluated for their ability to predict acutal cve occurring after kidney txp. pts with coronary artery disease (cad) were excluded from the frs analysis. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/transient ischemic attack. frs factors were evaluated by cox proportional hazards in univariate (uva) and multivariate (mva) models. rho kinase (rok) modulates calcium sensitivity of vascular smooth muscle cells and contributes to the regulation of peripheral vascular tone in man. in essential hypertension increased rok-activity contributes to the generation of vascular resistance. arterial hypertension is a common complication in renal transplant recipients. in this study we were interested in the role of rok for systemic hemodynamics in hypertensive renal transplant recipients (tx). we tested the specific inhibitor of rok fasudil. tx and matched control subjects (c) received either fasudil ( g/min) or placebo over a period of minutes intravenously. peripheral blood pressure and heart rate were recorded every min over a total of minutes. measurements for pulse wave analysis (sphygmocor vt) were performed every minutes during this period. statistics by anova for repeated measurements.compared to placebo fasudil significantly reduced peripheral mean arterial pressure p< . ; figure ) and increased heart rate (+ . . bpm, p< . ) in tx but not in c. likewise, central systolic pressure(p= . ; (figure ), augmented pressure and augmentation index were decreased in tx only.we conclude that acute inhibition of rok by fasudil consistently and effectively lowers blood pressure in tx with a calcineurin inhibitor-based immunosuppression. interestingly, rok-inhibition also reduces central blood pressure and arterial stiffness in these patients. improvement of both these parameters has been linked to a reduction in cardiovascular morbidity and mortality in large trials. hence rok inhibition might prove beneficial for the treatment of hypertension in renal transplant recipients. use death with function causes half of late ktx failure, and cardiovascular disease (cvd) is the most common cause of death. chronic kidney disease (ckd) is a cvd risk equivalent, justifying aggressive risk reduction with blood pressure (bp) control, statins, aspirin, and use of angiotensin converting inhibitors (acei) and angiotensin receptor blockers (arb). dekaf is an nih-sponsored prospective observational study examining causes of ktx failure at transplant centers in the us and canada, with current enrollment of over subjects. we examined the use of cardioprotective medications among patients transplanted after / / with at least mos follow-up, focusing on subgroups with preexisting diabetes (dm) and/or cvd. we conducted a retrospective cohort study to asses the prevalence and the predictors for the development of hyperuricemia at months after kidney transplantation and the association between hyperuricemia and clinical outcomes including patient and graft survival, new cardiovascular events and chronic allograft nephropathy (can). adult patients who underwent kidney transplantation at mount sinai medical center between . . - . . were included. patients who died or lost the allograft within months after transplantation were excluded from analysis. of the patients with a functioning allograft at months after transplantation, patients ( %) had normal uric levels and patients ( %) had hyperuricemia. after age, race, sex adjustment, receiving a cadaveric kidney, having an egfr< ml/min, and taking diuretics and cyclosporine were associated with a higher odds ratio of hyperuricemia. over a mean of . years of follow-up, patients had one, or more, of the pooled outcomes; had new cardiovascular events, developed biopsy-proven can, patients died, and had graft failure. kaplan-meier survival curves demonstrated that the pooled outcomes of events occurred more frequently in hyperuricemic patients (figure, p < . ). due to association between low egfr and hyperuricemia, we analyzed the clinical outcomes in patients with low and normal egfr. while . % of hyperuricemic patients with an egfr< ml/min had one of the pooled outcomes, it was . % in patients with normal uric acid levels (p= . ). among patients with an egfr ≥ ml/min, . % of normouricemic and % of hyperuricemic patients had one of the events. these results suggests an important association between hyperuricemia at months after transplantation and the new cardiovascular events, biopsy-proven can, and graft loss in kidney transplant recipients with decreased allograft function. background: long-term survival after liver transplantation (lt) is now the rule rather than the exception. hence, assessment of outcomes for children after lt must consider not only the quantity, but also the quality, of life years survived and restored. aim: to examine key hrqol themes after pediatric lt raised by both recipients and their parent proxies, with evaluation by time ( - yrs, - yrs, - yrs, and > yrs) since lt. methods: semi-structured : item generation interviews were conducted in person with children (c) and parents (p) at time of ambulatory lt follow-up at pediatric lt programs in canada and uk. all interviews were audio-taped, transcribed verbatim, and subjected to content analysis utilizing qsr nvivo . software for hrqol related theme generation. the participants interviewed were part of a larger research program aimed at developing a disease-specific instrument to assess hrqol for children after lt. results: data representing ( % male) pediatric lt recipients was obtained from a total of ( c, p) item generation interviews. median recipient age at lt was . (range, . to ) yrs, for primary indications including biliary atresia ( %), fulminant liver failure ( . %), metabolic liver disease ( , %), malignancy ( . %) and others ( . %). median patient age at time of interview was . (range, . to . ) yrs. themes emerging at all time points post-lt included infection risks, limitations on physical activities, side effects from immunosuppression meds, educational supports, and ongoing bloodwork. themes identified within the medium ( - yrs) follow-up included worries about rejection episodes, need for future re-transplantation, school absenteeism, and altered sibling and family dynamics. the impact of living with a surgical scar was a more frequent theme with recipients > yrs from lt. as time from lt increased to > yrs, themes suggest a focus on normalization and health promoting behaviours, along with expressed desires to be like healthy peers. conclusions: unique hrqol themes emerged from item generation interviews not captured by currently available generic hrqol tools. hrqol themes identified after pediatric lt suggest the importance of considering time trajectories from lt, and a focus on elements of 'everyday life' apart from lt. the shortage of cadaveric donors has led many transplant centers to expand their criteria for accepting life-saving organs. utilization of donation after cardiac death (dcd) donors has been estimated to increase the number of cadaveric donors. we report our experience with a recently established dcd program at a pediatric hospital and the outcome with the transplanted grafts. methods: in a protocol for dcd was established at a free standing pediatric hospital. from to all patients undergoing withdrawal of care were evaluated for dcd. patients meeting criteria for dcd underwent withdrawal by the critical care team and organ retrieval was initiated if asystole was reached in less than minutes. in addition, one dcd liver was imported and was included in the liver results. results: during the year study period patients ( % of total donors) underwent dcd resulting in organs ( kidneys and livers) transplanted. the cases had a mean donor age of yrs (range - ), wit of min ( - ), time sbp < of min ( - ), and time from asystole to aortic flush of min ( ) ( ) ( ) ( ) ( ) ( ) ( ) . four kidneys were transplanted locally with cit - hrs, no dgf, and one month creat . - . . the remaining kidneys were exported for transplant. four livers were transplanted locally with donor age mth- yrs, wit - min, recipient age mth- yrs, cit - hrs, ast peak - , ast day - , inr peak . - . , inr day . - . , total bili one month . - . , and graft survival . - . yrs. there were no vascular or biliary complications. conclusions: a protocol for dcd at a pediatric hospital increased the number of pediatric donors by %. liver and kidney grafts from pediatric dcd donors demonstrated excellent graft function and survival. liver retransplantation in children. a year single centre experience. christophe bourdeaux, andrea brunati, magda janssen, jean-bernard otte, etienne sokal, raymond reding. pediatric liver transplant program, université catholique de louvain, saint-luc university clinics, brussels, belgium. when graft failure occurs in liver recipients, secondary transplantation represents the only chance of long-term survival. in such instance however, several surgical and immunological aspects should be carefully considered, with respect to their impact on final outcome.in the present study, the epidemiology and outcome of graft loss following primary pediatric liver transplantation (lt) were analysed, with the hypothesis that early retransplantation (relt) might be associated with lower immunologial risks when compared to late relt. between march and december , liver grafts were transplanted to children at saint-luc university hospital, brussels. among them, a total of children ( %) underwent relt, and were categorized into two groups (early relt, n= ; late relt, n= ), according to the interval between both transplant procedures (< or > days).ten-year patient survival rate was % in recipients with a single lt, versus % in recipients requiring relt (p= . ). ten-year patient survival rates were % and % for early and late relt, respectively (p= . ), the corresponding graft survival rates being % and % (p= . ). along the successive eras, the rate of relt decreased from % to %, whereas progressive improvement of outcome post-relt was observed. no recurrence of chronic rejection (cr) was observed after relt for cr ( / ). two children developed a positive cross-match at relt ( / , %), both retransplanted lately for cr secondary to immunosuppression withdrawal following a post-transplant lymphoproliferative disease.in summary, the current need for relt has been decreasing over years, with a parallel improvement of its outcome. the results presented could not evidence better results for early relt when compared to late relt. the latter did not seem to be associated with higher immunological risk, except for children with immunosuppression withdrawal following the first graft. the background: a serum conjugated bilirubin greater than umol/l (cb ), in neonates who receive parenteral nutrition (pn), has been demonstrated to be a predictor of end-stage liver disease requiring transplantation. given the recent interest in the role of omega- lipids in the development of parenteral nutrition associated liver disease (pnald), we sought to examine in a multiple variable model the role of days of maximal lipid (> . g/kg/day), in the development of this outcome. method: between and , data were collected prospectively on all neonates undergoing an abdominal surgical procedure. univariate logistic regression models for the prediction of cb were developed with the following predictors: gestational age, percentile weight, percent predicted small bowel and colonic length, resection of the ileocecal valve, presence of a stoma, post-operative enteral tolerance, number of septic episodes, days of pn amino acid > . g/kg/day, days of pn lipid > . g/kg/day, and total days of pn. univariate predictors significant at the . level were entered into a backward stepwise multiple variable logistic regression. results: infants received pn post-operatively, and developed cb . predictors that met criteria for consideration in the multiple variable model were: age (p= . ), weight (p= . ), small bowel length (p= . ), presence of a stoma (p= . ), proportion of enteral feeds post-operatively (p= . ), days of pn amino acid > . g/ kg/day (p= . ), days of lipid > . g/kg/day (p= . ), and total days of pn (p= . ).the final multiple variable model which had a negative predictive value of . % and positive predictive value of . % is presented in the table below. our model suggests a key role of pn lipids and intercurrent septic events in the development of cb from pnald. these data may provide targets, such as careful line care, reduction in maximal lipid dose, or the use of alternate lipids such as omega- fatty acids, to prevent cb an identified marker for the need of subsequent liver transplantation in infants with pnald. terminal renal failure occurs in more than % of liver transplant recipients after years. we have previously shown that, beside renal toxicity of calcineurin inhibitors, renal lesions may be related to diabetes, arterial hypertension, accumulation of hydroxyethylstarch (elhoes), and the etiology of the liver disease. we made the hypothesis that these lesions may be already present at the time of liver transplantation (lt), a finding that could lead to adapt the perioperative management. this work investigated prospectively whether renal histopathological lesions were present before lt by performing systematically a renal biopsy by endovenous route in candidates to lt with end-stage liver disease. these patients were ± years old, males ; / had a diabetes, and an arterial hypertension ; the liver disease was related to alcohol in cases, hcv in cases, hbv in cases, and to a cholestatic disease in cases. at the time of the pre-lt workup, the biochemical parameters were : child score ± , meld score ± , prothrombin rate ± , creatinin serum level ± umol/l, proteinuria . ± . g/ h. severe side effects related to the procedure were limited to cases of macroscopic hematuria, lasting less than hours. in cases, the material obtained during the procedure did not allow the histological analysis. among the samples available, were considered as normal ; in cases, lesions related to mesangial iga glomerulonephritis ( cases), diabetic glomerulosclerosis ( cases), elhoes accumulation ( cases), thrombotic microangiopathy ( case) were found, often associated ; in cases, the lesions were severe and lead to combined kidney/liver transplantation in cases. in conclusion, significant renal lesions are detectable in more than % of the candidates to lt. interestingly, histological findings often combined lesions related to the liver disease and to an associated cause (diabetes, previous treatment by elhoes or interferon). results of histological analysis could help to decide either to perform a combined renal/liver transplantation, to adapt the immunosuppressive regimen, or to abandon the lt project. . because serum creatinine is one of the components of meld, liver candidates with renal insufficiency have been transplanted in increasing numbers, with some candidates receiving a kidney along with the liver transplant. we aimed to compare the liver graft outcomes for liver alone (lta) transplants with those from combined liver-kidney transplants (clkt). a propensity score analysis was used to reduce the impact of selection bias in the comparison of outcomes in the two groups. methods. demographics, clinical factors and outcomes on lta and clkt recipients from / / to / / (n= , ) obtained from the optn database were used for the analysis. univariate post-transplant survival rates were estimated using kaplan-meier survival, and multivariable post-transplant outcomes were analyzed using a cox regression model with and without stratification by categories of the propensity score. the propensity score (probability of receiving a clkt) for each recipient was estimated using a logistic regression model. several donor and recipient factors were included in both the cox and logistic regression models. in this cohort, liver graft outcomes for clkt were significantly better than those for lta based on the multivariable analysis. the results were similar, although slightly less significant, when the model was adjusted for propensity. the superior outcomes of clkt may be due to unobserved differences between these groups of recipients, reflecting data not currently captured by the optn. effect of liver the tgfβ to bmp- ratio was higher in the ar group (median ratio: ) compared to recipients with stable graft function & normal biopsy (median ratio: , p= . ).our observations that bmp- is specifically down-regulated during an episode of ar and that the balance between tgfβ & bmp- is in favor of emt advance a mechanism for the deleterious impact of ar on the long-term outcome of human renal allografts. the non-statistical bioinformatic approach identified leader genes which define the highest interaction genes derived from the sam-gene list. an interaction map between the genes identified has been calculated. this network is formed around majors clusters: a network of interleukins and a network of signal transduction which allow us the identification of key genes such as bank , a negative modulator of cd mediated akt activation, thereby preventing hyperactive b cell response in blood from patients with operational tolerance and il r, a specific marker absent on potentially regulatory cd + cd +high t cells in blood from patients with chronic rejection. we have identified by a non-statistical analysis of the peripheral blood gene expression in human kidney recipients a cluster of genes which are strongly interconnected and which could be a starting point for further analysis of the molecular mechanisms of kidney graft operational tolerance and chronic rejection. fecal algorithm based on multiparameter mixed lymphocyte reaction assay for tailoring maintenance immunosuppressants after living donor liver transplantation. yuka tanaka, hideki ohdan, toshimasa asahara. department of surgery, hiroshima university, hiroshima, japan.background: no reliable immunological parameters exist for identifying liver allograft recipients in whom immunosuppressants can be safely withdrawn. for minimizing maintenance immunosuppressants, we established an algorithm determining anti-donor alloreactivity based on multiparameter mixed lymphocyte reaction (mlr) assay, wherein the number and phenotype of alloreactive precursorscan be quantified. we enrolled adults undergoing living donor liver transplantation (lt). the initial immunosuppressive regimen comprised tacrolimus/cyclosporine and methylprednisolone, which were gradually tapered off by months after lt. thereafter, therapeutic adjustments were determined by a policy of slow tapering off in the case of normal liver function. mlr assay was performed at month intervals to monitor immune status. in this assay, cfse-labeled pbmcs from recipients were used as responders. irradiated donor and third-party pbmcs were used as stimulators. after coculture, the responder cells were stained with cd or cd mabs along with cd mab, followed by fcm analyses. the proliferation and cd expression of cd + and cd + t cell subsets in response to anti-donor and anti-third-party stimuli were analyzed; the immune status of lt patients was categorized as hypo-response, norm-response, or hyper-response for cd + t cells and as hyper-response for cd + t cells. of the patients, had normal liver function at > months after lt. we examined the fluctuation of immunosuppressants at months after mlr in these patients. in patients whose immune status was categorized as hyper-response for cd + or cd + t cells (n= ), immunosuppressants had to be increased. in patients with norm-response immune status (n= ), immunosuppressant tapering was abandoned. immunosuppressant therapy was successfully tapered off in patients with hypo-response immune status (n= ). of patients with hypo-response immune status at > years after lt, immunosuppressants were completely discontinued in . in these "operational tolerance" patients, the precursor frequency of anti-donor cd + t cells (mean= . ± . %) was not reduced compared to that in non-tolerance patients, suggesting that donor-specific immune tolerance is maintained via inhibitory/ suppressive mechanisms rather than via clonal deletion. conclusion: multiparameter mlr assay can provide a clinically validated rule predicting the success of tailoring/weaning immunosuppression. psychological factors associated with non-adherence among adolescents before and after kidney transplant. nataliya zelikovsky. dept. of pediatrics, div. of nephrology, the children's hospital of philadelphia, philadelphia, pa.purpose: little is known about psychosocial risk factors for poor adherence among pediatric transplant patients. identification of variables that impact illness management can guide targeted interventions to improve adherence. methods: a longitudinal study was conducted to determine whether quality of life (pedsql), family functioning (fad), and parent adjustment (pip) would predict adherence in adolescent transplant patients. psychological questionnaires were administered prior to and months after the transplant. medical adherence measure (mam), a semi-structured interview was used to assess adherence. adherence was calculated as % missed and % late doses of those prescribed. results: patients (m = . years + . , % male, % caucasian) and their parents were evaluated at the time of listing for kidney transplant. the rate of non-adherence prior to transplant was high, with % of patients reporting some degree of nonadherence. of these patients, % missed and % took late > % of prescribed doses. on the quality of life measure, behavior issues were associated with missed (r=-. , p=. ) and late doses (r=-. , p<. ), and mental health issues were associated with late doses (r=-. , p<. ). adolescent reports of problems in affective responsiveness among family members was associated with missed (r=. , p=. ) and late (r=. , p=. ) doses. missed doses were also associated with mother reports of difficulties with overall family functioning (r=. , p<. ), communication (r=. , p<. ) and role definitions (r=. , p<. ) among family members. of the families were re-evaluated one year after the kidney transplant. % had been on dialysis prior to transplant and % received living-related transplants. % of the patients reported some degree non-adherence post-transplant, and using more stringent criteria, % of patients reported missing and % reported taking late > % of prescribed doses. worse quality of life such as limitations due to emotional problems (r=-. , p<. ), behavioral problems (r=-. , p<. ), and difficulties with family cohesion (r=-. , p<. ) was related to worse adherence. discussion: adolescent quality of life in behavioral and emotional domains, and family functioning play a significant role in adherence both before and after transplant. programs to improve adherence among transplant patients should incorporate psychosocial supports and behavioral interventions to improve adjustment of patients and families. kidney transplantation leads to marked improvements in health, yet transplant (tx) recipients often have difficulty with sexual functioning, which can affect quality of life. specific sexual concerns of tx recipients remain under investigated. the purposes of this study were to ) further establish the psychometric properties of the sexual concerns questionnaire (scq) including reliability and preliminary construct validity and ) identify the sexual concerns of kidney tx recipients. the scq was answered by kidney tx recipients who rated each item on a (not at all) to (extremely) scale. a cronbach's alpha correlation coefficient was calculated to determine the reliability of the scq. an alpha value of . was calculated for the questionnaire indicating it was reliable. exploratory factor analysis (efa) was performed to establish preliminary construct validity of the scq. as a result from the efa, items were dropped and a factor structure was accepted. examples of items and responses include question : "how difficult is it for your vagina to get or stay wet or moist?" (for women) and "how difficult is it for you to get or keep an erection?" (for men) and question "how comfortable are you talking about sexual concerns with your doctors and nurses?"participants were also asked to indicate how important their sexuality was to them on a (not at all) to (extremely) rating scale. twenty-six percent of participants rated their sexuality as quite a bit important, % rated their sexuality as very important, and % rated their sexuality as extremely important. the findings provide evidence of a reliable questionnaire with evidence for preliminary construct validity. they also indicate that sexuality is an important issue for a majority of kidney tx recipients. a cross-sectional study of fatigue before and after liver transplantation. james r. rodrigue, timothy antonellis, p= . # (none) to (extremely high); ♣ higher score = more fatigue, poorer sleep quality, or more mood disturbance; ¶ higher score = better qol one-third of pre-lt ( %) and post-lt ( %) patients reported severe fatigue. poor sleep quality was reported by % and % of pre-and post-lt patients, respectively. pre-lt fatigue was predicted by higher bmi (ß = . ) and meld (ß = . ), depression (ß = . ), and poor sleep quality (ß = . ), adj r = . , f = . , p < . . post-lt fatigue was predicted by older age (ß = . ), tension-anxiety (ß = . ), anger-hostility (ß = . ), and poor sleep quality (ß = . ), adj r = . , f = . , p < . . more fatigue was associated with lower sf- physical (r = - . ) and mental (r = - . ) qol. conclusions. fatigue and poor sleep quality are clinically significant problems for lt candidates and recipients. bmi, psychological functioning and sleep quality are modifiable variables that predict fatigue severity and should be targets of intervention when addressing fatigue symptoms. health literacy in kidney transplant recipients. elisa j. gordon, michael s. wolf. medicine, albany medical center, albany, ny; medicine, northwestern university. background: in order to successfully manage the transplant long-term, kidney recipients must have a basic understanding of key transplant-related concepts and terms indicative of their condition and treatment to properly communicate with health care providers and manage their health. kidney recipients must also possess numeracy skills to enable proper medication-taking and to monitor serum creatinine levels, bodily temperature, etc. the objective of this study was to examine health literacy levels among kidney transplant recipients. methods: we surveyed consecutive adult renal transplant recipients using the test of functional health literacy in adults (s-tofhla), and a modified version of the rapid estimate of adult literacy in medicine, called the "realm-transplant," which measured patients' knowledge of kidney transplant-related terms that patients are expected to have familiarity with. open-ended and multiple choice questions assessed numeracy related to kidney survival. results: most kidney recipients ( %) had adequate health literacy (s-tofhla), but % were unfamiliar with at least kidney transplant-related term (realm-t). patients who were less educated (p< . ), had lower income (p< . ), and were single or without a partner (p= . ) had significantly lower health literacy levels (s-tofhla). patients less familiar with transplant-related terms (realm-t) had less education (p< . ), lower income (p< . ), and were nonwhites (p= . ). the five least familiar terms were: sensitization ( %), urethra ( %), trough level ( %), blood urea nitrogen ( %), and toxicity ( %). sixteen percent wanted more information about their transplant. numeracy levels varied: % knew the likelihood of -year survival; % knew that half of kidney recipients have problems with the transplant in the first months; % knew the normal range of creatinine for kidney recipients; and % were aware of the risk of death within the first year of transplantation. conclusion: at this clinic, kidney transplant recipients generally had high levels of health literacy. however, most had difficulty recognizing frequently used transplantrelated terms, which could impede their understanding of health information and self-care management. greater efforts are needed to educate kidney recipients about transplant concepts, which may foster better self-care management, and ultimately transplant outcomes. abstracts by a single pathologist using the banff, cadi and cnit classifications. all indication biopsies with clinical acute rejection (ar; . % for sf and . % for sb) were excluded from this analysis. the histological and clinical parameters were assessed using multivariate generalized-estimating-equations statistical analysis. results: subclinical ar was present in . % sf vs. . % sb bx at mo and % sf vs % sb bx at mo (p=ns); borderline ar was seen in . % sf vs. . % sb bx at mo and % sf vs. % sb bx at mo (p=ns). despite the pristine condition of the kidneys at implantation, regardless of steroid exposure, there was a significant trend increase (p< . ) in chronic tubulo-interstitial damage; % of mo bx and % of mo bx demonstrated ifta; with moderate/severe changes (ifta grade - ) in . % and % of and mo bx respectively. the prevalence of biopsies with ischemic glomerular changes (p< . ), tubular microcalcifications (p= . ), vascular intimal thickening (p= . ) and the number of sclerosed glomeruli (p< . ) increased over the first year after transplantation, without any difference between the sb and sf group. a critical risk factor for ifta injury by multivariate analysis, independent of time after transplantation, was smaller recipient size. in this first ever serial histological analysis, embedded in a randomized multicenter pediatric study of steroid avoidance, we found significant progression of chronic graft injury in the first year post-transplantation in both study arms. small recipient size is the primary risk factor for tubulo-interstitial damage, likely related to vascular size discrepancies between recipient and the graft, resulting in chronic graft ischemia. in the -year symphony core study, a regimen with g mycophenolate mofetil (mmf) + low-dose tacrolimus ( - ng/ml) + daclizumab + steroids resulted in less acute rejections and better glomerular filtration rate (gfr) compared with g mmf + steroids and either standard-dose cyclosporine (csa), low-dose csa ( - ng/ml) + daclizumab or low-dose sirolimus ( - ng/ml) + daclizumab. methods: patients participated in an optional follow-up of years. gfr data from % of included patients ( % of the core itt population) were available at years.here we present results in the itt population. results: at inclusion into follow-up %, % and % of patients received csa, tacrolimus or sirolimus, and at years %, % and %, respectively. many follow-up patients had been switched to tacrolimus in the st year, including % of patients randomized to sirolimus. at years % of patients were on mmf and % on steroids.over the nd and rd year all arms had a low rate of biopsy-proven acute rejection (bpar; - %) and of graft loss ( - %). low-dose tacrolimus remained clearly superior in terms of bpar ( % vs. %- % in the other arms). uncensored -year graft survival was % with low-dose tacrolimus and low-dose csa, % with standarddose csa and % with low-dose sirolimus (p= . ). patient survival was between % and % (p= . ). in the four arms, the mean gfr change over the nd and rd year was between + and - ml/min and low-dose tacrolimus still had the highest gfr ( vs - ml/min, p= . ). observational follow-up results based on approximately half of the core symphony population indicate that during the nd and rd year renal function was stable, bpar and graft loss rates were low and many patients changed treatment regimen substantially. still, the itt arm with g mmf + low-dose tacrolimus + daclizumab + steroids was superior at years with respect to renal function and graft loss but differences were less marked and statistically not significant. discovering histological evaluation of time zero donor kidney biopsies has not conclusively predicted graft outcome. we hypothesize that gene expression analysis could provide additional information to determine graft outcome in the first year of transplantation. to this end, we evaluated all implantation biopsies obtained post reperfusion in deceased donors (dd) and living donors (ld) at our center. biopsies were evaluated and scored using banff criteria. low density real time pcr arrays were utilized to measure intragraft expression of genes associated with programmed cell death, fibrosis, innate and adaptive immunity, and oxidative stress signaling. results of expression were defined as folds compared to a pool of normal kidney biopsies. in dd, histological features of atn were more common ( %) than in ld grafts ( %; p< . ), whereas arteriosclerosis was infrequent in both groups ( % and %, respectively), as well as the extent of glomerular sclerosis ( % and %). there was no association between these histological features and renal function at year post transplant. not surprisingly, dd grafts displayed a pattern of gene expression remarkably different from ld, including an increased expression of complement protein c ( background: isa is a novel calcineurin inhibitor (cni), developed using a pharmacodynamic approach for use in autoimmune disease and solid organ transplantation. in moderate to severe plaque psoriasis, a canadian phase iii trial has demonstrated that isa is efficacious with minimal changes to renal function and a european trial is presently underway comparing isa to cyclosporine a (csa).in renal transplantation, a phase iia study comparing isa to csa in stable renal transplant recipients demonstrated isa to be efficacious and well tolerated. a phase iib study in de novo renal transplant patients comparing isa to tacrolimus is ongoing and final data will be available may . we hypothesize that isa is non-inferior to tacrolimus in terms of efficacy.methods: this is a month, randomized, multicenter, open-label, concentrationcontrolled study comparing three oral isa dosing groups ( . , . , or . mg/kg bid) to tacrolimus in north american transplant centres. all cni's were titrated to target trough concentrations. inclusion criteria included males and (non-pregnant) females between the ages of - who were receiving a first deceased or living donor renal transplant. cold ischemia times were to be ≤ hours, and peak panel reactive antibodies ≤ %. the primary efficacy parameter of the trial is non-inferiority (in at least one dose group) in biopsy proven acute rejection (bpar) at months as compared to tacrolimus. secondary objectives include: renal function; pk/pd relationships; patient and graft survival; and proportion of patients with hypertension, hyperlipidemia or new onset diabetes mellitus (nodm).results: interim data, as previously presented at the atc, demonstrated that isa had rejection rates similar to tacrolimus (isa . mg/kg bid %, isa . mg/kg bid %, isa . mg/kg bid %, tacrolimus %) and confirmed previous results indicating an improved safety profile. patients have now been enrolled between january and june , with an optional extension to months added to the trial. a recent approval by both fda and health canada has allowed continued use of isa in these patients until commercialization. the six month final results will be available for presentation at atc . key: cord- -er irjw authors: powell, fiona; rothwell, lisa; clarkson, michael; kaiser, pete title: development of reagents to study the turkey's immune response: cloning and characterisation of two turkey cytokines, interleukin (il)- and il- date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: er irjw the cdnas of two turkey cytokines, interleukin (il)- and il- , were cloned using oligonucleotide primers designed from their chicken orthologues. the coding regions of the chicken and turkey genes are highly conserved, with il- and il- exhibiting . % and % nucleotide and % and . % amino acid identity respectively. both showed consistent mrna expression in turkey lymphoid and gut tissues. expression in non-lymphoid tissues was more variable but generally highest in the skin and trachea. recombinant turkey il- was expressed and bioactivity demonstrated by inhibition of ifn-γ synthesis from activated splenocytes. chicken and turkey il- cross-reacted in functional assays. the immune system of the chicken has been characterised to a far greater extent than that of the turkey, primarily due to its greater economic status and stimulating more research interest. however, the increasing consumption of poultry meat other than chicken is driving a concomitant increase in the economic importance of other poultry species, including turkeys. there is a subsequent need for improved vaccination strategies, and therefore a greater understanding of the immune systems of these species (kaiser, ; meyerhoff et al., ; schultz and magor, ) . the release of the chicken genome sequence (international chicken genome sequencing consortium, ) revolutionised our under-standing of the repertoire and biology of immune-related molecules, including cytokines and chemokines, resulting in a large and expanding chicken immunological "toolbox" of reagents kaiser, kaiser, , kaiser and stäheli, ) . the availability of genome sequences for other avian species, including the zebrafinch (warren et al., ) , the turkey (dalloul et al., ) and the duck (http://pre.ensembl.org/anas platyrhyncos/info/), should allow identification and subsequent characterisation of their immune gene repertoires. dalloul et al. ( ) catalogued the majority of the turkey immune gene repertoire, which unsurprisingly is very similar to that of the chicken. however, only five cytokines have been biologically characterised in the turkey: type i ifn (suresh et al., ) , ifn-␥ (lawson et al., ; loa et al., ) , il- ␤ (wu et al., ) , il- (lawson et al., ) , and il- (kaiser, ) , and a single chemokine, il- (also known as cxcli ) (wu et al., ) . a partial sequence for turkey il- ␤ had also been identified pre-genome (balu and kaiser, ) . all of these share high nucleotide (nt) and amino acid (aa) identities with their chicken orthologues. only three (ifn-␥, il- and cxcli ) have been tested for cross-reactivity. all three cytokines cross-reacted, somewhat surprisingly in the case of il- as chicken and turkey il- share only . % aa identity (lawson et al., (lawson et al., , wu et al., ) . this leads to the hypothesis that other chicken and turkey cytokines would also show cross-reactivity. in addition, powell et al. ( a) identified and cloned three turkey t cell surface markers and demonstrated cross-reactivity of monoclonal antibodies recognising chicken cd ␣ and cd with the respective turkey orthologues. to date, reagents have been developed to measure mrna expression of turkey cytokines of the induced innate response (il- ␤ and ifn-␣), an innate pro-inflammatory chemokine, cxcli , and cytokines of the t helper (th) adaptive response (ifn-␥, il- , il- ). the aim of this work was to clone examples of turkey th cytokines and regulatory cytokines, and develop reagents to them. il- is an anti-inflammatory cytokine and is a crucial mediator of immune-regulation following infection with a range of pathogens, including viruses, bacteria, fungi, protozoa and helminths in mammals (reviewed by couper et al., ; ding et al., ) . chicken il- inhibits ifn-␥ production from mitogen-activated splenocytes, and increased expression of il- has been linked with susceptibility to infection with the intracellular protozoan parasite, eimeria maxima . il- expression is also significantly increased (along with il- and il- ) in chickens during the lytic phase of infection with marek's disease virus (heidari et al., ) , indicating that il- plays a role in the immuno-regulation of th responses in the chicken. il- is a th signature cytokine which is critically involved in protective immunity, being particularly important in anti-helminthic responses. these effects are mediated through a variety of mechanisms, including inhibition of inflammatory cytokine responses and promoting ige class-switching (reviewed by wynn, ) . it was cloned in the chicken through genomic analysis based on the conservation of synteny with the rest of the th cytokine gene cluster (avery et al., ) . we and others have shown that during infection with extracellular pathogens, chicken il- is induced to far greater levels than chicken il- (degen et al., ; powell et al., b; schwarz et al., ) , the canonical th cytokine in man and mouse. given that chickens and turkeys are closely related in evolutionary terms (dalloul et al., ) , it seems likely that il- and il- , as well as other immune-related molecules, will have similar roles in the two species. turkeys were obtained at hatch from sun valley foods ltd. (ludlow, shropshire, uk) and maintained at the institute for animal health (iah). turkeys used to produce a tissue panel were obtained at hatch from british united turkeys (cheshire, uk) and maintained at the university of liverpool, leahurst. chickens were produced and maintained at iah. turkey splenocytes were isolated, cultured and stimulated with phorbol myristate acetate (pma) (sigma, poole, uk) as previously described (lawson et al., ) . cells were harvested , , and h post-pma stimulation and mrna isolated with an oligotex direct midi kit (qiagen, crawley, uk). primers were designed to chicken il- ( rothwell et al., ) and il- (avery et al., ) . for il- , nested forward primers (f : -taaagaataaagaccccaggatg- (in utr to the start codon) and f : -atgcagacctgctgccaag- (from the start codon)) and reverse primers in the utr (r : -gctgagagcggctgtgc- and r : -ctcgtctggtgtttgcagtt- ) were used. for il- a forward primer from the start codon ( -atgcaccgcacactgaaggc- ) and nested reverse primers (r : -atggggccgggcatcag- (in utr) and r : -tcagtttgcagctgtggc- (from the stop codon)) were used. first strand synthesis was carried out for h at • c in a l reaction containing pmol reverse primer, u superscript ii (invitrogen, paisley, uk) and ng template mrna. after denaturation of the reverse transcriptase at • c for min, l of this template cdna were added to a l pcr reaction, containing pmol of each primer, . mm dntps, and . units of taq polymerase (invitrogen). thermal cycling conditions were cycles of • c for min, • c for min and • c for min using a mastercycler pcr machine (eppendorf, cambridge, uk). pcr products were ligated into pgem-t easy (promega) and the sequence of representative clones determined using capillary electrophoresis on a ceq sequencer (beckman coulter, high wycombe, uk). sequence analysis was carried out using dsgene and vector nti (invitrogen) software. the cdnas were then subcloned from pgem-t easy, using ecori or noti, into the mammalian expression vector, pci-neo (promega). the resulting clones were transfected into cos- cells using a deae-dextran-based method as previously described to produce high expression of the relevant gene of interest. the supernatant (containing the relevant recombinant cytokine) and rna from the transfected cos- cells were harvested after - days of culture. rna was isolated from the cells with an rneasy mini kit (qiagen) for use as a positive control in real-time qrt-pcr. tissue panels were taken from three turkeys post mortem. lymphoid tissues included spleen, bursa of fabricius, thymus, caecal tonsil, harderian gland, meckel's diverticulum and bone marrow. non-lymphoid tissues taken were brain, trachea, heart, liver, lung, pancreas, kidney, ovary, skin and muscle. tissues from the digestive tract were also taken, and these were the crop, proventriculus, gizzard, upper and lower ileum, caecum and large intestine. tissue samples were homogenised separately using a bead mill (retsch mm , haan, germany) and qiashredders (qiagen). rna was extracted from homogenates using an rneasy mini kit (qiagen) following the manufacturer's instructions. eluted rna was stored at − • c prior to use. expression of il- and il- in the turkey tissue panel was quantified using a well-described method (e.g. avery et al., ; rothwell et al., ; eldaghayes et al., ) . primers and probes were designed from the turkey cdna sequences using the primer express software programme (applied biosystems) and were as follows: il- forward primer -cgacctgggcaacatgct- ; il- reverse primer -cctctcgcaggtgaagaagt- ; il- probe -(fam)-cctgaagatgacaatgaagcgctgtca-(tamra)- ; il- forward primer -cctgcacggccagatga- ; il- reverse primer -ggcaagaagttccgcaggta- ; il- probe -(fam)-tgccagctgagcaccgacaacg-(tamra)- ; ifn-␥ forward primer -aaccttcctgatggcgtgaa- ; ifn-␥ reverse primer -cttgcgctggattctcaagtc- ; ifn-␥ probe -(fam)-aaagatatcatggacctggccaagcttca-(tamra)- . the s and chicken ifn-␥ primer-probe sets were as previously described . real-time qrt-pcr was performed, in triplicate, on rna from the tissue panels described above, using the reverse transcriptase qpcr master mix rt-pcr kit (eurogentec, southampton, uk), and amplification was performed using the abi prism sequence detection system (applied biosciences, warrington, uk), with the following cycling conditions: cycle of • c for min, • c for min and • c for min followed by cycles of • c for s and • c for min. results are expressed as -c t , after normalising each sample using the c t value for the s rrna product for the same sample, as described previously (avery et al., ; rothwell et al., ; eldaghayes et al., ) . the il- bioassay was carried out as previously described . briefly, splenocytes from -week-old chickens or -week-old turkeys were isolated and resuspended at × cells/ml in dmem containing mg/ml bsa, % l-glutamine, u/ml penicillin and g/ml streptomycin. they were added to round-bottomed well plates or -well plates containing serial two-fold dilutions of recombinant chicken or turkey il- (ex-cos), in a final volume of l/well ( -well plates) or ml/well ( -well plates), in the presence of . g/ml pha (sigma) or no mitogen. negative controls included serial two-fold dilutions of supernatant collected from cos- cells transfected with pci-neo alone, or media alone, with or without pha. cells were incubated at • c, % co for h for analysis of ifn-␥ content in the supernatant. assays were carried out in triplicate, and repeated three times. supernatants were assayed (in triplicate) for ifn-␥ content using a quantitative chifn-␥ capture elisa (biosource, nivelles, belgium), as previously described (lambrecht et al., ) , and by bioassay, using the macrophage activation factor assay, again as previously described lawson et al., ) . there were insufficient cells to carry out the bioassay on supernatants from turkey splenocytes. total rna was isolated from cells in -well plates using the rneasy mini kit (qiagen) following the manufacturer's instructions. purified rna was eluted in l rnase-free water and stored at − • c. real-time qrt-pcr was used to determine ifn-␥ mrna expression levels in pha-stimulated splenocytes (from chicken and turkey) treated with recombinant chicken or turkey il- or mock-transfected cos cell supernatant at various dilutions. results are expressed as fold-difference from levels in untreated pha-stimulated splenocytes. the turkey th cytokines ifn-␥, il- ␤ and il- have all been characterised previously (balu and kaiser, ; kaiser, ; lawson et al., ) but no reagents have been available to investigate the turkey th response or regulatory response. turkey il- cdna was amplified from rna from pmastimulated turkey splenocytes by rt-pcr, using primers based on the chicken il- cdna sequence. the coding sequence of nt from start to stop codon was identified and encodes a aa polypeptide (fig. ) . sequence identity between turkey il- and human il- is . % and . % at the nt and aa levels, respectively. turkey and murine il- share . % and . % nt and aa identity, respectively. identity between chicken and turkey il- is high, with . % and % identity at the nt and aa levels respectively, with complete conservation of the il- family signature motif, g-x-x fig. ). all cysteine residues in il- are conserved between the chicken and turkey and all hydrophobic aa of the heptad repeats are conserved, suggesting that turkey il- has six ␣-helices, as do chicken and mammalian il- . the cdna sequence was submitted to ensembl with the acc. no. am . il- mrna expression ( fig. a) was consistent in all lymphoid tissues with lowest expression in bone marrow. expression in non-lymphoid tissues was variable with relatively high expression in brain, lung and ovary, followed by the heart and liver. the profile of il- mrna expression in digestive tissues was fairly consistent, presumably as this is an antigenic environment and a potential site of pathogen entry. no expression was seen in the proventriculus and there was lower expression in the gizzard relative to other digestive tissues. little or no il- mrna expression was seen in the remaining tissues. when comparing il- mrna expression in the chicken and turkey, differential expression was seen in chicken lymphoid tissues while il- mrna was constitutively expressed in turkey lymphoid tissues. il- mrna expression was not detected in the kidney or muscle of either species but was detected in the liver and lung of both . we assessed the ability of recombinant chicken and turkey il- (ex-cos) to inhibit ifn-␥ expression by chicken and turkey splenocytes following pha-stimulation . comparison of the predicted aa sequences of human (hu), murine (mu), chicken (ch) and turkey (tu) il- . shaded areas represent conservation of aa similarity -the darker the shading, the more conserved the residue across species. dashes indicate gaps in the alignment. conserved cysteine residues in the avian predicted proteins are indicated with an asterisk. the ␣-helical assignments are based on the structure of human il- , as indicated by overlying lines (zdanov et al., ) . the predicted signal peptide cleavage site is indicated by an arrow. double underlined bases indicate the il- family signature motif. acc. nos. of the cdna sequences are: human, nm ; murine, nm ; chicken, aj ; turkey, am . at the mrna (real-time qrt-pcr, fig. a and b) and protein levels (elisa, fig. c and d and bioassay, fig. e) . at the mrna level ( h post-stimulation), both chicken and turkey il- inhibited ifn-␥ expression by chicken splenocytes after stimulation with pha in a dosedependent manner (fig. a) . similarly, there was inhibition of ifn-␥ production from pha-stimulated turkey splenocytes by recombinant chicken and turkey il- , although expression patterns of (a) il- and (b) il- mrna, as measured by real-time quantitative rt-pcr, in lymphoid tissues, non-lymphoid tissues and digestive tissues of the turkey. the average expression in tissues from each of three birds is shown, with results expressed as mean -ct values ± sem. , spleen; , bursa of fabricius; , thymus; , caecal tonsil; , harderian gland; , meckel's diverticulum; , bone marrow; , brain; , trachea; , heart; , lung; , liver; , pancreas; , kidney; , ovary; , skin; , muscle; , crop; , proventriculus; , gizzard; , upper ileum; , lower ileum; , caecum; , large intestine. the effect was less marked than that observed on chicken cells. this effect had titrated out by : dilution of the recombinant protein. at the protein level ( h post-stimulation), recombinant chicken and turkey il- inhibited the production of ifn-␥ by both chicken and turkey splenocytes at high concentrations, as measured by elisa, and this effect titrated out with increasing dilution of recombinant il- ( fig. c and d respectively). the bioassay measures the ability of ifns, including ifn-␥, to stimulate nitric oxide (no) production by a chicken macrophage cell line (hd ). both recombinant chicken and turkey il- inhibited production of no-inducing agents (presumably ifn-␥) following stimulation of splenocytes with pha, the inhibition titrating out with increasing dilution of recombinant il- (fig. e) . ifn-␥ production by pha-stimulated splenocytes was not inhibited by the presence of supernatant from cos cells transfected with pci-neo alone. our attempts to clone and sequence turkey il- failed (data not shown), but turkey il- cdna was amplified from turkey rna by rt-pcr using primers designed to chicken il- . the coding sequence of nt from start to stop codon was identified (fig. ) , which included the insertion of an additional serine residue, at position (cca at - bp), compared to the chicken sequence. sequence identity between turkey il- and human il- is . % and . % at the nt and aa levels, respectively, with similar levels of identity between turkey and murine il- . mammalian and avian il- each has five cysteine residues, four of which are conserved in location. identity between chicken and turkey il- is high, with . % and . % identity at the nt and aa levels, respectively. amino acid changes show evidence of some clustering, with seven aa changes from positions to , which is the region directly following the predicted cleavage site for processing of the signal peptide of mammalian il- . five aa changes occur between positions and which lie within helix a of human il- and four changes between positions and , three of which lie within helix b of human il- . five aa changes between positions and lie between helix c and the second ␤-sheet (avery et al., ) . since the exon boundaries are consistent between species (http://www.ensembl.org/meleagris gallopavo/), this shows that a high proportion of replacements occurs in exons , and . all of the cysteine residues in il- are conserved between the chicken and turkey. with the relatively high level of aa identity and the conservation of cysteine residues, we could speculate that the biological activities exhibited by chicken il- are conserved between the two avian species, and that turkey and chicken il- will cross-react. the sequence was submitted to ensembl with the acc. no. am . expression levels of il- mrna (fig. b ) were generally lower than those of il- in the panel of tissues analysed. il- mrna was detected in all lymphoid organs but to the highest degree in the thymus. the highest expression of il- in non-lymphoid tissues was seen in the trachea, followed by ovary, skin and muscle. il- mrna was also expressed in all digestive tract tissues to similarly low levels and to varying extents in the non-lymphoid tissues. interestingly, expression of il- in the chicken, compared to the turkey, seems more restricted (avery et al., ) , particularly in the non-lymphoid tissues, where helix a human mhpllnplllalglmall lttvial tclggfaspgpvp pst----a lre lieelvn itqn murine malw vtavlal aclgg laapgpvp rsvslp ltlke lieelsn itqd chicken mhrt lkaalal lclae lvastpl amn-lsk lklsd itqgiqk lnrg turkey mhrt lkaalal lclae liastplp tssmpk mklsd itrs iqq lnkg * helix . comparison of the predicted aa sequences of human (hu), murine (mu), chicken (ch) and turkey (tu) il- . shaded areas represent conservation of aa similarity -the darker the shading, the more conserved the residue across species. dashes indicate gaps in the alignment. conserved cysteine residues in the avian predicted proteins are indicated with an asterisk. the ␣-helical and ␤-sheet assignments are based on the structure of human il- and indicated by overlying lines (bamborough et al., ) . the predicted signal peptide cleavage site is indicated by an arrow. acc. nos. of the cdna sequences are: human, nm ; murine, nm ; chicken, nm ; turkey, am . expression of chicken il- was restricted to the lung and brain. the turkey cytokines described herein, and the reagents developed to them, make it possible to extend the characterisation of the turkey's immune response. we now have the means to measure key cytokines involved in the innate immune response, a th or th -type response, as well as a regulatory t cell response. in addition to the immune response of turkeys to foot pad dermatitis (mayne et al., ) and histomonosis (powell et al., b) , it would be interesting to investigate the immune response to other commercially important turkey pathogens, including turkey rhinotracheitis, an avian pneumovirus which causes up to % mortality ( - % morbidity), avian flu, coccidia and bacteria (including salmonella, campylobacter and escherichia coli). also of interest are re-emerging turkey diseases, such as bluecomb disease, caused by a turkey coronavirus, and poult enteritis mortality syndrome, an emerging disease whose etiology remains unclear. the reagents developed during this study will aid these efforts. characterisation of the first non-mammalian t cytokine gene cluster: the cluster contains functional single-copy genes for il- , il- , il- , and gm-csf, a gene for il- that appears to be a pseudogene, and a gene encoding another cytokine-like transcript avian interleukin- ␤ (p ): cloning and characterization of the cdna and gene predictive modelling of the -d structure of interleukin- il- : the master regulator of immunity to infection multi-platform next-generation sequencing of the domestic turkey (meleagris gallopavo): genome assembly and analysis th /th polarization by viral and helminth infection in birds interleukin- infectious bursal disease virus: strains that differ in virulence differentially modulate the innate immune response to infection in the chicken bursa international chicken genome sequencing consortium, . sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution turkey and chicken interleukin- (il ) share high sequence identity, but have different polyadenylation sites in their utr the avian immune genome-a glass half-full or halfempty? advances in avian immunology-prospects for disease control: a review a genomic analysis of chicken cytokines and chemokines differential cytokine expression in avian cells in response to invasion by salmonella typhimurium, salmonella enteritidis and salmonella gallinarum avian cytokines and chemokines production of antibodies against chicken interferon-␥: demonstration of neutralizing activity and development of a quantitative elisa turkey and chicken interleukin- cross-react in in vitro proliferation assays despite limited amino acid sequence identity turkey and chicken interferon-␥, which share high sequence identity, are biologically cross-reactive molecular identification and characterization of turkey ifn-␥ gene foot pad dermatitis in growing turkeys is associated with cytokine and cellular changes indicative of an inflammatory immune response comprehensive analysis of commercially available mouse antichicken monoclonal antibodies for cross-reactivity with peripheral blood leukocytes from commercial turkeys development of reagents to study the turkey's immune response: identification and molecular cloning of turkey cd , cd ␣ and cd the turkey, compared to the chicken, fails to mount an effective early immune response to histomonas meleagridis in the gut cloning and characterisation of monoclonal antibodies specific for chicken interleukin- cloning and characterization of chicken il- and its role in the immune response to eimeria maxima comparative immunology of agricultural birds molecular and functional characterization of turkey interferon immunopathogenesis of ascaridia galli infection in layer chicken the genome of a songbird sequence and phylogenetic analysis of interleukin (il)- ␤-encoding genes of five avian species and structural and functional homology among these il- ␤ proteins structural and functional homology among chicken, duck, goose, turkey and pigeon interleukin- proteins il- effector functions crystal structure of human interleukin- at . Å resolution and a model of a complex with its soluble receptor research association. further financial support came from the bbsrc, in the form of institute strategic programme grants to iah and the roslin institute. key: cord- -q wx k authors: resende, lucilene aparecida; aguiar-soares, rodrigo dian de oliveira; gama-ker, henrique; roatt, bruno mendes; de mendonça, ludmila zanandreis; alves, marina luiza rodrigues; da silveira-lemos, denise; corrêa-oliveira, rodrigo; martins-filho, olindo assis; araújo, márcio sobreira silva; fujiwara, ricardo toshio; gontijo, nelder figueiredo; reis, alexandre barbosa; giunchetti, rodolfo cordeiro title: impact of lbsapsal vaccine in canine immunological and parasitological features before and after leishmania chagasi-challenge date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: q wx k dogs represent the most important domestic reservoir of l. chagasi (syn. l. infantum). a vaccine against canine visceral leishmaniasis (cvl) would be an important tool for decreasing the anxiety related to possible l. chagasi infection and for controlling human visceral leishmaniasis (vl). because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of leishmania parasites, their inclusion in an anti-leishmania vaccine has been investigated in past decades. we investigated the immunogenicity of the “lbsapsal” vaccine (l. braziliensis antigens, saponin as adjuvant, and lutzomyia longipalpis salivary gland extract) in dogs at baseline (t( )), during the post-vaccination protocol (t( rd)) and after early (t( )) and late (t( )) times following l. chagasi-challenge. our major data indicated that immunization with “lbsapsal” is able to induce biomarkers characterized by enhanced amounts of type i (tumor necrosis factor [tnf]-α, interleukin [il]- , interferon [ifn]-γ) cytokines and reduction in type ii cytokines (il- and tgf-β), even after experimental challenge. the establishment of a prominent pro-inflammatory immune response after “lbsapsal” immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism ( . %) and indicating long-lasting protection against l. chagasi infection. in conclusion, these results confirmed the hypothesis that the “lbsapsal” vaccination is a potential tool to control the leishmania chagasi infection. leishmania infantum (syn. l. chagasi) is an intracellular protozoon that cause a severe systemic disease named visceral leishmaniasis (vl) [ ] . it is widely distributed in the mediterranean basin, middle east, and south america. vl has an annual incidence of approximately , cases [ ] . brazil declared a total of , clinical vl cases between and , and this number accounts for % of all reported vl cases in the americas; however, it is subject to substantial under-reporting [ ] . different geographical regions of the globe where visceral leishmaniasis is endemic, present the dogs as main reservoir for the l. chagasi that play a relevant role in transmission of the parasite [ , ] . dogs are also excellent models for the study of vl because the natural history of the disease in dogs and in humans is similar [ ] , especially regarding parasite-host interaction, immune response, and the development of new vaccines [ ] . the natural history of canine vl (cvl) has been well-described, particularly in regard to the parasite load in different tissues and the immunopathological changes according to progression of clinical forms [ , , , , , , , , , ] . a vaccine against cvl would be an important tool in the control of vl and would decrease the anxiety associated with l. chagasi infection in humans [ ] . the induction of long-lasting cell-mediated immune response triggering high levels of protection against cvl is considered as prerequisite of an ideal vaccine for controlling the l. chagasi transmission. different vaccine candidates against cvl have been reported showing the ability to induce immunoprotective mechanisms [ , , , , , , , , ] . in this sense, it has been shown that leishmune (fucose manose ligand plus saponin as adjuvant; zoetis, são paulo, brazil) presented % protection in a phase iii study [ ] . leishmune provided protection associated with the ability to trigger early and persistent activation of neutrophils and monocytes, in addition to activation of t cd + and t cd + lymphocytes displaying high levels of ifn-γ in the cd + t-cell subset [ , ] . additionally, the leish-tec vaccine (hertape calier, juatuba, brazil; a antigen plus saponin as adjuvant) has also been shown to induce increased levels of ifn-γ in four out of seven dogs with l. chagasi-infected bone marrow [ ] . more recently, a vaccine composed of excreted/secreted antigens of l. infantum promastigotes, referred as liesap has been shown to induce increased levels of ifnγ and nitric oxide (no), thus supporting its leishmanicidal effect [ ] . the efficacy of the lie-sap vaccine was reported as % in a double-blind randomized study [ ] . liesap is commercially available as canileish (virbac, carros, france) and is able to induce a th profile and reduce the parasitic load of infected macrophages co-cultured with lymphocytes from immunized dogs [ ] . the lbsap (l. braziliensis crude antigens plus saponin as adjuvant) and "lbsapsal" (l. braziliensis crude antigens plus saponin as adjuvant and lutzomyia longipalpis saliva) vaccines against cvl has also shown to induce a strong immune-mediated response. the lbsap and "lbsapsal" presented higher levels of circulating t-cell subsets (cd + , cd + ) and b lymphocytes (cd + ), as well as leishmania-specific cd + and cd + t cells [ , , , ] . furthermore, lbsap-vaccinated dogs presented high ifn-γ and low interleukin (il)- and transforming growth factor (tgf)-β expression in the spleen, resulting in a significant reduction of parasite load in this tissue [ ] . additionally, lbsap has been shown to induce a prominent pro-inflammatory immune response characterized by increased levels of both il- and ifn-γ and decreased levels of tgf-β by peripheral blood mononuclear cells (pbmcs), which were associated with parasite control in dogs [ ] . the incorporation of salivary proteins of sand flies have been widely used in experimental challenge studies, and the results suggest that this could be a good strategy to protect against leishmania infection [ , , , , , , , , , ] . previous studies of dogs using the "lbsapsal" vaccine displayed higher counts of circulating and leishmania-specific cd + t cells in addition to high nitric oxide (no) production [ ] and reduction of splenic parasite load [ ] . considering the inclusion of saliva from sand flies as a promising compound in vaccines against leishmania infection, we describe additional biomarkers induced by the "lbsap-sal" vaccine in dogs. we considered the previous vaccine protocol (t ), post-vaccine protocol (t rd ), and early (t ) and late (t ) periods of the l. chagasi-challenge. all research involving dogs was carried out according to the regulations of the brazilian society of science in animal research, adopted by the federal university of ouro preto ethics committee in animal experimentation, that approved the technical procedures using dogs under protocol number / . all dogs included in this study, were born and bred in the kennel facility at universidade federal de ouro preto in ouro preto, minas gerais, brazil. this kennel facility was established with male and female mongrel dogs, with negative serological and molecular/parasitological diagnosis for leishmania infection, donated by the local zoonosis control center from belo horizonte, minas gerais, brazil. the filial generations, comprising of offsprings resulting from the cross between the original mongrel dogs pairs were used further maintain the mongrel dog colony. in the present study, twenty mongrel dogs ( males and females), with seven months of age and negative results for indirect fluorescence immunoassay to anti-leishmania antibodies, were selected and received an anti-helmintic treatment and were submitted to polyvaccination protocols, including rabies (tecpar, curitiba-pr, brazil), leptospira, parvovirus, canine distemper, type ii adenovirus, parainfluenza and coronavirus (vanguard htlp / cv-l; pfizer animal health, new york, ny, usa). prior the experimental onset, the animals were maintained in quarantine in kennel runs ( m length x m width x m height) completely covered with stainless steel wire mesh to prevent the entry of sand flies. the kennel facilities were sprayed with pyrethroid insecticide, every three months, as a measure to control insect access. each run was installed with an infrared lamp heating ( watts) to ensure the thermal comfort of animals during the night and on cold days. all the dogs were housed without environmental enrichment (e.g. toys, exercise regimes, etc). the dogs were monitored twice a day, as routine inspection, during which the responsible veterinary was in charge of stimulating and playing with them to aiming to avoid behavior problems and minimize their suffering or distress throughout the study. all the experimental procedures described in this study, including this aspect of animal care was approved by federal university of ouro preto ethics committee in animal experimentation (protocol number / ). the animals were maintained with water and food ad libitum throughout the experiment time. the euthanasia of all dogs was performed under the supervision of a veterinarian physician, using barbiturate anesthetic (thiopental sodium, mg/kg, iv) followed by intravenous injection of saturated solution of potassium chloride. four groups of dogs involved in experiments were treated as follows: (i) "control" (c) group (n = ; subcutaneous injections of ml of sterile . % saline); (ii) "sal" group (n = ; subcutaneous injections of salivary lutzomyia longipalpis gland extract (sge; obtained as previously described- [ ] ) in ml of sterile . % saline); (iii) "lbsal" group (n = ; subcutaneous injections of μg of l. braziliensis promastigote antigen and sge in ml of sterile . % saline); and (iv) "lbsapsal" group (n = ; subcutaneous injections of μg of l. braziliensis promastigote antigen plus mg of saponin and sge in ml of sterile . % saline). all animals received three injections in the right flank at days intervals. the l. chagasi-experimental challenge in all dogs was performed after days of vaccination protocol, using intradermal promastigotes during the stationary phase of cultivation. the challenge was performed in the inner side of the left ear including five lutzomyia longipalpis salivary gland acini. all the analyzed dogs were euthanized days after l. chagasi-experimental challenge and the spleens were collected to evaluate parasite loads. the rationale for choosing such a long endpoint ( days after challenge) was based on the chronic course of the experimental canine visceral leishmaniasis after intradermal l. chagasi-challenge as previously reported [ ] . the vaccine was obtained as previously described [ ] . briefly, l. braziliensis (mhom/br/ / m ) promastigotes were obtained by in vitro culture in neal, novy, nicolle/liver infusion triptose media [ ] , and the parasite was fully disrupted by ultrasound treatment ( w, min, °c), aliquoted and stored at − °c. protein concentration was determined according to the method of lowry [ ] . the sge used in this study as the antigenic component of the vaccine was obtained from lutzomyia longipalpis females that were not fed, aged days, and dissected in slightly hypotonic unbuffered saline . %, as described in [ ] . after collection, the glands were disrupted in a sonicator for seconds and centrifuged at , g for min. the supernatant was collected and stored in a freezer at - °c until use. jugular vein was used for collecting ml of peripheral blood in sterile heparinized syringes. we analyze distinct times: baseline before vaccination (t ), days after third immunization dose (t rd ) as well as early ( days-t ) and late ( days-t ) after experimental l. chagasi-challenge. the pbmcs were isolated as previously described [ ] . briefly, the whole blood samples were added over ml of ficoll-hypaque (histopaque ; sigma, usa), centrifuged at g for min at room temperature, and the pbmcs were washed twice with rpmi ( g for min at room temperature). the pbmcs were resuspended in rpmi at cells/ml. the pbmcs cultures were performed in -well flat-bottom tissue culture plates (costar, cambridge, ma, usa). the in vitro assays were performed using μl of pbmcs ( . × cells/well) with μl of vaccine l. braziliensis-soluble antigen (vsa; μg/ml) or μl of soluble l. chagasi antigen (slca; μg/ml). the control cultures (cc; unstimulated) were analyzed using μl of rpmi in place of the antigenic stimulus. incubation was performed in a humidified % co atmosphere at °c for days, after which time the supernatants were collected and stored in a freezer at − °c for detection of cytokine and no. the quantification of cytokines was carried out by enzyme-linked immunosorbent assay (elisa) according to the manufacturer's instructions (r&d systems, minneapolis, mn, usa), as previously described [ ] . minimum cytokine sensitivity detection levels were pg/ml (il- ), pg/ml (tnf-α and ifn-γ), pg/ml (il- and il- ) and pg/ml (tgf-β). the analysis of il- (anti-canine il- /il- p , catalog number dy ), ifn-γ (anti-canine ifn-γ, catalog number dy b), tumor necrosis factor (tnf)-α (anti-canine tnf-α/tnfsf a immunoassay; catalog number dy ) and il- (anti-canine il- , catalog number dy ) cytokines were performed using duoset elisa. quantikine kit (mouse/rat/porcine/canine tgf-β immunoassay, catalog number mb b) (r&d systems, minneapolis, mn, usa) was used to measure tgf-β levels. the analysis of il- levels employed the capture antibody (monoclonal anti-canine il- antibody-catalog number mab ); the standard curve was obtained using recombinant canine il- (catalog number cl); and streptavidin (dy ; r&d systems/usa), biotinylated anti-canine il- antibody (catalog number: baf ) and substrate solution ( : mixture of h o and tetramethylbenzidine; product code - - ) were used. all experiments were performed according to the instructions of r&d systems using -well plates (corning incorporated, costar , washington, dc, usa). the microplate automatic reader (el ; biotek, winosski, vt, usa) was employed at a wavelength of nm. the measurement of no levels in supernatants of pbmcs cultures was performed by indirect method that quantify the nitrite concentration by griess reaction [ , ] . briefly, μl of griess reagent ( % sulfanylamide, . % naphthylethylene-diamide-dihydrochloride, and . % phosphoric acid; all from sigma, usa) was mixed with μl aliquot of cell-free culture supernatant. the microplate reader (biotek, el ) was used to analyze the absorbance at nm, after min of incubation at room temperature in the dark. the final nitrite concentration was determined based on a standard curve interpolation constructed by using sodium nitrite solutions in the range of - μm. the interference of nitrites already present in the culture medium was discounted; data were calculated by taking into account the blank, as control reaction, assayed by using the pbmcs cultures medium. the data were expressed as nitrite concentration (μm). the spleen specimens ( mm) were collected during necropsy and stored at - °c until use for dna extraction. the wizard™ genomic dna purification kit (promega, madison, wi, usa) was used to extract total genomic dna in mg of spleen following manufacturer's recommendations. primers that amplified a -bp fragment of a single copy of the dna polymerase gene of l. chagasi were used to analyze the spleen parasite burden by quantitative polymerase chain reaction (qpcr). for the q pcr analysis nm forward and reverse primers, μl of template dna and syber green reaction master mix (applied biosystems, grand island, ny, usa) were used in a final volume of μl. as previously described [ ] , we used the targets the dna polymerase gene l. chagasi (genbank accession: af ) and the pair of primers (forward: ' tgt cgc ttg cag acc aga tg '; reverse: ' gca tcg cag gtg tga gca c '). the pcr reactions employed an initial denaturation at °c ( min), denaturation cycles at °c ( seconds), in addition to annealing and extension at °c ( min.). the pgemh t plasmids (promega) was used for constructing stardart curves, for each run, containing inserts of interest [ ] . the gapdh gene ( -bp fragment genbank accession number ab ) was used to analyze the integrity of the samples. the primers 'ttccacggcacagtcaag ' (forward) and ' actcagcaccagcatcac ' (reverse) were used for gapdh gene amplification. the spleen parasite load was performed in duplicate and calculated by interpolation from the standard curve included in the same experimental batch. the data was expressed as number of l. chagasi organisms/ ng of total dna. the prism . software package (prism software, irvine, ca, usa) was used to evaluate data distribution normality by kolmogorov-smirnoff test and for further statistical analyses. oneway analysis of variance (anova) followed by tukey's multiple comparison were used for comparisons of cytokine profiles, no levels and parasite load amongst the experimental groups. student's t-test was used for intra-group comparisons between the control cultures (cc) and antigen-stimulated cultures (vsa or slca-stimuli in vitro). pearson correlation analysis was further applied to evaluate the relationship between spleen parasite burden and no profiles. additionally, the cytokine networks were assembled using cytoscape software version . . (institute of systems biology, seattle, usa), for each experimental group ("control", "sal", "lbsal", and lbsapsal) in all times analyzed (t , t rd , t , and t ), based on the correlations indices obtained by pearson correlation analysis. the network constructed using distinct edges between nodes to identify negative or positive correlations, referred as moderate ( . <r> . ) or strong (r> . ). in all cases, the significance were considered at p< . . "lbsapsal" immunization induced increased levels of ifn-γ before and after experimental l. chagasi-challenge aiming to verify whether the immunization protocols were able to induce the production of pro-inflammatory cytokines, we evaluated the profiles of tnf-α, ifn-γ, and il- in cc or upon vsa or slca-stimuli in vitro. no significant differences were observed amongst the experimental groups at baseline (t ) ( fig a, b and c, upper panel) . data analysis performed at (t rd ) post-vaccination did not show any differences in the tnf-α levels amongst the experimental groups, regardless the culture conditions. analysis of il- demonstrated that the "lbsapsal" group displayed increased levels (p< . ) upon vsastimulation as compared with the same cultures in the "control", "sal", and "lbsal" groups ( fig b, middle panel) . moreover, the "lbsal" group showed reduced il- levels (p< . ) upon slca-stimulation as compared with the "control" group ( fig b, middle panel) . interestingly, the analysis of ifn-γ showed that "lbsapsal" group presented increased levels (p< . ) in cc as compared to the "control" group ( fig c, middle panel) . additionally, "lbsapsal" group displayed increased ifn-γ levels (p< . ) upon vsa and slca-stimuli in comparison with "control", "sal", and "lbsal" groups ( fig c, middle panel) . analysis of the cytokine profile early after l. chagasi-challenge (t ) demonstrated that "lbsapsal" group showed a significant increase of tnf-α levels (p< . ) upon vsa-stimulation as compared to "sal" and "lbsal" groups ( fig a, middle panel) . no differences in the il- levels were observed amongst the experimental groups, regardless the culture conditions. analysis of ifn-γ, "lbsapsal" group displayed increased levels (p< . ) upon vsa-stimulation as compared to "control" and "lbsal" groups at t (fig c, middle panel) . additionally, "lbsapsal" group showed a significant increase of ifn-γ (p< . ) upon slca-stimulation as compared with the "control" group ( fig c, middle panel) . data analysis late after l. chagasi-challenge (t ) demonstrated that the "lbsal" group displayed increased tnf-α levels (p< . ) upon slca-stimulation as compared with the "control" group ( fig a, bottom panel) . in addition, the "lbsapsal" group displayed higher levels of tnf-α (p< . ) upon vsa-stimulation as compared with "lbsal" group ( fig a, bottom panel) . no differences in the il- levels were observed amongst the experimental groups, regardless the culture conditions. interestingly, the analysis of ifn-γ revealed that "lbsapsal" and late ( days-t ) after experimental l. chagasi-challenge. the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars). the x-axis displays the different experimental groups ("control", "sal", "lbsal", and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the cytokine levels (pg/ml) for tnf-α (a), il- (b) and ifn-γ (c). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) between the cc, vsa or slca-stimulated cultures. the symbols c, sal and lbsal indicate significant differences in comparison to the "control", "sal" and "lbsal" groups, respectively. group showed higher levels (p< . ) upon vsa-stimulation as compared with the "control", "sal" and "lbsal" groups ( fig c, bottom panel) . "lbsapsal" induced lower levels of il- and tgf-β but while unvaccinated dogs presented higher amounts of il- after l. chagasichallenge aiming to evaluate whether the immunization protocols would induce regulatory/anti-inflammatory cytokines, we further quantified the levels of il- , il- , and tgf-β upon vsa or slca-stimuli in vitro. no significant differences were observed amongst the experimental groups at baseline (t ) (fig b, upper panel) . the results observed at the post-vaccination period (t rd ) demonstrated that the "lbsal" group showed a significant reduction in the il- levels (p< . ) upon vsa-stimulation as compared to the "sal" group ( fig b, middle panel) . no differences in il- and tgf-β levels were observed amongst the experimental groups, regardless the culture conditions. data mining performed early after l. chagasi-challenge (t ) revealed no differences in the il- production amongst the experimental groups, regardless the culture conditions. however, "lbsapsal" group showed a significant reduction of il- levels (p< . ) upon slca-stimulation as compared to the "sal" group (fig a, middle panel) . interestingly, "lbsapsal" group also presented a significant reduction of tgf-β levels (p< . ) upon slca-stimulation as compared the "control" group (fig , middle panel) . analysis carried out late after l. chagasi-challenge (t ) did not differences in il- levels amongst the experimental groups, regardless the culture conditions. the results showed that the "lbsal" group presented a significant reduction in the il- levels (p< . ) upon vsa-stimulation as compared with the "sal" group ( fig b, bottom panel) . interestingly, "lbsapsal" group displayed a significant reduction of tgf-β levels (p< . ) upon slca-stimulation as compared with the "control" group (fig , bottom panel) . the hallmark of the cytokine network in the "lbsapsal" group is a balanced immune response regarding positive correlation between proinflammatory cytokines (il- , ifn-γ, tnf-α) and regulatory cytokines (il- ) aiming to identify the overall balance of the evaluated cytokines, we have applied machinelearning approaches to analyze the cytokine network for all immunization protocols generated upon slca and vsa-stimuli in vitro (s fig and fig ) . the cytokine balance was calculated as the ifn-γ/il- and ifn-γ/il- ratio for all experimental groups and culture conditions and data provide in the s fig. the results re-enforce that ability of "lbsapsal" vaccine to induce a long lasting ifn-production over the synthesis of both il- and il- , regardless the antigen-stimuli (s fig). data mining by system biology tools were used to generate biomarker networks for each experimental group using both vsa and slca-stimuli. the results demonstrated that upon vsa-stimulation, the "control" group presented negative correlations between il- with tnfα and il- (vsa stimulus). positive correlation was also observed between tnf-α and ifn-γ. upon slca-stimulation, positive correlation was observed between il- and ifn-γ along with a negative correlation between tnf-α and il- (fig , upper left panel) . the "sal" group presented, upon vsa-stimulation, a range of positive correlations, including tnf-α with il- , ifn-γ and il- along il- with il- . in addition, negative correlation was observed between ifn-γ and il- . analysis upon slca-stimulation demonstrated data were analyzed at baseline before vaccination (t ), days after third immunization dose (t rd ) as well as early ( days-t ) and late ( days-t ) after experimental l. chagasi-challenge.the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars). the x-axis displays the different experimental groups ("control", "sal", "lbsal", and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the cytokine levels (pg/ml) for il- (a) and il- (b). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) between the cc, vsa or slca-stimulated cultures. the symbol sal indicates significant differences in comparison to the "sal" group. positive correlations amongst the pro-inflammatory cytokines (tnf-α, ifn-γ and il- ) along positive correlation of them with il- (fig , upper right panel) . analysis of the "lbsal" group upon vsa-stimulation demonstrated a single negative correlation between tnf-α and il- (fig , bottom left panel) . "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars).the xaxis displays the different experimental groups ("control" and "lbsapsal") according to the in vitro stimuli (control culture [cc] and slca). the y-axis represents the tgf-β levels (pg/ml). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) between the cc or slca-stimulated cultures. the symbol c indicates significant differences in comparison to the "control" group. data analysis demonstrated that upon vsa-stimulation, the "lbsapsal" group showed strong positive correlation among tnf-α and ifn-γ along with positive correlation between tnf-α and il- . negative correlations were also observed amongst tnf-α and ifn-γ with il- (fig , bottom right panel) . moreover, upon slca-stimulation, positive correlation was observed between il- with ifn-γ and il- (fig , bottom right panel) . a sustained prominent reduction in spleen parasite load was observed in "lbsapsal" group later on after l. chagasi-challenge the parasitological analysis performed in spleen samples later on (t ) following l. chagasichallenge and reported as number of l. chagasi organisms/ ng of total dna in spleen samples and presented in table . data analysis demonstrated that all vaccination protocols were able the groups are represented as follows: "control"; "sal" (salivary glands of lutzomyia longipalpis); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands) and "lbsapsal" (antigen of l. braziliensis plus saponin and lutzomyia longipalpis salivary glands). the letter "a" indicate significant difference in relation to the sal, lbsal and lbsapsal groups. reduction (%) in parasite load was calculated as the proportion of number of amastigotes organisms/ ng of total dna observed in "sal", "lbsal" and "lbsapsal" groups in relation to "control" group. no clinical signs and mortality were observed throughout the experimental design. doi: . /journal.pone. .t to induce a reduction in the splenic parasite load as compared to the "control group". indeed, "sal" and "lbsal" groups yielded . % and . % of reduction in the spleen parasite load ( . and . amastigotes/ ng of total dna, respectively) as compared to the "control" group ( . amastigotes/ ng of total dna). "lbsapsal" groups showed the lowest parasite load ( . amastigotes/ ng of total dna), leading to . % of reduction rate in relation to the "control" group (table ) . moreover, no clinical signs and mortality were observed throughout the experimental design (table ) . "lbsapsal" group showed after l. chagasi-challenge enhanced no production with negative association with the spleen parasite load the levels of nitric oxide produced by pbmcs were evaluated upon vsa or slca-stimuli in vitro early (t ) and late (t ) after l. chagasi-challenge. data analysis carried early (t ) after l. chagasi-challenge, demonstrated that the "lbsap-sal" group presented upon control culture condition enhanced no levels (p< . ) as compared to the "sal" group ( fig , upper left panel) . additionally, "sal", and "lbsapsal" groups showed increase in the no levels (p< . ) upon vsa-stimulation as compared to the "control" and "lbsap" groups ( fig , upper left panel) . the analysis performed late (t ) after l. chagasi-challenge showed the "lbsapsal" group displayed higher no levels (p< . ) upon slca-stimulation as compared to the "control" group ( fig , upper right panel) . additional analysis revealed that the no production at t displayed a negative correlation with the spleen parasite load selectively upon slca-stimulation (fig , bottom right panel) . interestingly, it was observed that in the "lbsapsal" group, four out of five animals ( %) showed simultaneous high no production and low spleen parasite load, compatible with a protection profile (fig , bottom right panel) . in contrast, in the "control" group, only one out of five ( %) showed high no production but all animals presented high spleen parasitism (fig , bottom right panel) . the control of leishmania chagasi (syn. leishmania infantum) infection in dogs is essential to stop the current spread of zoonotic visceral leishmaniasis. therefore, a vaccine against vl would be an important tool for controlling cvl and would dramatically decrease anxiety regarding l. chagasi infection in humans [ , ] . in this sense, the establishment of biomarkers of immunogenicity is considered critical in the rational approach for analyzing candidate vaccines against cvl, and it contributes to identifying the pattern of immune response in dogs and the search for vaccine candidates against cvl [ , ] . for this reason, in this work, the immunogenicity and protective effects of the "lbsapsal" vaccination in dogs were investigated using levels of no and cytokines and evaluations related to the spleen parasite load. furthermore, the addition of sand fly saliva extract to vector-based vaccines can enhance the ability of the host to control or block leishmania infection [ , ] . the major results observed after "lbsapsal" immunization revealed a reduction in il- levels during the early (t ) post-challenge period. importantly, previous studies have identified that the presence of il- in splenocytes from dogs naturally infected with l. chagasi would be an important biomarker during ongoing cvl [ , ] . however, in some reports, the role of il- was related to the resistance profile or susceptibility profile of cvl [ , ] ; the higher levels of il- are considered the hallmark of dogs naturally infected with l. chagasi [ ] and sustained reduction of il- has been also reported by other immunogen candidates. in fact, reduction in the levels of il- after vaccine immunization against cvl has been considered a biomarker for protection against leishmania infection [ ] . in the present study, the evaluation of il- demonstrated a possible association with events related to the susceptibility to infection by l. chagasi; the "control" group has presented increased amounts during the late (t ) post-challenge period. in fact, this cytokine has been fig . impact of distinct immunization protocols on the no production elicited early and late after l. chagasi-challenge. no levels (μm) were determined in supernatants from pbmcs cultures maintained upon vaccine-soluble antigen (vsa) or soluble leishmania chagasi antigen (slca) stimuli in vitro. data were analyzed early ( days-t ) and late ( days-t ) after experimental l. chagasi-challenge. the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars). top panels: the x-axis displays the different experimental groups ("control", "sal", "lbsal" and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the nitrite levels [μm] . data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) between the cc, vsa or slca-stimulated cultures. the symbols c, sal and lbsal indicate significant differences in comparison to the "control", "sal" or "lbsal" groups, respectively. bottom panels: correlation between no levels and spleen parasite load (# amastigotes/ ng of total dna) at t considering cc (bottom left panel) or the presence of a stimulus (vsa: bottom middle panel; or slca: bottom right panel) in all groups. the groups are distinguishable by colors as follows: as follows: "c" (white circles); "sal" (ligh gray circles); "lbsal" (dark gray circles) and "lbsapsal" (black circles). the quadrants represented in the bottom panels delimit the low and high no producers (y-axis) and the low and high spleen parasite load (x axis). associated with severity of vl [ ] and cvl [ , , , , , ] . importantly, the "lbsapsal" group did not have increased il- production, even after the late l. chagasi-challenge period. additionally, analysis of tgf-β revealed reduced production in the "lbsapsal"-immunized group during the early and late post-challenge periods. interestingly, the presence of tgf-β has been associated with inducing immunosuppression characteristics during the course of vl [ , ] . moreover, the presence of tgf-β in vitro has a protective effect for amastigotes in macrophages, favoring the maintenance of parasitism [ ] . in addition, alves et al. [ ] reported high levels of tgf-β associated with increased parasite load in lymph nodes from symptomatic dogs and concluded that tgf-β is associated with morbidity in cvl. in this context, it is possible to hypothesize that lower levels of tgf-β in the "lbsapsal" group would indicate the establishment of immunoprotective mechanisms, whereas higher amounts in the "control" group would be associated with the susceptibility pattern after l. chagasi-challenge. analysis of pro-inflammatory cytokines has been considered a prerequisite for composing immunogenicity analyses before and after experimental challenge with l. chagasi in clinical trials of anti-cvl vaccines [ , ] . the analysis of tnf-α before vaccine immunization (t ) demonstrated increased levels in slca-stimulated cultures in the "control" and "lbsapsal" groups and demonstrated a tendency for enhanced amounts in the "sal" and "lbsal" groups. similar results were observed for il- and ifn-γ at t . these results seem to indicate that slca stimulation would induce increased levels of these cytokines, pointing to an inherent feature of the antigenic stimulus. for this reason, we evaluated all groups and analyzed the same stimulus in each group to identify a cytokine profile regarding type i immune response. this strategy demonstrated enhanced levels of il- and ifn-γ post-vaccination and sustained production of both tnf-α and ifn-γ even after early and late post-challenge periods in the "lbsapsal" group. some studies have described tnf-α as being associated with a resistance profile in cvl [ , , , , ] or in dogs vaccinated against cvl [ ] , and has been associated with susceptibility when associated with high levels of il- and il- [ ] . results obtained by strauss-ayali et al. [ ] showed that after stimulation with exogenous il- , pbmcs from l. infantum−infected dogs were able to reverse an apparent state of anergy, resulting in increased production of ifn-γ. moreover, menezes-souza et al. [ ] found that low levels of il- concomitant with high levels of il- and tgf-β represent a favorable condition for the persistence and replication of parasites in cvl. it has been described that ifn-γ is linked to a resistance profile in different experimental models for vl [ , , , , ] and in dogs vaccinated against cvl [ , , , , ] . our data regarding the cytokine network indicated a balanced immune response in the "lbsapsal" group. in this sense, we describe positive (tnf-α versus ifn-γ and il- ; il- versus ifn-γ and il- ) and negative (il- versus tnf-α or ifn-γ) correlations demonstrating a prominent pro-inflammatory immune response in the "lbsapsal" group. in addition to a non-significant increase in il- or il- levels in the "lbsapsal" group, a balanced immune response was demonstrated by taking into account the positive correlations between il- and type i cytokines (ifn-γ, il- , tnf-α). these data should be related to the regulation of the prominent pro-inflammatory immune response induced by the "lbsapsal" vaccination and should aim to control potential tissue damage by type i cytokines. the parasitological evaluation revealed in "lbsapsal" group a remarkable reduction in spleen parasite load ( . %) after experimental l. chagasi-challenge, in concordance with a prominent pro-inflammatory immune response induced by this vaccine. in fact, we have been described a parasite load reduction of % in the lbsapsal group [ ] , indicating the capacity of this vaccine to control parasite replication even long after challenge ( days). sand fly saliva displays an important role in the first steps of leishmania infection, due to the vast repertoire of pharmacologically active molecules that surround host's hemostatic system [ ] . the re-exposure to the salivary components seems to display an immunogenic activity, eliciting antibody production and cell mediated immunity by the host that could be block or limit the leishmania infection [ ] . some studies have been shown that l. longipalpis salivary proteins induce an immune response associated with protection in dogs [ , ] . furthermore, in a hamster model, salivary proteins of a sand fly protects against the fatal outcome of visceral leishmaniasis [ ] . similarly, we observed . % parasite load reduction in "sal" group, showing that salivary components have a high potential to limit infection in dogs. the experimental challenge in vaccine studies against canine visceral leishmaniasis is considered as crucial to analyze the protection performance. distinct studies have been published aiming to determine the experimental l. chagasi-challenge plus sand fly saliva using intradermal route in dogs would be more similar to natural infection than intravenous challenge [ , ] . however, using intradermal challenge, the dogs would be asymptomatic during all the study, besides to present lower parasitism [ ] . since the "lbsapsal" vaccine presents saliva as antigenic compound, the ideal experimental challenge to test the protection should ideally be performed by intradermal route, as analyzed in our study. importantly, we observed increased no levels in the "lbsapsal" group during the early and late post-challenge periods. interestingly, four out of five dogs immunized with "lbsapsal" presented higher no amounts and low spleen parasite burden during the late post-challenge period, indicating long-term immunogenicity and resulting in reduction of parasitism. in fact, we have previously demonstrated that "lbsapsal" induced resistance biomarkers specifically related to expansion of circulating cd + and cd + t-cells and leishmania-specific subsets and lower levels of parasitism [ , ] . panaro et al. [ ] also observed an increase in no production and anti-leishmanial activity of macrophages, as well as increased levels of ifn-γ in pbmcs supernatants, in dogs immunized with a vaccine comprising crude antigens of l. infantum. taken together, our major data indicate that immunization with "lbsapsal" is able to induce a protection profile characterized by enhanced amounts of type i (tnf-α, il- , ifnγ) cytokines and reduction in type ii cytokines (il- and tgf-β), even after experimental challenge. the establishment of a polarized type i immune response after "lbsapsal" immunization supported increased levels of no production, favoring a reduction in parasitism and indicating long-lasting protection against l. chagasi infection. these results encourage further studies that can provide important information for a better understanding of the effectiveness of the "lbsapsal" vaccine and strategies for addressing leishmania antigens in combination with sand fly proteins such as those present in the saliva in the vector. supporting information s fig. impact of distinct immunization protocols on the pro-inflammatory/regulatory cytokine balance. the balance of inflammatory cytokine ifn-γ and regulatory/anti-inflammatory (il- and il- ) were analyzed in the supernatant of pbmcs maintained upon vaccinesoluble antigen (vsa) or soluble leishmania chagasi antigen (slca) stimuli in vitro. data were analyzed early ( days-t ) and late ( days-t ) after experimental l. chagasi-challenge. the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars).the x-axis displays the different experimental groups ("control", "sal", "lbsal" and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the cytokine ratio (ifn-γ/il and ifn-γ/il- ). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p < . ) amongst the cc, vsa or slca-stimulated cultures. the symbols c and sal indicate significant differences in comparison to the "control" or "sal" groups, respectively. 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high antibody production in balb/c mice caused by natural exposure to lutzomyia longipalpis bites a follow-up of beagle dogs intradermally infected with leishmania chagasi in the presence or absence of sand fly saliva experimental infection of dogs with leishmania and saliva as a model to study canine visceral leishmaniasis nitric oxide production by macrophages of dogs vaccinated with killed leishmania infantum promastigotes the authors are grateful for the use of the facilities at cebio, universidade federal de minas gerais, rede mineira de bioterismo (fapemig) and universidade federal de ouro preto. this work was supported by fundação de amparo a pesquisa do estado de minas gerais, bra- key: cord- -w hemznv authors: jans, jop; elmoussaoui, hicham; de groot, ronald; de jonge, marien i.; ferwerda, gerben title: actin- and clathrin-dependent mechanisms regulate interferon gamma release after stimulation of human immune cells with respiratory syncytial virus date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: w hemznv background: respiratory syncytial virus (rsv) can cause recurrent and severe respiratory tract infections. cytoskeletal proteins are often involved during viral infections, either for cell entry or the initiation of the immune response. the importance of actin and clathrin dynamics for cell entry and the initiation of the cellular immune response against rsv in human immune cells is not known yet. the aim of this study was to investigate the role of actin and clathrin on cell entry of rsv and the subsequent effect on t cell activation and interferon gamma release in human immune cells. methods: peripheral blood mononuclear cells and purified monocytes were isolated from healthy adults and stimulated in vitro with rsv. actin and clathrin dynamics were inhibited with respectively cytochalasin d and chlorpromazine. t cell receptor signaling was inhibited with cyclosporin a. flow cytometry was used to determine the role of actin and clathrin on cell entry and t cell activation by rsv. enzyme-linked immunosorbent assays were used to investigate the contribution of actin and clathrin on the release of interferon gamma. results: cell entry, virus gene transcription and interferon gamma release are actin-dependent. post-endocytic processes like the increased expression of major histocompatibility complex ii on monocytes , t cell activation and the release of interferon gamma are clathrin-dependent. finally, t cell receptor signaling affects t cell activation, whereas soluble interleukin is dispensable. conclusion: analysis of cell entry and interferon gamma release after infection with rsv reveals the importance of actin- and clathrin-dependent signaling in human immune cells. insights into the cellular biology of the human immune response against respiratory syncytial virus will provide a better understanding of disease pathogenesis and may prove useful in the development of preventive strategies. respiratory syncytial virus (rsv) is a negative-sense single stranded rna virus of the family paramyxoviridae and is a major burden on the current health care system. in healthy adults, rsv infections are limited to the upper respiratory tract, but remarkably do not generate long-term immunity [ ] . in children and elderly, rsv can cause severe lower respiratory tract infections requiring admission to an intensive care unit in a small percentage of cases. the first line of defense against rsv infection consists of epithelial cells. upon infection, epithelial cells attract antigen-presenting cells, including dendritic cells and monocytes. monocytes and macrophages are able to engulf pathogens leading to antigen-presentation. the monocytic cell is one of the major immune cell types that is susceptible to rsv infection and the role of monocytes and macrophages in the pathogenesis of rsv infections has been appreciated for decades [ ] [ ] [ ] [ ] [ ] [ ] . during rsv infection in mice, the recruitment of monocytes from the bloodstream limits viral replication and reduces disease severity [ ] . viral particles can interact with receptors at the membrane of monocytes resulting in attachment, uptake and initiation of the immune response [ ] [ ] [ ] . under many circumstances, actin or clathrin are essential for receptor-mediated internalization [ ] [ ] [ ] [ ] [ ] . internalization can be regulated differentially dependent on the cell type. uptake of transferrin occurs clathrindependent in macrophages and is not dependent on clathrin in epithelial cells [ ] . cell-specific differences in entry mechanisms between epithelial cells and fibroblasts have been shown for human cytomegalovirus [ ] . previous studies have studied the internalization of rsv in epithelial cells [ ] [ ] [ ] [ ] . no data is available regarding cell entry of rsv in monocytes, which raises the question whether internalization of rsv occurs differentially in innate immune cells. after internalization, immune cells are involved in antigen-presentation, t cell activation and the production of cytokines like interferon gamma (ifn-γ). ifn-γ, a type ii interferon, plays a critical role in the immune response against viral infections [ ] . t cell activation may occur through cytokines like interleukin or through stimulation of the t cell receptor (tcr). the relationship between cell entry, t cell activation and subsequent release of ifn-γ during rsv infection in primary human cells is unknown. peripheral blood mononuclear cells (pbmcs) provide a useful model to investigate the impact of cellular pathways on antiviral immunity. pbmcs contain important cells that reflect the immune response against rsv like dendritic cells, monocytes and t cells [ , [ ] [ ] [ ] [ ] . in this study, we aimed to investigate the regulation of ifn-γ by actin-and clathrin-dependent mechanisms after stimulation of human immune cells with rsv. for this, we used pharmacological inhibitors to inhibit actin and clathrin. hereby, the contribution of actin-and clathrindependent processes on cell entry, t cell activation and induction of ifn-γ in primary human immune cells during rsv infection was studied. we first examined the dynamics of cell entry of rsv into cd + monocytes by using pharmacological inhibitors. cytochalasin d (cytod) and wiskostatin (wisko) have been used in previous literature to inhibit actindependent entry and chlorpromazine (cpz) for clathrindependent entry [ ] [ ] [ ] . a representative figure of the gating strategy to determine internalization of rsv into monocytes is shown (fig. a) . disruption of actin filaments in monocytes with cytod or wisko significantly reduces the internalization of rsv whereas inhibition of clathrin with cpz has no effect on the internalization indicating that cell entry is an actin-dependent process (fig. b) . extracellular binding of rsv on membrane of monocytes occurred and is not reduced by pre-treatment of monocytes with cytod or cpz (fig. c) . to confirm the inhibitory effect of cytod on cell entry, pbmcs were stimulated with rsv for h and virus gene transcription was measured in monocytes. treatment of pbmcs with cytod abrogates virus gene transcription in monocytes whereas cpz has no effect (fig. d) . mean percentages of internalization, binding and virus gene transcription in untreated monocytes were respectively %, % and %. previous literature shows that t cells are the main producers of ifn-γ in our model of pbmcs and stimulation with rsv [ ] . to determine whether the adaptive immune response against rsv is actin-dependent, the ifn-γ release was measured after stimulation of pbmcs with rsv. inhibition of actin with cytod abrogates the rsvinduced ifn-γ release (fig. a) . although cpz has no effect on the internalization of rsv, inhibition of clathrin reduces the release of ifn-γ (fig. b) . both cytod and cpz have no effect on t cell activation after stimulation with the non-specific t cell activator phytohaemagglutinin (pha) (fig. a-b) . from this, we conclude that both cell entry and post-endocytic processes play a role in the adaptive immune response against rsv whereas mere binding of rsv to the cell membrane of monocytes is not sufficient to induce ifn-γ release. to address the discrepancy between clathrin-independent internalization and clathrin-dependent ifn-γ release, we examined whether post-endocytic processes like t cell activation and upregulation of antigen-presenting molecules are clathrin-dependent. cd was used as an activation marker on t cells [ ] . pha was used as positive control for t cell activation. rsv induces activation of cd and cd t cells and pre-treatment of pbmcs with cpz prevents t cell activation after rsv infection ( fig. a-b) . to investigate whether the upregulation of antigen-presenting molecules is inhibited by cpz, the expression levels of mhc-i and mhc-ii on monocytes was determined. rsv induces a significant increase of mhc-i and mhc-ii expression on monocytes ( fig. c-d) . upregulation of mhc-i expression is not affected by treatment of pbmcs with cpz (fig. c) . the rsv-induced upregulation of mhc-ii expression is not present after pre-treatment of pbmcs with cpz indicating that upregulation of mhc-ii is clathrin-dependent (fig. d) . to confirm the role of antigen-presenting molecules and thereby tcr signaling as its ligand, we evaluated whether il- , as a soluble t cell activator, or tcr signaling affects the ifn-γ release after rsv infection. neutralization of il- with il- bp does not inhibit rsv-induced ifn-γ release. as a control, we show that il- bp is able to significantly inhibit candida-induced ifn-γ response, which is consistent with previous literature ( fig. a ) [ ] . the release of ifn-γ is inhibited when tcr signaling is blocked with cyclosporin a (csa) (fig. b ). internalization and subsequent virus gene transcription of rsv in human monocytes and the induction of ifn-γ are actin-dependent. although clathrin is not involved in fig. cell entry and subsequent virus gene transcription of rsv in monocytes is actin-dependent. a gating strategy to determine the amount of internalization of rsv in monocytes. b monocytes were pre-treated with rpmi, cytod ( μg/ml), wisko ( μm) or cpz ( μg/ml). after pre-treatment, monocytes were incubated with rpmi or rsv at moi for h at °c and internalization was measured. c monocytes were pre-treated with rpmi, cytod ( μg/ml), or cpz ( μg/ml). after pre-treatment, monocytes were incubated with rpmi or rsv at moi for h at °c and binding was measured. d pbmcs were pre-treated with rpmi, cytod ( μg/ml) or cpz ( μg/ml). after pre-treatment, pbmcs were incubated with rpmi or rsv at moi for h and virus gene transcription was measured in monocytes. one-way analysis of variance with bonferroni's comparison test was used for statistical analysis. data are normalized to untreated cells and depicted as mean ± sem (n = - ). (**p < . , ***p < . ) (ns = no significant difference between untreated and cytod-treated cells or between untreated and cpz-treated cells) fig. ifn-γ release after rsv infection is actin-and clathrin-dependent. a-b pbmcs were pre-treated with rpmi, cytod ( μg/ml) or cpz ( μg/ml). after pre-treatment, pbmcs were incubated with rpmi, rsv at moi or pha ( μg/ml) for h and ifn-γ release was measured. lower limit of detection: pg/ml. wilcoxon-signed rank test was used for statistical analysis. data are mean ± sem (n = - ). (*p < . ) (nd = non-detectable, ns = no significant difference between untreated and cytod-treated cells or between untreated and cpz-treated cells) the internalization of rsv, upregulation of mhc-ii on monocytes, t cell activation and the release of ifn-γ after rsv infection are clathrin-dependent processes. finally, t cell receptor activation contributes to the rsv-induced ifn-γ response whereas the production of il- is dispensable. actin is required for cell entry of rsv in epithelial cells [ ] . we have shown for the first time, that primary human monocytes also require actin to internalize rsv for subsequent virus gene transcription to occur. actin plays an essential role in processes involving internalization, including receptor-mediated phagocytosis. for the nipah virus, a paramyxovirus like rsv, infection induces the co-internalization of nipah virus receptor ephrinb and, in general, many receptors are internalized in an actin-dependent manner [ ] [ ] [ ] . several receptors are involved in the recognition of rsv at the membrane, like toll-like receptor (tlr ), cd and nucleolin [ , ] . internalization of tlr and cd has been investigated upon bacterial ligand stimulation. in this process, internalization of tlr is dynamin-dependent and cd is actin-dependent [ , ] . clustering of nucleolin is dependent on actin [ ] . whether actin-dependent clustering of nucleolin occurs after rsv infection and whether it is necessary for internalization is unknown. our finding that intact actin filaments are required for internalization of rsv in monocytes narrows the scope of potential receptors that co-occur with cell entry and are required for internalization. besides cell entry, intact actin is required for virus filament formation, viral transmission and the production of cell-associated infectious virus by epithelial cells [ ] [ ] [ ] [ ] . we observed an inhibitory effect of cytod on the release of ifn-γ during an incubation period of h. inhibition of post-endocytic processes like virus filament formation and transmission could therefore explain the observed reduction of ifn-γ after treatment with cytod. however, in our model of pbmcs, monocytes exhibit abortive infection as no replicating virus is detected in the supernatant of pbmcs stimulated with rsv (data not shown). this is in line with the abortive infection of rsv in alveolar macrophages [ ] . therefore, the role of actin on virus filament formation and viral transmission may be applicable to epithelial cells but most likely would not play a role in our model. after inhibition of actin with cytod or wisko, approximately % of the monocytes still remain positive for rsv f protein. permeabilization of the cells in our assay will not exclude extracellular staining of rsv f protein and therefore the remaining rsv-positive monocytes after treatment could reflect extracellular binding of rsv. because cytod does not influence the extracellular binding of rsv, we conclude that actin dynamics are involved in the internalization of rsv by monocytes. actin-independent processes could also explain the incomplete prevention of internalization by cytod. fusion of the f protein of rsv with the cell membrane could result in entry of viral proteins and might act independently of actin filaments. clathrin has previously been implicated for internalization of rsv in an epithelial cell line after an incubation period of h [ ] . contrary, others have shown that internalization of rsv after h is not clathrin-dependent [ ] . the authors conclude that discrepancies between these studies may arise due to a difference in experimental design. the incubation period of h in our study excludes inhibition of postendocytic processes like viral replication and cell-to-cell transmission. in addition, differences between epithelial cells and monocytic cells have been described previously for internalization of nanoparticles and may explain our results compared to epithelial cells [ ] . the role of cellular mechanisms on t cell activation can be studied in our model of pbmcs. besides internalization, inhibition of actin dynamics reduces the ifn-γ response. these data suggest that, at least in part, the intracellular compartment is involved in the induction of an adaptive response and binding of rsv to the outer membrane is not sufficient to induce ifn-γ release. these results are in line with previous data indicating that intact rsv particles are not able to signal via membrane-associated receptors such as tlr [ ] . in our study, inhibition of clathrin has no effect on the internalization of rsv by monocytes, but effectively reduces the induction of ifn-γ. cpz at μg/ml was the highest tolerable concentration without inducing cytoxicity (data not shown). our data suggest that activation of t cells by rsv is clathrin-dependent. calabia-linares et al. observed the importance of clathrin in the formation of the immunological synapse between t cell and antigen-presenting cells [ ] . possibly, inhibition of clathrin reduces the ifn-γ release due to improper formation of the immunological synapse and thereby inefficient t cell activation. less than % of the t cells in the peripheral blood are rsv-specific t cells [ , ] . therefore, the relative high percentage of - % cd + t cells after stimulation with rsv in our experiments most likely indicates that a general activation of t cells occurs. we demonstrate that clathrin plays an essential role in the upregulation of rsv-induced mhc-ii expression on monocytes . the requirement of clathrin for mhc-ii trafficking has been observed previously [ ] . the role of mhc-ii in the induction of ifn-γ is strengthened by the observation that inhibition of tcr signaling, as a ligand for mhc-ii, reduces the ifn-γ response, whereas inhibition of il- has no effect. the combination of reduced t cell activation and mhc-ii expression suggests that clathrin plays a role in the interplay between innate and adaptive immunity against rsv. fig. ifn-γ release after rsv infection is dependent on t-cell receptor signaling. a il- bp was added to pbmcs simultaneously with rpmi, rsv at moi or heat killed candida. ifn-γ release was measured after h. b pbmcs were pre-treated with rpmi or csa ( nm). after pretreatment, pbmcs were incubated with rpmi or rsv at moi for h and ifn-γ release was measured. lower limit of detection: pg/ml. wilcoxon-signed rank test was used for statistical analysis. data are mean ± sem (n = - ). (*p < . ) (nd = non-detectable) (ns = no significant difference between untreated and cpz-treated cells) the simplification of in vivo rsv infections using our model of pbmcs could be a limitation of the study. although others have shown that monocytes can be become infected with rsv and serve as an in vitro model, alveolar macrophages or inflammatory macrophages are the most likely target of infection in vivo [ ] . therefore, there could be differences in the immune response in vivo compared to our model. pre-treatment of isolated monocytes with the inhibitors and performing a subsequent co-culture with autologous t cells could be considered as an alternative instead of simultaneous culture of all cells present in pbmcs. however, during rsv infection in vivo, multiple immune cells, including monocytes, natural killer cells and t cells are recruited to the lung tissue [ ] . our model can give more insights in the interplay between rsv and different immune cells that are present during rsv infection. in conclusion, this study underlines the importance of actin and clathrin dynamics during rsv infections for cell entry and t cell activation. currently, there is no effective treatment or vaccine against rsv infections. understanding the different aspects of rsv disease pathogenesis and immunity, from early virus-cell interactions to final induction of the adaptive immune response may contribute to the development of novel therapies and effective vaccines. the aim of this study was to investigate the role of actin and clathrin on cell entry of rsv and the subsequent effect on t cell activation and release of ifn-γ in human immune cells. cell entry in human monocytes, virus gene transcription and ifn-γ release are actindependent. post-endocytic processes like the increased expression of mhc-ii on monocytes , t cell activation and the release of ifn-γ are clathrin-dependent. finally, t cell receptor signaling affects the release of ifn-γ, whereas soluble interleukin is dispensable. insights into the cellular biology of the human immune response against rsv will provide a better understanding of disease pathogenesis. actin-dependent pathways were inhibited by pre-treatment of cells with cytod ( μg/ml) or wisko ( μm) and clathrin-dependent pathways were inhibited by cpz ( μg/ ml) (sigma-aldrich). csa (sigma-aldrich) and il- bp (r&d systems, united kingdom) were used to respectively inhibit t cell receptor signaling and il- . no cytotoxic effect determined as annexin v+/ -aad+ cells was observed when using the indicated concentrations of the inhibitor (data not shown). after obtaining informed consent, peripheral blood mononuclear cells (pbmcs) from healthy adults were isolated using lymphoprep and isolation of monocytes from the pbmc fraction was performed using an indirect negative selection magnetic labeling kit (monocyte isolation kit ii human; miltenyi biotec). each -well plate was filled with x pbmc per well or x isolated monocytes per well. the study was approved by the committee on research involved human subjects of the radboudumc. rsv a containing an additional transcription unit encoding gfp (rgrsv) was cultured as previously described [ ] . rgrsv was cultured on hela cells (atcc, ccl- ) in dulbecco's minimum essential medium (dmem) with % fetal calf serum (fcs) and % penicillin/streptomycin. near-confluent hela cells were infected with rgrsv and incubated for three days at °c. cells were scraped and the suspension was centrifuged to remove cellular debris. rgrsv was ultracentrifuged on a sucrose cushion for purification and titrated on hela cells. confluent hela cells ( - %) were infected with fivefold viral dilutions for - h. virus titer was determined by counting wells with ≥ and ≤ infected cells/view (ckx microscope; olympus, tokyo, japan). rgrsv was snapfrozen and stored at − °c until use. pbmcs or isolated monocytes were treated with cytod ( μg/ml), wisko ( μm) or cpz ( μg/ml) for min at °c. for quantification of cell entry, monocytes were permeabilized with cytofix/perm (bd biosciences) after incubation with rsv at moi ( x iu/ well) for h at °c. cells were incubated for min on ice with mouse anti-respiratory syncytial virus fusion protein antibody (ab ; abcam). goat anti-mouse igg pe (bd pharmingen) was used as secondary antibody. for the quantification of rsv binding, coincubations of pbmcs with rsv were performed for h at °c and cells were fixed with % paraformaldehyde before staining. pbmcs were treated with cpz ( μg/ml) for min at °c. pbmcs were stimulated with rsv at moi or with pha ( μg/ml) for h. cd expression, as t cell activation marker, was measured with flow cytometry. for gating, cd and cd or cd positive t cells were selected and the percentage of cd positive cells was determined. after pre-treatment with cpz ( μg/ml), pbmcs were incubated with rsv at moi for h and the expression of mhc-i and mhc-ii on monocytes was determined. for gating, cd positive cells were selected and the geometric mean fluorescence intensity (mfi) of mhc-i and mhc-ii was determined. cell surface markers were stained with cd v , cd af , cd v , cd apc-h , cd pe, cd percp-cy . , cd pe-cy , hla-abc pe and hla-dr pe (bd pharmingen). for quantification of rsv, mouse anti-rsv f protein (ab ; abcam) and goat anti-mouse igg pe (bd pharmingen) were used. virus gene transcription in monocytes was determined by gating cd positive cells as monocytes and calculating the percentage of gfp positive cells. cytoxocity was determined by calculating annexin v+/ -aad+ cells (pe annexin v apoptosis detection kit i, bd pharmingen). events were acquired on an lsr ii flow cytometer and analyzed using flowjo. pbmcs were treated with cytod ( μg/ml), cpz ( μg/ ml) or csa ( nm) for min at °c. after pretreatment, cells were stimulated with rsv at moi , pha ( μg/ml) or heat-killed candida albicans ( x / well) for h. il- bp ( μg/ml) was added simultaneously with rsv. concentration of interferon gamma were measured in the cell supernatants by enzymelinked immunosorbent assays (elisa) (sanquin blood supply, the netherlands) with a lower limit of detection of pg/ml. the authors declare that they have no competing interests. author's contributions jj carried out the design of the study, performed the immunoassays and the statistical analyses and drafted the manuscript. he performed the immunoassays and helped draft the manuscript. rdg and mdj participated in the design of the study and helped draft the manuscript. gf carried out the coordination and the design of the study, participated in the statistical analysis and drafted the manuscript. all authors read and approved the final manuscript. immunity to and frequency of reinfection with respiratory syncytial virus respiratory syncytial virus infection of human cord and adult blood monocytes and alveolar macrophages role of monocytes and eosinophils in human respiratory syncytial virus infection in vitro respiratory syncytial virus induced type i ifn 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clathrin and ap- in the trafficking of mhc class ii molecules to antigen-processing compartments antibodies enhance cxcl production during rsv infection of infant and adult immune cells we would like to thank dr. m.e. peeples, ohio state university, for kindly providing us with a transgenic rsv strain expressing renilla-gfp. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -u x jaw authors: abe, takayuki; shapira, sagi d. title: negative regulation of cytosolic sensing of dna date: - - journal: int rev cell mol biol doi: . /bs.ircmb. . . sha: doc_id: cord_uid: u x jaw in mammals, cytosolic detection of nucleic acids is critical in initiating innate antiviral responses against invading pathogens (like bacteria, viruses, fungi and parasites). these programs are mediated by multiple cytosolic and endosomal sensors and adaptor molecules (c-gas/sting axis and tlr /myd axis, respectively) and lead to the production of type i interferons (ifns), pro-inflammatory cytokines, and chemokines. while the identity and role of multiple pattern recognition receptors (prrs) have been elucidated, such immune surveillance systems must be tightly regulated to limit collateral damage and prevent aberrant responses to self- and non-self-nucleic acids. in this review, we discuss recent advances in our understanding of how cytosolic sensing of dna is controlled during inflammatory immune responses. the innate immune system, which is conserved across virtually all multi-cellular organisms on the planet (from sponges to insects, plants, and vertebrates), includes a diverse set of receptors capable of detecting molecular patterns like sugars, lipids, polymers, and nucleic acids that are principal in prompting protective responses (broz and monack, ; kieser and kagan, ; kumar et al., a) . in vertebrates, cytosolic detection of nucleic acids (derived from microbes like viruses, intracellular bacteria, fungi and parasites) is critical in initiating innate (characterized by production of type i ifns) and adaptive (characterized by t and b cell responses) immune responses (wu and chen, ) . recent efforts have focused on identifying relevant immune surveillance sensors and components of downstream signaling-toll-like receptors (tlrs) and their cognate ligands, cytosolic sensing of rna (primary mediated by the rig-i/ips- axis), cytosolic sensing of dna (primary mediated by the cgas/sting axis), and the inflammasome pathway (primary mediated by nod-like receptors; nlrs) (broz and monack, ; kieser and kagan, ; kumar et al., a ). yet, while the identities of immune surveillance systems have been revealed, inflammatory programs mediated by these sensors must be tightly regulated to prevent aberrant and inappropriate responses to selfderived ligands (like self rna and dna) that may be released from damaged cells, senescent cells, apoptotic cells, or during fertilization (barber, ; de oliveira mann and kranzusch, ) . indeed, rig-i, the cytosolic receptor for rna distinguishes self from non-self rna through interaction with a -triphosphate that is unique to viral rna (wu and chen, ) . though exceptions to this requirement have been reported, the ability of rig-i to distinguish self from non-self in this way ensures that anomalous immune responses to cellular rna do not occur. responses to dna are far more agnostic. cgas, in collaboration with sting, does not distinguish between cellular and foreign dna (barber, ; crowl et al., ) . indeed, ht-dna (herring testes-dna), as well as isd (interferon-stimulated dna) oligonucleotides, is known to act as potent stimulators of cgas/ sting in multiple mammalian cell types. it has also been suggested that chronic cgas/sting activation induced by self dna may be responsible for induction of aberrant inflammatory diseases like systemic lupus erythematosus (sle), aicardi-goutières syndrome (ags), and polyarthritis (barber, ; crowl et al., ) . while apoptotic cells represent a possible source of dna, the existence of dnases in cytoplasmic (e.g., dnase-iii, also known as trex ) and lysosomal compartments (e.g., dnase-ii) can clear potential ligands and ensure that inappropriate responses are not initiated (barber, ; crowl et al., ) . in the event of mitochondrial dna (mtdna) release into the cytoplasm (which occurs following mitochondrial damage), intracellular caspase activation controls the aberrant immune response (mcarthur et al., ; rongvaux et al., ; white et al., ) . similarly, cgas/sting and the necessary cofactors, and cellular dna are compartmentalized such that sensing of self-dna is avoided; the receptor in the cytosol and the ligand (dna) in the nucleus (barber, ) . however, recent publications have illustrated that cell cycle progression in the context of dna-damage may lead to the formation of micronuclei which elicit cgas/sting-mediated dna sensing (harding et al., ; mackenzie et al., ) . in addition, cellular dna can serve as a cgas/sting ligand following cellular senescence (defined as the senescence-associated secretory phenotype; sasp) (gluck et al., ; yang et al., ) . induction of sasp factors, like inflammatory cytokines and chemokines, may then reinforce senescent cells via autocrine and paracrine routes. similarly, during fertilization, sperm cell-derived dna can be found in oocyte cytoplasm may serve to activate cytoplasmic nucleic acid sensing (nas) pathway to induce inflammatory responses. thus, there are at least two contexts unrelated to infection where cytosolic responses must be repressed: during mitosis when chromosomal dna naturally exists in cytoplasm, and during fertilization when sperm-derived dna enters oocyte cytoplasm. such control can be achieved through the downregulation of molecules necessary for prompting responses to nucleic acids, and/or the upregulation of a negative regulators that act as molecular safeguards. in this review, we provide an update on cellular machinery that negative controls cytosolic sensing of dna and discuss therapeutic opportunities for the germ-cell specific nlr family member, nlrp , a recently identified inhibitor of dna-sensing during fertilization. in contrast to the identification of molecular machinery responsible for tlr and rna-sensing rig-like receptor (rlr) pathways, identification of a universally accepted cytosolic dna sensor and its related signaling pathway involved a more untidy trajectory (unterholzner, ) . since the existence of a cytosolic sensor for dna was first postulated (one that could elicit tlr-independent production of ifn to transfected double-stranded dna; dsdna) (ishii et al., ; stetson and medzhitov, ) , a great deal of effort has gone into revealing its identity. first, dnadependent activator of irf (dai; also referred to as dlm- /zbp ) was shown to participate in the activation of irf downstream of dsdna sensing (takaoka et al., ) . evidence suggests that following cellular exposure to synthetic dsdna or dna virus infection, dai can form a signaling complex together with tbk and irf to initiate production of type i ifns. however, reports also suggested that cells from dai-deficient mice did not exhibit impaired responses to synthetic b-form dsdna and dna genomes derived from bacteria (ishii et al., ) . ifi (ifn-γ-inducible protein ), a pyhin family member protein, was also shown to be involved in the recognition of synthetic dsdna and dna genomes of nuclearly replicating viruses (e.g., hsv- , kshv, hcmv, and ebv) (unterholzner et al., ) . in addition, ifi was also shown to participate in the dna damage response through promoting apoptosis and senescence, suggesting that such processes may mediate inflammatory diseases (unterholzner, ) . related to ifi is yet another putative cytosolic dna sensor, absent in melanoma (aim ), which, through an inflammasome dependent pathway, induces production and secretion of il- β and il- rather than type-i ifns after dna exposure (burckstummer et al., ; fernandes-alnemri et al., ; hornung et al., ) . more recently, aim -like receptors (alrs), consisting of members possessing a pyrin signaling domain and a dna-binding hin domain, have been shown to not contribute to type i ifn production downstream of dna sensing, nor to autoimmune diseases like as ags which are associated with responses to self-dna (gray et al., ) . the establishment of human ifi -deficient cells further illustrated competent type-i ifn responses to hcmv infection. nevertheless, ifi may participate in the recognition of cytosolic dna in a cell type and/or species-specific manner. an additional putative dna sensor, the ddx helicase, was identified through rnai screening and shown to be involved in dna recognition in immunocompetent cells rather than epithelial cells (zhang et al., ) . shortly after its discovery, ddx mediated signaling was shown to go through the bacterial-derived second messenger cyclic dinucleotide (cdn) molecules cyclic di-amp and cyclic di-gmp (parvatiyar et al., ) . solved crystal structures confirmed that the binding regions for these ligands overlapped, suggesting that ddx can recognize multiple ligands via the dead domain (omura et al., ) . of note, both ifi and ddx have been proposed to act upstream of sting (see below) through physical interactions. yet, their collaborative role in modulating cytosolic sensing of dna requires further investigation using genetically engineered mice. additionally, several dna damage inducible host factors like the catalytic subunit of dna-dependent protein kinase (dna-pkcs), and its binding co-factors ku / , or mre (meiotic recombination ) have also been implicated in cytosolic sensing through direct interactions with dna ligands (ferguson et al., ; kondo et al., ) . importantly, while dna-pkcs appear to have critical roles in dna-mediated immune responses, dna-pkcs deficient cells exhibit normal expression of many isgs upon stimulation with dna and dna virus infection. nonetheless, these observations reinforce the notion that dna damage response induces type i ifn production (brzostek-racine et al., ) . use of dna damage induced agents like , -dimethylbenz-α-anthracene (dmba) has helped shed light on the underlying events initiate the dna damage-induced immune response via cytosolic dna sensing pathway and implicates nucleosome leakage in eliciting cgas/sting-dependent signal activation via recognition of self-dna (barber, ) . finally, lrrfip , cytosolic protein, has been shown to recognize both dsdna and dsrna derived from pathogens, and may control the production of type-i ifns through the transcriptional co-activator β-catenin rather than irf -potentially acting as an amplifier of cytosolic nas (yang et al., ) . collectively, evidence indicates that while the long list of candidate cytosolic dna sensors may have redundant functions, they vary in ligand specificity as well as cell type and tissue distribution. between and , prior to the identification of several candidate dna sensors described the above, barber and colleagues reported that sting (stimulator of ifn genes, also referred as mita, mpys, or eris, and encoded by tmem ), an endoplasmic reticulum (er) localized protein consisting of multiple transmembrane regions, acts as an essential molecule for cytosolic sensing of dna (barber, ; ishikawa and barber, ; ishikawa et al., ) . several dna species appear to trigger sting-dependent signaling via tbk /irf and ikk/nf-κb axis in a length dependent manner and sting has also been attributed with a role in responses to plasmid-based vaccines and induction of long-term immunity. and though evidence suggests that sting may interact directly with dsdna, the physiological relevance of these observations remains to be explored (abe et al., ) . nevertheless, upon ligand stimulation, sting translocates from the er to the perinuclear-golgi region, forms a signaling complex with tbk and leads to phosphorylation and activation of irf . in addition, sting signaling appears to self-regulate by inducing protein degradation through a ubiquitin-mediated proteasome pathway and termination of signal transduction. a subsequent study indicated that sting dimers bind cyclic dinucleotides (cdns), suggesting that sting acts as the direct innate immune sensor for cdns (burdette et al., ) . other reports indicated that the type-i ifn and pro-inflammatory cytokine inducing flavonoid compounds, , -dimethylxanthenone- -acetic acid (dmxaa, also known as vadimezan or asa and initially identified as a potent tumor vasculature disrupting agent in mice), and -carboxymethyl- -acridanone (cma) may trigger sting dependent signal activation through direct interactions (prantner et al., ; roberts et al., ) . however, it was also known that these compounds displayed such sting activating properties in mice but not human cells. consistent with the unfortunate failure of these drugs in human clinical trials, these observations may be explained by structural differences in the dmxaa-binding sites across the human and mouse proteins (gao et al., ) . while the identification of sting undoubtedly contributed to our understanding of the underlying molecular events that control cytosolic dna-mediated immune responses, it was universally accepted that a sensor upstream of sting remained to be discovered. indeed, the breakthrough came in when chen and colleagues uncovered a role for cyclic gmp-amp (cgamp), and the catalytic enzyme (cyclic gmp-amp synthase (cgas), encoded by mb d and c orf ) in response to several cytosolic dna ligands (sun et al., ; wu et al., ) . cgamp is a cdns that consist of varying phosphodiester linkages (a -phosphodiester linkage and a canonical -phosphodiester linkage; the cgamp isomer is known as -cgamp) and cgas undergoes conformational rearrangement following direct binding to dna which leads to the synthesis of cgamp from cellular stores of atp and gtp. cgamp produced by cgas upon dna stimulation in turn acts as a cognate sting ligand to activate signaling and downstream production of type i ifn (barber, ) -it is interesting to note that similarly to other prrs (tlr, rlr and the inflammasome pathway) cgas activation of the sting pathway does not require direct interaction the sensor and adaptor that mediates signal transduction. importantly, the cgas/sting pathway plays a crucial role in the induction of autoimmune and inflammatory diseases (see section ), highlighting the need to properly control the dna sensing pathway for the maintenance of cellular homeostasis and immune response. taken together, while the functional and physiological relevance of several candidate dna sensors (e.g., ddx , ifi , and dna-pkcs) needs to be fully investigated, cgas and sting have emerged as bona fide players in cytosolic sensing of dna (fig. ). intracellular post-translational modifications (ptm), including phosphorylation, ubiquitination, and ubiquitin-like modifications like sumoylation and isgylation, glutamylation, acetylation or methylation, are critical in controlling cellular responses. these modulators of pathway activity participate in signal transduction by tuning enzymatic states, subcellular localization, protein stability and degradation, as well as protein-protein interactions. not surprisingly, recent evidence implicates several ptms in the cgas/sting pathway (summarized in fig. ). trim and trim , both e ubiquitin ligases, can positively regulate sting-dependent signal activation through conjugation of k -linked poly-ubiquitination of sting (trim at position k , and trim at k , k , and k (tsuchida et al., ; zhang et al., ) . similarly, k -linked poly-ubiquitination of sting at four lysine residues (k , k , k , and k ), mediated by e ubiquitin ligase complexes of amfr (autocrine motility factor receptor)-gp /insig (insulin-induced gene ), also leads to positive regulation of sting function (wang et al., ) . more recently, mul (mitochondrial e ubiquitin protein ligase ) was also shown to be responsible for k -linked poly-ubiquitination of sting at k , leading to specific enhancement of irf -dependent signaling but not nf-κb (ni et al., ) . conversely, two distinct ubiquitin ligases, rnf (ring finger protein ) and trim α (tripartite motif containing α), lead to termination of signal activation through k -linked polyubiquitination of sting at k and k which target it to subsequent degradation (wang et al., a; zhong et al., ). epstein-barr virus (ebv), a dna virus, coopts this regulatory property of the dna sensing pathway to evade host innate immune response. ebv induces expression of trim which in turn induces k -linked poly-ubiquitination of sting, and attenuation of sting-mediated antiviral response (xing et al., ) . proper regulation of signal transduction requires the poly-ubiquitin conjugation system to be reversible. indeed, inactive rhomboid protein (irhom ) contributes to stabilize sting protein by recruiting the deubiquitinating enzyme eif s (eukaryotic translation initiation factor subunit ) and de-conjugating rnf -mediated k -linked poly-ubiquitination (luo et al., ) . in addition, it appears that irhom may regulate the translocation of sting in response to dna through recruitment of er translocon-associated protein trapβ, which was previously identified as a sting-interacting protein (ishikawa and barber, ) . conversely, the ubiquitin specific protease (usp ) can counteract k -linked poly-ubiquitination and rnf -mediated k -linked poly-ubiquitination of sting, thereby facilitating sting-mediated signaling (zhang et al., ) . similarly, rnf catalyzes k -linked poly-ubiquitination of sting at the same conjugating site as rnf (k ), thereby preventing rnf -mediated poly-ubiquitination of sting, which positively regulates sting function (qin et al., ) . another ubiquitin specific protease, usp , also participates in this process, suggesting that collaboration between two distinct usps may catalyze de-ubiquitination of sting for signal activation. moreover, usp has also been shown to act as a de-conjugating enzyme of k -linked and k -linked poly-ubiquitination of sting, though the role of k -linked poly-ubiquitination of sting remains to be defined (sun et al., ) . nevertheless, de-ubiquitinated sting fails to form signaling complexes with tbk , thereby suppressing signal transduction, supporting a repressive role for usp . while multiple ubiquitin ligases have been implicated in directly regulating sting protein, much less is known about their modulatory role on cgas function. a recent report illustrated that the ubiquitin ligase rnf acts as a positive regulator of cgas via k -linked poly-ubiquitination at both k and k and may contribute to the enzymatic activity of cgas and promoting synthesis of cgamp (wang et al., a) . it was also reported that rnf expression is elevated in pbmcs of sle patients, suggesting that it could play a role in auto-inflammatory disorders. though an initial report described a role for trim in targeting sting for ubiquitination, it may also conjugate mono-ubiquitin to cgas at k (seo et al., ) . thus, negative regulation of cgas function by a ubiquitin ligase remains to be identified. in short, while the role of multiple lysine (k)-residues in sting protein has been implicated in regulating its activity, it remains unclear how ubiquitination of sting and cgas is regulated to orchestrate productive and appropriate immune responses. additionally, the biological significance and machinery regulating non-degradative poly-ubiquitin linkage (like k and k ) of sting remains to be fully resolved. recent studies have also implicated sumoylation in modulating signaling through the cgas/sting axis. the first report indicated that trim , an e ubiquitin ligase, may promote stabilization of cgas and sting by targeting them for sumoylation (hu et al., ) . in contrast, senp , a sumo-specific protease, was shown to mark these proteins for degradation via proteasomal and chaperone-mediated autophagy pathways. though cgas k and k (which are conserved between mouse and human), and sting k (corresponding to k in murine sting), were shown to regulate this process, a conflicting report suggested that sumoylation at k , k , and k residues of cgas suppressed functions including dna-binding, conformational rearrangement, and enzymatic activity-and that senp may reverse this suppression through catalyzing de-sumoylation of cgas (cui et al., ) . other ptms, including glutamylation, mediated by tubulin tyrosine ligaselike enzymes (ttlls), have also been shown to be involved in modulating cgas-mediated immune responses (xia et al., ) . poly-glutamylation and mono-glutamylation of cgas, mediated by ttll and ttll , respectively, negatively regulate cgas-mediated dna binding and enzymatic activity. conversely, the intracellular carboxypeptidases ccp and ccp counteract mono-glutamylation and poly-glutamylation of cgas, suggesting that cgas function is tightly regulated through cellular glutamylation and de-glutamylation. in addition to glutamylation, recent work also implicates palmitoylation, the covalent attachment of fatty acids (like palmitic acid) to cysteine (c) residues of a substrate proteins, in modulating sting (specifically, at positions c and c ) mediated signaling and type i ifn production downstream of dna sensing (mukai et al., ) . perhaps most recognized and best studied ptm is the phosphorylation of substrate proteins, and sting is no exception. recent studies have shown that sting possesses a number of serine (s) residues in the c-terminal region that can act as potential phosphorylation sites, though the primary phosphorylation site is serine- (s ) (konno et al., ; tanaka and chen, ) . while tbk , whose primary target is irf , was suggested to be involved in the phosphorylation of sting, in vitro experiments hinted otherwise (konno et al., ) . in addition, sting-phosphorylation and downstream degradation appear normal in tbk -deficient mouse embryonic fibroblasts stimulated with sting agonists, suggesting that if tbk does indeed target sting, its role is redundant (abe and barber, ) . subsequent studies reported that the autophagy-related serine/threonine protein kinases ulk and ulk as well as the ribosomal protein s kinase (s k ) are involved the phosphorylation of sting (konno et al., ; wang et al., ) . while multiple candidate sting kinases are being explored, only one has been implicated in directly controlling cgas function, and its identity came out of studies on hsv-host interactions. induced by hsv- , akt, also known as protein kinase b (pkb), phosphorylates cgas at s (s in murine cgas), thereby suppressing the synthesis of cgamp and attenuating cgas/sting-mediated antiviral responses (seo et al., ) . thus, hsv infection has coopted cellular machinery to negatively regulate cgas function and evade immune responses. taken together, while the ptms that control cgas/sting function remain to be fully defined, understanding the underlying network that controls these processes may contribute to the establishment of potent therapeutic approaches for tuning cgas/sting-mediated signaling (see section ). while cytosolic sensing of nucleic acids and induction of innate immune responses is critical for eliminating invading pathogens, inappropriate responses to dna from necrotic or apoptotic cells can result in the development of autoimmune diseases like sle, ags (characterized by high levels of anti-nuclear antibodies (ana) and high levels of circulating proinflammatory cytokines). to limit such aberrant inflammatory responses, vertebrates have evolved mechanisms to tightly control the availability of self dna and regulate inflammatory responses through cgas/sting dependent signaling (fig. ) . for example, mice lacking dnase ii, which acts in macrophages to degrade chromosomal dna derived from apoptotic cells, exhibit embryonic lethality due to anemia induced by the accumulation of undigested dna (kawane et al., ) . rescue of these mice through deletion of the type i ifn receptor, ifnar highlights the pathogenic potential of ifn, though aged mice develop tnfα-mediated severe polyarthritis (kawane et al., ) . additionally, mice lacking the three-prime repair exonuclease , trex (encoded by dnase iii), which degrades nicked dsdna as well as single-stranded dna, exhibit a significantly shortened lifespan due to constitutive activation of pro-inflammatory genes across multiple organs (yang et al., ) . similarly, using genetically engineered mice, dnase i has also been implicated in the development of sle (napirei et al., ) , though its role in nucleic acid sensing remains to be elucidated. importantly, the dramatic phenotypes observed in both dnase ii and trex deficient mice can be reversed through abrogation of cgas or sting gene expression, suggesting that the cgas/sting axis can initiate auto-inflammatory diseases (ahn et al., ; gall et al., ; gao et al., ; gray et al., ) . adding credibility to these studies, mutations in trex have also been implicated in ags and sle patients. furthermore, a recent report suggests that mutations in sting (specifically, n s, v m, and v l) may cause vascular and pulmonary syndrome (vaps), an auto-inflammatory disease characterized by abnormal inflammation across multiple tissues, including skin, vasculature, and lung (liu et al., ) -in vitro interrogation of these mutations revealed that they result in a gain-of-function phenotype and likely contribute to constitutive production type i ifn ( jeremiah et al., ) . furthermore, mutations in rnaseh , which degrades rna/dna hybrids, have also been implicated in the development of ags (crow et al., ) , and mutant mice possessing mutated human allele (g s) die perinatally (pokatayev et al., ) . collectively, these studies and observations highlight the importance of tightly regulating dna sensing and point to intracellular dnases like dnase ii and trex critical gatekeepers in controlling cgas/sting access to self-dna. strong selection pressure, coupled with high mutation rates and short generation times, has led to intricate strategies employed by viruses to counter host immune surveillance and establishment of infection. while the subject is more comprehensively covered by others, here we focus on a few examples from recent reports that highlight the roles of ptms and protein stability in regulating cytosolic sensing of dna (fig. ) . for example, the hepatitis b virus (hbv) polymerase suppresses cytosolic sensing of dna by interfering with k -linked poly-ubiquitination of sting (liu et al., ) . though the clinical significance of this observation remains to be resolved-given that hepatocytes lack functional sting dependent signaling (thomsen et al., ) -the data highlight the role of polyubiquitination in regulating sting function. indeed, other viruses have evolved machinery to modulate k -linked poly-ubiquitination of sting. the human coronavirus (hcov)-nl , severe acute respiratory syndrome (sars) cov, porcine epidemic diarrhea virus (pedv) papain-like protease, hepatitis c virus non-structural protein b (ns b), as well as the tax protein of human t lymphotropic virus type- (htlv- ), all suppress sting through either direct physical interactions or regulation of the polyubiquitination process ding et al., ; nitta et al., ; sun et al., ; wang et al., b; yang et al., ) . other examples include the human papillomavirus (hpv ) derived e protein and human adenovirus type- (had ) derived e a protein, both of which suppress cytosolic sensing of dna through direct interactions with sting (lau et al., ) . an lxcxe motif encoded in both proteins, and conserved among many dna tumor viruses, mediates disruption of sting-dependent signaling, suggesting a possible functional link between oncogenesis and antagonization of sting function. another oncogenic dna virus, kaposi sarcoma-associated herpesvirus (khsv) encodes a viral interferon regulatory factor (virf ) gene capable of preventing dna sensing via direct association with sting (ma et al., ) . additionally, though details remain to be resolved, the murine gammaherpesvirus (mhv ) encoded de-ubiquitination (dub) enzyme orf was shown to antagonize cytosolic sensing of dna (sun et al., ) . in addition to targeting sting (described above), the hcv employs the ns / a protease to target the mitochondrial adaptor ips- for cleavage, thereby short-circuiting the signaling cascade (chan and gack, ) . in a rather exquisite example of host restriction, dengue virus (denv), a mosquitoborne flavivirus which infects hundreds of millions of people annually, encodes the ns b/ns protease which cleaves human but not murine sting (aguirre et al., ) . importantly, as tbk mediated signaling is not suppressed by ns b/ns in murine cells, mice produce higher levels of type i ifns and effectively restrict denv replication. though far fewer examples exist of viral targeting of cgas, recent reports suggest that capsid proteins of hiv- and hiv- may dampen cgas function by recruiting cellular cpsf and cyclophilin-a (lahaye et al., ; rasaiyaah et al., ) . what is clear is that understanding virus-mediated immune evasion strategies can provide critical insights into regulatory functions that control cellular responses to dna in the cytosol. the nucleotide-binding domain leucine-rich repeat-containing receptor (nlr) family contains five subfamilies that are subclassified according to the composition of domains in their n-termini-the nlrc family contains a caspase activation and recruitment domain (card), and the nlrp family contains a pyrin domain (pyd). nlrs are known for their role in triggering caspases to cleave pro-il- β and il- into mature proinflammatory cytokines in response to various pathogen associated molecular patterns (pamps) and danger associated molecular patterns (damps) (saxena and yeretssian, ; zhong et al., ) . however, recent advances have revealed that some members of the nlr family may also regulate tissue homeostasis and modulate innate immune signaling pathways (kufer and sansonetti, ; saxena and yeretssian, ; zhong et al., ) . several nlrps (including nlrp , , , , , and ) are expressed primarily in mammalian oocytes, and nlrp , nlrp , and nlrp have been shown to have important roles in reproduction and development (mcdaniel and wu, ; murdoch et al., ; tong et al., ; westerveld et al., ; zhang et al., ) . additionally, nlrc and nlrc , as well as several nlrps, have been demonstrated to function as negative regulators of prrs (table ) . for example, nlrp suppresses negative regulator of tlr pathway cui et al. ( ) both tlr and tnfα-mediated nf-κb activation at the level of ikk complex formation (bruey et al., ; fontalba et al., ) . similarly, nlrp inhibits rig-i signaling by recruiting dtx , an e ubiquitin ligase which marks tbk for degradation (cui et al., ) . nlrp and nlrp suppress nf-κb signaling and are involved in the maintenance of intestinal homeostasis and tumorigenesis (allen et al., ; anand et al., ; lupfer and kanneganti, ; zaki et al., )-more recent reports also point to a role for nlrp in anti-viral immunity in mouse intestines (wang et al., b) . nlrx , the only mitochondrially localized member of this family, modulates sensing of dsrna and virus-induced ros production (guo et al., ; ma et al., ; moore et al., ; qin et al., a; tattoli et al., ) . in collaboration with iqgap , nlrc suppresses dna sensing lymphoid cells, myeloid cells, and epithelial cells through interactions with sting (tocker et al., ; zhang et al., ) . in addition, nlrc can suppress tlr signaling by modulating ikk activation (cui et al., ) , though nlrc deficiency does not affect this signaling pathway (kumar et al., b) . finally, the primate specific nlrp suppresses tlr-mediated activation of nf-κb by targeting traf for degradation in myeloid cells and b-cells (ellwanger et al., ; qin et al., b; wu et al., ) . together, these emerging properties call into question whether the established role for this family in activating proinflammatory responses may in fact represent an exception rather than a rule. as highlighted above, inappropriate activation of the nas pathway can result in highly destructive immune responses. yet, molecular dampeners, structural determinants and topological barriers described above as well as novel checks and balances yet to be discovered tightly regulate the sting/cgas axis and defend against such circumstances. one instance when distinguishing between self and non-self dna is particularly important is fertilization, when sperm cell derived dna can be found in oocyte cytoplasm. yet, while sting/cgas and rest of the nas pathway (which are all expressed in oocytes) are afforded access to this foreign dna, a nucleic acid sensing response is not triggered. in a recent publication, abe and colleagues reasoned that germ cells must therefore possess a robust mechanism to negatively regulate cytosolic nucleic acid sensing (abe et al., ) . through a combination of data-mining and experimental approaches, the group identified candidate genes that may serve as putative regulators of nas in oocytes. among these, four (trim , tgif lx, c orf , zpld ) significantly suppressed cgas-mediated signaling, and one, nlrp (nacht, lrr and pyd domains-containing ; a component of the inflammasome family of proteins), shut off nas virtually all together. what followed were a series of observations that delineate a role for nlrp as a rheostat for nucleic acid sensing, highlight the importance of controlling innate immune responses to foreign and endogenous ligands, and suggest that tight regulation of these processes is critical in maintaining proper immunologic homeostasis in germline. specifically, the authors demonstrate that nlrp associates with both sting and mavs and prevents downstream signal transduction (fig. ) . in turn, these adaptor degradation of lrr activation nlrp fig. nlrp -mediated inhibition of cytosolic nucleic acid sensing. upon activation of sting dependent signaling, conformational rearrangement from inhibitory-form to an active-form of nlrp is induced. targeting of tbk for k -linked poly-ubiquitination is then initiated and results in proteasome dependent degradation of tbk and termination of sting dependent as well as rig-i dependent signaling. similarly, nlrp is targeted for degradation to avoid persistent immunosuppression. abbreviations: nlrp , nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein ; pyd, pyrin domain; nacht, naip (neuronal apoptosis inhibitory protein), ciita (mhc class ii transcription activator), het-e (incompatibility locus protein from podospora anserine) and tp (telomerase-associated protein), lrr, leucine-rich repeat; ub, ubiquitin; p, phosphorylation. molecules induce the proteasomal degradation of nlrp , a feedback loop that may be critical in preventing persistent immunosuppression and proper induction of innate immune responses under appropriate conditions (for example, post fertilization of oocytes). importantly, several human diseases, including crohn's disease, celiac disease, blau syndrome, rheumatoid arthritis, type- and - diabetes, sle, cryopyrin-associated periodic syndromes (caps), inflammatory bowel diseases, colitis as well as colon cancer, have been shown to be associated with mutations in nlrps (saxena and yeretssian, ; zhong et al., ) . other nlrps (like nlrp , nlrp , and nlrp ) have been associated with miscarriage and infertility (docherty et al., ; huang et al., ) . in line with these observations ectopic expression of a k x nlrp allele (rs ; coding for a nonsense mutation that introduces an early stop codon in nlrp ) results in reduced suppression of tbk -mediated signaling (abe et al., ) . with an allele frequency of . % in the human population, and a minor allele frequency of % in east asian and ad mixed american populations, infertility associated with homozygosity of this gene may affect in , individuals. as an immunological rheostat, nlrp safeguards against inappropriate cytosolic responses to nucleic acids and the acquisition of such a function may have been a prerequisite to sexual reproduction. indeed, homologs of the inflammasome family of proteins have been found widely across life-arguing in favor of their involvement in immunity and broader cellular processes, as is observed with other expanded gene families. the past decade has seen rapid advancement in our understanding of how cells sense cytosolic dna, including the identification of receptors and molecular machinery that participate in this process as well as recognition of multiple diseases associated with this pathway. recent findings highlight the importance of controlling innate immune responses to dna and suggest that tight regulation of these processes is critical in maintaining proper immunologic homeostasis across multiple organs. thus, understanding the underlying network of regulators that control signaling through the cgas/sting axis will undoubtedly contribute to the development of potent therapeutic agents against auto-immune and inflammatory diseases, as well as certain cancers. cytosolic-dna-mediated, sting-dependent proinflammatory gene induction necessitates canonical nf-kappab activation through tbk sting recognition of cytoplasmic dna instigates cellular defense germ-cell-specific inflammasome component nlrp negatively regulates cytosolic nucleic acid sensing to promote fertilization denv inhibits type i ifn production in infected cells by cleaving human sting sting manifests self dna-dependent inflammatory disease nlrp suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical nf-kappab signaling nlrp negatively regulates innate immunity and host defence against bacterial pathogens sting-dependent cytosolic dna sensing pathways sting: infection, inflammation and cancer the birds, the bees, and innate immunity newly described pattern recognition receptors team up against intracellular pathogens pan /nalp /pypaf , an inducible inflammatory mediator that regulates nf-kappab and caspase- activation in macrophages the dna damage response induces ifn an orthogonal proteomic-genomic screen identifies aim as a cytoplasmic dna sensor for the inflammasome sting is a direct innate immune sensor of cyclic di-gmp viral evasion of intracellular dna and rna sensing role of nlrp and nlrp in the maintenance of intestinal homeostasis sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex mutations in genes encoding ribonuclease h subunits cause aicardi-goutières syndrome and mimic congenital viral brain infection intracellular nucleic acid detection in autoimmunity nlrc negatively regulates the nf-kappab and type i interferon signaling pathways nlrp negatively regulates type i interferon signaling by targeting the kinase tbk for degradation via the ubiquitin ligase dtx senp potentiates cgas activation by relieving sumo-mediated inhibition of cytosolic dna sensing cgas conducts micronuclei dna surveillance hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity mutations in nlrp are associated with reproductive wastage and multilocus imprinting disorders in humans the nlr family pyrin domain-containing protein contributes to the regulation of inflammatory signaling dna-pk is a dna sensor for irf- -dependent innate immunity aim activates the inflammasome and cell death in response to cytoplasmic dna nlrp , an inhibitor of the nf-kappab pathway, is transcriptionally activated by nf-kappab and exhibits a nonfunctional allelic variant autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease structure-function analysis of sting activation by c[g( , )pa( , ) p] and targeting by antiviral dmxaa activation of cyclic gmp-amp synthase by self-dna causes autoimmune diseases innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence cutting edge: cgas is required for lethal autoimmune disease in the trex -deficient mouse model of aicardi-goutières syndrome the aim -like receptors are dispensable for the interferon response to intracellular dna nlrx sequesters sting to negatively regulate the interferon response, thereby facilitating the replication of hiv- and dna viruses mitotic progression following dna damage enables pattern recognition within micronuclei aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc sumoylation promotes the stability of the dna sensor cgas and the adaptor sting to regulate the kinetics of response to dna virus a genetic association study of nlrp and nlrp genes in idiopathic recurrent miscarriage a toll-like receptor-independent antiviral response induced by double-stranded b-form dna tank-binding kinase- delineates innate and adaptive immune responses to dna vaccines sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity inherited sting-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations requirement of dnase ii for definitive erythropoiesis in the mouse fetal liver chronic polyarthritis caused by mammalian dna that escapes from degradation in macrophages multi-receptor detection of individual bacterial products by the innate immune system dna damage sensor mre recognizes cytosolic double-stranded dna and induces type i interferon by regulating sting trafficking cyclic dinucleotides trigger ulk (atg ) phosphorylation of sting to prevent sustained innate immune signaling nlr functions beyond pathogen recognition pathogen recognition by the innate immune system nlrc deficiency does not influence cytokine induction by virus and bacteria infections the capsids of hiv- and hiv- determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway activated sting in a vascular and pulmonary syndrome hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dna-sensing pathways irhom is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting unsolved mysteries in nlr biology modulation of the cgas-sting dna sensing pathway by gammaherpesviruses nlrx negatively modulates type i ifn to facilitate kshv reactivation from latency cgas surveillance of micronuclei links genome instability to innate immunity bak/bax macropores facilitate mitochondrial herniation and mtdna efflux during apoptosis identification of oocyte-selective nlrp genes in rhesus macaque monkeys (macaca mulatta) nlrx is a regulator of mitochondrial antiviral immunity activation of sting requires palmitoylation at the golgi mutations in nalp cause recurrent hydatidiform moles and reproductive wastage in humans features of systemic lupus erythematosus in dnase -deficient mice ubiquitination of sting at lysine controls irf activation hepatitis c virus ns b protein targets sting and abrogates rig-imediated type i interferon-dependent innate immunity structural and functional analysis of ddx : a bispecific immune receptor for dna and cyclic dinucleotide the helicase ddx recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response rnase h catalytic core aicardi-goutières syndrome-related mutant invokes cgas-sting innate immune-sensing pathway in mice -dimethylxanthenone- -acetic acid (dmxaa) activates stimulator of interferon gene (sting)-dependent innate immune pathways and is regulated by mitochondrial membrane potential rnf temporally regulates virus-triggered type i interferon induction by two distinct mechanisms nlrx mediates mavs degradation to attenuate hepatitis c virusinduced innate immune response through pcbp nlrp disrupts mavs signalosome to inhibit type i interferon signaling and virus-induced apoptosis hiv- evades innate immune recognition through specific cofactor recruitment the chemotherapeutic agent dmxaa potently and specifically activates the tbk -irf- signaling axis apoptotic caspases prevent the induction of type i interferons by mitochondrial dna nod-like receptors: master regulators of inflammation and cancer akt kinase-mediated checkpoint of cgas dna sensing pathway trim -mediated monoubiquitination of cgas for cytosolic dna sensing recognition of cytosolic dna activates an irf -dependent innate immune response coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway evasion of innate cytosolic dna sensing by a gammaherpesvirus facilitates establishment of latent infection usp negatively regulates antiviral responses by deubiquitinating sting dai (dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response sting specifies irf phosphorylation by tbk in the cytosolic dna signaling pathway nlrx is a mitochondrial nod-like receptor that amplifies nf-kappab and jnk pathways by inducing reactive oxygen species production lack of immunological dna sensing in hepatocytes facilitates hepatitis b virus infection the scaffolding protein iqgap interacts with nlrc and inhibits type i ifn production mater, a maternal effect gene required for early embryonic development in mice the ubiquitin ligase trim regulates innate immune responses to intracellular double-stranded dna the interferon response to intracellular dna: why so many receptors? ifi is an innate immune sensor for intracellular dna the e ubiquitin ligase amfr and insig bridge the activation of tbk kinase by modifying the adaptor sting trim alpha is a negative-feedback regulator of the intracellular dna and dna virus-triggered response by targeting sting nlrp regulates intestinal antiviral innate immunity s k-sting interaction regulates cytosolic dna-mediated activation of the transcription factor irf the e ubiquitin ligase rnf facilitates the cgas-mediated innate immune response htlv- tax impairs k -linked ubiquitination of sting to evade host innate immunity mutations in the testis-specific nalp gene in men suffering from spermatogenic failure apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production innate immune sensing and signaling of cytosolic nucleic acids cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna nlrp attenuates toll-like receptor signalling by targeting traf for degradation via the ubiquitin ligase rnf a glutamylation of the dna sensor cgas regulates its binding and synthase activity in antiviral immunity trim promotes dna virus infections by inhibiting innate immune response trex exonuclease degrades ssdna to prevent chronic checkpoint activation and autoimmune disease the cytosolic nucleic acid sensor lrrfip mediates the production of type i interferon via a beta-catenin-dependent pathway proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease cgas is essential for cellular senescence the nod-like receptor nlrp attenuates colon inflammation and tumorigenesis expression analysis of the nlrp gene family suggests a role in human preimplantation development the helicase ddx senses intracellular dna mediated by the adaptor sting in dendritic cells trim protein modulates type i interferon induction and cellular antiviral response by targeting mita/sting protein for k -linked ubiquitination nlrc , a member of the nlr family of proteins, is a negative regulator of innate immune signaling induced by the dna sensor sting usp recruits usp to promote innate antiviral response through deubiquitinating sting/mita the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita functions of nod-like receptors in human diseases we apologize to authors whose work could not be cited due to space constraints and narrow focus of the review. s.d.s. acknowledges funding from nih r (gm - ) and nih u (ca - ). key: cord- -ynionj r authors: hwang, mihyun; bergmann, cornelia c. title: alpha/beta interferon (ifn-α/β) signaling in astrocytes mediates protection against viral encephalomyelitis and regulates ifn-γ-dependent responses date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: ynionj r the contribution of distinct central nervous system (cns) resident cells to protective alpha/beta interferon (ifn-α/β) function following viral infections is poorly understood. based on numerous immune regulatory functions of astrocytes, we evaluated the contribution of astrocyte ifn-α/β signaling toward protection against the nonlethal glia- and neuronotropic mouse hepatitis virus (mhv) strain a . analysis of gene expression associated with ifn-α/β function, e.g., pattern recognition receptors (prrs) and interferon-stimulated genes (isgs), revealed lower basal mrna levels in brain-derived astrocytes than in microglia. although astrocytes poorly induced ifnβ mrna following infection, they upregulated various mrnas in the ifn-α/β pathway to a higher extent than microglia, supporting effective ifn-α/β responsiveness. ablation of the ifn-α/β receptor (ifnar) in astrocytes using mgfapcre ifnar(fl/fl) mice resulted in severe encephalomyelitis and mortality, coincident with uncontrolled virus replication. further, virus spread was not restricted to astrocytes but also affected microglia and neurons, despite increased and sustained ifnα/β and isg mrna levels within the cns. ifn-γ, a crucial mediator for mhv control, was not impaired in infected mgfapcre ifnar(fl/fl) mice despite reduced t cell cns infiltration. unexpectedly however, poor induction of ifn-γ-dependent major histocompatibility complex (mhc) class ii expression on microglia supported that defective ifn-γ signaling contributes to uncontrolled virus replication. a link between sustained elevated ifn-α/β and impaired responsiveness to ifn-γ supports the novel concept that temporally limited early ifn-α/β responses are critical for effective antiviral ifn-γ function. overall, our results imply that ifn-α/β signaling in astrocytes is not only critical in limiting early cns viral spread but also promotes protective antiviral ifn-γ function. importance an antiviral state established by ifn-α/β contains initial viral spread as adaptive immunity develops. while it is apparent that the cns lacks professional ifn-α/β producers and that resident cells have distinct abilities to elicit innate ifn-α/β responses, protective interactions between inducer and responder cells require further investigation. infection with a glia- and neuronotropic coronavirus demonstrates that astrocytes mount a delayed but more robust response to infection than microglia, despite their lower basal mrna levels of ifn-α/β-inducing components. lethal, uncontrolled viral dissemination following ablation of astrocyte ifn-α/β signaling revealed the importance of ifn-α/β responses in a single cell type for protection. sustained global ifn-α/β expression associated with uncontrolled virus did not suffice to protect neurons and further impaired responsiveness to protective ifn-γ. the results support astrocytes as critical contributors to innate immunity and the concept that limited ifn-α/β responses are critical for effective subsequent antiviral ifn-γ function. v iral infections of the central nervous system (cns) are rare but can lead to rapid mortality or long-term neurological disabilities even if acute encephalitis is resolved ( ) . early essential host defense mechanisms involve induction of alpha/beta interferon (ifn-␣/␤) and signaling through the ifn-␣/␤ receptor (ifnar) to upregulate ifn-stimulated genes (isgs). isg expression is associated with numerous biological activities, including antiviral and immunomodulatory pathways, e.g., interference with translation, apoptosis, and enhanced major histocompatibility complex (mhc) class i antigen (ag) presentation ( ) ( ) ( ) . while the ifn-␣/␤ response is critical in stemming cns viral replication and spread, it is rapidly downregulated and insufficient to eliminate virus in the absence of subsequent adaptive immune responses ( ) ( ) ( ) . moreover, the efficiency of the innate response in limiting viral spread sets the stage for the effectiveness of subsequent adaptive immunity ( ) ( ) ( ) ) . detection of viruses and induction of ifn-␣/␤ are initiated by pattern recognition receptors (prrs), which differ in subcellular localization and are specialized to recognize distinct molecular entities in virus structural components, genome, and/or replication intermediates ( , , ) . prrs which recognize rna viruses include endosome-associated membrane-bound toll-like receptors (tlrs), mainly tlr , and the cytoplasmic retinoic acid inducible gene (rig-i)-like receptors (rlrs) and melanoma differentiationassociated antigen (mda ). specialized prr adapter proteins transmit signals to activate ifn response factor (irf ), irf , and irf , which translocate to the nucleus to induce ifn-␤ and a subset of ifn-␣ genes ( ) . secretion of ifn-␣/␤ and signaling through ifnar subsequently activate isgs and the "antiviral state." prr activation also activates nuclear factor b (nf-b)-regulated genes, thereby inducing proinflammatory cytokines and chemokines to facilitate recruitment of innate and adaptive effector cells ( , ) . virtually all nucleated cells are able to induce and respond to ifn-␣/␤, including cns resident cells ( , ) . the cns does not harbor plasmacytoid dendritic cells, which are potent peripheral ifn-␣/␤ inducers ( , ) , and therefore has to rely on intrinsic induction of ifn-␣/␤. cns resident cells have been shown to differ widely in the repertoire as well as magnitude of basal and inducible transcripts encoding prrs and factors associated with the ifn-␣/␤ pathway ( ) ( ) ( ) ) . as ifn-␣/␤ signaling can rapidly induce genes involved in virus sensing and their signaling components, cell types which are not effective initial ifn-␣/␤ inducers may nevertheless contribute to the overall innate antiviral activity by efficient ifn-␣/␤ signaling and amplification of the ifn-␣/␤ response ( , , ) . the interdependence of cns cells in achieving an ifn-␣/␤-dependent antiviral state thus requires further investigation. microglia and astrocytes are the cns resident cells most prominently involved in early innate responses to injury, autoimmune attack, or infection ( , , , ) . in infections these early responses are essential to attract peripheral immune cells and limit viral dissemination ( , , ) . importantly, not only productively infected but also abortively infected astrocytes mount innate responses that contribute to protection ( , , ) . cns infections by several neuronotropic rna viruses, including la crosse virus, rabies virus, vesicular stomatitis virus (vsv), and theiler's murine encephalomyelitis virus (tmev), suggested that astrocytes, not neurons, are the main source of ifn-␤ expression and contributors to virus control through both tlr and rlr activation pathways ( ) ( ) ( ) . both microglia and astrocytes express various prrs, and their sentinel function is supported by their rapid response to microbial infection ( , , , , , ) . in addition to participating in direct antiviral functions, astrocytes regulate cns homeostasis, support neuronal function, and participate in formation and maintenance of the blood-brain barrier (bbb) ( , ) . both astrocytes and ifn-␣/␤ actively participate in bbb function to restrict cns entry of molecules, cells, or pathogens ( , ) . ifn-␣/␤ signaling specifically to astrocytes promotes bbb integrity in the cerebellum and protects from west nile virus infection ( ) . thus, both astrocyte ifn-␣/␤ induction and signaling regulate the outcome of infection within the cns at different functional levels. the essential role of ifnar signaling in limiting viral dissemination within the cns is supported by neurotropic virus infections of ifnar-deficient (ifnar Ϫ/Ϫ ) mice ( , , , ). however, caveats for ifnar Ϫ/Ϫ mice are the reduced basal levels of factors involved in the ifn-␣/␤ pathway as well as loss of innate myeloid cell dynamic activity in response to infection ( , , ) . further, while much information on cns innate responsiveness is derived from primary glial and neuronal cultures, the in vivo studies of ifn-␤ induction highlight how unsuspected players, such as abortively infected astrocytes, contribute to pathogen control ( ) . based on the prominent role of astrocytes in participating in innate responses, this study set out to assess how ifnar ablation in astrocytes affects pathogenesis in a gliaand neuronotropic coronavirus infection. the murine coronavirus mouse hepatitis virus (mhv) strain a infects microglia, astrocyte, neurons, and oligodendroglia following intracranial (i.c.) administration ( , ) . although mhvs are at best poor inducers of ifn-␣/␤ ( ) ( ) ( ) , they do induce ifn-␤ in microglia/macrophages ( ) . importantly, even the low levels of ifn-␣/␤ are essential to prevent viral dissemination and mortality ( , ) . the studies here reveal distinct patterns of basal and inducible levels of mrnas encoding components of the ifn-␣/␤ pathway in astrocytes and microglia isolated from naive and infected adult mouse brains. despite expressing lower baseline mrna levels, astrocytes upregulated ifn-␣/␤ pathway gene expression to a greater extent than microglia, supporting effective ifn-␣/␤ responses. ablation of ifn〈r in astrocytes using mgfapcre ifnar fl/fl mice resulted in severe encephalomyelitis and mortality by days postinfection (p.i.). this contrasted with mild clinical symptoms and no fatalities in infected control ifnar fl/fl mice. uncontrolled viral spread throughout the cns parenchyma of mgfapcre ifnar fl/fl mice not only was associated with increased astrocyte infection but also affected neurons and microglia, despite overall elevated and sustained levels of mrnas for ifn-␤ and ifn-␣ genes and isgs. ifn-␥, a crucial mediator of mhv control in the cns, was not impaired, despite reduced t cell cns infiltration. unexpectedly however, defective ifn-␥ signaling was implicated by impaired induction of ifn-␥-dependent mhc class ii expression on microglia. overall our results imply that ifn-␣/␤ signaling in astrocytes not only is critical in limiting cns viral spread but also promotes lymphocyte-derived protective antiviral ifn-␥ function. mhv strain a induces type i ifn in the cns coincident with viral replication. to evaluate the kinetics of mhv a replication relative to ifn␣/␤ and ifn␥ mrna levels in the cns, brains from uninfected and infected wild-type (wt) c bl/ mice were harvested out to day p.i. virus replication was monitored by expression of viral rna encoding the n protein (a n), which is present on genomic and all subgenomic rnas ( ) . viral n mrna was most abundant between days and p.i. and declined by day p.i. (fig. ), but it remained detectable out to day p.i. ifn␤, ifn␣ , and ifn␣ mrnas, the most abundant ifn␣ mrnas expressed following mhv a infection ( ) , also reached maximal levels between and days p.i. and dropped significantly by day p.i. (fig. ). while ifn␤ mrna was still elevated at day p.i., ifn␣ and ifn␣ mrnas had declined to basal levels by day p.i. ifn␥ mrna was already detectable at day p.i. but did not peak until to days p.i. expression levels declined by day p.i. but remained elevated above background out to day p.i. (fig. ) . ifn␣/␤ mrna levels thus peaked together with viral mrna, whereas the ifn␥ mrna peak was delayed and coincided with t cell infiltration ( ) . astrocytes exhibit distinct induction of and responsiveness to ifn-␣/␤ compared to microglia. although mhv a replicates in glia and neurons, it induces ifn-␣/␤ only in microglia, not astrocytes, using primary cell cultures ( ) . to assess the relative induction of and responsiveness to ifn-␣/␤ in astrocytes and microglia in vivo, we used infected gfap-gfp mice to isolate cd -negative, green fluorescent protein (gfp)-positive (cd Ϫ gfp ϩ ) astrocytes and cd int cd b ϩ microglia by fluorescenceactivated cell sorting (facs) ( fig. a ). measurement of viral n relative to glyceraldehyde- -phosphate dehydrogenase (gapdh) mrnas in either glia population revealed viral replication in both microglia and astrocytes at days and p.i. and a decline by day p.i. (fig. b ). while increased viral n mrna in microglia relative to astrocytes at days and p.i. did not reach statistical significance, overall viral n mrna levels mirrored those in total brain. the same populations of purified cells were used to measure transcript levels of the ifn␣/␤ genes, genes regulating ifn-␣/␤, signaling and selected antiviral isgs (fig. c) . in naive mice, constitutive expression of these genes was lower or undetectable in astrocytes compared to microglia. following infection, ifn␤ mrna was upregulated by day p.i. in microglia but was not upregulated until day p.i. and was less robust in astrocytes. in contrast, ifn␣ and ifn␣ mrnas were not significantly upregulated in microglia but were increased prominently in astrocytes by day p.i. consistent with the decline in viral rna, ifn␣/␤ mrnas dropped to baseline levels by day p.i. (fig. c ). transcripts for components required for ifn-␣/␤ signaling showed no significant differences between astrocytes and microglia throughout days to p.i. (fig. c) . while infection mediated a decline in ifnar mrna relative to basal levels in microglia by day p.i., it did not alter expression levels in astrocytes. stat mrna levels varied between cell preparations and showed no significant changes throughout infection in either cell type. irf transcripts were not affected by virus infection in microglia but increased modestly in astrocytes. in contrast, individual isgs were regulated distinctly not only between the glia populations but also within each glia type over time (fig. c) . mhv cns infection has been shown to strongly induce ifn-induced protein with tetratricopeptide repeats (ifit and ifit ) and =, =-oligoadenylate synthetase (oas ) transcripts ( , ) . although ifit mrna was increased in both populations by day p.i., the relative induction was significantly higher in astrocytes than in microglia at all time points. in contrast, ifit mrna was induced more prominently in microglia at day p.i., reached similar levels in both cell types at day p.i., and dropped thereafter in both populations. lastly, oas mrna showed peak upregulation in microglia by day p.i. and modest induction in astrocytes (fig. c) . under the assumption that viral mrna levels reflect similar replication in both glial populations, these data support microglia as superior initiators of ifn-␤ production relative to astrocytes following mhv a infection in vivo; these findings are consistent with results from primary neonatal cell cultures ( , ) . moreover, delayed ifn␤ upregulation in astrocytes suggested that their sensitivity to viral replication is enhanced once viral sensors are elevated via ifn-␣/␤ signaling. this notion was supported by delayed, yet more extensive, upregulation of the ifn␣ as well as the ifit and ifit genes compared to that in microglia. isg upregulation further appeared to be independent of direct changes in ifnar, stat , or irf transcript levels. many prr-encoding genes are themselves isgs and are upregulated during cns infections ( , , , ) . mda , rig-i, and tlr have all been established as prrs contributing to ifn-␣/␤ induction in mhv-infected microglia/macrophages and oligodendrocytes ( , , , ) . basal mda , rig-i, and tlr transcript levels were all significantly lower in facs-purified astrocytes than in microglia (fig. ) . nevertheless, astrocytes rapidly upregulated mda , rig-i, and tlr mrnas to levels observed in microglia as early as day p.i. (fig. ) , when total ifn␣/␤ mrna peaked in the brain (fig. ). relative to basal levels, the fold increase in astrocytes exceeded that in microglia. all three prr transcripts declined by day p.i., reflecting the decline in ifn-␣/␤. with the exception of mrna encoding ikk, a kinase involved in both induction and ifnar signaling, constitutive levels of transcripts for prr signal transduction factors represented by irf and irf were also lower in astrocytes (fig. ) . expression of the cellular kinase ikk mrna was gradually increased in both glial populations over time up to day p.i., with increases more pronounced in microglia (fig. ) . irf mrna expression was decreased in microglia following infection and remained unchanged in astrocytes, contrasting with upregulation of irf mrna in both cell types between days to p.i. and downregulation thereafter (fig. ) . these data are consistent with constitutive irf expression but inducible expression of both ikk and irf following infection or ifn-␣/␤ treatment of non-cns cells ( ) . overall, the data suggest that initial production of ifn-␤ in microglia results in upregulation of select isgs in astrocytes, thereby amplifying ifn-␣/␤. however, it remains unclear whether astrocytes induce ifn-␤ directly via virus-derived prr activators or via mediators released from necrotic or apoptotic cells in the inflamed cns environment. regardless, the results demonstrate that astrocytes are sensitive responders to ifn-␣/␤ and active participants in the ifn-␣/␤-induced amplification loop. ifnar signaling in astrocytes is essential to control mhv a infection. given the prominent response of astrocytes to ifn-␣/␤ following mhv a infection in vivo, we determined the protective role of astrocyte-mediated ifnar signaling against infection using mgfapcre ifnar fl/fl mice. disease onset was similar in mgfapcre ifnar fl/fl and ifnar fl/fl mice starting at day p.i. however, in contrast to mild clinical symptoms in ifnar fl/fl mice, mgfapcre ifnar fl/fl mice developed severe encephalitis and succumbed to infection by day p.i. (fig. a ). the -to -day-delayed mortality compared to that of ifnar Ϫ/Ϫ mice infected with a similar virus dose ( ) supported astrocytes as critical, but not the only, contributors to early ifnar-dependent protection. to evaluate whether disease severity and mortality in mgfapcre ifnar fl/fl mice were correlated with impaired viral control, infectious virus was measured in brain supernatants. the virus loads were comparable in both groups at day p.i. but reached ϳ -log -higher levels in mgfapcre ifnar fl/fl mice than in ifnar fl/fl mice by day p.i. (fig. b ). the anatomical localization and distribution of viral n protein were also evaluated by histology. compared to small isolated residual foci of viral ag-positive cells in brains of ifnar fl/fl mice at day p.i., mgfapcre ifnar fl/fl mice exhibited extensive dissemination with increased intensity of viral ag staining throughout the brain (fig. c ). high-magnification images indicated that virus was not limited to cells consistent with glial morphology but also was in cells with typical neuronal morphology in mgfapcre ifnar fl/fl mice (fig. c, insets) . as mhv a is hepatotropic following peripheral infection and can spread to the liver following i.c. infection of immunocom- by rt-pcr. data represent the means Ϯ sem from two experiments, each comprising or pooled brains per time point, and were analyzed by the unpaired two-tailed student t test and two-way anova. *, significance between microglia and astrocytes; #, significance compared to each respective naive population: * and #, p Ͻ . ; ** and ##, p Ͻ . ; *** and ###, p Ͻ . . promised mice ( ), we also surveyed liver for viral n mrna. the levels of viral n relative to gapdh transcripts were elevated in livers of mgfapcre ifnar fl/fl compared to ifnar fl/fl mice at day p.i. (values of Ϯ versus Ϯ ) (data not shown). however, overall viral n mrna levels were significantly higher in the cns ( , Ϯ versus Ϯ , respectively) (see fig. ). there was also no evidence of necrotic foci by gross visual examination (data not shown). mortality was thus attributed to overwhelming virus spread within the cns. mhv a infects multiple cns cell types, including microglia, astrocytes, and neurons ( , ) . to evaluate to what extent the absence of ifn-␣/␤ signaling in astrocytes predisposes astrocytes over other cell types to enhanced infection, brains from infected mgfapcre ifnar fl/fl and ifnar fl/fl mice were analyzed for viral n protein in combination with antibodies (abs) specific for neurons (neun), astrocytes (glial fibrillary acidic protein [gfap]), and microglia/macrophages (iba ). characteristic staining and colocalization patterns at day p.i. in wt mice are depicted in fig. a . microglia and neurons showed prominent perinuclear cytoplasmic n protein localization, with some extension into processes in microglia. in contrast, viral ag was only occasionally detected in the cytoplasm of astrocytes and was localized mainly proximal to, but not overlapping with, astrocytic processes. in this context it is critical to note that the anti-gfap ab used to identify astrocytes stains main stem branches but not the cytoplasm or fine processes of astrocytes and poorly stains gray matter astrocytes ( , ) , suggesting that viral protein may localize to astrocytic processes poorly detected by anti-gfap ab. total n protein staining revealed an ϳ -fold-higher positive area in mgfapcre ifnar fl/fl than in ifnar fi/fi mice as determined by pixels per observed field at day p.i.; the difference increased to ϳ -fold by day p.i., when virus already declined in ifnar fi/fi mice (fig. b) . keeping the limitations of gfap staining in mind, colocalization of viral ag with gfap was overall increased at day p.i. furthermore, when the relative proportion of the dual-positive area within the total viral ag-positive pixel area was assessed, percentages were similar in both mouse strains at day p.i. but were -fold higher in mgfapcre ifnar fl/fl mice than in controls at day p.i. (fig. c ). viral ag-positive microglia were also overall increased in mgfapcre ifnar fl/fl mice at day p.i. however, the relative proportion of the dually viral ag-and iba -positive area per total viral ag-positive area was comparable between groups at both days and p.i. (fig. d) . to evaluate the relative infection of astrocytes versus microglia, we also measured the proportions of viral ag-positive area relative to gfap-or iba -positive areas. while infection of microglia increased from ϳ % to ϳ %, it increased from Ͻ % to % in astrocytes in mgfapcre ifnar fl/fl mice between days and p.i. (fig. ) . although infected neurons were increased in mgfapcre ifnar fl/fl compared to ifnar fl/fl mice throughout the brain by day p.i., they declined significantly in both groups by day p.i. remaining elevated neuronal infection in mgfapcre ifnar fl/fl mice at day p.i. was still particularly evident in lower and distal parts of the brain (pons and medulla) (fig. e) . analysis of apoptosis to explain the decline in virus-infected neurons indicated and microglia in brain cortex of both mouse groups at day p.i. bar graphs show the proportion of n ϩ gfap ϩ or n ϩ iba ϩ per total n ϩ area, respectively. insets show infected astrocytes at higher magnification and arrows indicate infected microglia. (e) representative staining of viral n protein in neurons in brain cortex and brain stem at days and p.i. the bar graph shows average numbers of virus-infected neurons per brain section from or whole brain sections per mouse (n ϭ mice). arrows indicate virus-infected neurons. scale bar, m. (c to e) left and right panels indicate ifnar fl/fl and mgfapcre ifnar fl/fl mice, respectively. the data in panels b and c represent the means Ϯ sem from or fields in or slides from mice per group and were analyzed by the unpaired two-tailed student t test and two-way anova. *, significance between ifnar fl/fl and mgfapcre ifnar fl/fl mice; #, significance between days and p.i. in the same group: *, p Ͻ . ; ** and ##, p Ͻ . ; ***, p Ͻ . . overall sparse but more abundant neuronal apoptosis in mgfapcre ifnar fl/fl mice (fig. a ). occasional apoptotic cd ϩ lymphocytes were also observed with elevated frequency in mgfapcre ifnar fl/fl mice (fig. b ). while these results likely underestimate the extent of viral infection in astrocytes, they support that the absence of astrocyte ifnar signaling leads to increased viral dissemination most prominently within astrocytes but also to microglia and neurons. abrogated ifnar signaling in astrocytes does not impair overall expression of ifn-␣/␤, isgs, chemokines, and cytokines. to assess whether uncontrolled virus spread was attributable to overall impaired innate immune responses, we determined transcripts for ifn-␣/␤ and isgs in relation to viral n mrna levels in brains of both mouse groups (fig. ). viral n mrna levels were similar at day p.i. but increased significantly in mgfapcre ifnar fl/fl mice compared to ifnar fl/fl controls by day p.i. (fig. ) , consistent with increased infectious virus by day p.i. (fig. b) . elevated viral n mrna correlated with increased ifn␤, ifn␣ , and ifn␣ transcripts at day p.i. (fig. ) . to assess how elevated ifn-␣/␤ affected downstream ifnar signaling, we further compared mrna levels of the isgs (ifit , ifit , isg , mx , pkr, and oas ). while these transcripts were all similar in both mouse groups at day p.i., ifit , ifit , and mx mrnas were increased -to -fold in mgfapcre ifnar fl/fl mice by day p.i. isg , pkr, and oas mrnas were not affected or reduced relative to those in ifnar fl/fl controls (fig. ) . ifnar abrogation on astrocytes thus did not impair global ifn␣/␤ or select isg expression in the infected cns. however, innate immunity by ifnar-competent cells was clearly insufficient to stem viral dissemination throughout the cns in mgfapcre ifnar fl/fl mice. astrocytes are also potent inducers of chemokines and cytokines ( , ) , and a subset of chemokines, e.g., the neutrophil chemoattractant cxcl , can be negatively regulated by ifn-␣/␤ ( ). we therefore evaluated how the absence of ifnar on astrocytes modulates expression of select nf-b-dependent chemokines and the proinflammatory cytokine interleukin (il- ) ( , ) . transcripts for cxcl and ccl , potent chemoattractants for neutrophils and monocytes, respectively, were similar at day p.i. but were significantly upregulated at day p.i. (fig. ) , consistent with increased viral replication and ifn-␣/␤-mediated downregulation of cxcl by astrocytes ( , ) . increased il mrna expression at day p.i. (fig. ) further indicated that ifnar abrogation on astrocytes does not impact proinflammatory cytokine induction. in contrast, ccl mrna, which is prominently expressed by t cells during mhv infection ( ) , was significantly reduced compared to that in ifnar fl/fl mice at day p.i. dysregulation of these proinflammatory factors suggested that myeloid cells may be recruited in favor of lymphocytes in mgfapcre ifnar fl/fl mice, thereby contributing to morbidity and uncontrolled viral spread. infected mgfapcre ifnar fl/fl mice exhibit increased cns neutrophil accumulation and reduced t cell infiltration. flow cytometry revealed that altered chemokine expression patterns indeed affected the composition of inflammatory cells within the cns. consistent with elevated cxcl mrna, neutrophils in mgfapcre ifnar fl/fl brains were increased by ϳ -fold at day p.i. relative to those in ifnar fl/fl mice, although numbers in both groups were already reduced compared to those at day p.i. (fig. a) . differences in macrophages were not statistically significant (fig. b) . as both cd and cd t cells are essential to reduce infectious mhv, with ifn-␥ playing a prominent antiviral role ( , ) , we further assessed if impaired t cell recruitment and ifn-␥ production within the cns is associated with lack of virus control. indeed, both cd and cd t cells were reduced in the cns of mgfapcre ifnar fl/fl mice at day p.i. (fig. c) . however, similar ifn-␥ levels (fig. d) , despite reduced t cell numbers, suggested that increased viral replication leads to increased ag presentation, thereby triggering more or prolonged ifn-␥ production by t cells in mgfapcre ifnar fl/fl mice ( , ) . impaired access of t cells to the cns parenchyma was ruled out as an explanation of loss of viral control, as we found no differences in anatomical distribution of cd ϩ t cells within perivascular and parenchymal sites between the two groups (data not shown). uncontrolled viral replication in mgfapcre ifnar fl/fl mice despite similar ifn-␥ production thus implied that viral replication overwhelms the t cell response or that ifn-␥ signaling is impaired. determination of ifn-␥-specific responses in vivo is complicated by the fact that many factors upregulated by ifn-␥ are also induced by ifn-␣/␤ ( ). nevertheless, a prominent indicator of ifn-␥ signaling in microglia/macrophages is induction of the transcription factor class ii transactivator (ciita), a master regulator of mhc class ii expression ( , ) . interestingly, ciita transcripts were barely induced in brains of mgfapcre ifnar fl/fl mice, in contrast to robust upregulation in control mice, at day p.i. (fig. e) . analysis of mhc class ii expression on microglia by flow cytometry confirmed that the very modest ciita mrna induction impaired ifn-␥-dependent mhc class ii upregulation. only ϳ % of microglia expressed mhc class ii in mgfapcre ifnar fl/fl mice, compared to ϳ % in ifnar fl/fl mice, and the mean fluorescence intensity, reflecting expression levels, per cell was also lower (fig. f) . other ifn-␥-dependent factors include the chemokines cxcl and cxcl ( , ) . while cxcl expression is inducible mainly by ifn-␥, cxcl is inducible by ifn-␣/␤, ifn-␥, and tumor necrosis factor (tnf) ( , , ) . furthermore, cxcl is produced by macrophages/microglia and endothelial cells, but not astrocytes, whereas cxcl is induced prominently in astrocytes during inflammation ( , ) . assessment of cxcl and cxcl mrnas showed similarly low levels in both groups at day p.i. (fig. g) . however, unlike ifnar fl/fl mice, mgfapcre ifnar fl/fl mice failed to upregulate cxcl mrna by day p.i. in contrast, cxcl mrna levels were elevated by ϳ . -fold in mgfapcre ifnar fl/fl mice by day p.i. but did not increase in ifnar fl/fl mice. given that mgfapcre ifnar fl/fl mice do not express ifnar on astrocytes, cxcl mrna upregulation is likely driven by ifn-␥ and/or tnf. overall, these results implied that microglia, but not astrocytes, are significantly compromised in ifn-␥ responsiveness in mgfapcre ifnar fl/fl mice. preexposure to ifn-␣/␤ modifies ifn-␥ responsiveness in myeloid cells. one explanation for the differential responsiveness to ifn-␥ in microglia versus astrocytes in mgfapcre ifnar fl/fl mice is that prolonged ifnar signaling via elevated and sustained ifn-␣/␤ expression may skew subsequent ifn-␥ responsiveness. this is supported by an antagonistic effect of ifn-␣/␤ on ifn-␥ responses in macrophages following ifn treatment or listeria infection ( ) ( ) ( ) . we therefore used the viral rna mimic poly(i·c), a synthetic double-stranded rna (dsrna) analog recognized by tlr as well as rig-i/mda , to induce ifn␣/␤ mrna in bone marrow-derived macrophages (bmdm) and monitor ifn-␥ responsiveness. previous in vivo data revealed that poly(i·c) administration into the cns of mice induced ifn␣/␤ mrna by h, which was sustained out to h ( ). bmdm were thus pretreated with poly(i·c) for or h and subsequently treated with ifn-␥ for h. ifn-␥-only treatment was included as a positive control. poly(i·c) alone did not induce ciita mrna at h and induced it only sparsely by h. however, while ifn-␥ treatment alone effectively induced ciita mrna, it failed to induce ciita mrna in cells pretreated with poly(i·c) for either or h (fig. ) . similarly, poly(i·c) alone only sparsely induced cxcl mrna at h compared to induction by ifn-␥ treatment alone, and short poly(i·c) pretreatment suppressed cxcl mrna induction by ifn-␥. assessment of an inhibitory effect of prolonged poly(i·c) exposure was preempted by induction of cxcl mrna levels similar to those with ifn-␥. finally, treatment with poly(i·c) alone was superior to ifn-␥ alone in upregulating cxcl mrna, with saturation already achieved at h. these results are consistent with poly(i·c)-mediated upregulation of cxcl via ifn-␣/␤ and tnf and preempted use of cxcl as a target gene to assess suppressive functions on ifn-␥ signaling in this experimental setting. overall, these data indicate that preexposure of macrophages/ microglia to a virus-induced inflammatory milieu including ifn-␣/␤ has the potential to negatively regulate subsequent ifn-␥ signaling. however, the magnitude of ifn-␣/␤mediated suppression of ifn-␥-dependent gene induction depends on other regulatory elements in the respective target gene promoter. these results reveal that the duration and/or level of cell exposure to ifn-␣/␤ can substantially diminish responsiveness to temporally lagging ifn-␥, thereby reducing ifn-␥ protective functions. the ability to induce and respond to ifn-␣/␤ varies between cell types as well as between types of virus, but the contribution of different cns cell types to mount ifn-␣/␤-mediated protection in vivo are less well explored ( ) . our studies comparing the capacity of adult astrocytes relative to microglia to respond to mhv a infection revealed that astrocytes expressed lower basal levels of prrs and signaling components to induce ifn-␣/␤ and isgs than microglia, consistent with published gene array data ( ) . nevertheless, despite a delay in response to infection, astrocytes exhibited strong upregulation of prrs, ifn␣s, and select isgs, supporting that astrocytes are well positioned to respond to paracrine ifn-␣/␤ to amplify their innate antiviral response. the critical role of ifnar signaling in astrocytes to block viral spread was confirmed by rapid virus dissemination throughout the cns upon abrogation of ifnar signaling in astro-cytes. thus, although basal expression levels of ifn-␣/␤ signaling components have been shown to determine the responsiveness of cells to insults ( , ) , our data reveal that cells expressing low basal levels of ifn-␣/␤ pathway components can nevertheless trigger a protective innate immune response through paracrine ifn-␣/␤ signaling. delayed mortality of infected mgfapcre ifnar fl/fl mice compared to ifnar Ϫ/Ϫ mice implied that other cell types, including microglia, can produce and respond to ifn-␣/␤. this is clearly demonstrated by elevated and sustained ifn␣/␤ as well as select isg expression despite ifnar deletion on astrocytes. however, vast dissemination of virus throughout the cns clearly indicated that ifnar signaling specifically in astrocytes plays a dominant role in host protection, which could not be compensated by ifnarcompetent glia and neurons. while microglia appeared to exhibit some autocrine action as suggested by less overall infection relative to that in astrocytes, neurons in the brain stem appeared to be most susceptible and vulnerable to infection in the absence of astrocyte ifnar signaling. although we cannot exclude that inherently reduced activation of innate viral components in neurons may contribute to their susceptibility, our data indicate that overwhelming infection of ifnar-deficient astrocytes increases adjacent neuronal infection. west nile virus infection of neuronal cultures demonstrates that neurons have the capacity to mount innate viral responses via tlr and irf activations ( , ) . moreover, neurons have also been demonstrated to induce ifn-␤ following tmev and la crosse virus infection in vivo ( ) . nevertheless, these responses were very sparse and focal. in contrast, subsequent studies of la crosse virus infection in ifn-␤ reporter mice showed that apparently noninfected astrocytes and microglia in close proximity to infected neurons were prominent ifn-␤-producing cells, while ifn-␤ expression by infected neurons was rare ( ) . more recent data demonstrate that ifn-␤-producing astrocytes appeared to be transiently or abortively infected during various infections associated with neuronal tropism ( ) . the spatial extent of innate protection induced in an infected environment may thus depend on the prominent infected cell type. as both microglia and astrocytes have far-reaching processes, release of ifn-␣/␤ from these cells may provide more extensive protection than release from neuronal cell bodies. two independent studies with vsv indeed indicate that locally induced ifn-␤ in the olfactory bulb can trigger isgs in distal parts of the brain, although one indicates astrocytes and the other neurons as the prominent ifn-␤ inducers ( , ) . regardless, the vast increase of virus in mgfapcre ifnar fl/fl mice supports the notion that the inability of astrocytes to clear virus, rather than loss of direct isg effects on other cns cells, is responsible for loss of viral control. in addition to uncontrolled virus, elevated neutrophil accumulation may contribute to tissue damage and rapid morbidity of mgfapcre ifnar fl/fl mice. increased neutrophil cns infiltration correlated with increased mrna expression of the neutrophil chemoattractant cxcl ( ) and its downregulation by ifn-␣/␤ and ifn-␥ ( , , , ) . neutrophils are early innate responders, which contribute to bbb breakdown and tissue damage via secretion of matrix metalloproteinases (mmps) and a wide range of other degrading enzymes released upon degranulation ( , ) . during autoimmune-mediated cns inflammation, neutrophils have also been shown to release cytokines which mature antigen-presenting cells and subsequently reactivate t cells ( ) ( ) ( ) . elevated cxcl mrna as well as increased levels of mrnas encoding il- and ccl , two other proinflammatory factors prominently induced in astrocytes ( ) , further suggested that the nf-b pathway initiated by ppr activation was intact in infected mgfapcre ifnar fl/fl mice. finally, our results show that extensive virus spread in the absence of protective astrocyte ifnar signaling cannot be controlled by t cells. although previous studies of cxcl and cxcl monoclonal antibody (mab) blockade to reduce cxcr -dependent t cell recruitment showed a reduction in t cell migration and loss of viral control in a related mhv infection ( , ) , subsequent studies in cxcl -and cxcl -deficient mice indicated that cxcl is more critical for effective adaptive immunity ( ) . impaired t cell accumulation despite unimpaired cxcl mrna in mgfapcre ifnar fl/fl mice was thus unanticipated. while an overwhelming virus load may lead to aginduced t cell exhaustion and death ( ) , dysregulation of t cells requires future investigation. regardless of reduced t cell numbers, their initial activity as monitored by ifn-␥ secretion was not impaired. ifn-␥ signaling is essential for mhv control within the cns by both exerting direct antiviral effects and enhancing mhc class i and ii upregulation ( ) . however, reduced induction of ifn-␥-dependent ciita and cxcl mrnas showed that microglia were significantly impaired in ifn-␥ responsiveness. in contrast, increased cxcl mrna expression in the absence of ifnar on astrocytes implied that ifn-␥ signaling in astrocytes remained intact. the notion that elevated and sustained ifn-␣/␤ signaling acts as a negative regulator of ifn-␥ signaling was supported by earlier reports showing antagonistic effects of ifn-␣/␤ on ifn-␥-induced activation of macrophages ( - ). one possible mechanism may involve downregulation of the ifn-␥ receptor (ifngr), as shown during listeria infection ( ) . our in vitro studies further supported selected downregulation of ifn-␥ responsiveness by ifn-␣/␤ via pretreatment of bmdm with the dsrna mimic poly(i·c). ifn-␣/␤-dependent sensitization of microglia/macrophages to subsequent ifn-␥ responses may provide a mechanism to limit excessive immune responses leading to tissue damage. in this context it is of interest to note that both ifn-␣/␤ and ifn-␥ responses are tightly controlled by negative regulators, including phosphatases ( ) . cross talk between ifn-␣/␤ and ifn-␥ responses may thus provide additional levels to protect the host from exacerbated proinflammatory responses. overall, our data demonstrate a vital protective role of astrocyte-mediated ifn-␣/␤ signaling in host protection from neurotropic coronavirus-induced encephalomyelitis. further, the link between sustained elevated ifn-␣/␤ and impaired responsiveness to ifn-␥ supports the novel concept that temporally limited early ifn-␣/␤ responses are critical for effective antiviral ifn-␥ function. mes/j (gfap-gfp) mice were originally purchased from the jackson laboratory (bar harbor, me) and backcrossed to c bl/ mice for at least generations. b .cg-tg (gfap-cre) . mvs/j (stock number ; mgfapcre) mice were also purchased from the jackson laboratory (bar harbor, me). ifnar fl/fl mice engineered to contain loxp sites flanking exon of the ifnar gene were originally produced on the /ola background in u. kalinke's laboratory (paul-ehrlich-institut, langen, germany) as described previously ( ) and were backcrossed onto the c bl/ background in r. schreiber's laboratory (washington university school of medicine, st. louis, mo) as described previously ( ) . ifnar fl/fl mice were crossed with mgfapcre ϩ/Ϫ mice to generate mgfapcre ϩ/Ϫ ifnar fl/fl (mgfapcre ifnar fl/fl ) mice, which were crossed with ifnar fl/fl mice for experimentation. offspring were genotyped for both the presence of the cre recombinase gene and exon deletion from the ifnar gene by pcr using the following primers: cre, =-gtccaatttactgaccgtac acc- = (forward) and =-gttattcggatcatcagctacacc- = (reverse); ifnar flox, =-tgctttgaggagc gtctgga- = (forward) and =-catgcactaccacaccaggcttc- = (reverse). cre-negative ifnar fl/fl littermates were used as wt controls (ifnar fl/fl ). all mice were housed under specific-pathogen-free conditions at an accredited facility at the cleveland clinic lerner research institute. mice of both sexes were infected intracranially (i.c.) at to weeks of age with , pfu of the hepato-and neurotropic mhv a strain expressing enhanced green fluorescent protein (egfp), kindly provided by volker thiel (kantonal hospital, st. gallen, switzerland) ( ) . mhv a was propagated on delayed brain tumor (dbt) astrocytoma monolayers, and virus titers were determined as described previously ( ) . virus in the cnss of individual mice was measured by plaque assay on dbt cells using clarified supernatants from brain homogenates prepared as described previously ( ) . clinical disease severity was graded daily using the following scale: , no disease symptoms; , ruffled fur; , hunched back or inability to turn upright; , severe hunching/wasting or hind limb paralysis; , moribund condition or death ( ) . all animal procedures were approved by the institutional animal care and use committee of the cleveland clinic (phs assurance number a - ) and were conducted in compliance with the guide for the care and use of laboratory animals from the national research council. brains from mice perfused with cold phosphate-buffered saline (pbs) were homogenized in dulbecco's pbs (dpbs) (ph . ) using tenbroeck tissue homogenizers as described previously ( ) . homogenates were centrifuged at ϫ g for min at °c. cells were resuspended in rpmi containing mm hepes (ph . ), adjusted to % percoll (pharmacia, uppsala, sweden), underlaid with ml % percoll, and centrifuged at ϫ g for min at °c. cells were collected from the %/ % interface, washed with rpmi, counted, and suspended in fluorescence-activated cell sorting (facs) buffer ( . % bovine serum albumin in dpbs). fc␥ receptors were blocked with % mouse serum and rat anti-mouse cd / mab (clone . g : bd biosciences, san diego, ca) for min on ice prior to staining with fluorescein isothiocyanate (fitc)-, phycoerythrin (pe)-, peridinin chlorophyll protein (percp)-, or allophycocyanin (apc)-conjugated mabs specific for cd (clone -f ), cd (clone - . ), cd (clone gk . ), ly g (clone a ), cd b (clone m / ), and mhc class ii (clone m / . . ) (all from bd bioscience, mountain view, ca) and f / (serotec, raleigh, nc) in facs buffer. samples were analyzed on a bd accuri c plus instrument (bd biosciences). forward-and side-scatter signals obtained in linear mode were used to establish a gate containing live cells while excluding dead cells and tissue debris. data were analyzed using flowjo software (tress star inc., ashland, or). microglia and astrocyte isolation for gene expression analysis. brains from or naive or mhv a -infected gfap-gfp mice at days , , and p.i. were homogenized using a neural tissue dissociation kit (papain, miltenyi auburn, ca) following the manufacturer's protocol. brain homogenates were prepared for / % percoll gradients to isolate cells as described above. cns cells were blocked with mouse serum and anti-cd / mab as described above prior to staining with cd and cd b in facs buffer. microglia and astrocytes were sorted based on their cd int cd b ϩ and cd neg gfp ϩ phenotypes, respectively, using a facs aria iii (bd biosciences) and facs diva software (bd biosciences). sorted cells were resuspended in trizol (invitrogen, carlsbad, ca) and stored at Ϫ °c. gene expression analysis. rna from brains or sorted cells was extracted using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer's instructions. following treatment with dnase i using a dna-free kit (ambion, austin, tx), cdna was synthesized using moloney murine leukemia virus reverse transcriptase (invitrogen) in buffer containing mm deoxynucleoside triphosphate mix, ng random hexamer primers, and oligo(dt) ( : ratio) (invitrogen). rna expression was assessed using either sybr green master mix (applied biosystems, foster city, ca) or taqman fast master mix (applied biosystems, foster city, ca) as described previously ( ) . the following primers were used with sybr green master mix: for the gapdh gene, =-catggcct tccgtgttccta- = (forward) and =-atgcctgcttcaccaccttct- = (reverse); for cxcl , =-tgcac gatgctcctgca- = (forward) and =-aggtctttgagggatttgtagtgg- = (reverse); for cxcl , =-ga cggtccgctgcaactg- = (forward) and =-gcttccctatggccctcatt- = (reverse); for the viral nucleocapsid (n) gene, =-gccaaataatcgcgctagaa- = (forward) and =-ccgagcttagccaaaacaa g- = (reverse); for il , =-acacatgttctctgggaaatcgt- = (forward) and =-aagtgcatcatcgttgt tcataca- = (reverse); for oas , =-aaaaatgtctgcttcttgaattctga- = (forward) and =-tgtgcct ttggcagtggat- = (reverse); for ifnar , =-cccaaggcaagagctatgtc- = (forward) and =-tctgaa cggcttccagaact- = (reverse); for ikk, =-ccagaagattcagtgttgtttgg- = (forward) and =-tca ttgtagctgagccctg- = (reverse); for mda , =-gacaccagaattcaagggac- = (forward) and =-gc cacacttgcagataatctc- = (reverse); and for rig-i, =-gtcagcacaaaccacaacc- = (forward) and =-gtctcaaccactcgaatgtc- = (reverse). taqman fast master mix and taqman primers/probes gene expression in total tissue mrna or glial populations was normalized to the respective gapdh mrna expression in each mrna preparation and converted to a linearized value using the formula: e(c t gapdh Ϫ c t gene ) ϫ , . immunohistochemistry and immunofluorescence. brains from pbs-perfused mice were fixed with % neutral zinc-buffered formalin and embedded in paraffin for viral n protein analysis. the distribution of viral antigen (ag) was determined using mab j . specific, for the carboxyl terminus of the viral n protein, as the primary mab, biotinylated horse anti-mouse igg as the secondary ab, and streptavidinconjugated horseradish peroxidase and , =-diaminobenzidine substrate (vectastain-abc kit; vector laboratories virus-infected cells were identified using mab j . ( ) in combination with cell type-specific markers for microglia/macrophages using rabbit anti-iba astrocytes using rabbit anti-glial fibrillary acidic protein (anti-gfap all images were analyzed using fiji version . . quantification of viral n protein staining areas and the relative proportion of colocalization with gfap or iba reactivity were calculated per field of interest using image-pro plus version . . infected neurons were enumerated by counting entire brain sections. for immunohistological analysis, representative data are presented from or separate fields per mouse with or mice per group per time point. ifn-␥ elisa. ifn-␥ in brain supernatants was measured by enzyme bone marrow was collected from femurs and tibiae of -to -week-old mice, passed through a cell strainer, and centrifuged at ϫ g for min at °c. cells were suspended in bmdm medium (dulbecco modified eagle medium [dmem], % fcs, % l conditioned medium as a source of colony-stimulating factor , . % gentamicin, % sodium 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viral encephalomyelitis we thank michael diamond (washington university school of medicine, st. louis, mo) for kindly providing ifnar fl/fl mice on the c bl/ background. we also sincerely thank jennifer powers for exceptional facs purification and the cleveland clinic lerner research institute imaging core and john peterson for assistance with confocal microscopy. key: cord- -uwxk o d authors: mayer, karin; nellessen, cordula; hahn‐ast, corinna; schumacher, martin; pietzonka, sandra; eis‐hübinger, anna m.; drosten, christian; brossart, peter; wolf, dominik title: fatal outcome of human coronavirus nl infection despite successful viral elimination by ifn‐alpha in a patient with newly diagnosed all date: - - journal: eur j haematol doi: . /ejh. sha: doc_id: cord_uid: uwxk o d introduction: human coronavirus nl (hcov‐nl ) is one of four common human respiratory coronaviruses. despite high incidence, hcov infections are grossly understudied. we here report a case of hcov infection in a leukemia patient with fatal ards despite successful virus elimination by pegylated interferon‐alpha (peg‐ifn‐α). case: the ‐year‐old female pre‐t‐all patient was treated according to the german‐multicenter trial for adult all protocol. no relevant infectious complications were seen until day when the neutropenic patient developed fever without any clinical focus. antibiotic prophylaxis was switched to meropenem. the patient deteriorated rapidly with respiratory failure due to ards. anti‐infectious therapy was escalated to additional linezolid and liposomal amphotericine‐b. bal revealed a significant viral load of hcov‐nl . based on successful application of peg‐ifn against the related sars coronavirus in animal experiments, a single injection of μg peg‐ifn‐α b was applied. despite immediate initiation of treatment and elimination of virus in subsequent tests, the progressive lung failure led to death d after onset of fever with massive lung bleeding as a consequence of diffuse alveolar hemorrhage. conclusion: this report emphasizes the fatal consequences of common respiratory virus infections in immunocompromised patients. hcov‐nl replication can be reduced by treatment with peg‐ifn if diagnosed early. human coronavirus nl (hcov-nl ) is one of four common human respiratory coronaviruses ( ) . the emergence of sars (severe acute respiratory syndrome) and mers (middle east respiratory syndrome) caused by two recently identified coronaviruses has brought the global attention to the clinical significance of coronaviruses ( ) . clinical outcome data for non-sars/mers coronavirus infections in immunocompromised patients are very rare as are potential treatment options for this type of infection. we here report a case of hcov nl infection in a leukemia patient with fatal ards despite successful virus elimination by pegylated interferon-alpha (peg-ifn-a). the -year-old female pre-t-all patient was treated according to the german-multicenter trial for adult all (gmall) - protocol in january . the patient experienced mild mucositis during therapy, but no other infectious complications (especially no coughing or respiratory tract infection signs) were seen until day . infectious prophylaxis (fluoroquinolones, cotrimoxazole, aciclovir, and mycafungin) were applied according to our institutional recommendations since treatment initiation. in the morning of day , the patient developed fever ( . °c), blood cultures were immediately taken, and the antibiotic prophylaxis was switched to broad spectrum antibiotics (meropenem). in the clinical examination, no infectious focus could be found, particularly no signs of respiratory tract infection. two hours later, the patient collapsed in the bathroom, a positive shock index and decreased oxygen saturation immediately triggered icu referral after stabilization of the patient by oxygen and additive volume supply. antibiotic therapy was escalated to additional linezolid and antifungal therapy to liposomale amphotericine-b due to the rapid clinical worsening as a consequence of suspected pneumonia with sepsis. during the night, the patient deteriorated rapidly because of respiratory failure (partial pressure of oxygen (pao ) mmhg) even though immediate non-invasive ventilation was started after icu referral and the patient was intubated for mechanical ventilation in the following hours. bronchoalveolar lavage (bal) was carried out, and routine microbial analyses (cultures from urine and peripheral blood specimens) were repeatedly taken. clinical instability with highest catecholamine requirement and highly invasive respiration parameters did not allow ct scan for further diagnostic work-up of fulminant respiratory failure, but conventional x-ray of the lungs showed typical signs of acute respiratory distress syndrome (ards) (fig. ). bal revealed a significant viral load of human coronavirus nl (hcov-nl , . copies/ml, real-time rt-pcr), no other respiratory viruses were detected. the blood cultures were positive for multisensible staphylococcus epidermidis triggering plastic change resulting in sterile follow-up blood cultures. of note, coronavirus was neither detectable in the peripheral blood nor pleural effusion. as lung organ failure clinically represented the leading problem, hcov-nl infection was assumed to be the trigger for the ards. there is very limited literature for the treatment of hcov nl infection as this viral infection in otherwise healthy individuals is usually cleared by the functional immune response. based on the successful application of pegylated ifn against the related sars coronavirus in animal experiments ( ), a single injection of lg peg-ifn-a b was applied. intravenous immunoglobulins ( g/kg) were given because of the wide prevalence of anti-hcov-nl in the population. steroids were provided to limit inflammation ( mg/kg per day), based on experience with sars-cov. despite immediate initiation of treatment, the clinical picture significantly worsened throughout the next h and mechanical ventilation had to maximally escalated ( % oxygen supply and highest tolerable pressures) to maintain sufficient oxygenation. the option for ecmo was rejected due to the excessive bleeding risk (thrombocytopenia and already beginning endotracheal bleeding). of note, despite significant clinical worsening at this time point ( h after peg-ifn-a b application), hcov-nl was no longer detectable in a bal sample collected d after the specimen testing positive for nl (fig. ) . the clinical picture of septic shock with leading lung failure was progressive, and the patient died d after the first fever episode due to massive and uncontrolled lung bleeding as a consequence of diffuse alveolar hemorrhage probably triggered by the coronaviral infection. to the best of our knowledge, this is the first report in the literature ascribing ards with subsequent diffuse alveolar hemorrhage in an immunocompromised patient to respiratory infection with hcov-nl despite successful viral clearance by pegylated ifn. the two novel coronaviruses sars-cov and mers-cov, similarly to our case, cause ards, which is associated with high mortality rates. so far, no clinically approved vaccines or antiviral drugs are available for either of these highly pathogenic infections, and the development of novel effective therapeutic and preventive strategies is urgently needed to reduce mortality. in contrast, hcov-nl is an alphacoronavirus that usually induces only mild to moderate upper respiratory tract infections and is associated with croup in children. no specific therapy exists so far and classical antiviral drugs, such as ribavirin, have only limited efficacy in other coronaviruses, such as sars-cov and mers-cov. early reports emphasized the efficacy of ifn-a in severe ards as prophylactic treatment with pegylated ifn-a significantly reduced the viral load and protected rhesus macaques from sars-cov infection in a prophylactic setting ( ) . the importance of ifn-a is further underscored by the observation that both sars-cov and mers-cov infections induce an enhanced ifn type secretion by human plasmacytoid dendritic cells ( ) which also demonstrated therapeutic potential in murine cov-infection models. the efficacy of peg-ifn-a is also supported by our case, as after single injection of peg-ifn-a b, hcov-nl could not further be detected in the bal. however, rapid viral replication obviously severely damaged lung tissue (pneumocytes) and triggered severe inflammation which was not reversible even though the virus was successfully cleared. in the case presented here, the infection route remains elusive and excessive screening of the surrounding did not reveal any other infected individual. however, the case underlines the infection risk in hematological patients receiving chemotherapy and regularly steroids, which are known to be immunosuppressive. therefore, in patients undergoing chemotherapy and clinical signs of respiratory tract infections appear, particular vigilance is required and clinical work-up should include extensive screening for respiratory viruses to prevent excessive viral replication triggering irreversible end organ damage. evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndromerelated coronavirus is inhibited by interferon-alpha pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques high secretion of interferons by human plasmacytoid dendritic cells upon recognition of mers-cov the authors declare no conflict of interest. key: cord- -wmzq lkh authors: ahmed, ahmed a.; rasheed, zafar; salem, tarek; al-dhubaibi, mohammed s.; al robaee, ahmad a.; alzolibani, abdullateef a. title: tnf-α − g/a and ifn-γ + a/t gene polymorphisms in saudi patients with cutaneous leishmaniasis date: - - journal: bmc med genet doi: . /s - - - sha: doc_id: cord_uid: wmzq lkh background: cutaneous leishmaniasis (cl) is well linked with immunogenetic factors. this study was undertaken to test the association of tnf-α − and ifn-γ + gene polymorphisms with the susceptibility of leishmania (l) species among cl patients in central region of saudi arabia. methods: this is a case-control study involved saudi subjects with different l. species and healthy controls from central region of saudi arabia. all subjects were characterized by tnf-α − g/a and ifn-γ + a/t gene polymorphisms using pcr. results: evaluation of genotyping and allelic frequency of tnf-α − g/a in different l. species showed no significant association compared to controls (p > . ). except, in cases of l. tropica that showed significantly higher tnf-α − a versus g allele frequency (p = . ). evaluation of genotyping of ifn-γ + (tt versus aa+at recessive) and allelic frequency of ifn-γ + (t versus a) showed significant higher in l. major and also in total cl cases as compared to healthy controls (p < . ). furthermore, a strong association was observed between the susceptibility of l. major, l. tropica or total cl cases with synergistically combined high tnf-α /inf-γ alleles. conclusions: this is the first report that shows the gene polymorphisms of tnf-α − g/a and ifn-γ + a/t in saudi patients with different l. species infections. data showed that the tnf-α- g/a gene polymorphism is not associated with the susceptibility of cl in saudi subjects. the only correlation was found in between a versus g allelic frequency in l. tropica. importantly, ifn-γ + a/t polymorphism was found to be associated with the susceptibility of l. major and also with total cl subjects. moreover, data from synergistically combined high tnf-α /inf-γ alleles strongly suggest their potential role in the susceptibility of leishmania infection. leishmania is a one of the most common forms of trypanosomiasis protozoan, which is endemic throughout the tropical and subtropical regions of the globe [ ] . cutaneous leishmaniasis (cl) is the most frequent leishmania infection, known to induce dermal lesions that most likely produce permanent life-long scars. now it is well known that cl infection is primarily caused by the transmitting the parasites from the female phlebotomine sandflies bite [ ] . etiology of cl infection dependents on the number of factors such as the characteristics of the parasite, type of sand-fly species, the ecological regions, current/former exposure of infection, and also on the human behavior [ , ] . it is now well documented that the leishmania has more than different species but their prevalence varies from region to region [ ] . these l. species have a wide geographic distribution from south/north america to all over middle east with a significant increase of number of patients in last few years in the countries such as bolivia, brazil, peru, colombia, afghanistan, iran, syria, algeria and also in saudi arabia [ ] [ ] [ ] [ ] . furthermore, the distribution of l. species or cl also reported to have an association with the number of environmental factors including temperature and humidity. moreover, the urbanization and migration have also been documented to have an association with the global diffusion of leishmania infection [ , ] . in saudi arabia, despite of the several important efforts taken by the government, but cl remains to be a major heath issue of the country. several studies have shown that the l. major and l. tropica are the principal reservoirs of this infection, as these species are distributed in almost all region of the country [ ] . reports have also shown that the phlebotomine sandflies are commonly found in the desert regions around the farms, which assumed to be one of the responsible factors for the exposure of this parasitic infection among saudi population [ ] . it is now well established that the proinflammatory cytokines play several crucial roles in onset of cl, which varies from the resistance or the host immune response to infection, especially tumor necrosis factor alpha (tnf-α) and interferon-gamma (ifn-γ) [ ] . the role of tnf-α and ifn-γ was well reported in l. major as both of these cytokines are produced by t helper (th ) cells, which are synergistically activates macrophages [ ] . furthermore, an association of tnf-αor ifn-γ with the increase of tissue damage or ulceration was also reported in cl patients [ ] . moreover, the role of th response against l. major infection was also demonstrated in animal model of l. major [ , ] . out of all studied th cytokines, tnfα and ifn-γ were found to be critical for the initiation of preventive immunity against all studied l. species, particularly l. major [ ] [ ] [ ] [ ] . tnf-α- g/a polymorphism was well studied in several autoimmune/inflammatory diseases, the a allele of tnfα ( g/a) was reported to have higher transcriptional activity by - folds as compared with the common g allele [ , ] . therefore, the a allele frequency has been implicated in the pathogenesis of several infectious as well as autoimmune disorders [ , ] . on the other hand, ifn-γ- a/t polymorphism was also reported to have a genetic link with several autoimmune/inflammatory disorders through + t allele carriers [ , ] . however, it is still unclear whether tnf-α − g/a or ifn-γ + a/t polymorphisms play a role in the leishmania infection. in view of these, we hypothesized that tnf-α- g/a and ifn-γ + a/t gene polymorphisms may be associated with l. species infection. to test this hypothesis, dna samples were isolated from cl patients and the association between tnf-α- g/a or ifn-γ + a/t gene polymorphisms with the l. species susceptibility was studied. the study was carried out in accordance with the code of ethics of the world medical association (declaration of helsinki as revised in tokyo ) for humans and the study protocol was approved by national plan for science, technology and innovation of saudi arabia (nstip/kacst # -med - ). with the institutional review board (irb) approval, the participants were recruited from the clinical units and the qassim hospitals, ksa. all patients were recruited after thorough examination based on the patients clinical appearance, microscopic and specific pcr-based observations as described previously [ , ] . briefly, patients with compatible lesions (range table . genomic dna was extracted from the lymphocytes of peripheral blood from the patients and controls by the magna pure lc instrument (roche diagnostics gmbh, roche molecular biochemical, mannheim, germany) as described previously [ ] . the purity and quantity of dna was measured the by absorbance ration / as described previously [ ] . the genetic variants of tnf-α g/a (rs ), and ifn-γ t/a (rs ) polymorphisms were amplified by the pcr based amplification refractory mutation system (arms-pcr) using the primers sequence summarized in table . the arms-pcr for the tnf-α − g/a gene polymorphism was performed two times for all subjects including patients and healthy human controls. one time the arms-pcr was performed by using common forward primer ( `-tctcggtttcttctccatcg- `) and reverse primer represented for g genotype ( `-ataggt tttgaggggcatgg- ) and the second pcr was performed by the same common primer with the reverse primer that represented a genotype ( `-ataggt tttgaggggcatga- `). detection at the correct size band with reverse primer g that means the homozygote genotype gg, whereas detection of the same size band when repeated with reverse primer a that means the homozygote genotype aa. if detected the correct band from the first run with g and also with the second run with a for the same patients, that means the genotype represents heterozygote ga. similarly, the ifn-γ + a/t gene polymorphism was validated by calculation homozygote tt or gg alleles and heterozygote ta allele. the pcr amplification was carried out using primus ht dual thermal cycler pcr (mwg ag biotech, usa) in a μl total volume containing about ng of genomic dna template, μm of each primer, × go taq® green master mix (promega). the initial denaturation in pcr was carried out at °c for min followed by cycles of °c for s, °c for s, and °c for s. after pcr running, all amplified pcr products were electrophoresed on % agarose gel and fig. tnf-α − g/a polymorphism in cl patients. lane m indicates for dna ladder bp, lanes and 'represent ga genotype for sample , lanes and `represent ga for sample , lanes and `represent gg genotype for sample , lanes and `represent aa genotype for sample , lanes and `represent gg genotype for sample and lanes and `represent gg genotype for sample . all tnf-α − g/a genotype polymorphism detected at the same size ( bp) by the amplification refractory mutation system pcr products were visualized under uv light using ethidium bromide stain. data were analyzed using the statistical software program spss version . the frequencies of studied genotypic and allelic polymorphisms among cases were compared to those of controls using fisher's exact test and odds ratio (or) with the % confidence interval ( % ci). a p level of < . was considered significant. the amplified pcr product for tnf-α- was detected at base pair as shown in fig. , based on these results, different species of cl, the genotypes and the alleles of the host genes polymorphism were determined and evaluated in comparison with their respective healthy controls (supplementary file (table ). in contrast to l. . ) ]. the complete details of tnf-α g/a polymorphism in different l. species and healthy human controls are summarized in table . the amplified pcr product for ifn-γ + were detected at base pair as shown in fig. , based on these results, different species of cl, the genotypes and the alleles of the host genes polymorphism were determined and evaluated in comparison with their respective healthy controls. results show in table table . to further re-evaluate the potential role of genotype frequencies of tnf-α g/a and inf-γ + a/t in the susceptibility of cl infection among the studied saudi patients, the data were analyzed by the synergistically combined genotype frequencies of tnf-α g/a and inf-γ + a/t in all cl patients and normal human controls. analysis from the synergistically combined tnf-α g/a and inf-γ + a/t genotype showed a stronger association of the high tnf-α/inf-γ genotype with the susceptibility of l. ] respectively. the complete details of the synergistically combined genotype frequencies of tnf-α- g/a and inf-γ a/t in all studied l. species and human controls are summarized in table . these results further support a potential role of tnf-α- g/a and inf-γ a/t in the pathogenesis of cl infection among the studied saudi population. this study determined the association of the two important gene polymorphisms in saudi patients with cutaneous leishmaniasis. in one of our previous studies, we reported the prevalence of l. species among cl patients in qassim area of saudi arabia [ ] . our reported data fig. ifn-γ + a/t polymorphism in cl patients. lane m indicate dna ladder bp, lanes and 'represent aa genotype for sample , lanes and `represent at genotype for sample , lanes and `represent tt genotype for sample , lanes and `represent at genotype for sample , lanes and `represent aa genotype for sample and lanes and `represent tt genotype for sample . all ifn-γ + a/t genotypes detected at the same size ( bp) by the amplification refractory mutation system clearly pointed out that cl patients in this region were mainly infected by l. major and l. tropica [ ] . it is now well established that the parasite of leishmaniasis is endemic in all over the globe and now it becomes the major public health problem [ ] . in saudi arabia, the prevalence of cl infection is on the rise and now it remains a major unsolved health problem of the country [ ] . several previous studies reported an association between the susceptibility and resistance of leishmaniasis with the number of cytokines gene polymorphisms [ ] [ ] [ ] , but none of the study did not show direct association of cytokines gene polymorphism with the cl infection nor with the any of the l. species and the controversial role of their snps with leishmaniasis remains continued [ ] [ ] [ ] [ ] . the role of th cytokines such as tnf-α, ifn-γ was well studied in cl patients, as their levels were found to be higher in these patients [ , ] . in support of these, akhzari et al. found that the expression of these cytokines in cl lesion varies with the treatment response [ ] . furthermore, wilhelm et al. reported that treatment of cl patients with tnf-α improves the parasitic burden and decreased the cl lesion size [ ] . because of these important implications of these th cytokines for cl patients and frequency of occurrence of cl infection in saudi arabia, this study was designed to investigate the gene polymorphisms of tnf-α- g/ a and ifn-γ + a/t in patients of cl with different l. species in central region of saudi arabia. analysis of tnf-α- g/a polymorphism on saudi patients with different l. species and healthy controls from the same area, showed no significant association of tnf-α- genotype with cl patients. these results were fully supported with the other studies of tnf-α- polymorphism performed on different population of cl patients [ , ] . in contract of these results, our data also showed the cl patients infected with l. tropica, showed significantly higher tnf-α- a versus g allele frequency. this may be due the different genetic backgrounds of saudi populations. more specifically the frequency of tnf-α a allele in the saudi healthy controls was found in a range of . %, which seem to be statically significant as compared with other [ , ] . besides these, this study also demonstrated ifn-γ + a/t polymorphism on the same cl samples obtained from qassim region of saudi arabia. our results showed that ifn-γ + genotyping (tt versus aa+at recessive) and allelic frequency of ifn-γ + (t versus a) showed significant higher in cl patients infected with l. major and also similar results were obtained in total cl cases as compared with their respective control humans. these are novel findings which have not been reported before. the ifn-γ + tt polymorphism was reported to be associated with the transcription of ifn-γ gene in host cl, in which t allele was found to be involved in higher production of ifn-γ [ ] . in addition, the resistance role of ifn-γ as well as inf-γ a/t in immuno-response against leishmaniasis has also been reported in different animal models [ , ] . all these data either directly or indirectly supported our results. furthermore, a study by kamali-sarvestani et al. also supported our results by pointing out a significantly higher frequency of t allele or tt genotype of ifn-γ + a/ t polymorphism in patients infected with l. major in iranian population [ ] . in contrast of these results, matos et al. reported no association between ifn-γ + a/t polymorphism with the susceptibility leishmaniasis [ ] . despite of these, ifn-γ + a/t snp seems to be involved in the pathogenesis of leishmaniasis by influencing the amount of cytokine released in cl patients [ ] . in support of these, al-bushier reported an association of ifn-γ + a/t polymorphism with the susceptibility of visceral leishmaniasis [ ] . to further determine the potential of tnf-α g/a and inf-γ + a/t snps in the susceptibility of cl infection, the analysis was performed on the synergistically combined tnf-α g/a and inf-γ + a/t alleles. the calculated data showed strong association between the combined tnf-α- g/a and ifn-γ + a/t gene polymorphisms with the susceptibility of l. major, l. tropica and also with total studied cl patients. in short, the data demonstrated no association of tnf-α- gene polymorphism with the susceptibility of cl in saudi patients. the only association was found in between a versus g allelic frequency in l. tropica. whereas, ifn-γ + a/t polymorphism was found to be associated with the susceptibility of l. major and also with the total studied cl cases. moreover, the data from the synergistically combined high tnf-α /inf-γ alleles strongly suggested their potential role in the susceptibility of leishmania infection. who leishmaniasis control team. leishmaniasis worldwide and global estimates of its incidence world health organization: weekly epidemiological record (wer) cutaneous leishmaniasis in cuban immigrants to texas who traveled through the darién jungle, panama spatial epidemiology of cutaneous leishmaniasis in colombia: socioeconomic and demographic factors associated with a growing epidemic cutaneous leishmaniasis in saudi arabia: a comprehensive overview. vector borne zoonotic dis historical perspectives on tumor necrosis factor and its superfamily: years later, a golden journey combined neutralization of interferon gamma and tumor necrosis factor alpha induces il- production but has no direct additive impact on parasite burden in splenic cultures of human visceral leishmaniasis pro-and anti-inflammatory cytokines in cutaneous leishmaniasis: a review immunopathology of leishmaniasis: an update the immunology of susceptibility and resistance to leishmania major sp in mice t helper (h) /th and leishmania: paradox rather than paradigm meta-analysis of tnf-α promoter − a/g polymorphism and sle susceptibility in asian populations tnf promoter - g>a and lta a>g polymorphisms in portuguese patients with systemic lupus erythematosus the interferon gamma gene polymorphism + a/t is associated with severe acute respiratory syndrome a single nucleotide polymorphism in the first intron of the human ifn-gamma gene: absolute correlation with a polymorphic ca microsatellite marker of high ifn-gamma production interferon-gamma and interleukin- gene polymorphisms in pulmonary tuberculosis prevalence of leishmania species among patients with cutaneous leishmaniasis in qassim province of saudi arabia clinical manifestations and classification of old world cutaneous leishmaniasis acquired immunogenicity of dna after modification with malondialdehyde in patients with alopecia areata bisphenol a modified dna: a possible immunogenic stimulus for anti-dna autoantibodies in systemic lupus erythematosus tropical infectious diseases polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis genetics of host response in leprosy lesion size correlates with leishmania antigen-stimulated tnflevels in human cutaneous leishmaniasis the role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis expression of pro-inflammatory genes in lesions, spleens and blood neutrophils after burn injuries in mice treated with silver sulfodiazine rapidly fatal leishmaniasis in resistant c bl/ mice lacking tnf cytokine gene polymorphisms and susceptibility to cutaneous leishmaniasis in iranian patients ifn-gamma modulates the early development of th and th responses in a murine model of cutaneous leishmaniasis mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with leishmania major sp but mount a polarized t helper cell -type cd + t cell response ifng + t/a polymorphism is not associated with american tegumentary leishmaniasis susceptibility but can influence leishmania induced ifn-gamma production impact of ifn-(+ t/a) and il- (− g/a) on the susceptibility to visceral leishmaniasis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors gratefully acknowledged the mr. casimero a. victoria for data collection.availability of data and material the raw data file was uploaded as a supplementary file.authors' contributions aa, aaz participated in study design and coordination of data collection. msad, aar, aaz were responsible for samples collection and for obtaining patients written consents. aa, ts carried out experimentation and data collection. aa, aaz and zr consulted for data interpretation and manuscript drafting. all authors have read and approved the final manuscript. this study was funded by the national science, technology and innovation plan grant from king abdulaziz city for science and technology, ksa (nstip/ kacst # -med - ). the funding body played a role in the design of the study and collection, analysis, and interpretation of data and in the manuscript drafting. the study was approved by the ethical review boards committee of the national science, technology and innovation plan, ksa (nstip # -med - ) and the study was carried out in accordance with the principles outlined in the declaration of helsinki. informed written consent was obtained for all participants. this is the first report that shows the two most important gene polymorphisms of tnf-α- g/a and ifn-γ + a/t in saudi patients infected with different l. species. the study demonstrated no association between the host tnf-α- g/a gene polymorphism and cl infection in saudi patients. the only significant correlation was found in a versus g allelic frequency in cl patients infected by l. tropica. interestingly, ifn-γ- a/t polymorphism was found to be significantly correlated with the susceptibility of l. major and also in total leishmania subjects, suggesting its role in the production of ifn-γ, and thus enhance immune protection. furthermore, a notable association between the susceptibility of l. major, l. tropica or total studied cl patients with the synergistically combined high tnf-α /inf-γ alleles, strongly suggested their crucial role in the onset of cl infection among the studied saudi population. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file .abbreviations cl: cutaneous leishmaniasis; l. major: leishmania major; l. tropica: leishmania tropica; l. infantum/donovani: leishmania infantum/donovani; tnf-α: tumor necrosis factor alpha; ifn-γ: interferon gamma; th cells: type t helper cells; kacst: king abdulaziz city for science and technology; nstip: national plan for science, technology and innovation the authors declare that they have no competing interests. key: cord- -b e bbrp authors: li, shi-fang; zhao, fu-rong; shao, jun-jun; xie, yin-li; chang, hui-yun; zhang, yong-guang title: interferon-omega: current status in clinical applications date: - - journal: int immunopharmacol doi: . /j.intimp. . . sha: doc_id: cord_uid: b e bbrp since , interferon (ifn)-ω, a type i ifn, has been identified in many animals, but not canines and mice. it has been demonstrated to have antiviral, anti-proliferation, and antitumor activities that are similar to those of ifn-α. to date, ifn-ω has been explored as a treatment option for some diseases or viral infections in humans and other animals. studies have revealed that human ifn-ω displays antitumor activities in some models of human cancer cells and that it can be used to diagnose some diseases. while recombinant feline ifn-ω has been licensed in several countries for treating canine parvovirus, feline leukemia virus, and feline immunodeficiency virus infections, it also exhibits a certain efficacy when used to treat other viral infections or diseases. this review examines the known biological activity of ifn-ω and its clinical applications. we expect that the information provided in this review will stimulate further studies of ifn-ω as a therapeutic agent. interferons (ifns) were first reported by isaacs and lindenmann as antiviral proteins that are generated by cells in response to viral infection [ , ] . ifns are composed of three subgroups: type i, type ii, and type iii ifns. the type i ifns, including ifn-α, ifn-β, ifn-ε, ifn-ω, ifnκ, ifn-δ, ifn-τ, and ifn-ζ, exert biological activity through common receptors (interferon-α/β receptor [ifnar ] and ifnar ) [ , ] . type ii ifn, namely ifn-γ, is produced by t lymphocytes and natural killer cells in response to the recognition of infected cells [ ] . type iii ifns consist of ifn-λ , ifn-λ , and ifn-λ , which regulate the immune response via a distinct receptor complex that uses a signaling pathway that is similar to that of type i ifns [ , ] . ifn-ω genes were first found in humans in [ , ] . ifn-ω diverged from the ifn-α gene approximately million years ago, and it is produced primarily in leukocytes [ ] . human ifn-ω genes include four pseudogenes and one full gene that is expressed in leukocytes, and human ifn-ω shares % amino acid sequence homology and similar functions with ifn-α, and % amino acid similarity with ifn-β [ ] . similar to other ifns, ifn-ω is also produced by cells in response to viral infection, and it has biological activities because it binds to the same receptors and activates a pathway that is similar to that activated by ifnar [ ] , while the activation of phosphoinositide- -kinase/ protein kinase b (p k/akt) signaling is also vital for biological responses mediated by ifn-ω [ ] . however, its antigenic structure is distantly related to ifn-α, -β, and -λ, as it does not cross-react with antibodies against them [ ] . as a result, treatment with ifn-ω can be effective for patients who are resistant to ifn-α [ ] . although ifn-ω has been studied extensively, our knowledge of ifn-ω is still limited compared with that of interferon ifn-α and -β. here, we provide a broad review of what is known about the biological activities and potential clinical application of ifn-ω. overall, we hope that the information provided in this review will stimulate further studies of ifnω as a therapeutic agent. until now, ifn-ω has been identified in humans [ ] , felines [ ] , pigs [ ] , horses [ ] , rabbits [ ] , serotine bats [ ] , cattle [ ] , and sheep [ ] , while it has not been found in canines and mice [ ] ; however, the characteristics of ifn-ω differ in different species. the characteristics of ifn-ω from different species are summarized in table . despite such differences, there are common characteristics that exist in these species. generally, ifn-ω genes are intronless, and nglycosylation sites (not present in felines) and transcription factor binding sites, such as irfs, isres, and nf-κb, are present in the promoter regions of these genes [ , , , ] . in addition, all mammalian ifn-ω genes lack the proline codon that precedes the final conserved cysteine codon [ ] . the ifn-ω protein contains several conserved residues, such as arginine at position , cysteine residues at positions and and , seven prolines (four of which are located at positions , , , and of the mature protein), while positions and and form two disulfide bonds. these residues are present at similar locations in type i ifns, and they are vital for their biological activity [ ] . other conserved structural motifs, such as the regions between amino acid residues - , - , and - , are highly conserved among ifn-α proteins, and they play a key role in the biological activity of ifn-ω [ , , ] . in addition to having a structure that is similar to that of type i ifns, ifn-ω also displays the same physicochemical characteristics as type i ifns, such as high sensitivity to trypsin, insensitivity to temperature, and stability at ph [ , ] . some studies revealed that ifn-ω loses its antiviral activities after being treated with . % trypsin. however, it retains high antiviral activity against vesicular stomatitis virus after exposure to ph for h at temperatures ranging from °c to °c [ , , ] . based on analyses of phylogenetic trees obtained from alignments of the ifn-ω protein sequences from these mammal species, it was proposed that all these proteins evolved from a common ancestor [ , ] . type i ifns, including ifn-ω and ifn-α, display a common mechanism of action. ifns interact with specific cell-surface receptors, and then the expression of ifn-stimulated genes (isgs) is induced, some of which encode antiviral effectors or molecules, such as signaling proteins, transcription factors, and apoptotic proteins, while chemokines further regulate ifn signaling and other host responses in a positive or negative manner [ ] . ifn-ω is not an exception. it has been proposed that ifn-ω shares antiviral activities with ifn-α because it binds to the same type i ifn receptor complex. studies have revealed that ifn-ω induces the transcription of the mx , isg , ifit , and isg genes [ , ] . however, different from ifn-α, it shows certain degrees of cross-species activity; therefore, ifns could exhibit different physiological functions in the host. for example, bovine ifn-ω (boifn-ω) protects madin-darby bovine kidney cells, primary embryo bovine lung table ifn-ω in different species and the characteristics of the ifn-ω gene and protein. location characteristics of the ifn-ω gene and protein reference human human chromosome human ifn-ω has at least five members, but only one member of the human ifn-ω family is a functional gene that results in a functional and glycosylated protein. this protein has six additional amino acids located at its carboxyl-terminus ( amino acid residues) and a single polypeptide chain comprising two disulfide bonds and an n-glycosylation-linked site at amino acid . in terms of the primary structures of the type i ifns, there is approximately % amino acid sequence identity between ifn-α and ifn-ω. [ , , , , ] cat no data felines ifn-ω (feifn-ω) includes subtypes that contain an amino-terminal secretory signal sequence from residues - . the mature sequence of ifn-ω contains four highly conserved cysteines (positions , , , and ) and seven prolines (four of those at positions , , , and of the mature protein), while the feifn-ω protein has six additional amino acids at its carboxyl-terminus compared with feifn-α. among these subtypes, feifn-ω and feifn-ω contain a seven-amino-acid insertion at position , and the insert location is comparable to that of the feifn-α, while it has higher antiviral activity than the other subtypes. interestingly, different from that in other mammalian subtypes, these subtypes do not have an n-glycosylation recognition site. [ , ] pig chromosome porcine ifn-ω (poifn-ω) contains seven or eight members with variable open reading frame lengths. the most extensive similarity of the poifn-ω sequences are to bovine leukocyte ifn-ω, and the lowest one is to equine ifn-ω. it contains an ifabd functional domain, multiple putative binding sites for type i ifn receptor subunits, and one putative nglycosylation site (asn/asp-x-ser/thr). the subtypes of the ifn-ω protein, which have five alpha-helices, bear aminoterminal -to -amino-acid signal peptides and four conserved cysteine residues, cys , cys , cys , and cys , which are also found in ifn-α . in addition, all ifn-ω subtypes, except ifn-ω (whose carboxyl-terminus is residues shorter than the other poifn-ω subtypes), have or extended residues, while the pairs ifn-ω / and ifn-ω / share identical sequences. [ , , ] horse chromosome horse ifn-ω contains eight members plus four pseudogenes, which is a greater than the number of ifn-α genes and more diverse than the other type i ifn genes. the ifn-ω gene contains a putative glycosylation sequence, asn-thr-thr, at positions - . the ifn-ω protein has six alpha-helices and it contains ifabd (cd ), interferon (pfam ), and ifabd (smart ) domains, and multiple putative binding sites for the type i ifn receptor subunit and and an nglycosylation site. notably, the length of the open reading frame of each ifn-ω subtype is invariant. [ , ] rabbit no data the rabbit ifn-ω family comprises at least eight genes that display the highest degree of homology with human ifn-ω and the lowest with murine ifn-αii. there are two irf- binding sites at nucleotide positions and , the hexamer repeat sequence ′-gaaann- ′ in the ′ noncoding sequence, and a repetition of the ′-ttatttat- ′ motif, which is similar to many ′ downstream regions of genes encoding inflammatory cytokines. in addition, the immediate promoter region of the rabbit ifn-ω genes has a high proportion of purine residues. an atg sequence, ( ′-gaaatg- ′), was found only in the promoter region of rbifnw at nucleotide position . similarly, ifn-ω includes four cys residues at amino acid positions , , , and . [ ] cattle chromosome q the ifn-ω family in cattle (boifn-ω) consists of members that contain two pseudogenes and functional genes. among these subtypes, the nucleotide similarity is . %- . %, and the amino acid identity is . %- . %. most of these subtypes encode amino acids, expect boifn-ω and boifn-ω , which lack nine amino acid residues at their carboxyl terminus. in addition, cells, feline kidney cells, porcine kidney cells, rabbit kidney cells, and primary bovine testicular cells from vesicular stomatitis virus challenge. however, the antiviral activities of boifn-ω are low in madin-darby bovine kidney cells, but high in porcine kidney and bovine testicular cells. additionally, no protective effects were found on madin-darby bovine kidney cells and baby hamster kidney cells [ , ] . these results suggest that cells have a tendency to be insensitive to ifnω from distantly related species. the antiviral activities of ifn-ω also vary with the subtypes and viral strain used in challenges. in a previous study, the expression of poifn-ω /-ω , poifn-ω /-ω , and poifn-ω were upregulated, while poifn-ω (which has fewer residues at its carboxyl terminus than the other poifn-ω subtypes) was not detected in porcine kidney cells challenged with pseudorabies virus or poly (i):poly(c). interestingly, the level of poifn-ω also increased when peripheral blood mononuclear cells were challenged with pseudorabies virus, and poifn-ω /-ω was upregulated the most in these studies [ ] . similarly, recombinant serotine bats ifn-ω can inhibit european bat lyssavirus type , european bat lyssavirus type , and rabies virus replication in a dose-dependent manner, and different biological activities were elicited (in the order of european bat lyssavirus type < rabies virus < european bat lyssavirus type ) [ ] . the antiviral activities of ifn-ω have also been compared to those of other ifns. when a cells were respectively treated with ifn-ω and type i and type iii ifns, the cells treated with ifn-ω at a concentration of ng/ml obviously repressed the replication of influenza a virus in a dose-dependent manner, and it resulted in larger reductions in viral titers, compared with those obtained with ifn-α [ ] . however, its activity was much lower than the activity of ifn-β , and it was slightly lower than the activities of ifn-λ and ifn-λ . depending on the dose, pretreating caco- cells with human ifn-ω significantly reduced the loads of the h n influenza virus [ ] . interestingly, ifn-β and ifn-ω showed similar inhibitory activity against both ca/ and bj/ influenza viruses at every concentration tested. in addition, both were significantly more potent than ifn-α as far as the inhibitory effects on both stains at a given concentration. in addition to its in vitro activities, ifn-ω has been demonstrated to have antiviral activity in vivo as well. for example, a robust induction of mx mrna and the oas- a protein was found in animals treated by ifn-ω compared with controls [ ] . furthermore, following daily intravenous treatment with human ifn-ω (huifn-ω), the viral load of h n influenza virus decreased significantly in the lung tissues of guinea pigs, and ifn-ω exhibited an identical inhibitory effect against the ca/ influenza virus compared with ifn-α [ ] . although ifn-ω has demonstrated promising antiviral activity against some viral infections in vivo and vitro, several factors could limit its antiviral efficacy, such as poor pharmacokinetics and a short half-life [ ] . pegylation, a covalent conjugation of nontoxic polyethylene glycol (peg), has been demonstrated to improve the plasma half-life of a therapeutic protein and decrease its proteolytic sensitivity [ ] . recently, yu et al. [ ] found that pegylation improved the poor pharmacokinetics of recombinant huifn-ω (rhuifn-ω), despite the fact that its antiviral activity was decreased to some extent. it is worth noting that the bioactivity of pegylated rhuifn-ω was determined by examining its pegylation sites, in which residues at the amino-terminus had higher activities compared with those of residues lys and lys , while the poor pharmacokinetics were determined by the conjugated peg mass. in a previous study, amino-terminally pegylated rhuifn-ω with kda of linear peg, which exhibited . % of the rhuifn-ω biological activity with a half-life of . h, allowed some viral diseases to be treated by fewer doses at longer dosing intervals [ ] . the use of human igg fc fusion proteins has also been demonstrated to be a simple and effective method for prolonging serum half-life [ ] . some studies showed that compared with rhuifn-ω expressed in yeast with a specific activity of × iu/mg, rhuifn-ω-fc had a lower activity of . × iu/mg when it was expressed in chinese hamster ovary cells. furthermore, the terminal half-life of rhuifn-ω-fc was -fold higher than that of rhuifn-ω, and it exhibited better pharmacokinetics characteristics [ ] . perhaps rhuifn-ω-fc will become a new alternative antiviral drug for the treatment of chronic viral infections. several studies also revealed that glycosylation has an essential effect on the activity of ifns [ ] , and glycosylated ifn-ω was more potent than ifn-ω against bovine viral diarrhea virus, yellow fever virus, and west nile virus [ , ] . a possible explanation for its potency is that glycosylated ifn-ω could induce the activation of the sterol regulatory element binding transcription factor, the activating enhancer binding protein -like yy site, the interferon consensus sequence binding protein, the erythroid kruppel-like factor gene, the homeotic gene forkhead of drosophila /hepatocyte nuclear factor / mouse forkhead lung protein, hnf- α, the interferon conserved sequence-binding protein, and the lymphocyte-enriched dna binding protein lyf, which are not induced by ifn-α [ ] . glycosylated ifn-ω also exhibits good clinical applications. previous studies showed that glycosylated ifn-ω + ribavirin (rbv) had a synergistic effect in terms of its antiviral activity in hepatitis c virus (hcv)-infected patients [ ] . similarly, a synergy of the antiviral effects of glycosylated ifnω + rbv against bovine viral diarrhea virus was also obtained. interestingly, an antagonism of the cytotoxic effects of rbv by glycosylated ifn-ω was observed [ ] . thus, the combination of glycosylated ifn-ω and rbv appears to be favorable for the synergy of antiviral activities and the antagonism of the cytotoxic effects of rbv. increasing evidence suggests that ifn-ω may be associated with the nonspecific immune response based on increased survival time, the presence of acute-phase proteins (serum amyloid-a, α- -glycoprotein, and the c-reactive protein), the phagocytic activities of whole blood cells and macrophages, natural killer cell activities, and reduced concurrent viral excretion [ ] [ ] [ ] [ ] [ ] . in general, increasing the number of isgs, mx proteins, and zaps, which could enhance pathogen detection and innate immune signaling, indicates that ifn-ω elicits an immune response. when cells are treated with ifn-ω, mx- , isg , ifit , and isg expression was upregulated in a dose-dependent or time-dependent manner [ , ] . more recently, studies from leal et al. [ ] showed that interleukin (il)- production was affected considerably in feline immunodeficiency virus (fiv)-infected cats treated with recombinant feline ifn-ω (rfeifn-ω) by subcutaneous or oral protocols. specifically, il- plasma levels decreased and proviral loads increased in fiv-cats treated subcutaneously with rfeifn-ω, while il- mrna expression decreased in the oral group. however, the therapy did not affect viremia and the expression of other cytokines (il- , il- , il- , il- p , ifn-γ, and tumor necrosis factor-α) [ ] . thus, ifn-ω appears to be involved in innate immunity, and it could further inhibit viral replication and exert its antiviral activity. ifns have been demonstrated to have antiproliferative effects as a result of inducing cell-cycle arrest and apoptosis, which are independent pathways [ ] . similar to ifn-α, ifn-ω also inhibits cell proliferation in a dose-dependent manner [ , ] . however, ifn-α exhibits a higher antiproliferative activity than ifn-ω at elevated concentrations [ ] . ifn-ω also exhibits antitumor activity in vitro in a cell-specific manner. feline mammary carcinomas are among the most common feline tumors, and they represent an important cause of mortality. when used to treat feline and canine mammary carcinoma cells and derived putative tumor-initiating cells, rfeifn-ω exhibited a dose-dependent, species-specific, target cell-specific action, and an additive effect was observed between rfeifn-ω and conventional anticancer drugs such as mitoxantrone, doxorubicin, and vincristine [ ] . thus, rfeifn-ω may be used as a therapeutic agent in feline and canine mammary carcinomas. there is evidence showing that gene transfer would allow one to take advantage of the beneficial effects of type i ifns without their undesirable side effects [ ] . for instance, huifnβ gene lipofection produces an equal or even superior effect than that of high doses of exogenously applied huifnβ protein. recently, a study conducted by villaverde et al. [ ] demonstrated that felines feifn-ω gene lipofection exhibited an equal or higher cytotoxic effect than rfeifn-ω. this kind of effect was due to the induction of reactive oxygen species generation, mitochondrial membrane potential disruption, and calcium uptake, indicating that ffeifn-ω lipofection is an alternative approach for treating both sensitive and resistant phenotypes with an equal or superior outcome and with fewer adverse effects than recombinant ffeifn-ω therapy [ ] . similarly, injecting the gene encoding interferon ω via the tail vein into mice bearing a hep-a- liver tumor via liposomal gene delivery also significantly decreased tumor weights [ ] . interestingly, this effect was greater ( % tumor inhibition) only when the plasmid contained human thymosin alpha [ ] . a dna ladder (a hallmark of cells undergoing apoptosis) was observed in the tumor cells treated by ifn-ω. interestingly, the antitumor mechanisms involve increasing the expression of tα and ifn-ω, and a plasmid-liposome complex of ifn-ω and ifn-α together exerted greater potency in inhibiting the growth of hep-a- tumor cells than either compound alone [ ] . because of its antiviral, immunomodulatory, anti-proliferation, and antitumor activities, ifn-ω has been explored as a treatment option for some diseases and viral infections in humans and other animals. huifnω displays antitumor effects in several models of human cancer in vivo. in addition, it has also been used to diagnose some diseases [ , ] . when produced in silkworm larvae using a baculovirus vector that includes the feifn-ω sequence, rfeifn-ω, as an immunomodulator, has been registered in many countries (e.g., japan, australia, new zealand, and mexico) to treat canine parvovirus, feline leukemia virus (felv), and fiv infections (http://emea.europa.eu) [ , ] . despite the fact that it is not licensed to treat other viral infections, it exhibits efficacy when used to treat other viral infections such as bovine enterovirus, infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, vesicular stomatitis virus, pseudorabies virus, european bat lyssavirus, influenza virus, feline calicivirus (fcv), and feline herpesvirus- (fhv- ) [ , , , ] . the rfeifn-ω licensed protocol consists of three therapeutic cycles of five daily subcutaneous injections ( mu/kg), beginning respectively on days , , and [ ] . however, its widespread use is limited because this protocol is relatively expensive and time-consuming. some alternative subcutaneous and topical protocols, such as oral and intralesional administration, have been suggested [ , ] . interestingly, no adverse effects were found in cats treated with rfeifn-ω following mucosal administration [ , ] , while subcutaneous administration was accompanied by some mild adverse effects (e.g., fever, lethargy, vomiting, and diarrhea) in some trials [ ] . thus, rfeifn-ω could become an option for treatment in veterinary clinical practice. hcv is a major public health problem with approximately million infections worldwide [ ] . many hcv-infected individuals cannot eliminate the virus, and persistent infection with hcv causes chronic liver disease, including cirrhosis and hepatocellular carcinoma [ , ] . until now, a combination of ifn-α and rbv has been the standard of care for chronic hcv infections [ , ] . however, it has many deleterious side effects. furthermore, some hcv chronic carriers are not suitable for this therapy, and new antiviral approaches are needed. some studies showed that ifn-ω was equally effective at inhibiting hcv replication compared to ifn-α and ifn-β. interestingly, the combinations of ifn-ω and ifn-α or ifn-β exhibited primarily antagonistic interactions [ ] . in addition, other studies showed that the activity of non-glycosylated ifn-ω was comparable to that of ifn-α, while glycosylated ifn-ω was more potent in repressing hcv rna replicons. thus, ifn-ω can be used as a prospective antiviral candidate for the treatment of hcv infections [ ] . in addition to being a potential therapy for hcv, ifn-ω could also be used to diagnose some human diseases. aps- is a monogenic disease that is caused by mutations in the autoimmune regulator (aire) gene [ , ] . currently, many autoantibodies, including those targeting aromatic acid decarboxylase, tyrosine hydroxylase, nacht leucine-rich repeat protein , tryptophan hydroxylase, and ifn-ω, have been found in aps- patients [ ] . a study has shown an anti-ifn-ω antibody was highly prevalent in aps- patients with mutated aire alleles, even though the presence of anti-ifn-ω antibodies preceded aps- clinical symptoms and the development of other autoantibodies [ ] . therefore, antibodies against ifn-ω have become an essential diagnostic tool for aps- . numerous available assays, such as immunoassays, radioligand binding assays, and antiviral neutralization assays, were designed to detect anti-ifn-ω antibodies [ ] [ ] [ ] . however, all of them have drawbacks, including a lack of sensitivity, the use of radioisotopes, the requirement for facilities to grow viral cultures, as well as being time-consuming. some novels diagnosed methods were also developed. zhang et al. [ ] reported that an ipa based on i-labeled ifn-ω showed higher sensitivity to aps- than other anti-type ifn antibodies when aps- patients were examined. similar to the study by zhang et al., aps- patients were also investigated in a study by oftedal et al. [ ] , and these patients were also positive for anti-ifn-ω antibodies, as determined by an ipa based on s-labeled ifn-ω, which also exhibit a greater correlation to aps- than other anti-type ifn antibodies. these results suggest that out of the spectrum of anti-type ifn antibodies, testing for anti-ifn-ω antibodies is more suitable for patients suspected of having aps- . recently, larosa et al. [ ] developed a novel, highly sensitive, and specific assay to measure anti-ifn-ω antibodies. they found that i-labeled ifn-ω and s assays resulted in / and / discrepant samples, respectively, which agrees with aire gene results [ ] . regarding the measurement of anti-ifn-ω antibodies in the studied patients, the i assay seems to have a better performance, as it is disease-specific, more reliable, and more precise than the s assay, making it possible to assess patients with aps- and patients suspected of having aps- prior to an aire gene analysis. fcv, a member of the caliciviridae family, is an important and common pathogen for cats, which is characterized by clinical signs ranging from mild and severe oral ulcerations or upper respiratory tract disease to a severe fatal systemic disease [ , ] . unfortunately, there are still no direct-acting antiviral drugs for the treatment of fcv. previous studies demonstrated that rfeifn-ω had a positive therapeutic effect in fcv-infected cats in some experiments or trials [ , , ] . combination antiviral therapy has become a common practice. recently, researchers revealed that a combination treatment of rfeifn-ω and mefloquine resulted in additive effects with a reduction in the half maximal inhibitory concentration [ ] . another study conducted by lee et al. [ ] showed that the relative expression of ifn-ω, mx, and zaps mrna in crandell-reese feline kidney cells was upregulated significantly in fcv-infected cats. interestingly, after the cats were pretreated with a korean red ginseng (krg) extract or ginsenosides, the expression of these genes was higher than that in the group treated with fcv alone [ ] . it is desirable to find a combination of a krg extract or ginsenosides and ifn-ω that effectively treats fcv-infected cats. because of its similar structural characteristics and genomic structure, fcv could be a representative human norovirus surrogate [ ] ; thus, these therapies could also be used to treat human norovirus infections. fhv- , a double-stranded dna virus that replicates in the nucleus of host cells and produces intranuclear inclusion bodies, is one of the foremost causes of feline upper respiratory tract disease [ ] . it has been demonstrated that rfeifn-ω has a dose-dependent inhibitory effect on the replication of fhv- in vitro [ ] . several studies showed conflicting results about the biological activity of rfeifn-ω against these two viruses in cats. for instance, cats suffering from fcvassociated feline upper respiratory tract disease were treated with an intravenous dose of . or mu/kg rfeifn-ω three times per day every other day [ ] . while improved clinical signs were detected within days, no control group was used in this study. in another study, cats with fhv- -associated ocular keratitis were treated five times per day with a dose of . mu/ml rfeifn-ω, and although a significant improvement of ocular signs was observed after weeks of treatment, no control group was included [ ] . in , using a placebo as a control group, the effect of rfeifn-ω in cats that were pretreated with rfeifn-ω before challenge with fhv- was investigated [ ] . unexpectedly, no beneficial effects were observed in these cats. ballin et al. [ ] reported a similar finding; they found that compared to a control group treated with a placebo, cats treated with rfeifn-ω did not exhibit improved clinical signs of acute viral feline upper respiratory tract disease despite having lower fcv copy numbers than cats receiving rfeifn-ω [ ] . more studies are needed to verify that rfeifn-ω has a therapeutic effect in vivo. felv, an endogenous retrovirus that belongs to the genus gammaretrovirus of the family retroviridae causes a variety of degenerative and immunosuppressive disorders such as severe anorexia, cachexia, and progressive weakness [ , ] . studies indicated that rfeifn-ω affects the felv cycle at the post-transcriptional level. however, it did not alter protein synthesis [ ] . treatment with rfeifn-ω lowered reverse transcriptase activity, which is used to evaluate the amount of infectious viral particles, in a dose-dependent manner in felv-infected fl cells [ ] . furthermore, according to the half maximal inhibitory concentration, rfeifn-ω was more potent than rhuifn-α in inhibiting reverse transcriptase activity. in addition, this study showed that ifn-ω decreased the viability of felv-infected fl cells and increased apoptosis and mortality in infected cells [ ] . fiv is a feline lentivirus that was first described in , and many studies have focused on it because its complex genomic and immunopathogenic features that are similar to those of human immunodeficiency virus [ , ] . furthermore, fiv coinfection was found to occur in % of felv-infected cats [ ] . previous studies showed that rfeifn-ω significantly decreased clinical scores and rates of mortality, and improved abnormal hematologic parameters (red blood cell count, packed cell volume, and white blood cell count) in virus-infected cats [ ] . these studies demonstrated that rfeifn-ω had significant therapeutic effects on clinical signs. evidence also showed that an effective immune modulation in fiv-cats could be obtained by treating them orally with rfeifn-ω. in a previous study [ ] , fiv-infected cats that were orally administered rfeifn-ω at a dose of . mu/cat gained weight, although no dramatic changes were obtained regarding viral loads and the cd + /cd + ratio. however, this study did not assess any clinical benefits [ ] . recently, oral protocols were also used to administer rfeifn-ω in naturally fiv-infected cats, and initial clinical scores were obtained that were similar to those obtained using the licensed subcutaneous protocol [ ] . interestingly, similar to the use of the licensed protocol, rfeifn-ω also induced a significant clinical improvement of cats that were treated orally, despite viral excretion, while there were no significant variations in acute-phase proteins [ ] . these results suggest that oral administration of rfeifn-ω can be an effective alternative therapy for fiv-infected cats. fcgs is a multifactorial disease. some infectious viral diseases, such as fiv, fhv- , and felv, can also trigger fcgs by inducing immune suppression and dysregulation [ , ] . some studies showed that oral treatment with rfeifn-ω was a beneficial therapy for fcgs. a study conducted by hennet et al. [ ] found that the oral protocol correlated with an obvious clinical improvement of fcgs lesions (caudal stomatitis and alveolar/buccal mucositis). furthermore, the only difference between rfeifn-ω and corticosteroids were that better pain relief was obtained with the rfeifn-ω treatment. recently, two clinical cases showed that the use of oral rfeifn-ω was also effective in type ii diabetic cats with fcgs [ ] . in this study, a clinical improvement of oral lesions and a concurrent reduction of the required insulin dose had a positive relationship with rfeifn-ω therapy, which agrees with hennet's work showing that there was an overall relief of pain in refractory cases of fcgs [ , ] . parvovirus, a small, non-enveloped, single-stranded dna virus, infects humans and carnivores in a host-specific manner. canine parvovirus type- (cpv), feline panleukopenia virus, and mink enteritis virus share approximately % homology in their nucleotide and amino acid sequences [ ] . there are several therapies used to treat cpv infections; however, many of them have drawbacks [ ] . reports suggest that rifn-ω has a significant therapeutic effect on cpv-infected dogs [ , ] . for instance, it improved parvoviral clinical signs (such as pyrexia, vomiting, anorexia, and diarrhea), and reduced severe parvoviral enteritis and mortality in cpv infections. a study by kuwabara et al. [ ] showed that continuous immunoenhancement after rfeifn-ω treatment and whole blood cells counts > × /μl in dogs might be essential for improving severe clinical symptoms caused by cpv infection. cpxv, belonging to the orthopoxvirus genus, is most frequently found in domestic cats with > cases having been reported, and it is endemic in northern europe and western areas of the former soviet union [ , ] . however, until now, no antiviral drugs have been licensed to cure cats of cpxv infection. a recent study conducted by mcinerney et al. [ ] showed that treatment with rfeifn-ω had a certain protective effect in cats infected with cpxv, although two of the four cats in the study died. nevertheless, rfeifn-ω may be potentially useful for treating cpxv-associated pneumonia and dermatitis in cats. of note, additional research is needed before this can be confirmed. fibrosarcoma, a common disease in cats, accounts for over % of all skin tumors in this species [ ] . currently, few successful treatments for fibrosarcoma have been reported in cats, although treatment of feline fibrosarcoma with rfeifn-ω has been demonstrated to be safe, well tolerated, and easily performed. a study by hampel et al. [ ] revealed that rfeifn-ω increased the expression of major histocompatibility i molecules on feline fibrosarcoma cells, while eosinophilia, neutropenia, and weight loss were remarkably reduced by rfeifn-ω treatment. however, a placebo-controlled trial is needed. cad is a common, allergic skin disease. some therapeutic protocols have been reported, such as allergen-specific immunotherapy, the use of topical and oral glucocorticoids and calcineurin inhibitors, essential fatty acid supplementation, and bathing [ ] . however, none of them were highly efficacious, and some severe side-effects were observed occasionally. fortunately, a study showed that treating dogs with cad with subcutaneously administered rfeifn-ω had a comparable efficacy to orally-administered cyclosporin [ ] . in later studies [ ] , oral ifn therapy showed higher efficacy than the subcutaneous treatment in terms of the canine atopic dermatitis extent and severity index and total scores, although the improvement of pruritus and quality of life scores were not significant; however, in this study, serum antibodies against rfeifn-ω were not detected in any of the dogs. fip is a common immune-mediated disease that is triggered by infection with a feline coronavirus [ ] . previous studies showed that ifn-ω is a beneficial therapy in fip-infected cats. however, these studies used fewer than cats per group and did not include a control group. furthermore, the effect of ifn-ω was not reliably confirmed in fip [ , ] . for instance, in a study of cats suspected of having fip, cats were treated with u/kg of feline feifn-ω every other day until remission, followed by injections every week; four of the cats lived longer than years [ ] . in a subsequent study conducted by ritz et al. [ ] , a control group was used, but there were no significant differences between the survival times of cats treated with feifn-ω and a placebo, although the feifn-ω treatment resulted in a significantly lower lymphocyte count. interestingly, one cat survived > months in the feifn-ω group. these results suggest that feifn-ω could also have a certain therapeutic effect in fip-infected cats. as an ifn, ifn-ω has been demonstrated to have similar levels of biological activities as ifn-α and ifn-β, which are limited by their toxicity and side effects; thus, ifn-ω may be a useful and alternative antiviral agent. although rfeifn-ω has only been registered to treat cpv, felv, and fiv infections, it shows comparable efficacy when used to treat other viral infections or diseases. however, its safety (e.g., toxicity and side effects) when used at a high dose has not been investigated extensively. in addition, the antiviral activity of ifn-ω in some fatal infectious diseases also needs to be 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in germany: clinical and epidemiological aspects antiviral activity of the eb peptide against zoonotic poxviruses pulmonary cowpox in cats: five cases congenital infantile fibrosarcoma mimicking a cutaneous vascular lesion: a case report and review of the literature adjuvant immunotherapy of feline fibrosarcoma with recombinant feline interferon-omega international task force on canine atopic dermatitis, treatment of canine atopic dermatitis: clinical practice guidelines from the international task force on canine atopic dermatitis the use of recombinant omega interferon therapy in canine atopic dermatitis: a double-blind controlled study oral and subcutaneous therapy of canine atopic dermatitis with recombinant feline interferon omega feline coronaviruses, pathogenesis of feline infectious peritonitis comparison of different tests to diagnose feline infectious peritonitis effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis the authors declare no conflicts of interest. key: cord- -d sj v authors: ströher, ute; dicaro, antonino; li, yan; strong, james e.; aoki, fred; plummer, frank; jones, steven m.; feldmann, heinz title: severe acute respiratory syndrome-related coronavirus is inhibited by interferon-α date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: d sj v current treatment schemes for severe acute respiratory syndrome (sars) include broad-spectrum antibiotics, glucocorticoids, and ribavirin. we evaluated the susceptibility of the sars-related coronavirus (sars cov) to ribavirin and interferon (ifn)-α in vitro by use of cytopathic effect, plaque assay, and immunoblot analysis. ribavirin did not inhibit viral growth at concentrations attainable in human serum. in contrast, ifn-α showed an in vitro inhibitory effect starting at concentrations of iu/ml. in conclusion, ribavirin alone is unlikely to be beneficial in the prophylaxis or treatment of sars cov infections. clinical trials with ifn-α might be justified to determine a beneficial effect on the outcome of sars. lishing an antiviral treatment for patients with sars. sars is primarily diagnosed by a process of clinical/epidemiological exclusion. despite improvement in the recent past, laboratory diagnosis, particularly molecular detection of the virus by polymerase chain reaction, remains unreliable, especially in the first few days of the disease; in addition, the tests are not yet validated. serologic analysis is thought to be the confirmatory laboratory test, but most patients with sars develop detectable igg antibody levels - weeks after the onset of symptoms. thus, differential diagnosis remains a problem for the clinician at the time of initial presentation, particularly in individuals without known exposure to other patients with sars or history of travel to a region where sars is endemic. the case-fatality rate of . % reflects, in part, the lack of an effective specific treatment for this viral infection. broad-spectrum antibiotics to treat community-acquired bacterial pneumonia, glucocorticoids, and ribavirin have been administered to patients with sars, but their efficacy is unknown [ ] [ ] [ ] . recently, glycyrrhizin has been demonstrated to inhibit the sars-related cov (sars cov) in vitro [ ] . ribavirin has been used on the basis of its ability to inhibit other covs [ ] and also showed an inhibitory effect on several other rna viruses, such as bunyaviruses and arenaviruses [ ] . to support the search for effective antiviral treatments, we evaluated the susceptibility of sars cov isolates (detailed studies were performed with the tor isolate [toronto, canada]) to ribavirin and interferon (ifn)-a- b in vitro. our data indicate that ribavirin does not inhibit the virus at concentrations attainable in human serum but that ifn-a- b may be useful and deserves further evaluation as a therapeutic agent. materials and methods. studies were performed with the recently sequenced tor isolate, obtained from a toronto patient [ , ] . the general findings were confirmed by use of additional independently obtained isolates from toronto patients (tor , tor , and tor ). all isolates originated from nasopharyngeal swabs. vero e cells (atcc ) were used for the susceptibility studies. the cells were maintained in dulbecco's modified eagle's medium (dmem) containing % fetal bovine serum (gibco brl). vero e cells were infected at an moi of . with the sars cov. after h of adsorption, the supernatants were replaced with dmem containing various amounts of ribavirin ( - mg/ml) or ifn-a- b ( - iu/ml) (schering-plough) for or h. for immunoblot analysis, cell lysates were analyzed by sds-page on % tris gels. protein was electrotransferred to polyvinyl- idene difluoride membrane (immobilon p membrane; millipore). viral antigen was detected by use of enhanced chemiluminescence, using a patient serum sample and horseradish peroxidase-conjugated anti-human igg. for plaque assay, confluent vero e cells were infected in fold dilutions of sars cov; min after infection, the inoculum was removed, and cell monolayers were overlaid with . % lowmelting-point agarose in dmem, with or without ifn-a- b. titers were determined h after infection, following crystal violet staining. for microassay, vero e cells were infected with sars cov at an moi of . for min at Њc and then were incubated in dmem containing various amounts of ifn-a- b ( - iu/ml); h after infection, cells were formalin fixed, removed from biocontainment, and analyzed by use of phase-contrast microscopy, using an axiovert microscope (zeiss). results. in vitro studies were conducted in vero e cells, which is one of only a limited number of cell lines susceptible to sars cov. vero e cell monolayers were treated with ribavirin at various concentrations ( , , , , , , and mg/ml) for h. treatment was initiated concurrently with inoculation of the cell cultures with virus at an moi of . . inhibition of viral cytopathic effect (cpe) was used to determine the antiviral effect of the drug. no reduction in cpe was detected after days, compared with untreated control samples. viral titers determined from tissue culture supernatant were identical for treated and untreated cells and amounted to ∼ pfu/ml (data not shown). ribavirin was tested up to concentrations times greater ( mg/ml) than those that inhibit the replication of ribavirin-sensitive viruses, such as arenaviruses and bunyaviruses [ , ] . there was no evidence that the sars cov was susceptible to the action of ribavirin. ifn susceptibility has been demonstrated for several coronaviruses, such as mouse hepatitis virus, porcine transmissible gastroenteritis virus, feline infectious peritonitis virus, and human coronavirus [ ] [ ] [ ] [ ] . inhibitory concentrations ranged from iu/ml [ ] to iu/ml [ ] . therefore, we tested the susceptibility of the tor isolate to ifn-a- b (schering-plough). in brief, vero e cells were infected at an moi of . , either h after or at the time of treatment with ifna- b ( - iu/ml). treatment was continued for h. the cpe was used to demonstrate an antiviral effect. as indicated in figure , the cpe on day after infection was significantly decreased in tor -infected cultures treated with iu/ml of ifn-a- b in both treatment groups, compared with that in the untreated control group. this indicated that pretreatment was not necessary for the protective effect in tissue culture (data not shown). almost complete protection was achieved when infected vero e cells were treated with iu/ml of ifn-a- b. to quantify the effect of ifn-a- b on the replication of the sars cov, vero e cells were infected at an moi of . and were incubated in the presence ifn-a- b ( - iu/ml), as described above. after h, cells and supernatants were harvested to determine protein expression by immunoblot analysis and virus yield by plaque titration on vero e cells. as indicated in figure a , a concentration of iu/ml of ifn-a- b substantially reduced virus yields, and ∼ log decrease in virus yield was achieved at a dose of iu/ml of ifn-a- b. the reduction in virus yield seems to be related to a decrease in virus growth, as supported by the change in the plaque morphology ( figure b ). inhibition of viral protein expression was investigated by use of immunoblot analysis after sds-page of the cell lysates ( figure c ). a serum sample from a patient with sars who seroconverted to the sars cov was used to detect viral proteins. earlier studies had shown that this serum sample reacted strongly with the nucleocapsid protein of the sars cov [ ] . viral protein expression was reduced at a concentration of iu/ml and abolished at concentrations of and iu/ml of ifn-a- b. for confirmation, the effect of ifn-a- b on additional independent sars cov isolates (tor , tor , and tor ) obtained from canadian patients with sars was determined. all isolates showed the same pattern of inhibition by ifn-a- b as . b, analysis of cell-to-cell spread of sars cov in the presence and sd absence of ifn-a- b. vero e cell monolayers were infected with fold dilutions of the sars cov. after h of virus adsorption, the inoculum was removed, and cell monolayers were overlaid with . % low-meltingpoint agarose in dmem (b ) or dmem containing iu/ml of ifna- b (b ). on day after infection, cells were stained with crystal violet. c, protein analysis. cell lysates were subjected to % sds-page. protein was electrotransferred to polyvinylidene difluoride membrane (immobilon p membrane; millipore). viral antigen was detected by use of a patient serum sample and horseradish peroxidase-conjugated anti-human igg, by use of enhanced chemiluminescence. np, nucleoprotein. d, comparison of the effect of antiviral activity on different sars cov isolates. vero e cells were infected with independently isolated sars covs (isolates tor , tor , tor , and tor ) and were analyzed for their sensitivity to ifn-a- b, as described above. the tor isolate ( figure d ). for all isolates, the ic of ifna- b was ∼ iu/ml. the present study has demonstrated that the sars cov is not susceptible to ribavirin at high concentrations, even when added at the time of infection. in contrast, the virus is inhibited in tissue culture by ifn-a- b at concentrations у iu/ml. although the relationship between in vitro and serum ifn concentrations and biological effect is not known, peak serum ifn concentrations of at least iu/ml are observed after intramuscular (im) injection of iu/ml . ϫ [ ] . similar ifn serum concentrations ( - iu/ml) after im injection were reported from a study in patients with hepatitis c virus (hcv)-associated systemic vasculitis [ ] . doses up to iu/ml have been infused intravenously, which . ϫ might achieve serum concentrations in the range observed to inhibit the sars cov. at present, there is no data on the serum levels of ifn-a in patients with sars, which would be helpful before treatment. the in vitro studies have thus far only been performed in vero e cells. this is significant, because vero e cells have been shown to have an ifn i gene deficiency and thus are unable to express endogenous ifn i [ ] . however, the ifndependent pathways are functional and can be activated by exogenously provided ifn. we have tested a limited number of different cell lines (hela, t, t , and crfk) for susceptibility to the sars cov; thus far, we have failed to identify another line that supports viral replication (data not shown). the observation that the vero cell clone e propagates the virus may indeed be related to the lack of a functional ifn system and, thus, supports the findings in the present study. despite efforts by our group and others to grow the virus in different rodent species, a small animal model has not yet been established. recently, cynomolgus macaques (macaca fascicularis) have been infected with the sars cov, which induced similar clinical symptoms and pathological findings to those of humans with sars [ ] . because only a limited number of animals have been used in the present study, it remains to be determined in the future whether the cynomolgus macaque is a suitable model for sars and, thus, for antiviral in vivo studies. until an animal model is definitively established, it might be justified to consider clinical trials with ifn, which is already approved for human use, to determine whether ifn has a beneficial effect on the outcome of sars cov infection. combined with the observation of a lack of efficacy of ribavirin in patients with sars, our in vitro data further support the conclusion that ribavirin, at least alone, is unlikely to be beneficial in the prophylaxis or treatment of sars cov infection. whether combined therapy with ifn-a- b and ribavirin would inhibit the replication of the sars cov in vitro has not yet been evaluated; the combination is more effective than either agent used alone for the treatment of hcv infection in humans. the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada a cluster of cases of severe acute respiratory syndrome in hong kong glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug effect of ribavirin on bunyavirus reproduction in cell culture and in an experiment on white mice inhibitory effect of selected antiviral compounds on arenavirus replication in vitro the biological relationship of mouse hepatitis virus (mhv) strains and interferon: in vitro induction and sensitivities comparative susceptibility of respiratory viruses to recombinant interferons-alpha b and -beta inhibitory effects of recombinant feline interferon on the replication of feline enteropathogenic viruses in vitro antiviral action of interferon-alpha against porcine transmissible gastroenteritis virus mass spectrometric characterization of proteins from the sars virus: a preliminary report clinical pharmacokinetics of interferons plasma exchange and interferon-alpha pharmacokinetics in patients with hepatitis c virus-associated systemic vasculitis transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis koch's postulates fulfilled for sars virus we thank the severe acute respiratory syndrome task force at the canadian science centre for human and animal health for valuable discussions. key: cord- -yr jwm authors: karnam, guruswamy; rygiel, tomasz p.; raaben, matthijs; grinwis, guy c. m.; coenjaerts, frank e.; ressing, maaike e.; rottier, peter j. m.; de haan, cornelis a. m.; meyaard, linde title: cd receptor controls sex-specific tlr responses to viral infection date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: yr jwm immunological checkpoints, such as the inhibitory cd receptor (cd r), play a dual role in balancing the immune system during microbial infection. on the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. we studied the influence of the inhibitory cd -cd r axis on clearance and pathology in two different virus infection models. we find that lack of cd r signaling strongly enhances type i interferon (ifn) production and viral clearance and improves the outcome of mouse hepatitis corona virus (mhv) infection, particularly in female mice. mhv clearance is known to be dependent on toll like receptor (tlr )-mediated type i ifn production and sex differences in tlr responses previously have been reported for humans. we therefore hypothesize that cd r ligation suppresses tlr responses and that release of this inhibition enlarges sex differences in tlr signaling. this hypothesis is supported by our findings that in vivo administration of synthetic tlr ligand leads to enhanced type i ifn production, particularly in female cd (−/−) mice and that cd r ligation inhibits tlr signaling in vitro. in influenza a virus infection we show that viral clearance is determined by sex but not by cd r signaling. however, absence of cd r in influenza a virus infection results in enhanced lung neutrophil influx and pathology in females. thus, cd -cd r and sex are host factors that together determine the outcome of viral infection. our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of cd -cd r. to generate an appropriately controlled response during infections, the immune system is balanced by the action of activating and inhibitory receptors. lack of inhibition leads to excessive inflammation and autoimmunity and other severe disease symptoms. one of the receptors regulating this balance is cd receptor (cd r) [ ] . cd r was originally described as a myeloid receptor [ ] , being expressed on macrophages, granulocytes and dcs, but later we and others recognized that it is also expressed on t cells, b cells and nk cells [ , ] . the cd r intracellular domain is devoid of the classical immunoreceptor tyrosine-based inhibition motif (itim) present in most immune inhibitory receptors but it does have three tyrosine residues that can be phosphorylated, one of which is embedded in an npxy motif. cd r-downstream signaling is dependent on the recruitment of dok and rasgap [ ] . the signal that triggers cd r and results in delivery of an inhibitory intracellular signal to the cell is given by its ligand cd , which has a short intracellular tail devoid of any known signaling motifs. cd is expressed on thymocytes, activated t cells, b cells, dendritic cells (dcs), vascular endothelial cells, hair follicular cells, in the central nervous system and in the retina (reviewed in [ ] ). both in mice and humans, cd exclusively binds to the inhibitory cd r. in contrast to humans, the mouse cd r family contains several activating receptors, but these do not bind cd [ ] . cd / mice were first described to be more susceptible to autoimmune disorders [ ] . later its role in microbial infections was recognized. infection of cd / mice with the gram negative n. meningitides causes increased lethality, proinflammatory cytokine production and lymphocyte activation [ ] . we and others showed that in mouse influenza a virus infection cd deficiency aggravates immune pathology [ , ] . these studies were exclusively performed in female mice. they indicate that cd -cd r signaling controls the strength of the initial antimicrobial response and the return to homeostasis. we here studied the influence of cd -cd r blockade on clearance and pathology in two different virus infection models, coronavirus and influenza virus, in both male and female mice. mouse hepatitis coronavirus (mhv) is an accepted model for the most illustrious coronavirus (cov): severe acute respiratory syndrome (sars)-cov. host control of mhv infection is completely dependent on an immediate type i ifn response, initiated upon tlr triggering by viral rna. mice lacking this pathway show massive mhv replication and fatal infection within a few days [ , ] . as a model where a strong anti-viral response causes immune mediated pathology we studied influenza a virus infection in which immune pathology is known to be important for clinical outcome. we here report that lack of cd r signaling has a more profound effect on the beneficial but also on the pathological immune responses to viruses in female mice as compared to male mice, which can be attributed to the capacity of cd r to inhibit tlr responses. to determine the role of cd -cd r signaling in cov infection, we intraperitoneally inoculated male and female wild type (wt) and cd / mice with a recombinant mhv encoding luciferase (mhv-eflm). we monitored viral spread using bioluminescence imaging (bli) at day and after infection [ , ] . interestingly, at both time points we observed a decreased viral spread in female wt mice when compared to males. moreover, lack of cd resulted in a significantly lower level of viral replication in females ( figure a ,b and figure s a ,b,c). the viral rna load in the livers at day was assessed by quantitative pcr and confirmed the imaging results: wt female mice had significantly lower viral rna levels than wt male mice ( figure c) . again, cd -deficiency greatly decreased virus load in female mice. this was confirmed in histological liver sections stained with a monoclonal antibody against mhv (data not shown). the number of focal lesions in the liver, typical for mhv was also lower in female mice and again cd -deficiency had a significant effect on these lesions only in female mice ( figure d,e) . clearance of mhv critically depends on tlr -mediated type i ifn production by hematopoietic cells [ , ] . in wt mice, mhv infection resulted in detectable ifn-a production only in female mice ( figure a) . in cd / animals, all females and out of males produced detectable amounts of ifn-a and cd / female mice produced the highest amounts of ifn-a ( figure a ). ifn-a concentrations in serum inversely correlated with viral load at day (p = . ) (data not shown). thus, the combination of female sex and cd deficiency results in increased type i ifn production and decreased viral load and pathology upon mhv infection. enhanced tlr responses in female cd / mice sex differences in tlr responses have previously been reported for humans [ , ] . our observed sex difference in ifn-a production and viral clearance upon mhv infection in mice is likely due to a similar sex bias in tlr responses. we hypothesized that cd r signaling suppresses tlr responses in wt mice. release of this inhibition would further reveal the intrinsically higher tlr responses in females and result in more rapid viral clearance. to test this hypothesis, we first administered a tlr ligand in vivo. as described previously [ ] , intraperitoneal injection of a synthetic tlr ligand (imiquimod) leads to rapid release of type i ifn into the circulation. one-hour after injection we only detected significant amounts of ifn-a in the sera of female cd / mice, confirming the notion that cd r inhibits the intrinsic potential for a higher tlr response in females ( figure b ). we sacrificed the mice at hrs after ligand injection, at which time point no serum ifn-a was detectable (data not shown) but type i ifn mrna could be detected in the livers of these mice. again, females expressed more ifn-b mrna than males, with cd -deficient female mice displaying even more elevated ifn-b mrna levels ( figure c) . thus, release of cd -mediated inhibition leads to increased production of type i ifn in response to tlr ligands, particularly in female mice. we next tested whether cd r-mediated signaling directly inhibits signals transduced via tlr . we generated a chimeric construct containing a lair- receptor in which the intracellular tail was replaced by that of cd r. this allows for efficient cross-linking using anti-lair- antibodies to induce signaling via the cd r cytoplasmic tail. we transfected hek cells with plasmids encoding a lair- -cd r chimeric receptor, human tlr and a luciferase reporter under control of a nf-kb driven promoter. cross-linking of the chimeric receptor by anti-lair- antibody, but not by isotype-matched control antibody, resulted in robust inhibition of imiquimod-induced nf-kb activity ( figure a ). cd r contains three intracellular tyrosine residues. a chimeric lair- -cd r protein in which all three tyrosines are mutated to phenylalanine (fff) did not suppress tlr responses upon cross-linking, indicating that the observed inhibitory effect is indeed dependent on cd r-signaling ( figure a ). in a cell line with stable ectopic expression of tlr and transient expression of the nf-kb-reporter and the lair- -cd r constructs we also observed that tlr signaling was inhibited through cd r-mediated signaling. a similar effect was observed when luciferase expression was driven by an ifn-b promoter ( figure b) . thus, the enhanced type i ifn production and viral clearance of mhv in female cd / mice can be explained by the release of cd r-mediated inhibition of the intrinsically higher tlr responses in females. a strong anti-viral response can also cause immune mediated pathology that can be detrimental to the host. we therefore moved to a virus infection model in which immune pathology is known to immune responses need to be carefully orchestrated to prevent disease due to an overactive immune system. immunological checkpoints are provided by immune inhibitory receptors, which set a threshold for activation and dampen the immune system. in the case of a viral infection, this prevents pathology induced by the immune system, but on the other hand may prevent adequate removal of the virus. in this paper, we show that removal of such an immunological checkpoint in mice leads to rapid removal of corona virus, but also to more immuneinduced disease symptoms in case of influenza virus infection. we observe this predominantly in female mice. we demonstrate that this particular checkpoint inhibits anti-viral responses that are naturally stronger in females. release of this checkpoint enlarges these sex differences. our findings have major implications for therapeutic use of blockers of this pathway, which are currently in clinical trials for the treatment of cancer, as we predict that female patients will have a stronger response to such therapeutics. be important for clinical outcome. upon intranasal infection with influenza a virus we again observed a sex bias in the viral load, measured in the lungs at day post infection ( figure a ). female mice had lower viral loads compared to male mice, which was accompanied by enhanced ifn-a concentrations in the broncho-alveolar lavage (bal) fluid ( figure b ). however, as opposed to mhv infection, cd -deficiency did not enhance type i ifn production in influenza virus infection ( figure b ). confirming previous reports, we found a significantly enhanced body weight loss in female cd / mice compared to wt females ( figure c ) [ , ] . although male cd / mice lost more weight than wt males, the weight loss started later and the difference was not as prominent ( figure d ). this may indicate that lack of cd results in a more severe pathology in females. we observed an increased level of cellular infiltration in lung tissue of females ( figure s a ). therefore we determined lung cellular influx by differential cell count in bal fluid ( figure s b-d) . the total number of cells in bal fluid was higher in all female groups but differences were not significant ( figure s b ). the number of lymphocytes was increased in females of both genotypes ( figure s d ). at day after infection, the number of lymphocytes was increased in females of both wt and cd / groups. at day , the number of neutrophils in the bal fluid was decreased in all groups, still the wt females displayed elevated numbers over males. importantly, lack of cd resulted in significantly higher neutrophil counts in females but not in males ( figure e ). as an additional parameter of lung damage we measured the total protein content in the bal fluid. lack of cd resulted in increased protein levels in the bal fluid of both males and females, especially at day after infection ( figure f ). overall these data indicate that female mice experience increased lung pathology upon influenza a virus infection, which is aggravated by the lack of the cd r-regulatory pathway. in agreement with the increased neutrophil counts we measured elevated levels of kc (il- ) in the bal fluid ( figure g ). il- concentrations were increased in all females at and days after infection, which was further enhanced by the lack of cd only at day ( figure h ). tnf-a was hardly detectable, but significantly increased levels were measured in cd / female mice at day after infection ( figure i ). thus, from two different viral infection models we can conclude that sex has a profound effect on type i ifn production and viral clearance. this study is the first to report a significantly enhanced viral clearance in female mice due to a sex bias in tlr responses. sex differences in tlr induced type i ifn production have previously been reported for humans [ , ] and our data show that in mice this has a strong impact on the course of a viral infection. the mechanism for this is not understood. on the one hand, incomplete inactivation of the tlr gene, on the x chromosome, resulting in higher tlr expression in females has been proposed [ , ] . in our experiments, tlr mrna expression was equal in male and female mice ( figure s ). this is consistent with a prior report in which no evidence for escape from x-inactivation of the tlr gene in humans was found [ ] . there are conflicting reports concerning the influence of sex hormones on tlr responses [ , ] . alternatively, sex-dependent epigenetic mechanisms may contribute [ ] . we demonstrate direct inhibition of tlr signaling through cd r. previously, cd r-mediated inhibition of lpsinduced cytokine production was reported [ , , ] . this suggests that cd r affects proximal events in the tlr signaling pathway. cd r is a unique inhibitory receptor, since its intracellular tail does not contain itims. cd r does contain three intracellular tyrosine residues. mutation of all three tyrosine completely abrogates its inhibitory function [ ] . the most distal tyrosine is located in an npxy motif, to which the adaptor molecule dok is recruited [ ] . dok activates rasgap and knockdown of these proteins diminishes the inhibitory action of cd r [ ] . however, the down-stream targets for cd r mediated inhibition are not yet identified. upon influenza a virus infection, cd -deficiency strongly enhances neutrophil influx into the lungs of female mice possibly leading to pathology, but it does not affect viral clearance and type i ifn production. this implies that, for influenza virus, the sexbiased type i ifn production and viral clearance are not regulated by cd r, while the events leading to increased neutrophil recruitment and lung pathology are. neutrophil responses to influenza virus infection were shown to be dependent on tlr [ ] . since neutrophils express cd r, the strongly increased neutrophil influx in female cd / mice is in line with our finding that cd r inhibits sex-biased tlr responses. in contrast to mhv infection, clearance of influenza a virus is not dependent on plasmacytoid dendritic cells [ ] . although influenza rna triggers tlr [ ] , the main source of type i ifn is the infected respiratory epithelium [ ] . these cells do not express cd r and hence are not influenced by cd -deficiency, explaining the lack of effect of cd -deficiency on type i ifn production. there is emerging evidence that tumor cells employ immunological checkpoints for their benefit. as a result of this, inhibitory immune pathways have become therapeutic targets to strengthen anti-tumor responses and develop (adjuvant) therapeutic strategies in cancer treatment. the successful application of anti-ctla (cytotoxic t-lymphocyte antigen ) in melanoma is followed up with blocking agents for other checkpoints, among which the cd -cd r immune inhibitory pathway. strong evidence for a role for cd in tumor progression comes from studies in patients. expression of cd is an independent prognostic factor for multiple myeloma and acute myeloid leukemia predicting worse overall survival of these patients [ , ] . a clinical trial with a blocking anti-cd antibody aims to enhance anti-tumor responses towards cd -expressing malignancies (clinical-trials.gov identifier: nct ). on the basis of our data, one of the predicted side effects would be severe immune pathology to infections. our finding that the combination of lack of cd r signaling and female sex has such a profound impact on the control of virus infection as well as on immune pathology raises some important issues. we are the first to demonstrate a strong sex bias in type i ifn production and viral clearance in mice utilizing two different models of virus infection. this is of importance for scientists studying these widely used models and may result in a completely different interpretation of data obtained, depending on the sex of the mice used. moreover, sex biased clinical responses to virus infections have been reported in humans [ ] . for influenza a virus (h n ) a significantly enhanced case-fatality rate was found in women [ ] . in agreement with these findings we now show increased lung damage, enhanced neutrophil influx and elevated il- , il- levels in bal fluid of female mice upon influenza a virus infection. a few reports discuss the possibility of a sex bias in severity of sars-cov infection [ , ] . also for hiv- infection, sex-related differences have been well-established [ , ] . our results underscore the importance of the issue of sex bias in scientific research, clinical trails, and vaccine studies, previously raised by others [ , ] . particularly, cd blocking antibodies are currently entering clinical trials for cancer treatment. our data point to a possible pathological outcome of e.g. influenza virus infection in women as a result of cd blocking therapies. wild-type c bl/ j mice and cd / mice, which were made and maintained on a full c bl/ j background [ ] , were bred at the specified pathogen free (spf) unit at the utrecht university central animal laboratory and used between and weeks of age. mice were injected intraperitoneally with tcid of mhv strain a expressing the firefly luciferase (fl) reporter gene (mhv-eflm) [ ] in ml pbs. intranasal infection with . tcid of influenza strain a/hk/ / was performed as described [ ] . mice were monitored once every hours for symptoms of illness. in additional experiments we injected the mice intraperitoneally with the tlr agonist imiquimod (invivogen; mg in ml pbs). the utrecht university ethical committee for animal experimentation approved the animal study protocols, in accordance with the advice of the central committee on animal experimentation ( januari ) and the dutch law on animal experimentation (art. a). after mhv-eflm injection (day ), mice were imaged at day and day as described previously [ ] with minor modifications. briefly, mice were anaesthetized with isoflurane and subsequently injected with ml of the fl substrate d-luciferin (synchem figure . stimulation of cd r directly inhibits tlr mediated nfkb activity. a hek t cells were transiently transfected with tlr , nf-kb luciferase reporter and lair- -cd r chimera or signaling-defective lair- -cd r-fff constructs. twenty-four hours later cells were stimulated with control or anti-lair- antibody. forty-eight hours after transfection cells were stimulated with the tlr ligand imiquimod ( . mg/ml). seventytwo hours after transfection cells were harvested, luciferase activity, and total protein content were determined. protein normalized, luciferase activity from independent experiments is shown. b hek t cells with stable expression of tlr were transiently transfected with a nf-kb luciferase reporter construct or an ifnb luciferase reporter construct and a lair- -cd r chimera. tlr stimulation and cd r crosslinking was performed as in (a). protein normalized with luciferase activity from independent experiments is shown. mean sem is shown. statistical significance was calculated with mann-whitney test. ns = not significant. * = p, . . doi: . /journal.ppat. .g laborgemeinschaft ohg) dissolved in pbs ( mg/kg). mice were positioned to the ventral side in a specially designed box and placed onto the stage inside the light-tight photon imager (biospace laboratory). five mice were imaged simultaneously exactly min after the injection of d-luciferin. the biolumines-cence signals were acquired with photovision software (biospace laboratory) over a -min interval and are expressed as integrated light intensity (photons/min). a low-intensity visible light image was generated and used to create overlay (heatmap) images for each individual animal. tissue homogenization and isolation of total rna whole livers or left lungs were dissected from the mice. the tissues were processed in lysing matrix d tubes (mp biomedical), containing ml of pbs, using a fastprep instrument (mp biomedical). the tissues were homogenized at g for sec and immediately placed on ice. subsequently, the homogenates were centrifuged at g for minutes at uc and supernatants were harvested and stored at uc. total rna was isolated from the homogenates using the trizol reagent (invitrogen) according to manufacturer's instructions. gene expression levels of ifn-a, ifn-b , and tlr were measured by quantitative pcr using lightcycler rna master hydrolysis probes in combination with a lightcycler system (both from roche applied science), according to the manufacturer's instructions. the housekeeping gene gapdh was used as a reference in all experiments, and expression of this gene was found relatively constant among samples. the amounts of mhv rna were determined by quantitative rt-pcr using primers and probe directed against the n gene of mhv-a [ ] . for the influenza rna quantification the primers mapping to the influenza a nucleoprotein (n) gene were used. amplification and detection were performed with an abi prism system. samples were controlled for the presence of possible inhibitors of the amplification reaction by internal control (murine encephalomyocarditis virus dna). bronchoalveolar lavage (bal) fluid was obtained by flushing the lungs two times with ml pbs using a canula inserted into the trachea, yielding around . ml bal fluid. pelleted cells from bal fluid were counted and cytospins were prepared and stained with may-grunwald/giemsa and neutrophils were scored on the basis of morphology (dade behring, switzerland). bal fluids were kept on ice or stored at uc until further processing. bal fluid was centrifuged, and ml of aliquot was used to determine the protein concentration with a bca kit (pierce) according to the manufacturer's instructions. to measure the interferon concentration in the sera, blood was sampled from naïve mice or four days after mhv infection or one hour after imiquimod treatment. sera were separated by spinning the blood at g for livers of mhv-infected mice were sampled, fixed in % neutral buffered formalin, and embedded in paraffin. seven mm liver sections were stained with hematoxylin and eosin. total liver sections were examined by light microscopy and foci of hepatocellular necrosis and inflammation were scored in a semiquantitative manner. hek t cells were transiently co-transfected with: human tlr (kindly provided by rogier sanders, amc, amsterdam, the netherlands), and nf-kb-reporter or ifna-reporter constructs, kindly provided by dr paul moynagh (national university of ireland). a chimeric construct containing the extra cellular region of human lair- (amino acids - ) fused with the transmembrane and intracellular rat cd r (rcd r) (amino acids - ) was cloned into pcdna . /zeo (invitrogen, breda, the netherlands). a tyrosine (y) to phenylalanine (f) mutant of tyrosines , , and in the intracellular rcd r tail were generated with pcr-based mutagenesis. the mutant was cloned into the same vector and all sequences were confirmed by automated dna sequencing. the lair-cd r plasmid was co-transfected with the tlr and reporter constructs. twentyfour hours after transfection cells were trypsinized and seeded in -well plates coated with ug/ml anti-lair- monoclonal antibody (clone a ). forty-eight hrs after transfection cells were stimulated with imiquimod mg/ml (invivogen) in pbs. on the next day, cells were lysed with passive lysis buffer (promega), luciferase activity was measured on a luminometer (berthold technologies centro lb ), and data were analyzed with microwin software. total protein content was determined with a pierce bca protein assay (thermo scientific). all luciferase values were normalized to protein concentration. alternatively, we used hek cells stably expressing human tlr (invivogen). significance was calculated with mann-whitney test using graphpad prism software. online supplemental material figure s shows bli measurement in naïve mice and viral spread of mhv at day after infection. figure s depicts the quantification of lung pathology and differential cell count in the bal fluid in influenza a virus infected mice. figure s is a quantitative analysis of tlr mrna in male and female mice. the nuclear lung surface area was used as a benchmark of the inflammatory response, its color range selections were measured thrice. the relative nuclear surface area (nuclear surface area/lung surface area * ) of the lung sections was used as a measure of the tissue response to exposure to the influenza virus (b) total cell count in bal fluid. quantification of monocyte (c) and lymphocyte (d) numbers in bal fluid by differential cell count. in all panels mean sem is shown, statistical significance was calculated with mann-whitney test. * = p, . , ** = p, . . (doc) figure s no sex difference in expression of tlr mrna. four days after mhv injection mice were sacrificed, rna was isolated from livers and tlr mrna expression was quantified by qpcr in male and female wt and cd / mice. mean sem is shown. (doc) cd and its receptors as targets for immunoregulation the leukocyte/ neuron cell surface antigen ox binds to a ligand on macrophages characterization of the cd receptor family in mice and humans and their interactions with cd the inhibitory cd r is differentially expressed on human and mouse t and b lymphocytes essential roles for dok and rasgap in cd receptor-mediated regulation of human myeloid cells cd and membrane protein interactions in the control of myeloid cells recombinant cd protein does not bind activating proteins closely related to cd receptor downregulation of the macrophage lineage through interaction with ox (cd ) immune inhibitory ligand cd induction by tlrs and nlrs limits macrophage activation to protect the host from meningococcal septicemia a critical function for cd in lung immune homeostasis and the severity of influenza infection lack of cd enhances pathological t cell responses during influenza infection control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon hematopoietic cell-derived interferon controls viral replication and virusinduced disease non-invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo the proteasome inhibitor velcade enhances rather than reduces disease in mouse hepatitis coronavirus-infected mice tlr ligands induce higher ifn-alpha production in females sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv- small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway the xs and y of immune responses to viral vaccines x-inactivation profile reveals extensive variability in x-linked gene expression in females mice lacking cd r show absence of suppression of lipopolysaccharide-induced tumor necrosis factor-alpha and mixed leukocyte culture responses by cd long term potentiation is impaired in membrane glycoprotein cd -deficient mice: a role for toll-like receptor activation identification of tyrosine residues crucial for cd r-mediated inhibition of mast cell activation molecular mechanisms of cd inhibition of mast cell activation toll-like receptor-mediated activation of neutrophils by influenza a virus clearance of influenza virus from the lung depends on migratory langerin+cd b-but not plasmacytoid dendritic cells innate antiviral responses by means of tlr -mediated recognition of single-stranded rna differential type i interferon induction by respiratory syncytial virus and influenza a virus in vivo cd as a prognostic factor in acute myeloid leukaemia cd is a new prognostic factor in multiple myeloma the impact of sex, gender and pregnancy on h n disease the epidemiological and molecular aspects of influenza h n viruses at the human-animal interface in egypt sars in singapore-predictors of disease severity do men have a higher case fatality rate of severe acute respiratory syndrome than women do? the x-files in immunity: sex-based differences predispose immune responses sex differences in hiv- viral load and progression to aids sex bias in trials and treatment must end cyclooxygenase activity is important for efficient replication of mouse hepatitis virus at an early stage of infection we thank hans clevers, lewis lanier, louis bont, josé borghans and erik hack for critical discussions and review of versions of the manuscript. we also thank eva rijkers, rogier sanders and paul moynagh for providing plasmids, guus rimmelzwaan for influenza a virus, julia drylewicz for advice on statistics and alan rigter, robert de vries and marco viveen for their technical assistance. conceived and designed the experiments: gk tpr mr pjmr camh lm. performed the experiments: gk tpr mr gcmg fec. analyzed the data: gk tpr mr pjmr camh lm. contributed reagents/ materials/analysis tools: mer fec. wrote the paper: gk tpr mr camh lm. key: cord- -a pixbuc authors: zhi, yan; kobinger, gary p.; jordan, heather; suchma, katie; weiss, susan r.; shen, hao; schumer, gregory; gao, guangping; boyer, julie l.; crystal, ronald g.; wilson, james m. title: identification of murine cd t cell epitopes in codon-optimized sars-associated coronavirus spike protein date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: a pixbuc the causative agent of severe acute respiratory syndrome (sars) has been identified as a new type of coronavirus, sars-associated coronavirus (sars-cov). cd t cells play an important role in controlling diseases caused by other coronaviruses and in mediating vaccine-induced protective immunity in corresponding animal models. the spike protein, a main surface antigen of sars-cov, is one of the most important antigen candidates for vaccine design. overlapping peptides were used to identify major histocompatibility complex class i-restricted epitopes in mice immunized with vectors encoding codon-optimized sars-cov spike protein. cd t-cell responses were mapped to two h- (b)-restricted epitopes (s – and s – ) and one h- (d)-restricted epitope (s – ). the identification of these epitopes will facilitate the evaluation of vaccine strategies in murine models of sars-cov infection. furthermore, codon and promoter optimizations can greatly enhance the overall immunogenicity of spike protein in the context of replication-defective human and simian adenoviral vaccine carriers. the optimized recombinant adenoviral vaccine vectors encoding spike can generate robust antigen-specific cellular immunity in mice and may potentially be useful for control of sars-cov infection. severe acute respiratory syndrome (sars) is an emerging infectious disease associated with a novel coronavirus, sars-associated coronavirus (sars-cov), which caused worldwide outbreaks. the case fatality rate has been as high as % for patients younger than years old and can be higher than % for patients years or older. nearly % of patients developed respiratory failure that required assisted ventilation (de groot, ) . the severe morbidity and mortality associated with sars make it imperative that effective means to prevent and treat the disease be developed and evaluated, especially since it is not known whether the virus will exhibit a seasonal pattern or whether it will be reintroduced into the human population through animal reservoirs, or laboratory accidences, or acts of terrorism. the successful development of effective treatments and vaccines against sars-cov depends on understanding the roles of various immune effectors in protective immunity and identifying protective antigens recognized by these effector cells. like other covs, sars-cov is an enveloped plus-stranded rna virus with a¨ kbs genome encoding replicase (rep) gene products and the structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n) (marra et al., ; rota et al., ) . s protein is responsible for binding to specific cellular receptor , e protein plays a role in viral assembly, m is - /$ -see front matter d elsevier inc. all rights reserved. doi: . /j.virol. . . important for virus budding, and n protein is associated with viral rna packaging (holmes, ) . no conclusive information is available on the immune correlates of protection to sars in patients. however, it has been reported that antibodies against sars-cov were detected in patients infected with sars . recently, using pseudotyped lentiviral particles bearing the sars-cov spike protein, it has been shown that spike-mediated infection could be inhibited by sera from sars patients, demonstrating that spike is a target for neutralizing antibodies (hofmann et al., ) . in addition, two identified cd t-cell epitopes in sars-cov spike protein have been shown to elicit specific t-cell responses in hla-a + sars-cov-infected patients (wang et al., ) . therefore, we initially focused on sars-cov spike protein as the target antigen for our vaccine development. a number of animal models have emerged for studying sars pathogenesis and evaluating therapies including macaques, ferrets, and mice hogan et al., ; kuiken et al., ; martina et al., ; subbarao et al., ) . however, for the early stage of product evaluation, a small animal model, such as mice, would be very useful. we (hogan et al., ) and others (subbarao et al., ) have shown that sars-cov replicates in mice although the infection is self-limited. to study antigen-specific immune responses in mice, mhc irestricted cd t-cell epitopes need to be identified. vectors based on replication-defective adenoviruses are capable of high-level gene transfer and activation of t and b cells to the transgene product (van ginkel et al., ; yang et al., ) . these properties have been exploited in the development of genetic subunit vaccines (sullivan et al., ; xiang et al., ) . therefore, several versions of replication-defective adenoviral vectors expressing spike protein were created to induce spike-specific t cell responses in mice and to screen for cd t-cell epitopes using an overlapping peptide library spanning the entire spike protein in ifn-g elispot and intracellular ifn-g staining assays. here, we report the detailed mapping of both h- b -and h- d -restricted cd t-cell epitopes from codon-optimized spike protein. these results provide critical information for analyzing cd t-cell responses in murine models of sars-cov infection and for developing spike-based sars-cov vaccines. more importantly, a single administration of the optimized sars-cov spike vaccine vectors based on replication-defective human and simian adenovirus can generate strong spikespecific cd t-cell responses in mice. to identify cd t-cell epitopes in sars-cov spike protein, c bl/ mice were injected im with adhu . cmvspike, a replication-defective human adenovirus serotype -based vaccine vector encoding wild-type spike protein. splenocytes were harvested days after immunization and stimulated in vitro with pools of overlapping peptides corresponding to spike protein. no pool resulted in specific stimulation of an ifn-g response in t cells using elispot assay (data not shown). we then used adhu .cmvnspike, another replicationdefective human adenovirus serotype -based vaccine vector encoding codon-optimized spike protein, to immunize c bl/ mice im. splenocytes were harvested at days after immunization and subjected to ifn-g elispot assay with pools of overlapping peptides from spike protein. of all peptide pools screened, pools , , and showed specific ifn-g responses in c bl/ . the remaining pools, as exemplified by pool , showed no specific response (fig. a) . pools and were selected for mapping the individual peptide(s) responsible for stimulating ifn-g expressing t cells. peptide (tstgnyny-kyrylrh, corresponding to s - ) and peptide (ynykyrylrhgklrp, corresponding to s - ) within pool and peptide (knqcvnfnfngltgt, corresponding to s - ) and peptide (nfnfn-gltgtgvltp, corresponding to s - ) within pool were identified as the major positive peptides responsible for specific stimulation of t cells to produce ifn-g (fig. b) . based on the syfpeithi algorithm (rammensee et al., ) , several potential cd t-cell epitopes for sars-cov spike protein in c bl/ mice were identified. table showed their sequences, positions, and scores. indeed, ynykyryl (s - ) completely present in peptides and was predicted to have strong binding affinity for h -k b ; while vnfnfngl (s - ) completely present in peptide and partially present in peptide was predicted to have weak binding affinity for h -k b . since elispot assay cannot readily distinguish antigen-specific cd t-cell responses from antigenspecific cd t-cell responses, the immunized splenocytes were further subjected to intracellular ifn-g staining after stimulating in vitro with peptides and (fig. ) . subsequently, the predicted optimal cd t-cell epitopes, ynykyryl resided in peptide and vnfnfngl resided in peptide , were synthesized and used to stimulate immunized splenocytes in vitro for ifn-g secretion (fig. ) . as predicted, ynykyryl and peptides gave much stronger cd t-cell responses than vnfnfngl and peptides . furthermore, optimal epitopes, ynykyryl and vnfnfngl, were able to induce ifn-g responses in cd t cells either as effectively as or more effectively than their parental mers, respectively. in conclusion, a single administration of a replication-defective human adenoviral vaccine vector encoding codon-optimized spike can generate strong spike-specific cd t-cell responses. we also identified two optimal cd t-cell epitopes of sars-cov spike in c bl/ mice. determining the dose responses of two h- b -restricted cd t-cell epitopes for t cell activation immunized c bl/ splenocytes were in vitro stimulated with the two identified optimal cd t-cell epitopes at different concentrations ranging from ag/ml to eÀ ag/ ml, respectively. t cells activation was measured by intracellular ifn-g staining (fig. ) . it appeared that the minimal amounts of peptides needed to fully activate t cells in vitro were similar for both epitopes, even though the magnitude of t cell response with ynykyryl was much higher than that with vnfnfngl. mapping of h- d -restricted cd t-cell epitopes in sars-cov spike protein balb/c mice were immunized in a similar fashion as c bl/ mice. using elispot assay, pools , , and showed specific ifn-g responses in balb/c. the remaining pools, as exemplified by pool , showed no specific response (fig. a ). we focused on pools and because of the stronger responses observed with these pools. subsequently, peptide (fstfkcygvsatkln, correspond-ing to s - ) and peptide (cygvsatklndlcfs, corresponding to s - ) within pool and peptide (tstgnynykyrylrh, corresponding to s - ) and peptide (ynykyrylrhgklrp, corresponding to s - ) within pool were identified as the major positive peptides responsible for specific stimulation of t cells to produce ifn-g (fig. b ). based on the syfpeithi algorithm (rammensee et al., ) , several potential cd t-cell epitopes for sars-cov spike protein in balb/c mice were identified. table showed their sequences, positions, and scores. indeed, cygvsatkl (s - ) completely present in peptides and was predicted to have strong binding affinity for h -k d ; while nyny-kyryl (s - ) completely present in peptide and partially present in peptide was predicted to have weak binding affinity for h -k d . the immunized splenocytes were also subjected to intracellular ifn-g staining after stimulating in vitro with these positive mers. notably, there was no significant ifn-g production from cd t cells stimulated with peptides and by intracellular ifn-g staining (fig. a ), even though both peptides gave strong responses in ifn-g elispot assay (fig. b ). this suggested that peptides and may contain a cd t- table selected candidates of cd t-cell epitopes for sars-cov spike protein in c bl/ the candidates of cd t-cell epitopes present in the positive mers were shown in red. cell epitope that stimulated cd t-cell responses in ifn-g elispot assay. in contrast, peptide can significantly stimulate cd t-cell responses (fig. b ). subsequently, the predicted optimal cd t-cell epitope, cygvsatkl resided in peptide , was synthesized and used to stimulate immunized splenocytes in vitro for ifn-g secretion (fig. b ). the results indicated that peptide indeed contained an h- d -restricted cd t-cell epitope. as expected, optimal epitope was able to induce ifn-g responses in cd t cells more effectively than its parental mer. collectively, we have identified one optimal cd t-cell epitope of sars-cov spike in balb/c mice. the dose responses of this fig. . intracellular ifn-g staining to confirm cd t-cell epitopes of sars-cov spike protein in c bl/ mice. mice were immunized with  particles of adhu .cmvnspike via im injection. days after immunization, splenocytes were harvested and pooled from mice and stimulated with either mers positive peptides or optimal mers cd t-cell epitopes, as indicated, for h. the immune response was evaluated by intracellular ifn-g staining with pe-anti-ifn-g and fitc-anti-cd antibodies. numbers in the upper right corner of each graph represent the frequencies of ifn-g-producing cd t cells. experiments were done in duplicate and representative results were shown. fig. . dose responses of h- b -restricted cd t-cell epitopes for t cell activation in vitro. c bl/ mice were immunized with  particles of adhu .cmvnspike via im injection. days after immunization, splenocytes were harvested and pooled from mice and stimulated with two identified cd t-cell epitopes at different concentrations, as indicated, respectively, for h. t cell activation was evaluated by intracellular ifn-g staining. experiments were done in duplicate and representative results were shown. cd t-cell epitope for t cells activation in vitro were also determined as described above (fig. ) . the results suggested that about -fold more peptides were needed to fully activate t cells in vitro with this h- d -restricted cd t-cell epitope compared to those with two h- brestricted cd t-cell epitopes (fig. ) . furthermore, peptides and may contain a cd t-cell epitope of sars-cov spike in balb/c mice. to confirm the presence of cd t-cell epitope(s) in peptides and , the adhu .cmvnspike-immunized splenocytes of balb/c mice were harvested and in vitro stimulated with three mers individually, including peptides , , and . the cd t-cell response was directly evaluated by intracellular cytokine staining with fitc-anti-cd and pe-anti-ifn-g antibodies (fig. ) . as expected, when stimulated with peptide , there was no detectable ifn-g secretion from immunized cd t cells, even though a significant ifn-g production from immunized non-cd t cells (cd t cells) was observed. more importantly, peptides and were able to stimulate immunized cd t cells to product ifn-g. these results indicated that peptides and indeed contained a cd t-cell epitope of spike protein in balb/c mice. increasing the immunogenicity of sars-cov spike protein in the context of replication-defective simian adenoviral vaccine carrier to circumvent the potential problem that neutralizing antibodies to human adenovirus serotype vector by previous natural infections will impair its efficacy as vaccine carrier, our lab recently developed a series of novel replication-defective adenoviral vaccine carriers based on simian serotypes pinto et al., ) . adc , one of those carriers, is able to induce robust transgene-specific cd t-cell responses in immunized mice (kobinger et al., submitted for publication) . three replication-defective simian adenovirus serotype -based vaccine vectors encoding either wild-type spike driven by cmv promoter (adc .cmvspike) or codon-optimized spike driven by cmv promoter (adc .cmvnspike) or codonoptimized spike driven by a hybrid promoter cag (adc .cag nspike) were created and used to immunize mice. cag promoter was created by deleting a -bp table selected candidates of cd t-cell epitopes for sars-cov spike protein in balb/c the candidates of cd t-cell epitopes present in the positive mers were shown in red. apai/aflii fragment from the original caggs promoter (niwa et al., ) . similar to the negative results observed when adhu .cmvspike was used to immunize mice, there was no detectable stimulation of an ifn-g response in t cells with any of the identified positive pools using elispot assay when adc .cmvspike was used for immunization (data not shown). in contrast, spike-specific cd t-cell responses were observed when adc .cmvnspike vector was used to immunize c bl/ mice (fig. ) . however, the magnitude of spike-specific cd t-cell responses was much lower than that observed in mice immunized with adhu .cmvnspike vector (fig. ) . in order to further improve the immunogenicity of spike protein delivered by the simian adenoviral vaccine carrier, adc .cag nspike vector was created. chicken h-actin promoter has been shown to increase expression of sars-cov spike protein in transfected cells (simmons et al., ) . spike-specific cd t-cell responses in c bl/ mice elicited by adc .cag nspike vector were examined by intracellular ifn-g staining (fig. ) . the result indicated fig. . dose responses of h- d -restricted cd t-cell epitope for t cell activation in vitro. balb/c mice were immunized with  particles of adhu .cmvnspike via im injection. days after immunization, splenocytes were harvested and pooled from mice and stimulated with the identified cd tcell epitope at different concentrations, as indicated, for h. t cell activation was evaluated by intracellular ifn-g staining. experiments were done in duplicate and representative results were shown. fig. . intracellular ifn-g staining to confirm cd t-cell epitopes of sars-cov spike protein in balb/c mice. mice were immunized with  particles of adhu .cmvnspike via im injection. days after immunization, splenocytes were harvested and pooled from mice and stimulated with either mers positive peptides or optimal mers cd t-cell epitope, as indicated, for h. the immune response was evaluated by intracellular ifn-g staining with pe-anti-ifn-g and fitc-anti-cd antibodies. numbers in the upper right corner of each graph represent the frequencies of ifn-g-producing cd t cells. experiments were done in duplicate and representative results were shown. that codon and promoter optimizations can greatly enhance the overall immunogenicity of sars-cov spike protein in the context of simian adenoviral vaccine carrier. more importantly, a single administration of an optimized sars-cov spike vaccine vector based on a replication-defective simian adenovirus can generate strong spike-specific cd t-cell responses in mice. it has been shown that increased expression of codonoptimized hiv gag protein is responsible for its enhanced immunogenicity in mice (deml et al., ; gao et al., a) . therefore, it was of interest to determine whether optimization of transgene expression cassette, including codon and promoter optimizations, in the context of replication-deficient adenoviral vaccine vectors also resulted in the increased expression of spike protein in vitro. expression of spike protein in ad vector-infected t cells was examined by western blot analysis and the bands representing spike protein were quantified by image-quant . . forty-three-fold increase of protein expression was achieved when cag promoter, instead of cmv promoter, was used in the simian adenoviral c vector (fig. , lanes and ), while -fold increase of protein expression was achieved when codon optimization of spike protein was applied (fig. , lanes and ) . overall, the results supported that the enhanced immunogenicity of spike in mice injected with adhu .cmvnspike and adc .cag nspike vaccine vectors (fig. ) indeed correlated to high expression of spike protein in cells infected with these vectors. sars-cov represents an emerging threat. currently, no effective therapies or vaccines exist, and relatively little is known about the pathogenesis of the virus. it has been shown that t cells transfected with a functional receptor for sars-cov, angiotensin-converting enzyme (ace ), formed multinucleated syncytia with cells expressing spike protein . more significantly, in the postmortem lung tissue samples from patients who died from sars, multinucleate giant cells of macrophage and epithelial origins have been observed within the damaged alveoli (nicholls et al., ) . in addition, cd t-cell responses are necessary for clearance of other covs, such as mouse hepatitis virus (sussman et al., ; williamson and stohlman, ) . therefore, eradication of sars-cov may not be achieved by humoral response alone and t cellmediated immunity may be also required to clear infection. to study more closely cellular immune responses in murine model, mhc i-restricted cd t-cell epitopes need to be identified. in this study, we have identified two octamers (s - and s - ) as h- b -restricted cd t-cell epitopes and one nonamer (s - ) as an h- d -restricted cd t-cell epitope. in c bl/ mice, one epitope is more dominant than the other in immunized mice. the strength of these epitopes appeared to correlate with the binding affinity to h -k b predicted by syfpeithi algorithm. in the preliminary study, splenocytes were collected from c bl/ mice immunized with adc .cag nspike vector and in vitro cultured in the presence of the dominant h- brestricted cd t-cell epitope for a week. subsequently, the cultured splenocytes were subjected to elispot assay stimulated with mc sv cells (h- d b , a cell line derived from b mice) previously infected with either adhu .null or adhu .cmvnspike vector. the initial results indicated that cultured splenocytes were able to induce higher level of ifn-g response after stimulated with adhu .cmvnspike vector-infected mc sv cells than after stimulated with adhu .null vector-infected mc sv cells. these data suggested that t cells raised to the epitope were able to recognize cells expressing the spike protein and that the identified cd epitope is naturally processed and presented by cells. currently, additional experiments are underway to address this issue more thoroughly. coronaviruses are common and worldwide pathogens that infect a variety of mammals and birds. these viruses have been classified into three groups. although sars- fig. . intracellular ifn-g staining to confirm the presence of cd t-cell epitope of sars-cov spike protein in balb/c mice. mice were immunized with  particles of adhu .cmvnspike via im injection. days after immunization, splenocytes were harvested and pooled from mice and stimulated with mers, as indicated, for h. the immune response was evaluated by intracellular ifn-g staining with pe-anti-ifn-g and fitc-anti-cd antibodies. numbers in the upper right corner of each graph represent the frequencies of ifn-g-producing cd t cells. experiments were done in duplicate and representative results were shown. fig. . promoter optimization can further increase the immunogenicity of sars-cov spike protein delivered by the replication-defective simian adenoviral vaccine carrier. c bl/ mice were immunized with  particles of vaccine vectors, including adhu .cmvnspike, adc .cmvnspike, and adc .cag nspike, via im injection. days after immunization, splenocytes were harvested and pooled from mice in each group and stimulated in vitro with positive peptide for h. the immune response was evaluated by intracellular ifn-g staining with pe-anti-ifn-g and fitc-anti-cd antibodies. numbers in the upper right corner of each graph represent the frequencies of ifn-g-producing cd t cells. experiments were done in duplicate and representative results were shown. cov exhibits a similar genome structure, it is only distantly related to known covs and fmost like_ group covs, which includes bovine, murine, and human viruses (snijder et al., ) . the most commonly studied coronavirus is mouse hepatitis virus (mhv), in part because the natural host for this infection, the mouse, is more easily managed in the laboratory. two spike-specific cd t-cell epitopes (dominant s - and subdominant s - ) were identified in mhv-infected c bl/ mice (bergmann et al., ; castro and perlman, ) , while no spikespecific cd t-cell epitope has been recognized in mhvinfected balb/c mice. the dominant s - epitope is located in a hypervariable region of spike protein that appears to be readily deleted without loss of viability of mhv (parker et al., ) . in contrast, it was impossible to recover infectious virus with mutations in the subdominant s - epitope (personal communication, m.m. chua and s.r. weiss). examination of sars-cov spike sequence reveals that three cd t-cell epitopes are present in the s domain of the spike protein. specifically, the dominant cd t-cell epitope in c bl/ (s - ) and the cd t-cell epitope in balb/c (s - ) both reside in the minimal region of s required for interaction with its cellular receptor (babcock et al., ; wong et al., ) . several spike-specific cd t-cell epitopes have also been reported in mhv-infected mice (heemskerk et al., ; xue and perlman, ) . cd t cells are needed to help cd cells for viral clearance (stohlman et al., ; sussman et al., ; williamson and stohlman, ) . in this study, we clearly showed that both peptide and peptide were able to specifically stimulate immunized cd t cells to produce ifn-g in vitro. however, the exact sequences of cd t-cell epitope in the - region of spike protein remain to be further elucidated. the first step in designing a genetic vaccine is to enhance the intrinsic immunogenicity of the selected target gene. in this study, we demonstrated that codon and promoter optimizations can greatly enhance the overall immunogenicity of spike protein in replication-defective human and simian adenoviral vaccine vectors. we showed that the expression of spike protein in vitro was greatly increased by expression cassette optimization. this provided useful information for developing spike-based sars-cov genetic vaccines. more importantly, this may provide us a platform to quickly generate genetic vaccine vectors based on simian adenovirus. namely, cag but not cmv promoter should be used in simian adenoviral vaccine vector. currently, we are testing this hypothesis with other viral antigens. since the cmv promoter has been widely used in human adenoviral vectors, it is surprising to see the inferior performance of cmv promoter in simian adenoviral vector. one possibility is that certain viral sequences only present in simian adenoviral vector have negative effects on cmv promoter activity. the other possibility is that certain inflammatory cytokines induced by simian adenoviral vector may inhibit cmv promoter activity. recently, a strategy involving priming with dna vaccine and boosting with adenoviral vaccine vectors, each expressing a similar antigen, has resulted in the generation of unparalleled levels of specific immunity and afforded protection against infectious agents in animal models (shiver et al., ; sullivan et al., ) . nevertheless, dna vaccines have performed poorly in clinical trials so far (macgregor et al., ; wang et al., ) , and it is thus uncertain whether dna vaccine prime followed by adenoviral vaccine vector boost will be as efficacious in humans as in preclinical experimental animal studies. therefore, we developed an additional replication-defective adenoviral vaccine vector of chimpanzee origin to increase our repertoire of vaccine carriers that can be given sequentially pinto et al., pinto et al., , reyes-sandoval et al., ; roy et al., ; xiang et al., xiang et al., , a xiang et al., , b xiang et al., , c . the toxicity of adenoviral vectors in vivo is directly related to the dose of injection. therefore, a major benefit of prime/boost strategy is the potential to significantly reduce the dose for immunization in order to achieve a high level of immune responses. in this study, we showed that both adhu .cmvnspike and adc .cag nspike vaccine vectors could induce robust spike-specific cd t-cell responses in immunized mice alone. currently, we are studying whether strong cd t-cell responses can be achieved when mice were primed and boosted with these heterologous adenoviral vaccine vectors at a much lower dose. complementary dna (cdna) of spike gene for sars-cov was isolated by rt-pcr from the viral rna of the sars-cov (tor isolate). the pcr fragment was topocloned (invitrogen, ca) and characterized by sequencing at seqwright (seqwright, tx), and was found to be % identical to the published sequence (marra et al., ) . subsequently, tor spike protein sequence was used as a template to design a synthetic spike gene sequence with human pattern of codon usage, according to entelechon backtranslation software tool. finally, cloned tor spike cdna was used as a template and amplified with overlapping oligonucleotides in which human codon usage was introduced. resulting overlapping pcr fragments were fused and full-length codon-optimized spike cdna (nspike) was created. to generate the molecular clones of adhu vectors, wild-type or codon-optimized spike insert was cloned into a pshuttle plasmid, followed by homologous recombination in bacterial cells using padeasy system (invitrogen, ca). molecular clones of all adc vectors used in the study were created through a direct cloning and green-white selection procedure as described elsewhere . all of these molecular clones of replicationdefective adhu and adc vectors were transfected into cells for virus rescue. the rescued vectors were expanded to large-scale infections in cells and purified by the standard cscl gradient sedimentation method. genome structures of the vectors were confirmed by restriction analysis. infectivity of the vectors was determined by the standard plaque assay on cells and levels of replication competent adenovirus (rca) contaminants in the vector preparations were inspected as described previously (gao et al., ) . and t cells were maintained in dmem (gibco-life technologies, grand island, ny) supplemented with antibiotic and % fbs (hyclone, logan, ut). c bl/ and balb/c mice ( - weeks old) were purchased from charles river laboratories (wilmington, ma) and kept at the animal facility of the wistar institute (philadelphia, pa). peptide library derived from the sars-cov spike protein sequence was synthesized as mers with -amino-acid overlap with the preceding peptide (mimotopes, victoria, australia) and dissolved in dmso at approximately mg/ ml. pools of consecutive peptides were made and stored at À -c. peptides were used at the concentration of ag/ml in all experiments except the dose response studies and dmso concentrations were kept below . % (v/v) in all final assay mixtures. groups of three to five mice were immunized with recombinant adenoviral vectors diluted in al phosphatebuffered saline (pbs) given im. t cells were infected at particles/cell with each recombinant adenoviral vector encoding spike protein. twenty-four hours later, cells were harvested and resuspended in lysis buffer and frozen at À -c. all samples were normalized to the lysate with the lowest total protein concentration by diluting with  sds sample buffer (invitrogen, carlsbad, ca) plus % h-mercaptoethanol. the total protein content for all lysates was determined using the bradford assay (biorad, melville, ny). diluted samples were heated to -c for min and loaded onto an sds -polyacrylamide gel. after electrophoresis, proteins were transferred onto pvdf membrane. blots were blocked with % milk diluted in tbs/tween (tbs/t) for min, washed three times ( min each) with tbs/t, and probed with primary antibody at room temperature for h. primary antibody consisted of whole serum isolated from rabbits inoculated with purified spike protein diluted in % milk/ tbs to a final concentration of : . the blots were then washed three times with tbs/t before being incubated with secondary antibody for min at room temperature and washed again three times with tbs/t. secondary antibody was anti-rabbit hrp diluted in % milk/tbs to a final concentration of : (santa cruz biotechnology, santa cruz, ca). protein bands were developed using super-signal west pico chemiluminescent substrate (pierce, rockford, il) and exposed on kodak biomax film. the western blot image was scanned and the interested protein bands were quantified by imagequant . (molecular dynamics). assay was performed using elispot mouse set (bd pharmingen, san diego, ca) following the protocol provided by the vendor. briefly, -well elispot plate was coated with . ag/ml anti-mouse ifn-g capture antibody overnight at -c. next day, wells were washed and blocked with complete culture medium for h at room temperature. splenocytes from immunized mouse were added to microwells along with spike-specific peptides. cells were incubated at -c and % co for - h. control cells were incubated either without peptide or with nonspecific stimulator, seb ( ng/ml). then wells were extensively washed with pbs containing . % tween and subsequently incubated with . ag/ml biotinylated anti-mouse ifn-g detection antibody for h at room temperature. after washing, wells were incubated with ag/ ml streptavidin-horseradish peroxidase antibody for h at room temperature. wells were washed again, and final substrate was added to wells. color development was monitored and stopped by washing with water. after drying overnight at room temperature, wells were counted using an elispot reader. splenocytes from immunized mice were stimulated with spike-specific peptides for h at -c and % co in the presence of al/ml brefeldin a (golgiplug, bd pharmingen, san diego, ca). control cells were incubated without peptide. after washing, cells were stained with either a fitc-labeled anti-mouse cd antibody (bd pharmingen) or a fitc-labeled anti-mouse cd antibody (bd pharmingen). then, cells were washed and permeabilized in cytofix/ cytoperm (bd pharmingen) for min on ice. subse-quently, cells were washed again and stained with a pelabeled anti-mouse ifn-g antibody (bd pharmingen). after extensively washing, cells were examined by two-color flow cytometry and data were analyzed by winmdi software. amino acids to of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor the jhm strain of mouse hepatitis virus induces a spike 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mouse hepatitis virus from the central nervous system requires both cd + and cd + t cells a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme a replicationdefective human adenovirus recombinant serves as a highly efficacious vaccine carrier novel, chimpanzee serotype -based adenoviral vaccine carrier for induction of antibodies to a transgene product mucosally delivered e -deleted adenoviral vaccine carriers induce transgene product-specific antibody responses in neonatal mice t helper cellindependent antibody responses to the transgene product of an e -deleted adenoviral vaccine require nk . t cells oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier antigen specificity of cd t cell response in the central nervous system of mice infected with mouse hepatitis virus immunology of gene therapy with adenoviral vectors in mouse skeletal muscle this work was supported by glaxosmithkline pharmaceuticals. jmw is an inventor on patents licensed to various commercial entities. key: cord- - c ut authors: nan title: cis annual meeting: immune deficiency & dysregulation north american conference date: - - journal: j clin immunol doi: . /s - - -z sha: doc_id: cord_uid: c ut nan abstract/case report text oral lichen planus (olp) is a t-cell mediated chronic inflammatory tissue reaction in which presentation can range from asymptomatic plaques to painful, erosive, bullous, or ulcerative lesions. here, we present a year-old female with a novel ctla- variant, multiple autoimmune conditions, and unusual tongue lesions. our patient was healthy until years of age when she developed hashimoto's thyroiditis. at , she developed psoriasis. at , she was diagnosed with alopecia totalis and epstein-barr virus (ebv) with resultant and persistent anemia, thrombocytopenia, lymphopenia and neutropenia. she had chronic abdominal pain and diarrhea since age . esophagogastroduodenoscopy revealed lymphocytic esophagitis and active duodenal inflammation with increased intraepithelial lymphocytes. colonoscopy revealed mildly active chronic colitis with eosinophils. whole exome sequencing revealed a heterozygous c. dela (p.q rfs* ) pathogenic mutation in exon of ctla- . family history is remarkable: father (splenomegaly and psoriasis) and brother (autoimmune hemolytic anemia) have ctla haploinsufficiency with the same mutation. abatacept was initiated with re-growth of hair, improvement in cytopenias, improvement in psoriasis, and some reduction of gastrointestinal symptoms. since her abdominal pain persisted repeat endoscopies after six months of abatacept revealed persistent active lymphocytic esophagitis with some improvement in inflammatory injury in her duodenum and colon. physical exam revealed glossitis with a gel-like coating and ulceration on her tongue, xerosis along her face and scalp without other abnormalities ( figure) . she denied recent dental procedures, appliances, or tongue biting. her wbc ranged from - x ^ cells/l and hemoglobin . - . g/dl. absolute lymphocyte count ranged from . - . x ^ cells/l. immunologic evaluation revealed low iga and pan-low lymphocyte subsets (table) . ebv pcr ranged from - , copies/ml. tongue scraping revealed candida dubliniensis and she responded to days of fluconazole. two months later, she developed painful white patches along her tongue and subsequent kilogram weight loss recalcitrant to viscous lidocaine, antacids, and days of fluconazole. incisional tongue biopsy revealed ulceration with underlying granulation tissue with lymphocyte and plasma cell infiltration consistent with olp ( figure) . periodic acid-schiff diastase stain and grocott stain were negative. aerobic culture was normal. no fungus was isolated within days. epstein-barr encoding region in situ hybridization was negative. two weeks of topical dexamethasone lead to temporary improvement. her tongue lesions waxed and waned over the following months. due to persistent psoriasis, methotrexate was initiated without worsening in her tongue lesion. to our knowledge, this is the first case of olp reported in a patient with ctla- haploinsufficiency. ctla- haploinsufficiency may present with variable clinical phenotypes including increased risk of ebv viremia and malignancies. therefore, after ebv and malignancy are ruled out, olp may be a prudent diagnosis to consider in a ctla insufficient patient with unusual oral lesions. mutations of bcl b appear to be associated with lymphoproliferation and autoimmunity as well as susceptibility to herpes virus infections. additional research focusing on characterization of dna binding sites of bcl b as well as the downstream expression of associated target genes is needed. these data combined with longitudinal analysis of additional patients with confirmed bcl b mutations, will help clarify determinants of bcl b pathogenesis and highlight potential therapeutic strategies. consanguineous marriages in tribal cultures, such as that in the united arab emirates significantly increase the prevalence of autosomal recessive disorders. premarital genetic screening and counseling, thus, are expected to reduce the frequency of these diseases. in this pilot study, diagnostic exome sequencing was used in the premarital screening program to identify recessive pathologic variants preventable by premarital counseling. a total of pathologic or likely pathologic variants were identified in studied emiratis ( couples), averaging . variants per person. four percent of the persons had negative diagnostic exome sequencing; the remaining had one to eight variants per person. of the distinct variants, ( %) were novel. twenty ( %) couples had pathologic or likely pathologic variants of inborn errors of immunity (iei). two couples ( %) had iei pathologic or likely pathologic heterozygous variants in both partners imposing risk for autosomal recessive disease in the offspring. other eighteen couples ( %) had pathologic/ likely pathologic heterozygous variants present in only one person of the couple. total of sixteen ( . %) iei variant identified and eight ( %) were novel. fourteen known phenotypic iei diseases were recognized (table. ). these preliminary results support a need for nationwide premarital genetic screening, and primary immunodeficiency registry to identify common and novel pathogenic variants with high heritability rate. these results will aid adopting a preand post-connectional reproductive carrier counseling to reduce autosomal recessive diseases. also, it will assist the diagnosis of these complex diseases in our community. table pathologic or likely pathologic variants of primary immunodeficiency (pid). abstract/case report text background: chronic granulomatous disease (cgd) is a primary immunodeficiency disorder caused by defects in the phagocytic nadph oxidase complex, leading to increased susceptibility to infection and inflammatory or autoimmune disease. up to % of patients have gastrointestinal (gi) involvement and meet diagnostic criteria for inflammatory bowel disease (cgd-ibd). objectives: we analyzed cgd patients from the united states immunodeficiency network (usidnet) registry to determine whether ibd may change the presentation, treatment, and outcomes of cgd patients, as compared to those without ibd. methods: a retrospective evaluation of cgd cases from the usidnet registry was completed. cgd-ibd was defined as the presence of any major physician-reported inflammatory, non-infectious gi tract disease manifestation, including crohn disease, ulcerative colitis, ibd endoscopy findings, gi fistulas, gi strictures, gi obstruction, and proctitis. demographic information, genotypes, symptoms and conditions, infections, antimicrobial therapies, immunomodulator use, and allogeneic hematopoietic stem cell transplantation (hsct) data were analyzed. results: patients with a diagnosis of cgd were identified. met criteria for ibd; were categorized in the non-ibd group. crohn disease and colitis were the most common gi disease manifestations in the cgd-ibd group (n= ), followed by gi fistulas (n= ). cgd-ibd patients had an increased average frequency of infections ( . events/patient) compared to the cgdnon-ibd group ( . events/patient). in both groups, lower respiratory tract infections were the most common infection type and aspergillus was the most common organism. enteric organism infections were more common in ibd patients. temporal data regarding the timing of infections were not available. immunomodulators, including biologics and interferon-gamma, were used at a significantly higher rate in ibd patients compared to non-abstract/case report text down syndrome (ds) is characterized by the occurrence of three copies of human chromosome (hsa ). these patients often develop chronic mucocutaneous candidiasis (cmc) and autoimmune thyroiditis, mimicking patients with heterozygous gain-of-function (gof) stat mutations, which enhance cellular responses to the three types of interferon (ifn). hsa contains a cluster of four interferon receptor (ifn-r) genes: ifnar , ifnar , ifngr and il rb. a gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with ds. we report high levels of ifn-αr , ifn-αr and ifn-γr expression on the surface of monocytes and ebv-transformed-b (ebv-b) cells from ds patients. levels of ifn-ɣr , encoded by a gene on chromosome , were similar in the immune cells of ds patients and healthy controls. total and phosphorylated stat (stat and pstat ) levels were constitutively high in unstimulated and ifn-α-and ifn-γ-stimulated monocytes from ds patients, although less so than those in patients with gof stat mutations. following stimulation with ifn-α or -ɣ, but not with il- or il- , pstat and ifn-ɣ activation factor (gaf) dna binding activities were significantly higher in the ebv-b cells of ds patients than in controls, this response resembling the dysregulated responses observed in patients with stat gof mutations. plasma type i ifns concentrations were high in about % of the ds patients tested. a genome-wide transcriptomic analysis involving principle component analysis and a comparison of interferon modules was performed on circulating monocytes. it showed that ifn-stimulated genes (isgs) were expressed more strongly in ds than in controls. ds monocytes have intermediate levels of ifn-α-and ifn-γ-induced isgs relative to monocytes from healthy controls and from patients with gof stat mutations. by contrast to patients with gof stat mutations, circulating th counts were normal and the proportion of terminally differentiated cd + t cells was high in ds patients. the constitutive upregulation of type i and type ii ifn-r, at least in monocytes of ds patients, may therefore contribute to the autoimmune diseases observed in these individuals. scid screens (positive screen defined as trec values less than units/ul per statewide criteria). results forty nine neonates were identified with low trec values. ( %) of these infants had repeat trec screening, ( %) of which were found to have a positive second trec screen. lymphocyte subsets were evaluated in of these infants and of which were noted to have lymphopenia (defined as absolute lymphocyte count less than ). infants were noted to have low cd levels (defined as < cells/ul) and were noted to have low cd levels (defined as less than cells/ul). of note, % of the infants with cd and cd lymphopenia had normal repeat trec levels. of the infants were noted to have low b cell levels (defined as < cells/ul). infants had quantitative immunoglobulin levels and of these two were noted to have igg levels less than . of note one infant was diagnosed with partial digeorge syndrome via microarray. in our study population, no infants were diagnosed with scid. discussion our study shows that testing for trec levels on newborn screen may be beneficial in identifying not only scid, but also other immunologic conditions. infants in our study had evidence for both cell mediated and humoral immunodeficiency which necessitated further workup and follow up from allergy and immunology specialists. it may be beneficial to develop further programs to track infants identified with abnormal trec levels on newborn screens to determine if they develop signs of immunodeficiency syndromes later in life. abstract/case report text the patient was transferred to our adult clinical immunology transition clinic for low igg and iga, elevated igm and b cell lymphopenia, treated with subcutaneous gamma globulin. his medical history was relevant for recurrent respiratory infections since two years-old, failure to thrive and developmental delay. he also developed chronic auto-immune hemolytic anemia (aiha) at ten years old, accompanied by prominent lymphoid hyperplasia. our initial evaluation at the age of twenty years old showed massive polyadenopathy and splenomegaly. work-up confirmed flair-up of aiha. at that point, the diagnosis of apds was raised. he also had mild intellectual impairment and dysmorphic features such as a mild degree of ocular depression, deep-set eyes, vaguely triangular face, small chin, but had normal stature. a customized panel for usual genes involved in classic hyperigm syndromes, apds, noonan and kabuki syndromes came back negative. at years old, an urgent coloscopy was performed because of acute abdominal pain and showed diffuse ileal lymphoid hyperplasia. biopsies confirmed reactional lymphoid hyperplasia without infection nor malignancy. a second genes ngs panel associated with pid identified a heterozygote mutation in tap ; expression of hla class was normal on flow cytometry. the patient was then started on sirolimus for an "apds-like syndrome" despite the lack of genetic confirmation. six months after introduction of mtor inhibitor, his abdominal pain had completely disappeared. tep scan showed complete resolution of axillar, retroperitoneal and inguinal lymph nodes and significant regression of splenomegaly. a third large non-biased + genes ngs panel revealed a pb de novo deletion that included the splice site of pik r exon typically involved in apds : c. _ + del p.(asp glufs* ) , which was missed by the first two panels. indeed, oligonucleotide-selective sequencing technology used for the previous panels was associated to mapping errors of short reads and difficult detection of large deletions. interestingly, the patient also presented some but not all dysmorphic features of short syndrome which is related to pik r haploinsufficiency. in this new era of genetic testing, this case is a reminder that we need to be aware of the pitfalls of genetic tests and that clinical judgment is still our best diagnostic tool. abstract/case report text intro: patients with chronic granulomatous disease (cgd) are theorized to have a lower risk of malignancy related to their lack of free radical formation. there are relatively few reports of malignancy described in patients with cgd. we report three cases of malignancies in the large cohort cgd population at the national institutes of health followed between - to add to the seven cases described in the literature. case : a -year-old man with x-linked chronic granulomatous disease with a history of severe inflammatory bowel disease, who presented with progressive left-sided chest pain in , decreased appetite and weight loss. transthoracic lung biopsy showed atypical cells and a pet/ct showed abnormally dense mesentery and widespread hypermetabolic abnormalities. a mesenteric biopsy showed metastatic pancreatic adenocarcinoma. palliative care was initiated. patient expired four months after diagnosis. case : a -year-old man with x-linked chronic granulomatous disease and severe inflammatory bowel disease requiring total proctocolectomy and who had been remotely treated with infliximab, presented in with right upper quadrant pain. abdominal ultrasound and mri of the liver showed multiple liver lesions. biopsy of these lesions revealed hepatocellular carcinoma. patient underwent two courses of radiolabeled itrium spherules. however, his disease progressed and he expired approximately five months after diagnosis. c a s e : a n -y e a r-o l d m a n w i t h x -l i n k e d c h r o n i c granulomatous disease and inflammatory bowel disease who presented in with fevers, abdominal pain and pancytopenia. during the course of his hospitalization, he developed sepsis which led to his demise. on autopsy, an incidental finding of papillary thyroid carcinoma was made. discussion: these three patients all had poorly controlled inflammatory bowel disease. additionally, patients with cgd are typically exposed to higher doses of radiation, leading one to expect higher rates of radiation induced malignancies. however, there are still relatively few case reports of cancer in the cgd population. tissue biopsy is necessary for diagnosis. due to end organ damage secondary to the underlying disease in the first two cases, treatment options were limited. managing infections during chemotherapy can be complex due to drug interactions with chemotherapeutic agents. abstract/case report text myeloperoxidase (mpo) deficiency is the most common inherited defect of phagocytes that impairs microbial killing since the toxicity of the respiratory burst is dampened without myeloperoxidase release from the azurophilic granules. a significant portion of these patients remain asymptomatic, however there is a clinically variable phenotype that can present if they do become symptomatic. fungal infections with candida strains appear to be the most frequently reported. we present an adulthood case of recurrent invasive candidal disease due autosomal recessive myeloperoxidase deficiency from a pathogenic missense variant in the mpo gene (c. c>t (p.arg trp)). a -year-old caucasian male was in his normal state of health without any major illnesses until years of age when he was diagnosed with candida osteomyelitis of the heel, followed by cryptococcal meningitis the following year which ultimately required a ventriculoperitoneal shunt. in , he had a prolonged hospitalization after presenting with lethargy, headache and vomiting that culminated in seizure activity and prompted an emergency room visit. imaging at the time showed ventriculomegaly, and fluid from the shunt revealed yeast, but no bacteria. he was started on broad spectrum antifungal therapy and admitted for further management. cerebral spinal fluid and blood cultures confirmed invasive candida albicans meningitis. during this hospitalization, he also developed sepsis secondary to serratia marcescens. because of the pathogens that were being isolated, our service was consulted. of note, our patient does not have diabetes mellitus. a neutrophil oxidative burst assay showed an absent respiratory burst compared to control. a primary immunodeficiency panel to identify genetic variants was also sent to invitae. variants in cyba, cybb, ncf , and ncf were not identified, making chronic granulomatous disease less likely. peroxidase staining was negative on neutrophils and normal on eosinophils, suggesting a diagnosis of mpo deficiency. this led to mpo gene sequencing for deletion and duplication analysis. a homozygous pathogenic variant consistent with a molecular diagnosis of a mpo related condition was identified. immunoblotting of patient-derived immune cells demonstrated an absence of mature enzyme. although not typically indicated, given the severity of his presentation, our patient remains on fluconazole for long term prophylaxis. his younger brother also had a history of invasive disease with candidaosteomyelitis and meningitis. a neutrophils oxidative burst assay showed similar results in his brother and similar results with peroxidase staining, also suggesting a diagnosis of mpo deficiency. confirmatory genetic testing has not been performed yet. their father, who reported severe skin infections with candida, had peroxidase stains performed on neutrophils and eosinophils which were both normal. we have presented a patient without a significant history of diabetes mellitus who developed invasive disease from candida and serratia and was ultimately diagnosed with myeloperoxidase deficiency. abstract/case report text introduction: juvenile xanthogranuloma (jxg) is an often benign, histiocytic proliferative disorder of the mononuclear phagocytic system. patients typically present with localized cutaneous lesions. systemic disease, especially central nervous system involvement, rarely occurs but has significant morbidity and mortality risk. no standard evaluation nor therapy regimen exists for systemic jxg and little is known about the genomic alterations underlying its pathology. case report: a full-term male infant presented at months of age with post-prandial abdominal pain, fevers, altered mental status and weight loss. abdominal ultrasound and ct identified renal masses. a chest ct was obtained showing a paraspinal mass with possible neural foramina extension. mri brain and total spine was consistent with diffuse leptomeningeal disease involving the left frontal convexity, brainstem, cerebellum, and multiple cranial nerves. abnormal enhancement was also present along the entire surface of the spinal cord extending into the cauda equina with additional enlargement of the cervical/upper thoracic cord with intramedullary enhancing masses and a right paraspinal mass. renal biopsy yielded a pathologic diagnosis of disseminated jxg. integrative clinical sequencing of the mass identified a somatic driving alk rearrangement (kif b-alk in-frame fusion). tumor and matched germline dna sequencing did not detect any alterations in the ras/mapk pathway. bone marrow biopsy was negative for disease with cerebrospinal fluid analysis showing numerous monocytes and macrophages consistent with jxg. the patient was started on therapy consisting of systemic dexamethasone, intrathecal methotrexate/hydrocortisone and systemic intravenous cytarabine. his first cycle was complicated by pseudomonas aeruginosa bacteremia and gangrenous cellulitis of the perianal region, treated with systemic/topical antibiotics and topical gm-csf. following completion of the initial cycle of therapy, the patient was noted to have declining neurologic status, including seizure-like activity. repeat mr imaging revealed worsening cns disease with new subdural fluid collection and progression of leptomeningeal enhancement and intramedullary cervical lesion. in light of disease progression, the decision was made to continue dexamethasone treatment, but add adjunct intrathecal cytarabine, and transition to targeted alk inhibition via daily oral ceritinib, given its predicted cns penetrance followed by ceritinib in combination with systemic intravenous clofarabine. significant clinical and radiographic improvement was noted with the new targeted treatment regimen. ceritinib therapy was tolerated well overall after a % dosing reduction made for initial grade gastrointestinal toxicity and grade hypertriglyceridemia (non-life threatening but level > mg/dl). following continued treatment with daily ceritinib and completion of cycles of clofarabine therapy, our patient experienced complete disease remission. he continues to do well on daily ceritinib monotherapy with plan to complete an additional year of therapy. conclusion: our report highlights the potential benefit of real-time integrative clinical sequencing in the management of systemic histiocytic lesions, specifically non-langerhans cell conditions. it has the potential to identify novel somatic genetic alterations, other than the typical lchassociated braf mutations of the mapk pathway, that may be therapeutically targetable. treatment with nd generation alk-inhibition in our pediatric disseminated jxg patient was a novel, biologicallyrationale management approach with minimal toxicity and potentially contributed to his complete remission. abstract/case report text background: c glomerulonephropathy (c gn) is a progressive kidney disease with the predominant pathological feature of c deposits around the glomerular capillaries. c gn patients suffer from dysregulated activation of the alternative pathway as the result of autoantibodies or congenital genetic defects that stabilize cleavage of c . despite therapy involving immunosuppression and complement-pathway inhibition, the prognosis for c gn is poor. we report a patient with autoantibody-mediated, refractory c gn who demonstrated no improvement on rituximab but achieved sustained remission on bortezomib. follow up studies after one year demonstrated clearance of the culprit autoantibody, normalization of c levels, and improved pathologic appearance of the kidneys. this case supports the idea that c gn is frequently driven by pathogenic autoantibodies that may not clear with rituximab alone. plasma cell directed therapy has the potential to clear these autoantibodies and halt the progression of disease. case presentation: we report the case of a hispanic male with chronic renal dysfunction initially diagnosed with membranoproliferative glomerulonephritis on renal biopsy at years of age. despite cellcept and prednisone, over the next years, he had worsening proteinuria and an increase in protein-to-creatinine ratio. renal biopsy suggested c gn, and lab studies revealed a factor-h autoantibody and c level below the assay limit of detection. after initiating eculizumab, the proteinuria temporarily improved; however, the proteinuria eventually worsened, and he was referred to immunology. we hypothesized that the factor h-binding autoantibody was the cause of dysregulated c cleavage and disease progression, and blocking the terminal complement pathway with eculizumab would not halt upstream c -mediated kidney injury. at the age of , rituximab and plasmapheresis were administered to clear the factor-h autoantibody. three months after rituximab administration, the factor-h autoantibody level decreased to the normal range, but he continued to have significant proteinuria with low serum albumin and undetectable c level. we concluded that the relevant autoantibody was not solely produced by differentiating memory b cells, so we decided to target the plasma cell compartment. bortezomib was started at the age of , and eculizumab was continued given his initial response to treatment. after adding bortezomib, factor h autoantibody levels dropped below prior levels and serum c level normalized. renal biopsy at the age of showed evidence of imp r o v i n g c d e p o s i t i o n a n d l e s s p r o m i n e n t g l o m e r u l a r hypercellularity, with stable mesangial hypercellularity, interstitial fibrosis, tubular atrophy, and sclerotic glomeruli. although his proteinuria did not worsen, it remained persistent, suggesting that earlier introduction of bortezomib could have prevented disease advancement. there has been no further progression of kidney failure. conclusions: the majority of c gn patients harbor autoantibodies to components of the alternative pathway of complement. this case provides evidence that at least some of these autoantibodies are indeed the cause of complement dysregulation, and thus are prime targets for therapy. b cell targeting therapies may be inadequate to decrease autoantibody levels for some patients. early initiation of bortezomib, or other plasmacell directed therapy, may effectively induce complement normalization and disease remission in these cases. inhaled corticosteroid and a long-acting bronchodilator. he has no family history of immunodeficiencies or congenital disorders. computed tomography(ct) chest showed bronchiectasis. his complement studies and isohemagglutinin titers were also normal. his serum immunoglobulin(ig) and lymphocytes on presentation are shown in table . he had a poor response to polysaccharide pneumococcal vaccination. he was diagnosed with combined igg /igg subclass/iga deficiency and was started on immunoglobulin replacement therapy and prophylactic rotating antibiotic therapy. thereafter, his clinical course markedly improved with a reduction in the frequency of rti's as well as the number of bronchiectasis exacerbations. there was high suspicion for an underlying genetic disorder based on his constellation of neurodevelopment disorders and immunodeficiency. cytogenetic evaluation with array comparative genomic hybridization(cgh) analysis showed duplication of xq and xq consistent with mds. discussion: mds is caused by duplications involving the mecp gene locus of the x chromosome at xq . it has a % penetration rate in males whereas females act as carriers and are usually unaffected. rarely, cases of de novo mutations causing mds have been reported. chromosome microarray analysis is currently the best initial clinical test when mecp duplication syndrome is suspected. management needs a multidisciplinary approach involving geneticists, neurologists, ophthalmologists, physical medicine and rehabilitation specialists, psychologists, gastroenterologists, and allergy and immunology specialists. prophylactic treatment with ivig and antibiotics has been the standard of care for immunodeficiency in these patients. prognosis is guarded and most male patients die in the mid to late 's because of severe rti's secondary to immunodeficiency. conclusions: this case confirms the association of mds with combined iga and igg subclass deficiencies. clinicians should consider pursuing genetic evaluation for mds in patients with neurodevelopmental disorders and immunodeficiency because the diagnosis of the syndrome can change the overall approach to management and expectations in prognosis. abstract/case report text introduction: c nephritic factor is an autoantibody that binds to the alternative pathway c convertase (c bbb). this results in unchecked overactivation of the alternative complement pathway, which can lead to renal disease, partial lipodystrophy, retina disease, and frequent infections. in this case, we present a patient with partial lipodystrophy and low c , subsequently found to have c nephritic factor. case description: a year old female presented with a month history of low c levels. she was diagnosed months ago with poststreptococcal glomerulonephritis (psgn) after presenting with hematuria and elevated aso titers. she had c levels drawn - months after time of diagnosis and c level was low at (normal range - ), which was consistent with psgn. it was rechecked months after time of diagnosis and was still low. she was referred to rheumatology at this time and was found to have a positive ana titer : . tests for lupus and anti-phospholipid syndrome were negative. c normalized to the low-normal range at months after time of diagnosis to . her pediatrician checked to make sure it remained normal around months after initial diagnosis and c was low again at . c was normal at . she was referred to immunology for further evaluation. during this time she was asymptomatic with no fevers, infections, hematuria, rashes, joint pain, or joint swelling. she has no history of hospitalizations other than the first for psgn. mother denied family history of autoimmune disorders. physical exam: physical exam was notable for abnormal subcutaneous facial fat with normal fat distribution in the rest of her body. the rest of the exam was unremarkable with normal cardiac, pulmonary, abdominal, and skin exam. testing: c level was rechecked and low at . c nephritic factor was elevated at . (normal range . - . ). alternate pathway complement (ah ) was confirmed twice and was undetectable, < (normal level greater or equal to ). total hemolytic complement (ch ) was low at (normal level - ). other complement levels were checked and c q, c , c , c , c , c , c , and c complement were within normal range. discussion: the overactivation of the alternative complement pathway by c nephritic factor can result in various clinical manifestations, such as c glomerulopathy and acquired partial lipodystrophy in predominantly the face and the upper torso. the exact mechanism of how c nephritic factor is related to facial and upper body lipodystrophy is not known. one proposed mechanism is that adipocytes in the face and upper body produce more factor d, which is a complement protein utilized by c nephritic factor. overactivation of the alternative complement pathway on the adipocyte then leads to formation of the membrane attack complex, resulting in adipocyte lysis. eye disease, such as retinitis pigmentosa and macular degeneration can develop. c nephritic factor can also lead to more frequent infections and renal disease. patients need to be closely monitored. if patients develop c glomerulopathy, they may need to be considered for immunomodulatory therapy, such as steroids and other immunosuppressants. project manager/ucsf benioff children's hospital senior clinical research associate/ucsf benioff children's hospital associate professor/department of clinical pharmacy, ucsf staff research assistant iv/ucsf benioff children's hospital senior supervisor/ucsf benioff children's hospital laboratory specialist/ucsf benioff children's hospital research specialist/ucsf benioff children's hospital assistant professor/ucsf benioff children's hospital clinical professor/ucsf benioff children's hospital assistant professor/ucsd rady children's hospital staff pediatrician/tuba city indian health service staff pediatrician/phoenix children's hospital associate professor/seattle children's hospital chief, genetic immunotherapy section/niaid, nih professor/university of minnesota professor/ucsf benioff children's hospital abstract/case report text background: artemis-deficient scid (art-scid) represents % of all scid, but occurs in / births in navajo and apache native americans. artemis protein, encoded by dclre c, is essential for repairing dna double-stranded breaks, including those generated during v(d)j recombination of antigen receptor genes as t and b cells develop. artemisdeficiency causes not only t-b-nk+ scid, but also increased sensitivity to alkylating drugs and radiation. art-scid is the most difficult scid to treat with allogeneic hematopoietic cell transplantation (hct) due to high rates of rejection and gvhd, incomplete immune reconstitution, and toxicity following intensive conditioning regimens. as an alternative, we developed a self-inactivating lentiviral vector containing the human artemis promoter and dclre c cdna (aproart). we are evaluating its toxicity and efficacy in a phase i/ii gene transfer trial in art-scid patients. methods: newly diagnosed infants with art-scid and older patients with insufficient immunity despite prior allogeneic hct were eligible if organ function was acceptable. infants needed to have no matched sibling donor and be at least months old at conditioning. cd + cells were isolated from bone marrow or cytokine-mobilized peripheral blood, cultured with cytokines, transduced x with aproart, and cryopreserved. patients received daily doses of busulfan, targeted for a cumulative exposure (cauc) of mg*hr/l, with infusion of thawed cells on the following day. results: we treated newly diagnosed infants (art - & - ) with median age . m (range . - . ) and previously-treated patients (art - ) ( . y, . y and . y), with a median follow-up of . m (range . - . ). the mean (sd) bu cauc was . ± . mg*hr/l. patients received a median of . x aproart-transduced cd + cells/kg (range . - . ). the average vector copy number (vcn) and transduction efficiency in the marrow grafts exceeded those in the pbsc grafts: . ± . copies/cell vs . ± . (p= . ) and ± % vs ± . % (p= . ), respectively. there were no serious busulfan side effects. all patients had transduced peripheral blood leukocytes by w and of developed gene marking in t, b, nk and myeloid cells by w (fig. ) . gene-corrected cd , cd , cd / ra/ccr , cd and cd cells appeared in of patients (fig. ) , with art having t, nk and myeloid marking without b cells at m post infusion. normalization of lymphocyte proliferation to pha occurred in the evaluable (> w) infants (fig. ) , all now outpatients off isolation. two infants and previously treated child developed autoimmune hemolytic anemia (aiha), with requiring immunosuppressive therapy. infections included rhinovirus at presentation in art that resolved with t cell reconstitution. after discharge art acquired and recovered from norovirus and art acquired and recovered from cmv and rotavirus. analyses of insertion sites and t cell receptor diversity are pending. conclusion: infusion of aproart-transduced autologous cd cells into art-scid patients pretreated with very low exposure busulfan resulted in multilineage engraftment of transduced cells with evidence for t and b cell immune development. aiha, the only complication to date, occurred early and appears to resolve following restoration of t cell immunity. these encouraging results suggest potential effectiveness of ex vivo gene therapy for art-scid. ( ) submission id# mailan nguyen, md , susan canny, md, phd , andrea ramirez, md , ivan chinn, md abstract/case report text background: systemic lupus erythematosus is a heterogeneous disorder of the immune system. systematic genetic evaluation of patients with childhood-onset sle (csle) has begun to identify phenotypic clusters of csle patients with classic sle-causing genetic variants, as well as revealed unexpected genetic mimics of lupus. we report patients diagnosed with csle with similar typical and atypical lupus features, who were subsequently found to carry pathogenic nras variants that are the cause of ras-associated autoimmune leukoproliferative disorder (rald). cases: all patients ( females, male) presented at < years of age (average age . months, range - months) with antinuclear antibodies, anti-double-stranded dna antibodies, autoimm u n e h e m o l y t i c a n e m i a , s e v e r e t h r o m b o c y t o p e n i a , antiphospholipid antibodies, hypocomplementemia and nephritis. additionally, the patients all displayed fevers, organomegaly, lymphadenopathy and hypergammaglobulinemia. two out of patients had a malar rash, leukopenia, lymphopenia, anti-smith antibodies, serositis or arthritis. no patient had oral or nasal ulcers or photosensitivity. despite the fevers, lymphoproliferation and systemic autoimmunity, the patients did not display overwhelming immune dysregulation (peak ferritin - ng/ml). interestingly, the patients were found to have monocytosis ( - %), as has previously been reported in rald. double negative t cells were within normal range in the patients in which this was tested. all patients required aggressive immune modulation for control of their disease manifestations. two of the developed severe infections, specifically pneumococcal sepsis, during therapy. current follow-up covers an average of . years (range . to years). the patients responded to corticosteroids and were given sequential trials of various steroid-sparing therapies. in general, they appeared to benefit from both b cell depletion and t cell-directed modalities (cyclosporine, rapamycin), which are not first line therapy in csle. unfortunately, patient developed a fatal pulmonary infection while on treatment; her underlying disease was felt to be quiescent. due to the early-onset of disease, each patient was selected for genetic evaluation ( by exome sequencing, by gene panel). this lead to the discovery of pathogenic nras variants (c. g>a, p.g d) in all patients, assumed to be somatic, although this was confirmed in only case. conclusion: ras-associated autoimmune leukoproliferative disorder can present indistinguishable from csle with positive autoantibodies, immune cytopenias, arthritis, nephritis and hypocomplementemia. clinicians should consider evaluating for rald in csle patients who present at an early age ( < years) with predominant features of lymphoproliferation and hematologic abnormalities, particularly monocytosis. t cell-directed therapy with cyclosporine or rapamycin should be considered for rald. ( ) human ctla loss-offunction causes dysregulation of foxp + regulatory t (treg) cells, hyperactivation of effector t cells, and lymphocytic infiltration of target organs. patients also exhibit progressive loss of circulating b cells, associated with an increase of predominantly autoreactive cd (lo) b cells and accumulation of b cells in non-lymphoid organs. inherited human ctla loss-of-function demonstrates a critical quantitative role for ctla in governing t and b lymphocyte homeostasis. ( ) this case highlights the importance of next generation sequencing (ngs) in diagnosing and managing complex presentations with multi-system involvement. case presentation: patient was diagnosed with diffuse large b cell lymphoma at age and treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop) in / . he underwent autologous stem cell transplant with preparative carmustine, etoposide, cytarabine, and melphalan (beam) in / . he subsequently developed recurrent giant condyloma acuminata following transplantation requiring surgical resections, refractory immune thrombocytopenic purpura (itp) requiring aggressive systemic steroids and high dose ivig at least yearly, experiencing hypogammaglobulinemia, recurrent sinopulmonary infections, disseminated herpes zoster, and kaposi sarcoma. in / , he underwent ct/pet, revealing extensive hypermetabolic lymphadenopathy and splenomegaly. bone marrow biopsies were negative for lymphoma, although showed a slightly hypocellular marrow ( - % cellularity), % blasts, and eosinophilia without peripheral eosinophilia. repeated evaluations for hiv, syphilis, histoplasma, cmv, hhv , hhv , bartonella, coxciella, brucella, htlv, toxoplasma were negative. htlv and antibodies had been negative prior to transplantation. tonsillectomy / due to progressive enlargement showed reactive follicular hyperplasia with focal acute tonsillitis without granulomas or viral inclusions. repeated lymphocyte enumeration and proliferation studies were normal. a repeat pet scan / revealed persistent diffuse lymphadenopathy involving the neck, chest, abdomen, and pelvis. left lung biopsy revealed non-caseating granulomas without lymphoma. stains for ebv were negative. repeat pet scan / indicated disease progression prompting a left axillary excisional lymph node biopsy, revealing ebv lymphadenitis with large, reactive follicles with interspersed inflammation and loosely formed granulomas and cd positive b cells within the follicles. ebv blood pcr was negative. afb and fungal stains were negative on all biopsies. in / , his igg was ( - ), iga ( - ), and igm ( - ) with only out of protective serotypes to pneumococcus post-vaccination at . or greater. in / , igg was ( - ). custom ngs panel showed a heterozygous missense variant in ctla c. c>g (p.s r) located in the transmembrane domain. this variant of uncertain significance is suspicious and strongly suggests the diagnosis of ctla -related autoimmune lymphoproliferative syndrome . patient was referred to the national institute of health, where he received a bone marrow transplant. conclusion: primary immunodeficiency diseases comprise a group of highly heterogeneous immune system diseases and around forms of pid have been described. ngs has recently become an increasingly used approach for gene identification and molecular diagnosis of human diseases guiding treatment to patients who may otherwise have poor outcomes. ( ) ( ) submission id# erik newman, md , cullen dutmer, md allergy and immunology fellow/university of colorado and children's hospital colorado assistant professor of pediatrics/section of allergy & immunology, children's hospital colorado, university of colorado school of medicine, aurora, co, usa abstract/case report text introduction: inherited defects of the complement system are rare disorders that can result in unique susceptibility to infections with select bacteria. patients with a deficiency of a complement protein early in the complement pathway (affecting c qrs, c , c , factor h, or factor i) have increased susceptibility to infection with encapsulated bacteria, most notably streptococcus pneumoniae and neisseria species. in contrast, patients with a deficiency of a complement protein at the terminal end of the complement pathway (affecting c , c , c , c alpha/beta/gamma, or c ) almost universally present with severe, recurrent, or disseminated neisseria species infections. most genes encoding complement proteins are found on autosomes, in which specific complement deficiencies result from biallelic mutations. although particular complement deficiencies occur at higher frequencies in certain populations, the prevalence of specific complement deficiencies is unknown in many parts of the world, especially in underdeveloped regions, including sub-saharan africa. herein, we describe a young congolese boy with an atypical presentation of c alpha deficiency. case description: a -month-old congolese boy with consanguineous parents (first cousins) presented with recurrent infections. prior to an evaluation of his immune system, he was hospitalized five times. his infections included episodes of acute otitis media, bacterial pneumonia, and viral pneumonitis. a bronchoscopy revealed diffusely edematous airways and growth of candida albicans, moraxella catarrhalis, and streptococcus pneumoniae in bronchoalveolar lavage cultures. concurrently, his respiratory pcr panel was positive for adenovirus. his initial immune evaluation included assessments of his serum immunoglobulin levels, vaccine titers (tetanus, diphtheria, haemophilus influenzae, and streptococcus pneumoniae), neutrophil oxidative burst, lymphocyte subsets, and ch , in which only his ch was abnormal ( u/ml). his ch remained low on repeat assessment ( u/ml), at which time an ah was pursued and also returned with a low result ( % of normal). a complement system genetic panel identified a homozygous intronic variant in c a (c. - g>a). functional confirmation of the variant revealed that the patient had a significantly decreased c level ( mcg/ml) and absent c function. discussion: we present a case of a young congolese boy with c alpha deficiency and a clinical presentation atypical for defects in terminal complement proteins. our patient presented primarily with recurrent respiratory infections, including streptococcus pneumoniae pneumonia, but without a preceding history of meningococcal disease. while it is well established that patients presenting with terminal complement pathway defects have an increased susceptibility to meningococcal disease, it is less clear if they have increased susceptibility to pneumococcal infections. occurring between exons and , the homozygous intronic variant in c a identified in our patient is predicted to result in abnormal splicing. while the allele frequency of this mutation is relatively high in the african population ( . ), functional confirmation of the variant demonstrated a decreased c level and absent c function that support the pathogenesis of the mutation. the discrepancy between the allele frequency and reported disease cases could be explained in part by varying clinical manifestations seen in c deficiency or underrecognized disease in sub-saharan africa. abstract/case report text rationale: scid is a syndrome characterized by profound t, b, and (in some cases) nk cell defects that is universally fatal unless immune reconstitution is achieved. a total of scid infants have been given allogeneic bone marrow transplantation at duke university medical center without pre-transplantation chemotherapy or post-transplantation graft-versus-host disease (gvhd); % received t cell-depleted haploidentical parental marrow and ( %) are known to be deceased. post-transplantation follow-up ranged from months to years. the aim of this cross-sectional study is to characterize the clinical status of a large cohort of survivors treated at a single medical center. methods: clinical status was assessed by detailed questionnaires delivered by mail or electronically. adult (≥ years old) and pediatric questionnaires were based on patients' age. patients were also contacted by telephone and evaluated at clinic visits. molecular type of scid, demographics, type, date and age at transplant were obtained from a clinical database. results: fifty questionnaires were completed to date from survivors ranging in age from to years. twenty-nine/ were adults ≥ years at the time of the questionnaire. genetic defects were known for all patients-xlinked scid was the cause in about half ( figure ). twenty-three of patients were on immunoglobulin replacement. thirty of reported having received immunizations, and about half of those received live vaccines. fifteen of reported they were taking no regular medications; reported taking prophylactic antibiotics. >we found substantial scholastic achievement, with / adult patients reporting college attendance. two had post graduate education including doctorate level degrees. occupations included physician, nurse, factory worker, musician, teacher, and engineer. one patient had children. twenty-seven / adult patients shared their height and weight and % ( / ) had a healthy bmi (bmi . - . ), while % ( / ) were overweight, and % ( / ) were underweight (< . ) . in pediatric patients, the average age and sex-adjusted bmi was at the th percentile and only had a bmi that was < th percentile. thirty-four/ patients reported seeing an immunologist regularly. in the adult group, % reported no longer seeing an immunologist. the health conditions reported were similar to those common in the general population, and included rashes, warts and mouth ulcers. most reported these were transient, self-resolving issues. thirteen of ( %) reported having adhd, higher than nih reported rates which estimate adhd in . % of adults and % of children). ten of ( %) reported having anxiety, similar to the nih reported prevalence of . % in the general population. / (~ %) reported having no active concerns about their health. conclusions: overall, our findings are consistent with those in the last update done by railey et al, j. peds. : [ ] [ ] [ ] [ ] [ ] [ ] [ ] in this population. patients are doing well with most problems similar to those common in the general population. most have a healthy bmi. adhd had a higher prevalence than in the general population. more than / of scid patients are not seeing an immunologist regularly, and a majority do not have any active concerns. . genetic causes of scid in the patients whose questionnaire data are presented. x-linked scid was the most common, followed by ada and il- r deficient scid clinical fellow/national institute of allergy and infectious diseases (niaid/nih) research nurse/nih-nhgri professor of pediatrics and allergy and immunology/ann & robert h. lurie children's hospital of chicago chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih abstract/case report text rationale: myopathy has been occasionally documented in patients with primary immunodeficiency (pid). however, data on frequency and patient characteristics associated with myopathy are lacking. we performed a descriptive analysis of patients with primary immunodeficiency (pid) in the usidnet having myopathy as a feature of their primary disease. methods: the usidnet registry was queried for the spectrum of myopathic disorders in pid patients that had been entered into the registry as of november , . results: a total of pid patients with myopathy were identified, of which ( . %) were female. median age at onset of symptoms related to pid was years (range . - years, iqr . - years). median age of diagnosis of pid was . years (range . - years, iqr - . years). age at onset of myopathic disorders was not known. twenty-eight ( . %) patients had a diagnosis of common variable immunodeficiency (cvid), ( . %) had agammaglobulinemia, patients each ( . %) were diagnosed with severe combined immunodeficiency, hypogammaglobulinemia, or 'hlh and pigmentary disorders', while patients ( . %)were reported in each category of combined immunodeficiency (cid), autoimmune lymphoproliferative syndrome (alps), and autoinflammatory disease. thirty-five patients ( . %) had a causative gene variant identified attributable to pid. the most common variant identified was btk ( patients) followed by aire, lyst, cybb ( patients each) and pi kcd ( patients). eighteen individual patients had other variants identified ( figure ). patients had cellulitis or skin/ subcutaneous tissue infection, patients had a 'skin or subcutaneous tissue abscess', had pyoderma gangrenosum, and patient with eczema herpeticum. within this cohort of patients, the most common myopathy listed was myositis ( ) followed by 'muscle weakness' ( ), dermatomyositis ( ) , myalgia/s ( ) , myalgia/myositis ( ), myopathy ( ), polymyositis ( ) , steroid-induced myopathy ( ) . no patient had an infectious myositis or muscle abscess listed. eighteen patients ( . %) had a myopathic disorder at the time of diagnosis of their pid. thirtyone patients ( . %) received prednisone, ( . %) received hydrocortisone and ( . %) received dexamethasone. two patients had a diagnosis of adrenal insufficiency. nine patients ( . %) underwent hematopoietic stem cell transplantation. nine patients ( . %) died; median age of death years (range . - . years, iqr . - . years). ). one patient with chronic granulomatous disease had myopathy due to duchenne muscular dystrophy, which was listed as a cause of death. no other myopathic disorders were listed as a cause of death for the other patients. conclusion: myopathy and inflammatory myopathic disorders occur at relatively high frequency in pid, and may be present even at the onset of clinical symptoms. the underlying etiology can be speculated to be multifactorial. further subgroup analysis is warranted to elucidate possible variant-specific or treatment-associated characteristics of myopathy in pid. laboratory studies at years revealed normal igg/iga/igm but markedly elevated serum ige ( , ku/l), anemia (hb . g/dl), thrombocytopenia ( x /l) and lymphopenia ( cells/l), with low t and b cell counts, very low proportion of naïve t cells, skewed repertoire of cd + t cells, undetectable trec levels, and impaired t cell proliferation to mitogens and antigens. there was an elevated percentage of circulating plasmablasts ( . %) and of dysreactive cd low cd low b cells ( . %). at the age of , hsct with reduced intensity conditioning was performed from her phenotypically hla-matched father, with improvement of t and b cell count and function. whole exome sequencing (wes) identified a homozygous missense variant in the mannosidase alpha class b member (man b ) gene (p.asp asn), that segregates with disease in the pedigree. the man b asp residue is evolutionary conserved. the p.asp asn allele has a minor allele frequency of . in gnomad, with no homozygotes. the cadd score for this variant is . , significantly higher than the mutation significance cutoff score ( . ). man b is involved in the lysosomal degradation of glycoproteins and demannosylation of free n-glycans. in particular, man b cleaves man glcnac to generate man glcnac . serum n-glycan profiling revealed elevated man /man and man / man in the patient. n-linked and free glycan profiling by mass spectrometry (ms) showed accumulation of man glcnac , man glcnac and man glcnac glycans in patient fibroblasts as compared to control cells, consistent with defective lysosomal glycoprotein degradation. lentiviral transduction of wild-type man b into patient fibroblasts led to normalization of the n-linked glycan profile, with reduction of man glcnac from . to . times control levels, and of man glcnac from . to . control levels, indicating rescue of the impaired deglycosylation ( figure a ). western-blotting demonstrated defective n-glycosylation of lamp and icam proteins in patient fibroblasts ( figure b) , which were corrected upon lentiviral transduction of wild-type man b ( figure c ). overall, our results indicate that loss of man b enzymatic activity leads to dysregulation of deglycosylation and abnormal mannosylation of glycans. in conclusion, we have demonstrated that man b deficiency accounts for a novel autosomal recessive cdg with prominent features of immune deficiency and immune dysregulation. abstract/case report text heme oxygenase- (hmox ) is a rate-limiting enzyme that catalyzes the degradation of heme to carbon monoxide, ferrous iron, and biliverdin, which becomes bilirubin. these byproducts are implicated in inflammation, cell homeostasis, and antioxidant defense( ). hmox -deficiency is an extremely rare autosomal recessive disorder with a complex presentation of a wide spectrum of symptoms, including hemolytic anemia and hyperinflammation, requiring genetic testing for confirmed diagnosis ( ) . we report the fifth known case of hmox -deficiency ( ) ( ) ( ) , a boy who presented at years of age with aspects of the characteristic phenotype, but also had early onset asplenia, interstitial lung disease, and previously undocumented immune deficiency. patient's presentation was notable for hyperinflammatory exacerbations triggered by viral and bacterial infections as well as vaccinations. episodic flares occurred every few months lasting weeks to months with fevers of - f, hypoxia, leukocytosis above , /mm , hemolytic anemia with negative coombs, thrombocytosis exceeding million/ mm , transaminitis, hemoglobinuria, hyperferritinemia to , ng/ml, and elevated ldh to , iu/l. immune evaluation revealed normal immunoglobulin levels and adequate vaccine titers to both protein and carbohydrate antigens. although class switched populations were normal, b-cell phenotyping showed absent immature and transitional b-cells, low mature memory, and reduced cd + memory b-cells at % (normal > %). mitogen stimulation with phytohemagglutinin and anti-cd were decreased ( . % of control and . % of control, respectively). t-cell phenotyping demonstrated cd population heavily skewed to immaturity with % of cells with naïve phenotype cd ra+cd +ccr +. there were few effector-memory t cells and the cd population was skewed towards immaturity with > % of the cells naïve. liver biopsy was performed secondary to hepatomegaly yielding mild to moderate sinusoidal fibrosis. bone marrow biopsy revealed a normocellular marrow with % blasts, increased megakaryocytes, and extensive hemophagocytosis. natural killer cell function was very low, while soluble il- ra level was normal. further workup for hemoglobinopathies, metabolic defects, congenital disorders of glycosylation, lysosomal storage disorders, wilson's disease, autoimmune hepatitis, inherited and autoimmune hypercoagulability disorders, connective tissue disorders, myositis, and myopathies were all unremarkable. imaging demonstrated asplenia and howell-jolly bodies were present. hemophagocytic lymphohistiocytosis (hlh) genetic testing showed no variants. he was suspected to have systemic juvenile idiopathic arthritis (sojia) with episodes of macrophage activation syndrome. the frequency of his autoinflammatory flares increased such that he was corticosteroid dependent by age , having failed methotrexate, azathioprine, and anakinra. he was started on tocilizumab with laboratory improvements, but his lung disease progressed and became oxygen dependent. lung biopsy confirmed nonspecific interstitial pneumonitis (nsip) with cholesterol granulomas also seen in sojia. ultimately, chronic lung disease led to his death at age . whole exome sequencing yielded a paternal frame shift hmox and maternal splice donor hmox resulting in absence of protein. bone marrow transplantation (bmt) in hmox deficient mice have rectified phagocytotic defects and thereby their autoinflammatory phenotype, but no human reports for bmt treatment of hmox -deficiency has been described. here we describe a phenotype expansion for hmox deficiency to include not only asplenia and hepatomegaly, but also interstitial lung disease with cholesterol granulomas and inflammatory flares. abstract/case report text introduction: mutations in the gene encoding signal transducer and activator of transcription (stat ) cause autosomal dominant hyperimmunoglobulin e syndrome (ad-hies) characterized by recurrent skin and sinopulmonary infections, atopic dermatitis, and elevated serum immunoglobulin e (ige) levels. treatment is largely aimed at controlling symptoms and preventing infections with no standard of care. there is a paucity of literature describing the utilization of biologic therapies in the ad-hies patient population. we present patients from one family with ad-hies successfully treated with monoclonal antibody therapies targeted at il- , il- and il- . case descriptions: patient : -year-old female with stat lof c. c>t (p.arg trp) with a history of atopic dermatitis and asthma requiring - steroid courses per year with frequent school absences. she developed a severe pruritic rash covering her upper body months ago that failed to respond to antihistamines and topical antibiotics prescribed by her primary care provider. given her poorly controlled asthma and our concern for a follicular type morphologic variant of atopic dermatitis, dupilumab was initiated. her scoring atopic dermatitis (scorad) prior to initiation of biologic therapy was . and improved to . following doses ( weeks) of dupilumab with clear dramatic improvement in her skin and quality of life ( figure ). she also reports decreased asthma severity with no steroid courses, reduced albuterol usage, and significant decline in school absences since initiation of dupilumab. patient : -year-old female with stat lof c. c>t (p.arg trp) who is the sister of patient . she has a history of severe asthma requiring frequent emergency department visits, hospitalizations, and - steroid courses per year despite therapy with high-dose fluticasone-salmeterol. spirometry prior to april demonstrated an obstructive pattern with an fev ranging from - %. she was initiated on mepolizumab in april . subsequent spirometry demonstrates an fev average of % with a range of - %. she had one hospitalization in early but otherwise no hospitalizations for asthma since initiation of biologic therapy. patient : -year-old female with stat lof c. c>t (p.arg trp) who is the paternal first cousin of patients & . she has a history of severe atopic dermatitis with associated pruritus and picking behaviors, poorly controlled despite daily triamcinolone application. she previously failed ultraviolet therapy and crisaborole. she also has a history of severe asthma requiring - steroid courses per year despite high-dose fluticasone-salmeterol. dupilumab was started in may . scorad prior to initiation monoclonal antibody therapy was . and declined to . following weeks ( doses) of dupilumab therapy with marked improvement in skin appearance and pruritus ( figure ). discussion: we present three cases of ad-hies caused by stat loss-of-function mutations treated successfully with monoclonal antibody therapies targeted at il- or il- and il- . to the best of our knowledge, there is no published data describing the use of these biologic agents in the treatment of ad-hies. future studies are needed to clarify the role of these cytokines in the pathogenesis of ad-hies and to elucidate clinical indications for biologic therapy in this patient population. informed consent was obtained from all individual participants included in the study. abstract/case report text ctla- is a potent inhibitor of t cell proliferation that competes with costimulatory receptor cd for its ligands cd and cd expressed on antigen presenting cells. heterozygous loss-offunction mutations in ctla- have been identified in patients with lymphocytic infiltration of multiple nonlymphoid organs (lo et al). the patient is a -year-old jordanian male born at term to nonconsanguineous parents, hospitalized at mo for lll pneumonia, and at mo and at mo he was evaluated in the ed and diagnosed with non rsv-bronchiolitis with lll infiltrate thought to be secondary to atelectasis. at yo he developed lll pneumonia and respiratory failure requiring picu admission. he was treated with ceftriaxone. after discharge, he had weeks of intermittent fever, progressive fatigue, productive cough, and ftt. he received courses of cefdinir, clindamycin, and tmp-smx without improvement. chest ct revealed left lung consolidation, lll bronchiectasis, lul tree in bud opacities, and hilar lymphadenopathy. bronchoscopy with bal revealed no bacterial growth and no acid-fast bacilli. srrna ngs was positive for h. influenzae. lung biopsy demonstrated acute and chronic bronchiolitis with bronchiolitis obliterans and intraluminal polyps, with lymphocytic infiltration involving the bronchi and bronchioles. the lung parenchyma showed airspace filling with foamy macrophages and chronic interstitial inflammation. acid fast and fungal stains were negative. he was treated with systemic steroids for bronchiolitis obliterans with noted improvement. the severity of lung disease at such an early age prompted an immune evaluation. sweat test, anca, anti-pr and hiv were negative. total immunoglobulins were normal for age, and titers to s. pneumoniae, diphtheria and tetanus were protective. lymphocyte enumeration revealed elevated t and nk cell numbers for age. lymphocyte proliferation to pha, pwm, candida and tetanus were normal. dihydrorhodamine assay was normal. b cell phenotyping was normal. there was normal expression of cd , hla-dr and cd on activated t cells; of note the patient was on systemic steroids when tested. invitae gene pidd panel revealed a variant in ctla : c. g>a (p.gly glu) that has been shown to be pathogenic in one patient (schawb et al). flow cytometry showed normal frequency of t follicular helper cells and t regulatory cells compared with controls, however ctla- expression by t regulatory cells was lower than control. due to the severe and progressive nature of the patient's lung disease, therapy with x weekly azithromycin and abatacept mg sq weekly was initiated. we report a case of ctla- haploinsufficiency presenting with recurrent pneumonia and bronchiolitis obliterans in a -year-old child. based on patient registry data, our patient appears to be the youngest child diagnosed with ctla- haploinsufficiency reported in the literature to date (schawb et al). notably, our patient l a c k s o t h e r f e a t u r e s c o m m o n l y d e s c r i b e d i n c t l a - haploinsufficiency, including autoimmune cytopenias, gastrointestinal disease, lymphoproliferation, and hypogammaglobulinemia. this case illustrates the importance of consideration of this diagnosis in young children with severe lung disease without other evidence of immune dysregulation. our hope is that prompt recognition and early treatment administration will prevent disease progression and further decrease in pulmonary function. splenectomy, he had an episode of pneumococcal meningitis at age , and sepsis of unknown origin at age . over the past several years he has developed chronic tinea corporis, onychomycosis, and otitis externa infections despite numerous antimicrobial regimens. at age , the patient developed urinary retention, walking, and balance difficulties. he was found to have diffuse white matter changes on mri, elevated wbc, and positive oligoclonal bands. initially, he was diagnosed as progressive ms treated with steroids with partial improvement. csf microbiology studies including afb stains, bacterial, fungal, mycobacterial cultures, cryptococcal antigen, vdrl, t. pallidum particle agglutination (tppa), as well as, pcr for cmv, ebv, vzv, enterovirus, hsv - , jc virus and t. pallidum were all negative. peripheral blood studies included mycobacterial blood culture, pcr for cmv, ebv, hhv- , in addition to serology for cryptococcal antigen, and coccidioides species, all of which were negative. additional neurological complications include granulomatous uveitis and oscillopsia, which he developed around age . immune evaluation performed at age revealed low igg and igm, and the patient was started on grams of monthly ivig. cbc with differential was notable for normal monocyte count and thrombocytopenia, mild neutropenia (table ). immunophenotyping revealed absent b cells and nk cells, while the cd t cells were elevated. cd t cells were normal (table ) . at age , whole-exome sequencing identified a heterozygous missense mutation in gata c. c>t, p.(arg trp). after the diagnosis of gata haploinsufficiency, he was found to have myelodysplastic syndrome with multilineage dysplasia (mds-mld) on bone marrow biopsy. he is currently awaiting bone marrow transplant discussion: we present a -year-old male with cytopenias, splenomegaly, leukoencephalomyelopathy, granulomatous uveitis, and recurrent fungal infections found to have a pathogenic heterozygous missense mutation in gata . leukoencephalomyelopathy in gata haploinsufficiency has been associated with jc virus and ebv infection. our patient did not have any evidence of a chronic csf infection. to our knowledge, myelopathies have not been reported with gata c. c>t, p.(arg trp). this case highlights the variable nature of presentation in gata haploinsufficiency, and the need for clinical awareness of this entity in order to facilitate early diagnosis and appropriate therapy. immunoglobulins* white blood cells . x * /l (n: - ) magnetic resonance imaging (mri) of the brain and spine showed numerous enhancing parenchymal nodules (figure one). brain or spinal biopsy was requested, but not recommended by our neurosurgery service. lumbar puncture evaluation was performed. cerebral spinal fluid showed no bacterial or fungal elements. both quantiferon gold for tuberculosis and three consecutive sputum cultures for acid fast bacilli were negative. he continued his sirolimus and the intravenous immunoglobulin replacement was increased to two grams/kg. the patient was cleared from respiratory isolation and discharged after two weeks in our facility with mild improvement in his neurologic status. within five days he was sent to a nationally renowned hospital. at this facility, extensive evaluation for his neurologic deficits were performed including culture and pcr for bacteria, virus, mycobacteria from csf bone marrow, lymph node, blood, and induced sputum. these were noncontributory. he was given high dose corticosteroids for two days. one of our facility's sputum cultures was reported with acid fast bacilli. but sputum mycobacterium tuberculosis pcr was negative. repeat mri scans of the brain and spine showed improvement (figure ), so no brain biopsy was performed. he was sent back to our facility with the recommendation to start a targeted pi kinase inhibitor on compassionate grounds as he was not eligible for the clinical trial because his weight was less than kg. but pretreatment abdominal ct revealed multiple low-density lesions scattered throughout the liver (figure ) not previously seen on prior noncontrast ct four months prior. discussion: although this gain in function mutation of the pi kδ signaling pathway disorder has been well characterized, this is a rare report of a patient with pasli immunodeficiency with central nervous system and later liver lesions pet imaging showed subcarinal, mediastinal, retroperitoneal lymphadenopathy, splenic enlargement to cm and bilateral lung nodules ( figure ). excisional biopsies of left axillary and left lower lobe of lung were performed and showed low-grade b-cell lymphoma (mucosal) and underlying lymphoproliferative disease. invitae alps and cvid panels ( genes) revealed a heterozygous variant of unknown significance in exon of fas (c. a>g(p.asp gly) unlike most fas mutations causing alps, this mutation is in the extracellular region rather than the death domain . the c. a>g variant has been reported in a single patient with alps phenotype, affecting fas protein function by inhibiting binding to fas ligand (fas-l), reducing fas-l induced apoptosis . this fas c. a>g mutation was found in alps affected brother and was absent in the unaffected father. the patient's mother passed away prior to testing. since diagnosis, the patient's malt lymphoma has been treated with rituximab weekly for the first month and then monthly. he continues to receive monthly ivig for hypogammaglobulinemia. after two years, ct demonstrates a significant decrease in pulmonary nodules and splenomegaly ( figure ). the patient's forced vital capacity (fvc) improved from . l ( % predicted) to . l ( % predicted). conclusion: we report the first case of malt lymphoma seen in a patient with alps. it is unknown whether the unique fas c. a>g mutation in the non-death domain contributes to malt lymphoma progression. we propose malt lymphoma is a malignant transformation of chronic inflammation that has the potential to occur in patients with alps. in the future, improved knowledge of mechanistic pathways of inflammation in lymphoma development and progression is important in the optimal management of alps. abstract/case report text background wiskott-aldrich syndrome (was) is a rare x-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and increased risk of hematological malignancies. gene therapy (gt) using autologous cd + cells is an emerging alternative treatment with possible advantages over standard allogeneic hematopoietic stem cell transplant. we report the outcomes of a phase i/ii clinical trial in which was patients underwent gt using a self-inactivating lentiviral (sin-lv) vector expressing the human was cdna under the control of a . kb fragment of the human was promoter. subjects and methods: five patients with severe was (clinical score - ) were enrolled (table ) . cd + cells were transduced ex-vivo and reinfused after conditioning with busulfan and fludarabine. two subjects (p , p ) had autoimmunity pre-gt, manifested as skin vasculitis and autoimmune cytopenias. results: all subjects were alive at median follow-up of . (range . - . ) years. multi-lineage vector gene marking was sustained over time. all had clinical improvement of eczema, infections and bleeding diathesis. was protein (wasp) expression was increased over baseline but remained below normal levels. proliferation of t cells in response to anti-cd improved post-gt. humoral immune deficiency improved, with normalization of igm, and independence from ig replacement and vaccine responses in those tested. platelet levels increased to > x cells/ul in only the two subjects with a vcn ≥ in transduced stem cells. podosome formation in monocyte-derived dendritic cells was near absent pre-gt and improved in all subjects post-gt, but only reached healthy control levels in the subjects with highest vcn. in contrast to other trials using this sin-lv, two patients (p and p ) had flares of autoimmunity post-gt, offering the opportunity to study the poorly understood mechanistic features of immune dysregulation in this disease. selfreactive vh - -expressing b cells and cd lo b cells remained elevated in most patients. however, despite wasp expression in foxp + tregs, those with autoimmunity had poor numerical recovery of t cells and tregs at the time of clinical symptoms ( fig a) . in addition, il- producing regulatory b cells (bregs) were highly deficient pre-gt, recovered in subjects who did not experience autoimmunity, but failed to recover in p and p ( fig b) . moreover, transitional b cells, which are enriched in bregs and are potent inducers of treg populations, also recovered poorly in those two subjects ( fig c) . there have been neither severe gt-related adverse events nor abnormal clonal expansion in transgene-marked cells to date. conclusion in summary, our data confirm and extend the safety and efficacy of gt in correcting disease manifestations associated with was, with the longest overall follow-up reported so far in studies using sin-lv. in addition, our findings suggest that higher vcn is needed in order to correct myeloid compartments such as platelets and monocytes. finally, we report the novel finding of the restoration of bregs and suggest that recovery of this compartment, along with tregs, is protective against development of autoimmunity post-gt. overall, these data suggest a mechanism for breakdown of immune tolerance in was with important therapeutic implications and prognostic value. this is an -year-old hispanic female who initially presented with failure to thrive, recurrent fevers and intermittent cough with episodes of perioral cyanosis. symptoms started at age months and were attributed to recurrent viral and bacterial infections. at months old, she was hospitalized with fever and hypoxemia (o saturations %). cxr showed prominent interstitial lung markings and she was diagnosed with pneumonia. ct scan confirmed cxr findings and ruled out anatomical anomaly. she was lost to follow up for years, and re-presented with worsening respiratory status. a repeat ct scan demonstrated worsening interstitial thickening. immune workup, including quantitative immunoglobulins, ch , lymphocyte subsets and vaccine response titers (pneumococcal and tetanus), was unremarkable, except for elevated igg levels. genetic testing for surfactant dysfunction mutations was negative. thoracoscopic lung biopsy revealed interstitial fibrosis, pas-positive granular alveolar proteinosis, type ii cell hyperplasia, and lymphoid follicles. at age , she was admitted for a pericardial effusion. rheumatology was consulted for evaluation frequent fevers and persistently elevated inflammatory markers, with concern that the pericarditis was autoinflammatory. she had an elevated ana (> : homogeneous pattern), il- ( . pg/ml), and igg ( mg/dl) at that time. she had an atypical anca pattern with positive myeloperoxidase antibodies. anti dsdna, smith and scl were negative. she was treated with steroids and hydroxychloroquine with some improvement in her oxygen requirement. one year later she had an additional episode of pericarditis, treated with colchicine. a few months later, she was admitted with newonset gross hematuria and elevated serum creatinine (to mg/ dl). kidney biopsy showed anca vasculitis with glomerulonephritis ( % crescents, no scarring or fibrosis). she provisionally received a diagnosis of microscopic polyangiitis, with lung and kidney involvement. she did not have peripheral vasculopathy. she was started on cyclophosphamide, rituximab, and iv steroid pulses. cyclophosphamide was discontinued due to recurrent episodes of posterior reversible encephalopathy syndrome (pres) after infusion. her igg level decreased as she developed nephrotic range proteinuria. a primary immunodeficiency genetic panel was sent to evaluate for monogenic immune dysregulation syndromes and revealed a tmem gene mutation (c. g>a) which has previously been reported in other subjects with savi (stingassociated vasculopathy of infancy syndrome). sting is a cytosolic dna sensor that leads to type i interferon production upon stimulation. this gain-of-function mutation was confirmed by measuring interferon signature gene expression at the nih (fig ) , and her diagnosis was revised accordingly. the patient was started on a jak-inhibitor (tofacitinib) to block interferon signaling. unfortunately, the patient is now deceased, due to overwhelming infection and multi-organ system failure. conclusion: genetic testing can be crucial in aiding the diagnosis of complex patients with immune dysregulation and can provide an opportunity for targeted therapy, which should be employed as soon as able to stop disease progression. abstract/case report text background: activated phosphoinositide -kinase δ syndrome (apds- ) was first described in as a monogenetic immune dysregulation syndrome with a variable phenotype. increased sinopulmonary and herpesvirus infections are well described, but fungal infections such as candidiasis have been rare. to date, disseminated histoplasmosis has not been described. history: a yo caucasian male who was previously diagnosed with common variable immunodeficiency (cvid) in late childhood due to recurrent sinopulmonary infections presented with recurrent fever, pancytopenia, severe splenomegaly, and lymphadenopathy. urine histoplasmosis antigen and beta-d-glucan were elevated. a bone marrow biopsy demonstrated granulomatous inflammation. transbronchial biopsy of a subcarinal lymph node was consistent with granulomatous disease. this led to a diagnosis of disseminated histoplasmosis. he was treated with amphotericin b and then months of itraconazole, with improvement of his symptoms. he was admitted to the hospital about years later when he presented with fatigue, fever, chills, dark urine, and scleral icterus. he was found to have an acute worsening of chronic anemia with a hemoglobin of . g/dl. due to elevated ldh, presence of schistocytes on peripheral smear, and undetectable haptoglobin, he was diagnosed with autoimmune hemolytic anemia, despite a negative direct coombs. a bone marrow biopsy specimen was hypercellular with marked erythroid predominance, with normal flow cytometry and no blasts identified. infectious workup was negative. ct chest during the workup revealed new right hilar and mediastinal lymphadenopathy, in addition to calcified right hilar and subcarinal lymph nodes, bronchiectasis, and stable hepatosplenomegaly. transbronchial biopsy of lymph nodes showed benign lymph nodes with calcified necrotizing granulomata and presence of non-viable fungal species, presumably "old" histoplasmosis. family history: family history was significant for mom dying at years-old from undefined cns infection. immune labs: · panlymphocytopenia: absolute lymphocyte count of /ul, cd + t cells /ul, cd + /ul, cd + /ul, cd + /ul, cd +cd + /ul. cd +/cd + ratio . · decreased class-switched memory b cells and plasmablasts · elevated t central memory cells and activated (hla-dr+) cd + and cd + t cells · hemoglobin . g/dl, platelets , /ul, anc ranging from /ul to /ul · iga mg/dl, igm mg/dl; reportedly had low igg prior to initiating ivig in childhood · ebv pcr and cmv pcr negative genetics: · pik cd (c. g>a), consistent with diagnosis of autosomal dominant apds- . discussion: gain-of-function variants leading to increased pi kδ activity have been shown to cause both b and t cell dysfunction, leading to impaired immunologic responses to bacterial and viral infections. recurrent sinopulmonary infections and herpesvirus infections are commonly seen and while mucocutaneous candidiasis has been reported in cohorts of patients with pik cd, other fungal infections are not common. severe disseminated histoplasmosis infections have been described in primary immunodeficiencies characterized by signaling defects in the il- /ifn-γ pathway, stat deficiency, cd l deficiency, gata deficiency and in stat gain-of-function mutations. to our knowledge, disseminated histoplasmosis has not been previously reported in patients with pik cd immunodeficiency. abstract/case report text the reported case represents the first case of nbas disease detected by newborn screening program for primary immunodeficiency, based on krec assay. the patient came to our attention due to the complete absence of krecs and normal trecs on dbs (dried blood spot) while hospitalized for low weight at birth ( , g), intolerance for enteral feeding, hepatosplenomegaly, slightly elevated liver transaminase, head and face eczematous dermatitis. during the st month, he also presented klebsiella pneumoniae urinary tract infection and methicillin-resistant staphylococcus aureus sepsis. peculiar phenotypic features including triangular face, proptosis, flat philtrum, mild retrognatia, hirsutism, loose and slightly wrinkled skin, and apparent reduction of subcutaneous fat were noticed at birth. complete blood count showed lymphocytopenia, marked hypereosinophilia. serum immunoglobulin g (igg) were markedly decreased, iga and igm were undetectable. extended immune-phenotyping showed complete absence of cd + cells, low count of cd + lymphocytes, and reduced natural killer (nk) levels. at month of age a colonoscopy was carried out for persistent diarrhea and reduced tolerance to enteral feeding. the histological examination of mucosal intestinal biopsies showed signs compatible with autoimmune enteropathy. for this reason immunosuppressive therapy with rapamycin was started without consistent clinical amelioration. many cvc-sepsis occurred in the last months, associated with persistent gastrointestinal symptoms and severe growth restriction. despite the absence of experience data in literature for nbas syndrome, we retain that hsct represents the only resolutive therapy for him. abstract/case report text case: a -year-old female with asthma and allergies presented to immunology clinic with a history of chronic fatigue and sinusitis. fatigue occurred daily every - weeks and was described as not feeling rested even after hours of sleep. chronic sinusitis required - prolonged antibiotic courses per year. nasal cultures grew methicillin-sensitive and -resistant staphylococcus aureus and haemophilus influenzae type b (hib). three separate sinus surgeries over the prior few years reduced her sinus symptoms. other infectious history was significant for recurrent urinary tract infections with e. coli and klebsiella, recurrent otitis media as a child, and a diagnosis of transient hypogammaglobulinemia of infancy that resolved at years of age. review of systems revealed axillary lymphadenopathy for - days twice per year not related to infection. she had longstanding eczema that responded to topical tacrolimus, multiple environmental allergies, and recently diagnosed asthma that improved with inhaled budesonide/formoterol. as a teenager she received allergy immunotherapy for a few years but stopped due to frequent adverse reactions. family history revealed that father died from cancer. physical exam was unremarkable. laboratory evaluation demonstrated normal igg mg/dl, igm mg/dl, iga mg/dl, elevated ige mg/dl, normal t and nk cell enumeration, mildly low total b cells ( cells/mcl, . %), and normal b cell subsets (cd +igm+cd - %, cd +igm+cd + %, cd +igm-cd + %). tetanus antibody titer was protective, but hib antibody titer was undetectable at < . mcg/ml with marginal response after vaccination ( . mcg/ml). pneumococcal serotype specific igg levels (mayo) were mostly undetectable with of serotypes protective at baseline and only protective post vaccination with pneumovax . cd / cd blastogenesis was poor. due to poor antibody response and continued sinus infections she was started on igg replacement. her fatigue and sinus symptoms improved moderately but she continued to require antibiotics and sinus ct scans continued to demonstrate significant disease. a focused exome sequencing panel was pursued and a novel heterozygous card variant was found (c. g>t, p.r l). discussion: the card /bcl /malt (cbm) complex is a critical signaling adapter that facilitates several downstream immune responses predominately through nf-kb. mutations in several different domains of card result in a clinical entity collectively referred to as card -associated atopy with dominant interference of nf-kb signaling (cadins). cadins is associated with a broad range of clinical manifestations but most have marked atopy with infections, poor t cell proliferation, and varying levels of poor antibody response. both our variant (p.r l) and a previously reported pathogenic variant in the same amino acid (p.r g) involve a change from a charged arginine to a non-polar amino acid in the critical bcl / card binding interface. iκbα degradation was not present in b cells from our patient ( figure ) confirming the functional defect in nf-kb signaling. thus, we present a novel variant that fits cadins both clinically and genetically. clinicians should be aware of cadins when patients present with recurrent infections in the setting of significant allergic disease. i b degradation assay. stimulation: μl of whole blood was stimulated in a ml facs tube with ng/ml phorbol -myristate -acetate (pma; sigma, cat# p ) at °c for , , or min, at which point ml of pre-warmed x lyse/fix buffer (bd, cat# ) was added. cells were fixed for min at °c, centrifuged and washed twice with facs buffer (pbs supplemented with % fbs and mm edta). staining and permeabilization: fc receptors were blocked for min at rt (human trustain fcx; biolegend), followed by a min stain on ice with anti-cd af (clone rpa-t ; biolegend), and anti-cd bv (clone hib ; biolegend). cells were washed with facs buffer and permeabilized for min on ice with ml phosflow perm buffer ii (bd biosciences, cat# ) that had been precooled to - °c. after permeabilization, two ml facs buffer was added and the samples were centrifuged. after three additional washes, the cells were stained with anti-iкbα pe (clone /ikba/mad- ; bd biosciences) for min at rt. samples were washed three times and data were collected on a cytek dxp flow cytometer. data were analyzed with flowjo software. abstract/case report text introduction: wiskott-aldrich syndrome (was) is a rare, but well-defined x-linked disorder. loss-of-function mutations in the was gene result in classic was and x-linked thrombocytopenia (xlt), while gainof-function mutations lead to x-linked neutropenia (xln). classic was phenotypic features include recurrent infections, microthrombocytopenia and eczema along with increased susceptibility to autoimmune disorders and malignancy. most males with classic was are diagnosed in early childhood and early death can result from its various clinical manifestations. case: we present a -year-old male who was referred to immunology for hypogammaglobulinemia. as an infant he had moderate eczema, and at the age of two was diagnosed with immune thrombocytopenia (itp) with baseline platelets of - x ^ /l. infectious history was notable for one episode of pneumosepsis and recurrent otitis media, influenza, and herpes labialis infections. around the age of , he was diagnosed with common variable immunodeficiency (cvid) based on the finding of low immunoglobulins. he developed diffuse large b cell lymphoma at age , and was treated with cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab (chop-r). at age he developed abdominal pain with bloody stools. investigations confirmed an endoscopic and pathologic diagnosis of ulcerative colitis. due to the severity of his disease, he has required maintenance therapy with vedolizumab. he was again noted to have hypogammaglobulinemia at which point he was referred to immunology at our centre. there was no significant family history of immunodeficiency, malignancy or autoimmunity. blood work was notable for normal white blood cell and lymphocyte counts, platelets of x ^ g/l, low igg at . g/l ( . - . g/l) with normal iga, igm and ige. lymphocyte subsets including t, b and nk cells were within the normal range. genetic testing was performed and he was found to have a known pathogenic mutation in the was gene (c. g>a, p.asp asn) which has been previously reported in association with was and xlt. he has since been placed on immunoglobulin replacement and has been referred for consideration of hematopoietic stem cell transplantation. discussion: was is a rare syndrome that can have a similar phenotype to other immunodeficiency disorders including cvid, omenn syndrome and ipex (immune dysregulation, polyendocrinopathy, x-linked). individuals with cvid present with hypogammaglobulinemia and recurrent infections, and these individuals also have an increased susceptibility to autoimmune disorders, gastrointestinal disease and malignancies, especially lymphoma. although eczema is a common disorder, its presence in addition to features of early onset thrombocytopenia, immunodeficiency, autoimmunity and/or malignancy in male patients should heighten the suspicion for was. it is important to make the diagnosis of was as hematopoietic cell transplantation and gene therapy are potentially curative treatment options. abstract/case report text secondary immune deficiencies (sid) are caused by varied mechanisms and are common in patients with hematological malignancies such as chronic lymphocytic leukemia (cll) and multiple myeloma (mm). in this setting, both the disease and its treatment (such as b cell ablation therapy) contribute to the development of secondary antibody deficiency. infections remain a major cause of morbidity and mortality in cll and mm patients. this underscores the need for early recognition and stratification of risks in order to guide appropriate treatment, including immunoglobulin replacement therapy (igrt). new guidelines for the use of human normal immune globulins in sid patients were implemented by the european medicines agency (ema) in . despite these new guidelines, significant variations remain across european countries in the assessment and approaches aiming to achieve reduction in infection burden, including different strategies for initiation, dosing and discontinuation of igrt. the same is true for north america where igrt is widely used off-label to prevent infections in patients with sid due to hematological disease or other reasons. in order to address this variability, a task force comprising both immunologists and hemato-oncologists drafted statements aiming to test for consensus. statements were related to six major areas: definition of infections, measuring igg levels, initiating igrt, igrt dosing, scig usage and discontinuing igrt. this was followed by an international delphi consensus exercise in three rounds which aimed to develop recommendations on how to diagnose, treat and follow-up patients with antibody deficiency associated with hematological malignancies. the first delphi round consisted in testing the statements with a panel of sid specialists and subsequently their comments were used by the task force to refine the statements. in the second delphi round, the refined statements were presented via phone interviews to the same panel to assess their level of agreement with each statement (ranging from "i totally disagree" to "i totally agree"). consensus was considered to be reached per statement if % of the experts agreed with each statement overall. the cut-off for overall agreement was "i somewhat agree". if the expert chose level or less the reasons underpinning his/her choice were discussed. consensus was achieved for all statements on level ("i somewhat agree"). only statements did not achieve consensus on level "i mostly agree". in delphi round , panelists who had not "mostly agreed" with these five statements were given the opportunity to reconsider their assessment based on the feedback from other panelists, which was shared with them. the panelists then chose to maintain or refine their assessment. analysis of the full results on the six key areas identified by the task force will be presented at the conference to offer recommendations and help guide the management of sid in patients with hematological malignancies. abstract/case report text introduction: mast cells (mcs) are hematopoietic-derived immune cells, whose precursors migrate within tissues reaching maturation and differentiation. masitinib, a selective tyrosine kinase inhibitor, is efficient in controlling the survival, differentiation, and degranulation of mcs. aim: to optimize mast cell-differentiation from human bone marrow (bm) hematopoietic stem cells, and to find best cell culture conditions for proliferation, differentiation, and maintenance of mcs, which is important when studying particularly mcs' response to cytotoxic compounds. material-methods: to produce mcs in vitro, the first method (m ) we used was a modified semi-solid culture method ( ). briefly; human bm mononuclear cells (mncs) were obtained with ficoll gradient from bm sample of a patient with idiopathic thrombocytopenic purpura. colonyforming unit (cfu)-mast was developed from mncs in methylcellulose medium supplemented with scf ( ng/ml) + il- ( ng/ml), and il- ( ng/ml; only first week). - weeks later mast cell colonies were transferred into suspension cultures, in which mcs matured and multiplied up to - weeks and were used in experiments till th week of culture. on the other hand, in our second method (m ); mncs were separated by ficoll, seeded in well-plates with imdm containing fbs %, pen/ strep, and a little amount of methylcellulose, and incubated at o c, %co . cultures were then supplemented with imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) on day ; and imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) + il- ( ng/ml) on day . beginning on day till the end, imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) were added to cultures. for both methods, morphological assessment of colonies/cells were evaluated under an inverted microscope (figure and ). verification of mcs was performed by immunoflorescence staining for anti-tryptase andchymase antibodies, and by toluidine blue staining. macrophages were verified by anti-cd- immunoflorescence staining. mcs were exposed to masitinib or dmso for the evaluation of dose-related effects of masitinib, and cytotoxicity was evaluated by mtt assay. results: in m , culture conditions were easier to handle compared to m . in m , high amounts of mcs in immature and pre-mature forms were appeared as early as - days, and peak levels of proliferation rate was around - weeks of culture, which was about weeks earlier than m . culture could be maintained till weeks in both methods. although mcs are non-adherent cells, in liquid method adherent bm cells such as fibroblasts, endothelial cells and mesenchymal stem cells have adhered to the plate and grown up, providing an attachment site for mcs and serving as a natural bm nest, mimicking in-vivo environment, for mcs to grow and proliferate ( figure ). attachment of mcs has provided medium exchange available without changing culture dishes. when mcs were exposed to masitinib ( . , , and μm/μl), approximate survival rates were %, %, %, respectively. discussion: in our liquid medium method, the adherent bm cells not only provided a natural nest supporting mc development and differentiation, they also served as an attachment site for mcs. as the cells slightly adhered, when trypsinized shortly, they easily detached and used for experiments. and we also report for the first time that adding a little amount of methylcellulose to the liquid medium provides ease of aggregation of cfus, and easy development of mcs. we suggest that our liquid culture may be superior to semi-solid method, that it is faster and easier to handle. in studies subjects crossed over to subcutaneous (sc) igiv-c %, and in the third study crossover was to immune globulin sc (human), % caprylate/chromatography purified (igsc %). a total of pi patients from these studies were included in the poppk analysis and serum igg concentrations were included in the final pk analysis. the pk of igg following iv and sc administration was adequately described by a two-compartment model with first-order elimination from the central compartment. administration of igiv was modeled as an infusion directly into the central compartment. absorption of exogenous igg from the depot site of sc infusions into the central compartment was modeled as a first-order process with an absorption rate constant (ka). the full model was constructed by incorporation (forward selection process) of covariates of interest into the model. after completion of the covariate model development, the final model showed that igg pk was not influenced by (a) the igsc formulation used in the different studies ( % vs. %), (b) gender, and (c) age (pediatric vs. adult). body weight was identified as a significant covariate having an effect on clearance and volume of distribution. based on the final pk results, serum clearance of igg for the reference population was estimated to be . l/day. the volume of distribution of the central and peripheral compartments accounted for . l and . l, respectively. the intercompartmental clearance was . l/day, and the absorption constant from the depot (ka) was . day- . the absolute bioavailability of igg after sc administration was calculated as . %. the developed method was used to evaluate alternative dosing intervals following sc administration. the equivalent of a weekly igsc maintenance dose administered , , , , or times per w e e k , o r b i w e e k l y p r o d u c e d o v e r l a p p i n g s t e a d y -s t a t e concentration-time profiles and similar area under the concentration versus time curve (auc), maximum concentration (cmax), and minimum concentration (cmin) values. the results of the evaluation and simulations for igg exposure following a switch from igiv-c % dosing (every -or -weeks) to sc dosing further suggest that a range of dose-adjustment factors (daf), from : to : . would be sufficient to provide clinically effective trough igg concentrations throughout the course of treatment at various treatment frequencies. current us product labeling for igsc % specifies a daf of : . for transitioning immune globulin dosing from iv to sc, and specifies igsc % dosing frequencies of weekly or more frequently ( - times per week). in this poppk analysis all sc dosing regimens evaluated theoretically would provide viable alternative administration options for maintaining adequate immunoprotection in pi patients with dosing flexibility over a range of regimens. however, in % or more, no causative gene can be found going down to undefined inflammatory syndromes (uis). anti-il drugs (ail d) revolutionized some il- mediated diseases, such as traps, caps, hyper-igd/mkd and fmf. nevertheless, treatment response among disorders are not the same as well as no specific study was designed for uis. papa et al, , recently suggested the use of anakinra for the treatment of uis, especially those refractory/intolerant to colchicine with severe or very symptomatic phenotype. this paper aims to retrospectively report for the first time the experience with canakinumab in monogenic and multifactorial disorders in a single, private center in brazil. patient and methods: patients's records that received canakinumab from january to december at clinica croce, ima-brazil, were revised. demographic and clinical data were extracted and descriptively described. all statistical analysis are presented as: average (minimal; maximum; standard deviation). results: a total of patients with autoinflammatory diseases were enrolled and % (n= ) are female. of them, % (n= ) patients had a monogenic disease: % (n= ) caps, % (n= fmf), % (n= ) mkd, % (n= ) homozygous nlcr and % (n= ) pami syndrome. multifactorial disorders were % (n= ) patients : % (n= ) recurrent idiopatic pericarditis, % (n= ) schnitizler syndrome and % (n= ) uis. the average age of the first symptoms was , years ( ; ; , ) and the average age of diagnosis was , years ( ; ; , ) while the aveage of diagnosis delay was , years ( ; ; , ). all patients had used, prior to anti-il , corticosteroids with % prevalence of cushing syndrome and % (n= ) tried at least one steroid sparing agent without clinical success due to: intolerance or non-effective disease control or side effects. in the fmf group (n= ) % tried colchicine prior to canakinumab and this drug was not effective to % because of amyloidosis status and in % colchicine-induced hepatitis was observed. canakinumab was effective for disease control in % (n= ) considering: control of clinical manifestations, amyloidosis reversion and normalization of acute reactants markers. the only side effect observed during the follow up were acute flu-like symptoms and psicomotor agitation ( , % , n = ). the average time of follow up is of , months ( ; ; , ). canakinumab could be discontinued in just one patient with uis. conclusions: this is the first report of canakinumab use for autoinflammatory disorders in brazil. canakinumab is an effective and safe drug for monogenic and multifactorial disorders control. no serious adverse effect could be observed in the years maximum follow up of this drug. neither, no specific infectious disease more prevalent in south america, such as yellow fever, dengue, zika or chikungunya was observed. abstract/case report text a -year-old caucasian male with autosomal recessive hyper igm syndrome type (higm ) due to aicda mutation, diagnosed at age , presented with a newly developed mediastinal mass. he receives routine ivig, pulmonary function tests (pft's) and chest x-rays. at age , patient was noted to have cervical and inguinal lymphadenopathy. ct scan indicated left mediastinal, hilar and pleural lymphadenopathy with soft tissue infiltration around the descending thoracic aorta and esophagus. biopsy indicated no evidence of a lymphoma or infection. years after initial workup, routine pft's showed a declining diffusion capacity by %. patient complained of intermittent chest pain but displayed no clinical symptoms of cough, dyspnea, dysphagia or reflux. ct scan which revealed an extensive illdefined soft tissue mass extending from the thoracic outlet to the level of the esophageal hiatus that encased vascular structures resulting in narrowing and occlusion of left upper lobe pulmonary artery and left lower lobe pulmonary arteries respectively. imaging demonstrated homogenous ventilation to bilateral lungs and decreased perfusion in the left lung compared to the right lung. infectious workup was negative for atypical infections. biopsy revealed miced cellular infiltrate with no predominenant cell type or evidence of malignancy, consistent with previous lymph node biopsy years prior. cd and cd stains revealed aggregates and scattered b-cells and t-cells, respectively. removal of mass was proposed but due to the ambiguous borders and location, surgical excision was not possible. patient was given doses of rituximab ( g), doses mg/kg pulse steroids hours apart, and daily sirolimus (level was adjusted based on sirolimus level). follow-up ct scan indicated significant interval improvement with - % reduction of the soft tissue mass. blood flow in the left lower lobe pulmonary artery has still not returned. this may be due to collaterals and may be a separate problem from compression due to the mass. cytotoxic t-lymphocyte antigen (ctla- ) is an inhibitory immune regulator critical for governing t and b cell homeostasis. heterozygous ctla mutations can cause a syndrome of immune dysregulation with a variable clinical phenotype including hypogammaglobulinemia , autoimmune cytopenia and endocrinopathies, lymphoproliferation, predisposition to malignancy, tissue specific lymphocytic infiltration of brain, lung and gi tract as well as colitis. methods: we retrospectively reviewed medical records of all patients with ctla haploinsufficiency evaluated at the nih between - . a pathologic variant in ctla was confirmed in all patients. we analyzed frequency of campylobacter species detected in the stool samples by pcr based biofilm rapid array as well as reflex bacterial stool and blood cultures when available. results: forty-six patients aged - years were evaluated at the nih between and . six of patients ( %) had at least one episode of campylobacter species associated acute or worsening diarrhea, with one patient also having campylobacter bacteremia. all patients with positive campylobacter species in stool samples had clinical histories and/or endoscopic biopsy findings consistent with enteropathy or colitis predating the incidence of campylobacter infection. two of the six patients ( %) had recurrent or chronic campylobacter infection, while four of the six patients ( %) had multiple gastrointestinal pathogens detected by stool pathogen screening at various times. conclusions: campylobacter species infection of the gastrointestinal tract seem to occur at an increased incidence in our ctla haploinsufficient cohort. to the best of our knowledge, this is the initial report for the association between ctla haploinsufficiency and campylobacter species infection of the gastrointestinal tract. although ctla- is a critical immune checkpoint involved in mucosal immune homeostasis and gut microbiota-immune system cross talk, the underlying mechanism predisposing to campylobacter infection in ctla- deficient patients remains to be explored. our study suggests screening of stool for campylobacter species in patients with ctla haploinsufficiency associated enteropathy. ( ) submission id# abstract/case report text ikaros transcription factor and ikaros family members are critical for development of lymphocytes and other blood cell lineages. full length ikaros (isoform ) contains six c h zinc fingers (zf), four nterminal dna binding zf and two c-terminal dimerization zf. somatic ikaros mutations and deletions have been associated with increased predisposition to b-acute lymphoblastic leukemia (all) as well with poor disease prognosis. recently, germline ikaros mutations affecting the n-terminal dna binding domain and acting in a haploinsufficiency or dominant negative manner were reported to be associated with common variable immunodeficiency (cvid) and combined immunodeficiency (cid), respectively. herein we describe a novel set of germline heterozygous ikaros allelic variants affecting the c-terminal dimerization domains in four unrelated families. clinical manifestations include hematopoietic cytopenias presenting as evans syndrome, and hematologic malignancies including t-cell all and burkitt lymphoma; other manifestations observed were b-cell lymphopenia and hypogammaglobinemia, but recurrent or severe infections were not prevalent or characteristic. we demonstrate that mutants affecting dimerization abolish ikaros homodimerization as well as heterodimerization with ikaros family members aiolos and helios. these variants also affect dna binding at dimerization sites and pericentromeric targeting. opposed to previous allelic variants reported, dimerization changes alter post-translational sumoylation and gene transcription regulation. our data show that mutations affecting ikaros dimerization are mainly associated with cytopenias and/or malignancies, have a different mechanism of action than previously reported variants, present with incomplete clinical penetrance, and contribute to the growing spectrum of genotype-phenotype ikaros associated diseases. introduction: there has been much discussion regarding the return of secondary findings in genetic sequencing research. opinions differ on whether researchers should return secondary findings to participants at all and if so, what the best method is to do so. we have opted to systematically identify and return pertinent secondary findings to participants in our cohort of patients with immune-mediated diseases that undergo exome sequencing. additionally, exome sequencing may determine multiple or other genetic diagnoses in addition to the primary diagnosis, which we call "incidental findings." here, we discuss the secondary and incidental findings discovered in our cohort thus far. methods: individuals in our protocol underwent consent for exome sequencing, including a discussion of the possibility of secondary findings. exome sequencing data was analyzed, and variant pathogenicity was scored using the acmg criteria (richards et al); variants determined to be likely pathogenic, pathogenic, or otherwise clinically important were confirmed via clia-certified sanger sequencing. confirmed variants were returned to participants. we then queried internal databases for cases involving secondary and incidental findings. results: as of november , exome sequencing, interpretation and reporting had been completed for participants. we detected a total of secondary findings in ( . %) participants, including variants in apob, brca ( ), brca ( ), dsp, fbn , kcnh , ldlr, mybpc ( ), ryr , pkp ( ), and vhl. additionally, we detected possible dual/multiple genetic diagnoses in ( . %) participants, some of which explained an unusual clinical presentation or symptom. these included individuals with variants in multiple immune-related genes, including one individual with variants in gata and tnfrsf a, and those with variants in genes related to multiple organ systems, including an individual with variants in ifngr and sco . discussion: exome sequencing in this cohort detects not only important secondary findings, but also discovers a significant portion of individuals with multiple genetic diagnoses. notably, exome sequencing may provide further context or explanation for unusual phenotypic presentation and help determine specific symptom etiology even when a primary genetic etiology is already known. additionally, these secondary and incidental finds may be important to consider when delineating risks and symptoms of novel or recently-discovered conditions. abstract/case report text background: immune dysregulation and lymphoproliferative disorders including alps like disease, hlh ebv driven lymphoproliferative disease leading to rare lymphomas require a multidisciplinary approach utilizing expertise in immunology and hematology/oncology to care for these patients as we learn the molecular etiology of their underlying disorders. at texas children's hospital, the immunology lymphoproliferative evaluation and diagnostic (ilead) clinic was created to provide a comprehensive clinical and research approach to caring for patients with these rare disorders. in an effort to streamline care and access, we recently on-boarded an advanced practice provider (app) . methods: a chart review was conducted months before and after onboarding the app for ilead patient visits. we reviewed the following patient care and access parameters to determine increase in efficient and effective patient care as well as improved access to the clinic. these parameters included: referral process, time of referral placement to appointment, number of patient visits, wait time in clinic, lab interpretation and reporting time for disseminating results to families, and collaboration process with other specialties. results: within months of the app starting our average wait from placing the referral to first appointment fell by an average of %. in addition, we created an algorithm to prioritize patients with immediate need to be seen. by streamlining the referral process and patient priority, we developed a "pre-clinic" conference process by which all patients are reviewed and preliminary plans are made prior to the patient's arrival. this has translated into our ability to increase the number of patients seen in clinic from to and decreased the wait time in clinic by approximately minutes. since the app started, no patient has been in clinic for more than minutes. this has also led to an increase in rvu generation. in terms of efficiency in patient care, all labs are now ordered while in the room with the patient by the app and physician providers. in turn, all labs are resulted directly to the app who reviews labs, collaborates with physicians for care and reports to families in a timely fashion within - weeks of labs being resulted compared to greater than month previously. to improve collaborator communication and post visit plans, a post-visit clinic summary was created. this has been effective in reducing the time to other specialty referrals, follow up visits and effective care for ongoing clinical needs. conclusions: the addition of an app in our ilead multidisciplinary clinic which provides specialized care for patients with immune dysregulation and lymphoproliferative disorders effectively increases work productivity of providers and enhances patient care by increasing access to care, decreasing wait time in clinic and time of reporting of results and future plans. the app with knowledge and expertise in immunology and immune dysregulation is a cost effective way to enhance provider and patient support. with the overwhelmingly positive results, future plans include expanding our multidisciplinary clinic to other services that care for patients with suspected immune deficiency. abstract/case report text introduction: pediatric lymphoproliferative disorders represent a clinically and genetically heterogeneous group of conditions. misdiagnosis and delayed diagnosis can contribute to substantial morbidity and mortality. identification of molecular etiologies and underlying disease mechanisms may facilitate timely interventions and guide targeted or curative therapies. methods: the study was performed through retrospective chart reviews in accordance with all local ethics and irb committees. the study was designed to investigate a cohort of pediatric patients who met criteria for non-malignant lymphoproliferative disorders from texas children's hospital and collaborating centers for underlying genetic etiologies. results: a total of affected individuals from families met criteria. distribution between male and females was nearly equivalent: males (n = ) and females (n = ). approximately half of the cohort was hispanic (n = ). overall kaplan meier survival was % (n = ). whole exome sequencing was performed in all subjects and available family members. likely disease-causing genetic defects were identified in of families ( %). within these families, ( %) carried variants in genes in international union of immunological societies established primary immunodeficiency diseases. potential novel genetic causes of immune deficiency or immune dysregulation were also discovered. mechanistically, all of the implicated genes had roles in modulating lymphocyte activity; initial activation, cytoskeletal organization, or apoptosis of lymphocytes; or regulation of inflammation. all subjects less than one year of age had an identified gene in one of the three mechanistic categories with the dominant mechanistic genetic category being defective control of lymphocyte signaling ( %). in addition, % of patients between and years of age were found to have a potential genetic diagnosis underlying the lpd, with a more equal distribution of mechanistic categories compared to patients greater than years of age where only % have a genetic cause. other important disease manifestations identified were ebv-associated disease in subjects ( %) and subjects ( %) met hlh- criteria. conclusion: primary immunodeficiency diseases and other genetic abnormalities of the immune system underlie a significant percentage of pediatric lymphoproliferative disorder cases. greater than % of patients less than years of age have a genetic etiology underlying the lymphoproliferative disorder. many of these gene defects can be treated with targeted therapies or hematopoietic stem cell transplantation. genetic testing therefore plays an essential role in the diagnosis and management of children with these conditions. abstract/case report text a -year-old gentleman with a history of immune dysregulation polyendocrinopathy enteropathy x-linked (ipex) syndrome with known pathogenic variant in foxp presented to our emergency department with two witnessed episodes of tonic-clonic seizures earlier that day. he has had a longstanding history of recurrent infections and autoimmune conditions since birth, and was being treated with monthly ivig infusions and sirolimus while awaiting bone marrow transplantation. his symptoms on admission included foaming at the mouth, generalized shaking, bladder incontinence, and tongue biting that lasted about five minutes. family reported recent sores inside his mouth and lips, but denied any recent fevers, neck pain, headaches, chest pain, abdominal pain, nausea, vomiting, and sick contacts. he lives on a farm with livestock and reportedly had recent tick exposure. his last ivig infusion was two weeks prior to admission, at which time he also received inactivated flu vaccine. in the ed, a third seizure was witnessed by multiple medical providers. he subsequently received lorazepam and lacosamide with interval improvement. he underwent diagnostic lumbar puncture, as well as extensive evaluation for infections. he was started on empiric antibacterial and antiviral meningitis coverage. analysis of the csf showed a lymphocytic pleocytosis; bacterial cultures and hsv / pcr were negative, as was a -pathogen meningitis/ encephalitis panel performed by pcr. eeg was negative for seizure-like activity, and brain mri showed mild atrophy without sclerosis in the left hippocampus. subsequently, anti-infectious therapy was stopped, and patient was discharged with outpatient followup scheduled for suspected non-infectious aseptic meningitis that was potentially triggered by flu vaccination versus ivig. on day four post-discharge, however, pcr for ehrlichia chaffeensis in the serum returned positive, and he was started on oral doxycycline. ehrlichiosis is a rare tick-borne illness that may cause various nonspecific symptoms including fever, headaches, myalgias, and generalized malaise. most prevalent in the mid-atlantic regions of the united states, tick-borne ehrlichia spreads through the mononuclear phagocytic system and can infiltrate many organs including the kidney, liver, lungs, and heart. csf penetration can cause sometimes fatal meningoencephalitis. aseptic meningitis due to ehrlichiosis has been described in recent literature. cases in hiv patients and transplant patients on chronic immunosuppressive therapy have been severe, resulting in organ dysfunction in many instances and death in a few. however, this case marks the first documented ehrlichia infection in a patient with primary immunodeficiency. this patient's presentation of aseptic meningitis and clear exposure history fits the clinical picture. his relatively benign course could be due to preserved t effector function not seen in persons with hiv or transplant patients with significant immunosuppression. patients with ipex usually present with autoimmunity and allergies, but are also prone to significant infections. it is important to perform a comprehensive workup, including testing for atypical infections, in patients with immune dysregulation syndromes who present with symptoms of unclear etiology. special attention should be paid to patients who live in areas with known endemic exposure risks. empiric antibiotic therapy may need to be considered early to prevent delays in treatment. abstract/case report text introduction autosomal recessive hypomorphic mutations in pgm have been described to result most commonly in either hyper-ige or severe combined immunodeficiency (scid) clinical phenotypes in humans, with one report of an individual with combined immunodeficiency without atopy. herein, we describe a series of individuals newly diagnosed with pgm deficiency functionally confirmed using lectin-based flow cytometric analysis of peripheral blood mononuclear cells, that broadens the associated clinical phenotypes to confirm cid without atopy and childhood evans syndrome. in addition, we present new disease-causing pgm variants, and functionally confirm the pathogenicity of a fourth (p.i t). classical hies phenotype cases . and . identify sisters of spanish descent with a classical hyper-ige phenotype. the younger sibling demonstrated severe atopic dermatitis, mild-moderate asthma, multiple food allergies, one episode of itp, and adhd. the older sibling demonstrated atopic dermatitis, skin infections, and c. albicans otomastoiditis. the siblings were found to have the damaging compound heterozygous variants p.t i and p.q x in pgm . case is a year-old guatemalan boy with prominent atopy including asthma, allergic rhinitis, food allergy, elevated ige, atopic dermatitis, as well as oral hsv who was found to be homozygous for the damaging pgm variant p.i t. cid phenotype with a paucity of atopy case is a -year-old turkish girl who is the daughter of a consanguineous union. she presented with infantile nephrotic syndrome at months of age, and subsequently developed leukopenia, neutropenia, and low igg. complications include bronchiectasis, sinusitis, pseudomonas urinary tract infection, and inflammatory skin lesions without atopy. she was found to be homozygous for the damaging pgm variant p.r h evans syndrome case is a -year-old girl from guatemala. she developed multilineage autoimmune cytopenias including immune thrombocytopenic purpura (itp), autoimmune hemolytic anemia (aiha) and autoimmune neutropenia (ain) at the age of years, refractory to multiple treatments and finally responding to mycophenylate mofetil. she has a history of mild eczema but is without other atopy and suffered from multiple invasive bacterial infections. an additional patient, case , was diagnosed with coombs positive aiha and itp at age years refractory to multiple treatments and finally responsive to cyclosporine. cytopenias recurred year later, resulting in hypoxic brain injury. he died of infectious complications at the age of years. both patients were found to be homozygous for the damaging pgm variant p.i t. discussion this is the first report of pgm deficient individuals presenting with evans syndrome as a primary presentation without additional pathology. while disease-associated mutations appear to cluster around the key conserved domains of the protein, no clear genotype-phenotype correlation is readily observed. in addition to autoimmune cytopenias, pgm deficient individuals have also been reported with splenomegaly, lymphoma, and ebv viremia. thus, in particular for children with lymphoproliferative disease, pgm deficiency should also be considered in the differential diagnosis. abstract/case report text introduction: meningitis is a life-threatening manifestation of cryptococcus neoformans (c. neoformans). it occurs in increased frequency in those with impaired cell-mediated immunity, especially those with hiv/aids. infection with c. neoformans has been seen in previously healthy individuals diagnosed with idiopathic cd lymphopenia (icl). icl is defined by an absolute cd + count of less than cells/m on multiple occasions, usually to months apart, without other apparent cause such as hiv infection, immunodeficiency, or immunosuppressive medications. case description: our patient is a previously healthy -yearold female with cryptococcus meningitis and fungemia. her course was complicated by elevated intracranial pressure requiring extraventricular drain. she was treated with amphotericin and flucytosine for month. notably, the patient was also found to have moraxella catarrhalis (m. catarrhalis) bacteremia without identifiable source. she denied history of environmental risk factors, was not up to date on cancer screening, and recently returned from a trip to italy. initial evaluation revealed lymphopenia ( cells/ul), low cd + ( cells/ul), cd + cells ( cells/ul), and cd / + ( cells/ul), but normal cd + ( cells/ul) and cd + cells ( cells/ul). hiv, ana, leukemia/ lymphoma flow cytometry panel was negative. she also had a normal lymphocyte proliferative responses to pha ( . %), normal cd ra:ro, and protective tetanus titers ( . iu/ml), but only / protective pneumococcal serotypes. initial immunoglobulins demonstrated slightly low igg ( mg/dl). laboratory studies months after presentation demonstrated improved lymphopenia ( cells/ul) continued low cd + cells ( cells/ul), but normalized igg levels ( mg/dl). followup labs also demonstrated decreased cd + b cells ( cells/ ul) and insufficient response to polysaccharide vaccine ( / pneumococcal serotypes). three months after discharge, she is continued on daily fluconazole without recurrence of infections although she still has diplopia and headache. discussion: in a review of cryptococcosis in patients with icl, of them had cryptococcal infection in both the cns and blood. of these patients, was cured, improved, relapsed and then improved, and died. three of these patients were treated with amphotericin and flucytosine. five of these patients had underlying disease and had notable infections with vzv, tb, or hpv, however other infections such as m. catarrhalis were not mentioned. m. catarrhalis bacteremia has been described in children with underlying immune dysfunction and respiratory infection as well as secondary to pneumonia with m. catarrhalis. in cases of m. catarrhalis bacteremia in adults, most had underlying malignancy and/or neutropenia, predisposing respiratory factors, or source for infection. conclusion: this report of c. neoformans meningitis and m. catarrhalis bacteremia in the setting of icl is unusual in that to our knowledge, m. catarrhalis bacteremia has not been reported in icl. cases like this also raise the question as to whether some laboratory abnormalities are secondary to infection, treatment, or underlying disease. it is important to report these cases with icl in order to group disease phenotypes, as continued monitoring and data collection of these cases may lead to discovery of new disease processes. abstract/case report text cytokines play critical roles in regulating the development, survival, differentiation and effector function of immune cells. cytokines exert their function by binding specific receptors on the surface of immune cells and typically activating intracellular jak/stat signaling pathways, resulting in induction of specific transcription factors and regulated expression of target genes. in order to differentiate into an appropriate effector fate, lymphocytes need to integrate multiple signals often provided concomitantly by numerous cytokines that activate shared transcription factors. how these signals are balanced and regulated to yield the optimal class of immune response remains to be completely determined. inborn errors of immunity, or primary immunodeficiencies (pids), result from germline mutations in defined genes, leading to loss-of expression, loss-of function, or gain-of function of the encoded protein. pids are characterised by defects in immune cell development, or their differentiation into effector cells during immune responses, thereby rendering patients not only highly susceptible to infectious diseases, but also autoimmunity, autoinflammation, allergy and cancer. pids are thus an unprecedented model to link defined monogenic defects to immune dysregulation in clinical settings. indeed, pids have unequivocally revealed non-redundant roles of single genes, molecules, signaling pathways and lymphocyte subsets in host defense and immune regulation, and formed the basis of better therapies for immunopathologies. our indepth analysis of inborn errors of immunity of cytokine signalling pathways have identified fundamental requirements for generating long-lived humoral immune responses in humans. here, i will present data relating to our recent studies of how inactivating mutations in il r, il r, znf , stat , stat , and stat , disrupt or dysregulate the generation and function of human memory b cells and tfh cells, thereby precipitating humoral immunity, as well as allergic disease and autoimmunity. abstract/case report text introduction: flow cytometry is a powerful diagnostic tool for detecting hematologic malignancies in a variety of patient specimens including body fluids and lymph node aspirates. cytopathologists are frequently confronted with lymphocyterich effusions, and the definite decision of whether the lymphocytosis is of a purely reactive nature or a presentation of an indolent lymphoma may be an extremely difficult based on microscopy alone. moreover, small proportions of malignant cells that may be missed out by routine morphology can be detected by flow cytometry. objective: the purpose of this study was to evaluate the usefulness of multiparametric flow cytometry immunophenotyping (fci) to confirm the presence of leukemia or lymphoma cells in body fluids and fna specimens. methods: body fluids and fna specimens simultaneously obtained for fci, cytologic analysis and real time pcr from patients were submitted to our flow cytometry laboratory from january to september . the samples studied were body fluids ( pleural fluids and ascitic fluids) and fna samples ( enlarged lymph nodes and lung mass).four color fci method was performed and the following fluorescent monoclonal antibodies were used: cd , cd , cd , cd , cd , cd , cd b, fmc , kappa and lambda light chains, cd , cd , cd , cd c, cd , cd a, cd , cd , cd , cd , cd , tdt, cd , cd , cd , cd , cd , cd , bcl , cd . fci analysis was performed on a beckman coulter cytomics fc flow cytometer using software cxp to analyze data. the cases were diagnosed as leukemia or lymphoma as per tuberculosis ( case). ascitic fluid (n= ) samples showed positivity for angioimmunoblastic t-cell lymphoma ( cases) and dlbcl ( case). fna of lymph nodes (n= ) were positive fort-lymphoblastic lymphoma ( cases), angioimmunoblastic tcell lymphoma ( cases), dlbcl ( cases), hodgkin lymphoma ( case), nodular lymphocyte predominant hodgkin lymphoma ( case), peripheral t-cell lymphoma (nos) ( case), splenic b-cell marginal zone lymphoma( case), tuberculosis ( cases). one fna of lung mass were tumor of neural cell origin. both immunophenotype and cytomorphology positive for malignancy were in / ( . %) cases.cytomorphology was negative/ suspicious in / ( . %) cases, of which both cytomorphology a n d i m m u n o p h e n o t y p e n e g a t i v e w e r e ( % ) a n d cytomorphology negative but immunophenotype positive cases were ( . %). mtb dna was detected in pleural fluid in case and fna sample in cases. conclusion: multiparametric flow cytometry by using comprehensive panel of monoclonal antibodies is a useful diagnostic test to evaluate body fluids or fna as it can demonstrate small malignant populations that may be missed out by routine cytomorphology. clinical laboratory geneticist/department of genetics, university of groningen, groningen, the netherlands abstract/case report text the phenotypes of primary antibody deficient (pad) patients range from milder (e.g. specific antibody deficiency) to severe (e.g. x-linked agammaglobulinemia) deficiency of the immune system. pad patients form a clinically, immunologically as well as genetically heterogeneous group. often, the genetic background has not been elucidated; it probably is not monogenetic in a large subgroup of patients. pad patients suffer most frequently from recurrent bacterial infections of the respiratory or gastrointestinal tract due to immune deficiency, but may also have varying degrees of autoimmune and lymphoproliferative comorbidities due to immune dysregulation. unfortunately, a standardized description of pad phenotypes is lacking rendering robust definitions of pad-subtype diagnoses, including cvid, difficult. this impairs the formation of homogeneous cohorts that can form the starting point for future clinical and genetic research. the pad subgroup of the human phenotype ontology (hpo) immune mediated disorders consortium supported by ern rita and esid is addressing the gaps in standardized phenotypic description of pads. using the hpo dataset, literature mining, and esid, iuis and omim classifications, we aimed to reevaluate and complete the pad-related hpo terms to allow efficient data exchange and matching of phenotypically similar pad patients. as a principle, it was decided to avoid the ongoing variance in pad-subtype definitions and to build the pad-related hpo tree based as much as possible on unambiguously interpretable items. 'hypogammaglobulinemia' was deleted as hpo term, and replaced by separate hpo terms such as 'decreased total igg in blood', subdivided in 'transient' vs. 'chronic', and '(near) absent' vs. 'partially decreased' (the same for igg , igg , igg , igg , iga and igm). 'decreased specific antibody level in blood' was specified further into 'decreased natural antibody level to blood group antigens in blood', subdivided in '(near) complete' vs. 'partial' absence (the same for protein, polysaccharide and protein-conjugated polysaccharide vaccination). relevant hpo terms related to infection and to specific organ manifestations like bronchiectasis, autoimmunity and lymphoproliferation were re-evaluated and completed, and will be linked to pad diseases in the hpo online system by the pad subgroup experts. once finalized, existing pad cohorts will be classified according to the new hpo pad-related terms, and studied by clustering technologies (example of two patients shown in figure ; white = absent, color = present). acceptance and widespread use of this pad-related hpo tree for standardized phenotyping will be essential to empower future multicenter clinical research and related genetic discoveries as well as support clinicians in diagnosing pad through the linkage of hpo terms to pad disease entities. senior investigator oral immunity & infection section/nih/nidcr abstract/case report text leukocyte adhesion deficiency type (lad ) is an autosomal recessive disorder characterized by the inability of granulocytes to emigrate from the bloodstream to sites of inflammation. lad is caused by mutations in the itgb gene ( q . ), encoding the beta- -integrin, cd , which is essential for firm adhesion of leukocytes to the endothelium. in lad survival is compromised, morbidity from inflammatory lesions is high, and treatment is poor. the moderate form of lad is often managed with antibiotics for prophylaxis and during acute infections. after infancy severe gingivitis and chronic periodontitis are universal. periodontal findings affect primary and permanent teeth, causing intense oral mucosal (gingival) inflammation and destruction of tooth supporting bone, which are hallmarks of the disease periodontitis. blocking the il- /il cytokines, which are up regulated in lad gingiva, can reduce bacterial load and resolve inflammatory gingivitis. ustekinumab binds to the shared p subunit of human il- and il- , cytokines that modulate lymphocyte function, including t helper (th) cells and th subsets, thereby blocking them. objective: explore the effect of ustekinumab on lad inflammatory disease. method: prospective study using ustekinumab for oral inflammation. patients receive five doses over year, -or mg depending of weight. results: (two patients have enrolled, p is > year post treatment, p is still on study) patient characteristics · patient : age at diagnosis, yrs.(itgb mutation c. delt (null)); cd (%pmn control): . %; cd a(%pmn); . %. at the initiation of the protocol ( yrs old) wcc: . k/ul; crp: . · patient : age at diagnosis, yrs.(itgb mutation c. a>g,p.g s c. c>t,p.a v) cd (%pmn control): %; cd a(%pmn): . %. at the initiation of the protocol ( yrs old) wcc: . k/ul; crp: . response: patient : oral ulcers before treatment: episodes every two months. during ustekinumab therapy: · oral ulcers: episode in a year · reduction in bleeding on probing: . % · gingival index reduction: % patient : oral ulcers before treatment : monthly. during ustekinumab therapy: · oral ulcers: none in first months · reduction in bleeding on probing : . % · gingival index reduction: . % safety: no significant adverse events were documented during the therapy p had a previous skin lesion that flared leading to iv antibiotics. p had a previous sebaceous cyst drain spontaneously. discussion: two patients showed improvement in chronic periodontitis and a substantial decrease in oral ulcers while on ustekinumab. no clear safety signals were seen. durability of these findings is still unknown. ustekinumab in lad deficiency appears to be safe and potentially effective. post doctoral fellow/servicio de inmunología, inst. multidisciplinario de investigación en patologías pediátricas (imipp), hospital de niños ricardo gutiérrez. chief resident/servicio de inmunología-hospital de niños "dr. r.gutierrez" immunologist/centro de inmunología clínica "dra. liliana bezrodnik y equipo"-servicio de inmunología htal. de niños "dr. r.gutierrez" immunologist/centro de inmunología clínica "dra. liliana bezrodnik y equipo" immunologist/servicio de inmunología-hospital de niños "dr. r.gutierrez" abstract/case report text introduction: primary immunodeficiencies with dysregulation associate defects in the immune homeostasis leading to inappropriate immune response (lack or excess) that causes autoimmunity, allergy and/or inflammation. impairment of different subsets of t and b compartments may be associated with these pids. aim: ) describe t and b memory compartment of pid patients (pts) with dysregulation: cd deficiency, stat gof, stat b deficiency, ctla variant, pi kcd variant and cvid-like (with no molecular defect) and compare them with a group of healthy donors (hd). ) associate ctfh profile with b cell compartment impairment. results: ) pts showed a significant decrease of naïve cd + t cells (cd ra+cd +) ( , % vs , %) (p < . ) with expanded central memory t cells (cd ra-cd +) ( . % vs . %) (p < . ); cd + t cells had higher levels of activation markers (cd +hla-dr+) ( , %vs , %) (p < , ). pts showed a significant increase of circulating follicular t cells (ctfh) (cd ra-cxcr +) compared with hd (mean , % vs , %) (p < , ) with pd- overexpression (p < . ). stat gof, ctla , pi kcd and cvid-like pts showed a skew towards ctfh (cxcr +). regulatory t cells (cd +cd ++ foxp +) were absent in cd and stat b deficiency and decreased in the other pts. within cd + cells, although effector memory (cd ra-cd -) (p < . ), temra (cd ra+ cd -) (p < . ) and hla-dr+ cd + (p < . ) subsets showed a significant increase compared with hd, the behaviour was variable between different mutations. regarding b cell compartment, pts with stat gof, pi kcd and cvid-like showed a severe impairment of switched-memory b cells (sw-mbl) (cd +igd-igm-); the stat b deficient patient had increased frequencies of this subset, while ctla pts had a variable b defect. ) lower sw-mbl values were significantly associated with lower values of ctfh cells (p < . ) (r= . ). cd low b cells were exclusively high in cvid-like pts, and transitional b cells were increase in pi kcd and almost all cvid-like pts. discussion: in summary, patients with dysregulatory syndromes associate a defect of t and b homeostasis (survival, activation and differentiation). specific mutations can differentially affect the quantity and/or the quality of ctfh. there is a strict association between the differentiations of tfh with th profile with the generation of sw-mbl. these alterations may play a role in the pathophysiology of primary immunodeficiencies with b lymphocyte functional impairment. immune monitoring of lymphocyte subsets of patient with dysregulation may approach to the diagnosis of specific monogenic mutations. objective: the purpose of this study is to increase awareness and improve diagnosis of primary immune deficiency (pid) in the heterogenous group of patients with autoimmune cytopenia (aic) by identifying clinical characteristics and laboratory biomarkers that distinguish those with underlying pid, disease activity and guide mechanism-based targeted therapy. methods: patients with aic (autoimmune hemolytic anemia (aiha), immune thrombocytopenia (itp), or autoimmune neutropenia (ain)) were referred to our immune dysregulation team and prospectively enrolled during - . detailed immune phenotyping (igg, iga, igm, lymphocyte subsets, vaccine titers, lymphocyte proliferation to mitogens/ antigens), serum lipopolysaccharide (selps) and autoantibodies were measured and/or collected by chart review and genetic testing for pid was pursued. results: from to , patients were enrolled; two subjects were removed due to parental request or lack of aic diagnosis. of the remaining patients, ( %) were classified as "aic-pid" based on genetic testing and/or immune phenotyping; ( %) were classified as aic-only, and ( %) were asymptomatic family controls. the patients were predominantly children (ages - years, average age . years); % ( / ) were male. among patients who have had genetic testing to date (n= )( %), pathogenic genetic mutations were confirmed in / ( %) of patients. mutations include fas/fasl (n= , including family members without aic), ctla (n= ), q (n= ), and one patient each with nfkb , was, pole- , pi k, casp , card , and cgd; the remainder of aic-pid patients were classified as combined immune deficiency or common variable immune deficiency based on immune phenotyping. lymphocyte subsets (cd +t, cd +t, cd +b, cd + nk) and immune globulins (igg, iga, igm) tended to be lower in aic-pid patients vs aic-only (p < . ). evans syndrome was more common in aic-pid patients ( / , %) compared to aic-only ( / , %). lps was elevated in the serum of aic patients compared to healthy controls (mean vs pg/ml, p < . ). excluding partial digeorge syndrome patients (average lps pg/ml), selps levels were significantly higher in genetically-defined untreated pid patients (average pg/ml) vs. other pid (average pg/ml)(p= . ) or patients with aic alone (average pg/ml)(p= . ). studies are ongoing on specific subsets that are linked to immune dysregulation (switched memory b cells, t-regulatory cells, double negative t cells, t follicular helper cells) and the use of soluble il- as a biomarker of disease activity. conclusions: a high fraction of aic patient were identified with underlying pid in our study. basic immune evaluation with immunoglobulin levels and lymphocyte subsets expedited diagnosis of pid. genetic evaluation distinguished a group of patients with aic-pid and highly elevated lps level, reflecting high bacterial load, which may distinguish them from the rest the aic cohort. the source of bacterial lps can be multifactorial and is yet to be determined. our studies continue focusing on biomarkers that can be applied to the heterogenous group of patients with aic. this will allow early detection and timely initiation of targeted therapies. investigator/dermatology branch, niams, nih head, dermatology consult service/dermatology branch, niams, nih chief, fungal pathogenesis section/laboratory of clinical immunology and microbiology, nih abstract/case report text introduction/background: autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) is a monogenic autoimmune disease resulting from biallelic mutations in the aire gene. although typically characterized by the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal insufficiency, we recently reported that the clinical spectrum of the syndrome is far broader and that incorporation of an adjunct triad of apeced rash, intestinal dysfunction, and enamel hypoplasia in the classic triad could lead to earlier diagnosis (ferre et al., jci insight, ). among the adjunct triad manifestations, apeced rash occurs in % of american apeced patients by age , most often developing in the first year of life. objectives: to report and describe the clinical features of apeced rash as the first manifestation in a -month old patient with apeced. methods: following enrollment in a niaid irb-approved protocol ( -i- ) the patient was evaluated with history and physical examination, aire sequencing, measurement of interferon-autoantibodies, and skin biopsy with immunohistochemical analyses. results: a -month-old girl with a family history of genetically confirmed apeced in her -year old sister developed discrete circular, maculopapular erythematous lesions on her torso that spread to the face, arms, and legs while sparing the palms and soles. the rash was partially blanching, non-painful and non-pruritic and was preceded by low-grade fever ( □c) without other accompanying symptoms. she had not received medications or vaccinations prior to the rash onset. the lesions increased in size with associated central clearing and resolved . months after onset. the rash recurred with similar appearance times over months with each recurrence lasting between days and . months. as with the first rash episode, recurrences were often preceded by fever ( - □c) without accompanying symptoms or inciting factors. neither topical nor oral antihistamines improved the rash. aire sequencing identified the same compound heterozygous mutations (c. _ del and c. c>t) that the sister has. high titers of interferon-□ autoantibodies were measured in serum. skin biopsy revealed superficial perivascular chronic inflammation and intraepidermal lymphocytes composed predominantly of mixed cd and cd t lymphocytes with few perivascular b lymphocytes. no eosinophils or vasculitis was observed. myeloperoxidase immunostaining revealed extensive karyorrhexis. laboratory studies revealed normal white count and esr, negative anti-ige receptor antibody, and positive anti-ige antibody. at months, she developed oral candidiasis as second manifestation of apeced, thus reaching a diagnostic dyad when applying our proposed expanded diagnostic criteria. she has not developed hypoparathyroidism or adrenal insufficiency; thus, she has not yet reached a classic diagnostic dyad. systematic screening for these endocrinopathies will be needed to avoid lifethreatening complications of acute endocrine failure. conclusions: we report the clinical and histologic features of apeced rash manifesting as the first disease component of apeced in a month old girl. apeced should be considered in the differential diagnosis of recurrent erythematous maculopapular urticaria-like eruptions characterized by mixed lymphocytic and neutrophilic infiltration unresponsive to antihistamines. our case illustrates the clinical utility of incorporating the expanded diagnostic criteria of apeced rash, enamel hypoplasia and intestinal dysfunction into the classic diagnostic triad, which can lead to earlier apeced diagnosis. who presented with prolonged severe neutropenia despite g-csf and seven hospitalizations for febrile neutropenia in the span of ten months. prior to his neutropenia, patient was on monthly ivig, with igg trough - in the past year. he was evaluated for bmt in but declined. patient was first found to be neutropenic in aug when he was admitted with pseudomonas thigh abscess, hsv stomatitis and rhinovirus infection. he was treated with broad-spectrum antibiotics with improvement in neutropenia. the following month, he was hospitalized again with neutropenic fever, left axilla pseudomonas abscess and rhinovirus infection. he underwent bone marrow biopsy revealing left shifted myeloids with decreased maturing forms and t cell predominant lymphoid aggregates, suggestive of autoimmune neutropenia vs. hyper igm syndrome associated with neutropenia. anti-neutrophils antibodies were negative. he was then admitted the following month ( / ) with febrile neutropenia with cxr concerning for viral pneumonitis vs. atypical pneumonia. he was started on g-csf therapy with significant initial response in anc. however, this response was short-lived as he was again admitted in / with febrile neutropenia and upper respiratory rhinovirus infection. he was continued on daily g-csf. due to persistently normal anc for approximately three weeks, he was weaned off g-csf in / . in / and / , he had two more hospitalizations for febrile neutropenia. g-csf was restarted with dose uptitrated to mcg/kg during his hospitalization in april. he was found to be thrombocytopenic with splenomegaly on abdominal ultrasound. anti-platelet antibodies and repeat anti neutrophil antibodies were not detected. he was discharged with close follow up with immunology and hematology. due to his age, he was transitioned to penn allergy/immunology in / . there was close communication between chop allergy/immunology, chop hematology and penn allergy/immunology during this transition period. patient was admitted to hup in / with febrile neutropenia (despite higher dose of g-csf), rhinovirus infection, pseudomonas sinusitis and ct chest findings suggestive of possible fungal pneumonia. due to persistent neutropenia refractory to g-csf treatment, hematology was consulted and repeat bone marrow biopsy showed hypercellular bone marrow with markedly left shifted granulocytic hyperplasia, compatible with g-csf therapy. flow cytometry showed no evidence of plasma cell neoplasm. dose of ivig was adjusted and increased based on his weight. per hematology, he also received an additional high dose ivig g/kg x days for presumed immune mediated neutropenia with immediate increase in anc. despite anc of for days,anadditional g/kgofivigimprovedhisancto> within hours of his first dose. thrombocytopenia also improved to normal range. since then, patient has been on monthly - mg/kg ivig with no recurrence in neutropenia. this patient's prolonged persistent neutropenia with immediate response to high dose ivig is suggestive of autoimmune neutropenia, which should be taken into consideration in hyper igm patients with persistent neutropenia. abstract/case report text background: children with digeorge anomaly (dga) represent a heterogenous group, often classified as either partial dga (pdga) or complete dga (cdga) based upon the degree of thymic hypoplasia. this paucity of t-cell parameters and function has serious implications for infection risk, autoimmunity, and malignancy. however, there are limited studies stratifying children with dga by these subgroups, especially in regard to immune function and subsequent infection risk. study design: single-center, retrospective cohort analysis evaluating the relationship between pdga and cdga to infection risk with particular focus on infection-related hospitalization, pathogenic organism identification, and antimicrobial resistance profiles. the source population includes all pediatric patients < years of age diagnosed with either pdga or cdga while receiving care at duke university from january , to june , . the final analysis sample included patients. methods: to evaluate the differences in immune function between dga subgroups, we will report the proportion of low ( < th percentile for age) t cell immune biomarkers for both subgroups and compare populations using a chi-squared test. to compare per year incidence of infectionrelated hospitalization for dga subgroups, a poisson model with number of hospitalizations per patient as the outcome, an offset equal to the time at risk for hospitalization, and either pdga or cdga diagnosis as the exposure will be used. models will be bivariate. we will report an incidence rate ratio (irr) and % confidence interval ( % ci). to evaluate the impact of cellular and humoral immune function on infection-related hospitalization, we will use poisson models where the outcome is the number of hospitalizations per patient, an offset equal to the time at risk for hospitalization, and low immune biomarker as the exposure. all models will be bivariate. we will report an irr and % ci. infection type and resistance profiles will be completely descriptive. results: as expected, children with cdga had a significantly higher probability of a low ( < th percentile for age) values for total t cells (cd +), helper t cells (cd +cd +), cytotoxic t cells (cd +cd +), and naïve helper t-cells (cd +cd ra+cd l+) as well as a significantly lower probability of low pan memory t-cells (cd +cd ro+) compared to children with pdga. no differences were detected in the percentage of low natural killer (nk) cells (cd +cd +) or b cells (cd +) between subgroups. cdga patients had a significantly higher incidence of hospitalization per year ( . ( . , . )) compared to pdga patients ( . ( . , . )). the irr is . ( . , . ). across both subgroups, the incidence of hospitalization was higher in dga patients who had low helper and naïve t-cells. there is ongoing analysis into hospital-related infection and resistance profiles. notable frequencies include bacteremia ( > %), invasive viral disease ( > %), and opportunistic infections ( > %). conclusions: children who had cdga were % more likely to have an infection requiring hospitalization than children who had pdga, emphasizing the need for thymus transplant for cdga. further analysis of infection type and patient outcomes is critical to enhancing management of this unique patient population. abstract/case report text antibody cross-reactivity among flavivirus has been documented. in recent times zika virus has been emerging in pockets of the mosquito-infested regions, while southwestern saudi arabia is known for arthropod-borne viral diseases and we do not know the incidence or even presence of zika virus in this region. it is restricted to predict the igm and igg antibody detection ranges owing to limited data and colossal cross-reactivity among the zika and other flaviviruses. we tested sera from pregnant women irrespective of their clinical presentation for zika and dengue igm, igg respectively. the zika positive samples were further confirmed by plaque reduction neutralization tests (prnt). from our results, . % ( ) cases were positive for zika igm against . % ( ) positivity to igg. when these samples were assessed for dengue igm and igg, we observed . % ( ) seropositivity for igm and igg respectively. there was no single sample positive for both igm and igg of zika or dengue. however, we observed one sample positive for both zika and dengue igm. upon mapping the overlapping serotiters, there was no significant correlation observed between the dengue igm and igg. whereas zika igg positive sample showed high serotiter for dengue igg indicating the contribution of cross-reactivity for observed zika positivity. screening for the incidence of zika, therefore, becomes particularly hard in a population that has the presence of pre-exposure of dengue and this cross-reactivity makes it hard to determine the zika incubation and antibody prevalence confounded with other flaviviruses. abstract/case report text introduction humoral pid diagnostic protocol includes the analysis of the immune response to different protein and polysaccharide antigens (ags) ( ) . although the analysis of the immune response against the polysaccharides vaccine from pneumococca has been the standard method, the use of s. typhim vi vaccine has appeared as a good alternative ( ) . in this report we show the results obtained with the use of s.typhim vi in adults patients attending the pid outpatient clinic. material and methods patients with humoral-suspected pids were challenged with typhoid polysaccharide vaccine (typhim vi®; sanofi-pasteur). serum was obtained on basal and after weeks of vaccination. specific igg levels against s.typhim were measured using "vacczyme tm human anti-salmonella typhi vi igg enzyme immunoassay kit" (binding-site). results a total of adult patients attended the pid clinics during nov -nov . from those, patients were fully evaluated using a humoral-suspected pid algorithm that includes the s.typhi vaccination. in total male and female patients completed the protocol and were analyzed. twenty patients were considered as responders (ratio pre/ post > x) whereas patients were non-responders. discussion the main advantage of assessing polysaccharide immune response using s.tyhpim is the usual lack of specific igg at the moment of the initial evaluation. in this serie, just one patient has a high basal level (# -vaccinated in the past). thirteen patients had basal levels below the detection limit of the test ( , u/ml) and patients between , and , u/ml, that has been described as a cut otf level for nonimmunised individuals (personal experience, , ). regarding the polysaccharide immune response as a tool to distinguish pid vs non-pid patients, the results showed a good correlation between those non-responders with more clinical relevant pid diagnostics. seven non-responders patients were subsequently diagnosed with a primary (* on table i ) and/or secondary id (** on table i ). despite this, there were patients that we could have classified as strong responders (ratio > x, absolute specific igg postvaccination level > u/ml) and patients considered as weak responders (ratio post/pre > x, absolute specific igg postvaccination level < u/ml). strong responders were considered non-pid after including other clinical investigations and laboratory tests (cell subpopulation study, pcp response) whereas weak responders group consisted in some "minor" forms of pid, like isolated igm or ig subclasses deficits. more patients are needed to confirm this functional classification of pid patients regarding their s.typhi immune response. abstract/case report text activated pi kδ syndrome (apds) is a primary immunodeficiency characterized by recurrent respiratory infections, as well as increased risk of chronic viremia with herpes family viruses, benign lymphadenopathy and b cell lymphoma. it is caused by heterogeneous germline gain-of-function mutations which ultimately lead to the hyperactivation of the phosphoinositide- -kinase δ (pik δ). pik δ exists as a heterodimer composed of a catalytic and a regulatory subunit. it interacts with b cell receptors, t cell receptors, costimulatory and cytokine receptors, and is a key player in a signaling pathway involved in cell growth, proliferation and survival. apds is caused by mutations in the pik cd gene, affecting its protein product p δ (catalytic subunit). apds is caused by mutations in the pik r affecting p a (regulatory subunit). short syndrome is a rare multisystem disorder characterized by short stature, hypertextensible joints, ocular depression, reiger anomaly and tooth eruption delay. the primary causes of short syndrome are heterozygous loss-of-function mutations in the pik r gene. the combination of apds and short syndrome is very rare, with only few cases described in the literature. in this report we present a teenager with a pathogenic variant in the pik r gene, and phenotypic characteristics of both apds and short syndrome. our patient is a -year-old female with a history of growth delay and short stature, delay tooth eruption, recurrent sinopulmonary infections and hypogammaglobulinemia. evaluation performed at a prior institution for recurrent infections revealed low igg levels. she did not initiate therapy at that time and was lost to follow up for several years. at the time of our initial evaluation she reported continued recurrent episodes of upper respiratory infections and sinus infections requiring antibiotic treatment that often did not clear the infections. her physical exam was relevant for short stature ( %ile, z=- . ), low weight for age ( < %ile, z=- . ) and hyperextensibility. her facial features were significant for prominent forehead and triangular face. given concern for immune deficiency, a complete immune evaluation was obtained. her workup revealed low igg levels, with igm and iga within normal limits. she did not have protective titers to s. pneumoniae, h. influenza or diphtheria and tetanus. after administration of vaccine boosters, she was able to generate a response to all vaccines except for tetanus. she had remarkably low absolute b cells ( cells/ul) and percentage ( %), and low cd :cd ratio ( . ). she was started on amoxicillin prophylaxis and monthly ivig replacement therapy. invitae immunodeficiency panel genetic testing was sent and revealed a pathogenic loss of function variant in an intronic splice site in the gene pik r (c. + g>c). after initiating treatment with ivig, her sinus infections significantly improved and she has not had any further episodes. igg levels have remained within normal limits with monthly ivig therapy. this pathogenic variant had been previously associated with apds ; however, it had not been associated with short syndrome. the mechanisms that link both conditions is yet to be identified. this case report emphasizes the importance of screening for comorbidities associated with short syndrome in apds patients, and vice versa. finding the genetic diagnosis for patients with suspicion of primary immunodeficiency (pid) is becoming increasingly important in the management of primary immunodeficiency and estimating the risk for family members. we constantly increase the diagnostic yield for pids by improving the sequencing technology, updating the panels with new genes discovered related to pid, and finding diagnoses from difficult to sequence regions and regions with high homology. here we report our experiences with nearly patients suspected with pid. moreover, we provide a case example, how we increase the diagnostic yield by developing unique techniques for specific genes which cannot be reliably analyzed by ngs alone. diagnostic yield including all immunology related panels was . % ( / ). the majority of the tested individuals were males ( / , . %) and the most common age of testing was between to years ( / , . %). the highest diagnostic yield . % ( / ) is in children from ages to years, whereas in patients over years of age the diagnosis was found for only . % ( / ) of the patients. in two patient cases, our cnv detection algorithm indicated a homozygous deletion in the index patient samples potentially covering the whole ncf gene. additional bioinformatic analysis targeting specifically two coding positions that differ between the ncf gene and the two pseudogenes showed that all reads in those positions originated from the pseudogenes. homozygous deletion in the ncf gene was further confirmed by sanger sequencing two regions in ncf with primers that specifically bind to either ncf or the pseudogenes. while clean ncf sequences from both regions were obtained for a control sample, no ncf -specific amplification product was obtained for the index patient samples. pseudogenes were amplified and sequenced successfully in both index patient samples and positive control samples. loss-of-function of ncf is a well-established mechanism leading to cgd and by overcoming the difficulties regarding ncf deletion detection by ngs, we can improve diagnostic rate in individuals affected with cgd. gata deficiency can lead to a broad spectrum of clinical and hematological phenotypes; in some cases, nk cell deficiency is the primary manifestation, resulting in a greatly increased susceptibility to viral infections and malignancy. gata -deficient patients, particularly those who suffer from severe viral infections, have reduced frequencies of peripheral blood nk cells and loss of function in the existing nk cells. specific loss of the less mature (cd ^bright) nk cell subset is a hallmark of the immune phenotype in gata deficiency, suggesting that generation or survival of nk cell precursors is impaired. given the remarkable spectrum of clinical phenotypes in gata -deficient patients and the poorly understood biology underlying their nk cell defect, we sought to characterize circulating nk cells on a single-cell level. we performed single-cell (sc)rnaseq of lineage-depleted innate lymphocytes from a patient with gata deficiency. as expected from flow cytometric phenotyping of peripheral blood cells from this and other patients, scrnaseq revealed decreased representation of canonical cd ^bright cells. within the cd ^dim population, we identified two nk cell populations that were seemingly unique to the gata deficient patient relative to a healthy donor. pathway analysis defined the first of these populations (population ) by the expression of genes associated with cellular response to stress, extracellular stimulus and inflammation, as well as programmed cell death and regulation of proliferation and apoptosis. the second population (population ) was defined by genes associated with nk cell chemotaxis, cytokine responses and interferon signaling. to extend our findings, we performed scrnaseq of additional healthy donors and analyzed an additional gata -deficient individual who was clinically asymptomatic (yang et al. ). of note, we detected population in seemingly healthy cmvnegative individuals, suggesting it was not uniquely a result of gata deficiency but associated with an inflammatory response and not related to adaptive nk cells generated in response to cmv infection. population , on the other hand, only appeared in our symptomatic gata -deficient patient. we additionally performed bulk gene expression analyses from an unrelated gata -deficient patient that confirmed the altered expression of genes associated with both novel cell populations. current efforts are focused on better defining the functional response of nk cells in these patients and confirming the identification of our novel populations by mass cytometry (cytof). together, our data define the heterogeneity and complexity of nk cells in gata deficient and healthy individuals. perforations have been reported with tocilizumab, a monoclonal antibody of the interleukin (il- ) receptor, suggesting that il- signaling plays a role in intestinal wall integrity. as il- signals through stat , we sought to investigate the potential association between lof stat and intestinal perforations, as well as the incidence and outcome in our patient cohort. methods: we performed a retrospective chart review of patients with lof stat (n= ) followed at our institution, looking for those with non-malignancy associated spontaneous gastrointestinal perforations. the demographic information, stat mutation, comorbidities at the time of perforation, clinical presentation, management, and clinical outcomes were compiled results: ten lof stat patients were identified as having documented intestinal perforations, an approximate rate of %. one perforation was the initial presentation of diffuse large b cell lymphoma (dlbcl) of the duodenum and liver, and was excluded from the rest of the analysis. the other nine perforations occurred between to years old (mean: ), and % were female. stat mutations were localized to the dna binding domain (n= ) and the sh domain (n= ). two of the perforations occurred while inpatient for lung infection. another occurred while recovering from pneumonia at home. two perforations were associated with the initial diagnosis of diverticulitis (at age and ). one perforation occurred in the terminal ileum, one in the cecum, one in the transverse colon, and six in the sigmoid. five patients underwent primary closure of the bowel. four patients required a temporary ostomy, with subsequent successful ostomy reversal. only one patient has since died of pulmonary hemorrhage, the other patients are alive with a mean of years post perforation follow-up, with no recurrence of perforation. one patient with prior sigmoid resection required ileal resection post perforation due to as massive intestinal bleed. conclusion: spontaneous gastrointestinal perforations occurred in our lof stat cohort at a rate of approximately %. one case was associated with malignant infiltration of the gastrointestinal tract and two cases were associated with diverticulitis both known risk factors for perforation. although the pathogenesis of the perforations in lof stat remains unclear, the connective tissue phenotype likely contributes as well as the association with diminished il- signaling, as has been demonstrated with the perforations and tocilizumab. abstract/case report text background granulomatous and lymphocytic interstitial lung disease (glild) is a life-threatening complication that occurs in patients with common variable immunodeficiency (cvid) and monogenic cvid-like disorders, but the optimal treatment is unknown. objective to determine if the use rituximab and azathioprine (rtx-aza) or rituximab and mycophenolate mofetil (rtx-mmf) would improve the radiographic abnormalities as determined by high-resolution computed tomography (hrct) of the chest and/or pulmonary function tests (pfts) in patients with cvid and glild. methods this is a retrospective study of patients seen from july to december with cvid and glild who completed immunosuppressive therapy (rtx ( mg/m ) for weeks, repeated at -month intervals for or total courses, and aza ( . - . mg/kg/day) or mmf ( mg- mg-bid) for months). complete pfts and hrct scans were performed prior to therapy, at the conclusion of therapy, and periodically thereafter. hrct scans were blinded, randomized, and scored independently (in pairs) by two radiologists. all patients underwent whole exome sequencing (wes). number (percentage) and median (interquartile range) were reported for categorical and continuous variables, respectively. differences between pre-and post-treatment and between relapse and post-relapse hrct scores and pft parameters were analyzed with wilcoxon signed ranks test. kaplan-meier survival curves were also done. unadjusted one-sided p-values < . were considered statistically significant. results the glild cohort (n= ) had a : female predominance, and age at glild diagnosis was ( - ) years (table ) . autoimmunity was present in the majority of patients, with thrombocytopenia ( ( %)) the most common manifestation. enteropathy ( ( %)), inflammatory bowel disease ( ( %)), and nodular regenerative hyperplasia of the liver ( ( %)) were also present. splenomegaly ( ( %)) was present in the majority, but polyarthritis ( ( %)) was notably absent. twenty ( %) patients had been previously treated with systemic steroids. hrct scores substantially improved between pre-and posttreatment for rtx-mmf (p= . ) and rtx-aza (p < . , figure ). fev (p= . ), fvc (p= . ), and tlc (p= . ) also improved, but dlco (p= . ) was unchanged (figures and ). excluding two ( %) patients who died . and years after therapy of respiratory failure ( ( %)) and septicemia ( ( %)) respectively, / ( %) patients relapsed . ( . - . ) years following therapy with an estimated % relapse rate after years ( figure ). as of december , of patients that relapsed showed improvement in hrct scores (p= . ), and the remaining patients are still undergoing retreatment ( figure ). four ( %) pneumonias occurred during immunosuppressive therapy, all with severe restrictive lung disease . eight ( %) patients had a damaging mutation in a gene known to predispose (tnfrsf b, n= ( %)) or cause a cvid-like primary immunodeficiency (ctla : ( %); kmtd : ( %); birc : ( %)). immunosuppressive treatment improved the hrct scores regardless of the absence (p < . ) or presence of a damaging mutation (p= . ) ( figure ). conclusion combination chemotherapy appeared to be effective in improving the radiographic abnormalities and pulmonary function of patients with cvid and glild. a majority of patients had sustained remissions, regardless of the presence or absence of a monogenic disorder. iqr=interquartile range, cvid=common variable immunodeficiency, glild=granulomatous and lymphocytic interstitial lung disease, vats=video-assisted thoracoscopic surgery, tbx=transbronchial biopsy, ms=mediastinoscopy excluding b cell malignancy, pft=pulmonary function test, h/o=history of, dz=disease, nrh=nodular regenerative hyperplasia, ibd=inflammatory bowel disease table . baseline patient characteristics since its first description the number of cases has increased progressively [ ] . although described as a predominant antibody deficiency [ ] , various complex phenotypes have been associated with mutations in this gene [ ] . case description. female patient with no remarkable history until years old, when she suffers from a persistent fever associated with purulent abscesses in venipuncture areas and hyperleukocytosis with neutrophilia, so she was treated for about months in hospitals in the city of barranquilla before she was referred to our institution. the patient's clinical picture consisted of persistent fever unresponsive to broad-spectrum antibiotic treatments, skin abscesses, left subphrenic abscess and toes osteomyelitis. the microbiological studies documented a bacteremia by acinetobacter baumannii and isolation in bone marrow of candida parapsilosis. during her care stay in barranquilla, she was approached as a chronic granulomatous disease versus job's syndrome, she received two doses of immunoglobulin with partial control of symptoms. due to the recurrence o f fever, abs ces ses and hyperleukocytosis, they decided to refer to our institution for further studies. upon admission to our institution, the patient presented nutritional compromise, with spontaneous resolution of fever but persistence of high acute phase reactants, with significant improvement of leukocytosis. all the cutaneous lesions she presented were debrided at the site of remission. immunoglobulin levels, lymphocyte populations and dyhidrorhodamine test were normal. she remained with no weight gain, constipation, abdominal distension and hepatic involvement with elevated liver enzymes and prolonged coagulation times. new bone compromise was documented. inflammatory bowel disease, neoplastic or chronic infectious disease involvement was ruled out. during the stay in our institution no microbiological isolation was documented. skin, colon and bone tissue biopsies were performed and extra-institutionally performed liver biopsy were examined, showing as a single common finding leukocytoclastic vasculitis in all tissues. given the heterogeneous nature of the condition, the diagnostic possibility of an immune dysregulation disorder was considered and a therapeutic trial with nsaids and prednisolone at mg/kg/day was started, as well as genetic studies by exome sequencing. the exome results documented a novel mutation in the pik cd gene [c. t> a (p.phe tyr)] as probably pathogenic. the patient has presented a clinical improvement and a significant decrease in inflammation markers. at the moment, we are waiting for the performance of functional tests to define the definitive therapy for this patient. conclusions. this case description highlights the diagnostic difficulties that face in developing countries, where the nonavailability of functional testing has implications on the diagnosis opportunity and establishment of optimal therapeutic for patients with complex diseases such as primary immune regulatory disorders abstract/case report text background: autoinflammatory syndromes, a wide family of diseases, defined as attacks of inflammation that are unprovoked (or triggered by a minor event) and are primarily related to dysregulation of the innate immune system. periodic/recurrent fever syndromes were the former name of these diseases. however, only in two conditions: cyclic neutropenia (cn) and periodic fever, aphthous stomatitis, pharyngitis, and adenitis (pfapa) are febrile episodes truly periodic. for pfapa, although diagnostic criteria differ and there is no consensus research definition, patients are usually not difficult to recognize based on clinical course and presentation . high index of suspicion and understanding the parental experiences and descriptions of febrile episodes is imperative in facilitating early recognition and timely diagnosis. aims: to standardize and summarized the clinical presentation of pfapa, based on parental descriptions and providers observation of febrile episodes. methods: utilizing a query for the icd- diagnosis code m . ( + ) we identified a cohort of children diagnosed and managed for periodic fever, excluding those with monogenetic mutation (e.g. blau, majeed) and those with chronic illness. we reviewed the charts for documented parental report and provider observation of febrile episodes. standardized signs and symptoms were recorded for each patient [ table ]. results: a cohort of children, boys ( %) and girls ( %) with documented, cyclic episodes of fever > , was identified. the average age at diagnosis was . +/- . years. classic symptoms were reported or observed in % of patients ( ). more than half ( %, patients) had documentations of other symptoms, usually reported by parents to occur sporadically during some fever episodes. decreased oral intake and general "ill appearance" was reported by parents in % of patients. when reporting the time intervals, parents usually reported similar length for each episode, typically between - days, and regular interludes, typically between - weeks. the findings are summarized in table . discussion: the findings presented here are in concordance with previously published data describing pfapa as a syndrome affecting young, generally healthy children with identical episodes of fever lasting for a few days, recur with regularity. our data support the approach that parental observation is fundamental in identifying the unique pattern of illnesses. engaging the parents with directed interview is crucial to establish this clinical diagnosis in a timely fashion, prevent misdiagnoses of future febrile episodes as presumptive infections, and avert unnecessary antibiotics courses. this cohort adds the observation that up to % of patients who display this identifiable pattern of illnesses, do not present with aphthous stomatitis, pharyngitis, or adenitis. these classic symptoms although common, are not the rule. the cyclic chronicity of febrile episodes associated with general ill appearance (but not lethargy), and decreased po, in an otherwise healthy child is the clinical gold-standard of this condition. abstract/case report text heterozygous mutations in nfkb are frequently identified among immunodeficient patients with highly variable clinical symptoms. in a world-wide collaborative effort, we characterized the clinical and cellular phenotype and the management of of these patients harboring distinct nfkb variants. nfkb encodes the transcription factor precursor p which is processed to p (canonical pathway). known pathogenic variants cause p haploinsufficiency (due to protein decay) or p -skipping (with expression of p -like forms). most variants however are single amino acid changes with yet unknown effects. all sequence changes were assessed in silico for their probability of pathogenicity including variants which were additionally tested in vitro. these analyses include the sub-cellular protein localization (microscopy), protein expression, stability and processing (western blotting), transcription factor activity (reporter assay) and dna-binding ( figure ); the associated unadjusted odds ratio (or) was not significant, suggesting igrt had restored cases to a similar baseline infection risk as the controls. in a multivariate conditional logistic regression model adjusting for the significantly higher occurrence of risk factors in cases compared with controls in the preindex period, cases were associated with a % lower adjusted odds of major/severe infections in the post-index period versus controls (or= . ; p= . ). conclusions: patients who required treatment with igrt (privigen®/ hizentra®) had previously experienced more major/severe bacterial infections than those not needing treatment with igrt. however, igrt was associated with a reduction in the risk-adjusted odds of major/severe bacterial infections compared with non-igrt treated sid patients with haematological malignancies. abstract/case report text introduction: real-world data are lacking as far as identifying patients with secondary immunodeficiency (sid)/hypogammaglobulinemia who may benefit most from interventions to protect them from potentially fatal infections. this study aimed to identify risk factors for major/severe infections in patients with sid with underlying haematological malignancies. methods: a retrospective database analysis was conducted using the iqvia real-world data adjudicated claims -us database (study period: january -september ). inclusion criteria were adults newly diagnosed with sid (first diagnosis termed the index date), with ≥ months continuous health plan enrolment pre-index (baseline period) and a minimum of months' data post-index (mean: days), with chronic lymphocytic leukaemia, multiple myeloma and/or non-hodgkin's lymphoma and without claims for any ig therapy in the -month baseline period. patient characteristics in the -month baseline period were assessed. over the post-index period, antibiotic/antiviral use and frequency of infections were assessed. the frequency of major/severe infections was determined using diagnosis codes for bacterial, viral, fungal, parasitic, other or unspecified causal pathogen infections. major/severe infections were defined as those requiring inpatient hospitalisation with an infection diagnosis code and/or use of intravenous (iv) antibiotics or iv antivirals in an outpatient setting. a multivariate cox proportional hazards (ph) model evaluated baseline patient characteristics associated with risk of major/severe infections post-index. results: a total of , patients met the inclusion criteria. the mean age of patients was years and . % were male. in the -month baseline period: . % of patients received cancer treatments and . % of patients received antibiotics ( . % iv antibiotics). a total of . % of patients experienced any infection, . % experienced ≥ infections and . % experienced major/ severe infections. the mean number of infections over the baseline months was . for any infection (at the unique diagnosis code level) and . for major/severe infections (unique hospitalisations with any infection diagnosis code and/or unique days with an outpatient iv antibiotic or iv antiviral). in the post-sid diagnosis period, . % of patients had major/severe infections; of the major/ severe infections, . % were identified as bacterial, . % were viral, . % were fungal, while . % did not have a causal pathogen specified. a total of . % of patients experienced one severe/ major infection and . % experienced ≥ severe/major infections ( figure ). the mean annualised number of major/severe infections post-index was . . receiver operating characteristic (roc) curve analysis to optimise sensitivity versus false positives in identifying those at risk of major/severe infections post-index identified a cutoff point of three bacterial infections in the baseline pre-index period as a potential optimal trigger to consider treatment to avoid major/severe infections post-index ( figure ). the multivariate cox ph analysis suggested that hospitalisations, infections (≥ ), or antibiotic use in the -months pre-index (prior to sid diagnosis) were predictive of major/severe infections post-index (post-sid diagnosis) (all p < . ). conclusion: infections are common in patients with haematological malignancies and sid. key baseline predictors for major/severe infections in patients with an sid diagnosis were a history of infections, hospitalisations or antibiotic use. unfortunately, ms/ms detection is limited by the extremely low (e.g., pmol/l) protein concentrations in blood cells. peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-srm) is a robust method for quantification of low abundance proteins in complex matrices, including dried blood spots (dbs). in a study of patients, immuno-srm reliably identified wiskott-aldrich syndrome (was) and x-linked agammaglobulinemia (xla) patients using direct quantification of proteins responsible for disease (front. immunol., ). we further expanded our approach for x-linked chronic granulomatous disease (x-cgd), ada and dock deficiency. marker proteins representing platelets, nk cells, and t-cells have also been analyzed to provide additional information about disease processes. these results demonstrate the utilization of immuno-srm as a sensitive platform for multiplexed signature peptide quantification and its potential for pidd newborn screening and clinical diagnosis from dbs. methods: candidate peptides were selected based on ms/ms sensitivity and uniqueness in the proteome. anti-peptide monoclonal antibodies (mabs) were then generated for peptide enrichment from dbs. blood from normal controls, xla, was, xl-cgd, dock and ada deficiency patients was collected after consent on filter paper, dried, and stored at - °c. proteins were extracted from dbs, digested with trypsin, and enriched using mabs bound to magnetic beads. the enriched peptides were then eluted and analyzed with a waters xevo tq-xs. results: a multiplexed immuno-srm panel has been generated for screening eight signature peptides representing five pidd-specific and three cell-type specific proteins from dbs. limits of detection and quantification were femtomoles of peptide, the assay showed a broad linear range, and intra-assay and inter-assay coefficients of variation were < %. in samples from xla, was, xl-cgd, dock and ada deficiency patients, signature peptides are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. also included in the multiplex panel are cell specific markers for platelets (cd ), t-cells (cd ɛ), and nk cells (cd ). diagnostic cutoffs for each peptide concentration have been established. in was patients, cd levels were significantly reduced consistent with characteristic thrombocytopenia. immuno-srm also has the ability demonstrate the effects of pidd treatment. a was patient analyzed before and after bone marrow transplant showed normalized was protein and cd after treatment. two ada deficiency patients showed normal levels of ada enzyme after rbc transfusion. finally, a high-throughput (ht) immuno-srm method screens pidd-specific peptides in a . -minute runtime meeting high volume nbs workflow requirements. this ht method returned identical results to the standard immuno-srm pidd panel. conclusions: the data herein demonstrate the feasibility of using immuno-srm as a broad clinical diagnostic for identifying and studying pidd patients from easily collected and shipped dbs. significantly, ht immuno-srm workflows represent a promising potential option for nbs of pidds and other congenital disorders. chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih abstract/case report text we have previously used the artificial thymic organoid (ato) system, based on the d aggregation and culture of a delta-like canonical notch ligand -expressing stromal cell line (ms -dll ) with cd + cells, to study t cell differentiation from cd + cells obtained from patients carrying defects that are intrinsic to hematopoietic cells (rag - , ak , il rg) or that affect thymus development (digeorge syndrome). we now report results of in vitro t cell differentiation of cd + cells obtained from patients with either haploinsufficiency or dominant negative (dn) mutations of the ikzf gene. ikzf is an essential transcription factor expressed throughout hematopoiesis and involved in both lymphocyte and myeloid differentiation. heterozygous germline mutations in ikzf give rise to distinct clinical phenotypes, depending on the nature of the mutation. in particular patients with ikzf haploinsufficiency present with common variable immunodeficiency (cvid) associated with b cell immune deficiency, b-all susceptibility, and autoimmune manifestations. no clinical t cell defects are evident among these patients, except for elevated naive and central memory cd +cd + t cells. in contrast, patients carrying dn ikzf mutations present with combined immunodeficiency (cid) characterized by the presence of an increased proportion of naïve t cells, associated with defective generation of memory t cells, impaired t cell activation, signaling and proliferation, reduced t-helper (th) polarization, and susceptibility to pneumocystis pneumonia. different mouse models of ikzf mutations have been developed, however their phenotype does not fully match what reported in patients, and in some models indicates a more severe defect in t cell development. to address these controversies and to gain novel insights into the effects of distinct ikzf mutations on human t cell development, we used the ato system to analyze progression of t cell development from cd + cells obtained from one patient with ikzf haploinsufficiency and one patient with dn ikzf mutation. both patients showed a similar early block in t-cell differentiation a t p re -t c el l s ta g e. ho w ev e r, th e p a ti e nt wi t h i kz f haploinsufficiency showed a more pronounced leakiness, with a residual production of cd +tcrab+ cells, which could account for the milder t-cell phenotype presented in this type of patients. interestingly, the dn patient presented an increased accumulation of cd -cd b-cd aa+ cells. these results show an unexpected role for ikzf in humans in early stages of t-cell differentiation and indicate ikzf as a necessary factor for the induction of cd b expression in t cells. abstract/case report text background: pediatric acute liver failure without an identifiable cause (indeterminate palf/ipalf) is associated with increased rates of liver transplant and mortality. aplastic anemia (aa) may develop weeks after the diagnosis. the immunologic mechanisms that contribute to disease pathogenesis have not been clearly elucidated. we report detailed immunophenotyping of a patient with ipalf/aa. case: a previously-healthy -year-old male was admitted for acute hepatitis presenting with jaundice and hepatosplenomegaly. evaluation for infectious, toxic, metabolic, autoimmune, and rheumatologic disorders was negative. he was pan-lymphopenic (cd +alc cells/μl) with an inverted cd :cd ratio of . on admission. liver biopsy showed severe portal, interface, and lobular inflammation characterized by activated sinusoidal macrophages and perforinexpressing cd +t-cells. compared to a healthy control, the percentage and number of peripheral blood cd +t-cells expressing perforin ( %v. %, v. cells/μl) and granzyme-a/b ( %v. %, v. cells/μl) was also increased, while percentages of perforin+ ( %) and granzyme-a/b+( %) nk cells were normal. bone marrow (bm) showed % cellularity with rare hemophagocytosis. serum cytokine analysis demonstrated il- pg/ml, il- -binding-protein pg/ml, cxcl pg/ml, and sil- rα u/ml, consistent with smoldering hemophagocytic lymphohistiocytosis (hlh), but he did not meet hlh diagnostic criteria. genetic sequencing did not identify pathogenic variants in genes associated with primary immunodeficiencies. he was diagnosed with ipalf and treated with three doses of anakinra and two weeks of ruxolitinib, followed by prednisone - mg/kg/day and intravenous immunoglobulin g/kg/month. immunophenotyping performed after two months of therapy showed persistent inversion of the cd :cd ratio with small expansions of cd +cd -cd -cells and tcr··+t-cells. in the cd +t-cell subset, there was a substantial paucity of naïve cells, with effector memory t cells (tem) being more abundant than central memory t cells (tcm). in the cd +t-cell subset, the majority of cd ro+cells were tem with no detectable tcm, and % of all cd +t-cells were cd +temra. in both cd + and cd +t-cell subsets, activated (hla-dr+) and senescent (cd +) subpopulations were increased, and the majority of cells expressed the exhaustion marker pd- . the hepatic inflammatory infiltrate similarly reflected repetitive antigenic stimulation, with expansion of cd +cd +t-cells. quantitative immunoglobulins and total memory b-cells and plasmablasts (cd +cd +) were normal for age. however, there were no circulating iga-memory b-cells and a reduced number of iggswitched memory b-cells (table ) . given the severity of his phenotype and bm hypocellularity ( %), allogeneic hct was performed using a matched-related-donor ( / ) with conditioning of flu+cy+alemtuzumab. at d+ , he shows improved liver function but persistent pancytopenia, with transfusion-dependence for platelets. discussion: to our knowledge, this is the first description of detailed immunophenotyping in blood from a patient with ipalf/ aa. other studies have identified distinguishing hepatic infiltrates and cytokine/chemokine profiles that suggest excessive activation of cytotoxic t-lymphocytes and macrophages contribute to disease pathogenesis (alonso et al, ). our preliminary data supports this hypothesis and expands the spectrum of immune dysregulation in the t and b cell compartments, proposing a primary immune etiology. immune dysregulation may be concordant with hyperinflammation and cytokine storm, the latter offering potential therapeutic targets. early diagnosis and treatment of immune dysregulation may prevent development of aa. background: x-linked agammaglobulinemia (xla) is one of the first inborn errors of immunity identified, with thousands of patients described to date. infections originally dominated the clinical phenotype, but early diagnosis and immunoglobulin replacement allowed for long term survival as well as recognition of late-onset complications. nodular regenerative hyperplasia (nrh) of the liver is a silent cause of non-cirrhotic portal hypertension. nrh underlying pathophysiology remains blurry and the disease has no specific treatment. nrh has been increasingly reported in primary immunodeficiency but data in xla are very limited. objectives: to assess and characterize nrh in patients with xla. methods: we retrospectively reviewed the medical records of all xla patients referred to the nih between and . hepatology evaluation and liver biopsies were performed when clinically indicated. patients were stratified into nrh+ or nrhgroups, according to their nrh biopsy status (patients with no liver biopsies were classified as unknown). laboratory values are presented as medians. fisher's exact test and mann-whitney test were used to compare categorical and continuous variables, respectively. results: twenty-one xla patient records were reviewed, with a median age at start of follow-up (f/u) of y and a median duration of f/u of years. eight patients underwent at least one liver biopsy of whom ( % of nih xla cohort) were nrh+. the median age at nrh diagnosis was y ( - ). among patients who had liver biopsies, alanine aminotransferase (alt) levels were mildly elevated in all, while alkaline phosphatase (alp) levels were only increased in nrh+ patients (p= . ). both nrh+ and nrhgroups had similar aspartate aminotransferase (ast) levels at baseline but higher values were observed at the end of f/u in the nrh+ group ( vs. u/l, p= . ). persistently low platelet count ( < k/μl for more than months), mildly to highly elevated hepatic venous pressure gradient (hvpg) and either hepatomegaly and/ or splenomegaly were present in all nrh+ patients. in opposition, neither persistently low platelet counts, nor hepato-or splenomegaly were present in the two nrh-patients evaluated. hvpg was normal in the only nrh-patient tested. all-cause mortality was higher among nrh+ patients ( / , %) than in the rest of the cohort ( / , % among nrh-and unknown patients, p= . ). conclusions: based on our retrospective analysis, nrh appears as an underreported, frequent and severe late-onset complication in xla, which is highly associated with increased mortality. persistent thrombocytopenia, elevated alp, elevated hvpg, hepato-and/or splenomegaly were common in liver biopsyproven xla/nrh+ patients and distinguish them from xla/ nrh-patients. based on nrh prevalence, severity, lack of specific treatment and poor outcome in xla, immune-reconstitution (rather than igg replacement and infectious prophylaxis) should be considered early in this population in order to prevent fatal long term complications. abstract/case report text introduction: barth syndrome (bths) is an x-linked recessive disorder caused by a mutation in the tafazzin (taz) gene resulting in an inborn error of cardiolipin phospholipid metabolism (an important mitochondrial inner membrane lipid). it is commonly characterized by intermittent neutropenia and cardiac and skeletal myopathies. we present a case of bths with associated lymphopenia and hypogammaglobulinemia, which has not been previously described in the literature. case report: a two-month old male, born full term with normal newborn screening, was first admitted for rsv bronchiolitis. at this time, patient underwent an echocardiogram given his older brother with hydrops had died hours after birth and on autopsy was found to have dilated cardiomyopathy (dcm). patient was similarly noted to have dcm and thus had whole exome sequencing done that showed a hemizygous mutation in the taz gene (c. g>a). this novel variant resulted in early termination of the protein (p.trp ter) with concern for loss of function. in regard to patient's first year of life, he had frequent uri symptoms, episodes of acute otitis media requiring tympanostomy tubes, but no documented pneumonias or other serious bacterial infections. patient also had gross developmental delay, particularly motor, and feeding difficulties with persistent failure to thrive requiring g tube placement. his absolute neutrophil count ranged from - cells/mm in the first year. at age months, patient was found to be in acute decompensated heart failure with concern for myocarditis (ck , u/l, troponin i . ng/ml) as well as acute hypoxic respiratory failure with respiratory cultures growing pseudomonas. he was incidentally found to have an igg level of mg/dl (normal for age - ) and treated empirically with ivig. when seen by immunology, further workup showed persistent b cell lymphopenia (absolute cd of - /mm ). he also had a low initial nk cell count ( - /mm , later normal) with normal cd and cd t cell counts. tetanus and hib titers could not be assessed as he had recently received ivig. his igg trended up to mg/dl a few days after initial ivig and then subsequently dropped to mg/ dl, with a level of mg/dl two weeks following initial dose. workup for gastrointestinal or renal losses of immunoglobulin were negative. he also shortly after developed enterobacter bacteremia. his igg levels at this time continue to remain around mg/dl. he subsequently required a heart transplant at age months for his dcm. after transplant, he continued to improve from a cardiac standpoint, but his lymphopenia persisted and each time he was weaned off ivig, his hypogammaglobulinemia persisted at - mg/dl thus requiring additional ivig replacement over the course of the next months. the remainder of immunoglobulins were normal initially, but the igm slowly dropped over time to - . mg/dl. patient was started on weekly subcutaneous immunoglobulin replacement at months, doing well clinically at age -month follow up. conclusion: here we present a patient with bths, with a novel variant, who had b-cell lymphopenia as part of his presentation with persistent hypogammaglobulinemia requiring ivig replacement. year fellow/ucla associate professor/division of allergy, immunology, and rheumatology, university of california los angeles chief of pediatric allergy and immunology/harbor-ucla chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih biologist/laboratory of clinical immunology and microbiology, division of intramural research, national institute of allergy and infectious diseases, national institutes of health project scientist/ucla abstract/case report text a -year-old female presented for combined immunodeficiency. at years of age she was diagnosed with rag hypomorphism and started on ivig. as a child, she was hospitalized for pneumonias and cryptococcal meningitis. she suffered sinusitis, hepatitis, tooth abscess, cmv and herpes stomatitis. later, she experienced recurrent cutaneous abscesses, utis, vaginal yeast infections, and hidradenitis. she twice hospitalized recently for pneumonias and diagnosed with mycobacterium abscessus on bronchoscopy. she suffers onychomycosis, osteomyelitis and oral and esophageal candidiasis with odynophagia. on exam, she had white plaques on tongue and buccal mucosa. she had hyperpigmented plaques on forehead and cheeks and thickened nails. immune evaluation was significant for lymphopenia with alc and thrombocytopenia with platelets k. b cells were nearly absent ( absolute count) and nk cells were low at absolute count. ige was absent, igm mg/dl, iga mg/dl and igg mg/dl (on replacement). her total cd + count was , cd + t cells were low at %, but cd + cells normal at %. the cd + t cells were mostly memory phenotype, which probably reflects lymphopenia-induced proliferation of a small number of clones. her cd + t cells also had an elevated amount of memory cells for age, but still had presence of naive cd + t cells. as expected with perpetual lymphopeniainduced proliferation, there was evidence of terminal memory (temra) in the cd + lineage. proliferative responses of t cells were modest. cd + t cells did respond to pokeweed, but less to pha and cona. there were no antigen specific responses. trecs were normal. esr was mildly elevated at . of note, her liver enzymes were elevated with alkaline phosphatase and ast , presumably secondary to prolonged fluconazole use. w es r e v e a l e d a k n o w n p a t h o g e n i c v ar i a n t i n s tat (nm_ . : c. c>g (p.n k)) as well as a heterozygous variant in rag p.m t. the stat mutation is de novo and was previously published as a gain of function mutation. however, when we performed validation studies to evaluate cd + cells with stimulation to ifna, the patient had decreased pstat as compared with control. va . analysis was performed to evaluate rag defect and showed % of t cells with va . expression confirming that the rag defect is not clinically significant. she developed severe thrombocytopenia refractory to platelet transfusions and ivig. she was started on ruxolitinib which improved platelet counts. however, she presented with shortness of breath, persistent tachycardia and was found to have cmv carditis and hepatitis significant for echocardiogram with ef %. cmv pcr is improving with last check iu/ml after month of therapy with ganciclovir. we now are looking for evidence of socs to explain the decreased stat phosphorylation. genetic testing is critical when evaluating a patient with immunodeficiency. our patient demonstrates that genetic mutations cannot be taken at face value and should be evaluated and validated fully to optimize patient care. fellow/university at buffalo / oishei children's hospital abstract/case report text opportunistic infections (oi) are commonly seen in patients undergoing hematopoietic cell transplantation (hct). different strategies for antimicrobial prophylaxis are often employed in the transplant setting to reduce the likelihood of encountering infection. the predisposing risks for infections include the expected neutropenia and lymphopenia following conditioning, prolonged defects in cell-mediated and humoral immunity during the engraftment period, and iatrogenic immunosuppression by medications for graft versus host disease (gvhd). we report the case of a -year-old male with acute lymphoblastic leukemia, which relapsed to chronic myelogenous leukemic blast crisis, and failed a subsequent allogeneic hct with central nervous system relapse. he was subjected to a second allogeneic hct. his immediate post-second transplant course was complicated with skin and gut gvhd, and infection and/or reactivation of coronavirus, respiratory adenovirus, epstein-barr virus, and human herpesvirus . while the herpesviral infections were controlled with antivirals and rituximab, adenovirus c infection proceeded to involve the gastrointestinal tract, and proved persistent over several months despite use of cidofovir. the patient's gvhd and transplant-associated thrombotic microangiopathy necessitated use of further immunosuppressants, including the complement protein c -binding eculizumab (an inhibitor of formation of the terminal c b- complex), ruxolitinib (a janus kinase [jak] / inhibitor) and low-dose interleukin- . h i s c l i n i c a l c o u r s e w a s f u r t h e r c o m p l i c a t e d b y stenotrophomonas maltophilia gut colonization and subsequent bacteremia, as well as multiple gram-positive bacteremia courses. at around day + , there was a life-threatening pericarditis with pericardial effusion and respiratory distress, associated with pneumocystis and stenotrophomonas being isolated from bronchoalveolar lavage. this occurred despite the patient being on pentamidine prophylaxis. the patient eventually recovered on trimethoprim/sulfamethoxazole therapy. we discuss the various risk factors potentially contributing to each oi in this illustrative case. in particular, complement and jak inhibitor therapy are fairly new drugs approved for other indications, whose off-label use in transplant patients is increasing. both have recently been associated with certain oi in the literature, as they are in this patient. abstract/case report text background: autoimmune lymphoproliferative syndrome (alps) is characterized by chronic nonmalignant lymphadenopathy, splenomegaly, hepatomegaly, cytopenias, and other autoimmune manifestations. typically, the biomarker profile of patients with alps includes elevated tcr αβ+ dnt cells, serum igg, serum b , serum il and soluble fas ligand (sfasl). hdl cholesterol can also be significantly low. alps is caused by lymphocyte accumulation due to defects in the fas-mediated apoptosis signaling pathway. these defects cause resistance to physiological apoptosis in lymphocyte populations that results in chronic lymphoproliferation. the molecular defect underlying most alps etiologies is attributed to heterozygous germline or somatic (limited to dnt cell subpopulation) pathogenic single nucleotide variants (snv) in fas. we describe copy number variants (cnvs) at the fas locus underlying alps in unrelated families. methods: through the centralized sequencing initiative at at the national institute of allergy and infectious diseases (niaid), patients undergo genomic workup to identify molecular defects contributing to clinical phenotypes of immune system disorders. all patients receive exome sequencing and a subset of patients also receive array-cgh analysis. patients and results: we performed exome sequencing on patients with a clinical diagnosis of alps. for patients with no molecular defect through exome, we performed cnv analysis. in this cohort, we identified three patients with a copy number variant involving the fas locus. all patients presented with splenomegaly and lymphadenopathy in childhood with ages of onset ranging from months to years old. all patients experienced anemia, autoimmune neutropenia, and thrombocytopenia. they had biomarker evidence showing elevated serum b levels, sfasl levels, and elevated αβ+dnt cell populations. they were found to have very low hdl cholesterol in early childhood ranging from - mg/dl ( - mg/dl). all patients had negative family histories for lymphoproliferative disorders and immunodeficiency. these patients had clinical presentations and biomarker profiles similar to alps patients with germline and somatic fas variants. patient : we detected a~ . mb copy number loss encompassing all of fas. parental studies were not performed. patient : we detected a~ . mb copy number loss encompassing all of fas. parental studies showed this to be maternally inherited. in addition, prior karyotype testing of the bone marrow showed the same deletion. patient : we detected a~ . mb copy number loss encompassing exons - of fas. parental studies were not performed. these results are consistent with the pathogenic nature of copy number variant losses involving fas. the mechanism of disease in these patients is consistent with haploinsufficiency. in family , the mother harboring the fas deletion is unaffected. this is consistent with prior observation of reduced penetrance within a family in alps. conclusion: these three cases harbored causative deletions in fas in the presence of biomarkers indicative of alps and negative results for germline and somatic genetic variant testing. these patients demonstrate that copy number variant analysis should be pursued if there is robust clinical and biomarker evidence of alps as it can lead to a molecular diagnosis and appropriate treatment when exome or next generation panel based fas sequencing is inconclusive. abstract/case report text rationale: the thymus is essential for the development of tcells. patients with thymoma have decreased aire expression and have an abnormal thymic microenvironment where the negative selection of t-cells is compromised, resulting in a broad spectrum of autoimmune-mediated diseases. besides myasthenia gravis, which is found in to % of patients with thymoma, other autoimmune diseases have been reported including erythroblastopenia, systemic lupus erythematosus, inflammatory myopathies, thyroid disorders and good's syndrome. recent studies have described additional autoimmune conditions such as pneumonitis in thymoma patients. we identified a patient who developed chronic cough post-thymectomy and was found to have lymphocytic pneumonitis with associated autoantibodies against lung antigen kcnrg and lung immunopathology consistent with apeced pneumonitis, which implies a common pathogenic mechanism between these conditions. methods: we describe a patient with thymoma who developed autoimmune pneumonitis associated with kcnrg autoantibodies and a characteristic pattern of immunopathology recently described in patients with monogenic disorder caused by primary aire deficiency (apeced) and secondary aire deficiencies (thymoma, rag deficiency). results: patient is a -year-old male with no significant past medical history who was in good state of health until age when he was diagnosed with and received treatment for guttate psoriasis (resolved with uv therapy) and alopecia areata. at age , he developed severe abdominal pain and weight loss. he had an abdominal ct performed that showed chronic pancreatitis and thymoma. one month later, the patient underwent thymectomy and subsequently, underwent ercp and pancreas biopsy, revealing atrophic pancreatitis with negative staining for lgg and lgg . at that time, he was started on pancreatic enzymes with improvement of abdominal symptoms. following thymectomy, he developed persistent dry cough and recurrent symptoms of sinusitis which did not respond to several courses of oral antibiotics to treat his positive culture for pseudomonas. he had a negative work up for vocal cord dysfunction and cystic fibrosis, and negative autoantibodies against ifn-gamma, il- a, and gm-csf. for work up of chronic cough, the patient underwent ct imaging of the chest which revealed diffuse peri-bronchial thickening, mucus plugging, and tree-in-bud nodularity through most of his lungs. he underwent bronchoscopy with bal which revealed n o r m a l b r o n c h i a l m u c o s a a n d a i r w a y n e u t r o p h i l i a . endobronchial biopsies showed basement membrane thickening and dramatic lymphocyte infiltration in intraepithelial and submucosal areas. his bal cultures revealed mycobacterium intracellulare/chimaera. patient was also tested for autoantibodies against lung-specific bactericidal/permeabilityincreasing fold-containing b (bpifb ) and the potassium channel regulator kcnrg that have been associated with the development of pneumonitis in patient with apeced, thymoma and rag deficiency, and was found to have kcnrg-targeted autoantibodies. conclusions: thymoma is a disease associated with secondary aire deficiency. this case illustrates common clinical, radiographic, histological, and autoantibody features in thymomaassociated and apeced-associated pneumonitis, indicating that disorders with primary and secondary aire deficiencies may have common pathogenetic mechanisms. bpifb and kcnrg should be included in the autoantibody profile testing of patients with thymoma and lung disease. immune suppression and antimycobacterial antibiotic treatment are planned. abstract/case report text introduction/background: activated phosphoinositide -kinase δ (pi kδ) syndrome (apds) is a primary immunodeficiency caused by a gain-of-function mutation in the pik cd gene that encodes the p δ catalytic subunit of pi kδ. it is characterized by recurrent respiratory tract infections, lymphoproliferation, nodular mucosal lymphoid hyperplasia, enteropathy, ebv and/or cmv infection, reduced t cell function and high levels of igm. there is not evidence of this disease in peruvian patients. methods: a case series of two pediatric patients with apds. results: the first patient is a girl of non-consanguineous parents. family history shows four maternal uncles died at pediatric ages with unknown diagnosis. at the age of , she presented lymphadenopathy and fever being treated as cat scratch disease without improvement of symptoms. months later, she was hospitalized due to anemia, mild hepatosplenomegaly, ascites and chronic diarrhea and diagnosed with gastrointestinal tuberculosis (tb). a hepatic biopsy only showed reactive hepatitis. however, the patient did not improve her symptoms despite anti tb treatment. years later, she was hospitalized for lymphadenopathy, pancytopenia, chronic diarrhea, ascites and severe hepatosplenomegaly. cmv igg was positive and lymph node biopsy revealed paracortical and follicular lymphoid hyperplasia due to ebv infection without neoplastic proliferation. low cd + t and cd + b cells and high igg levels were found (table ) . at this time, it was suggested the diagnosis of apds which was confirmed by next generation sequencing (ngs) identifying a heterozygous mutation in the pik cd gene (c. g>a, p.glu lys). she was treated with sirolimus and ivig for years. the symptoms persisted despite treatment and died at the age of . the second patient is a -year-old girl also of non-consanguineous parents. family history includes eczema (father) and colorectal cancer (mother). she has had recurrent respiratory infections, chronic diarrhea and poor weight gain since months old receiving symptomatic treatment only. at the age of , she was hospitalized for persistent pneumonia ( p s e u d o m o n a s p o s i t i v e ) , l y m p h a d e n o p a t h y a n d m i l d hepatosplenomegaly. a ct scan showed bilateral bronchiectasis and the sweat chloride test was negative. based on this, a diagnosis of cystic fibrosis was made and treatment was started. however, a genetic study only showed heterozygous mutations in the cftr gene (g d and g x). year later, she presented a neck-located skin abscess. at the age of , she was hospitalized for complicated pneumonia, diarrhea, lymphadenopathy, ascites and severe hepatosplenomegaly. multiple polyps in the duodenum and colon with lymphoid hyperplasia were detected, ebv igm and igg were positive and a lymph node biopsy showed paracortical hyperplasia without neoplastic proliferation. cd + t cells and igm levels were increased (table ) . a diagnosis of apds was suspected and ivig was started. ngs showed the same mutation as the first patient (c. g>a, p.glu lys). conclusion: apds should be considered in patients with recurrent respiratory tract infections, lymphoproliferation, enteropathy and abnormal immunologic function without another explanation. ngs is a useful tool to identify these cases in low-income countries. acknowledgments: we thank drs. raif geha and janet chou, division of immunology, boston children's hospital, harvard medical school for the genetic diagnosis. background: granulomatous-lymphocytic interstitial lung disease (glild) is an increasingly recognized pulmonary complication associated with common variable immunodeficiency (cvid) but the natural history and long term prognosis remains poorly defined. imaging findings with computed tomography (ct) are heterogeneous and visual features do not consistently predict a patient's progression to fibrotic lung disease. computer-aided lung informatics for pathology evaluation and rating (caliper) provides an objective analysis of lung parenchymal texture and quantifies the extent of normal lung, along with abnormal features such as honeycombing, reticular/consolidative and groundglass opacity. this may be useful in cvid patients to monitor changes in character or extent of disease and may facilitate early intervention before the disease becomes more aggressive or advanced. case description: our patient is a -year-old non-smoking female with cvid who has been followed for her cvid and associated interstitial lung disease. for more than twenty years, she has had varying abnormalities found on chest ct and these appear consistent with glild. specifically, she has had variable regions of mixed consolidation, ill-defined nodularity and septal thickening. the changing morphology and distribution made assessment of overall severity and extent of fibrosis versus parenchymal infiltration inconsistent. for clinical decision support we used caliper to analyze the current ct ( ) and compared caliper results for previous ct data. caliper provided a comprehensive analysis of the extent and characteristics of parenchymal features, and objectively determined normal and abnormal regions, some of which were not visually apparent. the caliper color overlay was able to highlight subtle regions of ground-glass opacity in areas that visually were regarded as uninvolved lung and quantify the extent of the reticular densities/ consolidation over time. caliper does not differentiate reticulation from consolidation, does not detect nodularity or septal thickening, and ct imaging cannot distinguish inflammation from fibrosis. however, caliper has the power to quantitatively assess overall disease extent and demonstrate subtle abnormalities that would otherwise have been dismissed as normal, given relative sparing compared to other regions. caliper may also provide evidence for disease progression or therapeutic response that is not otherwise radiographically apparent. conclusion: caliper assessment may be a useful tool as an adjunct for a patient with glild to help quantify the extent and character of lung parenchymal involvement. this information may serve as an important guide for clinicians in the assessment of successful management and early intervention to prevent irreversible fibrosis. patients had a trial of fingolimod without any beneficial changes in immune status. both patients receive pneumocystis jirovecii pneumonia prophylaxis with sulfamethoxazole-trimethoprim. conclusions: these results indicate that s pl deficiency due to sgpl mutations is a syndromic primary immunodeficiency leading to profound lymphopenia and hypogammaglobulinemia. our data emphasize the importance of sphingolipid metabolism for an efficient immune response and the need for more studies to delineate the exact mechanisms on how this happens in humans. after d at °c there was a median - % (range - % to + %; p= . ) change in activity. after d - % (range - % to + ; p= . ), d - % (range - % to + %; p < . ), d - % (- % to + %; p < . ) and after d - % (- % to + % p < . ). a °c stability of d was determined from the median percentage reduction; total allowable error adjusted stability data indicated a °c stability of d. samples stored at - °c following repeat freeze thawing saw a freeze/thaw cycle dependent decrease in ch activity. after freeze/thaw cycle there was a median - % (range - % to + %; p= . ) change, cycles - % (range - % to + %; p= . ), cycles - % (range - % to + %; p < . ), cycles - % (range - % to - %; p < . ) and after cycles - % (range - % to - %; p < . ). allowable error adjusted stability data indicated a maximum of freeze/thaw cycles. conclusion: sample storage and handling can have a significant impact on functional complement assessments. room temperature storage should be avoided unless samples will be analysed on the day of collection, °c storage is tolerable providing that assessment is within d; freezing samples at - °c with limited freeze/thaw analysis would be optimal. however, further investigations into longer-term storage at - °c and - °c would be beneficial. conclusions: gi disease is common in cvid affecting % of patients in our cohort. gi+ cvid patients have a higher frequency of autoimmune manifestations than those without gi complaints. the odds of itp, hypothyroidism, and evans syndrome all showed significantly increased odds in the gi+ group. the results of our study may have implications for both gastroenterologist and immunologist. recurrent infections especially those of the sinopulmonary tract are often the trigger for cvid evaluation. autoimmune and gi symptoms however may be the initial presentations of cvid and overlooked until other more recognizable manifestations evolve. the combination of gi issues and autoimmunity especially thrombocytopenia, evans syndrome, and hypothyroidism should include cvid in the gi differential. for the immunologist, a cbc is standard in the work-up of cvid and may reveal autoimmune cytopenia. evaluation for autoimmune disease and in particular hypothyroidism is not. given our findings an initial immune work up specifically for thyroid disease may be indicated. is the transcriptional factor for many cytokines such as il- , responsible for t cell and neutrophil defense again fungal infection. stat mutation leads to defect of neutrophil proliferation and chemotaxis to inflammatory site as well as production of antimicrobial peptides by respiratory epithelial cells. the poor tissue repair in the cavitary lesions and bacterial superinfection in patient's lung created a culture dish for fungal growth and dissemination. traveling to the endemic area and patient's noncompliance to antifungal prophylactic treatment further increased the risk of histoplasmosis infection. pediatric immunologist/john hunter children's hospital abstract/case report text we present the case of a month old boy, the first child to his nonconsanguineous parents of european descent. he first presented at months of age with a cellulitis of his right fourth finger culture positive for staphylococcus aureus which responded to a prolonged course of flucloxacillin. at months he presented with norovirus positive gastroenteritis leading to a brief admission and slow resolution. the first of two severe episodes of oral stomatitis and respiratory distress occurred at months of age. hsv was isolated from the oral lesions and blood culture during that admission was positive for kingella kingae. no cardiac or bone involvement was identified. a more severe episode of oral stomatitis occurred two months later (age months) swab positive for an enterovirus (not typed). due to airway compromise and rapid deterioration he was admitted to the pediatric intensive care unit. again, kingella kingae was cultured from blood cultures with no obvious focal systemic source. the only notable clinical finding was rapid deterioration and, in retrospect, the absence of any significant recorded fever ( < oc). crp elevation was observed (max. mg/l) and neutropenia was found with each of the more severe infectious presentations but recovered in the interval. baseline immunological investigations were normal (lymphocyte subsets, naïve t cell populations, lymphocyte proliferation, serum immunoglobulins and vaccine responses). serial measurement of circulating neutrophils did not identify a cyclical pattern and they were morphologically normal. a panel of genes relevant to primary immunodeficiency (invitae©) revealed a homozygous mutation in irak ((c. c>t (p.gln *)) which leads to a premature stop codon. this is a known pathogenic mutation leading to disease and is most prevalent in the european population (allele frequency (gnomad) = . ). prophylaxis with sulfamethoxazole / trimethoprim and amoxicillin was commenced along with monthly ivig. he has been well since diagnosis with no further severe infectious presentations. functional testing is underway to assess in vitro host viral defence in our patient and potential novel mechanisms relevant to this rare innate immunodeficiency. case studies will be presented on the five cases of fmp that were diagnosed and treated in . potential exposures were identified in four out of five cases: gardening exposure in one case and vaping exposure in three cases. all five were male, age range - . four were gp deficient, and one was p -phox deficient. historically, the vast majority of cases of fmp could be traced to a significant gardening exposure such as lawn mowing or spreading mulch. this was the first year that we saw patients with no identifiable gardening exposure in the setting of significant vaping exposure. with vaping at epidemic levels, especially among teenagers and young adults, it is important to consider that a vaping history is potentially a risk factor for fmp and counseling regarding the potential risks of vaping should be included in infection risk modification for all patients with cgd. abstract/case report text background: granulocyte-macrophage colony-stimulating factor (gm-csf) plays a critical role in macrophage and dendritic cell maturation and host defense against fungus. autoantibodies to gm-csf are associated with susceptibility to cryptococcus and nocardia infections as well as pulmonary alveolar proteinosis (pap) in otherwise healthy individuals. we report a case of a -year-old previously healthy female who presented with cryptococcal meningitis and was found to have autoantibodies against gm-csf. case presentation: weeks prior to admission, our previously healthy -yearold taiwanese female developed a headache associated with tinnitus and visual changes. the headache worsened over the next few weeks and she developed photophobia, phonophobia, and severe nausea/vomiting. at presentation, her exam was notable for papilledema, bilateral cn vi palsy and right foot & left hand paresthesia. mri brain showed ring-enhancing lesions in the anterior frontal lobe, caudate head, and the inferior globus pallidus. she underwent a diagnostic and therapeutic lp. opening pressure was elevated at and csf studies were notable for low glucose, elevated protein, pleiocytosis ( % lymphocytes) and positive cryptococcal antigen. csf culture grew cryptococcus gattii. ct chest revealed a right upper lobe and a left lower lobe nodule. workup: cbc with diff was unremarkable. hiv was negative. lymphocyte subsets were unremarkable with only mildly decreased nk cells, normal immunoglobulin panel including ige, protective titers to tetanus, diphtheria, and ppsv . targeted genetic sequencing did not identify any known mutations in primary immunodeficiency. notably, anti-gmcsf autoantibodies were detected by elisa and were able to neutralize gm-csf phosphorylation of stat detected by flow cytometry. autoantibodies to ifn-γ were not detected. management: patient was initiated on a -week course of liposomal amphotericin b and flucytosine. her csf cultures were cleared of cryptococcus after days of treatment, but her hospital course was complicated by persistently symptomatic intracranial hypertension, worsening pleiocytosis, and elevated cytokine levels in the csf, all of which were consistent with post-infectious inflammatory syndrome (piirs). she received therapeutic lps - x/week until subsequent ventriculoperitoneal shunt placement. concurrently, methylprednisolone was administered for days with a gradual prednisone taper. these interventions led to improvements in her symptoms, including diplopia, and reduction in opening pressures and inflammatory markers in the csf. lifelong fluconazole prophylaxis was recommended. from a pulmonary standpoint, she remained asymptomatic without signs of pap and has had normal pulmonary function tests (normal dlco) and stable chest imaging. conclusion: in otherwise healthy hiv-negative patients presenting with extrapulmonary cryptococcus or nocardia infections, autoantibodies to gm-csf should be suspected and testing for functional autoantibodies to gm-csf (and ifn-γ) should be sent, as genetic testing will not pick up this disease entity. genetic testing should be considered to rule out gata deficiency and x-linked cd l deficiency. idiopathic cd lymphopenia can be ruled out with lymphocyte enumeration. immediate treatment of cryptococcosis is not necessarily different from patients without gm-csf autoantibodies. long-term prophylaxis (fluconazole if presenting with cryptococcus; trimethoprim-sulfamethoxazole if with nocardia) is likely warranted in addition to monitoring for the development of pap. recognizing piirs in patients with cryptococcal meningitis and management with corticosteroids are critical steps. abstract/case report text background: non-infectious complications cause most morbidity and mortality in common variable immunodeficiency (cvid). cvid with complications (cvidc) is defined by elevated t helper (th ) responses attributed to increased circulating microbial products resulting from mucosal iga deficiency. however, complications do not uniformly occur in those with iga deficiency. objective: we tested whether cvidc occurs preferentially in those with hyper-responsiveness to microbial stimuli, manifested by elevated nf-κb-driven cytokines and resultant th responses in cvid patients with increased circulating microbial products. methods: we applied unbiased high-throughput seromics and mass cytometry, cellular and molecular biology approaches, and clinical record review in a subject cvid cohort. results: cvidc was defined by increased nf-κb-driven cytokines that promote th immunity in blood in association with elevated soluble cd , a marker of circulating microbial products, and elevated tnf production by peripheral blood mononuclear cells stimulated with lipopolysaccharide. this cytokine upsurge was associated with mutation of full-length nfkb p gene product ( delt) but not mutations that also involved the nfkb p product involved in transactivation. cytokine elevation corresponded with increased cd +cd -monocytes expressing higher cd and hla-dr and more central and effector memory cd + t cells, t cell chemoattractants, and t cellpredominant tissue pathology. those with granulomatous or neutrophilpredominant, rather than t cell, pathology had the highest tnf. tnf antagonism improved neutrophilic gastritis in cvid with nfkb delt after t cell targeted therapy failed. conclusion: nf-κb dysfunction underlies th immunopathology and tnf-associated innate inflammation in cvidc. both forms of nf-κb immune dysregulation may divergently shape cvid immunopathology. staff clinician/laboratory of clinical immunology and microbiology, immunopathogenesis section, national institute of allergy and immunology, national institutes of health, abstract/case report text introduction: patients with autoantibodies to ifn-γ develop severe and progressive infections with intracellular pathogens, despite aggressive antimicrobial treatment. we describe the use of daratumumab (anti-cd , targeting plasma cells) in a patient with autoantibodies to ifn-γ and progressive disseminated mycobacterium avium infection. she had progressive disease despite treatment with multi-drug antimycobacterials rituximab, and bortezomib. methods: clinical symptoms, total cd /cd , anti-ifn-γ autoantibody titers, and specific imaging were obtained before and after treatment with daratumumab. anti-ifn-γ autoantibody titers were determined by serial -fold dilutions of plasma and measuring anti-ifn-γ autoantibody levels by a particle-based technique as previously described. results: a -year-old filipino woman had progressive disseminated m. avium with extensive bone and soft tissue involvement (calvarium, ribs, bilateral arm soft tissue, paraspinal muscles, bilateral glutei, left inferior pubic ramus, bilateral iliac bones, sacrum, and bilateral humeri) and a tracheo-esophageal fistula. she received bedaquiline, azithromycin, ethambutol, tedizolid, moxifloxacin, clofazimine and meropenem as well as rituximab g once monthly for months. despite these she had progression of clinical and radiographic disease. bortezomib . mg/m twice weekly for weeks was added, but discontinued for ast and alt elevations. rituximab was continued to maintain cd numbers undetectable but clinical and radiographic disease progressed. while on rituximab, total igg level and anti-ifn-γ autoantibody levels decreased from mg/dl to mg/dl and to , respectively. while on bortezomib, total igg levels remained stable ( mg/dl to mg/dl) and anti-ifn-γ autoantibody levels fell slightly ( to ). after starting daratumumab, there was clinical and radiographic improvement, with reduced pain and disappearance of multiple soft tissue lesions. igg levels decreased from mg/dl to mg/dl and anti-ifn-γ autoantibody levels decreased from to . adverse effects of daratumumab were urticaria, pruritus and shortness of breath after the first infusion and aseptic meningitis after the th infusion. conclusions: daratumumab resulted in clinical and radiographic improvement of disseminated m. avium in a patient with rituximab and bortezimib-refractory autoantibodies to ifn-γ. daratumumab is another potentially effective therapeutic agent for anti-ifn-γ autoantibodies. abstract/case report text next-generation sequencing (ngs) is now routinely used as a clinical diagnostic tool. however, regions of high sequence homology continue to be a major challenge for short-read technologies. regions within ikbkg, ncf , sbds, c a, c b, coro a, fcgr a, fcgr b, pms , slfn , slfn , stat b, unc b , and ups are not available by standard ngs. we discuss strategies for analysis of these special regions. we have developed a strategy for supplementing our disease targeted panels which are performed using capture chemistry and a standard reference file. the supplemental method uses gene specific long range amplicon and a special gene specific reference file for alignment. the genes of interest are separated from their homologous counterparts using specific long range amplification primers. multiple amplicons may be pooled together and prepared for sequencing on an illumina miseq instrument using truseq nano dna library prep. bioinformatic analysis proceeds with a custom reference file in which non-specific regions of homology have been removed. this allows reads to be uniquely mapped despite significant homology; a requirement for variant calling. we prepared specific amplicon for several homologous gene targets including the ikbkg gene and the ikbkg pseudogene (ikbkgp ). both amplicons were sequenced in separate reactions and were compared with the standard capture method. variants which are not called in the standard-capture method due to poor mapping scores (non-uniquely mapped reads) are called in the amplicon method. in the capture method, the variants are visualized in the bam as a mixture of gene and pseudogene, while gene and pseudogene variants are clearly separated and identified in the amplicon method. due to high variability in alignment, many homologous regions do not provide reliable copy number variant (cnv) results and must be removed from cnv analysis. however in some situations, we are able to creatively leverage cnv analysis to identify alleles that mis-align to the pseudogene. the pathogenic ncf gt deletion in exon appears to resemble a copy number deletion event when present as reads from one allele mis-align to the ncf b and ncf c pseudogenes. complement genes, c a and c b, share alignment due to their high homology with each other. cnv analysis in normal samples represents four alleles rather than two alleles. cnv events may have a weak signal with no indication of which gene is affected. variant frequencies from the capture and supplemental pcr analysis can be used in tandem with cnv analysis to detect events and may indicate which gene is affected. we plan to include these strategies in our new inborn errors of immunodeficiency gene panel (ieigp) which will enable us to provide a more comprehensive analysis than is currently available. the im diagnosed were inflammatory bowel disease-like (n = , with perianal fistula in / ), mouth ulcers (n = ), discoid lupus (n = ), autoimmune dermatitis (n = ) and eczema (n = ), chronic lung disease (n = ) and granulomas (pulmonary n = ; ocular n = ; bladder n = ; oropharynx n = ). three patients presented more than one site of inflammatory disease. all patients were treated with systemic or topical immunosuppressive or immunomodulatory therapy, most of them corticosteroids. five patients underwent hematopoietic stem cell transplantation (hsct), median age at hsct was years ( - ), and two died month after hsct. conclusions: although infections are more frequent and have a major impact on patient morbidity and mortality, im are increasingly prevalent in patients with cgd. awareness regarding this possible comorbidity is of major importance, since earlier diagnosis and adequate treatment may be crucial for patients survival and quality of life. there is a gap in clinical knowledge regarding associations between specific pid and different rheumatological diseases. in this study, we are reporting the incidence of various rheumatological conditions reported in a large pid population using the usidnet (united states immunodeficiency network) registry. methods: we used the retrospective usidnet registry to conduct the analysis. we included all primary immunodeficiency patients with physician diagnosed rheumatological diseases. results: the total number of pid patients in our query was . ( . %) patients had a diagnosis of rheumatological disease. this cohort included ( . %) female and ( . %) male patients. rheumatologic complications were highest in the interferonopathies ( . %), complement deficiencies ( . %) and autoimmune lymphoproliferative syndrome (alps) ( . %). additionally, disease patterns were noted to be different in each pid. dermatomyositis was found to be the most common rheumatologic condition in patients with x-linked agammaglobulinemia (xla) with a rate of . %, which was remarkably higher than the reported prevalence in the united states ( . %). alps patients had a higher ( . %) numbers of sjogren syndrome diagnoses as compared to the general population ( . - . %). systemic lupus erythematosus was increased in patients with mucocutaneous candidiasis ( . %) as compared to the general population ( . %) and other pids. rheumatoid arthritis (ra) was reported in patients with specific antibody deficiency ( . %), common variable immunodeficiency (cvid) ( . %) and alps ( . %). wiskott-aldrich syndrome patients had the highest numbers of cases diagnosed with vasculitis ( . %). . % of patients with severe combined immunodeficiency (scid) had reported rheumatologic disease. juvenile rheumatoid arthritis (jia) and systemic sclerosis were reported in . % of patients with digeorge syndrome. conclusions: this study reports that higher numbers of rheumatologic diseases are diagnosed in pids compared to the general population. the incidence of different rheumatological disease was variable based on the pid diagnosis. early diagnosis of these diseases is crucial, given the high risk of irreversible complications. limitations of our study include possible selection bias as majority of cases were enrolled from tertiary care centers. abstract/case report text background disorders of immune dysregulation are associated with autoimmune features. this feature could potentially have an impact on the outcome post hematopoietic stem cell transplantation (hsct). hsct, although curative, can be challenging with the underlying immune dysregulation resulting in significant morbidity and mortality. we present the journey through hsct for these children and the factors affecting the outcome. we analysed the data on children up to the age of years diagnosed to have a disorder of immune dysregulation through gene mutation analysis and who underwent hsct at our centre from to . results . xiap mutation a -year-old boy underwent a haploidentical hsct from his father using fludarabine, treosulfan, and gray radiotherapy with post-transplant cyclophosphamide. after initial complete chimerism and cytomegalovirus reactivation responsive to valganciclovir, he developed progressive diarrhoea almost months post-hsct. a rectal biopsy confirmed cmv reactivation and features of inflammation. he has since been treated for the same and is on follow up for inflammatory bowel disease. his chimerism had dropped to % and has remained stable. the second child is a -year-old girl who underwent tcr alpha/beta depleted haplo sct and is months post-hsct, with no features of gvhd or infections, and is doing well with complete chimerism. . il r deficiency three boys aged eight months, one year, and two years of age, diagnosed to have il r deficiency underwent hsct. all three children needed nasogastric tube feeding, parenteral nutrition, and vigilant monitoring for electrolyte disturbances. in the first two children, we had performed tcr alpha/beta depleted pbsc transplants from their haplo matched fathers. the -year-old engrafted by d+ and is doing well two years post hsct with complete chimerism, no gvhd, and infections. his autoimmunity, including recurrent skin scarring, has resolved entirely. the -month-old, however, had primary graft failure and succumbed to his illness. the -year-old boy underwent matched unrelated donor hsct and engrafted by d+ with completed chimerism documented on three occasions. he, however, had secondary graft failure around d+ , and he succumbed to the illness. . lrba deficiency an -month-old girl with lrba deficiency had presented at four months of age with excessive sweating, hepatosplenomegaly, and recurrent chest infections. she was started on monthly intravenous immunoglobulin replacement and abatacept. she received myeloablative conditioning with thiotepa, treosulfan and fludarabine and underwent a matched sibling donor hsct. she engrafted by d+ and has been well ten months post hsct with complete chimerism, no gvhd, and infections. conclusion disorders of immune dysregulation are a heterogeneous group with a varied spectrum of immune dysfunction. myeloablative conditioning is essential, and there is a high risk of cytokine release syndrome and the need for supportive care. the autoimmune features need to be followed for progression in organs other than the hematopoietic system and may require interventions. as long-term data evolves, more precise definitions for patient and donor selection will enable improving outcomes. we performed a retrospective observational analysis of case records of children up to years of age, diagnosed to have variants of scid, and underwent hsct at our centre from to . results . zap deficiency a -month-old girl presented with oral thrush and submandibular cellulitis from one week of life with failure to thrive. she underwent a tcr alpha/beta depleted haploidentical hsct. conditioning included treosulfan/thiotepa/fludarabine/anti-thymocyte globulin. she engrafted by d+ ; now three years post-hsct with complete donor chimerism without gvhd or infections. . orai- mutation a -month-old girl presented with failure to thrive, generalized hypotonia, oral thrush, and recurrent respiratory infections. she underwent haplo-sct with post-transplant cyclophosphamide with pbsc from her haplo-matched father. conditioning included fludarabine/treosulfan. she had cytokine release syndrome grade , which responded to tocilizumab. she had hypertension throughout the peri-engraftment period and had an episode of pres with seizures. her symptoms abated with neutrophil engraftment by d+ . the post-transplant period was complicated by grade skin gvhd and cytomegalovirus reactivation. she has remained disease-free with complete chimerism three years post-hsct. her hypotonia is steadily improving with physiotherapy. . cernunnos-xlf deficiency a -year-old male presented with recurrent infections from years of age, aplastic anemia diagnosed at years of age, subsequent transformation to acute myeloid leukemia at years of age. he had developed multiple fusarium abscesses during the neutropenic period post-chemotherapy for aml. he was referred for a matched sibling sister hsct when in remission. conditioning included fludarabine/treosulfan. he engrafted by d+ with complete chimerism. he developed progressively worsening skin, gut, and liver toxicity secondary to chemotherapy and succumbed to the illness two months post-hsct. . ikzf mutation an -month-old girl presented with failure to thrive, massive splenomegaly, persistent pneumonia, anemia, and thrombocytopenia. she underwent a matched sibling donor pbsc transplant after myeloablative conditioning with thiotepa/treosulfan/fludarabine. she engrafted by d+ , following which all her symptoms abated. she had secondary graft failure two months post-hsct and succumbed to her illness. . mhc class ii deficiency (bare lymphocyte syndrome) three children, aged months, two years, and four years underwent matched sibling donor hsct. myeloablative conditioning with thiotepa/treosulfan/fludarabine resulted in engraftment. the first child died of invasive intestinal aspergillosis days post-hsct. the other two children are well months post-hsct with complete chimerism without gvhd or infections. the two-year-old girl received one cycle of pre-transplant immunosuppression with fludarabine/dexamethasone to prevent graft rejection pre-hsct as she was referred for a second transplant. conclusion children with scid have traditionally been transplanted using reduced intensity (ric) conditioning with immunomodulation. scid variants require myeloablative conditioning with a vigilant follow up for the detection of graft rejection. radiation sensitive scid associated with dna breakage repair defects require ric and close monitoring for gvhd. advances in hsct, including supportive care and haplo-sct, have provided a ray of hope for these hitherto rare conditions. j clin immunol abstract/case report text immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) (omim # ) is a monogenic autoimmune disorder that occurs due to loss of function variation in foxp causing dysfunctional t regulatory cells. although immunosuppression is a mainstay of treatment for autoimmunity, ipex treatment is frequently limited by insufficient response to therapy or side effects of immune suppression. we present a year old male with ipex whose prior immunosuppressive treatment was complicated by inefficacy and medication side effects, requiring a new approach to treat his colitis and erosive dermatitis. he initially presented with infantile diabetes and subsequently developed dermatitis, squamous cell carcinomas, alopecia totalis, and colitis. his clinical diagnosis of ipex was confirmed by foxp sequencing, demonstrating known pathogenic variant c. g>a (p.ala thr). this variant has been described in ipex affected individuals in multiple publications (ref ). his variant affects at the frkhead domain of foxp and has been associated with others with severe psoriasiform dermatisis and alopecia universalis (ref ). his prior immunosuppressive therapies included at different times combinations of corticosteroids, tacrolimus, sirolimus, azathioprine, infliximab, adalimumab, rituximab, dupilumab, and oral mesalamine. the relative efficacy of these agents based on experiences in a cohort of ipex patients was reviewed in (ref ), with the exception of duplimab, which was not listed in that review. for our patient, management of his widespread autoimmunity has been limited by toxicity or lack of efficacy of medications. notably, his dermatitis had no improvement with duplimab, consistent his low total ige and lack of allergic manifestations. at age , after initiation of treatment with sirolimus, he had spontaneous colonic perforation requiring descending colectomy. after stabilization of his colonic perforation, his multi-disciplinary team of allergy-immunology, gastroenterology, and dermatology initiated tofacitinib. tofacitinib is small molecule inhibitor of janus kinase (jak) signaling pathways that mediate cytokine driven autoimmune activation. it is fda approved to treat rheumatoid arthritis, psoriatic arthritis and ulcerative colitis. the decision to use this jak inhibitor was due to its fda approved use for ulcerative colitis, to target our patient's colitis and his other autoimmune manifestations, specifically his dermatitis. its off label for primary immune dyregulatory disorders including candle, stat -gain of function and stat -gain of function disorders has been published (ref ), but thus far its use to treat autoimmunity due to ipex has not been published. he experienced leukopenia while on mg of tofacitinib, which resolved after lowering his dose. currently, he has had improvement in his colitis and dermatitis, and partial improvement in alopecia. he has been on tofacitinib mg daily for months, with only prednisone mg daily as additional immune suppression. as the number and types of selective immune modulators increases, there is continued need to share the experiences of treating physicians of which therapies have been successfully able to decrease disease manifestations with tolerable side effect profiles. we present a year old male with ipex syndrome with severe dermatitis and colitis complicated by colonic perforation despite standard immunosuppressive therapy, who is safely and effectively being treated with tofacitinib. is not frequently associated with autoimmunity, likely due to impaired il- and il- pathways. however, in our relatively large cohort of lof stat patients, we have noted an increased incidence of systemic lupus erythematosus (sle) diagnoses and sle-like symptoms. herein, we characterized the clinical and laboratory features of the patients in our cohort with sle and sle-like disease, with the aim to better understand the pathogenesis by evaluating ifn stimulated genes and neutrophil net formation. methods a retrospective chart review was performed of patients with lof stat to identify those with sle and sle-like presentations, and included clinical features, laboratories including inflammatory markers, auto-antibodies, and complement levels. rt-pcr was performed for interferon stimulated genes (isgs) from neutrophils and pbmcs of lof stat patients with and without sle, and healthy controls. neutrophil net formation was assessed for lof stat patients with and without sle, and healthy controls. results out of a cohort of patients, five patients (ages - ) were identified who carried the diagnosis of sle, and with slelike disease (ages - ). for those with sle, age of presentation was - years, of were female. clinical features included nephritis ( ), alopecia ( ), autoimmune cytopenias ( ), arthritis ( ), discoid rash ( ), and raynaud ( ). all had positive auto-antibodies, and of had low c and/or c . for those with sle-like disease, age of presentation was - , and of were female. clinical features included alopecia ( ), autoimmune cytopenias ( ), raynaud( ), and nephritis ( ). all had positive autoantibodies, and of had low complements. lof stat patients with and without clinical features of sle had increased expression of isgs from both pbmcs and neutrophils. increased spontaneous net formation was observed for lof stat patients both with and without sle symptoms. discussion although autoimmunity is not a common finding in lof stat , we have identified sle or sle-like disease in about % of our cohort, with a high incidence of kidney disease, including one patient who required kidney transplant. the interferon signature and net formation were unexpectedly high in both the patients with and without the sle features. ongoing studies include whole exome sequencing for possible second mutations or modifiers, the role of ige in the kidney disease, and further autoantibody detection. the increased ifn signature raises the question about jak-stat modulation for therapy. chief, genetic immunotherapy section/niaid, nih abstract/case report text chronic granulomatous disease (cgd), a rare immunodeficiency with decreased reactive oxygen species (ros) production, increased susceptibility to infection, and increased mortality is caused by mutations in any one of distinct phagocyte oxidase (phox) components of the nadph oxidase, nox . in the past, identification of the specific protein defect was primarily determined by immunoblotting using specific antibodies to the phox proteins. recently, however, we have shown using fluorescenceactivated cell sorting (facs) analysis of neutrophils in whole blood permeabilized and stained with specific anti-p phox antibody that p phox protein expression was absent in p phox cgd patients and significantly reduced in p phox cgd carriers [kuhns et al. . blood adv. ( ): - ]. these findings demonstrated that determination of phox protein expression by facs analysis provide an alternative to immunoblotting and can aid in the identification of p phox cgd patients and carriers. we now have extended these studies to patients and carriers with p phox cgd. facs analysis of p phox expression in permeabilized neutrophils demonstrated that p phox expression was absent in four patients with different mutations in ncf [two patients homozygous for c. e (+ ) g>a, one patient homozygous for c. _ del aag, p.glu del; and one patient compound heterozygous for the mutations, e (+ ) g>a and c. _ del aagaaggac]. moreover, the expression of p phox in nine p phox cgd carriers was significantly reduced > % compared to expression in neutrophils from healthy volunteers. another cytosolic phox protein, p phox, has been shown to associate with p phox in a : molar ratio [tsunawaki et al. . biochem biophys res comm. ( ): - ]. the expression of p phox was reduced in both carriers and patients with mutations in ncf . despite reduced expression of p phox and p phox, neutrophils isolated from carriers of p phox cgd exhibited normal dihydrorhodamine (dhr) oxidation after stimulation with phorbol ester and fell within the normal range for ros production (measured by luminol-enhanced chemiluminescence) after stimulation with either fmlf, opsonized zymosan, or phorbol ester with one notable exception. included in this cohort of p phox carriers was a p phox cgd patient (homozygous for a gt deletion at the start of exon in ncf ) who also carried a heterozygous damaging mutation in ncf [c. a>t; p. asn ile]. normal ros production in the presence of reduced p phox and p phox expression suggest that these proteins are not rate-limiting components for maximum nox activity in neutrophils. finally, determination of the expression of specific phox components by facs analysis of permeabilized neutrophils from whole blood provides a rapid and alternative approach to immunoblotting to determine the specific protein defect in cgd, and, importantly, one that could be easily established in most clinical labs. funded by nci contract no. n d . the original clinical observation that defined patients with hyper-ige syndrome (hies) was the presentation of cold abscesses ("job's syndrome"), which indicated a deficient inflammatory response. mutations in the stat gene have now been identified in most classic autosomal dominant hies patients, but we do not fully understand how these mutations cause the clinical presentation. since the discovery of stat mutations, research on hies focused largely on the adaptive arm of the immune system and suggested that the innate immune defects could be secondary. for example, the discovery that there is a th cell and il- cytokine deficiency in hies provided a possible explanation to the neutrophil chemotaxis defects in hies, as il- is one of the chemokines critical for neutrophil recruitment in vivo. the goal of this study was to investigate myeloid cells from hies patients. first we used c a, fmlp, il- , cxcl , and cxcl to study neutrophil chemotaxis in vitro. responses to c a, fmlp, and il- were equally robust in hies compared to healthy controls, demonstrating that neutrophils from patients are capable of efficient directed migration in vitro. neutrophils from all hies patients responded to cxcl and cxcl significantly below that of the healthy controls. cxcl and cxcl are cxcr -specific chemokines. these results indicated a neutrophil intrinsic cxcr -specific defect. we also found that patient-derived cells express comparable levels of cxcr on the cell surface, suggesting a cxcr chemokine receptor signaling defect. after identifying a neutrophil defect in hies, we wanted to get a broader view of myeloid cells in hies in addition to identifying the cxcr specific defect. stat is a transcriptional regulator, therefore we performed transcriptional profiling of hies and healthy control-derived neutrophils and monocytes. as it was shown before, the expression of stat was not different between patients and controls, since hies is usually caused by the decrease in stat activity not by decrease in expression. we found, however, an increase in stat and stat expression as well as significant changes in the expression of genes regulated by interferons. increased expressions of stat / in both neutrophils and monocytes likely provide and explanation for the increase in interferon regulated genes. multiple genes were identified as potential regulators of cxcr signaling. the balance between the stat and stat signaling has long known to be a regulator of immune cell activation, especially in t cells, but less studied in myeloid cells. stat and stat / signaling pathways crossregulate each other in healthy cells. we propose that in hies the decreased stat signaling leads to not only changes in expression of effector (e.g. inflammatory) genes, but also decreases expression of genes in the regulatory (negative) feed-back loop, which are required for decreasing stat / activity. therefore, the immune cell defects caused by decreased stat activity are compounded by the increase in stat / activity. increase in stat / signaling can cause pathologies in the absence of stat defects, as well as further decrease stat signaling, thus contributing to hies. interfering with stat / signaling in hies may represent a therapeutic opportunity. abstract/case report text rationale: t-cell receptor excision circles (trecs) testing on newborn screening (nbs) has been vital for identifying patients with severe combined immunodeficiency (scid). we aimed to determine whether one or more abnormal trecs result on a nbs might predict higher mortality rates despite the absence of an identifiable underlying etiology. methods: newborns with a positive trecs nbs result without the diagnosis of scid or q . deletion syndrome born from october to december were included (n= ). newborns were divided into three groups: group infants had a subsequent normal repeat screen (n= ); group infants did not undergo repeat screening as the majority expired before a repeat screen could be conducted (n= ); group infants had a normal initial screen but subsequent abnormal screen (n= ). cases were matched : to controls on gestational age, birth weight, nicu status, race, birth quarter, and birth year. nbs records were linked to birth and death certificate records. demographic characteristics were compared and mortality rates were calculated between the groups. results: the mortality rate of group was . %, group was . % and group was . %. when compared with matched controls, there was no difference in the mortality rate of group when compared to the control group. there was a significant difference in the mortality rate between cases and controls in both group (p < . , % ci . , . ) and group (p < . , % ci . , . ). the apgar scores in group infants were comparable to their matched controls. infants in group (p = . ) and group (p = . ) had significantly lower apgar scores than the controls. the majority of the infants in all three groups were less than weeks gestation, however, group had a higher percentage of infants born very premature (less than weeks). there was no significant difference in maternal age, maternal education, prenatal care status, cigarette use, or maternal steroid use between the cases and controls in all three groups. conclusions: infants with an initial abnormal screen who had a subsequent normal repeat screen did not have an increased rate of mortality compared to their matched controls (group ). however, group infants (with unresolved repeat screen) and group infants (with a first abnormal value on a repeat screen) did have increased mortality rates when compared to their controls. overall, an abnormal trecs level on nbs without a confirmed negative repeat screen, was associated with higher mortality in our study population. further studies will be needed to determine if the trecs assay can serve as a predictor for mortality in newborns with an abnormal screen. abstract/case report text introduction: primary immunodeficiency refers to a heterogeneous group of diseases characterized by altered function or composition of the immune system, and are grouped into adaptive or innate system defect. immunoglobulin g subclass immunodeficiencies (iggscs) are classified as a b-cell-related adaptive system disorder and are therefore associated with recurrent sinopulmonary infections with encapsulated bacteria, presenting with pneumonia, recurrent bronchitis, rhinosinusitis, and herpes zoster. its primary mechanisms are still unclear, although the cause for this deficiency might be related to gene deletions, transcription errors, or be an effect of allotype. igg immunodeficiency reaffirms its association with the patient's clinical condition and is often associated with igg deficiency. objective: to evaluate the prevalence of igg immunodeficiency in ferraroni's clinic, classify it by gender, age, igg dosage and other subclasses, correlate it with igg immunodeficiency and the clinical presentations presented by the patients under analysis. method records of patients with igg immunodeficiency whose clinical pictures were followed throughout years were evaluated, patients aged from to years. all tests were done at the same laboratory and all patients have consented to be part of this study, which has been approved by the ethics committee. results twenty-four patients with igg deficiency, , % (n= ) were women and , % (n= ) were male, with average of and years, respectively. the average of igg was . mg/dl, and that of igg was mg/dl. of the patients evaluated, . % had upper airway infections (sinusitis, rhinitis, otitis and tonsillitis), % herpes simplex, . % asthma. less prevalent cases were reported as . % of patients had bronchiectasis, . % candidiasis and . % herpes zoster. . % presented the association of igg and igg deficiency. discussion: the role of specific igg deficiency in the infectious setting is still unknown, but it usually occurs in association with other isotypic deficiencies and sinopulmonary infections. furthermore, the igg subclass is relevant on the study of environmental antigens -suggesting its involvement with allergic disordersand has been described in association with other diseases, such as chronic mucocutaneous candidiasis, ataxia-telangiectasia and allergic colitis. igg deficiency is related to increased susceptibility to bacterial infections. studies show a correlation between igg and igg immunodeficiency that generally imply clinical features characterized by recurrent infections by encapsulated bacteria. the data obtained through the analysis of patients' charts corroborated this information, since it was evident that most of the patients had really similar clinical conditions. conclusion: igg deficiency has a direct correlation with higher prevalence of upper airway infections, such as rhinitis, sinusitis and pneumonia, and with an increased incidence of allergic disorders, here presented by our cohort. additionally, research suggests that hies may cause impaired cd + t cell function. we hypothesized that a low percentage of both th and th cells would be predictive of hies and would differentiate hies from atopic disorders. to evaluate this hypothesis, we examined the percentage of th , th , and ifng+cd + t cells, laboratory parameters, and genetic diagnoses from a large cohort of patients to determine which parameters distinguish patients with stat loss-of-function variants. methods: we conducted a retrospective, multi-institutional chart review of over patients who received a th assay at the medical college of wisconsin clinical immunology research laboratory. the th assay is performed by activating pbmcs with pma/ionomycin/brefeldin a and staining for cd , cd , ifng and il- a. the following parameters were included in the chart review: the percentage of th , th , and cd +ifng+ cells, immunoglobulin levels, atopy scores, infectious history, and genetic diagnoses. results: using logistic regression, we demonstrated that the percentage of th , cd +ifng+, and th cells were positively correlated with age, and percentage of cd +ifng+ cells was higher in females than males. we found that the percentage of th and th cells were decreased in both atopic disease and hies, with hies having the lowest values. interestingly, one subject with a stat gain-of-function (gof) variant had an elevated percentage of th and th . in addition, we determined that ige levels were inversely correlated with the percentage of th , cd +ifng+, and th cells, while iga and igm were positively correlated with the percentage of th cells. several different monogenic defects characterized by increased fungal infections exhibited a low percentage of th including tatton-brown-rahman syndrome and cornelia de lang syndrome. conclusions: we confirmed that the percentage of th cells is low in both hies and atopy in a large cohort of subjects, and that the percentage of th cells may be helpful in distinguishing hies from atopic disease. however, since the percentage of th , cd +ifng+, and th cells correlate with age, caution should be used when testing young children. the inverse correlation between ige levels with th and th responses suggests that similar pathway(s) may drive both hies and atopy. additionally, the decreased th responses in stat lof and increased th responses in stat gof hies raise questions about the role of stat in regulating ifng levels. we also identified patients with different genetic disorders with fungal infections in which the th percentages were low, suggesting that the th test may be useful in evaluating individuals with unusual fungal infections. abstract/case report text background:primary and secondary autoimmuune neutropenia (pan/san) are well described entities. several autoimmune neutropenias do not fit the criteria of either pan or san showing peculiar characteristics mainly for older age at onset and/or for duration of the disease; moreover they are not associated , at least at the beginning, with autoimmune markers/diseases aim of the study: to describe a cohort of subjects affected with autoimmune neutropenia, defined as "atypical" (aan), registered in the italian neutropenia registry (inr) and to compare these data with those from subjects diagnosed with pan still in the inr. patient and methods: subjects with neutropenia and positivity of indirect antibodies against neutrophils (registered in the inr from to ) lasting for more than years, or diagnosed after years of age ( up to y), without any associated autoimmune, signs/markers were considered eligible for the present study. results: data from patients were collected: / subjects ( %) were defined as aan and / ( %) as pan. among aan affected patients %, were "long lasting" aan, while % were defined as "late onset" aan .the degree of neutropenia in aan group was mild in %, moderate % and severe in % of the subjects . leukopenia at onset was a common hall mark seen in % of aan patients (median values /mm³ ; range - /mm³ ) especially in the "late onset" aan if compared with pan and " long lasting" one ( p= . ). as for clinical features, almost half of the aan cohort suffered from recurrent or "significative infections", while severe episodes (namely sepsis, meningitis , osteomyelitis , pneumonia, deep abscess or flemmon) were shown in % being more frequent, but non significantly higher than those reported in the pan group ( % )( p=ns) interestingly, recurrent apthae were significantly more seen in the "late onset" aan group if compared with the "long lasting" aan (p= . ). during follow up, markers and/or symptoms of autoimmunity appeared in % of the aan cohort, being another element of peculiarity in respect to pan (p < . ). as for immunological pattern in aan, immunoglobulin values were lower than the references for age in %,while were above them in % of the cohort . lymphocytes subsets evaluation showed decreased value of cd +cd + cells in % of cases, followed by depletion of cd -cd +cd + subtype in %, cd +cd + in % of cases and cd + cd + in % . preliminary study on b memory and t-reg cells values, showed a quantitative deficiency respectively of in % and % of the studied subjects. mutation analysis performed by ngs in % of the subjects identified pathogenic variants of : taci ( ), tinf ( ) and lrba ( ) .comparison between pan and aan is detailed in table . conclusions atypical neutropenia in childhood is a disorder which show many difference with pan; indeed appears an epiphenomenon of a complex immunological disturbances rather than a disease itself. occasionally mutations of genes of immunodeficiency/disimmunity can be demonstrated abstract/case report text background: medications treating ra typically include systemic corticosteroids used to treat inflammation flares, and disease modifying therapies (dmards). traditional dmards include methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine. recently, biologic/immuneresponse modifiers have come to the forefront for overall therapeutic benefit, however, an unfortunate side-effect may be the risk of increased immunosuppression. this study seeks to determine the occurrence rates of immune deficiencies among patients initiating ra therapies. methods: using the pharmetrics plus commercial claims database from - , ra patients (icd- and - codes: m , m ) over the age of were indexed on their first use of a new biologic therapy. all patients were required to have enrollment six-month pre and one-year post index. cohorts of patients were grouped by medication: methotrexate, adalimumab, etanercept, and rituximab. ra patients receiving adalimumab, etanercept, and rituximab were allowed concomitant use of methotrexate, but could not use any other biologic medications in the post period. a minimal adherence of % was required of all biologic treated ra patients. an additional cohort of ra patients untreated with biologic therapies was indexed on their first ra diagnosis within the time window and used as a control. ra patients with comorbid conditions who would also require biologic treatment were excluded including crohn's disease and ulcerative colitis. between group comparisons were made with the no treatment group as the referent. to account for differences in age, gender, and elixhauser comorbidity conditions patients in each cohort were matched : to the rituximab group. results: , ra patients met inclusion criteria: , in the methotrexate group, , receiving etanercept, , receiving adalimumab, and receiving rituximab. a total of , in the treated groups and , in the no biologic treatment group. demographic information including age and gender were significantly different but numerically similar between the groups, with rituximab group having the highest proportion of female patients but limited dispersion with the lowest proportion being in the etanercept group. healthcare utilization metrics highlighted a significantly higher average number of office visits ( . , sd: . vs no treatment . , sd: . , p < . ) and a higher proportion of rituximab patients being hospitalized ( . % vs no treatment . %, p < . ). the diagnosis of immune deficiency was highest among the rituximab group with . % followed by methotrexate . %, adalimumab . %, etanercept . %, and no treatment . %. after matching, similar rates were seen for healthcare utilization to the pre-match results. the post-match odds of being diagnosed with immune deficiency were significantly greater for the rituximab group (or . , ci: . - . ) than the no treatment group. conclusions: the purpose of dmards is to modulate the immune system and decrease autoimmunity in ra. however, this treatment may lead to significant immunosuppression. this study suggests that treatment with certain biologic/immune-response modifier therapies may be associated with higher rates of healthcare utilization. in particular, the increased post-treatment diagnostic coding of immune deficiency demonstrates the heightened awareness among healthcare providers of the chronic immunosuppressive potential of rituximab. evaluation of potential secondary immunodeficiency pre-and post-dmard use should be incorporated into routine practice. abstract/case report text introduction: autoimmune lymphoproliferative syndrome (alps) is a rare inherited disorder of lymphocyte homeostasis due to a fasmediated apoptosis and characterized by non-infectious and nonmalignant lymphoproliferation, autoimmunity, and secondary malignancies (national institute of health criteria). in spite of recent progress, one third of alps patients still remain gene orphan and they have been previously categorized as alps-u. in some cases, patients fitting alps diagnostic criteria have been shown to carry mutation on genes involved in other immune-dysregulation syndromes. aims: the aim of this study is to compare the clinical and immunological features, and the outcome of a cohort of alps patients with mutations on the typical causing genes (fas, fasl, fadd and casp )-here defined as alps-g -vs the ones without a molecular diagnosis or carrying mutations on other genes (both defined as alps-u). patients and methods the demographic, clinical, biochemical, genetic informations and details about treatment are derived from the alps italian network. search of mutations was performed with sanger pcr and/or next generation sequencing techniques (extended to immunodeficiency genes panel). results: alps patients were registered in our data base; the genetic analysis was performed in subjects ( %): / pts ( %) were alps-g and the remaining ( %) alps-u. six-teen out of ( %) alps-u patients resulted to carry mutations on other genes (lrba, stat +cecr, ctla , baffr, taci, nmlrc , ikbkg, gaucher), and the remaining ( %) were negative. the alps-u subjects showed a more complex phenotype compared to the alps-g group, which was characterized by multi-organ involvement (p= . ) and positivity of autoimmune markers (p= . ). (table ). cytopenia affecting one or more haematopoietic lineages was present in both groups ( % and %) with no significant difference, apart from lymphocytopenia that was more frequent in alps-u group (p= . ) ( table ) . as for lymphocyte subets and immunoglobulin dosage no differences were shown within the two groups. vitamin b and il- were more frequently raised in alps-g group (p= . , p= . ) (table ). four out of ( %) patients did not require any treatment. first-line treatment (steroid or intravenous immunoglobulins) controlled the disease only in / ( %) cases. the response rate to second line therapy -micofenolate mofetile (mmf) or rapamycin-was % and % in alps-g and alsp-u group, respectively. moreover, target therapies or drug combinations were more commonly applied in alps-u subjects (p= . ) ( table ) . conclusions: our study showed that alps-u subjects, despite the alps phenotype, represent distinct clinical entities and that genes associated with other immune-dysregulation syndromes are frequently represented in this group ( / , %). the identification of such disorders is crucial for the management of second-line treatment and/or the administration of target therapies abstract/case report text background: we have shown previously that allergic reactivity to ovalbumin (ova) could be regulated in mice following perturbation of immune networks using combinations of an immune ig along with antiidiotypic ig. we have explored features of this regulation including: its persistence after cessation of administration of combined igs; the ability of heterologous igs to produce immunoregulation; a role for treg induction in regulation; and the ability to attenuate responses in mice presensitized to an allergic stimulus. methods: balb/c mice were sensitized to ova. mice also received weekly injections of immune ig or anti-idiotype ig (at separate sites) from either homologous (mouse) or heterologous (human) sources. in the latter case pooled ivig (given im, hence hereafter imig) was used as a source of anti-idiotype ig, and human anti-tet as immune ig. injections of the ig were given from the time of ova sensitization (to attenuate development of immunity), or after pre-sensitization of mice (to attenuate existing allergic responses). all mice were assayed for development of ova-specific serum ige and igg, as well as the production of ova-induced il- , il- , il- , il- and il- in splenocytes cultured for hrs. in studies examining possible mechanism(s) responsible for inhibition of immunity mice received, in addition to the ig treatments described, infusion of depleting anti-cd , and/or anti-cd antibodies, or a mab to tnfsfr , known to expand tregs implicated in regulation of allo immunity. results: combinations of both heterologous and homologous immune igs and anti-idiotype igs attenuated ova allergic responses in both naïve and pre-sensitized mice. this attenuation persisted in mice greater than weeks after cessation of treatment with the igs used. finally, depletion of either cd or cd cells ameliorated the suppressive effect seen, while the combination of anti-cd and anti-cd essentially abolished suppression. suppression was further enhanced by anti-tnfsfr mab. conclusions: we conclude that the combine ig treatment protocols used produced a long-lasting suppression of allergic immunity, even in pre-sensitized animals. the effects seem to depend upon induction and expansion of tregs and represents a novel approach to treatment of allergic disease in humans and other animals. abstract/case report text background wiskott-aldrich syndrome protein (wasp) is found in the cytoplasm of hematopoietic cells but can transit to t lymphocyte nuclei at distinct developmental timepoints. wasp deficiency is a rare, x-linked combined immunodeficiency disease. affected patients display qualitative but not quantitative t cell defects. we report two immune deficient subjects with nearly identical exon frameshift mutations in was, the gene encoding wasp. one subject lacked circulating t cells, the other possessed several distinct cd t cell populations each expressing quantitatively different amounts of wasp. objective to determine how similar was mutations can cause scid in one person and generate b and t cells with heterogenous wasp expression in another. methods to identify somatic was mutations, we deeply sequenced was exons, introns, promoters and ' untranslated regions at , read depth in genomic dna from various b and t cell populations of each subject and their unaffected relatives. we confirmed genomic variants were transcribed and translated by sequencing was transcripts and analyzing wasp in primary cell lysates, both fractionated and not. to model our subjects' diseases we transfected primary cells and cell lines with mutant was transcripts and then measured viability and nuclear localization via confocal microscopy. results deep sequencing of genomic dna revealed all of subject one's cells carried the same germline exon frameshift was mutation. the mutation was incorporated into subject one's was transcripts and translated into a truncated form of wasp, which was relegated primarily to the cell nucleus. subject two possessed three distinct cd t cell subsets that each carried either the germline exon frameshift was mutation or a variety of somatic mutations that circumvented frameshift wasp expression. evasion strategies included exon skipping, adoption of a cryptic exon splice site and reversion to wild type amino acid sequence. subject two incorporated somatic mutations into was transcripts which encoded either stable near full-length proteins or unstable non full-length ones. subject one's sister and subject two's mother, who both carried the germline exon frameshift mutation, produced only wild type transcripts and proteins. conclusion we report two patients with was mutations encoding truncated wasp. if expressed, truncated wasp localized to the cell nucleus, and this was associated with t cell developmental arrest and severe combined immune deficiency. if, through a variety of epigenetic and somatic strategies, t cells could avoid expression of truncated wasp, they would survive but display phenotypical abnormalities and functional defects. patients with pidds show a higher susceptibility to hematopoietic malignancies, in particular to non-hodgkin lymphomas (nhl) that, generally, account for approximately - % of paediatric cancers and their incidence increases with age. recently new gene defects responsible for pidds with lymphoproliferation as a key clinical sign have been identified. our goal is to investigate possible immune-mediated mechanisms underlying malignant lymphoproliferation in children who did not show other typical symptoms of pidds. we retrospectively selected and reviewed the clinical history of nine patients with nhl ( burkitt lymphoma, large b cell lymphoma and lymphoblastic tcell lymphoma). immunophenotyping and exome analysis of known pidds genes were performed after lymphoma remission. six out of nine patients showed a mild hypogammaglobulinemia at time of presentation, not noticed before. moreover, one patient had history of recurrent respiratory infections, one of hematologic autoimmunity and two of nine were ebv-positive at diagnosis. preliminary results show an aberrant b cell phenotype in four patients; exome analysis reveals a novel heterozygous genetic variation in ikzf gene in one patient with burkitt lymphoma and autoimmune cytopenia was identified. concerning the remaining patients, further studies are ongoing. a detailed review of clinical history of paediatric patients affected from nhl as well as an impaired immunophenotyping can be important indicators of immune-mediated disorder underlying lymphoproliferation and helpful signs of possible pidds that should promptly be investigated by genetic analysis. this will allow an appropriate diagnosis and disease management. abstract/case report text introduction: caspase activation and recruitment domain (card ) encodes a scaffold protein that links antigen receptor activation to intracellular signaling. dominant heterozygous loss of function (lof) mutations in card cause a syndrome of severe atopic dermatitis, elevated ige, and allergic disease. atopic dermatitis can be difficult to control leading to substantial morbidity. dupilumab is a humanized monoclonal antibody that blocks il- and il- signaling approved for treatment of refractory atopic dermatitis. we present a case of a -year-old female with card deficiency successfully treated with dupilumab. case: a -year-old puerto rican female with history of recurrent sinopulmonary infections with episodes of pneumonia, moderate persistent asthma, food allergies, recurrent skin boils, and severe atopic dermatitis was referred for further management and evaluation for autosomal dominant hyper-ige syndrome (ad-hies). her atopic dermatitis was refractory to conventional therapy with topical corticosteroids, twicedaily emollient use, and bleach baths; it was also refractory to immunosuppression with mycophenolate mofetil and cyclosporine. on exam the patient exhibited coarse facial features and a high palate. she had eczematous lesions on the face, trunk, and extremities (scorad ). laboratory evaluation showed: eosinophilia ( cells/ul), elevated ige (> ku/l), low igm ( mg/dl), and elevated iga ( mg/dl). lymphocyte subsets and mitogen response were normal but antigeninduced proliferation was abnormal. autosomal dominant hyper ige score was indicating a high likelihood of ad-hies. no mutations in stat were identified and th cell expression was elevated. dedicator of cytokinesis (dock ) deficiency was also considered but dock protein expression was normal. further genetic testing revealed an base pair deletion in card (c. _ del) predicted to be pathogenic. the combination of the patient's phenotype and large deletion was consistent with card deficiency. despite continued immunosuppression with cyclosporine and aggressive skin care, the patient's atopic dermatitis was still severe and poorly controlled. off label (patient < years) treatment with subcutaneous dupilumab mg every weeks was initiated. at last follow-up, months after dupilumab start, the patient had substantial improvement in dermatitis with clear skin on the face, trunk, and extremities (scorad ). cyclosporine was discontinued and topical medications were applied less frequently. discussion: hypomorphic heterozygous dominant negative loss of function mutations in card have recently been associated with severe atopic dermatitis and allergic disease. treatment of atopic dermatitis in card deficiency remains challenging, but dupilumab appears to be an effective alternative to refractory disease. longer follow-up and a larger cohort of card -lof patients treated with dupilumab are necessary to understand the long-term efficacy and safety for use of dupilumab in these patients. abstract/case report text purpose: the micromilieu within premalignant respiratory papillomas supports persistent hpv / infection and disease recurrence in recurrent respiratory papillomatosis (rrp). these patients show polarized (th -/treg) adaptive immunity in papillomas and blood, enriched immature langerhans cell (ilc) numbers, and overexpressed cox /pge in the upper airway. to better understand the adaptive and innate dysregulation in rrp, we studied blood-derived monocytes, ilcs, and tissue-derived ilcs from rrp patients and controls. experimental design: monocyte subpopulations were isolated, differentiated into ilcs, activated, and then assessed by flow cytometry. monocytes were induced to differentiate into ilcs with/ without added pge , and then activated by il- γ, pge , pge +il γ, or lps. ilc cd expression was identified by flow cytometry. monocyte-derived ilcs, papilloma, foreskin, and abdomen skin ilcs, were also analyzed by qpcr for select chemokine/cytokine mrna expression after isolation, hrs later in culture, and again after poly(i:c) or tnfα stimulation. results: the three monocyte sub-populations differed between patients and controls, and patients' monocytes generated fewer ilcs. classical monocytes generated most, but not all ilcs. pge levels were higher in rrp plasma, and added pge reduced control, but not patients' monocyte-ilc differentiation. pge had no effect on ilc maturation identified by cd expression. papilloma-derived ilcs expressed low ccl- , and high ccl- mrna and were unresponsive to poly(i:c) or tnfα. tissuespecific cytokine/chemokine responses between ilcs from papillomas, foreskin and abdominal skin differed. only papilloma ilcs expressed il- γ after isolation, and they up-regulated ccl mrna hrs later without further stimulation. conclusions: monocyte/ilc innate immunity is impaired in rrp, in part due to increased pge exposure. the immunosuppressive papilloma micromilieu likely alters ilc responses that skew, hpv / -specific th /treg adaptive immunity in rrp. abstract/case report text introduction: familial mediterranean fever is a hereditary auto inflammatory disorder that typically manifests with recurrent fevers, abdominal pain and in some patients there is an associated with amyloidosis leading to eventual renal failure. while there are several common mutations in the mefv gene that when homozygous give these classic symptoms, patients with atypical mutations or heterozygous mutations often have a different clinical course. we present identical twin siblings with compound heterozygous mefv mutations but differing clinical phenotypes. case description: the index patient is a year old girl, conceived via ivf, who began having fevers at age . . her fevers occurred every weeks for months before she was referred to immunology for evaluation. her parents describe her as happy and otherwise not ill appearing during these episodes. genetic testing for familial mediterranean fever revealed compound heterozygous e q and p s mutations in the mefv genes. initiation of colchicine therapy in the affected sibling has resulted in a complete resolution of her symptoms. a trial off colchicine resulted in return of cyclic fevers. her identical twin sister was also tested, and carries the same mutation, but is still asymptomatic. this created great concern amongst their parents who had genetic testing prior to undergoing ivf that revealed no parental mutations in mefv. in consultation with genetics the mother was tested again through the same laboratory that had performed testing on the children. this revealed an identical mutation in mom who is also asymptomatic. conclusions: although classic homozygous mefv mutations have resulted in well described fever syndromes, there is considerably less data on heterozygous and compound heterozygous mefv mutations. in these two identical siblings only one patient has a classic manifestation of familial mediterranean fever. while it is possible that the other twin will develop similar symptoms later on in life, it is also possible that another factor is necessary to trigger symptoms in this unusual genetic presentation of fmf. in addition this case highlights the importance of understanding the testing method used by the laboratory performing the genetic testing. while the mother was initially reported as negative the laboratory that performed her testing only tested for the most common mefv mutations. more complete testing, that included the entire gene sequence, revealed that she did contain an mefv mutation in e q, which although more rare is thought to be pathologic when combined when combined with a second mutation. abstract/case report text there are several lines of evidence that link the pi k/akt/mtor signaling pathway to primary immunodeficiencies. hyperactivation of the pi k/akt/mtor/s k signaling pathway in immune cells can be the consequence of dominant gain-of-function mutations in the genes encoding for pi kδ that cause the activated pi kδ syndrome (apds). patients with these mutations may develop immunodeficiency and immune dysregulation as well as neurodevelopmental delay and growth retardation. in addition, mutations of genes within the pi k-akt-mtor pathway were also known to cause megalencephaly and segmental cortical dysplasia. mutations in akt , a member of the akt family of proteins and a downstream effector of pi k-mediated signaling, was shown to be associated with autosomal dominant megalencephalyassociated syndromes. here we describe a year old girl, born to consanguineous healthy parents, who presented with megalencephaly, developmental delay, hypotonia, cervical lymphadenopathy and hepatosplenomegaly. the patient had recurrent hospital and icu admissions for idiopathic thrombocytopenia (treated with ivig), recurrent laryngitis, recurrent peritonsillar abscess, preorbital cellulitis, conjunctivitis with purulent discharge, otitis media, pneumonia with pleural effusion (required drainage), metapneumovirus pneumonia with respiratory failure, recurrent skin cellulitis, and abscesses that grew mrsa (required drainage). in addition, the patient is known to have asthma and allergic rhinitis. mri of the brain showed megalencephaly, ventriculomegally, thin and dysplastic corpus callosum, a normal cerebellum, and myelination appropriate for age. immunoglobulin levels, lymphocyte subsets and the oxidative burst test were all within normal limits. cmvand ebv were not detected. bacterial cultures grew mrsa (skin), strept. pneumoniae, h. influenzae, and e. coli (urine). extensive metabolic workup was done, which was inconclusive (metabolic/mitochondrial diseases). whole exome sequencing identified an akt variant c. g>a; p.(asp asn) in exon . the variant was identified in the patient but not in the parents and it was confirmed by sanger sequencing. further molecular testing concluded that the variant is caused by a de novo mutation during early development. although pathogenic variants in akt gene were shown to be associated with megaloencephaly-associated syndromes, no associations with immune deficiency have be reported. functional studies will be pursued to confirm the link between the clinical phenotype and the identified variant in the akt gene. complex is a critical signalling adaptor that regulates lymphocyte activation, proliferation, survival, and metabolism. primary immunodeficiencies affecting each component (termed 'cbm-opathies') result in broad clinical manifestations ranging from combined immunodeficiency (cid) to atopic disease or lymphoproliferation. we present the laboratory and clinical findings of two canadian first nations patients found to be homozygous for the same novel card mutation (c. c>t; p.r *) causing complete card deficiency. results: we recently identified an -month-old boy who presented with a severe case of entero/rhinovirus bronchiolitis with interstitial lung disease and a -year-old boy with a history of severe pulmonary infections with bronchiectasis (including pjp), chronic sinusitis, candidiasis, invasive bacteremia, and severe ileo-colitis and oral ulceration requiring total colectomy. testing of both patients demonstrated absent tregs, elevated naïve b cells with absent memory b cells, and panhypogammaglobulinemia. next generation sequencing revealed that both patients were homozygous for the same novel variant of card (c. c>t; p.r *), which rendered card protein undetectable by immunoblot. card deficiency was confirmed by stimulating patient b cells with phorbol -myristate acetate (pma) and ionomycin and immunoblotting for signalling proteins in both the nf-κb (ikkα/β, iκbα, p ) and mapk (mek / , mkk , jnk / , erk / ) pathways as well as cleavage substrates of the malt paracaspase (relb, cyld, bcl , hoil ). nf-κb and jnk activation were completely absent and m a lt p a r a c a p a s e a c t i v i t y w a s l o s t . f u r t h e r m o r e , c oimmunoprecipitation experiments revealed that card was required for optimal malt association with bcl in response to stimulation. to define the impact of card deficiency on the b cell transcriptome, rna-seq experiments were performed. this revealed an inability to upregulate critical genes involved in immunity and tolerance (e.g. cd lg, ctla , il , il ), decreased enrichment in cytokine pathways (e.g. ifn-α, il- , tgf-β), and decreased enrichment in malt -dependent genes. furthermore, rna-seq confirmed the developmental block observed in patient b cells and suggested that b cells were halted at the centroblast to centrocyte transition. both patients ultimately underwent hematopoietic stem cell transplantation (hsct), which restored lymphocyte signalling and activation as measured by nf-κb, jnk, and malt paracaspase substrate cleavage. conclusions: we have presented the most comprehensive clinical and molecular characterization of human card deficiency to date. these two cases highlight the crucial role of card in regulating b cell development, function, and humoral responses, as confirmed by signalling and transcriptomic analyses. furthermore, hsct is potentially helpful for these patients as assays performed on post-transplant cells demonstrated restored signalling and activation. abstract/case report text introduction: primary immune deficiencies (pid) can have a significant impact on the quality of life of patients and their families. as more patients with pid are surviving to adulthood, the need to monitor them closely and ensure they are transitioned appropriately is even more crucial. we compared the perspectives of pediatric and adult immunologists toward the transition of patients with pid at our institution. methods: pediatric allergy/immunology providers at lurie children's hospital and adult allergy/immunology physicians at northwestern university both in chicago, il completed respective surveys anonymously (www.surveymonkey.com). questions were derived from the validated 'attitude' and 'quartt' instruments for transition. respondents were asked to rate their level of agreement on a -point likert scale, ranging from strongly disagree to strongly agree. results: overall, pediatric and adult providers participated (response rate . %). of total respondents, % thought the transition process should be initiated at age - . about % of the adult immunologists selected and older, whereas pediatric providers would begin earlier; . % of pediatric providers note they would initiate transition at age - . both pediatric and adult immunologists agreed that patients should be transferred when the provider felt they were ready ( %) and when they were in stable condition ( %). both adult and pediatric immunologists selected transfer of complete medical file as a preferred communication method for transition. other strategies preferred by adult providers were a referral letter with brief summary of medical history ( . %) and staff meeting with pediatric and adult immunologists ( . %), whereas pediatric providers would prefer a joint outpatient dedicated transition clinic ( . %). pediatric and adult immunologists, patient, parent, and transition liaison were considered the most important active participants in the transition process. the most prominent barriers to a formal transition were unavailability of a transition coordinator or nurse specialists ( %) or of all disciplines of the interdisciplinary team ( %), and limited time ( %). on the other hand, limited demand (too few patients) was strongly rejected as a barrier. all participants agreed during transition patients should be educated about medications and their side effects, their condition and related potential future complications, and symptoms that require seeking health care. over % of participants also agreed that education about how to set up ivig and further insurance needs were important. all participants agreed that the transition process should include assistance on how to promote the patients' independence and self-management skills, medication management/adherence, and understanding of immunoglobulin replacement and side effects. other important transition components included knowing how frequently lab draws are required for monitoring ( %) and having a written individualized transition plan ( %). pediatric providers also thought having an email or telephone help line would be beneficial. conclusion: this study adds to the growing body of literature examining attitudes of immunologists toward transition, and it highlights important transition components and barriers. further work is ongoing to determine the transition needs identified by patients and parents and define markers for successful transfer in order to build a transition policy at our institution specific to immunodeficiency patients. abstract/case report text introduction: bronchiectasis (bq) is an abnormal and irreversible dilatation of bronchi secondary to repeated cycles of airway infection and inflammation. predominantly antibody deficiency is the main group of primary immunodeficiencies (pid) in adults and had been reported up % of subjects with non-cystic fibrosis bronchiectasis (ncfb). hypergammaglobulinemia (igg level higher than , mg/dl) had been observed in . % of ncfb cases (retrospective data). diagnostic delay and inappropriate management of patients with predominantly antibody deficiency can lead to irreversible lung damage or even death from serious infections. the effect of hypergammaglobulinemia on ncfb is unknown. here we present the frequency of immunoglobulin abnormalities (pad and hyperigg) in adults with ncfb in cali, colombia. methods: we present preliminary data of a descriptive prospective study that will include patients with ncfb. women and men > and < years old will be included. all volunteers will be evaluated by a clinical immunologist, complete blood count and serum igg, iga, igm and ige levels will be determined. according with clinical suspicious, igg subclasses, anti-pneumococcal igg response and b cell subpopulations will be performed. the project will be executed in months. written informed consent has been obtained for all subjects included. this project count with irb approvals at universidad del valle and hospital universitario del valle. results: a total of ncfb cases have been included in the study. the mean age was . years ( - years) with a female:male ratio : . moderate-severe dyspnea was observed in / cases (medical research council -mrcdyspnea scale to ). recurrent pneumonia was found in / cases ( %). the main etiologies of bronchiectasis were: post-infection / ( %); idiopathic / ( %); autoimmunity / ( %); primary immunodeficiency / ( %) asthma / ( %); copd / ( . %); primary ciliary diskinesia / ( %); reflux / ( %) and others. primary immunodeficiencies, . % of ncfb cases, were classified as: predominantly antibody deficiency ( cases) including cvid cases, igm deficiency cases, igg subclasses deficiency case, selective iga deficiency case and hypogamaglobulinemia case. combined immunodeficiency (dock deficiency) case. interestingly igg hypergammaglobulinemia was observed in / cases ( . %) suggesting humoral immune response deregulation. conclusion to the best of our knowledge this is the first prospective study evaluating the etiology of non-cystic fibrosis bronchiectasis (ncfb) in colombia. hypergammaglobulinemia and predominantly antibody deficiencies affect % of adults with ncfb in colombia. our study reinforced the necessity to evaluate humoral immune response in patients with bronchiectasis. conflict of interest: authors disclosure any potential financial conflict of interest related to this abstract. acknowledgements: this investigator-initiated research was supported with a grant from baxalta us inc, a member of the takeda group of companies bt - /iir-col-bxlt- . abstract/case report text background: early-onset inflammatory bowel disease (eoibd) is defined as ibd diagnosis in children less than years of age. the occurrence of autoimmune disease in children (where it is relatively rare, compared to adults) may be caused by a highrisk predisposition gene (monogenic disorders). mayo clinic children's center has a unique care model, where a patient who is referred for eoibd meets with a team of physicians, including gastroenterology, immunology, genetics, and nutrition. we describe our experience of our eoibd clinic from an immunologic perspective. methods: we conducted a retrospective cohort study through emr chart review of pediatric patients who were referred to our eoibd program ( - ). first diagnosis of ibd under the age of was the inclusion criteria. we assessed the presentation, clinical correlates, and immunologic evaluation. approval was obtained from mayo's institutional review board. data abstraction and analysis was done using the software jmp. results: pediatric patients met the inclusion criteria, with ( %) males and ( %) females. the median age of ibd diagnosis was years ( - years range). median values and the distribution of variables used in the nutritional and immune evaluation were assessed (fig ) . nutritional assessment was remarkable for low to low normal hemoglobin and ferritin levels. vitamin d and albumin levels were overall within the normal range. growth parameters indicated that the median bmi percentile was ( - ). with immune and genetic screening, one patient was found to have x linked chronic granulomatous disease (cgd). immune evaluation of other patients was overall within normal limits. fecal calprotectin served a reliable non-invasive biomarker for inflammation with the median being . ( . - . ). of these patients underwent gi pathogen panel testing of which ( %) tested negative, four ( %) tested positive for c. diff, and two ( %) others to shiga toxin-producing e. coli. it was also noted during the chart review that most patients had poor disease control despite undergoing treatment with various anti-inflammatory and immunosuppressive drugs. the patient diagnosed with cgd underwent bone marrow transplantation. a higher proportion of patients referred to our program in recent years underwent a more comprehensive multispecialty evaluation. conclusion: awareness of monogenic causes of inflammatory disorders in children has increased in recent years. it is also important to rule out intestinal infections that can act as ibd mimic. identifying monogenic disorders and other ibd mimics helps with targeted therapy and symptom improvement in these patients who have a difficult-to-treat disease. the group of children with eoibd, regardless of whether there is an inborn error of immunity, suffers from very high morbidity and a high burden of disease. comprehensive immune-nutrition assessment of eoibd patients paves way for further in-depth immunogenic assessments and allows for global management. abstract/case report text background: chronic granulomatous disease (cgd) is a primary immunodeficiency (pid) affecting the nadph oxidase system in phagocytes resulting in increased susceptibility to catalase-positive organisms. holland presented the first report of dual impact of cgd and hiv in a patient with disseminated nocardiosis. because their cgd patient admitted to history of iv drug use, he was frequently screened for hiv. case: a -year-old african american male with known cgd tested positive for hiv by western blot in the ed in when he presented with complaints of intermittent fever and cervical lymphadenopathy. his cgd was diagnosed by nbt blood testing at years of age. he had frequent skin infections and fever prior to diagnosis. clinically, he did so well that his cgd diagnosis was questioned by his immunologists. however, cgd was confirmed by additional abnormal nbt tests and, ultimately, dhr flow cytometry testing. during his second infectious disease consultation for hiv, at age , he disclosed that he was bisexual. previously, the patient was screened for hiv and hiv antibodies in due to anal fissure. he was screened again in for marked cervical and supraclavicular lymphadenopathy. his cd + t cell absolute count was noted to be low ( /mm ) in at age . until , his prior hiv screenings were negative. during his cgd treatment course as an adult, he was known to be variably adherent with administration of interferon gamma due to adverse effects, particularly pain at the site of injection and malaise. at the time of his positive hiv western blot in , his cd + t cell count was / mm . after starting hiv antiretroviral treatment, his viral load became undetectable. at age , he had burkholderia cepacia pyelonephritis resulting in left nephrectomy. sepsis from b. cepacia was fatal (positive blood cultures without known primary source) in at age . his recent viral load was still undetectable and cd + count was /mm . summary: our case reveals the complexities of treating a patient with both primary and acquired immune deficiencies. it illustrates the importance of taking a thorough social and sexual history starting in adolescence, including those patients with pid. patients with pid should be followed closely by a primary care physician, in addition to an allergist-immunologist and infectious diseases specialist, to ensure age appropriate medical and developmental screening. the recognition of pids is improving due to better screening, awareness, and treatment. currently, the rate of hiv infection is highest among young homosexual african american males. it remains important to understand the epidemiology of primary and acquired immunodeficiencies to best identify those at highest risk. ( ) submission id# phosphatase and tensin homolog (pten) hamartoma tumor syndrome identified by newborn tcell receptor excision circle screening for severe combined immunodeficiency δ) subunits that are critical for cellular signaling. heterozygous gain-offunction (gof) mutations in pik cd (encoding p δ) result in activated pi k δ syndrome (apds ), while heterozygous loss-of-function (lof) mutations in pik r (encoding p α) result in activated pi k δ syndrome (apds ). given its role as a negative regulator of the pi k signaling pathway, heterozygous lof mutations in pten (encoding phosphatase and tensin homolog, pten) result in a clinical phenotype that approximates that of apds /apds and is therefore referred to as activated pi k δ syndrome-like (apds-l). however, sequelae of heterozygous pten lof mutations extend beyond the immune system and include a group of disorders collectively known as pten hamartoma tumor syndrome (phts). although severe t cell lymphopenia at birth would be unexpected in apds , apds , or apds-l, below normal t cell receptor excision circle (trec) counts have been reported in apds , but only in individuals outside of the neonatal period. herein, we describe an infant girl with a low trec count at birth who was found to have phts. case description: a -day-old girl, born at a gestational age of weeks, was found to have a low trec count of /microliter (normal => ). a second trec count obtained at weeks of age resulted as /microliter. arguing against a diagnosis of severe combined immunodeficiency (scid), flow cytometric analyses performed at weeks of age revealed only a modestly diminished cd + t cell count ( /microliter; cd + and cd +) with a normal percentage of naïve and memory cd + t cells ( % and %, respectively). by months of age, her cd + t cell count dropped to /microliter, which was accompanied by a significantly decreased percentage of naïve cd + t cells ( %). sequencing and deletion/duplication analysis was pursued via a commercially available -gene panel aimed at genetically defined primary immunodeficiency (pid), in which no clearly pathogenic mutations were identified. over the following months, the patient was noted to have macrocephaly, tall stature ( th percentile), axial hypotonia, and gross motor delays. sequencing and deletion/duplication analysis was then pursued via a commercially available -gene panel aimed at genetically defined macrocephaly and overgrowth syndromes, in which a hemizygous pathogenic mutation in pten (c. a>g, p.gln arg) was identified. subsequent flow cytometric analyses demonstrated findings characteristic of apds-l, including expanded transitional and cd lo b cells, decreased isotype switched memory b cells, increased effector memory t cells, a lowered threshold for intracellular calcium mobilization upon b cell receptor engagement, and increased basal akt (protein kinase b) and s (ribosomal protein s ) signaling. discussion: we report the first case of phts identified by newborn trec screening for scid. as pten is not included in most commercially available, scid-or pid-tailored gene panels, phts would be missed by conventional genetic testing. therefore, analysis for variants in pten should be considered in neonates with low trec counts, macrocephaly, developmental delay, and other suggestive sequelae. abstract/case report text primary (or familial) hemophagocytic lymphohistiocytosis (hlh) is a rare, life-threatening hyper-inflammatory syndrome affecting mainly young children. it is caused by mutations in genes involved in the granule-dependent cytotoxic pathway, inducing extreme inflammation and massive tissue infiltration by activated t cells and macrophages. standard chemotherapy-based treatment regimens are toxic and induce remission in only % of patients. to this day, hsct is the only available curative treatment, but the inability to efficiently control the inflammation in many patients prior to transplantation often leads to graft failure, with transplant-related mortality around %. thus, the development of new, more potent and less toxic anti-inflammatory regimens would be a major advancement in the treatment of hlh. here, we hypothesize that combination therapies targeting several jak-dependent cytokines will be more effective than monotherapy to reduce the life-threatening symptoms induced by this pathology. using a perforin-deficient (pko) mouse model, we first tested the effects of blocking antibodies against ifnγ, the dominant cytokine secreted during hlh, in combination with antibodies targeting other highly elevated cytokines, such as il- and il- , on the manifestations of the disease. we found that anti-il- r and anti-il- antibodies, when used in combination with anti-ifnγ antibodies, did not significantly improve the symptoms of hlh compare to anti-ifnγ antibodies alone. further, we found that targeting the jak-stat signaling pathway with ruxolitinib, a specific inhibitor of jak and jak , molecules downstream of ifnγ and il- , but not il- signaling, was as beneficial as anti-ifnγ monotherapy. next, we tested the efficacy of ruxolitinib in combination with anti-il- antibody, as this later cytokine is not jak-dependent and was shown to drive macrophage activation syndrome in other contexts. unfortunately, this combination did not result in better symptom resolution than the use of ruxolitinib only. in contrast, combination therapy using ruxolitinib and anti-ifnγ antibodies showed a striking synergistic effect on the resolution of most disease manifestations, to such an extent that our pko mice presented a clinical phenotype indistinguishable than that of a c bl control mice. our findings demonstrate that jak-dependent cytokines are the main cytokines driving the progression of hlh in pko mice. collectively, our results suggest that anti-ifnγ antibodies and ruxolitinib, although effective independently, should be used in combination to more efficiently suppress hlh progression. these results are particularly relevant since the emapalumab, an anti-ifnγ monoclonal antibody was recently approved by the fda for the treatment of hlh while ruxolitinib will soon be in clinical trials for this indication. this project was supported by funds from the fondation de cancérologie charles bruneau and the canadian institutes of health research (mop- ). abstract/case report text introduction: transcription factor (tcf ), also known as transcription factor e -alpha (e a), is a helix-loop-helix transcription factor which plays a critical role in lymphopoiesis. tcf is required for b and t lymphocyte development. defects in tcf have been associated with agammaglobulinemia , autosomal dominant, characterized by low levels of immunoglobulin and early onset recurrent bacterial infections. deletion or diminished activity of tcf may also play a role in lymphoid malignancies. runs of homozygosity (roh) are contiguous stretches of homozygous genotypes at consecutive polymorphic dna marker positions. roh are important reservoirs of homozygous deleterious variation. the homozygosity heterogeneous hmm (h m ) algorithm was specifically developed for analyzing whole exome sequencing (wes) data. the branch point sequence (bps) is an essential splicing signal located - bases upstream of splice acceptor sites. while bps variants are rare, they may result in aberrant pre-mrna splicing and genetic disorders. these variants may be overlooked by standard wes analysis methods because they are intronic and the mammalian bps is a degenerate motif. objective: describe the method used to identify a bps variant in consanguineous brothers with immunodeficiency, including early onset recurrent infections and b-all, hypogammaglobulinemia, t and nk lymphocytosis, low b cells, and low naïve t cells. methods: wes was performed for all family members. data were analyzed using standard read mapping, variant calling and annotation methods. roh were analyzed using the h m algorithm (magi et al. ). roh from the siblings was intersected (bedtools) and the output was submitted to the genomic oligoarray and snp array evaluation tool (v . ). variants were confirmed by sanger sequencing. results: no candidate disease variants were detected in the coding regions, ' or ' splice sites, or utrs for both brothers; however, analysis of intersected roh revealed a homozygous tcf intronic variant within a putative bps (tcf c. - a>t). sanger sequencing of the mutant cdna revealed activation of a cryptic splice site. heritability for routine childhood vaccines has been shown to range from - %. the genetic component of vaccine response suggests we should be able to predict vaccine response in infants with biomarkers. methods: multi-center study of infants born vaginally at full term and followed through months of life. cord blood was collected at birth & peripheral blood was collected at and months of life. six-month collection was weeks post administration of routine vaccinations, while -month collection was immediately prior to receiving the -month booster vaccines. b cell subsets were analyzed with flow cytometry. vaccine titers and cytokines were measured via multi-plex elisa. study was irb approved. results: our data confirmed the immaturity of the newborn humoral immune system with a lower overall b-cell abundance, a predominance of naïve b cells, an inability to class-switch and produce igg or iga, and a th bias. maturation was observed over the first year with increasing overall b-cell abundance, frequency of memory b-cells producing iga, igg, and igm, and frequency of plasmablasts. scd levels also increased throughout the first year due to microbial translocation reflecting establishment of the microbiome. conversely, cord blood contained high levels of baff, april, scd l, il- , and il- , with levels decreasing thereafter. all infants displayed evidence of humoral immune system activation after getting -month vaccines. total plasmablast levels peaked weeks after receipt of month immunizations. a decrease in total plasmablasts was evident between and months, although levels remained above those at birth, corresponding with the need for -month booster vaccinations to maintain long-lasting immunity. il- and ifn-gamma had a significant positive correlation with memory b cells and plasmablasts at subsequent time points suggesting that these cytokines play a role in b cell differentiation and vaccine response. baff and april cytokines were elevated at birth, consistent with germinal center formation and underwent a compensatory decrease thereafter. april & scd levels in cord blood significantly correlated with higher tetanus titers at months suggesting that vaccine response may be predicted by cytokine biomarkers at birth. response to vaccines was also dynamic with il- levels being significantly correlated with tetanus titers at weeks after receipt of month immunizations. conversely, scd l levels did not correspond to b cell development consistent with a known b cell hyporesponsiveness to cd l in infants. conclusion: humoral immune development is both predictable and dynamic. biomarkers in the cord blood, produced by the infant, are predictors of b cell development and vaccine response in infancy. background: adult-onset immunodeficiency with anti-ifnɣ autoantibodies is a newly described immunodeficiency syndrome characterized by disseminated nontuberculous mycobacterial and other opportunistic infections in previously healthy middle-aged individuals typically from southeast asia. it is caused by the presence of autoantibodies directed against the cytokine ifnɣ, which is required for intracellular pathogen killing by macrophages as well as phosphorylation of the transcription factor stat , which is involved in cell survival gene expression. successful treatment of the immunodeficiency has been described in prior case reports with immunomodulatory therapies, including rituximab. however, there are no standard recommendations for dosing or timing of these agents, or recommendations for longterm monitoring of disease activity. case presentation: a -year-old laotian woman with a history of type diabetes and possible prior hepatitis c infection presented to the immunology clinic for evaluation of immunodeficiency. in the two years prior to presentation, the patient was diagnosed with mycobacterium avium infection involving the parotid gland and lymph nodes of the neck, mycobacterium avium complex bacteremia, histoplasma capsulatum involving the lymph nodes of the neck, and leukocytoclastic vasculitis of the lower extremities. as a child and young adult, she had no severe or recurrent illnesses and did not suffer from any chronic disease. preliminary immunologic testing demonstrated normal t, b, and nk cell subsets, elevated immunoglobulin g, a, and m levels, and protective titers to tetanus, diphtheria, and / pneumococcal serotypes. measurement of anti-ifnɣ autoantibodies was positive, which led to the diagnosis of adult-onset immunodeficiency with anti-ifnɣ autoantibodies. the patient was treated with four doses of monthly rituximab with resolution of the anti-ifnɣ autoantibodies, restoration of normal stat phosphorylation, and depletion of cd -positive b cells. after a -year period of being lost to follow-up, during which she continued to receive rituximab every months at the direction of a local provider, the patient re-presented to the immunology clinic to re-establish care. at that time, the patient had no evidence of anti-ifnɣ autoantibodies based on titers and normal stat phosphorylation. cd -positive b cells remained depleted. the patient also confirmed subjective clinical improvement and denied any interim infectious complications. conclusion: this case provides an example of successful treatment of a patient with adult-onset immunodeficiency with anti-ifnɣ autoantibodies with rituximab. it also highlights the utility of ifnɣ functional testing with stat phosphorylation, which may be used to monitor disease activity and to make decisions about ongoing immunomodulatory treatment. it is necessary to monitor additional immunological markers to refine the diagnosis of a secondary post-lt immunodeficiency to take preventive measures before infections occur. we sequentially measured t lymphocytes and antibodymediated immunity in a -year-old male receiving a lung transplant for idiopathic pulmonary fibrosis to determine which immune indicators could improve the identification of a secondary immunodeficiency. methods: t and b cell numbers, igm, igg, iga, ige and igg subclasses and specific antibodies to s. pneumonia capsular polysaccharides were assessed over a months-long post lt period. the clinical progress, infections and pulmonary function were monitored prior to transplantation and at regular intervals thereafter. results: the patient had progressive idiopathic pulmonary fibrosis starting with an episode of pulmonary hypersensitivity years earlier. he developed increasing respiratory failure progressing to complete dependency requiring a lt in june . he was treated with prednisone, mg/ day continuously for months, then decreased to mg/day. other immunosupressants included mycophenolic acid and tacrolimus and on/ off antibiotics that eventually led to severe tendinitis at - months post lt. at ½-month post lt he developed an early onset bronchiolitis obliterans syndrome (bos) that was controlled by increasing the prednisone dose. sequential immunologic evaluation showed his igg dropping from , mg/dl to after two weeks and then remaining stable at that level for the rest of the observation period. igm and iga had minor variations and ige remained very low. igg fell from - mg/ml pre-lt to - in weeks and remained stable at that level thereafter. antibodies against s. pneumoniae polysaccharides started high, between - ·g/ml for all serotypes and fell rapidly in the first weeks post lt, then continued a steady decline with > % serotypes falling < . ·g/ml at months. twelve of pneumococcal serotype antibodies increased above . ·g/ ml after igg replacement at months. cd t lymphocytes decreased from ± , cells/ul to - at month, remaining at that number after that. cd /th cells increased from - cells/ul to at months when prednisone was tapered down and then decreased to after increasing the prednisone dose again. this decrease coincided with a reduction in bos manifestations. conclusions: immune monitoring revealed an independent decrease of immunoglobulins with a stronger decrease in igg and specific pneumococcal antibodies. the role of th cell increase in developing bos needs further investigation. stat is a frequent target of cancer therapies due to its role in certain malignancies for cell proliferation and metastasis. with lof stat , decreased incidence of some cancers may be expected, however increased rates of lymphoma are described. we sought to describe the incidence and spectrum of malignancy in our relatively large lof stat cohort. methods: we performed a retrospective analysis of lof stat patients evaluated at the nih clinical center to determine the type of malignancies diagnosed, treatments received, and outcomes following therapy. results: a total of patients with malignancies were identified (cancer incidence %). six patients ( %) were diagnosed with non-hodgkin lymphoma (nhl); with diffuse large b-cell lymphoma (dlbcl) and with burkitt lymphoma (bl) with age at diagnosis ranging from years to years with median age of years. pathology staining for ebv was available in four patients; all of whom were negative by eber. all dlbcl patients received da-epoch-r for - cycles, and all achieved complete remission. five of patients with lymphoma are alive and disease-free. one patient died of heart failure years post chemotherapy without disease relapse. two patients were diagnosed with papillary thyroid carcinoma at ages and , one of whom was subsequently diagnosed with nhl. two other patients were diagnosed with basal cell carcinoma of the skin at ages and ; both of whom had prior voriconazole exposure. conclusion: malignancy, most commonly nhl, occurs in patients with lof stat mutations. nhl should be considered in patients with progressive lymphadenopathy, and thyroid carcinoma should be considered in patients with thyroid nodules. patients treated with voriconazole are at an increased risk of skin cancer and require careful skin monitoring. as survival increases, it will be important to monitor the incidence of malignancies diagnosed, as it is possible that decreased stat signaling may prove to be protective of some cancers, such as colon and breast carcinoma, in which increased stat signaling is implicated in pathogenesis. abstract/case report text introduction: recurrent pneumonia is defined as or more episodes of pneumonia in one year or more than pneumonias throughout life (with radiological resolution between episodes). in retrospective studies, up to % of adult subjects with recurrent pneumonia coursed with primary immunodeficiencies (pid). prospective studies evaluating the etiology of recurrent pneumonia are scarce. diagnostic delay and inappropriate management of patients with pid (predominantly antibody deficiency for example) could lead to irreversible lung damage or even death from serious infections. here we present the frequency of primary immunodeficiencies in adults with recurrent pneumonia in cali, colombia. methods: we present preliminary data of a descriptive prospective study that will include patients with recurrent pneumonia. women and men > and < years old will be included. all volunteers will be evaluated by a clinical immunologist, complete blood count and serum igg, iga, igm and ige levels will be determined. according with clinical suspicious, igg subclasses, anti-pneumococcal igg response and b cell subpopulations will be performed. the project will be executed in months. written informed consent has been obtained for all subjects included. this project count with irb approvals at universidad del valle and hospital universitario del valle. results: a total of recurrent pneumonia cases have been included in the study. the mean age was . years ( - years) with a female:male ratio : . moderate-severe dyspnea was observed in / cases (medical research council -mrcdyspnea scale to ). non cystic fibrosis bronchiectasis was found in / cases ( %). the main etiologies of recurrent pneumonia were: primary immunodeficiency / ( %); asthma / ( . %); autoimmunity / ( . %); primary ciliary diskinesia / ( . %) and others. hypergammaglobulinemia represented / ( %) of cases. primary immunodeficiencies, % of recurrent pneumonia cases, were classified as: predominantly antibody deficiency ( cases) including cvid cases, igm deficiency cases, selective iga deficiency cases, igg subclasses deficiency case, hypogamaglobulinemia case and agammaglobulinemia case. combined immunodeficiency: dock deficiency case and ataxia telangiectasia case. conclusion: to the best of our knowledge this is the first prospective study evaluating the etiology of recurrent pneumonia in colombia. predominantly antibody deficiencies and igg hypergammaglobulinemia affect % of adults with recurrent pneumonia in colombia. this study allows us diagnosed more than new cases of adult onset pid. immunological evaluation is critical in the assessment of patients with recurrent pneumonia. conflict of interest: authors disclosure any potential financial conflict of interest related to this abstract. acknowledgements: this investigator-initiated research was supported with a grant from baxalta us inc, a member of the takeda group of companies bt - /iir-col-bxlt- . abstract/case report text introduction: lysinuric protein intolerance (lpi) is an autosomal recessive metabolic disorder due to pathogenic mutations in slc a . it is distinguished by decreased plasma concentrations and increased urinary excretion of lysine, arginine and ornithine and can present with multiorgan involvement and a spectrum of immune deficiency. we present a five-year-old female with lpi, early-onset juvenile systemic lupus erythematous (sle), hemophagocytic lymphohistiocytosis (hlh), and granulomatous skin lesions that were positive for vaccine-strain rubella. methods: retrospective chart review was conducted. laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogen, quantitative serum immunoglobulins, vaccine titers, autoantibodies, metabolic studies, and genetic evaluation by next generation and whole exome sequencing. results: a five-years-old female of mixed native american and african american race presented at years of age with severe failure to thrive, history of recurrent fevers, joint swelling, recurrent skin lesions, severe anemia and neutropenia, and hypergammaglobulinemia. upon further evaluation, she demonstrated hyperferritinemia and ana, rnp, smith, and ss-a autoantibodies and was diagnosed with early-onset juvenile sle. laboratory immune evaluation revealed age-appropriate lymphocyte subpopulations and lymphocyte proliferative responses to mitogens and antigens, markedly elevated igg, iga, igm with no associated monoclonality, and protective tetanus and pneumococcal titers. given her severe clinical manifestations at an early age, concern for immunodeficiency prompted further genetic evaluation with next generation dclre c sequencing, which was negative, and whole exome sequencing, which revealed two heterozygous mutations in slc a , consistent with lpi. laboratory metabolic evaluation was also consistent with a diagnosis of lpi. she continued to experience recurrent cutaneous lesions on her upper and lower extremities. biopsy findings were consistent with a granulomatous lesion and subsequently identified by the cdc to have vaccine-strain rubella infection. due to recurrent pneumonia and concern for pulmonary alveolar proteinosis (pap), pulmonology was consulted and eventually confirmed pap, and she has required home oxygen supplementation. given her history of recurrent infections and vaccine-strain rubella infection, supplemental ivig was initiated. her sle has been fairly refractory to medical management, including systemic corticosteroids, mycophenolate, rituximab, and cyclosporine. she recently developed hlh at years of age and is currently maintained on canakinumab, mycophenolate, and systemic corticosteroids, yet continues to have sle and pap that has been difficult to control. conclusion: the range of clinical and immunologic findings in lysinuric protein intolerance has varied widely in the literature. our patient presented with early-onset juvenile sle and did not develop hlh and pap until years after initial presentation. despite relatively normal cellular immunity by laboratory evaluation, our patient was identified to have chronic infection with vaccine-strain rubella virus, indicating severe t cell dysfunction, and poses challenges for future immunomodulatory treatment. introduction: x-linked immunodeficiency with magnesium defect, ebv infection, and neoplasia (xmen) disease is caused by lossof-function (lof) mutations in the magnesium transporter (magt ) gene. it is a rare x-linked combined immunodeficiency and selective congenital disorder of glycosylation. clinical manifestations include chronic ebv viremia, recurrent bacterial and viral infections, lymphadenopathy, splenomegaly, autoimmunity, liver and central nervous system (cns) abnormalities. magt deficiency was first noted to result in chronic ebv infection and an increased susceptibility to ebv+ lymphomas. we recently recognized merkel cell carcinoma at a very young age in two xmen patients, leading to our review of the malignancies in this cohort. methods: we reviewed the records of male patients ( seen at the nih) with confirmed hemizygous lof mutations in magt for diagnosis of malignancy, therapy, and outcome. results: we identified malignancy in patients of with magt deficiency ( %). four patients had hodgkin's lymphoma (hl) (ages - years), three had non-hodgkins lymphoma (nhl) (ages - years), one had kaposi sarcoma ( years) , one patient developed eber-negative liposarcoma (age years) after receiving chemo and radiotherapy for severe lymphoproliferative disease (lpd) at age years and two had merkel cell carcinoma at exceedingly young ages ( and years). all patients had chronic ebv viremia. they all received treatment according to established protocols. currently, all except for three patients are alive and in remission, including one post-hsct. overall malignancy survival of %. it is important to note that three patients who did not have malignancy had ebv lpd so severe that it warranted treatment with a malignancy protocol, with one mistaken as having lymphoma. conclusion: xmen immune deficiency, an x-linked glycosylation disorder, is a multisystem disease associated with increased susceptibility to malignancies. initially, ebv driven lymphoproliferation and lymphoma was described with xmen; however, with increasing diagnoses, more malignancies are being recognized. all the recognized malignancies are associated, at least in part, with dnaviruses, including ebv, hhv- , and merkel cell virus. understanding the clinical phenotype and pathogenesis of this disease will improve monitoring and early diagnosis of malignancies for patients with magt deficiency. abstract/case report text background: primary immune deficiencies (pid) constitute a heterogeneous group of over individually rare congenital diseases that involve genes coding for proteins of the immune system, and which result in increased susceptibility to infection, inflammation, autoimmunity, allergy and cancer. the complexity of the diagnostic task, and the intrinsic biases and limitations of the human mind, can be aided by computational tools. among the available machine learning approaches, decision tree algorithms select the best node to split based on entropy and information gain; random forests build dozens or thousands of decision trees randomly to improve accuracy and reduce overfitting. aim: to implement a machine learning-assisted clinical decision support system for the diagnosis of pid. methods: with a local database of patients with suspected iei, we built a decision tree using c . dtc, and a random forest on python . (jupyter notebook, scikit, mathplotlib, pandas, numpy). the database was obtained by conducting an electronic search on medsys of patients with the term "immunodeficiency" in their electronic medical records, and then hand-picking cases in which a pid had been confirmed or ruled out. it consisted of patients, of which had been diagnosed with iei. we first split the dataset randomly into training ( %) and testing ( %) sets. the decision tree was tasked with classifying correctly pid or not. after running the algorithm in the training set, we evaluated in the testing set through cross-validation. results: accuracy was greater than % for the dataset (pid/not). . for the dtc with levels. the attribute with the lowest gini coefficient was low iga ( . ). accuracy for the random forest classifier was . with trees. feature importance was highest for lung infection ( . ), high igg ( . ), low iga ( . ), skin infection ( . ), no isolate ( . ), and allergy ( . ); it was lowest for consanguinity, high igm, central nervous system infection, parasites and no infections. during the random generation of trees, accuracy reached up to %. discussion: we built two classification models. decision trees lend themselves more easily to learning and deriving rules of thumb from their sequences. random forests are more robust and better suited for categoric (as opposed to binary) classification. we next want to develop a chatbot, currently under construction, that will ask relevant questions in optimal sequence, and extract undiagnosed patients with suspected iei, based on statistical "red flags". we also have preliminary results of this process applied to a usidnet database with over , patients, and are also working on multinomial logistic regression and naïve bayesian classifiers for this and other databases. abstract/case report text objectives: acute viral respiratory infections (avri) are associated with significant healthcare resource use and cost. the use of intravenous immunoglobulin (ivig) may be an effective treatment for immunosuppressed patients and reduce overall healthcare resource utilization. the goal of this study was to assess hospital resource utilization associated with ivig use among patients hospitalized for avri. methods: using data from the - premier hospital database, we identified patients hospitalized with a diagnosis of avri [respiratory syncytial virus (rsv), parainfluenza virus, rhinovirus, or metapneumovirus], and who had an immune deficiency (chemotherapy treatment, transplant, primary immunodeficiency disorder (pidd), specific antibody deficiency, other immunodeficiency, or disorders of the immune or lymphatic systems). patients receiving ivig within the first hours were compared to patients who did not receive ivig at all. due to the nature of the need to better understand the treatment effect associated with ivig, we used an inverse probability weight-based regression model. since there were substantially more controls than cases, we randomly drew , controls. a logistic regression model was developed to adjust for factors associated with the probability of ivig use within hours of admission. this propensity score was then used to weigh subsequent models to assess length of stay (total and icu) using negative binomial models and logistic regression for inpatient death. results: a sample of , immunocompromised inpatients were identified, receiving ivig within the first hours of admission and , who did not receive ivig. the ivig group was older (mean age vs , p < . ), had more antiviral use ( % vs %, p < . ), and had less cancer ( % vs %, p < . ). after adjustment for immunity type (transplant, cancer), rsv, pidd, age, prednisone, antiviral use, ribavirin use, urban hospital setting, teaching status, intubation and lung disease, patients with ivig use had . less days of hospitalization (p= . ) and . less days in the icu (p= . ) than non ivig users. conclusions: this data analysis suggests that hospital length of stay and icu length of stay were significantly shorter for immunocompromised patients hospitalized for acute viral respiratory infections who were administered ivig within the first hours of admission, as compared to patients who did not receive ivig. it is possible that ivig use may have an impact on hospital resource utilization and costs. future prospective studies would help further assess the role of ivig in patients hospitalized with acute viral respiratory infections. associate professor/yale university abstract/case report text background: cd ligand deficiency is an x-linked combined immunodeficiency associated with opportunistic infections and increased risk of malignancies. expansion of memory cd + t-cells with senescent features is known to be associated with chronic immune stimulation including aging, chronic infection and malignancy. cd + t-cell characteristics of cd l deficient (cd ld) patients in relation to their clinical history have not been described. objective: we studied correlation between cd + t-cell senescence with clinical histories of cd ld patients. methods: we analyzed the frequency and phenotypic characteristics of peripheral cd + t-cell subsets in four cd ld patients ( , , and years old (yo)) and healthy controls (hcs). t cell excision circle (trec) counts and telomere lengths of the patients and hcs were measured using quantitative pcr. in-depth analysis of cd + t-cells of the yo patient and hcs was done using high-dimensional cytometer time of flight analysis (cytof). results: three patients ( , and yo) with histories of recurrent infections and poor compliance with immunoglobulin therapy (ivig) showed an increased frequency of effector memory cd + t-cells with the senescent phenotype compared to age matched hcs. whereas yo patient with excellent ivig compliance starting at infancy did not show any senescence phenotypes of the cd + t-cells. the telomere length and trec count of each patient correlated with the degree of cd + t-cell senescence and their current ages, respectively. in-depth analysis showed similar expression patterns of molecules related to senescence and cytotoxicity in cd + t-cells including cd , t-bet, eomes, granzyme b and perforin in the yo patient and mid-elderly hcs. conclusion: our findings suggest that prompt diagnosis and compliance with ivig starting at the infancy may prevent early onset cd + t-cell senescence in cd l deficiency. abstract/case report text introduction: immunoglobulin g -related disease (igg -rd) is an immune-mediated fibroinflammatory condition that affects multiple organs. when igg -rd is found in the ocular adnexa, the term "igg related ophthalmic disease (igg -rod)" is used. objective: our case describes a patient with igg -rod without systemic involvement. case: mr. x is a -year-old male with a pmh of cml (on imatinib) and allergic rhinitis who presented to clinic with orbital swelling for twenty years. his swelling had always been responsive to steroids, but would return once steroids were tapered. patient was diagnosed with biopsy proven cml in and is currently taking imatinib. because his peri-orbital edema persisted, a right lacrimal gland biopsy was done which showed "marked lymphocytic infiltrate of soft tissue with lymphoid follicles, many plasma cells, and eosinophils. no atypical histiocytes." flow cytometry was negative for malignancy. results: crp . mg/l. esr mm/hr. igg elevated at mg/dl. ct chest from and ct chest, abdomen, pelvis from were without fibrotic changes. assessment: when diagnosing igg -rd, we categorize diagnosis into three levels (possible, probable, or definite) by three criteria (clinical manifestation, elevated serum igg , and histopathology). this is detailed as follows: clinical exam showing organ specific swelling or masses, elevated serum igg (> mg/dl), and histopathology with either lymphocyte and plasmacyte infiltration and fibrosis or infiltration of igg + plasma cells (ratio of igg +/igg+ cells ≧ % and ≧ igg + plasma cells per high power field). not all these components are required for diagnosis, but meeting histopathologic criteria makes diagnosis more probable. our patient's disease was localized to his eye, and patients with igg -rod have unique diagnostic criteria. these criteria are similar to the criteria for igg -rd, but emphasize enlargement of the ocular adnexa, less frequent fibrosis, and ≧ igg + plasma cells per high power field. our patient's histopathology revealed a lymphoplasmacytic infiltrate, but lacked storiform fibrosis or obliterative phlebitis. his serum igg level was mg/dl, and his biopsy was positive for an igg +/igg+ ratio of % and more than igg + plasma cells per high power field. based on this, he meets criteria for igg -rod. conclusion: igg -rod is a rare condition that is usually associated with systemic organ involvement. our case is unique, as no systemic disease has been detected. we also suspect our patient has been living with igg -rod for several years, as his orbital swelling began in high school. it is important to note that he has been on imatinib, a tyrosine kinase inhibitor, for treatment of his cml. imatinib inhibits c-abl and platelet-derived growth factor receptor, tyrosine kinases involved in profibrotic pathways. patient's lack of fibrosis could also be due to his longstanding use of this drug. it is also possible that he has a rare form of igg -rod without systemic involvement. a limited number of such cases have been reported, but no consensus has been made on why disease course was localized. our patient was started on rituximab, and his serum igg decreased to mg/dl after the first cycle. we hope his disease achieves remission. informed consent: informed consent was obtained from all individual participants included in the study. abstract/case report text platelet abnormalities with eosinophilia and immune-mediated inflammatory disease (plteid) is a recently discovered combined immunodeficiency with inflammatory and allergic manifestations with few cases reported. we describe a female patient with compound heterozygous mutation in arpc b gene with suggestive clinical findings of plteid. a -year-old girl presented with chronic diarrhea since neonatal period, with bloody stools and failure to thrive. she also presented atopic dermatitis, recurrent cutaneous and mucosal ulcers, recurrent respiratory infections ( episodes of otitis media, pneumonias) and many episodes of mucocutaneous candidiasis. family history revealed a sibling deceased in the second month of life, who presented a similar clinical picture and a paternal uncle and second degree cousin that died in the first year of life. there is no history of consanguinity. laboratory evaluation revealed peripheral eosinophilia ( /mm ), normal platelet numbers with low platelet volume ( , fl -reference value , - , fl), normal igm levels with elevated igg ( mg/dl-rv - mg/dl), iga ( mg/dl -rv - mg/dl) and ige ( iu/ml -rv a) associated with plteid. t h e i n f a n t r e c e i v e s a n t i m i c r o b i a l p r o p h y l a x i s w i t h sulfamethoxazole-trimethoprim and fluconazole, intravenous immunoglobulin replacement and was referred to hematopoietic stem cell transplantation (hsct). this case was the first one described in brazil and highlights the importance of seeking for a genetic diagnosis in patients with complex clinical phenotypes. precise diagnosis can impact on treatment approach. live vaccines are generally contraindicated in patients with combined immunodeficiency (cid). however, in less severe cid, such as partial dgs, those vaccines can be considered depending on the immunologic status of the patient. there are recommendations regarding to measles, mumps, rubella (mmr) and varicella vaccines, but yellow fever vaccine (yfv) is generally contraindicated in this population. considering the severity of the yellow fever disease and the absence of specific treatment, the use of this vaccine is an important topic for debate in cases of patients from endemic areas. objective: this study aimed to describe the use of yfv and other live attenuated vaccines in patients with dgs, associating it with their immunological profiles and the presence of adverse effects. methods: retrospective study of medical records of patients with dgs confirmed by mlpa or fish, followed in a pediatric reference center for primary immunodeficiencies between and . collected data included: demographic characteristics, medical history, history of immunization with live vaccines, postvaccination adverse reactions and immunological profile, including immunoglobulins levels, serologic vaccination responses, lymphocyte immunophenotyping, lymphocyte proliferation responses to mitogens and prophylactic treatments (antibiotic or immunoglobulins). results: thirty-five patients with confirmed dgs and median age of years ( - y) were included ( m: f). thirty-three children ( %) received mmr vaccine: nine presented t lymphopenia. two of the patients had cd < , one of them with normal mitogenic proliferation response and the other was not tested. three of the patients had low immunoglobulins levels ( / low igg, / low igm and / low iga), and one of them received intravenous immunoglobulin (ivig). twentynine of had normal serologic vaccination responses. adverse effect was only reported by one patient, who had one episode of fever after the administration of all vaccines. yellow fever vaccine was administrated to children ( %): had t cell l y m p h o p e n i a ( b u t c d > ) , a n d a n o t h e r p a t i e n t h a d hypogammaglobulinemia and received ivig and prophylactic antibiotics. twelve of showed adequate serologic responses to mmr and hepatitis b. only patient reported mild reaction (tremors) two days after the yfv administration. the same patient had normal t cells, immunoglobulins and vaccine responses. twenty patients ( %) received bacillus calmette-guerin vaccine (bcg), ( %) received oral polio, ( %) rotavirus and ( %) received varicella vaccine. no severe adverse events were documented in any patient that received live vaccines, and no patient developed measles, mumps, rubella or yellow fever diseases as a consequence of administration of the vaccine. conclusions: in this cohort of pediatric patients with dgs, yfvand other live vaccines were well tolerated, and no severe adverse events were reported, suggesting that widespread contraindication of yfv may endanger unvaccinated patients with less severe phenotype living in endemic areas. immunological evaluation and individualized decisions are always recommended, and further studies are needed to assess the safety of the yfv in this pediatric population. abstract/case report text introduction: lad-i is a rare inherited disorder of leukocyte (primarily neutrophil) adhesion to endothelial cell surfaces, migration, and chemotaxis resulting from itgb gene mutations encoding for the β -integrin component, cd . severe lad-i (i.e., cd expression on < % of neutrophils) is characterized by recurrent serious infections, impaired wound healing, and childhood mortality. although allogeneic hematopoietic stem cell transplant (allohsct) is potentially curative, its utilization and efficacy are limited by hla-matched donor availability and risk of graftversus-host disease (gvhd). rp-l - (clinical trials.gov # nct ) is a phase / open-label clinical trial evaluating the safety and efficacy of autologous cd + cells transduced with a lentiviral vector (lv) carrying the itgb gene encoding for cd (chim-cd -wpre) in severe lad-i. methods: pediatric patients ≥ months old with severe lad-i (demonstrated by cd expression on < % neutrophils and at least one prior significant bacterial or fungal infection) are eligible. peripheral blood (pb) hematopoietic stem cells are collected via apheresis after mobilization with granulocyte-colony stimulating factor (g-csf) and plerixafor. cd + hspcs are selected, transduced with chim-cd -wpre lv, and cryopreserved. myeloablative conditioning with busulfan (therapeutic drug monitoring (tdm) dosing with adjustments to enable target area under the curve (auc)) is administered over days, followed by infusion of the thawed investigational drug product (rp-l ). patients are followed for safety assessments including replication competent lentivirus (rcl) and insertion site analysis (isa), and for efficacysurvival to age ( months) and at least -year post-infusion without allohsct, increase in neutrophil cd expression, pb vector copy number (vcn), decrease in infections and/or hospitalizations, and resolution of skin or periodontal abnormalities. results: an initial lad-i patient (age years) with recurrent severe infections and documented itgb mutations has been treated as of november . baseline cd , cd a, and cd b expression were < %. mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target auc. investigational product was comprised of . x e cd + cells/kg with vcn of . copies/cell (liquid culture), and was infused without complications. no serious treatment-emergent adverse events were reported. neutrophil engraftment ( consecutive days of anc ≥ ) was observed days post-infusion. pb pmn cd expression months posttreatment was . % with comparable cd a and cd b expression levels; pb cd (myeloid) vcn at . months was . . safety and efficacy data months post-treatment will be available at the time of presentation, in addition to preliminary data regarding a potential additional patient. conclusion: preliminary evidence demonstrates that rp-l enables itgb genetic correction with robust cd /cd neutrophil expression in this frequently fatal primary immunodeficiency. abstract/case report text introduction: the complement system plays an integral role in the innate immune system and links innate and adaptive immunity. complement deficiencies, hereditary or acquired, are rare. acquired deficiencies are more prevalent, occurring in nephrotic syndrome, reduced hepatic synthesis or transiently in sepsis/viremia. they are also seen in the presence of autoantibodies known to cause depletion of complement factors, such as c nephritic factor (c nef). c deficiency is associated with infection susceptibility, particularly to encapsulated bacteria, and immune complex disease. case description: a year old male was evaluated for recurrent infection. in childhood, he had recurrent sinusitis, otitis media requiring tympanostomy tube placement and persistent pharyngitis despite tonsillectomy. as a teenager, he developed glomerulonephritis, progressing to end stage renal disease and requiring transplant at age . the kidney allograft failed years later, with biopsy demonstrating recurrent glomerulonephritis. the patient was transitioned to peritoneal dialysis and later hemodialysis, due to recurrent pd-related infections. his adult course was complicated by recurrent methicillin sensitive staphylococcal aureus (mssa) catheter and soft tissue infections (cellulitis and abscess), sinusitis, sepsis (streptococcal, mssa and tularemia), multifocal pneumonia and a left below knee amputation for osteomyelitis that required revision surgery. patient reported other autoimmune phenomena including a presumptive diagnosis of vasculitis and possible lupus-like syndrome. the constellation of recurrent infections and autoimmune features was most concerning for an early complement deficiency. prior work up was notable for low c , ch and ah with normal c , factor h and factor i. extensive laboratory work up revealed normal c q, c level and function, serum immunoglobulins, vaccine titers, factor b and factor d levels. atypical hus (ahus) panel revealed a heterozygous silent variant in exon of cfh and a heterozygous polymorphism within an intron in mcp/cd , seen with increased prevalence in the patient population with ahus. wes was notable for a variant of uncertain significance in the vcl gene only. c level and function were markedly decreased, alongside low ch and ah . both sc b- level and c nephritic factor were elevated. a diagnosis of acquired c deficiency due to c nef was made and patient was started on bactrim prophylaxis. he has remained free of serious infection since starting antibiotic prophylaxis. discussion: c nef stabilizes the alternative pathway c convertase, c bbb, increasing its half-life and blocking dissociation. this leads to unregulated consumption of c with subsequent deficiency. c nef has been associated with c glomerulopathy, infection and partial lipodystrophy. however, there is marked heterogeneity in clinical phenotypes with reported asymptomatic individuals. our patient's glomerulonephritis likely represents c glomerulopathy. case reports and series of successful treatment of c glomerulopathy with rituximab and eculizumab have not commented on immune outcomes beyond the kidney. other potential therapeutic strategies include plasma cell depletion with either bortezomib or daratumumab. further study is needed to evaluate these therapies influence on both reversal of c depletion and overall impact on immune function in the setting of c nef. abstract/case report text introduction: patients with heterozygous signal transducer and activator of transcription (stat ) gain of function (gof) pathogenic variants exhibit an array of phenotypes including susceptibility to viral, bacterial, fungal and mycobacterial infections, autoimmunity, and cancer predisposition. progressive disseminated histoplasmosis (pdh) is well-described to affect infants. however, no reports have evaluated underlying monogenic immune dysregulation in previously healthy infants presenting with pdh. we report an infant who presented with pdh and associated hemophagocytic lymphohistiocytosis (hlh) leading to the diagnosis of a heterozygous stat gof mutation. case report: a previously healthy -month old male presented with persistent fever, pancytopenia, transaminitis, elevated ferritin, hepatosplenomegaly and coagulopathy. his clinical and laboratory evaluations were concerning for hlh syndrome. he had no prior history of immune hyperactivation or atypical infections. secondary causes of hlh were investigated, and patient was diagnosed with pdh based on marked histoplasma antigenemia. targeted genetic testing did not reveal a genetic etiology of familial hlh. he was successfully treated with a pulse and taper of dexamethasone as well as liposomal amphotericin b with transition to itraconazole. immunologic evaluation at the time of initial presentation demonstrated increased mean channel fluorescence for both perforin and granzyme noted in his nk cells. his nk function was decreased; however, he had a normal cd a degranulation assay. his b-cell panel demonstrated low non-switched memory b-cells, low switched memory b-cells and low total memory b-cells. given his extreme immune activation with histoplasmosis, abnormal immunologic testing, and persistent lymphopenia despite resolution of his infection, a primary immunodeficiency next generation sequencing panel was sent. the results demonstrated a pathogenic variant in stat (c. c>t; p.ala val). this single nucleotide variant has been previously shown to be pathogenic (clinvar). abstract/case report text introduction: cytotoxic t lymphocyte antigen- (ctla- ) is known to have an important role as a negative regulator of immune responses, participating in the control of regulatory t cells and effector t cells. in mice its absence is associated with fatal autoimmunity and several ctla- mutations, leading to low or absent ctla- expression, have been shown in humans to be associated with a phenotype that includes hypogammaglobulinemia (with recurrent respiratory infections) and several manifestations of autoimmunity (enteropathy, granulomatous lymphocytic interstitial lung disease, organ infiltration, splenomegaly, autoimmune cytopenias, lymphadenopathy, amongst others), in an autosomal dominant mode of transmission. one of the published mutations, c.c t, that results in an alanine to valine substitution (p.a v), with a highly conserved alanine at that position, had a cadd score of and was associated with the phenotype above, and was shown to be associated with a low expression of ctla- on regulatory t cells and with low ctla- function (reduction of ctla- -mediated transendocytosis). methods: after irb approval, we searched for ctla- mutations present in the biome biobank· biorepository, containing whole exome sequencing data on patients, with data obtained using illumina· v hiseq sequencing platform. sifting through all the ctla mutations in the data, we identified four patients with the c.c t mutation described above. extensive chart review of the four patients was performed. results: four patients were found with the ctla- c.c t mutation. none of them had any of the described phenotypical characteristics of ctla- deficiency. patient is a -year-old male with history of coronary artery disease, atrial fibrillation, stroke, hypertension, brain aneurysm, chronic kidney disease, gout and depression. patient is a -year-old female with history of morbid obesity. patient is a -year-old female with history of hypertension, obesity, pre-diabetes, dyslipidemia and iron deficiency anemia. patient is a -year-old female with history of peripheral artery disease, hypertension, dyslipidemia, chronic kidney disease and lung cancer. conclusion: prior literature has attempted to characterize the clinical penetrance of ctla- mutations, suggesting it to be around %, with that number applying to different mutations in ctla- mutation carriers. we screened a large biorepository of more than thousand patients for ctla- patients and identified four patients that carry one of the best described ctla- mutations, previously validated from a functional standpoint and associated with a severe phenotype. none of the four patients demonstrated any of the previously described phenotypical characteristics, and all four have ages above the median age of onset of years. with the increasing use and broad population application of genetic studies, it is crucial to define the value of identifying presumed pathogenic variants in the absence of the adequate phenotype, with all the prognostic, therapeutic and ethical considerations it may imply. prior case reports of pil patients with b-cell malignancies have discussed treatment regimens with chemotherapy, radiation, and/or surgery, but neither the use nor the outcomes of allogeneic hematopoietic stem cell transplantation (hsct) in the management of recurring b-cell malignancies have been readily reported. case description: a -year-old man with pil and an accompanying history of lymphopenia, hypoproteinemia, hypoalbuminemia, and hypogammaglobulinemia was diagnosed with diffuse large b-cell lymphoma (dlbcl) of the liver following a preceding history of burkitt lymphoma of the ileum at years of age and dlbcl of the liver at years of age, in which each malignancy was genetically distinct. in addition, the patient had a history of benign nodular adenomatoid hyperplasia of the thyroid at years of age that required a hemi-thyroidectomy. treatment considerations for the patient included chimeric antigen receptor t-cell therapy, autologous hsct, and allogeneic hsct, in which allogeneic hsct was ultimately pursued. prior to hsct, the patient was lymphopenic ( cells/microliter) with significant t-cell lymphopenia ( cells/microliter) and an increased proportion of memory t-cells ( % of his cd + t cells were cd ro+), as well as hypogammaglobulinemic (igg mg/dl; iga mg/dl; igm mg/dl). immediately following treatment of his dlbcl with rituximab, ifosfamide, carboplatin, and etoposide, the patient underwent a matched-related sibling donor hsct with a preparative regimen of busulfan, thiotepa, and fludarabine. now months status-post hsct, the patient has maintained full-donor chimerism and has no evidence of graft-versus-host disease or malignancy. as expected, hsct has not corrected abnormalities in certain parameters associated with his pil, as he continues to display significant hypoproteinemia, hypoalbuminemia, and hypogammaglobulinemia, but he has an improved lymphocyte count ( , cells/microliter). discussion: there is no definitive or curative treatment for pil; furthermore, the genetic etiology of pil remains unknown. supportive regimens to help mitigate or offset manifestations of pil exist, such as adherence to a low-fat diet with medium-chain triglyceride supplementation, but there are no therapies available to prevent or reduce the risk of developing bcell malignancies in this patient population. although previous case reports have detailed successful treatment of b-cell malignancies in pil patients with chemotherapy, radiation, and/or surgery, there are no published consensus guidelines regarding management of b-cell malignancies in the setting of pil, especially if recurrent in nature. for non-pil patients with chemotherapy-refractory disease, or recurrent disease following autologous hsct, allogeneic hsct is a potentially curative option. herein, we describe a pil patient with a history of multiple b-cell malignancies who underwent a successful allogeneic hsct, indicating that allogeneic hsct may be an effective treatment option for similarly affected patients. abstract/case report text introduction diarrhea in young infants is common and generally self-limited. in persistent cases, the differential diagnosis is broad and includes infections, food protein-induced allergic proctocolitis, congenital diarrheas and enteropathies. in addition to monogenic inflammatory bowel diseases, many cellular, humoral, and combined immunodeficiencies should be considered, including but not limited to cvid, ipex and ipex-like phenotypes, lad, dyskeratosis congenita, intestinal lymphangiectasia, omenn syndrome, cartilage hair hypoplasia, cgd, il- axis defects, aid deficiency and wiskott-aldrich syndrome. case presentation a full term infant born after an uncomplicated pregnancy to nonconsanguineous honduran parents presented with non-bloody, non-bilious vomiting and dehydration at days of life. the infant later developed frequent loose stools, some of which were bloody, and failure to thrive. his family and prior medical history, including newborn screen, were normal. an extensive workup was initiated which showed: -persistent and severe anemia with a hemoglobin nadir of . mg/dl -hypoalbuminemia requiring multiple infusions -elevated alpha- -antitrypsin and calprotectin level in stool -profound hypogammaglobulinemia with normal iga, igm and ige for age -normal gross and histologic findings on esophagogastroduodenoscopies and colonoscopies besides a gastric ulcer thought not be the cause of his anemia -normal abdominal imaging including ultrasound, ct angiography and mri -no source of bleeding on meckel scan or exploratory laparotomy -normal dhr assay, g pd level and positive myeloperoxidase stain -immunophenotyping showing t cell lymphocytosis affecting cd + more than cd + compartment, with normal lymphocyte proliferation to mitogens -normal sweat chloride level genetic testing was initiated with a targeted immunodeficiency panel which showed variants of unknown significance in adar, dock , lyst, ptprc and tbx genes, none of which adequately explained his presentation. whole exome sequencing showed that he was a compound heterozygote in the dgat gene. a pathogenic variant c. + t>c (ivs + t>c) was inherited from the father and a likely pathogenic variant c. g>c (p.r p) was inherited from the mother. patient was diagnosed with dgat deficiency, an inborn error of lipid metabolism resulting in protein-losing enteropathy (ple). under gastroenterology's guidance, a low fat diet was initiated, resulting in rapid improvement in stool consistency, weight gain, albumin level and stool alpha- -antitrypsin level. he remains on subcutaneous immunoglobulin replacement therapy for ongoing hypogammaglobulinemia. conclusion: protein-losing enteropathies commonly present with intractable diarrhea and significant laboratory derangements due to malabsorption including hypogammaglobulinemia. as a result of these findings and since many of the etiologies are immunologic in origin, immunologists are an integral part of the evaluation of such cases. in cases where immune system interrogation reveal normal results, genetic testing is crucial in guiding the diagnosis. in our case, whole exome sequencing not only provided the diagnosis but also characterized a variant that was previously of unknown significance as likely pathogenic. dietary management provided rapid improvement in growth and nutritional status. ongoing monitoring will reveal if this management also assists in igg level maintenance and hematologic abnormalities or if even more stringent control of dietary fat will be required abstract/case report text background: foxp gene mutations are associated with immune dysregulation polyendocrinopathy x-linked (ipex) syndrome, a rare xlinked monogenic disease of immune dysregulation and autoimmunity. the classic presentation consists of severe enteropathy, dermatitis, and endocrinopathies (commonly early onset insulin dependent diabetes mellitus). clinical presentation and severity can be variable even in family members with the identical variant. we present a patient with ipex symptomatology and a hemizygous variant in the polyadenylation (polya) signal of foxp that is classified as a variant of uncertain significance (vus). this specific variant was reported in a single case study in which the patient improved after hematopoietic stem cell transplantation (hsct). case presentation: a month-old ex -week gestation boy was admitted with lethargy, hypovolemia, electrolyte disturbances, and acute kidney injury. he developed persistent diarrhea and vomiting after receiving rotavirus vaccine. his family history is significant for early deaths of three maternal unclesone stillborn, one death at months and another at years from unknown gastrointestinal problems. he demonstrated peripheral eosinophilia (to . k/ul), elevated ige, and anemia requiring multiple transfusions. he developed severe enteropathy with hypoproteinemia requiring total parenteral nutrition, continual albumin infusions and maintenance of npo. he had generalized edema, respiratory distress requiring high flow nasal cannula, and repeatedly spiked fevers with negative infectious evaluation. acute kidney injury improved but renal ultrasound showed persistent nephrocalcinosis. endoscopy yielded biopsies demonstrating duodenitis with severe villous atrophy, scanty isolated intraepithelial eosinophils and neutrophils, a few crypts with mucin, reactive epithelial changes and increased lamina propria eosinophils. colon biopsies showed mucosa with focally increased lamina propria eosinophils with scanty neutrophils and surface epithelium without cryptitis. esophagitis with reactive epithelial changes, spongiosis, and many intraepithelial eosinophils was also present. the patient's lymphocytes showed unremarkable proliferation to pha and pwm. cd + cd + t cells demonstrated intracellular foxp expression by flow cytometry. a commercially-available immunodeficiency targeted panel revealed that he was hemizygous for a vus in foxp (exon , c.* a>g non coding). this variant is also referred to as an aauaaa>aauaag or aataaa>aataag change in the polya site. he was also heterozygous for vus at these additional loci: cd a c. c>t abstract/case report text introduction: implantation of allogeneic cultured thymus, partially depleted ex vivo of t cells, can result in naïve t cell development in patients with complete digeorge syndrome (dgs). in a few patients, early and transient skin rash, often characterized as "atypical dgs" or late autoimmune manifestations have been reported following implantation. here we describe a patient with complete dgs who developed immune reconstitution inflammatory syndrome (iris) or atypical dgs following thymus implantation. case description: a female patient was diagnosed at birth with complete dgs due to absent t cell receptor excision circles (trec), hypoplastic thymus, profound hypocalcemia with hypoparathyroidism and cardiac defects. the patient also had microretrognathia, oral motor dysfunction, sialorrhea, recurrent aspirations and reflux requiring a gastro-jejunum feeding tube, low-set ears with right ear microotia, semicircular canals atresia, alopecia and mal-rotated kidneys. prior to thymus implantation, the patient was thriving, had no skin rash, no eosinophilia and no t cells. detailed genetic analyses, did not reveal a cause for her syndrome. at months of age pulmonary aspergillosis was diagnosed presumptively. at months of age the patient received an allogeneic t-cell depleted thymus implant from a male donor, without prior conditioning or post-implantation immune suppressive medications. the procedure was uneventful and the patient returned home after days. results: four months after implantation, a pruritic maculopapular rash appeared on the head and trunk that spread to the extremities including the palms and soles. there was no lymphadenopathy or splenomegaly. an infectious etiology could not be found. eosinophilia and an increase in liver enzymes were noted. there was an increase of cd + and cd + t cells with predominantly memory phenotype, which had been undetectable month earlier. analysis of t cell diversity showed a restricted repertoire with expansion of two v-beta families. there was no evidence of donor cells to suggest graft versus host disease. skin biopsy showed minimal superficial perivascular inflammatory infiltrate composed mainly of cd + histiocytes and rare cd + t cells. the patient was treated with prednisone and cyclosporine. a liver biopsy was performed weeks after initiation of treatment that showed moderate and diffuse peri-portal ductular reaction but no duct associated lymphocytic infiltrate or significant duct epithelial injury or ductopenia. the skin rash rapidly resolved with desquamation, while the liver enzyme abnormalities persisted for two more months. cyclosporine and prednisone were weaned over months. t cell numbers, their response to stimulation and diversity have since normalized, as well as trec and naïve t cell production. the patient is producing appropriate antibodies to protein and polysaccharide vaccines. sixteen months after implantation the patient developed grave's disease with markedly elevated free-t , undetectable tsh and elevated antibodies to the thyroid receptor, which rapidly normalized with ongoing methimazole treatment. the patient is currently months after the implantation and is free of infections, thriving and developing appropriately. conclusions: this patient developed atypical dgs or iris, often associated with autologous and allogeneic hematopoietic stem cell transplants, organ transplants or effective treatment of hiv, after successful thymus implantation for complete dgs. abstract/case report text congenital disorders of glycosylation are a rare group of genetic disorders due to defects in protein glycosylation. phosphoglucomutase (pgm ) is an enzyme necessary for the synthesis of uridine diphosphate n-acetylglucosamine, an important precursor for protein glycosylation. patients with autosomal recessive pgm deficiency have a multisystemic disorder characterized by a neurologic impairment and clinical features classically observed in autosomal dominant hyper-ige syndrome due to stat mutations; including recurrent pneumonias, skin abscesses, elevated levels of ige, and abnormalities in connective tissues and bones. we hypothesized that gp , a highly glycosylated protein and coreceptor of the cytokine il- , would be weakly expressed on pgm deficient cells, due to impaired glycosylation. we studied pgm -deficient patients from kindreds and showed that il- -driven stat phosphorylation was impaired in their pbmcs and ebv-transformed b cells. accordingly, the induction of socs target gene was significantly decreased. in contrast, the patients had normal stat phosphorylation and socs induction downstream of il- , a cytokine whose signaling is independent of gp . flow cytometry and immunoblotting showed significantly lower gp expression in peripheral t-cells and ebv-transformed b cells from pgm -deficient patients compared to healthy donors. we did also show that in vitro inhibition of n-glycosylation, using tunicamycin in ebv-transformed b cell line from healthy donor, alters gp -mediated signaling. collectively, our findings demonstrate that defective glycosylation in pgm -deficient patients results in reduced expression of gp and consequently, impaired gp dependent stat phosphorylation and defective il- signaling. this may account for the overlapping clinical features shared by pgm and stat deficient patients. abstract/case report text introduction: there are no known effective therapeutic modalities for patients hospitalized with moderate to severe acute viral respiratory infections, and treatment is primarily supportive. intravenous immunoglobulin (ivig) has been reported in limited cases to be used in this setting, especially in immunocompromised patients. the primary objective of this retrospective study is to compare clinical and economic outcomes among immunocompromised patients hospitalized with viral respiratory infections who received ivig to those who did not receive ivig at a large academic center hospital. methods: we performed a double-center, retrospective cohort study of all immunocompromised patients who were hospitalized for acute documented respiratory viral infections between and . we divided patients into two groups: those who received ivig therapy for respiratory infections, and those who did not receive ivig therapy. data on age, gender, immune status, viral type, immunosuppression type, respiratory support, microbiological data, length of hospital stay (los), icu los, as well as death and readmission rates were extracted from medical records. in order to adjust for severity bias typically present in observational data such as these, we employed inverse probability weighting (ipw) using all collected baseline covariates. outcomes (death, length of stay in hospital and icu, readmission) were examined using a series of logistic and poisson regression models adjusting for baseline covariates and employing ipw. results: a total of individual hospital admissions were analyzed; patients received ivig and did not receive ivig. there were no significant differences between the two groups in terms of mean age, gender . average age was . , % were female, . % were transplant patients of which . % had lung transplant, . % had liver transplant, . % had bone marrow transplants (bmt), . % had kidney transplant, . % had heart transplant and . % had both solid organ and bmt. . % of patients had a hematologic malignancy, and . % had a primary immunodeficiency. the most common isolated respiratory virus w a s r h i n o v i r u s ( . % ) , f o l l o w e d b y r s v ( . % ) , parainfluenza ( . %) and metapneumovirus ( . %). overall, the use of ivig as associated with a significantly shorter icu length-of-stay, with an (or=- . , p= . ), and a higher hospital readmission rate. in the sub-analysis of patients who received ivig within the first hours of hospitalization (n= ), ivig use was associated with a significantly shorter icu los (or=- . , p= . ), significantly shorter overall hospital los (or=- . , p= . ), and no significant change in readmission rate. conclusions: to our knowledge, this is the first retrospective cohort analysis evaluating the effect of ivig in immunocompromised patients hospitalized with respiratory viral infections. the results suggest that immunocompromised patients receiving ivig may have a shorter hospital and icu los, especially if ivig is provided within the first hours of admission. this may result in reduced healthcare costs. this study is limited by its retrospective nature, and the potential bias that patients treated with ivig are sicker to start with. future prospective studies are suggested to further evaluate these findings. ( , ) . the most common precipitant in children is medication, followed by infection ( , ) . although a clear association between mycoplasma pneumoniae and sjs has been established, there is a scarcity of literature exploring the role of this infection in recurrent sjs in children ( ) ( ) ( ) . case presentation: a -year-old female with prior history of sjs was admitted for mucosal and skin lesions in the setting of community acquired pneumonia. her past medical history included sjs with eye involvement, secondary to mycoplasma pneumoniae (ig m positive), occurring five years prior to this admission. she also had frequent episodes of acute otitis media and sinusitis in early childhood. family history was negative for immunodeficiency. her clinical presentation included respiratory symptoms and fever for days treated with ceftriaxone, followed by cefdinir and levofloxacin. her fever improved the day prior to admission, but she developed conjunctival injection, ocular pain, and ulcerative lesions in her mouth and nares. on physical examination, she had low grade fever with mucosal lesions including conjunctival erythema with serous discharge, painful blisters and denudated skin in lips, perioral area, nares, tongue and oropharynx. initial testing included negative blood hsv pcr, blood culture, rapid antigen testing for group a streptococcus and influenza a/b, and elevated crp in . mg/dl and esr mm/hr. right lower lobe p n e u m o n i a w a s c o n f i r m e d w i t h a c h e s t r a d i o g r a p h . nasopharyngeal pcr and serum igm were positive for mycoplasma pneumoniae. she had a mildly elevated anticardiolipin igm ( mpl), a mildly decreased c ( mg/dl) and a negative ana. she was diagnosed with recurrent sjs secondary to mycoplasma pneumonia infection. she completed treatment with levofloxacin for mycoplasma pneumonia, and received cyclosporine and high-dose methylprednisolone. she had bilateral amniotic membrane transplantation to prevent corneal ulceration . she was discharged after clinical improvement, and recurrent oral lesions were noted at followup. immunological work up as an outpatient revealed normal serum immunoglobulins, normal lymphocyte subsets and low pneumococcal titers with adequate response post-vaccination. sjs secondary to mycoplasma pneumonia infection has predominance of mucosal involvement over rash, which was observed in our patient ( , ) . some case series reported a recurrence of sjs up to % within a -year follow up. almost half of patients with recurrent sjs developed multiple sequelae ( , ) . early diagnosis of sjs, especially in those with prior history of sjs, helps to provide appropriate supportive care, monitoring of complications and treatment of possible superinfections ( , ) . conclusions: there is limited information in the literature regarding the role of mycoplasma pneumoniae associated recurrent sjs in children. it is possible that these episodes are triggered by and/or immune predisposition. there is ongoing discussion regarding whether these clinical presentation should be labeled sjs secondary to mycoplasma pneumonia infection or, depending of the skin involvement, m. pneumonia-associated mucositis (mpam) and m. pneumonia-induced rash and mucositis (mirm) ( , , ). mycoplasma should be treated appropriately in patients with recurrent sjs. abstract/case report text background: growing access to genetic testing has facilitated the genetic evaluation of primary immunodeficiencies but has also greatly increased the number of variants of uncertain significance (vus) encountered in clinical practice. interpreting the significance of vus requires multiple lines of evidence. w e d e s c r i b e a n e u t r o p e n i c i n d e x p a t i e n t w i t h hypogammaglobulinemia, unusual hpv susceptibility, and dual heterozygous pathogenic loss-of-function nfkb and heterozygous missense cxcr vus. family analysis showed the nfkb variant was inherited from his mother, while the novel cxcr variant was present in his father and sister. all four patients presented with recurrent infections, warts, and hypogammaglobulinemia. (figure ) the nf-κb gene encodes p /p transcription factor of the canonical nf-κb pathway, the most common autosomal dominant monogenic cause of common variable immunodeficiency (cvid). cxcr is a g-protein-coupled chemokine receptor with cxcl as cognate ligand. autosomal dominant pathogenic gain-offunction cxcr variants lead to impaired receptor downregulation and retention of neutrophils and other leukocytes in the bone marrow defining whim (warts, hypogammaglobinemia, infections, and myelokathexis) syndrome. all cxcr pathogenic variants truncate the carboxyl-tail of the cxcr receptor, a region responsible for receptor internalization, with the exception of one missense non-truncating variant p.e k. case series: the proband (p ) is a -year-old male with history of recurrent bacterial respiratory tract infections, warts, moderate neutropenia, thrombocytopenia and hypogammaglobulinemia requiring immunoglobulin replacement therapy (igrt). bone marrow biopsy didn't show myelokathexis. next-generation panel sequencing identified a novel heterozygous missense cxcr (c. c>a, p.s y) vus. the serine residue is highly conserved up to zebrafish. this variant was present in heterozygous form in two cases in gnomad database ( , alleles). additional whole-exome sequencing revealed a heterozygous pathogenic nfkb variant (c. dup, pa sfs* ) located in the nterminal rel homology domain, consistent with nfkb loss-offunction. both, the patient's sister (p ) and their father (p ), carry the heterozygous cxcr vus but not the pathogenic nfkb variant, and have history of warts, hypogammaglobinemia, and recurrent infections. the hpv susceptibility is particularly striking in p manifesting by genital warts and hpv-positive oropharyngeal cancer. bone marrow evaluation didn't identify myelokathexis in p (p is pending). the mother of the index case (p ) has cvid requiring igrt and immunomodulation. she shares the nfkb variant with p but is negative for the cxcr vus. extensive t and b cell phenotyping revealed low class-switched memory b cell count ( - counts/ul) in all subjects, and loss of transitional and mature naïve b cells in p and p with nfkb variant. proband b cells showed the highest tendency for apoptosis ( - %) within the family. we describe members of a family with similar presentation (infections, hypogammaglobinemia, warts), however variable combination of nfkb and cxcr variants, where either genetic defect or their combination could explain the clinical phenotype. biochemical consequence of our novel cxcr variant is pending. as the proband showed the most severe immune phenotype and neutropenia, we hypothesize that cxcr has a synergistic effect on nkfb loss-of-function. the contribution of cxcr vus of the clinical phenotype of the two other family members is yet to be determined. background: the yield of diagnosis by exome sequencing for some primary immunodeficiencies (pid) has been less than the typical diagnostic rate for clinical exome analysis (~ - %). the relatively low diagnostic rates for certain subtypes of the pids may be attributed to variable expressivity and/or an incomplete understanding of the genetic basis, among others. additionally the extent of multiple diagnoses and phenotypyic expansion in pid is not well explored. cohorts with highresolution clinical and genetic data are instrumental for exploring these questions. we evaluated the use of human phenotype ontology (hpo)annotated datasets to systematically address the prevalence of these issues using a cohort of individuals with pid who participated in research exome sequencing at the niaid. results: we generated a phenotype dataset of individuals with pids by annotating the clinical features of these subjects obtained from electronic health records (ehr) with hpo terms. exome sequencing of these individuals identified probands with a pathogenic or likely pathogenic (p/lp) variant in a gene associated with the respective clinical presentation. we identified probands where the same gene harbored a p/lp variant in at least three unrelated individuals. we used the clinical and genetic data of individuals in the following areas: ) we identified p/lp variants in aire, pik cd, nlrp , fas, ctla , gata , cybb, stat and tnfrsf b in at least ten patients that explained their clinical presentations. this dataset allowed us to characterize variable expressivity of diseases associated with these genes by capturing the variability in the observed hpo terms among probands with p/lp variants in the same gene. dimensional reduction of clinical features of probands allowed us to cluster patients sharing similar phenotypic profiles. we found clinical presentation of individuals with monoallelic p/ lp variants in aire were relatively less variable and clustered more compactly compared to that of individuals with gata variants. ) the extent of multiple diagnoses in pid is not well explored. the benchmark cohort we developed allowed us to identify candidates for multiple diagnosis or phenotype expansion by comparing the phenotype profile of each patient expressed in hpo terms to the hpo terms typically observed for a given pid. for example, we identified gain-of-function pathogenic variant in pik cd in a patient that explained the clinical features of the pid observed in this patient. however, the patient also displayed developmental delay, congenital hemiplegia, cerebral palsy and absent speech. these features are not known to be associated with pik cd variants, making this individual a candidate for > genetic diagnoses. conclusions: we developed a benchmark dataset where clinical features of patients were described using hpo terms. this dataset allowed us to quantify variable expressivity for certain pid subtypes and to systematically identify potential candidates for multiple diagnosis or phenotypic expansion. abstract/case report text warts, hypogammaglobulinemia, recurrent infections and myelokathexis syndrome is a rare combined immunodeficiency due to autosomal dominant gain-of-function mutations of cxcr chemokine receptor. the late diagnosis of whim syndrome in two ukrainian adolescents highlights the diagnostic challenges in this disease. patient , year-old girl, had recurrent pneumonia since the first year of age; overall she had episodes of pneumonia. she has suffered from chronic bronchitis for last several years. she had recurrent otitis media and chronic pyelonephritis. neutropenia was revealed when she was year old. during episodes of bacterial infections she occasionally had normal value of neutrophils. the girl does not receive any treatment. patient , year-old boy, had three episodes of pneumonia when he was , and year old. others symptoms include recurrent herpetic infection, warts on the hands. since years of age he has haven persistent low neutrophil counts. the child was followed by hematologist and since years of age he has received g-csf ( mg/kg) twice a month. both children have leukopenia - cells/mm , neutropenia - - cells/mm , lymphopenia - cells/mm , low number of bcells - - cells/mm . hypogammaglobulinemia was not prominent in both children, they have slightly decreased level of igg ( , g/l), normal level of igm ( , - , g/l), patient has low level of iga , g/l. patient does not have protective level of antibodies to diphtheria and tetanus anatoxin, and anti-hbs antibodies were absent despite complete immunization. bone marrow aspirate revealed hypercellular marrow with granulocytic hyperplasia which was characterized by hypersegmented nuclei and cytoplasmic vacuolization of neutrophils. on molecular analysis of cxcr , heterozygous mutation c. c>t (p.arg *), known as r x mutation, was detected in both patients, confirming the diagnosis of whim syndrome. replacement therapy with intravenous immunoglobulin was started in both children together with antibacterial prophylaxis and g-scf. vaccination with -valent vaccine against hpv infection was recommended for both patients. whim syndrome is very rare immunodeficiency but may be underdiagnosed. the awareness about rare forms primary immunodeficiency is very important in clinical practice for early diagnosis and treatment. methods: clinical providers recruited from nicer institutions electively completed web-based survey questions related to provider characteristics as well as initial diagnostic evaluation of itp, aiha, ain and es via securequestionpro® software. likert scales ranging from ("rarely" < %), ("sometimes" to %), ("half the time" % to %), ("frequently" to %), and ("almost always" to %) were used to ascertain frequency of evaluation for each diagnostic study. statistical analysis and plotting was done using rv . . . plots were created using packages ggplot , v . . and ggiraphextra v . . . mean likert scale scores were calculated for each study for each suspected disease and plotted on radar charts. results: the survey was completed by providers, including hematology/oncology ( . %), rheumatology ( . %), allergy/ immunology ( . %) and other sub-specialties ( . %). a slight majority of physicians ( %) were fellows or within years of graduation; physician extenders and clinical pharmacists were also respondents. the majority ( . %) of respondents indicated that ≤ new immune-mediated cytopenia patients were seen at their institution annually. the vast majority of respondents ( . %) reported evaluating ≤ new es patients per year at their institution with % evaluating ≤ cases annually. collated data from all respondents showed that in all disease states, the primary evaluation was focused on peripheral destruction mechanisms; the majority of patients are only "sometimes" or "rarely" evaluated for bone marrow failure syndromes, connective tissue disease, immunodeficiency and non-malignant lymphoproliferative disorders, but when done were more likely in es ( figure ). evaluations were biased by sub-specialty with higher degrees of connective tissue focus by rheumatology and immunodeficiencies by allergy/immunology (table ) . genetic sequencing was "frequently" or "almost always" sent in . % of itp, . % of aiha, . % of ain and . % of es p a t i e n t s . p e r s o n a l o r f a m i l y h i s t o r y o f a u t o i m m u n e / hyperinflammatory disease, malignancy or cytopenias most strongly influenced the decision to send genetic testing. lack of insurance coverage/negative financial impact on the patient and concerns about the inability to resolve variants of uncertain significance were the biggest barriers for obtaining genetic testing. conclusions: current practices in the evaluation of immunecytopenias are heterogeneous by sub-specialty and globally limited in scope with few patients being evaluated for underlying etiologies. in particular, despite a known high frequency of pathogenic variants in es, less than a third of patients are undergoing sequencing, highlighting a need to reduce barriers to genetic testing. development of a consensus guideline with multi-disciplinary engagement to harmonize an optimal evaluation for patients with immune-mediated cytopenias is needed. interferon regulatory factor- (irf ) binding protein- (irf bp ) was originally identified as a transcriptional co-repressor of irf ( ). mutated irf bp was identified in a -member family with recurrent sinopulmonary infections, progressive hypogammaglobulinemia, and poor response to protein vaccines ( ) . we have now identified additional families ( subjects) with irf bp mutations. clinical histories show an expanded phenotype with / having chronic gastrointestinal disease; with gastrointestinal manifestations as the initial clinical complaint. five had granulomata in liver(x ), spleen, lung(x ) and gastrointestinal tract. five out of six tested had poor pneumococcal vaccine responses and four patients reported viral infections including varicella zoster(x ), influenza a and sapovirus. irf bp is a amino acid protein containing a highly conserved cterminal protein-protein interaction ring domain (rd). constraint metrics from gnomad indicate mild tolerance to missense changes and intolerance to loss-of-function alleles. we identified categories of mutations: rd mutation or deletion (n= patients), null alleles (n= ) and non-rd missense changes (n= ). functional studies assessing the ability to affect nfatdriven luciferase expression were performed. rd mutations ( / ) had more profound loss-of-repression than wild-type, while missense changes had lesser, but still measurable effects. further, mutation categories and functional studies correlated with clinical phenotypes. of patients with rd mutations, / had infections as presenting symptoms, / tested had hypogammaglobulinemia and / were diagnosed with cvid. one patient with a missense rd mutation had only an infectious phenotype (pulmonary mycobacterium avium) with slight decrease in immunoglobulins; in functional studies this mutation had the least effect of the rd mutations. haploinsufficient patients reported respiratory infections ( / ), recurrent urinary tract infections ( / ), gastrointestinal disease ( / ) and hypogammaglobulinemia ( / ). in contrast, / patients with non-rd missense changes presented with gastrointestinal complaints while only patients had infections (recurrent bronchitis, shingles). gi disease prevalence is consistent with high levels of irf bp expression in the colonic crypt cells (human protein atlas). to confirm this, immunohistochemical staining of colon biopsies from two patients was performed, identifying epithelial and glandular cells of the colon. irf bp is involved in multiple processes, including the negative regulation of nfat signaling( ), tcr signaling( ), inflammatory macrophages ( ) , and pd-l transcription ( ) . interaction with the glucocorticoid receptor affecting anti-inflammatory and metabolic transcription ( ) has also been reported. these observations highlight the irf bp response to type-i interferons (irf ) and tcr stimulation (nfat), regulation of inflammatory macrophages and co-regulation of glucocorticoid receptor mediated signaling. the expanding role of irf bp in multiple biologic systems correlates with the broad clinical presentation we observed in our patients. further studies utilizing irf bp mutation knock-in mice will help characterize the gastrointestinal, lung and immune pathology seen in our cohort. abstract/case report text common variable immunodeficiency (cvid) is a disorder of antibody deficiency arising from over genetic lesions. the clinical presentation of patients with cvid varies from recurrent, severe infections to autoimmunity. the immune dysregulation in cvid is especially difficult to treat and the lifespans of patients suffering from autoimmunity are much shorter than those without such complications. unfortunately, we have no way to identify which patients fall into which categories, or even know how many sub-categories of cvid there are. therefore, the field requires a method to classify patients into categories to precisely recognize and aggressively treat the more severe phenotypes. we address this goal by integrating analyses of patient exomes with analyses of cellular signaling. by analyzing stimulation assays with phospho-protein mass cytometry and high-dimensional data analytics, we aimed to elucidate signaling and phenotyping deficiencies in patients with cvid. importantly, our panel identifies all circulating immune cell subsets in whole blood. in eosinophils, we found amplified responses of pp , pstat , and cleaved caspase- in response to tlr / stimulation. we found additional amplified responses of pstat and pstat in cd lo monocytes. this finding suggests a previously unidentified role for eosinophils and cd lo monocytes to contribute to the pathophysiology of cvid. we found abormal numbers of memory b cell counts, total switched b cell counts, and igm+, cd + b cell (plasmablasts) counts between cvid patients and healthy controls. cd expression on b cells was significantly reduced in cvid patients as well. these b cell results mirror findings from prior, seminal studies on cvid. notably, we have found higher pd- expression in the effector cd t cells of patients. integrating phenotype data, genetic analysis, and mass cytometry data will provide a deeper understanding of each patient's phenotype and how the are clustered. we also expect that a better understanding of alterations in the exomes and functions of the circulating immune cells of cvid patients will lead to new therapeutic approaches. abstract/case report text objectives: primary atopic disorders are monogenic disorders leading to profoundly dysregulated allergic responses. studying patients with these disorders has been instrumental in expanding our understanding of the pathogenesis of allergic inflammation with therapeutic implications for common polygenic versions of allergic disease. clinical findings: we have identified a now -year old boy who presented with severe eczema, extremely high blood eosinophil counts ( . x cells/l, normal range: - . x cells/l) after birth and very high serum ige levels ( υg/l, normal range: - ug/l) since birth. known allergic disorders and parasitic infections are ruled out. given the extreme phenotype, whole exome sequencing was performed on the trio of patient and parents, and the patient was found to have a homozygous mutation in the evolutionarily conserved fibronectin iii domain of the osmr gene (c. t>a, p.v d) (figure ). osmr encodes oncostatin m receptor-beta, a component of both the osm type ii receptor and the il receptor, and is important for keratinocyte cell proliferation, differentiation, apoptosis and inflammation. mutations in osmr have been reported in association with familial primary localized cutaneous amyloidosis, however this condition was ruled out in this patient through skin biopsy which showed no amyloid deposits. methods and results: we modelled the c. t>a osmr mutation in hek cells and observed a loss of expression of the osmr receptor on the cell surface (with normal intracellular protein levels). this observation was mirrored in primary fibroblasts obtained from the patient. signal transduction through phosphorylation of stat and stat and gene expression (il and ccl measured via qpcr) was absent after stimulation with osm in patient fibroblasts. these signaling defects were rescued using a lenti-viral transduction approach to introduce the wild-type (wt) osmr gene. whole transcriptome analysis using rna sequencing confirmed that osm mediated jak-stat signalling pathways were deficient in the patient fibroblasts and were rescued after lenti-viral transduction of wt osmr. rna sequencing analysis also suggested significantly enhanced expression of genes in the nf-κb signalling pathway (e.g.: il and cxcl ) and decreased expression of genes in the tgf-β signalling pathway (e.g.: smad and smad ) in patient fibroblasts at baseline. this was also rescued upon lentiviral transduction. conclusion and future directions: our findings shed light into the disease mechanism of a novel primary atopic disorder, caused by a homozygous missense mutation in osmr. abstract/case report text -year-old caucasian female presented to immunology clinic with hypereosinophilia, eosinophilic esophagitis, peptic ulcer disease, severe gi bleeds, and chronic hepatitis. healthy throughout childhood, with minimal infectious history. in adolescence developed chronic severe myalgias and nsaid overuse, to which the peptic ulcer disease and bleeding were attributed. parents healthy and non-consanguineous. son with severe bleeding episodes and small stature. on exam she weighed lb, bmi . sclerae anicteric. tongue deeply furrowed. cervical nodes palpable. heart and lung exam normal. no hepatosplenomegaly. no clubbing of the digits or edema. skin was clear. wbc , /ul, eosinophils /ul, hemoglobin g/dl, normal platelet count. however, platelet aggregation testing abnormal. bone marrow normocellular, and flow cytometric and molecular analysis did not show hematolymphoid malignancy, primary hypereosinophilic syndrome, or systemic mastocytosis. lymph node biopsies did not show lymphoma or aberrant t cell populations. noted to have chronically elevated creatine phosphokinase, ranging from - u/l over two years at our institution. deltoid muscle biopsy showed non-specific myelopathic changes. an adult dystrophy immunostaining panel was normal. ultrastructure examination showed no abnormal storage material. a genetic panel for metabolic myopathies failed to reveal a cause. total igg, iga and igm normal. ige elevated at ku/l, and igg subclasses showed igg elevated at mg/dl. flow cytometry showed normal t, b and natural killer cell numbers. normal proportions of naïve, mature and activated t cells. vaccine response assessment was normal. evaluation for autoimmune/rheumatologic diseases was negative. liver biopsy demonstrated findings consistent with primary or secondary sclerosing cholangitis (without increased igg staining). given her inflammatory phenotype, additional genetic analysis was sent, assessing for primary immunologic disorders. this identified heterozygous variants of uncertain significance in ctla (c. t>a; ps t), zap (c. c>g; p.d e), and stim (c. t>c; p.l s). analysis of the ctla variant in vitro revealed that it was expressed normally. foxp expressing regulatory t cells were present in normal proportions in vivo and appeared phenotypically normal. this variant was found in her unaffected father. the zap variant is present in population databases (rs , exac . %), and was felt unlikely to be clinically relevant. the stim l s variant, although not shown previously in human patients, has been previously shown in vitro to be a gain of function mutation [ ] [ ] [ ] . furthermore, familial analysis revealed that this was a de novo mutation arising in the patient, and present in her son. humans with other gain of function mutations in stim and the orai channel it activates have overlapping syndromes including storkmorken syndrome, tubular aggregate myopathy and york platelet syndrome, characterized by chronic myopathy and platelet aggregation defects [ ] . the stim l s mutation is predicted to cause constitutive stim activation and calcium influx and likely provides an explanation for the patient's chronic myopathy and abnormal platelet aggregation. neither eosinophilic disease, nor cholangitis, have been described previously in stim gain of function-related diseases. it is unclear whether these issues are related to this novel stim mutation, or to other genetic or environmental influences. treatment of diseases caused by overactive crac channels is challenging as no pharmacologic inhibitors are yet clinically available. nomid/cinca syndrome is one of the periodic syndromes associated with cryopyridines. it is a defect in the innate immune system causing excessive activation of the inflammasome, with consequent il- secretion and neutrophil recruitment. clinically, damage occurs to organs such as the skin (neutrophilic urticaria), central nervous system (meningitis and deafness) and joint (arthritis). levy et al. ( ) evaluated a large series of patients and median onset age was . years, while the median age at diagnosis was years, although the symptoms initiate in the first days of life. treatment includes corticosteroids, which act by nonspecifically blocking all inflammatory cytokines, or by blocking il- specifically. if early diagnosis and treatment of the disease is not made, natural evolution leads to motor and adaptive disability and death in % of cases already in adolescence due to infection, neurological complications or secondary amyloidosis. we report a -month-old male child from nonconsanguineous parents who presented shortly after birth, multiple scaling and erythematous lesions throughout the body, evolving with following symptoms: abdominal abscess, hepatitis, meningitis and pioarthritis. laboratory tests showed elevation of inflammatory tests (esr, crp, amyloid protein a) and leukocytosis. the diagnosis was suspected at the nursery where the patient remained hospitalized for days. a personalized multigene panel was requested. it was identified the variant p.gly val, heterozygous for nlrp gene, not described in the literature, confirming the diagnosis of cinca/nomid syndrome. after discharge, it was introduced prednisolone ( , mg/kg/day) and antiinterleukin- (il- ). after the second dose, skin lesions and joint edema regressed, weight gain, and neuropsychomotor development improved. this case reports a very early diagnosis of nomid/cinca syndrome. it warns neonatologists and pediatricians about the need of precocious recognition of the syndrome, probably improving the prognosis of the patient. professor/university center health abc abstract/case report text background: leprosy affects more than , people worldwide. brazil represents the rd. country in the world in leprosy frequency and maranhão state is an hyperendemic region. the city of imperatriz (ma) stands out as a reference center in the care of these patients. according to few reports, lectin pathway of complement system may play a role in susceptibility to leprosy. mannose binding lectin (mbl) and ficolins (fcns) recognize patterns of sugars and acetylated residues (pamp), respectively, in a wide variety of pathogens, including m. leprae. high levels of ficolins and mbl may act unfavorably promoting the spread of m. leprae. the present study evaluated the role of ficolin and mbl in m.leprae patients and contacts. methods: a cross-sectional case-control analytical study was carried out, evaluating clinical and epidemiological data and serum levels of mbl and fcn (elisa) from july to april . the study was approved by ethics committee and informed consent forms were signed before sample collection. data analysis was performed using the spss . for windows statistics program. results: we evaluated serum samples ( patients and healthy family contacts), . % were female, % under years old, % african-brazilian, % of the families had more than contacts at home. clinical data showed multibacillary forms in . %; dimorphic ( %) and virchowian clinical forms ( . %), up to affected nerves in ( . %) and more than lesions in ( . %). it was observed that ( . %) had a reaction, being type ( %) more predominant. disability grade was found in patients ( . %). in children under years, . % were multibacillary, . % dimorphic and % undetermined; ( . %) also had reactions, % type reaction and degree of disability in . % of children with the disease. the evaluation of serum fcn and mbl levels for the patients (n = ) and contacts (n = ) were . ng/ml and . ng/ml, (p = . ), and . ng/ml (p) and . ng/ml (c) (p = . ), respectively. there were lower values of fcn in patients with type reaction (sudden and intense inflammatory processes) versus no reaction ( . ng/ml vs . ng/ml) (p = . ) and in patients with disability grade (severe sequelae) versus disability grade ( . ng/ml vs . ng/ml) (p = . ). higher fcn values was observed in patients with no disability ( . ng/ml) (p = ). mbl concentrations were higher for patients above years in comparison with patients below that age ( . ng/ml vs . ng/ml)(p = . )) and correlated with the occurrence of a multibacillary clinical form. conclusions: mbl and fcn levels were not different in the patients and contacts of m. leprae, nevertheless the presence of severe forms with sequelae (reaction type and disability grade ) were associated with lower levels of fcn . in addition, it is possible that lower mbl levels could influence the higher frequency of multibacillary disease below years old. abstract/case report text introduction: hyper ige syndrome (hies) is a primary immunodeficiency characterized by elevated ige levels. symptoms can range from severe eczema, recurrent skin infections or pneumonias, and typical dysmorphic facies. there have been wide non-immunologic presentations in patients with hies, including retained primary teeth, scoliosis, craniosynostosis, arterial aneurysms and joint hyperextensibility. an association between hies and autoimmune hemolytic anemia (aiha) has further been described in the literature. however, there have been no reported cases of hies in association with iron deficiency anemia and concurrent pica. we present a unique case of a patient with a history of eczema, recurrent skin infections and pica found to have hies and iron deficiency anemia. case presentation: a -year-old boy with a history of allergic rhinitis presented to the allergy & immunology clinic for evaluation of chronic eczema and recurrent skin infections. the patient had a history of multiple hospitalizations requiring intravenous antibiotics for cellulitis and superinfected eczema since he was an infant. symptoms were refractory to the use of multiple skin barrier ointments and oral antihistamines. his mother further noted that for the past two months prior to initial evaluation, he developed a fixation with eating crayons, baby powder and chewing on drywall. physical exam was notable for a dysmorphic face, broad based nose, pale nasal mucosa with ample clear discharge, high-arched palate and lower incisor supernumerary teeth. his skin was characterized by generalized dryness, lichenification and scaly desquamation with boils on extensor surfaces of knees and elbows. initial screening for hies via t-helper functional assay was consistent with decreased expression of il- . genetic testing revealed stat s g missense pathogenic variant consistent with hies. cbc was also notable for decreased hemoglobin at . g/l and mcv of fl. patient was diagnosed with concurrent hies and pica in the setting of iron deficiency anemia. iron supplementation was started and patient's pica improved. discussion and conclusion: our patient with hies had a peculiar initial presentation with the classic signs and symptoms of hies and pica. the diagnosis of hies can often be delayed due to the wide range of clinical presentations. to our knowledge, the association of hies with iron deficiency anemia and pica has been underreported in literature. screening for anemia should be considered when evaluating patients with hies in order to rule out comorbid iron deficiency anemia which can be easily treated with iron supplementation. abstract/case report text introduction: common variable immunodeficiency is a primary immunodeficiency with variable and diverse phenotypic presentations. the two main phenotypes include a group which primarily exhibits recurrent infections and a group with or without infections and primarily inflammatory and autoimmune complications. the latter, may lead to a delay in diagnosis and is associated with poorer outcomes and higher morbidity and mortality. ( ) another group of patients present with t-cell defects, lung disease, autoimmunity, and infections and may be diagnosed as having cvid but instead can have mutations in lrba or pi kinase. this subset of patients has been referred to as "cvid-like" in the literature. ( ) case presentation: patient is an year old female who initially presented to an outside facility due to days of fatigue, fever, and abdominal pain. upon presentation, she was found to have massive splenomegaly, hepatomegaly, and an abnormal chest x-ray showing mediastinal lymphadenopathy and pleural effusion. laboratory results demonstrated pancytopenia, hypogammaglobulinemia, and low b cells, t cells, and nk cells via flow cytometry. she was transferred to our institution for further work up. she did not have any prior history of recurrent infections, asthma/lung disease, or autoimmune conditions. initial ct of the chest was consistent with granulomatous lymphocytic interstitial lung disease. patient was diagnosed with common variable immunodeficiency with granulomatous lymphocytic interstitial lung disease and was treated initially with high dose ivig, corticosteroid taper, rituximab, and imuran. she had interval worsening of pft and lung disease as shown by ct scan. genetic panel for cvid and related conditions revealed variants of unknown significance. one heterozygous mutation in blnk gene (c. g>a) and one heterozygous mutation in lrba gene (c. g>a). she was started on infliximab with plans to repeat ct scan in months. discussion: mutations in both blnk and lrba have been associated with primary immunodeficiency. mutations in blnk, which is located on chromosome , have been associated with autosomal recessive agammaglobulinemia. homozygous or compound heterozygous mutations in lrba on chromosome , can lead to lrba deficiency which encompasses a wide range of clinical presentations including hypogammaglobulinemia, autoimmune disease, inflammatory bowel disease, antibody deficiency, organomegaly, and recurrent infections. ( ) without genetic testing, the clinical presentation can be difficult to distinguish from common variable immunodeficiency. the patient presented has clinical features that can be seen with mutations in both blnk and lrba, however she is heterozygous for both mutations. further analysis, including measurement of lrba protein expression, is needed to further define her underlying immunodeficiency so appropriate treatment can be administered. abstract/case report text a month-old, previously healthy, unvaccinated male presented with one week of diarrhea and cough and was admitted for dehydration and hypoxemia. his mother and sister both had a history of incontinenti pigmenti (ip). on physical exam, he was alert, afebrile, with tachypnea and subcostal retractions. enterovirus/rhinovirus and parainfluenza were detected, but he became progressively hypoxemic and eventually required intubation and high-frequency oscillatory ventilation. chest x-ray showed multifocal bilateral airspace opacities. empiric treatment for pjp with trimethoprim/ sulfamethoxazole and glucocorticoids was started. tracheal aspirate pcr confirmed p. jiroveci. hiv rna pcr was negative. ivig was started due to suspicion for primary immunodeficiency. although his respiratory status gradually improved, he subsequently developed multiple skin lesions. skin biopsy grew mycobacterium szulgai. m. szulgai osteomyelitis of the right fibula and the left nasal bone was also detected, indicating hematogenous spread of the infection. he was started on four-drug anti-mycobacterial therapy and interferon-gamma (actimmune) at doses ranging from μg/m^ three times weekly to μg/m^ qod. immune work-up revealed t-cell lymphopenia [cd +/cd + /μl ( - , /μl) and cd +/cd + /μl ( - , /μl)] with an abnormally increased proportion of memory cd t-cells compared to naïve cells for age. b-cell numbers were normal, and nk cells were decreased [cd +cd +/cd - /μl ( - /μl)]. nk cell lytic function by k lysis was normal, whereas cd a degranulation was decreased. the serum igm level was normal [ mg/dl ( - mg/dl) whereas iga [ mg/dl ( - mg/dl)] and igg [ mg/dl ( - mg/dl)] were elevated. mononuclear cell cytokine response to ligands for tlr -tlr , tlr -tlr , tlr , tlr , and tlr -tlr was normal. dna sequencing revealed a novel nonsense mutation in exon of the ikbkg (p.gln ter (q x) (cag>tag): c. c>t, confirming the diagnosis of nemo deficiency, which was suspected based on the infectious disease presentation and the maternal history of ip. the diagnosis was further supported by signs of ectodermal dysplasia of teeth that appeared starting at months of age. he underwent hsct using bone marrow from a / matched unrelated donor after conditioning with atg, busulfan, fludarabine and rituximab. actimmune therapy was continued until days prior to transplant. for gvhd prophylaxis, he received tacrolimus and low-dose methotrexate. he achieved full donor chimerism post-transplant and has had no significant gvhd. interesting features of this case include the prominence of ip in mother and sister, which is usually due to female heterozygosity for an ikbkg null allele. such null alleles when inherited by the male fetus are embryonic lethal. our patient's nonsense mutation would be expected to result in severely impaired ikbkg protein expression and function. however, the fact that he had was born at term and initially was healthy coupled with his preservation of normal tlr function suggests that his ikbkg allele is likely to be a hypomorphic mutation. studies are in progress using ebv-transformed b-cell lines from the patient to evaluate ikbkg expression and function. also of interest, our patient was able to tolerate relatively high doses of interferon-gamma therapy without inflammatory side effects or an adverse impact on engraftment or gvhd. abstract/case report text background: primary atopic disorders are caused by genetic mutations that skew the immune system towards severe allergic disease. germline gain-of-function (gof) mutations in jak are a newly described monogenic cause of severe atopy, with affected patients demonstrating profound eosinophilia and allergic inflammation. our initial report of this novel condition identified a dramatic clinical response to the combined jak / inhibitor ruxolitinib. we aimed to determine the long-term clinical response to ruxolitinib in patients carrying a germline jak gof mutation, and to characterize the effect of enhanced jak signaling on t lymphocyte effector functions and hematopoiesis. methods: clinical outcomes were evaluated in two pediatric patients carrying the c. c>a (p.a d) gof mutation in jak after . years of ruxolitinib treatment. t cell phenotyping was performed using extracellular surface marker and intracellular cytokine staining by flow cytometry, and by gene expression signature profiling of rna sequencing data. to evaluate the effect of enhanced jak activity on myelopoeisis, we reprogrammed jak gof patient-derived peripheral blood mononuclear cells into induced pluripotent stem cells (ipsc) and performed directed myeloid differentiation. rna sequencing was performed on rna collected during ipsc myeloid differentiation and from whole blood of affected patients before and after ruxolitinib treatment. results: long-term use of ruxolitinib was associated with improved growth, reduced eosinophilia, and control of allergic inflammation without significant infectious complications, however, anemia represented a dose-limiting adverse effect. t cell immunophenotypic analysis revealed severe t helper (th) cell skewing towards a th phenotype preruxolitinib treatment, in keeping with the allergic clinical manifestations. analysis of myeloid differentiation revealed an increased myeloid to erythroid ratio in colonies derived from jak gof ipscs compared to controls. rna sequencing analysis of jak gof human whole blood and ipscs compared to controls revealed upregulation of cytokine and cytokine receptor genes implicated in allergic inflammation and early eosinophil precursor commitment, including csf- and the interleukin- receptor. reactome pathway analysis of genes upregulated in both jak gof ipsc and whole blood compared to controls showed enrichment of several pathways including interferon alpha/beta, interleukin- /- and interleukin- signaling. conclusions: this work demonstrates a critical role for jak in atopic immune dysregulation, specifically driving a th phenotype and eosinophilia. combined jak / inhibition can reverse much of the allergic inflammation, with dramatic clinical effects. this has important implications for our understanding of the pathogenesis and potential therapeutic targets for early life allergic immune dysregulation. had severe combined immunodeficiency (scid) and/or severe disease in association with their combined immunodeficiency (cid) necessitating haematopoietic stem cell transplantation (hsct). we present clinical and laboratory features of new zealand patients from the same family with a novel heterozygous missense variant in rac [c. t>g, p.ile ser (i s)]. the index patient (p -age y, m) has a history of infectious gastroenteritis, staphylococcal aureus conjunctivitis, recurrent otitis media and recurrent herpes simplex virus (hsv)- cutaneous infections. his siblings (p age y, m; p -age y, f) and his mother (p age y, f) all have a history of recurrent viral (hsv- ) and bacterial (staphylococcal aureus, streptococcal pyogenes) cutaneous infections and/or recurrent sinopulmonary infections that respond to empiric antimicrobial therapy. their neutrophils all had enhanced superoxide production in response to stimulation by fmlp and pma as compared to healthy controls'. these findings suggest that rac i s is an activating mutation causing notable abnormalities in neutrophil morphology and nadph oxidase activation similar to other recently reported mutations. this novel mutation expands the phenotypic spectrum of rac activating mutations. clinical management of affected patients needs to be tailored to their phenotype and disease severity. background and aims: heterozygous mutations in cytotoxic tlymphocyte antigen- (ctla ) are associated with recurrent infections, lymphoproliferation, autoimmunity and lymphocytic infiltration of target organs. disease penetrance can be highly variable even among related family members carrying the same ctla mutation. our evaluation of a subset of the ctla patient cohort followed at the national institutes of health (nih) revealed that % of ctla mutation carriers have gastrointestinal (gi) manifestations which include diarrhea and diffuse lymphocytic enteropathy. our aim was to determine whether the intestinal microbiome, metagenome and metabolome could distinguish patients with ctla haploinsufficiency (ctla -h) based on disease severity, and the presence or absence of gi manifestations. methods: clinical metadata and fecal samples were collected from healthy individuals (n= ) and patients with ctla -h (n= ). patients with ctla -h were classified as having minimal (n= , only endocrine and/or dermatological manifestations) or systemic disease (n= , hematological and multi-organ involvement). they were further classified based on whether they had a history of enteropathy (n= ) or active gi disease ( < bowel movements per day and/or blood or mucus in stool) at time of sampling (n= ). metabolomic profiling (using a panel of metabolites) and s rrna gene sequencing (v region) was performed on fecal samples (total samples: ; number of reads/sample: , to , ; median: , ). a subset of samples were subjected to shotgun metagenomic sequencing based on findings from the s rrna gene sequencing analysis. results: all patients with ctla -h and a history of enteropathy or active gi disease also had systemic disease. fecal samples from patients with a history of enteropathy had a distinct microbial community structure (fig. ) which was significantly less diverse (fig. ) compared to healthy individuals and patients with minimal vs. systemic ctla -h. patients with a history of enteropathy had significantly higher relative abundance of bacterial taxa including shigella-escherichia (fig. ) . shotgun metagenomic sequencing confirmed that samples from patients with a history of enteropathy were dominated by subsets of identified escherichia coli strains, all of which share genes coding for specific types of virulence factors such as curli fibers (facilitate uptake into host cells), flagellar proteins (increase motility) and enterobactins (increase bacterial iron transport). meanwhile, samples from patients without active gi disease at the time of collection were enriched for several taxa including bacteroides nordii and akkermansia muciniphila compared to patients with ctla -h and active gi disease (fig. ) . metabolomic analyses showed that asparagine, -hydroxybutyrate, cytosine and cystine were enriched in samples with abundant e. coli, whereas samples without e. coli were enriched in metabolites involved in pyrimidine (holm p= . ), purine (holm p= . ), and alanine/aspartate/glutamate metabolism (holm p= . ) (fig. ) . conclusions: fecal samples from ctla -h patients with a history of enteropathy were heavily colonized with e. coli strains that are associated with a specific metabolomic profile and that share virulence factor genes that may facilitate host invasion. these data suggest that the microbiome and metabolome can distinguish patients with ctla -h and gi disease, and support the potential use of antibiotics or even antimetabolites to treat ctla -hrelated enteropathy. the dna polymerase delta (pol δ) complex is essential for leading and lagging dna strand synthesis. its catalytic subunit (pold ), carries both polymerase and exonuclease activities and plays a crucial role in dna replication and repair. heterozygous pold mutations have been associated with inherited colorectal cancer and mandibular hypoplasia, deafness, progeroid features and lipodystrophy (mdpl) syndrome. more recently a biallelic loss of function mutation in pold (p.r c) that impairs the stability of the pol δ complex, has been reported in related subjects with recurrent infections, deafness and combined immunodeficiency (cid) with t-cell lymphopenia, cd + t cell oligoclonality but preserved b cell proliferation. we report here a second family in which a novel biallelic missense mutation in pold gene was associated with cid. the proband is a -year-old boy born to consanguineous pakistani parents. since infancy he suffered from failure to thrive and recurrent infections, including episodes of pneumonias, multiple otitis media, sinusitis, recurrent cellulitis at the g tube site, bk viruria and shingles. live and dead vaccines were well tolerated. at years of age sensorineural hearing loss together with profound leukopenia (anc cell/μl, alc cells/μl) and hypogammaglobulinemia ( mg/ dl) were identified. intermittent ivig replacement and antimicrobial prophylaxis were initiated. immunophenotyping at years of age showed severe t cell lymphopenia ( cd + cells/μl, cd + cells/μl, cd + cells/μl, figure . volcano plot of metabolites present in fecal samples enriched with e. coli vs. samples without e. coli. c e l l s / μ l , c d + c d h i f o x p + c e l l s / μ l ) , a n d hypogammaglobulinemia (igm mg/dl, igg mg/dl, iga < ). physical exam was remarkable for multiple acquired nevi in the groin area, teeth abnormalities and global developmental delay. whole exome sequencing analysis revealed a homozygous pold missense variant (nm_ c. c>g, p.q e) absent in public databases (cadd score of ). parents were heterozygous. tcr-vβ family expression was normal in both cd + and cd + t cells, but the proportion of t cells expressing vα . (encoded by the distal trav - gene) was markedly reduced (less than %), consistent with impaired vdj recombination at the tra locus and/or with defective thymocyte survival. constitutive expression of γh ax was observed in t and nk cells after h and h of culture in unirradiated conditions. at h post-irradiation ( gy), reduced levels of p-atm were detected in t and nk cells, and lack of atm, smc and h ax phosphorylation was observed in a subset of b cells, suggesting inability of these cells to mount an effective dna repair response. bone marrow examination showed normal trilineage hematopoiesis but decreased proportion of cd -cd + mature b cells and increased proportion of pre-b cells. conclusion: we report the second mutation associated with autosomal recessive pold deficiency. our findings broaden the understanding of the mechanisms underlying the immune defect in this disease to include b cell maturation arrest in the bone marrow and a dna repair defect that may support the generation of a restricted tcr repertoire in the thymus and increased malignancy risk. abstract/case report text following allogeneic hematopoietic cell transplantation (hct) for scid, the development of a diverse t cell repertoire is essential for optimal immune recovery. high-throughput sequencing (hts) of the trb repertoire is the best tool for the evaluation of clonotype dynamics during immune reconstitution as compared to cdr spectratyping and staining of vβ families. we investigated whether longitudinal hts analysis of trb would accurately assess development of tcr repertoire diversity over time and reflect the quality of t cell reconstitution following hct for scid. we wanted to study the effect of conditioning regimen, scid genotype, donor type on tcr diversity post hct. we hypothesized that repertoire diversity may represent an early biomarker to predict long-term immune reconstitution vs. need for a second intervention. we assessed if the trb repertoire post-hct carried a molecular signature of selfreactivity. methods: the composition and diversity of trb repertoire of scid infants, pre-hct and at d, and mo and yearly posttreatment(s) was studied by hts. median time of follow-up was mo. subjects were part of a prospective study of scid by the primary immunodeficiency treatment consortium. equal amounts of total rna extracted from peripheral blood was used as template to semi-quantitatively amplify trb rearrangements. the vdj statistics file (past program) was used to calculate a shannon entropy (h) index of repertoire diversity and simpson ( -d) index of repertoire clonality. results: trb sequence analysis of scid patients showed poor diversity at baseline, followed by improvement to normal complexity (h index > . ) after hct. similar kinetics of development of trb diversity were seen in patients with il rg, jak , and il r defects (n= ) as in those with rag and artemis defects (n= ). in the latter group, however, hct with no conditioning or immune suppression only was associated with persistently lower diversity than hct with conditioning (p < . ), a difference not found in the il rg/jak /il r group (fig. ) . hct from a matched donor ( / conditioned) correlated with higher diversity than hct from a mismatched donor ( / conditioned) (p= . ). having > cd + t cells/ul at mo post-hct correlated with higher trb diversity at and mo post-hct (p < . ). the trb repertoire d post-hct was enriched for the presence of central cysteines at the apex of the cdr (p < . ), a biomarker of self-reactivity ( fig. ). an h-index of . or lower at d after hct predicted need for second intervention (hct or gt) (fig. ) . conclusions: analysis of trb diversity allows for detailed assessment of development of a diverse t cell repertoire following cellular therapies for scid and confirms the need for patienttailored treatment strategies based on scid genotype. t-cell repertoire d post-hct is characterized by a molecular signature that may contribute to the increased rate of autoimmunity early post-transplant. furthermore analysis of trb diversity at d post-hct may identify patients at risk for failure of sustained immune reconstitution, thus prompting a second intervention without delay. abstract/case report text background atopic dermatitis is a chronic, multifactorial, relapsing inflammatory skin condition which is one of the main known health problem worldwide. atopic dermatitis lesions are frequently colonized by staphylococcus aureus and staphylococcus epidermidis. their susceptibility to form biofilms, ability to form adhesive skin colonies which lead to extremely resistant to antibiotics and immune responses. formation of skin biofilm resulted in complex bacterial communities that have unique effects on human keratinocytes, mouse fibroblasts and host immunity. aims: the aims of this study to confirm the specificity of s. aureus or its secreted factors in induction of pro-inflammatory cytokines il- , tslp and toxicity on human keratinocytes and mouse fibroblast. the second aim to study the inhibitory effect of co-culture of s. epidermidis with s. aureus in term of production of pro-inflammatory cytokines and toxicity. method and materials: human epidermal keratinocytes and mouse embryonic fibroblasts cell lines from t were used as a control strain to examine production of inflammatory response (il- and tslp) and cell death induced by s. aureus in the presence and absence of s. epidermidis. tslp and il- were detected by elisa and the apoptosis of s. aureus and s. epidermidis on these cells was evaluated by flow cytometry. result: recent findings propose the important role of skin biofilms in the pathogenesis of atopic dermatitis. s. aureus have been found to induce secretion of pro-inflammatory cytokines and cause apoptosis of human keratinocytes and mouse fibroblasts. presence of s. epidermidis as skin biofilm found to protects the human keratinocytes and mouse fibroblasts from induction of proinflammatory cytokines and cytotoxicity. conclusions and future work: s. aureus are essential in production of inflammatory response and cell death of mouse fibroblasts and human keratinocytes. future work will be carried out to identify the soluble factors that responsible in induction of pro-inflammatory cytokines. in addition, more studies are needed to be able to understand the mechanism by how s. epidermidis reduce the induction and cytotoxicity caused by s. aureus. j clin immunol in adult patients, in whom arbitrarily defined diagnostic criteria for antibody deficiency syndromes are not fulfilled, is subject to interpretation and decision differences reported by immunologists world-wide. in this study, we explored whether training in one particular program would decrease the variability in diagnostic and treatment approaches seen in the responses to two nationwide questionnaires in the uk and the usa. methods: a -minute online survey originally administered to a cross-sectional sample of us allergists/immunologists (usa/i) in january, , was also answered by a/i subspecialists who had trained in the last years at the louisiana state university health science center allergy immunology training program in new orleans (laa/i). respondents were asked questions on patient assessment, antibiotic use, initial igrt, and immune response assessment in decisionmaking to prescribe igrt. usa/i participants were recruited from the dynata physician professional panel. laa/i participants were recruited by the louisiana primary immunodeficiency network (lapin). results: overall, laa/i had consensus responses to the various practice questions close to % of the time, but outliers were always present, as was also observed in the usa/i. there was a higher frequency in the reported care of patients as described in the questionnaire by laa/i. over % of laa/i assessed vaccine responses prior to commencing igg replacement vs only % of usa/i p < . . all la a/i used the pneumococcal vaccine for assessment purposes while few used tetanus and hemophilus influenza, and none used meningitis or salmonella vaccines. these vaccines were still used by some of usa/i. a high level of concordance was observed among all respondents in that only few regarded pneumococcal antibody testing as the definitive test to commence igrt. high resolution chest ct scan was used more often by laa/i before starting igrt. assessment of effectiveness of igrt was decided after only months by more usa/i, vs laa/i, who tended to wait months to decide to continue or discontinue igrt. conclusions: all a/i responders saw a significant number of patients who do not conform to strict diagnostic criteria for antibody deficiency syndromes. there is diversity in the approach of usa allergists/ immunologists in determining the indication for igrt for non-classical antibody deficient patients. laa/i responses made it obvious that post graduate influences always play a role in shaping the way a/i practice evolves after graduation. drawing on clinician experiences through questionnaires offers a valid contribution to developing consent approaches to improve patients' clinical conditions. diagnostic criteria and treatment guidelines would benefit from practice-based realistic recommendations based on a/i experience. abstract/case report text background: autoimmune lymphoproliferative syndrome (alps) is a rare genetic disorder secondary to a defective fas-mediated apoptotic pathway of mature lymphocytes. it is characterized by chronic nonmalignant lymphoproliferation in the form of lymphadenopathy and/or splenomegaly, autoimmune manifestations such as cytopenias, increased risk of lymphoma, and expansion of tcrαβ+ cd -/cd -(dnt)t-cells. germline or somatic pathogenic variants in fas, fasl, and casp are well described genetic defects associated with alps. the definitive diagnosis for alps, based on the revised nih diagnostic criteria, include both required criteria (chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, or both and elevated tcrαβ+ dnt t-cells) and one of the primary accessory criteria (defective lymphocyte apoptosis or mutation in the genes mentioned above). patients who do not meet the current diagnostic criteria are considered for alps-related disorders. case presentation: we report a -year-old male who presented with recurrent infections, splenomegaly and chronic lymphadenopathy since month of age. due to its chronicity he was evaluated by multiple specialists for malignant and infectious causes. hematological workup including bone marrow biopsy was unremarkable except for an elevated ldh level. infectious workup identified a past cmv infection. clinical course is pertinent for chronic splenomegaly which was identified incidentally at . years of age during an evaluation for intussusception. family history is pertinent for a father with recurrent infections, paternal grandmother with thrombocytopenia of unknown cause requiring platelet transfusions, and paternal cousin with neutropenia. there is no family history of lymphomas. history of chronic lymphoproliferation and recurrent infections prompted an evaluation for lymphoproliferative disorder. full immune workup was notable for elevated plasma il- and il- , normal immunoglobulin levels, lymphocytes subsets, vitamin b level, soluble fasl, and relative frequency (%) but borderline increased absolute count of tcrαβ+ (dn) t-cells. in addition, he was noted to have presence of anti-platelet antibodies, poor lymphocyte proliferation to antigens, and low pneumococcal antibody titers. genetic testing with a pid gene panel identified a likely pathogenic heterozygous variant in prf c. del (p.his thrfs* ), a heterozygous variants of uncertain significance in casp c. c>t (p.pro leu) and stim c. a>g (p.thr ala). the casp variant is present in alleles in gnomad ( k total allele count) and reported deleterious by sift. discussion: unlike the typical alps presentation, characterized by dominantly lymphoproliferation and autoimmunity, our patient's clinical phenotype is striking for recurrent infections, abnormal t-cell function, and poor antibody response. our patient does not the meet diagnostic criteria for alps due to normal relative frequency of dn t-cells. however, presence of elevated of il- , il- , platelet autoantibodies raise concern for alps-related disorder. in addition, family history of recurrent infections and cytopenias raises concern for familial autoimmunity and alpslike phenotype. although casp is associated with autosomal dominant and autosomal recessive alps, the role of this vus is yet to be determined. conclusion: we continue to investigate the pathogenicity of our novel casp vus. further studies include pedigree analysis, fas apoptosis assay and apoptosis pathway testing to assess for the etiology of this alps-related disorder. (word count , max ) abstract/case report text rationale: ocrelizumab is a recombinant anti-cd monoclonal antibody, which binds to a different, but overlapping cd epitope than rituximab. there have been increasing reports evaluating hypogammaglobulinemia and morbidity and mortality in patients receiving rituximab, but there is a paucity of data on hypogammaglobulinemia in patients treated with ocrelizumab. methods: we performed a retrospective review of patients who received ocrelizumab in our healthcare system. we evaluated the demographics, indication for ocrelizumab, frequency of immunologic evaluation, and h y p o g a m m a g l o b u l i n e m i a p r e -a n d p o s t -o c r e l i z u m a b . hypogammaglobulinemia was stratified as mild (igg < mg/ dl or less than lab reference range), moderate (igg < mg/ dl) or severe (igg < mg/dl). results: we identified patients who received ocrelizumab for multiple sclerosis (average number of ocrelizumab cycles = ; range - cycles). there were ( %) female patients, with a mean age of years old (range - ; standard deviation ± t-cells. tnfα, ifnγ, and il- were not statistically different between cgd patients and healthy controls. tnfα, ifny, il- , and il- a expression in patients with cgd who had active colitis or history of colitis were increased as compared to cgd patients without a history of colitis but did not reach statistical significance. in two patients, il- a expression that was elevated pre-hct normalized post-hct. discussion: the mechanism for increased susceptibility to inflammatory disorders in patients with cgd has not been well elucidated. our results agree with previous studies demonstrating increased il- and il- a production from cd + t-cells in patients with cgd indicating a proinflammatory state in these patients at baseline. also, there appears to be an increase in tnfα, ifny, il- , and il- a expression from cd + t-cells that correlates with presence of inflammatory disease vs. those without inflammatory disease indicating that these cytokine perturbations may be able to serve as biomarkers of disease activity. a larger sample size with prospective collection will be analyzed in the future. abstract/case report text background: glucose- -phosphatase catalytic subunit (g pc ) deficiency, is characterized by severe congenital neutropenia, recurrent bacterial infections, mild intermittent thrombocytopenia and a high incidence of congenital cardiac and uro-genital defects. we report the case of a -yo male with chronic neutropenia and thrombocytopenia, who was found to have homozygous pathogenic variants in g pc (c. del, p.phe serfs* ). unique to this case is the patient's long history of misdiagnosis of evans syndrome (chronic autoimmune neutropenia with thrombocytopenia). case presentation: a -yo male with reported diagnosis of chronic autoimmune neutropenia and thrombocytopenia since age was referred to our a b clinic with concern for an underlying immune dysregulation syndrome. he had a history of oral ulcers, gingivitis, recurrent bacterial infections (otitis media, pneumonia, skin abscess) concomitant with severe neutropenia ( < cells/ul), for which he had received treatment with systemic steroids and g-csf since he was years old. he also had history of asthma and short stature, thought to be secondary to his chronic systemic steroid use. we present the first chilean patient with stat gof immunedysregulation . moreover, to our knowledge this is the first stat gof patient presenting with lymphomatoid granulomatosis. this is a severe pulmonary disease in which primary immunodeficiencies including stat gof should be considered in the differential. in this case rituximab successfully resolved pulmonary nodules and respiratory symptoms. there was persistence of mild hepatosplenomegaly but otherwise clinical stability and monitored expectantly until the age of . at that time he presented with fever, left knee and ankle arthritis. he underwent arthrocentesis of the left knee and left ankle, both aspirates were sterile, with notable leukocytosis with heavy neutrophilic predominance. an extensive rheumatologic and infectious workup was non-diagnostic. both sil- r and il- were elevated, , units/ml ( < ) and pg/ml ( < ), respectively. he was treated with systemic corticosteroids, ultimately arthritis resolved after months. at age he presented for the first time with periorbital pain and conjunctival injection of the left eye that persisted after minor trauma. he was found to have nongranulomatous uveitis, which responded ultimately to systemic corticosteroid. he then presented at age with fever and right knee and great toe arthritis. again he underwent arthrocentesis which revealed aseptic arthritis, and at that time was started on anakinra (anti il- β) and prednisone. there was clinical improvement over several weeks followed by return of right knee arthritis, coupled with onset of symptomatic uveitis of the left eye. despite systemic corticosteroids and anakinra and il- blockade, the patient was again admitted shortly thereafter to the hospital with arthritis, fevers, rash and abdominal pain. there was concern for evolving hlh and the patient was ultimately transferred to cincinnati children's for further evaluation and treatment. pertinent inflammatory biomarkers at that time included sil- r of , units/ml and il- level of , pg/ml. in addition to anakinra and systemic corticosteroids, the patient was started on tadekinig alfa (recombinant human il- binding protein) as part of a prospective study. hlh flare ultimately resolved without use of antineoplastic agents, and the patient was discharged home. soluble il- r levels since normalized, and il- levels decreased to less than pg/ml. the patient has been doing well on anakinra and tadekinig alfa, though continues to experience mild to moderate right knee effusion. this case suggests il- inhibition may be an effective therapeutic approach for patients with xiap deficiency. in the absence of neurological involvement and infectious trigger, ruxolitinib was initiated at a dose of mg/m /day, in combination with dexamethasone ( mg/m /day). this treatment led to rapid normalization of the neutropenia ( hours), complete resolution of the splenomegaly ( days) and disappearance of hlh biological markers (triglycerides levels in week, activated hla-dr+ cd t cells in weeks, fibrinogen levels in month), without the need for etoposide or serotherapy. dexamethasone was weaned every two-weeks and stopped after weeks. ruxolitinib was well-tolerated with no side effects. while in complete remission of her hlh, the patient then received alemtuzumab ( . mg/kg total dose) and a fludarabine-based myeloablative conditioning regimen. ruxolitinib was weaned over one week, and a / unrelated transplant was performed with success. the immediate posttransplant period was complicated by a veno-occlusive disease that responded rapidly to defibrotide and a corticosteroidresistant skin and ocular graft-vs-host disease (gvhd) despite a prophylaxis with ciclosporine and mycophenolate mofetil. gvhd was controlled by the reintroduction of ruxolitinib. at months post-hsct, her chimerism is % donor. to our knowledge, this case is the first description of a patient with primary hlh successfully treated in first intent by a combination of dexamethasone and ruxolitinib prior to hsct. our observation suggests that this targeted and less-toxic treatment regimen, that does not include etoposide nor high-dose alemtuzumab, is effective, well-tolerated and could be used in first intent to treat primary hlh. abstract/case report text presentation a -year-old girl, presented in ambulatory consultation, with a -year history of recurrent fever, influenza-like symptoms (sore throat, malaise), associated with self-limited painful genital ulcers (just within the period of fever). the first episode was characterized for an fournier's infection, requiring in-hospital treatment, multiple surgical procedures, antibiotics and hyperbaric oxygen therapy. after that catastrophic debut, she was diagnosed approximately episodes per year pharyngitis (with fever, malaise and sore throat) treated with corticoids, antibiotics and topic medication. the last year, noticed that every episode of fever (total of ) were associated with one or several genital lesions. the patient had no relevant medical history, she didn't receive long-term medication, she received all immunizations, she was sexually inactive, and hadn't apply any topic medication or product on the vulva, there was no trauma history, psychological medical history or sexual abuse. episodic gynecologic examination showed her labia minor several lesions, fibrinous, soft ulcerations on their inner aspect, these lesions had a symmetrical appearance, known as kissing lesions; no vulvar swelling, vaginal discharge or lymphangitis were noticed. there were no other skin or mucous membrane lesions (figure ). investigations viral (hiv, hbv, hcv, ebv, cmv) and treponemal (tpha-vdrl) serologies were negatives. erythrocyte sedimentation rate (esr) and pcr analysis within ulcers episodes were positive. specific antibodies (cardiolipin, anti ro/ss-a, anti la/ss-b, anti ccp, anti ena, anti gliadin, anti tpo anti tpo) serologies were negative. otherwise, important elevation immunoglobulin d was observed ( , mg/dl, twice the normal value): mild elevations of immunoglobulin m and immunoglobulin a were observed. serum subtypes of immunoglobulin g and immunoglobulin e were normal. leukocytosis with monocytes elevation and an increase of lymphocytes b were present. (table ). discussion lipschütz ulcers are uncommon and an often unknown entity for physicians, but it is important to recognize and include it in the differential diagnosis of vulvar ulcerations. this condition is characterised by self-limited painful ulcerations of the vulva or lower vagina in adolescent or young women, non-sexually transmitted, and usually preceded by influenza or mononucleosis-like symptoms. hyperimmunoglobulin d syndrome (hids) is characterized for unremitting fever lasting four to seven days and the presence of palpable tender lymphadenopathy, splenomegaly, arthralgia/arthritis, abdominal pain, and mucocutaneous manifestations. laboratory findings suggestive of hids include elevated age-specific serum immunoglobulin d (igd) and/or immunoglobulin a (iga) levels, elevation of acute phase reactants, and urinary excretion of mevalonic acid during, but not between, attacks. the diagnosis is established if an elevated age-specific level of igd is detected. iga levels are typically measured at the same time but are not required for diagnosis. elevated serum igd is not specific for hids and can occur in patients with certain neoplastic, infectious, heritable, and idiopathic disorders. in the present case report, the patient was treated with colchicine, with favorable evolution and free from new events. levels of ig d, platelets and monocytes remain high. we describe a young female patient presenting recurrent lipschütz ulcers, fever and elevation of serum immunoglobulin d, suggesting that hids could be associated with genitalia ulcers. ( ), and transmission in vivo in extremely low birth weight infants was considered ( ) . based on these considerations, the risk/benefit was considered favorable for restarting pasteurized donor breastmilk feeds. given his small size and young age, we had significant concerns about using ganciclovir prophylaxis and opted to hold this and monitor weekly cmv pcr. the child has been titrated up on these feeds, is gaining weight appropriately, and has had weekly cmv pcrs which are negative x since restarting donor breastmilk. t cells, last checked at weeks of age ( weeks gestational age) remain essentially absent. given the lower risk of nec in premature infants with breastmilk-based enteral feeds, a broader, multi-institutional study is warranted to best examine the safety of pasteurized donor breastmilk in infants with scid and complete digeorge syndrome. transfected with a reporter plasmid (for luciferase), wt or mutant-stat plasmids. nk cell cytotoxicity was measured by cr release assay. we used multiparametric immune profiling to dissect the effect of stat -gof mutations on nk cell developmental phenotype. results: similar to our previous studies, we observed higher levels of stat phosphorylation after two hours of stimulation from the dbd mutation compared to the ccd mutations. the stat activity assay confirmed gain of function observed by flow cytometry, but this activity was higher in k q mutant and d e mutant (ccd-closer to dbd) than v i mutant. all patients demonstrated low nk cell lytic unit compared to healthy donors. interestingly, we observed a correlation between low lytic unit and lower numbers of cd dim perforin + cd + nk cells; much lower in patient with k q mutation. stat -gof patients showed a significant decrease in total nk cell numbers and impaired nk cell maturation was characterized by low expression of cd , and higher levels of immature nk cell markers (cd , nkg a, cd b). conclusions: these data suggest that impairment of nk cell function is affected by the location of the stat mutation and continues to be the case in novel mutations identified. the identification the genotype/ phenotype correlation in the spectrum of the nk cell defect in stat gain-of-function mutants may help to better understand the molecular basis for stat activation and/or function to predict clinical manifestations of disease and ultimately treatment regimens. mutation specific analysis after an amniocentesis showed that one twin was a carrier (l m) and the other was a compound heterozygote (r c and l m). after birth, twin a had a mildly low erythrocyte ada level ( . nmol/h/mg; normal range + ) with normal metabolites and a normal immunophenotype, similar to both parents. twin b showed a normal absolute lymphocyte count and mitogen proliferation, normal t lymphocyte subsets, mildly low b and nk cells with % naïve t cells and a normal trec assay. erythrocyte ada levels were absent in peripheral blood, with mildly elevated metabolites [daxp= . μmol/ml rbc (normal < . ) and %axp= . (normal < . )]. weekly recombinant ada enzyme replacement therapy (ert) was started at week of life with subsequent normalization of the metabolites by week . absolute lymphocyte, t cell subsets were normal at birth but continued to rise slightly above normal range after starting ert. b and nk cell counts were mildly low at birth but normalized by week . genetic testing confirmed the prenatal genotypes in the twin girls. the patient is now months old and doing well with no history of infections. her twin was not an hla match and family is currently awaiting gene therapy approval. discussion: ada deficient patients show substantial clinical and metabolic heterogeneity that tends to correlate with the genotype but phenotypic discordance occurs even within the same genotype. we describe an infant with prenatally diagnosed compound heterozygous mutations in the ada gene (grade-i: r c and grade-ii: l m). ada alleles are graded from -iv with increasing ada expression and decreasing severity respectively. there are reports of children with grade i/iii allele combinations with delayed, late and partial phenotypes. two siblings have been reported with l m allele (grade iii) in combination with a different grade i allele (r q), presenting with combined immunodeficiency at and months. the specific allele combination from our patient has not been previously reported, however, we expected that the grade-i allele likely would be more deleterious than the grade-iii allele. in our case, predicting a future phenotype remains a challenge, creating a dilemma regarding management strategies. however, with only mild metabolite elevations in our patient after birth, we may speculate whether the prenatal diagnosis with early ert precluded the development of a full immunophenotype and it remains to be seen whether nonimmune sequeli may be prevented. conclusion: children with compound heterozygous mutations in the ada gene can pose diagnostic and therapeutic challenges, especially due to the associated metabolic and clinical phenotypic variability. early recognition and treatment may potentially alter long-term morbidity and mortality. (ipex)-like phenotype. immunodeficiency is often combined with impairment of the humoral and cellular compartments. hematopoietic cell transplant (hct) can resolve disease-related manifestations in stat -gof, but overall survival is poor and there is a high rate of secondary graft loss in transplanted patients. jakinibs are a class of medications that block cytokine-induced jak/stat activation. ruxolitinib preferentially inhibits jak and jak and has been used as precision-directed therapy for treatment of stat -gof related manifestations with success in stabilizing and in some cases reversing organ-specific manifestations. the utility and safety of jakinibs for long term treatment of stat -gof and in the prevention of disease-related manifestations is not known. as such, hct is often pursued for patients once disease-related manifestations are controlled with jakinibs. we present a patient with stat -gof mutation with gradual secondary graft loss following hct years ago, that has had continued disease progression despite chronic ruxolitinib treatment. case presentation this is a years-old male diagnosed with a de novo heterozygous stat mutation (c. a>g/a) at age , years following hct for ipex-like disease. he has been treated with ruxolitinib for the last three years. this patient initially presented at months of age with wasting enteropathy, failure to thrive, early-onset type diabetes and hypothyroidism. he had frequent upper respiratory infections during childhood including mycobacterium fortuitum mediastinal lymphadenitis. at years he underwent / matched, unrelated bone marrow transplant following reduced-intensity conditioning. mixed donor chimerism was present in the first days following hct, and he continued to have a slow progressive decline of donor chimerism with full graft loss ( % whole blood donor chimerism) by age . at age , enteropathy returned leading to cachexia and tpn dependence. concurrently, he had recurrent upper respiratory tract infections, lymphopenia, and hypogammaglobulinemia. imaging showed bronchiectasis and lung function was consistent with obstructive lung disease (fev : . l fvc: . l dlco: . ml/min/mmhg). initiation of ruxolitinib at age resolved his enteropathy with discontinuation of tpn and > -pound weight gain. enteropathy has not returned. pulmonary clearance measures have also been employed. dlco initially improved (dlco: . ml/min/mmhg) but obstructive lung pattern continued (fev : . l fvc: . l). after initial improvement, dlco began to decline. over the last years and despite treatment with ruxolitinib, lung function has deteriorated with worsened fev ( . l), fvc ( . l), and dlco ( . ml/min/mmhg). with this progressive decline, the family is now pursuing second hct. discussion jakinibs apply precision-directed therapy for immune dysregulatory features of stat -gof. their use leads to substantial disease control and clinical improvement but does not prevent disease progression. jakinibs should be used as a bridge to definitive therapy with hct in patients with stat -gof mutation. a recent large registry study showed a higher risk of infections (hr . , % c.i . - . ) in children with thymectomy as compared to surgery controls, in addition to demonstrating differences in the risk of cancers, autoimmunity and atopy. limited small studies have described some risk factors for altered immune consequences; however, specific predictors of infections among children with congenital heart disease (chd) undergoing thymectomies have not been systematically assessed. among children with chd and thymectomy, we sought to characterize children with and without reported infections within years postthymectomy and identify predictors of bacterial and viral infections. methods: using a retrospective chart review (institutional irb approved) from / / and / / , we identified children with chd that underwent thymectomy and excluded any known conditions associated with immunodeficiency and those with less than -month follow-up post-thymectomy. first absolute lymphocyte count (alc) after thymectomy was stratified using a cutoff at % of the lower limit of age-adjusted normal values (alc value < % vs alc value > % of the lower limit of age-adjusted normal levels). we sought to assess predictors of reported bacterial (positive blood, cerebrospinal fluid, respiratory cultures and chest-x-ray confirmed pneumonia) and viral infections (positive viral pcr tests) within years postthymectomy. results: we identified children with chd who had thymectomies, of which, % ( / ) were male. the median age at thymectomy was months (interquartile range months- . years); % ( / ) underwent a complete thymectomy; and % ( / ) developed a chylothorax within week post-thymectomy. a substantial proportion of children had an alc below % of the lower limit of age-adjusted normal levels after thymectomy ( % [ / ] pre-thymectomy vs % [ / ] postthymectomy). among children with chd post-thymectomy, % ( / ) and % ( / ) reported bacterial and viral infections within years, respectively. children with post-thymectomy alc values below % of the lower limit of age-adjusted normal levels had higher odds of reported bacterial (or . , % c.i . - . , p= . ) and viral (or . , % c.i . - . , p= . ) infections post-thymectomy as compared to those with an alc greater than % of the lower limit of ageadjusted normal levels (multivariate logistic regression). there was no association with the type of thymectomy (partial vs complete), age at thymectomy, weight at thymectomy, sex or prematurity. conclusions: among children with congenital heart disease with no known immunodeficiency undergoing thymectomy, alc below % of age-adjusted normal levels post-thymectomy may be associated with higher odds of bacterial and viral infections. a retrospective study design with a small sample size poses several limitations; however, this study suggests that post-thymectomy absolute lymphocyte values may be a potentially useful marker to identify higher risk patients in this population. radiological assessments esp. in the ct chest is commonly performed, but has associated radiation exposure and pulmonary function testing, at times, maybe insensitive to small changes in lung pathophysiology. many pids may have overlapping features with short telomere syndromes (sts) a, which are accelerated aging syndromes affecting hematopoietic, pulmonary, hepatobiliary and/or immunological systems, unified by a high cell turnover in these organs. clinical assessment of ageappropriate telomere length (tl) is performed using flow cytometry & fluorescence in-situ hybridization (flowfish). methods: we retrospectively analyzed telomere lengths in lymphocytes and granulocytes using the flow cytometry and fish method .flowfish testing was done at reference laboratories in johns hopkins university (jhu, usa).approval was obtained from mayo's institutional review board. data abstraction and analysis was done using the software jmp. results: patients were included in our analysis with females ( %) and males ( %).the median lymphocyte count of our cohort was . ( . - . ).the telomere length was strongly associated with the presence of lung disease (p= . *) and the presence of interstitial lung disease closely paralleled the changes in telomere length (delta-as compared to age adjusted normal percentiles lengths). shorter lymphocytic telomere length was associated with more severe reduction on total lung capacity (tlc; p= . *). conclusion: shorter lymphocytic telomere length served as a reliable biomarker for interstitial lung disease in pid patients. this may open up newer avenues for assessment of aging pathways in pid and may offer the option of using senolytic therapies in pids. mutations in the il- receptor common gamma chain gene (il rg) result in x-linked severe combined immunodeficiency (scid). the common gamma chain is shared by il- , il- , il- , il- , il- and il- receptors. x-linked scid typically presents with low or absent t and nk cells and normal or elevated numbers of b cells. we report a case of x-linked scid with elevated b and nk cell numbers (t-b+nk+). the male patient had an abnormal newborn screen for scid in north carolina. lymphocyte enumeration performed at days of life showed cd + cells/mm , b cells/mm , and nk cells/mm . he had no naïve t cells. repeat lymphocyte enumeration two weeks later showed that the cd + count had increased to /mm . only . % ( cells/mm ) were cd ra+ naïve t cells. he continued to have elevated b cell and nk cell numbers. chimerism studies revealed the presence of % female cells in mitogen-stimulated pbmc by fluorescence in situ hybridization, indicating the presence of transplacentally transferred maternal cells. lymphocyte proliferation responses to pha and cona mitogen stimulation were very low (less than % of normal). immunoglobulin levels were igg mg/dl, igm mg/dl, and undetectable iga and ige. genetic studies revealed a missense mutation in il rg, c. c>t, resulting in an amino acid substitution (p.ala val) in the extracellular domain. family testing showed that the patient's mother was a carrier for this variant. the father and the two healthy older brothers did not have this variant. of note, the family history was significant for lateral maternal male early deaths. at weeks of age, the patient received an unfractionated bone marrow transplant from his hla-identical brother without conditioning or gvhd prophylaxis. at the time of this report's submission, he is weeks post-transplantation and has had successful engraftment (whole blood-cd + fraction was composed of > % donor cells). he also now has normal t cell proliferation in response to mitogens and normal levels of all immunoglobulins. genetic defects that cause primary immunodeficiency can have variable phenotypic presentations. the patient's phenotype was atypical in that he had elevated nk cell numbers. to further evaluate these cells, we checked for stat phosphorylation following il- stimulation of abstract/case report text background: stat gain-of-function (gof) mutations cause a multisystem disease of early onset autoimmunity and lymphoproliferation, severe post-natal growth restriction, and recurrent and/or invasive infections. treatment of the autoimmune and auto-inflammatory features of stat gof patients relies heavily on immunosuppression and is often challenging. the full scope of phenotypes, treatments and outcomes may be broader when analyzing a substantially larger cohort than those already reported. methods: we gathered and analyzed data on patients from centers world-wide with confirmed gof mutations in stat . retrospective chart reviews were performed in accordance with all local ethics and irb committees to determine clinical manifestations, immunophenotype, treatment regimens, success of treatment methods, and overall survival. funcitonal transcriptional activity was assessed by luciferase reporter assay on each individual mutation. results: fifty-nine individual mutations were identified and all conferred gof by a validated luciferase assay. there were mutations in the nterminal domain, in the coiled-coil domain, in the dna binding domain, in the sh domain, and in the transactivation domain with the overwhelming majority being missense mutations. median age at presentation was approximately years; % of subjects are male and % are female. immunodysregulatory features presented in all patients. autoimmune cytopenias were the most common occurring in % of subjects (n= ), followed by lymphoproliferation in % (n= ) with increased frequencies of double negative (cd -cd -)t cells being found in % of of patients tested, enteropathy in % (n= ), endocrinopathy in % (n= ), interstitial lung disease in % (n= ), dermatitis in % (n= ), and inflammatory brain disease in . % (n= ). growth failure was present in % (n= ) with half of those patients having concurrent enteropathy. infections were reported in % of the cohort to include recurrent and/or invasive viral, bacterial, opportunistic, fungal, and mycobacterial infections. prominent abnormalities of immunophenotyping included t cell ( %) and b cell ( %) lymphopenia with reduced t cell proliferation in response to mitogens or antigens in % of those evaluated patients. fifty-nine percent of the patients hypogammaglobulinemia while % exhibited poor specific antibody responses to recall antigens. overall survival was % at data collection.treatment of stat gof patients often included multiple agents: ivig , chronic and pulse steroids, mtor inhibitors, calcineurin inhibitors, rituximab, mycophenolate mofetil, alemtuzumab, tocilizumab, and jakinibs. those started on jak inhibition showed improvement in clinical symptoms and, to date, there are stat gof patients on targeted jak inhibition. thus far, patients have undergone bone marrow transplant with a % survival rate. discussion: stat gof mutations were first reported in to cause a heterogeneous syndrome of autoimmunity and lymphoproliferation with immunodeficiency and infection susceptibility. earlier treatment with targeted therapy such as jak inhibitors has led to reduced disease morbidity. we report the largest cohort of stat gof patients collected through a multi-national collaboration of the longitudinal data and natural history of stat gof disease. understanding the heterogeneity of presentation and key features that will lead to proper diagnosis and early treatment in an effort to prevent long term disease associated sequelae. we present the case of month old male with a novel heterozygous mutation in tcf and two previously unreported phenotypes: ) absent circulating cd + b cells yet preserved immunoglobulin synthesis and vaccine responses and ) significant thrombocytopenia that improved with immunosuppression. there is also a striking family history of two half-sisters who died during early infancy with similar clinical and lab findings and the same genetic change. the infant boy was born at term with respiratory failure and generalized rash. at birth he had thrombocytopenia ( k/ul) and lymphopenia ( / ul). initial absolute cd + t cell count was low ( /ul), yet he had normal thymic output and proliferative responses to mitogens ruling out scid. cd + b cells were < /ul and bone marrow biopsy revealed decreased hematagones, yet he had a normal igm level ( . mg/dl) elevated iga ( mg/dl), and elevated ige ( iu/ml). igg levels were initially obscured by maternal igg and ivig; in turn he made positive titers to diphtheria and tetanus vaccination. the infant has maintained his own igg production. rapid genome sequencing revealed a heterozygous predicted deleterious vous in tcf , in the second transactivation domain (c. c>t, p.pro ser). the same change in tcf was identified in the deceased half-sisters as well as the father: all infants had different mothers, suggesting autosomal dominant inheritance. the sisters had similarly severe thrombocytopenia and absent circulating b cells; their causes of death were not completely understood. the year-old father has normal platelet levels, very low cd + b cells ( /ul), elevated igg ( , mg/dl) and ige ( , iu/ml), and normal levels of iga and igm. the father also has an elevated number of cd + t cells ( , /ul) with an increased percentage of t cells expressing hla-dr ( %). in the months after birth, the infant boy continued to require frequent platelet transfusions. despite the persistent t lymphopenia, there was evidence of increased t cell activation with elevated levels of soluble il- r ( pg/ml) and increased percentage of cells expressing hla-dr ( %) cd ( %), cd ( %), cd ( %), and cd ( %). a -day trial of prednisone was associated with an increase in his platelet count to > k/ul. he was switched to rapamycin as a steroid-sparing agent, and his platelet count has remained > k/ul for several weeks without transfusions. interestingly, his b cell counts also improved after the steroid trial ( /ul) and his absolute lymphocyte count is normalizing on rapamycin. a potential mechanism could be rapamycin decreasing t cellmediated destruction of platelets or b cells. reassessments of t cell activation markers and b cell phenotyping while on rapamycin will be done in the future. in contrast to multiple published cases of tcf mutations associated with complete agammaglobulinemia and absent b cells, we present a case of an infant with absent b cells yet preserved humoral function as well as severe thrombocytopenia responsive to rapamycin. in collaboration with colleagues at nih, studies are underway to understand whether/how the unique change in tcf is related to either phenotype described above. abstract/case report text background: childhood-onset, chronic, multi-system inflammatory diseases are increasingly being characterized as monogenic inborn errors of immunity. arpc b deficiency is a recently described, rare combined immunodeficiency characterized by recurrent/severe infections, a variety of autoimmune manifestations and platelet defects. we describe a case of arpc b deficiency identified in an adult patient with recurrent ulcers/ bechet-like disease, non-malignant lymphoproliferation and intermittent microthrombocytopenia. patient case: at year of age, our female patient was diagnosed with behcet disease based on a history of bloody stools at months, oral ulcers at months and vulvar lesions at year. she underwent rheumatology evaluations for inflammatory arthritis, episcleritis, eczema, vasculitic ulcerating nodules of the trunk, perineum and extremities, and verrucae forming flat plaques similar to epidermodysplasia verruciformis without a unifying diagnosis. other infections include otitis media, sinusitis, pseudomonas ecthyma gangrenosum, cervical lymphadenitis, and pneumonia. at years old, the patient was referred to our immuno-hematology comprehensive program clinic with a concern for malignancy versus a primary immune regulatory disorder (pird). she had a -month history of drenching night sweats, urticarial plaques, edema in her extremities and diffuse cervical, axillary and inguinal lymphadenopathy. past complete blood counts showed intermittent mild microthrombocytopenia. lymph node biopsies were negative for a neoplastic process but identified plasmacytosis, including focally increased iga-kappa+ plasma cells. expert review of the lymph node biopsy, and further evaluation excluded multicentric castleman disease. consideration was also given to autoimmunune lymphoproliferative syndrome (alps)-like disorders; however, her alps flow cytometry panel was nondiagnostic. her basic immune evaluation showed severe t cell lymphopenia (cd + cells/ cm, cd + cells/cm, cd + cells/cm) with adequate b and nk cells, normal lymphocyte proliferation to pha and pwm, and dysgammaglobulinemia with igg g/dl, iga g/dl, igm g/ dl and ige g/dl. due to concern of an underlying pird, a primary immunodeficiency panel was sent for gene analysis with negative results. however, trio clinical exome sequencing identified biallelic variants in the gene arpc b. one allele has a truncating, nonsense pathogenic variant in exon denoted as c. g>t, p.glu ter. the other allele has a likely pathogenic variant in intron denoted as c. - a>g, resulting in disruption of the canonical splice acceptor for exon . this is predicted to cause exon skipping, with an in-frame deletion of amino acids coded by exon . conclusion: this case highlights the value for evaluation for pirds in patients presenting with behcet-like disease, particularly in the context of other autoimmune manifestations and/or microthrombocytopenia. it also underscores that patients with arpc b deficiency may present with chronic non-malignant lymphoproliferation. moreover, this patient emphasizes the value of exhaustive genetic testing for complex immunologic phenotypes. abstract/case report text lipoyltransferase gene defect is associated with severe mitochondrial dysfunction disrupting lipoic acid biogenesis. clinical manifestations associated with early seizures, hypotonia, cardiomyopathy and pulmonary hypertension and encephalopathy. early neonatal death due to sepsis and cardiovascular collapse is commonly seen. the patient is a week preemie male with congenital heart disease who developed severe intractable lactic acidosis on day of life with increased excretion on organic acids of -methyl- , dihydroxybutyric acid. a mitochondrial disorder , echs or hibch deficiency was suspected. at mo of age the patient was admitted for apneic spells and respiratory compromise. he was found to have elevated crp associated with rhinovirus infection and gram-negative bacteremia. due to the history of failure to thrive and sepsis, immunology was consulted. immunologic work up indicated normal b, t and nk cells with normal dhr, but showed agammaglobulinemia. the patient was started on ivig and whole exome sequencing was done. molecular analysis showed compound heterozygote mutations in the lipt gene: c. g>a (p.arg gln) and c. t>g (pval gly). subsequent biochemical analysis also showed biochemical abnormalities consistent with lipt defect. lipoyltransferase is an enzyme involved in activation of a number of enzymes requiring lipoic acid. it is involved in lipoic acid synthesis. lipoic acid is required for the activity of pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain alpha-ketoacid dehydrogenase. the literature indicates that most patients with lipt defect have a severe, often fatal course. the patient is now almost years old and has stable clinical course without any major infections. he certainly has significant hypotonia and developmental delay. in conclusion, we are presenting the first case of lipt gene mutations associated with agammaglobulinemia who responded well to ig supplementation therapy. our immunologic findings in this case highlights the importance of immunodeficiency work up in challenging cases. as we see more cases lipt gene mutations, we will better understand the clinical spectrum. abstract/case report text a now -year-old male was initially evaluated for concerns regarding food allergy, eczema, food protein-induced enterocolitis syndrome, and failure to thrive. he had reactions of varying severity to multiple foods. these usually involved immediate urticaria or prolonged vomiting, diarrhea, and abdominal pain. ige and skin prick testing was performed to suspected foods and was positive to milk, egg, pork, wheat, peanut, pecan, coconut and corn. these foods had historically caused reproducible immediate symptoms. testing was negative to other suspected foods. he developed an oral aversion and extremely restricted diet. symptoms of abdominal pain, hematochezia, rashes, arthralgias, headaches, fatigue, dyspnea, and palpitations increased. urticaria and severe abdominal pain with vomiting and diarrhea continued intermittently without identifiable triggers on a restricted diet. laboratory markers demonstrated elevated inflammatory markers, anemia, iron deficiency, vitamin b deficiency, and vitamin c deficiency (scurvy). gastroenterology work up did not identify any pathology. gastrointestinal symptoms did not respond to treatment with multiple gerd medications or oral steroids. baseline tryptase was elevated. low histamine diet was initiated and repeat tryptase remained elevated. fractionated tryptase revealed normal mature (beta) tryptase with elevated total tryptase, negative genetics for c-kit mutation, normal urine prostaglandins. family members had tryptase levels drawn. one parent and sibling had elevated tryptase levels, while the other parent's tryptase was normal. hereditary alpha tryptasemia syndrome is defined by elevated blood tryptase levels and symptoms involving multiple organ symptoms. patients with elevated tryptase levels without symptoms are defined as having hereditary alpha tryptase trait. there is significant variability regarding which patients are symptomatic. organ symptoms that may be involved include skin, gastrointestinal, neurologic, connective tissue, cardiac, neuropsychiatric. severe allergic reactions such as anaphylaxis can occur. increased blood levels of the protein tryptase are caused by extra copies of the alpha tryptase gene (tpsab ). treatment is usually directed at specific symptoms, antihistamines, and mast cell stabilizers. research continues into additional treatment options. this patient was started on cromolyn and long-acting antihistamine. his gastrointestinal symptoms and rash/urticaria improved, and he began tolerating a small, but increased, variety of foods. the majority of his constitutional symptoms of fatigue, arthralgias, weakness resolved as he began gaining weight, and hemoglobin, vitamin c and b normalized. his sibling was evaluated and noted to have food allergy, asthma, abdominal pain, gerd, and eczema. she was also started on cromolyn and antihistamines which improved her gastrointestinal symptoms. parent with elevated tryptase was recommended to be evaluated further with allergist. this is an example of a patient with elevated tryptase and multiple organ system involvement. some of his signs and symptoms responded to mast cell stabilizing and antihistamine medications. patients with history of recurrent episodes of allergic reactions to foods and multiple constitutional symptoms would benefit from baseline tryptase levels. family members should also be tested if the patient has elevated tryptase. multiple studies have been published looking at the rates of scid in the united states. the estimated rate of scid prior to screening was per live births. post screening implementation, on average rates of scid were found to be closer to in live births. results : development of a t cell receptor excision circles (trec) scid screen in alberta involved the screening of anonymous term neonates using quantitative pcr for trecs. the cycle threshold for the control gene, rnasep, was set at . as % of our population had a cycle threshold < . ( % ci [ . , . ]). from those bloods spots with adequate dna, a final trec cut off of was chosen, as it would give an accuracy of . %, and fairly low false positive rate of . % ( % ci [ . , . ]). since starting a population based screen for scid in june of , we have identified cases of scid and cases of low trec not caused by scid. to date we have detected one case of reticular dysgenesis, cases of ada scid and one case of x-linked scid. other causes of lymphopenia in the neonatal period detected with abnormal trecs include one syndrome associated with variably affected cellular immunity (charge) and cases of secondary lymphopenia including four cases of prematurity, three cases of diaphragmatic hernia or gastroschisis, four patients with underlying cardiac disease, and one patient with severe hydrops. discussion : canada has multiple unique populations with increased risk of scid. the estimated rate of scid in canada prior to implementation of a population based screen was . per live births. the rate within canada's first nations, métis and inuit populations is . per live births. prior to scid screening, alberta had cases of scid identified between - with an estimated rate of per live births. to date, our screen in alberta has identified cases of scid with a rate of per live births which is significantly higher than previously estimated. given that early diagnosis and definitive management through bone marrow transplant or gene therapy has been shown to reduce mortality this screen will help reduce morbidity and mortality in this vulnerable population. abstract/case report text introduction: human herpesvirus (hhv- ) has the ability to integrate its genome into host telomeres. if this integration occurs in gametes, then the virus can be genetically transmitted and offspring will carry a copy of chromosomally integrated hhv- (cihhv- ) in each somatic cell. this can lead to false attribution of infectious and non-infectious presentations of hhv- , and make the diagnosis of active hhv- infection difficult. we present the case of a patient with meningoencephalitis attributed to hhv- and persistently elevated blood levels of hhv- by pcr concerning for primary immunodeficiency who was discovered to have cihhv- . case description: a -year-old female who carried a past diagnosis of hhv- meningoencephalitis was seen in immunology clinic for follow up of persistently elevated levels of hhv- dna in her blood by pcr. she was born at weeks and as an infant had failure to thrive (ftt), anemia and a varicella like rash after varicella immunization. at the age of , she received flumist vaccine and developed a fever the following day. over the next few days, she developed lethargy, altered mental status, headache, photophobia, seizure, papular rash and oral ulcers. she was admitted to the hospital and csf studies were consistent with viral meningoencephalitis ( wbc, l, m, rbc) although hsv, cmv, ebv and enterovirus were negative. she was treated for presumed hsv encephalitis with days of iv acyclovir and days of high dose steroids. one week after discharge, she again developed papular rash on feet, headache, oral ulcers and lethargy. she was admitted and csf studies this time showed only wbc but positive hhv- . blood and skin swab were also positive for hhv- . immunology was consulted while admitted and work up for primary immunodeficiency was initiated. her work up was normal including responses to vaccine titers, complement studies, moderate in t cells, monocytes, neutrophils, lung and muscle, and low in skin, liver, heart and kidney tissues. analysis of each individual gene expression level by nanostring in comparison with healthy controls demonstrated significantly higher levels of the following irgs: ddx , epsti , gbp , ifi , isg , ly e, oas , oas , oas , rsad , rtp and socs . whole blood rna-seq was performed in patients and pathway analysis with the differentially upregulated genes demonstrated an enrichment of intraluminal vesicle formation and negative regulation of apoptotic signaling pathways. stimulation of pbmcs with the tlr ligands poly i:c, odn, and lps induced a fold increase in ifi and a -fold increase in ifna and ifnb transcription compared to baseline. one patient had constitutive upregulation of stat and stat in monocytes and of stat and stat in t cells. conclusion: we describe a novel immunedysregulatory disease caused by de novo truncating variants in samd l that presents similar to candle with neutrophilic pannicultis and points to an important role of samd l on regulation of adaptive and innate immune responses. acknowledgements: this work was supported by the nih irp of niaid abstract/case report text introduction: ataxia telangiectasia (at) is caused by a defect in the atm gene which is responsible for repair of damaged dna. it is a rare, devastating neurodegenerative disease that results in ataxia and telangiectasias, particularly of the sclera and skin. those with at are at increased risk for immunodeficiency and cancer. the immunodeficiency is variable and may result in deficiency in humoral and cellular immunity in some patients. here we present a patient with ataxia telangiectasia and hypogammaglobulinemia on immunoglobulin therapy who developed recurrent urinary tract infections (uti) and sepsis. case presentation: the patient is a -year-old female with ataxia telangiectasia and hypogammaglobulinemia who presented with three episodes of uti, one of which resulted in prolonged hospitalization due to sepsis and acute kidney injury (aki). she presented with days of flank and back pain and was hospitalized for days for e coli uti. she improved on iv antibiotics and was discharged home to complete treatment with oral ciprofloxacin. due to persistent emesis, she was readmitted weeks later with urosepsis and aki with a creatinine of . mg/dl, over times her baseline creatinine. after additional antibiotics and iv fluids, she improved clinically, renal function normalized and she was discharged home. renal ultrasound was unremarkable with no anatomical abnormalities. she was relatively healthy prior to this with only one episode of bacterial pneumonia in . she receives weekly subcutaneous immunoglobulin therapy dosed at mg/kg with normal igg levels ( , , mg/ dl). at baseline, she had high igm ( mg/dl) and low iga ( < mg/ dl) levels, as well as decreased t cells but normal nk and b cells ( cells/ul cd +, cells/ul cd +, cells/ul cd +, cells/ul cd +cd +, and cells/ul cd +). she has hyperglycemia (on metformin), hypertriglyceridemia (on atorvastatin) and hypertension (on losartan). she is thin and wheelchair bound with bilateral telangiectasias to the sclera, neck, and chest. she has occasional eye bleeding and epistaxis, presumably from her telangiectasias. she has good hygiene and good adherence to medications. she voids voluntarily, has no indwelling urinary catheter and is not sexually active. discussion: patients with ataxia telangiectasia may have frequent viral and bacterial infections, most frequently upper and lower respiratory tract infections, as well as wart and skin infections. based on our review, this is the first reported case of ataxia telangiectasia with hypogammaglobulinemia on immunoglobulin therapy with recurrent utis complicated by urosepsis and aki. despite adequate igg levels on immunoglobulin therapy, our patient continued with recurrent utis. it is uncertain whether her non-ambulatory status, hyperglycemia or related immunodeficiency are the causes for her increased susceptibility to utis. the literature reports patients with at and bladder wall telangiectasias can result in significant hematuria, and perhaps this may be a source of entry for bacteria and consequent development of uti. this suggests that patients with ataxia telangiectasia and recurrent utis may benefit from renal ultrasound and possible cystoscopy to better visualize telangiectasias. we recommend consideration of workup for recurrent utis in patients with ataxia telangiectasia. abstract/case report text objectives: patients with partial rag deficiency frequently present with humoral autoimmunity suggesting breach in tolerance mechanisms and subsequent expansion of autoreactive b cell clones. here we aim to trace polyreactive b cells and their descendants at b cell developmental stages through our in-house bioinformatic pipeline, immchaintracer (ict). methods: the b cell receptor (bcr) was expressed as monoclonal antibodies from single sorted mature naive b cells (n= - per donor) from patients with hypomorphic rag deficiency and healthy donors. xthe recombinant monoclonal antibodies were screened for polyreactivity (dsdna, insulin, lps and ifnα) by elisa. in parallel, igh repertoires were deep sequenced from sorted mature naïve, activated naïve and memory b-cell compartments. our in-house assembled bioinformatic pipeline called immchaintracer (ict) was applied to track down the descendants of cloned autoreactive igh sequences in repertoires of subsets above. results: igh sequences (n= including polyreactive, nonpolyreactive clones) from mature naive b cell from six patients with partial rag deficiency and healthy donors (n= including polyreactive, non-polyreactive) were analyzed with our novel inhouse bioinformatic approach to track lineage fate in repertoire at specific developmental stages. interestingly, . % of the patients' sequences and their descendants were identified in their mature naive, activated naive or memory b cell repertoires, while none of the analyzed healthy donor clones were found at later subsets. furthermore, genealogical analyses of related clones revealed lineage expansion and progressive positive antigen selection of the autoreactive clones in the patients. conclusions our findings demonstrate that peripheral tolerance checkpoint is broken in hypomorphic rag patients. our novel method enables tracing the fate of autoreactive naive b cells in the effector repertoires. we have shown that impaired b cell tolerance allows the expansion and combination of sirolimus and rituximab therapy controlled his autoantibody production which was an important goal for his autoimmune condition. we present a new treatment approach for anti-nmda receptor encephalitis. rituximab was tried on these cases before but the combination of sirolimus and rituximab therapy was never given before. we now recommended that on refractory cases of anti-nmda receptor encephalitis, combination of rituximab and sirolimus therapy can be tried. ( ) submission id# mosaic variants in immune function genes identified through exome sequencing stable with no interim infections and is doing well on thyroid hormone replacement. the current plan is for the patient to undergo a stem cell transplant for the arpc b deficiency as he is at high risk for recurrent infections and severe disease. although this gene mutation is rare, review of the current literature describes patients with this condition that have undergone stem cell transplant and have done well. at this time, this seems to be the best option for management, and it may potentially be curative. abstract/case report text introduction: common variable immunodeficiency (cvid) is a disorder characterized by impaired immunoglobulin production and frequent or recurrent infections, but also associated with an increased risk for developing malignancies such as lymphomas. although intravenous and subcutaneous immunoglobin g replacement has been successful in reducing the number of bacterial infections and prolonging survival, it fails to address other complications that arise from this disorder. we report a case of a patient with cvid who developed mycosis fungoides (mf). mf is a rare form of cutaneous t-cell lymphoma, occurring in about in , to , individuals, lack of treatment could potentially be fatal. case presentation: a -year-old caucasian woman with a history of ulcerative colitis, allergic rhino-conjunctivitis and tonsillectomy was referred to the immunology clinic for evaluation of low serum immunoglobulins. there was no family history of infections or immune deficiencies, but paternal grandfather had colon cancer and maternal grandmother had lung cancer. the patient reported frequent episodes of bronchitis, and sinus infections. immunizations were up to date for her age. medications included azathioprine, cetirizine, fluticasone nasal spray, hyoscyamine, montelukast, lactobacillus, omeprazole, and olopatadine ophthalmic solution. no history of frequent use of systemic steroids. initial serum immunoglobulins revealed normal ige and igm but low iga ( mg/dl, normal range - mg/dl) and low igg ( mg/dl, normal range - mg/dl). cbc with differential, lymphocyte subsets, c and c levels were normal. while she had adequate protective titers against haemophilus influenza type b, diphtheria and tetanus, titers against pneumococcus were < % protective and she failed to mount an adequate response to pneumococcal polysaccharide vaccine. given her diagnosis of cvid, she started scig ( mg/kg every weeks). a year later, she developed a bilateral nonpruritic rash in the abdomen and upper trunk. initial skin biopsy suggested a drug reaction. a subsequent biopsy revealed a superficial perivascular lymphoid infiltrate with focal epidermotropism and positive t cell receptor gamma gene rearrangement, consistent with mf. testing for cell t receptor beta gene rearrangement was negative. while mf treatment consisted of triamcinolone . % ointment, treatment for ulcerative colitis transitioned from azathioprine to vedolizumab. conclusions: cutaneous t cell lymphomas, although uncommon, can be seen in cvid. there are several reasons for the increased risk of lymphoma in cvid. the role of chronic infections and the development of lymphoma as of yet, is not clear. skin reactions to scig products in the areas of infusion are relatively common and resolve promptly. high index of suspicion is crucial in obtaining tissue sample to confirm or rule out malignancy therefore avoiding delaying proper treatment. abstract/case report text patients with lipopolysaccharide responsive beige-like anchor protein (lrba) deficiency present with a plethora of immune related defects including a defective humoral response characterized by low numbers of switched memory b cells and plasma cells, as well as an impaired production of antibodies, leading to recurrent infections. however, the molecular mechanisms behind the defective b cell response remain unknown. to gain better insights into the possible roles of lrba in b cell physiology, we screened for lrba-interacting proteins using computational predictions. twenty-seven proteins involved in vesicle trafficking and autophagy were identified as potential lrba-interacting partners. to validate those potential lrba interactions, we performed coimmunoprecipitations and proximity ligation assays (pla), finding that endogenous lrba interacts with the phosphoinositide kinase regulatory subunit (pik r ) in b cells. pik r (aka vps ) is the regulatory subunit of vps , the catalytic subunit of the pi k-iii complex, which acts as a positive regulator of autophagy by producing phosphatidyl inositol- phosphate (pi( )p). autophagy is a catabolic mechanism essential for cell survival and plasma cell differentiation. in fact, we observed that reduced lrba impaired the production of pi( )p upon autophagy induction. in addition, we observed in both lrba-deficient hela and b cells reduced mobility, abnormal accumulation and increased size of autophaghosomes, accompanied by an atypical lysosomal positioning. these abnormalities are due to a blockade of the autophagosome-lysosome fusion, as detected by reduced lc -ii lipidation upon autophagy induction in the presence of lysosome inhibitors. interestingly, lrba-deficient hela and b cells exhibited enhanced activity of mammalian target of rapamycin complex (mtorc ) signaling, a key suppressor of autophagy whose activation possibly contributes to defective autophagy. taken together, b lymphocytes lacking lrba can form autophagosomes but they fail to fuse with lysosomes. thus, we propose a role of lrba at late stages of autophagy through the binding to pik r . abstract/case report text apds caused by gain-of-function mutations (gof) in the genes (pik cd and pik r ), encoding for the p δ and p subunits of phosphoinositide -kinase δ (pi kδ), results in hyperactivation of the pi k/akt/mtor/s k pathway and lead to immune dysregulation, lymphoproliferation and immunodeficiency. apds manifests with respiratory tract infections, bronchiectasis, susceptibility to herpes group viruses, autoimmunity, cytopenia, lymphoproliferation and lymphoma. gastrointestinal system manifestations include enteropathy, colitis, and liver disease. eosinophilic esophagitis (eoe) or eosinophilic gastrointestinal disease (egd) have been under diagnosed in reported apds cohorts. objectives: to review the incidence, demographics and relevant clinical data for eosinophilic gastrointestinal disease in a single center apds cohort. methods: review of clinical and laboratory findings from apds patients followed at the nih clinical center, from to . results: patients were either historically diagnosed or actively studied at our center for egd. incidence of all egd is % in our cohort and all patients had mutations in pik cd, none in pik r . most patients also had multiple gi manifestations. conclusion: immunopathology and genetic predisposition leading to eoe is complex. eosinophilic gi disease including eoe appears to represent significant gi pathology in apds. this implies that activation of pi k pathway may be directly involved in the etiology of eoe. abstract/case report text introduction: dominant negative mutations in stat (lof stat ; job's syndrome) cause a primary immune deficiency characterized by eczema, recurrent skin and lung infections, and connective tissues and skeletal abnormalities. over the last several years, vascular abnormalities causing tortuous and aneurysmal middle-sized arteries have increasingly been recognized. our institution has been imaging prospectively the coronary and cerebral arteries since - for brain imaging and from - for heart imaging. the purpose of this review is to provide an update on the extent of clinical manifestations noted in hies (continued) pain improved but rapidly worsened with tapering of steroids. the pet/ ct at that time, showed extensive hypermetabolic areas in lymph nodes, femurs, left acetabulum, left pubic ramus, right ischium, sacrum, both iliac bones, eight rib, t and t , both clavicles, both humeral, manubrium, and extension into musculature. initial biopsies were culture negative, s was positive for mac. she was referred to the national institutes of health where she was diagnosed with autoantibodies to interferon-γ . prior to referral, she initially was treated with azithromycin, ethambutol (need to discontinue due adverse effects), amikacin, rifampicin, linezolid, with not no evidence of clinical response. she underwent debridement of epidural anterior abscess to t . surgery involved t - laminectomy, and curettage on several bones. she presented unable to walk secondary to pain and neuropathies. initial laboratory: crp: mg/l; wc . ; hgb: . g/dl; ana : . (strongly positive) cd : ul her first course of rituximab consisted of doses of gm at d , , and monthly thereafter for a total of doses with clinical and radiographic improvement. she was maintained on optimal antibiotics therapymeropenem, rifampin, azithromycin, moxifloxacin, and clofazimine. two years after she completed rituximab, she presented to her home hospital with increased left hip pain and biopsy grew mac. retreatment with rituximab failed to show clinical improvement. her medical regimen was augmented with tedizolid and bedaquiline, however no iv antibiotics were added. rituximab was reinitiated, after the progression of symptoms despite treatment with rituximab; bortezomib, was trialed using the schedule based on the multiple myeloma literature. she completed full cycles (two at the nih, three at home). she subsequently has had clinical improvement and is working again and has not had progressive neurologic decline. the titers of antibodies to interferon-· did not follow the clinical improvement. however, we plan to keep her cd + cells zero and continue the bortezomib given her clinical and radiologic improvement. abstract/case report text for patients with primary immunodeficiencies (pid), finding a genetically defined diagnosis can be critical for prognosis, treatment, and counseling. however, for many patients, determining a genetic etiology remains elusive despite routine gene panel and exome sequencing because of an inability to resolve variants of uncertain significance (vus). crispr-based genome editing could be used to address this need by introducing patient-derived vus into primary human immune cells for further study; however, existing techniques are limited by poor efficiency. we recently developed novel non-viral techniques for large gene editing in primary human t cells and hematopoietic stem cells (hscs). we achieve up to fold greater efficiency than existing tools using crispr cas ribonucleoprotein nanoparticles that are non-covalently linked to homology directed repair (hdr) template dna. whereas mutation analysis has previously been limited to expensive and time-consuming animal models and transformed cell lines, we now have the ability to rapidly recreate any mutation in the native gene locus in otherwise healthy primary human cells. we demonstrate this ability by using our technique to knock-in well characterized loss-of-function mutations in jak and il rg and gain-of-function mutations in jak that are known to cause severe combined immunodeficiency and tumor growth, respectively. we show that these recreated mutations have the expected effects on t cell proliferation and intracellular signal transduction in the setting of il- stimulation. we then use our technique to investigate a prototype case of an adult patient with the unusual combination of common variable immunodeficiency, inflammatory arthritis and uveitis, and neutrophilic urticaria. genetic testing in this patient had previously revealed heterozygous coding vus's in four genes previously associated with a pid disease, including jak , but none of the specific variants have been previously reported. it is thus not clear which mutation (if not more than one) causes this patient's dysregulated cell activation, which limits targeted treatment options with kinase inhibitors or future gene or cell therapy. by knocking our patient's jak variant into primary human t cells and comparing these cells to those carrying wild-type, known loss-of-function, and known gain-of-function mutations, we are able to rapidly characterize the functional impact of our patient's variant and isolate its effect on his complex phenotype. this in vitro genetic engineering approach thus allows patient-specific vus to be modeled directly in primary human immune cells with a rapid turnaround time that is relevant for clinical applications, including molecular diagnosis and screening of pharmacologic or gene therapies. further, similar strategies could be leveraged as a potential basis for future gene correction therapy. abstract/case report text definition : "at variants" comprise a heterogeneous group characterized by the later onset of clinical symptoms, a slower progression, a prolonged lifespan compared to most patients with at and decreased levels of chromosomal instability and cellular radiosensitivity. in these patients, telangiectasia and / or immunodeficiency may be absent, while neurological features are present.( ). material and methods :a years old girl, born out of a non-consanguineous parents with clinical picture consisting in progressive alteration of the march ( months), associated to exotropia, no weight gain or height, alteration of balance while she is sitting, she walks by herself. she has acute bronchiolitis. results and discussion: brain magnetic resonance without alterations. abdominal ultrasonography and cpk normal. ophthalmologist assessment found exotropia, he did not find telangiectasia. motor and sensitive neuro-conduction were reported normal. alpha fetoprotein ng / ml increased. karyotype xx, non-structural alterations. normal auditory-visual evoked potentials. whole exome sequencing (wes): identified the small homozygous pathogenically deletion ¬c. + _ + del taag; p.? have a spelling effect in the splicing. it is not present in the population database or not as a known variant in function mutation of smad has been shown to increase smad phosphorylation in the nucleus in fibroblasts. further research is needed to examine the role of this mutation in t and b lymphocytes, given the interesting immunological phenotype of this patient. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. outbreak of mycoplasma pneumoniae-associated stevens-johnson syndrome characterization of children. with recurrent episodes of stevens johnson syndrome recurrent stevens-johnson syndrome secondary to mycoplasma pneumoniae infection recurrence and outcomes of stevens-johnson syndrome and toxic epidermal necrolysis in children syndrome after mycoplasma pneumoniae infection a review of causes of steven-johnson syndrome and toxic epidermal necrolysis in children submission id# expanded phenotype and genotype-phenotype correlations bsc(hons) msc bmbs mrcp dominant activating rac mutation with lymphopenia, immunodeficiency, and cytoskeletal defects an autosomal dominant scid form due to a gain of function mutation in the rac gene national institute of allergy and infectious diseases (niaid) professor biochemistry/molecular pharmacolog/nyu langone health canada head of the ircm bioinformatics core facility/montreal clinical research institute (ircm) associate investigator/national human genome research institute (nhgri) division of intramural research (dir), national institute of allergy and infectious diseases (niaid) translational and functional genomics branch/chief and senior investigator of the translational and functionanational human genome research institute (nhgri) division of intramural research (dir), national institute of allergy and infectious diseases (niaid) formula versus donor breast milk for feeding preterm or low birth weight infants cytomegalovirus (cmv) inacƟvaƟon in breast milk: reassessment of pasteurizaƟon and freeze-thawing prevenƟon of cytomegalovirus transmission via breast milk in extremely low birth weight infants submission id# the costa rican registry for primary immunodeficiencies department chief/department of pediatric immunology and rheumatology, national children´s hospital "dr. carlos sáenz herrera national children´s hospital "dr. carlos sáenz herrera" cid % (n= ), cid with syndromic features % (n= , at ), antibody deficiencies % (n= , xla ) ada deficiency, cd l-, flh- (syntaxin deficiency), ipex and osteopetrosis. nineteen patients developed malignancies, mainly non-hodgkin lymphomas among at patients conclusions: during the study period, pid were registered, mainly at and osteopetrosis cases. scid represented % and xla % of patients allergy and immunology/ department of pediatric immuunology and texas children's hospital director immunogenetics program/department of immunology, allergy and rheumatology at baylor college of medicine associate professor, director food allergy program/department of pediatric immuunology shearer center for human immunobiology associate professor/department of pediatric immuunology, allergy, and retrovirology granuloma faciale: successful treatment of nine cases with a combination of cryotherapy and intralesional corticosteroid injection intralesional steroid injection: a novel method to treat the symptoms of idiopathic granulomatous mastitis long-term effectiveness of intralesional triamcinolone acetonide therapy in orofacial granulomatosis: an observational cohort study sarcoidosis in the periocular scar as the first finding of systemic sarcoidosis: clinical-radiological characteristics a case of scar sarcoidosis of the eyelid submission id# null mutations: a gene dosage effect shubham goel, phd , hye sun kuehn clinical centre, nih, staff scientist/immunology service certified molecular geneticist/national institute of allergy and infectious diseases, national institutes of health genetic counselor/national institute of allergy and infectious diseases, national institutes of health division of intramural research; chief, immunopathogenesis section/laboratory of consultant/university hospital freiburg center for pediatrics and adolescent medicine cci -center for chronic immunodeficiency medical director professor/division of blood and marrow transplantation and cellular therapies new south wales, australia. professor of medicine and pediatrics/icahn school of medicine at mount sinai senior investigator/laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health functional nk assay, t/b/nk panel, tlr assay phd staff scientist/translational autoinflammatory disease section, lcim, niaid, nih, bethesda, md research fellow/translational autoinflammatory disease section post-baccalaureate fellow/translational autoinflammatory disease section pediatric rheumatologist/department of pediatric infectious diseases and immunology shupyk national medical academy for postgraduate education alder hey children's nhs foundation trust hospital, liverpool, uk pediatric rheumatologist/department of pediatrics dra. liliana bezrodnik y equipo dra. liliana bezrodnik y equipo"-servicio de inmunología htal. de niños hospital de niños ricardo gutiérrez pathology department/children's hospital of philadelphia, philadelphia, pa post-doctoral fellow/translational autoinflammatory disease section results: we identified patients with de novo frameshift variants in samd l (c. dela, p.i lfs* reduced toxicity allogeneic hct with a busulfan, fludarabine regimen: a promising approach for non-cgd primary immune deficiencies requiring myeloablation? phd , blachy davila saldana college of medicine assistant professor/division of bone marrow transplant, immunedysregulation and immuno-hematology program, aflac cancer and blood disorders center instructor of pediatrics/division of bone marrow transplantation and immune deficiency, cincinnati children's division of bone marrow transplantation and immune deficiency, cincinnati children's national institutes of health certified molecular geneticist/national institute of allergy and infectious diseases genetic counselor/national institute of allergy and infectious diseases, national institutes of health operations manager and genetic counselor/national institute of allergy and infectious diseases collaborative bioinformatics resource/national institute of allergy and infectious diseases genome in a bottle analysis team/genome in a bottle consortium division of intramural research (dir), national institute of allergy and infectious diseases (niaid) medical genomics and metabolic genetics branch/national human genome research institute, national institutes of health table clinical and demographic characteristics, severity measures, and previous treatments for the pid cohort geographic region, n (%) north central , ( . ) ( . ) , ( . ) northeast , ( . ) ( . ) , ( . ) south ) ( . ) , ( . ) high-potency oral antibiotics , ( . ) ( . ) , ( . ) systemic high-dose corticosteroids copd, chronic obstructive pulmonary disease cd / cd : . , lymphocytes b , natural killer cell/mm . conclusions: although the atm activity corresponding to this new splicing mutation is unknown, it is presumed that it has some residual function, since splicing mutations is associated with better neurological prognosis have been reported (pidd) . at the immunodeficiencies research unit we discuss and pursue the molecular and genetic diagnoses for patients with suspected pid from all around the country. starting this year, we are processing and analyzing our own patients' wes results at the unit. evaluating the diagnostic yield of wes, as a measure of effectiveness or quality control, may result in process optimization and perhaps allow for better patient selection and resource allocation. objective: to describe and characterize our patients with suspected pidd whose dna samples were sent out for whole-exome sequencing; to analyze and compare our wes diagnostic yield after the first batches of patients; to identify patient attributes that may predict a positive diagnostic wes result. methods: genomic dna was obtained from whole-blood samples of patients with suspected pidd from hospitals in mexico city, monterrey and puebla. wes was performed using a ng sequencer (illumina hiseq) in new jersey (admera health, llc), with % coverage and a x depth of the idt xgen library, human genome version (december ). two fastq files for each patient sample were transferred back to our unit, where the bioinformatic workflow was completed. we used galaxy in the cloud for quality control, mapping & alignment, and detection of variants; variant effect predictor to process, map, annotate and filter variants; and igv (broad institute) and genome browser (ucsc) for visualization. we defined diagnostic yield as the proportion of patients with a genetic diagnosis after analysis of their wes results. we performed multivariate logistic regression, tree partitioning algorithm and linear abstract/case report text a -year-old male with x-linked chronic granulomatous disease (cgd) complicated by severe perianal disease and proctocolitis presented with two weeks of open draining lesions on the thighs, bilateral inguinal regions, and gluteal cleft. the wounds became excruciating and prevented normal ambulation. the patient was admitted for iv antimicrobial therapy, local wound care and systemic steroids. wound cultures, throughout his course, yielded growth of klebsiella pneumonia, candida parapsilosis, malassezia globose, escherichia coli, enterococcus faecalis and staphylococcus epidermidis allowing for directed antibiotic and antifungal therapies. despite improvement, the wounds persisted after several weeks of treatment. faced with recalcitrant cutaneous lesions despite aggressive systemic and topical therapies, we looked to alternative options. noting that other granulomatous diseases show response with intralesional corticosteroid therapy, we considered this for our patient ( , ) . for example, patients with idiopathic granulomatous cheilitis had a complete response after three monthly injections of intralesional corticosteroids ( ) . sarcoidosis patients also improve with intra-granuloma corticosteroid injection ( , ) . our patient received mg triamcinolone acetonide injections two separate occasions, administered in multiple open lesions at eight-week intervals. the cutaneous lesion improvement was gradual and complete resolution of the first open wound was noted fifty-two days from initial steroid injection. to our knowledge, intralesional glucocorticoid therapy has not previously been used to treat cutaneous disease in cgd patients. we are reporting the first cgd patient with successful lesion resolution following steroid injection as part of therapy. as such, we believe this case is significant and suggests that direct lesion injection with glucocorticoids can add to treatment options for cgd patients with recalcitrant cutaneous disease. serum igg, alone or in combination with iga and/or igm, were reduced in all patients. t-follicular helper cells were reduced only in patients carrying biallelic mutations (a.ii. , a.ii. and a.ii. ) but not in any patients with monoallelic tcf null mutation. t cell enumeration and function by means of proliferation was normal in all mutation+ individuals. no mutated (truncated) protein expression was detected from patients with either biallelic or monoallelic tcf null mutations. however, wildtype tcf protein was detectable in about half amount in heterozygous patients. cdna data showed either / or / wt/mutated transcripts ratios in homozygous or heterozygous individuals, respectively, suggesting mutated proteins instability; and all together, protein haploinsufficiency for the heterozygous cases. ex-vivo, cd l and il -induced plasmablast differentiation was found to be reduced in / patients tested ( biallelic patient a.ii. and monoallelic patients a.i. , b.i. and c.i. ). moreover, decreased igg, iga and igm production in vitro correlated with reduced plasmablast cell differentiation.in conclusion, all individuals carrying either mono-or biallelic null mutations have immunological penetrance of the b cell defect. however, while clinical penetrance was complete in patients with biallelic mutation, it was partial for those with monoallelic tcf null mutation suggesting a gene dosage effect for clinical penetrance. in addition, our study emphasizes that tcf is relevant to the plasmablast differentiation process as well as for ig production. further studies are being conducted to evaluate the individual roles of e and e on the immune and clinical features. background: heterozygous gain-of-function (gof) mutations in the stat gene result in a hyperphosphorylated state where patients develop recurrent or persistent chronic mucocutaneous candidiasis (cmc), other cutaneous mycosis, bacterial infections, disseminated dimorphic fungal infections and autoimmune disease. furthermore, the nk defect we characterized illustrated an immature cd dim nk cell subset with decreased expression of cd , perforin, cd , and impaired cytotoxic capacity associated with increased susceptibility to viral infections observed in these patients. methods: in this study, we evaluated patients with novel stat mutations (d e mutation, located in coiled-coil domain and k q mutation, located in dna-binding domain). a third patient with the previously reported v i mutation (ccd) was also recruited for this study. in vitro, pbmcs from these patients were stimulated with ifn-α for , , and minutes and levels of phospho-stat on cd dim nk cell subset were measured by flow cytometry. the stat activity (firefly and renilla luciferase activities) was evaluated in u a-stat deficient cells abstract/case report text background: t cell lymphopenia associated with genetic syndromes can be identified with low t cell receptor excision circle (trecs) up on the newborn screen for severe combined immune deficiency (scid). jacobsen syndrome (js), q terminal deletion, is a rare genetic disorder seen in / , births characterized by facial dysmorphisms, platelet abnormalities, neurologic complications, immune system abnormalities including t and b cell defects. we report an infant with js found to have low trecs on nbs and review the immune phenotypes of our cohort of patients with js. methods: a retrospective chart review of all patients with js seen by the allergy immunology service at a large tertiary referral center from / / - / / was performed in accordance with irb standards. result: the index patient had two newborn screens hours after birth and two weeks later in accordance with texas state law. the first nbs resulted normal trecs while the second nbs had low trecs. a third nbs was done per protocol and again showed low trecs. subsequently, lymphocyte subsets at months of age showed severe t-cell lymphopenia: cd cells/ dl ( %), cd cells/dl ( %), cd cells/dl, and low recent thymic emigrants (cd +cd ra+ccr +cd +) cells/dl ( %) with normal lymphocyte mitogen proliferation. a chromosomal microarray (cma) revealed a q deletion known to cause js.over the five year study period we evaluated seven patients with js referred to our center. the majority of patients ( %) presented to clinic with history of recurrent infections including recurrent pneumonia, sinusitis, otitis media, skin abscesses and warts. t-cell lymphopenia was found in of ( %), / ( %) had abnormal lymphocyte proliferation (mitogens and antigens) and met criteria for pjp prophylaxis. in addition, / ( %) had antibody deficiency requiring igg replacement therapy. of the cases reviewed, only patients were born during the period of time that texas was performing the nbs. conclusion: jacobsen syndrome can present with a spectrum of immune defects most notably t cell lymphopenia and antibody deficiency. these patients can present at birth with low trecs. this cohort analysis highlights the importance of considering chromosomal genetic syndromes with features of primary immunodeficiency in evaluating patients with low trecs. further evaluation of larger cohorts gathered from neurology or genetics clinics at multiple centers would be helpful for future study in identifying those who need close immunology care. abstract/case report text introduction: recognized as sentinels of the immune system, mast cells (mcs) and dendritic cells (dcs) are both derived from hematopoietic/ progenitor stem cells in bone marrow (bm). the crosstalk and direct contact between these cells have been well documented and play an important role in modulating immune response. we showed that presence/absence of il- directs cell fate; whether progenitor cells will be differentiated into mcs or dcs. we also report an easy method in which in vitro generation of dcs is possible without external induction of gm-csf or il- . material-methods: to produce mcs and dcs in vitro, briefly bm samples were collected from patients with idiopathic thrombocytopenic purpura., bm mononuclear cells (mncs) were seperated by ficoll gradient, and seeded in plates with imdm medium containing fbs %, pen/ strep, and a little amount of methocult, and incubated in oc, % co (day ). the treatments on the following days were as follows: day , imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml); day , imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) + il- ( ng/ml). on day , groups were formed: group i: imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) + il- ( ng/ml), group ii: imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml), group iii: dmem + fbs %. the cultures were evaluated every days under an inverted microscope. verification of mcs was performed by toluidin blue and tryptaseimmunoflorescence staining. macrophages were verified by cd- immunoflorescence staining. dendritic cells of different stages of abstract/case report text introduction a reduced toxicity busulfan, fludarabine regimen with alemtuzumab or anti-thymocyte globulin (gungor et al.) was efficacious in patients undergoing allogeneic hct for cgd. we report our experience with a similar approach for patients with non-cgd primary immune deficiencies needing a reduced toxicity myeloablative approach. methods we retrospectively reviewed records of consecutive patients who underwent allogeneic hct for primary immune deficiencies with a preparative regimen containing busulfan, fludarabine and alemtuzumab or anti-thymocyte globulin(atg), at three transplant centers between - . busulfan was given either every hours over days with target auc of to μmol/min (based on q hr. dosing) or twice daily over days with target auc of to μmol/min (based on q hr. dosing) or once daily over days with a target auc of - μmol/min (based on q hr. dosing). fludarabine mg/m to mg/ m was given divided over - days. serotherapy included alemtuzumab . - . mg/kg or atg . mg/kg given divided over days. gvhd prophylaxis consisted of cyclosporine and mycophenolate mofetil. results forty patients (was= , hlh= , cd l deficiency= , ipex/veoibd= , scn= , ifngr def./cid/x-scid/msn / lad= ) received busulfan, fludarabine and alemtuzumab or atg for allogeneic hct (first hct in patients and second hct in the patients with hlh). median age was . years (range, . years - . years). patients received a graft from an hla-matched related (n= ), unrelated (n= ), or single allele mismatched related or unrelated donor (n= ). all except one patient engrafted at a median of days (range, - days). one patient developed veno-occlusive disease and two patients developed diffuse alveolar hemorrhage. notably, it was the second transplant for all patients. eight patients ( %) developed grade - acute gvhd and patients ( %) developed chronic gvhd. one patient developed primary graft failure and two patients secondary graft failure. nineteen patients ( %) maintained full donor (> %) chimerism following allogeneic hct. twenty patients ( %) developed mixed chimerism, predominantly in the t-cell lineage, but t-cell donor chimerism progressively increased post-hct. at -year post-transplant, of patients ( %) with mixed chimerism had donor myeloid chimerism > % and t-cell chimerism > %. two patients underwent a second transplant for graft failure. there were deaths in the cohort. overall survival was %( of ) and event free survival was %( of ) at year. conclusion our experience suggests that a reduced toxicity busulfan, fludarabine regimen with alemtuzumab or atg as serotherapy offers a promising approach with low toxicity, durable myeloid engraftment, low incidence of grade - gvhd and excellent survival and can be considered for a variety of primary immune deficiencies where myeloablative hct is desired. abstract/case report text this is an -year-old patient who has the diagnoses of anti-nmda receptor encephalitis with associated catatonia. the patient has history of a pineal gland germinoma diagnosed in august after months of double vision. however, after several months, the patient developed difficulty in sleeping, anxiety and nightmares. the patient presented to emergency department in february with personality changes. he was diagnosed with nmda encephalitis based on the clinical findings as well as presence of elevated serum and csf anti-nmda antibodies. the patient was initially treated with high dose systemic steroids with poor response. due to the worsening of his clinical condition, he was started on plasmapheresis, but had poor response to this therapy as well. these treatments are known standard treatment options for this condition. during his hospital stay, different therapies were discussed. cyclophosphamide was one of the treatment options, but because of the side effect profile and severe toxicity, we recommended different treatment modality. we started the patient on rituximab as well as sirolimus therapy to suppress both t and b-cell responses. after receiving two doses of rituximab in addition to daily sirolimus, the patient showed improvement of his symptoms. and declining nmda receptor antibody titers. this treatment plan was chosen for autoantibody mediated encephalomyelitis due to the fact that rituximab has inhibitory effect on naive b cells, but not on the proliferation of memory b cells and sirolimus has profoundly inhibiting role on memory b cells as well as a t-cell responses. a mosaic gene variant is one which is present in some, but not all, cells within an individual. they are increasingly associated with a number of diseases, including a number of primary immunodeficiency disease (pid). here we systematically analyzed mosaic variants in known or putative immunodeficiency genes from exome sequencing data from individuals. lofreq was used to identify mosaic variants with a variant allele fraction (vaf) of . - . ( . is the vaf for a non-mosaic, heterozygous variant). we removed variants from extreme read-depth (> standard deviations), unmappable, repeat-rich, and duplicated genomic regions. the average number of detected variants per mb was . and the total number of genomic locations with variants was , . mosaic variants were underrepresented in exonic regions, suggesting that coding variants may be deleterious. more mosaic variants were detected in saliva exomes compared to peripheral blood exomes (p-value < x - ), suggesting tissue-specific mosaic variants in buccal epithelial cells and white blood cells of saliva. to understand the clinical relevance of these findings, the variants were further filtered to include only nonsynonymous variants found in fewer than % of samples, leaving a total of , variant locations. of the remaining variant locations, had a pathogenic assertion in the human gene mutation database, and were in international union of immunological societies pid genes. further variant interpretations and clinical correlations are underway. these data suggest that mosaic variants in pid genes are common, vary by location of collection, and may have clinical diagnostic relevance. abstract/case report text introduction/background: the arpc b (actin related protein / complex subunit b) gene is a protein coding gene prominently expressed in blood cells and is necessary for the assembly and maintenance of the human actin-related protein / complex (arp / ). actin polymerization plays a central role in many immune functions including proliferation and differentiation of immune cells, migration, intercellular and intracellular signaling and activation of both innate and adaptive immune responses. defects in the actin cytoskeleton affect hematopoietic cells in the bone marrow and the immune response giving rise to a distinct primary immune deficiency, which is phenotypically similar to wiskott-aldrich syndrome (was). arpc b deficiency clinically presents as a severe multisystem disease which includes platelet abnormalities, recurrent infections, failure to thrive, inflammatory changes in the intestine, eczema, cutaneous vasculitis, eosinophilia, and elevated inflammatory markers. arpc b deficiency is rare and has only recently been described in the literature. here we present a clinical case of a patient found to have a pathogenic arpc b mutation via whole exome sequencing. case report: here we describe a month old somalian boy who presented to the immunology team at months of age with hematemesis, hematochezia, melena, failure to thrive, atopic dermatitis, hypothyroidism, autoimmune thrombocytopenia and recurrent infections concerning for a primary immune deficiency. family history was notable for parental consanguinity and an older sibling with a similar presentation of hemorrhagic gastroenteritis who died in kenya around months of age due to complications of his symptoms. the initial primary immunodeficiency evaluation revealed normal inflammatory makers, normal igg and protective vaccine (prevnar and tetanus) response. iga and ige were elevated at mg/dl and . ku/l respectively. flow cytometry was remarkable for t cell lymphopenia (cd , cd , and cd ) with reduced naïve cd and cd t cells. b and nk cell count were normal. alps panel and was protein expression was unremarkable. whole exome sequencing was performed and revealed homozygotic mutation of arpc b c. t>c, ivs + t>c which was predicted to be a pathological variant. subsequently, dhr flow cytometry with fmlp showed significant increase of dhp fluorescent (mfi . when compared to control mfi . ) consistent with findings from other arpc b deficiency patients. at the time of the most recent clinic visit, the patient has remained abstract/case report text introduction: activation of the nod-like receptor family cardcontaining protein (nlrc ) leads to the formation of an inflammasome. the inflammasome, a large cytosolic multiprotein complex of innate immunity, promotes proteolytic cleavage, maturation, and secretion of pro-inflammatory cytokines, including il- and il- . a gain-of-function mutation (gof) in the gene encoding nlrc or nlrc -inflammasomopathy is characterized by hyperinflammation with persistent elevated il- , infantile enterocolitis, and early-onset macrophage activation syndrome (mas). objectives: to describe phenotypic variation among three siblings with a novel nlrc gof variant and to expand our current understanding of the clinical manifestations of the disease methods: clinical and laboratory features were studied in three siblings of a hispanic family with a novel nlrc gof variant. results: a novel variant, c. g>a (p.arg gln) on the nlrc gene, was identified in a -year-old male with recurrent febrile episodes since one year of age. his laboratory findings showed highly elevated esr, crp, il- , and fecal calprotectin. his endoscopic finding was unremarkable. the recurrent fever partially responded to canakinumab. a -year-old sister with ileocolonic crohn's disease for two years was found with the same nlrc variant and highly elevated il- . crohn's disease was well controlled after adding infliximab infusion to methotrexate therapy. a -year-old sister, who has been asymptomatic and healthy, was tested with the same positive nlrc variant and highly elevated il- . the nlrc variant is inherited from their father, who currently has a diagnosis of psoriasis vulgaris. the il- levels of the three siblings show in the figure patients with flh- develop the classic hlh phenotype early in life, with periods of remission. the pathogenesis is associated with a defect of perforin-dependent cytotoxicity. t-ctl and nk cells fail to remove abnormal cells, consequently, an uncontrolled proliferation and activation of cd + t cells and macrophages develops and generates an inflammatory cytokine storm and a solid organs infiltration. mortality of fhl- is very high without treatment; allogeneic hsct is the only curative therapy. we report the outcome of a child with fhl- , four years after his treatment with an allogeneic hsct using an hla haploidentical donor. case report: a boy born in december , previously healthy, the only child of parents without consanguinity. he has a normal family and perinatal history. when he is years old, he begins suffering from with fever, arthralgia, hepatosplenomegaly and pancytopenia. he meets the diagnostic criteria for hemophagocytic lymphohistiocytosis (hlh) and received treatment with iv methylprednisolone and entered in remission of its hlh. six months later, he restarts his clinical picture of hlh and j clin immunol receives treatment with the hlh- protocol, which leads to remission. ayear later he presents a new episode of hlh that responds to ivig, cyclosporin a and dexamethasone. the possibility of primary hlh is then suggested. when he is years old, the molecular diagnosis is made with the identification of a mutation in the stx gene, the case was classified as fhl- due to syntaxin deficiency. in october , a hsct was performed. at the age of , his clinical status is evaluated and laboratory studies show that his immunodeficiency due to syntaxin deficiency was cured. his -year-old mother, hla haploidentical was used as a donor and a protocol developed for the treatment of osteopetrosis was applied. the hsct protocol did not use a tcell depleted graft. hstc conditioning was done with melphalan days - and - , fludarabine days - to - , anti-thymocyte globulin days - and - , and cyclophosphamide day - . (figure) . clinical and demographic characteristics, including the use of a novel claims-based weighted algorithm (risk vital sign; rvs) and those initiating ivig and scig, were described. stratified analysis based on pid diagnosis codes was performed. probability of receiving available ig treatments based on baseline characteristics was evaluated by logistic regression and propensity score methods. results: selected clinical and demographic characteristics, severity measures, and previous treatments between the overall pid population (n= , ), pid patients initiating scig (n= , ), and pid patients initiating ivig (n= , ) are presented in the table. patient characteristics and previous treatments tended to be stable, although hypertension, obesity, and corticosteroid use increased during the study period. new ig users tended to be older and female, with increased depression, dyslipidemia, and hypertension than all pid patients. new scig users had more diagnoses of respiratory (e.g., asthma, copd) and inflammatory (e.g., arthritis, fibromyalgia, inflammatory bowel disease) comorbidities and less cancer than all pid patients. new scig users compared with new ivig users had increased asthma and copd, fibromyalgia, and inflammatory bowel disease and decreased cancer and peripheral vascular disease. previous corticosteroid use was higher in ig users than all pid patients. among scig users, prior pid treatments of iv antibiotics, and oral high potency antibiotics were similar to all pid patients. ivig users had higher iv antibiotic and antifungal use. rvs was initially developed to identify patients likely to have undiagnosed pid. this analysis applied rvs to patients diagnosed with pid to assess severity. rvs based on -year history in the overall pid cohort was predominantly low, with only . % of patients scoring in the medium and high ranges. rvs was increased in incident ig users, with ( . % medium/high for scig; . % medium/high for ivig). in other markers of severity, scig users had more sinusitis and ivig had more pneumonia than all pid. ig users had fewer abscesses, cellulitis, and otitis media than the full pid cohort. conclusions: this exploratory analysis showed a trend toward increased hypertension, inflammatory and respiratory comorbidity, higher rvs, and previous corticosteroid treatment in patients initiating on ig compared with all pid patients. results could be confounded based on pid diagnosis codes used and warrants further research. author disclosures: mp, cas, and zh are employees and stockholders of the takeda group of companies. jo is a consultant to takeda. jbl is an employee of rti health solutions, an organization funded by takeda to conduct this research. mer was an employee of rti health solutions at the time this research was conducted.presenting author: colin anderson-smits submission topic: immunoglobulin replacement therapy patients in a larger cohort group and to determine if there are patterns of disease not previously reported. methods: we performed a retrospective chart review of patients with lof stat (n= ) followed at the national institutes of health. we specifically looked at tortuosity, aneurysms, and dilation of both coronary and cerebral arteries. epidemiologic information, stat mutation, co-morbidities, and laboratory information were reviewed along with imaging studies, specifically brain mri, brain mra, heart mri, and coronary ct. results: most recently, patients with hies are found to have vascular abnormalities including tortuosity, dilatation, narrowing, and aneurysms of middle sized, cerebral, and coronary arteries. in an effort to determine the extent of vascular involvement in addition to miscellaneous organ involvement, we are reviewing a cohort of patients with hies who were evaluated at the nih. of these patients, are women and are men. of these patients, two have passed away due to vascular events leading to their deaths. there are four patients under the age of , patients between the ages of and , patients between the ages of and , and three patients above the age of (age range - , mean age . ).of the patients, five of these patients were found to have abnormal brain mri/mra at an approximate rate of . %. two of these patients were found to have at least one cranial aneurysm, two of these patients were found to have a level of narrowing or stenosis, and one patient was found to have dilatation.in terms of coronary abnormalities, of the ( . %) patients were noted to have at least one coronary abnormality including dilatation, aneurysm, or tortuosity on heart mri or coronary ct. eight patients ( . %) were found to have dilatation of which four patients were female and patients were male. of the patients, ten patients ( . %) were found to have at least one aneurysm. there were patients ( %) that were found to have at least mild tortuosity. conclusion: vascular abnormalities in our lof stat patients occurred at an exceedingly high rate-cerebral and coronary artery, . % and . % respectively. due to this, patient's with lof stat should be considered for screening with brain and heart imaging. currently, there are no guidelines which outline the appropriate timeline for screening in these patients however following these patients over time will allow us to determine the most appropriate interval for imaging follow up. abstract/case report text year old woman with personal history of severe and recurrent upper and lower respiratory infections, chronic pulmonary disease with bilateral bronchiectasis and several micro nodules, chronic diarrhea without diagnosis (colonoscopy with mild colitis, without cmv. no bacterias no parasits were found in stools), mild osteopenia, focal lesion in right hepatic lobe, atopic dermatitis and anemia. she was followed up in other center and in she was diagnosed with common variable immunodeficiency (cvid) and started treatment with intravenous immunoglobulin (ivig), but with low adherence to it. she did not have referred history of lymphoproliferation nor significant viral infections. she had a daughter with spherocytosis who required esplenectomy and also had bronchiectasis and cvid diagnosis, she deceased at years old due to pulmonary infection. one years old son has anemia. her other daughter and son are healthy. objective: to describe unique management of recurrent viral respiratory infections in a qds patient. case: -year-old male with a history of qds complicated by truncus arteriosus, vsd, hypoparathyroidism, asthma, and autism was followed for a history of "frequent pneumonias." he had daily rhinitis and a history of frequent otitis media which resolved after tympanostomy tube placement at age y. however, over the next two years, he had admissions with various respiratory viral infections resulting in respiratory distress and prolonged oxygen need. viruses detected during these separate admissions varied and included parainfluenza , metapneumovirus, rsv, b and coronavirus nl , coronavirus hku , and rhino/enterovirus ( times total). he was previously on a prophylactic course of antibiotics which made no difference in his symptoms. laboratory evaluation showed protective adaptive immunity with normal immunoglobulin numbers for age (igg mg/dl), along with normal b and t cell numbers. he had protective pneumococcal titers and mounted a normal mitogen response. while his nk cell numbers were normal, his nk t cells were low. his tlr functioning also appeared normal. in order to decrease his overall illness burden and to keep him out of the hospital, ivig infusions (~ mg/kg) were initiated monthly. shortly after initiation of treatment, his nasal purulence and drainage resolved and his family noted that he became more active and playful. treatment was continued for months, during which time he had only one episode of influenza infection needing inpatient management. he has been off of ivig for months without recurrence of his viral infections. conclusion: in this patient, severe recurrent viral respiratory infections despite apparently normal adaptive and cellular immunity presents a unique management dilemma. this was not an issue of recurrent bacterial infections as prophylaxis did not make a difference in the frequency of his infections. his nk t cells were low, which could have contributed to his frequent viral infections as nkt cells are known to play a role in viral immunity. the successful use of ivig treatment in his case points to a different use for ivig, namely for the anti-respiratory virus antibodies which are presumably contained within the formulation.given this finding, it may be prudent to consider ivig in management of qds patients, even with normal immune evaluation, in order to decrease risks of complications associated with severe and recurrent viral respiratory infections. abstract/case report text introduction: smad is a critical downstream signaling molecule for transforming growth factor-β (tgf-β) and bone morphogenic protein (nmp ). initially, smad , also known as dpc deleted in pancreatic cancer locus , was described as a tumor suppressor gene, and somatic deletion of the smad is seen in % of pancreatic carcinomas. subsequently, a germline point mutation in the smad gene (p. i v) was reported to cause myhre syndrome (mim# ). myhre syndrome is an autosomal dominant disease characterized by cognitive impairment, hearing loss, and musculoskeletal anomalies. the immunological phenotype of these patients has not been previously described, despite the critical role of tgf-β in regulating t cell response and the prevention of excessive inflammation. case report: a -year-old boy with myhre syndrome was referred to immunology clinic for evaluation of recurrent ear infections. he developed acute otitis media infections as an infant and had tympanostomy tubes placed at one year of life. he also had a recurrent sinus infections. at two years of age, he was diagnosed with autism and sensory neuronal hearing loss. brain mri showed a mildly hypoplastic pituitary gland, and a thickened corpus callosum with decreased myelination. given these findings, whole-genome sequencing was performed, which revealed a heterozygous de novo mutation in smad (p.i v / c. a>g) consistent with the diagnosis of myhre syndrome. through age , he was in the th percentile for height until he was started on growth hormone, which he responded to robustly. he is now he is in the th percentile for height. he had adenoidectomy due to sleep-disordered breathing at the age of years. he is maintained on montelukast and inhaled corticosteroids for treatment of rhinitis and mild persistent asthma. he is on atenolol for the treatment of primary hypertension. on physical exam, he has facial dysmorphisms, thickened skin, and contraction of the fingers consistent with myhre syndrome. immunologic elevation showed significant hypogammaglobulinemia (igg mg/dl), low iga ( mg/dl) and normal igm mg/dl. igg subclasses showed low igg and igg at mg/dl and mg/dl respectively. although he was fully vaccinated, his tetanus antibody was low at . iu/ml. however, this improved after repeat vaccination to . iu/ml. total t and b lymphocyte counts were normal; however, his memory cd and cd t cells were low for age at . % and . %, respectively. additionally, his switched memory b cell count was low at . %. conclusion: smad gain of function (myhre syndrome) can lead to impaired memory t and b cell formation with significant hypogammaglobulinemia and low iga. although the patient was able to respond to protein vaccination (tetanus), it is not clear if he will be able to maintain a long-term response. in a previous study, a similar gain of key: cord- -bx tq y authors: mikola, emilia; elenius, varpu; saarinen, maria; palomares, oscar; waris, matti; turunen, riitta; puhakka, tuomo; ivaska, lotta; rückert, beate; aab, alar; vahlberg, tero; vuorinen, tytti; allander, tobias; camargo, carlos a.; akdis, mübeccel; akdis, cezmi a.; jartti, tuomas title: tonsillar cytokine expression between patients with tonsillar hypertrophy and recurrent tonsillitis date: - - journal: clin transl allergy doi: . /s - - -z sha: doc_id: cord_uid: bx tq y background: tonsils provide an innovative in vivo model for investigating immune response to infections and allergens. however, data are scarce on the differences in tonsillar virus infections and immune responses between patients with tonsillar hypertrophy or recurrent tonsillitis. we investigated the differences in virus detection and t cell and interferon gene expression in patients undergoing tonsillectomy due to tonsillar hypertrophy or recurrent tonsillitis. methods: tonsils of surgical patients with tonsillar hypertrophy (n = ) or recurrent tonsillitis (n = ) were analysed. patients were carefully characterized clinically. standard questionnaire was used to asses preceding and allergy symptoms. respiratory viruses were analysed in tonsils and nasopharynx by pcr. quantitative real-time pcr was used to analyse intratonsillar gene expressions of ifn-α, ifn-β, ifn-γ, il- , il- , il- , il- , il- , il- , tgf-β, foxp , gata , rorc and tbet. results: median age of the subjects was years (range – ). patients with tonsillar hypertrophy were younger, smoked less often, had less pollen allergy and had more adenovirus, bocavirus- , coronavirus and rhinovirus in nasopharynx (all p < . ). only bocavirus- was more often detected in hypertrophic tonsils (p < . ). in age-adjusted analysis, tonsillar hypertrophy was associated with higher mrna expressions of il- (p < . ). conclusions: intratonsillar t cell and interferon gene expressions appeared to be relatively stable for both tonsillar hypertrophy and recurrent tonsillitis. of the studied cytokines, only newly discovered anti-inflammatory cytokine il- , was independently associated with tonsillar hypertrophy showing slightly stronger anti-inflammatory response in these patients. electronic supplementary material: the online version of this article ( . /s - - -z) contains supplementary material, which is available to authorized users. tonsillar disease is one of the most common disorders in the field of otorhinolaryngology. different types of tonsillar disease include recurrent tonsillitis and tonsillar hypertrophy, with both leading to symptoms of mouth breathing, snoring, dyspnea, apnea or dysphagia. treatment is usually antibiotics or tonsillectomy. tonsils are secondary lymphoid organs, which are centrally located at the beginning of the respiratory and gastrointestinal tracts where the immune system first comes into contact with infections agents and allergens [ ] . surgically removed palatine tonsils provide a conventional accessible source to study the interplay between foreign pathogens, allergens and the host immune system. our previous studies demonstrated that tonsils are organs where immune regulation takes clinical and translational allergy *correspondence: tuomas.jartti@utu.fi † varpu elenius and maria saarinen contributed equally as second author department of paediatrics and adolescent medicine, turku university hospital and turku university, p.o. box , turku, finland full list of author information is available at the end of the article place in which allergen-specific regulatory t cells can be generated by mechanisms depending on plasmacytoid dendritic cells [ , ] . the expression of t cell-and interferon specific genes in tonsils has been shown to be closely related to existing viral infections, age, and allergic illnesses [ , ] . however, data are scarce on the differences in tonsillar virus infections and immune responses between patients with tonsillar hypertrophy or recurrent tonsillitis [ ] [ ] [ ] . tonsils appear to provide a good in vivo model for investigating the mechanisms of inflammatory processes and infections in lymphoid organs [ ] [ ] [ ] [ ] . therefore, we studied whether virus detection and t cell and interferon gene expressions differed between the two main indications of surgery, tonsillar hypertrophy or recurrent tonsillitis. human tonsil samples used in this study were acquired from consecutive tonsillectomy patients who underwent tonsillectomy in satakunta central hospital, pori, finland between april and march . the inclusion criteria were elective tonsillectomy according to clinical indication and written informed consent from the study patient and/or his/her guardian. the study protocol was approved by the ethics committee of satakunta central hospital, pori, finland. study was initiated only after obtaining written consent from the participant or his/her guardian. a standard questionnaire was used to obtain information on allergic diseases and respiratory symptoms within days before the operation (additional file : table ) . tonsillectomy was performed according to routine clinical procedure. internal tonsillar tissue was immediately cut in - mm cubes, stored in rnalater rna stabilization reagent (qiagen, hilden, germany), incubated at + °c until next working day and finally stored at − °c after removal of the non-absorbed reagent. for viral analyses, a part of the tonsils and a nasopharyngeal aspirate were stored in dry tubes at − °c [ ] . nasopharyngeal aspirate samples were obtained during anaesthesia using a standardized procedure [ ] . serum total (oh)d measurement was done using an immunoassay (abbott architect, chicago, usa) and bioavailable levels of (oh)d were estimated using additional serum measurements (d-binding protein and albumin) and published formulae. tonsillar hypertrophy group was defined as patients who underwent tonsillectomy because of obstructive symptoms such as snoring, breathing difficulties or swallowing problems. there were no tonsillar infection problems in this group. recurrent tonsillitis group was defined as patients who underwent tonsillectomy because of recurrently infected tonsils (viral or bacterial) during the past - months. those operated because of acute infection or peritonsillar abscess were excluded. in-house real-time pcr assays were used to detect human bocavirus- , rhinovirus, enterovirus, and respiratory syncytial virus as described previously [ ] . seeplex rv ace detection (seegene, seoul, korea) multiplex pcr assay was used for detection of adenovirus, coronaviruses ( e/nl and oc /hku ), influenza a and b viruses, metapneumovirus, parainfluenza virus types - , respiratory syncytial virus group a and b, and rhinovirus [ , ] . virus diagnostics were carried out in the department of virology, university of turku, turku, finland, and in the department of clinical microbiology, karolinska university hospital, stockholm, sweden. to isolate total rna from palatine tonsils, tissues (previously stabilized in rnalater) were homogenized in grinding tubes containing ck ceramic beads by using a precellys homogenizer (bertin technologies, montigny le bretonneux, france) two times at rpm for s [ ] . total rna from cell samples was isolated using the rneasy mini kit (qiagen, hilden, germany). reverse transcription was performed with the revert aid m-mulv reverse transcriptase (fermentas, st. leon-rot, germany) using random hexamer primers according to the manufacturers protocol. gene expressions of ifnα, ifn-β, ifn-γ, il- , il- , il- , il- , il- , il- , tgf-β, foxp , gata , rorc and tbet were analysed by quantitative real-time pcr using itaq sybr green supermix with rox (bio-rad, hercules, ca, usa) on a ht fast real-time pcr instrument (applied biosystems, foster city, ca, usa). housekeeping gene elongation factor α (ef α) was used for normalization. data are shown as relative expressions, which show −(Δct) values multiplied by , where Δct corresponds to the difference between the ct value for the gene of interest and ef α. continuous variables are described as medians and interquartile ranges, and were analysed using mann-whitney u test due to skewed distribution. categorical variables are expressed as frequencies and percentages, and were analysed using chi square test or fisher exact test. clinical, viral and immunological differences between study groups were analysed using unadjusted and multivariable linear regression analysis. the adjustments for immunologic analyses were chosen using backward stepwise multivariable models that initially included clinical factors and virus infections which significantly differed between the groups (age, self-reported pollen allergy, self-smoking, both adenotomy and tonsillectomy performed, respiratory symptoms one month prior to the operation and bioavailable (oh)d level). the final model was adjusted only for age. before regression analyses, cytokine and transcription factor values were logtransformed because of positively skewed distributions. the mean difference was computed for log-transformed values: a recurrently infected group minus hypertrophic group. statistical analysis was completed using jmp version . . software (sas institute inc. cary, nc, usa). a two-sided p < . was considered statistically significant. originally, patients participated in the study. of them, subjects did not have remaining tonsil and/ or nasopharyngeal samples for the current analysis, and had no intratonsillar virology done in their samples. another subjects were excluded for having mixed indications of operation other than hypertrophy or tonsillitis ( fig. ) . thus, patients comprised the analytic cohort. forty-seven ( %) of them had tonsillar hypertrophy and ( %) had recurrent tonsillitis. all operations were performed during afebrile period of chronic tonsil condition. respiratory symptoms on the operation day were present equally in the hypertrophy group and the recurrent tonsillitis group ( vs. %, respectively; p = . ). the median age of the patients was years (range - ) and years (range - ), respectively (p < . ) ( table ). in addition to being younger, patients in the hypertrophy group had more often adenotomy and tonsillectomy done, had had hypertrophy and another indication (recurrent otitis media n = ; recurrent otitis media and fever n = ; recurrent fever n = ). had recurrent tonsillitis and another indication (recurrent fever n = ; recurrent otitis media n = ). had hypertrophy and tonsillitis; had hypertrophy, tonsillitis, and another indication (recurrent fever, n = ; recurrent otitis media, recurrent fever n = ; recurrent otitis media n = ). less self-reported pollen allergy, smoked less, and had less throat pain, but had more often rhinitis and cough month prior the operation and higher bioavailable (oh)d level than patients in the recurrent tonsillitis group (all p < . ) ( table ) . otherwise no significant differences were found between the two groups. significantly more patients in the hypertrophy group, compared to the recurrent tonsillitis group, had a virus in their nasopharyngeal aspirates ( vs. %, respectively; p < . ). in addition, patients in the hypertrophy group had more often adenovirus, bocavirus- , coronavirus or rhinovirus in nasopharyngeal aspirate (all p < . ) ( table ). however, intratonsillar virus detection didn't show statistically significant differences, except for bocavirus- which was detected in tonsils in % of patients with hypertrophy and only % of patients in recurrent tonsillitis group (table ) . patients in the hypertrophy group were more often positive for one virus in their nasopharyngeal aspirates ( vs. %) or two viruses in their tonsils ( vs. %, respectively) (both p < . ) ( table ) . in unadjusted analysis, patients in the hypertrophy group had stronger tonsillar expression of tbet (p = . ) and il- (p = . ) than patients in the recurrent tonsillitis group (tables , ). in the multivariable regression analysis, only age remained as a significant co-factor (table ) . after adjustment for age, the expressions of only il- was independently associated with tonsillar hypertrophy group (p < . , fig. ) . no other differences in cytokine or transcription factor expression were found between the groups. this study shows differences in virus detections and t cell and interferon gene expressions in patients undergoing tonsillectomy due to tonsillar hypertrophy or recurrent tonsillitis. patients with tonsillar hypertrophy were typically younger, and had more viral findings, but only bocavirus- was more often found in tonsils when compared to patients with recurrent tonsillitis. respectively, they also had less self-reported pollen allergy, but no differences were found in food allergies between the groups. after age-adjusted analysis, tonsillar hypertrophy was associated with higher tonsillar mrna expressions of il- . other than age, no other significant co-factors were found. il- (formerly il- family member ) is a fundamental inhibitor of innate immunity [ , ] . it has been shown to be expressed in macrophages, monocytes, plasma and epithelial cells [ ] . after ligand activation, il- inhibits inflammatory cytokines (especially il- β, but also il- , il- , ifn-γ, and tnf-α) and augments the level of antiinflammatory il- and t regulatory cells [ ] . we have previously shown that the expression of il- is closely and positively associated with other "immune activation/regulatory" cytokines (il- , il- , il- , tgf-β, foxp , gata , rorc , tbet) in tonsils [ ] . the current analysis adds that tonsillar expression of anti-inflammatory cytokine il- is also independently and positively associated with tonsillar hypertrophy. interferons (ifn-α, ifn-β, ifn-γ, il- , il- ) are cytokines with antiviral activity and their expression is induced by viral infection. il- and il- are members of ifn-λ family [ , ] . they are produced by dendritic data are expressed as mean differences as a recurrently infected group minus hypertrophic group. the data were analysed using backward stepwise linear regression analysis after logarithmic transformation. only significant co-factors were used as adjustments in the final model [ ] [ ] [ ] . we expected to see differences in ifn expression (lower responses in recurrent tonsillitis than in tonsillar hypertophy group), since they have antiviral properties and they up-regulate the expression of mhc class ii molecules on cells which increases the immune system's ability to recognize viruses [ , ] . however, we did not observe these differences. we speculate that tonsillar hypertophy may be a consequence of chronic inflammation in tonsils and the same interferon pathways are equally activated in both conditions. we have previously found strong intragroup correlations of tonsillar ifn expression(ifn-α, ifn-β, ifn-γ, il- ) [ ] . age was the main clinical characteristic differentiating the tonsillectomy indication groups. in agreement with previous findings [ ] , we found that obstruction due to the hypertrophy is more common with younger children where as adults have more recurrent tonsillitis. the age difference between the groups also explains the differences in smoking and in additional adenotomy performed. interestingly, reis and colleagues found no difference between the age distribution of hypertrophy and tonsillitis patients, but the narrow age range of their subjects (ages - years) may explain the lack of difference [ ] . virus was found in the nasopharynx of % of patients with tonsillar hypertrophy group and % in recurrent tonsillitis group. most often detected viruses were adenovirus, bocavirus- , coronavirus and rhinovirus. however, intratonsillar virus detection was low and did not show any statistically significant differences except for bocavirus- . the results of nasopharyngeal and intratonsillar virus detection vs tonsillar cytokine responses are discussed in detail in our previous report [ ] . small differences in cytokine expression may partly be explained by differences concerning tonsillar germinal centers. the mean follicular area has been found to be larger, and the number of germinal centers higher, in the hypertrophy group compared to the recurrent tonsillitis [ ] [ ] [ ] . in our study, the samples were taken from inside of the tonsils to minimize the margin of error and the possibility to misinterpreted differences between the groups. seasonal changes, e.g. pollen and influenza seasons, may affect the expression of peripheral t cells [ ] , but we found no differences in tonsillar expression of cytokines between the seasons of the surgery. also, respiratory viruses are continuously detected in children with chronic tonsillitis throughout the year [ ] [ ] [ ] . circulating serum (oh)d level has been shown to been positively associated with il- level [ ] , but here it did not confound the results. a limitation of the current study is that we did not investigate bacterial colonization of the tonsils in these patients due to fact that the operation was done during an afebrile period of their chronic tonsil condition. the downstream signaling of il- is a complex process and to show functionality of il- by downstream mediators was not in the scope of this study. in addition to forming cell-surface receptor complexes, il- translocates to the nucleus where it binds to nuclear dna and participate in transcription. [ , ] il- is regarded as a "dual function" cytokine, similar to il- α and il- . in summary, this study provides new insights about t cell research in lymphoid tissue from the clinical aspect of the surgical indication for tonsillectomy. we found tonsils as a good in vivo model for investigating the mechanisms of inflammatory processes and infections in lymphoid organs. our data suggest that t-cell and interferon gene expressions appear to relatively stable over the two main indications of tonsillectomy, tonsillar hypertrophy and recurrent tonsillitis. however, anti-inflammatory immune responses, namely il- , might be slightly stronger in patients with tonsillar hypertrophy than with patients with recurrent tonsillitis. additional file . health questionnaire. mucosal immune response in the ear, nose and throat induction and maintenance of allergen-specific foxp + treg cells in human tonsils as potential first-line organs of oral tolerance triggering of specific toll-like receptors and proinflammatory cytokines breaks allergen-specific t-cell tolerance in human tonsils and peripheral blood distinct regulation of tonsillar immune response in virus infection the relationship of serum vitamins a, d, e and ll- levels with allergic status, tonsillar virus detection and immune response concomitant in vivo production of different cytokines in human tonsils increased cellular proliferation and inflammatory cytokines in tonsils derived from children with obstructive sleep apnea compartmentalization of total and virus-specific tissue-resident memory cd + t cells in human lymphoid organs il- is a fundamental inhibitor of innate immunity from il- to il- : the expanding spectrum of anti-inflammatory cytokines potential therapeutic use of il- : a key suppressor of innate immunity and allergic immune responses mediated by mast cells the role of il- in immunity and cancer il- a, il- b, and il- : promising cytokines with type i interferon-like properties interleukins, from to , and interferon-γ: receptors, functions, and roles in diseases the extended il- superfamily: il- , il- lymphocyte subsets in human tonsils: the effect of age and infection tonsillar hyperplasia and recurrent tonsillitis: clinical-histological correlation comparison of histology between recurrent tonsillitis and tonsillar hypertrophy the production of immunoregulatory cytokines is localized to the extrafollicular area of human tonsils association between cd + cd (high) t cells and atopy in children respiratory viruses are continuously detected in children with chronic tonsillitis throughout the year detection of the epstein-barr virus, human bocavirus and novel ki and ku polyomaviruses in adenotonsillar tissues hhv- infection of tonsils and adenoids in children with hypertrophy and upper airway recurrent infections the il- family member b translocates to the nucleus and down-regulates proinflammatory cytokines role of caspase- in nuclear translocation of il- , release of the cytokine, and il- inhibition of innate immune responses springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. the study protocol and manuscript were written by the investigators. tonsil the authors declare that they have no competing interests. the study protocol was approved by the ethics committee of satakunta central hospital, pori, finland. study was initiated only after obtaining written consent from the participant or his/her guardian.. key: cord- - hgku e authors: wong, hui hui; fung, to sing; fang, shouguo; huang, mei; le, my tra; liu, ding xiang title: accessory proteins b and ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: hgku e severe acute respiratory syndrome coronavirus (sars-cov) is an inefficient inducer of interferon (ifn) response. it expresses various proteins that effectively circumvent ifn production at different levels via distinct mechanisms. through the construction of recombinant ibv expressing proteins a, b and ab encoded by sars-cov orf , we demonstrate that expression of b and ab enables the corresponding recombinant viruses to partially overcome the inhibitory actions of ifn activation to achieve higher replication efficiencies in cells. we also found that proteins b and ab could physically interact with irf . overexpression of b and ab resulted in the reduction of poly (i:c)-induced irf dimerization and inhibition of the ifn-β signaling pathway. this counteracting effect was partially mediated by protein b/ ab-induced degradation of irf in a ubiquitin-proteasome-dependent manner. taken together, we propose that sars-cov may exploit the unique functions of proteins b and ab as novel mechanisms to overcome the effect of ifn response during virus infection. when challenged by a viral infection, the host mounts an immediate innate immune response, leading to the production of type i interferons (ifn-α and ifn-β) and the expression of hundreds of downstream ifnstimulated genes (isgs) (stetson and medzhitov, ; suhara et al., ) . central to the induction of type i ifn is interferon regulatory factor (irf ) (hiscott, ; taniguchi et al., ) . the initial step of the signaling cascade leading to irf activation is recognition of specific viral pathogen-associated molecular patterns (pamps) by host pattern recognition receptors (prrs) in two major pathways. these include cell surface toll-like receptors (tlrs), such as tlr , tlr , tlr and tlr , which sense viral components (kumagai et al., ; kumar et al., a kumar et al., , b , and cytosolic rna helicases such as retinoic acidinducible gene i (rig-i) and/or melanoma differentiation-associated gene (mda ), which detect viral rna (loo et al., ; onomoto et al., ) . upon binding to their ligands, prrs recruit adaptor proteins to set off a series of signaling cascades to phosphorylate and dimerize irf (fitzgerald et al., ) . the activated irf homodimer then translocates to the nucleus, switching on ifn synthesis (lin et al., ; suhara et al., ; thanos and maniatis, ) . many viruses have also evolved strategies to counteract the ifn action, including mechanisms that allow virus to evade recognition by the immune surveillance system, so as to inhibit ifn induction by hijacking molecules involved in ifn activation pathways or by inhibiting downstream signal transduction (goodbourn et al., ; versteeg et al., ; weber et al., ) . severe acute respiratory syndrome coronavirus (sars-cov) was the etiological agent of the sars epidemic in (guan et al., ; marra et al., ) . apart from four typical structural proteins, nucleocapsid (n), envelope (e), membrane (m) and spike (s) proteins, and approximately non-structural proteins (nsp - ) involved in viral replication, sars-cov encodes an exceptionally high number of accessory proteins that bear little resemblance to accessory genes of other coronaviruses narayanan et al., b) . similar to other coronaviruses, sars-cov is an inefficient inducer of ifn-β response in cell culture system (spiegel et al., ) and is sensitive to the antiviral state induced by ifns (spiegel et al., ; zheng et al., ) . its genome may therefore encode proteins that allow this virus to effectively circumvent ifn production in order to overcome limitations imposed by the ifn action. together with a few structural proteins and nsps, many coronavirus accessory proteins could suppress ifn production by targeting different aspects of the ifn signaling cascade (lim et al., ; liu et al., ; zhong et al., ) . for instance, sars-cov papain-like protease (plpro) attenuates ifn synthesis by abrogating irf phosphorylation and nuclear translocation by physically interacting with irf . ifn induction mediated by a constitutively active irf is also inhibited by the de-ubiquitination activity of sars-cov plpro (matthews et al., ) . on the other hand, nsp of pedv does not interfere irf phosphorylation and nuclear translocation, but interrupts the enhanceosome assembly of irf and creb-binding protein (cbp) by promoting proteasomal degradation of cbp (zhang et al., ) . targeting further upstream, sars-cov m protein prevents irf phosphorylation by inhibiting the assembly of tbk /ikk complex (siu et al., ) , whereas mers-cov m protein interacts with traf and disrupts traf -tbk association, leading to reduced irf phosphorylation (lui et al., ) . likewise, the accessory protein orf b of mers-cov has been shown to specifically bind to tbk and ikkε, thereby inhibiting irf phosphorylation and ifn-β production . signaling molecules downstream of ifn synthesis are also targets of sars-cov proteins. for instance, sars-cov nsp inhibits stat mediated transcription of isgs by inhibiting its phosphorylation (wathelet et al., ) , apart from inducing degradation of a wide range of host mrnas (kamitani et al., ; narayanan et al., a) . sars-cov orf blocks stat nuclear translocation by trapping the nuclear import factors in the endoplasmic reticulum and golgi apparatus . among other ifn antagonists identified are nucleocapsid (n) protein of sars-cov and pedv ding et al., ) , accessory protein a of mers-cov (siu et al., ) , and plpro domain (plp ) of mhv-a (wang et al., ) . ifn antagonism mediated by sars-cov n protein seemed to target a very early step of rna recognition (lu et al., ) . overexpression of sars-cov accessory protein a was found to down-regulate type i ifn receptor by promoting its ubiquitination and subsequent degradation via the lysosomal pathway (minakshi et al., ) . although both b and ab are encoded by sars-cov orf , they are expressed under distinct conditions. ab is expressed as a single protein encoded by the single continuous orf (orf ab) found in sars-cov isolated from animals and early stage human isolates. in contrast, as a consequence of a -nt deletion that results in two separate overlapping orfs (orf a ⁄ orf b), most human isolates obtained at the middle to later phase of the epidemic encode instead a and b as two distinct proteins (guan et al., ; oostra et al., ) . orf is presumably acquired from sars-related coronavirus from greater horseshoe bats through recombination (lau et al., ) , and the -nt deletion may be an evolutionary adaptation for enhancing viral pathogenesis in the human host. proteins a, b and ab may possess different biochemical properties and possibly cellular functions (law et al., ; le et al., ) . protein ab was shown to be a glycosylated er resident protein which can activate atf to modulate the unfolded protein response (sung et al., ) . protein a was found to enhance viral replication and induce cell death (c.y. , while b induces dna synthesis and down-regulates sars-cov e protein via a proteasomeindependent pathway (keng et al., ) . sars-cov b and ab were also shown to bind to both mono-and poly-ubiquitin when expressed in cell culture (keng et al., ) . whether these ubiquitin-binding properties allow them to interact with host cell proteins remains unknown. interestingly, when an in vivo attenuated recombinant sars-cov lacking the full-length e gene is passaged in mice, the orf sequence mutates to encode a pdz-binding motif in protein a, and the virus regained virulence (jimenez-guardeño et al., ) . in this study, we show proteins b and ab as novel ifn antagonists. evidence presented supports the direct interaction between these two proteins and irf . the two proteins were also found to partially suppress ifn induction by limiting irf activation and/or promoting the proteasome-mediated degradation of irf . african green monkey kidney cos- and vero cells, human nonsmall cell lung carcinoma h cells and human hepatocellular carcinoma huh cells were cultured in dulbecco's modified eagle's medium (dmem) supplemented with % fetal calf serum (hyclone) and % penicillin/streptomycin dmem (invitrogen) and maintained at °c with % co . to inhibit the proteasome activity, mg (sigma) at a final concentration of µm was added to cells h prior to harvest. to compare the growth kinetics of various recombinant virus strains, vero cells were infected with the respective recombinant ibv strains at a multiplicity of infection (moi) of . , and harvested at a h interval within h post-infection for virus titration through plaque assay. a monolayer of vero cells seeded on -well plates a day prior to infection was infected with µl of -fold serially diluted virus stock. after h of incubation at °c with regular shaking to ensure even distribution of the virus, cells were washed with pbs and cultured in ml of dmem containing % carboxymethyl cellulose (cmc) for days. the cells were then fixed with % paraformaldehyde and stained with . % toluidine. the number of plaques was counted in duplicates and the virus titer was calculated as plaque-forming unit (pfu) per ml. the pkto-flag- b and pkto-flag- ab plasmids were previously described (le et al., ) . the coding sequences of b or ab were also subcloned to the pxj -flag vector, which contains the cmv promoter for expression in mammalian cell lines. for the construction of the pxj -myc-irf plasmid, human irf gene was amplified from cdna of h cells using the forward primer ′-aacgcctcgacggaaccc caaagccacggat- ′and the reverse primer ′-gccggtaccttattg gttgaggtggtgggg- ′ prior to ligation into pxj -my plasmid at xhoi and kpni restriction sites. truncated mutants were then constructed based on the pxj -myc-irf construct using the following primers for pcr amplification: for irf ( − ), forward ′-ccgct cgagcggatgatgggaaccccaaagccacg- ′and reverse ′-gggg tacccctcaagaagtactgcctccaccat- ′; for irf ( - ), forward ′-ccgctcgagcggatggatacccaggaagacattct- ′ and reverse ′-ggggtacccctcatccaggcagcgtcctgtctc- ′; for irf ( − ), forward ′-ccgctcgagcggatgtggccagtcacactgc caga- ′ and reverse ′-ggggtacccctcagctctccccagggccct- ′; for irf ( − ): forward ′-ccgctcgagcggatggatacccagg aagacattct- ′ and reverse ′-ggggtacccctcagctctccccagg gccct- ′. constitutively active mutant pxj -myc-irf - d was generated by performing sequential site-directed mutagenesis pcr (quik-change ii site-directed mutagenesis kit; stratagene) to replace amino acids at positions , , , , and with the phosphomimetic aspartic acid. cells were lysed in ripa buffer in the presence of protease inhibitors (roche diagnostics) and phosphatase inhibitors (pierce). protein lysates were separated by electrophoresis in % sds polyacrylamide gels and transferred to nitrocellulose membrane (amersham biosciences) via wet transfer (bio-rad). the membranes were blocked overnight at °c with % non-fat milk in pbst before probing with specific primary antibodies, followed by horse-radish peroxidase (hrp)-conjugated antimouse, anti-rabbit or anti-goat igg secondary antibodies (dako), respectively. the following commercial primary antibodies were used: βtubulin (sigma), irf (santa cruz biotechnology), pirf ( ) (cell signaling) and β-actin (santa cruz biotechnology). polyclonal antibodies against ibv n were raised in rabbits by this laboratory (li et al., ) . monolayer cells grown overnight in -well plates (nunc) were infected with recombinant vaccinia virus encoding the bacteriophage t rna polymerase before transfection of plasmids using effectene reagent (qiagen), as previously described inglis, , ) . briefly, at h post-transfection, cells were harvested with µl of ripa buffer in the presence of protease (roche diagnostics) and phosphatase inhibitors (pierce). lysates were centrifuged at , ×g at °c for min, and the supernatant obtained was immunoprecipitated directly with antibody-conjugated agarose beads for h or with appropriate antibodies followed by incubation with protein a agarose beads (sigma) for another h at room temperature. the immunoprecipitated proteins were separated on sds-page and analyzed by western blot using appropriate antibodies. the hrp-conjugated anti-myc and anti-flag antibodies were purchased from sigma, while antibodies against igg, isg , β-actin and full-length irf were from santa cruz biotechnology. cells were lysed in buffer ( mm tris-hcl (ph . ), mm nacl, mm edta, % np- ) containing protease inhibitors and phosphatase inhibitors for min at °c. proteins were then separated by electrophoresis in % non-denaturing polyacrylamide gels, with % sodium deoxycholate (sigma) in the cathode buffer. irf monomers and dimers were detected by western blot analysis using polyclonal antibodies against full-length irf (santa cruz biotechnology). huh cells seeded on a -well plate were transfected with a total of μg of the appropriate plasmids using lipofectamine (invitrogen) according to manufacturer's instructions. pifn-β-luc and prl-tk were purchased from promega. µg of poly (i:c) complexed with µl lipofectamine was then introduced into the cells h later. cells were lysed h post treatment in passive lysis buffer (promega) and an aliquot of the lysates was measured for firefly and renilla luciferase activities according to the manufacturer's instruction (promega). construction of recombinant ibvs (ribvs) was carried out essentially as previously described le et al., ; tan et al., ) . to generate the b mutant ( bm) containing lysine to arginine mutations at all three positions, standard pcr site-directed mutagenesis was performed using the construct containing orf insertion as a template. the genotypes of ribvs were validated by sequencing. total rna was isolated using the trizol reagent (invitrogen) as described by manufacturer's protocol. three µg total rna was reversed transcribed (roche). the relative abundance of ifn-β, isg , isg and rantes mrnas in treated samples with respect to their mock treated counterparts was determined by real-time quantitative rt-pcr using the sybr green method (roche). briefly, a µl pcr reaction containing cdna template, the respective primers and lightcycler fast start sybr green i dna mastermix (roche) was prepared and subjected to a qpcr program using the lightcycler (roche). pcr cycling conditions comprised of an initial denaturation step at °c for min followed by an amplification program for cycles of s at °c, s at °c, and s at °c with fluorescence acquisition at the end of each extension. the relative expression of each gene is calculated using the comparative ΔΔc t method, using the mock treated sample as calibrator and housekeeping gene gapdh as internal control. the following primer pairs were used: for gapdh forward ′-gacaactttggtatcttggaa- ′ and reverse ′-ccaggaaatgag cttgaca- ′; for isg forward ′-tctcag aggagcctggctaag- ′ and reverse ′-ccacactgtatttggtgtctagg- ′; for isg forward ′-tggtggacaaatcgcacgaa- ′ and reverse ′-caggcgcagattc atgaac- ′; for rantes forward ′-ggcacgcctcgctgtcatcc tca- ′ reverse ′-cttgatgtgggcacggggcagtg- ′; and for ifn-β forward ′-ctctcctgttgtgcttctccac- ′ and reverse ′-tagtct cattccagccagtgct- ′. in a previous study, we reported the construction of two recombinant ibv (ribv b and ribv ab) expressing sars-cov proteins b and ab, respectively (le et al., ) . to assess the role of sars-cov b and ab in modulating viral replication, the growth properties and kinetics of the recombinant viruses were characterized and compared to wild type ibv (wtibv). a new recombinant ibv expressing the a and b in separate orfs (ribv a/b) was also constructed as a control. vero cells, known to lack the expression of type i ifns, were infected with wild type and recombinant ibv at an moi of~ . and harvested at every h over a time course of h for plaque assay to determine virus titers. consistent with our previous report on the impediment of virus replication by b expression, recombinant viruses expressing b, ab and a/b replicated at a slightly slower rate, compared to wild type virus during the first h of infection ( fig. a & b) . at h post-infection, however, the three recombinant ibvs were able to attain titers comparable to that of wild type virus (fig. b) . expression of protein b or ab could not be detected in cells infected with ribv a/b using western blot analysis (data not shown), further supporting our previous observation that b is not expressed from this construct (le et al., ) . taken together, these results demonstrate that the inclusion of b and ab does not render detectable enhancement effects on the replication and growth of ibv in culture cells. the slightly slower growth rates observed for the recombinant viruses at early time points of the infection cycle may be due to the introduction of extra sequences into the ibv genome. this is consistent with our previous observations that such genetic manipulations may alter the replication of ibv in cells (le et al., ; shen et al., ) . as expression of proteins a, b and ab did not render growth advantages to ibv in normal cultured cells, these proteins may not have direct functions in viral replication, especially in a heterogeneous genome context. . . expression of b and ab confers growth and replication advantages to ribv b and ribv ab over wtibv and ribv a/b in the presence of poly (i:c) the ability to subvert the host innate immune response is a critical factor for establishing effective virus replication. to determine if b and ab may have a role in counteracting the action of ifn, one of the most common and potent host anti-viral defense mechanisms, we examined the relative replication efficacy of ribv b, ribv ab and ribv a/b in the presence of poly (i:c). for this purpose, h cells were infected with wild type or the recombinant ibvs for h prior to mock or poly (i:c) transfection. cells were then further incubated for h before lysates were harvested for analysis of viral protein expression (fig. a) . in agreement with our growth kinetics studies earlier, the recombinant viruses replicated to similar levels by h (fig. a) in the absence of poly (i:c) treatment although the wild type virus was observed to replicate slightly faster as indicated by a higher abundance of ibv n protein (fig. a) . not surprisingly, replication of wild type and the three recombinant viruses were severely suppressed in cells stimulated by poly (i:c) (fig. a) , reflecting the sensitivity of coronavirus to interferon intervention. compared to wild type and ribv a/b, however, ribv b and ribv ab were observed to replicate significantly better and express higher levels of n protein in cells stimulated by poly (i:c) (fig. a) . using plaque assay, viral titers attained in infected cells exposed to poly (i:c) treatment were compared to their respective mock-treated counterparts. while poly (i:c)-treatment reduced the virus titers of wtibv (from . × to . × ) and ribv a/b (from × to × ) drastically by . % and %, respectively, titers of ribv b (from . × to . × ) and ribv ab (from . × to . × ) were reduced by a more modest % and %, respectively (fig. b) . these results suggest that proteins b and ab may have a functional role in modulating the ifn pathway. as expression of protein a (from ribv a/ b) did not show a similar effect, we hypothesized that this effect may be unique to the b region of the two proteins. in view of the central role of irf in regulating ifn activation during virus infection, b and ab with flag epitope-tagged to their ntermini were co-expressed with myc-tagged irf (fig. a) in cos- cells using the vaccinia/t expression system (anderson et al., ; lim and liu, ) for co-immunoprecipitation assays to determine if there is any physical interaction between the proteins. irf was precipitated from the cell lysates prepared from cells harvested at h post-transfection using anti-myc antibody-coated agarose beads, followed by western blot analysis with antibodies against the flag epitope. as shown in fig. b , proteins b and ab were consistently pulled down with irf in samples where they were co-expressed with irf . similarly, when the experiment was repeated using anti-flag coated agarose beads, irf co-precipitation was detected with both proteins. these results demonstrate that irf could physically interact with protein b and ab. to confirm that this interaction occurs in the context of coronavirus infection, total lysates prepared from cells infected with wtibv, ribv b and ribv ab together with mock infected controls were also subjected to immunoprecipitation with either polyclonal antibodies raised against sars-cov b or igg controls. immunoprecipitated proteins were then analyzed for the presence of endogenous irf using antisera against the protein. the detection of irf in cells infected with ribv b and ribv ab, but not in mock-and wtibv-infected cells (fig. c) indicates that protein b and ab formed complexes with irf during the virus replication process. it was noted that apart from the kda band corresponding to the endogenous monomeric irf , an additional band with the apparent molecular weight of approximately kda was the number of infectious particles released in the supernatants was quantified using plaque assay in triplicates, and the average number of pfu for each treatment was calculated. the relative amount of virus produced after poly (i:c) treatment was expressed as a percentage of their respective control cells not treated with poly (i:c). error bars showed standard deviation from independent experiments. error bars showed standard deviation from independent experiments. h.h. wong et al. virology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] detected ( fig. c) in cells infected by ribv- b. the identity of this band is not certain, but it may represent a modified form of irf . irf is well documented to be heavily targeted for post-translational modifications such as phosphorylation, ubiquitination, sumoylation, and neddylation (bibeau-poirier et al., ; hiscott, ; kubota et al., ; ran et al., ) . since proteins b and ab exhibited comparable efficiency in terms of pulling down irf , just the b region was then used for subsequent pull-down experiments to pinpoint the domains in irf essential for the interaction. four deletion constructs of irf either with or without a myc-tag at the n-termini were constructed (fig. a) and co-expressed with the flag-tagged b. co-immunoprecipitation experiments showed that the n-terminal region covering the first residues of irf (myc-irf ( - )) failed to interact with protein b (fig. e) , suggesting that the n terminal dna-binding domain (dbd) is dispensable for the interaction. immunoprecipitation with fragments covering residues - and - , respectively, resulted in effective pulldown of b (fig. e) , suggesting that the - region is likely to be important for the interaction. this region spans across several functional domains, including the nuclear export signal (nes), proline-rich region (pro), and the first residues of the irf association domain (iad). interestingly, immunoprecipitation experiments repeated with just the n terminal fragment comprising of only the first residues, thus excluding the residues of iad, abolished the binding (fig. d) , indicating that just the nes and pro regions alone were insufficient for the binding. consistently, the full-length irf with the n-terminal myctag and the n-terminal region covering the first residues could be efficiently pulled down by protein b (fig. d) . we next sought to test the effect of b and ab expression on irf activation. cells were transfected with either plasmid encoding flag- b, flag- ab or a corresponding control vector, before subjected to poly (i:c) treatment. western blot analysis coupled with native page revealed that the ectopic expression of proteins b and ab resulted in markedly decreased levels of poly (i:c)-induced irf dimerization (fig. a) . however, analysis of the same samples by sds-page showed no observable difference in the levels of hyper-phosphorylated irf (p-irf ) (appearing as more slowly migrating bands) in cells over-expressing proteins b and ab, compared to that in the control (fig. a) . it was also noted that the total amounts of irf were approximately comparable in these transfected cells (fig. a) . the concomitant decreased in irf dimerization with b and ab fragments that immunoprecipitated with protein b are denoted with (+). b. co-immunoprecipitation of myc-tagged irf with flag-tagged b and ab, respectively. cos cells were infected with the recombinant vaccinia/t virus at an moi of approximately per cell. after incubation for , cells were transfected with pmyc-irf , pflag- b, pmyc-irf +pflag- b, pflag- ab and pmyc-irf +pflag- ab, respectively. cells were harvested at h posttransfection, lysates prepared, and subjected to immunoprecipitation with either the myc antibodyconjugated agarose beads (top two panels) or the flag-antibody-conjugated beads (bottom two panels). the precipitates were separated on sds-page and analyzed by western blot with either anti-myc (top and bottom panels) or anti-flag ( nd and rd panels) antibodies. c. pull down of the endogenous irf protein with protein b. h cells were infected with wtibv, ribv b and ribv ab, respectively. cells were lysed h post-infection and immunoprecipitated with either polyclonal antibodies raised against sars-cov b or control igg antibodies. the precipitates were probed with antibodies against the full-length irf . d. co-immunoprecipitation analysis of the flag-tagged protein b co-expressed with the untagged irf from - , - and - (full-length). h cells were infected with the recombinant vaccinia/t virus at an moi of approximately per cell. immunoprecipitation was performed as described above. total lysates and the precipitates were separated on sds-page and analyzed by western blot with either anti-myc or anti-flag antibodies. e. coimmunoprecipitation analysis of the flag-tagged protein b co-expressed with the myc-tagged irf from - , - and - . h cells were infected with the recombinant vaccinia/t virus at an moi of approximately per cell. immunoprecipitation was performed as described above. total lysates and the precipitates were separated on sds-page and analyzed by western blot with either anti-myc or anti-flag antibodies. expression lead us to examine the impact of such phenomenon on the activation of ifn-β and other known irf downstream effectors. for the study on the effect on ifn-β promoter activity, a reporter construct expressing firefly luciferase driven by the ifn-β promoter was cotransfected with either b, ab or a control plasmid prior to poly (i:c) stimulation. at h post poly (i:c) treatment, lysates were assayed for luciferase activity. relative to cells transfected with control plasmid, poly (i:c)-induced ifn-β promoter activation in cells expressing b and ab were reduced to % and % respectively (fig. b) . this data mirrored the results obtained from the analysis of endogenous ifn-β transcript levels examined by quantitative real-time rt-pcr where relative levels of ifn-β mrna in poly (i:c)-treated cells expressing b and ab were reduced to approximately - % (fig. c ) of control. similar to the effect on ifn-β expression, the mrna levels of other irf downstream target genes (grandvaux et al., ) , including isg , rantes and isg , were significantly lower in cells over-expressing proteins b and ab than those of the control. the relative levels of isg (fig. d) , rantes (fig. e) and isg (fig. f) were reduced to approximately - , - and - %, respectively, in poly (i:c)treated cells transfected with b and ab, compared to those in cells transfected with the empty vector. the negative modulation of b and ab expression on the activation of irf was further verified from the analysis of irf activation and stability in virus-infected cells shown in fig. a . in the absence of poly (i:c) treatment, infection by wild type and all the three recombinant viruses did not induce a detectable level of irf dimerization using native page analysis (fig. a) . while irf dimerization was detected in all infected cells in the presence of poly (i:c) treatment (fig. a) , less poly (i:c)-induced irf dimers could be detected in cells infected with ribv- b, compared to those infected by wtibv (fig. a) . reduction in fig. . suppression of poly (i:c)-induced irf activation by protein b and ab. a. suppression of irf dimerization by proteins b and ab. huh cells were transfected with μg of empty vector, flag- b and flag ab, respectively, followed by stimulation with poly(i:c) for h. whole cell lysates were subjected to either native page or sds-page and probed with anti-irf . tubulin was included as a loading control. the ratio of dimeric irf to monomeric irf was calculated as the band intensity of monomer divided by the band intensity of dimer. b. suppression of poly (i:c)-induced ifn-β promoter activity by proteins b and ab. huh cells were transfected with control vector pcdna . , pcdna- b and pcdna- ab, respectively, together with a luciferase reporter construct under the control of ifn-β promoter. at h post-transfection, cells were then further transfected with poly (i:c). at h post-stimulation, cells were lysed and measured for the firefly luciferase activity. prl-tk was also co-transfected to serve as an internal control. data were represented as mean of triplicates from independent experiments. c-f. huh cells were transfected with μg of empty vector, flag- b and flag ab, respectively, followed by stimulation with poly(i:c) for h. total rna was then extracted for quantitative real-time rt-pcr with specific primers for ifn−β (c), isg (d), rantes (e) and isg (f). the expression of each gene was expressed relative to their respective control sample transfected with empty vector. data were represented as mean of replicates from independent experiments. gapdh was used as internal control. h.h. wong et al. virology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] irf dimerization, although to a lesser extent, was also observed in cells infected with ribv ab (fig. a) . interestingly, analysis of the same lysates by denaturing sds-page revealed that hyper-phosphorylated irf was invariantly detected in the poly (i:c) treated cells regardless of whether they are mock infected, infected with wild type virus or the recombinant ibvs (fig. a) . these observations were in agreement with the data we described in fig. a . however, we did observe that the overall level of irf expression was significantly reduced in cells infected with ribv b, both in the presence and absence of poly (i:c) treatment (fig. a) . a more moderate reduction of irf was also detected in cells infected with ribv ab (fig. a) . when the mrna levels of ifn-β from these samples were analyzed, negligible ifn-β induction was observed in virus-infected cells in the absence of poly (i:c) stimulation (data not shown). among the samples treated with poly (i:c), ifn-β induction in cells infected with ribv b and ribv ab infection was suppressed ( % and % relative to control wtibv, respectively) (fig. b) . taken together, these data suggest that the observed enhanced replication of ribv b and ribv ab in the presence of poly (i:c) may be due to the diminished ifn activation owing to the down-regulation of irf levels coupled to reduced irf dimer formation and ifn-β induction. . degradation of irf by b in a ubiquitin/proteasome-dependent manner. a. degradation of irf by b but not by a lysine-knockout mutant b. a lysine knockout mutant of ribv, ribv bm, was created by substituting the three lysine residues (k , k and k ) with arginine. cells were then infected up to h with ribv- b and ribv bm, respectively, before analyzing for irf expression. the same membrane was also probed with anti-ibv n, sars-cov protein b and actin antibodies. the relative amount of irf was calculated as the band intensity of the protein divided by the band intensity of actin. b. ubiquitin-dependent degradation of irf . cells transfected with either myc-tagged ubiquitin alone or co-transfected with flag-tagged b, in the presence or absence of mg , were subjected to immunoprecipitation with antibodies against myc. immunoprecipitates were then probed for pull-down of endogenous irf with specific antibodies. the relative amount of irf was calculated as the band intensity of the protein divided by the band intensity of actin. h.h. wong et al. virology ( ) - . . degradation of irf mediated by proteins b and ab is ubiquitindependent we had previously demonstrated that b binds to both poly-and mono-ubiquitin (le et al., ) . to address if the down-regulation of irf in ribv b-infected cells is linked to its ubiquitin-binding activities, lysine knockout mutant of b, bm was generated by mutating all three lysine residues (k , k and k ) in b to arginine. mutation of these lysine residues enhanced the stability of the b protein. as shown in fig. a , while the mutant bm protein was readily detected in ribv bminfected cells at and h post-infection, protein b was not detected under the same conditions (fig. a) . this is consistent with previous reports of the instability of b protein and that protein b could only be detected in ribv b-infected cells in the presence of proteasome inhibitors (le et al., ) . irf was down-regulated in cells infected with ribv b at both and h post-infection (fig. a) . infection of cells with ribv- bm did not result in similar reductions in irf protein levels, despite a similar replication efficiency of the two recombinant viruses (fig. a) . as mentioned previously, irf levels are regulated by ubiquitination during virus infection. however, some viruses exploit this mode of regulation by expressing viral proteins that promote untimely proteasomal degradation of irf (z. saira et al., ; sen et al., ) . to affirm the involvement of the ubiquitin-proteasome pathway in the down-regulation of irf observed with b, irf stability was studied in cells overexpressing protein b in the presence of ubiquitin. over-expression of b together with ubiquitin resulted in a reduction of detectable levels of irf compared to cells expressing ubiquitin alone (fig. b) . this degradation was partially suppressed in the presence of proteasome inhibitor mg (fig. b) . in a previous study, we found that proteasome-mediated rapid degradation of protein b could be much more efficiently inhibited by nlvs than did lactacystin (le et al., ) . as the supply of nlvs was discontinued, we chose to use mg , a product shown similarly mild inhibitory effect as lactacystin in the suppression of proteasome-mediated degradation of protein b, in this study. finally, to address the observation of the negligible impact exerted by b and ab on irf phosphorylation despite their capacity to suppress irf dimer formation, we study the effect of b on activated irf . a construct expressing the phosphomimetic form of irf (irf - d) was constructed, which contains amino acid substitutions at positions , , , , and by the phosphomimetic aspartic acid. irf - d undergoes spontaneous dimerization leading to ifn-β induction (grandvaux et al., ; lin et al., ) . over-expression of proteins b and ab was able to reduce the irf - d-induced ifn-β promoter activity to approximately % and % to that of control, respectively (fig. a ). this suggests that b can act on irf at step(s) that is downstream of its activation. when protein b is co-expressed with irf - d, b reduces the expression of irf - d in a dose-dependent manner (fig. b) . for reasons yet to be known, we observed that the expression of a monomeric form of irf - d seemed to be more affected by the presence of b than its homodimeric counterpart. similar to results presented earlier with endogenous irf , irf - d levels was partially rescued with the addition of mg , indicating the role of the ubiquitin-proteasome pathway (fig. c) . the sars-cov genome encodes an exceptionally high number of accessory genes that bear little resemblance to other known coronavirus accessory proteins. it is believed that while these unique proteins may not participate directly in viral replication, they possess biological functions that may enhance sars-cov pathogenesis in cells. by demonstrating that recombinant ibv expressing b or ab replicates more efficiently in the background of ifn activation, data presented in this study demonstrate that expression of sars-cov orf b and orf ab contributes positively to viral pathogenesis. as the expression of b helped to overcome partially the potent inhibitory effect of ifn induction on coronavirus replication in cell culture, it suggests that b and ab are novel ifn antagonists. orf failed to show up in the screen for interferon antagonist in a previous study , likely due to the incomplete inhibition of ifn activation exhibited by orf . other reasons could include the low expression of b in their system owing to the inherent instability of the protein (le et al., ) , and/or that the antagonistic activity of b is not apparent in their ndv model because the ifn antagonistic activity of this protein may be specific to coronavirus infection due to the requirement of other viral proteins. however, this is control vector pcdna . , pcdna- b or pcdna- ab together with a luciferase reporter construct under the control of the ifn-β promoter were transfected into huh cells. at h post-transfection, cells were transfected with pirf - d and co-transfected with prl-tk to serve as an internal control. at h post-stimulation, cells were lysed and measured for the firefly luciferase activity. data were represented as mean of triplicates from independent experiments. b. cells were co-transfected with ng of pxj -irf - d and either , , , ng of pkto- b. the total dna transfected was made up to ng using empty pkto vector. lysates harvested h post-transfection were either subjected to native or sds page and probed with anti-irf antibodies. actin was also probed to serve as a loading control. c. irf - d was co-transfected pkto- b. h post transfection, co-transfected cells was either left untreated or treated with mg h prior to harvest. lysates were then probed for irf , b and actin expression. the relative amount of irf - d was calculated as the band intensity of the protein divided by the band intensity of actin. h.h. wong et al. virology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] quite unlikely because the inhibitory effect could also be observed with ectopic expression of b and ab in the absence of viral replication. the other significant finding from this study is that b and ab directly physically interact with irf and that b-irf interaction involves part of the iad domain that is responsible for the formation of irf homodimers. over-expression of b and ab appears to have a more profound effect on irf dimerization than on its phosphorylation status. although it is still unclear whether this is a consequence of assay insensitivity or that the inhibitory actions of b and ab bypass the step of phosphorylation and targets specifically on the event of irf dimerization, we are inclined to believe that it is the latter owing to the ability of b to suppress ifn-β induced by constitutively active phosphomimetic irf - d. while disruption to the dimerization event as a result of direct steric interference brought about by b interaction at the iad domain is an attractive model, the exact mechanisms can only be ascertained through more in-depth studies such as structural analysis. we previously reported that the b region of sars-cov proteins b and ab consists of domains that allow for ubiquitin binding, ubiquitination and glycosylation (le et al., ) . based on these finding, we proposed that the b region may mediate the binding of b and ab to ubiquitinated cellular proteins, such as p and iκbα (le et al., ) . here we showed that irf , another protein regulated by ubiquitination (siu et al., ; spiegel et al., ) , interacts with proteins b and ab, suggesting that the ubiquitin-binding properties of b region could allow them to interact with multiple cellular proteins. it would be interesting to find out what other cellular targets bind to b and ab and whether they have a regulatory role during sars-cov infection. furthermore, expression of b and ab appears to regulate the stability and function of irf by promoting degradation of irf in a ubiquitin/ proteasome-dependent manner. several viral proteins have been reported to cause proteasomal degradation of irf (z. saira et al., ; sen et al., ). irf degradation is typically triggered post-infection, when viral infection-induced irf activation leads to the ubiquitination of the protein targeting it for proteasomal degradation liu et al., ) . this serves to regulate type i ifn production as excessive ifn is detrimental to the cells. in this study, we observed irf degradation even in the absence of strong irf activation in cells infected with ribv b and ribv ab, suggesting that the b-and ab-mediated irf degradation is not the result of a typical negative feedback mechanism to bring the ifn level back to the physiological level at the end of viral infection as observed with other virus infection, but may be an active step undertaken by the virus to limit irf activation during its course of replication. at this stage, while we confirm the involvement of the proteasome, we do not know if other cellular factors are also recruited by b to mediate irf degradation. cellular factors, such as peptidyl-prolylisomerase pin (saitoh et al., ) , ro /trim (pin + ro ) and e ubiquitin ligase rbcc protein interacting with pkc (rbck ) (zhang et al., ) , were identified to participate in the negative regulation of irf by targeting it for ubiquitination. additional experiments such as mass spectrometry could perhaps help elucidate if b interaction with irf also involves any of these reported proteins. considering the fact that b expression is unstable and that it is only expressed during the late stages of sars-cov infection, it seems counter-intuitive why the virus would express such a late stage ifn antagonist as b. this is especially true since sars-cov has a strong inhibitory effect on ifn induction plausibly owing to the expression of multiple viral proteins that antagonize the pathway in myriad ways during the earlier stages of infection. a possible explanation may come from a study carried out by spiegel and coworkers who reported that while nuclear translocation of irf remains unabated by sars-cov infection during the early stages of infection at h post-infection, irf activation is specifically blocked during later stages of sars-cov infection at h post-infection (spiegel et al., ) . this coincides with the late expression of b during sars-cov infection (keng et al., ) . hence, we hypothesize that the growth advantage conferred by the expression of b in recombinant ibv was due to the role of b in latestage viral pathogenesis, when the expression of b aids in dampening the activation of irf that may occur during the later phase of infection. nevertheless, we do not rule out the possibility that b and ab may regulate irf via mechanisms independent of its ubiquitin-binding activity as it has also been shown to down-regulate e protein via a ubiquitin-independent proteasomal pathway (keng et al., (keng et al., , . using an infectious clone system based on the urbani strain of sars-cov, the -nt deletion is inserted to fuse orf a/b back into the single orf . compared with the wild type control, this recombinant virus replicates similarly in both cell culture and in the murine model (yount et al., ) . theoretically, only protein ab is produced in cells infected with this virus, whereas both proteins a and b are produced in the wild type control. because both b and ab can antagonize ifn signaling by mediating irf down-regulation, it is no surprise that the recombinant virus replicates similarly as the wild type control. deletion of accessory proteins , , b, a, b and b altogether in recombinant virus rsars-cov-Δ[ - b] (dediego et al., ) showed that the recombinant virus replicates as well as wild type control in both cell culture and transgenic mice expressing the sars-cov receptor human angiotensin converting enzyme- (hace- ). since ifn antagonist function is also possessed by other sars-cov proteins (such as nsp , plpro, m and n), it is possible that loss of functional b in rsars-cov-Δ[ - b] is compensated, and thus the recombinant virus is not attenuated in vivo. further studies using recombinant sars-cov with only b or ab deleted should be performed in cell culture and in appropriate animal models, to better characterize the detailed mechanisms of their involvement in modulating viral replication and pathogenesis. finally, poly (i:c) is known to be able to induce both ifn and a subset of ifn-stimulated genes through activation of irf in the absence of ifn. we believe that the observed antiviral effects of poly (i:c) on wild type and recombinant ibv in this study would be the combined action of poly (i:c)-induced ifn-stimulated 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type i interferon production through both cytoplasmic and nuclear targets severe acute respiratory syndrome coronavirus groupspecific open reading frames encode nonessential functions for replication in cell cultures and mice negative feedback regulation of cellular antiviral signaling by rbck -mediated degradation of irf suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp potent inhibition of sars-associated coronavirus (scov) infection and replication by type i interferons (ifn-alpha/beta) but not by type ii interferon (ifn-gamma) recent progress in studies of arterivirus-and coronavirus-host interactions this work was partially supported by an academic research fund (acrf) tier grant (acr / ), ministry of education, singapore and by guangdong province key laboratory of microbial signals and disease control grants msdc- - and msdc- - , guangdong, people's republic of china. key: cord- - tkhjiwl authors: gómez-laguna, j.; salguero, f.j.; barranco, i.; pallarés, f.j.; rodríguez-gómez, i.m.; bernabé, a.; carrasco, l. title: cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus date: - - journal: j comp pathol doi: . /j.jcpa. . . sha: doc_id: cord_uid: tkhjiwl porcine reproductive and respiratory syndrome (prrs) is caused by a virus that predominantly replicates in alveolar macrophages. the aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by the prrs virus (prrsv). expression of interleukin (il) α, il- and tumour necrosis factor (tnf)-α correlated with the severity of pulmonary pathology and the numbers of pulmonary macrophages. significant correlations were observed between prrsv infection and the expression of il- , between the expression of il- p and interferon (ifn)-γ, and between the expression of tnf-α and ifn-γ. these findings suggest that prrsv modulates the immune response by the up-regulation of il- , which may in turn reduce expression of cytokines involved in viral clearance (e.g. ifn-α, ifn-γ, il- p and tnf-α). the results also suggest that expression of ifn-γ is stimulated by il- p and tnf-α, but not by ifn-α. all of these cytokines were expressed mainly by septal macrophages with weaker expression by alveolar macrophages, lymphocytes and neutrophils. there appears to be differential activation of septal and alveolar macrophages in prrsv infection, with septal macrophages being the major source of cytokines. macrophages play a significant role in the defence against pathogens by phagocytosis following recognition by surface pattern recognition receptors (prrs), by antigen presentation involving class ii molecules of the major histocompatibility complex (mhc ii) and by production of cytokines (mitchell and kumar, ) . cytokines may also be synthesized by several other immune or non-immune cells including lymphocytes, neutrophils and fibroblasts. the expression of cytokines following engagement of prrs by pathogen-associated molecular patterns (pamps) constitutes the main pathway involved in the activation of macrophages (zhang and mosser, ) . some cyto-kines may also act as inhibitors of macrophage activation. for example, interleukin (il)- , tumour necrosis factor (tnf)-a, interferon (ifn)-a and ifn-g act as potent activators of macrophages, whereas il- inhibits activation of these cells (mitchell and kumar, ) . ifn-g and il- are classically involved in the subtype of immune response mediated by th lymphocytes, with both cytokines working in parallel (biron and sen, ) . the proinflammatory cytokines, including il- a, tnf-a and il- , are of greatest importance during the innate immune response (biron and sen, ) . ifn-a also participates in the innate response through antiviral activity, by inducing the differentiation of na€ ıve t cells into ifng secreting effector cells and by down-regulation of il- expression (biron and sen, ; tizard, ) . in contrast, il- is considered to be an immunosuppressive cytokine as it down-regulates the expression of several other cytokines including il- a, tnf-a, il- , il- itself, il- and ifn-g (biron and sen, ; moore et al., ) . porcine reproductive and respiratory syndrome (prrs) is one of the most economically significant diseases of the swine industry (neumann et al., ) . the syndrome is characterized by interstitial pneumonia in growing pigs and reproductive failure in gilts (rossow, ) . prrs is caused by a positive-strand enveloped rna virus, known as prrs virus (prrsv), which belongs to the family arteriviridae in the order nidovirales (fauquet et al., ) . prrsv replicates mainly in porcine alveolar macrophages and, to a lesser extent, in monocytes and dendritic cells (molitor et al., ; bautista and molitor, ) . several studies have examined the role of cytokines in the pathogenesis of prrs (van reeth and nauwynck, ) ; however, it is not clear how cytokines participate in macrophage activation during prrsv infection or how they regulate development of the immune response to the virus. thanawongnuwech et al. ( ) suggested that expression of ifn-g by macrophages and lymphocytes may have an inhibitory effect on the replication of prrsv. another study of a prrsv modified-live vaccine has shown that upregulation of il- expression is associated with a lower number of ifn-g secreting cells amongst peripheral blood mononuclear cells (pbmcs) (dı´az et al., ) . the role of cytokines in the interstitial pneumonia described in prrs has not yet been determined. accordingly, the aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by prrsv. thirty-two specific pathogen free, -week-old pigs from a prrsv seronegative farm were used in this study. twenty eight animals were randomly assigned to groups of four and inoculated by the intramuscular route with ml of the third passage of prrsv field isolate (kindly provided by dr. e. mateu) at . tcid . the virus was initially isolated in porcine alveolar macrophages from the serum of naturally infected piglets during a respiratory outbreak of prrs affecting a spanish farm. this field isolate has an open reading frame- sequence similarity of % with lelystad virus (genbank accession number ef ). the inoculated animals were killed at , , , , , and days post-inoculation (dpi). another group of four pigs were sham-inoculated controls. these animals were injected intramuscularly with ml of sterile rpmi medium and killed at the end of the study ( dpi). all animals were sedated with tiletamine-zolazepam (zoletilÔ; virbac, barcelona, spain) followed by a lethal dose of % sodium thiopental (thiovetÔ; vet limited, leyland, lancashire). the experiment was carried out according to the guidelines of the european union (directive / /eec) and was approved by the local ethical committee of centro de investigacio´n en sanidad animal (cisa-inia; valdeolmos, madrid, spain). the pigs were monitored daily for clinical signs, including rectal temperature and a clinical respiratory score, as previously described (halbur et al., ) . during post-mortem examination, gross lung lesions were evaluated by visual inspection and each lung lobe was scored to reflect the approximate volume or percentage of the lung tissue affected (halbur et al., ) . samples from the medial lobe of the right lung were fixed in % neutral buffered formalin and in bouin's solution, processed routinely and embedded in paraffin-wax. sections ( mm) of formalinfixed tissue were stained with haematoxylin and eosin (he) for microscopical examination. since prrsv is most frequently detected in the apical and medial lung lobes (halbur et al., ) , the medial lobe was selected for immunohistochemical examination. the avidinebiotineperoxidase complex technique (abc) was used for the detection of prrsv, macrophages and cytokine proteins as described previously (hsu et al., ) . formalin-fixed tissue was used for detection of macrophages and tissue fixed in bouin's solution for all other immunohistochemical reactions. briefly, the sections were dewaxed and dehydrated through graded ethanol and the endogenous peroxidase activity was quenched in h o % in methanol for min. the sections were washed with phosphate buffered saline (pbs; ph . , . m) and incubated for min at room temperature with ml per slide of blocking solution in a humid chamber. table describes the primary antibodies and antigen retrieval methods applied. primary antibodies were incubated overnight at c in a humid chamber. in each case the corresponding biotinylated secondary antibody was incubated for min at room temperature. an avidineperoxidase complex (vector laboratories, burlingame, california) was applied for h at room temperature. labelling was 'visualized' by application of the novaredÔ substrate kit (vector laboratories). sections were counterstained with mayer's haematoxylin, dehydrated and mounted. for negative controls, the primary antibody was replaced by blocking solution, normal serum and isotypematched reagents of irrelevant specificity. the number of labelled cells was determined as described previously (salguero et al., ) . briefly, the labelled cells were counted in non-overlapping and consecutively selected high magnification fields of . mm . results are expressed as the number of cells per mm . immunolabelled cells were identified and counted morphologically as macrophages, lymphocytes or neutrophils. pulmonary intravascular macrophages and interstitial macrophages were grouped together and described as 'septal macrophages'. the numbers of macrophages, prrsv-infected and cytokine-expressing cells were expressed as a mean ae sd. these values were evaluated for approximate normality of distribution by the kolmo-gorovesmirnov test. differences between the means of control and inoculated animals were assessed by the kruskalewallis test followed by the manne whitney-u non-parametric test (graphpad instat . , san diego, california). correlation between the presence of lung lesions and the expression of virus, macrophages and cytokines was assessed by the spearman test (graphpad instat . ). p < . was considered to represent a statistically significant difference. control animals did not display clinical signs or significant gross or microscopical lung lesions. although inoculated animals displayed no significant respiratory distress, they did develop dullness, weight loss and mild hyperthermia from dpi. from dpi until the end of the study, almost % of the pulmonary parenchyma of the inoculated animals was affected by interstitial pneumonia, and this was confirmed by microscopical examination of the tissue samples (figs. a, a). mac antibody defined macrophages in sections of lung tissue. the total number of macrophages increased in the lung of inoculated animals from dpi onwards (fig. b) . this related primarily to an increase in the number of septal macrophages (figs. b and b). the number of alveolar macrophages decreased to dpi and recovered thereafter (fig. b) . the number of macrophages, as determined by expression of mac , was significantly correlated with the microscopical score of lung lesions (r ¼ . ; p < . ) ( table ) . there was no expression of prrsv antigen in the lung of control animals. prrsv antigen was detected in the lung of infected pigs from dpi until the end of the study, peaking at dpi (fig. c) . this antigen expression was detected mainly in the cytoplasm of macrophages, and was significantly higher in alveolar macrophages than in septal macrophages (p < . ) (figs. c, c and a ). immunolabelled cells were observed not only in areas of interstitial pneumonia, but also in lung parenchyma without lesions. il- a was observed in the cytoplasm of alveolar and septal macrophages and neutrophils, the latter appearing to be a significant source of this cytokine (fig. d) . expression of il- a was always higher in inoculated animals than in controls, and had a bimodal peak at and dpi (p < . ) (fig. d) . the increase in il- a at dpi was attributed primarily to neutrophils (p < . ) (figs. d and d ). prrsv, il- a, il- and tnf-a respectively. *indicates statistically significant differences (p < . ) between the inoculated and control group. **indicates statistically significant differences (p < . ) between the numbers of alveolar and septal macrophages at a given time point. pams, alveolar macrophages. expression of il- and tnf-a also had a bimodal peak at and dpi (p < . ) (fig. e and f) . il- expression remained elevated until the end of the study (fig. e ), but the expression of tnf-a was no different to that of control animals from dpi (fig. f ). septal macrophages were the main cell population involved in the expression of both il- and tnf-a (p < . ) (figs. e, f, e and f). alveolar macrophages and lymphocytes also expressed these cytokines, but to a lesser extent ( fig. e and f) . the labelling of proinflammatory cytokines was observed mainly in areas of interstitial pneumonia with moderate to severe thickening of the alveolar septa. few immunolabelled cells were observed in areas of the lung without lesions (fig. def) . the correlation between the lung lesion score, macrophage count and expression of proinflammatory cytokines is shown in table . table shows the correlation between the expression of tnf-a and ifn-g. tissue expression of ifn-a, ifn-g, il- and il- p ifn-a was expressed in the cytoplasm of alveolar and septal macrophages and lymphocytes. septal macrophages were the main cell type involved in the expression of this cytokine, which was significantly increased at dpi (p < . ) and decreased thereafter (figs. a and f). the number of ifn-a-expressing alveolar macrophages was also increased at dpi (p < . ). ifn-a expression was always higher in inoculated animals than in controls (fig. a) . the expression of ifn-a was significantly correlated with virus expression (r ¼ . ; p < . ) ( table ) . the kinetics of labelling for ifn-g and il- p were similar throughout the study (r ¼ . ; p < . ) (table ) , with both cytokines peaking at dpi and decreasing thereafter ( fig. b and c) . these cytokines were expressed not only mainly by septal macrophages, but also by alveolar macrophages and lymphocytes (fig. c, e and g) . inoculated animals always had more ifn-g-expressing cells than controls. the expression of il- peaked at dpi and decreased thereafter (fig. d ). il- was expressed mainly in the cytoplasm of septal macrophages ( fig. b and d) . the kinetics of expression of il- were significantly correlated with that of the virus (r ¼ . ; p < . ) ( table ) . consecutive sections immunolabelled for prrsv antigen, ifn-g and il- showed co-localization of ifn-g and prrsv antigen, whereas the expression of il- occurred in areas without expression of ifn-g (fig. aec) . the number of septal macrophages expressing these cytokines was always greater than the number of labelled alveolar macrophages (fig. ) . immunolabelling for ifn-a, ifn-g, il- p and il- was associated with areas of mild to moderate interstitial pneumonia and was much less in areas of pulmonary parenchyma without lesions (fig. ) . the correlations between the expression of prrsv, ifn-a, ifn-g, il- , il- p and tnf-a in the lung of prrsv-infected pigs are shown in table . several reports have described changes in cytokine expression during prrsv infection, but these have not addressed local cytokine production within pulmonary lesions. the present study has characterized expression of cytokines by pulmonary macrophages in order to determine the role of these molecules in the pathogenesis of the respiratory form of prrs. the experimental infection did not lead to the animals developing respiratory symptoms, but dullness, weight loss, mild hyperthermia and lesions of the pulmonary parenchyma were observed. prrsv replication peaked at dpi and was mainly localized to alveolar macrophages, which are considered as the target cell for viral replication (molitor et al., ; bautista and molitor, ) . no correlation was observed between the presence of viral antigen and the severity of the microscopical lung lesions. however, the microscopical lung lesions were significantly correlated with marked inflammatory infiltration of the septa and the number of infiltrating macrophages. moreover, the lung lesions showed significant correlation with the expression of both il- a and il- , but not of tnf-a, and macrophage counts were correlated with the expression of il- a and tnf-a, but not of il- . these observations suggest that il- a may play a significant role in the development of interstitial pneumonia during prrs. nonetheless, when all the three proinflammatory cytokines were considered, a highly significant correlation was observed between both microscopical pulmonary lesions and macrophage counts. although prrsv replicated mainly in alveolar macrophages, proinflammatory cytokines were expressed mainly by septal macrophages, especially il- and tnf-a, from dpi onwards. this fact indicates activation of septal macrophages, which may be induced by the synthesis of cytokines (zhang and mosser, ) . similar findings have been reported for other porcine viral diseases, including african swine fever, which triggers activation of interstitial macrophages expressing il- a and tnf-a after viral replication (carrasco et al., ) . in the present study there was marked intra-alveolar infiltration of neutrophils expressing il- a at dpi. the earlier increase of both il- a and tnf-a may have been responsible for the induction of this infiltration and the subsequent activation of these cells, since these cytokines are considered as neutrophil-chemoattractant and stimulant agents (van reeth and nauwynck, ) . furthermore, il- a and tnfa may induce the synthesis of il- (van reeth and nauwynck, ; mitchell and kumar, ) ; however, in our study no correlation was observed between the expressions of these cytokines, although the maximum expression of il- temporally coincided with higher expression of il- a and/or tnf-a. isolate . *indicates statistically significant differences (p < . ) between the inoculated group and controls. **indicates statistically significant differences (p < . ) between the counts of alveolar and septal macrophages at a given time point. pams, alveolar macrophages. the interferons are known to play a significant role in the host immune response against viruses (van reeth and nauwynck, ; biron and sen, ) . ifn-a participates in the innate immune response and is able to induce synthesis of ifn-g (biron and sen, ; tizard, ) . in the present study, a significant correlation was observed between prrsv replication and ifn-a expression, suggesting that prrsv directly induces expression of ifn-a by macrophages. however, prrsv induces lower levels of ifn-a when compared with other porcine respiratory viral diseases, such as those caused by swine influenza virus or porcine respiratory coronavirus (van reeth and nauwynck, ) , which indicates that ifn-a expression may be insufficient to induce clearance of prrsv. the expression of ifn-g by macrophages and lymphocytes has been previously reported in the lung of prrsv-infected pigs (thanawongnuwech et al., ) . in that study, an increase in expression of ifn-g was observed at dpi for infection with highly virulent strains, whereas strains of low virulence induced a higher expression at the end of the study ( dpi). in the present study, the expression of ifn-g was undulating, showing a peak at dpi, just when prrsv replication was maximal. ifn-g is known to protect macrophages in vitro against prrsv replication (bautista and molitor, ) ; however, that viral replication occurred throughout the period of the present study may suggest that in this experimental infection the ifn-g response was not strong enough to eliminate prrsv infection. the production of ifn-g by pulmonary macrophages is induced by the expression of other cytokines including il- , tnf-a and ifn-a (nguyen and benveniste, ; mitchell and kumar, ; tizard, ) . in the present study there was good correlation between the expression of il- p , tnf-a and ifn-g, but poor correlation between expression of ifn-a and ifn-g. therefore, il- p and tnf-a might be the most significant cytokines involved in the induction of synthesis of ifn-g in this experimental infection. royaee et al. ( ) reported correlation between virus-specific ifn-a secreting cells and virus-specific ifn-g secreting cells in pigs vaccinated with an attenuated, modified-live prrsv vaccine. high antigenic and pathogenic differences have been related to european and north american prrsv genotypes, and suggested to occur within a given genotype (halbur et al., ; mateu et al., ; stadejek et al., ) , which may be the cause of the discrepancies between the present study and that of royaee et al. ( ) . despite the expression of ifn-a, ifn-g, il- p and tnf-a, prrsv was still replicating in the lung of infected pigs at the end of the study. il- is an immunomodulatory cytokine that is able to inhibit the synthesis and release of other cytokines (biron and sen, ; moore et al., ) . therefore, the expression of il- observed in the present study might be responsible for reduced expression of cytokines such as ifn-a, ifn-g, il- p and tnf-a, that in turn may impair prolonged viral replication in the lung of infected animals. interestingly, the expression of il- was observed in areas of lung that showed no expression of ifn-g. moreover, the expression of il- was significantly correlated with prrsv replication. these results suggest that prrsv may induce the expression of il- and, therefore, the expression of il- might inhibit the expression of other cytokines, allowing a prolonged viral replication in the lung. this idea is supported by the observed immunolabelling for il- and ifn-g in consecutive sections of the lung and by the correlation observed between the expression of il- and ifn-a. in conclusion, the results of the present study indicate that activation of septal and alveolar macrophages differs throughout prrsv infection, and that the septal cells are the main source of cytokines. proinflammatory cytokines are actively expressed at the onset of the interstitial pneumonia and there is direct correlation between this expression and the infiltration of the pulmonary interstitium by macrophages. additionally, prrsv appears able to modulate the local immune response by inducing the expression of il- by macrophages, which may in turn reduce the levels of cytokines involved in viral clearance such as ifn-a, ifn-g, il- p and tnf-a. ifn gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages interferons and other cytokines african swine fever: expression of interleukin- alpha and tumour necrosis factor-alpha by pulmonary intravascular macrophages different european-type vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs comparison of the antigen distribution of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus use of avidinebiotineperoxidase complex (abc) in immunoperoxidase techniques: a comparison between abc and unlabeled antibody (pap) procedures genetic diversity and phylogenetic analysis of glycoprotein of europeantype porcine reproductive and respiratory virus strains in spain immune diseases immunity to prrsv: double-edged sword interleukin- and the interleukin- receptor assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states critical role of tumor necrosis factor-a and nf-kb in interferon-g-induced cd expression in microglia/macrophages porcine reproductive and respiratory syndrome deciphering the involvement of innate immune factors in the development of the host response to prrsv vaccination proinflammatory cytokines induce lymphocyte apoptosis in acute african swine fever infection porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes cell signaling: cytokines and their receptors proinflammatory cytokines and viral respiratory disease in pigs macrophage activation by endogenous danger signals the authors thank dr. e. mateu (universitat auto`noma de barcelona, spain) for providing the prrsv field isolate and dr. k. van reeth (universiteit gent, belgium) for her kind gift of f mab. j.g-l. was supported by a doctoral grant from the spanish ministry of education and science (ap- - ). this work was supported financially by the spanish ministry of education and science project number agl - /gan. supplementary data associated with this article can be found, in the online version, at doi: . /j. jcpa. . . . the authors declare no competing financial interests. key: cord- -i fp rys authors: wang, mengmei; zhao, yang; hu, weihua; zhao, dong; zhang, yunting; wang, tao; zheng, zhishui; li, xiaochen; zeng, shaolin; liu, zhenlian; lu, li; wan, zhihui; hu, ke title: treatment of covid- patients with prolonged post-symptomatic viral shedding with leflunomide -- a single-center, randomized, controlled clinical trial date: - - journal: clin infect dis doi: . /cid/ciaa sha: doc_id: cord_uid: i fp rys objective: to evaluate the efficacy and safety of leflunomide, an approved dihydroorotate dehydrogenase inhibitor, to treat covid- patients with prolonged post-symptomatic viral shedding. methods: we conducted a prospective, randomized, controlled, open-label trial involving hospitalized adult covid- patients with prolonged pcr positivity. patients were randomly assigned to receive either leflunomide ( mg, q h, three consecutive times, orally; then mg, once daily for days), in addition to nebulized interferon alpha a (ifn α- a, million iu each time, twice daily for days), or nebulized ifn α- a alone for days. the primary end point was the duration of viral shedding. results: a total of covid- patients with prolonged pcr positivity were randomized into groups; were assigned to the leflunomide group, and were assigned to the interferon alone group. treatment with leflunomide was not associated with a difference from the interferon alone group in the duration of viral shedding (hazard ratio for negative rt-pcr, . ; % confidence interval, . - . ; p= . ). in addition, the patients given leflunomide did not have a substantially shorter length of hospital stay than patients treated with interferon alone, with median (iqrs) durations of . ( . - . ) days and . ( . - . ) days, respectively, p= . . two leflunomide recipients were unable to complete the full -day course of administration due to adverse events. conclusions: in covid- patients with prolonged pcr positivity, no benefit in terms of the duration of viral shedding was observed with the combined treatment of leflunomide and ifn α- a beyond ifn α- a alone. although clinical trials of compassionate or off-label uses of several drugs have been conducted, there is no specific and effective medication to treat patients with covid- [ , , ] . partial clinical trial results of lopinavir-ritonavir, remdesivir, chloroquine and hydroxychloroquine have already been performed in different countries, but have shown only moderate and controversial effects [ , ] . therefore, it is still necessary to seek safe and solid strategies to treat covid- when facing the increasing number of patients worldwide [ ] . the pandemic of covid- has been under control in wuhan, china since march, , but some patients remained viral rna-positive after their symptoms had resolved and their abnormal ct imaging had improved significantly [ , , ] . long-term covid- positive patients cause many problems [ ] , for example, they have to stay in the hospital for a long time and require more medical resources. in addition, they often had psychological disorders. moreover, no specific therapeutic agents have been recommended for covid- patients with prolonged post-symptomatic shedding [ ] , which has become a great concern [ ] . acute rna virus replication, including sars-cov- , largely depends on intracellular pyrimidine resources, and antagonists of dihydroorotate dehydrogenase (dhodh), a rate-limiting enzyme in the fourth step of the de novo pyrimidine biosynthesis pathway, can efficiently prohibit viral genome replication in infected cells [ ] . leflunomide, an approved dhodh inhibitor, has been widely used to treat patients with autoimmune diseases [ ] , but whether leflunomide can be used to treat covid- patients is unknown. as covid- patients also suffer from excessive inflammations similar to autoimmune patients [ ] , leflunomide may benefit covid- patients through its antiviral and antiinflammation effects. a small-scale study of leflunomide treatment for confirmed patients with covid- was conducted by our team, in which, leflunomide resulted in beneficial virologic clearance and length of hospital stay [ ] . based on that background, we conducted a prospective randomized, controlled, open-label trial, to evaluate the efficacy and safety of oral leflunomide to treat hospitalized covid- patients with prolonged post-symptomatic viral shedding. from march , to april , , a total of consecutive patients with confirmed covid- with prolonged viral shedding were enrolled as study candidates. all patients were referred from other covid- the inclusion criteria were as follows: ( ) aged - years with a diagnosis of covid- conforming to the chinese guidelines [ ] ; ( ) hospitalized for prolonged post-symptomatic viral shedding; ( ) able to orally take medication; ( ) non-pregnant women; ( ) effective contraception for days after taking the last medication. candidates were excluded based on the following: ( ) presence of any condition that would not allow the protocol to be followed, including known allergy to leflunomide, use of medications that are contraindicated with leflunomide and that could not be replaced or stopped during the trial period; ( ) pregnant or breast-feeding; ( ) known other serious comorbidities, such as liver, cardiovascular, cerebrovascular diseases, severe renal insufficiency or advanced cancer; ( ) had received interferon before enrollment; ( ) unwilling to participate in the study. this clinical trial received approval from the ethics committee of the renmin hospital of wuhan university (no.wdry -k ) and written informed consent was obtained from each participant. the study was registered at the chinese clinical trial registry (chictr ). patients were assessed for eligibility on the basis of the inclusion and exclusion criteria (figure ). at the first interview, each candidate completed a comprehensive questionnaire including demographics, comorbidities, initial-episode syndromes and disease severity at the first admission, length of virus shedding from onset to enrollment, duration of post-symptomatic viral shedding, antiviral medication before enrollment,etc.however,the original protocol had been amended,which was for a multicenter, randomized, double-blind, controlled clinical trial. due to few new covid- patients in wuhan, china since early march , only convalescing patients with prolonged post-symptomatic viral shedding rather than those in the acute stage were enrolled in single center,with a small sample size. fifty eligible patients were randomly assigned to a combination treatment group that received leflunomide ( mg, q h, three consecutive times, orally; then mg, once a day for days; a total course of days) plus nebulized ifn - a ( million iu each time, adding ml of sterilized water, atomization inhalation twice daily for days), or to a control group that received nebulized ifn - a this was an open-label, prospective randomized, controlled trial, which was conducted at east campus, renmin hospital of wuhan university. the enrollment was initiated on march , and ended on april , . the last patient studied was discharged on april , and was followedup until may , . m a n u s c r i p t since there is no standard definition, we adopted the following definition of covid- patients with prolonged post-symptomatic viral shedding, which refers to laboratory confirmed patients with covid- who continued to have nasopharyngeal rt-pcr positivity at least two weeks after symptom resolution and after their abnormal ct imaging improved significantly. after enrollment, serial nasopharyngeal swab specimens were obtained at the baseline (before leflunomide or ifn - a was administered) and once every two days until nucleic acid tests were negative twice consecutively with an interval of ≥ hours. rt-pcr for sars-cov- was performed using a commercial kit (geneodx biotech co., ltd, shanghai, china). clinical symptoms of patients were assessed once daily by trained nurses using diary cards, analysis of peripheral blood cells, biochemical indicators and chest imaging studies performed at the baseline, on day , one day after treatment and or one day before discharge for patients meeting discharge criteria within ten days of enrollment. data were recorded on paper case record forms, then were entered into an electronic database and validated by the clinical trial staff. discharge criteria were as follows [ ] : having a normal temperature for > days, significant improvements of respiratory symptoms and ct imaging, nucleic acid tests negative twice consecutively with an interval of ≥ hours. after discharge, the patients were isolated at a designated place for days as recommended [ ] , which was arranged by community committees where the patients resided. they were followed-up by primary health-care facilities and were retested for viral nucleic acid on days and . after that, they stayed in their homes for a second isolation period of days, and were then retested for viral nucleic acid by the end of this quarantine period. we collected each patient's medical information during the isolation, which was shared with permission. in our study, enrolled patients with five consecutively negative nucleic acid tests were considered as having "true negative" results (two times during hospitalization, two times during the first isolation, and one time at the end of the second quarantine). if any patient at any time-point had a positive test for sars-cov- , they were sent to a designated site for isolation and medical observation. the primary end point was the duration of viral shedding, which was defined as the time from randomization to the first negative nucleic acid test of five consecutive rt-pcr results. other clinical outcomes included clinical status, i.e. progressive rate to severe illness, syndromes, peripheral blood cells and biochemical parameters, c-reactive protein and inflammatory cytokines, length of hospital stay, etc. safety outcomes included adverse events that occurred during treatment, serious adverse events, and premature discontinuation of treatment. continuous variables are presented as medians (iqr). the normality of the distribution of variables was performed using the kolmogorov-smirnov test and statistical comparisons using a t-test. categorical variables are expressed as absolute numbers or percentages and are compared by the χ² test, fisher's exact test or one-way anova. the time to negative rt-pcr test was developed using the kaplan-meier method and was compared with a log-rank test. a p˂ . was considered statistically significant. all statistical analyses were performed using sas . software (sas institute inc, cary, nc). the characteristics of the patients in this study are summarized in tables and . of the patients who underwent randomization and treatment assignment, were assigned to the combination treatment group that orally received leflunomide plus nebulized ifn - a, and were assigned to the control group that received nebulized ifn - a alone. in the combination treatment group, patients ( . %) received all treatments as assigned, but two patients did not complete the day treatment regimen, one due to serious diarrhea days after taking the drug, and the other due to impaired liver function. there were no significant differences in age (table ) . there were no important between-group differences in baseline laboratory test results at enrollment, except for the level of creatine kinase in the control group, the level of tumor necrosis factor in the combination group was slightly higher, although both were within the normal range (table ) . twenty-four of the patients in the combination treatment group and all patients in the control group completed this study and were discharged. no deaths or severe illness occurred and the illness severity was not worse in either group. in terms of the duration of viral shedding after treatment, patients assigned to the combination treatment group had a time to negative rt-pcr results that was not different from patients assigned to the control group (figure ), the median time laboratory examinations were conducted before and after treatment for all patients ( table ) . of the post-treatment test results, there were no differences between the two groups except that the lymphocyte count in the control group was slightly higher than in the combination treatment group for safety, a total of patients in the combination treatment group and in the control group reported adverse events (table ) but that was not significantly different between the two groups ( . % vs. . %, p= . ). there was one serious gastrointestinal adverse event that caused the discontinuation of treatment in the combination treatment group but none occurred in the control group, which was judged by the investigators to be related to the trial medication. for laboratory results, the absolute number of increased liver enzymes in the combination treatment group was higher than in the control group but was not statistically different (table ) persistent viral shedding is a serious problem [ ] . cao and colleagues reported that sars-cov- rna was detected in . % of their patients on day after a -day treatment regimen with lopinavir-ritonavir [ ] . another report showed that the median duration of viral shedding was days in patients with covid- and could be as long as days [ ] . an analysis of the transmission of covid- revealed that % of subjects in china in january-february potentially contracted the virus from patients with no or minimal symptoms [ ] . the prolonged existence of virus presents difficulties in attempts to control the community spread of sars-cov- . partial in vitro studies or clinical trials have suggested the potential therapeutic activity of several compounds against coronaviruses [ ] , however, there are no specific antiviral pharmaceutical treatments available for patients with covid- [ ] . the results of those studies did not show clinical improvement or the clinical trial results were controversial, including lopinavir-ritonavir [ ] , remdesivir [ ] , favipiravir [ ] and chloroquine or hydroxychloroquine [ ] . we evaluated the efficacy and safety of leflunomide on sars-cov- infection in this study and compared it with the roles of interferon treatment alone. interferon is recommended to be used for patients with covid- by the chinese guidelines [ ] , for it has broad-spectrum antiviral activity [ ] , has been widely used for the treatment of virus infections [ , , ] , and is also effective for treating patients with covid- [ , ] . leflunomide is capable of inhibiting viral rna genome replication and rescues mice from advanced influenza infections [ ] . leflunomide directly targets dhodh, the host's de-novo pyrimidine synthesis enzyme, to cut off intercellular pyrimidine resources required as the starting step of building the viral rna genome [ ] . like chloroquine and hydroxychloroquine, leflunomide has a dual mechanism of antiviral and immunoregulation and has been approved to treat arthritis for many years [ , ]. leflunomide has a clear-cut drug target of dhodh and has few off-target effects [ ], whereas chloroquine and hydroxychloroquine are multitargeted and have more severe adverse effects [ ] . therefore, dhodh inhibitors may be attractive drugs for treating acute and severe virus infection diseases [ ] . in a preliminary trial, we found that leflunomide resulted in beneficial virologic clearance and length of hospital stay for patients with covid- [ ] . a c c e p t e d m a n u s c r i p t in the present study, the baseline characteristics of the patients at enrollment were generally balanced across the two groups that did not differ with regard to duration, severity of illness and majority baseline laboratory results. however, differences in the negative conversion of virus nucleic acid between the combination treatment group and the control group were not observed. as compared to treatment with nebulized ifn - a only, the combination of oral leflunomide and nebulized ifn - for safety, two leflunomide recipients discontinued treatment due to gastrointestinal adverse events or abnormal liver function, however, there was no statistical difference in the total number of adverse events between the two groups. the side-effect profile observed in the current trial arouses concern about the use of higher or more prolonged leflunomide dose regimens in efforts to improve outcomes. ＊ length of virus shedding from onset to enrollment in patients with initial cough and expectoration. ※ patients with initial cough and expectoration in patients with initial cough and expectoration. # lianhua qingwen capsule is a kind of chinese traditional medicine and is recommended for patients with covid- [ ] . a c c e p t e d m a n u s c r i p t who launches global megatrial of the four most promising coronavirus treatments potential antiviral drugs for sars-cov- treatment: preclinical findings and ongoing clinical research the epidemic of -novel-coronavirus ( -ncov) pneumonia and insights for emerging infectious diseases in the future treatment options for covid- : the reality and challenges race to find covid- treatments accelerates clinical features of covid- convalescent patients with re-positive nucleic acid detection probable causes and risk factors for positive sars-cov- test in recovered patients: evidence from brunei darussalam south korea's covid- infection status: from the perspective of re-positive test results after viral clearance evidenced by negative test results recurrence of positive sars-cov- in patients recovered from covid- prolonged viral rna shedding duration in covid- pcr assays turned positive in discharged covid- patients novel and potent inhibitors targeting dhodh are broad-spectrum antiviral against rna viruses including newly emerged coronavirus sars-cov- leflunomide and teriflunomide: altering the metabolism of pyrimidines for the treatment of autoimmune diseases clinical features of patients infected with novel coronavirus in wuhan, china a small-scale medication of leflunomide as a treatment of covid- in an open-label blank-controlled clinical trial released by national health commission & national administration of traditional chinese medicine persistence and clearance of viral rna in novel coronavirus disease rehabilitation patients a trial of lopinavir-ritonavir in adults hospitalized with severe covid- clinical course and risk factors for mortality of adult in patients with covid- in wuhan, china: a retrospective cohort study substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov- ) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro characteristics of and public health responses to the coronavirus disease outbreak in china remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial experimental treatment with favipiravir for covid- : an open-label control study. engineering (beijing) a rapid systematic review of clinical trials utilizing chloroquine and hydroxychloroquine as a treatment for covid- treatment with lopinavir/ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset bc hepatitis testers cohort team. sustained virological response from interferon-based hepatitis c regimens is associated with reduced risk of extrahepatic manifestations combination group: leflunomide plus ifn - a; control group: ifn - a alone ldh = lactate dehydrogenase; ultra-tni = ultratroponin i; aptt = activated partial thromboplastin time tnf = tumor necrosis factor. a: comparison of baseline data between the two groups. b: data comparison between after treatment the two groups we respectfully thank all patients enrolled in this study. this work was supported by the national key the funding agencies had no role in the study design and clinical medications; collection, analysis, and interpretation of the data; preparation, written, review, or approval of the manuscript. zhao y and hu wh contributed equally with wang mm. the authors have no competing interest to declare for this study. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t figure key: cord- -ee qyw h authors: monette, anne; mouland, andrew j. title: t lymphocytes as measurable targets of protection and vaccination against viral disorders date: - - journal: int rev cell mol biol doi: . /bs.ircmb. . . sha: doc_id: cord_uid: ee qyw h continuous epidemiological surveillance of existing and emerging viruses and their associated disorders is gaining importance in light of their abilities to cause unpredictable outbreaks as a result of increased travel and vaccination choices by steadily growing and aging populations. close surveillance of outbreaks and herd immunity are also at the forefront, even in industrialized countries, where previously eradicated viruses are now at risk of re-emergence due to instances of strain recombination, contractions in viral vector geographies, and from their potential use as agents of bioterrorism. there is a great need for the rational design of current and future vaccines targeting viruses, with a strong focus on vaccine targeting of adaptive immune effector memory t cells as the gold standard of immunity conferring long-lived protection against a wide variety of pathogens and malignancies. here, we review viruses that have historically caused large outbreaks and severe lethal disorders, including respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic fevers. to observe trends in vaccinology against these viral disorders, we describe viral genetic, replication, transmission, and tropism, host-immune evasion strategies, and the epidemiology and health risks of their associated syndromes. we focus on immunity generated against both natural infection and vaccination, where a steady shift in conferred vaccination immunogenicity is observed from quantifying activated and proliferating, long-lived effector memory t cell subsets, as the prominent biomarkers of long-term immunity against viruses and their associated disorders causing high morbidity and mortality rates. the world was forever changed by the introduction of vaccine against smallpox in the late s, at the time protecting its first , individuals. this was the first demonstration that a vaccine could successfully eradicate viruses causing disorders and diseases that even when not lethal, still had the potential to cripple both surviving populations and their surrounding geographical economies. since that time, the occurrence of epidemics and outbreaks are now at lower risk, following the introduction of massive vaccination programs able to induce immune system targeting of viruses causing severe disorders affecting distinct geographical locations, and with many epidemiological reports demonstrating long-term efficacy of viral control of non-naïve populations. outbreaks of existing and emerging viral diseases or disorders vary widely in duration and frequency across geographical populations. some can be predicted annually, while others may see decades between outbreaks, therefore driving the continuous epidemiological surveillance of associated infectious disorders, the development and implementation of targeting vaccines, and development of immune-monitoring strategies measuring vaccine efficiency in target populations. despite vaccination-mediated protection of numerous nations documenting complete eradication of the causative agents of viral disorders in endemic populations, there is threat of re-emergence of pandemic proportions of these agents. threats to virus re-emergence are caused by contemporary choices made to not vaccinate children, by strain recombination, by viral spread to naïve populations by world-travelers, migrants, or climate change, causing redistribution of viral vectors, by potential use of these agents in acts of bioterrorism or war upheaval, or by depletion of vaccination stocks required for protection against pandemics. other factors include the increasing and aging global population and increased number of immunocompromised individualsd factors strongly supporting the maintenance of herd immunity against existing viruses, despite lower incidence of outbreaks in industrialized countries. in the event of a re-emergence of previously eradicated viruses or the acquisition of increased pathogenicity by existing viral strains, there is an urgent need for vaccine development strategies that can rapidly and effectively arrest global spread. important advances are continuously being made in vaccine development strategies toward the control of viruses and associated disorders. vaccine design has been modified from the use of attenuated viruses to use of more precise viral protein subunits specifically targeted by t cells. historically, vaccination immunogenicity was documented by measures of serum immunoglobulin (ig) classes and antigen-specific antibodies produced by humoral immunity. more recently, quantification of cellular components of innate immunity at the interface between innate and adaptive immunity are made, in addition to more precise measurements of adaptive immunity. long-term protection achieved by adaptive immunity can be quantified by measuring levels of circulating cytokines, along with specific phenotypic profiles of effector memory, antigen-specific t cells. though both humoral and cellular arms of immunity are integrally linked during the initial induction of immunity against pathogens, these can become disconnected with developing pathology due to their individual needs for survival factors, unequal declines in immune function, and differential cellular lifespans. this loss of correlation between memory t cells and neutralizing antibody responses varies according to different viruses, suggesting that independent time course measures of these separate immune responses are required over time for adequate recording of biomarkers of natural infection and vaccine efficacy or suggesting that t cell status may be most crucial measure of conferred long-term immunity. from the standpoint of fundamental or clinical research, it has become established that the targeted induction of specific pathogen-and tumorclearing effector memory t cell subsets is our endgame armor toward long-term human survival against infectious diseases and cancers. this chapter provides an overview of viruses that have historically caused severe lethal disorders, including those of the respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic types. the features of viruses and associated disorders that we herein describe include viral genetics and replication cycles, transmission modalities, cell and organ tropism, host-immune evasion strategies, associated viral disorders and diseases, and epidemiology. we also report on well-accepted and other important documented instances of viral control by t cells, currently available and successful vaccines, and recorded measures of vaccination immunogenicity. we focus on quantification of vaccine-induced effector memory t cellemediated immunity, representing the gold standard of successful vaccination. just as it is for advances in vaccinology, investigations into the biology of t cells are currently at the forefront of many research fields examining various disorders, diseases, and malignancies not formerly considered to be controlled by immunity. although many viral infections are limited to the upper respiratory tract, it is lower respiratory tract infections (lrti) that most predominantly cause enormous disease burden in children and immunocompromised adults suffering from human immunodeficiency virus (hiv) infection or in patients having received stem cell or solid organ transplants for which immunosuppressive therapies were administered (henrickson et al., ; pavia, ; kim et al., ) . acute lower respiratory illnesses (alris) are a major cause of morbidity and mortality, accounting for approximately . million deaths, globally, per year (black et al., ) . frequently overlapping lrti syndromes include bronchiolitis, asthma exacerbation, wheezing, croup, and pneumonia. although certain specific syndromes can be more precisely associated with infection by specific viruses, syndromes overlaps can complicate diagnosis of these numerous viruses, and quite often, difficulties in differentiating between viral and bacterial pneumonias symptoms can also result in antibiotics being mistakenly prescribed during viral disorders. several viruses are normally, however, considered to be primarily responsible for lrtis, beginning with upper respiratory tract infections, most commonly caused by respiratory viruses that are typically spread from person-to-person by contact with infected respiratory droplets, and including respiratory syncytial virus (rsv), epidemic influenza a and b, h n and h n avian influenza a viruses (iavs), parainfluenza viruses through , adenovirus, human metapneumovirus (hmpv), severe acute respiratory syndrome coronavirus, human coronaviruses nl and hku , rhinoviruses, and bocaviruses (pavia, ; mahony, ; nichols et al., ) (table ) . currently, vaccines for human influenza viruses, human parainfluenza viruses (hpivs), and adenoviruses causing upper and lower respiratory infections are used to control these infections and the resulting propagation of their morbid symptom derivations. human influenza viruses make up three of the five genera of the family orthomyxoviridae and are classified as a, b, and c types, based on their highly conserved matrix protein (m ), membrane matrix protein (m ), and nucleoprotein (np). type a influenza viruses can be further sub-subtyped by the antigenicity of their hemagglutinin (ha) and neuraminidase (na) surface glycoproteins (gps). antigenic drift, caused by point mutations in ha and na and recombination of the ha genes, results in the generation of new strains that can escape pre-existing immunity, causing both the prediction of circulating strains difficult and antigenic mismatch by existing vaccines. approximately ha and na subtypes of influenza a are documented in aquatic birds, representing their natural hosts (i.e., vectors). influenza a h and h subtypes cocirculate seasonally, and influenza b viruses can only infect humans, via two distinct, seasonally cocirculating, lineages. type c influenza viruses are more rarely documented to infect humans and pigs (berlanda scorza et al., ) . influenza viruses cause acute upper and lower respiratory infections, and due to their rapid and unpredictable genetic drift, represent the most likely of pathogens to cause a human pandemics. annually, human influenza viruses have the potential to cause up to million cases of severe illness, with an associated , deaths worldwide (who_influenza_(seasonal), ), causing great economic burden. four influenza pandemics have occurred over the past century, as a consequence of the h n ( ), h n ( ), h n ( ), and h n ( ) variants (palese, ) . since the most recent outbreak in , an estimated , people globally have succumbed to the h n variant of swine origin (dawood et al., ) . epithelial cells that are infected with influenza virus produce inflammatory cytokines acting as chemoattractants for homing macrophages and dendritic cells (dc). dcs take up influenza viral particles to trigger their maturation and pursuant migration to the lymph, where they initiate antigen-specific t cell maturation. these influenza-specific effector t cells then enter the respiratory tract to counteract viral titres through cytokine expression and the direct lysis of infected cells, with activated cd þ effector cytotoxic t cells (ctls) representing the main constituents of this response by their release of perforins and granzymes, and the engagement of tumor necrosis factor (tnf) receptors (spitaels et al., ) . influenza-specific cd þ t helper cells can act directly and indirectly in viral clearance, primarily by producing cytokines that induce the functions of b cells and cd þ t cells and which have also been reported to directly eliminate infected cells themselves (topham and doherty, ; hua et al., ) . while preexisting cd þ t cell immunity has not yet been demonstrated to prevent infection from occurring, it is hypothesized to be the result of the loss of granzyme expression by memory cd þ t cells and populations of iavspecific cd þ t cells are still importantly correlated with the control of spread and recovery in healthy populations (grant et al., ) . the most currently administered influenza vaccines are inactivated (iv) trivalent (tiv) or quadrivalent formulations containing equal amounts of ha of two influenza a strains (h n and h n ) and one of two influenza b strains (yamagata and victoria lineage). these are derived from viruses typically grown in fertilized chicken eggs, are mainly focused on eliciting a strainmatched humoral immune responsedrequiring yearly updatesdand are unable to provide protection to all vaccinated individuals. the requirement of memory t cell immunity for long-term protection against influenza virus promotes the development of vaccines that elicit both humoral and cellular immunity: a strategy expected to overcome the inadequacies of current vaccines against influenza and other viruses (spitaels et al., ) . there is broad interest in the development of a universal influenza vaccine, considered to be the "holy grail" of influenza vaccine research. this approach is being developed to use virus-infected cell-killing antibodies that produce an antiviral environment; these termed antibody-dependent cellular cytotoxicity (adcc)-mediating antibodies, which are predicted to link innate and adaptive immune responses, and is becoming possible due to new technologies for rapid isolation and characterization of monoclonal antibodies targeting conserved regions of influenza virus, reviewed in jegaskanda et al. ( ) . this approach has been postulated to work, in part, from reports of iavspecific cd þ t cells, promoting viral clearance in the absence of neutralizing antibodies, and can also mediate cross-reactive immunity against distinct iavs to drive a rapid recovery from severe influenza disorders (grant et al., ) . the induction of infection-permissive immunity is both protective and allows virus-induced cross-reactive immune responses. vaccines targeting the conserved ectodomain of m deliver this kind of non-neutralizing immunity since these antibodies rely on fc receptors and innate immune components (el bakkouri et al., ) . antagonizing antibodies inhibiting na activity represents another promising strategy, not by blocking viral entry and eliciting sterilizing immunity, but by contributing to immunity against a virus possessing a similar na type (wan et al., ) . there is also progress being made in the development of recombinant t celleinducing vaccines, with the most advanced version of this strategy demonstrated by modified vaccinia ankara (mva) viruses expressing influenza virus np and m antigens (berthoud et al., ) , with vaccinated individuals demonstrating increases in interferon-gamma (ifn-g) expressing cd þ t cells and increased protection against influenza infection (antrobus et al., ; powell et al., ) . co-administration of this mva-based vaccine with tiv formulations results in increased influenza strainespecific antibody responses and the generation of memory t cells that recognize a range of influenza a subtypes (antrobus et al., ) . additional research demonstrates production of antigen-specific t cell responses using alternate prime/boost regiments of combinations of vaccination regimens employing recombinant replication-deficient adenovirus or mva, expressing iav np and matrix protein (lambe et al., ) . quality and clonal t cell receptor (tcr) characteristics of influenza-specific cd þ t cells, in addition to in silico predicted and peptide-based approaches for pools of minimal iav epitopes, are investigated for their induction of cellular immunity and recognition by cd þ t cells (reviewed in grant et al., ) . hpiv are enveloped negative-sense rna genome viruses of e nm, belonging to the large and rapidly growing paramyxoviridae family, causing significant human and veterinary disorders (henrickson, ) . hpiv is divided into serotypes to , with hpiv- representing the most common etiologic agent of associated disease, but with hpiv- , - , and - representing common causative agents of respiratory illness in pediatric, geriatric, and immunocompromised populations (schmidt et al., ) . hpivs are a common cause of acute respiratory illness throughout all stages of human life (schomacker et al., ) , causing acute respiratory infections in children (cooney et al., ) . second, only to human respiratory syncytial virus, hpivs are the major contributors to hospitalization due to alris and global pneumonia mortalities in young children, and up to % of children are seropositive for hpivs by the age of years (murphy, ; weinberg et al., ) . hpiv infections can induce potent humoral and cellular immune responses, including innate immune responses, local and systemic igg and iga responses, and adaptive cd þ and cd þ t cell responses (gitlin et al., ; hou et al., ) . though cellular responses can restrict hpiv replication dynamics and clear primary infections, neutralizing antibodies against virus envelope hemagglutininneuraminidase (hn) and fusion (f) gps are required for early infection (suzuki et al., ; zhang et al., ) and confer long-term protection against hpiv-related disorders (murphy, ; spriggs et al., ; schmidt et al., ) . currently, there is no vaccine to protect against human hpiv infection. progress in the development of hpiv vaccines using reverse genetics for serotypes À to À has generated several live-attenuated, intranasal hpiv vaccines evaluation in adults and in children, two of which, hpiv- are well tolerated in hpiv -seronegative pediatric populations (schmidt et al., ) . ongoing pediatric trials testing live-attenuated hpiv vaccines for hpiv- and hpiv- predict these will replicate in the upper respiratory tract of infants to induce the full spectrum of humoral and cellular immune responses (karron et al., ) . heterologous (i.e., jennerian) vaccine design strategies using the sendai virus (sev) to control infections by hpivs and rsv are discussed in the following section. human respiratory syncytial virus (rsv) types a and b are found within the genus orthopneumovirus, family pneumoviridae, of the order mononegavirales. rsv is an enveloped, spherical virus of w nm in diameter, and reaching up to several micrometers in length (gower et al., ) . the negative-sense rna genome encodes for outer structural, np, polymerase, ns, transmembrane, and regulatory proteins (griffiths et al., ) . rsv is a major cause of alris, resulting in numerous pediatric hospitalizations globally, where by years of age, most children have been exposed and are at risk of developing life-threatening bronchiolitis and pneumonia (glezen et al., ; hall et al., ; henrickson, ) . up to , infants are hospitalized due to rsv infection in the united states (marks, ) , and million episodes of rsv-associated alri in children globally represent at least million cases resulting in hospitalization, and approximately , associated deaths per year (nair et al., ) . a balance of adaptive immune ctls and neutralizing antibodies of the humoral immune response mediate protection and clearance of rsv infection (griffiths et al., ) . though neutrophils are the highest proportion of leukocytes found in the airways of those infected with rsv (everard et al., ) , and despite observations that natural killer (nk) cells are first to attain infected airways (hussell and openshaw, ) , it is cd þ helper and cd þ ctls that correlate with the early clearance of rsv-infected cells (anderson et al., ) . in infants and immunocompromised populations, fatalities resulting from rsv infection are associated with deficiencies in cd þ and cytotoxic cd þ t cells (hall et al., ; welliver et al., ) . later in infection, increases in neutralizing antibodies prevent reinfection by opsonizing viral epitopes required for rsv entry and infection, and rsv clearance can be associated with rsv-neutralizing nasal immunoglobulin a (iga) (mcintosh et al., ) . more recently, enhanced rsv clearance with reduced disease severity has been associated with vaccine-elicited memory cd þ t cells (lee et al., ) . while memory cd þ t cells can mediate protection against rsv infection, in the absence of antibodies and memory cd þ t cells, these cause mortality via systemic proinflammatory cytokine storms and local ifn-g production (stoley et al., ; schmidt et al., ) . other recent studies however indicate that the cd þ t cell response may not be the major determinant of severity of rsv-related pathology (collins and melero, ) . there are currently several recombinant rsv subunit vaccines in clinical trials, including the novavax rsv f vaccine representing the most promising candidate for licensing. this rsv f subunit targeting vaccine has been demonstrated to elicit the expression of circulating neutralizing antibodies against rsv (glenn et al., ) . toward the development of future adaptive immunity-inducing rsv vaccines, it has been demonstrated that the transfer of airway-resident t cells protect against rsv, where it has been suggested that, to lessen the burden of t cellemediated damage to airways, the induction of lung tissue t cells should be the focus of vaccine development (kinnear et al., ) . recently, the sev has been used as a component of a jennerian vaccine model for hpiv- and as a backbone for other viruses causing serious lower respiratory infections (lris), including other hpivs, rsv, and hmpv. sev-based vaccines have proven to be effective toward inducing b cell and t cell immune responses, and in the protection from hpiv- , - , and - , and rsv, where they can also be used in combination with other vaccines to primeeboosts or to target one or more than one paramyxovirus pathogen (russell and hurwitz, ) . sev is attractive for use in human vaccination because it is a murine pathogen unable to infect humans (bousse et al., ) and therefore does not require attenuation as it can never revert to a human pathogenic phenotype (schickli et al., ) . another important feature supporting the use of sev as a pan-virus vaccination agent stems from its ability to grow transiently in mammalian cells, accommodating the endogenous expression of antigens with posttranslational modifications matching those of the target antigens and neutralizing epitopes (henrickson et al., ) , with endogenous expression of antigens ensuring robust activation of cd þ t cells able to destroy antigen-producing cells and terminate virus amplification (york and rock, ; russell and hurwitz, ) . in murine studies, sev could elicit rapid and durable respiratory mucosa and systemic hpiv-specific b cell and t cell responses (sealy et al., ; rudraraju et al., ) . clinical testing of human populations infected with rsv and hpivs is underway (adderson et al., ) . for a full review of current development of antiviral compounds and vaccine candidates tested against rsv, see costello et al., . . adenovirus classification, epidemiology, immunology, and vaccinology human adenoviruses (hadvs) are classified in the mastadenovirus genus, containing seven known hadv species, from hadv-a to hadv-g, and with at least unique known human serotypes (buckwalter et al., ) . adenoviruses are nonenveloped double-stranded dna viruses ranging from to nm in diameter and are composed of a protein capsid, a np core, and internal proteins. dna homology between hadv subgroups ranges from % to % (walls et al., ) . hadv infection rarely causes serious or fatal illness in immunocompetent individuals but may cause severe disease in immunocompromised, pediatric, and geriatric populations (lynch et al., ) . clinical disease symptoms associated with hadvs are dependent on hadv genotypes, with at least recognized, and assigned to subgroups a through g. clinical symptoms include fever, rhinorrhea, pharyngitis, conjunctivitis, gastroenteritis, bronchitis, pneumonia, acute hemorrhagic cystitis, and meningoencephalitis (lynch et al., ) . recombination between hadvs are largely responsible for outbreaks of acute febrile respiratory disease in immunocompetent military recruits, where serotypes and type have been documented to account for approximately % of these respiratory illnesses (hilleman et al., ; dudding et al., ) , and are associated to other frequently occurring disorders including upper and lower respiratory illnesses, gastroenteritis, hepatitis, keratoconjunctivitis, meningoencephalitis, cystitis, and myocarditis in these immunocompetent populations, reviewed in lion ( ) . adenoviruses are endemic in pediatric populations (echavarria, ) . the incidence of adenovirus infection peaks in infants and children, where, globally, %e % of respiratory tract infections in pediatric patients are ascribed to hadv (ghebremedhin, ) . recently, re-emergence of type d hadv has caused fatal outbreaks due to severe pneumonia syndromes in children from high-density populations . immune responses to adenovirus infection are dependent on primary sites of inoculation, methods of transmission, viral serotypes, and secretory ig antibody status of the infected host; igas are present in respiratory tract early following infection, and igg is present in serum and nasal secretions at later time points, reviewed in walls et al. ( ) . histopathological changes resulting from infection can be divided into two phases: the first phase of immune histopathology predominantly involves nonspecific, cytokine-mediated inflammatory recruitment of monocytes and macrophages, while the second phase involves t cell infiltration (prince et al., ) . t cellemediated immunity is believed to be required for hadv recovery from acute infections, and individuals lacking adaptive immunity are found to be at elevated risk of infection, with cd þ t cells as primary mediators of response to respiratory viruses, with relatively little contribution by cd þ cells (woodland et al., ) . however, following adenovirus exposure, cd þ t cells have been shown to be responsible for increasing proliferation status of peripheral blood mononuclear cells (pbmc), and cd þ t cells represent the major ctl subsets produced and recognize conserved antigens across adenovirus serotypes (flomenberg et al., ; regn et al., ) . adenoviruses have, however, evolved several hostevasion strategies, including inhibition of apoptosis, responses to ifn-g and tnf-a, and major histocompatibility complex (mhc) class i expression (mahr and gooding, ; wold et al., wold et al., , . the live, oral adenovirus vaccine was licensed in the s for active immunization toward the prevention of febrile acute respiratory disease in military populations, where it initially reduced adenovirus-associated respiratory illnesses by over five-fold (dudding et al., ) . vaccine stock depletion and associated epidemics led to the manufacture of another vaccine in , again denting adenovirus-associated disease burden by approximately -fold among recruits within the first years of its introduction (radin et al., ) . these oral lyophilized vaccines replicate asymptomatically in the gut, inducing humoral and cell-mediated immunity, to confer longlasting protection from infection (berg et al., ) . due to their abilities to induce potent transgene product-specific t-and b-cell responses, adenovirus vectors are explored for use as vaccine carriers against a variety of many other pathogens (chen et al., ; harro et al., ; hill et al., ; radosevic et al., ) . alris by respiratory viruses are a major cause of morbidity and mortality, accounting for over . million deaths globally each year, and are predominantly resulting from human transmission of virus containing respiratory droplets. licensed vaccines are useful against several viruses causing these severe, often lethal, associated disorders. influenza has no borders and causes great economic burden, making it a prominent international concern. its rapid and unpredictable genetic drift causes human pandemics, where it has an annual potential of causing million infections and , deaths worldwide. influenza vaccinology requiring constant yearly updates has stimulated interest in the development of universal t cell vaccines that can elicit both humoral and cellular immunity, whereby influenza-specific memory cd þ t cell responses against a range of influenza subtypes could be induced to clear infection in absence of neutralizing antibodies. rsv causes million alris in children annually, resulting in million hospitalizations and almost , deaths per year. rsv is cleared by balance of adaptive immune ctls and humoral neutralizing antibody responses, correlating most highly with cd þ helper and cd þ ctls during natural infections, and with memory cd þ and cd þ t cells following vaccination, with research endeavours targeting their strengthening by specific induction of lung tissue rsv targeting t cells. second, only to rsv, hpivs cause many ari-and lri-associated mortalities in children. hpivs can induce potent humoral, innate, and adaptive cd þ and cd þ t cell responses able to restrict their replication and where neutralizing antibodies can confer long-term protection against their associated disorders. hadvs infect both immunocompetent and immunocompromised humans and have been shown to cause up to % of respiratory disorders in hospitalized military personnel. despite their having evolved convoluted host-evasion strategies, adaptive t cell immunity against hadvs starts early in diseases phases and is key to recovery from acute natural infection, with its greatest contributions by cytotoxic cd þ t cells that require stimulating by cd þ t cells for their expansion. vaccines against hadvs induce both humoral and adaptive immunity, including potent transgene virus-specific t-and b-cell responses conferring long-term protection. success from hadv vaccinology has influenced explorations of adenovirus vectors as target carriers for vaccination against numerous other pathogens. diarrheal disorders remain a leading cause of morbidity and mortality worldwide, with these listed in the top five causes of death worldwide, and which are associated with global estimates at e million deaths per year; reviewed in clark and mckendrick ( ) . the majority of gastric infections are viral in origin, and viral gastroenteritis is one of the most common illnesses in all age groups and an important cause of morbidity in industrialized countries (chang et al., ) . the human risk of viral gastroenteritis in the united states alone is at least one per individual per year, with , adults and , children hospitalizations recorded and an associated mortalities per year (mead et al., ; mounts et al., ) . several viruses are responsible for viral gastroenteritis, where their transmission typically occurs from person-to-person by the oral-fecal route. viruses commonly causing gastroenteritis include rotavirus (rv; causing the most serious gastric disorders), norovirus, astrovirus, adenovirus, and coronavirus-like agents (table ) . rvs are classified as a genus within the family reoviridae. these are nonenveloped viruses measuring nm in diameter and have inner and outer capsids surrounding their cores containing double-stranded rna viral genomes encoding viral capsid (vp- to vp- , and vp- ; vp outer capsid protein mediates virus attachment to cells) (bishop et al., ) and nonstructural (nsp- to nsp- ) proteins, reviewed in desselberger ( ) . rvs classify into seven serotypes (aeg), based on antigenic properties of the inner capsid vp protein, where subtypes aec represent human pathogens and are further subclassified into serotypes within these groups on the basis of differing outer capsid composition (anderson and weber, ; wilhelmi et al., ) . diarrhea is a major cause of death among children globally (liu et al., ) , and rv is the leading cause of severe diarrhea, globally causing an estimated , deaths in developing countries and . million pediatric patient hospitalizations parashar et al., ) . rv also represents a significant cause of disease in industrialized countries, with greater numbers of hospital admissions reported relative to developing countries (chang et al., ) . though group a rv causes the majority of endemic infections and can also lead to significant outbreaks in infant and geriatric populations (villena et al., ; marshall et al., ) , group b rvs are less common but can also lead to outbreaks and epidemics (sanekata et al., ; ahmed et al., ) , whereas group c rv is less often observed causing sporadic diseases. of the existing g and eight p rv group a serotypes, g to , p (kostouros et al., ; clark and mckendrick, ) . studies of t cell responses to rv infection in humans have reported that most healthy adults and children have circulating rv-specific t cells, with approximately % of rv-cd þ t cells expressing the intestinal homing receptor a b , and with circulating rv-cd þ and rv-cd þ t cells secreting ifn-g or interleukin (il)- (makela et al., ; offit et al., ; yasukawa et al., ; rott et al., ; parra et al., ) . frequencies of circulating ifn-g þ rv t cells are comparable to those specific for other mucosal respiratory viruses (mesa et al., ) , but these often possess profiles of terminally differentiated effector cells that are usually associated to those unable to provide long-term immunity (parra et al., ) . (yen et al., ) . as in the case of natural neonatal rv infection, fair protection rates are achieved via humoral immunity using these vaccines. though these are unable to protect against rv reinfection, they do offer protection against severe associated clinical symptoms causing patient hospitalization. these vaccines offer both homotypic and heterotypic immunity, and protection often correlates with increases in rv typee specific igg or iga antibodies, reviewed in desselberger and huppertz ( ) . although rv vaccineeinduced humoral immunity substantially decreases disease burden, these vaccination strategies are less effective and difficult to implement in low-income countries requiring them most (patel et al., ) . as with natural rv infection, vaccines provide nonsterilizing immunity to children (angel et al., ) , where lack of establishment of long-term immunity against rv causes half of children's guardians to be at risk of becoming infected and presenting with severe associated disorders (rodriguez et al., ) . this further demonstrates that rv-specific t (rv-t) cells are crucial for the development of overall, long-term, protective immunity against rv (franco et al., ; offit et al., ) . indeed, in models of rv infection, vaccine-induced protective immune responses are dependent on antiviral cytokine production and by direct killing of rv-infected cells by t cell and b cell adaptive immune subsets (jiang et al., ; wen et al., ) . in addition, with observations that gut cd þ t cells may become tolerogenic or anergic in response to rv infection, stimulating t cells with rv antigen in the presence of il- , il- , or r , a pharmacological diacylglycerol kinase alpha inhibitor, causes increased pbmc frequencies of rv antigen-specific t effector cells, including rv-cd þ tnf-a þ , rv-cd þ ifn-g þ , and rv-cd þ ifn-g þ cells (parra et al., ) . diarrheal disorders cause an annual e million deaths worldwide. rv is the leading cause of severe diarrhea outbreaks in infant and geriatric populations, with global annual estimates of , deaths in developing countries and . million pediatric hospitalizations. most immunocompetent individuals have circulating rv-specific ctls at comparable frequencies to those elicited by other respiratory viruses, but which have terminally differentiated effector profiles rendering them incapable of conferring long-term protection against the reoccurrence of associated disorders. rv vaccineemediated protection from severe disorders is from humoral nonsterilizing immunity unable to protect against reinfection, yet vaccination programs are challenging to implement in countries requiring them the most. rv-specific t cells are key to long-term protection, and vaccineinduced protection is dependent on cytokine production and direct killing of infected cells by t cells. countermeasures against crucial helper cd þ t celledeveloping anergic states may assist the development of vaccines conferring long-term protection. an exanthem is a widespread eruptive skin rash that may be associated with fever or other systemic symptoms. more than infectious agents causing exanthems have been identified (cherry, ) , where more than % of recorded cases of combined fever and widespread rash in pediatric populations were caused by viral infections, relative to the % resulting from bacterial infections (goodyear, laidler, price, kenny and harper, ) . correct diagnosis of these skin manifestations, resulting from direct inoculation of the infectious agent onto the cutaneous surface, or by dissemination from a distant site, is a main research theme on viral exanthems. this is because, while infections by many viral (i.e., paraviral) exanthems are benign and resolve spontaneously, others may rapidly lead to fatal conditions, reviewed in drago et al. ( ) . thus, special attention in diagnosing even vaccine-preventable viral exanthems must be applied to avoid the arising of serious complications in nonimmune pregnant women and their fetuses from the more harmful classes of viruses causing exanthems (white et al., ) . common exanthematous infections are typically caused by transmission of viruses from person-to-person (with exception of alphaviruses having a mosquito vector), and where a multitude of viruses are their causative agents, including rubeola virus, rubella virus, human parvovirus b , human herpesvirus (hhv) type , varicella-zoster virus (vzv), variola, alphaviruses, and molluscum contagiosum virus (table ) . numerous other exanthematous disorder causing viruses are not covered in this section, including ebola and zika, but which are becoming classified as emerging viral exanthems due to the increasing numbers of at-risk populations and the critical need to classify these diseases to minimize outbreaks and risk to pregnant women and fetuses (keighley et al., ) . rubeola, or measles virus (mev), belonging to the morbillivirus genus of the paramyxoviridae family, is a negative-sense rna virus having a nonsegmented genome and a lipid envelope, and measuring up to nm in diameter, reviewed in griffin et al. ( ) . the kb genome encodes eight proteins: the viral envelope is composed of hemagglutinin (h) and fusion (f) gps projecting from the matrix (m) protein lining its interior. the helical nucleocapsid is composed of the rna and nucleocapsid (n) protein packed within the envelope as a coil with the phosphoprotein (p) and large polymerase (l) proteins attached. the two ns proteins, c and v, regulate cellular response to infection and modulate ifn signaling (bellini et al., ) . humans are the only natural host of highly contagious mev virus spread by the respiratory route. despite the availability of a safe and efficacious vaccine, measles remains one of the most important viruses causing child morbidity and mortality worldwide (moss and griffin, ; wolfson et al., ) . infection by mev is associated with up to % of mortality rates in african children (grais et al., ; nandy et al., ) , and with % in unvaccinated refugee camp and virus-naive population mortalities (moss, ; shanks et al., ) . female mortality is a dominant feature disorders resulting from infection (garenne, ) , and many acute mortalities from secondary infections resulting from immune suppression induced by mev are also observed (beckford et al., ) . mev has a persistent and long latency infection period, often resulting in the development of subacute sclerosing panencephalitis (sspe) in males, causing fatal neurologic disease presenting itself many years following the original infection (bellini et al., ) . adaptive cellular immune responses are generally regarded as most important for clearance of mev. children with low plasma ig may recover from mev infection, while those with defects in cellular immunity develop progressive infections (albertyn et al., ; mcquaid et al., ) . mev-specific antibody and t cell responses coincide with the onset of the rash, whereby rash biopsies of mev-replicating, infected epithelial cells, have high levels of cd þ and cd þ t cell infiltrates (polack et al., ) . cd þ t cell subsets appear to be particularly important for control and clearance of infectious mev, where expanded circulating virus-specific ctls are found in the blood of patients suffering rash, and increases in cd þ t cells are also found in mev-induced pneumonias (jaye et al., ; mongkolsapaya et al., ; myou et al., ) . in addition, depending on the target tissue and cell type analyzed with regards to mev infection, though differentially rated, both cytotoxicity and ifn production have been implicated as key effector mechanisms for mev clearance (patterson et al., ; stubblefield park et al., ; finke et al., ) , with specific combinations of cd þ t cells, cd þ t cells, and b cells recorded as required for the control of primary mev infection (tishon et al., ) . protection against measles is based on mev-specific humoral, antibodybased, immunity. diagnostically, the current gold standard of protection is via quantification of neutralizing antibodies against the viral hemagglutinin (h) and fusion (f) surface gps (bouche et al., ; haralambieva et al., ; plotkin, ) . mev, however, triggers an aggressive immune response, involving both the humoral and cellular arms of the immune system (moss and griffin, ; de vries et al., ; buchanan and bonthius, ) . once measles has been cleared, it is memory t cells that can provide lifelong immunity against reinfection by mev (bester, ) . importantly, during mev infection, immune reactions to other pathogens are suppressed from weeks to years, leading to risk and susceptibility to secondary infections, and which is believed to be a driver of complications and mortality long after measles had been cleared. conversely, this measlesinduced immune "amnesia," sometimes disabling immune memory for up to years, has been suggested to work toward herd protection against other infections and is supported by the association of measles vaccination with lowered mortality rates from other childhood infections (mina et al., ) . occasional spontaneous tumor regressions have also been observed to occur during natural measles infection, suggesting that mev infection may be adopted in the generation of safe and effective oncolytic viruses (russell and peng, ). rubella virus belongs to the togaviridae family and is the sole member of the rubivirus genus. rubella contains a single-stranded, positive-sense rna genome (frey, ) , and its viral particles measure between and nm in diameter (oshiro et al., ) and have a pleomorphic nucleocapsid surrounded by a host-derived lipid membrane (battisti et al., ) . the e and e rubella protein spikes are anchored to the external layer of the membrane, with membrane-bound e proteins bridging rows of e proteins, considered as the main immunodominant antigens responsible for controlling receptor-mediated endocytosis (petruzziello et al., ; katow and sugiura, ) . antibody levels against the neutralizing domain of e correlate with protection against rubella virus (mitchell et al., ; cordoba et al., ; wilson et al., ) . rubella virus is spread from person-to-person via the respiratory route and is the causative agent of rubella disease, commonly known as german measles (lambert et al., ) . although rarer in the united states, rubella infection remains a major health concern in developing countries (tosh et al., ) . although acquired rubella infection is not severe in adults, transplacental transfer of the virus to the developing fetus during maternal viremia can cause devastating consequences of congenital rubella syndrome (crs) (watson et al., ) , where more than , infants worldwide are born with crs each year . common crs symptoms include spontaneous abortion, premature delivery, fetal death, ocular abnormalities, neurological problems, abnormal cardiac development, and deafness (white et al., ) . congenital malformations due to crs may be present at birth, while other conditions such as diabetes mellitus, deafness, intellectual disability, and/or subacute encephalitis may develop months to years later (watson et al., ; white et al., ) . from mass immunization programs, the number of rubella cases has progressively declined and was no longer endemic in the united states as of but remains endemic in other countries, with a dramatic increase in reported cases the last decade (reef et al., ) . recently, africa and asian have seen -fold increases in rubella cases, representing a significant proportion of the over , global cases reported, but where neither of these regions has immunization policies in place to control rubella outbreaks (white et al., ) . a recent, rubella epidemic in japan reporting over , cases, with at least crs cases (minakami et al., ) , has also served to demonstrate that partial vaccination strategies can lead to major outbreaks. in this case, vaccination was only provided to young women, while outbreaks affected the adult male populationsda phenomenon which has also been observed in other countries applying such vaccination strategies (paradowska-stankiewicz et al., ; janta et al., ) . once measles is cleared, memory t cells can provide lifelong immunity to mev (bester, ) , and distinct patterns of cellular immunity to rubella virus are observed and related to the time elapsed following vaccination (lambert et al., ) . predominant biomarkers of early cellular measles immunity are characterized by an immunosuppressive phenotype, with increases in il- and tnf-a and decreases in ifn-g and proliferative properties of circulating peripheral lymphocytes (pukhalsky et al., ) . late immunity is shifted to predominantly proinflammatory cytokine profiles via increased concentrations of il- , granulocyte-macrophage colony-stimulating factor, and tnf-a, in combination with decreases in il- (dhiman et al., ) . human leukocyte antigens (hlas), known to play critical roles in immune response to viruses, contribute to the heterogeneity of the immune response to rubella virus as a result of their polymorphic nature, whereby hla class i and ii polymorphisms restrict the available repertoire of rubella antigens presented to t cells and therefore influence the subsequent immune response (mitchell et al., ; ou et al., ou et al., , . current efforts are placed on deciphering the immunogenetics of antirubella humoral and cell-mediated immune responses, with a focus on better understanding hla polymorphisms toward the development of vaccine candidates that utilize constructs comprised of hla-specific epitopes that can induce immunity across heterogenetic populations, reviewed in lambert et al. ( ) . both natural infection and vaccines induce humoral and cellular immune responses conferring protection against rubella (tosh et al., ) . while humoral responses have been conventionally used to measure and record protective immunity in human populations, cellular immune responses are intrinsic to humoral immunity (bautista-lopez et al., ; horstmann et al., ; ovsyannikova et al., ; nepom et al., ; vesikari et al., ; akaboshi et al., ; farzaneh et al., ) . since its induction into healthcare systems, immunization with live attenuated rubella virus vaccine has been demonstrated to be safe and effective at preventing infection, crs, and to interrupt endemic rubella transmission (lambert et al., ) . the live attenuated rubella vaccine strain ra / has a proven track record for safety and immunogenicity efficacy (hilleman et al., ; plotkin, ) , where single doses have been demonstrated to potently induce humoral immunity and lifelong protection against infection, and where the vaccine has also been demonstrated to boost previously immunized persons (diaz-ortega et al., ) . from their safety and efficacy, use of recombinant rubella vectors has also been tested toward enhancing immune responses against siv and hiv epitopes, where increases in memory b cell repertoires have been observed upon re-exposure to rubella vectors (virnik et al., ) . durable hiv-specific cellular immunity has been observed from rubella vector boosting, with cytotoxic antigenspecific responses by central and effector memory cd þ and cd þ t cell subsets (rosati et al., ) . vzv, also known as hhv- , is a virus of the varicellovirus genus from the herpesviridae family. humans are its only vector (hambleton and , where it specifically infects t cells, epithelial cells, and ganglia (gershon et al., ) . vzv viruses have diameters measuring up to nm and are encoded by a linear double-stranded dna genome consisting of approximately kb and encoding at least unique genes, with all but the exception of , having homologs in herpes simplex virus (cohen, ) . vzv virions are composed of the viral dna, the capsid, the tegument surrounding the capsid, and the envelope surrounding the tegument and which incorporates the major viral gps (arvin, ) . during lytic infection phases, vzv produces at least gps expressed on both virions and human cell surfaces. during this process, and which is common to other herpesviruses, gene expression is believed to proceed in an orderly cascade of immediate early genes, early genes, and late genes. during latent vzv infection, gene expression is restricted until reactivation for additional rounds of lytic infection (gershon and gershon, ) . vzv has extraordinarily high transmission rates and is highly communicable via the airborne transmission route, with concentrated virus coming from vesicles shedding from skin lesions, leading to cell-free contagious airborne viruses, and as evidenced by the fact that infected children without skin lesions are not contagious (tsolia et al., ; chen et al., ) . primary vzv infection causes varicella, also commonly known as chickenpox. as cellular immunity to vzv wanes in the elderly and immunocompromised populations, latent vzv becomes reactivated and causes zoster (i.e., shingles, herpes zoster), which is usually associated with chronic pain but also numerous other serious neurological and ocular disorders, as well as multiple visceral and gastrointestinal disorders, including ulcers, hepatitis, and pancreatitis (gershon et al., ; gilden et al., ) . available antiviral drugs and vaccines against varicella and zoster are safe and effective for treatment and prevention strategies (gershon and gershon, ) . varicella is globally endemic and is transmitted year-round, with frequent epidemics occurring every to years. outbreaks most commonly occur in nurseries and schools, in hospitals and other medical institutions, and in refugee camps and military and correctional facilities (izurieta et al., ; levy et al., ; longfield et al., ) . although it can often be a self-limiting disease, varicella can also result in death, where in developed countries, an estimated of patients are hospitalized with serious complications, with up to three deaths per , patients (galil et al., ; rawson et al., ) . complications from varicella requiring hospitalization include bacterial superinfections of the skin, blood, bones, and lungs, as well as encephalitis and hemorrhagic manifestations in pediatric and immunocompromised populations (gershon et al., ) . importantly, acquiring vzv during early pregnancy often results in severe congenital defects in % of newborns (enders, ) . vzv is a great example of success through herd vaccination programs for children, dramatically influencing its epidemiology, and causing % declines of hospitalization cases in the united states (gershon et al., ) . following its transmission to the respiratory mucosa, vzv proliferates in the oral pharynx, where it infects human tonsillar activated memory cd þ t cells and induces their tissue-homing properties (sen et al., ) . vzv can be propagated to t cellerich regional lymph nodes for rapid proliferation and is then disseminated by the circulation to infect dermis, epidermis, and other organs (ku et al., (ku et al., , . lymphopenia is typically observed in patients during viral incubation, followed by an increase in leukocyte counts, correlating with the onset of rash until the resolution of viremia. during infection, vzv can be recovered from pbmcs in children exhibiting rash (ozaki et al., ; koropchak et al., ; sawyer et al., ) , is extensively observed in thymic lymphocytes (levin, ) , and observed in all t cell subsets examined (moffat et al., ) . though innate skin immunity can cause delays in multiplication of skin-bound vzv while the adaptive immune system mounts an attack, however, aggressive vzv replication in the skin results in characteristic varicella rash (ku et al., ) . high vzv titre-skin vesicles from rash provide cell-free virus for person-to-person transmission (chen et al., ) . vzv also latently infects neurons of cranial nerve ganglia, dorsal root ganglia, and enteric and autonomic ganglia (gershon and gershon, ) . vzv reactivation causes ganglia to become necrotic and hemorrhagic (head et al., ) , with vzv proteins found in neurons and non-neuronal cells, and where this vzv-induced ganglionitis is often also marked by the upregulation of mhc class i and ii proteins associated infiltration of cd þ and cd þ t cells (schmidbauer et al., ; steain et al., ; gowrishankar et al., ) . before vzv vaccines became available, approximately % of infected adults later developed shingles (yawn et al., ) . the single dose, lyophilized, live, attenuated vzv vaccine (i.e., zoster vaccine live (zvl), zostavax, merck) is indicated for prevention of latent vzv reactivation leading to shingles in individuals older than years. zvl is licensed in over countries, with million distributed doses globally (willis et al., ) , and which has associated efficacy rates of over % in all ages tested (oxman et al., ; schmader et al., ) ; consistent with original clinical trial datasets (tseng et al., ; langan et al., ; marin et al., ) . however, increases in vzv susceptibility have arisen due to increasing aging populations, and in immune-suppressed organ transplant recipients, chemotherapy patients, hiv-infected individuals, and those suffering from chronic illnesses (forbes et al., ) . in these patients, earlier exposure to exogenous vzv protects against shingles by boosting cellular immunity (arvin et al., ; thomas et al., ) . the memory immune response following naturally acquired primary vzv infection is characterized by vzv igg and iga antibodies, as well as vzv-specific cd þ and cd þ t cells, where vzv-specific igg antibodies bind many vzv proteins and mediate virus neutralization and antibodydependent cytotoxicity, reviewed in arvin ( ). the frequency of vzv-specific memory proliferating t cells is estimated to be approximately one in , pbmc (hayward et al., ) . vsv-specific memory cytotoxic mhc class i-or class ii-restricted t cells producing ifn-g and tnf-a can recognize the vzv ge, gb, gc, gh, gi, ie , and ie proteins and can be found to persist for over years after varicella exposure (jenkins et al., ; huang et al., ; asanuma et al., ; diaz et al., ; hayward et al., ; sharp et al., ; sadzot-delvaux et al., ) . the ability of the live attenuated varicella vaccine to elicit vzv-specific igg and t cell immunity in naive hosts was established during its prelicensing clinical evaluations (gershon et al., ) , and where, as expected from its design, the magnitude of these vzv-specific immune responses correlated with infectious virus content and with antigen content of individual vaccine formulations (bergen et al., ; watson et al., ) . importantly, it was later discovered that providing two doses to children resulted in higher igg antibody titres and increased t cell proliferation and where experimental evidence suggesting that memory responses were sustained more effectively from such regimens (watson, ) . these observations led to the more recent recommendation of implementing of a two-dose regimen of varicella vaccine for all vaccine recipients (arvin, ) . studies of how regimens affect long-term protection by the adaptive t cell immune response to vaccination, as exemplified by vzv vaccination studies, have the potential to modify dosages and timelines to maximize overall and persisting beneficial long-term effects from vaccination against many other viruses. historically, smallpox was a severe human disease caused by the variola virus (varv), which was both highly lethal and highly contagious prior to its eradication from human populations in (moore et al., ) . varv belongs to the genus orthopoxvirus of the family poxviridae, which also includes zoonotic species: vaccinia virus (vacv), monkeypox virus, cowpox virus, and camelpox virus (shchelkunov, ) . orthopoxviruses are enveloped, brick-shaped viruses measuring by nm, and containing a double-stranded dna genomes encoding e genes, and measuring approximately kb (garon et al., ) . unlike other dna viruses, these replicate as 'virus factories' in the cytoplasm of infected cells (pauli et al., ) . varv encodes approximately proteins, where over of these are found at terminal regions of the genome and are associated with host immune evasion. the origin of smallpox is unknown, but varv is considered to be one of the most deadly diseases of human history, decimating populations to such an extent that it significantly altered the course of human civilizations. smallpox is believed to have first appeared in , bc in africa, with the oldest credible confirmation found in sanskrit writings from bc and where smallpox lesions are believed to be observed on the mummified egyptian ruler ramses v ( bc) (ristanovic et al., ) . prior to its eradication in , varv circulated in the human population for many centuries and repeatedly caused large-scale epidemics. in the th century for instance, smallpox caused the death of more than , europeans per year (babkin and babkina, ; smith and mcfadden, ) . despite varv eradication from the human population more than two decades ago, fears about its potential re-emergence or the threat of its use as a potential bioterrorism agent have not subsided. this has led to numerous debates concerning the destruction of existing viral stocks, currently maintained in the united states and russia. destruction of these stocks has been postponed for the benefit of further research elucidating varv mechanisms of pathogenesis toward the design of therapeutics as well as on efficacious vaccine strategies that may be required for potential future outbreak (smith and mcfadden, ; stone, ) . immune-evasion mechanisms by varv are the least understood among the orthopoxviruses due to difficulties of finding an appropriate host animal model (turner and moyer, ) , along with limited availability of authentic variola proteins since its eradication (massung et al., ) . thus only two variola proteins, namely smallpox inhibitor of complement enzymes (spice) and vaccinia virus complement control protein (vcp), have been characterized and are similar in structure (dunlop et al., ) . these viral antigens regulate the human complement system (yadav et al., ) , are important for stimulating innate immunity, and also have important features for adaptive immunity, shown to bolster antiviral t cell responses including ifn and cytokine expression (noris and remuzzi, ; moss and shisler, ) . vacv has been used more extensively for human immunization than any other vaccine and what was employed to provide cross-protection against varv toward smallpox eradication (jacobs et al., ). the first generation vacv/varv vaccines produced in the s and s (dryvax, apsv, lancyevaxina, l-ivp) contained live vacv , and induced robust humoral immunity is characterized by high antibody titers, neutralizing and opsonizing viral particles, fixing complement, hemagglutination, and antibody-dependent cell cytotoxicity (amanna et al., ; panchanathan et al., ) . these vaccines have since been observed to generate adaptive immune responses over many concentrations (frey et al., ; rock et al., ) , including the secretion of effector cytokines (e.g., ifn-g) and the lysing of infected cells (amanna et al., ; hammarlund et al., ) . most second-generation vaccines created for biodefense contain replication competent viruses (artenstein and grabenstein, ) and have comparable efficacies to dryvax. thirdgeneration vaccine formulations using attenuated vacv strains (lc m , mva, nyvac, dvvl) have increased safety profiles (artenstein, ; kennedy et al., ) . proof that adaptive cellular immunity is essential in preventing the spread of varv following immunization and in its generating overall protective immunity against smallpox comes from observations that individuals having t celledeficiency disorders suffered serious and sometimes fatal infections after vaccination, but that agammaglobulinemic children were not at risk of these adverse complications (rock et al., ) . varv vaccine induces strong cd þ and cd þ t cell responses, peaking after immunization and then contracting to provide stable memory t cell populations that remain detectable for decades (amanna et al., ; hammarlund et al., ) and with memory cd þ t cells persisting the longest (amara et al., ) . defects in cellular immunity lead to uncontrolled vaccinia infection (lane et al., ) , where cd þ and cd þ t cells are able to prevent mortality of b celledeficient animals infected with vacv (belyakov et al., ) and where cd þ t cells have the most protective overall effects (xu et al., ) , and are essential for optimal ctl function and memory formation (sun and bevan, ; kennedy et al., ) . vacv-specific cd þ and cd þ t cells recognize a diverse array of viral proteins, and cd þ t cell epitopes are predominantly found in early, non-structural genes and transcription factors (terajima et al., ) . cd þ t cell epitopes are from late viral products including membrane, structural proteins, and replicative enzymes (jing et al., ) , and linkage of b cell and cd þ t cell epitopes to varv proteins suggests t helper celleb cell interactions are those required for generation of robust vacv-specific antibody responses (sette et al., ) . in humans, varvspecific cd þ and cd þ t cells have been observed to persist for over years following immunization (rock et al., ) . exanthem disorders by viruses represent more than % of cases of combined fever and widespread rash in pediatric populations, and their correct diagnosis is especially critical for the distinguishing of benign versus lethal viral strain variations that can cause lifelong morbidities in children born from infected mothers. mev is transmitted via human respiratory routes, and despite vaccine availability, still causes % of african children mortalities, and severe risk of sspe-derived fatalities years later in survivors. historically, in common with many other viruses, the gold standard diagnostic of protection is made by quantification of humoral neutralizing antibodies. adaptive cellular immune responses are, however, those most critical for mev clearance, where mev-specific t cell responses coincide with rashes densely infiltrated by cd þ and cd þ t cells. combinations of cd þ t cells, cd þ t cells, and b cells control primary infection, where cd þ t cells dominate for control and clearance, and memory t cells are able to provide lifelong protection. mev infection induces general longterm immunosuppression leading to vulnerability to other pathogens causing secondary infections, but this immunosuppression is believed, by some, to be simultaneously conferring herd protection and have been observed to induce spontaneous tumor regression. rubella virus infection has progressively declined from immunization programs but continues to be endemic in many countries, as a result of complete absence of or problematic or partial vaccination programs, still causing severe crs cases in , infants worldwide, per year. while humoral responses are conventionally used to measure protective immunity, it is adaptive immunity that confers protection. both natural infection and vaccination induce humoral and cellular immune responses, where memory t cells can provide lifelong immunity, with presence of cytolytic t cell biomarkers from vaccine-induced immunogenicity. vaccines in development can comprise hla-specific epitopes inducing immunity across heterogenetic populations. since rubella vaccines can boost the previously immunized, their vectors are being investigated for use toward immunization programs for unrelated viruses. vzv has extraordinarily high human transmission rates. primary vzv infection causes varicella, and before vaccination programs were initiated, would re-emerge from declines in adaptive immunity to cause zoster in % of in immunocompromised populations to cause the hospitalization of of every and the death of three per , patients. vzv represents a poster child of herd vaccination programs that led to % declines in hospitalization events. vzv infection rates are again on the rise in immunocompromised and immune-suppressed populations. it infects human tonsillar activated memory cd þ t cells that home to the lymph to then infect cd þ and cd þ t cells, followed by a lymphopenia resolved at rash onset. innate immunity controls vzv spread until adaptive immunity develops to fully counter the infection. vsv-specific memory ctls persist years after varicella exposure, and observations that increased t cell proliferation with better-sustained memory responses result from multiple booster doses of vaccine have caused modifications in vaccination programs. smallpox by varv was one of the most deadly diseases in human history, causing more than , european casualties annually prior to its vaccine-mediated eradication. viral stocks are maintained from the necessity of developing new vaccines to counter potential future re-emergence of varv from natural-or bioterrorism-derived sources. characterized variola proteins amplify and strengthen t cell responses. first-generation vacv vaccine induced robust humoral immunity and adcc, in addition to generating adaptive immune responses marked by cytokines and cell lysis. varv vaccine induces strong initial effector cd þ and cd þ t cell responses having b cell linkage, then contacting to generate stable memory populations of varv-specific cd þ and cd þ t cells that can persist for over years. accordingly, second-and third-generation vaccines created for biodefense are designed to stimulate adaptive cellular immunity. globally, liver cancer is the fifth most common of cancers, with an average of , cases per year, representing . % of all cancers, and with mortality rates reflecting geographic incidence rates. almost % of liver cancers occur in developing countries, with over of , individuals affected by these diseases. hepatocellular carcinoma (hcc) is the most common form of liver cancer, and approximately % of cases are associated with chronic infection by hepatitis b virus (hbv) or hepatitis c virus (hcv) (el-serag, ) . hepatitis viruses are so named because they display hepatotropism by preferentially infecting hepatocytes to cause liver inflammation, also known as viral hepatitis. infection by hbv and hcv promotes liver cirrhosis in most affected, leading to the development of hcc in up to % of patients (fattovich et al., ) . approximately % of the global population ( e million people) are chronically infected with hbv and strong correlations between hbv prevalence and hcc incidence and mortality. chronic hbv infection accounts for approximately % of hcc cases in adults and for all hcc cases in children (el-serag, ) . hepatitis transmission is from person-to-person contact with infected blood or body secretions or by the fecal-oral route and involving at least five specific viruses, namely hepatitis a, b, c, d, and e viruses (table ) . infectious viral hepatitis is an important challenge to health worldwide: hepatitis a virus (hav) and hepatitis e virus (hev) are acute and endemic in many low-income countries, usually causing self-limiting hepatitis, whereas hbc and hcv also cause acute illness but usually lead to chronic and progressive liver fibrosis, cirrhosis, and an increased risk of hcc (stanaway et al., ) . hbv is controlled in adults but is chronically persistent from neonatal infection (shin et al., ) . hepatitis viruses differ in their virology. hbv is an enveloped dna virus that belongs to the hepadnaviridae family. it contains a bp, partially double-stranded relaxed-circular dna genome that is reverse transcribed via a pregenomic rna intermediate and encodes four overlapping open reading frames, which are translated to produce viral core protein, surface proteins, reverse transcriptase, and hbx (nguyen et al., ) . transmission of hbv results from exposure to infectious blood or body fluids containing blood, and hbv can integrate into the human genome, contributing to its genomic instability and ultimately to hcc (zhao et al., ) . hcv is also transmitted by infected blood; but unlike hbv, hcv does not integrate into the host genome . hcv is also a positivestranded rna virus but is classified in the hepacivirus genus within the flaviviridae family. its genome is . kb in length, includes an internal ribosome entry site, and encodes structural and ns proteins. the structural proteins form the viral particle and include the core protein and the envelope gps e and e . the ns proteins include the p ion channel, the ns - protease, the ns serine protease and rna helicase, the ns a polypeptide, the ns b and ns a proteins, and the ns b rnadependent rna polymerase (moradpour et al., ) . hepatitis d virus (hdv) is also transmitted by contact with infected blood or other body fluids. hdv is an enveloped, negative sense, singlestranded, closed circular rna virus, and requires hbv coinfection for its propagation, where infection with both viruses commonly results in severe liver pathologies. hdv genomic rna of hdv is composed of approximately bp, packaged with approximately molecules of hepatitis delta antigen to form viral particles. hdv envelope surrounding its genome and hdag protein is composed of the three hbv small, medium, and large hbv hbsag envelope proteins. hdv also does not encode its own replicase or polymerase, and rather utilizes host cellular machineries for its replication (abbas and afzal, ) . hav and hev are positive-stranded nonenveloped rna viruses transmitted via the fecal-oral route, and unlike chronically persisting hbv and hcv, are typically cleared after acute infection of immunocompetent individuals (park and rehermann, ). hav is a hepatotropic virus belonging to the hepatovirus genus within the picornaviridae family. its genome consists of approximately bp and encompasses a single open reading frame coding for a single polyprotein, which is post-translationally processed into structural and ns proteins. the structural proteins of hav are divided into the polypeptides vp , vp , vp , and vp , forming the icosahedral capsid of the virus. ns proteins b, c, a, b, c, and d are involved in rna replication and viral polyprotein processing (martin and lemon, ) . hev of the family hepeviridae and genus orthohepevirus has a . kb genome having three opening reading frames encoding for the viral replicase, the capsid protein, and a small phosphoprotein required for the secretion of viral particles (debing et al., ) . hav and hev are waterborne viruses that usually cause acute hepatitis without progressing to chronic liver disease (joon et al., ) , where annually, over million cases of hav and million cases of hev infections have been recorded globally (makiala-mandanda et al., ) . hev outbreaks are reported in africa nearly every year, with some involving over , cases (kim et al., ) . hav is highly endemic in africa, infecting most children, conferring long-term immunity to reduce serious epidemics (jacobsen, ) . hbv, hcv, and hdv can be sexually, parenterally, or vertically transmitted and usually evolve into chronic hepatitis, liver cirrhosis, and hcc causing high morbidity and mortality rates, where globally, over million people are chronically infected with hbv, million with hcv, and million with hdv (kramvis and kew, ; hughes et al., ; thursz and fontanet, ) . superinfection of hbv patients with hdv frequently accelerates the progression of hbv disease to liver cirrhosis, considerably increasing the burden of chronic liver disease (hughes et al., ) . hav, hbv and hcv are responsible for the majority of viral hepatitis cases, and there are similarities and differences in immune responses to infections by these three viruses, possibly explaining the distinct disease courses and outcomes of each hepatitis virus infection (shin et al., ) . type i and iii ifns, major components of the antiviral innate immune system, induce the expression of ifn-stimulated genes (isgs), observed to be much more highly induced by hcv than hav, and not at all by hbv, indicating that this virus is not recognized by the innate immune system (su et al., ; lanford et al., ; wieland et al., ) . another component of the innate immune system, nk cells, are also believed to be responsible for protection against hcv, where increased nk cells in protected individuals coincide with increased ifn-g and cytotoxicity (shin et al., ) . though virus-specific antibodies are produced by all viral hepatitis infections, these have differing roles according to the hepatitis virus infection. hav-specific antibodies with virus-neutralizing activity are induced by natural infection and vaccine immunization and confer lifelong protective immunity (walker et al., ; martin and lemon, ) . hbv surface antigen hbsag-specific antibodies are induced by infection and immunization with the recombinant protein and have virus-neutralizing activity conferring protective immunity (guidotti and chisari, ) . hcv-specific antibodies produced after infection do not offer long-term protection as these do not persist, are subject to loss of neutralizing activity from virus mutation, and are ineffective for cellto-cell hcv transmission (takaki et al., ; dowd et al., ; timpe et al., ) . t cells play critical roles during acute hcv and hbv infections, where robust and multiple epitope-specific cd þ t cell responses are assisted by cd þ t cells for spontaneous resolution of infection (shin et al., ) . this is supported by observations that the depletion of cd þ or cd þ t cells in chimpanzees delays rapid clearance and recovery from infection by these viruses (grakoui et al., ; thimme et al., ) . when hcv and hbv infections become chronically persistent, virus-specific t cells become exhausted and functionally impaired. in acute hcv infection, virus-specific t cells are only detected in the blood and liver after weeks postinfection, and their appearance coincides with large declines of virus titres shin et al., shin et al., , . hbv virusespecific t cell responses are also important for spontaneous resolution of hbv infection, where their responses are observed to be vigorous, broad, and polyclonal in patients resolving primary infections and where their absences are associated with prolonged infection and delayed viral clearance (chisari et al., ; thimme et al., ) . cd þ t cells also play important roles in hav infection, where these have been observed to target multiple epitopes of hav, despite more recent results suggesting that hav is controlled by virus-specific cd þ t cells and not cd þ t cells (walker et al., ; shin et al., ) . finally, hepatitis virus infection results in liver injury, not directly caused by these viruses but rather by immunemediated mechanisms (guidotti and chisari, ) . liver injury biomarkers correlate with acute hav, hab, and hac infection (guidotti and chisari, ; park and rehermann, ; walker et al., ) and may result from cytotoxic activity of cd þ t cells, believed to induce apoptosis of hepatocytes in close proximity to their targeted cells (guidotti and chisari, ) , by il- producing th -differentiated t cells, and by recruitment of nonspecific mononuclear cells by hbvspecific cytokine secreting cd þ t cells (iannacone et al., ; shin et al., ) . effective vaccines controlling hav and hbv have been available for over decades, and an hev vaccine has also been licensed for use in china since (zhu et al., ; stanaway et al., ) . neonatal hbv vaccination has proven to be highly effective in inducing protective antibodies and preventing perinatal and horizontal transmission of hbv (lee et al., ) . however, observations that hbsag-specific ifn-g-or il- -secreting pbmcs are absent in many adolescents suggest that booster vaccines should be administered to provide continued hbv immunization (lu et al., ) . hav vaccination provides long-term immunity in the general population and in immunocompromised patients infected with hiv (crum-cianflone et al., ). there is no existing vaccine for hcv, despite ongoing efforts toward their design and testing for their ability to generate prolonged cellular and humoral immune responses, reviewed in naderi et al. ( ) . in the absence of a vaccine, progress in hcv treatment includes oral treatments achieving cure in most patients, including those previously considered as difficult to treat cases (poordad et al., ; lawitz et al., ) . liver cancer is the fifth most common cancer, representing . % of all cancers, with , annual cases from which in , mortalities occur. hcc is the most common liver cancer, with % of cases resulting from chronic infection by hbv or hcv, with a significant and million chronically infected, respectively. in contrast, hav and hev cause acute hepatitis but do not progress to chronic liver diseases, and hdv infection depends on pre-existing hbv infection. hcv induces the expression of type i and iii isgs and nk cells, not at all present from hbv infection unrecognized by innate immunity. virus-specific antibodies are produced by all viral hepatitis infections but have differing roles across infections. robust and multiple epitope-specific cd þ t cell responses and dominant but depend on assistance from cd þ t cells for resolution of acute hav, hbv, and hcv infections. in chronic infections, cytolytic t cells either cause extensive liver injury to hepatocytes and/or become tolerant and functionally impaired. effective vaccines controlling hav and hbv provide protective antibodies. hav vaccination provides longterm immunity to the immunocompromised, but booster vaccination programs are required for persistence of hbv immunization. no vaccine is licensed for highly variable and rapidly mutating hcv, despite numerous ongoing efforts to generate those which will provide robust cellular and humoral immune responses. historically, the central nervous system (cns) has been considered to be an immunologically privileged site within the body (bailey et al., ; galea et al., ; engelhardt, ; prendergast and anderton, ). by definition, immunologically privileged sites, also including the brain, cornea, testis, and pregnant uterus, have a reduced or delayed ability to reject foreign tissue grafts compared with conventional sites within the body, such as skin (streilein, ; bailey et al., ; carson et al., ; mrass and weninger, ; kaplan and niederkorn, ) . though the cns is protected by a highly complex barrier system, a wide variety of viruses still manage to gain access to it and induce diseases. due to their sizes and tissue penetration strategies, the number of cns viral infections outweigh bacterial, fungal, and protozoa cns infections combined (romero and newland, ) . following cns infection, inflammatory events can arise in distinct anatomical regions such as the meninges (meningitis), brain (encephalitis), and spinal cord (myelitis) or can also simultaneously arise in multiple regions (meningoencephalitis, encephalomyelitis). for many neurotropic viruses, viral cytopathology plays a major role in cns dysfunction, reviewed in swanson and mcgavern ( ) . virus can breach the protective barriers of the cns in many ways, with the main route mechanism being via the blood, where inhaled or ingested viruses can move past the mucosa to establish infection in secondary lymphoid tissues and later be shed into circulating blood to cause broad systemic infections (swanson and mcgavern, ) . the cns parenchyma is protected from a plethora of agents carried in the circulation via an elaborate network called the blood-brain barrier (bbb) and the blood-cerebrospinal fluid barrier (ransohoff et al., ) . viruses have evolved and adapted to overcome these barriers (mcgavern and kang, ) , where some viruses can infect vascular endothelial cells, permitting direct passage across the bbb into the cns (verma et al., ; moses et al., ; coyne et al., ) . in addition, parts of the cns that are not completely protected by the bbb permit more rapid entry of several viruses (van den pol et al., ; wolinsky et al., ) . infected hematopoietic cells in circulating blood can also serve as "trojan horses" that can transport undetected virus into the cns (clay et al., ; tabor-godwin et al., ) . other mechanisms can include systemic viral infections leading to massive systemic inflammation and an ensuing bbb breakdown which opens the floodgates to cns infection by a variety of otherwise restricted infectious agents (arsenio-nunes et al., ; eugenin et al., ) . there are more than recognized distinct virus strains that cause human neurological disease, and the majority of documented cases are caused by viruses that are transmitted to humans by blood-eating arthropod vectors, also known as arboviruses, that are mainly transmitted by mosquitoes and ticks and include polioviruses (pvs), alphaviruses, mosquito-borne flaviviruses, tick-borne orthobunyaviruses, mosquito-borne mammarenaviruses, and rabies virus (rabv; table ). pv, the causative agent of poliomyelitis, more commonly referred to as polio, is a human enterovirus and member of the family of picornaviridae. typically spherical, nonenveloped picornaviruses range in diameter between and nm and have a positive-strand rna of e nucleotides, translated into a polyprotein (i.e., vp -vp -vp -vp - a- b poliovirus c- a- b- c- d), which yields proteins upon its cleavage by viral proteases. picornavirus replication occurs in the cytoplasm of infected cells in association with intracellular membranes, where virions are released by cell lysis, ultimately killing cells and causing extensive damage to tissues. the host immune response against picornaviruses includes cytokine release, antibody production, and ctl activation, reviewed in dotzauer and kraemer ( ) . the viral genome of pv is a single-stranded rna of approximately nucleotides, enclosed in a nonenveloped capsid comprising copies of four different polypeptides arranged with icosahedral symmetry (racaniello, ) . all three pv serotypes cause paralytic disease, and cd is the cellular receptor for all three serotypes, whereby pv interaction with cd , expressed by many different cell types, leads to a conformational change of the virus particle and the following release of the rna genome into the cellular cytoplasm (mendelsohn et al., ; hogle, ) . once in the cytoplasm, the viral rna genome is translated, and the production of new infectious virions begins. pv infection results from ingested virus that replicates in the oropharyngeal and intestinal mucosa (sabin and ward, ) . from the primary sites of multiplication in the mucosa, pv virus drains into cervical and mesenteric lymph nodes and then into the blood, causing transient viremia symptoms. the person-to-person transmission of pv virus is through the fecal-oral route. once pv is shed in the feces, the majority of the natural human infection ends at this stage with a modest symptoms including sore throat, fever, and malaise (dotzauer and kraemer, ) . however, in %e % of pv-infected individuals, the virus gains entry to the cns through neurons at neuromuscular junctions (nmjs) and replicates in motor neurons within the spinal cord, brain stem, or motor cortex and leads to the pv-characteristic flaccid muscle paralysis disorder poliomyelitis (racaniello, ; koyuncu et al., ) . approximately % of these paralytic cases result in death (roush et al., ) . following both pv infection and vaccination, neutralizing antibodies are generated to clear the virus, and these can be detected for many years, providing lifelong protection (libbey and fujinami, ) . vaccination with the injected inactivated pv vaccine prevents viral spread to the cns, whereas vaccination with the live-attenuated oral pv vaccine protects against infection of the intestinal tract and also prevents person-to-person spread of the virus (nathanson, ; griffin, ) . pv remains an important cause of neurologic disease as the three live-attenuated vaccine strains are at risk of recombining their genomes to revert to virulent form (griffin, ) . pv is endemic in afghanistan, pakistan, india, and nigeria, where political reasons, in part, are the most significant modality toward achieving pv eradication via vaccination. pv can usually be cleared by the adaptive immune response. under conditions of antibody deficiencies in humans, however, continuous fecal shedding of pv contributes to the establishment of persistent infection cycles (martin, ; nathanson, ; libbey and fujinami, ) . though less is known about the roles of adaptive t cell responses in controlling pv infections relative to that of neutralizing antibody responses, it is known that pv-specific cd þ t cells are induced in vaccinated individuals, where key epitopes have also been identified (graham et al., ; simons et al., ) . the induction of pv-specific cd þ t cells has been suggested to be the result of stimulating by pv-infected dcs and macrophages (wahid et al., ; dotzauer and kraemer, ) , where it has been demonstrated that hla class ii presentation remains intact in infected, antigen-presenting cells (apcs), and that cytolytic cd þ t cells produce ifn-g to lyse pv-infected cells for virus clearance. pv-specific cytotoxic, ifn-g-secreting cytotoxic cd þ t cell responses induced by infected macrophages have also been documented (wahid et al., ) , suggesting that both cd þ and cd þ cytolytic t cells partake in the adaptive immune reaction against pv (dotzauer and kraemer, ) . approximately viruses are included in the flavivirus genus of the flaviviridae family of viruses, with of its species associated with dengue, yellow fever (yf), japanese encephalitis (je), tick-borne encephalitis, and west nile encephalitis as the most important arboviruses causing extensive global morbidity and mortality (diamond, ) . flaviviruses are enveloped viruses with single-stranded rna genomes that are translated in the cytoplasm to generate a single polyprotein that is then cleaved into structural and ns proteins by virus and host proteases. the various encoded viral proteins assemble to generate the capsid, the envelope for receptor binding, membrane fusion and viral assembly, and the transmembrane proteins (prm) that assist in protein folding and function. the entry of flaviviruses into their target cells is mediated by the interaction of the e gp with host cell surface receptors (perera-lecoin et al., ) . flaviviruses are believed to evade the immune system to enter the brain and spinal cord via circulating blood (johnson and mims, ) , where these may cross the bbb by passive transport across the endothelium, by active replication in endothelial cells, or by a "trojan horse" mechanism, where virus hides in inflammatory cells during their transit into the brain (solomon and vaughn, ) . ifn-dependent and complement system innate immune responses, along with humoral neutralizing antibodies, protect against virus dissemination and spread, reviewed in diamond ( ) . adaptive cellular immunity is also important toward the destruction of infected cells, whereby virus-specific ctls become activated, proliferate, and release inflammatory cytokines following exposure to flavivirus-infected cells (kesson et al., ; kurane et al., a; liu et al., ; murali-krishna et al., ) . there is also evidence that flavivirus replication is enhanced by myeloid cells, as observed for dengue, yf, west nile, tick-borne, and je viruses (diamond, ) . the je flavivirus is the leading cause of encephalitis and is amplified by waterfowl and only transmitted to humans by mosquito vectors, with no possibility of human-to-human transmission (erlanger et al., ) . pediatric and geriatric populations are at higher risk of infection by je (burchard et al., ) . while % of je infections remain asymptomatic, je can be devastating in symptomatic patients, causing mortality rates of % in these patient populations (batchelor and petersen, ) . after approximately days of je incubation, these patients suffer from high fever, chills, headache, myalgia, and confusion, where pediatric patients also have symptoms of gastrointestinal pain, vomiting, and seizures. post-je infection, handicaps from persistent neurological deficits can last a lifetime in up to % of these survivors. as there is no treatment against je, the only method of prevention is avoidance of mosquitos and vaccination (batchelor and petersen, ) . the four available je vaccines are registered worldwide and used in national immunization programs for different age groups, including inactivated vero cell culture vaccine (je-vc) (ixiaro), inactivated mouse braine derived vaccine (je-mb), a cell cultureederived (primary hamster kidney) live-attenuated vaccine based on the sa - - strain manufactured in china, and a live-attenuated chimeric vaccine based on the genes of yf d backbone combined with vero cellepropagated sa - - strain (imojev) (chen et al., ) . t cell responses to je vaccination have been reported, where for sa - - , t cell responses were detected in the majority following vaccination, and these cross-reacted with other flaviviruses (turtle et al., ) . je-specific t cell responses are observed in pbmcs isolated from je-infected patients and vaccinated individuals. cd þ and cd þ t cells directed against structural viral proteins were identified in vaccinated individuals, in contrast to specific cd þ and cd þ responses against ns or c proteins in infected patients (nathanson and cole, ) . these findings indicate that ns proteins, and especially ns have important roles in the initiation of t cell responses, as the main target of je-specific t cellemediated immune responses. in addition, cytolytic cd þ t cells clones that cross-reactive with other flaviviruses have been generated from individuals immunized with inactivated je vaccine (aihara et al., ) . finally, cd þ and cd þ and th t cells are believed to be primary determinants of protection from je infection (kumar et al., a (kumar et al., , b ). viruses from the alphavirus genus are members of the togaviridae family of viruses, a group of enveloped positive-sense rna viruses. these are mosquito-borne viruses causing two major types of human disease. the old world alphavirusesdsindbis, chikungunya, and ross river virusdcause arthritis and arthralgia, while the new world alphavirusesdeastern (eeev), western (weev), and venezuelan equine encephalitis virus (veev)dcause encephalitis (trobaugh and klimstra, ) . alphaviruses are small, icosahedral-shaped, enveloped viruses and are approximately nm diameter in size (mancini et al., ; morgan et al., ; fuller, ) . alphavirus virions acquire host cell lipid membranes during viral assembly (fuller, ; acheson and tamm, ; vogel et al., ) , with e and e viral gps spike protrusions, arranged in an icosahedral pattern embedded within their membranes and interacting with nucleocapsid (fuller, ; vogel et al., ; owen and kuhn, ) . alphavirus single-stranded, positive-sense, rna genomes are kb long and consist of two large open reading frames encoding the ns and structural polyproteins that are subsequently cleaved by both viral and host proteases to create four ns proteins (nsp to ) and five structural proteins (c, e , e , k, e ) (strauss et al., ; hardy and strauss, ) ; reviewed in leung et al. ( ) . of major concern are the new world eeev, weev, and veev alphaviruses, which are naturally transmitted by mosquitos, but where veev is also highly infectious via the aerosol route (zacks and paessler, ) . precise mechanisms of entry of alphaviruses into the cns remains elusive, however, once in, alphaviruses infect humans and equines neurons, causing neurologic symptoms from mild febrile illness to severe encephalitis resulting in death (ramakrishna et al., ; zacks and paessler, ) . development of severe encephalitis is believed to result from neuronal cell death from accelerated viral spread and host neuroinflammatory viral responses (paessler et al., (paessler et al., , . antibodies are protective against lethal meningoencephalitis when the virus is transmitted by insects, and virus-specific cd þ t cells are found to be important for protection from lethal meningoencephalitis from aerosol transmission routes (paessler et al., ; yun et al., ) ; reviewed in libbey and fujinami ( ) . veev remains an emerging disease threat by natural transmission as well as via its usage as a biological weapon. of the new world alphaviruses, veev is the most important human and equine pathogen, it having caused outbreaks of febrile and neurological disease primarily in latin america during the past century. past outbreaks have lasted several years and have involved up to , equine and human cases over large geographical regions, with the largest outbreaks on record were from the s, where central colombia saw over , human cases and an estimated , equine deaths. more recent outbreaks in mexico and south america are behind the classification of veev as a re-emerging disease (weaver et al., ) . because veev can also be developed as a biological weapon amenable to use in warfare or terrorism, current global emphases on biological defenses have renewed interest in its virology (hawley and eitzen, ; weaver et al., ) . veev infection in humans typically causes nonlethal, incapacitating symptoms including fever, headache, malaise, myalgia, sore throat, and vomiting. up to % of rarer cases of cns involvement usually follow acute febrile phases, with associated severities of neurological disease ranging from somnolence and mild confusion, to seizures, ataxia, paralysis, and coma, with mortality rates ranging as high as % in infected children and % in infected adults (bowen et al., ) . veev has also been reported to cause long-term neurological deficits, abortions, and teratogenic effects (de la monte et al., ; rivas et al., ; weaver et al., ) . like veev, though the majority of human infections with eeev are asymptomatic, cns involvement results in severe neurological signs, lesions, and sequelae, with an estimated associated human mortality rate of %, and with its neurological manifestations including facial edema, paresis, paralysis, respiratory impairment, altered mental state, and seizures in children, many of these symptoms persisting long-term in surviving patients. in fatal cases of eeev, gross lesions in the brain include edema, meningeal congestion, hemorrhage, and malacia (deresiewicz et al., ) . as with veev and eeev, natural human cases of weev typically show an early, flu-like illness with associated fever, malaise, and headache. similar to eeev, weev results in cns involvement in a significant proportion of cases, including symptoms of somnolence, seizures, coma, and motor neuron dysfunction. ninety percent of infants infected with weev have severe cns symptoms (calisher, ) . human mortality rates from weev infection range from % to %, and neurological sequelae may become permanent features in survivors (steele and twenhafel, ) . alphavirus expression vectors based on sindbis, semliki forest, and veev have been demonstrated to induce strong cd þ t cell responses against their antigens (rayner et al., ; lundstrom, lundstrom, , riezebos-brilman et al., ; schlesinger and dubensky, ; polo et al., ) . both innate and adaptive immune responses can control viruses targeting cns neurons (griffin, ) . viral disruption of the type i ifn signaling pathways interferes with survival from veev, as well as of those infected with sindbis and west nile viruses (ryman et al., ; samuel and diamond, ; white et al., ) . virus-specific antibody responses are critical in limiting viral spread and facilitating clearance of infectious virus from neurons within the brain levine et al., ) . both alpha beta (ab) and gamma delta (gd) t cell responses have been demonstrated as being important for the control of veev (paessler et al., ) . t cell responses reduce mortality rates by direct killing of infected cells, producing antiviral cytokines and increasing production of virusspecific antibodies (bilzer and stitz, ; patterson et al., ; shrestha et al., ; sitati and diamond, ) . veev replicon particles delivered as an adjuvant have been demonstrated to induce activation of cd þ t cell responses (thompson et al., ) . more recently, t cells have been demonstrated to facilitate recovery from veev-induced encephalomyelitis in absence of antibodies, responsible for dramatic reduction in viral titres in cns, where cd þ t cells were the best t cell producers of ifn-g response and were more efficient at controlling veev in cns lesions than cd þ t cells, facilitating recovery from severe viral encephalomyelitis (brooke et al., ) . commercial equine vaccines marketed in the united states are generated with inactivated tc- , which produces viremia, fever, and leukopenia in horses but generates robust neutralizing antibodies and veev protection from rechallenge (walton et al., ) . u.s. army special immunization programs provide inactivated c- to individuals failing to seroconvert in response to tc- boosters (pittman et al., ) ; however, neither of these vaccines can be shown to completely protect nonhuman primates against aerosol exposure (pratt et al., ) . a more stably attenuated veev vaccine candidate called v has been produced, where preclinical testing has demonstrated it to be safe and immunogenic and possibly superior to tc- (pratt et al., ; hart et al., ; ludwig et al., ) . adaptive immune pbmc-derived biomarker signatures have been identified and able to efficiently stratify tc- vaccinated from naïve or nonresponding individuals (erwin- cohen et al., ) . rabv is the type species of the genus lyssavirus, within the rhabdoviridae family. rhabdoviruses are negative-sense, single-stranded rna viruses having a distinctive bullet-shaped structure. up to viruses of lyssaviruses have the potential to cause rabies in humans. these have a , nucleotide genome encoding five proteins: nucleoprotein (n), phosphoprotein (p), matrix (m), glycoprotein (g), and rna-dependent-rna polymerase (l) (marston et al., ) . rabv causes acute encephalitis in mammals, causing fatality rates of almost %. rabv commonly infects many animals, including bats, skunks, foxes, and dogs and can also infect insects and plants. rabv in animal saliva spreads between hosts via bites or scratches. infected animals can survive for years, secreting infectious particles in their saliva, but untreated infection in humans generally results in rapidly fatal acute myeloencephalitis (koyuncu et al., ) . rabid dogs are the most important reservoirs for rabv, where dog bites account for more than % of human infections. rabv, like all members of lyssaviruses, is neurotropic and infects peripheral nerves close to the primary site of the bite. rabv then rapidly moves by retrograde axonal transport to the dorsal root ganglia where virus replication begins . rabv particles enter axons of motor neurons at the nmj via their binding to nicotinic acetylcholine receptors (e.g., nachr) and neural cell adhesion molecules (ugolini, ) . transneuronal rabv spread occurs between synaptically connected neurons, whereby viruses move from postsynaptic to presynaptic neurons. in humans, a relatively long asymptomatic incubation period after initial rabv infection can occur, sometimes lasting up to year, and providing some time for cns infection intervention. however, death almost always ensues after rabv infection reaches the cns, with marked behavioral and neurological symptoms (koyuncu et al., ) . once rabv has entered the cns, it rapidly moves to the brain and is associated with an explosive increase in virus replication. initial symptoms include pain or paraesthesia close to the bite site and are often associated with fever, fatigue, and weakness in associated limbs. nonspecific neurological symptoms including headache and anxiety occur days prior to acute encephalitis (morrison and wenzel, ) . currently, there are no available therapies against disease symptoms once they develop, and death ensues within a number of days following cns-associated symptoms (jackson et al., ) and reviewed in johnson et al. ( ) . rabv replication begins following cns penetration, thereby limiting earlier possible detection of low-level primary antigens in the peripheral circulation. this delays antigen presentation, where antigens later but rapidly drain from the cns to local lymphoid tissues (knopf et al., ) . once b cells are stimulated, the next delaying obstacle is re-entry into the cns, but experimental models have demonstrated t and b cell infiltration of dorsal root ganglia, spinal cord, and brain (johnson et al., ) , with t cells as the major immune subsets, but where most of these cns-infiltrated t cells have fas-mediated apoptotic phenotypes (baloul and lafon, ) . further intrinsic complexities in immune responses are present in the cns, including tight mhc expression regulation (irwin et al., ) , and the expression of immunosuppressive factors by neuronal cells. additionally, the bbb remains intact during rabv infection (roy et al., ) . numerous studies have suggested that the virus suppresses the adaptive immune response, believed to be in part due to a deficit of adaptive immune effector cell accumulation within the cns due to a virally induced reduction in bbb permeability (libbey and fujinami, ; roy et al., ) . two rabv vaccines are licensed for human application, the human diploid cell vaccine manufactured by aventis pasteur and the purified chick embryo cell vaccine manufactured by chiron . pre-exposure vaccination given to healthcare personnel, laboratory workers, and travelers to endemic areas causes detectable igm and igg antibodies within a week following exposure, and long-term studies have provided evidence that igg antibodies provide the most effective protection against rabv due to its ability to penetrate tissues, in contrast to igm which cannot penetrate tissues (turner, ) . a multifaceted approach for human rabies eradication involving government support, disease awareness, and vaccination of at-risk humans and dogs will be required to achieve the goals of the world health organization in eradication of rabies by (fooks et al., ) . the cns is immunologically privileged and protected by a highly complex barrier system. viruses that have evolved to overcome these barriers can cause cns infections greatly outnumbering those from all bacterial, fungal, and protozoa infections combined. ingested pv multiplies in the oropharyngeal and intestinal mucosa and drains to cervical and mesenteric lymph nodes and then into the blood ahead of penetrating the cns to cause polio, with % of cases resulting in death. both neutralizing antibodies and the adaptive immune system can clear pv infection and may provide lifelong protection. vaccination combinations can induce pv-specific cytolytic cd þ and cd þ t cells for virus clearance, but their coadministration can pose the risk for reversion to virulence by recombination. the je flavivirus is amplified by waterfowl and transmitted to humans by mosquitoes, and while % of its infections are asymptomatic, mortality rates in % of infected individuals cause associated disorders that leave its survivors a lifetime of associated morbidities. as there is no existing je treatment, prevention involves either avoidance of mosquitoes or vaccination. flaviviruses evade the immune system to cross the bbb by an inflammatory celle mediated "trojan horse" mechanism. je dissemination is limited by innate immune responses, neutralizing antibodies produced by humoral immunity, and by virus-specific ctls. je vaccines are licensed worldwide, and the majority of vaccinated individuals have circulating je-specific cd þ and cd þ t cells that can cross-react with other flaviviruses. alphaviruses are transmitted by mosquito bites to infect neurons, causing mild to severe encephalitis resulting in death, with past outbreaks numbering in the hundreds of thousands. veev infection causes up to % mortality in children, % of which involve cns penetration, causing severe long-term neurological disorders. veev is not only a naturally emerging disease threat but is also a highly developed biological weapon amenable to warfare or terrorism due to its aerosol transmission route and associated lethal meningoencephalitis. ifn signaling pathways and ab and gd t cell response from innate and adaptive immunity can control veev targeting of cns, where virus-specific antibody responses are critical in limiting viral spread. in the absence of antibodies, veev replicon particles can induce t cell responses able to induce recovery from veev-induced encephalomyelitis, where cytotoxic cd þ t cells control veev in cns lesions. veev vaccines induce robust neutralizing antibodies for protection against rechallenge. tc- vaccine responders have circulating pbmc biomarkers, and military programs give boosters of c- to those failing to seroconvert. in contrast to these other viruses, rabv replication only begins after cns penetration, as facilitated by depth of bite by its canine vector, thereby limiting possible detection of primary viral antigen in the periphery and resulting in delayed and minimal innate and humoral responses. once rabv-related acute encephalitis symptoms begin, fatality is sure to follow due to absence of cns infiltration by adaptive immune effector cells as a result of virus-induced decreases in bbb permeability. other countermeasures against protection are tight mhc expression regulation and apoptotic phenotypes of bbb-infiltrated t cells. rabv vaccines cause increases in ig, but little is known concerning vaccine-associated adaptive immune responses. viral hemorrhagic fever (vhf) classification originates from the study of hantaviral hemorrhagic fever (hf) and was later extended to include crimeanecongo hf and omsk hf. vhf can results from infection by enveloped rna viruses from four families: flaviviridae, filoviridae, arenaviridae, and bunyaviridae. vhf designation is given to severe febrile illnesses with abnormal vascular regulation and vascular damage (peters and zaki, ) . vascular dysregulation occurs early in the course of disease, visible as skin flushing, hypotension, and conjunctival vasodilation, whereby vascular damage with capillary leakage occurs as disease progresses, causing edema and serous effusions of pleural and peritoneal cavities. the terminal phase of vhf, or shock, arises from increased disease severity from combinations of vascular dysregulation and damage from capillary leakage (paessler and walker, ) . detailed mechanisms of hemorrhage and plasma leakage during vhf include endothelial injury, activation of the mononuclear phagocytic system, cytokine storm, platelet aggregation and consumption, activation of the coagulation cascade, and insufficiency of coagulation factors from severe hepatic damage (schnittler and feldmann, ; chen and cosgriff, ) . these mechanisms vary among diseases, cell and organ tropism of causative viruses, and host responses (paessler and walker, ) . flaviviruses, filoviruses, arenaviruses, and bunyaviruses are the main causes of hf (table ) . these viruses continue to propagate as part of the life cycles of primates, bats, rodents, farm animals, mosquitoes, and ticks. infection by these viruses can cause mild vascular instability to fatal shock, with hemorrhage ranging from unnoticeable to life-threatening. pathogenic mechanisms of hfv are diverse and include hepatic necrosis leading to deregulation of coagulation factors, cytokine storm, increased permeability, and complement activation. overall disease severity by these viruses is varied, whereby ebola and marburg hf can cause high fatality rates, whereas yf and dengue infections can be asymptomatic. severe vhf is commonly correlated with ineffective immunity and high viral loads, and severe plasma leakage can occur from viral clearance and fever breaks in dengue hf (dhf). approximately viruses are included in the flavivirus genus of the flaviviridae family of viruses, with of these species associated with dengue, yf, je, tick-borne encephalitis, and west nile encephalitis as the most important arboviruses causing extensive global morbidity and mortality (diamond, ) . flaviviruses are enveloped viruses with single-stranded rna genomes that are translated in the cytoplasm to generate a single polyprotein that is then cleaved into structural and ns proteins by virus and host proteases. the various encoded viral proteins include capsid, envelope for receptor binding, membrane fusion and viral assembly, and transmembrane proteins (prm) that assist in protein folding and function. although the precise mechanism is unclear, flaviviruses are believed to evade the immune system to enter the brain and spinal cord via circulating blood (johnson and mims, ) , where these may cross the bbb by passive transport across the endothelium, by active replication in endothelial cells, or by a "trojan horse" mechanism utilizing inflammatory cells (solomon and vaughn, ) . ifn-dependent and complement system innate immune responses and humoral immunity, producing neutralizing antibodies limit dissemination of infection and protect against viral spread, reviewed in diamond ( ) . cellular immunity is also important toward eradication of infected cells, whereby infection induces the recognition of flavivirusinfected cells by virus-specific ctls, which then become activated, proliferate, and release inflammatory cytokines (kesson et al., ; kurane et al., a; liu et al., ; murali-krishna et al., ) . however, there is also evidence that flavivirus replication is enhanced by myeloid cells and has been observed for dengue, yf, west nile, tick-borne, and je viruses (diamond, ) . yf virus (yfv) is important both historically and currently. it was once one of the most globally feared diseases terrorizing africa, europe, and the americas. hundreds of thousands were killed in the americas over a -year spandcrippling economies (watson and klimstra, ) . yfv is a member of the genus flavivirus of the flaviviridae family and contains a single-stranded rna genome of approximately kb. yfv virions are icosahedral and are composed of nucleocapsid, composed of capsid (c) protein subunits and a surrounding lipid bilayer derived from host membranes. the viral envelope is studded with dimers of envelope (e) and membrane (m) proteins, for a total diameter of approximately nm. as the major component of the virion surface, the e protein is responsible for cell-surface receptor binding, virion assembly, fusion, and immunogenicity. viral proteins are encoded in a single open reading frame and produced as a polyprotein later processed by proteolytic cleavage into structural (c, m, and e) and ns proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) , reviewed in gardner and ryman ( ) . during most yfv infections, the virus is transmitted by the bite of an infected aedes aegypti mosquito found in urban areas. infected patients often develop severe acute illness hemorrhagic yf disease, with associated symptoms of fever, nausea, vomiting, epigastric pain, hepatitis, jaundice, renal failure, hemorrhage, and shock, with %e % of cases resulting in death (watson and klimstra, ) . yf is the prototypical vhf, sharing many pathophysiological features with other viral disorders only associated via similarities in syndromes, but with the exception that yf causes the most severe symptoms of hepatic dysfunction (monath and vasconcelos, ) . yfv remains endemic in south american and african countries, with monkeys as its reservoir, causing regular outbreaks of jungle yf, and resulting in as many as , infections per year causing , deaths. millions are at risk for infection in africa, where vaccination prevalence is low. the outbreak in angola serves as an example of yfv traveler-associated spreading to neighboring countries, where it reached as far as china, then naïve for virus (watson and klimstra, ) , and representing a prime population for a major outbreak of epic proportions (wasserman et al., ) . geographical shifting of mosquito populations to north america is also creating new risk for yfv, dengue, and zika infection of naïve populations (monaghan et al., ) . despite the availability of vaccination against yfv since the s, large epidemics have still arisen, with dramatic surges of yfv in africa in the s and the late s, with each reporting over , cases. recent outbreaks have also affected brazil, paraguay and argentina, uganda, and sudan and ethiopia. immunity is the critical for reducing and eliminating viral infections, but other contributing factors to virus amplification are multifactorial and elusive, including the emergence of new viral strains and prolonged periods of hot and humid weather promoting insect propagation, reviewed in monath and vasconcelos ( ) . fifty-seven million people were vaccinated against yf across africa between and . five hundred million doses of the live-attenuated yf d vaccine, representing the most effective vaccine ever created, have been distributed over the last years (monath and vasconcelos, ) . both humoral and cellular immunity elicited by d are observed and well characterized, where neutralizing antibodies provide protection, but d also provides a robust, long-lived, and polyfunctional adaptive t cell immune response (watson and klimstra, ) . neutralizing antibodies remain the accepted correlate of protection against yfv, with % or greater of d immunized individuals developing neutralizing antibodies (gotuzzo et al., ) . d also elicits a complex modulation of innate immune cytokines, with elevated levels of plasma ifn-g days postvaccination (neves et al., ) . restimulation of innate immune cell cultures of nk cells, neutrophils, and monocytes from d vaccinated humans with yf antigen results in the increased production of ifn-g, il- beta, il- , il- , tnf-a, and il- (neves et al., ; gardner and ryman, ; luiza-silva et al., ; silva et al., ) . since its development, humoral immunity, as a gold standard of general vaccine development, was the most studied aspect of human immunity to d. however, recent studies of adaptive t cellemediated immunity to d have demonstrated that both cd þ and cd þ t cells strongly respond to d, with activated cd þ t cells detected as days after vaccination , and cd þ t cells detected several days later kohler et al., ; blom et al., ) . increased cd þ t cell proliferation correlates directly with the levels of virus genomes in plasma, which peaks once virus is eliminated . cd þ t cell clones responding to d differentiate into central memory and effector memory subpopulations (dewitt et al., ) and are still detectable years following vaccination (wieten et al., ) . d-specific cd þ t cells respond to epitopes contained from every protein product generated by the d polyprotein, and upon peptide restimulation, these d-specific cd þ t cells have activated cytotoxic profiles including increased expression of ifn-g, tnf-a, and mip -b and il- granzyme b and cd a (blom et al., ; akondy et al., ) but are not exhausted and retain long-lived memory and polyfunctional phenotypes for at least years following d rechallenge (akondy et al., ) . dengue virus (denv), also a member of the single-stranded positivesense rna viruses from the flaviviridae family, causes visceral and cns disease in humans and is closely related to yfv, where denv fever has often been mistaken for yfv infection. far more serious is dhf, where additional symptoms develop, including hemorrhage and shock, and have mortality rates exceeding % if left untreated (rogers et al., ) . denv is a spherical, -nm virion, comprising of three structural proteins: capsid (c), premembrane and membrane (prm and m), and envelope (e). the e protein directs several critical steps of the viral replication cycle, including engagement with cellular attachment and entry factors, membrane fusion, and virion assembly. denv binds to target cells via glycosaminoglycans, c-type lectins such as dc-sign, the mannose receptor cd , and immunomodulatory proteins (tim and tam receptors; diamond and pierson, ) . thus targets for denv infection include monocytes, macrophages, dcs, mast cells, and possibly hepatocytes and endothelial cells. following its entry into the cellular cytoplasm, the viral genomic . kb rna is translated into a single polyprotein, later cleaved into three structural and seven ns proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) by viral ns and host cell proteases. twenty-five percent of denv infections cause both mild symptoms including dengue fever (df) to more severe and lethal dhf, causing shock via hemorrhagic and capillary leak syndrome. df can be characterized by abrupt onset febrile illness causing headache, severe muscle and joint pain, and rash, whereas dhf is characterized by rapid onset capillary leakage accompanied by significant thrombocytopenia and liver injury (halstead, ) . as with yf, denv origins are believed to be that of a sylvatic virus, with a natural life cycle involving multiple mosquito and vertebrate species from asia and africa (diallo et al., ) . denv adaptation to human demography is via mosquito vector aedes aegypti, breeding in urban areas (trpis and hausermann, ) . cases of denv infection have increased since the s, with an estimated million cases of df, and , cases of dhf occurring globally every year. there are no cures for denvassociated disorders, and vaccine development has been complicated by antibody-dependent enhancement of future heterotypic infection induced by vaccination (vaughn, ; halstead and deen, ) . thus avoidance and control of aedes aegypti is the best approach for limiting denv infection (rogers et al., ) . adaptive immune cd þ t cells vigorously and frequently recognize denv ns , ns b, and ns proteins, whereas the capsid, envelope, and ns proteins are the dominant targets for cd þ t cells (simmons et al., ; duangchinda et al., ; weiskopf et al., weiskopf et al., , rivino et al., ) . both cd þ and cd þ t cells are believed to contribute to protection against denv, as denv-specific cd þ t and cd þ t cells proliferate, produce ifn-g, and lyse target cells, from primary denv infection (kurane et al., b; mathew et al., ; gagnon et al., ; livingston et al., ) . higher frequencies of denv-specific ifn-gproducing t cells are present in children with asymptomatic denv infection (hatch et al., ) . both cd þ and cd þ t cells contribute to protection against denv challenge (yauch et al., (yauch et al., , zompi et al., ; zellweger et al., ) , and hla alleles associated with increased risk of denv severity correlated with weak cd þ t cell responses and vice versa, implying a protective role for cd þ t cells against severe denv disease in humans (weiskopf et al., ) . in , the first dengue vaccine (dengvaxia) was licensed in asian and south american countries for protection against all four denv serotypes, and while it demonstrated an good safety and efficacy in clinical trials, it has recently been withdrawn in the philippines due to its causing elevated disease severity if administered following infection (wichmann et al., ) . it has been suggested that failure of this and other live-attenuated tetravalent dengueeyf chimeric virus vaccines (guy et al., ) is the result of their lacking the ns proteins ns , ns b, and ns , otherwise dominantly targeted by cd þ t cells (simmons et al., ; duangchinda et al., ; weiskopf et al., weiskopf et al., , rivino et al., ) , making it critical to accurately assess not only antibody responses but rather t cell responses in the context of denv vaccine development (weiskopf and sette, ) . lassa virus (lasv), causing lassa fever (lf), is an enveloped virus with two single-stranded rna segments and is another virus causing hf. lasv is an old world member of the arenavirida family of viruses. the single-stranded arenavirus genome consists of a small (s) and a large (l) rna segment, measuring . and kb, respectively. the large segment encodes a small zinc-binding (z) protein which regulates transcription, replication, and viral budding, along with the rna polymerase (l). the small segment encodes the np and the two envelope glycoproteins (gp and gp ) responsible for cell entry, reviewed in russier et al. ( ) . lasv disorder is endemic in africa and its neighboring countries (safronetz et al., ; gunther et al., ) , and though infection rates are difficult to quantify due to limited survey infrastructure, classification of its clinical symptoms is common to other diseases. lasv is predicted to be responsible for approximately , infections and up to resultant deaths each year (ogbu et al., ; mccormick et al., ) . transmission to humans is via the rodent host mastomys natalensis (mccormick et al., ) . apcs, dcs, and macrophages are believed to be the first cells targeted by lasv infection (baize et al., ; mahanty et al., ) , which can rapidly speed up dissemination of lasv to multisystem organs due to their widespread physiological distributions in mucosal tissues and skin. due to their ease in motility across various organs and tissues, apcs are believed to the responsible for the spread of lasv for the establishment of systemic infection (hensley et al., ) . apc infection results in substantial virus release in the secondary lymphoid organs, the liver, hepatocytes, fibroblasts, and endothelial cells that are subsequently infected. lymphopenia of cd þ and cd þ t cells, nk cells, and b cells is observed early during disease onset, reviewed in russier et al. ( ) . lasv infection severities range from asymptomatic infection to fatal hf (fisher-hoch et al., ) and commonly resulting from other viral infections, nonspecific symptoms beginning several days after infection include fever, headache, arthralgia, myalgia, and severe asthenia. these early symptoms are typically followed by more severe symptoms of pharyngitis, conjunctivitis, cough, abdominal pain, diarrhea, and vomiting. in severely affected patients, cervical and facial edema, hemorrhages, renal and liver failures, and encephalopathy occur, and death follows systemic shock (edington and white, ) . survivors of lasv-related disorders have persisting lifelong morbidities and disabling conditions including deafness (cummins et al., ) . no vaccine has been licensed against lasv, and ribavirin is the only existing treatment, but is only effective if administered very early after infection and is not available for broad distribution in countries where lasv is endemic (mccormick et al., ) . t cells play a crucial role in the outcome of severe lasv infection, which has been associated with defective t cell responses since the very cells responsible for stimulating t cell antigen responses are those infected by the virus. however, t cell responses have been demonstrated to play critical roles in the control of lasv, where strong memory cd þ t cell responses directed against lasv np and gp proteins are observed in lasvseropositive healthy individuals from endemic regions (ter meulen et al., ) . high serum concentrations of il- and cxcl chemokines that attract and activate t cells are associated with nonfatal lasv infections (christensen et al., ; dufour et al., ) and vice versa in fatality cases (mahanty et al., ) . the control of acute lf has been correlated with increases in circulating activated cd þ and cd þ t cells in response to lasv infection or antigen (baize et al., ) . in vaccine studies, protection against a lethal lasv rechallenge is associated with the induction of t cell immunity (fisher-hoch et al., ; geisbert et al., ) . however, in comparison to other viruses, lasv-infected dcs are unable to mount effector t cell responses (pannetier et al., ) . human and nonhuman primate studies have demonstrated that lasv np and gp proteins are the main viral antigens recognized by activated t cells (meulen et al., ; ter meulen et al., ; fisher-hoch et al., ; fisher-hoch and mccormick, , ; geisbert et al., ) , suggesting that vaccines using these proteins to induce long-term memory t cell expansion will best control the spread of lf. ebola virus (ebov) causes a rapidly fatal hf for which there is currently no treatment (muyembe-tamfum et al., ; team et al., ) . ebov is a member of the filoviridae family, which are filamentous, negative-stranded rna viruses that cause severe human disease. filoviruses viruses are variable, with long filaments measuring nm in diameter and which can reach lengths of up to nm, with many turns and branches and which have tendency to curve to resemble the number . viruses are composed of nucleocapsid, matrix, and envelope proteins, whereby seven genes encode np, the viral proteins vp -vp -vp -vp , l (polymerase), and the gp (hoenen et al., ) , expressed as gp and gp , and regulating virus production and release (mohan et al., ) . nps embed the genome in complex with vp and vp for rna synthesis. vp and vp proteins are localized in virus matrix space (watanabe et al., ; hoenen et al., ) . ebov is transmitted to humans via mucosal surfaces, skin injury, and vertical transmission (feldmann and geisbert, ) , and with the exception of t cells, can infect almost all human cells using various different attachment mechanisms, reviewed in falasca et al. ( ) . both innate and adaptive immune responses are involved in ebov pathogenesis, where innate immune deregulation involves inhibition of type-i ifn response and perturbation of cytokine signaling, along with impairment of dc and nk cells, and adaptive immune deregulation involves both humoral and cell-mediated immunity (falasca et al., ) . because high levels of ebov replication are associated to multiple cell types, its associated systemic dissemination results in a highly complex pathogenesis model, including detrimental immune suppression and hyperactivation, and leading to disordered coagulation and tissue damage, that, in the absence of treatment, results in rapid multiple organ failure and death within days of symptomatic infection (baseler et al., ) . for years, ebov and related filoviruses have been repeatedly re-emerging to cause large epidemics of highly fatal hf. ongoing ebov outbreaks in africa has brought this virus to the forefront of research, with over , reported cases of infection and an associated deaths (mcelroy et al., ) . natural serologic response to ebov infection involves virus-specific igm and igg antibody responses sometimes detected early, but usually later, once symptoms begin rowe et al., ) . ebovinfected dcs are impaired in cytokine production required for t cell activation (mahanty et al., ) , whereas infected macrophages are unable to mature (bosio et al., ) . ebov is classified as an immunosuppressive virus since numerous of its proteins interfere with immune responses by inducing t cell apoptosis, lymphopenia, and absence of antibody responses in fatal cases (basler and amarasinghe, ). classification of ebov-targeting mechanisms has been compromised by lack of infrastructure for adequate biosafety containment level facilities required to analyze this deadly virus. however, observations of the adaptive t cell immune response have shown that ebov correlates with fatal outcomes by causing aberrant cytokine responses (baize et al., (baize et al., , wauquier et al., ; villinger et al., ; ansari, ) , decreased cd þ and cd þ t cells, and increased apoptotic t cell phenotypes (baize et al., ; wauquier et al., ; geisbert et al., ; bradfute et al., ; gupta et al., ) . in recent work, ebov induced increased cd þ and cd þ t cell activation against the viral np, with cd þ t cells demonstrating the largest increases in expression of activation and proliferation biomarkers, with sustained activation following ebov clearance and following patient discharge, suggesting continued antigen stimulation after resolution of the disease (mcelroy et al., ) . recently, an rvsv-zebov recombinant, replicationecompetent vesicular stomatitis virusebased vaccine expressing a surface gp of zaire ebolavirus, demonstrate a % efficacy in preventing ebov disease in contacts and contacts of contacts of recently confirmed cases in guinea, west africa (henao-restrepo, # ). this vaccine produced rapid innate immune responses after a single dose, suggested to lead to longerterm full protection by providing an essential period of restricted virus replication during the development of specific adaptive responses (marzi, # ) . crimean-congo hemorrhagic fever virus (cchfv) is a tick-borne virus causing hf resulting in human fatalities. cchfv is a member of the nairoviridae family of viruses from the genus of orthonairovirus and the order of the bunyaviridae viruses. it has a single-stranded, negative-sense rna genome possessing three segments: the large (l), medium (m), and small (s) segments (casals, ; clerx et al., ) . the l segment encodes the viral rna-dependent rna polymerase responsible for mrna synthesis and rna replication (honig et al., ) . the m-segment encodes numerous ns and two structural gps (gn and gc) responsible for cell tropism and attachment and are targets for neutralizing antibodies. the s-segment encodes the viral np binding the rna segments toward formation of ribonucleoprotein complexes (altamura et al., ; sanchez et al., ) . though hf by cchfv infection in humans is not among the most common viral disorders reported, it remains important because it is fatal in up to % of cases (bente et al., ; goedhals et al., ) . transmission of cchfv to humans occurs through contact with infected animal blood, or ticks, belonging to the genus hyalomma, as its primary vectors and providing transit from one infected human to another (mousavi-jazi et al., ) . cchfv human infection involves sudden onset of acute symptoms, including high fever, headache, myalgia, and petechial rash, followed by hemorrhage progressing to multiorgan failure, with leukopenia, thrombocytopenia, and elevated liver enzymes as hallmarks of the overall disorder (begum et al., ; sanchez et al., ) . outbreak-associated fatality rates are varied but can reach % (mousavi-jazi et al., ) . there is currently no licensed vaccine, and use of ribavirin as treatment has been investigated but remains controversial (begum et al., ; bente et al., ) . distribution of cchfv infection and associated disease follows geographical spread of the principal vectors (bente et al., ; whitehouse, ) . clinical cchfv disorders are described in africa, asia, the middle and east eastern europe, and have recently emerged in other countries including turkey, india, spain, and greece, and with almost , cases reported in turkey between and (maltezou et al., ; papa et al., ; leblebicioglu et al., ) . typically, transient igm and igg antibody responses develop within days following primary cchfv infection and can persist long-term (shepherd et al., ; burt et al., ) , but where lack thereof usually results in fatality (shepherd et al., ) . igm and igg antibodies have however not been correlated with clearance, viral load, or outcomes (duh et al., ) , implying that innate and t cell immunity must be critical for viral clearance. neutralizing antibodies also do not cause protection, and nonneutralizing antibodies may assist in antibody-dependent cell-mediated cytotoxicity (bertolotti-ciarlet et al., ) . thus, as immune correlates of protection for cchfv are not well documented, vaccine design has aimed at targeting the cchfv np or gps. only an inactivated vaccine is available, and even though the attaining of immunogenicity has required its administration in multiple doses, it was demonstrated to reduce infections and induce both neutralizing antibody responses and t cell responses to np peptides (mousavi-jazi et al., ) . another promising approach is the use of modified vacv ankara recombinant vaccine expressing the viral gps, which induce cellular and humoral responses and which are observed to provide protection from lethal disease in mice (buttigieg et al., ; dowall et al., ) . although t cell responses are known to play a role in protection from and clearance of viral infections, it was only recently that specific cd þ t cell epitopes against gn and gc were shown to stimulate ifn-g production, whereby responses were detectable several years after the acute cchfv infection, even in the absence of continued antigenic stimulation, and where ifn-g-producing cd þ t cells were confirmed as responsible for providing long-term protection (goedhals et al., ) . vhfs causes high fatality rates and are commonly correlated with ineffective immunity and high viral loads. yfv is important for both historical and current reasons. historically, it crippled economies and families by killing hundreds of thousands over years. yfv is transmitted by mosquitos in urban areas, causing as many as , infections and , deaths annually. like other mosquito vector viruses, yfv is an existing and re-emerging threat due to increased geological spread by both world travelers and shifting mosquito breeding geographies. flaviviruses evade immunity to enter the brain and spinal cord via circulating blood, crossing the bbb via "trojan horse" mechanism. dissemination of infection and protection against yfv is elicited by ifn-signaling, complement system, and other innate, humoral, and adaptive immune responses. neutralizing antibodies are still the gold standard correlates of protection, but evidence that a robust, long-lived, and polyfunctional adaptive cd þ and cd þ t cell immune response is what is provided, early after vaccination by the million doses of effective yf d vaccine distributed globally. importantly, these are nonexhausted, polyfunctional central memory and effector memory t cell subsets that are detected for over years following vaccination. far more serious is dhf by infection by denv, contributing to either mild or severe hf, with mortality rates exceeding % in the untreated. as with yfv, denv uses mosquitos adapted to urban areas as their vectors. fifty million cases of df and , cases of dhf occur globally each year, with no available cures for its associated disorders and where vaccine development has been complicated by antibody-dependent vaccineinduced enhancement of future heterotypic infections. cytotoxic cd þ and cd þ t cells, however, vigorously and frequently recognize denv proteins and are believed to contribute protection against primary infection. the denv vaccine dengvaxia has been recently withdrawn, with its failure posited to result from its lack of denv proteins specifically targeted by cd þ t cells, and demonstrates that is essential to accurately assess t cell responses in the context of denv development. lasv also causes mild and severe vhf and may cause , infections and up to deaths each year. lasv is transmitted to humans by a rodent vector and first targets apc, dc, and macrophages, contributing to rapid multisystem and organ lasv dissemination due to their widespread physiological distributions. lasv also causes lymphopenia of cd þ and cd þ t cells, nk cells, and b cells, and survivors of lasv-related disorders can be expected to maintain lifelong morbidities. there is no licensed lasv vaccine as of yet, complicated by the fact that the very cells responsible for stimulating t cell antigen responses are those infected by the virus. however, control of acute lf is correlated with increased circulating strong memory cd þ and cd þ t cells in response to lasv infection or rechallenge and lasv antigen. ebov also causes mass hf. it can infect almost all human cells with exception of lymphocytes and has no known treatment despite documented involvement of innate and adaptive immune responses. classification of ebov targeting mechanisms are compromised by lack of infrastructure for adequate biosafety containment level facilities. ebov causes dcs to be impaired in cytokine production for t cells activation and inhibits macrophages maturation, thus classifying it as an immunosuppressive virus, with fatalities correlating with its induction of lymphopenia the absence of antibody responses. ebov, however, causes increased cd þ and cd þ t cell activation, with recorded persistence of activated cd þ t cells in survivors. the knowledge that cd þ and cd þ t cell responses are against the ebov np will be useful for the design of efficient targeting vaccines. cchfv also causes hf yielding fatalities in up to % of cases. there is no current vaccine, and cchfv has recently reemerged in naïve countries in response to geographical relocation of its primary tick vector. infection by cchfv causes transient ig antibody responses, inversely correlating with sure fatality, but not correlating with clearance, viral load, or outcomes. no protection is granted by neutralizing antibodies either, implying that innate and t cell arms of immunity are critical for viral clearance. with little to no immune correlates of protection for cchfv, vaccine design has aimed at targeting the cchfv np or gps, where immunogenicity requires the administration of multiple doses to induce both neutralizing antibody responses and t cell responses. only recently, specific cd þ t cell epitopes against these cchfv proteins have been confirmed to stimulate cytotoxic cd þ t cells providing protection in survivors. despite many documented instances of virus eradication from populations by earlier vaccination strategies, there is a continuous imminent risk of outbreaks of pandemic proportions by existing and emerging viruses, as a result of anti-vaccination campaigns, unforeseen viral strain recombination, exposure of naïve populations to viruses by infected world travellers, a rapidly growing and aging and immunocompromised world population, acts of bioterrorism, and use of viruses as biological weapons in war. past strategies in the development of certain vaccines have often been lucky in their efficacies due to their eliciting both humoral and long-lasting adaptive t cell immunity, whereas others have failed and have only induced shorter lived humoral responses that do not always confer long-lasting protection. the precise and likely overlapping mechanisms dictating lifelong immunity conferred by successful vaccines against smallpox, measles, mumps, rubella, polio, and yf are not completely understood. what has become clear however is that innate immunity usually primes adaptive immunity to confer this long-term protection. it is not a lack of existing immune-monitoring methodologies but rather perhaps a lack of their correct implementation in the continued analysis of vaccinated individuals that are hampering the gaining of this important knowledge. therefore it seems there is an urgent need for the standardization of vaccine methodologies adequately measuring effector memory t cell responses and host-immune evasion mechanisms in appropriate animal models and humans and through the use of multiparametric immune-monitoring platforms able to simultaneously document numerous immune cell phenotypes across time postexposure, vaccination, or rechallenge. the importance of global development of standardized procedures of biospecimen banking from vaccinated individuals cannot be understated and will provide 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protection from secondary dengue virus infection in a mouse model reveals the role of serotype cross-reactive b and t cells key: cord- - hzovg authors: galani, i. e.; rovina, n.; lampropoulou, v.; triantafyllia, v.; manioudaki, m.; pavlos, e.; koukaki, e.; fragkou, p. c.; panou, v.; rapti, v.; koltsida, o.; mentis, a.; koulouris, n.; tsiodras, s.; koutsoukou, a.; andreakos, e. title: untuned antiviral immunity in covid- revealed by temporal type i/iii interferon patterns and flu comparison date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: hzovg a central paradigm of immunity is that interferon (ifn) mediated antiviral responses precede the pro-inflammatory ones, optimizing host protection and minimizing collateral damage. here, we report that for covid- this does not apply. by investigating temporal ifn and inflammatory cytokine patterns in covid- patients hospitalized for pneumonia and longitudinally followed for the development of respiratory failure and death, we reveal that ifn-{lambda} and type i ifn production is both diminished and delayed, induced only in a fraction of patients as they become critically ill. on the contrary, pro-inflammatory cytokines such as tnf, il- and il- are produced before ifns, in all patients, and persist for a prolonged time. by comparison, in flu patients hospitalized for pneumonia with similar clinicopathological characteristics to covid- and milder non-hospitalized flu patients ifn-{lambda} and type i ifn are robustly induced, earlier, at higher levels and independently of disease severity, while pro-inflammatory cytokines are only acutely and transiently produced. notably, higher ifn-{lambda} levels in covid- patients correlate with lower viral load in bronchial aspirates and faster viral clearance, and a higher ifn-{lambda}:type i ifn ratio with improved outcome of critically ill patients. moreover, altered cytokine patterns in covid- patients correlate with longer hospitalization time and higher incidence of critical disease and mortality compared to flu. these data point to an untuned antiviral response in covid- contributing to persistent viral presence, hyperinflammation and respiratory failure. disease severity, while pro-inflammatory cytokines are only acutely and transiently produced. notably, higher ifn- levels in covid- patients correlate with lower viral load in bronchial aspirates and faster viral clearance, and a higher ifn-:type i ifn ratio with improved outcome of critically ill patients. moreover, altered cytokine patterns in covid- patients correlate with longer hospitalization time and higher incidence of critical disease and mortality compared to flu. these data point to an untuned antiviral response in covid- contributing to persistent viral presence, hyperinflammation and respiratory failure. of the worst pandemics of our time, causing high incidence of pneumonia, acute respiratory distress syndrome (ards) and death , . one of the most notable features of sars-cov infection is that it goes unnoticed for a remarkably prolonged period of time, running a course of a mild or uncomplicated illness for weeks until sudden and severe symptoms develop, in a subgroup of patients, requiring hospitalization, oxygen support and/or admission to an intensive care unit (icu) , . this is consistent with an unusually long incubation period of the virus, ranging from to days, and an unusually long presence of it in the respiratory tract, often being detectable for over a month after initial infection by conventional molecular diagnostic tests , . by comparison, influenza virus infection, the main respiratory virus accounting for pneumonia hospitalizations till now, has an incubation time of to days, a short window of virus positivity of a few days, and an abrupt onset of symptoms causing pneumonia within - days , . other frequent respiratory viruses such as respiratory syncytial viruses, rhinoviruses, parainfluenza viruses, metapneumonoviruses and regular human coronaviruses have also shorter incubation times (ranging from - days) and more rapid and acute manifestation of symptoms , rendering sars-cov quite unique in that respect. the basis of this is unknown but is likely to be a key driver of the pathophysiology of covid- underlying its distinctive disease course and clinical manifestations. the hallmark of covid- is the development of a hyper-inflammatory response, also known as 'cytokine storm', impairing the gas-exchange function and leading to acute respiratory distress syndrome (ards), multi-organ failure and death - . we and others have previously shown that a finely tuned antiviral response, orchestrated by ifn- (type iii ifn) and type i ifn is critical for balancing immunity for optimal protection and minimal damage [ ] [ ] [ ] . deviation from this can unleash a detrimental 'cytokine storm' with devastating consequences for human health. a recent study suggested that in covid- patients type i ifn and ifn- are not produced as they could not be detected in the sera of a small covid- cohort of otherwise unspecified clinical characteristics . in contrast, another one reported that type i ifn is induced in covid- patients, and indicated that their levels might be reduced in those that are critically ill . such discrepancy could be due to the fact that each of these studies focuses on a single and likely distinct snapshot of an apparently heterogeneous disease process. therefore, pursuing kinetic analyses is pertinent to delineating the course of the immune response, especially given that cytokines are transiently produced . this is particularly true for ifns which are expressed early during infection and are rapidly down-regulated thereafter. here, we have performed a comprehensive temporal analysis of type i and type iii ifn, and major inflammatory cytokine patterns in covid- and influenza a virus infected (flu) patients hospitalized for community acquired pneumonia and longitudinally followed up according to current who guidelines . both groups of patients exhibited similar clinicopathological characteristics and comparable disease severity on admission (table ) . we have also analyzed milder flu patients with no radiological findings of pneumonia and no need for hospitalization (referred to as mild flu; table ), as well as healthy individuals. using high sensitivity luminex and elisa assays, we quantified cytokines and chemokines relevant to antiviral immunity and hyperinflammation in patient sera collected at defined time intervals following hospital admission ( fig. a and s aa). this aligns patients on the basis of the same clinical criteria of disease symptoms and severity, mainly the presence of pneumonia and the requirement for oxygen support. we found that covid- patients had profoundly impaired induction of both ifn- and type i ifns. median levels of ifn- and type i ifns were not detectable in most covid- patients (fig. b interestingly, despite their limited ability to make ifns, covid- patients robustly expressed pro- inflammatory cytokines such as tnf, il- , il- , il- , il- , ifn- and ccl that were maintained at high levels for a prolonged time (fig. b) . other cytokines such as il- , il- , il- and ccl were also significantly up-regulated at specific time intervals compared to healthy individuals reflecting the heterogeneity of the disease course (fig. s ) . a similar pattern emerged when comparisons were made according to disease symptoms onset (fig. s b). covid- patients exhibited markedly delayed and reduced ifn- and type i ifn levels which were detectable only in a fraction of the patients and from days - onwards of symptoms onset ( fig. s , a-b). by comparison, all flu patients exhibited high levels during the first days ( fig. s , a-b). although covid- patients made little ifn during the first days of symptom onset, they potently produced pro-inflammatory cytokines and chemokines such as tnf, il- , il- , il- and ccl at levels similar to flu (fig. s , b-c). moreover, they exhibited prolonged expression of pro-inflammatory mediators, with high levels of tnf, il- , il- , il- , il- and ccl remaining detectable for over three weeks of onset, whereas in flu patients a number of these were by that time down-regulated (fig. s ). notably, covid- patients were admitted to hospital with similar markers of systemic inflammation such as crp levels, white blood cell (wbc) and neutrophil counts, and neutrophil/lymphocyte (n/l) ratio to flu patients (table and fig. s , a-f). they even had lower fever and a lower curb- score, a commonly used measure of pneumonia severity (fig. s , g-h). however, during follow up covid- patients developed a much higher incidence of ards requiring icu support. in our cohort, out of patients ( %) developed critical disease, of which died, compared to only out of flu patients ( . %) none of which died (fig. , a-b) . strikingly, covid- patients became critically ill over a much broader time window (up to nine days after admission) than flu patients which manifested critical disease within the first day post admission. this is in agreement with the high incidence and protracted all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint course of severe respiratory failure described for covid- , . interestingly, among covid- patients those who became critically ill had higher crp levels, wbc and neutrophil counts, and n/l ratio on admission (fig. s , a-f), but not curb- or fever (fig. s , g-h and table s ). critically ill flu patients also had a tendency for higher wbc and neutrophil counts and a n/l ratio, as well as significantly raised curb- , whereas non-hospitalized flu patients did not exhibit any of these increases (fig s , a- h). we thus examined whether temporal cytokine patterns differ between the various patient groups. surprisingly, we observed that although covid- patients that do not become critically ill produce little type i or iii ifn, the ones that become critically ill make ifn- which are significantly higher at the day - time interval compared to healthy and non-critically ill patients ( fig. c and s ). some of the critically ill patients also make ifn-α ( fig. d and s ), albeit at significantly lower levels to mild non- hospitalized flu patients (fig. d ) or the total of hospitalized flu patients (both critically and non- critically ill; p< . ). on the contrary, all covid- patients make pro-inflammatory cytokines such as tnf, il- , il- , il- and ifn-, with critically ill patients exhibiting also higher levels of il- , il- and tnf at specific time intervals, and a tendency for higher ifn- consistent with the increased hyper- inflammatory state they are in (fig. e , s and s ). individual patient data further confirmed these trends (fig. s ). interestingly, ccl is significantly higher than healthy controls in non-critically ill covid- patients but not in the critically ill ones (fig. s ). by comparison, critically ill and non- critically ill flu patients did not differ in their ability to make type i and type iii ifns nor pro- inflammatory cytokines such as tnf, il- or il- (fig. , c-e, s and s ). similarly, non-hospitalized flu patients with mild disease exhibited strong production of type i and type iii ifns, indicating that across the spectrum of flu disease severity the antiviral response remains robust. visualizing these patterns on radar plot reveals a major imbalance in the induction of antiviral and pro-inflammatory responses of covid- patients that does not occur in flu (fig. e) . we next sought to determine whether imbalanced cytokine patterns in covid- patients are related to systemic immune effects, and parameters linked to disease severity. to that end, we obtained temporal white blood cell transcriptomes from healthy individuals and covid- patients, non- critically and critically ill, starting from day of entry to the ward or icu and at different timepoints thereafter. in total, comprehensive rnaseq gene expression datasets were analyzed, clustering according to the clinical phenotype and indicating this as the main source of variation ( fig. a and s a). focusing at day as the most relevant timepoint, we found that genes were differentially expressed (degs) in covid- patients compared to healthy individuals (table s ) . when critically and non-critically ill patients were compared separately to healthy controls, and degs were observed, respectively, of which were common whereas the rest were uniquely found in one or the other patient group (fig. s b , table s and table s ). volcano plots pointed out notable differences in the most highly regulated genes between the groups with critically ill patients exhibiting a stronger immune and antiviral response gene patterns (fig. s , c-e). pathway analysis of degs indeed revealed that the most significant pathways over-represented in critically ill patients were related to the positive regulation of the immune system, the activation of the innate immune response, the defense response to virus and the cellular response to ifn ( fig. b and table s ). by contrast, in non- critically ill patients these pathways were not significant; the ones over-represented instead included the regulation of the cellular component size, il-  production and nk cell cytotoxicity (fig. b) . all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . fcgr a) in the critically ill group that was milder and not significant in non-critically ill patients (fig. , c-d). on the contrary, t and b lymphocyte lineage and related genes (cd d, cd e, cd , cd a, cd , cd ) were markedly down-regulated in critically ill patients. these data are consistent with lymphopenia, high neutrophil counts, and a high n/l ratio also present in these patients (fig. s ) and previously reported to be associated with more severe disease and worse outcomes in covid- patients , . cytokines such as tnf, il- and il- may directly account for these effects, as they are well known to trigger the mobilization and activation of neutrophils, the development of lymphopenia and the induction of innate immune responses and systemic inflammation , . notably, a long set of ifn- stimulated genes (isgs) was also strongly induced in critically ill patients compared to only a fraction of them being up-regulated in the non-critically ill group (fig e and s f ), in agreement with the patterns of ifn- and type i ifn production in these patients. this cannot be attributed to differential expression of ifn receptors as no differences between ifnlr , il rb, ifnar and ifnar mrna levels were observed among patient groups and healthy individuals with the exception of a -fold up- regulation of the already high levels of ifnar in critically ill patients (table s ) . interestingly, imbalanced cytokine patterns in covid- patients with pneumonia were associated with a much worse disease outcome compared to flu. first, the covid- group exhibited higher incidence of critical disease and mortality (fig. b) . second, covid- patients overall, as well as when grouped as critically and non-critically ill, required longer hospitalization time than their flu counterparts (fig. , a-c). for non-critically and critically ill covid- patients, median time was and days, respectively, compared to flu that was and days (fig. , b-c). prolonged hospitalization could be attributed to the untuned antiviral responses, leading to a more protracted clinical course of covid- relative to flu and a need for longer recovery even for the non-critically ill group. to identify cytokines and cytokine combinations that can predict hospitalization time and therefore be of prognostic value, we generated a correlation matrix of the cytokine levels at admission (days - interval) and the duration of hospital stay (fig. d ). we found that higher il- and il- , and lower ccl levels, are directly proportional to the duration of hospitalization (fig. , d-f). the value of tnf and il- as biomarkers for monitoring covid- severity has been reported , , but for ccl this is new. interestingly, ifn- levels also correlated with higher il- , and longer hospitalization, consistent with their almost exclusive induction in critically but not non-critically ill patients ( fig. d and c) . a question that arises is whether ifn levels induced in critically ill patients are beneficial as delayed type i or type iii ifn production has been shown in animal models to cause immunopathology , , or interfere with epithelial repair , , respectively. we found that higher ifn- concentrations during icu entry were associated with lower sars-cov viral load in the respiratory tract and faster viral clearance (fig. , g-h) . moreover, a higher ifn- to type i ifn ratio at that time was linked to a shorter stay in the icu (fig. i) , with the two patients with the highest ifn- levels also exhibiting the longest stay (both days over a median of days). these data suggest that delayed ifn- induction may still be protective in critically ill covid- patients whereas ifn- may do more harm than good, at least in a subset of patients. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint taken together, our findings demonstrate that sars-cov infection does not follow the conventional paradigm of antiviral immunity. instead of activating first the antiviral response followed by the pro- inflammatory process as a second line of protection, it does the opposite; it triggers the pro- inflammatory response long before ifn-mediated antiviral defenses are induced-if at all. this is a major paradox and helps explain many of the unique or unusual features of covid- . the long virus incubation time and persistence in the respiratory tract, giving positive sars-cov tests for weeks, can be attributed to the delayed and/or reduced production of type i and iii ifns. the absent or very mild symptoms of patients for an unusually extended period of time, can be attributed to the lack or impaired and delayed expression of type i ifns, principal mediators of flu-like disease and symptoms such as runny nose, coughing, fatigue, dyspnea and fever in humans . finally, the early and persistent expression of pro-inflammatory cytokines culminating into prolonged hyper-inflammation can promote the sudden development of respiratory failure requiring hospitalization and frequently icu admission. noteworthy, in flu the swift induction of the type i and iii ifn response, across the spectrum of disease severity, correlates with quicker recovery, and markedly lower incidence of critical disease or mortality , . the recent demonstration in a retrospective cohort study of covid- patients that early administration of ifn- (interferon-a b) is linked to reduced in-hospital mortality whereas late ifn- therapy leads to increased mortality and delayed recovery leaves little doubt that the timing of ifn production is also crucial in covid- patients . conceivably, late production of type i or iii ifn production might confer no viral resistance, but instead promote immunopathology. whether this unique clinical course of covid- is related to the presence of sars-cov -derived ifn inhibitors as previously proposed for sars-cov , and mers-cov is not known but is a possibility. as with other viruses, inhibition may be overcome once higher viral loads are reached, e.g. after incubation of the virus and eventual spread in susceptible individuals. in our study, we did not see significant differences in virus levels between non-critically and critically ill patients at the time ifns were measured (fig. s ) . however, higher virus load in severe over mild disease has been described in one study but not been confirmed in another , . moreover, higher virus load can overcome sars- cov dose-dependent suppression of ifn production in cultured respiratory epithelial cells . our study is not without caveats. first, it characterizes cytokine patterns in the circulation, and although these are commonly used to analyze 'cytokine storms' in response to infection, how well they correlate to immune responses in the respiratory tract is difficult to know. second, it is relatively small, and our findings await validation in other cohorts. still, our study is uniquely informative as it addresses the production of ifns and the activation of the 'cytokine storm' in covid- in a temporal manner, from hospital admission to icu entry, and should therefore be particularly useful for the design of clinical trials testing ifn therapies. finally, it provides a side-by-side comparison of covid- with flu, studying patient populations with similar genetic, demographic and clinicopathological characteristics, and therefore uncovers important differences in the antiviral immune response between these two diseases that have not been previously suspected. study participants in this non-interventional study thirty two patients with diagnosis of covid- pneumonia according to who guidelines and positive sars-cov- reverse transcription polymerase chain reaction (rt-pcr) all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . covid- patients upon admission (table ). all subjects included in the study were clinically evaluated and followed longitudinally during the whole period of hospitalization (from admission to discharge). all blood specimens were processed immediately for serum collection and aliquots were stored at - o c. the study conforms to the principles outlined in the declaration of helsinki, and received approval by perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . data were analyzed on graphpad prism software. statistical significance of differences was assessed using the mann-whitney u (mww) test for non-parametric data. associations between cytokines and hospitalization time (in days) were tested using spearman rank-order correlation coefficient and all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint time intervals. for flu, n= , and , respectively. for healthy, n= . grey shading marks the limit of quantification of the assay. p values were determined by a two tailed mann-whitney u test for non- parametric comparisons. *p < . , **p < . and ***p < . show significance over healthy controls. # p < . , ## p < . and ### p < . show significance between covid- and flu groups. shown in the healthy control. for days - n= , , , and for each of the five consecutive groups, respectively. for days - n= , , , and , respectively. for healthy individuals, n= . p values were determined by a two tailed mann-whitney u test for non-parametric comparisons. *p < . , **p < . and ***p < . show significance over healthy controls. # p < . , ## p < . and ### p < . show significance between covid- and flu subgroups. correlation of ifn- concentration levels with time required for viral clearance assessed as the first - all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in correlation coefficient for non-parametric data. *p < . , **p < . and ***p < . all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . ifi socs gbp p siglec ifi l oas oas eif ak ifit ifi ly e rsad ifit ifi cmpk oas oasl mx batf gbp irf irf innate immune viruses and interferons type i interferons in infectious disease clinical characteristics of coronavirus disease in china clinical features of patients infected with novel coronavirus in wuhan, china virological assessment of hospitalized patients with covid- viral load dynamics and disease severity in patients infected with sars-cov- in zhejiang province, china the pathology of influenza virus infections incubation periods of acute respiratory viral infections: a systematic review covid- : consider cytokine storm syndromes and immunosuppression risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, 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coronavirus pneumonia (trial version detection of novel coronavirus ( -ncov) by real-time rt-pcr moderated estimation of fold change and dispersion for rna-seq data with deseq key: cord- -wl d gs authors: morgenstern, birgit; michaelis, martin; baer, patrick c.; doerr, hans w.; cinatl, jindrich title: ribavirin and interferon-β synergistically inhibit sars-associated coronavirus replication in animal and human cell lines date: - - journal: biochemical and biophysical research communications doi: . /j.bbrc. . . sha: doc_id: cord_uid: wl d gs abstract initial in vitro investigations demonstrated type i interferons (ifns: ifn-α, ifn-β) to inhibit replication of sars coronavirus (sars-cov), but found the nucleoside analogue ribavirin ineffective in vero cells. in this report, ribavirin was shown to inhibit sars-cov replication in five different cell types of animal or human origin at therapeutically achievable concentrations. since clinical anti-sars-cov activity of type i interferons or ribavirin is limited, we investigated the combination of ifn-β and ribavirin. determination of the virus yield indicated highly synergistic anti-sars-cov action of the combination suggesting the consideration of ribavirin plus ifn-β for the treatment of sars. from the end of a new form of non-typical pneumonia, the severe acute respiratory syndrome (sars), emerged from the guangdong province in southern china, spread throughout the world, infected people in countries, and resulted in more than deaths (http://www.who.int/csr/sars). the causative agent was found to be a new member of the cororonavirus family, the sars-coronavirus (sars-cov) [ ] . corticosteroids, antibiotics, and antiviral agents were empirically used for the treatment of sars. the rapid emergence of the epidemic did not permit the conduction of controlled studies documenting the efficacy of therapies. ribavirin ( -b-d d-ribofuranosyl- , , -triazole- -carboxamide), a synthetic guanosine analogue and broad-spectrum inhibitor of rna and dna viruses, was frequently used for treatment of sars patients [ ] . however, reports about treatment success are con-troversial and therapy-limiting side effects have been reported in sars patients [ ] . in first studies, ribavirin did not inhibit sars-cov replication in vero (african green monkey kidney) cells [ , ] . newer experiments demonstrated anti-sars-cov activity of ribavirin in foetal rhesus kidney cells (frhk- ) in concentrations of about lg/ml [ , ] being still above mean plasma levels which are in the range of lg/ml after i.v. administration of mg ribavirin [ ] . to date, no systematic investigation of the effect of ribavirin on sars-cov replication in human cells lines had been performed. in numerous in vitro studies effects of interferons (ifns) on sars-cov replication had been observed [ ] . our investigations performed in caco -(human colon carcinoma) cells and vero cells demonstrated type i ifns (ifn-a, ifn-b) to have superior anti-sars-cov activity compared to that of type ii ifn-c. although ifn-b- b (betaferon) was shown to inhibit sars-cov replication more effectively compared to ifn-a- b (intron a) [ ] , some other ifn-a subtypes and human leukocyte ifn-a were highly effective [ , ] . preliminary clinical studies showed treatment with synthetic recombinant type i ifn-a (alfacon- ) to be beneficial for sars patients [ ] . clinical evaluation of the combination of type i ifns including ifn-b with ribavirin in patients with chronic hepatitis c demonstrated the superiority of the combination to either single treatment and resulted in decreased pharmacological active plasma levels for either drug [ ] . here, we tested the anti-sars-cov activity of the combination of ribavirin with ifn-b in different human and animal cells. vero, ma- , pk- , and caco cells were grown at °c in mem supplemented with % fetal bovine serum (fbs) containing iu/ml penicillin and lg/ml streptomycin. cl- cells were grown in °c in hamÕs f supplemented with % fbs containing iu/ml penicillin and lg/ml streptomycin. human primary epithelial kidney (hpek) cells were prepared from human renal tissue after nephrectomy from portions of the kidney (carcinoma free) and immunomagnetically isolated as described before and grown in medium with % fbs at °c [ ] . sars-cov strain ffm [ ] and strain (courtesy of prof. wilina lim, government virus unit, hong-kong) were prepared by infecting vero cell cultures. supernatants from infected cultures were collected days post-infection and aliquots were stored at À °c. virus titres were determined by % tissue culture infective dose (tcid ) in confluent cells in -well microtitre plates as described [ ] . antiviral assay. confluent cell cultures were infected with sarscoronavirus strain ffm or strain , for h in -well microplates. after adsorption period, cells were washed with pbs and incubated in mem supplemented with % fbs. cytopathogenic effect (cpe) was assessed visually h after infection. in some experiments virus yield reduction assay was performed as described before [ ] . confluent cell layers grown in . cm cell culture flasks were infected with sars-cov strain ffm or . after h incubation period, cells were washed four times with pbs and incubated ( °c) in mem supplemented with % fbs. after h cultures and supernatants were freeze-thawed and viral titres were determined by the % tissue culture infective dose (tcid ) in confluent vero cells on -well mictrotitre plates [ ] . the inhibitory effects were expressed as effective concentrations of compounds required to inhibit infectious virus titres by % (ec ), % (ec ) or % (ec ) of the control value, respectively. combination studies. combination indices (cis) indicating whether both substances acted synergistically, additively, or antagonistically when used in combination were calculated using calcusyn for windows. the calculation is based on the method of chou and talalay [ ] . a ci lower indicates synergistic action, a ci higher indicates antagonistic action, and a ci of indicates additive action. cells were inoculated with sars-cov strain ffm at a moi of . . maximum virus titres were . · tcid /ml in caco , . · tcid /ml in cl , . · tcid /ml in hpek cells, . · in ma cells, and . · in pk- cells. infection of caco and cl with the sars-cov strain resulted in infectious virus titres of . · and . · tcid /ml, respectively, which is similar to titres determined for strain ffm . cpe, detected h post-infection with ffm or strain, was characterised by considerable morphological changes such as numerous rounded, enlarged, and detached cells (representative photograph shown in fig. for caco cells). as shown before [ , ] ribavirin did not inhibit sars-cov-replication and cpe formation in vero cells at concentrations up to lg/ml. however, ribavirin inhibited sars-cov replication in other permissive cell lines tested (table ) . ec s ranging from . to . lg/ml, ec s ranging from . to . lg/ ml, and ec s ranging from . to . lg/ml were found in ffm -infected cells by determination of virus yield (moi . ). high concentrations of ribavirin ( lg/ml) completely suppressed cpe formation h p.i. in all cell lines infected with ffm or (moi . ). ribavirin inhibited sars-cov strain -replication similarly to strain ffm (not shown). the influence of the infectious dose on the anti-sars-cov activity of ribavirin was investigated in caco cells infected at different mois by measurement of virus titre. ec -values were . ± . lg/ml for moi . , . ± . lg/ml for moi . , and . ± . lg/ml for moi , showing no significant influence of the infectious dose on ribavirin-caused sars-cov replication inhibition. combinations of ifn-b and ribavirin were tested in a fixed ratio of : (iu/ml for ifn-b, lg/ml for ribavirin) starting with , iu/ml ifn-b and lg/ml ribavirin. starting concentrations were diluted in ratios of : . the combination synergistically inhibited sars-cov-induced cpe in caco and cl cells (representative photographs shown in fig. ) as well as production of infectious virus ( table ) . ec , ec , and ec of ribavirin, ifn-b or their combination in infected human intestinal cell lines caco and cl- were determined by measurement of infectious virus titre. for example, in caco cells infected with sars-cov ffm strain (moi . ), ribavirin concentrations inhibiting virus production in combination with ifn-b were at least -fold lower when compared with cultures receiving single treatment with ribavirin. inhibitory concentrations of ifn-b were -to -fold decreased in combination relative to single treatment with ifn-b. calculation of cis revealed highly synergistic antiviral effects of the drug combination. in ffm -infected caco cells the ci values were . ± . at the ec , . ± . at the ec , and . ± . at the ec . in ffm -infected cl cells (moi . ) the ci values were . ± . at the ec , . ± . at the ec , and . ± . at the ec . ribavirin was initially found not to inhibit sars-cov replication at concentrations up to lg/ml [ , ] . these results had been obtained in vero cells (or its e subclone), in which ribavirin is known to be of low antiviral activity, most probably because of insufficient phosphorylation to its active triphosphorylated form. more recent studies showed that ribavirin inhibits sars-cov replication in frhk- cells at concentrations of about lg/ml [ , ] . these findings suggest that multiple cell culture systems should be used to evaluate the activity of antiviral agents against emerging viruses such as sars-cov. in the present study, the effects of ribavirin on sars-cov replication were systematically investigated in a panel of sars-cov permissive cell lines. vero cells, human colon carcinoma cell lines caco and cl , and pig kidney cell line pk- had already been described to be permissive to sars-cov infection by us and by others [ , , ] . african green monkey kidney cell line ma- and primary epithelial human kidney (hpek) cells were demonstrated to be permissive to sars-cov replication in this report. hpek cells represent the first normal diploid cell type of human origin permissive to sars-cov infection. ribavirin inhibited sars-cov replication in all newly tested cell lines at concentrations at least -fold lower when compared with vero cells. the clinical value of ribavirin for the treatment of sars patients is regarded with scepticism. we observed about % viral replication inhibition at concentrations up to lg/ml ribavirin which is at reasonable therapeutical plasma levels after an intravenous dose of mg ribavirin [ ] . this may be insufficient to improve clinical symptoms. in concordance, randomised clinical trials showed low dose ( - mg/day) ribavirin to be ineffective [ ] . on the other hand, the most recently published study reported that ribavirin reduced the viral load in of the patients [ ] . moreover, this study suggested that the peak inflammatory cytokine (il- and il- ) levels concurred with or after the peak viral load and preceded or concurred with the maximum pulmonary infiltrates. thus, it is probable that viral replication leads to the activation of proinflammatory cytokines that, together with other factors, contribute to disease progression. these clinical findings together with the observation of antiviral activity in different sars-cov-infected cell lines encourage the testing of treatment strategies using ribavirin in combination with other antiviral agents such as ifns to increase inhibitory effects on virus replication and subsequently minimised immunopathological damages. type i ifns had been shown to inhibit sars-cov replication in vitro most effectively in cells that had been pre-treated before virus inoculation [ ] . a pilot clinical report demonstrated the effectiveness of synthetic recombinant ifn-a for the treatment of sars patients [ ] . overall, these findings suggest that only high doses of ifns exhibit anti-sars effects and are mainly effective for prophylaxis during sars epidemics. to reduce the drug concentrations needed for sars-cov inhibition, the anti-sars-cov activity of the combination of ribavirin with type i interferon was tested previously. tan et al. [ ] reported that ribavirin in combination with ifn-b did not demonstrate observable synergistic effects whereas chen et al. [ ] showed highly synergistic activity. however, both studies reported solely on cpe formation in vero cells. our results demonstrate that both drugs act highly synergistically in combination on formation of cpe as well as on production of infectious virus titres in two human cell lines. since antiviral drugs were added after virus adsorption, the results show that ifn-b in combination with ribavirin may be highly effective not only as prophylactic agent but also for the treatment of already infected sars patients. in conclusion, the combination of ribavirin with ifnb inhibits sars-cov replication in drastically reduced concentrations compared to either single treatment. this may enable the achievement of therapeutic plasma levels sufficient to suppress sars-cov replication during the early phase of sars, and thus to prevent subsequent immunopathological damages. moreover, efficient early inhibition of virus replication may reduce virus shedding and consequently, the risk of transmission. identification of a novel coronavirus in patients with severe acute respiratory syndrome ribavirin in the treatment of sars: a new trick for an old drug? glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus severe acute respiratory syndrome related coronavirus is inhibited by interferon alpha in vitro susceptibility of clinical isolates of sars coronavirus to selected antiviral compounds hku/uch sars study group, role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings role of interferons in the treatment of severe acute respiratory syndrome treatment of sars with human interferons inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study safety of interferon beta treatment for chronic hcv hepatitis isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation quantitative analysis of dose effect relationship: the combined effects of multiple drugs or enzyme inhibitors retroviral vectors pseudotypes with acute respiratory syndrome coronavirus s protein severe acute respiratory syndrome: scientific and anecdotal evidence for drug treatment ntu) college of medicine/ntu hospital, temporal relationship of viral load, ribavirin, interleukin (il)- , il- , and clinical progression in patients with severe acute respiratory syndrome the authors thank gabriele bauer for technical assistance. key: cord- -c w bw u authors: lui, pak-yin; wong, lok-yin roy; fung, cheuk-lai; siu, kam-leung; yeung, man-lung; yuen, kit-san; chan, chi-ping; woo, patrick chiu-yat; yuen, kwok-yung; jin, dong-yan title: middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk -dependent phosphorylation of irf date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: c w bw u middle east respiratory syndrome coronavirus (mers-cov) infection has claimed hundreds of lives and has become a global threat since its emergence in saudi arabia in . the ability of mers-cov to evade the host innate antiviral response may contribute to its severe pathogenesis. many mers-cov-encoded proteins were identified to have interferon (ifn)-antagonizing properties, which correlates well with the reduced ifn levels observed in infected patients and ex vivo models. in this study, we fully characterized the ifn-antagonizing property of the mers-cov m protein. expression of mers-cov m protein suppressed type i ifn expression in response to sendai virus infection or poly(i:c) induction. this suppressive effect was found to be specific for the activation of ifn regulatory factor (irf ) but not nuclear factor-κb. mers-cov m protein interacted with traf and disrupted traf –tbk association leading to reduced irf activation. m proteins from mers-cov and sars-cov have three highly similar conserved n-terminal transmembrane domains and a c-terminal region. using chimeric and truncation mutants, the n-terminal transmembrane domains of the mers-cov m protein were found to be sufficient for its inhibitory effect on ifn expression, whereas the c-terminal domain was unable to induce this suppression. collectively, our findings suggest a common and conserved mechanism through which highly pathogenic mers-cov and sars-cov harness their m proteins to suppress type i ifn expression at the level of tbk -dependent phosphorylation and activation of irf resulting in evasion of the host innate antiviral response. middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in september as a novel coronavirus that causes severe acute respiratory disease. since then, this virus has caused recurrent outbreaks in the arabian peninsula and has spread, occasionally, to other parts of the world. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] according to the world health organization, laboratory-confirmed cases were reported between september and january with related deaths in countries. in particular, people were infected and were killed in one recent outbreak in south korea. mers-cov is classified into lineage c of betacoronavirus and is most phylogenetically related to two bat coronaviruses, hku and hku , providing insight on its evolutionary origin. , mers-cov is a polycistronic positive-sense single-stranded rna virus with a genome of~ kb in size. the ′ most two-thirds of mers-cov genome encodes polyproteins a and ab, which are further cleaved to yield non-structural proteins, whereas the ′ end of the genome encodes several structural or lineage-specific proteins. upon infection, these proteins are expressed to facilitate viral replication and propagation in the host. mers-cov infection has been widely reported to mildly induce type i interferons (ifns), including ifn-α and -β, in patients as well as in animal and cellular infection models. [ ] [ ] [ ] [ ] [ ] [ ] [ ] this has been attributed to the ifnantagonizing property of some mers-cov-encoded proteins, which directly perturb the host ifn production mechanisms, [ ] [ ] [ ] [ ] [ ] lending support to the notion that mers-cov uses multiple strategies to evade the innate immune response. in non-specialized epithelial cells as well as a subset of specialized immune cells that are susceptible to mers-cov infection, , , type i ifn production is an important part of the host innate immune response and is initiated by ubiquitously expressed cytoplasmic viral sensors in the retinoic acid-inducible gene-i (rig-i)-like receptor (rlr) family in response to the detection of viral pathogen-associated molecular patterns such as double-stranded rna (dsrna). , stimulated rlrs mobilize downstream signal transducers that lead to the activation of the transcription factors ifn regulatory factor (irf ) and nuclear factor-κb (nf-κb) that drive ifn-β expression. the transduction events within this signaling cascade are prone to negative regulation by many mers-cov proteins. in a comparative analysis of mers-cov structural and accessory proteins, it has been shown that m, orf a, orf b and orf possess ifn-antagonizing properties. we, and others, have characterized the orf a protein as a dsrna-binding protein that interferes with the activation of rlr by either a dsrna ligand or the protein co-activator pact. , however, the molecular mechanisms through which other mers-cov proteins manipulate the rlr signaling pathway to disrupt ifn-β expression have not been elucidated. in this study, we focused on the characterization of the mers-cov m protein in ifn antagonism. coronavirus m protein is a transmembrane glycoprotein localized predominantly to the golgi complex and is required for virion assembly. [ ] [ ] [ ] mers-cov m protein is of particular interest because sars-cov m protein also inhibits ifn production through a mechanism by which the formation of traf ·tank·tbk /ikk-ε complex is impeded to ablate the activation of irf transcription factor. in contrast, m protein encoded by human coronavirus hku associated with common cold has no influence on ifn production. here we reported that the mers-cov m protein also specifically inhibited irf activation but not nf-κb signaling. mers-cov m protein was capable of interacting with traf adapter protein and hampered traf -tbk interaction leading to diminished irf activation. using a chimeric protein containing the mers-cov m protein n-terminal transmembrane domains and a dormant sars-cov m protein c-terminal domain, we confirmed that the n-terminal transmembrane domains of mers-cov m protein sufficiently account for its inhibitory effect. although another chimera containing sars-cov m protein n-terminal transmembrane domains and a mers-cov m protein c-terminal domain was fully competent in ifn antagonism, a truncation mutant lacking the functional first transmembrane domain of sars-cov m was not, suggesting that the c-terminal domain of the mers-cov m protein is largely dispensable for its immunosuppressive activity. the ifnβ-luc reporter plasmid and rig-i expression plasmid are gifts from professor takashi fujita (kyoto university, kyoto, japan). the expression plasmids for tbk , irf and traf were generous gifts from dr genhong cheng (university of california, los angeles, ca, usa), , whereas those for rig-i n, ikk-ε and mavs and iκb-α as well as irf -luc and κb-luc reporter plasmids have been described elsewhere. , [ ] [ ] [ ] viral rna was extracted from mers-cov-infected vero-e cells. the m gene was pcr-amplified from complementary dna and cloned into the ecori/xhoi sites of pcagen plasmid with the addition of a c-terminal v -tag with the following primers: ′-atg tct aat atg acg caa ctc act ga- ′ (forward) and ′-agc tcg aag caa tgc aag ttc- ′ (reverse). the sars-cov m protein expression plasmid has been described elsewhere. , the expression plasmids for the sn and mn chimeras were constructed by assembly pcr with the following forward primers covering the breakpoints: ′-agg ctg ttt gct cgt acc cgc tca tgg tgg tca ttc aat cct gag- ′ (sn) and ′-ccg gct gtt tat gag aac tgg atc aat gtg gtc att caa ccc a- ′ (mn). the reverse primers were complementary to their respective forward primers. the truncation mutant of the sn chimera lacking the first transmembrane domain was constructed using the forward primer: ′-atg gta aca ctt gct tgt ttt gtg ct- ′. mouse anti-flag (m ) and anti-β-actin antibodies were purchased from sigma-aldrich (st. louis, mo, usa). mouse anti-v and anti-ha (y ) antibodies were purchased from life technologies (grand island, ny, usa) and santa cruz biotechnology (dallas, tx, usa), respectively. rabbit anti-irf and anti-phospho-irf (ser ) antibodies were purchased from ibl-america (minneapolis, mn, usa). cell culture and sendai virus hek- human embryonic kidney cells were maintained in dulbecco's modified eagle's medium with % fetal bovine serum (life technologies) at °c in a humidified chamber supplemented with % carbon dioxide. plasmid transfection was performed with genejuice (merck millipore; billerica, ma, usa). poly(i:c) was purchased from sigma-aldrich and was transfected with lipofectamine (life technologies). sendai virus (cantell strain) was purchased from american type culture collection (manassas, va, usa). dual-luciferase reporter assay, co-immunoprecipitation and western blotting were performed as previously described. , particularly, relative luciferase activity in arbitrary units was calculated by normalizing firefly luciferase activity to renilla luciferase activity recovered from cell lysate. non-denaturing native polyacrylamide gel electrophoresis (page) was performed as previously described. , , bioinformatic analysis sequence alignment was performed using cluster omega, an online tool based on the hidden markov model, and hosted by the embl-ebi server (http://www.ebi.ac.uk/tools/msa/clustalo/). transmembrane domain prediction was performed using tmfinder, which considers hydrophobicity and helicity of the amino-acid sequence (http://tmfinder.research.sickkids.ca/). to characterize the mers-cov m protein in terms of its ifn antagonism, m protein was ectopically expressed in cultured cells for functional assays ( figure a) . a luciferase reporter construct driven by ifn-β promoter was used to reflect ifn-β promoter activity stimulated during infection. sendai virus was used as a model virus to potently induce ifn expression in transfected cells. when increasing doses of m proteins were expressed in advance, a dose-dependent inhibition of ifn-β promoter activity was observed ( figure b ; bars - compared with bar ). a similar observation was also noted when synthetic dsrna analog poly(i:c) was used as an alternative inducer that specifically stimulates the rlr pathway of ifn production ( figure c ; bars - compared with bar ). these two pieces of data are generally consistent with a previous report, and they further strengthen the current model of the ifn antagonism of mers-cov m protein. cellular ifn-β expression is under the control of multiple transcription factors, which work cooperatively to form a large enhanceosome complex. in particular, irf and nf-κb are two transcription factors that are primarily activated by rlr signaling. , , mers-cov m protein has previously been shown to have no influence on nf-κb activation induced by sendai virus infection. however, it remains to be seen whether the mers-cov m protein could preferentially inhibit irf and nf-κb signaling after rig-i activation. to address this issue, two different luciferase reporter constructs, in which tandem copies of either irf -or nf-κb-binding elements were inserted into their promoter region, were used. the truncation mutant of rig-i known as rig-i n that contains only the n-terminal card domain was chosen to be the inducer in these assays because it is constitutively active and highly competent to induce these two pathways. the mers-cov m protein was able to suppress the promoter activity of the irf -driven luciferase construct in a dose-dependent manner (figure a ; bars - compared with bar ), but no inhibitory effect was observed with the nf-κb-driven construct ( figure b ; bars - compared with bar ) although the canonical inhibitor iκb-α could efficiently blunt its activation as a positive control ( figure b ; bar compared with bar ). hence, the suppressive effect of the mers-cov m protein is specific for irf signaling but not nf-κb activation. to delineate the action point of the mers-cov m protein in ifn antagonism, we tested the ability of the m protein to inhibit the activation signal induced by different signal transducers of the rlr pathway individually. the activation signal will be mostly unaffected if the activation event mediated by that transducer is downstream of the action point where m protein exerts its inhibitory effect. as described above, rig-i n is a constitutively active mutant that resembles ( μl/ml) in c for h before harvest for dual-luciferase reporter assay. bars represent the mean of three biological replicates (n = ) and error bars indicate their s.d. the statistical significance between selected samples was evaluated using a two-tailed student's t-test for unpaired samples with equal variance and p-value (p) was indicated. figure a ; bars - compared with bar ). a similar result was also obtained using mavs as an activator ( figure b ; bars - compared with bar ), which is a mitochondrial adapter that diverts the activation signal from rig-i to the irf and nf-κb pathways. [ ] [ ] [ ] [ ] when activators committed to the irf pathway were used, greater inhibitory effects were observed, as in the cases of tbk ( figure c ; bars - compared with bar ) and ikk-ε ( figure d ; bars - compared with bar ), which are kinases which recognize and phosphorylate irf as direct substrate. [ ] [ ] [ ] [ ] surprisingly, when a constitutively active mutant of irf transcription factor (irf d), with five inducible phosphorylation sites at ser/thr residues mutated to asp, was employed, the expression of the m protein no longer quenched the irf -induced activation of ifn-β promoter ( figure e ), suggesting that the inhibitory effect of m protein occurs upstream of irf activation. to further analyze the molecular mechanism and consequences through which mers-cov m protein exerts its inhibitory effect, we first investigated what signal transducer molecule might interact with the m protein. several rlr transducers were ectopically expressed with mers-cov m protein in cultured cells for a coimmunoprecipitation experiment. when the transducers were precipitated with an anti-flag antibody, the m protein was only detected in traf -containing precipitate ( figure a ; lane compared with lanes - ) even though m protein was abundantly expressed in all samples with other transducers (figure a ; lower panel for input), traf functions as an adapter that bridges the mitochondrial transducer mavs with the downstream signaling complex containing tbk and ikk-ε kinases that are essential for irf activation. , the physical association of the mers-cov m protein with traf ( figure a ) prompted us to test whether the adapter function of traf would be particularly affected by m protein. we performed another co-immunoprecipitation experiment to explore the possibility that m protein could perturb the interaction of traf with the downstream transducer complex. when traf and tbk were ectopically expressed in cultured cells, the detection of tbk in traf -immunoprecipitate confirmed the specific recruitment of tbk to traf in the absence of m protein ( figure b ; lane compared with lane ). however, when m protein was added to the system, the interaction between traf and tbk was significantly disrupted ( figure b ; lane compared with lane ), demonstrating that the physical association of mers-cov m protein with traf perturbs traf -tbk interaction. it was observed that mers-cov m protein disrupted traf -tbk interaction ( figure b ), which is required for irf activation. we then evaluated whether irf activation would be affected by the expression of m protein. irf dimerization visualized by non-denaturing native page is a sensitive assay for evaluating direct irf activation. therefore, we ectopically expressed irf and m protein with the inducer rig-i n in cultured cells and subjected cell lysates directly to native page to check for irf dimerization. when the inducer rig-i n was exclusively co-expressed with irf , the detection of an additional slow-migrating band indicated the activation and dimerization of irf , which would otherwise be entirely in its monomeric form in the absence of any activator ( figure c ; lane compared with lane ). interestingly, when m protein was expressed, the signal reflecting the dimeric form of irf molecules was significantly diminished, even though the total irf level expressed in all samples was highly comparable as detected by conventional denaturing sds-page ( figure c ; lower panel for sds-page), suggesting that irf activation was greatly inhibited by the mers-cov m protein. irf phosphorylation was also suppressed with the expression of mers m protein in a similar experimental setup ( figure d ; lane compared with lane ). together with other results, mers-cov m protein was thought to interact with traf to perturb traf -tbk interaction, which, in turn, affects irf phosphorylation and activation. given that the m proteins of both sars-cov and mers-cov were capable of antagonizing ifn production through highly similar innate immunosuppression by mers-cov m protein p-y lui et al mechanisms, it will be of interest to analyze the sequence and domain architecture of the two proteins. sequence alignment of sars-cov and mers-cov m proteins revealed a strikingly high sequence similarity ( %) and the presence of three transmembrane domains at the n-termini ( figure a ). according to the prediction results, we have initially constructed two truncation mutants for mers-cov m protein, an n-terminal transmembrane domain-containing mutant and a c-terminal mutant, and tested their inhibitory capacity in suppressing ifn-β expression using a luciferase reporter assay. however, neither exhibited an inhibitory effect (data not shown), possibly due to unstable expression or aberrant localization. to overcome the inactivity of truncation mutants and to define the inhibitory activity of different domains, we decided to create chimeric proteins using domain swapping between mers-cov and sars-cov m proteins. particularly, the sn chimera contains the n-terminal transmembrane domains from sars-cov m protein and the c-terminal domain from mers-cov m protein, whereas the mn chimera contains the n-terminal transmembrane domains from the mers-cov m protein and the c-terminal domain from the sars-cov m protein ( figure b ). the breakpoint was designed to occur immediately after the third predicted transmembrane domain at residue and before the conserved ser residue in both proteins at residue ( figure b ). we next compared the inhibitory effect of these two chimeras and the full-length m proteins on ifn-β expression using the luciferase reporter assay. our previous study showed that the ifn-antagonizing activity of the sars-cov m protein is mediated by its n-terminal transmembrane domains, but the c-terminal domain has no effect. when we swapped the c-terminal domain of the sars-cov m protein with that of the mers-cov m protein in the sn chimera, a similar suppressive effect on ifn-β promoter activity was observed ( figure c ; bar compared with bar ), consistent with our previous results. likewise, when we swapped the c-terminal domain of mers-cov m with that of sars-cov m protein in mn, the chimera was capable of suppressing ifn-β promoter activity to comparable level ( figure c ; bar compared with bar ). given that the c-terminal domain of sars-cov m protein possesses no suppressive effect, the inhibitory activity of the mn chimera would be predominantly due to the n-terminal domains of mers-cov m protein. a biochemical assay also confirmed that the mn chimera maintained the ability to interact with the traf adapter protein in a co-immunoprecipitation experiment ( figure d ; lane compared with lane ). therefore, we concluded that the n-terminal transmembrane domains of the mers-cov m protein are required and sufficient for its innate immunosuppressive activity. to further determine whether the c-terminal domain of the mers m protein also possesses ifn-antagonizing activity, we utilized the knowledge that the first transmembrane domain of sars-cov m protein is fully responsible for its suppression effect in this study, we reported that the mers-cov m protein inhibited irf activation, hence ifn expression, by disrupting traf -tbk interaction. this innate immunosuppressive activity of the mers-cov m protein was due to its conserved n-terminal transmembrane domains. our mechanistic study complemented the previous work that showed that mers-cov m protein had ifn-antagonizing activity. both studies demonstrated that mers-cov m protein suppressed irf activity but not nf-κb signaling. it is known that the activation of rig-i and mavs results in the activation of both irf and nf-κb. , [ ] [ ] [ ] our results indicated that the mers-cov m protein was capable of differentially suppressing the rig-i-induced activation of irf (figure ). this provides further support to the bifurcation of irf and nf-κb signaling subsequent to rig-i and mavs activation. further investigations should elucidate the mechanism by which the mers-cov m protein preferentially modulates irf activators such as tbk , while sparing nf-κb activators such as card . we provided evidence that the traf -tbk interaction as well as irf phosphorylation and dimerization were affected by the mers-cov m protein (figure ). our findings fill the knowledge gap by providing novel mechanistic insight into the innate immunosuppressive activity of mers-cov m protein. in our study, traf was also shown to interact with the mers-cov m protein ( figure a ). in line with this, the adapter function of traf in tbk recruitment and subsequent irf activation was perturbed by mers-cov m protein ( figures b-d) , which plausibly contributed to the ifn antagonism of the mers-cov m protein (figures - ) . the mers-cov m protein is a transmembrane protein that was shown to co-localize with markers of the golgi apparatus and endoplasmic reticulum (er)-golgi intermediate compartments in the perinuclear area. although traf is known to adapt the mitochondrial transducer mavs, it is not associated with mitochondria but rather with the golgi apparatus and er-golgi intermediate compartments in unstimulated conditions, , rendering it susceptible to interaction with the mers-cov m protein. upon stimulation with ligands or viral infection, traf appears on membrane-bound fragments originating from golgi. retention of traf in er-to-golgi compartments and inability to form golgi fragments rendered ifn-β expression inefficient. therefore, whether traf -containing golgi fragment formation is affected by mers-cov m protein warrants further analysis. this may serve as a novel mechanism by which virus-encoded proteins counteract host ifn production. mers-cov and sars-cov are two highly pathogenic coronaviruses that have caused hundreds of deaths. on one hand, the development of relevant prophylactic and therapeutic agents has been well under way. [ ] [ ] [ ] on the other hand, the identification of the pathogenic factors in these viruses is also in full swing. the m protein is a pathogenic factor by virtue of its ifn-antagonizing property. both the mers-cov and sars-cov m proteins were found to suppress ifn production with a highly similar mechanism in which the irf phosphorylating complex of traf ·tank·tbk /ikk-ε was affected by their n-terminal transmembrane domains. , interestingly, in the case of community-acquired human coronavirus hku , which normally causes common cold in infected individuals, its m protein showed no ifn antagonistic property, further supporting the importance of the m protein in sars-cov and mers-cov pathogenesis. using a side-by-side comparison of sars-cov and mers-cov m proteins, we discovered that the extent by which the mers-cov m protein suppressed ifn-β promoter activity was lower than that by sars-cov m protein. this difference might be explained by the strengths of the interaction of mers-cov m protein with other transducers. whereas the mers-cov m protein was found to be strongly associated with traf , its interaction with tbk or ikk-ε was undetectable (data not shown). this distinguished mers-cov m protein from the sars-cov m protein, which interacts potently with every component of the traf ·tank·tbk /ikk-ε complex. further investigations are required to shed light on how the interaction of the m protein with traf complex might influence mers-cov pathogenesis. coronaviruses encode multiple proteins to counteract the host innate antiviral response. - mers-cov is no exception. several mers-cov-encoded proteins have been identified to be ifn antagonists. we, and others, have characterized at least three ifn-antagonizing proteins encoded by mers-cov. in addition to the m protein reported in this study, orf a is a dsrna-binding protein, which directly inhibits rlr activation induced by dsrna and/or the protein co-activator pact. , in addition, our unpublished data also revealed that orf b is a potent ifn antagonist. this is in line with findings by other groups although the mechanistic details of its action have not well documented. , one recent report suggested that orf b might not only interact directly with tbk / ikkε in the cytoplasm but also perturb ifn production in the nucleus through an as yet unknown mechanism. nevertheless, how m, orf a, orf b and the other ifn-antagonizing proteins of mers-cov coordinate with each other to modulate the host innate antiviral response to facilitate viral replication and propagation remains elusive. severe respiratory illness associated with a novel coronavirus-saudi arabia and qatar severe respiratory illness caused by a novel coronavirus first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in a 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coronavirus infection and disease the structural and accessory proteins m, orf a, orf b, and orf of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists the orf b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling middle east respiratory syndrome coronavirus accessory protein a is a type i interferon antagonist middle east respiratory syndrome coronavirus a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda in the innate antiviral response proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses cell type-specific involvement of rig-i in antiviral response severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf ·tank·tbk / ikkε complex a structural analysis of m protein in coronavirus assembly and morphology suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain irf mediates a tlr /tlr -specific antiviral gene program modulation of the interferon antiviral response by the tbk /ikki adapter protein tank mip-t is a negative regulator of innate type i ifn response the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response suppression of type i and type iii interferon signalling by nss protein of severe fever-with-thrombocytopenia syndrome virus through inhibition of stat phosphorylation and activation comparative analysis of the activation of unfolded protein response by spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus hku induction of irf- /- kinase and nf-κb in response to double-stranded rna and virus infection: common and unique pathways suppression of pact-induced type i interferon production by herpes simplex virus us protein fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega tm finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales virus induction of human ifnβ gene expression requires the assembly of an enhanceosome identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf ips- , an adapter triggering rig-i-and mda -mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-β signaling recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin β production ikkε and tbk are essential components of the irf signaling pathway ifnregulatory factor -dependent gene expression is defective in tbk -deficient mouse embryonic fibroblasts ikk-i signals through irf and nfkappab to mediate the production of inflammatory cytokines phosphorylation of innate immune adapter proteins mavs, sting, and trif induces irf activation virus-dependent phosphorylation of the irf- transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation regulation of antiviral responses by a direct and specific interaction between traf and cardif proteomic profiling of the traf interactome network reveals a new role for the er-to-golgi transport compartments in innate immunity yeast-expressed recombinant protein of the receptorbinding domain in sars-cov spike protein with deglycosylated forms as a sars vaccine candidate structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon to sense or not to sense viral rna-essentials of coronavirus innate immune evasion a molecular arms race between host innate antiviral response and emerging human coronaviruses middle east respiratory syndrome coronavirus orf b protein inhibits type i interferon production through both cytoplasmic and nuclear targets this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . / key: cord- - ldpz m authors: chen, chi-yuan; lin, chin-yu; chen, guan-yu; hu, yu-chen title: baculovirus as a gene delivery vector: recent understandings of molecular alterations in transduced cells and latest applications date: - - journal: biotechnol adv doi: . /j.biotechadv. . . sha: doc_id: cord_uid: ldpz m baculovirus infects insects in nature and is non-pathogenic to humans, but can transduce a broad range of mammalian and avian cells. thanks to the biosafety, large cloning capacity, low cytotoxicity and non-replication nature in the transduced cells as well as the ease of manipulation and production, baculovirus has gained explosive popularity as a gene delivery vector for a wide variety of applications. this article extensively reviews the recent understandings of the molecular mechanisms pertinent to baculovirus entry and cellular responses, and covers the latest advances in the vector improvements and applications, with special emphasis on antiviral therapy, cancer therapy, regenerative medicine and vaccine. baculoviruses are a diverse group of dna viruses capable of infecting more than insects, among which autographa californica multiple nucleopolyhedrovirus (acmnpv) is the best characterized and most extensively employed, thus baculovirus discussed in this article refers to acmnpv unless otherwise noted. acmnpv contains a circular dsdna genome of ≈ kb and replicates in a bi-phasic fashion. the viral proteins polyhedrin and p are expressed abundantly in infected cells and are dispensable for virus replication, thus recombinant baculovirus can be constructed by placing the foreign gene under the control of polyhedrin or p promoter, and used to infect insect cells for foreign gene expression. such baculovirus-insect cell expression system has been exhaustively utilized for the production of numerous recombinant proteins (kost and condreay, ) and commercial vaccine products such as cervarix ® and provenge ® (hitchman et al., ; . in addition to insect cells, baculovirus is able to transduce animal cells of human, rodent, rabbit, porcine, bovine, fish and avian origins (hu, (hu, , as well as relatively primitive cells including embryonic stem cells, adult stem cells (table ) and induced pluripotent stem cells (fig. ) . within the baculovirus-transduced cells, the transgene can be expressed as long as it is driven by an appropriate promoter (e.g. cytomegalovirus immediate-early (cmv) or hybrid cag promoter consisting of the cmv early enhancer and chicken β-actin promoter). baculovirus cloning capacity is as large as kb (cheshenko et al., ) , thus allowing for the insertion of multiple genes and regulatory elements (kost et al., ; kost and condreay, ) . baculovirus neither replicates inside the transduced cells nor integrates its dna into host chromosomes in the absence of selective pressure (chen et al., a; merrihew et al., ) , hence easing the safety concerns. humans do not possess pre-existing antibody and t-cells specifically against baculovirus (strauss et al., ) , therefore baculovirus may circumvent the pre-existing immunity problem faced by other viral vectors. finally, recombinant baculovirus can be readily constructed and propagated to high titers in biosafety level facilities by infecting its natural host insect cells (hu, ) . these attributes have fueled growing interests to explore baculovirus for a wide variety of applications, ranging from protein production (jardin et al., ; liu et al., ) , virus production lesch et al., lesch et al., , lucifora et al., ; mccormick et al., ; nakowitsch et al., ; zheng et al., ) , virus-like particle production (chen et al., ; matsuo et al., ; wang et al., ) , eucaryotic protein display (ernst et al., , grabherr and ernst, ) , vaccine development madhan et al., ; tani et al., ) , cancer therapy (wang and balasundaram, ) , cell-based assay development (condreay et al., ; condreay and kost, ; kost et al., ) to tissue engineering (lin et al., b) . this article will focus on recent understandings of intracellular events after baculovirus transduction and latest applications of baculovirus for antiviral therapy, cancer therapy, tissue regeneration and vaccine development. baculovirus entry into mammalian cells was initially suggested to depend on electrostatic interactions (duisit et al., ) , heparin sulfate (duisit et al., ) and phospholipids (tani et al., ) , but the exact cell surface molecules for baculovirus docking remained unknown. it was also proposed that clathrin-mediated endocytosis (long et al., ; matilainen et al., ) and macropinocytosis (matilainen et al., ) play roles in baculovirus entry. contradictorily, a recent study (laakkonen et al., ) discovered that ( ) baculovirus entered hek and hepg cells along fluid-phase markers from the raft areas into vesicles devoid of clathrin; ( ) macropinocytosis-related regulators (e.g. eipa, pak , rab and rac ) imparted no significant effects on virus transduction and ( ) the internalization and nuclear uptake were affected by the regulators of clathrin-independent entry. these data unveiled a baculovirus entry pathway independent of clathrin-mediated endocytosis and macropinocytosis and suggested table selected types of cells permissive to baculovirus transduction. human cells hepg (gerner et al., ; laakkonen et al., ; laakkonen et al., ) hek laakkonen et al., ; lavdas et al., ; sollerbrant et al., ) huh ) vero e cells (liu et al., a) coronary smooth muscle and endothelial cells (grassi et al., ) a (guo et al., ) colon cancer cells (paul and prakash, ; yin et al., ) glioblastoma u (lackner et al., ) osteosarcoma saos- (song et al., ) glioblastoma u (balani et al., ) astrocytes (balani et al., ; boulaire et al., ) neurons derived from es cells (zeng et al., ) bone marrow mesenchymal stem cells (bak et al., ; ho et al., ; lin et al., a; lo et al., ) embryonic stem (es) cells (du et al., ; zeng et al., ) non-human primate cells cos- (pan et al., ) cv- (tani et al., ) vero (zheng et al., ) coronary smooth muscle cell (grassi et al., ) rodent cells bhk (zheng et al., ) cho hu et al., ; pan et al., ) c c (shen et al., ) l (airenne et al., ; cheng et al., ) sol (shen et al., ) rat hepatic stellate cells (gao et al., ) primary mouse osteoblasts and osteoclast (tani et al., ) rat articular chondrocyte (ho et al., ) mouse amniotic fluid-derived stem cells (liu et al., b) rabbit cells aortic smooth muscle (raty et al., ) intervertebral disk cells ) primary articular chondrocyte (chen et al., c; lee et al., ; sung et al., (ping et al., ) chicken embryonic fibroblast (ping et al., ) chicken embryonic cells ) duck embryonic cells canine cells mdck norden laboratories feline kidney (nlfk) that phagocytosis might play a role (laakkonen et al., ) , which echoed the observations reported previously (abe et al., ) . moreover, other recent studies reported that baculovirus transduction related to direct fusion pathway induced by a short ph trigger (dong et al., ; paul and prakash, ) . these conflicting data underlined the need for more in-depth studies to elucidate the underlying mechanism and might suggest that baculovirus entry pathway varies with cell types. nevertheless, one consensus is that baculovirus envelope protein gp is pivotal for entry because blocking gp can abrogate the baculovirus ability to transduce mammalian cells (abe et al., ; niu et al., ) and activate dendritic cells (dcs) (schutz et al., ) . once inside the cells, baculovirus is transported to the endosome, followed by endosomal escape via acid-triggered gp fusion (kukkonen et al., ) and subsequent nuclear transport van loo et al., ) with the aid of actin filament (matilainen et al., ; salminen et al., ) . a major component of type iii intermediate filaments, vimentin, also participates in intracellular trafficking (mahonen et al., ) . vimentin is reorganized in the optimized culture medium and is linked to enhanced nuclear entry of baculovirus, underscoring the importance of culture medium in the cytoskeleton network assembly and in baculoviral gene delivery. baculovirus encodes ≈ genes and a number of viral genes (e.g. orf , ie , p and gp ) are expressed in transduced mammalian cells (fujita et al., ; kitajima et al., ) . among the baculoviral genes, ie is expressed early in insect cells and transactivates downstream gene expression. forced expression of ie by the minimal cmv promoter in vero e cells also markedly activates gp and pe , and upregulates ie , he , pcna, orf , orf and orf (liu et al., ) . the critical role of ie for transactivating downstream genes was further unraveled in a recent study , which showed that baculovirus deficient in ie gene mitigates residual baculoviral gene expression -to -fold (when compared with wild-type baculovirus) in transduced mammalian cells, thus enhancing safety features to baculovirus-based gene therapy. in contrast to ie , ie overexpression driven by the cmv promoter only upregulates baculoviral genes (pe and orf ), but baculovirusmediated co-expression of ie and ie acts in concert to upregulate out of baculovirus genes in mammalian cells (liu et al., ) . strikingly, ie is a strong transactivator of cmv promoter in both vero e and u- os cells (liu et al., a) . when overexpressed within the baculovirus context, ie forms the nuclear foci and develops into large nuclear bodies (nbs) with a hollow center. the ie nbs structure contains abundant g-actin, closely associates with rna polymerase ii, promyelocytic leukemia (pml) and small ubiquitin-like modifier- (sumo ) and is the site of active transcription, thereby contributing to the ie -associated gene stimulation (liu et al., a) . furthermore, the nbs formation and cmv promoter activation require the n-terminal ring finger and c-terminal coiled-coil domains of ie (liu et al., a) . since cmv promoter is exhaustively used for baculovirus-mediated gene transfer, the transactivating activity of ie may be useful for baculovirus-mediated protein production in mammalian cells . due to the complex cascade of events during baculovirus transduction, it is not surprising that baculovirus can alter the cell morphology and trigger cellular responses. for instance, baculovirus transduction of hepg cells alters the size of pml nbs, induces remodeling of the host cell chromatin and arouses extensive ruffle formation on the cell surface (laakkonen et al., ) . shotgun proteomics also attests that baculovirus-transduced hepg cells exhibit a slight induction of proteins related to inflammation, cell survival and chromatin function (gerner et al., ) . the most well-characterized baculovirus-induced cellular response is the innate immune response, as manifested by the induction of such cytokines as interferon α/β (ifn-α/β), interleukin- (il- ), il- , il- β and tumor necrosis factor-α (tnf-α) (abe and matsuura, ; tani et al., ) . baculovirus transduction of rat chondrocytes also elicits transient expression of ifn-α/β, which attenuates the transgene expression (lee et al., ) . not only the cytokine secretion, in vitro baculovirus transduction also activates human (schutz et al., ) and mouse (abe et al., ) dcs. moreover, wild-type baculovirus transduction of mouse bone marrow-derived dcs (bmdcs) upregulates the major histocompatibility complex (mhc) i and ii and co-stimulation molecules (e.g. cd , cd and cd ) (suzuki et al., a) . the activated bmdcs can further stimulate natural killer (nk) cells upon coculture, as evidenced by the ifn-γ production, cd upregulation and cell proliferation. in contrast to these differentiated, specialized cells, stem cells appear to be less sensitive to baculovirus transduction. upon wildtype baculovirus transduction, human bone marrow-derived mesenchymal stem cells (bmscs) secret il- and il- , but no detectable levels of ifn-γ, ifn-β, tnf-α, tnf-β, il- β, il- , il- , il- , il- and il- (chen et al., b) . human leukocyte antigen i (hla-i) is slightly upregulated, but the expression of hla-ii and other surface markers are barely disturbed (bak et al., ; chuang et al., ) . neither does baculovirus transduction compromise the immunosuppressive properties of bmscs . conversely, wildtype baculovirus transduction of mouse induced pluripotent stem cells (ipscs) elicits only the chemokine ip- , but not other wellknown cytokines (unpublished data). bmscs and ipscs are promising cell sources for regenerative medicine. the lack of these cytokine responses reduces the risk of mounting strong immune responses after transplantation of baculovirus-transduced bmscs and ipscs. in vivo, baculovirus administration triggers innate immune responses, activates macrophages (abe et al., ) , dcs (hervas-stubbs et al., ; schutz et al., ) and nk cells (facciabene et al., ; kitajima et al., ) . the baculovirus-induced innate immunity gives rise to antitumor activity (suzuki et al., a) and is sufficient to confer protection against influenza virus in mice (abe et al., ) , infectious bronchitis virus in chickens (niu et al., ) and foot-and-mouth-disease virus in mice (molinari et al., ) . the innate immunity also confers baculovirus the adjuvant activity to promote humoral and cellular immune responses against co-administered antigens (hervas-stubbs et al., ) . moreover, intramuscular (i.m.) injection of baculovirus triggers t-cell responses against the vector, but the magnitude of anti-baculovirus t cells response is lower than that of anti-adenovirus response (hervas-stubbs et al., ) . toll-like receptors (tlrs) are a family of pattern recognition receptors essential for initiating the innate immunity and substantiating the adaptive immunity (ishii et al., ) . for instance, tlr recognizes virus-derived dsrna while tlr recognizes unmethylated cpg dna motifs. upon engagement with cognate ligands, tlrs are activated and recruit adaptor molecules such as myeloid differentiating factor (myd ) and tir domain-containing adaptor inducing ifn-β (trif) to transduce signals to downstream molecules. besides, stimulator of interferon genes (sting) is a cytoplasmic sensor that activates irf and ifn-α/β in response to viral dsdna while retinoic acid-inducible gene (rig-i) and melanoma differentiation-associated gene (mda ) are tlr-independent cytoplasmic rna detectors that induce the ifn-α/β production through ifn promoter-stimulator- (ips- ). baculovirus-induced innate immunity has been ascribed to the recognition of cpg motifs in viral dna and ensuing activation of tlr / myd -dependent pathway (abe et al., ) . the detection activates nuclear factor-κb (nf-κb) and leads to subsequent ifn-α/β production. however, ifn-α/β are still produced in peritoneal cells derived from myd -or tlr -deficient mice (abe et al., ) . in mouse embryonic fibroblasts (mefs), baculovirus triggers ifn-β and ifn-inducible chemokines through tlr-independent and irf -dependent pathways and endosomal maturation is required for induction (abe et al., ) . these data suggest that baculovirus dna might be recognized by at least two different pathways: tlr -dependent endosomal recognition and tlr independent cytoplasmic recognition. the latter was suggested to be related to sting (abe and matsuura, ) because baculovirus-induced ifn-α/β production was impaired in sting-deficient mefs (ishikawa et al., ), yet ifn-α/β stimulation was found to be independent of other cytoplasmic rna detectors such as rig-i and mda (abe et al., ). however, a recent study noted that rig-i and mda mrna levels were elevated in baculovirus-transduced cells . addition of dna methytransferase inhibitors (dnmti) prior to transduction retarded such upregulation and enhanced baculovirus-mediated gene expression, suggesting that dnmti may somehow facilitate the baculovirus evasion from the cellular recognition and thus ameliorate the transgene expression. using the cdna microarray, wang and coworkers discovered that baculovirus injection into the striatum in the rat brain perturbed the expression of genes, which represented . % of the total gene probes on the microarray . the same study also identified gene probes ( . %) in human astrocytes and gene probes ( . %) in human neuronal cells that were disturbed in response to baculovirus. despite the disparity between cell types, in all samples baculovirus altered the expression of genes associated with tlr signaling pathway (e.g. tlr , tlr , ccl , cxcl and stat ) and cytokine-cytokine receptor interaction (e.g. cxcl , cxcl and ccl ). moreover, genes associated with interferon induction-related genes (e.g. cxcl , mx , mx , oas and stat ) and antigen processing and presentation pathway (e.g. cd and rt- ba) were affected. as such, wang and coworkers proposed that baculovirus recognition by tlrs triggers the expression of ifn-α/β, which initiates the subsequent signaling cascade involving stats, upregulating the expression of ifn-responsive genes and hence confers the cells the antiviral state. concurrent with the aforementioned findings, we also discovered that baculovirus transduction of human bmscs disturbed the expression of genes, most of which were related to signaling pathways: cell adhesion molecules, tlr, jak-stat, apoptosis as well as antigen processing and presentation (chen et al., b) . of note, baculovirus triggered the tlr pathway, resulting in downstream nf-κb and irf- activation and il- /il- production. however, how baculovirus containing the dsdna genome activated the dsrna sensor tlr remains an enigma. also, the transduction did not arouse the secretion of ifn-β (chen et al., b) , a signature cytokine associated with tlr activation, implying a signaling cascade somewhat distinct from that in immune cells. interestingly, the budded form of another baculovirus, antheraea pernyi nuclear polyhedrosis virus (apnpv), triggers the tlr signaling in chicken macrophage-like cells (hd ) but not in chicken b cell-like cells (han et al., ) . apnpv transduction of hd cells concomitantly induces the production of ifn-γ, il- p and nitric oxide (no) (niu et al., ) , which is accompanied by the phosphorylation of extracellular signal-related kinase / (erk / ), p mitogen activated protein kinase (mapk) and c-jun n-terminal kinase (jnk) as well as activation of p -nf-κb (han et al., ). inhibition of p mapk and nf-κb by their respective inhibitors abrogates the expression of cytokines and no, whereas inhibition of jnk abolishes only the induction of cytokines. since in mammals tlrs signaling activates the downstream nf-κb and mapk cascade comprised of at least p mapk, erk / , and jnk (ishii et al., ) , these data altogether suggest that apnpv transduction of hd cells activates tlr and signals though nf-κb, p mapk and jnk pathways, and chicken tlr might play a role similar to mammalian tlr (han et al., (han et al., , . baculovirus envelope protein gp comprises an n-terminal signal peptide and a mature domain that encompasses the transmembrane domain and cytoplasmic domain (ctd). heterologous protein/peptide has been inserted in between the signal peptide and mature domain, which after expression under the polyhedrin or p promoter as a fusion protein is translocated to the plasma membrane and incorporated into the viral envelope upon virus budding. such feature has been exploited for surface display of protein/peptide to improve the virus transduction (grabherr and ernst, ; grabherr et al., ; raty et al., ) or for ligand-directed targeting if an appropriate ligand is chosen (kitagawa et al., ; makela et al., , . for instance, baculovirus poorly transduces b lymphocytes (cheng et al., ; condreay et al., ) . via gp fusion, the transduction has been enhanced by displaying the short peptide motif from gp / of epstein-barr virus (ebv, which naturally infects b cells) on the baculovirus envelope (ge et al., ) . alternatively, the cytoplasmic transduction peptide (ctp) has been fused to gp to enhance the baculovirus transduction of vero e , u- os and cho-rd cells (chen et al., b) . another paradigm is the display of the fragment crystallisable (fc) region of antibody on the baculovirus surface (martyn et al., ) . fc receptors (fcrs) are membrane proteins that bind to the fc region of antibody and mediate the phagocytosis and antigen presentation. the fc display allows for specific baculovirus targeting to cell lines and antigen presenting cells (apcs) expressing fcrs, hence augmenting the vaccine effect (martyn et al., ). the display system also allows for the surface presentation of functional membrane proteins to simplify subsequent isolation . aside from the gp -aided display, expression of vesicular stomatitis virus g protein (vsvg) (chapple and jones, ; makela and oker-blom, ) , influenza virus neuraminidase (borg et al., ) , spodoptera exigua multiple nucleopolyhedrovirus f protein (yu et al., ) , single chain antibody fragments (kitidee et al., ) and human endogenous retrovirus envelope protein (lee et al., ) in insect cells also leads to incorporation of the protein into baculovirus envelope. among these strategies, display of vsvg or heterologous peptide/protein via the vsvg anchor is the most widely adopted and can tremendously enhance baculovirus transduction in vitro and in vivo (facciabene et al., ; kaikkonen et al., ; kaneko et al., ; kitagawa et al., ; lu et al., ; tani et al., tani et al., , zhou and blissard, ) . serum complement proteins (e.g. c b- ) inactivate baculovirus, hence constituting a major hurdle in the in vivo use of baculovirus. the inactivation problem has been circumvented by the use of complement inhibitors such as compstatin (georgopoulos et al., ) and soluble complement inhibitor (hoare et al., ; hofmann et al., ) , avoiding exposure to the complement (airenne et al., ) or administrating the vector to immunoprivileged sites (kinnunen et al., ; lehtolainen et al., ) . alternatively, the baculoviral vector can become complement-resistant by displaying human daf (decay accelerating factor) via gp fusion (huser et al., , kaname et al., . more recently, baculoviral vectors displaying different complement regulatory proteins (e.g. daf, factor h-like protein- , c bbinding protein and membrane cofactor protein) were generated by fusion with the membrane anchor of vsvg (kaikkonen et al., ) . these surface-modified vectors exhibited varying degrees of complement resistance in vitro and daf yielded the highest level of protection. intraportal delivery of the daf-displaying baculovirus increased the survival and gene expression in immunocompetent mice. the daf-displaying baculovirus provoked lower levels of inflammatory cytokines il- β, il- , and il- p in macrophages and mitigated liver inflammation in mice when compared with the control virus. these results prove that daf display offers protection to the baculoviral vector against complement inactivation and attenuates complement-mediated inflammation injury. other than the display on the envelope, heterologous protein has been displayed on the capsid by fusion with the major capsid protein vp . the vp fusion with enhanced green fluorescent protein (egfp) neither interferes with the virus assembly nor affects the virus tier, thereby enabling intracellular baculovirus trafficking and biodistribution monitoring (kukkonen et al., ) . similarly, the zno binding peptide has been fused to the n-terminus of vp while retaining the viral infectivity and conferring the ability to bind nanosized zno powders (song et al., ) . besides, by fusing the protein transduction domain (ptd) of human immunodeficiency virus (hiv) tat protein (a protein responsible for nuclear import of hiv genome) with vp , the engineered baculovirus results in improved transduction of various mammalian cells (chen et al., b) . in addition to surface modification by genetic engineering, baculovirus has been labeled with tracers for tracking (li et al., ) or biodistribution imaging raty et al., ) . chemical coupling of antigenic peptides also enables rapid modification of baculovirus particles and delivery of multiple epitopes (wilson et al., ) . baculovirus can also be chemically conjugated with polyethylene glycol (peg) alone (kim et al., ) or together with folate (kim et al., (kim et al., , to improve the transduction of folate receptor-positive kb cells. additionally, baculoviral vectors have been coated with positively charged polyethylenimine ( kda) through electrostatic interactions (yang et al., ) . the modification imparts baculoviral vectors resistance to human and rat serum-mediated inactivation in vitro and elevates in vivo transduction in the liver and spleen after tail vein injection into mice. rna interference (rnai) is a phenomenon that mediates sequence-specific post-transcriptional gene silencing and can be artificially elicited by the expression of short hairpin rnas (shrna) or microrna (mirna) precursors from an expression vector (garzon et al., ) . since the initial demonstrations of baculovirus-mediated shrna delivery (nicholson et al., ; ong et al., ) , baculovirusmediated rnai has been explored for antiviral therapy. one baculovirus expressing the shrna specific for the c-terminal nucleoprotein of porcine reproductive and respiratory syndrome virus (prrsv) genome inhibits the viral replication in vitro (lu et al., ) . the baculovirus harboring a bispecific shrna targeted against influenza virus a and b can inhibit the production of both virus types in transduced cell lines (suzuki et al., b) . another baculovirus expressing shrna against the peste des petits ruminants virus (pprv) represses pprv replication in vitro and the inhibition effect is superior to that mediated by the adenovirus expressing the same shrna (nizamani et al., ) . hepatitis b virus (hbv) covalently closed circular (ccc) dna is the source of hbv transcripts in chronically infected patients. baculovirus expressing the hbv-specific shrna is able to hinder the formation of hbv ccc dna (starkey et al., ) . additionally, the baculovirus expressing shrnas specific for the highly conserved core region of hepatitis c virus (hcv) genome dramatically impedes the target protein expression (suzuki et al., c) . the replication and segregation of ebv genomes to daughter cells is coordinated by the binding of ebv nuclear antigen (ebna ) to orip, an origin of replication derived from ebv. to prolong the transgene expression, baculoviral vectors incorporating the orip/ ebna sequences were developed (shan et al., ; wang et al., ) . based on this concept, suzuki et al. devised a baculoviral vector that accommodated the orip/ebna sequence and encoded the hcvspecific shrna. this vector inhibited hcv core protein expression for n days, which considerably outlasted the days of inhibition conferred by the conventional vector (suzuki et al., a) . recently, we also exploited the baculovirus vector for mirna delivery and gene regulation. the baculovirus vectors carried artificial gene-specific mirna sequences within the mir scaffold, which after expression under the cmv promoter could undergo the mirna processing pathway and knocked down the target gene (e.g. egfp and tnf-α) expression (unpublished data). the gene suppression effect was extended by flanking the mirna expression cassette with the inverted terminal repeat sequences/direct repeats sequences (ir/dr) recognized by the sleeping beauty (sb) transposase. co-transduction of cells with the hybrid baculovirus/transposon vector and another sb transposase-expressing baculovirus resulted in the integration of mirna expression cassette into the chromosome, giving rise to prolonged target gene suppression (unpublished data). these data altogether implicate the potential of baculovirus-based rnai shuttle for antiviral therapy and treatment of indications necessitating prolonged gene regulation (e.g. arthritis). the feasibility of baculovirus-mediated cancer therapy was first explored by wang et al. ( ) , who developed a baculoviral vector expressing the diphtheria toxin a and demonstrated that the baculovirus impeded the growth of cultured glioma cells and glioma xenograft in the rat brain. since then, baculovirus has been utilized for the treatment of mouse hepatoma (kitajima et al., ) , murine melanoma, lung cancer and brain cancer (kim et al., ) . recently, wang and coworkers constructed another baculovirus that selectively expressed herpes simplex virus thymidine kinase (hsvtk) in tumors for glioma suicide gene therapy (balani et al., ). the hsvtk gene was driven by a truncated human high mobility group box (hmgb ) promoter, which allowed hsvtk expression in glioblastoma tissues but not in normal brain tissues. this vector triggered death of glioblastoma cells in the presence of ganciclovir, but did not affect the survival of human astrocytes and neurons. in a mouse xenograft model, intratumoral injection of this baculovirus at days after tumor inoculation suppressed the growth of human glioblastoma and prolonged the mouse survival. however, the tumor mass was not completely eradicated after one single baculovirus injection, presumably due to the transient hsvtk expression (balani et al., ) . alternatively, the hsvtk gene was driven by the gfap (glial fibrillary acidic protein) promoter whose activity is similar in normal and tumor cells of the same lineage (wu et al., a) . to limit the transgene expression in glioma cells, repeated target sequences of mirnas (has-mir , has-mir and has-mir ) that are enriched in astrocytes but are sparse in glioblastoma cells were appended to the ′ untranslated region of the hsvtk gene. the baculovirus vector markedly improved in vivo selectivity when compared with the control vector without mirna regulation, effectively inhibited human glioma xenograft and imparted negligible toxicity to normal astrocytes. the incorporation of selected mirna sponge thus provides an additional safety switch to prevent off-target transgene expression (wu et al., a) . in addition to hsvtk, the baculovirus expressing nes (normal epithelial cell specific- ) can inhibit the growth of gastric cancer cells (sgc- ) in vitro and repress the sgc- xenografted tumor growth after intratumoral injection, implicating the possibility for the therapy of gastric and colon cancers (huang et al., ) . baculovirus can also carry tumor suppressor genes such as p or pdcd (programmed cell death ) for cancer treatment. in the mouse glioma xenograft models, the antitumor effect of the baculovirus-expressed p was substantiated by surface coating of the baculovirus envelope with polyethylenimine (yang et al., ) or by co-administering sodium butyrate (guo et al., ) , a histone deacetylase inhibitor that ameliorates baculovirus-mediated gene expression (guo et al., ; hu et al., ; yin et al., ; zhou et al., ) . the pdcd expressing baculovirus conjugated with folate-peg efficiently expressed pdcd protein and induced apoptosis in human epidermal carcinoma cells (kim et al., ) . in a tumor xenograft, intratumoral injection of the pdcd -expressing baculovirus significantly suppressed tumor growth and induced apoptosis. moreover, apoptin is a chicken anemia virus-derived protein that specifically triggers apoptosis in tumors. the vsvg-pseudotyped baculovirus expressing apoptin efficiently provoked apoptosis in mammalian cells, repressed the growth of xenograft tumors and prolonged the mice survival after intratumoral injection (pan et al., ) . one determinant to the baculovirus-mediated antitumor effects is the innate immune responses elicited by the virus (suzuki et al., b) . takaku and coworkers demonstrated that wild-type baculo-virus, after intravenous (i.v.) injection into immunocompetent b mouse, was taken up by the liver and spleen, preferentially entered dcs and b cells, activated dcs, induced nk cells proliferation in the liver and spleen, and enhanced the antitumor immunity in mice with b liver metastases (kitajima et al., ) . baculovirus administration also increased the survival of c bl/ , jα −/− and ifn-γ −/− mice bearing the b tumor, but did not enhance the survival of nk cell-depleted mice. these results prove that wild-type baculovirus efficiently induces nk cell-mediated antitumor immunity (kitajima et al., ) . besides direct injection, baculovirus has been employed in conjunction with cell therapy for cancer treatment. bone marrowderived dcs (bmdcs), after ex vivo transduction with wild-type baculovirus and i.v. injection into mice, significantly suppressed the lung cancer (suzuki et al., a) . in a mouse melanoma model, baculovirus-transduced bmdcs inhibited tumor growth and improved animal survival at least partly due to the induction of cd + t cell-and nk cell-dependent, cd + t cell-independent antitumor immunity. importantly, bmdcs administration did not provoke evident signs of damage to the liver or kidney, as judged from the negligible disturbance of serum alanine aminotransferase (alt), aspartate aminotransferase (ast) and creatine levels (suzuki et al., a) . another promising cell courier is bmscs thanks to their intrinsic tumor homing property. in this regard, bmscs were transduced with a baculovirus expressing hsvtk and injected via tail vein into the mice pre-inoculated with human u glioma cells (bak et al., ) . after ganciclovir injection, tumor growth was significantly repressed and the life span of animals was considerably prolonged. more recently, the same group generated msc-like cells from human embryonic stem (es) cells which, after transduction with the baculovirus expressing hsvtk and injection into the brain, were capable of inhibiting tumor growth and prolonging the survival of tumor-bearing mice in the presence of ganciclovir (bak et al., ) . these data implicated the feasibility of human es cells-derived mscs as a viable and attractive alternative for cancer therapy. gene therapy has converged with tissue engineering, by which the therapeutic genes stimulating tissue regeneration can be administered to cells either in vivo or ex vivo with an appropriate vector. given the highly efficient gene delivery, baculovirus has been used for cartilage and bone engineering (lin et al., b) . articular cartilage is a weight-bearing tissue comprising chondrocytes and extracellular matrix (ecm) composed of proteins (e.g. collagen ii) and glycosaminoglycans (gags) such as aggrecan. articular cartilage may be damaged due to direct trauma or joint diseases, but its ability to self-repair is limited, ultimately leading to debilitating pain and physical impairment. therefore, cartilage tissue engineering that combines cells, scaffolds and biological signals has emerged for cartilage repair. given that chondrocytes are pivotal in the synthesis, composition modulation, structural arrangement of ecm components and hence the mechanical properties, we developed a protocol for baculovirus transduction of rat (ho et al., ) and rabbit chondrocytes. this protocol involves the incubation of cells with the virus at - °c for - h using dulbecco's phosphate-buffered saline (d-pbs) as the surrounding solution and confers efficiencies higher than % . the key to such high transduction efficiencies is the absence of nahco in d-pbs, which hinders the baculovirus transduction (shen et al., ) . consequently, the surrounding solution has been replaced with nahco -deficient dmem medium . critically, chondrocytes transduced with an egfp-expressing baculovirus remain capable of producing cartilage-specific collagen ii and gags, and grow into cartilaginous tissues when seeded into the porous, poly (l-lactide-co-glycolide) (plga) scaffold and cultivated dynamically . these data demonstrate that baculovirus transduction of chondrocytes does not obstruct the chondrocytes differentiation. therefore, we further constructed baculovirus vectors each expressing one growth factor (tgf-β , insulin-like growth factor- (igf- ) or bone morphogenetic protein- (bmp- )) known to stimulate chondrogenesis, and confirmed the protein expression in chondrocytes isolated from new zealand white (nzw) rabbits . among the baculovirus vectors, only the bmp- -expressing baculovirus (designated bac-cb) remarkably enhanced the aggrecan and collagen ii production by partially de-differentiated passage (p ) cells and restored the differentiation. the baculovirus expressing tgf-β (designated bac-ct) modestly augmented the chondrogenesis but was insufficient to reverse the de-differentiation status of p cells . nonetheless, co-transduction of de-differentiated p chondrocytes with bac-cb and bac-ct synergistically modulated the re-differentiation program (sung et al., ) . albeit the chondrogenic potential, bac-cb transduction alone was insufficient to support uniform d cartilage growth in the static culture due to the lack of mechanical stimulation and poor oxygen/ nutrient transfer. to tackle these problems, p chondrocytes transduced with bac-cb were seeded into plga scaffolds and cultured in a rotating-shaft bioreactor (rsb), which grew into cartilage-like tissues after -week dynamic culture . implantation of the engineered cartilages into the osteochondral defects of nzw rabbits resulted in the regeneration of hyaline cartilages at week and improved the integration of the host and engineered cartilages (chen et al., c) . massive segmental bony defects often occur following trauma or tumor resection and remain a clinical problem. for bone regeneration, bmscs have evolved to be a promising cell source as they are immunoprivileged, immunosuppressive and capable of differentiating into osteoblasts . in this regard, baculovirus transduces bmscs with efficiencies exceeding ≈ % under optimized conditions ), but the transduction efficiencies for bmscs-derived progenitors varies widely with the differentiation states at which the committed progenitors are transduced lee et al., b) . furthermore, transduction of human bmscs with bac-cb (which expresses the potent osteogenic factor bmp- ) directed in vitro commitment of naïve bmscs into osteoblasts (chuang et al., ) . after injection into the back subcutis of immunodeficient nude mice, the transduced bmscs resulted in progressive mineralization and ectopic bone formation (chuang et al., ) . implantation of the bac-cb-engineered human bmscs into rat calvarial defects stimulated mineralized bone matrix deposition and initiated the bone island formation at week , but without immunosuppression the xenogeneic cells were rejected and eradicated at week (chuang et al., ) . to circumvent the rejection, bmscs isolated from nzw rabbits were used for baculovirus transduction and allotransplantation. given the important roles of vascular endothelial growth factor (vegf) in angiogenesis, ossification and callus maturation, rabbit bmscs were transduced with bac-cv (expressing vegf) or bac-cb, mixed at a number ratio of : , seeded into plga scaffolds and implanted to the critical-size ( mm in length) femoral segmental defects of allogeneic, immunocompetent nzw rabbits, which represent a fairly rigorous bone healing scenario (lin et al., a ). x-ray radiography, positron emission tomography (pet), micro computed tomography (μct), immunohistochemical staining and biomechanical testing illustrated that the baculovirus-engineered cell/scaffold constructs not only accelerated the bone healing, but also gave rise to prominent angiogenesis and improved mechanical properties at week . adipose-derived stem cells (ascs) are another promising cell source for regenerative medicine thanks to the ease of isolation in abundance and capacity of osteogenic differentiation (levi et al., ) . however, ascs appear to be inferior to bmscs in osteogenic differentiation and ascs engineered by the conventional baculovirus transiently expressing bmp- /vegf (s group) led to significantly poorer healing of segmental femoral bone defects than bmscs engineered by the same vectors (fig. ) . to use ascs for repairing large, segmental bone defects, we surmised that sustained expression of factors promoting osteogenesis (bmp- ) and angiogenesis (vegf) are necessary. as such, we employed a hybrid baculovirus system developed previously for persistent expression . the dual vector system constitutes two baculoviruses whereby one expresses the flippase recombination enzyme (flp) while the other accommodates the bmp- or vegf cassette flanked by the flippase recognition target (frt) sequences. within the mammalian cells cotransduced with the hybrid baculoviruses, flp catalyzes the recombination of the frt-flanking cassette, resulting in the cassette excision off the baculovirus genome, formation of episomal circle and substantially prolonged transgene expression . likewise, the flp/frt-mediated recombination occurred efficiently in the nzw rabbit ascs, enabling persistent transgene expression for n days . allotransplantation of the nzw rabbit ascs transduced with the hybrid baculoviruses expressing bmp- /vegf into the critical-size femoral segmental defects accelerated the healing, improved the bone quality and angiogenesis when compared with transplanting ascs engineered with the conventional baculoviruses (fig. ) . therefore, ascs engineered by the hybrid baculovirus vector hold promise for repairing massive segmental defects . the safety profile of the hybrid baculovirus vectors was recently assessed using human bmscs as the host (chen et al., a) . transduction of human bmscs with the hybrid baculovirus neither compromised the cell viability/differentiation, nor resulted in transgene integration into the host chromosome. the transduction did not disrupt the bmscs karyotype, nor did it disturb the expression of wellknown proto-oncogenes (c-myc, n-ras, k-ras and h-ras) and tumor suppressor genes (p and p ). furthermore, the transduced bmscs did not induce tumor formation in nude mice. these data altogether ensure the safe use of the flp/frt-based hybrid vector for stem cell engineering and tissue regeneration. as discussed in section . , wild-type baculovirus triggers the innate immunity and potentiates the adaptive immune responses, which protect the animals from the infection of several viruses. these attributes have sparked explosive interests to develop baculovirus as a vector vaccine candidate, in which the antigens can be ( ) expressed by the vector within the host cells, ( ) displayed on the baculovirus surface or ( ) displayed and expressed by the vector (table ) . the feasibility of using baculovirus as a vaccine expression vector was first tested by in vivo inoculation of the baculovirus expressing the glycoprotein gb of pseudorabies virus (aoki et al., ) or hemagglutinin (ha) of h n influenza virus (abe et al., ) into mice, which raised antigen-specific antibody. moreover, baculovirus expressing the e glycoprotein of hcv or carcinoembryonic antigen could elicit antigen-specific t cell responses (facciabene et al., ) . similarly, subcutaneous (s.c.) and intraperitoneal (i.p.) immunizations of mice with a baculovirus expressing the antigens of severe acute respiratory syndrome coronavirus (sars-cov) induced humoral immune responses and th -biased cellular immunity (bai et al., ) . the efficacy of baculovirus-based vaccines has been potentiated by surface display of vsvg protein on the envelope. the vsvgpseudotyped baculovirus expressing the gp and m proteins of porcine reproductive and respiratory syndrome virus (prrsv) under the cmv promoter elicited anti-prrsv neutralizing antibodies and ifn-γ, and conferred better immunogenicity than the dna vaccine expressing the same antigens . conversely, i.m. immunizations of a mixture of vsvg-pseudotyped baculovirus expressing pseudorabies virus (prv) proteins triggered th -biased immune responses, as manifested by the induction of ifn-γ and prvspecific igg a antibodies (grabowska et al., ) . the immunization also provoked nk cell activity (accompanied by the production of ifn-α and ifn-γ) and protected mice against lethal prv challenge. i. m. immunization of chickens with another vsvg-pseudotyped baculovirus expressing ha of h n avian influenza virus (aiv) also evoked significantly higher levels of h -specific antibody and cellular immunity than those receiving dna vaccines, and conferred protection against lethal challenge with the homologous virus strain (wu et al., b) . similar vsvg-pseudotyped baculovirus vectors have been constructed to express the japanese encephalitis virus (jev) envelope protein (li et al., b) , the capsid protein of porcine circovirus type (pcv ) (fan et al., ) , and the toxoplasma gondii sag protein . all these studies showed that i.m. immunization of the vsvg-pseudotyped baculovirus vector results in stronger vaccine effects than the dna vaccine counterparts, which can be attributable to the adjuvant properties of baculovirus and more efficient transduction of muscle cells in vivo. the effective transduction of muscle cells is desired as the chance of antigen presentation to dcs is increased and baculovirus-mediated expression is considerably prolonged in myogenic cells (shen et al., ) . note, however, that vsvg pseudotyping induces cytotoxicity and impairs the baculovirus titer, hence a truncated version of vsvg comprised of the amino acid ectodomain, the transmembrane and ctd domains was designed for baculovirus pseudotyping (kaikkonen et al., ) . the truncated vsvg reduced the cytotoxicity and was exploited to construct two pseudotyped baculoviruses: one expressing the capsid protein of footand-mouth disease virus (fmdv) and the other encoding the capsid and a t-cell immunogen . i.m. inoculation of the fig. . the ascs engineered with the hybrid baculovirus augment the healing of massive bone defects. the nzw rabbit ascs were transduced with the hybrid baculovirus vectors conferring sustained expression of bmp- or vegf, mixed at a number ratio of : , loaded into cylindrical plga scaffolds ( . × cells/scaffold) and implanted to the critical-size segmental defects at the femora of nzw rabbits ( constructs/defect, designated l group). the s group contained ascs that were transduced with conventional baculoviruses transiently expressing bmp- /vegf and implanted in a similar fashion. the mock group comprised the mock-transduced ascs as the negative control. x-ray radiography, gross appearance examination, micro computed tomography (μct), hematoxylin and eosin (h&e) staining and cd -specific immunohistochemical staining (to detect blood vessel formation) performed at weeks post-implantation collectively demonstrated that the l group (persistently expressing bmp- and vegf) resulted in significantly improved bone healing and angiogenesis in comparison with the s group (transiently expressing bmp- and vegf) and mock group. stars indicate the new bone while arrows indicate the blood vessel formation. baculoviruses into mice induced the fmdv-specific neutralizing antibodies and ifn-γ, and expression of the additional t-cell immunogen augmented the immunogenicity. besides vsvg, human endogenous retrovirus (herv) envelope protein was used for pseudotyping the baculovirus (designated acherv-hp l ) that encoded the l capsid protein of human papillomavirus (hpv ) (lee et al., ) . after i.m. injection and booster injections at -week intervals, the mice developed similarly high levels of igg, iga and neutralization titers, as well as tremendously higher levels of ifn-γ when compared with the mice immunized with the commercial virus-like particle vaccine gardasil ® ( μl/dose). thus acherv-hp l holds promise as a cost-effective and efficient hpv vaccine. as mentioned in section . , surface display of heterologous protein/peptide has been achieved by fusion with gp gene and expression driven by either polyhedrin or p promoter. taking advantage of this technology, baculovirus displaying the rodent malaria plasmodium berghei circumsporozoite protein (pbcsp) (yoshida et al., ) , the antigenic site a of fmdv vp protein (tami et al., ) or the sars-cov spike protein (chang et al., ; feng et al., ) has been shown to induce potent immune responses. more recently, baculovirus vectors displaying the pfs protein of plasmodium falciparum (mlambo et al., ) and -kda carboxyl terminus of merozoite surface protein (pymsp ) of plasmodium yoelii were also developed as potential vaccines against malaria. i.m. and intranasal (i.n) immunizations of mice with the pymsp -displaying baculovirus induced mixed th /th -type immune responses, but i.n. immunization yielded higher pymsp -specific antibody titers and natural boosting after challenge. i.n. immunization also conferred complete protection thanks to the th /th -type immunity associated with tlr -dependent pathway . ha protein is the major antigen of influenza virus and, similar to gp , is a class i transmembrane protein on the viral envelope. gp based fusion was harnessed to display the ha of aiv using the ctd derived from ha or from gp (yang et al., ) . in comparison with the ha ctd, gp ctd endowed more efficient ha display and stimulated stronger hemagglutination inhibition (hi) titers crucial for neutralizing the live aiv (yang et al., ) . the profound effect of gp ctd on ha display was attested in a recent study which delineated that the signal peptide and ctd of gp could enhance the display of influenza ha on baculovirus surface, while the gp transmembrane domain impaired ha display (tang et al., ) . based on the finding, a baculovirus simultaneously displaying has derived from subclades of h n aiv was constructed. i.m. immunization of mice with this tetravalent baculovirus elicited hi titers against all homologous h n viruses, significantly reduced viral lung titers of challenged mice, raised high levels of ifn-γsecreting and ha-specific cd + t cells, and provided % protection against lethal challenge with homologous h n viruses (tang et al., ) . the same design concept exploiting the gp ctd was also adopted to display the σc and σb proteins of avian reovirus (lin et al., ) , e rns envelope glycoprotein , e (xu and liu, ) or ns protein (xu et al., ) of classical swine fever virus (csfv) and the e glycoprotein of jev (xu et al., ) . these studies collectively attested that the antigen can be efficiently displayed on the baculoviral envelope and induce humoral/cellular immune responses against the lethal viral challenge. other than displaying the antigen via genetic engineering, cd + t helper epitope (sferfeipke) and the major b cell epitope (wltekegsyp) derived from the ha of influenza virus a/pr/ / have also been chemically conjugated to baculovirus envelope (wilson et al., ) . i.n. administration of such baculovirus to mice elicited antigen-specific igg a/igg a, iga and ifn-γ, thus chemical coupling allows for the delivery of multiple epitopes to baculovirus. in addition, ha of aiv (h n ) has been displayed on another baculovirus bombyx mori npv (bmnpv) via fusion with gp (jin et al., ) . the virus was produced and purified from silkworm pupae infected with the recombinant virus. immunization of rhesus monkeys with aluminum hydroxide as the adjuvant at doses of mg/kg and . mg/kg elicited neutralizing antibodies and protected monkeys against influenza virus challenge. the vaccine did not cause appreciable toxicity at the dose as large as . mg/kg in cynomolgus monkeys and . mg/kg in mice, indicating the safe vaccine doses (jin et al., ) . food-and-mouth disease virus (cao et al., ) gp and m proteins porcine reproductive and respiratory syndrome virus hpv- l protein human papillomavirus (lee et al., ) toxoplasma gondii (fang et al., ) display hemagglutinin protein influenza virus (jin et al., ; tang et al., ; yang et al., ) xu et al., ; xu and liu, ) -kda carboxyl terminus of merozoite surface protein plasmodium yoelii pfs protein plasmodium falciparum (mlambo et al., ) dual hemagglutinin protein influenza virus he et al., ; lu et al., ; prabakaran et al., ; prabakaran et al., a prabakaran et al., , b prabakaran et al., , c plasmodium falciparum (strauss et al., ) . . baculovirus as a dual vector for antigen expression/display it is perceivable that the antigen-expressing baculovirus mimicks the dna vaccine. following vector injection into the hosts, the antigen is expressed and presented via mhc i pathway in the transduced cells, and activates cd + t cells. conversely, the antigen-displaying baculovirus mimicks the subunit vaccine. antigens on the envelope are internalized by professional antigen presenting cells (apcs) such as macrophages and dcs, and presented by the mhc ii pathway to stimulate humoral immune responses. to fully exploit both mechanisms, a dual baculovirus vector that simultaneously displays and expresses the plasmodium falciparum circumsporozoite (cs) protein was developed as a human malaria vaccine (strauss et al., ) . this vector contained two expression cassettes, one encoding the cs-gp fusion protein under the polyhedrin promoter while the other accommodating the cs gene under the cmv promoter, such that cs can be displayed on the envelope and expressed after transduction into mammalian cells. upon i.m. injection into mice, the dual vector induced higher anti-cs antibody titers and higher frequencies of cs-specific cd + and cd + t cells than the vectors that only displayed or only expressed cs. recently, we adopted a similar approach and constructed vectors: bac-ha harbored the ha gene of aiv under the p promoter for ha display; bac-cha expressed ha under the cmv promoter while the dual vector bac-cha/ha encompassed both expression cassettes in opposite orientations, such that bac-cha/ ha displayed and expressed ha in the transduced cells . all vectors, after administration (i.m., i.n. and s.c.) into balb/c mice, provoked ha-specific humoral (igg , igg a and hi titers), mucosal (iga titers) and cellular (ifn-γ and il- producing t cells and ifn-γ + /cd + t cells) immune responses. the strong cellular immunity provoked by bac-ha , which in theory favors the mhc ii pathway and preferentially elicits humoral immune responses, was likely due to the potent adjuvant effects of baculovirus. regardless, via either administration route the dual vector bac-cha/ha triggered superior or at least comparable ha-specific immune responses than the other two vaccine forms, demonstrating the advantages of the dual form for vaccine design . instead of using two separate cassettes as above, yoshida et al. ( ) devised a single cassette system whereby the pbcsp-gp fusion gene was driven by a tandem promoter consisting of the cmv and polyhedrin promoters, so that pbcsp was expressed in insect cells and mammalian cells. i.m. immunization with this baculovirus elicited both th and th responses as evidenced by the high pbcsp-specific igg / igg a titers and pbcsp-specific cd + t-cells responses, and conferred % protection against sporozoite challenge (yoshida et al. ). the same dual expression system was subsequently utilized to express plasmodium vivax transmission-blocking immunogen (pvs ) for the development of malarial transmission-blocking vaccines against the sexual stages of the parasite (blagborough et al., ) . both i.n. and i.m. immunizations of mice induced a mixed th /th response as evidenced by high pvs -specific igg and igg a/igg b titers as well as a strong transmission-blocking response after challenge. another simple approach to developing the dual vector is to drive the antigen expression using the white spot syndrome virus (wssv) ie promoter, which is active in insect, mammalian and avian cells (gao et al., ; he et al., ) . baculovirus encoding the wssv vp envelope protein driven by the wssv ie promoter was able to display vp on the envelope (syed musthaq et al., ) . immunization of shrimp with this baculovirus resulted in vp expression in shrimp tissues, elevated the survival rate and reduced the wssv viral load after wssv infection. wssv ie promoter was also used in conjunction with vsvg pseudotyping. i.m. injection of the vsvg-pseudotyped baculovirus that expressed the e protein of csfv under the ie promoter induced csfv-specific neutralizing antibodies and lymphoproliferative responses in mice (li et al., a) . wssv ie promoter has been most extensively employed by the kwang group to drive the expression of ha of h n aiv, which enabled ha display on the envelope and conferred the viral particle hemagglutination activity . i.m. injection of this vector (designated bacha) into mice elicited the antibodies with hi titers against the tested influenza virus. in sf- cells, wssv ie promoter was more active than cmv promoter, thereby giving rise to more efficient ha incorporation into the baculoviral envelope . the ie promoter also resulted in strong ha expression in the lung (after i.n. inoculation) and thymus (after i.m. inoculation) in chickens. as a result, immunization of chickens with the baculovirus bearing the ie promoter (bacha) elicited higher anti-ha antibody levels than that bearing the cmv promoter . however, i.n. inoculation of bacha stimulated low anti-ha igg titers (prabakaran et al., ) . to enhance the vaccine efficacy via the i.n. route, bacha was co-administered with recombinant cholera toxin b subunit (rctb) as the adjuvant, which significantly enhanced the serum igg, mucosal iga and serum microneutralization titers. with the adjuvant, bacha also triggered higher ha-specific humoral and mucosal immune responses than the inactivated h n virus adjuvanted with the same dose of rctb, and conferred complete protection against challenge with homologous and heterologous h n strains (prabakaran et al., ) . additionally, gastrointestinal delivery of bacha into mice by oral gavage led to transduction in vivo and remarkably boosted the ha-specific igg, hi and mucosal iga titers (prabakaran et al., c) . the live bacha triggered stronger cross-clade neutralization against heterologous h n strains than the inactivated bacha, and provided % protection against challenge with lethal doses of homologous and heterologous h n . moreover, after challenge the immunized mice exhibited only minimal bronchitis in lungs and regained their body weight more rapidly (prabakaran et al., c) . the oral vaccine efficacy was further potentiated by encapsulating the live bacha within a reverse micelle structure of phosphatidylcholine, which provided protection against the destructive environment in the intestinal lumen (prabakaran et al., b) . in comparison with the non-encapsulated bacha, gastrointestinal delivery of the encapsulated baculovirus into mice led to significantly ameliorated ha-specific igg and iga responses, and higher hi titers. the encapsulated vaccine induced strong cross-clade neutralization titers against heterologous h n strains and conferred protection against infection with highly pathogenic, heterologous h n viruses (prabakaran et al., b) . using the same wssv ie approach, more recently kwang and coworkers selected ha proteins from different vaccine strains for expression and display on the baculovirus surface (prabakaran et al., a) . the ha proteins covered the entire variants in the neutralizing epitopes among the h n lineages. s.c. immunization of mice with a mixture of baculoviruses displaying the ha proteins (tri-bacha) induced antibodies capable of neutralizing viruses from clades , . , . , , , and of h n viruses. in contrast, s.c. immunization with a single ha-displaying baculovirus (mono-bacha) or a single strain of inactivated whole virus vaccine neutralized only clade (homologous), clade . , and clade . viruses. also, the tri-bacha vaccine protected % of the mice against challenge with three different clades (clades . , . , and . ) of h n viruses. since the discovery in that baculovirus effectively transduces mammalian cells and mediates transgene expression, baculovirus has emerged as a promising gene delivery vector. albeit the rapidly growing lists of permissive cells and applications, relatively little is known about what happens inside the cells after baculovirus transduction. evidence accumulating in recent years has indicated that after entry, baculovirus can translocate into the nucleus through a complex cascade of steps and express baculoviral genes and the transgene. the entire process results in the recognition of baculovirus by the cells via the tlr -dependent and -independent pathways, and disturbs the expression of a small percentage of host genes, particularly those pertaining to the innate immune responses. however, disparity does exist between reports and the exact intracellular events remain elusive. the lack of comprehensive knowledge about the events governing the virus entry, intracellular transport and cellular responses will constitute a roadblock to future applications in the clinical setting, thus entailing extensive research to elucidate the underlying mechanisms. although the innate immune responses stir up concerns regarding the safety of baculovirus in gene therapy, these responses represent opportunities to harness baculovirus as a vector to defend against infectious agents and tumors. indeed, baculovirus-based vaccine has captured explosive interests over the past years and in one case has entered into trials in primates (jin et al., ) . thanks to the intrinsic adjuvant properties, in most studies baculovirus-based vaccines were able to trigger potent immune responses in the absence of additional adjuvants. due to the widespread use and encouraging preclinical data in comparison with other vaccine forms (e.g. dna vaccine, viruslike particle vaccine and whole virus vaccine), it is envisioned that baculovirus-based vaccines can progress into next phase in the near future. the capability of shrna/mirna delivery also confers baculovirus an edge for target gene regulation and for future applications in antiviral therapy and immunotherapy. furthermore, baculovirus transduction of neural cells (kenoutis et al., ) and adult stem cells (bak et al., ; ho et al., ; zeng et al., ) does not markedly alter the inherent properties and mitigate the differentiation capacity, warranting baculovirus a promising vector for cell therapy and tissue engineering. recent progresses in the baculovirus vectors engineering with respect to surface modification, minimization of in vivo inactivation, transgene targeting and prolongation of expression further corroborate the potentials of baculovirus in these applications. to advance the baculovirus technology from bench to bedside, other roadblocks still stand in the way. over the past few years, a wealth of literature has addressed the problems in baculoviral vector production carinhas et al., carinhas et al., , dojima et al., ; tsai et al., ) , quantification (chan et al., ; ferris et al., ; kärkkäinen et al., ; lo et al., ; lo and chao, ; roldao et al., ) , purification (chen et al., a; kaikkonen et al., ; transfiguracion et al., ; vicente et al., vicente et al., , a vicente et al., , b wu et al., ) and quality assurance (ihalainen et al., ; jorio et al., ) . new baculoviral vectors taking advantage of hybrid promoters (gao et al., ; keil et al., ; lackner et al., ; pan et al., ) and new regulatory elements (du et al., ; mahonen et al., ) are also being developed and evaluated for their potential applications. additionally, the transduction conditions (keil et al., ; pan et al., ) , supplements (guo et al., ; yin et al., ) and parameters (lee et al., a (lee et al., , b shen et al., shen et al., , dictating the transduction efficiencies have been evaluated. these technological progresses undoubtedly will facilitate the production of baculoviral vectors in compliance of cgmp regulations, and advance baculovirus from a research tool to clinical applications. host innate immune responses induced by baculovirus in mammals baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice involvement of the toll-like receptor signaling pathway in the induction of innate immunity by baculovirus baculovirus induces type i interferon production through toll-like receptor-dependent and -independent pathways in a cell-type-specific manner baculovirus-mediated periadventitial gene transfer to rabbit carotid artery induction of antibodies in mice by a recombinant baculovirus expressing pseudorabies virus glycoprotein b in mammalian cells vaccination of mice with recombinant baculovirus expressing spike or nucleocapsid protein of sars-like coronavirus generates humoral and cellular immune responses baculovirus-transduced bone marrow mesenchymal stem cells for systemic cancer therapy human embryonic stem cell-derived mesenchymal stem cells as cellular delivery vehicles for prodrug gene therapy of glioblastoma high mobility group box promoter-controlled suicide gene expression enables targeted glioblastoma treatment cell density effect in the baculovirus-insect cells system: a quantitative analysis of energetic metabolism intranasal and intramuscular immunization with baculovirus dual expression system-based pvs vaccine substantially blocks plasmodium vivax transmission amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase gene expression profiling to define host response to baculoviral transduction in the brain a pseudotype baculovirus expressing the capsid protein of footand-mouth disease virus and a t-cell immunogen shows enhanced immunogenicity in mice baculovirus production for gene therapy: the role of cell density, multiplicity of infection and medium exchange improving baculovirus production at high cell density through manipulation of energy metabolism determination of the baculovirus transducing titer in mammalian cells induction of il- release in lung cells via activator protein- by recombinant baculovirus displaying severe acute respiratory syndrome-coronavirus spike proteins: identification of two functional regions non-polar distribution of green fluorescent protein on the surface of autographa californica nucleopolyhedrovirus using a heterologous membrane anchor baculovirus-mediated production of hdv-like particles in bhk cells using a novel oscillating bioreactor combination of baculovirus-mediated gene transfer and rotating-shaft bioreactor for cartilage tissue engineering combination of baculovirus-mediated bmp- expression and rotating-shaft bioreactor culture synergistically enhances cartilage formation concanavalin a affinity chromatography for efficient baculovirus purification baculovirus transduction of mesenchymal stem cells triggers the toll-like receptor (tlr ) pathway the repair of osteochondral defects using baculovirus-mediated gene transfer with de-differentiated chondrocytes in bioreactor culture baculovirus as an avian influenza vaccine vector: differential immune responses elicited by different vector forms biosafety assessment of human mesenchymal stem cells engineered by hybrid baculovirus vectors membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells a rapid and efficient method to express target genes in mammalian cell by baculovirus a novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus baculovirus as a new gene delivery vector for stem cells engineering and bone tissue engineering baculovirus transduction of mesenchymal stem cells: in vitro responses and in vivo immune responses after cell transplantation xenotransplantation of human mesenchymal stem cells into immunocompetent rats for calvarial bone repair baculovirus expression vectors for insect and mammalian cells transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector baculoviruses and mammalian cell-based assays for drug screening production of scfv-displaying bmnpv in silkworm larvae and its efficient purification autographa californica multicapsid nucleopolyhedrovirus efficiently infects sf cells and transduces mammalian cells via direct fusion with the plasma membrane at low ph the combined use of viral transcriptional and posttranscriptional regulatory elements to improve baculovirus-mediated transient gene expression in human embryonic stem cells baculovirus vector requires electrostatic interactions including heparan sulfate for efficient gene transfer in mammalian cells baculoviruses deficient in ie gene function abrogate viral gene expression in transduced mammalian cells improving baculovirus transduction of mammalian cells by surface display of a rgd-motif baculovirus vectors elicit antigen-specific immune responses in mice construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type in mice construction and immunogenicity of pseudotype baculovirus expressing toxoplasma gondii sag protein in balb/c mice model baculovirus surface display of sars coronavirus (sars-cov) spike protein and immunogenicity of the displayed protein in mice models evaluation of the virus counter ® for rapid baculovirus quantitation expression of autographa californica multiple nucleopolyhedrovirus genes in mammalian cells and upregulation of the host β-actin gene high efficiency gene transfer into cultured primary rat and human hepatic stellate cells using baculovirus vectors efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie promoter, a novel shuttle promoter between insect cells and mammalian cells targeting micrornas in cancer: rationale, strategies and challenges a surface-modified baculovirus vector with improved gene delivery to b-lymphocytic cells preclinical evaluation of innate immunity to baculovirus gene therapy vectors in whole human blood indications for cell stress in response to adenoviral and baculoviral gene transfer observed by proteome profiling of human cancer cells expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced nlfk cells baculovirus for eukaryotic protein display developments in the use of baculoviruses for the surface display of complex eukaryotic proteins new baculovirus recombinants expressing pseudorabies virus (prv) glycoproteins protect mice against lethal challenge infection comparison between recombinant baculo-and adenoviralvectors as transfer system in cardiovascular cells sodium butyrate enhances the expression of baculovirusmediated sodium/iodide symporter gene in a lung adenocarcinoma cells antiglioma effects of combined use of a baculovirual vector expressing wild-type p and sodium butyrate upregulation of proinflammatory cytokines and no production in bv-activated avian macrophage-like cell line (hd ) requires mapk and nf-κb pathways involvement of tlr in baculovirus-induced interleukin- gene expression in avian macrophage-like cell line hd wssv ie promoter is more efficient than cmv promoter to express h hemagglutinin from influenza virus in baculovirus as a chicken vaccine insect baculoviruses strongly potentiate adaptive immune responses by inducing type i ifn baculovirus expression systems for recombinant protein production in insect cells highly efficient baculovirus-mediated gene transfer into rat chondrocytes transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells baculovirus transduction of human mesenchymal stem cell-derived progenitor cells: variation of transgene expression with cellular differentiation states complement inhibition rescued mice allowing observation of transgene expression following intraportal delivery of baculovirus in mice protection of baculovirus-vectors against complement-mediated inactivation by recombinant soluble complement receptor type baculovirus as a highly efficient expression vector in insect and mammalian cells baculovirus vectors for gene therapy baculoviral vectors for gene delivery: a review enhancement and prolongation of baculovirusmediated expression in mammalian cells: focuses on strategic infection and feeding baculovirus as an expression and/or delivery vehicle for vaccine antigens combination of baculovirus-mediated gene delivery and packed-bed reactor for scalable production of adeno-associated virus suppression of gastric cancer growth by baculovirus vector-mediated transfer of normal epithelial cell specific- gene efficient gene delivery into fish cells by an improved recombinant baculovirus incorporation of decay-accelerating factor into the baculovirus envelope generates complement-resistant gene transfer vectors morphological characterization of baculovirus autographa californica multiple nucleopolyhedrovirus host innate immune receptors and beyond: making sense of microbial infections sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity expression of seap (secreted alkaline phosphatase) by baculovirus mediated transduction of hek cells in a hollow fiber bioreactor system safety and immunogenicity of h n influenza vaccine based on baculovirus surface display system of bombyx mori analysis of baculovirus aggregates using flow cytometry truncated vesicular stomatitis virus g protein improves baculovirus transduction efficiency in vitro and in vivo targeting and purification of metabolically biotinylated baculovirus screening of complement inhibitors: shielded baculoviruses increase the safety and efficacy of gene delivery acquisition of complement resistance through incorporation of cd /decay-accelerating factor into viral particles bearing baculovirus gp inhibition of hiv- replication by vesicular stomatitis virus envelope glycoprotein pseudotyped baculovirus vector-transduced ribozyme in mammalian cells a -well format for a high-throughput baculovirus generation, fast titering and recombinant protein production in insect and mammalian cells novel vectors for simultaneous high-level dual protein expression in vertebrate and insect cells by recombinant baculoviruses baculovirus-mediated gene delivery into mammalian cells does not alter their transcriptional and differentiating potential but is accompanied by early viral gene expression regulation of transduction efficiency by pegylation of baculovirus vector in vitro and in vivo direct vaccination with pseudotype baculovirus expressing murine telomerase induces anti-tumor immunity comparable with rna-electroporated dendritic cells in a murine glioma model suppression of tumor growth in xenograft model mice by programmed cell death gene delivery using folate-peg-baculovirus baculovirus is an efficient vector for the transduction of the eye: comparison of baculovirus-and adenovirus-mediated intravitreal vascular endothelial growth factor d gene transfer in the rabbit eye ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses characterization of baculovirus autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells induction of natural killer cell-dependent antitumor immunity by the autographa californica multiple nuclear polyhedrosis virus baculovirus display of single chain antibody (scfv) using a novel signal peptide recombinant baculoviruses as expression vectors for insect and mammalian cells recombinant baculoviruses as mammalian cell gene delivery vectors baculovirus as versatile vectors for protein expression in insect and mammalian cells baculovirus gene delivery: a flexible assay development tool baculovirus capsid display: a novel tool for transduction imaging mesenchymal stem cells expressing osteogenic and angiogenic factors synergistically enhance bone formation in a mouse model of segmental bone defect baculovirus-mediated immediateearly gene expression and nuclear reorganization in human cells clathrin-independent entry of baculovirus triggers uptake of e. coli in non-phagocytic human cells a bicistronic baculovirus vector for transient and stable protein expression in mammalian cells soluble forms of the cell adhesion molecule l produced by insect and baculovirus-transduced mammalian cells enhance schwann cell motility baculovirus transduction of rat articular chondrocytes: roles of cell cycle variation of baculovirus-harbored transgene transcription among mesenchymal stem cell-derived progenitors leads to varied expression baculovirus transduction of chondrocytes elicits interferon-α/β and suppresses transgene expression development of a novel viral dna vaccine against human papillomavirus: acherv-hp l baculoviruses exhibit restricted cell type specificity in rat brain: a comparison of baculovirus-and adenovirus-mediated intracerebral gene transfer in vivo generation of lentivirus vectors using recombinant baculoviruses production and purification of lentiviral vectors generated in t suspension cells with baculoviral vectors human adipose derived stromal cells heal critical size mouse calvarial defects axonal transport of recombinant baculovirus vectors immune responses induced by a bacmam virus expressing the e protein of classical swine fever virus in mice immunization with pseudotype baculovirus expressing envelope protein of japanese encephalitis virus elicits protective immunity in mice baculovirus surface display of σc and σb proteins of avian reovirus and immunogenicity of the displayed proteins in a mouse model the healing of critical-sized femoral segmental bone defects in rabbits using baculovirus-engineered mesenchymal stem cells baculovirus as a gene delivery vector for cartilage and bone tissue engineering recent patents on the baculovirus systems adipose-derived stem cells engineered with the persistently expressing hybrid baculovirus augment the healing of massive bone defects efficient and stable gene expression in rabbit intervertebral disc cells transduced with a recombinant baculovirus vector stimulation of baculovirus transcriptome expression in mammalian cells by baculoviral transcriptional activators ring and coiled-coil domains of baculovirus ie are critical in strong activation of the cytomegalovirus major immediate-early promoter in mammalian cells baculovirus-transduced mouse amniotic fluid-derived stem cells maintain differentiation potential maximizing baculovirus-mediated foreign proteins expression in mammalian cells rapid titer determination of baculovirus by quantitative real-time polymerase chain reaction development of a hybrid baculoviral vector for sustained transgene expression rapid baculovirus titration based on regulatable green fluorescent protein expression in mammalian cells functional entry of baculovirus into insect and mammalian cells is dependent on clathrin-mediated endocytosis suppression of porcine arterivirus replication by baculovirusdelivered shrna targeting nucleoprotein baculovirus surface-displayed hemagglutinin of h n influenza virus sustains its authentic cleavage, hemagglutination activity, and antigenicity initiation of hepatitis b virus genome replication and production of infectious virus following delivery in hepg cells by novel recombinant baculovirus vector baculovirus as vaccine vectors post-transcriptional regulatory element boosts baculovirus-mediated gene expression in vertebrate cells culture medium induced vimentin reorganization associates with enhanced baculovirus-mediated gene delivery baculovirus display: a multifunctional technology for gene delivery and eukaryotic library development enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides tumor targeting of baculovirus displaying a lymphatic homing peptide surface display of igg fc on baculovirus vectors enhances binding to antigen-presenting cells and cell lines expressing fc receptors baculovirus entry into human hepatoma cells characterization of hcv-like particles produced in a human hepatoma cell line by a recombinant baculovirus efficient delivery and regulable expression of hepatitis c virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors chromosomal integration of transduced recombinant baculovirus dna in mammalian cells functional immunogenicity of baculovirus expressing pfs , a human malaria transmission-blocking vaccine candidate antigen baculovirus treatment fully protects mice against a lethal challenge of fmdv optimization of baculovirus transduction on freestyle (™) cells for the generation of influenza b/lee/ rna interference mediated in human primary cells via recombinant baculoviral vectors baculovirus up-regulates antiviral systems and induces protection against infectious bronchitis virus challenge in neonatal chicken potential of adenovirus and baculovirus vectors for the delivery of shrna against morbilliviruses hybrid cytomegalovirus enhancer-h promoter-based plasmid and baculovirus vectors mediate effective rna interference efficient gene delivery into mammalian cells by recombinant baculovirus containing a hybrid cytomegalovirus promoter/semliki forest virus replicon antitumor effects of a recombinant pseudotype baculovirus expressing apoptin in vitro and in vivo baculovirus reveals a new ph-dependent direct cell-fusion pathway for cell entry and transgene delivery a chimeric baculovirus displaying bovine herpesvirus- (bhv- ) glycoprotein d on its surface and their immunological properties baculovirus-mediated gene expression in chicken primary cells protective immunity against influenza h n virus challenge in mice by intranasal co-administration of baculovirus surfacedisplayed ha and recombinant ctb as an adjuvant neutralizing epitopes of influenza virus hemagglutinin: target for the development of a universal vaccine against h n lineages reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against h n infection in mice gastrointestinal delivery of baculovirus displaying influenza virus hemagglutinin protects mice against heterologous h n infection enhanced gene delivery by avidin-displaying baculovirus magnetic resonance imaging of viral particle biodistribution in vivo spect/ct imaging of baculovirus biodistribution in rat error assessment in recombinant baculovirus titration: evaluation of different methods improvement in nuclear entry and transgene expression of baculoviruses by disintegration of microtubules in human hepatocytes an orip/ebna- -based baculovirus vector with prolonged and enhanced transgene expression the autographa californica nuclear polyhedrosis virus acnpv induces functional maturation of human monocyte-derived dendritic cells baculovirus-mediated gene transfer is attenuated by sodium bicarbonate sustained baculovirus-mediated expression in myogenic cells a novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors effective transduction of osteogenic sarcoma cells by a baculovirus vector transduction of avian cells with recombinant baculovirus baculoviral capsid display of his-tagged zno inorganic binding peptide hepatitis b virus (hbv)-specific short hairpin rna is capable of reducing the formation of hbv covalently closed circular (ccc) dna but has no effect on established ccc dna in vitro baculovirus-based vaccination vectors allow for efficient induction of immune responses against plasmodium falciparum circumsporozoite protein modulation of chondrocyte phenotype via baculovirus-mediated growth factor expression baculovirus-mediated growth factor expression in dedifferentiated chondrocytes accelerates redifferentiation: effects of combinational transduction stable replication of the ebna /orip-mediated baculovirus vector and its application to anti-hcv gene therapy baculovirus-mediated bispecific short-hairpin smallinterfering rnas have remarkable ability to cope with both influenza viruses a and b suppression of hepatitis c virus replication by baculovirus vector-mediated short-hairpin rna expression baculovirus activates murine dendritic cells and induces non-specific nk cell and t cell immune responses induction of antitumor immunity against mouse carcinoma by baculovirus-infected dendritic cells localization of vp on the baculovirus envelope and its immunogenicity against white spot syndrome virus in penaeus monodon immunological properties of fmdv-gp fusion proteins expressed on sf cell and baculovirus surfaces hemagglutinin displayed baculovirus protects against highly pathogenic influenza characterization of cell-surface determinants important for baculovirus infection in vitro and in vivo gene delivery by recombinant baculoviruses baculovirus vector for gene delivery and vaccine development high yield purification of functional baculovirus vectors by size exclusion chromatography factors influencing the production and storage of baculovirus for gene delivery: an alternative perspective from the transducing titer assay baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids purification of recombinant baculoviruses for gene therapy using membrane processes analysis of adsorption of a baculovirus bioreaction bulk on an ion-exchange surface by surface plasmon resonance modeling electrostatic interactions of baculovirus vectors for ion-exchange process development baculovirus-mediated gene expression in zebrafish potential cancer gene therapy by baculoviral transduction baculovirus as a highly efficient gene delivery vector for the expression of hepatitis delta virus antigens in mammalian cells recombinant baculovirus containing the diphtheria toxin a gene for malignant glioma therapy construction and immunogenicity of pseudotype baculovirus expressing gp and m protein of porcine reproductive and respiratory syndrome virus inhibition of nasopharyngeal carcinoma growth by rta-expressing baculovirus vectors containing orip dna methyltransferase inhibitors increase baculovirusmediated gene expression in mammalian cells when applied before infection delivery of vaccine peptides by rapid conjugation to baculovirus particles ion-exchange membrane chromatography method for rapid and efficient purification of recombinant baculovirus and baculovirus gp protein combinatorial control of suicide gene expression by tissuespecific promoter and microrna regulation for cancer therapy a pseudotype baculovirus-mediated vaccine confers protective immunity against lethal challenge with h n avian influenza virus in mice and chickens baculovirus surface display of e envelope glycoprotein of classical swine fever virus and immunogenicity of the displayed proteins in a mouse model baculovirus surface display of e rns envelope glycoprotein of classical swine fever virus baculovirus surface display of ns nonstructural protein of classical swine fever virus baculovirus surface display of e envelope glycoprotein of japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models establishment of medakafish as a model for stem cell-based gene therapy: efficient gene delivery and potential chromosomal integration by baculoviral vectors avian influenza virus hemagglutinin display on baculovirus envelope: cytoplasmic domain affects virus properties and vaccine potential polyethylenimine coating to produce serum-resistant baculoviral vectors for in vivo gene delivery baculovirus vector-mediated transfer of nis gene into colon tumor cells for radionuclide therapy baculovirus virions displaying plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection a baculovirus dual expression system-based malaria vaccine induces strong protection against plasmodium berghei sporozoite challenge in mice baculovirus-based nasal drop vaccine confers complete protection against malaria by natural boosting of vaccine-induced antibodies in mice transduction of vertebrate cells with spodoptera exigua multiple nucleopolyhedrovirus f protein-pseudotyped gp -null autographa californica multiple nucleopolyhedrovirus high-efficiency transient transduction of human embryonic stem cell-derived neurons with baculoviral vectors a novel method for isolation of membrane proteins: a baculovirus surface display system baculovirus expression of cloned porcine arterivirus generates infectious particles in both insect and mammalian cells display of heterologous proteins on gp null baculovirus birions and enhanced budding mediated by a vesicular stomatitis virus g-stem construct the feasibility of using a baculovirus vector to deliver the sodium-iodide symporter gene as a reporter the authors acknowledge the financial support from the national tsing hua university booster program ( n e , n e , n e ), cgmh-nthu joint research program ( n e , cmrpg , cmrpg ) and national science council ( - -e- - -my , - -e- - -my , nsc - -e- - -cc , nsc - -b- - ), taiwan. key: cord- -jpw hen authors: byeon, jung hye; lee, jin chul; choi, ic sun; yoo, young; park, sang hee; choung, ji tae title: comparison of cytokine responses in nasopharyngeal aspirates from children with viral lower respiratory tract infections date: - - journal: acta paediatr doi: . /j. - . . .x sha: doc_id: cord_uid: jpw hen aim: to determine whether nasopharyngeal aspirates (npas) cytokine response is different according to the causative viruses in children with lower respiratory tract infections (lrti). methods: npas from children with lrti caused by respiratory virus were evaluated. based on the proven viral agents, lrti patients were divided into four groups. levels of il‐ , il‐ and ifn‐γ were determined by elisa. results: patients with influenza virus infection demonstrated significantly lower il‐ and il‐ levels than those with other three groups. patients with respiratory syncytial virus (rsv) infection showed an increase in production of il‐ and il‐ , and a decrease in the ifn‐γ level when compared to patients with influenza virus infection. interestingly, a similar th response was seen in patients with parainfluenza virus or adenovirus infection. conclusion: these results demonstrate that respiratory viruses can induce different local cytokine responses. however, th biased responses are not unique for rsv but seem to be predominant in respiratory viruses of young children. respiratory viruses are important causes of lower respiratory infections in infants and children. cytokines are thought to be important in the pathogenesis of inflammation in the respiratory tract ( ) . several studies ( - ) have investigated cytokine profiles in nasopharyngeal aspirates (npas) during viral lower respiratory tract infections (lrti) in children. previous studies have suggested that respiratory viruses could stimulate both t helper cell type (th ) cytokines and t helper cell type (th ) cytokines. despite the scanty data available, it is apparent that cytokine responses of lrti are quite different according to the causative organisms. previous studies ( , ) were demonstrated that patients with respiratory syncytial virus (rsv) infection and those with influenza virus infection induce different cytokine profiles. influenza virus infection leads to a rapid proinflammatory reaction, which may condition the infected host for the subsequent virus antigen-specific th defense. while, cytokine profiles of rsv infections in children demonstrate variable results. infants with rsv infection elicit a predominant th response such as interleukin (il)- or il- ( , ) . however, it has been shown that in addition to il- and il- , th cytokines such as interferon (ifn)-γ are significantly elevated in rsv infection ( ) . in addition, an in vitro model of respiratory virus infection showed different responses of the cells with infected viruses ( ) . these results have demonstrated that cytokine responses of the cells infected with different respiratory viruses depend on infectious agents and on the host characteristics. cytokines are thought to be important in the pathogenesis of inflammation in the respiratory tract. lower respiratory tract secretions are usually obtained for cytokine measurement by bronchoalveolar lavage (bal), which is an invasive technique and requires the use of an anesthetic in young children. on the other hand, npa is often used for determining cytokine responses of viral respiratory infections in children. the present study was performed to determine whether clinical responses in children with lrti would differ according to causative viruses and to examine the differential patterns of il- , il- and ifn-γ concentrations in npa during acute viral lower respiratory infections according to the causative organisms. a total of children with signs and symptoms of acute viral lrti who were admitted to korea university anam hospital during a period of months (from july to june ) were enrolled in the present study. characteristic lrti symptoms included runny nose, coughing, wheezing and difficulty in breathing, with or without fever. of a total of cases with positive viral culture, two or more viruses were isolated from the same specimen in patients ( . %). of these children, cases with a single viral culture were selected as the study group. based on the proven viral agents, lrti patients were divided into four groups: patients with influenza virus (the influenza virus group), patients with parainfluenza virus (the parainfluenza virus group), patients with rsv (the rsv group) and patients with adenovirus (the adenovirus group). complete history taking, physical examination and routine laboratory tests were performed on all subjects. patients with bacterial or mycoplasmal infections or those under weeks of age were excluded. bacterial or mycoplasmal infections were identified as a positive blood culture for bacteria or a -fold or greater increase in the mycoplasma antibody titer. those who have a past history of chronic lung diseases such as asthma or bronchopulmonary dysplasia were also excluded. parents gave written informed consent for their children to participate in the study. the study protocol was approved by the hospital ethics committee. collection and preparation of specimens npas were collected usually within the first h of admission as part of the routine hospital procedure. in a small number of cases, which were admitted at nighttime or on a sunday, npas were collected sometime after but within h of admission. a polyethylene suction catheter was placed into the nasopharyngeal cavity via the nostril. aspiration was then conducted using a suction pump. npas collected from each patient were used for both virus culture and cytokine analysis. the specimen for culture was immediately placed in ml of hh medium after collection, transported to the virus laboratory on ice and kept at − • c until cultures were performed. npas from patients were cultured by using the cryopreserved r-mix cultures (diagnostic hybrids inc., athens, oh, usa) for four respiratory viruses (influenza virus, parainfluenza virus, rsv and adenovirus). identification of rhinovirus, coronavirus, bocavirus and metapneumovirus was not performed, as these were not a part of the routine service offered by the hospital virology department. analysis of viral culture positive cases we analysed characteristics including age, sex and the clinical diagnosis of the culture proven cases. the clinical diagnosis of acute lrti included tracheobronchitis, bronchiolitis and pneumonia. measurement of the total serum ige, serum eosinophil cationic protein (ecp) and blood eosinophil counts total serum ige levels were measured using coat-a-count total ige irma (diagnostic products co., los angeles, ca, usa). the serum ecp level was measured using a commercially available fluoroimmunoassay kit (pharmacia ecp unicap system feia; pharmacia diagnostics, uppsala, sweden) which had a detection limit of less than . μg/l. blood sample collection, serum preparation and serum ecp analyses were performed according to the manufacturer's instructions. the number of peripheral blood eosinophils was counted with blood samples containing ethylenediaminetetraacetic acid (edta) using an automated haematology analyzer (coulter counter stks; beckman coulter, fullerton, ca, usa). before cytokine measurement, each specimen was weighed and diluted to a volume of ml using phosphate-buffered saline (pbs). the final volume of npa/pbs solution was recorded and the exact dilution of npa was calculated. il- , il- and ifn-γ levels in npas were measured using a commercial elisa kit (bd inc., san diego, ca, usa) according to the manufacturer's instructions. the detection limit was pg/ml for il- , il- and ifn-γ , respectively. all assays were run in duplicate, and the mean value was used for statistical analysis. cytokine levels in npas were expressed per milliliter and were presented as means ± sd. we assigned cytokine values that were below the limit of detection a value of zero for facilitating statistical analyses ( ) . the values for serum total ige, serum ecp and blood eosinophil counts were logarithmically transformed before analysis and expressed as geometric means (range of sd). the variables were compared between the four groups using one-way anova and were followed by the tukey multiple comparison test. the frequencies were examined using the chi-square test. all statistical analyses were performed using the spss statistical package (spss inc., chicago, il, usa). a p-value of < . was considered statistically significant. the clinical characteristics of the four study groups are summarized in table . the mean age of rsv group was significantly lower than that of the other three groups. there was no significant difference in gender between the four groups. however, there were more male patients in all age groups. the geometric mean (range of sd) of the total ige level in the influenza virus group [ . ( . monthly distributions of the isolated viruses are shown in figure . lrti by influenza virus, parainfluenza virus and rsv occurred in epidemics and lrti by adenovirus occurred during all seasons of the year. the distribution of principal viral agents identified from patients with different respiratory syndromes is depicted in figure . influenza virus mainly accounted for pneumonia and tracheobronchitis, and parainfluenza virus caused pneumonia. rsv was most frequently associated with bronchiolitis, and adenovirus was associated with tracheobronchitis and pneumonia. the mean (±sd) il- level was . (± . ) pg/ml in the influenza virus group, which was significantly lower than that of the parainfluenza virus group ( . ± . pg/ml), the rsv group ( . ± . pg/ml) and the adenovirus group ( . ± . pg/ml) (p < . for each). the mean (±sd) il- level was . (± . ) pg/ml in the influenza virus group, which was also significantly lower than that of the parainfluenza virus group ( . ± . pg/ml), the rsv group ( . ± . pg/ml) and the adenovirus group ( . ± . pg/ml) (p < . for each). in contrast, the mean (±sd) ifn-γ level was . ± . pg/ml in the rsv group, which was significantly lower than that of the other three groups (the influenza virus group, . ± . pg/ml; the parainfluenza virus group, . ± . pg/ml; the adenovirus group, . ± . pg/ml) ( table ) . the present study of viral lrti children indicated that as seen in npas local inflammatory cytokine responses differed according to the causative organisms. patients with influenza virus infection have shown the lowest levels of il- and il- , and the highest level of ifn-γ . patients with rsv infection showed an increase in production of il- and il- , and a decrease in the ifn-γ level when compared to patients with influenza virus infection. interestingly, a similar th response was also seen in patients with parainfluenza virus or adenovirus infection. however, these two groups showed significantly higher ifn-γ production than the rsv group. these results suggest that respiratory viruses can induce different local cytokine responses and that th biased responses are not unique for rsv but seem to be predominant in young children. previous studies have shown variable relationships between viral respiratory infection and cytokine responses. however, because of the small number of cases involved ( ) and a relatively short study period ( ), a larger group of patients and a longer study period are necessary in order to better delineate the clinical and immunological behaviour of respiratory viruses. furthermore, since different monthly distributions of respiratory virus infection cases have been reported in our country ( ), we have examined cytokine responses involving a large series of lrti patients during a period of months. lower respiratory tract secretions are usually obtained for cytokine measurement by bal, which is an invasive technique that makes it very difficult to obtain appropriate samples for analysis. in the present study, we used npas instead of bal for cytokine analysis. however, this factor is unlikely to affect our results because a previous study ( ) has demonstrated that cytokines in npas are comparable to those in the lower respiratory tract of infants with respiratory virus infections. a few studies have investigated cytokine profiles in npas of patients with rsv lrti. infants with rsv infection have significantly elevated local production of th cytokine, il- and a lower level of ifn-γ ( ) . in addition, more severely infected patients are associated with less local productions of ifn-γ ( , ) . conversely, other studies have shown that levels of both ifn-γ and il- are significantly elevated in the nasal secretions of rsv bronchiolitis ( , ) . these data suggest that host-response factors, such as age, atopy or severity of infection, as well as causative viruses are important for determining local cytokine responses. allergic disease was caused by stimulation of a th response with consequent secretion of cytokines such as il- and il- ( ) . since il- stimulates ige synthesis to induce type hypersensitivity, it is indicated that an allergic mechanism may contribute to the pathogenesis of wheezing in acute bronchiolitis during rsv infection ( ) . compared to rsv infection, cell-mediated immunological responses of influenza virus infection have attracted less attention. while a predominant th cytokine and its related immunological responses are observed in infants with rsv infection, a predominant th cytokine response is observed in infants with influenza virus infection ( ) . this may explain different clinical manifestations between the two viral infections in infants. the role of ifn-γ in the pathogenesis of viral respiratory infections has been relatively well elucidated. hall et al. ( ) have already speculated that the direct antiviral activity of ifn-γ as well as cell-mediated immunity may be important in the pathogenesis of rsv ltri. ifn-γ is considered to be a key cytokine in inducing protective responses against viral pathogens ( ) . we have found that rsv ltri is associated with the reduced local level of ifn-γ . owing to the lower ifn-γ level, patients with rsv infection were most frequently associated with severe bronchiolitis and a longer duration of hospitalization. although the mean age of patients with rsv infections are significantly lower than that of the other three groups, it is unlikely that the difference in age could affect the difference in the ifn-γ level between this study groups. we excluded patients younger than three months of age from the study because t-cell mediated responses are delayed during the first - weeks of postnatal age and are accompanied by an impaired capacity to produce ifn-γ ( ) . in our study, it was found that levels of il- and il- were higher in the parainfluenza virus group or the adenovirus group than in the influenza virus group. there have been quite a few reports regarding these two viruses. kristjansson et al. ( ) have demonstrated that there is no significant difference in the th cytokine level between infants with parainfluenza virus infection and those with rsv infection or influenza virus infection. they concluded that infections with parainfluenza virus or rsv in early infancy preferentially promote a th -like response with local production of il- and il- . adenovirus is also a well-known cause of lrti in children, and most cases of infections are self-limited, whereas several cases of this infection may be associated with severe infections or chronic sequelae such as hyperlucent lung and bronchiolitis obliterans. cytokines are partly responsible for chronic sequelae of these infections. since authors have proposed that adenovirus produces a significantly higher ifn-γ level and induces a th immune response ( , ) , whereas other authors have shown no difference when compared to rsv ( , ) . in concordance with the study by fernandez et al. study ( ) , we observed that the ifn-γ level was higher in the adenovirus group than in the rsv group. these results indicate that adenovirus and rsv infections in children differ significantly with regard to the magnitude of production of ifn-γ . we found significant increases of eosinophil counts and serum ecp concentrations in the rsv group. our findings were in concordance with those reported by oymar et al. ( ) in that the serum ecp concentration was significantly higher in patients with rsv lrti than in patients with non-rsv lrti. this potentially explained the tendency of eosinophilic inflammation in patients with rsv infection. the cytokine responses to acute respiratory viral infections are known to vary according to the duration of infection ( , ) . however, the duration of the symptoms before admission was not significantly different between the four study groups. in this study, laboratory diagnoses of lrti have been made by virus culture instead of pcr. given that all patients included in the study proven with positive virus culture, it is possible that subject with negative culture might have had a viral infection that was not detected. we were unable to detect any viral etiology other than four kinds of respiratory viruses from npas in children recruited for this study. this may be attributed to the limitation of viral cultures in npa samples, which was not a part of the routine service offered by our hospital virology department. nevertheless, we were able to obtain significant results from patients with proven viral etiology. the present study was performed in patients with lower respiratory infection over a period of one year. we have obtained results from a relatively large number of patients. finally, we now prepared prospective study to determine how long after respiratory virus infection the cytokine imbalance may persist. in summary, we found a predominant th cytokine response in children with rsv infection and a predominant th cytokine response in children with influenza virus infection. children with parainfluenza virus infection and those with adenovirus infection elicited both th and th cytokine responses. these suggest that as seen in npas, local inflammatory cytokine responses may differ in children with acute viral respiratory infections according to causative viruses and that a th biased response may be not unique for rsv infection but it seems to be predominant in respiratory viral infections in young children. investigation of local cytokine responses evolved by common respiratory viruses is essential for understanding of the pathogenesis of distinct respiratory viral infections and development of their proper treatment modalities. interleukin- , interleukin- , interleukin- , and interferon-gamma levels in nasopharyngeal aspirates from wheezing children with respiratory syncytial virus or influenza a virus infection interferon-gamma levels in nasopharyngeal secretions of infants with respiratory syncytial virus and other respiratory viral infections a comparison of cytokine responses in respiratory syncytial virus and influenza a infections in infants respiratory syncytial virus infection in infants is associated with predominant th -like response the relationship of rsv-specific immunoglobulin e antibody responses in infancy, recurrent wheezing and pulmonary function at age - years association of cytokine responses with 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in children with bronchial asthma and acute bronchiolitis local and systemic cytokine responses during experimental human influenza a virus infection key: cord- -hkvurb k authors: menachery, vineet d.; gralinski, lisa e.; mitchell, hugh d.; dinnon, kenneth h.; leist, sarah r.; yount, boyd l.; graham, rachel l.; mcanarney, eileen t.; stratton, kelly g.; cockrell, adam s.; debbink, kari; sims, amy c.; waters, katrina m.; baric, ralph s. title: middle east respiratory syndrome coronavirus nonstructural protein is necessary for interferon resistance and viral pathogenesis date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: hkvurb k coronaviruses (covs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. in this study, we focused on highly conserved nonstructural protein (nsp ), a viral ′o-methyltransferase ( ′o-mtase) that encodes critical functions in immune modulation and infection. using reverse genetics, we disrupted a key motif in the conserved kdke motif of middle east respiratory syndrome cov (mers-cov) nsp (d a) and evaluated the effect on viral infection and pathogenesis. while the absence of ′o-mtase activity had only a marginal impact on propagation and replication in vero cells, dnsp mutant mers-cov demonstrated significant attenuation relative to the control both in primary human airway cell cultures and in vivo. further examination indicated that dnsp mutant mers-cov had a type i interferon (ifn)-based attenuation and was partially restored in the absence of molecules of ifn-induced proteins with tetratricopeptide repeats. importantly, the robust attenuation permitted the use of dnsp mutant mers-cov as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted mers-cov strain. these studies demonstrate the importance of the conserved ′o-mtase activity for cov pathogenesis and highlight nsp as a conserved universal target for rapid live attenuated vaccine design in an expanding cov outbreak setting. importance coronavirus (cov) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome cov (sars-cov), mers-cov, porcine epidemic diarrhea virus, and swine delta cov in the st century. these studies describe an approach that effectively targets the highly conserved ′o-mtase activity of covs for attenuation. with clear understanding of the ifn/ifit (ifn-induced proteins with tetratricopeptide repeats)-based mechanism, nsp mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent cov strains. importantly, other approaches targeting other conserved pan-cov functions have not yet proven effective against mers-cov, illustrating the broad applicability of targeting viral ′o-mtase function across covs. t he emergence of middle east respiratory syndrome coronavirus (mers-cov) in represents the second severe cov to strike the human population since the beginning of the st century ( ) . similar to its predecessor, severe acute respiratory syndrome cov (sars-cov), mers-cov is characterized by severe lung infection and high mortality rates ( ) . associated with elderly patients and nosocomial spread, mers-cov is likely harbored in camel populations with periodic reintroductions into humans, followed by periodic nosocomial outbreaks in hospital settings ( ) . importantly, with the continued rate of globalization, mers-cov remains an ongoing threat for future outbreaks both in and outside the middle east, as evidenced by the large outbreak in south korea ( ) . together, these factors highlight the importance of examining cov pathogenesis and developing conserved therapeutic targets for the treatment of current and future emergent strains. like all members of the cov family, mers-cov maintains a balance of conserved and novel viral proteins within its genome ( ) . it is a member of the group c cov family, and a wealth of distinct accessory open reading frames and nonstructural proteins (nsps) have already been established to have important roles in modulation of the host immune response ( ) . similarly, a number of viral proteins highly conserved in structure, replication, and fidelity are also maintained in the cov backbone ( ) . among these, mers-cov nsp provides a potent target for therapeutic development. a =omethyltransferase ( =o-mtase), cov nsp has been implicated in the capping of viral rna and prevention of its recognition by the intracellular sensor mda and antiviral effectors, including members of the ifit (interferon [ifn]-induced proteins with tetratricopeptide repeats) family ( ) . generation of mutants with changes in the nsp kdke active site resulted in ifn-mediated in vitro and in vivo attenuation of both mouse hepatitis virus (mhv) and sars-cov ( , ) . therefore, an approach targeting mers-cov nsp might be anticipated to result in attenuation and potentially provide a universal platform for cov vaccines against future emergent strains. using reverse genetics to target residues in the highly conserved active site, we evaluated mers-cov infection outcomes in the context of inactive nsp (dnsp ). consistent with previous studies of sars-cov ( ), the dnsp mers-cov mutant maintained no significant attenuation in terms of replication or the initial host immune response. however, both primary human airway epithelial (hae) cells and in vivo studies in a mers-cov mouse model demonstrated robust attenuation of dnsp mutant growth and pathogenesis. notably, attenuation was both ifn and ifit dependent, providing a clear mechanism for attenuation. importantly, the dnsp mutant also provided robust protection against a lethal mers-cov challenge and maintained attenuation in the mouse-adapted backbone. together, the results illustrate the broad conservation and necessity of nsp in cov pathogenesis and highlight the targeting of this protein as a rapid-response platform for future emergent cov strains. a combination of structural and biochemical approaches has established a critical role for cov nsp in =o-mtase activity (fig. a) . stabilized by interactions with nsp (orange), nsp has been identified as a structurally conserved adomet-dependent methyltransferase ( ) ; despite divergence in protein sequence across organisms, an invariant kdke motif (red) within the methyltransferase core is required to mediate its activity ( ) . this kdke motif is highly conserved within all of the nsp sequences examined in the cov family (fig. b) . importantly, mutation of any of the kdke residues has been shown to ablate =o-mtase activity ( ) . in addition, previous alteration of this motif in both group b sars-cov ( ) and group a mhv ( ) disrupted =o-mtase activity and attenuated various aspects of infection. on the basis of high conservation in the cov family, we hypothesized that disruption of the kdke motif would also attenuate other emerging cov families, including group c mers-cov. utilizing a mers-cov reversed genetic system ( ), we disrupted the kdke motif by mutating two nucleotides to produce a d a change (fig. a) . the resulting disrupted-nsp (dnsp ) mutant had no significant defect noted in stock titer generation (not shown); similarly, infection of both vero cells and calu- b cells, a respiratory epithelial cell line, at a low multiplicity of infection (moi) demonstrated only modest attenuation at late time points (fig. c and d) . together, these results indicate that nsp activity is not required for replication. similar in vitro host responses of sars-cov and mers-cov dnsp mutants. having established replication competence in both vero and calu- b cells, we next evaluated the induction of host pathways following infection. calu- b cells infected at an moi of demonstrated no differences in replication (not shown) and only modest differences in host induction (zero genes with a log change in expression of Ͼ . -fold), similar to observations with nsp mutant sars-cov compared with wild-type (wt) sars-cov ( ). however, unlike in studies with sars-cov, a rapid cytopathic effect (cpe) by h limited the analysis to early time points with wt and dnsp mutant shown is nsp (gray) highlighting the conserved kdke motif (red) required for =o-mtase activity. also shown is the nsp scaffold required for mers-cov (orange). the inset displays conserved kdke (left) and the d a mutation (right) that disrupts function. homology models were created with modeler in the max-planck institute bioinformatics toolkit. the known crystal structure of the nsp -nsp complex ( r in the rcsb protein data bank) was used as the template structure ( ) . homology models were then manipulated with macpymol. (b) heat maps were constructed from a set of representative covs from all four genogroups by using alignment data paired with neighbor-joining phylogenetic trees built in geneious (v. . . ) and visualized in evolview david-based analysis compared the network host responses to the mers-cov and sars-cov dnsp mutants (fig. ) . over the first h of infection, both mers-cov and sars-cov dnsp mutant infections showed no significant functional enrichment of any categories relative to corresponding wt infections, consistent with the lack of replication attenuation. however, at late times (Ͼ h postinfection), sars-cov produced robust changes in several host pathways, including cytokine responses, inflammation, and extracellular activity. similarly, changes in apoptosis, transcription repression, and regulation of phosphorylation indicated a host response more hostile to viral infection. while a more rapid cpe following both wt and dnsp mutant mers-cov infections precluded an equivalent finding at late time points, the sars-cov results suggest that the absence of nsp activity eventually initiates host response changes that contribute to attenuation at late time points. mers-cov dnsp mutant attenuated in primary and in vivo models. to further examine the replicative capacity of the dnsp mutant, we infected both hae cells and mice expressing human dipeptidyl peptidase , the receptor for mers-cov. primary hae cell cultures were challenged with wt and dnsp mers-cov at a low moi (fig. a ). while robust replication was observed following wt infection, dnsp mers-cov had significant attenuation that corresponded well to previous results obtained with dnsp mutant sars-cov ( ). we next examined dnsp mutant mers-cov replication phenotypes in the context of in vivo infection by using an adenovirus balb/c mouse transduction model ( ) . while neither infection produced weight loss (not shown), wt mers-cov replicated efficiently at both days and postinfection (fig. b ); in contrast, no detectable replication was seen following infection with dnsp mutant mers-cov. the lack of replication may be due to residual ifn responses associated with initial adenovirus infection. for greater clarity, we next infected crispr-cas -targeted mice that include mutations in dpp at positions and ( - ϩ/ϩ ) conferring efficient wt mers-cov infection and growth in mice but no clinical disease ( ) . following infection, no changes were observed in weight loss in either group of mice, consistent with previous findings (data not shown). however, absence of nsp activity severely attenuated dnsp mutant virus replication at both days and postinfection (fig. c ). coupled with data from hae cell cultures and the adenovirus model, the results demonstrate clear attenuation of dnsp mutant mers-cov relative to the control. dnsp mutant mers-cov attenuation is mediated by ifn and ifit . having established a deficit in dnsp mutant mers-cov replication in relevant in vitro and in vivo models, we next sought to evaluate the mechanism of attenuation. previous work by our lab and others had established increased susceptibility of dnsp mutants to type i ifn ( , ); however, dnsp mutant sars-cov had not shown augmented type i ifn stimulation following infection ( ) . consistent with this finding, infection with dnsp mutant mers-cov produced stimulation of type i ifn transcript similar to that seen following in vivo infection of dpp mutant ( - ϩ/ϩ ) crispr mice with the wt virus (fig. a ). in contrast, while both the wt and mutant viruses were sensitive to ifn treatment, dnsp mutant mers-cov had a significant reduction in replication relative to the control virus (fig. b) . these attenuation results are consistent with reports of nsp mutants of other covs and are in contrast to the equivalent replication observed without pretreatment (fig. c) ( ) . extending this analysis further, we examined the role of ifit and ifit gene expression in this attenuation phenotype in previously con- structed stable short hairpin rna (shrna) knockdown cell lines ( ) . similar to sars-cov, knockdown of ifit augmented replication of dnsp mutant mers-cov in the context of type i ifn pretreatment (fig. c ). in addition, knockdown augmented wt mers-cov infection, suggesting sensitivity to ifit activity despite the presence of nsp . notably, ifit knockdown had only a modest, nonsignificant impact on replication, contrasting with results obtained with sars-cov. overall, the data indicate that dnsp mutant mers-cov attenuation is driven by sensitivity to type i ifn mediated by the activity of ifit rather than augmented ifn responses. nsp mutant vaccination protects from a lethal mers-cov challenge. on the basis of ifn and ifit attenuation phenotypes, targeting of nsp offers a potential platform strategy for live attenuated vaccine generation. while previous work by our group had shown that the dnsp mutant sars-cov conferred protection from a lethal challenge, similar phenotypes in other more distant covs are essential for establishing universal principles of attenuation across a virus family. to test this hypothesis, dpp - ϩ/ϩ mutant mice were vaccinated with dnsp mutant mers-cov and subsequently challenged with a mouse-adapted mers-cov strain (fig. ) ( ) . following the challenge, dnsp mutant-vaccinated mice showed only modest weight loss, in significant contrast to the severe disease seen in the control group (fig. a ). in addition, both viral replication and lung hemorrhage were significantly reduced in the context of the dnsp mutant vaccine (fig. b and c) . importantly, serum analysis revealed robust virus neutralization with values similar to those seen in serum from wt virus-infected mice (fig. d) . together, the results indicate that dnsp mutant mers-cov can function as a vaccine platform that not only induces high levels of neutralizing antibodies but provides compete protection from a lethal mers-cov challenge. nsp mutation attenuates mouse-adapted mers-cov. despite conferring protection in the wt mers-cov backbone, it was unclear if the nsp mutant would be sufficiently attenuated in a virulent mers-cov backbone. to address this question, we inserted the dnsp mutation (d a) into the mouse-adapted mers-cov backbone ( ) . following infection, mouse-adapted mers-cov produced rapid weight loss and death (fig. a) . in contrast, the mouse-adapted dnsp mutant produced only modest weight loss and % survival following infection. in addition, the replication of the dnsp mutant was significantly attenuated relative to that of the wt at days and postinfection (fig. b) . finally, hemorrhage scoring of the lung revealed minimal disease in dnsp mutant-immunized mice relative to control mice at day postinfection (fig. c) . overall, the results demonstrate robust attenuation of mers-cov pathogenesis in the context an nsp mutation. in the context of the ongoing mers-cov outbreak, the development of universal platform strategies to attenuate emerging and contemporary covs is a significant priority. in this study, we demonstrate the critical importance of nsp function to mers-cov pathogenesis in vitro and in vivo. similar to mhv and sars-cov ( , ), the disruption of =o-mtase activity in mers-cov had no significant impact on replication or early host response patterns in vitro. however, dnsp mutant mers-cov demonstrated attenuated replication and growth in primary hae cell cultures, as well as reduced disease in vivo, relative to the wt virus. further examination revealed increased sensitivity to type i ifn in an ifit-dependent manner. notably, this ifn/ifitbased attenuation phenotype provided robust protection from a lethal challenge following vaccination with the dnsp mutant. importantly, the nsp mutant also was fully attenuated in a highly virulent mouse-adapted mers-cov backbone. these results, coupled with previous work with other covs, highlight the viral =o-mtase activity as a potential universal platform for therapeutic treatment and vaccine development for current and future emergent human and animal covs. similar to previously viral methyltransferase mutants of both flaviviruses and covs, substitution with the highly conserved kdke motif alters the pathogenicity of mers-cov. like sars-cov mutants ( ), dnsp mutant mers-cov had normal replication but was attenuated in the context of type i ifn and ifit activity. notably, the absence of =o-mtase activity did not result in an increased type i ifn response, suggesting that loss of nsp produces a sensitivity to ifit activity not seen in the wt virus. while the mutant rna should induce greater ifn activation through mda , multiple other cov proteins have been shown to disrupt immune recognition ( ) . similarly, viral processes including replication within double membrane vesicles may also limit ifn stimulation. together, the results indicate that even without nsp , covs are able to control ifn stimulation and replicate as efficiently as the wt virus. however, nsp is needed to protect the viruses once the ifn-stimulated gene (isg) effector response is initiated. the attenuation of the mers-cov nsp mutant also provides evidence supporting the targeting of viral =o-mtase as a global therapeutic strategy against emergent rna viruses. previous work with both flaviviruses and covs has demonstrated robust attenuation by targeting the conserved kdke motif of the viral =o- mtase ( , , ) . importantly, =o-mtase activity is the only known function for nsp and it appears completely dispensable for cov replication in the absence of a strong ifn response ( ) . in contrast, flaviviruses without nsp -encoded methyltransferase activity are much more sensitive to innate immune responses ( ) ; mutants of west nile virus, dengue virus, and japanese encephalitis virus are highly replication attenuated at early time points ( , ) . while diminished virus yields likely reduce flavivirus vaccine utility in vivo, robust propagation of both mers-cov and sars-cov dnsp mutants provides a useful parameter for vaccine generation. for both viral families, drugs targeting =o-mtase activity may have great efficacy if paired with stimulation of the ifn-responsive genes, including that for ifit ( , ) . overall, the results indicate that viral =o-mtase activity is a critical determinant of pathogenesis and can be leveraged to develop therapeutic treatments. nsp also represents the third highly conserved cov protein targeted as the basis of a vaccine platform. previous work by our group demonstrated the importance of cov fidelity for infection and pathogenesis ( ) ; disruption of the exonuclease activity of nsp rendered attenuated, reversion-proof versions of both mhv and sars-cov ( ) ( ) ( ) . for sars-cov, the disruption served as the basis of a successful live attenuated vaccine ( ) . similarly, groups have targeted the envelope protein of sars-cov and defined a key inflammatory role for e protein in pathogenesis. sars mutants targeting e function also conferred protection from a lethal challenge ( , ) . for both nsp and e mutants, fidelity and inflammation induction are partially responsible for attenuation. however, both viral mutants also maintain replication attenuated in terms of kinetics and yields, potentially complicating their use as vaccine platforms, especially in outbred populations ( , ) . importantly, the nsp mutant has not yet been recovered within the context of mers-cov and failure has been reported in group i covs ( ) . similarly, disruption of the e protein renders mers-cov, transmissible gastroenteritis virus, and mhv replication deficient without exogenous complementation ( ) ( ) ( ) ; this broad attenuation of the delta e mutant potentially limits its use as a live attenuated vaccine. in contrast, dnsp mutant mers-cov maintains robust propagation, as well as ifn/ifit-based attenuation phenotypes. together, these results suggest that targeting ofnsp may be the most broadly applicable platform for cov attenuation. despite the success of nsp mutants in protection studies with sars-cov and mers-cov, several additional parameters must be considered in the context of cov vaccination. previous work by our group has demonstrated failures of cov subunit vaccines in aged animals and in the context of a heterologous challenge ( ) . with the aged representing a population with high mortality rates and marginal vaccination success, the efficacy of the nsp mutant in this population is paramount in its pursuit as a platform. similarly, the existence of numerous covs in animal populations raises concerns about heterologous challenges from emergent viruses ( , ) . prior reports had also demonstrated vaccine-induced disease following heterologous challenges with related sars-like viruses ( , ) . with this in mind, nsp -vaccinated mice will need to be examined in the context of a heterologous challenge to determine if vaccine-induced pathology occurs. together, these two factors represent important checkpoints in the pursuit of nsp as a universal cov vaccine platform. in addition to aging and heterologous challenges, reversion and baseline pathogenesis also represent important risks that must be evaluated in the context of an nsp vaccine. while the nsp sars vaccine was absent sterilizing immunity in immunodeficient mice, the lack of reversion over time in vivo indicates safety in the approach ( ) . in contrast, passage of the e mutant rendered a novel mutant that transplanted a critical ion channel function from another viral protein and thus restored partial virulence ( ) . studies examining the reversion potential of nsp are critical prior to its use as a vaccine; these concerns are especially important considering that both the sars-cov and mers-cov dnsp mutants have robust replication, permitting additional opportunities for reversion ( ) . in addition, equivalent host immune responses during early infection may produce substantial damage prior to ifn/ifit-based attenuations, most notably in aged and immunocompromised mice. therefore, despite the promise of successful attenuation in multiple cov backbones, several additional metrics must be examined prior to the use of nsp mutants as a universal cov vaccine platform. overall, the present study demonstrates that targeting of =o-mtase activity is a robust strategy to attenuate mers-cov and other emergent covs. in the absence of nsp activity, the mers-cov mutant is sensitive to type i ifn in an ifit-dependent manner, providing a clear attenuation mechanism. importantly, unlike other conserved cov platforms, the nsp mutant is both viable and robust enough to be used as an effective live attenuated vaccine. while further vaccine characterization is required, the results indicate that disruption of cov nsp activity can be the basis of therapeutic strategies for both current and future emergent cov infections in both human and animal populations. cells and viruses. the wt, mutant, and mouse-adapted versions of mers-cov used in this study were previously described ( , ) and were cultured on vero cells grown in dulbecco's modified eagle's medium or opti-mem (gibco, carlsbad, ca) and % fetal bovine serum (hyclone, south logan, ut) along with antibiotic/antimycotic (gibco, carlsbad, ca). growth curves in vero, calu- b , and hae cells were performed as previously described, with examination of multiple samples (n Ն ) at each time point ( , ) . briefly, cells were washed with phosphate-buffered saline (pbs) and inoculated with virus or mock diluted in pbs for min at °c. following inoculation, cells were washed three times and fresh medium was added to signify time zero. samples were harvested at the time points described. for ifn pretreatments, u/ml recombinant human ifn-␤ (pbl laboratories) was added to cells h prior to inoculation, and the cells were infected as described above. stable shrna knockdown vero cell lines for both ifit and ifit were previously described for previous cov studies and had phenotypic validation ( ) . all virus cultivation was performed in a biosafety level laboratory with redundant fans in biosafety cabinets as described previously by our group ( , ) . all personnel wore powdered air purifying respirators ( m breathe easy) with tyvek suits, aprons, and booties and were double gloved. construction of wt and nsp mutant viruses. both wt and mutant viruses were derived from either mers-cov emc or a corresponding mouse-adapted (ma ) infectious clone as previously described ( ) . for nsp mutant construction, the d a mutation changed the sequence from the mers-cov e fragment, which was cloned into the psmart vector (lucigen) and used for alanine scanning mutagenesis of conserved residues in nsp . for the d a change, a product was generated by pcr with primers against mers-cov nsp [fragment , emc:e# (ϩ) (tgaactacctgtagctgtag) and emc:emuc(Ϫ) (nnnnnngctcttctcgcggaaataacaagatccacttg); fragment , emc:emuc(ϩ) (nnnnnngctcttc cgcgatgtatgatcctactactaag) and emc:e# (Ϫ) (caacctcaatacaagcagac)]. the two resulting products were digested with sapi (in boldface) and ligated overnight with t dna ligase. this product was then digested with ppumi and nsii and used to replace the region of the emc e plasmid (puc ) that had been similarly digested. thereafter, plasmids containing wt and mutant mers-cov genome fragments were amplified, excised, ligated, and purified. in vitro transcription reactions were then performed to synthesize full-length genomic rna, which was transfected into vero e cells. the medium from transfected cells was harvested and used as seed stock for subsequent experiments. viral mutants were confirmed by sequence analysis prior to use. synthetic construction of nsp mutants was approved by the university of north carolina institutional biosafety committee. rna isolation, microarray processing, and identification of de. rna isolation and microarray processing, quality control, and normalization from calu- b cells were carried out as previously described ( ) . differential expression (de) was determined by comparing virus-infected replicates with time-matched mock-treated replicates. criteria for de in determining the consensus isg list were an absolute log change of Ͼ . -fold and a false-discovery rate-adjusted p value of Ͻ . for a given time point. clustering and functional enrichment. genes identified as differentially expressed were used to generate clustered expression heat maps. hierarchical clustering (using euclidean distance and complete linkage clustering) was used to cluster gene expression according to behavior across experimental conditions. the david online resource (https://david.ncifcrf.gov/) was used to acquire functional enrichment results for the genes in each cluster. david output was manually summarized for each cluster. plots were generated with r. ethics statement. this study was carried out in accordance with the recommendations for the care and use of laboratory animals of the office of laboratory animal welfare (olaw), national institutes of health. the institutional animal care and use committee (iacuc) of the university of north carolina at chapel hill (unc; permit a- - ) approved the animal study protocols used in this study (iacuc protocols - and - ). mouse infections and vaccinations. ten-to -week-old balb/c (envigo/harlan) or crispr-cas targeted - ϩ/ϩ c bl/ mice were anesthetized with ketamine and xylazine (in accordance with unc iacuc guidelines) and intranasally inoculated with a -l volume containing pfu of wt mers-cov, dnsp mutant mers-cov, mouse-adapted variants, or pbs (mock inoculation) as indicated in the figure legends. infected animals were monitored for weight loss, morbidity, and clinical signs of disease, and lung virus titers were determined as described previously ( ) . in vivo adenovirus transduc-tion with dpp were performed as previously described ( ) . for vaccination experiments, -to -week-old - ϩ/ϩ mice were infected with pfu of dnsp mutant mers-cov as described above, monitored for clinical symptoms for days, and then challenged weeks postvaccination with pfu of mouse-passaged mers-cov ma . animal housing, care, and experimental protocols were in accordance with unc iacuc guidelines. data availability. raw microarray data for these studies were deposited in publicly available databases at the national center for biotechnology information (ncbi) gene expression omnibus ( ) and are accessible through geo series gse . middle east respiratory syndrome and severe acute respiratory syndrome middle east respiratory syndrome middle east respiratory syndrome coronavirus vaccines: current status and novel approaches mortality risk factors for middle east respiratory syndrome outbreak jumping species-a mechanism for coronavirus persistence and survival sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon the nonstructural proteins directing coronavirus rna synthesis and processing coronavirus non-structural protein : evasion, attenuation, and possible treatments ribose =-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking =-o-methyltransferase activity coronavirus nonstructural protein is a cap- binding enzyme possessing (nucleoside- =o)-methyltransferase activity an rna cap (nucleoside- =-o-)-methyltransferase in the flavivirus rna polymerase ns : crystal structure and functional characterization reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus a mouse model for mers coronavirus induced acute respiratory distress syndrome coronavirus nonstructural protein mediates evasion of dsrna sensors and limits apoptosis in macrophages the dengue virus ns protein as a target for drug discovery polymerases of hepatitis c viruses and flaviviruses: structural and mechanistic insights and drug development evasion of early innate immune response by =-o-methylation of dengue genomic rna =-o methylation of internal adenosine by flavivirus ns methyltransferase a crystal structure of the dengue virus ns protein reveals a novel inter-domain interface essential for protein flexibility and virus replication a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease infidelity of sars-cov nsp -exonuclease mutant virus replication is revealed by complete genome sequencing coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics a live attenuated severe acute respiratory syndrome coronavirus is immunogenic and efficacious in golden syrian hamsters immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in e protein protects against lethal respiratory disease mutagenesis of coronavirus nsp reveals its potential role in modulation of the innate immune response engineering a replicationcompetent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly a doubleinactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv -cov poised for human emergence successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus identification of the mechanisms causing reversion to virulence in an attenuated sars-cov for the design of a genetically stable vaccine host regulatory network response to infection with highly pathogenic h n avian influenza virus release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells biochemical and structural insights into the mechanisms of sars coronavirus rna ribose '-o-methylation by nsp /nsp protein complex this research was supported by grants from the niaid of the nih (u ai to r.s.b., u ai to r.s.b., hhsn i-hhsn to r.s.b., and k ag to v.d.m.). support for primary hae cell cultures was obtained from the nih through the unc cystic fibrosis research and translation core center cell models core (nih p dk ). the content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the nih. the pacific northwest national laboratory is operated by the battelle memorial institute for the department of energy under contract de-ac - rlo . key: cord- -djjtgbh authors: zhou, bei-xian; li, jing; liang, xiao-li; pan, xi-ping; hao, yan-bing; xie, pei-fang; jiang, hai-ming; yang, zi-feng; zhong, nan-shan title: β-sitosterol ameliorates influenza a virus-induced proinflammatory response and acute lung injury in mice by disrupting the cross-talk between rig-i and ifn/stat signaling date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: djjtgbh β-sitosterol ( -ethyl- -cholestene- -ol) is a common phytosterol chinese medical plants that has been shown to possess antioxidant and anti-inflammatory activity. in this study we investigated the effects of β-sitosterol on influenza virus-induced inflammation and acute lung injury and the molecular mechanisms. we demonstrate that β-sitosterol ( – μg/ml) dose-dependently suppresses inflammatory response through nf-κb and p mitogen-activated protein kinase (mapk) signaling in influenza a virus (iav)-infected cells, which was accompanied by decreased induction of interferons (ifns) (including type i and iii ifn). furthermore, we revealed that the anti-inflammatory effect of β-sitosterol resulted from its inhibitory effect on retinoic acid-inducible gene i (rig-i) signaling, led to decreased stat signaling, thus affecting the transcriptional activity of isgf (interferon-stimulated gene factor ) complexes and resulting in abrogation of the iav-induced proinflammatory amplification effect in ifn-sensitized cells. moreover, β-sitosterol treatment attenuated rig-i-mediated apoptotic injury of alveolar epithelial cells (aec) via downregulation of pro-apoptotic factors. in a mouse model of influenza, pre-administration of β-sitosterol ( , mg·kg(− )·d(− ), i.g., for days) dose-dependently ameliorated iav-mediated recruitment of pathogenic cytotoxic t cells and immune dysregulation. in addition, pre-administration of β-sitosterol protected mice from lethal iav infection. our data suggest that β-sitosterol blocks the immune response mediated by rig-i signaling and deleterious ifn production, providing a potential benefit for the treatment of influenza. the annual spread of seasonal influenza a virus (iav), particularly the sporadic transmission of highly pathogenic avian influenza (hpai) viruses (e.g., the h n and h n subtypes) continues to constitute a major threat to public health. as of december , avian influenza a h n viruses have caused an estimated individual infections, with an overall mortality rate of . % (http://www.fao.org/ag/againfo/programmes/en/empres/h n / situation_update.html). recently, the na r k and ns g a substitutions in the h n virus have been reported to be associated with increased resistance to oseltamivir and to confer enhanced viral replication in mammalian cells [ , ] . this raises concerns about a potential human pandemic. iav initiates infection in humans via entry through the upper respiratory tract, eliciting symptoms that can be mild and self-limited, but in some patients, it can lead to acute respiratory distress syndrome (ards), which is characterized by the impairment of gas exchange resulting in a fatal outcome [ , ] . a series of reports have indicated that a robust and dysregulated innate immune response is primarily responsible for the high mortality rate associated with influenza [ , ] . however, there are few alternative medicine strategies available for the treatment of influenza virus-infected patients who have an intense inflammatory response. the innate immune system is armed with an array of pattern recognition receptors (prrs) that provide the principal barrier defense against infectious agents [ ] . during the process of viral replication, influenza virus synthesizes viral rna containing a ′triphosphate end and is then transported to the cytoplasm [ ] . this pathogen-associated molecule (pam) is detected by the cytosolic sensor retinoic acid-induced gene i (rig-i) and subsequently activates multiple cellular signal cascades [ ] . ultimately, these signaling pathways lead to the activation of transcription factors including nf-κb, ap- (atf :c-jun), and irf , which together bind to specific sites of the ifn-β promoter and initiate ifn-β synthesis for the antiviral response [ ] . targeting p map kinase with a specific inhibitor affects the induction of ifn-β [ ] , which inhibits the downstream phosphorylation of atf by p map kinase [ ] . studies have demonstrated the crucial role of ifn production mediated by rig-i signaling in protecting against lethal iav challenge [ , ] . in vivo studies have shown that the increased susceptibility of both rig-i-and ifn-deficient mice to several rna viruses [ ] [ ] [ ] , including vesicular stomatitis virus (vsv), newcastle disease virus (ndv), and iav, is associated with the failure of ifns levels to increase or a lack of ifn signaling. there is evidence that infection with hpai h n viruses triggers the hyperinduction of proinflammation via rig-i signaling cascades despite the crucial role of rig-i signaling in defense against iav [ ] . the induction of downstream genes of rig-i signaling, such as il- , tnf-α, il- , and il- β, is significantly upregulated in the bronchoalveolar lavage fluid (balf) of patients with sustained ards [ , , ] . type i and type iii ifns secreted by infected cells bind to distinct receptors but trigger similar signal transduction through the jak/ stat cascades [ , ] . upon ifn stimulation, the phosphorylation of the stat :stat heterodimer leads to its interaction with irf and the formation of the interferon-stimulated gene factor (isgf ) complex [ ] . then, the complex moves from the cytoplasm to the nucleus where it binds to the interferonstimulated response element (isre) and drives the expression of interferon-stimulated genes (isgs). these genes possess direct antiviral and immunomodulatory activity [ ] . for example, the mx protein, which was the first isg identified to restrict influenza virus infection to be identified, has been demonstrated to impair vrnp nuclear import [ ] . however, despite the abundant production of ifns and hundreds of isgs in response to viral infection, the ifnmediated antiviral response is blocked by various viral products. the non-structural (ns ) protein of iav is a typical multifunctional protein involved in the blockade of the antiviral effect of isgs, such as ′, ′-oligoadenylate synthetase (oas) and dsrnadependent protein kinase r (pkr) [ , ] . in addition, iav infection has also been shown to suppress the antiviral response mediated by type i and iii ifns by upregulating the negative regulators socs and socs [ , ] . given that viruses can overcome the ifn-mediated antiviral response through various mechanisms, it is possible that excessive ifn production during the antiviral response may contribute to the harmful effects. surprisingly, studies have found that increased host susceptibility to secondary bacterial co-infections correlates with ifn induction during iav infection [ ] . moreover, a deficiency in ifn-α/β signaling increases survival through a reduction in excessive inflammation and apoptotic injury to lung epithelial cells [ ] . similarly, high levels of trail in balf collected from patients with influenza-associated ards may be a result of increased trail expression induced by macrophage-derived ifn-β [ ] , which contributes to lung alveolar epithelial cell injury. in addition, recent evidence has also revealed that ifn-mediated signaling drives immunopathologic injury in response to various viral infections, including respiratory syncytial virus infection (rsv) [ ] and severe acute respiratory syndrome cov (sars-cov) [ ] . given these findings, it is clear that the disease-promoting effects of ifn contribute to deleterious outcomes, and should be limited by pharmacological intervention. the identification of agents derived from medicinal plants that can prevent influenza is valuable. chinese medicinal plants, including lonicera japonica [ ] , chrysanthemum morifolium [ ] , taraxacum mongolicum [ ] , forsythia suspense [ ] , and isatis indigotica [ ] have been prescribed for the common cold, heatclearing, and detoxication for thousands of years, but the bioactive ingredients of these plants that mediate these pharmacological effects is unknown. phytosterols contain structural features that resemble those of cholesterol and are abundant in vegetables, fruits, and medicinal plants [ , ] . among phytosterols, β-sitosterol ( -ethyl- -cholestene- -ol) is the most common sterol and has been shown to possess antioxidant, antiinflammatory, antitumor, and antiasthmatic effects [ ] [ ] [ ] [ ] . in the present study, we hypothesized that β-sitosterol is the bioactive component of five types of medicinal plants. to test this hypothesis, we investigated the effects of β-sitosterol and the underlying mechanisms by which it may exert a therapeutic effect against influenza-mediated injury and dysregulated inflammation. preparation of extracts and quantitative analysis of β-sitosterol samples of four kinds of different heat-clearing and detoxifying traditional chinese medicines samples (l. japonica, c. morifolium, t. mongolicum, and f. suspense) were purchased from local markets in bozhou, china. i. indigotica was supplied by hutchison whampoa guangzhou baiyunshan chinese medicine co., ltd (guangzhou, china). a β-sitosterol standard was purchased from sigma (san francisco, usa), and hplc-grade methanol was purchased from fisher scientific (fisher, usa). a sample of each of the five medicinal materials was crushed into a coarse powder, and . g was placed in a -ml flask. extraction was performed using ultrasonic waves for min and the addition of ml of chloroform and was repeated three times. the samples were then centrifuged at × g for min. the supernatants were combined and condensed to a proper volume under reduced pressure, and then the concentrates were dissolved with chloroform. the samples were transferred to -ml volumetric flasks, diluted with chloroform to ml, and mixed. a total of . mg of the β-sitosterol standard was accurately weighed and dissolved in ml of chloroform to produce individual stock solutions. hplc analysis of β-sitosterol was performed at °c on an hplc instrument (shimadzu a, japan) with a dad detector at nm. chromatographic separation was performed on a shimadzu ods column ( . × mm, μm, tokyo, japan). the mobile phase was methanol, and the injection volume was μl. the samples were subjected to quantitative analysis, which was performed using the external standard method. the results are expressed as mg/g, and all analyses were performed in triplicate. influenza a/puerto rico/ / (h n ) and a/fm/ / (h n ) mouse-adapted viruses were stored in our laboratory and propagated in the allantoic cavities of -day-old specific pathogen-free embryonated chicken eggs at °c. freshly collected allantoic fluids were clarified by low-speed centrifugation at h postinoculation and then stored in small aliquots at − °c. the virus titers were determined using a plaque forming assay in monolayers of madin-darby canine kidney (mdck) cells as previously described. mouse experiments and viral challenge four-to six-week-old female balb/c mice (weighing - g) were purchased from guangdong medical laboratory animal center. all mice were housed and cared for under specific pathogen-free conditions at the state key laboratory of respiratory disease or guangdong laboratory animal monitoring institute. all animal experimental procedures in this study were approved by the ethics committee of the first affiliated hospital of guangzhou medical university and conducted in strict accordance with the approved guidelines. the % lethal dose (ld ) of the mouse-adapted h n virus was estimated in mice after the stock virus was serially diluted. the mice were treated intragastrically with β-sitosterol ( mg·kg − ·d − , mg·kg − ·d − ) or pbs (vehicle group) days prior to viral challenge. the mice were anesthetized ( % isoflurane inhalation) and challenged intranasally with ld of mouse-adapted h n virus. cell culture and viral infection human alveolar epithelial a cells and t human embryonic kidney cells were grown in dulbecco's modified eagleʼs medium (dmem/f , : mixture) (gibco) supplemented with % fetal bovine serum (fbs) (gibco) in a humidified incubator at °c and % co . for the viral infection, a cells ( × cells/ml) grown in well tissue culture plates (guangzhou jet bio-filtration co., ltd, tcp- - ) were inoculated with a/pr/ / (h n ) in serumfree dmem/f medium at an indicated multiplicity of infection (moi). after h absorption, the inoculum was discarded and replaced with fresh serum-free dmem/f medium containing diluted compounds. antibodies and recombinant protein the following primary antibodies were used in the current study: anti-rig-i, anti-nf-κb p , anti-phospho-nf-κb p (ser ), anti-p mapk, anti-phospho-p mapk ( protein preparation and immunoblot analysis excised lungs and cells were lysed using ice-cold ripa buffer ( mm tris·hcl ph . , mm nacl, % np- , % sodium deoxycholate, and . % sds) supplemented with protease inhibitors (sigma). the crude lysates were centrifuged at , rpm ( , × g) for min at °c, and the supernatant was collected. equal amounts of cell or tissue extracts were loaded onto % sds-polyacrylamide gels for separation. then, proteins were transferred to . μm pvdf membranes (bio-rad), which allowed subsequent probing with primary antibodies. after overnight incubation at °c, the membranes were washed with . % tbst (tbs/ . % tween ) and incubated with corresponding horseradish peroxidase (hrp)conjugated secondary antibodies (multisciences). the signals were visualized using enhanced chemiluminescence (ecl) reagents (perkin-elmer). quantification of the relative band intensities was performed using imagej software version . . ppprna generation, rna isolation, and real-time rt-pcr viral rna ( ′-triphosphate rna, ′ppp-rna) and cellular rna were generated as previously described [ ] . briefly, total rna was isolated from a lung epithelial cells infected with a/pr/ / (h n ) for h (viral rna) and uninfected a lung epithelial cells (cellular rna) using trizol reagent (takara, usa). then, the '-phosphate group was dephosphorylated by treatment with calf-intestinal alkaline phosphatase (ciap) (takara). a lung epithelial cells were transfected with the indicated rna using lp (thermo, usa). for qpcr, total rna ( μg) was reverse transcribed into cdna and subjected to real-time quantitative pcr with gene-specific primers and probes on an abi real-time pcr system. relative gene expression was calculated using the −ΔΔct method [ ] . the specific primer and probe sets are shown in supplementary table s . small interfering rna (sirna), plasmid transfection, and reporter gene assays transient transfection of plasmids into cells, including an isre luciferase reporter plasmid (beyotime) and a flag-rig-i overexpression plasmid (geneppl), was performed by using lipofectamine (invitrogen). after isre luciferase reporter plasmid ( . μg) and flag-rig-i ( . μg) overexpression plasmid were transfected into a cells for h, the transfected cells were stimulated with ifns or iav (moi = . ). in the other experiment, hek cells stably co-transfected with pnf-κb-tata-f-luci and pqcxip-egfp plasmid were stimulated with tnf-α ( ng/ml) or iav for the indicated times. firefly luciferase activity was assayed using the luciferase reporter system (promega, usa) and normalized to renilla luciferase activity or the levels of gfp expression. rig-i-specific sirnas (rig-i # sirna and, rig-i # sirna) were purchased from guangzhou ribobio co., ltd. and transfected into cells with lipofectamine according to the manufacturer's instructions. histology and immunohistology lung tissue was excised, fixed in % formalin and embedded in paraffin using routine procedures. tissue sections ( μm) were stained with hematoxylin-eosin for histological examination. for immunohistochemical staining, the sections were deparaffinized with xylene and rehydrated in a graded alcohol series. endogenous peroxidase was blocked with % hydrogen peroxide (h o ) in methanol for min at room temperature and antigen retrieval was performed in . mm citrate buffer (ph . ). afterward, the sections were blocked with % normal serum and incubated with primary antibody at °c overnight. the sections were then incubated for h in hrp-labeled secondary antibody solution and then visualized using a dab reagent kit (maixin, china). the sections were counterstained with mayer's hematoxylin for min before mounting. apoptosis detection by annexin v and flow cytometry cells collected from both suspension and adherent were washed twice with cold pbs, and subsequently resuspended in × annexin binding buffer (bioscience, usa) at a concentration of × cells/ml. one hundred microliters of cell suspension was stained with μl of annexin v-fitc and μl of propidium iodide (pi) for min at room temperature in the dark. the cells were analyzed using a novocyte flow cytometer within h. bronchoalveolar lavage and flow cytometry mice were euthanized intraperitoneally (i.p.) with an overdose of sodium pentobarbital at the indicated time point. the trachea was cannulated via ventral middle incision. the lungs were lavaged three times with μl of sterile saline. bronchoalveolar lavage fluid (balf) was centrifuged at × g for min at °c and the supernatants were collected and stored at − °c for biochemical analysis. the balf pellets were resuspended in pbs with . % bsa and stained with fluorochrome-conjugated monoclonal antibodies (mabs) (bioscience, usa) that bound to surface molecules for min at °c. all samples were analyzed on a novocyte flow cytometer. statistical analysis all data are presented as the mean ± sem. statistical analysis was performed using spss software version . . one-way anova followed by the student-newman-keuls (snk) test was carried out to assess differences between the study groups, and p values less than . were considered significant. quantitative analysis of β-sitosterol in five medicinal materials medicinal plants including l. japonica, c. morifolium, t. mongolicum, f. suspense, and i. indigotica are traditionally used for heatclearing and detoxification. we hypothesized that β-sitosterol is the common substance in these medicinal plants that mediates their pharmacological actions against respiratory diseases, such as β-sitosterol ameliorates iav-induced inflammation and ali bx zhou influenza. first, we quantified the content of β-sitosterol in these plants. as shown in table , β-sitosterol was detected in all samples at concentrations ranging from . to . mg/g. β-sitosterol treatment inhibits the activation of the cellular signaling pathway in iav-infected cells to clarify whether β-sitosterol possesses antiviral activity for the treatment of iav infection, cytopathic effect (cpe) inhibition assays and plaque reduction assays were performed to investigate the antiviral effects of β-sitosterol. as shown in supplementary table s , β-sitosterol showed no activity against a/gz/gird / (h n ), a/pr/ / (h n ), a/hk/ / (h n ), a/hk/y / (h n ), or b/lee/ (flub). these findings were further confirmed by plaque reduction assays ( supplementary fig. s ). the activation of multiple cellular signaling events has been implicated in the molecular pathogenesis of iav infection [ ] . to identify the pharmacological effects exerted by β-sitosterol during iav infection, we assessed the impact of β-sitosterol on the activation of cellular signaling pathway in cells infected with iav. hek cells in which an nf-κb-luc reporter was stably expressed were stimulated with either tnf-α ( ng/ml) (fig. a) or a/pr/ / (h n ) (fig. b) , and then incubated with β-sitosterol for h. we observed that hek cells stimulated with tnf-α or a/pr/ / (h n ) exhibited robust nf-κb activation, on which β-sitosterol treatment had a dose-dependent suppressive effect ( fig. a, b) . furthermore, to test whether β-sitosterol possesses other pharmacological properties, we assessed the activation of signaling pathways in iav-infected a cells in the presence or absence of β-sitosterol by immunoblotting. nf-κb and mapk (p , erk / , and jnk/spak) signaling was activated by iav infection (fig. c) . as expected, β-sitosterol inhibited the iav-induced p mapk activation and p phosphorylation, which is consistent with the data gathered using the nf-κb-luc reporter cell line. however, βsitosterol had no inhibitory effect on the erk / or jnk mapk pathway. together, these data demonstrate that β-sitosterol has the potential to inhibit the activation of nf-κb and p mapk signaling in response to iav infection. β-sitosterol treatment decreases the expression of iav-induced proinflammatory mediators both the nf-κb and p mapk signaling cascades have been implicated as major contributors to hypercytokinemia during human hpaiv h n infection [ , ] . therefore, we next investigated the effect of β-sitosterol on the expression of proinflammatory mediators in iav-infected cells. a cells were infected with the pr /h n virus (moi = . ) in the presence of increasing concentrations of β-sitosterol ( - μg/ml) for h. subsequently, the transcript levels of proinflammatory mediators were measured by qpcr. our data showed that the gene expression of an array of cytokines and chemokines, including il- , tnf-α, ip- , il- , mcp- , mip- β, and rantes, was elevated dramatically following iav infection, and that this elevation was attenuated by β-sitosterol treatment (fig. a) . the kinetics of iavinduced proinflammatory mediator release showed that the protein levels of these proinflammatory mediators (including il- , tnf-α, il- , ip- , rantes, and mcp- ) reached their expression peak at h after viral infection (fig. b) , and that the increases in the levels of these mediators were reversed by β-sitosterol treatment (fig. c) . the production of cyclooxygenase- (cox- ) and its derivative prostaglandin e (pge ), has been shown to occur via nf-κb and p mapk signaling and play a pathogenic role during iav infection [ , ] . indeed, increasing the expression of iav-induced cox- at both the mrna and protein levels at h p.i., while treatment with β-sitosterol profoundly reduced cox- production (fig. d, e) . furthermore, we measured the effect of β-sitosterol on the iav-induced expression of iav-induced cox- and carried out elisa to quantify cox- -derived pge in the culture supernatants. as expected, β-sitosterol treatment of iav-infected cells led to a significant dose-dependent reduction in pge levels (fig. f) . these results indicate that β-sitosterol treatment decreases the expression of iav-induced proinflammatory mediators through the inactivation of the nf-κb and p mapk signaling pathways. β-sitosterol treatment suppresses the iav-mediated induction of interferon expression and signal transduction by targeting rig-i in response to iav infection, transcription factors including nf-κb, atf /c-jun, and irf / bind cooperatively to the promoter regions of ifn genes, which are secreted to establish an antiviral state, to initiate their expression [ ] . the activation of p , which mediates the activation of its downstream target atf , is a prerequisite for optimal ifn-β induction [ ] . since β-sitosterol inhibits the iav-induced activation of p , we hypothesized that β-sitosterol treatment might reduce the expression of ifns. to test this hypothesis, culture supernatants from iav-infected a cells treated with or without β-sitosterol were collected at h p.i. and transferred to uninfected a cells. after min of incubation, we assessed ifn levels in the culture supernatant by monitoring the phosphorylation and downstream signaling of ifns by immunoblot analysis. as shown in fig. a , supernatants from infected a cells stimulated the phosphorylation of stat tyr and stat tyr , while supernatants from donor cells treated with β-sitosterol attenuated the phosphorylation of stat tyr . our results indicated that the suppression of stat tyr phosphorylation by β-sitosterol was associated with reduced ifns production. to further confirm that β-sitosterol inhibits the iav-induced expression of ifns, ifn concentrations in supernatants collected at h p.i. were measured using luminex. as demonstrated in fig. b , the iav-induced elevation of ifns, including type i ifn (ifnβ), type ii ifn (ifn-γ), and type iii ifn (ifn-λ ), was significantly decreased in the supernatants of β-sitosterol-treated cells. these observations led us to ask whether β-sitosterol directly affects the ifn signaling transduction pathway. to address this question, we tested whether signal transduction induced by exogenous ifn-β is altered by β-sitosterol treatment. then, we pretreated iav-infected a cells with β-sitosterol for h before stimulating them with ifn-β for min. the phosphorylation of stat tyr , but not stat , was markedly inhibited by β-sitosterol treatment (fig. c) . however, at later time points ( h p.i.), the ifnβ-mediated phosphorylation of stat tyr was reduced in iavinfected a cells regardless of whether they were pretreated with β-sitosterol or not (fig. d) . our data demonstrated that β-sitosterol reduced ifn production through the inhibition of nf-κb and p map kinase and that it blocks ifn-mediated signal transduction. we next sought to elucidate the mechanism by which β-sitosterol disrupts ifn production and signaling. the intracellular prr rig-i senses iav ′ppp-rna, resulting in the activation of nf-κb and p map kinase which preferentially promote the initiation of ifn transcription [ ] . it is worth mentioning that rig-i, an interferon-stimulated gene (isg), has been reported to augment stat activation and inhibit leukemia cell proliferation [ ] . thus, it is reasonable to speculate that cross-talk between the rig-i and ifn signaling pathways is affected by β-sitosterol. to confirm this assumption, we investigated the effect of β-sitosterol on the iavinduced expression of rig-i. our results show that β-sitosterol decreased the induction of rig-i in infected a cells (fig. e, f) . furthermore, cells transfected with the flag-rig-i overexpression plasmid transfection were found to have elevated p-stat levels, which were diminished by β-sitosterol treatment (fig. g) . the tyrosine kinases jaks are well recognized to be involved in the activation of stat . therefore, it is possible that β-sitosterol affected stat activation by inhibiting jaks. interestingly, we found that a cells pretreated with β-sitosterol for h prior to ifn-β ( ng/ml) stimulation did not exhibit decreased ifn-βmediated activation of jak , stat , and stat levels (fig. h) . together, these data indicate that β-sitosterol can exert an inhibitory effect on rig-i, which leads to decreased iav-induced ifn production and ifn-β signal transduction. β-sitosterol treatment attenuates the ifn-mediated amplification of the proinflammatory response during iav infection via the inhibition of rig-i it is widely recognized that ifn signaling plays a vital role in host antiviral immunity [ ] . furthermore, ifns possess immunomodulatory activities on the induction of both chemokines and cytokines and on the recruitment of various immune cells [ ] . to examine the effect of rig-i inhibition by β-sitosterol on the transcription of ifn signaling-related molecules, we analyzed iav-mediated ifn-stimulated response element (isre) activation with an assay that utilized a transient isre reporter plasmid. the results presented in fig. a show that iav-mediated isre-dependent transcription was significantly inhibited by βsitosterol treatment. rig-i knockdown in iav-infected cells by specific sirnas in iav-infected cells was confirmed to significantly inhibit the iav-mediated isre transcriptional activity (fig. b, c) . to further determine the effects of β-sitosterol on ifn-induced proinflammatory responses during iav infection, isre reporter plasmid-transfected cells were stimulated with ifn-β ( ng/ml) h prior to iav infection. prestimulation with ifn-β led to much higher isre activity than iav infection alone (fig. d) . similarly, ifn-β stimulation following iav infection also resulted in increased isre activity, but to a lesser extent than what was seen after ifn-β prestimulation (fig. d) . this indicates that ifn-β signaling contributes to the amplification of isre activity during iav infection. nevertheless, this increase in isre activity was diminished in a dose-dependent manner by β-sitosterol treatment representative results from at least three independent experiments are shown. d the band intensities of p-p , p-p , p-erk / , and p-jnk were semiquantified using imagej (normalized to the loading control gapdh). the data are presented as the mean ± sem (n = - ). **p < . , ***p < . versus the group treated with tnf-α or iav. β-sitosterol ameliorates iav-induced inflammation and ali bx zhou immunoblot analysis was performed to evaluate the protein expression of cox- . gapdh was used as the internal control (e). f quantification of pge in the culture supernatants using elisa. *p < . , **p < . , ***p < . versus the iav control group. ( fig. d) . we next assessed the effects of β-sitosterol on the expression of proinflammatory genes in iav-infected cells pretreated with or without ifn-β. consistent with the previously observed increase in isre activity, the mrna and protein levels of cytokines and chemokines, including il- , ip- , tnf-α, il- , mcp- and gm-csf, were robustly increased in iav-infected cells after stimulation with ifn-β (fig. e, f) . a similar cytokine and chemokine expression pattern was observed in response to stimulation with ifn-λ (data not shown), which signals through the jak/stat pathway leading to isre activation. as expected, the elevation in cytokine and chemokine levels induced by ifn stimulation was decreased by β-sitosterol treatment (fig. e, f) . to understand whether the attenuation of the amplified proinflammatory response in ifn-β pretreated cells was solely due to a reduction in ifn-β levels, cells with ifn-β prestimulated for h were treated with an ifn-β neutralizing antibody ( . μg/ml) prior to virus infection. however, we observed that the amplification effects of proinflammatory mediators (il- and ip- ) in cells prestimulated with ifn-β were not abrogated by ifn-β neutralizing antibody treatment (fig. g) . this indicates that cells prestimulated with ifn-β become sensitized and thereby amplify proinflammatory responses. to understand whether the observed inhibitory effect of βsitosterol was due to stat inhibition, we measured the phosphorylation of stat tyr in a cells stimulated with ifn-β ( ng/ml). stat was significantly activated in cells pretreated with ifn-β (lane ), but not in cells infected with iav prior to ifn-β stimulation (lane ). treatment with β-sitosterol abrogated stat activation in a dose-dependent manner (lanes - ) (fig. h) , indicating that the inhibitory effect of β-sitosterol on stat reduced the ifn-mediated amplification of cytokine and chemokine expression. notably, pretreatment with ifn-β significantly increased the iav-triggered expression of rig-i in a cells, which was inhibited by β-sitosterol treatment (fig. i, j) . together, these data demonstrate that β-sitosterol blocks the iav-induced amplification of the proinflammatory response in ifn-β-activated a cells, which is due to inhibition of rig-i levels by β-sitosterol, leading to the inactivation of stat , and thereby diminishes the transcriptional activity of interferon-stimulated gene factor (isgf ). in addition to playing a role in ifn induction, rig-i signaling has been demonstrated to be involved in apoptosis [ ] , which is implicated in iav-induced lung epithelial cell damage and injury. therefore, we investigated whether β-sitosterol affects rig-imediated apoptosis using intracellular viral rna (vrna, ′ppp-rna) stimulation. stimulation with cellular rna (crna) or vrna and treatment with calfintestine alkaline phosphatase (ciap) to fig. effect of β-sitosterol treatment on iav-induced ifn production and ifn signal transduction. a after h, iav-infected cells with or without the indicated concentration of β-sitosterol treatment were harvested, and the supernatants were transferred to uninfected cells. after min of incubation, the cells were lysed, and the expression of phosphorylated stat and stat was analyzed by immunoblotting. equal loading of protein was verified by immunoblotting for gapdh. b ifns (ifn-β, ifn-γ, and ifn-λ ) secretion into the culture media was measured at h p.i. using a multiplex luminex assay. c, d after allowing h for iav absorption, a cells were incubated with the indicated concentration of β-sitosterol for h (c) or h (d). then, the cells were stimulated for an additional min with human ifn-β ( ng/ml). the cells were lysed, and total extracts were processed for immunoblotting. e, f effect of β-sitosterol on the expression of rig-i. rig-i mrna expression levels were assayed by quantitative real-time pcr (e). rig-i protein levels were assessed by western blotting (f). g a cells were transfected with a flag-rig-i overexpression plasmid, and then treated with β-sitosterol. after h, the cell lysates were collected for immunoblotting. h a cells were pretreated with β-sitosterol for h, stimulated with ifn-β ( ng/ml) for min, and immunoblotted for the indicated proteins. *p < . , **p < . , ***p < . versus the iav control group. dephosphorylate ′-triphosphate did not induce apoptosis, excluding the possibility that crna or the non-phosphate at the ′ end of rna has a pro-apoptotic effect (fig. a) . strikingly, the apoptosis of vrna-transfected cells was reduced by β-sitosterol treatment (fig. a) . we confirmed the anti-apoptotic effect of βsitosterol by measuring the active caspase- and its substrate parp, observing that these products were found in cells transfected with vrna but not in those treated with β-sitosterol fig. (continued) β-sitosterol ameliorates iav-induced inflammation and ali bx zhou (fig. b) . to determine whether the inhibitory effect of β-sitosterol on rig-i-mediated apoptosis is related to alterations in the expression of pro-apoptotic factors, we quantified trail and sfas ligand levels in the supernatants of viral rna-transfected cells. trail and sfas ligand levels were significantly reduced by βsitosterol treatment (fig. c ). next, we determined the effect of βsitosterol on the apoptosis of iav-infected cell. β-sitosterol treatment reduced iav-mediated apoptosis and active caspase- and parp (fig. d, e) . last, we observed that β-sitosterol treatment blocked the increase in the release of trail and sfas ligand in iavinfected cells (fig. f) . furthermore, rig-i knockdown by specific sirnas was demonstrated to significantly decrease iav-mediated apoptosis and the release of trail (fig. g, h) . however, the combination of rig-i sirnas and β-sitosterol did not have an additive effect on the inhibition of iav-mediated apoptosis and trail release, which indicated that β-sitosterol decreased iavmediated apoptosis via the inhibition of rig-i. given that vrna ( ′ ppp-rna) recognition is associated with the rapid induction of ifns, we wondered whether β-sitosterol treatment affects ifn induction in the context of vrna transfection. as shown in fig. i , the upregulated expression of both type i ifn (ifn-β) and type iii ifn (ifn-λ ) in response to vrna was reversed by β-sitosterol treatment in a dose-dependent manner. to further explore whether the signaling cascade underlying rig-i-mediated apoptosis and ifn induction is affected by βsitosterol, we performed immunoblot analysis h after the fig. effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory mediators. a the effect of β-sitosterol on isre luciferase reporter activity in iav-infected cells. a cells were co-transfected with . μg of pisre-ta-luc reporter plasmid and . μg of prl-tk plasmid as described in the materials and methods. at h posttransfection, the cells were infected with iav and treated with βsitosterol. after h, the cells were lysed, and luciferase activity was measured. ## p < . versus the control group. *p < . , **p < . versus the iav control group. b rig-i knockdown by specific sirnas in iav-infected cells was confirmed by immunoblotting. c the effect of rig-i knockdown by specific sirnas on isre luciferase reporter activity in iav-infected cells. ### p < . versus the control group. ***p < . versus the iav control group. d the effect of β-sitosterol on isre luciferase reporter activity induced by stimulation with a combination of ifn-β and iav. after h of transfection, a cells were pretreated with ifn-β ( ng/ml) (columns - ) for h or infected with iav prior to ifnβ stimulation (columns - ) for h. ifn-β-pretreated cells were infected with iav in the presence or absence of β-sitosterol ( - μg/ml). iav-infected cells were stimulated with ifn-β ( ng/ml) in the presence or absence of β-sitosterol ( - μg/ml). the cells were harvested and subjected to a luciferase assay at h p.i. # p < . versus the iav control group (column ). *p < . versus the ifn-β-pretreated group (column ). § p < . versus the group infected with iav before being stimulated with ifn-β ( ng/ml) stimulation group (column ). e the effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory cytokines and chemokines at the mrna levels was determined by real-time pcr. ### p < . versus the iav control group (column ). *p < . , **p < . , ***p < . versus ifn-β-pretreated group (column ). § p < . , § § p < . versus the group infected with iav before being stimulated with ifn-β ( ng/ml) (column ). f the effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory cytokines and chemokines at the protein level was determined by a multiplex luminex assay. # p < . , ## p < . versus the iav control group (column ). *p < . , **p < . , ***p < . versus ifn-β-pretreated group (column ). § p < . , § § p < . , § § § p < . versus the group infected with iav before being stimulated with ifn-β ( ng/ml) (column ). g the effect of ifn-β neutralization on the ifn-β-mediated amplification of the iav-induced proinflammatory response. a cells prestimulated with ifn-β ( ng/ml) for h were treated with an ifn-β neutralizing antibody ( . μg/ml) prior to infection with iav. after h, the culture supernatants were collected to measure proinflammatory mediator levels by a multiplex luminex assay. ***p < . versus iav control group. ns, not significant. h the effect of β-sitosterol on the ifn-β-mediated activation of stat . lanes - : a cells were treated with either ifn-β ( ng/ml) or with iav for h. lanes - : a cells were pretreated with ifn-β ( ng/ml) for h prior to iav infection. lanes - : a cells were infected with iav for h and then stimulated with ng/ml ifn-β. after the indicated treatments, the cells were incubated with or without β-sitosterol for h. the cell lysates were analyzed by immunoblotting for the expression of phospho-stat and phospho-stat . i, j the effect of β-sitosterol on the expression of rig-i. a cells were pretreated with ifnβ ( ng/ml) for h and infected with iav in the presence or absence of β-sitosterol ( - μg/ml) (columns - , lanes - ) . meanwhile, a cells were infected with iav for h prior to ifn-β ( ng/ml) stimulation (columns - , lanes [ ] [ ] [ ] [ ] . i the expression of rig-i was determined by quantitative real-time pcr. ## p < . versus the iav control group (column ). *p < . , **p < . , ***p < . versus ifn-βpretreated group (column ). j the expression of rig-i was determined by immunoblotting at h p.i. the band intensities of rig-i were semiquantified using imagej (normalized to the loading control gapdh). *p < . versus ifn-β-pretreated group (column ). § p < . versus the group infected with iav before being stimulated with ifn-β ( ng/ml) (column ). (fig. j) (lane ) . notably, the dephosphorylation of the ʹtriphosphate of rna led to the inactivation of p nf-κb and p mapk but not stat (lane ). moreover, the vrna-triggered activation of p nf-κb, p mapk, and stat was inhibited by βsitosterol (lanes - ). the stimulation of rig-i with vrna initiated signaling events that ultimately led to the expression of proinflammatory cytokines. we detected increased expression of proinflammatory mediators after h of vrna stimulation, and observed that β-sitosterol treatment blocked this effect (fig. k) . meanwhile, these proinflammatory mediators induced by iav were effectively reduced by specific rig-i sirnas (fig. l) . the levels of il- , mcp- , rantes, and gm-csf were further reduced by the combination of rig-i sirnas and β-sitosterol (fig. l) . furthermore, the further increased levels of il- , tnf-α, and ip- in flag-rig-i overexpression plasmid-transfected cells with iav infection is dose-dependently decreased by β-sitosterol treatment (fig. m) . interestingly, the viral rna-induced upregulation of cox- expression was decreased following β-sitosterol treatment (fig. n) . to further confirm that β-sitosterol suppresses viral rna-mediated cox- upregulation, we quantified the level of its downstream product pge in the culture medium using elisa. our results showed that treatment with β-sitosterol dosedependently suppressed the viral rna-induced production of pge (fig. o) . collectively, these data suggest that β-sitosterol inhibits the activation of rig-i signaling and downstream apoptosis in response to vrna. considering that β-sitosterol exhibited immunomodulatory properties in vitro, we next sought to investigate whether β-sitosterol has a protective effect in a mouse model of influenza. balb/c mice were pretreated with β-sitosterol for days prior to intranasal challenge with ld of iav. at days p.i., histological analysis of lung sections showed that infection with iav resulted in extensive inflammation characterized by massive leukocyte recruitment to the lung parenchyma (fig. a, upper panel) . pre-treatment with mg·kg − ·d − β-sitosterol decreased leukocyte infiltration, and as a result, relatively few inflammatory cells were observed surrounding the bronchioles (fig. a, upper panel) . we next performed immunohistochemical staining for cd (pan t lymphocytes) and observed that the infiltration of cd + t lymphocytes was more pronounced in the lungs of mice with iav analysis of active caspase- and parp cleavage in a cells transfected with vrna at h. c luminex assay for trail and sfas ligand levels in the supernatants of a cells transfected with vrna. *p < . , **p < . versus the vrna transfection group (column ). d flow cytometry analysis of the effect of β-sitosterol on apoptotic a cells infected with iav at h. *p < . , **p < . versus iav control group. e analysis of active caspase- and parp cleavage in a cells infected with iav at h. f luminex assay for trail and sfas ligand levels in the supernatants of a cells infected with iav at h. *p < . versus the iav control group. g flow cytometry analysis of the effect of rig-i knockdown by specific sirnas on iav-induced apoptosis. h luminex assay for trail in the supernatants of a cells with rig-i knockdown. ***p < . versus iav control group (column ). i luminex assay for ifn-β and ifn-λ levels in the supernatants of a cells transfected with vrna at h. *p < . , **p < . , ***p < . versus the vrna-transfected group (column ). j a cells were transfected with vrna in the presence or absence of the indicated concentration of β-sitosterol; h later, the cells were lysed and analyzed by immunoblotting with the indicated antibodies. gapdh was used as a control for equal loading. k the effect of β-sitosterol on the protein expression of cytokines and chemokines in the supernatants of vrna-transfected cells. *p < . , **p < . , ***p < . versus the vrna transfection group (column ). l luminex assay for proinflammatory mediators in the supernatants of a cells with knockdown of rig-i or in combination with β-sitosterol treatment. ***p < . versus the iav control group (column ). # p < . , ### p < . versus the rig-i # sirna transfection group (column ). § p < . , § § p < . , § § § p < . versus the rig-i # sirna transfection group (column ). m luminex assay for cytokines and chemokines in the culture supernatants of iav-infected a cells transfected with flag-rig-i overexpression plasmid with or without β-sitosterol treatment. ## p < . versus the iav control group (column ). *p < . , ***p < . versus the flag-rig-i overexpression plasmid transfection control group (column ). n the effect of β-sitosterol on the expression of cox- in vrna-transfected cells. the cells were transfected with vrna in the presence or absence of the indicated concentration of β-sitosterol; h later, the cells were lysed and analyzed by immunoblotting using a specific antibody to cox- . o quantification of pge in the culture supernatants of vrna-transfected cells in the presence or absence of the indicated concentrations of β-sitosterol at h. ***p < . versus the vrna-transfected group (column ). β-sitosterol ameliorates iav-induced inflammation and ali bx zhou infection alone compared with those that received β-sitosterol treatment (fig. a, lower panel) . consistent with the immunohistochemical results, a significantly greater percentage of cd + cd + cytotoxic t lymphocytes (ctls) was detected in balf from iav-infected mice compared with balf from mice treated with β-sitosterol (fig. b) . moreover, the quantification of granzyme b, a pro-apoptotic enzyme secreted by ctl, in lung homogenates revealed a significant increase in its expression in iav-infected mice that was significantly reduced by β-sitosterol administration (fig. c) . similar results were obtained for the levels β-sitosterol ameliorates iav-induced inflammation and ali bx zhou of active caspase- measured by immunoblotting (fig. c) . although influenza antigen-specific cd + t cells are recruited to the sites of infection and contribute to viral clearance, immunemediated lung injury can be elicited by aberrant t-cell responses. lung index and total protein levels in balf can be used as an assessment of lung damage. our results showed that compared with iav infection alone, β-sitosterol treatment produced a significantly lower lung index and protein levels in balf (fig. d , e), suggesting that β-sitosterol treatment alleviated lung injury likely through the suppression of cd + t-cell recruitment. last, iav infection resulted in % ( / ) mortality by days p.i. and rapid and continuous weight loss (fig. f, g) . remarkably, the survival rate of mice that were treated with and mg/kg βsitosterol was significantly increased to . % ( / ) and . % ( / ), respectively. furthermore, β-sitosterol-treated mice exhibited less initial weight loss after viral challenge and a gradual recovery of body weight (fig. g) . together, these data reveal that β-sitosterol attenuates iav-induced lung injury and reduces mortality. β-sitosterol protects against iav by abrogating the iav-mediated activation of multiple signaling cascades to investigate the mechanisms underlying the protective effect of β-sitosterol against iav in vivo, we focused on inflammationassociated signal transduction in the lung. the phosphorylation levels of stat , stat , p , and erk / in lung homogenates were significantly increased in mice challenged with iav relative to uninfected mice (figs. a), whereas the phosphorylation levels of these molecules were decreased in mice treated with β-sitosterol. to address whether β-sitosterol inhibits the signaling events mediating the iav-induced expression of proinflammatory cytokines, we quantified cytokine and chemokine levels using luminex. the expression of cytokines and chemokines in balf (il- , tnf-α, rantes, kc, mcp- and mip- α) and lung homogenates (il- , tnf-α, ip- , and rantes) was reduced in mice treated with β-sitosterol (fig. b, c) . the iav-induced elevation of serum cytokines including ifn-γ, ip- , and rantes, was blocked in β-sitosterol-treated mice (fig. d) . given that β-sitosterol inhibited iav-mediated stat / activation in vivo and decreased the expression of rig-i and ifn in vitro (fig. b, f) , it was necessary to examine the impact of β-sitosterol treatment on the expression of rig-i and ifns in vivo. as expected, iav-induced expression of rig-i in lung homogenates was decreased by β-sitosterol treatment (fig. e) . similarly, the expression of ifns in balf (ifn-α, ifn-β, and ifn-γ) (fig. f) and lung homogenates (ifn-β) (fig. g) was also decreased. collectively, these data provide evidence regarding the mechanism by which β-sitosterol modulates dysregulated signaling cascades and proinflammatory responses linked to severe influenza. patients with influenza are frequently afflicted with severe pneumonia characterized by excessive infiltration of leukocytes and proinflammatory cytokine production [ ] [ ] [ ] , leading to a high risk of death. recent studies have revealed that iav-mediated ifns play a disease-promoting role in the pathogenesis of influenza [ ] . chinese herbal medicines, including l. japonica [ ] , c. morifolium [ ] , t. mongolicum [ ] , f. suspense [ ] , and i. indigotica [ ] , have a long history of being used for the treatment of the common cold and for heat-clearing. in the current study, we demonstrated that β-sitosterol derived from these herbal medicines has the ability to block iav-mediated ifn and proinflammatory mediator production through the inhibition of rig-i signaling. furthermore, our data showed that β-sitosterol attenuates the amplification of the iav-mediated proinflammatory response in ifn-sensitized cells by disrupting rig-i-mediated stat activation. furthermore, we showed that β-sitosterol abrogates the recruitment of cytotoxic t lymphocytes (ctls) in the lung, thereby significantly improving lung injury and survival in mice challenged with iav (fig. ) . rig-i detects viral rna ( ′ppp-rna) in the cytoplasm, which is then ubiquitinated by trim (tripartite motif-containing protein ) and subsequently interacts with ips- (ifn-β promotor stimulator ) to initiate the activation of nf-κb, p , and irf / [ , ] . we asked whether rig-i signaling is affected by βsitosterol during iav infection or in cells transfected with viral rna. we found that rig-i expression was increased following iav infection or prestimulation with ifn-β prior to iav infection, and that this increase in expression was significantly reduced in the presence of β-sitosterol. furthermore, the activation of nf-κb and p in both iav-infected and viral rna-transfected cells was inhibited by β-sitosterol treatment. these results suggest that βsitosterol treatment antagonizes the rig-i signaling cascades. the activation of rig-i and its downstream targets nf-κb and p contributes to the induction of proinflammatory cytokines in response to influenza virus infection [ ] . in previous studies, the upregulation of rig-i during fatal h n infection was shown to cause an amplification of inflammatory responses [ ] . the hyperinduction of proinflammatory cytokines via rig-i, nf-κb, and p signaling has been suggested to contribute to severity of symptoms in patients with h n infection [ , ] . we measured nf-κb activation using an nf-κb luciferase reporter system and found that β-sitosterol treatment downregulated the transcriptional activation of nf-κb following the administration of tnf-α and iav infection. consistent with these findings, the inhibitory effects of β-sitosterol on rig-i signaling led to reduced production of cytokines, such as il- and tnf-α, and chemokines such as il- and ip- in iav-infected and viral rna-transfected cells. rig-i or nf-κb and p activation in response to viral or bacterial infection, respectively, has been reported to induce cox- expression [ , ] . furthermore, it has been shown that decreased expression of cox- and its derivative pge has beneficial effects during influenza virus infection that lead to reduced hypothermia and enhanced type i ifn antiviral immunity [ ] . we observed that iav infection and viral rna stimulation were associated with increased expression of cox- and pge and that β-sitosterol treatment reversed the increase in a dosedependent manner. fig. β-sitosterol prevents iav-induced lung pathology in mice. two days prior to infection with ld of a/fm /h n virus, pbs or β-sitosterol ( mg·kg − ·d − or mg·kg − ·d − ) was intragastrically administered to mice for consecutive days. a on day p.i., the lungs were harvested and subsequently subjected to histological analysis by h&e staining (original magnification, × ) or cd antigen (t-cell marker) staining (original magnification, × ). b representative flow cytometry quantification of cd + cd + t cells in balf on day p.i. the histograms represent the percentage of cd + cd + t cells in balf (right panel). the data are representative of three independent experiments using - mice per group. *p < . , **p < . versus the iav-infected group. c on day p.i., the lungs were removed and homogenized. lung homogenates were subjected to immunoblot analysis of granzyme b and active caspase- . the ratios of the relative band intensities of granzyme b and active caspase- normalized to gapdh are shown (n = - mice per group) (right panel). *p < . , ***p < . versus the iav-infected mice group. d the lung index (lung/body weight ratios) of mice treated with pbs (n = ) or β-sitosterol (n = ) on day p.i. *p < . , **p < . versus the iav-infected group. e on day p.i., the total protein concentrations in balf was measured by bca assay (n = - mice per group). *p < . , **p < . versus the iav-infected group. f, g survival rate (f) and weight curves (g) of iav-infected mice treated with or without β-sitosterol (n = mice per group). β-sitosterol ameliorates iav-induced inflammation and ali bx zhou nf-κb, atf (a downstream target of p ), and irf form a transcriptional complex that drives the expression of the antiviral factor ifn-β [ ] . in addition, the viral-induced expression of type iii ifn requires the involvement of rig-i, ips- , tbk , and p signaling [ ] [ ] [ ] , suggesting that the expression of type i and iii ifns is promoted via a common mechanism. interestingly, some studies have indicated that nf-κb is crucial for ifn-β production when irf activation is weak but not when irf activation is strong [ ] . although we did not detect significant activation of irf at h, we were able to detect an inhibitory effect of β-sitosterol on the expression of ifns, including ifn-β and ifn-λ , in cells infected with iav or in those subjected to viral rna transfection. these findings may be attributable to the inactivation of rig-i, nf-κb, and p signaling. a clear link between rig-i expression and stat activation has been established by previous studies. experiments involving rig-i overexpression or knockdown have suggested that rig-i is essential for stat activation in leukemia cell lines [ , ] . in accordance with these findings, our results show that the augmentation of stat activation by rig-i overexpression was suppressed by β-sitosterol or the inhibition of iav-mediated isre transcriptional activity by specific rig-i sirnas. in addition, we observed that the activation of jaks was not affected by β-sitosterol. therefore, the inhibition of stat phosphorylation in response to ifn-β treatment for min or h can be attributed to the downregulation of rig-i by βsitosterol. moreover, the activation of rig-i, but not of mda- , has been shown to involve double-stranded rna (dsrna)-induced stat phosphorylation [ ] . our data showed that β-sitosterol treatment abrogated stat phosphorylation in cells stimulated with vrna ( ′ppp-rna), which binds to and activates rig-i. however, the dephosphorylation of viral rna with ciap did not reduce stat phosphorylation. a possible explanation for these findings is that the dsrna that is generated following viral rna dephosphorylation is also a ligand for rig-i and induces stat phosphorylation in an ifn-dependent or ifn-independent manner, as described previously [ ] . the important role of ifns in the defense against viral infection is widely recognized [ , ] . however, ifn receptor deficiency does not lead to a detrimental outcome, which is perhaps due to decreased ifn-induced immune injury [ , , ] . recent studies have clearly revealed the pathogenic potential of ifn-β-and ifn-λ -mediated immunopathology in viral infectious diseases and autoimmune diseases [ , , , ] . here, we have proposed a model in which ifns (including type i and iii ifn) secreted from iav-infected cells bind to their receptors and sensitize uninfected neighboring cells, leading to the amplification of proinflammatory responses driven by isgf following infection by progeny viruses. β-sitosterol treatment blocked the amplification of this proinflammatory response and the concomitant expression of proinflammatory cytokines through the inhibition of isgf complexes. the inhibitory effect on isgf complexes was due to the failure of downregulated rig-i to exert a converse effect on stat activation in β-sitosterol-treated cells (fig. ) . the activation of rig-i-mediated apoptosis via type i ifn-dependent and type i ifnindependent mechanisms has been considered a promising strategy for cancer therapeutics [ , ] . the ifn-induced activation of isgf leads to trail expression, resulting in substantial alveolar epithelial cell (aec) apoptosis and lung injury [ , ] . aec apoptosis has been found to play a critical role in the pathogenesis of h n and pandemic h n in patients with ards [ , ] . our data suggest that β-sitosterol prevents iav-induced apoptosis associated with decreased ifn-driven expression of trail. the loss of rig-i signaling has been correlated with a reduction in antigen presentation in bone marrow derived dendritic cells (bmdcs), and in the antiviral function of cd + cytotoxic t cells [ ] . thus, it is clear that the rig-i pathway is important for mediating the production of ifns during antiviral responses. in contrast, the rig-i-mediated expression of inflammatory mediators has been shown to induce the recruitment of monocyte-derived dcs (modcs) to support viral replication [ ] . antiviral effector cd + t cells and nk cells eliminate invading pathogens through fig. β-sitosterol effectively abrogates iav-triggered signaling in vivo. a mice were infected with ld of a/fm /h n virus and treated with pbs or β-sitosterol (intragastrically administered for consecutive days beginning days prior to viral infection). the lungs were harvested and homogenized on day p.i. and the processed for immunoblotting with the indicated antibodies. the relative band intensities of the indicated proteins were normalized to that of gapdh (n = - mice per group) (right panel). *p < . , **p < . versus the iav-infected group. b-d detection of cytokine and chemokine production in balf (b), lung homogenates (c), and serum (d) by luminex analysis. *p < . , **p < . versus iav-infected group. e on day p.i., lung homogenates were subjected to immunoblot analysis of rig-i. the rig-i band intensity normalized to that of gapdh is shown (n = - mice per group) (right panel). *p < . versus iav-infected group. f, g detection of ifns (ifn-α, ifn-β, and ifn-γ) production in balf (f) and lung homogenates (g) by luminex analysis. *p < . , **p < . versus the iavinfected group. β-sitosterol ameliorates iav-induced inflammation and ali bx zhou several mechanisms, including the expression of pro-apoptotic proteins such as trail and fas and the secretion of granzyme b (grb) and perforin. through these mechanisms, the apoptotic caspase cascade is activated in virus-infected cells [ ] [ ] [ ] . studies have reported that ifn-γ plays an important role in modulating cd + t-cell recruitment and that the production of granzyme b during the recruitment of cd + t cells is dependent on the ifn-βinduced activation of stat [ , , ] . accordingly, our data show that β-sitosterol administration decreases the levels of ifn-γ and ifn-β and concomitantly induces a low level of cd + t-cell recruitment and granzyme b secretion in the lungs. cytotoxic cd + t lymphocytes (ctls) seem to be essential for viral clearance. however, severe pneumonia in patients with pandemic influenza a (h n ) virus infection is apparently related to high levels of cd + t cells [ ] . interestingly, one study showed that ha-transgenic mice develop lethal lung injury following treatment with influenza ha-specific cd + cytotoxic t cells [ ] . deficiency of a (tnf alpha-induced protein , tnfaip ), which is a negative feedback ubiquitin-editing protein that inhibits nf-κb signaling, protects mice against viral challenge by reducing the population of grb + cd + t cells [ ] . consistent with these studies, our data showed that iav-induced acute lung injury and mortality are attenuated in mice treated with β-sitosterol and that this attenuation is associated with decreased cd + t-cell recruitment and granzyme b secretion in the lung. furthermore, we observed the inhibition of stat / and p phosphorylation by β-sitosterol in mouse lung tissue and made a similar observation in iavinfected a cells. the expression of cytokines driven by stat / and p , which exacerbate iav-induced immunopathology, in balf and lung tissue was decreased following β-sitosterol administration in balf and lung tissue. in contrast to our in vitro results, the phosphorylation of erk / was attenuated in the lung tissues of β-sitosterol-treated mice. the inhibition of erk / signaling is involved in the retention of viral rnp in the nucleus, but its activation also correlates with cytokine expression [ ] . it is likely that the decrease in phosphorylated erk / expression in vivo was a result of the immunoregulatory effects of β-sitosterol. fig. schematic diagram showing the mechanism by which βsitosterol attenuates iav-induced proinflammatory responses and injury. invading viruses are sensed by rig-i, leading to the activation and that of rig-i, nf-κb, and p , which initiates the expression of proinflammatory mediators and ifns. secreted ifns, including type i and iii ifns, bind to their receptors via an autocrine or paracrine mechanism and then exert their antiviral effects. subsequently, previously uninfected ifn-sensitized neighboring cells become infected by progeny viruses, which triggers the amplification of the inflammatory response. the inhibition of rig-i signaling by βsitosterol attenuates rig-i-linked proinflammatory ifn production, which results in a reduction in stat activation and thus a decrease in the amplification of proinflammatory responses driven by isg complexes in ifn-sensitized cells. furthermore, the inhibition of rig-i signaling by β-sitosterol also suppresses the recruitment of cd + t cells and granzyme b release in vivo, thereby blocking lung immune injury during 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upregulation of trail pathway in breast cancer cells antiviral response by natural killer cells through trail gene induction by ifn-alpha/beta molecular biology, and pathogenesis of avian influenza a (h n ) infection in humans rig-i signaling is critical for efficient polyfunctional t cell responses during influenza virus infection efficient influenza a virus replication in the respiratory tract requires signals from tlr and rig-i type i interferons regulate cytolytic activity of memory cd + t cells in the lung airways during respiratory virus challenge role of tumor necrosis factorrelated apoptosis-inducing ligand in immune response to influenza virus infection in mice distinct roles of nk cells in viral immunity during different phases of acute friend retrovirus infection intracellular ifn-gamma expression in natural killer cells precedes lung cd + t cell recruitment during respiratory syncytial virus infection production of interferon-gamma by influenza hemagglutinin-specific cd effector t cells influences the development of pulmonary immunopathology cd + /cd + t lymphocytes imbalance in children with severe pandemic influenza a (h n ) pneumonia structural and functional consequences of alveolar cell recognition by cd + t lymphocytes in experimental lung disease a deficiency in lung epithelial cells protects against influenza a virus infection inhibition of influenza virus-induced nf-kappab and raf/mek/erk activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo zfy and nsz conceived the study; bxz, zfy, and nsz designed the study; bxz and xll conducted the in vitro experiments; jl and xpp isolated and analyzed the compound β-sitosterol; bxz, xll, hmj, ybh, and pfx performed animal experiments; and bxz and jl wrote the paper. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -kgpvdb authors: sa ribero, margarida; jouvenet, nolwenn; dreux, marlène; nisole, sébastien title: interplay between sars-cov- and the type i interferon response date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: kgpvdb the severe acute respiratory syndrome coronavirus- (sars-cov- ) is responsible for the current covid- pandemic. an unbalanced immune response, characterized by a weak production of type i interferons (ifn-is) and an exacerbated release of proinflammatory cytokines, contributes to the severe forms of the disease. sars-cov- is genetically related to sars-cov and middle east respiratory syndrome-related coronavirus (mers-cov), which caused outbreaks in and , respectively. although ifn treatment gave some encouraging results against sars-cov and mers-cov in animal models, its potential as a therapeutic against covid- awaits validation. here, we describe our current knowledge of the complex interplay between sars-cov- infection and the ifn system, highlighting some of the gaps that need to be filled for a better understanding of the underlying molecular mechanisms. in addition to the conserved ifn evasion strategies that are likely shared with sars-cov and mers-cov, novel counteraction mechanisms are being discovered in sars-cov- –infected cells. since the last coronavirus epidemic, we have made considerable progress in understanding the ifn-i response, including its spatiotemporal regulation and the prominent role of plasmacytoid dendritic cells (pdcs), which are the main ifn-i–producing cells. while awaiting the results of the many clinical trials that are evaluating the efficacy of ifn-i alone or in combination with antiviral molecules, we discuss the potential benefits of a well-timed ifn-i treatment and propose strategies to boost pdc-mediated ifn responses during the early stages of viral infection. the severe acute respiratory syndrome coronavirus (sars-cov- ) is a beta-coronavirus that emerged at the end of in china and rapidly spread around the world, causing a pandemic [ , ] . sars-cov- infection is responsible for covid- , a disease associated with mild symptoms in the majority of cases but that can progress to an acute respiratory distress syndrome [ , ] . so far (july th, ), the virus has infected more than million people and caused more than , deaths worldwide. sars-cov- is genetically related to other betacoronaviruses that have caused epidemics: sars-cov and mers-cov (for middle east respiratory syndrome-related coronavirus), in phosphorylation, irf and/or irf dimerize and translocate into the nucleus, where they induce the expression of ifn-i and a subset of isgs referred to as early isgs (reviewed in [ ] ). secreted ifn-i then bind to the interferon alpha and beta receptor (ifnar, composed of the ifnar and ifnar subunits), leading to the activation of the jak tyrosine kinases tyrosine kinase (tyk ) and janus kinase (jak ), which in turn phosphorylate the signal transducer and activator of transcription (stat) and stat [ , ] . phosphorylated stats heterodimerize and associate with the dna binding protein irf to form a complex known as ifn-stimulated growth factor (isgf ). the isgf complex translocates into the nucleus and binds to interferon-stimulated response elements (isres) in isg promoters, thus inducing the expression of hundreds of isg products that establish the antiviral state at the site of viral infection [ ] . the antiviral response is intensified by various signaling factors, including sensors and transcriptional regulators, which are themselves isgs induced by isgf and/ or directly by the irf /irf transcriptional activators. aside from the ifn-i response, the recognition of double-stranded viral rna elements by the protein kinase receptor (pkr) triggers a translational arrest in infected cells (reviewed in [ , , ] ). this host response is highly connected to the ifn-i response because pkr is also an isg (reviewed in [ , ] ). ifn-i response requires fine-tuning because its overactivation is deleterious to the host. notably, some isgs are involved in the regulation of cell metabolism, intracellular rna degradation, translation arrest, and cell death, for which changes can be potentially detrimental to the host. ifn-i also potentiates the recruitment and activation of various immune cells. thus, although a robust ifn-i response is required as a first line of defense against viral infections, systemic/uncontrolled or prolonged ifn-i production can lead to inflammatory diseases. for example, an exacerbated ifn-i response contributes to the development of autoimmune diseases [ ] . covid- is no exception to the rule, and it is therefore critical to understand the regulation of the ifn-i response upon infection. sars-cov- is a poor inducer of ifn-i response in vitro and in animal models as compared with other respiratory rna viruses [ , ] . ifn-i levels in the serum of infected patients are below the detection levels of commonly used assays, yet isg expression is detected [ , ] , thus suggesting that a limited ifn-i production could be sufficient to induce isgs. alternatively, ifn-i production could be restricted to specific immune cells, such as plasmacytoid dendritic cells (pdcs). despite a more efficient replication in human lung tissues, sars-cov- induced even less ifn-i than sars-cov [ ] , which is itself a weak inducer in human cells [ ] [ ] [ ] ]. an ineffective ifn-i response seems to be a hallmark of other coronavirus infections, as observed with mers-cov in ex vivo respiratory tissue cultures [ ] and with animal coronaviruses such as the porcine epidemic diarrhea virus (pedv) or the mouse hepatitis virus (mhv), which are alpha-and beta-coronaviruses, respectively [ , ] . indeed, coronaviruses have developed multiple strategies to escape and counteract innate sensing and ifn-i production. sars-cov encodes at least proteins that allow the virus to either escape or counteract the induction and antiviral action of ifn (fig and table ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . initial observations already suggest that the sars-cov- anti-ifn arsenal is at least as efficient as that of sars-cov [ , , ] , although detailed mechanistic studies are required to determine whether the ifn antagonists identified in other coronaviruses have equivalently competent counterparts in sars-cov- . a virus-cell protein interaction map performed with of the sars-cov- proteins expressed in human embryonic kidney (hek) t identified several innate immune signaling proteins as partners of viral proteins cells (fig ) [ ] . sars-cov- orf b, like sars-cov orf b, interacts with mavs through its association with tom , thus suggesting a conserved mechanism of ifn-i evasion [ , ] (fig ) . furthermore, sars-cov- nsp and nsp were found to interact with tbk and the tbk activator ring finger protein (rnf )/nrdp , respectively [ ] (fig ) . nsp , which is a highly conserved endoribonuclease encoded by various coronaviruses, including sars-cov [ , , ], antagonizes the induction of ifn-i by cleaving the -polyuridines of the negative-sense viral rna, as demonstrated for mhv and pedv in various cellular models [ , , ] (table and fig ) . if further validated, the interaction between sars-cov- nsp and tbk may reveal that the viral endoribonuclease antagonizes ifn induction via at least mechanisms. sars-cov orf b was reported to inhibit ifn induction and to act either on irf or possibly on mavs because it translocates to mitochondria when overexpressed in vero cells [ , ] . despite the fact it encodes a shorter protein than sars-cov, sars-cov- orf b was recently found to suppress ifn induction even more efficiently [ ] . by screening , sars-cov- sequences, the authors identified a natural variant encoding a longer orf b and displaying an even greater inhibitory activity [ ] . finally, sars-cov- nsp was also recently found to bind s ribosomal subunits (fig ) , thus inhibiting host mrna translation, including that of ifn-i on this cartoon are schematically represented the signaling pathways triggered by sars-cov rna recognition by the cytoplasmic rna sensors rig-i and mda , which leads to ifn induction (a) and subsequent ifn signaling in surrounding cells, resulting in the expression of isgs (b). sars-cov proteins that have been reported to interfere with these pathways are indicated. ifn, interferon; ifnar, interferon alpha and beta receptor; iκb, inhibitor of nuclear factor κb; ikkε, iκb kinase-ε; irf, ifn regulatory factor; isg, ifn-stimulated gene; jak, janus kinase; m, membrane; mavs, mitochondrial antiviral signaling protein; mda , melanoma differentiation-associated gene ; n, nucleocapsid; nsp, nonstructural protein; orf, open reading frame; p, phosphate; plp, papain-like protease; rig-i, retinoic acid-inducible gene ; sars-cov, severe acute respiratory syndrome coronavirus; stat, signal transducer and activator of transcription; tank, traf family member associated nf-κb activator; tbk , tank-binding kinase ; traf , tumor necrosis factor receptor-associated factor ; tyk , tyrosine kinase . https://doi.org/ . /journal.ppat. .g [ ], a feature that was previously demonstrated for other coronavirus-encoded nsp , including sars-cov [ , ] (fig and table ) . another viral strategy to inhibit ifn-i signaling is to enhance the host retrocontrol of this pathway. several isgs are themselves repressors of the ifn-i response, and their regulatory functions operate at the viral and host mrna transcription and translation steps, acting via a wide-range of mechanisms (reviewed in [ , ] ). for example, the inducible negative regulators such as the suppressor of cytokine signaling (socs and socs ) act at various levels of the jak-stat pathway or by targeting irf for degradation [ ] . in the context of sars-cov, the s protein induces the expression of socs expression in b cells [ ] . induction of socs / expression is also detected in sars-cov-infected cells, albeit to a lower extent as compared with other respiratory viruses [ ] . recent genomic screen approaches identified a set of repressors of the ifn-i response depending on the cell type and activation pathway involved [ ] [ ] [ ] . hence, one might anticipate that distinct repressors of the ifn-i response are induced depending on the cell type targeted by sars-cov- , the level of replication, and the microenvironment. for example, in the context of coronaviruses, an inefficient detection of mhv infection likely results from an inhibition of the basal levels of sensors mrna expression in several cell types [ ] . it is conceivable that this inhibition might involve negative regulators such as the ifn-inducible rnf , which targets signaling components such as rig-i, mda , and mavs for degradation [ ] . inhibition of protein synthesis is a conserved host response to prevent viral infections. the host translation is dynamically regulated by pkr, activated via recognition of viral rna (reviewed by [ ] ). activated pkr inhibits the eukaryotic initiation factor (eif α), a major regulator of the initiation phase of mrna translation, by phosphorylating its α subunit. the pkr-induced translational arrest shuts down the negative feedback on the ifn-i response, which can thus result in a prolonged and/or amplified ifn-i response [ ] . because pkr is an isg, the translational arrest is, in turn, potentiated by the ifn-i response (reviewed in [ ] ). this highlights a paradoxical situation in which translation arrest prevents viral replication but also set a threshold of viral detection to commensurate the host transcriptional antiviral response to the level of infection [ ] . whether the pkr pathway is modulated by sars-cov is unknown, yet different coronaviruses regulate pkr-eif α axis and host translation. for example, the mers-cov protein a (p a) accessory protein impedes pkr activation [ , ] . future studies are needed to further uncover the relationship between ifn-i response and host translation and their dynamics in the context of sars-cov infection. the ifn-i response varies among different cell types and within different microenvironments. studies at the single-cell level suggest that the amplitude and kinetic of the response is also heterogeneous for a given cell type. mathematic modeling revealed that ifn-i response is, at least in part, stochastic because only a fraction of cells are able to produce ifn-i upon activation by agonists of the sensors and are sensitive to the paracrine stimulation by ifn-i [ ] [ ] [ ] [ ] [ ] . the heterogeneity of the ifn-i response can be imprinted by the state of the cell at the activation time, including its global translation activity, metabolism, expression levels of signaling molecules (sensors, adaptors, and receptors) [ ] [ ] [ ] [ ] . additionally, the distinct onsets of the ifn-i induction depend on the rapidity and amplitude of viral replication. this heterogeneous responsiveness at the individual cell level consequently shapes the dynamics of the host antiviral response at the whole population level [ ] [ ] [ ] . this model of the ifn-i response dynamics yielded in the context of various rna viruses provides a framework likely at play for coronavirus infections. a delayed induction of the isg expression via virus-induced modulation of the basal activity of transcriptional activity of stat and pkr pathways leads to a peak of coronavirus replication preceding the isg response [ ] . additionally, in vivo study of the dynamic of mhv infection showed that a fast and robust ifn-i production by pdcs down-regulate the ifn-i response by other cells [ ] . this suggests that the ifn-i response at play in different cell types might drive the control of coronavirus infection and potentially contribute to the progression of the disease. as mentioned above, coronaviruses possess various mechanisms to defeat the ifn-i response within infected cells, and this inhibition ability is associated with clinical severity (reviewed in [ ] ). clinical studies showed that coronaviruses evade innate immunity during the first days of infection, which corresponds to a period of widespread inflammation and steadily increasing viral load [ , ] . elevated virus replication eventually leads to inflammation and hypercytokinemia, referred to as a "cytokine storm" [ ] [ ] [ ] [ ] (fig ) . the delayed ifn-i response indeed promotes the accumulation of pathogenic monocyte-macrophages [ , ] . this cell infiltrate results in lung immunopathology, vascular leakage, and suboptimal t cell response [ , ] . immune phenotypic profiling in peripheral blood mononuclear cells (pbmcs) of covid- patients similarly revealed that high viremia is associated with an exacerbated ifn-i response, an aggravated cytokine secretion, and inflammation, driving clinical severity [ ] . although the ifnar signaling pathway was up-regulated at an earlier disease stage, down-regulation of isgs, together with exacerbated nf-κb activation, promotes a cytokine storm and hyperinflammation, found in critically ill patients [ ] . collectively, these findings highlight the negative impact of a delayed ifn-i response on viral control and disease severity. however, the underlining mechanisms that drive the temporal control of the ifn-i response in patients are still elusive. in particular, the host and viral determinants driving the on/off switch of the ifn-i response in infected cells, noninfected cells, and/or stimulated immune cells need to be investigated. such studies will certainly benefit from longitudinal studies of immune profiling in sars-cov infected patients at the single-cell level and in combination with the clinical data. by producing , -fold more ifn-i than any other cell types, pdcs are at the heart of the antiviral ifn-i response [ , ] . they also produce other proinflammatory cytokines, which contribute to modulate the function of several immune cells, such as the mobilization of natural killer (nk) cells or the licensing of virus-specific t cell responses [ ] [ ] [ ] [ ] . because pdcs are refractory to most viral replication, their antiviral response cannot be inhibited by viral proteins [ , ] . this unopposed response likely contributes to the exceptional magnitude of pdc ifn-i production [ , [ ] [ ] [ ] . pdcs are circulating immune cells; nonetheless, their response is mostly localized at the infection site because their activation requires physical contact with infected cells [ , , ] . the contact site between pdcs and infected cells, which we named the interferogenic synapse, is a specialized platform for pamp transfer from infected cell to the toll-like receptor (tlr ) sensor in pdc, leading to an antiviral response [ ] . previous studies on sars-and mers-cov demonstrated that the rapid production of ifn-i by pdcs is essential for the control of potentially lethal coronavirus infections in mouse models [ , ] . pdcs migrate into the lungs at the early phase of infection (i.e., pdc number peaks at day ), temporally coinciding with the peak of ifnα production [ , ] . the pdc number was found to be reduced in blood of covid- patients as compared with control patients [ ] , potentially resulting from a prior response followed by a vanished number of circulating pdcs and/or their mobilization to the infected site. future studies are needed to address how pdc responsiveness evolves in the course of sars-cov- infection and how pdcs respond to contact with coronavirus-infected cells. despite the abovementioned viral inhibitory mechanisms of ifn-i response (table ) , exogenous ifn-i in cell cultures efficiently inhibit sars-cov, sars-cov- , and mers-cov spread [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . consistently, ifn-i was shown to have a protective effect against sars-cov and mers-cov, alone or in combination with other antivirals, in various animal models including mice, marmosets, and macaques [ , , ] . ifn-i and iii interfere with virus infection by inducing the expression of several hundred isgs [ ] . numerous welldescribed isgs exhibit direct antiviral activities by targeting specific stages of the viral life cycle, including entry into host cells, replication, protein translation, and assembly of new virus particles. as mentioned above, many signaling regulators are themselves isgs, thus leading to amplification of the antiviral ifn-i pathway. as a first step towards identifying isgs able to restrict sars-cov- replication, transcriptomic responses to infection have been analyzed in different cellular models, including primary cells, organoids, and clinical samples [ , [ ] [ ] [ ] , as summarized in table . these studies demonstrate that, despite triggering very little to no ifn expression (table ) , sars-cov- replication induces moderate levels of a limited number of isgs. a small subset of infected cells may be refractory to the antagonistic mechanisms of sars-cov- , producing minute but sufficient amounts of ifns to trigger isg induction in larger population of cells. alternatively, isgs may be up-regulated in noninfected cells, which were analyzed together with infected ones. indeed, interpretation of genome-wide investigations of virus-pathogen interactions are often obscured by analyses of mixed populations of infected and uninfected cells [ ] . of note, by contrast to low-multiplicity of infection (moi) infection of a cells expressing angiotensin i converting enzyme (ace ), normal human bronchial epithelial (nhbe) cells, and patient samples, high-moi infections of a -ace and calu- cells led to the high induction of ifns and isgs, including isgs with broad antiviral activities [ ] ( table ). this discrepancy of ifn production/signaling between the levels of viral replication and/or proportion of infected cells might reflect that the counteraction measures employed by sars-cov- are less potent at high moi. alternatively, as suggested by blanco-melo and colleagues, high-moi infections in cell culture may generate more pamps, such as defective noninfectious viral particles, than low-moi infections [ ] . despite being expressed at moderate levels in vitro and in vivo, several up-regulated isgs identified by these transcriptomic studies ( table ) exhibit well-characterized broad-spectrum antiviral activities and could thus have additive restrictive effects on sars-cov- replication. for instance, the members of the interferon-induced protein with tetratricopeptide repeats (ifitm) family, known to inhibit entry of numerous enveloped rna viruses [ ] , similarly restrict entry of sars-cov, mers-cov, and the globally circulating human coronaviruses e and nl in t and a cell lines [ , ] . oas and mycovirus resistance protein (mx)a could also contribute to the ifn-i-mediated inhibitory effect on sars-cov- because a clinical study revealed that single nucleic polymorphisms in the oas -utr and mxa promoter region appear associated with host susceptibility to sars-cov in the chinese han population [ ] . moreover, the fact that mers-cov nonstructural protein b (ns b) is a - - oligoadenylate synthetase (oas)-rnase l antagonist [ ] suggests that the oas pathway contributes to the antiviral effects of ifns on coronavirus replication. isgs positively potentiating ifn signaling, such as ifih /mda , tank, irf , and stat , were also increased in the bronchoalveolar lavage fluid (balf) of covid- patients as compared with healthy controls [ ] and could potentially contribute to the amplification of ifn-i response against sars-cov- replication. zinc finger antiviral protein (zap), which is encoded by an isg, contributes to the anti-sars-cov- effect of ifns in human lung calu- cells [ ] . zap is known for restricting the replication of numerous viruses such as retroviruses and filoviruses [ ] . the protein recruits the cellular mrna degradation machinery to viral rna via -c-phosphate-g- (cpg) dinucleotide recognition [ ] . to further determine which individual isg or combination of isgs mainly restricts sars--cov- replication in vitro, several previously established approaches could be used, such as, for example, screening for single or combined isg activity using a lentiviral vector-based library, as successfully performed by schoggins and colleagues for other viral infections [ ] [ ] [ ] [ ] . indeed, this library of around human isgs was recently screened in human hepatoma cells for antiviral activity against hcov- e [ ] . the screen identified ifn-inducible lymphocyte antigen complex, locus e (ly e) as a potent inhibitor of the replication of multiple coronaviruses, including sars-cov, sars-cov- , and mers-cov, by blocking fusion of viral and cellular membranes [ ] . mice studies revealed that ly e directly protects primary b cells and dendritic cells from murine coronavirus infection [ ] . pursuing the identification and characterization of ifn effectors with potent anti-sars-cov- activities will reveal weakness points in the life cycle of sars-cov- and may lead to the design of drugs that activate antiviral isgs or either mimic or amplify their action. recent advances in systematic screening strategies have revealed the existence of a small subset of isgs exhibiting proviral activities [ , ] . these proviral isgs act either by exhibiting direct proviral activities such as facilitating viral entry [ ] or via their abilities to negatively regulate ifn signaling and facilitate the return to cellular homeostasis. the receptor tyrosine kinase axl is a well-characterized example of an isg that is used by enveloped virus for cellular internalization [ ] [ ] [ ] . alternatively, isgs that possess antiviral activities against a viral family can be hijacked by unrelated viruses to favor infection. this is the case for ifitm and ifitm , which potently block entry of a broad range of enveloped viruses [ ] while promoting entry step of human coronavirus oc (hcov-oc ) in human cells [ ] . sars-cov- uses ace and transmembrane serine protease (tmprss ) to enter cells [ ] . viral tropism is thus largely dictated by ace and tmprss coexpression. analysis of human, nonhuman primate, and mouse single-cell rna-sequencing (scrna-seq) data sets generated from healthy or diseased individuals revealed that expression of ace is primarily restricted to type ii pneumocytes in the lung, absorptive enterocytes within the gut, and goblet secretory cells of the nasal mucosa [ ] . interestingly, this meta-analysis identified an association between ace expression and canonical isgs or components of the ifn-signaling pathway in different tissues. independent analyzes of publicly available data sets concluded that ace expression pattern is similar to isgs [ ] . in vitro validations were performed by treating primary human upper airway cells with numerous inflammatory cytokines. ifnα , and to some extent ifny, led to greater and more significant ace up-regulation compared with all other tested cytokines [ ] . substantial up-regulation of ace was also observed in primary skin and primary bronchial cells treated with either ifn-i or ifn-ii. moreover, ace expression was also up-regulated upon ex vivo influenza a infection in human lung explants isolated following surgical resection [ ] . because the majority of cells robustly up-regulating ace were epithelial, this observation potentially explains why previous analyses to define canonical isgs within immune populations did not identify ace as an induced gene [ ] . finally, stat , stat , irf , and irf binding sites were identified within − , to + bp of the transcription start site of ace [ ] . despite need for direct evidence that ifns up-regulate ace in target cells in vivo, altogether these studies suggest that ace could be an isg that enhances sars-cov- internalization in human epithelial cells [ , ] . elucidating tissue and cell type specificity of isgs, as well as their mechanisms of action, is essential for understanding the potential dual role of ifns during human sars-cov- infection. it may also guide the use of ifns in clinical trials. although ifn-i treatment gave some encouraging results against sars-cov and mers-cov in vitro and in animal models, including mice, marmosets, and macaques [ , , , , ] , additional knowledge to optimize its therapeutic efficiency in humans is required [ ] [ ] [ ] [ ] [ ] . previous information yielded from these animal studies provided guidance for treating the current pandemic virus. first, it became clear from these former studies that ifnβ is a more potent inhibitor than ifnα as shown both in vitro and in patients [ , ] . second, the timing of ifn-i treatment seems determinant for infection outcomes. indeed, as shown in mice and in macaques, ifn-i is protective when administered prior to sars-cov or mers-cov infection or early in the course of infection, whereas late administration could be either ineffective or detrimental [ , ] . in humans as well, ifn-i-based therapies were not beneficial to critically ill patients with multiple comorbidities and who were diagnosed late with mers-cov, thus pointing out that ifn-i has to be administered early after infection [ , ] . the first clinical trials using ifn-i alone or in combination with other antivirals are currently carried out in covid- patients in several countries. for instance, the multicenter, adaptive, randomized, open clinical trial discovery evaluates, among other treatment, the efficacy of ifnβ as a treatment for covid- in hospitalized adults in europe. a recent openlabel, randomized, phase trial performed in adults with covid- in hong kong showed that the triple combination of ifnβ- b, lopinavir-ritonavir, and ribavirin was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate covid- [ ] . it has to be noted that the patients were treated in the early stages of the disease because the median number of days from symptom onset to start of study treatment was days, further reinforcing the fact that the timing of ifn-i treatment is key [ ] . other therapeutic approaches are under investigation to avoid the adverse effects of ifn-i therapy and/or its potential inefficacity when administrated too late postinfection. one strategy is to use aerosol formulations of recombinant ifn to deliver the cytokine directly inside the lung [ , ] . this approach has several benefits because it is a noninvasive route of administration, and the local concentration reached in the tissue can be higher than through systemic injection and is thus expected to minimize the adverse effects of ifn. nebulized ifnα- b was used on covid- patients in wuhan, alone or in combination with arbidol [ ] . the study, performed on adults, showed a significant reduction of the duration of detectable virus in the upper respiratory tract in ifnα- b-treated patients, with or without arbidol [ ] . another study currently ongoing in beijing aims at evaluating the efficacy and safety of recombinant human ifnα spray in preventing sars-cov- infection in highly exposed medical staffs (chictr ). type iii ifns (ifnλs or ifn-iii) are gaining an increased interest in antiviral therapies [ ] [ ] [ ] . like ifn-i, they activate the jak-stat signaling pathway. they do so via a receptor that is largely restricted to cells of epithelial origin, including respiratory epithelial cells (reviewed in [ ] ). ifn-iiis are induced upon viral infections, and they are growing evidence that they provide important first-line defense against viral infections of the respiratory and gastrointestinal tracts [ ] [ ] [ ] . in mice, ifn-iii was shown to protect epithelial cells of the respiratory and tract from infections with several respiratory viruses, including mers-cov [ ] . a study investigating sars-cov- infection of intestinal epithelial cells, using both colon-derived cell lines and primary colon organoids, showed that ifn-iii response was more efficient than ifn-i at controlling viral replication [ ] . however, ifn-iiis produced by dendritic cells in the lung were recently shown to cause barrier damage and to compromise host tissue tolerance and predispose to lethal bacterial superinfections [ ] . therefore, although the antiviral properties are promising, the benefit of ifn-iii to treat covid- patients awaits careful evaluation. the first clinical trials using ifn-iii are ongoing, including one launched at the massachusetts general hospital to evaluate the safety and efficacy of pegylated ifnλ on a small number of covid- patients (nct ). besides the use of recombinant ifn as a therapeutic treatment, one interesting alternative strategy would be to boost the natural innate immune defenses of covd- patients at early stages of the disease. because pdcs are seemingly crucial to control coronavirus infections [ , ] , a possibility would be to either amplify or prolong their activation to make them produce more ifn-i and ifn-iii. a number of negative feedback loops prevent an exacerbated activation of pdcs, which can be deleterious for the organism in the long term. thus, transitorily inhibiting these negative retrocontrols may increase the antiviral activity of pdcs. for instance, the bone marrow stromal cell antigen (bst ) is an isg that activates the immunoglobulin-like transcript (ilt ) inhibitory receptor expressed by pdcs to interrupt the ifn-i response [ ] . the blockade of this interaction using either antibodies or inhibitory molecules should thus increase the duration of pdc activation. one could also envisage to take advantage of viral proteins that counteract the antiviral activity of bst , such as hiv- viral protein u (vpu) [ ] . other pdc inhibitory molecules include natural monamines such as histamine, dopamine, or serotonin, which bind to the c-x-c motif chemokine receptor (cxcr ) at the surface of pdcs [ ] . because the cxcr antagonist amd (also known as plerixafor) blocks the binding of monoamines to pdcs, it can prevent the amine-dependent inhibition of pdc activation [ ] . amd is already used in clinics as an immunostimulatory molecule able to mobilize hematopoietic stem cells in cancer patients [ ] . finally, we recently reported that the peptidyl-prolyl isomerase peptidyl-prolyl cis-trans isomerase nimainteracting (pin ) switches off the ifn-i expression by pdcs by inducing irf degradation [ ] . a number of pin inhibitors have been developed and could be tested for their potential activity on human pdcs [ ] and could represent another possible therapeutic strategy to boost pdc-mediated ifn-i production. sars-cov- emerged in the human population around months ago, yet it seems well adapted to avoid and inhibit the ifn-i response in its new host. such efficient strategies allow the virus to replicate and disseminate in infected individuals without encountering the initial host defense. this modest ifn response could explain why viremia peaks at early stages of the disease, at the time of symptoms appearance, and not around to days following symptoms, like during sars-cov and mers-cov infections. ifnβ treatment would be expected to improve the antiviral response of patients at the early stage of covid- and, if possible, at plos pathogens the site of infection. indeed, ifnβ appeared to be pivotal to improve patient states in a combined therapy regiment of ifnβ, lopinavir-ritonavir, and ribavirin [ ] . nonetheless, ifnresistant viral mutants may arise and be able to control ifn even more efficiently than parental viruses. the exacerbated production of proinflammatory cytokines observed at later stage of covid- might challenge the efficiency of an ifnβ treatment administrated after appearance of symptoms. there is indeed an increasing appreciation of the detrimental effects of inappropriate, excessive, or mistimed ifn-i responses in viral infections [ ] . the underlying mechanisms by which ifn-i promote disease 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bst and ilt receptor interaction epstein-barr virusencoded ebna modulates the ap- transcription factor pathway in nasopharyngeal carcinoma cells and enhances angiogenesis in vitro natural amines inhibit activation of human plasmacytoid dendritic cells through cxcr engagement mozobil(r) (plerixafor, amd ), years after its approval by the us food and drug administration trim is required for virusinduced ifn response in human plasmacytoid dendritic cells development of pin inhibitors and their potential as therapeutic agents disease-promoting effects of type i interferons in viral, bacterial, and coinfections a critical role for the sphingosine analog aal-r in dampening the cytokine response during influenza virus infection endothelial cells are central orchestrators of cytokine amplification during influenza virus infection targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals we thank nathalie j. arhel for helpful discussion. key: cord- - uwhxs authors: plaisted, warren c.; weinger, jason g.; walsh, craig m.; lane, thomas e. title: t cell mediated suppression of neurotropic coronavirus replication in neural precursor cells date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: uwhxs neural precursor cells (npcs) are the subject of intense investigation for their potential to treat neurodegenerative disorders, yet the consequences of neuroinvasive virus infection of npcs remain unclear. this study demonstrates that npcs support replication following infection by the neurotropic jhm strain of mouse hepatitis virus (jhmv). jhmv infection leads to increased cell death and dampens ifn-γ-induced mhc class ii expression. importantly, cytokines secreted by cd + t cells inhibit jhmv replication in npcs, and cd + t cells specifically target viral peptide-pulsed npcs for lysis. furthermore, treatment with ifn-γ inhibits jhmv replication in a dose-dependent manner. together, these findings suggest that t cells play a critical role in controlling replication of a neurotropic virus in npcs, a finding which has important implications when considering immune modulation for npc-based therapies for treatment of human neurologic diseases. transplantation of multipotent neural precursor cells (npcs) is emerging as a feasible therapeutic strategy for the treatment of a variety of neurological disorders. recent studies have demonstrated both short and long-term clinical benefits following npc engraftment within the context of rodent models of alzheimer's disease, parkinson's disease, huntington's disease, and acute spinal cord injury (blurton-jones et al., ; mcbride et al., ; van gorp et al., ; yasuhara et al., ) . furthermore, in murine and non-human primate models of the neuroinflammatory disease multiple sclerosis (ms) the ability of human npcs to function as modulators of the immune system in addition to replacing lost or damaged neural cell populations has been suggested (aharonowiz et al., ; pluchino et al., pluchino et al., , . however, despite the clinical and histological benefits of npc transplantation in pre-clinical animal models of neurologic disease, there is limited evidence addressing the capacity of neural grafts to act as reservoirs for viral replication. studies using the non-polio enterovirus coxsackievirus b (cvb) demonstrate the ability of cvb to preferentially replicate in murine npcs (ruller et al., ) . the ensuing carrier-state infection results in increased cell death and impaired differentiation potential in vitro, as well as inflammation, microgliosis, and a variety of cns developmental defects in vivo (ruller et al., ; tsueng et al., ) . intracerebral infection of neonates with murine cytomegalovirus (mcmv) results in the loss of neural stem cells and their neuronal progeny, as well as a decrease in the production of neurotrophins imperative to normal brain development (mutnal et al., ) . borna disease virus (bdv) infection of human fetal human npcs results in cell death upon differentiation and impaired neurogenesis (brnic et al., ) . thus, the role of neural stem and progenitors as targets for a variety of neuroinvasive viruses is evident, while the consequences of infection within the context of cellular therapy remain to be elucidated. complicating npc-based therapies is the controversial issue of antigenicity of transplanted cells and immune-mediated recognition. a growing body of evidence suggests npcs are not immunoprivileged, as has previously been reported (hori et al., ) . indeed, we have shown that npcs derived from post-natal c bl/ brains express the co-stimulatory molecules cd and cd and up-regulate major histocompatibility complex (mhc) molecules in response to the pro-inflammatory cytokine interferon gamma (ifn-γ) (weinger et al., ) . furthermore, allogeneic npcs are rapidly rejected via a t cell mediated mechanism following intraspinal transplantation into mhc-mismatched recipients (weinger et al., ) . similarly, human npcs have the capacity to express mhcs i and ii and induce t cell proliferation (goya et al., ) . the apparent antigenicity of npcs suggests successful engraftment may require the use of immunomodulatory agents and lifelong suppression of the immune system, as with solid organ transplants. however, an unintended consequence of immune suppression is the potential for latent viruses to become activated, or for uncontrolled viral replication to occur following opportunistic infection (crough et al., ; jordan et al., ; wynn et al., ; young et al., ) . therefore, it is imperative to understand the consequences of neurotropic virus infection of npcs as cellreplacement therapies continue to move into the clinic (gupta et al., ; riley et al., ) . in this study, we demonstrate that cultured murine npcs are infected by the neurotropic jhm strain of mouse hepatitis virus (jhmv), which induces acute encephalomyelitis and chronic demyelination when injected intracranially into immunocompetent mice. jhmv-infected npcs support replication that ultimately results in increased cell death over time. importantly, cd þ t cells kill npcs pulsed with viral-peptides, and jhmv replication in npcs was suppressed, in part, by ifn-γ secreted from virus-specific cd þ t cells. npcs express the mhv receptor ceacam a and are infected by jhmv jhmv is a neurotropic coronavirus with relatively restricted tropism for glial cells through recognition and binding to the receptor carcinoembryonic antigen-cell adhesion molecule a (ceacam a) (hirai et al., ; thorp and gallagher, ) . ceacam a expression in mouse tissues is widespread and can be detected on the surface of a variety of epithelial cells in the gastrointestinal, respiratory, and reproductive tracts, as well as on small vascular endothelia and hematopoietic cells (hemmila et al., ) . however, ceacam a expression is not ubiquitous, and although it is known to be located at the surface of resident cells of the cns including glia, expression by neural stem or progenitor cells has not been evaluated. to determine if npcs derived from c bl/ transgenic mice engineered to express gfp (gfp-npcs) express ceacam a, mrna was isolated from cultured npcs and receptor expression was evaluated by pcr. using ceacam a-specific primers, pcr amplicons were detected in npcs, as well as mixed splenocytes from c bl/ mice acting as controls (fig. a) , and nucleotide sequencing confirmed homology with the specified region of the gene (data not shown). furthermore, cell surface expression of ceacam a was confirmed with more than % of npcs expressing the receptor as determined via flow cytometric analysis (fig. b) . we next infected sox þ gfp-expressing npcs with jhmv to assess susceptibility to infection. infected npc cultures were fixed h post-infection (p.i.) and stained with an antibody specific for the carboxyl terminus of the jhmv nucleocapsid (n) protein and subsequently imaged using fluorescence microscopy. compared to non-infected npcs that form a confluent monolayer when grown in tissue culture-treated, matrigel-coated vessels, sox þ npcs infected at a multiplicity of infection (m.o.i.) of . displayed jhmv-specific syncytia formation by h post-infection ( fig. a) . correspondingly, increasing viral titers were detected when plaque forming unit (pfu) assays were performed on supernatants harvested from jhmv-infected npc cultures at , , and h p.i. (fig. b) . furthermore, determination of lactate dehydrogenase (ldh) released into the supernatants of infected cultures at defined time p.i. revealed increased npc death over time, ranging from . . % at h p.i., increasing to . . % at h p.i., and peaking at . . % by h (fig. c ). as jhmv replication has been reported to occur via ceacam a-dependent and independent mechanisms (nakagaki and taguchi, ) , we performed a monoclonal antibody blockade to determine the role of ceacam a in the spread of jhmv infection in cultured npcs (fig. d ). by h p.i., significant (p o . ) inhibition of viral replication was observed in anti-ceacam a-treated cells ( .  .  pfu/ml, n¼ ) when compared to non-treated, jhmvinfected npcs ( .  .  pfu/ml). under normal culture conditions, expression of mhc classes i and ii is undetectable on npcs, yet mhc expression can be induced by treatment with ifn-γ (chen et al., ; weinger et al., ) . to investigate if jhmv infection alters mhc class i and/or ii expression on npcs, we compared surface expression levels of these molecules on non-infected and infected cells in the absence or presence of u/ml ifn-γ. our findings indicated r % of npcs were found to be positive for mhc class i ( fig. a fig. a and c). however, mhc class ii was detected on a significantly (p o . ) lower fraction ( . . %, n ¼ ) of infected, ifn-γ-treated npcs compared to non-infected, ifn-γ-treated npcs ( . . %, n¼ ) (fig. c) . furthermore, mhc class ii could not be detected on the majority of jhmv-infected npcs as determined by dual staining for viral antigen and mhc class ii (fig. d ). cd þ and cd þ t cells are pivotal in controlling jhmv replication within the infected cns (sussman et al., ; williamson and stohlman, ). virus-specific effector cd þ t cells help control replication in infected astrocytes and microglia through cytolytic activity (bergmann et al., ) . in addition to secreting ifn-γ that limits viral replication in oligodendrocytes, cd þ t cells carry out perforin-dependent cytolysis of astrocytes and microglia (bergmann et al., ; williamson and stohlman, ) . we co-cultured virus-specific ctls at diminishing effectorto-target (e:t) ratios with npcs pulsed with the immunodominant cd peptide specific for jhmv spike (s) glycoprotein spanning amino acids - (s - ), and treated with ifn-γ to induce mhc class i expression. subsequently, ldh released in the supernatants was evaluated to quantify ctl-mediated npc lysis; rma-s cells, a murine lymphoma cell line that presents viral peptides to ctls in an mhc class i dependent manner, were used as positive control (debruijn et al., ) . npcs pulsed with s - peptide were specifically lysed by virus-specific ctls at an e:t ratio of - (p o . , n ¼ ), indicating that virus-specific cd þ t cells are capable of recognizing and directly killing jhmv-infected npcs in vitro (fig. ) . importantly, this cytolytic effect waned as the e:t to target ratio declined. cd þ t cells have both indirect and direct antiviral roles during acute jhmv-induced encephalomyelitis, which include inducing the effector functions of virus-specific ctls, along with ifn-γ secretion (savarin et al., ; stohlman et al., fig. . virus-specific cd þ t cells target s - pulsed npcs for lysis. ctls were harvested from mice immunized with the dm variant of jhmv and co-cultured at varying effector:target ratios with s - pulsed, ifn-γ-treated npcs for h, and lactate dehydrogenase released into the supernatant was subsequently measured. non-ifn-γtreated rma/s cells pulsed with μm s - were used as a positive lysis control. negative selection was performed to purify the respective t cell populations. npc media was conditioned with cd þ t cell cytokines for h and then added to jhmv-infected npcs. supernatants from either naïve or virus-specific cd þ t cells suppressed viral replication in npcs at and h post-infection, with the most significant inhibitory effects observed in groups treated with media enriched with virus-specific cd þ t cell cytokines (fig. a) . however, while the suppressive effects of naïve t cell media appeared to wane by h p.i. ( .   pfu/ml), supernatants from npcs treated with virusspecific cd þ t cell conditioned media maintained low viral titers ( .  .  pfu/ml) in comparison to non-treated controls ( .  .  pfu/ml; fig. a ). t cell derived ifn-γ is critical in controlling jhmv replication in the cns (bergmann et al., ; smith et al., ) . furthermore, treatment with ifn-γ specifically inhibits jhmv replication in oligodendrocyte progenitors (opcs) derived from c bl/ npcs, and inhibition of ifn-γ signaling in oligodendrocytes is associated with increased viral loads and mortality (parra et al., ; whitman et al., ) . we evaluated levels of ifn-γ in naïve-versus-dm specific cd þ t cell conditioned media by enzyme-linked immunosorbent assay (elisa); absorbance values from media conditioned with dm-cd þ t cells were increased $ -fold when compared to naïve t cell conditioned media (p o . ; fig. b ). we subsequently treated jhmv-infected npcs with varying amounts of mouse recombinant ifn-γ for h and determined its effects on viral titers. npcs treated with or u/ml ifn-γ maintained high jhmv titers ( .  .  pfu/ml and .  .  pfu/ml, respectively) in relation to non-treated groups ( .  .  pfu/ml; fig. c ). however, jhmv replication in npcs was reduced in cultures treated with or u/ml ifnγ ( .   pfu/ml and .  .  pfu/ml, respectively; fig. c ). we next performed a -h time course to further probe the effects of ifn-γ ( u/ml) on jhmv-infected npcs. a reduction from .  .  pfu/ml to .  .  pfu/ml was observed in ifn-γ-treated cultures by h post-treatment when compared to non-treated groups (p o . , n ¼ ), and jhmv levels were reduced to .  .  in ifn-γ treated cultures, versus .  .  in non-treated cultures, by h post-treatment (po . ) (fig. d ). we previously showed that multiple pro-inflammatory cytokines secreted by dmspecific t cells have synergistic effects with ifn-γ (weinger et al., ) . to confirm the role of ifn-γ as the major cytokine contributing to suppression of jhmv replication in infected npc cultures, monoclonal antibody blockade against the ifn-γ receptor was performed on npcs before and during treatment with virusspecific cd þ t cell enriched media. as expected, by h p.t. jhmv levels were significantly (po . ) reduced in conditioned media treated cultures compared to npcs grown in non-conditioned media ( .  .  and .  .  pfu/ml, respectively; fig. e ). however, treatment with anti-ifn-γ receptor resulted in higher (po . ) viral titers ( .  .  ) compared to cd þ t cell media treated cultures, thereby confirming the pivotal role of ifn-γ in cd þ t cell mediated suppression of jhmv in npcs. we have previously shown that ifn-γ treatment of jhmvinfected opcs increases ifn-α/β secretion, and treatment with ifn-β suppresses jhmv replication (whitman et al., ) . type i interferon (ifn-β) levels in jhmv-infected, ifn-γ treated npc supernatants were assessed by elisa and ifn-β was not detected above background levels (data not shown). we evaluated the expression of the jhmv receptor ceacam a on npcs following treatment with u/ml ifn-γ and did not observe a change in the frequency of cecam aþ npcs between treated and non-treated groups at h p.t. (fig. a and b) (matthews et al., ) . to determine if m transcripts were decreased following ifn-γ treatment, gene-specific quantitative pcr (qpcr) was performed on total rna extracts from jhmv-infected npcs and m transcript levels were normalized to β-actin. m expression was significantly reduced in ifn-γ treated npcs compared to non-treated npcs at and h p.t. (po . ; fig. c ). these findings suggest that the ifn-γinduced inhibitory effect on jhmv replication within npcs is related to both muted expression of ceacam a and inhibition of viral rna synthesis. this study demonstrates that npcs derived from the brains of post-natal c bl/ -gfp mice express the jhmv receptor, cea-cam a, and support viral replication following ceacam adependent infection. additionally, jhmv infection of cultured npcs induces cytopathic effects over time as evidenced by syncytia formation and elevated ldh levels. within the context of jhmv infection of the cns, these findings demonstrate that resident npcs present within defined anatomical niches may be susceptible to viral infection. moreover, we have previously shown that intraspinal transplantation of npcs into mice persistently infected with jhmv results in clinical recovery associated with remyelination (carbajal et al., ; totoiu et al., ) . data presented within this report argues that transplanted npcs may be susceptible to jhmv infection, a finding that highlights important clinical implications for emerging therapies utilizing npcs to treat human neurologic disease as engrafted cells may be susceptible to infection by persistent neurotropic viruses. jhmv infection has previously been shown to inhibit constitutive expression of mhc class i in mouse primary astrocyte cultures and to block ifn-γ-induced mhc class ii expression on murine cerebral endothelial cells (correale et al., ; joseph et al., ) . here, we show that jhmv does not significantly affect mhc class i or ii expression following infection of cultured npcs in the absence of ifn-γ. however, ifn-γ-induced expression of mhc class ii was reduced following jhmv infection. mhc expression plays an important role in immune surveillance during viral infection, and control of jhmv replication within the cns requires antigen recognition by mhc class i and mhc class ii restricted cd þ and cd þ t cells (bergmann et al., ; sussman et al., ; williamson and stohlman, ) . impaired expression of mhc class ii following ifn-γ-treatment of infected npcs may be a mechanism employed to subvert detection by infiltrating virus-specific cd þ t cells. nonetheless, conditioned medium from virus-specific cd þ t cells was able to suppress jhmv replication within npcs, likely due to the effects of ifn-γ. supporting this notion, treatment of infected npcs with recombinant mouse ifn-γ had a dose-dependent inhibitory effect on virus replication, and blocking ifn-γ receptor abrogated the observed suppressive effects. ifn-γ treatment resulted in fewer ceacam a-expressing npcs with a concomitant decrease in jhmv membrane glycoprotein transcripts, suggestive of viral entry inhibition and reduced virion assembly. we also observed that npcs pulsed with the cd -specific viral peptide s - were detected and killed by virus-specific cd þ t cells, indicating that virallyinfected npcs may be targeted for lysis by ctls infiltrating into the cns in response to infection. collectively, our findings argue that t cells are important for controlling viral replication within npcs through both cytolytic activity and ifn-γ secretion. lineage fate mapping of neural stem/precursor cells residing within the subventricular zone of lateral ventricles and subgranular zone of the hippocampus demonstrates the ability of these cells to differentiate into neurons and glia throughout development (doetsch, ; gage, ) . furthermore, endogenous npcs have been shown to proliferate, migrate, and differentiate in response to acute cns inflammatory events, such as with spinal cord injury, stroke, and experimental models of chronic inflammatory demyelinating disorders (picard-riera et al., ; yagita et al., ; zhang et al., ) . though viewed as a glial tropic virus, this study highlights the potential for npcs to serve as a reservoir for jhmv infection and replication. ctl-mediated lysis of jhmv-infected npcs may be detrimental to npc-mediated repair during cns inflammation, and a loss of npcs destined to become oligodendrocytes could contribute to limited remyelination observed in the jhmv-infected cns. additionally, our findings have clinical relevance, as npcs are currently being employed in clinical trials for spinal cord injury as well as for treating the pelizaeus-merzbacher disease, a genetic disorder that affects the growth of the myelin sheath (gupta et al., ; mayor, ) . as npcs used for clinical trials are unlikely to be "self-derived", they would be subject to immune recognition and potential destruction by both innate and adaptive immune responses, necessitating long-term immune suppression to prevent graft rejection (chen et al., ; swijnenburg et al., ; weinger et al., ) . several classes of immunosuppressive drugs used during transplantation, including calcineurin inhibitors i.e. cyclosporine and fk , inhibit the activation and/or proliferation of t cells. such immunosuppressive drugs would foster an environment whereby opportunistic infection or reactivation of latent virus might occur. this raises the possibility that transplanted npcs may be subject to infection, and in the absence of adequate immune surveillance of the cns, could lead to damage/ death of engrafted cells. with this in mind, careful consideration should be given to potential viral infection when contemplating npc grafting for treating neurological disease. the jhm strain of mouse hepatitis virus (j . v- ) was added to npc cultures at a multiplicity of infection (moi) of . pfu/cell. virus was allowed to absorb overnight ( - h) before media were replaced. supernatants of infected cultures were collected at defined time p.i. and viral titers were determined using the dbt astrocytoma cell line as previously described (hirano et al., ) . npcs derived from the striatum of post-natal day transgenic c bl/ mice expressing enhanced green fluorescent protein (gfp) were cultured as previously described (carbajal et al., ) . npc media consisted of dmem/f with glutamax (gibco), n supplement ( x, gibco), ciprofloxacin hydrochloride ( μg/ ml, cellgro), gentamicin ( μg/ml, sigma-aldrich), fungizone ( . μg/ml, gibco), penicillin/streptomycin ( u/ml, gibco), and human epidermal growth factor ( ng/ml, sigma-aldrich). recombinant mouse ifn-γ was purchased from cell sciences. for studies involving blockade of ceacam- a, npcs were infected overnight and monoclonal antibody cc (ebiosciences) was subsequently added at a concentration of μg/ml. media were harvested h p.i. and plaque assay performed to determine viral titers. experimental blockade of ifn-γ receptor was performed using jhmv-infected npcs incubated with nm (final) antimouse cd (ifn gamma receptor ; ebiosciences) or nm purified rabbit igg (control; bd pharmigen) for h before media were replaced with non-conditioned or cd þ t cell conditioned media þ/ À anti-mouse cd or rabbit igg. supernatants were harvested h post-treatment and viral titers determined. cultured npcs were dissociated using . % trypsin-edta and suspended in pbs containing . % bsa and mm edta (invitrogen). cells were subsequently treated with blocking antibody (purified rat igg b anti-mouse cd /cd monoclonal antibody, : ; bd biosciences) for min at c before being incubated with antibodies specific for ceacam a (apc-conjugated, . μg/ test, ebioscience), mhc class i (pe-conjugated, : , ebioscience), or mhc class ii (pe-conjugated, : , bd biosciences), for - min. in experiments where facs analysis of jhmv was performed, npcs were fixed with % paraformaldhyde for min before being permeabilized using bd perm/wash buffer (bd biosciences). the anti-jhmv mab j. . specific for the carboxyl terminus of the viral nucleocapsid (n) protein was conjugated to alexa fluor using the apex labeling system (life technologies) and used at a final concentration of . ng/ml. detection of fluorescence was performed using a lsr ii flow cytometer (bd biosciences) and analysis of facs data was performed with flowjo software (tree star). total rna was isolated from c bl/ splenocytes and npcs using trizol reagent (invitrogen) and purified by phenol-chloroform extraction. cdna was reverse transcribed from rna according to manufacturer's instructions using the superscript iii first-strand synthesis system (invitrogen) and random hexamers. standard pcr for ceacam a expression was performed with an eppendorf mastercycler using the platinum taq dna polymerase kit (invitrogen) and the following primers purchased from integrated dna technologies: ttccctggggaggactactg (forward primer) and tgtatgcttgcc ccgtgaaat (reverse primer). gene products were run alongside a kb plus dna ladder (invitrogen) on a % agarose gel containing ethidium bromide before being imaged using the bio-rad geldoc system. for quantitative rt-pcr experiments, primers specific for the jhmv membrane protein (forward: cgagccgtagcatgtttatcta; reverse: cgcatacacgcaattgaa-cata) were designed using primerquest software (integrated dna technologies, inc.). sybr green real-time pcr master mix (life technologies) was used according to manufacturer's specifications and rt-pcr was performed using the applied biosystem viia real-time pcr system. c t values of m protein transcripts were normalized to β-actin c t values (forward: ggcccagagcaa-gagaggtatcc; reverse: acgcacgatttccctctcagc) and compared using the ΔΔc t method. to evaluate jhmv infection of cultured npcs, cells were dissociated and plated on slides or cover slips coated with reduced growth factor matrigel (bd biosciences). npcs were infected with jhmv overnight and fixed h p.i. with % paraformaldehyde for min at room temperature. immunofluorescence staining was performed as previously described (whitman et al., ) using the anti-jhmv mab j. . ( : dilution) specific for the carboxyl terminus of the viral nucleocapsid (n) protein and the alexa fluor goat anti-mouse igg secondary antibody (life technologies), as well as rabbit monoclonal anti-sox (epitomics) and alexa fluor goat anti-rabbit igg secondary antibody (life technologies). slides were imaged using a nikon eclipse ti inverted microscope. npc death due to jhmv infection was evaluated at , , and h p.i. by measuring lactate dehydrogenase released by lysed cells according to manufacturer's recommendations using the cytotox non-radioactive cytotoxicity assay (promega). briefly, spontaneous and virus-induced ldh levels were determined using the following formula: % lysis¼ (experimental ldh release)/(maximum ldh release). ldh levels from jhmv-infected cultures were then normalized to spontaneously released ldh and expressed as cell death due to infection (%). cd þ t cell isolation for npc media conditioning c bl/ mice were infected with an i.p. injection of .  pfu of a demyelinating (dm) variant of jhmv. on day p.i., cd þ t cells were isolated from spleens by negative selection according to manufacturer's specifications using the easysep mouse cd þ t cell isolation kit (stemcell technologies). briefly, red blood cell depleted splenocytes were suspended at a concentration of  cells/ml in pbsþ % fbs with mm edta. normal rat serum was added at the appropriate concentration and cells were incubated with a cocktail containing a combination of biotinylated monoclonal antibodies directed against cd a, cd b, cd c, cd , cd r/b , cd b, tcrγ/δ and ter , for min. subsequently, a suspension of streptavidin-coated magnetic particles in pbs was added and incubated with the cells for . min; buffer was added to the appropriate volume, and cells were incubated in the easysep magnet for . min to foster binding of magnetically-labeled unwanted cells to the tube walls before cd þ t cells were poured off. to generate cd þ t cell conditioned npc media, the magnetically-labeled fraction following depletion of total t cells was collected using the easysep mouse t cell isolation kit (stemcell technologies). this enriched fraction was treated with μg/ml mitomycin-c (ag scientific), and  cells were co-cultured with  cd þ t cells in ml npc media containing μm cd -specific membrane (m) glycoprotein spanning amino acid residues - (m - , bio-synthesis) for h. cd þ t cell conditioned media were administered to jhmvinfected npcs and supernatants were harvested , , and h p.i. for determination of viral titers. levels of ifn-γ in cd þ t cell conditioned media were determined by elisa using the mouse ifn-γ duoset according to manufacturer's recommendations (r&d systems). interferon-β levels in jhmv-infected npc cultures were evaluated using the verikine mouse interferon beta elisa kit (pbl assay science). the animal protocols and procedures used for these studies were reviewed and approved by the institutional animal care and use committee of the university of california, irvine. npcs were seeded at a density of , cells/well in a flatbottom -well format tissue culture plate (corning life sciences) and pulsed overnight with μm of the immunodominant cd peptide specific for mhv spike (s) glycoprotein spanning amino acids - (s - , bio-synthesis). npcs were simultaneously treated overnight with u/ml ifn-γ to induce mhc class i expression for the presentation of s - . cd þ t cells isolated from dm-infected c bl/ mouse splenocytes (as mentioned for cd þ t cells) using the easysep mouse cd þ t cell isolation kit (stemcell technologies) were then plated with npcs at effector-to-target (e:t) ratios ranging from : to . : . cocultures were incubated for h at c in % co at a final volume of μl/well. the amounts of lactate dehydrogenase released from lysed cells were determined using a cytotox non-radioactive cytotoxicity assay (promega). the percentage of ctlmediated lysis was determined as specified by the manufacturer's protocols. rma-s 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encephalomyelitis in non-human primates injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis intraspinal stem cell transplantation in als: a phase i trial, cervical microinjection and final surgical safety outcomes neural stem cell depletion and cns developmental defects after enteroviral infection memory cd þ t-cell-mediated protection from lethal coronavirus encephalomyelitis the role of gamma interferon in infection of susceptible mice with murine coronavirus cd t cells contribute to virus control and pathology following central nervous system infection with neurotropic mouse hepatitis virus t-cell-mediated clearance of mouse hepatitis-virus strain jhm from the central nervoussystem immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts requirements for ceacams and cholesterol during murine coronavirus cell entry remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the mhv model of multiple sclerosis coxsackievirus preferentially replicates and induces cytopathic effects in undifferentiated neural progenitor cells amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation mhc mismatch results in neural progenitor cell rejection following spinal cord transplantation in a model of viral-induced demyelination ifn-gamma-mediated suppression of coronavirus replication in glial-committed progenitor cells effective clearance of mouse hepatitis-virus from the central-nervous-system requires both cd þ and cd þ t-cells narrowing of t-cell receptor beta variable repertoire during symptomatic herpesvirus infection in transplant patients neurogenesis by progenitor cells in the ischemic adult rat hippocampus transplantation of human neural stem cells exerts neuroprotection in a rat model of parkinson's disease resurrection of endogenous retroviruses in antibody-deficient mice stroke transiently increases subventricular zone cell division from asymmetric to symmetric and increases neuronal differentiation in the adult rat this work was supported by the national institutes of health (nih) grant r ns to t.e.l. c.m.w. is supported by the california institute for regenerative medicine (cirm) grants rm - and tr - , the national multiple sclerosis society (nmss) collaborative center research award ca -a- , and the gleis family foundation. w.c.p. is supported by nih predoctoral training grant t ns - and j.g.w. is supported by nmss post-doctoral fellowship fg -a- . key: cord- -dnsdg n authors: nan title: poster sessions date: - - journal: eur j immunol doi: . /eji. sha: doc_id: cord_uid: dnsdg n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx , the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th -oriented adaptive response. actually, ptx -deficient mice are highly susceptible to aspergillosis and develop th skewed responses; moreover, ptx -deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx , which does not show direct activity on fungal cells. finally, ptx alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx -mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx -opsonised conidia, cd b activation, internalization, recruitment to the phagocytic cup and cd b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx , fcgriia/cd mediates inside-out activation of cd b and consequently phagocytosis of c b-opsonised conidia. in vivo phagocytosis experiments performed with c q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx ) and the cellular arm (fcgrs, cr ). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx -/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd , immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx . we found high concentration of ptx in human colostrum ( . ± . ng/ml at day post-delivery) compare to the one found in human serum ( x ng/ml). the presence of ptx in human colostrum seems to be due to the secretion of ptx by human mammary gland since we report the production of ptx by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx supplying by breast feeding: (i) soluble ptx in colostrum (ii) hmc that can secrete ptx upon stimulation in the specific tissue, (iii) an increase of ptx production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx may participate to the beneficial role of breast feeding on the newborn health. a. m. baru , j. stephani , h. wagner , t. sparwasser twincore, institute for infection immunology, hannover, germany, technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of receptors in humans (tlr - ) and in mice (tlr - , - ). as transmembrane receptors, tlrs are expressed on the cell surface (tlr , , , , , ( ) ( ) ( ) ( ) and at endosomal membranes (tlr , , and ) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about years later to this, toll-like receptor (tlr ) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr (hutlr ) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr (mutlr ) is also expressed on conventional dendritic cells (cdcs). consequently, tlr ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr (henceforth called as hut mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr knock-out background. we expect that hut -mutlr -/mice mimic the human specific expression pattern of tlr , i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr . by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin- is a heterodimeric cytokine consisting of the two subunits p and p . the main inducers of il- p are microbial components activating toll-like receptors with the magnitude of il- p induction depending on the specific tlr engaged. differential induction of il- p upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il- p due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il- p promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il- p promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone and acetylation in specific regions of the proximal promoter region. acetylation of h showed a specific distribution pattern and occured mainly in regulatory elements within the il- p promoter, whereas acetylation of h took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue on h turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue , w. ellmeier medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi , m. buxadé , j. minguillón , r. berga , j. aramburu , c. lópez-rodríguez universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat , the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat . our work indicates that nfat is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr , tlr , tlr and tlr and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p and p nfxb subunits. interestingly, we observed that tlr stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr , tlr and tlr stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl- expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr , tlr , tlr and tlr . finally, in vivo tlr stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr , tlr or tlr triggering can directly favor tumor development whereas tlr signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms- ) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd + and cd + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd + and cd + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd + and cd + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: . innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . . adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni , r. oatuni , m. collini , m. caccia , p. castagnoli , g. chirico , f. granucci university of milano-bicocca, biotechnology and bioscience, milan, italy, university of milano-bicocca, physics, milan, italy, singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin- (il- ) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il- production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca +/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca + influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr engagement, depending instead exclusively on cd . we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase- (cox- ) that, by generating prostaglandins (pgs), such as pge , regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd . given the essential involvement of cd in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd represents a major step towards the development of potential treatments with modes of action involving interference with cd functions. we have examined the interaction of cd , a -kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd . human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd antibody coupled to biotin visualised by qd- -streptavidin and anti-cd antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd and cd , suggesting a new functional role of cd as a member of a multimeric lps receptor complex. l. lundvall , r.r. schumann charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase- induction leads to an increased release of mature il- b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d ) was established for assessing il- b induction during this disease. the murine raw . cell line, the human astroglial u- mg and the murine microglial cell-line bv- were stimulated with the tlr ligand lps, the tlr ligand pam cys, the nod ligand mdp, or the nod ligand c -ie-dap, and, as control, atp alone or in combination. we assessed il- b by elisa and caspase- and pro-il- b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il- b induction pathway. s. pneumoniae (d ) infected mice showed a significant increase in il- b release after hours. in vitro, an increase in il- b levels after costimulation with lps or pam cys, and mdp or c -ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il- b by tlr-and nlr-ligands was observed in raw cells, bv- cells, u- cells and primary astrocytes. active caspase- (p ) was induced by mdp or c -ie-dap, corresponding with high il- b responses when lps or pam cys was added. sirna experiments show that a knock-down of nod leads to a diminished il- b release after lps-and mdp-stimulation. the precursor forms of il- b and caspase- seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il- b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il- b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il- b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd and cd activation model system -tlr presence on cd + cells is found in mouse t cells, human t cells and jurkat cell lines. following cd /cd activation for hours we have identified a small but distinct populationof tlr + cells. further characterization indicates these cells to be cd +cd + cells. further characterization of the expression and functional acitvity of the tlr + t cells indicates co-expression of tlr with md- indicating a functional tlr receptor. in addition lps activiation did not lead to upregulation of tlr expression in t-cells. the data indicate that tcr activation leads to tlr expression. the expression appears to be associated with cd +cd + cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann , d. kaul , c. krüger , f. zipp , r. nitsch , s. lehnardt charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna ) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr (and human tlr ) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr is expressed in these cells. incubation of microglia with all three of the above mentioned tlr ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il -b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr knock out (ko) microglia by real- because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin- was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin- is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin- -transfected plb cells, coronin- overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd b labeling. concerning apoptosis, increased coronin- expression in dmf-differentiated plb significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin- significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase- and caspase- activities, but not caspase- or bid truncation suggesting that coronin- interfered with mitochondria-related events. to validate the prosurvival role of coronin- in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin- expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin- immunolabeling. we concluded that coronin- could constitute a potential target in resolving inflammation. p.-n. hsu national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw . macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf- sirna and traf- decoy peptide, indicating this pathway is traf- dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b -h and b -dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il- was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il- and ifn-g which could not be overcome by restimulation with acd . this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd t cells into t helper (th ) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il- receptor-related protein st could be implicated in lps tolerance. the natural ligand of st was recently identified as interleukin- (il- ), a new member of the il- family. in this study, we investigated whether il- triggering of st was able to induce lps desensitization of mouse macrophages. we found that il- actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il- enhancing effect of lps response is mediated by the st receptor since it is not found in st ko mice. the biochemical consequences of il- pretreatment of mouse macrophages were investigated. our results show that il- increases the expression of the lps receptor components myeloid differentiation protein- (md ), cd and tlr- and the myeloid differentiation factor (myd ) adaptor molecule. in addition, il- pretreatment of macrophages enhances the cytokine response to tlr- but not to tlr- ligands. thus, il- treatment preferentially affects the myd -dependent pathway activated by the tlr. c. klotz , b. lenz , r. lucius , s. hartmann humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., ) . the cystatin effect was blocked by the application of anti-il- receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin- (il- ). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il- in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il- production after avcystatin stimulation. application of specific inhibitors revealed that the il- induction was p and erk dependent, and inhibitor titration indicated a higher sensitivity towards p . western blotting experiments confirmed the phosphorylation of p and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il- is also regulated by the phosphatidylinositol- -kinase (pi k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il- and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose- -phosphate (s- p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose- p and xylulose- p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p /jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s- p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts , y. morias , p. de baetselier , a. beschin vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m ) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd , ifng, il- , ccr and nf-kb to the development and/or recruitment of pathogenic m in the liver was investigated using knock-out mice or by delivering il- in infected mice. results: we established that cd b+ly c+cd c+ tnf and inos producing dcs (tip-dcs) represent the major m liver subpopulation. tip-dcs differentiated in an ifng/myd -dependent manner from cd b+ly c+ inflammatory monocytes in the liver of infected mice. ccr promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il- enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il- in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p and il- play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m , in particular tip-dcs. the inflammatory activity of liver m is controlled by il- and/or p nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov , j. driesen , z. abdullah , a. niño castro , t. chakraborty , m. krönke , o. utermöhlen , c. wickenhauser , j.l. schultze limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, institute of molecular medicine and experimental immunology, bonn, germany, institute of medical microbiology, university of giessen, giessen, germany, institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine , -dioxygenase, cyclooxygenase- and cd and production of prostaglandin e (pge ) and interleukin . all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd , expressed by regulatory myeloid cells, acts as an il- scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd + ido + cox- + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin (il- ) and il- in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il- and il- or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il- and il- . in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il- and il- . therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel , m. rodriguez gomez , m. niedermeier , y. talke , n. göbel , k. schmidbauer , m. mack unversity hospital regensburg, internal medicine ii, regensburg, germany, university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il- and il- . activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp- cells infected with m. bovis bcg, the avirulent m. tuberculosis h ra strain and the virulent m. tuberculosis h rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva , l.d. miteva , s.a. stanilova trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il- is a heterodimeric cytokine composed of a p subunit associated with the il- / p subunit. like il- , il- is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il- p -related cytokines controls the appearance of normal th and pathological th mediated immune responses. in this study, we examined the dynamics of inducible il- p and il- p mrna expression and protein production in purified human monocytes and how jnk and p mapks inhibitors influenced il- p and il- production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il- p gene expression was higher than those observed for il- p gene at all time-points. the level of il- p mrna increased after th h and reaching a maximum level at th h ( . fold for c bgp and . fold for lps). c bgp and lps triggered il- p gene transcription were almost equal at the rd h ( . and . fold) and at th h ( . and . fold, respectively) after stimulation. the higher level of il- p gene expression was detected at th h in lps compared to c bgp stimulated monocytes ( . vs. . fold). however, il- p and il- protein production was increased in the highest level after c bgp stimulation. the inhibition of p led to the statistical significant augmentation of c bgp stimulated il- p production. the inhibition of the same map kinase enhanced lps stimulated il- p production without significant difference. the inhibition of jnk and p mapks significantly decreased c bgp stimulated il- production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c bgp and lps to induce the expression of il- p and il- p mrnas in purified human monocytes. we showed that inhibition of p mapk down regulated il- and upregulated il- p production in stimulated monocytes. we concluded that in human monocytes p map kinase activation has an opposite effect on the il- p and il- p expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd , cd and cd , and secrete il- , thus acquiring the ability to induce proliferation and th polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd c, bdca- , and cd . by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd + subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd + dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd + dc, including their survival and their ability to produce il- p . besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il- , il- and mcp- involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg , s. wolke , a. brakhage , m. gunzer otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and -photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor (irf ), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr . in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens , s. vander beken , p. bogaert , j. grooten ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th -driven immunological counterpart of the th -driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th -and th -driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd . + ram remained constant in cell number for the first days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker , a. stojanovic , a. cerwenka german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr- + cd b + f / + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas , f. marchesi , m. fabbri , s. schiarea , c. chiabrando , a. mantovani , , p. allavena istituto clinico humanitas, rozzano, italy, istituto mario negri, milano, italy, università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m and m . m macrophages act as a first line of defence against pathogens whereas m cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about % of the monocytes differentiated after days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd , cd and low levels of mhc class ii. tc-macro produced il- , il- , tnf but not il- , even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl , cxcl , ccl and cxcl . the transcriptional profile of tc-macro revealed that several genes in line with an m polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g . kda). il- and il- were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl- h rat mast cell line. we demonstrate that gr nuclear translocation begins within minutes and completes after minutes in dx treated rbl- h cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within minutes as non-genomic. studying gc-caused apoptosis, rbl- h cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl- h cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. minutes of dx treatment inhibited ca + -signaling in antigen stimulated rbl- h cells in the concentration range of nm - mm. moreover, minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl- h cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box (hmgb ) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin ( ) . extracellular hmgb can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb exerts antibacterial functions in human adenoid and testis ( ) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity ( ). we asked whether hmgb from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for or minutes with nm phorbol ester (pma), ng/ml interleukin (il- ), or ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h a-histone h b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb localizes on nets. we hypothesize that net-bound hmgb might exert a direct antimicrobial function, or that nets might concentrate hmgb locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation ( °c, h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor- induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr- and anti-vegfr- antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il- . altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd a and cholesterol-dependent lipid rafts impact on cd a surface expression and cd a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd -restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd molecules in multiple sclerosis. we first analyzed cd expression on monocytes from ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd a was not expressed on ms patient monocytes, while the other members of the cd family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd molecules in this context. c. ohnmacht , d. voehringer ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il- reporter ( get) mice as well as in vivo depletion of basophils are used to uncover their role during type immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th cells. these results demonstrate that basophils play a crucial role as effector cells in type immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl- in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il- beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl- -transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water ( mg and mg daily) for days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il- and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il- and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant ). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type- macrophages, mph ), and during resolution of inflammation (type- macrophages, mph ). methods: mph / were generated by culturing cd + human monocytes for days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph / of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph / were stimulated with lps and secreted il- , il- , il- , il- p and tnf were quantified by elisa. results: mph are relatively efficient phagocytes for apoptotic neutrophils whereas mph are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph and mph which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il- , il- and tnf production is suppressed while il- secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of years old and older (long-livers). intracellular ros and hsp levels were registered by flow cytofluorimetry upon labeling with ', '-dichlorofluorescin diacetate (invitrogen) and anti-hsp antibody (brm- , sigma), respectively. intracellular level of hsp was also estimated in neutrophils after heat shock (hs) performed at °c for min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at - min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for min at °c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp (hsp basal ) and ros level, both intracellular and extracellular. at the same time increased hsp level immediately after hs (hsp ( min)) correlated negatively with intracellular ros (initial and after hs). the hsp increase value (hsp ( min) -hsp basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp increase was registered in min after hs (hsp ( min) -hsp basal ). thus it was found that within this age group the alteration in hsp induced by hs in neutrophils but not basal hsp itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant # . d. goyeneche-patiño , z. orinska , f. mirghomizadeh , s. bulfone-paus forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type ifn. two novel genes, receptor transporter protein (rtp ) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during and h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine . stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr and , as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd and trif and the pathways that they represent are not relevant in the expression of rtp and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano , e. erba , r. frapolli , m. d'incalci , a. anselmo , s. pesce , p. allavena , a. mantovani , humanitas clinical institute, rozzano, italy, mario negri institute, milan, italy, institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et- ) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl , cxcl and the inflammatory protein pentraxin (ptx ) either at transcriptional and protein level, especially after tnfa/il b stimulation. down-regulation of ccl , cxcl , ptx and also of il and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd + vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin- (il- ) is a key cytokine of the t helper cell response. il- has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il- production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il- producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day - after birth, a small population of il- producing cells was observed in the absence of any stimuli. the il- + population was not detectable at or weeks of age. other cytokine producing cells (ifn-g, il- ) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il- + cell population. phenotyping of the neonatal il- + cells showed that they were ige + /mhcii -/cd cells. the occurrence of cd + il- producing cells after pma stimulation increased slowly with age and did not reach adult levels by weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il- at day after birth. neonatal pbmc collected before colostrum uptake did not produce il- in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il- responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il- response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel , d. voehringer ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat -dependent manner when stimulated with the th cytokines il- or il- . alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat -dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat -deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat -deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il- exposed macrophages from wild-type and stat -deficient mice. candidate genes will be expressed using retroviral transfections of stat -deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of pleural effusions, including malignant and nonmalignant tumors. the following human malignant cell lines were tested: a , ht , hct , sw , mcf , mda-mb , jurkat and hl . results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m , and the tams isolated from the malignant effusions similar to m . among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms a a and ms a a: highest trascriptional level after h of stimulation with - m dexamethasone. ms a murine genes are differently expressed respect to the human counterpart and only the homologs of ms a a (ms a b, c and d) have a similar regulation. finally, egfp-tagged ms a a, ms a a, and ms a expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein- (mcp- )/ccl and the gpcr ligand fmlp or the tlr agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il- b or ifn-g, significantly and dose-dependently synergized with ccl in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin- /cxcl , but not of the ccl receptor ccr in monocytic cells. in turn, the induced cxcl synergized with ccl for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr /cxcr , because cxcl receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, ) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for min at °c with gentle shaking, followed by spinning down of pmns for min, at °c and g. the supernatant was sedimented at g for min, °c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd low cd + blood monocytes (mo) in healthy individuals and the increase in cd +cd + blood mo in patients with inflammation has been reported. the functional activity of human cd + blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd and activation markers such as hla-dr and trem- . percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a . fold increase (p x . ) in the total number of circulating cd low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd + blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd + blood mo expressed increased levels of cd on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem- +) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd on cd high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m -and m -polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m and m macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m type and were cd negative but cd and mhcii positive and produced high levels of il- , tnfalpha and il- following lps stimulation. culture in the presence of mcsf resulted in induction of the m phenotype. these cells were cd positive with intermediate expression of cd and mhcii expression and produced high levels of il- , il- and il- following lps stimulation. interestingly, already baseline il- production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m -and m -polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma ( ng/ml) treatment for h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl cells expressed antigen receptors cd , tlr , tlr and cd , and displayed enhanced phagocytic activity and production of ros. expression of cd , cd and cd was also maintained however the cells were hla-dr and cd a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl cells to differentiate into macrophages in response to pma. hl may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta , n. ferrer , a. gómez , j. gonzalo , a. arbués , a. anel , c. martín , j. pardo , apoptosis, immunity and cancer university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so , a m. tuberculosis phop mutant strain that was shown (perez et al ) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al ) . in the present study, we compare the time course and phenotype of cell death induced by so , bcg and wild type m. tuberculosis on the murine macrophage cell line j and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase- activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl x deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type receptor for vip (vipr ) gene is highly conserved through species and, in humans, is highly polymorphic. vipr has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr after , , and h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from donors that had been typed for the relevant vipr snps. the down-regulation of vipr correlates with the presence of a t at rs mapping in the '-end of the gene (p= . ). the vipr protein level was decreased about % in monocytes of subjects typed as t/t at rs whereas subjects typed as c/c at rs maintained a high level of expression after h of lps treatment. the data show that different haplotypes of the vipr gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska , t. vavrochova , d. filipp , immunobiology institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e . - . ) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd in the early mouse embryo (me). facs analysis of cell suspension prepared from . day me showed that about . - % of cells were positive for cd b. these cells exclusively were also positive for cd , tlr , and cd antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days , - , . reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e , ), embryo-derived mhcii + macrophages start to appear in the embryo around day . multicolor facs analysis of cd b, cd , cd , f / , tlr , tlr , c-kit and mhcii surface markers revealed differential expression of tlr and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd b + tlr + cells isolated from the e , embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il /il -dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in out of these patients mutations in several ifng pathway genes, other candidate genes are being investigated for the other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp . we did focus on the study of genes which are considered as important pathogen recognition receptors of the innate immune system: tlr is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il- cytokine. using a case/household-contact cohort we did investigate polymorphisms of these genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to - % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, . hsp are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp were used. exogenous hsp was used in concentration - ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio : and : directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp on their surface. results demonstrating effect of exogenous hsp on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp on amplitude of respiratory burst. the cells expressing surface hsp impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd /cd and cd /cd double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd / subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd / subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević , i. pilipović , s. stanojević , k. mitić , k. radojević , v. pešić , g. leposavić , institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h o ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to -day-long propranolol treatment and measured both no and h o production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b -and a -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h o production. an increase in h o production in the presence of the a -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h o production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b -and a -adrenoceptors on peritoneal macrophages (a stimulatory effect on b -adrenoceptors and a suppressive effect on a -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd ++ cd -('classical') and cd + cd + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd + cd + monocytes display higher tlr and - expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd ++ cd monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr stimulation. methods: cord blood (n= ) and peripheral-blood (n= ) mononuclear cells were stimulated in vitro for hrs with peptidoglycan and subsequently analysed for cd and cd and intracellular il- p and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il- p , both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il- p was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il- p (% positive cells and geomfi) was significantly higher for cd + cd + cells than for cd ++ cd cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd ++ cd cells were positive for tnf to a significantly higher extent than the cd + cd + cells. in particular the tnf response to tlr stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd ++ cd monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a -year-old boy who admitted with complaints of seizures during the previous months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g ( mgr/m /day, subcutaneously every other day) was started. after months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf gene concerns the well-known gt deletion in the second exon of ncf gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = ) subjected to moderate brain injury were randomized, and received either . mg/kg ubiquitin or vehicle (placebo) intravenously within min after cci. levels of tnf-a, il- b, il- , il- and il- receptor antagonist were analyzed in brain tissue using real time rt-pcr at and hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at h and days. data were analyzed with the mann-whitney u test and a two-tailed p x . was considered significant. all cytokines were highly up-regulated hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il- were significantly lower in the ubiquitin treated animals, whereas the levels of il- and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day . the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight ( ) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p= . ), tpp (p= . ), osi (p = . ), mda (p = . ) were significantly raised but taa (p = . ) was significantly reduced in ptb patients compared with controls. the levels of mda (p = . ), neopterin (p= . ) and tpp (p= . ) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p= . ), mda (p= . ), neopterin (p= . ), osi (p= . ) were significantly reduced while taa (p= . ) was significantly raised in c+m after weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas , e.m. cunha , m.j. oliveira icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles ( nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage ( %) of macrophages contained the metal particles than neutrophils ( %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap- is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap- regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., ; sivalenka and jessberger, ) . swap- -/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., ; sivalenka and jessberger, ; sivalenka et al., ) . crucial regulators of these processes are members of the rho family of small gtpases such as rac and rhoa. swap- interacts with rac in vitro and preferentially binds the active gtp-bound rac . swap- supports the increase of active rac in vitro by a yet to be defined mechanism (shinohara et al., ) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap- and rac . it was found that fulllength swap- preferentially binds to constitutively active rac (rac q l) but not to its dominant negative form (rac t n). binding assays with swap- truncated mutants showed interaction of swap- 's n-terminus with gtpgs rac or rac depleted of guanine nucleotide, whereas swap- central or c-terminal regions do not bind to any form of rac . preliminary competitive-binding assays with overlapping mer peptides, spanning the entire swap- sequence, mapped the rac binding site near the n-terminus of swap- . full-length swap- site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap- with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap- . v. c. barbosa , c. d. polli , m.c. roque-barreira , m.c. jamur , c. oliver , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl- h cells were sensitized with ige anti-tnp and stimulated with dnp -hsa or artinm. artinm binding to rbl- h cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp- and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp- release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand (psgl- ), b and b -integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il- b, il- and tnf-a. herpes simplex virus (hsv- ) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il- and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv . methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) ( ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv- for h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot . software. results: in the first itraq experiment over human proteins were identified in the hsv infected cell supernatants. from these proteins had at least fold increase after poly(i:c) + hsv infection compared to the uninfected cells. hsv infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of . fold increase in the protein amount in the poly(i:c) + hsv infected cell supernatant and a . fold increase in the hsv infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand , il- , tnf-a induced protein , complement factor b, galectin- and mxa. at present, further experiments are on-going for more detailed analysis of the hsv infected macrophage secretome. h. p. prakash , german cancer research centre, translational immunology, heidelberg, germany, max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein- (ciap- ) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap- and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal- ( mg/ml) binding to monocytes, in the presence or absence of mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal , laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal or rpmi and monocytes ( x ) were added into each insert. when necessary, hrgal was pre-incubated with mm lactose or sacarose. mcp- ( ng/ml) was used as positive control. we observed that hrgal- binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal- is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal- , we observed that the association between these glycoproteins and hrgal- resulted in a % increase in the number of migrating cells. both n-and c-terminal domains of hrgal- are involved in the association between laminin or fibronectin and hrgal- , since the presence of lactose resulted in % and % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal- induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for weeks in the presence of bmp ,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd +cd + cells. the sorted cd +cd + cells were further cultured for - days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd , m-csf receptor cd and the scavenger-receptor cd , we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd and cd (fcgammariii) : the cd lowcd -and cd + cd +. trancscriptional, phenotypic and functional assays suggest the alternative (m ) polarization of cd +cd + embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi , p. kropf imperial college london, immunology department, london, united kingdom the balance between t helper (th) and th cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th or th responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg vd t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f -atpase) is expressed on many cell types and is a possible specific ligand for the vg vd tcr. the present study aims at understanding the role of f -atpase in antigen regognition. using video microscopy calcium imaging in single vg vd t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f -atpase. purified f -atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg vd cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f -atpase. thus the f -atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg vd t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f -atpase. by using purified f -atpase and peptides derived from vg vd tcr sequences, interaction sites between f -atpase and tcr were identified on both ligands. based on these findings a generalized model for vg vd t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg d activation in vivo. by transiently overexpressing the nkg d ligand rae- -beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma vdelta gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae induction. the primary systemic th response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg d receptor suggest that different ones may play unique roles. a novel nkg d-ligand, h c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae- induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th (ifn-g, tnf) and th (il- , il- ) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd -nk . -inkt cells producing high levels of the pro-inflammatory cytokine il- has been identified (inkt cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt cells in type diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il- as compared to c bl/ mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt cells present in nod mice were mainly cd -nk . -, express the ror-g transcription factor and il- receptor, both molecules being usually associated with th commitment. we are currently analyzing, using co-transfer experiments, whether these inkt cells play a beneficial, a deleterious, or any role in the development of type diabetes in nod mice. j. s. dodd , r. muir , s.s. affendi , p.j. openshaw imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd d-deficient mice with poor nkt cell responses have inefficient induction of cd t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th immunity (increasing il- and il- ), promoting pulmonary eosinophilia and ablating cd t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd t cell activity (as measured by cd expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d ) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d ) weight loss by a cd t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd + ab t cells, are regarded as immature or t h biased. vg + vd + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)- -hydroxy- -methyl-but- -enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg + vd + t cells towards these activators. because il- is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg + vd + t cells. herein, we observed that zoledronate induced neonatal vg + vd + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h -like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h -like cytokines such as il- and il- were not induced. addition of il- to zoledronate selectively costimulated ifn-g production from neonatal vg + vd + t cells. furthermore, zoledronate/il- treatment resulted in neonatal vg + vd + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il- (il- r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il- resulted in a further increase of t-bet expression in neonatal vg + vd + t cells. these changes in the expression of il- r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il- treatment. of note, in contrast to adult peripheral blood vg + vd + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg + vd + t cells. altogether, these observations show that neonatal vg + vd + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il- is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e ligases such as hhv encoded k , k or mhv encoded mk . these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e ligases manipulate them. we discovered that both the cellular march and viral e ligases ubiquitinate cd molecules. however, whereas viral molecules inhibit cd -antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd molecules. in contrast mhc class ii was only targeted by cellular and not by viral e ligases. furthermore cd molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd neg clones in vivo, we generated hypodermal ht tumors in immunodeficient mice. concomitant injections of vd neg clones, in contrast to vd + cells, prevented the development of ht tumors. vd neg clones expressed chemokine c-c motif receptor (ccr ) and migrated in vitro in response to chemokines secreted by ht cells, among which were the ccr ligands macrophage inflammatory protein (mip)- d and monocyte chemoattractant protein (mcp)- . more importantly, a systemic intraperitoneal (i. p.) treatment with vd neg clones delayed the growth of ht subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd neg clones were not able to inhibit the growth of a hypodermal tumors. our findings suggest that cmv-specific vd neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht cells expressing the luciferase and realized orthotopic injection of ht -luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint , currently the only known determinant of a canonical iel compartment, that is selectively required for vg vd + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint function; for example, we demonstrate that the constitutive expression of wild-type skint fully restores detc development in a skint mutant mouse, but does not rescue normal detc function. thus, skint provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd + cd hi cd lo cd c -) and splenic conventional dendritic cells (cdcs) (cd c hi cd a +/-cd b +/-b -) from wt and cd d -/mice as apcs for nkt cells from va -ja transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il- by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il- producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h response, whereas cdc-primed nkt cells rather favor a t h response. objectives: il- is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il- give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd d -/-) mice. we set out to investigate the activated b cells in il- injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il- ( mg) for days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day was evaluated by flow cytometry and immunohistology. results: mice injected with il- developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il- was increased in inkt cell deficient (cd d -/-) mice, in contrast to published data. an increased response to il- was also observed in ja -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il- induced antibody responses. further characterization of the recruitment of b cells in il- injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il- induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd d, are prone to autoantibody production and often involved in early immune responses. the il- induced antibody response in mzb deficient (cd -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il- induced antibody responses. we conclude that the role for inkt cells in il- induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c bl/ mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha -/-and cd d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il- . moreover, ifn-g production required the presentation of ehlppg by cd d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il- . similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd in cmv-infected individuals. cd is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that . ± . % of gamma-delta t cells from cmv-infected ktr expressed cd , when compared with only . ± . % in non cmv-infected ktr (p x . ). similarly, . ± . % of gamma-delta t cells from cmv-seropositive blood donors expressed cd compared to . ± . % in cmv-seronegative donors (p x . ). cd + gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd -dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il- and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd a on cd + gamma-delta t cell lines. cd is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd + gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a skin carcinoma cell line pre-incubated either with rituximab (anti-cd ) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd -dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il- , ifn-g, tnf-a, osm, ccl , cxcl , cxcl , and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd , cd , hla-dr, and dc-sign, and lost cd , ccr , ccr , and cxcr . addition of further microbial stimuli (lps, peptidoglycan) induced ccr and enabled these inflammatory dcs to trigger antigenspecific cd + effector ab t cells expressing ifn-g and/or il- . importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg /vd t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg /vd t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg /vd t cells stimulated with hmb-pp in the presence of il- express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il- regulate expression of the b cell attracting chemokine cxcl /bca- , its receptor cxcr , and co-stimulatory molecules involved in b cell help. purified peripheral vg /vd t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to days with and without hmb-pp, in the absence or presence of il- or il- , or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl protein were detected in co-culture supernatants only when both il- and hmb-pp were provided, implying an il- -dependent and tcr-dependent expression. vg /vd t cells were confirmed as producers of cxcl by flow cytometry and immunofluorescence. under the same conditions, activated vg /vd t cells expressed cd , cd , cd l, cd , icos and ox . in contrast, neither cxcr nor ccr changed markedly by il- stimulation of peripheral vg /vd t cells. conclusion: our findings confirm on the protein level that stimulation of vg /vd t cells with hmb-pp and il- induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto , m. emoto gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)- . although downmodulation of surface t cell receptor (tcr)/nkr-p c (nk . ) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il- stimulation. to determine whether failure to detect inkt cells after il- stimulation is caused by dissociation/internalization of tcr and/or nkr-p c or by block of de-novo synthesis of these molecules, and to examine the role of il- in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p c by inkt cells after stimulation with a-galcer or il- , and influence of il- neutralization on down-modulation of stcr/snkr-p c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il- neutralization. whereas s/cnkr-p c + inkt cells became undetectable after in-vivo administration of il- , s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p c remained unaffected by a-galcer or il- treatment, despite the down-modulation of ctcr and/or cnkr-p c protein expression. in contrast, ctcr + cnkr-p c + stcr -snkr-p c -inkt cells and cnkr-p c + snkr-p c -inkt cells were detectable after in-vitro stimulation with a-galcer and il- , respectively. our results indicate that tcr and nkr-p c expression by inkt cells is differentially regulated by signaling through tcr and il- r. they also suggest that il- participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg -gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region d (cdr d ) and cdr d repertoire, with a striking enrichment in a specific germline-encoded cdr d sequence. differentiated gd t cells and the enriched cdr d sequence were detected as early as after weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd -jd and vgi-jg . / . rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd -jd and vg -jg . selection was seen in pb tcrgd cells. detailed analysis of the cdr motifs revealed selection determinants in both vg -jg . (canonical length and cdr motif) and vd -jd (minimal cdr length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚ ) and tcrgd cb cells ( - ) to tcrgd pb cells (˚ or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type diabetes (t d). we have previously shown that transgenic expression of a cd d-restricted, va . -vb tcr in nod mice lead to an increase in cd d-restricted type ii nkt cells ( abnkt cells), and prevention of the development of t d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc . cd + t cells, in the presence or absence of selected cells from abnkt cell transgenic mice. results: in ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, abnkt cells expressed a high level of cxcr and a low level of ccr and cd l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd + bdc . t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from ab transgenic mice with bdc . cd + cells resulted in the prevention of diabetes development. the protection from disease required a minor cd + subset of ab+ nkt cells, but was independent of cd + t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs , induces in vitro inkt cell expansion and ifng and il- secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il- with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th -type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th immune response is characterized by the secretion of il- a and il- f. the il- locus encodes the highly conserved il- a and il- f cytokines that are syntenic in kb distance to each other. besides cd + th and nkt cells, approximately % of the il a producers are gd t-cells. like cd + th cells, il- producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il- production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il- or ifn-g. combined with the well known classification of il- producing gd t-cells along the markers cd and cd , our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th -type immune responses in vivo. in this context, the potential redundancy of il- a and il- f may complicate the analysis. so far, most studies were carried out with il- a single-deficient or il- f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il- a and il- f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg vgd t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b and il- differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp ) and, similarly to ab tregs, suppress the proliferation of anti-cd /anti-cd stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il- was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd b in the liver following a-galcer treatment, numbers of cells lacking cd b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma /vdelta t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma /vdelta t cells expansion upon stimulation with zoledronic acid (za) and interleukin- (il- ). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by ) the bioinformatic analysis of gene expression profiling data ) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in patients ( %) (responders, r), whereas patients ( %) were non-responders (nr). vgamma /vdelta t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt , whereas temra of r patients had an higher expression of the costimulatory molecule nkg d. the proliferative response of vgamma /vdelta t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, % of r patients were m, whereas % of um patients were nr (p x . ). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c bl/ mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il- p (clone r - f ; atcc) or anti-cd- d (clone b ) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il- p , a component of both il- and il- ; pretreatment with anti-cd d mab had no effect. il- rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il- was not a critical factor. this suggestion is supported by the increased expression of il- p and il- p (but not il- p ) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il- production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb ) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd expression. gd + cells secrete interferon-g, while gd cells are capable of producing il- . this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd -defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine . cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚ % of gd + cells but not at all in gd cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, - % of gd + cells were positive for pgp activity, although their gd counterparts remained largely negative (p x . ). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd . as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd + and gd cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma vdelta t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma vdelta t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma vdelta t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma vdelta t cells from healthy donors were stimulated with different compounds (zol, ipp) for hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma vdelta t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma vdelta t cells. moreover, mcp- depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma vdelta -mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma vdelta tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma vdelta t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg vd t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg vd t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg vd t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg vd t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg vd t cells and their effects on the viability of glioma cells, we expanded in vitro vg vd t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg vd t cell lines to kill three different glioma cell lines (t , u , u ) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg vd t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg vd t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg vd t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg vd t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e -induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e -expressing transplanted skin is dependent on interactions between populations of cd d-expressing cd c+/f + myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il- and a-galcer. expression of cd a, cd d and the costimulatory molecules cd and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il- ) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd d expression on the cell surface in comparison with control cells. in contrast cd a expression was decreased. cd and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd a, cd b and cd c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il- producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, . - % of circulating lymphocytes express a vg vd t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg -enriched genes compared to conventional mhc-restricted cd + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf & crtam in gd and ab t cells respectively. because igsf binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd + ab t cells express crtam, and that engagement of igsf on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf /crtam. we therefore sought to answer: . what is the function of igsf /crtam on gd t cells? . how is the igsf -crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf enrichment on resting gd t cells, with expression also detected on˚ % of ab t cells. the properties of those cells are being examined. however, igsf generally correlates with markers of activation/antigen experience such as cd ro. thus, igsf cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf and crtam within hours. however, engagement of igsf by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf interactions are sufficient for cd t cells to kill gd t cells and/or vice versa. instead, crtam-igsf interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd z is a kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately - % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd tcr variable segment associated with the vg segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg vd t cells without antigen presentation. in vitro stimulated vg vd t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg vd t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg vd t cell anergy observed in hiv+ patients. to this aim, cd z expression and ifn-g production by vg vd t cells from hiv+ and hiv-subjects were analyzed. we show that vg vd t cells from hiv-infected patients expressed lower level of cd z compared with healthy donors. a direct correlation between cd z expression and ifn-g production capability by vg vd t cell was found. however, pkc activation by pma is able to restore cd z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg vd t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner , m. swamy , s.l. clarke , a. hayday king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd aa or cd -cd cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to days. the cells are initially activated by plate-coated acd antibody and a cytokine cocktail and maintained further in medium containing low levels of il- . after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il- ) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg molecules in heterodimer with cd , we screened the presence of these receptors on t cell subsets in bd. the expression of nkg a/c/d molecules on gd and cd + t cells were analyzed in active and inactive patients with bd and healthy controls. expression of nkg molecules was evaluated on cd +, gd t and cd + nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls ( . vs. . %, p= . ). in addition to the increase of gd t cells, increased expression of activating nkg c molecules was also observed on gd t cells ( % vs. %, p= . ). nkg a expression on gd t cells was found to be higher than nkg c expression in patients and controls; but nkg a expression on the t cells was not statistically different in both groups ( . vs. %). nkg d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd + cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th and th cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg /vd + lymphocytes. we have screened a panel of lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg /vd + cells, and selected two susceptible ("target") cell lines (over % death in the assay) and two vg /vd + resistant ("non-target") cell lines (under % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs (non-target), and validated the results by rt-qpcr quantification. results: we identified commonly up-regulated and commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp , ifitm and prame, for example, are up-regulated, whereas cd and clec d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg /vd + target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd d, in the rat. mice and rats have very similar cd d and inkt tcr genes, with the exception of the va gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd d revealed a very similar pattern of cd d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il- production was - fold lower in the best responder rat strain (f ) compared to mouse (c /bl ). since nkrp a (rat homologue of mouse nk . ) and tcr are not appropriate markers for rat inkt, cd d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g d t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g d t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g d t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g d t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp- . a significant (p x . ) reduction in intracellular bacterial numbers was observed (n= ) in the presence of pbmcs cultured with picostim+il- in comparison with pbmcs cultured with il- or media alone. picostim+il- caused significant expansion and activation of gd t cells following culture of pbmcs for - days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers -fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il- , reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma /vdelta (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: ) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; ) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); ) to establish whether the same issues could be solved using a simplified protocol of dc generation. ) dc were generated from cd + cells of healthy donors/mm patients; immaturedc on day were induced to fully mature by incubation for hours with tnfa + il- b + pge in the presence or absence of mm za. after days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; ) idc generated from cd + cells of hla-a* + healthy donors/patients were pulsed with sv-peptide and stimulated for hours with tnfa + il- b + pge in the presence or absence of mm za; after rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd + t cells was determined by svpentamers staining; ) the same experiments were performed both with dc generated following a standard protocol and a h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [ - h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd + cd + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin- on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd + have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n= ) and inactive (bdna) (n= ), versus healthy controls (hc) (n= ) and patients with recurrent oral ulcerations (ru) (n= ). to determine gd cytokine profile and surface markers treg-related in bd (n= ) and hc (n= ). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta , vdelta , cd alpha, cd beta, nkg d, nkg a and cd . -intracellular expression of ctla- , and foxp . -intracellular expression of il- , il- , ifngamma, il- and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta + cells were significantly increased in ru. vdelta + and gdcd + lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg d was slightly increased in gd from bda. -nkg a expression by gdcd + was not different in bd versus hc. -most of gdcd + presented cd alpha-alpha homodimers in bd and hc and were negative for cd , foxp and ctla- . gdcd + and gdcd -subsets were (in bd and hc): -high ifngamma-producers without differences. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd + than in gdcd --very low producers of il in most of cases. the hallmark in bd was the increase of gdcd /vdelta +. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd +, except a lower percentage of il- + cells than in the gdcd -subset. gdcd + from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd in humans. a subgroup, the invariant nkt cells (inkt), expresses the va vb tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd + cd + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from healthy umbilical cord blood samples and stimulated with ifn-g ( ng/ml), anti-cd ( ng/ml) and il- ( ui/ml). these cells were cultured for days and the expanded cd + cd + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd + c + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x , was considered significant. results: we could significantly expand cord blood cd + cd + nkt cells from , ± , % to achieve an enrichment of , ± , % (p= , ). table shows the percentage (mean±sd,n= ) of phenotypic markers in cd + cd + cells at baseline (day ) and after days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb and vb families (p x , ). conclusion: our results show that cord blood-derived nkt cells are mainly cd + and cd + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb and vb families in these cells. l. marischen , d. wesch , p. rosenstiel , a. till , d. kabelitz institute of immunology, kiel, germany, institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod . peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp and pvama proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd + and cd + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd a + (lysosomal associated membrane proteins: lamp- ) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th and th cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th -like and th -like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n= ) and healthy controls (n= ) were determined by flow cytometry using anti-cd and monoclonal antibody specific for the cdr loop of the invariant tcr a chain of inkt cells (clone: b ). furthermore, after pma/ionomycin stimulation for hours, intracellular ifng and il- cytokines were detected in cd +cd -, cd -cd -(dn), cd -cd + and cd +cd + subsets of inkt cells by five colour flow cytometry in patients with ad (n= ) and healthy controls (n= ). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x . ) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x . ). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r= . and p x . ) and healthy controls (r= . and p x . ). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il- level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x . ). the frequency, the number of inkt cells and the cytokine producing capacity of the cd /cd inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il- that promotes the th differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting . %, . %, . %, . %, . % and . % of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells ( . %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd + nkt and cd -cd -nkt cells. nk . + nkt cells may release large amounts of il- , il- , ifn-g and il- after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il- had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il- . there was a significantly increase in the percent of cd + nk . + and tcrvb + nk . + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd molecule existed in . % of the cells from large-scale selection. the percent of cd + nk . + and tcrvb + nk . + nkt cell subsets in the giant lymphocytes were enhanced to . and . folds, respectively. under a light microscope at x magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than . and they are non-adherent cells. the differentiation pathway of the seb-activating cd + and tcrvb + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd + nkt cell and tcrvb + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo- am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h begfp reporter mice. stimulation with anti-gd clone gl or anti-cd clone c elicited activation of gd t cells suggesting that tcr gd and cd molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl in vitro produced ccl , ifng and tnfa. therefore, we were interested whether the ccl production of gd iiel influenced the homing of ccr cells such as lamina propria (lp) cd + foxp + cells (tregs). to test this, wt mice were i. p. treated with gl mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il- production in g reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il- gene. in the absence of any manipulation gfp was expressed from the il- locus in populations of immature thymic nkt cells (predominantly cd +cd lotcrhi cells on a balb/c background, and cd +cd lonk . -on a c bl/ background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il- production was induced predominantly in cd + nkt cell subsets of the liver and spleen, and after i. n. administration, in cd + nkt cells of the airways. spontaneous and a-galcer-induced expression from the il- locus occurred in the absence of stat signalling, and did not require initial exposure to il- protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin- (il- ) plays an important role in neutrophil recruitment. herein, we investigated the role of il- receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c bl/ (wt) and il- receptor gene-deficient (il- r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated hours after surgery. the ability of il- mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il- r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il- induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il- r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il- receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il- showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il- also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il- receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin- receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il- r associated kinases to activate nfxb and p mitogen-activated protein kinase. the il- -induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il- in different mast cell subsets. methods: different mast cells subsets (hmc- , human cbmcs and murine bmmcs) were stimulated with il- . the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk / , pakt, pnfxb, p and pjnk). additionally, we studied the signal transduction of il- in il- r transfected hek t cells. results: we found, that a tir family member, il- r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il- -induced cytokine production depends on c-kit transactivation. il- r and il- r accessory protein (il- racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il- r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il- -induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il- in mast cells in absence of iger activation. ( ) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase- activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease- (ho- ) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho- expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin and . recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr shows a bimodal kinetic, with the first peak at ''/ ' and the second at ' after stimulation. rna interference-mediated depletion of beta-arrestin specifically inhibits the occurence of the second wave of rap activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin is involved in rap activation and that the oscillations in the formation of rap -gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c g (rap gef) and spa (rap gap): preliminary results suggest that spa- has probably a role in the early activation peak. since this oscillatory chemokine-induced rap activation is present on other myeloid cell lines (hl , d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin , localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of different sh domains that revealed over putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)- and il- stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il- and il- receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il- , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: ( ) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna- expression is induced by activation of the toll-like/interleukin- receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna- a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna- a transfected cells showed significantly reduced levels of the active proapoptotic caspases and (casp / ). in line with this, mirna- a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna- a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna- a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna- a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il- , il- r a-chain and il- r a -chain genes in hiv disease progression. methods: we studied antiretroviral treated patients (progressors) and long term non progressors (ltnp). we analyzed snps in the il- gene, snps in the il- r gene and snps in the il- r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il- r aa and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il- r and il- r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd and cd t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art , and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art transcripts by rt-pcr analysis in human blood leukocytes. soluble art , released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek cells with art and tnf resulted in modification of tnf at at least distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r , a site that has previously been implicated in binding to tnfr . binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik , t.v.poroshina institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and control men were assessed for slpi, tnf-alpha, il- and free tgf-b in ejaculate by elisas using stat-fax plus. a to day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at - degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients ( . ± . pg/ml, p x . ) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid ( . ± . pg/ml; p x . ). the il- concentration was elevated in all patients ( . ± . pg/ml; p x . ) in seminal fluid. free tgf-b was present in normal seminal plasma in high concentrations ( . ± . pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b concentrations were . ± . pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il- and free tgf-b are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi , e. a. elgaaeid faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th or th cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod /card protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod /card gene in cd. we performed a cases /controls study upon cd patients and healthy controls. this study suggests that in northen tunisian population, insc mutation in nod /card gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il- polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il- cytokine genetic profile in patients with ibd. we examined the contribution of il- gene promoter polymorphisms (- and - ) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card /nod gene mutations in cd presentation and location. in tunisian population, the insc insertion in nod /card gene is a marker of susceptibility to cd, while the a allele at position - in the il- promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il- gene polymorphisms and card /nod gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il- -dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il- -driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il- molecule by ganglioside. but gangliosides apparently can also form complexes with il- r; such complexes influence on the signal transduction through il- r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il- r subunits. in our work we use il- -dependent cytotoxic t-cell murine line ctll- . two different approaches for study of possible interaction types between exogenous gangliosides and il- r subunits were applied: antibody staining of il- r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with i-dcp-gm followed by immunoprecipitation of il- r subunits. the fluorescence intensity of the antibody-labeled il- r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by . % after cells incubation with ganglioside gm , and by . % after incubation with gm . labeling of the cells with antibodies to the il- r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm ( . %) or with gm ( . %). to determine the mode of this impressive masking influence of ganglioside gm , photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm with subunits of il- r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il- r, but not for il- r a-subunit. these results demonstrate that exogenous ganglioside gm can interact with a-and bsubunits of il- r in different modes. interaction of il- r b-subunit with ganglioside gm requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa , j. bukur , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak . results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak expression due to a gene deletion on chromosome , whereas the expression and functionality of other ifn-g signal transduction components like stat and jak were not affected. jak blockade by two jak -specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak leading to a lack of jak expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of controls of the spanish population. methods: two il polymorphisms located on the il- promoter region, snps - a/c (rs ) and - g/c (rs ) were genotyped using taqman snp genotyping assays. the functional il- gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il- - p= , . il - p= , ) but may contribute to disease onset and aggressiveness: the genotype il- - cc genotype, which is associated with higher il- production, was significantly associated with higher tumor size (p= , ), grade (p= , ), t (p= , ), m (p= , ) and stage (p= , ). the influence of the il- - gg genotype was less relevant, however was correlated with higher tumor size (p= , ), grade (p= , ), t (p= , ) and stage (p= , ). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il- polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il- gene (il- - and - ) may be associated with an worse prognosis of rcc. high levels of il- production can play an important role in grow, invasion and metastasis of renal cancer. interleukin- (il- ) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il- is its ability to induce the production of ifn-gamma in presence of il- . moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l . then it seems that il- has a crucial role in immunity against brucella infection. since the expression of il- can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il- gene polymorph isms and brucellosis. methods: a total of patients with brucellosis and healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il- polymorphisms at positions - , - , , + , + and codon / using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il- polymorphisms at position - g/+ t/+ c (correlated with high production of il- ), codon / c and - g/- c (correlated with higher production of il- ) were significantly higher in the healthy controls than the patients (p= . , p= . and p= . , respectively). discussion: as data revealed genotypes that have correlation with higher production of il- are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il- at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor (ifngr ) mouse mutant. we have generated a mouse line carrying a conditional ifngr allele using the loxp -flp recognition target (frt) approach. a targeting vector with loxp sites flanking exons - and frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez , r. carretero , p. saenz-lopez , j. cantón , j. carretero , f. ruiz-cabello , l.m. torres , cts- hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca) allele of ifgn is significant more frequent in healthy control than in cin patient (p= , ) in contrast homozygosis for (ca) allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p= , and p= , respectively). conclusions: our study suggest that ifng (ca) allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin- (il- ), a th -related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il- single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: patients with brucellosis and healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il- (c t, g a, c a and a t) polymorph isms using pcr-rflp. results: at position , the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p= . and p= . , respectively). at position , the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p= . , p= . ). discussion: as shown the frequency of cc genotype and c allele at position were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position is lower in patients than the healthy controls and the frequency of c allele at position is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf- and skbr- , as well as their ability to respond to several cytokines and to the g- gpr agonist. the mcf- and skbr- human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il- ra, il- rg, il- /il- /gm-csfr, il- /il- r, il- ra, il- ra, il- r (gp ), il- ra, il- r, tnfr i, tnfr ii, ifngra, cxcr , cxcr , cxcr , cxcr , cxcr . cells were incubated with il- , ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr agonist. cytosolic ca + concentrations [ca + ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr- cells were found positive for a larger number of receptors than the mcf- cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf- cells with il- reduced the calcium response to g- , while pretreatment with ifn-g and tnf-a potentiated the calcium response to g- . tnf-b had no effect on mcf- g- stimulation. no direct effect on basal [ca + ] i stimulation could be noticed when administering the cytokines alone. in skbr- cells, pretreatment with il- or tnf-b had no effect on basal [ca + ] i and did not significantly alter the calcium response to g- , while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g- . pretreatment with tnf-a produced calcium oscillations and reduced the response to g- . conclusions: mcf- and skbr- cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd a -pdcs and cdcs were infected with mcmv, whereas after h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag -deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg , respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt -l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f / + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd t cells in response to an adova infection due to an impaired cd t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il- . to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp and vacv-gp expressing the gp epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p t cell receptor recognizing the gp epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p ifnar -/mice. upon adoptive co-transfer of p ifnar -/and p ifnar wt/wt t cells and subsequent challenge with mva-gp , a massive expansion of p ifnar wt/wt t cells was observed, whereas p ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp induced a rather similar expansion of ifnar competent and ifnar deficient p t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h n -specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h n induced an exceptionally nf-kb dependent antiviral response. irf is essential part of this interferon-response of human endothelia. furthermore, we identified hmga as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h n . finally, nfatc was found to be a transcriptional regulator for specifically h n -induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h n which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns , delta-ns influenza virus (a recombinant influenza virus lacking the ns gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting days post-infection, reached its maximum at day , and triggered t cell priming in vivo. a direct comparison of delta-ns virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns protein. thus, we propose that the virally encoded ns protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that - - proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of - - target proteins. thus, these results suggest that - - proteins have a regulatory role in host defence against viral infections. i. wessels , d. fleischer , l. rink , p. uciechowski institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)- b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il- b expression is not elucidated, yet. it is known that the il- b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il- b when stimulated. b-cells which are il- b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il- b promoter and the impact of methylation on il- b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl- cells were differentiated into monocytic cells after dihydroxyvitamine d treatment. the monocytic phenotype was confirmed by flow cytometry. the il- b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with -aza- -deoxycytodine (aza) and changes in il- b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl- cells, differentiated cells displayed upregulation of cd antigen and acquired the ability to express il- b. by comparing the accessibilities of il- b promoter we detected that the il- b promoter was not accessible in undifferentiated hl- cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl- cells, demonstrating that the chromatin remodeling of the il- b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il- b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il- b expression were found. our data indicate that the il- b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il- b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg , g. wetzel , h. arnold , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il- b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp , , , and mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. ( )). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. aug ; ( )). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. dec; ( ) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il- b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory t breast cancer cells over expressing il- b ( t /il- b) tumor cells, but rarely in untransfected t cells. secretion of il- b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il- ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array . having p x - in two independent cohorts each consisting of blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for h and we measured the levels of il- , il- , il- , tnf-alfa and il -ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed snps with p x , * - . to identify/replicate the association of cytokine production for these we reanalysed these on a cohort. a combined analysis revealed snps with p x , * - .these results are not genome wide significant. the snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp on adipocytes. for the first time we could show a hsp -stimulated release of the proinflammatory cytokines il- , cxcl and mcp- in a time-and concentration-dependent manner from murine t -l adipocytes. analyses of hsp -signalling in these adipocytes revealed that members of the mapk-family (erk / , p ) and the transcription factor nfkb are involved in hsp -mediated induction of the mediators il- , cxcl and mcp- . binding-studies with fluorescence-labelled hsp demonstrated that the interaction of hsp with adipocytes exhibits basic features of a receptor-mediated binding. hsp -binding to adipocytes was saturable and reached its maximum at . mm. binding was inhibitable only by the unlabelled ligand ( %), but not by unrelated proteins, thereby proving the specificity of hsp -binding. further analyses to characterize hsp -receptor structures on adipocytes revealed the presence of toll-like receptor (tlr) on adipocytes. tlr has been found to be expressed on macrophages and to interact with hsp , therefore suggesting tlr as a potential receptor candidate for hsp on adipocytes. in order to identify the responsible binding-epitope of hsp we investigated the effect of specific antibodies directed against different epitopes of the hsp -molecule. incubation with antibodies directed against the n-terminus of hsp (aa - ; - mg/ml) were capable of inhibiting the hsp -binding to adipocytes ( - %) indicating that the n-terminal region of hsp is involved in receptor binding. our experiments demonstrate that hsp stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp -mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf . we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with % of fetal calf serum at °c and % co . bone marrow nonadherent cells were removed after h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd , cd , cd , cd and stro- and were negative for cd . intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost % of msc. interleukin- , ifn-gamma and tnf (th cytokines) increased msc contractility, whereas il- (a th cytokine) decreased msc contractility. by immunofluorescence, we observed that il- , ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il- decreased that incorporation. our results suggest that th and th cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca + -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of × /ml in complete rpmi- medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as nm and nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents ( mm) at the -h culture interval reached the values of approximately ng/ml and ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of - h in rat pecs. the -h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p and erk / . it was not suppressed by the calcium chelating agents bapta-am and tmb- . the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr / / . pin is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin substrates and targeting sites. recent data show that pin interacts with apo-bec g (a g). the pin /a g interaction results in a reduced a g expression and a diminished a g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin gene (- g/c and - t/c) modulate pin expression; in particular, the - gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, ; - , ) . the - c/g and - t/c polymorphisms in the promoter of pin gene as well as pin protein levels were analyzed in exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; hiv-infected patients (hiv) and healthy controls (hc). the genotype and allele distributions of the - snp was skewed in esn (genotype: p= . ; allele: p= . ). in particular esn showed a significantly lower frequency of the - gg genotype compared to hiv and hc (p= . and p= . , respectively) and consequently a lower g allele frequency (p= . and p= . , respectively). no significant differences were found for the - snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, -(r)- [ -(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, -[ -(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of × /ml in complete rpmi- medium. secretion of cytokines was determined after the -h culture by elisa. production of no was assayed at the interval of h using griess reagent. approximately compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. -[ -(phosphonomethoxy)ethlyl]- , -diaminopurine derivatives, c) -[ -hydroxy- -(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) -heteroalkyl substituted -amino- -guanidinopurines, and f) -amino- -(purin- -yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip- a and cytokines tnf-a and il- . although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as to mm. the remarkably enhanced secretion of chemokines was reached within - h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant m . host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and million units of ifn-a treatment three times a week for months was initiated. before and after treatment: percentages of the il- and ifn-g in cd + t cells were assessed to determine intracellular t helper cell (th ) type cytokine expression. similarly, percentages of intracellular il- and ifn-g were detected to verify cytotoxic t cell (tc ) type cytokine expression in cd + t cells. percentage of th and tc type cytokine expression, (il- and il- ) were determined in cd + and cd + t cells, respectively. six ( %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th cells with respect to healthy controls before treatment. tc percentages, both tc and tc , were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il- expression was higher and the percentages of th type cells were significantly low. il- expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency ( , %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects ( , % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt , f. juengerkes , b. schumak , g. gielen , j. kalff , p. knolle , b. holzmann , a. limmer university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, university hospital bonn, department of surgery, bonn, germany, department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il- , the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il- and il- in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, h. pylori-infected du patients ( patients were positive for anti-caga antibody and patients were negative for anti-caga antibody), h. pylori-infected as carriers ( subjects were positive for anti-caga antibody and subjects were negative for anti-caga antibody) and healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il- and il- was measured by elisa method. the mean serum levels of il- in total du patients ( . pg/ml ± . ) was significantly higher than those observed in total as subjects ( . pg/ml ± . , p x . ) and healthy control group ( . pg/ml ± . , p x . ). in du group, it was found that the mean serum levels of il- in subjects with positive test for anti-caga ( . pg/ml ± . ) was significantly higher than those observed in subjects with negative test for anti-caga ( . pg/ml ± . ; p x . ). the mean serum levels of il- in du ( . pg/ml ± . ) and as groups ( . pg/ml ± . ) was significantly higher than those found in uninfected control group ( . pg/ml ± . , p x . and p x . , respectively). however, no significant difference was observed for mean serum levels of il- between du and as groups. moreover, in both du and as groups the mean serum levels of il- was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il- and il- in h. pylori-infected subjects as compared with control group. in du group the expression of il- influenced by the bacterial caga factor. a. aral , , a. atak gazi university faculty of medicine, department of immunology, ankara, turkey, gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il- levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il- elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il- levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the th hour and reached to maximum levels at the st week and decreased again at the rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the st week and started to decrease at the rd week. il- reached to its peak at the rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic , m. jurisic university of kragujevac, school of medicine, kragujevac, serbia, university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in radicular inflamed cysts and odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd , cd and cd . tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x . ) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. ) drawings blood from patients and healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. ) detection of the gene expression of prolactin and tlr- in cd + peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p . ) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr- and peripheral prolactin expression in cd + monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table- ). when tb household contacts and healthy controls were compared, cfp and esat seemed to be more useful than tst in tb contacts for displaying ltb (table- ) . although cfp spot numbers were much more than esat spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: , )( table- ). both esat and cfp spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g analytes immediately prior to liver transplantation and at sequential time points up to days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip- , il- and il- . notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/ / (h n ) (hk) differs from its putative avian precursor by amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk- aa-i r, n d, k n, s n, g a, human ( - ); rhk-r -l q, s g and rhk- aa-i r, n d, k n, s n, g a, l q, s g, avian ( - )). among these variants, the double mutant rhk-r and the seven mutant (rhk- aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l q and s g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip- and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r and rhk- aa as compared to rhk and rhk- aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il- , shedded adhesion molecules (cd , vcam- , icam- ), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk- aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il- , and neutrophilic cd levels, procalcitonin and il- were measured by elisa technique while, neutrophilic cd by single colour flowcytometric technique. of these "infected" infants, had positive blood culture (subgroup ia: culture-positive sepsis), and infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il- , cd , and crp. il- had the highest sensitivity and specificity, % and %, respectively, using cutoff n . pg/ml. for pct, the highest sensitivity and specificity, % and %, respectively, were at a cutoff value of n . pg/ml. neutrophilic cd had maximal sensitivity and specificity of % and %, respectively, at cutoff value of . %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il- and neutrophilic cd , which together provided sensitivity and specificity of % and %, respectively, and npv %. the combination of il- and crp had high sensitivity and moderate specificity, % and %, respectively. conclusions: il- and neutrophilic cd levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp , naip or ipaf and the adaptor asc are involved in caspase- activation in response to bacterial infection, triggering the processing and secretion of il- b and il- . recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi k, vps and can be inhibited with the pi k inhibitors wortmannin and methyladenine ( ma). in contrast, activation of akt, via class i pi k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il- b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with ma, wortmannin or an akt inhibitor. supernatants were analysed for il- b by elisa. results: ma enhanced il- b secretion by bmdc treated with the tlr and tlr ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il- b secretion was greatly reduced in bmdc from nalp -/mice compared to wild type c /bl controls. treatment with the akt inhibitor had no effect on lps-induced il- b secretion by bmdc. tlr-dependent secretion of il- a was also enhanced by treatment with ma. conclusions: these data demonstrate that il- b secretion by bmdc in response to treatment with pi k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp inflammasome. this response is limited to tlr and tlr agonists. inhibition of akt had no effect on lps-induced il- b production, suggesting that the effect of wortmannin and ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr signaling by the inflammatory lipid mediator sphingosine -phosphate (s p) through receptors and in human monocytes-macrophages, which could explain some of the s p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s p, and later analyzed by flow cytometry. a pharmacological analysis of the s p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam- expression was increased following lps and s p concomitant stimulation in both venous and arterial cells, suggesting that tlr and s p receptors cooperate in the expression of icam- . conversely, no cooperation was observed when tlr ligands were used. in order to elucidate which s p receptor subtype was involved in the increase of icam- expression, we used a pharmacological approach with s p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam- after lps and s p challenge was significantly reduced by blocking s p receptor and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s p receptors and , which suggest that s p receptor might be involved in the effect. conclusions: altogether these data demonstrate that tlr and s p receptors can interact to increase adhesion molecules such as icam- in human endothelial cells, and the s p receptor subtype involved in the effect differs between arterial and venous cells. with ssc without pah) and a pool of sera of healthy controls (hc) were tested. results: in dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with - , - and - protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized ± , ± , ± and protein spots respectively. twenty one protein spots were recognized by more than % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein and peroxyredoxin and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: patients suffering from different diseases were enrolled in our study. patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam- and vcam- were assesed by an enzyme immunoassay (elisa). results: out of the patients ( , %) had elevated levels of the intercellular adhesion molecule. out of the patients suffering from bone diseases ( , %) had raised values (mean value ng/ml) whereas patients out of the suffering from soft tissue diseases and diabetes ( , %) had raised values (mean value , ng/ml). reference value for icam- was - ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients ( %) having increasd levels of icam- . high icam- levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes ( , %) than in patients with bone diseases ( . %). mean values were found , ng/ml and ng/ml accordingly. those findings verify the positive correlation between icam- and inflammatory diseases and tissue damage but not for vcam- . colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: cases of ulcerative colitis (urc), adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, tubular or tubulo-villous adenomas with low grade dysplasia, and infiltrating adenocarcinomas. immunohisto- objectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). mg of pravastatina oral hours they were administered before the procedure (group study, n= ) or placebo (group placebo, n= ), and control (group control, n= ). samples of outlying veined blood were extracted to the hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in degrees: degree . without expression. degree . weak; degree . moderate; degree . intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree - . the placebo group: mixed pattern, degree - . group study ( mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree - . the percentage of cells that expressed cd was greater in the group study ( mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam and adam . we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam . these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam activity in the tissue. in the presence of the adam inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam -mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf- / vegf+ is associated with rcc risk ( p= , ), metastases ( p= , ), nuclear grade ( p= , ), tumor stage ( p= , ), and tumor size (p= , ). on the other hand, the polymorphism vegf - a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol , , -trisphosphate. therefore, pten is one of the main antagonists of the pi -kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il- as well as il- levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il- as well as il- and il- mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th- t cells, we measured th- cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il- and a strong reduction of il- mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il- exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il- ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il- . based on our previous observation that support a direct role of il- -activated stat in the enhancement of il- ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il- ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il- . quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il- ra promoter. crosslinked nuclear lysates were immunoprecipitated and min after il- addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il- ra promoter. chip assays showed that the pol ii recruitment to the il- ra promoter induced by lps is significantly increased by il- , further strengthening the concept that the rapid enhancement of lpsinduced il- ra gene expression by il- initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat immunoprecipitated dna showed statistically significant levels of stat binding to the il- ra promoter only in cells stimulated with lps in the presence of il- . surprisingly, anti-p and anti-p chip assays revealed enrichment of both p and p recruitment to the il- ra promoter when il- was added to lps-stimulated cells, suggesting that il- enhances the recruitment of nf-kb to the il- ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il- -treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il- ra promoter site is dependent on il- -activated stat , since it is greatly reduced when stat activation by il- is impaired. the molecular mechanism through which il- -activated stat promotes the recruitment of nf-kb to the il- ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il- acts as a specific negative transcriptional regulator of mouse mast cell protease- (mmcp- ). we examined the mechanisms underlying the repression of mmcp- gene expression. our data show that the "repressor" effects of il- on mmcp- promoter activity are still operating on the mmcp- bp long minimal promoter. moreover, il- deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy . furthermore, chromatin immunoprecipitation revealed that il- promoted specific reciprocal recruitment of c/ebpb but not yy to the mmcp- promoter. finally, il- deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp- . thus, we proposed that the expression of mmcp- and possibly other immunoregulatory genes may be regulated by il- through epigenetic modification and by balancing the content and binding of c/ ebpb and yy in mast cells. i. nagy , k. filkor , a. vörös , l. kemény , a. szász bzaka, baygen, szeged, hungary, university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir- , mir- a and mir- , which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir- expression; in contrast, pgn re-stimulation had no further effect on mir- a and mir- expression. next, we investigated the correlation between the expression of mir- and its two known direct targets: regulatory protein p and suppressor of cytokine signalling- (socs- ). although the gene-expression profile of neither p nor socs- changed, we found that the expression of mir- reversibly correlates with both p and socs- protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir- prior to pgn-treatment completely abolished both p and socs- down-regulation, revealing the involvement of mir- in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir- expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb +/-had a lower incidence and burden of benign papillomas when compared to tgfb +/+ animals. however, more scc developed in the tgfb +/-mice. after acute and chronic promotion, tgfb +/-skin showed a reduced proliferative response with no increase in epidermal tgfb or nuclear p-smad compared to tgfb +/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb gene dosage. further, pharmacological inhibition of alk suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb +/-skin, tgfb +/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb +/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb +/+ but not tgfb +/-keratinocytes, indicating that tgfb switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp- ) and fibroblast (mrc- ) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of ra patients and controls the levels of survivin correlated to urokinase (upa) (r= . ), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that / ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc- and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol , , -trisphosphate -kinase type b (itpkb) phosphorylates inositol ( , , ) trisphosphate (ins( , , )p ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca + responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h and h receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il- b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il- b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase- to generate the mature secreted active form. caspase- is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of genes involved in splicing with an average -fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified genes that significantly affect the production of il- b by thp- cells after a h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il- b secretion. tissue transglutaminase (tg ) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg inhibitor, in a transgenic mouse model cf and in the taz transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg in generating inflammation in two very different pathologies. this work underlines the critical role of tg in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge stimulated a-dc. preliminary results indicate no accumulation of hif- alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif- alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p but not erk / or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il- and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt a -but not wnt a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp in te. after infection with t. gondii, mice lacking neuronal gp (synapsin-cre gp fl/fl ) died significantly earlier in the chronic phase of infection than control gp fl/fl mice. death of synapsin-cre cre gp fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp -deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il- b, il- and il- have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il- , il- and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb further may generate more effective therapies. as tnfa mrna ' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb suppresses tnfa protein production by upregulating the rnabinding protein fxr , which can bind to tnfa mrna and inhibit translation. methods: using raw . cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb , we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb and luciferase expression was quantified. cells treated with lps and tgfb were also examined for fxr expression using pcr and western blot. following fxr knockdown using sirna, the influence of tgfb and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb . using the luciferase-tnf- 'utr vector we show that tgfb targets the 'utr of tnfa. furthermore, tgfb and il- both upregulate fxr mrna and protein; and treatment with tgfb and lps can synergistically upregulate mrna expression, more than tgfb alone. following sirna inhibition of fxr , tgfb can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp- and mmp- secretion from saecs, nhbes and fibroblasts to a peak of . +/- . ng/ml, at hours. interleukin- augmented comtb-stimulated up-regulation of mmp- and mmp- secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin- down-regulated mmp- secretion from saecs by %. interleukin- up-regulated mmp- and mmp- secretion from fibroblasts but not from saecs. timp secretion from saecs was enhanced by interleukin- but there was no effect of interleukin- . mmp up-regulation by interleukin- and comtb was inhibited by the pi kinase inhibitor ly and on western analysis akt (protein kinase b) was phosphorylated at minutes. chemical inhibition of the p d isoform of pi kinase with ic abrogated the il- and comtb driven secretion of mmp- from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome ) accentuated mmp- secretion. these inhibitory effects were confirmed with sirna. mmp- up-regulation was secondary to increased gene expression with promoter activity peaking h after stimulation. in summary, interleukin- and interleukin- drive transcription dependent mmp- and mmp- secretion from airway epithelial cells and fibroblasts. interleukin- also increases timp but down-regulates mmp- gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi kinase pathway is central in interleukin- driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto , l.s. moreira , e. gonzalez-rey , m. delgado institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper response. cholesterol metabolism is regulated by factors such as pparg (proliferator activated receptor g), srb (a class b scavenger receptor), cd or abca , that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg , srb , cd , and abca . we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il- in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il- and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il- , tnf-a, il- b, il- and il- , and the anti-inflammatory cytokine il- , in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il- was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il- and il- , and in some cases, of il- . the maximal plasma levels of il- and il- were found at hours and of il- between - hours. modest plasma levels of il- were also observed, with maximal production at various time points ( - hours). by contrast, production of tnf-a, il- b and il- did not occur to a significant extent, while production of il- occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il- , il- , il- and il- also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il- , il- , il- and il- , i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il- , activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il- is a novel il- cytokine family member that is expressed as an intracellular precursor (pro-il- ) and is thought to be cleaved by caspase- to yield a mature bioactive form of the molecule (mat-il- ). to date however, evidence of cell-associated proteolytic processing and caspase- dependent secretion of mat-il- has not been reported. here we show that pro-il- but not mat-il- is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il- to the il- r and also il- r-dependent bioactivity of pro-il- on mast cells. we propose a previously unrecognized role for pro-il- as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il- p mrna expression in myeloid cells, and markedly increase secretion of il- , but not il- , by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il- promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop . chromatin immunoprecipitation (chip) assays using anti-chop and isotype control mab were performed using nuclear lysates from u cells and il- promoter dna measured by qpcr. chop binding on the il- promoter was detected following stimulation of u cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il- promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop in il- gene transcription, u cells expressing shrna's specific for chop or non-specific gene target were tested for their ability to express il- following tlr and er stress stimulation. u expressing three independent shrna targets for chop exhibited significant reductions in il- p mrna (up to % reduction of the response to lps+tp) compared to u expressing a control shrna. chop shrna expression did not affect the expression of other lpsresponsive genes, including il- , il- , ccl and sod . to identify if er stress induction of il- mediated by chop expression plays a role in a more physiological setting, we examined the role of chop in the induction of il- p gene expression following chlamydia trachomatis (ct) infection. infection of u cells with live but not g-irradiated ct induced expression of er stress response genes, including chop . u infected with live ct exhibited increased il- p mrna expression compared to u infected with nonviable bacteria. chop silencing significantly reduced the ability of live ct to induce il- p mrna, confirming the important role of chop in this response. these data suggest that er stress induction of chop could contribute significantly to the pathogenesis of diseases in which il- plays an major role, through induction of il- and il- producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd b maintained a sustained activation of p kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd b-deficient mice showed only transient p activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd and cd were elevated on gadd b-deficient thymocytes. thus, we provide evidence that gadd b and a resulting persistent activation of p constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo institut pasteur, paris, france, monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il- is recognized as an essential factor for thymopoiesis, the nature of the thymic il- niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il- promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il transcripts (il- hi cells). il- hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl , ccl , cxcl ) and cytokines (il ) that are critical for normal thymopoiesis. in the adult thymus, il- hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il- hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il- levels. conversely, the frequency of il- hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il- -expression by tecs. > together, our temporal-spatial analysis of il- -expressing cells in the thymus suggests that thymic il- levels are dynamically regulated under distinct physiological conditions. this novel il- reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il- expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl -tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl -tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl -tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg plasma cells and serum igg levels were˚ - fold increased. importantly, serum from young slc-tg;em-bcl -tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in / and / cases, respectively. these values were increased when compared with control groups: / in fcgriib -/and / in bcl -tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd + cd + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd hi cells, representing multipotent progenitors, or cd + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb and ephb expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b and/or ephrin b , the ligands of ephb and ephb receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb or ephrinb genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb /b double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd + cells in the cortex, increased proportion of k +k + cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb and ephrinb in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink , d. vanhecke university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op -dl cell culture system ( ) . using this in vitro assay we obtain large numbers of human cycd + and cd + cd + double positive thymocytes starting from umbilical cord blood (ucb) derived cd + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il- and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd + cells upon notch signaling is cd followed by cd . t cell specification is accompanied by the induction of cd a and loss of cd on cd + cd + cells. these cd -cd a + cd + cells become dependent on continuous il and notch signaling for sustained survival and further differentiation into cd + cd ab + dp thymocytes. we found that flt l is not essential for the differentiation of cd + cd + human thymocytes but that addition of exogenous flt l in the co-cultures increases the number of cd + precursors and consequently result in higher yields of developing cd + cd ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd + cd + cd ab + dp subset in this in vitro assay suggesting that op stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn to dn stage, where bdnf and its receptor p are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga , c. lópez-rodríguez pompeu fabra university, barcelona, spain nfat is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat -null mice are unable to mount cd +-and cd + -immune responses. data from our laboratory indicate that nfat -null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat during the development of t lymphocytes, we developed mouse models that delete nfat at early (lck-cre + /nfat flox/flox ) or late (cd -cre + /nfat flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p , is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues - of the proline-rich domain of p . methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p null (p -/-) and wild type (p +/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p -/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p is not important for preventing thymic t-cell tumors. s. myrczek , r. pardi , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, vita-salute san raffaele university school of medicine, milano, italy jab is the catalytic subunit of the highly conserved cop signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab regulates the activity of ap transciption factors. to date jab is thought to be essential for every cell type as jab knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab . to investigate the function of jab in b cells we established a mouse strain deficient for jab selectively in b cells. mice with floxed alleles of jab kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb -locus (m. reth, freiburg). ablation of jab expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b and b cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab -deficient, bcl -transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab when apoptosis is prevented. t. nitta , s. murata , k. tanaka , y. takahama university of tokushima, tokushima, japan, university of tokyo, tokyo, japan, rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th and th antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd + t cells or functional deficits that selectively interfere with th or germinal centre responses. in this talk i will present data from some of the first strains that have been identified including the first strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn c-kit high (etp), dn and dn thymocyte populations was hybridised to affymetrix mouse a- . genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors ( out of genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included signal transducers (out of genes) such as acvr , bmpr , fzd , chemokine receptors cx cr , cxcr and integrins a , a , a , ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata- , tcf- , notch- , rag- , rag- and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks out of of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by - days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd + cd ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged month to years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd + cd precursors with recombinant wnt a ( ng/ml) or with licl ( mm) for hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab ( e ) under conditions of phosphatase activity inhibition. wnt a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il- and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st cells suplemented with il- and flt l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il- and flt l and modify the transcription factor profiles of cd + cd thymocytes mainly increasing hes- and id expression levels. human th clones and circulating th cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th and th clones or circulating th oriented t cells, respectively. accordingly, human th cells exhibited lower expression of clusterin, and higher bcl- expression and reduced apoptosis in the presence of tgf-beta, in comparison with th cells. umbilical cord blood naï ve cd (+)cd (+) t cells, which contain the precursors of human th cells, differentiated into il- a-producing cells only in response to il- beta plus il- , even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd (+) t cells but it increased the relative proportions of cd (+) t cells differentiating into th cells in response to il- beta plus il- , whereas under the same conditions it inhibited both t-bet expression and th development. these data suggest that tgf-beta is not critical for the differentiation of human th cells, but indirectly favors their expansion because th cells are poorly susceptible to its suppressive effects. m. irla , w. reith university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd (+) or cd (+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd (+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd on mtecs by cd l induced on the positively selected cd (+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd (+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu , i. ravens , g. bernhardt hannover medical school, institute of immunology, hannover, germany cd is originally identified as human poliovirus receptor (pvr) and as rodent tage , which is overexpressed in rodent colon carcinoma. cd is also known as necl- , a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd is overexpressed in transformed cells and promotes the cell cycle. thus, cd seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd and cd ; the dn subset is further subdivided into four stages (dn - ) by differential expression of cd and cd . thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd + sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd lineage. here we show that the frequency of terminally matured cd + sp cells but not that of cd + sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd , a selective deficiency of mature cd + sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd + sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd + t cells. in cd deficient animals, a shift in the tcr repertoire displayed by the pool of cd + sp cells was found demonstrating that cd is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. , , . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht , propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd + ter + erythroid and cd + cd b + myeloid cells are simultaneously cycling (s/g /m) in the post-gastrulation mouse embryo (e - ). the peak of lymphohematopoietic cell proliferation occurs at e in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence - hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e - that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd -cd interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd , we hypothesised a role for cd -cd interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd -/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd on reconstitution of b cell numbers following depletion. we show that cd is expressed by pro-b cells, and these cells proliferate in response to cd signalling in vitro. pcr identified a source of cd , negative for cd eta, in the bm of wt mice showing this cd is not provided by activated re-circulating t cells. we have shown that when cd -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn (cd -/cd -/cd + /cd -) to the dp (cd + /cd + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn stage on. in the bone marrow we found yfp + /b + and yfp + /b populations. thus these pta expression analyses show closely similar pattern to those observed with hucd preta-reporter transgenic mice (gounari f. et al. , martin et al. . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. , - ( ) martin c. h. et al., nat. immunol. , - ( . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz , g. klein zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm- which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x kda.mmp- , a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm- . in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp- . by western blotting the zymogen and the activated form of mmp- can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm- and mmp- can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp- which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp- , timp- and timp- , are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp- plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of - % compared to only % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd /cd expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd +cd + double positive (dp) tcrab low cells entering selection, and their cd +cd + dp tcrab-immediate precedents followed by underrepresentation of the selected cd +cd + dp tcrab high and the most mature cd -cd + and, particularly, cd +cd -single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd -cd -double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd +cd -/cd -cd + sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd / cd /tcrab), in adult wistar rats subjected to -day-long treatment with a -ar blocker urapidil ( . mg/kg body weight/day s. c.). the a -ar immunoreactivity was found in both thymocytes (mainly less mature cd and cd low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed -postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd / cd /tcrab expression were observed. these changes comprised of an increase in the percentage of cd + + tcrabthymocytes, which was accompanied by the reduction in that of cd + + tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd + -tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd - + tcrab high was reduced. in addition, the percentage of cd + t regulatory and cd +tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c bl/ mice infected with m. avium ( cfus, iv) were sacrificed at different time points after infection ( , and weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il- ) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il- in this organ. in the spleen, ifn-g reaches a peak of expression earlier ( weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm and ai from nih and grant i- from the robert a. welch foundation to wtg, and by grants hl and hl from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. ) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c bl/ x balb/c)f mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski , s. lang , m. stein , t. winkler friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h ac) or methylation of h on lysine (h k me / ). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h ac and h k me / ) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar # ) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 'rlm race at the ivar # element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo locus, is thought to be accessible only during the double-negative (dn) and thymocyte stages based on mrna expression, implying that translocations between lmo and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx (hox ), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed ) to evaluate lmo and tlx breakpoint-site accessibility during thymocyte development; ) to determine in which stage of development there is an increased chance for lmo or tlx translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo and tlx loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn and dn development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn , dn , dn and immature single positive (isp) stages for both lmo and tlx . conclusion: our findings show that both the lmo and tlx loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn and dn stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that to successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e - ), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd cell depletion. however, cd depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd t cells has important implications for vaccine development against neonatal infections. ( ) showed that irf knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs . conclusions: although recent findings indicated that irf function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez , t. boehm max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd and cd t cells from tuberculosis patients highly express pd- when compared to healthy uninfected individuals. in addition, analysis of pd- expression in lung biopsies from tuberculosis patients revealed that pd- is expressed on cd and cd t cells confined to lung granulomatous lesions. finally, blocking of the pd- /pd-l axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd- /pd-l pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p to p . among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th and th cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd + t cells, nfatc and c are predominantly expressed. nfatc is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc /a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc /c. as demonstrated by y h screen and co-ips, nfatc /c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc /c -but not the unsumoylated nfatc /a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc target gene interleukin- . other lymphokines like ifng and il are reversely regulated. interestingly, ntreg cells which do not express il exerted only nfatc /c, but no nfatc /a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc function. therefore, especially ntreg cells and anergized cd + t cells might be regulated by the long sumoylatable isoform nfatc /c. lnk/sh b and aps/sh b , two members of the lnk/sh b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b- cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il- signalling in pre-b cells overexpression of lnk dramatically inhibits il- -dependent growth demostrating that lnk negatively regulates il- pathways. furthermore, we showed that il- stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav . to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh or sh domains known to mediate protein-protein interactions are key players in these processes. sly (sh domain protein expressed in lymphocytes ) was identified as a putative adaptor protein containing a sh and a sam domain as well as a bipartite nls. sly belongs to a family of three molecules: sly , sly and sash .in humans, the sly gene is located on chromosome , in mice on chromosome . sly is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin -associated polypeptide p (sap ) as a putative interaction partner of sly . sap is a conserved member of the sin a-hdac corepressor complex that contains histone deacetylase (hdac ) and histone deacetylase (hdac ) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected t cells. in addition, we could show a direct interaction between sly and hdac . to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly increases the activity of hdac in whole cell lysates and, more precisely, in nuclear extracts of t cells. the interaction of sly with sap and hdac indicates a transcriptional function of this protein. within the sin a-hdac corepressor complex sly might act as a switch for the activity of hdac . cd -cyt and cyt are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd (b cell marker) fused to the transmembrane and intracellular domain of cd -cyt or cyt in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt coengagement controlled il- secretion, while cyt coligation inhibited ifng production. moreover, our preliminary data suggest that cd -cyt inhibits the phosphorylation of several molecules known to be activated by cd stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh domain and three sh domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut cells with gst fusion proteins containing full length nck, the three sh domains or the individual sh and sh domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp and cd epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam ) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g , results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g ) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g ) degranulation as measured by cd a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose- -phosphate receptor which exhibits structural and functional similarity to the vps p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose- -phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla- (sctla- ) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla- in immune responses. using a specific anti-human soluble ctla- monoclonal antibody, jmw- b that selectively binds the soluble isoform but not membrane bound ctla- , or cleaved fragments of it, we demonstrate that sctla- plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla- , secreting increased amounts of cytokines including interferon-g, il- and tnf-a, but lower amounts of il- . soluble ctla- was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla- induced secretion of the immunoregulatory cytokine il- by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine , dioxygenase enzyme cascade was also initiated by sctla- . it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla- it is crucial to t cell inhibition. membrane-bound ctla- exists as a homo-dimer on t cells but sctla- is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b ligands on antigen presenting cells. a third important observation from this study is that sctla- exists both in serum and culture supernatants as a natural kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla- , concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il- dependent regulation is most critical, boosting sctla- secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin- /efhd- , in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin- /efhd is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin- /efhd- in the immature murine b cell line wehi enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin- /efhd- impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g cell cycle arrest. to understand how swiprosin- /efhd enhances pro-apoptotic bcr signals, we analyzed whether swiprosin- /efhd is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin- /efhd enhanced bcr-induced calcium flux in wehi cells, whereas shrna-mediated down-regulation of swiprosin- / efhd impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin- /efhd interacts with phospholipase cg (plcg ) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg and swiprosin- /efhd was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin- /efhd silenced wehi cells with swiprosin- /efhd- was inhibited by the syk inhibitor bay - . in analogy, swiprosin- /efhd regulated syk activity positively. moreover, swiprosin- /efhd re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg and of syk tyrosine residue , which is involved in syk activation. finally, reconstitution of swiprosin- /efhd knock-down cells with swiprosin- /efhd mutants revealed that the n-terminal putative sh -binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi cells. interestingly, swiprosin- /efhd re-expression in swiprosin- /efhd -silenced cells induced already in unstimulated cells raft partitioning of syk, plcg and the bcr, which was reversed after min of bcr stimulation. in summary, swiprosin- /efhd is an accelerator of proximal bcr signalling and acts through syk and plcg by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p mice die within hours after a second challenge with p peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e ligase dead mice can spontaneously reject tc tumors. conclusion: cbl-b e ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd + cd ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal ) and co-stimulatory signals (signal ), provided by beads coated with anti-cd /cd antibodies. gene expression patterns were compared for cells stimulated with anti-cd /cd beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip , itac). the enhanced expression of granzyme-b, ifn-g, trail and ip were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd -coated p cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal- to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd + foxp + and cd + cd high t cells. moreover, t cell receptor activation with combined anti-cd and anti-cd stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk- , but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv- infection leads to immune dysfunction owing to a successive loss of the cd + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv- virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk / . this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk / directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap- , nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk / is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal , t. brdicka , v. horejsi institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd are key regulators of src-family kinases in leukocytes. while cd is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh domain of csk binds phosphotyrosine of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd -csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie- kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa- integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi , s. parusso , b. frossi , g. gri , c. pucillo university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il- -/-mcs and anti-il- receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il- derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd on naï ve b cells and the interaction of cd on b cell surface and cd l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd l e cd on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves , n. bercovici , a. caignard inserm u , paris, france inhibitory killer ig-like receptors (kir dl - / ) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd + t cell subsets. these receptors suppress cd + t cell activation through recruitment of the src homology domain-containing protein tyrosine phosphatase (shp- ). to further analyse the yet largely unclear role of inhibitory kir receptors on cd +t cells, kir dl transfectants were obtained from a cd + t cell line and primary cells. the transfection of cd + t cells with kir dl dramatically increased the t cell receptor (tcr)-induced production of il- independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir dl -itim phosphorylation, shp- recruitment, zap- and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp- and p-pkc-v but not of shp- . in contrast, the kir dl /hla-cw interaction led to a strong synaptic accumulation of kir dl and the recruitment of shp- / , inhibiting tcr-induced il- production. kir dl may induce two opposite signaling outputs in cd + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir dl receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg antibody during these conditions. the observed igg switching behaviour mimics that of b cells responding to lps and il- , but is mediated by a different, stat -independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana , c. schwindling , m. pasche , c. junker , c. kummerow , u. becherer , e.c. schwarz , j. rettig , m. hoth saarland university, biophysics, homburg, germany, saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of days. under tolerogenic conditions (ova alone), cd + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, - bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd + t cell expansion and contraction kinetics in the early phase of the t cell response (days - ). in the late phase of the primary response (days - ), under immunizing conditions, the large majority of transgenic cd + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il- , ifn-g, and il- a, and only few ova-specific foxp + regulatory t cells ( x %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells ( %) was substantially increased. on day , both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells ( %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il- a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin- , interferon-g and macrophage inflammatory protein- by cd tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog - protein. our results provide direct evidence that mc contribute to cd -specific priming in eae and show that the tc proliferation failure is specific for cd tc from mog - -immunized w/w sh mice. the role of mc-cd tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla- , but not via any conventional motifs in this region. overexpression of trim augments ctla- surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla- localisation, mainly restricted to the tgn. ctla- vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla- trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla- expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla- transport to the cell surface. it is imperative to reveal the mechanisms by which ctla- is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd molecules, nkt cells are activated and release cytokines, including ifn-g, il- and il- . nkt cells are efficiently recruited to the liver via cxcr -dependent chemotaxis toward cxcl and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd t cell tolerisation via interaction with lsec. to this end we analysed cd d expression on lsec and their ability to activate nkt cells by presentation of the cd d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd d, as agalcerpresenting-lsec were capable to induce tnf-a, il- , il- and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd and b -h on lsec. as naï ve cd t cell tolerisation by lsec critically depends on b -h , we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg , jg . , and vd gene products. they recognize nonpeptide antigens like (e)- hydroxy- -methylbut- -enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd- . one of the inhibitory receptors, pd- , is a member of cd /ctla- family and contains a single ig v-like domain in its extracellular region. pd- can bind to two b homologue molecules, pd-l and pd-l . it has been reported that interaction of pd- with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd- is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with -methyl- -butenyl- -pyrophosphate plus il- to obtain gd t cells. pd-l + and pd-l -human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l mabs, the pd-l extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd- in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd- /pd-l interaction. results: gd t cells expressed pd- upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l . we first examined whether or not the engagement of pd- receptor could modulate the cytotoxic activity of gd t cells. pd-l -expressing tumor cells tempered cytotoxic activity of pd- + gd t cells, and cytokine production such as tnf-a was down-regulated by pd- engagement. in addition, inclusion of anti-pd-l mab reversed cytotoxic activity and cytokine production when pd-l -expressing tumor cells were challenged by pd- -expressing gd t cells. conclusion: pd- delivers inhibitory signals in gd t cells upon engagement with pd-l . peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il- production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova - peptide)-specific t cells from do . tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler , p. aichele immh, university freiburg, immunology, freiburg, germany interleukin (il- ) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il- has a direct influence on cd + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal ) and costimulation (signal ). we analysed direct il- signaling to cd t - signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd t cells lacking il- signaling failed to up-regulate klrg and to down-regulate cd in the context of listeria but not viral infections. thus direct il- signaling to cd t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd + t cells are tightly controlled in their effector functions by cd (ctla- ). we demonstrate that signals induced by cd reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd + t cells. for this novel function cd specifically represses the transcription factor eomes, but not t-bet. a cd mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd -mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd + t cells differentiated in the absence of cd signaling could be demonstrated in vivo. the novel insights that cd -mediated signal transduction in vivo indeed alters cd + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p receptors. p x - receptors open to non-selective ion channels, whereas p y , , , , - are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p x antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca( +)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek , a. brouckova , d. filipp institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih t cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y flck. comparative -d gel analyses followed by ms/maldi identified rack as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih t cells of ha-tagged rack with either a wild type lck or constitutively active y flck revealed a significantly enhanced complex formation between y flck and rack compared to that of wtlck. ectopic expression of y flck with its domain-inactivating mutations showed that lck-rack interaction depends on functional sh , sh and the c-terminal tail sequence of lck. lck-rack interaction is readily detectable also in primary cd + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack in the context of lck translocation to lr is further strengthen by the observation that rack is associated with elements of cytoskeleton. these results are the first to characterize rack as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin (il- ) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd +cd +cd -cd -igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th response to alumova inducing large early germinal centres and massive plasma cell formation with more than % of these switching to igg . the plasma cells up-regulate cxcr , but not cxcr , a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th -associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚ %), igg a (˚ %), igg b (˚ %) or igg (˚ %). in addition to cxcr , some % of these plasma cells strongly express cxcr . the induction of cxcr in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg a switching during th responses. this is functionally significant for oti-dependent cxcr expression, as well as induction of switching to igg a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg b. t-bet is known to be induced in b cells exposed to ifng or tlr stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd . objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd ) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd + t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd + t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla- pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa- and cd in the psmac of untransformed human t-cells. in marked contrast, tcr/cd accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl- regulates interclonal t cell competition during acute and chronic immune activation. we found p -independent noxa gene induction and mcl- downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl- , which was delayed in noxa -/cells. using ot- cells and altered peptide ligands we observed that the level of mcl- downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl- -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np ) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl- axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen (ebag ) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag , which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g -adaptin in t cells. both interactions suggested an involvement of ebag in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag -/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag -deficient ctls. these data imply a role for ebag in regulating the formation of mature ctl granules and identify ebag as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag -related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd / and il- . in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd /acd or pma/ionomycin could not significantly activate cd and cd t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca + influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena , s. carrasco , i. merida centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp -ras-erk signal intensity is critical to determine the final cell outcome. rasgrp is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. . analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. . develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd cells expressing gfp-c domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h , cd ), a cd -like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th biased responses and high production of the anti-inflammatory cytokine il- , and was essential to the development of germinal centres. however, icos can also help in the il- -dependent differentiation of inflammatory th cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p subunit of class ia pi- kinases. these can complex with one of the three kda catalytic subunits (p a, p b, and p d) expressed by leukocytes that generate pip affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi -k regulatory (p a, p b, p a) and catalytic (p a, p b, and p d) pi -kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p a g p a g g p b and p a g p d g g p b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi -kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd -induced secretion of il- and il- . during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap , a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap construct that acts as a dominant negative, or sirna knockdown of endogenous akap expression in t cells prevents the correct organization of cd z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa- localization was also analyzed to assess p-smac architecture and, interestingly, confocal d reconstruction revealed that lfa- ring was not clear in the akap -disrupted cells. moreover, akap was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap decrease the phosphorylation of molecules such as lat, plcg and pkcv. these defective activation events as reflected in a reduction of il- production. together, our results underscore a key role for akap in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin /cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca + mobilization and cell activation involving the pi k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc d c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr ) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as ) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. ) we then studied the phenotype of carabin kd (shrna expressing) a b cells after bcr stimulation. ) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t tot b cells and to follicular mature b cells. ) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. ) bcr simulation, but not lps stimulation, of carabin kd a b cells shows an acceleration of ras target erk / phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk / pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor (tlr ) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd + t cells express tlr and respond to the well characterized synthetic tlr ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr . tlr stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor (irf ) as revealed by realtime-rcr analysis of ifn b and irf , whose transcription depends on the activity of irf . combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd + t cells. this study was supported by dfg spp "innate immunity" (ka / - ). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd e cytoplasmic domain (cd e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd e cd and a fluorescent membrane dye (r ). with this assay, we show that the cd e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd e cd to lipid bicells. membrane binding by the cd e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia , y. ge , u. quitsch , p. beckhove german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd + t cell help is believed to contribute to optimal cd + memory expansion via cd l on cd + t cells binding cd on dendritic cells. however, a few reports suggest that cd l-cd engagement may mediate direct cell-cell contacts between cd + and cd + t cells. in this study, we investigated the importance of cd -cd co-operation and cd l-cd interactions for t em proliferation. methods: we isolated human cd + and cd + t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd + and cd + populations were activated in vitro using anti-cd /cd beads. proliferation was measured by [ h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd or cd l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd + or cd + t em cells, demonstrating that optimal t em expansion requires direct cd -cd interactions. surprisingly, not only cd + but also cd + t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd + t em proliferation depends on signals from cd + t em cells. activation induced the expression of cd on both populations and cd l on subsets of cd + and cd + t cells. blocking of cd l on cd + t em cells impaired significantly cd + t em proliferation, which confirms that the improved expansive potential of cd + t em cells in mixed populations depends on cd l co-stimulation by the cd t em subset. conclusions: our data demonstrate for the first time that activated cd + t em cells deliver help to the cd + t em subset via cd l-cd signalling and may play an important role for cd + t em expansion upon stimulation. the t cell surface glycoprotein cd , a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd localization upon t cell:apc conjugation. we have questioned which domains of cd mediate the localization within the is, and for this we have expressed cd mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd depends on sequences within the cytoplasmic domain, as a cd deletion mutant lacking most of the cytoplasmic tail, cd .k stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd translocation was mapped within amino acids glu and his since the cd .h stop mutant, just short of aa is still able to translocate to the is, whereas cd .e stop , that lacks important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu -his ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna- is missing, but lmp- is still expressed. using a hl derived cell line, we have shown that the cytokine il- can induce lmp- expression in vitro and can replace ebna- . we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is , or . a high affinity stat binding site is spaced by nucleotides. we found three potential stat binding sites in the lmp- promoter, which we named lrs, tr and edl . they were spaced by , and nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il- -treated or non-treated kmh -ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat binding site, or lrs-stat . a stat complex binding to the gl-epsilon promoter and lrs-stat was induced by il- . the specificity of the stat complex was shown by supershift experiments with anti-stat , but not anti-stat antibodies. when gl-epsilon or lrs-stat was used as cold competitors in a -fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat contains a functional stat binding site. oligonucleotides, corresponding to lrs in which the stat site had been mutated, could not compete for stat binding. interestingly, the unlabeled lrs-tr with nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by nucleotides (lrs-edl ), it could not compete. thus, expression the transforming protein lmp- can be induced directly by the t cell derived cytokine il- in a stat dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp- and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji (ji . : . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd + t cells primarily affect the proportion of cd + t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd , cd and cd on parameters of cd + t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd , a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd leads to an enhancement of ig and cytokine production. the current dogma postulates that these signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd l (rmcd l) can increase proliferation induced by tlr (poly ic) and tlr (lps) agonists. by contrast, we never observed any synergy between rmcd l and tlr / (pam csk ) or tlr / (pam csk ) agonists. to go further in the study of cd l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd l, named mini-cd ls, based on a c -symmetry core holding cd -binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd ls bind to immobilized human cd and compete with the binding of cd l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd l, mini-cd ls synergize tlr (lps), tlr (poly ic) and tlr / (r ) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd ls and tlr / (pam csk ), tlr / (pam csk ) and tlr (odn ) agonists. synergy between cd l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd + t cell tolerance. we have defined a phenotypic profile for cd + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd and cd , and high levels of expression of cd l and ly c. whereas, cd + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd and cd , and loss of cd l and ly c expression. ly c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly c, expressed on naïve cd + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to days after infection. results: daily injection of paraoxon induced g % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining weeks of treatment. mice exposed to paraoxon exhibited g % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with % of mice surviving the infection compared to % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th and th cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide -kinases (pi k) constitute a family of enzymes that generate -phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi k are divided into two types: class ia p /p heterodimers, which are activated by tyr kinases, and the class ib p g (p gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p g deletion affects tcr-induced t cell stimulation. mice lacking p g show a partial defect in t cell differentiation, activation and survival. p g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd +/cd + t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi kg-specific inhibitor causes a reduction in the number of cd + memory t cells that mediate renal injury. similarly, pi kg deletion in p pi k transgenic mice also reduces the numbers of cd + memory t cells. there is therefore evidence that pi kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi kg regulates this process is not well understood. we studied the specific role of p g in t cell activation. methods: we studied whether the tcr activates p g and the consequences of interfering with p g expression or function on t cell activation. results: we found that after tcr engagement, p g interacts with and forms a complex with ga q/ , lck and zap . tcr stimulation activates p g, which affects -phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p g controls rac activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p g-/-t cells. our observations clarify the activation mechanism and mode of action of p g in the control of t cell activation, confirming a crucial role for p g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a b and a , expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c bl/ j mice. they were stained with fluorescently labeled igm-, cd -or cd specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by h-thymidine incorporation upon stimulation with anti-cd and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a or b nachr subunits inhibited binding of igm-and cd -specific antibodies but facilitated that of cd -specific antibody. in contrast, antibody against a subunit prevented binding of anti-cd but not of anti-igm or anti-cd suggesting that a nachrs are located close to cd , while a b ones are close to bcr/cd . consequently, anti-cd -induced b lymphocyte proliferation was increased by mla much stronger than by a b -specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine- . in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd -mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd + t h -lymphocytes to antigen presenting cells or of cd + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim and trpc ), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about %. down-regulation of stim was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, ) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be - pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein (hsp ) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd + cytotoxic t-lymphocyte (ctl) and cd + t-helper cell (th ) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k cells as well as targetindependent cytotoxicity. cd + cells exhibited a strong increase in proliferation after stimulation with hsp , with rates of up to %. in the presence of target cells, a -fold up-regulation of granzyme b mrna was observed after stimulation of cd + t-helper cells with hsp in combination with il- , - and - . the target cell-independent secretion of granzyme b by cd + cells was greatly augmented after stimulation with hsp plus il- or il- , - and - . in this study, we have shown that hsp is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd + and cd + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd , which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk overexpression in a model system was achieved by transfection. pjnk / expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk , but not jnk activation. cd ligation on primary tonsillar b cells also resulted in jnk activation. however, bcr crosslinking did not affect the level of jnk / phosphorylation. cd -mediated jnk activation was independent from sh d a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase (hpk ) was associated with cd in primary b cells as well as in b cell lines. cd -hpk association was independent from cd tyrosine phosphorylation and sh d a expression. overexpression of hpk in a model system significantly enhanced cd mediated jnk phosphorylation. it is known that tnf family receptors such as cd , cd , rank trigger survival signals in hrs cells. we observed the expression of pjnk / in hrs cells of primary classical hl. cd could be involved in sustained jnk activation in primary hrs cells, and this may reflect the role of cd receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk is activated via cd in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk is involved in cd -mediated jnk activation. objectives: cd has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd are involved in c-cbl phosphorylation and association. methods: el thymoma cell line was stably transfected with wild-type human cd or hcd cytoplasmic tail mutants: cd .k stop (maintaining only a pseudo itim); cd .h stop (lacking the distal s and y in the carboxy-terminal region); cd . ¿ e -l stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd in combination or not with anti-human cd biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py ) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x . ). in murine thymocytes, co-crosslinking of cd with cd induces an increase in c-cbl phosphorylation compared to cd alone. analysis of the el- transfectants showed that mutants cd .k stop and cd .h stop lost the ability to costimulate cd -mediated phosphorylation of c-cbl. in contrast, cd . ¿ e -l stop mutant, was able to efficiently costimulate cd -mediated c-cbl phosphorylation, similarly to the hcd wt. our results indicate that the absence of the pseudo itam in cd does not interfere with c-cbl phosphorylation in response to cd plus cd crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd cytosplasmic tail, but rather, may indirectly associate with cd through the interaction with other sh -sh domain-containing molecules, that may be recruited to cd through its carboxy-terminal region. l. kolly , s. narayan , j. tschopp , a. so , n. busso chuv, rheumatology, lausanne, switzerland, unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase- into inflammasomes and thus plays a key role in regulating capase- -dependent il- b and il- production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd + and cd + t cells were activated in vitro through anti-cd stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd activated asc -/-t cells predominantly displayed a more th phenotype, producing more il- ( vs. pg/ml; asc +/+ vs. asc -/-t cells respectively; p= . ) and less ifn-g ( , vs. , pg/ml; asc +/+ vs. asc -/-t cells respectively; p = . ). when asc +/+ and asc -/-t cells were purified into cd + and cd + t cell fractions and activated individually using anti-cd , no inhibition in proliferation was observed amongst activated asc -/-cd + and cd + t cells. interestingly, the activated asc -/-cd + t cell fraction produced significantly more il- when compared to activated asc -/-cd + t cells and asc +/+ cd + and cd + t cells (asc -/-cd + t cells = pg/ml il- ; asc -/-cd + t cells = undetectable il- ; asc +/+ cd + t cells = pg/ml il- ; asc +/+ cd + t cells = undetectable il- ). cd + and cd + t cell mixing experiments revealed that asc -/-cd + t cells are able to inhibit the proliferative ability of asc -/-cd + t cells, asc +/+ cd + and cd + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd + t cells. collectively, these results demonstrate that the absence of asc drives cd + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge and pge suppress some t-cell functions including proliferation, activation and cytokine production. pge signals through four types of gpcrs called the ep receptors. at low concentrations, pge is believed to be necessary for t cell function, whereas at higher concentrations, pge inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd + t cells with ep receptor antagonists was found to impair cell surface expression of cd , cd , cd and ox but not cd . suppression of t cell proliferation by pge has already been widely studied. however, blocking ep receptors in cd + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd + t cells to the chemokine sdf- b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd + cd + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd + t cells. our results also suggest that considering pge -mediated camp signaling in cd + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd + human t-cells was examined. oxidation affects several ca + signalling pathways by altering the activity of ip receptors, trp channels and store operated ca + channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca + signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd knockout mouse possess enhanced mixed lymphocyte reactions and cd monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd in jurkat t cells augments the secretion of the t cell growth-factor interleukin- (il- ) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il- promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd , we identified the immunomodulatory sub-domain of cd . supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd and il (simulates t cell-dependent activation). microarray assays identified about mirnas in unstimulated b cells. of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor- (irf- ). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf- transcripts differs from that observed for irf- protein abundance after b cell stimulation. further analysis identified the irf- transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk and the dfg forschergruppe for . objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey , f. hauck , i. berberich , f. berberich-siebelt , gk -immunomodulation institute for virology and immunobiology, university of wuerzburg, würzburg, germany, university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd + t lymphocytes, the transcription factor is predominantly expressed in t helper (th ) compared to t helper (th ) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp- encoded by prdm is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp- is expressed in differentiated effector t cells where it is higher in th than th cells. the regulation of the blimp expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm promoter and activates blimp- expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp- in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp- in b cells using cre recombinase. moreover we found a new putative blimp- isoform lacking exon . currently, we analyze the expression of c/ebpb and blimp- in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd + t cells but not transitional b cells or cd + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein- a (mip- a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy revealed constitutively high background levels in cd low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas , v. courtois , k. de luca , r. sodoyer sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il- , il- or il- could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il- , il- and il- . furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a- - - ). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen- (ctla- ) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla- during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il ) in the tissue and the released eggs and first stage larvae (l ) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla- during the infection, a neutralysing antibody (a-ctla- ; f ) was administered intraperitoneally ( mg) two hours before subcutaneous infection with s. ratti il . the in vivo neutralisation of ctla- -signalling by applying a-ctla- during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th cytokines, such as il- and il- and a reduction of the proinflammatory cytokines ifn-g and il- . the investigation of the humoral response showed a remarked increase of the igg -titer in the serum during secondary infection in mice that had been treated with a-ctla- during primary infection. furthermore, the blockade of ctla- resulted in a diminished worm burden as indicated by reduced release of l in the faeces. these results suggest that the blockade of ctla- during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg during secondary infection also reflects the induction of a potent th response. objectives: cd is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd expression. cd knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd ko compared to heterozygous mice. since reduced levels of cd are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd ko mice. after one injection of apoptotic cells, cd ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd on b-cells are involved in setting this threshold. conclusion: our data suggest that cd is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd acts as a major check-point of immune responses, but the mechanism by which cd controls peripheral t cell responses is unknown. the consequences of cd signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd in th cells reduced migration towards ccl , cxcl and ccl . crosslinking of cd together with cd and cd stimulation on activated th cells increased expression of the chemokine receptors ccr and ccr , which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd expression and cd -mediated migration-enhancing signals. importantly, migration of cd positive th lymphocytes in in vivo experiments increased, as compared to cd negative counterparts, showing that indeed cd orchestrates specific migration of selected th cells to sites of inflammation and antigenic challenge in vivo. these data show that cd signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd adds to the already significant role of cd in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd signaling on the longevity of cd null t cells. using a sensitive staining method for cd , we show that human cd + cd null and cd + cd null t cells rapidly express surface cd . serological inactivation of cd using specific fab fragments or blockade of cd ligands using ctla ig in cd + cd null and cd + cd null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd crosslinking on activated cd null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd is mediated by pi 'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd act directly on activated cd null t lymphocytes and, due to its exclusive expression as a receptor for cd /cd on cd null t cells, prevention of cd mediated signaling is likely a major target mechanism taking place during therapy with ctla ig. objectives: cd is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd (ptprj, dep- ) which acts as a positive regulator of sfk in cd -/-b cells and macrophages and can compensate for cd deficiency in these cells. indeed, combined deficiency of cd and cd in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd -/mice were reported so far. however, in human t cells the role of cd may be different since naive human t cells express cd at a level comparable to b cells. using cd -/-/cd -/human t cell line (jurkat-derived js- cells) we tested the ability of cd to complement cd deficiency in t cells. we used retroviral transduction to express human cd or cd in js- cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js- cells. expression of wild type cd or cd in js- cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js- cells expressing cd when compared to control cells. finally, cd substrate trapping mutant expressed in js- cells interacted with lck in vivo suggesting that lck is a direct substrate of cd in js- cells. the results suggest a level of redundancy between cd and cd in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam . ) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr and tyr we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca + release-activated ca +) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa- (leukocyte function-associated antigen ) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa- prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al. ) we describe a non-viral vector based approach (vockerodt et al. ) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd signaling cascade was conducted. after activating this particular signaling cascade (cd ) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. ). a rat thymic epithelial cell (tec) line (r-tnc. ) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc. cell line in vitro. it was found that a number of adhesion molecules, such as cd , cd , cd , cd a, cd , cd , cd was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc. line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il- activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc. cells. we found that both the adhesion ( min and h) of activated thymoytes were partially blocked by mab to cd and cd molecules (ifn-g unstimulated and ifn-g stimulated r-tnc. cells). early adhesion was inhibited by mab to cd , abtcr, mhc class i molecule (ifn-g stimulated r-tnc. cells) and cd molecule (ifn-g unstimulated r-tnc. cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc. cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay ( h), namely mab to cd , cd , cd , cd molecule (ifn-g unstimulated and ifn-g stimulated r-tnc. cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc. cells). our results also suggest the involvement of cd a/cd dependent -cd independent pathway in adhesion and cd a/cd dependent -cd dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc. cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin- (il- ), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il- favours naï ve cd t cell differentiation into th cells to the detriment of th or th differentiation. the il- receptor (il- r) is a heterodimer composed of tccr, which confers ligand specificity, and gp , a signal transducing chain that is utilized by several other cytokines. il- has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd t cells, but the potential impact of il- on human cd t cells has not been elucidated. our goal is to investigate the impact of il- on human cd t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il- r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd than cd t cells expressing the complete surface il- r (gp +tccr). however, we detected high amounts of intracellular tccr in both, cd and cd t cells, but only polyclonal activation (anti-cd ) of cd t cells led to an actual increase of il- r surface expression. purified cd t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il- activated stat and stat signalling with rapid kinetics in both cd and cd t cells, indicating the capacity of il- to signal through these molecules. addition of il- to anti-cd activated cd t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il- on human cd t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd- /pd-l pathway is associated with production of the immunoregulatory cytokine il- , the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l and pd , as well as myelin basic protein (mbp)-stimulated il- production, pakt inhibition, and apoptosis (annexin v), in ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); had a diagnosis of stable disease (sms). results showed that: ) pd-l -expressing cd + and cd + cells are reduced in ams compared to sms individuals (p= . ); and ) pd expression is increased in cd + t cells of sms individuals and is comparable on cd + t cells of ams and sms patients. this is associated with a significant decrease in il- production by mbp-stimulated cd + and cd + cells of ams patients (p= . ). additionally, cd + anexin v+ (av+) cells were diminished and cd + pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd +av+ and cd + pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l /pd- pathway seen in ams patients result in a reduced mbp-specific il- production by cd + and cd + cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd + t. the pd /pdl pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti- - bb in cd cells. this difference could be due to down regulation of cd by activated lymphocytes and possible preferential response of cd cells to anti- - bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin (stx ) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx . rna interference technique was also used to down regulate stx expression in primary human ctls. results: we identified stx in ctls by pcr and immunocytochemistry. stx accumulates at the is after ctl/target cell contact. when stx function was blocked by overexpression of a dominant negative stx mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the years history of mouse t h and t h subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h , t h or t h phenotypes, based on their cytokine production (il- , ifng or il- ). a comparative analysis of t-bet, ifng and il- mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca +response, membrane raft expression/organization, k + -and ca + -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h g t h g t h , although the membrane cholesterol level (detected with anti-cholesterol ab, ac ) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h cells upon activation than in t h cells. t h cells produced a more sustained calcium response with higher amplitude than t h cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav . and kv . ion channels, major functional determinants of the sustained calcium influx. t h cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd -dynabeads. cd + isolated cells were facs sorted into cd + cd naïve b or cd + cd + memory b cells. dna synthesis was measured by h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd + cd naï ve b cells, of which bmp- and - were most efficient ( % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd + cd + memory b cells by - %. to induce differentiation, both naï ve and memory b cells were stimulated with cd l and il- . this increased the production of igm, iga and igg - -fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad / / in cd + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd l/il- -induced production of igs in mature human b cells. s. gutenberger , k. warnatz university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases and (erk / ). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of hd and cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk / phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk / . to increase the signal intracellular phosphatases were inhibited by h o . as markers of activation and initiation of proliferation, cd and ki expression were measured after days of in vitro stimulation. k. theil , p. aichele immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p cd t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b -recipient mice and compared their expansion with wild-type (wt) p t cells after viral infection. we could demonstrate a severe impairment in the capacity of p t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p t cells into h mice. h mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp - . therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h mice are infected with lcmv . . this is a lcmv variant that has got a point mutation in gp - and consequently cannot be recognized by the p t cells. s. frischbutter , r. baumgrass deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap which are important for expression of cytokines such as il- , ifng and il . it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl- was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl- phosphorylation but rather an inhibition of bcl- dephosphorylation. furthermore, calcineurin and bcl- co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl- /malt signaling complex and dephosphorylates bcl- and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop , j. lamoureux , c. fortin université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla- has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla- expression was higher after stimulation in t cells of elderly. there was differences between cd and cd t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh and ww domain proteins. since fasl surface expression is regulated by adam -mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh domain proteins that potentially interact with the riped fasl prd, we used a sh domain phage display library containing all sh domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conse- optimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b family members on antigen-presenting cells with cd on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound , with classical gcs regarding their suppressive effect on cd -costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd and anti-cd , and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd -costimulated human memory/effector cd + t cells by compound than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound than prednisolone. when evaluating possible mechanism for the increased activity of compound in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound which was not prevented by the partial gc receptor antagonist, ru- , in vitro. moreover, in vivo we observed less induction of il- beta and tnf-alpha by pre-treatment with compound than with prednisolone. our data indicate that the non-steroidal segra, compound , may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b mice elicits robust cd + t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il- + cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa -specific cells also express tnfa, but only about half of the ifng+ np -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd + t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd + t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova - peptide shows a close correlation with division in vivo. early after antigen encounter ( - divisions) the vast majority of cells express only tnfa. after - divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions ( - divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd + t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd + t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd + t cells, expansion plays a very important role in the regulation of cd + t cell effector function. in addition to its chemo-attractant function, sdf- a (stromal-cell derived factor- a, cxcl ) has been described to costimulate cd + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd + t cells was increased by immobilized sdf- a to a level similar to that obtained with the costimulatory molecule cd . as visualized by real time confocal microscopy, t cells entering in contact with sdf- a formed a tether and displayed an active scanning activity. since sdf- a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd mabs, it is conceivable that the sdf- a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf- a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf- a in the context of cd + t activation by antigen-presenting cells secreting sdf- a. this study should help us to better define how sdf- a modulates cd + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd and anti-cd antibodies and cape for days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il- production and lymphoproliferation in cd + t cells stimulated by anti-cd /cd . cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd t cells express a variety of molecules on their surface, such as mhc-class ii, cd , cd , cd , whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd t cells might induce t cell proliferation and differentiation from cd resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd , cd , and cd . analysis of the cytokine profile of these cells revealed the differentiation of il- -and ifn-g-double-producing cells in response to contact with th effector cells, and il- -producing cells in response to contact with th effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd /cd stimulation. whereas neutralization of ifn-g or il- during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd , cd , and cd could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to - %. conclusion: interaction of cd memory t cells with activated t cells resulted in the production of the cytokines il- , il- , and ifn-g. given the immunomodulatory capacity of il- and il- , these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir , s. thompson , j.j. murphy king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il- and il- and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp l by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp l has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl differentiation was observed to be associated with downregulation of zfp l protein. in an attempt to determine whether zfp l downregulation was directly linked to bcl differentiation, a zfp l shrna expressing lentivirus (psicor) was employed to knockdown zfp l expression. this reagent downregulated zfp l expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp l shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp l downregulation in promoting bcl plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd (fas, apo- ) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd /cd -triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il- and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk / phosphorylation, the expression of various activation markers, the il- production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb ) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb , galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd + and cd + t cells. in addition, mitogenic stimulus increase -fold the all binding to cd + t cells. previous studies in human pbmc showed that all binds to human cd + t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd + t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd -dependent activation of purified cd + t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd expression, but enhanced the anti-cd -dependent proliferation and cd expression of purified cd + t cells. analisis of calcium influx showed that all enhanced anti-cd dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd + t cells by up-regulating t cell activation mediated by anti-cd stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in ) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd receptor. it has been previously demonstrated that cd ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp- , slap- and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il- r, il- r, il- r, il- r, il- r, il- r, cd , cd , cdw ( - bb), cd (fas) and cd (fasl) on hpbls in different cultured time, i. e. d, d, d, d, d, d, d and d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. . the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. ) the expressions of membrane immune molecules before cultured. ) expressions of the membrane molecules on hpbls during culturing. % fbs rpmi group ( group), il- group, pha group... ( ) mtt assay. ( ) proliferative times and growth curves of hpbls... . during cultured in vitro, there are expression changes of the il- rs (il- ra, il- ra, il- rg, il- r, il- r, il- r, il- r), co-stimulatory molecules (cd , cd , - bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in group, il- group and pha group, but the rests are different. . our data also suggest that the hpbls cultured in cd mcab+cd mcab+il +il a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. . celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun , r. tauber zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p lck and the ras/rac signalling pathway ( ) followed by mitogen-activated protein kinases ( ) and c-jun n-terminal kinase ( ), which leads to an enhanced binding of l-selectin to soluble ligands ( ). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues ( ). here we show that the protein phosphatase a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer , e. glasmacher helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago - genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm and rck or expression of dominant-negative gw . interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd + t-cell activation and differentiation. naïve cd l hi cd lo cd + t cells were sorted and stimulated by anti-cd and anti-cd antibodies. results: firstly, we show that the activation and differentiation of cd + t cells require il- provided by activated cd + t cells at the initial priming stage after stimulation. secondly, this critical il- signal is delivered through il rbg of cd + cells and is independent of il- ra. besides promoting cell proliferation, il- stimulation increases the amount of ifng and granzyme b produced by cd + t cells. conclusion: therefore, our studies demonstrate that a full cd + t-cell response is elicited by a critical temporal function of il- released from cd + t cells, providing mechanistic insights into the regulation of cd + t cell activation and differentiation. most antigenic peptides recognized by cd t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a - is a tumor antigenic peptide presented by hla-a and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a - we setup an in vitro digestion assay using a -mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a - by tumor cells. by electroporating hla-a cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart- /melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart- /melan-a - specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart- /melan-a - ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart- expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart- /melan-a - epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart- /melan-a - ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev : - , ) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h -m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity : - , ) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide - , whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from - peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the - epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, ), we found, after an initial induction at hrs p. i., a strong inhibition of tapasin transcription at hrs p. i. furthermore, also reduction of tap and tap transcription was observed contrasting to the elevated levels of erp and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß , ß and ß , which are replaced by their ifng-inducible counterparts ß i, ß i and ß i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß i is replaced by a thymus-specific subunit ß t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß i, ß i, ß i and ß in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß ,ß and ß i (single intermediate proteasome), and the other comprises ß i, ß and ß i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent - % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about % of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a* ) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a* binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a* from tapasin, but only ttp-ha dissociated hla-a* from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl- . -a showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a* from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a* . m. basler , , c. lauer , m. groettrup , biotechnology institute thurgau, kreuzlingen, switzerland, university of constance, division of immunology, department of biology, konstanz, germany two lmp -dependent antigens have been described that relied on the 'structural presence' of lmp in the proteasome but not on the activity of lmp . here we have investigated processing of the h- d b -restricted uty - epitope of the male minor antigen uty reported to be lmp -dependent. using splenocytes from lmp -/-, lmp -/and mecl- -/mice we found that the uty - epitope requires lmp and lmp but not mecl- . curiously, a selective lmp inhibitor did not interfere with uty - presentation. objective: we investigated why the deletion but not the inhibition of lmp interferes with uty - presentation. we hypothesized that the 'structural' requirement for lmp is based on replacement of the caspase-like activity of b in the proteasome. methods: it was determined if t a mutants of lmp and/or b can rescue the uty - epitope. we used a b -selective inhibitor to determine if the inhibition of the caspase-like activity of b preserves the epitope. finally we determined by mass spectrometry if the uty - epitope embedded within a mer precursor peptide is differentially cleaved by lmp -deficient and proficient immunoproteasomes in vitro. results: we found that t a mutants of lmp and b rescue presentation of uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . also inhibition of cells with a b -selective inhibitor preserves uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] presentation. an aspartate in position of the uty - sequence wmhhnmdli is preferentially used as a cleavage site by lmp -deficient but not half as frequently by lmp -proficient immunoproteasomes. the generation of the uty - epitope relies on the replacement of the caspase-like activity of b by lmp because the b activity destroys the uty - epitope. this is the first example for the 'structural' requirement of lmp for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp and perhaps also lmp and mecl- exert their function in antigen processing. a. linnemann , a. musiol , r. lindner hannover medical school, cell biology, hannover, germany, hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf -regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf ) were overexpressed in t fibroblasts. we show that antibody-mediated oligomerization of mhc i in t fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf : endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf -regulated to a novel, arf -independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv virus and since sv binding triggers mhc i oligomerization, this novel pathway may be involved in sv uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab b, c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta -microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab b and rab c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab b or c positive vesicles. furthermore, the rab b, c positive compartment were colocalizd with a fraction of rab a at a juxtaposition of phagosomes. together these data demonstrate that rab b and rab c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd + t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf a and bglf have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf , has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens ). this represents a novel function for a virally-encoded gpcr. bilf is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf can hinder the recognition of virally-infected cells by cytotoxic cd + t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe natural ligands, including two peptides derived from semenogelin- , a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg is transcribed in thymus from both male and female individuals. finally, we detected the semg mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta , beta , beta ) and immuno-subunits (beta i, beta i, beta i). deficiency in beta i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta i-deficient cells could be restored by treatment with d t, which increases the amount of proteasomes independent of beta i via induction of mixed proteasomes containing beta i, beta i and beta . consequently, not the lack of the specific proteolytic activity of beta i or immunoproteasomes, but the reduced proteasome quantity in beta i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr , and , which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd + t cells. the presence of exogenous tlr and tlr ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james , i. bailey , t. elliott university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct colon carcinoma. this response is long lasting and mediated by both cd and cd t cells. importantly, the treg depleted ct specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a , c , bcl , renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw , which is h -d d restricted. analysis of the generation of gsw in ct revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct cells substantially increased the amount of gsw present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct and immunised balb/c mice. greater than % of mice injected with eraap kd ct were found to reject the tumour. analysis of t cell responses revealed the presence of gsw -specific t cells, however, these responses were small ( . - %). this compared to a much larger response to ct (˚ %). preliminary results indicate that the majority of the t cell responses (non-gsw -specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [ ] . the di-allelic hla-a restricted minor histocompatibility antigen ha- locus codes for the highly immunogenic ha- his and the non-immunogenic ha- arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha- arg immunogenicity was the estimated numbers of cell surface presented copies i. e. /cell for ha- his and less than / cell for ha- arg [ ] . as ha- his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha- his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha- mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha- allelic peptides, we investigated the impact of the ha- his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha- his and ha- arg peptides were performed. moreover, the crystal structures of hla-a in complex with either ha- his , ha- arg or a ha- variant with a citrulline residue at position were determined in order to obtain atomic level insights into the conformation of the hla-a /ha- peptide complexes. our results exclude a role for antigen processing in preventing ha- arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha- arg allele in the hla-a peptide binding groove [ ] . they provide a rationale for the lack of ha- arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd t cell response. on the other hand, immunosubunit expression is not essential for development of cd t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp (ib ) and mecl- (ib ) develop a variety of autoimmune responses, including a latent form of t d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd but not cd t cells. a cd t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose- -phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr and dr . analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type (hsv- ) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd + and cd + t cells in hsv- immunity have yet to be clearly elucidated. to better understand the role of hsv- -specific cd + t cells in immune control we have identified a amino acid epitope derived from glycoprotein d of hsv- . following flank infection, gd-specific cd + t cells were first detected in the draining brachial and axillary lymph nodes (ln) -days post-infection (pi), peaking at day and declining thereafter. gd-specific cd + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day pi and peaked at day . while hsv-specific t cells were first observed in the draining ln at day pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as days pi, with peak activity days pi before declining to background by day . however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd + t cells was observed up to days post-infection in the brachial ln. ex vivo analyses suggest that only cd c + cells were involved in gd antigen presentation at days , and post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd a + dcs can present the gd antigen to cd + t cells at day pi, whereas by day pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv- infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap and erap unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap /erap in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp . upon assembly of the heavy chain with b m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp . both crt and erp are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp point mutant that fails to bind to cnx or crt was just as effective as wild type erp in normalizing rates of disulfide formation. we conclude that erp does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp point mutant, class i heavy chains, crt and the tapasin-erp disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp have no effect on plc stability or class i surface expression, suggesting that erp plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies - % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb * /dqb * haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr* transgenic ab nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd + and cd + t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr* transgenic mice (cd + t cell competent) develop aip even unprovoked, similar to ab nod mice (cd + t cell deficient), while hla-dr* , hla-dq or hla-dr* /dq (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr* fails to protect from aip, likely due to defects in the induction of cd + regulatory t cells. our results identify hla-dr* as a prominent risk factor for aip on the hla-drb * /dqb * haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan , c. britten , h. overkleeft , g. van der marel , k. melief , d. filippov , f. ossendorp leiden university medical center, section tumorimmunology, leiden, netherlands, leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd and cd t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi , , x. hu , , a. kawana- tachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping -mer and -mer epitopes (rypltfgwcf (nef - ) and rypltfgw (nef - )) were found to be presented by hla-a* and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y f, y w, t c, f l, w r, f r, f y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef - (wild type) and its four mostly common immune escape mutants (y f, t c, y f&t c, f l), and also nef - (wild type). we found that there was little difference between the nef - (wild type) and nef - (y f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y or f . interestingly the central bulge region of the peptide was becoming very flexible for the nef - (t c) and nef - (y f&t c), which may affect the binding of peptide and the recognition of t cell receptor. for nef - (f l), the side chain of l was more flexible compared to the nef - (wild type). alignment of the nef - and nef - showed that the nef - became flat and the side chain of f was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef - was featured, while the peptide nef - was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef - was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd + and cd + t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a restricted cmvpp derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a - log reduction of activation efficiency. this pattern was seen for / epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to - log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b restricted cmvpp rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv -mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd + t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp , and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor- (fgf- ) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf- peptide was produced in vitro after incubation of proteasomes with a -amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf- spliced peptide by cells transfected with mutant constructs encoding fgf- proteins where the intervening segment was shortened from amino acids to , or residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b* , are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b* , appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b* and b* are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us protein. to address this hypothesis, us was stably coexpressed in b lymphoblastoid cell lines expressing hla-b* or hla-b* . in the presence of us , the surface expression of hla-b* was substantially reduced whereas hla-b* expression was relatively unaffected. although us was able to form complexes with both hla class i allotypes, only hla-b* was retained intracellularly in an immature form whereas hla-b* was transported to the cell surface. accordingly, in the presence of us , hla-b* , but not hla-b* , constitutively presented a hla-b restricted alloantigen to reporter t cells, suggesting that us binds hla-b* without interfering with peptide loading. us has been reported by others to bind the plc but surprisingly we have not detected such us -plc complexes in our system. rather, in the presence of us we identified a pool of class i molecules distinct from the plc and only present in us expressing cells, implying that us may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa , b. seliger martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il p . since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for h with the gold standard of maturation (tnfa, il b, il and pge ) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase- (lap ) had a more than -fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap and erap and of the immunoproteasome subunits lmp and lmp was enhanced between and -fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr ligand mpla in comparison to the tlr- and / ligand polyi:c and r . these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il- induced surface expression of mature mhc class ii molecules and cd . when ifn-g/il- primed pcmc were used as antigen presenting cells for cd + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type -diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b - to specific cd + t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd + t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd + tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd + t cell clones, faure, , eur j immunol ( ): - ). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd + t cells after their in vitro differentiation into dc, days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv- genome contains arfs within gag, pol and env genes encoding for a panel of hla-b* restricted epitopes. qprsdthvf (q vf/ d) is one such epitope but its parental epitope qprsnthvf (q vf/ n) has a significant higher frequency among hiv- isolates. strikingly, q vf/ d-or q vf/ n-specific ctls recognize apcs infected with hiv strains encoding for q vf/ d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad ) encoding for q vf/ n do not activate ctl responses raising the possibility that q vf/ n epitope is not presented by infected cells. we asked whether introducing mutations within q vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q vf/ n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q vf mhc-i presentation. we performed in vitro proteasomal digestions of mer peptides encompassing q vf/ d or q vf/ n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q vf/ n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q vf/ n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q vf/ d or q vf/ n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q vf/ d peptide. we cloned and sequenced hiv- genomes from the three donors. surprisingly, out of hiv proviral genomes isolated from pbmcs of q vf/ d reactive donors, we could not find any virus bearing the q vf/ d sequence. the isolated hiv sequences either encoded for q vf/ n or had a stop codon within the epitope. in contrast, viruses encoding for q vf/ d were isolated from pbmcs of the q vf/ d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv- seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch , y. wang , d.c. tscharke the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h- bxd f mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f progeny, with some peptides being almost equally immunogenic in f and inbred mice, while others elicit responses that are reduced by more than % in f mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd + t cells with a restricted vb usage in f mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr agonist, s-[ , -bispalmitoyiloxy-( r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp- ), as an adjuvant for cross-priming against cellular and soluble antigens. malp- has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd + and cd -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr / results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap ) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, and % as compared to control rma cells, were injected s. c. in the flank of c bl/ syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to days after injection. all mice injected with rma clone with a % level of eraap expression developed a tumor and died within days after injection. surprisingly, any animal injected with rma clone with a % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd + t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor ( bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor ( . bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb * -restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with -mer synthetic fviii peptides to stain epitope-specific cd + cells.cd + t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a domain: fviii - , fviii - and fviii - , as well as the c domain peptide fviii - , but not any c domain peptides. the c domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost. : - , ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni , s. albini , m.z. limongi , l. cifaldi , d. fruci ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap and erap , we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap , erap and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap and erap . this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p nf-kb subunit, we demonstrated that nf-kb is involved in erap and erap expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap and erap and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap , erap and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch , s. tenzer , h. schild , e. von stebut-borschitz uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c bl/ mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th immunity, whereas ifng secretion of both cd + th and cd + tc cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il- -dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm (lmp -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c bl/ mice. in addition, ex vivo co-cultures with infected dc from either lmp -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc cell ifng secretion and the dc restimulatory capacity of cd + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd + t cells, isolated from low dose infected c bl/ wildtype or lmp -/mice, were not detected. in summary, our data indicate that despite the fact that cd responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd + t cells against l. major is independent of the lmp subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a , generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd + t cell epitope ( - ) from the g domain of aggrecan , times more efficiently than non-specific b cells and over times more efficiently than the macrophage line j . however, despite this highly efficient aggrecan capture, processing and presentation of the - epitope took at least hours, comparable to the time required for presentation of aggrecan by j . treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the - epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions , and at the peptide binding groove. position also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b* , peptide loading is relatively independent of this chaperone. we analyzed the effect of b subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b subtypes. the association of b heavy chain with tapasin was analyzed in c r cells transfected with hla-b subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta- , which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me monoclonal antibody that recognizes b properly folded b /peptide complexes. the formation of fully assembled b molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc , which recognizes mhc class i free heavy chains (hc), or with me . maturation was analyzed by pulse-chase analysis, immunoprecipitation with me and treatment with endoglycosidase h (endo h). hla-b polymorphic positions other than , both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c bl/ mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately % of vacv global presentation in the context of h- b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd + and cd -dc, which differ in their mhci antigen presentation capacities. cd + dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd -dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd + and cd -dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd + and cd -dc was determined. surprisingly, cd + dc exhibit rapid surface mhci turnover compared to cd -dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd + and cd -dc were stabilized and no longer underwent rapid turnover. this suggests that cd + and cd -dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd + and cd -dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h- kb loaded with the ova-derived peptide, siinfekl. cd + and cd -dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd + dc, but not the cd -dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd +, and not cd -dc. this data further validates the role for cd + dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp- cytosol using gelatinase b/mmp- as a model enzyme. in the first dimension, ion exchange chromatography separated the thp- proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp- . in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp- on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp- cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp- -cleaved or intact thp- cytosol. proliferation was assessed by measuring incorporated hthymidine. results: - mmp- candidate substrates were isolated, of which were identified, revealing many novel mmp- (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about / of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp- decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e - k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue in hla-a and residue in e - k are highly critical for association of both proteins. this defines a putative interaction surface between e - k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e - k and the class i antigen presentation pathway. moreover, because soluble e - k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap and tap . tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap and tap were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap on tap was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap mutants suggested that the lack of tap protein expression is associated with a strong reduction of tap protein levels, which could be restored by tap gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap on tap different shrna plasmids specifically targeting tap or tap , respectively, were stably transfected into constitutively tap and tap expressing hacat keratinocytes and colo melanoma cells. in both cell types the shrna-mediated tap and tap inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap caused not only an almost complete loss of tap , but also a strong decrease of tap protein expression. in contrast, the tap knock down exhibited no influence on the tap expression. specific inhibition of the proteasome prevented the degradation of tap in the tap knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap on tap protein expression is not restricted to rare tap mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of healthy volunteers was exposed twice to . minimal erythema dose of uv. blister roofs were then collected before and , and days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd + cd + and of a macrophagic cd -cd + cell subsets that emerge and days post exposition, respectively. more importantly whereas classical cd a hi cd + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd a low cd and cd a low cd + that emerged and days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd and hladr. finally, days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey , e. reeves , t. elliott , e. james university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd + cd + regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct , stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct -derived cross-protective antigen, gsw , which was found to be encoded within the ectotropic murine leukaemia virus (emv- ) envelope protein, gp . this protein has previously been shown to encode ct -specific cd and cd antigens, implicating it as a 'hot-spot' for ct tumour antigens. interestingly, we have identified a truncated version of gp which may be responsible for generation of gsw . expression studies have revealed increased gp expression in ct compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw ) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p , p and p tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp - , bound to hla-dp with high affinity ( - nm). binding studies of mbp - derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f a, f a and k a) were affected, and only by a - fold reduction in affinity for hla-dp . the observation that none of the substitutions resulted in a complete loss of binding to hla-dp indicates that ) hla-dp binding to peptides does not depend on a large hydrophobic residue accomodated in p , or ) mbp - can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp molecule binds the immunodominant epitope in ms, mbp - , possibly in more than one register. additional studies are required to resolve the hla-dp peptide binding properties, and to determine whether expression of hla-dp affects the disease course in ms patients. results: depletion of mncs for cd + cells abrogated the tg-induced cytokine production and proliferation of cd + t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd + t cell proliferation was significantly reduced in cd /cd -depleted mnc cultures, as compared to cultures depleted for cd + monocytes alone. the same applied to the production of il- , il- and tnf-a. production of ifn-g and il- was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa , , p. pala , g. miiro , j. todd , p. kaleebu , , n. imami , f. gotch , mrc uganda, basic science, entebbe, uganda, imperial college, london, united kingdom, mrc uganda, entebbe, uganda background: hiv- -specific t-cell responses are preserved in hiv- infected individuals with non-progressing hiv- disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv- for g years, maintaining cd + t-cell counts g , and with minimal cd + decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: art naï ve hiv- infected patients from the entebbe cohort in uganda were recruited and stratified by cd + t-cell count, cd + decline slopes, and time of enrolment, into groups - ltnp and rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd t-cell count, and ifn-g, il- and il- elispot responses to pools of to peptides ( -mers overlapping by aa). peptides were based on consensus sequences of gag and nef from hiv- clades a , a and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il- responses were significantly higher in the ltnp than in rp (p= . , ifn-g responses to gaga pool ; p= . , il- responses to gaga pool ). il- responses were low and not significantly different between ltnp and rp. there was a positive correlation between il- responses to gaga pool and cd t-cell counts (r = . , p= . ), but no correlation between either il- or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv- specific responses were higher in ltnps than in rps confirming previous results. non-specific il- responses were high possibly reflecting baseline th responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her /neuand her /neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using -aad for dna analysis and specific antibodies directed against the proliferation marker ki- , the m-phase specific phistone h as well as for the h- l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her /neuversus her /neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g /g -, s-and g /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g /g -phase, which continuously raised and peaked within the s-phase. however, h- l d surface antigen expression was not altered in her /neucells during cell cycle progression. in contrast to her /neufibroblasts the her /neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h- l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd ) and ccr were cultured from cd + stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd + derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il- -dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mØ). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mØ than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mØ have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, ) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, , ) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x , ), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd +, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for of them. for of these patients hla-drb data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p= . ). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb- a preparations, whereas no differences were shown for ifnb- b. an association between hla-drb * and nab-negativity was detected (p= . ). the known associations between hla-drb * and csf-positive ms and hla-drb * and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb- a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb- b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart- and gp more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni , s. lorenzi , f. mattei , f. spadaro , l. gabriele istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg thymoma line, we show that type i ifn promote the ability of cd a + dc to capture apoptotic eg cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd a + dc and the persistence of apoptotic eg -cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg -derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd a + dc. as a result, eg -loaded dc become competent at inducing ot-i cd t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. ( ). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd + dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam csk , and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd + dc but not cd -dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm , which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a human lung cancer cell line. methods: dm induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a cells. we examined the morphological change using optical microscope. and gfp-lc punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm was showed high cytotoxicity on various cancer cell line, especially a cells. dm treated a cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc and beclin- protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc was increased concentration and time-dependently. beclin- also was increased. and then, we examined gfp-lc punctation on a / gfp-lc stable cells using confocal. a cells were formed gfp punctuation after dm treatment. to confirm these data, we measured cell death ratio using -methyladenine ( -ma), an autophagocytosis inhibitor. after -ma treatment, dm induced cell death was decreased comparing with dm treatment. conclusion: dm did not induce apoptotic cell death. but dm showed several characteristics of autophagic cell death. taken together, dm induced autophagocytosis on a cells. these finding indicated that therapeutic potential of dm by triggering autophagic cell death. s. b. rasmussen , s. r. paludan university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr) , which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv- infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv- , we found that tlr dependent ifn regulatory factor activation was abrogated. in addition, inhibition by -methyladenine of phosphoinositol -kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr dependent recognition. in the ongoing project we are examining how hsv- triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko , i. berberich , gk -immunomodulation universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl- family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl- family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a and its human homologue bfl- are anti-apoptotic members of the bcl- family. a is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a . unless the c-terminal ends of other bcl- proteins the tail of a does not function as a strong membrane anchor. nevertheless, the last amino acids of a are important for the protein. in fact, the c-terminus of a serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl- factor bimel results in increased stability of a . this is due to reduced ubiquitination of a after binding of bimel. we conclude that the cterminus of a /bfl- serves as a docking site for e ubiquitin ligase(s) that control the stability of a by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e ubiquitin ligase that interacted with a in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs- in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs- is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs- significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs- was manifest not only by the suppression of jaks acting upstream of p mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak / activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs- overexpressing cells. the results strongly suggest that socs- acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl /sdf- a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u- , and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p and fas-l expression was carried out by western-blot. results: cxcl induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p -ser levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after h of tnf-a treatment. conclusions: cxcl induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p function/levels. s. y. demiroglu , r. dressel universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp does not protect against cell death mediated by ctl. acute overexpression of hsp can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res : ) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp on granzyme b-induced apoptosis. methods: hsp was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type . results: hsp did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase- were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g peak measurements (p= . ). in contrast, a partial protection of ge-tet cells was observed after acute hsp overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood : ) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk and dr / - . the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr and tnfr , both belonging to the tnf receptor superfamily. tnfr -mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr -associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr , resulting in depletion of traf from the cytoplasm. here we confirm that tnfr induced depletion of cytosolic traf by the use of tnfr -and tnfr -specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf in the alternative nfkb pathway, we show that tnfr induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr and membrane tnf. j. c. morales , d. ruiz-magaña , d. carranza , c. ruiz-ruiz universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase- . in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp l is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp l may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl- protein is an important cell survival protein at different stages of b cell development. bcl- mrna also contains are regions that could possibly be targeted by zfp l protein. in the present study, we have tested the ability of zfp l protein to bind to a bcl- mrna are probe and to degrade bcl- mrna. recombinant bacterially expressed zfp l protein was shown to bind specifically to a bcl- are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp l also provided evidence that endogenous zfp l in b cells could bind to the bcl- are. in order to examine whether zfp l binding to bcl- are resulted in bcl- mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp l knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl- mrna was expressed in both wild-type and knockout mefs. the half-life of bcl- mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp l does play a role in degradation of bcl- mrna. overall, our data are consistent with a role for zfp l in degradation of bcl- mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp . since lmp is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi -kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te ) and lymphocytic (nc ) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes or tes (transforming effectors site) derived from the c-terminal intracellular part of lmp , in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp 's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase- . using this nc tumorigenic model in scid mice, we showed that induction of the dn lmp -tes prevent development of tumours and mouse death. these dominant negative derived from lmp could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs and socs differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p mapk activities, while socs promoted apoptosis with an increase in p activities. in contrast, both socs and socs display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs or socs induced a slight decrease in g or s phase cells and a prominent increase in g /m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g /s transition and g /m arrest induced by socs or socs . socs promoted dna damage-induced p induction and g /m cyclin b expression, while socs induced decrease in g cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein (hsp ) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp is released during necrotic cell death and proinflammatory effects of extracellular hsp have been observed. thus, we asked whether apoptozing cells cleave hsp during apoptosis or how hsp is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp protein content. we observed that hsp is degraded during apoptosis resulting in the formation of a fragment of about kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp (hsp alpha and hsp beta) we could show that the kda fragment is formed after degradation of the alpha isoform of hsp . further, we were able to show, that hsp cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp was inhibited or if exogenous hsp was added. these results demonstrate that cleavage of hsp represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc -i is processed to lc -ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf- gfp-lc cells were therefore incubated in starvation medium for hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl- /beclin- control of autophagy. recently, jnk was shown to be necessary for beclin- upregulation, and jnk-mediated phosphorylation of bcl- is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf- cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol -angelate (pep ), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb turned their response to pep from an increased to decreased rate of apoptosis. furthermore, our data show that pep inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl- and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p , trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch and analysed by flow cytometry. expression of the caspase inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases and . diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase- abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor (tnf-r ), and tumor necrosis factor (tnf)-a receptor (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid -o-octadecyl- -o-methyl-rac-glycero- -phosphocholine (et- -och ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd independent of its ligand fasl/cd l. fas/cd is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin- , an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl -family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment ( -fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo , sw , lovo, caco- , ht- ) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki- , pcna, p , bcl- ) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl- , bax, caspase , , , nfkb, parp / kda, tnfr /cd a and cd /fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl- , bax, nfkb, parp was significantly lower and expression of caspases, tnfr as well as percentage of cd cells significantly higher as compared with control group. caspase expression was significantly higher as compared with nfkb, parp and tnfr . in comparison to the standard nutrition preoperative immunonutrition increased bcl- and nfkb expressions and decreased caspases and parp expressions. in addition, we found a significant down-regulation of bcl- expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega- fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x . ). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x . ). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with healthy volunteers receiving either la ( cfu/day) or placebo, during days prior to uv ( × . med). blister roofs, liquid and skin biopsies were collected , and days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p ). while a similar decrease of lc for both groups was observed on day after uv exposure compared to placebo, la- group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd a low cd -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il- and a tendency to inhibit il- was observed in la- group compared to placebo. on day , la- group presented a greater recruitment of early lc precursors and a trend to increase cd a low cd + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il- , tnfa, il- , and il- ) was observed in la group compared to placebo. we show that la limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la supplementation for photoprotection at a lower dose ( log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn appears to be expressed in all epithelial cells of the early thymic rudiment starting around e . . previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn in a nude (foxn -deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn . if true, it should be possible to target this cell type by use of foxn -promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either or glutamine residues under the control of foxn promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei , , y. toriumi , k. kimura european university viadrina, frankfurt (oder), germany, shimane institute of health science, izumo, japan, shimane university faculty of medicine, department of pediatrics, izumo, japan, japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a -minute-rest period, followed by a -minute yoga exercise called asana, a -minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a -minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp ). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r= . , p x . ), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r= . , p x . ) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd (r= . , p= . ). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd , within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd + cd + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox l expressed on mcs and the constitutive expression of ox (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca + responses and degranulation. the cross-talk between t reg cells and mcs through ox -ox l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th or th effector cells and the induction of antigen specific foxp + regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th and treg subset, gata and foxp respectively, counter regulate each other (mantel y et al., ) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp + tregs and high gata + th cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost million allergic patients are sensitized to the major birch pollen allergen bet v , which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g %) of patients' ige binding to bet v was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa - (p ) and aa - (p ) of bet v . cross-reactive ige epitopes between bet v and related pollen allergens and plant food allergens involved primarily p . p and p are not adjacent peptides in the bet v sequence but define a surface-exposed patch on the three-dimensional structure of bet v . as determined by surface plasmon resonance, monoclonal antibody mab specific for p and mab specific for p showed high affinity, i. e., dissociation constants, k(d) = . e- m and k(d) = . e- m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p and anti-p antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th cells as well as tc cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th -mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th /tc cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd + and cd + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd (gk . ) or anti-cd ( . . ) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr- monoclonal antibody rb - c or monoclonal antibody nimp-r . one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd + t cells, cd + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd + or cd + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th and tc cells operative in the elicitation of airway inflammation. conclusions: robust type immune responses, although highly effective in the counter-regulation of local th -mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h . the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p , we were able to show that the major allergen of phleum pratense, phl p , although sharing molecular similarities with der p , does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p . chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il- and il- from nci-h cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p . in contrast to hdm extract grass pollen allergens induce the release of mcp- from respiratory epithelial cells, as well as moderate levels of il- . detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr ) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): . ± . ) and of il- (mean±sd: ± pg/ml), and reduced il- (mean±sd of il- -spot forming units/ x cells (sfu): ± ), compared to healthy subjects ( . ± . si, p x . ; ± pg/ml, p x . ; ± sfu, for proliferation, il- and il- , respectively); children with non-ige mediated cma had a significant reduction of il- ( ± pg/ml), compared to ige patients (p x . ) and an increased, although not statistically significant, production of ifn-g ( . ± . sfu) compared to control ( . ± . sfu) and to ige-allergic patients ( . ± . sfu). finally, tolerant patients showed reduced il- ( ± pg/ ml, p x . ) and proliferation ( . ± . si), compared to acute ige-cma children. interestingly, the high level of il- observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il- was undetectable in all patients even blocking the il -receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th -like response with il- and proliferation is dominant in ige-mediated cma patients; by contrast a th -skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang , b. chua , f.c. lew , a. ho , k. wong , k.l. wong , d. m. kemeny national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd th t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd t cells inhibits the induction of the th response. here we have investigated the effect of cd t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd + t cells ( . %± . % to . %± . %). when ifn-gamma -/-ot-i cd t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced ( . %± . %), suggesting an important role for cd t cell ifn-gamma. cd c + cd + cd blung dcs from cd transferred mice secreted higher levels ( pg/ml) of il- p following ex-vivo stimulation as compared with animals that were not given cd t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd t cells can subsequently divert the local lung environment to one that favors th immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in % of patients with hiv infection. this reaction is strongly associated with hla-b* and appears to be mediated by cd + t cells producing inflammatory cytokines. we show that cd +t cell responses can be primed in vitro, in normal blood donors who are hla-b* +, but not in non-b* + donors. cd t cells, but not cd t cells, are expanded by abacavir pulsed autologous apc over a -day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b* . similar responses were detected in abacavirhypersensitive hlab* + patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b* since they were undetectable using apc expressing closely related hla-b or b allotypes. responses to apc expressing mutants of hla-b* demonstrated a crucial role for residue . isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b* triggering cd t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b* may be relevant to the role of other disease-associated class i allotypes such as hla-b and b . a. jenckel , s. bulfone-paus , n. föger research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin a (coro a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro a during mast cell activation. cell fractionation experiments demonstrated that the association of coro a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro a phosphorylation. a functional correlation between coro a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro a. thus, coro a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin a (coro a) deficient mice have demonstrated that coro a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin b (coro b) is a closely related homolog of coro a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro b deficient mice and crossed them with coro a deficient mice to obtain coro a/coro b double deficient mice. analysis of t lymphocytes from coro a/coro b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro a/coro b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro a/ b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro a/ b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn (also known as hacs or sly ) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology (sh ) domain and a predicted bipartite nuclear localization signal. the samsn gene is located on mouse chromosome and encodes a well conserved protein with amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn in all tested hematopoietic cell types. the highest expression level of samsn mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd + and cd + t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly (hacs ) and sly (sash ) -were expressed only at a very low level in mast cells. the high level of samsn mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn deficient mast cells. in additional studies we are now investigating the effects of samsn deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., ). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st siai-v; st galnac i-iv, st gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg abs. results: we observed that the expression of st gal i, iii and v coding genes was similar in both hybridomas, while the st gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg . in addiction, the expression levels of st sia and st galnac genes in the hybridoma producer of anaphylactic igg were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg . conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il- and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il- and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf mediates the differentiation into th cells by activating multiple genes which are independently crucial for the development of naive t cells into th cells. because irf is expressed in many different tissues, it can be considered as a master switch factor for th cell differentiation. methods: here, we tested mice deficient in irf in the murine acute asthma model to evaluate its importance in this th cell-mediated disease. the protocol setup was the following: sensitizations s. c. with ova, followed by challenges via ova inhalation and adoptively transferred wildtype cd + t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf deficient mice with the help of adoptively transferred cd + t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by . fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. ) were also significantly higher in irf deficient mice. conclusion: interferon regulatory factor plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd + t cells. mechanistically, a potential counterregulation of asthma by th cells is not available in irf knockout mice. together with our previous report that irf represents a susceptibility gene for allergy in the human, our data highlight irf as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c bl/ mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk -receptor and ppt mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd c expressing apc substets characterized by cd and cd expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th- cytokine profile and increased levels of il- mrna. further the number of cd + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: ) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase , ) asthma and ee are prototypic th diseases in which the cytokines il- and il- play a principal role through the activation of the transcription factor stat , and ) we have recently demonstrated that some chloromethyl ketones can downregulate stat by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat , we analyzed its effect in the activation of stat by il- and il- . we found that treatment of cells with ppis inhibited the ability of il- and il- to signal stat activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd and cd high (treg)), that might inhibit the development of a th response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd or cd at hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h r. the level of intracellular ccl production in human lc was reduced after stimulation with h r agonists and basal production could be restored when h r was blocked with the specific antagonist jnj . moreover histamine and a h r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h r in allergic skin diseases and encourage further exploration of the h r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g p , an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g p was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g p which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th to chronic th -predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received weekly intravenous infusions of rituximab at a dose mg/m body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g , ; g , ; g , mg/dl, respectively). all patients underwent prior treatment with omalizumab for months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the nd week after initiation of rituximab therapy up to year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up year), ige levels decreased from , to , mg/dl. the other two patients are in the and -months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) and th cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either %dncb, %tma or to vehicle alone for . - h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il- (mean= pg/ml, n= , p x . ), il- (mean= pg/ml, n= , p x . ) and il- a ( pg/ml, n= , p x . ) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of ng/ear of murine recombinant il- or of the known regulator of lc migration; il- b. interleukin- b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after ( %) and h ( %) (n= , p x . ). in contrast, il- or control injections were without effect. however, il- administration caused an increase in cutaneous il- production ( pg/ml, n= , p x . ) compared with control injection and naï ve tissue ( and pg/ml, n= , ns). in addition, systemic treatment with anti-il- antibody failed to impact on lc migration provoked by dncb ( % reduction; n= , p x . ). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il- a ( pg/ml, n= , p x . ) and il- ( pg/ml, n= , p x . ) expression was reduced to baseline levels by anti-il- treatment. these data demonstrate that il- is not involved in the regulation of lc migration, unlike il- b and tnf-a. however, il- is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il- may influence lc and dermal dc maturation, via the expression of il- a and il- . allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey ), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p -mapk-pathway related protein (p prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct- - , www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il- receptor antagonist (il- ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il- b and of il- -like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il- -related genes by real-time pcr on mrna from blood cd + monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il- , il- bp, and caspase- , both before and after therapy with kineret. il- b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il- b, tnfa, il- ) nor anti-inflammatory cytokines (il- , tgfb) were detected. as expected, il- ra was only detectable after therapy. il- was detectable in schnitzler's sera at higher levels than in controls ( . vs. . pm) and decreased after therapy ( . pm). the circulating il- inhibitor il- bp was lower than in controls and not affected by therapy. thus, free il- levels were increased in schnitzler's patients as compared to controls ( . pm vs. . pm in controls) and decreased after therapy ( . pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il- -related cytokines (il- b, il- ), nor of the il- /il- -converting enzyme caspase- in blood monocytes. however, the high circulating levels of il- suggest an increased activity of caspase- , as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr signal via myd to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c bl/ myd -deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd -/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg -titers in ova-treated myd -/-mice compared to wildtype mice, although the nacl-treated myd -/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over , species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies ( mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = ) or from non-infested contra-lateral sites. expression of mip- a, igf- , mcp- and ip- genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd , cd , cd , cd , mhc class ii, and p than that of holsteins. lymphocytes expressing wc and cd antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x . ). infested skin of nelores contained significantly more message for mip- a, igf- , mcp- and ip- than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd + t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il- plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa- in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam- /lfa- binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th than a th local response pattern by virtue of il- secretion and icam- /lfa- interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th like micromilieu in acute towards a th dominated milieu in chronic lesions. the genotyping of ccl l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi / ). psoriasis is an inflammatory dermatosis with % prevalence among caucasians. hla-cw allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in - and - positions. strong association was found between polymorphisms in the - region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b , and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to years of age) were selected. hla-a -b -c -dr -dq alleles and tnf- and - snp were differentiated by pcr/ssp. analyzing the total group, patients ( . %) were male, ( . %) were female. in the male group, the mean age at disease onset was , years. severe forms were seen in this group (psoriatic arthritis in cases and erythroderma in ). seven patients ( , %) had a favorable evolution of the disease, but ( , %) developed extensive psoriasis, covering over % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw* allele was positive in cases ( , %), tnf g/a genotype was found in ( , %) and tnf g/a in ( , %). in the female group, the mean age at disease onset was , years, one case of psoriatic arthritis and one of erythroderma. twenty-nine ( , %) had a favorable evolution of the disease and ( , %) an unfavorable evolution. cw* allele was positive in cases ( , %), tnf g/a genotype was presented in ( , %) and tnf g/a in ( , %). severe disease was seen in male patients. there was no difference in frequency of cw* allele between male and female groups, but there was a tendency of significant difference in tnf g/a genotype. we found that c bl/ mice were more susceptible than balb/c and dba/ mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il- and mrp / , among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: cases, which were studied retrospectively, included children ( boys and girls), aged - years, who had an anaphylactic reaction, out of the that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food ( %-particularly sea food and dried fruit), drugs ( %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites ( %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema ( %), and gastrointestinal symptoms ( %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g was found in out of the severest cases ( , %), in which the adequate control was held, while in their vast majority ( out of ) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in , % ( cases) there was a hereditary family history of atopy, while in children ( %) there was also a personal history of asthma. finally, at a great percentage ( %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that )the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. ) in particular, in many cases it is proved that there is a personal but also a family history of atopy. ) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd we could observe dose dependent upregulation of programmed death ligand (pdl- ) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since years. in march a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters ( mg) every second day. in april the patient presented himself in the consult with multiple livid papules with a diameter of mm in the area of the auricle. the histological examination showed an hhv- positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs , socs and foxp in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n: ), the patients who showed recurrence or progression after treatment (non-responders, n: ) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n: ) were evaluated for socs , socs ve foxp mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd , cd , cd , foxp , cd + cd high , cd + foxp + . expression of socs and foxp- mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd , foxp , cd +cd high , cd + foxp + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs , socs and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs and socs molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of silicosis patients, with mild ( ), moderate ( ) and severe ( ) silicosis, coal workers, exposed to inorganic dust containing fcs (exposed), and healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than . was considered significant.neopterin level was significantly elevated in exposed ( , ± , ng/ml) compared to controls ( , ± , ng/ml; p= , ). moreover, the neopterin level in exposed was similar to silicosis patients ( , ± , ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls ( , ± , au vs , ± , au p= , ) and to silicosis patients ( , ± , au p= , ). in contrast, igmcic was significantly elevated in silicosis than in exposed ( , ± , au vs , ± , au; p= , ).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p= , and , respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a* , b* , drb * (these of good prognosis) * /* /* /* ) or tbc (drb * /* /* /* and associations with dqa * /* /* ) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a* , b* and drb * (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a* , drb * . as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd + , cd ++ , foxp + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein (hhsp ). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il- , il- , il- , tgf-b and ifn-g production prevailed, pointing to a th /th weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp . importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th /th cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices ( ) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c -complement, myeloid related protein and two new proteins, integrin-ß and fibrinogen. data from more than patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than % and %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least mice per group with distinct cytokine pattern: th (c bl/ , c bl/ ) and th (balb/c). muscular injury was performed by injection of bupivacaine. both th -dominant strains presented more areas with regenerating myofibers and macrophages at dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at dpi. balb/c mice showed increased collagen expression and decrease of mmp- activity associated with more mrna for tgf-b . this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th cytokines) or myonecrosis and collagen deposition (th cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß -adrenergic receptor (anti-ß ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß ec ii -specific rat monoclonal antibody (clone f ), and showed by elisa that it binds to the linearized ß ec ii peptide. additionally, we confirmed with flow cytometry that f also binds the ß ec ii in its native conformation, i. e. directly labeled circular ß ec ii (dyl -ß ec ii ) peptide. moreover, we demonstrated activation of the ß -adrenoreceptor by f using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß ec ii -specific peptide-based therapy, by intravenously applying a circular ß ec ii peptide in the dcm lewis rat model to neutralize the anti-ß ec ii antibodies. while the peptide therapy strongly reduced the anti-ß ec ii titers in the serum by up to % and consecutively lead to clinical remission, elispot assays for the detection of ß ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca - epitope elicits autoreactive cd + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca - peptide. in a first step, hybridoma cells were generated by fusing bw tcra -cd lymphoma cells with myhca - -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca - -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle , a. schwarting , p.r. galle university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase (pr ), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr -positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il- . pr -mrna from the murine cancer cell line wehi- was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr -promoter in the second intron of the mouse pr gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr transcript and its promoter indicates that the murine pr may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n= ) or pbs (n= ) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr activation, in the presence/ absence of och. subsequently we transferred . x mdcs (n= ) or och-primed mdcs (n= ) ( times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a . % reduction in plaque size compared to mice treated with mdcs (p x . ). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant . % (p x . ) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a to -fold increase in these cells was detected days after the last treatment with och-primed dcs (p x . ). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il- . discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c bl/ background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c bl/ and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg c were isolated only from fcgriib deficient mice, but not from c bl/ control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box protein (hmgb ), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb . we investigated if hmgb -containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb remains associated with ncs released from late apoptotic cells in vitro. hmgb -ncs complexes were detected also in the blood of patients with sle. hmgb containing ncs from apoptotic cells induced secretion of il-b, il- , il- , and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd and toll-like receptor . neither hmgb -free ncs from living cells nor from apoptotic hmgb -or hmgb / -deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il- release by macrophages. immunizations with hmgb -containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr -dependent manner. in conclusion, hmgb in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher , k.p. hoefig , e. kremmer , v. heissmeyer stretches and helps in the selection of the correct splicing borders. a allele of (r h) creates a strong binding site for a splicing enhancer protein srp according to bioinformatics. our findings indicate that, the putative branch point, r h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank . finally, we believe that bank delta protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta protein may contribute to the observed reduction in sle susceptibility. s. beermann , r. seifert , d. neumann hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h receptor (h r), h r, h r, and h r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or -methylhistamine, a h r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and -methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c bl/ mice induced by either a-cd antibodies or cpg-odn. this histamine effect was completely inhibited by the h r-specific antagonist famotidine, while h r-, h r-, and h /h r-selective antagonists had no or only moderate effects. interestingly, the h r-selective reagent jnj , which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h r, jnj may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h r and, to a much lesser extend, the h r. using this assay system, cells obtained from control c bl/ mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega- fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of ng/ml resolvin e (rve ) on the release of tgfb, il- and il- in the culture of peripheral mononuclear cells ( x /ml) stimulated by phorbol ester (pma) ( nm), and the combination of pma and ionomycine ( mg/ml) for hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from healthy subjects and from patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = . ) and sjögren's (p= . ) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p= . ) or pma+ionomycin (p= . ), however rve was ineffective at reducing tgfb release in the sle and ss patients. rve caused a non-significant decrease in il- release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il- was not significantly modified by rve in any of the groups tested. the release of tgfb by ng/ml of rve can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve tested, il- and il- release were not significantly affected in healthy or autoimmune patients. omega- fatty acid derived rve may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis , a.k. tsirogianni , m. herrmann , h. m. moutsopoulos , m.n. manoussakis university of athens, dpt pathophysiology, athens, greece, university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included with primary ss (american-european criteria ) and with sle (acr criteria ). age-and sex-matched healthy blood donors to the ss and sle groups ( donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, ) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma ( patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd -staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p= . ). in ss patients, defective mb-phagocytosis involved only monocytes (p x . ) and significantly correlated with the presence of extraglandular manifestations (p= . ). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x . ) and of ss (p= . ). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: healthy persons have been included in our study and sle patients ( women and men) with various clinical signs ( persons had st degree of disease activity, persons - nd degree of pathological process activity). women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in , %, aphl of igg class -in , % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x = , ; p x , ). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x = , ; p x , ). the increased levels of anti-ada were revealed in of women with hnp, and the combination of anti-ada and aphl ( / ) was found out more often than isolated anti-ada ( / , x = , ; p x , ) or isolated aphl ( / , x = , ; p x , ). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc and mdc , and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc , mdc and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from pss patients fulfilling the american european consensus group criteria (aecc) and gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p= , ) and mdc (p x , ) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd + b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin (il- ) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd + b cells and increased il- levels. we therefore hypothesized that il- may have similar biological effects on cd + b cells in autoimmune diseases. here we demonstrate that the amount of il- in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to % of cd + b cells in sle individuals expressed grb. in-vitro experiments revealed that il- was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor . importantly, il- significantly decreased the cd +/cd -b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd + b cell death. these results suggest that il- -induced grb may play a regulatory role for cd + b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il- and grb levels in sle and showing that il- reduces the cd +/ cd -b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il- may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il- in sle and other autoimmune diseases. r. de palma , e. d'aiuto , s. vettori , g. abbate , g. valentini second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd + t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il- beta, il- and il- , cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed igg antibodies from single facs purified cd +cd +cd +cd + bone marrow plasma cells of patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran , e. moazemi godarzi , e. kamali sarvestani , e. aflaki shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin (il- ) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il- gene at positions - g/c and - g/c have been described that are key regulators of il- gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il- gene polymorphism at - position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the - position significantly differed in sle patients and controls. at this position gg genotype was observed in . % of patients compared to . % in the control group (p x . ). the frequency of - g allele in patients ( . %) was also higher than in controls ( . %) (p= . ). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. - polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x . ). at - polymorphism, a significant difference with regard to photosensitivity in male patients (p= . ) was found. in conclusion, results of this study showed that - polymorphism plays an important role in susceptibility to sle and that - polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained patients with ibd ( with cd, with uc, with gluten sensitive enteropathy, gse) and healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd -ve cd b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd (+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling- (rgs- ) protein, we have now found substantial over-expression ( - fold) of rgs- in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs- decreases primary t cell responses to cxcl and ccl , strongly implying that it may regulate t cell localisation. thus, rgs- may be a novel target for modulating t cells in ibd, consistent with which snps in rgs- have been associated with both coeliac disease and type diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c bl/ j mice were divided in control (c) and experimental (e) groups. c -c received peanut protein immunization, animals of the control groups c were sham immunized, and control group c received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a day peanut containing cd, the experimental group was divided into groups (e -e ). ova feeding began days prior cd (e ) on day (e ), (e ), (e ) and (e ) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e ) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase and secretion of bioactive il -b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il -b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying % destrane sulphate sodium (dss) in the drinking water for days to wildtype mice. when ultrafine nanoparticles were administered on day and by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl , n. schneiderhan-marra , m. blum , g. stein , m. schmolz , t. joos nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [ ] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco- cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of -well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h -associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h -like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il- production and t h polarization and decreases recruitment of cd + cd + foxp + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen , , g. van assche , p. rutgeerts , a. liston , j. l. ceuppens k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, k. u. leuven, experimental immunology lab, leuven, belgium, k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp- allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced types of colitis dss (th /th ), tnbs (th ) and oxazolone (th ) in hp ko mice. neutralizing anti-il- mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th /th cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. ) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; ) in dss but not in oxa colitis mice, il- , ifn-g, tgf-b and il- were significantly increased (p x . , dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x . , ko vs wt). in tnbs colitis, we found elevated il- and ifn-g (p x . , tnbs vs control). although not significant, il- was also somewhat upregulated; ) in dss colitis we observed that il- enhanced differentiated th cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il- and il- , more mln-t cells from hpko mice differentiated into th cells; ) anti-il- mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il- showed reduced il- , il- and ifn-g in both mln and lp (p x . , anti-il- vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il- , supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann , g. talabér , g. süt" o , p. németh , t. berki university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that . million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd +cd hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th and th cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd + and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd + inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells ( . ± . %, mean ± sem), while cd + inkt cells ratio was moderate ( . ± . %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: . ± . %; cd: . ± . %, mean ± sem), while the percentage of cd + inkt cells was elevated (uc: . ± . %; cd: . ± . %, mean ± sem) in both disease groups. proportions of foxp + treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b adenovirus (tgf-b -exo). the t cell inhibitory function of tgf-b -exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b -exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b -exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd + foxp + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b -exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b -exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd + foxp + regulatory t cells (treg) revealed that the relative numbers of cd + foxp + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd + foxp + treg. tgf-b -exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd + t-cells was similar in the four vaccinees as observed by ifng, il- production-profiles. however, the killing capacity of expanded cd + t-cells was distinct as observed by the competence to kill ns -peptide presenting transfectants in vitro. as depicted in figure , cd + t-cells cells from both vac (cleared ) and vac (chronic) produced il- and ifng after stimulation with ns -peptide . however, specific killing of the peptide loaded transfectants was only observed in vac , who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure ] killing of ns peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd + t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr -expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns or eda-ns under the control of cmv promoter were prepared. recombinant ns and the fusion protein eda-ns were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns . results: immunization of mice with the plasmids expressing eda-ns , but not ns alone, induced strong t cell responses against the main hla-a restricted cytotoxic t cell determinants from ns . the recombinant eda-ns fusion protein, but not ns , was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp- monocyte cell line. immunization of hhd mice with eda-ns fusion protein induced both cd + and cd + t cell responses against ns and, when immunized with poly(i:c) and anti-cd antibodies, was able to down-regulate the intrahepatic expression of hcv-ns rna. the recombinant eda-ns fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd +cd a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd + responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd + t cells by mature dcs transduced with melan-a-recombinant human coronavirus e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin (il ) and interleukin (il ) will be involved, and fms-like tyrosin kinase ligand (flt l) will be expressed to modulate dendritic cells. il is known to enhance early t cell expansion and limits t cell overshoot, whereaes il guarantees survival of high affinity t cells during memory phase. on the other hand flt l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik , ag waisman uniklinik mainz, . med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il- production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il- produced by b cells by conditional deletion of the il- gene in these cells. we found that b cell secreted il- has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for days in the presence of il- and il- with exposure to overlapping peptide pools of latent ebna- and lytic bzlf- antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd , cd , cd , ccr , cd ra, il- , tnfa, ifng and cd a. the majority of ex vivo ebv-reactive cd + t cells as well as ebna- -reactive cd + t cells were il- and tnfa-producing memory cells, the later being more frequent in bone marrow (cd +, median, ebna- : bm . %;pb . %; bzlf- : bm . %;pb . %, p= . ). after in vitro expansion a major subset of ebv-specific cd + and cd +t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd + and cd + t cells retained, however, a tnfa single, tnfa/il- or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd + and cd + t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr /cd ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il- delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd + and cd + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd + and cd + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd + and cd + t cells in peripheral blood with a hexon protein-spanning pool of synthetic -mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to - % in the t cell lines and the absolute numbers of both hexon-specific cd + and cd + t cells were to log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd + and cd + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype (ad ) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad vaccine vectors expressing whv core protein was first examinated in c bl mice. groups of mice were immunized with a dna prime-ad boost regimen or with dna and ad alone. ad was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd + t cell responses against peptide pools and in spleens of dna and dna-ad immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd + t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad very weak cd + t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad level of antibodies did not change. those antibodies were only igg a subclass what indicates th t helper type of response. ad -immunized mice had over -fold lower level of anti-whc: both igg a and igg subtypes were detected. the weak response induced by ad may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal ) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m e serum ab titers against peptide and native m e. this correlated with a large number of m -specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h n ), which contains a variant m e-sequence different in amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x (h n ) and the highly pathogenic pr/ (h n ) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m e as a potential "universal" influenza vaccine. this research is supported by a nih t fellowship ca - , the nih grant ai and a grant from the commonwealth of pa. l. yu zhejiang university, zhangzhou, china interleukin- (il- ) is a cytokine produced by stimulated mononuclear macrophage system. in this report, -day-old chicken embryos were vaccined with the plasmid dna (pci-chil- ) encoding chicken interleukin- and the copy numbers of chil- in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil- in -day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil- could accelerate high concentrations of chil- in nonage peripheral blood, accelerate high expression of chil- in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil- could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil- (p x . ). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on volunteers and a phase i study on volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv- neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) and the transmembrane protein gp . two sites on gp and gp are attractive targets for vaccine design: the epitope in the third hypervariable region (v ) is recognized by the human monoclonal antibody - d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies e and f . in order to elicit anti-hiv- neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp -v region or the gp -mper. the vlps are based on the acyltransferase component (e chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e chain self-assembles into a nm protein scaffold resembling a vlp and that contains copies of e . efficient display and refolding of the v and mper regions in e vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the ' of the gene coding for e . the priming and boosting with a combination of vlps and specific hiv- envelope dna were used to immunize mice and rabbits. results: the v -e and mper-e vlps were purified as stable mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of - d, e and f antibodies to hiv-e monomers was confirmed by western blot. we obtained high titers of hiv- gp -specific antibodies in mice immunized with a combination of vlps plus dna (hiv- sf gp ). these antibodies generated a low ( %- %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e vlps plus dna elicited a low titer of hiv- gp specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e vlps are able to display antigenic determinants of hiv and to induce high titers of hiv- -specific antibodies. the e vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h n whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h n influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [ ] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h n whole virus wild-type vaccine, could induce an immune response and protect mice against h n influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h n antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h n virus. the protective efficacy of the trivalent vaccine derived mainly from the h n component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h n virus, suggesting that antibodies are the main contributor to protection. h n specific serum did not inhibit neuraminidase activity of h n virus suggesting that protection was not mediated by neuraminidase n -specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h n whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h n vaccine enhanced anti-h n antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h n vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h n candidate vaccine. [ ] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of children in the first grade of primary school in the area of central and west macedonia. there were greek and foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following ) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to %. ) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). ) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. ) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om- , a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om- . cytokine secretion, activation and proliferation of b cells and foxp + tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr , tlr or the myd adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om- . two synthetic tlr agonists used as adjuvant in human (om- -dp and om- -mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om- -induced protection of diabetes required natural tregs, as nod-cd -/mice were not protected. remarkably, om- activated b cells and not t cells, promoting their proliferation and il- secretion, two phenomena that were tlr -and myd -dependent. om- -dp and om- -mp-ac two synthetic murine tlr agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd + cd + foxp + t cells and the proliferation of il- secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr signaling in the protective effect of om- on development of diabetes and show that two other tlr agonists induce proliferation of b cells and their secretion of il- as well as stimulation of regulatory cd + cd + foxp + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit , a. legat , s. thomas , m. van mechelen , p. hermand , m. goldman institute for medical immunology/université libre de bruxelles, gosselies, belgium, glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide -phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor (tlr ). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd (scd ) for its tlr ligand activity, we investigated the involvement of both forms of cd in the responses elicited by crx- , a prototypical agp. first, we found that crx- efficiently induces nf-kb and irf activation in hek cells transfected with tlr and md- genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd . likewise, crx- efficiently induces the synthesis of nf-kb and irf- dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd respond to crx- but not to lps in serum-free medium. the addition of the soluble form of cd did not modify the levels of il and tnf produced by crx- stimulated dc but increased the levels of interferon-b (ifn-b). when scd was added to hek cells expressing tlr /md- , nf-kb activity was not modified but irf activity was increased in a dose-dependent manner in response to crx- . we will further compare the responses induced by crx- in wild-type and cd deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h- d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled and days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin- gene polymorph isms (c t, g a, c a and a t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [ ] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n= ) have higher amount of both nonactivated (subtype infg+/cd -, p= . ) and activated (subtype infg+/cd +, p= . ) cbmc, producing ifn-g, as compared with newborns from urban mothers (n= ) exposure to pets and the risk of allergic symptoms during the first years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc / to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity ( - %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: months - years (n= ), - years (n= ) and - (n= ) tb has the highest diagnostic value in children n years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp- antigen is more sensitive for detection of tb-specific t cells compared to esat- antigen. . in children with tst - mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr and tlr in the development of spontaneous lupus disease by creating tlr or tlr deficient c bl/ lpr/lpr mice. methods: tlr and tlr deficient lupus prone mice have been generated by crossing c /bl -tlr -/-or c /bl -tlr -/-mice with c /bl lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr or tlr deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr or tlr in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr deficient mice suggesting an important role of tlr in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr pc / expression of full length mcl- and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl- , an anti-apoptotic protein. a splice variant of mcl- arises by removal of exon and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl- (mcl- l) and its splice variant (mcl- s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl- . method: neutrophils were isolated from children (diagnosed x years) with jsle (n= ) and non-inflammatory conditions (control, n= ) and incubated with control serum, jsle serum alone or with jsle serum plus pg/ml gm-csf. quantitative real time pcr was used to assess mcl- l and mcl- s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl- s to mcl- l was also higher in jsle patients compared to controls (p x . ). the addition of gm-csf to jsle serum was associated with an increase in mcl- l ( . ± . ) and a decrease in mcl- s ( . ± . ) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl- compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl- s, and less anti-apoptotic full length mcl- cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex / mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd + cyld ex / t cells were transferred into rag -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex / cd + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex / mice are capable to control inflammatory responses. for this purpose cd + cd + cells were co-transferred with naïve wt cd + t cells into rag -/-recipients. interestingly, rag -/-recipients of cyld ex / t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc / the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a* in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb * , * and * alleles, and a decreased allele frequency of drb * in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb * is associated with the development of severe disease and positive association of cd with drb * and drb * . indeed, drb * was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb * frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb * in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb * were strongly increased with respect to cd and controls. however, the frequency of drb * was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb * is associated with uc in european, north american, japanese and korean populations methods: a total of children were studied, ( boys and girls), up to years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, positive cases of children were found ( %) with active infection : boys ( x years of age, - years of age and g years of age) and girls ( x years of age, - years of age and g years of age). pharyngitis was present in children ( , %), had fever ( , %) and had lymphadenitis ( %). the lab tests revealed leukocytosis up to . leukocytes in cases ( , %) and leukocytosis g . in cases ( , %). the most frequent complication documented was streptococcal superinfection in children ( , %) and thrombocytopenia in children ( , %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and ) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il- ra , previously believed to be a decoy for il- only, is able to transmit a signal via il- . our results support this and may suggest that il- / il- ra signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il- ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il- or il- ra and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd + cd -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il- and il- b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd + cd -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi- , l. acidophilus ncfm tm and b. bifidum bi- ) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi- strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il- or il- b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed pediatric cd patients ( active, remission), pediatric uc patients ( active, remission), and age-matched non-ibd controls. nkg d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x . was considered significant. results: nkg d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd +cd + nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg d ligands on circulating monocytes is also observed. the dramatic increase of nkg d+ lymphocytes, and the strong upregulation of nkg d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc / peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp + t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd + t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec- antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa - ) or hcv-ns (aa - ) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns and core we successfully optimized culture and protein purification conditions. briefly, ns was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec- to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec- / hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd / mva-nef vaccination induces polyfunctional cd t-cells and increases the proliferative capacity of cd t-cells in hiv- infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv- protein nef (mva-nef) was safe in hiv- infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il- and mip- b, the expression of the activation marker cd and the differentiation marker cd ra in nef-specific cd and cd t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il- and mip- b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd t-cell response and a significant increase of polyfunctional nef-specific cd t-cells, simultaneously expressing ifn-g, il- and cd . using the standard ics no increase of nef-specific cd t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il- and cd expressing cd t-cells and the increase of proliferating cd t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd t-cells in hiv- infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv- mediated membrane fusion and p -detected replication. db was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd + and cd + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns ( - aa) and rns a ( - aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/ j mice were immunized intraperitoneally times at a month interval with different doses ( . - mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns protein. immunization with rns a-immunomax conjugate and rns in cfa ( . mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd / degree of cross-genotype reactivity of hcv-specific cd t cells directed against ns the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd t cell response targeting hcv genotype (gt ) and genotype (gt ) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: subjects ( with gt , with gt and anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns from gt or gt . individual reactive peptides and the degree of cross-reactivity between the gt and gt variants were determined by ics. complete ns is sequenced from all viremic patients pd / anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes , , or suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes , or , but not led to a significant reduction in viral loads. decreased splenic viral load after ifna treatment correlated with an expansion of activated fv-specific cd + t cells and nk cells in the spleen, whereas in ifna -and ifna -treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna , and are under investigation pd / elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a* -or k b -restricted cd t cell responses by these dna vaccines differed. k b /ova - -and k b /s - -specific cd t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd + and cd + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least healthy, randomly chosen blood donors were cultured for days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. out of tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il- -exposed cd + t cells showed at least five-fold increase of survival rate in vivo than il- -exposed cd + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il- -exposed cd + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il- and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x . ) . there was a significant elevation of t ada and ada activities in iga deficient patients as compared to healthy individuals (p x . ) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd / hiv- sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after - weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in out of patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, -day-old ross broiler chickens divided randomly into groups, experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/ strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/ and h strains, via the drinking water at days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, & days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd t cells from patients with active tb and patients with successfully treated tb were analysed for simultaneous expression of ifng and il at the single cell level using multi-colour flow-cytometry after hours stimulation with ppd. moreover, cells were stimulated with esat- and cfp- receiver operator characteristics analysis revealed that a percentage of ifng /il dual positive cells x % served as an accurate marker for active tb patients (specificity %, sensitivity %), while frequencies g % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd / necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately inhabitants) is . to . among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, out of children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently . to inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of to led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd / novel analogues of thalidomide inhibit cd expression and production of tnf-a, il- , ifn-g, cxcl- this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta -dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta r k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd and cd . these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il- a and il- b, while il- levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il- b production. using bmdc from nlrp -/-mice we examined il- b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il- b production is dependent on nlrp . collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho- expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from % in wild type (hmox +/+ ) mice to % in ho deficient (hmox -/-) mice. hmox -/-but not hmox +/+ mice developed end-stage multiorgan failure. mortality of hmox -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h o or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb . similarly, circulating and cytoplasmic hmgb was increased in hmox -/-relative to hmox +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b- , -linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at - years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and - weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were . and . iu/ml and those of dtwp-pasteur were . and . iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were . eu/ml for dtwp-local and . eu/ml for dtwp-pasteur vaccines, respectively (p x . ). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd / united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b: , a gram-negative coccobacillus. jrmt , an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of cfu of jrmt . a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b: on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for h before adding concanavalin-a (cona) pd -vaccination and immunotherapy against parasitic diseases pd / evaluation of simian adenoviral vector adch expressing msp- as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype (adhu ) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp- and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho- expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho- is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp expression was confirmed by sds-page and elisa using monoclonal antibody against gp . discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a - recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a - recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at - °c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r= . , p= . ) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il- also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il- on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il- to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il- and il- -related inhibition of cfu-e growth were dependent on p mapk activity, since the p mapk inhibitor, sb , markedly downregulated il- -induced activation of nos and reversed the growth inhibitory effects of il- on cfu-e. the in vivo stimulating effect of il- on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il- effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il- acts on bone marrow cells and also revealed a link between the il- , no and hematopoiesis. further studies on il- -mediated induction of both inos and enos methods: a total of blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of samples ( , %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of iu/l anti-hbs in all the blood units, which also goes for our study pd / neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen , national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa- a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa- a iggs we monitored children ( boys and girls) in ages from . to . years with average age of . years. in of them all was diagnosed for the first time. subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest ( subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc h gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. , nature , - ). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 'utr of icos, in which a bp sequence, containing a possible mir- binding site, was sufficient (yu et al. , nature, , - ). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. , j biol chem , - ). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia- -and ago -deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r ai . we have recently reported that -hydroxyl- -methylindole- -acetonitrile ( -hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw . macrophages. in this report, we investigated the effect of -hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin- (il- ), monocyte chemotactic protein- (mcp- ) in ht- human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed -hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated -hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. -hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p . in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of -hma significantly reduced the mrna levels of il- and mcp- stimulated by tumor necrosis factor-a (tnf-a) in the ht- cells. pretreatment of -hma also significantly blocked the ixba degradation and nf-xb p nuclear translocation stimulated by tnf-a in the ht- cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of -hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, -hma could be new potential therapeutic agent for inflammatory bowel disease.cd serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd igg immune response in hiv- -exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp -binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp , reacted with the external domains of soluble human cd , in the absence of the target cells expressing the co-receptor ccr . the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd -gp complex and trigger the membrane fusion between effector (gp expressing) cells and target (ccr expressing) cells. thus, in the absence of ccr we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv- infections. a conventional protocol for the generation of monoclonal antibodies was used. db- (igg , x), one of the anti-cd antibodies obtained, recognized preferentially cd complexed to gp , as compared to cd alone, not competed for the gp binding site on cd and was specific for the second extracellular domain of cd . g. röder , l. geironson , a. darabi , m. harndahl , c. schafer-nielsen , k. skjödt , s. buus , k. paulsson copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, lund university, immunology bmc d , lund, sweden, lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, schafer-n, copenhagen, denmark, department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a* . to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn - / , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn / was demonstrated to be located on tapasin [ ] [ ] [ ] [ ] [ ] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd + t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region - of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b* -restricted np-flu epitope (aa - ) was replaced by hla-b or hla-a -restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard cr release assay and through the ifn-g production. results: using hla-b or hla-a restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b as a remarkable hla-class i molecule that, differently from hla-a , can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv- )-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv . consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p -gag and p region was determined in vitro. mer peptides were digested with i s and c proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv- p -and p -gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers , g. koster , o. landsverk , f. skjeldal , o. bakke university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi kinases and thus ptdins( )p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier , l. visvabharathy , j. fu university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e - k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e - k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type e - k and class i molecules.results: we showed that e - k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a molecules, with the mature form being more tightly associated. we also provided evidence that e - k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e - k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e - k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il- , il- , tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands and , but also with the t-celldependent stimulus cd l (cd ) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd , cd , and cd was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p and erk- / . strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd + t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th (il- +) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th / th balance and to identify potential t cell target antigens (ags), we analyzed circulating cd + t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il- expression in peripheral blood cd +cd + lymphocytes were measured in ccs patients and healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il- /ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il- and lower ifn-g intracellular expression were detected in cd + t cells from css patients, as compared to hs (il- : . ± . % vs. . ± . %, p x . ; ifn-g: . ± . % vs. . ± . %, p x . ). similar results were obtained by elisa (il- : . ± . vs. . ± . pg/ml, p x . ; ifn-g: . ± . vs. . ± . pg/ml, p x . ). elispot counts of hexavalent vaccine-stimulated cd + cells were positive for il- in / ( %) css patients and in / ( %) hs, and for ifn-g in / ( %) css patients and / ( %) hs. mpo stimulation determined significant ifn-g release in / ( %) css patients, but not in hs ( / ) no il- response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd + cells from css patients show a th -polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd + t cells, and responses towards it are instead th -polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd + t cells in css. a. voigt , e. opitz , k. savvatis , k. klingel , k. stangl , u. kuckelkorn , p.-m. kloetzel charité -universitätsmedizin berlin campus mitte, berlin, germany, charite -campus benjamin franklin, berlin, germany, universität tübingen, tübingen, germanymurine models of coxsackievirus b (cvb )-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp of the stress-induced ip, were infected with x e pfu cvb nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp as early as day p. i. in lmp -deficient mice. however, lmp -deficiency was linked to less severe myocarditis at day p. i. (he stain of cardiac tissue sections: wt . ± . vs. lmp -deficiency . ± . (grade of myocarditis; scale - ; p x . ). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x . ), there was no difference in lmp -deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb infection in lmp -deficient mice. likewise, diastolic function was preserved in lmp -deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp -deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp -immunosubunit function in regulatory processes of viral replication. absence of lmp confers host protection in enterovirus myocarditis. h. w. liao , j. xu , j.q. huang sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype (den- ) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr and tnfr - were constantly very low whereas trailr - decreased after den- infection. fasl was expressed at similar levels on huvecs throughout den- infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase , caspase were also observed by western blot after by den- infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for days into wis rat paw web tissue in saline containing . x cells. group ii. s.epidermis was injected as in group after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day .they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors , and , respectively (p x . ). immunohistochemical pictures showed increase in percentage of ox (migrating dendritic cell), mhc ii, his (granulocytes), ox (stem cells) and cd (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed (macrophages) and ox cells. popliteal lymph nodes contained evidently less of ox , his and mhcii cells than in group i (p x . ). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega- fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega- fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega- fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega - fatty acids consumption. the results indicate that omega- fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega- fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega- fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density . and , g, top % layer contained lipoproteins only and - % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin- . objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e f mutation, and other affecting b cell apoptosis control, such as bcl- over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c bl/ (b ) genetic background. e f -/-bcl- tg were obtained backcrossing e f -/-and e f +/+hbcl- tg mice. e f -/-bcl- tg, e f -/-, e f +/+hbcl- tg and control mice (e f +/+) were followed up to mo-old. serologic studies: serum samples obtained at , and month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of , and mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h ]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl- tg in b lymphocytes of e f -/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e f -/-bcl- tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e f -/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e f or the overexpression of a bcl- tg in the b genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor (tlr ) abolishes the generation of antinucleosome igg a and igg b autoantibodies but exacerbates lupus disease. however, the tlr -dependent tolerance mechanism is unknown. here we show that loss of tlr in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th and th t cells. transfer of a synthesized monoclonal polyreactive igm to tlr deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr -dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n= ) (vitali et al. ) . ninety-seven of ( %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g (fs+), while biopsies from / ( %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in / ( %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x . ). interleukin (il) , il- ra, il- beta, il- p , il- , macrophage inflammatory protein (mip) alpha, mip- beta, eotaxin, interferon (ifn) alpha, and il- levels were significantly increased in the gc+ patients (n= ) compared with the gc-patients (n= ). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il- found in these patients (gao et al. ) . increased titers of th -associated cytokines, il- , il- beta and the il- subunit il- p , may indicate a higher activity of these cells in gc+ patients (nguyen et al. ) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il- . the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg . production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd -dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd -dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/- baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il- . objectives: increased levels of il- , an innate and inflammatory cytokine of the il- family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il- and other genes of the il- /il- r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from patients and healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il- and il- bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il- are higher than in controls. serum il- correlates with disease activity and decreases upon remission. monocyte expression of the receptor il- rb is increased and correlates with disease severity, while expression of tir /sigirr (a down-regulatory receptor of the il- r/il- r family) is reduced. in mrl lpr/lpr mice, expression of il- , caspase- and il- rb genes precedes disease onset in lymph-nodes. in other organs, changes in il- -related genes (il- and tigirr- up-regulation, tir /sigirr down-regulation) occur after disease onset. free il- levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il- levels correlate with autoimmune lupus both in mice and humans. free il- may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il- rb is a marker of pathology, suggesting increased il- -dependent activation. both in mouse organs and human monocytes, tir /sigirr expression decreases with disease, suggesting impaired control of il- r activation. thus, il- may be involved in autoimmune lupus pathology, and il- -related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f (amino acid (aa) - ) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f was recognized by all patients. fragment f (aa - ) was recognized by of dcssc patients. fragment f (aa - ) was recognized by of sle patients. analysis of clinical data revealed a significant difference between the f negative and f positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f and f could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril- . by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd + t cells enriched in tregs were co-cultured with activated cd + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd + t cells anergy was also evaluated based on cbl-b, grail and foxp mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b- cells through targeting p d by shrnas strategy. methods: we used the drugs, ly and wortmannin, pan-specific inhibitors against pi ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p d. we then introduced either pan-specific inhibitors against all pi ks or p d-targeting shrnas into an sle-prone animal model, nzb/w f mice, for therapeutic purposes. the results suggested that pi ks are not only important for the development of b- cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p d. either pan-specific inhibitors against pi ks or p d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f mice. one inhibitor, ly , and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi ks are critical for the maintenance of b- cell populations might shed light on future treating other diseases associated with b- cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool , m. alarcón-riquelme , s. kozyrev institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs , r h). we identified that bank gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon (delta ). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs ), which is in complete ld with r h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia , which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a -year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti- gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow cnrs , strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed patients, with minor clinical and/or biological manifestations of their disease, for at least monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below . b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd surface protein for all patients. this cd lower expression is associated with cd lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf lacking an exon in the c -domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf in t-cells. smurf expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf expression was upregulated by tgf-beta stimulation in t-cells and smurf was markedly upregulated in tumor infiltrating cd + lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day , smurf transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf showed an upregulation of the tgfbrii and an activation of smad and as compared to wild-type t-lymphocytes, which were previously described as typical smurf targets for degradation. in addition the transfection of smurf and a caga-luc plasmid into cos-cells for smad -promotor studies yielded the same effect as shown by upregulation of the smad activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf has beneficial effects on mucosal inflammation and tumor development. smurf thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat has important functions in cytokine signalling in a variety of tissues. however, the role of stat in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat activation in dss colitis is dependent on il- rather than il- . il- was secreted by colonic cd c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat activity were highly susceptible to experimental colitis, indicating that epithelial stat regulates gut homeostasis. stat iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il- and epithelial stat was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat activation regulates immune homeostasis in the gut by promoting il- -dependent mucosal wound healing. stat seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd and tlr expression at day and day after infection. mycobacterial infection did not result in differential tlr expression as compared to uninfected cells. cd is involved in stimulating th pro-inflammatory responses, although map may interfere with cd signalling ( ) . tlr signalling elicits anti-inflammatory responses, which can contribute to bacterial replication ( ) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd and tlr receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd t cells by exogenous antigen leads to colitis. methods: eight million naïve cd + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h -kb, were transferred i. v. into b mice. at day and , mice were treated intra-rectally (i. r.) with % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day after the injection of ot-i cells. the phenotype of effector cells was evaluated at day by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd , the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd + t cells. our study demonstrates that the local activation of antigen-specific cd t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of patients, ( male and female), with age average , years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x and t-test programmes. results: in patients ( , %,with age average , years of age) no antibodies were detected. on the contrary, in the remaining , male and female, ( , %, with age average , years of age), antibodies were detected. out of them, in cases the results were strong positive ( male and female) and weak positive in cases ( male and female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: ) helicobacter pylori infection is relatively common in the general population ( , %). ) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women). ) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova , t. ulmannova , k. stechova , k. tesarova-flajsmanova , v. stavikova , j. nevoral charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of mothers whose infants were diagnosed with allergic colitis and mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis ( - weeks, average . weeks of infant's age) or at the age of weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il- , il- , il- , il- , il- , ifn-gamma, tgf-beta , egf and eotaxin. man-whitney u test was used for statistical analysis, p x . was considered statistically significant.results: il- as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x . ) in breast milk of mothers whose infants were suffering from allergic colitis (range - . pg/ml, mean . pg/ml) than in control group (range - . pg/ml, mean . pg/ml). higher concentrations of il- and lower concentration of tgf-beta were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. - . background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il- (murine homologs kc and mip- ) and its receptor cxcr are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at , , and h post-transfer. anti-kc antibody or its isotype control was administered at mg/mouse one hour before transfer followed by whole body and organ imaging hours post-transfer. results: facs analysis revealed % neutrophil purity, % of which were cxcr + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model ( . % dss in drinking water for week, followed by pure drinking water for week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell ), treatment of commensal-depleted mice with the tlr ligand lps in drinking water protected against the lethality of % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of . % they may become pathogenic and drive an intestinal inflammation. at % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by . % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il- production which is an important pathological factor. neutralizing il- or il- prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th cytokines in colitis remain undefined, we used mice deficient in il- /il- or the key receptor through which they signal, il- ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il- ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il- /il- double deficient mice which were protected from colitis. removing il- production from il- ra -/mice, by using il- ra/il- double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il- mediates susceptibility in an il- ra independent manor. recent evidence pc / introduction: the activation of cd + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th -like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th cytokines, such as il- ,- and il- . however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc was found in uc and cd colonic tissue compared to control specimen. transmitted to the th -mediated oxazolone-induced colitis model, nfatc -production is significantly increased in both diseases, too. nfatc deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc deficient mice compared to control mice, which can be observed by tunel assays, caspase and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl- and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il- , ifn-gamma, il- and il- by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc regulates il- /il- in an indirect way. last, administration of il- blocked the protective effects of the nfatc deficiency in experimental colitis, suggesting that nfatc through il- signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc in colitis by controlling mucosal t cell activation in an il- dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd cd rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd cd rb high t cells also regulatory cd cd rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at different time points during colitis progression. after weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at weeks. microrna was isolated from colons of mice in different stages of colitis progression ( , and weeks) and control mice that do not induce colitis (n= for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from micrornas that demonstrated an induction during the development of disease we selected micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd cd rb high transfer model. objective: the purpose of this clinical trial (id: nct of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in groups: group a (n= ) receiving haart+ gh (for months) + tt+hva/b vaccines (at month post gh adminsitration); group b (n= ) receiving haart+gh but not vaccines; and group c (hiv control group, n= ) with haart+vaccines (at month ) but without gh. all patients are followed up months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after weeks of administaring hormone the absolute numbers of cd incresase from ± to ± cells per mm (mean and sem; p x . ). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd absolute numbers from ± to ± cells per mm (p x . ). viral load remained undetectable in all patients. despite the increase in cd counts the percentage of recent thymic emigrants (as assessed by the expression of cd ) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv ( , - , ), hepatitis c virus (hcv; . - . million) and hepatitis b virus (hbv; - million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv- infected patients with g cd + t-cell counts and plasma hiv-rna levels of x . log copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g and when the responses were g times the standard deviation above the mean of replicate negative controls (mock electroporated dc). / patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in / patients before and / patients after vaccination. for rev, / patients showed a pre-existing rev specific response and / patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already / patients responding before and / patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in out of patients before vaccination and / patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus (ev ) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev inactivated virus. methods: mice were immunized intraperitoneally with mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after , , and weeks. blood samples were collected at , , , and days. the total serum anti-ev igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il- from splenocytes was also measured. results: immunization with ev /ps-g showed that the anti-ev igg levels were significantly increased compared with ev alone or ev /cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev /ps-g group compared to ev alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev /ps-g-immunized mice compared to those of ev or ev /cfa/ifa-immunized mice, indicating a th cells response elicited by heat-inactivated ev vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd + t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd + t-cell responses against hcv-ns , studying induction of il- , ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il- and ifng production by cd + t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv- -recognizing cd + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv- replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd + t cells by electroporation, and chose tcrs which were able to recognize the hla-a restricted hivpol-peptide iv , or the hivgag-peptide sl . results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il- , tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd , after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt l pretreated balb/c mice were incubated for hrs with hcv ns -coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g % dcs and was used for immunization. dc expression of the maturation markers cd , cd and cd was determined before and after ingestion of ns -coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns expressing sp / cells). results: in immunized animals, the ctl activity was increased -fold compared to mock immunized mice. accordingly, tumor challenge with ns expressing tumor cells showed a significant reduction in tumor growth. the number of cd + ifn-g + cells was increased g -fold and the number of cd + il- + increased g fold in the dc-ns -bead immunization group. these results paralleled the proliferative response of splenocytes to ns protein obtained from immunized animals with the most significant response in the cd + population of dc-ns -bead immunized animals. the use of ns coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec- antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec- induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec- promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec- antibody can be developed. we screened the anti-dec- sequence computationally for putative hla dr -restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec- antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec- as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih r ai a live oral vaccine based on human adenovirus (ad) has proved safe and effective in us military recruits for nearly years. in these experiments, we have investigated whether replication-deficient ad can be an efficient potential vaccine carrier for oral vaccination. ad vectors were used throughout to provide a benchmark for efficacy. we generated novel ad and ad vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad than ad . ad routinely infected and provided transgene expression in˚ % of human cd + and cd + t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to - % of cd + t cells present, showing that ad infected a surprisingly large proportion of t cells. in comparison, ad provided egfp expression in x % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad and ad vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad and ad induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day after dosing, and transgene expression being reduced below detectable levels by day . interestingly, when delivered together ad and ad vectors targeted the same subset of cells. together, these data show that ad is a viable alternative to ad -based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies ( recently commercialized; g in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that -treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, ; gros et al, ; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp ) of rhdv at two different locations: ) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp- ), and ) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp- ). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp- ) was more immunogenic than insertion at position for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp- at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr- ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd + t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd + t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np - specific cd + t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr- targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd +, cd +, cd +, cd + t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by , - -fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns and ns a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h n influenza virus inhibits the ifn-g synthesis (mibayashi et al., ) and causes a decrease in cd + and cd + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., ) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions - (tat'kov c. i. et al., ) . deltaferon was i. p. administered to male non-inbred mice in doses of - * iu once or twice at an interval of hours, alone or in combination with double-stranded yeast rna preparation ( mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., ) . results: when deltaferon was administered in doses of - * iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of * iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon ( * iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd responses (tetramer+ hiv- gag p cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd responses after pla-p immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas , c. prego , s. vicente , m.j. alonso , a. gonzález-fernández university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer , with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with or immunizations to balb/c female mice ( weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day to post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease ( miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigen- background: infection with human immunodeficiency virus type (hiv- ) is characterized by dysfunction of hiv- -specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p antigen from hiv- and the tlr ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p microcapsules containing p and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p peptides. intracellular staining of interferon-gamma (ifn-g), interleukin- (il- ) and cd a after p stimulation was also performed. results: mddc from hiv(-) subjects, incubated for hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il- , upon p peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd b between our model cytokine il- and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il- dependent proliferation assays of il- variants. results: murine il- attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il- dependent cell line ht- . we found that the membrane anchors comprising one and four ig-like domains (il- :: iggpi and il- :: iggpi) resulted in severely reduced vlp production by producer cells and despite of an increased targeting of il- :: iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il- fused to the minimal gpi-anchor acceptor sequence of hcd b (il- ::gpi). il- :: iggpi, however, showed comparable particle production and biological activity in vitro when compared to il- ::gpi. furthermore, il- fused to ig::gpi showed an increased capacity to co-stimulate primary p tcr transgenic t-cells specific for lcmv-gp - in the context of h -d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il- ::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f -b of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild , interdisciplinary transplant laboratory charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects - % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of - months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from , month to days. t cell lines are composed of cd and cd cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker , m. assenmacher , a. richter miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp -specific cd + and cd + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd ro -cd -) cd + and cd + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp peptide pool and cd -depleted autologous pbmc as feeder cells in the presence of il- , il- , and il- . already - days after primary activation pp - /a -tetramer + cd + t cells were detectable for hla-a + blood donors. to analyze cd + t cells having other specificities than for the peptide pp - as well as probably primed cd + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to . % of the cd + t cells and up to . % of the cd + t cells after restimulation with pp peptide pool, but not with either irrelevant ie- peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for days led to a - fold expansion of pp -specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd and cd only if restimulated with the pp peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il- , indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd + and cd + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf , k. mozdzanowska , l. otvos , j. erikson the wistar institute, philadelphia, united states, temple university, philadelphia, united statesthe influenza virus a matrix protein ectodomain (m e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine ; virology journal ), we generated a novel peptide and investigated its efficacy in inducing an anti-m e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd + t cells, have been considered. clinical data confirm a crucial role for antiviral cd + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a* and hla-b* previously. considering other frequent hla alleles cmv specific cd + t cells were monitored longitudinally in hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a* /pp ( - ) ( . x cells/l) and hla-b* /pp ( - ) ( . x cells/l) specific cd + t cells are significantly lower than those for hla-a* /pp ( - ) ( . x cells/l) and hla-a* /pp ( - ) ( . x cells/l) specific cd + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. ( , - mcg) . no adverse effects were indicated during trials (up to month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after th immunization with mcg of vichrepol.no differences were detected in cd + and cd + t cell counts and cd +/cd + ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp or gp alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb f hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb f mice with adenoviral nanoparticles expressing fusion proteins containing gp resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted - % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf did not show any increase in their levels of ifn-gamma. conclusion: caf enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from children aged - years old ( boys and girls) -group , and children ( boys and girls) - years old -group were isolated on a gradient of density before vaccination ( or revaccination) with priorix, week, and months after and incubated with cfse. then million/ ml pbl were incubated in rpmi- supplemented with % fcs (the negative control), at presence of mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing % cÎ at °c within day. intensity of a fluorescence estimated on fl by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control % pbl in both groups did not enter mitosis. in the positive control % of cells have passed one and more mitoses. in group measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in months - % of lymphocytes demonstrated antigen-specific proliferation. in group , on the contrary, before the vaccination the most part of cells ( - %) has not entered division, but - % of cells have passed and more mitoses. in a week specific lymphocyte proliferation decreased and in months it was increased up to - %. production of the interleukin (il) , ifn-g, tnf-a, il- , il- was more informative than il- , il- , il- , il- . measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp cd -binding site (cd bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv- neutralizing polyclonal iggs directed against the cd bs. the mabs were shown to react with an anti-cd bs human neutralizing mab (b ), to elicit antibodies that recognize the gp molecule and an anti-hiv- neutralizing response in rabbits, confirming them as cd bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p and p ) reacted in elisa only with the cd bs-directed igg fraction. the clones were also recognized in elisa by b . p and p -immunized rabbit sera showed a strong anti-gp titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb strain glycoproteins. in particular, / rabbits in the p group and / in the p group showed an % hiv neutralization at dilutions ranging from : to : . the immunoenzymatic assay used, allowed to detect a p and p reactivity in hiv-positive sera and was able to detect a b concentration equal to ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines ( d and dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a -year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following d- vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd , cd and cd ) along with increased levels of nkt-cells (cd + cd +/-cd +/-/cd + ratio) and activated t-cells (cd + and cd + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il- , il- , il- , il- and il- ) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il- + ), cd + t-cells (il- + and il- + ) and b-lymphocytes (tnf-a + , il- + and il- + ). the analysis of cd + t-cells revealed a complex profile with increased frequency of il- + and ifn-g + and decreased percentage of tnf-a + , il- + and il- + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester , h.-g. rammensee , s. stevanović eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd t cell epitopes for the frequent mhc class i alleles a* , a* , and a* from the proteins pii (hexon), pviii, and e a of adv strains ad and ad by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a -day recall stimulation of pbmcs from at least healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd t cells. we could identify new peptides eliciting ifn-g responses, several of which were confirmed as novel cd t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle ( ara criteria) was diagnosed in a -year-old african female patient with hiv- (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd t-cell count x cells/mm . initiation of mg mmf bid was associated with biological and clinical remission of sle and cd t-cell increase. no opportunistic infections or cancers were noted during a -year follow-up and the patient remained always naive to art. hiv- -specific cd and cd t-cell responses were analyzed after months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il- production following stimulation with a panel of hiv- -derived optimal epitopes ( / -mers) covering various hiv regions and a pool of hiv- -derived peptides ( -mers overlapping by aminoacids) encompassing the entire gag protein. all peptides are derived from hiv- consensus strain iiib. results: highly polyfunctional hiv- specific cd and cd t-cell responses against gag were detected. epitope-specific cd t-cell responses were identified: except for one response restricted by hla a* and another one by hla cw* , all the others were restricted by hla-b alleles and mostly by b* (n = ). seven out of responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il- +) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv- specific cd and cd t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv- t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of dl /ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf- of - g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt- , cofaa and cgpi- . . state of vaccine-induced measles immunity was determined by means of elisa in - , - and - years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in - years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p . ) measles immunity and antibody level much lower (p p . ) than among children with mt conversion. in - years the comparison group kept decreased (p p . ) measles immunity, the majority ( ± . %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p . ) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p . ), the majority ( . ± . %) of persons lost protected antibody level; among children with mt conversion in - years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p . ) to low values; among children with hyperergic mt igg level decreased (p p . ) and reached low (in - years), minimal protected (in - years) and lower than protected (in - years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il- producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein (dnahsp ). methods: balb/c female mice were infected by intra-tracheal route with h rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp genetic vaccine was done at days , , and post-infection. each dose consisted of micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd +, cd + and gamma-delta t cells from lungs were determined and days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x . were considered significant (t test). results: at day after the end of the immunotherapy, dnahsp treated mice exhibit increased numbers of absolute cd + and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il- producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp treated mice showed more ifn-gamma producing cells in both cd + and cd + cell populations. at day after the end of the therapy, the main observation in mice which received dnahsp treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd + and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd + cells, together with a more frequency of gamma-delta t cells producing il- . finally, the immunotherapeutic effects of dnahsp vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd + cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il- , are the main effects of dnahsp immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il- and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of - log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput ( -well format), requires only ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to % in pb, % in rx and % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd l, a co-stimulatory molecule preferentially expressed on activated cd + t cells, is the ligand of cd . cd -cd l interaction induces the production of il- and the initiation of a th -type immune response. several studies show that cd l is required for the activation of macrophages and the maturation of dcs. moreover, cd l enhances the capacity of cd + t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd l. we have constructed the recombinant bcg strain expressing cd l (rbcg ) by electroporation of bcg with pgfm /signalsequenceag b-cd lec and an another recombinant strain with empty vector pgfm (rbcg ) as a control. the expression of cd l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd l weeks after vaccination but not at and weeks. rbcg seems to be more protective against paratuberculosis than rbcg , but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il- a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x . ) of tnfa, ifng and il- a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x . ) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd mab and ag a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd binding on cd transfected l fibroblasts. the conjugates were tested in vivo in wild type and cd + cell-depleted mice for the induction of specific anti-ag a serum antibodies. splenocytes were challenged ex vivo with ag a and were examined for their ability to produce th -related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il- . we developed a method to successfully crosslink anti-cd mab to ag a. serum antibodies against ag a were detected after immunisation with this conjugate vaccine in both wild type and cd + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag a alone. production of two other th -related cytokines, tnfa and il , was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer , r. burger robert koch-institute, cellular immunology, berlin, germany, robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd -positive and cd -positive t-lymphocytes. cd -positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd + t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd + t-lymphocytes in vivo. similarly, the cd -positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd + and the cd + subpopulation depended on the presence of apc. stimulation oft cd + cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd -positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target (esat- ) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, iranian and afghan adults ( patients with sputum smear and culture positive tuberculosis, recovered patients during months after full course of chemical treatment and healthy individuals) were recruited to quantify the frequency of esat- and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of spot forming unit ( g spots per million), we found detectable response to esat- in almost % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and % of these individuals had detectable esat- specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in %, % and % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th immune response, against esat- , in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal , p.k. upadhyay national institute of immunology, pdc- , delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c bl/ mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: . % alginate, . % pva concentration gives particles with size of - micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with % trehelose and . % pvp (poly vinyl pyrollidone). there was fold increase in proliferation index of spleenocytes and releases pico-gram of interferon gamma after week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by % increase in cd and . % in ccr expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b s. by cell cycle analysis we determined that b s is able to induce apoptosis in up to % of jurkat and molt- t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase is the first to be cleaved, followed by caspases and , thus suggesting that b s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd , cd and cd , and of the inflammatory cytokines il- , il- and mcp- . using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th and th /th skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd t cell responses. microcapsule based vaccination resulted in a marked induction of il- secreting th cells, without inducing strong th (cfa) or th (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg , igg c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd , cd and cd and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il- and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr -dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr -defective c h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il- b secretion by dc, through its effects on caspase- processing, which is also tlr -independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc (st ) or lactobacillus rhamnosus ncc (lpr), each at x cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week to week of life. fresh feces were collected at , and weeks of life for determination of iga levels (assessed by elisa). pups were bled and weeks after immunization for determination of measles-specific igg and igg a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st -fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, ( - )- -amino- -deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at : ration and washed times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw . by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of extracts from euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc /propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that up to euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd + cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il- and ige serum levels were increased on animals from group i when compared to control.the enhancement on th immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd + ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd +t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg a and igg b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren , , n. almqvist , a. lönnqvist , s. Östman , c. rask , e. telemo , a.e. wold university of göteborg, department of clinical bacteriology, göteborg, sweden, university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; . mg/ml) in the drinking water for days and, days later, mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin- and interleukin- . examination of gut sections from sea treated donor mice revealed increased density of cd a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp , proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna , a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp , or groel together with cpg-dna reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp together with cpg-dna . the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp microspheres induced protective ifng-secreting cd + t-cells and raised pulmonary pmp -specific iga levels in vivo. also, pmp microspheres caused lower il- serum levels upon administration than the injection of pmp together with cpg-dna , indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in cases of s. aureus bacteraemia in iv drug users and cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen ).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day ) and four weeks thereafter (day ). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter ) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st (spa-type t , agr , and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p= . ).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at and years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in and ds children, respectively. samples were taken before and - weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg , igg and igg ). at years, ds children had lower postvaccination geometric mean igg anti-tt-titers only. post-vaccination igg -anti-tt avidity levels were decreased in / and / ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. kda; , kda; , kda; , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda and , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an -month period. sera were obtained from healthy subjects from the same community ( months to years of age). the highest incidence of salmonella-associated diarrhea, / , occurred in children under years of age. the lowest incidence, / , was observed in the population aged to . whereas serum from individuals ranging - years of age showed maximum igg , igg and iga anti-s. typhimurium titres, children less than years-old did not show detectable igg and igg titres and had weak igg , iga and igm antibody levels; only their igg levels were comparable to those detected in adults. moreover, the levels of igg and igg antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs - , a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses ( %), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a- , linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after -fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková , s. bystrický institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd , expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age - years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg , igg and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after to days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers : ). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones ( : vs. : ). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was . fold of normal control). subcutaneous immunization gave a weaker stimulation: . fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv - and vega / / grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a- , -linked d-mannopyranose units and many branches composed of a- , a- , and/or b- , -linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant ( mg oligosaccharide per one conjugate dose) two times in days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs ( g- g) were treated orally with mg respivax five consecutive days. after the last application, on days , , , , and six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on mm thick serial sections, stained with hemalauneosin. the populations of cd , cd and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd positive cells, which number reached maximum at the end of the second week. on day b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd and cd positive cells. on days and the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta , s. majumdar bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein- (ip- ) and interleukin- (il- ) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip- mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase (inos ) expression and was linked to the mapk signaling pathway via antagonistic regulation of p mapk and erk / . further, ip- was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th cytokines and no. thus this study strongly demonstrates that ip- , like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th /th cytokines mediated by cd + t cells and activating cd + t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x . ) following immunization and after challenge with leishmania major. il- values were increased in all groups, but there was no statistical difference between the groups(p g . ) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x . ) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x . ) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x . ). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at week intervals. three weeks after booster injection, × stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg a/igg was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin (il- ), and cellular expression of cd and cd . results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd + lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with , infective n. brasiliensis larvae on day post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg and igg a). this was independent of level of me supply. feeding regime did not affect levels of the type- cytokines il- and il- . conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp of l.major strain mrho/ir/ /er. methods: l.major promastigotes were grown in rpmi supplemented with % fcs. l.major rna extraction and cdna synthesis were carried out. gp gene segment was amplified by specific primers and cloned into ptz r to construct ptz r/gp . the presence of gp into ptz r was confirmed by pcr. then, ptz r/gp was sent to determine the sequence of its nucleotides. after that the gp gene segment was sub-cloned into pet a (+) expression vector and transformed into e.coli bl (de ) plyss and gp protein was expressed in presence of mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, ) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including volunteers. male volunteers ( and years) for the adult group, and children of both sexes ( - years) were enrolled. subjects received virosomal formulations containing mg of ama -c (pev t), an apical membrane antigen- derived synthetic phospatidylethanolamine (pe)-peptide conjugate and ug of uk (pev t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days , and . results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling % of the treated mice to survive till days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only . % of the amount fed and it remained in circulation in the blood only for minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to . % ( of the amount fed but it's circulation was sustained till hrs post feeding. under such conditions when mgm of curcumin bound to mgm of chitosan nano particles were fed one time daily for days post infection to plasmodium yoelii infected mice % of mice were cured and survived atleast for days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma vdelta t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il- responses, to epitopes on the dominant rbc autoantigen, anion exchanger- (ae- , or band ) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla- + regulatory t cells or soluble forms of ctla- , may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =- . ) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b (r medium =- . ) and il- (r medium =- . ) in isolated splenic hscs ex vivo. analysis of effector cd + t cells in spleen showed decrease of t h cells quantity and simultaneous t h cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd + cd + ctla- + -and cd + cd -il- + il- regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h , t h and regulatory t cells were correlated (r th =- . , r th = . and r th = . and r tr = . ) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th = . and r tr = . ) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b -and il- -produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p , jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin- , t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il- , as compared to non-infected controls. the infection also altered the effect of il- on mapk activation by preventing its stimulating effect on p mapk. moreover, in s.obvelata-infected animals il- markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il- induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. .-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from ⁄ to / with a normal serum pool. .-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r g . . the analytical range was from g/l to g/l. the coefficient of variation (cv) was x % for [mc] n g/dl, and x % for [mc] x g/dl. this procedure was successfully implemented to quantify the mc in serum samples between march and february, . among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: . we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. . the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from day and reached plateau level after day of teratocarcinoma insertion. moreover, cd + cd cells showed in spleen and main lymph nodes from day and achieved plateau level after day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at day and had a maximum pick at day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than , times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b , il- , pge and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number = . and r activity = . ) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd -ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd l is cleaved to soluble (s)cd l. we sought to examine the levels of scd l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd l -along with other products -were assayed by quantitative elisa. levels of il , cd p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l . to examine if scd l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il- secretion. the il- concentration was consistently below - pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il- . the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il- production over control (p x . ) at d after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l (p x . ). pre-incubation of b cells with cd -blocking antibodies substantially abrogated il- secretion, unlike isotype-matched control. the partial blocking of cd binding on cd + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd l (under investigation) and indicates a sustained role for pc-derived scd l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule- (dnam- ) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from aml patients before specific anti-leukemia therapy and healthy donors. all results were analyzed statistically using spss version . results: aml patients under years showed a significant reduction in the expression of dnam- , nkp and nkp compared with age-matched controls. both healthy individuals and aml patients older than years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam- on nk cells and its ligand cd on aml blasts has been found in aml patients under years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam- expression on aml patients and confirm previous reports showing a significant decrease of nkp and nkp on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel , e. gorska , u. demkow , m. wasik medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for or hours with or without dexamethasone in concentration - m. analysis of: p-gp surface expression, p-gp function (rhodamine test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was , %± , . after hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine efflux, which characterized p-gp activity, was , %± , . after hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine ( , %± , , p= , ). average phi in lymphoblasts was , ± , . acidification of cells incubated hours with dexamethasone was seen in - % percentage of cells ) . rest of lymphoblasts showed alkalization (phi - , ).the percentage of lymphoblasts in early stage of apoptosis after hours incubation with dexamethasone (annexin-v test) was higher than in control cells ( , % vs , %; p= , ). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd d/cd integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd and anti-cd (clon huts ) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures ( hours). results: cd active form was expressed in the majority of normal and tumoral bm pc from healthy subjects ( . ± . , n= ) and mm patients in the early stages of the disease ( . ± . , n= ). in these cells, huts epitope was clearly upregulated by mn + . in contrast, circulating pc were almost all huts negative, and levels did not significantly augment when these cells were exposed to mn + . moreover, not only pb but also bm malignant cells from pcl patients were also huts negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index ( . ± . brdu+ cells, n= ) in comparison to pc from mm patients in the first stages of disease ( . ± . brdu+ cells, n= ). these results suggest that the active form of cd must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either % fetal bovine serum (fbs) with and without fgf ,or % human platelet lysate (hpl), %hpl, ( % fbs + % hpl), ( % fbs + % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day ). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: ) ten cases with fa diagnosed on the basis of dna breakage analysis, ) ten cases with aaa, and ) ten normal control cases. the presence of p dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p dna was demonstrated in bone marrow of % of children with fa, compared to % in children with aaa (p x . ), while, no p dna was seen in normal control. a positive correlation between dna breakage and presence of p dna was seen in bone marrow from fa (p x . , r . ). the presence of p tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord- -f b t ts authors: kabeerdoss, jayakanthan; danda, debashish title: understanding immunopathological fallout of human coronavirus infections including covid‐ : will they cross the path of rheumatologists? date: - - journal: int j rheum dis doi: . / - x. sha: doc_id: cord_uid: f b t ts severe acute respiratory syndrome coronavirus (sars‐cov‐ ) infection causing coronavirus disease (covid‐ ) is the biggest pandemic of our lifetime to date. no effective treatment is yet in sight for this catastrophic illness. several antiviral agents and vaccines are in clinical trials, and drug repurposings as immediate and alternative choices are also under consideration. immunomodulatory agents like hydroxychloroquine (hcq) as well as biological disease‐modifying anti‐rheumatic drugs (bdmards) such as tocilizumab and anakinra received worldwide attention for treatment of critical patients with covid‐ . this is of interest to rheumatologists, who are well versed with rational use of these agents. this brief review addresses the understandings of some of the common immunopathogenetic mechanisms in the context of autoimmune rheumatic diseases like systemic lupus erythematosus (sle) and covid‐ . apart from demographic comparisons, the role of type i interferons (ifn), presence of antiphospholipid antibodies and finally mechanism of action of hcq in both the scenarios are discussed here. high risks for fatal disease in covid‐ include older age, metabolic syndrome, male gender, and individuals who develop delayed type i ifn response. hcq acts by different mechanisms including prevention of cellular entry of sars‐cov‐ and inhibition of type i ifn signaling. recent controversies regarding efficacy of hcq in management of covid‐ warrant more studies in that direction. autoantibodies were also reported in severe acute respiratory syndrome (sars) as well as in covid‐ . rheumatologists need to wait and see whether sars‐cov‐ infection triggers development of autoimmunity in patients with covid‐ infection in the long run. there are demographic, immunological and therapeutic similarities and dissimilarities between hcov infections and autoimmunity. in general, adult women have stronger immune response and they are protected more often from infectious disease compared to men of similar age. women appears to have robust antimicrobial immune responses, especially against viral infections. x chromosomes and sex hormones are thought to be responsible for this phenomenon. in addition, negative regulators of immune response are less marked in woman as compared to men, for example lower number of circulating t-regulatory cells and lower expression of immune checkpoint inhibitors like pd-l in t-cells of women. coronavirus strains sars-cov and sars-cov- utilize angiotensin i converting enzyme (ace) as a receptor for entry into host cells. ace is differentially expressed in different organs; high levels are reported in the small intestine, colon, heart, muscle, kidney, testis and moderate levels in lungs. expression of ace is also higher in males compared to females, especially in liver and lung tissues even though its gene is present in the x chromosome. ace activity and expression is regulated by β-estradiol. messenger rna (mrna) expression of ace correlates with immune signatures in lungs and it is dependent upon age and gender. there is positive correlation between ace expression and immune signatures in the lungs of men and older individuals, whereas negative correlation is observed in women and younger individuals. this might be the reason for excessive immune response in the form of the cytokine storm observed in older-aged males that results in severe respiratory complications. however, sars-cov- infects both genders equally, although higher mortality is observed in males. , in an animal model studies, males were found to be highly susceptible to sars-cov infection and more severe lung pathology. mortality of male mice was higher than that of female mice and it was dependent on viral load. blocking estrogen receptor signaling, however, led to an increase in mortality even among sars-cov in- similar to animal studies, male population is predominantly susceptible to sars-cov- infection and accounts for nearly % of all cases of covid- with higher mortality. similar gender bias was also observed in mers-cov infection, although it was attributed to social activities and religious customs that involved more men than women in the middle east. african american women are at times higher risk and latino american as well as asian women are at times higher risk for developing sle than european american women. sle disease severity, number of clinical manifestations, prevalence of autoantibodies and nephritis as well as mortality are higher in african american, asian and hispanic populations as compared to the white populations. however, socio-economic and environmental background may also be confounding factors that influence ethnicity-based prevalence and phenotypic differences in sle. as on june , mortality rate (deaths/ total cases) of covid- infection in europe, north america, asia and africa are . %, . %, . % and . % respectively. however, mortality rate of covid in the usa is disproportionately higher among blacks ( . deaths per population) and hispanics/latino americans ( . deaths per population) than the white american population ( . deaths per ). , blacks, asians and minority ethnic (bame) groups are also found ethnicity also influences type i ifn secretion, as asians and african americans have higher type i ifn expression in peripheral blood cells as compared to caucasians. , this observation goes hand in hand with prevalence of lupus in these ethnicities, a predominantly type ifn-driven disease. type i ifn expression is also lower in male individuals compared to females, again keeping in line with high female predominance in lupus. body mass index (bmi) and smoking also increases type i ifn expression and inflammatory cytokines. these risk factors tend to adversely affect outcome of covid- disease as well as lupus and other systemic autoimmune diseases. [ ] [ ] [ ] these reports suggest that gender, ethnicity, bmi and smoking affect type i ifn secretion. hence, male gender, african ancestry, high bmi and smoking are risk factors for severe or critical covid- disease ; interestingly, these are also risk factors for severe lupus. this mechanism of type i ifn induction is strikingly similar to that of sle. early secretion of type i ifn by pdcs is important for controlling viral replication and preventing dissemination of virus to major organs. depletion of pdcs results in loss of antiviral type i ifn response and impaired survival of virus-specific natural killer (nk) or cd + t-cells. numbers of pdcs gradually decrease as age increases, whereas there is no change in conventional dc (cdc) cells. this may be the reason for reduced antiviral ifn responses in older age and increased mortality among the elderly due to covid- . there is nearly -fold higher secretion of type i ifn by pdcs upon recognition of mers-cov compared to that of sars-cov. this may be one of the factors contributing to the higher mortality rate observed in mers than sars and covid- . type iii ifn (ifn-λ) secretion is also higher by coronavirus-infected pdc and its levels were similar to that of type i ifn. signal in summary, both hcov-mediated diseases and sle have robust production of type i ifn. in both conditions, pdc is a major cellular source for type i ifn via tlr and cgas-sting signaling, as well as via the tlr pathway, a relatively lesser known mechanism. early response to sars-cov- infection is mediated by cd cells. dramatic reduction in the number of cd and cd t-cells during the acute phase of infection in patients of sars and covid- are uniformly reported. decrease in activated cd t cells and increase in antibody- during convalescence phase of covid- are other encountered observations. low levels of t-cells (cd +), both cd + t-cells and cd + t-cells, are also associated with severity and hospital death in covid- . , , neutrophil to lymphocyte ratio as a predictor for severity in covid- has also been studied. type i ifn is required for activation of cd th cells that sustain antiviral response of cd ctls. in parallel, type i ifn is also required for differentiation of tfh cells that mediate b cell differentiation and antibody production. therefore, type ifn are crucial for immediate and long-term protection against covid- . wide variability in cytokine secretion patterns that is noticed among patients with sars and covid- determines the course of disease. pre-existing comorbidities of these patients also synergistically alter cytokine levels and decide outcome. ards, disseminated intravascular coagulation and multiple-organ failure rapidly progress in severe covid- patients, leading to death within to days of intensive care unit admission. increased infiltration of neutrophils and macrophages as well as secretion of high levels of pro-inflammatory cytokines result in a condition called cytokine storm. cytokine storm could be the leading cause for respiratory complications and multi-organ failure in patients with covid- . reduced lymphocytes, increased cytokine levels and abnormal coagulation parameters are frequent in these cases. reduced ifn levels and increased viral load are higher in critical and severe cases as compared to mild to moderate patients with covid- . in view of diminished lymphocyte and dendritic cell function, neutrophils and macrophages take over antiviral defense response. interleukin (il- ) and tumor necrosis factor alpha (tnf-α) are major pro-inflammatory cytokines secreted by these cells that induce tissue damage, eventually leading to alveolar flooding and fibrosis. cytokine storm is also a relatively common complication in pa- age-dependent altered innate immune response to hcov was studied in a mice model. young mice infected with sars-cov efficiently cleared the virus. in contrast, aged mice showed exacerbated immune response to virus with increased lymphocyte infiltration in lungs. during initial infection in young mice, activation of innate immune cells, namely pdc, macrophages and nk cells were involved in viral clearance. however, this response was not effective in aged mice to contain the virus; instead, a robust cytokine storm and altered lung pathology followed the immunological war against the virus in older mice. in a macaque model of sars-cov infection too, aged macaques had more severe lung pathology, lower expression of type i ifn and higher expression of pro-inflammatory cytokines as compared to younger macaques. as mentioned earlier, sars-cov infects hosts equally across all age groups, but complications and fatality are noted much more commonly in older populations. early type i ifn response is important for preventing hcov-mediated inflammation and severe disease. both ifn secretion from innate cells like pdc and response threshold by its receptor in t-cells are impaired in older age as compared to young individuals. , , delayed ifn response causes apoptosis of t-cells and recruitment of monocytes and neutrophils, leading to cytokine storm and lung injury. , inflammation in the form of chronic subclinical systemic inflammation and immune senescence, that is reflected by blunted and impaired immune response, are other factors contributing to age-related differential covid- pathogenesis. on the other hand, sars-cov- -associated multisystem inflamma- kawasaki-like diseases have also been reported in some immunodeficiency states. it is likely that children with mis-c may have some underlying immunodeficiency state that is triggered into an auto-inflammatory syndrome by covid- infection; only future studies can reveal exact immunological mechanisms behind this unique mimic of kd. during recent times, hcq has been subjected to huge discussions in relation to its benefit and harm in covid- ; its role in autoimmune diseases like rheumatoid arthritis (ra) and lupus are already established. antimalarial agents, chloroquine (cq) and hcq are drugs of choice for various connective tissue diseases. both are immunomodulatory agents; unlike immunosuppressants, these drugs are safer for patients with chronic diseases. role and mechanism of hcq in management of autoimmune rheumatic diseases is discussed in a recent review. here we discuss its perspectives in covid- ( figure ). cq and hcq not only interfere with tlr / -mediated signaling, there is evidence that it has an effect on tlr also. cq inhibit ifn-β secretion and phosphorylation of stat in human mesangial cells treated with tlr agonist polyinosinic-polycytidylic acid (poly i:c). this may be one more effective mechanism of cq and hcq in treatment of lupus nephritis patients. as mentioned above, tlr also recognize sars-cov dsrna and activates transcription factors in a small randomized controlled trial, treatment with hcq was associated with reduced viral load and improvement in radiological progression on computed tomography images in patients with covid- . however, another clinical study involving patients showed failure of hcq to clear viral load and no clinical benefit. in an observational study, administration of hcq in severe covid- patients has shown no benefit in preventing intubation or death. in other randomized and clinical observational studies, hcq was not shown to be beneficial for patients who are transferred to the intensive care unit. these studies were different in terms of dose of medication, day of starting treatment, primary endpoint and selection bias of severity (table s ). therefore, randomized clinical trials with larger sample size is warranted for testing its efficacy. on the other hand, hcq has been recommended as prophylactic regimen specifically for healthcare workers in india. to patients with covid- that it is a mild immunomodulatory f i g u r e hydroxychloroquine (hcq) inhibits sars-cov- entry and inhibits virus-induced type i interferon (ifn) signaling and proinflammatory cytokines production. here are the various pathways: . angiotensin i converting enzyme (ace ) is an inducible gene. hcq inhibit type i ifn, thereby inhibit ace expression. also hcq may inhibit n-terminal glycosylation of ace . . hcq can also inhibit viral entry by disrupting endosomal acidification. . hcq alters endosomal ph, there by disrupts ligand binding to toll-like receptor (tlr ) and tlr . . hcq inhibit cgas-sting (stimulator of interferon genes) signal and thereby reduce type i ifn and pro-inflammatory cytokines expression agent and not an immunosuppressant. while its use in hospitalized covid- patients are not yet proven to be beneficial, several developing nations are using it in early disease and as a pre-exposure prophylaxis for frontline healthcare workers exposed to sars-cov- , as recommended by indian council of medical research (icmr) (table s ). presumable viral etiologies have been known for many connective tissue diseases. we might very well expect sars-cov- triggered development of autoantibodies. some available evidence to support this notion are discussed in the following paragraphs. injection of convalescent sera from sars patients to rhesus macaques also causes lung injury with similar histopathological features as in human disease. thus convalescent sera containing autoantibodies or other antiviral antibodies might cross-react with antigens expressed in the lung. this has to be kept in mind while recommending over enthusiastic use of convalescent sera/plasma therapy in critically ill covid cases. high titers of anticardiolipin antibodies are also seen in sars patients presenting with osteonecrosis. in vitro experiments also reveal that sera of sars patients contain autoantibodies targeting pulmonary epithelial cells and endothelial cells. another in vitro experimental study demonstrated anti-s spike antibodies in sera of sars patients that can bind to epithelial cells inducing cytotoxic injury. sera from patients with autoimmune diseases like mixed connective tissue disease, and ra were found to have higher positivity for anti-sars-cov igg and igm as compared to healthy controls. false positivity was also reported for anti-sars-cov antibodies in patients with sle. cross-reactivity between anti-sars-cov antibodies and autoantibodies targeting the same antigenic target is possible. however, autoantibodies in sars specifically bind to antigens expressed in lung tissue, and are not expected against cell nuclei (like ana), against smooth muscles (like sma) or against parietal cells (like pca). these studies show that both sars-cov and sars-cov- infections might induce expression of ana and apl antibodies. further studies are needed to delineate the spectrum and pattern of developing autoantibodies in patients with covid- . systemic vasculitis was noted in an autopsy study from patients who died from sars. simultaneous diagnosis of covid- and kd was also made in a -month-old child. outbreak of kawasaki-like diseases is reported in children, of them were positive for either sars-cov- by nasal swab or antibodies. pediatric patients who were positive for sars-cov- igg also developed cutaneous vasculitis lesions. development of cutaneous small vessel vasculits was seen in elderly female covid- patients also on the th day after onset of symptoms. sars-cov- infects endothelial cells and induces apoptosis as well as pyroptosis resulting in multi-organ dysfunction. these show hcov not only targets lungs, but can infect blood vessels, thereby causing multi-organ damage. in summary, ana and apl autoantibodies can be seen in patients with sars and covid- . theoretically, these patients may have higher chances to develop autoimmune diseases in future, like aps or a lupus spectrum disorder. rheumatologists will have to wait for the post-covid- era to witness any unfolding of events towards a rising prevalence of lupus, vasculitic process or aps. complications and fallouts of covid- disease have some similarities as well as dissimilarities with autoimmune diseases like sle. while male gender, older age and people with metabolic syndrome seem to be at a higher risk of contracting more severe sars-cov- infection, younger females of african and asian ancestry have higher risk for developing sle; male gender among lupus patients, however, is an independent risk factor for severe disease. johns hopkins coronavirus resource center clinical characteristics of coronavirus disease (covid- ) in china: a systematic review and meta-analysis sex differences in immune responses sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor landscape of x chromosome inactivation across human tissues estrogen regulates the expression of sars-cov- receptor ace in differentiated airway epithelial cells expression of the sars-cov- cell receptor gene ace in a wide variety of human tissues suppressed t cell-mediated immunity in patients with covid- : a clinical retrospective study 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mcp- and il- ra during sars-cov- infection is associated with disease severity and fatal outcome. medrxiv complex immune dysregulation in covid- patients with severe respiratory failure clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study on the alert for cytokine storm: immunopathology in covid- potential effect of blood purification therapy in reducing cytokine storm as a late complication of critically ill covid- genomic analysis reveals age-dependent innate immune responses to severe acute respiratory syndrome coronavirus exacerbated innate host response to sars-cov in aged non-human primates inflamm-aging: why older men are the most susceptible to sars-cov- complicated outcomes an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study clinical characteristics of children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov- paediatric multisystem inflammatory syndrome temporally associated with sars-cov- mimicking kawasaki disease (kawa-covid- ): a multicentre cohort pediatric kawasaki disease and adult human immunodeficiency virus kawasaki-like syndrome are likely the same malady coagulopathy and antiphospholipid antibodies in patients with covid- novel paediatric presentation of covid- with ards and cytokine storm syndrome without respiratory symptoms characteristics of ischaemic stroke associated with covid- lupus anticoagulant is frequent in patients with covid- lupus anticoagulant and abnormal coagulation tests in patients with covid- neutrophil extracellular traps in covid- covid- and apl ab -hematology.org [internet mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology chloroquine attenuates tlr /ifn-β signaling in cultured normal human mesangial cells: a possible protective effect against renal damage in lupus nephritis poly(i:c) induces human lung endothelial barrier dysfunction by disrupting tight junction expression of claudin- small molecules targeting the innate immune cgas-sting-tbk signaling pathway cutting edge: antimalarial drugs inhibit ifn-β production through blockade of cyclic gmp-amp synthase-dna interaction sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro chloroquine is a potent inhibitor of sars coronavirus infection and spread a pilot study of hydroxychloroquine in treatment of patients with moderate covid- . zhejiang xue xue bao yi xue ban no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection observational study of hydroxychloroquine in hospitalized patients with covid- clinical efficacy of hydroxychloroquine in patients with covid- pneumonia who require oxygen: observational comparative study using routine care data hcq) as prophylaxis for sars-cov- infection (in supersession of previous advisory dated clinical course of coronavirus disease (covid- ) in a series of patients with systemic lupus erythematosus under long-term treatment with hydroxychloroquine human line endonuclease domain as a putative target of sars-associated autoantibodies involved in the pathogenesis of severe acute respiratory syndrome long interspersed nuclear element- retroelements are expressed in patients with systemic autoimmune disease and induce type i interferon high prevalence of antinuclear antibodies and lupus anticoagulant in patients hospitalized for sars-cov pneumonia safety of convalescent sera for the treatment of viral severe acuterespiratory syndrome: an experimental model in rhesus macaque osteonecrosis in patients after severe acute respiratory syndrome (sars): possible role of anticardiolipin antibodies autoantibodies against human epithelial cells and endothelial cells after severe acute respiratory syndrome (sars)-associated coronavirus infection antibody to severe acute respiratory syndrome (sars)-associated coronavirus spike protein domain cross-reacts with lung epithelial cells and causes cytotoxicity cross-reaction of sars-cov antigen with autoantibodies in autoimmune diseases analysis of false-positive associated with antibody tests for sars-cov in sle patients detection of autoimmune parameter of sars patients. zhonghua shi yan he lin chuang bing xue za zhi zhonghua shiyan he linchuang bingduxue zazhi the clinical pathology of severe acute respiratory syndrome (sars): a report from covid- and kawasaki disease: novel virus and novel case images in practice: painful cutaneous vasculitis in a sars-cov- igg-positive child cutaneous small-vessel vasculitis associated with novel coronavirus sars-cov- infection (covid- ) endothelial cell infection and endotheliitis in covid- key: cord- -wyanwvxa authors: sen, adrish; ding, siyuan; greenberg, harry b. title: chapter the role of innate immunity in regulating rotavirus replication, pathogenesis, and host range restriction and the implications for live rotaviral vaccine development date: - - journal: mucosal vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: wyanwvxa abstract rotaviruses (rvs) are important causative agents of viral gastroenteritis in the young of most mammalian species studied, including humans, in which they are the most important cause of severe gastroenteritis worldwide despite the availability of several safe and effective vaccines. replication of rvs is restricted in a host species-specific manner, and this barrier is determined predominantly by the host interferon (ifn) signaling and the ability of different rv strains to successfully negate ifn activation and amplification pathways. in addition, viral attachment to the target intestinal epithelial cells also regulates host range restriction. several studies have focused on the role of the innate immune response in regulating rv replication and pathogenesis. the knowledge accrued from these efforts is likely to result in rational attenuation of rv vaccines to closely match circulating (and host species-matched) virus strains. in this chapter, we review prevalent models of rv interactions with innate immune factors, viral strategies employed to regulate their function, and the implications of these findings for improved rv vaccine development. rotaviruses (rvs) remain one of the two most important viral causes of gastroenteritis despite the availability of several safe and effective live attenuated vaccines [ , ] . rotavirus infection has its biggest health impact on children under the age of years, in whom it still accounts for approximately , deaths annually, almost entirely in less-developed countries [ ] . rvs can infect many cells of the nonimmune host, but the overwhelming bulk of viral replication occurs in the mature villus tip cells of the small intestine [ ] . in this review, we focus on the regulation of rotavirus replication by the host innate immune system, the host-restricted nature of the innate immune response to specific rotavirus strains, and the practical utility of these host range barriers in the development of safe and effective rv vaccines. infection with rv results in the immediate activation of a conserved cellular innate immune signaling pathway that involves multiple pattern recognition receptors (prrs) recognizing discrete rv-encoded pathogen-associated molecular patterns (pamps). a primary purpose of this diverse host-signaling system is to induce different types of interferons (ifns) and a set of virus-induced stress genes (visgs) through two principal transcriptional factors: nuclear factor-κb (nf-κb) and ifn regulatory factor (irf ) [ , ] . the induced ifns and visgs then function to restrict rv replication and pathogen-induced cell injury [ ] . of note, rvs, like virtually all other viral pathogens, have evolved a set of countermeasures to inhibit the host innate immune response, and these countermeasures are most pronounced during homologous rv infection (rv infection with a strain routinely isolated from that specific host species) [ ] . interestingly, rv strains that differ in their ability to regulate the secretion of ifns similarly induce this early recognition pathway, as indicated by the transcriptional upregulation of ifns and several visgs [ ] . based on the collective evidence, initial rv transcription engages the two related prrs rig-i and mda- (members of the family of rig-i-like receptors, or rlrs) [ , ] , which then trigger activation of the mitochondrial antiviral-signaling protein (mavs). these receptors are likely to be stimulated by early rv transcriptional by-products such as exposed -phosphate groups, incompletely methylated -cap structures, and local dsrna structures such as panhandle loops in viral transcripts [ ] . in addition to inducing the secretion of different ifns, rlr responses to rv are likely to orchestrate other host responses. rotavirus activation of mda- results in apoptosis, which occurs mostly in the pancreas of rv-infected mice, indicating that such prrdependent consequences can occur in a cell or organ type-specific fashion [ ] (chapter : innate immunity at mucosal surfaces). in addition to rig-i-and mda- -dependent host responses to rv rna, other sensors are also recruited by the innate immune machinery to trigger early anti-rv responses. among these is a third member of the rlr family: lgp , which seems to exert a proviral effect on rv replication [ ] and whose activation during rv infection may represent a viral strategy to dampen this pathway. yet another player in the innate recognition of rv is the dsrnadependent protein kinase pkr, which is essential for rv-infected cells to secrete ifn [ ] . the molecular basis for pkr's role during rv infection is not well understood, but given the importance of pkr in antiviral signaling in general and its inhibition by a majority of viruses, pkr is likely to be important for rv pathogenicity. distinct from the cytosolic receptors discussed above, rv recognition also involves the toll-like receptors (tlrs), a class of viral receptors that function in the context of cellular membranes, including surface and endosomal membranous components. this aspect of innate rv recognition possibly reflects the rv entry process that exploits endosomal vesicle transport to gain access into host cells. so far, tlr , tlr , tlr , and tlr have all been implicated as potential players in the innate rv detection cascade [ À ] . the ability of tlr to recognize and regulate rv and thus perpetuate an antiviral effect has been tied to tlr 's agedependent expression in the intestine [ ] . since rv typically causes severe disease and replicates in the intestine of infants and young children (in many mammalian species), coincidental with lower levels of tlr expression in infants [ ] , one possible implication is that age-restricted rv intestinal replication is partly due to enhanced tlr signaling with age in this mucosal compartment. other tlrs play specialized roles in discrete types of cells during rv infection. the rvencoded enterotoxin nsp may function as a pamp and in macrophages triggers inflammatory cytokine secretion by a tlr -dependent pathway [ ] . during rv infection in human enteroid cultures [ ] and in different species of mammals [ À ], different types of ifns are secreted, and as will be discuss below, antiviral actions of these ifns are actively countered in a host-range-specific manner by pathogenic rvs. of the ifns, type i ifn is mostly expressed in the intestinal hematopoietic cell compartment rather than in the epithelium where rv primarily replicates [ ] . studies to date have implicated tlrdependent signaling in dendritic cells in the type i ifn secretion process during rv infection [ , , ] . infection of plasmacytoid dendritic cells with rv, which are a major source of type i ifn secretion during viral infections, leads to endosome-dependent (and possibly tlr -mediated) type i ifn secretion that is triggered by viral genomic dsrna (or, potentially, a rv structural protein) [ , , , ] . a central role for tlr-dependent defense against rv is also indicated by the finding that the absence of myd , an essential convergent adaptor in signaling from the different tlrs, results in increased rv infectivity, severity of diarrhea, and impaired humoral immunity [ ] . in addition, rv is susceptible to the antiviral effects of tlr , since activation of this receptor by bacterial flagellin prevents or cures rv infection by a process that involves the secretion of il [ , , ] . inflammasomes are cytosolic multiprotein complexes that remain quiescent at resting state [ ] . upon activation, apoptosis-associated speck-like protein containing card protein, named asc (encoded by pycard) and caspase- (encoded by casp ), oligomerize and mediate the proteolytic processing of proinflammatory cytokines such as pro-il- β and pro-il- and the pore-forming protein gasdermin d, ultimately leading to a lytic form of cell death known as pyroptosis [ ] . these events not only contribute to the host defense against bacterial and other microbial infections, but also regulate the homeostasis of the immune system and the development of various inflammatory diseases and cancer [ ] . although it is known that aim and ifi inflammasomes recognize dna viruses and that nlrp inflammasome responds to general stress and breach of plasma/endosomal membrane integrity [ , ] , how inflammasomes control rna virus replication is less well understood. in addition, whether noncanonical inflammasomes operate in cell types other than myeloid cells is largely unknown. recently, we found that oral infection of suckling mice with murine rv-induced robust activation of casp in the small intestinal tissue, indicating a potential role for inflammasomes in rv pathogenesis [ ] . of note, in contrast to other nod-like receptors, including nlrp , nlrp , nlrc , and naips, targeted deletion of nlrp b in intestinal epithelial cells (iecs) of suckling mice led to an increase in diarrhea, rv shedding in the feces, and intraintestinal viral replication compared to wild-type pups, highlighting a crucial role of nlrp b in controlling rv replication. mechanistically, we found that during rv infection, dexh-box helicase (dhx ) binds to viral rna pamp and interacts with nlrp to activate the downstream signaling pathway ( fig. . ) . furthermore, primary mouse intestinal enteroids generated from dhx -or nlrp deficient mice produced less il- and underwent less pyroptosis compared to wildtype enteroids upon rv infection, confirming a role for dhx in the activation of the nlrp b inflammasome during rv infection [ ] . identification of the dhx -nlrp b-asc-casp cascade as a novel rv-sensing pathway opened up new research directions. are there other inflammasome sensors of rv or other enteric viruses? how do different rnabinding proteins (dhx , rig-i, mda- , etc.) coordinate in the cytoplasm? what is the physiological relevance of theses sensors in the human intestine? addressing these fundamental issues will provide new insights into the biological functions of host innate immune recognition during acute rv infection and, more generally, in overall human enteric health and disease. in addition, answering these basic questions is likely to inform more practical considerations, such as the development of better therapeutics and preventive strategies for enteric infectious diseases. the concept of host range restriction (hrr) is central to many aspects of rv replication and disease, including the development of several of the currently available safe and effective rv vaccines [ ] . rvs are distinguished by being highly pathogenic and infectious to their homologous host species (i.e., the species of host normally infected by the particular rv strain and the species in which that rv strain spreads efficiently) [ ] . rvs are also subject to very severe species-specific restriction of replication and transmission in heterologous host species [ ] . these fundamental properties of rvs are not only of great importance for viral pathogenicity. they also form the basis for several licensed live attenuated orally administered rv vaccines (which are attenuated by virtue of their hrr). in traditional continuous cell line culture systems, most rv strains efficiently block the induction of type i ifn and have evolved to target several different host factors that regulate the ifn pathway [ ] . this multipronged subversion of the ifn response is accomplished primarily by the versatile rv nonstructural nsp protein, the product of rv gene (see below). most ifn-sensitive rv strains encode forms of nsp that exhibit defective ifn inhibition and therefore elicit enhanced ifn secretion [ À ] . although such strains are still viable infectious agents, their ability to replicate is substantially hampered. in addition, ifn sensitivity of rvs encoding full-length "functional" nsp proteins also occurs in specific cell lines, possibly reflecting nsp 's inability to target host innate factors across different species [ ] . enteric infection of suckling (i.e., -to -dayold) mice with a homologous murine rv compared to a heterologous simian rv strain reveals a substantial (b À log) host restriction of the simian rv replication in the intestine [ , , , ] . this host restriction phenotype is substantially reduced (down to log or less) in mice lacking and three ifn receptors (ifnrs) or stat , a key transcription factor relaying antiviral signals from different ifnrs [ , , , ] . the replication phenotype strongly cosegregates with the genetic origin of the vi. mucosal vaccines for viral diseases murine or simian rv nsp encoding gene segments. the suckling mouse thus presents a highly tractable model in which ifn-specific effects on rv replication can be studied within the biologically relevant framework of intestinal rv replication in a natural host and in a host-range-restricted manner. ectopic parenteral injection of purified ifn types i, ii, or iii in many species, including mice, results in the activation of the key downstream transcription factor stat in small iecs (the predominant site of rv replication) [ ] . multiple lines of evidence indicate important roles for ifn types i, ii, and iii in restricting rv replication in the gut and in cell culture [ , , , , ] . in mouse embryonic fibroblasts lacking both types i and ii ifnrs, the replication of several nonmurine rv strains is substantially enhanced (by four to five orders of magnitude). in suckling mice lacking the types i, ii, and iii ifnrs (either singly or in combination) significant enhancement of simian rv intestinal replication occurs demonstrating the sensitivity of heterologous nonmurine rvs to different ifn types in the mouse gut [ , , , ] . in contrast, replication of the homologous murine rv is quite resistant to these ifns (b log or less replication gain). the collective evidence thus highlights the important role of different types of ifns (and their inhibition by the rv nsp protein) in rv pathogenicity and attenuation [ , , , , ] . deciphering the mechanisms underlying these interactions is key for rational rv strain attenuation and designing improved third-generation live attenuated rv vaccines. in a manner analogous to that of other rna viruses, rv-induced ifn activation is dependent on an intact rna sensing pathway [ ] . postrecognition of viral rna by the cytoplasmic sensors rig-i and/or mda- , epithelial cells activate the mavs, a mitochondriaresident adaptor protein that is alternatively known as ifn-β promoter stimulator (ips- ), card adapter inducing ifn beta (cardif), or virus-induced signaling adapter (visa) [ À ] . mavs serves as a central hub in the ifn induction pathway by activating further downstream kinases including tank-binding kinase (tbk ) and inhibitor of kappa light polypeptide gene enhancer in b cells, kinase epsilon (ikk-ε) that phosphorylate irf and nf-κb, respectively [ ] . these molecules translocate into the nucleus upon phosphorylation and function as transcription factors, which ultimately leads to the expression of different ifns and the activation of ifnstimulated response elements (isres). in addition to irf , irf has been characterized as an important transcription factor for type i ifn induction in immune cells, in particular dendritic cells [ ] . similar to irf , irf undergoes phosphorylation and subsequent translocation into the nucleus in response to rv infection and activates ifn expression by functioning as transcription factors. to block such an important pathway, the rv-encoded nsp protein efficiently degrades both irf and irf in a virus-strain-dependent manner [ , ] . this process takes place first through the recognition of irf using an ellis motif localized at the c-terminal end of nsp present on the nsp molecule derived from simian, murine, and some other nonhuman rv strains [ ] . the nsp Àirf interaction subsequently results in a rapid and efficient degradation of irf at the proteasome and suppression of ifn production in rv-infected cells (fig. . ) . besides the irf family members, nf-κb has been shown to be another key arm of the host innate immune response downstream of mavs in many virus-infected cells [ ] . nf-κb signaling is robustly activated by virus infection as well as proinflammatory cytokines, including il- β and tnf-α, the latter of which has recently been shown to be directly antiviral against rv [ ] . for rv infection of ht- cells, the secretion of il- is dependent on the nf-κb activation [ ] . in a suckling mouse model, other chemokines such as ccl , ccl , cxcl , and gm-csf were also upregulated following rv infection [ ] , although whether these canonical nf-κb cytokines are activated through mavs or tlrs remains unknown. similar to irf , β-trcp, a critical protein essential for degrading cellular nf-κb inhibitor iκb, is also targeted by nsp for proteasomal degradation [ ] . in the case of β-trcp, the binding domain within nsp maps to a c-terminal dsgis motif in human and porcine rv strains [ ] . importantly, this is the same region as the ellis motif responsible for irf binding mentioned above. this interesting dichotomy of nsp Àsubstrate interaction may stem from the distinct contribution of irf versus β-trcp in ifn induction in different human and animal rv species [ ] . in contrast to the previous speculation of nsp as a viral e ligase due to the presence of an n-terminal ring finger domain, we recently discovered an interesting codestruction mechanism, in which nsp localizes to the golgi apparatus and hijacks the host cullin Àring box protein e ligase complex to induce the proteasomal degradation of both β-trcp and nsp itself [ ] . chemical blockade or sirna knockdown of cullin- components impaired nsp 's ability to degrade β-trcp, leading to a significant increase in the levels of β-trcp and reduced rv replication (fig. . ) . interestingly, the cullin complex did not appear to be required for nsp -mediated irf degradation, suggesting an alternative mechanism of action at work. more recent unpublished data from our lab indicate that in addition to irfs and β-trcp, mavs itself is also subject to rv inhibition. mavs levels were significantly reduced during rv infection, and this process is mediated, surprisingly, by the rna methyl-and guanylyltransferase vp protein (fig. . ) . by localizing to the mitochondria and binding to mavs through an n-terminal domain, vp induced efficiently proteasomal degradation of mavs in a host-species-and virus-strain-specific manner. mavs inhibition by vp is another striking manifestation of rv's ability to subvert host innate immune signaling. this is the first example of mavs degradation by an enteric virus, and it would be of interest to further test other enteric rna viruses such as norovirus. the antiviral ifn response to rv infection follows a biphasic pattern consisting of an pathway. rv rna pamp activates the rna sensing pathway, which is antagonized by rv-encoded proteins at multiple steps. in unpublished studies, vp has been shown to directly induce the proteasomal degradation of mavs. in addition, nsp from different rv strains degrades either irf or β-trcp, the latter of which is mediated by the host cullin Àe ligase complex. together, these rv proteins work in concert to dampen ifn induction in rv-infected cells. vi. mucosal vaccines for viral diseases initial ifn induction phase followed by a ligand-mediated (and ifn receptor-relayed) amplification phase [ , ] . as was discussed above, rvs are adept at inhibiting ifn induction and the rv nsp protein functions to degrade the essential factors β-trcp and/or irf during ifn induction in a rv strainspecific manner [ ] . interestingly, in spite of viral antagonism of ifn induction, infection with rv leads to the transcriptional induction and secretion of different ifn types in both cell culture and in vivo [ , , , , , ] . at least two factors contribute to the failure of rv to completely suppress the induction of ifn secretion. first, synthesis of the ifn antagonist nsp occurs only after viral entry, uncoating of the virion, rv transcription, and translation. during this initial infection process, several byproducts of viral transcription are generated that act as potent triggers of the ifn induction pathway [ , ] . second, rv entry into different types of cells may not always result in productive infection. for example, rv exposure to primary human pdcs results in two distinct populations of cells that differ in their level of viral infectivity [ ] . dendritic cells that are not productively infected nevertheless exhibit robust activation of the ifn induction response [ , ] . given the remarkable efficiency of ifn secretion in this cell type, they are a likely source of substantial ifn secretion from a nonepithelial cellular compartment where rv does not replicate efficiently. in suckling mice, infection with rv leads to significant induction of different types of ifns, of which the type i ifns are induced primarily in intestinal immune cells rather than being derived from iecs, where viral infectivity is maximal [ ] . the secretion of ifns from different cell types poses a unique challenge to successful viral propagation and spread to uninfected bystander iecs. this is because ifn binding to its cognate cell surface receptor activates a positive feedback loop that amplifies the expression of ifns as well as more than different ifn-stimulated genes [ ] . this ifn release then efficiently amplifies the expression of antiviral proteins targeting a variety of viral replication steps in uninfected bystander cells. each of the three major ifn types (i, ii, and iii) that are found to be induced in the intestine following rv infection is capable of mediating phosphorylation of the key convergent transcription factor stat (at y , an event that is critical for unlocking the transcriptional program resulting in an antiviral state) (fig. . ) . each of these ifn types is biologically relevant in the context of modulating rv infection and spread [ , ] . several lines of evidence indicate that rv employs potent countermeasures to subvert the antiviral state mediated by secreted ifns during initial infection [ , , , , ] . in cell culture, addition of purified exogenous ifns after rv adsorption does not significantly hamper viral replication; instead, ifn treatment of cells prior to rv infection is required to achieve efficient rv replication restriction [ ] . in the rv suckling mouse model, infection with a homologous murine rv and infection with a heterologous simian rv strain result in comparable levels of induction of different ifns from the intestine [ , ] . however, as was noted above, the presence of ifns during infection has a negligible effect on murine rv replication (b -log restriction in titer) but has a potent effect on heterologous simian rv replication ( -to -log restriction in titer) [ , ] . these observations suggest that in order to replicate successfully, homologous rvs have evolved strategies to induce resistance to the actions of different secreted ifn types in cells prior to their actual infection (bystander cells). classical reassortment genetic studies of the ifn-mediated replication phenotype of murine and simian rvs implicated a constellation of rv genes (encoding the vp , nsp , nsp , and nsp viral proteins) in determining the resistance to ifn signaling [ ] . of these, nsp is a necessary and the major determinant of efficient intestinal rv replication in an ifn-dependent fashion. direct evidence for rv subversion of the antiviral state mediated by exogenous ifns comes from the finding that rv-infected ht- cells (a human iec colonic cancer cell derived line) are able to efficiently block stat -y phosphorylation in response to exogenously added purified ifns i and ii [ , ] . using single-cell analytic techniques, ifn-mediated stat inhibition is found to occur within rvinfected cells. remarkably, stat responses to exogenous ifn ligand are also potently inhibited in rv uninfected bystander cells, which do not express any detectable viral antigen [ ] (fig. . ) . although initially described for a porcine rv strain sb -a, this bystander inhibitory effect has now been observed in vitro with several other rv strains, albeit with lower efficiency (sen and greenberg, unpublished observations). the ability of rv to block ifndependent signaling has also been observed in vivo. suckling mice infected with murine rv are able to significantly suppress iec stat -y phosphorylation and subsequent transcription that occurs in response to parenterally figure . rv regulation of the ifn amplification pathway. following infection, rv nsp efficiently blocks exogenous ifn-directed phosphorylation of stat at y , shown here for the ifnar ( ). in the virus-infected cell, rv also mediates the lysosomal degradation of receptors for types i, ii, and iii ifn by an unknown process ( ) . along with irf and irf , the viral nsp protein also proteasomally degrades irf and irf in the infected cells, which are required for optimal ifn amplification responses ( ). in addition, rv can inhibit the nuclear translocation of stat -py by an unknown mechanism ( ) . remarkably, in addition to these viral effects in infected cells, rv also potently inhibits stat phosphorylation in uninfected bystander cells in response to different types of ifns ( ) . the viral and host factors underlying this bystander inhibition of ifns are unknown. vi. mucosal vaccines for viral diseases administered purified ifns i or iii [ ] . although ifna , ifna , and ifna transcripts are induced in the intestines of murine rv-infected mice, transcriptional analysis of isolated mature villous enterocytes revealed that within this compartment (where rv replication predominantly occurs), both infected and bystander cells fail to amplify the type i ifn genes [ ] . in the villous epithelium, rv bystander cells also do not express elevated levels of transcripts encoding irf , which is upregulated in response to stimulation of cells with secreted ifns and is critical for the optimal expression of several antiviral genes. the effectors in rv-infected cells that mediate stat inhibition in bystander cells and the rotaviral factors responsible have not been identified. in contrast, rv inhibition of ifndirected stat activation in rv-infected cells is well characterized [ , , ] . recent findings demonstrate that at the single-cell level, rv infection results in the efficient depletion of type i, ii, and iii ifnrs within rv (vp antigen ) infected cells [ ] . such rv-mediated ifnr degradation is unlikely to be directed by secreted ifns (i.e., by a ligand-dependent pathway) and despite prolonged infection of cells is restricted exclusively to the subset of cells expressing vp . the depletion of ifnrs in rv-infected cells occurs from to hours postinfection (hpi) onwards by a lysosomalÀproteasomal pathway of protein degradation and is not observed in the rv (vp antigen) uninfected bystander cells, which are nevertheless highly refractory to ifn-directed stat activation (fig. . ) . the relevance of rv-mediated ifnr degradation is shown in vivo by the significant decrease in intestinal type i and ii ifnr protein expression in the small intestine following murine rv infection [ ] . degradation of different types of ifnrs by rv represents an ingenious strategy to ensure that any autocrine ifn antiviral amplification is inhibited, thus allowing viral replication and cell to cell spread to proceed efficiently [ ] . interestingly, these findings indicate the likelihood that rv targets a common hostsignaling pathway that is responsible for the expression of all three ifnrs. continuing to unravel the mechanisms by which rv also inhibits the response to different ifns in bystander cells is important for several reasons. first, since the level of rv replication substantially determines host pathogenicity, such information will enable more rational attenuation of homologous and heterologous rv strains and their use as candidate thirdgeneration live vaccines. second, for several diseases (including sepsis and systemic lupus erythematosus), an excessive ifn response is undesirable and implicated as a causative and/or exacerbation trigger of disease. in these situations, discovering novel therapeutic modalities that can dampen ifn signaling is potentially valuable. other rv strategies have also been identified that are directed at disrupting stat signaling during infection. the ability of rv to perturb stat signaling was first reported by holloway and colleagues [ , ] , who observed that as early as hpi, several rv strains inhibited the nuclear translocation of phosphorylated stat -y in response to exogenous ifn stimulation (fig. . ) . although viral factors responsible for this inhibitory effect downstream of stat activation have not yet been identified, it is possible that redundancy in rv inhibition of the stat pathway exists, perhaps indicative of the vital role of inhibiting ifn signaling in enabling rv replication. the ability of the rv nsp protein to target irfs for proteasomal degradation extends to irf and irf , two additional factors that are critical for the optimal amplification of ifndependent antiviral responses [ , ] . whereas early induction of different ifns and antiviral transcripts is mediated primarily by irf , ifnmediated signaling results in an increase in irf expression, which subsequently orchestrates the amplification of ifns and of isgs. the ifn-mediated effects on transcription of several genes (including irf ) require the assembly of a transcriptional complex isgf , which includes stat and irf . the role of irf and irf degradation in ifn-dependent responses during rv infection has not yet been well studied. nevertheless, the degradation of irf and irf by nsp is likely to be an additional weapon in the rv arsenal that can be used to halt an efficient ifn amplification response (fig. . ). in addition to the ifn induction and amplification pathway, rv is equipped with the ability to block further downstream effector antiviral proteins. one such example is ribonuclease l (rnasel), a key enzyme in the ifn-inducible - -oligoadenylate synthetase (oas)-rnasel pathway responsible for potent rna degradation, including both host and viral rna molecules [ ] . the rv rna capping enzyme vp encodes a c-terminal phosphodiesterase (pde) domain that was recently shown to induce the degradation of , -oligoadenylate, the second messenger responsible for rnasel activation and dimerization [ ] . the rv vp pde domain functionally replaced the comparable domain in the murine coronavirus ns protein and inhibited rnasel activity. a more recent study suggests that another vp -independent, yet-to-be identified, mechanism also exists and contributes to rv inhibition of rnasel [ ] . however, the actual physiological roles of rnasel in modulating rv replication and of vp in antagonizing rnasel in vivo remain to be demonstrated. there are currently two time-honored and demonstrably successful approaches to developing safe and effective human viral vaccines [ ] . in the first case, a wide variety of current viral vaccines rely on the parenteral administration of replication-incompetent inactivated whole virus, the parenteral administration of a viral protein(s) component, or the administration a molecularly produced virus-like particle (vlp). all of these entities are selected because immunity to the individual protein, inactivated whole virus, or vlp induces protective immunity to the host and, at the same time, is both safe and well tolerated. there are numerous highly successful examples of this category of viral vaccine (e.g., the inactivated polio vaccine, the various preparations of inactivated influenza hemagglutinin-based vaccines, the human papilloma vlp vaccine, the hepatitis b virus vaccine, and the recently licensed herpes zoster ge protein-based vaccine). in all cases, these vaccines are administered parenterally. several are administered with adjuvants of one kind or another to enhance immune responses, and in all cases, they appear to function primarily by stimulating systemic immunity, with the primary effector function generally mediated by systemic antibodies. of note, none of these types of vaccines are directed at a predominantly enteric pathogen, although an investigation of a potential parenterally administered norovirus vlp vaccine is currently under way [ ] , and there are plans to study the utility of a heat-inactivated rv virion-based vaccine in humans [ ] . the greatest advantages of the inactivated/recombinant protein-based vaccines are their general safety and the fact that they can be produced even when the actual pathogen cannot be readily propagated. the general disadvantage of such vaccines is that they are almost always less effective at stimulating t-cell-based immune responses, they are not very efficient at stimulating mucosal immune responses, that highly effective mucosal immune adjuvants are not yet readily available, and in some cases, immune responses to these inactivated vaccine preparations tend to diminish over time more rapidly than do responses to several live viral vaccines. other potential disadvantages of inactivated vaccines become apparent when the antigen or antigens required to induce protective immunity are difficult to synthesize artificially or when immunity is most potent when it is directed at multiple antigens that are correctly folded only on the actual or recombinant multiprotein viral particle. of note, rvs have at least two separate proteins (vp and vp ) that are targets of protective antibodies, and vp is cleaved by enteric trypsin into two separate protein components: vp * and vp * [ ] . both vp * and vp * are individually targeted by protective antibody responses. vp is correctly folded into its proper antigenic trimeric form only within the context of the rv virion, and a similar issue likely is true for vp *. on the other hand, the rv vp * protein can be relatively simply and accurately synthesized in several prokaryote and eukaryote systems, and because of this feature, it is currently being examined as a potential inactivated vaccine to be administered parenterally [ ] (chapter : development of oral rotavirus & norovirus vaccines). the second highly successful approach to human viral vaccine development has been the production of live attenuated viral vaccines that actually infect susceptible people but are attenuated to such a degree that their level of reactogenicity and pathogenicity is acceptable, while they reliably generate protective immunity. as with inactivated vaccines, a number of highly effective replication-competent viral vaccines are currently available (e.g., oral sabin polio vaccine, measles vaccine, rubella vaccine, yellow fever vaccine, smallpox vaccine, live attenuated influenza vaccine, and, of course, several licensed rv vaccines, such as rotateq, rotarix, and rotavac) [ ] . most but not all of the live attenuated viral vaccines are administered by parenteral injection; however, oral polio, rotavirus, and live attenuated influenza vaccine are all administered to a mucosal surface (the gi tract and the respiratory tract, respectively). the general thinking is that live attenuated viral vaccines more closely mimic the type and level of immunity induced by natural infection than parenterally administered inactivated vaccines do. if natural infection is an effective preventative of severe secondary infection, then reproducing it without undue reactogenicity can be desirable. this feature is present when natural immunity is operative primarily at a mucosal surface, as is the case for rvs. natural rv infection efficiently protects against severe reinfection, and for this reason, a variety of experimental and licensed live attenuated rv vaccines have been developed or proposed. the key issue to overcome in developing a live viral vaccine is to discover a method that reliably attenuates viral pathogenicity while retaining the ability of the viral infection to induce protective immunity. traditionally, such modification has been accomplished by multiple passage of virulent viral strains in cell culture with the hope that such multiple passages will lead to the acquisition of sufficient mutations in the viral genome (acquired to enhance cell culture replication) and that these cell culture adaptations will attenuate viral pathogenicity in the target host. this strategy is vi. mucosal vaccines for viral diseases time honored and has been used to develop multiple vaccines (e.g., live polio, measles, mumps, rubella, yellow fever, and some rv vaccines). while this approach is frequently successful, there is no way to determine how many passages are needed to eliminate residual virulence while retaining immunogenicity, so it is often tedious and always an inexact approach. in addition, concerns about reversion to virulence are always present. that said, this approach was used successful to develop a highly effective human rv vaccine (r , rotarix), which consist of a single human rv strain that was repeated passed in several cell culture systems and, over the passage history, became attenuated in people [ ] . while the genomic sequence of both the virulent wildtype parental rotarix strain and the eventual vaccine strain are known, the exact genomic mutations responsible for attenuation are unclear. what has been established, however, is that, given the very substantial and decade-long safety record, this human rv vaccine strain has sufficient attenuating mutations to ensure a high degree of genetic stability in humans. of note, the r rotarix vaccine represents a single g and [p] serotype yet reliably induces protective immunity to virtually all frequently circulating human rv strains. this finding strongly supports the conclusion that serotype-specific immunity is not a major determinant of immunity to severe rv disease in humans [ ] . the other strategy that has proven highly successful for the reproducible attenuation of the rvs used in live attenuated rv vaccines has been to take advantage of the hrr (see above) of heterologous (nonhuman origin) rvs as vaccine candidates for humans [ ] . several currently licensed rv vaccines (e.g., the r vaccines rotateq and rotasil and the r vaccine rotavac) consist of either natural or experimentally selected reassortants between animal rvs (in these cases, all bovine rvs) and human rvs. both r vaccines are pentavalent constructs derived experimentally on the basis of bovine rv genomes but containing individual vp -or vp s-encoding genes derived from various human serotypes. rotateq is broadly licensed around the world, while rotasil is currently licensed only in india [ ] . rotavac is, interestingly, a naturally occurring reassortant rv derived from a human rv and a bovine rv. it contains only a bovine vp , with all other ten rv gene segments derived from a human rv strain. this virus was originally isolated from a neonatal nursery where asymptomatic rv infection was endemic [ ] . finally, of relevance to this review, an entirely lamb origin rv strain is currently licensed for rv prevention in china. this vaccine is also highly attenuated, presumably because of the hrr of a lamb origin rv in humans. while this vaccine seems safe, the data on its efficacy are limited [ ] . the key point here is that all these animal virus origin based rv vaccines are highly attenuated in humans, and this attenuation, as was discussed above, is most likely based on their host range replication restriction in humans. this hrr is primarily due to the inability of heterologous rv to efficiently suppress the human intestinal innate immune response, primarily the human ifn response, owing to the presence of heterologous nsp s in the vaccine candidates. the active human ifn response to these heterologous rv vaccines suppresses their replication sufficiently to restrict pathogenicity and reactogenicity but not so much that the generation of effective rv immunity is suppressed. however, in the case of the indian rotavac vaccine, attenuation might be based on the heterologous bovine origin vp , which might be expected to reduce rv binding to human iecs. the big question for the future is whether, with the advent of a tractable reverse genetic system and our improved understanding of the genetic determinants of hrr, we will be able to better fine-tune the replication competence and immunogenicity potential of rv vaccine candidates to 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discovered by their strong and direct antiviral activity [a. isaacs, j. lindenmann, virus interference. i. the interferon, proc. r. soc. lond. b biol. sci. ( ) – ]. (see review by j. lindenmann on p. , in this issue). nevertheless, only very recently it was entirely realized that viruses would not succeed without efficient tools to undermine this potent host defense system. current investigations are revealing an astonishing variety of viral ifn antagonistic strategies targeting virtually all parts of the ifn system, often in a highly specific manner. viruses were found to interfere with induction of ifn synthesis, ifn-induced signaling events, the antiviral effector proteins, or simply shut off the host cell macromolecule synthesis machinery to avoid booting of the antiviral host defense. here, we will describe a few well-characterized examples to illustrate the sophisticated and often multi-layered anti-ifn mechanisms employed by viruses. in most nucleated body cells, viral infections activate transcription of the ''classic'' ifn-b gene [ ] by a signaling chain which is initiated by the rna sensors rig-i and mda- , which in turn act trough the adaptor ips- and the kinases tbk- and ikk- to activate the transcription factor irf- (see reviews by p. pitha and by t. fujita on pages and this issue respectively). a parallel pathway involves the dsrna-binding kinase pkr, the traf adaptor molecules and the nf-kb kinase ikka/b (see review by garcía et al., on p. this issue). most viruses investigated so far interfere with one or several steps in these important signaling chains [ e ]. fig. provides a schematic overview over the ifn induction pathway and some selected viral counterparts. until very recently, it was thought that the only ifn-inducing molecule which clearly distinguishes viruses from their host (i.e. self vs. non-self) is double-stranded rna (dsrna). many rna and dna viruses therefore express proteins which bind this key molecule to avoid both ifn induction and activation of dsrna-dependent antiviral enzymes [ , ] . well-investigated examples are the ns protein of influenza a virus [ e ], the e l protein of poxviruses [ , ] , the vp protein of ebola virus [ , ] , the sigma protein of reoviruses [ ] , and the us protein of herpes simplex virus [ , ] . the murine cytomegalovirus encodes two proteins, m and m which together block dsrna-mediated signaling pathways [ , ] . however, in the case of the influenza virus ns and the ebola virus vp dsrna-binding appears only to contribute to the ifn antagonism without being essential [ , , ] . in addition, we have recently shown that some viruses do not produce detectable amounts of dsrna at all [ ] , indicating that in these cases other molecules ifn-eliciting molecules are important. indeed, viral ssrnas bearing a triphosphate group are a potent trigger of ifn induction, acting through rig-i [ , ] . in line with this, it was shown that the ns of influenza a virus can bind ssrna as well, and is able to form complexes with rig-i [ , ] . similarly, a dsrna-binding defective vp mutant can still block ifn induction [ ] , suggesting a similar mode of action. thus, rna binding by these viral ifn antagonists appears to be contributing to their ifn antagonism without being sufficient. an ifn induction antagonist devoid of any rna-binding activity is the v protein of the paramyxovirus sv . this small protein inhibits ifn induction by sequestering the rig-i-related rna sensor mda- [ , ] , raising the question how sv deals with the parallel rig-i pathway. this paramyxovirus-specific problem can be avoided by blocking components of the signaling pathway which are situated further downstream and therefore needed by both rig-i and mda- . the next in line, the adaptor protein ips- , connects the rna sensors with the irf- kinases tbk- /ikk- and is specifically cleaved by the ns - a protease of hepatitis c virus [ , ] . the activation of irf- by tbk- is prevented by the phosphoprotein p of rabies virus [ ] and the g glycoprotein of the hantavirus ny- [ ] . irf- itself is degraded by the npro proteins of classical swine fever virus and of bovine viral diarrhea virus [ e ]. also, the e protein of human papilloma virus binds and inactivates irf- [ ] , and human herpes virus (hhv- ) expresses several irf homologues, termed virfs, which exert a dominant-negative effect [ e ]. target-specific ifn-escape strategies are often pursued by viruses causing persistent infections, e.g. herpes viruses. by contrast, many viruses which lytically infect the host cell simply impose a general block on host cell transcription and translation. for example, the non-structural nss proteins of the rift valley fever virus and bunyamwera virus interfere with the basic cellular transcription machinery [ e ]. although this strategy appears to be unspecific, in vivo experiments clearly demonstrated that the biological purpose of this broad-band shut-off is to inhibit ifn synthesis [ , ] . the matrix (m) protein of vesicular stomatitis virus (vsv) is also a potent host cell shutoff factor which inhibits basal transcription [ ] , impairs nuclear-cytoplasmic transport of rnas and proteins [ ] , and inactivates translation factors [ ] . as is the case with bunyavirus nss, the biological significance of vsv m-mediated shutoff is to suppress ifn induction [ , ] . also, proteinases of picornaviruses (e.g. foot and mouth disease virus, theiler's virus, polio virus) and pestiviruses (e.g. classical swine fever virus) cause a shutoff-of the host cell metabolism to interfere with the ifn response [ , e ] . interestingly, the non-structural protein ns of influenza a virus also impairs the post-transcriptional processing and nuclear export of cellular pre-mrnas [ e ] in order to counteract the antiviral host response [ , ] . thus, ns is a versatile protein with the ability to prevent ifn induction both by ifn pathway-specific and by less specific means, and recent studies suggest that there is a surprisingly great strain-specific variation in these activities [ ] . ifn-b and the multiple ifn-a subspecies activate a common type i ifn receptor (ifnar) which signals to the nucleus through the so-called jak-stat pathway (fig. ) . the stat proteins are latent cytoplasmic transcription factors which become phosphorylated by the janus kinases jak- and tyk- [ ] . phosphorylated stat- and stat- recruit a third factor, irf- , to form a complex known as ifn-stimulated gene factor (isgf- ) which translocates to the nucleus and binds to the ifn-stimulated response element (isre) in the promoter region of interferon-stimulated genes (isgs). the ifn signal transduction pathway represents another important target of viruses (fig. ) . members of the paramyxovirus family, which contains mainly important pathogens, encode two different (but genetically related) proteins named c and v which interfere with stat function. depending on the virus species, these ifn antagonists act either by binding the stat proteins, by inducing their degradation, or by inhibiting the jak kinases [ e ]. the p protein of rabies virus binds to activated stat and stat and retains them in the cytoplasm [ ] . thus, the paramyxoviral v protein as well as the rabies virus p protein have a dual anti-ifn function as they block both ifn induction (see above) and stat signaling. ebola virus, by contrast uses a different protein, vp , to block nuclear import of stat by interacting with the transporter protein karyopherin alpha [ ] . stat signaling is also disturbed by viruses causing persistent infections, such as hepatitis c virus [ the dsrna-binding proteins mentioned above also serve a second purpose, namely the inhibition of the dsrna-activated antiviral enzymes. this has been demonstrated for the influenza virus ns [ , , e ] , the poxvirus e l [ ] , the reovirus sigma [ ] , the herpesvirus us [ , ] , and the dsrna-binding proteins of human and murine cytomegaloviruses [ , , ] . importantly, also for this anti-ifn effector function more that just dsrna binding appears to be necessary, since in many cases a direct interaction with e.g. pkr has been demonstrated (reviewed in ref. [ ] ). sequestering dsrna may also inhibit the e oas pathway and adar, although this has only been shown in a few cases [ , ] . dsrna-independent inhibition of the rnasel system is achieved by the icp protein of herpes simplex virus [ ] and by upregulation of rli, a cellular inhibitor of rnasel, in hiv-and picornavirus-infected cells [ , ] . pkr is also attacked by other means. the g . protein of herpes simplex virus triggers the dephosphorylation of eif- a, thus reverting the translational block established by pkr [ ] . the e protein of hepatitis c virus [ ] , the tat protein of hiv [ ] and the k l protein of vaccinia virus [ ] act as pseudosubstrates for pkr. another strategy is to encode small, highly structured rnas which compete with dsrna and inactivate pkr. this was demonstrated for adenoviruses [ ] , hepatitis c virus [ ] , epstein-barr virus [ ] , and hiv [ ] . however, for epstein-barr virus it was shown that the pkr inhibition by the so-called eber rnas observed in vitro does not occur in vivo [ ] , suggesting that ebers are important for other activities such as inhibition of apoptosis. it is obvious from the listings above that viruses have evolved multiple means to disrupt the ifn response. in some cases, there are specialized anti-ifn factors such the non-structural proteins of influenza viruses. in many other cases, however, viral gene products with a defined function in virus replication cycle can additionally acquire the ability to block the ifn system. important examples include the v, w and c proteins of paramyxoviruses [ , ] , the p protein of rabies virus [ , ] and the vp protein of ebola virus [ ] , which are regulators of the viral polymerase. also, the matrix proteins of thogoto virus [ ] and vesicular stomatitis virus [ ] , the nucleoprotein of arenaviruses [ ] , and the glycoprotein of hantaviruses [ ] not only have structural functions, but are ifn antagonists as well. some viruses such as dengue virus or sars-coronavirus encode a multitude of anti-ifn factors which together may strongly contribute to an enhanced virulence [ e ]. apparently, modulating the ifn system can be achieved either by ''inventing'' one or several specialized factors or by expanding the function of existant gene products. understanding the interplay between viruses and the ifn response can help to design new strategies for prevention and therapy. viruses unable to counteract the ifn response are excellent candidates for live virus vaccines. they can be grown to high titers in ifn-deficient cell cultures but are attenuated in vivo since they elicit a robust innate and adaptive immune responses. this concept has been proven for influenza viruses [ e ], human parainfluenza virus type [ ] , human and bovine respiratory syncytial viruses [ e ], and may likewise apply to other viruses. oncolytic viruses designed for the targeted destruction of tumors is another promising application. tumor cells often eliminate one or several parts of the ifn system during the transformation process [ e ]. for example, tumor cells were shown to acquire specific mutations leading to resistance of cellular translation to inhibition by pkr [ ] . the payoff is an increased susceptibility to infection [ , , ] , and the tumor selectivity of viruses can be further increased by using mutants with defective ifn antagonists. the inability of these mutant viruses to fight the ifn response is complemented by the ifn-deficiency of the tumor cells. at the same time, these viruses are unable to infect the ifn-competent body cells. this concept is proven by an ifn-inducing vsv mutant [ ] and a herpes simplex virus lacking the anti-pkr gene g . [ , ] which specifically destroyed tumors in immunocompetent hosts. thus, unravelling the strategies by which viruses counteract the ifn system not only helps to better understand viral pathogenesis but can also result in novel vaccination strategies and therapies. virus interference. i., the interferon viruses and the type i interferon antiviral system: induction and evasion transcriptional activation of alpha/beta interferon genes: interference by nonsegmented negative-strand rna viruses type interferons and the virus-host relationship: a lesson in detente the interferon response circuit: induction and suppression by pathogenic viruses inverse interference: how viruses fight the interferon system inhibition of pkr by rna and dna viruses characterization of viral doublestranded rna-binding proteins influenza a virus lacking the ns gene replicates in interferon-deficient systems inhibition of interferon-mediated antiviral responses by influenza a viruses and other negative-strand rna viruses binding of the influenza virus ns protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf- translation initiation factor the primary function of rna binding by the influenza a virus ns protein in infected cells: inhibiting the - oligo (a) synthetase/rnase l pathway replication of modified vaccinia virus ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene e l blockade of interferon induction and action by the e l double-stranded rna binding proteins of vaccinia virus ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling reverse genetic generation of recombinant zaire ebola viruses containing disrupted irf- inhibitory domains results in attenuated virus growth in vitro and higher levels of irf- activation without inhibiting viral transcription or replication reovirus sigma protein: dsrna binding and inhibition of rna-activated protein kinase neutralizing innate host defenses to control viral translation in hsv- infected cells inhibition of pkr activation by the proline-rich rna binding domain of the herpes simplex virus type us protein double-stranded rna binding by a heterodimeric complex of murine cytomegalovirus m and m proteins murine cytomegalovirus m and m are both required to block protein kinase r-mediated shutdown of protein synthesis the n-and c-terminal domains of the ns protein of influenza b virus can independently inhibit irf- and beta interferon promoter activation a recombinant influenza a virus expressing an rna-binding-defective ns protein induces high levels of beta interferon and is attenuated in mice double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses -triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing phosphates inhibition of retinoic acid-inducible gene-i-mediated induction of interferon-{beta} by the ns protein of influenza a virus the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter mda- , but not rig-i, is a common target for paramyxovirus v proteins dissociation of a mavs/ ips- /visa/cardif-ikkepsilon molecular complex from the mitochondrial outer membrane by hepatitis c virus ns - a proteolytic cleavage cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein p interferes with phosphorylation of interferon regulatory factor the pathogenic ny- hantavirus g cytoplasmic tail inhibits rig-i-and tbk- -directed interferon responses role of double-stranded rna and npro of classical swine fever virus in the activation of monocyte-derived dendritic cells the npro product of bovine viral diarrhea virus inhibits dna binding by interferon regulatory factor and targets it for proteasomal degradation loss of interferon regulatory factor in cells infected with classical swine fever virus involves the n-terminal protease, npro n(pro) of classical swine fever virus is an antagonist of double-stranded rna-mediated apoptosis and ifn-alpha/beta induction human papillomavirus e oncoprotein binds to interferon regulatory factor- and inhibits its transcriptional activity functional analysis of human herpesvirus -encoded viral interferon regulatory factor and its association with cellular interferon regulatory factors and p unique properties of a second human herpesvirus -encoded interferon regulatory factor (virf- ) inhibition of interferon signaling by the kaposi's sarcoma-associated herpesvirus full-length viral interferon regulatory factor protein kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor characterization of a novel human herpesvirus -encoded protein, virf- , that shows homology to viral and cellular interferon regulatory factors kaposi's sarcoma-associated herpesvirus-encoded virf- stimulates the transcriptional activity of cellular irf- and irf- human herpesvirus encodes an interferon regulatory factor (irf) homolog that represses irf- -mediated transcription nss protein of rift valley fever virus blocks interferon production by inhibiting host gene transcription tfiih transcription factor, a target for the rift valley hemorrhagic fever virus inhibition of rna polymerase ii phosphorylation by a viral interferon antagonist genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein nss bunyamwera bunyavirus nonstructural protein nss counteracts the induction of alpha/beta interferon inhibition of host rna polymerase iidependent transcription by vesicular stomatitis virus results from inactivation of tfiid inhibition of ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus vesicular stomatitis virus infection alters the eif f translation initiation complex and causes dephosphorylation of the eif e binding protein e-bp the vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter vsv strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response cytopathogenesis and inhibition of host gene expression by rna viruses, microbiol classical swine fever virus interferes with cellular antiviral defense: evidence for a novel function of n(pro) the leader protein of theiler's virus inhibits immediate-early alpha/beta interferon production influenza a virus ns protein targets poly(a)-binding protein ii of the cellular -end processing machinery influenza virus ns protein inhibits pre-mrna splicing and blocks mrna nucleocytoplasmic transport the -end-processing factor cpsf is required for the splicing of single-intron pre-mrnas in vivo human influenza viruses activate an interferon-independent transcription of cellular antiviral genes: outcome with influenza a virus is unique cellular antiviral responses against influenza a virus are countered at the posttranscriptional level by the viral ns a protein via its binding to a cellular protein required for the end processing of cellular pre-mrnas variation in the ability of human influenza a viruses to induce and inhibit the ifn-beta pathway stats: transcriptional control and biological impact degradation of stat and stat by the v proteins of simian virus and human parainfluenza virus type , respectively: consequences for virus replication in the presence of alpha/beta and gamma interferons the v protein of simian virus inhibits interferon signalling by targeting stat for proteasome-mediated degradation all four sendai virus c proteins bind stat , but only the larger forms also induce its mono-ubiquitination and degradation the v protein of human parainfluenza virus antagonizes type i interferon responses by destabilizing signal transducer and activator of transcription stat ubiquitylation and degradation by mumps virus suppress cytokine and oncogene signaling the stat activation process is a crucial target of sendai virus c protein for the blockade of alpha interferon signaling rinderpest virus blocks type i and type ii interferon action: role of structural and nonstructural proteins stat protein interference and suppression of cytokine signal transduction by measles virus v protein newcastle disease virus (ndv)-based assay demonstrates interferon-antagonist activity for the ndv v protein and the nipah virus v, w, and c proteins nipah virus v protein evades alpha and gamma interferons by preventing stat and stat activation and nuclear accumulation hendra virus v protein inhibits interferon signaling by preventing stat and stat nuclear accumulation nipah virus v and w proteins have a common stat -binding domain yet inhibit stat activation from the cytoplasmic and nuclear compartments, respectively nuclear localization of the nipah virus w protein allows for inhibition of both virus-and toll-like receptor -triggered signaling pathways sendai virus c protein physically associates with stat measles virus suppresses interferon-alpha signaling pathway: suppression of jak phosphorylation and association of viral accessory proteins, c and v, with interferon-alpha receptor complex inhibition of interferon signaling by rabies virus phosphoprotein p: activation-dependent binding of stat and stat ebola virus vp binds karyopherin alpha and blocks stat nuclear accumulation expression of hepatitis c virus proteins interferes with the antiviral action of interferon independently of pkr-mediated control of protein synthesis expression of hepatitis c virus proteins inhibits signal transduction through the jak-stat pathway herpes simplex virus gene products occlude the interferon signaling pathway at multiple sites induction of suppressor of cytokine signaling- by herpes simplex virus type contributes to inhibition of the interferon signaling pathway a cytomegalovirus inhibitor of gamma interferon signaling controls immunoproteasome induction a cytomegaloviral protein reveals a dual role for stat in ifn-{gamma} signaling and antiviral responses vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity the vaccinia virus soluble alpha/ beta interferon (ifn) receptor binds to the cell surface and protects cells from the antiviral effects of ifn vaccinia virus-encoded cytokine receptor binds and neutralizes chicken interferon-gamma vaccinia virus encodes a soluble type i interferon receptor of novel structure and broad species specificity influenza virus ns protein counteracts pkr-mediated inhibition of replication mutant influenza viruses with a defective ns protein cannot block the activation of pkr in infected cells inhibition of pkr by vaccinia virus: role of the n-and c-terminal domains of e l binding of the influenza a virus ns protein to pkr mediates the inhibition of its activation by either pact or double-stranded rna double-stranded rna binding of influenza b virus nonstructural ns protein inhibits protein kinase r but is not essential to antagonize production of alpha/beta interferon inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype sigma protein inhibition of pact-mediated activation of pkr by the herpes simplex virus type us protein double-stranded rna binding by human cytomegalovirus ptrs icp prevents rnase l-independent rrna cleavage in herpes simplex virus type -infected cells rnase l inhibitor is induced during human immunodeficiency virus type infection and down regulates the e a/rnase l pathway in human t cells rnase l inhibitor (rli) antisense constructions block partially the down regulation of the e a/rnase l pathway in encephalomyocarditis-virus-(emcv)-infected cells the gamma( ) . protein of herpes simplex virus complexes with protein phosphatase alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor and preclude the shutoff of protein synthesis by double-stranded rna-activated protein kinase inhibition of the interferon-inducible protein kinase pkr by hcv e protein control of the interferon-induced -kilodalton protein kinase by the hiv- tat gene product the vaccinia virus k l gene product potentiates translation by inhibiting double-stranded-rna-activated protein kinase and phosphorylation of the alpha subunit of eukaryotic initiation factor adenovirus virus-associated rna and translation control inhibition of the protein kinase pkr by the internal ribosome entry site of hepatitis c virus genomic rna regulation of the double-stranded rna-dependent protein kinase pkr by rnas encoded by a repeated sequence in the epsteinebarr virus genome tat-responsive region rna of human immunodeficiency virus can prevent activation of the double-stranded-rna-activated protein kinase protection from interferon-induced apoptosis by epsteinebarr virus small rnas is not mediated by inhibition of pkr interferons: cell signalling, immune modulation, antiviral response and virus countermeasures the ebola virus vp protein inhibits activation of interferon regulatory factor thogoto virus lacking interferon-antagonistic protein ml is strongly attenuated in newborn mx -positive but not mx -negative mice inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus sars coronavirus proteins orf b, orf , and nucleocapsid function as interferon antagonists inhibition of interferon signaling by dengue virus inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor immunogenicity and protection efficacy of replication-deficient influenza a viruses with altered ns genes influenza virus evades innate and adaptive immunity via the ns protein vaccination of pigs against swine influenza viruses by using an ns -truncated modified live-virus vaccine influenza a and b viruses expressing altered ns proteins: a vaccine approach attenuating mutations in the p/c gene of human parainfluenza virus type (hpiv ) vaccine candidates abrogate the inhibition of both induction and signaling of type i interferon (ifn) by wild-type hpiv role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking ns proteins the interferon antagonist ns protein of respiratory syncytial virus is an important virulence determinant for humans recombinant respiratory syncytial virus that does not express the ns or m - protein is highly attenuated and immunogenic in chimpanzees negative regulation of the alpha interferon-induced antiviral response by the ras/raf/mek pathway differentially regulated interferon response determines the outcome of newcastle disease virus infection in normal and tumor cell lines exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus the molecular basis of viral oncolysis: usurpation of the ras signaling pathway by reovirus defective translational control facilitates vesicular stomatitis virus oncolysis reovirus therapy of tumors with activated ras pathway attenuated, replication-competent herpes simplex virus type mutant g : safety evaluation of intracerebral injection in nonhuman primates attenuated multi-mutated herpes simplex virus- for the treatment of malignant gliomas our own work described in the text was supported by grants from the deutsche forschungsgemeinschaft. key: cord- -ap wfjhq authors: lewis, toby c.; henderson, tiffany a.; carpenter, ashley r.; ramirez, ixsy a.; mchenry, christina l.; goldsmith, adam m.; ren, xiaodan; mentz, graciela b.; mukherjee, bhramar; robins, thomas g.; joiner, terence a.; mohammad, layla s.; nguyen, emily r.; burns, mark a.; burke, david t.; hershenson, marc b. title: nasal cytokine responses to natural colds in asthmatic children date: - - journal: clinical & experimental allergy doi: . /cea. sha: doc_id: cord_uid: ap wfjhq background: the mechanisms by which viruses induce asthma exacerbations are not well-understood. objective: we characterized fluctuations in nasal aspirate cytokines during naturally-occurring respiratory viral infections in children with asthma. methods: sixteen children underwent home collections of nasal aspirates when they were without cold symptoms and again during self-reported respiratory illnesses. the presence of viral infection was ascertained by multiplex pcr. cytokines were measured by multiplex immune assay. mrna expression for selected markers of viral infection was measured by rt-pcr. a cumulative respiratory symptom score was calculated for each day of measurement. generalized estimated equations were used to evaluate associations between viral infection and marker elevation, and between marker elevation and symptom score. results: the patients completed a total of weeks of assessment ( “well” weeks; self-assessed “sick” weeks). viral infections were detected in three of the “well” weeks and of the “sick” weeks ( rhinovirus, coronavirus, influenza a, influenza b, respiratory syncytial virus, parainfluenza). compared to virus-negative well weeks, nasal aspirate ifn-γ, cxcl /il- , cxcl /ip- , ccl /rantes, ccl /eotaxin- , ccl /mcp- , ccl /mip- β, ccl /mcp- and ccl /mip α protein levels increased during virus-positive sick weeks. only a subset of cytokines (ifn-γ, cxcl , ccl , ccl , ccl and ccl ) correlated with self-reported respiratory tract symptoms. while many aspirates were dilute and showed no mrna signal, viral infection significantly increased the number of samples that were positive for ifn-λ , ifn-λ / , tlr , rig-i and irf mrna. conclusions & clinical relevance: we conclude that, in children with asthma, naturally-occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, ifns and ifn-responsive genes. epidemiological studies have uncovered a strong association between viral infections, especially those caused by rhinovirus, and exacerbations of asthma attacks [ ] [ ] [ ] . however, the mechanisms by which viruses induce exacerbations of chronic airways disease are not well understood. in response to viral infection, the airway epithelium, and to a lesser extent immune cells such as monocytes, macrophages, and dendritic cells [ ] [ ] [ ] [ ] , release pro-inflammatory cytokines, interferons (ifns), and antimicrobial substances, which in turn, promote clearance of microorganisms, and activation of the adaptive immune system. however, in patients with chronic respiratory illnesses, the innate immune response may also be responsible for disease exacerbation [ ] . although the inflammatory response to experimental rhinovirus infection has been monitored [ ] [ ] [ ] [ ] [ ] [ ] , few studies have examined the innate immune response of patients with asthma to natural colds. increases in respiratory tract cxcl /il- , ccl /rantes, ccl / mip- a, il- , ccl /mcp- , and ccl /mcp- have been detected [ ] [ ] [ ] [ ] . to further examine the innate immune response to viral infection in children with asthma, we measured nasal aspirate cytokine levels in asthmatic children before and after upper respiratory tract infections. in contrast to the previous studies, we examined at least three time points before and in association with symptomatic illness. we evaluated the associations between viral infection and cytokine expression, and between cytokine expression and symptom score. we also examined the effects of upper respiratory tract infection on mrna levels of selected markers of viral infection, including ifns. finally, we evaluated a new method of virus detection using a single polymerase chain reaction-ligation detection reaction (pcr-ldr) multiplex assay. we hypothesized that respiratory viral infection of children with asthma causes robust elaboration of pro-inflammatory cytokines and ifns, and that the level of pro-inflammatory cytokines correlates with symptom severity. we conducted an observational cohort study of children with asthma. children were eligible for the study if they received care from a university of michigan physician for asthma, were aged - years, and lived within a mile radius of ann arbor. outpatients were enrolled for - months, with the goal of assessing cytokines at baseline and in association with at least one viral-induced exacerbation during that period. initial consent and first evaluation was conducted at the university of michigan, but all subsequent evaluations were performed in the participant's home. this study was approved by the university of michigan institutional review board (approval number hum ). all clinical investigations were conducted according to principles expressed in the declaration of helsinki (http://www.wma.net). at the initial assessment, parents or guardians completed an extensive questionnaire, which included standardized questions about presence and frequency of asthma symptoms, an inventory of a child's asthma medications and current usage, and queries about environmental exposures, and child and family demographic information. baseline asthma severity was assessed using national asthma education and prevention program guidelines [ ] incorporating an adjustment for asthma controller therapy use [ ] . we performed home measurements of respiratory symptoms and collected nasal secretions (for detection of viral rna by pcr and host biomarkers by pcr and elisa) on days during a week when children were healthy (not reporting upper respiratory tract infection or asthma symptoms), and again during a week when they experienced cold or flu-like symptoms. families were given a calendar and a simple respiratory symptom scale and asked to mark the level of their symptoms. we used a modified version of the respiratory symptom score developed by the child origins of asthma study [ ] , which assessed fever, cough, nasal symptoms, wheezing, difficulty breathing, waking up at night with cough, and interference with usual activities. when the patient experienced a symptomatic respiratory illness, as defined by a symptom score of two or higher on a scale of - , the family notified the study staff and scheduled a visit within h of the beginning of symptoms (defined as day of the illness). information regarding impact of respiratory symptoms on daily activities and recent use of asthma medicines was also collected. as noted above, study technicians visited the home every - days to retrieve additional nasal washing samples, for a total of three visits during the week following reporting of symptoms. the three specific days of the week selected for analysis were based on the convenience of the subjects and laboratory technician. although specimens were most often collected on days , , and of a given week, specimen collection was sometimes compressed into a or -day period. to collect a nasal lavage sample, we utilized a protocol developed by powell and shorr [ ] and modified by james gern (university of wisconsin, personal communication). two squirts of isotonic . % sodium chloride were instilled into the child's nostrils (estimated at < ml per nostril) using a commercially available nasal saline spray (b.f. ascher, lenexa, ks, usa). the subject then blew their nose into a zippered plastic bag, and ml of m rt viral transport medium (remel, lenexa, ks, usa) was added. in general, samples were collected in the presence of study staff and were transported from homes to the laboratory within h of collection in a cooler with ice packs. however, due to logistical issues scheduling home visits, families were also provided with kits for independent collection of nasal washings specimens at home. if a visit could not be scheduled within h, families were instructed to collect the sample, double bag the sample, place in a tightly sealed collection box, and to keep it in the refrigerator until the staff could collect the sample. we required a week interval between sample collections for sequential illnesses. nasal aspirates were homogenized using a handheld homogenizer to allow pipetting of viscous samples and frozen at À °c to allow for batched analysis. viral rna and dna were extracted using a minelute kit (qiagen, valencia, ca, usa). samples were assessed for the presence of viral rna or dna via pcr using the seegene seeplex rv- detection kit (seegene, rockville, md, usa). this kit detects human parainfluenza viruses , , and , human metapneumovirus, human coronavirus e/nl , and oc , human adenovirus, influenza viruses a and b, human respiratory syncytial virus (rsv) a and b, and human rhinovirus a. we also analysed specimens for respiratory viruses using a novel polymerase chain pcr-ldr multiplex assay [ ] . the original system, which was designed to detect influenza viruses, was expanded to include parainfluenza , , , a and b, coronaviruses e and oc , influenza a and b, rhinoviruses a, b, and c, adenoviruses a-e, metapneumovirus, and rsv a and b. we have detected all the viruses included in this multiplex system in clinical samples (i.e. nasal aspirates), with the exception of the adenoviruses, for which we used laboratory positive controls. the viral sequences for the multiplex assay are listed in table . protein levels of ifn-c, cxcl /il- , cxcl /ip- , ccl /rantes, ccl /eotaxin- , ccl /mcp- , ccl / mip- b, ccl /mcp- , ccl /mip- b, and ccl /mip- a were determined using a magnetic bead-based multiplex immune assay (bio-rad, hercules, ca, usa). our interest on the above cc chemokines was prompted by gene array results from rv b-infected, ovalbumin-sensitized, and -challenged mice (data not shown). ifn-a and ifn-b levels were measured using standard elisa (r&d systems, minneapolis, mn, usa). aspirates were homogenized and mrna extracted as described above, using the rneasy or the rneasyplus kit (qiagen). mrna was analysed for cxcl /il- , cxcl / ip , ccl /rantes, ccl /eotaxin, ifn-k , ifn-k / , ifn-a, ifn-b, icam- , tlr , mda- , rig-i, and irf using quantitative real time pcr using specific primers and probes. signals were normalized to gapdh. we assessed feno in exhaled breath on days during a week when the child was well, and on at least days during a viral infection. to measure feno, we employed the niox mino (aerocrine, new providence, nj, usa). because feno measurement can be influenced by diet and exercise, participants were asked to refrain from eating or drinking within h of their exhaled breath assessment. triplicate samples were obtained to assess reliability. mean, standard deviation and interquartile range were used to describe measured values of nasal cytokine levels, nasal mrna, and symptom score before and during viral illnesses. box plots were used to represent the change in biomarkers at different points of the cold relative to the baseline. whiskers of the boxplots represent the minimum and maximum values. distributions of these continuous outcome variables were examined and appropriate transformations taken to achieve normality. the effects of viral illness, as defined by the seegene kit results, on biomarker protein levels, and of biomarker level on symptom score were determined using a generalized mixed models (proc mixed), with an auto-regressive correlation structure using sas statistical software (sas, cary, nc, usa). we evaluated and adjusted for relevant covariates such as age, gender, ethnicity/race, and nasal steroid use. other potential explanatory variables, such as family income, baseline asthma severity, tobacco smoke exposure, and oral antihistamine use were evaluated, but not included in final models as they were not significant predictors. for all mrna outcomes except ifn-b, levels were undetectable in a large number of samples. we therefore categorized our results into detectable/non-detectable levels, and analysed as a binary variable using the generalized estimating equation (gee) approach with the tobit link [ ] . we analysed ifn-b mrna levels as a continuous variable using the gee approach with identity link. unadjusted models are shown. sixteen children with physician-diagnosed asthma were recruited. participants ranged in age from to years old, were % boys, % non-white, and reported symptoms or medication use consistent with persistent asthma ( table ) . participants completed a total of weeks of assessment ( table ). the current report focuses on the weeks where there was concordance between self- reported illness and viral detection using the seegene kit ( virus-positive 'sick' weeks, and virus-negative 'well' weeks). we re-tested eight positive and seven negative samples (by seeplex) using the pcr-ligation detection multiplex assay. twelve of samples were concordant between the two assays. two samples were positive by seeplex and negative by pcr-ldr. one sample showed rsv by seeplex assay and coronavirus oc by pcr-ldr. symptom scores, which qualitatively evaluated the severity of illness, rose during the sick weeks (fig. a) . respiratory symptoms were highest at the first assessment during sick weeks, - days after initial report of illness. children with confirmed viral illnesses experienced significant increases in cough, wheeze, and chest tightness, indicative of asthma exacerbation (table ) . selected nasal aspirate cytokines were measured using magnetic bead-based multiplex immune assay. there were significant increases in all cytokines examined during weeks of reported symptoms, with the single exception of ccl (table , fig. b) . this pattern was more striking when samples with documented viral infections were compared to those confirmed to be virus-negative. unadjusted estimates of the association of confirmed viral infection and cytokine level were highly significant (not shown) and did not change appreciably when adjusted for age, gender, race, and nasal steroid use. changes in cytokines over the course of the illness are shown in fig. . in most cases, cytokines increased during the first days of infection and persisted throughout the week. we also examined the relationship between cytokines and symptom score. unadjusted estimates of the association of confirmed viral infection and cytokine level with symptom score were significant for ifn-c, cxcl , ccl , ccl , ccl , and ccl (table ) . cxcl , ccl , ccl , and ccl cytokine levels did not correlate with symptom score. we also attempted to measure ifn-a and ifn-b protein levels in the nasal aspirates by elisa. ifn-a levels over the lower detection limit ( . pg/ml) were found in only five of specimens (all virus-positive weeks). for ifn-b, levels over the detection limit ( pg/ml) were found in only samples (five virus-negative, seven virus-positive). many aspirates were dilute and a number of specimens showed no mrna signal (table ) . nevertheless, we detected transcripts for icam- , cxcl- , ifn-k , cxcl- , rig-i, mda- , tlr , ifnk / , irf , ifn-a, and ifn-b from patients both before and during viral infections. we could not detect ccl and ccl mrna in any specimens. finally, infection significantly increased the number of samples that were positive for cxcl , ifn-k , ifn-k / , tlr , rig-i, and irf mrna. we measured feno before and during virus-positive sick weeks (fig. ) . there was no significant change in feno measurement with viral infection. viral infections are the most frequent cause of asthma attacks [ ] [ ] [ ] . in theory, chemokine production by virus-infected epithelial cells induces the recruitment of inflammatory cells to the airways, which in turn elaborate cytokines and mediators capable of increasing airway responses. data suggest that immune cells may also be infected by respiratory viruses and produce chemokines [ , [ ] [ ] [ ] [ ] [ ] [ ] . in the present study, we found that, during natural colds, respiratory tract cytokine levels significantly increase in children with asthma. levels of a subset of cytokines correlated with the degree and time course of respiratory symptoms. the chemokines detected are responsible for recruitment of a broad array of inflammatory cells including neutrophils, monocytes, macrophages, and eosinophils. finally, we detected changes in nasal lavage specimens, which were collected at home on a repeated basis, both before and in association with infection. although we did not measure cytokine levels in normal subjects, these data show that children with asthma are apparently capable of a robust cytokine response to viral infection, even during mild exacerbations. in addition, home collection of nasal lavage specimens appears to be a practical tool for studying the natural history of viral infection in children. our results expand upon prior work characterizing cytokine responses detectable in respiratory secretions of asthmatics in association with viral illnesses. pizzichini et al. [ ] found that, compared to days after infection, sputum levels of cxcl , eosinophil cationic protein and fibrinogen from six adult asthmatics were significantly elevated days after the start of a coldinduced exacerbation. furthermore, cxcl levels correlated with the number of sputum neutrophils. teran et al. asthma [ ] found that, compared to asymptomatic periods, levels of major basic protein, ccl and ccl were increased in the nasal aspirates of children during acute viral-induced exacerbations. grissell et al. asthma [ ] found that compared to a group of stable asthmatics, levels of ccl and il- were significantly elevated in the induced sputa of older children and adults suffering from acute viral-induced asthma exacerbations, along with neutrophils and eosinophils. cxcl and ccl were also increased, although not significantly. finally, santiago et al. [ ] found that compared to control samples, ccl and ccl were increased in the nasal aspirates of asthmatic children - h after upper respiratory tract infections. chemokine levels correlated with macrophages in the nasal aspirate and upper respiratory symptoms scores. in the present study, in which patients were used as their own controls, we confirmed the above changes in cxcl , ccl , ccl , and ccl , and provide new data showing significant increases in cxcl , ccl , ccl , and ccl . importantly, we found that increases in these chemokines were sustained throughout the week following viral infection. we also examined the relationship between cytokines and respiratory symptom score. interestingly, we found that some cytokines correlated with symptom score (ifn-c, cxcl , ccl , ccl , ccl , and ccl ) whereas others did not (cxcl , ccl , ccl or ccl ). although we did not examine lower airway cytokine levels, it is tempting to speculate that the former set of cytokines is responsible for the acute asthmatic response to viral infection, whereas the latter set may modulate future immune responses. based on differences in ifn-b and ifn-k production between cells isolated from controls and asthmatic subjects [ ] [ ] [ ] , it has been proposed that patients with asthma are prone to rhinovirus-induced exacerbations due to deficient ifn production on the other hand, other in vitro studies showed no differences in rhinovirus-induced gene expression in epithelial cells isolated from asthmatic and healthy subjects [ , ] , and con-trol and asthmatic subjects experimentally infected with rhinovirus show no difference in respiratory tract viral titre or copy number [ ] . we found that viral infection during natural colds significantly increased the levels of ifn-c protein and ifn-k mrna in the nasal aspirates. viral infection also significantly increased the expression level of rig-i and irf- , each of which are inducible by type i ifns [ , ] . together, these data suggest that asthmatic children are capable of a functional ifn response. on the other hand, we failed to detect ifn-a or ifn-b protein in nasal aspirates, and there was no significant change in mrna levels during natural colds. detection of type i ifns in respiratory secretions is made difficult by the low sensitivity of commercially available assays, physiologically low levels of these cytokines in biological fluids (particularly in fluids without sufficient numbers of plasmacytoid dendritic cells) and presence of natural inhibitors (e.g. soluble receptors). also, we could not compare the results from our experimental subjects to children without asthma. our finding that irf mrna is elevated after respiratory viral infection in children with asthma is validated by a recent study analysing patterns of gene expression in nasal lavage samples from children experiencing picornavirus-induced asthma exacerbations [ ] . consistent with our previous work examining the requirement of irf for rhinovirus-induced gene expression in cultured airway epithelial cells [ ] , coexpression analysis demonstrated irf to be a major hub connecting ifn-mediated responses in virus-induced asthma exacerbations. in this study, we piloted a new pcr-ldr-based method for respiratory virus detection. twelve of samples were concordant between the two viral detection techniques; in two cases, samples were positive by seeplex and negative by pcr-ldr. the precise cause of the discrepancy between the two tests is not certain, but the seeplex test, which relies on visual detection of bands on agarose electrophoresis gels, may be liable to false positives. we also optimized our pcr-ldr test for maximum specificity, with the goal of avoiding false positives. this may have resulted in a reduction in sensitivity if copy number of viral nucleic acid was low. we measured the effect of natural colds on feno. previous studies have shown feno to be a reliable measure of eosinophilic airway inflammation, steroid responsiveness, and clinical control in children and adults with allergic asthma [ ] [ ] [ ] [ ] . feno levels have also been shown to increase during emergency steroid treatment of acute asthma exacerbations [ , ] . in contrast, we found that despite increases in airway cytokines and respiratory symptoms, there was no increase in feno during natural colds in children relative to their baseline state. these data are consistent with the notion that airway inflammation in the context of viral-induced asthma exacerbations is different in character than that associated with chronic asthma. there are several limitations to our study. first, although nasal sampling has the advantage of increased safety and decreased participant burden, sputum speci-mens more closely reflect the response of the lower airways to viral infection. however, as noted above, levels of ifn-c, cxcl , ccl , ccl , ccl , and ccl significantly correlated with respiratory symptoms, including lower respiratory tract symptoms such as cough, wheeze, chest tightness, and shortness of breath, each of which are indicative of asthma exacerbation. in addition, previous studies have found similar changes in nasal aspirate and sputum cytokine concentrations following viral exacerbations of asthma [ , ] . finally, observations demonstrate consistent effects of nasal and bronchial provocation [ ] . second, we did not measure inflammatory cells in the nasal aspirates. we therefore could not assess the functional effects of chemokine release in our subjects. previous studies have shown recruitment of neutrophils, eosinophils, and macrophages to the respiratory tract following rhinoviral infection of asthmatic subjects [ , , , , [ ] [ ] [ ] [ ] . also, we cannot determine the cellular source of the mrnas measured in our study. pilot analysis showed primarily epithelial cells and neutrophils in the nasal aspirates, suggesting that epithelial cells were the primary source of mrna. third, as noted above, we did not compare responses of asthmatic subjects to controls. a recent study showed that asthmatics and non-atopic subjects experience similar colds, including chemokine responses, following experimental rhinovirus infection [ ] . fourth, although we were able to detect significant differences in many cytokines following upper respiratory tract infection, a fraction of the samples were too dilute to detect mrna. (there may have been other reasons for the loss of rna, for example sample ribonucleases). dilution may also affect the concentration of measured biomarkers. we did not adjust measurements by total protein content, as vascular leakage due to inflammation may alter protein concentrations in children with upper respiratory infections [ ] . in our experience, total protein is difficult to measure in many nasal (and tracheal) aspirate specimens, even when cytokines are readily detectable, and therefore normalization for total protein does not increase the reliability of the concentrations. finally, studying naturally occurring colds in children using home-based methods inherently involves some variability in sample timing and handling that is not present in the controlled environment of the laboratory. yet this approach also offers an opportunity to observe 'real world' scenarios. our ability to detect statistically significant changes in specific mrnas and proteins using the current study design suggests that this is a viable approach for use in future investigations, for example, studies examining whether an early adjustment of asthma medications could abort or temper the impact of respiratory viral infections. we conclude that in children with asthma, naturally occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, ifns, and ifn-responsive genes. cytokine responses are sustained the first week following viral infection. future analyses of controls and asthmatics which combine the determination of cytokine responses and asthma health outcomes will provide further insight into the cellular pathways responsible for virus-induced asthma exacerbations. respiratory viruses and exacerbations of asthma in adults community study of role of viral infections in exacerbations of asthma in - year old children persistence of rhinovirus rna after asthma exacerbation in children respiratory syncytial virus infection 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il- production gene expression profiles during in vivo human rhinovirus infection: insights into the host response similar colds in subjects with allergic asthma and nonatopic subjects after inoculation with rhinovirus- inflammatory indices in induced sputum: a feasibility study rantes, macrophage inhibitory protein -a and the eosinophil product major basic protein are released into upper respiratory secretions during virus-induced asthma exacerbations in children il- gene expression in acute virus-induced asthma role of monocyte chemotactic protein- and - in children with virus exacerbation of asthma lung and blood institute. national asthma education and prevention program expert panel report : guidelines for the diagnosis and management of asthma. bethesda, md: national heart, lung and blood institute identification of gaps in the diagnosis and treatment of childhood asthma using a community-based participatory research approach rhinovirus illnesses during infancy predict subsequent childhood wheezing improved method for collection of nasal mucus an integrated microfluidic device for influenza and other genetic analyses linear mixed models for longitudinal data rhinovirus enters but does not replicate inside monocytes and airway macrophages rhinovirus replication in human macrophages induces nf-jb-dependent tumor necrosis factor alpha production the role of p mapk in rhinovirus-induced monocyte chemoattractant protein- production by monocytic-lineage cells human rhinovirus induces robust ip- release by monocytic cells, which is independent of viral replication but linked to type i interferon receptor ligation and stat activation rhinoviruses induce interleukin- mrna and protein production in human monocytes respiratory virus induction of alpha-, beta-and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus role of deficient 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declare no conflicts of interests. key: cord- -qm h qui authors: jeon, young joo; yoo, hee min; chung, chin ha title: isg and immune diseases date: - - journal: biochim biophys acta mol basis dis doi: . /j.bbadis. . . sha: doc_id: cord_uid: qm h qui isg , the product of interferon (ifn)-stimulated gene , is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains. isg is synthesized as a precursor in certain mammals and, therefore, needs to be processed to expose the c-terminal glycine residue before conjugation to target proteins. a set of three-step cascade enzymes, an e enzyme (ube l), an e enzyme (ubch ), and one of several e ligases (e.g., efp and herc ), catalyzes isg conjugation (isgylation) of a specific protein. these enzymes are unique among the cascade enzymes for ubiquitin and other ubiquitin-like proteins in that all of them are induced by type i ifns or other stimuli, such as exposure to viruses and lipopolysaccharide. mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by isg . some of them are type i ifn-induced proteins, such as pkr and rig-i, and some are the key regulators that are involved in ifn signaling, such as jak and stat , implicating the role of isg and its conjugates in type i ifn-mediated innate immune responses. however, relatively little is known about the functional significance of isg induction due to the lack of information on the consequences of its conjugation to target proteins. here, we describe the recent progress made in exploring the biological function of isg and its reversible modification of target proteins and thus in their implication in immune diseases. since the discovery of type i interferons (ifnα and ifnβ) in , they have widely been used as clinical drugs [ , ] . for example, ifnα has been used for the treatment of chronic hepatitis b and hepatitis c viruses and of several cancers, such as leukemia, and ifnβ is effective for treating multiple sclerosis. type i ifns exert their effects through the activation of janus tyrosine kinase (jak)/signal transducer and activator of transcription (stat) signaling pathway [ ] . upon binding of the ifns to their cell surface receptors (ifnar), the receptorassociated kinases jak and tyrosine kinase (tyk ) become activated and phosphorylate tyrosine residues in the cytoplasmic tails of the receptor subunit, ifnar . the phosphorylated subunit provides specific docking sites for the activation of stats by jak /tyk mediated phosphorylation [ , ] . activated stats dissociate from the receptor and translocate into the nucleus, where they act as transcription factors that bind to the promoter regions of ifn-stimulated genes (isgs) [ ] . stat /stat heterodimers associate with ifn regulatory factor (irf- ) to form the transcription complex ifn-stimulated gene factor (isgf ), which binds to ifn stimulatory response elements (isres) within the promoters of isgs [ ] . on the other hand, homodimers and heterodimers of stat and stat bind to gammaactivated sequence (gas) response elements [ ] . the isg proteins generated by these pathways play key roles in the induction of innate and adaptive immune responses [ , ] . protein modifications by ubiquitin and ubiquitin-like proteins (ubls), including sumo and nedd , have emerged as critical regulatory processes, such as in the control of cell cycle, stress response, signaling transduction, and immune response [ ] [ ] [ ] [ ] [ ] . moreover, deregulation of the modification systems gives rise to numerous human diseases, such as cancers, neurodegenerative diseases, and immune diseases. conjugation of ubiquitin and ubls to target proteins involves threestep cascade enzymes: ubiquitin-and ubl-activating e enzymes, ubiquitin-and ubl-conjugating e enzymes, and ubiquitin-and ubl e ligases. protein modifications by ubiquitin and ubls are reversible processes that are catalyzed by isopeptidases, called deubiquitinating enzymes (dubs) and ubl-specific proteases (ulps), respectively. isg , a member of the ubl family, shows a significant sequence homology to ubiquitin. like ubiquitin, isg is conjugated to numerous cellular proteins via isopeptide bonds. this isg conjugation (isgylation) utilizes ube l as e enzyme, ubch as e enzyme, and some ubiquitin e ligases, such as efp and herc , as e enzymes (fig. ) . on the other hand, ubp (also called usp ) acts as a major isg -specific deconjugating enzyme. isg is not present in yeast, nematode (caenorhabditis), or insect (drosophila), indicating that the functions of isg and its modification are restricted to higher animals with evolved ifn signaling. isg is strongly induced by type i ifns [ , ] . viral infection also strongly induces isg [ , ] because one of its major host responses is the production of type i ifns. a number of proteins that are involved in antiviral signaling pathways, including rig-i, mda- , mx , pkr, stat , and jak , have been identified as target proteins for isgylation. moreover, recent studies have explored the biological functions of isg and its conjugation to target proteins. isg and its conjugation impair viral replication in vivo [ ] [ ] [ ] [ ] [ ] [ ] [ ] . conversely, certain viruses induce viral specific proteins that can deconjugate isg from its target proteins or prevent the genera-tion of isgylated proteins, thus abrogating the antiviral response [ , , ] . however, functional consequences of reversible isg modification of most target proteins, which are induced by viral infection, are still largely unknown. one of the key components of the innate immune response in regulating cancer development is the activation of type i ifn signaling pathways [ , ] . type i ifns suppress cell proliferation and promote apoptosis and, therefore, have been used in the treatment of several cancers, such as leukemia [ ] . notably, isg appears to function as an oncogenic protein as well as a tumor suppressor protein [ ] . the findings that cancer chemotherapeutics cause an increase in the level of isg conjugates suggest the role of isg as a tumor suppressor [ ] [ ] [ ] [ ] [ ] . conversely, the observations that deregulated overexpression of isg and enhanced isgylation are positively correlated with carcinogenesis implicate the oncogenic potential of isg protein [ ] [ ] [ ] . however, since ifns are induced on the development of many cancers and isg is an interferon-inducible protein, the enhanced expression of isg and its conjugation could be a side product of ifn response to cancer and may not play a general role in carcinogenesis. thus, further studies are required for clarification of the functional relationship between isg and carcinogenesis. . isg , the product of interferon-stimulated gene isg was originally identified by farrell et al. [ ] . because some antibodies directed against ubiquitin also react with isg , it was initially named as an ubiquitin cross-reactive protein (ucrp) [ ] . this cross-reactivity is explained by the fact that the -kda isg protein consists of two domains, each of which bears high sequence homology to ubiquitin. the primary sequences of the two ubiquitinlike domains that correspond to the n-and c-terminal regions of isg share % and % identities with ubiquitin, respectively (fig. ) . furthermore, both the n-and c-terminal domains of isg show a striking similarity in their tertiary structures to ubiquitin (fig. ) [ ] . like isg , fat , which is also a member of the ubl family, fig. . protein modification by isg . isg -specific proteases cleave off the c-terminal extensions from isg precursors to generate matured isg molecules. isg is activated by ube l (e ) at the expense of atp and is subsequently linked to the activating enzyme via thiosester bond. isg linked to ube l is transferred to ubch (e ) and then to a target protein with the aid of an isg e ligase, such as efp and herc . ubp functions in the reversal of the isgylation process by cleaving off isg molecules that are conjugated to the substrate proteins via isopeptide bonds. comprises two ubiquitin-like domains [ ] . fat is conjugated to a limited number of cellular proteins [ , ] . like ubiquitin, isg proteins in some species are synthesized as precursors that need to be processed before conjugation to target proteins (fig. ) [ ] . while isg molecules in human, mouse, and rat are translated as precursors containing the extensions of - amino acids in their c-termini, isg proteins in most other species, including fish and bovine, are synthesized as matured forms [ ] . the c-terminal lrlrgg sequence of isg , as that of ubiquitin, is essential not only for its conjugation to substrates but also for its recognition by the relevant processing proteases. however, this hexapeptide sequence is absent in the analogous position of the n-terminal domain. instead, a pro residue is present at the position of isg , thus preventing the incorrect processing that might generate the two cleaved ubiquitinlike domains of the -kda polypeptide [ ] . the c-terminal ubiquitin-like domain is sufficient for its activation by ube l and subsequent thioesterification of ube l and ubch [ ] . when overexpressed, the c-terminal domain of isg can be conjugated to cellular proteins. however, the level of cellular proteins conjugated by the c-terminal domain alone is much lower than that by full-length isg , suggesting that both domains of isg are required for efficient conjugation to cellular proteins. interestingly, the replacement of arg in human isg (arg in mouse) by ala ablates the binding of isg to ube l and subsequent transesterification of ubch [ ] . accordingly, the ability of the mutant isg to form protein conjugates in t cells is markedly diminished. furthermore, while expression of wild-type isg protects ifnar −/− mice from lethality after sindbis virus (snv) infection, expression of the arg-to-ala mutant form of isg confers no survival benefit. this mutation also attenuates the ability of isg to decrease snv replication in ifnar −/− mice or prolong the survival of isg −/− mice. thus, arg appears to serve as a binding site for ube l. type i ifns are capable of inducing isg [ ] . notably, the induction of isg shows biphasic kinetics. after ifnβ treatment, the increase in the level of free isg molecules is first observable within h, continues for the next h, and becomes maximal by about h. on the other hand, isg conjugates are observable at least h after the exposure to ifnβ, and their level dramatically increases between and h and continuously increases thereafter although at a slower rate. this delayed induction of isg conjugates indicates the requirement of isg -conjugating machinery that is expressed in the later periods after the treatment with type i ifns. isg is strongly induced by viral infection [ , ] . like other ifn-stimulated genes, the isg gene has a ′-cis-regulatory sequence called isre (interferon-stimulated response element) [ ] . a number of irfs, including irf- and irf- , bind to the isre for isg induction (fig. ) [ ] [ ] [ ] . irf- forms a complex with cbp/p coactivators for isg induction [ , ] . on the other hand, irf- interacts with stat and stat , leading to the formation of isgf complex that also induces isg . upon dsrna treatment, however, irf- induces isg independently of ifns [ ] . isg is also strongly induced by lipopolysaccharide (lps) [ ] [ ] [ ] . when macrophages are stimulated by lps, isg can be detected as early as h and its level becomes maximal at about h [ ] . infection of mice with bacille calmette-guérin (bcg) markedly induces isg in macrophages. moreover, this bacterial infection leads to isgylation of serpin a (serine protease inhibitor a), which was the first identified target protein, although its biological significance remains unknown. isgylation of serpin a may protect macrophages from lysosomal enzymes because a variety of lysosomal proteases are upregulated by ifnγ for the destruction of ingested bacteria and other pathogens. in addition, it has been shown that trif/ irf- signaling pathway is responsible for lps-mediated induction of isg and its conjugation to target proteins [ ] . isg is a target gene of nf-κb. lps induces isg in z/ cells that recapitulate aspects of the pre-b to immature b-cell transition in response to nf-κb activation, but not in . e, which is a nf-κb signaling defective mutant cell line, indicating that nf-κb activation is required for isg induction [ ] . ataxia telangiectasia (at) is a multifaceted autosomal recessive genetic disorder, characterized by the loss of coordination, progressive neuronal degeneration, immunodeficiency, and cancer proneness [ ] . nf-κb is constitutively activated in human fibroblasts derived from at patients [ ] . moreover, the level of isg is constitutively elevated in the at cells [ ] . because activated nf-κb is capable of inducing the expression of type i ifns, it appears that the elevation of isg level in the at cells is due to abnormal production of type i ifns. isg also is a target gene of p [ ] [ ] [ ] . activation of temperaturesensitive p leads to isg induction in the absence of exogenous stimuli. since cycloheximide treatment does not influence the increase in the level of isg mrna, isg induction appears to be a primary response to p . while type i ifns induce the accumulation of isg mrna in both wild-type or p -deficient cells, dsrna induces it only in wild-type cells, indicating that p is required for isg induction by dsrna but not for that by type i ifns. in addition, sequence analysis of the human isg gene has led to the identification of a putative p -responsive element that is located adjacent to the isg -specific isre [ ] . thus, in virus-infected cells, p seems to exert its antiviral function by the induction of isg in addition to the induction of apoptotic signaling [ ] . jnk induces the expression of isgs, such as isg , isg , and igtp [ ] . swiss t cells expressing constitutively active mkk -jnk β fusion protein show increased resistance to apoptosis induced by vesicular stomatitis virus (vsv) infection, suggesting the involvement of jnk signaling pathway in antiviral response. recently, pi k has been shown to control both ifnα-and ifnγ-induced expression of isgs, including isg , at both transcriptional and translational levels [ ] . ifn-mediated antiviral response is defective in cells lacking p α/p β, the regulatory subunits of pi k, suggesting the involvement of pi k signaling pathway in innate immune response. isg is induced by certain genotoxic stresses. for example, treatment with camptothecin, an inhibitor of topoisomerase i, leads to an increase in the level of isg mrna, and this increase requires protein synthesis and a functional p protein [ ] . interestingly, ifns and jak/stat are dispensable for camptothecin-mediated induction of isg . isg conjugates generated by camptothecin are distinct from those produced by type i ifns, suggesting that different protein substrates are targeted for isgylation. however, camptothecin markedly increases ifn-induced isgylation of cellular proteins. furthermore, treatment with both ifns and camptothecin causes synergistic killing of colorectal cancer xenografts in nude mice, suggesting that the combination of the drugs can be an effective therapeutic strategy [ ] . retinoic acid upregulates the levels of isg , its conjugates, and ube l in acute promyelocytic cells [ , ] . the retinoic acid-mediated accumulation of isg and its conjugates occurs in retinoic acid-sensitive leukemic cells but not in retinoic acid-resistant cells and the pattern of accumulated isg conjugates is similar to that observed by the exposure to type i ifns. in addition, several of the type i ifninduced proteins, such as irf- and ( ′- ′) oligoadenylate synthetase, are induced by retinoic acid [ ] [ ] [ ] . treatment with retinoic acid also leads to an increased secretion of type i ifns into culture media. blockade of ifnar with a neutralizing antibody prevents retinoic acidmediated accumulation of isg and its conjugates. thus, retinoic ns b inhibits the thioesterification of ube l and thereby the generation of isg conjugates that are required for antiviral response. plpro, otu protease, and viral e protein mediate the removal of isg from its conjugates. acid seems to elevate the levels of isg and its conjugates by stimulating cells to secrete ifns. ube l is a -kda protein that shows a % identity in amino acid sequence to the human ubiquitin-activating e enzyme (ube ) [ ] . ube l expressed in baculovirus forms a thioester bond with isg but not with ubiquitin [ ] , indicating that it is a specific isg activating e enzyme. in addition, ube l has a c-terminal ubiquitinfold domain that is required for the transfer of isg from ube l to ubch as well as for the binding of ube l to ubch [ ] . as expected, ube l-deficient mice are not capable of producing isg conjugates upon stimuli [ ] . in ube l −/− macrophages treated with lps and in ube l −/− mouse embryonic fibroblasts (mefs) treated with ifns, ubiquitination of cellular proteins occurs normally, but isgylation of proteins is completely abolished, confirming the strict requirement of ube l for isgylation. the ube l gene is located in the d f s locus of chromosome q [ ] . ube l can be detected in the jejunum, colon, lung, liver, tonsils, testis, and skin but not in the spleen and pancreas. significantly, ube l is not detectable in all tested human lung cancer cell lines [ , ] , implicating a tumor-suppressive role of ube l. this repressed expression in lung cancer cell lines is intriguing because a region of chromosome q, in which the ube l gene resides, is often deregulated in preneoplastic or neoplastic epithelial tissues [ ] . however, upon generation of ube l −/− /k-ras la mice, it has recently been shown that the loss of isgylation does not affect tumor spectrum, tumor pathology, or survival of k-ras la mice [ ] . nonetheless, it is possible that ube l deficiency and k-ras mutation are two separate pathways in lung cancer development. additional works with other lung cancer mouse models are necessary to clarify the potential tumor suppressor function of ube l in k-ras mutation-independent human lung cancers. ubch , an isg -conjugating e enzyme, is induced by type i ifns and viral infection [ ] [ ] [ ] . ubch is also induced by type i ifns and forms a thioester intermediate with isg , suggesting that ubch has the potential to function as an isg -conjugating e enzyme [ ] . however, the amount of the thioester intermediate formed by ubch is much lower than that by ubch , indicating that ubch is a major e enzyme for isg . ubch was originally identified as an ubiquitin-conjugating e enzyme by a yeast two-hybrid screening for its interaction with e ap [ ] . ubch was later shown to also interact with other ubiquitin e ligases, such as parkin, dorfin, staring, efp, and rlim and function as an ubiquitin-conjugating e enzyme [ ] [ ] [ ] [ ] [ ] [ ] . thus, ubch serves as an e enzyme for both ubiquitin and isg . however, although ubch is the most closely related e to ubch , it does not function in isg conjugation [ ] . moreover, ubch shows a higher affinity to ube l than ubch , while ubch binds more strongly to ube than ubch . in addition, it has been shown that two structural elements within the e n-terminal region are responsible for the differential interaction of ubch and ubch with ube l. thus, ubch may preferentially, but not exclusively, function as an e enzyme for isg . interestingly, ubch and isg can act as functional regulators of rnf , an ubiquitin e ligase of rig-i [ ] . in the absence of isg , ubch interacts with rnf and interferes with rig-i ubiquitination. upon overexpression of isg , however, the interaction of ubch with isg leads to the dissociation of ubch from rnf , thus allowing the association of rnf with ubch for rig-i ubiquitination. the overlapping function of ubch as an e enzyme for both ubiquitin and isg raises a possibility that some ubch -interacting ubiquitin e ligases can also function as isg e ligases. indeed, the ubch -interacting protein efp (estrogen-responsive finger protein) that has a ring finger domain serves as an isg e ligase [ ] . in the absence of isg , ubch and efp may function as e and e enzymes for ubiquitination, respectively. upon isg induction, however, isg may compete with ubiquitin for binding to ubch , allowing ubch and efp to serve as the enzymes for isgylation of - - σ. replacement of the active site cys residue in the ring domain disrupts efp-mediated isgylation of - - σ. like ubch , efp is a type i ifninducible protein [ ] . interestingly, efp can isgylate itself on the lys residue and this auto-isgylation negatively regulates the isg e ligase activity of efp toward - - σ, suggesting that a feedback loop is operating for the control of the protein isgylation [ , ] . an additional ring-containing ubiquitin e ligase, called hhari (human homolog of drosophila ariadne), also serves as an isg e ligase for ehp [ ] . herc (hect domain and rcc -like domain containing protein ) is an isg e ligase that contains the hect domain [ ] [ ] [ ] . like efp, herc is a type i ifn-inducible protein. knockdown of herc by using sirna prevents ifn-mediated increase in the total level of isgylated cellular proteins, unlike that of efp, which blocks the isgylation of - - σ with little or no effect on the total level of isgylated proteins. thus, it appears that herc serves as a general ifn-induced isg e ligase. like efp, herc itself is a target for isgylation, but its functional significance is unknown. . . . ubp and other isg -specific proteases ubp cleaves off isg from its protein conjugates that are linked via isopeptide bonds [ ] . ubp can also cleave the peptide bonds immediately after the lrlrgg sequence in isg fusions [ ] . thus, ubp appears to also function in the processing of isg precursors to generate matured isg molecules. however, apart from ubp , additional isg -specific proteases must exist because isg precursor is processed to its matured form in ubp -deficient mice [ ] . the overlapping function of some e and e enzymes in the conjugation of both isg and ubiquitin also implies the existence of promiscuous dubs that can serve as isg -specific proteases. indeed, several dubs, including usp , usp , usp , and usp , have been identified as the candidates that can function as isg -processing and/or deconjugating enzymes [ ] . in addition, it has been reported that recombinant isg precursors can be properly processed by a -kda enzyme in the extracts of human lung fibroblasts cell line a , whose activity is unaffected by type i ifn stimulation [ ] . this -kda enzyme is a cysteine protease and shows a partial similarity in its amino acid sequence with that of the human ortholog of yeast ubp or ubp -related protein. however, deletion of the ubp gene in mouse leads to a massive increase of isg conjugates in tissues without affecting the level of ubiquitin conjugates, indicating that ubp is the major isg -specific protease that deconjugates isg from its target proteins. thus, it appears likely that the above-mentioned dubs as well as the -kda cysteine protease function primarily in the processing of isg precursors to generate matured isg molecules. ubp is induced by type i ifns, and this induction requires a functional jak/stat signaling pathway [ ] . ifnβ induces ubp more strongly than ifnα and dsrna, but ifnγ barely induces it [ ] . ubp is also induced by lps [ ] . irf- is responsible for lps-mediated induction of ubp , while irf- is involved in its induction to a basal level. however, both irf- and irf- are required for optimal responsiveness to lps. interestingly, ubp induction is upregulated by the acute myelogenous leukemia (aml )-eto fusion protein that is created by t( ; ), suggesting a possible involvement of ubp in hematopoiesis [ ] . however, it is unknown how aml -eto affects the up-regulation of ubp induction. in addition, ubp has been identified as a substrate for skp [ ] . skp promotes the ubiquitination of ubp and subsequent degradation by the proteasomes, suggesting that the scf skp may be involved in the regulation of type i ifn signaling by controlling the stability of ubp . the human ubp gene maps to the chromosome q . [ ] . this region, known as digeorge syndrome critical region, is consistently deleted in digeorge syndrome and related disorders, suggesting that ubp may be involved in the development of thymus or differentiation of t cells. ubp -deficient mice are viable and resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that develop in wildtype mice upon intracerebral inoculation of lymphocytic choriomeningitis virus (lcmv) and vsv, respectively [ , ] . furthermore, survival of ubp −/− mice after lcmv infection correlates with severe inhibition of lcmv replication as well as with an increase in the level of isg conjugates in the brain. none of the ubp −/− mice infected with lcmv died or developed clinical symptoms by day after infection, whereas all lcmv-infected wild-type mice died by day infection. these findings strongly suggest that ubp deficiency causes an unfavorable environment for lcmv replication. in addition, ubp −/− mef cells exhibit enhanced type i ifn-mediated resistance to cytopathic effect caused by vsv and snv. these findings strongly suggest that ubp serves as a negative regulator of innate immune response against viral infection. however, the enhanced resistance to viral infection in ubp −/− mice cannot be rescued in ubp −/− / isg −/− or ubp −/− /ube l −/− double knockouts, indicating that the phenotypic alterations are not associated with the protein modification by isg [ , ] . thus, ubp , independently of its isopeptidase activity, may have another biological function. ubp −/− cells are hypersensitive to type i ifns, resulting in a dramatic increase in the level of isgylated proteins, which is associated with the enhanced and prolonged jak/stat signaling [ ] . however, ubp , independently of its catalytic activity, also functions as a negative regulator of type i ifn signaling [ ] . this ubp action is achieved through a direct interaction between ubp and ifnar , a subunit of type i ifn receptor. binding of ifnar to ubp interferes with the interaction between jak and the receptor, leading to the inhibition of downstream phosphorylation cascade and other signaling events. in addition, complementation of ubp −/− cells with a catalytically inactive mutant of ubp leads to the inhibition of stat phosphorylation to a level seen by that with wild-type ubp . moreover, down-regulation of the total level of isg conjugates by sirnamediated knockdown of ube l in ubp −/− cells shows little or no effect on stat phosphorylation in comparison with that seen in ubp +/+ cells, indicating that the deisgylating activity of ubp is not required for its inhibitory effect on type i ifn signaling. this conclusion is further corroborated with the findings that phenotypic alterations in ubp −/− mice are not influenced by the lack of isg in ubp −/− /isg −/− double knockout mice [ , ] . the isopeptidase-independent action of ubp is also involved in the replication of hbv (hepatitis b virus) [ ] . ubp −/− cells show an increased induction of isgs in response to type i ifns, indicating that the lack of ubp results in a strengthened immune response. consistently, the steady-state level of hbv dna is substantially reduced in ubp −/− mice in comparison with that in ubp +/+ mice. thus, approaches that modulate ubp level could be used as therapeutic potentials in treating viral infection, especially for viruses sensitive to type i ifn signaling. in addition to the protection against hbv, ubp deficiency increases the resistance to oncogenic transformation by bcr-abl (breakpoint cluster region abelson leukemia protein), a fusion protein consisting of the n-terminal portion of bcr joined to most of the abl tyrosine kinase [ ] . this resistance to leukemia development is heavily dependent on the activation of type i ifn signaling pathway. loss of type i ifn signaling through the loss of ifnar results in a reversal of the original resistance to the leukemia development observed in mice that received a transplant of bcr-ablexpressing ubp -deficient donor cells. thus, it appears that inhibition of the negative effect of ubp on type i ifn signaling could enhance innate immune response against cancer development. by using a combination of affinity purification and mass spectrometry, hundreds of target proteins for isgylation have been identified. some of them are type i ifn-induced proteins, including pkr, mxa, hup , and rig-i [ ] . some are key regulators that are involved in type i ifn signaling, such as plcγ , jak , erk , and stat [ ] . most other targets are constitutively expressed and function in diverse cellular pathways, including translation, glycolysis, cell motility, protein modification, intracellular protein trafficking, rna splicing, chromatin remodeling, cytoskeletal organization, and stress responses [ , , ] . these findings implicate the role of protein isgylation in type i ifn-induced immune responses as well as in the control of numerous fundamental cellular pathways. isg is synthesized in many cell types and secreted from human monocytes and lymphocytes [ ] . both native and recombinant isg induce the synthesis and secretion of ifnγ from b-cell-depleted lymphocytes, implicating the role of isg as a cytokine that modulates immune response [ ] . treatment with human isg , but not its precursor form, leads to an increase in dna synthesis in cultured primary blood lymphocytes in a dose-dependent fashion, suggesting that isg can act as a mitogen and that the processing of isg precursor is required for the generation of biologically active isg [ ] . furthermore, isg has been identified as a member of the cytokine cascades. upon viral infection, type i ifns produced in infected cells induce the synthesis of isg . these isg molecules can be secreted or released by lysis of the infected cells. they may then induce the production of ifnγ from t cells, augment nk cell proliferation, activate non-major histocompatibility complex-restricted cytolytic lymphocytes, and activate monocytes and macrophages via the induced ifnγ. filamins are nonmuscle actin-binding proteins that comprise a family of three members: filamin a, b, and c [ , ] . these filamin isoforms are large cytoplasmic proteins that play important parts in cross-linking cortical actin filaments into a dynamic three-dimensional structure. recently, it has been shown that filamin b functions as a scaffold that links between activated rac and a jnk cascade module for mediating type i ifn signaling [ , ] . filamin b interacts with rac , mekk , mkk , and jnk and enhances their sequential activation in response to type i ifns, thereby promoting jnk activation and apoptosis. this acceleration of jnk-mediated apoptosis provides a biological basis for antitumor and antiviral functions of type i ifns. furthermore, it has been revealed that type i ifns induce isgylation of filamin b, which leads to dissociation of rac , mekk , and mkk from the scaffold protein and thus to the prevention of prolonged activation of type i-induced jnk signaling pathway [ , ] . in contrast, blockade of filamin b isgylation by substitution of the isg acceptor site lys with arg or by sirna-mediated knockdown of ubel prevents the release of the signaling molecules from filamin b, resulting in persistent promotion of jnk activation and jnk-mediated apoptosis. these findings indicate that isgylation of filamin b serves as a negative feedback regulatory gate for desensitization of type i ifn-induced jnk signaling, thus providing an important mechanism for the survival of uninfected bystander cells. pp cβ dephosphorylates tak and suppresses tak /tab mediated iκbα degradation, thus controlling nf-κb signaling pathway, which plays a critical role in innate and adaptive immunity and cancer [ ] [ ] [ ] [ ] . pp cβ is a target for isgylation, and this modification blocks the suppressive function of the protein phosphatase against tak / tab -mediated nf-κb activation [ ] . this conclusion is based on the observation that overexpression of ube l, ubch , and isg blocks the suppressive effect of pp cβ on nf-κb activation, but not that of its mutant, in which the isg acceptor sites lys and lys are replaced by arginine. thus, isgylation of pp cβ seems to play a role in the control of nf-κb pathway. ubc , an ubiquitin-conjugating e enzyme, is a target for isgylation [ , ] . isg is conjugated to lys of ubc , which is very closely located to its active site, thus preventing the formation of a thioester bond with ubiquitin. since mms , which forms a heteromeric complex with ubc , interacts with both unmodified and isgylated ubc , the inhibitory effect of ubc isgylation on the thioesterification of ubiquitin seems to be achieved by the inability of isgylated ubc to accept ubiquitin to its active site or by blocking the recognition of ubiquitin e enzyme and subsequent transfer of ubiquitin from the e enzyme to ubc . ubc is known to mediate the generation of lys -linked poly-ubiquitin chains that are conjugated to a variety of signaling molecules [ , ] . thus, it is possible that isgylation of ubc may play an important role in the control of signal transduction pathways, such as nf-κb pathway, which are associated with lys -linked poly-ubiquitination [ ] . ubch and ubch also are targets for isgylation [ ] . isgylation of ubch occurs in response to type i ifns and blocks the thioesterification of ubiquitin, like that of ubc , suggesting that isgylation of ubch may also lead to the suppression of its ubiquitin-conjugating activity. initial efforts that intend to determine the role of isg in antiviral responses appeared unsuccessful. ube l −/− mice are fertile and exhibit normal antiviral responses against vsv and lcmv infection, indicating that ube l and protein isgylation may not be essential for ifn signaling [ ] . furthermore, isg −/− mice are also fertile and display no obvious abnormalities [ ] . lack of isg does not affect the development and composition of the main cellular immune system. the ifn-induced antiviral and immune responses directed against vsv and lcmv are not significantly altered in the absence of isg . in addition, ifn-or endotoxin-induced stat phosphorylation as well as the expression of typical stat target genes remain unaffected by the lack of isg , indicating that isg is dispensable for stat -mediated ifn signaling. however, the function of isg as an antiviral effector has come to the front. unlike the infection by vsv and lcmv, ube l −/− mice display markedly increased susceptibility to influenza b virus infection, with only % survival of ube l −/− mice for days, compared to % survival of ube l +/+ mice [ ] . furthermore, both of the kinetics of lethality and the overall survival of ube l −/− mice are identical to those of isg −/− mice, indicating that the antiviral activity of isg against influenza b virus is mediated by its conjugation to target proteins. the predominant site of isg action during influenza virus infection resides within a stromal cell population. a likely candidate is the respiratory epithelium, since it is the site of influenza virus replication. this is the first phenotype described for ube l −/− mice, which were previously found to have no defect in response to vsv or lcmv infection [ ] . in addition, it has recently been shown that both the synthesis of influenza a virus protein and the early rate of the virus replication are inhibited by isg conjugation of cellular proteins [ ] . isg −/− mice also exhibit increased susceptibility to infection by a number of other viruses, such as influenza a/wsn/ and b/lee/ viruses, herpes simplex virus type and murine gamma herpes virus , and snv [ ] . in addition, isg is induced after snv infection, and this induction is markedly attenuated in ifnar −/− mice, indicating that induction of isg by snv infection is dependent on type i ifns [ ] . isg induction protects against snv-induced lethality and decreases the virus replication in multiple organs. the increased susceptibility of ifnar −/− mice to snv infection can be rescued by the expression of wild-type isg having the c-terminal diglycine residues but not by that of an isg mutant, of which the c-terminal diglycine is replaced by dialanine, again indicating that the antiviral action of isg requires its conjugation to target proteins. type i ifn-mediated inhibition of human immunodeficiency virus (hiv) assembly and release has been shown to correlate with the induction of isg [ ] . furthermore, overexpression of isg mimics the ifn effect and inhibits the release of hiv- virions without having any effect on the synthesis of the viral proteins in infected cells [ ] . in cells infected with hiv- provirus, overexpression of isg and ube l causes a complete inhibition of hiv- replication. on the other hand, coexpression of ubp can partially rescue the release of hiv- in isg -expressing cells. intriguingly, isg expression specifically inhibits the ubiquitination of gag and tsg and disrupts their interaction, thereby preventing assembly and release of virions from infected cells. expression of isg alone (i.e., without ube l) does not block the viral release, indicating that isgylation of target proteins, but not isg itself, is required for the inhibition of gag ubiquitination, although isgylation of either gag or tsg could not be detected. thus, isgylation of certain unknown viral and/or host proteins appears to play a critical role in the ifn-mediated inhibition of hiv- assembly and release. overexpression of isg or ube l with ubch has been shown to inhibit budding of ebola virus vp virus-like particles (vlps) [ , ] . ebola virus is a member of the filoviral family of negative-sense rna viruses. the vp matrix protein is a key structural protein that is critical for the virion release. the efficient budding of vlps requires the interaction of overlapping l-domains (late-budding domains) in the vp protein with nedd , an ubiquitin e ligase, as well as with other members of the escrt pathway (e.g., tsg ) [ ] [ ] [ ] [ ] . isg overexpression decreases not only the level of vp detected in vlps but also the levels of endogenous nedd incorporated into budding vp vlps. nedd interacts with and is conjugated by isg . moreover, isg overexpression blocks the ability of nedd to ubiquitinate vp , leading to the prevention of l-domain-mediated budding of vp vlps. in addition, it has been shown that free isg specifically binds to nedd and inhibits the interaction of the e ligase with ubiquitin e enzymes (e.g., ubch ), thus preventing the transfer of ubiquitin from the e enzymes to nedd [ ] . these findings reveal a mechanism for the antiviral function of isg that involves the isgylation and inactivation of the host nedd e ligase. irf- is a target for isgylation, which can be induced by both type i ifns and viral infection [ ] . this isgylation prevents the ubiquitination and degradation of irf- in ndv (newcastle disease virus)-infected human fibrosarcoma ftgh cells, and promotes the translocation of irf- to the nucleus, where it binds to the ifnβ promoter. the relative level of irf- is significantly lower in ndvinfected isg −/− cells than in isg +/+ cells, indicating that the subversion of antiviral response is mediated by proteolysis of irf- . moreover, the degradation of irf- can be counteracted by the induction of isg . this finding provides a positive feedback mechanism for the induction of host antiviral response by isgylation-dependent stabilization of irf- . however, isg is typically conjugated to only a small fraction of target proteins. therefore, an important issue is how the small fractions of isgylated proteins, including irf- , can exert their biological functions. it has been proposed that if the small fractions of proteins that are modified by ubls (e.g., sumo and isg ) are localized to some functionally unique cellular site or the transient modification is sufficient to switch the protein into new state, their functions could efficiently operate [ , , ] . intriguingly, overexpression of isg leads to the appearance of irf- as punctates in the nucleus [ ] . this nuclear retention could allow irf- to exert its profound antiviral function, although only a small portion of irf- is isgylated upon the viral infection. an additional example is that upon overexpression of filamin b, ubch is recruited to membrane ruffles where filamin b is also concentrated with actin [ , ] . this recruitment of ubch , which otherwise resides throughout the cytoplasm and the nucleus, should allow the e enzyme to efficiently function in filamin b isgylation and thus in desensitization of type i ifn-induced jnk signaling (see above). the inducible nitric oxide synthase (inos) is not expressed under normal conditions, but like isg , it is induced by various stimuli, such as exposure to cytokines, microbes, or microbial products, resulting in the sustained production of no [ ] . upon stimuli, no as well as the products generated by its interaction with ros, such as peroxynitrite and peroxynitrous acid, are accumulated and utilized for the antibacterial or antiviral effects [ ] [ ] [ ] . no is also covalently attached to the thiol group of cysteine residues of proteins, causing their s-nitrosylation. this posttranslational modification serves as an important mechanism that mediates antibacterial and antiviral effects through the alteration in enzymatic activity, protein-protein interaction, and signal transduction [ ] . interestingly, the cysteine residue in isg can also be modified by no and this s-nitrosylation decreases the dimerization of isg , thereby increasing the availability as well as the solubility of monomeric isg for its conjugation to cellular proteins [ ] . moreover, treatment with s-ethylisothiourea, an inos inhibitor, reduces the level of isg conjugates, whereas overexpression of inos increases it. thus, inos may play a role in the enhancement of innate immune responses by mediating isg s-nitrosylation. ehp is an mrna ′cap structure-binding protein and acts as a translation suppressor by competing with eif e for binding to the cap structure [ ] . ehp is a target protein for isgylation and this modification substantially increases its cap structure-binding activity [ ] . this is the first report that shows "gain of function" by ifn-induced isgylation, thus providing a mechanism by which a small fraction of any isgylated protein can generate profound biological effects in response to ifns or pathogen infections. interestingly, ehp fused with isg at either of its n-or c-terminus dramatically enhances the cap structure-binding activity, indicating the isg fusion protein can mimic the isgylated ehp. since ifns inhibit the translation of viral mrnas while allowing normal translation of the majority of cellular mrnas [ ] , it is possible that isgylated ehp acts as a viral mrnaspecific translation inhibitor in a cap binding-dependent manner. rig-i senses intracellular virus-specific nucleic acid structures and as an early antiviral response induces the production of type i ifns, which in turn activates the expression of rig-i, thus generating a positive feedback loop for further accumulation of rig-i [ ] [ ] [ ] [ ] [ ] . intracellular dsrna activates nf-κb and irf- /irf- through rig-i and the mitochondrial adaptor protein ifn promoter stimulator (ips- ) [ ] [ ] [ ] [ ] . furthermore, rig-i is a target protein for isgylation [ , ] . however, isgylation of rig-i leads to a reduction in the levels of both basal and virus-induced ifn production. consistently, the basal mrna and protein levels of rig-i are significantly higher in ube l −/− cells than in ube l +/+ cells. based on these observations, it has been proposed that a negative feedback loop is operating for fine-tuning the strength of rig-i-mediated signaling to maintain a balance between innate immune response and hypersensitivity during antiviral responses. in addition, ubiquitination of rig-i by trim is required to mediate antiviral signaling responses, whereas that by rnf results in the proteasomal degradation of rig-i [ ] . thus, multiple positive and negative mechanisms appear to contribute to the maintenance of optimal rig-i level and thus to the control of rig-i-mediated signaling. viruses escape from the antiviral activity of isg by using different mechanisms. influenza b virus strongly induces isg during infection but specifically blocks isgylation of host proteins [ ] . this inhibition is mediated by the viral ns b protein, which interacts with isg and prevents the generation of isg conjugates under in vitro conditions as well as in infected cells (fig. ) . purified ns b inhibits the formation of isg -ube l-like thioester intermediate. isg conjugates accumulate in ifn-treated cells, but not in cells infected by influenza b virus, although similar amounts of isg are synthesized in both cells. moreover, isg conjugates are not detectable in cells infected with the virus expressing full-length ns b but are markedly accumulated in cells infected with the virus expressing truncated ns b, which cannot bind to isg , indicating that the interaction of ns b with isg is responsible for the inhibition of isg conjugation and thus of the antiviral function of isg . sars-coronavirus (sars-cov) produces a papain-like protease, called plpro, which can generate an authentic isg molecule by cleaving off a protein that is fused to the c-terminus of isg [ ] . it is possible that the activity of plpro may mimic the isg -deconjugating activity of ubp , thus favoring viral replication by counteracting protein isgylation. vaccinia virus (vacv) e protein is an early protein that interacts with isg through its c-terminal domain [ ] . whereas sirnamediated knockdown of isg enhances the viral replication, complementation of isg to isg −/− cells attenuates it. notably, the level of isg conjugates is much higher in isg -complemented isg −/− cells infected with the e -deficient virus than that with the wild-type virus, suggesting that vacv e protein is directly or indirectly involved in deconjugation of isg from cellular proteins. moreover, the virus lacking e protein that is unable to grow in isg +/+ cells can replicate in isg −/− cells. these findings suggest a new strategy for poxviruses to evade the host antiviral response through the viral e protein-mediated control of protein isgylation. the ovarian tumor domain (otu)-containing proteases from nairoviruses and arterviruses, two unrelated groups of rna viruses, are capable of deconjugating ubiquitin and isg , but not sumo, from cellular target proteins [ ] . purified otu protease cleaves off ubiquitin from both lys -and lys -linked poly-ubiquitin chains in vitro. remarkably, the expression of otu protease antagonizes the antiviral effects of isg and enhances the susceptibility to snv infection. it has been shown that approximately % of ifnar −/− mice survive after infection of the virus expressing both isg and an otu variant, of which the catalytic cys residue is replaced by ala. in contrast, only % of the mice survive when infected with the virus expressing both isg and wild-type otu protease, indicating that the antiviral response is mediated by isg , but not by other isgs, and that the immune-evading effect of otu protease requires its catalytic activity. these findings indicate that snv uses otu protease as a unique strategy to evade the host antiviral response. hepatitis c virus (hcv) is an enveloped virus that causes liver diseases, including cancer [ , ] . rig-i signaling induces a host response that controls hcv rna replication [ ] . however, hcv can evade this response in part through its ability to antagonize the relay of rig-i signaling to irf- [ ] . ns / a, a major serine protease expressed by hcv [ ] , cleaves ips- , causing the release of cleaved ips- from the mitochondrial membrane [ , ] . this cleavage results in the subcellular redistribution of ips- and loss of its interaction with rig-i, thereby preventing downstream activation of irf- and ifnβ induction. intriguingly, in liver tissues chronically infected by hcv, the ips- cleavage and its subcellular redistribution are associated with the lack of isg and its conjugates. among the hcvinfected tissues, isg and its conjugates can be detected only in the liver from patients, which has predominantly uncleaved, full-length ips- protein. these findings indicate that ns / a plays a critical role in evading the host antiviral response by attenuating the ifn amplification loop of rig-i and thus the expression of isgs, including isg , which are normally induced by rig-i-and irf- -dependent pathways. . . isg in tumorigenesis . . . isg as a tumor suppressor ube l has been shown to play an important role in the suppression of lung cancer growth. ube l overexpression inhibits the growth of human bronchial epithelial cells and lung cancer cells. furthermore, ube l promotes cyclin d isgylation and this modification leads to the destabilization of cyclin d , indicating that ube l confers growth suppression by targeting cyclin d [ ] . these findings provide a mechanism for the tumor-suppressive role of ube l [ , , , ] , although it is unknown how the stability of cyclin d is affected by its isgylation. acute promyelocytic leukemia (apl) is characterized by the accumulation of oncogenic pml/rarα fusion protein [ , ] . retinoic acid induces ube l and subsequent isgylation of pml/rarα [ , , ] . moreover, ube l-mediated isgylation of pml/rarα leads to a decrease in the level of pml/rarα, thus overcoming the oncogenic effects of the fusion protein [ , ] . on the other hand, treatment with n-acetyl-leu-leu-norleucinal, a proteasome inhibitor, prevents the degradation of isgylated pml/rarα, indicating that the proteasomes are involved in the control of pml/rarα stability. these findings implicate an important role of ube l in the suppression of leukemia. in many tumors and tumor cell lines, isg is highly elevated and extensively conjugated to cellular proteins [ ] . the level of isg mrna is significantly higher in cancerous mammary epithelial cells lines, such as bt , mda-mb , mda-mb , t d, and mcf , than in nonmalignant mammary cell lines, including hmec and mcf a. the level of isg protein is also higher in breast tumors than in normal tissues [ ] . in addition, a correlation between increased isg level and unfavorable prognosis in the survival of patients has been reported, suggesting a potential role of isg in breast cancer development, although the role of isg in breast carcinogenesis is unknown. significantly, the increased level of isg in tumor cells is associated with the decreased levels of ubiquitinated proteins, suggesting that isg plays an antagonistic role against ubiquitin-mediated proteolysis and that deregulation of ubiquitin-proteasome pathway is related with tumorigenesis [ ] . since some of the e (e.g., ubch ) and e enzymes (e.g., efp) can utilize both ubiquitin and isg for conjugation to target proteins, it seems possible that isg may potentially interfere with the ubiquitination pathway at the level of e and e enzymes, thus decreasing the level of ubiquitinated proteins in tumor cells. however, it has been shown that the level of ubiquitin conjugates in ubp −/− cells is nearly the same as that in ubp +/+ cells [ ] , despite the fact that treatment with type i ifns leads to a dramatic accumulation in the level of isg conjugates in ubp −/− cells as compared with that in ubp +/+ cells. furthermore, treatment with lactacystin, an inhibitor of the proteasome, shows little or no effect on the level of isg conjugates in either of ubp +/+ or ubp −/− cells whether or not exposed to ifnβ, while as expected the level of ubiquitinated proteins markedly increases in both cells. thus, it appears that isg and its conjugates by themselves do not interfere with the ubiquitination of cellular proteins and their subsequent degradation. the reason for the tumor-specific overexpression of isg in different tumors is unclear. one possibility is that the elevated expression of isg may be due to constitutively activated nf-κb in many tumor cells. this possibility is supported by an observation that isg overexpressing zr- - cells show a greater nf-κb activity than isg -underexpressing bt breast cancer cells [ ] . alternatively, the deregulated expression of ube l and ubp in certain tumors could contribute to the variation in the levels of isg conjugates in tumor biopsy samples. androgen receptor has been implicated in the initiation and progression of prostate cancer [ , ] . significantly, ube l and ubch are upregulated in prostate cancer lesions as compared to those in corresponding nonmalignant tissues [ ] . moreover, overexpression of ube l in lncap cells leads to a marked increase in the mrna and protein levels of androgen receptor in addition to an increase in the rate of cell proliferation. on the other hand, sirna-mediated knockdown of isg and ube l in lncap cells results in a significant decrease in the transcript and protein levels of androgen receptor. these findings suggest that isgylation machinery participates in a positive control of androgen receptor expression during the onset of prostate cancers, thereby promoting the tumor growth. since the discovery of ifns, a vast knowledge on the role of the cytokines in innate immune responses has been accumulated. isg is one of the most strongly induced isgs upon exposure to type i ifns, virus, lps, and other stresses, including certain genotoxic stresses [ , , , , , ] . considering that type i ifns play critical roles in innate immune responses regulating the antiviral responses as well as the cancer development, there is no doubt that isg and its conjugation to target proteins play critical roles in the type i ifninduced immune responses. however, the biological significance of protein modification by isg has been established only in several cases. therefore, much studies are required for clarification of the role of isg in innate immune responses against viral infection and cancer and thus for the control of immune diseases as well as for resolving the contradictory findings, such as the role of isg as a tumor suppressor versus an oncogenic protein. clinical uses of interferons virus interference: i. the interferon jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins contribution of stat sh groups to specific interferon signaling by the jak-stat pathway a single phosphotyrosine residue of stat required for gene activation by interferon-gamma stat proteins and transcriptional responses to extracellular signals the jak-stat pathway: cytokine signalling from the receptor to the nucleus stats and gene regulation antiviral actions of interferons type i interferons in host defense evolution and function of ubiquitin-like protein-conjugation systems isg , not just another ubiquitin-like protein the ubiquitin system protein regulation by monoubiquitin ubiquitin and its kin: how close are the family ties? accumulation of an mrna and protein in interferon-treated ehrlich ascites tumour cells interferon induces a -kilodalton protein exhibiting marked homology to ubiquitin influenza b virus ns protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg protein induced expression of the endogenous beta interferon gene in adenovirus type -transformed rat fibroblasts mice lacking the isg e enzyme ube l demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza b virus infection interferon-induced isg conjugation inhibits influenza a virus gene expression and replication in human cells virgin, ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses role of interferon-stimulated gene isg- in the interferon-omega-mediated inhibition of human immunodeficiency virus replication innate antiviral response targets hiv- release by the induction of ubiquitin-like protein isg isg inhibits ebola vp vlp budding in an l-domain-dependent manner by blocking nedd ligase activity isg inhibits nedd ubiquitin e activity and enhances the innate antiviral response ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses vaccinia virus e protein prevents the antiviral action of isg interferons alpha and beta as immune regulators-a new look apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis cancer immunotherapy: the interferon-alpha experience the interferon regulated ubiquitin-like protein, isg , in tumorigenesis: friend or foe? involvement of ube l in isg conjugation during retinoid-induced differentiation of acute promyelocytic leukemia ube l is a retinoid target that triggers pml/ raralpha degradation and apoptosis in acute promyelocytic leukemia camptothecin induces the ubiquitinlike protein, isg , and enhances isg conjugation in response to interferon interferon potentiates antiproliferative activity of cpt- against human colon cancer xenografts ube l represses pml/rar{alpha} by targeting the pml domain for isg ylation stage-associated overexpression of the ubiquitin-like protein, isg , in bladder cancer elevated expression of isg in tumor cells interferes with the ubiquitin/ s proteasome pathway exploration of global gene expression patterns in pancreatic adenocarcinoma using cdna microarrays crystal structure of the interferon-induced ubiquitin-like protein isg a mhcencoded ubiquitin-like protein (fat ) binds noncovalently to the spindle assembly checkpoint protein mad the ubiquitin-like protein fat forms covalent conjugates and induces apoptosis fat , a ubiquitinindependent signal for proteasomal degradation a -kda interferon-induced protein is derived by cooh-terminal processing of a -kda precursor molecular cloning of the fish interferon stimulated gene, kda (isg ) orthologue: a ubiquitin-like gene induced by nephrotoxic damage the interferon-inducible -kda ubiquitin homolog conjugates to intracellular proteins different roles for two ubiquitin-like domains of isg in protein modification virgin, isg arg and the isg -conjugating enzyme ube l are important for innate immune control of sindbis virus interferoninduced transcription of a gene encoding a -kda protein depends on an upstream enhancer element gene induction pathways mediated by distinct irfs during viral infection identification of a member of the interferon regulatory factor family that binds to the interferonstimulated response element and activates expression of interferon-induced genes characterization of specific dna-binding factors activated by double-stranded rna as positive regulators of interferon alpha/beta-stimulated genes interferon regulatory factor and crebbinding protein/p are subunits of double-stranded rna-activated transcription factor draf direct induction of interferon-gamma-and interferon-alpha/beta-inducible genes by double-stranded rna novel nemo/ikappab kinase and nf-kappa b target genes at the pre-b to immature b cell transition bacterial lipopolysaccharide and gamma interferon induce transcription of beta interferon mrna and interferon secretion in murine macrophages lipopolysaccharide activates the expression of isg -specific protease ubp via interferon regulatory factor serpin a is induced in activated macrophages and conjugates to a ubiquitin homolog enhanced antibacterial potential in ubp -deficient mice against salmonella typhimurium infection by up-regulating type i ifn signaling ataxia-telangiectasia: an inherited disorder of ionizing-radiation sensitivity in man. progress in the elucidation of the underlying biochemical defect correction of radiation sensitivity in ataxia telangiectasia cells by a truncated i kappa b-alpha elevation of interferon beta-inducible proteins in ataxia telangiectasia cells - - sigma is a p -regulated inhibitor of g /m progression a model for p -induced apoptosis role for p in gene induction by doublestranded rna integration of interferon-alpha/beta signalling to p responses in tumour suppression and antiviral defence differential gene regulation by specific gain-offunction jnk proteins expressed in swiss t fibroblasts dual regulatory roles of phosphatidylinositol -kinase in ifn signaling gene expression profiling during all-trans retinoic acid-induced cell differentiation of acute promyelocytic leukemia cells gene expression networks underlying retinoic acid-induced differentiation of acute promyelocytic leukemia cells stat is induced and activated by all-trans retinoic acid in acute promyelocytic leukemia cells a gene in the chromosomal region p with greatly reduced expression in lung cancer is similar to the gene for ubiquitin-activating enzyme the basis for selective e -e interactions in the isg conjugation system ube l and protein isgylation are not essential for alpha/beta interferon signaling deletions of the short arm of chromosome in solid tumors and the search for suppressor genes the ubiquitin-activating enzyme e -like protein in lung cancer cell lines alteration of tumor spectrum by isgylation in p -deficient mice deficiency of a potential p . tumor suppressor gene ube l (uba ) does not accelerate lung cancer development in k-rasla mice proteome analysis reveals ubiquitin-conjugating enzymes to be a new family of interferon-alpharegulated genes interferon-inducible ubiquitin e , ubc , is a conjugating enzyme for protein isgylation the ubch ubiquitin e enzyme is also the e enzyme for isg , an ifn-alpha/beta-induced ubiquitin-like protein link between the ubiquitin conjugation system and the isg conjugation system: isg conjugation to the ubch ubiquitin e enzyme physical interaction between specific e and hect e enzymes determines functional cooperativity staring, a novel e ubiquitin-protein ligase that targets syntaxin for degradation parkin suppresses unfolded protein stress-induced cell death through its e ubiquitin-protein ligase activity the histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of hdac a novel centrosomal ring-finger protein, dorfin, mediates ubiquitin ligase activity efp targets - - sigma for proteolysis and promotes breast tumour growth parkin functions as an e -dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicle-associated protein, cdcrel- ubch regulates ubiquitin and isg conjugation to rig-i the interferon-inducible ubiquitin-protein isopeptide ligase (e ) efp also functions as an isg e ligase novel growth and death related interferon-stimulated genes (isgs) in melanoma: greater potency of ifn-beta compared with ifn-alpha a ubiquitin e ligase efp is up-regulated by interferons and conjugated with isg negative regulation of isg e ligase efp through its autoisgylation isg modification of the eif e cognate ehp enhances cap structure-binding activity of ehp identification and herc -mediated isgylation of novel target proteins herc is an ifn-induced hect-type e protein ligase that mediates type i ifn-induced isgylation of protein targets herc , an interferon-induced hect e enzyme, is required for conjugation of isg in human cells ubp (usp ) specifically removes isg from conjugated proteins role of isg protease ubp (usp ) in innate immunity to viral infection screen for isg -crossreactive deubiquitinases precursor processing of pro-isg /ucrp, an interferon-beta-induced ubiquitin-like protein rnase-l-dependent destabilization of interferon-induced mrnas. a role for the - a system in attenuation of the interferon response cloning and characterization of human ubiquitin-processing protease- from terminally differentiated human melanoma cells using a rapid subtraction hybridization protocol rash a novel ubiquitin-specific protease, ubp , cloned from leukemia fusion protein aml -eto-expressing mice, functions in hematopoietic cell differentiation the isg isopeptidase ubp is regulated by proteolysis via the scfskp ubiquitin ligase cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine ubp (usp ) dysregulation of protein modification by isg results in brain cell injury reexamination of the role of ubiquitin-like modifier isg in the phenotype of ubp -deficient mice protein isgylation modulates the jak-stat signaling pathway ubp is a novel regulator of interferon signaling independent of its isg isopeptidase activity the level of hepatitis b virus replication is not affected by protein isg modification but is reduced by inhibition of ubp (usp ) expression ubp regulates bcr-abl leukemogenesis via the type interferon receptor signaling human isg conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways high-throughput immunoblotting. ubiquitiin-like protein isg modifies key regulators of signal transduction proteomic identification of proteins conjugated to isg in mouse and human cells ifn-induced -kda protein is released from human lymphocytes and monocytes a human -kda ifn-induced protein induces the secretion of ifn-gamma immunoregulatory properties of isg , an interferon-induced cytokine filamins as integrators of cell mechanics and signalling structural and functional aspects of filamins filamin b serves as a molecular scaffold for type i interferon-induced c-jun nh -terminal kinase signaling pathway filamin b: a scaffold for interferon signalling isg modification of filamin b negatively regulates the type i interferon-induced jnk signalling pathway regulation of the tak signaling pathway by protein phosphatase c protein phosphatase cbeta association with the ikappab kinase complex is involved in regulating nf-kappab activity signaling to nf-kappab nf-kappab: linking inflammation and immunity to cancer development and progression negative regulation of protein phosphatase cbeta by isg conjugation isg modification of ubiquitin e ubc disrupts its ability to form thioester bond with ubiquitin isg modification of ubc suppresses its ubiquitinconjugating activity molecular insights into polyubiquitin chain assembly: crystal structure of the mms /ubc heterodimer crystal structure of the human ubiquitin conjugating enzyme complex isg , an interferonstimulated ubiquitin-like protein, is not essential for stat signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus virgin, identification of interferon-stimulated gene as an antiviral molecule during sindbis virus infection in vivo a ppxy motif within the vp protein of ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies role for amino acids klr of ebola virus vp in assembly and budding overlapping motifs (ptap and ppey) within the ebola virus vp protein function independently as late budding domains: involvement of host proteins tsg and vps- isg enhances the innate antiviral response by inhibition of irf- degradation modification of proteins by ubiquitin and ubiquitin-like proteins protein modification by sumo inos (nos ) at a glance perspectives series: host/pathogen interactions. mechanisms of nitric oxide-related antimicrobial activity reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens protein snitrosylation: purview and parameters nitrosylation of isg prevents the disulfide bond-mediated dimerization of isg and contributes to effective isgylation a new paradigm for translational control: inhibition via ′- ′ mrna tethering by bicoid and the eif e cognate ehp mechanisms of type-i-and type-ii-interferon-mediated signalling pathogen recognition and innate immunity species-specific recognition of single-stranded rna via toll-like receptor and small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway ′-triphosphate rna is the ligand for rig-i tlr in antiviral immunity: key player or bystander? ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf visa is an adapter protein required for virus-triggered ifn-beta signaling negative feedback regulation of rig-imediated antiviral signaling by interferon-induced isg conjugation negative regulation of the rig-i signaling by the ubiquitin ligase rnf the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme global epidemiology of hepatitis c virus infection unravelling hepatitis c virus replication from genome to function regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i regulation of interferon regulatory factor- by the hepatitis c virus serine protease control of antiviral defenses through hepatitis c virus disruption of retinoic acid-inducible gene-i signaling viral and therapeutic control of ifnbeta promoter stimulator during hepatitis c virus infection ube l causes lung cancer growth suppression by targeting cyclin d proteasomes modulate conjugation to the ubiquitinlike protein, isg pulsed-field gel electrophoresis analysis of retinoic acid receptor-alpha and promyelocytic leukemia rearrangements. detection of the t( ; ) translocation in the diagnosis of acute promyelocytic leukemia chromosomal translocation t( ; ) in human acute promyelocytic leukemia fuses rar alpha with a novel putative transcription factor accelerated degradation of pml-retinoic acid receptor alpha (pml-rara) oncoprotein by all-trans-retinoic acid in acute promyelocytic leukemia: possible role of the proteasome pathway caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia pml/raralpha fusion protein the ubiquitinlike molecule interferon-stimulated gene (isg ) is a potential prognostic marker in human breast cancer isg as a novel tumor biomarker for drug sensitivity prostatic intraepithelial neoplasia in mice expressing an androgen receptor transgene in prostate epithelium androgeninduced differentiation and tumorigenicity of human prostate epithelial cells expression, regulation and function of the isgylation system in prostate cancer identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays we thank mr. sangman michael kim for his critical reading of this article. this work was supported by grants from the krf (krf- - -c ) and kosef (m - n ). we apologize for the event that any relevant publications were inadvertently omitted. key: cord- -yhdj m e authors: yang, li-tao; peng, hui; zhu, zhao-ling; li, gang; huang, zi-tong; zhao, zhi-xin; koup, richard a.; bailer, robert t.; wu, chang-you title: long-lived effector/central memory t-cell responses to severe acute respiratory syndrome coronavirus (sars-cov) s antigen in recovered sars patients date: - - journal: clin immunol doi: . /j.clim. . . sha: doc_id: cord_uid: yhdj m e the role of cell-mediated immunity in human sars-cov infection is still not well understood. in this study, we found that memory t-cell responses against the spike (s) protein were persistent for more than year after sars-cov infection by detecting the production of ifn-γ using elisa and elispot assays. flow cytometric analysis showed that both cd (+) and cd (+) t cells were involved in cellular responses against sars-cov infection. interestingly, most of sars-cov s-specific memory cd (+) t cells were central memory cells expressing cd ro(+) ccr (+) cd l(−). however, the majority of memory cd (+) t cells revealed effector memory phenotype expressing cd ro(−) ccr (−) cd l(−). thus, our study provides the evidence that sars-cov infection in humans can induce cellular immune response that is persistent for a long period of time. these data may have an important implication in the possibility of designing effective vaccine against sars-cov infection, specifically in defining t-cell populations that are implicated in protective immunity. long-lived effector/central memory t-cell responses to severe acute respiratory syndrome coronavirus (sars-cov) s antigen in recovered sars patients introduction severe acute respiratory syndrome (sars) is a newly emerged infectious disease that led to thousands of human infections and hundreds of deaths. the etiologic agent of the syndrome, a novel coronavirus termed sars-associated coronavirus (sars-cov) has been identified [ ] [ ] [ ] . although the epidemic was eventually controlled, the high morbidity and mortality make it extremely urgent to develop effective means to prevent this disease. therefore, a successful vaccine against sars-cov remains a high priority. the unique opportunity of studying survivors of the infection allows for delineation of the t-cell populations implicated in the protective immunity, and those t cells would be the target population for expansion via a vaccine. the spike (s) glycoprotein is a major structural protein of sars-cov and a potential target for sars-specific humoral immunity and/or cell-mediated immune responses. recent studies in animal models have demonstrated that vaccines based on the s protein of sars-cov seem to induce a considerable neutralizing antibody response [ ] [ ] [ ] [ ] [ ] and provide protection via an inhibition of viral replication after challenge with live sars-cov [ , ] . in addition, sars-covspecific igg antibody can be detected at week after the onset of syndrome in sars patients and persistent for a long period of time [ , ] . polyclonal immune sera from convalescent sars patients were passively transferred to treat sars patients during the outbreak in [ ] . these findings revealed that humoral immunity may provide immediate protection against sars-cov infection in mice and humans. in addition to humoral immune responses, the cellmediated response likely plays a major role in eliminating virus. although it has been demonstrated that dna vaccine encoding the s protein of sars-cov can induce antigenspecific t-cell responses in animal models [ , ] , the protective effect of t cells specific for the s protein in humans remains undefined [ ] . thus, it is essential to determine whether recovered sars patients maintained cell-mediated immunity against sars-cov and how long this immunity will persist. in addition, the phenotypic characteristics of memory cd + or cd + t cells specific for sars-cov have not been extensively studied. in this study, we analyzed cd + and cd + t-cell responses in peripheral blood of recovered sars patients to a pool of overlapping sars-cov s peptides and characterized the phenotype of memory t-cell subpopulations. our study demonstrated that sars-cov s-specific memory cd + and cd + t cells in peripheral blood of recovered sars patients were persistent for more than year after infection. these memory t cells revealed both effector and central memory phenotypes. eight fully recovered sars patients, male and female, age to years, worked at the third affiliated hospital of the sun yat-sen university, guangzhou, china. all were diagnosed with the infection of sars-cov between march and april, . controls for the study were consisted of five healthy donors, male and female, age to years, without any contact history with sars-cov patients, and had no detectable antibodies specific to sars-cov in their sera. a total of peptides spanning the entire sars-cov s protein (each peptide was - amino acids in length and over-lapped by amino acids) were kindly provided by the vaccine research center, niaid, national institutes of health, usa. anti-cd percp and anti-cd percp were purchased from bd bioscience immunocytometry systems (san jose, ca, usa). mouse anti-human cd , anti-human cd d, anti-cd fitc, anti-cd pe-cy , anti-cd ro fitc, anti-ccr pe, anti-cd l pe, anti-ifn-g pe, anti-ifn-g apc, anti-il- apc and isotypes matched control antibodies were obtained from bd bioscience pharmingen (san jose, ca, usa). rpmi medium, penicillin-streptomycin and -mercaptoethanol were purchased from gibco (grand island, ny, usa). peripheral blood mononuclear cells (pbmcs) were isolated from heparinized venous blood that was mixed with an equal volume of hank's balanced salt solution using ficoll-hypaque density gradient centrifugation. the cells were washed twice in rpmi medium supplemented with % heat-inactivated fetal calf serum (fcs) (hyclone), u penicillin/ml, ag streptomycin/ml and am -mercaptoethanol. the cells were finally adjusted to a concentration of  /ml in complete rpmi medium. cell culture conditions and ifn-g g g g g detection by elisa fresh pbmcs were isolated from recovered sars patients and healthy donors, suspended in complete rpmi medium and stimulated with a pool of overlapping sars-cov s peptides in the presence of ag/ml anti-cd and ag/ml anti-cd d mabs. the final concentration of each peptide was ag/ml. pbmcs were plated into a flat-bottomed -well plate at a total of  cells/well in triplicate and incubated for h at c with % co . the supernatants were harvested and assayed for ifn-g production by elisa according to the manufacturer's protocol (bd pharmingen, san diego, ca). in each experiment, pbmcs were cultured with ag/ml anti-cd plus ag/ml anti-cd d in the absence of sars-cov s peptides as a negative control. in addition, the cells were stimulated with ng/ml phorbol- mristate- -acetate (pma) (sigma, st. louis, mo, usa) and ag/ml ionomycin (sigma) as a positive control. ifn-g g g g g elispot assay human ifn-g elispot assays were performed using a commercially available set (bd pharmingen) according to the manufacturer's instructions. briefly, -well nitrocellulose plates (millipore) were coated overnight at c with ag/ml anti-human ifn-g capture antibody. the wells were then washed and blocked for h at room temperature with % fcs-rpmi medium. a total of  pbmcs were added to microwell in triplicate and incubated for h with a pool of sars-cov s peptides ( ag/ml), ag/ml anti-cd and ag/ml anti-cd d mabs. similarly, the cells supplemented with anti-cd ( ag/ml) and anti-cd d ( ag/ml) mabs in the absence of sars-cov s peptides were considered as negative controls, and the cells were cultured with polyclonal stimuli ( ng/ml pma plus ag/ml ionomycin) as positive controls. the plates were washed and subsequently incubated with ag/ml biotinylated anti-human ifn-g detection antibody for h at room temperature. after washing, wells were developed for another h with streptavidin-hrp followed by addition with aec substrate reagent. spot development was monitored, and substrate reaction was stopped by washing wells with water. the spots were counted automatically using immunospot image analyzer (cellular technology ltd, usa). a spot represents an ifn-g-producing cell. the average of spots in triplicate wells was calculated and considered as the number of specific spot-forming cells (sfc)/  cultured pbmcs. cell surface, intracellular cytokine staining and flow cytometric analysis pbmcs from recovered sars patients and healthy individuals were stimulated with or without a mixture of overlapping sars-cov s peptides in the presence of ag/ml anti-cd and ag/ml anti-cd d mabs in ml tubes. after the first h incubation, brefeldin a (sigma) at a final concentration ag/ml was added to cultures to enable intracellular protein to accumulate in all stimulations. after incubation for a total h, the cells were washed, fixed, permeabilized using saponin (sigma) and blocked with ag/ml human igg for min at c. the cells were then stained with anti-cd , anti-cd , anti-ifn-g, anti-il- , anti-cd ro, anti-ccr and anti-cd l. after staining, all samples were washed twice in pbs buffer (containing . % saponin, . % bsa and . % nan ) and resuspended in al pbs for flow cytometry. more than , cells were acquired on a facs aria flow cytometer (becton dickinson, san jose, ca), and facs data were analyzed using cellquest software (becton dickinson, san jose, ca). the levels of ifn-g and numbers of ifn-g-producing cells were compared respectively using student's t test between recovered sars patients and healthy donors in the same condition. value of p b . was considered significant. we first focused on understanding whether recovered sars individuals could generate and maintain memory t-cell responses against the s protein of sars-cov more than year after sars-cov infection. fresh pbmcs were isolated from fully recovered sars patients and healthy donors and cultured in vitro with or without a pool of peptides spanning the entire s protein of sars-cov in the presence of ag/ml anti-cd and ag/ml anti-cd d mabs. the final concentration of each peptide was ag/ml. the pbmcs were cultured with polyclonal stimuli ( ng/ml pma plus ag/ml ionomycin) as a positive control. after stimulation for h, cell-free culture supernatants were harvested and assayed for the production of ifn-g by elisa. the results showed that pbmcs from five healthy donors secreted very low levels of ifn-g ( . - . ng/ml) when cultured either with or without the sars-cov s peptides. statistic analysis indicated that the production of ifn-g between these two culture conditions was not significantly different ( p n . ) (fig. a) . in contrast, pbmcs from eight recovered sars individuals produced significantly higher levels of ifn-g ( . f . ng/ml) when stimulated with sars-cov s peptides ( p b . ) compared with the levels of ifn-g from the cells cultured without sars-cov s peptides ( . f . ng/ml) (fig. a) . in addition, no markedly difference in the production of ifn-g between recovered patients and healthy individuals was observed when the pbmcs were stimulated with pma plus ionomycin ( p n . ) (data not shown). to determine the frequency of sars-cov s-specific t cells at the single cell level, fresh pbmcs isolated from recovered sars patients and uninfected healthy individuals were cultured in the presence or absence of sars-cov s peptides. the frequency of ifn-g-producing cells was detected by elispot assay after stimulation for h. as shown in fig. b in recovered sars patients with an average number of spotforming cells (sfc) /  pbmcs (range to ) (fig. b) , which were significantly higher ( p b . ) than spotproducing cells in healthy donors ( - spots in  pbmcs) when pbmcs were stimulated with sars-cov s peptides. moreover, the pbmcs from recovered sars patients revealed similar frequency of ifn-g-forming cells ( /  pbmcs) compared with the pbmcs from healthy individuals ( /  pbmcs) when stimulated with pma plus ionomycin ( p n . ) (data not shown). taken together, sars-cov s-specific memory t cells were persistent in peripheral blood of recovered sars individuals and displayed the capacity to secrete ifn-g upon subsequent stimulation with sars-cov s peptides in vitro. we next analyzed the subpopulation of ifn-g-producing t cells by flow cytometry. pbmcs were isolated and stimu- figure expression of intracellular ifn-g and il- in pbmcs of recovered sars patients after stimulation with sars-cov s peptides in vitro. fresh pbmcs from recovered sars patients were cultured for h in vitro with a mixture of sars-cov s peptides and stained with anti-cd , cd , ifn-g and il- mabs. the expression of ifn-g and il- on cd + (a) and cd + (b) t cells was analyzed. the numbers in each quadrant were indicated as percentage. data were representative of two of five recovered sars individuals with similar results. the percentage (mean f se) of ifn-g or il- -secreting cells from independent experiments was shown within cd or cd t cells (c). student's t test was used for statistical analysis. statistical significance was set at p b . (*), ns, not significant. lated with a pool of sars-cov s peptides ( ag/ml), ag/ ml anti-cd and ag/ml anti-cd d mabs in the presence of ag/ml brefeldin a. after stimulation for h, the cell surface markers and intracellular cytokines were stained with anti-cd fitc, anti-cd percp, anti-ifn-g pe, anti-il- apc and their corresponding isotype controls. the expression of ifn-g and il- was assessed when cells were gated on cd + or cd + t cells. the results showed that both cd + figure the correlation of intensity of ifn-g and il- within sars-cov s-specific cd t cells. the subsets of sars-cov s-specific cd + t cells based on the expression of ifn-g and il- (a), the expression of il- in both ifn-g high or ifn-g low subpopulations (b) and frequency as well as intensity of ifn-g-producing cells in il- high and il- low subsets (c) (a, top panels, unstimulated cells; bottom panels, s-peptide-stimulated cells; b-c, left panels top quadrant, unstimulated cells; left panels bottom quadrant, s-peptidestimulated cells). percentage of indicated cells and the mean fluorescent intensity (mfi) were shown. data were representative of two of five recovered sars individuals. and cd + t cells from recovered sars patients failed to secret ifn-g and il- in the absence of sars-cov s peptides. however, addition of sars-cov s peptides to cultures resulted in the expression of ifn-g and il- in both cd + and cd + t-cell populations. two representative examples, patient and patient , are shown in figs. a and b. the percentage of ifn-g and il- -producing cd + and cd + t cells from recovered sars patients in sars-cov s-peptide-stimulated cultures was significantly higher than that from unstimulated cells ( p b . ) (fig. c) , whereas no difference was observed in the proportion of il- -producing cd t cells between those two groups ( p n . ). taken together, the results demonstrated that both memory cd + and cd + t cells participated in cellular immune responses to sars-cov s antigen in humans. we next analyzed the expression of ifn-g and il- in cd + t cells following stimulation of pbmcs from sars recovered patients with a pool of sars-cov s peptides. based on the expression of ifn-g and il- , antigen-specific cd + t cells could be divided into three distinct subpopulations: ifn-g + il- À , ifn-g + il- + and ifn-g À il- + (two examples, patient and patient , shown in fig. a) . we further analyzed the simultaneous expression of high or low levels of ifn-g and il- at the single cell (two examples, patient and patient , shown in figs. b, c). a total of four patients have been assessed for cytokine production. a representative of patient where approximately - % cd + ifn-g high cells showed high level expression of il- and - % cd + ifng low cells was il- low . - % of cd + il- high displayed phenotype of ifn-g high and - % cd + il- low were ifng low . similar tendency was also found in the mean fluorescence intensity (mfi). however, in patient , il- low cells accounted for only % of cd + ifn-g low but still showed similar results as seen in other patients. these results suggested that the majority of ifn-g + il- + t cells were confined to ifn-g high il- high subset. with regard to memory cd + and cd + t cells in humans, multiple phenotypes and a broad spectrum of functions have been observed in different viral infections. in this study, we performed phenotypic characterization of antiviral t-cell responses specific for sars-cov s peptides on the basis of their ability to secrete ifn-g and the expression of cd ro, ccr and cd l. consistent with previous reports, our results indicated that the majority of total cd + t cells (fig. a ) expressed high levels of ccr ( %) (fig. c ) and cd l ( %) (fig. d) , whereas approximately % of cd + t cells expressed cd ro (figs. c, d. sars-cov s-specific ifn-g-producing cd + t cells (fig. b) had an increased expression of cd ro ( fig. c ) but had decreased expression of cd l (fig. d ) compared with total cd + t cells. furthermore, the majority of antigen-specific cd + ifn-g + cells were cd ro + ccr + (fig. c ) or cd ro + cd l À (fig. d) . in contrast, a total of cd + t cells (fig. a ) expressed low level of ccr (fig. c ) and cd l (fig. d ) compared with total cd + t cells. moreover, ifn-gpositive cd + t cells (fig. b) were predominantly cd ro À ( %) and mainly defined as cd ro À ccr À cd l À (figs. c, d) . these results emphasized the heterogeneity of memory t cells and also demonstrated difference between memory cd + and cd + t-cell populations. in the current study, we found that antigen-specific memory t cells were capable of secreting high levels of ifn-g by elisa assay upon stimulation in vitro with a pool of sars-cov s peptides. these memory t cells persisted for more than year after recovery in peripheral blood of sars individuals. these data were further confirmed by ifn-g elispot assay showing a high frequency of sars-cov s-specific ifn-gproducing t cells in recovered sars patients. our results are consistent with the report that two newly identified hla-a* -restricted cd + t-cell epitopes from sars-cov s protein, s and s , elicit memory t-cell responses in pbmcs from hla-a* + recovered sars patients up to months post-infection [ , ] . to date, most studies have been focused on mapping of cd + t-cell epitopes from patients who were fully recovered from sars infection [ ] [ ] [ ] . however, little is known about memory cd + t-cell responses to sars-cov. consistent with observation in mouse model [ ] , we found that both memory cd + and cd + t cells participated in cellular responses to sars-cov. it is known that naive cd + t cells can be divided into two major subpopulations, th and th , based on cytokine production and biological functions [ ] . the th cells are composed of ifn-g + and ifn-g À cells. the ifn-g À th cells may differentiate into memory cells in vivo [ ] . based on the production of ifn-g and il- , sars-cov s-specific cd + t cells could be divided into three distinct populations: single ifn-g-secreting cells, double ifn-g/il- -secreting cells and single il- -secreting cells. interestingly, ifn-g/ il- double positive cd + t cells were mainly restricted to ifn-g high il- high subset. these results further demonstrated the heterogeneity of memory th cells and also indicated that distinct subpopulations of memory cd + t cells have different capacities to develop into short (ifng + ) and long-lived (ifn-g + il- + and il- + ) memory cells. it is generally accepted that cd ro expression defines to activated or memory t cells. here, we found that most of sars-cov s-specific memory cd + t cells were cd ro + , whereas the majority of memory cd + t cells displayed cd ro À phenotype. recently, ssp- , an hla-a* -restricted cd + t-cell epitope, was identified from sars-cov s protein. the ssp- -specific ctl cells displayed the cd ra + ccr À cd l À phenotype, after pbmcs from recovered sars patients were stimulated in vitro with heat-inactivated sars-cov for days [ ] . consistent with these observations, we found that the majority of sars-cov s-specific memory cd t cells were confined to cd ro À ccr À cd l À population. our results also demonstrated that the majority of memory cd + t cells exhibited effector memory phenotype of cd l À . this observation is in agreement with recent studies showing that only central memory cd + t cells produce il- following a short-peptide stimulation in lymphocytic choriomeningitis virus (lcmv)-infected mouse model [ ] . generally, the expression of cd l is associated with the expression of ccr [ ] . surprisingly, sars-cov sspecific memory cd + t cells expressed a mixed ccr + cd l À phenotype that differs from phenotypes of classical ccr + cd l + t cm and ccr À cd l À t em cells. some groups have also found memory cd + or cd + t cells expressing a mixed ccr + cd l À phenotype in mouse model [ , ] . these studies suggest that the expression of ccr and cd l in memory cd + or cd + t cells may overlap only partially. based on the expression of chemokine receptor ccr , memory t cells can be divided into two distinct subsets: central memory t cells (t cm ) expressing ccr and effector memory t cells (t em ), which lack the expression of ccr . in our study, the majority of ifn-g-producing cd + t cells were ccr + . this is supported by evidence that memory th cells are mainly ccr + in mice [ ] . recent report has identified two functional distinct populations of memory cd + t cells: double ifn-g/il- -secreting cells and single ifn-g-secreting cells. virus-specific ifn-g/il- -secreting cd + t cells were cd ra À ccr À , whereas single ifn-g-secreting cd + t cells were either cd ra À ccr À or cd ra + ccr À [ ] . consistent with observations in hiv infection, we showed that sars-cov s-specific cd + t cells produced ifn-g but not il- and predominantly displayed cd ro À ccr À phenotype which might represent terminally differentiated effector memory t cells [ ] . this may reflect difference in the inherent stability of cd + and cd + memory t cells that are ccr À in normal steady-state conditions [ ] . in conclusion, sars-cov infection elicits a cellular response mediated by both cd + and cd + t cells to the s protein. sars-cov s-specific memory cd + and cd + t cells are persistent for more than year in peripheral blood of recovered sars patients. this study provided an opportunity to delineate the memory t-cell response in survivors of a disease with high morbidity and mortality. the conclusions implicate a target population of t cells as a correlate for protection for effective vaccines. coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the severe acute respiratory syndrome a dna vaccine induces sars coronavirus neutralization and protective immunity in mice severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia 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hiv- -specific cd t cells skewed maturation of memory hiv-specific cd t lymphocytes similarities and differences in cd + and cd + effector and memory t cell generation key: cord- -yy g npi authors: zhu, xinyu; fang, liurong; wang, dang; yang, yuting; chen, jiyao; ye, xu; foda, mohamed frahat; xiao, shaobo title: porcine deltacoronavirus nsp inhibits interferon-β production through the cleavage of nemo date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: yy g npi porcine deltacoronavirus (pdcov) causes acute enteric disease and mortality in seronegative neonatal piglets. previously we have demonstrated that pdcov infection suppresses the production of interferon-beta (ifn-β), while the detailed mechanisms are poorly understood. here, we demonstrate that nonstructural protein (nsp ) of pdcov, the c-like protease, significantly inhibits sendai virus (sev)-induced ifn-β production by targeting the nf-κb essential modulator (nemo), confirmed by the diminished function of nemo cleaved by pdcov. the pdcov nsp cleavage site in the nemo protein was identified as glutamine , and was identical to the porcine epidemic diarrhea virus nsp cleavage site, revealing the likelihood of a common target in nemo for coronaviruses. furthermore, this cleavage impaired the ability of nemo to activate the ifn response and downstream signaling. taken together, our findings reveal pdcov nsp to be a newly identified ifn antagonist and enhance the understanding of immune evasion by deltacoronaviruses. porcine deltacoronavirus (pdcov), a new swine enteropathogenic coronavirus, belongs to the genus deltacoronavirus in the family coronaviridae (woo et al., (woo et al., , . it is an enveloped virus with a single-stranded, positive-sense rna genome of nearly kb, initially detected in pigs in hong kong, china in (woo et al., ) . the clinical significance of pdcov has been highlighted since outbreaks of this virus, which causes severe diarrhea and mortality of piglets, occurred in multiple states of the united states in homwong et al., ; hu et al., hu et al., , jung et al., ; ma et al., ; marthaler et al., ; thachil et al., ) . subsequently, reports of pdcov in china, south korea and canada have caused considerable attention to be paid to the strategy employed by this emerging coronavirus to manipulate the host immune response (dong et al., ; lee et al., ) . the interferons (ifns) are vital proteins in innate immune signaling, playing a key role in the initial stages of virus invasion. through the recognition of pathogen-associated molecular patterns by pattern recognition receptors, such as cytoplasmic rig-i and mda , adapter molecules such as ips- can be recruited and subsequently transfer the signal to the ikkα, ikkβ, ikkγ (also called nf-κb essential modulator (nemo)), tbk and ikk-ε. as an essential adapter, nemo is responsible for the recruitment of tbk and ikkε and the activation of ikk complex, leading to the triggering and transportation of nf-κb and irf to the nucleus, both of which induce downstream ifn-β production (kawai and akira, ; loo and gale, ; ramos and gale, ; seth et al., ; yoneyama and fujita, ) . notably, recent studies have demonstrated that several viral proteins encoded by coronaviruses (cov), such as severe acute respiratory syndrome (sars) cov, middle east respiratory syndrome cov and mouse hepatitis virus, can modulate innate antiviral signaling lui et al., ; thornbrough et al., ) . relying on its proteinase activity, cov non-structural protein (nsp ), also called c-like protease or main protease, is responsible for processing the viral polyprotein to produce most of the non-structural proteins during viral replication (lai and cavanagh, ; masters, ; perlman and netland, ; ziebuhr et al., ) . the c-like protease of the arterivirus porcine reproductive and respiratory syndrome virus (prrsv), and the c proteases of foot-and-mouth disease virus (fmdv) and hepatitis a virus (hav), both of which belong to the picornaviridae family, are reported to impair innate immune signaling (huang et al., ; wang et al., wang et al., , . a study published by our lab also demonstrated that nsp , the c-like protease of porcine epidemic diarrhea virus (pedv), which is classified into the alphacoronavirus family, antagonizes ifn-β production by cleavage of nemo . recently, our research has shown that infection with pdcov inhibits rig-i-mediated ifn signaling, although the specific inhibition mechanism remains poorly understood . as such, we were extremely interested to discover whether nsp of pdcov could disrupt type i ifn signaling. in this study, we reveal that nsp of pdcov antagonizes the type i ifn signaling pathway through the cleavage of nemo, a critical constituent of the ikk complex, thus representing a newly identified mechanism by which pdcov evades the innate immune response. human embryonic kidney cells (hek- t) and porcine kidney cells (pk- ) were obtained from the china center for type culture collection. llc-pk cells for pdcov infection were purchased from the atcc (atcc number cl- ) and cultured at °c in % co in dulbecco's modified eagle's medium (invitrogen, usa) supplemented with % fetal bovine serum. pdcov strain chn-hn- (genbank number kt ) isolated from a suckling piglet with severe diarrhea in china in , was the object of this research . sendai virus (sev) was acquired from the center of virus resource and information at the wuhan institute of virology. the utilized luciferase reporter plasmids of ifn-β-luc, nf-κb-luc and irf -luc have been previously described (wang et al., (wang et al., , . the rig-i, ips , mda , nemo, and the activated mutant of nemo (nemo-k a) expression plasmids were constructed as previously described (wang et al., (wang et al., , . nemo and its mutants were cloned into the plasmid pcaggs-flag with an nterminal flag tag (wang et al., , . pdcov nsp was amplified and cloned with a c-terminal hemagglutinin (ha) tag into the expression plasmid pcaggs-ha-c. reporter and various expression plasmids were transfected into hek- t cells or pk- cells in -well plates. twenty-four hours after transfection, cells were stimulated for h with sev. firefly luciferase and renilla luciferase activities of lysed cells were verified with a luciferase reporter assay system (promega, madison, wi), and normalized to prl-tk (promega). trizol reagent (invitrogen) and avian myeloblastosis virus reverse transcriptase (takara, japan) were utilized for rna extraction and reverse transcription of cdna. each quantitative real-time pcr (qpcr) experiment, evaluated by sybr green, was performed three times and normalized with glyceraldehyde- -phosphate dehydrogenase (gapdh). primers used for qpcr are detailed in table . in western blot analyses, h after transfection, the cells were treated with lysis buffer (beyotime, china) in -mm dishes. the lysates were separated by sds-page and transferred to polyvinylidene difluoride membranes (millipore, usa). following this, an anti-flag antibody (macgene, china), was applied to analyze the expression of proteins such as rig-i, mda , ips- , and nemo. pdcov nsp and its mutant were tested with an anti-ha antibody (mbl, japan) in western blot analyses. the endogenous nemo in pdcov-infected cells was tested with an anti-nemo polyclonal antibody (abclonal, china). the expression of pdcov n-protein was assessed with an anti-pdcov n-protein monoclonal antibody . the anti-β-actin mouse monoclonal antibody (beyotime, china) was applied to distinguish the expression of β-actin and to determine equal loading of each sample. coronavirus nsp plays an indispensable role in viral replication and immunoregulation. to characterize pdcov nsp with regards to type i ifn signaling, we constructed an expression vector encoding the nsp of pdcov and determined its impact on sev-induced ifn-β synthesis. firstly, the cytotoxicity of pdcov nsp in transiently transfected pk- and hek- t cells was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (mtt) assay. as shown in fig. a and fig. b , no detectable cytotoxicity could be observed in cells transfected with nsp expression plasmid equal to or less than . μg in -well cell culture plates. further, the data presented in fig. c and d revealed that nsp exhibited strong inhibition of sevinduced ifn-β promoter activity in both cell types. the sev-induced ifn-β protein in the supernatant was also strongly decreased under the ectopic expression of pdcov nsp in hek- t cells (fig. e) . moreover, nsp inhibited the activity of both irf and nf-κbdependent promoters in a dose-dependent manner ( fig. f and g). these data revealed the antagonistic role of pdcov nsp in type i ifn signaling. as the nsp of cov contains the catalytic residue cys , a point mutation here could disrupt protease activity (anand et al., ; hsu et al., ; ye et al., ) . consistent with this, sequence alignment showed that cys (numbering based on pdcov nsp ) residues are highly conserved among other cov subfamilies ( fig. a) . conversely to the activity of wild-type pdcov nsp , the activity of sev-induced ifnβ promoter was strongly restored upon the overexpression of nsp c a (fig. b) , implying that the protease activity of pdcov nsp participated in ifn-β antagonism. to verify at which point in the signaling cascade nsp facilitated its inhibitory role, we assessed several crucial molecules in the mda / rig-i signaling pathway, which play an important role in induction of ifn-β production (yoneyama and fujita, ) , including rig-i, mda , ips- , nemo, and tbk . similar to previous studies, overexpression of these crucial molecules significantly activated the ifn-β promoter compared with cells transfected with the empty vector control (siu et al., ; wang et al., wang et al., , . however, pdcov sequences ( ′ to ′) notably impaired the activation of the ifn-β promoter upon stimulation by rig-i, mda , ips- and nemo. in contrast, tbk induced activation of the ifn-β promoter was not affected by nsp (fig. a) , suggesting that pdcov nsp inhibits rig-i/mda signaling by targeting nemo or other upstream proteins. due to the indispensable role of pdcov nsp protease activity in ifn-β antagonism, we speculated that pdcov nsp cleaved nemo or an upstream molecule to impair type i ifn signaling. thus, rig-i, mda , ips- or nemo were co-transfected with nsp into hek- t cells. western blotting showed a smaller band (~ kda) in the nemo/pdcov nsp coexpression samples, while no similar cleavage products were detected with rig-i, mda or ips- co-transfections (fig. b) . furthermore, the cleavage of nemo increased gradually with increasing transfection dose of pdcov nsp (fig. c ) and the cleaved product could not be observed following transfection with the nsp c a mutant lacking protease activity (fig. d) . these data indicate that pdcov nsp , relying on its protease activity, targets the essential molecule nemo for cleavage. in order to evaluate the real effect of this cleavage on endogenous nemo protein in viral infection, llc-pk cells were infected with pdcov at a multiplicity of infection of . and harvested at h, h, and h post-infection. the results in fig. e indicate that endogenous nemo was clearly reduced within h of infection and the reduction in nemo correlated with the duration of pdcov infection. however, no difference was detected in nemo mrna levels between mock-and pdcov-infected cells at any time during infection (fig. f) . the above results indicate that degradation of nemo occurs only in pdcov infection, without affecting transcription. with expression plasmids encoding pdcov nsp or its protease-defective mutant c a, for analyzing ifn-β promoter activity followed by stimulation and harvest as described in fig. d . ns, p > . ; *, p < . ; **, p < . ; ***, p < . . virology ( ) - the specific preference for substrate cleavage by cov nsp has been previously reported as glutamine (q) at the p position (chuck et al., (chuck et al., , . as such, we examined the p position residue of nemo recognized by pdcov nsp . considering the kda molecular weight of the cleaved product (n-terminal), a series of nemo mutants were constructed between amino acids and (fig. a) . the wt or mutated nemo proteins were co-transfected into hek- t cells with pdcov nsp . the results indicated that q a, q a/q a or q a mutations were cleaved by pdcov nsp normally, while q a or q a mutations was resistant to cleavage (fig. b ). as previously found, the common substrate preference of cov nsp consists of leucine (l) at the p position and glutamine, lysine (k) or isoleucine (i), all of which contain a long side-chain and positive charge, at the p position (chuck et al., (chuck et al., , wang et al., ) . we speculated that if q is the p residue, then the recognition and cleavage of q by pdcov nsp could be affected by a q a mutation (fig. c) . therefore, two q mutants at the speculated p position were substituted with lysine or arginine (r) which also has a long side- chain and positive charge. evidently, q k and q r mutation can be cleaved to produce the same cleavage product as wt nemo (fig. d) , revealing that q , but not q , is the p position residue cleaved by pdcov nsp . docking studies between cov nsp and substrate showed that through van der waals interactions, the side chain of p residues could interact with the side chain of glutamic acid at residue (xue et al., ) . the homology model for pedv nsp and the nemo peptide substrate also illustrated that a long side chain in the p position strongly supported nsp -substrate recognition . these studies provide important clues for the pdcov nsp -nemo interaction and give a possible explanation for the finding that mutant q a, but not q k or q r, significantly impaired cleavage by pdcov nsp . to assess the influence of nemo cleavage by pdcov nsp in type i ifn signaling, a constitutively active nemo mutant, nemo-k a, was used, leading to more efficient activation of the ifn-β promoter compared with wt nemo (bloor et al., ; wang et al., wang et al., , . the k a mutation has the same cleavage site as wt nemo, with the pdcov nsp p recognition site also being q (fig. a) . hence, the ifn-β promoter activated by nemo-k a, was suppressed by pdcov nsp in a dose-dependent manner and this suppression was lost upon the expression of the protease-defective nsp c a mutant ( fig. b and c) . to evaluate the activity of the cleavage fragment in activation of the ifn response, nemo-k a ( - aa), nemo-k a ( - aa) or nemo-k a (full) expression plasmids were transfected into pk- cells. quantitative pcr showed that mrna levels of immune-related molecules, such as isg , isg , mx , oas , and rantes, were not successfully induced by the cleaved fragments of - or - (fig. d) . a previous study of pedv nsp showed that cleavage fragments of nemo did not activate ifn-β, irf or nf-κbdependent promoters . as the cleavage site q is identical for both pedv and pdcov nsp , these results together demonstrate that pdcov nsp -induced cleavage fragments lack the capacity to activate the type i ifn response and downstream signaling, revealing that the role of nemo in type i ifn signaling can be significantly inhibited following cleavage by pdcov nsp . so far, evidence presented by recent studies has shown that the c and c-like proteases of different viruses cleave nemo at multiple residues, such as q and q targeted by fmdv and hav c proteases, e targeted by prrsv c-like proteases and q targeted by pedv c-like protease, leading to disruption of ifn-β production (huang et al., ; wang et al., wang et al., , wang et al., , and implying that nemo is a common target easily hijacked by viral proteases. in particular, the conserved q residue recognized by both pdcov nsp and pedv nsp prompted us to speculate that the same ifn antagonistic mechanism may be common to members of the coronaviridae family. it is worth investigating whether nsp from different cov genera, including alpha-, beta-, gamma-, and deltacoronaviruses, retain the ability to cleave nemo and comparing this property between the different cov genera. in summary, our research demonstrated that pdcov nsp has the capacity to impede generation of ifn-β. as a new ifn antagonist encoded by a deltacoronavirus, pdcov nsp induced the cleavage of nemo at the conserved residue q , leading to suppression of the type i ifn signaling pathway. our research provides new insights into strategies and tactics employed by pdcov in host innate immune evasion. after h, pk- cells were analyzed for isg , isg , mx , oas , rantes mrna levels in qpcr, using sybr green. ns, p > . ; *, p < . ; **, p < . ; ***, p < . . coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs signal processing by its coil zipper domain activates ikk gamma pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in -day-old neonatal piglets profiling of substrate specificity 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protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda in the innate antiviral response development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies middle east respiratory syndrome coronavirus ns b protein inhibits host rnase l activation molecular cloning and functional characterization of porcine ifn-beta promoter stimulator (ips- ) foot-andmouth disease virus leader proteinase inhibits dsrna-induced type iinterferon transcription by decreasing interferon regulatory factor / in protein levels foot-and-mouth disease virus c protease cleaves nemo to impair innate immune signaling porcine epidemic diarrhea virus c-like protease regulates its interferon antagonism by cleaving nemo hepatitis a virus c protease cleaves nemo to impair induction of beta interferon coronavirus diversity, phylogeny and interspecies jumping discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus c-like protease rig-i family rna helicases: cytoplasmic sensor for antiviral innate immunity rna recognition and signal transduction by rig-i-like receptors virus-encoded proteinases and proteolytic processing in the nidovirales key: cord- -fiqx tll authors: mäkelä, m. j.; halminen, m.; ruuskanen, o.; puhakka, t.; pirhonen, j.; julkunen, i.; ilonen, j. title: lack of induction by rhinoviruses of systemic type i interferon production or enhanced mxa protein expression during the common cold date: journal: eur j clin microbiol infect dis doi: . /s sha: doc_id: cord_uid: fiqx tll to study whether mxa protein expression is systemically upregulated during rhinovirus infection, blood specimens were collected from patients with common cold and mxa expression in mononuclear cells analyzed by flow cytometry. none of the patients with a confirmed rhinovirus infection (n= ) or with an infection of unknown etiology (n= ) had elevated expression of the mxa protein (median fluorescence intensity, and , respectively) when compared to healthy controls (n= , median ). patients with influenza infections had significantly elevated values (n= , median ), and interferon could be detected only in serum samples from influenza patients. in conclusion, expression of mxa in blood lymphocytes and an apparently systemic type i interferon response is not induced during rhinovirus infection or during most other cases of common cold in young adult patients. interferons (ifns) form the first line of defence against viral infections. type i interferons (ifn-a/b) are often produced by many types of cells, and ifn-a can be detected in serum. measurement of ifn-a in serum has been used as a marker to differentiate viral and bacterial infections [ ] [ ] [ ] . the sensitivity of these assays for indicating viral infection ranges from to %. therefore, other assays based on the expression of ifn-inducible genes have been developed. one of these ifn-a/b-inducible genes, mxa, encodes for a cytoplasmic gtpase. this protein has antiviral activity against many viruses [ , ] and is expressed in lymphocytes and in some tissues under strict regulation by type i ifns [ ] . previously, we showed via flow cytometry that nearly all febrile children with a documented viral infection have elevated mxa levels [ ] . recent studies suggest that rhinovirus is the single most important respiratory virus because of its role in the common cold and in the pathogenesis of otitis media, sinusitis and exacerbations of asthma [ ] . little is known about the significance and function of the ifn system in rhinovirus infections. in the present study, the induction of a systemic ifn response as reflected by the expression of mxa protein in blood lymphocytes of patients with rhinovirus infection was examined. a total of subjects were recruited for the study. blood was drawn from patients with common cold within h of the onset of symptoms. the median age of the patients was . years (range . - . years). diagnosis of viral infection was performed as described previously [ ] . briefly, antigen detection or culture from nasopharyngeal aspirate and serological tests were used for detection of adenovirus, respiratory syncytial virus, influenza viruses a and b and parainfluenza virus types , and . rhinoviruses were detected both by culture and by polymerase chain reaction (pcr). based on the viral diagnosis, the patients were divided into three groups. there were rhinovirus-positive subjects ( culture positive, of whom were also pcr positive; serum type i ifn levels were also analyzed by two different assays as described below. since we were not able to detect elevated mxa levels in the rhinovirus-positive patients in the first phase of the study, we wanted to study whether these patients would have type i interferon in serum. for this purpose, two assays were used as described below. there was no serum left of the influenza virus-positive patients analyzed in the mxa assay, and, therefore, the serum specimens from influenza virus-positive patients whose lymphocytes were used for mxa expression analysis were not available. therefore, in our analysis, we used paired serum samples from patients with a marked rise in serum antibodies against influenza a. these frozen serum samples were obtained from a collection of serum samples stored at the department of virology, university of turku (j.i.). mxa protein was analyzed from peripheral blood mononuclear cells by indirect immunofluorescence and flow cytometry as described previously [ ] . the lymphocyte population was identified by the typical pattern of side scatter (intracellular structure) and forward scatter (size) parameters in flow cytometry. the mxa level was indicated by the median channel of logarithmic fluorescence counts from the cytometer. the nonparametric mann-whitney u test was used for comparisons between the groups. serum ifn-a levels were determined using a commercial assay kit (human ifn alpha elisa; endogen, usa) according to the manufacturer's instructions. the sensitivity of the assay was theoretically pg/ml, which corresponds to approximately . - iu/ml of ifn-a. only two of the influenza virus patients were positive by this assay, suggesting inadequate sensitivity. therefore, serum specimens were also analyzed by a biological ifn assay. the presence of biologically active ifn-a/b in sera was measured in hep- cells by a vesicular stomatitis virus plaque reduction assay as described previously [ ] . the results are expressed as international units (iu/ml), using an international control ifn-a prepared as a laboratory standard. the ethical committee of the turku university hospital approved the study. informed consent was obtained from all patients. the median intensity values of mxa-specific fluorescence in peripheral blood mononuclear cells of different patient groups are shown in figure . based on earlier data from healthy children and patients with bacterial infections, the cut-off value was set at , which is two standard deviations above the mean of healthy children [ ] . in the present study, the median fluorescence intensity value for the healthy adults was ( table ) . none of the patients with rhinovirus infection (np ) had values above , whereas all influenza virus-positive patients (np ) had values above this limit, with an upper range of . the median value in the rhinovirus-negative group (np ) was , which does not differ significantly from the median value in the rhinovirus-positive group (pp . ) but is significantly lower than the value in the influenza group (pp . ). to study whether the capacity of lymphocytes to produce mxa protein was blocked by some repressors or other inhibitory factor, we also cultured the cells for h in the presence of exogenous ifn-a ( iu/ml). in all groups the induced values were significantly higher than the baseline values (table ) . ifn-a/b levels were measured in serum, since mxa expression is regulated by type i ifns. none of the patients with rhinovirus infection had detectable ifn-a/b in serum as measured by either enzyme immunoassay or by a biological assay. a similar observation was made in the rhinovirus-negative patients and in the control group. ifn-a/b was detectable in the serum of seven of the . in six of the seven patients, ifn was detected in the acute-phase serum but not in the convalescent serum, and in one patient, ifn was detected only in the convalescent serum sample. low mxa values in patients with common cold were not expected, particularly in light of our earlier findings in children with viral infections in whom mxa was induced in practically all patients with influenza, adenovirus, respiratory syncytial virus and rotavirus infection [ ] . moreover, rhinovirus has been shown to induce ifn in vitro [ ] , and ifn has been found in the nasal secretions of volunteers infected with rhinovirus [ ] . stimulation with exogenous ifn-a, on the other hand, resulted in significant induction, suggesting that there is no functional defect associated with the rhinovirus infection per se. it is known that viruses and, probably, virus strains differ in their ability to induce ifn production, and there is also significant individual variation [ ] . ifn synthesis is often short-lived, but mxa is a stable protein with a half-life of several days [ ] . since all patients were examined within h after the onset of symptoms, it is unlikely that potential mxa upregulation would have been missed due to delayed analysis [ , ] . the mxa gene is under the strict control of type i ifns, and thus expression of the mxa protein indirectly reflects the presence of ifn in the host. serum ifn-a/b levels were therefore measured as well. the findings were in accordance with the study of parry and parry [ ] , who found interferon in the sera of nine of their patients with influenza but in none of the patients with rhinovirus infection. our data suggests that type i ifn production in serum is not comparable to that seen in most other respiratory viral infections in vivo, since the rhinovirus-positive patients had only low or no expression of the mxa protein and no ifn-a/b was detectable in serum samples. there is still uncertainty about the presence of rhinovirus in different parts of the airways, but according to recent studies with experimental infections in humans, rhinoviruses seem to infect epithelial cells both in the upper and lower airways [ ] . it is possible that, in most cases, the rhinovirus infection is limited mainly to the respiratory epithelium without major systemic influence. in conclusion, this study suggests that the systemic type i ifn response and subsequent upregulation of ifninducible mxa protein expression is poor in rhinovirus infection. mxa protein expression may, therefore, not be a suitable marker of viral infection in the common cold. it is still unknown whether the lack of a systemic ifn response plays a significant pathophysiological role during rhinovirus infection. although we did not find ifn or clearly induced mxa protein expression in the peripheral blood of any subject with rhinovirus infection, we cannot rule out the possibly significant role of ifn in local epithelial response against the virus. interferon assay as a viral diagnostic test serum alpha-interferon in lower respiratory tract infections of children detection of serum interpheron-alpha by dissociation-enhanced lanthanide fluoroimmunoassay. studies of patients with acute viral and bacterial infections inhibition of puumala and tula hantaviruses in vero cells by mxa protein resistance to influenza virus and vesicular stomatitis virus conferred by expression of human mxa protein control of ifninducible mxa gene expression in human cells expression of mxa protein in blood lymphocytes discriminates between viral and bacterial infections in febrile children what's new with common colds? complications and management viruses and bacteria in the etiology of the common cold rapid production of interferon-gamma in uninduced human leukocyte suspensions induction of interferon in human leukocyte cultures by natural pathogenic respiratory viruses interferon and resistance to upper respiratory virus illness mxa protein in infants and children with respiratory tract infection interferon assay as a diagnostic test detection of rhinovirus rna in lower airway cells during experimentally induced infection acknowledgements this work was supported by the finnish cancer foundation, the maud kuistila memorial foundation and the academy of finland. dr. david smith is acknowledged for revising the language of the manuscript. key: cord- - e pws authors: pitkäranta, anne; nokso-koivisto, johanna; jäntti, virva; takala, aino; kilpi, terhi; hovi, tapani title: lowered yields of virus-induced interferon production in leukocyte cultures and risk of recurrent respiratory infections in children date: - - journal: journal of clinical virology doi: . /s - ( ) - sha: doc_id: cord_uid: e pws abstract objective: to study the correlation between the yield of virus-induced interferon (ifn) production in leukocyte cultures and the risk of recurrent respiratory infections. methods: a sample of consecutive children enrolled in the finnish otitis media cohort study were selected. children suffering from frequently recurring respiratory infections (frris) were defined as the highest quintile of the entire cohort of children, as regards the number of upper respiratory infections (uris) and/or episodes of acute otitis media (aom) during the follow-up period from to months. results: in the sample of children, there were children with frri (≥ uri and/or ≥ aom). leukocyte cultures, prepared from blood drawn from these children at months of age, produced lower yields of ifn than those of the remaining children, when stimulated with adenovirus (p< . ), coronavirus (p< . ) or rhinovirus (p= . ). the difference in ifn yields was even greater (p< . with all three viruses) if the comparison was made between children with frri and those with no or maximally one uri during the follow-up period. when the ifn production capacity induced by rhinovirus was measured at the age of months, a statistically significant difference between the children with frri and the others was also seen (p= . ). influenza a virus-induced ifn production capacity did not differ between the groups at either age (p= . ). conclusions: lowered ifn responses in children suffering from recurrent uris and/or aom may, in a subgroup of the children, be due to a genetic property of the child. however, because of the great interindividual variations, we cannot use the ifn production capacity as such for prediction of forthcoming respiratory infections and/or otitis media. recurrent upper respiratory tract infections (uris) and acute otitis media (aom) are common problems in children, and some children seem to suffer from apparently continuous or frequently recurring uris and aoms. despite intensive study of risk factors of recurrent respiratory infections, relatively little is known about the factors that could, with some certainty, predict the proneness to respiratory infections. certain socio-environmental factors, like attendance at day-care centers, are known risk factors for recurring respiratory infections and otitis media, but no unifying biological cause has been discovered for the situation except in very rare cases of primary immunodeficiency (lyall et al., ) . interferons (ifn) are considered to have a key role in animal host defense against virus infections (muller et al., ) . type i ifns are synthesized in most nucleated cells as a response to virus infection and comprise several subtypes of ifn-a, a closely related ifn-v and the more distantly related ifn-b. production of ifn-g (type ii ifn) is limited to cells of bone marrow origin. it is produced by activated t-lymphocytes and nk cells, but, according to recent reports, also by cells of the myeloid line. previous studies have shown that induction of ifn-a expression is under genetic control in mice (muller et al., ) , whereas regulation of ifn-a production in humans has not been studied extensively. leukocyte cultures derived from different donors are known to vary remarkably with respect to the yields of ifn induced by standard amounts of viruses (katschinski et al., ) . reduced yields of virusinduced ifn by leukocyte cultures were previously associated with recurrent respiratory infections in children (isaacs et al., ; bondestam et al., ; vanecek and lehovcova, ; pitkäranta et al., pitkäranta et al., , . however, in these case-control studies the baseline ifn-responsiveness of the leukocytes of these children was not available and the question remained uncertain whether the decreased ifn response reflected an underlying predisposition or was secondary to the recurrent respiratory infections. we have now investigated prospectively a cohort of children to determine if the lowered ifn-producing capacity is present before the onset of the occurrence of frequent respiratory infections and to evaluate the possible predictive value of ifn responsiveness in relation to respiratory infection and otitis media development. this study was carried out in the context of the finnish otitis media (finom) cohort study. a total of children born in the hervanta area, tampere, were enrolled consecutively in the study at their second routine visit to the hervanta well baby clinic at months of age. the entire cohort comprised children. the enrolment of this subcohort took place from may through august , and the clinical follow-up continued until july . written informed consent was obtained from the parents during the enrolment visit. the study protocol and consent form were approved in advance by the ethical committees of the finnish national public health institute, tampere university hospital and the departments of social and health care of tampere city. the children were followed from their nd to their th month of age at a special study clinic in which one or two physicians and two or three study nurses worked full time to carry out the clinical follow-up. the children were examined at regularly scheduled monthly visits up to the age of months, thereafter every months up to the age of months, and finally at months of age. a blood sample for preparation of mononuclear cells was scheduled to be obtained from this subcohort at the age of and months. in addition to the regular visits, the study children had access to the study physician at any day of the week in case of respiratory infection. the parents were asked to bring their child to the study clinic whenever the child needed medical care for acute respiratory infection, especially if they suspected aom. on these sickness visits, history was taken in a structured way and physical examination was performed. the definition of an infection-prone child or a child with frequently recurring respiratory illnesses is difficult and, consequently, the prevalence figures of infection-prone children vary in the literature according to the definitions used and population under study (westbom and kornfält, ; tupasi et al., ) . our aim was to identify the most infection-prone quintile of the entire cohort of children. the number of uris and aom episodes experienced by each child was counted. after reviewing these data, we concluded that the highest quintile had experienced at least nine uris and/or at least four aom episodes during the follow-up. these criteria were used to define frequently recurring respiratory infections (frris) in this study. aom was defined as a visually abnormal tympanic membrane (with regard to color, position and/or mobility) suggesting middle ear effusion with at least one of the following symptoms or signs of acute infection: fever, earache, irritability, diarrhea, vomiting, acute otorrhea not caused by otitis externa, or other symptoms of respiratory infection (karma et al., ) . moreover, in this analysis only such events were taken into account as aom events, in which the presence of middle ear effusion was confirmed by collection of at least one middle ear fluid sample. in the finom cohort study, myringotomy was a routine procedure for aom with marked symptoms. consequently, one or two middle ear fluid samples were obtained from % of the clinically diagnosed cases of aom. uri was diagnosed if the patient had symptoms and/or signs of acute infection and if a nasopharyngeal aspirate (npa) sample was obtained. aom and/or uri episode was considered to start when the respective diagnosis was made by the study physician. a new episode could start when at least days had elapsed since the beginning of the previous episode. preparation of peripheral blood mononuclear leukocytes from heparinized blood has been de-scribed earlier (pitkäranta et al., (pitkäranta et al., , . mononuclear leukocytes were stored frozen at − °c in rpmi- supplemented with % fetal calf serum containing % dmso until assayed for ifn responsiveness. to study virus-induced ifn production, leukocytes were thawed rapidly, sedimented to remove dmso containing medium and resuspended in growth medium at × cells/ml. leukocyte cultures were then inoculated with standard amounts of adenovirus type a, human coronavirus e, human rhinovirus (a clinical isolate provided by t. ziegler), and influenza a beijing/ / /h n (a gift from r. pyhälä) as reported earlier (pitkäranta et al., ) . a single preparation of each virus was used throughout the study stored at − °c as small aliquots. culture medium for ifn assay was harvested on day and clarified by centrifugation. the supernatants were stored at − °c until assayed. for logistic reasons, we could not use freshly isolated leukocytes in the induction assays. the feasibility of using frozen cells was tested in advance with cells from several volunteers. the absolute yields of ifn were usually two-fold lower from frozen-thawed cell preparations than those in the corresponding fresh cultures, but the donorspecific pattern of ifn yields induced by different viruses was retained (data not shown). ifn concentration was measured by a micromethod based on the reduction of cytopathic effect caused by a standard amount of vesicular stomatitis virus in monolayers of a continuous bovine cell line (nbl), which is not sensitive to human ifn-g. hence, we assume that most of the measured ifn activity was ifn-a. a standardized leukocyte ifn preparation (a gift from k. cantell) was included in all assays so that results could be expressed in iu/ml. differences between groups in ifn production were tested with a mann-whitney u-test. the demographic and clinical data of the children studied are shown in table . among the children, ( %) children had ] uri and/or ] aom during their first years and thus belonged to the frri group. a total of eight ( %) children did not have any uri or aom at all, and seven ( %) children had only one uri during their first years of life. there were altogether boys and girls in the sample. there was no trend for either sex being overrepresented among the children with frri. the first uri occurred at a mean age of . months (range . - months) in the frri group and at . months (range . - . months) in the remaining children (excluding the eight children who had no respiratory infections during the follow-up). altogether, these children experienced uri and aom episodes during their first years of life: % of all uri and % of all aom episodes occurred in the children with frri. leukocytes of children were examined for ifn production after stimulation with adeno-, corona-, rhino-and influenza a viruses at months of age. ifn yields from cultures stimulated by common respiratory viruses were lower for the children with frri than for the remaining children (table ). significant differences existed between these two groups of children with adenovirus, coronavirus, rhinovirus, but not with influenza a virus (table ). when ifn production by virus-stimulated leukocytes from frri children was compared with the children who had none or only one uri, the differences were even more striking (table ). this was due to an enhanced ifn responsiveness in the subgroup of children with no or only one uri episodes. at the age of years, leukocytes were available from children with frri and from other children. again, the ifn yields induced by rhinovirus were statistically significantly lower among the frri children than among the children with less frequent illness. no significant difference was seen when ifn was induced by influenza a virus ( table ). the leukocyte amounts recovered from the frozen specimens did not allow testing for other viruses. when the comparison of ifn production capacity at the age of months was carried out only for those children whose first uri and/or aom started after months of age, there was a tendency of the leukocytes from the seven frri children to produce less ifn than those from the non-frri children when stimulated with adeno-, corona-and rhinovirus. statistically significant difference was seen with cultures stimulated with coronavirus (table ). the production of ifn was not influenced by the genderofthechildren. spontaneous ifnproduction was not detected in leukocyte cultures of children. our previous studies (pitkäranta et al., (pitkäranta et al., , indicated that impaired virus-induced production of ifn by cultured leukocytes ex vivo showed a correlation with a record of frequently recurring respiratory illnesses. the studies did not determine whether this deficiency reflected a genetic trend to increased susceptibility to respiratory infections or was a secondary consequence of the frequent illnesses. the present study was designed to investigate the correlation in a prospective set-up. as a group, leukocytes from the children with frequent illnesses ( ] uri and/or ] aom) produced decreased ifn amounts when compared to the ifn production by leukocytes from children with less frequent illnesses ( uri and/or aom). a similar pattern existed at months and years of age with respect to ifn responsiveness to rhinovirus. the findings suggest that the impaired ifn production in some frri children might indeed be a genetic property. we also made a comparison of ifn yields between the children with frri and those with no or maximally one uri during the followup period. the differences in ifn responsiveness were even greater than in the primary comparison, a finding which further supports the view that the inherent intensity of the virus-induced ifn response might play a role in the susceptibility to uri and/or aom. of note, there are many examples of genetically determined aberrations in the production of other cytokines that result in increased severity of other infections (mcguire et al., ; stuber et al., ; westendorp et al., ) , including such a common disease as periodontitis (kornman et al., ) . it is remarkable that this difference was seen when the leukocytes were stimulated with adeno-, corona-or rhinoviruses but not with influenza a virus. this virus-specific difference was also seen in our earlier studies (pitkäranta et al., (pitkäranta et al., , and similar observations have been reported by others (neustock et al., ) . it is noteworthy, that the overall ifn yields obtained with the influenza a virus in our study are much higher than those seen with the other viruses revealing the difference between the groups of children. influenza viruses are very effective ifn inducers which may mask differences in production capacity. in addition to the virus inducer-specific differences, there were also great interindividual variations in ifn yields. this makes it difficult to use the ifn producing capacity as such as a prognostic marker for recurrent respiratory infections at the individual level. in conclusion, we have shown that reduced ifn-responsiveness in the leukocytes of children suffering from recurrent respiratory infections and/or otitis media may, in a subgroup of children, be due to a genetic predisposition of the child. unfortunately ifn production capacity cannot presently be used to predict the risk of future respiratory illnesses because of great interindividual and virus-specific variations. nevertheless, our results encourage the search for ways to identify children who might have genetic predisposition to recurrent respiratory infections and otitis media. interferon production in children with undue susceptibility to infections deficient production of leukocyte interferon (interferon-a) in vitro and in vivo in children with recurrent respiratory tract infections finnish approach to the treatment of acute otitis media. report of the finnish concensus conference influence of various factors on interferon-a production in culture of human leukocytes the interleukin- genotype as a severity factor in adult periodontal disease assessment of a clinical scoring system for detection of immunodeficiency in children with recurrent infections variation in the tnf-a promoter region associated with susceptibility to cerebral malaria functional role of type i and type ii interferons in antiviral defense deficient interferon-alpha response of newborn in comparison to adults virus-induced interferon production in human leukocytes: a low responder to one virus can be a high responder to another virus deficiency in interferon production by leukocytes from children with recurrent respiratory infections virus-induced interferon production in leukocyte cultures from children with recurrent respiratory infections. a follow-up study a genomic polymorphism within the tumor necrosis factor locus influences plasma tumor necrosis factor-a concentrations and outcome of patients with severe sepsis community-based studies of acute respiratory tract infections in young children interferon production in children with respiratory diseases chronic illness among children in a total population. an epidemiological study in a swedish primary health care district genetic influence on cytokine production and fatal meningococcal disease we wish to thank all the children and their parents who participated in the study. we thank personnel in the special study clinic in tampere: ritva syrjänen, md, study doctor, and the study nurses mervi martola, rn, marja-leena hotti, rn, päivi tervonen, rn, study nurse. kirsi ijäs and riitta heino are acknowledged for their expert technical assistance. key: cord- - uop z authors: montoya, maría; foni, emanuela; solórzano, alicia; razzuoli, elisabetta; baratelli, massimiliano; bilato, dania; córdoba, lorena; del burgo, maria angeles martín; martinez, jorge; martinez-orellana, pamela; chiapponi, chiara; perlin, david s.; del real, gustavo; amadori, massimo title: expression dynamics of innate immunity in influenza virus-infected swine date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: uop z the current circulating swine influenza virus (iv) subtypes in europe (h n , h n , and h n ) are associated with clinical outbreaks of disease. however, we showed that pigs could be susceptible to other iv strains that are able to cross the species barrier. in this work, we extended our investigations into whether different iv strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. for this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a h n swine iv and four different h n iv strains circulating in different animal species. pigs had been clinically inspected and four subjects/group were sacrificed at , , and days post infection. in the present study, all groups but mock exhibited antibody responses to iv nucleoprotein protein. pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. interestingly, pigs infected with avian and seal h n strains also showed moderate lesions and viral replication, whereas equine and canine ivs did not cause overt pathological signs, and replication was barely detectable. swine iv infection induced interferon (ifn)-alpha and interleukin- responses in bronchoalveolar fluids (balf) at day post infection, as opposed to the other non-swine-adapted virus strains. however, ifn-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of ifn-alpha genes. remarkably, the equine strain gave rise to a serum amyloid a response in balf despite little if any replication. each virus strain could be associated with expression of cytokine genes and/or proteins after infection. these responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system. the current circulating swine influenza virus (iv) subtypes in europe (h n , h n , and h n ) are associated with clinical outbreaks of disease. however, we showed that pigs could be susceptible to other iv strains that are able to cross the species barrier. in this work, we extended our investigations into whether different iv strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. for this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a h n swine iv and four different h n iv strains circulating in different animal species. pigs had been clinically inspected and four subjects/group were sacrificed at , , and days post infection. in the present study, all groups but mock exhibited antibody responses to iv nucleoprotein protein. pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. interestingly, pigs infected with avian and seal h n strains also showed moderate lesions and viral replication, whereas equine and canine ivs did not cause overt pathological signs, and replication was barely detectable. swine iv infection induced interferon (ifn)-alpha and interleukin- responses in bronchoalveolar fluids (balf) at day post infection, as opposed to the other nonswine-adapted virus strains. however, ifn-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of ifn-alpha genes. remarkably, the equine strain gave rise to a serum amyloid a response in balf despite little if any replication. each virus strain could be associated with expression of cytokine genes and/or proteins after infection. these responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system. keywords introduction the first line of defense after viral infections is based on innate immunity, which has the capacity to respond to pathogens by sensing pathogen-associated molecular patterns (pamps) through germline-encoded pattern recognition receptors. this process leads to the production of several cytokines such as type i (α and β) interferons (ifns). these bind to the type i ifn receptor to signal the induction of hundreds of interferonstimulated genes in the local uninfected and virus-infected cells, thus creating an antiviral state that suppresses virus infection and serves to promote the onset of adaptive immunity ( , ) . innate immune responses are crucial not only for blunting viral replication in the first instance but also for orchestrating effective adaptive immune responses. therefore, a transient induction of cytokines and chemokines is required for efficient antiviral responses. however, overreacting and prolonged immune responses may lead to undesired secondary effects, contributing to immunopathology. as for influenza virus (iv), the innate immune response may vary between mild and severe infections, and include widely different soluble innate immune inhibitors: sialic acid-based inhibitors, ca-dependent lectin inhibitors, anti-microbial peptides, complement, natural igm, ifns, to name a few ( ). toll-like receptors and (tlr- and tlr- ) have been found to play an important role in influenza a virus recognition and initiation of the immune response in human respiratory epithelial cells and plasmacytoid dendritic cells (pdcs) ( ). activation of both tlrs and cytoplasmic receptors leads to a potent type i ifn release and simultaneous pro-inflammatory cytokine expression. type i ifns, ifn-γ, and pro-inflammatory cytokines, such as interleukin (il)- , il- , il- , il- , and tumor necrosis factor-alpha (tnf-α), have been shown to be upregulated in lung tissue and lung lavage after experimental infection of pigs with swine iv ( - ). three iv subtypes (h n , h n , and h n ) are currently circulating in swine herds in europe ( ) ( ) ( ) and have been associated with disease occurrence and gross lesions in swine ( ) . also, pigs are susceptible to infection with low pathogenic and highly pathogenic avian ivs (lpaiv and hpaiv, respectively) ( ) . most lpaiv subtypes diagnosed in field samples possess a h or h hemagglutinin usually restricted to birds. hpaiv are able to infect pigs under natural and experimental conditions ( ) . besides, it is well known that some ivs are able to infect humans and pigs, as it was the case in the last h n pandemic infection ( , ) . in vitro, different iv strains can interact with porcine dendritic cells (dcs) and induce sequential waves of cytokine production that are dependent on time and virus strain ( ) . this effect could account for the different immune responses generated by iv strains. on the other hand, we have previously examined the potential of h n iv from canine, equine, avian, and seal origin to productively infect pigs. our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation ( ) . however, no studies in vivo have addressed whether innate immune responses of pigs to swine-adapted and non-adapted iv strains might be different. the above findings outline the main working hypothesis of our study, which implies that swine iv strains might give rise to peculiar innate immune responses and time courses thereof in pigs, clearly different from those triggered by other iv strains. in order to answer these questions, samples from pigs infected with a swine (h n ) and four different non-swine-adapted h n iv strains circulating in different animal species (dogs, horses, wild aquatic birds, and seals) from our previous study ( ) were analyzed and innate immune responses in the respiratory tract were thoroughly investigated. in line with the above operational framework, six groups of pigs ( - weeks old, landrace × pietrain, free from common pathogens) were housed in separate isolation rooms and adapted to the new environment and stockmen over a -week period under veterinary supervision. the animals used in our study were seronegative at that time to influenza a viruses by competition elisa (id screen ® influenza a antibody competition elisa, id-vet, france). also, they had been always healthy and thrifty before the present study. the experimental infection of pigs with different iv strains was described in our previous paper ( ) . pigs were infected with the following iv strains: at day , each pig in groups , , , , and was intratracheally infected with ml of virus suspension in pbs, containing × chicken embryo infectious doses % (eid ) of the corresponding virus strain. animals were clinically inspected on a daily basis. rectal temperature and weight were measured at the same times. four pigs of each virus-infected group were euthanized at day , , and post infection (p.i.), respectively. in the mock-infected group, two pigs were euthanized at each of the same times. blood serum samples in vacuum tubes were collected from jugular veins at day and just before sacrifice at the aforementioned days. after sacrifice, bronchoalveolar fluids (balf) were collected by lung lavage with pbs, as described by busquets et al. ( ) . briefly, the right lung of sacrificed pigs was used to perform a bronchoalveolar lavage (bal) using around ml of pbs, and the left one was sampled for histopathological and virological studies. a complete necropsy was performed. lung lesions were classified depending on the extent of pneumonia. mild lesions were recorded when affecting small areas (< cm ) of cranial or medial lung lobes, moderate lesions when affecting extended areas ( - cm ) of cranial or medial lobes, and severe lesions when affecting large areas (> cm ) of cranial, medial, diaphragmatic, and accessory lobes. histopathology samples from nasal turbinates, trachea, and lungs were collected, fixed in % buffered formalin and processed for histopathology, i.e., they were dehydrated through graded alcohols and embedded in paraffin. the -µm thick sections were cut, stained with hematoxylin-eosin and examined in a "blind-fashion" manner. in particular, we aimed to evaluate bronchiolar epithelial changes and peribronchiolar inflammation in large, medium, and small or terminal bronchioles, as well as inflammatory changes in alveoli. in the lung, bronchointerstitial pneumonia intensity was assessed by means of a semi-quantitative lesion score. the pathological scores for tracheal and pulmonary tissues were as follows: : no lesions, : mild lesions, : moderate lesions, : severe lesions. in order to check iv replication in the lower respiratory tract of pigs, balf samples collected from pigs killed at day , , and were tested for gene m of the influenza a virus using a quantitative real-time pcr procedure ( ) . the sensitivity of the test was equal to tissue culture infectious doses % (tcid )/ microliters. anti-influenza a virus nucleoprotein (np) antibody levels were investigated in serum using the id screen ® influenza a antibody competition elisa (id-vet, france) following the manufacturer's instructions at days , , and p.i. as previously described ( ) . the threshold level was set at < % of the od level observed in the control wells without any swine serum. the kit detects antibodies against all influenza a subtypes and antigenic variants thanks to the use of a monoclonal antibody against a highly conserved epitope of the influenza a virus np and it was used with pig serum as previously described ( , ) to avoid background. acute phase proteins (app) of pigs (haptoglobin and serum amyloid a, saa) were investigated in balf samples by commercial colorimetric kits (haptoglobin kit, tridelta development, code tp . multispecies saa elisa kit, tridelta development, code tp ), according to the manufacturers' directions. interferon-alpha was measured in balf samples by two different assays. first, a sandwich elisa with monoclonal antibodies (mab) f and k to porcine ifn-alpha was used as previously described ( ) . ifn-alpha was also measured by a cytopathic effect inhibition assay on mdbk cells with vesicular stomatitis virus ( ) . the test was calibrated with a preparation of porcine recombinant ifn-α (pbl biomedical laboratories, cat. - ). the units of this preparation are determined with respect to the international reference standard for human leukocyte ifn (ga- - ) provided by national institutes of health (bethesda, md, usa). this assay for porcine ifn-α in serum had been also validated in a previous study ( ) . identification of the cytokine was performed by a neutralization assay on mdbk cells of ifn α-positive balf samples ( ) , using monoclonal antibody (mab) g to porcine ifn-α (serotec, cat. mca z). the assay was calibrated with porcine recombinant ifn-α . interleukin- and tnf-α were determined in balf samples by bioassays on td and wehi cells, respectively, as previously described ( , ) . cytokine concentrations were determined from a standard curve created with reference preparations of human recombinant il- and tnf-α (pierce endogen, rockford, il, usa). immunophenotyping of balf cells was carried out by flow cytometry. this was performed using indirect labeling of cells by hybridoma supernatants for cd (clone ppt ), cd a (swc ) (clone - - a), and swine mhc (sla) ii (clone f ) ( ) . the antibody against γδ tcr (clone pgbl a) was purchased from euroveterinaria s.a. and the one against cd (clone - - ) was purchased from serotec s.a., both conjugated with r-phycoerythrin (pe). as for the hybridoma supernatants, the concentration of antibody was empirically calculated after titration of each preparation and the secondary antibodies were either pe or apc-conjugated goat anti-mouse igg (jackson immunoresearch, suffolk, uk) used following the manufacturer's instructions. each primary antibody was compared with its relevant isotype control in each sample using the same concentrations. briefly, . × cells/ μl/well were labeled for h at °c with µl of each monoclonal antibody (relevant and irrelevant) at a pre-established, optimal dilution. after -h incubation at °c, cells were washed with cold pbs with % fcs by centrifugation at × g, °c, min. then, the secondary antibody conjugated to r-phycoerythrin diluted : was added. cells were incubated for a further h at °c, then washed as before and resuspended in pbs with % fcs. stained cells were acquired using facsaria i (becton dickinson ® ) and analyzed by software facsdiva v. . . . a gating strategy was applied to living cells using their forward and side scatter (fs/ss) characteristics. staining with isotype controls did not exceed % in all the samples. total rna was extracted from balf cells using rneasy mini kit (qiagen, milan, italy) by the qiacube system (qiagen, milan, italy) in accordance with the manufacturer's instructions. the protocol included a dnase treatment for eliminating genomic dna. rna concentration was evaluated by uv absorbance (biophotometer, eppendorf, milan). three µl of rna ( ng/µl) were added to the reaction mix for cdna synthesis. this was performed in the presence of random hexamers as previously described ( ) . then, the expression of porcine ifn-α, il- , il- , il- β, and tnf-α genes was determined using the primer sets shown in table . the choice of the porcine ifn-α subtypes shown in table was dictated by their important role in constitutive expression of type i ifns ( ) and, therefore, by their possible expression before iv infection. porcine beta -microglobulin was used as housekeeping control gene ( table ) to normalize the test results at different sampling times. evagreen real-time pcr amplification was performed over cycles in a cfx real-time system (bio-rad, milan, italy) as described by razzuoli et al. ( ) . in each sample, the relative expression of the selected genes was calculated using the formula Δct = ct (target gene)-cycle threshold (ct) (housekeeping), where ct (cycle of threshold) values were the mean of three test replicates ± sd, as previously described ( ) . negative samples were given a ct fictitious value for further statistical examination. gene expression data (Δct values) obtained on balf cells were checked for normality by the kolmogorov-smirnov test. the data sets passing the normality test were checked for significant differences between time points (days , , p.i.) by one-way anova for unpaired data, whereas the others were evaluated by the non-parametric kruskal-wallis procedure (graphpad prism . , graphpadsoftware, san diego, ca, usa). the threshold for significance was set at p < . . statistical analyses on flow cytometry data were carried out using the sas system v. . . (sas institute inc., cary, nc, usa). for all analyses, the individual pig was used as the experimental unit. shapiro wilk's and levene tests were used to evaluate the normality of the distribution of the continuous variable and the homogeneity of variances, respectively. a statistical analysis was performed to study the association between the different variables with the different experimental groups at the different time points ( , , and days post-infection). to analyze the association between continuous, normally distributed variables and the different experimental groups, an anova test was used at , , and days post-infection. finally, a wilcoxon rank-sum test was used for the continuous, non-normally distributed variables at the different time points. no clinical signs were shown during the experimental procedure, such as fever, weight loss, anorexia, depression, nasal discharge, or altered breathing. temperature was not higher than . °c in any animal at any time point. there were no traumatic lesions as a result of intratracheal virus inoculation. gross lesions were mostly shown by animals infected with the swine iv, but some lesions were also detected in lungs from pigs infected with other iv strains. pulmonary lesions ( table ) were consistent with bronchointerstitial pneumonia at different grades of severity ( - ). the pigs infected with swine iv presented the highest level of severity followed by those infected with seal iv, avian iv, and either equine or canine iv in descending order. histopathology results are shown in figure . nasal turbinates sections did not differ between iv and mock-infected animals. tracheal lesions of iv-infected pigs consisted of variable grades ( - ) of lymphoplasmacytic infiltration in the lamina propria. in the most severe cases, focal ulceration of the mucosa with fibrin deposition and neutrophilic infiltration was observed. a lymphoplasmacytic infiltration was observed in the bronchioli with extension to surrounding alveolar septa. in the most severe cases, at day p.i., necrosis of the bronchiolar epithelium was observed with accumulation of necrotic cells in the bronchiolar lumen and alveolar spaces. in agreement with the gross lesion scores shown in table , histopathological lesions were much more severe in pigs of the swine group at day p.i. followed by the animals infected with seal or avian iv. the pigs infected with either equine or canine iv showed the least severe lesions ( table ) . to confirm the virological data (iv titration in embryonated chicken eggs) shown in our previous paper ( ) , viral replication was evaluated by quantitative real-time pcr on balf samples of animals sacrificed at days , , and p.i. (figure ) . the swine-adapted iv actively replicated in the pig respiratory tract at both day and p.i., as shown by ct values ranging from to . instead, we could detect only one iv-positive balf sample in the equine group at day p.i. with a ct value of and none in the canine group, indicating very little replication of those viruses. on the contrary, the avian and seal virus strains showed a moderate replication at day in most pigs, with ct values ranging from to . one animal from the seal group was still iv-positive at day p.i. with a ct value of . the above pcr data are in agreement with iv titrations in embryonated chicken eggs, reported in our previous paper ( ) . total anti-np antibody levels were measured by a competition elisa kit in serum samples from all animals at the beginning of the assay (day ), days , and p.i. all animals were seronegative at the beginning of the experiment (day , figure ) . also, no antibody response was observed in mock-infected animals. on the other hand, all iv-infected animals seroconverted (od < the threshold) by day p.i. most of them seroconverted by day p.i. in groups infected with swine, avian, and seal viruses, whereas the response was delayed in groups infected with the equine and canine viruses (figure ). the above findings confirmed a different invasiveness in pigs of swine-adapted and non-adapted iv strains in terms of virus replication and pathological lesions. on this basis, we investigated innate immune responses during iv infection in the respiratory tract that might differ between pathogenic and non-pathogenic iv strains in pigs. protein cytokine levels were measured in all animals from all groups by different assays. firstly, levels of ifn-α in balf were analyzed by elisa. all samples were negative except those collected from animals infected with swine iv at day p.i. (figure a) . these findings were confirmed by a bioassay for type i ifn on mdbk cells, and the specificity of the response was confirmed by an ifn-neutralization assay based on a mab to porcine ifn-alpha (data not shown). there was only one pig (sw- ) with a slight tnf-α response in balf at day p.i. in the swine iv-infected group. balf samples were always tnf α-negative in the other groups (data not shown). on the other hand, il- was clearly detected at day p.i. in the balf samples of swine iv-infected pigs. in the other groups, there was no significant difference with respect to mock animals, with the exception of pig se- in the seal group (day p.i.) ( figure b) . the high il- response in balf of the swine group at day p.i. led us to investigate haptoglobin and saa in balf samples of the mock, swine, and equine groups at day p.i. all the samples were haptoglobin-negative (< . mg/ml). on the contrary, all the equine iv-infected pigs were saa-positive (range: . - . mg/ml), as also was one sample of the swine group ( . mg/ml). all the other samples were saa-negative (< . mg/ml). the expression of some cytokine genes was investigated by quantitative real-time pcr on balf cells. results are shown in figure and detailed hereunder. mock group: there was no significant difference between the different sampling times for each cytokine gene under study. swine group: there was no significant difference of gene expression at the three time points. only tendencies (p < . ) were shown for il- , il- , and ifn-gamma genes. equine group: significant differences were shown for ifna , ifn-gamma, and il- genes. in particular, at day p.i., an increase was observed of ifn-gamma and il- and a decrease of ifna gene expression. canine group: significant differences were shown for ifna / , ifna / , and ifn-gamma genes. at day p.i., there was a downregulation of ifna / and / , as opposed to a significant increase of ifngamma gene expression at day p.i. no other significant change of expression was detected. avian group: there were significant differences for ifna / , ifna , ifna , and ifn-gamma gene expression and tendencies for ifna / and ifna genes. in particular, a downregulation of these genes occurred at day p.i. seal group: ifna / was downregulated at day p.i., whereas ifn-gamma and il- genes were upregulated at the same time. based on the fact that local responses in balf from swine iv-infected animals were different from those of the other iv-infected pigs, we investigated whether there was any difference in cells infiltrating the lungs of infected pigs (figure ) . therefore, cells from balf were analyzed by flow cytometry to detect changes among animals of each group. swine iv-infected animals had significantly more t cells (cd +) at day and p.i. than the rest of the infected groups (p < . ), and the highest concentrations also corresponded to the appearance of a low concentration of the γδ t cell receptor phenotype. at day p.i., γδ t cell percentages were significantly different from those of the other infected pigs (p < . ). also, only swine iv-infected animals showed a clear increment of pdcs (defined as cd a + cd + cells) at day p.i., with a statistical tendency (p = . ). no major changes were detected concerning the surface expression of sla-ii or cd in balf cells (data not shown). in this study, the pathogenicity of iv strains with different host specificity was characterized in swine in terms of clinical symptoms and tissue lesions. also, we aimed to define a possible association between innate immune responses to iv strains, and clinical and postmortem findings. the main findings of our study are outlined in table . despite wide variations among individual pigs, the iv strains under study could be discriminated in terms of pathogenicity. our results confirmed a widely different replication of iv strains in the lower respiratory tract of swine: the swine-adapted virus replicated to a large extent, as opposed to the other iv strains under study. these pcr results were fully in agreement with virus titration results in our previous study ( ) . interestingly, the avian and seal h n strains replicated more than the equine and canine strains. replication of the canine and equine strains could have transiently taken place at days - post infection, before the first sampling. this is consistent with low-grade postmortem lesions and histopathological analyses on samples at day p. i., as reported in tables and . these findings highlight different invasiveness of swine, seal and avian, equine, and canine viruses, in descending order (figure ) . the replication to a higher level of swine, avian, and seal iv was associated with an earlier antibody response compared with groups equine and canine (figure ) , where a possible low-titered replication could account for the late antibody response to np. also, we cannot rule out that defective or partially assembled particles in the viral inoculum could also have induced antibodies to np, and that different iv strains could elicit antibodies with different reactivity in the assay. please notice, however, that amino acid identity of nps with respect to swine-adapted iv was very high, ranging between % (canine strain) and % (avian and seal strains). therefore, the combination of an ab response to iv np with macro and microscopic lesions and the clear discrimination from mock-infected animals indicate that pigs can be productively infected by h n viruses, which is also in agreement with the replication of both seal and avian h n strain in tracheal explants ( ) . most importantly, the pathogenic role of swine-adapted iv strain was confirmed by both gross and histological lesions in lungs, not induced by the other viruses under study. in turn, the presence of lesions could be temporarily associated with local innate immune responses in the lower respiratory tract (balf samples): ifn-α and il- ( figure ) . these findings are in agreement with those of other experimental infections of pigs with ivs ( , ) . moreover, previous results in our group showed that ifnα secretion was only detected in vitro when swine iv-infected myeloid porcine dcs but not when other ivs from human or avian origin were used ( ) . interestingly, the ifn-α titers in bal fluids of swine iv-infected pigs were not associated with the expression of ifn-α genes, in agreement with our previous data in vitro ( ) and another study in vivo ( ) . but for one animal, the il- local response in balf samples of the swine group at day p.i. was not in agreement with the presence of app. unexpectedly, saa was clearly expressed in balf samples of the equine group at day p.i. this indicates that saa responses can be induced despite poor virus replication in the lower respiratory tract. also, the equine strain widely affected the expression of cytokine genes (see figure ). this result suggests that innate immune responses can be triggered by some iv strains regardless of their replication efficiency. also, our findings confirm the possible extrahepatic expression of app observed in other disease models ( ) . the swine iv strain also caused detectable changes of the balf-infiltrating leukocyte populations. in particular, some cd +cd a+ cells were observed in balf from swine iv-infected animals but not in balf from the other iv groups. these are considered pdcs in swine ( ) , and their main function is the release of high levels of type i ifns. this is in agreement with the fact that ifn-α was only observed in balf samples of the swine iv-infected group (figure a) . ifn-α was detected at day p.i., whereas the increased prevalence of pdcs was detected at day p.i. this apparent discrepancy could be explained by our detection limit for pdcs in balf, as a certain minimum concentration would probably be required for detection by flow cytometry. also, we do not rule out a distinct or overlapping release of ifn-α by alveolar macrophages or other cells (e.g., epithelial cells) at an early time point after infection. despite the lesions caused by the swine iv strain, no disease signs were observed. this result is in contrast with an experimental infection of pigs with a danish h n iv strain, which led to the development of typical clinical signs of influenza ( ) . the absence of clinical signs was probably associated in our study with the reduced infectious challenge ( × eid ), which was necessary to standardize the challenge infection with iv strains showing high or poor replication in embryonated chicken eggs ( ) . yet, our data are in agreement with the fact that many pigs become infected with one or more iv subtypes without showing clinical signs ( ) . the gene expression data point at a crucial neglected issue: iv strains seem to cause a long-term regulation of the innate immune response, by far beyond the actual period of virus replication and persistence in organs and tissues. instead, the modulation of cytokine genes was more related to tissue damages, as indicated by the gross and hystopathological lesions persisting in some pigs until day p.i. retrospectively, earlier sampling of lung tissues and/or balf samples could have provided a more clear picture of cytokine gene expression levels, which were probably in a descending phase at day p.i. after a possible major peak at day ( ) . this is particularly true of ifn-β, which is highly up-regulated for a short time after iv infection ( ) . indeed, our previous results actually showed that ifn-β gene upregulation was only detected h after swine iv infection of myeloid porcine dcs in vitro ( ) . this is also in agreement with its role as immediate-early type i ifn gene in non-lymphoid cells ( ) . some innate immune responses were triggered by the swineadapted iv strain, only. diverse iv strain-specific factors could account for these responses, such as hemagglutinin receptor specificity and affinity, polymerase efficiency and activity of iv anti-ifn proteins, which may show additive or synergistic interactions with figure | rt real-time pcr for some cytokine genes was carried out on bronchoalveolar fluids cells collected at the indicated times after infection. for each sample, the relative expression of the selected genes was calculated using the formula Δct = ct (target gene)-cycle threshold (ct) (housekeeping), where ct values were the mean of three test replicates. results are shown as n-fold change in gene expression ( −Δct ). the same superscripts (a, b, c) on the bar indicate significant differences (p < . ) in one-way anova or kruskal-wallis test. samples from day p.i. are shown as gray bars, from day p.i. as striped bars, and from day p.i. as black bars. the host's immune system. on the basis of our previous results on tracheal explants of pigs ( ) , components other than affinity for sialic acid receptors of swine iv strains are likely to account for high innate immune responses. in particular, gross and histological lesions are probably induced following recognition of pamps of the swine-adapted iv strains and activation of the nlrp inflammasome (cryopyrin) ( ) . such a response is probably less intense after infection with other iv strains, which can be accounted for by different causes: (a) amino acid changes of some viral pamps. (b) suppressive regulations by odns of non-swine iv strains [as shown, e.g., for porcine circovirus ( ) , and/or by ifn-α subtypes with anti-inflammatory control activity ( ) ]. (c) third, the innate immune response to the swine-adapted iv could be simply due to the much higher and/or quicker replication of this strain. in this respect, viral replication measured by quantitative real-time pcr correlated with the degree of lesions. beyond the above hypotheses, a biological explanation of the observed results could be obtained by reverse genetics studies: changes or disruptions should be engineered in the viral genome, and effects of such alterations should be checked in vitro or in vivo. on the whole, the emerging picture outlines a host/virus relationship characterized by a strict control of the innate immune responses of pigs to non-adapted iv strains. the swine-adapted ones can circumvent these controls, in the framework of a rapid, effective, non ab-dependent containment of the primary virus infection in the lower respiratory tract. interestingly, the uncomplicated exposure to the swine iv strain did not induce clinical signs in our study. this finding points at a crucial role of the infectious pressure caused by common pathogens on farm for disease onset. this feature probably exerts additive and/or synergistic effects with common environmental stressors (animal density, seasonal changes, hierarchy fights after commingling) and genetic traits. as author contributions mm supervised the entire study. er performed real-time pcr assays for cytokine genes and data analysis thereof. mb and pm-o performed flow cytometry experiments and data analysis thereof. db, lc, and jm supervised sample collection, storage and distribution, as well as cytokine, clinical immunology, histopathology, and antibody assays. ef and cc performed realtime pcr assays for ivs and data analysis thereof. as, dp, mab, and gr provided scientific support. ma supervised clinical immunology assays, cytokine protein assays, and the manuscript writing. acknowledgments regulation of adaptive immunity by the innate immune system type i interferons in host defense the 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characterization of biologically active murine interleukin- produced in escherichia coli increased levels of tumor necrosis factor and interleukin in bronchoalveolar lavage fluids from pigs infected with mycoplasma hyopneumoniae characterization of five monoclonal antibodies specific for swine class ii major histocompatibility antigens and crossreactivity studies with leukocytes of domestic animals characterization of the interferon-alpha response of pigs to the weaning stress reverse transcription real-time pcr for detection of porcine interferon alpha and beta genes ipec-j cells as reporter system of the anti-inflammatory control actions of interferon-alpha analysis of relative gene expression data using realtime quantitative pcr and the (-delta delta c(t)) method expression of innate immune genes, proteins and micrornas in lung tissue of pigs infected experimentally with influenza virus (h n ) rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs ) for the invaluable help in measuring some clinical immunology parameters, dr. l. fraile (udl, spain) for assistance in statistical analysis dendritic cells in innate and adaptive immune responses against influenza virus porcine innate and adaptative immune responses to influenza and coronavirus infections differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor- caspase- : the inflammasome and beyond identification of a sequence from the genome of porcine circovirus type with an inhibitory effect on ifn-alpha production by porcine pbmcs differential biological activities of swine interferon-alpha subtypes we would like to thank dr. jaime maldonado the research leading to these results has received funding from: the european community's seventh framework programme (fp , (fp , - , research infrastructures action, under the grant agreement no. fp - (nadir project), and from the project agl - -c - of spanish ministry of science and innovation. key: cord- -g y kjju authors: konstantinova, p; de vries, w; haasnoot, j; ter brake, o; de haan, p; berkhout, b title: inhibition of human immunodeficiency virus type by rna interference using long-hairpin rna date: - - journal: gene ther doi: . /sj.gt. sha: doc_id: cord_uid: g y kjju inhibition of virus replication by means of rna interference has been reported for several important human pathogens, including human immunodeficiency virus type (hiv- ). rna interference against these pathogens has been accomplished by introduction of virus-specific synthetic small interfering rnas (sirnas) or dna constructs encoding short-hairpin rnas (shrnas). their use as therapeutic antiviral against hiv- is limited, because of the emergence of viral escape mutants. in order to solve this durability problem, we tested dna constructs encoding virus-specific long-hairpin rnas (lhrnas) for their ability to inhibit hiv- production. expression of lhrnas in mammalian cells may result in the synthesis of many sirnas targeting different viral sequences, thus providing more potent inhibition and reducing the chance of viral escape. the lhrna constructs were compared with in vitro diced double-stranded rna and a dna construct encoding an effective nef-specific shrna for their ability to inhibit hiv- production in cells. our results show that dna constructs encoding virus-specific lhrnas are capable of inhibiting hiv- production in a sequence-specific manner, without inducing the class i interferon genes. supplementary information: the online version of this article (doi: . /sj.gt. ) contains supplementary material, which is available to authorized users. rna silencing or rna interference (rnai) is an evolutionary conserved sequence-specific post-transcriptional gene regulation mechanism that plays an important role in cell differentiation and development. [ ] [ ] [ ] in addition, rnai serves as a defence mechanism against invading viruses and transposons. [ ] [ ] [ ] rna interference is triggered by double-stranded rna (dsrna) molecules, which are processed in the cytoplasm by the dsrnaspecific endonuclease dicer into - nucleotides (nt) small interfering rnas (sirnas) or micro-rnas (mirnas). these si/mirnas are incorporated into the multiprotein rna-induced silencing complex (risc) that guides the recognition and ultimately the cleavage or translational repression of complementary singlestranded rna, such as messenger rna or viral genomic rna. [ ] [ ] [ ] rna interference has been employed to inhibit the replication of a wide range of viruses, including the human immunodeficiency virus type (hiv- ), hepatitis c virus (hcv), hepatitis b virus (hbv), dengue virus, poliovirus, influenza virus a, coronaviruses, herpesviruses and picornaviruses. human immunodeficiency virus type virions contain a single-stranded rna genome that is a putative rnai target. after entry into a host cell the genomic rna is reverse transcribed into dsdna, which is integrated into the host chromosomal dna. newly synthesized unspliced genome-length and spliced subgenomic viral rnas are possible targets for rnai in the cytoplasm. it has recently been reported that hiv- encodes a suppressor of rnai, the tat protein, indicating that hiv- replication is controlled by rnai in human cells. due to its sequence specificity, rnai is a potentially powerful and selective method for intracellular immunization against hiv- infection. rna interference-mediated suppression of hiv- replication has been accomplished by synthetic sirnas in a transient manner [ ] [ ] [ ] [ ] and by short-hairpin rna (shrna)-expression vectors in stably transfected cells. [ ] [ ] [ ] despite potent inhibition, the use of sirna/shrna as a therapeutic antiviral is limited, because of the rapid emergence of hiv- escape mutants. [ ] [ ] [ ] minor sequence changes in the target sequence, sometimes even a single point mutation, are sufficient to abrogate the inhibition of virus replication. strategies to reduce the chance of viral escape include the simultaneous use of multiple sirnas , or the use of long-hairpin rna (lhrna, a single-hairpin molecule) or long dsrna (two complementary molecules that form a duplex). , another possibility is the use of mirna-based approaches, which do not require perfect sequence complementarity. , several reports describe efficient rnai induction by lhrna and long dsrna as in vitro generated transcripts that are transfected into cells or as gene constructs that produce the transcripts intracellularly. transfection of pre-implantation mouse embryo cells, undifferentiated embryonic stem cells and embryonic carcinoma cells with in vitro synthesized long dsrna confers specific gene silencing. , however, exposure of non-embryonic mammalian cells to dsrnas longer than basepairs (bp) leads to rapid induction of a specific set of cytokines, including the class i interferons (ifns). during natural virus infections, the ifn response is activated by virusproduced dsrnas, and acts as an innate defense mechanism. viruses counter this response by encoding ifn antagonists, which are also responsible for the fact that antiviral ifn therapy is often not successful. , so far, virus-encoded rnai suppressor factors, like the hiv- tat protein, do not appear to be able to suppress induced antiviral rnai. strong induction of rnai by intracellular expression of virus-specific dsrnas is likely to outcompete the inhibiting effects of rnai suppressors. efficient rnai-mediated gene silencing has been shown in mammalian cells by endogenously expressed long dsrnas. , , in chinese hamster ovary (cho) cells, a dna construct encoding a bp long dsrna specifically inhibits luciferase expression in a sequencespecific manner. complete and specific gene silencing was achieved in different mammalian cell types by expression of , , or even bp long dsrnas. , [ ] [ ] [ ] interestingly, intact dsrna could not be detected in these cells, suggesting that it is rapidly processed by dicer in the cytoplasm. recently, ski knockdown mice have been produced using a dna construct encoding long dsrna-specific for the murine ski gene. these results suggest that dsrna is tolerated in mammalian cells, most likely because it is rapidly processed by the rnai machinery. several antiviral approaches using extended dsrna have been reported in plant and insect cells lacking the innate antiviral ifn response. although plants and insects lack the ifn response, they also have potent innate antiviral responses, comparable to those in mammals. transient expression of dna constructs encoding virus-specific dsrna in plant protoplasts or insect cells partially protects the cells from infection by the homologous virus. , stable expression of such constructs in plant or insect cells renders the cells completely resistant or immune to infection. , in vitro made long dsrnas have been used to inhibit hiv- production under certain conditions without induction of the ifn response. , we have previously demonstrated potent inhibition of hiv- replication in t cells that stably express an shrna targeted to viral nef gene sequences. to test whether endogenously expressed lhrna and long dsrna can inhibit hiv- at least as potently as sh-nef, we constructed and tested a series of dna constructs. it has previously been demonstrated that hiv- replication can be inhibited by sirnas and shrnas directed against viral targets. , , , , , most of the active sirnas against hiv- are targeted to the early regulatory tat, rev and nef genes. , , , interference with an early stage of the hiv- replication cycle may be beneficial. for this reason, the dna constructs encoding lhrnas (a single-hairpin molecule) and long dsrnas (two complementary molecules that form a duplex) were designed to target tat, rev and nef sequences as indicated in figure . inhibition of human immunodeficiency virus type by in vitro transcribed ds-nef rna and its diced product we initially tested whether in vitro transcribed and annealed nef dsrna and its in vitro diced product si-nef , a mixture of nef-specific sirnas, can inhibit hiv- . nef dsrna of bp was diced in vitro to create si-nef rnas of approximately bp (figure a) . we cotransfected ng of the hiv- molecular clone plai with and without ng inhibitory rna in human embryonic kidney (hek) t cells. dna of prl expressing renilla luciferase was included in the transfection mixtures to monitor cell viability and possible non-specific effects, for example, due to ifn induction by dsrna. virus production was measured by ca-p enzyme-linked immunosorbent assay (elisa) in the culture supernatant days after transfection. the amount of virus production without an inhibitory rna, generally in the - ng/ ml ca-p range, was set at %. nef dsrna induced a significant decrease in ca-p production, but even more pronounced level of inhibition was obtained with diced si-nef ( figure b ). this can be explained by the fact that si-nef bypasses the intracellular dicing step, which may be a limiting factor in the rnai pathway. one of the hallmarks of the rnai is its sequence specificity. therefore, we tested if nef dsrna and its in vitro diced product si-nef would inhibit pgl -nef reporter, in which nt from the nef target sequence was placed downstream of the luciferase reporter gene. nef dsrna induced a decrease in luciferase expression, but an even more pronounced level of inhibition was figure scheme of the human immunodeficiency virus type (hiv- ) plai proviral genome and target sequences used for the design of long-hairpin rnas (lhrnas). the target sequences are indicated as bars below the hiv- coding regions. lhrna ( basepairs (bp)) tat fuses tat exon (gray bar, - ) and tat exon (black bar, - ) sequences, rev fuses rev exon (gray bar, - ) and rev exon (black bar, - ) and nef contains nef-ltr sequences ( - ). double-stranded rna nef is a duplex of two separate, complementary sense and antisense nef sequences ( - ). the positive control sh-nef is a -bp hairpin consisting of nef sequences ( - ). inhibition of hiv- by lhrna p konstantinova et al obtained with diced si-nef ( figure c ). the prl expression was not influenced (results not shown). in fact, in vitro diced si-nef is a much more effective inhibitor than the in vitro synthesized short-hairpin inhibitor sh-nef rna, which was included as a positive control. we next tested the inhibitory potential of nef dsrna in the - ng range. at amounts above ng, non-specific decrease of renilla reniformis luciferase (rl) expression was observed, which is most likely due to ifn induction (results not shown, see also figure ). the high level of inhibition obtained with low amounts of dsrna convinced us to design and test a series of dna expression plasmids encoding long hiv- -specific dsrnas. low-level inhibition of human immunodeficiency virus type by long-hairpin rna expression plasmids to make lhrna constructs, we cloned the hiv- tat, rev and nef gene sequences as inverted repeats under the transcriptional control of the constitutive ef a promoter ( figure ). these vectors should produce lhrna, a longhairpin structure consisting of an approximately bp stem and a nt loop. during transcription of the inverted sequences, an rna molecule is made, which folds back on itself to form a hairpin structure with a stem of approximately bp. in silico rna analysis with the mfold program confirms the folding of these extended hairpins (data not shown). we tested inhibition of hiv- in several mammalian cell lines (c a, hek t and vero). cotransfection of plai with the pef atat, pef a-rev or pef a-nef vectors resulted in marginal inhibition of hiv- production in c a and hek t cells, and only the pef a-tat vector was inhibitory in vero cells ( figure a ). we also designed a control pef a-green fluorescent protein (gfp) plasmid that expresses a similar extended lhrna against gfp mrna. no inhibition of virus production was observed with the control pef a-gfp vector, thus providing additional evidence for the specificity of lhrna-mediated inhibition of hiv- production. we previously demonstrated potent inhibition of hiv- replication in t cells that stably express an shrna targeted to viral nef gene sequences. therefore, if lhrna inhibits hiv- potently, it should be at least as active as the sh-nef control. unlike the results with in vitro synthesized nef dsrna, either diced or not, all lhrna constructs (tat, rev and nef ) were much less potent inhibitors than the control sh-nef construct, which produces the short hairpin from a polymerase iii promoter. because pef a-tat and pef a-nef were slightly more effective than pef a-rev in c a and t cells, we focused on these lhrnas in the subsequent experiments. in order to avoid innate viral responses or possible other side effects and to obtain controllable expression of lhrna, we placed the expression of the lhrnas tat and nef under control of inducible promoters: (i) the doxycycline (dox) inducible tet system and (ii) the tat-inducible long-terminal repeat (ltr) promoter/enhancer of hiv- (figure ). the latter system seems ideally suited to restrict lhrna expression to cells that are infected by hiv- , thus providing a unique safety feature. furthermore, we replaced the bp spacer between the repeats by the bp ef- a intron, since it has been shown that this improves the inhibitory potential of lhrna-encoding dna constructs. the tet-on system is based on the specific, highaffinity binding of the rtta trans-activator to the tet operator (teto) in the presence of dox, triggering transcription of downstream genes. this system has recently been used to express synthetic mirna precursors in mouse or human genomes, following trans- inhibition of hiv- by lhrna p konstantinova et al duction with retroviral or lentiviral vectors. vector plai was cotransfected with p teto-tat or p teto-nef ( figure ). we also included pcmv-rtta and the control prl, and dox was added h later. p teto-tat or p teto-nef conferred no or very poor inhibition of hiv- as compared to the sh-nef control (figure b ). the potency of the lhrnas was not increased by varying the ratio of lhrna-expression vector to pcmv-rtta or the dox concentration. moreover, a non-specific decrease of rl expression was observed at higher pcmv-rtta concentrations, which is probably due to promoter squelching (results not shown). in order to restrict lhrna expression to cells that are infected with hiv- , the inverted tat and nef repeats were cloned downstream of the tat-inducible ltr promoter/enhancer of hiv- , resulting in plasmids pltr-tat and pltr-nef ( figure ) . a tat-inducible pol ii promoter expressing anti-hiv shrna has been described for inhibition of hiv- gene expression in mammalian cells. in this setting, lhrna or shrna expression is activated in trans by the tat protein encoded by hiv- . to avoid self-targeting, we deleted a large part of the u region (up to position À ) in the ltr promoter of both ltr expression plasmids that overlaps with the nef coding domain ( nt). human embryonic kidney t cells were cotransfected with plai and pltr-tat or pltr-nef . both expression plasmids inhibit virus production by approximately % (figure a ). the poor inhibitory potency of the different lhrna constructs could be due to expression problems, but the lhrna may also encounter difficulties in entering or proceeding along the rnai pathway. in order to increase the efficacy of the lhrna molecules, we cloned the hiv- leader sequence (c) between the ltr and nef in pltrc-nef ( figure ). control plasmid pltrasc-nef was made with the c element inserted in antisense orientation. previously, we reported that sequences from the untranslated leader of the hiv- genome, such as the rna dimerization signal, can be used to inhibit hiv expression in trans. we presumed that c should bring the antiviral rna along viral pathways, conferring a stronger inhibitory effect. indeed, in hek t cells transfected with plai and pltrc-nef , hiv- production was almost completely inhibited ( figure a ). the level of inhibition conferred by pltrc-nef is comparable to that of the positive control sh-nef. inhibition is specific and not due to a more general cell toxicity problem because no significant decrease in rl expression was observed (results not shown). control plasmid pltrasc-nef was much less effective in inhibiting hiv- production, with a potency comparable to that of the original pltr-nef construct. plasmid pltrc, in which the nef sequence has been deleted, failed to inhibit hiv- production (figure b ). this result indicates that the presence of the c element enhances lh-nef -mediated inhibition of hiv- , but the presence of the nef sequence is essential for the potent effect of pltrc-nef . transcript c-nef expression from the ltr promoter is induced by plai-encoded tat protein. because plai gene expression is strongly inhibited by pltrc-nef , a negative feedback loop may have been established, which leads to an underestimation of the inhibitory potential of pltrc-nef . we therefore added a tatexpression plasmid (pcdna -tat) in trans to secure pltrc-nef expression. human embryonic kidney t cells were transfected with ng plai and - ng pltrc-nef with or without pcdna -tat. as shown in figure c , pronounced hiv- inhibition was obtained with as little as ng pltrc-nef . the presence of figure expression vectors for long-hairpin rna (lhrna), double-stranded rna (dsrna) and short-hairpin rna (shrna). longhairpin rnas were created by cloning the -nucleotide (nt) inverted repeats from tat, rev and nef (see figure ) downstream of the ef a, teto or long-terminal repeat (ltr) promoters. the kb ef a intron is positioned downstream of the ef a promoter. a schematic representation of the final hairpin structures is shown on the right. in pef a constructs, the two complementary rna strands are separated by a nt loop. in the p teto and pltr constructs, the complementary sequences are separated by a kb spacer that contains splice donor and acceptor sites. vector pltrc-nef is a derivative of pltr-nef in which the human immunodeficiency virus type (hiv- ) leader sequence (c, - , marked as a gray box) was inserted. the predicted transcript will have the c domain upstream of the rna hairpin. all transcripts contain a polyadenylation signal (pa) downstream of the hairpin sequences. vector pt -nef has basepairs (bp) long doublestranded nef sequences flanked by t promoters (t ) and terminators (f) at both and ends. two separate complementary rna chains, potentially capable of forming dsrna, are transcribed from the convergent promoters by t rna polymerase (encoded by expression plasmid pt -pol). vectors pt sh-nef and ph sh-nef express sh-nef from the t and h promoters, respectively. inhibition of hiv- by lhrna p konstantinova et al additional tat did not significantly improve the inhibition conferred by pltrc-nef , indicating that the background ltr promoter activity produces sufficient amounts of the inhibitory transcript. we next wanted to test if the potent pltrc-nef construct was able to inhibit hiv- variants that escaped from the sh-nef inhibitor. we described previously a series of viral escape variants with mutations or deletions in the targeted nef sequence. we selected mutant r with a nt deletion that includes the complete sh-nef target sequence and mutant r with two point muta- figure marginal inhibition of human immunodeficiency virus type (hiv- ) production by pef a-and p teto-driven longhairpin rna (lhrna) constructs. (a) cells (c a, human embryonic kidney (hek) t and vero) were lipofectamine-transfected with ng plai, ng inhibitory construct, ng pcmv-rtta and . ng prl. vector pef a-green fluorescent protein (gfp) was used as a control expressing an irrelevant lhrna against gfp. vectors ph sh-nef and the empty vector were used as negative and positive controls, respectively. virus production was determined as described in the legend to figure . standard error bars represent the means of three independent experiments. the sh-nef control construct was not tested (n.t.) in c a cells. (b) hek t cells were cotransfected with ng plai, ng p teto-tat, p teto-nef or ph sh-nef, ng pcmv-rtta and . ng prl as an internal control. culture medium was refreshed after h and mg/ml doxycycline was added. virus production was determined as described in the legend to (table ) . human embryonic kidney t cells were cotransfected with ng hiv- molecular clone plai (wild-type, r or r ) and ng pltrc-nef or ph sh-nef plasmid. only the wild-type plai construct was equally inhibited by the pltrc-nef and sh-nef constructs. the deletion mutant r is completely resistant to sh-nef, but was potently inhibited by pltrc-nef . mutant r partially escapes from sh-nef, but is potently inhibited by pltrc-nef . rna interference is mainly a cytoplasmic process, and putative problems in nuclear export of lhrnas may thus hamper their inhibitory activity. in order to test whether there is a difference between the rnai-inducing capacity of dsrnas produced in the nucleus or cytoplasm of cells, we expressed dsrna targeted to the nef-coding domain (nef in figure ) using the cytoplasmic t rna polymerase system. to generate pt -nef , we cloned a bp nef-specific dna fragment between convergent t promoters and terminators ( figure ). in addition, the control expression plasmid pt sh-nef was constructed. cytoplasmic transcription of pt -nef and pt sh-nef is performed by t -polymerase encoded by pt -pol. expression of the firefly luciferase (fl) reporter from the t promoter (pt -luc) was measured as a positive control in all transfection experiments with pt -pol ( figure a ). human embryonic kidney t cells were transfected with plai and pt -nef or pt sh-nef, with and without pt -pol. virus production was measured in the culture supernatant days after transfection by ca-p elisa. in the absence of t -polymerase, pt -nef and pt sh-nef confer a low level of inhibition on virus production probably due to leaky rna expression from the plasmids (figure b ). with t -polymerase, pt -nef and pt sh-nef confer up to % inhibition of hiv- production (figure b ). this level of inhibition is comparable to that obtained with the sh-nef control expression plasmid. renilla luciferase expression was not affected (figure b, gray bars) . to demonstrate that the effect of pt -nef is sequence-specific, we cotransfected it with the pgl -nef reporter (figure c ). in the presence of t -polymerase, pt -nef potently inhibited luciferase expression. we next tested the inhibitory potential of both pt -nef and pt sh-nef plasmids in the presence of an increasing amount ( - ng) of pt -polymerase. the inhibition was consistently strong even at low amounts ( ng) of the pt -pol plasmid (figure d ). when more than ng pt -pol was used in the transfection experiments, a non-specific decrease in rl reporter expression was observed. a high amount of t -polymerase accumulating in the cytoplasm of cells may be toxic or high amounts of dsrna accumulating in the cytoplasm of cells may induce the innate antiviral ifn response. to measure the rna expression levels of selected lhrna-expression constructs, we performed a reverse transcriptase-polymerase chain reaction (rt-pcr) assay with nef-specific primers. all constructs expressed nef rna (figure ) . notably, pt -nef produced low amounts of rna even in the absence of the pt pol inducer. this can explain the slight inhibition of gene expression obtained with pt -nef in the absence of pt pol (figure b and c) . no nef-specific fragment was detected in mock-transfected cells or in cells transfected with poly(i:c). endogenously expressed long-hairpin rna and long double-stranded rna do not induce the interferon response it has been reported that introduction of dsrnas longer than bp in mammalian cells induces the innate antiviral ifn response. we determined ifn-b induction by rt-pcr in hek t cells upon transfection with in vitro made nef dsrna and dna constructs encoding pef a-nef , pltr-nef , pltrc-nef , pt -nef or ph shnef. transfection of mg poly(i:c), a known inducer of ifn and other cytokines, was used as a positive control. the lhrna-and sh-nef-expression plasmids did not induce detectable levels of ifn-b mrna (figure ). both nef dsrna and poly(i:c) controls induced high amounts of the ifn-b mrna. the use of synthetic sirnas or shrna-expression plasmids as inducers of rnai-based therapy against hiv- faces the major obstacle of the emergence of virus escape variants. similar to the combined use of antivirals in highly active antiretroviral therapy (haart), one could design an rnai therapy with extended lhrna/ dsrna. endogenous expression of two long complementary rnas, with the potential to form an extended dsrna duplex, leads to specific suppression of gene expression in mammalian cells. , , , interestingly, fulllength dsrnas could not be detected, suggesting that inhibition of hiv- by lhrna p konstantinova et al processing by dicer precludes their accumulation in the cytoplasm. long dsrnas are indeed processed in vitro by the rnai machinery into multiple active sirnas. , several lines of evidence show that lhrnas induce gene silencing by the rnai mechanism. inhibition of dicer abrogated the gene silencing induced by lhrna against gfp, indicating that silencing was mediated by rnai. these results suggest that long dsrna is well tolerated in mammalian cells, most likely because it is processed rapidly by the rnai machinery. it is also relevant to mention that mammalian cells may naturally produce dsrna derived from repetitive and transposable elements. , a recently performed bioinformatics study revealed the presence of at least full-length transcripts, which form sense-antisense gene pairs in the human genome. we designed a set of anti-hiv- lhrna/dsrnaexpression constructs and compared their ability to inhibit virus production with a very potent shrnabased inhibitor that we described previously. ideally, a single lhrna should provide more potent inhibition of hiv than an shrna. an additional advantage of lhrna is that it does not require predetermination of optimal shrnas and hiv- target sequences because multiple effective sirnas will be produced. a potential disadvantage of the use of lhrna as therapeutic is that the multiple sirnas are more likely to cause off-target effects. (a) pt -luc ( ng) construct was cotransfected in human embryonic kidney (hek) t cells with or without ng pt -pol. at days after transfection, cells were lysed and the expression of firefly luciferase was measured. (b) pt -nef ( ng) and pt sh-nef vectors were linearized of the t termination signal with xbai and bamhi, respectively, and cotransfected with ng plai, ng pt -pol and . ng prl in hek t cells. two separate complementary rna chains, potentially forming dsrna, are transcribed in the cell from convergent t promoters. equal amounts of a ph sh-nef expression vector and the empty vector were added as positive and negative controls, respectively. cotransfections were also performed without pt -pol to check for non-specific effects of the t plasmids. virus production was determined as described in the legend to figure . standard error bars represent the means of four independent experiments. cells were lysed days after transfection to measure renilla luciferase. (c) sequence-specific inhibition of the pgl -nef reporter, containing the -nucleotide (nt) nef target sequence downstream of the luciferase coding domain, by pt -nef . hek t cells were cotransfected with ng of pgl -nef, ng pt -nef , ng pt -pol and . ng prl. ph sh-nef ( ng) expression vector and the empty vector were added as positive and negative controls, respectively. (d) titration of t polymerase. plai ( ng) was cotransfected with increasing amounts ( - - - - ng) of pt -pol and ng pt -nef or pt sh-nef. inhibition of hiv- by lhrna p konstantinova et al long-hairpin rnas expressed from constitutive (ef a) and inducible ( teto, hiv- ltr) promoters inhibited hiv- production marginally, but we demonstrated potent and specific hiv- silencing with modified dna constructs. the most active constructs either link the lhrna to viral rna sequences (c) or express the long dsrna directly in the cytoplasm, suggesting that translocation of dsrna from the nucleus to the cytoplasm is a crucial step for these molecules to enter the cytoplasmic rnai pathway. it is widely accepted that rnai is a cytoplasmic process, as most protein components of the rnai pathway, including argonaute , trbp and dicer, assemble and function in the cytoplasm. , [ ] [ ] [ ] [ ] [ ] putative problems in nuclear export of lhrna may thus hamper its inhibitory activity. modification of the lhrna by addition of the hiv- leader sequence (c) or direct cytoplasmic expression of long dsrna by the t polymerase created potent inhibitors. the viral c sequence may provide the transcript with a non-self signature and thereby boost rnai. it has previously been suggested that the inhibitory effect of c-containing transcripts is sequence-specific, possibly due to premature formation of rna dimers, but the exact mechanism of inhibition remains to be elucidated. a major advantage of an lhrna inhibitor over shrna is the reduced chance of viral escape because a larger segment of the viral genome is targeted, as has been shown recently. as an initial test of this idea, we used two hiv- mutants that escaped from sh-nef by two point mutations or complete deletion of the target sequence. these escape mutants could be inhibited by the lhrna construct pltrc-nef , confirming the hypothesis that lhrna may delay or prevent the evolution of hiv- escape variants. we showed that endogenously expressed lhrna and dsrna do not activate the innate antiviral response. a similar result has recently been described in literature. , exposure of cells to a bp in vitro synthesized dsrna induces the production of class i ifns, but not when such molecules are expressed in the cell from a dna construct with the u promoter. modification of lhrnas expressed from a pol iii promoter by inclusion of multiple g:u wobbles induced rnai without any non-specific effects. in fact, most reports on ifn induction by long dsrnas in mammalian cells are based on transfection of cells with in vitro synthesized dsrnas. , apparently, endogenously produced dsrna is less active than exogenous dsrna in inducing the ifn response. this finding may have a major impact on the further development of rnai-based antiviral strategies. rna interference-based gene therapy against hiv- seems to be a viable option, either as mono-therapy or combined with traditional drug therapy. , the outgrowth of rnai-resistant virus mutants presents a major obstacle for all sequence-specific inhibitory strategies. the simultaneous targeting of multiple conserved targets by lhrna may confer increased robustness to future rnai therapies. we will focus on optimization of the dna constructs encoding hiv- -specific lhrnas, for example, the type of promoter used and the structure of the lhrna. we will also test stable expression of these constructs in hiv-susceptible cells. these cell lines will be extensively tested for the emergence of rnai-resistant virus variants. figure in vivo rna expression from long-hairpin rna (lhrna) constructs. hek t cells were transfected with ng of the indicated lhrna/double-stranded rna (dsrna)-expression constructs using lipofectamine . constructs pltr-nef and pltrc-nef were cotransfected with ng ptat. construct pt -nef was cotransfected with ng pt -pol. the puc plasmid and poly (i:c) were used as negative controls and pt -nef plasmid dna was used as a positive control. total rna was isolated from the cells h after transfection. the nef expression level was determined by reverse transcriptase-polymerase chain reaction (rt-pcr) with primers nef-b/x and antiu -att, which create a basepairs (bp) amplification product. b-actin mrna expression was analyzed as an internal control. pt -nef rtÀ is a control reaction without rt step. t cells ( .  ) were cotransfected with plai and the indicated lhrna/double-stranded rna (dsrna)-expression constructs using lipofectamine . construct pt -nef was cotransfected with pt -pol. renilla luciferase (prl) was used as an internal control. the puc plasmid was used as a negative control. in vitro transcribed ds-nef rna and poly (i:c) act as positive controls for ifn-b induction. two separate transfections were performed, which were processed for either ifn-b mrna expression or renilla measurement. no significant differences in renilla expression were measured, except for the toxic treatment with ds-nef rna and poly(i:c) (results not shown). in addition, we measured ca-p in the supernatant, which showed the inhibition characteristics described earlier. total rna was isolated from the cells h after transfection. the ifn-b expression level was determined by reverse transcriptase-polymerase chain reaction (rt-pcr). b-actin mrna expression was analyzed as an internal control. puc rt-and poly (i:c) rt-are control reactions without rt step. inhibition of hiv- by lhrna p konstantinova et al the full-length hiv- molecular clone plai was used to produce wild-type virus and to study inhibition by lhrnas directed against the tat, rev or nef sequences. a detailed description of the construction of all lhrnaexpression constructs is available as supplementary information. plasmid pcdna -t pol, expressing bacteriophage t polymerase (pt -pol, a kind gift of dr jean-marc jacque, university of massachusetts medical school, worcester, ma, usa), pgl -nef and ph sh-nef have been described previously. , the mfold program (http://www.bioinfo.rpi.edu/ applications/mfold) was used to check the correct folding of extended hairpins. in vitro transcription and dicing of ds-nef rna the ds-nef rna was in vitro transcribed with the megashortscript t transcription kit (ambion, austin, tx, usa) from the nef pcr template that contains convergent t promoters and terminators. the sh-nef rnai inducer was transcribed in vitro from the bamhilinearized pt sh-nef vector. ds-nef rna ( mg) was diced in vitro at c for - h using recombinant dicer enzyme (stratagene, cedar creek, tx, usa). the fulllength ds-nef rna, the cleaved si-nef (a mix of sirnas derived from ds-nef ) and sh-nef were purified through microspin g- column (amersham biosciences, piscataway, nj, usa). to remove undigested dsrna from the dicing reaction, the si-nef was purified further on a microcon ym- column (millipore, billerica, ma, usa). si-nef rna was analyzed on % metaphor (bma, sanver tech, the netherlands) alongside a bp dna marker (gensura, san diego, ca, usa). human embryonic kidney t, c a cervix carcinoma and african green monkey kidney vero cells were grown as a monolayer in dulbecco's modified eagle's medium (dmem) (gibco brl, carlsbad, ca, usa) supplemented with % fetal calf serum (fcs) (hybond, escondido, ca, usa), minimal essential medium with non-essential amino acids, penicillin ( u/ml) and streptomycin ( mg/ml) at c and % co . at day before transfection, cells were trypsinized, resuspended in dmem and seeded in -well plates at a density of .  cells per well. cells were cotransfected with - ng plai and - ng in vitro transcribed ds-nef rna and in vitro diced si-nef or - ng lhrna (tat, rev, nef ) expression constructs using lipofectamine (invitrogen, carlsbad, ca, usa). virus production was determined by measuring the ca-p levels in the culture supernatant by elisa (abbott, abbott park, il, usa) as described previously. for firefly luciferase measurements, cells were cotransfected with ng pgl -nef and ng in vitro transcribed ds-nef rna and in vitro diced si-nef or ng pt -nef and ng pt -pol. in all experiments, vector prl ( . ng) (promega, madison, wi, usa), expressing rl under the control of the cmv promoter, was added to the transfection mix as a control for variation in transfection efficiency and cell viability. equal amounts of the ph sh-nef expression vector or the empty vector were added as positive and negative controls, respectively. vectors pt -luc and pt -pol were cotransfected in equimolar amounts ( ng) and fl reporter expression was measured. briefly, cells were lysed - h after transfection in ml  passive lysis buffer (promega) by shaking for min at room temperature. the cell lysate was centrifuged for min at r.p.m. and firefly and renilla luciferase expression was measured in ml supernatant with the dual-luciferase reporter assay system or renilla luciferase assay system (promega). induction of the ifn system was measured by a sensitive rt-pcr on the ifn-b mrna. rna was isolated from hek t cells with the mirvana mirna isolation kit (ambion) h after transfection with the long dsrna constructs. first-strand cdna was reverse transcribed from approximately mg rna with random hexamer primers (invitrogen) using the mmlv-rt enzyme (invitrogen) according to the manufacturer's instructions. approximately ng cdna was pcr amplified with primers ifn-bf and ifn-br using standard conditions. amplification of the b-actin gene was used as an internal control. transfection of mg poly(i:c) (amersham pharmacia biotech, piscataway, nj, usa), a synthetic inosine/cytosine polymer that mimics viral dsrna, was used as a positive control for ifn-b induction. rna of transfected cells was subjected to pcr with nef-specific primers nef-b/x and antiu -att, yielding a bp fragment (supplementary table ). induction of stable rna interference in mammalian cells ribo-gnome: the big world of small rnas rnai: the nuts and bolts of the risc machine rna silencing: the genome's immune system sequence-specific inhibition of microrna-and sirna-induced rna silencing a potential role for rna interference in controlling the activity of the human line- retrotransposon dicer functions in rna interference and in synthesis of small rna involved in developmental timing in c. elegans potent and specific genetic interference by double-stranded rna in caenorhabditis elegans an rnadirected nuclease mediates post-transcriptional gene silencing in drosophila cells atp requirements and small interfering rna structure in the rna interference pathway inhibition of virus replication by rna interference evidence that hiv- encodes an sirna and a suppressor of rna silencing inhibition of hiv- infection by small interfering rna-mediated rna interference modulation of hiv- replication by rna interference sirna-directed inhibition of hiv- infection prevention of hiv- infection in human peripheral blood mononuclear cells by specific rna interference bispecific short hairpin sirna constructs targeted to cd , cxcr , and ccr confer hiv- resistance enhanced gene silencing of hiv- specific sirna using microrna designed hairpins human immunodeficiency virus type escapes from rna interference-mediated inhibition human immunodeficiency virus type escape from rna interference hiv- can escape from rna interference by evolving an alternative structure in its rna genome rna interference as an antiviral approach: targeting hiv- a novel approach for inhibition of hiv- by rna interference: counteracting viral escape with a second generation of sirnas double-stranded nef rna interferes with human immunodeficiency virus type replication changes in microrna expression profiles in hiv- -transfected human cells targets for human encoded micrornas in hiv genes specific interference with gene function by double-stranded rna in early mouse development specific interference with gene expression induced by long, doublestranded rna in mouse embryonal teratocarcinoma cell lines specific inhibition of gene expression by small double-stranded rnas in invertebrate and vertebrate systems hiv- tat directly interacts with the interferoninduced, double-stranded rna-dependent kinase hiv-i tat inhibits pkr activity by both rna-dependent and rnaindependent mechanisms stable suppression of gene expression by rnai in mammalian cells analysis of double-stranded rna-induced apoptosis pathways using interferon-response noninducible small interfering rna expression vector library sensitive assay of rna interference in drosophila and chinese hamster cultured cells using firefly luciferase gene as target long endogenous dsrnas can induce complete gene silencing in mammalian cells and primary cultures specific and potent rna interference in terminally differentiated myotubes control of specific gene expression in mammalian cells by co-expression of long complementary rnas generation of ski-knockdown mice by expressing a long double-strand rna from an rna polymerase ii promoter plant pathogens and integrated defence responses to infection inhibition of viral gene expression and replication in mosquito cells by dsrnatriggered rna interference dissecting rna silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense rna rna silencing of dengue virus type replication in transformed c / mosquito cells transcribing an inverted-repeat rna derived from the virus genome rna interference of hiv replication expression of small interfering rnas targeted against hiv- rev transcripts in human cells choosing ccr or rev sirna in hiv- mfold web server for nucleic acid folding and hybridization prediction tight control of gene expression in mammalian cells by tetracycline-responsive promoters tat trans-activates the human immunodeficiency virus through a nascent rna target total silencing by intron-spliced hairpin rnas a lentiviral microrna-based system for single-copy polymerase ii-regulated rna interference in mammalian cells negative feedback inhibition of hiv- by tat-inducible expression of sirna inhibition of human immunodeficiency virus expression by sense transcripts encoding the retroviral leader rna rna interference in human cells is restricted to the cytoplasm rnai: doublestranded rna directs the atp-dependent cleavage of mrna at to nucleotide intervals role for a bidentate ribonuclease in the initiation step of rna interference antisense transcripts in the human genome effects of length and location on the cellular response to double-stranded rna antisense transcription in the mammalian transcriptome ribonuclease activity and rna binding of recombinant human dicer argonaute is the catalytic engine of mammalian rnai assembly and function of rna silencing complexes trbp recruits the dicer complex to ago for microrna processing and gene silencing trbp, a regulator of cellular pkr and hiv- virus expression, interacts with dicer and functions in rna silencing intracellular-diced dsrna has enhanced efficacy for silencing hcv rna and overcomes variation in the viral genotype escape from the interferon response associated with rna interference using vectors that encode long modified hairpin-rna rna interference is mediated by -and -nucleotide rnas gene therapy progress and prospects: novel gene therapy approaches for aids changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of hiv- lai , hiv- mal , and hiv- eli functional differences between the long terminal repeat transcriptional promoters of hiv- subtypes a through g double-stranded rna-exposed human keratinocytes promote th responses by inducing a type- polarized phenotype in dendritic cells: role of keratinocyte-derived tumor necrosis factor alpha, type i interferons, and interleukin- gt) inhibition of hiv- by lhrna p konstantinova et al rnai research in the berkhout lab is sponsored by nwo-cw (top grant) and senter-novem (ts grant with viruvation). we thank stephan heynen for performing ca-p elisa; dr p midoux (centre de biophysique moleculaire, orleans, france) for providing us with the pt -luc plasmid; dr jean-marc jacque (university of massachusetts medical school, usa) for providing the pcdna -t pol expressing vector and dr e de jong (university of amsterdam, the netherlands) for the kind gift of ifn-b primers. key: cord- -bch v authors: singanayagam, aran; joshi, priya v; mallia, patrick; johnston, sebastian l title: viruses exacerbating chronic pulmonary disease: the role of immune modulation date: - - journal: bmc med doi: . / - - - sha: doc_id: cord_uid: bch v chronic pulmonary diseases are a major cause of morbidity and mortality and their impact is expected to increase in the future. respiratory viruses are the most common cause of acute respiratory infections and it is increasingly recognized that respiratory viruses are a major cause of acute exacerbations of chronic pulmonary diseases such as asthma, chronic obstructive pulmonary disease and cystic fibrosis. there is now increasing evidence that the host response to virus infection is dysregulated in these diseases and a better understanding of the mechanisms of abnormal immune responses has the potential to lead to the development of new therapies for virus-induced exacerbations. the aim of this article is to review the current knowledge regarding the role of viruses and immune modulation in chronic pulmonary diseases and discuss avenues for future research and therapeutic implications. chronic diseases are the leading cause of death worldwide and the third most common group of chronic diseases are chronic pulmonary diseases that account for an estimated four million deaths annually [ ] . the most prevalent diseases of the respiratory tract are chronic obstructive pulmonary disease (copd), asthma, tuberculosis and lung cancer, and the most common genetic disease is cystic fibrosis (cf). copd is estimated to be the fourth leading cause of mortality by [ ] and an estimated million people suffer from asthma. copd, asthma and cf are all chronic inflammatory conditions but their etiology and pathogenesis differ markedly. copd and asthma are believed to be caused by exposure to relevant environmental agents (mainly cigarette smoke and aeroallergens, respectively) in patients with a susceptible genetic background, whereas cf is caused by mutations in the cf transmembrane regulator gene. the typical clinical course of these conditions is of chronic symptoms that are punctuated by periods of increased symptoms termed 'acute exacerbations'. acute exacerbations are now recognized to be significant events in the course of the disease and have enormous implications for patients, their caregivers and for healthcare providers. exacerbations accelerate disease progression, impair quality of life, cause significant morbidity for patients and are the major cause of mortality. in addition they are the major drivers of excess healthcare costs as they often result in unscheduled healthcare visits, treatment costs and above all hospitalizations. therefore, preventing exacerbations is a major therapeutic goal in all three diseases and one that has not been achieved with currently available treatments. despite the differences between copd, asthma and cf, all three have in common that respiratory virus infections are a major trigger of acute exacerbations. an important mechanism underlying this may be impaired host immune responses to virus infection and a better understanding of these mechanisms has the potential to lead to the development of new therapies that may be beneficial in different chronic pulmonary diseases. the aim of this article is to review the current knowledge regarding the role of viruses and host immune responses in asthma, copd and cf, and discuss avenues for future research and therapeutic interventions. although this article primarily focuses on acute exacerbations of chronic respiratory diseases, virus infection has also been implicated in the induction of asthma. asthma is strongly related to a genetic predisposition to develop allergic reactions to aeroallergens. however, not all individuals with atopy develop asthma and, therefore, it has been proposed that other environmental factors may act as 'triggers' to the development of asthma in genetically susceptible individuals. one such factor that has attracted much research interest is respiratory virus infections, in particular infection with respiratory syncytial virus (rsv). in the majority of cases rsv causes a self-limiting upper respiratory tract infection, but in infants under the age of one year it can cause a more serious infection of the lower respiratory tract -bronchiolitis -and studies have linked rsv bronchiolitis with an increased frequency of subsequent wheezing and asthma [ ] . recently, it has been reported that rhinovirus (rv) infection is also related to the development of asthma [ ] . however, these studies are unable to ascertain the direction of the relationship between viral infections and asthma, that is, whether infections cause asthma or infections occur more frequently in individuals predisposed to asthma. recent evidence has emerged supporting the later hypothesis. a study using data on hospitalization due to rsv infection for all twins born in denmark between and found that rsv hospitalization and asthma were positively associated but that a model in which asthma 'causes' rsv hospitalization fitted the data significantly better than a model in which rsv hospitalization 'causes' asthma [ ] . a study of the temporal relationship between sensitization to aeroallergens and viral wheeze showed that allergic sensitization led to an increased risk of wheezing illnesses but viral wheeze did not lead to increased risk of subsequent allergic sensitization [ ] . therefore, the link between asthma and virus infection may be due to genetically determined alterations in airway or immune responses that predispose infants both to infection and asthma, rather than virus infections causing asthma [ ] . this will be discussed later in light of recent developments regarding innate immune responses in asthma but it is clear that the relationship between respiratory virus infections and the induction of asthma is complex and requires further study. asthma is the most common chronic respiratory disease affecting up to % of adults and % of children in the western world [ ] . the global initiative for asthma (gina) defines asthma as 'a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. the chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. these episodes are usually associated with widespread, but variable, airflow obstruction within the lung that is often reversible either spontaneously or with treatment'. this definition refers to the key physiological marker of asthma -reversible airflow obstruction, and the key pathological characteristic -airways inflammation. the characteristic pattern of inflammation of allergic diseases and also in asthma involves eosinophils, mast cells and t helper lymphocytes (th ) and a wide range of inflammatory mediators. asthma exacerbations are episodes characterized by progressive increase in shortness of breath, cough, wheezing and chest tightness, or some combination of these, and increased airflow obstruction that is manifested by reductions in measurements of lung function such as peak expiratory flow (pef). acute exacerbations are a common occurrence in asthma and the social and economic burden of asthma exacerbations is substantial, due to both the direct costs of healthcare utilization and the indirect costs associated with lost productivity. current therapies for asthma consist of bronchodilator and antiinflammatory medications, the mainstay of which are inhaled β -agonists and inhaled corticosteroids, respectively. these are highly effective in relieving symptoms and reduce exacerbations by approximately % in clinical trials [ ] . however, in 'real life' surveys of asthmatics a significant proportion of patients continue to experience acute exacerbations despite therapy and, therefore, prevention/treatment of exacerbations remains a major unmet clinical need in asthma [ ] [ ] [ ] . it has long been recognized that viral respiratory tract infections are triggers for exacerbations of asthma in both adults and children but early studies reported low detection rates of viruses in asthma exacerbations casting doubt on this association. the development of highly sensitive and specific molecular diagnostic techniques using polymerase chain reaction (pcr) technology led to a reappraisal of the role of virus infections in asthma. studies using pcr detected viruses in approximately % to % of asthma exacerbations in school-aged children and % to % of exacerbations in adults. although respiratory virus infection can be detected in stable asthma patients detection rates are consistently lower than in exacerbated patients [ , ] . therefore, these studies suggest that the majority of asthma exacerbations are associated with respiratory virus infections and that the low detection rates in earlier studies were a consequence of diagnostic methods with a low sensitivity. the most common viruses detected in these studies were rv. rvs are members of the picornaviridae family and are the most common cause for the common cold in both children and adults. more than serotypes exist. virus typing classified rvs into rv-a and rv-b groups based on susceptibility to anti-viral drugs and on genetic sequence similarity. more recently a newly identified group termed rv-c has been identified based purely on sequencing data [ ] . other respiratory viruses have been detected in subjects with asthma exacerbations including influenza, rsv, coronaviruses, human metapneumoviruses, parainfluenza viruses (piv) and adenoviruses. however, in a recent study in children the only virus type significantly associated with asthma exacerbations was rv [ ] . the risk of exacerbation following virus infection is influenced by other factors such as allergy and environmental pollution. allergen sensitization, exposure to sensitizing allergens, and respiratory virus infection act in a synergistic manner to significantly increase the risk of hospitalization with acute asthma in both adults [ ] and children [ ] . the presence of high ambient levels of nitric oxide(no) is also associated with an increased risk of exacerbation following rv infection [ ] . following discovery of the role of rv in asthma exacerbations research attention has focused on the mechanisms of susceptibility to virus infection in asthmatics. rv infection in healthy individuals results in a predominantly upper respiratory symptom syndrome ('common cold'), whereas in asthmatics infection results in lower respiratory symptoms and airflow obstruction ('acute exacerbation'). a study of co-habiting partners discordant for the presence of asthma demonstrated that asthmatics do not have a higher frequency of rv infections but have more severe lower respiratory symptoms and changes in airway physiology [ ] . similar results have been reported in experimental rv infection studies in asthmatics and nonasthmatic control subjects [ ] . therefore, it would appear that the consequences of virus infection in asthmatics are more severe than in non-asthmatics. understanding the mechanisms underlying increased disease severity is crucial to developing new strategies to treat virus-induced exacerbations. most research into virus-induced asthma exacerbations has focused on rv as these are the most common viruses detected in asthma exacerbations and well-characterized models of rv infection exist both in vitro and in vivo. rvs primarily enter and replicate in epithelial cells in the respiratory tract and trigger a cascade of immune and inflammatory responses. following viral entry into a cell, uncoating of the virus leads to the release of viral rna that is recognized by pattern recognition receptors including toll-like receptors (tlr)- , - and - , and the cytosolic rna helicases, retinoic acid inducible gene i (rig-i) and melanoma differentiation-associated protein- (mda- ) [ , ] . the interactions between ligand and receptor trigger signaling cascades ultimately resulting in the activation of transcription factors such as interferon regulatory factor (irf)- and- , nuclear factor-b (nf-b) and activating transcription factor (atf ). these activated transcription factors translocate to the nucleus and induce transcription of the type i interferons (ifn-α and -β) and pro-inflammatory cytokines including interleukin (il)- /cxcl , il- , epithelial-derived neutrophilactivating peptide (ena- /cxcl ) and ifn-γinduced protein kda (ip- /cxcl ) [ ] [ ] [ ] [ ] [ ] . ifn-α and -β have both a direct antiviral effect through inhibition of viral replication in cells and an indirect effect through stimulation of innate and adaptive immune responses. the direct antiviral activity of type i ifns is mediated by various mechanisms including blocking viral entry into cells, control of viral transcription, cleavage of rna and blocking translation. these effects are mediated through the up-regulation of interferon stimulated genes (isgs) and the production of antiviral proteins. the indirect antiviral effect is mediated through induction of natural killer cell cytotoxicity [ ] , up-regulation of the expression of major histocompatibility complex (mhc- ) on cells and up-regulation of co-stimulatory molecules on antigen-presenting cells. therefore, a robust interferon response is central to effective antiviral responses and resolution of virus infections. recently a novel class of interferons termed type iii interferons, or interferonlambda (ifn-λ) has been described. the type iii ifns consist of ifn-λ , , (respectively, il- , il- a and il- b) [ ] . the ifn-λs utilize a different receptor than ifn-α/β but appear to have functional similarities, however much more is known about the mechanism of action of ifn-α/β. the pro-inflammatory mediators and cytokines induced by rv infection lead to chemoattraction of inflammatory cells such as neutrophils, lymphocytes and eosinophils. this inflammatory response contributes to virus clearance but is also responsible for the pathology induced by rv infections. the balance between antiviral and inflammatory responses following virus infection is likely to determine the clinical outcome of the infection. an effective antiviral response rapidly controls viral replication with a minimal inflammatory response and limited clinical illness. if antiviral responses are inadequate this is likely to result in uncontrolled viral replication, greater inflammatory response and more severe clinical illness ( figure ). the evidence that clinical illness following virus infection is more severe in asthmatics has stimulated research into possible dysregulation of antiviral and inflammatory responses in asthmatics. in wark et al. examined the kinetics of virus replication in bronchial epithelial cells obtained from asthmatics and healthy volunteers and reported that viral replication is increased in cells from asthmatics compared to non-asthmatic subjects [ ] . this was the first report indicating that the innate immune response to virus infection may be impaired in asthma. furthermore, the authors demonstrated that production of ifnβ was impaired in asthmatics and administration of exogenous ifn-β resulted in restoration of a normal antiviral response, and this was confirmed in a subsequent study [ ] . deficient ifn-β production by bronchial epithelial cells [ ] , as well as deficient ifn-α production by peripheral blood mononuclear cells [ ] [ ] [ ] and dendritic cells [ ] has also been reported in asthma. our group has also shown that ifn-α and ifn-β production by alveolar macrophages is impaired in asthmatics (manuscript submitted). furthermore, deficient ifn-λ production by epithelial cells and alveolar macrophages in asthmatics has also been reported and related to clinical outcomes following experimental rv [ ] . however, other groups have not reported deficient ifn induction in epithelial cells from asthmatics [ , ] . in experimental rv infections virus loads were higher and virus shedding prolonged in asthmatics but this was not statistically significant [ , ] . therefore, although interferon deficiency is an exciting new mechanism underlying increased severity of virus infection in asthma it has not been conclusively demonstrated to occur in all asthmatic subjects studied. the studies in question were small with different experimental conditions such as cell culture techniques and virus dose. it is also possible that interferon deficiency occurs in some asthmatics only and it may also be related to disease severity, disease control or degree of atopy. further studies with more subjects and careful patient selection and characterization are required to provide answers to these ongoing research questions. up-regulation of interferonstimulated genes if interferon production in response to virus infection is impaired in asthmatics what are the possible molecular mechanisms underlying this? the discovery that ifn-α, ifn-β and ifn-λ are all deficient suggests that it is not a genetic defect as ifn-α and ifn-β are on different genetic loci than ifn-λ. a key family of proteins regulating both interferon production and allergic inflammation are the suppressor of cytokine signaling family (socs), and one member of this family, socs , is a potent negative regulator of type i and type ii interferons and of th inflammation [ , ] . socs is induced by type ii cytokines such as il- [ ] and, therefore, persistent th inflammation may result in chronic up-regulation of socs and impaired interferon responses, but this hypothesis requires further investigation. in vitro infection of airway epithelial cells with rv induces the production of inflammatory mediators and this has also been reported in vivo in both experimental and naturally-acquired viral infections. chemokines and cytokines such as il- , il- and regulated on activation, normal t-cell expressed and secreted (rantes) have been detected during virus infections in asthmatic patients [ ] [ ] [ ] [ ] [ ] . however it remains unclear whether the inflammatory response following virus infection differs quantitatively or qualitatively in asthmatics. one experimental rv infection study reported increased nasal lavage levels of il- and il- β in asthmatics [ ] but not in control subjects; however, another study reported no differences in il- , il- , il- and granulocyte-monocyte-colony stimulating factor (gm-csf) levels in either nasal lavage or sputum between asthmatics and nonasthmatics [ ] . increased sputum levels of il- but not rantes or il- have been reported in asthmatics [ ] . these conflicting results highlight the need for further studies evaluating the inflammatory profile (preferably in the lower airway) in well-characterized patients and nonasthmatic controls following virus infection. many of the inflammatory mediators produced are chemoattractants and, therefore, following virus infection inflammatory cells are recruited to the lungs. a number of different inflammatory cells have been identified in both naturally-occurring and experimental virus infections in asthma. although stable asthma is characterized by eosinophilic inflammation, a number of studies have identified neutrophils as the key inflammatory cell in virus-induced asthma exacerbations [ , [ ] [ ] [ ] . neutrophils release bioactive mediators such as the protease neutrophil elastase that have effects such as stimulation of mucous production and, therefore, are likely key contributors to the pathogenesis of asthma exacerbations. another key cell involved in immune and inflammatory responses in the lungs is the macrophage. there is evidence that rvs can infect macrophages and that in asthmatics macrophage responses to virus infection are altered. our group has reported that rvs infect macrophages and induce tnf-α production [ ] and that production of the cytokine il- , that plays a key role in linking innate and adaptive antiviral immune responses and promoting t cell anti-viral immune responses, is impaired in asthmatics [ ] . as described previously, macrophage production of ifns in response to virus infection is also impaired in asthma [ ] . therefore, there is evidence of impaired antiviral responses in asthmatics in macrophages as well as respiratory epithelial cells. increased lymphocyte numbers in bronchoalveolar lavage (bal) and bronchial biopsies in experimental rv infection in asthmatics has been reported, with increases in cd +, cd + and nk cells [ , ] . abnormalities of the acquired immune system in stable asthma have been well described with skewing of acquired immune responses towards a th profile. as robust antiviral responses require an adequate th response it is possible that in diseases such as asthma with predominant th cells antiviral immunity is impaired. impaired levels of the th cytokines il- , - , - and ifn-γ have all been reported in asthma [ , [ ] [ ] [ ] . in human experimental rv infection lower respiratory symptoms, bronchial hyperreactivity, reductions in blood total and cd + lymphocytes and virus load are related to deficient ifn-γ, il- or il- responses and to augmented il- , il- , and il- responses [ , ] . sputum ifn-γ/il- messenger rna ratio following virus infection is inversely related to both peak cold symptoms and the time to viral clearance [ ] . therefore, augmented th and deficient th immune responses are associated with greater clinical illness following rv in asthma. the identification of impaired innate immunity in asthma suggests a possible mechanism not only for virus-induced asthma exacerbations but also for the link between respiratory virus infections and the subsequent development of asthma. it is possible that infants who will develop asthma in later life have impaired immune responses from birth and, therefore, are more likely to develop more severe disease manifestations (for example, bronchiolitis) following respiratory virus infection. most studies to date have focused on the role of the acquired immune system and identified reduced ifn-γ production as a significant risk factor both for subsequent wheezing illness and allergic sensitization [ ] [ ] [ ] . no studies have investigated innate immune responses in infants prior to the development of symptomatic asthma but impaired ifn-α production has been reported in older children with atopic asthma [ ] . in conclusion there is evidence that both innate and acquired immune responses in asthmatics are impaired and this may be a key mechanism underlying virusinduced asthma exacerbations and the link between virus infections and the subsequent development of asthma in infants. further studies are needed to determine whether these deficiencies are common to all asthmatics, whether they represent a specific asthma phenotype and how they relate to conventional measures of asthma control. another important research question is whether new interventions targeting the interferon pathways can prevent asthma exacerbations and even potentially prevent the development of asthma in infants. chronic obstructive pulmonary disease (copd) is the most common chronic respiratory condition in adults. the global initiative for obstructive lung disease (gold), a collaboration between the world health organization and the national heart lung and blood institute, defines copd as 'a preventable and treatable disease with some significant extrapulmonary effects that may contribute to the severity in individual patients. its pulmonary component is characterized by airflow limitation that is not fully reversible. the airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious inhaled particles or gases' [ ] . the main etiological agents linked with copd are cigarette smoking and biomass exposure and the inflammatory response consists of neutrophils, macrophages and cd + t cells and, therefore, differs from the allergic inflammation seen in asthma. pulmonary inflammation is further amplified by oxidative stress and excess proteases released by inflammatory cells recruited to the lung. as in asthma, acute exacerbations are a common occurrence in copd and become more frequent as the disease progresses [ ] . exacerbations are a major cause of morbidity, mortality and healthcare costs and accelerate decline in lung function [ ] and quality of life [ ] in copd patients. historically, bacterial infections have been considered the predominant infectious etiology, however epidemiological data showing a greater frequency of exacerbations in the winter months [ ] and frequent coryzal symptoms preceding exacerbations suggest a causal role for viruses [ ] . older studies using cell culture and serologic diagnostic tests detected viral infection in only approximately % to % of exacerbations [ , ] . however, these diagnostic methods have low sensitivity for virus detection especially for rvs that are the most common cause of upper respiratory tract infections. more recent studies using modern pcr-based techniques have allowed a re-evaluation of the importance of viruses in copd exacerbations and these studies have shown the presence of viruses in % to % of exacerbations [ ] [ ] [ ] [ ] . a recent systematic review evaluated weighted mean prevalence of respiratory viruses detected by pcr in patients with acute exacerbations of copd. eight studies were included with an overall prevalence of . %, with picornaviruses including rvs being the most frequently detected pathogen, followed by influenza, parainfluenza, rsv and adenoviruses [ ] . although these studies have higher detection rates they are likely to have underestimated the role of viral infections in copd exacerbation as they evaluated patients at the time of presentation to healthcare services which often occurs considerably later than the onset of exacerbation and by which time virus may no longer be detectable. although viruses are frequently detected in copd exacerbations, their presence during exacerbations does not prove a definite causative role. experimental infection using rv provides a novel tool for investigating relationships between virus infection and exacerbations. such studies have been previously conducted in asthma and yielded important insights into the mechanisms linking virus infection to exacerbations in asthma. a recent study from our group reported the first experimental rv infection study in copd [ ] . copd patients and nonobstructed controls were infected with rv with sequential measurement of symptoms, lung function, inflammatory markers and virus load. following rv infection, copd subjects developed symptomatic colds followed by the typical lower respiratory symptoms of an acute exacerbation. symptoms were accompanied by objective evidence of airflow limitation and airways inflammation and inflammatory markers correlated with virus load. virus was detected in airway samples prior to the onset of symptoms and viral clearance was followed by symptom resolution and return of inflammatory markers to baseline levels. therefore, this study directly links respiratory virus infection to lower respiratory symptoms, airflow obstruction and airways inflammation in copd and provides novel evidence supporting a causative role for rv infection in copd exacerbations. much less is known regarding mechanisms of virusinduced exacerbations in copd compared to asthma. in the experimental infection study, symptoms, airflow obstruction and airways inflammation were more severe in the copd subjects compared to non-obstructed controls [ ] . therefore, as is the case in asthma it would appear that clinical illness following rv infection is more severe in copd subjects, but the mechanisms underlying this are poorly understood. copd exacerbations are associated with increased levels of inflammatory mediators including tumor necrosis factor-alpha (tnf-α) [ ] , il- [ , ] , il- [ ] , and leukotriene b [ ] and inflammatory cells such as neutrophils [ , ] and eosinophils [ ] . however, few studies have examined the inflammatory response specific to virus-induced exacerbations. virus infection has been associated with high levels of il- [ , ] and ip- [ , ] and papi et al. reported that elevated sputum eosinophils were only seen in exacerbations in which a virus was present [ ] . others have reported that the presence of rv is not associated with significant airway inflammation [ ] and that only exacerbations associated with purulent sputum (presumed bacterial infection) are associated with airways inflammation [ ] . from the data available no clear conclusions can be drawn regarding the inflammatory response to virus infection in copd and there are no studies comparing the effects of naturally-occurring virus infections in copd patients and non-copd controls. there is evidence from animal models that the inflammatory response to virus infection may be exaggerated in copd. in a mouse model of copd utilizing intranasal administration of lipopolysaccharide and elastase, infection with rv resulted in increased levels of tnf-α and il- compared to control mice [ ] . this was accompanied by increased airway hyper-responsiveness and increased mucus production. similarly, in the human copd rv challenge study, increased levels of il- and neutrophil elastase were reported in copd subjects when compared to non-obstructed controls [ ] . these studies suggest that copd is associated with an exaggerated inflammatory response to viral infection and this may explain the increased severity and duration of symptoms seen in these patients. in vitro studies have shown that cigarette smoke impairs release of ifn-β and ifn-α [ ] . bal cells from copd patients infected ex vivo with rv demonstrated deficient induction of ifn-β with similar trends for deficient induction of ifns-α and -λ, associated with deficiency of the interferon stimulated gene cxcl [ ] . similar findings have been reported in a mouse model where persistence of rv, increased airways inflammation and deficient induction of ifns-α, -β and -γ were reported in copd mice compared to controls [ ] . however in vitro rv infection of epithelial cells from copd patients resulted in higher virus load and increased inflammatory mediators, but no differences in interferon production compared to cells from control subjects [ ] . further studies examining the role of interferon deficiency in viral exacerbations are required as this may lead to potential future therapeutic application of interferon therapy in reducing exacerbation severity in copd. rvs bind to cells via intercellular adhesion molecule- (icam- , major group rvs) or members of the low-density lipoprotein receptor family (minor group rvs). icam- is upregulated on the bronchial epithelium of patients with copd [ , ] and, therefore, it is possible that increased icam- levels may permit greater virus binding and increased viral entry into epithelial cells in copd patients. the majority of studies have detected viruses at a greater frequency during acute exacerbations compared to the stable state. one study indicated that rsv is detected in nasal lavage at a similar frequency of around % in the stable state and during exacerbations [ ] . this was followed by a similar study reporting detection of rsv in about % of sputum samples, with detection being related to greater airway inflammation and to a faster decline in lung function [ ] . however, other studies have not reported increased rsv detection in stable copd [ , ] . a study comparing virus loads between infants with acute respiratory infections and adult copd patients found that virus loads were -fold higher in the infants, suggesting low-grade virus infection in copd [ ] . the disparity between these findings is likely to be due to a combination of factors including differing sensitivity of the pcr techniques used, differences in severity of copd patients included or differences in the populations studied [ ] . latent infection by adenovirus has also been proposed to be involved in the pathogenesis of copd. lung tissue from copd patients has been demonstrated to carry more group c adenoviral dna than matched nonobstructed smokers [ ] . latent adenoviral infection in combination with cigarette smoke exposure in a guinea pig model caused an increase in lung volumes, airspace volume and reduced surface to volume ratio compared to smoke exposure alone [ ] . additionally, adenovirus detection has been shown to be similar in exacerbated and stable copd patients [ ] . some authors have postulated that the presence of rsv and adenovirus in stable copd may contribute to the pathogenesis of the disease as there are some common pathologic features between respiratory viral infection and copd including a predominance of cd + t lymphocytes. however, this remains a largely unproven hypothesis. cystic fibrosis (cf) is an autosomal recessive disease caused by mutations in the gene for the cystic fibrosis transmembrane regulator (cftr) protein. defective cftr function leads to abnormal transport of chloride and sodium across the pulmonary epithelium, resulting in viscous secretions in the lungs, recurrent bacterial infections and progressive loss of lung function. pulmonary involvement is the most common manifestation of the disease and respiratory failure the most common cause of death. respiratory infections are the leading cause of morbidity, decline in lung function and hospitalizations due to acute exacerbations. the major cause of infectious complications in cf has always been considered to be bacterial infection, with pseudomonas aeruginosa the most common organism detected. there has been relatively little research on the role of virus infections in cf but recent studies have suggested that viruses have a significant impact on the cf patient. the role of respiratory viruses in cf exacerbations is likely to have been under-appreciated in the past because older studies investigated only one virus type and the detection methods used were not sufficiently sensitive. newer pcr techniques have helped to improve detection and it is now becoming clear that viruses are implicated in exacerbations in cf. previous studies using serology, culture and immunoflourescence detected viruses in % to % of exacerbations in cf patients [ ] [ ] [ ] [ ] . in contrast, studies using pcr for virus detection have reported detection rates of % to % [ ] [ ] [ ] . a number of different viruses have been detected in cf patients with the most common being rvs, influenza and rsv. the incidence of viral infections in children with cf is not elevated in comparison to healthy children but the severity of clinical illness associated with infection is greater [ ] . viral infections are associated with deterioration in lung function and more severe clinical illness indicating that they contribute to disease progression thus demonstrating the clinical importance of research within this field [ , ] . the mechanisms of viral-induced cf exacerbations and increased clinical illness are poorly understood with conflicting results from published studies. some authors have reported increased production of pro-inflammatory cytokines and chemokines by epithelial cells obtained from cf patients compared to healthy controls [ , ] . however, others have failed to detect any differences in cytokine production between cf and normal cells [ , ] . these differences may be due to different viruses used (rv, rsv, piv) and differences in cell culture techniques, but it remains unclear whether the cf epithelium is intrinsically pro-inflammatory in response to virus infection. another mechanism that has been postulated is a deficiency in antiviral innate immune responses in cf cells. increased replication following piv infection of cf cells has been reported and this was corrected by administration of ifnα [ ] . ifn responses were not impaired but induction of nitric oxide synthase (nos ) was impaired in cf. nos is required for production of no that has potent antiviral effects and, therefore, impaired no synthesis may be one mechanism of impaired antiviral host responses in cf. our group has reported reduced ifn-β and ifn-λ production and reduced isgs in cf epithelial cells [ ] and, therefore, ifn deficiency may be relevant to cf as well as in asthma and copd. holtzman has proposed that 'hypersusceptibility' to virus infection, via defective interferon pathways, is a unifying pathway in asthma, copd and now cf [ ] . both bacterial and virus infections are common in cf and copd and, therefore, co-infections are likely to be common. there is now increasing evidence that both viral and bacterial infections can modulate host immune responses and increase susceptibility to subsequent infection. there is abundant evidence from both human studies and animal models that influenza infection impairs antibacterial immunity and this can result in secondary bacterial pneumonia [ , ] . however, much less is known regarding the effect of other respiratory viruses, such as rvs, on susceptibility to bacterial infection. in vitro studies have reported that rv infection increases bacterial adhesion to epithelial cells [ ] [ ] [ ] and impairs macrophage immune responses to bacterial products [ ] . we have found that experimental rv infection in copd is followed by secondary bacterial infection in % of patients and this is related to deficiency of the antimicrobial peptides elafin and secretory leukoprotease inhibitor (slpi) (submitted manuscript). there are also studies indicating that virus-bacteria interactions influence host immune responses in cf. chattoraj et al. reported that rv infection of cf cells liberates planktonic bacteria from biofilm [ ] . planktonic bacteria express virulence factors and stimulate inflammatory responses more readily compared to biofilm bacteria and this was manifested by increased cytokine responses. evidence is also emerging that bacterial infection can increase susceptibility to viral infection. infection of epithelial cells by haemophilus influenzae (a common organism in copd) increases susceptibility to infection by rv, possibly by up-regulation of icam- [ ] . cf cells infected with mucoid p.aeruginosa and then with rv produced less ifn and viral loads were higher compared to cells infected with the rv alone [ ] . this effect was not seen in normal epithelial cells infected with pseudomonas and was related to the inhibition of akt phosphorylation and irf- activation -both prerequisites for the ifn response to rv infection. it is widely acknowledged that the main infectious cause of asthma exacerbations is virus infection and it is believed that bacteria play only a minor role. however, a recent study using culture-independent molecular methods for bacterial detection reported that the bacterial flora in the airways of asthmatics is closer to that of copd patients than of non-asthmatics [ ] . the role of bacteria in asthma exacerbations needs to be revisited as virus-bacterial interactions may play a role in the pathogenesis of asthma exacerbations. this is a fertile area for further research. our knowledge of the interactions between respiratory viruses and bacteria, and how these influence host immune responses in pulmonary diseases, is still at an early stage. further research is required to understand better these complex relationships and to explore the implications they may have for the development of new therapies. there is now convincing data implicating respiratory viruses as a major cause of acute exacerbations in asthma, copd and cf. in all these conditions there is evidence that host immune responses to virus infection are impaired, but whether this occurs through a common mechanism, or whether mechanisms differ between the different diseases is unclear. further research is needed to elucidate the exact mechanisms of increased susceptibility to virus infection in pulmonary diseases, the interactions between viruses and bacteria and how these impact on host immune responses. a better understanding of these mechanisms has the potential to lead to the development of novel therapies that will reduce the impact of acute exacerbations in chronic pulmonary diseases. list of abbreviations atf: activating transcription factor; bal: bronchoalveolar lavage; cf: cystic fibrosis; cftr: cystic fibrosis transmembrane regulator; copd: chronic obstructive pulmonary disease; ena- : epithelial-derived neutrophilactivating peptide ; icam- : intercellular adhesion molecule; ifn-α: interferon-alpha; ifn-β: interferon-beta; ifn-λ: interferon-lambda; ifn-γ: interferon-gamma; il: interleukin; ip- : ifn-γ-induced protein- ; irf: interferon regulatory factor; isg: interferon stimulated genes; mda- : melanoma differentiation-associated protein- ; nf-κb: nuclear factor-kappa b; no: nitric oxide; nos : nitric oxide synthase ; pcr: polymerase chain reaction; pef: peak expiratory flow; piv: parainfluenza virus; rantes: regulated on activation: normal t-cell expressed and secreted; rig-i: retinoic acid inducible gene i; rsv: respiratory syncytial virus; rv: rhinovirus; slpi: secretory leukoprotease inhibitor; socs: suppressor of cytokine signaling family; th / : t helper / ; tlr: toll-like receptors; tnf-α: 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rhinovirus infection on the adherence of streptococcus pneumoniae to cultured human airway epithelial cells rhinovirus disrupts the barrier function of polarized airway epithelial cells rhinovirus exposure impairs immune responses to bacterial products in human alveolar macrophages rhinovirus infection liberates planktonic bacteria from biofilm and increases chemokine responses in cystic fibrosis airway epithelial cells hershenson mb: h. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing icam- and tlr expression pseudomonas aeruginosa suppresses interferon response to rhinovirus infection in cystic fibrosis but not in normal bronchial epithelial cells disordered microbial communities in asthmatic airways all the authors contributed equally to writing the manuscript. all authors read and approved the final manuscript. pre-publication history key: cord- -b y authors: vanleuven, james t.; ridenhour, benjamin j.; gonzalez, andres j.; miller, craig r.; miura, tanya a. title: lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: b y the severity of respiratory viral infections is partially determined by the cellular response mounted by infected lung epithelial cells. disease prevention and treatment is dependent on our understanding of the shared and unique responses elicited by diverse viruses, yet few studies compare host responses to viruses from different families while controlling other experimental parameters. murine models are commonly used to study the pathogenesis of respiratory viral infections, and in vitro studies using murine cells provide mechanistic insight into the pathogenesis observed in vivo. we used microarray analysis to compare changes in gene expression of murine lung epithelial cells infected individually by three respiratory viruses causing mild (rhinovirus, rv b), moderate (coronavirus, mhv- ), and severe (influenza a virus, pr ) disease in mice. rv b infection caused numerous gene expression changes, but the differential effect peaked at hours post-infection. pr altered an intermediate number of genes whose expression continued to change through hours. mhv- had comparatively few effects on host gene expression. the viruses elicited highly overlapping responses in antiviral genes, though mhv- induced a lower type i interferon response than the other two viruses. signature genes were identified for each virus and included host defense genes for pr , tissue remodeling genes for rv b, and transcription factors for mhv- . our comparative approach identified universal and specific transcriptional signatures of virus infection that can be used to distinguish shared and virus-specific mechanisms of pathogenesis in the respiratory tract. viruses from several different families, including picornaviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, and adenoviridae, infect and cause diseases in the respiratory tract. these diseases range from mild infections of the upper respiratory tract, to severe diseases when viruses infect the lungs. respiratory viruses commonly target epithelial cells of the a a a a a airways and lungs. these epithelial cells are responsible for detecting viral pathogens and initiating antiviral responses at the level of infected cells and the immune system, and therefore their response to infection has an important role in determining disease outcomes. knowledge of the shared and virus-specific responses of respiratory epithelial cells to infection by diverse viruses is fundamental to understanding viral pathogenesis and developing therapies to treat severe respiratory infections. murine models of respiratory viral infections have been widely used to identify the mechanisms that determine disease severity in the respiratory tract. while these models are invaluable for evaluating pathology and host responses to infection, parallel in vitro studies can be used to identify gene expression and signaling pathway changes that occur in infected cells to mediate pathogenesis. in this study, we compare the gene expression response of murine lung epithelial cells to infection by three respiratory viruses used in murine models: rhinovirus (serotype rv b) in the family picornaviridae, mouse hepatitis virus (mhv strain ) in the family coronaviridae, and influenza a virus (strain pr ) in the family orthomyxoviridae. viruses from different families and with different replication strategies were chosen to identify which responses to infection in respiratory epithelial cells are shared and which are virus-specific. in the following paragraphs, we describe some of the key features of these three viruses in murine models. minor group rhinoviruses, including rv b, use low-density lipoprotein receptor to enter either human or murine cells. because rv b can replicate in mouse cells, it has been used to study immune responses and mechanisms of asthma exacerbation in infected mice [ ] [ ] [ ] [ ] [ ] . intranasal inoculation of mice with a high dose of rv b results in viral replication and an early inflammatory response in the respiratory tract without clinical signs of disease [ , [ ] [ ] [ ] . rv b antigens have been detected in cells of the epithelia and lamina propria of the upper respiratory tract of infected mice [ , ] . mhv- is a strain of mouse hepatitis virus that preferentially replicates and causes disease in the respiratory tract of specific mouse strains and thus serves as a model for respiratory coronaviral infections [ , ] . mhv- causes severe disease in a/j-strain mice and milder pathology in other mouse strains [ , ] . mouse strain-dependent disease severity corresponds to inflammatory responses, fibrin deposition, and reduced type i interferon (ifn) production [ ] . further, type i ifn-mediated signaling is required for protection from severe disease during mhv- infection of resistant mice [ ] . mhv- has been detected in pulmonary macrophages of infected mice, but has not been reported to infect respiratory epithelial cells in vivo [ ] . although primary murine alveolar epithelial cells are susceptible to mhv- infection in vitro, their role during in vivo infection is not clear [ ] . mice have been used for decades to study the pathogenesis of influenza. one of the most commonly used strains of influenza a virus, pr , has been serially passaged in mice to produce a model for pulmonary infection. pr infection results in a range of disease severities that is mouse strain-dependent [ ] . although susceptible mice mount a type i ifn response to pr infection, lethal infection is associated with spread of virus to the alveoli and an excessive inflammatory response [ ] [ ] [ ] [ ] . pr replicates in bronchiolar and alveolar epithelial cells of the lower respiratory tract in vivo and in primary murine respiratory epithelial cells in vitro [ , , , ] . in this study, we used a murine lung epithelial cell line (la ) to compare the gene expression response to these three unrelated viruses. la cells were derived from neoplastic lung epithelia from strain a (a/he) mice and have some properties of alveolar type ii cells, although they do not maintain a highly differentiated type ii phenotype [ ] . strain a (a/j) mice are highly susceptible to respiratory viral infections, including mhv- and influenza a viruses [ , ] . other studies have demonstrated that la cells are susceptible to infection by pr and rv b [ , ] . in this study, we show that la cells are also susceptible to infection by mhv- (hereafter referred to as mhv). the gene expression response of la cells to infection by mhv, pr , and rv b (hereafter referred to as rv) differed in timing and magnitude of the changes. while we expected to see highly divergent transcription responses to these three viruses, they induced expression of a surprisingly large overlapping set of shared genes, including genes involved in antiviral responses. however, and more in line with our expectation, each virus also altered expression of unique genes, which highlight their different replication strategies and mechanisms of pathogenesis in murine hosts. virus stocks were generated by h of growth from a low dose inoculum in the cell lines described below. supernatant medium from infected cells was centrifuged to remove cell debris, aliquoted, flash frozen and stored at - ˚c. pr (a/puerto rico/ / (h n )), obtained from bei resources (nr- ), was grown and titrated by plaque assay in mdck cells from atcc (ccl- ) [ ] . mhv, obtained from atcc (vr- ), was grown and titrated by plaque assay in cl. cells [ ] (provided by dr. kathryn holmes, university of colorado denver school of medicine). rv, obtained from atcc (vr- ), was grown and titrated by tissue culture infectious dose % (tcid ) assay in hela (atcc: ccl- ) cells [ ] . la , a murine lung epithelial cell line from atcc (ccl- ), was cultured in ham's f k medium (mediatech) with % fbs (atlanta biologicals) and x antibiotic/antimycotic (gibco). our experimental approach was to inoculate la cells with the three viruses at times t = h and t = h and harvest rna for microarray analysis at t = h. controls were mock-inoculated at both time points. preliminary experiments were done to establish the growth kinetics of each virus and determine a multiplicity of infection (moi) that resulted in comparable numbers of cells positive for viral antigen at h post-infection (s fig) . based on this, la cells were inoculated with tcid /cell rv, pfu/cell pr , or pfu/cell mhv. triplicate wells of la cells in -well plates were inoculated with each virus diluted in serum-free medium or were mock-inoculated with serum-free medium for h at ˚c. viral inocula were removed and the cells were rinsed twice with serum-free medium. the cells were incubated in ham's f k medium with % fbs until the h time point at which time rna was isolated from cell cultures using an rnaeasy plus kit (qiagen) and transcript levels were measured by microarray (nimblegen mus musculus mm expression array). for the h samples, the media were removed and replaced with fresh media h after inoculation, which is the same time that h samples were inoculated. raw and processed data are available from ncbi gene expression omnibus under accession number gse . nimblescan v . software (niblegen, madison, wi) was used to extract raw intensity data for each probe on each array. intensity data were read into the r statistical computing environment and checked for quality [ ] . data were prepared for processing using the pdinfobuilder package and then normalized using the robust multichip average (rma) function in the oligo package [ ] . statistical tests for differences in expression between treatments were conducted on the normalized expression data using a linear mixed-effect model followed by linear contrasts corrected for multiple comparisons. more specifically, expression was modeled as a function of treatment, while probes for a particular gene were treated as random effects. the models used the nlme::lme function in r. the data contained seven treatments: three viruses tested at two time points ( h and h) each plus the mock treatment (rv , rv , mhv , mhv , pr , pr , and mock). the following nine post hoc, two-sided contrasts were then performed on the fitted models using the multcomp::glht function in r: each virus-time combination vs. mock (rv vs. mock, rv vs. mock, mhv vs. mock, mhv vs. mock, pr vs. mock, pr vs. mock) and each pairwise combination of viruses at the h time point (rv vs. mhv , rv vs. pr mhv vs. pr ). these tests had their p-values adjusted by the multcomp::summary.glht function according to their joint distribution. any factors detected to be significant at the family (gene) level were then subsequently corrected using the benjamini-hochberg algorithm [ ] with a false discovery rate set at %. genes associated with type i ifn responses were identified among the sets of genes with differential expression for each virus compared to mock at h and h using the interferome v. . database (http://interferome.its.monash.edu.au; [ ] ). this database was queried using the search criteria: type i ifn (all), in vitro, mus musculus, . fold change (up or down). interferome genes were manually sorted into functional categories: antiviral, ifn signaling, viral detection, immune response, mhc class i, inhibitory, apoptosis, ubiquitination, miscellaneous, and unknown. the significance of each virus having genes in the specific categories was tested using a chi-squared test. gene expression responses to rv b were compared between our data from mouse cells and published data using human cells [ ] using the mgi vertebrate homology database provided by the jackson laboratory [ ] as well as the annotate package in r. mhv, pr , and rv alter cellular gene expression by different magnitudes and with different timing our goal was to evaluate the gene expression response of murine respiratory epithelial cells to infection by three unrelated respiratory viruses studied in murine models. a preliminary study was undertaken to identify a murine cell line that was susceptible to infection by the three viruses. la cells were chosen because mhv, rv, and pr established productive infections in this line, as shown by increased viral titers released from infected cultures over a h infection (s fig). additionally, comparable numbers of viral antigen-positive cells were observed for the three viruses h after inoculation of la cells (s fig) . to compare how unrelated respiratory viruses (mhv, pr , and rv) alter gene expression of murine epithelial cells, we inoculated la cells with each of the three viruses and evaluated cellular gene expression by microarray analysis after and h of infection compared to mock-inoculated controls. fig shows the log -fold change in expression level of genes that were differentially expressed in virus-infected, compared to mock-inoculated, cells. by plotting the changes in gene expression at vs. hours, we observed differences in magnitude and timing of gene expression changes mediated by the three viruses. the genes with significantly different expression in mhv-infected cells had low fold change values (fig a) . at h, when gene expression changes were the highest, genes that were up-regulated by mhv infection had log -fold change values of less than five. in contrast, pr and rv induced expression of many genes by greater than five log -fold at h, and genes were spread consistently across the full range of values. by h, the genes most strongly up-regulated by pr and rv induced changes of - . log -fold and - . log -fold compared to mock, respectively. this same pattern was observed with the down-regulated genes (fig ) . in addition to the differences in fold change values, the three viruses also differed in the timing of gene expression changes. mhv altered expression of relatively few host genes, most of which were only significantly different from mock at h ( fig a) . while both pr and rv induced expression of large subsets of host genes, they did so with different timing. pr -induced changes in gene expression occurred at a constant rate: the expression level of most genes at h was approximately twice the expression value at h ( fig b) . in contrast, rv infection altered expression of a large number of genes by h and the expression levels were maintained at approximately the same levels at the h time point (fig c) . taken together, we observed differences in magnitude and timing of gene expression changes mediated by the three viruses. the limited response to mhv infection is in agreement with studies of other coronaviruses, such as mhv-a [ ] and sars-cov [ , ] . in addition to inducing minor transcriptional up-regulation of host genes, mhv-a shuts down host gene expression by enhancing mrna degradation [ ] . a related coronavirus, sars-cov, also induces degradation of host mrnas [ ] . the low numbers of host mrnas that were altered in response to mhv infection in our study could be due to one or both of these mechanisms. while rhinoviruses are known to down-regulate host gene expression by inhibiting transcription [ ], upon rv infection we saw robust, early increases in host mrna expression ( fig c) . this is in agreement with other transcriptome studies of major and minor serogroup rhinoviruses in human respiratory epithelial cells and experimental infections of humans [ , [ ] [ ] [ ] [ ] . the plateau in gene expression changes in rv-infected cells at h may be due to transcriptional inhibition later in infection, or the death of infected cells. pr infection induced a strong transcriptional response in la cells, which has also been seen with multiple strains of influenza a viruses in primary human and mouse airway or lung epithelial cells [ ] [ ] [ ] [ ] . interestingly, the differences in kinetics of host cell gene expression did not correspond with differences in the kinetics of viral replication in this cell line (s fig). despite inducing ) . these data also demonstrate that the limited response of cellular gene expression to mhv infection was not due to limited infection of la cells (s fig). host genes have shared and unique responses to rv, pr , and mhv infection we identified which genes were altered by each virus at h compared to mock and the degree of overlap among the differentially expressed genes. at h rv infection resulted in up-regulation of the largest number of genes, followed by pr then mhv (fig a) ; a similar pattern was seen with down-regulated genes (fig b) . while one might worry that some of the small number of significant genes that were altered by mhv could be false positives, the majority of these genes ( % of up-regulated and % of down-regulated genes) were also significantly altered by at least one other virus suggesting that most of these genes are true positives. for both up-and down-regulated gene sets, rv had the largest proportion of unique genes, while the majority of genes affected by pr and mhv were shared by at least one other virus. s table contains the list of genes whose expression was significantly up-regulated by all three viruses compared to mock-inoculated cells. these genes may reflect a global response of epithelial cells to viral infection. several of the genes with the highest fold change values are involved in antiviral defense at the level of infected cells (eg., mx , bst , oas , gbp ) or recruitment of immune cells (eg. , cxcl , cxcl , cxcl ) . these genes are up-regulated by type i ifns, suggesting that induction of a type i ifn response is shared by these viruses. in contrast to the shared up-regulated genes, genes that were significantly down-regulated by all three viruses have diverse functions (s table) . some examples of genes that were down-regulated by all three viruses included genes that encode transmembrane proteins (tmem , , , a, and c), extracellular matrix proteins (spon , ogn, aspn), and apoptotic signaling proteins (sdpr, bmf, bnip l). as a measure of validation, the expression levels of five genes (tnf, cxcl , bst , icam , and oas a) were also quantified by rt-qpcr at h post-infection (s fig). a strong correlation was observed between rt-qpcr and microarray measurements of gene expression (slope = . , r = . ). identification of signature genes that were uniquely altered by each virus comparing the number of genes altered by each virus provides insight into shared and unique cellular responses elicited by the viruses, but it does not provide information on the relative magnitudes of gene expression changes between viruses. to compare gene expression changes between viruses, we plotted the log -fold change of each gene at h for mhv vs. rv vs. pr (fig a) . we only included genes that were differentially expressed in at least one viral infection compared to mock. like fig , this d plot illustrates that pr and rv not only caused a larger number of genes to be up-regulated compared to mhv, but they also induced higher fold change values (fig a) . for each of the three viruses, we defined a signature gene as a gene that is both differentially regulated at h compared to the mock treatment and has an effect size significantly larger than the other two viruses (i.e. fold change on the x axis is significantly different from y-axis, z-axis, and mock). these genes are colored in fig a and appear along the diagonal in fig b. as expected, rv had the largest number of signature genes, followed by pr , then mhv ( fig b) . interestingly, the genes with the highest fold change values compared to mock were not signature genes, but were up-regulated by both pr and rv infection. a pairwise analysis was performed to identify the number of genes with altered expression compared with mock in two viruses compared with the third. this analysis, shown in fig b, reveals that rv and pr had the most similarities in both up-and down-regulated genes (fig b, purple blocks) . the pattern of up-regulated gene expression changes during mhv infection was more similar to pr ( genes) than rv ( genes). this may reflect the fact that pr and mhv cause severe pathogenesis in mice, whereas rv-infected mice do not exhibit clinical signs of disease. among the six genes co-upregulated by mhv and pr was tnf-α, a key proinflammatory cytokine (not shown). several host defense genes were identified as signature genes uniquely up-regulated by pr infection (s table) . these genes included cytokines and chemokines (cxcl , ccl , il b, ccl ), ifn response genes (ifitm , ifi l a, ifna , ifit , ifitm , ifna ) , and genes involved in processing mhc class i antigens (psmb , tap , h -q , h -k , psmd , psme , psme ) . the significant up-regulation of host defense genes in response to pr in the la cell line corresponds with the expression profile of murine type ii alveolar epithelial cells in response to pr infection in mice [ ] . pr infection in highly susceptible mouse strains results in dramatic up-regulation of inflammation-associated genes when compared to resistant mouse strains [ ] . many studies in murine models of influenza a virus infection have demonstrated a relationship between an excessive inflammatory response and disease severity [ ] [ ] [ ] ] . furthermore, infection of tlr -/-mice with influenza a virus results in a reduced inflammatory response and increased survival [ ] . our data support the role of alveolar epithelial cells in generating this excessive inflammatory response in vivo. several genes that were uniquely down-regulated by pr are involved in cellular metabolic pathways (cdo , aldh a , acad , hsd b ) or intracellular transport (myl b, ift , anxa ). although rv induced expression of several genes involved in host defense, these were largely shared by pr so were not identified as signature genes. the signature genes up-regulated by rv included kallikrein- and ten kallikrein- -related peptidases and additional proteins involved in tissue remodeling (s table) . although tissue remodeling is not likely to be relevant in murine models of rhinovirus infection alone, due to the limited damage, it may be an important factor in murine models of rhinovirus-induced allergic asthma [ , , ] . rhinovirus infections are a significant cause of asthma exacerbations, which correspond with inflammatory responses in the airways. kallikreins generate kinins and contribute to many disease processes, including inflammation. kinins are induced by rhinovirus infections and kallikrein- is up-regulated by rhinovirus infection in humans, especially those with asthma [ , ] . upregulation of these genes in mouse cells upon rv infection would provide a tractable animal model in which to study the roles of kallikreins in rhinovirus-induced asthma exacerbations. mucins, which contribute to mucus hypersecretion, are up-regulated by rhinovirus infection of airway epithelial cells in vitro and in mice [ , ] . muc was the only mucin gene up-regulated by rv in our study, and was unique to rv infection (s table) . mhv infection resulted in regulation of a small set of signature genes (fig b, s table) . signature genes that were uniquely up-regulated by mhv infection included multiple transcription factors from the double homeobox (duxf , dux, dux ) and zinc finger and scan domain (zscan d, zscan c, zscan -ps , and ) families. despite up-regulating expression of transcription factors, mhv infection had a minor impact on the host cell transcriptome. this may be due to enhanced degradation of mrnas as discussed above, which has been shown to occur during other coronaviral infections [ , ] . therefore, la cells may be up-regulating transcription in response to mhv infection through expression of various transcription factors while mhv causes degradation of these transcripts, which would reflect the small number of up-regulated transcripts in mhv infected samples. in contrast to mhv-a , mhv- infection did not cause down-regulation of a substantial number of host genes. differences could be due to virus strain, host cell type, and timing differences between the studies. in contrast to the robust up-regulation of genes involved in innate immunity and inflammatory responses by pr and rv, the limited response of infected epithelial cells to mhv infection may reflect the ability of mhv to replicate (s fig) without being detected by the host cell. coronaviruses, including mhv, delay induction of antiviral responses. multiple mechanisms have been proposed to account for this, including replicating within double membrane vesicles, ribose '-omethylation of viral mrna, and endonuclease activity within the rna polymerase complex [ ] [ ] [ ] [ ] . this would allow mhv to replicate to higher levels before triggering antiviral defenses, which might promote pathogenesis in the murine respiratory tract [ ] . alternatively, the reduced response to mhv by epithelial cells may reflect the different cellular tropism of mhv. in contrast to pr and rv, which are known to infect epithelial cells in the murine respiratory tract, mhv- has only been reported in alveolar macrophages [ , , ] . type i ifn-related genes had increased expression in la cells infected by pr , rv, and mhv as described above, several of the genes with up-regulated expression in response to all three viruses, as well as those that were unique to pr , are induced by type i ifns. to specifically evaluate how ifn response genes were altered by the three viruses, genes that were significantly up-regulated by each virus at the h time point were used to query the interferome v . database (see materials and methods). a venn diagram was generated to visualize the degree of overlap in ifn-related genes whose expression was induced by at least one of the three viruses (fig ) . pr induced expression of the greatest number of ifn-related genes, a majority of which were shared by at least one other virus. rv up-regulated slightly fewer ifninduced genes compared to pr and mhv infection resulted in up-regulation of the fewest ifn-induced genes. it was somewhat surprising that pr induced a higher type i ifn response than rv, given that rv induced expression of nearly twice as many genes than pr (fig ) . however, some of these genes contribute to inflammatory responses, which could explain the excessive inflammatory response to pr infection vs. the self-limited inflammation during rv infection in mice [ ] [ ] [ ] ] . there was strong overlap between the ifn-induced genes up-regulated by each virus. the timing of ifn-related gene expression followed the same trend as was seen for all significant genes in fig (data not shown) . most of the ifn-related genes up-regulated by mhv were only increased at h. pr induced expression of ifn-related genes at h and these genes were a subset of the genes up-regulated at h. in contrast, rv infection induced expression of more ifn-related genes at h ( genes) than at h ( genes). relative to up-regulation, few ifn-related genes were down-regulated at the h time point (mhv = , pr = , rv = ). type i ifns induce expression of genes with different functions during an antiviral response. to determine whether there were specific patterns in expression of ifn-induced genes that correspond with function, the ifn-induced genes that had significantly increased expression by any of the three viruses were separated into functional groups. heatmaps that demonstrate differences in fold change (color scale) and significant differences (outlined boxes) in expression compared to mock-inoculated controls were generated (figs and s ) . as shown in the venn diagram, this analysis also demonstrates that pr infection resulted in up-regulation of the most genes involved in type i ifn responses, followed by rv then mhv. the fold change values induced by pr infection were also generally higher than the other two viruses. however, there was not a significant association between virus identity and functional group. for most of the functional groups, mhv up-regulated expression of a smaller subset of the same genes as pr and rv, with the exception of the mhc class i pathway (fig ) . mhv significantly up-regulated expression of only one gene involved in the mhc class i pathway (blmh), which was not significantly up-regulated by the other two viruses. this observation suggests that cytotoxic t cell responses may differ in mhv infections compared to pr and rv. t cell responses have complex roles in mhv- infections, as they contribute to protection in resistant mouse strains but mediate pathology in susceptible strains [ ] . however, mice with the cd + t cell repertoire of a resistant strain in the background of a susceptible strain remain susceptible to severe mhv- infection [ ] . the failure of mhv- to activate processing and presentation of mhc class i antigens could explain the inability of a broadly reactive cd + t cell response to protect these mice. the interferome analysis focuses on ifn-induced gene expression, but not expression of the type i ifns that induce these responses. multiple type i ifns exist, including ifn-β and subtypes of ifn-α, all of which signal through the type i ifn α/β receptor [ ] . type i ifns can induce autocrine and paracrine signaling; thus the ifn-induced genes we detected could be from both infected and uninfected cells in the cultures. to determine if differential expression of type i ifns explains the differences in ifn-induced gene expression upon infection by pr , rv, and mhv, we analyzed the expression of type i ifn and receptor genes for each virus compared to mock (fig ) . probes for ifn-β and ten subtypes of ifn-α were present on the arrays. in agreement with expression of ifn-induced genes, pr induced expression of the largest set of type i ifns, followed by rv. both viruses induced expression of ifnb and ifna , which encode type i ifns known to be expressed early during antiviral responses [ , ] . five subtypes of ifna were up-regulated by both pr and rv, while three ifna subtypes were uniquely up-regulated by pr and only ifnab was uniquely up-regulated by rv. only pr induced expression of ifnar , which encodes the high affinity chain of the type i ifn α/β receptor [ ] . this may allow for enhanced positive-feedback signaling and account for the larger number of ifn-induced genes up-regulated by pr infection. differential signaling through mda- and rig-i pathways may contribute to the differences in type i ifn responses by rv and pr . rhinoviruses and influenza a viruses are known to induce type i ifn responses through recognition by mda- and rig-i, respectively [ , ] . furthermore, both viruses are recognized by tlr in infected epithelial cells [ , ] . however, tlr predominantly induces expression of pro-inflammatory genes, rather than type i ifn-dependent genes, during influenza a virus infection [ ] . zaritsky et al. have demonstrated that the type i ifn response to sendai virus differs when cells are infected by different doses [ ] . they further showed that these differences were mediated by differential signaling through the ifn α/β receptor, with robust signaling in uninfected cells. this supports our findings that pr induces expression of ifnar and additional type i ifn genes that are not up-regulated by rv (fig ) . none of the type i ifns or receptors had significantly altered expression upon mhv infection (fig ) , despite up-regulation of a modest number of ifn-stimulated genes (fig ) . this could be due to ifn-independent expression of these genes, or induction by a type i ifn that was not represented on the microarray. coronaviruses are notorious for being able to replicate within cells without triggering type i ifn responses, or delaying ifn induction until late in the replication cycle [ , [ ] [ ] [ ] . other studies have shown that the ifn response to mhv- is a critical determinant of susceptibility. severe disease in a/j mice compared to c bl/ mice correlates with lower type i ifns detected in the lungs of a/j mice upon mhv- infection [ , ] . similarly, the expression of various type i ifns in response to mhv- infection in vitro is cell line-dependent [ ] . because the cell line we used, la , was derived from the lungs of a/ he mice, we would expect it to have a similar response as a/j mice. thus the lack of type i ifns induced by mhv- in la cells in vitro corresponds with pathogenesis observed in a/j mice in vivo. the finding that la cells mount a stronger response to pr than rv or mhv infection may be due to differences in the viral recognition and signaling pathways used to detect these different viruses and amplification of the type i ifn response as discussed above. alternatively, it could be due to the timing of our analysis. rv-infected cells have started dying by the h time point (not shown), therefore expression of host genes may be declining by that time point. in contrast, coronaviruses are known to delay cellular responses to infection [ ] , so the h time point may be too early to evaluate the innate response to mhv infection. alternatively, the cells may detect mhv and up-regulate transcription of ifn response genes, but mrna degradation would mask this process. by quantifying mrna transcripts at two time points after viral infection, our study cannot distinguish between these possibilities. one limitation of our study is the analysis of three viruses that do not share a natural host. mhv is a natural pathogen of mice and pr is a highly mouse-adapted strain of influenza a virus. however, rv b is a human rhinovirus whose receptor is conserved between mice and humans. rv b is increasingly being used in mouse studies [ ] [ ] [ ] [ ] ] . despite the difference in host, we found similar changes to gene expression in murine cells as studies with rv b in human cells [ ] . of the , and , genes represented on our mouse microarray and the human microarray chip used by chen et al., respectively, , genes are shared. using the same -fold increase in expression cut-off and restricting our list only to homologous human genes studied by chen et al., we found that mouse genes were up-regulated by rv b infection. comparing this list of genes to the up-regulated human genes identified by chen et al., we found that genes (s table) were up-regulated by rv b infection in both human and mouse cells. a chi-squared test confirmed the significance of this shared number of up-regulated genes (χ = . , d.f. = , p< . ). interestingly, all of the shared genes we identified are involved in type i ifn responses. while far from identical, the similarity of the responses in the two cell types suggests conserved activation of type i ifn responses by these different hosts and supports the validity of a murine model for studying rhinovirus infections in humans. alveolar epithelial cells have a key role in alerting the immune system to infection by respiratory viruses and shaping immune responses [ , , ] . as viruses from several different families all target respiratory epithelial cells, it is important to understand the similarities and differences in how these cells respond to a diverse set of viruses. a significant number of genes were up-or down-regulated in response to infection by three distinct viruses from different families. genes that were associated with a shared response to the three viruses included those involved in defense against viruses and particularly genes induced by type i ifns. however, there were differences in the timing, numbers of genes altered, and expression levels of these genes. this may reflect differences in viral replication cycles and signaling pathways that are activated by infection, which may reflect differences in pathogenesis of these viruses in murine models. la cells were inoculated with virus using the same moi's as for the microarray study and rna was extracted at h post-infection using trizol (ambion). rna was converted to cdna using random hexamers and super-script vilo (invitrogen). five genes with differential expression by microarray analysis were validated by qpcr analysis using sybr green (powerup, applied biosystems) and the primer pairs listed in the figure on a stepone plus instrument (applied biosystems). ct values from triplicate qpcr reactions were averaged and normalized to β-actin before calculating the fold change values of virus-infected samples vs. mock. linear regression was used to compare fold change values between qpcr and microarray with removal of outliers ( Ã ) with cook's distance > . . (pdf) genes with significantly up-regulated expression compared to mock at h (see fig ) were used to query the interferome v. . database. interferon-regulated genes were divided into functional groups and heat maps were generated using log -fold change values for each virus at h compared to mockinoculated controls. heat maps of additional functional groups can be found in fig . gene names are indicated to the right of each row and statistically significant values are outlined in black. (pdf) s table. genes whose expression was significantly up-regulated by all three viruses compared to mock-inoculated cells. these genes were from the center of the venn diagram in fig a. (xlsx) s table. genes whose expression was significantly down-regulated by all three viruses compared to mock-inoculated cells. these genes were from the center of the venn diagram in fig b. (xlsx) s table. signature genes for pr . these genes were significantly different in pr infection compared to mock, rv, and mhv. (xlsx) s table. signature genes for rv. these genes were significantly different in rv infection compared to mock, pr , and mhv. (xlsx) s table. signature genes for mhv. these genes were significantly different in mhv infection compared to mock, rv, and pr . (xlsx) s table. genes up-regulated by rv in both murine and human cells. these genes were identified to be up-regulated in our study by rv b in murine cells and also were found by chen et al. ( ) to be up-regulated by rv b in human cells. 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their receptors-distribution and regulation cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells role of double-stranded rna pattern recognition receptors in rhinovirus-induced airway epithelial cell responses virus multiplicity of infection affects type i interferon subtype induction profiles and interferon-stimulated genes murine coronavirus cell type dependent interaction with the type i interferon response group coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna distinct signature type i interferon responses are determined by the infecting virus and the target cell dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection toll-like receptor -expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation host-pathogen interactions during coronavirus infection of primary alveolar epithelial cells virus-infected alveolar epithelial cells direct neutrophil chemotaxis and inhibit their apoptosis the authors are grateful to dr. kathryn holmes, university of colorado at denver school of medicine, dr. elizabeth fortunato, university of idaho, and dr. julian leibowitz, texas a&m university for cells and antibodies that were used in this study. the following reagents were key: cord- -d p nbgq authors: lasfar, ahmed; abushahba, walid; balan, murugabaskar; cohen-solal, karine a. title: interferon lambda: a new sword in cancer immunotherapy date: - - journal: clin dev immunol doi: . / / sha: doc_id: cord_uid: d p nbgq the discovery of the interferon-lambda (ifn-λ) family has considerably contributed to our understanding of the role of interferon not only in viral infections but also in cancer. ifn-λ proteins belong to the new type iii ifn group. type iii ifn is structurally similar to type ii ifn (ifn-γ) but functionally identical to type i ifn (ifn-α/β). however, in contrast to type i or type ii ifns, the response to type iii ifn is highly cell-type specific. only epithelial-like cells and to a lesser extent some immune cells respond to ifn-λ. this particular pattern of response is controlled by the differential expression of the ifn-λ receptor, which, in contrast to ifn-α, should result in limited side effects in patients. recently, we and other groups have shown in several animal models a potent antitumor role of ifn-λ that will open a new challenging era for the current ifn therapy. despite the early discovery of interferon (ifn) in , ifn lambdas were just identified during the recent years and classified as a new group, type iii ifn. in human, distinct proteins called ifn-λ , ifn-λ , and ifn-λ have been identified [ , ] . they are also named interleukin- (il- ), il- a, and il- b, respectively [ ] . the members of this new ifn family were found to interact through unique receptors that are distinct from type i (ifn-α/β) and type ii (ifn-γ) ifn receptors. the receptor for type iii ifn is composed of the unique ifn-λr chain also called il- ar and the il- r chain, which is shared with il- , il- , and il- receptor complexes. although type iii ifns bind to a specific receptor, the downstream signaling is similar to that induced by type i ifns. both type i and type iii ifns stimulate common signaling pathways, consisting of the activation of jak and tyk kinases and leading to the activation of ifn-stimulated gene factor (isgf ) transcription complex. isgf is composed of stat and stat and the interferon regulatory factor irf (isgf -γ or p ) ( figure ). although there are three genes encoding highly homologous but distinct human ifn-λ proteins (ifn-λ , ifn-λ , and ifn-λ ), our search of the mouse genome revealed the existence of only two genes, representing mouse ifn-λ and ifn-λ gene orthologues, located in chromosome and encoding intact proteins. the mouse ifn-λ gene orthologue is a pseudogene containing some variations in addition to a stop codon in the first exon and does not code for an active protein [ ] . we have cloned the mouse ifnλs (mifn-λ and mifn-λ ) and ifn-λ receptor (mifn-λr ) orthologues and found them to be quite similar to their human counterparts. experiments showed that similar to their human counterparts, mifn-λ and mifn-λ signal through the ifn-λ receptor complex, activate isgf , and are capable of inducing antiviral protection and mhc class i antigen expression in several cell types. the results showed that murine type iii ifns (ifn-λs) engage a unique receptor complex, composed of ifn-λr and il- r subunits, to induce signaling and biological activities similar to those of type i ifns. interestingly, in contrast to type i and type ii ifns, type iii ifns demonstrate less species specificity. the intensity of cell signaling as measured by stat activation appeared to be significantly lower for type iii ifns [ ] . in comparison with type i ifn, only restricted cell types respond to type iii ifn ( figure ). interestingly, we did not find a strict correlation between the intensity of cell signaling induced by ifn-λ and the level of biological activity. for example, in b melanoma cells, although ifn-λ induced a very weak stat activation in comparison with ifn-α, we observed a robust stimulation of mhc class i expression at the cell surface, indicating the potential contribution of cellspecific modulators of the ifn-λ activity. antiviral studies performed in vitro and in vivo have shown that both ifn-α and ifn-λ contribute to the overall host antiviral defense system [ , , [ ] [ ] [ ] [ ] . it has been demonstrated that ifn-λ induces antiviral activity against vsv (vesicular stomatitis virus) and emcv (encephalomyocarditis) in many human cell lines [ , , , ] . however, by using different mouse models of viral infection, ank et al. demonstrated that ifn-λ was effective against dna virus, simplex virus hsv but not rna viruses such as emcv and lymphocytic choriomeningitis virus lcmv [ ] . several other studies demonstrated that type iii ifns can also inhibit replication of hepatitis c virus (hcv) and hepatitis b virus (hbv) in vitro [ ] [ ] [ ] [ ] [ ] . these studies were important since they underlined the fact that ifn-λ could be used as an alternative to ifn-α for hcv patients who are resistant to ifn-α treatment. it has been reported that ifn-λ has the ability to inhibit human immunodeficiency virus type (hiv- ) infection of blood monocyte-derived macrophages that expressed ifn-λ receptors [ ] and the herpes simplex virus type (hsv ) infection of human astrocytes and neurons [ ] . however, in most other cases, the antiviral potency of ifn-λ against several viruses seems to be lower than that of ifn-α [ , , , , , ] . in addition, ifn-λ and ifn-α may induce distinct signal transduction and gene regulation kinetics [ , ] . moreover, type i ifn-α activates a plethora of innate and adaptive immune mechanisms that help eliminate tumors and viral infections. ifn-α immunoregulatory functions include major histocompatibility complex (mhc) class i expression in normal and tumor cells, activation of nk cells, dendritic cells (dcs), and macrophages, resulting in the promotion of adaptive immune responses against tumors and virally infected cells [ , ] . the role of ifn-λ in the immune system is currently being investigated by several groups. so far, data suggests that ifn-λ exerts immunomodulatory effects that overlap those of type i ifn. it has been recently demonstrated that human ifn-λ (il- ) modulates the human plasmacytoid dcs function and cytokine response [ , ] . ifn-λ treatment of whole peripheral blood mononuclear cells (pbmcs) upregulated the expression of il- , il- , and il- but not il- or tnf. this ifn-λ-induced cytokine production was inhibited by il- . by examination of purified cell populations, it was also shown that ifn-λ activated monocytes, rather than lymphocytes, resulting in the secretion of the above panel of cytokines, suggesting that ifn-λ may be an important activator of innate immune responses particularly at the site of viral infections [ ] . ifn-λ was also shown to possess immunoregulatory functions on t helper (th ) responses by markedly inhibiting il- . however, only moderate effect was observed on il- and il- , the neuron [ , ] endothelial cell [ ] adipocyte [ ] fibroblast [ ] macrophage [ ] melanocyte [ ] keratinocyte [ ] colon cell [ ] hepatocyte [ ] lymphocyte [ ] dendritic cell [ , ] ifn-α ifn-λ other important cytokines in the th response [ ] [ ] [ ] . this immunoregulatory function was enhanced through the expression of ifn-λr on cd + t cells [ ] . these findings correlate with data suggesting that ifn-λ may have an immunoprotective role against asthma, the allergy disease caused by an exaggerated th response [ , , ] . similar to ifn-α, ifn-λ produced by dcs, in response to toll-like receptor (tlr) stimulation, was found to have specific effects on dc differentiation and maturation [ ] , which include only partial maturation of dcs, upregulation of mhc class i and ii molecules, and no induction of costimulatory molecules [ , ] . during their differentiation from monocytes, dcs acquire ifn-λ responsiveness through the expression of ifn-λr . interestingly, dcs treated with ifn-λ promoted the generation of tolerogenic dcs and the il- -dependent proliferation of foxp -expressing cd + cd + regulatory t cells (tregs) [ ] . more recently, morrow et al. have demonstrated, through dna vaccination with plasmids encoding ifn-λ (il- b) and il- , that ifn-λ , just like il- , is able to enhance adaptive immunity. however, in contrast to il- , ifn-λ reduces regulatory tcell populations. they also showed that unlike il- , ifn-λ is able to increase the percentage of splenic cd + t cells in vaccinated animals and that ifn-λ can completely protect mice from death following a lethal influenza challenge [ ] . these studies altogether highlight the strong candidacy of ifn-λ as a potential novel immunotherapeutic agent. in addition to antiviral and immunomodulatory activities, type i ifns demonstrate antiproliferative activities in most cell lines, while this activity seems to be restricted with ifn-λs [ , ] . type i ifns have been shown to induce apoptosis in tumor cells, yet the molecular mechanisms mediating cell death in response to these ifns remain to be fully explained. by binding to their corresponding cellular receptor complexes, ifns induce a quick and potent signaling which leads to the expression of more than ifn-stimulated genes (isgs) [ , , ] . many isgs encode proteins that have been implicated in apoptosis [ , ] . unlike ifn-α, ifn-λs do not inhibit the proliferation of several cell lines including the daudi cells (a b-lymphoblastoid cell line from burkitt's lymphoma), which strongly respond to type i ifns in an antiproliferative assay [ , , , ] . however, it was demonstrated that ifn-λs do inhibit the proliferation of few tumor cell lines, such as the ln human gliobastoma cell line [ ] and of cells constitutively expressing high levels of ifn-λr [ ] . the antiproliferative effects of ifn-λ have been demonstrated in various tumor cell lines that express ectopic or endogenous ifn-λ receptors [ , , ] . therefore, the ability of ifn-λs to induce antiproliferative activity in cells depends on the level of ifn-λr expression. it has been recently reported that ifn-λ signaling in colorectal adenocarcinoma ht cells led to caspase activation, externalization of phosphatidylserine (ps), and dna fragmentation, resulting in subsequent apoptosis [ ] . this study provided evidence for the first time that type iii ifns, alone or in combination with other stimuli, have the potential to induce apoptosis. moreover, another recent study revealed that ifn-α and ifn-λ differ in their antiproliferative effects and this was correlated with a difference in the duration of jak/stat signaling activity between the two ifns and prolonged isg expression upon ifn-λ treatment [ ] . using the human keratinocyte hacat cell line that expresses receptors for both ifn-α and ifn-λ, they found that ifn-λ induced a more pronounced growth inhibitory effect than ifn-α. ifn-λ was also more efficient than ifn-α in inducing an antiproliferative effect that overlapped with the activation of apoptosis. prolonged duration of ifn-λinduced stat activation, and isg expression could account for the enhanced antiproliferative and proapoptotic effects observed in hacat cells, effects not seen upon treatment with high doses of ifn-α [ ] . interestingly, a study has shown that ifn-λ can induce the growth of human multiplemyeloma cells and antagonize the dexamethasone-induced cell death in these cells [ ] . ifn-λ-mediated cell growth of multiple myeloma cells was mapk dependent [ ] . high level of ifn-λ was found in the malignant bone marrow microenvironment, implying that ifn-λ may play a direct role in multiple myeloma development. by using a plasmid electrotransfer approach, sommereyns and coworkers reported a differential response to ifn-λ in mice, with a very low response to ifn-λ for the liver, central nervous system, and spleen. however, a high response to ifn-λ was observed in the stomach, intestine, heart, kidney, and lung [ ] . the ifn-λ response was restricted to epithelial cells and correlated with the expression of ifn-λr (il- ralpha). paradoxically in mice, in spite of the epithelial nature of the hepatocytes, the liver expressed low levels of il- ralpha and responded poorly to ifn-λ [ , ] . however, a significant response to ifn-λ was reported in human hepatocytes [ , ] , suggesting the existence of some variations in the response to ifn-λ between mice and humans, at least in the liver. although the main ifn-λ targets are the epithelial cells, the presence of potential tissuespecific factors may modulate the ifn-λ response through the ifn-λ receptors. recently, it has been shown in mice that in contrast to the hepatocytes, prominent response to ifnλ was observed in intestinal epithelial cells. in comparison with ifn-α, this response is higher and plays a critical role in protecting the intestinal epithelium from viral infection [ ] , strongly suggesting the prominent role of ifn-λ in organs with mucosal surface at least in mice [ , , ] . in addition to the direct effect of ifn-λ on the mucosal epithelium, local immunomodulations can also be promoted [ ] . the functional ifn-λr is formed by two chain proteins, ifn-λr (also called il- ralpha) and il- r . ifn-λr is unique for the ifn-λs, and its tissue distribution is highly restricted. in contrast to ifn-λr , il- r is shared by il- , il- and il- and ubiquitously expressed in all tissues. unlike ifn-α, only few cell types respond to ifn-λ ( figure ). in contrast to the epithelial-like cells, fibroblasts and endothelial cells were completely unresponsive to ifn-λ [ ] . although the hematopoeitic system is not the primary target of ifn-λ, the response of some subpopulations to ifn-λ is not excluded. in mice, we found that ifn-λ induces stat activation in both plasmacytoid and myeloid dendritic cells [ ] . these results are in accordance with those obtained by mennechet and uzé [ ] , who proposed the acquisition of an ifn-λ response by monocytes after their differentiation into dendritic cells. therefore, the response to ifn-λ may be controlled by the induction of the ifn-λr expression. different levels of ifn-λr were found in different tissues [ , , ] . the highest levels were found in the gastrointestinal tract and lung. the brain showed the lowest level of receptor expression. the ifn-λr expression was also analyzed in different cell types. the expression of cell populations isolated from human skin showed a high expression of ifn-λr in keratinocytes and melanocytes. however, dermal fibroblasts, endothelial cells, and subdermal adipocytes did not express significant amounts of ifn-λr . significant expression of ifn-λr was detected in primary human hepatocytes in comparison with the chondrocytes, isolated from the hyaline cartilage of the knee joint [ , ] . although the expression of ifn-λr was significantly high in lymphoid tissues, the ifn-λ response was very weak, implying the presence of specific mechanisms in the lymphoid tissues that may inhibit the ifn-λ response. for example, ifn-λr levels in b cells are threefold those detected in keratinocytes, which exhibit one of the highest responses to ifn-λ. witte et al. proposed the potential role of soluble ifn-λr , highly released by the immune cells, in this weak response to ifn-λ [ ] . although all the ifn-λs interact with the same receptor, ifn-λr , the binding characteristics for each ligand are still under investigation. in the future, it will be important to analyze the ifn-λ activity in light of the ifn-λ binding to the cells and understand particularly the role of ifn-λ , which possesses the highest activity as compared with the other ifn-λs [ , ] . analysis of the ligand binding in combination with the activity induced by ifn-λ will be also important in understanding the impact of ifn-λ in epithelial cells, particularly in comparison with the immune cells expressing ifn-λr . besides several carcinomas, originating from epithelial cells, which respond to ifn-λ, other tumors not arising from epithelial cells may become more sensitive to ifn-λ. it was reported that multiple myeloma cells, which originate from b-cell plasmocytes, showed high binding and response to ifn-λ [ ] . studying the ifn-λ binding in transformed cells versus normal cells may be very helpful for tumor targeting and for the establishment of the optimum dose of ifn-λ to be used for the in vivo treatment. ifn-λ can also be used as a drug carrier, to specifically target a drug to tumors expressing high ifn-λ binding sites. clinical and developmental immunology the availability of ifn-λr knock-out mice allowed for the investigation of the role of type iii ifns in vivo. by using those mice, mordstein et al. showed for the first time the contribution of ifn-λ in the innate immunity against the influenza virus [ ] . later, they found that ifn-λ played an important role in the defense against other pathogens that infect the respiratory tract, such as the respiratory syncitial virus, the metapneumovirus, and the severe acute respiratory syndrome (sars) coronavirus. however, the lassa fever virus which replicates in the liver, was not affected by the lack of ifn-λr [ ] . although this study clearly demonstrated that ifn-λ played an important role in protecting the respiratory and gastrointestinal tracts against virus infection, in comparison with type i ifn, the protection provided by type iii ifn remains limited. however, in combination, type i and type iii may provide a better viral protection. when the response to both type i and type iii is deficient, the mice are not able to clear the sars coronavirus from the intestine as compared with mice in which type i or type iii remains functional, implying that ifn-λ may strengthen the antiviral activity by acting as a first line of defense for the mucosa [ , ] . use of type iii ifn. the first use of ifn-λ in the clinic has started for hepatitis c. the phase b study has been conducted in patients with chronic genotype hepatitis c virus infection ((hcv) [ ] ). pegylated ifn-λ in combination or not with ribavirin (rbv, which belongs to a class of antiviral medications called the nucleoside analogues) has been used in this study to assess the efficacy and the potential cytotoxicity. the study was performed in parts. the first part evaluated the pegylated ifnλ as single agent for relapsed patients after ifn-α-based treatment. the second part concerned the combination of pegylated ifn-λ and rbv in treatment-relapse patients. the third part evaluated pegylated ifn-λ in combination with rbv in treatment-naïve patients. in addition, different doses (from . to microg/kg) of pegylated ifn-λ were used. fifty-six patients were enrolled. , , and patients were used, respectively, for part to . the data showed an antiviral activity in all doses of pegylated ifnλ tested. % of treatment-naïve patients achieved rapid antiviral response. as expected, due to the limited ifn-λr distribution, the treatment was well tolerated with few adverse effects. minimal flu-like symptoms and limited hematologic suppression were reported. in summary, the authors concluded that weekly pegylated-ifn-λ with or without daily rbv for weeks is associated with a clear antiviral activity in patients with chronic hcv. however, this study lacks a direct comparison between ifn-λ and ifn-α and the influence of viral and patient genotypes. now it is well accepted that the response to ifn-α or the natural clearance of hcv infection is depending on singlenucleotide polymorphisms (snps), upstream of ifn-λ , which could be used as biomarkers to help determine the treatment outcome [ ] . the first genome-wide association studies (gwas) in hcv infection were reported by ge et al. they evaluated the treatment outcome in a group of patients of mixed ethnicity, receiving pegylated ifn-α and ribavirin. an association was discovered between sustained viral response (svr) to treatment and a cluster of seven snps linked to the ifn-λ gene, with the most significant snp (rs ) demonstrating high statistical significance [ ] . many other studies have replicated these findings, demonstrating the high link between ifn-λ and treatment outcome [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however the mechanisms explaining this link remain to be determined. it is not clear yet if this snp is associated with a constitutive production of ifn-λ that may play a role in hcv clearance and the success of ifn-α treatment. these results also suggest the therapeutic potential of the ifn-α and ifn-λ combination therapy as demonstrated for the hepatocellular carcinoma (hcc) mouse model [ ] . antitumor agent although they engage distinct receptors, ifn-α and ifn-λ induce similar cell signaling (figure ). since ifn-α is widely used in the clinic to treat cancer (table ) , we have investigated the potential antitumor activity of ifn-λ by using the mouse b melanoma model. we have chosen this cancer model because melanoma is a very aggressive cancer, and one of the therapeutic agents frequently used in the treatment of melanoma is ifn-α. significant improvements in relapsefree and overall survival, with postoperative adjuvant ifnα therapy, have been reported by large and randomized studies [ ] [ ] [ ] . however, the beneficial effect of ifn-α was only obtained when the patients received high doses ( miu/m intravenously five times per week). studies with low doses of ifn-α have not shown significant increase in overall survival [ , ] . usually, the dose for optimal antitumor activity is higher than the maximally tolerated dose. this dose dilemma profoundly affects the acceptance of ifn-α treatment by both the clinicians and the patients. the adverse effects associated with high doses of ifn-α include myelosuppression and nervous system disorders. these effects often compromise the beneficial antitumor effect, with premature discontinuation of the treatment or the reduction of the dose of ifn-α. since virtually all the cells of the body respond to ifn-α, it is not surprising that the patients develop numerous side effects. making a dissection between the beneficial and harmful effects of ifn-α is a very challenging task, which requires more investigation of the interferon system. to investigate the antitumor effect of ifn-λ in melanoma, we have used a gene therapy approach, consisting on the delivery of the ifn-λ gene to tumor cells. gene transfer into tumor cells is very useful approach to test the effectiveness of cytokines in animal cancer models. this approach does not require production and purification of the protein. the secretion of constant amounts of various cytokines by transduced tumor cells at the site of tumor growth could elicit more to investigate the potential antitumoral role of ifnλ, we first evaluated the response of b melanoma cells to ifn-λ, by analyzing stat activation and mhc class i antigen expression. in comparison with ifn-α, we have found that ifn-λ induces weak stat phosphorylation but strong stimulation of mhc class i antigen expression, indicating a difference between ifn-α and ifn-λ in the link intensity of cell signaling/biological activity. this result warrants further investigation in comparing the response to ifn-α and ifn-λ. by using gene transfer, we engineered b cells, which constitutively produced mifn-λ (b .ifnλ cells). in response to their secretion of ifn-λ, b .ifnλ cells exhibited constitutively high levels of mhc class i antigen expression. all the c bl/ syngeneic mice injected with parental b cells developed tumors. however, the constitutive production of mifn-λ by b .ifn-λ cells markedly affected tumorigenicity of the cells. b .ifn-λ cells were either rejected by the host or grew at a slower rate than control parental b cells. the antitumor effect of ifn-λ was dose dependent. b .ifn-λ cells also inhibited the growth of parental b cells when both cell types were injected together [ ] . we also used the engineered b .ifn-λ res. cells, which, in addition to their constitutive ifn-λ secretion, are completely resistant to ifn-λ, as demonstrated by the lack of ifn-λ-induced mhc class i antigen expression. interestingly, similar to b .ifn-λ cells, we have found a reduction of the tumorigenicity of b .ifn-λ res. cells, implying the involvement of host antitumor mechanisms induced by ifn-λ [ ] . following our report on the characterization of the mouse ifn-λ system and the potent antitumor activity of ifn-λ in the b mouse melanoma model, independent groups confirmed the role of ifn-λ as an antitumoral agent in melanoma and other tumor models. to demonstrate the antitumor activity of ifn-λ, sato et al. [ ] used the mouse melanoma b f and b f and the colon cell lines transfected with ifn-λ cdna. the ifn-λ-transduced b f cells showed an increased activity of caspase / , an induction of p and a dephosphoryation of rb, which triggered a cell cycle arrest and apoptosis. these events, obtained, in vitro, were apparently associated with a growth delay, observed in vivo after the injection of the b f transduced with ifn-λ. a delay in tumor growth was also observed after the administration of the colon cells transduced with ifn-λ. by using the b f cell line, which represents metastatic mouse melanoma cells, the authors showed that the overexpression of ifn-λ significantly inhibited lung metastasis. in another study, to evaluate the antitumor activity of ifn-λ, numasaki et al. [ ] first transduced the mouse fibrosarcoma cells, mca , with the retroviral vector pa il- (ifn-λ ). following the injection of the engineered tumor cells to mice, the authors observed a significant antitumor and antimetastatic effect in mice inoculated with the mca il- in comparison with those injected with the parental tumor cells. hcc is the most prevalent type of liver cancer. it is the fifth most common solid tumor and the third leading cause of cancer-related death worldwide. it is also the second most lethal cancer with the five-year survival rate below % [ ] [ ] [ ] . treatment options for hcc are limited mainly because of the inefficiency of existing anticancer chemotherapeutic drugs against hcc. unfortunately, due to a lack of biomarkers and screening for hcc, most patients are diagnosed at advanced stages of the disease and do not meet strict selection criteria for potentially curative surgical tumor resection or orthotopic liver transplantation (olt) [ ] [ ] [ ] . in patients with unresectable hcc and preserved liver function, transarterial chemoembolization (tace) has been shown to prolong survival. however tace is rarely curative, and progression-free survival beyond months is not frequent [ , ] . for patients with advanced disease, systemic chemotherapy is of limited benefit because of the resistance of hcc to existing anticancer drugs and the fact that about % of patients with hcc die secondary to liver failure from cirrhosis [ , ] . hcc occurs most frequently in patients with cirrhosis as a result of chronic hbv (hepatitis b virus) and hcv (hepatitis c virus) infections, and alcohol abuse [ , ] . although the link between the cancer and the viral infection is not fully understood yet, there is some suggestion that viral infection interferes with signal transduction and consequently disrupts the normal, controlled growth of cells. since ifn-α is used in the clinic for the treatment of chronic hcv and hbv infections, several studies evaluated the effect of ifn treatment on the incidence of hcc [ ] . it was previously shown that the systemic administration of high doses and long-term ifn-α into nude mice bearing human hcc with high metastatic potential, following curative resection, inhibited tumor metastatis and recurrence [ ] . the majority of clinical studies also concluded that ifn therapy, alone or in combination with ribavirin, decreased the incidence of hcc, particularly in patients with sustained virological response [ ] [ ] [ ] [ ] . therefore, ifn alone or, perhaps, in combination with other drugs can be used as a preventive therapy against the development of hcc in hcvand hbv-infected patients. however, numerous side effects limit the overall tolerability of ifn-α, particularly in patients with cirrhosis [ ] [ ] [ ] . in the following part of this section, we describe our findings on the antitumor properties of ifn-λ in the bnl mouse model of hcc. to evaluate the antitumor activities of both ifn-λ and ifn-α, we used a gene therapy approach as previously described [ ] . we expressed ifn-λ and ifnα genes under a strong constitutive promoter in bnl cells and selected stable cell lines, bnl-ifn-λ and bnl-ifn-α, constitutively expressing ifn-λ and ifn-α [ ] . since the constitutive expression of ifn-λ at the tumor site was found to affect the tumorigenicity of b melanoma cells in vivo [ ] , we examined whether similar effects of ifn-λ would be displayed in the case of bnl hepatoma. mice injected with bnl vector or parental bnl cells developed tumors in to weeks, whereas the tumor appearance for bnl-ifn-λ cells was significantly delayed. similar effects were obtained in mice inoculated with bnl-ifn-α cells. these experiments demonstrated that constitutive expression of ifns at the tumor site resulted in the delay of tumor growth in vivo. interestingly, we found that ifn-α and ifn-λ exhibited similar antitumor activities [ ] . despite the antiproliferative effects of ifn-α, it seems that the direct effects on tumor cells may not be the major mechanism by which ifnα displays its antitumor activity. ifn-α can act indirectly on the tumor by inhibiting angiogenesis which is induced by the tumors and is required to promote their growth and metastasis [ ] . in mice bearing human tumors, it was clearly demonstrated that the antitumor activity of ifn-α is associated with the inhibition of tumor angiogenesis in bladder carcinoma [ ] and prostate cancer [ ] . the involvement of the immune system in the antitumor mechanism of ifnα was strongly suggested by gresser et al. [ , ] . early studies in tumor models have shown that an intact immune system was essential in ifn-α-induced antitumor activities. the inhibition of friend leukemia cells (flc) by ifn-α in mice was shown to depend on the activation of host cells, such as nk cells and macrophages [ ] . both host humoral and cellular immune mechanisms were involved in the continued suppression of friend erythroleukemia metastases after ifn-α treatment in mice [ ] . in addition, effective adaptive immunotherapy was observed in a t-cell lymphoma model, after the injection of tumor-sensitized spleen cells and ifn-α. by using antibodies against different immune cell populations, it has been shown that cd + t lymphocytes and cd + t lymphocytes were the major effectors in the antitumor activities induced by ifn-α [ , ] . although ifn-α and ifn-λ signal quite similarly (figure ), the mechanisms underlying the antitumor activity of ifn-λ may be qualitatively different from ifn-α. as previously described, we initially investigated whether type iii ifns also possessed antitumor activities utilizing a gene therapy approach in the b melanoma model. since secreted ifn-λ did not affect the proliferation rate of b melanoma cells in vitro, studies in the b melanoma model suggested that ifnλ acted through host mechanisms to elicit its antitumor activity [ ] . however, we did not observe a significant longlasting immunity, implying that there may be a lack of effective adaptive immunity in the mice which rejected the tumor. on the other hand, we noticed a reduction in tumor vascularity in the presence of ifn-λ, suggesting a potential role of ifn-λ in the tumor microenvironment [ ] . since we found that keratinocytes are highly sensitive to ifn-λ and they are known to interact with melanocytes, the cells from which the melanoma originates, we suggested that ifn-λ delivery to the tumor microenvironment may affect the function of the keratinocytes as well as other stroma cells thereby promoting inhibition of tumor growth [ ] . nk cells, the major effectors of innate immunity, could also be recruited to the tumor microenvironment and help destroy the tumor cells. two groups have reported that nk cells played a role in the antitumor mechanisms of ifnλ. sato et al. [ ] have described the involvement of nk cells in melanoma and colon cancer antitumor responses. they have shown that transient transduction of b cells with mouse ifn-λ cdna enhanced mhc class i and fas expression, suppressed cell proliferation by inducing increased caspase- / activity, increased p waf /cip levels, and dephosphorylated rb (ser ) in vitro [ ] . this meant that ifn-λ was able to induce cell cycle arrest and apoptotic cell death in vitro. in addition, they have demonstrated that overexpression of ifn-λ inhibited local and pulmonary metastatic tumor formation in vivo. depletion of nk cells, by injecting an anti-asialo gm antibody before tumor cells injection, revealed that nk cells are important in this ifnλ-mediated tumor growth inhibition in vivo, suggesting that ifn-λ activated the innate immune response [ ] . numasaki et al. [ ] have also implicated nk cells, polymorphonuclear neutrophils, and cd + t cells in the antitumoral activity are induced by ifn-λ in the mca murine fibrosarcoma mouse model. inoculation of mca -ifn-λ cells into mice enhanced ifn-γ production and cytotoxic t-cell activity in the spleen. the antitumor activity of ifn-λ was partially dependent on ifn-γ. in addition, ifn-λ increased the total number of splenic nk cells in severe combined immunodeficiency (scid) mice, enhanced il- -induced ifn-γ production in vivo, and expanded spleen cells in c bl/ mice. furthermore, they reported that il- augmented the ifn-λ-mediated antitumor activity in the presence or absence of ifn-γ. based on their findings, they suggested that ifn-λ is able to induce both innate and adaptive immune responses to suppress in vivo tumor growth [ ] . our recent study in the bnl hepatoma model also revealed that nk cells are implicated in the antitumor activity induced by ifn-λ and probably more potently than ifnα. however, in contrast to ifn-α, we did not detect any response after in vitro treatment of nk cells by ifn-λ, suggesting that ifn-λ may activate other cells, which then mediate nk cell activation [ ] . there was also a marked nk cell infiltration in ifn-λ-producing tumors. in addition, ifn-λ and, to a lesser extent, ifn-α enhanced immunocytotoxicity of splenocytes primed with irradiated bnl cells. splenocyte cytotoxicity against bnl cells was dependent on il- and ifn-γ and mediated by dendritic cells. in contrast to nk cells, isolated from spleen, cd c + and mpdca + dendritic cells responded directly to ifn-λ, suggesting that the effects of ifn-λ on nk cells are mediated by other ifn-λ-responsive cells, such as dcs [ ] . on the other hand, a significant decrease in cd + cd + foxp + tregs was observed in mice inoculated with bnl cells secreting ifn-α, whereas the moderate decrease in tregs observed in mice receiving bnl cells secreting ifn-λ was not statistically significant [ ] . therefore, antitumor mechanisms activated by ifn-α and ifn-λ may differ; ifn-λ increased the number of nk cells at the tumor site whereas ifn-α had a stronger effect on tregs in the bnl model. these studies altogether suggest that although ifn-α and ifn-λ signal quite similarly, differences exist in their biological potency, kinetics, and the sets of target cells sensitive to ifn-λ and ifn-α. therefore, these two types of ifns may have distinct physiological functions. higher antitumor activities? unlike ifn-α, only a small subset of cells are sensitive to ifn-λ, implying that its potential clinical use may be associated with limited side effects. this presumption raises the question whether ifn-λ could be an alternative to ifn-α in cancer therapy. however, despite the severe and numerous side effects inherent to ifn-α treatment [ ] , we believe that alternative treatment to ifn-α should be weighed first against the real benefits to patients in terms of overall survival and their tumor clearance. we have demonstrated in the bnl hepatoma model that the combination of ifnλ and ifn-α could achieve a marked antitumor activity in comparison with the use of each ifn alone [ ] . the benefits of the combination therapy of ifn-λ and ifn-α have been demonstrated both by using a gene therapy approach and by direct administration of ifns to the mice bearing the tumors. the mice injected with bnl cells secreting both ifn-λ and ifn-α can completely reject the tumor, in contrast to the mice that only received the bnl-ifn-λ cells or the bnl-ifn-α cells. furthermore, mice bearing established tumors and treated with exogenous ifn-λ and ifn-α showed a drastic tumor repression. this effect was observed when the ifns were delivered locally and even at low doses. therefore, we believe that ifn-λ is not simply acting like ifn-α, with reduced side effects, but can be combined with ifn-α to achieve efficient antitumor activity. combination of ifn-λ with low doses of ifn-α, which are subtherapeutic but less toxic [ ] , may improve ifn therapy and benefit cancer patients. combinational therapy of ifn-λ and ifnα may achieve ultimate antitumor activity by inducing complementary mechanisms directly on the tumor cells or by indirectly modulating the tumor microenvironment, thereby leading to the stimulation of the immune response against the tumor and the inhibition of tumor angiogenesis. by acting with different intensities on the same targets, ifn-λ and ifn-α may generate a high level of synergy, leading to a potent antitumor activity. similarly to ifn-α, ifn-λ has been shown to play an important role in cancer and viral disease treatment. although the two ifns act through an identical signaling pathway in the cell, the pattern of their activity seems to be different in vivo, implying that ifn-λ and ifn-α are not redundant cytokines. by acting on some targets with different intensities, we believe that ifn-λ and ifn-α act in concert to better control tumor development in vivo. therefore, to achieve better treatments for viral diseases or cancers, we believe that the development of a combination therapy rather than the use of each ifn alone will be more beneficial for the patients. the combination of ifns with other cytokines, growth factors, or their antagonists could also be an important strategy for the improvement of the ifn therapy. transforming growth factor-beta (tgfβ) which plays a dual role in cancer, mediating tumor-suppresive activities at early stages and prooncogenic activities at later stages of tumor progression [ , ] , could represent one potentially important modulator or mediator of the ifn response. understanding the potential crosstalks between ifn-α, ifnλ and other cytokines or growth factors, such as tgfβ, could be rewarding and lead to new preclinical studies in animal models and new clinical trials resulting in better cancer treatments. interferon-lambda: a new addition to an old family ifn-λs mediate antiviral protection through a distinct class ii cytokine receptor complex il- , il- and their class ii cytokine receptor il- r characterization of the mouse ifn-λ ligand-receptor system: ifn-λs exhibit antitumor activity against b melanoma an important role for type iii interferon (ifn-λ/il- ) in tlr-induced antiviral activity lambda interferon (ifn-λ), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo intranasal administration of alpha interferon reduces seasonal influenza a virus morbidity in ferrets interferon-λ contributes to innate immunity of mice against influenza a virus but not against hepatotropic viruses interferon-λs: the modulators of antivirus, antitumor, and immune responses il- and il- : newcomers to the interferon family effect of interferon-lambda on replication of hepatitis b virus in human hepatoma cells hepatitis c virus replication in transfected and serum-infected cultured human fetal hepatocytes interferons α and λ inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics lambda interferon inhibits hepatitis b and c virus replication lambda interferon inhibits human immunodeficiency virus type infection of macrophages interferon lambda inhibits herpes simplex virus type i infection of human astrocytes and neurons biological activity of interleukins- and - : comparison with type i interferons ifnα and ifnλ differ in their antiproliferative effects and duration of jak/stat signaling activity interferons α and β as immune regulators-a new look links between innate and adaptive immunity via type i interferon modulation of the human cytokine response by interferon lambda- (ifn-λ /il- ) modulation of human plasmacytoid dc function by ifn-λ (il- ) ifn-λ (il- ) inhibits gata expression and suppresses th responses in human naive and memory t cells human interferon lambda- (ifn-λ /il- ) modulates the th /th response interferon-λ (interleukin- ) preferentially down-regulates interleukin- over other t helper type cytokine responses in vitro type iii ifn-λ mrna expression in sputum of adult and school-aged asthmatics innate immunity in the pathogenesis of virusinduced asthma exacerbations viral infection and toll-like receptor agonists induce a differential expression of type i and λ interferons in humans plasmacytoid and monocyte-derived dendritic cells interferon-λ-treated dendritic cells specifically induce proliferation of foxp -expressing suppressor t cells comparative ability of il- and il- b to regulate treg populations and enhance adaptive cellular immunity identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays interleukin- uses a type interferon-like program to promote antiviral responses in human hepatocytes interferons and apoptosis the grims: a new interface between cell death regulation and interferon/retinoid induced growth suppression role of the interleukin (il)- receptor tyrosine residues for antiviral and antiproliferative activity of il- /interferon-λ . similarities with type interferon signaling il- a and il- mediate antiproliferative and antiviral signals in intestinal epithelial cells and murine cmv infection increases colonic il- a expression novel interferonλs induce antiproliferative effects in neuroendocrine tumor cells regulation of apoptosis by type iii interferons a role for ifn-λ in multiple myeloma b cell growth ifnlambda (ifn-λ) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo ifn-λ determines the intestinal epithelial antiviral host defense lambda interferon is the predominant interferon induced by influenza a virus infection in vivo temporal and spatial resolution of type i and iii interferon responses in vivo il- a (ifn-λ ) modulates lung dc function to promote th immune skewing and suppress allergic airway disease antitumor activity of type i and type iii interferons in bnl hepatoma model despite ifn-receptor expression, blood immune cells, but not keratinocytes or melanocytes, have an impaired response to type iii interferons: implications for therapeutic applications of these cytokines maturing dendritic cells are an important source of il- and il- that may cooperatively increase the innate immunity of keratinocytes human interferon-λ is a potent member of the type iii interferon family structural linkage between ligand discrimination and receptor activation by type i interferons lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections phase b study of pegylated interferon lambda with or without ribavirin in patients with chronic genotype hepatitis c virus infection interferon lambdas: the next cytokine storm genetic variation in il b predicts hepatitis c treatment-induced viral clearance il (interferon lambda ) gene polymorphisms and response to ifn-alpha treatment in patients infected with hepatitis virus c an il b polymorphism determines treatment response of hepatitis c virus genotype or patients who do not achieve a rapid virologic response replicated association between an il b gene variant and a sustained response to pegylated interferon and ribavirin interleukin- b genetic variants and hepatitis virus infection by different viral genotypes large-scale candidate gene analysis of spontaneous clearance of hepatitis c virus genetic variation in il b is associated with chronic hepatitis c and treatment failure: a genome-wide association study il b is associated with response to chronic hepatitis c interferon-α and ribavirin therapy genome-wide association of il b with response to pegylated interferon-α and ribavirin therapy for chronic hepatitis c ifn therapy in tib hcc model: combination of ifn-lambda and ifn-alpha induces complete remission high-dose interferon alfa- b does not diminish antibody response to gm vaccination in patients with resected melanoma: results of the multicenter eastern cooperative oncology group phase ii trial e interferon alfa- b adjuvant therapy of high-risk resected cutaneous melanoma: the eastern cooperative oncology group trial est interferons in the treatment of solid tumors effect of long-term adjuvant therapy with interferon alpha- a in patients with regional node metastases from cutaneous melanoma: a randomised trial final results of the eortc /dkg - randomised phase iii trial: rifn-α b versus rifn-γ versus iscador m versus observation after surgery in melanoma patients with either high-risk primary (thickness > mm) or regional lymph node metastasis antitumor activity of ifn-λ in murine tumor models il- elicits antitumor responses against murine fibrosarcoma hepatocellular carcinoma pathogenesis: from genes to environment hepatocellular carcinoma: current management and recent advances hepatocellular carcinoma: epidemiology, risk factors, and screening liver transplantation for hepatocellular carcinoma predicting 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interferon/ribavirin reduces hepatocellular carcinoma and improves survival in chronic hepatitis c: a nationwide, multicentre study in taiwan randomized controlled trial of interferon treatment for advanced hepatocellular carcinoma a randomized, controlled trial of postoperative adjuvant interferon therapy after resection of hepatocellular carcinoma combination therapy with s- and pegylated interferon alpha for advanced hepatocellular carcinoma inhibition of angiogenesis by interfersons: effects on tumor-and lymphocyte-induced vascular responses inhibition of basic fibroblast growth factor expression, angiogenesis, and growth of human bladder carcinoma in mice by systemic interferon-α administration ifn-γ regulation of the type iv class ii transactivator promoter in astrocytes antitumour effects of interferons: past, present and future interaction of ifn α/β with host cells essential to the early inhibition of friend erythroleukemia visceral metastases in mice successful immunotherapy of the highly metastatic murine esb lymphoma with sensitized cd + t cells and ifn-α/β host cd + t lymphocytes are required for the synergistic action of interferonα/β and adoptively transferred immune cells in the inhibition of visceral esb metastases transforming growth factor-β in cutaneous melanoma resistance to transforming growth factor β-mediated tumor suppression in melanoma: are multiple mechanisms in place the authors thank dr. sergei kotenko and dr. andrew de la torre for their helpful discussions. key: cord- -ousbar c authors: horman, william s. j.; nguyen, thi h. o.; kedzierska, katherine; butler, jeffrey; shan, songhua; layton, rachel; bingham, john; payne, jean; bean, andrew g. d.; layton, daniel s. title: the dynamics of the ferret immune response during h n influenza virus infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ousbar c as the recent outbreak of sars-cov- has highlighted, the threat of a pandemic event from zoonotic viruses, such as the deadly influenza a/h n virus subtype, continues to be a major global health concern. h n virus strains appear to exhibit greater disease severity in mammalian hosts compared to natural avian hosts, though the exact mechanisms underlying this are somewhat unclear. knowledge of the h n host-pathogen interactions have mainly been constrained to natural sporadic human infections. to elucidate the cellular immune mechanisms associated with disease severity and progression, we used a ferret model to closely resemble disease outcomes in humans following influenza virus infection. intriguingly, we observed variable disease outcomes when ferrets were inoculated with the a/anhui/ / (h n ) strain. we observed relatively reduced antigen-presenting cell activation in lymphoid tissues which may be correlative with increased disease severity. additionally, depletions in cd (+) t cells were not apparent in sick animals. this study provides further insight into the ways that lymphocytes maturate and traffic in response to h n infection in the ferret model. in recent years cases of zoonotic strains of avian influenza (ai) causing severe disease in humans have caused significant global concern, with fears that these viruses may lead to devastating pandemic events in future ( , ) . one such group of viruses are strains of h n influenza virus, which have caused over , cases of infection (with a ∼ % mortality rate) in humans since their first detection in ( , ) . of most concern with the h n viruses has been the ability for this virus to present as a low pathogenicity virus in its native avian hosts and yet present with severe clinical symptoms and death in humans, without obtaining virulence factors such as multi-basic cleavage sites usually required for high pathogenicity infections. deciphering the immune cellular mechanisms associated with disease severity progression will provide a better understanding as to why h n infections can produce severe disease in humans. changes in leukocyte subsets have previously been shown to correlate with more severe outcomes during ai infection, with decreases in t cell populations commonly reported in both human (h n virus) and avian (h n virus) hosts ( , ) . decreases in t lymphocytes are often accompanied by upregulation of several pro-inflammatory cytokines such as interferons (ifn), most notably ifn-γ, as well as interleukins (il) such as il- ( ). overproduction of cytokines or hypercytokinemia has also been identified as a key contributing factor in severe ai pathogenesis in both chickens and macaques, where induction of pro-inflammatory cytokines is associated with cellular apoptosis and tissue damage ( , ) . furthermore, macrophages have been shown to be predominantly pro-inflammatory responders to h n strains in a mouse model ( ) , though highly pathogenic strains such as h n and h n display attenuated macrophage inflammation responses compared to seasonal strains such as h n and h n ( , ) . while hypercytokinemia is common amongst many ai virus infections, h n strains such as the human infecting a/anhui/ / virus have been associated with dampened ifn responses in humans ( , ) . furthermore, they have previously been associated with an attenuated humoral immune response in the mouse model ( ) . these studies demonstrate the unpredictable nature and wide spectrum of pathogenicity of ai viruses. investigations into the pathogenesis and transmission of many human-infecting influenza viruses have been conducted in ferrets, as the clinical presentation in these animals is considered a more robust representation of human illness when compared to mice ( ) . however, only a limited number of studies exist looking at influenza-specific immunity in ferrets, which have primarily focused on seasonal influenza strains ( ) ( ) ( ) . in this study, we aimed to examine the ferret immune response to h n influenza virus infection by analyzing leukocyte population variation associated with disease pathogenesis. this study found the ferret model may allow for increased knowledge of the outcomes of h n infections and help in boosting our understanding of both this model and of these viruses in readiness for potential future outbreaks. all procedures described here were reviewed and approved by the commonwealth scientific and industrial research organization (csiro), australian center for disease prevention (acdp) animal ethics committee (aec# ) and were performed in accordance with the australian code for the care and use of animals for scientific research ( th edition ). influenza virus a/anhui/ / (h n ) used in this study was propagated by allantoic cavity inoculation of - -days of embryogenesis specific-pathogen-free (spf) embryonated chicken eggs. the virus stock was titrated in chicken eggs and the % egg infectious dose (eid ) /ml was calculated according to reed and muench ( ) . all in vitro and in vivo work involving live virus was conducted within biosafety level three facilities at acdp. animal work was performed using personal protective equipment and powered air purifying respirators. fitch ferrets that were ∼ months of age (csiro werribee animal facility) and serologically negative by hemagglutination inhibition (hi) assay for h n were used in this study. prior to initiation of the study, all ferrets were free from signs of clinical disease. body temperatures were measured using an implantable subcutaneous microchip (destron fearing, delray beach, fl, usa). baseline body weights and temperatures were obtained for consecutive days before challenge (i.e., day − , − , and − ) and on the day just prior to the challenge (day ). six ferrets were infected intranasally with × eid of virus diluted in . ml of sterile pbs, and four ferrets were mockinfected with an equivalent dilution of allantoic fluid collected from spf chicken eggs in sterile pbs as non-infected controls. following viral challenge, ferrets were monitored daily for body weight, temperature, and clinical signs of illness (including sneezing, lethargy, nasal discharge, diarrhea, and neurological dysfunction) for the duration of the study. blood samples were collected every second day. animals were anesthetized with ketamine/xylazine, and blood samples of - µl were taken from the jugular or axillary vein on days , , , and from the heart at the time of euthanasia for the terminal bleed. for virus titration, nasal washes were collected on days , , , and upon euthanasia, as described previously ( , ) . all ferrets were euthanised on day (study endpoint) or earlier due to ethical endpoints (≥ % weight loss or escalation of clinical signs). histological analysis of ferret tissues following infection was performed as previously described ( ) . tissues were fixed in % neutral-buffered formalin for at least h, processed into paraffin wax, cut and stained using haematoxylin and eosin for examination for histopathological lesions. consecutive tissue sections were stained in an immunohistochemistry (ihc) test for influenza a virus nucleoprotein ( ) . lung, spleen, and mediastinal lymph nodes were harvested and processed, as previously described ( ) . briefly, lung samples were manually minced using a scalpel followed by enzymatic digestion ( u/ml collagenase i and u/ml dnase i) while single-cell suspensions of spleen and lymph node samples were prepared by passing the tissue though a µm strainer. peripheral blood mononuclear cells were isolated by hypotonic lysis of red blood cells using erythrocyte lysing solution ( . m nh cl, mm khco , and mm edta ph . ). viral titers in tissues were measured on mdck cells by standard tcid assay. relative expression of ferret immune genes was assessed using a steponeplus tm real-time pcr system and the comparative threshold cycle (ct) method according to manufacturer's instructions (applied biosystems, foster city, ca, usa). relative gene expression was calculated using mean values obtained from ct relative to the housekeeper gene (gapdh), with each ferret compared to the average of the control ferrets for each gene. primers for ferret cytokines, as well as relative gene expression calculations, were obtained from carolan et al. ( ) . cells processed from ferret tissues were stained with anti-cd (alexafluor , csiro acdp sourced from wehi ( ) , geelong, vic, australia), anti-cd (pe, clone okt , ebioscience, ca, usa), anti-gl (alexafluor , clone gl , bd phamingen, san diego, ca, usa), anti-mhc-ii (biotin, clone cat a, kingfisher, saint paul, mn, usa), and anti-cd b (alexafluor , clone m / , bd pharmingen, san diego, ca, usa). cells were stained for h at • c, washed in facs buffer (pbs, % fcs, . % sodium azide) and analyzed using the lsr ii (becton-dickinson, franklin lakes, nj, usa). flow cytometry data were analyzed using flowlogic software (version . . , inivai technologies, mentone, vic, australia). to assess antibody responses, serum was collected prior to infection and at the point of euthanasia. haemagglutinin inhibition assays were performed on rde treated sera by using homologous antigen (influenza a/anhui/ / ) as per the standard method. hi titers were expressed as the reciprocal of the highest serum dilution causing complete inhibition of hemagglutination. ferret lung and spleen cells were pelleted by centrifugation at , × g max for min and washed in pbs. cells ( . × / well) were cultured with or without live h n virus for h at • c/ % co . the ferret ifn-γ elisa development kit (alp) (mabtech, stockholm, sweden) was used to determine the quantity of ifnγ secreted by cells ex vivo at endpoint and after restimulation with live h n virus (moi . ). elisa plates were measured using a multiskan ascent plate reader with ascent software version . (thermofisher, waltham, ma, usa). ifn-γ-producing cells were detected using a ferret ifn-γ elispot assay, as per the manufacturer's instructions (mabtech, stockholm, sweden). for analyzing the infected ferrets compared to the non-infected controls, all six ferrets were grouped regardless of timepoint to give an overall view of the infection time course. for these analyses, student t-tests were conducted between the two groups. time course data was analyzed by -way anova. to assess the impact of h n virus in ferrets, viral pathogenicity was firstly assessed based on observation of clinical signs of infected ferrets throughout the -day study (figure ) . from the six h n -infected ferrets, three ferrets showed little to no clinical signs and survived until the study endpoint (day ). of the remaining ferrets, two showed mild clinical signs but severe weight loss, leading to euthanasia at day post-infection. one ferret, however, showed moderate to severe clinical signs consistent with those seen in highly pathogenic avian influenza infections ( ) , and was euthanised at day due to escalation of these clinical signs ( figure a ). this ferret's increase in clinical signs was identified by a play score of two, with play scores > signifying moderate-to-severe clinical signs ( figure b ). significant weight losses (> % compared to baseline, p < . ) were observed in all the infected ferrets when compared to the control ferrets from day until the study endpoint ( figure c) . similarly, infected ferrets also showed a significant increase (p < . ) in body temperature at day post-infection, with an average temperature of . ± . • c compared to the controls at . ± . • c ( figure d) . consequently, h n infection in ferrets resulted in variable clinical symptoms, but overall body weight losses and heightened body temperatures at h postinfection, which are within the typical timeframe for onset of influenza illness in humans. to further examine the clinical progression between the infected ferrets, viral titres from the nasal washes were determined to assess whether differences in viral replication or clearance were correlative with worsened disease progression. titres > . log tcid /ml were obtained for all infected ferrets at day postinfection and virus was still detected in all ferrets at day postinfection (> . log tcid /ml, figure a ). one ferret had cleared the virus by day post-infection, while the other two ferrets that survived until the end of the study showed viral clearance at day (figure a ). neither the day nor the day ferrets showed viral clearance by the point of euthanasia. only one ferret euthanised on day showed live virus in the lung (> . log /ml, figure b ). additionally, antibody titres were measured in the ferret sera, with animals euthanized on day showing the highest hi titres (all > ) and the ferret euthanized on day showed a hi titer > . the two ferrets euthanised at day had no detectable hi titer ( figure c ). infected ferrets showed a variety of pathological outcomes following viral challenge. challenged ferrets exhibited epithelial metaplasia in the nasal turbinates (figure a) , with ferrets euthanised at the earlier day time point exhibiting viral infection of the nasal epithelium (figure b) . the day ferret showed the most severe lung pathology, with the lungs of this ferret showing diffuse interstitial pneumonia, with severe alveolar oedema and inflammatory cell infiltration in the alveolar spaces and around the blood vessels (figure c) . other infected ferrets showed broncho-interstitial pneumonia and interstitial inflammation (figure d ) and bronchitis, with infected epithelial cells and early stage lesions observed in one of the day ferrets (figure e) . bronchial adenitis was also present in some of the infected ferrets, with necrosis of the bronchial glands observed along with viral antigen (figure f) . these changes in pathology contrast to what is seen in the healthy nasal turbinates (figure g) , and lung (figure h ) of uninfected ferrets where no lesions were present. the number of localized lesions and qualitative assessment of epithelial metaplasia was also recorded to support the histopathological findings (supplementary figure ) . here, we observed epithelial metaplasia in the turbinates of all ferrets sampled, as well as several occurrences of bronchio-interstitial pneumonia and bronchio-adenitis. we also observed viral antigen in a number of tissues sampled (supplementary figure ) , though interestingly, viral antigen was not observed in the diffuse interstitial pneumonia of lung lesions associated with the ferret euthanized due to clinical disease. changes in certain pro-inflammatory cytokines (including il- , tnfα, and ifnγ) have been associated with h n disease severity in humans and animal models ( ). we measured levels of mrna transcripts of several pro-inflammatory cytokines commonly associated with influenza infections. in the blood there were few differences between the groups, with mcp the only tested cytokine showing an average fold increase > fold compared to the controls at day post-infection (data not shown). however, intriguingly the ferret euthanised on day showed a large decrease in il- ( figure a , -fold) and ifn-γ (figure b , -fold) transcript levels on day . this also coincided with a lack of detectible ifn-γ in terminal serum by elisa, though this trend was consistent across all tested ferrets (data not shown). in the spleen at endpoint, there appeared to be variable outcomes for the day ferrets, with somewhat increased pro-inflammatory cytokine levels such as type i ifns (ifn-α and ifn-β) in the day ferrets, and decreased levels in the day ferret compared to the controls (figure c) . in the lung, tnfα was significantly reduced (figure d , p < . ) in the infected animals, though no other consistent trends were seen. the ability to produce ifn-γ ex vivo or after restimulation with live h n virus was also assessed in spleen ( figure e ) and lung cells (figure f ). there were no significant differences except for in the infected lungs, where ifn-γ-producing cells were significantly higher compared to the control ferrets ex vivo (p = . ). based on these findings, our ferret infection model showed observable changes in the cytokine profiles at a key site of infection in the lung compared to non-infected controls. depletions in t cell numbers in the spleen and lungs have previously been reported for highly pathogenic avian strains h n and h n in both chickens and mice ( , ) . similarly, patients that died from h n infection were found to have lower t cell numbers in the blood compared to those that survived ( ) . therefore, in order to assess lymphocyte involvement in disease progression we investigated the cellular responses associated with h n -infection both in the blood at several timepoints, and in the tissues at endpoint. here, we found that in the blood the numbers of cd + and cd + t cells in infected ferrets were generally lower than the control ferrets, particularly for the cd + t cell population at day (p < . ). nevertheless, these t cell populations remained low but relatively stable over the days of infection ( figure a) . the gl monoclonal antibody can be used as a marker activated t lymphocytes. however, gl expression was absent on the t cell subsets examined. interestingly we observed significantly higher numbers of cd + t cells in the spleen (p = . ) and in the lungs (p = . ) of infected ferrets, and a trend toward higher numbers of cd + t cells in the lungs (figures b,c) , indicating a t cell response to h n infection at a key site of infection. antigen presenting cells (apcs), such as macrophages residing in the lungs, have previously been recognized as key proinflammatory responders during h n , h n , and h n infections in mice ( ) . cells expressing higher levels of cd b + have been shown to directly kill virally-infected cells in humans, and act as important antigen-presenting cells by upregulating mhc class ii expression in the ferret model correlating to reduced pathology associated with influenza infection ( , ) . in general, h n -infected ferrets showed higher levels of cd b/mhc-ii dual-expressing cells (cd b + mhc-ii + ) in the spleen, lung and lymph nodes compared to the control ferrets (figures a-c) . at the site of infection in the lungs, infected ferrets showed significantly higher levels of mhc-ii single-positive cells (mhc-ii + , p = . ). conversely, cd b single-positive cells (cd b + ) from infected ferrets were found at elevated levels in the spleen and lymph nodes. individually, we did observe that the ferrets euthanised on days and had higher cd b + cells in the spleen, suggesting a less activated and immature phenotype, and a trend toward lower cd b + mhc-ii + cells in the lymph nodes when compared to the other ferrets. when analyzed by time point, there are statistically greater proportions of these cells in the ferrets that survived until trial endpoint compared to the controls (p = . ) and the earlier timepoint ferrets (p = . , figure d ). as such, as these markers are often attributed to antigen-presenting cell maturation in the mouse model ( ) , apc activation in response to h n infection may be associated with disease severity seen in the ferret model. the recent sars-cov- outbreak has brought a sharp focus onto zoonotic viruses causing severe global pandemics. while sustained human-to-human transmission of avian-origin h n viruses has yet to occur, if a zoonotic outbreak of these highconsequence viruses were to occur, the results could be a pandemic much like the sars-cov- outbreak but with a virus which has potential for much higher mortality rate. as such, avian influenza viruses continue to present as a major global health concern, with one of the key concerns being what makes these viruses cause such severe consequences in mammalian hosts. influenza-related symptoms of ferrets more closely resemble human clinical symptoms than that of mice ( , ) , with outbred ferret populations giving a more accurate representation of the genetic variability seen in human populations compared to clonally inbred mouse colonies. however, husbandry requirements and a general lack of reagents for ferrets limits the scope of experimental research using these animals to date. these issues are exacerbated by biosafety level (bsl ) requirements for pathogens such as h n , which make immunological experiments in ferrets difficult to undertake. as a result, ferret cellular immunology is still a developing field of research in the context of influenza viruses, with only a handful of studies investigating changes in leukocyte populations following influenza virus infection ( ) ( ) ( ) . for avian-origin influenza viruses such as h n , little work has been done to investigate how cellular subsets are affected by viral infection, with the bulk of experiments using ai in the ferret model focusing on classifying viruses through clinical outcome-based pathogenesis and transmission studies ( ) ( ) ( ) . thus, our study aimed to give new insight into how h n affects mammalian hosts in the ferret model with a focus on cellular subsets. the ferrets in our viral challenge presented clinical outcomes with different severities. five of the six ferrets showed little to no clinical signs, nevertheless only three survived until study endpoint without complications. two ferrets reached the ethical weight loss cut-off of ≥ % by day post-infection, giving us two different time points to assess the immune response. furthermore, one ferret showed a quite different disease outcome, in which the ferret presented an escalation of clinical signs and was euthanised for ethical reasons at day post-infection. other studies have previously shown clinical variation between strains of h n ( ) , however most studies with a /anhui/ / (h n ) show ferrets presenting only mild clinical signs ( , , ) . to our knowledge, this is the first reported result showing variable disease outcome in an animal study using a single low pathogenicity h n strain, in which one ferret showed severe clinical outcomes. while the findings of this study are novel for this model, we were limited by the number of animals that could be used in this study and thus limited by our statistical power. furthermore, there are limitations in directly comparing ferrets euthanised on different days as, particularly for our serological data, it is difficult to discern whether the results are a function of observed severity or differences in the sampling time. as such, we believe the results from our observational study can provide a rationale for developing future ferret studies with greater numbers. it is also worth noting that ferrets used for these experiments are outbred animals, which may suggest that other combinations of host factors may be contributing to the variability seen in clinical presentation. the airway pathology in these infected ferrets was variable, with different degrees of severity observed in the six animals. these findings were generally consistent with pathology observed in other studies in which ferrets were infected with lpai h n or pdm (h n ) viruses ( , , , ( ) ( ) ( ) . while the day ferret which showed worsened disease progression did exhibit the most severe lung pathology, the lack of viral antigen in and around these lesions (supplementary figure ) means they cannot conclusively be classified as being caused by the influenza infection, and thus we are hesitant to classify disease presentation in this ferret as like a high pathogenicity infection based on the histopathology findings regardless of the increase in clinical signs. hypercytokinemia has frequently been associated with worsened disease progression in cases of severe ai infections, therefore we aimed to assess the induction of pro-inflammatory cytokines in this ferret model of infection. upregulation of proinflammatory cytokines such as tnf-α and il- commonly observed following h n infection in both cell culture ( , ) and in severe human cases, which follow similar patterns of pathogenesis to infections with hpai h n viruses ( , , ) . whilst the localized pro-inflammatory cytokine response to influenza infection has been characterized for circulating influenza strains in ferrets ( ) , examination into these responses for avian influenza viruses remains limited. our study found limited induction of cytokine responses at the site of infection in the lungs and in lymphoid tissues, and while attenuated ifn responses have been previously reported in human bronchial epithelial cells infected with h n ( ), and in ferrets severely infected with seasonal influenza strains ( ) , the overall lack of cytokine induction was an unexpected finding. a limitation of this study was that these tissues could only be sampled at study endpoint, which may suggest the pro-inflammatory response had abated at the site of infection by the time of sampling given that these responses typically occur in short timeframes of - h post-infection ( ) , though a lack of an evident cytokine response in the day ferret still presenting clinical signs and shedding live virus was surprising. furthermore, levels of ifn-γ were below the threshold of detection by elisa in the serum of infected ferrets, and a previous study has shown ifn-γ detectable in the lungs of seasonal influenza-infected ferrets from day post-infection at low levels, with greater detection observed from days - ( ) . these results suggest both timing and sampling may be critical for future studies to best capture the overall ifn-γ response to h n , if there is such a response occurring. sampling in the blood of infected ferrets over the course of the study also found no large-scale upregulation of the cytokines tested, with only the early innate cytokine mcp showing an average fold increase > fold compared to the controls at day post-infection. however, in the day ferret, decreases in both ifn-γ ( -fold) and il- ( -fold) where observed at day post-infection, coinciding with an escalation in clinical signs in this animal. the decrease in il- in particular was unexpected, as previous studies have implicated higher levels of il- correlating to worsened disease progression ( ) . our findings here suggest an immune dysregulation, rather than an over-activation, for worsened disease progression, and may be attributed to other factors in the cellular response. immunological work to determine the innate and adaptive responses to these viruses has mostly been conducted from infected patients or in the mouse model. human cases are often marked by leukopenia, though these measurements are routinely made via hematology rather than flow cytometric analysis ( , ) . in other species however, we have previously described the lymphopenia for highly pathogenic strains of avian influenza, measuring splenic cd + t cells following infection with highly pathogenic h n in chickens via facs analysis ( ) . in this study, cd + t cells levels were relatively stable. a loss of lymphocytes detected via hematological analysis at day (data not shown) was reconciled with our facs data showing a significant difference between cd + t cells in the blood, in which control animals had much higher levels compared to the infected animals. however, this loss was short-lived, as no further significant differences were observed across the study time points in the blood. this data is suggestive of a potential early "transient lymphopenia" of circulating leukocytes, which has previously been identified in one study during influenza infection in ferrets, and is also a phenomenon seen in humans following influenza a infection ( , ) . our data appears to show cd + t cells levels in the blood of infected animals remaining relatively and consistently low across time points, whilst greater variation was observed in the uninfected control levels. furthermore, reagents for facs analysis in the ferret are still relatively novel, often not well-characterized, and heavily reliant on cross-reactivity from other species. while cd + and cd + t cells have previously been examined in influenzainfected ferrets ( ) , little work has been conducted for innate cell subsets. myeloid-derived and antigen-presenting cells (apcs) have only been identified and analyzed in a handful of ferret studies, with one such study speculating that cd b + cells are likely granulocytes such as neutrophils ( , ) . as such, we have used these studies, together with mouse and human studies ( ) ( ) ( ) ( ) , as a basis for possible classification of apcs, with cd b + mhc-ii + cells suggested as likely "mature" or "activated" apcs. however, further work is needed to classify these innate cell subsets in the ferret model. apcs such as alveolar macrophages have been recognized as key early responders against influenza virus infection, as they mount robust pro-inflammatory cytokine responses within h of infection ( ) . while our study found no significant differences in apc numbers in infected lungs compared to control numbers, there was a trend toward much greater levels of mhc-ii + apcs in the lung, especially in our cell counts. rather, it was in the peripheral tissues that we found variability in the apc subsets. cd b + mhc-ii + "mature" cells were found in the spleens of infected ferrets at significantly higher levels (p = . ), however the trend toward higher cd b + cells in the earlier time point ferrets suggests a higher level of immature myeloid cells, or the presence of pro-inflammatory granulocytes; however, the latter is less likely due given the lack of pro-inflammatory cytokines detected in the spleen. moreover, in the draining lymph node a significant increase in cd b + cells (p = . ) was observed. interestingly, in the lymph node the day seven ferrets showed noticeably higher levels of cd b + mhc-ii + "activated" cells, with statistically greater proportions of these cells compared to the controls (p = . ) and the earlier timepoint ferrets (p = . ), which may suggest that these apc responses do not occur as early as day . this study found increased pathology in the ferret model diverges from the commonly observed pathogenesis markers such as lymphopenia and hypercytokinemia, suggesting another varied pathway that h n viruses can cause severe disease in mammalian hosts. further investigation into the ferret model may allow for better characterization of these outcomes and assist in increasing our knowledge of these viruses in preparation for any potential pandemic events. all datasets generated for this study are included in the article/supplementary material. the animal study was reviewed and approved by australian animal health laboratories (aahl) animal ethics committee, csiro. characterization of h n influenza a viruses isolated from humans the drivers of pathology in zoonotic avian influenza: the interplay between 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and hypercytokinemia characterization of the localized immune response in the respiratory tract of ferrets following infection with influenza a and b viruses severe seasonal influenza in ferrets correlates with reduced interferon and increased il- induction timing and magnitude of type i interferon responses by distinct sensors impact cd t cell exhaustion and chronic viral infection human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome comparison of patients hospitalized with influenza a subtypes h n , h n , and pandemic h n avian influenza virus a h n infects multiple mononuclear cell types in peripheral blood and induces dysregulated cytokine responses and apoptosis in infected monocytes identification of myeloid cell subsets in murine lungs using flow cytometry distinct macrophage subpopulations characterize acute infection and chronic inflammatory lung disease all authors agreed on the final draft of the manuscript prior to submission. wh wrote the first draft and revised the manuscript. tn, kk, jbu, ss, jbi, jp, ab, and dl reviewed and edited the manuscript. wh was supported by a joint csiro/university of melbourne onehealth scholarship. this project was supported by csiro strategic funding. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © horman, nguyen, kedzierska, butler, shan, layton, bingham, payne, bean and layton. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -apdyaujt authors: coulter-mackie, marion; adler, richard; wilson, greame; dales, samuel title: in vivo and in vitro models of demyelinating diseases xii. persistence and expression of corona jhm vims functions in rn - schwannoma cells during latency date: - - journal: virus research doi: . / - ( ) - sha: doc_id: cord_uid: apdyaujt abstract the coronavirus jhmv persistently infects rat schwannoma cells rn - at . °c and enters a host-imposed reversible, latent state at . °c. jhmv can remain up to days in the latent state and about days before the cultures lose the capacity to resume virus production upon return to . °c. although persistently and latently infected rn - cells display resistance to superinfection by a heterologous agent vsv, these cells do not release detectable soluble mediators (e.g., interferon) of the antiviral state. nevertheless, rn - cells are competent to synthesize and release interferon when treated with the appropriate inducers. these observations suggest that interferon does not play any role or may not be the major factor in the control of latency in the schwannoma cell. hybridization with virus-specific cdnas shows that all viral mrnas are present during latency and that viral mrnas are present in the polysomes of infected cells at . °c. western immunoblotting with hybridoma antibodies demonstrates that viral specific proteins are produced at the restrictive temperature. these results the coronavirus jhmv persistently infects rat schwannoma cells rn - at °c and enters a host-imposed reversible, latent state at °c. jhmv can remain up to days in the latent state and about days before the cultures lose the capacity to resume virus production upon return to °c. although persistently and latently infected rn - cells display resistance to superinfection by a heterologous agent vsv, these cells do not release detectable soluble mediators (e.g., interferon) of the antiviral state. nevertheless, rn - cells are competent to synthesize and release interferon when treated with the appropriate inducers. these observations suggest that interferon does not play any role or may not be the major factor in the control of latency in the schwannoma cell. hybridization with virus-specific cdnas shows that all viral mrnas are present during latency and that viral mrnas are present in the polysomes of infected cells at . "c. western immunoblotting with hybridoma antibodies demonstrates that viral specific proteins are produced at the restrictive temperature. these results suggest that despite the absence of production of infectious virus at s"c, there is active transcription and translation into virus-specified products. latent persistent infection, coronavirus, rat, schwannoma cell viruses of many types are associated with the etiology of neurological diseases in animals and man. among the rna agents the coronaviruses have sparked considerable interest in connection with demyelinating diseases of the cns. the murine agents of the mouse hepatitis virus (mhv) group, constituting several serotypes, have in particular been studied extensively in persistent infections in vitro (holmes and behnke, ; stohlman and weiner, ; lucas et al., ) and in vivo (knobler et al., ; hirano et al., ; nagashima et al., ; sorensen et al., sorensen et al., , sorensen et al., , . the epidemiological surveys which revealed that in some countries essentially % of the human population is seropositive (hovi et al.. ; hasony and macnaughton, ; macnaughton, ) attest to the ubiquity of these agents. in various studies we have described a model involving the persistent infection of rn - rat schwannoma cells by the neurotropic coronavirus jhmv (lucas et al., (lucas et al., , . with this agent, virus production, measured by the quantity of extracellular infectious particles, occurs in a cyclical fashion when cultures are maintained at . "c. elevation of temperature to a restrictive . "c interrupts jhmv production. however, infectious particle formation may be resumed when the cultures are returned to the permissive . "c. in these cell-virus systems the control on replication, related to alterations in the temperature, appears to be imposed by the host cell because thermosensitivity of either virus is insufficient to account for the observed temperature-related restriction of virus production, termed hereafter latency. on the assumption that during prolonged restriction imposed by the host rn - cell, latent jhmv are somehow perpetuated, we focussed our attention on the nature of the incomplete virus expression, particularly the detection of viral genomes, mrna and proteins. data from studies presented here reveal that both viral mrna and protein are expressed during latency. the source and characterization of the rat schwannoma cell line, rn - , and mouse l- cells has been previously reported (lucas et al., (lucas et al., , . primary rat embryo cells (re) were derived from -day embryos of the wistar rat. cells were released from tissues by mincing and dispersal into phosphate-buffered saline, containing mm glucose and . % trypsin, employing agitation at °c for min. the monodisperse re cells and continuous lines were grown in nutrient medium (nm) consisting of eagle's minimal essential medium supplemented with or % fetal bovine serum (fbs). the origin and propagation methodology for jhmv has been published (lucas et al., ) . virus titres, expressed as plaque-forming units per millilitre (pfd/ml) were measured by assay on l- cells for jhmv, according to lucas et al. ( ) . in order to determine the fraction of cells yielding pfu in the persistently or latently infected rn - cultures, the monolayers were trypsinized, free cells counted and then plated at -fold serial dilutions onto monolayers of the indicator l- cells, in -well microtitre plates. the co-cultures were incubated at . "c for - days, then fixed with formaldehyde and stained with crystal violet. the minimum number of test cells added that were required to cause a cytopathic effect (cpe), i.e. the end point, became a measure of the frequency of infectious centers in the population of the culture. the initiation of persistent or latent infections by jhmv in rn - cells has been described (lucas et al., ) . inocula are millipore-filtered before adding to cells (coulter-mackie et al., ) . interference with vesicular stomatitis virus (vsv) production by jhmv was tested in persistently or latently infected rn- cells using as the controls, uninfected rn - cells. for this purpose test monolayers, in mm dishes, were inoculated with lo pfu of vsv, incubated for h, then fixed and stained for plaque counting. the efficacy of induction of an antiviral state was measured as interference with vsv replication. to produce an antiviral state, some uninfected cells were pre-incubated with nm containing o- pg/ml poly(i) : poly(c) (sigma) for h. the medium was removed, cells were washed and then infected with vsv, as above. specifically the sampled medium was adjusted to ph with n hcl, kept for h at °c then neutralized and assayed for content of interferon. two-fold serial dilutions of the test medium were added to rn - or re cells in -well microtitre plates. after h exposure at °c the medium was removed, cells were washed with pbs, then challenged with vsv. seven cm culture plates (gibco) of rn - cells were infected with jhmv at a m.o.i. of . five plates were maintained at . "c and two at . "c. at h intervals titrations were made for virus production. cultures sampled at intervals were extracted for total rna. using guanidine-hcl (strohman et al., ) . purified rnas were denatured with glyoxal and electrophoresed on agarose gels (mcmaster and carmichael. ) six -cm culture flasks of rn - cells were infected with millipore-filtered jhmv. three of these were maintained at . "c and the other three continuously at . "c. days post-infection (pi) cells at . "c were yielding virus at ca. ' pfu/ml and those at . "c at pfu/ml. ['h]uridine ( ci/mmol, new england nuclear) was added to a concentration of yci/ml and cells were incubated a further h. cell extracts containing polyribosomes (polysomes) were prepared according to gielkens et al. ( ) . cells which had been treated with pg/ml cycloheximide for min were trypsinized, then lysed in np . the extract was layered on % . %' isokinetic gradients and centrifuged lo x g for min. then the gradient was divided into aliquots and the labelled rna measured in a scintillation counter. the polysome-rich fractions were pooled, concentrated by ethanol precipitation and the pellets formed were taken up in a small volume of water. in preparation for dot-blotting analysis (white and bancroft. ) the solution was made % with respect to formaldehyde and % with respect to ssc giving a total volume of . ml. samples were heated min at °c and then applied to nitrocellulose (biorad). using a schleicher and schuell 'minifold' filtration apparatus. nitrocellulose blots were hybridized with [j p]cdna (jhmv), washed and subjected to autoradiography. preparation of the cdna and hybridization conditions have all been described (sorensen et al., ; coulter-mackie et al., ) . extracts of both uninfected and infected rna- cell cultures were prepared by washing twice with ice-cold phosphate-buffered saline (pbs), followed by solubilization for min at °c in lysis buffer: . m tris-hcl, ph . / % glycerol/ % -mercaptoethanol/ % sodium dodecyl sulfate (sds). proteins were separated by sds-polyacrylamide gel electrophoresis (sds-page) in . % acrylamide gels (laemmli, ) . replicas were prepared by electrophoretic transfer for h at v/cm onto . pm nitrocellulose filters (schleicher and schuell). the replicas were cut into strips, placed in pbs + . % tween for h at °c and incubated for h at °c with murine monoclonal antibodies (kindly supplied by dr. michael buchmeier, scripps clinic and research foundation, la jolla), according to towbin et al. ( ) . the blots were washed six times in pbs-tween , and rabbit anti-mouse kappa chains (miles laboratories) were added to serve as links between jhmv polypeptides and protein a. subsequently the strips were washed six times in pbs-tween , reacted for h at °c with ' -labelled protein a, specific activity pci/pg (new england nuclear) and finally washed six times in pbs-tween , dried, and exposed to x-ray film to detect radioactive bands by autoradiography. the phenomenon of coronavirus persistence at . "c and profound suppression of virus production at . "c in rn - cells, and other continuous rat cell lines, is quite striking (lucas et al., ; ) . similar cell-virus interactions were shown, more recently, to occur in primary rat neural (sorensen et al., ; beushausen and dales, ) and embryonic re cells (m. coulter-mackie, unpublished), indicating that in appropriately selected cells, temperature restriction on virus replication is under host control. to ascertain the extent to which jhmv can be maintained latently at . "c, a long-term study of events prevailing at the elevated temperature was undertaken. at intervals of - days, prior to change of nm, the extracellular virus titre was determined. at weekly intervals cells in monolayers were released by trypsinization, transferred into new culture flasks and perpetuated at . "c. the fraction of cells which could function as virus yielders was determined by an infectious centre assay. the duration for which the latently infected cells maintained capacity to resume virus production at . "c was also determined. resumption of virus production after temperature shift down from . "c was usually detectable within days at . "c. under coculture conditions with indicator l- cells, cytopathic effects (cpe) due to jhmv became evident within - days after return to . "c. the results of the infectious centre assays, summarized in fig. , revealed that there was a progressive decrease, with time, in the fraction of infectious centres. rn - cultures persistently infected with jhmv at . "c usually contained o.l- % virus yielding cells during the course of a well-established infection. since upon infection of rn - cultures with jhmv at . "c and maintenance in a state of latency at that temperature for several days, the cells commenced yielding pfu upon shift down to . "c (lucas et al., ) , these data suggest that penetration and eclipse of the virus inoculum must have occurred normally at . "c and restriction most probably developed at a subsequent stage. of jhmv in rn - cells. infectious centres assays, as described in materials and methods, were carried out at weekly intervals on samples from cultures at s"c ( ) or s"c ( ). the data, plotted semiiogarith~cal~y, indicate a decline to zero (arrow), at the time infectious centres were no longer detectable. in the data shown, the experiment was initiated days pi, i.e. with a well-established persistent infection. although infectious centres were detectable for about days at . "c the capacity of latently infected rn - cultures to resume virus production, upon temperature shift down to . 'c, was retained for only about days. apparently the infectious centres assay is the more sensitive measure of potential infectiousness. incubation of rn - cells at s"c, "c, or °c for - h prior to infection had no effect on the outcome of the infection, indicating that the observed inhibition of jhmv replication does not result from induction of factors in the uninfected cells when they are cultured at the elevated temperature. one approach towards rescue of jhmv from latently infected rn - cells was by means of co-culture with the indicator line l- , host cells which do not restrict virus production at °c. upon co-cultivation for l- days cell-cell fusion became evident, typical of that seen during lytic infection of l- cells. these observations revealed the presence of latent jhmv which could be transferred to indicator cells upon close cell-cell contact and therein established a lytic infection. to test whether some factor such as interferon (ifn), or an interferon-like effect, prevailed during persistence or latency in rn - cells, jhmv-infected cultures were superinfected with vsv at either . or °c. such cells were, indeed, resistant to challenge with vsv showing % inhibition of vsv inoculated at the m.o.i. of . the procedure is described in materials and methods. cells were inoculated with x pfu of vsv and checked for plaque counts h later. results are given as the percent reduction in vsv titer compared to untreated controls. control cells gave plaque counts in the range of - on re cells and - on rn - cells. a uninfected cells were treated with pg/ml poly ( ) : poly(c). ' poly ( ): poly(c)-containing medium, collected from cells being tested for the cell-associated effect, was rnase treated and tested on fresh rn - and re cells for its ifn inductive effects. these results revealed that although only a small fraction of the cells in a population were scored as positive for jhmv, the ongoing infectious state of the culture was sufficient to inhibit totally the replication of vsv. this finding suggests that persistent or latent infections of only a few cells were adequate to protect the entire culture against vsv. the above results led us to initiate tests for the presence of antiviral material i.e. ifn. for this purpose, 'conditioned' nm from latently or persistently infected rn - cells was used to treat uninfected rn - cells prior to inoculation with vsv, as described in materials and methods. there was no significant reduction in the titer of vsv produced. nor was evidence found for presence of extracellular mediators in 'conditioned' nm from which the protein had been concentrated by procedures used to concentrate rat ifn, described by schellekens et al. ( ) . these findings indicate that, within the limits of detection of our assay, persistently or latently infected schwannoma cells were not producing any soluble interferon or ifn-like material. failure to detect ifn during persistence and latency led us to investigate whether the schwannoma line, like primate vero cells (desmyter et al., ) is deficient for ifn inducibility, as defined by lockart ( ) . the analyses involved both the ability of test cells to become resistant to vsv and to produce a transferable, soluble factor which conferred resistance against vsv. our results, summarized in table , showed that primary rat embryo (re) cells were highly sensitive to the interferon inducer poly( ) : poly(c), both with respect to acquisition of resistance to vsv and production of a soluble antiviral material. by comparison, rn - cells were affected to a lesser extent by this inducer. however, exposure of either re or rn - cells to uv-inactivated reovirus type produced maximal responses within h in both cell types and some ifn was already detectable by - h after treatment. following addition of inactivated reovirus, equivalent to - pfu/cell of live virus, the same amount of ifn in the nm was produced by either re or rn - cells, about . units/ml by comparison with a standard ifn preparation. this demonstrated that both the rat cell types tested had equal responsiveness to induction of ifn by reovirus. from this one can conclude that rn - cells are not lacking in capacity to make ifn. despite absence of detectable ifn in jhmv-infected culture medium, the rn - cells were resistant to superinfection with vsv. this led us to test the influence of exogenously added ifn by exposing persistently or latently infected cultures to purified rat ifn, characterized as the (y type (lee biomolecular). preliminary tests revealed that addition of units/ml of this ifn reduced by over % the titer of vw in previously uninfected rn - cells. on the other hand, treatment of this line, with - units/ml of the rat ifn, at the time the cells were already undergoing persistent infections with jhmv, failed to decrease significantly the yields of jhmv. therefore, jhmv persistence in this system does not appear to be sensitive to or under the control of ifn. 'northern' transfer hybridization with isotopically labelled cdna probes, specific for jhmv was conducted using extracts of rna from cultures maintained after infection at either . or . "c. bands were evident in the autoradiograms at positions corresponding generally to those associated with mrnas of jhmv (fig. ) . the number and approximate m,'s of the mrna bands (l- ) agreed with findings from lytic infection of l cells, as reported by cheley et al. ( b) , except that the band related to the s nucleocapsid mrna (mrna ) was evident at a position of slightly lower m, than anticipated. this was most probably due to a displacement by the presence of relatively much more abundant s rrna in the vicinity, which could have produced a distortion of the migration of the viral mrna in the agarose gels. it was noted that mrna , which has an approximate molecular weight of . x lo", showed a secondary faint band of slightly lower molecular weight. the lower molecular weight band predominated when jhmv was grown lytically in l- cells (data not shown). these findings are of interest in light of those of taguchi et al. ( ) who observed alterations in the molecular weights of mrnas and in brain-derived vs. culture cell-derived material. throughout persistent infection of rn - cells, at . "c. all species of jhmv mrna were detected, as illustrated with a -day pi sample in channel a of fig. . judging by the unchanged intensity of the bands with time of sampling, the quantity of mrna did not vary appreciably. by contrast, during latency at . "c, the intensity of the bands decreased with time elapsing pi as evident in channels b-e. thus, by day pi at the elevated temperature, the bands became faint. an extract of rna from a culture, kept in a state of latency for days, apparently contained -!i- -- s *l es fig. . 'northern' transfer and cdna hybridization to rna extracted from latently or persistently jhmv infected rn - cells. data after incubation at . "c for days pi are in channel a; data from infection at . "c for , , , or days pi in channels b-f, respectively. material in f was from a separate experiment and involved a x longer exposure time for the autoradiogram. rna extracted from uninfected cells in channel g. on the left-hand side are indicated the m,'s of jhmv mrnas to , which are respectively . x x , . ~ , . x , . x , . x , . x ' and . x '. location of the s and s ribosomal rnas is shown by arrows on the right side of the figure. mrna corresponding to the nucleocapsid gene. these results suggested that early during latency all or most of the jhmv mrnas were being expressed but later than days pi the nucleocapsid mrna, the most abundant viral mrna (stern and kennedy, ) was the only mrna detectable by these means. the data suggest that transcription at the restrictive temperature continued but the abundance of the mrnas decreased rapidly within a few days. the results from cdna probing of transcripts are consistent with the observed decline in the number of infectious centers which became evident as the duration of latency was prolonged (see fig. ). to ascertain whether active translation was underway at . "c, polysomes from infected rn cells were isolated, fractionated and subjected to hybridization with jhmv-specific [ p]cdna and autoradiography, as described in materials and methods. the autoradiograms, shown in fig. , indicated that mrna specified by sucrose isokinetic gradients and centrifuged at ' x g for min. the gradients were fractionated and acid insoluble cpm determined. fractions from major areas of each gradient were pooled (a, b and c), precipitated with ethanol and used for 'dot-blotting', as described in the materials and methods. the incubation temperatures for the persistently or latently infected rn - cells, from which the extracts were made, are indicated. the 'dot-blot' hybridization autoradiograms of the pooled gradient fractions are shown above the gradient profiles. the columns designated a, b and c correspond to similarly identified pooled fractions, shown below. the lower dot in each panel represents l/ of the total material in the pooled fractions. the upper dot is a l/ dilution of material present in the lower dot. the centre panel of the autoradiogram shows hybridization results with polysome extracts from uninfected rn - cells. jhmv was present in the gradient fractions enriched for polysomes, isolated from cells kept at either . or . "c for days pi. this finding suggests that active translation of coronavirus messengers was also taking place at the restrictive temperature, in the absence of infectious virion formation. since it was apparent from work with polyribosomes that some jhmv transcrip- tion occurred during latency at .s°c, analyses were made to ascertain whether presence of viral mrna could be related to translation into viral poi~~ptide(s). as already mentioned, incubation of infected rn - cells at the permissive s"c was associated with only minimal cytopathology, implying that detection of viral polypeptide might be different, especially during latency at s"c. to maximize the sensitivity of detection, virus related antigens were identified by immunob~otting and autoradiography. for this purpose, infected rn - cells, used as controls, were cultured at the permissive or restrictive temperatures. samples were removed at intervals and extracts prepared from them, then subjected to sds-page, immunoblotting with hybridoma antibodies and autoradiography. the results from the permissive conditions, after tagging with anti-nu~leocapsid antibodies, are presented in fig. . it is evident from channel b, representing material sampled h after inoculation, that there was a significant quantity of material in a band at the da ( k) nucleocapsid position, corresponding to the m, of primary product of viral nucleocapsid (cheley and anderson, a) . the other bands with apparent m,'s k and k, evident in these gels, were presumably irrelevant and artifactual, since they occurred in other channels, including those representing uninfected controls whenever the anti-mouse kappa chain antibody was utilized as a linker. lanes c-i of fig. represent immunoblots of extracts from, respectively, uninfected rn - cells kept at . "c those sampled min after infection ( h) at . "c or respectively at , , , h and days post-inoculation. in samples prepared min after inoculation the antibodies could identify a predominant polypeptide band of k and a minor one of k. in samples taken h after inoculation the antigen(s) recognized by the hybridoma was k and k, as evident in lane e. previous work by cheley and anderson ( a) , confirmed by our own studies, established by means of pulse-chase labelling experiments that a precursor-product relationship exists between the k and ok polypeptides. prominent k and k antigens were detected also in cell extracts for days or longer after inoculation, revealing that production of nucleocapsid protein was continuous throughout the course of the persistent infection. there was therefore, good correspondence between data on jhmv transcription (figs. and ) translation and formation of infectious jhmv. by contrast, identical immunoblotting utilizing extracts of latently infected rn - cells, maintained at °c gave somewhat different results. as evident in channel b of fig. , in the infected l- cells, used as controls, the predominating antigen was k. with rn - cells sampled at h ( min after inoculation), the predominant form of nucleocapsid species was the k antigen (fig. , channel d) . similar antigenic bands were identified in the h samples (fig. , channel e). it is. however, not clear from these data alone whether the k band material is nascent antigen or the residual antigen introduced with the inoculum. judging by the bands corresponding to the nucleocapsid, the antigen which accumulated at . "c, when infection was extended beyond h, was the k precursor polypeptide (channels f-i of fig. ). it is, therefore, most probable that the k polypeptide evident in channel d was an intracellular cleavage product of nascent k since, with time, the k component decreased at a relatively faster rate than the k antigen. it is very significant that during incubation at the restrictive temperature for as long as days and beyond, when rn - cells had multiplied through - generations, the k antigen was readily detectable (channel i), implying that during latency, in the absence of infectious virus production, translation into jhmv nucleocapsid protein continued. this finding is consistent with the presence of jhmv-specified mrna in the polyribosomes isolated from rn - cells, latently infected for days (fig. ) . probing for the expression of the other structural components, the e, and e, glycoproteins, of apparent m, k and isok, respectively, was also undertaken. employing anti-e, and anti-e, hybridoma antibodies of collins et al. ( ) for immunoblotting, it was found that at the permissive temperature both antigens were expressed in rn - cells, but during latency at . "c neither glycopeptide was detectable (data not shown). these findings suggest that the temperature-related inhibition of infectious virus formation might be due either to the selective suppression of the synthesis or rapid turnover of jhmv envelope components, while production of the nucleocapsid polypeptide is much less affected. on the other hand, the normally greater abundance of the nucleocapsid polypeptide than of the other structural components could account for our inability to detect e, or ez in rn - cells at . "c. the present study was feasible because it is possible upon infection of rat schwannoma cells rn - with jhmv, to establish rapid and reproducible persistence or latency, influenced by elevated temperature, as demonstrated in an earlier report by lucas et al. ( ) . thus, when infections are initiated at . "c the virus is able to establish itself without formation of infectious virions. the temperature restriction on the replication process is most probably controlled by a host-imposed function and depends on the continued presence of the virus in state of latency, since susceptibility to reinfection is restored at . "c, after rn - cells become 'cured' of the infection. the prolonged latency of jhmv under restrictive conditions is remarkable. in comparison, measles virus which also establishes a persistent infection in rn - cells (lucas et al., ) has shown a latency period of up to days (coulter-mackie and dales, unpublished observations). the duration of latency with jhmv during which reactivation remains possible is shorter, perhaps only - days. the difference between the two viruses is probably related to the proportion of cells in a culture which carry viral information, the fraction involved with jhmv persistence being much lower than with measles virus persistence. the conversion of jhmv infection from the persistent into latent states upon temperature elevation to s"c, and the prolonged latency which can occur in the in vitro cultures, could have direct bearing on understanding the infectious process during jhmv infection of the rat central nervous system (cns). since symptom-free rats. surviving an initial intracerebral infection with jhmv, can be caused, following treatment with the immunosuppressive agent. cyclophosphamide, to develop a chronic. demyelinating disease (sorensen et al., ) . it is quite possible that this virus can be perpetuated in a covert form within the cns. consistent with this view is the finding that rats which remain asymptomatic for as long as days after inoculation may harbour jhmv rna in the cns (sorensen et al.. ). in the present study several approaches were taken towards an understanding of the nature of the block in virus production at the elevated temperature. as previously discussed by lucas et al. ( ) , involvement of defective-interfering particles or development of thermosensitive virus variants was not demonstrable and, therefore, is unlikely to account for the inhibition observed. host factors might be responsible for the observed virus suppression. one possible candidate for the host factor in question is ifn. it is' well known that during chronic in vitro infections with neurotropic agents, such as rabies virus, there is an inverse correlation between the cycling virus titre and ifn levels in the medium (wiktor and clark, ) . it may also be significant in the present context that ifn activity can be enhanced at elevated temperature (heron and berg, ) . thus, in our system it was postulated that ifn or interferon-like substance might influence the control of the persistence at °c and suppression of infectious particle formation at . "c. although in the present study the development of resistance to infection of rn - cells by a heterologous virus. vsv. could be demonstrated during persistent and latent infections with jhmv and likewise with measles virus (coulter-mackie and dales, unpublished observations), no evidence was obtained to demonstrate that such persistently or latently infected cells produce ifn or an ifn-like substance. these two viruses differ in that measles virus is known to be an ifn inducer (demaeyer and enders, : mckimm-breschkin and rapp, ) while jhmv probably does not induce ifn in cultured cells. although replication of this coronavirus is sensitive to exogenous ifn (garlinghouse et al., ) . however, since these schwannoma cells are inducible for ifn with uv-inactivated reovirus and can respond to exogenously added ifn. yet continue to produce jhmv at a normal rate after the addition of purified rat ifn to persistently infected rn - cultures. it is questionable whether ifn exercises any control in this system or that involving rat neural cells in general. therefore, the interference with vsv replication during latency or persistence remains unexplained. prqbing for jhmv-specific rna by means of 'northern' transfer indicated that transcription can occur at both . or . "c. while at . "c the mrna specifying the nucleocapsid was the predominant species and was still evident on the in vivo and in vitro models of demyelinating disease xi. tropism and differentiation regulate the infectious process of coronaviruses in primary explants of the rat cns to the other viral functions could be detected only during the first few days post-inoculation.however, the corresponding immunoblots made on extracts from latently infected cells could detect only the k nucleocapsid protein, suggesting that preferential translation of the nucleocapsid mrna occurs in the repressed state. the situation regarding the other major jhmv structural polypeptides e, and e, remains unresolved. while these glycoproteins were expressed normally at the permissive temperature, we were unable to detect their presence during latency, despite the presence of mrna species corresponding to e, and e, during the initial - days of latency. these data suggest that infection at the elevated temperature may lead to either a differential inhibition of the synthesis of envelope polypeptides, or their rapid turnover, or both. the western blot analysis also revealed a processing step connected with the nucleocapsid protein, which is blocked during latency. the nucleocapsid is initially produced as the k form and integrated into the virus. soon after infection, however, the nucleocapsid material of the inoculum exists as a k molecular weight polypeptide (figs. and ) . this precursor-product relationship has been documented by cheley and anderson ( a) . both sizes of this antigen were found to be phosphorylated (our data, not shown, and siddel et al., ) . the k proteins found in infected rn - cells incubated at . "c are likely. therefore, to represent the product of processing which may occur during cell-to-cell spread of progeny virus. upon incubation of rn - cells at °c assembly and/or spread of the virus progeny are probably inhibited. this is consistent with our preliminary observations (adler et al., unpublished) , which reveal that compounds interfering with jhmv entry into l- cells also affect processing of k nucleocapsid of inoculum virions.from the above it is clearly evident that in latently infected rn - cells the viral genomes are not dormant and merely segregated to daughter cells but are instead, active in transcription and translation. it remains to be shown, first of all, whether genome duplication is reduced or stopped. secondly, whether the expression of some functions is specifically reduced during latency and thirdly, why the production of infectious progeny ceases. . . schellekens, h., dewilde. g.a. and weimar. w. ( ) production and initial characterization of rat interferon. j. gen. virol. , . siddell. s.. wege, h. and ter meulen. v. ( ) the biology of coronaviruses.j. gen. virol. , - . sorensen, .. percy, d. and dales, s. ( ) in vivo and in vitro models of demyelinating disease iii. jhm virus infection of rats. arch. neurol. , - x . sorensen. .. dugre, r.. percy. d. and dales. s. ( ) key: cord- -wl wlxh authors: wang, ling; xu, xiaopeng; ruan, junshan; lin, saijin; jiang, jinhua; ye, hong title: quadruple therapy for asymptomatic covid- infection patients date: - - journal: expert rev anti infect ther doi: . / . . sha: doc_id: cord_uid: wl wlxh introduction: the novel coronavirus (covid- ) is currently in epidemic stage. after large-scale interpersonal infection, asymptomatic patients appear. whether asymptomatic patients are contagious or not and whether they need medication are the arguments among clinical experts. areas covered: this paper reports a special asymptomatic couple with covid- , of which the male patient is an intercity bus driver but has not induced confirmed infection of his passengers. the patients were treated with four combinations of lopinavir/ritonavir tablets, arbidol tablets, lianhuaqingwen granules, and recombinant human interferon-α b (ifn-α b) injection via aerosol. their clinical characteristics and medication were summarized and analyzed. expert opinion: the two asymptomatic patients far away from wuhan did not seem to be highly contagious. they improved obviously, after treatment with the quadruple therapy, but the effective drug is still unknown. it should be noted that lopinavir/ritonavir tablets have many drug interactions and are the most likely drugs to cause hyperlipidemia and hyperglycemia in these two patients. ifn-α b is more effective in the early stage of virus infection. arbidol instruction dose may not be sufficient to inhibit the novel coronavirus in vivo. the evidence-based medicine of lianhuaqingwen granules for treating various viral infections is just based on chinese patients. since december , the novel coronavirus (covid- ) has been an epidemic. it is highly infectious and spreads rapidly. according to covid- global cases by the center for systems science and engineering (csse) at johns hopkins university (jhu), more than million cases have been diagnosed worldwide, and over thousand patients have died. the world health organization (who) listed the outbreak caused by covid- as 'public health emergencies of international concern (pheic).' this virus belongs to the same coronavirus as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronavirus but has higher replication ability, stronger interpersonal transmission ability, and higher pathogenicity. the population is generally susceptible, and there are numerous severe patients. however, after large-scale interpersonal infection, it is surprising that 'asymptomatic persons' appeared with no clinical symptoms, but pathogen detection is positive. the emergence of asymptomatic persons poses challenges to the prevention and treatment of the epidemic. whether asymptomatic patients are contagious and whether they need treatment are controversial issues for clinical experts. on the one hand, in terms of the law of infectious diseases, asymptomatic persons are also capable of spreading the virus, but the viral load and transmission harm need to be investigated. on the other hand, the progress of asymptomatic patients and the effect of drug therapy are still unclear. not long ago, a german asymptomatic patient was reported. the patient had chills, fever, and cough but recovered the next day and infected two colleagues in close contact with him in the latent period [ ] . here, we report two special asymptomatic patients, a couple, without definite contact history. moreover, the male patient is an intercity bus driver; however, within days after contact with him, no case of covid- infection was confirmed in the passengers he had carried. only one passenger developed cough days after taking the bus, whose covid- nucleic acid tests have been negative for many times so far, and influenza b was once weakly positive, waiting to be excluded covid- infection. this paper discussed the clinical characteristics of these two asymptomatic patients and summarized the pharmacological effects, clinical studies, and other precautions of the antiviral therapy on them, so as to provide a reference for the identification, diagnosis, and medication of asymptomatic patients in the epidemic prevention and control. on january , the patient went to a hospital in songxi county, fujian province, china, with 'soreness of loins' as the main complaint. the patient was in good health without previous medical history, medication history, and smoking or drinking habits. the physical examination showed the body temperature of °c, blood pressure of / mmhg, pulse rate of beats per minute, respiratory rate of breaths per minute, and oxygen saturation of % while the patient was breathing ambient air. lung auscultation did not reveal rough breathing sounds, rhonchi, or pleural friction rub. blood test results demonstrated his c-reactive protein (crp) of . mg/l, procalcitonin of . ng/ml, glutamic oxaloacetic transaminase of u/l, glutamic-pyruvic transaminase of u/l, and glutamyl transpeptidase of u/l, and other indicators were normal. chest ct examination showed multiple ground-glass appearance ( figure ). since the patient works as a bus driver, covid- pneumonia cannot be ruled out, although he claimed no known contact with anyone from hubei. the hospital notified the local center for disease control and prevention (cdc) immediately and designated him as a 'suspected person.' the patient was isolated in the hospital. his throat swab specimen was obtained for covid- nucleic acid detection. on the same day, the local cdc confirmed that the patient's covid- test was negative. on january , the patient did not have fever or any other discomforts, and the covid- nucleic acid throat swab test was still negative. on january, the patient had no discomfort. in order to exclude covid- infection, double throat swab specimens of the patient were collected and sent to both local and provincial cdc for reexamination. on january, the results showed positive. with the coordination of the local health commission, the patient was sent to the isolation ward of nanping first hospital for treatment on january . on the first day of admission, the patient was suffered from soreness of waist, without cough, fever, shortness of breath, vomiting, or other discomforts. his vital signs appeared normal. the physical examination showed rough breathing sounds, and other monitored parameters were generally normal. from january to february , the patient was given quadruple therapy, including lopinavir/ritonavir tablets ( / mg every h), arbidol tablets ( . g every h), lianhuaqingwen granules (a chinese patent medicine, g every h) orally, and recombinant human interferon-α b injection via aerosol ( . × iu with ml of sterilized water for injection every h). the patient had stable vital signs and normal oxygen saturation, without any discomfort. his clinical laboratory indicators are shown in table . the patient's white-cell count, absolute neutrophil count, absolute lymphocyte count, platelet count, and crp were all in normal quantity. fasting blood glucose and triglyceride continued going up to . and . mmol/l, respectively, significantly higher than normal values. cholesterol rose to . mmol/l eventually. alanine aminotransferase ( - iu/l) and glutamyl transpeptidase ( - iu/l) persisted a bit higher. total bilirubin, fibrinogen, lactate dehydrogenase, and blood urate normalized after slightly exceeding the normal. on february, compared with the previous chest ct result, the scattered patchy fuzzy shadows of both lungs slightly decreased. the patient was taken covid- nucleic acid throat swab tests on february and february , respectively, and the results were both negative. on february , the patient discharged without any discomfort and in good condition. the patient is a -year-old housewife and has close contact with her husband, the patient of case . • asymptomatic patients are controversial and concerning issues of covid- . the infectivity of some asymptomatic patients does not appear to be strong. • quadruple therapy, which is lopinavir/ritonavir tablets, arbidol tablets, lianhuaqingwen granules, and recombinant human interferon-α b (ifn-α b) injection via aerosol, is a common regimen for patients with covid- in china. the lung imaging of asymptomatic patients improved obviously after quadruple therapy, but which drug takes effect needs further studies. • lopinavir/ritonavir tablets cause high risks of drug interactions, hyperlipidemia, and hyperglycemia. • inhalation of ifn-α can raise its lung concentration. ifn-α b is the most active type of ifn-α. . - × iu/day is the appropriate inhalation dose. on january , the patient had no discomfort and accompanied her husband to a hospital in songxi county, fujian province, china. as her husband was suspected to be infected with covid- , she was also hospitalized and isolated. the patient was in good health without previous medical history, medication history, and smoking or drinking habits. the physical examination showed her body temperature of . °c, blood pressure of / mmhg, pulse rate of beats per minute, respiratory rate of breaths per minute, and oxygen saturation of % while the patient was breathing ambient air. auscultation of both lungs was normal. her clinical laboratory results were within normal range, except crp of . mg/l ( table ). on the same day, the local cdc confirmed that the patient's covid- nucleic acid throat swab test was negative. on january , the patient had no discomfort, but her covid- nucleic acid throat swab test was positive. on january , her throat swab was collected again and the result was also positive. chest ct showed that her two lungs had diffuse patchy shadows and ground-glass appearance ( figure ). she and her husband were sent to the isolation ward of nanping first hospital for treatment on january . on the first day of admission, the patient had no discomfort, and the vital signs were normal. the physical examination revealed rough breathing sounds, and other monitored parameters were generally normal. from january to february , the patient was treated with four drugs, which are oral administration of lopinavir/ritonavir tablets ( / mg every h), arbidol tablets ( . g every h), and lianhuaqingwen granules (a chinese patent medicine, g every h) and atomization inhalation of recombinant human interferon-α b injection ( . × iu with ml of sterilized water for injection every h). the patient's vital signs were stable, and oxygen saturation was normal. there were no discomfort and any abnormal quantity of white-cell count, absolute neutrophil count, or absolute lymphocyte count. fasting blood glucose ( . -- . mmol/l) and triglyceride ( . - . mmol/l) remained relatively high. crp and fibrinogen dropped back to normal from . mg/l and . g/l, respectively. platelet count increased slightly to × /l finally. on february, from chest ct, it can be seen that the ground-glass shadow in her lungs has narrowed. the patient's covid- nucleic acid throat swab tests became negative on february and thus discharged from the hospital without any discomfort. covid- infection presents a cluster attack. most patients were male, with an average age of . years [ ] . fifty-one percent of patients were suffered from chronic diseases, including cardiovascular and cerebrovascular diseases, endocrine system diseases, digestive system diseases, respiratory system diseases, malignant tumors, and nervous system diseases. elderly men with complications are more likely to be infected and may lead to serious or even fatal respiratory diseases [ ] . although the two patients in this paper were about years old, they had good health and no basic medical history, which may be one reason why they had no obvious clinical symptoms of covid- pneumonia. however, whether there are other possibilities need to be discussed and confirmed. for example, the male patient of the first case, saying no contact with wuhan people, might be infected unconsciously by other people who got in touch with wuhan people. after repeated reproduction and transmission, the virus is getting weak. or, the viral load entering the body is relatively low; thus, under the condition of a good immune system, it will not emerge that large and rapid virus proliferation and migration in vivo lead to serious damage to lungs and even other organs. all these conjectures need further investigation. asymptomatic cases are easy to miss diagnosis or misdiagnose, due to the lack of specific covid- pneumonia manifestations and normal inflammatory indexes such as white blood cells, lymphocytes, and crp. at present, there have been some asymptomatic cases in china, which should be paid attention to. it is worth noting that the first male patient in this paper, complaining of soreness of loins, is similar to the atypical covid- pneumonia german patient with myalgias as a complaint [ ] . the male patient of case also showed transient elevation of serum lactate dehydrogenase. with respect to muscle pain, there are similarities between asymptomatic infection of covid- and influenza to some extent, and sometimes, the rise of serum creatine kinase, lactate dehydrogenase, and myoglobin can be seen. at present, the guidelines of covid- pneumonia in various countries do not list muscle pain as one of the clinical manifestations, which may lead to the omission of some patients, especially the mild patients. what should be taken seriously is not only that asymptomatic patients did not have typical covid- infection symptoms but also that the covid- nucleic acid throat swab tests showed false negative in these patients, delaying the diagnosis of covid- infection. this may be caused by two reasons, one is that asymptomatic patients may have a low viral load, and the other is that the detection rate of nucleic acid throat swab may be not high enough. for mild cases of covid- infection, the use of lung imaging as a supplement or even an alternative to throat swab detection may also be an effective means to reduce the omission diagnostic rate. under certain circumstances, lower respiratory tract specimens such as tracheal aspirates, bronchoalveolar lavage, and sputum, with higher viral load, can be tested to raise the detection rate. on the other hand, interpretation of positive results is equally important. at present, in china, two consecutive negative results of covid- nucleic acid throat swab test are the necessary standard for patients to leave the hospital. in this paper, the female patient has no symptoms all the time, but the duration of positive nucleic acid test results is longer than that of the male patient, so she cannot be discharged from the hospital for a longer time. that provokes questions that how long the course of antiviral treatment should be and whether the sign of stopping antiviral treatment should be the clinical manifestation or nucleic acid turning negative. what is worth thinking about is whether it is a better choice for asymptomatic patients to rest at home instead of medication at hospital in order to prevent adverse drug reactions, such as the increase of triglyceride in cases and as described below. in this paper, two patients were treated with quadruple therapy combining chinese and western medicines. covid- , sars, and mers belong to the same coronavirus, and their protein structures have many similarities. the antivirus drugs used in this paper have been proved to have an inhibitory effect on sars/mers in vitro or in vivo, but their adverse reactions and efficacy/safety in special populations such as liver and renal insufficiency still need to be paid attention to by clinicians. lopinavir/ritonavir is recommended by the national health commission of china for the treatment of covid- at present [ ] . lopinavir is used to prevent the hiv gag-pol polyprotein from splitting. the action site of ritonavir is aspartyl protease of the virus, blocking the precursor of hiv gag-pol polyprotein. the two components work together, resulting in immature virus particles without regeneration capability. moreover, ritonavir is also an enhancer of lopinavir, which can inhibit the cyp a-mediated degradation of lopinavir in the liver [ ] . two sars-based clinical studies [ , ] and a mers-based clinical case report [ ] suggested that lopinavir/ritonavir has an anticoronavirus effect. in addition, lopinavir/ritonavir was listed as a candidate therapeutic drug in an anti-mers guideline written by clinical experts in south korea [ ] . these studies are of great significance for the treatment of covid- with lopinavir/ritonavir. however, the clinical studies on sars published by chan and chu both pointed out that lopinavir/ritonavir as the initial treatment can remarkably improve the total mortality rate, oxygen saturation reduction rate, intubation rate, or glucocorticoid usage. but for treating the terminal stages, there was no obvious curative effect [ , ] . this may be because lopinavir/ritonavir mainly acts by inhibiting the formation of new virus and cannot prevent the virus from entering cells, but numerous viruses have entered cells in the endstage patients, causing organ damage and poor outcome. in terms of adverse reactions, lopinavir/ritonavir induces diarrhea, hyperglycemia, hyperlipidemia, arrhythmia, liver dysfunction, etc. its serious adverse reactions also include fatal diseases such as pancreatitis [ ] . in this paper, the triglyceride and fasting blood glucose levels of the two patients exhibited a huge rise far above normal after treatment. according to naranjo's adverse drug reaction probability scale score, points were obtained, indicating that lopinavir/ritonavir probably caused adverse reactions of hyperlipidemia and hyperglycemia in both the patients. the adverse drug reactions may be common in patients because these two patients, as good representatives, had normal basic monitoring indexes and good liver/kidney function and physical condition and took lopinavir/ritonavir in a short time, thus metabolized and excreted lopinavir/ritonavir efficiently. for severe patients with liver function injury, risk of the adverse drug reactions may become higher, possibly leading to serious consequences, including fat overload syndrome, pancreatitis, other pathogen infection, and even prolonged hospitalization or death for reasons other than covid- infection. besides, lopinavir and ritonavir, both inhibitors of cyp a, may increase the plasma concentration of drugs metabolized mainly by cyp a, such as amiodarone, fentanyl, and midazolam, commonly used in severe patients. the drug action time will prolong and the occurrence of adverse drug reactions may increase. lopinavir and ritonavir also have certain inhibition effects on atp-binding cassette subfamily b member (abcb ), but the effects of abcb were only detectable in the presence of cyp a, suggesting that the simultaneous use of abcb substrates should also be cautious [ ] . the severe pneumonia incidence of covid- infection is relatively high, especially in the elderly. for severe patients with hepatic or renal insufficiency, it should be noted that blood drug concentration of lopinavir showed a rapid increase in the patients with liver dysfunction [ ] , suggesting the necessity of therapeutic drug monitoring of lopinavir. therapeutic concentration of lopinavir was - mg/l, and toxic concentration was above mg/l [ ] . however, patients with renal insufficiency can continue to take lopinavir/ritonavir at the conventional dosage because the renal clearance rate of lopinavir and ritonavir is extremely low. moreover, both lopinavir and ritonavir have strong protein-binding capacity, so hemodialysis will not affect their clearance greatly [ ] . but lopinavir/ritonavir was reported to be strongly related to kidney impairment, so its potential nephrotoxicity should be concerned [ , ] . there are few reports of old people treated with lopinavir/ritonavir, yet the lopinavir plasma level in the elderly seems to be higher than the recommended value [ ] . atomization inhalation of interferon-α (ifn-α) is also recommended by the national health commission of china as medication for covid- . ifn-α is bound to receptors on the cell surface to induce the production of various antiviral proteins, thereby inhibiting the replication of viruses in cells. ifn-α has a broad antivirus spectrum. in addition, it can enhance the specific cytotoxic effect of macrophages and lymphocytes by regulating immune function and thereby stop the invasion and infection of the virus effectively. at present, there are three types of ifn-α subtypes approved for clinical use, namely ifn-α b, ifn-α a, and ifn-α b . it is found that ifn-α b used in this paper tends to have a relatively higher activity [ ] . ifn-α is mainly administered subcutaneously and intramuscularly, which is easy to cause influenza-like symptoms, myelosuppression, mental abnormalities, and other adverse reactions. inhalation will not inactivate ifn-α and can raise the lung concentration [ ] . . × iu/day is the minimum inhalation dose of ifn-α that can induce biological effects without side effects [ ] . below the dose of × iu/day, ifn-α hardly enters the systemic circulation through inhalation [ ] . as for the treatment of ifn-α on coronavirus, early therapy of ifn-α inhalation combined with antiviral drugs has been shown to be associated with better outcomes [ , ] . while the clinical research of omrani et al. demonstrated that the combination therapy of ribavirin and ifn-α a inhalation can improve the early and intermediate survival rate of severe mers patients significantly, it was useless for the late survival rate [ ] . however, the sample sizes of the above investigations were small, and the effectiveness of ifn-α inhalation on coronavirus still lacks sufficient evidence-based medical evidence. inhalation of ifn-α may induce bronchospasm reaction and decrease the peak expiratory flow rate [ , ] but few systemic adverse reactions such as influenza-like symptoms [ ] . there are no data about the application of ifn-α inhalation on the elderly and patients with liver or renal insufficiency. however, the usage of ifn-α injection for inhalation is offlabel, so the patient-informed consents are required. besides, attention should be paid to the possibility of allergy caused by ifn-α and auxiliary materials. the two patients in this paper also used hemagglutinin inhibitor, arbidol, which is not recommended by the national health commission of china for covid- . arbidol can specifically inhibit the contact, adhesion, and fusion of virus lipid membrane and host cell membrane and block virus gene from penetrating into nucleus, by activating , -oligoadenylate synthetase (antiviral protein) [ , ] . at present, there is no clinical report of arbidol on coronavirus pneumonia, but it has a good inhibition effect on sars, even ebola virus (ebov) and lassa virus (lasv) in vitro [ ] [ ] [ ] . an arbidol concentration of μg/ml was required to achieve a % reduction in virus proliferation and hemagglutinin levels [ ] . according to china news, cell experiments in vitro showed that arbidol can effectively inhibit covid- up to times at the concentration of - μm and significantly suppress the pathological effect of the virus on cells [ ] . however, the concentration of - μm is equivalent to . - . µg/ml of arbidol, which is far above the peak concentration ( . μg/ml) that can be achieved in vivo by oral administration of single-and multipledose arbidol [ , ] . therefore, whether to apply a higher dose of arbidol for covid- pneumonia needs to be concerned and studied. liver and intestine are the main metabolic organs of arbidol in the human body, and cyp a is the major isoform enyzme, indicating possible drug interactions between arbidol and cyp a substrates [ ] . additionally, the adverse event rate of arbidol is approximately . %, mainly manifested as nausea, diarrhea, dizziness, and increased serum transaminase [ ] , which is similar to the adverse reactions of the digestive system in covid- -infected patients, probably increasing difficulties in differential diagnosis. in the bioequivalence experiment of arbidol preparation conducted in china, h after taking the medicine, some healthy subjects had bradycardia (heart rate less than beats/min and reduced - beats per minute). but the subjects had no adverse symptoms and the correlation with arbidol was unclear. arbidol can be used for the elderly patients and improve their cellular immunity [ ] . for patients with hepatic or renal insufficiency, the safety of arbidol is not clear. in view of the fact that arbidol is mainly removed by feces, accounting for . % of the dose, and its glucuronide and sulfuric acid metabolites are excreted in urine, accounting for . % of the dose, therefore, patients with severe liver dysfunction need to use arbidol with caution or reduction, while patients with renal insufficiency may consider the original dose [ ] . in the guideline for covid- pneumonia of china, traditional chinese medicine has equal status with western medicine. lianhuaqingwen preparation has broad antiviral spectrum, antipyretic effect, and anti-inflammatory effect and is even superior to oseltamivir in improving the symptoms of influenza infection [ , ] . lianhuaqingwen preparation has been recommended by the national health commission of china repeatedly in a series of infectious public health events such as sars, mers, h n , avian influenza, and hand-footmouth disease, for more than years, and plays an important role in prevention and treatment. lianhuaqingwen preparation can obviously inhibit the replication of sars virus with ic of . mg/ml and therapeutic index of . [ ] . lianhuaqingwen preparation has compounds or more, in which high-content compounds are salidroside, chlorogenic acid, forsythoside e, cryptochlorogenic acid, amygdalin, sweroside, hyperin, rutin, forsythoside a, phillyrin, rhein, and glycyrrhizic acid [ ] . the common adverse reactions of lianhuaqingwen preparation were mostly related to digestive system, mainly manifested as nausea, diarrhea, vomiting, and abdominal pain. skin allergy reaction and clinical laboratory results are rare and can be relieved after drug withdrawal. the treatment of lianhuaqingwen preparation on covid- pneumonia has yet to be proved by clinical trials. the best medication for covid- infection has not yet been confirmed. in this paper, two patients were treated with quadruple therapy combined with chinese and western medicines. the symptoms and chest x-rays of both patients have been greatly improved. it can be said that quadruple therapy has a certain curative effect, but the possibility of spontaneous improvement cannot be ruled out, and it is impossible to tell whether one or more drugs work. the reason why the two patients were selected is that they were the only asymptomatic patients in northern fujian province of china, and their medication was analyzed by the authors, who often discuss difficult cases in the form of multidisciplinary team. the treatment of two patients met the guideline for covid- pneumonia of china and got the ethical approval of relevant hospitals. this paper has limitations. it is a report of individual cases, and we have not quantified the viral load, leaving the virus dynamics unclear. more clinical data are needed for asymptomatic patients as well as this combination medication. asymptomatic patients have no fever or respiratory tract symptoms, but pneumonia can still be seen on imaging. for asymptomatic patients, pharmaceutical care should be strengthened while virus detection and antiviral treatment are carried out, including the discovery, solution, and prevention of potential or actual drug problems, so as to ameliorate the safety, effectiveness, and economy of medication and achieve the ideal goal of improving the quality of life for patients with covid- . the lack of effective drugs directly targeting covid- is the major challenge to doctors and the treatment of patients. whether the western medicine lopinavir/ritonavir, remdesivir, arbidol, and chloroquine or traditional chinese medicine with antiviral activity has caused the public to expect the control of covid- but lacks 'effectiveness' and 'safety' from multicenter, randomized, double-blind, controlled studies. in fact, it takes quite a long way from in vitro experiments to actual clinical use. in the design of clinical trials for covid- , the trial efficiency is of great importance; thus, phase i trials with extended cohorts, trials based on pattern recognition, machine learning, and other technologies should be utilized. further study of covid- is not limited to antiviral drugs. anti-inflammatory (anti-il- , anti-tnf-α, etc.) treatment should be actively carried out to quickly neutralize the cytokine storm in vivo and reduce the mortality of critical patients. adoptive reinfusion of cured patients' serum and the immune regulation of mesenchymal stem cells may also show a certain effect. with the help of multidisciplinary cross-analysis, to control symptoms quickly and reduce mortality effectively is the main task at present. a reasonable analysis of existing clinical data to make treatment decisions is the only choice in the absence of effective covid- antiviral drugs. the efficient utilization of clinical data needs the establishment of big data and the promotion of scientific, networked, and shared data collection and management modes. specifically, many complicated clinical data are cleaned and integrated, and the terminology is unified. the outliers or small-sample data are eliminated. repeated researches are conducted in clinical manifestations, laboratory test results, lung imaging, combined basic diseases, drug therapy and clinical outcomes, etc., via statistical analysis to find out characteristic data, helping physicians to realize accurate differential diagnosis and treatment of covid- . in the future, the following aspects should be emphasized for the control of covid- : ( ) strengthen epidemiological research, especially the prevention and control of asymptomatic and mild cases as infection sources; ( ) carry out indepth research on structures and functions of various virus proteins and then conduct drug prediction and highthroughput screening; and ( ) enhance international cooperation. for example, traditional chinese medicine has played a positive role in preventing and treating covid- , but further discussion is needed on how to standardize the use, how to reasonably evaluate the curative effect, and how to integrate with western medicine. this paper was not funded. transmission of covid- infection 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oseltamivir for influenza a virus infection: a meta-analysis of randomized, controlled trials systematic review of efficacy and safety of lianhuaqingwen capsules in treatment of viral influenza • lianhuaqingwen capsules have therapeutic effect on viral influenza, but the documents included are few and of low quality. therefore, the efficacy and safety of lianhuaqingwen capsules should be confirmed by more high-quality clinical studies inhibitory effects of three prescriptions of traditional chinese medicine on sars-associated coronavirus in vitro qualitative and quantitative analysis of the major constituents in chinese medical preparation lianhua-qingwen capsule by uplc-dad-qtof-ms the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. key: cord- -ko knslk authors: fu, weihui; liu, yan; liu, li; hu, huiliang; cheng, xiaobo; liu, ping; song, zhigang; zha, lijun; bai, shimeng; xu, tingting; yuan, songhua; lu, fengru; shang, zhiying; zhao, yihong; wang, jing; zhao, jun; ding, longfei; chen, jun; zhang, lin; zhu, tongyu; zhang, xiaoyan; lu, hongzhou; xu, jianqing title: an open-label, randomized trial of the combination of ifn-κ plus tff with standard care in the treatment of patients with moderate covid- date: - - journal: eclinicalmedicine doi: . /j.eclinm. . sha: doc_id: cord_uid: ko knslk background: epidemic outbreaks caused by sars-cov- are worsening around the world, and there are no target drugs to treat covid- . ifn-κ inhibits the replication of sars-cov- ; and tff is a small secreted polypeptide that promotes the repair of mucosal injury and reduces the inflammatory responses. we used the synergistic effect of both proteins to treat covid- . methods: we conducted an open-label, randomized, clinical trial involving patients with moderate covid- . patients were assigned in a : ratio to receive either aerosol inhalation treatment with ifn-κ and tff every h for six consecutive dosages in addition to standard care (experimental group) or standard care alone (control group). the primary endpoint was the time until a viral rna negative conversion for sars-cov- in all clinical samples. the secondary clinical endpoint was the time of ct imaging improvement. data analysis was performed per protocol. this study was registered with chictr.org.cn, chictr . findings: between march and may of , covid- patients with symptoms of moderate illness were recruited, and patients were excluded due to not matching the inclusion criteria (patients with pneumonia through chest radiography). among the remaining patients, patients were assigned to experimental group, and the others were assigned to control group to only receive standard care. efficacy and safety were evaluated for both groups. the time of viral rna negative conversion in experimental group (mean, · days, % ci · – · ), was significantly shorter than that in control group ( · days, % ci · to · ) (p = . ), and difference between means was · days. the percentage of patients in experimental group with reversion to negative viral rna was significantly increased compared with control group on all sampling days (every day during the -day observation period) (p = · ). for the secondary endpoint, the experimental group had a significantly shorter time until improvement was seen by ct (mean · days, n = / , % ci · – · ) than that in control group ( · days, n = / , % ci · – · ) (p = . ), and difference between means was · days. no discomfort or complications during aerosol inhalation were reported to the nurses by any experimental patients. interpretation: in conclusion, we found that aerosol inhalation of ifn-κ plus tff in combination with standard care is safe and superior to standard care alone in shortening the time up to viral rna negative conversion in all clinical samples. in addition, the patients in experimental group had a significantly shortened ct imaging improvement time than those in control group. this study suggested that this combination treatment is able to facilitate clinical improvement (negative for virus, improvement by ct, reduced hospitalization stay) and thereby result in an early release from the hospital. these data support the need for exploration with a large-scale trial of ifn-κ plus tff to treat covid- . funding: funding was provided by the national natural science foundation of china, national major project for control and prevention of infectious disease in china, shanghai science and technology commission, shanghai municipal health commission. background: epidemic outbreaks caused by sars-cov- are worsening around the world, and there are no target drugs to treat covid- . ifn-k inhibits the replication of sars-cov- ; and tff is a small secreted polypeptide that promotes the repair of mucosal injury and reduces the inflammatory responses. we used the synergistic effect of both proteins to treat covid- . we conducted an open-label, randomized, clinical trial involving patients with moderate covid- . patients were assigned in a : ratio to receive either aerosol inhalation treatment with ifn-k and tff every h for six consecutive dosages in addition to standard care (experimental group) or standard care alone (control group). the primary endpoint was the time until a viral rna negative conversion for sars-cov- in all clinical samples. the secondary clinical endpoint was the time of ct imaging improvement. data analysis was performed per protocol. this study was registered with chictr.org.cn, chictr . findings: between march and may of , covid- patients with symptoms of moderate illness were recruited, and patients were excluded due to not matching the inclusion criteria (patients with pneumonia through chest radiography). among the remaining patients, patients were assigned to experimental group, and the others were assigned to control group to only receive standard care. efficacy and safety were evaluated for both groups. the time of viral rna negative conversion in experimental group (mean, ¢ days, % ci ¢ À ¢ ), was significantly shorter than that in control group ( ¢ days, % ci ¢ to ¢ ) (p = . ), and difference between means was ¢ days. the percentage of patients in experimental group with reversion to negative viral rna was significantly increased compared with control group on all sampling days (every day during the day observation period) (p = ¢ ). for the secondary endpoint, the experimental group had a significantly shorter time until improvement was seen by ct (mean ¢ days, n = / , % ci ¢ À ¢ ) than that in control group ( ¢ days, n = / , % ci ¢ À ¢ ) (p = . ), and difference between means was ¢ days. no discomfort or complications during aerosol inhalation were reported to the nurses by any experimental patients. interpretation: in conclusion, we found that aerosol inhalation of ifn-k plus tff in combination with standard care is safe and superior to standard care alone in shortening the time up to viral rna negative conversion in all clinical samples. in addition, the patients in experimental group had a significantly shortened ct imaging improvement time than those in control group. this study suggested that this combination treatment is able to facilitate clinical improvement (negative for virus, improvement by ct, reduced hospitalization stay) and thereby result in an early release from the hospital. these data support the need for exploration with a large-scale trial of ifn-k plus tff to treat covid- . covid- sars-cov- tff ifn-k clinical tiral aerosol inhalation treatment the recent emergence of the coronavirus disease (covid- ) pandemic caused by the novel pathogenic severe acute respiratory syndrome coronavirus (sars-cov- ) has affected more than million patients, with more than , deaths in more than countries [ ] . at present, many therapeutic approaches are being evaluated, since there is no specific treatment that has been proven effective. current clinical management includes symptomatic treatment or supportive care, with supplemental oxygen and mechanical ventilatory support when indicated, so there is an urgent need to identify active antiviral agents. chloroquine (cq) and hydroxychloroquine (hcq) are considered the most promising agents against covid- [ ] . however, the clinical efficacy of hcq in the treatment of patients with covid- is controversial. on the one hand, several clinical studies have shown that hcq treatment had a favorable effect, including shortened body temperature recovery time and cough remission time; decreased pneumonia proportion, as assessed by computed tomography (ct) scan [ ] ; alleviated symptoms; and decreased c reactive protein concentration [ ] . on the other hand, a french study conducted in covid- patients with relatively severe illness did not show any difference regarding transfer to the icu or death [ ] . furthermore, in a large-scale observational study involving hospitalized covid- patients, hydroxychloroquine administration was not associated with either intubation or death [ ] . in addition, compassionate use of a nucleotide analog, remdesivir, contributed to clinical improvement in patients with severe covid- in a small study [ ] . however, two recent larger clinical trials showed that remdesivir in adults hospitalized with covid- was not associated with statistically significant clinical benefits, except for shortening the time to recovery, which was accompanied by serious adverse events [ , ] . other antiviral agents, such as lopinavir-ritonavir, were also shown to have no benefit in hospitalized adult patients with severe covid- [ ] . therefore, new effective antiviral agents need to be developed to combat covid- . blood from patients with severe covid- had impaired type i interferon activity and exacerbated inflammatory responses [ ] . an in vitro study has shown that sars-cov- was sensitive to type i interferon pretreatment [ ] . in a small-scale clinical trial, treatment with ifn-a b significantly reduced the duration of detectable virus in the upper respiratory tract and elevated the blood levels of the inflammatory markers, il- and c-reactive protein (crp) [ ] . in addition, early triple-antiviral therapy with a combination of interferon beta- b, lopinavir-ritonavir, and ribavirin was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospitalization stay in patients with mild to moderate covid- [ ] . however, the persistent inflammatory responses resulting from ifn-a/b may cause damage to infected patients. in the study, interferon beta- b was injected only before day from symptom onset to avoid its proinflammatory effects [ ] . interferon kappa (ifn-k) is a relatively mild type i interferon that can effectively inhibit the replication of enveloped viruses, including encephalomyocarditis virus (emcv), influenza avian virus (iav), hepatitis c virus (hcv), and others, by activating the interferon-stimulated response element signaling pathway [ ] . however, unlike ifn-a or ifn-b, the antiviral activity of ifn-k is cell-associated [ ] ; ifn-k inhibits the replication of influenza virus largely through the ifnar-mapk-fos-chd axis [ ] , whereas the effects of ifn-a or ifn-b are mainly through the stat pathway. trefoil factor (tff ) is a small secreted polypeptide that promotes the repair of injury and reduces the inflammatory response [ , ] . our previous clinical pilot study indicated that aerosol inhalation of ifn-k plus tff is a safe treatment and is able to significantly facilitate clinical improvement, including cough relief, ct imaging improvement, and viral rna reversion, thereby resulting in an early release from hospitalization without induction of a proinflammatory response [ ] . to further optimize the therapeutic efficacy, we launched a clinical trial that combined standard care with aerosol inhalation (ifn-k plus tff ) to evaluate the efficacy and safety in patients with moderate covid- . this was an open-label, randomized clinical trial conducted from march , , for virologically confirmed covid- patients evidence before this study we searched pubmed and china national knowledge infrastructure database for articles that use tff , ifn-k, or the combination of both, to treat covid- patients on march , using the search terms ("novel coronavirus" or "sars-cov- or "covid- ) and ("tff " or "ifn-k") with no language or time restrictions. however, no published works were found about the treatment of tff , ifn-k, or the combination of both, in adult patients with covid- . it is urgently needed to develop an effective treatment regimen for covid- . ifn-a/b treatment could suppress viral replication, however, may also trigger persistent inflammatory responses and cause the damage to patients. this study employed a combination approach of a mild type i interferon ifn-k to suppress viral replication and a host factor tff to reduce inflammatory responses and promote the repair of damaged respiratory mucosa. this strategy has been demonstrated as a safe and effective regimen in our previous pilot clinical study, this study is a randomized and scaled-up one to further to prove its effectiveness and safety. this study demonstrated that the combination inhalation of ifn-k and tff is able to shorten the time of viral rna negative conversion and ct improvement, and facilitating patients early discharge from the hospital, in the absence of induction of a proinflammatory response and treatment-related adverse events. overall, the data from our two clinical trials validated it is worth to test this regimen in large scale clinical trials and in severe patients. recruited from the shanghai public health clinical center, which is a designated and authorized hospital to receive and cure adult patients with covid- in shanghai, china. hospitalized patient that was positive on rt-pcr for sars-cov- in throat swabs was enrolled in this study. the inclusion criteria were as follows: patients gave written consent for participation in the study. male and nonpregnant female patients at years of age or older were eligible after they were confirmed as sars-cov- positive by rt-pcr. in addition, patients were included if their peripheral capillary oxygen saturation (spo ) was > % on room air at screening. symptoms of infection include fever, cough, and myalgia, with diarrhea, with the subsequent development of dyspnea or of pneumonia on chest ct. patients with moderate pneumonia were then included following diagnosis and treatment protocol for novel coronavirus pneumonia (trial version ) released by national health commission & state administration of traditional chinese medicine on march , ) [ ] . the exclusion criteria included a physician's decision that involvement in the trial was not in the patient's best interest, presence of any condition that would not allow the protocol to be followed safely, known allergy or hypersensitivity to ifn-k and tff , known severe liver disease (e.g., cirrhosis, with an alanine aminotransferase level > £ the upper limit of the normal range ( À u/l) or an aspartate aminotransferase level > £ the upper limit of the normal range ( À u/l)). breastfeeding and pregnant patients were also excluded. the randomized treatment was open label. moderate covid- patients showing fever, respiratory symptoms and radiological findings of pneumonia were recruited by clinical doctors. after admission, the doctor first introduced the study to the patients. if a patient agreed to join in the study, he/she would voluntarily sign the informed consent form. patients who met the inclusion criteria were enrolled in the study, assigned with randomized numbers, the random allocation sequence was generated through the website "https://www.randomizer.org/#randomize", and then sorted into either experimental group or control group. based on the purpose of this study and our previous explorative pilot study [ ] , considering % of shedding cases, we calculated that a sample of approximately participants per group was required for approximately % statistical power in using a two-sided, two-sample t-test by pass v software, assuming the true difference between the means to be - , with standard deviation of . the significance level (alpha) of the test is ¢ . therefore, eligible patients were enrolled and randomly assigned to either ifn-k plus tff or control group with standard care at a ratio of : by a simple randomization with no stratification. consequently, patients were assigned to experimental group, and patients for control group. the age, sex, baseline demographics, and laboratory test results in each group were comparable. fever and unproductive cough were the most common presenting signs and symptoms. no significant differences were observed between two groups at baseline (table ) . no discomfort or complications during aerosol inhalation were reported to the nurses by the patients in the study. ct imaging improvement was evaluated and interpreted by radiologists who are blinded to the arm of the patients. all data were collected from electronic data files. all enrolled patients started to receive standard care once they were admitted to the hospital, standard care included symptomatic treatment with hydroxychloroquine, antibiotic agents, vasopressors, antifever medicine, vitamin c, immune enhancers, or traditional chinese medicines. the therapy of aerosol inhalation was started on the second day after admission only in experimental group. aerosolized substances were made of purified mature ifn-k and tff proteins produced by the novoprotein company under conditions in accordance with good manufacturing practices (gmp); the purity of the proteins was more than %, and the biological activities of the two proteins were verified in vitro. in addition, both proteins ( mg tff plus mg ifn-k) were dissolved in ml sterilized water, and the combination aerosol was delivered to the patient for À min by a nasal mask driven by a medical compressed air atomizer (yuwell, m). the aerosol inhalation treatment started from the first day of hospitalization and was administered times every h. after treatment, a survey was implemented to evaluate whether there were any adverse reactions during the course of aerosol inhalation. the use of a placebo group was generally not accepted among the specialists responsible for the treatment of covid- . our previous study showed that aerosol inhalation of ifn-k plus tff is a safe treatment and is able to significantly facilitate clinical improvement [ ] . clinical findings, including medical history and physical, laboratory and radiological examination results were entered into a predesigned database. chest radiographs were taken at baseline and at regular intervals for monitoring patient progress. the initial diagnosis of sars-cov- infection was made upon admission. all recruited patients underwent daily collection of nasopharyngeal swabs, throat swabs, and stool swabs to test for the presence of sars-cov- by rt-pcr until discharged from hospital. complete blood count, liver and renal function tests, crp, and creatine kinase were regularly determined until discharge. blood and urine samples for bacterial culture were taken when clinically indicated. all patients were followed up at the infectious disease clinic within days after discharge. all the data of biochemical and blood indexes were used to analyze the kinetic changes. lymphocyte subset counting was performed. cd + t, cd + t, cd + t, and cd + cd + natural killer (nk) cells were stained by using bd multitest tm -color tbnk reagent in trucount tubes and analyzed using the bd facs-canto tm Ⅱ flow cytometer. we collected plasma samples from covid- patients at three time points, including before, among, and after aerosol inhalation. patients' plasmas ( from each group) were randomly selected for emergency testing and thereby unavailable for this assay. all left patients' plasmas were analyzed for biomarkers (ifn-g, il- b, il- , il- , il- , il- , il- , il- p , il- , tnf-a) using the simoa cor-plex human cytokine panel kit (cat no: - ). the assay was carried out according to the manufacturer's (quanterix, billerica, ma, usa) protocols. briefly, ¢ ml of plasma sample was diluted -fold with sample diluent from the kit. multiconstituent calibrators were prepared and added, together with the diluted samples, to -well microplates prespotted with analyte-specific capture antibodies. the microplate was incubated for h. after incubation, unbound proteins were washed away, and biotinylated detection antibody reagent was added for min. after the unbound detection antibody was washed away, streptavidin-horseradish peroxidase (sa-hrp) was added for min. the microplate was then washed, and the substrate was added. the microplate was imaged on the sp-x platform within ~ min. each microplate contained calibrators that were used to calculate cytokine concentrations for the plasma samples. the viral loads of sars-cov- in nasopharyngeal swabs, throat swabs and stool swabs were determined by quantitative real-time rtÀpcr according to previously report [ ] . simply, they were assayed by using takara one step primescript rtÀpcr kit (takara rr a) following the manufacturer's instructions. reverse transcription was carried out at °c for min, °c for min, followed by cycles of °c for s and °c for min. the reactions were performed by abi real-time pcr systems. ct values were collected and viral loads were analyzed according to the ct value of the standard samples. the primary endpoint was the timing to achieve viral rna negative conversion for sars-cov- in all three specimens, including nasopharyngeal swabs, throat swabs and stool swabs. when a covid- patient was discharged from hospital, the viral rna negative conversion of all three specimens needed to be considered. the secondary clinical endpoint was the timing when ct imaging improvement was observed, which was mainly based on the size and density reduction of lesions. safety data included adverse events during treatment, severe adverse events, and premature discontinuation of treatment. adverse events were classified according to the national cancer institute common terminology criteria for adverse events, version . [ ] . baseline comparisons of clinical and demographic characteristics according to study group allocation were done using student's t-test for parametric continuous data or the mannÀwhitney u test for nonparametric data. categorical data were compared using x test. the primary endpoint, secondary endpoint and safety were assessed in all randomized patients. the quantitative indexes were described as the mean values (mean), and comparisons between experimental group and control group were analyzed using the t-test. all statistics were conducted using a two-sided scale, and a p-value less than or equal to ¢ was considered statistically significant. statistical the funding source of this study had no role in the study design, data collection, data analysis, data interpretation, or other report writing. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit the manuscript for publication. between march and may of , covid- patients with symptoms of moderate illness were screened; patients did not fulfill the inclusion criteria (of patients with pneumonia on chest radiograph), and the remaining patients were randomly divided into two groups, each group for patients (fig. ) . the primary endpoint was a significantly shorter time (mean ¢ days, % ci ¢ À ¢ ) from the start of the study treatment to viral rna negative conversion for sars-cov- in all clinical samples, including nasopharyngeal swabs, throat swabs and stool swabs, in experimental group than in control group ( ¢ days, % ci ¢ À ¢ ) (p = ¢ ), and difference between means was ¢ days (fig. a) . the percentage of patients in experimental group who had reversion to a negative viral rna was significantly increased compared with that in control group on any sampling day (every day during the -day observation period) (p = ¢ ) (fig. b) . the second endpoint was improvement of patients with confirmed pneumonia by chest ct after days of treatment (the start time of hospitalization). experimental group had a significantly shortened time (mean ¢ days, n = / , % ci ¢ À ¢ ) until the improvement of their ct results than control group ( ¢ days, n = / , % ci ¢ À ¢ ) (p = ¢ ), and difference between means was ¢ days (fig. c) . there were patients in experimental group and patients in control group with unchanged ct imaging from the start of treatment to discharge. pulmonary ct imaging changes were defined as three levels: exacerbated, unchanged, and improved. after continued aerosol inhalation treatment for days, the rates of improvement in ct imaging in experimental group and control group were ¢ % ( / ) and ¢ % ( / ), respectively, with no statistical significance. after aerosol inhalation treatment for days, the rates of improvement in ct imaging in experimental group and control group were % ( / ) and ¢ % ( / ), respectively, p < ¢ . after days of treatment, the rates of improvement in ct imaging rose to ¢ % ( / ) in experimental group and ¢ % ( / ) in control group, respectively, p < ¢ . after days of treatment, the rates of improvement in ct imaging rose to ¢ % ( / ) and ¢ % ( / ), respectively, with no statistical significance. in addition, experimental group was significantly less hospitalized (mean ¢ , % ci ¢ À ¢ ) in comparison with control group ( ¢ , % ci ¢ À ¢ ) (p = ¢ ), and the difference between means was ¢ (fig. d ). the hospitalization time for patients staying in the hospital was longer than the our last study [ ] , because the clinical research at the shanghai public health clinical center had revised the discharge evaluation criteria (including nasal ct improvement which is usually taking prolonged times). in this trial, we found that aerosol inhalation of ifn-k plus tff is a safe treatment. compared with the control group, total bilirubin (t-bil), alt, ast and creatinine showed no significant differences, p > . , there were also not clinical significance at every following up time-point, and they all fell into normal ranges for both groups (fig. aÀd) , indicating that ifn-k plus tff treatment did not negatively impact the liver, gallbladder, or heart. interestingly, a significant less crp was noticed in experimental group on day when compared with that in control group, p = . (fig. e) , suggesting the administration of ifn-k plus tff might facilitate rapid containing of inflammation. we then analyzed the kinetic changes in the effectiveness index in the peripheral blood of control group and experimental group. the total white blood cell (wbcs) absolute counts, blood platelet counts and lymphocyte counts were all not significantly different between two groups, which suggested that ifn-k plus tff treatment had no significant effect on total blood parameters (fig. fÀh) . after treatment for days, all blood indexes returned to values in the normal range. we further determine the kinetic of different lymphocyte subsets (cd + t, cd + t, cd + t and cd + cd + nk) in the peripheral blood of two groups of patients. cd + t, cd + t, cd + t cell counts in both groups were continuously increasing whereas nk cells remained stable during the whole clinical observation with no significant differences at every time point between two groups, and all were fallen in the normal ranges for both groups (fig. aÀd ). in addition, the level of plasma cytokines from those patients with moderate covid- was low, and the inflammatory cytokines (such as il- b, tnf-a, il- , il- ) in the early stage ( ~ days) in experimental group showed a slight fluctuation temporarily; however, after days of aerosol inhalation treatment, the level of plasma cytokines gradually decreased, and showed lower than that in control group, except for il- (fig. ) . no serious from this randomized open-label clinical trial in patients with covid- , we found that aerosol inhalation of ifn-k plus tff in combination with standard care is effective in suppressing and clearing sars-cov- , not just in throat samples but also in all clinical specimens. most patients treated with aerosol inhalation of ifn-k plus tff were rt-pcr negative in all specimens by ¢ days. furthermore, ct improvement time in experimental group was significantly shorter, by ¢ days, than that in control group. in addition, ifn-k plus tff treatment significantly shortened the duration of hospitalization. the side effects of the combined aerosol treatment were generally mild and self-limiting. the results were highly consistent with the first clinical study of ifn-k plus tff in patients with moderate covid- [ ] . specific highly active antiviral drugs for any emerging infectious disease are always needed because the development of a new antiviral drug through the preclinical and clinical stages takes years before its approval for clinical use. when the sars-cov- epidemic suddenly emerged, most of the antiviral research focused on therapeutic agents with some in vitro activity against betacoronavirus, including favipiravir, remdesivir, lopinavir-ritonavir, chloroquine, hydroxychloroquine and interferons [ À ]. these drugs have known pharmacokinetic and pharmacodynamic properties, side effects, and dosing regimens. unfortunately, most studies have shown limited therapeutic effects in covid- patients, and some effective therapeutics in published papers have been questioned for various reasons [ ] . lopinavir-ritonavir was shown to have similar effects to placebo on reducing viral load, despite some improvement in symptoms [ ] . in patients with severe covid- receiving compassionate-use remdesivir, clinical improvement was observed in of patients ( %) [ ] . in addition, the clinical efficacy of hcq in the treatment of patients with covid- is controversial. in summary, there has been no strong evidence reported so far for specific effects in the treatment of covid- . interestingly, early triple-antiviral therapy with a combination of interferon beta- b, lopinavir-ritonavir, and ribavirin was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate covid- [ ] . interferon-a b treatment for covid- significantly reduced the duration of detectable virus in the upper respiratory tract [ ] . these findings suggest that interferons should be further investigated as a therapy in covid- . in our study, we observed that ifn-k plus tff added to standard supportive care was effective in clearing sars-cov- in the throat, which implied that ifn-k plus tff might inhibit sars-cov- replication in vivo and thereby promote negative reversion of viral rna, similar to hydroxychloroquine, remdesivir and interferon treatments [ , , ] . in addition, the prognosis of covid- patients has been associated with the levels of pro-inflammatory cytokines in the peripheral blood [ ] , and tocilizumab therapy has been applied to counteract the cytokine storm in patients with severe covid- [ ] . thus, it is critical to examine whether treatment with ifn-k plus tff influences inflammation in covid- patients. crp was monitored as an indicator of inflammatory responses and showed a lower level in experimental group than in control group, indicating that treatment with ifn-k plus tff may eventually reduce inflammatory responses. in addition, plasma inflammatory cytokine test results showed that aerosol inhalation of ifn-k plus tff did not significantly induce inflammation in patients with moderate covid- . the synergistic effect of ifn-k and tff proteins shortened the duration of hospitalization of covid- patients. surprisingly, the higher level of il- expression was maintained in experimental group. as known, tff is able to improve the mucosal reconstitution while il- is also known to facilitate the mucosal recovery [ ] , it is possible that tff could up-regulate il- and exerts its functionality. indeed, previous studies showed that il- reduced lung inflammation and promoted lung epithelial repair during influenza virus infection [ , ] . however, the relationship between il- and prognosis in covid- patients need to be further investigated. in this study, the combination of ifn-k plus tff with standard care was a safe treatment for patients with moderate covid- . safety evaluations, including wbc levels, lymphocytes, crp, hemoglobin, alt, ast, blood platelets and t-bil, all fell within normal ranges, and no severe adverse effects were observed during hospitalization or follow-up after release, suggesting that ifn-k plus tff did not affect the function of the liver, blood, gallbladder, kidney and heart of covid- patients. recent reports show that patient lymphocyte counts exhibit a graded decline from mild, moderate and severe covid- [ , ] whereas mild and moderate covid- patients remain normal [ ] , which was consistent with our results. in addition, ifn-k and tff are small proteins that could be endogenously induced to respond to respiratory viral infections [ ] , therefore, they are safe and effective, convenient for transportation, and easy to prepare and use. our clinical survey showed that there was no discomfort during aerosol inhalation. overall, this therapeutic strategy could be quickly popularized and implemented. cautions should be taken to interpret these data. first, this trial was open label in the absence of a placebo group. second, the trial was only single centered and not blinded, it was possible that knowledge of the treatment assignment might have influenced clinical decision-making that could have affected the ordinal scale measurements we used. in the future, a larger, multicenter, randomized, double-blind, placebo-controlled clinical trial needs to be carried out to further verify the effectiveness of the treatment strategy in covid- patients. in conclusion, we found that aerosol inhalation of ifn-k plus tff in combination with standard care is safe and superior to standard care alone in shortening the times for viral rna conversion of sars-cov- and for ct improvement and facilitating clinical recovery, thereby resulting in early release from hospitalization. this aerosol inhalation strategy should be developed with priority to provide emergency reserves for the prevention and early treatment of acute respiratory infectious diseases emerging in the future. at present, more clinical data are needed to promote clinical application as early as possible and contribute to sars-cov- epidemic prevention and control. jx configured this study. jx, hl, xz, and tz designed the study, had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. wf and yl contributed to analyzing data and writing the report. jx contributed to critical revision of the report. wf and jw provided the ethics files. yl contributed to the statistical analysis, and jx, xz, wf, and sb provided the purified proteins. ll, pl, zs, jz, jc are the clinical doctors responsible for carrying out this trial by communicating with the patients and their families, and lz, tx, fl, yz, lz are the nurses responsible for the implementation of the aerosol inhalation by patients. hh, xc, and zs were responsible for collecting the clinical data of the enrolled patients and providing viral load data. sy, ld monitored the serum of covid- patients. all authors reviewed and approved the final version. data are available on various websites and have been made publicly available. additional materials may be requested after approval from the corresponding author and national health commission. jianqing xu, xiaoyan zhang, weihui fu, and songhua yuan have applied for the patent . (pending), and the patent pct/cn / (pending) base on these data. all authors reviewed the signed the conflict of interest forms. who. coronavirus disease (covid- ) situation report review: hydroxychloroquine and chloroquine for treatment of sars-cov- (covid- ) efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial hydroxychloroquine in patients mainly with mild to moderate covid- : an open-label, randomized, controlled trial no evidence of clinical efficacy of hydroxychloroquine in patients hospitalized for covid- infection with oxygen requirement: results of a study using routinely collected data to emulate a target trial observational study of hydroxychloroquine in hospitalized patients with covid- compassionate use of remdesivir for patients with severe covid- remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for the treatment of covid- -preliminary report a trial of lopinavir-ritonavir in adults hospitalized with severe covid- impaired type i interferon activity and exacerbated inflammatory responses in severe covid- patients sars-cov- sensitive to type i interferon pretreatment interferon-alpha b treatment for covid- triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial interferon-kappa, a novel type i interferon expressed in human keratinocytes antiviral activity of transiently expressed ifn-kappa is cell-associated ifn-kappa suppresses the replication of influenza a viruses through the ifnar-mapk-fos-chd axis trefoil factor- reverses airway remodeling changes in allergic airways disease trefoil peptide tff (spasmolytic polypeptide) potently accelerates healing and reduces inflammation in a rat model of colitis a clinical pilot study on the safety and efficacy of aerosol inhalation treatment of ifn-k plus tff in patients with moderate covid- diagnosis and treatment protocol for novel coronavirus pneumonia (trial version ) a new coronavirus associated with human respiratory disease in china common terminology criteria for adverse events (ctcae), version . in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) therapeutic options for the novel coronavirus ( -ncov) broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus in vitro susceptibility of clinical isolates of sars coronavirus to selected antiviral compounds role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment with lopinavir/ritonavir or interferon-beta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease discovering drugs to treat coronavirus disease (covid- ) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov- infected patients tocilizumab treatment in covid- : a single center experience healing of intestinal inflammation by il- interleukin- reduces lung inflammation during influenza a virus infection and protects against secondary bacterial infection il- is essential for lung epithelial repair following influenza infection clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china viral and host factors related to the clinical outcome of covid- the authors thank xuanyi wang and honglu zhou for the statistical assistance and the measurement of sample size, and xiujing hong for the perfect of the pictures. in addition, we thank all patients who participated in this trial and their families. and all the clinical, technical and paramedical staff of the hospitalization units and laboratories for support in this difficult context. supplementary material associated with this article can be found in the online version at doi: . /j.eclinm. . . key: cord- -m vpizuh authors: kindler, e.; thiel, v.; weber, f. title: interaction of sars and mers coronaviruses with the antiviral interferon response date: - - journal: adv virus res doi: . /bs.aivir. . . sha: doc_id: cord_uid: m vpizuh severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) are the most severe coronavirus (cov)-associated diseases in humans. the causative agents, sars-cov and mers-cov, are of zoonotic origin but may be transmitted to humans, causing severe and often fatal respiratory disease in their new host. the two coronaviruses are thought to encode an unusually large number of factors that allow them to thrive and replicate in the presence of efficient host defense mechanisms, especially the antiviral interferon system. here, we review the recent progress in our understanding of the strategies that highly pathogenic coronaviruses employ to escape, dampen, or block the antiviral interferon response in human cells. coronaviruses have made a remarkable career. originally recognized as viral pathogens of veterinary importance but little medical (i.e., human) relevance, the appearance of sars-cov causing a worldwide epidemic with a large number of fatalities has changed everything. in , the virus emerged in chinese animal markets to circle the world in just a few weeks, teaching us important new lessons on perceived "differences" between animal and human pathogens. just in case someone did not get the message, mers-cov repeated the coronavirus wake-up call years later, providing yet another example for how easily animal viruses may be transmitted and adapt to new hosts including humans. often, the tricks and strategies that viruses evolved to propagate in specific animal hosts may only need some fine-tuning (if at all) to enter the wide world of human crowds, air travel, and camel races. here, we will summarize the insights gathered so far on an important aspect of virulence and host adaptation, the interactions of sars-cov and mers-cov with antiviral interferon (ifn) responses of human cells. the coronavirus genome is composed of a linear, single-stranded, monopartite rna with a cap structure at its end and a polya tail at the end . the -terminal two-thirds of the cov genome contain the open reading frames (orf) a and b that together constitute the viral replicase gene. translation is initiated at the start codon of orf a and may continue to orf b via a ribosomal frameshift mechanism, ultimately giving rise to two overlapping replicase polyproteins pp a and pp ab perlman and netland, ; snijder et al., ; thiel et al., ) . virus-encoded proteinases, namely two papain-like cysteine proteases (pl pro and pl pro ), residing in nonstructural protein (nsp) and a c-like cysteine protease ( cl pro ) associated with nsp , proteolytically process the polyproteins into nsps - (anand et al., ; schiller et al., ; thiel et al., ; ziebuhr et al., ) . a multitude of functions and enzymatic activities associated with specific nsps have been identified over the past years (for reviews, see masters and perlman, ; ziebuhr, ) . moreover, orf b harbors several rna-processing enzymes, including a - exonuclease and a guanosine n -methyltransferase (associated with the n-and c-terminal domains, respectively, of nsp ), an endoribonuclease (nsp ) and a -o-methyltransferase (nsp ) (chen et al., ; decroly et al., ; ivanov et al., ; kindler and thiel, ; minskaia et al., ; perlman and netland, ; snijder et al., ; thiel et al., ; zust et al., ) . the orfs are translated from a set of subgenomic (sg) rnas and yield on one hand four canonical structural proteins like the spike protein (s), the envelope (e), the membrane (m), and the nucleoprotein (n). moreover, sgrnas express accessory genes, which vary in function and number between different cov strains and are interspersed between the structural genes perlman and netland, ; snijder et al., ; thiel et al., ) . specifically, the genome of sars-cov expresses eight different accessory genes ( a, b, , a, b, a, b, and b), while mers-cov encodes five accessory genes ( , a, b, , and b). the schematic overview of the genome organization of sars-cov and mers-cov is depicted in fig. . the cov life cycle starts with the attachment of the viral spike protein to particular cellular receptors, subsequently leading to fusion between the viral envelope and the plasma membrane or the endosome membrane of the host. cov uses a range of receptors, with sars-cov employing angiotensinconverting enzyme (ace ) and mers-cov employing dipeptyl peptidase (dpp ) (li et al., ; raj et al., ) . following membrane fusion, the viral rna genome is delivered into the host cytoplasm, where translation of the two -terminal orfs a and b is accomplished by the cellular translation machinery. most of the newly synthesized nsps assemble with the n protein into a replicase-transcriptase complex (rtc) responsible for viral genome replication and transcription. at the site of replicative organelles , the rtc initiates minus-strand synthesis using the full-length genome as template, thereby either copying the entire template to generate full-length minus strands or to move discontinuously along the template to produce a nested set of sgrnas with negative polarity. the minus strands of genomic and sgrnas are subsequently used as templates to synthesize positive sense strands (mrnas), specifically the genomic rna (genome replication) and sg mrnas (transcription) (sawicki et al., ) . the n protein then encapsidates the newly synthetized rna genome and thereby forms a helical nucleocapsid. virion assembly is triggered by the action of the m protein, which assists in incorporating the nucleocapsid, the envelope and the spike into virus particles. budding takes place between the endoplasmic reticulum and the golgi and new viruses are released by exocytosis (mcbride et al., ; neuman et al., ; ruch and machamer, ; ujike and taguchi, ) . the antiviral ifn (ifn-alpha/beta) system confers an important part of the innate immune defense in chordates (tenoever, ) . ifns are cytokines that are produced and secreted by cells encountering viruses or parts thereof (fig. ) . humans are able to express one ifn-beta, subtypes of ifnalphas, and one each of ifn-kappa and ifn-omega (schneider et al., ) . all nucleated cells are able to respond to them as they express the ifn receptor (composed of the two subunits ifnar- and ifnar- ) on their surface (bekisz et al., ) . the docking of ifn-alpha/beta onto its cognate receptor activates the so-called jak-stat pathway. thereby, the janus kinases jak and tyk are waiting to be activated on the cytoplasmic side of ifnar and , respectively. the activated kinases then phosphorylate the signal transducers and transcription factors stat and stat , which form a complex with irf (isgf ) that enters the nucleus to transactivate promoters of an antiviral gene expression program. genes that are specifically upregulated by ifns are collectively called isgs (ifn-stimulated genes). the alpha/beta-ifns are classified as type i ifns, since they had been discovered first (isaacs and lindenmann, ) . the type ii ifns use a different receptor and consist of only one member, ifn-gamma. ifn-gamma also confers some antiviral activity but is regarded more of an immunoregulator (produced by specialized immune cells) than a general antiviral mediator (schneider et al., ) . it signals through a jak/stat pathway that partially overlaps with one of the type i ifns. ifn-gamma will not be further discussed here as it is not to the core of the antiviral ifn response to coronaviruses. recently, the ifn family was extended by the newly discovered type iii ifns, consisting of ifn-lambda - (schneider et al., ) . type iii ifns resemble type i ifns in that they also trigger stat / phosphorylation via jak and tyk . they employ however a different receptor which is only expressed by epithelial cells (sommereyns et al., ) . thus, type i and type iii ifns trigger largely overlapping sets of isgs, but while the former constitute a major, general antiviral cytokine system, the latter are mainly restricted to mucosal sites (galani et al., ) . the molecular events leading to the upregulation of type i ifns are well established. as indicated earlier, molecular structures that are specific for virus infections (often called pamps, for pathogen-associated molecular patterns) are sensed by pathogen recognition receptors (prrs) of the host, that in turn are triggering the upregulation of ifn genes. a prototypical pamp relevant for coronaviruses is double-stranded rna (dsrna), a by-product of genome replication and transcription (weber et al., ; zielecki et al., ) . dsrna can be sensed by toll-like receptor (tlr ) in the endosome, and in the cytoplasm by the rna helicases rig-i (retinoic acid-inducible gene i) and mda (melanoma differentiation antigen ), as well as by the kinase pkr (protein kinase, rnaactivated) (rasmussen et al., ; yim and williams, ; yoneyama et al., ) . rig-i is thereby specific for long dsrna molecules and short dsrnas bearing a tri-or di-phosphorylated end, whereas mda senses long dsrnas, preferentially with a higher-order structure (binder et al., ; goubau et al., ; pichlmair et al., ; schlee, ) . pkr is activated by dsrna as well as by short stem-loop rnas bearing a triphosphate end (dabo and meurs, ; nallagatla et al., ) . also specific single-stranded rnas (ssrnas) can act as pamps, either if they are in the wrong location or if they display particular features. tlr senses gu-rich ssrna in the endosome (heil et al., ) . rig-i can be triggered by polyu/uc rich or monophosphorylated ssrnas stretches, and mda was found to bind ssrna stretches of negative-sense rna viruses and hypomethylated capped mrnas (luthra et al., ; malathi et al., malathi et al., , rasmussen et al., ; runge et al., ; saito et al., ; zust et al., ) . depending on the particular prr, various-partially cross-talking-signaling pathways lead to the transactivation of promoters for antiviral genes (o'neill et al., ) . the endosomal prr tlr engages the intracellular adapters trif (tir domain-containing adapter protein inducing ifnbeta) and traf (tnf receptor-associated factor ) to activate the kinases tbk (tank-binding kinase ) and ikkepsilon (inhibitor of nf-kappab kinase epsilon). the kinases tbk and ikkepsilon then phosphorylate irf (ifn regulatory factor ), a transcription factor that activates genes for ifns and other immunoregulatory cytokines. signaling by tlr , by contrast, requires the adaptor proteins myd (myeloid differentiation primary-response protein ) and traf to channel to the kinase ikkalpha. this kinase then phosphorylates irf , a transcription factor that covers a gene spectrum similar to irf . tlr /myd also recruits the adaptor protein traf that eventually activates the transcription factor nf-kappab via the kinases ikkalpha and ikkbeta. nf-kappab drives transcription of genes for proinflammatory cytokines but also enhances ifn gene expression. in the cytoplasm, rna sensing by the two prrs rig-i and mda (collectively termed rig-i-like receptors, rlrs) converges on the adaptor protein mavs (mitochondrial antiviral signaling protein) that uses various trafs (traf , , , ) to trigger tbk /ikkepsilon and ikkalpha/ ikkbeta. these kinases then activate irf and nf-kappab, respectively (belgnaoui et al., ; liu et al., ) . besides the rlrs, pkr contributes to ifn induction in the cytoplasm. pkr is a master regulator of mrna translation (see later), but several lines of evidence indicate a role in activation of nf-kappab and irf via traf / , ikkalpha/beta, antiviral stress granule formation, and ifn-alpha/beta mrna stability (gil et al., ; onomoto et al., ; pfaller et al., ; pham et al., ; schulz et al., ; zamanian-daryoush et al., ) . thus, several types of prrs are constantly surveying the extracellular and intracellular space to detect virus infections in a timely and sensitive manner. importantly, tlrs are preferentially expressed by immune cells, especially myeloid dendritic cells (mdcs) and plasmacytoid cells (pdcs), whereas rlrs and pkr are thought to be active in all nucleated cells. detection of viral rna in mdcs is mainly mediated by tlr (and some tlr ), and in pdcs by tlr (and tlr in human pdcs) (schreibelt et al., ) . pamp sensing by prrs eventually culminates in activation of irf , irf , and nf-kappab, as described earlier, the transcription factors driving the expression of genes for ifn-beta, ifn-alpha, and various proinflammatory and immunomodulatory cytokines (belgnaoui et al., ) . signaling by both type i and type iii ifns triggers the formation of isgf (see section . ), the heterotrimeric transcription factor complex consisting of phosphorylated stat and stat , and irf (schneider et al., ) . isgf binds to the isres (for ifn-stimulated response element), specific promoter sequences of the so-called isgs. of note, there are actually several types of "isres" that are responding to different types of triggers and transcription factors. first, there are the isres that purely respond to ifn signaling and isgf , as it would be expected from the name. a prominent example is given by the promoter of the human antiviral protein mxa (holzinger et al., ) . second, there are the-somewhat mislabeled-isres that do not respond to ifn at all, but only to the irf -, irf -, and nf-kappab-related signal transduction that occurs much earlier, directly after virus infection has triggered a prr. the ifn-beta promoter belongs to this class of isres (freaney et al., ; schmid et al., ) . third, there are mixed-type isres that can be activated by both virus infection and ifns. an example is the promoter of the gene for the antiviral protein ifit (also known as isg ) (fensterl and sen, ) . the different isre classes can be distinguished as irf-specific isres (responding only to prr signaling), isgf -specific isres (responding only to type i or type iii ifns), and universal isres (responding to both infection and ifns) (schmid et al., ) . many isgs are controlled by additional promoter elements ensuring basal levels of expression already in the absence of ifn. moreover, low levels of ifn itself are constitutively secreted by many tissues (tonic ifn), ensuring physiological homeostasis and priming of cells for a rapid response against pathogens (gough et al., ) . it is estimated that, depending on the ifn subtype, dose, and cell type, ifns regulate hundreds, if not thousands of genes (rusinova et al., ) . many of the isgs (i.e., those genes that are upregulated by ifns) are known to have antiviral, immunomodulatory, or antiproliferative function (samuel, ; stark and darnell, ) . the broad antiviral activity of ifns occurs on several levels, namely virus entry, viral polymerase function, host cell translation, rna availability, rna stability, particle budding, apoptosis, or general boosting of innate and adaptive immune responses. high-dose ifn treatment (type i and type iii) has clear effects against sars-cov and mers-cov in cell culture cinatl et al., ; falzarano et al., ; kindler et al., ; spiegel et al., ; stroher et al., ; zielecki et al., ) , in animal experiments (channappanavar et al., ; frieman et al., ; haagmans et al., ; mahlakoiv et al., ; mordstein et al., ) , and possibly also in patients (loutfy et al., ; omrani et al., ; strayer et al., ) . remarkably, mers-cov was found to be substantially more ifn sensitive than sars-cov in cell culture . the cellular basis for the relatively low (sars-cov) and high (mers-cov) ifn sensitivity is currently unknown. several prominent (i.e., potent) isg products were studied in the context of human pathogenic coronaviruses, but only some of them were found to have an effect. the ifn-induced transmembrane (ifitms) proteins , , and restrict the entry of many enveloped viruses including sars-cov (huang et al., b) as well as reoviruses . they act by altering the site of membrane fusion, but the exact mechanism remains to be elucidated . strikingly, while ifitms are inhibitory for the highly pathogenic sars-cov, they appear to boost infection with the related, low pathogenic coronavirus hcov-oc (zhao et al., ) . in particular, ifitm or ifitm acts as entry factor for hcov-oc by facilitating-rather than impeding-membrane fusion. human mxa (for myxovirus resistance protein a) is a well-known antiviral host factor with activity against a wide range of (mostly) rna viruses (haller et al., ) . it blocks early replication steps of influenza viruses but was found have no effect on sars-cov (spiegel et al., ) . the kinase pkr is an isg product acting as a signaling prr on one hand (see earlier), but its main function in antiviral defense is the inhibition of protein synthesis. after binding viral dsrna, pkr undergoes autophosphorylation to activate itself, and subsequently phosphorylates eif- alpha that is thereby converted from a translation initiation factor to a translation inhibitor (yim and williams, ) . pkr has a broad antiviral spectrum. nonetheless, pkr has no bearing on the replication of sars-cov, although it is involved in virally induced apoptosis (krahling et al., ) . also the - oligoadenylate synthetase (oas) family members are triggered by viral dsrna (chakrabarti et al., ) . in the dsrnabound state they synthesize short chains of - oligoadenylates that activate the latent rnase l. rnase l then cleaves virus and host ssrnas, predominantly at single-stranded ua and uu dinucleotides (wreschner et al., ) . interestingly, the small -monophosphorylated cleavage products of rnase l are recognized by the prrs rig-i and mda , thus amplifying the ifn response in an infection-dependent manner (malathi et al., ) . polymorphisms of the oas- gene might affect susceptibility to sars-cov (hamano et al., ) , but to our knowledge, there is no direct data on antiviral effects of the oas/rnase l system on human coronaviruses. for the mouse coronavirus mhv-a , however, it was shown that mutants deficient in the ns gene are highly sensitive against rnase l (zhao et al., ) (see also later). as mentioned, there are several hundreds of isgs, of which about were characterized as being antiviral (schneider et al., ) . it is in a way remarkable that relatively little is known about isgs that impede human pathogenic coronaviruses. most likely, active and passive evasion mechanisms such as the ones described later are responsible for the relative insensitivity of at least sars-cov against ifn and potent antiviral isgs. although our review will focus on the human pathogenic coronaviruses sars-cov and mers-cov, we will draw additional conclusions from well investigated other coronaviruses whenever adequate. viral evasion strategies against the ifn response can act on several levels, namely the induction of ifn, ifn signaling, or antiviral action of individual isg products (gack, ; kindler and thiel, ; vijay and perlman, ; weber and weber, ; wong et al., ; zinzula and tramontano, ) . the viruses can thereby actively sequester or destroy key regulators, or otherwise interfere with the ifn system. moreover, several aspects of the viral replication cycle can be regarded as a passive ifn evasion. the strategies described later are also summarized in three tables. both sars-cov and mers-cov induce very little-if any-ifn in most cell types cheung et al., ; kindler et al., ; lau et al., ; menachery et al., a; spiegel et al., ; zhou et al., ; ziegler et al., ; zielecki et al., ) . in fact, it was recently shown in a mouse model of sars that the delay in ifn induction is responsible for the activation of proinflammatory monocyte-macrophages and cytokines in the lung, resulting in vascular leakage and impaired adaptive immune responses (channappanavar et al., ) . thus, the high levels of dsrna that are produced during replication (weber et al., ; zielecki et al., ) do not result in an adequate ifn induction. one of the reasons (besides the active measures described later) is certainly the storage of coronaviral dsrna inside double-membrane vesicles van hemert et al., ; versteeg et al., ) . moreover, the n protein sequesters ifninducing rna pamps lu et al., ) . however, the fact that infection with coronaviruses activates the cytosolic dsrna-sensing host factors pkr and oas (birdwell et al., ; krahling et al., ; zhao et al., ) , as well as the existence of numerous mechanisms dedicated to suppress dsrna-dependent ifn induction (see later) strongly suggest that dsrna stashing alone is not sufficient and that some dsrna or other pamps are exposed to prrs, thus necessitating the presence of additional, active mechanisms. while most cell types remain ifn-silent after infection, a notable exception are pdcs, which express high levels of ifn-alpha/beta in response to infection with both sars-cov and mers-cov (cervantes- channappanavar et al., ; scheuplein et al., ) . for the mouse coronavirus mhv-a it was shown that ifn induction in pdcs occurs through tlr (cervantes- , suggesting the same to be true for sars-cov and mers-cov. indeed, gu-rich ssrnas from the sars-cov genome were shown to activate an excessive innate immune response via tlr . moreover, the membrane (m) protein and the envelope (e) protein of sars-cov are able to activate a tlr-like pathway and nf-kappab signaling, respectively (dediego et al., ; wang and liu, ) . the mouse coronavirus mhv-a also naturally induces ifn in brain macrophages/microglia, with mda being the responsible prr (birdwell et al., ; roth-cross et al., ) . also in oligodendrocytes ifn induction by mhv occurs through both mda and rig-i (li et al., ) . interestingly, a general (i.e., not restricted to particular cell types) mda -dependent ifn induction can be obtained by ablating the ribose -o-methylation activity of the nsp . as it was shown for mhv-a , sars-cov, and the mildly human pathogenic coronavirus hcov- e, nsp -mediated -o-methylation of viral mrna cap structures prevents recognition by mda (menachery et al., b; zust et al., ) . besides these "hiding" or "disguising" strategies, active mechanisms targeting specific host factors are in place (table ). sars-cov was shown to inhibit irf by preventing its hyperphosphorylation, dimerization, and interaction with the cofactor cbp (spiegel et al., ) . curiously, irf initially enters the nucleus of infected cells, but later returns to the cytoplasm. sars-cov also inhibits the nuclear import of the related transcription factor irf (kuri et al., ) . in this context, the papain-like protease (pl pro ) domain of nsp (the largest coronaviral protein) of sars-cov and the mildly pathogenic hcov-nl both interact with irf and block its activation (devaraj et al., ; frieman et al., ) . moreover, pl pro was shown to drive the deubiquitination (or inhibit ubiquitination) of rig-i, tbk , and irf (clementz et al., ; devaraj et al., ; frieman et al., ; sun et al., ) . irf activation is also prevented by the m protein of sars-cov through inhibiting complex formation between traf and tbk (siu et al., ) . since m was also found to activate a tlr-like signaling pathway (wang and liu, ) , a final picture of m protein function in the context of ifn induction/inhibition remains to be provided. ifn induction is also disturbed by the sars-cov nsp , nsp , nsp , orf b, orf , and orf b proteins, respectively (frieman et al., ; kopecky-bromberg et al., ; shi et al., ; zust et al., ) . the anti-ifn function of nsp is based on its ability to mediate host mrna degradation, while sparing viral mrnas at the same time, and to block host mrna translation (huang et al., a; narayanan et al., ; tanaka et al., ) . nsp also has a function in evasion from ifn signaling (see later), providing a possible reason why nsp mutants are particularly ifn sensitive (wathelet et al., ; zust et al., ) . while the mechanisms of other sars-cov ifn induction antagonists like nsp , nsp , orf b, and orf proteins remain to be characterized, for the orf b protein it was shown that it drives degradation of mavs, traf , and traf by interacting with the host factors pcbp and the e ubiquitin ligase aip (shi et al., ) . also for mers-cov, the reason for the low levels of ifn produced by infected cells kindler et al., ; lau et al., ; menachery et al., a; zhou et al., ; zielecki et al., ) was further investigated. the orf a protein inhibits ifn induction by interaction with dsrna and the rlr cofactor pact (niemeyer et al., ; siu et al., ) . like the orf a, the orf b, , and m proteins of mers-cov were shown to prevent irf translocation (yang et al., ) . the orf b protein, in particular, inhibits ifn induction by binding to tbk and ikkepsilon (matthews et al., ; yang et al., ) . in agreement with the data on sars-cov, the pl pro of mers-cov has deubiquitinating activity and inhibits ifn induction (bailey-elkin et al., ; mielech et al., ) , and the nsp mediates host mrna degradation (lokugamage et al., ) . in contrast to sars-cov, however, infection with mers-cov additionally activates repressive histone modifications that downregulate isg expression (menachery et al., a) . several proteins of sars-cov and mers-cov were found to interfere with the signal transduction chain that leads from ifn docking onto its receptor to the upregulation of isgs by isgf , the stat /stat / irf complex ( table ). the orf a protein was shown to decrease levels of ifnar, most probably by ubiquitination and proteolytic degradation (minakshi et al., ). the orf protein was the first factor described for sars-cov that affects ifn signaling in infected cells, disrupting nuclear import of stat kopecky-bromberg et al., ) . the orf protein binds to the nuclear import factor karyopherin alpha and tethers it (together with karyopherin beta ) to intracellular membranes . there, they become unavailable for their normal cellular function, the import of, e.g., stat . the phosphorylation of stat is impeded by the multifunctional nsp protein of sars-cov, which otherwise drives degradation of host mrnas and inhibits translation (see earlier) (wathelet et al., ) . for mers-cov, the orf a, b, and m proteins inhibit isre activation after stimulation with ifn (yang et al., ) . the mechanisms are currently unknown. the orf a protein, which also acts as an inhibitor of ifn induction (see earlier), had the strongest activity. lastly, the repressive modifications that are imposed by mers-cov onto the cellular histones are also a strategy to dampen isg expression (menachery et al., a) . despite having some sensitivity toward ifn, especially mers-cov (see section ), viral strategies to increase ifn resistance are also in place ( table ). the sequestration of viral dsrna in dmvs van hemert et al., ; versteeg et al., ) not only reduces cytoplasmic exposure to prrs and hence ifn induction but also limits activation of antiviral dsrna-responsive isg products like pkr. however, pkr is eventually activated by sars-cov infection, but has no effect on viral replication (krahling et al., ) . interestingly, other coronaviruses kuri et al. ( ) cope differently with pkr. the avian infectious bronchitis virus (ibv) expresses a weak inhibitor or pkr (nsp ) and additionally upregulates the phosphatase subunit gadd to reduce phosphorylation of the pkr downstream target eif- alpha (wang et al., ) . by contrast, the porcine reproductive and respiratory syndrome virus (prrsv; a member of the arteriviridae that are related to the coronaviridae and other nidoviruses) does not inhibit but rather requires pkr for optimal replication and gene expression . thus, the interactions and interdependencies of coronaviruses with pkr are complex and far from being understood. with respect to the antiviral oas/rnase l system that is also activated by dsrna, the mouse coronavirus mhv-a was shown to expresses an ns protein that antagonizes by degrading the product of the oas enzyme, - oligoadenylate that would activate rnase l (zhao et al., ) . sars-cov and mers-cov do not possess an ns homolog (silverman and weiss, ) , but the mers-cov ns b was recently demonstrated to cleave - oligoadenylate (thornbrough et al., ) . although ns b-mutated mers-cov was not attenuated in cell culture, it provoked increased rnase l activity in infected cells (thornbrough et al., ) . a critical factor for ifn resistance of sars-cov (and of the low pathogenic hcov- e) is the adp-ribose- -monophosphatase (adrp) domain that is contained within the nsp protein (kuri et al., ) . virus mutants lacking a functional adrp domain (also called macrodomain) display an increased ifn sensitivity. adrp-like macrodomains are encoded by other coronaviruses and several other positive-strand rna viruses (gorbalenya et al., ) . also for mhv-a , a role of the adrp domain in pathogenesis was shown (eriksson et al., ; , but this seems not be related to ifn sensitivity. the last + years have seen tremendous progress toward the identification of ifn antagonists of human coronaviruses (de diego et al., ; gralinski and baric, ; kindler and thiel, ; perlman and netland, ; thiel and weber, ; totura and baric, ; vijay and perlman, ; wong et al., ) . for sars-cov, the catalogue of ifn antagonists may be nearly complete by now and that of mers-cov may follow soon. nonetheless, we are still far from comprehensively understanding the manifold interactions of human pathogenic coronaviruses with the ifn system. many of the factors described here were identified by overexpression studies, and still lack the final biological assessment through generation and characterization of adequate virus mutants. it would also be interesting to see at which infections stage, in which subcellular compartment, and with which comparative intensity the ifn antagonists act, and whether and how they interact with each other. it is however safe to state that coronaviruses, which have the largest rna genome known to date, do not rely on single virulence factors but employ several layers of anti-ifn strategies. otherwise they would not be able to exist, thrive, and even expand to new 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coronavirus fails to activate cytokine-mediated innate immune responses in cultured human monocyte-derived dendritic cells human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit coronavirus non-structural protein is a major pathogenicity factor: implications for the rational design of coronavirus vaccines ribose -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda acknowledgments f.w. is supported by the sfb and grant we / - (spp ) of the deutsche forschungsgemeinschaft. e.k. and v.t. were supported by the swiss national science foundation (snf grant ).disclosures: no conflicts of interest declared. key: cord- -vhmi authors: lannes, nils; python, sylvie; summerfield, artur title: interplay of foot-and-mouth disease virus, antibodies and plasmacytoid dendritic cells: virus opsonization under non-neutralizing conditions results in enhanced interferon-alpha responses date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: vhmi foot-and-mouth disease virus (fmdv) is a highly infectious member of the picornaviridae inducing an acute disease of cloven-hoofed species. vaccine-induced immune protection correlates with the presence of high levels of neutralizing antibodies but also opsonising antibodies have been proposed as an important mechanism of the immune response contributing to virus clearance by macrophages and leading to the production of type-i interferon (ifn) by plasmacytoid dendritic cells (pdc). the present study demonstrates that the opsonising antibody titres mediating enhanced ifn-α responses in pdc were similar to neutralizing titres, when antigenically related viruses from the same serotype were employed. however, sera cross-reacted also with non-neutralized isolates of multiple serotypes, when tested in this assay. both uncomplexed virus and immune complexed virus stimulated pdc via toll-like receptor . an additional finding of potential importance for strain-specific differences in virulence and/or immunogenicity was that pdc activation by fmdv strongly differed between viral isolates. altogether, our results indicate that opsonising antibodies can have a broader reactivity than neutralizing antibodies and may contribute to antiviral responses induced against antigenically distant viruses. foot-and-mouth disease virus (fmdv) is a highly contagious infectious agent inducing disease of cloven-hoofed animals including cattle, swine, goats and sheep. due to the significant economic impact on livestock, a tight disease control is required. however, its high mutation rate contributes to immune escape and the presence of seven serotypes (o, a, c, asia- , south african territories , and ) each containing a large variety of isolates with high antigenic variability. current conventional vaccines, consisting of inactivated virus, provide a short-term serotype specific protection. however, vaccination does not induce protection against all isolates within one serotype [ ] . protection is related to the presence of high level of neutralizing antibody in serum. however, animals with low levels of neutralizing antibodies can also be protected [ , ] . furthermore, non-neutralizing concentrations of monoclonal antibodies (mab) can induce protection in mice [ ] . thus, other mechanisms than neutralization could be involved in protection. it has been shown that opsonisation of fmdv enhances phagocytosis by monocytes and macrophages in vitro [ ] . more recent in vivo data emphasize the potential role of opsonising antibodies in a mouse model, in which protection was mediated in a macrophage-dependent manner [ ] . while these studies indicate that immune complexed virus could be eliminated after phagocytosis by macrophages bearing fc receptors (fcr), other studies also indicate a participation of dendritic cells (dc), at least in vitro. immune complexes induce the activation of porcine plasmacytoid dc (pdc) resulting in the production of interferon (ifn)-α [ ] . the possible involvement of opsonising antibodies in fmdv immunity was also confirmed with bovine pdc [ ] . pdc represent . - . % of porcine peripheral blood mononuclear cells (pbmc), with similar functional characteristics to their human and murine counterparts [ ] . they are characterized by producing high amount of antiviral type-i ifn in response to a wide range of pathogens. pdc activation is mediated by sensing pathogen-associated molecular patterns (pamp) through pattern recognition receptors (prr). by expressing prr, such as toll-like receptors (tlr) and c-type lectin receptors, pdc contribute to the innate and adaptive antiviral immunity [ ] . antibody-dependent fmdv entry in pdc occurs via the fcγrii receptor (cd ) [ ] , linking pdc to the adaptive immunity [ ] . considering the possible importance of opsonising antibodies and pdc in the protection against fmdv, the main aim of this study was to characterize the relationship between neutralizing and opsonising activities of polyclonal sera from immunized pigs. although neutralization and opsonisation occurred at similar serum dilutions when antigenically related viruses were employed, opsonisation also occurred in the absence of neutralization and across different serotypes. we also discovered differences in the ability of various fmdv isolates to activate pdc. for pdc enrichment, monoclonal antibodies against following cell surface markers were used: cd a (mab - - a), cd (mab cam a), cd (mab e ) and cd (mab pt a). for phenotyping, mab against cd a and cd were used. hybridoma for mab - - a was kindly provided by dr a. saalmüller (veterinary university, vienna, austria). mabs cam a, e and pt a were purchased from vmrd (pullman, wa, usa). unsorted and sorted (see below) pbmc were cultured in dulbecco's modified eagle's minimal essential medium (dmem) plus glutamax ™ -i (gibco, life technologies, basel, switzerland) supplemented with μm of βmercaptoethanol (life technologies) at °c at % co . baby hamster kidney (bhk) cells were grown in glasgow's minimum essential medium (gmem, life technologies) supplemented with % v/v fetal bovine serum (fbs, south america origin, biowest, nuaillé, france). for virus preparation and serum neutralization test, cells were cultured in fbs-free gmem at °c, % co . blood was collected alternatively from a total of specific pathogen-free (spf) pigs of - months old kept at our institute. pbmc were isolated from citrated blood using ficoll paque ( . g/l, amersham pharmacia biotech ag, dubendorf, switzerland) density gradient [ ] . for pdc enrichment, pbmc were separated using magnetic sorting system (macs) with depletion (ld) and selection (ls) columns (miltenyi biotech gmbh, bergisch-gladbach, germany). pdc were enriched either using cd a positive selection with ld columns or by a first depletion of cd + cells with a subsequent positive selection for cd a + cells. alternatively, pbmc were isolated using ficoll paque and optiprep ( % w/v solution of oidixanol in water, sigma-aldrich, saint louis, mo, usa) density gradients followed by a depletion of cd + cells and a final enrichment of cd + cells [ ] . purity of the sorted population was verified by flow cytometry detection, after staining with anti-cd a and anti-cd mabs and isotype-specific r-phycoerythrin (r-pe) and fluorescein isothiocyanate (fitc) conjugates (southern biotechnology associates, birmingham, al, usa) as described [ ] . the pdc population was identified as cd high cd a low cells by flow cytometry [ ] . isolates of fmdv were propagated in bhk- cells as previously described [ ] and viral titres were determined by end-point titration on bhk- cells [ ] . o ukg , c noville, o bulgaria / , o vietnam / , a brazil / , a turkey/ and asia- turkey/ were kindly provided by drs. nigel ferris and satya parida (institute for animal health, pirbright, uk). in order to avoid heparin sulphate adaptation of the virus, the isolates were not passaged more than three times in bhk- cells [ ] . mock antigen was prepared from uninfected bhk- cells in the same manner as fmdv. two spf pigs were immunized intra-muscularly with the full-dose of a monovalent fmdv vaccine consisting of inactivated type-o manisa antigen (merial, lyon, france). animals received prime injection at day and a booster at day . at day , , , , and , blood samples were taken and serum prepared for storage at - °c. serum samples from a naïve animal of the same litter were used as controls. in certain experiments, serum was complement inactivated by heat-treatment at °c for min. serial dilutions of heat-treated serum were incubated at room temperature (rt) for min with tcid of fmdv isolate to form immune complexes in final volume of μl/well and then added to confluent bhk- cells, grown in -well plate for days. the neutralization titre was calculated according to kaerber [ ] based on fmdv-induced cytopathogenic effect (cpe). isolated pbmc and sorted cells ( × cells/ml) were stimulated in μl of serum-free medium with fmdv, fmdv/immune serum mixture or fmdv/naïve serum mixture. sera were used either untreated or heat-treated. fmdv/serum mixtures were previously formed for min at °c. as control, cells were stimulated with cpg d ( μg/ml, invitrogen, basel, switzerland). influenza virus strain pr was employed to test the specificity of the tlr inhibitor irs ( '-tgcttgcaagcttgcaagca- '). as control, the oligonucleotide (odn) sequence ( '-tcctgcaggttaagt- ') was used [ ] . irs and ctrl-odn were purchased from eurofins mwg operon (ebersberg, germany). influenza virus was grown on days embryonated chicken eggs and titrated as previously described [ ] . pdc activation by influenza virus employed an multiplicity of infection (moi) of tcid / cell [ ] . supernatants from enriched pdc were harvested after h and ifn-α was detected by specific elisa as described [ ] . data were collected using a versamax photometer with softmax pro software. significant differences were determined with sigmaplot v using the sum rank test (p < . ). we previously documented that pdc activation by fmdv occurs only in presence of specific antibodies [ ] . however, these experiments were performed with pdc, which were only enriched about~ - fold from pbmc using a cd a positive selection by magnetic cell sorting ( figure a ). therefore, more efficient purification methods were evaluated in this study. pdc are non-t cells (cd ) [ ] and are found within the cd alow cd high cd pbmc [ ] . based on this, pbmc were first depleted from cd + monocytes and subsequently enriched using cd a. this resulted in a frequency of - % of pdc (~ fold enrichment), depending on the experiment. a third protocol employed a cd depletion and subsequent cd enrichment of pbmc resulting in a pdc population of - % ( figure a ). type a cpg oligonucleotides, like cpg odn d , are potent activators of ifn-α secretion from porcine pdc [ ] and were employed as positive controls. while cpg induced only a few hundred units of ifn-α from unpurified pbmc, after magnetic cell sorting the production was increased by~ fold but with little differences between the different purification protocols ( figure b ). this contrasted with pdc stimulation by fmdv o ukg . as reported previously, no stimulation of pbmc or cd a-enriched pbmc in terms of ifn-α production was observed. only the protocols employing either the cd -depleted and subsequent cd a-enriched pbmc or the cd -depleted and subsequently cd -enriched pbmc permitted the induction of ifn-α after stimulation with fmdv o ukg ( figure c ). it is important to note that these responses were in the range of times lower than those seen after cpg stimulation. as the ifn-α level was not significantly higher when cd -depleted/cd -enriched pbmc were compared with the cd -depleted/cd a-enriched pbmc while the number of cells obtained upon enrichment was higher with the latter protocol, the cd depletion/ cd a enrichment method was employed for the following experiments. while testing for the capacity of different fmdv isolates to activate pdc, we found remarkable differences, exemplified for three different fmdv isolates employed at mois of , . and tcid /cell (figure a ). at the lowest moi, ifn-α was only detected after stimulation with o bulgaria / . with the moi of . tcid /cell, asia- turkey induced a weak response and o bulgaria / a relatively strong response. these responses were further increased when the virus dose was doubled, but o ukg remained a poor ifn-α inducer ( figure a ). considering these results, we tested more fmdv isolates for their ability to activate pdc at an moi of tcid /cell ( figure b ). the results confirmed that the ability of fmdv to activate pdc varies considerably, even within one serotype. relatively strong inducers were o bulgaria / and asia- turkey ; weak inducers were o vietnam / and o ukg , and the most inefficient inducers were c noville, a brazil / and a turkey . as previous studies used purified subtype-specific antibodies [ ] or high concentration of serotype-specific immune serum [ ] to form fmdv immune complexes, we were interested to determine the relationship between serum concentration and ifn-α enhancement. opsonisation of fmdv o ukg by a serum with a neutralization titre (nt) of . log at dilutions below log did not induce pdc activation ( figure a ). in fact, such high serum concentrations showed suppressive effects on pdc stimulation by fmdv, which were independent on the immune status of the donor animal (data not shown). they were therefore omitted in the remaining experiments. by increasing serum dilutions, immune complexes enhanced pdc-produced ifn-α. even with an moi as low as . tcid /cell, immune complexes activated pdc ( figure a) . the same approach was applied for o bulgaria / , a high ifn-α inducing fmdv isolate ( figure b) serum employed showed a nt of . log against this virus. with o bulgaria / at an moi of tcid /ml, immune serum enhanced pdc-derived ifn-α response in the expected concentration-dependent manner with maximum ifn-α level at a serum dilution of . log and a gradual loss of the enhancing activity with further serum dilutions ( figure b ). in contrast, when o bulgaria / was used at mois of . and tcid /cell, this serum concentration-dependent relationship was not observed ( figure b ). in particular, with the highest virus dose a clear enhancement of ifn-α responses was not observed. we concluded that low mois have to be employed to measure antibody-dependent enhancement of ifn-α responses by pdc when high ifn-α-inducing fmdv isolates are used. results obtained with various serum dilutions and various fmdv isolates indicated that other serum factors than specific immunoglobulin (ig) influenced pdc activation. for this reason a naive serum was titrated in parallel to the immune serum, and the possible impact of complement tested by heat-treatment of the sera before forming immune complexes. figure shows a representative experiment in which immune serum enhanced the ifn-α response induced by fmdv o ukg relative to naive serum. nevertheless, this enhancement was only clear at a serum dilution up to log ( figure a ). with heattreated sera, immune complexes enhanced pdc-derived ifn-α were more clearly visible with statistical differences at serum dilutions up to log . we concluded that complement is not required for the enhancement of ifn-α by immune-serum, and heat-labile serum factors could even have a suppressive effect. considering this, all further experiments were performed with heat-treated serum. our previous work demonstrated that fmdv immune complex-mediated pdc activation required live virus and was associated with expression of non-structural fmdv proteins. this indicated that a virus replication cycle is initiated with formation of double stranded rna, a potential trigger of ifn-α responses [ ] . although tlr is known to be the main sensor for rna viruses in pdc, it represents a receptor for single stranded rna. we were therefore interested to determine the role played by tlr in sensing fmdv and fmdv immune complexes. to this end we used the immuno-regulatory sequence (irs ) representing an odn inhibitor of tlr which had been previously established for human and murine immune systems [ ] . we employed influenza virusstimulated pdc, a known ligand for tlr to test the efficiency of irs to inhibit the tlr pathway of porcine pdc. as shown in figure complexes were employed irs inhibited pdc responses by - % ( figure b ). similar levels of inhibition were also observed with other fmdv isolates (data not shown). the control odn did not influence ifn-α production of fmdv-stimulated pdc (data not shown). pdc cultures permit to detect efficient opsonisation of fmdv in the absence of neutralization we were next interested to determine whether opsonising activity can also be detected in the absence of neutralizing activity. to this end, an anti-o manisa serum was employed and tested against fmdv o ukg (nt of . log ) and against a brazil / (nt below detection limit). interestingly, despite the inability to neutralize the a serotype virus, opsonising activity against both viruses was observed at a similar serum dilution between to log . also the maximum ifn-α production was reached at the same serum dilution of log with both fmdv isolates ( figure a ). these experiments were repeated with five different batches of pdc preparations, and the enhanced ifn-α responses by the immune serum compared to the effect of naive serum were consistently observed despite some variability in the height of the ifn-α responses (figures b and d) . similar results were also obtained with another serum sample obtained from a second vaccinated pig (data not shown). furthermore, both sera were also able to opsonize a/turkey/ confirming the existence of opsonizing antibodies cross-reacting with other serotypes. considering these surprising results, neutralizing and opsonising activities of an anti-o manisa serum were measured against a collection of fmdv isolates of serotypes o, a, c and asia- and the results are summarized in table as nt and opsonisation titres (ot) representing the highest serum dilution able to opsonise fmdv for enhanced ifn-α responses. as expected the immune serum was only able to neutralize the o serotype viruses o ukg and o vietnam / . with these viruses we found a similar ot of and log , respectively. as shown in figure c , d, non-neutralized a brazil / could be opsonised to a titre of log . also fmdv a turkey and asia- turkey/ , albeit to a lesser extent, were opsonised although not being neutralized. only, c -noville was neither neutralized nor opsonised by an anti-o manisa serum. these results demonstrate that opsonisation of fmdv can occur in the absence of neutralization. pdc are professional sensors of viruses producing large amount of type-i ifns. however, in particular nonenveloped viruses such as fmdv have been shown to be unable to trigger pdc activation in vitro, unless the cells are stimulated with immune complexed virus [ , ] . however, in the present study we demonstrate that fmdv can activate pdc also in absence of specific antibodies if improved purification methods are employed. type-c c -noville < . * opsonizing titres representing minimum serum dilution able to enhance ifnα responses by pdc (calculated from - independent experiments as described in figure ). we also demonstrate that both immune-complexed fmdv and uncomplexed fmdv activate pdc via tlr . as expected, the responsiveness of pdc to fmdv is possible with more pure populations of pdc but our results also indicate that other factors could be important such as the percentage of other cell types present in the cell culture. the observation that in vivo ifn-α responses occur in the early innate phase of the immune response of cattle and pigs [ , ] support the idea of an early activation of pdc by fmdv in absence of specific antibodies and support the in vivo relevance of our data. in fact, in cattle it has been demonstrated that pdc are the source of early ifn-α responses [ ] . our study also demonstrates remarkable differences of different fmdv isolates to activate pdc. the reason for the differences in activation of pdc could be either related to the uptake of the virus by pdc or to the function of viral genes such as the ifn antagonist l pro , the viral proteinase. fmdv interacts with immune and nonimmune cells [ ] via arg-gly-asp (rgd)-dependent and rgd-independent mechanisms, after cell culture adaptation to heparin sulphate binding [ ] . certainly, the differences we observed with pdc are not related to a cell culture adaptation of certain fmdv isolates to heparin sulphate binding since all viruses employed in this study had a maximum of three passages. furthermore, we have compared cell culture adapted heparinsulphate binding virus to its non-adapted original wild-type fmdv (viruses as described in [ ] ), and found no differences in the ability to activate pdc (data not shown). on the other hand, the leader proteinase l pro inhibiting the type-i ifn pathway [ ] , is variable among serotypes [ ] and could contribute to a isolate-specific fmdv counteraction on the ifn-α induction in pdc. the potential of some fmdv isolates to consistently induce ifn-α also at low mois would support this hypothesis. overall, it is important to note that in comparison to the ifn-α levels in response to influenza viruses [ ] and cpg [ ] , fmdv is a poor stimulator of pdc. nevertheless, considering that ifn-α is known to efficiently inhibit fmdv replication in vitro and in vivo [ ] , it is now important to investigate the relationship of the ability of an fmdv isolate to induce ifn-α in vitro and to promote in vivo innate and adaptive immune responses. previous studies have demonstrated that antibodydependent internalization of fmdv via the fcγrii represents an important pathway to enter macrophages [ ] and pdc [ , ] . one of the main objectives of this study was to determine the relationship between opsonisation and neutralization. non-neutralized serotype-a and asia- isolates could be efficiently opsonised using an anti-o serum while others were not, such as a serotype c isolate. from phylogenetic studies based on vp amino acid sequence, which contains the most antigenic site [ ] , serotypes o and asia- belong to the same branch [ ] . serotypes a and c are more distant, partially explaining our findings [ ] . interestingly, this opsonisation efficiently occurred with serum dilutions similar to those required for neutralization, when antigenically related viruses from the same serotypes were employed. this would indicate that the type of antibodies and their specificities is similar for both functions. however, we also found opsonisation in nonneutralizing conditions contradicting this idea. it could be speculated that opsonising activity is possible at lower avidity compared to neutralization, in which the antibody binding to the virus is in competition to virus binding to the cellular receptor. this competition can only occur indirectly for opsonisation, since the cellular fcr will bind to the fc portion of the antibody. the observation that opsonisation can occur at similar serum dilutions with a neutralized and non-neutralized virus would favour the presence of non-neutralizing epitopes, which can be conserved between different serotypes. the answer to these questions needs to be addressed using monoclonal antibodies rather than a polyclonal serum such as in the current study. the broad activity of opsonising antibodies is interesting in the frame of vaccine-induced immune responses. non-neutralizing vaccine-induced antibodies could enhance innate immune responses against antigenically less related fmdv challenge strains and restrict their ability to replicate. furthermore, other fcr-mediated functions such as virus phagocytosis and more efficient antigen presentation of viral antigens to t cells could be promoted. although this is not sufficient to mediate complete protection against disease, it could limit virus replication, the severity of clinical diseases and transmission within the heard. future studies are required to determine how such responses correlate to protection (partial or complete), and if this is the case how vaccines can be improved to promote broadly reactive antibody responses able to link innate and adaptive immunity to fmdv via opsonising antibodies. foot and mouth disease virus vaccines relationship between the anti-fmd virus antibody reaction as measured by different assays, and protection in vivo against challenge infection confidence in indirect assessment of foot-and-mouth disease vaccine potency and 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mononuclear cells producing ifn alpha following induction by coronavirus-infected cells (porcine transmissible gastroenteritis virus) natural interferon-alpha producing cells: the plasmacytoid dendritic cells novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease aspects of the persistence of foot-and-mouth disease virus in animals-the carrier problem innate immune responses against foot-and-mouth disease virus: current understanding and future directions foot-and-mouth disease virus: a long known virus, but a current threat the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase the non-structural leader protein gene of foot-and-mouth disease virus is highly variable between serotypes foot-and-mouth disease submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this present work was supported by eu fp project fmd-disconvac (grant agreement ) and r research foundation switzerland (project - ). we thank drs. nigel ferris and satya parida for providing some of the fmdv isolates. we also thank heidi gerber for mab preparation and help with virus preparation as well as andreas michel and hans-peter lüthi for regular bleeding and care of the animals. http://www.veterinaryresearch.org/content/ / / the authors declare that they have no competing interests. key: cord- -zolwjl u authors: xiao, shuqi; jia, jianyu; mo, delin; wang, qiwei; qin, limei; he, zuyong; zhao, xiao; huang, yuankai; li, anning; yu, jingwei; niu, yuna; liu, xiaohong; chen, yaosheng title: understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: zolwjl u porcine reproductive and respiratory syndrome (prrs) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. prrs virus (prrsv) replicates mainly in porcine alveolar macrophages (pams) and dendritic cells (dcs) and develops persistent infections, antibody-dependent enhancement (ade), interstitial pneumonia and immunosuppression. but the molecular mechanisms of prrsv infection still are poorly understood. here we report on the first genome-wide host transcriptional responses to classical north american type prrsv (n-prrsv) strain ch a infection using solexa/illumina's digital gene expression (dge) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after n-prrsv infection and infection pathology. our results suggest that n-prrsv appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ade. upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during n-prrsv infection processes. n-prrsv-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. our systems analysis will benefit for better understanding the molecular pathogenesis of n-prrsv infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to prrs. porcine reproductive and respiratory syndrome (prrs), also called ''blue ear'' disease due to a typical, but not often observed hallmark of ''blue ears'', is widely accepted as being one of the most economically important diseases affecting swine industry. since its first appearance in the late s in the us and europe, prrs has spread worldwide [ , , ] . prrs is characterized with high mortality in piglets, reproductive failure (late-term abortions and stillbirths, premature farrowing, mummified pigs) in pregnant sows and respiratory disease (interstitial pneumonia, respiratory difficulties) in nursery and grower/finishing pigs, causing highly significant economic losses to the swine industry worldwide, resulting in .$ . million losses each year in the us alone [ ] . the etiologic agent of prrs is prrs virus (prrsv), a small enveloped, linear, single, positive-stranded rna virus, which is a member of the family arteriviridae which includes lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus and enters in the newly established order delayed and their levels remain low, which can not eliminate effectively prrsv-infected cells [ , ] . because of these features of prrsv infection, prrs has been one of the most challenging subjects of research in veterinary viral immunology [ ] . regulation of immune responses and genetic resistance to infectious viral diseases is an area of concern for human and swine [ ] . prrsv strongly modulates the host's immune responses, and changes the host's gene expression. studies showed that prrsv inhibits type i interferons (ifn-a/b, spi ifn), especially ifn-a [ ] , and induces interleukin- (il ) [ , ] . because the primary cellular target of prrsv is the porcine alveolar macrophages (pams) of the lung, several studies have analysed the immune responses of pams to prrsv infection. one group [ ] used differential display reverse-transcription pcr to identify molecular genetic changes within prrsv-infected pams over a h pi period. their results suggest that myxovirus resistance (mx ) and ubiquitin specific proteases (usp) genes may play important roles in clinical disease during prrsv infection. notably, one recent paper on genome-wide transcriptional response of pams following infection with the lelystad prrsv strain (european type, eu prrsv) using affymetrix microarrays has been published during the preparation of our manuscript [ ] . they found that the expression of beta interferon (ifn-b) was strongly upregulated while the expression of il- and tnf-a was weakly upregulated. almost in the same time, the other group employed serial analysis of gene expression (sage) to examine the global expression of genes in vr- prrsv strain (north american type, na prrsv)-infected pams. they identified over unique tags with significantly altered expression levels [ ] . in vitro studies will be useful for investigating how prrsv modifies genes expression in primary target cells, such as pams. however, many of the outstanding issues will be answered only in the context of prrsv-infected animals. hence, the characterization of host immune response under in vivo environment to prrsv is still an area in urgent need of investigation. lung pathogenesis is a major feature of prrsv infection. moreover, in addition to serving as a source of protein in the human diet, the pig is also an excellent biomedical model for humans because of the similarity in size and physiology, and in organ development and disease progression [ ] . thus, understanding the host's immune response to prrsv infection is important not only for swine production but also for human consumption. however, to date, the immune response to prrsv in porcine lung has not been analyzed by transcriptome profiling. next generation high-throughput sequencing technology has been adapted for transcriptome analysis because of the inexpensive production of large volumes of sequence data [ , , , ] . the technology developed by illumina (formerly solexa sequencing) [ ] , which is also referred to as digital gene expression (dge) tag profiling, allows identification of millions of short rnas in a sample and of differentially expressed genes without the need for prior annotations. here we employed the illumina genome analyzer platform to perform a digital gene expression analysis of the porcine lung transcriptome response to n-prrsv infection, and used histopathology examination to analyze the pulmonary pathological changes of the infected-porcine lungs. the relationship between pulmonary gene expression profiles after n-prrsv infection and infection pathology was systematically analyzed. the comprehensive analysis of the global host response induced by n-prrsv suggested an inflammatory response, mediated by multiple inflammatory molecules early during infection that induced tissue injury, an immunosuppressive state, mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. our systems analysis will benefit for better understanding the molecular pathogenesis of n-prrsv infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to prrs. our study had been approved by animal care and use committee of guangdong province, china. all animal procedures were performed according to guidelines developed by the china council on animal care and protocol approved by animal care and use committee of guangdong province, china. nine conventionally-reared, healthy -week-old, crossbred weaned pigs (landrace yorkshire) were selected from a highhealth commercial farm that has historically been free of all major pig diseases, such as prrsv, porcine circovirus type , classical swine fever virus, porcine parvovirus, pseudorabies virus, swine influenza virus and mycoplasma hyopneumoniae infections. all pigs were prrsv-seronegative determined by elisa (herdchek prrs xr; idexx laboratories) and absence of prrsv tested by rt-pcr. pigs were randomly assigned to two groups in the experiment and raised in isolation rooms. six pigs were inoculated with ml viral suspension ( ml intranasally and ml intramuscularly) of classical north american type prrsv (n-prrsv) strain ch a, isolated from china in , gifted by dr. zhang guihong, south china agricultural university) at a dose of . tcid ml on day . three uninfected negative control (unc) pigs were treated similarly with an identical volume of dmem culture media from uninfected marc- cells day prior to experimental infection, and were immediately necropsied. n-prrsv-inoculated pigs were clinically examined daily and rectal body temperatures were recorded from days to pi. viral reisolates were performed after the pigs were killed. the infected group showed positive, and the unc group was negative. tissue homogenates and serum were examined by n-prrsv-specific quantitative pcr (qpcr). the oligonucleotide primers used were nsp f( -gtgggtcggcaccagtt- ) and nsp r , designed in the gene segment encoding for nsp . the taqman probe, fam-cacagttctacgcggtgcagg -tamra , was synthesized. three infected pigs randomly chosen were necropsied at each time point of h pi and h pi. lung samples were collected from unc group (c), three pigs at h pi (n ), three pigs at h pi (n ) and immediately frozen in liquid nitrogen for rna isolation or fixed in % neutralized buffered formalin for histological processing. lungs of unc and experimentally infected pigs were processed routinely for haematoxylin and eosin (h&e) and immunohistochemistry staining, as described previously [ ] . total rna was extracted from frozen lungs using standard protocols (trizol) and then treated with dnase to remove potential genomic dna contamination according to the manufactures's protocols. rna integrity and concentration were evaluated by agilent bioanalyzer (agilent technologies). for rna library construction and deep sequencing, equal quantities of rna samples from three unc individual lungs were pooled, rna samples from the three infected pig lungs (n ) were pooled, and rna samples from the three infected individual lungs (n ) were pooled. approximately mg of rna representing each group were submitted to solexa (now illumina inc.) for sequencing. sequence tag preparation was done with illumina's digital gene expression tag profiling kit according to the manufacturer's protocol. in brief, mrna was isolated from mg total rna by binding the mrna to a magnetic oligo bead. first-and secondstrand cdna were synthesized while the mrna was attached to the beads. the double stranded cdnas were digested with nlaiii to wash away all fragmens other than the catg fragment attached to the oligo bead. then gex nlaiii adapter was ligated at the site of nlaiii cleavage. in addition, gex nlaiii adapter contains the sequence for the restriction enzyme mmei, subsequently, we applied the restriction enzyme mmei to create the bp tag. the gex adapter was ligated at the site of mmei cleavage. a pcr with cycles was performed with two primers that anneal to the ends of the adapters to enrich the adapterligated cdna construct. the resulting bp fragments were purified from % novex tbe page gel. subsequently, the purified cdna tags were sequenced on the illumina cluster station and genome analyzer. image recognition and base calling were performed using the illumina pipeline. all data is miame compliant. the raw data (tag sequences and counts) has been submitted to gene expression omnibus (geo) under series gse . for the raw data, we filtered adaptor tags, low quality tags and tags of copy number = to get clean tags. subsequently, we classified the clean tags according their copy number in the library and show their percentage in the total clean tags and analyzed saturation of the library. the preprocessed database of all possible catg + -nt tag sequences was created, using sus scrofa unigene (http://www.ncbi. nlm.nih.gov/unigene/ugorg.cgi?taxid = , unigene build # sus scrofa, nov, th, ) from ncbi. for monitoring the mapping events on both strands, both the sense and the complementary antisense sequences were included in the data collection. information on the position of polyadenylation signals was also collected from the transcript dababase. then we aligned all clean tags to the reference sequences, and unambiguous tags were annotated. we counted the clean tag number corresponding to each gene. to compare the de of gene across samples (n /c, n /c, n /n ), the number of raw clean tags in each library was normalized to tags per million (tpm) to obtain normalized gene expression level. de detection of gene or tag across samples was performed according to the previous description [ ] . genes were deemed significantly differentially expressed with a p-value , . , a false discovery rate (fdr) , . and an estimated absolute log -fold change . . in sequence counts across libraries. in order to verify the dge results, we used qpcr analysis. the rna samples used for the qpcr assays were both the same as for the dge experiments and independent rna extractions from biological replicates. qpcrs were done on the lightcycler (roche), with sybr-green detection (sybr primescript rt-pcr kit, takara biotechnology co., ltd.), according to the manufacture's instruction. each cdna was analyzed in triplicate, after which the average threshold cycle (ct) was calculated per sample. the relative expression levels were calculated with the ddct method. the results were normalized to the expression level of hprt and relative to the c sample. through browsing all health traits of pig quantitative trait locus (qtl) database (pigqtldb, http://www.animalgenome. org/qtldb/pig.html) by trait classes, we obtained mapping details of qtl on the corresponding pig chromosome. then pig affymetrix elements corresponding to health trait qtl regions were downloaded to an excel file. by matching the id of de genes to all genes in the qtl regions, we obtained de genes of the corresponding qtl region. pathway analysis was mainly based on the kyoto encyclopedia of genes and genomes (kegg) database. two-side fisher's exact test with a multiple testing and x test were used to classify the pathway category. the false discovery rate (fdr) was used to correct the p-value. we chose only pathway categories that had a p, . . within the significant category, the enrichment re was , where n f is the number of flagged proteins within the particular category, n is the total number of proteins within the same category, nf is the number of flagged proteins in the protein reference database list, n is the total number of proteins in the gene reference database list. stc is implemented entirely in java. the clustering algorithm first selects a set of distinct and representative temporal expression profiles. these model profiles are selected independent of the data. the clustering algorithm then assigns each gene passing the filtering criteria to the model profile that most closely matches the gene's expression profile as determined by the correlation coefficient. since the model profiles were selected independent of the data, the algorithm can then determine which profiles have a statistically significant higher numberthan genes assigned using a permutation test. this test determines an assignment of genes to model profiles using a large number of permutations of the time points. it then uses standard hypothesis testing to determine which model profiles have significantly more genes assigned under the true ordering of time points compared to the average number assigned to the model profile in the permutation runs. significant model profiles can either be analyzed independently, or grouped together based on similarity to form clusters of significant profiles [ , ] . stc-go supports gene ontology enrichment analyses for sets of genes having the same significant temporal expression pattern. we select random samples of s a (s a is the number of genes assigned to the same model temporal expression profile r.) genes at each iteration and compute fisher's exact test p-values for the selected genes in all go biological categories [ ] . the two-sided fisher's exact test p-value for a category reflects a test of the null hypothesis that the category is enriched in genes assigned to profile r with respect to what would have been expected by chance alone. to decide whether or not to follow up a category that appears enriched in these genes, we would know the statistical reliability of the apparent enrichment. to assess the significance of a particular category, we need to know the distribution of p-values that would occur by random chance. the percentage of false positives to be tolerated will generally depend on the relative costs of false positives and false negatives in whatever follow-up study is to be done. this way of framing the question leads us to specify the false discovery rate (fdr) for a set of categories, rather than significance level (p-value) for each category. with the significance at the . level, for a given category, the enrichment r e is given by r e~( i=m)=(s a =n) where i is the number of genes assigned to profile r within the go category of interest, m is the total number of genes within the go category of interest, and n is total number of unique genes in the gene reference database list. after n-prrsv infection, the affected pigs exhibited the following clinical symptoms within - days: fever of . - . uc, depression, anorexia, rough hair coats, dyspnoea, reddening of skin, oedema of the eyelids, conjunctivitis, mild diarrhoea, shivering. those unc pigs did not show any obvious changes in body temperature and clinical signs. qpcr assay showed that n-prrsv virus was present in each of the infected pigs. but n-prrsv nsp gene was not differentially expressed at h pi and h pi (table s ). histopathology examination of n-prrsv-affected pigs showed interstitial pneumonia in lungs with thickening of alveolar septa accompanied with infiltration of immune cells ( figure b ). most viral antigen was detected in alveolar cells and bronchiolar epithelial cells in lesions ( figure c ). to investigate the regulation of the host response to the n-prrsv virus, we considered the global gene expression profiles in lungs using solexa/illumina's dge system, a tag-based transcriptome sequencing method. we sequenced three porcine lung dge libraries from c, n , n using massively parallel sequencing on the illumina platform. major characteristics of these three libraries were summarized table . we obtained approximately . million total sequence tags per library with distinct tag sequences. prior to mapping these tag sequences to reference sequences, we filtered adaptor tags, low quality tags and tags of copy number = , producing approximately . million total clean sequence tags per library with distinct clean tag sequences. the c library had the highest number of both total sequence tags and distinct sequence tags; this was followed by the n , n libraries. moreover, the c library had the highest ratio of number of distinct tags to total tags and the lowest percentage of distinct clean high copy number tags. the data showed that more genes were detected in the c library than other two libraries and more transcripts were expressed at lower levels in the c library. saturation analysis of capacity of libraries showed that new emerging distinct tags were gradually reduced with increasing of total sequence tags when the number of sequencing tags was big enough. when the number of sequencing tags reached million, library capacity approached saturation ( figure s ). for tag mapping, we preprocessed one reference tag database that included sequences from sus scrofa unigene. to get the reference tags, we used nlaiii to digest all the samples and took all the catg+ tags in the gene as the gene's reference tags, not only the most one. we obtained total reference tag sequences with unambigous tag sequences. considering polymorphism across samples, tolerances were set to allow one mismatch in each alignment. by the criteria, . %, . % of distinct clean tags mapped to the unigene virtual tag database, . %, . % of the distinct clean tags mapped unambiguously to the unigene, and . %, . % of the distinct clean tags didn't map to the unigene virtual tag database ( table ). the occurrence of unknown tags was probably due to the incompleteness of pig genome sequencing. most solexa experimental tags matched to the st or nd catg site in high-confidence transcripts ( figure s ). for solexa sequencing can distinguish between transcripts originating from both dna strands, employing the strand-specific nature of the sequencing tags obtained, we found evidence for bidirectional transcription in to of all detectable unigen clusters and to antisense-stand specific transcripts (table s ). by comparison, the ratio of sense to antisense strand of the transcripts was approximately . : for all libraries. this suggests that in spite of the high number of antisense mapping events detected, the transcriptional regulation in the n-prrsv-induced immune response acts most strongly on the sense strand. to analyze the depth of transcriptome sampling in the dge libraries, we studied the rate of increase of the number of genes (sense+antisense strand) identified as the size of the corresponding library increases. when the library size reached one million, we could identify % and % all genes and genes identified by unambigous tags, respectively ( figure s ). at this time, library capacity approached saturation. to gain the global transcriptional changes in n-prrsv infected porcine lungs, we applied the method described previously [ ] to identify de genes from the normalized dge data by pairwise comparisons between all differential time points (n /c, n /c, n /n ) during infection. results showed that genes had p values , . , false discovery rate (fdr) , . and estimated absolute log -fold change . . in at least one of the pairwise comparisons, which were declared to be differentially expressed during infection course (table s ) . to characterize the functional consequences of gene expression changes associated with infection with n-prrsv, we performed pathway analysis of de genes based on the kegg database by two-side fisher's exact test. we chose only significant pathway categories that had a p-value of , . and an fdr of , . . as shown in figure s , the significant signaling pathways include cell adhesion molecules (cams), t cell receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, biosynthesis of unsaturated fatty acids, pantothenate and coa biosynthesis, etc (table s ) . to validate de genes identified by solexa sequencing, we selected genes for qpcr confirmation. the set included two down-regulated genes (epithelial chloride channel protein (aecc) and hyaluronan and proteoglycan link protein (hapln )) and six up-regulated genes (inflammatory response protein (irg ), dead (asp-glu-ala-asp) box polypeptide (ddx ), usp , cxcl , cytochrome p (cyp a ), and cd ). data were presented as fold changes in gene expression normalized to the hprt gene and relative to the c sample. pearson's correlation coefficient (r) showed that both the dge and qpcr data (pooling samples) were highly correlated, for the genes modulated by n-prrsv had a high consistency and r values ranging from . (cyp a ) to . (aecc) between the two methods ( figure ). qpcr analysis (both pooling samples and independent rna extractions from biological replicates) confirmed the direction of change detected by dge analysis. this correlation indicated the reliability of dge results. qtl play a central role in linking genomic information with phenotypes. the ultimate goal of qtl studies is to identificate the actual gene(s) that are responsible for the phenotypic variation observed in a particular trait [ ] . in the present paper, we mapped the de genes to pig qtl regions of health traits in pig qtldatabase (pigqtldb). our search found that de genes were distributed in different known qtl regions related to pig health traits ( figure s ; table s ). among the de genes, and were located in qtl regions of the cd -positive leukocytes and cd -positive leukocytes, respectively; were distributed in the qtl region of the band-formed neutrophils and cd -positive leukocytes. immune responses against pathogens depend in part on the generation of fully differentiated 'killer' (or effector) and memory cd + t cell. effective priming and maintenance of cd + t cell responses to viral infection require 'help' from cd + t cells, the latter play also a critical role in programming cd + t cell memory development [ ] . moreover, recent study showed that cd + t cells guide effector cytotoxic t lymphocytes (ctls) to virally infected tissues where they can destroy infected cells [ , ] . in order to profile gene expression time series and search for the most probable set of clusters generating the observed time series, we used stc algorithm of gene expression dynamics, which explicitly took into account the dynamic nature of temporal gene expression profiles during clustering and identified the number of figure s ; table s ) with (profile , , , ) significant cluster profiles which have significantly more genes assigned under the true ordering of time points compared to the average number assigned to the model profile in the permutation runs ( figure s ). then gene ontology (go) based on biological process (bp) enrichment analyses for sets of de genes having significant cluster profiles was performed by two-side fisher's exact test (table s and table s ; figures s , s , s s ). we chose only significant go categories that had a p-value of , . . the most prominently overrepresented go terms of significant cluster profile ( , , ) and profile ( , , ) , which are down-regulated genes, involved in regulation of lipid, cholesterol biosynthetic and metabolic process; regulation of skeletal muscle development, muscle cell differentiation; digestion; negative regulation of neuron apoptosis and neurological system process (table s ; figures s and s ) . the most prominently overrepresented go terms of significant cluster profile ( , , ) and profile ( , , ), which are up-regulated genes, included negative regulation of fibroblast proliferation; natural killer cell, macrophage, lymphocyte, mononuclear cell, leukocyte and t cell proliferation, differentiation and activation; complement activation, immune response, inflammatory response, defense response, and apoptosis; response to stimulus(stress); lipid and fatty acid metabolic process and oxidation; positive regulation of ubiquitinprotein ligase activity and protein proteolysis, protein targeting to mitochondrion (table s ; figure s and s ). these results are consistent with these genes and their associated processes playing important roles in n-prrsv replication and pathogenesis. viral infection of host leads to the initiation of antiviral innate immune responses, which results in the induction of expression of the type i interferons [ ] . meanwhile, many viruses have also developed strategies to evade and subvert the immune response. as shown in figure a , transcripts of the ifn c was significantly induced in n-prrsv-infected pigs at days through pi, but short type i interferon (spi ifn) gene expression was suppressed, and interferon alpha (ifna ) gene expression was markedly down-regulated. lipid rafts, lipid microdomains of the cell membrane enriched in sphingolipids, cholesterol and associated proteins, play critical roles in the life cycle of many viruses [ ] . some viruses enhance their replication by modulating host cell lipid metabolism [ ] . dge analysis of pigs infected with n-prrsv showed significant increase of transcript abundance in many genes involved in lipid metabolism, including those for apolipoprotein b receptor (apob r), apolipoprotein-e (apoe), low density lipoprotein b (ldlb), phosphatidylinositol -kinase catalytic subunit type (pik c ) ( figure b ). perhaps n-prrsv alters hosts' lipid metabolism to create a lipid-rich intracellular environment to facilitate its own multiplication. moreover, we also observed that n-prrsv induced upregulation expression of anti-apoptotic genes in n-prrsv infected lungs, including myeloid cell leukemia sequence (bcl -related) (mcl ), nuclear factor kappa-b (nfkb ), nfkb , adrenomedullin (adm), and interleukin (il ), and downregulation expression of pro-apoptotic genes, including bak protein (bak), (apoptosis-related protein ) (apr ) ( figure d ) to inhibit apoptosis, which might prolong cell life and increase the yield of progeny virions. n-prrsv infection caused anorexia and subsequent slow growth. accordingly, we observed that transcript abundance of genes involved in digestion, such as gastric mucin (muc ac) and cytochrome p (cyp a ), was significantly decreased ( figure e ). simultaneously, transcript abundance of the genes associated with cell, muscle and cartilage development was markedly decreased ( figure f ). these genes include insulin-like growth factor binding protein (igfbp ), collagen, type ii, alpha isoform (col a ), connective tissue growth factor (ctgf), epidermal growth factor (egf). fever and heat shock fever is frequently the host's initial response to infection [ ] . after viral infection, pathogen-associated molecular patterns (pamps) in viral proteins and nucleic acids were recognized by host pathogen-recognition receptors (prrs), such as toll-like receptors (tlrs), which trigger gene expression and synthesis of the il- b precursor. active caspase- (casp ) cleaves the il- b precursor into mature, bioactive il- b, which is an inflammatory cytokine most responsible for fever [ , , ] . as shown in figure a , transcript abundance of tlr , , , , il- b and casp was significantly increased in n-prrsv infected porcine lungs. moreover, transcript abundance of genes involved in the activation of casp and il- b secretion including apoptosisassociated speck-like protein containing a card (asc), prostaglandin e synthase (pge ) and phospholipase a , group vii (pla g ) was significantly increased ( figure a ). the expression of heat shock proteins (hsps), known as stress proteins, can be markedly upregulated by all cells under conditions of stress, such as increased temperature (fever) and viral infection [ ] . transcript abundance for most of these heat shock genes, including -kda hsp (hsp ), hsp , and heat shock protein beta- (hsp ) was significantly elevated in n-prrsv infected lungs relative to unc lungs ( figure b ). viral infection results in an inflammatory response, which is an essential component of the antiviral innate immune response [ ] . after recognizing the pamps, either surface or intracellular prrs trigger intracellular signaling cascades that results in the activation of transcription factors, including nuclear factor-kb (nf-kb), interferon-regulatory factors (irfs), and signal transducer and activator of transcription (stats). as shown in figure a , transcripts of the toll-like prrs tlr , tlr , tlr , tlr , were significantly increased in n-prrsv-infected pigs at days through pi, but no change in tlr which specializes in the recognition of viral dsrna was detected. cytoplasmic prrs ( figure a ), retinoic-acid-inducible protein i (rig-i, ddx ) and melanoma differentiation-associated gene (mda ), the two most relevant for defense against viruses, were expressed at a high level after n-prrsv infection. cell surface prrs such as cd , md- protein (md ) and cd (which is probably involved in prrsv entry during uncoating [ ] ) were likewise up-regulated expression after n-prrsv infection ( figure a ). after binding to n-prrsv viral pamps, prrs initiate intracellular signaling cascades that activate transcription factors, including irf , irf , irf , but not irf and stat , stat , stat ( figure b ). activated transcription factors and stats in turn induce the transcription of specific sets of interferon-stimulated genes (isgs) [ , ] , and expression of multiple inflammatory genes [ ] , which induce a pro-inflammatory response and attract cells, such as neutrophils and macrophages, to sites of infection. accordingly, we observed significant increase of transcript abundance in many genes involved in isgs ( figure c ), pro-inflammatory cytokines (such as il b, il ) ( figure d ), chemokines (ccl , cxcl , cxcl ) ( figure e ), adhesion molecules (vcam, icam , sell), and other inflammatory molecules (such as mmp- ,) ( figure f ). moreover, immunoglobulin (such as igg b, igg ) ( figure g ), three categories of fc receptors and mannose receptor c (mrc ) ( figure h ), and complement proteins ( figure i ) were also significantly induced in the n-prrsv-infected lungs. however, several complement inhibitors that possess inhibitory and/or decay-accelerating acitivity, such as decay-accelerating factor cd , complement component binding protein, alpha (c bpa), c bpb, were significantly repressed in the n-prrsvinfected lungs ( figure i ). cytotoxic t lymphocytes (ctls) detect cells infected with a virus and destroy them through perforin-mediated apoptosis [ ] . cd + t cells activation require t cell receptors (tcrs) to recognize cognate antigenic peptides for presentation on mhc class i molecules displayed on the surface of antigen presenting cells (apcs) [ ] . accordingly, we observed that transcript abundance of ubiquitin specific peptidase (usp) and ubiquitin enzyme ( figure a ), proteasomes, and aminopeptidases ( figure b ) was significantly increased in n-prrsv-infected lungs. the transcript abundance of b m, mhc class i antigen (sla- ), sla- , tap , and chaperones (such as grp ) was markedly increased after infection with n-prrsv while the transcript abundance of sla-b was significantly decreased ( figure c ). in addition to recognization of cognate peptides presented by mhc class i molecules, cd + t cells activation needs also to receive 'costimulatory' signals and help by helper cd t + cells [ ] . as shown in figure d and e, cathepsins and mhc class ii antigens were significantly induced in n-prrsv-infected lungs. the transcript abundance of costimulatory molecules (such as cd , icos), cams, and tcrs/cd complex as well as co-receptor molecules (such as cd a, table s for full gene names. doi: . /journal.pone. .g cd b) was remarkably increased after infection with n-prrsv ( figure f and g) . activated ctls release perforin (pfr) and granzymes, which two effectors act collaboratively to induce apoptosis of target cells. as shown in figure h , prf and granzyme b (gzmb) transcript abundance was significantly elevated in n-prrsv infected lungs relative to unc lungs. in addition to cytotoxins released from ctls, the transcript abundance of other pro-apoptotic members ( figure i ), such as nfkbia, growth arrest and dna-damage-inducible protein alpha (gadd a), bh interacting domain death agonist (bid), xiap-associated factor (xaf ), cytochrome c (cycs), casp , was also significantly increased after infection with n-prrsv, which can induce apoptosis of virus-infected cells. in addition, we also identified the upregulated expression of cytochrome b heavy chain (gp -phox), a critical component of the membrane-bound oxidase of phagocytes (macrophages and neutrophils), and the downregulated expression of heme oxygenase (hmox ) during n-prrsv infections, which might result in the oxidative stress response and subsequent oxidative damage of tissues ( figure j ). from the data presented in the paper, a model for the relationship between pulmonary gene expression profiles and infection pathology can be surmised in figure , n-prrsv virus replicates and spreads by subverting host innate immune response and hijacking host lipid metabolism as well as inducing an antiapoptotic and anti-inflammatory state, as indicated by suppression expression of spi ifn, ifn-a, down-regulation expression of proapoptotic genes for bak, apr- , sarp , high levels expression of genes involved in lipid metabolism, such as apoe, ldlb, pik c , anti-apoptotic genes for mcl , bcl a , chfr, adm, nfkb, il , and anti-inflammatory molecule pge as well as cd . infections of n-prrsv viruses resulted in fever and inflammatory response, as indicated by high expression of proinflammatory cytokines and chemokines, adhesion molecules, inflammatory enzymes and receptors, such as il- b, il , sell, icam, ccl , cxcl , cxcl , b m, proteasomes, cathepsins. this was compounded by cell death and elevated expression of nfkbia, xaf , gadd a, perforin, granzymes, and cytochrome c, coupled with increased ros-mediated oxidative stress, as indicated by by up-regulation expression of cytochrome b . taken together, the n-prrsv infections may have resulted in an excessively immune and inflammatory response that contributed to tissue damage. infection of pigs with n-prrsv caused fever, dyspnoea, reddening of skin, oedema of the eyelids, conjunctivitis, depression, anorexia, mild diarrhoea. histopathology examination showed interstitial pneumonia in lungs with thickening of alveolar septa accompanied with infiltration of immune cells [ ] (figure ). although great efforts have been made by many researchers, the molecular basis of n-prrsv infection is largely unknown. here we report on the first genome-wide host transcriptional response to n-prrsv infection using solexa/illumina's digital gene expression (dge) system, a tag-based novel high-throughput transcriptome deep sequencing method. given the nature of the methodology of illumina's dge system, we have pooled biological replicates from three pigs for each group to make representative samples for deep sequencing analysis. we could reach a sequencing depth of . - . million tags per library (table ) and found over genes to be differentially expressed during n-prrsv infection processes (table s ) . although others studies have also pooled biological replicates for library construction and deep sequencing [ , ] , resulting in the lack of biological replicate, one may blur the impact of variations in pooling samples. because of the variations of pigs in response to prrsv infection, it is possible that one pig could significantly affect results without independent libraries. but we performed the qpcr validation both on the same pooled material that was used for deep sequencing and on independent rna extractions from each pig, which all confirmed the direction of change detected by dge analysis (figure ). our dge analysis showed massive changes in the transcript abundace of known immune response genes and of genes that have been implicated in prrsv infection [ , , ] . we also identified many interesting genes that had not been linked to prrsv infection in previous studies. for example, transcript abundace of lipid metabolism-related genes including apob r, apoe, pik c, was significantly increased during n-prrsv infection processes. alterations in lipid metabolism, perhaps including those with significant upregulation in this study, have been observed in response to infection by a range of viruses including sars-cov, hcv, influenza a virus, or dengue virus [ , , , ] . although in vitro studies have investigated how prrsv modifying genes expression in pams [ , , ] , many of the outstanding issues will be answered only in the context of prrsv-infected animals [ ] . hence, we characterized the genome-wide transcriptome response to prrsv infection in porcine lung by deeping sequencing. but studies of transcript abundance in lung tissues have also their intrinsic limitations. for example, the transcriptome of lung tissues is actually a merging transcriptional responses of a wide range of cell types, some of which are infected, some of which are responding directly to the infectious process and others of which are bystanders. moreover, increased cellularity of tissues may be confused as biologically important increased transcript abundance. despite such limitations, our dge study offers a broad, system-wide window into molecular processes that regulate gene expression and also provides new leads for functional studies of candidate genes involved in host-virus interaction, as illustrated in this paper. the induction of expression of type i interferons (ifns; including ifn-a and ifn-b) is a well-known innate antiviral immune reaction in the virus-infected cells [ , ] . however, n-prrsv infection suppressed spi ifn gene expression and decreased the transcript abundance of ifn-a ( figure a ). previous studies [ , , ] , both in vitro and in vivo, have also showed that prrsv elicited only a minimal ifn-a production or even suppressed it's expression. the suppression of spi ifn, in particular of ifn-a, is probably a crucial step in the pathogenesis, because ifn-a has been shown to inhibit prrsv replication [ ] . other viruses infection, such as the influenza virus [ ] , hepatitis c virus (hcv) [ ] , ebola virus [ ] , also suppressed type i ifn gene expression which led to extensive viral replication and increased pathogenesis. irf plays an important role for type i ifn gene expression. the transcript abundance of irf was decreased intensively in n-prrsv-infected pigs by h pi ( figure b ). one study [ ] indicated that prrsv nsp b inhibited irf , and then down-regulated ifn-b gene expression. it is worth mentioning that the nsp of the influenza a can also suppress innate immunity by inhibiting irf activation, and subsequently disrupting the induction of a/b-interferon [ ] . research has indicated that the expression of cd , a prrsv receptor [ ] , on macrophages in different microenvironments, in vivo, may determine the replication efficiency and subsequent pathogenecity of prrsv [ ] . transcript abundance of cd was significantly increased after n-prrsv infection ( figure a ). the internalization of prrsv via cd in the target cells may induce the expression of il , and in turn induce the expression of cd on neighboring undifferentiated monocytes and increased overall prrsv susceptibility [ ] . moreover, infected pigs develop a strong and rapid humoral response but these initial antibodies do not confer protection and can even be harmful by mediating an ade [ ] . these antibodies enhance prrsv viral replication by coating the virus and enhancing the internalization of viral particles into macrophages [ ] . as shown in figure g and h, igg and fcc receptors were significantly induced during n-prrsv infection processes. interestingly, the presence of antibodies during feline enteric coronaviruses (fecvs) infection does not also provide sterilizing immunity, instead, these antibodies opsonize virus particles and facilitate their entry to monocytes and/or macrophages through fcc receptors, resulting in disease enhancement [ ] . the activation of pro-inflammatory transcription factor nf-kb induces robust activation of the casp inflammasome and subsequent release of il- b that cause fever and inflammation [ , , , ] . accordingly, we identified upregulation expression of casp , nf-kb, and il- b genes during n-prrsv infection processes ( figure a ). nf-kb activation also enhanced the expression of matrix metalloproteinases (mmp ) and mmp , two cytotoxic substances in prrsv-infected cells [ , ] . similarly, transcript abundance of mmp and ngal ( kda alpha- -microglobulin-related subunit of mmp- ) was significantly increased in the lungs of n-prrsv-infected pigs ( figure f ). upregulation expression of mmps would likely facilitate infiltration of inflammatory cells and increase inflammation. upregulation expression of il (also known as cxcl ), which is an attractant for neutrophils and other polymorphonuclear leukocytes produced after acute infection, in prrsv-infected pams [ , ] and lungs of n-prrsv-infected pigs ( figure d ), was observed. other chemokines such as ccl (also known as mcp ), cxcl , cxcl (also known as ip ), which were significantly increased ( figure d ), may be also crucial for lymphocyte and macrophage infiltration into the sites of n-prrsv infection. ccl , il and ip expression were upregulated during sars-cov [ , ] , and murine coronavirus [ ] infections process, which may recruit monocytes and/or macrophages to sites of infection and be a major cause of lung pathology. although the present study indicates that upregulation expression of pro-inflammatory molecules contributes to the pathogenesis of n-prrsv, increased transcript abundance of anti-inflammatory molecules, such as il , pge , was also detected in the study. upregulation of il gene expression was found previously in prrsv-infected porcine leukocytes, pams, dcs, and in vivo in prrsv infected pigs [ , , , ] . perhaps an increase in pro-inflammatory molecules followed by increased anti-inflammatory molecules is the normal progression of events in inflammation [ ] . the upregulation expression of il might skew the immune response away from a protective th -cell response towards a non-protective th -cell response, therefore impairing clearance of virus, which benefits viral infections [ ] . upregulation expression of anti-inflammatory molecules and proinflammatory molecules occurring concurrently was also observed after sars-cov and fipv infection [ , ] . antibodies might also contribute to immunopathogenesis through increasing the uptake of virus by macrophages, resulting in activation of these macrophages and secretion of pro-inflammatory cytokines and chemokines. antigen-antibody complexes might increase transcript abundance of complement ( figure i ), which leads to generation of the classical inflammatory response through the production of potent proinflammatory molecules [ ] . furthermore, complement activation might also contribute to the development of pulmonary edema and oedema of the eyelids. further understanding the roles complement plays in the hostpathogen interactions may help to develop more effective pharmacological agents against infection. moreover, damage to the lungs of n-prrsv-infected pigs seems to occur directly by viral destruction of alveolar and bronchial epithelial cells and macrophages ( figure c) , as well as indirectly through production of immune mediators. activated ctls and nk cells release perforin (pfr) and granzymes, which two effectors act collaboratively to induce apoptosis of target cells. transcript abundance of pfr and granzymes increased in the lungs of n-prrsv infected pigs ( figure h ). pro-apoptotic molecules xaf , bid, cyto c, casp , aifm , were significantly up-regulated after infection with n-prrsv, which may induce apoptosis of virus-infected cells ( figure i ). simultaneously, we also observed upregulation expression of anti-apoptotic genes in n-prrsv infected lungs, including bcl a , mcl , chfr, nfkb, adm, il etc ( figure d ). upregulation expression of anti-apoptotic genes and pro-apoptotic genes occurring concurrently after n-prrsv infection seems in contradiction of each other. however, this may reflect a balance between apoptotic and anti-apoptotic mechanisms. perhaps prrsv actively induces an anti-apoptotic state to complete its virus replication cycle through delaying cell death while induces apoptosis of virus-infected cells after completion of virus replication to increase rate of spread of virus [ , , ] . anti-apoptotic and pro-apoptotic activaties were also observed in prrsv-infected marc- cells, in which prrsv stimulated anti-apoptotic pathways early in infection while caused apoptosis of prrsv-infected cells late in infection [ , ] . infection with n-prrsv also increased transcript abundance of nfkbia ( figure i ), an inhibitor of the tnf receptor activated transcription factor nf-kb. loss of nf-kb activity has been shown to increase the cytotoxic effects of tnf which resulted in increased cell death [ ] . an increase of transcript abundance in proapoptotic genes might result in disruption of the mitochondria transmembrane potential, thereby inducing release of cyto c from mitochondrial membranes to induce apoptosis and secondary necrosis [ ] . the production of ros, especially superoxide radicals, and the subsequent oxidative damage of cells and tissues are recognized as key contributors to the viral pathogenesis [ , ] . ros-mediated oxidative stress might also be involved in inducing apoptosis by prrsv [ ] . accordingly, we identified the remarkable upregulation of cytochrome b heavy chain (gp -phox) ( figure j ), a critical component of the membrane-bound oxidase of phagocytes (macrophages and neutrophils), after infection with n-prrsv, that generated superoxide radicals that killed both infected and normal cells at sites of infection, which would further exacerbate the immunopathological response. infection of macrophages, monocytes and dcs that are essential for immune function, is likely to be a key component in n-prrsv-induced pathogenesis [ , , , ] . apoptosis of infected cells causes immune suppression by two mechanism: apoptosis either induces a decrease in the numbers of immune cells that compromises both the innate and adaptive immune response in which they are unable to eradicate the primary infection, or impairs immunity by inducing immunosuppressive effects in the surviving cells [ ] . for example, uptake of apoptotic cells by normal macrophages and dcs stimulates immune tolerance by inducing the release of anti-inflammatory cytokines, such as il , and suppressing the release of pro-inflammatory cytokines [ ] . histopathological analysis of the lymphnodes of pigs infection with n-prrsv revealed a profound depletion of immune cells compared with those of unc (data not shown). in summary, the data presented in this study suggest that n-prrsv appears to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ade. after infection of macrophages and possibly dcs, prr-pamp interactions triggered signaling cascades that increased the transcript abundance of multiple inflammatory molecules, including cytokines, chemokines, adhesion molecules and inflammatory enzymes that induce a proinflammatory response, activate and recruit immune cells, such as macrophages and neutrophils, to sites of infection for virus elimination and thereby produce the clinical symptoms of viral infection, such as fever, dyspnoea, interstitial pneumonia in lungs. further, antibodies and complement activation might exacerbate inflammatory response. n-prrsv might induce an immunosuppressive state, mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. figure s signaling pathways of de genes. pathway analysis was mainly based on the kegg database. a p-value of , . and an fdr of , . in the two-side fisher's exact test were selected as the significant criteria. the vertical axis is the pathway category and the horizontal axis is the log (p value) of these significant pathways. found at: doi: . /journal.pone. .s ( . mb tif) figure s genes that distributed in the known pig qtls of health traits. the x axis represents the qtl symbol, and the y axis indicates the number of genes associated with health traits. see table s for full qtl names. figure s biological process go terms of profile . functional classification of the de genes was performed according to go biological processes. a p-value of , . in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. found at: doi: . /journal.pone. .s ( . mb tif) figure s biological process go terms of profile . functional classification of the de genes was performed according to go biological processes. a p-value of , . in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. found at: doi: . /journal.pone. .s ( . mb tif) figure s biological process go 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inflammation in sepsis death-defying immunity: do apoptotic cells influence antigen processing and presentation? we thank the beijing genomics institute (bgi) shenzhen and genminix informatics ltd.,co for their providing us with technical assistance in dge and bioinformatics analysis. key: cord- -cy y vnt authors: kumar, matam vijay; nagineni, chandrasekharam n; chin, marian s; hooks, john j; detrick, barbara title: innate immunity in the retina: toll-like receptor (tlr) signaling in human retinal pigment epithelial cells date: - - journal: j neuroimmunol doi: . /j.jneuroim. . . sha: doc_id: cord_uid: cy y vnt toll-like receptors (tlrs) are crucial components of innate immunity that participate in host defense against microbial pathogens. we evaluated the expression and function of tlrs in human retinal pigment epithelial (rpe) cells. real time pcr analysis revealed gene expression for tlrs – , , and in rpe cells. tlrs and were the most highly expressed tlrs. protein expression for tlrs , , and was observed on rpe cells and this expression was augmented by treatment with poly i:c or interferon-γ (ifn-γ). tlr is the receptor for dsrna, an intermediate of virus replication. because rpe cells express tlr and are frequently the site of virus replication within the retina, we evaluated tlr signaling. rpe cells treated with poly i:c produced ifn-β but not ifn-α, and this was inhibited by the treatment of rpe cells with anti-tlr antibody. human recombinant ifn-β was shown to be biologically active on rpe cells by inhibiting viral replication. poly i:c treatment of rpe resulted in an increase in the production of il- , il- , mcp- , and sicam- . the presence of tlrs on rpe cells and the resultant tlr signaling in rpe cells suggest that these molecules may play an important role in innate and adaptive immune responses within the retina. toll-like receptors, tlrs, are a family of evolutionary conserved innate immune recognition molecules that recognize molecular patterns associated with microbial pathogens (medzhitov and janeway, ) . they constitute a first line of defense against a variety of pathogens and play a critical role in initiating the innate immune response. tlr recognition of these specific microbial patterns leads to a signal transduction cascade that generates a rapid and robust inflammatory response marked by cellular activation and cytokine release (gordon, ; schnare et al., ) . to date, mammalian tlrs have been identified and each receptor appears to be involved in the recognition of a unique set of microbial patterns (gordon, ; zuany-amorim et al., ) . for example, tlr recognizes various ligands expressed by gram-positive bacteria, whereas tlr engages dsrna and tlr is specific for gram-negative bacteria lipopolysaccharides (lps; alexopoulou et al., ; campos et al., ; johnson et al., ; opitz et al., ) . tlr , on the other hand, recognizes bacterial flagellin, while tlrs and interact with antiviral compounds and tlr binds bacterial dna (bauer et al., ; gewirtz et al., ; ito et al., ) . recently, tlrs were observed to influence the development of the adaptive immune response presumably through the activation of antigen-presenting cells (apc; schjetne et al., ) . the most compelling evidence comes from studies on dendritic cells (banchereau et al., ) . it has previously been shown that multiple tlrs are found on dendritic cells. signaling through these receptors augments antigen presentation by driving cell maturation, upregulating expression of costimulatory molecules on the cell, and inducing cytokines (palucka and banchereau, ) . thus, tlrs may serve as a unique link between innate and adaptive immunity. the retinal pigment epithelium consists of a single layer of cells of neural ectoderm origin, which lie between the photoreceptors of the neural retina and the blood-rich choroid. these vitally important cells phagocytize the shed discs from the photoreceptor outer segments, recycling their components, such as retinoids. additional functions of this monolayer of cells include the transport of nutrients from the choroid into the retina and the transport of waste in the reverse direction. this cell also adsorbs light and provides adhesive properties for the retina (bok, (bok, , . the retinal pigment epithelial (rpe) cell also plays a key role in a variety of retinal pathologic processes. inherited retinal degenerative diseases can be associated with mutations of rpe cellular genes (hamel et al., ; morimura et al., morimura et al., , . moreover, degenerative diseases, such as age-related macular degeneration and diabetic retinopathy, can be associated with early damage to the rpe cell (cai et al., ; lutty et al., ) . in addition, a variety of in vivo and in vitro studies have identified this cell as an ideal target for infectious agents such as cytomegalovirus (cmv), toxoplasma gondii and coronavirus hooks et al., ; nagineni et al., nagineni et al., , . furthermore, this cell is a rich source of cytokines, chemokines, and growth factors that may contribute to or limit pathologic processes (chin et al., ; momma et al., ) . recently, the rpe cell has been shown to play a pivotal role in the immune system. it has been demonstrated that interferon-g (ifn-g) treatment up-regulates the expression of both mhc class-i and -ii molecules on rpe cells (percopo et al., ) . moreover, rpe cells have been reported to act as apc in the retina (percopo et al., ) . hence, rpe cells can incorporate pathogens, produce a variety of cytokines, and present pathogen-derived peptides to sensitized t cells. this finding, that rpe cells function as apc in the retina, extends the activities of this cell beyond its participation as a first line defense cell and underscores its important role in adaptive immunity. therefore, based on these observations, it was of interest to further define the role of this epithelial cell in innate and adaptive immune responses within the retina. to date, there is no information about the presence of tlrs within the retina. the results of this study demonstrate that rpe cells do indeed constitutively express distinct types of tlrs and that their expressions are modulated in the presence of dsrna and cytokines. tlr is a receptor for dsrna, and dsrna is a common replication intermediate for many viruses. moreover, tlr has been described as a specific tlr because it displays the most restricted cellular expression pattern (janssens and beyaert, ) . because tlr gene expression was identified in rpe cells and because the rpe cells are a site of replication for both rna and dna viruses, we further investigated tlr signaling in these cells. our data suggest that the binding of poly i:c, an analog of dsrna, to tlr on human rpe cells resulted in the production of ifn-h and other cytokines, chemokines, and adhesion molecules. thus, tlr signaling within the retina may provide additional protective molecules to mediate viral infections. affinity purified, monoclonal, antihuman tlr and tlr antibodies were purchased from imgenex (san diego, ca) while antihuman tlr (hta ) antibodies were purchased from ebiosciences (san diego, ca). polyclonal antibodies to tlr were obtained from santa cruz biotechnology (santa cruz, ca), lps (salmonella typhosa), poly i:c, and poly di:dc were purchased from sigma (st louis, mo). the recombinant human (ifn-g) and ifn-h were procured from roche molecular biochemicals (indianapolis, in). rna stat- was obtained from tel-test (friendswood, tx). minimal essential media (mem), fetal bovine serum (fbs), penicillin/streptomycin/ fungizone, nonessential amino acids, and normal horse serum (nhs) were purchased from life technologies/gibco (gaithersburg, md). geneamp rna pcr kits and taqman reagents were obtained from perkin elmer (branchburg, nj) . human rpe cell cultures were prepared from donor eyes and grown in mem supplemented with % fbs, nonessential amino acids, and penicillin/streptomycin/fungizone in a % co , humidified jc incubator. characterization of these cells has been described previously (li et al., ; nagineni et al., nagineni et al., , briefly, these cells demonstrated a hexagonal morphology when grown to confluence and formed monolayers with distinct intracellular boundaries. homogeneity of the cultures was established by positive immunostaining with monoclonal antibodies to cytokeratin, an epithelial cell-specific cytoskeletol protein. for the experiments described in this paper, human rpe cultures, at passages to , were used. a human monocyte cell line, u , was grown in an rpmi- medium supplemented with % fbs (atcc, manassas, va). total rna prepared from confluent monolayers of human rpe cells and from suspension cultures of u was used to evaluate the constitutive expression of tlr mrna. to study the effects of tlr activators, human rpe cells were washed with serum-free media (sfm) and incubated in sfm for h in the presence of poly i:c ( ag/ml ), lps ( ag/ml), or ifn-g ( u/ml). total rna was prepared from the cell cultures by using the rna stat- extraction solution. one ag of total rna was used for each rt-pcr reaction. the rt-pcr procedure was performed using an rna pcr kit (perkin-elmer) according to the manufacturer's instructions. pcr products were separated by gel electrophoresis, photographed under uv light, and integrated with an image acquisition system (eagle eye, stratagene, san diego, ca). the following primer pairs were used for the analysis of tlrs, costimulator molecules, and gapdh by rt-pcr: tlr ( bp) v-ctatacaccaagttgt-cagc- v and v-gtctccaactcagtaaggtg- v; tlr ( bp) v-gccaaagtcttgattgattgg- v and v-ttgaagttctccagctcctg- v; tlr ( bp) v-gatctgtctcataatggcttg- v and v-ga-cagattccgaatgcttgtg- v; tlr ( bp) v-tggatacgtttccttataag- v and v-gaaatg-gaggcaccccttc- v; tlr ( bp) v-tagctcc-taatctgatg- v and v-ccatgtgaagtctttgct gc- v; tlr ( bp) v-tctacctgggccaaaact gtt- and v-ggcacatgctgaagagagtta- v; tlr ( bp) v-gccagcgagtctcactgaact- v and v-gccagggcagccaacata- v; tlr ( bp) v-gt ccccacttct ccatg- v and v-gg c aca - (faure et al., ; ito et al., ) , jarrossay et al., ; bauer et al., and tabeta et al., ) . total rna was prepared from quiescent rpe and u by using an rna stat- reagent. quantitative rt-pcr analysis of tlr in rpe and u was performed on an abi prism (applied biosystems, foster city, ca) by using taqman master mix reagent kits according to the manufacturer's instructions. fam-labeled taqman probes and primers for human gapdh and toll-like receptors - (assays-on-demand gene expression products) were obtained from applied biosystems. standard curves were generated to gapdh and tlrs to by ten-fold serial dilutions of rpe and/or u rna. rna samples were analyzed in triplicate under similar conditions as those of the standards in the same -well plates for cycles. fluorescence intensities obtained for the samples were used to calculate relative fluorescence units by normalizing to gapdh fluorescence intensities. results are expressed as relative fluorescence units of tlrs - mrna levels in rpe and u cells. the rpe cells were seeded onto lab-tek tissue culture chamber slides (nalge nunc international, naperville, il). after h, the cells were washed with sfm and stimulated with media, poly i:c ( ag/ml), lps (s. typhosa, ag/ ml), or with ifn-g ( u/ml) for h. the slides were then fixed in equal parts of acetone/methanol and stored at À jc until analyzed. the slides were washed twice with pbs for min and then treated with % nhs in pbs for h at room temperature. the slides were overlaid with primary mouse antihuman tlrs , , or monoclonal antibodies ( ag/ml in pbs with % nhs) or with mouse igg (control) and incubated for -h followed by five washes in pbs containing % nhs. cells were then incubated for h with biotin-labeled horse antimouse igg (h + l; vector laboratories, burlingame, ca). the cells were washed again five times as described previously. fitc-labeled streptavidin ( ag/ml) was added and the cells were incubated for min in the dark (vector laboratories). the slides were then washed twice, mounted, and examined with a fluorescent microscope. . . eia assays for cytokines, chemokines, and adhesion molecules rpe cultures were grown to confluence in -well dishes. cultures were washed with sfm and incubated in the presence of various concentrations of poly i:c or poly di:dc for h at jc. supernatants were collected and stored at À jc until analyzed. the concentration of il- , il- , mcp- , sicam- , ifn-a, and ifn-h in the cell culture supernatant fluids was determined by eia. the assay was performed according to manufacturer's instructions (quantikine eia kits, r&d systems, minneapolis, mn). the data were analyzed using the versamax data analysis program (molecular devices, sunnyvale, ca). results from two representative experiments are presented as the means f s.d. of triplicate cytokine measurements. . . neutralization of poly i:c effects on rpe by tlr antibody rpe cultures were grown to confluence in -well plates in % fbs media. media were removed and replaced with serum free media (sfm). after h, media were removed and replaced with fresh sfm ( ml per well) and polyclonal antibody to tlr ( ug igg/ml). after a -h incubation at jc, poly i:c was added to the wells to obtain a final concentration of or ug/ml. the cultures were further incubated for h and culture supernatants were collected. the levels of secreted ifn-h were determined by eia. rpe cultures were grown to confluence in -well plates. the cultures were washed and incubated for h with serial ten-fold dilutions of recombinant human ifn-h or with media. the monolayers were washed and challenged with approximately plaque-forming units (pfu) of vesicular stomatitis virus. one hour later, the virus inoculum was removed and the cells were washed and refed with ml of mem containing . % methylcellulose and % fbs. after a -h incubation at jc, the overlay medium was removed, the cells were fixed with ethanol and stained with giemsa's solution, and the viral plaques were counted. preliminary studies using rt-pcr analysis indicated that human rpe cells contained detectable amounts of mrna for tlrs , , , , and . u cells are a human monocyte cell line that was used as a control. u cells contained detectable levels of mrna for tlrs , , , , , and . tlr mrna was barely detected in the u cells. in contrast, tlr mrna was highly expressed in human rpe cells. moreover, mrna for two coreceptors for tlrs, cd , and md were detected in human rpe whereas the u cells expressed only cd . in order to more accurately define the constitutive expression of tlrs, we analysed mrna obtained from the two cell types using real time rt-pcr analysis. as shown in table , real time rt-pcr analysis of rpe cells revealed the constitutive expression of varying levels of mrna for tlrs , , , , , , , , and . tlr was not detected and low levels of expression were noted for tlrs , , , and . tlrs and were the most highly expressed tlrs in rpe cells. when compared to u cells, rpe cells contained times more mrna for tlr . we next wanted to determine if poly i:c (dsrna) treatment altered tlr gene expression in rpe cells. the effect of poly i:c treatment on tlrs , , and gene expressions in rpe cells is shown in fig. . nih image analysis of this data indicated that poly i:c treatment increased gene expression of tlr by %, tlr by %, and tlr by %. gene expression for the coreceptors, cd and md , was also evaluated. poly i:c treatment had no effect on cd and md . rpe cells propagated on culture slides were exposed to media alone or media containing poly i:c ( ag/ml) or ifn-g ( u/ml; fig. ). after h, the cells were washed and fixed with equal volumes of acetone and methanol and reacted with normal mouse igg or with antibody directed to tlrs , , or . staining was not observed in cells treated with normal mouse igg (control). the intensity of tlr reactivity was weak on untreated rpe cells, but this was enhanced by pretreatment with poly i:c or ifn-g (data not shown). intensity of tlr reactivity was moderate in untreated rpe cells. this intensity was enhanced with poly i:c ( fig. a and b) or ifn-g. intensity of tlr reactivity was moderate on untreated rpe cells but was augmented following pretreatment with ifn-g ( fig. c and d) or with poly i:c. these data demonstrate that rpe cells express varying amounts of tlrs , , and and that these receptors can be modulated by selected tlr activators. the pattern of staining for tlr and tlr in rpe cells was different. tlr staining appears dispersed throughout the cytoplasm whereas tlr staining appears to be more localized at the cell boarders. tlr has only been detected on a limited number of cell types and recent studies indicate that tlr is a receptor for dsrna produced by viruses. moreover, dsrna binding to tlr on the cell surface results in the production of type- ifns. therefore, we next investigated the possibility that treatment of rpe cells with synthetic dsrna, poly i:c, would induce ifn. rpe cells were treated with varying concentrations of poly i:c or with poly di:dc ( mu/ml). poly di:dc is a synthetic double-stranded polydeoxyinosine/ deoxycytosine (dsdna) and is used as a negative control for poly i:c (dsrna). the cells were incubated for h and supernatant fluids were collected and assayed for ifn-a and ifn-h by eia. as seen in fig. a , poly i:c induced ifn-h in a dose-dependent manner. ifn-a was not detected in these samples or in untreated cells. moreover, cells treated with poly di:dc did not release ifn-a or ifn-h. because we have shown that rpe cells can produce ifnh, we next wanted to determine if ifn-h was biologically active on rpe cells. cell cultures were treated with varying concentrations of recombinant human ifn-h. after incubation for h, cells were challenged with approximately pfu of vsv. virus replication in rpe was evaluated by plaque assay. as seen in fig. b , rpe cells were sensitive to the antiviral action of ifn-h. one unit of ifn-h inhibited vsv by %, whereas and units inhibited virus replication by to %. in order to evaluate the specificity of the poly i:c induction of ifn-h through tlr , we next performed cultures were washed with sfm twice and incubated in the presence of various concentrations of poly i:c or poly di:dc for h. the culture supernatants were collected and analyzed for ifn-a and -h by eia. results from three experiments conducted in duplicate are presented as the means f s.e. (b) human rpe cultures were grown to confluence and were infected with vesicular stomatitis virus (vsv) as described in the materials and methods section. after h, cultures were fixed and stained with giemsa, and the plaques were counted. the data are presented as the means f s.e. of triplicate cultures obtained from a representative experiment. fig. . the effect of anti-tlr antibody on poly i:c-induced ifn-h production in rpe cells. rpe cell cultures grown to confluence were washed with sfm and incubated with media or anti-tlr antibody ( ug/ ml) for h. then, poly i:c was added to the appropriate wells to give a final concentration of or ug/ml. after h of incubation, supernatant fluids were collected and the concentration of ifn-h was determined by eia. results presented were obtained from one representative experiment with quadruplicate samples. antibody inhibition assays. rpe cells were pretreated with anti-tlr antibody for h and were then incubated with poly i:c for h. supernatant fluids were harvested and then analyzed for the presence of ifn-h. as seen in fig. , treatment with or ug of poly i:c induced . f . and . f . units of ifn-h, respectively. pretreatment of the cells with anti-tlr antibody reduced the levels of ifn-h produced to . and . ( p < . ). . . evaluation of poly i:c treatment of rpe cells: cytokines, chemokines, and adhesion molecules we next investigated whether poly i:c treatment of rpe cells resulted in the modification of additional cellular functions such as the production of cytokines, chemokines, and adhesion molecules. rpe cells were incubated with varying concentrations of poly i:c for h. the supernatant fluids were collected and evaluated by eia for il- , il- , mcp- , and sicam- production. as seen in fig. a -d, supernatant fluids from untreated rpe cells did not contain il- or il- and only very low levels of mcp- and sicam- . poly i:c treatment of rpe cells resulted in a dose-dependent enhancement of il- , il- , mcp- , and sicam- . following poly i:c treatment at , , or ag/ml, the concentration of il- increased from to ng/ml, il- increased from to ng/ml, mcp- increased from to ng/ml, and sicam- increased from . to ng/ml. in contrast, treatment of rpe cells under similar conditions with poly di:dc did not enhance the secretion of il- , il- , mcp- , and sicam- (fig. ) . tlrs are critical elements in the host defense against microbial pathogens (takeda et al., ) . in this report, we demonstrate for the first time the presence of tlrs on human rpe cells. real time pcr analysis of tlr gene expression identified tlrs - , and in human rpe cells. furthermore, human rpe cells highly expressed tlr . protein expression for tlrs , and was also observed on rpe cells and this expression was augmented by treatment with poly i:c or ifn-g. because tlr is found on a limited number of cells and was highly expressed in fig. . the effects of poly i:c treatment on the production of cytokines by human rpe cells. rpe cell cultures grown to confluence in -well plates were washed with sfm and incubated without (control) or with sfm containing various concentrations of poly i:c or poly di:dc. after h, the culture supernatants were collected and the concentrations of il- , il- , mcp- , and sicam- were determined by eia. results presented for il- (a), il- (b), mcp- (c), and icam- (d) were obtained from duplicate samples of two representative experiments. rpe cells, we performed studies to analyze signaling through tlr . the interaction of poly i:c with rpe cells resulted in the secretion of ifn-h as well as il- , il- , mcp- , and sicam- . moreover, we show that ifn-h is highly effective in inhibiting virus replication in rpe cells. specificity for tlr signaling was demonstrated by the inhibition of poly i:c induction of ifn-h by pretreatment of rpe cells with anti-tlr antibody. mucosal cells such as gastrointestinal, airway, and urinary epithelial cells are considered as the front line of defense against pathogenic microorganisms (schulz et al., ; hornef et al., ; pitman and blumberg, ; tsuboi et al., ) these cells have developed specific mechanisms for microbial protection that contribute to the innate immune response. recently, it has been demonstrated that these epithelial cells contain several tlrs and respond to microbes by secreting cytokines and chemokines. for example, a murine intestinal epithelial cell line is highly responsive to lps and expresses both tlr and cd . corneal epithelial cells have been reported to express tlr and cd , and the lps treatment of these cells resulted in the secretion of multiple proinflammatory cytokines and chemokines (song et al., ) . like other epithelial cells, the rpe cell can also be considered as a front line defense against invading organisms. it is strategically located between the neural retina and the blood-rich choroid. the rpe forms a barrier, limiting access to photoreceptors and other neuronal cells within the retina. earlier studies by our laboratory and others have revealed that rpe cells can be stimulated to produce a variety of cytokines, chemokines, and adhesion molecules (elner et al., (elner et al., , momma et al., ; nagineni et al., ) . in this report, we show that rpe cells possess a variety of tlrs and the costimulatory molecules, cd and md (tabeta et al., ) . tlr and tlr are the most widely studied members of the tlr family (johnson et al., ; faure et al., ; opitz et al., ) . both of these tlrs were found on the rpe cell. thus, rpe cells may defend against infections by sensing microbial invasion through multiple tlrs and the costimulatory molecules, cd and md . to date, tlr expression has been limited to a small number of cells types. earlier studies demonstrate that tlr is constitutively expressed on intestinal epithelial cells, dendritic cells, and mast cells (cario and podolsky, ; muzio et al., ) . the dendritic cell is a potent antigenpresenting cell that expresses multiple tlrs including tlr (banchereau et al., ; jarrossay et al., ) . several studies suggest that tlr signaling on dendritic cells amplifies antigen presentation by producing proinflammatory cytokines, up-regulating co-stimulatory molecules, such as cd , cd , and cd , and by increasing migration of cells to the lymph node (palucka and banchereau, ) . the dendritic cell and the rpe cell share some common features. both cells express tlr and are apc. the dendritic cell responds to tlr signaling by producing both ifn-a and ifn-h, whereas the rpe cell produces only ifn-h. this difference is probably a reflection of different cellular origins. presently, dendritic cells are considered to be of bone marrow origin while rpe cells are of neural ectoderm origin. additional studies are required to determine if the tlrs on the rpe cell also can augment apc functions. the binding of dsrna or poly i:c, an analog of dsrna, to tlr results in the production of type- ifns and other cytokines and chemokines. specificity for dsrna interactions with tlr was demonstrated by matsumoto et al. ( ) . their studies pointed out that poly i:c-induced ifn-h was suppressed by pretreatment with monoclonal antibody to tlr . additional studies on tlr knockout mice revealed that poly i:c treatment up-regulated the production of type- ifns (ifn-a, ifn-h) in wild-type mice but not in tlr ko mice (alexopoulou et al., ) . in this report, we demonstrate that the poly i:c treatment of human rpe cells results in the production and release of ifn-h and not ifn-a. moreover, these rpe cells are highly sensitive to the antiviral actions of human ifn-h. clearly, dsrna-mediated signaling in rpe cells can have a protective role in viral infections in the retina. in light of this, numerous in vivo and in vitro studies have identified that rpe cells are one of the principle targets for infectious agents, such as cmv, t. gondii, and murine coronaviruses (bodaghi et al., ; detrick et al., ; hooks et al., ; nagineni et al., ) . it is important to point out that additional tlrs may also participate in selected virus infections and further work is needed to better define this interaction (bieback et al., ; compton et al., ; kurt-jones et al., ) . when an infecting agent enters the retina, it is critically important for the host to have a rapid response system to limit damage to nonregenerating retinal cells. the rpe cells are strategically placed to function as protective cells. clearly, the innate immune system composed of tlrs is a primary rapid response system to infection. this study demonstrates that the rpe cell expresses tlrs - , and and therefore can initiate signaling pathways that stimulate host defenses. moreover, these cells respond to tlr stimulation by producing ifnh, il- , il- , mcp- , and sicam- . these cytokines, chemokines, and adhesion molecules together then participate in initiating adaptive immune responses. the demonstration that rpe cells express tlr and release ifnh represents a hitherto unrecognized biological role for the rpe cell. recognition of double-stranded rna and activation of nf-kappab by tolllike receptor dendritic cells: controllers of the immune system and a new promise for immunotherapy human tlr confers responsiveness to bacterial dna via species-specific cpg motif recognition hemagglutinin protein of wild-type measles virus activates toll-like receptor signaling entry of human cytomegalovirus into retinal pigment epithelial and endothelial cells by endocytosis retinal photoreceptor-pigment epithelium interactions. friedenwald lecture the retinal pigment epithelium: a versatile partner in vision oxidative damage and protection of the rpe activation of toll-like receptor- by glycosylphosphatidylinositol anchors from a protozoan parasite differential alteration in intestinal epithelial cell expression of toll-like receptor (tlr ) and tlr in inflammatory bowel disease cyclooxygenase- gene expression and regulation in human retinal pigment epithelial cells human cytomegalovirus activates inflammatory cytokine responses via cd and toll-like receptor cytomegalovirus replication in human retinal pigment epithelial cells. altered expression of viral early proteins inhibition of human cytomegalovirus replication in a human retinal epithelial cell model by antisense oligonucleotides neutrophil chemotactic factor (il- ) gene expression by cytokine-treated retinal pigment epithelial cells interleukin- (il- ) gene expression and secretion by cytokine-stimulated human retinal pigment epithelial cells bacterial lipopolysaccharide activates nf-kappab through toll-like receptor (tlr- ) in cultured human dermal endothelial cells. differential expression of tlr- and tlr- in endothelial cells cutting edge: bacterial flagellin activates basolaterally expressed tlr to induce epithelial proinflammatory gene expression pattern recognition receptors: doubling up for the innate immune response a developmentally regulated microsomal protein specific for the pigment epithelium of the vertebrate retina retina and retinal pigment epithelial cell autoantibodies are produced during murine coronavirus retinopathy toll-like receptor resides in the golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells interferonalpha and interleukin- are induced differentially by toll-like receptor ligands in human blood dendritic cell subsets role of toll-like receptors in pathogen recognition specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by toll-like receptor pattern recognition receptors tlr and cd mediate response to respiratory syncytial virus interferon-gamma signaling in human retinal pigment epithelial cells mediated by stat , icsbp, and irf- transcription factors changes in choriocapillaris and retinal pigment epithelium in age-related macular degeneration establishment of a monoclonal antibody against human toll-like receptor that blocks double-stranded rna-mediated signaling innate immunity: the virtues of a nonclonal system of recognition differential expression of chemokines by human retinal pigment epithelial cells infected with cytomegalovirus mutations in the rpe gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis mutations in rgr, encoding a light-sensitive opsin homologue, in patients with retinitis pigmentosa differential expression and regulation of toll-like receptors (tlr) in human leukocytes: selective expression of tlr in dendritic cells synergistic effects of gamma interferon on inflammatory mediators that induce interleukin- gene expression and secretion by human retinal pigment epithelial cells mechanisms of interferon-induced inhibition of toxoplasma gondii replication in human retinal pigment epithelial cells toxoplasma gondii infection induces gene expression and secretion of interleukin (il- ), il- , granulocyte-macrophage colony-stimulating factor, and intercellular adhesion molecule by human retinal pigment epithelial cells transforming growth factorbeta expression in human retinal pigment epithelial cells is enhanced by toxoplasma gondii: a possible role in the immunopathogenesis of retinochoroiditis toll-like receptor- mediates treponema glycolipid and lipoteichoic acid-induced nf-kappab translocation how dendritic cells and microbes interact to elicit or subvert protective immune responses cytokine-mediated activation of a neuronal retinal resident cell provokes antigen presentation first line of defense: the role of the intestinal epithelium as an active component of the mucosal immune system cutting edge: link between innate and adaptive immunity: toll-like receptor internalizes antigen for presentation to cd + t cells and could be an efficient vaccine target toll-like receptors control activation of adaptive immune responses differences in lps-induced activation of bronchial epithelial cells (beas- b) and type ii-like pneumocytes (a- ) the expression of functional lps receptor proteins cd and toll-like receptor in human corneal cells toll-like receptors confer responsiveness to lipopolysaccharide from porphyromonas gingivalis in human gingival fibroblasts toll-like receptors roles of toll-like receptors in c-c chemokine production by renal tubular epithelial cells toll-like receptors as potential therapeutic targets for multiple diseases key: cord- - zyoddl authors: hu, xiaoliang; tian, jin; kang, hongtao; guo, dongchun; liu, jiasen; liu, dafei; jiang, qian; li, zhijie; qu, juanjuan; qu, liandong title: transmissible gastroenteritis virus papain-like protease antagonizes production of interferon-β through its deubiquitinase activity date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: zyoddl coronaviruses (covs), such as human coronavirus nl (hcov-nl ), severe acute respiratory syndrome cov (sars-cov), murine hepatitis virus (mhv), porcine epidemic diarrhea virus (pedv), and middle east respiratory syndrome coronavirus (mers-cov), encode papain-like (pl) proteases that inhibit sendai virus- (sev-) induced interferon (ifn-β) production. recently, the crystal structure of transmissible gastroenteritis virus (tgev) pl has been solved, which was similar to that of sars-cov pl (pro), which may antagonize host innate immunity. however, very little is known about whether tgev pl can antagonize host innate immune response. here, we presented evidence that tgev pl encoded by the replicase gene could suppress the ifn-β expression and inhibit the nuclear translocation of interferon regulatory factor (irf ). the ability to antagonize ifn-β production was dependent on the intact catalytic activity of pl . furthermore, tgev pl exerted deubiquitinase (dub) activity which strongly inhibited the retinoic acid-induced gene i- (rig- -) and stimulator of interferon gene- (sting-) dependent ifn expression. our data collectively suggest that tgev pl can inhibit the ifn-β expression and interfere with rig- - and sting-mediated signaling through a viral dub activity. our study has yielded strong evidence for the tgev pl mechanisms that counteract the host innate immunity. the innate immune system is the first line of defense that protects the host against viral infection, and the induction of ifn-/ is a crucial antiviral mechanism of the innate immune system [ ] . the initiation of ifn expression is triggered by pathogen-associated molecular patterns (pamps) through host pattern recognition receptors (prrs) [ ] . after viral rnas are sensed by prrs, signals are transmitted to different downstream adaptor molecules (such as ifnpromoter stimulator (ips- )); and then i b kinase-(ikk-) related kinases are recruited. next, interferon regulatory factor (irf ), nuclear factor b (nf-b), and atf- /c-jun are activated by the kinase complexes and translocate to the nucleus and directly induce the expression of type i ifns [ ] . tgev is an enveloped virus belonging to the coronaviridae (cov) family and the nidovirales order. covs are positive-strand rna viruses that replicate in the cytoplasm of infected cells [ ] . covs encode two types of cysteine proteases, m pro , and papain-like proteases, pl and pl , which contained nonstructural protein (nsp ) and nsp , respectively. pl pro is served mainly as in processing of the replicase pp a and pp ab polypeptides [ ] . other than their role in replicase polyprotein processing, pl domains possess an additional but related enzymatic activity, in hcov-nl [ ] , mhv [ ] , sars-cov [ , ] , and mers-cov [ ] , through their deubiquitination (dub) enzymes, which play a key role in antagonizing ifn induction. however, tgev pl processes the nsp /nsp site and is capable of hydrolyzing isopeptide bonds in both lys -and biomed research international lys -linked polyubiquitin chains [ ] . whether tgev pl could antagonize the production of ifns was unknown. in the present study, we found that tgev pl encoded by the replicase gene could suppress the ifn-expression and inhibit the nuclear translocation of interferon regulatory factor (irf ) and exerted deubiquitinase (dub) activity which strongly inhibited the retinoic acid-induced gene i-(rig- -) and stimulator of interferon gene-(sting-) dependent ifn expression. cells and viruses. hek t cells and pk- cells were cultured in dulbecco's modified eagle's medium (hyclone, logan, usa) containing % (v/v) fetal calf serum supplemented with penicillin ( u ml − ) and streptomycin ( g ml − ). sendai virus (sev) was obtained from the centre of virus resource and information (wuhan institute of virology, chinese academy of sciences). plasmids and agents. ifn--luc, x prdiii/i-luc (referred to as irf -luc), x ap- -luc, and x nf-b-luc luciferase reporter plasmids were constructed according to an earlier protocol [ ] . accession numbers of sting, irf , and mavs were kc , kc , and kc , respectively. expression plasmids for rig- (p-flag-rig- ) and tbk- (p-flag-tbk- ) were generated with the following primers: rig- forward, -tttggatccatgacagca-gagcagcggcggaat- , rig- reverse -tttaag-cttcactcaaggttcgggattccctg- ; tbk- forward, -tttgaattcatgcagagcacttctaatcat-cttt- , tbk- reverse -tttagatcttaaagacag-tcaacattgcgaa- . to construct the dna expression vector, pmyc-pl , pflag-pl , and ppl -myc, encoding tgev pl , standard reverse transcription-(rt-) pcr was applied to amplify cdna of the total rna extracted from pk- cells infected with the tgev strain hx, using the following primers: pl -forward, -gtacaagaa-gctgaacaatttaa- ( - bp), pl reverse, -atcgtttttaggactttgaattt- ( - bp). all constructs were validated via dna sequencing. pdsred -mito was purchased from clontech (tokyo, japan). transfection agent was performed with x-tremegene hp (roche, switzerland) per the manufacturer's instructions. luciferase reporter gene assay. hek- t cells grown in -well plates were cotransfected with . g/well reporter plasmid, . g/well prl-tk plasmid (promega, madison, usa) as an internal control for normalization of transfection efficiency, and the indicated expression or empty control vector plasmid. where indicated, cells were also mockinfected or infected with sev ( hemagglutinating activity units/well) at h after cotransfection. cells were subsequently lysed, and firefly and renilla luciferase activities were determined with the dual-luciferase reporter assay system (promega, madison, usa), according to the manufacturer's protocol. data are presented as mean relative luciferase units ± standard deviation from triplicate samples. for statistical analysis, data were compared between empty vector-and tgev pl -transfected groups with the unpaired, two-tailed student's t-test using spss . software. values < . were considered statistically significant [ ] . elisa. cell supernatants of transfected pk- cells were centrifuged at , for min to remove cell debris and stored at − ∘ c until use. secreted ifn-in the cell supernatants was determined using commercial porcine ifn-(interferon beta) elisa kit (elabscience, china) according to the manufacturer's instructions. immunoblot analysis. hek t cells were cultured in well plates and mm dishes were transfected with the appropriate plasmids. after h, cells were harvested by the addition of lysis buffer and protein concentrations in whole cell extracts measured. equal amounts of samples were subjected to sds-page and analyzed for tgev pl , sting, tbk- , and irf proteins via immunoblotting using ha, flag, or gfp-tagged antibodies (sigma, st louis, usa). expression of p-irf , irf , and gapdh was detected with the rabbit-anti p-irf (ab ), irf (ab ) (abcam, cambridge, uk), and a mouse anti-gapdh monoclonal antibody (sigma, st louis, usa). assay of deubiquitinase activity in cultured cells. hek t cells were cotransfected with pcdna . -ha-ub plus the indicated amounts of tgev pl , p-flag-rig- , and p-flag-sting constructs. the effect of tgev pl on ubiquitinated proteins in cultured cells was assessed by immunoblot analysis. coimmunoprecipitation analysis. coimmunoprecipitation experiments were performed on hek t cells transfected with the indicated expression plasmids as described in an earlier report [ ] . immunofluorescence assay. hek t cells were plated on fibronectin-treated glass coverslips in -well plates. to evaluate the localization of tgev pl , cells were cotransfected with plasmid dna expressing flag-pl ( ng per well) and pdsred-mito ( ng per well) using x-treme gene hp, according to the manufacturer's protocol. hek t cells were cotransfected with irf -gfp ( ng per well) and empty vector ( ng per well) or irf -gfp ( ng per well) and flag-pl ( ng per well). h after transfection, cells were infected with sev ( hemagglutinating activity units/well) for h. next, cells were fixed with % paraformaldehyde for min and permeated with . % triton x- for min at room temperature. after three washes with pbs, cells were blocked with pbs containing % bovine serum albumin for h, followed by incubation with a mouse monoclonal antibody against flag ( : ) for h at room temperature. cells were treated with fluorescein isothiocyanate-labeled goat anti-mouse (sigma, st louis, usa) for h, and subsequently with , -diamidino- -phenylindole (dapi) for min at room temperature. samples were washed with pbs, and fluorescent images were acquired under a confocal laser scanning microscope (tcs sp ; leica, solms, germany). to assess the formation of sting dimers, hek t cells were transfected with flag-sting ( ng per well) and the lysates were subjected to western blot, as described earlier [ ] , with the indicated antibodies. is an ifn antagonist. the crystal structure of tgev pl has been determined [ ] . the structure of tgev pl is similar to that of sars-cov pl pro . in order to determine whether tgev pl is capable of blocking ifn-production, we assessed ifn-promoter activity in the presence of pl (figure (a) ). hek t cells were cotransfected with tgev pl and ifn-luciferase or renilla luciferase reporter plasmids for h and subsequently infected with sev to activate the rig- -dependent ifn-expression pathway. we observed the inhibition of sev-induced ifn-promoter activation in the presence of pl , similar to the antagonistic function of nl plp and porcine epidemic diarrhea virus (pedv) plp , clearly indicating that tgev pl could act as an interferon antagonist. to establish the mechanisms by which pl inhibits ifnexpression, transcriptional activities of nf-b, irf , and ap- were analyzed using the luciferase assay to identify the precise transcription factor involved. notably, the luciferase activities of all three transcription factors were significantly inhibited by tgev pl in a dose-dependent manner ( figures (b) , (c), and (d)). furthermore, flag-pl also significantly inhibited ifn-production in pk- cells at protein level ( figure (e) ), which was further confirmed with the result that tgev pl could block the production of interferon. to further establish whether tgev pl affects irf phosphorylation or migration from the cytoplasm to nucleus, hek t cells were transfected with tgev pl and/or irf -egfp. then the result was analyzed using western blot and confocal microscopy. in figure (f) , the level of p-irf was decreased significantly by tgev pl compared with that of sev-induced. furthermore, irf -egfp was located in the cytoplasm compared with mock-infected hek t cells but translocated to the nucleus when the cells were inoculated with sev. in contrast, after being inoculated with sev, it was found that irf -egfp was translocated from cytoplasm to nuclear in mock infected hek t cell, which was not observed in cells transfected with tgev pl (figure (g) ). our results collectively suggested that tgev pl suppressed ifn-transcription by interfering with nf-b-, irf -, and ap- signaling-mediated ifn expression. to determine whether tgev pl is capable of blocking stingmediated activation of the ifn-promoter, we assessed promoter activity in the presence of sting along with increasing amounts of tgev pl . stimulation of hek t cells with sting alone resulted in a robust increase in ifnpromoter activity. coexpression of sting and tgev pl induced a dose-dependent decrease in ifn-activity, clearly indicating antagonistic activity of tgev pl on stingmediated activation of the ifn-promoter (figure (a) ). sting dimerization is reduced in the presence of hcov-nl . sting dimmers are visualized as an kd band on sds-page. to further determine whether tgev pl inhibits sting-mediated signaling through disrupting the stability of sting dimers, hek t cells were cotransfected with plasmid dna expressing sting in the presence or absence of tgev pl and sev, and cell lysates were evaluated for dimmers via immunoblotting (figure (b) ). interestingly, the results indicated that sting dimerization was not affected by tgev pl . inhibiting ifn-expression. to determine whether catalytic activity is required for tgev pl -mediated inhibition of ifn-expression, hek t cells were cotransfected with alanine mutants of three conserved catalytic residues of tgev pl (c a, h a, and d a) with or without rig- , mavs, sting, or tbk- , and ifn--luc and prl-tk plasmids, followed by infection with sev to activate ifnpromoter activity. tgev pl mutation at two of the catalytic sites (c a and h a) led to almost complete loss of ifn antagonistic activity, relative to wild-type tgev pl , but the d a mutant showed a little inhibition for ifn-promoter activity (figures (a), (b) , (c), (d), and (e)). based on the results, we conclude that the intact catalytic triad of tgev pl is required to inhibit activation of the ifn-promoter driven by sting and tbk- . recent studies have revealed that sting acts as a scaffold protein for tbk- and irf and links them to the mavs complex in mitochondria upon viral infection [ ] . moreover, activation of sting is critical for stimulation of irf- activity. here, we observed that tgev pl protein inhibits sting-and tbk- -induced activation of ifn-. additional localization experiments showed that pl existed in mitochondria (figure (f) ). modification of signaling molecules by ubiquitin (ub) plays a critical role in activation of the ifn response. tgev pl has been shown to possess dub activity. here, we investigated the dub activities of tgev pl and its catalytic mutants. hek t cells were cotransfected with pcdna ha-ub and tgev pl , and the level of ubiquitinated proteins was assessed via western blot. the level of ub-conjugated proteins was reduced dramatically in cells transfected with wild-type tgev pl , while the ubiquitinated ub-ha level was not reduced in the presence of the c a, h a, and d a mutants (figure (a) ). next, we investigated whether tgev pl recognizes and deubiquitinates the key regulators, rig-i and sting, in the ifn signaling pathway. tgev is known to induce robust expression of ifn-at the late step of the replication and is distinct from covs [ , ] . moreover, tgev infection activates transcription factors nf-b, irf , and ap- in porcine kidney cells and a delayed activation of the ifn response in intestinal epithelial cells [ , ] . however, the mechanism of its evasion of the innate immune system has never been reported. the current study firstly showed antagonistic function of the tgev pl protein against the irf signaling pathway to inhibit ifninduction through its dub activity. to combat the host antiviral effects, coronaviruses likely take advantage of pl activity to escape from the host innate antiviral response. hcov-nl (pl -tm) and sars-cov (plpro-tm) inhibit sting-mediated activation of irf- nuclear translocation and induction of irf- -dependent promoters [ , ] . pl of mhv strongly inhibits cardif-, tbk -, and irf -mediated ifn-reporter activities and prevented nuclear translocation of irf [ ] . pedv plp negatively regulated rig-i and sting-mediated ifn-expression [ ] . moreover, tgev pl displays a similar structure to sars-cov pl [ ] and gives rise to the speculation that tgev pl may similarly act as an ifn antagonist. in the present study, we first found that overexpressed tgev pl inhibited sting-and tbk- -mediated ifntranscription and antagonized the type i ifn response stimulated by sev in pk- cells. the catalytic activity of tgev pl is essential for inhibiting ifn-transcription. furthermore, sting dimerization is reduced in the presence of hcov-nl pl -tm, which was not affected by tgev pl . these results suggested that tgev pl acted as an ifn antagonist to negatively regulate host antiviral innate immunity. ubiquitination and deubiquitination are critically involved in regulation of virus-induced type i ifn signaling pathways [ , ] . recently, dubs have been reported in a variety of viruses, such as foot-and-mouth disease virus, lpro [ ] , human cytomegalovirus, ul [ ] , herpes simplex virus type , ul [ ] , and porcine reproductive and respiratory syndrome virus, nsp [ , ] . interestingly, all covs have evolved to encode dub enzymes, which may contribute to modulation of the innate immune response. plp of hcov-nl , sars-cov, mhv, pedv, and mers-cov dramatically reduced the levels of ubiquitinated sting, rig-i, tbk , and irf- to negatively regulate host antiviral innate immunity. here, we showed that tgev pl interferes with and significantly inhibits ubiquitination of rig- and sting, which are essential activators of type i ifn signaling. then, the levels of phosphorylated irf- were reduced, which blocked nuclear translocation of irf to activate the transcript of ifns. three catalytically inactive mutants of tgev pl (c a, h a, and d a) found to be defective in dub activity failed to inhibit virus-induced inf-expression, indicating that the dub function of tgev pl is directly involved in inhibition of type i ifn induction. however, the membrane protein m and envelope protein e of tgev were translated at the late step of the replication as the major inducing component of ifns. further studies are required to establish the precise functions of pl protease/dub activity in coronavirus interactions with the host innate immune response. our results are the first report identifying tgev pl that is responsible for inhibiting the induction of ifn-. we found that tgev pl displayed ifn antagonist activity dependent on the intact catalytic triad (c , h , and d ) and interfered with rig- -and sting-mediated signaling through a viral dub activity. these characteristics of tgev pl served as a multifunctional protein with a critical regulatory role in tgev interactions with the host antiviral innate immune response. moreover, these findings contribute to our understanding of the molecular mechanisms of innate immunity evasion strategies utilized by tgev. antiviral signaling through pattern recognition receptors rna recognition and signal transduction by rig-i-like receptors rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity development of protection against coronavirus induced diseases: a review virus-encoded proteinases and proteolytic processing in the nidovirales deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity mers-cov papain-like protease has deis-gylating and deubiquitinating activities papainlike protease from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates molecular cloning and functional characterization of porcine ifn-promoter stimulator (ips- ) replicates and repeatswhat is the difference and is it significant? a brief discussion of statistics and experimental design the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase eris, an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization the adaptor protein mita links virus-sensing receptors to irf transcription factor activation interferon-response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes transmissible gastroenteritis virus infection induces nf-b activation through rlr-mediated signaling transmissible gastroenteritis virus does not suppress ifn-induction but is sensitive to ifn in ipec-j cells ubiquitylation in innate and adaptive immunity ubiquitination, ubiquitinlike modifiers, and deubiquitination in viral infection the leader proteinase of foot-andmouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase cleavage specificity of the ul deubiquitinating protease activity of human cytomegalovirus and the growth of an activesite mutant virus in cultured cells a deubiquitinating enzyme encoded by hsv- belongs to a family of cysteine proteases that is conserved across the family herpesviridae immunodominant epitopes in nsp of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions this work was supported by the national key technology support program ( bak b - ), the state of international science and technology cooperation projects ( dfb ), and the national natural science foundation of china ( ). interferon regulatory factor dub:deubiquitinase rig- :retinoic acid-induced gene i sting:stimulator of interferon gene prr:pattern recognition receptors pamp:pathogen-associated molecular patterns mda :melanoma differentiation gene ips- :ifn-promoter stimulator ikk: i b kinase tbk :tank binding kinase nf-b:nuclear factor b nsp :nonstructural protein . the authors declare that they have no conflicts of interest regarding the publication of this paper. key: cord- -amzzdh t authors: mochizuki, m.; nakatani, h.; yoshida, m. title: inhibitory effects of recombinant feline interferon on the replication of feline enteropathogenic viruses in vitro date: - - journal: veterinary microbiology doi: . / - ( ) - sha: doc_id: cord_uid: amzzdh t abstract antiviral activities of a recombinant feline interferon (rfeifn) kt- were evaluated against feline enteropathogenic viruses in feline and canine cell lines. sensitivity to antiviral activities of the rfeifn varied with cell types; felis catus whole fetus (fcwf- ) cells were more sensitive than crandell feline kidney cells, but no sensitivity was found for madin-darby canine kidney cells when vesicular stomatitis virus was used as a challenge virus. reductions were generally ifn dose-dependent and were more consistent when the cells were continuously treated with the rfeifn than when they were pretreated only before viral challenge. compared with each virus control culture of fcwf- cells, yields of rotavirus, feline panleukopenia virus (fplv), feline calicivirus and feline infectious peritonitis coronavirus were reduced by ranges of . to ⩽ . log , . log , . to . log and . to . log , respectively, in the cultures continuously treated with to u of the rfeifn. the yield reduction of fplv was considered to be in part attributable to inhibition of cell growth by the rfeifn supplemented in the medium. antiviral activities of human and feline interferons (ifn) against some feline viruses of veterinary importance have been demonstrated in vitro: human alpha-ifn (huifn-a) against feline leukemia virus (felv) (jameson and essex, ; sen et al., ) ; huifna or feline ifn-fl (feifn-fl) against feline herpesvirus (fhv), feline calicivirus (fcv) or feline infectious peritonitis (fip) virus (fulton and burge, ; weiss, ; weiss and oostrom-ram, ; weiss and toivio-kinnucan, ); feifns-a or -/ against felv (rodgers et al., ; yamamoto et al., ) ; felfn-y against fcv (engelman et al., ) . furthermore, some in vivo experiments which used hulfn-a alone for fip (weiss et al., ) and feline viral rhinotracheitis (fvr) (cocker et al., ) or in combination with antiviral drugs such as zidovudine for felv infected cats (cummins et al., ; zeidner et al., ) have been described (for review, rollinson, ) . results obtained in such studies have indicated that: hulfn-a may be a useful drug for therapeutic use in fvr cats; it can be applied as an auxiliary, rather than primary, treatment for fip; or the continued efficacy of hulfn-a therapy for felv infections appeared to be limited due to the induction of cytokine-specific neutralizing antibodies. recently, yanai et al. ( yanai et al. ( , first produced recombinant felfn (rfelfn) in silkworm larvae (bombyx mori) using a baculovirus vector. they cloned a cdna fragment coding for ifn from a cdna library constructed from mrna extracted from the feline lymphoblastoid cells established previously by yamamoto et al. ( ) . the amino acid sequence of the rfelfn has about % homology to hulfn-a , suggesting that it is a-type (nakamura et al., ) . thus, mass production of this rfelfn has now become possible and evaluation experiments are now being carried out. the purpose of this study was to evaluate antiviral activities of this rfelfn on feline and canine cells subsequently challenged with feline enteropathogenic viruses. crandell feline kidney (crfk) cells (atcc no. ccl ), fells cams whole fetus (fcwf- ) cells (horzinek et al., ) , and madin-darby canine kidney (mdck) cells (atcc no. ccl ) were used for determination of antiviral activities of rfeifn. cells were cultured in a growth medium (gm) consisting of eagle's minimum essential medium (emem) supplemented with % fetal bovine serum (fbs), u of penicillin g per ml, /xg of streptomycin per ml, and . /~g of amphotericin per ml. fetal macacus rhesus monkey kidney ma cells was used for rotavirus infectivity assay (mochizuki et al., ) . the following viruses were used for viral challenge: ( ) vesicular stomatitis virus (vsv) as reference; ( ) fhv c strain (mochizuki et al., ) as reference for a feline pathogenic virus; ( ) rotavirus, feline fip coronavirus (fipv), fcv, and feline panleukopenia virus (fplv) as feline enteropathogenic viruses. the new jersey strain of vsv was provided by the national institute of animal health with the permission of the ministry of agriculture, forestry and fisheries of the japanese government. vsv was grown once in either crfk or mdck cell cultures before use. canine rotavirus rs strain, a member of the k canine-feline interspecies genogroup , was serially passaged times in fcwf- cell culture for adaptation, and then used for the challenge. the kuk-h strain of fipv used here was our isolate from a spontaneous fip case, and it had properties corresponding with the criteria for type ii-fip inducing feline coronavirus (pedersen et al., ) . the fc strain of fcv which was considered to be an enteric-type fcv ( mochizuki, ) and fplv tu strain ( mochizuki et al., ) were used. the rfelfn (kt- ; lot no. f - ) produced in silkworm larvae (yanai et al., ) was supplied by toray ind., inc., - , nihonbashi muromachi -chome, chuo-ku, tokyo. it was stored lyophilized in a vial at °c, containing × units (u) which was determined by using vsv and feline fc cells (yanai, et al., ) . stock soluion ( u per ml) of rfelfn was prepared by adding ml of emem with % fbs (mm) into the vial and stored at - °c until further diluted for use. preformed monolayers of fcwf- , crfk or mdck cells in -well cell culture plates (coming, new york) were treated with the rfelfn at °c for h before challenge of vsv, fhv, rotavirus, fipv and fcv. serial tenfold dilutions of the ifn were made in mm, the cells were once washed with mm, and then . ml of the diluted rfelfn was added to each well. for fplv, . × cells in gm were seeded in the plate, cultured for h at °c, and then the medium was replaced with the gm containing the rfelfn. eight wells were treated for each ifn dilution. virus control (vc) cultures receiving no ifn were included under the same condition in each experiment. after h pretreatment, the ifn was removed from each well, the cells were once washed with emem, and challenge virus in . ml of emem was added to all wells. virus dose was pfu for vsv, fhv, rotavirus, fipv and fcv, and approx. hemagglutination (ha) units (hau) for fplv. after h of adsorption at °c, the monolayer was washed twice with emem, and . ml of mm (gm for fplv) alone and mm (gm for fplv) containing the same amount of the rfelfn as the pretreatment was added into half of wells for one ifn diltuion, respectively. for rotavirus, mm contained . /zg per ml of trypsin (type , sigma chemical comp., st. louis) instead of fbs. the plates were then incubated at °c. when the maximum cpe appeared in the wells of vc cultures, the plate was frozen at - °c until assayed for virus titers. the plate for fplv was frozen h post inoculation (p.i.). after thawing, the culture fluid was centrifuged ( rpm for min) to remove cell debris and the resultant superuatant was applied to the assays. the virus titers of vsv, fhv, rotavirus, fipv and fcv were determined by the plaque assay, using crfk (for vsv, fhv and fcv), ma (for rotavirus) or fcwf- (for fipv) cells, as reported previously (mochizuki, et al., and ) . the virus titer of fplv was determined by the ha assay described previously (mochizuki and hashimoto, ) because it is difficult to determine the infectivity of fplv precisely. titers were expressed, throughout the present paper, as pfu per . ml and hau per /zl of culture fluids, and the titer represented the geometric mean of eight vc wells or four ifn treated wells. the inhibitory effect of rfeifn on growth of fcwf- and crfk cells was examined. four ml of the ceils suspended at × per ml of cell density in gm were put into -mm plastic cell culture dishes. after h incubation at °c, the medium was changed to gm alone and gm supplemented with , , and u of the rfelfn, respectively. the dishes were incubated for further h and the number of viable cells in each dish was counted by the trypan blue exclusion method. antiviral activities of the rfelfn on cell-types were compared by using vsv. both feline fcwf- and crfk cell lines treated with the rfelfn were protected from vsv challenge (table ) : vsv did not grow at all even in fcwf- cells pretreated with only u of the rfelfn, and crfk cells appeared to be slightly less sensitive than fcwf- cells against antiviral activities of the rfelfn. no antiviral effect was generated in canine mdck cells pretreated and maintained after viral challenge (continuous treatment) with to u of the rfelfn (data not shown). the yields of fhv in fcwf- cells continuously treated with to u of rfelfn was reduced by ranges of . and . log~o, but it did not reduce further even when the cells were treated with u amount. there was no difference in yield reductions between cells pretreated only and continuously treated cells with the rfelfn. the maximum infective titer obtained in fcwf- cell culture after the serial passage for adaptation was only around pfu. however, anti-rotavirus effects of fcwf- cells treated with the rfelfn were obviously demonstrated. compared with the yield in vc cultures, the virus did not grow in the cells continuously treated with the rfelfn, but it grew a little in the cells pretreated as shown in table . ( ) fplv growth of the tu strain in both fcwf- and crfk cell cultures was slightly but ifn dose-dependently inhibited when the cells were continuously treated with the rfelfn for h (table ) . this slight reduction obtained in the cells treated with the rfelfn was reconfirmed in separate experiments. slight or no yield reductions were obtained in the cells only pretreated with the rfelfn. compared with the vc cultures, the yields were ifn dose-dependently reduced by ranges of . to . , and . to . loglo pfu in the continuously treated cultures of fcwf- and crfk cells, respectively. the reductions in yield found in the cell cultures receiving only pretreatment were less than those in the cell cultures continuously treated. since the kuk-h strain of fipv did not grow efficiently in crfk cell culture, antiviral activities of the rfelfn could be determined only in fcwf- cell culture. when vc cultures showed maximum cpe h p.i., which was scored + as all the cells detached from the surface, the cultures continuously treated with , , and u of the rfeifn decreasingly showed ~ +, ~ +, + and ~ + of cpe, respectively. the yield in vc cultures was . log~o pfu, but those in the cells treated with , and u were unexpectedly . , . and . log~o pfu: thus, the reductions were . , . , and . loglo, respectively, as shown in table . this inconsistency between the degree of cpe and the virus yield was observed repeatedly in separate experiments. in the cells pretreated alone, no inhibitory effect against replication of fipv was detected. four ml of the cells suspended at x per ml of cell density in eagle's mem supplemented with % fbs (gm) were put into -mm plastic cell culture dish, and after h incubation, the medium was changed to gm alone and gm + rfelfn, respectively. after further h incubation, viable cell numbers were counted and obtained as the mean of two dishes per each amount of rfelfn, and reduction was obtained by (a-b)/a x (a:cell numbers in gm alone, b:cell numbers in gm + rfelfn). data are expressed as the mean of the results from two separate experiments. compared with the control cultures, the growth of both fcwf- and crfk cells in gm supplemented with the rfelfn were dose-dependently inhibited (table ) . however, the inhibitory effect was different between the cell types. the growth inhibition of fcwf- cells was obviously detected when the medium contained u or more amount of the rfelfn, but it required to times amount of the rfelfn for crfk cells to cause a similar degree of growth inhibition. host-species specificity of ifns is generally accepted. however, most hulfns-a show specific activities also on heterologous cells. because they can be produced in large quantities at a relatively low cost by recombinant dna techniques, such rhuifns are a potential broad spectrum antiviral agent for veterinary use as indicated by both in vitro and in vivo experiments. the rhuifn-a may be a useful drug for therapeutic use in cats with some viral diseases such as felv, fip and fvr (cocker et al., ; cummins et al., ; weiss et al., , zeidner et al., , but a disadvantage is its heterogeneity which elicits neutralizing antibodies in animals (zeidner et al., ) . the rfeifn kt- , examined in the present study, is the first mass-produced ifn as a drug for cats (yanai et al, ) , and it was demonstrated that antiviral activities of the cells treated with the rfeifn are in no way inferior to those against fhv and fcv by feifn and rhuifn-a reported previously (fulton and burge, ; weiss, ) . more antiviral effects were detected in the cells continuously exposed to the rfelfn than in the cells transiently treated before viral challenge: an appropriate example of interest was the experiment for fplv. the growth inhibition of fplv was shown in the cells continuously treated with u of the rfeifn, but little if any, in the cells pretreated with u to u of the rfelfn (table ) . however, wiedbrauk et al. ( ) claimed that slight reductions in yield of mink parvoviruses in cells treated with high concentrations ( to u per ml) of the ifn were not considered significant, for the reasons that such high ifn concentration inhibit cell growth and delay the entry of cells into the s phase. this may result in reduction of virus replication in such cells because these autonomously replicating parvoviruses require one or more cellular functions generated during the s phase of the cellular division cycle. although some experimental conditions of the present study differed from the previous one particularly in the use of actively dividing cells, it is reasonable to consider that the yield reduction detected in the present study is also partly attributable to the inhibition of cell growth by the rfelfn as presented in table . the cell line of fcwf- cells may possess more ifn-receptors than crfk cells, and thus be more sensitive against antiviral activities and cell-growth inhibitory effects of the rfelfn, as demonstrated here. this is consistent with the observation in which enhanced sensitivity to the anti-fipv effects of rhulfn-a or felfn-fl in fcwf- cells was noted when compared with other feline diploid cell types such as fc lu or crfk cells (weiss and toivio-kinnucan, ) . however, the replication of fipv in the fcwf- cells treated with the rfelfn was not inhibited so much reported previously (weiss and toivio-kinnucan, ). this may be influenced by some factors, such as the type of ifn used or strain differences of fipv in ifn sensitivity as pointed out by the same authors. another finding of interest is the inconsistency between the fipv yield and the degree of cpe observed in fcwf- cell culture treated with the rfelfn. unexpectedly, high infectivities were obtained in the cultures showing low degrees of cpe. this peculiar finding is in agreement with the previous description: fip coronaviral particles may persist intracellularly without showing cpe in ifn-treated cells (weiss and toivio-kinnucan, ). in the present study, the whole culture including infected cells and media was subjected to the viral assay. therefore, one of the probable explanations is that the test samples contain not only extracellular but also intracellular viruses from the disrupted cells which did not show cpe. in conclusion, we evaluated the antiviral activities of newly produced rfelfn kt- against feline enteropathogenic viruses in vitro. the results indicate that certain degrees of antiviral effects against rotavirus, fipv, fcv and fplv can be generated in the feline cells by treatment with the rfelfn. thus, it will be worthwhile to know its potential as an antiviral drug given orally for prophylaxis of enteritis, because this administration route of ifn has already been described in cats (cummins et al., ) . oral use of human alpha interferon in cats suppression of gamma interferon production by inactivated feline leukemia virus susceptibility of feline herpesvirus and a feline calicivirus to feline interferon and recombinant human leukocyte interferons antigenic relationships among homologous structural polypeptides of porcine, feline and canine coronaviruses inhibition of feline leukemia virus replication by human leukocyte interferon different stabilities to bile among feline calicivirus strains of respiratory and enteric origin growth of feline panleukopenia virus and canine parvovirus in vitro comparison of feline parvovirus subspecific strains using monoclonal antibodies against a feline panleukopenia virus studies on cytopathogenic viruses from cats with respiratory infections iii. isolation and certain properties of feline herpesviruses hemagglutinin activity of two distinct genogroups of feline and canine rotavirus strains characterization of canine rotavirus rs strain and comparison with other rotaviruses micro-neutralization test with canine coronavirus for detection of coronavirus antibodies in dogs and cats molecular cloning of feline interferon cdna by direct expression pathogenic differences between various feline coronavirus isolates cat interferon inhibits feline leukaemia virus infection in cell culture prospects for antiviral chemotherapy in veterinary medicine: . feline virus diseases antiviral and protein-inducing activities of recombinant human leukocyte interferons and their hybrids synergistic antiviral activities of acyclovir and recombinant human leukocyte (alpha) interferon on feline herpesvirus replication effect of interferon or propionibacterium acnes on the course of experimentally induced feline infectious peritonitis in specific-pathogen-free and random-source cats inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro inhibition of feline infectious peritonitis virus replication by recombinant human leukocyte (a) interferon and feline fibroblastic (/ ) interferon mink parvoviruses and interferons: in vitro studies a feline retrovirus induced t-lymphoblastoid cell-line that produces an atypical alpha type of interferon synthetic plasmid, transformant, feline interferon gene and method for producing feline interferon. european patent application feline interferon and process for production thereof by a silkworm virus vector. european patent application alpha interferon ( b) in combination with zidovudine for the treatment of presymptomatic feline leukemia virusinduced immunodeficiency syndrom we thank toray industries, inc., for the generous contribution of rfeifn kt- used in this study. cocker, f.m., howard, p.e. and harbour, d.a., . effect of human o~-hybrid interferon on the course of feline viral rhinotracheitis. vet. rec., : - . key: cord- -k jssr authors: volk, aaron; hackbart, matthew; deng, xufang; cruz-pulido, yazmin; o’brien, amornrat; baker, susan c. title: coronavirus endoribonuclease and deubiquitinating interferon antagonists differentially modulate the host response during replication in macrophages date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: k jssr coronaviruses (covs) encode multiple interferon (ifn) antagonists that modulate the host response to virus replication. here, we evaluated the host transcriptional response to infection with murine coronaviruses encoding independent mutations in one of two different viral antagonists, the deubiquitinase (dub) within nonstructural protein or the endoribonuclease (endou) within nonstructural protein . we used transcriptomics approaches to compare the scope and kinetics of the host response to the wild-type (wt), dubmut, and endoumut viruses in infected macrophages. we found that the endoumut virus activates a focused response that predominantly involves type i interferons and interferon-related genes, whereas the wt and dubmut viruses more broadly stimulate upregulation of over , genes, including networks associated with activating the unfolded protein response (upr) and the proinflammatory response associated with viral pathogenesis. this study highlights the role of viral interferon antagonists in shaping the kinetics and magnitude of the host response during virus infection and demonstrates that inactivating a dominant viral antagonist, the coronavirus endoribonuclease, dramatically alters the host response in macrophages. importance macrophages are an important cell type during coronavirus infections because they “notice” the infection and respond by inducing type i interferons, which limits virus replication. in turn, coronaviruses encode proteins that mitigate the cell’s ability to signal an interferon response. here, we evaluated the host macrophage response to two independent mutant coronaviruses, one with reduced deubiquitinating activity (dubmut) and the other containing an inactivated endoribonuclease (endoumut). we observed a rapid, robust, and focused response to the endoumut virus, which was characterized by enhanced expression of interferon and interferon-related genes. in contrast, wild-type virus and the dubmut virus elicited a more limited interferon response and ultimately activated over , genes, including players in the unfolded protein response and proinflammatory pathways associated with progression of significant disease. this study reveals that endou activity substantially contributes to the ability of coronaviruses to evade the host innate response and to replicate in macrophages. responses or of later, adaptive responses that aim to limit virus replication. by understanding how these viral antagonists regulate the immune response, we can fine-tune the rational design of therapeutics and vaccines to control existing and emerging viral pathogens. coronaviruses (covs) are characterized in part by their large (ϳ kb), positive-sense single-stranded rna genomes that yield a nested set of subgenomic mrnas during viral replication ( , ) . these large genomes encode the replicase polyprotein, the canonical structural proteins (spike, envelope, membrane, and nucleocapsid), and, depending on the cov, a number of accessory genes. notably, many of these different genes (replicase, structural, and accessory) encode proteins that regulate the antiviral response, indicating that viral antagonists of host defenses play an important role during infection ( ) ( ) ( ) ( ) ( ) . in this report, we investigate the role(s) of two highly conserved enzymatic domains within the replicase polyprotein of mouse hepatitis virus (mhv)the viral protease/deubiquitinase (dub) and the endoribonuclease (endou)-in altering host immune responses. the replicase polyprotein machinery must be processed into its functional parts during viral replication, which, in the case of mhv, requires the activity of three proteases, two papain-like proteases (plp and plp ) and one chymotrypsin-like protease ( clpro, sometimes referred to as mpro). mhv plp is structurally similar to the single papain-like protease (termed plpro) encoded by severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) . previous studies demonstrated that cov plp s/plpros are multifunctional, with catalytic residues that mediate protease, deubiquitinase (dub), and deisgylating (deconjugating interferon-stimulated gene [isg ] molecule from modified substrates) activities ( ) ( ) ( ) ( ) ( ) . in our companion manuscript ( ) , we describe the x-ray structure-guided identification of a residue in mhv plp that is required for dub activity, but not protease activity. using reverse genetics, we generated an mhv that expressed the dub mutant enzyme, and we found that dubmut virus is mildly attenuated in mice. however, the role of dub activity during replication in macrophages, a cell type critical for virus replication and pathogenesis, was not known. we wanted to determine the host response to a dubmut virus and compare it to that against a virus expressing a mutant form of another recently identified antagonist, the coronavirus endoribonuclease (endou). endou, a highly conserved enzyme within the coronavirus family, was initially thought to play a role in coronavirus rna synthesis ( , ) . recent studies revealed that endou acts as an interferon (ifn) antagonist by preventing activation of host sensors by viral double-stranded rna (dsrna) ( , ) . viruses encoding a catalytically inactive endou undergo initial rna replication; however, viral dsrna intermediates accumulate during viral replication and are detected by the host sensors mda , pkr, and oas. activation of these dsrna sensors initiates the innate immune response, including activation of type i interferons, interferon-responsive genes, and apoptosispromoting caspases, all of which collectively limit virus replication. we previously reported that mhv-endou mutant viruses are profoundly attenuated in mice and elicit a protective immune response ( ) . in the current study, we use transcriptome profiling to evaluate the kinetics of host gene expression in macrophages upon infection with mhv wild-type (wt), dubmut, and endoumut viruses. our analyses of the respective transcriptional response to each virus reveal significant differences in the kinetics, magnitude, and breadth of host gene expression during infection and provide new information on the extent to which viral interferon antagonists manipulate the overall host response to viral infection. transcriptome profiling reveals differences in kinetics and magnitude of host responses to mutant viruses. we sought to determine if viruses that contain inactivating mutations in distinct interferon antagonists (dub versus endou) would exhibit unique host transcriptional signatures in response to virus infection. briefly, bone marrow-derived macrophages (bmdms) were infected with the designated virus at a multiplicity of infection (moi) of , and total rna was harvested for transcriptome profiling at , , , and h postinfection (h p.i.). rna was isolated from all cells in the well, both infected and uninfected bmdms. using the rna-sequencing (rna-seq) data, we identified differentially expressed genes by applying a cutoff of at least -fold elevated expression in wild-type-infected cells at h p.i. over mock (q Ͻ . ). we then utilized cluster . software to visualize overall patterns of gene expression between all groups across all time points (fig. ) . these analyses reveal striking differences in overall patterns of gene expression between the endoumut-infected cells and wild-type virusinfected cells, whereas the gene expression profile induced by the dubmut virus is remarkably similar to that in wild-type virus infection (fig. ). more specifically, , genes are significantly transcriptionally activated (Ͼ -fold; q Ͻ . ) in wt-and dubmut-infected macrophages by h p.i. compared to those in mock-infected macrophages. interestingly, our analyses also identified a subset of genes, indicated by the bracket in fig. , that were most highly upregulated in endoumut-infected cells. it is important to note that these genes were also induced during wt and dubmut infections, but not to the same magnitude as that detected in the endoumut-infected cells. we note a transient downregulation of a subset of genes related to transcriptional responses in the endoumut-infected cells at h p.i., which may be related to the apoptotic response of these cells ( ) . we next sought to functionally cluster the genes most highly upregulated by each mutant virus as a means of investigating the respective host transcriptional response to the different viruses. endoumut infection activates genes associated with an antiviral response. we used an online tool called database for annotation, visualization and integrated discovery (david) to cluster the genes highly upregulated by endoumut virus infection (bracket in fig. ) based on functional similarities ( , ) . we found that the protein products of these genes are predominantly involved in antiviral response and signaling pathways. notably, the david analyses reveal a subset of unique genes, including several interferon isoforms, which are clustered together into statistically significant pathways associated with the immune response and signaling cascades. interestingly, the heat map of these genes reveals a similar temporal trend in upregulation of ifn gene expression during infection with each of the three viruses; however, this expression profile is by far the most pronounced in the endoumut-infected cells ( fig. a) . in this heat map view, as in fig. , it is difficult to ascertain any difference in expression of ifn genes between wild-type-and dubmut-infected cells. therefore, to obtain more information on the kinetics of transcriptional activation between groups, we quantitated the reads associated with each gene and graphically displayed the normalized read counts (fig. b ). using this method of plotting the read counts over time, we detected statistically significant transcriptional upregulation of ifn gene expression in the dubmut-infected cells. however, the magnitude of dubmut-induced ifn gene expression was dwarfed by ifn activation in endoumut-infected cells. endoumutinfected macrophages exhibited markedly elevated levels of ifn transcripts as early as h p.i. by h p.i., expression of the ifn gene transcripts upon endoumut infection was significantly higher than that during wild-type or dubmut infection, with our assay detecting , to , more reads per gene in the endoumut-infected cells. this focused response to endoumut is successful in limiting virus replication in cultured macrophages, as demonstrated in our previous report ( ) . notably, we evaluated the transcriptional response during the first h p.i., prior to the onset of apoptosis, which we and others have shown to occur in bmdms upon endoumut infection ( , ) . we note that the differential expression of ifn genes in the endoumut-infected cells influence the row z-score in fig. a to directly compare ifn gene expression between wild-type and dubmut infections, we generated a heat map of the comparative response of type i ifns, with the row z-score calculations reflecting the relative expression between these two infections (fig. c) . overall, we observe a significant increase in expression of all of these ifns, particularly at h p.i., during dubmutinfection relative to that during wt-virus infection. interestingly, despite of this elevated ifn expression during dubmut infection, we did not observe a similar increase in isg mrna, which is upregulated by endoumut virus infection (fig. b) . dubmut-infected cells exhibit downregulation of proinflammatory cytokine and chemokine genes compared to wild-type virus-infected cells. although we observed an enhanced ifn response during dubmut infection of bmdms, this phenotype did not lead to dramatic differences in pathogenesis during infection of mice ( ) . to further investigate the differences between the host responses to dubmut versus wild-type virus infection, we reduced the cutoff stringency to at least -fold differential expression with a significant q value (q Ͻ . ) and a normalized read count of at least . we identified genes upregulated in dubmut-infected cells at and h p.i. compared to wild type-infected cells (fig. a) ifna ifnb ifna ifna ifna ifna ifna ifna ifna ifna ifnab ifna ifna ifna ifna row z-score color key infected cells compared to wt-infected cells (fig. b ). our analysis identified dysregulation relative to the wild type of multiple genes that encode proinflammatory cytokines and chemokines, including il , il ␤, and il ␣ (fig. c) ccl , cx cl , and cxcl , compared to wt-infected cells. we note that the host response to wild-type and dubmut virus infections trended in the same direction and that these cytokine and chemokine responses were much more robust compared to the response to the endoumut virus infection. the results of proinflammatory cytokines gene expression shown in fig. prompted us to evaluate secretion of proinflammatory cytokines in response to the wild-type, dubmut, and endoumut virus infection. to determine the concentrations of pro-and anti-inflammatory cytokines released during infection, we performed cytometric bead array (cba) analysis on supernatants collected from virus-infected bmdms at h p.i. we performed the cba at h p.i. to allow for the cytokine mrna to be translated into protein and the protein to be secreted from the cells into the supernatant. we report that dubmut-and endoumut-infected cells have reduced levels of the inflammatory cytokines tumor necrosis factor alpha (tnf-␣), interleukin (il- ), and macrophage inflammatory protein ␣ (mip- ␣) relative to those in wild-ϩ type-infected cells (fig. a) . we also found that endoumut-infected cells exhibit lower levels of kc, il- , and mip- ␤ proteins relative to those in wt-and dubmut-infected cells (fig. b) . endoumut viruses induce apoptosis in bmdms ( ) , which likely limits their ability to produce proinflammatory cytokines. the results of these analyses indicate that wild type-infected bmdms have a heightened proinflammatory profile compared to those of dubmut-and endoumut-infected cells. this proinflammatory cytokine response has been shown to contribute to increased immunopathogenesis during in vivo infections ( ) . wild-type and dubmut coronavirus replication activate the unfolded protein response and host response to cell damage. in contrast to the robust and specific antiviral gene expression response detected in endoumut-infected macrophages, we uncovered a more complex response involving , genes in both wild-type-and and ) . in addition to a similar proinflammatory signature ( fig. and ) , we found that wild type-and dubmut-infected cells also exhibit hallmarks of activation of the host unfolded protein response (upr). upr pathways are activated when unfolded proteins accumulate in the endoplasmic reticulum (er), at which point the host cell initiates measures to resolve this overload ( ) . in the context of virus infection, viral glycoproteins accumulate in the er, triggering the release of bip from three sensor proteins: ire , atf , and perk. activation of these sensors triggers signaling cascades resulting in transcriptional activation of genes encoding er chaperones and proteins involved in lipid synthesis and amino acid transport (fig. a ). our previous study documented the activation of these pathways after mhv infection of fibroblasts ( ) . this study provides new information on the early transcriptional response to mhv in macrophages. based on our analyses of differential data were subjected to statistical analysis using two-tailed student's t tests. *, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ; ****, p Ͻ . ; ns, not significant. data are presented as means Ϯ sd. gene expression, we report significant transcriptional upregulation of multiple genes associated with activation of er sensors ire , atf , and perk, such as edem , hspa (encoding bip protein), and ddit (encoding chop), in response to virus replication, with the most robust response detected in cells infected with the wild-type or dubmut virus infection (fig. b, c, and d) . to further evaluate the activation of ire- during virus infection, we analyzed the splicing of xbp- mrna by reverse transcription-pcr (rt-pcr) and visualized the products by electrophoresis on agarose gels. we found that the wt and dubmut viruses exhibit higher proportions of spliced xbp- mrna at h p.i. and h p.i. compared to that in endoumut-infected cells (fig. e) , consistent with activation of the ire- arm of the unfolded protein response. overall, these differential gene expression analyses in macrophages reveal similar host responses to the wild-type and dubmut viruses that include activation of upr pathways and proinflammatory genes, whereas a distinct transcriptional profile during infection with the endoumut virus is predominately defined by a focused, robust antiviral response. we report that inactivation of a coronaviral interferon antagonist, endou, profoundly alters the host response to viral replication in macrophages compared to the response to wild-type coronavirus infection. we find that the endoumut virus elicits a rapid, robust, and specific antiviral response that is effective in limiting virus replication. in contrast, our data show that the wild-type and dubmut viruses ultimately elicit very similar host responses that are both characteristic of an unfolded protein response and consistent with a proinflammatory profile that is associated with viral pathogenesis ( ) . our studies of the dubmut-infected macrophages indicate that mere induction of type i ifn is not a sufficient marker for attenuation of the virus. instead, these results suggest that the timing and the magnitude of the host antiviral response are critical for determining the outcome of infection in macrophages (fig. ) and for pathogenesis in infected animal ( ) . our observation that the endoumut virus induces an earlier and more profoundly elevated type i interferon response than even the dubmut virus implies that there is a threshold of ifn expression that must be breached before the cell mounts an effective antiviral response. a remarkable finding from this study is the distinct transcriptional profile elicited by the endoumut virus during infection of bone marrow-derived macrophages compared to the profiles of wild-type-and dubmut-infected cells (fig. ) . we detected elevated levels of interferon and interferon-responsive genes as early as to h p.i., with endoumut-infected cells exhibiting by far the highest expression levels of these genes. we and others found that an antiviral response to endoumut infection results in activation of apoptosis, which subsequently limits virus replication in cell culture and in infected mice ( , ) . the current study indicates that screening mhv mutants in interferon-responsive cells may be an effective approach to identifying mutations that stimulate a robust innate immune response, which may then restrict virus replication in animals. previous studies of coronavirus-encoded interferon antagonists focused on evaluating the host transcriptional response to infection at later time points (such as and h p.i.) and in a variety of cell types or the tissues of infected animals. for example, a study comparing infection of mice with wild-type sars-cov versus a virus containing a mutation in the interferon antagonist nsp , termed the dnsp cov, revealed that the transcriptional profile in the lungs of the dnsp -infected mice mirrored the response to wt virus ( ) . however, combining the dnsp mutation with a mutation in another interferon antagonist, exon, was shown to reduce disease in mice and elicit protective immunity. it would be interesting to determine if the transcriptional profile elicited by the double mutant sars-cov is altered compared to that of the wild-type virus, particularly at early times after infection. a study of mers-cov comparing the host response to wt virus with the response to a mutant virus containing deleted accessory open reading frames (orfs) (mers-cov dorf - ) provides evidence of significant differences in the early transcriptional responses to infection in calu b cells ( ) . this study revealed that mers-cov dorf - infection prompted earlier ( , , and h p.i.) and more robust type i and type iii interferon responses, and that the mutant virus was more sensitive than the wild type to pretreatment with interferon. the mers-cov interferon antagonist mutant virus was attenuated in mice and elicited a protective response against subsequent lethal challenge. one difficulty that arises when evaluating the role(s) of virally mediated modulation of the host response is that multiple viral proteins, including both structural and nonstructural proteins, have been shown to antagonize the innate immune response in vitro and/or in vivo. these include: nsp - =o mtase, nsp -exon, nsp , nsp , e protein, n protein, m protein, sars-cov-orf , mers-cov-orf - , mers-cov- a, and mers-cov- b ( , ( ) ( ) ( ) ( ) ( ) . each of these interferon antagonists may play either a cell-or tissue-type-specific role, or may act in concert with other factors during viral replication to mitigate the innate immune response. further studies are needed to fully understand if all or only a subset of these antagonists must be silenced to generate an effective live-attenuated vaccine. the results presented here, and from studies of the mers-cov dorf - mutant virus ( ) upon infection of a bmdm with endoumut, host double-stranded rna (dsrna) sensors (including mda , pkr, and oas) are activated, resulting in robust transcription of type i ifn genes and rapid induction of apoptosis, the latter of which precludes the development of a potent inflammatory response. viral replication is severely restricted in these apoptotic macrophages as early as h p.i. although dubmut induces significantly higher type i ifn during infection than wt-mhv, infection with either dubmut or the wt yields same outcome, accumulation of er stress and subsequent activation of the upr, which then likely leads to the establishment of a robust nf-b-mediated proinflammatory response. bmdms infected with either of these viruses acquire a potently activated inflammatory phenotype, are unable to attenuate viral replication, and likely contribute to severe immunopathology in vivo. and inactivating nsp in pedv ( ) , suggest that inactivating interferon antagonists and screening for an early and robust antiviral transcriptional profile may represent an efficient and informative approach to evaluating live-attenuated vaccine candidate strains for existing and emerging coronaviruses. the dysregulation of innate immune signaling in myeloid cells, such as macrophages, is a key component of coronavirus-associated immunopathogenesis ( ) ( ) ( ) . using viral infection of macrophages, we can identify and evaluate the viral factors involved in antagonizing innate immune pathways. we and others have found that endou is important for the inhibition of innate immune signaling in macrophages ( , ) . loss of endou activity significantly alters gene expression and cytokine production in macrophages, which results in the attenuation of endoumut viruses in vivo ( ) . in contrast, we report mild attenuation of the dubmut virus in mice ( ) and show here that dubmut and mhv-a generate similar patterns of transcriptional activation in macrophages. while macrophages have been documented to be important for controlling cov infections, it is also important to appreciate that covs infect multiple cell types, including as epithelial cells in the lung and intestines. viral dub activity may play a more significant role during infection of epithelial cells, and further studies are needed to provide further insights into the role of dub activity during coronavirus infection. our results demonstrating upregulation of the unfolded protein response (upr) in response to wild-type and dubmut coronavirus replication confirm and extend the work of earlier studies that documented activation of the er sensors perk, ire , and atf- during coronavirus infection ( , , ) . heavy utilization of the endoplasmic reticulum for generating coronavirus replication complexes and of the er-golgi intermediate compartment for assembling virus particles places a substantial load on the host translational machinery during infection. host sensors ire , atf- , and perk are situated in the er to sense and respond to such overload by prompting upregulated expression of genes encoding er chaperones, amino acid transporters, and activators of lipid biosynthesis. ultimately, many of these proteins may facilitate virus replication and assembly. notably, it has been demonstrated that upr pathways that promote apoptosis are blocked during coronavirus replication ( , ) . the ability of viruses to modulate the upr has important implications for the innate immune response to such viruses because the upr has been shown to attenuate antiviral defenses by way of degrading the type i interferon receptor ( ) . to our knowledge, the data presented here provide the first transcriptomic evidence of upr activation in coronavirus-infected macrophages, underlining an important role for upr pathways in the coronavirus life cycle. our observation that endoumut-infected macrophages exhibit significantly lower expression of several genes involved in upr pathways compared with wild type-and dubmut-infected cells is consistent with the reduced levels of virus replication detected in endoumut-infected macrophages. the notion of inactivating viral interferon antagonists as a means of generating live-attenuated vaccines is supported by recent reports of screening for inactivation of influenza a virus-encoded interferon antagonists ( ), as well as studies that revealed that the classic vaccine strain of yellow fever virus encodes an interferon antagonist in the ns protein ( ) . for coronaviruses, it is not yet clear if disabling a single interferon antagonist, such as the highly conserved endou, will be sufficient to attenuate viruses that infect different cell types in different species. promisingly, our studies of a coronavirus that causes lethal disease in piglets, porcine epidemic diarrhea virus (pedv), revealed that inactivation of endou activity is associated with attenuated disease ( ) . we believe the information gained from studying coronaviruses containing inactivated interferon antagonists can be directly applied to the recently emerged coronavirus designated severe acute respiratory syndrome coronavirus (sars-cov- ) ( ) ( ) ( ) . more research is needed to determine if inactivating multiple interferon antagonists, including endou, is an effective approach for generating safe and protective live-attenuated coronavirus vaccines. ethics statement. the harvesting of bone marrow for obtaining bone marrow-derived macrophages in this study was carried out in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the experimental protocol was reviewed and approved by the institutional animal care and use committee (iacuc) at loyola university chicago (iacuc no. - ). c bl/ female mice were purchased from the jackson laboratory and maintained in the comparative medicine facility of loyola university chicago. bmdms and viruses. bone marrow-derived macrophages (bmdms) were prepared and cultured as described previously ( ) . differentiated bmdms were maintained in bone marrow macrophage (bmm) media containing dulbecco's modified eagle's medium (dmem) (product no. - -cv; corning) supplemented with % l cell supernatant, % fetal bovine serum (fbs), % l-glutamine, % sodium pyruvate, and % penicillin-streptomycin. wild-type mhv strain a (genbank accession number ay ) and endoumut (h a) were previously generated by reverse genetics and confirmed by deep sequencing. dubmut (d a) was generated as described in the companion paper ( ) . rna-seq data analysis pipeline. rna-seq data have been deposited in the ncbi geo database (accession number gse ). raw rna-seq reads were subjected to analysis using galaxy's online platform to generate differential gene expression data between infection groups ( ) . reads were clipped to remove any residual unique barcode sequences that were added during preparation of each sample for sequencing. clipped reads were concatenated to combine multiple files per sample into a single file per sample. these files were groomed to ensure that all reads were in sanger fastq format. fastq reads were aligned to the grcm ensembl build of the c bl/ j mouse genome using the hisat aligner, which locates the region of the genome to which each read corresponds, resulting in an output bam file ( ) . all reads that did not align to the mouse genome (i.e., reads that originated from viral rna) were discarded. the bam files contained the alignment information for each read in that sample and were used as inputs into featurecounts, which quantifies the number of reads in each sample that corresponds to each gene in the mouse genome ( ) . finally, the output count data from featurecounts was used as the input for deseq to calculate differential expression for each gene across all samples and treatment groups. deseq was used to generate normalized count values for each gene in all samples ( ) . these normalized counts are intended to correct for size differences between samples that might otherwise skew differential expression calculations if some samples contained substantially different numbers of total reads. the normalized count values were plotted and visualized in heat maps, generated using r, and line graphs, generated using prism software. identifying differentially expressed genes. to identify and analyze differentially expressed genes (degs) between infection groups, we used as a starting point the list of degs between mock-and wild-type mhv-infected bmdms at h p.i. that was generated as an output by deseq . this list of genes was filtered based on the statistical significance associated with the fold change differential expression value. a q value (adjusted p value, calculated by deseq for each gene in each comparison using the benjamini-hochberg procedure) of Ͻ . was chosen as the cutoff for statistical significance; genes whose differential upregulation values did not meet this cutoff in wild-type mhv-infected bmdms at h p.i. compared with mock-infected cells were removed from the list ( ). next, a differential expression magnitude cutoff of Ͼ was applied to the remaining genes. genes that were not more highly expressed by at least -fold in wild-type mhv-infected cells at h p.i. compared to mock were removed from the list. after applying these cutoffs, , genes remained and were arranged in order of most to least highly upregulated in wild-type mhv-infected cells at h p.i. compared to mock. cluster . software was then used to apply mathematical clustering to the z-score-standardized log -normalized mean normalized count values associated with each gene at each time point and in each infection group ( ) . specifically, the default settings-the similarity metric "pearson correlation (uncentered)" and the clustering method "centroid linkage"-were applied to the list of , genes and the corresponding expression values for each gene across all samples to produce a hierarchically clustered gene list based on how similar or different the expression patterns were between groups of genes across all samples. this new list of clustered genes and their associated expression values were then visualized as a heat map using java treeview software. functional clustering analyses using david. the bracketed set of genes identified in fig. were subjected to david analyses. specifically, under the "gene ontology" category, the data from the chart associated with the "goterm_bp_direct" result were reported here. after excluding all functional cluster terms that were not statistically significant using a cutoff of q Ͻ . , we identified a total of unique genes that appeared in at least one cluster of statistical significance. expression values of these genes were plotted in heat map and line graph forms as described above. xbp- splice analysis. c bl/ bmdms were infected with wild-type-, dubmut-, or endoumut-mhv at an moi of . at designated time points, intracellular rna was isolated from cells with rneasy kit (catalog no. ; qiagen). isolated rna was reverse transcribed into cdna with an rt first strand kit (catalog no. ; qiagen). xbp- splicing was analyzed by pcr following the protocol from bechill et al. ( ) . briefly, pcr was performed with the forward primer =-gttgagaaccaggagttaag- = and the reverse primer, =-agagaaagggaggctggtaag- =. pcr conditions were (i) °c for min, (ii) °c for min, (iii) °c for min, and (iv) °c for min, repeating steps ii to iv for cycles. pcr products were separated on % polyacrylamide-tbe ( mm tris-borate and . mm edta [ph . ]) gel, stained with sybr green ii stain, and imaged. quantification of band density was performed with image lab software (bio-rad). cytometric bead array. c bl/ bmdms were infected with wild-type-, dubmut-, or endoumut-mhv at an moi of . at h p.i., supernatants were collected and centrifuged at , ϫ g for min at cba staining was performed according to the manufacturer's instructions (cba flex set mouse soluble proteins, catalog no. ; bd biosciences) molecular mechanisms of innate immune inhibition by non-segmented negativesense rna viruses vaccinia virus protein c inhibits type i ifn signalling in the nucleus and binds to the 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spliced aligner with low memory requirements featurecounts: an efficient general purpose program for assigning sequence reads to genomic features moderated estimation of fold change and dispersion for rna-seq data with deseq open source clustering software we thank qunfeng dong for assistance with the bioinformatic analyses. we also thank robert mettelman for assistance with quantitative pcr (qpcr) setups and help editing the manuscript. we thank francis alonzo iii and zack resko for assistance with the cytometric bead array analyses. key: cord- -v dr hm authors: albert, manuel; bécares, martina; falqui, michela; fernández-lozano, carlos; guerra, susana title: isg , a small molecule with huge implications: regulation of mitochondrial homeostasis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: v dr hm viruses are responsible for the majority of infectious diseases, from the common cold to hiv/aids or hemorrhagic fevers, the latter with devastating effects on the human population. accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. interferon-stimulated gene (isg ) plays an important antiviral role during viral infection. isg catalyzes a ubiquitin-like post-translational modification termed isgylation, involving the conjugation of isg molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. numerous biomedically relevant viruses are targets of isg , as well as proteins involved in antiviral immunity. beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. in this review, we give an overview of the biological consequences of isgylation for virus infection and host defense. we also compare several published proteomic studies to identify and classify potential mitochondrial isgylation targets. finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of isg in the regulation of mitochondrial processes, specifically oxphos and mitophagy. the innate immune response is the first line of defense against microbial and viral infections. invading microorganisms produce danger-and pathogen-associated molecular patterns that interact with host pattern-recognition receptors, triggering several intracellular signaling cascades that activate nuclear factor kappa-b (nf-κb), mitogen-activated protein kinases (mapks) and interferon (ifn) regulatory factors (irfs), resulting in the expression of a broad array of proteins involved in host defense such as type-i ifns and proinflammatory cytokines [ , ] . the release of type-i ifns has both autocrine and paracrine effects via ifnα/β receptors (ifnars) on the cell surface. binding to ifnars leads to the activation of the janus kinase-signal transducer and activator of transcription proteins (jak-stat) signaling pathway and the formation of the interferon-stimulated gene factor (isgf ) complex, with the subsequent expression of ifn-stimulated genes [ ] that establish an antiviral state and play important roles in determining the host innate and adaptive immune responses [ ] . one of the most highly induced genes in the type-i ifn signaling cascade is isg (interferon-stimulated gene ), which encodes a small ubiquitin-like protein involved in a post-translational modification ( ) . intracellular isg can be processed into its mature form and conjugated to de novo synthesized proteins in a process termed isgylation. isg processing exposes its carboxy-terminal lrlrgg motif, allowing its conjugation to lysine residues in target proteins to modulate their function. in addition, isgylation is reversible due to the action of the protease usp , which also regulates ifnar-mediated signaling ( ) . isg can remain unconjugated within the cell, regulating protein activity ( ), or be secreted as a cytokine, acting as a chemotactic and stimulating factor for immune cells ( ) . binding of isg to lfa- integrin receptor on the surface of nk cells promotes the activation, production and release of ifn-γ il- after il- priming. moreover, extracellular isg is able to form dimers/multimers through cysteine residues, to modulate cytokine levels. isg conjugation to target proteins is a covalent and reversible process through the action of a -kda deisgylase enzyme, ubiquitin-specific protease (usp ) [ , ] . interestingly, both isg and its conjugating and deconjugating enzymes are upregulated by type-i ifn [ ] , as well as by other stimuli such as type-ii and type-iii ifns [ ] [ ] [ ] , lipopolysaccharide [ ] , retinoic acid [ ] , dna damage or genotoxic reagents [ ] . usp not only acts as a deconjugating enzyme, but also as a negative regulator of the type-i ifn pathway (figure ) , with important implications in antiviral and antibacterial responses, immune cell development, autoimmune diseases and cancer [ ] . in humans, isg binds to usp , increasing its stability and leading to a decrease in ifn-α/β signaling. consequently, isg deficiency results in low usp levels, and therefore a sustained elevation in figure . intracellular and extracellular activities of isg . different stimuli trigger the expression of isg , which is produced as a precursor of kda with two ubiquitin-like domains linked by a hinge region ( ) . intracellular isg can be processed into its mature form and conjugated to de novo synthesized proteins in a process termed isgylation. isg processing exposes its carboxy-terminal lrlrgg motif, allowing its conjugation to lysine residues in target proteins to modulate their function. in addition, isgylation is reversible due to the action of the protease usp , which also regulates ifnar-mediated signaling ( ) . isg can remain unconjugated within the cell, regulating protein activity ( ), or be secreted as a cytokine, acting as a chemotactic and stimulating factor for immune cells ( ) . binding of isg to lfa- integrin receptor on the surface of nk cells promotes the activation, production and release of ifn-γ il- after il- priming. moreover, extracellular isg is able to form dimers/multimers through cysteine residues, to modulate cytokine levels. isg conjugation to target proteins is a covalent and reversible process through the action of a -kda deisgylase enzyme, ubiquitin-specific protease (usp ) [ , ] . interestingly, both isg and its conjugating and deconjugating enzymes are upregulated by type-i ifn [ ] , as well as by other stimuli such as type-ii and type-iii ifns [ ] [ ] [ ] , lipopolysaccharide [ ] , retinoic acid [ ] , dna damage or genotoxic reagents [ ] . usp not only acts as a deconjugating enzyme, but also as a negative regulator of the type-i ifn pathway (figure ) , with important implications in antiviral and antibacterial responses, immune cell development, autoimmune diseases and cancer [ ] . in humans, isg binds to usp , increasing its stability and leading to a decrease in ifn-α/β signaling. consequently, isg deficiency results in low usp levels, and therefore a sustained elevation in isg expression. this role for isg , which is absent in mice, seems to be predominant in humans, since patients appear not to be more susceptible to viral infections [ , ] . beyond the above-mentioned forms of isg -conjugated to target proteins or unconjugated within the cell-isg is also secreted into the serum, mainly by granulocytes via their secretory pathway [ ] . lymphocyte function-associated antigen receptor (lfa ) has recently been identified as the cellular receptor for isg ( figure ). isg binding to lfa triggers the activation of src family kinases, promoting ifn-γ and interleukin- (il- ) secretion in natural killer (nk) cells and, likely, also t-lymphocytes [ ] . the role of isg as an inductor of ifn-γ secretion seems to be the basis for the increased susceptibility to mycobacterial diseases in patients lacking a functional form of isg [ ] . secreted isg has also been described to promote nk [ ] and dendritic cell [ ] maturation, and to act as a chemotactic factor for neutrophils [ ] . along this line, a recent study highlighted the presence of dimeric and multimeric forms of extracellular isg important for its cytokine activity during parasite infection, and speculated on the existence of an unknown isg receptor on dendritic cells that mediates chemotaxis of these cells to the site of infection and il- β production [ ] . although there are several features of isg that are shared with ubiquitin, specially its structure, conjugation and deconjugation mechanisms [ ] , isgylation has not been shown to stimulate proteasomal degradation of its substrates [ ] . furthermore, some of the isgylation consequences are exerted by restricting the ubiquitin system, what might be mediated through the conjugation of isg to different e and e ubiquitin-conjugating enzymes [ ] , or even through the formation of mixed ubiquitin-isg chains [ ] . as a result, isgylation can decrease the polyubiquitylated proteins levels and downregulate protein turnover by the proteasome system [ ] . additionally, unlike ubiquitin, no poly-isg chains or specific isg -interacting motifs have been identified yet. in the following sections, we discuss the antiviral mechanisms mediated by isgylation of both viral and cellular proteins, with a focus on mitochondrial proteins, as we recently showed that isg modulates essential mitochondrial metabolic processes such as respiration and mitophagy in macrophages, with important implications for innate immune responses [ ] . the antiviral activity associated with isg and/or isgylation has been widely described since the first observation that isg -/mice were more susceptible to viral infections than their wild-type counterparts, albeit the role of isg and isgylation in viral life cycles is specific to the virus involved [ ] . early studies using isg -/mice demonstrated that isg has a protective effect against lethal infection by influenza virus, herpes simplex virus (hsv- ) and sindbis virus (sinv) [ ] . similarly, mice deficient in ube l-the e enzyme of isg -were also more susceptible to lethal infection by sinv [ ] . moreover, exogenous expression of wild-type isg by recombinant chimeric sinv protected ifnar-/-mice against systemic and lethal infections, whereas expression of isg mutants unable to conjugate to proteins did not show this protective effect [ ] , indicating an intrinsic antiviral role for isgylation. it should be noted that such an antiviral effect could be due to the conjugation of isg to viral and/or cellular proteins. by contrast, free isg , but not isgylation, has been described to promote antiviral responses against chikungunya virus (chikv) infection [ ] . to date, an antiviral effect mediated by isg or isgylation has been described using in vitro and/or in vivo systems for many other dna and rna viruses, including hepatitis b virus [ ] , vesicular stomatitis virus [ , ] , respiratory syncytial virus [ , ] , human immunodeficiency virus type (hiv- ) [ ] , and ebola virus [ ] . the antiviral effect of isg and isgylation has also been described against viruses of the genera novirhabdovirus, birnavirus and iridovirus in zebrafish, an example of the evolutionary conservation of the antiviral role of isg among vertebrates [ ] . given the importance of the antiviral response governed by isg , it is not surprising that viruses have evolved strategies to counteract its antiviral effects. for example, influenza b virus (ibv) ns protein [ ] , vaccinia virus e protein [ ] , and human cytomegalovirus (hcmv) ie and pul proteins obstruct isg antiviral action by preventing isgylation [ ] . similar mechanisms are also described for orthonairovirus and arterivirus otu-domain-containing proteases [ ] and for coronavirus papain-like proteases (plpro), which cleave isg from target proteins. remarkably, a plpro inhibitor was shown to protect mice from lethal infection in vivo [ ] . surprisingly, it has been reported that isgylation is necessary for robust production of hepatitis c virus (hcv), conferring a novel role for isg as a proviral factor that promotes virus production. indeed, in human hepatocytes, sirna silencing of isg was sufficient to both inhibit hcv replication and increase ifn expression [ ] . several reports have now highlighted a role for isg in the monitoring of hcv replication in cell cultures, as well as in the maintenance of hcv in liver, and pinpoint isg as among the predictor genes for non-response to ifn therapy [ ] . regarding the direct antiviral effect of isgylation via conjugation to viral proteins, perhaps the best-known example is the influenza a virus (iav) ns protein. this non-structural protein is abundantly expressed in infected cells and acts in multiple stages of the viral cycle, with important roles in ifn antagonism including sequestering double-stranded rna (dsrna), inhibiting dsrna-activated protein kinase (pkr) and contributing to the nuclear export of viral mrnas while blocking the splicing and export of cellular mrnas [ ] . seven lysine (k) residues in the ns protein were identified as potential target sites of isgylation [ ] . specifically, isg binding to k , which is part of the ns nuclear-localization signal, prevents its interaction with importin-α, inhibiting the translocation of ns to the nucleus and therefore repressing iav replication and viral rna processing [ ] . moreover, isgylation of the iav ns protein blocks its ability to counteract the innate immune response, prevents its interaction with pkr and, therefore, restores ifn-induced antiviral activities against iav [ ] . beyond ns , influenza virus nucleoprotein (np) and matrix protein (m ) have also been reported as targets of isg conjugation. isgylated np hinders the oligomerization of the more abundant unconjugated np, acting as a dominant-negative inhibitor of np oligomerization, impeding the formation of viral ribonucleoproteins and causing decreased viral protein synthesis and virus replication [ ] . interestingly, this study also identified a new role for influenza b virus ns in the sequestration of isgylated viral proteins, especially isgylated nps, which is perhaps an evolutionary mechanism to block the antiviral effect of isgylation. another example of isgylation of a viral protein with antiviral effects is the a protease ( apro) of coxsackievirus b (cvb ). isg conjugation to apro inhibits its ability to cleave the eukaryotic initiation factor eif g in cardiomyocytes, hindering the translational shutoff induced by cvb infection [ ] . consequently, isg conjugation to cvb leads to a reduction in virus titers and limits inflammatory cardiomyopathy, heart failure and lethality [ ] . similarly, isgylation of the hcmv scaffold protein pul interferes with the viral modulation of the innate immune response. specifically, isgylation of pul at k and k inactivates its function in the downregulation of tnfα-mediated nf-κb activation, suppressing hcmv growth [ ] . finally, another example of an isgylated viral protein is the human papillomavirus (hpv) l capsid protein. isgylated l proteins were shown to be incorporated into hpv pseudoviruses, resulting in a reduced infectivity; the precise mechanism that mediates this inhibitory effect remains elusive [ ] . knowledge about the impact of host protein isgylation in virus replication and cell homeostasis is still scant. in contrast to ubiquitylation, the molecular effect of isg conjugation on target proteins is not always clear. protein isgylation has been reported to increase protein degradation by selective autophagy [ ] , but there are also many examples where isgylation inhibits ubiquitylation, frustrating proteasome-mediated degradation of target proteins [ ] [ ] [ ] . with regard to proteins involved in antiviral response, many effectors of ifn signaling such as pkr [ ] , retinoic acid-inducible gene-i (rig-i) [ ] and myxoma resistance protein (mxa) [ ] have been reported to be targets of isgylation. pkr isgylation at k and k , both located in the dsrna-binding motif, triggers its activation. this modification occurs in the absence of viral rna and leads to the phosphorylation of eif α, preventing protein translation [ ] and suggesting that isgylation might mediate the activation of pkr in response to stressful stimuli beyond viral infection. further, isg conjugation to rig-i decreases rig-i cellular levels and downregulates rig-i-mediated signaling. accordingly, isgylation of rig-i represents a negative feedback loop that might control the strength of the antiviral response [ ] . interestingly, free isg also regulates rig-i levels by promoting the interaction between rig-i and the autophagic cargo receptor p , mediating rig-i degradation via selective autophagy [ ] . the interferon-induced mxa protein is also a target of isgylation, though the effect of this modification is not clear. other proteins involved type-i ifn signaling and regulation, such as components of the jak-stat pathway or regulators of signal transduction (e.g., jak and extracellular signal-regulated kinase [erk ]), are also bound by isg , although the functional consequences of isgylation remain unknown [ , ] . moreover, interferon regulatory factor (irf ), stat and the actin-binding protein filamin b are also targets for isg conjugation, with implications in the development of the innate immune response. irf is isgylated at k , k and k , which attenuates its interaction with the peptidyl-prolyl isomerase pin , preventing irf ubiquitylation. thus, isgylation of irf sustains its activation and enhances irf -mediated antiviral responses by inhibiting its degradation [ ] . in a similar manner, isgylation of phosphorylated stat (pstat ) inhibits its polyubiquitylation and further proteasomal degradation, supporting sustained stat activation [ ] . isgylation of filamin b, which acts as a scaffold of ifn signaling mediators, negatively regulates ifnα-induced c-jun n-terminal kinases (jnk) signaling, preventing apoptosis induction [ ] . beyond antiviral response, isgylation has been described to block the process of virus budding by interfering with the endosomal sorting complexes required for transport (escrt) machinery. for example, isgylation of chmp triggers its aggregation and the sequestration of the vps cofactor lip , impairing the membrane recruitment of vps and its interaction with the gag budding complex of avian sarcoma leukosis virus and hiv- , leading to the inhibition of virus release from the cell [ ] . similarly, isgylation of tumor susceptibility gene protein (tsg ), another component of the escrt sorting complex, inhibits the trafficking of viral hemagglutinin to the cell surface during iav infection [ ] , blocking virus release. isg has also been described to inhibit the interaction of hiv- gag protein with tsg , underscoring a critical role of isg in the ifn-mediated inhibition of hiv- budding and release [ ] . this sorting mechanism is also used in the generation of exosomes, which are small vesicles secreted to the extracellular environment by most cell types. interestingly, isgylation of tsg has been recently reported to inhibit exosome secretion [ ] . the above examples serve to illustrate the relevance of isgylation in the induction and regulation of the antiviral response (for a more complete review of isgylated cellular proteins see reference [ ] ), and highlight the complexity of fully understanding the consequences of isgylation in the regulation of biochemical processes where it is involved. although the significance of isgylation of host proteins has been elucidated for only a small set of cellular proteins, isgylation has a broad target specificity, and there is increasing evidence for its role in regulating many cellular functions. to address this concept, several proteomic studies have been performed to determine isgylated host proteins. zhao et al. [ ] transfected a tagged isg protein into ifn-stimulated hela cells, and used affinity selection to identify isgylated proteins. in a similar approach, giannakopoulos et al. [ ] used ifn-stimulated usp -/-mouse embryonic fibroblasts and human u cells to detect up to proteins conjugated to endogenously-expressed isg . a third proteomic study [ ] identified isgylated cellular proteins in ifn-stimulated a human lung adenocarcinoma cells stably expressing flag-isg . more recently, peng et al. [ ] examined isgylated proteins in influenza virus-infected a cells, identifying a total of cellular proteins in addition to viral ns protein. we have surveyed the proteins identified by these four studies, which rendered up to cellular proteins. identified proteins include, as previously outlined [ ] , abundant constitutively expressed proteins as well as diverse interferon-induced proteins. interestingly, there is only a low degree of overlap between the studies, and only four proteins are common to all four analyses (the glycolytic enzymes aldo and eno , the peroxiredoxin prdx , and stat ). these discrepancies may reflect the different transcriptional/translational patterns of the different cell lines included in each study, as it is believed that the biological effects of isgylation are dynamic and cell type/tissue-specific [ ] . we used david bioinformatics resources [ , ] to determine the subcellular localization of the proteins identified as isgylation targets in the aforementioned studies, with the aim to obtain a comprehensive picture of the broad range of actions of isg . in agreement with a previous report [ ] , our analysis ( figure ) shows that isg -targeted proteins are found almost throughout the cell, including nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum and cell membranes [ ] . moreover, a similar percentage of isgylation targets were predicted to be located in the nucleus, cytoplasm, extracellular space or as secreted proteins (figure ) . interestingly, proteins associated with cytoskeleton and cell junctions represent a significant percentage of the isg target proteins. other cell structures such as the melanosome or myelin sheath were also represented in the study, perhaps accounting for a specific role of isgylation in these organelles. the potential role of isg in mitochondria seems to be relevant, as a recent study predicted that % of free isg was localized to mitochondria [ ] . in our own analysis of the above proteomic studies, fifty-two isgylated proteins were predicted to localize to mitochondria, representing about % of the total isg target proteins ( figure ). further examination of these potentially isgylated proteins indicate that different mitochondrial processes could be affected by isg conjugation (table ) . remarkably, several subunits of the atp synthase (complex v of the respiratory chain) appear to be isg targets, which may be of relevance as mitochondrial atp production is the main source of energy for the cell. in line with these observations, our recent work linked isg to the control of the mitochondrial oxidative metabolism in macrophages in the context of viral infection [ ] . based on the evident association between isg and mitochondria, we will briefly review the role of mitochondria as antiviral mediators and targets of ubiquitin-like modifiers, focusing on the current knowledge about isg -and isgylation-mediated regulation of these multifunctional organelles. included in each study, as it is believed that the biological effects of isgylation are dynamic and cell type/tissue-specific [ ] . we used david bioinformatics resources [ , ] to determine the subcellular localization of the proteins identified as isgylation targets in the aforementioned studies, with the aim to obtain a comprehensive picture of the broad range of actions of isg . in agreement with a previous report [ ] , our analysis ( figure ) shows that isg -targeted proteins are found almost throughout the cell, including nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum and cell membranes [ ] . moreover, a similar percentage of isgylation targets were predicted to be located in the nucleus, cytoplasm, extracellular space or as secreted proteins (figure ) . interestingly, proteins associated with cytoskeleton and cell junctions represent a significant percentage of the isg target proteins. other cell structures such as the melanosome or myelin sheath were also represented in the study, perhaps accounting for a specific role of isgylation in these organelles. the potential role of isg in mitochondria seems to be relevant, as a recent study predicted that % of free isg was localized to mitochondria [ ] . in our own analysis of the above proteomic studies, fifty-two isgylated proteins were predicted to localize to mitochondria, representing about % of the total isg target proteins ( figure ). further examination of these potentially isgylated proteins indicate that different mitochondrial processes could be affected by isg conjugation (table ) . remarkably, several subunits of the atp synthase (complex v of the respiratory chain) appear to be isg targets, which may be of relevance as mitochondrial atp production is the main source of energy for the cell. in line with these observations, our recent work linked isg to the control of the mitochondrial oxidative metabolism in macrophages in the context of viral infection [ ] . based on the evident association between isg and mitochondria, we will briefly review the role of mitochondria as antiviral mediators and targets of ubiquitin-like modifiers, focusing on the current knowledge about isg -and isgylation-mediated regulation of these multifunctional organelles. [ , [ ] [ ] [ ] predicted to locate to mitochondria. proteins are grouped according to biological functions. acyl-coa thioesterase (acot ) [ ] complement c q binding protein (c qbp) [ ] receptor for activated c kinase (rack ) [ ] solute carrier family member (slc a ) [ ] solute carrier family member (slc a ) [ ] staphylococcal nuclease and tudor domain containing (snd ) [ ] negative regulation of apoptotic process nme/nm nucleoside diphosphate kinase (nme ) [ ] annexin a (anxa ) [ , ] glutathione s-transferase pi (gstp ) [ ] heat shock protein family a (hsp ) member (hspa ) [ ] interferon-induced protein with tetratricopeptide repeats (ifit ) [ ] positive regulation of protein insertion into mitochondrial membrane involved in apoptotic signaling pathway stratifin (sfn) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein beta (ywhab) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein épsilon (ywhae) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein gamma (ywhag) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein theta (ywhaq) [ ] tyrosine -monooxygenase/tryptophan -monooxygenase activation protein zeta (ywhaz) [ ] atp biosynthetic process atp synthase, h+ transporting, mitochondrial f complex, alpha subunit , cardiac muscle (atp a ) [ , ] atp synthase, h+ transporting, mitochondrial f complex, beta polypeptide (atp b) [ , ] atp synthase, h+ transporting, mitochondrial fo complex subunit g (atp l) [ ] oxidation-reduction process aldehyde dehydrogenase family member a (aldh a ) [ ] fatty acid synthase (fasn) [ , ] glutathione-disulfide reductase (gsr) [ ] lactate dehydrogenase b (ldhb) [ ] malic enzyme (me ) [ ] peroxiredoxin (prdx ) [ , , ] peroxiredoxin (prdx ) [ ] sorbitol dehydrogenase (sord) [ ] superoxide dismutase , soluble(sod ) [ ] thioredoxin reductase (txnrd ) [ , ] thioredoxin (txn) [ ] aminoacyl-trna synthetase alanyl-trna synthetase (aars) [ ] glycyl-trna synthetase (gars) [ ] phenylalanyl-trna synthetase , mitocondrial (fars ) [ ] tricarboxylic acid cycle malate dehydrogenase (mdh ) [ ] malate dehydrogenase (mdh ) [ ] glycolisis oxoglutarate dehydrogenase (ogdh) [ ] pyruvate kinase, muscle (pkm) [ , , ] chaperone chaperonin containing tcp subunit (cct ) [ ] heat shock protein alpha family class b member (hsp ab ) [ , , ] heat shock protein family a (hsp ) member a (hspa a) [ , ] heat shock protein family d (hsp ) member (hspd ) [ , ] ion channel chloride intracellular channel (clic ) [ , ] annexin a (anxa ) [ ] other functions creatine kinase, mitochondrial b (ckmt b) [ ] ubiquitin-like modifier activating enzyme (uba ) [ ] leucine aminopeptidase (lap ) [ ] -aminoimidazole- -carboxamide ribonucleotide formyltransferase/imp cyclohydrolase (atic) [ , ] clathrin heavy chain (cltc) [ , ] queuine trna-ribosyltransferase accessory subunit (qtrt ) [ ] enoyl-coa hydratase and -hydroxyacyl coa dehydrogenase (ehhadh) [ ] atp binding cassette subfamily f member (abcf ) [ ] mitochondria have myriad functions in the cell although they are best known for providing energy in the form of atp and for controlling metabolism to maintain energy homeostasis. owing to their endosymbiotic origin, mitochondria have their own genome, a single -kb circular dna which codes for mitochondrial proteins, ribosomal rnas and transfer rnas [ ] . the remainder of mitochondrial proteins are encoded by nuclear dna and are then transported to the mitochondria through the recognition of amino acid sequences known as mitochondrial targeting signals [ ] . as double-membrane organelles, mitochondria have an outer mitochondrial membrane (omm), where proteins responsible for transport of different molecules are embedded [ ] ; an intermembrane space (ims) and an inner mitochondrial membrane (imm), where electron transport chain (etc) proteins are localized and oxidative phosphorylation (oxphos) and atp production takes place [ ] , and a mitochondrial matrix (mm), compartment, where many metabolic pathways occur, such as the tricarboxylic acid cycle, fatty-acid oxidation, synthesis of biomolecules and regulation of apoptosis [ ] . the proper development of mitochondrial processes is critical for immune response, as the susceptibility to microbial infections and the risk of systemic inflammatory responses increases considerably when these organelles malfunction [ , ] . mitochondria are important for antiviral signaling. during rna-virus infection, viral rnas are initially recognized by cytoplasmic sensors, mainly rig-i-like receptors (rlrs) [ ] , whose interaction with mitochondria is essential for the coordination and development of an adequate antiviral response. the common structure of rlrs consists of a carboxy-terminal regulatory domain, a central rna helicase domain and amino-terminal caspase recruitment domains (cards) [ ] . after binding to viral rna, rlrs trigger ifn-mediated antiviral responses through their interaction with mitochondrial antiviral-signaling protein (mavs), a card-containing omm protein [ ] . the card-card interaction between rlrs and mavs causes mavs polymerization and consequent recruitment of a variety of downstream effectors, including tumor necrosis factor receptor-associated factor family proteins, ikb kinase epsilon (ikkε) and tank binding kinase , among others [ ] . this "mavs signalosome" activates nf-κb, irf and irf , promoting the expression of type-i ifn and antiviral molecules [ ] . given the central role of mavs in mitochondrial antiviral signaling, mavs and both upstream and downstream molecules are under tight regulation to ensure an adequate response [ , ] . mitochondria are dynamic organelles that undergo constant fusion and fission to regulate their morphology, activity and turnover according to the metabolic needs of the cell [ ] , and these mitochondrial dynamics are involved in the regulation of mitochondrial immune functions. mitofusins and optic atrophy protein are responsible for mitochondrial fusion, whereas the cytosolic gtp-ase dynamin-related protein (drp ) mediates mitochondrial fission through its interaction with adaptor proteins in the omm [ , ] . interestingly, these proteins have been shown to be implicated in the regulation of various mitochondrial immune-relevant processes, such as rlr signaling [ , , ] , apoptosis [ , ] , autophagy and mitochondrial bioenergetic conditions [ ] , which are important mechanisms to combat viral infections. mitophagy is a selective autophagic process in which defective mitochondria are engulfed in autophagosomes and eliminated by fusion with lysosomes [ ] . damaged mitochondria constitute a signal for the recruitment of pten-induced putative kinase protein (pink ), which surrounds the mitochondrial surface. the accumulation of pink and its kinase activity promote the translocation of the e ubiquitin ligase parkin from the cytosol to dysfunctional mitochondria, triggering the ubiquitylation of omm proteins. the formation of ubiquitin chains by parkin favors the binding of adaptor proteins (e.g., p and optineurin), which mediate the interaction with autophagosomes and the further degradation of dysfunctional mitochondria [ , ] . mitophagy is closely related to mitochondrial dynamics, as mitochondrial fragmentation promotes mitophagy whereas mitochondrial fusion hinders this process [ , ] . interestingly, defective mitochondria can play either positive or negative roles against viruses. for example, alterations in mitochondrial respiration trigger the production of reactive oxygen species (ros) which, in addition to being harmful for the cell in high levels, play an important role as second messengers in diverse intracellular signaling pathways [ ] . in the context of antiviral signaling, ros are involved in the regulation of the rlr pathway, potentiating rlr-mavs signaling and the production of type-i ifn [ ] . because healthy mitochondria are required for an adequate metabolic state and the activation of apoptotic processes [ , , ] , mitophagy must be finely regulated to modulate the mitochondria-mediated antiviral response. the implications of mitochondria in innate immunity are enormous. we have briefly discussed the interplay between different mitochondrial pathways, such as rlr signaling, mitochondrial dynamics, mitophagy, ros production and apoptosis, in the protection against viruses. however, mitochondria perform a plethora of functions in the establishment of a defensive state of the cell, which have been thoroughly reviewed by others [ , [ ] [ ] [ ] [ ] [ ] . moreover, the regulation of mitochondrial function is not only carried out by the host, but also by viruses with the aim to shut down defense mechanisms, complete their life cycle and spread [ ] , underscoring the relevance of mitochondria in antiviral response. such critical roles of mitochondria in the control of pathogen invasion and maintenance of cellular homeostasis must be strictly coordinated. as we previously discussed, ubiquitin and ubiquitin-like ptms are key regulatory processes of the innate and adaptive immune response against viruses, and both processes are finely regulated by mitochondria. in this regard, there is a broad spectrum of ptms [ ] , some of which occur within mitochondria, which are responsible for modifying their internal state and function [ , ] . thus, mitochondria are targets of ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifiers (sumos) and isg . ubiquitin is a highly conserved . -kda protein known as a master regulator of cellular processes. its covalent conjugation to target proteins has proteolytic and non-proteolytic or regulatory outcomes, which fine-tune protein function and recycling [ ] . indeed, ubiquitylation is essential for the regulation of many mitochondrial processes, such as mitophagy [ , [ ] [ ] [ ] , mitochondrial dynamics [ , ] , and mitochondria-related immune signaling [ , , ] , establishing the importance of this modifier in the homeostasis of these organelles. sumos are a family of highly conserved -kda proteins that are essential in eukaryotic cells. similar to ubiquitin, sumo conjugation to specific lysine residues of target proteins (sumoylation) alters their function, their interaction with other proteins, and their stability [ , ] . although the major role of sumoylation is in the regulation of nuclear processes [ ] , it also targets mitochondria. sumos have been proven to be involved in the regulation of mitochondrial dynamics by binding to drp [ ] , with an important implication in programmed cell death [ ] [ ] [ ] . furthermore, sumoylation of the mitochondrial oxidative stress sensor dj- results in its stabilization and full activation, reinforcing its protective role against parkinson's disease [ ] , where mitochondrial dysfunction has great significance. although isg has been associated with mitochondria, its functions, both free or conjugated to mitochondrial proteins, are still being examined. one exception is the protein parkin. while not strictly a mitochondrial protein, its translocation to the omm from the cytoplasm is essential for parkin-mediated mitophagy [ ] . isg conjugation to parkin enhances its e ubiquitin ligase activity and its cytoprotective effect in parkinson's disease [ ] , an example of how isgylation affects mitochondrial processes. isg and isgylation for the regulation of mitochondrial metabolism [ ] . we undertook a comprehensive analysis of bone marrow-derived macrophages (bmdms) from wild-type and isg -/mice to interrogate how isg and isgylation could modulate the regulation of mitochondria in the context of stressful stimuli. monomeric isg and isgylated proteins were observed in mitochondrial fractions from wild-type bmdms after type-i ifn pre-treatment, and these proteins were preferentially located to the ims and imm (figure ) . given their localization, we hypothesized that isg and isgylation could impact mitochondrial respiratory metabolism, and we focused our study on oxphos and atp production. this analysis revealed that oxygen consumption and atp production were lower in isg -/-bmdms than in equivalent wild-type cells, indicative of defective oxphos. in accord with these observations, a clear difference in the distribution of etc supercomplexes was observed between the two groups, pointing to a possible role for isg in the correct assembly of etc proteins ( figure ). as recently reported by yoshizumi et al. [ ] oxphos activity is required for rlr-mediated antiviral signaling, and mice with oxphos defects showed increased susceptibility to viral infections. similarly, knockout mice for isg or the isg -activating e enzyme (ube l) were more susceptible to infection with many viruses than wild-type mice [ ] . since the lack of isg seems to cause alterations in oxphos, such increase in the sensitivity to viral infections in isg -/and ube l-/-mice might be explained as a result of defects in rlr-mediated antiviral responses, supporting the role of isg as a regulator of mitochondrial functions. regarding mitochondrial respiration byproducts, isg -/-bmdm also produced lower levels of ros. because ros production is tightly controlled by mitochondrial membrane potential [ ] , low levels of ros in isg -/-bmdm might be the result of abnormalities in the transmembrane proton gradient due to the absence of isg , and could affect the immune response against viral infections, as discussed earlier. mitochondrial ros also participate in the regulation of macrophage polarization [ ] and, interestingly, isg -/-bmdms displayed mixed features of m and m phenotypes, suggesting that alterations in mitochondrial oxphos could drive changes to immune cell function. finally, bmdms lacking isg accumulated non-functional mitochondria with an absence of parkin, suggesting that isg is also implicated in the regulation of mitophagy, perhaps through the control of parkin translocation from the cytosol (figure ) . taken together, these findings establish a relevant role for isg and isgylation in the control of mitochondrial oxphos and recycling, at least in murine bmdm, expanding the range of functions of this ptm and underscoring its importance in the regulation of essential cellular processes. viruses , , x for peer review of figure . impact of isg on mitochondrial activities. mitochondria are targets of isg and isgylation in murine bone marrow-derived macrophages (bmdms). isgylated proteins can be found in all mitochondrial localizations, mainly in the mitochondrial intermembrane space (ims) and inner mitochondrial membrane (imm), where free isg is also present. isg and isgylation are involved in the regulation of mitochondrial metabolism. absence of isg leads to alterations in oxphos, with lower oxygen consumption rates and atp production levels, in addition to aberrant etc supercomplexes assembly. such disruption of oxphos mechanisms decreases ros production, with repercussions for macrophage polarization. mitophagy is also altered in cells lacking isg . finally, isg -/-bmdm accumulate defective mitochondria and parkin cannot be found in mitochondrial extracts, suggesting that isg is important during the translocation of parkin from the cytoplasm to mitochondria. the functional significance of ptms in disease etiology, and the pathologic response to their disruption, is the subject of intense investigation. many of these reversible modifications act as regulatory mechanisms in mitochondria and show promise for mitochondria-targeted therapeutic strategies. with the advent of mass spectrometry-based screening techniques, there has been a vast increase in our current state of knowledge on mitochondrial ptms and their protein targets. detecting isgylated proteins in different organelles remains challenging, as it typically occurs in only a small portion of the total protein pool of the cell, albeit with essential roles in regulating protein fate and function. understanding the consequences of isgylation of mitochondrial proteins will require much work, but should be rewarding not only for developing new strategies to combat viral infections, but also for future applications in other biomedically relevant processes/diseases, for example inflammation, cancer and neurodegeneration. . impact of isg on mitochondrial activities. mitochondria are targets of isg and isgylation in murine bone marrow-derived macrophages (bmdms). isgylated proteins can be found in all mitochondrial localizations, mainly in the mitochondrial intermembrane space (ims) and inner mitochondrial membrane (imm), where free isg is also present. isg and isgylation are involved in the regulation of mitochondrial metabolism. absence of isg leads to alterations in oxphos, with lower oxygen consumption rates and atp production levels, in addition to aberrant etc supercomplexes assembly. such disruption of oxphos mechanisms decreases ros production, with repercussions for macrophage polarization. mitophagy is also altered in cells lacking isg . finally, isg -/-bmdm accumulate defective mitochondria and parkin cannot be found in mitochondrial extracts, suggesting that isg is important during the translocation of parkin from the cytoplasm to mitochondria. the functional significance of ptms in disease etiology, and the pathologic response to their disruption, is the subject of intense investigation. many of these reversible modifications act as regulatory mechanisms in mitochondria and show promise for mitochondria-targeted therapeutic strategies. with the advent of mass spectrometry-based screening techniques, there has been a vast increase in our current state of knowledge on mitochondrial ptms and their protein targets. detecting isgylated proteins in different organelles remains challenging, as it typically occurs in only a small portion of the total protein pool of the cell, albeit with essential roles in regulating protein fate and function. understanding the consequences of isgylation of mitochondrial proteins will require much work, but should be rewarding not only for developing new strategies to combat viral infections, but also for future applications in other biomedically relevant processes/diseases, for example inflammation, cancer and neurodegeneration. a virological view of innate immune recognition innate immune pattern recognition: a cell biological perspective a virological view of innate immune recognition innate immune pattern recognition: a 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redox regulation of macrophage polarization we thank diego sanz for his technical assistance and kenneth mccreath for reviewing and correcting the manuscript. the authors declare no conflicts of interest. acknowledgments: we thank diego sanz for his technical assistance and kenneth mccreath for reviewing and correcting the manuscript. the authors declare no conflicts of interest.viruses , , key: cord- -egvdvvtx authors: damsky, william; peterson, danielle; king, brett title: when interferon tiptoes through covid- : pernio-like lesions and their prognostic implications during sars-cov- infection date: - - journal: j am acad dermatol doi: . /j.jaad. . . sha: doc_id: cord_uid: egvdvvtx nan william damsky md, phd ,* , danielle peterson md brett king md, phd ,* has suggested that sars-cov- infection is sometimes characterized by a muted anti- viral type i and iii interferon (ifn) response, , which may explain progression to severe clinical manifestations in some patients; a robust type i ifn response was associated with rapid viral clearance and bland disease course. here, we describe pernio-like lesions as they have been reported in the literature and consider other settings where pernio is observed, including familial chilblains lupus, an interferonopathy syndrome. together, these data suggest that covid toes may be a marker of patients that are able to mount a robust anti-viral immune response to sars-cov- and prognosticate a milder course of covid- . sporadic pernio (also known as chilblains) is an idiopathic cold-sensitive inflammatory disorder that presents with red-to-violaceous macules or papules on acral sites; vesiculation and ulceration may occur. these lesions are typically located on the distal toes, but can also occur on fingers, heels, and even the nose and ears. histopathology reveals edema in the superficial dermis and marked superficial and deep perivascular and peri-eccrine lymphocytic inflammation (figure a) . interface change and/or vasculopathic changes (e.g. focal thrombosis) may be present. type i ifn response is associated with early viral control and a mild course, while an insufficient type i ifn response may be associated with progression to more severe disease ( figure b) . , therefore, we hypothesize that pernio-like lesions, which can occur with elevated type i ifn signaling, are the result of a robust anti-viral response in patients with covid- , and, therefore, are associated with a favorable disease course, as observed in these patients. characterization of acute acro-ischemic lesions in non-hospitalized patients: a case series of patients during the covid- outbreak chilblains in children in the setting of covid pandemic covid- ) infection- induced chilblains: a case report with histopathologic findings classification of the cutaneous manifestations of covid : a rapid prospective nationwide consensus study in spain with cases pernio-like skin lesions associated with covid- : a case series of patients from countries type i ifn immunoprofiling in covid- patients imbalanced host response to key: cord- -xemarhlv authors: goswami, biswendu b.; kulka, michael; ngo, diana; cebula, thomas a. title: apoptosis induced by a cytopathic hepatitis a virus is dependent on caspase activation following ribosomal rna degradation but occurs in the absence of ′– ′ oligoadenylate synthetase date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: xemarhlv we have presented previously evidence that the cytopathogenic f strain of hepatitis a virus (hav) induced degradation of ribosomal rna (rrna) in infected cells [arch. virol. ( ) – ]. in contrast, the non-cytopathogenic parent virus hm clone had no effect on rrna integrity. we present here data showing that rrna degradation is followed by apoptosis accompanied by characteristic dna laddering in the cytoplasm of f infected cells. the dna laddering coincided with the detection of caspase and parp- cleavage and was dependent upon activation of the caspase pathway, since treatment with z-vad-fmk, a pan-caspase inhibitor, inhibited both events. rnase l mrna was present in both virus-infected and uninfected cells. messenger rna for the interferon inducible enzyme ′– ′ oligoadenylate synthetase ( ′– ′ oas), which polymerizes atp into ′– ′ oligo adenylate ( – a, the activator of rnase l) in the presence of double-stranded rna, was not detected following virus infection. ′– ′ oas mrna was induced by treatment of the cells with interferon-β (ifn-β). ifn-β mrna was marginally induced following infection. however, phosphorylated stat , a key regulator of interferon-stimulated gene transcription was not detected in virus infected cells. stat phosphorylation in response to ifn treatment was lower in virus-infected cells, compared to uninfected cells treated with interferon, suggesting that f virus infection interferes with interferon signaling. the results suggest that f infection causes the induction of a – a independent rnase l like activity. hepatitis a virus is a picornavirus and is the only known member of the genus hepatovirus. it is the major causative agent for infectious hepatitis worldwide and is easily disseminated by person to person contact as well as via contaminated foods and water, due to its resistance to environmental factors such as heat, low ph, etc. (siegl et al., ; gust, ) . most of the known hav strains establish a slowly replicating and non-cytopathogenic persistent infection in permissive cells in vitro (provost and hillemen, ; flehmig, ; binn et al., ; vallbracht et al., ) . this is in contrast to in vivo infection, where persistence is restricted to a few weeks (lemon, ; vallbracht et al., ) , although sporadic cases of chronic infection have been reported (fagan et al., ; inoue et al., ) . several laboratories have isolated rapidly replicating cytopathogenic (cp) strains that produce visible morphological changes primarily in continuous cell lines of monkey kidney origin (divizia et al., ; venuti et al., ; anderson, ; cromeans et al., ; nasser and metcalf, ; zhang et al., ; brack et al., ) . the mechanism of the cytopathogenic effect (cpe) produced by the rapidly replicating hav strains has been intensely investigated during the past few years to understand why wild type (wt) and cell culture adapted (cc) strains replicate slowly and are unable to induce cpe in these cell lines. these studies have revealed that the genome of the cpe-inducing (cp) strains contain point mutations, compared to the wt strains from which they were derived, scattered throughout the genome, as well as a -bp repeat in the non-translated region (ntr), which harbors the internal ribosomal entry site or ires (lemon et al., ; brack et al., ) . the biological significance of these mutations for the rapid replication phenotype of the cp strains is not completely understood. experiments with chimeric viral genomes containing mutated regions of cp strains on a genetic background of cc strains suggested an effect of these mutations on the expression of the viral genome, possibly due to altered interaction of the mutated genomes with cellular proteins that either positively or negatively regulate viral gene expression whetter et al., ; zhang et al., ; funkhouser et al., ; yi et al., ) . however, the view that hav ires is inefficient compared to other picornaviruses is not universally accepted (jia et al., ; graff and ehrenfeld, ; gauss-müller and kusov, ) . we have recently reported that in the permissive monkey kidney cell line frhk infection with the cp strain hm f (hereafter referred to as f) resulted in degradation of ribosomal rnas (rrnas), whereas viral genomic rna was not degraded. in contrast, the parental cc strain did not cause such degradation either after an acute infection or extended persistent infection (kulka et al., ) . based on the pattern of rrna degradation in intact ribosomes, we suggested that the f virus activates the interferon (ifn) controlled - oligoadenylic acid-dependent rnase l pathway (for recent reviews, see stark et al., ; barber, ; sen, ) . since this pathway is essentially an antiviral mechanism mounted by cells in defense against viral infection, the usurpation of this pathway by the f virus could be a mechanism employed by this virus to reduce competition from cellular mrnas for the host protein synthetic machinery. this virus lacks an active a protease (schultheiss et al., ; harmon et al., ) capable of degrading either eif- g or other cellular translation factors required for translation of capped cellular mrnas, therefore, the importance of such an rna degradative mechanism cannot be overstated. the activation of the rnase l pathway is controlled by ifn (stark et al., ; barber, ; sen, ) through the induction of - oligoadenylate synthetase ( - oas), which synthesizes - oligoadenylic acid or - a, the proximal activator of the rnase l from atp, in the presence of double stranded rna (dsrna). overexpression of rnase l has been shown to inhibit the growth of diverse rna viruses (zhou et al., ) . whether hav infection can induce synthesis of type i ifn (ifn ␣/␤) in cultured cells or intact organisms and consequently activate the - oas/rnase l pathway remains unclear. cell cultures persistently infected with culture-adapted hav do not induce type ifn, and the replication of the virus in persistently infected cells remains sensitive to exogenously added ifn (vallbracht et al., ; brack et al., ) . acute infection with cc or cp strains of hav did not cause induction of ifn ␣/␤ mrna transcription and cc strains also blocked ifn mrna transcription and secretion of ifn in response to dsrna in frhk and mrc- cells (brack et al., ) . exogenous ifn inhibited virus replication when added either before or after exposure to virus in a dose dependent manner in plc/prf/ cells (crance et al., ) . inhibi-tion of virus replication in the above study also showed a strong dependence on the multiplicity of infection (moi). a significant increase in the activity of the ifn-induced - oas was observed following ifn treatment, suggesting that the rnase l pathway may be involved in the inhibition of virus replication (crance et al., ) . constitutive expression of - oas results in the inhibition of picornavirus replication (chebath et al., ) , and ectopic expression and activation of rnase l induces apoptosis in animal cells (diaz-guerra et al., ; castelli et al., ) . the goal of this investigation was to study the role of the ifn -regulated - a activated rnase l on rna degradation and apoptosis in f infected cells. frhk monkey kidney cell line was a kind gift of dr. g. kaplan (fda, center for biologics evaluation and research, bethesda, md). the cells were grown in eagles minimal essential medium (emem) containing % heat inactivated fetal bovine serum (fbs), mem non-essential amino acids and sodium pyruvate (all from invitrogen, carlsbad, ca), with routine weekly sub-culturing. under these conditions the cells increase about -fold in - days. the cell line is contact inhibited and can be kept in growth medium or maintenance medium ( % fbs) for several weeks without degeneration of the monolayer. hav strains hm / f (cytopathogenic in frhk cells) and hm /clone (non-cytopathogenic in frhk cells) were obtained from atcc. viruses were grown in frhk cells. virus stocks were prepared as described before (kulka et al., ) . f was titered by plaque assay, while clone virus was titered by eia in -well plates as described previously (goswami et al., ) using the havab eia diagnostic kit (abbott laboratories, abbott park, il). human ifn-␤ and the pancaspase inhibitor z-vad-fmk were obtained from sigma (st. louis, mo). confluent cultures of frhk cells in cm flasks ( × cells) were infected with a multiplicity of infection (moi) of pfu/cell of f or tcid /cell of clone in . ml of mem containing % heat inactivated fbs or were mock infected. after h of adsorption, . ml of the same medium was added to each flask and incubation continued until the desired time of harvest. cells were harvested by scraping, centrifuged to remove medium, and the pellets washed once with pbs (ca + and mg + free) and stored frozen at − • c or used immediately. rna isolation was carried out as previously described (kulka et al., ) . a persistent infection of frhk cells with clone virus was established as described (kulka et al., ) . the cells were maintained by routine subculturing in the same manner as for normal frhk cells. to monitor virus replication, cells were periodically seeded into -well plates along with uninfected cells, and viral antigen in methanol fixed cells measured by the havab eia procedure (goswami et al., ; kulka et al., ) . all experiments with this cell line (hereafter referred to as clone ) were carried out between months and year of routine subculture. cell pellets of uninfected or virus infected cells were resuspended in mm tris-hcl, mm edta, ph . and lysed by the addition of triton x- to . % (brack et al., ) . cell lysates were kept on ice for min with intermittent vortex mixing, followed by centrifugation at , × g for min. the supernatant was carefully removed and digested with proteinase k ( . mg/ml) in the presence of . % sds. the digest was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (pcia), and the aqueous phase removed to a fresh tube. nucleic acids were precipitated by the addition of sodium acetate to . m and . volume of ethanol. the precipitates were collected by centrifugation, washed once with % ethanol, dried and dissolved in rnase/dnase free water. nucleic acids were quantitated by a measurement. equal amounts of sample were incubated at • c for min in l te with or without u of cloned rnase (ambion, austin, tx), and analyzed by % agarose gel electrophoresis in tbe. denaturing agarose gel electrophoresis was performed as described before (kulka et al., ; chomczynski and mackey, ) . the gel was photographed under uv light. the induction of apoptosis was investigated by the incorporation of biotin labeled deoxyribonucleotides and detection of biotin incorporation according to the manufacturer's instructions (deadend colorimetric tunel system, promega). cells were seeded in four well chamber slides, and infected at confluency with f at pfu/cell. cells were fixed for tunel assay at h pi. persistently infected cells were seeded in four well chamber slides, and processed when the monolayers reached confluence. viral antigen-positive cells were displayed by immunohistochemistry, using an antibody to vp (kulka et al., ) . briefly, cells were fixed in buffered formalin, permeabilized with . % tween in pbs, and reacted with : dilution of rabbit anti-vp antibody in pbs containing % nonfat dry milk (nadala and loh, ) . after several washes with pbs containing . % tween , the cells were incubated with hrp labeled anti-rabbit igg diluted in % non-fat dry milk. following several washes to remove unbound antibody, color was developed using cn-dab substrate (pierce chemical co.). total cytoplasmic rnas were digested with rnase free dnase (promega) in the presence of rnasin rnase inhibitor followed by pcia extraction and ethanol precipitation, as above, to remove any contaminating dna. rna samples ( g) each were reverse transcribed using amv reverse transcriptase in a total volume of l using g oligo(dt) as primer as described previously (goswami et al., ) . ifn-␤ mrna was amplified using the primer pairs accaacaagtgtctcctcca (sense) and gaggtaacctgtaagtctgt (antisense), with an annealing temperature of • c. this primer pair amplifies a -bp fragment (brack et al., ) . amplification of the p / and the p / forms of - oas were carried out with the primers p / sense ( gaagcctgtcaaagagagag ) and p / antisense ( tgtgttttcatgctccctcg ) which amplifies a -bp fragment (nucleotides - ), p / sense ( caatcagcgaggccagtaatc ), and p / antisense ( cttgacgattttgtgccgctc ), which amplifies a -bp fragment (nucleotides - ), with annealing at and • c, respectively (takahashi et al., ) . to amplify rnase l mrna, the following primers were synthesized based on the sequence of human rnase l (genbank accession number nm ): rnase l sense ( agatgaggaaactaaggacctc ), and rnase l antisense ( gaggtttgtttggactgtggg ), which amplifies a -bp fragment (nucleotides - ). the annealing temperature was • c. ␥-actin mrna levels were used to normalize the rt-pcr signal generated by the other mrnas. the primers for ␥-actin were aagtaccccattgagcatggc (sense) and cacagcttctccttgatgtcgc (antisense). the annealing temperature was • c. viral rna in total or cytoplasmic rna samples were amplified using primers ccgtttgcctaggctataggcta (sense) and cagctccatgctaatcatggagt (antisense). this primer pair is directed to the end of the hav genome and can differentiate between the genomes of f and clone strains of hav based on the presence of an additional bases in the ntr of the f genome (goswami et al., ; kulka et al., ) . all pcr amplifications were carried out in a total volume of l, and contained - l of the cdna pool, . mm mgcl , m of each deoxynucleoside triphosphate, pmol of each primer, and u of taq dna polymerase (promega) for - cycles. frhk cells were mock infected or infected with pfu/cell of f, tcid /cell of clone or pfu/cell of coxsackievirus b (cbv ) in mem containing % heat inactivated fbs. after h adsorption period, medium was added to each flask and incubation continued for the indicated time period. for infected cultures treated with z-vad-fmk, the virus adsorption media was removed prior to the addition of mem/ % fbs containing - m inhibitor and subsequently incubated for h. at the indicated times post-treatment, cell culture monolayers were scraped into their media and pelleted by centrifugation ( × g, • c). cell extracts were prepared and the protein content estimated as described previously (kulka et al., ) . equal concentrations of each protein extract were adjusted to equivalent volumes in denaturing sample buffer and heated at • c for - min and subjected to sds-page under reducing conditions, followed by electrophoretic transfer onto nitrocellulose membranes in transfer buffer containing methanol. the membranes were blocked with tbs-t (tris buffered saline/ . % tween ) containing % nonfat dried milk (nfdm) for min at room temperature (rt) and washed four times with tbs-t. membranes were incubated either overnight at • c or h at rt with primary antibody diluted in tbs-t containing either % bsa or % nfdm. primary antibodies were used that recognize (i) stat (phosphorylation state independent) or stat phosphorylated on tyrosine at position (cell signaling technologies), used at a : dilution ( % bsa) for incubation overnight at • c; (ii) full length/large (cleaved) fragment caspase- (cell signaling technologies), used at a : dilution ( % nfdm) for overnight incubation at • c; (iii) full length/large (cleaved) fragment parp (sigma), used at a : dilution ( % bsa) for h incubation at rt, and (iv) ␤-actin (sigma), used at a : dilution ( % nfdm) for h incubation at rt. secondary antibody-hrp conjugates (pierce-endogen) were used at a : , dilution. all other steps including detection were performed as previously described (kulka et al., ) . confluent cultures of frhk cells in -well plates were infected with f at . , , or pfu/cell, as described previously. replication of virus was assessed by the level of viral antigen in each well as described before using the havab eia kit (goswami et al., ; kulka et al., ) . previous investigations have demonstrated that a cp strain of hav, havcyt/hb . , derived from the cc strain hm , induced apoptosis in - % of the cells by h post-infection (pi), increasing to . % by days pi (brack et al., ) . similar slowly developing cpe and apoptosis were reported in cells infected with another cp strain, hm / a (gosert et al., ) . in contrast, cells infected with the f strain develop extensive cpe in approximately % of the cells by h, and an almost total cpe is observed by h (kulka et al., ) . using a colorimetric tunel assay at h pi we investigated whether cpe induced by the f strain is a result of apoptosis (fig. ) . tunel positive cells were present in the f infected monolayers, whereas positively stained cells were only occasionally observed in the absence of virus infection. persistently infected clone cells at days or days pi also showed an occasional positively stained cell. the culture remained viable, however, and f like cpe was not observed until more than a year of weekly passages. immunohistochemical staining was performed on the persistently infected clone cells to determine if the persistently infected status of these cells is due to the lack of actively replicating virus in a majority of the cell population (fig. ) . almost all of the cells showed positive staining for viral antigen. in addition, replication of the virus as measured by eia confirmed the presence of actively replicating virus (data not shown). taken together, these results show that the f strain causes apoptosis in infected cells much earlier than cells infected with other cp strains, and that the lack of apoptosis in persistently infected cells is not due to lack of active virus replication. we have previously shown that rrna degradation is dependent upon replication of f, since treatments that inhibited replication also resulted in inhibition of rrna degradation. however, certain cell lines are sensitive to perturbation of cellular macromolecule synthesis and can be driven to apoptotic cell death by treatment with metabolic inhibitors. in such instances, apoptosis causes s rrna fragmentation (houge et al., ) . therefore, to determine whether rrna degradation is a cause, or the effect of apoptosis in f infection, it was necessary to establish the temporal sequence of these two events. frhk cells were infected with f and total nucleic acids from the cytoplasm were prepared at different times as described in section . as control, cytoplasmic nucleic acids were also isolated from mock infected cells and persistently infected (clone ) cells. nucleic acids were analyzed by agarose gel electrophoresis before and after digestion with rnase (fig. ) . degradation of the s and s molecules into discrete products was fig. . rapid induction of apoptosis in f infected frhk cells. confluent cultures of frhk cells were mock infected or infected with the cytopathic f strain of hav. cells were processed for the detection of tunel positive cells at h pi as described in section . persistently infected cultures following infection by the non-cytopathic hm (clone ) strain of hav were established as described in section . cells were processed for tunel positive cells at days or days post infection. evident at h pi, and by h pi very little intact rrnas could be detected. the appearance of apoptotic dna laddering in the cytoplasm could be detected following elimination of rrnas from total nucleic acid preparations by rnase and clearly lagged behind rrna degradation as a result of f infection (fig. , panel a) . neither mock infected cells nor persistently infected (clone ) cells showed any evidence of rna or dna degradation (fig. , panel b) . no evidence of rna or dna degradation could be seen in nucleic acid preparations from cells infected with f virus, at or h pi (data not shown). the data indicate that rrna degradation in f infected cells occurs prior to apoptosis, and is not a consequence of apoptosis or dna fragmentation. previous studies have shown that rrna degradation in cells over-expressing the ifn regulated - a activated rnase l results in apoptosis due to activation of caspase (rusch et al., ) . caspase is the terminal enzyme in the caspase cascade, and activation of caspase by upstream caspases occurs due to proteolytic cleavage (for a review, see roulston et al., ) . to investigate if a similar activation of caspase takes place due to infection with the f virus, we investigated caspase activation by western blotting experiments (fig. ) . proteolytic cleavage of caspase could be detected in f infected cells at h pi, which coincided with the appearance of a dna ladder in the cytoplasm (compare fig. , panel a with fig. , panel a) . proteolysis of caspase was not detectable at earlier times, nor in persistently infected (clone ) cells (fig. , panel a) , confirming that caspase activation follows rrna degradation (fig. ) . activation of caspase was accompanied by the degradation of parp- (poly adp-ribose polymerase ), a target for caspase during the apoptotic response (kaufmann et al., ) . cleavage of parp- was evident at h pi and coincided with the appearance of activated caspase ; parp was no longer detectable in f infected cells at h pi (fig. , panel b) . commensurate with the lack of caspase activation or rna degradation, there was no cleavage of parp in persistently infected cells. having shown the proteolytic activation of caspase in f infected cells, it was of interest to determine if apoptotic dna degradation was a consequence of caspase activity. frhk cells were infected with f and then treated with different concentrations of the caspase inhibitor z-vad-fmk for h. cultures were processed for the analysis of caspase activation by western blotting as described in section . caspase activation was prevented in a dose dependent manner by treatment with z-vad-fmk (fig. ). significant inhibition of caspase activation was achieved with m z-vad-fmk, and complete inhibition was observed at m of the inhibitor. rna and dna analyses by agarose gel electrophoresis in the cytoplasm of f infected cells were carried out to establish a connection between caspase activation and apoptotic dna laddering (fig. ) . treatment of f infected cells with m z-vad-fmk did not prevent rrna degradation (panel a). the appearance of apoptotic dna laddering in the cytoplasm was completely prevented (panel b). these results show that rrna degradation is independent of caspase induced dna degradation, fig. . effect of the caspase inhibitor z-vad-fmk on proteolytic activation of caspase in response to f infection. cells were infected with f ( pfu/cell), and incubated in growth medium containing - m of the inhibitor for h, prior to protein extraction. electrophoresis, transfer, and immunodetection with antibody against caspase were performed as described in section . the lane marked m contains protein extracts from mock infected cells harvested and processed h following mock infection in the absence of inhibitor. fig. . effect of z-vad-fmk on rrna degradation and apoptotic dna laddering in the cytoplasm of infected cells. cells were infected with f ( or . pfu/cell) or mock infected, and further incubated in growth medium containing m z-vad-fmk for h, prior to isolation of total cytoplasmic nucleic acids. samples were separated in an % agarose gel before (a) or after (b) digestion with rnase . and is not a consequence of apoptosis due to perturbation of cellular macromolecule synthesis as a result of virus infection. we have previously shown that despite rrna degradation, cellular protein synthesis remains unaffected in f infected cells between and h pi (kulka et al., ) . in agreement with the lack of effect of z-vad-fmk on rrna degradation which requires replication of the virus (kulka et al., ) , virus replication was not inhibited by treatment with this compound (table ) . ribosomal rna degradation is one of the three antiviral mechanisms controlled by ifn. it is brought about by the activation of a latent ribonuclease rnase l by - a, and requires ifn treatment of cells prior to virus infection fig. . effect of ifn on rrna degradation and dna ladder formation in response to f infection. cells were either pretreated with u/ml ifn-␤ for h ( h pre) before infection or treated with ifn following virus infection ( h post). at h pi, cytoplasmic nucleic acids were isolated as described in section and analyzed by agarose gel electrophoresis before (a) or after (b) treatment with rnase to assess rna or dna degradation. the bracketed areas indicate the position of the degradation products of s and s rrna. (stark et al., ; barber, ; sen, ) . in contrast, rna degradation or apoptosis induced by f did not require ifn pretreatment of the cells (figs. and ) . to investigate whether ifn is involved in f induced rrna degradation and apoptosis, cells were pretreated with u/ml ifn-␤ (human) for h, prior to exposure to f virus at a moi of or . pfu/cell. dna and rna from the cytoplasm of infected cells were analyzed at h pi as described in section (fig. ) . pretreatment with ifn had a modest inhibitory effect on both rrna degradation and apoptotic dna degradation, particularly at low moi. when ifn was added to the cells following the h virus absorption period, neither rna degradation nor dna laddering was affected by ifn treatment (fig. ) . ifn treatment had a significant growth inhibitory effect on f only at low moi ( . pfu/cell) infection (table ) , and had no effect on virus replication in clone cells over a h treatment period (data not shown). thus, the involvement of the canonical ifn regulated - a system seemed unlikely. the relatively slow replication of even the f strain might allow the infected cells to synthesize and excrete ifn, however, which may stimulate the - a pathway in neighboring uninfected cells causing rna degradation. we addressed this question by two different approaches. first, the level of ifn, - oas and rnase l mrnas were investigated by rt-pcr to determine if any of these rnas were induced following virus infection (fig. ) . in agreement with previous reports that hav infection does not induce ifn, we did not observe significant induction of ifn message up to h pi with f virus (fig. , panel a) . fig. . rt-pcr amplification of selected cellular and viral mrnas. two microgram of rna isolated from virus infected, ifn-␤, or dsrna treated cells were reverse transcribed in a volume of l using oligo(dt) as primer as described in section . five microliter of each rt reaction was amplified for cycles with sense and antisense primers for ifn-␤ (a), - oas (b), ␥-actin (c), rnase l (d), or hav genomic rna (e) as described in section , and l pcr reactions were analyzed by % agarose gel electrophoresis. rna from uninfected cells (ui) and cells transfected in the absence of dsrna (trfn. control) were included in the rt-pcr analyses. takahashi et al. ( ) successfully demonstrated the application of rt-pcr for the detection of mrnas, which encode the - oas isoforms that synthesize the biologically active forms of - a. using these primers, we determined that the mrnas for either the p / form (fig. , panel b) or the p / form (data not shown) of - oas was not induced following virus infection. treatment of the cells with u/ml ifn-␤ induced the message for the / form (panel b), but not the / form, although the primer pair is capable of amplifying this mrna from both mouse and human cells (data not shown). thus, the frhk cells are competent to mount an ifn response by inducing the / form of - oas. rt-pcr experiments with primers for rnase l, on the other hand, revealed that this mrna was present in cells constitutively, with some increase in the level of this mrna following virus infection (panel d). to ascertain that both the f and clone cells were actively replicat- fig. . stat phosphorylation in response to virus infection and/or ifn-␤ treatment. (a) frhk cells were treated with ifn-␤ for or h prior to total protein extraction. (b) frhk cells were mock infected (m), infected with f for various times, or infected with coxsackie b (cbv ) prior to protein extraction. (c) frhk cells were infected with pfu/cell f for h followed by the addition of growth medium. after h, cells were treated with u/ml ifn-␤ in growth medium. nt means not treated with ifn-␤. proteins were extracted after or h of ifn treatment. clone cells were used at d pi. western blotting with an antibody to unphosphorylated stat (anti-stat), phosphorylated stat (anti-stat-p), or actin was performed as described in section . ing virus, viral genomic rna was amplified using primers directed to the ntr (panel e). the results clearly show the presence of undiminished amount of viral genome in clone cells after several months of cultivation, with no indication that the virus has acquired the base repeat present in the genome of f and other cp strains (brack et al., ; zhang et al., ) . second, we determined whether stat phosphorylation occurs following virus infection. phosphorylation of stat on y is indicative of the activation of the jak-stat pathway, and is a prerequisite for the transcriptional induction of isgs by ifn (for a review, see stark et al., ) . to investigate if this signaling pathway is activated following viral infection due to synthesis of low levels of ifn, phosphorylation of stat was investigated by immunoblot analysis (fig. ) . in ifn treated frhk cells, stat phosphorylation was observed as early as min post-treatment (data not shown), and the peak level of phosphorylation was detected at h post-treatment (panel a). these results confirm that frhk cells are capable of responding to ifn (panel a). however, no phosphorylation was detectable following infection with f, up to h pi (panel b), when rrna degradation is occurring. previously, we followed stat phosphorylation for up to h pi and found no evidence of activation of this pathway (kulka et al., ) . surprisingly, cells infected with the f virus for h showed diminished phosphorylation of stat when treated with ifn, compared to uninfected or clone cells treated with ifn (panel c). thus, the cp strains of hav not only interfere with the induction of ifn by dsrna (brack et al., ) , but can also interfere with ifn signaling. similarly, coxsackievirus b , a fast-replicating picornavirus that induces similar rna degradation in frhk cells, did not induce phosphorylation of stat (panel b). the data clearly suggest the existence of a virus inducible, - a independent rna degradative pathway that is activated by f infection in the apparent absence of ifn induction of - oas. in this study, we report a novel pathway for rrna degradation in cytopathogenic hepatitis a virus infected cells. the observed cleavage of both species of rrna suggests that this phenomenon is different from the cleavage of s rrna in mouse hepatitis a virus (a coronavirus) infected cells (banerjee et al., ) . we have also shown that the virus induced rrna cleavage is not a consequence of apoptosis, as has been reported to occur as a result of perturbation of cellular macromolecular synthesis (houge and døskeland, ) . in a previous study, we have shown that the cleavage of rrnas is also accompanied by a reduction in the levels of some cellular mrnas, while viral rna was not significantly affected (kulka et al., ) . the appearance of specific sizes of rrna degradation products is reminiscent of ifn regulated - a activated rnase l. the activation of this pathway, which is part of the cellular defense against viral infection, requires prior exposure of cells to ifn (stark et al., ; barber, ; sen, ) . rna degradation during f infection, however, occurs without prior treatment of the cells with ifn. it is conceivable that a slowly replicating virus such as hav could induce production of low levels of ifn to activate the - a pathway in surrounding uninfected cells. we showed that this is not the case, since the jak/stat pathway was not activated following virus infection, even though ifn-␤ mrna was marginally induced in f and clone infected cells. these results make it unlikely that the canonical - a dependent rna degradation pathway is activated during f infection. we also showed that rna is not degraded in persistently infected cells despite the presence of comparable levels of viral rna and antigens (proteins). our results complement the results of brack et al. ( ) and vallbracht et al. ( ) , that ifn-␤ does not play a major role in hav infection. the rnase l enzyme is one member of a multi-component system for rna degradation, consisting of ifn, - oas (the enzyme that synthesizes - a from atp in the presence of dsrna), and the latent endoribonuclease rnase l. rnase l is generally present in most cells and may function in situations other than virus infections, particularly during differentiation, apoptosis, hormonal regulation, chronic fatigue syndrome, and certain types of prostate cancer (for a review, see silverman, ) . rnase l levels are usually unaffected by virus infection or ifn treatment, but it is activated only in the presence of the proximal activator - a, a series of unique - phosphodiester linked oligoadenylic acid molecules. the synthesis of - a is dependent upon the induction of the enzyme - oas which is activated by dsrna or other activator molecules such as the hepatitis c virus core protein (naganuma et al., ) . we showed that - oas was not induced in f infected cells, despite the occurrence of massive rna degradation. we also showed that the failure to detect - oas mrna was not due to a failure of the rt-pcr protocol or the genetic background of the frhk cells, since treatment of this cell line with ifn-␤ resulted in an induction of the / form of - oas mrna. we also showed that the mrna for rnase l was present in both virus infected and uninfected cells, with some increase in the level of its mrna following virus infection. we conclude that either rnase l may be activated by factors other than - a, or that the enzyme responsible for rna degradation is a novel rnase specifically induced or activated in f and coxsackievirus infected cells. in this regard, ifn treatment has been shown to stimulate the expression of a second ribonuclease, identified as isg (nguyen et al., ) . however, this enzyme is an exonuclease, and the negative effect of ifn treatment on f-induced rna degradation (fig. ) argues against the role of this enzyme. unlike most picornaviruses, persistent infection is the norm rather than the exception for hav, at least in vitro in tissue culture cells. persistent infection by most cc strains of hav has been suggested to be the result of inefficient translation initiation from its ires (whetter et al., ; funkhouser et al., ; yi et al., ) . recent results suggest that hav ires is as efficient as the ires of other picornaviruses in initiating translation, and the switch from translation to negative strand synthesis is the rate-limiting step in hav replication (gauss-müller and kusov, ) . it has also been suggested that the slow replication (even the replication of cp strains are slow compared to other picornaviruses) of hav is a result of down regulation of its replication by the virus itself by unknown mechanisms (de chastonay and siegl, ) . in the discussion of slow replication of hav, the lack of a functional a protease has often been overlooked. picornaviral a protease is responsible for promoting cap-independent translation by inactivating eif g, and possibly other translation factors (ziegler et al., ; kerekatte et al., ) . a protease is also a strong inducer of apoptosis when expressed ectopically (goldstaub et al., ) . in the absence of a functional a protease, hav viral rna has to compete with cellular mrnas for the host cell translation machinery. we suggest that induction of an rnase activity may partially compensate for the absence of an active a protease, by reducing competition from cellular mrnas and contributing to the rapid replication phenotype of f and possibly other cp strains of hav. apoptosis is generally thought to be a host defense response to virus infection (for a review, see barber, ) . premature induction of apoptosis results in abortive virus replication. however, a late induction of apoptosis might favor the virus by allowing it to evade the host immune system. many viruses have thus evolved mechanisms to delay the onset of an apoptotic response (roulston et al., ) . similarly, induction of ifn mediated antiviral pathways is a defensive response of the host to virus infection, and many viruses have evolved mechanisms to block the induction of ifn mediated antiviral pathways (for reviews, see barber, ; sen, ) . it is therefore not surprising that the two major ifn mediated antiviral pathways, namely the - a/rnase l pathway and the dsrna activated pkr pathway, are also mediators of apoptosis. among the picornaviruses, hav is unique in its slow replication in permissive cells, and also in its ability to initiate and maintain a persistent infection. persistent infection is known for other picornaviruses, but usually requires restriction of virus replication and gene expression, through the use of inhibitors in cultured cells and presumably the host ifn regulated system or the immune system in the intact organism (agol et al., ) . moreover, cp strains of hav kill cells by apoptosis in cell culture, while in the whole organism, a role for apoptosis in the destruction of infected cells, or evading the host immune response is uncertain. the late depletion of parp- clearly indicates that hav is capable of avoiding the induction of necrosis in infected cells and evade a host immune response. in conclusion, we present evidence that unlike some other picornaviruses, notably encephalomyocarditis (emc) virus, hav is relatively insensitive to the antiviral effect of ifn. furthermore, the degradation of rrna in hav f infected cells occurs in the absence of - oas, the key enzyme that regulates the - a/rnase l antiviral pathway. the f virus is able to suppress the expression of isgs including - oas by interfering with jak/stat signaling by an unknown mechanism, and it utilizes the degradation of rna as a means to augment its replication and subsequently induce apoptosis in the host cell. competing death programs in poliovirus infected cells: commitment switch in the middle of the infectious cycle cytopathology, 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disease antigenic and genetic variation in cytopathic hepatitis a virus variants arising during persistent infection: evidence for genetic recombination immunofluorescence and immunoperoxidase assays for the titration of infectious hepatitis a virus (hav) activation of the interferon-inducible - -oligoadenylate synthetase gene by hepatitis c virus coat protein production of cytopathology in frhk cells by bs-c- passaged hepatitis a virus the human interferon and estrogen regulated isg /hem gene product degrades single stranded rna and dna in vitro propagation of human hepatitis a virus in cell culture in vitro viruses and apoptosis caspase-dependent apoptosis by - oligoadenylate activation of rnase l is enhanced by ifn-␤ proteinase c of hepatitis a virus (hav) cleaves the hav polyprotein p -p at all sites including vp / a and a/ b viruses and interferons stability of hepatitis a virus implications for rnase l in prostate cancer biology how cells respond to interferons quantitative analysis of p / and p / forms of , -oligoadenylate synthetase mrna by competitive pcr and its clinical application persistent infection of human fibroblasts by hepatitis a virus hepatitis a virus infection and the interferon system liver derived cytotoxic t cells in hepatitis a virus infection isolation and molecular cloning of a fast growing strain of human hepatitis a virus from its double stranded replicative form low efficiency of the nontranslated region of hepatitis a rna in directing cap-independent translation in permissive monkey kidney cells functional significance of the interaction of hepatitis a virus rna with glyceraldehydes -phosphate dehydrogenase gapdh: opposing effects of (gapdh) and polypyrimidine tract binding protein on internal ribosome entry site function an infectious cdna clone of a cytopathic hepatitis a virus: genomic regions associated with rapid replication and cytopathic effect impact of rnase l overexpression on viral and cellular growth and death picornavirus a proteinase-mediated stimulation of internal initiation of translation is dependent on enzymatic activity and the cleavage products of cellular proteins we thank dr. joseph e. leclerc and dr. david reese for critical reading of the manuscript, ms. marcia meltzer for the graphics, and ms. mobolanle ayodeji for her excellent technical assistance. key: cord- - hiiw authors: keshavarz, mohsen; solaymani-mohammadi, farid; namdari, haideh; arjeini, yaser; mousavi, mohammad javad; rezaei, farhad title: metabolic host response and therapeutic approaches to influenza infection date: - - journal: cell mol biol lett doi: . /s - - - sha: doc_id: cord_uid: hiiw based on available metabolomic studies, influenza infection affects a variety of cellular metabolic pathways to ensure an optimal environment for its replication and production of viral particles. following infection, glucose uptake and aerobic glycolysis increase in infected cells continually, which results in higher glucose consumption. the pentose phosphate shunt, as another glucose-consuming pathway, is enhanced by influenza infection to help produce more nucleotides, especially atp. regarding lipid species, following infection, levels of triglycerides, phospholipids, and several lipid derivatives undergo perturbations, some of which are associated with inflammatory responses. also, mitochondrial fatty acid β-oxidation decreases significantly simultaneously with an increase in biosynthesis of fatty acids and membrane lipids. moreover, essential amino acids are demonstrated to decline in infected tissues due to the production of large amounts of viral and cellular proteins. immune responses against influenza infection, on the other hand, could significantly affect metabolic pathways. mainly, interferon (ifn) production following viral infection affects cell function via alteration in amino acid synthesis, membrane composition, and lipid metabolism. understanding metabolic alterations required for influenza virus replication has revealed novel therapeutic methods based on targeted inhibition of these cellular metabolic pathways. influenza virus infection (ivi) is one of the most common infectious agents, capable of infecting a variety of avian and mammalian species. the virus is responsible for seasonal epidemics, leading to - million severe infections and , - , deaths annually [ , ] . despite the annual vaccination program, the high mortality rate caused by influenza infection and its various complications, including chronic lung disease, cardiac disease, asthma, and metabolic disorders, is yet to be adequately addressed [ ] [ ] [ ] . in , eagle et al. first indicated that the addition of glucose to hela cell medium could promote the generation of poliovirus progeny [ ] . results of a published study showed that the replication of the influenza virus depends on host cellular metabolism such that metabolites including nucleic acids, proteins, glycoproteins, and lipids are crucially required for the life cycle of the influenza virus [ ] . recent research on a mouse model showed that influenza infection could affect more than metabolite markers in serum, lung, and bronchoalveolar lavage fluid [ ] . acquiring the required materials from the host cell to self-replicate, the virus can disrupt biochemical processes such as glycolysis, fatty acid (fa) synthesis, and glutamine pathways [ ] . it is of particular importance for scientists to broaden their horizon on the metabolic changes during influenza infection, which in turn paves the way for preventing life-threatening consequences. influenza infection actively provokes the pro-oxidant condition in the host cell to facilitate viral proliferation and pathogenesis. increased expression of influenza m protein can activate protein kinase c and increase reactive oxygen species (ros) production [ ] . on the other hand, pb -f decreases superoxide anion dismutase (sod ) expression and consequently disrupts the ros scavenging process [ ] . in people with influenza infection, increased levels of dna, lipid, and protein oxidation products are found in plasma and urine [ ] [ ] [ ] . also, increased levels of ros and inducible nitric oxide synthase (inos) have been observed as markers of oxidative stress in the lungs of people who died due to pandemic influenza infection [ ] . ros-producing enzymes induced by influenza infection mainly include nadph oxidase (nox) and xanthine oxidase, upregulation of which causes the impaired defensive function of antioxidants [ ] . an increase in ros production, along with impaired antioxidant function, ultimately leads to a profound change in redox homeostasis of the cell [ ] [ ] [ ] [ ] . nox is a phagocytic enzyme that is involved in the production of ros induced by influenza virus [ ] [ ] [ ] [ ] , and impaired nox expression results in a lack of increased rns and ros production following influenza infection [ ] . xanthine oxidase is also an ros-producing enzyme that is induced by influenza infection [ , ] , and its inhibition can hinder ros increase in the cell. on the other hand, increased expression of sod reduces influenza virus titers within the cell [ ] . it is also reported that influenza infection significantly increases ros production by inducing nox , and the proliferation of this virus in lung epithelial cells is dependent on redox-sensitive pathways activated by nox -derived ros [ ] . glutathione (gsh) is a vital antioxidant in the cell, and its cellular content is inversely related to influenza virus replication in the cell [ , ] . it is indicated that higher levels of gsh, antioxidant enzymes such as glutathione peroxidase, and the anti-apoptotic protein bcl- in the lungs of female mice result in superior resistance of these mice to influenza infection. in contrast, male mice are more susceptible to this infection due to higher expression of nox . this difference is due primarily to the higher ability of female mice to maintain cellular redox homeostasis [ ] . amatore et al. also showed that an increase in gsh content in organs by affecting gsh-dependent antiviral pathways strengthens the immune system, in particular th cell response, and decreases viral replication [ ] . however, gsh depletion results in a deviation of the response towards th cells [ ] . furthermore, oxidative stress following infection can induce the transcription factor nf-kb, which subsequently leads to increased levels of inflammatory cytokines, including interleukin (il)- β, il- , ifn, and tnf [ , ] . ifns are one of these cytokines that trigger and affect t cell metabolism via mediating glucose uptake, glycolysis, and lipid synthesis. ifn can also exert its function on metabolic changes by producing several mediators including indoleamine- , -dioxygenase (ido) and nitric oxide (no), both of which appear to have either an inducible or an inhibitory role in viral replication [ ] . since tryptophan is critical for t cell proliferation, depletion of this amino acid by ido suppresses the immune system through the stimulation of t regulatory cells. no, on the other hand, inhibits viral replication via changes in the structure of viral proteins [ ] . in this review, we first discuss the metabolic abnormalities during influenza infection and then shed light on the role of immunometabolites that regulate cellular metabolism. the following sections summarize recent evidence about the novel therapeutic approaches that target metabolic pathways in influenza infection. viruses take advantage of various cellular mechanisms to replicate efficiently. vital metabolic pathways of host cells are one of the most widely used mechanisms targeted by viruses, resulting in considerable changes. in this context, studies have revealed that various human viruses, such as cytomegalovirus [ ] [ ] [ ] , rubella [ , ] , dengue [ ] , mumps [ ] , poliovirus [ , ] and reovirus [ ] can strongly affect host cell glycolysis, lipid metabolism, and glutaminolysis. furthermore, a review by sanchez and lagunoff delineated the activation of these metabolic processes by several viruses [ ] . as will be discussed in this section, influenza as a highly pathogenic human virus interferes tremendously with the host metabolic cycles and thereby forces them to produce viral particles more efficiently. the metabolism and concentration of glucose in the cell play a cardinal role in the homeostasis of cellular metabolic procedures [ ] . shortly after the onset of ivi, the rate of glucose uptake by infected cells increases continually, and the subsequently enhanced glycolysis results in higher glucose consumption, production of viral particles [ , ] , and extracellular concentration of lactate. the relationship between glycolysis and ivi has shown that influenza infection at a higher multiplicity of infection (moi) raises the glycolytic activity of the cells [ ] . in a study by kohio and adamson, a dose-specific increase in influenza infection was associated with higher glucose levels, whereas the treatment of cells with glycolysis inhibitors remarkably suppressed the viral replication. however, the viral infection could be retriggered by adding atp to the cell environment. this study revealed that enhancing vacuolar-type atpase (a proton pump essential for influenza uncoating) via increasing glucose metabolism and, as a result, higher available atp resources, augments the virus infection [ ] . the observations, as mentioned above, reveal a significant increase in atp and glucose consumption within cells following influenza infection and also highlight the dependence of the influenza virus on the glycolysis pathway for energy production. the viral replication has the highest use of atp during influenza infection, releasing large quantities of energy in the form of heat. this process can increase the temperature of infected cells by - °c. as viral proliferation increases, the cellular atp level drops sharply, resulting in reduced potential and stability of the mitochondrial membrane [ ] . based on available results, patients with metabolic disorders can develop more severe influenza infection compared to healthy hosts. there have been several studies showing that diabetes can increase the risk of influenza infection, the severity of the disease, and the fatal consequences of this infection [ ] [ ] [ ] [ ] . about % of patients with type diabetes are overweight, and obesity is a significant risk factor for severe influenza infection [ ] . this reinforcing effect of diabetes on influenza may be due to the inhibitory effect of hyperglycemia on the immune system [ ] [ ] [ ] . it has been shown that this hyperglycemia-associated immunosuppression and susceptibility to influenza infection can be alleviated by insulin administration and diabetes control [ ] . on the other hand, the pentose phosphate pathway (ppp), as another glucose-consuming pathway reported to be enhanced by ivi [ ] , contributes to a higher yield of nucleotides and atp for viral replication [ ] . significant up-regulation of the ppp key enzymes in influenza-infected cells, including glucose -phosphate dehydrogenase (g pd) and -phosphogluconate dehydrogenase ( pgd), was reported by janke et al. [ ] . g pd enzyme is also responsible for the generation of nadph [ ] , a critical component of fatty acid biosynthesis. the level of g pd activity specifies the ability of the cell to clear the accumulated ros. cells with an average g pd level can retain the appropriate gsh/gssg ratio and keep the ros production at a tolerably low level, indicating that g pd activity has an inverse correlation with cellular ros level [ ] . disruption of redox balance has been shown to contribute to replication and virulence of several viruses [ ] [ ] [ ] , and g pd deficiency can cause this disruption. despite the results reported by janke et al., g pd activity seems to have an inverse relation with some other respiratory viral infections. for example, in an in vitro study, after infection with human coronavirus (hcov) e, the production of viral particles in g pd-deficient or g pd-knockdown cells was higher than in healthy cells, and this was correlated with increased oxidant production [ ] . molecular mechanisms through which the virus can control the metabolic pathways have been thoroughly identified. smallwood et al. have shown that an increase in glucose uptake, glycolysis, and glutaminolysis following influenza infection may be related to the loss of pi k/akt/mtor pathway homeostasis and subsequent increase in c-myc expression in the infected cells [ ] . regarding the available results, the mechanistic target of rapamycin complex (mtorc ) and mtorc signaling can be activated by a variety of influenza virus proteins. the viral hemagglutinin (ha) protein, along with virus replication, can upregulate pdpk -mediated phosphorylation and activate akt, which is required for induction of the mtorc signaling pathway by the influenza virus. on the other hand, influenza m protein is capable of down-regulation of the mtorc inhibitor redd , thereby enhancing the mtorc activation [ ] . mtorc signaling, in turn, promotes c-myc expression at the translational level [ ] . additionally, the ns protein can effectively promote the activity of mtorc , which, in turn, upregulates c-myc through foxo inhibition [ ] . moreover, akt-dependent inactivation of foxos can increase glycolysis [ , ] by removing the suppressive force of c-myc [ ] [ ] [ ] . myc enhances glycolysis by upregulating expression of the glucose transporter glut , glycolytic genes, and lactate dehydrogenase (ldh), as the converter of pyruvate to lactate [ , ] . mtorc also mediates upregulation of hypoxia-inducible factor- α (hif- α), a factor that increases the expression of various genes [ ] , including several glycolytic enzymes, glucose transporters, and ldh [ ] [ ] [ ] . akt is able to promote the expression and membrane localization of glut as well as the function of phosphofructokinase [ , ] . furthermore, akt is demonstrated to activate srebp in an mtorc -dependent manner [ ] and to upregulate srebp by enhancing the stability of its processed form [ ] . srebp is shown to be required for the mtorc induced increase in the expression of g pd, which is the rate-limiting enzyme of the ppp oxidative branch [ ] . in addition to the above-mentioned metabolic pathways, the influenza virus exhibits disruptive effects on some other metabolic processes, which gives rise to metabolic disorders and atp crisis. previous studies have found that the influenza infection increases the cellular synthesis of fatty acids [ ] , with some of their derivatives, including eicosanoids [ ] , and these molecules are natural endogenous ligands and stimulators of peroxisome proliferation-activated receptors (ppars) [ ] [ ] [ ] . ppars are a group of nuclear receptor proteins that act as transcription factors and regulate the expression of different genes [ ] involved in cellular differentiation and metabolism of carbohydrates, lipids, and proteins [ ] . furthermore, all types of ppars discovered so far are able to suppress the activity of the pyruvate dehydrogenase (pdh) enzyme (known as a catalyzer of the oxidative decarboxylation of pyruvate leading to acetyl-coa production) in various organs through the upregulation of pyruvate dehydrogenase kinase (pdk)- [ ] . in this respect, extremely low pdh enzyme activity has been found after influenza infection in vitro. enhanced pdk -mediated inhibition of pdh has been found in the lung tissue of influenza-infected mice. this enzyme inhibition contributes to an erroneous process, which causes significant disruption of glucose oxidation, cellular respiration, and lipid metabolism ( fig. ) [ , ] . following infection, levels of phospholipids and several lipid derivatives undergo perturbations. several lines of evidence have shown that in obese mice due to abnormalities in the fig. metabolic changes caused by influenza infection and related mechanisms. several anabolic and catabolic processes can be affected: higher glucose uptake and metabolism in glycolysis and pentose phosphate pathways, higher nucleotide catabolism, increase in biosynthesis of fatty acids including arachidonic acid, the precursor of proinflammatory lipids, and also enhanced glutaminolysis and protein synthesis. activation of mtorc & signaling and downstream factors by influenza infection may have an essential role in the upregulation of these metabolic processes. in addition, high atp consumption and reduced β-oxidation, as well as glucose oxidation by influenza infection, contribute to the atp crisis and hence influenza-related multi-organ failure [ ] . this influenza-mediated elevation of aa, consistent with inflammation, has also been reported in the same study [ ] . the accumulation of the above-mentioned proinflammatory lipids in the cell leads to the promoted synthesis of eicosanoids and inflammatory mediators, thus exacerbating post-infection necrosis, inflammation, and tissue damage in the lungs [ ] . in a prospective cohort study on influenza-infected subjects, lipid inflammatory mediators in serum samples of patients were mainly aa-derived oxylipins, including txb , -deoxy- , -pgd , -hete, , -dhet, -oxoete, lte , and -hpete. although all of these metabolites were shown to be elevated shortly after the infection, , -dhet and -oxoete levels remained considerably high up to weeks postinfection, indicating a constant pulmonary inflammation [ ] . the pulmonary surfactant system, which is involved in suppressing influenza infection in the respiratory tracts [ ] , can be disrupted due to significant influenza-induced changes in the abundance of different types of pc and pe species as the major components of surfactants [ , ] . tanner et al. proposed a principal correlation between influenza replication and choline lipids metabolism. they found an ivi-mediated reduction in ester-linked pc species as well as an increased level of sphingomyelin (sm) [ ] , probably connected with expending cellular choline stores for sm synthesis. this led to an increase in sm species within the infected cell. sm and short-chain fatty acid-containing ether-linked pc (epc) species were found in higher amounts in both infected cells and virions and therefore appeared to be involved in viral morphogenesis. on the other hand, long-chain fatty acidcontaining epc was increased in infected cells while having low levels in the structure of the virion, highlighting the role of this phospholipid in replication of the virus [ ] . evidently, higher production of these complex lipids in the cell will require increased biosynthesis of fatty acids. in this regard, the results of a study revealed that influenza infection could induce fatty acid biosynthesis and cholesterol metabolism in human lung basal epithelial tumor cells [ ] . since srebps are thoroughly identified as stimulators of expression of many genes involved in lipid and sterol biosynthesis, including fatty acid synthase [ ] [ ] [ ] , their upregulation by the influenza virus (through induction of mtorc signaling, as discussed earlier) may logically explain the increased rate of lipogenesis [ ] . a coincidence between increased fatty acid synthesis and a decline in fatty acid β-oxidation has been found during influenza infection, which is attributed to a variety of mechanisms directly or indirectly related to viral replication. for instance, the sharp increase of proinflammatory cytokines during influenza infection [ ] causes decreased hepatic fatty acid β-oxidation both in vitro and in vivo [ , ] , most likely through excessive nitric oxide and other related free radicals [ ] . in addition, increased temperature of cells during infection (which could be the result of virus replication and fever) causes heat stress which in turn can considerably downregulate carnitine palmitoyltransferase ii (cpt ii) activity and reduce the β-oxidation and atp levels in fibroblasts of influenza-associated encephalopathy patients and healthy volunteers [ ] . a study on influenza-infected mice demonstrated a significant depression in long hepatic chain fatty acid β-oxidation at both the mrna and protein level, as several β-oxidation essential enzymes were reduced by > % [ ] . a significant decrease in mitochondrial fatty acid β-oxidation simultaneously with increased biosynthesis of fatty acids and membrane lipids may reflect the fact that the virus stores structural lipids to produce more infectious particles. in addition to impaired metabolism in mitochondria, influenza infection induces severe peroxisomal lipid metabolism disorders, which can be inferred from abnormal levels of several specific long-chain fatty acids [ ] (fig. ) . the aforementioned metabolic processes are not the only pathways affected by influenza virus infection. this virus has the ability to induce higher consumption rates of glutamine during glutaminolysis, which can be attributed to transient c-myc overexpression [ ] . myc acts to regulate glutamine uptake and its utilization in the cell [ ] . it has been demonstrated that catalytic activity of glutaminase, as the key enzyme in glutaminolysis, greatly increases following the infection [ ] . moreover, essential amino acids, especially tryptophan, are other materials whose quantities have been shown to decline in infected tissues [ ] . mtorc can up-regulate protein synthesis through several downstream factors [ ] . thus, induction of mtorc signaling by the influenza virus leads to higher usage of essential amino acid storages for concurrent production of large amounts of viral and cellular proteins. infection of influenza virus can also alter the cellular level and metabolism of purines and pyrimidines [ , , ] , and is associated with both increased activities of nucleotide catabolism core enzymes including adenosine deaminase (ada) and xanthine oxidase (xo) and elevated levels of inosine, hypoxanthine, xanthine, and uric acid in serum and bronchoalveolar lavage fluid. enhanced catabolic degradation of nucleotides and their metabolites can facilitate the production of superoxide and contribute to the pathogenesis of influenza infection [ ] . interferons are well-known cytokines with a powerful capability of altering the cellular functions following viral infections. these alterations affect protein synthesis, composition of the membrane, cellular proliferation, and nutritional status [ ] . interferon stimulated genes (isgs) are the effector components whose transcription could be induced by type i ifns and ifnγ [ , ] . studies have recently underscored the general effect of ifns on the energy metabolism of cells, mostly by promoting glycolysis. for instance, ifnβ has been shown to induce the glucose uptake of embryonic fibroblasts in a pi /akt-dependent manner, thereby increasing atp production [ ] . it has also been demonstrated that type i ifn can stimulate oxygen consumption in a range of cells, including conventional dendritic cells (dcs), keratinocytes, and memory t cells [ ] . indeed, the high yield of atp and mitochondrial fitness guarantee the host cell's need for energy in plasmacytoid dcs (pdcs) and non-hematopoietic cells following challenges with viral pathogens [ ] . these studies emphasized the mediatory effect of type i ifn on glycolysis induction via ifnar , tyk , and stat . it has also been shown that influenza infection stimulates pdcs to enhance their glycolysis and develop a warburg-like remodeling of energy metabolism. this enhanced glycolysis leads to higher ifn production and, consequently, more potent antiviral activity [ ] . ifnγ induces metabolic reprogramming of m macrophages as a rapid increase in aerobic glycolysis, followed by a reduction in oxidative phosphorylation. this metabolic reprogramming maintains cell viability and the inflammatory response while reducing dependence on mitochondrial oxidative metabolism. excessive production of pro-inflammatory cytokines and chemokines in human monocytes/macrophages can be blocked by inhibition of aerobic glycolysis [ ] . also, activation of macrophages by ifnγ induces expression of the atp-citrate lyase enzyme (acly), and blockage of acly activity reduces the production of ros and nitric oxide [ ] . there is a strong consensus that influenza replication is crucially dependent on fatty acids [ ] , which makes it a fascinating target for therapeutic modalities [ ] . thus, the ability of ifn to channel the fas from biosynthesis to catabolism via fatty acid oxidation (fao) is currently known as a promising antiviral strategy in pdcs [ ] , which requires further research for more elucidation. several lines of current evidence have revealed the antiviral activity of type i ifn to be exerted through hampering glucosederived cholesterol and fatty acid synthesis [ , ] . sterol regulatory binding protein (srebp ), along with srebp , is known as the leading transcription factor which orchestrates the biosynthesis pathway of sterol, whose inhibited transcription and expression can be strongly mediated by ifns via ifnar [ ] (fig. ) . [ ] . this recognition can lead to the induction of an inflammatory response that, in turn, controls the replication and spread of the virus [ ] . h n , h n , and h n were correlated with increased transcription of the cytokine response in mice. severe infection with h n , h n , h n , or virus can lead to upregulation of inflammatory cytokine genes along with downregulation of lipid metabolism and coagulation genes [ ] . this uncontrolled proinflammatory response accompanied by an inadequate anti-inflammatory response is referred to as the cytokine storm [ ] . monocytes/macrophages, neutrophils, and lung epithelial cells have useful roles in the cytokine storm developed by influenza infection [ ] . severe cytokine storm, with greater levels of interferons and tumor necrosis factors, has been recognized in patients hospitalized due to influenza infection [ ] . such influenza-induced cytokine storms, together with viral virulence, can develop severe lung injury in patients [ , ] . it is believed that the level of the cytokine storm is directly associated with the severity of the disease caused by influenza infection [ , ] . some specific polymorphisms in immune system genes have determinative roles in the outcome of influenza infection. our previous studies have shown a relationship between cytokine gene polymorphisms and severity of the influenza disease. several cytokines were evaluated after influenza a/h n virus infection, among which il- rs gg and ag, gg and gt of il- (rs ) and il- (rs ) genotype tt polymorphisms were associated with increased risk of influenza infection. in contrast, il- β (rs ) (gg) and il- (rs ) gg and tg genotypes were associated with reduced risk of infection [ ] . in another study, an association between il- β rs and il- rs genotypes and severe influenza disease was found while il- rs and il- rs polymorphisms were not associated with influenza disease. also, lacking an a allele in il- rs could increase the risk of influenza a (h n ) infection [ ] . such polymorphisms in immune system genes may be associated with some metabolic changes and, in turn, may reinforce the metabolic disorders following influenza infection. however, additional studies are needed in this field to confirm or reject this opinion. ifns are known to be capable of depletion of polyamines to limit virus replication. polyamines are small ornithine-derived polycationic molecules which encompass three molecules: putrescine, spermidine, and spermine. spermidine and spermine depletion is one of the compelling mechanisms through which ifns produce their antiviral effects on the replication of rna viruses. mechanistically speaking, polyamines appear to play a pivotal role in the processes of rna transcription and protein translation of viruses, making them a promising target to combat viral infections [ ] . l-tryptophan is one of the nine essential amino acids with a remarkable role in immunosuppression and tolerance and is also essential in protein, kynurenine, and serotonin synthesis [ ] . ido is an intracellular enzyme that induces production of kynurenine from l-tryptophan, thereby acting to deplete tryptophan and modulate the immune system following viral infections [ ] . having two ifn-stimulated response elements and three ifnγ-activated sites in the promoter of ido, ifnγ acts as the most powerful inducer of ido expression [ ] . ido has also been shown to be expressed during influenza infection [ ] . dendritic cells, macrophages, and epithelial cells can express ido [ , ] , and since the primary target for replication of influenza is primarily found to be respiratory epithelial cells, understanding the role of ido during influenza infection is of particular importance. there exists a coincidence of peak ido and ifn-k expression during influenza infection. also, mouse lung airways considerably express ifn type i and iii following infection with influenza [ ] . these findings emphasize that there is upregulated expression and enhanced function of ido during influenza infection, which is found to be induced by ifn-i. moreover, ifn-i is thought to signal the adjacent cells via ifn-ir and stimulate them to produce ido [ ] . nonetheless, the ifn-mediated ido induction during influenza infection generally has undesirable consequences and establishes immune tolerance [ ] . indeed, an inhibitory effect of tryptophan depletion on t cell responses has been confirmed. also, ido induces kynurenine derivation from tryptophan, leading to stimulation of regulatory t cells [ ] . nowadays, ido is hypothesized to be part of the "metabolic, immune regulation," which plays a protective role in immune responses and inhibits the overreaction of these responses against influenza infection. a pleiotropic role has been attributed to ido during infections, which gives rise to the opposing outcomes (fig. ) [ ] . research on the role of ido in influenza infection has been mainly focused on the murine models of influenza infection, emphasizing the increased ido activity and its maximum expression correlated with increased lymphocyte numbers in the respiratory tract [ ] . nitric oxide (no) is a gaseous free radical with accessible vasodilatory and microbicidal functions [ ] . however, the antiviral effect of no has also been documented, leading to reduced viral load and more efficient clearance of infection [ ] . nitrosylation of viral molecules has been offered as an antiviral mechanism employed by no [ ] . also, no synthase-mediated generation of no leads to the depletion of l-arginine, thereby reducing the level of polyamines. thus, ifn-induced nos represents antiviral activities, and this, in turn, may exhibit another mechanism through which ifns hinder viral infections such as influenza [ ] . despite existing data regarding the antiviral activity of no, many studies have considered no as a double-edged sword with both pathogenic and viricidal effects. the role of no in the pathogenesis of pneumonia caused by influenza virus infection has been described in mice. the ifnγ response induces greatly increased levels of inos in the lungs of infected mice, leading to the production of a significant amount of no and peroxynitrite species, which are among the most important pathogenic factors in influenza virus-induced pneumonia in mice [ ] . uetani et al. also observed overexpression of the inos gene in human airway epithelial cells induced by influenza a virus infection [ ] . in addition, no produced by phagocytic cells has antiviral activity that is simultaneous with nonspecific damage of host cells and viral pathogenesis [ ] . in a survey by nin et al. on pandemic a/h n influenza infection, all cases showed increased levels of inos protein, tyrosine nitration, and oxygen free radicals, indicating the production of peroxynitrite. their results revealed the involvement of oxidative and nitrative stress in the pathogenesis of h n influenza virus-induced acute respiratory distress syndrome (ards) [ ] . influenza-induced cytokines such as ifnγ stimulate no release from human airway epithelial cells [ ] [ ] [ ] . as mentioned previously, the influenza infection induces upregulation of hif- α. interestingly hif- α-knockout macrophages show decreased expression of inos after ifnγ stimulation [ ] , indicating the possible involvement of hif- α in influenza pathogenesis. it has been shown that infection of h n and viruses induces higher levels of no in mice compared to the seasonal h n virus and, as a result, they develop more intense pathogenic outcomes, while mice with inos deficiency showed reduced morbidity, mortality, and cytokine production in the lungs following h n and virus infection. also, systemic administration of nos inhibitor could postpone weight loss and death among virus-infected mice [ ] . in another survey, the delivery of no to influenza-infected mice could not improve the lung infection and survival of mice, indicating that no administration was not a suitable treatment strategy for influenza although this was probably due to the difficulty of determining concentrations of no that are both viricidal and safe in host airways [ ] . also, it is reported that no released from s-nitroso-n-acetylpenicillamine (snap) reduces the replication of influenza virus in a dose-dependent manner. the production of no in airway epithelial cells can lead to antiviral rather than harmful effects following influenza infection provided that its production is precisely controlled [ ] . since the influenza virus affects about % of the world population annually, preventive and therapeutic approaches require much closer attention. therapeutic drugs for influenza infection fall into three groups: ) neuraminidase inhibitors (zanamivir, oseltamivir, laninamivir, peramivir), ) m inhibitors (rimantadine and amantadine), and ) polymerase inhibitors (favipiravir) [ , ] . antiviral drug resistance has recently emerged as a global problem which can bring about a remarkable financial and social burden [ ] . therefore, further research is urgently needed to develop novel and promising antiviral drugs. many of the metabolic pathways in influenza infections are increasingly changing, dampening of which appears to hamper the virus replication. one of the newly developed strategies aiming to hinder influenza infection is targeting metabolic pathways and restoration of hemostasis in cells ( table ). the pi k/mtor signaling pathway has been shown to play a pivotal role in a variety of cellular pathways, including proliferation and nutrient uptake, and its activation increases the glucose uptake through the up-regulation of cell surface glucose transporter [ ] . bez alters glucose metabolism via blockage of the pi k/mtor pathway, and some clinical trials are underway to assess this strategy in cancer therapy (smith et al., ). on the other hand, several lines of evidence have demonstrated that sirna targets the pi k-akt-mtor pathway, thereby warding off influenza infection [ ] . in a new study by smallwood et al., it was found that although bez did not interfere with the early stages of the infection, it could finally reduce the viral progeny and result in prolonged survival in mice challenged by the influenza virus. indeed, bez induced hemostasis in the pi k/mtor pathway via phosphorylation of p and e-bp and through reconstitution of metabolic status, which was already altered by the virus [ ] . it has been found that there is an elevated level of pdk in lung, liver, and heart during influenza infection, while the levels of atp and pdh, a key enzyme in the regulation of glucose, lipid and atp levels in human cells, are shown to be reduced [ ] . furthermore, dichloroacetate (dca) is a pyruvate dehydrogenase kinase inhibitor with anti-tumor activity in a variety of carcinomas. studies have also indicated that diisopropylamine dichloroacetate (dada) could ameliorate the metabolism of hepatocytes in chronic liver disease [ ] . in a study by yamane et al. attempting to evaluate the effect of dada on influenza-infected mice (pr ), oral administration of dada was found to not only restore the activity of pdh and atp in affected organs but also suppress cytokine storm and viral replication [ ] . sterols are intermediate metabolites that play an essential role in a broad spectrum of biological pathways, including inflammation. research has shown that interferon production following the immune response in viral infection regulates sterol production paths. blanc et al. revealed that sterol metabolism pathway regulators such as simvastatin, zometa (zoledronic acid), and fpt inhibitor iii could effectively hinder h n influenza replication and cytokine production, which makes them promising therapeutic candidates in acute patients [ , ] . on the other hand, as mentioned earlier, srebps are transcription factors that have a critical role in the process of lipogenesis. studies have shown that these factors can play a variety of roles, such as energy supply and post-translational protein modification, as well as in the propagation of various groups of viruses such as influenza viruses. a study has shown that the am compound, which is a retinoid derivative, inhibits srebp-linked pathways, and it has antiviral activity against influenza a and coronavirus in vitro and in vivo [ ] . concerning the fact that mitochondria and glycolysis are two sources of energy production, they play vital roles in the regulation of innate immunity responses. during the immune system response, and especially the cytokine storm, following influenza infection, atp synthesis in the mitochondria decreases, leading to weakened innate immune responses (dengbing yao). studies have revealed that traditional herbal medicines have an important role in improving influenza-like symptoms in infected patients. results of a study demonstrated that pre-treatment of infected cells with hochuekkito (a traditional japanese herbal medicine) could activate both mitochondrial and glycolytic energy metabolism and thereby intensify symptoms [ ] . also, the effects of traditional chinese medicine (modified jiu wei qiang huo) on h n infected mice were evaluated in another study. the results showed that this herbal medicine could ameliorate weight loss and inflammatory mediators in infected mice through the regulation of amino acid, fatty acid, and arachidonic acid pathways [ ] . based on recent studies, influenza virus infection can interfere with cellular metabolic pathways either directly or indirectly via stimulation of immune system mediators. through enhancing the activity of the mtorc complex, the influenza virus strengthens several metabolic pathways, including glycolysis, glutaminolysis, pentose phosphate, and fatty acid synthesis, to provide more atp and structural materials for viral replication. on the other hand, β-oxidation suppression following viral infection can help to supply essential fatty acids for the synthesis of structural lipids. however, exhausting cellular atp resources due to virus replication, as well as an increase in pro-inflammatory lipid synthesis, will ultimately lead to irreversible cell damage. innate immune responses following influenza infection play a crucial role in metabolic alterations. ifn is one of these mediators that acts on several metabolites such as ido and no and thereby affects lipid and amino acid pathways. since the drug resistance in influenza infection is a global concern, research on designing novel therapeutic modalities to tackle pandemics is of particular importance. thus, a clear understanding of the metabolic alterations during influenza infection would be tremendously helpful for therapeutic purposes. induction of protective immune response to intranasal administration of influenza virus-like particles in a mouse model association of polymorphisms in inflammatory cytokines encoding genes with severe cases of influenza a/h n and b in an iranian population acute 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pluripotency and cell fate determination inhibition of influenza a virus replication by antagonism of a pi k-akt-mtor pathway member identified by gene-trap insertional mutagenesis superior anti-tumor efficacy of diisopropylamine dichloroacetate compared with dichloroacetate in a subcutaneous transplantation breast tumor model key: cord- -bjqwwzam authors: zhang, lei; wang, hao; zhang, yi-qing title: against ebola: type i interferon guard risk and mesenchymal stromal cell combat sepsis date: - - journal: journal of zhejiang university-science b doi: . /jzus.b sha: doc_id: cord_uid: bjqwwzam the ebola outbreak in west africa triggered a global crisis. nine countries have reported more than infection cases in total and nearly lives have been lost. the actual death toll is likely much higher than this figure; the death rate is as high as %, considering confirmed cases. the ebola virus launches its destruction by shutting down the host’s innate and adaptive immune systems. the virus then replicates itself out of control and causes a cytokine storm in the host. consequently, the host’s overdriven immune system attacks its own endothelial cells and this leads to multiple organ hemorrhagic damage, the host dies of septic shock finally. under current circumstances where no specific interventions have shown effectiveness against the virus, our opinions are justified in applying a non-specific anti-viral approach during the incubation period of virus infection as an essential protection to put the host’s immune system into an alert state and henceforth to slow down the viral replication. when the viral infection proceeds to the terminal stage, the key factor would be applying a non-specific immune modulation approach to suppress the cytokine storm that causes multiple organ failure, in an attempt to open a time window for the host’s immune system to recover. the ebola outbreak in west africa triggered a global crisis. nine countries have reported more than infection cases in total and nearly lives have been lost. the actual death toll is likely much higher than this figure; the death rate is as high as %, considering confirmed cases. the ebola virus launches its destruction by shutting down the host's innate and adaptive immune systems. the virus then replicates itself out of control and causes a cytokine storm in the host. consequently, the host's overdriven immune system attacks its own endothelial cells and this leads to multiple organ hemorrhagic damage, the host dies of septic shock finally. under current circumstances where no specific interventions have shown effectiveness against the virus, our opinions are justified in applying a non-specific anti-viral approach during the incubation period of virus infection as an essential protection to put the host's immune system into an alert state and henceforth to slow down the viral replication. when the viral infection proceeds to the terminal stage, the key factor would be applying a non-specific immune modulation approach to suppress the cytokine storm that causes multiple organ failure, in an attempt to open a time window for the host's immune system to recover. the ebola virus invades antigen presenting cells (apcs) quietly and turns their alarm system off essentially, so that the immune system remains inactivated toward the virus. the virus then grows uncontrollably and invades many organs. eventually, many premature cells start dying and exploding, and these cells release all their contents, including signal molecules, into the blood. the signals eventually trigger the extreme immune attacks, which cause arteries, veins and capillaries to leak blood and plasma. the host's body temperature drops and blood pressure falls, causing the host to go into a severe septic shock. it has been shown that the ebola virus primarily targets macrophages and dendritic cells (dcs); later, the membrane-associated glycoprotein (gp) in the virus can bind to endothelial cells, causing cell death and vascular permeability. the latter allows the virus to spread to vital organs and execute severe and extensive damage to the body. the symptom of bleeding appears when the virus gains entry into hepatocytes and causes liver failure. although neutrophils, lymphocytes, and natural killer (nk) cells appear to remain uninfected, those cells undergo 'bystander apoptosis', presumably induced by inflammatory mediators and/or the loss of support signals from dcs. dissemination to regional lymph nodes results in further rounds of virus replication, followed by a spread through the bloodstream to dcs, fixed and mobile macrophages in the liver, spleen, thymus, and other lymphoid tissues, resulting in an extensive tissue necrosis (mohamadzadeh et al., ; ansari, ) (fig. ). the ebola virus applies multiple physical and biological mechanisms to evade host innate and acquired humoral and cellular immune responses. interferon (ifn) belongs to the cytokines that are used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. inhibition of the ifn alarm seems to be one of the most important aspects in the pathogenesis of ebola. the virus primarily antagonizes both the ifn-α and ifn-β responses in target cells, especially macrophages, monocytes, and dcs, by utilizing the viral protein vp to block the phosphorylation of the ifn regulatory factor (irf ), which acts as a transcription factor for the ifn production (cárdenas et al., ) , whereas vp to block the ifn-mediated antiviral response (xu et al., ) . macrophages play a critical role in nonspecific defense, and they also help to initiate specific defense mechanisms by recruiting other immune cells such as lymphocytes. massive infection of macrophages and related cells by the ebola virus indicates that it is able to block or evade the cells' innate antiviral mechanisms. the nk cells respond in an antigen-independent manner to viral infections and kill infected cells. nk cell numbers dramatically drop during the course of infection and almost all undergo apoptosis. dcs have a crucial role in both innate and adaptive immunity; however, infected dcs do not produce proinflammatory cytokines or costimulatory molecules, their ability to support t-cell proliferation is thus impaired and the infected dcs undergo anomalous maturation (hoenen et al., ) . impaired apcs function and lymphocyte apoptosis contribute to the failure of specific immune responsiveness. ebola triggers the systemic dysregulation of immunity that likely results in an uncontrolled virus replication. macrophage-produced pro-inflammatory cytokines are the key players of the host defense system against pathogens. in most patients, ebola viral burden elevates by time and triggers an extremely strong immune attack-a phenomenon called 'cytokine storm' (sullivan et al., ) , during which monocytes and/or macrophages produce a massive amount of pro-inflammatory cytokines, including tumor necrosis factor-α (tnf-α) and interleukins (ils), and this figure was created in reference to mohamadzadeh et al. ( ) in combination with our own considerations. nk: natural killer; tnf: tumor necrosis factor; il: interleukin; m-csf: macrophage colony-stimulating factor; mip: macrophage inflammatory protein; mcp: macrophage/monocyte chemotactic protein; no: nitric oxide; sgp: soluble virus glycoprotein chemokines, such as macrophage inflammatory protein α/β (mip- α/β), macrophage/monocyte chemotactic protein- (mcp- ), macrophage colonystimulating factor (m-csf), nitric oxide (no), and eotaxin (baize et al., ) . by now, the body response to ebola infection would be too severe and too late, and the cytokine storm leads to exaggerated inflammatory responses that contribute to lymphoid cell apoptosis and sepsis (ansari, ) . the virus eventually disables the vascular system and causes blood leakage combined with massive viremia and intravascular coagulopathy. the terminal stage of the virus infection usually includes diffusive bleeding and hypotensive shock, which would eventually kill the patients. ebola infection is generally composed of the incubation period, the symptomatic/acute stage, and the convalescent/terminal stage. the virus burden, inflammatory response, and specific antibodies are the main contributors to different outcomes: mortality, survival, or symptomless infection (fig. ) , suggesting that the appropriate intervention strategy in each stage would accordingly be able to control the ebola virus. whether an inflammatory response executes protective or damaging effects depends not only on the specific cytokine profile, but also on a delicate balance between the individual host immune response and the incoming virus (sullivan et al., ) . some patients have a delayed and prolonged inflammatory response that leads to a cytokine storm characterized by extremely high circulating levels of numerous pro-inflammatory cytokines; these patients generally cannot survive in the viral infection (misasi and sullivan, ) . the average levels of the proinflammatory cytokines in these patients' blood have been found to be - times higher than those in the patients who recovered from the viral infection (wauquier et al., ; ansari, ) . survivors tend to have a short-lived, balanced pro-and anti-inflammatory responses characterized by the presence of il- β, il- , and tnf-α in the early clinical course (bray and mahanty, ) , but no ifn-α was detected in either survivors or nonsurvivors (wauquier et al., ) . some viral infection cases are asymptomatic. an immediate, proper inflammatory response is critical to these cases, which is characterized by transient high levels of il- β, il- , tnf-α, mcp- , and mip- α/β in plasma within d of the first putative infectious contact (baxter, ; leroy et al., ; baize et al., ) . it is believed that in these cases, viral replications are suppressed by effective inflammatory responses. why some infected patients can develop a proper inflammatory response, whereas the others cannot, remains unclear. nonetheless, this phenomenon does give clues to the treatment against ebola virus disease (evd)-early activated innate immune responses may prevent the viral infection. prompt and appropriate adaptive immune responses to ebola seem to be important to the resolution of infection. both virus-specific humoral and cellular mechanisms are required for clearing the viral infection; the humoral immunity is more important at the acute stage for halting viral spread, whereas the cellular immunity is more important for eliminating virus-infected cells that could continuously serve as a source of virus (ansari, ) . fatal cases have been found to associate with a defective humoral response without specific igg production, in these cases only low levels of specific igm were detected in % of the patients (baize et al., ; ksiazek et al., ) . survivors were generally associated more significantly with the early emergence of specific humoral responses and regulated activation of cytotoxic cells that coincided with clearance of viral antigens from the blood. survivors produced specific igm as early as in the first d of symptoms onset and specific igg in - d (baize et al., ; ksiazek et al., ) . asymptomatic individuals produced specific igm and igg within - weeks after initial exposure to viral sources, these antibody productions reached only moderate levels one month later (leroy et al., ) . the outcome of an early viral load is unclear as the viral titers in the plasma were undetectable during the incubation period, whereas the virus load was similar between fatalities and survivors during the first days of the symptom stage. typically, million or more copies of the ebola virus per milliliter in plasma can be reached as early as two days after the onset of symptoms in fatal cases. the level of circulating antigens keeps rising until death. moreover, the virus load at or near the time of death can be -fold greater in fatal cases in comparison with nonfatal cases (schieffelin et al., ) . survivors' plasma often contained fewer than copies of the virus per milliliter of plasma. the level of circulating antigens began declining d after the onset of symptoms and dropped to an undetectable level when the patients recovered (baize et al., ; hoenen et al., ) . the viral load was the lowest in asymptomatic individuals, but the virus rna was detectable in these subjects for up to three weeks after initial exposure (leroy et al., ; baize et al., ) . ebola virus, but they undergo massive 'bystander apoptosis' (hoenen et al., ) . a strong depletion of both cd + and cd + lymphocytes and plasma cells was found in fatal cases (geisbert et al., ) , followed by extensive intravascular apoptosis, vascular dysfunction, and loss of endothelial barrier function, which kill the patients (baize et al., ) . in the symptomatic stage, the supportive care is mainly toward aggressive prevention of intravascular volume depletion, correction of profound electrolyte abnormalities, and prevention of the shock complications (fowler et al., ) . there is neither precaution for the incubation period nor effective medication for the terminal stage. we propose that a preventative antiviral intervention for the incubation period may lower the consequent virus burden. furthermore, we propose that an immunomodulatory strategy for the terminal stage may reduce the damages caused by the cytokine storm, thus prolonging the survival time of the patients, making it possible for the patients' adoptive immunity to recover and beat the infection. type i ifns are cytokines that are secreted by infected cells. they induce cell-intrinsic antiviral states in infected and neighbor cells, they also modulate innate immune responses in a balanced manner and activate the adaptive immune system (le bon and tough, ; ivashkiv and donlin, ) (fig. ) . this figure was created in reference to ivashkiv and donlin ( ) in combination with our own considerations ifn: interferon; dc: dendritic cell; isg: interferon-stimulated gene type i ifns have a broad spectrum of antiviral capability, which is able to fight most virus infections. to give an example, the recombinant ifn-α b has been shown to have a significant nonspecific inhibition of herpes simplex virus, influenza virus, and severe acute respiratory syndrome (sars) coronary virus replication (cao et al., ) . the ebola virus blocks the production of type i ifn by apcs including dcs and macrophages; it also blocks the ifn-mediated antiviral response by virus proteins. it is a crucial mechanism whereby the viruses evade attacks from the host immune system directly. however, recombinant ifn-α b ( iu/ml) can suppress ebola replication by -folds in vero cells in vitro, early treatment of ebola-infected cynomolgus with recombinant ifn-α b delayed onset of viremia and death by several days (jahrling et al., ) . in addition, ifn-β treatment was associated with reducing the plasma and tissue viral burden; it thus significantly increased survival time in macaques infected with the ebola virus in vivo (smith et al., ) . collectively, these results indicate that type i ifn may have therapeutic potential: it is reasonable to postulate direct effects on viral replication as well as adaptive immune response. the observation that the virus titers were not detectable in the incubation period indicates that the vast majority of cells were not infected by the ebola virus. based on this fact, we suggest that exogenous administration of type i ifn may induce uninfected cells into an antiviral state. type i ifn might limit the spread of the ebola virus and prolong survival if administered immediately after exposure to ebola viruses. some patients indeed recovered from the ebola infection without receiving specific interventions despite ebola infection's high mortality rate. this fact suggests that appropriate immune responses can help the body heal itself. the outcome highly depends on two cellular-level competing mechanisms: the apoptosis of endothelial cells executed by autoimmune attacks and vascular regeneration by stem cells. interestingly, the patients under the age of years had a lower fatality rate of %, whereas the fatality rate was up to % in those over the age of years, between these two age groups, the fatality rate was % (schieffelin et al., ) . stem cells are the essential for the regenerative processes of almost all tissues and organs. the significant differences indicate that patients' cellular-level healing capacity in different age groups may be relevant to the consumption of age-related changes in the stem cell reservoir. we suggest that treatment with mesenchymal stromal cells (mscs) during the terminal stage of evd should be considered. mscs are mesoderm-origin, multipotent cells that exist in many tissues and are capable of differentiating into several different cell types, and the numbers or potential of msc populations in adult organs decline during aging (stolzing et al., ; toledano et al., ) . after exogenous administration, mscs migrate to injured tissue sites where they can inhibit the release of pro-inflammatory cytokines and promote the survival of damaged cells. mscs operate through a variety of effector mechanisms on key cells of the innate and adaptive immune systems, mostly through manipulating the cell cycle or inducing maturation arrest without apoptosis (tyndall and pistoia, ) . the therapeutic effects of mscs may depend largely on the capacity of mscs to regulate inflammation and tissue homeostasis via an array of immunosuppressive factors, cytokines, growth factors, and differentiation factors. mscs reduce inflammation by shutting down the tnf-α pathway for immune cell activation and prevent a cytokine storm by inhibiting or disabling t-cell response. mscs reprogram macrophages, neutrophils, nk cells, dcs, t lymphocytes, and b lymphocytes, all of which counteract sepsis (plock et al., ) (fig. ) . mscs can specifically communicate with the inflammatory microenvironment and this immunoregulatory function of mscs is highly plastic . while stimulating tissue repair by mitogenic and angiogenic effects, mscs inhibit ongoing inflammation, apoptosis, and later fibrosis of injured tissue, and support endothelial cell growth and blood vessel repair; this strategy can help avoid the abuse of steroid hormones and various sequelae. it has been reported that graft-versus-host disease (gvhd), systemic lupus erythematosus (sle), and sepsis can be successfully treated by mscs (kebriaei et al., ; sun et al., ; wannemuehler et al., ; pedrazza et al., ) . it would be interesting to use the available nonhuman primate models of evd to test such a therapeutic hypothesis. it has been almost years since the first ebola outbreak in . to date, there are no effective therapeutic or prophylactic interventions available to prevent this infection. several experimental interventions are in early stages of development, and their availability is limited and intermittent (zhang and wang, ) . positive results were observed in several cases where zmapp and tkm-ebola were administrated, but it was unclear whether the positive outcome was due to the drugs or due to better supportive care in western countries than that of africa. clinical trial on an ebola vaccine developed by merck and newlink has been suspended due to unexpected side effects recently. meanwhile, médecins sans frontières (msf) has selected three existing interventions for clinical trials, they are favipiravir approved in japan for treating influenza, brincidofivir approved in the usa for virus treatment, and ebola convalescent serum, and they might all be present less of a supply challenge or are already approved for other purposes. however, preliminary data show that their potency against the ebola virus is limited. the outlook for the development of ebola therapeutics is not optimistic. there are many different kinds of viruses. these viruses mutate their dna/rna signatures and evolve rapidly. there has been no specific therapy for even the most common human viral infection such as hbv, hcv, hiv, hpv, and avian influenza. realistically, the non-specific treatment is an essential strategy against viral diseases in the foreseeable future. when our immune system is given sufficient time for intentional activation when we are exposed to deadly plock et al. ( ) in combination with our own considerations. nk: natural killer; il: interleukin; pge : prostaglandin e ; ido: indoleamine , -dioxygenase; shla-g : soluble human leukocyte antigen-g ; tnfr: tumor necrosis factor receptor; ifn: interferon; egf: epidermal growth factor viruses, there is a good chance that it can gear up and eliminate the viruses by itself. clinical features and pathobiology of ebolavirus infection defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in ebola virus-infected patients inflammatory responses in ebola virus-infected patients symptomless infection with ebola virus ebola hemorrhagic fever and septic shock antivirus evaluation and clinical evaluation of recombinant human interferon α b spray ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling caring for critically ill patients with ebola virus disease. perspectives from west africa apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses ebola virus: unravelling pathogenesis to combat a deadly disease regulation of type i interferon responses evaluation of immune globulin and recombinant interferon-α b for treatment of experimental ebola virus infections adult human mesenchymal stem cells added to corticosteroid therapy for the treatment of acute graft-versus-host disease clinical virology of ebola hemorrhagic fever (ehf): virus, virus antigen, and igg and igm antibody findings among ehf patients in kikwit, democratic republic of the congo links between innate and adaptive immunity via type i interferon human asymptomatic ebola infection and strong inflammatory response early immune responses accompanying human asymptomatic ebola infections camouflage and misdirection: the full-on assault of ebola virus disease how ebola and marburg viruses battle the immune system mesenchymal stem cells decrease splenocytes apoptosis in a sepsis experimental model perspectives on the use of mesenchymal stem cells in vascularized composite allotransplantation clinical illness and outcomes in patients with ebola in sierra leone interferon-β therapy prolongs survival in rhesus macaque models of ebola and marburg hemorrhagic fever age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies ebola virus pathogenesis: implications for vaccines and therapies mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans the let- -imp axis regulates ageing of the drosophila testis stem-cell niche mesenchymal stem cells combat sepsis plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications advances in mesenchymal stem cell research in sepsis human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis ebola virus vp targets a unique nls binding site on karyopherin alpha to selectively compete with nuclear import of phosphorylated stat forty years of the war against ebola we thank paul leufkens of neurophyxia b.v. (the netherlands) for critical reading of the manuscript. lei zhang, hao wang, and yi-qing zhang declare that they have no conflict of interest.this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord- -ov bf ff authors: janowicz, anna; caporale, marco; shaw, andrew; gulletta, salvatore; di gialleonardo, luigina; ratinier, maxime; palmarini, massimo title: multiple genome segments determine virulence of bluetongue virus serotype date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: ov bf ff bluetongue virus (btv) causes bluetongue, a major hemorrhagic disease of ruminants. in order to investigate the molecular determinants of btv virulence, we used a btv strain minimally passaged in tissue culture (termed btv (l) in this study) and a derivative strain passaged extensively in tissue culture (btv (h)) in in vitro and in vivo studies. btv (l) was pathogenic in both ifnar(−/−) mice and in sheep, while btv (h) was attenuated in both species. to identify genetic changes which led to btv (h) attenuation, we generated reassortants between btv (l) and btv (h). we found that partial attenuation of btv (l) in ifnar(−/−) mice was achieved by simply replacing genomic segment (seg , encoding vp ) or seg (encoding ns ) with the btv (h) homologous segments. fully attenuated viruses required at least two genome segments from btv (h), including seg with either seg (encoding vp ), seg (encoding vp and ns ), or seg (encoding ns ). conversely, full reversion of virulence of btv (h) required at least five genomic segments of btv (l). we also demonstrated that btv (h) acquired an increased affinity for glycosaminoglycan receptors during passaging in cell culture due to mutations in its vp protein. replication of btv (h) was relatively poor in interferon (ifn)-competent primary ovine endothelial cells compared to replication of btv (l), and this phenotype was determined by several viral genomic segments, including seg and seg . this study demonstrated that multiple viral proteins contribute to btv virulence. vp and ns are primary determinants of btv pathogenesis, but vp , vp , vp , vp , and vp also contribute to virulence. importance bluetongue is one of the major infectious diseases of ruminants, and it is listed as a notifiable disease by the world organization for animal health (oie). the clinical outcome of btv infection varies considerably and depends on environmental and host- and virus-specific factors. over the years, btv serotypes/strains with various degrees of virulence (including nonpathogenic strains) have been described in different geographical locations. however, no data are available to correlate the btv genotype to virulence. this study shows that btv virulence is determined by different viral genomic segments. the data obtained will help to characterize thoroughly the pathogenesis of bluetongue. the possibility to determine the pathogenicity of virus isolates on the basis of their genome sequences will help in the design of control strategies that fit the risk posed by new emerging btv strains. b luetongue is one of the major infectious diseases of ruminants and is caused by bluetongue virus (btv), an arbovirus transmitted by culicoides spp. ( ) ( ) ( ) . btv is a member of the orbivirus genus within the reoviridae. the double-stranded rna (dsrna) genome of btv is composed of segments encoding seven structural (vp to vp ) and four nonstructural (ns to ns ) proteins ( , ) . the btv virion is an icosahedral particle assembled in a triple layer of capsid shells ( , ) . the viral core contains linear dsrna genomic segments associated with replication complexes formed by three minor proteins, vp (rna-dependent rna polymerase), vp (capping enzyme including methyltransferase), and vp (rna-dependent atpase and helicase) enclosed by layers of vp (subcore) and vp (core) ( , , , ) . the outer capsid is formed by two structural proteins, vp and vp , that are responsible for cell attachment and entry ( ) ( ) ( ) . ns , the largest of the nonstructural proteins, forms tubules in the cytoplasm of btv-infected cells and is a positive regulator of viral protein synthesis ( ) ( ) ( ) . ns is a major component of viral inclusion bodies (vib) which are the sites of btv morphogenesis and rna replication ( ) ( ) ( ) . ns , the only btv glycoprotein, is involved in virus exit from infected cells and is thought to play a role in counteracting the innate immune response of the host cell ( ) ( ) ( ) . the recently discovered ns is the smallest of the btv proteins, and it also confers a replication advantage in cells in an antiviral state ( ) . at present, there are different btv serotypes circulating worldwide. the serotype is determined predominantly by vp , which is the most variable of btv proteins and a main target of neutralizing antibodies ( ) ( ) ( ) ( ) . bluetongue is remarkably variable in its clinical manifestations, which can range from asymptomatic infection to a lethal hemorrhagic fever ( , ( ) ( ) ( ) . this variability is due to several factors related both to the infected host and the virus ( , ( ) ( ) ( ) ( ) ( ) . over the years, btv has been used extensively as a model to study the orbivirus replication cycle, structural biology, and interaction with the host cell. however, we have little understanding of the molecular determinants of btv virulence. the northern european strain of btv was the cause of one of the largest outbreaks in the history of bluetongue. the virus emerged in in an area between the netherlands, belgium, and northern germany and then spread throughout the continent, causing high mortality in naive sheep flocks and, occasionally, also clinical disease in cattle ( ) ( ) ( ) . interestingly, no clinical signs were detected in sentinel sheep when the virus reached northern italy and sardinia in . experimental infections with the northern european strain of btv (btv net ) and the italian strain of btv (btv it ) confirmed a marked reduction in the virulence of the latter virus ( ) . a comparison of the consensus sequences of both viruses showed nucleotide differences between the two strains, resulting in eight amino acid residue substitutions in vp , vp , vp , vp , ns , and ns . genetic drift occurring during natural transmission cycles plays a significant role in the diversification of btv strains and their pathogenicity. similarly, in vitro passage of btv was shown to have an impact on virulence in vivo ( ) ( ) ( ) . in particular, strains isolated from severe clinical cases and consequently adapted to mammalian tissue culture have been reported to have reduced virulence in experimentally infected animals ( , ) . interestingly, a decrease in pathogenicity was shown to occur even in minimally passaged strains and was attributed to a genetic bottleneck that occurs after a single passage in mammalian cells rather than to mutations in the consensus sequence ( ) . conversely, live attenuated vaccines and tissue culture-adapted strains of btv with a history of multiple passages in vitro show increased accumulation of nucleotide substitutions correlating with increasing numbers of passages in mammalian cells ( ) . genomic segments , , and (seg , seg , and seg , respectively, encoding vp , vp , and ns ) were shown to be consistently mutated in high-passagenumber strains of btv , btv , and btv viruses and to be attenuated in murine models of bluetongue ( ) . here, in order to determine the roles played by specific btv genomic segments in virus adaptation to tissue culture and attenuation in vivo, we compared genetic and phenotypic differences between btv net minimally passaged in tissue culture, a derivative of this strain extensively passaged in cell culture, and reassortants between the two viruses. cell lines. all cell cultures were grown at °c in % co humidified atmosphere. bsr cells (a variant of bhk- cells; kindly provided by karl-claus conzelmann) were propagated in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) and % penicillin-streptomycin (p-s). transfections in bsr cells were performed in dmem with reduced fbs and no antibiotics. cpt-tert cells ( ) are sheep choroid plexus cells immortalized with simian virus (sv ) t antigen and human telomerase reverse transcriptase. cpt-tert cells were propagated in iscove's modified dulbecco's medium (imdm) supplemented with % fbs and % p-s. the cho cell line is derived from adult chinese hamster ovary, and pgsa- (atcc number crl- ) is a cho-derived cell line deficient in xylotransferase that does not produce glycosaminoglycans (gags). both cell lines were propagated in ham's f- medium supplemented with % fbs and % p-s. primary endothelial cell cultures. primary ovine aortic endothelial cells (ovec) were isolated directly from aortas harvested from euthanized animals as recently described ( ) . cells were maintained at °c in % co and % o for a maximum of three passages. virus strains. btv net (pirbright reference collection number net / ) was originally isolated from a naturally infected sheep during the outbreak in northern europe and has been previously described ( , ) . in this study, we refer to this virus as btv l . the subscript "l" is used to indicate that this virus has a low number of passages in cultures. btv h (h, high passage number) was obtained by serial passages of the btv l strain in bsr cells, followed by plaque purification. reverse genetics. plasmids used for the rescue of btv l (resulting in rgbtv l ) were described previously ( , ) . plasmids used for the rescue of btv h by reverse genetics (resulting in rgbtv h ) were derived by site-directed mutagenesis of nonsynonymous sites in btv l . reassortants between rgbtv l and rgbtv h are denoted with the name of virus forming the backbone (either btv l or btv h ) followed by the substituted segment marked with the "l" or "h" subscript to indicate the virus of origin. for example, btv l ϩs h is a reassortant containing the rgbtv l backbone with seg from rgbtv h . rescued versions of both btv l and btv h (rgbtv l and rgbtv h , respectively) and reassortants between both parental viruses were obtained using established procedures for btv reverse genetics ( , , ) . virus titrations and replication curves. titers of viral stocks were determined by standard plaque assays in cpt-tert cells as previously described ( , ) . virus replication in cpt-tert cells and ovec was assessed by infecting cells at a multiplicity of infection (moi) of . and collecting supernatants at , , , and h postinfection (p.i.). to compare replication kinetics of selected viruses in cho and pgsa- cells, infections were performed at an moi of . , and supernatants were collected only at h p.i. supernatants were centrifuged for min at ϫ g to remove cell debris and then titrated by endpoint dilution assays. titers were expressed as the log % tissue culture infective doses/ml (tcid /ml). all virus titration experiments were performed at least three times independently. statistical calculations were carried out using graphpad prism. btv genome sequencing. the full genome sequence of btv h was obtained using the illumina platform. bsr cells were infected, and total rna was extracted using trizol reagent (invitrogen). single-stranded rna was precipitated using lithium chloride, and double-stranded rna was harvested from the supernatant by precipitation with isopropanol in the presence of sodium acetate. double-stranded rna was used as a template for full-length amplification of cdna (flac) by reverse transcription-pcr (rt-pcr) using established methods ( ) . samples were analyzed using an illumina genome analyser. the libraries were constructed from the pcr samples using a truseq dna sample preparation kit (illumina) according to the manufacturer's instructions. briefly, the dna samples were fragmented, the fragment was end repaired, and the = ends were adenylated. after adaptor ligation steps, the fragments were purified by size selection on agarose gel, and the fragments containing adaptors on the = and = ends were enriched by pcr. sequencing was performed on a gaiix sequencer (illumina) according to the manufacturer's protocol. genomes were assembled using maq software ( ) with btv l used as a reference sequence. the assemblies were manually curated using tablet for sequence visualization ( ) , and consensus sequences were generated as fasta files. in vivo experiments. animal experiments were carried out at the istituto zooprofilattico sperimentale dell'abruzzo e del molise g. caporale (teramo, italy) in accordance with locally and nationally approved protocols regulating animal experimental use (protocol no. / ). experiments in sheep were conducted in an insect-proof isolation unit with veterinary care. prior to the experiments the animals were confirmed to be seronegative for btv using a blocking enzyme-linked immunosorbent assay (elisa), as described previously ( ) . groups (n ϭ animals per group) of dorset poll sheep were infected intradermally with a total of ϫ pfu (in ml) of btv l and btv h by multiple inoculations in the inner leg and in the prescapular areas. control animals were inoculated with ml of mock-infected cell supernatant. blood samples were collected from all animals daily for the first days and at , , , and days p.i. and then examined for the presence of viremia by quantitative rt-pcr (qrt-pcr). sera were tested by virus neutralization assay for the presence of btv-specific antibodies in the samples collected at days p.i. all sheep were examined for the presence of clinical signs and fever, starting week prior to inoculation and continuing for days p.i., with further observations on days , , , and p.i. clinical signs were scored using a clinical reaction index (cri) as previously described ( ) . transgenic mice deficient in type i interferon (ifn) receptor ( sv ifnar Ϫ/Ϫ ) were maintained at biosafety level . for each experiment, groups of adult mice matched for sex and age (n ϭ per group) were infected intraperitoneally or subcutaneously with pfu or , pfu of virus or mock-infected cell culture medium as a control. mice were examined for clinical signs daily until the experiment was concluded at days p.i. serum neutralization assays. the presence of neutralizing antibodies was assessed by neutralization assays as previously described ( , ) . qrt-pcr. levels of viremia in infected sheep were assessed by qrt-pcr as described before ( ) . red blood cells were lysed with water for min on ice and centrifuged at °c for min at , ϫ g. armored west nile rna (asurage, usa) was spiked into each sample as an internal control for nucleic acid extraction. total rna was extracted using a high pure nucleic acid extraction kit (roche, nutley, nj) according to the manufacturer's instructions. for all samples, ng of total rna was amplified by one-step qrt-pcr using primers and probes for btv seg . armored rna and ␤-actin were amplified as control reactions. levels of glyceraldehyde- -phosphate dehydrogenase (gapdh), ␤-actin, ifn-␤, mx , and rsad expression in infected ovec were measured by qrt-pcr. briefly, cells were seeded in -well plates and infected h afterwards with rgbtv l , rgbtv h , or selected reassortants (moi of ). the medium was replaced after h, and the cells were incubated for a further h at °c. next, supernatants were collected, and monolayers were directly lysed in . ml of trizol (life technologies). phase separation was performed according to the manufacturer's instructions, whereupon the aqueous phase was mixed with ethanol and purified using an rneasy minikit (qiagen), including an rnase free/dnase set on-column dnase treatment step. residual contaminating genomic dna was removed using a turbo dna-free kit (ambion) according to the manufacturer's conditions. reverse transcription was performed using ng of rna using random hexamers and superscript iii (life technologies) for h at °c. quantitative pcr (qpcr) was performed using brilliant iii ultra-fast qpcr mastermix reagents (agilent) and in-house designed primers/probes (sequences available upon request) targeting ovine gapdh, ␤-actin, ifn-␤, mx , and rsad . samples were run on an mx p pcr machine with rgbtv l -infected cells set as a calibrator. gapdh was used as normalizing gene against which fold induction was determined for ifn-␤, mx , rsad , and ␤-actin. ifn protection assays. measurement of ifn levels in cell supernatants was based on methods described previously ( , ) . briefly, ovec were seeded in -well plates and infected after days at an moi of with selected viruses. medium was replaced after h of incubation, and supernatants were collected h after infection and treated for min with uv light to inactivate infectious virus. cpt-tert cells were seeded in -well plates, and h later -fold serial dilutions of uv-treated supernatants or serial dilutions of a known concentration of universal interferon (uifn) were added to the cells. after h of incubation, supernatants were removed, and cells were infected with encephalomyocarditis virus (emcv) and incubated for further h. cells were then inspected for emcvinduced cytopathic effect. the levels of ifn in supernatants collected from btv-infected ovec were calculated based on the number of wells protected from emcv-induced cell death compared to those of the uifn control wells. cell protection by pretreatment with uifn was performed using cpt-tert cells. cells were seeded in -well plates and h later treated with , units of uifn. after h of incubation, uifn was removed, and cells were infected with selected viruses at an moi of . . in parallel, untreated cpt-tert cells were infected with the same set of viruses. inocula were replaced by fresh tissue culture medium . h later. at h p.i. supernatants were collected, and viral titers were determined by endpoint dilution assay and expressed as the log tcid /ml. phenotype of tissue culture-adapted btv . btv l was serially passaged times in bsr cells and plaque purified. the resulting virus, btv h , and the parental btv l were then titrated in sheep cpt-tert cells and in primary ovine endothelial cells (ovec). cpt-tert cells are unable to initiate an ifn response following virus infection ( , ) , while ovec are ifn competent ( ) . both viruses replicated very efficiently in ifn-deficient cpt-tert cells, but btv h reached titers approximately -fold higher than its parental virus (fig. a) . both viruses produced lower yields in ovec than in cpt-tert cells, and btv l replicated better than btv h in these cells (fig. b) . next, we determined the pathogenicity of btv l and btv h in ifnar Ϫ/Ϫ mice. btv l was highly virulent in this mouse model, inducing % mortality within days p.i., whereas btv h was completely attenuated (fig. c ). next, we assessed the virulence of btv l and btv h in sheep, the natural host of btv infection, in order to characterize further the phenotype of these viruses in vivo. sheep infected with btv l showed fever and general clinical signs, including depression and mild respiratory distress. viremia was detected in all btv l -infected animals (n ϭ ) from approximately day p.i. until the end of the experiment at day p.i. on the other hand, sheep infected with btv h did not show any clinical signs of bluetongue or viremia ( fig. d and e) . as expected, both btv l -and btv h -infected sheep developed neutralizing antibodies although the antibody titers were lower in the btv h -infected animals (fig. f) . genotype and phenotype of rgbtv l and rgbtv h . the data obtained so far indicate that btv h had accumulated mutations that affected virus replication in ifn-competent cells and resulted in attenuation in vivo both in ifnar Ϫ/Ϫ mice and in sheep. fullgenome sequencing of btv h revealed nucleotide mismatches compared to the sequence of btv l , out of which resulted in amino acid residue substitutions ( fig. a) . nonsynonymous mutations were present in each of the genomic segments and affected all characterized btv proteins with the exception of ns . in order to identify btv genomic segments affecting virulence, we rescued both btv l and btv h by reverse genetics. we reconstructed btv h by substituting the nucleotide mutations of btv h into the btv l vectors by site-directed mutagenesis. hence, we rescued rgbtv l with the same nucleotide sequence as the original virus (btv l ) while rgbtv h was identical to the original high-passage-number virus at the amino acid level but did not contain the silent mutations present in the genome of btv h . we then inoculated ifnar Ϫ/Ϫ mice with rgbtv l and rgbtv h in order to test whether the phenotypes of the rescued viruses were identical to those of the parental btv l and btv h . groups of five mice were inoculated intraperitoneally with pfu of each virus and monitored over a -day period (fig. b) . all mice infected with rgbtv h survived the challenge while rgbtv l caused % mortality. these data showed that both rescued viruses retained the phenotypes of the original viruses. pathogenicity of reassortants between rgbtv l and rgbtv h . in order to define the genomic segments carrying attenuating mutations, we rescued in total reassortants. a set of reassortants was based upon the btv l backbone with a single genomic segment or a combination of segments from btv h . four monoreassortants showed reduced mortality in ifnar Ϫ/Ϫ mice (fig. a) . in particular, btv l ϩs h and btv l ϩs h caused no mortality in mice at an inoculation dose of pfu and caused % mortality with , pfu. full attenuation using both inoculation doses was achieved with double reassortants btv l ϩs / h (which contains seg and seg of btv h ), btv l ϩs / h , and btv l ϩs / h . these data suggest that vp and ns primarily, in addition to vp and vp , were major factors of btv h attenuation in this mouse model. we next aimed to identify the minimal number of btv l segments that would restore full virulence to a reassortant based upon the btv h backbone (fig. b) . none of the single or double reassortants was virulent in ifnar Ϫ/Ϫ mice. furthermore, btv h ϩs / / / l also possessed an attenuated phenotype. these data suggested that other viral proteins besides vp , vp , vp , and ns play a role in the virulence of btv l . to achieve % mouse mortality at an inoculation dose of either or , pfu, it was required that at least five proteins of btv h be replaced by the btv l equivalents, including either vp or vp in conjunction with the aforementioned vp , vp , vp , and ns (fig. b) . replication kinetics of btv l monoreassortants in cpt-tert cells. as shown above, the replacement of specific genome segments of btv l with the homologous segments from btv h resulted in attenuation in vivo. to establish whether the replication kinetics of these reassortants were compromised in vitro, we assessed the full set of monoreassortants containing the btv l backbone in cpt-tert cells (fig. a) . overall, of the monoreassortants displayed similar replication kinetics to those of the parental rgbtv l . remarkably, btv l ϩs h showed replication kinetics essentially identical to those of rgbtv h and reached over -fold higher titers than rgbtv l at h p.i. both rgbtv h and btv l ϩs h produced larger plaques than those induced by rgbtv l and all the other monoreassortants (fig. b) . overall, these data indicated that vp was the sole determinant of increased replication efficiency of btv h in vitro in cpt-tert cells. cells cultured in vitro tend to display increased expression of glycosaminoglycans (gags) at the cell membrane. interestingly some viruses, like foot and mouth disease virus, ( ) show an increased affinity for heparan sulfate after being passaged in vitro. we hypothesized that vp might have acquired higher affinity for gags during passage in tissue culture, given that vp is the main determinant of btv serotype and mediates viral entry ( , , ) . therefore, we performed viral replication kinetic assays in cho cells and in a derived cell line (pgsa- ) deficient in xylotransferase and thus lacking heparan sulfate gags. rgbtv l and a btv h reassortant with the vp of btv l (btv h ϩs l ) grew equally well in both cell lines. however, rgbtv h and btv l ϩs h reached approximately -fold higher titers in cho cells (p Ͻ . ) (fig. a) and induced a more pronounced cytopathic effect (fig. b ) than rgbtv l and btv h ϩs l , respectively. hence, btv h vp has a higher affinity for gags and facilitates btv replication in vitro but not in vivo. replication of rgbtv l , rgbtv h , and btv monoreassortants in ifn-competent ovec. btv h does not replicate efficiently in ifn-competent primary ovec. thus, we further explored this model in order to characterize the interplay between rgbtv l /rgbtv h genomic segments and the cell-autonomous innate immune system. first, we compared the replication kinetics of btv l and the btv h monoreassortants in ovec (fig. ). btv l ϩs h reached higher titers in culture than the other viruses tested, which suggested that mutations in vp h conferred an advantage to the replication of this reassortant in vitro, irrespective of the ability of the cell to produce ifn. several reassortants demonstrated delayed growth compared to that of rgbtv l . in particular, btv l ϩs h and btv l ϩs h yielded substantially lower titers than the parental btv l at and h p.i. (fig. ) . none of the monoreassortants, however, replicated as poorly as rgbtv h , which indicated that the growth restriction of this virus was the cumulative result of mutations in several of its genome segments. next, we wanted to establish whether btv h was a more potent ifn inducer than the parental btv l . to this end, we measured ifn production in the supernatants of ovec infected with the same set of viruses as above. rgbtv h and most of the monoreassortants induced similar amounts of ifn. however, statistically significant differences (p Ͻ . ) were observed between rgbtv l and btv l ϩs h (fig. a) . we also assessed the relative quantities of the ifn-␤ mrna, selected ifn-stimulated gene (isg) mrnas (mx and rsad ) , and ␤-actin in ovec infected with either rgbtv h , rgbtv l , or one of the various monoreassortants as above. mock-infected cells and cells treated with universal ifn (uifn) were used as controls. we detected no ifn-␤ mrna in either of the control samples, while it was readily detectable at similar levels in cells infected with rgbtv l , rgbtv h , and the rgbtv l /rgbtv h monoreassortants (fig. b ). mx and rsad were also upregulated in virusinfected cells, but no significant differences were found between cells infected with the various monoreassortants and the parental viruses (fig. b) . ␤-actin levels were consistently uniform in all samples analyzed. effect of ifn pretreatment on the replication of rgbtv l , rgbtv h , and rgbtv l /rgbtv h monoreassortants. the data described above, showed that the reduced replication kinetics of rgbtv h , btv l ϩs h , and btv l ϩs h in ovec were not due to increased ifn induction in these cells. in light of these data, we wanted to establish whether rgbtv l , rgbtv h , and the rgbtv l /rgbtv h reassortants were better or worse equipped to overcome restriction in cells already in an antiviral state prior to infection. for these experiments, we used cpt-tert cells as they do not produce ifn but can respond to ifn treatment ( , ) . we pretreated cpt-tert with either , units of uifn or control medium for h prior to infection with rgbtv l , rgbtv h , and monoreassortants with a btv l backbone at an moi of . . at h p.i. supernatants of infected cells were collected and titrated by endpoint dilution analysis. comparison of viral yields in cells treated and untreated with uifn showed that the replication of all viruses was inhibited by at least -fold by uifn (p Ͻ . ) (fig. ) . the reduction in yield of rgbtv l in uifn-treated cells compared to that in untreated cells was approximately ( ϫ )fold. strikingly, this ratio was more than a millionfold ( . ϫ ) for rgbtv h . in uifn-treated cells, most reassortants reached yields similar to those obtained by rgbtv l under the same conditions. a notable exception was btv l ϩs h , which had yields -fold lower than those of rgbtv l (p Ͻ . ) in the uifntreated samples. btv l ϩs h showed the highest degree of inhibition in treated cpt-tert cells among all monoreassortants. however, the yield of btv l ϩs h in treated cells was equivalent to the one obtained by rgbtv l under the same conditions. several btv incursions have occurred in europe in the last years. some strains, such as the northern european btv strain used in this study, have caused major animal health and economic problems while others did not cause any clinical signs in the field ( , ) . although the concept of btv serotype/strain-related virulence is often quoted in the literature, only a few studies have attempted to understand what the viral molecular determinants that under- score this property are ( , , ) . in this study, we aimed to map the molecular determinants of virulence of the northern european strain of btv . we found that nonsynonymous mutations in four segments (seg , - , - , and - ) encoding vp , vp , vp , and ns contributed to the attenuation of btv in vivo in ifnar Ϫ/Ϫ mice. reassortants where segments encoding the vp or ns of the attenuated rgbtv h were used in the context of the virulent rgbtv l backbone caused no mortality in mice inoculated at the lower dose ( pfu) used in this study. the combination of seg with either seg , seg , or seg of rgbtv h in the context of the rgbtv l backbone resulted in a fully attenuated reassortant that was nonpathogenic at either the low or high doses. the multigenic nature of btv attenuation has been previously suggested by us as we showed that tissue culture-attenuated strains of different btv serotypes that show reduced virulence in ifnar Ϫ/Ϫ mice tend to accumulate mutations consistently in segments , , and ( ) . interestingly, seg with either seg , seg , or seg of rgbtv l did not restore virulence in the rgbtv h backbone. the fact that a degree of virulence in rgbtv h was achieved only after combining seg with seg , seg , and seg of rgbtv l might suggest that interactions between specific viral proteins (or genomic segments) could play a role in the pathogenicity of btv. both vp and vp have previously been shown to interact with vp trimers in btv virions ( ) . additionally, other studies demonstrated the importance of ns interactions with outer capsid proteins in virus trafficking and assembly ( , ) . it is therefore possible that while mutations in high-passage-number vp or vp did not directly affect the functions of these proteins, they did influence virus virulence through their interactions with vp and ns . interestingly, two mutations found in vp of btv h (positions and ) were located in the same region that was previously identified as being associated with attenuated btv strains and as a target for neutralizing antibodies ( , , ) . this external and highly exposed area of vp was also implicated in attachment to a host cell receptor, and the mutations that arise in this region could be due to the changes in affinity for binding to specific ligands ( ) . cryo-electron microscopy showed that vp possesses a sialic acid binding region located in its hub domain, which is one of two sites suggested to interact with the cell surface receptor ( ) . an increased affinity for gags has often been cited in the context of tissue culture-adapted strains ( , ( ) ( ) ( ) . here, we found that btv h or reassortants with the vp of btv h had, indeed, a greater affinity for gags and were attenuated in ifnar Ϫ/Ϫ mice. previous studies have shown that viruses acquir-ing mutations conferring a higher affinity for heparan sulfate proteoglycans are often attenuated in vivo ( ) ( ) ( ) ( ) ( ) . btv is a potent inducer of type ifn in vivo and in vitro ( , , ( ) ( ) ( ) ( ) . ifnar Ϫ/Ϫ mice, due to the lack of expression of the type ifn receptor, are a suitable tool to study determinants of viral virulence unrelated to ifn expression ( , , ) . however, it is most likely that other viral factors that are used to counteract the ifn system contribute to btv virulence. pathogenicity studies of multiple reassortants in sheep are not feasible; hence, we used primary ovec as an in vitro surrogate model to identify the genome segments that confer higher sensitivity to the cell-autonomous ifn response. btv h (and its derivative rescued by reverse genetics) replicated poorly in primary cells. these data showed that the ifn response played a role in the inhibition of btv h replication. both btv l and btv h induced similar amounts of ifn in infected primary cells. these data suggest that primary ovec sense and respond to infection with both viruses in a similar manner but that btv l was able to counteract this response better than btv h . most of the btv l monoreassortants containing btv h genomic segments were able to replicate to similar titers as the parental low-passage-number virus in primary cells. two notable exceptions included btv l ϩs h and btv l ϩs h . however, the vp (encoded by seg ) of btv h also influenced the viral phenotype in ifnar Ϫ/Ϫ mice, suggesting that mutations in this protein could also have affected its functions unrelated to the type i ifn response. vp of btv acts as a capping enzyme, and therefore the mutations in vp h and, in particular, the mutation in the amino acid residue at position of the predicted =-o-methyltransferase ( = omtase) domain could have an adverse effect on the efficiency of viral mrna capping ( , ) . moreover, recent studies have shown that viral mrna that lacks =-o-methylation at its = cap structure induces a more potent innate immune response through mda activation or direct interactions with proteins members of the ifit (interferon-inducible protein with tetratricopeptide repeats) family ( ) ( ) ( ) . an inefficient capping mechanism would therefore explain the slight, yet consistent, decrease in virus yields reached by btv l ϩs h in ifn-deficient cpt-tert cells and the more pronounced growth inhibition in ovec. this hypothesis was further supported by comparing the growth kinetics of monoreassortants in the btv l backbone in cpt-tert cells either untreated or pretreated with uifn. of all reassortants, btv l ϩs h showed the lowest yields in uifn-pretreated cells, which suggested that vp h was one of the proteins contributing to the inability of rgbtv h to replicate in cells primed with ifn. these data are in concordance with other studies showing that west nile virus, coronaviruses, and poxviruses mutants with deficient = omtase activity were not able to escape ifit- induced restriction in cells stably expressing ifit- ( ) . it is therefore possible that through viral mrna capping, the vp of btv could play a role in evading host restriction factors in order to allow the virus to replicate in host cells in an antiviral state. btv l ϩs h also replicated less efficiently than the parental virus in primary endothelial cells. vp is encoded by seg of the btv genome, as is ns , which is a nonstructural protein shown to counteract the ifn system ( ; unpublished data). we found no mutations in the ns open reading frame, but we detected a nonsynonymous mutation in vp (rna-dependent atpase and helicase). we are not aware of viral helicases being involved in interactions with the innate immune system. we noticed a slight reduction in the replication kinetics of btv l ϩs h (compared to those of btv l ) in cpt-tert cells. therefore, this reassortant might possess a suboptimal replication capacity that is amplified in the ifn-competent ovec. it is important to stress that although the btv monoreassortants displayed an array of intermediate growth patterns in ovec, none of them replicated as poorly as rgbtv h . this indicated that mutations in segments other than seg and seg contributed to the restricted replication of the high-passage-number virus. altogether, these data reinforce the concept that passaging viruses in an environment with no constraints from the ifn system allows greater flexibility of the viral genome, which in turn leads to the emergence of viruses with optimal replication efficiencies. the mutations that arise under such conditions might not necessarily involve major ifn antagonists but can involve proteins that are normally fine-tuned to evoke a minimal immune response while allowing sufficient (yet suboptimal) transmission in the natural host ( ) . a recent study looking into interserotype determinants of btv pathogenesis in sheep showed that replacement of btv seg , - , and - with homologous btv segments resulted in reassortants that showed a similar phenotype in sheep as btv ( ) . however, this study was performed using btv and btv serotypes that are both virulent in sheep. consequently, a clear distinction between levels of pathogenicity of the parental viruses and their reassortants was difficult to assess. in conclusion, our data show that multiple genome segments determine the virulence of btv . our study reinforces the concept that a constellation of genome segments determines the virulence of viruses with a segmented genome, as suggested in other studies in rotaviruses, influenza viruses, and bunyaviruses ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . vp and ns were found to be primary determinants of btv pathogenesis although vp , vp , vp , vp , and vp were also found to contribute to virulence. given the high diversity of btv, it is possible that different determinants of pathogenicity will be found in other serotypes. more studies aimed to map the molecular determinants of btv virulence, using different strains/serotypes and experimental systems, will help to build an intellectual framework to characterize thoroughly btv pathogenesis. the possibility to determine the pathogenicity of viral isolates on the bases of their genome sequences could help in designing control strategies that fit the risk posed by new emerging viruses. isolation of the bluetongue virus from texas sheep-culicoides shown to be a vector the pathology and pathogenesis of bluetongue bluetongue virus assembly and morphogenesis identification and characterization of a novel non-structural protein of bluetongue 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are grateful to mariana varela for useful suggestions. key: cord- -v l o va authors: li, yang; wilson, heather l.; kiss-toth, endre title: regulating sting in health and disease date: - - journal: j inflamm (lond) doi: . /s - - - sha: doc_id: cord_uid: v l o va the presence of cytosolic double-stranded dna molecules can trigger multiple innate immune signalling pathways which converge on the activation of an er-resident innate immune adaptor named “stimulator of interferon genes (sting)”. sting has been found to mediate type i interferon response downstream of cyclic dinucleotides and a number of dna and rna inducing signalling pathway. in addition to its physiological function, a rapidly increasing body of literature highlights the role for sting in human disease where variants of the sting proteins, as well as dysregulated sting signalling, have been implicated in a number of inflammatory diseases. this review will summarise the recent structural and functional findings of sting, and discuss how sting research has promoted the development of novel therapeutic approaches and experimental tools to improve treatment of tumour and autoimmune diseases. cellular stresses or infections lead to the release of dna molecules into the cytoplasm which may threaten the stability of the host genome [ ] . the intracellular appearance of naked dna molecules triggers a doublestranded (ds)dna sensing mechanism which consequently induces innate immune responses including the production and release of type i interferons (ifn-i). this response is central to the resolution of dna-induced cellular stress [ ] [ ] [ ] and prevents the emergence of autoimmunity [ , ] . a recently described protein named sting (stimulator of interferon genes, also known as tmem , eris, mita and mpys) is a critical regulator of these innate immune responses [ ] [ ] [ ] [ ] . sting is an endoplasmic reticulum (er)-resident transmembrane protein and was first recognised as part of the er translocon system [ , ] . suppression of components of the translocon-associated protein (trap) complex such as trap-β and sec β have been found to impair dna-induced type i interferon (ifn-i) signals downstream of sting [ ] . the trap complex has been shown to be involved in two of the er's major responses: protein n-glycosylation [ ] and endoplasmic reticulum-associated degradation (erad) [ ] . although the functional relevance of sting in the er translocon system has not yet been fully elucidated, it has been proposed that sting can interact with trap component ssr /trapβ to enable its migration from the er to perinuclear membranes, a process key to ifn-β promoter activation [ , ] . recent reports have demonstrated that cytoplasmic dna released by microbes and viruses can trigger dsdna-sensing pathways which activate sting [ , [ ] [ ] [ ] . sting then signals to the tank binding kinase (tbk ) / interferon regulatory factor- (irf ) axis to upregulate type i interferon production [ , ] . as an ikk (iκb kinase) -related kinase, tbk can also interact with iκb kinases to induce phosphorylation and thus degradation of iκb, thereby liberating nf-κb (nuclear factor kappa b) subunits allowing their nuclear translocation resulting in upregulation of type i interferon and other pro-inflammatory cytokines such as il- (interleukin- ), cxcl (c-x-c motif chemokine ), ccl (c-c motif chemokine ligand ) and ccl [ ] . table summarises the pathogens that have been shown to activate sting [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . of note, sting knockout mice generated by ishikawa and barber were highly susceptible to infection by the single stranded rna viruses vesicular stomatitis virus (vsv) and sendai virus [ ] , suggesting sting activation pathways may overlap with rna sensing mechanisms or reverse transcription of viral rna [ , , ] . whilst the sting-mediated dsdna-sensing mechanism is critical for successful cellular protection against infections and disease progression, dysregulated sting activity leads to the excessive production of inflammatory mediators with potentially detrimental effects on surrounding cells and tissues. recent studies revealed some important functions for sting in autoinflammatory diseases [ ] [ ] [ ] , cancer [ ] [ ] [ ] [ ] and lipid regulations [ , ] , highlighting the importance of this protein in health and disease. here we review the recent insights into sting function in human pathologies and discuss the potential of sting-targeted therapies which are of considerable scientific and clinical interest. whilst sting acts as an adaptor protein in the dsdna sensing pathway, it is not activated directly by dna molecules. instead, sting responds to dna sensing proteins and molecules known as cyclic dinucleotides (cdns) [ , [ ] [ ] [ ] (figure ). cdns are derived from infectious agents exogenously, or are produced by the mammalian dsdna sensor cgas (cyclic guanosine monophosphateadenosine monophosphate synthase; cyclic gmp-amp synthase). the canonical cdns, or microbial secretory cdns, are molecules made of ′- ′ phosphodiester bonds joining two adenosines (a)cyclic di-amp [ , ] , two guanosines (g)cyclic di-gmp [ ] or one of eachcyclic gmp-amp [ ] . one of the sting-activating universally expressed dna sensors, cgas, is capable of catalysing a unique form of cdn endogenously upon dna recognition [ ] . this molecule is comprised of one ′- ′ phosphodiester bond and a non-canonical ′- ′ linkage between adenosine and guanosine, and is thus named ′- ′ cgamp to distinguish from the secretory cyclic dinucleotide cgamp ( ′- ′ cgamp) which contains two ′- ′ bonds [ ] . previous literature [ ] [ ] [ ] has suggested that ′- ′ cgamp is ten-to a thousand-fold more potent than ′- ′ cgamp in activating sting. a number of studies reported that the change of phosphodiester linkage in ′- ′ cgamp results in a higher binding affinity to sting and thus leads to an augmented type i interferon [ ] kaposi's sarcoma-associated herpesvirus enveloped dsdna virus dsdna activates cgas-sting pathway (cytoplasmic) and ifi -sting pathway (nuclear) [ ] herpes simplex virus enveloped dsdna virus dsdna activates cgas-sting pathway (cytoplasmic) and ifi -sting pathway (nuclear) [ , ] epstein-barr virus dsdna virus dsdna activates ifi -sting pathway (nuclear) [ ] human cytomegalovirus dsdna virus dsdna activates cgas-sting pathway (cytoplasmic), dai-sting (cytoplasmic), and ifi -sting pathway (nuclear) [ ] [ ] [ ] sendai virus negative strand ssrna virus possibly via rig-i -dependent rna detection which may in turn induce sting [ ] vesicular stomatitis virus negative strand ssrna virus unknown [ ] human immunodeficiency virus negative strand ssrna virus dsdna reverse transcribed from viral rna induces cgas-sting pathway [ ] influenza a virus negative strand ssrna virus possibly via membrane fusion or unknown mechanism independent of dna recognition [ ] mycobacteria tuberculosis bacteria producing c-di-gmp c-di-gmp [ , ] streptococcus pneumoniae bacteria dsdna bacterial dsdna [ ] streptococcus pyrogenes bacteria dsdna bacterial dsdna [ ] staphylococcus aureus bacteria producing c-di-amp c-di-amp [ ] listeria monocytogenes bacteria producing c-di-amp c-di-amp [ ] vibrio cholera bacteria producing ′- ′ cgamp ′- ′ cgamp [ , ] abbreviations: dsdna double-stranded dna, ssrna single-stranded rna the type i interferon signal adaptor protein sting is responsible for mediating double-stranded dna sensing responses and the detection of bacterial cyclic dinucleotides c-di-amp, c-di-gmp, and ′- ′ cgamp. many dna viruses, rna viruses and bacteria have been implicated in the activation of sting response [ , ] . it is also possible that hydrophilic secretory cyclic dinucleotides are excluded by the selectively permeable plasma membrane [ ] , and thus cannot be recognised by sting. in addition to being activated by cyclic dinucleotides, sting also mediates antiviral responses downstream of dna sensors including dna-dependent activator of ifn-regulatory factor (dai) [ ] , ifnγ-inducible protein (ifi ) [ ] , dead-box helicase (ddx ) [ ] , and components of the rna-sensing pathways [ , ] ( figure ). the z-form dna sensor dai was the first identified activator for sting, whose expression is highly cell-type and tissue-specific and therefore could not fully account for the widespread ifn-i induction during viral infection [ , ] . further research identified that the dna sensor ddx could interact with bacterial cyclic dinucleotides, in addition to dna molecules [ ] , prior to activating sting signalling [ ] . another interferon-inducible dna sensor ifi is a pyrin-containing protein which also induces sting activation downstream of dna detection. ifi , as well as its mouse orthologue p , is a universally expressed dna sensor, which forms multimers prior to sting activation in response to hsv viral infections [ , ] . evidence shows that ifi is responsible for detecting foreign dna both in the cytoplasm and in the nucleus and therefore it is capable of combating nuclearreplicating viruses such as kaposi's sarcoma associated herpesvirus [ , ] and human cytomegalovirus (hcmv) [ , ] , implying an ability to discriminate between "self" and "non-self" dna molecules. a recent report by diner and colleagues showed dynamic regulation of ifi oligomers at different cellular compartments in response to altered viral infections [ ] . throughout hcmv infection, ifi oligomers are densely gathered nuclear "punctate" structures, whereas in herpes simplex virus- (hsv- ) infection these "puncta" become gradually dispersed across the whole nucleoplasm and are eventually degraded. in contrast to previous studies, diner et al. also found that ifi knockout cells do not impede tbk activation upon immune stimulation, whereas both sting and cgas knockout cells will strongly suppress tbk activity, suggesting that nuclear dna detection mediated by ifi is independent of the sting/tbk- /irf axis. other studies indicate that antiviral cytokine production occurs in the absence of ifi via an unknown mechanism [ ] . recent reports have revealed an interesting relationship between ifi and cgas during dna detection. it was shown that hsv infection can induce both dna sensors in various cell types, and that cgas is partially nuclear, thus is able to regulate the stability of nuclear ifi oligomers during detection of viral dna [ ] . this provides a molecular mechanism by which cgas regulates nuclear dna sensing. further evidence also suggests a dna dose-dependent interaction between ifi and cgas in keratinocytes [ ] . although this interaction does not affect cgas's ability to generate cyclic dinucleotides, evidence indicates that ifi can facilitate the detection of these ligands by sting, and the loss of ifi can significantly impair downstream type i interferon and pro-inflammatory signalling [ ] . therefore, interestingly, several rna viruses such as human immunodeficiency virus (hiv) [ , ] , influenza a virus [ ] , sendai virus and vesicular stomatitis virus [ ] have been found to activate sting signalling, via mechanisms both dependent and independent of dna detection. complementary dna (cdna) produced from reverse transcription of negative-stranded rna in retroviruses such as hiv, murine leukemia virus (mlv) and simian immunodeficiency virus (siv) can induce a cgas-dependent dna sensing pathway and sting activation [ , ] ( figure ). however, hiv, in particular, is capable of inhibiting transcription of immediate antiretroviral factors [ ] and exploits the host sting blocker nod-like receptor nlrx to aid the establishment of virus latency [ , ] . it was also reported that cationic liposomes and nucleic acid-free herpesvirusderived virus-like particles can directly induce sting/ tbk relocation regardless of dna sensing pathways, suggesting that the membrane fusion mechanism may be an alternative route for enveloped dna and rna viruses to activate sting. enveloped rna virus influenza a virus has been shown to release haemagglutinin fusion peptide which induces sting but not cgas activation [ ] , thus indicating another sting activation mechanism independent of cyclic dinucleotide recognition. it remains unclear whether the fusion particles alone are direct sting ligands or if activity requires facilitation by unidentified co-regulator(s). the rna-inducing adaptor mavs (mitochondrial antiviral signalling protein; also known as visa, cardif, ips- ) can also interact with and activate sting [ , ] ( figure ). the mitochondria-resident adaptor mavs is the major molecular platform through which the rlrdependent rna sensing pathway elicits the type i interferon response. mavs responds to the cytoplasmic rna sensors rig-i (the retinoic acid-inducible gene i) and mda (melanoma differentiation-associated protein ) [ ] , and in turn induces proinflammatory transcription factors nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells), irf , irf , irf and irf [ ] [ ] [ ] [ ] . castenier and colleagues [ ] found that rig-i induced mavs activation can modulate mitochondrial dynamic changes promoting signalling to sting at mams, "mitochondria-associated membranes", where mitochondria and er are closely associated [ ] . this adaptor interaction was found to be dependent on a mitochondrial fusion mechanism which induces mitochondrial elongation towards the er, and hence mam formation. virus-induced mitochondria fragmentation disrupts membrane association and hence abolishes mavs activation and secondary sting signalling [ ] . another report also suggests that rna virus-induced release of stress-associated mitochondrial (mt)dna activates the sting-dependent dsdna sensing pathway [ ] . this process, which is also likely to involve mitochondrial stress-induced apoptosis, may provide an effective means to remove damaged cells for infection control. ablasser [ ] and chiu [ ] noted that the sting inducer and a b-form dsdna sensor rna polymerase iii (pol iii) activate the rig-i -dependent rna sensing pathways ( figure ). rna pol iii reversely transcribes dsdna into dsrna molecules that are activators of rig-i. however, it is unclear how sting is involved in this process. activation of sting induces adaptor dimerization [ ] and subsequent migration from er membranes to punctate membranes of the golgi by mechanisms similar to autophagy [ ] . it remained difficult to identify the role of sting in autophagy regulation as little evidence was found to link sting with the recycling process during cell starvation. yet many autophagy-related proteins such as autophagyrelated gene (atg) a and vps are key to sting interorganelle trafficking [ ] , and that the loss of early autophagy marker lc ii can significantly impair the stingdependent innate response to viral and bacterial infections [ ] [ ] [ ] . this autophagy-like behaviour could also be related to the er-originated pre-autophagosome formation, where sting is located [ ] . the sting-dependent cellular responses are mainly dependent on two transcription factors, irf and nf-κb ( figure ). sting activation first induces adaptor dimerisation [ ] and trim -dependent ubiquitination to enable tbk docking [ ] . together, sting and tbk migrate to the perinuclear membranes of the golgi via autophagy-like processes [ , , ] . the association between sting-tbk leads to autophosphorylation of tbk at s , a residue known to induce tbk activation [ , ] . this further allows tbk to phosphorylate sting at s and s (s and s , respectively, in mouse sting). phosphorylated s , together with l , are important for the recruitment of irf in close proximity to tbk at the cterminus of sting, thereby enabling tbk to phosphorylate and activate irf by exposing its nuclear localisation signal [ ] . activated irf then translocates into the nucleus and promotes expression of type i interferons. via a rapid feedback mechanism, ifn-i is released and binds to cell surface interferon receptors (ifnαrs), which then induces the expression of interferon-stimulated genes (isgs) via tyk /jak [ ] [ ] [ ] and stat -stat dimers [ , ] . sting ligands have also been shown to activate the canonical nf-κb pathway (figure ), leading to the production of pro-inflammatory cytokines including il- α, tnf-α (tumour necrosis factor-α), il- , and numerous chemokines such as cxcl and ccl- [ , ] . the mechanism of this was found to be dependent on sting-tbk activation, which in turn regulates the activation loop of ikkα/β releasing p to form active dimers with p . hence, the functional nf-κb complex can translocate into the nucleus and promote transcription of pro-inflammatory genes. abe and barber also suggested that traf may be involved upstream of tbk , which likely facilitates nf-kb activation [ ] . however, the ifi -dependent sting pathway can only induce irf but not nf-κb activation, which would serve to preserve the survival activities regardless of the antiviral response during infection [ ] . the sequence and topology of sting has been studied in parallel to its function. it is known that sting is a amino acid long er transmembrane protein encoded by the human tmem gene (accession np_ , xp_ ) and homologous genes in other mammalian species. the sting structure is highly conserved between mammalian species, with the n-terminal forming a putative multi membranespanning region, a middle cdn-recognition domain, and a cytoplasmic tail ( figure ). it is understood that the n-terminal amino acids of sting form four transmembrane helices [ , ] that are mainly responsible for membrane anchorage and inter-organelle trafficking [ ] (figure ). an additional helix, named helix α , was previously considered as the fifth transmembrane domain [ , , , ] but has more recently been proposed to form a distinct domain with different functions [ ] . helix α is formed between residues - and has been reported to play an essential role in protein folding and dimerisation. compromising the integrity of helix α causes sting precipitation in the cytoplasm and abolishes the homo-dimeric structure due to the loss of abundant hydrophobic interactions between dimers [ ] . ouyang's group reported that helix α also supports strong hydrogen force between sting dimers and cyclic di-gmp, suggesting its importance in multiple sting functions [ ] . in addition to helix α , the rest of the cytoplasmic tail also contributes to sting dimerisation [ ] , cyclic dinucleotide recognition [ ] , and tbk and irf recruitment [ , ] (figure ). in the absence of ligands, sting dimers show an open structure susceptible to cyclic dinucleotides [ ] . upon recognition of cyclic di-gmp, residues - are rearranged to expose a docking site for tbk [ ] , enabling tbk to phosphorylate sting at serine [ ] . this phosphorylation in turn enhances tbk -sting attachment. tanaka's group found that a -residue fragment at the carboxyl end of the sting protein was sufficient to activate irf signalling in response to dna challenge [ ] , and the loss of this tail region, encoded by exon , creates a dominant negative sting isoform for tbk -irf signalling [ ] . further truncation of the sting c-terminal fragment revealed that s and l are the key residues for irf recruitment and activation [ ] . therefore, tbk and irf are recruited to sting in a amino acidspanning region to facilitate their interaction by close proximity. crystallisation of the sting protein has revealed that key residue substitutions or segment deletions significantly alter its structure, resulting in a dysfunctional protein [ , , , , , ] . mutations of single or multiple amino acids of sting have been found to influence its dimerisation capability [ , [ ] [ ] [ ] , ligand binding capacity [ , [ ] [ ] [ ] , or the ability to be posttranslational modified by regulatory proteins [ , ] . some of these mutations occur naturally in humans to cause lethal autoinflammatory diseases [ , , ] , and some were generated experimentally in order to understand the structure-function relationship of sting [ , , , ] . sting variations exist between mammalian species; the amino acid sequences of human and mouse sting are % identical and % similar [ ] . whilst this may not lead to dramatic differences in their three-dimensional structures, the structural differences at certain amino acid residues may be responsible for species-specific immune responses to some viral infections. for instance, dengue virus (denv) can inhibit sting signalling in human but not in mouse [ , ] . denv encodes for the protease ns b that cleaves human sting at a highly-conserved putative cysteine motif (c xxc ) to disable the adaptor. the equivalent cleavage target in mouse sting (msting) harbours a mutation that prevents ns b cleavage, thus mouse sting can avoid denv evasion. mutant mouse embryonic fibroblasts transfected with hsting re-constructed with a mouse ns b cleavage site sequence blocks the type i interferon response against denv. specifically, a substitution of s a in hsting sequence results in inhibition of ns b cleavage, suggesting that protecting sting at this residue may provide the basis for a novel anti-dengue treatment [ ] . in humans, single nucleotide polymorphisms (snp) of sting were found to result in different levels of ifn-i signalling modulation (table ). substitution at r m can greatly stabilize the sting dimer, indicating its strong potential to cause chronic sting-dependent autoimmunity [ ] . substitution at r q can strongly reduce the c-di-gmp -induced ifn-i signals and completely abolishes the signals induced by other ′- ′ bond cyclic dinucleotides [ ] . other single residue polymorphisms such as r h and g a may affect the ligand binding pocket of sting, thereby reducing its response to bacterial ligands [ ] . in particular, a loss-offunction triple sting mutant, r h-g a-r q (named haq), abolishes almost % of the interferon response to all cyclic dinucleotides [ ] . the haq mutant occurs in one-fifth of the population from a thousand genome screen [ ] . homologous haq mutant knock-in mice have demonstrated that a variety of immune cells, including lymphocytes and ly c hi monocytes, express significantly less sting protein compared to the wildtype, and the mutant animals completely failed to respond to cdn challenge [ ] . this suggested that the haq mutant may be a de facto null allele of sting, thereby reducing the availability of the sting protein to mediate a dsdna sensing response. the function of several sting residues has been characterised using experimental point mutations ( table ) , some of which were also found to occur naturally in mammals. a c bl/ -derived, goldenticket (gt) mouse strain harbouring a single i n mutation in sting leads to the complete abolishment of ifn-i activity to listeria monocytogenes infection or stimulation of cyclic di-gmp and cyclic di-amp [ ] . the human equivalent mutation i n was also considered to have the same effects, but no such spontaneous mutant has been discovered. only a few gain-of-function hsting mutants have been identified clinically [ , ] (table ) . patients with these sting mutations showed early on-set of severe systemic inflammation in blood vessels and various organs, displaying chronic inflammatory symptoms that are highly similar to pathologies of sle (systemic lupus erythematous) and ags (aicardi-goutières syndrome) [ , ] . all of these sting mutants have shown considerable structural resemblance to the active conformation, presumably leading to constitutive adaptor dimerization and signalling to type i interferon production. both liu and könig's groups suggested that inhibition of the interferon signalling adaptor jak could significantly dampen ifn-i over-expression as measured in biopsy samples from these patients, indicating that jak inhibitors could be a promising avenue to therapeutically control disease progression. as evidenced by the above studies, sting variants are likely to be associated with increased susceptibility to certain infections and autoimmune diseases, emphasising the value of genetic analysis of individual mutations to reveal novel targets for developing personalised therapy and immunisations. as a critical coordinator of the innate immunity, sting is tightly regulated by a variety of signalling molecules. except that sting is post-translationally modified to enable dimerisation and activation, some regulators are essential for the prevention of constitutive type i interferon signalling which have been shown to cause autoimmunity both in animal models [ , ] and in human [ , ] . negative regulation of sting signalling is necessary for the resolution of inflammatory responses post infection ( figure ). post-translational modifications contribute to the spatiotemporal regulation of sting signalling. sting is commonly modified by ubiquitination and phosphorylation. upon ligand binding, the e ubiquitin ligase trim is recruited to initiate k- linked ubiquitination on sting, a prerequisite for sting dimerisation and activation [ ] . another e ligase amfr (autocrine motility factor receptor), together with its interacting partner insig (insulin-induced gene ), catalyses k- linked poly-ubiquitination, which is critical for tbk recruitment [ ] . in contrast, trim α-dependent k ubiquitination [ ] and rnf (ring finger protein ) -dependent k ubiquitination [ ] degrade sting dimers and negatively regulate antiviral signalling ( figure ). this process is likely to involve the antiviral adaptor protein mavs on mitochondria at the mams where the closely associated er and mitochondrial membranes single nucleotide polymorphisms of sting have been discovered in human and mouse which are implicated in dysregulation of type i interferon signalling and the proinflammatory innate immune response. sting mutations highlighted in green manifest as loss-of-function characteristics, mutations highlighted in red manifest as gain-of-function characteristics, and mutation in black lacks any gain-of-function or loss-of-function characteristic of sting. gain-of-function mutations of v m, n s and v l have been identified in human autoinflammatory disease called sting-associated vasculopathy with onset in infancy (savi), and substitution of g e is the major cause of another human autoimmune disease known as familiar chilblain lupus (fcl). the most predominant loss-of-function sting mutant named haq is considered to compromise host innate response against infection, yet no clinical evidence is available for further discussion. others sting mutations are experimentally created to study type i interferon signalling pathways, but they are potentially pathological bring sting and mavs in close proximity [ , ] . since ubiquitination is critical to sting regulation, some viruses can secrete proteases to specifically disrupt this process to suppress innate immune recognition, as summarised below (table ) . some sting activities are also critically dependent on its phosphorylation. one such example is the phosphorylation of s on the c-terminal domain to provide docking sites for irf prior to its phosphorylation by tbk [ , , ] . ulk -dependent phosphorylation on s post golgi trafficking blocks irf binding to sting and thus prevents chronic sting activation [ ] (figure ). ulk acts upon release from its repressor ampk (adenosine monophosphate activated protein kinase) which is induced by production of ′- ′ cgamp from cgas. interestingly, both loss-of-function (s a) and gain-offunction (s d) mutations can abrogate irf signalling [ ] , suggesting that either the phosphorylation of s is temporally and spatially regulating irf docking, or that alternative post-translational modifications are responsible for this functional regulation. in particular, s -dependent inhibition of irf does not impair the nf-κb pathway, indicating that these two pathways act independently of each other, which has likely evolved to prevent dysregulated antiviral responses from affecting survival activities [ ] . sting can be negatively regulated by the nlr family inflammasome components nlrc [ ] and nlrx [ ] (figure ). both of these have been shown to sequester sting to prevent tbk recruitment; in particular the latter is strongly enhanced in hiv infection to suppress sting-dependent recognition of reversetranscribed dsdna [ ] . depletion of nlrx not only impedes nuclear transportation of viral dna, but also restores the sting-mediated interferon response to stall progression of infection. this suggests that pharmacological suppression of intrinsic sting inhibitors could potentially support the re-establishment of stingmediated innate immunity against rna viruses, and thus may offer promising adjuvant therapy to combat retrovirus infection. however, guo and colleagues also noted that such suppression must be finely controlled to avoid excessive inflammatory responses that may lead to autoimmunity [ ] . a primate specific microrna (mir)- - p has been identified as a novel sting regulator that promotes virus replication [ ] (figure ). over expression of this microrna promotes the spread of vesicular stomatitis virus, whereas its inhibition protects against virus growth. further studies show that mir- - p is an irf -induced gene that can target multiple genes of fig. negative regulation of sting-mediated response. sting-mediated signalling can be negatively regulated via multiple mechanisms, including e ubiquitin ligase trim α-and trim -mediated degradation of sting and its upstream dna sensor ddx , respectively. certain phosphodiesterases (pdes) also specifically hydrolyse bacterial cyclic dinucleotides to prevent them being sensed by sting. akt kinase is also capable of inhibiting cgas detection of cytoplasmic dna. activated cgas produces ′- ′ cgamp to release ampk-mediated inhibition of ulk , which in turn blocks irf recruitment downstream of sting activation. ′- ′ cgamp produced by cgas can also activate beclin- which can sequester cgas as well as induce degradation of dsdna. in a negative feedback loop, the product of the irf -dependent antiviral response, microrna- - p (mir- - p), can prevent further sting activation. some viruses can encode proteases or protein inhibitors to interfere in sting signalling, while others may enhance the activity of inflammasome complexes nlrc and nlrx to block sting/ tbk interaction interferon-stimulators, including sting, mavs, traf and stat , thereby reducing their levels [ ] . since irf is a downstream signalling molecule in the sting-tbk axis, upregulation of mir- - p serves as a negative feedback loop to prevent sustained inflammatory response during and post infections. a recent discovery suggests that certain phosphodiesterases (pdes) may specifically degrade bacterial cyclic dinucleotides to halt excessive sting activation [ ] [ ] [ ] [ ] (figure ). the pathogen mycobacterium tuberculosis secretes phosphodiesterases mtbpde (rv ), cnpb and cdnp to remove cytosolic cdn, thus avoiding sting mediated detection [ ] [ ] [ ] . although this may appear to undermine bacterial virulence and growth signals that are critical to infection [ ] , it can also significantly reduce early type i interferon induction. in particular, the enzyme cdnp can degrade both bacterialderived and host-derived cyclic dinucleotides, critically promoting survival of m. tuberculosis at early stages of infection [ ] . in addition to the above regulatory mechanisms, akt kinase (or protein kinase b, pkb) has been shown to phosphorylate cgas at residues s or s to stall signalling via sting [ ] , while the e ubiquitin ligase trim can specifically target the dna sensor ddx at residues k and k for proteasome degradation and hence prevent recognition of dna and sting-dependent type i interferon expression [ ] (figure ) . the autophagy protein beclin- may also terminate sting-dependent immunity by sequestering cgas and promoting autophagy-dependent digestion of dsdna [ ] (figure ). this is thought to prevent prolonged dna recognition, which could lead to autoimmunity. immune responses to malaria infection are highly strain specific; the lack of understanding linked to these strain specific responses makes the disease clinically difficult to manage [ ] [ ] [ ] . recent studies on host-parasite interaction have revealed distinct roles for stingdependent type i interferon responses during crosstalk with other pro-inflammatory pathways. cd receptors expressed on antigen-presenting cells are understood to initiate the cellular and humoral response of the adaptive immunity, specifically enhancing the generation of immunoglobulins against pathogens [ , ] . mice infected with plasmodium yoelii nigeriensis, infected red blood cells, tlr ligands or parasitic dna/rna upregulate both cd and type i interferon expression, whereas the loss of cd can reduce the level of sting further impairing the early type i interferon response in macrophages and dendritic cells [ ] . although cd induced sting upregulation leads to reduced cd levels and thus downstream nf-κb signalling, type i interferon immunity has been suggested to be highly inducible in certain strains of parasitic infection [ ] . a number of dna and rna viruses have been found to encode and secrete sting-targeted proteases or inhibitors to prevent innate immune detection to help establish the of latent phase of infection. viruses of the same family tend to adopt similar strategies/mechanisms to block sting activation. some viruses also express multiple inhibitors to target both dna sensors and sting, or release viral oncogenes in parallel to further compromise immunity, which consequently increases their chance of survival in the host the establishment of malaria infection in the host is critically dependent on early innate immune mechanisms. yu et al. suggested that depletion of plasmacytoid dendritic cells, rather than canonical dendritic cells and macrophages, can significantly impair type i interferon signalling at h post plasmodium yoelii infection [ ] . the ubiquitous sting-cgas pathway has been shown to enhance type i interferon production, and also potently induces socs to inhibit the myd / irf dependent type i interferon production which specifically acts on pdcs to protect against early malaria infection. the loss of sting and cgas in mice augments the immediate type i interferon response from pdcs following malaria infection, and therefore protects the animal against early mortality [ ] . therefore, sting's activity and its crosstalk with other proinflammatory pathways may be variable in complex diseases such as malaria, and thus the manipulation of sting signalling axis for therapeutic benefits may be difficult to achieve. it is evident that many viruses can evade sting signalling to establish a biological niche in mammalian cells, and that those within the same family tend to adopt similar evasive strategies [ , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table ) . for instance, several members of the gammaherpesviridae family were found to express orf protein homologs to disrupt cgas activities, preventing subsequent production of ′- ′ cgamp [ ] . species of the flaviviridae and coronaviviridae families tend to secrete proteases that directly cleave or block sting [ ] ; typical examples include the ns b/ and ns b proteases released by flaviviridae dengue virus and hepatitis c virus, respectively, which can both degrade hsting. in particular, ns b protease shows strong homology to the er-embedded n-terminal domains of sting, leading to colocalisation and direct protein-protein interactions with sting [ ] . it has also been suggested that tumour associated viruses such as human papillomavirus and adenovirus could potentially release oncogenic proteins to block cgas/sting interactions with tumour suppressors, hence compromising innate immunity and supporting cancer progression [ ] . the impairment or absence of interferon responses often seen in hiv infection has been proposed as one of the mechanisms by which this virus is capable of suppressing host immunity [ ] [ ] [ ] [ ] . recent research suggests that hiv- enhances the action of nlrx to dampen sting activity [ ] and recruits host ′ exonuclease trex to degrade excessively produced, reverse-transcribed, viral dna thereby avoiding detection by the cgas/sting pathway [ , ] . the capsid of hiv also regulates its association with host protein cyclin a, which controls the masking of viral cdna from cgas recognition in the cytoplasm and its exposure in the nucleus to facilitate genome integration [ ] . mutations in the hiv- capsid sequence enhance its binding to cyclin a prematurely in the cytoplasm enabling dc sensing of double-stranded dna and a potent innate immune response against viral infection [ ] . type i interferons are key cytokines induced by antimicrobial and antiviral immunity. this family of cytokines consists of the predominantly produced interferon-α and interferon-β, and the less abundantly expressed subtypes such as ifn-ε, −κ, −τ, and -ζ [ ] . type i interferons are ubiquitously expressed by a variety of cells including macrophages, lymphocytes, dendritic cells, fibroblasts and haematopoietic plasmacytoid dendric cells, with a widespread role in cellular biology [ , ] . "basal" expression of type i interferons is regulated via an autocrine mechanism [ ] , whereas the activation of interferon-inducing regulators such as sting can significantly boost their expression by activating the transcription factor irf . type i interferons are released extracellularly for detection by self or nearby interferon receptors, ifnαrs, which are coupled to jak (janus kinase ) and tyk (tyrosine kinase ) [ , ] . this activation further promotes the formation of stat -stat heterodimers [ , , ] and the subsequent recruitment of irf to assemble the transcription complex isgf to upregulates the expression of a series of interferon-stimulated genes (isgs) [ ] . a broad range of isgs have been found to control chemotaxis, cell migration, apoptosis, cell proliferation, and to regulate immune detection and defense against infection; many of which have been thoroughly reviewed previously [ ] [ ] [ ] [ ] [ ] (table ). thus, dysregulation of type i interferon signalling can cause an excessive production of isgs, in turn over-activating the immune system. the persistent or excessive presence of cytoplasmic dna is one of the major causes for chronic inflammation and autoimmune diseases. chronic production of type i interferons, termed "type i interferonopathy", is a key indication of immune dysregulation predominantly associated with dna-induced autoimmunity. the overactive ifn-i response alerts cytotoxic immune cells systemically via isg production. this in turn promotes sustained release of proinflammatory cytokines including il- α/β, il- , and tnf-α, causing excessive inflammation and tissue damage [ ] [ ] [ ] . autoimmunity also induces aberrant cell death, which releases cellular components to t and b lymphocytes and leads to the production of self-reacting antibodies that congest in capillaries [ ] . unresolved b cell activation predisposes individuals to the development of systemic lupus erythematosus (sle) that is clinically challenging to treat [ , , ] . systemic lupus erythematosus is a systemic chronic autoimmune disease. [ , , ] (figure ). sle is often diagnosed by the accumulation of serological antinuclear antibodies (ana) against nucleic acids released from dead cells, which cause multiple tissue and organ damage [ ] . chronic activation of dna and rna sensing pathways triggered by infection and cell death can contribute to the type i interferonopathy that predisposes individuals to sle [ ] . mutations in nucleic acid sensors (such as endosomal toll-like receptors [ ] , rig-i [ ] , and dai [ ] ), interferon regulatory factors [ ] and dnases [ , ] can also increase sle susceptibility. recently, ding and colleagues proposed that the cgas-sting axis could be another pathway potentially exacerbating sle, via the upregulation of type i interferon production downstream of cytoplasmic dna sensing cascades [ ] . however, sting deficiency in macrophages in fact renders hyperresponsiveness to endosomal tlr ligands, and sting knockout mice have shown accelerated lymphocyte accumulation and expansion of an ifn-α -responding cell population [ ] . this therefore suggests inhibitory roles for sting in sle development. sharma et al. also showed that sting suppression can restrict the expression of regulatory t cell activation factor ido- and tlr negative regulators such as a , socs and socs , contributing to uncontrolled systemic inflammation [ ] . since this effect is not seen in cells lacking irf , the transcription factor mediating most of dna sensing responses downstream of the sting-tbk axis, it is possible that sting is immunosuppressive in inflammatory pathways independent of cytoplasmic dna recognition. therefore, autoimmune therapies targeting the sting pathway should be considered with caution and an awareness of the resultant sting downregulation, which may have opposing effects in certain diseases. ags (aicardi-goutières syndrome) is another genetically-based autoimmune disease, characterised by dna-triggered type i interferonopathy [ ] (figure ). patients with ags often carry mutations in dna restriction factors including the ′ exonuclease trex [ , ] , dntp restriction factor samhd (sam domain and hd domain) [ , ] , rnase h (ribonuclease h ) [ , ] , dsrna sensor ifih (ifn-induced helicase c domain containing protein ) [ , ] , and the dsrna-specific adenosine deaminase adar [ ] . these regulators maintain a balance between the production and degradation of nucleic acids, providing intrinsic protection against immune activation due to "self-recognition". mutations of dna or rna restricting factors cause nucleic acids to accumulate in the cytoplasm leading to sle and ags. recent studies show that the type i interferonopathy associated with sle and ags is potentially cgas-sting dependent, and that the aberrant ifn-i response can be suppressed by the loss of dna sensor or sting in cells or animals expressing mutated trex or samhd [ , ] . gain-of-function mutations in sting have been identified in infants who suffer from severe and chronic vasculopathy and pulmonary inflammation, a condition known as sting-associated vasculopathy with onset in infancy (savi) [ ] (figure ). mutations of sting v l, n s, v m, and v r were found to direct it to an active conformation enhancing dimerisation and inducing tbk -irf signalling ( table ) . this results in an excessive ifn-i response in fibroblasts, keratinocytes and immune cells to attract and amass proinflammatory cells and regulators in capillaries and tissues, ultimately causing lesions in these regions. sustained ifn-i signals activate interferon receptors and promote expression of interferon-stimulated genes via jak -tyk signalling and stat -stat dimers. in vitro experiments and pioneering clinical studies suggest that jak adaptor inhibition effectively dampens stingmediated ifn-i over-activity. for instance, the elevated ifn-i levels in biopsy samples from savi patients can be restored close to that of the normal controls with treatment using the jak inhibitor, tofacitinib [ ] . further investigation is required to examine the potential adverse effects of interferon suppression, which is likely to increase host susceptibility to infection. gain-of-function mutations of sting (table ) have also been linked to the autoimmune disease familial chilblain lupus (fcl) [ , ] , a rare hereditary form of sle commonly associated with cytoplasmic dna accumulation in monogenic mutations of exonucleases trex [ , ] or samhd [ ] (figure ) . a recent discovery reports flc in five members of a four-generation family sharing the same tmem (sting) variant that encodes a single polymorphism of g e. structural analysis of mutated sting dimers reveals strong hydrogen attractions between e on one monomer and two threonine residues on the associating monomer, hence leading to enhanced adaptor dimerization and constitutive ifn-i -activated signalling [ ] . although limited treatment data is currently available for fcl, the authors showed that continuous administration of the jak inhibitor tofacitinib can markedly ameliorate type i interferonopathy and associated symptoms in two patients. a similar therapeutic strategy was previously proposed by liu and colleagues for treatment of sting-associated vasculopathy with onset in infancy (savi) [ ] . therefore, this therapeutic approach, based on mechanistic data, could be adapted to treat type i interferonopathy found in various diseases. a new insight into sting research was recently provided by york and colleagues who suggested that this protein is a crucial element of cholesterol metabolism [ ] . previous studies indicate that high cholesterol levels in the plasma membrane correlate with viral loads and host susceptibility to infections [ , ] . virus and microbial infections have been shown to modulate lipid metabolism in the plasma membrane to facilitate infectivity. for instance, influenza virus encodes fusion protein haemagglutinin that is specialised in manipulating membrane lipid to permit penetration into the cytoplasm, a central step to viral infectivity and survival [ ] . in addition, membrane lipids can also form signalling microdomain named lipid rafts which are frequently hijacked by hiv for attachment, signalling and budding to further promote infection [ , ] . the antiviral type i interferon response reduces cholesterol availability in membranes to prevent viral infection; however the underlying mechanism remains largely unknown [ ] [ ] [ ] . york's group recently identified that sting/tbk signalling is critical to the production of type i interferons to reprogram lipid biosynthesis in pathogenic infection [ ] . they demonstrated that the shift from lipid biosynthesis to lipid uptake not only affects the plasma membrane but also er membranes, a cue to activate sting, bypassing the dsdna sensing pathway. however, since sting is not the only adaptor for innate immunity against pathogenic dna in the cytoplasm, it is conceivable that additional dna sensors may further enhance sting actions to modulate cholesterol metabolism and promote antiviral processes. in light of the membrane fusion theory that potentially mediates sting activation [ ] , virus-host lipid regulation at the plasma membrane offers a promising and novel future research direction. studies on sting regulatory pathways provide novel insights into antiviral and anti-inflammatory therapies. activating sting-dependent pathways has been developed therapeutically for antiviral and, more recently, antitumour benefit. the predominant approach taken has been to introduce sting ligand cyclic dinucleotides to promote the ifn-i response, to combat infection or to prevent tumour progression. in contrast, type i interferonopathy associated with sting over-activity represents another set of pathologies underlying autoimmunity. counteracting these disease processes requires potent suppression of sting signalling; attenuation of the interferon receptor adaptor jak is used as a current target, whilst inhibiting immune cell activation may also help ameliorate symptoms. certain types of cancer cells express molecular structures specifically recognised by cd α + dendritic cells (dcs), which subsequently interact with cytotoxic t cells to induce cancer cell death. this event, known as t cell priming, is a prerequisite for anti-tumour adaptive immunity relying on activation of cd α + dendritic cells to promote ifn-i signaling in immature t cells [ ] . however, cancer cells also boost anti-inflammatory immune cells and regulatory t cells (treg) to restrict cd α + dc activity thereby attenuating the activation of tumoursuppressive t cells [ , ] . therefore, a potent and long-acting adjuvant that can promote cd α + dc activities is highly desirable to enhance t cell priming and subsequent anti-tumour immunity. it has been reported that the ifn-i response critical to t cell priming during tumourigenesis is dependent on the cgas / sting pathway [ , ] . loss of sting in dendritic cells abolishes antigen cross-presentation from cd α + dc to t cells, whereas neither myd nor trif knockouts can significantly affect dc ifn-i signalling, suggesting that sting may be the only adaptor central to this process [ ] . in both immunogenic and irradiation-induced tumour models, tumour-derived dna was engulfed and recognised by the universal dna sensor cgas prior to sting activation [ , ] . however, direct stimulation with sting ligands also enhanced dc production of type i interferons, suggesting that sting-inducing therapies may offer potential as anti-tumour adjuvants. preclinical studies published recently suggest that dmxaa-derived cyclic dinucleotides have been successfully applied in established mouse models of malignant tumours achieving sustained tumour regression [ ] . in malignant tumour b cells, sting also induces an er stress response through the ire- / xbp- (x-box binding protein ) pathway [ ] . in the presence of ′- ′ cgamp sting dimers are phosphorylated and aggregate, rather than undergoing degradation. this consequently enables prolonged sting signalling to induce apoptosis of tumour cells. similarly, tang et al. showed administration of ′- ′ cgamp induces rejection of chronic lymphotic leukemia in mouse models [ ] . another promising vaccine candidate is the recently developed stingvax which combines granulocytemacrophage colony-stimulating factor (gm-csf) and formulated ′- ′ - ′- ′ linked cyclic dinucleotide [ ] . in various established in vivo tumour models, stingvax has shown notable positive effects on dendritic cell activation and in promoting tumourinfiltrating t cells. interestingly, activated cytotoxic t cells also upregulate the expression of pd-l (programmed death ligand ), which enhances the therapeutic action of pro-apoptotic ligand pd- to promote tumour cell death. although these sting-based vaccines have recently been developed and have been tested in mouse models, the synergistic effect of the combined sting agonist and immune promoting therapy represents a novel strategy to combat tumours by reinforcing both adaptive immunity and anti-tumour targets. inhibition of sting signalling can be achieved by targeting interferon receptors. as previously mentioned, gain-of-function mutations in tmem (the gene encoding sting) underlie type i interferonopathies that manifest in the autoimmune diseases, savi [ ] and fcl [ ] . constitutive sting signalling was detected in both of these diseases resulting in dysregulated ifn-i signalling via interferon receptors and the adaptors jak and tyk. this leads to the accumulation of activated stat / dimers in the nucleus, promoting transcription of interferon-stimulated genes. both liu and könig's groups demonstrated that treatment with the jak / inhibitor tofacitinib in patient biopsy samples suppresses stat activation and restores the sting response to immune stimuli similar to healthy control cells [ , ] ( figure ). following the work of liu and colleagues, fremond's group conducted an -month clinical investigation on three patients expressing sting-mutations to assess the efficacy of the jak / inhibitor ruxolitinib [ ] ( figure ) , which was previously found to partially inhibit stat activation in in vitro studies of sting mutants [ ] . marked amelioration of systemic inflammation and reduction of interferon-stimulated gene expression was consistently observed in all three patients and in the subsequently recruited additional four patients with sting gain-offunction mutations. however, suspension of jak inhibition in one patient resulted in a dramatic inflammatory relapse, though rescued by re-introducing ruxolitinib, demonstrating that this approach may be unsustainable and requires continuous monitoring [ , , ] . nonetheless, it is arguable that the partial inhibition of jak-stat pathway has the advantage of preserving sting-dependent immune protection against infection in these patients, since no excessive infection incidents were observed during these clinical trials [ ] . thus, it is timely to determine whether such jak inhibitors can be modified and adapted for future treatment of type i interferonopathy. anti-inflammatory biologics represent a further opportunity to suppress interferon responses in order to control type i interferonopathy in autoimmune diseases, including but not limited to sle, a major manifestation linked to sting over-activity. for instance, the anti-ifn-α drug sifalimumab is effective in controlling cutaneous and joint pain in sle patients [ ] (figure ). another approach to control sle in sting gain-offunction mutations is to deplete or inhibit b-cell responses to prevent the over-production of autoantibodies. the effective anti-sle biologics, belimumab, targets the blys protein of b lymphocytes, preventing b cell activation and expansion critical to the production of autoantibodies and downstream activation of t lymphocytes [ , ] (figure ) . a series of stage ii and stage iii clinical trials have shown effective b-cell inhibition and significant improvement of clinical symptoms in combination with traditional care therapy of sle in the treatment group compared to placebo control, whilst drug tolerance and immunosuppression-induced infection susceptibility were not markedly increased [ ] [ ] [ ] . this suggests an effective drug efficacy and safety of belimumab over another b-cell depleting drug rituximab, which failed to ameliorate sle symptoms in phase ii and iii trails [ ] . unlike jak inhibitors and anti-ifn biologics, b-cell targeted therapies are much less effective in controling type i interferonopathy that consequently cause complex inflammatory responses in sting mutant patients. however, they are still commonly used to treat the sle-related consequences of sting over-activation to stall symptom deterioration, while they have also been considered suitable adjuvant candidates for sting-targeted therapeutics. accumulating evidence link sting-mediated ifn-i signaling to anti-tumour activity, and thus sting ligands have been proposed to offer promising immuneenhancing therapies to defend against dna infections and tumourigenesis [ , ] . however, targeting intracellular proteins such as sting remains challenging as the plasma membrane is a highly hydrophobic and sizeselective barrier that resists passive entry of chemicals [ ] . in contrast to the de novo sting ligand ′- ′ cgamp [ ] , other cyclic dinucleotides are produced exogenously by pathogens and introduced into the cytoplasm by active transport or particle fusion [ , , ] . since the hydrophilic phosphate groups in dinucleotide compounds are strongly repelled by membrane lipid bilayers, designing an effective delivery system for cyclic dinucleotides would provide a significant step forward in the development of sting-specific therapeutics. under in vitro studies, transfection of microbial cyclic dinucleotides is aided by digitonin [ , , , ] or liposomal-based systems such as lipofectamine® [ , ] (figure ). the former method, first described by woodward's group [ ] , aims to achieve reversible permeabilisation of cellular membranes to increase uptake of chemicals [ ] [ ] [ ] , but is limited to in vitro studies due to high toxicity in vivo. in contrast, the liposomalbased delivery system is based upon the principle of encapsulating drugs in artificial double-layered liposomes for cytoplasmic delivery via liposomal fusion. the system is highly adapted for a variety chemicals and has been used in both laboratory studies and clinical practice [ ] . in light of the liposome-based designs, miyabe's group has reported that the ysk -based lipid particles were able to entrap and deliver cyclic di-gmp into the raw . macrophage cell line [ ] (figure ). these particles induced cellular expression of type i interferon genes which were effectively blocked by the tbk inhibitor bx , suggesting that the interferon response was specifically induced via the sting/tbk pathway. furthermore, the ysk -based particles also express high levels of the antigen-presenting molecule mhc class i and t cell co-stimulating molecules cd and cd that are a prerequisite of t lymphocyte activation; all of these characteristics suggest they offer potential as adjuvants in anti-tumour therapies. preliminary tests carried out by miyabe and colleagues showed that mice immunized with cyclic di-gmp containing ysk liposomes reject tumour implantation compared to matched controls [ ] . subsequently, nakamura's group demonstrated that in vivo injection of these particles greatly enhances the expression of ifn-i and mhc class i molecules in tumourigenic mice, resulting in augmented nk cell activation and potent innate immune protection against lung melanoma metastasis [ ] . therefore, the ysk liposomes offer a potential vehicle to assist delivery of sting ligands and to develop sting-based adjuvants for cancer immunotherapy. as an alternative approach, a nanoparticle-based delivery system developed by lee and colleagues also enables in vitro delivery of cyclic dinucleotides to target sting pathways for anti-tumour effects [ ] (figure ). this method employs polyethyleneimine / hyaluronic acid (lh) -based hydrogels to enclose dinucleotide drugs into micron size spheres, which are selectively taken up by phagocytic cells such as raw . macrophages, l fibroblasts and bone marrow-derived macrophages (bmdms), but not by non-phagocytic fibroblasts. thus, the micron-sized particles appear to target phagocytosis specifically to gain entry into the cytoplasm. furthermore, lee et al. showed that lh-cgamp hydrogel can induce ifn-i spikes in raw . macrophages, which were more than twice the magnitude of that induced by lipofectamine-delivered cgamp at the same dose [ ] . administration of ovalbumin-containing lh-cgamp particles in mice activates both the type i interferon response and humoral production of igg to protect against ovalbumin challenge. taken together, miyabe and lee's work indicates that the modified liposome-based delivery systems can markedly enhance in vitro and in vivo delivery of cyclic dinucleotide to cytoplasmic sting, and this success will promote the development of novel cancer vaccination and immunotherapies dependent on sting signalling. cytoplasmic dna has been implicated in many human pathologies, many associated with chronic inflammation. research has revealed that the er transmembrane protein sting is a crucial player in dsdna pathogen -sensing pathways whose dysregulation contributes to the development of several diseases. by responding to dna sensors and cyclic dinucleotides, sting induces irf -and nf-κb -dependent pathways to elicit proinflammatory responses against infection and cancer progression. sting also directly crosstalks with several other regulators to modulate critical biological processes including autophagy and fig. in vitro and in vivo delivery of sting agonists. the plasma membrane is a selectively permeable barrier that prevents cytoplasmic entry of large or hydrophilic molecules, including naked cyclic dinucleotides (cdns) ( ). in vitro (blue background) delivery of dinucleotide compounds could be achieved by the liposomal delivery system ( ), or via reversible permeabilisation of plasma membrane to allow diffusion of naked cdns into the cytoplasm ( ). recently designed ysk -containing liposomes ( ) could carry c-di-gmp across plasma membranes to induce ddx mediated sting activation as well as enhance the expression of mhc class i molecules and t cell co-stimulatory receptors (not demonstrated), and thus it is considered to be a potential adjuvant for cancer immunotherapy. in addition, the polyethyleneimine/ hyaluronic acid (lh) hydrogel-based vesicles use phagocytosis to deliver both sting ligands and antibody-stimulating agents such as ovalbumin (dark triangles) to cells ( ) , and enhance both sting-dependent innate immunity and mhc class ii-activated adaptive immunity to suppress cancer growth. both ysk particles and lh hydrogel-based particles have been tested in vivo (green background) to stall tumour progression in mice ( ) cholesterol biosynthesis. it is evident that activating sting results in the type i interferon response to protect against infection and tumour formation, while dysregulated gainof-function sting mutations lead to detrimental consequences of autoimmunity. understanding the molecular signalling mechanisms of sting activation has provided new insights to advance therapeutic strategies in treating infection, cancer and autoimmune diseases. it has also prompted the development of intracellular delivery systems to administer sting agonists. despite the fact that sting has only been studied for a decade, this adaptor protein will continue to attract attention in immunology research and clinical practice into the future. dna recognition in immunity and disease recognition of cytosolic dna attenuates glucose metabolism and induces ampk mediated energy stress response liposome-based dna carriers may induce cellular stress response and change gene expression pattern in transfected cells long non-coding rnas and control of gene expression in the immune system intracellular dna recognition sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization the adaptor protein mita links virus-sensing receptors to irf transcription factor activation sting/mpys mediates host defense against listeria monocytogenes infection by regulating ly c(hi) monocyte migration sting recognition of cytoplasmic dna instigates cellular defense sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity palmitoylated calnexin is a key component of the ribosome-translocon complex simultaneous induction of the four subunits of the trap complex by er stress accelerates er degradation the sting pathway and regulation of innate immune signaling in response to dna pathogens ifi is an innate immune sensor for intracellular dna the helicase ddx senses intracellular dna mediated by the adaptor sting in dendritic cells dlm- / zbp ) is a cytosolic dna sensor and an activator of innate immune response sting specifies irf phosphorylation by tbk in the cytosolic dna signaling pathway cytosolic-dna-mediated, sting-dependent proinflammatory gene induction necessitates canonical nf-κb activation through tbk adenovirus detection by the cgas/sting/ tbk dna sensing cascade modulation of the cgas-sting dna sensing pathway by gammaherpesviruses hsv- degrades, stabilizes, requires, or is stung by sting depending on icp , the us protein kinase, and cell derivation rig-i mediated sting up-regulation restricts hsv- infection viral evasion of intracellular dna and rna sensing human cytomegalovirus induces the interferon response via the dna sensor zbp cgas senses human cytomegalovirus and induces type i interferon responses in human monocyte-derived cells innate nuclear sensor ifi translocates into the cytoplasm during the early stage of in vitro human cytomegalovirus infection and is entrapped in the egressing virions during the late stage the cytosolic exonuclease trex inhibits the innate immune response to human immunodeficiency virus type influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses cyclic gmp-amp synthase is an innate immune dna sensor for mycobacterium tuberculosis the cytosolic sensor cgas detects mycobacterium tuberculosis dna to induce type i interferons and activate autophagy streptococcus pneumoniae stimulates a sting-and ifn regulatory factor -dependent type i ifn production in macrophages, which regulates rantes production in macrophages, cocultured alveolar epithelial cells, and mouse lungs type i interferon production induced by streptococcus pyogenes-derived nucleic acids is required for host protection cyclic di-amp released from staphylococcus aureus biofilm induces a macrophage type i interferon response c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response structural biochemistry of a vibrio cholerae dinucleotide cyclase reveals cyclase activity regulation by folates coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for v. cholerae virulence sting mediates neuronal innate immune response following japanese encephalitis virus infection activated sting in a vascular and pulmonary syndrome sting manifests self dna-dependent inflammatory disease sting: infection, inflammation and cancer sting-dependent cytosolic dna sensing mediates innate immune recognition of immunogenic tumors sting-dependent cytosolic dna sensing promotes radiation-induced type i interferondependent antitumor immunity in immunogenic tumors sting-mediated dna sensing promotes antitumor and autoimmune responses to dying cells limiting cholesterol biosynthetic flux spontaneously engages type i ifn signaling how low cholesterol is good for anti-viral immunity sting is a direct innate immune sensor of cyclic di-gmp cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna cyclic gmp-amp synthase is an innate immune dna sensor for mycobacterium tuberculosis sting-dependent recognition of cyclic di-amp mediates type i interferon responses during chlamydia trachomatis infection cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway cyclic dinucleotides bind the c-linker of hcn to control channel camp responsiveness structurefunction analysis of sting activation by c[g( ′, ′) pa( ′, ′)p] and targeting by antiviral dmxaa the innate immune dna sensor cgas produces a noncanonical cyclic dinucleotide that activates human sting cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting hydrolysis of ′ '-cgamp by enpp and design of nonhydrolyzable analogs recent approaches to intracellular delivery of drugs and dna and organelle targeting ifn?? responses induced by intracellular bacteria or cytosolic dna in different human cells do not require zbp (dlm- /dai) bruton's tyrosine kinase phosphorylates ddx and activates its binding of dsdna and sting to initiate type interferon response ifi senses dna forms of the lentiviral replication cycle and controls hiv- replication ifi acts as a nuclear pathogen sensor to induce the inflammasome in response to kaposi sarcoma-associated herpesvirus infection nuclear innate immune dna sensor ifi is degraded during lytic reactivation of kaposi's sarcoma-associated herpesvirus (kshv): role of ifi in maintenance of kshv latency human cytomegalovirus tegument protein pul inhibits ifi -mediated dna sensing for immune evasion viral dna sensors ifi and cyclic gmp-amp synthase possess distinct functions in regulating viral gene expression, immune defenses, and apoptotic responses during herpesvirus infection cgas-mediated stabilization of ifi promotes innate signaling during herpes simplex virus infection sting during dna sensing in human keratinocytes nlrx sequesters sting to negatively regulate the interferon response, thereby facilitating the replication of hiv- and dna viruses cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses rapid inflammasome activation following mucosal siv infection of rhesus monkeys ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-beta signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kb and irf mitochondrial dynamics regulate the rig-i-like receptor antiviral pathway mam (mitochondria-associated membranes) in mammalian cells: lipids and beyond mitochondrial dna stress primes the antiviral innate immune response rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway atg a controls dsdna-driven dynamic translocation of sting and the innate immune response activation of autophagy by α-herpesviruses in myeloid cells is mediated by cytoplasmic viral dna through a mechanism dependent on stimulator of ifn genes extracellular m. tuberculosis dna targets bacteria for autophagy by activating the host dna-sensing pathway structure of sting bound to cyclic di-gmp reveals the mechanism of cyclic dinucleotide recognition by the immune system the ubiquitin ligase trim regulates innate immune responses to intracellular doublestranded dna use of the pharmacological inhibitor bx to study the regulation and physiological roles of tbk and iκb kinase ε ikk-i and tbk- are enzymatically distinct from the homologous enzyme ikk- : comparative analysis of recombinant human ikk-i, tbk- , and ikk- the receptor of the type i interferon family interferon-??-dependent activation of tyk requires phosphorylation of positive regulatory tyrosines by another kinase tyk kinase activity is required for functional type i interferon responses in vivo formation of stat -stat heterodimers and their role in the activation of irf- gene transcription by interferon-?? transcriptional regulation by stat and stat in the interferon jak-stat pathway interferon γ-inducible protein (ifi) transcriptionally regulates type i interferons and other interferon-stimulated genes and controls the interferon response to both dna and rna viruses sting and the innate immune response to nucleic acids in the cytosol mpys, a novel membrane tetraspanner, is associated with major histocompatibility complex class ii and mediates transduction of apoptotic signals cutting edge: novel tmem allele reveals importance of sting n terminus in trafficking and type i ifn production cyclic di-gmp sensing via the innate immune signaling protein sting molecular basis of dna recognition in the immune system the structural basis for the sensing and binding of cyclic di-gmp by sting structural analysis of the sting adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-gmp binding backbone resonance assignments of the ??kda dimeric c-terminal domain of murine sting in complex with dmxaa ligand-induced ordering of the c-terminal tail primes sting for phosphorylation by tbk an alternative splicing isoform of mita antagonizes mita-mediated induction of type i ifns xcyclic dinucleotides trigger ulk (atg ) phosphorylation of sting to prevent sustained innate immune signaling single amino acid change in sting leads to constitutive active signaling single nucleotide polymorphisms of human sting can affect innate immune response to cyclic dinucleotides familial chilblain lupus due to a gain-of-function mutation in sting the common r h-g a-r q human tmem is a null allele denv inhibits type i ifn production in infected cells by cleaving human sting inherited sting-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations dengue virus targets the adaptor protein mita to subvert host innate immunity identification and characterization of a loss-of-function human mpys variant the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides trim α is a negativefeedback regulator of the intracellular dna and dna virus-triggered response by targeting sting the e ubiquitin ligase amfr and insig bridge the activation of tbk kinase by modifying the adaptor sting the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita the endoplasmic reticulum-mitochondria connection: one touch, multiple functions activation of stat by sting is critical for antiviral innate immunity phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation. science ( -) nlrc , a member of the nlr family of proteins, is a negative regulator of innate immune signaling induced by the dna sensor sting primate-specific mir- - p sets host defense signalling threshold cyclic di-amp impairs potassium uptake mediated by a cyclic di-amp binding protein in streptococcus pneumoniae identification and characterization of a cyclic di-gmp-specific phosphodiesterase and its allosteric control by gtp the ubiquitous protein domain eal is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive eal domains v-cgaps: attenuators of ′ ′-cgamp signaling gyanu lamichhane hos& wrb. deletion of the cyclic di-amp phosphodiesterase gene (cnpb) in mycobacterium tuberculosis leads to reduced virulence in a mouse model of infection inhibition of innate immune cytosolic surveillance by an m. tuberculosis phosphodiesterase twostep synthesis and hydrolysis of cyclic di-amp in mycobacterium tuberculosis akt kinase-mediated checkpoint of cgas dna sensing pathway the e ubiquitin ligase trim negatively regulates the innate immune response to intracellular double-stranded dna crosstalk between the cgas dna sensor and beclin- autophagy protein shapes innate antimicrobial immune responses strain-specific innate immune signaling pathways determine malaria parasitemia dynamics and host mortality strain-specific humoral response to a polymorphic malaria vaccine immunity to malaria: more questions than answers molecular regulation of cd gene expression in macrophages and microglia increased cd expression enhances early sting-mediated type i interferon response and host survival in a rodent malaria model cross-regulation of two type i interferon signaling pathways in plasmacytoid dendritic cells controls anti-malaria immunity and host mortality the hepatitis c virus ns b can suppress sting accumulation to evade innate immune responses hepatitis c virus ns b protein targets sting and abrogates rig-imediated type i interferon-dependent innate immunity herpes simplex virus ubiquitin ligase icp interacts with pml isoform i and induces its sumo-independent degradation message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection e proteins of high risk human papillomaviruses down-modulate sting and ifn-?? transcription in keratinocytes dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway. science ( -) hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dnasensing pathways viral evasion of dna-stimulated innate immune responses inhibition of cgas dna sensing by a herpesvirus virion protein the capsids of hiv- and hiv- determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hsv- icp targets the tbk -activated sting signalsome to inhibit virus-induced type i ifn expression type i interferon production is profoundly and transiently impaired in primary hiv- infection hiv infection of dendritic cells subverts the ifn induction pathway via irf- and inhibits type ifn production type i interferon responses are impaired in latently hiv infected cells hiv- and interferons: who's interfering with whom? type i interferon in the pathogenesis of lupus type i interferon: friend or foe? regulation of type i interferon responses constitutive type i interferon modulates homeostatic balance through tonic signaling interferon-stimulated genes and their antiviral effector functions functional classification of interferon-stimulated genes identified using microarrays interferon-stimulated genes: a complex web of host defenses interferon-inducible antiviral effectors apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis the role of cytokines in the generation of inflammation and tissue damage in experimental gram-positive meningitis anti-and proinflammatory cytokines in the pathogenesis of tissue damage in crohn's disease autoimmune t cell responses in the central nervous system apoptosis in autoimmune diseases the role of extracellular dna in autoimmunity in sle interferon alpha in systemic lupus erythematosus development of autoantibodies before the clinical onset of systemic lupus erythematosus the role of toll-like receptors in systemic lupus erythematosus genetic polymorphisms of dsrna ligating pattern recognition receptors tlr , mda , and rig-i. association with systemic lupus erythematosus and clinical phenotypes dna-dependent activator of interferon-regulatory factors (dai) promotes lupus nephritis by activating the calcium pathway interferon regulatory factors in human lupus pathogenesis trex gene variant in neuropsychiatric systemic lupus erythematosus dnase and systemic lupus erythematosus the regional function of cgas/sting signal in multiple organs: one of culprit behind systemic lupus erythematosus? med hypotheses suppression of systemic autoimmunity by the innate immune adaptor sting aicardi-goutières syndrome and the type i interferonopathies rpa and rad constitute a cell intrinsic mechanism to protect the cytosol from self dna activation of cyclic gmp-amp synthase by self-dna causes autoimmune diseases mutations involved in aicardi-goutières syndrome implicate samhd as regulator of the innate immune response restriction by samhd limits cgas/sting-dependent innate and adaptive immune responses to hiv- ribonuclease h mutations induce a cgas / sting-dependent innate immune response rnase h catalytic core aicardi-goutieres syndrome-related mutant invokes cgas-sting innate immune-sensing pathway in mice aicardi-goutières syndrome is caused by ifih mutations autosomal dominant ifih gain-of-function mutations cause aicardi-goutieres syndrome mutations in adar cause aicardi-goutières syndrome associated with a type i interferon signature cutting edge: cgas is required for lethal autoimmune disease in the trex -deficient mouse model of aicardi-goutières syndrome familial chilblain lupus due to a novel mutation in the exonuclease iii domain of ′ repair exonuclease ( trex ) familial chilblain lupus -a monogenic form of cutaneous lupus erythematosus due to a heterozygous mutation in trex autosomal dominant inheritance of a heterozygous mutation in samhd causing familial chilblain lupus interaction of vesicular stomatitis virus with lipid vesicles: depletion of cholesterol and effect on virion membrane fluidity and infectivity lipid raft disruption by cholesterol depletion enhances influenza a virus budding from mdck cells the mechanisms of lipid-protein rearrangements during viral infection evidence for budding of human immunodeficiency virus type selectively from glycolipid-enriched membrane lipid rafts lipid rafts and hiv pathogenesis: host membrane cholesterol is required for infection by hiv type the transcription factor stat- couples macrophage synthesis of -hydroxycholesterol to the interferon antiviral response role of cholesterol in parasitic infections high cholesterol may protect against infections and atherosclerosis virus-cell fusion as a trigger of innate immunity dependent on the adaptor sting type i interferon is selectively required by dendritic cells for immune rejection of tumors dendritic cells and immunity against cancer tumor-infiltrating dendritic cells in cancer pathogenesis direct activation of sting in the tumor microenvironment leads to potent and systemic tumor regression and immunity agonist-mediated activation of sting induces apoptosis in malignant b cells sting agonist formulated cancer vaccines can cure established tumors resistant to pd- blockade efficacy of the janus kinase / inhibitor ruxolitinib in the treatment of vasculopathy associated with tmem -activating mutations in three children jak inhibition in sting-associated interferonopathy sifalimumab, an anti-interferon-α monoclonal antibody, in moderate to severe systemic lupus erythematosus: a randomised, double-blind, placebo-controlled study efficacy and safety of biologic therapies for systemic lupus erythematosus treatment: systematic review and meta-analysis belimumab: review of use in systemic lupus erythematosus a phase ii, randomized, double-blind, placebo-controlled, dose-ranging study of belimumab in patients with active systemic lupus erythematosus a phase iii, randomized, placebo-controlled study of belimumab, a monoclonal antibody that inhibits b lymphocyte stimulator, in patients with systemic lupus erythematosus efficacy and safety of rituximab in moderately-to-severely active systemic lupus erythematosus: the randomized, double-blind, phase ii/iii systemic lupus erythematosus evaluation of rituximab trial the sting pathway and the t cell-inflamed tumor microenvironment mpys is required for ifn response factor activation and type i ifn production in the response of cultured phagocytes to bacterial second messengers cyclic-di-amp and cyclic-di-gmp nuclear import of dna in digitonin-permeabilized cells nuclear import in digitonin-permeabilized cells reversible membrane permeabilization of mammalian cells treated with digitonin and its use for inducing nuclear reprogramming by xenopus egg extracts liposomal drug delivery systems: from concept to clinical applications a new adjuvant delivery system "cyclic di-gmp/ysk liposome" for cancer immunotherapy liposomes loaded with a sting pathway ligand, cyclic di-gmp, enhance cancer immunotherapy against metastatic melanoma submicron-sized hydrogels incorporating cyclic dinucleotides for selective delivery and elevated cytokine release in macrophages funding hlw was supported by funds from the biotechnology and biological sciences research council (grant number bb/l / ) and the british heart foundation (grant number pg/ / / ). ekt was supported by funds from the british heart foundation (grant number pg/ / / ). authors' contributions all authors were involved in drafting and editing, they all read and approved the final manuscript. the authors declare that they have no competing interests. ethics approval and consent to participate n/a springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - k zseao authors: cinatl jr., j.; hoever, g.; morgenstern, b.; preiser, w.; vogel, j.-u.; hofmann, w.-k.; bauer, g.; michaelis, m.; rabenau, h. f.; doerr, h. w. title: infection of cultured intestinal epithelial cells with severe acute respiratory syndrome coronavirus date: journal: cell mol life sci doi: . /s - - - sha: doc_id: cord_uid: k zseao to identify a model for the study of intestinal pathogenesis of severe acute respiratory syndrome (sars) we tested the sensitivity of six human intestinal epithelial cell lines to infection with sars coronavirus (sars-cov). in permissive cell lines, effects of sars-cov on cellular gene expression were analysed using high-density oligonucleotide arrays. caco- and cl- cell lines were found to be highly permissive to sars-cov, due to the presence of angiotensin-converting enzyme as a functional receptor. in both cell lines, sars-cov infection deregulated expression of cellular genes which may be important for the intestinal pathogenesis of sars. cells infected with sars-cov at multiplicity of infection (moi) and moi were collected at different times post infection (p. i.) by trypsinization of adherent cells. non-adherent cells (the numbers of which increased with time after infection) were collected by centrifugation of culture supernatants. both adherent and non-adherent cells were fixed on glass slides with / methanol/acetone for min. immune peroxidase staining was performed using human immune serum obtained from a sars patient as described previously [ ] . to investigate whether ace is a functional receptor for sars-cov in intestinal epithelial cell cultures, the cells were pre-treated for min at °c with goat antibody directed against the human ace ectodomain (r&d systems; wiesbaden-nordenstadt, germany). after treatment, the cells were washed three times with phosphatebuffered saline (pbs) and infected with one of the sars-cov strains at moi . twenty-four hours p. i. the cells were fixed and stained for viral antigens as described above. goat anti-ace antibody (r&d systems) was used as control. both antibodies were added at a concentration of µg/ml. to investigate expression of cell surface ace , intestinal cell lines were washed twice with pbs and incubated for min with goat anti-ace antibody (r&d systems). after washing with pbs, the cells were incubated with fitc-conjugated anti-goat igg (becton dickinson, heidelberg, germany) for min. as controls, cells were stained with irrelevant primary antibody (goat antimouse igg; sigma biochemicals, seelze, germany) or without a primary antibody to determine unspecific and background fluorescence, respectively. instrument settings of the flow cytometer (facscan; becton dickinson) were adjusted to obtain background mean fluorescence in the histogram mode between and on the logarithmic scale. caco- cells were infected - days after reaching confluence with sars-cov at moi . one day p. i., the cells were processed for ultrastructural analysis as described previously [ ] . briefly, cells were pelleted and fixed with . % glutaraldehyde, post-fixed in % osmium tetroxide, dehydrated in ethanol and embedded in durupan-epon. thin sections were contrasted with uranyl acetate and lead citrate and viewed with a jeol jem, cx electron microscope (arishima, japan). to assess effects of sars-cov infection on caco- cell viability, confluent cell layers in -well plates were infected at moi and moi . the viability was measured at different times p. i. using the mtt assay performed as described previously [ ] . gene array analysis was done according to the principles of miame [ ] we used the affymetrix hg-u a chip (affymetrix, santa clara, calif.). this oligonucleotide microarray targets , genes. sample preparation was done by the rneasy mini kit (qiagen, hilden, germany) standard protocol. generation of biotin-labelled crna, hybridization and staining were done according to standard protocols available from affymetrix. data analysis was performed using microarray analysis suite (affymetrix) and genespring software version . (silicon genetics, san carlos, calif.) as published previously [ ] . in brief, the lowest raw data value was arbitrarily defined as ' ' in order not to eliminate genes which are expressed only in one sample. to eliminate false j. cinatl jr. et al. 'fold-change' calls, genes that were classified as 'up-regulated' had to be flagged as 'present' in the infected samples, while genes that were classified as 'down-regulated' had to be flagged as 'present' in the mock-infected samples. within those parameters, genes were selected if they were either up-or down-regulated at least threefold in duplicate. following microarray analysis, genes related to apoptosis, cytokines, chemokines or interferons were confirmed by rt-pcr, according to standard protocols [ ] . pcr primer and amplification conditions were determined by the software primer (whitehead institute for biomedical research, cambridge, mass.) [ ] . previously, we demonstrated that caco- cells are highly permissive to infection with sars-cov strain ffm- [ ] . since ace was identified as a functional sars-cov receptor in different cell types [ ] we measured whether its expression may correlate with the sensitivity of intestinal cell lines to sars-cov infection. caco- and cl- expressed ace mrna and protein which were not detectable in the other cell lines tested ( fig. a the relative abundance of specific mrna in sars-covinfected cells was compared to mock-infected confluent caco- cell cultures (same passage and identical culture conditions) h p. i. when cell viabilities were similar ( fig. b) . all gene expression experiments were done in duplicate and only genes which were up-or down-regulated in both samples underwent further evaluation. after applying strong restrictions as described in materials and methods, resulting genes were grouped according to their function (table ). we focussed on genes related to apoptosis, chemokines, interferon-induced genes and transcription factors, since these gene groups may play an important role in the pathogenesis of sars. expression of the selected genes was confirmed by rt-pcr ( fig. ). in the infected cells, we found an up-regulation of some anti-apoptotic genes including bcl- (only in caco- but not in cl- cells) and a , while several pro-apoptotic genes including bid, bad, caspase- and caspase- were down-regulated. on the other hand, the anti-apoptotic programmed cell death gene (pdcd ) was down-regulated in infected cells. increased levels of mrna of members of the ap- family of cellular transcription factors including c-jun . some viral particles attach onto the microvilli, whereas some detach from the cell surface (e). c, cytoplasm; n, nucleus. and c-fos were observed in the infected cells. concerning cytokine/chemokine-related genes, our results showed an up-regulation of several cxc chemokines; among the down-regulated genes we found interleukin (il)- and macrophage migration inhibitory factor (mif). several interferon-induced genes were up-regulated, including the human ¢- ¢ oligoadenylate synthetase gene (oas ) and human myxovirus resistance- gene (mxa). sars-cov strains ffm- and influenced similarly the expression of the selected genes ( fig. ). neither uv-inactivated virus nor virus-free filtered cell culture supernatants caused any changes in gene expression pattern compared to mock-infected cells (data not shown). to provide an experimental model for the study of sars gastrointestinal pathology, we tested the sensitivity of six intestinal cell lines to sars-cov infection. in addition to caco- cells which were previously shown to be per-missive to sars-cov [ ] , only cl- cells promoted sars-cov replication. cl- cells show features of well-differentiated enterocytes [ ] while caco- cells show an undifferentiated phenotype with the ability to undergo spontaneous enterocytic differentiation after reaching confluence [ ] . we infected caco- cells - days after confluence, i. e. when electron microscopy identified mostly poorly differentiated enterocytes and only few well-differentiated (villus) enterocytes. both poorly and well-differentiated enterocytes supported sars-cov replication suggesting that the sensitivity of intestinal epithelial cells does not depend on a particular stage of cellular differentiation. ace has recently been shown to be a functional receptor for sars-cov [ ] and surface ace is abundantly present on enterocytes of the small intestine [ ] . while it down-regulated pro-apoptotic genes such as bid, bad, caspase- and caspase- . in a murine model, bcl- overexpression in gut epithelial cells, decreased the apoptosis [ ] and protected against intestinal injury [ ] . although bcl- was detectable only in caco- cells, other regulators of the apoptotic mitochondrial pathway including pro-apoptotic bcl- homologues bid and bad were down-regulated by sars-cov in both caco- and cl- cells. since bid and bad are involved in the regulation of intestinal epithelial cell survival [ ] , their role in sars-cov intestinal infection should be studied further. in both cell lines, sars-cov up-regulated a which may protect different cell types against tumour necrosis factor (tnf)-mediated programmed cell death and is critical for limiting inflammation by terminating tnf-induced nuclear factor (nf)-kb responses in the intestine and other organs [ ] . in addition, down-regulation of caspase- and caspase- in infected intestinal cell lines may be of interest as these caspases were shown to be important mediators of apoptosis in gastrointestinal epithelium [ , ] . the results show that sars-cov-infected epithelial cells develop an anti-apoptotic response which may be important to inhibit or delay destruction of infected enterocytes. these findings are consistent with clinical observations demonstrating a relatively normal endoscopic and microscopic appearance of the intestine in patients with sars [ ] . on the other hand, sars-cov suppressed expression of the anti-apoptotic gene pdcd which is constitutively expressed in most normal tissues including lung and intestine [ ] . apart from its effects in the regulation of apoptosis, pdcd was shown to play a role in inhibition of translation by direct interaction with eukaryotic translation initiation factor a (eif a) [ , ] . this finding is of interest since activity of eif e (together with eif a and eif g forming the translation initiation factor complex eif f) was shown to be important for replication of murine coronavirus [ ] . moreover, pdcd has the ability to suppress transactivation of activator protein (ap)- [ ] . since the sars-cov nucleocapsid was shown to activate the ap- transduction pathway [ ] , it will be of interest to show whether there may be a mechanistic link between pdcd suppression and enhancement of ap- tranactivation in sars-cov-infected cells. the present observations demonstrate that sars-cov infection elevated mrna levels of ap- subunits c-fos and c-jun in intestinal cells which could also increase ap- transactivation. infection with most viruses up-regulates different interferon (ifn)-induced genes which may establish an anti-viral state within cells. its major effectors and indicators include double-stranded rna-dependent protein kinase (pkr), oas and mx proteins [ ] . sars-cov infection of caco- cells up-regulated oas and mxa but not pkr genes. the discrepancy between transcriptional activation of ifn-induced genes and the ability of sars-cov to replicate in caco- cells could be explained by the existence of a specific viral mechanism for escaping ifninduced anti-viral effects common to most viruses [ ] . the enteropathogenic potential of hcov-oc (strain paris) has been suggested to be due to its inability to induce ifn-a [ ] . recently, we showed that ifn-a and ifn-b (type i ifn) inhibited sars-cov replication in caco- cells while ifn-g (type ii ifn) was not effective [ ] . moreover, ifn-b was - times more potent than ifn-a against different sars-cov strains. the differences in anti-viral activity of different types of ifn could result from their ability to differentially influence expression of cellular genes important for anti-viral activity. for example, treatment of the human fibrosarcoma cell line ht (expressing both type i and type ii ifn receptors) with ifn-b stimulated pkr which was not stimulated by ifn-a or ifn-g [ ] . since pkr was not up-regulated in the infected caco- cells and virus replication progressed despite up-regulation of oas and mxa, a role for pkr in sars-cov replication must be elucidated. although enteric pathogens such as viruses, protozoans, multicellular helminths and enteroinvasive bacteria vary in their mode of infection, enterocytes display a common chemokine/cytokine profile in response to infection. several elr+cxc chemokines (containing a conserved glutamate-leucine-arginine sequence) including cxcl (groa), cxcl (grob), cxcl (grog) and cxcl (il- ) were up-regulated in sars-cov infected caco- cells. these chemokines mainly regulate neutrophil trafficking [ ] . in addition, sars-cov induced in caco- cells non-elr cxc chemokines cxcl /ifn-g inducible protein- (ip- ) and cxcl /ifn-inducible t cell alpha chemoattractant (i-tac) which are potent cd + t cell chemoattractants [ , ] . rotavirus infection was shown to induce cxcl , cxcl and cxcl in intestinal cell lines [ ] . mucosal inflammation associated with rotavirus infection is predominantly mononuclear, i. e. consists of monocytes and t lymphocytes [ ] , although neutrophil infiltration is found in some cases [ ] . in contrast, biopsy specimens taken from the colon and terminal ileum of patients with sars failed to demonstrate any inflammatory infiltrates [ ] . neutrophil infiltration in the intestine of sars patients may be limited despite neutrophilia due to changes of cytokine/ chemokine levels in the intestinal environment. we observed that sars-cov infection of caco- cells inhibited expression of il- which is constitutively expressed in intestinal epithelial cells [ ] . suppression of il- levels reduces neutrophil accumulation in liver and lungs [ ] . the absence of t lymphocyte infiltration of the intestine in sars may be a consequence of the profound decline of both cd + and cd + lymphocytes in the blood [ ] , possibly resulting from lymphocyte apoptosis [ ] . although macrophage counts were increased in lungs [ ] , macrophage infiltration was absent from the gut of sars patients [ ] . in caco- cells, sars-cov down-regulated mif. recently, mif was identified as a major factor produced by intestinal cells in response to microbial infection regulating macrophage emigration, inflammation and cell metabolism [ ] . some of the chemokines we found up-or down-regulated in vitro were also changed in serum samples from sars patients. for example, serum levels of cxcl and il- were increased whereas il- was decreased [ , ] . this justifies the use of intestinal cell lines as a model to study the direct effects of sars-cov infection on gene expression in permissive human cells. given the intestinal tropism of sars-cov, the results presented here provide several important hints at possible mechanisms of intestinal pathogenesis and potential novel therapeutic targets in sars. identification of a novel coronavirus in patients with severe acute respiratory syndrome critically ill patients with severe acute respiratory syndrome angiotensin-converting enzyme is a functional receptor for the sars coronavirus clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study enteric involvement of severe acute respiratory syndrome-associated coronavirus infection clinical features and short-term outcomes of patients with sars in the greater toronto area viral replication in the nasopharynx is associated with diarrhea in patients with severe acute respiratory 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enteritis il- , a novel immunoregulatory cytokine, is up-regulated in crohn's disease: j. cinatl jr. et al. sars-cov infection of intestinal cells expression and localization in intestinal mucosal cells interleukin- and host defense against infection expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome sars: understanding the coronavirus. apoptosis may explain lymphopenia of sars lung pathology of fatal severe acute respiratory syndrome ubiquitous production of macrophage migration inhibitory factor by human gastric and intestinal epithelium dynamic changes in blood cytokine levels as clinical indicators in severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome acknowledgements. the authors wish to acknowledge valuable technical support by g. meincke, l. stegmann and c. sippel. the authors are grateful to h. kabickova for transmission electron microscopy experiments. key: cord- -mbrw og authors: flego, michela; frau, aldo; accardi, luisa; mallano, alessandra; ascione, alessandro; gellini, mara; fanunza, elisa; vella, stefano; di bonito, paola; tramontano, enzo title: intracellular human antibody fragments recognizing the vp protein of zaire ebola filovirus inhibit the protein activity date: - - journal: bmc biotechnol doi: . /s - - - sha: doc_id: cord_uid: mbrw og background: ebola hemorrhagic fever is caused by the ebola filovirus (ebov), which is one of the most aggressive infectious agents known worldwide. the ebov pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. the multifunctional viral vp protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (ifn-α/β) response, and represents a suitable target for the development of strategies to control ebov infection. phage display technology permits to select antibodies as single chain fragment variable (scfv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. scfv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scfv is expressed as intracellular antibody (intrabody) or delivered into the cells. results: monoclonal antibodies (mab) in scfv format specific for the ebov vp were isolated from the eth- library of human recombinant antibodies by phage display technology. five different clones were identified by sequencing, produced in e.coli and expressed in cho mammalian cells to be characterized in vitro. all the selected scfvs were able to react with recombinant vp protein in elisa, one of the scfvs being also able to react in western blot assay (wb). in addition, all scfvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in a cells showed that two of the scfvs can significantly hamper the inhibition of the ifn-β-induced rig-i signaling cascade mediated by ebov vp . conclusion: five antibodies in scfv format recognize an active form of ebov vp in elisa, while one antibody also recognizes vp in wb. two of these scfvs were also able to interfere with the intracellular activity of vp in a cell system in vitro. these findings suggest that such antibodies in scfv format might be employed to develop therapeutic molecules able to hamper ebov infections. ebola hemorrhagic fever caused by the ebov is one of the most aggressive zoonoses affecting humans, leading to death within a few days of the exposure [ ] . six species of ebov are known to date, named after the geographical region in which they were first isolated: bundibugyo, reston, sudan, taï forest (formerly côte d'ivoire ebov), zaire and bombali ebov. sudan, taï forest, and zaire ebov are responsible for outbreaks in humans, whereas reston ebov infects non-human primates, and bombali virus was recently discovered in bats [ , ] . fatal ebov infections are characterized by rapid viral replication combined with an inadequate antiviral response. hallmarks of fatal cases are immune suppression with t cells levels below the normal level, no cd t cell activation, delay of antibody response in the blood, and high viremia ( genome copies/ml serum). the ebov pathogenesis starts with the subversion of both innate and adaptive immune response and the consequent induction of harmful inflammatory responses and tissue necrosis due to disseminated infections [ , ] . ebov has a linear, single-stranded, negative rna genome about , nucleotides in length. it is composed of seven genes coding for eight proteins in this order: np (encoding the nucleoprotein), vp , vp , gp (encoding the glycoproteins), vp , vp , l (encoding the polymerase). the gp gene codes for the two molecular forms gp and gp , generated by rna editing [ ] . vp is a conserved multifunctional protein which is a cofactor of the viral rna polymerase complex along with the np, vp , and l protein. its activity starts at an early stage of the ebov infection; it is also a doublestranded rna-binding protein shown to be implicated in hampering the innate immune response [ ] by blocking the ifn-mediated antiviral activity through multiple inhibitory effects which include disruption of the rig- pathway by preventing irf- phosphorylation [ , ] , and inhibition of activation of the ifn-inducible dsrna and dicer-dependent protein kinase r [ ] . antibody phage display technology makes it possible to select from an artificial immune system human antibodies in scfv format capable of specifically recognizing an antigen of interest. scfvs, consisting of the vh and vl chain regions of a whole immunoglobulin (ig), are the smaller portion still retaining the binding properties of the parental ig. this format is ideal for genetic manipulation in order to obtain antibody constructs potentially useful for diagnostic and therapeutic applications [ ] . furthermore, scfv antibodies can be expressed inside the cell as intrabodies so as to bind to their intracellular target antigen [ ] . the two main mechanisms underlying the efficacy of intrabodies are: ) knockdown of the activity of cytosolic antigens through cytosolic intrabodies [ ] [ ] [ ] ; ) diverting of antigens from their natural intracellular compartment by scfv binding due to an extra-signal for intracellular localization [ ] [ ] [ ] . intrabodies can be used to reveal the function of proteins by interfering with their function, although the possibility of targeting intracellular antigens gives them a therapeutic potential for a number of viral infections [ , ] , neurological diseases [ , ] and cancers [ , , , ] . this study reports the selection by phage display and characterization of different human scfv antibodies binding to an active form of the zaire ebov vp [ , ] . the ability of these scfvs, expressed as cytosolic intrabodies, to reverse the inhibition of type i ifn induction by intracellular expression of vp was tested by a luciferase reporter gene inhibition assay in a cells treated with dsrnas [ ] . in order to isolate antibodies specific for the recombinant vp expressed in e.coli and purified in an active form [ , ] , an approach based on the phage display technology was used. in the eth- library which we used, the diversity (about clones) has been introduced in the complementary-determining region (cdr ) of both the variable heavy chain (vh) and variable light chain (vl) domains [ ] . to recover antigen-specific antibody phages, an aliquot of the eth- antibody library containing cfu phage was used for the panning procedure as described elsewhere [ , ] . in fig. soluble scfvs derived from iptg-induced colonies were screened by elisa to find those specific for the vp protein. all the e. coli colonies corresponding to the clones exhibiting an od λ value in elisa higher than . , were grown and subjected to dna extraction and sequence analysis. several clones had identical nucleotide sequences and five different clones, namely b , a , e , f and h , were identified; the amino acid composition of the complete sequences of the vh and vl domains is shown in fig. along with the schematic representation of a scfv gene in the phagemid cassette. the scfv reactivity towards vp was further characterized by elisa (fig. , panel a) and wb (fig. , panel b) using vp recombinant antigen. the protein glucose oxidase (go) and an anti-go scfv for detection were used as negative controls. the anti-vp reactivity in elisa was confirmed for all b , a , e , f and h scfvs, while in wb the positivity was only observed for scfv a , which reacted with a kda protein identified as the recombinant his-tagged vp protein also detected by the anti-his mab. the scfvs b , e , f , h recognized their antigen in elisa but showed no reactivity in denaturing conditions of wb, suggesting that they probably recognize conformational epitopes. for its part, a is still reactive in wb and probably recognizes a linear epitope. the specificity of the anti-vp reactivity of b , a , e , f and h scfvs is confirmed by the observation that they did not react with the irrelevant go antigen either in elisa or in wb (fig. ) . in order to use the scfvs in the ebov vp luciferase reporter gene inhibition assay, the scfv genes selected were pcr amplified with opportune oligonucleotides and cloned into the ptarget vector for expression in the eukaryotic system. in view of the cytoplasmic vp localization, it was not necessary to provide the scfvs with signal sequences for expression in specific cell compartments. transfection experiments were performed in the cho cells to evaluate the functionality of the scfv ptarget constructs as described in methods. the a , h , b , f and e constructs were all able to express scfvs of the expected molecular mass in eukaryotic cells (fig. , panel a) . evaluation of the anti-ebov vp scfvs ability to restore the ifn-β activity by a luciferase reporter gene inhibition assay to evaluate the capability of the scfvs b , a , e , f and h to block the vp activity, the cell-based miniaturized luciferase reporter gene inhibition assay, previously described [ ] , was used. the assay measures the capacity of the vp expression to inhibit the ifn-β induced by dsrna treatment, in a cells. before performing the ebov vp luciferase reporter gene inhibition assay using the scfvs, it was crucial to exclude that the expression of an irrelevant scfv could influence the ifn-β induction, either in the absence or in the presence of vp expression. to this end, a cells, transfected with the pgl ifn-β luc and the pcdna -ebov-vp expression plasmid, were co-transfected with the irrelevant anti-go scfv expression plasmid. further, to exclude any non-specific effect due to the transfection procedure, the cells were co-transfected in parallel with the pgl ifn-β luc expression vector and an empty pcdna vector. the luciferase signal emitted in the case of pgl ifn-β luc and anti-go scfv concurrent expression, was comparable to that obtained with the ifn-β positive controls. also, in the case of ebov vp and anti-go scfv simultaneous expression, the measured ifn-β signal was comparable to that obtained with the ebov vp expressed alone (fig. , panel b) . the results confirmed that ifn-β induction was affected by neither the transfection procedure nor the irrelevant scfv either in the presence or in the absence of vp expression. next, we tested all the scfv ptarget constructs in the dsrna rig-i-mediated luciferase reporter gene inhibition assay. two of the five scfvs tested, f and e , showed a significant ability (p = . and p = . , respectively) to subvert the inhibition of the ifn-β production generated by dsrna, after the vp expression. by contrast, scfv h , a and b showed no ability to counteract the inhibition of ifn-β induction mediated by ebov vp in the cellular assay (fig. , panel c). competitive elisa using the scfv-expressing phage to verify whether the two different scfvs e and f , able to subvert the inhibition of the ifn-β production, targeted different epitopes, we performed a competitive elisa (fig. ). this relies on detection of the scfv-expressing phage particles that compete with soluble non-phage-fused scfvs for binding to the antigen immobilized on elisa plate. it was not possible to detect the soluble non-phage-fused scfvs using the anti-tag flag ab because the tag is also present on the phage. to determine the best scfv expressing-phage concentration for competition tests, identified as × tu/ml, we first performed a phage elisa experiment in which different scfv-expressing phage concentrations were tested on vp coated plates (data not shown). in a competitive assay, elisa plates coated with vp or with the control antigen go were blocked; they were then incubated with purified scfv-expressing phage both in the absence and in the presence of the competitor soluble non-phage-fused scfv at the maximum concentration of μg/ml. the binding of the scfv-expressing phages was measured. the detection step was via the phage coat protein using an anti-m mab conjugated to hrp. the anti-go scfv-expressing phage clone was assayed against the anti-go soluble nonphage-fused scfv as a positive control for competitive measurement (fig. , panel a) . it was found that μg/ml of soluble non-phage-fused scfv could inhibit the binding of the scfv-expressing phage in a dose-dependent manner. binding in the presence of specific anti-go soluble non-phage-fused scfv was compared: to binding in the presence of e anti-vp soluble non-phage-fused scfvs (p value = . ); to binding in the presence of f anti-vp soluble non-phage-fused scfvs (p value = . ); to binding in the absence of soluble non-phage-fused scfvs (p value = . ) using student's t-test. next, we assayed the binding of e scfv-expressing phages both in the absence and in the presence of the competitor soluble non-phage-fused scfv against itself as an intrinsic positive control, against anti-go soluble non-phage-fused scfv as a negative control and against f soluble non-phage-fused scfv for competitive measurement. the inhibiting activity was determined at a single fixed concentration of μg/ml. f soluble nonphage-fused scfv shows a clear competitive effect at the concentration used (fig. , panel b) . binding in the presence of f soluble non-phage-fused scfv was compared to the binding in the presence of anti-go soluble non-phage-fused scfv (p value = . ) and in the absence of soluble non-phage-fused scfvs (p value = . ) using student's t-test. as a further confirmation, we performed a one-shot experiment by detecting the competition suffered by the f scfv-expressing phages when co-incubated with e soluble non-phage-fused scfvs and with the control anti-go soluble non-phage-fused scfv at the concentration of μg/ml. also in this case we observed that the control anti-go soluble non-phage-fused scfv has no competitive binding effect. the e soluble non-phage-fused scfv competes with the binding of f scfv expressing-phages. binding in the presence of e soluble non-phage-fused scfvs was compared to the binding in the presence of anti-go soluble non-phage-fused scfv (p value = . ) and to binding in the absence of soluble non-phagefused scfvs (p value = . ) using student's t-test (data not shown). the urgency to find effective counter measures for the ebov disease (evd) was strengthened by the west africa outbreaks occurring in - , which resulted in , cases of ebola with , deaths [ ] , pushing the international scientific community to investigate the widest possible range of defense strategies to counteract the virus. however, the current ebola outbreak, which started in may in democratic republic of the congo (drc) and has caused cases and deaths to date [ ] , received only minor benefits from the use of new diagnostic assays, vaccines and drugs. the reason is that to control ebola outbreaks, transversal coordination between health facilities and communities is essential to achieve rapid isolation of the cases of the disease and to stop the transmission chain. detection of new cases at an early stage by rapid diagnostic tests and ring vaccination strategy with experimental vaccines on volunteers are therefore crucial for controlling ebov in the current drc outbreak [ ] . due to the variable onset of antibody response in the ebola-infected subjects, serology is not used in the acute evd diagnosis. conversely, virus and viral proteins accumulate in blood to detectable levels within a few days from disease onset. molecular tests based on the detection of viral proteins were developed and proved to be effective for diagnosis in acute infection [ ] . currently, in the case of suspected ebov infection, the world health organization (who) recommends a list of validated tests for detection of either viral rna by rt pcr or viral antigens by immunological tests [http://www.who. int/medicines/ebola-treatment/emp_ebola_diagnostics/en/]. most of the antigen-capture tests used in national reference laboratories [ ] utilize mabs generated in mice immunized with the recombinant np [ ] , vp [ ] or gp [ ] ebov proteins. during the recent outbreak, lateral flow immunoassays (lfis) emerged as powerful tools for rapid antibody-mediated antigen-capture practicable at the point of care [ ] . this confirmed the advantages of tests based on antigen-antibody reaction over rt-pcr methodology, which requires significant laboratory infrastructures often lacking in low-income countries. furthermore, in order to control the transmission-chain of the infection and limit virus spread, it is important to have tests that can be easily automated; as such, antigen capture tests meet this requirement. ebov vp is a validated drug target for which only a few small molecules have been reported to be active [ , , ] , albeit no drug has yet been approved. here, we present mabs in scfv format, which are able to react with the zaire ebov vp protein. this is a key viral protein whose action starts at an early stage of the infection and is based on interference with the host immune response by blocking the ifn-mediated antiviral activity. the antibodies presented here enrich the list of available anti-vp antibodies. they could be used individually or in combination to develop novel reagents for ebov vp detection and therapeutics. regarding therapy, pools of neutralizing antibodies have been used in passive immunization of individuals with acute infection [ ] ; nevertheless, antibodies specific for the vp would act with a different mechanism with respect to neutralizing antibodies targeting surface glycoproteins. recent studies showed that targeting vp by either nucleic acid mimics or sirnas, provides protection against the ebov infection in murine [ ] as well as in non-human primate models [ ] . however, the effectiveness of antibodies targeting the vp has not yet been demonstrated in vivo. here, we show that two out of five scfv antibodies selected against the vp significantly hindered the inhibition of the rig-i signaling cascade mediated by vp , in a cellular system. we characterized the binding of these two scfv clones to the recombinant vp antigen by competitive elisa, and found that their epitope is at least partly shared. a more detailed analysis of the binding epitopes could further define whether it is a total or partial sharing and which antigenic region of ebov vp is involved in the inhibition of the interferon pathway. regarding the other anti-vp scfvs selected, we cannot exclude that the observed lack of functionality is due to incorrect scfv folding in the cytoplasmic environment. as antibodies are usually produced in an oxidizing biochemical environment with the help of er-based chaperones, only a fraction of naïve antibodies can be folded correctly in the reducing cytoplasmic environment which prevents the formation of disulfide bridges. however, many examples of successful intrabodymediated proteins knockdown in vitro, obtained using cytosolic intrabodies, have been reported in literature [ ] . scfvs selected in the extracellular environment were previously reported to work intracellularly [ , , ] depending on intrinsic biophysical characteristics such as stability, mainly ascribable to the scaffold. however, several methods have been developed to address the issue of cytosolic intrabodies functioning [ ] . interestingly, anti-vp scfvs able to interfere with vp activity, were recently isolated from a phage library different from the eth- and were linked to a cell-penetrating peptide for intracytoplasmic delivery [ ] . therefore, although we used a different delivery system, our data strengthen the idea that intracellular antibodies in scfv format can be used to counteract ebov vp activity. scfvs against different intracellular ebov targets could be used either to develop a well-defined cocktail of antibodies with different specificities or also in combination with other drug molecules for therapeutic purposes, provided that an appropriate delivery system is developed. furthermore, the vp amino acid sequence is highly conserved among the zaire ebov isolated in several outbreaks (> . % amino acids identity). therefore, it can be hypothesized that the scfvs selected retain a broad-spectrum activity. nevertheless, the efficacy of these new antibodies should be further evaluated either in ebolavirus infected cells or in animal models of ebolavirus infection. five scfv antibodies against an active form of the zaire ebov vp were isolated and characterized. their specific reactivity in elisa and wb suggests the possibility of developing novel reagents for ebov vp detection during the virus life cycle. the two anti-vp scfvs f and e proved to be able to interfere with the vp -depending inhibition of ifn activity in a cell system, so suggesting that such antibodies also represent potential therapeutic agents. further investigations into an ebov infection system in vitro and in animal models are necessary to validate these reagents. the synthetic library of recombinant human antibodies (eth- ) consists of about scfv polypeptides displayed on the surface of the m phage. the library was built by random mutagenesis of the complementarity-determining region (cdr ) of the variable domains of both the heavy (vh) and light (vl) chain of immunoglobulins, using only three antibody germline gene segments (dp for the vh, dpk , and dpl for the vl). in the vh, diversity was created by randomizing four to six positions replacing the pre-existing positions to of the cdr ; in the vl, diversity was obtained by randomizing six positions ( to ) of the cdr [ ] . an aliquot of the eth- library, containing cfu phage, was used to isolate specific human antibodies in scfv format against the recombinant vp protein ( ) . immunotubes (nunc maxisorp; denmark) were coated overnight (on) at room temperature (rt) with purified recombinant vp protein ( μg/ml in pbs). after panning, phages were eluted with ml of mm triethylamine and the solution was immediately neutralized by adding . ml of m tris-hcl ph . . the eluted phages were used to infect an e. coli tg strain in a log phase, and amplified for the next round of selection, as described in flego et al. [ ] . three rounds of panning were performed to recover vp -specific antibody phages from the eth- library. for the preparation of soluble anti-vp scfvs, individual colonies were grown in flat bottomed wells (nunc) for h at °c in μl of . % glucose xyta medium and induced with μl of mm iptg/ xyta medium. the following day, the plates were spun down at g for min, and the supernatants containing soluble scfvs were recovered and tested for specificity with purified vp in elisa and wb, as described in flego et al. [ ] . plasmid dna from individual bacterial colonies clones was extracted using the quiaprep spin miniprep kit and subjected to enzymatic restriction; sequence analysis of the cdr regions was then performed with an automated dna sequencer (biofab, roma, italy) using the fdseq ( ′-gaa ttt tct gta tga gg- ′) and pelbback ( ′-agc cgc tgg att gtt att ac- ′) primers. the scfv gene clones reacting with the recombinant vp in elisa, were pcr amplified using the following primers: eth nco as a forward primer: ′ gcgc acc atg gcc gag gtg cag ctg ′. nhe i stop his as a reverse primer: ′ gcgc gct agc cta atg atg atg atg atg atg tgc ggc cgc gcc tag gac ′ containing the xhis tag sequence. for transient expression in eukaryotic cells, the amplimers were cloned in ptarget (promega) under the strong viral promoter (cytomegalovirus immediate-early enhancer). the clones obtained were sequenced to check for mutations possibly introduced by pcr, and used to transfect cho cells using jetpei dna transfection reagent, according to the manufacturer's instructions. transiently transfected cells were lysed after h with sds-loading buffer ( mm tris-hcl ph . , % sds, % -mercaptoethanol, % glycerol), loaded onto % sds-page, and then transferred to a nitrocellulose membrane using standard procedures. the membrane was blocked in % mpbs on at rt. blotted proteins were incubated for h in % mpbs with μg/ml of tet-ra·his antibody (qiagen), which was the only anti-his mab able to recognize the tag at the scfv cooh terminus, in our experimental conditions. after an additional incubation for h at rt in the presence of goat anti-mouse antibody hrp-conjugate ( μg/ml, dako), the reaction was developed and visualized with a chemiluminescence detection kit (pierce; il, usa). luciferase reporter gene assay ifn-β induction luciferase reporter gene assays a cells ( × per well) were transfected in -well plates with t-pro p-fect transfection reagent (t-pro biotechnology) with the construct pgl ifn-β luc, kindly provided by prof. stephan ludwig (institute of molecular virology, münster, germany). twenty-four hours after transfection, cells were additionally transfected using iav pr vrna and incubated for a further h at °c with % co . cells were harvested with lysis buffer ( mm na-mes ph . , mm tris-hcl ph . , mm dithiothreitol, . % triton x- ). the crude cell lysates were cleared by centrifugation and μl of cleared lysates were added to μl of luciferase assay buffer ( mm na-mes ph . , mm tris-hcl ph . , mm magnesium acetate, . mg/ ml atp) in a white -well plate. immediately after addition of μl of mm d-luciferin into each well, the luminescence was measured in victor luminometer (perkin elmer). the relative light units (rlu) were normalized as the fold activity of the unstimulated control. each assay was carried out in triplicate. the above described luciferase reporter gene assay was also performed for evaluating the ifn-β induction inhibition mediated by ebov vp . twenty-four hours after co-transfection with pgl ifn-β luc and pcdna or pcdna ebov wtvp expression vectors, cells were transfected with the ctrl anti-go scfv ptarget as an irrelevant scfv and with the different scfv p target vectors the next day, cells were additionally transfected with iav vrna. inhibition of luciferase expression was indicated either as the fold activity of the unstimulated control or as a percentage of the induced control. each assay was carried out in triplicate. competitive elisa using scfv-expressing phages for elisa competition assay, we used soluble nonphage-fused scfvs produced and purified as in gellini et al. [ ] . scfv-expressing phages were produced from a monoclonal bacterial culture grown to od = . - . and infected with m k helper phage in a ratio of around : phage/bacteria. one hundred ml of supernatant containing scfv-expressing phages were x concentrated by precipitation with peg and resuspended in pbs. phage titer was determined by plating of bacteria infected with phages at scalar dilutions. coating was performed with vp or go antigen as described in the elisa section. the following day, the plate was blocked with % mpbs for h at rt and washed with tpbs. twenty-five μl of soluble non-phage-fused scfvs at two-fold the desired final concentration were pre-incubated in % mpbs with the antigen for min prior to the addition of μl of the scfv-expressing phage mix at two times the desired final tu/ml in % mpbs, and incubated for h at rt. the plate was washed with tpbs and incubated with anti-m pviii coat protein mab conjugated to hrp (amhersham) diluted : , for h rt. a washing step was conducted followed by the addition of peroxidase substrate as described above. ebola haemorrhagic fever ebola virus disease the discovery of a new ebolavirus, bombali virus, adds further support for bats as hosts of ebolaviruses ebola virus: new insights into disease aetiopathology and possible therapeutic interventions how ebola and marburg viruses battle the immune system strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit the ebola virus vp protein functions as a type i ifn antagonist ebola virus protein vp impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk- ebola virus vp antagonizes pkr activity through its c-terminal interferon inhibitory domain production technologies for monoclonal antibodies and their fragments expression and targetingof intracellular antibodies in mammalian cells generation and functional characterization of intracellular antibodies interacting with the kinase domain of human egf receptor intracellular antibody capture technology: application to selection of intracellular antibodies recognising the bcr-abl oncogenic protein effects of intrabodies specific for rotavirus nsp during the virus replicative cycle intracellular anti-e human antibodies in single-chain format inhibit proliferation of hpv -positive cervical carcinoma cells in vivo antitumor effect of an intracellular single-chain antibody fragment against the e oncoprotein of human papillomavirus a novel intracellular antibody against the e oncoprotein impairs growth of human papillomavirus -positive tumor cells in mouse models characterization and binding of intracellular antibody fragments to the hepatitis c virus core protein generation and characterization of single-chain anti-body fragments specific against transmembrane envelope glycoprotein gp of maedivisna virus isolation of a human single chain antibody fragment against oligomeric α-synuclein that inhibits aggregation and prevents α-synuclein-induced toxicity direct in vivo intracellular selection of conformation-sensitive antibody domains targeting alzheimer amyloid-β oligomers human singledomain neutralizing intrabodies directed against etk kinase: a novel approach to impair cellular transformation a nanobody targeting the f-actin capping protein capg restrains breast cancer metastasis purification and functional characterization of the full length recombinant ebola virus vp protein expressed in e. coli dsrna binding characterization of full length recombinant wild type and mutants zaire ebolavirus vp a luciferase reporter gene assay to measure ebola virus viral protein -associated inhibition of double-stranded rna-stimulated, retinoic acid-inducible gene -mediated induction of interferon β identification of myricetin as an ebola virus vp −doublestranded rna interaction inhibitor through a novel fluorescence-based assay design and use of a phage display library. human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel design and use of phage display libraries for the selection of antibodies and enzymes generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (n) sars-cov protein using a phage display approach ebola: anatomy of an epidemic who regional office for africa. ebola virus diseases new ebola outbreak declared in democratic republic of the congo rapid diagnosis of ebola hemorrhagic fever by reverse transcription-pcr in an outbreak setting and assessment of patient viral load as a predictor of outcome identification of essential outstanding questions for an adequate european laboratory response to ebolavirus zairewest africa detection of ebola viral antigen by enzyme-linked immunosorbent assay using a novel monoclonal antibody to nucleoprotein development, characterization and use of monoclonal vp -antibodies for the detection of ebola virus production of monoclonal antibodies and development of an antigen capture elisa directed against the envelope glycoprotein gp of ebola virus strategies in ebola virus disease (evd) diagnostics at the point of care antiviral agents against ebola virus infection: repositioning old drugs and finding novel small molecules insights into ebola virus vp and vp interferon inhibitory functions and their initial exploitation as drug targets, infectious disorders -drug targets a randomized, controlled trial of zmapp for ebola virus infection vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-of-concept study specific in vivo knockdown of protein function by intrabodies human transbodies that interfere with the functions of ebola virus vp protein in genome replication and transcription and innate immune antagonism generation of human single-chain antibody to the cd cell surface determinant specifically recognizing ewing's sarcoma tumor cells publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank philochem for the generous permission to use of the eth- antibody phage display library. we thank martin bennett for his help in revising the manuscript for grammar and style. authors' contributions mf isolated and characterized the scfvs, participated in the design of the research and drafted the manuscript. aa and mg performed wb analyses and scfv production. am worked on the design and the genetic construction of the anti-vp scfvs in ptarget vector and their expression in cho cells. pdb and la expressed the anti-vp scfvs in the ptarget vector and critically reviewed the manuscript. af and ef produced the recombinant vp , carried out ebov vp luciferase reporter gene inhibition assay and analyzed the data. sv coordinated the study design on the basis of the scfv isolation. pdb and et conceived, coordinated and supervised the study. all authors have read and approved the manuscript. this work was supported by sardinia regional government grants lr / (crp- /f i ), and intramural fund of istituto superiore di sanità. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable the authors declare that they have no competing interests. key: cord- -pol qm authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: pol qm nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on - march . when, in the mid- s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in provided stateof-the-art information and consensus on factors involved in injury and sepsis. in , the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until or . however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il- to il- , il- and il- and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in , to be dealt with at this symposium and congress? ( ) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. ( ) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. ( ) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. ( ) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. ( ) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. ( ) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~ ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha- macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro -iarg] ]aprotinin (bay ) or the b kinin antagonist, hoe on the hypotensive response to dxs. in the first study, anesthetized miniature pigs ( pigs/group, randomly assigned) were given one of the following treatment protocols: ) dxs ( mg/kg), - ) dxs plus bay ( , , , or rag), or ) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c adesarg and fibrin mom)mer were all increased. bay produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay . llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs ( /group) were given either dxs alone ( mg/kg) or dxs minutes after a bolus injection of hoe ( #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse , d- munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion ( , mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to u/ . in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to u/ . in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs ( ). when very small plasma kailikrein activities ( , u/kg bodyweight) were given intravenously a % decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c -inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin- ( x iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of , or u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion ( - u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c -inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; thrombosis research center, temple university, penn., usa; oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c b which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c b/c and c b/c, and a significant reduction was observed in animals that had received a lethal dose of e. coli together with mab c (treatment group), compared to animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-= antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c a, c , and c a were measured in patients from an internal intensive care unit. patients were clinically septic defined by the criteria of bone et al.(l) . the remaining patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l days period. during the first days blood samples were drawn every h, on day - every h and the last days once daily. mean plasma concentrations of c a within the first h after clinical onset of sepsis were + pg/ml, whereas non-septic-patients exhibited mean values of only +_ p_g/m/. c levels were lower for septic-patients ( + lag/ml) than for non-septic-patients ( _+ lag/ml). the most profound difference between both groups was found, when the c a/c ratio was compared ( . + . for septic-patients and . _+_ . for the control group). no significant differences between both patient groups were observed in c a plasma levels ( . + . ng/ml in septic-patients vs. . _+ . ng/ml in control patients). in of cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n= ) and non-survivors (n=l ) within the septic-group revealed, that the c a/c ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c a and c in only - minutes ( ) and the significant difference of the c ajc ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of amino acids with cysteine residues (mr daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw , behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of . p.g/kgxh thromboplastin for hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to . % and , % respectively, and by an increase of fibrin monomers from . to > . ~tg/ml. we administered rec. hirudin to rabbits in different concentrations ( . , . and . mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin ( . , . and . mg/kg i.v. bolus) as late as hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, marburg, germany $ novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r- ) and its effect on sepns-lnduced organ injury in rat atsuo murata , hitoshi toda , ken'ichi uda , hidewaki nakagawa , takesada mori , hideaki morishita , tom yamakawa , jiro hirese , atsushi ni~ , nariaki matsuura osaka university medical school, osaka, mochida pharmaceutical co. ltd. tokyo, wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je which lacks the membrane lipoprotein. the recombinant second domain (r- ) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r- was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r- significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai., ) . the cathepsin l-like and cathepsin b-like activity were measured in serum of patients with chronic bronchitis ( -with obstructive, -with nonobstructive bronchitis),acute bronchitis ( ) and healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai., ) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls ( - fold) .after treatment there was a tendency to normalization of indices,but the increase was about - % more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis - - -times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/ - diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il- / , ifn-a, il- , il- , il- , paf, pge, ltb and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur ( , ), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/ selectively t cells to produce lymphokines including i - and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/ + t cells accumulate within - min mrna for the ii- , i - , tnfai~ and ifn- ,. parallel in time high concentrations of bloodborne i - and tnf are noted. anti tnf t~// neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within - h distinct levels of tolerance to seb become discernable on ligand reactive v/ t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd , cd , cd accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c h/hej mice. the absence of sensitization to lps in lps d c bl/ sccr mice was identified as being due to their inability to produce interferon- (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c bl/ sccr mice concerns only the ifn response to bacteria, since the response to the t-cell mitogen con a and to cd monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c bl/ sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il- levels, whereas treatment with aztreonam resulted in progressively increasing levels of il- and a significant increase in mortality. the increased mortality in pbp- specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as -to lo -fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf- dgaln-treated mice with e. coli olll:b resulted in an lds of approximately . x cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection ( fold vs fold respectively, p< . ). parallel studies with endotoxin hyporesponsive c h/hej mice indicate significantly greater levels of protection with imipenem (> fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since , when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe ; pollack ) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: ) enhancing of opsonization and phagocytosis(antibactericidai activity) ) synergistic effects with [ - actam antibiotics ) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis ) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il- release in mice (baumgartner , corriveau and danner ) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger (berger , , in animal models (stephan , berger and also in prospective, randomized, controlled clinical trials (schedel , poynton , behre . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg ak) has been selected and chimerized into a human igglk (sdz - ). in elisa and in immunoblots on purified lps both sdz - and wni - show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz - and wni - have biological activity as they inhibit the lal assay and the secretion of monokines (il- and tnf) by mouse and human macrophages. moreover, sdz - and wni - inhibit the release of il- and tnf in vivo. in vivo sdz - as well as wni - neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni - as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch basel, $chweiz $ presentation of lps to cd by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd (mcd ). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd (scd ). these complexes of lps with scd are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd , either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd . these results show that the delivery of lps to scd is catalysed by lbp, i.e., lbp is not included with the lps-scd complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd . the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm , ai , ai , gm , and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm- , n. torrey pines rd. la jolla, ca usa . modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d- borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i _- , and il- , which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound ) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin- and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a % amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd receptors. a recombinant protein (rbpi ), corresponding to the amino terminal kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi will have to successfully compete with relatively high serum levels of lbp ( - ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of . nm for rbpi and nm for rlbp. in a competition assay format rbpi was approximately -fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi at . ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a -fold weight excess of rlbp over rbpi . and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions - , - and - . a single synthetic peptide ( - ) was bactericidal. these results suggest that rbpi contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p and p receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only % of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il- [ -inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered ( kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in % plasma +/-bpi ( ng/ml). pbmc were incubated for hours at °c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n= , mear~+sem, *p< . vs control). control . _+ + bpi . + % plas. . _+ + bpi . _+ pmp (ng/ml) lps (ng/ml) . _+ . _+ . _+ . _+. . +. . _+. . _+. " _+ " . _+ " . _+ " . _+. . + _+ _+. * _+ " . +. " . + -+ . -+ " . _+. " cba, c bl/ , balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection - % of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats ( - g) were studied [no operation (n= ) , sham operation (n= ), and bile duct ligation for days (bdl)(n= )]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n= ), sham op(n= ) or bdl(n=i ). blood was taken at this time (to) and days later(t at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp< . , mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t [p< , ,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp< . , paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than . pglml. polymyxin b was administered at a dose of , u every hours. plasma endotoxin rapidly decreased to the normal range in of the patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. - uchimaru, morioka , japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, n o r w e g i a n b r e d landrace pigs ( - kg) of either sex, pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a / air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e . m g / k g h a n d d i a z e p a m . m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n . x l cfu of e colt intraperitoneally as a bolus in ml saline, the a n t i b o d y g r o u p received in a d d i t i o n m g / k g e a n t i e n d o t o x i n i n t r a v e n o u s l y over h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for hours. results : a t a n d hours, the o x y g e n c o n s u m p t i o n increased by % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po . in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h- szeged~ hunbary involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit des toxines microbiennes and "unit d'immuno-allergie, institut pasteur, , rue du docteur roux - paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il- d and ~, tnf ~ and il- in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il- and il- . however, the most preponderant cytokine was tnf~, which peaked at i ng/ml after stimulation with i ~g eta. comparable amounts of tnfd (ca ng) were induced by .i ~g eta and .i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i, -glucan (ram ) was injected twice and h before the shock i.v. in a dose of ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli bs, mg/kg. aiiofcmg pretreated ruts survived during first haher ¢ndotoxine, while in controi shock group the lethality was %. the concentration of ~col)terin in serum was significantly elevated hafterthc second cmginjection (appare~tly % if compare with the control rats), but didu't chartged rain and s rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i and min after endotoxine onset ( i % trod ~, respectively of initial level) than in the control shock group ( % and % at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; . modify records; . erase records; . searches; . reports; . configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd ) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of organisms and the chance of identifyin organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group ( cases), the total effective rate was , %, in the contral group (combined administration of gentamycin and dexamethasone, cases) the total effective rate was %. however the obviously effective rate in sa group % was significantly higher than in contral group % (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o ). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and -glucuronidase ( -gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c h/hen mice at i and h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n= /grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at h the thymic cell yield from clp mice was significantly decreased (sham: . ! . x l vs clp: . i . x * cells; *,p< . ), the % of cells apoptotic by facs analysis increased from . i . % in sham to . ± . %* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from to patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). patients ( %) out of patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all patients with chronic septic knee joints showed poor results. additionally, in of these patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco- cell line) is grown on a micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco- monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco- system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco- monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. ) caco- cells are actively involved in the transcellular passage of bacteria across the caco- monolayer, since inhibition of caco- microtubule or microfilament function decreases the magnitude of st. ) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco- cell monolayer. ) caco- cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco- bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco- cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers ( . x colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about . % within the first minutes and are totaiy cleared within minutes. % offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about cfu of viable e.eoli per ml blood could still be counted hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with % respectively % of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at (n~ ), g (n ~ ), and (n~ ) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker - % at hour~ with return to % of gut leugth (similar to ~m) at hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at ,; hours with return to sham control at hours ( log cfu/g veraus log cfu/g ia ileum). translocation to mesenteric nodes - , , and percent at , , and fi hours compared to b in sham controls (n=l of ). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il- , soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs ( mmhg mean arterial pressure: map for - h terminated at an accumulated oxygen debt of - ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of - mmhg - rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at min and were still markedly increased at rain postshock. plasma tnf, il- , and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at min following resuscitation in rats subjected to hs for min, similarly at h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at min, while in baboons higher levels up to pg/ml were found at the end of shock and h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the -hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a (txa ) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky- , a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup , an a- receptor antagonist as a posthum treatment modality was evaluated. treatment with dup prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup from % to % (p< . , fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf -dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il- and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus , lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. male swiss (icr) mice were used weighing - g elimination of liver and spleen macrophages was accomplished by intravenous injection of / suspension of ci mdp-liposome suspension. control mice received an i.v. injection with pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either mg/g, . mg/g or . mg/g body weight (n= in each group), signs of st and bacterial translocation bt were measured hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n= in each group) were tested to verify that neither paraffin nor pbs or ci mdpqiposome suspension alone induced bt or st. furthermore survival over a -day period was assessed in a control and macrophage-depleted group with a dose of d mg/g z. results. neither the paraffin nor ci mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: / vs. / at . mg/g zymosan (p~o. ); vs. at mg/g zymosan (p~q ); / vs. / st . mg/g zymosan (p~o.o ).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of . mg/g zymosan ( . -+ . vs. . -+ , p~o. ) and . mg/g zymosan ( . -+ . vs. . -+ . , p<_.o. ) . the day mortality after . mg/g zymosan was % in the control group and % in the macrophage depleted group (p=o. ). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n= ) and two experimental (n= each). a portal vein catheter was placed and remained in situ hrs. a femoral artery and vein catheter were in place hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of . hrs., given more hours of anesthesia, and studied for further hrs. % of the estimated blood volume was withdrawn from each experimental animal over min. these animals were kept in shock for . hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for hrs. samples from portal blood for cultures and measurement of leukotriene b (ltb ), prostaglandin e (pge ), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to hrs. after hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb , pge and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha- a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c hb-iej mice). five days post-transfusion the animals were gavaged with lxl viable escherichia coil and randomized into groups (n= /group) to receive a sham burn (sb group) or a % thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies ( mg/kg) (ha- a group) or a % burn injury and administration of equal volume of sterile saline (c group). all groups were resuscitated by intraperitoneal injection of . ml of saline. the animal survival rate was observed for days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n= /group) were gavaged with viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline . and hr prior to sacrifice, group ih treated mice receiving ip of utinastatin . hr and endotoxin ( mg/kg) hr prior to sacrifice,group ilk sham reated mice receiving ip of saline . hr and endotoxin ( mg/kg) hr prior to sacrifice,group iv: saline control mice receiving ip twice during rain intervals,group v: mice received ip of utinasmtin, and min later they were received ip of endotoxin ( mg/kg), group vi: mice received ip of saline,and min later they received ip of endotoxin ( mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu - ,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (> points at days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range . - . mind) was studied after i.v. injection of mbq rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results . + + liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r= . ) and between l/m and ela (r= . ). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il- release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and , , , min, . the plasma concentrations (,pc) of -keto-pgft,, txb: and-ki- -pgf ~ (stable metabolites of pgi , txa and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il- levels by elisa. data are given as mean+sem (* p< . placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t . + . e[ . + . ] eu/ml). il- pc demonstrated an increase continuously from t to t (t + [ + ] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of -keto-pgf~,, pc up to h after mt in untreated patients with a peak of *[ ] ng/ at tl. also txb: and kh pge pc showed a considerabe increase up to h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il- release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into groups. group received standard laboratory chow feeding ad lib. in group a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group % of the calculated daily required calory intake (drci) ( /kcal/kg) was given by iv-tpn ( % glucose, , % amino acids) and % by limited chow administration. groups and received % and % of the drci by i.v. tpn and % and % respectively by chow feeding. group received iv-tpn only. after days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. , + , , - , , / + ~ " , -+ , , -+ ~ - , / +~ + _+ , + , , - , -+ + , ~ , , -+ ~ conclusions: the administration of % of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal ( %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and % in animals subjected to % bepatectomy and lreatment with fermented oatmeal, while - % and - %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < . vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < . ) in salinetreated animals following % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. _+ " +_ " . " data=mean_+sd, * stats anova p< . vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<. ) in lps gut translocation compared to control and l+co . this correlated with a significant increase in peritoneal inflammatory responses (o -,tnf) above that of the control and l+co groups, while mac- and cr opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin , ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), -keto-prostaglansin, leueotriene c , interleukin and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of patients showed a significant increase of endotoxin plasma levels during colonoscopy (p= . ), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after hours. ~hromboxane b o levels also increased after five min. from to pgyml peaking at min. with pg/ml. -keto-prostaglandin,leucotriene c , ii- and crp remained unchanged. a control group of i volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into groups were subjected to , and minutes of %, % and % of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p . ). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p . ). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: .long term( - days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. .in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. athol. . . .) . there was a increasing infiltration of the mucosa c m~nating at the d to th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. ~in isehemia was followed by a hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at m_in, i, ~ , and hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the th hour in the arterial blood .and at the d and th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for and minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion , and hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr ). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats hours after reperfusion in ischemia- rain group and and hours after reperfusion in the ischemia- rain group. delayed small intestinal transit time was seen from hours and on after intestinal ischemia for both and rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < . ). the distribution of radioactivity in segments and in the group with repeffusion hours after intestinal iscbemia for rain was significantly higher than that noted in the group with repeffusion hours after intestinal ischemia for min (p < . ). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy ( %) in the rat. water-soluble ehec was administered orally and hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a - % translocation rate to mlns or blood and hours after operation, while only - % of rats subjected to sham operation or animals with % hepatectomy and pre-treatment with ehec (p < . ). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal -hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s- lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca ÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca + concentration, [ca *]i, and ca + fluxes were measured by the use of fluorescent ca + chelating dyes (fura- or indo- ) and ca respectively. to assess sepsis-related changes in ca + dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca + i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response ( % ¢) in the liver, and a blunted insulin mediated sugar utilization ( % ) and increased proteolysis ( % ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca +]i by - % was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm ). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca + from intracellular stores. this protein kinase dependent process of [ca+ ] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca + signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn- (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute biochemistry, albert-ludwigs-university, hermann-herder-str. , d- freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a and kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: "-gggpunnf'ypycc- ") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il- b, tnf, [l- , il- , gm-csf etc), cell adhesion molecules (icam- , elam- , vcam- ), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p . upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a / kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g cells showed that lbp transcripts could be induced to ford within hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter . kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il- responsive elements hstf-hsp . and h-apf- rs as well as the transcription factor hnf- and the glucocrticoid-responsive elements oct- -d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g cells by il- , il- and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd .transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd , a kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd , which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd (cho/cd ). stimulation of cho/cdi with as little as ng of lps/ml resulted in the release of h-arachidonic acid ( h- : ) into the culture supernatant. in addition to h- : release, we examined lps-imiuced activation (transl cation) of the transcription factor nf-~b. similar to lps-induced h- : release, these responses were observed only in cho cells lransfected with cd closely resembling the same response in the marine macrophage cell line raw . . maximum nf-~cb expression occurred at minutes. in contrast to lps-induced h- : release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd , suggesting that nf-r,b activation begins early after lps binding to cd and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd , lps-induced tk activity in cho/cd was not observed. although the tk inhibitor herbimycin inhibited the release of h- : in cho/cd and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd -related lps signal transducer in cho/cd is not a tymsine ldnase. dimished cardiac inotropic function and response to -adrenergic receptor ( -ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin ( : gm/kg). to stimulate various components of the transmembrane signal cascade of -ar was isoproterenol (i -ar), acetylcholine (m ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia ( °c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c b and cdb was given to rats at a dose of mg/kg by intravenous bolus, min before reperfusion of the ischemic gut. a control group was given pbs buffer. at h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri ( . ± . to . _+ . ,p< . ) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced ( . ± . to . ± . u/g). c was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced ( . ± . x i to . ± . x , p<o. ). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia ( hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc , interleukin-i ( l-i) and il- plasma levels increased significantly, reaching maximum levels after hours ofreperfusion. polyclonal antibodies to tnfet and l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by % and % respectively and mpo was reduced by % and %. antml- yielded a protection in muscle and lung vascular permeability of . °, and . % respectively whereas muscle mpo was reduced by . % and lung mpo by . °, . these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using mabeled antmcam-i, constititive expression of icam- was evident only in lung but not in muscle. during reperfusion icam- expression increased by . %. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by . °, and . % respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following min of ischemia (mi) and . h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb . to p-selectin in a feline model of mi+r. pb . ( mg/kg) administered rain post-occlusion ( min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp . ) for p-selectin ( + vs + % of area-at-risk, p< . ). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb . compared to nbp . ( + vs + , p< . ). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb . treated cats ( + vs + pmns/mm z, p< . ). also, normal coronary endothelium activated with thrombin ( u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p< . ) blocked by pb . , but not by nbp . . furthermore, p-selectin is markedly upregulated - min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested , , , , , , , and h after transplantation (n= /time point) . snap frozen sections of organs were stained for cd- (w / ), cd- (ox ), t-lymphecytes (tl) (ox- ), macrophages (ed , ed ), neutrophils (pmn) (mom), icam-i (iap ), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w / , ox , ox , ed , ed and mom, staining for icam , lain and fib was assessed visually using a scale (ve= - ). as early as h after transplantation, fibrinogen and laminin expression increased in i, peaking h after transplantation (ve= . ; nc: ve=i. ) . at and h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i. ) but rose again at h, peaking at h (ve= ) and remaining elevated thereafter (ve= ). the high expression at h correlated well with the maximal pmn infiltration at that time (cf= ) in i returning to baseline at h (cf= . ) and thereafter. the second peak at h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il- , tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the minute interval (il-lb: , __+ , to , __+ , pg/ml; il- : . - , to , __+ , pg/ml; tnf-a: , __+ , to , + , pg/ml; p< . ). immediately after the beginning of reperfusion, soluble icam- and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam- , lfa- or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n= /group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within rain, ischemic hearts were stored in cold ringer's solution for hours before revascularization. representative grafts were removed after . , hrs, and days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts ( vs rain). after hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked ( vs c/f in non-ischemic grafts), whereas edl+macrophages ( vs c/f) and tnfe expression peaked by hrs. by hrs t-lymphocytes began to enter ischemic myocardium and icam- was moderately increased. after days cellular infiltration had returned to baseline, and no differences were seen among both groups after days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il- for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd treatment in the same model. in other species similar controversial results with anti-cd therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a -fold increase in neutrophil numbers associated with dispersed lullg tissues hours after lps treatment, while macrophage levels increased by -fold at the hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin- (il- ). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by hours, while mip-i levels were maximal at hours ( , ng/ml), with a second peak at hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a -fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip- . in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by -integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of -integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib and dreg , baseline values were determined immediatley after drawing blood. in addition cells were incubated min at °c to allow for spontaneous regulation of adhesion molecules. blood specimens from septic patients were obtained during the course of their illness. control values were determined in healthy volunteers. results: baseline expression of -integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g -integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to pg/ml tnfc~ or nm fmlp (but not at pg/ml tnfc~ or nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n = , bacteremia = ); group b : sepsis syndrome without septic shock (n = , bacteremia = ). serial plasma samples obtained on day , , , , and , were assayed using elisas method (british biotechnology), normal control levels of soluble icam- and e-selectin, obtained from healthy volunteers, were respectively ± . ng/ml and ± . ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis, ) . compared to control mean values, initial levels of groups a and b were significantly higher for icam- (p < - ) and e-selectin (p < - ). comparisons of group a and [] (* = p< . ; ** = p< . t) soluble icam- levels of group a enhanced significantly (p< . ) during the first hours, and a sustained high levels was of bad prognosis ( % of survivors at day ). the evolution of soluble icam- and e-selectin levels were significantly correlated with murray's score (spearman test : p < . ). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam- and e-selectin are correlated with endothelial lung damage, and loam- seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il- and il- , govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il- , have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam- . such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: ) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, ) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and ) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room ; barnhill drive; indianapolis, in usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd) chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd is expressed on all leukocytes, whereas cd b/cd and cd c/cd . are restricted to cells of myeloid origin. cd i / cd interacts with intracellular adhesion molecule- (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd , however, would affect all leukocytes, as would antibodies to cdlla/cdi . targeting cdllb/cd would affect cells of the myeloid lineage only, which could prove to be beneficial. cd b/cd is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd lb/cd may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt gs r.he, t focus on replacing slslic ~sd ~nd fuc s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c .an does sle:, s=h compounds are m ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the ~peutics for i~fi~anmat ry disease. lr:rng maimai models of acute lung lu ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi or its endothelial ligaud, intercellular adhesion molecule- (icam- , cd ). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two x -mm zones were produced by applying brass probes heated to °c to the animals' backs for sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd r . prior to burn injury (pre-r . ); animals given r . min after burn injury (post-r . ); animals given a monoclonal antibody to icam-i, r . prior to burn (pre-r . ); and animals given the r . min postburn injury (post-r . ). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies min after injury was as effective as preburn administration in preserving blood flow. at hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to . _+ % of baseline (p < . by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy- , a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but patients who died during the first hours were excluded, as this was part of the protocol. cy- was administered as a single intravenous bolus of . mg/kg (n= ), . mg/kg (n= ) or i mg/kg (n= ) mg/kg. the antibody was well tolerated. none of the patients died during the day follow-up period. organ failure was assessed for organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially . ± . , decreased to . ± . at the end of the study (p<o.o ). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin- (il- ) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh /sh domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb- and shc, via direct binding of the shc sh domain to the tyrosine-phosphorylated chain of the tcr/cd complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/ t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~// t cell population further segregates into cd t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a// t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of /$ t cells. moreover, these studies show that the relative contribution of cd or cd t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl- and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl- does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il- , il- and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format -d page, autoradiography of s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at and hrs. following clp. serum il- levels were determined at , , and hours by the tdi bioassay. by hrs. % of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant % elevation (p < . ) in serum il- levels at hrs. there was no difference in il- at and hrs. when compared to the control groups. d page studies examined splenic macrophage total protein distribution and biosynthetic activity at and hrs. post-clp. at hrs. macrophages from clp rats showed a substantial decrease in total protein pattern ( protein spots vs protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< kd) was observed. at hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early ( hrs.) staining with cd and cd mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd antigen (cd ++) and a minor population with weak expression of cd and expression of the cd antigen lcd +/ cd + cells). in healthy control donors(n= ) the latter cells account for + cells/mm and %+ % of the monocytes. in sepsis patients, the cd] +/cd + cells can become a major population with more than % of all monocytes in of patients and with more than cells/mm in of cases. there was no correlation of cd +/ cd + cells to any clinical parameter except for cd +/cd + percentage and body temperature (p= . ). the cd ++ monocytes showed a substanttial decrease in cd antigen density in of patients. three-color-if shows that the cd +/ cd + cells in sepsis patients when compared with the cd ++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd antigens, consistent with a more mature nature of the cd +/cd + cells. levels of il- were increased in sepsis patients: of patients with high numbers of cdi +/cd + cells ( /mm ) had high levels of il- ( u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il- . human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl ) and production of the cytokines il-lb, il- , tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il- production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il- and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il- -production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps ) on mo mediator release with a second endotoxin stimulus (lps ). methods: in vitro cultures of marine peritoneal exudate mo were exposed to hr. of hypoxic or normoxic conditions, then incubated hr under identical normoxic conditions _+ ng/ml of lps pretreatment. during the final hours all m~l were exposed to a range of lps concentrations. m~i supernatants were assayed for tnf, il- , il- , pge , nitric oxide (no), and superoxide release. results: lps markedly inhibited mo tnf release by lps but hypoxia had no effect on lps -triggered tnf release. hypoxia increased mo il- production in the absence of lps , but inhibited lps augmentation seen under normoxic conditions. pretreatment with lps increased no production from lps under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps signifcantly augmented mo release of il-i and pge , but hypoxia had no significant effect (data not shown) superoxide production was increased by lps under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. ( ) . h materials and methods. different groups of icu patients, postoperative (n= ), sepsis (n= ), septick shock (n= ), and healthy control (n= ), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf pl oflps ( ng/ml) to id of whole blood in presence of various concentrations of ptx ( , " , - , " , i " m). after hrs incubation at °c, the tubes were centrifuged and supematants frozen at - °c. tnfa and/l were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h . the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and , , corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being %, ) stimulation by lps. the control group is much more stimulated (> % for il , > % for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (> % for il , > % for tnfa ). the lowest stimulation was observed in patients with septic shock (median = %), some patients being not stimulated at all. )effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at - m (reduction to less than '¼ of the median values), and is almost complete at " m. the septic shock group has a decreased sensitivity to ptx. il production exhibits a lesser reduction at - m (~ 'a to ½ of the median values), further increased at - m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at - m and the inhibition is nearly complete at - m. surprisingly, there is a lesser, but significant suppressive action of ptx on il , not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. ( ) lymphokine research ( ) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp- was induced with phorbol ester ( h) and activated for h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il- and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received ml of either saline or blood from brown norway donors,iv. the animals were killed or days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day ; -+ vs + , p< . ). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure ( + , p< . vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from % in ileum on day to %, p< . ). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb i, hb nijmegen, the netherlands. leukemia cell line, teip- . robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. . adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa , an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa may alter monocyte adhesiveness. we selected the thp- cell line, a human monocytic leukemia cell line to further investigate the effect of txa on adhesion. we tested the hypothesis that txa alters lpsinduced adhesion of thp- cells and that txa exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa mimetic i-bop ( . .tm,) or txa receptor antagonists bms and l ( ~m). cells were allowed to adhere for hours and adherent protein/well was determined. lps-induced a significant (p< . ;n= ) increase in adherence of thp- cells (basal, . + . gg protein/well; lps, . +_ . p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between . - . mm, and is reduced in septic patients up to % ( . - . mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than %. subsequently cells were cultured in phenolred-free rpmi medium with various concentrations of glutamine ( . , . , . , . , . , , mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between and %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam- /cd on monocytes in a dose-dependent manner. the receptor for fc'/rucd as well as the receptors for complement cr /cdllb and cr /cdllc were down-regulated. cr /cd which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi , the receptor for transferrin cd and fc'friii/cd were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up - times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> days) of low hla-dr expression (< %) predicts fatal outcome (mortality > %). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [ of patients recovered from "immunoparalysis" following repeated plasmapheres; of them survived ( %). patients recovered temporarely and patients did not respond (all died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was of ( %; p< , ). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study : in vivo stimulation:cd- mice (n= ) were randomized to receive taurolidine ( mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after hours and were assessed for pm function [superoxide anion generation (o -), nitric oxide (no), tumor necrosis factor (tnf), fc/cr -mediated phagocytic function (phago) study : control pm were harvested and cultured in vitro with taurine ( . mg/ml for hrs), after which time they were assayed for -and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m . release of the inflammatory mediators -and tnf, and fc/cr -mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & , and nitric oxide. but injury and inflammation als cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi + % fetal calf serum and stimulated with i ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced ! fm of et/ cells (vs. unstimulated controls of < ). this calculates to - % of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal , germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il- receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i , which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il- message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il- message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l , which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il- production and the effects of il- on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med : , ) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il- production. macrophages were obtained from peritoneal exudate of thioglycollate treated c heb/fej mice and cultured in rpmi medium containing % fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b ) for hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for hours. the amount of il- in the supernatant was measured by il- dependent cell line (b and td ) proliferation assay. il- was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il- production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il- during the initial incubation with lps and subsequent desensitization. il- , like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il- shared the similar potentiational pathway, but differed by the fact that only il- was inhibited. (supported by r ai and po a .) department of microbiology, molecular genetics and immunology and the cancer center, wahl east, university of kansas medical center, kansas city, ks - . korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, ~g/ml, min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice - days after (peak at nd day for i.v. and th day for i.p. mode of administration of the same dose of mg/ g b.w.).the preliminary injection of cryelan ( mg/ g, or h before) to mice with acute cold stress (- ° c, h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al., ) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium ( - fold); bcg possessed similar effect.cmg in the same concentrations ( /~g/ml) increased release of these enzymes only saightly ( . times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren, ) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased - fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, , , prostaglandin e , and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et- (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. . x ucells/ ml in rpmi + % fcs were incubated i min., & hrs. with - m et-i, - m vic or i ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps ) for hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin- (il-i) in response to a subsequent endotoxin stimulus (lps ). in this study we examined the kinetics of lps inhibition of tnf and augmentation ofil- . methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or ng/ml of lps for intervals of to hours. culture medium was then replaced with , or ng/ml of lps for hrs. tnf and il- in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin- (il- ), interleukin-i (il-i) and prostaglandin e (pge ). we showed that preexposure to lps (lps ) alters the response of murine m~i to subsequent lps stimulation (lps ). we hypothesized that in vitro cytokine release by lps in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n= ), cultured in vitro hrs, then pretreated hr _+ lps -cultures were then stimulated with lps and mediators in mo supernatant measured: tnf, il-i, and il- by specific bioassays, pge by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps = ng/mh the table shows that all mediators were increased, in the absence of lps . pretreatment with lps resulted in complete inhibition of lps -triggered tnf release. in contrast, lps significantly increased mo secretion of il- , il- and pge (data not shown). serum cytokine levels were as follows: tnf _+ , il-i + , and il- . -+ . ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps -triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps . provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing - g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd , cdllb, icam- and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd , and icam- , but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi on activated neutrophils and icam- on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il- and il- as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps hrs following endotoxin infusion followed at hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p integrln (cdlla, b,c/cd ) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~ integrlns, but not cd , is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (> ~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng- p.g/ml) or f'mlp ( " to " m) et oc for mln, in ~; human ab serum, the expression of the ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase ( fold) in the expression of cd b on pmns from burned patients compared to an and fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = and total occlusion of blood flow to the lower extremities was maintained for g mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h production was determined by a previously described ' '-dichlorofluorescein flowcytametric assay. _+ amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < . vs. baseline; "p < . vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h production was increased by . -fold above baseline values by hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at rain. post-ischemia, however after release the ligature a significant reactive hyperemia was observed by mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from burned patients (mean age , +_ . years, mean burn size . % _+ . %) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at °c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and ,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ , s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of . u/ml (recommended by manufacturer is the range of - o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns ( . % of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin- (il- ) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il- and tgf- in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il- and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il- and tgf-i~ on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll- up to u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf- . in conclusion, these data suggest a contribution of il- in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at h followed by return of this parameter to normal levels at h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at h. this hi~her index persisted until the end of experiments ( days). the early ( - h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes ( ). there are reports of nentrophil dysfunction in inflammatory disorders of the skin ( ), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score ± ) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously ( ) . in parallel, granulocytes from healthy individuals were tested. two granulocyte functians were studied in vitro: . migration of the ceils in a boyden chamber through a filter matrix following stimulation with different receptor dependent stimuli (c a, intefleukin- , platelet-activating-factor, leukotrien b , fmlp). . release of glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of -glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure ( / ) and nonsurvivors ( / ), whereas / patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. : - , klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg , kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i ~g/ml) on granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load ( % of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in h of the recovery and cytosol proteolytic activity (ph . ; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was - -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by h of the recovery (the comparison was done with the sedentary rats). in h cytosol proteolytic activity decreased and then increased again by h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate ( - %) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il ,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s mdrome. the presence of interleukin (il)- in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il- in animal models can attenuate lung injury. collectively, evidence to date suggests that il- attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il- is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il- can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, .albumi n leak was quantitated by measuring the counts which crossed the monolayer, using a gamma counter. il- (lpg/ml) was incubated in the culture medium with .albumi n for hrs. the il- dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to .- addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt expression and primes pmn's for subsequent generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd expression and pmn priming via the [ adrenergic signal transduction pathway. methods: normal human pmns were primed with paf ( ng/ml for min) or pre-treated with - m of isoproterenol (i) or forskoklin (f) for rain and then primed with paf. cd expression was measured by flow cytometry (fig.l) and -generation in response to -rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig. holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf x on cytokine-and endotoxin induced expression of intercellular adhesion molecule (icam- ) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo -and lipopolysaccharide (lps)-induced icam- expression on human endothelioma celts (eahy ) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf x significantly reduced icam- expression induced by interferon-y (ifn-?) and interleukin- (il- ). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il- induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il- driven icam- expression on eahy cells as well as the il- induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody ( f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b (ltb , x " tool/l) or intravenous injection of interleuldn- (il- , - iu/kg b.w.) and lipopolysaccharide (lps, mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of mg/kg b.w., superoxid-dismutase (sod, . iu/kg b.w.) was able to decrease the ltb effect, and the immuumodulating drug leflunomide (hwa ) exerted inhibitory effects on il- -induced permeability at a dose of mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb or il- induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam- in neutrophil migration. blocking antibodies to human pecam- , in which the antibodies are crossreactive with rat pecam- , were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam- / integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs ( - kg) were subjected to % arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added ( , , , i.tg/ml) ex vivo. after hr incubation at - °c, %cd (+) pmns were determined with fitc-ib and flow cytometry from mean channel fluorescence histograms. ± # p< . vs baseline * p< . vs sham $p< . vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd expression was depressed for at ]east hr after trauma relative to sham but by hr, was enhanced, relative to sham, and because fentanyl anesthesia at hr had a greater effect on cd expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps ( ng/ml), tnf( ng/ml), and lps/tnf( ng/ ng/ml). cultures of the human ec tine(ecv- ) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for , , , and hrs. ec necrosis was assessed by a cr release cytotoxicity assay. pmn activation was assessed by cd lb receptor expression and respiratory burst activity hr _+ . -+ -+ . _+ _+ . _+ _+ . _+ . hr + . _ _+ . _+ _+ _+ " +_ +-- . " lghr - . _+ +_ - " o:fo , " ~ +- . * hr _+ . - -+ +_ * _+ _+ * _+ _+ " data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p< . vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received ml/ g bw normal saline. at hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, xi - m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at hr post-clp. after pre-contraction of blood vessels in the isolated gut with xl m ne, vascular responses to ach ( x m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, xl - m), were determined. total vascular resistance (tvr, mmhg/mi/min/ g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n= /group) in the aortic rings were: ach lxl i~s, st-in ~ ~ significantly at hr post-clp (i.e., increased *p(o vs. sham; n- per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin- on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. - g) shook was induced by fractionated withdrawl of arterial blood within rain and maintained for h (map at mm hg, cardiac output % of baseline). rats were adequately resuscitated with % of shed blood and twice the volume in ringer's solution additionally. following h of reperfusinn (map > mm hg, co > % of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n= ) received the il-lra as pretreatment beginning min prior to shock induction. in the group t (n= ) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of mg/kg and was followed by continuons administration of mg/kg/h. the control group c (n= ) received equal volumes in nac , %, the sham-operated group s (n= ) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: , % + , %; c: , % _+ , %) was significantly reduced by pretreatment or treatment with il-lra (p: , % -+ , %; p< . , t: , % -+ , %, p< . , anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: iam /s + um /s, p: llm /s + }am /s, t: ams/s -+ lam /s, s: am /s -+ am /s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il- . pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to retool/ asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc ( g/kg bw bolus, followed by . g/ kg-h infusion) on the endotoxin-induced ( . ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p< . at mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p< . at mmol/l asc) and reduced the adhesion dose-dependently (p< . at mmol/l asc); ec damage was also reduced (p< . at retool/ asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p< . ), decreased the protein clearance (p< . ) as well as the zymosan-induced rom production (p< . ), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi on neutrophils and lymphocytes. methods: from blood samples (n= ) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after , , , , and minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at min was observed in wbc-cultures (n/r: min / , min / , min / , min / , min / ). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to min followed by a decrease to normal values at min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl -expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c -delicient mice in a model of zymesan indt~ed mof. materials and methods. c -deficient b d /oid and c -sufficient b d /new mice were used in this study. on day all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of mg/g body weight. between day and , biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score= ). on day all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x ) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day - ) and a late phase (day - ). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c deficient mice (all p< . ). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was / ( %) in c deficient mice and / ( %) jn c sufficient mice (p= . ), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c bl/ mice received zymosan ( mg/g body weight) intraperitoneally. groups of animals were killed after , , , and h and subsequently at each day until day . plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin -ct, and - (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c were measured in plasma and culture fluid by means of a ria (detection limit . ng/ml). interleukin- (il~) levels were assayed using the b hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day , they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within h after zymosan injection and disappeared within h. from day onwards, tnf levels started to rise again. plasma il- behaved almost similarly in the acute phase, but in the mof phase plasma il- remained low. no circulating il- could be detected at any time point. macrophage lps-stimulated production of il-lcq il- ~ and tnf--c~ was suppressed immediately after zymosan injection. production of il- and tnf-~ was normalized within h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day did production of il-i~ reach control values. il- production was higher than control values from day onwards. il production was similar to that of ili-il the production of tnf-ct was strongly elevated between days and and again during days to . the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il- or il- . also, the ability of macrophages to produce excessive amounts of il- and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~ -acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with antisense dna's (as) complementary to the ' region of the il- gpl receptor. the antisense were '-agtttagggatgagg- ' (asl), '-atcttcatcttctgaat- ' (as ), '-aagtgaatgattaaaacact- ' (as ), '-aaacctttataggcg- ' (as ), and '-cgttctacaactgcaacgt- ' (as ). using hepg , the biological effects of these antisense complexes on the high affinity il- receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for h. underivatized asl was less effective and the complex, asgp-pll-as , had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il- (i units) asgp-pll-asi alone showed a dose dependent ( .i- . ~m) inhibition of ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem , israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l female merino-sheep were included (mean weight: kg). day : hemorrhagic shock (mean arterial pressure (map) mmhg for hrs.), closed femoral nailing (ao-technique), day - : bolusinjection of endotoxin (et) ( , ~tg/kgbw) und zymosan-activated plasma (zap) ( ml) every hrs., day - : observation. bronchoalveolar lavage (bal): day , , . the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm- ), pap -putm.art.pressure (mmhg), pap -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov ), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p< ) results: baseline day day day day heart: co , _+ , , _+ , , _+ , , _+ , * , _+ , * svr _+ + _+ +_ " +- " lung: pap , _- , , _+ , " , +- , " , + , " , +- , ' pap , + , , +- , , _+ , , +- , , +_ , * liver: bill , _+ , , _+ , ' , _+ , ' , _+ , " , _+ , " kidney:crcl , +_ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , _+ , , + , , + , histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il- and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg responses, t cell proliferation, and il- synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il- and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after - rain even without cyclooxygenase inhibition although pgi levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated patients undergoing major abdominal surgery as compared to ibuprofen ( rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time , , , , , , , rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of -keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of -k-pgf~,tp~, peaked minutes after m.t. ( _+ , ibu: _+ , to: +i ng/l) and declined monotonously over h ( +_ , ibu: _+ ng/ ). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after rain ( _+ , ibu: + , to: -+ /~u/ml); aldosteronc also presented a maximum after rain ( + , ibu: -+ , to: +- pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum h after m.t. ( +_ , ibu: -+ , to: +_ ~g/ ). adh pc peaked min after m.t. ( + , ibu: -+ , to: +_ pg/ml) and showed analogously to -k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. rats of them received sma clipping and others wee used as control. control and treated rats at , , , hours were perfused and fixed. the brain were sectioned at pm and stained by abc method using c-fos antibody. plasma endotoxin level of rats were measured at , , , , hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of , , , , hours after the treatment were . ± . , . _- - . , . _+ . , . ± . and . ± . pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi ). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed , , , , . tile plasma concentrations (pc) of -keto-pgft, (stable metabolite of pgi ) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < . placebo vs. [ibuprofen] [ ] mmhg (*p< . i). these changes were accompanied by a marked increase of -keto-pgf~ pc up to rain after mt in arterial and mixed venous blood of untreated patients with a peak of *[ ] ng/l tl (*p< . ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao while -keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for -keto-pgft~ was not detectable. a splanchnic vascular source for pgi release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e , ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day , two groups of n= week old aj mice were subjected to either a % scold burn (ti), or were untreated (c) n= . on day , mice (ti+clp) and mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days , and after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il- and adherent splenocytes (as) were cultured with lps for il- , tnf, il- and pge . results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi ) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. ( /ml) at rates increased incrementally from . to . mi/kg/hr over hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n= ); pga (n= ), graded bacteremta plus anti-pgiz antibody, ml/hr iv, beginning at hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po , mmhg, cardiac index (ci), l/min/m , oxygen delivery index (do i) and consumption index (vozi), ml/min/m , and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg -keto f alpha ; in the first instance~ peak values of lt ~ after i~ hrs post infarction were times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets , ~ev , ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical ( patients) and experimental material( rats and dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period ( - days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin ( microgram per kilogram of body mass intrap'eritoneally within days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [ ] . peep can affect rv function by reducing preload and ejection fraction (ef) [ ] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this -year-old patient (apache-ii: ) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [ ] . the therapeutic procedure included volume replacement, vasopressors (dopamine , dobutamine gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf- , od= mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id= ram, bennett a, pressure controlled mode, pm~x= mbar, peep= mbar, i:e=i:i, fio = . ). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [ ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new , dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [ ] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing mg% glucose & gm% bovine albumin and incubated in dubnoff shaking water bath for min at °c the results revealed that there was an enhancement in release of free fatty acids (ffa) ( %) and lactate ( %) and in glucose uptake ( %) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition ( ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by %. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a , but not with a direct vasodilator (nano ). this failure to relax to ach and a reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by weeks. exogenous no donors (e.g., c - or spm- ), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor ( p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at days. rat carotid artery rings relaxed only + % and + % to gm ach in vehicle treated rats and in inactive no donor treated rats respectively days following injury compared with + % in no donor rats (p< . ). relaxation to gm nan was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name ( mm) was markedly reduced by injury, but was protected by no donors (p< . ). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il- , ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin- and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde- -phosphate dehydrogenase (gapdh) and the cytochrome p (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to mmhg for hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, nm) was reduced by % compared to control sma segments (p< . ) while relaxation to sodium nitrite ( gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p< . l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p< . ), attenuated mesenteric mpo (p< . ) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm- (schwarz pharma), following regional myocardial ischemia ( hour) and reperfusion ( . hours) . treatment with spm- ( rim) significantly reduced myocardial necrosis by % (p< . ) compared to an no-deficient analog of spm- , spm- . furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p< . ) reduced following treatment with spm- compared to spm- ( . + . vs. . + . u/ mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps ( mg/kg, n= : mg/kg, n= ; or mg/kg, n= ) and treated with l-nma ( , , , , mg/kg/hr) for hours following a , , or mg/kg loading dose. animals were resuscitated with iv ringers solution ( ml/kg/hr). hemodynamic data were collected at , , , , , and hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p= . ), and systemic vascular resistance index (p= .ol) and decreased cardiac index (p= . ) and oxygen delivery index (p= . ). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p< . ), but this differential effect on the venous and pulmonary circulation occurred, > hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied ( , , , or mg/kg/h) in either the low ( mg/kg) or high dose ( mg/kg) lps-challenge groups. a nonsignificant (p> . ) trend toward a beneficial effect on survival ol low dose l-nma ( mg/kg/h) in animals given the mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study ( mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p< . ). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad . recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [ ]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no ( and ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad [ ] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation ( - ppm) consistently lowered the pap and augmented the pao /f.o for up to days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below . %. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [ ] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately - ppm, the improvement of oxygenation had a maximum at ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad , thus allowing reduction of the f.o . no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd- -phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il- , ifnj and lipopolysacchafide (lps). hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of , -diphosphoglycerate to glyceraldehyde- -phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by _+ %. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only _+ %. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il- , tnf, ifn, and lps for hours. , , and ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no ); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n= .) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont . + . + . # stim . + . + stim+ o tzm nmma . + . + . # stim+ ~m nmma . _..+ . * + . # stim+ pm nmma . + . * + . # stim+ )lm nmma . + . * + . # anova , . (* p < . versus stim, # p < . versus stim; anova with neuman-keuls) gsh in cont+ i~m l-nmma was equivalent to that of cont ( . vs. . ). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c bl/ mice and lew rats underwent a % and % ph, respectively. sc were prepared - days after ph and plated at to x ecells per well. after h incubation at °c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient days aider liver partial resection ( . nmol nitrite/ cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: ) encletoxin and l-name, ) endotoxin, ) naci and l-name, ) nach preliminary results from groups (n= ) and (n= ) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin ( . gg/kg/h) was given as a continous portal infusion over the entire observation period of hrs. l-name ( mg/kg) was given as a bolus after hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by %* and hepatic artery flow (hal e) by %*, while l-name caused a further and lasting reduction in flow (pvf %, haf %)*. transhepatic (portal-hepatic vein) vascular resistance increased to times baseline value during endotoxemia while l-name caused a further marked increase in resistance to times initial value. portal oxygen saturation (so ) decreased by %* during endotoxemia. l-name caused a reduction in portal so by %*. arterial so was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a % reduction in cardiac output (co). the addition of l-name reduced co by a total of %*. *: p < . . conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; mg kg -i s.c., at h prior to lps) or with an il-i receptor antagonist (il-ira; mg/kg bolus and . mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at - min after lps. for instance, endotoxaemia for min resulted in a fall in map from -+ (control) to -+ mmhg (p< . ; n= ). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at min when compared to lps-control: -+ mmeg (n= ) and -+ mmhg (n= ), respectively (p< . ). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, min, x - m). pretreatment of rats with tnfab or il-ira significantly (p< . ) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at min after lps there was an induction of calcium-independent nos activity in the lung ( . -+ . pmol citrulline/mg/min, n= ), which was attenuated by tnfab and !l-ira by -+ % and -+ %, respectively (n= ; p< . ). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with live e. coli, which consistently increased cardiac output - % above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name, mg/kg iv), prevented this increase and elevated map by - %. in the first groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are rains after e. cob. n= - /group. * p< . vs bl by remanova and § p< . vs e. coli alone by anova. ec+l-name -+ - _+ " § - _+ * § - _+ * § - + * - + * § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 'untranslated region (utr) of ferritin mrna and 'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j ) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k cells, where nos was stimulated by increasing the availability of the essential nos cofactor , , , -tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn- or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group , no, ppm, was added to the inhaled gas from the start of dialysis. in group no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group , from minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats ( - g, n= - /group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received rrd/ g bw normal saline. the animals were killed at , , or h post-clp ( h post-clp=early sepsis; - h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, to - m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, /~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at and h after clp. in contrast, l-nmma administration produces an % increase (p< . ) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a -years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of mg of l-nmma, a continuous infusion was installed ( . mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose ( to mmhg}, heart rate stayed stable ( + b/rain), systemic vascular resistance increased ( to dyne.sec/cm ), cardiac output decreased ( to . l/rain), and cardiac index declined ( . to . l/min/m }. before and after minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after matutes. neopterine increased from . to . ng/ml, tumour-necrosis-factor-a increased from . to . pg/ml and intedeukin- increased from . to . pg/ml. immunoglobulines a, g, and m ( . to . , . to . , . to . g/i), complement factor c- c and c- ( . to . , . to . g/i), alpha-l-antitrypsine ( . to . g/i), c-reactive-protein ( . to . rag/i), interleukin- ( pg/ml) and soluble interleukin- ( to units/ml) did not change significantly. pulmonary-shuntvolume decreased from . % to . % within one hour and to . % after minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse , zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. . microbiol.immuno : ) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc ,um, ifn-,/ u/ml, lps fig/m/, ngmonomethyl-l--arginine (mmla) ram. nitrite (no -) content of the supematants was determined by a standard griess reaction, and h release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x- , and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with . ml of a suspension of . ~" s.typhimurium, strain c , and injected with aminoguanidine (ag) mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from % in the control group to % in the treated group, days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom % to %, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no -accumulation, . nmole/well v.s. nmole/well. conversely h production is enhanced from nmole/well up to , nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, % in iron exposed macrophages v.s, % in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity ( % v.s. %) whereas the addition of afc to these macrophagas restores an elevated ( %) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit de microbiologie. bp . la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at °in tcm- / % fcs, with or without additional rat serum. after h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of u cobra venom factor/kg b.w. at days and . significantly higher (p<o.o ) lps-stimulated no-generation was found in spleen cells prepared at day ( . - . nmol nitrite/ cells, n= ) compared to spleen cells from non-treated animals ( . ± . nmol, n= ) complement activation was effective also in vitro. when incubated in % zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli ( qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l bioassay. survival rate (%) qrql/,p n hr hr dav control (normal saline . ml/body i.p.) n (l-name mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to min of partial liver ischemia ( %) and min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i mg/kg). sod, when used, was given as mn-containing preparation at a dose of u. i.v. i min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : . ± . "#/ . ± . / . ± . . alt (u/ ) • ± ~#/ ± / ± . gldh (u/l): . ± . e#/ . ± . #/ . ± . . in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous ~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [ ] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [ ] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no -chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of . - . parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non . . . . , parts per million (ppm) no, mean, n = ; ~ p < . (t-test) between smokers and non-smokers; p < . (t-test) between in-and expiration. about % of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than % {o. ppm before intubation vs. . ppm after intubation, ~, < . ), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [ ] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi , yoshihiro inoue , masao yoshida critical care and emergency center, and department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il- contribute to the occurrence of septic shock (circ shock : ; . the present study investigated the in vitro effects of lps, tnf-o~, il- and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il- , tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il- and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il- and tnf-c( enhanced lps-facilitated no production to a greater extent than il- or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. - uchimaru, morioka . japan. t. yolk , , i. ioannidis , w.j. kox and h. de groot objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: -morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin- ( mm), which releases both no. and o -., caused a time-dependent cytoreduction of % and % after and hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes --to form h and . in contrast, the addition of catalase (cat), which removes h to form h and , diminished the cytotoxic effect ot sin- to %, equivalent to high concentrations of snp ( mm), which solely releases no.. in addition, the amount of h formed by mm sin- equaled the amount ot enzymaticany produced h by mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration ( mm) alone were not toxic to endothelial cells, the combination caused an % reduction of viability, comparable to the effect observed with sin- ( mm) alone. moreover, toxicity mediated by high concentrations of snp ( mm and ram) was reduced by % under hypoxic conditions indicating synergism with no-and . conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with -. however, toxicity increases dramatically in the presence of h . this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no and no levels were measul*d sliectrophotometricallymonocyte cultures were done for itrs with or without lps+ifn-t stimulation mid no and no levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. )both no levels in the supematmlts of monocytes cultured with lps+ifn-t mid no , no levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. )based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [ u/ml]. after hours of incubation no-release was determined as nitrite (no ) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d , that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no -production (p<o, ). although we observed no toxic effect on cell viability, no -production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo nm] released % less no in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [ }jm] and bim [ioijm] reduced the no -formation by % and %, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d cells pretreated with d [ ~g/mt] released % less no than stimulated controls the inhibitory effect on no production decreased the later the inhibitore were added after stimulation; e.g. bim added and hours after stimulation reduced no -production only by % and %, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr , erlangen interleukin- (il- ) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il- ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the ' utr of the human il- ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable ( %) gc rich region. the translation of the type i il- r appears under a significant suppression and may limit the biological activities of il- by low level of expression. on the other hand, the type ii il- r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il- activity; inhibition of il- converting enzyme which cleaves pro-il-l[ , anti-il- ri, the il-i receptor antagonist (il- ra) and soluble (extracellular) il- ri and il- rii. il- ra is structurally related to il- and binds at °c to the il- ri with near equal affinity as that of bona fide il-i; however, il- ra does not transduce a signal. il- ra has been given to humans in pharmacologic levels (mean plasma levels of gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by % and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il- ra are in -fold molar excess to those of il-i [ . it is unclear whether these endogenous levels of il- ra are sufficient to block il- activity in disease. a single injection of ]l- or ifna in humans does not induce il- but rather high levels of il- ra ( ng/ml). prof.dr.l.a. aarden interleukin (il ) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il -anti-il complexes, suggesting that plasma il measurements represent a gross underestimation of il production in the patient. until now no natural antagonists of il have been described. the biological activities of il results from binding to a lowaffinity il -receptor (il r). the il /il r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp . studies on the structure-function relation of il revealed that disruption of the gpl -interaction site on il gives rise to a mutant il that, by virtue of its intact binding to the il r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin- (il- ), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il- and the ability of il- to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il- during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il- was found to be a potent angiogenic factor at concentrations ranging from - ng/ml. sequential fluorescein angiograms demonstrated that il- induced neovascularization by days, which regressed by weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il- . in addition, an il- antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il- has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th , thl and th cd ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il- are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il- also directly inhibits t-cell proli/eration induced by cross]inked anti-cd or anti-cd mabs (e.g. in the absence of professional apc), by specifically inhibiting il- gene transcription and il- protein production. il- also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il- , and tnf-a and the chemokines il- and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il- enhances the production of the il- receptor antagonist, a cytokine with anti-inflammatory activity. il-l produced by monocytes downregulates class ii mhc expression and il- production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il- is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il- also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il ) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd and cd memory t cells. ip- chemoattracts only preactivated (not resting) cd or cd t cells. hip-l~ preferentially chemoattracts gd t calla, while hip- more readily attracts the cd t call subset. the chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla- . these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln ', peaked by hr and was completed in hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin- (il- ) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of kd and kd. il- was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il- . phagocytic cells produce il- in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il- exert biological activity are t and nk cells. the major direct biological function of il- on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il- , produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il- to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il- , other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il- has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type (th-i) response and suppressing a th- response. the presence of il- during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il- , and inhibits the generation of th- cells producing il- . il- induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il- , however, il- needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il- -mediated inhibition of il- producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il- for thl cells and an inhibitory effect of ifn-y on th cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il- on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il- appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. , interleukin- and its role in the generation of t e cells, imm~ol. on peripheral blood monocyte/macrophages, il- behaves like il- in a variety of assays, and shows both similar and contrasting, activties with either il- or ifn-y. il- reduces inflammatory monokine and il- production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, , febs lett. , ) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi expression. il- produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il- . it inhibits niv-i production (montaner et el, , i. exp. mad. , ) , and interferes with hiv-induced cell fusion. the activities of il- and il- differ on t-ly•ocyte and large granular lymphocyte population. in particular il- does not exhibit the suppressive effects of il- on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il- and il-l share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. , embo j. . - ), explaining both thai common and divergent activities. the levels of il- ~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il- mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t cytotoxic lymphocytes express markedly higher levels of il- ~rna. in vivo, il- may thus be contributing, significantly to some of the activites previously ascribed to il- il- . recently, we cloned the human cdna encoding interleukin- (il- ). human il- is a non-glycosylated protein of aa with a molecular mass (mr) of kd and has biological activities on monocytes and b cells, il- , like il- , strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd (fc~rii). furthermore, il- , like il- and il~ , inhibited the production of pro-inflammatory cytokines il- , ]l- , tnfo~ and chemokines miplc~ and tl- by lps activated monocytes. both il- and il- enhanced the production of il-lra by monocytes. in contrast, il- lacked the t cell stimulating activities of il- . the responses of monocytes to il- and il- were not additive or synergistic, supporting the hypothesis that il- and il- receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il- , il~ and il- have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il- is unique in that it can modify inflammatory reactions without stimulating (as does il- ) or inhibiting (as does il- ) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of amino acids that is inactive until processed to a disulfide linked homodimeric molecule of t amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf- has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf- isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[ family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il- , pge and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il- and il- , have been characterized as critical in regulation of mo tnfa and il- , as well as in induction of t lymphocyte proliferation and ifn production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score > ) were analyzed for their il- levels, their in vitro proliferation to mitogen (pha) and their response after il- addition. since il- produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il- levels. increased patient il- production might also be resulting from the high levels of tnfa a known stimulator of il- . conversely, since il- augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il- deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il- found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il- production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il- were explored. increases in tgf[ may have partially contributed to the patients' depressed il- level, but elevated pge had no effect. addition of il- to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il- , pge ) and a dearth of other monokines (il- , il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells ( mmc) may result in the production of different cytokines including the interleukins (il) , , , and as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il- , l- , tnf-c as well as various mast cell growth factors (il- , il- , il- , il- , ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl bmmc/ml; ll.um ionomycin; - h). the actixdties of bmmc supernatants were assessed in specific biological (il- , il- il- , l- ) and/or elisa assays (il- , il- ). here we show that homogeneous populations of bmmc (> %alcian blue+/safranln-; in vitro age: weeks) generated in the presence of recombinant (r) rail- from normal balb/c mice produced modest amounts of l- and low or undetectable levels of il- , - , and - after induction with lp.m ionomycin only. however, a dramatic increase ( -to -fold) of these cytokine activities was noted, when in addition to ionomycin also human ( ) rll-la was provided during the induction period. this il- effect was dose dependent with a maximgm at - u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with ng/ml hrll- receptor antagonist abolished the action of u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll- or rmtnf-~. both mrll- and hrll- substantially enhanced ionomycin-induced l- production of bmmc in the absence or presence of il- . il- significantly enhanced il- and il- production while decreasing il- activities to abont - % of control levels, when il-i was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t antibody (ascites : ) abrogated the effects of mrll- . other mast cell-active cy~okines (] ,- , il- , l- , ngf, or kl) added to ionomycia-or l- /ionomycin-treated bmmc had no major effects on cytokine production. il- and il-i did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of " m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il- /ll- induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il- inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il- previously recognized as a product of th type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il- , and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of different httman cytokines, ilia, ill [ , illra, il , il , il , il , il , ils, ill , gm-csf, tnfa, ifn and tgf[ . , was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with % paraformaldehyde and permeabilized by . % saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[ _ , could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il- in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n= ) were incubated at °c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after , , , , and minutes plasma levels of tnf-alpha, il-i beta and il- were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from , pg/ml at min to pg/ml, pg/ml, pg/ml after , and min. il-ibeta ( , pg/ml, , pg/ml, , pg/ml, pg/ml and pg/ml after , , , and min) and il- ( , pg/ml, l,lpg/ml, , pg/ml, pg/ml and , pg/ml after , , , and min) plasma levels were increased min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il- to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr. , ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf- ) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf- ( . - . ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf- (> . ng/ml) potentiate production of the immunedepressive prostaglandin e . other investigators have shown that tgf-] can cause the appearance of cd (fc immunoglobulin receptor) on monocytes exposed to ng/ml of tgf-[~i for hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd is depressed in septic patients and therefore we hypothesized that tgf- could account for the down-regulation of cd observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[ for and hours at °c and examined ceils for cd and cd expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at °c alone. one displaying high density of cd had increased fluorescence over the homogeneous expression of cd in cells maintained at °c (baseline). the other population displayed decreased cd expression relative to the baseline cells. tgf-i~i ( - ng/ml) caused a shift of ceils from the high density into the low density cd population. this trend was observed within hours of incubation and was complete by hours. we observed a net decrease in cd expression f % for all subjects studied (p< . vs controls). phorbol myristate acetate ( ng/ml) also caused down-regulation of cd to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd after incubation with tgf- ( ng/ml) for hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd by tgf- deplete their surface expression of cd while generating cd . this down-regulation of cd by tgf- correlates with our clinical observations of lower cd expression on monocytes obtained from septic patients. for over years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw . and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw . cells, mif secretion was induced by as little as pg/ml of lps (e.coli l:b ), peaked at ng/ml, but was not detectable at lps concentrations > txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn . production of mif by lps-stimulated (l ng/ml) macrophages peaked at hr. expression ofmif mrna and tnf mrna by lps-stimulated raw . macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations ( pg/ml to p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately -fold after lps induction ( pg/ml). mif secretion also was induced by tnfoc ( ng/ml) and ifn? ( iu/ml), but not by il- and il- (up to ng/ml). lps and ifn had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif ( gg/ml) were found to secrete bioactive tnf~ (> pg/ml by l cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il- , il- , and tgf , have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il- , il- , il- , tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il- by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at °c for h in the absence or presence of lipopolysaccharide (lps) or tnfa. il- release was measured in the supernatants using a specific elisa. among tested cytokines, il- was the most efficient inhibitor of il- production by lps-activated pmn. il- was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il- production. northern analysis are under investigation to precise the level of the il- -and il- -induced inhibition of il- production by pmn. our data illustrate that il- and il- possess the capacity to down regulate the production of il- by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il- superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. female, wistar ( - gm) rats received either or pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with mg/kg ibuprofen lp prior to pg/kg bw cntf. rats treated with ,ug/kg bw cntf lost . _+ . gms bw as compared to freely-fed controls which gained . _+ . gms (p<o. by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (- . _+ . ). animals pair fed to the high dose cntf gained body weight ( . -+ . ), although weight gain was less than seen in freely-fed controls. rats treated with pg/kg bw had reduced body weight gain (+ . +_ . gin). food intake was also reduced by % in pg/kg cntf (from . +_ . gm/day to . _+ . gm/day; p<o. ) and was also unaffected by prior ibuprofen treatment ( . +- . gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight ( . + , gms vs. . _+ . gins), total protein ( . +- . gms/l vs . +- . gins/l) and albumin ( . + . gms/l vs. . +_ . gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il- and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box , gainesville, fl. . of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il- , have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il- are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp signal transd,uction protein, including il- and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il- and cntf also induced hepatic acute-phase proteins. thus il- and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il- -and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of to pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound ) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound ) or the phosphono-oxyethyl analogue pe- , structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd ÷-as welt as cd ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between / to / cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il- and ifnf, but not for il- or il- (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd ÷ as well as cd + t cells express cd , the high affinity il- receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il- receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t, cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)- when stimulated with calcium ionophore (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was re/eased into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il- in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il- concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il- in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by - % within days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. versus kin/day). they developed elevated plasma corticosterone levels ( _+ gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il- a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il- and tnf activity in plasma was not affected. after administration of .tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il- activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin (il- ). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il- ) and those of their specific antagonists interleukin- receptor antagonist (il- ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from patients with acute myocardial infarction at presentation in the coronary unit, every three hours for hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il- and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within hr whereas stnfr and l-lra peaked at - hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin- (il- ) is a multifunctionai cytokine known to be involved in important homeostatic processes. il- levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il- binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il- receptor (sll- r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil- r and il- are measured using elisa's. both elisa's are tested for interference of sil- r mid il- . the effect of sll- r in the il- bio-assay (b ) is tested. results: baseline levels are (mean + sd): . -+ . ng/ml in serum, . -+ . ng/ml in urine, . _+ . ng/ml in csf and . + . ng/ml in sf. there is no interference of sll- r in the il- elisa or bio-assay and no interference of l- in the sil- r elisa. conclusions: in all investigated biological fluids sll- r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin (il ) and to turnout necrosis factor ~x (tnfc . methods. bum patients (n= , -> % total body surface area) were studied weekly up to days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il ~-and tnfcz-binding t(cd ) lymphocytes was assessed by flow cytometry analysis. levels of il receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p< . ) increased up to weeks post-bum (means . + in non-surviving and . + . u/ml in surviving patients vs < u/ml in the control). within weeks of bum, the percentage oft ceils expressing receptors for tnfa and il [~ constitutively was elevated (by - fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range . - x j pg/ml vs < . x j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range - x - pg/ml vs - x j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl ~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of l lra may affect il -regulated biological responses including specific immune reactions. while studies suggest that il- is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il- contributes to depressed t-cell responses seen following hem; and ) how other agents, known to play a role in hem, effect il- release. to study this, c h/hen mice were bled to and maintained at a map of mmhg for h and then adequately resuscitated. mice were killed h post-hem to obtain splenic t-cells (nylon-wool purified). il- 's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il- to markedly improve the t-cell proliferative response [ . #g the marked increase in capacity of t-cells from hem mice to produce il- was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il- are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il- release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il- down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il- has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n= ) were randomized to groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received % etoh or ns daily for days by gavage. a % full thickness bum was induced hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed days post bum. splenic lymphocytes were cultured for days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma . ug/ml, ug/ml). splenocyte production of il- , interferon-gamma, il- , pge were measured, and lymphocyte proliferative response examined. results: il- production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of .- in a dose-dependent manner, indicating nitric oxide may modulate il- and interferon-gamma production in thermal injury. il- production is normal in etoh-burn animals. conclusion: il- and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il- production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin- (i - ), interleukin- (i - ), the interleukin- receptor antagonist (i - ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn , , , , , , and hours following operation. a portion of the results are shown (mean -+ sem). + -+ _+ one and two factor anova; *p< . , #p< . , §p< . , ¶p< . , for differences between groups i - was not detected at any time point. both ii-ira and i - increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for hours; the b-ira increase was a thousand fold greater than the rise in i - levels. i - levels increased up to hours after surgery. crp levels reflected maximum ii-ira and i - levels (r =. , p< . and r =. , p< . respectively). high ii- ra and i - levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i - ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin , ireland. change of il- and soluble il- receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il- plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il- receptor (sll- r) is considered to be agonistic to il- , unlike other soluble type receptors of cytokines. here we measured il- and sll- r levels in the serum and drain fluid from surgical field in order to investigate the changes of il- and sll- r after surgery and their origins. [materials and methods] serum and drain fluid samples from cases ( of esophagectomy and of gastrectomy ) were serially collected before and after surgery. il- and sll- r levels were measured by elisa. [results] ( ) serum il- : all cases reached the maximum level on pod-l, more precisely - hours after operation. ( ) il- in the drain : maximal il- levels in the drain were recognized - hours after operation, at almost the same time as serum il- . furthermore the il- values in the drain were much higher, about times, than those in serum. ( ) sll- r in the serum : all cases reached minimum levels - hours after operation and recovered to the preoperative levels a few days later (decrease ratio : . + . ~,, range : - ~'). ( ) sll- r in the drain : sll- r levels in the drain showed almost the same value and change as serum sll- r. [conclusions] ( ) il- is produced from the cells gathering around operative fields whereas sll- r is considered to be produced in the cells which do not gather around the operative fields. ( ) there may be a mechanism that down-regulates sll- r in the early stage of surgery. [objectives] il- plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il- concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il- . [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il- was measured by elisa and il- mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] ( ) serum il- levels reached the maximum concentration on the st postoperative day in all patients. ( ) the il- peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r= . , p< . , r= . , p< . , respectively). ( ) the peak concentration of serum il- in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p< . ), despite a similar degree of surgical trauma. ( ) peak l- concentration observed in a patient who underwent esophagectomy was about fold greater in the drain fluid of thorax than in the peripheral blood. ( ) il- mrna was demonstrated in leukocytes from thoracic and abdominal exudate at , and hours after surgery. in contrast, il- mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il- is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over years old show a different serum level cytokine pattern than pts under after a standard surgical procedure considered as a "medium strength trauma". patients and methods: pts( females males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a: pts under years(mean age: . +- )gr.b: pts over years(mean age: . _+ ). all pts underwent cholecystectomy and cholangiography. pts in gr.a and pts in gr. b underwent common duct exploration. spbintercctomy was performed in each group. on the day of surgery (pre) and on the st and th postoperative day(leo, po) : percentages of cd , cd , cd , cd and cd cells we measured by means of flow cytometry using moab. and levels of il- , il- , il- and tnf "in vivo" by elisa using moab. results: ere: cd % was . _+ in gr.a and . objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin (il- ) levels to determine a role of g-csf and il- in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il- levels were measured before and after surgery in patients with esophageal cancer (ec), and patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p< . ) and peripheral pmn chemiluminescence (p< . ) of patients with ec were significantly decreased compared to those of patients with gc at and hours after surgery. serum g-csf levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at and hours after surgery. serum il- levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at , and hours after surgery. significant inverse correlations (p< . l) between peripheral pmn count and serum g-csf and il- levels were seen at hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il- , may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin- (il- ) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system ( , , ). in our study we monitored il- and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il- and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) < and ct abnormatities or gcs < over hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over hours csf and serum. il- and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described ( ) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il- and il- antibody. astrocyte migration was tested in a chemstaxis chamber. results head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il- were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il- could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il- antibodies, purified il- exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il- was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o il- on astrocyte functions, il- promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il- stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il- represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of - mmhg for pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. animals received a bolus dose ( mg/kg) of tnf mab (celltech, berkshire, uk) at min after resuscitation (tn ). the control group (n = ) was treated similar to the tn group but received ringer's lactate (con). results: at min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph ( . + . ), hco -( . ___ . mm), and base excess (- . + . ram) values with pco ( . + . mmhg) and po ( . + . mmhg) in the tn with no difference to the con group. immediately after resuscitation ( min) plasma endotoxin levels were found to be increased in both groups ( . + . in tn vs . _ . pg/ml in con group) . prior to the treatment with tnf mab ( min) there was also no difference between plasma tnf levels of the two groups ( . + . in tn vs + . pg/ml in con group). treatment with the tnf mab at rain post-hs improved the hour survival rate to . % as compared to . % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy animals in the tn vs in the con group. no bleeding in the kidneys was found in the tn but in rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = ) was prevented in animals which died in the tn (n = ) group (( . +_ . vs . +_ . g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. -+ n=ll*$ -+ n= _+ n= * * p< . vs baseline :~p< . no anesthesia vs anesthesia thus ) tnf production increased - fold by - hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; ) anticd had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd does not involve tnf suppression; ) fentanyl anesthesia at hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from to yeas of age. the percentages of bum ranged from % - %. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of pgjml - pg/ml. peaks in the tnfa values were above pg/ml and were also associated with a peak in the stnfr levels. these levels began at < , pghnl within the in,st ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl there is a significant corresponding peak in il-trn (> ~ /ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin- beta l-ii ), intefleukin- (il- ) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the hours. patients' ages ranged from to years. iss varied from to and transfusion requirement from to units. five patients died after the study period. ] ,- was raised in / patients (max level , pg/ml) but was unrelated to condition or outcome. / showed a rise in il- b (max level pg/ml) which was negatively correlated to iss (i=- . , p< . ). tnfa was raised in / (max level pg/ml). peak tnfc~ was positively correlated with iss ( = . , p< . ) and haemorrhage (i= . but p< . ). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak ,- and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [ ] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [ ] . radioligand binding studies with ~ odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc ) for hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./( + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o o (mm h¢i): _k, (nm}: n (molecules/cell): - . ± . _+ - . ± . + - , ± . -+ - . + . -+ presented are calculated values on the idealized curve + % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [ ] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il- and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c, see, ° v bsa, ld ) and ( c, see, ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il- , - hr following injury and of try activity hr after scald, followed by elevated levels of il-i and il- at hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and postburn. il- increase peaked - hr after scalding and levels remained elevated - days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il- activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska , belgrade~yu. asadullah k ( ), woiciechowsky c ( ), liebenthai c ( ), doecke wd ( ), volk hd ( ), vogel s ( ), v. baehr r ( ); depts. of med. immunology ( ) and neurosurgery ( ) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l patients undergoing neurosurgery from the first preoperative day until at least day after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)- levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il- ( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il- and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit ( - ml/kg) hypotension phase (mean arterial pressure = map of - mmhg for - hours followed by adequate resuscitation). rats subjected to hs (map of - mmhg for rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin ( ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin ( ng/ml) for hrs at °c. results: at min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group ( _+ vs + pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats ( + post-hs vs + ng tnf/ml pre-hs) and baboons ( ± post-hs vs ± pg tnf/ml pre-hs). in contrast, the il- productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs ( ± pre-vs ±_ pg il- /ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., , new biol. : ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin- (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et- /et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et- on the release of histamine, serotonin, and leukotriene c (ltc ) by primary mouse bmmc (in vitro age: weeks) caltured with different recombinant mttrine cytokines (interleukin (il- ) and/or kit ligand (kl) in the presence or absence of il ) for two weeks prior to activation. et- ( x - to lxl - m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to % and % of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il- and il- . only when cultured in the additional presence of il- rather than in medium containing kl or kl plus il- alone, bmmc released histamine and serotoaln upon challenge with et- .furthermore, et- stimulated ltc biosynthesis up to -fold in bmmc cultured in the presence of il- . in contrast, the peptide was without major effect on ltc biosynthesis in bmmc culturd without il- . the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il- prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l- . et- ( " m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of - m. crude peritoneal cells (containing - % serosal mast cells) obtained from normal balb/c-mice released % histamine upon challenge with et- ( - m; rain). taken together, these resu/ts demonstrate that et- can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc production in mast cells. il- appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a (type ii pla ) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea , endotoxin, tnf-c~, il- and il- were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla and tnf-a in these patients were . ± . ng/ml and . ± . pg/ml, respectively, the two parameters being significantly correlated (r = . , p = . ). there were also a significant correlation between the level of type ii pla an il- (r = . , p = . ). there was no significant correlation between the level of type ii pla and il- . these in vivo findings suggest that tnf-a may be involved in the production of type ii pla . a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)- down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. , j. immunol. : ) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. , cancer res. : ) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. , embo j : ) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il- , tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il- the additional presence of rmull- for or days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack- ) nor kl-mediated proliferation or chemotaxis. in contrast, il- syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes . pre-incubation of bmmc with rhutgfl~ ( . , . , or . ng/ml) for h had no effect on kit receptor expression, although tgffil at concentrations of . - . ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il- , i,- , il- , and/or il- ). moreover, pre-incuhation ( h) of bmmc with tgf-gl (> pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il- -or kl-induced chemetaxis. when bmmc were preancuhated with rmukl ( ng/ml) for , . or days, a transient down-modulation of kit receptors with a maximum effect on day was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il- nor tgfi affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi cells from tnf-mediated cytolysis. material and methods: wehi subclone ceils ( x ), that are highly sensitive to the cytolytic activity of tnf, were grown on -well culture plates in rpm medium. after hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn ( - bp) were added ( . , and pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at nm. results: wehi ceils incubated with tnf ( - ng/ml) were completely lysed after hours ( % abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with . ng/ml tnf and jm odn showed still % of the absorbance observed in control ceils without tnf ( % abs). the protective effect of odn started at . pm, reached a maximum at ,um, and diminished at jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e , d- erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il- ), in the sepsis model of lipopolysaccharide (lps, mg/kg i.p.) induced hepatitis in galactosamine (gain, rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at hr and hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, mg/kg i.p.), rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il- levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, x mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, x unit/mouse i.v) r spectively. pretreatment with al and sod ( and hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il- into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee b'~ th~ recombinant mmlse tnf-o' ( . ~/rno~e j.p.) ~d oi~lps ~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il- release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients ( + years) in severe sepsis (sepsis score > according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma ( + years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. pl of whole blood were incubated either with / of a standard medium or with pl of a standard medium and pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours plasma concentrations of tnf-a, il-la, il- and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if- ,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients ( males, females, mean age +_ ) with severe sepsis (sepsis score > , elebute and stoner} were drawn on day , , , , and of disease. blood was immediately placed on ice and processed within hour. pl of whole blood were incubated with pl rpmi-medium supplemented with antibiotics and l-glutamlne or with pl of rpmi-medium and pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours samples were centrifuged and plasma was harvested and stored at - ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < , ) for circulating mean arterial tnf-a concentration ( pg/ml _+ sem} and central venous tnf-a ( pg/ml +_ sem). upon in vitro stimulation there was also a significant difference (p < , ) between released arterial tnf-~' { pg/ml _+ sem) and venous tnf-a ( pg/ml +_ semi. conclusions: these results are difficult to interprete but could reflect the influence of pao and sao on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il- ), tumor necrosis factor-a (tnf-c and interleukin- (il- ) the multifunctional cytokine intedeukin- (il- ) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il- -concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il- may be a potential substrate of these preteases. the serine preteases elastase (ec . . . ) and cathepsin g (ec . . . ) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin- (il- ) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il- leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin- . furthermore the loss of the biological activity of both, recombinant and natural human il- , was demonstrated by determination of the capacity of protease-treated il- to stimulate hybddoma growth ( td bioassay). these data suggest a possible downregulation of pathologically elevated il- levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il- , il- , _- -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il- , _- and il- were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to days, for a total of four tests performed (to, tl, t , t ). i .- was not detected in any samples. the serological profiles of the two cytokines .- and _- were similar; augmented levels were found at study entry and throughout the observation period, peaking at t and decreasing at t . however, in patients with sepsis, il- and _- concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il- and il- release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il- and il- production appears more significantly elevated, suggesting a role of il- and _- in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc and interleukin (il- ) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after hours and every hour. tnfct and il- levels were measured using the bioassays with cell lines wehi- ci and td , respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il- was found in all the patients' plasma samples and five patients' ( . %) ultrafiltrate samples. the il- blood level ranged from . to . u/ml (mean . , sd . ). the il- level in positive ultrafiltrate samples ranged from . to . u/ml (mean . , sd . ). conclusions: our preliminary results suggest that il- is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il- and il- and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd- mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first h with a peak h after clp at a significantly lower level than after lps. il- was measurable in serum only after h, significantly increased in spleen and liver after and h and in mesenteric lymphonodes from to h after clp compared with shammice. il- was significantly increased in serum throughout the first h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= ld ) to the sublethal (= / ld ) of three different lps (e.coli oiii:b and :b ; p.aeruginosa r ) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps :b induced as much tnf~ as the sublethal dose of lps :b ; furthermore, lps r , whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r ) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to h post lps administration moreover, the treatment with mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. ) circulating and organ levels of if are proportional to the dose and degree of toxicity of all the administered lps even if lps r was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if . to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if production. together with the total ablation of if , the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin- (il- ) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd- mice were randomized to mg/kg e.coli :b lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at , ', lh, . h, h, h, h, and h ( mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit pg/ml) and expressed as ng/g weight + sem (lowest detectable amount ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [ ~- p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present ( . + . ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced -fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was . + . ng/gwt at lh, reached maximal levels at . h ( . -+ . ng/gw-i) and returned to baseline at h. saline controls maintained their constitutive il-i~ levels. sb had fold more il- ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r = . ). separate mice were pretreated with saline i.p. orthe il- receptor antagonist (irap, mg/kg bolus i.p.) and were challenged rain later with . mg/kg lps i.p. or saline i.p. specific blockade of il- by irap decreased intestinal secretion at h and h post-lps challenge (p<_. . , student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content ( . % of total pituitary protein) and peak serum mif levels ( - ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps ( mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone ( % vs. %, p = . ). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps ( . mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from % to % (p = . ). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by . _+ . % in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin- (il- ) antibodies and il- receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il- in the serum does not correlate well with outcome. we hypothesized that this may be because il- acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il- levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n= in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. , j. inununol. : ) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after and hours of lps injection ( btg/ . ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels iron after injection amounting to only to % of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in % mortality in w/w v versus % mortality in +/+ mice . days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin- (il- ) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli :b , gg ip) into balb/c mice induces the rapid release of circulating il- ( ± u/ml at min). blocking endogenous il- using monocional antibody (jes - a , mg, h before lps) resulted in a massive increase in tnf production ( ± in lps+anti-il- treated mice vs ± ng/ml in lps alone, p< . , n= to mice per group) and an enhanced lps-induccd lethality ( % vs % in anti-il- +lps or lps alone respectively, p= . , n= mice per group). irrelevant igg rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il- during human septicemia. plasma samples were obtained from patients with gramnegative (gns, n= ) or gram-positive septicemia (gps, n= ) and from healthy volunteers. among these patients, suffered from septic shock at the time of sampling. il- levels were measured by elisa (detection limit: i pghrd). we found that patients ( %) had increased il- plasma levels (range to pg/nd). patients with gps had il- levels similar to the ones observed in gns (median: vs . pg/m, respectively). patients with septic shock had higher il- values (median: pg/ml) than septicemic patients without shock ( pg/ml, p= . ). no il- was detected in plasma from healthy volunteers. we conclude that il- is produced daring human septicemia. our experimental data suggest that il- might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, route de lennik, brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache i and sss). patients and melhods: septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i ( kda) were measured by enzyme immuneassays (norms values = + pg/ml and . _+ a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were + pg/ml and . + . ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p< . ). mean tnf concentrations for each patient were significantly greater in non survivors ( + vs _+ pg/ml p< . ); stnfr-i levels also were greater in this group, but the difference was not statistically significant ( . + . vs . _+ . ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = . p< . ). mean tnf levels were significantly correlated with apache ii (r = . p< . ) and sss (r = . p<o. ); stnfr-i levels were likewise correlated with both scores (r = . p< . and r = . p< . respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il- ). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of pg/ml and ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in and out of the patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il- . multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il- . peak plasma levels of both lif and il- correlated inversely with patients survival duration. cox's analysis for plasma levels of lif > pg/ml yelded a hazard ratio of [exp ( . )= . ]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human kda tnf receptor (stnfri) and the kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: points). serial blood samples were obtained within h after diagnosis of sirs, every hrs for the first hrs and every hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o ) compared to healthy controls (normal value: tnfrh , ng/ml; tnfrii: , ng/ml). the serum concentration of tnfri ( , ng/ml) as well as tnfrii ( , ng/ml) were significantly higher in nonsurvivors (n= ) compared to survivors ( , and , ng/ml; n= ) at the onset of sirs and during the course of the inflammation response (p< , ). a positive correlation exists between both receptors (r- , ; p < , ). increasing levels and maximal values of stnfri ( ng/ml) and i ( ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in out of patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. - is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first hours after administration of il- . in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il- and il- using immuno assays. in these plasma samples we also measured phospholipase a activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il- therapy the cytokines tnf, il- , and il- are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il- . objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin- (il- ) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il- by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of healthy volunteers and cultured at a concentration of . • ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after hours. using elisa, tnf-c~, il-ib and il- concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il- ] and il- by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il- in ng/ml as mean _+ standard error. ca/ml . . ( ) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity ( , x - m) as the fab-fragment ( , x - m) but bind the antigen with a eightfold lower avidity compared to the parental mab ( , x - ). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl- and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l , the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a -fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies ( ) reveales that the glucocorticoid antagonist ru , nufepristone, sensitizes both endotoxin responder and nonresponder, c h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and hrs following lps injection, ru significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru . however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru treatment. these studies suggest that ru aggravates lps lethality via factors other than tnf in endotoxin non-responder, c h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi response in - % of the pbmc after - hours of cell stimulation. in contrast, il and tnfc~ containing cells was only detected in - % of the pbmc. the induction ofil , il , il , and ill , was weak or completely absent. thus, the response indicates activation of th cells. however, illc~, illl , illra and il , were consistently found and gradually produced, peaking at hems in approximately - % of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of rag , , -trinitrobenzenesulphunic acid in . ml % ethanol (n= ). control animals received an isovohlmetric instillation of normal saline (n= ). faecal pellets were collected before induction of colitis (day ) and throughout the experiment. after days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day -#p= alld the total ftnf measured during the study correlatcs with the cms oll day -p= . there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice ( - g) were primed intraperitoneauy with either lipopolysaccharide (lps) ug]anlmal or saline. five days later, the animals were subjected to hypoxia ( % ) for , followed by of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at , , and and assayed for tumor necrosis factor (tnf), prostaglandin e (pge ) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia ( %o disease , berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl . we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il- receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps ( ng/ml) supplemented with different concentrations of mps ( , pg/ml, mg/ml). after hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il- ra, and tnfbp-i (tnfsrp ). il-la, il- ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to . + . %( mg/ml) by mps in a dose dependent fashion, whereas about % decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased times more in the supernatant of lps-stimulated pbmc with gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional -adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that -ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected times with ! -ar agohist isoproterenol ( . mg/kg i.m. and . mg/kg s.c.) , and hours prior to a lethal dose of lps injection ( mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. ) . these data suggest that -ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at °c in rotating polystyrene tubes. samples were taken before addition of x ng/l e. col± endotoxin and after hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin- (il- ) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of mg/ml or mg/ml or with the monoclonal lipid a igm antibody ha- a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il- values were found.in the plasma. cytokine values at hours: tnf-ec pg/ml (mean) and l- pg/ml (mean). preincubalion with mg/ml standard immune globulins reduced il- values by % (mean) while there were no effects on tnf-c~ values at hours. a slight reduction in il- values was also found with the lower concentration, mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il- nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha- a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced l- release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha- a had no effect on the il- or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto % of the estimated blood volume was withdrawn over - rain, to reduce bp to - mmttg and co by / mad withheld for rain. shed blood ( %) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of %. zymoeen activated plasma ( , gg/ml) was given rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r , rat igg , mgkg i.e. n= ) or emine (nffi ), rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p< . ) higher in saline compared to r -treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p< . ) higher in ealine ve r animals at and h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p< . ) attenuated in r treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin- (il- ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il- in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, sprague-dawley rats were studied. anesthetized rats were divided in groups, implanted with alzet r minipumps and randomized to receive the following treatment for days:i: gg/kgbw/min of il-lcc; ii: ~tg/kgbw/min of il- receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a % bsa, full thickness scald burn and the wounds then infected with + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first days and improved food intake between days and . we conclude that blockade of il- after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il- . ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il- producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx ( mg/day) in septic patients resulted in tnf and il- productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( ml/kg/day ). the prevention of lpsinduced in vitro tnf and il- production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam- in sera of septic patients was significantly higher ( - ng/ml) than in normal individuals ( - ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy rgyi medical university, institute of microbiology, szeged, hungary h- ddm t. . e~i~im~/tii, septic sh ~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit ' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il- concentrations. methodology female wistar rats (wt - g) (n- per group) were prefed with % tanrine, . % glyciuc ( in tap water) or tap water (tw) for days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received . % saline subcutaneously ( mls/ g) post-procedure. animals were venosected by direct cardiac puncture at or hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at - °c was completed within hours. tnf and il- were measured using wehi and b bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il- estimation revealed a statistically significant reduction at hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin (il- ), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after days systemic blood was harvested to determine the circulating levels of il and tnf. a second group was treated with thy for days and then received a gavage wide lxl escherichia coli and a % burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had mice/group. in separate groups (n= /group), receiving the same treatments, mortality was observed for days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av difference. it was mclean in a study reported in that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are , neutrophils per cubic millimeter of blood, a kg man would have ~ . xi u circulating neutrophils. when activated ixl ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate - . xi nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of - . ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il- , il- , and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m populations; it decreases splenic m but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon- offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin- , which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il- and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il- and il- +m-csf treatments increased the production of tnf in burn day macrophages and progenitor cells compared to normal cells. il- +m-csf+il- increased the production of il- by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il- +m-csf increased the mrna for tnf in the -day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il- /~ actin mrna indicated that il- , il- +m-csf, and m-csf increased the il- ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~ servojf of appr m ~ separating i~ eucayotic ceils of the hosl ttom l ~ bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (. u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= %' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h -receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h -receptors by h -agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,- decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il- , and , and shared mscrophags funclion with loss nr f and c b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we g~ven in doses of ~ . to ~ , u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~ animals, interferon ~amma was a so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to ,s uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< , ). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c , interleukin- (il-ii ), interleukin- (il- ), and interleukin- (il- ) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin- (il- ), interleukin- (il= ), transforming growth factor- (tgf-j ), and the newly sequenced interleukin- (il-i ). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u . in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il- b and tnf-a. the il- receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p ) and type ii (p ). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il- , il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il- and l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, ) early decreases in total t and helper t cells in the peripheral bloo~ } lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and ) severe and prolongei deficiency of interleukin- (il- ) prcductlon by peripheral blood mononuclear cells (pbmo). this il- deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il- deficiency end shown that the defect lies post-transcrlptionally since mrna for il- is also depressed following thermal injury. we have also investigated the potential of a defect in ii- receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il- r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd +) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: ) occurrence of activation-induced cell death (aicd), ) activation-related phenotypic and functional_ diversity in the pool of peripheral cd + t cells. initially, (up to days post-bum), peripheral blood lympliocytes from severely injured (> % total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l (cd ) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd +/cd ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il- secretion and downregulating excessive il- and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either . g/kg of e in a % solution by garage (ge), or . g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a % body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p and p were both - fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p and p during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. decker, d., sch ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages -ii according to me sherry), were - years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn h before surgery, immediately before incision (after anaesthesia), h and h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in laparoscopic and open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were . the interleuldn- receptor and im soluble form (cd /scd ); . the activation antigen fd- and its soluble form (cd /scd ), a member of the nerve-growth-factor receptor family; . the cd ro epitope which characterizes t memory ceils; . the trausferrin receptor cd ; . the soluble adhesion molecule icam- ; and . the cytokines interieukin- and interleuldn- . on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to h before surgery. h after incision the tra index in open cholecystectomy showed a distinct - fold increase, whereas in laparoseopic surgery a mere - fold increase was noted. h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was . % (range %- %) of total body surface and mean age was years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd and most strongly positive for cd . this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd and cd positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd +) was unchanged and nk (cd +) cells were decreased by ahnos[ % compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd were unchanged. cd (il receptor) and cd (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days ( ) t, , , and post injury. we analyzed the cumulative data for interleukin- beta (il-i[ ), il- , il- as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d post-trauma and recovery thereafter. *p < . , ** p < . vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il- and ,- , suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r= . ). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss> , n= ). i - , - , g-csf, fpa, and c a -levels differed significantly (p< . ) between the iss-groups (>-< iss ) at the time of admission, whereas on day tnfa, c a, - , and ealpi showed significant differences. beyond the first week, major differences were restricted to pge and c a. the formation of two groups with respect to later multiple organ failure (mof < ; mof > n= ) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd (monocytes), cd /cd (i - r + t-cells), and cd /cd (th calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving % or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma ( % tbsa). following a % tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a % tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of % tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with % and % tbsa thermal injuries, respectively. a % tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a % tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm and the office of navy research n - -j- . division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut . post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. cd- mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, cd- mice were randomised into control (c), l or s groups and peritoneal cells studied at hours for bacterial phagocytosis and killi:~g, superoxide ( -) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd- b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac- was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac- expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in ,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. - mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a ,c chamber for recording by real-time video confocal microscopy. the cells were subjected to msec, v/era, a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was , .c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with mm mg%. experiments were repeated with mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. patients ( males females,mean age: . _+ ) with gallstone disease and with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the st and th postoperative days (pre, lpo, po) percentages of cd , cd , cd , cds, cdi were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il- , il- , il- , il- , tnf) were measured in pts. of each group by means of elisa using moab. _r. esults:variations of il- and il- were not s.s.. il- increased but differences between groups were not statistically significant (s.s). il-i decreased on po and increased on po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p< . ).tnf decreased in the l.g. and increased in the th.g. on the po, the difference was s.s(p< . ); on po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on po, except for %cd cell that increased on lpo and decreased on po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< . i), but differences between them were not s.s. in contrast, igg decreased on po (p< . ) and increased on po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, % nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia, and hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound or hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd ) and helper-t-cells (cd ) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd ) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd ) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd +hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd +cd +leu -pos) was increased just after anaesthesia being decreased at g and hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples hours after the operation with recovery at hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at hours (p<o. ) and hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in trauma patients on hospital admission and x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age -+ years ( m, f), mean injury severity score (iss) + , sepsis ( patients), sepsis syndrome ( patients), ards ( patients), renal failure ( patients). ige increased in patients ( %). the mean increase was ng/ml (range to ng/ml; % ci was to ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p= . ) and renal failure (p< . ). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p> . ). plasma ige increase was not related to severity of injury by iss score (p = . ). the mean day to highest ige was . -+ . . the day sepsis was first observed preceded the day of highest ige by . + . days. there was a significant association between the day of sepsis onset and the day of highest ige (p= . ). eight of nine patients with sepsis syndrome had > % increase in plasma ige from admission. one patient's ige levels were normal ( - ng/ml) for days and then increased to ng/ml over the next days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il- , which concurrently regulate production of ige and il- receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during hrs. adult male wistar rats ( +_ gin) were submitted to hs (sbp tort) and after t hr (shock i hr) and hrs (shock hrs) hsl (nac . %, . ml/kg) treatment, e. coli (i ) was injected into the portal vein ~tci (n_> ). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p< . , ~" p< . and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung uptake was increased and liver and spleen uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after hr of hs hsl reatment, diminishing by hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after hr hs hsl treatment, returning to normal values by hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of rabbits, labelled with indium-tropolene and reinjected intravenously into the rabbits, i rabbits received an infusion of escherichia coli endotoxin ~g/kg, while i rabbits were subjected to a major sham operation and i rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and , and hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd , cd , cdlla/b, cdis, cd , cd , mhcii and cd /cd ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd /cd ratio while the cd positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to % of initial values following endotoxaemia and to % following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd cells compared to cd positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin- (il- ) and interleukin- reeeptor(il- r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at , i, , , days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il- stimulated by concanavalin a(con a): the sera from the rabbits days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, kd,milipore; centricon- , kd,amicon), that are less than kd, between i and kd, and more than kd. each group of the substances also was tested for the expression of il- and il- r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il- by the con a-stimulated splenocyte in mice; ) the sera from the rabbits days after injury had more profound suppression than other injured sera; ) there was a marked band at about kd in sera from the rabbits days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; ) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il- and il- r than other groups substances, of which molecular weights are more than kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; ) there is kind of substance, of which molecular weight is about kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; } the substance can depress the expression of mrna of both il- and il- r. research institute of surgery daping, chongqing, p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m( results in decreased antigen presentation, induces tgf- and pge while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il- . consequently, here we investigated rnonokine production in trauma patients (n= ) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t ) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days - post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the - days post injury period became higher in alcoholic trauma patients. furthermore, over days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml - days post-injury days post injury stimulus ale. pt. pt . . . . immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol ( - mm) exposure resulted in a significant down-regulation of tnfc~ (p< . ) and il- (p< . ) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd + receptor, the proinflammatory mediator il- , the macrophage activating factor ifn- ,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days , , , and after mechanical trauma of and after bum trauma of patients (mean age ~ years; mean iss ± pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il- and ifn- in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly ( + u/ml) compared to controls ( + u/ml) as well as mechanical trauma ( + u/ml). il- showed a significant suppression following mechanical trauma ( + u/ml) vs control ( + u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than % of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l- and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il- agonist) in eutrophic rats subjected to either % tbsa cutaneous scald injury (bi), muscle scald injury of equivalent % tbsa (mbi), sham muscle bum (resection of skin only, up to % tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation ( mukg i.p.). separate rats were infused with mg/kg e.coli :b lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount . ng/gwt). il-lo~ was constitutively present only in the skin ( + . ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at . h (in the liver, bi = . _+ . ng/gwt, sbi = . + . ng/gwt, p _< . ; in the lung, bi = . + . ng/gwt, sbi = . + . ng/gwt, p -< . ). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint already ( min real-time). these levels increased in parallel until min and became eventually different by log at - . h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il- ~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il- c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma . j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of mmi-ig for rain. the animals were then resuscitated with either times (x) the shed blood vohune as lactated ringer's solution (lrs) or x lrs + lx % lies. sham mice were neither hemorrhaged nor resuscitated. at or hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il- ( alternatively pmqb showed no differences in il- release between all groups at and h, while sm~ from the lrs + hen group showed a depression at h. tnf production by pm~ was depressed in all groups at h and remained so in the lrs + hes group at h. sm~b showed decreased tnf release values in both hem groups at and h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il- ) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in patients with multiple trauma (median injury severity score = , ). blood samples.were collected shortly after injury and after , , , , s and l days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p< , s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury ( - h) at , eki/ml (median), decreased thereafter to , eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day revealing the lfighest level to la followed by pa (= % of la-sac), ec (= % of la-sac) and vc (= % of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day ). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day and . however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r> , ; p< , ) most often between antibodies of igm-elass ( %) followed by igaclass ( %) and lgg class ( %]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin- (ii- ) and c-reactive protein (crp) were measured in patients. i female and male, ageing from to with a median age of . blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, and hours after the operation and then every morning until the th postoperative day. blood was drawn into heparinized tubes (i iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. - levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p . ) in endotoxin plasma occurred during surgery, culminating in a peak (median value of . eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the th day after surgery, whereas those of interleukin- rose at the end of the operation and were at their highest hours later (median value of . pg/ml). crp levels were also high postoperatively with a median value of mg/l, and were markedly raised on day ( mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and - during the operation was establisthed (p . ), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of - , and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or - plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map ± mm hg) for minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at and hours following hs for quantitation of slga by ria. animals were sacrificed at hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled and days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day in the control . + . , in group . + . , in group ii . + . , in group iii . + . , in group iv . _+ . , in group v . _+ . x ( ). the evident increase in bmc yield in all groups continued until day . increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from through , and who had serum cholesterol assayed during the first hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< . level. results: trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first hrs. patients had cholesterol levels less than mg/dl; of these ( . %) died, ( . %) developed ards, ( . %) developed acute renal failure, and ( . %) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was times greater and risk of multi-organ failure times greater in this group than in those with a normal serum cholesterol (>if mg/dl; patients; p< . ). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n= ) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n= ) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi ) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f ~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p< . ) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa which was more pronounced in c (p< . ) and most in b (p< . ). similar results were found for pge~ and pgf =. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p< . vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r= . , p<o.o ) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt . + . tt , +_ , n.s. pla-ca(s) mt . +_ . tt . _+ . n.s. goris mt . - . tt . _+ . n.s. iss mt - tt - p< . we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim nez f j, l pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha- antitrypsin (aat), alpha- macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of patients, males and females (mean age: years), with compound fractures. % of them were located in tibia and fibula. intercurrent infection developed in patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days , , , , , and . a control group of healthy volunteers was also evaluated. aat and cp showed increased values from day which kept elevated untill the end of the study. amg elevated from day . when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il- , il- , tnf, pge and tgf-b have all been implicated in these immune system changes. in vitro, pge causes many of the immune system changes following hs, yet the time course of pge release has not been well deliniated. the aim of this study was to determine the time course of pge release by peritoneal and splenic macrophages following hs. c h/hen mice were bled to a mean bp of + mm hg for minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at , , , , and hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for hours. the supernatants were collected and frozen until assayed. pge was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge release by peritoneal macrophages was significantly elevated at , and hours over control. it was maximal at hours, but by hours it had started to decline. splenic macrophage pge release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge is significantly elevated at hours following hs, and reaches a peak at hours. pge release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in patients ( male, female, mean age . ), admitted to our icu following serious trauma (mean iss= ) and in control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was . (range - ). patients didn't develop organ failure(nof-group), patients survived, developing mof (s,mof-group), patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days - . n and crp levels showed a parallel release peaks on the days - and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values - h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats ( g) divided in groups (observation time hrs): a: mild hemorrhage by sequential blood sampling ( . ml each)( , , , , , , , , hrs). b: a with hemoperitoneum (hp)( ml/ g), c: hemorrhagic shock: blood withdrawal of ml/ g over min and resuscitation with ringer's lactate ( x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); fe labeling of erythrocytas (application of ci fe i.a. into a donor rat, days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at and hrs, respectively ( + vs. + ; +_ vs. +_ ). hp reduced this increase ( +_ vs. + ; + vs. +- , p<. ). i-ip significantly increased hemoglobin between to hrs in mild ( . +_. vs. . +. ) and severe hemorrhage ( . +. vs. . +. ). in mild and severe hemorrhage comparable maximal resorption ( fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at hrs (mild: +_ vs. severe: +- counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: / ; hp: / ). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at hrs a significant higher transferrin level than with hp alone ( . +_. vs. . + . ). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during days after cct. antigenic challenge was made on day i, , or . number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day and reached a maximum when antigen was presented on day after trauma (pfc number . times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, & , prostaglandin e (pge ) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (> %.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output > cc's per hour had et levels drawn on admission, at i, , , and hrs. et levels were measured by radioimmunoassay. mean levels were elevated at ± pg/ml at all time points versus levels in healthy controls of ± . in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin these processes since it eradicates the influence of infectious factc~rs. dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il- , saa and neopterin, the percentage of cd + cells, the in-vitro il- synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of patients with cholecystolithiasis undergoing either laparascopic ( ) or open (i ) cholecystectomy on consecutive perioperative days - , , and . there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients ( versus minutes, versus days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i + lng/ml, d + ng/ml) versus lap group (d- + ng/ml, d + ng/ml), while in serum il- levels we saw a less steep increment in the lsc group ( -fold from d- to d ) compared to the lap group ( -fold from d- to d ). the analysis of cd + receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d - : . + . cpm, d : . + . cpm) compared to the lsc group (d -h . + . cpm, d h . + . cpm). in both groups il- synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors ( - and kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il- mrna and il- production was detected. the nucleotide sequences of the human il- gene regulatory region bounding by the splenic nuclear proteins were determined between + - b.p. the il- trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il- production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il- gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania non-pregnant patients from rural areas were followed. of preoperative patients % had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied patients of which % had a parasitaemia on admission and % had postoperative malaria. half of the surgical patients came from rural areas, whilst only % of those with malaria lived in the city (with much less exposure and immunity). % underwent major surgery and % minor. bloodtransfusions ( % with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about % of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that % was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co derived from [i- c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol . ~g/ml, glucagon #g/m , epinephrine ~g/ml, insulin t~u/ml; traumaeortisol . ~g/ml, glucagon /*g/ml, epinephrine ~g/ml, insulin ~ij/ml. analysis of twenty subjects showed a reduction of ° ~mp shunt activity by lymphoeytes and a ] % reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < . . lymphocyte glucose uptake was also reduced by trauma hormones p~ . . it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence f.neutr phils. there~bre, neutr pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln- (i ..- ) and mo~tocyte chemotacde pop, de (x¢cp- ) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from ipf and sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii. arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i ( . ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf ( . ng!mg) in comparises to . normal individuals ( . ng/mg) and to patients w~th obreic bronentis (cb) (~, rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp- e.qu/va]ent) was strongly increased in sareoidosis ( . ngjmg) as well as ~n f pag,nts ( . ng/mg). norra.al indlvidu~s and cb patiants hzd a . or -fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il- ievei~ ( . and . rig/rag, respe~veiy; nomzls: . rig/rag; cb: . ng/mg) mad nvatropmi ohemotax~ ( . ~'~d . nnmg, res!z~ztiveiy; aormals: . ng,'mg; cb: l ngmg). these data suggest that increased ievels of born mcp. ~d il- may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ ( fold vs. lps or paf alone), ll-lg ( fold), kc ( fold) and il- . taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day , hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays - and further observation and measurements at days - . from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight kg) neutrophil counts ( e pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood ( e cpm/ , pmn), and of blood-and bal-isolated pmn ( e cpm/ , pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca + second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in polytranmatized patients (mean injury severity score = ) arterial and venous blood samples during days. daily evaluation of horowitz-quotiant (po /fio ), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day and (day : +. ; day : . +. ). plasma lactate was significantly increased at day l ( + ) and day ( . + ). hurowitz-quotient (day : + ) was low at day ( + ) and day to ( + )(p<. ). at day a substantial rise in venous pmnl-superoxide production (day : . +_. , day : . +. , day : . +_. ), oecured with significant increase in plasma lipid peroxidation (day : . + . ; day : . + . ). pivin~-myeloperoxidase activity was high at day ( . +--. ) and then continuously declined (day : . +. ). plasma antiexidant activity (glutathione pemxidase) was reduced by % at day (day : . +. ; day : . +_. ; day : . +. ). whereas basal ca + concentration remained unchanged (day : +_ , day : +_ ), fmlp-stimulated cytosolic ca + mobilization increased at day (day : + , day : , day : + ). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca + mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, homburg/saar donnelly sc, haslett c, dransfield i, robertson ce, grant is, carter c, ross ja, tedder tf. dept's of respiratory medicine, accident & emergency, intensive care, surgery, university of edinburgh, scotland and dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n= ), perforated bowel (n= ), and multiple trauma (n= )), of whom progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o. ) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity ( _+ pmol/i vs _+ ; p - . ), but not the release of neutrophil related mediators ( . _+ . ng/i vs . _+ . ; p= . ). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb ( . + . % vs . _+ . ; p = . ). although not significant, nac prevented also the increase in elastase levels in lavage fluid ( . + . pmol/i vs . + . ; p = . ). it is concluded that nac is able to protect against the inactivation of a -proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion ( rain) and repeffusion ( min). sprague dawley rats were randomized into: control (n=i ); ischaemia repeffusion (ir) (n= ); and ir treated with gtn ( ug/kg/min) during repeffusion (n= ). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n= per group). control _+t "* _+ _+ ** bal pmn/mm l+- " + # _~ *p< . , #p< . v control/gtn;**p< . v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from . + . to . _+ . mcf after minutes repeffusion (p<. ). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin , ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, #g in . ml saline). three groups (n= each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh ( ~tg in . ml saline) at , , and hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f , = . , p<. ), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<. ). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<. ), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia ( mg/kg, i.p.) in female balb/c mice ( - g) that were then randomly assigned ( - mice per group) to receive at time and hr later: control saline thjectioas (i.p.), injections of ~-msh ( ~tg), gentamicin sulfate (i mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in patients ( female, + years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and min after declamping. arterial pla concentrations were higher than venous ( + vs. + ). while cytosolic basal pmnl ca + concenlxation was not altered at the end of ischemia (v: . + ; a: _+ ) and during reperfusion (v: + ; a: + ), fmlp-stimulated cytosolic ca + mobilization showed a significant arterial increase as compared to venous ( _+ vs. + , p < . ). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by ) pulmonary membrane damage possibly due to pmnl-degranulation, ) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and ) a significant arterial increase of pmnl cytosolic ca + mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca + messenger system by inflammatory mediators (e,g. ii- , tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p cell) is not only an important target, but as recently shown it is also capable of producing h . the aim of this study was to investigate the influence of endotoxin on h release of p cells. we obtained p cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h release of fresh isolated cells and on cells cultured for days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for hours with p.g/ml lps and tested the conditioned medium (mcm) on cultured p cells. p cells h ex vivo were _> % pure, _> % vital and had an basal h release of . _+ . nmol h per hour and million cells. adding p.g/ml lps to the incubation medium the h release decreases slightly but significantly to . _+ . nmol. adding . p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h release increased -fold to . _+ nmol. pma induced h release decreased to . + . nmol after addition of p.g/ml lps. after culture days the p cells were _> % pure and showed a pma inducible h release of . _+ . nmol addition of p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h release up to . _+ . nmol. addition of mcm to cultured p cells increases pma-stimulated h release to . +_ . nmol. the release decreased to . _+ . nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > % in all experiments. the results show that lps has an direct effect on p cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h release of p cells could be important for generating internal oxidative stress in p cells before external oxygen radicals exceed. the produced h did not necessarily damage p ceils, but it can effect surfactant metabolism, especially when extracellular h release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled patients with severe lung injury after sepsis ( ) or polytrauma ( ) and obtained multiple blood samples ( days) for endotoxin, tumor necrosis factor e (tnfa), interleukin (il- ) and interleukin (il- ) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l and il- were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, . ng/ml) for hours at c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao /fiao quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < ( . ± . eu/ml, mean values ± sem) versus the patients with a sli > ( . ± . eu/ml, p<o. ). the basal plasma concentration of ll- also correlates with the sli index (p<o.o ). the delta tnfe and delta il- values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n: ), the deceased patients (n= ) showed higher endotoxin level, a reduced tnfa and il- releasing capacity of the blood and higher basal il- levels. we can conclude that endotoxemia, high il- plasma level and a decreased capacity of the blood to release tnfa an il- under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il- in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado ) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($ or $ ) of the right and left lung through a bronchoscope on days l, , and days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado unexceptionally deteriorated postoperatively and the mean aado in patients reached the maximum (mean ± sem; _+ mmhg) on the rd postoperative day. the mean concentrations of il- , ne and neutrophil count in balf also increased postoperatively and reached their maximum level ( + pg/ml, -+ gg/l, and ± x cells/ml, respectively) on the rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il- and ne levels in balf (r= . , p= . ). also, significant correlations between aado and the neutropbil count, and the concentration of ne or l- were observed. in contrast to il- , neither il- , il- nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il- increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes ( mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats h prior to pma exposure ( pg/kg, intratracheally) and treated rats were killed h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp- ,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n- ) weighing .d- . kg oaselinepao> kpa and mean pulmonary arterial pressure (mpap)<i. kpa were included in this study. anaesthesia was induced with thiopental - mg/kg bw and "blind" jntubation was performed. a f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose mg iv or metylprednisolone mg iv min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap( . -+o.~ kpe ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp- or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients ( sepsis, trauma) with an fie of . + .( , a peep of . ± . , and a murray score of . ± . were included in this study. healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) . ~: . * . ± . *= < . , **=p< . condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io ), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of serum samples were collected from patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n= ) and non-septic (n= ) patients. patients with ards (n= , . + . ng/ml) and with respiratory disease other than ards (n= , . +- . ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n= , . _+ . ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using -hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase ( .i- . %) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej a - ~ia~ystok, poland, tel/fax + . in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) - ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. : , ) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n- -and n- -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf ( um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of -series lts and -hete (total sum of - ipoxygenade products rd. and rd. pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of - ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. pmofml). eicosapentaeooic acid ( um) elicited exclusive generation of -series lts and -hepe (total sum rd. pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived - ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. pmol/ml). we conclude that the paf-evoked - ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n- -versus n- enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr- , incubated with either pmn or mnc for hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin (cx ) which forms channels between cells capable of pas.~ing any molecule smaller than d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > adjacent hepatocytes within seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx is markedly decreased while the secondary gap junction protein cx is either unchanged or even increased. while cx readily effects electrical coupling, molecules > d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $ although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., and h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci , peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for , and hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after minutes of hepatic ischemia and hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to min of hepatic ischemia followed by reperfusion (rp) and injection of . mg/kg salmonella enteritidis endotoxin (et) at min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from + u/l (i/rp only) to + u/l at h rp. inhibition of kupffer cells (kc) with gadolinium chloride ( mg/kg) attenuated liver injury in this model by %, however, monoclonal antibodies (cl , wt ) directed against adhesion molecules ( integrins, cd ) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time ( + pmns/ hpf; baseline: + ). isolation of kc and neutrophils from the postischemic liver indicated a -fold increase of the spontaneous superoxide formation only in the kc fractions [ . + . nmol o -/h/ %elts (kc ); . _+ . (kca) ] at h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments ( h), the antibody wt ( mg/kg) attenuated liver injury by % at h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es- and gm- ) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type (scri), mg/kg iv after vascular occlusion (n= ) or by depleting the complement system using cobra venom factor (cvf), . mg im, and hours before ischaemia (n= ). non-ischaemic rats (n= ) and ischaemic non-treated rats (n= ) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c and c were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p< . ) and haemoglobin saturation (p< . ). c and c were present in the endothelium of the ischaemic control group. no deposits of c or c were found in the cvf group. few c and no c were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, control and cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to + mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by %.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [ mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < . ) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < . ); fhm ( < . ); and liver blood flow (p < . ) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < . ) in cirrhotic rots. tnf levels were higher (p < . ) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin (cx ) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx to % of sham controls. immunostaining of liver sections using anti-cx revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles hrs after animals were treated with lps, suggesting the internalization of cx without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at hr post injection . % ofpixels exceeded threshold, compared to . % in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca ÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca ÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats ( g, n>_ /group; pentobarbital mg/kg) with hemorrhage for rain at mm hg. reperfusion by ringer's lactate ( x maximal bleed out/ min) and % of citrated shed blood. tnfcz-moab (tn , ceutech, mg/kg in . % nac ) infused during flrst min of reperfusion. at baseline, end of ischemia and min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " hepatocyte incubation ( mg w.w./ml) with caci ( . + + + mbq/ml) for rain (ca influx [slope, /mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca + (ca +ex) [nmol ca +/mg protein]; ca + membrane flux [nmol ca +/mg protein'min]). during incubation, aliquots ( ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca +ex and membrane ca + flux were significantly increased at both, the end of ischemia ( . +. ; . +. ) and reperfusion ( . +. ; . +. ), as compared to sham-operated animals ( . +_. ; . +. )( <. ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca +ex ( +. ) and membrane ca + flux ( . +. )(p<. ). fast ca + influx was significantly increased by epinephrine in hepatecytes from sham-operated rats ( . +. vs. epi: . +. , p< . ). this hormone effect was not observed in isehemia ( . +. , epi: . !-_. ) or reperfusion (untreated: . +. , epi: . +. ; tnft~-moab: . _+. , epi: . +. ). conclusion. the present study clearly demonstrated hepato-cellular ca + overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca + fluxes and hormone ca + mobilization suggests disturbances of membrane ca + transport mechanisms, e.g. through ca +-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca + buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing - kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. . fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive % of tbs a third degree burn. eighteen hours after burn. gg/kg e. coli lps was intravenously administered over rain. ali animals were studied for additional hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc ) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc -indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc . inositol(l, , ) triphosphate (ip ) has been proposed as a second messenger for calcium mobilization. the addition of ip at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip receptor binding was performed with tridium label ip . the results shewed that the ip binding was significantly depressed by - % (p< . ) during late sepsis ( hrs after clp), but not in early sepsis ( hrs after clp). the ip binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p< . ), but not on smooth subfraction. since ip binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h ) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h# a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was consecutive patients with liver cirrhosis admitted to the department of surgery over a year period from january to december were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c , c ) were measured and functional assay for complement (ch ) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c , c and the functional chs complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch ) showed marked impairment in child's c patients (p< . ) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p= . ) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o per . ml medium fi +dmem ( : ) with % fetal calf serum for days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by - % (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than . times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., ) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., (kudryavtseva et al., , the ability of metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in consumption supported by nad+-iinked substrates, but showed almost no change in consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state oxidation of site i substrates and with the increase in state oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca + release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca + cycling was prevented, either by chelating ca + with egta or by inhibiting ca + re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca ÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally mmol/kg and mmollkg, respectively, and lps ( mg/kg) given h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase h after lps injection. the degree of hydrogen peroxide (h ) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [ ]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: .evaluation nfthe course of the ees due to the biochemic changes .determination of the influence of the ees on the activity of lysosomal enzymes in liver cell .deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: .activity of acid phosphatase in the venous blood serum. - .total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after hours of the experiment). conclusions . essential increase of activity of lysosomal enzymes was observed in the ees. . increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia ( min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. : g , ) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. : , ) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. : , ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats ( - g) were subjected to rain of hemorrhage (mean arterial pressure mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from . + . txmol/g to . _+ . ( min shock) indicating severe ischemia, and recovered to . -- . at min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by % compared to baseline levels ( . !-_ . i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was . + . nmol o -/min/ ecells (kc ) and . + . (kc ) in controls and did not change significantly after shock and rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after min of hemorrhagic shock and up to rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after weeks animals were sacrificed and livers were "perfusion fixed" with % glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as -fold of basal levels at the end of min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: ( ) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda ( ) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. ( ) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan , r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing - m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps( .gg. ) . rag/kg intraperitoneally. at the end ,:,f l,gand hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at - hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a c in wlstar rats was developed by tigatlng the common bite duct and injecting d. mt solution of e. goli % b, (g× ' efm/mt) into the bite duct. the mortality rate was ~ at h after aoc. at , , , h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at h after aoc. at h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p< . ), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at h after a c (p< . og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho ensis of hepatic dysfunction and m f following h . " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il- , il- , l- , i , tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il- and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: ( ) the same immurzoassay (elisa) yielded comparable results in different laboratories; ( ) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; ( ) assays for soluble receptors yielded less discrepant results; ( ) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately % for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p and tnf-r p . these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin- and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta- -microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, ) increasing neopterin levels predict rejection episodes in allograft recipients ) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv- infection ) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease ) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv- infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ -proteinase inhibitor (a pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage , m nchen, germany procalaitonin (proct) a aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as hours after the andoto:edn release and readied maximal level with a to b-fold increase h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii- , that reached peak levels at h and h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about daltons is unable to cross the kidney and the c_n barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau . ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i to fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp ) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin and interleukin (ill, il ). class acute phase genes are mainly controlled by ill and by combinations of ill plus il ; class genes mainly by ii, . the human c-reactive protein(crp), serum amyloid a(saa) and complement c genes will be discussed as examples of class genes, and rat c~ macroglobulin as a prototypical class gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class genes use the transcription factor nf-il or c/ebp as the key factor to mediate cytokine responsiveness; class genes use a different, so far unidentified factor to achieve responsiveness to il . ongoing efforts to purify this factor, called the il -response factor (il -rf), from rat liver nuclear protein extracts will be described. the il -rf apparently mediates the effects of il in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il as nf-il . the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex ~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf sioi to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to % and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than % when sepsis is present or when at least organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of to ml/ g/min are reflected in isoelectric eeg, and values about t ml/ g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about ml/ g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm , berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e synthesizing m~ and a simultaneous decrease of functionally competent cd + and cd + lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin- (il- ) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il- , most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper- (thl) secreting principally il- and ?-ifn and t-helper- (th ) acting principally in inducing antibody formation with a secretion of il- , il- , il- and tnf-[ has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u , tnf-c~, il- , il- ) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: % of all pmn's in this compartment show downregulated or non existent ~-integrins within hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr + receptors (cdll/cd complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+ ) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in- (il- ) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge . administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il- production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il- after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen - hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc gradients. supranorma[ delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. . myocardial depression with a drop in delivery (do~) . redistribution of blood flow between the different organ systems . inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ supply may therefore be inadequately matched with cellular needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april to august patients, aged from months to years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in out of these patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between - s and an activated clotting time between - s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four days. in the conventionally treated group % survived and in the additionally with ecmo treated group % survived, representing a % survival rate in patients whose risk of death is considered more than %. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med ) . in contrast, mean skeletal muscle po was found to be abnormally high in patients with sepsis ( _+ , mmhg, n= ) compared to patients with limited infection ( , + , mmhg, p< . , n= ) , to patients with cardiogenic shock ( , + , mmhg, p< . , n=ls) and to healthy subjects ( , _+ , mmhg, p< . , n= ). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po was higher ( , _+ , mmhg) on days with severe sepsis than on days with intermediate state ( , _+ mmhg) and than on days without sepsis ( , _+ , i mmhg) . thus, a decrease of abnormally high skeletal muscle po was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po with the decrease of apache ii score or elebute score as well as interleukin- serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il- and c a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on consecutive pat. after elective open-heart surgery, we had determined ele daily for at least pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n = patient-days, both ele and sirs showed significant (p< . ) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: . vs. . ; icu treatment days: , vs. . ; days on mechanical ventilation: , vs. . ; days with vasopressor requirement: . vs. . ; number of microbiological findings: . vs. . ), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of . for ele (ele >_ on >_ days, accuracy: %) compared to . for sirs (sirs present on > days, accuracy: %). accordingly, ele roc curve areas for both overall ( . ) as well as sepsis-related prognostic evaluation ( . ) were significantly (p< , ) larger compared to sirs ( . and . , resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate ( % and %, compared to % and %, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. , d- munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score , while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation . four of the nine patients died. apache iii scores at h were lower in survivors (s) than in non-survivors (ns), ( -+ vs -+ p< . ), while apache iii scores at admission were not significant different between s and ns ( -+ vs -+ ). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an fold increase of tnf-a bioactivity with[r~ hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il- [ and il- immunoactivity. tnf was measured by wehi bioactivity, il- [~ and il- immunoactivity were determined by elisa. the sensitivity was pg/ml for tnf, pg/ml for il-ll and pg/ml for il- , respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day , p< . , the pattern of cytokine production by pbmc upon lps stimulation over the first h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin- (il- ), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, polytraumatized patients were prospectively studied (mean age y, % male, iss ). serum and edta-plasma samples were taken in h intervalls until the patient left the icu. il- , tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, ) with at least of clinical signs: ( ) tachycar-dia> /min, ( ) temperature > , °c, ( ) blood pressure < mmhg, ( ) mechanical ventilation, ( ) leukocytosis > . /ml, ( ) thrombocytopenia < . /ml and ( ) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day after trauma. results: of patients developed a systemic sepsis ( . %), and died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il- ( vs. ng/l; p= . ), crp ( vs. mg/l; p= . ) and tnf ( vs. ng/l; p= . ) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il- , tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n= ) was determined by the injury severity score (iss). lidocaine is given at a dose of mg/kgbw over rain. i.v. and is metabolized in the liver by a cytochrome p- mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the ~t and the ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin- (il- ), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~ , stnfr ) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il- , tnfot, stnfr , stnfr ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il- was detected in an biological assay using the/l- dependent -cell line / . detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (> mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with ( ± mg/l) or without ( a: ) multiple organ failure (mof) nor in survivors ( ± ) or non-survivors ( :t: ). in contrast, l- was elevated in patients wilh mof (mean pg/ml, range - pg/ml). il- levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il- and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r= . ). patients could be divided into two groups by values for stnfr~ and stnfr : the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il- correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg , kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in consecutive patients with multiple injuries (iss . ) the inflammatory response to trauma was prospectively studied. the patients were divided into groups according to their iss points. additionally, the alterations after secondary operations (> hr) were determined (msteosynthesis of the femur (n= ), pelvic girdle (n=ll) and spine (n= ), facial reconstruction (n= ), smaller osteosynthesis (n= ) and others (n= )). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every h, beginning on admission of the patient to the emergency room for a period of hr, and in the operative patients on the morning of the operation, at the end of the procedure and every hr during the first two days. results: lactate, neutrophil elastase, heart rate, po /fio -ratio, and other parameters discriminated significantly between the injury severity groups during the first hr (kruskal-wallis-test, p<. ). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<. ) between the types of operations for lactate, heart rate, po /fio -ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss to ) or moderate (iss to ) accidental trauma for neutrophil elastuse, heart rate, po /fio -ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~ years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a -year period, patients were consecutively admitted into the icu; and during a -year period, patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o ), sepsis (p<o.o ), and mortality rate ( % vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o ). of the total group of , patients, there were ( %) patients years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o ). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients ( % vs. % in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of . ( % confidence limits, . - . ), age did not increased [odds ratio . (i. i.i )], while admission to the sicu reduced risk of death by a factor of . ( . - . ). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan , ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il- , l- ,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n= ) and non-survival group (n= ).tn these patients,ll- , l- ,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were points in survival septic group, l l points in non-survival septic group and points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p< . . results: il- levels in non-survival septic group were higher than in both survival septic and los group.ll- levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/ ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha- -maeroglobulin, c estemse inhibitor, antithrombin ill and alpha- -antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in patients and continued postoperatively in patients, % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication - days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by - days. dept. of surgery m'alm general hospital, s- i malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of patients admitted with burns covering more than per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: out of burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were . -- . eu/ml, significantly higher than that of patients without mof ( . - . eu/ml) on , , and days postburn (p< . -- . ). significantly more incidence of positive endotoxin test (>_ . eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p< . ). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp- , and gp ) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs expression) was significantly elevated (p< . ) and correlated well with severity of disease (f . ). no significant change in surface expression ofthrombospondin, gmp- or gp was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (! < . ) that correlated well with severity of mof (gmp- , r= . ; thrombospondin, r= . ).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a -year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period - i patients with abdominal sepsis were treated at the icu at our university hospital. patients were women and men with a mean age of ( - ) years. in cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for (- ) days out of which (- ) in the intensive care unit. out of patients ( %) died in median after ( - ) days. the primary cause of mortality was multiple organ failure ( / ; %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin ( %) and in % cultures grew multiple bacteria. patients bad organ failure and multiple organ failure. / patients ( %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about / of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january to september patients, mean age + years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for . _+ . days. diagnosis of sepsis was confirmed in cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, -patients with sepsis, the control group consisted of patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c ,c ), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c (mg/dl) c (mg/dl) k +- + _+ + +- -+ " -+ * _+ " -+ ' _+ " +_ '* -+ ** -+ "* -+ "* _+ " in a prospective study we investigated serum of severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n = ) was investigated as. well as serum of patients undergoing elective herniotomy (n= ) hours before (op i) and hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain :k :h . / suspension with a final concentration of - cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co . _+ . opi . _+ . opii . _+ . * tri . _+ . "* trii . + . ** (*:p< . ; **:p<o. ) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied patients with sls (group i: age . ~ . years); they were compared to control patients (group : age ± . years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone ( mg/ hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in pts. preoperatively pts in group revealed a significantly lower cardiac index ( . ± i/min/m ) compared to group ( . ± . i/min/m ; p < . ) higher left atrial pressure (group i: . ~ . mmhg; group : . ~ . mmhg; p < . ) and lower ejection fraction (group i: . z . %; group . ± . %; p < . ). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: , ± , /ui; group : , ~ , /ui; p < . ) and core temperature hours pop was higher (group i: . . °c; group : . ~ . °c; p < . ). serum lactate was already elevated on arrival on the icu pop (group i: , z . mmol/l, group : . ~ . mmol/l) but dropped within hours (p < . ). icu stay (group i: . ! . ; group : . ± . days; p < . ) and mortality over months (group i: . %; group : . %; p < . ) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. patients who were admitted to the intensive care units between january and july ' with polytrauma, sepsis or those requiring ventilatory support for more than hours were selected for the study. the patients were randomized into groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored hourly and the patients underwent an upper gi endoscopy hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the patients selected for the study, had undergone cardiac surgery, had polytrauma and had sepsis. the change in average pre and post drug gastric ph was + . in the omeprazole group, + . in the ranitidine group and -). in the placebo group (p < . ). endoscopic evidence of stress ulcers was seen in % of patients not on prophylaxis, . % of those on omeprazole and . % of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, % of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < . ). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin- (il- ) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn- receptors (sil- r). the aim of this study was to evaluate: the significance oftnf c~, il- and sll- r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il- and sll- r. we examined patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, ,- and sil- r were measured in septic patients and in adult healthy controls. tnf ct, il- and sil- r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il- and sil- r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll- r serum levels were significantly (p< . , student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ] ,- levels. we conclude that only the increased serum sil- r levels were predictive of bacterial sepsis within hours after the first septic signs. tnf c~ and il- levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed children with light and children with severe course(l- class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch , level of c -c , factors b told d, c a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group ( %- st and ioo%- nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c , c components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~ ) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr der j, braun j, rennekampff ha, schr der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in trauma patients with an injury severity score (iss) > (mean iss = ; mean age years) lymphocyte and neutrophil phenotypes cd (t-cells), cd (t-helper cells), cd (t-suppressor cells), ratio cd /cd , cd b (receptor for cr ) and cd (fcriii) were measured on day , , , , and post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd ) and il -receptors on t-helper cells (cd /cd were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd were significantly lower an day , , and in patients who developed mods (p< , ) compared to patients without mods and a healthy control (p<o,o ). the expression of il receptors on cd was significantly different only on day . no differences could be measured in lymphocyte phenotypes cd , , and cd /cd -ratio as well as in expression of cd b and cd on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > /zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the consecutively admitted patients to a surgical and a medical intensive care unit during -ye r period, ( %) had arf. arf mortality was %. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o, ). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p< . ), the number of osf before was higher compared to after arf in both groups of patients (p< . ). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p< . ). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p< . ). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama ; : z - ~ ) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of , recent admissions to hospitals in the united states and western europe, we identified , patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors ( % of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original , patients with a receiver d_perator characteristics (roc) area of . and in two independent databases, the first independent database contained hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc= . ); in the second, the placebo patients withtn the recently completed phase ill e~aluatlan of il- ra (rocffi . ). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low % to a very hlglt %. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il ( ) -to use a model for risk of death either from a score (apache ii, lii, ( ) saps ii) or directly (mpm ii system ( )) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system ( )) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii ) before any therapy. -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: ( ) toxicity models with injection of bacterial endotoxin or exotoxins; ( ) live or killed bacterial challenge models; ( ) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: ( ) hemodynamic parameters; ( ) modulation of host-derived inflammatory mediators; ( ) resolution of end organ injury; or ( ) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in %- % of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field ( ). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa pi, tat), cytokines (il- , il- ), and soluble intercellular adh sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il- , il- and tnfe) as well as breakdown products of complement proteins (e.g. c a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, ) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd mab wig was able to down-regulate production of il- , il- , ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin- receptor antagonist (il-lra) but not of il- ; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il- ), some only by solid-phase detection systems (anti-ll- , anti-ifn% anti-tnfc . infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il- in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il- , and il- . self limitiation of this process might be due to demonstrable instant release of il- ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il- , il- , tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. ( ) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi ,hokkaido,japan $ immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h mon departmen~ of surgery, san frandsco general hospital, urdversr , of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi is down-regu/ated, along wl~ cd and cd . thus responsiveness to lipopolysacchmdde ( ) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e . in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge . ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s! otlse. by examining m n cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il- was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than pg/ml (normal: less than pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il- level and the number of organs showing failure (r= . ). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p< . ). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i- - , kumamoto , japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock ( ). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of mg/ rain, followed by a continuous infusion ( mggh). with hemodynamic improvement (dopamine < gg/kg/min) the infusion rate nfhc was tapered over a period of days. phospbolipase a (pla ), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after days dopamine requirements decreased to less than gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day and ) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla , and finally by the relapse of septic shock in four patients on days , , and o. a second infusion of hc again resulted in shock reversal and in a decrease in pla , crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il- , il- , and il- is attenuated by- rag hc prior to endotoxin challenge in humans ( , ) . cytokines like tnf, il- , il- , and il- are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: patients with an iss nf more than points who could be treated with at iii administration within rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of % by using the following calculation: at iii to administer = ( % -at iii measured) x body weight. at iii was given every six hours for a period nf hours and on the th day. patients were followed up throughout their stay in the icu but at least days. in this abstract the data of the first patients are included. results: the patients' median injury severity score was points with a median age of years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received , units of the inhibitor on average during the first days. with our regimen, the at iii levels could be kept between % and % of normal, whereas control patients an at hi concentration of % to % was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to % of normal. white blood count, pmn--elastase, interleukin and and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii group ( . vs. . days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of %, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. patients with severe multiple trauma ( treatment [at-ill] -- controls [c]) were investigated. study cdteda were age > and < years, injury severity (iss) > points and glasgow-coma-scale > points; randomization and treatment has to be started within hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp- , a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp- in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. h later bk was injected ia and the pain score ranked from (no responses) to (vocalization). cp- at . umoles/kg completely inhibited the pain responses for a period of . - h. cp- at . umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a - h period by an intraplantar injection of . % carrageenan. the abdominal constriction response o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp- . when ul of . % formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp- significantly inhibited both the first ( - min) and second ( - min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at °c for sec the increase in paw volume was significantly reduced by pretreatment with cp- , . umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (- °c for sec) to the dural surface following a craniectomy. cp- at . umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp- , may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi , a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats ( - g.) received a % liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either . mg/kg/hr rbpi (phx-bpi, n= ; sh-bpi, n= ) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n= ; sh-con, n- ). various parameters were measured h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi . blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il- levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at - rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of - mmhg for rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. rbplg. was administered at a total dose of mg/kg i.v. ( . mg/kg at the -eginning followed by two doses of . mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi . results: imrffe?diately after resuscitation ( min) the detected plasma endotoxin levels in the control group (mean = , range = - pg/ml) were almost neutralized by rbpi treatment (mean = , range = - pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points and min of experiment (means: and in bpi vs , pg/ml in the control group). the -hour survival rate was improved from / ( . %) in the control to / ( %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. rabbits weighed + grams divided into two groups. group i included tabbits were treated with allopurinol mg/kg for seven days before induction of haemorrhage. group ii as a control included rabbits. all rabbits were subjected to % arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was out of while in group ii it was two out of . our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, pigs were first anesthetized, then injured with joules of energy to the posterior thigh, then hemorrhaged - % of their blood volume. after i hr of shock, all the shed blood plus x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine ( . mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after hrs, a septic challenge was administered ( bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for min. baseline values and units were cardiac index _+ ml/min/kg (ci), arterial po + mmhg(pao ), base excess . -+ meq (be), physiologic dead space fraction +_ % (pds), and tnf . + . units/ml. baseline gastric ph was . -+ . and . _+ . in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao * be* gastric* pds* peak* rain rain rain ph min tnf ranitidine _+ _+ - . ± . bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin- (ii- ). this study was designed to clarify the role of lps and - in the metabolic response to bum injury. twenty-five burn patients ( -+ %; + % ft bsa burn; _+ years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i - were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b ( , u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic ( . +_ . pg/ml; normal < pg/ml). i - did demonstrate a significant correlation with cole temperature (tr~ = . + . ogi - , p= . ) and with nitrogen excretion (nou t = - . - . ogi - + . tr, p= . ). administration of polymixin b had no effect on metabolic rate, temperature or i - levels but did reduce nitrogen excretion resulting in more positive nitrogen balance ( .t grn/day vs. - . gm/day, p= . ). although bum injury does not produce an obligatory endotoxemia, i - does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within rain and shock state was hold for h at a map of mm hg (cardiac output of %). following adequate resuscitation with % of shed blood and twice of this volume as ringer's solntion, animals recovered to map > mm hg and co > %. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at , , or hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [ mg/kg, celltech, uk) , pentoxffylline (ptx, mg/kg, hoechst, d), and a paf antagonist (web , boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n= - group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence - of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from , to about pglml, but did not abolish it. the inhibition of the lps( . ug/ml)-induced tnf production occured already in presence of , pg peptide/ml and peaked at the concentration of , ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp- and tp- ), thymocartin did not enhanced the pwm-induced pge production in pbmc cultures up to a concentration of ng/ml, inclusive. the selection of thymocartin (tp- ) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about % registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: patients (pts) formed control group a. group b pts received x mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d ) intravenously to block prostaglandin e induced suppression of cmi response as well as mg thymopentin (tp- ) subcutaneously preop., on d and d to augment cmi reactivity. in vitro investigations preoperatively, on dl and d included measurements of the percentage of cd + t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)- as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il- as one cytokine primarily produced by th -cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d were used as in vivo tests for thl-and th -induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd + th cells, il- /il- ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p< . vs.bl). in group b pts no change in cd + th cells, il- /il- ratio, or dth skin response was observed (p< . vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: .these results clearly indicate a significant suppression of th induced cmi response, while th induced response remains normal following ohs. .these alterations may gain clinical significance whenever a postoperative infection requires a th response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing - g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il- . they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from to . results.it was found that in % of infants with - classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then times, ratio th/ts was reduced to . or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c a subcomponent were increased but the level of c , c ,, c and c components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i- mcg/kg per day and mg/kg per day accordingly during a week in children with - classes el heaviness. aiter - days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of ( - )-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c h/hen or c h/hej mice, to wkolds of both sexes were used through experiments. highly purified ( - )-{ -d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b- . induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c h/hen and c h/hej mice, whereas lps only induced such activities in c h/hen mice. most effective dose of bgc was ug/ml. ) bgc-dependent proliferative response of c h/hen splenic cells was not detected under the culture condition used. ) induction of tnf~ prodction was apparently observed in bgc-stimulated c h/hej pd cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~ activation, intracellular signaling pathways of p~ by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il- in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il- . under anesthesia, -weekold male c h-hej mice were subjected to a % scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of organisms of p. aeruginosa hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells ( x ) intraperitonelly after septic challenge. the mice were scored for survival daily. on day after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il- and il- in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il (n= ) were raised to . _+ . and _+ pg/ml, while the levels in lak treated mice were reduced to . _+ . and _+ pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included operated patients, of which received mg indomethacin before and , and days after surgery (group a) and patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd ), suppressor (cd ), nk-cells and interleukin- and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day and , and days after operation. in group a we detected a significant (p< . ) increase in cd /cd ratio on day while no differences were ~oted in nk-cells or cytokine production in both groups (table ) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. female cd- mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p< . vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p< . ). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p< . ). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin , ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h -antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. -aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi )(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p< . ) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi and plasma cholesterol (p< . ) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, -aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, x u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. -hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the -aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in neurosurgical centers and included patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and groups of tirilazad in escalating dosage ( . - mg/kg/day). the clinical outcome was compared for these groups and cerebral a~giography was performed in % of patients on day - follovang sah, to study the effect f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z patients sufferin s from severe ap (c-reactlve protein > me/l) we determined on day and day after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i patients were randomly treated with ug/day of se for days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only / of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains % hes and % chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats ( - g, n> ; pentobarbital anesthesia mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate ( ) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and ( ) whether dfo-hes could prevent this tissue damage. methods: in rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to groups: one group was treated with ml dfo-hes ( mg/kg iv), the other rats received solely ml of the carrier starch solution. , , , and min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> . eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p< . ) in the lies group already min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p< . ). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p< . ) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca + levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose ( mg%) and bovine albumine ( gm%) for min at °c.it composed of groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo& mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were % in group ab and % in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a , leukotriene b and leukotriene c are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after , , , and minutes plasma levels of thromboxane b (txb ), leukotriene b (ltb ) and leukotriene c (ltc ) were determined via elisa technique. another volunteer had taken mg aspirin one day before taking the blood sample (no ). results: txb plasma levels increased from pg/ml at min to pg/ml, pg/ml, pg/ml and pg/ml at , , and min (p< , ) . ltb and ltc plasma levels showed an increase during the first few minutes (ltb : min: llpg/ml, min: pg/ml; ltc : min: pg/ml, min: pg/ml (p< , )) followed by a decrease to normal values at min. in the sample no the cyclooxigenase-pathway was completely inhibited, the txb plasma-levels did not alter at all, whereas ltb and ltc -plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il- secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il- productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early th century, however the protein was not isolaled until . the human gene was identified and cloned in , which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a - day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet ; : - ) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p< . ) in patients receiving allogeneic blood (n= ) or operated without blood transfusions (n= ). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n= ). in contrast, specific igg-levels increased sigmficantly (p< . ) in patients receiving allogeneie blood (from . + . to . _+ . ie/ml) whereas in patients receiving autologous blood a smaller increase (from . + . to . + . ; p= . ) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l . + . to . + . u/ml) compared to those patients with autologous blood ( . + . to . + . ). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin- (il-i), interleukin- (il- ) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during - hours after surgery in patients undergoing arthroplasty ( hips and knee). kf, il- , it- , thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il- concentrations ~ere analysed at the beginnlqg and end of the - hour blood retrieval period. in a separate study ( hip arthroplasties) f~f, il-i and il- ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il- coocemtratiems rose from a median value of to pg/ml, p<o. , during the blood ret.rieval period. in c was detectable in patients, range: - pg/ml. il-i was not detectable in the patients. in retrieved blood tag was > mg/ml in all samples (ref:< . mg/ml) and at was . - . units/ml (ref:o. - . ) . the il- concentrations in retrieved blood was > pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n= ), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: - pg/ml) and il-i (range: - pg/ml) ~re present in all samples but ii- (range:o- pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients ( . %) were perioperatively transfused. thirty-six ( . %) patients were given autologous blood , while ( . %) received homologous blood. no patients received both autologous and homologous blood. twenty eight ( . %) patients developed postoperative infections. non transfused patients had a . % infection rate , those receiving autologous blood had a . % infection rate, whi]e in the homologous blood group the infection rate was . % (p < . ). univariate analysis showed that infections were significantly related to operative blood loss (p< . ), length of operation (p< . ) blood transfusion (p< . ) and attending surgeon (p< . ) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, month old rats were transfused intravenously with syngeneic heparinized venous blood ( x ml, every other day), and , and days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a : : . : . mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of . microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over - hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of - -weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il- . we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium- m ( mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a % topload of leh ( mg of phospholipid, . g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, % at minutes and % by hours. the spleen accumulated activity at a slower rate, % at minutes and % at hours. at hours, autopsy biodistribution studies revealed that approximately . % of the dose is in the blood pool, . % in liver, . % in spleen, . % in lungs, . % in muscle and . % in urine, with trace levels in kidney, brain and heart (< °/o). in a hypovolemic model, rats were % or % exchange transfused with mtc-leh. in the % exchange model, mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at hours. with the % exchange, % of the leh was in circulation at hours. the interaction of leh with platelets labeled with indium- was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha ( ) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens ( ) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine ( ) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength ( ) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t - years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. o]~ga- pufa there continues to much interest in the application of the mega- pufa in clinical nutrition. the basic principle has been that the mega- pufa will displace arachidunic acid and result in a decrease in eic san id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega- incorporation with continuou~ enteral feeding both in control and endotoxic animals within days. this includes the liver, spleen, circulating and alveolar marc phages and the lung. this incorporation resuls in significant changes in the eicosan id production including pgf and ket -pgflalpha. there is improvement in the cardio-vascular reep nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of mega- pufa. clinical studies using short term entsral nutrition with mega- either alone or with other enteral supplements in a number of clinical settings have shown significant mesa- incorporation and decreased eicosan id production. these positive results must be discussed with the additional evidence that long term omega- supplementation decrease eic san id production but als induce a state of immune suppression that is capable of increasing transplant sunvival. these ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf- mice aged - wks received % tbsa fullthickness dorsal burns & were resuscitated with cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast hrs, chow hrs). at hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p< . , ** p< . compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of adult icu patients who required enteral feeding for > days. patients entered the study within hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least ml/hr by hr after event. results. both groups tolerated administration of formula well. for patients fed > days, the median los was % shorter (p=o.ol) for the--supplemented group ( days) compared to the conventional group ( days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o ). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia ( vs. days) and skin/soft tissue infection ( vs. days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into groups ( n= each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within hours following surgery ( ml/hour). it was progressively increased reaching a full regimen on day . on hospital admission and on post-operative day and , the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day , , . the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day . comparing post-operative day versus day a significant increase of prealbumin (p< . ) and rbp (p< . ) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p< . ) and a significant recovery in delayed hypersensitivity response (p< . ) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent ( % in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = % vs vivonex ten = %), mean mof score (immun-aid = l.b vs vivonex ten = . ), or mortality (immun-aid % vs vivonex ten = %) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> ), patients receiving immun-aid appeared to have fewer septic complications ( % vs %) and their mean mof was significantly lower ( . _+ . vs . + . , p = . , student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from patients in septic shock and heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for minutes. pmn (p{o, ) and monocytes (p<o, ) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the %-bcaa vs %-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o for all). clearances of bcaa increased with increasing bcaa dose (p<o.o ); arg and gln clearances were directly related to the bcaa clearances (rz=o. , p<o.o i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation ( gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with x t.spiralis larvae. thirty to fifty days later rats received gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o mucosal mast cells and their subsequent degranulation. mast cell-derived -ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group ( % vs %). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m- polyunsaturated fatty acids (pufa) from safflower oil (so) or (o- pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at % in the diets was adjusted to provide % each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for days, lipopolysaccharide (lps, mg/ g) was injected through the tail vein. the percent of animals surviving in the mo group was % ( / ), and did not differ significantly compared to % ( / ) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so ( . + . ng/ml) and those fed mo ( . + . ) did not differ significantly. the levels of prostaglandin(pg)e , -keto-pgflc~ and tumor necrosis factor-ct (tnf-c were determined min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and h after the lps administration (lps+). the data (mean_+sd; n= ) are as follows. group these data indicate that a marked reduction in the plasma levels of poe and -keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a . % reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf- outhred female mice ( - g) were fed purina # chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from to days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a- desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for weeks. the incorporation of dihomo-y-linolealc acid (dgla: : o)- ) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between - % compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps ( p.g/kg body wt), the plasma levels of both pge , and txb , were significantly decreased (p< . ) by nearly % for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a- desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a (pla ) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was % ( / ) which was markedly higher (p< . ) compared to only % ( / ) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly % reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c bl/ mice that have been stressed by immobilization. the proteins have been labelled with ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of ss-methionine reveal about protein spots, from which a small fraction -about are altered in both qualitative and quantitative manner. the major changes are in the range of to . d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery days and days preoperatively and on the ist and th postoperative days (study-days i, , , and ) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived days preoperatively an enteral nutrition enriched with omega- fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a ( . pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb (mean values . ng/l x i~ cells) compared to the placebo group ( . ng) at study-day [p, . ). in contrast, the capacity of neutrophils to produce ltb was not significantly different in both patients populations at study-days and . the omega-oxidation of ltb to its biologically less active metabolites oh-ltb and cooh-ltb was not significantly different in both groups. however, neutrophils from group f patients generate more ltc at study-day (p( / ). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega- fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega- fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega- fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution ( kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn ( , g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide ( , g n derived from ala-gin = % gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o, ). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p< , ). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p< , ). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o ~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai y erodes both fimetianal and stm~ tissue. wiu _b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~ po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh iu daily three days before the experiment (gh- ), another group treated with gh ie at the start of the surgical procedure (gh- ) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u- c-glutamine. substrate fluxes were measured one (lh) and five ( b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh- : . + . mg/kg, gh-i: . +_. . mg/kg, con: . +- . mg/kg, p= . ). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh- : . + . gmol/min at lh and - . + . in.tool/rain at h, gh-i: - . + . i.tmol/min at lh and - . + . pmol/min at h, con: - . +_ . g mol/min at lh and - . + . /amol/min at h, p= . , p= . ), whereas the absolute uptake of glutamine in hindleg was unchanged (p= . ). glucose uptake in the hindleg was decreased (p= . ). net glutamine uptake in liver was attenuated (p= . at h), whereas net glutamate release from liver was increased (p= . ). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of , ; , or , i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from . calories per kg body weight to . calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus . to plus . g n / days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either u of rhgh bid (sc) or no alternative medication. the study period lasted days. from daily hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days and to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf and igf , igfbp were measured daily during the study period, on day a hour profile of gh was.performed. samples were collected each minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf differed significantly between the groups for the whole study period, but not igf and igfbp- . the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n= , age range - yr, bmi . + . kg/m ) or for days additional gh ( x i.u.s.c.) (gh group, n= , - yr, . + . kg/m ). metabolic studies (calorimetry and lsc-leucine infusion) were performed (test a) and days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the -week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was + ; + (co) and + ; ± (gh) pmol/kg ffm/h (n.s.). protein breakdown was ± ; + (co) and + ; + (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was ± ; + (co) and ± ; + (gh) pmol/kg ffm/h (p< . for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days - : gh + vs co ± g; p< . ). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf- levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf- . this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: patients (both sexes, age: - years, bmi - kg/m ) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received ug rhlgf- /kg body weight/twice daily or placebo during treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition ( g chng, o. g aa/kg) troughout the whole study period. cumulated nitrogen balance, -methyl-histidin exretion in urine as well as serum-igf- levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to groups (n= /gronp): gh-high ( + mg/kg/day), gh-iow ( a mg/kg/day) or control (saline). following subcutaneous treatment (every hours for days) with gh or saline, mice were challenged i.p. with e.coli (lxl /body). survival rate was recorded every hours. in the next experiment, gh-high and control animals were sacrificed hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for hours with lipopolymccharide ( p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf- ) and the insulin like growth factor binding protein (igfbp- ) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf- and igfbp- . materials and methods: patients sheduled for esophagus resection were investigated once they had given written informed consent. patients showed acute alcohol abuse and were treated with clonidine postoperatively• patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf- and igfbp- were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf- and igfbp- levels showed no differences between the groups. discussion: igf- and igfbp- plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n= , mg/kg/day), low dose igf-i (n= , . mg/kg/day) or saline ( = ) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for hours with lipopolysaccharide ( ).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l bioassay. results igf-i improved the survival rate at a dose of mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than /~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated patients ( scn, cyclic neutropenia) with r-methug-csf (filgrastim). thirty of patients with scn and all cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than /~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between . and ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations hours after administration. these levels were -fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately - -fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day of gestation (parturition in rodents occurs on approximately day ) survived a lethal challenge of group b- hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res : , ) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood : , cairo, pediatr res : , ) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. infants < hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm ( - wk), preterm ( - wk), and term (> wk) and randomized to receive either a placebo or , , and gg/kg/qd or and p.g/kg/bid of rhg-csf infused by pump over hour for consecutive days. cbcs were obtained at , , , , and hrs. tibial bone marrow aspirations were performed hrs after study entry and differential counts and cfu-gm pools were determined. c bi expression was determined at and hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at , and hrs following administration of both and ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred hrs after and ~tg/kg/d ( - %) (p< . ) and ( % -+ %) (p< . ), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool ( _+ % vs. + %) (p< . ) (placebo vs. ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo ( . _+ . vs. -+ ) (colonies/l(p cells/plate). c bi expression was significantly increased hrs after bg/kg/d of rhg-csf ( + % vs. +- %) (p< . ). peak serum g-csf levels occurred at hrs and were dosedependent. the half-life of rhg-cse was . + . hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. of patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research. : , ) . methods: awake ylo beagles had serial cardiopulmonary and laboratory studies before and for up to days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until days after clot placement, animals received high (n= ) or low dose (n= ) g-csf or protein control (n= ) subcutaneously. results: survival in high dose g-csf animals ( / ) was significantly improved compared to low dose ( ) and controls ( ) (p< . wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day after clot, p< . ) and mean arterial pressure (day , p< , ) compared to low dose and controls. high dose rcg-csf increased (p< . ) peripheral neutrophil numbers both before and after clot implantation ( hours to days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p< . ) rise (day ) and fall (day ) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin ( to h, p< . ) and tumor necrosis factor (tnf, h, p< . ) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and or hours later. lps-inducible tnf, il- , stnf-r p and il-lra were assessed in the supernatant of whole blood incubations stimulated with ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about % hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p which was indetectable in blood incubations from untreated donors increased dramatically hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as /.tg/kg iv every h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either or days before or hours after cp. results. mortality was reduced from % to about % with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin and lactate were attenuated the first hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin and lactate are suppressed the first hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels h after lps administration. gm-csf pretreatment ( ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin- (il- ) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il- -tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in hs. continuous infusion for days. safetyandefyieacy will be assessed till to day . the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > (score corresponds to expected mortality rate of %).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than , were included in the study. filgrastim was given by daily continuous intravenous infusion for days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > apache i score points on day after onset of treatment. results: none of the patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to . + . /~tl (mean + sem) and reached a level of . +_ . /tal at day after onset offilgrastim therapy. the apache ii score initally was + . (mean + sem) and as an indicator of filgrastim response a decrease of points ~dthin days oceured in out ot patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= ) or colorectal (n= ) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il- , which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of pg/ml (preoperative < pg/ml) on day and day to after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < , ) correlated with infectious complications in patients whh gastric cancer (n= / ). secondly patients could be arranged into two groups according to an anergic (n= ) or normergi¢ (n = ) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= / ) or colorectal (n= / ) cancers. interestingly we found a significant correlation (p < , ) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= ) within the pre-and postoperative period resulted in reduced mean values (about %) for ifn-ff release (preoperative means llo pg/nfl), if compared to patients with normergic dth (n= , preoperative means pg/ml). similar, but less significant results were obtained for il- secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il- ) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s orl o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe -gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)( m$/k bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after weeks was significantly decreased by administratlon of rh~-csf(p< , ).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached . %. nextly,treatn%ent wlth rhg-csf(s ~ $/k body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p< . ). iv~oreover,functions of neutrophlis which were phagocytic activity and h p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~( ~ /ks b ody wt) (p< .( ). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < /mm ) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < /ram ) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of to mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within to days. in most of them, spectacular correction was observed within hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed ( cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, , rue de s~vres, hopital necker, paris, france. changes in serum g-csf and il- after surgical intervention hitoshi toda , atsuo murata , hidewaki nakagawa , takesada mori , nariaki matsuura osaka university medical school, osaka, wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin (il- ) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il- and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l- was less than pg/ml and g-csf less than pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il- levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il- (x) was y= . x+ . (r= . , n= , p< . ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il- was pg/ml and g-csf pg/ml respectively. the estimated value of g-csf was pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than were treated with filgrastim ( . - l.tg/kg/day) for prophylaxis of sepsis on days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced ( - reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of % +- % ( % - %) compared to pretreatment values of %. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p< . ). before initiation of csf treatment urinary neopterin was similar in both groups of patients ( +/- and +/- lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day the mean value was about % above the basal level (p< . ). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than fold induction was noted and the difference between g-csf and gm-csf was highly significant (p< . ). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp- human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r a and pot ca . john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, . at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i. ], dmpg[ . ], cholesterol [ . ] , and monophosphoryl lipid a [ - gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected - days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing , or gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds for control (liposomes with no lipid a) mice was x bacteria; ld for mice vaccinated with p.g was x ( -fold increase in resistance) and with ~tg was x bacteria ( -laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld for control (liposomes with no lipid a) mice was ng lps; the ld for gg lipid a was rig lps ( -fold increase in resistance) and for xg was ng lps ( -fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld of franciseua in normal mice or mice inoculated with liposomes without lipid a was - bacteria. in contrast, mice vaccinated with liposomal lipid a ( ggl survived challenges as high as , bacteria, ( logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il- , and il- responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd . to evaluate if blocking lps-cd interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a hour period. to evaluate the effect of treatment with anti-cd mab, animals were given either nothing, an isotype control or anti-cd mab ( mg/kg) rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il- , i,- , il- and il- , and liver enzymes during a hour period revealed that treatment with anti-cd mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi ). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a kd n-terminal fragment (rbpi; ) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi has equivalent antibacterial activity to bpi against rough gnb but is up to x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi block the cd -dependent lpsinduced synthesis of the cytokines tnf, il- , el- and il- in vitro. rbpi has also been shown to inhibit the lps-induced synthesis of reactive metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi was not toxic to rats after daily consecutive i.v. doses of mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd ) of myeloid cells or as a soluble serum protein (scd ) lacking the gpi-anchor. binding of lps to mcd triggers cell activation while binding of lps-scd complexes to cells such as endothelial or epithelial cells that normally do not express mcd activates these cells. these pathways are shown in schematic form below. ~di mcd plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: mg/kg --- . mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il- , or il- , but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of - m and has a t / of • h in normal humans. methods: normal volunteers were randomized to receive either tnfr:fc vehicle (n= ), or low dose (ld) tnfr:fc ( mg/m iv, n= ), or high dose (hd) tnfr:fc ( mg/m iv, n= ) infused over rain prior to endotoxin (e) (escherichia coil : ; ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point ( - h) in subjects given tnfr:fc compared to vehicle ( h) (p=. ). at h, h (post fluid load ), and h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=. ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<. ) and later leukocytosis (p< . ). peak tnf bioactivity in subjects given vehicle was + pg/ml and < pg/ml in the ld or hd tnfr:fc groups (p<. ). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=. ) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<. ). decreased levels of il- (p=. t), granulocyte colony stimulating factor (p=. ), and il- receptor antagonist (p=. ) were found after ld or hd vs vehicle. il- levels were lower after ld vs vehicle or hd (p=. ). il- levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the ' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥ ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c inhibitor complexes ( ). in this in vitro plasma model we also found that methylpredoisolone alone in doses of mg/ml induced contact activation with the formation of kallikrein-c inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade ( ) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et ( %) and interleukin- ( % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c - nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c - nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c - nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c - nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type- in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type- (cr- ) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c /c convertase formation. we have recently purified the human recombinant cr- in a truncated form (brl , tp-io, scr- ) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: ) endotoxin-induced ards in paf primed animals; ) .- induced ards; ) thermal injury ( % body surface)-induced ards; ) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr- , - mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il- ), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr- in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that ( ) ptx suppresses lps-induced tnf release from activated macrophages; ( ) ptx prevents tnf-induced pmn activation; ( ) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; ( ) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse , d- m nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il- most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il- and il- . moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons ( . +. kg), were randomized to a control group receiving ° cfu/kg of intravenously administered e. co/i (n= ) and a treatment group (n = ), pretreated with . mg/kg of paf receptor antagonist ro - two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving ng/kg of intravenously administered endotoxin (lps) (n= ) and a treatment group (n= ) pretreated with mg of ro - eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for hours. results: baboons pretreated with the paf antagonist had improved oxygenation ( + mm hg v -+ in controls, p < . ) and creatinine clearance hours post e.coli ( . + . ml/min v . _+ . in controls, p < . ). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at hour post lps. this was in concert with a diminished peak cortisol ( +_ mmot/l v +- mmol/l in controls, p < . ), epinephrine secretion ( + nmol/l v + nmol/l in controls, p < . ). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il- and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il- ra, a naturally occurring il- antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha- a and e . future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa , and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn , have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il- and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd)i chain covalently linked to one of three different alpha chains (cd a, cd lb, cd lc) and exist on the cell surface as three distinct heterodimers. cd l a/cdi is expressed on all leukocytes, whereas cd l b/ cd and cd lc/cd are restricted to cells of myeloid origin. cd i/cd interacts with intracellular adhesion molecule- (icam- ), its ligand on endothelial cells. icam- is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = ) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria endotoxin) during the first days after inciosion in the study. the other randomized group (n = ) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of < wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one ( %) of patients who received immunoglobulln. by comparison, nine ( %) of control group patients died during this period (p <. ). within the first hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; ) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_ (meaning a mortality risk > %) were accepted into this protocol. patients were randomized to receive . g/kg of ivig or placebo on day (when they reached sepsis score> ), repeated on day + and + . at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: % vs, % respectively; p< , ). in conclusion, the results of our study in patients with severe surgical sepsis were the following: ) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', ) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg ( ) - ) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in patients with sepsis and septic shock (infection ~ ) - ), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score - ) and severity of sepsis (elebute sepsis score - ) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo ( .i % albumin) or polyglobin n" - ml ( . g)/kg on day and ml ( . g)/kg on day i. with an anticipatedpopulation size of patients the study should comply with the statlstical requirements (estimated mortality: %, with a % reduction in -day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november , more than patients had been included; patient enrollment will be finished in . previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il- , increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled patients at academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) . or . mg/kg/hr by continuous intravenous infusion for hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over days utilizing a linear dose-response model, assuming a log-normal distribution. results: patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< . endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d ) and tolerance of bn ( iv infusion of mg x /day over days) in severe sepsis has enrolled pts. the day mortality rate was % for the placebo group and % for the bn group (p = . ). the efficacy of bn was greater in pts with gram-negative sepsis: the -day mortality rate was % for the placebo group and % for the bn group (p = . ). bn also reduced mortality among pts with gram-negative septic shock (mortality was % for placebo vs % for bn ; p = . ). using statistical adjusments for pronostic factors, the relative risk of death of the bn group was . ( . - . , % confidence interval; p = . ). this risk corresponds to an adjusted reduction in mortality of % for pts receiving bn . no differences in mortality rates were found between the placebo and the bn groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn groups. bn is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~ lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for -day mortality (jamb. ; : , - ) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin- receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x = . , p [] . , log.normal) for all patients in the trial• survival benefit began st approximately % baseline risk of -day mortality. for the $ patients with a predicted risk > %, there was a % reduction (p= , $ log normal). when we examined the variation in patients above and below the % risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < % risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > % risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp- , in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp- on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population ( . %) of pop. low-risk pat. (ap< ; mortality: %) and the small groups of pop. pat. at risk lap= - ) and high risk lap_ ) with a significantly higher mortality ( % and %, mainly due to sepsis). subsequently, among consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n = ): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day : ml/kg, day : ml/kg). second study period (n= ): iggma (pentaglobin r, biotest, dreieich, frg, ml/kg on days to ). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n= , high-risk: n- ), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n= : p<o. ; iggma, n= : p: . ). in this group, ig led to higher (p< . ) response rates, defined as an ap score decrease -> within four days (igg: %, iggma: %; con.: %), and reduction in mortality (igg: %, iggma: %; con.: %), statistically significant (p< . ) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, aminoacids et (et ) in rat abdominal sepsis. methods. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et were determined with amersham tm endothelin rias , and h after sepsis induction. experimental groups: . cp control, . volume replacement (vr); , % saline ml/kg/h continous iv infusion started after h, . antibiotic; imipenem mg/kg iv after h, . e ®; mg/kg iv after h, . vr + imipenem + es® after h. results. high concentrations of both big et and et could be demonstrated after h and lasting for h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e ® significantly reduced et both , and h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et was the same as for e ® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e ® reduces circulating lps and tnf which is the probable mechanism of the suppressed et synthesis. the unaltered big et fraction after e ® treatment indicates conversion of big et to et as the site of action responsible for reduced et . conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et . e ® anti-lps antibody significantly reduces plasma et while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e- in congestive heart failure has been demonstrated aumann, ). therefore, we investigated the effect of bu-e- in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [ ] [ ] [ ] [ ] [ ] [ ] were anesthesized with fentanyl/dormicum, ventilated (n :o = : ) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study ( animals) to evaluate the effective concentration of bue- in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of ~g lps/kgkg/h ( :b , fa. difco). the hypodynamic state was defined as a decrease of cardiac output by % of steady state levels. a wedge pressure of - mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) groups with animals per group were studied. the groups were treated as follows: group i, lps and , % nac ; group ii, lps and bu-e- ( #g/kgkg/h); group iii, famotidine (h -blocker) pretreatment ( mg/kgkg), lps and bu-e- . results: the pilot study in healthy pigs revealed, that bu-e- had positive inotropic effects. these effects were inhibited by the h antagonist famotidin. bu-e- however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do ) were not significantly influenced by bu-e- application (group i versus group ii). bu-e- did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e- led to a further significant decrease of lvsw (p < , ). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h -agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci . % (control without endotoxin, n= ). group e : endotoxin tjg kg " bolus i.v. + naci . % (control with endotoxin, n= ). group c : endotoxin pg kg - bolus i.v. + cll u kg - bolus + u kg " h "~ i,v. (treatment group, n= ). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m ) and after (m ) and rain. (m ). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p< . ) between group e (lung , liver ) and c (lung , liver ) (group k : lung , liver ). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july to june , patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of . years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the st, rd, th and th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk receptor is constitutive. the bk receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk (bradycor [cp- ]),bk~ (cp- ) and bkz/bk (cp- ) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk agonist) in the lps-treated ( ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk agonists increased with time and at time min appeared maximally induced. constant iv infusions of cp- blocked bk but not bk~ and cp- bk~ but not bk responses. cp- ,cp- +cp- and antril+cp- blocked both bk and bk~ responses. antril alone had no effect on bk or bk~ responses. within - min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk ,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk receptors. in vitro studies demonstrated that beth bradycor and cp- (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp- is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september and july olt's in patients were performed at our department. the actuarial -year patient survival is %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from to hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss - ), mean temp. was . f, leukocytosis was k, pan was , fin was . , and peep was . at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t am and a thickness of nm. the ami-fungal efficacy is - times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than nm with a mutina ld of > mg/kg. it has been used in dosages up to mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a % response rate for invasive candida infections to % response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was % among placebo treated patients compared to % for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from % to % ill placebo and ambisome treated patients respectively (p< . ). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of mg/kg at day , , , , and after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of elisa units during months. cmv infection occurred in / ~eronegetlve and in / seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower ( / ] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f t ) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection ( v / ), but no difference in incidence of disease ( vs / ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from patients with different types of leucemia after autologous and allogenzc bmt had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in % of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity - days after intranasal inoculation of influenza virus a (h n - ) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest . consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years and . in the same period of time pri~ total hlp replacen~nts were performed under i entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than %. in the selected i cases the septic rate was % indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c ) and fourth (c ) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate ( "= - . , p < . ). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c , c , crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il- ra, il- , il- r, il- r, tnf-ct, tnf-~r (p ) and icam- . in contrast, pmn-elastase, il- and il- closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd +/cd + ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il- and il- , however, closely correlate to the clinical course and may prove valuable for follow-up. the cd +/cd + ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and ( ) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of mu/ml xanthine oxidase (xod), mm hypoxanthine (hx), mm fec and mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il- . cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& ) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, ) the trant~ of vim.©n~e traits between ~renr~a.,dsms and ) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il- receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h pital erasme, route de lennik , bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin ) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il- by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il and monocyte chemotactic activity when cultured. these activies were significantly increased ( -fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il- in patients with bacteriologic proven abdominal infection (intraabdominal abscess , diffuse peritonitis , pancreatic necrosis , pancreatic abscess ) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a to hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il- determination were drawn preoperatively, intraoperatively, and until h postoperatively in regular intervals (min /pat), results: preoperative apache ii was in median (rain , max ). patients fulfilled the criteria of sirs. of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p= , i, spearman coefficient). preoperative cardiovascular (systol. rr< mmhg) and respiratory (pao < mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p= , i, wilcoxon; pulmonal: p= , ) and il- (cardial: p= , ; pulmonal: p= . ) overall, lps, tnfa and il- values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i , mean pg/ml) than in those who did not fulfill these criteria (group b; n= , mean pg/ml; p= , i, wilcoxon). il- was significantly higher in group a (mean pg/ml) than in group b (mean pg/ml; p= , o i wilcoxon). conclusion: perioperative tnfa and il- were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel - , wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= ) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after hours. stastistieal calculations were performed on a personal computer with the spss programm vers. . (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla -activities [endotoxin range from . eu/i, sd= . to . eu/ , sd- . (p < ), ifn-¥ levels, range from . pg/ml, sd- . , to pg/ml, sd= (p < . ), circulating pla -activities range from . , sd= . to . u/ , sd= . (p < . ) and biopterin range from . nmol/l sd= . to . nmol/l, sd= . (p < . )]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = . , p < . ) and bioptefin synthesis (rv= . , p < . ). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = p < . .. p • , . . ) and to the pla -actwtues (rp = . , p < . ). the biopterin synthes~s correlates slightly with the pla -actn,ities (rp= : . ; p < . ). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of %. anaerobes were found in . %, anaerobes were isolated in . %. there were aerobic and anaerobic associations in . % and microflora was not found in . % of the cases. express method of anaerobes discovering let to receive information on - days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of patients with abdominal sepsis treated at the surgical intensive care unit during a -year period, ( %) had an age of years or more. were women and were men, a sex distribution not differing with patients younger than years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was (- ) days including in median days in the icu. figures in patients less than years of age were comparable ( (- ) days out of which in median days in the icu). apache ii and sss-scores did not significantly differ ( . vs and . vs . , respectively), between the groups. neither did the incidence of organ failure differ ( / vs / ). however, the incidence of multiple organ failure was significantly lower in elderly patients ( / vs / (p < . )). the mortality rate, however, did not differ between the groups ( / vs / ). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n-- in each group). at , , , , and hours (n= /timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of : i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was % both at and hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from hours and on and in the proximal and distal small intestine from hours and on. different types of alterations in capillary permeability seem to appear: ) a temporary short increase e.g. in the proximal small intestine and spleen; ) a temporary longer increase e.g. in the colon and kidneys; ) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p integrins (cd l a, b,c/cd ), and the fc'?r (cd , cd , and cd ), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score > ) or thermal injury (> ~ total body surface area, % full thickness), and healthy controls. the expression of cd b and c and to a lesser degree cdi a was significantly reduced on pmns. the expression of cd and cd but not cd was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, ~ ethesda ave, cincinnalt, oh, usa, - s b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet , dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e ) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il- . the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =. , mean b state = . , mean ~ state = . ). the accuraay of this estimate was prospectively analyzed in this group of m~itlple patients of whom % had sepsis and % had ssptlo ards. the survivors had a mean p death of . and the deaths had a mean p death of . . the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of patients who were enrolled into one of four treatment groups: ( ) . mg/kg of anti-tnf mab, ( ) . mg/kg, ( ) mg/kg or ( ) . mg/kg at study entry and the second dose hours later. the small sample size in each group (n= ) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin- may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x ) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of mg/kg tnf~ mab, . mg/kg tnf~ mab or placebo ( . % human albumin).patients received standard aggressive medical/surgical care during the day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca , was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca . as a single infusion or in combination with ha- a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage was an open label trial in which groups of patients each with the clinical diagnosis of sepsis received ascending doses of ca ( . , , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of ha- a ( mg) and placebo or one of doses of ca ( , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of placebo or one of doses of ca ( . , , mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il- , and il- levels.a total of patients were enrolled from clinical sites ( in stage , in stage and in stage ). primary analyses were performed on patients in stage and . there were patients who received ca exclusively and patients received placebo. administration of ca was well tolerated at doses up to mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in % ( / ) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca . the mean half-life was - hrs ( - hrs). a dose related decrease in tnf concentration was observed hr post infusion of ca . tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak f. it is a f(ab') fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak f or placebo were administered ( , ; , and i mg/kg) over a perid of hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il , il and tnf), and outcome. at this time only an interim analysis of patients is available i indicating that mak f in all dosage groups resulted in a decrease in il . this contrasted to a further in crease of il in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all patients in the study will be presented and discussed.$ s staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a atom-chain spacer. the detoxification capacity was ug[et/ml column material. the biocompatbility resulted in ~ platelet recovery. the column contained ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine . iu/h in the inflowline after bolus injection of . iu. hp was performed on pigs at a rate of ml/min by means of a roller-pump until the animals succumbed (h). animals served as controls (c). serum et levels rose from . pg/ml to , pg/ml after hours in the c and from . pg/ml only to pg/ml in the h group after hours whieh was highly significant. survival time could be extrended from to min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b( rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from to consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in °, and by septic-toxic event in %. all patients required mechanical ventilation (fio > , ) . ° showed hyperdynamic shock. % had renal and % hepatic failure. medium appache ii score amounted to , points. cvvh was performed in postdilution mode with a polyamide membrane (fh ) and high volume exchange ( l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in %, hemodynamic in % and renal in % of the cases. % of the patients survived and were discharged from hospital. of non-survivors ( %) died because of fatal mof within h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web ), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin ( p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n= ), or after pretreatment with web ( xl -gm, n= ), or diclofenac ( #g/ml, n- ). furthermore, the application of specific antitoxin (mg/ml, n= ) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, . mg/ml) (n= ) and the combination of immunogiobulins, web and diclofenac (n= ). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of . times higher than the control, starting after min and a delayed onset of edema formation resulting in a mean weight gain of g after min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about times that of controls in the toxin treated lungs. pretreatment with web or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st ( -n,n,n trimethylammonium-(r)- -isovaleroyloxy-butanoic acid z- -( -chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of h-paf (ki= . x - m) to rabbit platelets. the present study shows that pretreatment of c bl/ mice with st , administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli :b injected at mg/kg intraperitoneally). very interestingly, st administered at the same doses as above (i.e. . , . , and mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia ( . °c) induces heat shock protein ('asp) and increases smvival - fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp- by sodium arsenite improves survival in a rat sepsis model (abstract a am. rev. resp. dis. vol. , ) . the effects of heat are complex and in addition to formation of lisp- include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp- in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite ( mg/kg) and ethanol ( ~ of % ethanol) caused marked induction of hsp- in lung, gut, kidney, and liver, which was comparable to heat-induced hsp- . female nd mice weighing - gms were pretreated with arsenite or alcohol hours prior to challenge with escherichia coli endotoxin (-ld ) and survival was compared to control mice. results: survival at hrs. for arsenite treated and alcohol treated mice was % and % respectively and was statistically different from the % survival for control mice. (p< . ) (n= mice per group). however, at days post endotoxin, there were no differences in survival in the groups, i.e., ~ % survival for all groups. in contrast, the protective effect of hyperthermia remains present at days, i.e., ~ % survival vs % survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp- formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered x per day from the day be ~ fore the operation until the th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in / after pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from . % in the placebo-group to . % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from . % in the placebo-group to . % in the treatment-group (p ~ . ;fisher's exact test). in march final results with more than patients will be presented for the first time. (= po < mm hg, b s-creatinin > mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only of deaths occurred during the st week, all other deaths occurred late (after - weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. -- ) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only % of deaths today occur eady in the course of the disease, whereas this percentage varied between - % just years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for to years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in patients with acute pancreatitis ( males, females; median age: years, range: - years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type (slcam- ) were determined using immunoassays (henning, bender, t cell sciences). / had increased concentrations of stnf-r compared to the th percentile obtained in healthy controls (> . ng/ml), and / patients had increased neopterin (> . nmol/i), / presented with elevated slcam- (> u/i). all patients with increased neopterin also had increased stnf-r, patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = . , p < . ). weak associations between age and stnf-r (rs= . , p=o. ) or neopterin (rs= . , p = . ) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as ( , ) and characterized by increased serum or urine amylase levels (in about % of patients) in the fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the ffs, studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery ( , , ) . in a recent study ( ), evidence of pancreatic cellular injury was found in out of patients undergoing cardiac surgery, with out of these patients presenting abdominal signs or symptoms and developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), hours alter surgery and in the folfowing days alter surgery (days , , , and ). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day . we measured abnormal levels of trypsin and pancteatic iso-amylase in % of patients and observed simultaneous releases of these enzymes, the fi,'st one in the hours after surgery and the second more intense from day and pa~icularly on day after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about % of our patients : pancrealic abnormalities were revealed by scan in patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion ( ). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin- (il- ) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii- (elisa), transferrin and crp (nephelometry) were analysed daily along the first i days of hospitalisation by patients with acute pancreatitis admitted to our hospital from / to / . it was judged whether the patients have either interstitial (aip) (n= ) or necrotizing (anp) (n=lg) pancreatitis. patients with anp have died during the course of pancreatitis (mortality= . %). results: -severity o~ pancreatitis: signifcant differences (p<o. , mann-whitney test) could be calculated between aip and anp for endotoxin, il- , transferrin and crp during the i days of monitoring. -mortality: except of il- on days , , and after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. -organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il- and crp along the i days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il- and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from / - / , patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n= ) and n (n=l ) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in patients. the etiology of ap was gallstones in , alcohol abuse in , hyperlipidemia in and other or unknown causes in patients.five of patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il- concentration in blood reached up to pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of pg/ml was correct in patients ( %, sensitivity (se): %= specificity (sp): %, (z: , ). high levels of il- over pg/ml correlated well with the prognosis ifri in the first week (accuracy: %, se: %, sp: %, c~: , ). the blood levels of il- reached a maximum of pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of l,- blood levels indicated correctly the severity of ap in out of patients using a cut offtevel of pglml (accuracy: %, se: %, sp: %, ~: , ). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il- and il- . in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il- , and to a lesser extent of [l- , can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n= ), around the pancreatic duct (group b, n= ) or around both ducts (group c, n= ). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a mtcalbumin colloid with a diameter of nm, was measured before, , and days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day , the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day . the opossums in groups a & had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day in group a & b (p<o, ) and at day it~ group c (p< ,ol). at day , res function in group c was significantly worse than that in group a & b (p< , ). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day (p< . ), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b ) in lewis rats weighing - g. there were five groups: group a (n= ) were untreated controls; group b (n= ) were given isotonic saline intraperitoneally and observed for hours; group c (n= ) ml x e. coli intraperitoneally and observed for hours; group d (n= ) were given ml x e. coli and observed for hours; group e (n= ) ml x i e. coli and observed for hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser ). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p= , ) and spleen (p= , )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p< , ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel - , wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar( - ocalized jo-diffuse, -~eneralized) and -hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen- , the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, -com:lissural ileus). he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; ) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and ) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding /co hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate - wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. grafts were cryo'd in % dmso, quick-thawed & sutured onto x cm excised full-thickness dorsal wounds on athymic mice. "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained > % cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in - , x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through days of followup. histologic exams on days - showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than % total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we ] as ready availability of urdform lis~ue~. on dec~mt~r , , the fda assumed control of as tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, " ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki- (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since when galllco et el reported theh'use in two brothers with greater than % total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day ), the full thickness wounds were covered with epigard, a synthetic dressing until day . after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from patients at the beginning of surgical treatment (day ) and after , , and days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf- , double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.( ) pharmatec gmbh, frankfurt ( ) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group , cells migrating through skin: + % t lymphocytes (cd ), + % langerhans and dendritic cells (cdla, hla dr, s ), + % cd , + % cd , no b cells (cd , ), % cd r (memory cells), + % il r. approximately % cells possessed cdlla and antigens. cd lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group , cells in skin: langerhans cells were found only in epidermis, cd , and , cd r , rb, ila/ cells around venules, cd (macrophages) uniformly dispersed, no il r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group , one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il r cells, perivenular infiltrates of cd , r but less cd cells, high density of cdlla/ cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht bioassay). the hollow lipid microcylinders ( microns in length and i micron in diameter) show an initial burst ( - ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed , , and days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd and cd , mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code , naval research laboratory, washington, dc. - . expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet ) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to go of the origional size (t %) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from juvenile human bum patients, immediately after wound debridemerit mad on day after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*' ~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of psoriatics and healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx t-lymphocytes were then incubated alone or in coculture with x irradiated autologous lymphocytes in culture medium containing - mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce -surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t si-hsa over a rain period, the chemotac~c peptide imlp ( . . ng) and bradykinin were used to induce cell.dependent and cell- ndependent leakage respectively, the antibodies used were . e (cdis), nri (cdlla) and antibody (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien ( .~. ffi . p.l to . z b.bttl and . ,- . ~ to . z . d respectively. . e ( . - . mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at . mk/kg, the plasma leakage was completely inhibited, antibody nr produced similar results, treatment with antibody did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . patients without infectious coz~lications and group - patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection patients of the group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i- days after injury revealed a considerable decrease of lymphocytes, ed ",$d ~ and cd cells amo~it in the blood, cd /cd ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the - days in pateents of+the i group. but stable ~eple$ion of cd and cd cells amount, lower cd /cd ratio and indexes of leukocyte migration inhibition test in patients of the group were observed~ besides that, these persons showed higher cd cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in % of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. , , st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p integrlns (cd a,b,c/cd ) and the fw receptors (cd , and cd ) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~ integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z ) or thermal injury (> ~; total body surface area, > ~ full thickness) and incubated /n v/tro with gm or g-csf. the j integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd lb, cd , and cd while gm bur not c-csf induced an increase in the percentage expressing cdi a, cd lc, and cd , gm-csf and to a lesser extent g-csf induced an increase in the density ( , fold) of the ~ integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, bethesda ave, cincinnati, oh, usa, - . funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in % and in association with bacterial infection(l %), viral infection( */.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a. of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e -ebiary, a. torres et al, reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of olt in adult patients olt were performed for hbsag positive liver disease (cirrhosis n= , fulminant liver failure n= , retransplantation n= ) in pat. all pat. received . u hig in the anhepatic phase and . u/per day for the first week. a small group of pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are % in pat. who were transplanted for fulminant hepatitis, % in pat. with cirrhosis and long term prophylaxis, and % ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was % under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in % in pat, negative for hbv dna. in hbv dna positive pat. infection rate was %, the total rate of reinfection for all pat. with long term prophylaxis was %the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢ capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il- , ltb , c a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi , poe, lt's), complement components (c a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il, , il- or tnf), and immunosuppressivc agents (poe , il- ). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, - th ave za- , seattle, wa usa key: cord- -t rua e authors: jung, kwonil; wang, qiuhong; kim, yunjeong; scheuer, kelly; zhang, zhenwen; shen, quan; chang, kyeong-ok; saif, linda j. title: the effects of simvastatin or interferon-α on infectivity of human norovirus using a gnotobiotic pig model for the study of antivirals date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: t rua e the lack of an animal model for human norovirus (hunov) has hindered the development of therapeutic strategies. this study demonstrated that a commonly used cholesterol-lowering statin medication, simvastatin, which increases hunov replication in an in vitro replicon system, also enhances hunov infectivity in the gnotobiotic (gn) pig model. in contrast, oral treatment with interferon (ifn)-α reduces hunov infectivity. young piglets, all with a or h histo-blood group antigens on enterocytes, were treated orally with mg/kg/day of simvastatin; days later, the pigs were inoculated orally with a gii. hunov (hs / /us strain) and then treated with simvastatin for more days. simvastatin induced significantly earlier onset and longer duration of hunov fecal shedding in treated pigs, frequently with higher fecal viral titers. simvastatin impaired poly (i:c)-induced ifn-α expression in macrophages or dendritic cells, possibly due to lowered toll-like receptor (tlr) expression; however, the mechanisms were not related to interferon regulatory factor or nuclear factor kappa b signaling pathway. thus, the enhanced, earlier infectivity of hunov in simvastatin-treated pigs coincided with the inhibitory effect of simvastatin on innate immunity. in contrast to the increased hunov shedding that simvastatin induced, viral shedding during the treatment period was reduced or curtailed in the hunov-inoculated pigs pre-treated/treated with human ifn-α. our findings are the first to indicate that ifn-α has potential as antiviral therapy against hunov. based on these intriguing and novel findings using the gn pig model, we confirmed that hunov infectivity is altered by treatment with simvastatin or ifn-α. collectively, these findings indicate that gn pigs are a useful model to test immunomodulators or efficacy of antivirals against hunov. human norovirus (hunov), a single-stranded, positive sense rna virus, is a member of the caliciviridae family. this virus is the leading pathogen causing food-or water-borne gastroenteritis [ ] . hunovs are estimated to cause million cases of illness annually in the us, and they account for approximately % of the foodborne illnesses caused by different bacteria, parasites and viruses [ ] . clinical and pathological features of hunov infections include: i) a short incubation period ( - hr) prior to onset of clinical signs such as vomiting and diarrhea, although asymptomatic infections occur frequently [ ] ; ii) acute and self-limiting infection, but often with prolonged fecal virus shedding [ ] ; and iii) lymphocytic, atrophic enteritis [ ] . hunovs are classified into distinct genogroups (gi, gii, and giv), which are further subdivided into or more different hunov genotypes [ , , ] . in the last years, the gii. hunovs have been responsible for the majority of hunov outbreaks, possibly due to several viral and host factors that have been reviewed recently [ ] : i) the broader binding of host receptor to gii. hunovs, ii) incomplete herd immunity against gii. or its variants, and iii) higher mutation rate of their polymerases. in the viral capsid protein, the p domain is the most protruding and variable region. it is believed to recognize host cellular receptors and to contribute to establishment of viral infection. histo-blood group antigens (hbgas) are considered as cellular receptors or coreceptors that determine host susceptibility to certain hunovs. individuals of blood type a or o (h) of secretors were more susceptible to gi. /norwalk/ /us virus infection than individuals who were non-secretors or secretors of blood type b [ , ] . however, an emerging hunov, gii. strain did not bind to hbgas when tested in vitro [ ] . these observations imply a host factor affecting infection by certain genogroups or genotypes of hunov. hunov infection is generally self-limiting, but it can induce severe illness and fatal disease in immunocompromised patients, specifically, organ recipients receiving long-term chemotherapy or hematopoietic stem cell transplantation [ , ] . the young and elderly are also at risk, due to their high exposure rates to hunov infection in community settings (child care centers, nursing homes, hospitals, etc). notably, the common use of statin medications, which lowers serum cholesterol levels and prevents cardiovascular disease, is a significant risk factor for exacerbating hunov disease severity and increasing the related fatality rates [ ] . these conditions require effective therapeutic strategies against hunov infection. however, the lack of small animal model for hunovs has hindered development and testing of hunov antivirals or vaccines [ ] . gnotobiotic (gn) pigs are susceptible to oral infection by a gii. hunov strain (hs / /us) [ , ] and an emerging gii. hunov strain (hs / /us) [ ] . most hunov-infected gn pigs shed virus in feces. the incubation period ( - hrs) for gii. hunov in gn pigs was similar to that ( - hrs) observed in humans experimentally infected with the gii. snow mountain virus [ , ] . the longer fecal hunov shedding in gn pigs infected with the gii. hs strain was similar to that observed in human cases [ ] . also, like humans possessing secretor phenotype, gn pigs express a or h hbga on enterocytes. different hbga phenotypes (a or h) were shown to influence susceptibility of gn pigs to hunov infection [ ] . in a norwalk virus (gi. ) replicon-harboring cell system, the viral rna and protein levels were increased after treatment with cholesterol lowering drugs, such as simvastatin, that act as hydroxy- -methylglutaryl-coenzyme a (hmg-coa) reductase inhibitors [ ] . after statin treatment, reduced cellular cholesterol levels are followed by high expression of low-density lipoprotein receptor (ldlr) gene that compensates for the lower cellular cholesterol levels. these results indicated that cholesterol pathways may be associated with enhanced replication of hunov in vitro, although the related mechanisms are unclear. statins are also involved in a variety of immune responses and are immunosuppressive. they inhibit major histocompatability complex (mhc) class ii expression on antigen presenting cells in mice, promote the generation of foxp + t regulatory cells in mice, and impair lipopolysaccharide-induced toll-like receptor (tlr) -mediated inflammatory responses in human embryonic kidney- cells (hek- ) [ , , ] . the aim of our study was to determine if statins also enhance hunov infectivity in vivo in our gn pig model of hunov infection [ ] . the gn pigs were treated with high-doses of simvastatin and then inoculated with gii. hunov (hs / /us strain). we further investigated if the enhanced, earlier infectivity of hunov seen in simvastatin-treated pigs correlated with inhibitory effects of simvastatin on innate immunity. finally, we investigated the effect of an innate immunity mediator, ifn-a on hunov infectivity in the gn pig model. simvastatin treatment lowered the serum cholesterol level in gn pigs and resulted in increased early ldlr gene expression in a porcine enterocyte cell line (ipec-j ) serum cholesterol levels were monitored to measure the pharmacological activity of simvastatin in gn pigs. simvastatintreated pigs had significantly decreased serum cholesterol levels ( . . to . . mg/dl) at to days after treatment began, which were . to . times lower than the levels in the untreated pigs ( . . to . . mg/dl) during the same period (fig. a) . we further investigated if a reverse relationship existed between the cholesterol level and the ldlr gene expression in a porcine jejunal epithelial cell line, ipec-j , because intestinal epithelial cells are a cell type critical for initiation of hunov infection [ ] . cells were treated with multiple concentrations ( mm, mm, mm, and mm) of simvastatin, and ldlr gene expression was analyzed by quantitative real-time rt-pcr (qrt-pcr). at hours after treatment with mm simvastatin, ldlr gene expression levels in ipec-j cells were significantly increased compared to to mm treated groups (fig. b) . at and hours after treatment with to mm simvastatin, the ldlr gene expression levels were significantly increased by . to . times compared to untreated groups (fig. b) . significantly earlier onset of hunov shedding was observed in simvastatin-treated pigs, which began shedding at mean postinoculation day (pid) . . , compared to mean pid . . in untreated pigs (p, . ) ( fig. a) . significantly longer duration of hunov shedding was also observed in simvastatin-treated pigs, which shed for a mean of . . days, compared to a mean of . . days in untreated pigs (p, . ) (fig. b ). the mean daily fecal hunov titers were compared statistically between simvastatin-treated and untreated pigs in each trial, due to high variability in mean viral titers among the independent trials. significantly higher viral rna titers were detected in simvastatintreated pigs than untreated pigs in trial ( . . log genomic equivalents (ge)/ml vs. . . log ge/ml] (p, . ) and trial ( . . log ge/ml vs. . . log ge/ml) (p, . ) (fig. c) . however, no such significant difference was observed in trial . immunohistochemistry (ihc) results showed hunov antigens in the cytoplasm or on the surface of enterocytes, but not in lamina propria cells (fig. ) , supporting that fecal virus shedding is a result of hunov replication and infection in the intestine. the ihcpositive cells were in the small intestine, but not the large intestine, in which epithelial cells also expressed similar levels of hbga as in the small intestine (fig. a ). however, after hunov inoculation of simvastatin-treated or untreated gn pigs, no pronounced histological changes were evident in the small and large intestines of gn pigs or as a side-effect after oral treatment with high-doses of simvastatin in controls. under our experimental conditions no hunov-infected pigs showed diarrhea, whereas mild diarrhea was noticeably observed in all of the statin-treated pigs up to days after statin treatment. both hbga a+ and h+ type pigs were equally susceptible to gii. hunov infection all gn pigs used in this study were positive for either hbga a or h (fig. a-c) . the hbga antigens were distributed on the surface or in the cytoplasm of epithelial cells lining the intestine, the salivary glands, and pulmonary (bronchial) and renal tubular epithelial cells (fig. a-c) . under similar ihc conditions, amounts of a antigens in a + pigs were greater in the intestine and other positive tissues, as compared to those of h antigens in h + pigs (fig. a) . however, no significant differences in the onset and duration of fecal virus shedding were found between the a + and h + pigs ( fig. a and b) . despite the higher expression levels of a antigens, significantly lower viral rna titers ( . . log ge/ml) were detected in the feces of a + pigs than that ( . . log ge/ml) in the h + pigs (p, . ), but no difference was observed between the simvastatin-treated, a + and h + pigs (fig. c ). simvastatin impaired tlr -mediated induction of ifn-a in macrophages or dendritic cells, possibly due to lowered expression of tlr after treatment our in vivo data showing enhanced early infectivity of hunov suggested potential subversion of innate immunity related to simvasatin treatment. thus, we further investigated if simvastatin inhibits the capacity of macrophages or dendritic cells (dcs) to produce ifn-a after stimulation with poly (i:c), which triggers tlr -mediated induction of ifn-a. in general, ifn-a was not detected in culture supernatants of macrophages or dcs treated with either simvastatin only or mock. in porcine pulmonary alveolar macrophages (pams), ifn-a levels ( . . u/ml) released in simvastatin + poly (i:c)-treated pams were significantly lower than those ( . . u/ml) of poly (i:c) onlytreated pams at hours after poly (i:c) treatment (p, . ) (fig. a) . peripheral blood mononuclear cell (pbmc)-derived macrophages responded less to treatment with mg/ml of poly (i:c), as compared to pams treated with mg/ml of poly (i:c) ( table ) , possibly due to lower ratios of harvested adherent macrophages from pbmc. similar to the observations for pams, significantly lower ifn-a levels ( . . u/ml) were observed in simvastatin-treated, enriched intestinal dcs at hours after poly (i:c) treatment (p, . ), as compared with those ( . . u/ml) after poly (i:c) treatment alone ( table ). the mean percentages ( sem) of tlr + cells ( . . %, n = ) from simvastatin + poly (i:c)-treated intestinal macrophages at hours after poly (i:c) treatment were significantly lower (p, . ), as compared to that ( . . %, n = ) from poly (i:c) alone. a representative flow cytometric profile is illustrated in fig. b and c. gene expression levels of irf and nfkb, mainly involved in poly (i:c)-induced, tlr -mediated ifn production, were analyzed to investigate possible mechanisms underlying the subversion of innate immunity induced by simvastatin in pams and intestinal dcs. although simvastatin alone did not induce ifn-a production in pams (fig. a ), increased expression of irf and nfkb genes was found in simvastatin-treated pams ( fig. a and b). cotreatment with simvastatin and poly (i:c) synergistically resulted in increased gene expression of irf and nfkb. at hours after poly (i:c) treatment, however, gene expression levels of irf and nfkb were reduced in poly (i:c) only-treated pams, but not in simvastatin + poly (i:c)-treated pams, as compared to no treatments. swine ifn-a levels were decreased in the poly (i:c) and simvastatin-treated pams or dcs, and hunov infection was enhanced in vivo. therefore, we investigated whether fecal hunov shedding, i.e. hunov replication in the gut of infected gn pigs, was altered by treatment with ifn-a. oral treatment of gn pigs with natural human ifn-a (nhifn-a) [ international unit (iu)/ kg/day] reduced or curtailed virus shedding in treated animals during the treatment period (pid to ), compared to untreated animals ( fig. a-c) . the treatment significantly delayed the onset of virus shedding by . day in treated pigs, which began shedding at mean pid . . , compared to mean pid . . in untreated pigs (p, . ) (fig. a ). during the nhifn-a treatment period (pid to ), a significantly shorter duration of hunov shedding was observed in the nhifn-a-treated pigs, which shed for a mean of . . days, compared to a mean of . . days in untreated pigs (p, . ) (fig. b ). during the treatment period, a significantly lower qrt-pcr-positive rate of the fecal samples tested (p, . ) was also observed in the nhifn-a-treated pigs ( / ; . %) than in the untreated pigs ( / ; %), with significantly lower viral rna titers in the feces ( . . log ge/ml in the treated pigs vs. . . log ge/ml in the untreated pigs) (p, . ) (fig. c) . however, at pid to after nhifn-a-treatment was discontinued, significantly increased viral shedding titers were noted in the nhifn-a-treated pigs ( . . log ge/ml), compared to the untreated pigs ( . . log ge/ml) (p, . ) (fig. c) . at pids - , there were no significant differences in the duration of fecal virus shedding and the qrt-pcr-positive rate of the fecal samples tested between the nhifn-a-treated pigs and untreated pigs (fig. b) . further repeated studies in additional pigs are needed to investigate how termination of nhifn treatment results in higher viral shedding titers post ifn-a treatment. no negative control pigs shed detectable viral rna in the feces throughout the experiment. we demonstrated that use of simvastatin enhances hunov infectivity in the gn pig model. thus, its use may also support growth of hunov in cell culture. we also verified that the increased infectivity of hunov may be associated with the inhibitory effect of statins on innate immunity (ifn-a). this observation might explain the exacerbated hunov disease and the related higher mortality described in statin-treated humans [ ] . because of the immunosuppressive effects, use of statins have been proposed for immunomodulatory therapy against severe influenza a virus infections in which large amounts of innate (ifna) cytokines are involved [ ] . in addition, we showed that oral treatment with nhifn-a can curtail early hunov fecal shedding in the gn pig model. because no hunov vaccines are available, use of effective antivirals such as ifn-a should be tested to control multiple genogroups and genotypes of hunovs, including the gii. variants that have emerged each year [ ] . our findings that hunov infectivity in gn pigs can be enhanced by simvastatin treatment or reduced by oral treatment with nhifn-a, suggest that gn pigs are a useful model to test efficacy of antivirals against hunov. as a surrogate model for hunovs, murine nov (mnv) infection of mice was useful for investigating the roles of specific immunologic factors such as type i or ii ifns in host defense [ ] . however, the different pathogenesis of mnv infection with its systemic spread raises concerns about extrapolation of these findings to the hunov restricted gastrointestinal infection. a chimpanzee model was recently established to evaluate the efficacy , and the daily viral titers (mean) as monitored by qrt-pcr are shown (c). monitoring continued until to weeks after infection and terminated when pcr results were negative (, . log ge/ml) for consecutive days. data from independent animal trials were combined, and the pcr test was performed in duplicate or triplicate. additional analysis was also conducted according to the hbga a or h phenotype of each animal. each bar represents the mean sem. *p, . ; **p, . for simvastatin + hunov vs hunov alone or for a+ pigs vs h+ pigs by the unpaired two-tailed mann-whitney test. animal numbers (n) are indicated at the bottom of each graph. the dotted line indicates the detection limit ( . log ge/ml) of the qrt-pcr. doi: . /journal.pone. .g of vlp-derived vaccines against infection with gi or gii hunovs [ ] . chimpanzees developed serum antibody responses after intravenous injection of gi. /norwalk virus or intramuscular injection with norwalk vlps and were protected from gi. / norwalk virus challenge (but not gii hunov infection). the chimpanzee model, however, is compromised by the lack of availability of chimpanzees, and the finding that oral infection of chimpanzees with hunovs failed to induce gastroenteric disease comparable to human cases [ ] , as well as by the intravenous route required for viral challenge. in our study, although hunov infection of gn pigs induced mild enteric disease, gn pigs were susceptible to oral infection by the gii. hs strain, which reaffirms the results of our previous studies using a closely related gii. hunov (hs strain) [ , ] and the emerging gii. hunov (hs strain) [ ] . fecal hunov shedding patterns in gn pigs, with peak viral titers during an early stage of infection are also typical for other acute enteric viral infections in pigs, such as rotavirus and porcine epidemic diarrhea virus [ , ] . however, how hunov shedding in infected gn pigs is maintained for or weeks after viral inoculation is unclear and requires further investigation. cholesterol biosynthesis and metabolism are mainly mediated by hepatic enzymes, such as hmg-coa reductase [ ] . statins act as competitive inhibitors of hmg-coa reductase and reduce production of cholesterol in the liver. as in humans, our study showed that statins lower serum cholesterol levels in gn pigs, possibly due to similar cholesterol pathways between swine and humans as reported previously [ ] . when hepatic cholesterol stores are depleted, the liver increases the expression of ldlr which leads to uptake of ldl from plasma to compensate for the lower cellular cholesterol levels. several rna viruses manipulate cholesterol pathways in diverse ways for more efficient viral infection and replication as exemplified for novs in comparion to hepatitis c virus (hcv) and coronavirus [ , , , ] . for example, low cellular cholesterol levels (or high cellular ldlr expression) following statin treatment contributed to increased gi. /norwalk virus replication, as verified in an in vitro replicon system [ ] . our study also showed that increased ldlr expression levels in ipec-j cells (a porcine jejunal cell line) treated with simvastatin might similarly contribute to enhanced hunov replication in the gastrointestinal tract. our other ongoing in vitro studies also found that hunov rna titers in supernatants or lysate samples of ipec-j cells treated with simvastatin were slightly increased in trials using gii. hs strain compared to those of controls (without simvastatin), but did not differ significantly in cell cultures using the gii. hs strain. the latter was previously reported [ ] . the data indicate a positive but inconsistent effect of simvastatin on hunov replication in vitro, possibly depending on the hunov strains or different environmental conditions for hunov replication in vitro versus in vivo. a paper describing more detailed and comprehensive in vitro cell culture findings is in preparation by takanashi et al. (unpublished data, ) . in addition to the cholesterol lowering effects, the inhibitory effects of statins on innate immunity also might influence the immunological and cellular microenvironment for more efficient hunov replication. in our study, simvastatin impaired tlr -mediated innate immunity and inhibited production of ifn-a induced by poly (i:c) in pams or intestinal dcs. these observations are similar to the results of an in vitro study using hek- cells, showing the inhibitory effect of simvastatin on tlr -mediated immune responses, such as tumor necrosis factor (tnf)-a and interleukin- [ ] . nevertheless, it is notable that observations for gn pigs and humans infected with hunovs [ ] are contrary to the effect of statins that reduced replication of hcv in replicon-harboring cells [ ] and a positive correlation between cellular cholesterol levels and entry of coronaviruses [ ] and of gv/mnv into host cells [ ] . further confirmatory data are needed to define the role of the cholesterol pathway in the pathogenesis of hunov. although simvastatin treatment inhibited ifn-a production, we found that gene expression of irf and nfkb in simvastatintreated pams was increased rather than being decreased. because activation of nfkb kinase is a shared property among tlrs, including tlr [ ] , simvastatin or its cellular byproducts could trigger other tlrs that stimulate nfkb gene expression. notably, at hours after poly (i:c) treatment, gene expression levels of irf and nfkb in poly (i:c) only-treated cells were reduced remarkably, as compared with other treatments or those at the earlier time-point. this observation could be explained by a cellular negative feedback effect to mediate production of ifna. it is also notable that a similar result did not occur in simvastatin + poly (i:c)-treated cells, possibly due to reduced ifn-a levels after simvastatin treatment. at least four families of transcription factors are activated by dsrna and relate to tlr : nfkb, irf- , c-jun, and activating transcription factor [ ] . besides lowered tlr expression by macrophages after simvastatin treatment, which was thought to be mainly responsible for reduction of ifn-a production in statin + poly (i:c)-treated cells, other tlr -or ifn-mediated signaling pathways might be involved in the impaired innate immunity by simvastatin. type i ifns are essential for early viral clearance and development of adaptive immune responses. as a crucial mediator of the innate antiviral immune responses, ifn-a has been an effective antiviral treatment for viral infections, such as hcv and influenza [ , ] . the signal transducer and activator of transcription- (stat- )-dependent ifn stimulation was essential for controlling murine nov (mnv) infection. although mnv did not cause disease in immunocompetent mice, oral mnv infection caused fatal systemic disease in mice lacking either type i and type ii interferon receptors or stat- , which is critical for ifn signaling [ , ] . several studies have suggested that repeated oral treatment with nhifn-a may be effective in treating acute viral gastroenteritis related to coronavirus and rotavirus in domestic pigs [ , ] . similarly, our study showed that oral administration of nhifn-a inhibits infection by or replication of hunov, as fecal hunov shedding is curtailed in the gn pig model. the nhifn-a is formulated to be stable at low ph of the stomach. degradation of nhifn-a by a variety of intestinal enzymes appears to be slow enough to allow nhifn-a to reach some ifn-a receptors of cells in mucosal lymphoid tissues of the oral cavity and intestine [ ] . the table . therapeutic effectiveness of nhifn-a might be related to its immunostimulatory effects. orally delivered nhifn-a promoted systemic innate immunity by increasing expression levels of innate immunity-related genes, such as ifn-stimulated genes (isgs) and tnf-a, and phagocytic capacity of phagocytes [ , ] . further studies with larger numbers of animals are needed to determine the most effective dose and regimen of nhifn-a to prevent or treat hunov infections, and to elucidate the immunological and molecular mechanisms related to the antiviral effects of ifn-a. based on the effectiveness of a combination of nhifn-a pre-and post-treatment, the nhifn-a treatments need to be tested therapeutically in future studies using the gn pig model or in five or six day-old piglets were treated orally with iu of nhifn-a once a day from pid - to pid . on day after nhifn-a treatment, they were inoculated orally with gii. hs hunov, and subsequently treated with nhifn-a ( iu) for more days. after nhifn-a treatment or hunov inoculation, clinical signs and fecal virus shedding were monitored daily until shedding terminated. data from independent animal trials were combined, and the pcr test was performed in duplicate or triplicate. duration of virus shedding in nhifn-a-treated and untreated pigs were analyzed based on nhifn-a treatment period, i.e. during treatment at pids - ; post-treatment at pids - ; and overall at pids - . each bar represents the mean sem. *p, . ; **p, . for nhifn-a + hunov vs hunov alone by the unpaired two-tailed mann-whitney test. animal numbers (n) are indicated at the bottom of each graph. the dotted line indicates the detection limit ( . log ge/ml) of the qrt-pcr. doi: . /journal.pone. .g clinical trials. the mechanisms by which fecal virus shedding recurred and the increased viral rna titers in nhifn-treated gn pigs compared to untreated pigs after nhifn treatment was discontinued need to be investigated. however, we hypothesize that during the period of nhifn treatment, ifn signaling pathways might be regulated by a negative feedback in some ifn producing cells in the intestine. thus, on the termination of treatment such a distinct condition of the ifn system might hinder induction or production of ifn-a in most ifn containing cells or its antiviral activity against hunov. in conclusion, simvastatin treatment increased hunov infectivity in the gn pig model, possibly due to its inhibitory effect on innate immunity as well as its cholesterol lowering effect as reported previously [ ] . these findings could partially explain the exacerbated hunov disease in statin-treated humans [ ] . testing of nhifn-a as an antiviral for hunov using the gn pig model also revealed that ifn-a has potential as a hunov antiviral therapy. development of hunov antivirals is important because hunovs cause large-scale epidemics with significant mortality in immunocompromised, elderly and young patients. thus, the gn pig model for hunov will allow testing of new treatment modalities for hunov infection and new knowledge on the antiviral mechanisms of innate and adaptive immunity. the ipec-j cells were kindly provided by dr. bruce d. schultz (kansas state university) [ ] . cells were maintained in dulbecco's modified eagle's medium/nutrient ham's mixture f- (invitrogen, carlsbad, ca) with % fetal bovine serum (fbs; hyclone laboratories, inc., logan, ut), % insulin-transferrinsodium selenite (roche, mannheim, germany), and epidermal growth factor ( ng/ml) (invitrogen). the gii. /hs / / us (hs ) strain (genbank accession number: gu ) used as viral inoculum in this study was isolated from stool samples of a young child with watery diarrhea [ ] . stool samples were screened for other enteric viruses, including gi hunov, rotavirus groups a, b and c, sapovirus, astrovirus, and adenovirus by reverse transcription (rt)-pcr or pcr, respectively, as described previously [ ] . near-term pigs were derived by hysterectomy and maintained in sterile isolator units [ ] . by ihc using monoclonal antibodies to human a (immucor, norcross, ca) and h (covance research products, inc., dedham, ma), we determined the a/h phenotype on fresh bucal cells or formalin-fixed, paraffin-embedded intestinal and salivary glandular tissues of gn pigs. the institutional animal care and use committee (iacuc) of the ohio state university approved all protocols related to the animal experiments in this study. all animals used in this study were also handled in accordance with the guidelines of the iacuc of the ohio state university. piglets were randomly assigned to one of four groups: simvastatin + hunov (n = ), hunov alone (n = ), mock (n = ), and simvastatin alone (n = ). to lower serum cholesterol levels prior to virus infection, five or seven day-old piglets were first treated orally with mg/day/pig (approximately kg of body weight) of simvastatin (zocor; merck and co, inc., whitehouse station, nj). on day after treatment, they were infected orally with . or ge of the gii. hunov hs , and then subsequently treated with the half doses of pre-treatment for more days. after simvastatin treatment or hunov inoculation, we monitored clinical signs daily. at an acute (pid to ) stage of hunov infection, to pigs per group were euthanized for histopahological examination. when virus fecal shedding terminated as determined by qrt-pcr, i.e. at a later (pid to ) stage of infection, to pigs per group were euthanized. the nhifn-a was kindly provided by dr. joseph cummins (amarillo biosciences, inc., amarillo, tx). piglets were randomly assigned to one of three groups and constituted independent trials: nhifn-a-treated, hunov-infected (n = ), nhifn-a-untreated, hunov-infected (n = ), and negative control (n = ). five or six day-old piglets (approximately kg of body weight) were treated orally with iu of nhifn-a once a day from pid - to pid . on day after nhifn-a treatment, they were infected orally with . ge of the gii. hs hunov, and subsequently treated with nhifn-a ( iu) for more days. after nhifn-a treatment or hunov inoculation, clinical signs and fecal virus shedding were monitored daily until shedding terminated. total serum cholesterol levels were assessed in simvastatin treatment trials by using an amplex red cholesterol assay kit (invitrogen), as described previously [ ] . total cholesterol was extracted in chloroform-methanol-double-distilled water [( : : ) (vol/vol/vol)]. the chloroform phase was separated, mixed with a : volume of polyoxyethylene -lauryl ether (sigma-aldrich, st. louis, mo), dried, and resuspended in the assay reaction buffer in the kit. each treatment was duplicated in additional sixwell plates, and cell lysates were prepared for the measurement of protein contents by using a bca protein assay kit (bio-rad, hercules, ca). the concentrations of total cholesterol were normalized with the protein contents. rectal swabs were collected daily from each animal throughout the experiment. the gii hunov fecal shedding titers were determined by the taqman real-time rt-pcr (cog f/ r primer set and ring probe), as described previously [ , ] . the detection limit of this pcr assay was ge per reaction determined based on the standard curve generated using serially diluted plasmid dna carrying hs -specific cog f/ r amplicons. the limit of viral rna detection in the qrt-pcr assay was . log ge/ml. the recombinant baculovirus carrying the capsid protein (vp ) gene (orf ) of hs strain (genbank accession number: gu ) was generated by using the baculodirect tm baculovirus expression system (invitrogen) according to the manufacturer's instructions. briefly, a gateway entry clone containing the orf of hs (pentr tm /sd/d-topo-hs ) was generated and used with the baculodirect tm linear dna to perform a lr recombination reaction to generate recombinant baculovirus dna carrying the orf of hs . the insect sf cells were transfected by the recombination reaction products and the cells containing the recombinant baculovirus dna were positively selected by ganciclovir. the expression of hs capsid proteins in the sf cells and culture supernatants were examined by immunoblot using the guinea pig antiserum against hu/nov/ gii. /hs / /us strain [ ] . the recombinant baculoviruses (rbac-hs ) were propagated to prepare virus stocks with high titers for routine vlp expression. the production and purification of hs vlps were performed as described previously [ ] . the protein concentration was quantified using the bradford method and the vlps were negatively stained with % phosphotungstic acid (ph . ) and examined by transmission electron microscopy as described previously [ ] . hyperimmune serum against hunov gii. hs vlps was generated using guinea pigs according to an approved iacuc protocol, as previously described [ ] . small (duodenum, proximal, middle and distal jejunum, and ileum) and large (cecum and colon) intestinal tissues and other major organs (lung, liver, heart, kidney, spleen, and lymph node) were examined grossly and histologically and tested by ihc for nov antigen detection. tissues from age-matched mock controls were tested for histological comparisons and as a negative control for ihc. the ihc was performed on formalin-fixed, paraffinembedded tissues or fresh frozen tissues using the guinea pig hyperimmune antisera to vlps of the gii. hunov hs , as described previously [ ] . the pams ( , cells/ml) were collected from the lungs of ten-day-old, uninfected gn pigs using aseptic techniques, as previously described [ , ] . cells ( , cells/well) were seeded onto -well cell plates, and the wells were randomly assigned to treatment groups: no treatment, simvastatin alone, simvastatin + poly (i:c), and poly (i:c) alone. pams were first treated with mm simvastatin (sigma), and hours later, treated with either mm simvastatin or poly (i:c) ( mg/ml) (sigma), or treated with both. the cell culture supernatants were harvested at and hours after poly (i:c) treatment to measure ifn-a levels released from pams. intestinal mononuclear cells or pbmc were isolated from ileum or blood of gn pigs, as previously described [ ] . gut or pbmc monocyte-derived macrophage or dc-enriched cell preparations were obtained by plating gut mononuclear cells or pbmc at , cells/ml in rpmi- supplemented with % fbs, % gentamicin, . % ampicilin, mm hepes, mm lglutamine, and mm sodium pyruvate for - days before harvesting adherent cells. non-adherent cells were used for dcenriched cells. macrophage-or dc-enriched cells ( , cells/well) were seeded onto -well cell plates, and the wells were randomly assigned to treatment groups: no treatment, simvastatin alone, simvastatin + poly (i:c), and poly (i:c) alone. cells were treated with simvastatin in the same manner as for pams. mardin-darby bovine kidney (mdbk) cells were grown in mem with % fbs and % antibiotic-antimycotic. the cell supernatant samples, serially diluted : in mem, were added to the confluent cell monolayers seeded in -well plates, as previously described [ ] . at hrs after incubation at uc, media was removed and ml of vesicular stomatitis virus ( plaque forming unit/ml) was added. at hrs after incubation, ml of alamar blue was added to each well and incubated for hrs at uc. fluorescence was measured at - nm. antiviral ifn-a levels (u/ml) were expressed as the reciprocal of the sample dilution which resulted in a % reduction in cytopathic effects. expression levels of porcine ldlr, nfkb, and irf mrna were measured in ipec-j cells or pams by taqman real-time pcr, as described previously [ , , ] , with slight modifications. the mrna expressions were normalized to the expression levels of porcine b-actin [ ] . primers and probes of ldlr and b-actin and probes of nfkb and irf were designed by geneious primer design software, as follows ( - ): ldlr f, cgccctccaaaacggtggct; ldlr r, acttcggc-gagcgtgggttg; and ldlr probe, fam-ac-ctgtgtctgccagctccaca- iabkfq. b-actin f, cccacgccatcctgcgtctg; b-actin r, gtagccccgctccgtcagga; and probe, fam-ggccgggacctgaccgacta- iabkfq. nfkb probe, fam-accaggctggcagctctcctcaaagcagca- iabkfq. irf probe, fam-ccggtctgccctgaaccg-gaa- iabkfq. all values are expressed as the means standard error of the means (sem). cholesterol level data among the treatment groups were analyzed by the kruskal-wallis test (nonparametric) using the statistical analysis systems. all gene expression and ifna level data and numbers of tlr + cells were analyzed by oneway analysis of variance (anova). virus titers that were undetectable (, . log ge/ml) during the shedding period were assigned as a value of . log ge/ml for statistical analysis. the mean onset and duration of virus shedding and viral titers between simvastatin + hunov and hunov alone groups, between a + and h + pigs, and between nhifn-a-treated and untreated pigs, were compared by unpaired two-tailed mann-whitney tests. specifically, duration of virus shedding and viral titers in nhifn-a-treated and untreated pigs were analyzed based on nhifn-a treatment period, i.e. during treatment at pids - ; post-treatment at pids - ; and overall at pids - . fisher's exact test was used to compare the pcr-positive rates of the fecal samples tested at pids - or pids - between the nhifn-atreated and untreated pigs. a value of p, . was considered statistically significant. 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green qpcr we thank dr. joseph cummins who kindly provided the natural human ifn-a and gave helpful suggestions for the ifn-a treatment of our experimental pigs. we also thank dr. j. hanson and r. mccomick for assistance with animal care and t. aubrecht, c. siegismund, a. vlasova, s. takanashi, and k. chatta for assistance with immunological assays. key: cord- -ixiam qr authors: zhu, xun; he, zhenjian; yuan, jie; wen, weitao; huang, xuan; hu, yiwen; lin, cuiji; pan, jing; li, ran; deng, haijing; liao, shaowei; zhou, rui; wu, jueheng; li, jun; li, mengfeng title: ifitm ‐containing exosome as a novel mediator for anti‐viral response in dengue virus infection date: - - journal: cell microbiol doi: . /cmi. sha: doc_id: cord_uid: ixiam qr interferon‐inducible transmembrane proteins , and (ifitm , ifitm and ifitm ) have recently been identified as potent antiviral effectors that function to suppress the entry of a broad range of enveloped viruses and modulate cellular tropism independent of viral receptor expression. however, the antiviral effect and mechanisms of ifitms in response to viral infections remain incompletely understood and characterized. in this work, we focused our investigation on the function of the extracellular ifitm protein. in cell models of denv‐ infection, we found that ifitm contributed to both the baseline and interferon‐induced inhibition of denv entry. most importantly, our study for the first time demonstrated the presence of ifitm‐containing exosome in the extracellular environment, and identified an ability of cellular exosome to intercellularly deliver ifitm and thus transmit its antiviral effect from infected to non‐infected cells. thus, our findings provide new insights in the basic mechanisms underlying the actions of ifitm , which might lead to future development of exosome‐mediated anti‐viral strategies using ifitm as a therapeutic agent. conceivably, variations in the basal and inducible levels of ifitms, as well as in intracellular and extracellular levels of ifitms, might predict the severity of dengue virus infections among individuals or across species. study for the first time demonstrated the presence of ifitm-containing exosome in the extracellular environment, and identified an ability of cellular exosome to intercellularly deliver ifitm and thus transmit its antiviral effect from infected to noninfected cells. thus, our findings provide new insights in the basic mechanisms underlying the actions of ifitm , which might lead to future development of exosome-mediated anti-viral strategies using ifitm as a therapeutic agent. conceivably, variations in the basal and inducible levels of ifitms, as well as in intracellular and extracellular levels of ifitms, might predict the severity of dengue virus infections among individuals or across species. dengue virus is an enveloped, single-stranded positive strand virus, belonging to the flavivirus genus of the family flaviviridae, with four related but distinct serotypes (denv- to ) (halstead, ; simmons et al., ) . denv is the aetiologic agent of dengue fever (df), the most prevalent arthropod-borne viral disease in humans with more than - million cases worldwide annually. denv is transmitted by mosquito aedes aegypti or aedes albopictus in the tropical and subtropical regions, where these mosquitoes are endemic, placing . billion people at risk of infection globally (guzman et al., ; simmons et al., ) . of note, % of infected individuals develop more severe, and often lethal, syndromes, designated dengue haemorrhagic fever (dhf) and dengue shock syndrome (dss), with children bearing most of the disease burden guzman et al., ) . while genotypic differences of denvs have been indicated to be associated with variations in viral virulence, heterotypic dengue virus antibodies are believed to be a risk factor for the development of dhf or dss in secondary infections (guzman et al., ) . despite the formidable toll imposed by denv on world health, no approved vaccine or effective specific anti-denv therapies are currently available for denv infection (halstead, ; guzman et al., ) . as a major host defence system against viral infection, the interferon (ifn) family, notably ifn-alpha and ifnbeta, initiates a potent antiviral response that activates the innate immunity mediated by induction of more than ifn-stimulated genes (isgs), leading to the establishment of an anti-viral state (sadler and williams, ) . ifn inducible transmembrane proteins , and (ifitm , and ) have recently been identified as antiviral mediators induced by ifn, to confer host cells resistance to a variety of pathogenic viruses such as influenza a virus (iav) (brass et al., ; feeley et al., ; huang et al., ; everitt et al., ) , west nile virus (brass et al., ; jiang et al., ) , denv (brass et al., ; jiang et al., ; chan et al., ) , vesicular stomatitis virus , marburg virus (huang et al., ) , ebola virus (huang et al., ) , sars coronavirus (huang et al., ) , human immunodeficiency virus (lu et al., ) and hepatitis c virus (yao et al., ) . mechanistically, ifitms are the only known isg products that act to restrict the entry step of viral infection process (feeley et al., ) . in particular, brass et al. demonstrated that the action of a single intrinsic immune effector, ifitm , profoundly effected as an essential barrier to iav infection in vitro, in a knockout mouse model and in humans, by blocking the virus-cellular membrane fusion and thus preventing cytosolic entry of the virus (brass et al., ; feeley et al., ; everitt et al., ) . interestingly, another study revealed that ifitm proteins could interfere with the antibody-dependent enhancement (ade) effect during secondary dengue virus infection, which bypassed the ifn-mediated restriction (chan et al., ) . taken together, these previous studies have defined ifitm as a mediator required for the anti-viral action of ifn, representing a key component of human anti-viral defence system, probably through targeting an early step of viral infection. open questions, however, remain concerning the detailed biological functions of ifitm in ifn-triggered cascades and the precise mechanisms how the host cellular defence system is involved in ifitm activation in response to viral infections. such knowledge will provide insights in designing new antiviral therapeutics using ifitm proteins. furthermore, it is worth noting that secreted or membrane-bound proteins are of particular interest in drug development, because their extracellular nature renders them more accessible for therapeutic intervention. as a novel alternative secretion system, exosomes are small vesicles ( - nm in diameter) of endocytic origin that are released from cell into the extracellular environment, under both normal and pathological conditions (thery et al., ) . exosomes are formed through the inward budding of late endosomal membrane that gives rise to intracellular multivesicular bodies (mvbs), which involve their fusion with the plasma membrane and release of mvbs into the extracellular environment as exosome (thery et al., ) . prior work has focused on exosome as a new family member of 'bioactive vesi-cles' that function to promote intercellular communication, by shuttling for proteins, lipids and rnas of the cells, and participate in various biological processes including immunomodulatory events (schorey and bhatnagar, ) . while shuttling for components of pathogenic microbes, exosome could stimulate immune responses. for instance, exosome isolated from cells infected with mycobacterium tuberculosis, have been shown to contain bacterial components and promote antigen presentation and macrophage activation (bhatnagar and schorey, a; bhatnagar et al., b) . moreover, exosome may also promote intercellular spreading of infectious cargo, such as the observed cell-to-cell transmission of hiv (gould et al., ; izquierdo-useros et al., ). on the other hand, despite these advances in our understanding of the functions of exosome, the physiological significance of exosome in shuttling bioactive molecules key to the host defence system remains enigmatic. more recently, atanu et al. demonstrated that apobec g, which belongs to an isg family of cellular cytidine deaminases that effect to restrict replication of a variety of exogenous retroviruses, was exported by exosome and conferred antiviral phenotype to recipient cells (khatua et al., ). li et al. propose an antiviral mechanism of ifn-α activity that involves the induction and intercellular transfer of antiviral molecules (such as lamp- protein, apobec g proteins, ifi mrna, ddit mrna, hsa-mir- , hsa-mir- and hsa-mir- etc.) from liver non-parenchymal cells to hepatocytes via exosomes, by using hbv infection as a model . they also observed similar exosome-mediated transfer of ifitm mrna from macrophages to hepg . . cells, but there is not in-depth study on whether the anti-hbv activity is dependent or independent of exosome-mediated transfer of ifitm mrna . it is therefore tempting to hypothesize that direct exchange of isgs proteins transferred by exosome among host cells might contribute to the establishment of anti-viral state in uninfected cells, in addition to the direct action of ifns stimulation. in this study, we focused our investigation on the function of the extracellular ifitm protein. in cell models of denv- infection, we found that endogenous basal protein levels of ifitms inversely correlated to denv- infection, and induction of ifitms in cells with low basal protein levels was sufficient to drive protection of host cells from denv- infection at entry. conversely, loss of ifitm in host cell pronouncedly enhanced denv- infection. most importantly, our study for the first time demonstrated the presence of ifitm-containing exosome in the extracellular environment and the function of these exosome vehicles for inter-cellular transmission of antiviral proteins. in an effort to investigate whether endogenous ifitm in host cells plays a role in the interaction between denv and the host cells, we first compared the expression of ifitm in denv-permissive cell lines with versus without denv- infection. notably, following infection with denv- at multiplicity of infection (moi) of in human cell lines permissive for denv- replication, expression of ifitm was found to be inducible by denv- infection in various cell lines as demonstrated by western blotting analysis (fig. a) . next, we analysed the correlation of ifitm level in cell lines with denv- infection. we found that while endogenous ifitm expression was varied in different cell lines, cells expressing low level of ifitm protein were more susceptible to denv- infection (fig. b) . further exponential regression analysis showed that the level of ifitm protein in host cells inversely correlated with their susceptibility to denv- infection significantly (r = . , p = . , n = , fig. c ), suggesting that ifitm might be an important cellular restriction factor for denv infection. to evaluate the role of ifitm proteins in denv infection, we generated permanent cell lines stably overexpressing human ifitm , or , or control vector, respectively, with u and hela cells ( fig. s a and c). as show in fig. s b and d, ifitm , ifitm or ifitm overexpression drastically diminished the number of denv- infected cells, as indicated by twofold to fourfold reduction of denv- viral e protein accumulation in virally infected u and hela cells. similar profound restriction was also seen when ifitm was transiently transduced in t, despite a lesser extent of ifitm overexpression than that of ifitm or ifitm ( fig. s e and f), demonstrating the potency of ifitm in conferring host cells resistance to denv infection. we next examined the effect of ifitm on the entry step of denv- , and found that entry of denv- was reduced by ifitm by approximately twofold (fig. a ), as controlled with the g monoclonal neutralizing antibody to demonstrate a successful entry blockage, suggesting that the disruption of denv replication by ifitm was associated with a targeted abrogation of viral entry. since ifn stimulated signalling plays a pivotal role in protecting cells from viral infection and damage, we investigated the functional significance of ifitm in ifnmediated anti-denv response. as shown in fig. s a and b, compared with huvec cells transfected with control scramble sirna, transfection of ifitm -sirna- and ifitm -sirna- efficiently depleted basal-level ifitm expression, and increased the permissiveness of the transfected cells to denv- infection, as indicated by a > -fold increase of infection, in huvec that constitutively express high basal level of endogenous ifitm expression. similar effect of ifitm depletion was reproducible in another cell model, in which the basal ifitm level is low but greatly inducible by ifn-α. sirna silencing of ifitm markedly attenuated the protective effect of ifn-α against denv- infection in hela cells, leading to profoundly increased infection by denv- in the presence of ifn-α, by . -or . -fold ( fig. s c and d) , indicating that the depletion of ifitm could decrease the antiviral actions of ifn-α. we also performed denv binding/entry assay following knockdown of ifitm expression. our results showed no effect of sirna silencing of ifitm on the binding of denv- to hela cells, but the penetration of denv- into cells was significantly promoted by the depletion of ifitm (fig. s e) . thus, our results suggest that ifitm is involved in mediating ifn-induced cellular response against denv infection. in an attempt to analyse the biochemical properties of ifitm , we found that the protein was present both intracelluarly and extracellularly. specifically, cell culture supernatants were collected from parental huvec cells or hepg cells that express high basal level of endogenous ifitm , from huvec or hepg cells transfected with ifitm sirna- (huvec-siifitm or hepg -siifitm ), from huvec or hepg cells transfected with non-targeting control sirna (huvec-sinc or hepg -sinc), from t cells transfected with pcdna ( t-vector), and from t cells transfected with the pcdna-ifitm , , -flag construct ( t-ifitm , , ) for h. supernatants derived from each of the above groups with different treatments were collected and centrifuged at g for min, and then g for min at °c to remove cells and cell debris, followed by filtration with . μm filters. then the supernatants were concentrated by -fold using an amicon ultra- centrifugal filter unit with kda cut-off value, and subjected to western blotting analysis using an anti-ifitm antibody. our data showed that ifitm protein was present both in the cytosol and in the culture medium ( fig. a-d) , suggesting the possibility that ifitm could be released to extracellular space. it was also noteworthy that the expression of ifitm in the antiviral effect of ifitm mediated by exosome supernatants collected from cultured huvec cells is lost when ifitm was knocked down or exosome secretion was inhibited by gw (an exosome-release inhibitor) (fig. d ). as the amino acid sequence of ifitm does not contain a putative signal peptide for protein secretion, we sought to explore the possibility that the exportation of ifitm could be through an exosome-mediated a-d. cell culture supernatants of huvec or hepg cells transfected with ifitm sirna- (huvec-siifitm or hepg -siifitm ), from huvec or hepg transfected with non-targeting control sirna (huvec-sinc or hepg -sinc), from t cells transfected with pcdna ( t-vector), or from t cells transfected with the pcdna-ifitm , , -flag construct ( t-ifitm , , ) for h, were collected and concentrated for sds-page and immunoblotting, using an anti-ifitm antibody, with cell lysates as positive control. e. electron micrographs of crude exosomes negatively stained with uranyl acetate and examined at kv are shown. f. purified ifitm -containing exosomes derived from each group of cells above described were analysed by immunoblotting with anti-ifitm , anti-flotillin- , anti-cd , anti-calnexin (endoplasmic reticulum, er marker) and anti-gm (golgi marker) antibodies. ifitm is identified in the exosomes derived from each group of cells as indicated. mechanism. to achieve this, exosomes were prepared from the culture media of huvec, huvec-sinc cells, huvec-siifitm cells, ifn-α-treated t cells ( t-ifn), t-ifitm cells, t-vector cells and t cells, and subjected to sucrose gradient ultracentrifugation. the electron microscopic examination of the exosomes revealed vesicles ranging in size from nm to nm (fig. e) . interestingly, ifitm was detected in exosomes purified from the culture supernatant of huvec and huvec-sinc cells, and was also abundant in those produced by t-ifitm or t-ifn cells. in contrary, however, exosomes isolated from the t cells, t-vector cells and huvec-siifitm cells did not contain detectable ifitm (fig. f ). purified exosomes were further characterized for the presence of conventional markers for exsosomes, namely, flotillin- and cd , and non-exosomal markers calnexin (endoplasmic reticulum marker) and gm (golgi matrix marker) using western blotting analysis. as shown in fig. f , exosomes collected from each group was shown to be positive for flotillin- and cd , but negative for calnexin and gm , verifying that there is no contamination by other subcellular fractions in the purified exosome preparations. the ifitm protein contained in exosome composition was further verified by mass spectrometry (fig. s ). together, our data demonstrate that secretion of ifitm in exosome is not limited to huvec that express high level of endogenous ifitm , but also by t cells undergoing ifn stimulation or transiently expressing exogenous ifitm . the finding that ifitm is contained in exosome and exported from cells in which it is expressed prompted us to investigate whether the ifitm -containing exosomes are internalized by other cells in the system so that the anti-denv activity can be transferred to the recipient cells. in this study, we tested such a possibility by incubating hela cells, which express a low-level of ifitm , with exosomes collected from huvec, huvec-sinc, huvec-siifitm , t-ifitm or t-ifn cells. indeed, as shown in fig. a , an increased abundance of ifitm was detected in the recipient hela cells treated with huvec-, t-ifitm cell-, or t-ifn cellderived exosomes ( μg ml − ), but not with those from t-vector cells. moreover, detection of ifitm proteins in the lysates of recipient hela cells treated with ifitm -laden exosomes derived from t-ifitm and huvec cells, as determined by immunoblotting, was dose-and time-dependent ( fig. c and d) . notably, as it was reported that exosomes might be capable of delivering exogenous small rna such as sirna and microrna in vitro and in vivo (van den boorn et al., ; pan et al., ; shtam et al., ) , in this experiment we preformed the protein transfer assay in recipient hela cells transfected with ifitm -sirna (hela-siifitm ) to rule out the potential side-effect of ifitm -sirna possibly delivered by exosomes on the endogenous or exosome-delivered ifitm mrna in the recipient cells, furthermore, as shown in fig. b and d, increased abundance of ifitm was detected in the recipient hela-siifitm cells treated with huvec-sincderived exosomes in a time-dependent manner, but not in those treated with huvec-siifitm cell-derived exosomes. consistent with the western blotting data, immunofluorescence staining and confocal microscopic analysis of ifitm -flag showed that the localization in the cytoplasm in the recipient hela cells was significantly higher following treatment with exosomes derived from t-ifitm cells (fig. e ). in contrast, our results showed that t-ifitm cell-derived exosomes did not change ifitm and ifitm expression in the recipient hela cells, when compared with treatments of t cell-or t-vector cells-derived exosomes (fig. s ) . moreover, when the exosome treatments were terminated at h, further incubation with blank culture media for a total length of or h did not increase the amount of endogenous ifitm within the recipient hela cells (fig. f) , suggesting that the exosome treatment procedure per se does not change endogenous ifitm expression in the recipient hela cells. in addition, in order to verify that the increased ifitm protein level in the transfer recipient cells was due to the exosomal transfer of ifitm , rather than a stimulated expression of endogenous ifitm , we measured ifitm mrna levels in cells treated with ifitm -exosomes using quantitative real-time rt-pcr, and we found that ifitm -exosome treatment did not induce an increase of endogenous ifitm mrna in hela cells (fig. g) . furthermore, to evaluate the half-life of ifitm in cell culture supernatant, we quantified ifitm protein in the supernatant of cultured huvec at various time points ( , , , , , , and h) using western blotting. specifically, supernatants derived from huvec cells were collected and centrifuged at g for min, and then further centrifuged at g for min at °c to remove cells and cell debris, followed by filtration with . μm filters. the obtained supernatants were then concentrated by -fold using an amicon ultra- centrifugal filter unit with kda cut-off value, divided into groups, and incubated at °c in a humidified atmosphere of % co for , , , , , , and h, respectively, before being subjected to western blotting analysis using an anti-ifitm antibody. as shown in the fig. s , there was no or little change of the ifitm protein in the inter-cellular transfer of ifitm -containing exosome. hela cells were incubated with purified μg ml − exosomes derived from supernatants of cultured huvec, huvec-sinc, t-ifitm or t-ifn cells in serum-free media for h (a), incubated with increasing amounts of exosomes derived from t-ifitm cells for h (c), incubated with μg ml − ifitm -exosomes derived from t-ifitm cells or huvec for h, h, h and h (d), or incubated with μg ml − ifitm -exosomes derived from t-ifitm cells for h, followed by washing exosomes after h treatment (f). '*' indicates that hela cells were maintained in dmem after treatment with ifitm -exosomes for h. hela cells with ifitm silenced (hela-siifitm ) were incubated with μg ml − exosomes derived from huvec, huvec-sinc and huvec-siifitm (b). cell lysates were analysed by immunoblotting for ifitm or ifitm -flag proteins. to investigate whether that ifitm -laden exosome internalized into recipient cells could confer resistance to denv- infection, hela cells were cultured in serum-free conditions and exposed to increasing amounts of purified ifitm -containing exosomes for h. purified ifitm containing exosomes were used to treat hela cells that were simultaneously infected with denv- (moi = ). at h post infection when the infected cells were examined by real-time rt-pcr assay, strikingly, denv- infection was suppressed potently by the ifitm -containing exosomes in a dose-dependent manner in parental hela cells (fig. a ) or in hela cells with endogenous ifitm knocked down (fig. c) , and the copy number of denv- viral rna was reduced in the culture supernatant ( fig. b and d), indicating that the observed antiviral effect was mediated by the exosome-transferred ifitm . we also performed immunofluorescence analysis of infected hela cells treated with, or without, ifitm -exosomes, by staining for delivered ifitm -flag and a denv protein e. as shown in fig. s , our results showed that denv protein e are present only in some cells that do not contain ifitm -flag. the data from the cpe reduction assay demonstrated that ifitm -laden exosomes also attenuated cpe triggered by denv- infection (fig. e) . moreover, our results showed no effect of ifitm -exosomes on the binding of denv- to host cells or post-entry steps during denv- infection, but the penetration of denv- into cells was significantly inhibited by the ifitm -containing exosomes, with a reduction of approximately fourfold (fig. b ). in contrast, the control exosomes prepared from t cells that contained only the vector plasmid without the ifitm expression cassette did not exhibit any anti-denv activity, suggesting that ifitm was the main exosomal component responsible for the detected antiviral activity of exosome. to verify that the enhanced antiviral effects caused by ifitm -exosome transfer was a result of ifitm action rather than a stimulated paracrine ifns response, the concentration of human ifn-α/β in culture supernatant of hela cells or hela-siifitm cells treated with ifitm laden exosomes was measured with elisa by using sendai virus (sev; hau ml − ) as a positive stimulus control. our results showed that ifitm -exosomes treatment did not change ifn-α and -β expression, as compared with the control-exosomes ( fig. f and g) , implicating that reduction of virus replication was not due to a non-specific effect of ifitm -containing exosomes mediated by interferon. ifns trigger a potent antiviral response that activates host innate immunity and leads to establishment of a cellular antiviral state, which is mediated by inducing expression of isgs. accordingly, isgs represent a key line of defence against viral infection, and ifitms are among the most potent isg products with broad anti-viral spectra. a key finding of our current study, namely, that ifitm can be transferred intercellularly via extracellular exosome, provides new insights in the functional significance of ifitm as an ifn-induced anti-viral protein. the discovery of the roles of free ifitm proteins and ifitm-laden exosome as innate cellular defenders present an opportunity developing exosome-based tools to actively combat existing or emerging pathogens. conceivably, variations in the basal and inducible levels of ifitms, as well as in intracellular and extracellular levels of ifitms, might predict the severity of dengue infections among individuals or across species. exosome is secreted from many types of cells and remain stable in various body environments. by transferring proteins, mrnas and micrornas to neighbouring or distant cells, exosome contribute to modulation of important physiological or pathological processes such as immunity, angiogenesis, cell proliferation, cell migration and invasion, and cell-to-cell signalling (schorey and bhatnagar, ) . here, we propose a novel model for ifn-inducible innate immune response against denv- infection mediated by ifitm-containing exosome. noteworthy in this context is that exosome released from ifntreated cells may remotely communicate with other cells through such a mechanism that the anti-viral phenotype can be transferred to the exosome recipient cell that might not have the chance to be directly stimulated by ifn. interestingly, this inter-cellular communication model might also be applicable to explain a pioneering observation on the antiproliferative activity transmitted from ifnstimulated cells to non-stimulated cells (lloyd et al., ) . at the subcellular level, following ifn induction of ifitm expression, ifitm is transferred to the multivesicular complexes and assembled into exosomal vesicles. upon transport to the surface of a recipient cell, fusion of mvcs with the plasma membrane allows assembly of ifitm with a pre-existing exosome receptor complex that is unknown thus far. potentially, the released exosome particles containing ifitm protein can travel to non-stimulated cells where receptor assembly also occurs. such a model is consistent with the finding that the antiviral state can be transferred from ifns induced cells to non-treated cells in culture. hypothetically, this process might require ifitm proteins to be processed or relocalized in the host cells by an unknown mechanism so that it can be targeted for fig. . the antiviral activity of ifitm -containing exosome. after h incubation with ifitm -containing exosomes, hela cells (parental) or hela cells with ifitm silenced were incubated with denv- virus for h. intracellular (a and c) and extracellular (in supernatant) (b and d) viral rna was measured by real-time rt-pcr assay, and the results are presented as relative ratios of total cellular rna. (e) ifitm -containing exosome suppressed cpe triggered by denv- infection. intracellular viral rna was measured in a real-time rt-pcr assay, and the results are expressed as relative fold of total cellular rna. conditioned media obtained from exosomes derived from each group of cells treated as indicated were quantitatively measured for contained ifn-α (e) and ifn-β (f) proteins by elisa, using sendai virus (sev; hau ml − ) as a positive stimulus control. data points are presented as means ± sd of triplicated experiments. student's two-tailed t test was performed, and statistical significance is shown with asterisks (*p < . , **p < . ). exosome secretion. interestingly, it has been reported that cytosolic proteins present with exosome including members of the rab family, are involved in the formation and secretion of exosome. rab b, , and gdi are found to promote exosome docking and the membrane fusion events (thery et al., ) . interesting, feeley et al. reported that ifitm primarily resides in the late endosomal compartment and partly colocalizes with rab and lamp (feeley et al., ) . in the context, therefore, it would be of great interest to further investigate the biogenesis of ifitm exosome as well as its regulation, which might provide important clues for a rationale development of optimal anti-viral strategies. while application of ifns has been highly appreciated in the clinic, problems associated with the use of ifns, such as suboptimal efficacies against many viruses and varied degrees of adverse effects caused by ifn treatment, remain unsolved (borden et al., ) . in the intensive efforts of identifying and developing new endogenous anti-viral factors with potential clinical applicability, isgs have been attractive candidate agents for therapeutic purposes. in such a context, exosome-carried ifitm , as discovered by our present study, appears to represent a novel, attractive potential anti-viral strategy, as both natural and specially engineered exosome can serve as nano-shuttle vehicles for drug delivery. animal studies to test the feasibility and efficacy of applying exosomebased delivery of ifitm for anti-denv purposes in vivo are therefore warranted. our findings provided the first set of evidence for the existence of an extracellular, exosome-packaged form of ifitm , which enables anti-viral activities to transmit from one cell to another, leading to efficient establishment of an anti-viral state. further studies to decipher the structural and cell biological basis for the antiviral activities of ifitmexosome will be of theoretical as well as practical importance in this area of research. thp- , a , huh , t, u , hepg , hela and bhk- cells were maintained in dulbecco's modified eagle's medium (dmem; invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs; hyclone, logan, ut), mm lglutamine, μg ml − streptomycin and units ml − penicillin (invitrogen). human umbilical endothelial cells (huvec) were grown in human umbilical endothelial cell serum-free medium (invitrogen), supplemented with g ml − endothelial cell growth supplements (upstate, billerica, ma, usa) . the cultures were maintained at °c in a humidified atmosphere of % co . c / aedes albopictus cells were cultured at °c and % co in dmem supplemented with % fbs, u ml − penicillin and mg ml − streptomycin. the denv- strain ngc (new guinea c, genbank accession number m ) was kindly provided by guangzhou centers for disease control and prevention (cdc) (tian et al., ; wu et al., ) and was propagated in the c / cell line. briefly, a monolayer of c / cells was infected with denv- at an moi of , and incubated at °c and % co for days. the supernatant was harvested and centrifuged for min at g to remove pelleted cellular debris. denv- titres were determined by facs assays in c / cells, as previously described (lambeth et al., ) . cells were harvested and lysed in × sampling buffer containing mm tris-hcl (ph . ), mm pmsf, % glycerol, % sds, % mercaptoethanol and . % bromophenol blue before sonication. the protein concentration of the lysate was determined using the bicinchoninic acid protein assay kit (thermo fisher scientific, rockford, il) according to the manufacturer's instructions with bsa as the standard. protein molecular weight standards (genstar biosolutions co. ltd, beijing, china) were used to determine molecular weights of sample proteins. an aliquot of the cell lysates containing μg of protein was subjected to sds-page, and then transferred to pvdf membranes. following a blocking step using the blocking buffer (tris-buffered saline, tbs, containing % non-fat milk) for h at room temperature, the membranes were incubated overnight at °c with the following specific primary antibodies: monoclonal anti-ifitm , monoclonal anti-ifitm , polyclonal anti-ifitm (proteintech group, chicago, il), monoclonal anti-flag (sigma-aldrich, st louis, mo), d - (anti-denv- e mouse monoclonal antibody; santa cruz biotechnology, santa cruz, ca), monoclonal anti-actin (sigma-aldrich), polyclonal anti-cd (h- ; santa cruz biotechnology), anti-flotillin (c-terminal; sigma-aldrich), anticalnexin (proteintech group), and monoclonal anti-gm (cell signaling, danvers, ma) antibodies. further incubation with appropriate horseradish peroxidase (hrp)-conjugated secondary antibodies, depending on the primary antibody used, was performed for h at room temperature. membranes were washed three times in tris-buffered saline containing . % tween- for min after each incubation step. the bands were detected using enhanced chemiluminescence kit (thermo fisher scientific) with a kodak film. actin was used as a loading control for quantification normalization. intensities of the bands of interest on the pvdf membranes were quantitatively calculated with the quantity one . . measurement software (bio-rad, hercules, ca). human ifitm , , cdna with a c-terminal ha epitope tag were amplified by rt-pcr using primers shown in table s . the resultant pcr fragments were cloned into the pmscv-puro vector (clontech laboratories, inc., mountain view, ca) at bglii and ecori sites to generate plasmids pmscv-ifitm -ha, pmscv-ifitm -ha and pmscv-ifitm -ha respectively. the cloned ifitm , , cdnas were sequencing verified. a blank pmscv-puro vector was generally used as a control. these constructs were used to establish stably transduced cell lines with u and hela cells to permanently express ifitms. briefly, retroviral particles were prepared by transfecting μg of each of the pmscv-puro-ifitm -ha, pmscv-puro-ifitm -ha and pmscv-puro-ifitm -ha plasmid dna into the packaging cells t, together with μg of the pik plasmid. recombinant retroviral particles were used to infect u or hela cells, and stably transduced cell lines were selected in dmem medium supplemented with puromycin ( μg ml − ). to transiently express ifitms protein, ifitms genes were separately subcloned into the pcdna vector (invitrogen) at kpni and bamhi sites and a flagtag sequence was introduced into the coding sequence as the c-terminus. t cells were transiently transfected with the resultant plasmid pcdna-ifitms-flag by a standard calcium phosphate co-precipitation method. hela cells were transiently transfected using lipofectamine (invitrogen) by following the manufacturer's instruction. the small interfering rna (sirna) duplexes against ifitm , whose sequences are provided in table s ) were published previously (brass et al., ) , and were purchased from ribobio inc. (guangzhou, guangdong, china). huvec and hela cells were transfected with sirnas at indicated concentrations, using lipofectamine reagent (invitrogen) following the manufacturer's instructions, followed by treatment with ifn-α ( , , u ml − ) and infection with denv- at an moi of . to assess the expression of ifitm proteins, cell lysates were harvested at h after transfection and examined by western blotting analysis with corresponding specific antibodies. dengue e protein was detected in cells infected with denv- by intracellular staining and flow cytometric (fcm) analysis, as previously described (brass et al., ; chan et al., ) . briefly, cells in growth dmem medium were seeded and subsequently cultured for h, followed by infection with denv- at an moi of . cells harvested at indicated time points were washed with cold × phosphate-buffered saline (pbs) and fixed with % paraformaldehyde at °c for min. fixed cells were scraped and then permeabilized in % methanol before being incubated with nyrdeng (anti-denv- e mouse monoclonal antibody; santa cruz biotechnology), and further with appropriate fitcconjugated goat anti-mouse igg secondary antibody (santa cruz biotechnology). the fitc-positive cells were acquired and scored by flow cytometry (becton-dickinson counter, san jose, ca). the percentage of denv-infected cells was defined by fitc-positive cells distribution in a fluorescence dot plot using the cytometry list mode data acquisition & analysis software (becton-dickinson counter) . denv in cell supernatants were quantified by determining the copy number of viral rna using quantitative real-time rt-pcr as previously described (mota and rico-hesse, ). cell supernatants were harvested at indicated time points, and total rna was extracted using qiaamp minelute virus spin kit by following the instruction of the manufacturer (qiagen, chatsworth, ca). cdna was synthesized, and quantitative real-time pcr was performed with standard curve analysis using a cfx real-time pcr detection system (bio-rad). for relative quantification, results are presented as ratios relative to the quantification for gapdh. primer pairs used are shown in table s . to estimate denv rna copy number, a standard curve was generated using in vitro-transcribed rna standards, which were produced as follows. a bp fragment of the denv- ngc strain was amplified by rt-pcr and cloned into the pmd -t simple vector (takara, dalian, china). the cloning plasmid was linearized with ecorv, and rna transcripts were generated with a t megascript kit (ambion) according to the manufacturer's instructions. concentration of transcribed rna was determined with the nanodrop c spectrophotometer (thermo scientific, rockford, il), and -fold serial dilutions were prepared and used to construct a standard curve. the binding and entry experiments were performed as described previously (kanlaya et al., ; weidner et al., ) . briefly, for virus binding experiment, cells were seeded in -well plates at a density of × cells per well and cultured for h, followed by inoculation with denv- at moi of and incubation on ice for h to allow binding but impede cell entry. such a multiplicity of infection ensures that at least % of cells were infected with a minimum of one infectious viral particle. unbound virus was removed and cells were harvested to determine the amount of viral rna accumulation by quantitative rt-pcr min later. to assess denv- virus entry into cells via endocytosis, after binding on ice for h, virus inocula were removed after h of binding on ice, and infected cells were washed with × pbs followed by incubation with pre-warmed dmem for min at °c to initiate virus cell entry. subsequently, cells were rinsed three times with × pbs and then treated with . % trypsin for min, and again washed three times with × pbs to remove any cell-associated virus that did not enter the cytoplasm. for assessments of post-entry infection steps, after virus binding and entry procedures were finished, hela cells were subsequently treated with ifitm -containing or control exosomes. total cellular rna was extracted to measure the quantity of viral genomes that had entered cells by using a real-time rt-pcr assay. seven groups of cells, including huvec (with high basal-level ifitm protein), non-targeting control sirna-transfected huvec (huvec-sinc), ifitm sirna- -transfected huvec (huvec-siifitm ), ifitm -transfected t cells ( t-ifitm ), control vector-transfected t cells ( t-vector), ifn-α-treated t cells ( u ml − ; t-ifn) and t cells, were cultured in medium supplemented with % exosome-free fbs (overnight centrifugation, g), and grown to monolayer with - % confluence. exosomes were isolated from cell supernatants by filtration steps and differential ultracentrifugation as previously described (khatua et al., ) . briefly, supernatants derived from huvec and t cell culture with different treatments were collected and centrifuged at g for min, and then g for min at °c to remove cells and cell debris, followed by filtration with . μm filters (pall life sciences, port washington, ny) and ultracentrifugation at g for h at °c to pellet crude exosomes. for further purification, the crude exosomes were mixed with ml of . m sucrose in pbs and placed on the bottom of a optiseal centrifuge tube (beckman coulter inc., munich, germany), overlaid with ml m sucrose and ml . m sucrose, and ultracentrifuged for h at g. the purified exosomes accumulating at the m/ . m interface were collected, washed twice by diluted in pbs, and pelleted by ultracentrifugation at g for min. pelleted exosomes were re-suspended in pbs and used immediately or kept at − °c. gw (sigma-aldrich) was employed as an inhibitor of exosome secretion. protein concentrations of exosome preparations were determined with the bicinchoninic acid protein assay kit (thermo fisher scientific) according to the manufacturer's instructions, using bsa as the standard. finally, the ifitm -laden exosome samples were characterized by western blotting analysis, and the protein band was excised from the gel and subjected to mass spectrometry (ms) analysis according to a standard procedure (khatua et al., ). purified exosomes were processed by negative staining and analysed by transmission electron microscopy (tem) as previously described (khatua et al., ) . briefly, exosomes resuspended in μl pbs were dropped onto a sheet of parafilm, and a formvar-carbon coated nickel grid were floated for min at room temperature to absorb exosomes, followed by wash with pbs twice and fixed with % paraformaldehyde after rinsing with pbs twice, the grids were negatively stained with % phosphotungstic acid for min. finally, dry samples were viewed using a tecnai transmission electron microscope at kv or stored in a grid box for future work. cells were seeded in -well plates at × cells per well h prior to viral infection or treatments described below. to evaluate the anti-denv ability of purified ifitm -laden exosomes, and μg ml − of ifitm -exosomes, respectively, was added to cultured cells and incubated for h. subsequently, mixture of ngc denv- (moi = ) and or μg ml − of ifitm -exosomes, respectively, which had been pre-incubated for h at °c, was added to cells. after three washes with pbs to remove the unbound virus, ifitm -exosomes was added to cells and incubated for the indicated lengths of time ( , , or h). the control-exosomes was prepared from t cells that contained only the vector plasmid without the ifitm expression cassette. ifn-α and ifn-β was quantified in the supernatants derived from cells treated with ifitm -exosomes, by using enzyme linked immuno-sorbent assay (elisa) kits, according to the manufacturer's instruction (keygen biotech, china; pbl interferon source, piscataway, nj) respectively. following a pre-lysis treatment with trypsin to remove adherent exosomes, infected cells and culture medium were harvested at various times after infection and subjected to rna extraction. the amounts of dengue viral rna were measured by using a quantitative or relative real-time rt-pcr assay. for cpe reduction assay, at h post infection, microscopic examination was performed to determine the antiviral effect, and the data were confirmed using a cell viability assay. the viability of bhk- cells was measured by using the mts [ -( , -dimethylthiazol- -yl)- -( -carboxymethoxyphenyl)- -( -sulfophenyl)- h-tetrazolium] assay according to the manufacturer's instructions. briefly, μl mts solution (celltiter aqueous one solution reagent, promega, madison, wi) was added to each well and incubated for an additional h at °c, and the absorbance was subsequently measured at nm using a microplate reader (bio-tek, winooski, vt). cells treated with control-exosome were used as the control group. cell growth inhibition rates were determined using the following formula according to a previously published method (muhamad et al., ) : cell viability (%) = (od of treated cells/od of control cells) × %. the experiment was repeated at least three times from which mean and standard deviation (sd) values were calculated. the data given in the text were presented as means ± sd. comparison between two groups was evaluated by the -tailed student's t test, using . as the cut-off p-value. correlations were determined with the exponential regression analysis using the ibm spss statistics software. additional supporting information may be found in the online version of this article at the publisher's web-site: fig. s . the ifitm protein family restricts denv- infection at entry. u (a) or hela (c) cells were transduced to express ifitm , or protein or control vector alone, as verified by western blotting analysis. ifitm protein expression in t cells was measured by western blotting analysis, using aliquots of the same cells assayed in (e) respectively. actin was included as a loading control. denv- -infected cells were detected using a monoclonal antibody (nyrdeng ) directed to the envelope (e) glycoprotein of the virus, and the numbers of denv- -infected u (b), hela (d) or t (f) cells were determined by flow cytometry. fig. s . ifitm is required for the antiviral effects of interferons and silencing ifitm enhances denv- infection. (a) huvec cells transfected with indicated sirnas, or a nontargeting control sirna (sinc), were assessed for ifitm levels by western blotting analysis. at h post transfection with indicated sirna, the percentage of infected cells was assessed h after virus addition by flow cytometry for e protein (b). hela cells were transfected with the indicated sirnas, or a control, non-targeting sirna. at h post transfection, cells were incubated with , or iu ml − ifn-α for h, and the levels of ifitm were examined by western blotting (c). (d) hela cells transfected with indicated ifitm -trageting sirnas were incubated with the indicated concentrations of ifn-α for h at h post transfection, followed by infected with denv- at an moi of . the percentages of infected cells were assessed h after virus addition by flow cytometry for the denv e protein. e, effect of sirna silencing of ifitm on the binding and entry of denv- to hela cells. data points are presented as means ± sd of triplicated experiments. student's two-tailed t test was performed, and statistical differences are shown with asterisks (**p < . ). fig. s . mass spectrometric analysis of ifitm peptides. results automatically generated by the mass spectrometer. briefly, purified ifitm -containing exosomes derived from t cells overexpressing ifitm were assayed by massspectrometric analysis of proteolytic peptides. the red vertical lines represent peptide ion intensities, and the horizontal axis shows the mass-to-charge (m/z) values around the peptide ions of interest. the green dash lines were automatically given by the spectrometer for pointing the labels to the red peaks. fig. s . t-ifitm cell-derived exosomes do not change the quantities of ifitm and ifitm in the recipient hela cells, as compared with the t cell-and t-vector cell-derived exosomes. fig. s . ifitm half-life in the cell culture supernatant. the levels of ifitm protein in cell culture supernatant of huvec at various times ( , , , , , , and h) was assayed with western blotting. briefly, supernatants derived from huvec cells were collected and centrifuged at g for min, and then further centrifuged at g for min at °c to remove cells and cell debris, followed by filtration with . μm filters. the obtained supernatants were then concentrated by -fold using an amicon ultra- centrifugal filter unit with kda cut-off value, divided into groups, and incubated at °c in a humidified atmosphere of % co for , , , , , , and h, respectively, before being subjected to western blotting analysis using an anti-ifitm antibody. intensities of the bands of interest on the pvdf membranes were quantitatively calculated with the quantity one . . measurement software. the results are presented as relative ratios of intensity of the band for the h treatment group. fig. s . immunofluorescence staining and confocal microscopic analysis of ifitm -flag and denv- viral e protein in hela cells treated with t-ifitm -exosome followed by infection with denv- . table s . primer sequences and sirna oligonucleatides. exosomes released from infected macrophages contain mycobacterium avium glycopeptidolipids and are proinflammatory exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo interferons at age : past, current and future impact on biomedicine the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus ifitm proteins restrict antibody-dependent enhancement of dengue virus infection ifitm restricts the morbidity and mortality associated with influenza ifitm inhibits influenza a virus infection by preventing cytosolic entry the trojan exosome hypothesis dengue: a continuing global threat more dengue, more questions the burden of dengue infection distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus capture and transfer of hiv- particles by mature dendritic cells converges with the exosome-dissemination pathway identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein and is important for viral replication and release exosomes packaging apobec g confer human immunodeficiency virus resistance to recipient cells flow cytometry-based assay for titrating dengue virus exosomes mediate the cell-to-cell transmission of ifn-alpha-induced antiviral activity cell-tocell transfer of interferon-induced antiproliferative activity the ifitm proteins inhibit hiv- infection humanized mice show clinical signs of dengue fever according to infecting virus genotype antiviral actions of flavanoid-derived compounds on dengue virus type- hepatic cell-tocell transmission of small silencing rna can extend the therapeutic reach of rna interference (rnai) interferon-inducible antiviral effectors exosome function: from tumor immunology to pathogen biology exosomes are natural carriers of exogenous sirna to human cells in vitro exosomes: composition, biogenesis and function identification and immunogenicity of two new hla-a* -restricted cd + t-cell epitopes on dengue ns protein interferon-induced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms ) mir- a facilitates replication of dengue virus by dampening interferon induction by targeting traf identification of the ifitm gene as an inhibitor of hepatitis c viral translation in a stable stat cell line we owe our special thanks to professor xi huang of sun yat-sen university for providing materials essential for the study. key: cord- -ktl jw v authors: coccia, eliana m.; battistini, angela title: early ifn type i response: learning from microbial evasion strategies date: - - journal: seminars in immunology doi: . /j.smim. . . sha: doc_id: cord_uid: ktl jw v abstract type i interferon (ifn) comprises a class of cytokines first discovered more than years ago and initially characterized for their ability to interfere with viral replication and restrict locally viral propagation. as such, their induction downstream of germ-line encoded pattern recognition receptors (prrs) upon recognition of pathogen-associated molecular patterns (pamps) is a hallmark of the host antiviral response. the acknowledgment that several pamps, not just of viral origin, may induce ifn, pinpoints at these molecules as a first line of host defense against a number of invading pathogens. acting in both autocrine and paracrine manner, ifn interferes with viral replication by inducing hundreds of different ifn-stimulated genes with both direct anti-pathogenic as well as immunomodulatory activities, therefore functioning as a bridge between innate and adaptive immunity. on the other hand an inverse interference to escape the ifn system is largely exploited by pathogens through a number of tactics and tricks aimed at evading, inhibiting or manipulating the ifn pathway, that result in progression of infection or establishment of chronic disease. in this review we discuss the interplay between the ifn system and some selected clinically important and challenging viruses and bacteria, highlighting the wide array of pathogen-triggered molecular mechanisms involved in evasion strategies. the ability of the host to respond to invading pathogens relies on the activation of the innate immune system that orchestrates adaptive immune responses for pathogen clearance. in recent years, our understanding of the mechanisms involved in the activation of the innate response has evolved significantly with the identification and characterization of the mammalian system of pathogen recognition. the innate immune system detects the presence of a pathogen through a set of germline-encoded membrane-associated or cytoplasmic receptors, termed pattern recognition receptors (prrs) that are engaged by microbial-derived products named pathogenassociated molecular patterns (pamps). major classes of prr include toll-like receptors (tlrs), nucleotide-binding oligomerization domain (nod)-like receptors (nlr), c-type lectin receptors (clrs), retinoic-acid inducible gene (rig)-i-like receptors (rlrs) and a growing list of cytosolic dna-sensing receptors [ ] [ ] [ ] [ ] [ ] [ ] [ ] . upon engagement, these receptors recruit a number of adaptor proteins to signal downstream and activate three major pathways: the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-b), the mitogen-activated protein kinases (mapks) and the ifn regulatory factor (irf) pathway [ , ] . downstream tlrs and rlrs, type i ifn and pro-inflammatory cytokines are mainly produced, while nlrs predominantly activate inflammasomes, resulting in the release of il- and multiple inflammatory cytokines (fig. ) . based on the structure of their receptors, interferons are broadly classified into three groups, type i, ii and iii ifns. type i ifn comprises the largest ifn class that includes ifn-␣, constituted by several partially homologous genes and ifn-␤, ifn-, ifn-, ifnrepresented by a single gene. the ifn-␣ and ifn-␤ are the best characterized as antiviral and the most broadly expressed [ ] [ ] [ ] and will be referred as ifn-i from now on. the ifn-␥, the only type ii ifn, is released by activated t and nk cells; type iii ifns, which include ifn- - , similarly to ifn-i are believed to regulate the antiviral response [ ] . according to the most common view, ifn-i exerts primarily an anti-viral action while ifn-ii acts predominantly on macrophages to induce a microbicidal state against ingested intracellular, non-viral pathogens. anti-microbial ifn-i activity is not intrinsic, but mediated, in both autocrine and paracrine manner, by a unique set of induced genes named ifn-stimulated genes (isgs) [ , , ] . secreted ifn-i, indeed, binds to a common heterodimeric ubiquitously expressed receptor composed of ifnar and ifnar chains, and initiates a signaling cascade that has been characterized in detail and reviewed elsewhere [ ] [ ] [ ] . briefly, canonical ifn-i pathway results in activation of the janus kinase (jak) family (cytoplasmic tyrosine kinases) that, in turn, activate by phosphorylation the signal transducers and activators of transcription (stats) [ ] . activated stats complex with irf to form the heterocomplex ifn-stimulated gene factor (isgf ), that translocates in the nucleus, binds to upstream sequence elements named ifn-stimulated response elements (isre), and activates the transcription of isgs (fig. ) . these genes act to promote viral clearance and establish an antiviral state in uninfected bystander cells, or to induce apoptosis and several anti-microbial mechanisms in infected cells. they also stimulate cells at the interface of innate and adaptive immunity, such as macrophages and dendritic cells (dcs) that trigger the adaptive response [ , ] . the ability to break in innate immunity before the onset of the adaptive response is, thus, crucial for the survival of virtually all mammalian pathogens. on the other side of the coin, an essential part of the early response to pathogens is aimed at limiting their ability to hijack host cellular machinery and evade the ifnmediated antimicrobial mechanisms. nowadays, while it is largely accepted that also bacteria can induce the production of ifn-i, the role of the ifn system in the pathogenesis of bacterial infections can be either detrimental (mainly in intracellular bacterial infections) or protective (mainly in extracellular bacterial infections). the route and tropism of bacterial infection, viral co-infections and last, but not least, the balance between ifn-i and ifn-ii effects are all determinants of the different outcome [ ] . the ratio between ifn-i and ii species produced in response to infection, might differ as consequence of the capacity of the pathogen to stimulate specific cell types in the infected tissues. moreover, taking into consideration that several antibacterial ifn-ii-induced genes are stimulated to a much lesser degree by ifn-i, it is quite difficult in vivo to separate the activities strictly dependent on one of the two ifn families. a more reliable view consists of a crosstalk between the two pathways: if the bacterium is able to stimulate ifn-i production, generally it occurs early after the infection as an immediate innate immune response, while ifn-ii intervenes later on when immune cells (such as t cell subsets and nk cells) are activated. also due to this complex picture, while a number of viral evasion strategies have been mechanistically defined so far, studies aimed at characterizing the bacterial components that inhibit the ifn system are only recently starting to be elucidated in molecular details [ ] [ ] [ ] . main strategies that pathogens have evolved to disarm the ifn-i response include: (i) blocking ifn-i production by modifying, curtailing or limiting production of their pamps to make them inaccessible to prrs and/or hitting the components of prrs signaling pathways; (ii) interfering with the ifn-i signaling by inhibiting signal transducers; (iii) blocking or disturbing the action of isgs; (iv) hijacking host proteins or components of the ifn system. each of these strategies involves a number of different molecular mechanisms and the combination of more strategies may be necessary to overcome the ifn-i response by a single pathogen. depending on the nature of the pathogen, these countermeasures that tip the balance toward the pathogen, may result in increased replication or in the establishment of a persistent infection. leaving out strategies that envision ifn-i repression due to a general inhibition of cellular gene expression, reviewed elsewhere [ , ] , here we summarize recent findings on evasion tricks utilized by pathogens to specifically subvert the ifn system. a special focus is put on few highly pathogenic bacteria and emerging or re-emerging viruses, which represent a major threat to human health due to the lack of effective vaccines and/or therapeutics. for an in-depth coverage of other pathogens and strategies to escape the host immune response, the reader is referred to more comprehensive and specific reviews [ ] [ ] [ ] [ ] [ ] [ ] [ ] . recent years have seen major advances in our understanding of the innate response to infectious pathogens and specifically of how pathogen recognition promotes ifn-i release [ , , ] . cytoplasmic and membrane-associated prrs recognize a variety of pathogen components. viral pamps mainly consist of nucleic acids originating from the uncoating of infecting virions, the transcription of viral genomes and the replication of genomic intermediates, in the form of single-stranded (ss) and double-stranded (ds) dna, and ss and ds rna, as well as viral glycoproteins. bacterial pamps include various molecules, ranging from lipoproteins, lipopolysaccharide (lps), flagellin and peptidoglycan to unique bacterial nucleic acid structures, such as cyclic dinucleotides (cdns). multiple endosome-associated tlrs (tlr , , and ) are specialized in the detection of viral and bacterial nucleic acids. tlr recognizes dsrna, tlr / are bound by ssrna and tlr by cpgcontaining dna. tlr , and , previously considered sensors only for bacterial components, are now been involved also in recognition of viral ligands and in the induction of ifn-i [ ] . all tlrs contain an intracellular domain, the toll-interleukin- receptor (tir), which recruits one or more tir-containing adaptor proteins to transmit signals downstream. tlr signals through the adaptor tir domaincontaining adaptor inducing ifn-␤ (trif) to activate the two related kinases, inhibitor of kb kinase (ikk)-and tank-binding kinase (tbk ) that mediates activation by phosphorylation of irf and irf . tlr / and tlr use, as adaptor, myeloid differentiation primary-response protein (myd ) that then initiates signaling cascades involving il- r-associated kinases (irak - ) and tnfrassociated factor (traf) / proteins, which finally converge at the activation of the ib kinase (ikk) family members ikk-␣, ikk-␤, ikk-and tbk responsible for activation of nf-b and irf / [ ] . in the cytoplasm, two closely related helicases, retinoic acidinducible gene (rig-i) and melanoma differentiation-associated gene- (mda ), recognize dsrna of many replicating viruses in a tlr-independent manner. rig-i preferentially senses short dsrna and ssrna with a -triphosphate ( -ppp rna), while mda recognizes long dsrna and poly i:c [ ] [ ] [ ] . like viral rnas, bacterial rnas can possess -ppp termini and secondary structures that make them rig-i agonists. consistent with this notion, rig-i can act as a sensor of bacterial rna and may help maintain homeostasis to gut microbiota [ , ] . upon recognition of non-self rna, rig-i and mda are recruited to the mitochondrial antiviral signaling protein (mavs; also known as cardif, visa or ips ), which triggers a signaling cascade that leads to the activation of ikk-and tbk and, in turn, irf / phosphorylation [ ] . stimulator of ifn genes (sting), initially identified as a cytosolic dna sensor, also participates in the rig-i signalling [ ] . sting interacts with the adaptor mavs at the mitochondrial associated membranes (mam) facilitating the recruitment of tbk and the activation of irf [ ] . a more general role of sting in innate immune responses, not only limited to its function as adaptor of rna and dna sensors, has been now established [ ] . in addition to these rna sensors, viral rnas may also be recognized by effector molecules that are themselves ifn-induced proteins with antiviral functions. these molecules include dsrnaactivated protein kinase (pkr) that binds and is activated by dsrna from viruses and bacteria [ ] , ifn-induced tetratricopeptide repeat proteins / / (ifit , ifit , and ifit ) that, as rig-i, bind -ppp rna and may recognize viral mrnas that lack -omethylation [ , ] . a growing list of cytosolic dna sensors then recognizes dna from different sources including viruses, bacteria and apoptotic cells [ , , ] . more than ten dna cytosolic receptors have been proposed so far that include dai (dna-dependent activator of irf), rna polymerase-iii, ifn-inducible interferon gamma-interferoninducible protein (ifi ), sting, extrachromosomal histone h b, leucine rich repeat (in flii) interacting protein (lrrfip ), ku , deah box protein (dhx) and dhx , cyclic gmp-amp synthase (c-gas). as other sensors, dna sensor activation results in the production of ifn-i and proinflammatory cytokines and chemokines via the sting-tbk -irf axis. in addition to the activation of irf -and nf-b-dependent signaling cascades, cytosolic dna can also promote an apoptosis-associated speck-like protein containing a card (asc)-dependent inflammasome-mediated response resulting in the secretion of proinflammatory cytokines [ , , , , ] . the intracellular nlrs scaffold large signaling complexes to mediate innate immunity and inflammatory responses. they may trigger the assembly of inflammasomes and modulate the nf-b, mapk and irf signaling pathways. in particular, nod and nod are important for immune detection of intracellular bacterial pathogens and are also involved in a variety of immune homeostatic functions [ ] . upon the recognition of bacterial peptidoglycans by nod and nod , receptor-interacting serine-threonine kinase (rip ) is activated via cellular inhibitors of apoptosis and (ciap and ), subsequently leading to ubiquitination of nf-b essential modulator (nemo) and the activation of the proinflammatory nf-b pathway. in parallel, the recognition of muramyl dipeptide present in all peptidoglycans, can also lead to the activation of mapk pathway via rip , which contributes to cytokine production. for the induction of ifn-i, nod activated by viral rna, signals through the mitochondrial mavs independently of rip . then, some sensors can aggregate with adaptors as apoptosisassociated speck-like protein containing a caspase recruitment domain (asc) and caspase to form multimeric structures named inflammasome [ ] (fig. ) . as first line strategy to face these sensing pathways, pathogens hide detection by modifying their pamps. they may also degrade/inactivate target key signal transduction hubs by counteracting host-induced post-translational modifications required for signaling molecule activity and use concomitant different strategies as better described below and also discussed by thomas kufer and igor brodsky in this issue. we focus here on some members of coronaviruses, flaviviruses, hepaciviruses, filoviruses and retroviruses as well as bacteria whose pamps could in principle activate the ifn system, but that efficiently betray host sensing and effector pathways (fig. ) . coronaviruses (cov) are large enveloped rna viruses, in the family coronaviridae, of both veterinary and clinical importance. two newly emerging viruses in the family, the severe acute respiratory syndrome cov (sars-cov) and the middle east respiratory syndrome cov (mers-cov), have been recently responsible for severe disease in humans (reviewed in [ ] [ ] [ ] [ ] [ ] ). a common trait in their pathogenesis is the lack of induction of a robust ifn-i response in infected cells [ , ] . to do this, cov have developed multiple strategies (recently reviewed in [ , ] ). rig-i, mda and the host isgs ifit and are critically involved in sensing of cov infection. however, as first line of hiding from recognition, cov encode several highly conserved nonstructural proteins (nsps) implicated in viral rna capping activity in order to examples of viral and bacterial antagonists that block subvert or exploit the ifn system. pathogens affect at every step the ifn system by multiple mechanisms. sites of intervention by several antagonists are indicated. ifn antagonists may prevent prr recognition by hiding or modifying pamps, may inhibit prr signaling by directly targeting adaptors and signaling effectors, may interfere with the ifn signaling by impairing signaling transducers and may block or disturb the action of isgs. some antagonists have more than one cellular target while others target common signaling molecules, effectively blocking ifn induction from a variety of pamps. see the text for more details and references. mimick n -and -o-methylated cap structure of cellular mrnas [ ] . these viral proteins include a rna-triphosphatase, a guanine-n -methyltransferase, and a -o-methyltransferase encoded by nsp , and , respectively [ , ] . consistently, human and mouse cov mutants lacking -o-methyltransferase activity induce higher expression of ifn-i and are highly sensitive to ifn-i effects [ , ] . sars-cov nsp also encode a , exoribonuclease that is involved in rna-proofreading, but probably it also functions in degrading viral pamps, further hiding immune detection [ , ] . similarly, nsp encodes a ribonuclease that may degrade viral pamps [ ] . another strategy used to avoid detection is the replication in protected sites as the double membrane vesicles (dmvs) that the virus induces in the host cytoplasm. in the case of sars-cov these membranes contain the replicase complex and the viral genomic rna suggesting that replication occurs in these sites and the generated nucleic acids are soon after shielded [ , ] . in addition, the highly basic nucleocapsid (n) protein of sars-cov has been reported to directly inhibit ifn-i production induced by both poly(i:c) or sendai virus, by a mechanism that involves steps upstream rig-i. thus, it is conceivable that by binding dsrna, n protein prevents rig-i/mda activation [ ] . others sars-covencoded proteins, as orf- b and orf , may also interfere with the rlr recruitment of the adaptor mavs based on their preferential localization at the mitochondrial membrane even if their mechanism of action is not yet elucidated [ , ] . the sars-cov membrane (m) protein blocks transcription of ifn-i when stimulated by dsrna or members of the rig-i signaling pathway including rig-i, mavs, ikk-, and tbk , but it does not influence the transcriptional activity of the ifn promoter when irf or irf are overexpressed. the physical association of sars-cov m with rig-i, tbk , ikk-, and traf suggests that m protein may prevent the formation of the functional complex with tbk thereby inhibiting activation of irf /irf and ifn-i transcription [ ] . similarly, the papain-like protease (plp) domain contained in the sars-cov nsp protein, an essential component of the viral replicase complex, interacts with the adaptor sting blocking the binding to mavs and the recruitment of the tbk /irf complex [ ] [ ] [ ] . finally, through its deubiquitinating activity, the plp protein removes ubiquitin from several components of the rlr pathway blocking their activation (reviewed in [ , , , ] ). recently, two mers-cov accessory proteins, the orf- a and orf- b products, have also been identified as immunosuppressive factors. mers-cov a is a rna-binding protein that interacts in a mrna-dependent manner with pkr-associated activator (pact), a cellular dsrna-binding protein, which potently stimulates rig-iinduced ifn production by binding to the c-terminal repression domain of rig-i [ ] . so, orf- a inhibits rig-i/mda pathway without a direct binding to the sensor, but by perturbing the function of a stimulator of rig-i signaling as pact [ ] . the orf- bencoded accessory protein is also able to inhibit ifn-i induction in vitro, however, the mechanism involved is not yet elucidated [ ] . the genus flavivirus, of the flaviridae family, includes west nile virus (wnv), dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), tick-borne encephalitis virus (tbev) and several other viruses all causing serious medical problems in humans [ ] [ ] [ ] [ ] . currently, vaccines for humans are available only for yfv, jev, and tbev and no clinically approved antiviral therapy is available for the treatment of flavivirus infections [ ] . in particular, denv and wnv are re-emerging as global life-threatening human pathogens [ , ] . both viruses are sensitive to ifn antiviral effects and the severity of the disease is mostly dependent on the ability to avoid and/or attenuate induction of ifn-i and its effector responses through several viral encoded ifn-antagonists [ , ] . interestingly, some of these strategies are shared with cov. the most relevant prrs for the detection of wnv and denv products, described so far, are the tlr , , and the rlrs, rig-i/mda- . suppression of both tlr-and rlr-mediated ifn induction has been shown to be important for viral replication [ , ] . as cov, flaviviruses contain a cap structure that is generated by a methyltransferase mapped to the n-terminal region of the ns protein [ , ] . through -o-methylation of the viral mrna cap wnv, denv and jev evade ifit -dependent and -independent mechanisms of host restriction in vitro and in vivo [ , ] . moreover, to hide their nucleic acids during the replicative cycle, both denv and wnv induce the formation of convoluted membranes in the endoplasmic reticulum (er) and golgi apparatus that envelop the virus replication complex [ , ] protecting viral nucleic acids from both tlr and rlr recognition, as it occurs along the infection with cov. so far, an antagonism at the level of sensing signaling has been clearly defined only for denv that fails to induce an ifn response in myeloid cells where it replicates, despite other pro-inflammatory cytokines and chemokines are produced [ , ] . the ability to inhibit ifn-i production is due to the viral ns b/ protease that binds and cleaves the adaptor/sensor sting [ ] [ ] [ ] . interestingly, first evidences indicate that the ns b/ proteases of other flaviviruses, as jev or yfv, are not able to cleave sting, while the ns b of yfv can do it [ ] . to the same flaviviridae family belongs the hepacivirus genus, distantly related to flaviviruses, that includes hepatitis c virus (hcv) a major cause of chronic liver disease [ , ] . hcv uses several strategies to efficiently evade innate immunity and this escape is considered the main determinant of viral persistence that leads to a chronic infection in - % of infected people [ , ] . hcv rna can be sensed by different prrs, namely rlrs, tlrs, nlrs, and, as recently reported, by protein kinase r (pkr). rig-i is the best described sensor for the poly u/uc region located within the untranslated region (utr) of the viral rna, along with a -ppp. this region is essential for viral replication and, thus, highly conserved among hcv genotypes. the key viral protein involved in the evasion strategies is the ns / a protease, which consists of ns and ns a. the complex is essential for several steps in the viral cycle including viral rna replication, polyprotein processing and viral assembly [ ] . taking advantage of its serine protease activity, ns / a cleaves the adaptor mavs, preventing its dimerization and downstream signaling [ , ] . after cleavage, mavs dissociates from the mitochondrial associated endoplasmic reticulum membranes (mam) where upon hcv-induced rig-i activation it is recruited and colocalizes with ikk- [ ] , thus impairing ifn-i expression [ ] . importantly, the cleavage of mavs by the viral protease has been confirmed in patients [ ] . to antagonize ifn-i production, ns b protein instead targets sting. hcv ns b, indeed, contains a sting homology domain and interacts with sting in the er blocking sting interaction with mavs and tbk [ , ] . even if the exact molecular mechanism involved in ns b inhibition of sting signaling has not yet been defined in the context of viral infection, ns b likely cooperates with ns / a in targeting the rig-i signaling pathway. interestingly, ns b/ and ns b of other members of the flaviviridae family including denv and yfv as mentioned above also block sting signaling and possess the same sting homology domain, indicating a conserved mechanism of sting antagonisms between flaviviruses and hepaciviruses. intracellularly, both tlr and tlr have been shown to sense hcv rna, depending on the infected cell type considered [ ] . sensing by tlr may occur in liver cells, as hepatocytes, and liver resident macrophages kupffer cells, while tlr sensing occurs predominantly in plasmacytoid dcs (pdcs) and macrophages. as reported above for the mavs adaptor, the serine protease ns / a also cleaves the key adaptor of tlr , trif [ ] , thus preventing an ifn response in productively-infected hepatocytes. in these cells, the hcv-induced mir- has been recently reported to be involved in evasion of ifn-i production and stimulation of hcv replication, upon suppression of myd and irak expression, that is required for the tlr -mediated sensing of the virus [ ] . in macrophages, myd signaling is instead targeted by the ns a protein that, upon the direct binding to the adaptor, impairs the recruitment of irak- and cytokine production in response to tlr ligands [ ] . interestingly, tlr and tlr levels are decreased in patients chronically infected with hcv [ , ] and this has been recently correlated with increased levels of mir- [ ] . hcv-infected cells, however, can trigger ifn-i production in non-productively-infected pdcs in a cell-cell contact-and tlr dependent manner depending on the intracellular hcv rna level of cocultured infected cells [ ] . this production of ifn-i by pdcs may thus account for the strong ifn-i response observed in the liver of infected people [ ] . interestingly, a robust expression of isgs correlated with a decreased response to ifn therapy [ ] supporting a pathogenetic role of a high ifn signature in chronically infected individuals where the virus has established a persistent infection. in contrast, in monocytes and macrophages upon clathrinmediated endocytosis and recognition of the virus by tlr , hcv activates the inflammasome and not ifn-i production in an infection-independent process [ ] . an association between tlr-polymorphisms and cytokine production in response to tlr agonist in vitro has also been reported, supporting a pathogenic role of tlr -mediated sensing in immune cells [ ] . interestingly, this cell-type dependent stimulation of ifn-i and inflammasome in response to hcv infection is also observed in human immunodeficiency virus type (hiv- ) infection that as hcv can establish a persistent infection (see discussion below). pkr has been shown to sense hcv rna very early in infection even prior to rig-i sensing [ ] . pkr is a dsrna binding protein that upon activation phosphorylates the ␣ subunit of the eukaryotic translation initiation factor (eif ␣) to inhibit translation of host capped mrna but not of non-capped mrna, as that of hcv. pkr, upon binding hcv rna and independently of its kinase-activity, interacts with mavs to induce the transcription of a number of early isgs including ifn stimulated gene (isg ), but not ifn-i. isg , in turn, deubiquitinates rig-i inhibiting its functions [ ] . by doing so, hcv blocks sensing by pkr and reinforces hcv evasion from rig- signaling. amongst rna viruses that, as hcv, can establish a persistent infection, hiv- , a lentivirus from the retroviridae family, represents a paradigm for its ability to prevent or circumvent the innate immune response mediated by ifn-i. in spite of evidence that a sustained ifn-i response occurs in hiv-infected patients, it fails to clear the infection in the first place and to prevent the early establishment of long-lived hiv- reservoirs [ ] [ ] [ ] . hiv- has, indeed, developed a number of strategies to block the ifn signaling and the activity of ifn-induced host restriction factors. here, we only briefly summarize these strategies some of which have been only recently discovered, leading to the identification of immune pathways, thus far, unrecognized (as recently reviewed elsewhere [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). in the past few years, the knowledge on innate immunity against hiv- has evolved enormously with the recent identification and the characterization of the molecular basis of retroviral recognition by prrs [ , , ] . retroviral replication generates several structural and intermediate molecules as ssrna, hybrids rna/dna, ss and ds dna produced upon reverse transcriptase, that are potentially available for recognition by cellular prrs as "non-self". with the exception of pdcs, which produce high levels of ifn-i upon detection of hiv- ssrna by tlr , in all other immune cells ifn-i production is prevented or barely detectable unless viral countermeasures are disabled. in conventional dcs (cdcs), monocytes and resting cd + t cells, indeed, hiv- sensing is prevented by the restriction factor sterile alpha motif (sam) and the hystidine/aspartic acid (hd) domain-containing protein (samhd ) that blocks reverse transcription or directly degrades genomic rna, thus preventing prr recognition [ , ] . this samhd mediated restriction is overcome by the viral protein vpx [ , ] that is a hiv- and siv accessory protein absent in hiv- . this apparent disadvantage is, however, effectively exploited by the virus that maintains a reward by not replicating in myeloid cells and by reducing the impact of ifn-i production in these cells. the block of productive infection in non-cycling cells where samdh is active, particularly in cdcs, results in lack of maturation and thus in impairment in priming of naive, hiv- -specific t cells for optimal anti-hiv- immunity [ ] . furthermore, in the small fraction of cdcs that become infected, the recognition of hiv- genomic rna by tlr paradoxically licenses hiv- transcription [ ] . in this case, productive dc infection allows an increased transmission to t cells while inhibiting ifn-i production. in monocytes instead, recognition of viral rna by tlr , does not trigger ifn-i production but, as in the case of hcv, leads to the formation of the nlrp inflammasome with activation of caspase- and il- ␤ production, favoring the establishment of an inflammatory milieu that fuels hiv- replication [ , ] . in contrast, in cells that are target of a productive infection, such as macrophages and t lymphocytes, ifn-i production is prevented by an escape mechanism mediated by the hiv- protease that drives rig-i to the lysosomes [ ] . the ssdna derived from proviral dna upon rt can, instead, be sensed by the newly identified dna sensor interferon gammainterferon-inducible protein (ifi ) [ ] , however, hiv exploits the host cytosolic nuclease repair exonuclease (trex ) to digest hiv- dna generated during infection that, thus, does not accumulate at levels sufficient to be detected by ifi , unless trex activity is blocked [ ] . finally, resting cd + t cells are not permissive to virus replication due to the expression of an active samhd that, as mentioned before, degrades genomic rna and prevents efficient rt and recognition of ssdna. however, this prevention of sensing may not be complete and partial recognition of rt intermediates by the ifi sensor not only leads to the initiation of ifn-i production, but also to the activation of the inflammasome, triggering cell death mechanisms including pyroptosis and apoptosis [ ] . finally, the viral capsid exploits two cellular proteins, cyclophilin a and cpsf , and binds just a right amount of both to allow opening of the capsid and rt process, while preventing sensing of the viral cdna before integration with the following production of ifn-i [ , ] . overall, viruses as hcv and hiv- have evolved nifty strategies to dampen the host innate response in cells where a productive infection may take place, while they induce infection-independent mechanisms in non-permissive cells to facilitate the viral life cycle and promote a chronic inflammation. the genus filoviruses from the filoviridae family are among the most virulent known human pathogens and comprise one species of marburg virus and five species of ebola viruses, including zaire ebolavirus (ebov) that is the most lethal and responsible of the recent severe outbreak of hemorrhagic fevers causing up to % mortality in untreated humans [ ] . several immunoevasion strategies that result in total impairment of the innate immune system are responsible for most of ebov virulence [ ] . a major target of these strategies that are exerted by few viral proteins, is the ifn-i response that controls in vivo filoviruses infection [ ] . at the level of viral sensing, the ebov vp inhibits rig-i signaling [ , ] . vp binds with high affinity to dsrna and -ppp dsrna in a sequence-independent manner. four crystallographic structures of ebov vp rbd/iid from zaire and reston viruses and of marv vp rbd/iid have elucidated how vp rbd/iid dimers bind to rna strands and how the dimers mimic the rlr shape, hiding the rna recognition site [ , ] . interestingly, the residues involved in both protein-protein interactions at the rbd/iid dimer interface and involved in dsrna binding are highly conserved among all known ebov and marv species. moreover, all residues that are important for dsrna binding are also crucial for ifn-i inhibition. as the knowledge on the importance of ifn-i in controlling the immunity against bacteria increases, studies aimed at characterizing the evasion mechanisms that these pathogens employ to evade, inhibit, or otherwise manipulate the innate immune response are arising. the mechanisms induced by bacteria for ifn-i expression in different cell types are various and reflect the heterogeneity of host-pathogen interactions established along bacterial infections. while the extracellular bacteria activate ifn signaling mainly through the interaction with molecules present on the cell surface, the intracellular bacteria are recognized as they enter a cell by cell-surface or endosome/phagosome bound receptors or by cytoplasmic pathogen sensors once they escape from these compartments [ ] . here, we report only few examples of bacterial evasion mostly related to signaling pathways converging in ifn-i stimulation. a more exhaustive discussion of bacterial evasion strategies from prr-signaling pathways and inflammasome is provided by the contribution of thomas kufer and igor brodsky in this issue. to elude prr recognition many bacterial pathogens, like viruses, have modified the molecular structure of their pamps. lps, an ubiquitous component of gram-negative bacterial cell wall, is a pamp that is recognized by the tlr /md- complex present on the cell surface. some bacterial species have evolved an alternative form of lps resulting in a weak antagonist of tlr /md- signaling. this strategy is utilized by yersinia pestis, the causative agent of pestis infection. the acylation status of lipid a from hexa-to tetra-acylated is reduced when the temperature increases from • c (flea temperature) to • c (human temperature). this alteration renders lipid a less recognizable lps by tlr and, following transmission from fleas to humans, contributes predominantly to the virulence of the bacterium [ ] . another example is shigella that, after internalization and proliferation within epithelial cells, hypoacylates lipid a to become less visible to the immune system once leave the infected epithelial cells [ ] . in addition to lps, other bacterial components that may induce ifn-i, include bacterial nucleic acids and peptidoglycans. these pamps can be recognized outside or inside the host cells, leading to the activation of distinct signaling pathways [ ] . intracellularly bacterial rna and dna nucleic acids are recognized by the several intracellular receptors that also sense viral pamps, while bacterial peptidoglycans are detected by nod and nod system. most of these pathways then converge in the activation of the sting/tbk /irf axis [ ] . as viruses, bacteria also encode proteins with enzymatic activity that interfere with the activation of adaptor molecules involved in prr signaling. this is the case of the yersinia pestis virulence factor yopj that, besides being a potent inhibitor of the nf-b and mapk signaling pathways, also inhibits tlr-mediated ifn response. as a deubiquitinating protease, yopj prevents or removes the k polymerized ubiquitin conjugates, which are required for traf and traf activation in the signal transduction pathway leading to irf activation [ ] . similarly, the type iii effector ospi of shigella flexneri inactivates by deamination the e ubiquitin ligase ubc , a factor important for traf auto-polyubiquitinylation and activation [ ] . another interesting example is represented by the translocated intimin receptor (tir), which is one of the first type iii effector proteins discovered in a/e pathogens including the enteropathogenic e. coli (epec), enterohemorrhagic e. coli o :h (ehec), and citrobacter rodentium. in addition to the role played in the attachment to the host membrane, this factor shares sequence similarities with conserved regions present in the cytoplasmic tails of inhibitory receptors of the host immune system, such as the immunoreceptor tyrosine-based inhibition motifs (itims). tir utilizes these itimlike motifs to mimic an endogenous innate immunoregulatory mechanism. in particular, tir recruits the host src-homologyregion- -domain-containing phosphatase to the adaptor traf and thus prevents polyubiquitinylation and activation of traf in the ifn-stimulated pathway [ ] . adaptor molecules downstream sensors transmit signals to classical ib kinase complex, including nemo/ikk-␥, tank and to the atypical ikk-related kinases ikk-and tbk that trigger activation of nf-b and irfs, respectively [ , ] . ifn-i production downstream of these signaling pathways depends essentially on the presence and activation of irfs and their contribution changes depending on the cell type considered [ ] . the irf family is presently composed of nine mammalian members namely irf to , coded by distinct but related genes that exert a number of functions in the regulation of innate and adaptive immune responses. the irfs with intrinsic antiviral function include irf and irf that are essential for the prr-mediated ifn gene transcription, but also induce some ifn effectors in an ifn-independent manner. irf , as mentioned before, is part of the heterocomplex isgf that drives the expression of most isgs including irf (fig. ) . although irf is itself an isgs, which affects different aspects of the immune response even independently from ifn-i production [ , ] , it also represents a positive regulator of ifn-i gene expression in response to specific stimuli in a cell type specific manner [ ] . moreover, irf plays a crucial role in regulating mavs-dependent signaling from peroxisomes [ ] . in this respect, irf regulates the transcriptional profile of antiviral genes unique to that induced by ifn-i and cooperatively promotes an effective antiviral program against a broad spectrum of viruses [ , ] . irf is instead specifically involved in inflammatory cytokines induction [ ] . given the unique functions exerted by irfs, viruses have evolved strategies aimed at the specific destruction of these transcription factors. with regard to the viruses covered here, most of them inhibit irf activation either indirectly by acting on sensors and elements of the signaling pathway that activate them, as described above, or directly by impairing/hijacking irf activity (fig. ) . as described above, the sars-cov plp affects the activation of both irf and nf-b not directly, as initially suggested [ , ] , but by targeting rig-i, mavs, traf [ ] and, as more recently reported, sting [ , ] . interestingly, this activity is independent from plp protease activity. recently, it has been reported that the plp of mers-cov also suppresses ifn-i transcription by interfering with irf phosphorylation and nuclear translocation [ ] . as sars-cov, mers-cov plp is a viral deubiquitinating enzyme that acts on both k -and k -linked ubiquitination and isg -linked isgylation, two posttranslational modifications that play important roles in regulating the rig-i and sting/irf and nf-b activation [ ] . whether the deubiquitination and deisgylation activity of mers-cov plp are directly responsible for inactivation of irf /nf-b or upstream signaling pathway, it remains unclear. the wnv ns protein inhibits the tlr -induced activation of ifn-i and il- transcription through inhibition of nuclear translocation of irf and nf-b [ ] . recent studies indicate that this effect seems to be dependent on ns domains that control viral replication [ ] . interestingly, in the draining lymph nodes the protein released predominantly from macrophages and dcs can inhibit the innate immune signaling pathways in uninfected cells and impairs cytokine production in response to infection [ ] , thus suggesting that ns could also influence the development of the adaptive immune response directed to wnv. the ns b/ serine protease of denv, instead, blocks the serine phosphorylation and nuclear translocation of irf by directly interacting with ikk-and masking the kinase domain [ ] . two tick-borne flaviviruses, lgtv and tbev, have been recently reported to inhibit irf independently of their ability to antagonize ifn signaling. in particular, a weak expression of irf protein and nuclear localization, without reduction in irf mrna expression, was observed in dcs, an early cellular target of infection [ ] . several hcv proteins interfere with irf activity. the hcv ns protease impairs irf activation by blocking the interaction with tbk and irf [ ] . in addition, the ns protein inhibits, in a dose-dependent manner, ikk--and especially tbk -induced irf phosphorylation [ ] . the basic amino acid region (br ) in the n-terminal region of the core protein is also crucial in inhibiting irf dimerization as well as phosphorylation induced by ndv infection and poly (i:c) [ ] . interestingly, this domain has been identified as the binding region for a dead box protein the ddx , which has been recently found to enhance the tbk /ikk--induced ifn-␤ promoter activity upon binding to the adaptor mavs [ ] . the hcv core also decreased the expression levels of ddx suggesting that the irf inhibition may be mediated by the core effect on ddx . moreover, through binding to ddx , the hcv core protein also promotes hcv replication. thus, the core protein appears to switch ddx from an ifn-inducing mode to an hcv-replication mode [ ] . irf expression is, instead, suppressed by the hcv core at the transcriptional level. this event blocks the expression of several antiviral and immunomodulatory genes of both innate and adaptive immunity and, in doing so, facilitates the establishment of hcv persistent infection [ ] . in line with this hypothesis, accumulating evidence suggests that hcv also targets dcs to control the host antiviral response and trigger persistence. as wnv ns , hcv core is, indeed, a secreted protein found in the peripheral blood of patients with chronic infection that may thus affect directly dc functions. in this context, it has been recently reported that the core protein suppresses ifn-i production in response to tlr agonists and to rig-i stimulated by hcv pamp in a cell culture model of pdcs, through the reduced levels of irf and of phosphorylated stat protein [ ] . the effect on irf is, however, not direct but probably mediated by the reduced levels of ifn-i production by core-stimulated pdcs. whether or not this also occurs along the natural infection and contributes to hcv persistency remains to be determined. to increase virus replication and establish viral persistence and latency, hiv- , besides to dismantle or exploit almost all cell intrinsic innate recognition pathways, as discussed above, also directly hits irfs [ , , ] . in t cells, the virion-associated accessory proteins, vif, vpr and vpu, directly target irf for ubiquitin-associated proteasome degradation [ ] [ ] [ ] . recently, this vpu effect on irf degradation has been, however, challenged and it has been reported that vpu, instead, mediates a partial cleavage of irf in a caspase-dependent manner. interestingly, this cleavage produces a c-terminal fragment that can act as a negative regulator of irf -dependent gene activation [ ] . thus, hiv- , as already reported for several viruses, can also exploit the apoptotic machinery to interfere with irf function [ ] . in myeloid cells, instead, hiv- does not inhibit but, rather, stimulates both irf and irf expression. irf activity is, however, exploited by the virus to induce a distinct subset of isgs that despite displays intrinsic and unique antiviral actions, does not restrict viral infection [ ] . irf is also induced in hiv- -productively infected t cells where it may regulate viral promoter activity even in the absence of the viral transactivator tat driving initial transcription of the viral genome [ , ] . later on, however, when viral replication is mostly accomplished by the viral transactivator, irf is sequestered by tat to accelerate proteasomal-mediated irf degradation (remoli al and battistini a, unpublished) and to quench irf transcriptional activity on target genes [ ] . by so doing, tat disarms the unique antiviral response against viral infections that irf could exert [ ] . a block of ifn transcription in primary cd + t cells may also depend on cd /cd -mediated activation of ikk-. we, indeed, recently reported that ikk-activation results in a peculiar pattern of irf phosphorylation in t cells, including a splicing isoform of irf , which may function as an inhibitor of ifn-␤ expression, and phosphorylation of irf that blocks its activity on ifn-i promoter [ ] . the ebov vp , in addition to prevent rlr recognition, inhibits ifn-i promoter activation mediated by tbk- /ikk-overexpression but not by a constitutively active irf , strongly suggesting a specific inhibition of the kinase activity of tbk- /ikk-. indeed, vp interacts with the tbk- /ikk-kinase domain and functions as a substitute substrate, thus inhibiting both the kinase activity and the binding of the physiological irf / substrate [ , ] . notably, mutations of vp residues, involved in this ifn antagonism, do not alter the function of vp in viral replication and transcription [ ] . in dcs, vp targets irf . by interacting with the small ubiquitinlike modifier sumo e enzyme ubc and the e ligase pias , vp promotes irf sumoylation, a post-translational modification that prevents irf translocation into the nucleus and, in turn, ifn-i gene transcription. a similar effect of vp was also reported for irf [ ] . this vp activity is independent of its ability to recognize dsrna and maps to the n-terminus, which is essential for interactions with both irf and pias . interestingly, sumo modification of irf / is a part of the negative feedback loop of normal ifn-i signaling [ ] that is exploited by ebov to weaken host innate immunity. downstream prrs, bacteria mainly target the mapk pathway and the nemo-ikk-nf-b signaling axis, which primarily induces inflammatory cytokines (reviewed in [ ] and thomas kuper and igor brodsky in this issue). as an example of bacteria that target the irf pathway. listeria monocytogenes suppresses ifn-i gene induction downstream of tlr-triggered myd signaling pathway, acting on irf . indeed, a mapk phosphatase renders irf hypophosphorylated by enhancing the formation of a mapk phosphatase-irf -tbk- ternary complex in response to infection [ ] . in fig. is illustrated the ifn-i signaling pathway downstream the ifn-i receptor (ifnar) (a heterodimer of ifnar and ifnar ) that is activated upon binding of virus-infected cell-secreted ifn-i, and some isgs, including positive and negative feed-back regulators. most of the viruses here covered and some bacteria also target these pathways (fig. ) . upstream the jak/stat pathway, the sars-cov a promotes serine phosphorylation within the ifnar degradation motif and increases ifnar ubiquitination [ ] . the plp from sars-cov has a complex mechanism of interference with the jak/stat pathway. through its de-ubiquitinase activity upregulates the expression of the ubiquitinating enzyme e - k, leading to degradation of the erk kinase that, in turn, interferes with stat phosphorylation [ ] . the orf -encoded protein, instead, antagonizes stat function by interacting and sequestering in the er components of the nuclear import complex, as karyopherin alpha and karyopherin beta . by doing so, orf- competes for the binding of the nuclear import complex to stat , thus inhibiting stat nuclear import [ ] . however, the majority of these evidences have been obtained by overexpression or stably expression of individual viral components in cell culture, which represents an experimental setting that may not accurately reflect the innate immune signaling occurring during sars-cov infection in vivo. wnv interferes with ifnar complex by promoting phosphorylation-dependent ubiquitination and degradation of ifnar . this effect is mediated by the hydrophobic ns a and ns b proteins that potently induce the unfolded protein response (upr). this pathway is physiologically induced by different stimuli, including the accumulation of misfolded proteins in the er [ , ] that, as mentioned before, represents the site of flaviviruses replication. activation of the upr pathway inhibits ifn activation and induces a general er stress response, thus facilitating viral replication. the methyltransferase domain of ns from langat virus and jev, instead, binds directly to ifnar through its methyltransferase domain and inhibits the activation of kinases associated to the receptor [ , ] . several wnv non structural proteins, as ns a, ns b, ns , ns a, ns b and ns , have been reported to prevent the phoshorylation of jak , tyk and, as a consequence, the activation of stat / (recently reviewed in [ ] ). likewise, expression of denv nonstructural protein ns a, ns a, or ns b proteins impairs the jak/stat signaling pathway by reducing the phosphorylation and nuclear translocation of stat [ ] . phosphorylation of stat is also blocked by denv ns through inhibition of jak and tyk activity [ ] . ns also binds to the coiled-coil region in the first half of the human stat protein and acts as a bridge between ubr- , a member of the n-recognin family, and stat leading to stat ubiquitination and proteasomal-mediated degradation [ , ] . interestingly, only proteolytically-processed ns can efficiently mediate stat degradation, though both unprocessed and processed ns proteins are able to bind stat . the jev ns protein greatly reduces tyk and, partially, stat phosphorylation, probably through its phosphatase activity [ ] . the tbev ns , instead, blocks stat phosphorylation by promoting the association with the pdz membrane protein scribble [ ] . by activating the ras/raf/mek pathway, hcv replication has been shown to increase the phosphorylation of a motif contained in the cytoplasmic tail of ifnar , which is involved in controlling the receptor ubiquitin-dependent endocytosis and attenuation of stat / phosphorylation [ ] . several hcv proteins have also been implicated in the regulation of the ifn response pathway interfering directly with the jak/stat signaling. however, contrasting results have been reported that probably stem from the use of different cell lines or different hcv expression/replication systems [ ] [ ] [ ] [ ] [ ] . the core protein has been reported to upregulate the expression of socs , thereby inhibiting tyrosine phosphorylation of stat [ ] , although the decreased stat phoshorylation has not been detected in other studies [ , ] . the hcv core protein, expressed alone, has been reported to directly bind to stat and to prevent its phosphorylation and subsequent expression of downstream isgs [ ] . the ns a, similarly, binds and prevents stat phosphorylation specifically in hepatocyte-derived cell lines [ ] . two strategies are used by ebov vp to limit the jak /tyk mediated activation of stat / and their subsequent nuclear localization. vp binds within the tyrosine-phosphorylated-stat binding region located in the c terminus of members of the npi- subfamily of karyopherin alpha nuclear localization signal receptors preventing their binding and shuttling to the nucleus of tyrosine-phosphorylated-stat [ , ] . the crystal structure of human kpnalpha c terminus in complex with vp has been recently resolved and a unique nonclassical nuclear localization signal binding site on kpna has been identified. this motif is necessary for binding and efficient nuclear import of tyrosinephosphorylated-stat [ ] . ebov vp can also directly bind stat ; whether the binding occurs with the unphosphorylated or phosphorylated stat or both isoforms it is not yet clear [ ] . however, also unphosphorylated stat enters the nucleus to activate and sustain the expression of a number of ifn-induced immune regulatory genes, which are distinct from those activated by the phosphorylated stat . thus, ebov vp binding and inhibition of either forms of stat may be important in the suppression of an antiviral state. notably, in spite of the high similarity of ebov and marv genome organization and high sequence homology between many of the ebov and marv encoded proteins, marv vp unlike ebov vp , does not inhibit jak/stat signaling [ ] . this is consistent with the observation that regions important for karyopherin alpha binding are different between these two vp s [ ] . in contrast, tyrosine phosphorylation of jak and stat is inhibited by the marv matrix protein vp that also inhibits this ifn-ii signaling. interestingly, also jak -dependent and il- -induced tyrosine phosphorylation of stat and stat are targeted by marv vp suggesting that marv may globally inhibit jak dependent cytokine signaling with mechanisms different from that employed by ebov [ ] . among the escape mechanisms used by bacteria to evade immune responses, some have been recently reported to target ifn-i signaling molecules. having found that influenza viruses replicate to a higher efficiency in cells co-infected with staphylococcus aureus, warnking et al. [ ] demonstrated that an impaired stat /stat dimerization is responsible for a poor induction of isg transcription in spite of an abundant secretion of ifn-i driven by the flu virus infection. similarly, the inhibition of the response to ifn-i by mycobacterium tuberculosis was observed in human macrophages and correlated with mycobacterial pathogenicity [ ] . in primary cells and thp- cells, indeed, mycobacterium tuberculosis specifically inhibits ifn-i signal transduction pathway by impairing the activation of stat , while the avirulent mycobacterium bovis bcg fails to do so. alteration in isgf- complex formation was instead observed in human macrophages infected with nonpathogenic lactobacillus rhamnosus gg where only stat homodimers were found. in contrast, the pathogenic streptococcus pyogenes led to formation of not only stat homodimers but also of isgf- [ ] . based on these finding the authors speculated that the efficient induction of ifn-i production and related transcription factor activation by streptococci would lead to fast and effective immune responses that, however, could play a role in the pathogenesis. although the first antiviral isgs were discovered decades ago, until recently the mechanisms of action was defined for only a limited number of isg-encoded proteins. the renewed interest in the innate immune response to retroviruses with the identification of how several host restriction factors may limit retroviral infection [ ] [ ] [ ] , as well as large-scale functional screening of isgs, have identified genes that coordinately control the infection of a range of rna and dna viruses and have begun to dissect their mechanism of action [ , ] . interestingly, some of the most potent antiviral effectors reinforce the system by further inducing ifn or isgs. thus, by directly disarming and/or making the use of individual ifn-induced effector proteins, the antiviral effect of host cells may still be attenuated even though ifn-i is induced, and viruses can ensure protection from both autocrine and paracrine effects of secreted ifn-i. here, we will primarily focus on isgs for which viral countermeasures have been identified in the context of viruses covered in the present review. recent reviews report more extensively on isg viral antagonists and specifically on ifn-induced hiv restriction factors that are not covered here [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pkr is one of the major effectors of the ifn-i-induced antiviral state. upon activation from binding dsrna molecules via a dsrna binding domain, pkr phosphorylates the translation initiation factor, eif ␣, thus blocking cellular and viral protein synthesis in infected cells. due to its ability to bind dsrna molecules pkr is also a nucleic acid sensor, as mentioned above [ ] . viruses have evolved specific mechanisms to inhibit pkr activity or escape its action downstream. these include: the production of small and highly structured rna molecules that prevent the dsrnainduced dimerization and activation of pkr; expression of proteins that bind directly to and inhibit the activity of pkr or pkr activators; proteins that behave as pseudosubstrate and competitive inhibitors of pkr [ ] [ ] [ ] . amongst viruses covered in this review, both antiviral and proviral roles of pkr have been reported for hcv. the hcv ns a and e proteins directly interfere with the antiviral action of pkr. ns a binds directly to pkr, while the glyco-protein e acts as competitive substrate with eif ␣ for pkr binding, resulting in inhibition of pkr kinase activity and in increased hcv replication. the full length ires of hcv rna, which is recognized by pkr, may mediate either activation or inhibition of pkr [ ] . moreover, ns a, by binding to various domains of the ires, can alter the activation of pkr [ ] . another indirect inhibitory strategy mediated by the hcv ires has been recently reported. upon hcv rna sensing, pkr activates the mavs/traf /irf pathway that, however, does not induce ifn but a set of isgs including isg . as mentioned, isg deubiquitinates rig-i to negatively control the rig-i/mavs pathway and prevent uncontrolled detrimental ifn-i expression in physiological conditions. thus, hcv hijacks a protective cellular pathway to curtail host innate response [ ] . through a specific ires-mediated inhibition of eif ␣-dependent translation, the hcv ires also regulates the translational activity of pkr [ ] . eif ␣ is, indeed, essential to the translation of capped mrna, as those of isgs, while non-capped mrnas, as those of hcv, are translated independently from this factor. a general attenuation of isgs expression by hcv ires can be achieved also through a direct activation of pkr [ ] . interestingly, this attenuation of isg expression is observed in acute but not persistent infection, where, instead, a sustained isg expression occurs [ , ] . other proteins from flaviviruses have been shown to inhibit pkr including the ns a protein of jev that physically interacts with pkr and blocks its activation in response to several stimuli [ ] . ebov vp also interferes with the pathway regulated by pkr by blocking and also reversing pkr activation, thereby preventing translational arrest of viral mrnas. this pkr antagonism seems to be functionally different from dsrna binding and irf inhibition [ ] [ ] [ ] . ebov vp also associates with pact (pkr-associated activator). this complex abolishes pact interaction with the cterminal domain of rig-i, which is required for full activation of rig-i [ ] . as for retroviruses, hiv- infection does not activate pkr due to both viral and cellular controls (reviewed in [ ] ). in vitro, pkr is activated by low amounts of tar rna, whereas high concentrations inhibit the kinase function. the viral tat protein also counteracts pkr activation by several other mechanisms: it sequesters the activating dsrna; it can act as a substrate homologue of eif ␣ preventing the pkr mediated inhibition of protein synthesis; it prevents pkr auto-phosphorylation and exploits pkr activity to get phosphorylated and increase its binding to tar rna. moreover, hiv- replicates only in cells that have high levels of the tar rna binding protein (trbp), a strong inhibitor of pkr activation. interestingly, during hiv- infection of lymphocytes, when hiv- replicates at high levels, increased amounts of adar , an ifn-induced rna editing enzyme that binds to pkr to inhibit its activation, have been observed. moreover, pact contributes to pkr dephosphorylation during hiv- replication probably due to its binding to adar [ ] . thus, hiv- has evolved to replicate in cells with high levels of trbp, to induce the expression of adar and to change the function of pact for pkr inhibition [ ] . isg is an ubiquitin-like modifier that is induced rapidly by ifn-i and possesses antiviral activity against a number of viruses. isg antiviral functions include inhibition of virus release, isgylation of both viral and host proteins and immunomodulatory cytokine-like properties in its unconjugated and secreted form, as recently reviewed [ , ] . more than host proteins that are isgylated have been identified including irf , pkr and rig-i. isgylation preferentially targets newly translated proteins and, as a consequence of isgylation, degradation of the target protein is reduced by competition with ubiquitin conjugation [ , ] . to date, only few viral proteins have been shown to be isgylated and functional characterizations of isg conjugation has not been always verified under conditions of endogenous protein expression [ ] . moreover, often, isg -mediated protection might not be a result of direct antagonism of virus replication. as an example, isg has been shown to inhibit the release of hiv- and ebov. this effect is mediated by an ubiquitin antagonism. isg disrupts the ubiquitin-mediated regulation of ebov vp , necessary to produce budding and release of vp vlps [ ] , as well as the ubiquitination of the hiv- gag protein, which is required for the interaction with the cellular protein tsg to mediate hiv- budding and release [ ] . several viruses have, thus, developed countermeasures against isg and/or its conjugation. strategies, identified so far, include viral proteins that bind isg or that remove isg from target proteins (reviewed in [ ] ). sars-cov plp has both deubiquitinating and deisgylating activities [ , ] . recently, the structural basis of recognition and processing of deubiquitin and isg by plp has been reported [ ] . interestingly, despite mers-cov encodes a single plp similar to sars-cov, there is little to no sequence conservation among residues important for the deubiquitinating and deisgylating activity, suggesting that mers-cov plp is likely to recognize and process ubiquitin and isg substrates differently than sars-cov plp [ , ] . however, by affecting this post-translational modification, both viruses may modify cellular protein localization, protein activity and stability as well as signal transduction in order to increase viral replication and severity of infection. nevertheless, although these results stem from in vitro overexpression or mutant studies, the direct evidence for isg antagonism by these proteins remains to be demonstrated during viral infection. despite the well-characterized role in restricting replication of several viruses, isg may actually also promote the replication of specific viruses, as hcv. in hcv infections both anti-viral or proviral effects exerted by isg could be related to the net effect of isgylation on the various viral and host proteins targeted by isg . an antiviral effect has been reported when isg could conjugate to hcv ns a, thereby enhancing the inhibitory effect of ifn-i on hcv replication [ ] . in contrast, several groups have reported that isg and isgylation promote hcv production in a cell culture model independently of upstream ifn signaling [ , ] . although counter-intuitive, this finding may be explained by the observed inhibition of ifn-i induction by hcv upon isg overexpression that negatively controls the rig-i/mavs pathway at the level of rig-i ubiquitination [ ] . a negative regulation of ifn-i expression may also be mediated by usp , an isg displaying a potent inhibitory effect on the ifn-i pathway, to prevent autoinflammatory consequences of uncontrolled ifn-i production. similarly, usp may dampen the detrimental role that the hyperactivation of ifn-i signaling plays in the pathogenesis of some viral and bacterial infections, including hiv- , hcv and mycobacterium tuberculosis. usp is, indeed, stabilized by isg in an unconjugated free form [ ] . in this respect, the suggested potential role of the isg /usp pathway in hcv persistence is consistent with the observation that increased expression of hepatic isgs before ifn treatment is associated with an absent or poor response in patients chronically infected with hcv [ ] . thus, isg /usp pathway might explain the paradox that the preactivation of the endogenous ifn system, while fails to clear the infection, instead, may stimulate hcv production and blunt the effect of exogenous ifn-i. usp is similarly induced during some bacterial infections, including salmonella and mycobacterium tuberculosis. the decreased survival of mice that carry a point mutation in usp results from higher salmonella load in the spleen and liver, an increased inflammatory response and increased ifn-i signaling. similarly, these usp mutant mice are more susceptible to mycobacterium tuberculosis infection and have increased bacterial load in the lung and spleen, elevated inflammatory cytokine production and more severe lung pathology [ ] . in line with these findings, the results of dorhoi et al. [ ] have shown that ifnar deficient mice were protected from death upon aerogenic infection with mycobacterium tuberculosis. moreover, a rather detrimental effect of ifn-i was also found in whole blood of patients with tuberculosis, where a neutrophil-driven, ifn-inducible transcriptional signature was associated with clinical severity [ ] [ ] [ ] . these results thus reveal that some viruses and bacteria, to push replication and persistence, utilize an opposite, but as much as effectual strategy, consisting in enhancing/perpetuating an ifn-i response by targeting negative regulators of ifn-i expression. anti-microbial molecules, such as nitric oxide (no) radicals and reactive oxygen species (ros) mediate the antibacterial properties of ifn-i. the key producers of no is inducible nitric oxide synthase (inos), an enzyme that can be induced by both ifn-i and -ii, although the latter is the conventional inducer that plays a crucial role in fighting the infection of intracellular bacteria [ ] . similarly, ifn-i and -ii induce the subunits of the phagocyte nicotinamide adenine dinucleotide phosphate (nadph) oxidase, which generates ros for killing organisms [ ] . thus, pathogens have evolved several ways of avoiding ros-and no-mediated killing. in spite of the fact that no data are available, so far, on the strategies exploited by bacteria to contrast the ifn-mediated transcriptional regulation of inos and nadph oxidase, a common theme for successful intracellular pathogens is the ability to avoid the colocalization with these harmful host enzymes. intracellular salmonella, which resides within a specialized membrane compartment called the salmonella-containing vacuole (scv) in macrophages, uses a t ss called salmonella pathogenicity island (spi ) to mediate protection from no and ros intermediates [ ] . intracellular organisms have also developed mechanisms to detoxify and repair no-mediated damage [ ] , as well as to avoid the induction of inos activity [ ] . given the crucial role played by these antimicrobial enzymes in contrasting bacterial infection, it is likely that strategies pinpointed by pathogens to inhibit the ifn-driven expression of these molecules will be discovered in the nearest future. to effectively resist the continual microbial threat from the environment, vertebrates possess several defense mechanisms including innate and adaptive immunity. as an essential component of the innate immunity, the ifn system constitutes the first line of defense against a number of pathogens to clear an incoming infection and instructing an ensuing adaptive response. successful pathogens have, thus, evolved sophisticated strategies to subvert and/or exploit the host immune system where blocking the ifn response, in the first place, is required to replicate and survive. the elucidation of some of these strategies has led to the identification of several, thus far, poorly recognized features of the innate immune response. in parallel, with the enormous recent advances in the comprehension of the molecular mechanisms of innate immune responses to pathogens, specific processes by which pathogenic microorganisms subvert these innate immune pathways, including the ifn system, is becoming progressively appreciated and it is reasonable to assume that many more will be discovered in the near future. by learning from the anti-immune strategies of pathogens we can, thus, not only identify key pathogen regulators as useful target to exploit to the host advantage, but we can also unveil weaknesses of host defenses and intervene to more precisely tune the immune response. the number and diversity of pathogen strategies for counteracting at each step the ifn system is stupefying. although beyond the scope of this review to discuss all antagonisms in detail, the ones that we have here reported, represent common and recurrent strategies used by a number of pathogens. this is illustrated by the existence of both viral and bacterial examples of pamp modifications as well as of viral and bacterial proteins that share cellular-like domain or cellular-like enzymatic properties that can compete with the host counterparts to dampen their physiological activities. in this respect, common hubs in the signaling pathways downstream pathogen sensors that trigger ifn-i, as few common adaptors or cofactors and transcription factors, are attractive targets of pathogen antagonism. the recognition of the mechanisms involved in microbial countermeasures may have several translation implications. the definition of microbial ability to elude the detection, as the methylation of their rna caps, can suggest strategies to utilize methyltransferase mutants as successful vaccine candidates against a number of different viruses as already suggested for denv [ ] . many of the pathogen proteins responsible for ifn-i antagonism are also determinants of virulence and pathogenesis and, as such, they are highly conserved and may, thus, constitute attractive targets for the development of promising therapeutics against various clinically relevant pathogens reducing the bias of resistance mutations. in turn, some of the cellular identified targets of pathogen proteins as well as protein interacting partners might turn out to be new drug targets for treating a range of different diseases that disarme common components of the ifn pathway. in this respect, the resolution of the crystallographic structures of viral antagonists in complex with their different viral and cellular ligands, is then crucial for the rational design of new drugs. the system biology approach and the ability to simultaneously investigate diverse pathways has led to appreciate the interconnection between these pathways also in terms of shared components and stimulation by different pathogens. thus, a single therapeutic strategy could modulate multiple pathways to the host benefit. nevertheless, each approach needs to be complemented with effective treatments that also overcome other concurrent strategies that often the same pathogen put in place. on the other side of the coin, recovery of a full innate response must be finely tuned. ifn-i is, indeed, not always protective but can instead play a pathogenic role as reported for some bacterial and viral infections, where an uncontrolled ifn production is a determinant of disease progression. even in these cases, however, insights in the mechanisms involved in turning an ifn protective response into a pathogenetic one, may be as well relevant for nonpathogen-induced diseases, as autoimmunity and inflammatory diseases. in this context, cell death and inflammasome activation have been described as crucial ifn-i-regulated events exploited by both pathogen and host to get their own advantage. despite inflammasome facilitates pathogen clearance and is beneficial to the host, in some instances, ifn-induced non-canonical nlrp inflammasome activation and pyroptosis appear to be detrimental due to excessive cell death, inflammation, and collateral tissue damage in vital organs [ ] . so pathogen proteins themselves or modified versions of them could be used as therapeutics working in suppressing inappropriate immune activation. this would be a bright way of hijacking molecules evolved during pathogen adaptation and associated fitness, to shift the balance to the host advantage. similarly, some identified targets of viral proteins in prr signaling pathways might be turned out to be new targets for treating a range of diseases. to find the way to generally induce an ifn response that is protective against a number of different infectious diseases, may be particularly relevant during an outbreak of unknown etiology or during the arising of newly emerging and re-emerging strains. likewise, finding the key to unlock the detrimental outcome of excessive ifn-i production during an infectious disease can open the way to cure autoimmune and inflammatory diseases. the authors declare no competing 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respiratory syndrome coronavirus open reading frame (orf) b, orf , and nucleocapsid proteins function as interferon antagonists molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame b protein severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk /ikkepsilon complex sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf and nf-kappab signaling strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response middle east respiratory syndrome coronavirus a protein is a double-stranded rnabinding protein that suppresses pact-induced activation of rig-i and mda in the innate antiviral response the orf b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling flaviviral rnas: weapons and targets in the war between virus and host immune evasion strategies of flaviviruses flavivirus rna cap methyltransferase: structure, function, and inhibition flaviviruses and flavivirus vaccines emerging viral diseases: confronting threats with new technologies innate immunity evasion by dengue virus west nile virus infection and immunity flavivirus methyltransferase: a novel antiviral target -o methylation of the viral mrna cap by west nile virus evades ifit -dependent and -independent mechanisms of host restriction in vivo ifit inhibits japanese encephalitis virus replication through binding to capped -o unmethylated rna the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex composition and three-dimensional architecture of the dengue virus replication and assembly sites dengue virus inhibits the production of type i interferon in primary human dendritic cells inhibition of the type i interferon response in human dendritic cells by dengue virus infection requires a catalytically active ns b complex denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity activation and evasion of antiviral innate immunity by hepatitis c virus the global burden of hepatitis c interferon-stimulated genes and their role in controlling hepatitis c virus nonstructural protein - a: the swiss army knife of hepatitis c virus cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity mitochondrial-associated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus dissociation of a mavs/ips- /visa/cardif-ikkepsilon molecular complex from the mitochondrial outer membrane by hepatitis c virus ns - a proteolytic cleavage cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system hepatitis c virus ns b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity immune evasion by hepatitis c virus ns / a protease-mediated cleavage of the tolllike receptor adaptor protein trif hcv-induced mir- contributes to evasion of host immune system by targeting myd and irak hepatitis c virus nonstructural protein a modulates the toll-like receptor-myd -dependent signaling pathway in macrophage cell lines expression of toll-like receptors in chronic hepatitis c virus infection distinct toll-like receptor and expression in peripheral blood mononuclear cells from patients with chronic hepatitis c infection hepatitis c virus infection decreases the expression of toll-like receptors and via upregulation of mir- plasmacytoid dendritic cells sense hepatitis c virus-infected cells, produce interferon, and inhibit infection recovery, persistence, and sequelae in hepatitis c virus infection: a perspective on long-term outcome interferon-induced gene expression is a stronger predictor of treatment response than il b genotype in patients with hepatitis c hiv and hcv activate the inflammasome in monocytes and macrophages via endosomal toll-like receptors without induction of type interferon sex-specific association between x-linked toll-like receptor with the outcomes of hepatitis c virus infection hepatitis c virus reveals a novel early control in acute immune response redefining the viral reservoirs that prevent hiv- eradication therapeutics for hiv- reactivation from latency hiv- latency: an update of molecular mechanisms and therapeutic strategies hiv- , interferon and the interferon regulatory factor system: an interplay between induction, antiviral responses and viral evasion innate antiviral immune signaling, viral evasion and modulation by hiv- innate immune recognition of hiv- the interface between the innate interferon response and expression of host retroviral restriction factors unmasking immune sensing of retroviruses: interplay between innate sensors and host effectors type i ifn -a blunt spear in fighting hiv- infection interactions between hiv- and the cellautonomous innate immune system innate immune sensing of hiv- by dendritic cells the ribonuclease activity of samhd is required for hiv- restriction samhd host restriction factor: a link with innate immune sensing of retrovirus infection samhd is the dendritic-and myeloid-cell-specific hiv- restriction factor counteracted by vpx vpx relieves inhibition of hiv- infection of macrophages mediated by the samhd protein dendritic cells in progression and pathology of hiv infection hiv- exploits innate signaling by tlr and dc-sign for productive infection of dendritic cells rig-i-mediated antiviral signaling is inhibited in hiv- infection by a proteasemediated sequestration of rig-i ifi senses dna forms of the lentiviral replication cycle and controls hiv- replication lieberman, the cytosolic exonuclease trex inhibits the innate immune response to human immunodeficiency virus type cell death by pyroptosis drives cd t-cell depletion in hiv- infection the capsids of hiv- and hiv- determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hiv- evades innate immune recognition through specific cofactor recruitment transmission dynamics and control of ebola virus disease (evd): a review characterization of host immune responses in ebola virus infections the role of the type i interferon response in the resistance of mice to filovirus infection evasion of interferon responses by ebola and marburg viruses filoviral immune evasion mechanisms marburg virus vp can both fully coat the backbone and cap the ends of dsrna for interferon antagonism structural basis for marburg virus vp -mediated immune evasion mechanisms the yin and yang of type i interferon activity in bacterial infection structural modifications of bacterial lipopolysaccharide that facilitate gram-negative bacteria evasion of host innate immunity intracellular shigella remodels its lps to dampen the innate immune recognition and evade inflammasome activation yopj targets traf proteins to inhibit tlr-mediated nf-kappab, mapk and irf signal transduction the shigella flexneri effector ospi deamidates ubc to dampen the inflammatory response inhibition of tlr signaling by a bacterial protein containing immunoreceptor tyrosine-based inhibitory motifs triggering the interferon antiviral response through an ikk-related pathway ikkepsilon and tbk are essential components of the irf signaling pathway regulation of immunity and oncogenesis by the irf transcription factor family interferon regulatory factors in immune cell development and host response to infection evidence for licensing of ifn-gamma-induced ifn regulatory factor transcription factor by myd in toll-like receptor-dependent gene induction program peroxisomes are signaling platforms for antiviral innate immunity a diverse range of gene products are effectors of the type i interferon antiviral response regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease immunity by ubiquitylation: a reversible process of modification west nile virus nonstructural protein inhibits tlr signal transduction abrogation of tlr inhibition by discrete amino acid changes in the c-terminal half of the west nile virus ns protein modulation of innate immune signaling by the secreted form of the west nile virus ns glycoprotein dengue virus subverts the interferon induction pathway via ns b/ protease-ikappab kinase epsilon interaction tick-borne flaviviruses antagonize both irf- and type i ifn signaling to inhibit dendritic cell function interaction between the hcv ns protein and the host tbk protein leads to inhibition of cellular antiviral responses hepatitis c virus ns protease inhibits host cell antiviral response by inhibiting ikkepsilon and tbk functions impairment of interferon regulatory factor- activation by hepatitis c virus core protein basic amino acid region dead/h box (ddx ) helicase binds the rig-i adaptor ips- to up-regulate ifn-beta-inducing potential hepatitis c virus core protein abrogates the ddx function that enhances ips- -mediated ifn-beta induction repression of interferon regulatory factor by hepatitis c virus core protein results in inhibition of antiviral and immunomodulatory genes hepatitis c virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor- and signal transducer and activator of transcription (stat) protein expression battistini, hiv- targeting of ifn regulatory factors human immunodeficiency virus type mediates global disruption of innate antiviral signaling and immune defenses within infected cells hiv- accessory proteins vpr and vif modulate antiviral response by targeting irf- for degradation vpu-deficient hiv strains stimulate innate immune signaling responses in target cells hiv- vpu induces caspase-mediated cleavage of irf apoptosis as an hiv strategy to escape immune attack hiv infection of dendritic cells subverts the ifn induction pathway via irf- and inhibits type ifn production irf- is required for full nf-kappab transcriptional activity at the human immunodeficiency virus type long terminal repeat enhancer modulation of human immunodeficiency virus replication by interferon regulatory factors intracellular hiv- tat protein represses constitutive lmp transcription increasing proteasome activity by interfering with the binding of irf- to stat ikappab kinase epsilon targets interferon regulatory factor in activated t lymphocytes the ebola virus vp protein inhibits activation of interferon regulatory factor ebola virus protein vp impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk- basic residues within the ebolavirus vp protein are required for its viral polymerase cofactor function ebola zaire virus blocks type i interferon production by exploiting the host sumo modification machinery virus infection triggers sumoylation of irf and irf , leading to the negative regulation of type i interferon gene expression beneficial innate signaling interference for antibacterial responses by a toll-like receptor-mediated enhancement of the mkp-irf axis the sars coronavirus a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type interferon receptor severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferoninduced responses through downregulation of extracellular signal-regulated kinase -mediated signalling pathways severe acute respiratory syndrome coronavirus orf antagonizes stat function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane virus-induced unfolded protein response attenuates antiviral defenses via phosphorylation-dependent degradation of the type i interferon receptor a conserved peptide in west nile virus ns a protein contributes to proteolytic processing and is essential for replication inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns as an interferon antagonist identification of residues critical for the interferon antagonist function of langat virus ns reveals a role for the rna-dependent rna polymerase domain inhibition of alpha/beta interferon signaling by the ns b protein of flaviviruses dengue virus ns inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription phosphorylation ns of dengue virus mediates stat binding and degradation dengue virus co-opts ubr to degrade stat and antagonize type i interferon signaling blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns through a protein tyrosine phosphatase-mediated mechanism tick-borne encephalitis virus ns associates with membrane protein scribble and impairs interferon-stimulated jak-stat signalling activation of the ras/raf/mek pathway facilitates hepatitis c virus replication via attenuation of the interferon-jak-stat pathway expression of hepatitis c virus proteins inhibits signal transduction through the jak-stat pathway ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling- hcv structural proteins interfere with interferon-alpha jak/stat signalling pathway expression of hcv structural proteins impairs ifn-mediated antiviral response identification of the nonstructural protein b of hepatitis c virus as a factor that inhibits the antiviral activity of interferonalpha hepatitis c virus inhibits interferon signaling through up-regulation of protein phosphatase a intracellular innate immune cascades and interferon defenses that control hepatitis c virus hcv ns a inhibits interferon-alpha signaling through suppression of stat phosphorylation in hepatocyte-derived cell lines ebola virus vp proteins inhibit the interaction of npi- subfamily karyopherin alpha proteins with activated stat ebolavirus vp binding to karyopherins is required for inhibition of interferon signaling ebola virus vp targets a unique nls binding site on karyopherin alpha to selectively compete with nuclear import of phosphorylated stat the ebola virus interferon antagonist vp directly binds stat and has a novel, pyramidal fold marburg virus evades interferon responses by a mechanism distinct from ebola virus super-infection with staphylococcus aureus inhibits influenza virusinduced type i ifn signaling through impaired stat -stat dimerization inhibition of response to alpha interferon by mycobacterium tuberculosis lactobacilli and streptococci activate nf-kappa b and stat signaling pathways in human macrophages interferon-stimulated genes: roles in viral pathogenesis host restriction factors in retroviral infection: promises in virus-host interaction intrinsic antiviral immunity evolutionary conflicts between viruses and restriction factors shape immunity protein kinase pkr and rna adenosine deaminase adar : new roles for old players as modulators of the interferon response the dsrna protein kinase pkr: virus and cell control inhibition of pkr by rna and dna viruses activation of the antiviral kinase pkr and viral countermeasures hepatitis c virus controls interferon production through pkr activation regulation of pkr by hcv ires rna: importance of domain ii and ns a hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation failure of innate and adaptive immune responses in controlling hepatitis c virus infection blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein a ebola virus vp antagonizes pkr activity through its c-terminal interferon inhibitory domain the vp protein of ebola virus inhibits the antiviral effect mediated by double-stranded rna-dependent protein kinase pkr ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling mutual antagonism between the ebola virus vp protein and the rig-i activator pact determines infection outcome multiple levels of pkr inhibition during hiv- replication the pkr activator, pact, becomes a pkr inhibitor during hiv- replication hiv- translation and its regulation by cellular factors pkr and pact the antiviral activities of isg interferon-induced isg pathway: an ongoing virus-host battle positive regulation of interferon regulatory factor activation by herc via isg modification the isg conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg isg inhibits ebola vp vlp budding in an l-domain-dependent manner by blocking nedd ligase activity innate antiviral response targets hiv- release by the induction of ubiquitin-like protein isg the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease inhibition of hepatitis c virus replication by ifn-mediated isgylation of hcv-ns a the interferon stimulated gene functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response isg , a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment human intracellular isg prevents interferon-alpha/beta overamplification and auto-inflammation activation of endogenous type i ifn signaling contributes to persistent hcv infection contribution of increased isg , isgylation and deregulated type i ifn signaling in usp mutant mice during the course of bacterial infections type i ifn signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics genome-wide expression profiling identifies type interferon response pathways in active tuberculosis an interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis common patterns and disease-related signatures in tuberculosis and sarcoidosis inducible nitric oxide synthase and control of intracellular bacterial pathogens nadph oxidases: an overview from structure to innate immunity-associated pathologies salmonella pathogenicity island -dependent evasion of the phagocyte nadph oxidase modulation of inducible nitric oxide synthase expression by the attaching and effacing bacterial pathogen citrobacter rodentium in infected mice rational design of a live attenuated dengue vaccine: -o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques role of type i interferons in inflammasome activation, cell death, and disease during microbial infection we apologize to the many colleagues whose data and influence have been overlooked due to space or our knowledge limitations. a special thank to members of the e.m. coccia's and a. battistini's laboratory for helpful discussion and critical reading of the manuscript and eugenio morassi for preparing drawings. our work is supported in part by grant rf- from italian ministry of health (to emc) and from istituto superiore di sanità (to ab). key: cord- -l re h j authors: sultana, shehnaz; venkata, kolla k; pranay, penagaluru k; usha, rani p; reddy, p.p. title: interferon gamma (ifnγ) + a/t gene polymorphism in south indian ischemic stroke patients date: - - journal: ann neurosci doi: . /ans. . . sha: doc_id: cord_uid: l re h j background: ischemic stroke is a complex vascular and metabolic process resulting in neuronal death and progression with time. cytokines play a role in immune response and also maintains the normal homeostatic environment of the central nervous system. ifn-γ is one of the key effector cytokines produced by nk and t cells that enhances microbicidal activity of macrophages and neutrophils. purpose: as the association of ifnγ + a/t gene polymorphism with stroke has not been investigated in indian population, we wanted to evaluate the association of this polymorphism with ischemic stroke in a south indian population. methods: we genotyped ischemic stroke patients and age-matched control subjects. results: statistical analysis showed a significant association of tt homozygote with ischemic stroke (or= . , % ci= . - . , p= . ), while aa (or= . , % ci= . – . , p= . ) and at(or= . , % ci= . - . , p= . ) genotypes were not significantly associated. a and t allele frequencies in stroke were . % and . % as against . % and . % in control group, respectively, thus, suggesting no statistically significant differences in the a (or= . , % ci= . – . , p= . ) and t (or= . , % ci= . – . , p= . ) allele frequencies between the two groups. conclusion: we conclude that the ifn-γ + tt genotype is associated with the increased risk of ischemic stroke. ischemic stroke is a complex vascular and metabolic process resulting in neuronal death and progresses with time. cytokines play a role in immune response and also maintain the normal homeostatic environment of the central nervous system. inflammatory cytokines play an important role in the etiology of cerebral infarction and they are under strong genetic control. as genetic traits contribute significantly to cerebral infarction variations in the genetic regulation of inflammatory system may increase the risk of the disease from individual to individual. ifn-γ has antiviral, immunoregulatory, and anti-tumor properties. atherosclerosis is an inflammatory disease, and plaque induced inflammation is considered a cause of intimal erosion and rupture and therefore leads to acute ischemia. , ifn-γ has important immunoregulatory roles and enhances both antigen specific and non-specific immune responses through actions on monocytes and macrophages. , complications related to infections such as chest and urinary tract infections, have been reported to occur in - % of all stroke patients within the first few days after stroke. , brain injury was identified as an independent risk factor for infectious complications in trauma patients due to a central nervous shutdown of the immune defense. , howard et al, reported association of immunosuppressive state with stroke. ifn-γ is one of the key effector cytokines produced by nk and t cells that enhances microbicidal activity of macrophages and neutrophils. several gene polymorphisms are associated with stroke in humans, association between the gene polymorphisms of inflammatory cytokines are meager. in the present study we have examined single nucleotide polymorphism in interferon gamma (ifnγ) at position + a/t in south indian ischemic stroke patients. fer containing triton-x was added to the whole blood sample, in-order to lyse the rbc and centrifuged to get the pellet. the pellet was lysed with wbc lysis buffer containing % sds, and then high molar concentration of nacl was added consecutively to separate out the protein fraction. finally, ice cold ethanol was added to get the dna which were separated and resuspended in te buffer and stored at - c until the pcr reaction was performed. the polymorphism in interferon gamma (ifnγ) at position + a/t was studied using amplification refractory mutation system polymerase chain reaction method (arms pcr). in brief, each reaction employed a generic antisense primer '-tcaacaaagctgatactcca- ' and one of the two allele-specific sense primers '-ttcttacaacacaaaatcaaat-ca- ' for 'a' allele and '-ttcttacaacacaaaatcaaatct- ' for 't' allele. for evaluation of the pcr amplification bp internal control was amplified using a pair of specific primers '-gccttccaaccattccctta- ' and '-tcacggatttctgtt-gtgtttc- '. the pcr incubation mixture in a total volume of µl consisted of mm tris-hcl, ph . ; mm kcl; mm dntps; . mm mgcl ; . units taq polymerase; . mm of each primer; . % gelatin and ng genomic dna. amplification was performed with an initial denaturation at °c for minutes, cycles were run with denaturation at °c for seconds, annealing at °c for seconds and extension at °c for seconds. the products were analysed on % agarose gel stained with ethidium bromide. the association between genotypes and stroke was examined by using odds ratio (or) with % confidence interval (ci) and chi square (χ ) analysis using epi info software (epi info cdc). all the statistical tests were two sided, and were considered significant at p value < . . genotypic frequencies were calculated according to the number of different genotypes observed and the total number of genotypes examined. yate's correction was applied wherever necessary. genotype frequencies were checked for deviation from hardy-weinberg equilibrium and were not significantly different from those predicted. the details on the demographic characteristics of the study population are shown in table . the mean age of the patients was . ± years as against the mean age of . ± years in the control group. the percentage of males among the stroke patients was . % (n= ), which was higher compared to controls . % (n= ), whereas the percentage of females was . % (n= ) in stroke patients and . % (n= ) in the control group. the percentage of hypertension was . % in stroke patients and . % in controls. the percentage of diabetes was . % among stroke patients and . % in control group. the percentage of smokers were more in patient group ( . %) compared to controls ( . %). the percentage of alcohol users in patients group ( . %) was more compared to controls ( . %). family history of hypertension in patients group ( . %) is more compared to controls ( . %). family history of diabetes in patients groups was . % as against . % in controls. family history of stroke was reported in . % of patients and . % of controls. in our case-control study, we genotyped ifn-γ + a/t polymorphism in ischemic stroke patients and in control subjects. the genotype frequencies of ifn-γ + a/t polymorphism among the patients and controls are shown in table . the distribution of genotypes was in hardy-weinberg equilibrium among controls. the frequencies of the "aa", "at", and "tt" genotypes of ifn-γ + a/t polymorphism in stroke patients were . %, . %, and . % as against . %, . %, and . % in controls, respectively. the genotypic frequency of "tt" homozygote showed a significant association with ischemic stroke (or= . , % ci= . - . , p= . ), while aa (or= . , % ci= . - . , p= . ) and at(or= . , % ci= . - . , p= . ) genotypes were nonsignificant. a and t allele frequencies in stroke were . % and . % as against . % and . % in control group, respectively, thus, suggesting no statistically significant differences in the a (or= . , % ci= . - . , p= . ) and t (or= . , % ci= . - . , p= . ) allele frequencies between the two groups. interferon gamma (ifn-) is an important cytokine in cellular immunity and the presence of thymidine at + correlates with microsatellite repeats associated with high cytokine production. in the present study we examined single nucleotide polymorphism in interferon gamma (ifnγ) at position + a/t and found a significant association of "tt" genotype with ischemic stroke. infectious complications in particular, bacterial pneumonia and their relevance for mortality are well known in acute stroke. the high incidence of infections in stroke patients is likely to be a result of an impaired immune function. a functional role of neutrophils in the development of strokeassociated injury remains controversial, and the contribution of specific lymphocyte subpopulations and their products to the pathogenesis of ischemic stroke are not clear. t-cell derived interferon-γ (ifn-γ) has been shown to contribute to the injury elicited by ischemia-reperfusion in other organs and ifn-γ mrna is increased in rat brain tissue after permanent focal cerebral ischemia. activation of the sns and the hpa by proinflammatory cytokines in systemic inflammation results in the release of glucocorticoids and catecholamines, which in- hibit further production of proinflammatory mediators. vagus nerve activation by inflammatory cytokines during endotoxemia was found to inhibit macrophage cytokine production through release of acetylcholine , and rapid activation of these pathways in inflammatory conditions protects the organism against any adverse effects of an overwhelming immune response. however, an excessive activation of inhibitory neuroendocrine pathways without systemic inflammation can inappropriately suppress the immune system and increase the risk of infections. intrathecal release of proinflammatory cytokines is associated with signs of systemic immunodepression and a high incidence of infections in neurosurgical patients. according to konstantin et al stress mediator blockade underlines the importance of functional defects in ifn-γ production in the control of infectious complications after stroke. γδ t cells are essential for pulmonary bacterial clearance and αβt cells are more critical in the peripheral blood in stroke induced infections. inflammation is an early and rate-determining step in the microvascular dysfunction and tissue injury associated with cerebral ischemia-reperfusion (i/r) is supported by several reports that describe a reduction in brain edema and infarct size in animal models of stroke treated with antibodies that block leukocyte adhesion. , the microvasculature of postischemic brain assumes an inflammatory phenotype that is manifested as endothelial activation and barrier dysfunction, enhanced generation of oxidants and inflammatory mediators, and the recruitment of adherent leukocytes and platelets. aspiration due to dysphagia is a known risk factor for pneumonia after severe strokes and other factors that might predispose stroke patients to pneumonia is an impaired immune responsiveness. , , . a study carried out in egyptian atopic patients showed a significant association of ifn-gamma gene polymorphism at position + a/t. study from china reported a significant association of ifn-gamma + a/t gene polymorphism and severe acute respiratory syndrome. a significant association was observed between interferon-gamma gene polymorphisms and systemic lupus erythematosus suggesting that elevated interferon gamma is associated with increased systemic erythematosus susceptibility. lai et al reported that genetic polymorphism of ifn-gamma gene is associated with individual susceptibility to cervical carcinogenesis. feher et al could not find any association between ifn-γ + a/t gene polymorphism and alzheimer disease. our study found a significant association of 'tt' genotype of ifn-γ + gene polymorphism and ischemic stroke in south indian population. the article complies with international committee of medical journal editor's uniform requirements for the manuscripts. genetics of inflammation and risk of coronary artery disease: the central role of interleukin- interferon-gamma: an overview of signals, mechanisms and functions atherosclerosis -an inflammatory disease recombinant interleukin suppresses the production of interferon gamma by human mononuclear cells il- inhibits the synthesis of ifn-gamma and induces the synthesis of ige in human mixed lymphocyte cultures complications after acute stroke medical complications after stroke: a multicenter study pneumonia: incidence, risk factors, and outcome in injured patients pneumonia following closed head injury acquired immunologic deficiencies after . trauma and surgical procedures stroke genetics update subtyping in ischemic stroke genetic research diagnosis and classification of diabetes mellitus; definition and . description of diabetes mellitus a non organic and non enzymatic extraction methods gives high yields of genomic dna from whole blood samples than do nine other methods tested interferongamma and interleukin- gene polymorphisms in pulmonary tuberculosis t-lymphocytes contribute to hepatic leukostasis and hypoxic stress induced by gut ischemia-reperfusion link h. il- and ifn-gamma mrna expression is increased in the brain and systemically after permanent middle cerebral artery occlusion in the rat pharmacological stimulation of the cholinergic antiinflammatory pathway the inflammatory reflex immunodepression following neurosurgical procedures stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke t helper cell type -like immunostimulation inflammatory responses to ischemia and reperfusion in the cerebral microcirculation hu f g, an antibody recognizing the leukocyte cd /cd integrin, reduces injury in a rabbit model of transient focal cerebral ischemia platelet-leukocyte-endothelial cell interactions after middle cerebral artery occlusion and reperfusion screening for dysphagia and aspiration in acute stroke: . a systematic review aspiration pneumonia aspiration pneumonitis and aspiration pneumonia interferon gamma gene polymorphism as a biochemical marker in egyptian atopic patients the interferon gamma gene polymorphism + a/t is associated with severe acute respiratory syndrome interferon-gamma gene polymorphisms associated with susceptibility to systemic lupus erythematosus genetic polymorphism of the interferon-gamma gene in cervical carcinogenesis association study of interferon-?, cytosolic phospholipase a , and cyclooxygenase- gene polymorphisms in alzheimer disease key: cord- -tgexahwd authors: van tol, sarah; hage, adam; giraldo, maria isabel; bharaj, preeti; rajsbaum, ricardo title: the trimendous role of trims in virus–host interactions date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: tgexahwd the innate antiviral response is integral in protecting the host against virus infection. many proteins regulate these signaling pathways including ubiquitin enzymes. the ubiquitin-activating (e ), -conjugating (e ), and -ligating (e ) enzymes work together to link ubiquitin, a small protein, onto other ubiquitin molecules or target proteins to mediate various effector functions. the tripartite motif (trim) protein family is a group of e ligases implicated in the regulation of a variety of cellular functions including cell cycle progression, autophagy, and innate immunity. many antiviral signaling pathways, including type-i interferon and nf-κb, are trim-regulated, thus influencing the course of infection. additionally, several trims directly restrict viral replication either through proteasome-mediated degradation of viral proteins or by interfering with different steps of the viral replication cycle. in addition, new studies suggest that trims can exert their effector functions via the synthesis of unconventional polyubiquitin chains, including unanchored (non-covalently attached) polyubiquitin chains. trim-conferred viral inhibition has selected for viruses that encode direct and indirect trim antagonists. furthermore, new evidence suggests that the same antagonists encoded by viruses may hijack trim proteins to directly promote virus replication. here, we describe numerous virus–trim interactions and novel roles of trims during virus infections. eukaryotes are constantly exposed to a variety of pathogens, including viruses. as with other environmental signals, viral invasion triggers tightly regulated intracellular signaling cascades to optimally respond to infection. mammals enact both an innate and an adaptive immune response to identify an infecting pathogen, to clear the foreign agent, and to protect against subsequent invasion. a primary mechanism for fine-tuning molecular pathways is utilization of post-translational modifications. altering the functional proteome influences protein interactions, transcriptional programs, translation, secretion, and cytoskeletal arrangement. a variety of molecules, including phosphates, sugars, lipids, or proteins, can be attached or removed enzymatically to modulate protein function. post-translational modifications thus enable rapid and reversible regulation. under positive selection [ ] . this species-specific pattern of positive selection of closely related trims suggests that individual trims play specific antiviral roles. in addition to differential mrna expression upon viral infection, several trim family members are intimately involved in the antiviral response. type-i ifns and other cytokines, such as pro-inflammatory cytokines induced via the nf-κb pathway, have been noted to differentially regulate the expression of a significant population of trims [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . likewise, trim overexpression influences the transcription of type-i ifn, pro-inflammatory cytokines, and ifn-stimulated genes (isgs) [ , ] . the roles of trims in viral infection include intrinsic restriction of viral pathogens, positive regulation of immune pathways that promote viral clearance, and negative regulation of antiviral pathways to limit immunopathology [ , , ] . the incorporation of trim antagonists into viral genomes exemplifies the importance of trims in antiviral responses [ ] [ ] [ ] [ ] [ ] [ ] . here, we will focus on the role of trims in the direct and indirect inhibition of viruses and novel mechanisms of viral-mediated antagonism and hijacking of trims. excellent reviews on the roles of trims in autophagy, cancer, and other diseases have been recently published [ ] [ ] [ ] . cells identify pathogen invasion due to the presence of pathogen-associated molecular patterns (pamps) contained in viral components, which are recognized by host pattern recognition receptors (prrs) [ ] . examples of viral pamps include some envelope or capsid proteins, viral nucleic acid, or intermediates of genome replication [ ] . upon pamp engagement of a prr, a signaling cascade is initiated that relies on post-translational modifications for proper coordination. these modifications include ubiquitination and phosphorylation, which facilitate the assembly of adaptor and enzymatic molecules needed to activate and inactivate transcription factors and other effector molecules [ , , ] . these transitions in the transcriptional profile and functional proteome enable the cell to respond optimally to the pathogen and to communicate (e.g., cytokine secretion) with neighboring cells to limit viral replication and promote clearance. examples of pathways critical in response to viral infection include ifn induction and signaling and nf-κb activation [ ] . trim e ligases regulate ifn production and signaling as well as nf-κb induction at multiple levels, from prr-mediated pamp recognition to regulation of transcription factors and from promotion of signaling complex assembly to degradation of inhibitors [ , ] . in this section, we discuss trim regulation of antiviral pathways (summarized in figures and ). viral double-stranded rna (dsrna) or single-stranded rna (ssrna) containing '-triphophates produced during virus replication, in the cytoplasm of a host cell, act as a retinoic acid-inducible gene i rig-i-like receptor (rlr) agonist [ ] [ ] [ ] [ ] . rig-i and melanoma differentiation-associated protein (mda ), encoded by ddx and ifih , respectively, bind distinct viral rna agonists yet they induce similar downstream antiviral pathways [ ] . rlrs are atp-dependent rna helicases that have two n-terminal caspase-activated recruitment domains (cards), a central dead box, and an auto-inhibitory c-terminal domain [ ] . the unique pamps recognized by these receptors enable the host to respond to a broader range of pathogens [ ] . upon engagement of the pamp with the rlr, a conformational shift exposes the cards, which allows homo-oligomerization and recruitment of the rlrs to their adaptor mitochondrial antiviral signaling protein (mavs) at the mitochondrial outer membrane (mom) [ , [ ] [ ] [ ] . a variety of factors influence the activation of rlrs downstream of pamp recognition including atp hydrolysis [ , ] , rlr oligomerization [ , , ] , and post-translational modifications [ ] [ ] [ ] [ ] [ ] . interaction of the n-terminal cards of both the rlrs and mavs induces the adaptor to form prion-like aggregates and exposes domains to recruit critical ring e ligases including tumor necrosis factor (tnf) receptor-associated factors (trafs) and [ , ] . downstream of traf , the ubd-containing adaptor tgf-β-activated kinase (tak )/mitogen activating protein k (map k )-binding protein (tab) / recruits the critical kinase tak [ ] [ ] [ ] . tak auto-phosphorylates to enable the phosphorylation of nf-κb essential modulator (nemo), the regulatory domain of inhibitor of nf-κb (iκb) kinase (ikk) complex, to activate the enzymatic domains of ikkα and β [ , ] . ikkα and β then phosphorylate iκb, resulting in the recruitment of another e ligase, β-trcp (β-transducin repeat containing e ubiquitin protein ligase). β-trcp ligates k -linked ubiquitin to iκb, inducing the proteasome-mediated degradation of the nf-κb inhibitor [ ] . once the inhibitor is destroyed, nf-κb is phosphorylated and its nuclear localization sequence is exposed allowing nuclear translocation [ , ] . inside the nucleus, nf-κb regulates the transcription of a variety of genes including pro-inflammatory cytokines and chemokines, such as pro-il- β, tnf-α, and il- , and negative regulators of the pathway to limit an exacerbated inflammatory response [ ] . vaccines , , of another e ligase, β-trcp (β-transducin repeat containing e ubiquitin protein ligase). β-trcp ligates k -linked ubiquitin to iκb, inducing the proteasome-mediated degradation of the nf-κb inhibitor [ ] . once the inhibitor is destroyed, nf-κb is phosphorylated and its nuclear localization sequence is exposed allowing nuclear translocation [ , ] . inside the nucleus, nf-κb regulates the transcription of a variety of genes including pro-inflammatory cytokines and chemokines, such as pro-il- β, tnf-α, and il- , and negative regulators of the pathway to limit an exacerbated inflammatory response [ ] . trims play an integral role in the positive and negative regulation of antiviral pathways. trims can act as pathogen prrs, as is the case for trim in the recognition of non-enveloped viruses bound by immunoglobulin (ig). additionally, these trims can regulate the activation of other prrs that recognize viral pathogen-associated molecular patterns (pamps) in the cytosol (ddx (dead-box helicase ), cyclic gmp-amp synthase (cgas), deah-box helicase (dhx ), nucleotide-binding oligomerization domain-containing protein (nod ), retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein (mda )) and at membrane surfaces (toll-like receptors, tlrs). downstream of the initial pattern recognition, trims also influence the recruitment and interaction of adaptor molecules (stimulator of ifn genes (sting), mitochondrial antiviral signaling protein (mavs), tgf-β-activated kinase (tak )/map k -binding protein (tab) , myeloid differentiation primary response gene (myd ), tir-domain-containing adapterinducing interferon-β (trif), nf-κb essential modulator (nemo), nucleosome assembly protein (nap- ), and tumor necrosis factor (tnf) receptor-associated factors (traf) family memberassociated nf-κb activator (tank)) and enzymes (traf , traf , tak , inhibitor of nf-κb (iκb) kinase (ikk) α,β,ε, tank binding kinase (tbk )) to signaling complexes in order to activate transcription factors. this includes ifn regulatory factor (irf) and irf , important in type-i interferon (ifn) signaling, and nf-κb, important in expression of pro-inflammatory genes, which regulate the expression of antiviral effectors. type-i ifn production is critical for an effective antiviral response. trims play an integral role in the positive and negative regulation of antiviral pathways. trims can act as pathogen prrs, as is the case for trim in the recognition of non-enveloped viruses bound by immunoglobulin (ig). additionally, these trims can regulate the activation of other prrs that recognize viral pathogen-associated molecular patterns (pamps) in the cytosol (ddx (dead-box helicase ), cyclic gmp-amp synthase (cgas), deah-box helicase (dhx ), nucleotide-binding oligomerization domain-containing protein (nod ), retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein (mda )) and at membrane surfaces (toll-like receptors, tlrs). downstream of the initial pattern recognition, trims also influence the recruitment and interaction of adaptor molecules (stimulator of ifn genes (sting), mitochondrial antiviral signaling protein (mavs), tgf-β-activated kinase (tak )/map k -binding protein (tab) , myeloid differentiation primary response gene (myd ), tir-domain-containing adapter-inducing interferon-β (trif), nf-κb essential modulator (nemo), nucleosome assembly protein (nap- ), and tumor necrosis factor (tnf) receptor-associated factors (traf) family member-associated nf-κb activator (tank)) and enzymes (traf , traf , tak , inhibitor of nf-κb (iκb) kinase (ikk) α,β,ε, tank binding kinase (tbk )) to signaling complexes in order to activate transcription factors. this includes ifn regulatory factor (irf) and irf , important in type-i interferon (ifn) signaling, and nf-κb, important in expression of pro-inflammatory genes, which regulate the expression of antiviral effectors. type-i ifn production is critical for an effective antiviral response. trims in cytokine signaling. downstream of the initial pathogen recognition and induction of pro-inflammatory cytokines, trims can regulate their cytokine signaling pathways through interactions with cytokine receptor adaptors (tab / ) and enzymatic proteins (ikkα, ikkβ and ikkε) within the signaling complexes, the activity and stability of pathway negative regulators (protein inhibitor of activated stat (pias ), suppressor of cytokine signaling (socs), and influence the transcription of various cytokine-effector genes (nf-κb-induced pro-inflammatory cytokines, signal transducer and activator of transcription (stat)-induced genes, interferon stimulated genes (isgs)) or cytokine signaling regulators (tumor necrosis factor (tnf) receptors (tnfr / , and stat-induced genes)). in addition to activation of nf-κb, rlr signaling induces type-i ifn [ ] . traf , in cooperation with nemo, recruits and stabilizes traf family member-associated nf-κb activator (tank) or nucleosome assembly protein (nap ) which are critical in linking tank binding kinase (tbk ), and in some cases inhibitor of kappa light polypeptide gene enhancer in b cells (ikkε), to the mavs signalosome [ , ] . once activated, tbk and/or ikkε phosphorylate the ifn regulatory factor (irf) and irf [ , ] . upon phosphorylation, the irfs homodimerize and translocate to the nucleus where they bind to dna regulatory regions [ , ] . to induce optimal ifn-β transcription, activated irf , nf-κb, and ap- (activator protein ) must translocate to the nucleus and bind to their respective regulatory regions of the ifnb promoter [ ] . the resulting ifn-β is then secreted and signals in a paracrine and autocrine manner. binding of ifn-β to its heterodimeric receptor results in the activation of tyrosine kinases, janus kinase (jak ) and tyrosine kinase (tyk ), which phosphorylate signal transducer and activator of transcription (stat) and stat . following phosphorylation, stat and stat heterodimerize and associate with irf to form ifn-stimulated gene factor (isgf ) and translocate to the nucleus [ ] . within the nucleus, isgf binds to genes with an ifn stimulated response element (isre) in their promoter to activate transcription [ ] . the resulting proteins expressed from these isgs, such as pkr (protein kinase r), mxa (myxovirus resistance gene a), isg , and trims, are involved in creating a cellular environment prohibitive to viral entry and replication [ ] . as with other immune pathways, isgf also promotes the transcription of type-i ifn negative regulators to mitigate deleterious effects [ ] . trims play a critical role in both the positive and negative regulation of the rlr pathway to ensure optimal virus restriction while minimizing self-inflicted damage ( figure ). several trims have been shown to positively regulate the receptors rig-i and mda [ ] [ ] [ ] . the best characterized example of trim-mediated rig-i activation involves trim . trim ligates k -linked poly-ub chains onto the n-terminal card at k , which induces downstream signaling [ ] . additionally, trim catalyzes the synthesis of unanchored k -linked poly-ub chains, which facilitate rig-i oligomerization and stabilization [ ] . both oligomerization and stabilization of rig-i promotes the interaction of its cards with mavs [ ] . adding complexity to this interaction, trim k -linked . trims in cytokine signaling. downstream of the initial pathogen recognition and induction of pro-inflammatory cytokines, trims can regulate their cytokine signaling pathways through interactions with cytokine receptor adaptors (tab / ) and enzymatic proteins (ikkα, ikkβ and ikkε) within the signaling complexes, the activity and stability of pathway negative regulators (protein inhibitor of activated stat (pias ), suppressor of cytokine signaling (socs), and influence the transcription of various cytokine-effector genes (nf-κb-induced pro-inflammatory cytokines, signal transducer and activator of transcription (stat)-induced genes, interferon stimulated genes (isgs)) or cytokine signaling regulators (tumor necrosis factor (tnf) receptors (tnfr / , and stat-induced genes)). in addition to activation of nf-κb, rlr signaling induces type-i ifn [ ] . traf , in cooperation with nemo, recruits and stabilizes traf family member-associated nf-κb activator (tank) or nucleosome assembly protein (nap ) which are critical in linking tank binding kinase (tbk ), and in some cases inhibitor of kappa light polypeptide gene enhancer in b cells (ikkε), to the mavs signalosome [ , ] . once activated, tbk and/or ikkε phosphorylate the ifn regulatory factor (irf) and irf [ , ] . upon phosphorylation, the irfs homodimerize and translocate to the nucleus where they bind to dna regulatory regions [ , ] . to induce optimal ifn-β transcription, activated irf , nf-κb, and ap- (activator protein ) must translocate to the nucleus and bind to their respective regulatory regions of the ifnb promoter [ ] . the resulting ifn-β is then secreted and signals in a paracrine and autocrine manner. binding of ifn-β to its heterodimeric receptor results in the activation of tyrosine kinases, janus kinase (jak ) and tyrosine kinase (tyk ), which phosphorylate signal transducer and activator of transcription (stat) and stat . following phosphorylation, stat and stat heterodimerize and associate with irf to form ifn-stimulated gene factor (isgf ) and translocate to the nucleus [ ] . within the nucleus, isgf binds to genes with an ifn stimulated response element (isre) in their promoter to activate transcription [ ] . the resulting proteins expressed from these isgs, such as pkr (protein kinase r), mxa (myxovirus resistance gene a), isg , and trims, are involved in creating a cellular environment prohibitive to viral entry and replication [ ] . as with other immune pathways, isgf also promotes the transcription of type-i ifn negative regulators to mitigate deleterious effects [ ] . trims play a critical role in both the positive and negative regulation of the rlr pathway to ensure optimal virus restriction while minimizing self-inflicted damage ( figure ). several trims have been shown to positively regulate the receptors rig-i and mda [ ] [ ] [ ] . the best characterized example of trim-mediated rig-i activation involves trim . trim ligates k -linked poly-ub chains onto the n-terminal card at k , which induces downstream signaling [ ] . additionally, trim catalyzes the synthesis of unanchored k -linked poly-ub chains, which facilitate rig-i oligomerization and stabilization [ ] . both oligomerization and stabilization of rig-i promotes the interaction of its cards with mavs [ ] . adding complexity to this interaction, trim k -linked polyubiquitination negatively regulates rlr activation, but the ubiquitin specific protease (usp ) can specifically disassemble these poly-ub chains to stabilize trim [ ] . trims , , and have also been implicated in positive regulation of the rig-i pathway. similar to trim , trim also catalyzes the ligation of k -linked poly-ub chains onto rig-i card [ ] . additionally, trim functions as an e sumo (small ubiquitin-like modifier) ligase and sumoylates both rig-i and mda to prevent the ligation of k -linked poly-ub chains thus stabilizing these prrs [ , , ] . the capacity of multiple trims to activate rig-i suggests that ubiquitination is crucial in rig-i signaling, but the relative contribution of each trim is not well understood. perhaps multiple trims allow for redundancy in the instance that one trim is inhibited or if trims play cell-type specific roles in rlr signaling. recently trim was identified as an e ligase of mda . unlike trim , trim ubiquitinates mda at the rna helicase domain [ ] . the covalent linkage of k -linked poly-ub onto k promotes mda oligomerization and downstream activation of irf [ ] . demonstrating the specificity of mda activation, trim only promotes the restriction of encephalomyocarditis virus (emcv), a picornavirus, and not vesicular stomatitis virus (vsv), a rhabdovirus [ ] . in mouse cells, trim was shown to impair mda -mediated activation of the ifn pathway through an unclarified mechanism [ ] . another trim inhibitor of the mavs pathway is trim , which interacts with evolutionarily conserved signaling intermediate in toll pathways (ecsit) and mavs and subsequently inhibits the transcription of irf and nf-κb target genes [ ] . although trims have not been identified as rig-i negative regulators, their role in mda inhibition suggests there may be unidentified trim-mediated rig-i inhibition. the role of trim in the regulation of rlr pathways and/or type-i ifn induction has been shown to be conserved among different species. in fact a diverse range of vertebrates encode rig-regulating trims. in salmonids, trim , mavs, mda , and rig-i were induced following infection with an alphavirus although the signaling pathways were not addressed directly [ ] . duck trim catalyzes the synthesis of unanchored poly-ub chains to activate rig-i [ ] . despite lacking lysine in duck rig-i, duck trim ubiquitinates rig-i's card domains and promotes rlr signaling [ ] . in chicken cells, despite lacking a functional rig-i gene, knockdown of chicken trim results in reduced ifn-β upon infection with specific strains of the influenza a virus (iav) [ ] , suggesting that trim is involved in activation of ifn signaling through a rig-i-independent mechanism, perhaps activation of mda or mavs. expression of chicken trim is induced after newcastle disease virus (ndv), poly(i:c) treatment, or poly(da:dt) treatment [ ] , probably via a type-i ifn signaling-dependent pathway. similar to human trim , trim -l stimulates ifn-β production in response to iav infection in ducks [ ] . this anti-viral benefit is absent in chickens and turkeys, as they do not carry the trim -l gene in their trim cluster [ ] . however, expression of duck trim -l and d card in chicken df cell lines was shown to facilitate ifn-β and mx expression [ ] . this difference may account for the different pathologies in avian species as waterfowl are typically more resistant to some strains of iav as compared to chickens. downstream of rlr activation, a variety of trims promote mavs signaling. trim has been implicated in the k -linked polyubiquitination of mavs, which results in its proteasome-mediated degradation and release of downstream signaling molecules (tbk , nemo, and possibly traf ) to induce type-i ifn production [ ] . recently, trim has been described to mediate the k -linked ubiquitination of mavs at lysines , , and [ ] . this ubiquitination promotes the prion-like aggregation of mavs needed for optimal signaling, exemplified by the decrease in tbk and ikkε phosphorylation [ ] . in a trim -deficient murine model, trim knock-out mice demonstrated an increased susceptibility to vsv infection and an upregulation in ifn-β production. although rig-i was demonstrated to be required for activation of trim function, its role in mda -mediated signaling was not investigated in-depth [ ] . trim has been demonstrated to play a crucial role in linking the nf-κb and irf branches of rlr signaling [ ] . despite lacking a ring domain, trim interacts with nemo via its pry-spry domain and promotes k -linked ubiquitination of nemo, which is critical in recruiting nemo to mavs [ ] . the role of trim stabilization of mavs has also been characterized [ , ] . although no trim inhibitors of mavs have been described, screens of trims that inhibit mavs-mediated type-i ifn production may reveal such trims. in addition to regulating rlrs and mavs, trims modulate downstream adaptors and enzymes. several of these trims can likewise mitigate signaling downstream of other prrs that converge on shared molecules such as nemo and tak , but trims identified to be involved at the level of signaling using rlr induction are described below. recently, the short isoform of trim (trim s) was shown to bridge gsk β ( glycogen synthase kinase beta) to phosphorylated tbk to promote tbk oligomerization and activation of irf [ ] . to facilitate the interaction between gskβ and ptbk , trim s must be auto-ubiquitinated [ ] . this activation of tbk -signaling occurs downstream of rlr and sting signaling [ ] and biases the immune response toward the type-i ifn pathway while limiting nf-κb-induced transcription [ ] . trim 's coiled-coil domain interacts with the coiled-coil domain of tbk to prohibit the kinase's interaction with adaptors nap or tank [ ] . impairing this interaction results in lack of ifn-β production [ ] . nap is targeted for degradation following trim -mediated k -linked poly-ub, which likewise decreases activation of ifn-β [ ] . interaction between the arf (adp-ribosylation factor domain) (c-terminus) domain of trim , and both the coiled-coil and lz domains of nemo, allow trim to facilitate k -linked ubiquitination of nemo [ ] . this ubiquitination of nemo facilitates the activation of irf and nf-κb signaling downstream of pathogen recognition, but not tnf-α signaling [ ] . similar to the described trim s mechanism, trim is able to interact with tbk and auto-phosphorylates k -linked poly-ub chains to bridge tbk and nemo [ ] . this interaction was demonstrated downstream of mavs signaling and promoted irf activation [ ] . indirectly, trim antagonizes ifn-β transcription [ ] . this trim is able to inhibit both toll-like receptor (tlr) and rlr-driven activation of the type-i ifn pathway [ ] . several trims are likewise implicated in the inhibition of the nf-κb activation branch of prr signaling. murine specific trim α negatively regulates the tab /tab complex, which impedes the recruitment and activation of tak , thus preventing the phosphorylation of nemo and subsequent nf-κb activation [ , ] . through a different mechanism in the brain, the long isoform of trim (trim l) inhibits β-trcp [ ] . perturbation of β-trcp inhibits both canonical and non-canonical nf-κb activation [ ] . the trim l protein sequence includes a degron motif that, when phosphorylated at serine residues and , recruits β-trcp [ ] . titrating β-trcp from the nf-κb inhibitors prevents their degradation and subsequent pro-inflammatory signaling [ ] . several other trims also inhibit nf-κb activation including trim [ ] , trim [ ] , and trim [ ] . the mechanisms for trim regulation have not been clearly elucidated, but trim targets nemo for degradation in alveolar macrophages [ ] and trim stabilizes cactin [ ] , a nuclear, negative regulator of nf-κb. the bias of trims in the negative regulation of the nf-κb branch of rlr signaling suggests that trims may play a role in promoting the type-i ifn pathway at the cost of nf-κb activation, and further supports the hypothesis that groups of trims may have evolved as part of the antiviral type-i ifn system [ ] . however, it is important to note that some studies showing negative regulatory roles of trims have only used overexpression assays with large concentrations of trim expressing vectors, which could lead to artifacts. however, it is now clear that overall trims act at several levels to regulate rlr signaling to balance viral clearance and cell survival. in addition to regulating cytosolic rna-stimulated responses, trims also regulate the pathways following cytosolic dna recognition. in the cytoplasm, the host expresses multiple dna and rna receptors aside from rlrs, including ifi (interferon gamma inducible protein ), cyclic gmp-amp synthase (cgas), and ddx . the listed double-strand (ds) dna receptors activate the adaptor molecule stimulator of ifn genes (sting) at the endoplasmic reticulum (er) [ ] [ ] [ ] [ ] [ ] . upon recognition of dsdna, which can result from infection with dna viruses, cgas oligomerizes and catalyzes cyclic dinucleotide (c-gmp-amp) synthesis [ ] . c-gmp-amp and other dinucleotides activate sting and induce sting dimerization [ , , ] . consequently, sting recruits tbk , which phosphorylates irf for type-i ifn induction [ ] . the dna helicase ddx recognizes both cytoplasmic dsdna and cyclic dinucleotides, both of which promote ddx activation of sting [ , ] . five trims are known to influence sting-mediated signaling ( figure ). the pry-spry domain of trim interacts with ddx 's helicase domain and catalyzes ubiquitination at lysine residues and , targeting the prr for degradation [ ] . this degradation pathway occurs in myeloid dendritic cells and restricts type-i ifn induction [ ] . the murine-specific trim, trim α, ubiquitinates sting following herpes simplex virus (hsv- ) and targets the adaptor protein for degradation [ ] . both trim and trim facilitate k -linked polyubiquitination of sting to promote dimerization and activation of sting-mediated antiviral responses [ , ] . finally, trim sumoylates cgas and sting similar to the sumoylation of rig-i and mda [ ] . sumoylation inhibits the ligation of k -linked ubiquitin and results in their stabilization [ ] . in addition to regulating cytosolic prr signaling, trims also modulate membrane-bound prrs including toll-like receptors (tlrs) ( figure ). several tlr family members recognize viral pamps. the main tlrs involved in virus recognition include endosome-localized tlrs , , , and [ ] . tlr recognizes both double-stranded rna and the viral rna mimic poly(i:c), tlr and tlr recognize single-stranded rna, and tlr recognizes cpg [ ] . tlr is associated mainly with the plasma membrane, although it can also be internalized in endosomes, and can recognize some viral surface antigens. tlrs , , , and signal through iraks (interleukin- receptor-associated kinase) and , which are recruited via the adaptor molecule myd (myeloid differentiation primary response gene ) [ ] . traf then re-localizes to the tlr signaling complex to activate tak , inducing nf-κb [ , ] similar to the pathway described in the aforementioned rlr section. tlr and also signal through iraks and , and interact with the adaptor trif (tir-domain-containing adapter-inducing interferon-β), resulting in the activation of traf . ubiquitination downstream of traf induces tbk -and ikkε-mediated phosphorylation of irf and irf to induce type-i ifn [ ] . trim 's pry-spry domain interacts with irfs, including irf , , and , to induce their degradation [ ] [ ] [ ] . although trim may promote degradation of irfs downstream of other prrs, the interaction between the two proteins may depend on the induction of a specific tlr pathway. trim targets tlr signaling at multiple points. downstream of tlr , , , or , trim targets traf for proteasome-mediated degradation following k -linked polyubiquitination [ ] . downstream of tlr and tlr signaling, trim also targets trif and nap for degradation [ , ] . finally, trim has been shown to promote tlr activation via interaction with trif in a ring ligase-independent manner [ ] . perhaps this interaction promotes the stability of trif to facilitate downstream signaling. disruption of adaptor protein availability thus bottlenecks antiviral signaling. nucleotide-binding domain and leucine-rich repeat-containing receptors (nlrs) are another class of cytosolic receptors that recognize both pamps and damage-associated molecular patterns (damps). damps are host-derived molecules that are expressed only after a cell experiences stress and/or damage commonly due to inflammation [ , ] . upon activation of a nlr, the receptor assembles an inflammasome in cooperation with the adaptor asc to recruit pro-caspases [ , ] . the nlrp (nlr family pyrin domain containing ) -induced inflammasome promotes the cleavage of pro-caspase to caspase , which then promotes a pro-apoptotic response involving the caspase- -mediated cleavage of pro-il- β and pro-il- to their active forms il- β and il- [ , ] . the secreted pro-inflammatory cytokines then promote further inflammatory responses, such as pyroptosis, which may be damaging to the host when uncontrolled [ , ] . in some instances another nlr, nod , may function as a cytosolic dsrna receptor and converge with the rlr pathway at the level of mavs [ , ] . at this point only a few trims have been described to interact with nlrs in the control of viral infections. trim binds to and ubiquitinates dhx , a cytosolic dsrna receptor that acts upstream of nlrp , at lysine k to facilitate inflammasome activation [ ] . the knockdown of trim diminishes the activation of caspase and likewise decreases the release of il- β and il- [ ] . the interaction of trim with dhx relies on the b-box and coiled-coil domains [ ] . in contrast, the murine-specific trim α impairs the nlrp inflammasome through an unknown mechanism [ ] . another nlrp inhibitor identified using a dextran sodium sulfate-induced colitis model is trim [ ] . the coiled-coil domain of trim interacts with the leucine rich and nacht domains of nlrp and ligates k -linked poly-ub chains to target nlrp for proteasome-mediated degradation [ ] . although the authors did not evaluate virus infection directly, trim -deficient cells stimulated with poly(i:c) express higher levels of nlrp compared to wild-type cells suggesting trim may regulate nlrp downstream of virus recognition [ ] . trim is able to promote degradation of nod [ ] , which may prohibit this receptor from recognizing dna and rna virus infection [ , ] . the further investigation of trim interactions with nlrs is important for understanding the immune response during bacteria-virus co-infections. several trims are also involved in regulating the cytokine signaling following initial pathogen recognition ( figure ). downstream of tnf-α engagement with its receptor tnfr , the tak complex is recruited to activate nf-κb [ ] . trim specifically promotes the activation of tnf-α-induced nf-κb signaling through inhibition of the nf-κb nuclear repressor protein inhibitor of activated stat (pias) [ ] . the trim -mediated repression of pias likewise promotes il- -dependent activation of stat [ ] . specifically downstream of il- β and tnf-α signaling in ifn-β-primed cells, trim targets tab for lysosomal degradation [ , ] . as described above, the degradation of tab inhibits the recruitment and activation of tak which blocks pro-inflammatory signaling. zheng and colleagues showed that trim catalyzes k -linked poly-ub at residues k and k of tbk to promote proteasome-mediated degradation downstream of ifn signaling [ ] . trim -tbk interactions require the coiled-coil and b-box trim domains [ ] . in endothelial cells, trim sustains the expression of tnfr and tnfr to promote the activation of pro-inflammatory pathways [ ] . the role of trim in the activation of the endothelium may play an important, unexplored role in immune cell trafficking in response to infection. in response to cytokines (i.e., il- ) secreted from activated dendritic cells (dcs), ifn-γ (type-ii ifns) is released from t cells and natural killer cells [ ] . after this, type-ii ifn binds its receptor, and the downstream kinases jak and jak promote stat phosphorylation and homodimerization to form gamma activating factor, which translocates to the nucleus to bind gamma-stimulated elements in gene promoters [ ] . trim inhibits stat transcription via binding to the stat promoter [ ] . in contrast, trim is able to destabilize socs- (suppressor of cytokine signaling ), a negative regulator of the ifn-γ signaling pathway, resulting in increased type-ii ifn signaling [ ] . the regulation downstream of ifn signaling suggests that this is a negative regulatory mechanism to prevent inflammatory response overactivation. in response to type-i ifns, trim catalyzes the formation of k -linked unanchored poly-ub chains to facilitate the activation of ikkε, which favors isgf formation due to stat phosphorylation at s [ ] . this modification increases stat -stat dimerization [ , ] . this trim -enahnced activation of ikkε may also play a role in activating ifn-β transcription and translation downstream of rlr activation [ ] . trim induces the lysosome-mediated destruction of foxo and consequently impairs the transcription of ifn-β downstream of tlr and rlr signaling [ ] . after the innate response to pathogen invasion, the host evolves an adaptive immune response. establishment of an adaptive response requires the presentation of pathogen antigens to t and b lymphocytes. although trim proteins have been mostly studied as regulators of innate immune responses and the role of trims in the regulation of antigen presentation has not been investigated intensively, several studies indicate that trims play an important role in regulating t cell activation. expression of trim , for example, has been shown to be down-regulated upon cd /cd -mediated activation despite being expressed at high levels in resting t cells [ ] . in contrast, trim expression in t cells is increased following il- and il- cytokine signaling [ ] , both of which act as pro-survival signals. recently, trim was shown to influence the expression of th -type cytokines but not other cd + t cell sub-types [ ] . as th cells predominantly act in allergy-and parasite-induced responses while th cells are more important for viral clearance, deregulation of trim expression may impact the cd + population composition and the capacity of the host to efficiently clear the virus. in the past, trim was described as impairing the activation of cd + t cells via k -linked poly-ub of pi kc b (phosphatidylinositol -phosphate -kinase c domain-containing subunit beta) [ ] . this impairment was observed specifically in cd + t cells downstream of t cell receptor (tcr) engagement and not cd + t cells [ ] . additionally, trim depletion in vivo promotes expansion of the th population resulting in an autoimmune phenotype [ ] . following tcr-mediated activation, trim was phosphorylated suggesting it plays a role t cell activation [ ] . throughout the lifespan of trim knockout mice, the ratio of cd + to cd + progressively increased and the cd + t cells proliferated abnormally [ ] . the role of trim is intrinsic to cd + t cells, because the same defect was observed upon adoptive transfer of knockout cd + t cells to wild-type mice [ ] . it remains to be seen whether other trims play important roles in adaptive immunity. it is likely that trims have unexplored functions in recognition of antigen presentation by the t cell receptor, and or in differentiation of cd + /cd + t cells. despite the numerous host evasion mechanisms pathogens employ, a variety of host encoded molecules, such as trims, are able to restrict viruses [ ] . in addition to conferring an antiviral state indirectly by regulating cytokine production downstream of prr signaling, trims are capable of restricting the effectiveness of pathogens through direct interactions with viral proteins crucial to their entry, dissemination, or life cycle [ ] . the categories of viral restriction include: inhibition of viral transcription, replication or translation, degradation or interference of viral proteins, and impairment of virus entry or exit. we next outline the various means by which hosts deploy trims to counter and clear pathogens. trim α is an example of a trim functioning as both a direct virus restriction factor as well as a pathogen-recognition receptor ( figure ). in old-world monkeys, such as african green monkeys and rhesus macaques, trim α enables natural resistance to hiv- while new-world monkeys, like owl monkeys, are protected from hiv- by fusion of trim with cyclophilin a (tcypa) [ ] [ ] [ ] [ ] . progress on the topic ranges from revealing the α isoform of trim as the restriction factor to discerning the role in restriction of each structural motif [ , ] . . the role of trims in retrovirus replication. trim α oligomerization into a hexagonal lattice associates directly with hiv- capsids to promote premature uncoating. recognition of hiv- capsid by trim α also triggers nf-κb/ap- -mediated innate immune signaling via synthesis of unanchored k -linked poly-ub chains that activate the tak kinase. one potential mechanism of trim αmediated restriction could be involved ubiquitination of trim α and proteasomal degradation of trim α-capsid complexes. trim mobilizes cellular microtubule formation to prematurely uncoat hiv- and facilitate rapid release of the vrna from the viral core, resulting in inhibition of virus replication. trim translocates to the cytoplasm and binds daxx to prevent its degradation by the proteasome, allowing for daxx-mediated disruption of hiv- reverse transcription (rt). trim inhibits sp , preventing ltr-mediated transcription. the connection between trim α-mediated restriction and proteasomal-mediated degradation of hiv- requires further characterization. however, one potential model may involve improved proliferation of hiv- -specific cd + t cells due to enhanced production of viral peptides from proteasome-mediated degradation of hiv- capsids. several components inherent to trim α play a role in hiv- capsid restriction, including dimerization, oligomerization, and ubiquitination [ ] [ ] [ ] . there have been different models proposed for the mechanism of restriction and all have been attributed to the interaction of the spry domain of trim α with the retrovirus capsid. binding of trim α to viral cores serves to prematurely uncoat the viral particle and induces early release of the viral genome. elucidation of the trim α higher order structure has revealed the significant contributions made by each component of the trim α protein [ , [ ] [ ] [ ] [ ] . monomeric trim α can dimerize in an antiparallel fashion through the coiled-coil domains, thereby generating the most fundamental component needed to bind the viral capsid [ , ] . once bound to its target, trim α can form higher-order structures resembling a hexagonal net [ , , , ] . trim α and trim variants from several primate species, including rhesus macaques, african green monkeys, and owl monkeys, are capable of forming flexible, hexagonal frameworks encompassing hiv- capsid surfaces [ ] . the hexagonal nets are formed from trim α trimers and are dependent on the trim α's b-box domain [ , ] . these trimers have been shown to be flexible and may allow for ideal binding of the spry domains on the highly variable hiv- capsid [ , ] . the ability of rhesus macaque trim α (rhtrim α) to form higher-order structures upon binding to the capsid also allows the formation of trim α ring dimers, which enhances its e -ubiquitin ligase activity and innate anti-hiv- activity [ ] . as one potential mechanism of rhtrim α-mediated restriction it was proposed that as the linker regions of the dimer change their conformation, the spry domains that are bound to the hiv- capsid in their hexagonal lattice formation disturb the viral structure [ ] . the induction of this conformational change disrupts the integrity of the viral core and may be responsible for pre-mature viral uncoating. capsid by trim α also triggers nf-κb/ap- -mediated innate immune signaling via synthesis of unanchored k -linked poly-ub chains that activate the tak kinase. one potential mechanism of trim α-mediated restriction could be involved ubiquitination of trim α and proteasomal degradation of trim α-capsid complexes. trim mobilizes cellular microtubule formation to prematurely uncoat hiv- and facilitate rapid release of the vrna from the viral core, resulting in inhibition of virus replication. trim translocates to the cytoplasm and binds daxx to prevent its degradation by the proteasome, allowing for daxx-mediated disruption of hiv- reverse transcription (rt). trim inhibits sp , preventing ltr-mediated transcription. the connection between trim α-mediated restriction and proteasomal-mediated degradation of hiv- requires further characterization. however, one potential model may involve improved proliferation of hiv- -specific cd + t cells due to enhanced production of viral peptides from proteasome-mediated degradation of hiv- capsids. several components inherent to trim α play a role in hiv- capsid restriction, including dimerization, oligomerization, and ubiquitination [ ] [ ] [ ] . there have been different models proposed for the mechanism of restriction and all have been attributed to the interaction of the spry domain of trim α with the retrovirus capsid. binding of trim α to viral cores serves to prematurely uncoat the viral particle and induces early release of the viral genome. elucidation of the trim α higher order structure has revealed the significant contributions made by each component of the trim α protein [ , [ ] [ ] [ ] [ ] . monomeric trim α can dimerize in an antiparallel fashion through the coiled-coil domains, thereby generating the most fundamental component needed to bind the viral capsid [ , ] . once bound to its target, trim α can form higher-order structures resembling a hexagonal net [ , , , ] . trim α and trim variants from several primate species, including rhesus macaques, african green monkeys, and owl monkeys, are capable of forming flexible, hexagonal frameworks encompassing hiv- capsid surfaces [ ] . the hexagonal nets are formed from trim α trimers and are dependent on the trim α's b-box domain [ , ] . these trimers have been shown to be flexible and may allow for ideal binding of the spry domains on the highly variable hiv- capsid [ , ] . the ability of rhesus macaque trim α (rhtrim α) to form higher-order structures upon binding to the capsid also allows the formation of trim α ring dimers, which enhances its e -ubiquitin ligase activity and innate anti-hiv- activity [ ] . as one potential mechanism of rhtrim α-mediated restriction it was proposed that as the linker regions of the dimer change their conformation, the spry domains that are bound to the hiv- capsid in their hexagonal lattice formation disturb the viral structure [ ] . the induction of this conformational change disrupts the integrity of the viral core and may be responsible for pre-mature viral uncoating. proteasomal-mediated degradation of viral components is a host restriction strategy characteristic of many trim family proteins involved in host innate immunity. however, the functional role of the proteasome in trim α-mediated hiv- restriction has been difficult to discern and has been subject of debate. early work with proteasomal inhibitors, like mg , suggested a two phase restriction of hiv- by trim α. in the proteasome-independent phase, trim α inhibits nuclear entry of the reverse transcription (rt) products [ , ] . in the second phase, trim α can inhibit late rt products in the presence of a functional proteasome, suggesting trim α may utilize the proteasome to disrupt the viral rt complex [ , ] . while proteasome inhibition can block disassembly and/or degradation of viral core components, it does not appear to rescue infectivity, which has led some investigators to conclude that degradation of capsid by the proteasome is not the mechanism of trim α-mediated restriction [ ] . in addition, thus far no study has described ubiquitination sites on the hiv- capsids, which could potentially link degradation of the capsid with proteasomal function. however, additional studies have shown proteasomal involvement in trim α restriction of hiv- by degrading trim α itself [ ] . trim α's e ligase activity can facilitate auto-ubiquitination, thereby signaling for its own destruction by the host proteasomal machinery [ , ] . these occurrences have led some to speculate on a potential model where trim α interacts with the hiv- capsid leading to auto-ubiquitination, capsid uncoating, and delivery to the proteasome [ ] [ ] [ ] [ ] . despite numerous investigations centered around proteasomal involvement, controversy within the literature surrounding the exact model connecting trim α, hiv- cores, and proteasomes remains, and proteasome-dependent and independent mechanism have been proposed [ , ] . polyubiquitination through the e ligase activity of trim α's ring domain may also be a factor involved in inhibition of retroviral replication [ ] . in addition to its restriction activity, trim α can induce antiviral type-i ifns. multiple trim orthologs induce ap- -mediated innate immune signaling [ , ] . in the presence of the hiv- capsid, the e ligase activity of trim facilitates the generation of unanchored k -linked poly-ub chains. these k -linked chains promote tak autophosphorylation and activation, resulting in the induction of ap- -and nfκb-mediated transcription [ ] . collectively, these recent findings serve to better characterize the mechanisms through which trim α achieves inhibition of hiv- replication. the restriction benefits conferred by trim α can be observed in the progression to disease upon infection with simian immunodeficiency virus (siv) in rhesus macaques. course of infection studies with either a trim α-sensitive or -resistant strain of siv revealed that inoculation with a trim α susceptible strain of siv provided higher survival rates for rhesus macaques. also, delayed development of the pathology associated with trim α-sensitive siv compared to subjects treated with a trim α-resistant strain was observed [ ] . in addition to these benefits, the restriction of the trim α sensitive siv strain correlated with prolonged maintenance of cd + central memory t cells [ ] . the preservation of cd + central memory t cells has been extensively characterized as important in resisting viremia and generating ctl (cytotoxic t lymphocytes) subsets [ ] [ ] [ ] . the prolonged presence of these cd + t cells in the trim α-susceptible siv-infected treatment may be responsible for the higher survival rate [ ] . in spite of this, trim α escape mutants were present [ ] . half of the subjects that received the trim α-sensitive strain of siv still developed aids, and a third succumbed to infection at a similar time as the trim α-resistant group [ ] . sequencing of the siv capsids from these macaques revealed two mutations in the region encoding the gag protein, which resulted in nonsynonymous substitutions that mimicked the alterations made by the authors to generate their trim α resistant strain [ ] . subversion of trims is a central evolutionary strategy for viral innate immune evasion, as demonstrated by the high mutational rate of retroviruses. although first described as a viral restriction factor against hiv- in old-world monkeys, humans and other species are capable of utilizing trim α to inhibit other retroviruses. human trim α is incapable of restricting hiv- , yet a single amino acid mutation in its pry-spry domain can confer resistance to the pathogen [ ] . instead, human trim α can bind to and restrict n tropic mouse leukemia virus (n-mlv) [ ] . interestingly, the -amino-acid stretch in the pry-spry domain of primate trim α is under strong positive selection [ ] . trim α paralogues have been identified in bovine [ , ] , ovine [ ] , and piscine [ ] species, and have also been shown to restrict retroviruses. overall, trim α plays a convergent role in the recognition and restriction of retroviruses. aside from its role in non-human primates, trim α has been described functioning in a cell-type specific manner [ ] [ ] [ ] . in contrast to conventional dc-sign + (dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin) dcs, langerhans cells (lcs) benefit from the anti-retroviral capabilities of trim α through an increased activity of the lc autophagocytic components [ ] . upon langerin-mediated hiv- uptake, the presence of cellular autophagosomes increases as a result of trim α-directed assembly, leading to targeting of the viral capsid for destruction [ ] . further evidence of cell-type specific hiv- restriction by trim α was found in rhesus macaque dcs, which in contrast to macrophages appear to be permissive to hiv- infection [ ] . the lack of trim α-mediated restriction in both human and macaque conventional dcs may be due to sumoylation of trim α, which promotes its sequestration in nuclear bodies [ ] . however, this lack of trim α-mediated restriction in dcs provides an innate immune sensing advantage by allowing recognition of hiv- reversed transcribed dna by cgas, resulting in type-i ifn production [ ] . it remains to be seen whether hiv- can actively promote or enhance sumoylation of trim α in dcs as mechanism to antagonize type-i ifn production. the ability of trim α to promote restriction of hiv- may go beyond direct intervention of the viral lifecycle. trim α has also been linked to the activity of other components of cellular innate immunity including antigen presentation to cytotoxic t lymphocytes (ctl). tcypa and rhtrim α enhanced the ability of cd + t cells to identify hiv- infected cells, and promoted a hiv- -specific immune response [ ] . the presence of these trim orthologs was associated with increased associations between hiv- particles and the host proteasome [ ] . it is speculated that this may facilitate improved hiv- -specific ctl development, as amplified peptide concentrations could support enhanced antigen presentation to cd + t cells. the mechanism linking direct restriction of viral capsids with heightened cd + t cell activation warrants further investigation. a plethora of trim-mediated restriction mechanisms targeting hiv- and other retroviruses have been proposed [ ] , and have been reviewed previously [ ] . more recent reports acknowledged trim as a potent host restriction factor of hiv- [ , ] . the mechanisms of trim -mediated restriction include curbing the amount of viral reverse transcription products allowed to accumulate in the host. through an interaction with the viral capsid-nucleocapsid protein (ca-nc) complexes, trim promotes premature uncoating and release of the viral genetic material, reducing transduction efficiency. neither proteasomal nor lysosomal inhibitor treatments recovered viral p protein in the pellets of trim overexpressing cells, suggesting that ubiquitin-mediated degradation by the proteasome or lysosomal acidification is not required for trim -mediated uncoating [ ] . furthermore, while rhtrim α-mediated inhibition of hiv- is rescued by proteasome inhibitors [ , ] , trim -mediated inhibition was not, indicating that trim and trim α restrict hiv- by different mechanisms [ , ] . in contrast, using the microtubule dynamics inhibitors nocodazole and taxol, the authors were able to demonstrate a restoration of hiv- capsid levels in cells supplemented with exogenous trim , suggesting that microtubules may contribute to trim -mediated hiv- restriction [ ] . earlier work with the use of nocodazole and taxol in a study examining hiv- and trim α also demonstrated recovered hiv- infectivity in the absence of functional microtubules [ ] . although the exact mechanism of trim -mediated restriction of hiv- in the aforementioned study has yet to be determined, the authors demonstrated that purified trim associated with in vitro assembled hiv- capsids [ ] , indicating that trim interacts directly with the capsid and it probably does not require trim α or other cellular proteins for promoting untimely capsid uncoating. microtubule involvement in viral uncoating has been implicated as both a host restriction and a viral propagation mechanism [ ] [ ] [ ] [ ] . functional microtubules and their associated motor proteins, like dynein, have been suggested as key components in genome release for both iav and hiv- [ ] [ ] [ ] [ ] . interestingly, in the case of iav, unanchored poly-ub chains contained in the virion are recognized by the host histone deacetylase (hdac ), a component of the aggresome-autophagy pathway, which interacts with dynein and microtubules to promote viral uncoating [ , ] . it will be interesting to examine whether hiv- utilizes a similar mechanism and whether this is mediated by trim . additionally, several members of the trim family (trims , , , , , and ) have been shown to possesses a c-terminal domain motif allowing for association with cytoskeletal elements like microtubules [ ] . so far only trim (also called mid ) and trim (also called mid ) have been shown to be directly involved in microtubule stabilization [ ] , and trim has been implicated in restriction of n-mlv [ ] . whether trim -mediated restriction is dependent on microtubules, or whether other microtubule-interacting trims may have viral restriction activity by microtubule-dependent mechanisms, remains to be seen. trim-mediated suppression of hiv- replication has revealed additional avenues through which trims subdue pathogens. trim prevents normal viral transcription events by regulating the effectiveness of the transcription factor sp to bind the hiv- long terminal repeat (ltr) promoter region [ ] . this restriction was independent of trim 's e ligase activity and did not involve direct interaction between trim and sp , implying that the observed reduction in hiv- ltr-mediated transcription requires additional unspecified factors [ ] . trim exhibits anti-retroviral functions through interference of hiv- infection, replication, and transcription, possibly interfering with dna synthesis [ ] . however further investigation into trim-mediated transcriptional inhibition will be required to reveal additional pathways in which trims play a significant role. another example of indirect inhibition of virus replication by trims is illustrated by the promyelocytic leukemia protein (pml)/trim . pml/trim interferes with hiv- infection in human and murine fibroblasts through the reduction of reverse transcriptase products [ , ] . this effect was dependent on two events; the translocation of pml/trim from the nucleus to the cytoplasm in the presence of hiv- infection, as well as association of the pml/trim cytoplasmic bodies (cb) with daxx [ ] . pml/trim interacts with daxx in a protective fashion in order to prevent its degradation by the proteasome, thereby making pml/trim a necessary component for daxx-mediated inhibition of hiv- rt [ ] . the inhibition of hiv- by pml/trim and daxx may be cell-type dependent, since studies using different cell types have shown different results [ , ] . another well characterized trim involved in pathogen recognition is trim , which has been shown to detect intracellular antibody-opsonized viruses (reviewed previously in [ , ] ). some immunoglobulin-coated non-enveloped viruses are internalized into the host cell. within most cells, a high affinity antibody receptor, trim , binds to the highly conserved fc region of virion-bound immunoglobulin (ig)g, igm, or iga [ , ] , and targets the virus for degradation by the proteasome before the virus can transcribe its genes [ ] . trim 's pry-spry domain interacts with fc residues conserved across mammalian species [ ] . trim binding with an immunoglobulin-virion (ig-v) complex triggers tightly regulated intracellular antibody neutralization and pro-inflammatory pathways [ , ] . upon engagement of trim with the ig-v complex within the cytoplasm, the e ube w (ubiquitin conjugating enzyme e w) monoubiquitinates trim , which promotes ube n/ube v e complex recruitment to trim for k -linked poly-ub [ ] . following poly-ub, trim is recruited to the proteasome to initiate degradation of the antibody-bound virus. concurrently, with proteasome-mediated degradation, poh de-ubiquitinates trim [ ] . this antibody-dependent intracellular neutralization appears to be limited to non-enveloped dna and rna viruses that can enter the host cytosol with immunoglobulin attached to the virion surface [ ] . the de-ubiquitinated trim is then able to promote both ifn induction and nf-κb activation [ , , ] , hypothesized to result from the release of the viral genome into the cytoplasm for prr-mediated recognition [ ] . however, some antiviral cytokines are induced independent of prrs [ ] , but the pathway is not well characterized. in addition, it has also been proposed that recognition of the ig-v complex in the cytoplasm by trim triggers the synthesis of unanchored k -linked poly-ub chains that activate nf-κb, ap- and irf pathways [ ] . notably, when the affinity of trim for the fc portion of an antibody is decreased, the viral neutralization efficiency is maintained while the activation of cytokine signaling is diminished [ ] . this suggests that the association of trim with ig-v complexes is important for triggering antiviral responses. trim -deficient mice infected with mouse adenovirus experienced lethal disease while wild-type mice were protected, exemplifying the importance of trim in viral neutralization in vivo [ ] . most likely, these mechanisms are shared in other species due to the highly conserved fc and trim interacting residues, however the activity of trim as an intracellular antibody receptor in non-human, non-murine models has only been demonstrated in pig cells infected with foot-and-mouth disease virus [ ] . trim family proteins can target several components of pathogens ranging from structural elements to factors essential for transcription and replication. a variety of influenza a virus (iav) and influenza b virus (ibv) proteins are targets of trim-mediated inhibition of virus replication (figure ). despite being a target of iav-ns (nonstructural protein )-dependent ifn antagonism, trim interacts with the n-terminus of ibv-ns preventing the viral protein's c-terminal component from binding viral rna [ ] . preventing ibv-ns from interacting with rig-i allows signaling through this rlr pathway to proceed [ ] . another trim-mediated anti-iav mechanism is polyubiquitination of iav nucleoprotein (np) by trim , which leads to np proteasome-mediated degradation and reduced virus replication [ ] . the polymerase basic protein (pb ) of iav is also a target of trim-mediated inhibition. pb is one of the three components that make up the rna-dependent rna-polymerase responsible for transcribing the eight segments of the iav genome [ ] . trim facilitates k -linked polyubiquitination of pb , resulting in enhanced turnover of this viral subunit, and a reduction in viral titers [ ] . aside from targeting pathogen components for proteasomal degradation via ubiquitination, some members of the trim family have been shown to enact their restriction factor capabilities through other means. the trim c-terminal domain, rather than its e ligase activity, is required to reduce iav and ibv replication. the mechanism trim employs to diminish vrna levels of both influenza viruses is currently unknown, although inhibition of translation through direct interaction between trim and the vrnas has been proposed [ ] . trims have also been reported to mediate restriction against flaviviruses ( figure ). the flavivirus genus comprises more than viruses including a number of important human pathogens such as dengue virus (denv), zika virus (zikv), west nile virus (wnv), tick-borne encephalitis virus (tbev), japanese encephalitis virus (jev), hepatitis c virus (hcv), and yellow fever virus (yfv) [ ] . flaviviruses are small enveloped viruses hosting a positive-sense single-stranded rna genome. several flaviviral proteins are associated with viral persistence, immune system evasion, or viral replication [ ] . influenza viruses is currently unknown, although inhibition of translation through direct interaction between trim and the vrnas has been proposed [ ] . trims have also been reported to mediate restriction against flaviviruses ( figure ). the flavivirus genus comprises more than viruses including a number of important human pathogens such as dengue virus (denv), zika virus (zikv), west nile virus (wnv), tick-borne encephalitis virus (tbev), japanese encephalitis virus (jev), hepatitis c virus (hcv), and yellow fever virus (yfv) [ ] . flaviviruses are small enveloped viruses hosting a positive-sense single-stranded rna genome. several flaviviral proteins are associated with viral persistence, immune system evasion, or ( ) . rna genome is released into the cytoplasm ( ). the positive-sense genomic ssrna is translated into a polyprotein, which is cleaved into all structural and non-structural proteins ( ). replication takes place at the surface of endoplasmic reticulum in cytoplasmic viral factories ( ) . in this step, the trims restrict virus replication, degrading viral proteins such as ns a in japanese encephalitis virus (jev) by trim and viral rna inhibition in jev and dengue virus (denv) by trim . trim and trim degrade ns protein in hepatitis c virus (hcv) and tick-borne encephalitis virus (tbev), respectively. virus assembly occurs at the endoplasmic reticulum. the virion buds at the endoplasmic reticulum and is transported to the golgi apparatus ( ) . the prm protein is cleaved in the golgi, thereby maturing the virion, which is fusion competent ( ) . release of new virions by exocytosis ( ) . trims and antagonism function also are used by flaviviruses. trim inhibits ifn-β production during jev infection. trim promotes yellow fever virus replication. denv short noncoding sfrnas bind trim to inhibit ifn expression. hcv encodes a nonstructural protein, ns a, which inhibits the phosphorylation and nuclear translocation of stat in the ifn-α -induced jak/stat pathway via their ifn sensitivitydetermining region [ , ] . trim 's spry domain specifically interacts with ns of hcv and induces ns a degradation [ ] , which is an example of trim-mediated ifn-independent inhibition. trim specifically binds the ns a-d protein (domain ) via its spry domain and the positive-sense genomic ssrna is translated into a polyprotein, which is cleaved into all structural and non-structural proteins ( ). replication takes place at the surface of endoplasmic reticulum in cytoplasmic viral factories ( ) . in this step, the trims restrict virus replication, degrading viral proteins such as ns a in japanese encephalitis virus (jev) by trim and viral rna inhibition in jev and dengue virus (denv) by trim . trim and trim degrade ns protein in hepatitis c virus (hcv) and tick-borne encephalitis virus (tbev), respectively. virus assembly occurs at the endoplasmic reticulum. the virion buds at the endoplasmic reticulum and is transported to the golgi apparatus ( ) . the prm protein is cleaved in the golgi, thereby maturing the virion, which is fusion competent ( ) . release of new virions by exocytosis ( ) . trims and antagonism function also are used by flaviviruses. trim inhibits ifn-β production during jev infection. trim promotes yellow fever virus replication. denv short noncoding sfrnas bind trim to inhibit ifn expression. hcv encodes a nonstructural protein, ns a, which inhibits the phosphorylation and nuclear translocation of stat in the ifn-α -induced jak/stat pathway via their ifn sensitivity-determining region [ , ] . trim 's spry domain specifically interacts with ns of hcv and induces ns a degradation [ ] , which is an example of trim-mediated ifn-independent inhibition. trim specifically binds the ns a-d protein (domain ) via its spry domain and utilizes its e ubiquitin ligase activity to target ns a for removal [ ] . wenchun and colleagues have shown that trim interacts with the ns a protein of jev and targets the protein for proteasome-mediated destruction [ ] . ns a is a small, hydrophobic transmembrane protein involved in the virus life cycle and subversion of host antiviral responses [ , ] , including inhibition of the double-stranded rna-activated protein kinase pkr during jev infection [ ] . trim -dependent inhibition of jev ns a protein occurs in bhk- and t cells and is therefore important for restricting jev replication [ ] . recent evidence also suggests that the ring and c-terminal domains of trim may be important in the restriction of other flaviviruses, including yfv and denv [ ] , but the mechanism of action remains elusive. trim might modulate post-translational modification of one or more viral proteins and/or host factors to suppress viral replication [ ] . although trim fails to restrict hcv replication when overexpressed in human hepatoma huh cells [ ] , trim overexpression in hek cells support some selectable hcv rna replicons at very low efficiencies [ ] . perhaps trim facilitates degradation of viral proteins similar to mechanisms observed with trims and against hcv and trim against jev. further studies are needed to elucidate the underlying molecular mechanisms of trim -mediated restriction of denv and yfv. interestingly, trim is capable of binding to the protease n pro of bovine diarrheal virus (bvdv), a pestivirus of the flaviviridae family [ ] . the restriction of this viral protein is critical considering that n pro is capable of mediating irf degradation, thus impairing production of ifn-β [ ] . trim -mediated restriction of bvdv is specific, as closely related viruses are not likewise impaired in their replication [ ] . trim α, also known as trim - or trim d, is present only in rodents. trim α is highly expressed in the spleen, lymph node, and bone marrow in a type-i ifn-dependent manner, and is required for effective restriction of tbev replication [ ] . trim α is an important mediator of the innate cellular response to restrict langat virus (a member of the tbev serogroup) infection by targeting the viral rna polymerase and major ifn antagonist, ns [ ] . ns has a methyltransferase and rna-dependent rna polymerase activity that associates with ns and ns b to form the viral replication complex. ns inhibits ifn-α/β-dependent responses by preventing jak-stat signaling and thus suppresses ifn-stimulated gene (isg) expression [ ] [ ] [ ] [ ] . taylor and colleagues demonstrated that trim α interacts with ns from lgtv and tbev and blocks the replication of these viruses via a lysosomal-targeting mechanism. despite ns being the most conserved of the flaviviral proteins, trim α did not target ns from wnv, nor could it inhibit wnv replication [ ] . besides flaviviruses, other positive-sense rna viruses have been reported to be inhibited by trim-mediated mechanisms and can occur through both direct and indirect means. for example, trim acts as a cofactor for the zinc-finger antiviral protein (zap), a member of the poly(adp-ribose) polymerase family that is known to bind and promote viral rna degradation [ ] . trim enhances zaps' ability to inhibit sindbis virus translation [ ] . however, although trim is capable of promoting k -and k -linked polyubiquitination of zap isoforms, ubiquitination does not appear to affect zap antiviral activity [ ] . it will be of interest to elucidate what other factors may be ubiquitinated by trim that could affect zap antiviral activity. trim -iv has been shown to confer resistance to encephalomyocarditis virus (emcv) hampering viral replication and protein synthesis [ ] . this ability was shown to originate from trim -iv's c-terminus, specifically through an interaction with the viral d polymerase ( dpol), leading to nuclear body sequestration of the viral polymerase [ ] . additionally, sumoylation of trim -iv was required to facilitate restriction of emcv [ ] . trims play an additional role in restricting dna virus replication. eight different trim proteins (trim , , , , , , , and ) were identified to inhibit hepatitis b virus (hbv) [ ] . in particular, trim was the only trim from this group of eight that specifically reduced both the enhancers i and ii components of hbv. this inhibition of viral transcription required trim 's ring and pry/spry domains, implicating its e ligase activity [ ] . trim is associated with hbv clearance in acutely infected chimpanzees, and possesses anti-hbv activity under physiological conditions [ ] . gao and colleagues also reported that trim was one of the most strongly induced trim family molecules in human hepatoma hepg cells after treatment with ifns [ ] . epstein-barr virus (ebv) replication and transcription activator (rta) protein activates ebv lytic genes for proliferation [ ] . trim α promotes the ubiquitination of rta upon interaction, thereby blocking the ebv lytic cycle [ ] . the involvement of trim α in a non-retroviral innate immune response implies trims possess diverse, situation-specific functions that have yet to be characterized. trims may also operate as recognition receptors for other components of host immunity. trim isoforms are able to capture varicella-zoster virus nucleoproteins to impede nuclear egress and thus block the release of new virions [ ] . comprehension of the multiple roles trim-family proteins play may become critical in discerning the impact that they have on the innate immune response. as we have described, trim family proteins play an important role in innate immunity and counter pathogens [ ] . in retaliation to this evolutionary pressure, pathogens have adapted to antagonize trims. mechanisms of viral-mediated trims restriction range from impairment of trim-promoted innate immune signaling complex assembly to direct hindrance of the trim proteins. interfering with trims, either directly or indirectly, undermines their intended function, and enables viruses to gain an early advantage over the host [ ] . the following section highlights several recent examples that elucidate viral manipulation of trims. a broad range of rna viruses have evolved effective evasion strategies to manipulate host antiviral immunity. influenza viruses employ well-studied examples of trim antagonism ( figure ). the nonstructural protein (ns ) of influenza a and b virus (iav/ibv) has been characterized as a viral antagonist of host innate immunity through interactions with trim [ , , , ] . as described above, trim plays a critical role in the activation of the rlr pathways [ , , ] . the ns protein from iav directly interacts with the coiled-coil domain of trim to impede its multimerization [ , ] . since dimerization is required for trim e ligase activity, ns binding to trim leads to impaired ubiquitination of the rig-i and downstream signaling, resulting in a reduced antiviral response [ , ] . the trim interaction with iav ns is species-specific. human trim interacts with iav strains isolated from many species while chicken trim binds only ns from avian strains and murine trim did not bind any ns [ ] . riplet, a close relative of trim , lacks a b-box domain but shares homologous ring and spry domains. its predicted coiled-coil structure also binds ns to inhibit rig-i signaling in mice and humans [ ] . aside from interacting with trim , iav ns also associates with host rig-i directly [ ] . as seen with iav, the n-terminal domain of ibv ns is also able to block the lys -linked ubiquitination of rig-i and subsequent antiviral signaling downstream of the rlr pathway [ ] . members of the coronaviridae, flaviviridae, and bunyaviridae families also encode viral proteins that inhibit trim-mediated regulation of rlr signaling. severe acute respiratory syndrome and middle east respiratory syndrome coronavirus (sars/mers-cov) are large, positive-sense single-stranded rna viruses. akin to the influenza virus ns protein, the nucleoprotein of sars-cov was demonstrated to interact with the spry domain of trim , preventing the necessary interaction and subsequent ubiquitination of the rig-i card domains [ ] . a similar loss of rig-i-induced ifn-β is achieved when mers-cov nucleoprotein associates with trim [ ] . for denv, manokaran et al. compared two viral sequences (pr and pr- b) and identified mutations that resulted in the increased production of subgenomic flavivirus non-coding rnas (sfrnas) by the pr- b strain [ ] ( figure ). the pr- b sfrnas were capable of binding to host trim and prevented usp -mediated deubiquitination [ ] , which is crucial for activation of rig-i [ ] . this data provides unique molecular insight into the epidemiological fitness of denv, suggesting that denv sfrnas can bind to host proteins to promote viral evasion of innate immunity [ ] . the nss protein of severe fever with thrombocytopenia syndrome virus (sftsv), a negative-sense rna virus in the family bunyaviridae, interacts directly with trim , and indirectly with rig-i and tbk to isolate these signaling molecules from associating with mavs [ ] . as with the other viral protein-trim interactions described above, downstream activation of irf and subsequent ifn-β production are impaired [ ] . trim is a common target of a diverse group of rna viruses, suggesting that other pathogens may also impair trim -mediated stimulation of the rlr pathway. similar to iav, hiv- encodes multiple proteins that restrict trim function ( figure ). for example, hiv- vpr protein has been suggested to play a role in trim manipulation [ ] . interestingly, protein expression levels of trim have shown to be under the control of vpr in a dose-dependent manner, where the presence of trim was decreased when the concentration of vpr was low [ ] . currently, the mechanism hiv- vpr employs to antagonize trim is unknown. following infection of human neural precursor cells (hnpcs) with hiv- , trim becomes upregulated, primarily due to the viral trans-activator of transcription protein (tat) [ ] . this tat-mediated upregulation of trim induces proliferation arrest in hnpcs, eventually leading to neurodegeneration [ ] . flaviviruses also encode viral proteins that antagonize trim-mediated innate immunity ( figure ). yfv and denv ns protein have amino acid residues on the n-terminus, which are essential for antagonism of type-i ifn signaling [ , ] . yfv ns binds stat only after ifn treatment, and appears to inactivate isgf within the nucleus [ ] . morrison et al. found in denv ns a glycine and a threonine residue within the n-terminus that are required for binding with ubr (ubiquitin protein ligase e component n-recognin ) to mediate stat degradation [ ] . ubr , a member of the n-recognin family, is a potential e ligase that recognizes and degrades proteins containing destabilizing n termini. [ ] . ubr interacts preferentially with proteolytically-processed denv ns , but not with yfv ns or wnv ns [ ] . although ubr does not belong to the trim family, it is possible that trim members may also be involved in stat degradation by ns . for example, trim was identified as an essential factor in yfv replication due to its interaction and poly-ub of residue k on yfv-ns , promoting binding with stat and inhibition of type-i ifn signaling [ ] . another flavivirus, japanese encephalitis virus (jev), induces expression of trim in human microglial cells, which attenuates jev-induced antiviral signaling [ ] . the study by manocha et al. demonstrated that trim overexpression suppressed phosphorylation of irf and activation of ifn-β, while silencing trim permitted efficient type-i ifn responses in jev-infected human microglial cells [ ] . this study provides evidence that jev suppress the ifn-i response due to induction of trim . finally, we recently showed that nipah virus (niv), a single-stranded negative-sense highly pathogenic rna virus (paramyxoviridae family, genus henipavirus) that causes fatal diseases in humans [ ] , can inhibit trim -mediated type-i ifn responses [ ] (figure ). mechanistically, the niv matrix (niv-m) structural protein, which is required for virus assembly and budding [ , ] , targets trim for degradation. interestingly, niv budding requires trafficking of niv-m from the cytoplasm to the nucleus before reaching the cell membrane for virus assembly [ , ] . the reason for niv-m trafficking to the nucleus is still unclear, however, a lysine residue (k ) in the niv-m bipartite nuclear localization signal that is conserved in divergent henipaviruses and is required for trafficking, is critical for the ifn antagonist function [ ] . consistent with this, the matrix proteins of ghana, hendra and cedar viruses were also able to inhibit ifn-β induction [ ] . it is currently unknown whether trim e -ligase activity affects niv-m trafficking or whether it directly interferes with niv-replication. niv-m-induced trim degradation did not appear to require the proteasome, however. although the precise mechanism of trim degradation remains to be elucidated, inhibitors that recover trim protein levels could potentially be used as therapeutic drugs against niv infections. these findings highlight the importance of trim as an antiviral factor of the type-i ifn system. a variety of dna viruses also encode viral proteins that interfere with trims. alternative methods for subverting trim-induced innate immune responses can be noted through alterations made to host transcription by hepatitis b virus (hbv). the hbv-encoded protein x (hbx) methylates a single cpg in trim 's promoter region [ ] . this viral-mediated epigenetic modification blocks ifn-α/γ-induced irf , a transcriptional activator, from binding the trim promoter and consequently facilitates viral proliferation and escape [ ] . in epstein-barr virus (ebv), kap (krab [kruppel-associated box domain]-associated protein )/trim regulates activation of the viral lytic cycle [ ] . in cells undergoing lytic stage activation, kap /trim becomes phosphorylated at serine residue by ataxia telangiectasia mutated (atm). this post-translational modification impairs kap /trim 's restriction factor function and allows ebv to transition from latency to lytic stage [ ] . additionally, the anti-malarial drug chloroquine acts as an activator for atm by phosphorylating kap /trim , which ultimately leads to promotion of the ebv lytic cycle and escape of the virus particles [ ] . mutations in gene expression serve as an additional focal point highlighting the role that trims play in a dysfunctional antiviral response. when a population of hbv-infected individuals was screened, chronic hbv infection was found to correlate to a single t to c silent mutation single nucleotide polymorphism (snp) in the trim ring domain [ ] . although the implications to patient outcomes are considerable, additional investigations into the basic biological mechanisms are warranted to discern the importance of these snps in trim-mediated innate immunity. figure . trim is targeted by nipah and ebola viruses to enhance virus replication. ebola virus (ebov) inhibits type-i ifn production by multiple mechanisms. ebov vp binds and inhibits rig-i, ikkε, and tbk- to inhibit ifn production. trim ubiquitinates vp on k and promotes vp activity as the cofactor of the viral polymerase and enhances virus replication. additional unidentified ubiquitination sites of vp exist. whether trim enhances ebov replication by promoting viral genome replication or viral gene transcription is not known. in nipah virus infection, the viral matrix protein (niv-m) promotes trim degradation, resulting in reduced synthesis of k -linked unanchored polyubiquitin chains, ikkε oligomerization, ikkε-t autophosphorylation, irf phosphorylation, and reduced ifn induction. these combined losses confer an impaired host-antiviral response. a variety of dna viruses also encode viral proteins that interfere with trims. alternative methods for subverting trim-induced innate immune responses can be noted through alterations made to host transcription by hepatitis b virus (hbv). the hbv-encoded protein x (hbx) methylates a single cpg in trim 's promoter region [ ] . this viral-mediated epigenetic modification blocks ifn-α/γ-induced irf , a transcriptional activator, from binding the trim promoter and consequently facilitates viral proliferation and escape [ ] . in epstein-barr virus (ebv), kap (krab [kruppel-associated box domain]-associated protein )/trim regulates activation of the viral lytic cycle [ ] . in cells undergoing lytic stage activation, kap /trim becomes phosphorylated at serine residue by ataxia telangiectasia mutated (atm). this post-translational modification impairs kap /trim 's restriction factor function and allows ebv to transition from latency to lytic stage [ ] . additionally, the anti-malarial drug chloroquine acts as an activator for atm by phosphorylating kap /trim , which ultimately leads to promotion of the ebv lytic cycle and escape of the virus particles [ ] . mutations in gene expression serve as an additional focal point highlighting the role that trims play in a dysfunctional antiviral response. when a population of hbv-infected individuals was screened, chronic hbv infection was found to correlate to a single t to c silent mutation single nucleotide polymorphism (snp) in the trim ring domain [ ] . although the implications to patient outcomes are considerable, additional investigations into the basic biological mechanisms are warranted to discern the importance of these snps in trim-mediated innate immunity. in addition to impairing trim gene expression, dna viruses can mimic host proteins to prohibit trim function. the immediate early protein (icp ) of herpes simplex virus (hsv- ) possesses a ring domain and facilitates the degradation of trim through ubiquitination [ ] . this destruction of trim is facilitated through the host's proteasome [ ] , mimicking the numerous instances of trim-mediated restriction. gammaherpesvirus mhv- similarly targets trim for proteasome-mediated degradation [ ] . the ie proteins of human cytomegalovirus (hcmv) mimics the coiled-coil domain of trims to recruit and sequester trim from nuclear bodies thus impairing the activation of ifn responses [ ] . so far, most studies on trims have focused on their roles as antiviral factors by directly restricting virus replication or indirectly by inducing antiviral cytokines, as described above. the fact that trims are targeted by viruses for immune evasion further highlights their important roles in protecting the host against infections. however, whether trims may directly act as host factors required for virus replication, or "pro-viral" factors, has not been addressed. some studies have shown that certain viral antagonists can hijack trims to activate their ifn antagonist activity (e.g., trim ubiquitinates yfv-ns for antagonism of stat function [ ] ), but these are indirect effects that provide an advantage to the virus by reducing host antiviral responses. since ubiquitination of viral proteins may positively influence specific steps of the replication cycle, it would not be surprising if trims are involved in directly promoting virus replication by non-degradative ubiquitination of viral proteins. indeed, we recently reported the first example of such a role for trim [ ] . as described above in section . , trim is involved in antiviral type-i ifn responses by catalyzing the synthesis of unanchored k -linked polyubiquitin chains that activate ikkε (figures and ). further evidence of trim as an antiviral factor is highlighted by our findings that the niv can inhibit ifn-i responses by targeting trim [ ] . furthermore, knockdown of trim in lung a cell lines and primary human monocyte derived dendritic cells has shown increased replication of multiple viruses, including iav, emcv, and sendai virus (sev), most probably due to reduced type-i ifn responses [ ] . in addition, trim a knockout cells showed reduced ifn responses [ ] . unexpectedly, despite a defect in the ifn response, infectious ebola virus (ebov: filoviridae family) replicated less efficiently in trim knockout cells as compared to parental wild type (wt) cells. this observation raised the question whether trim may be acting as a pro-viral factor or an enhancer of virus replication. in support of this hypothesis, we found that trim interacts with ebov-vp , which is a major viral ifn antagonist by targeting rig-i [ , ] and the kinases ikkε and tbk- [ ] . however, since vp also plays a critical role as the cofactor of the virus polymerase [ ] , trim could directly affect polymerase function. mass spectrometry analysis and co-immunoprecipitation assays demonstrated that trim ubiquitinates vp on k [ ] , a lysine residue located on its ifn antagonist domain. moreover, minigenome reporter experiments showed that trim can enhance vp -mediated polymerase activity, and this effect requires the e -ubiquitin ligase activity of trim . although vp is ubiquitinated in multiple (currently unidentified) residues in addition to k , the trim -dependent effects on vp minigenome activity required an intact k residue. vp was also able to inhibit rig-i induced trim -enhanced ifnβ in reporter assays, however the precise mechanism was not elucidated [ ] . collectively, these findings suggest that trim is a host factor hijacked by ebov-vp for both immune evasion and for promoting virus replication via ubiquitination of vp ( figure ). future studies will address whether ubiquitination of vp regulates virus rna replication or transcription. it remains to be seen if other trims that are targeted by viruses may also directly enhancing virus replication via ubiquitination of viral proteins. autophagy (self-eating) is a highly conserved catabolic mechanism through which eukaryotic cells deliver dispensable, or potentially dangerous, cytoplasmic material to lysosomes for degradation [ ] . the process is characterized by the formation of autophagosomes, which sequester the cytoplasmic structures targeted for destruction. autophagy has been linked to a wide range of physiological processes, including cell differentiation and development, the degradation of aberrant structures and turnover of damaged organelles, as well as innate and adaptive immunity [ , ] . a growing number of studies indicate that several trims are linked to autophagy and recent excellent reviews are available on the emerging roles of trims in autophagy [ , , , ] . a good example of the potential role of trims in autophagy and virus infections is rhesus trim α, which acts both as a regulator of autophagy by providing a platform for the assembly of activated ulk and beclin (key components of the autophagy regulatory complexes) and as a receptor for selective autophagy [ , ] . in its role as an autophagic cargo receptor, trim α directly recognizes viral capsid sequences via its spry domain [ , [ ] [ ] [ ] . this is an example of selective autophagy in mammalian cells, which could occur via direct substrate recognition by trims and connects autophagy with a role in defense against viral pathogens. the rhesus trim can execute precision autophagy of the hiv- capsid. in contrast, the weak affinity of human trim for the hiv- capsid precludes effective precision autophagy. as a consequence, rhesus trim , but not human trim , could contribute to defense against hiv- through precision autophagy. since this recognition depends on the c-terminal region of trims (e.g., spry domain of trim α), other types of c-terminal domains on trims could selectively recognize diverse protein targets. thus, trim proteins, as a group, could comprise a class of broad-repertoire, high-fidelity, selective autophagic receptors. given the breadth of the role of trims in various diseases, it will be important to explore precision autophagy-in addition to bulk autophagy-as a therapeutic target against viral infections. the proposed role of trims in autophagy raise questions. for example, how many trims act as autophagic receptors and what are their specific targets? do trim proteins function as hubs connecting different signaling pathways or different systems? how is the autophagic role of trims integrated with the other functions of trims, including regulation of gene expression and pro-inflammatory signaling? what is the interplay between the e ligase activity of trims and precision autophagy? it is important to determine inhibitory compounds of trim proteins for their use as therapeutic tools in infectious diseases. however, because some trim proteins have simultaneous dual functions in carcinogenesis and the immune response, it should be considered that putative drugs (inhibitors of some trim proteins) for cancer therapy may affect immunological reactions as a side effect. further detailed analysis of trim proteins is needed for their use as novel therapeutics with minimal side effects. this review highlighted the roles of trims in virus-host interactions. extensive reports detail trim involvement in immune signaling and direct virus restriction. in addition, viral antagonism of trims exemplifies the importance of this protein family in antiviral responses. despite these advancements, many trims have yet to be characterized. additionally, the molecular mechanisms underlying trim-mediated virus restriction or viral protein-mediated trim inhibition are not fully elucidated. the role of trims in regulating poly-ub chain topology is also of interest. in several examples, including trims [ ] , [ ] , [ ] , and [ ] , trims facilitate the synthesis of unanchored poly-ub chains. the relative contribution of trims in synthesizing specifically unanchored poly-ub chains, versus covalently linked poly-ub chains, is unclear. classically, the e is considered more important in determining poly-ub chain characteristics [ , ] , but post-translational modifications may influence the decision between covalent and non-covalent linkage [ ] as may the choice of the partnering e . perhaps trims play a unique role in the synthesis of unanchored chains. in the future, evaluating the factors that regulate e -trim pairing may add an additional layer of complexity to trim-mediated regulation and the ubiquitin code. although one study addressed this question by testing interactions between trims and the e -conjugases and a few trim-e pairs were identified [ ] , the complexity of potential transient interactions, and the possibility of cell-type specific expression for the combination of trims and e -conjugases makes this task a huge challenge. importantly, another question is how poly-ub chains of unconventional linkages may affect virus replication. for example, unanchored poly-ub chains can be incorporated into the iav virion to enable hijacking of the host's aggresomal pathway to facilitate viral replication [ ] . the e and e enzymes responsible for synthesizing these poly-ub chains have not been identified, but perhaps iav hijacks a trim to make these poly-ub chains critical in efficient infection. therefore, unanchored poly-ub chains may have both pro-viral and antiviral functions [ ] and elucidating how to tip the balance towards antiviral responses may help develop novel antiviral therapies. additionally, iav replication via host ubiquitin and aggresome systems relies on the host's cytoskeletal network [ ] . this may further implicate the role of trims in similar pathways as several trims associate with microtubules [ , ] . many trims assemble into cytoplasmic bodies, which can be dynamic structures as described with trim [ ] . the exact role of cytoplasmic bodies is not well characterized. possible trim-cytoplasmic body functions may include facilitation of signaling complex assembly [ ] , regulation of active trim solubility, and/or generation of autophagosome-like complexes to target proteins for degradation. viruses can also disrupt or reorganize trim-cytoplasmic bodies, further supporting a role in antiviral responses. understanding how trim sub-cellular localization influences activity will also benefit the field. the applicability of the information garnered from studying trim-virus interactions may facilitate the identification of novel antiviral targets. further studies will advance our understanding of the complexities involved with trim signaling such as isoform-specific roles, cell-type specific activity, cytoplasmic body assembly and function, and post-transcriptional modification-induced trim function. finally, future studies will need to address whether other trims, in addition to trim , may be hijacked by viruses to directly enhance replication, acting as pro-viral factors. the increasing complexity of the ubiquitin code ubiquitin enzymes in the regulation of immune responses ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions building ubiquitin chains: e enzymes at work ube d and ube n are essential for rig-i-mediated mavs aggregation in antiviral innate immunity the roles of the trim e -ubiquitin ligase family in innate antiviral immunity intrimsic immunity: positive and negative regulation of immune signaling by tripartite motif proteins ubiquitin acetylation inhibits polyubiquitin chain elongation ubiquitin modifications ubiquitin-binding domains unanchored k -linked polyubiquitin synthesized by the e -ubiquitin ligase trim stimulates the interferon-ikkepsilon kinase-mediated antiviral response sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of trim trim family proteins and their emerging roles in innate immunity trim protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity trim e ligases interfere with early and late stages of the retroviral life cycle the e -ligase trim family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors structural determinants of trim protein function the tripartite motif family identifies cell compartments trim acts as an e ubiquitin ligase and can heterodimerize with other trim family members solution structure of the rbcc/trim b-box domain of human mid : b-box with a ring functional role of trim e ligase oligomerization and regulation of catalytic activity the tripartite motif coiled-coil is an elongated antiparallel hairpin dimer structure and catalytic activation of the trim ring e ubiquitin ligase structural insights into the trim family of ubiquitin e ligases crystal structure of trim c-terminal coiled-coil/b . fragment: implications for the recognition of higher order oligomers a b-box surface patch important for trim α self-association, capsid binding avidity, and retrovirus restriction the trim alpha b-box domain promotes cooperative binding to the retroviral capsid by mediating higher-order self-association tripartite motif ligases catalyze polyubiquitin chain formation through a cooperative allosteric mechanism ring dimerization links higher-order assembly of trim alpha to synthesis of k -linked polyubiquitin trim is a mitochondrial adaptor that facilitates retinoic acid-inducible gene-i-like receptor-mediated innate immune response subclassification of the rbcc/trim superfamily reveals a novel motif necessary for microtubule binding genomic analysis of the trim family reveals two groups of genes with distinct evolutionary properties negative regulation of nf-κb activity by brain-specific tripartite motif protein trim prevents autoinflammatory t cell development in vivo - - proteins sequester a pool of soluble trim ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation rapid turnover and polyubiquitylation of the retroviral restriction factor trim the cellular level of trim , an rbcc protein overexpressed in gastric cancer, is regulated by multiple mechanisms including the ubiquitin-proteasome system genomics and evolution of the trim gene family identification of a genomic reservoir for new trim genes in primate genomes origin and evolution of trim proteins: new insights from the complete trim repertoire of zebrafish and pufferfish an evolutionary screen highlights canonical and noncanonical candidate antiviral genes within the primate trim gene family positive selection of the trim family regulatory region in primate genomes discordant evolution of the adjacent antiretroviral genes trim and trim in mammals positive selection of primate trim alpha identifies a critical species-specific retroviral restriction domain human trim gene expression in response to interferons induction of trim by ifn-gamma involves jak and pc-plc/pkc, but not mapks and pi k/akt/mtor pathways expression of the immune regulator tripartite-motif is controlled by ifn regulatory factors type i interferon-dependent and -independent expression of tripartite motif proteins in immune cells trim negatively regulates tlr / -mediated innate immune and inflammatory responses by two sequential and distinct mechanisms trim is a negative regulator of mda -mediated type i interferon production expression profiling of trim protein family in thp -derived macrophages following tlr stimulation tripartite-motif proteins and innate immune regulation regulatory role of trim in the type-i interferon pathway in japanese encephalitis virus-infected human microglial cells the interferon signaling antagonist function of yellow fever virus ns protein is activated by type i interferon dengue subgenomic rna binds trim to inhibit interferon expression for epidemiological fitness the matrix protein of nipah virus targets the e -ubiquitin ligase trim to inhibit the ikkepsilon kinase-mediated type-i ifn antiviral response influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways trim family proteins: roles in autophagy, immunity, and carcinogenesis precision autophagy directed by receptor regulators--emerging examples within the trim family trim proteins and diseases pattern recognition receptors and inflammation expanding role of ubiquitination in nf-κb signaling convergence of the nf-κb and irf pathways in the regulation of the innate antiviral response in vivo ligands of mda and rig-i in measles virus-infected cells the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses '-triphosphate rna is the ligand for rig-i reis e sousa, c. rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates differential roles of mda and rig-i helicases in the recognition of rna viruses rig-i-like receptor regulation in virus infection and immunity ubiquitin-induced oligomerization of the rna sensors rig-i and mda activates antiviral innate immune response structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner phosphorylation-mediated negative regulation of rig-i antiviral activity the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity negative regulation of the rig-i signaling by the ubiquitin ligase rnf ubch regulates ubiquitin and isg conjugation to rig-i mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response mavs recruits multiple ubiquitin e ligases to activate antiviral signaling cascades traf molecules in cell signaling and in human diseases activation of the iκb kinase complex by traf requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain tab and tab activate the nf-κb pathway through binding to polyubiquitin chains direct activation of protein kinases by unanchored polyubiquitin chains the regulation of nf-κb subunits by phosphorylation. cells type i interferons in infectious disease are the ikks and ikk-related kinases tbk and ikk-epsilon similarly activated? primary activation of interferon a and interferon b gene transcription by interferon regulatory factor trim -catalized ubiquitination is essential for mda -mediated antiviral innate immunity trim modulates type i interferon induction and cellular antiviral response by targeting rig-i for k -linked ubiquitination trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i the ubiquitin-specific protease usp promotes rig-i-mediated antiviral signaling by deubiquitylating trim multifaceted roles of trim in innate immune and inflammatory responses innate immunity to rna virus is regulated by temporal and reversible sumoylation of rig-i and mda trim interacts with ecsit and negatively regulates nf-κb and irf- / -mediated signal pathways de novo transcriptome analysis shows that sav- infection upregulates pattern recognition receptors of the endosomal toll-like and rig-i-like receptor signaling pathways in macrophage/dendritic like to-cells activation of duck rig-i by trim is independent of anchored ubiquitin species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns protein molecular characterization, tissue distribution and expression analysis of trim in gallus gallus domesticus duck trim -l enhances mavs signaling and is absent in chickens and turkeys mavs ubiquitination by the e ligase trim and degradation by the proteasome is involved in type i interferon production after activation of the antiviral rig-i-like receptors the ubiquitin e ligase trim promotes aggregation and activation of the signaling adaptor mavs through lys -linked polyubiquitination novel function of trim promotes an antiviral response by stabilizing visa trim short isoform preferentially promotes dna and rna virus-induced production of type i interferon by recruiting gsk β to tbk crosstalk between cytoplasmic rig-i and sting sensing pathways trim negatively regulates ifnβ production and antiviral activity by targeting tbk tripartite motif-containing protein negatively regulates tlr / -and rig-i-mediated ifn-β production and antiviral response by targeting nap polyubiquitin conjugation to nemo by triparite motif protein (trim ) is critical in antiviral defense autoubiquitination of trim links tbk to nemo in rlr-mediated innate antiviral immune response trim negatively regulates ifn-β production by degrading trk fused gene, a novel driver of ifn-β downstream of anti-viral detection systems trim α negatively regulates tlr-mediated nf-kappa b activation by targeting tab and tab for degradation the human antiviral factor trim is under the regulation of hiv- vpr identification of a role for trim in the control of innate immunity in the respiratory tract trim negatively regulates the nfκb-mediated signaling pathway through stabilization of cactin sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity sting is a direct innate immune sensor of cyclic di-gmp structure of sting bound to cyclic di-gmp reveals the mechanism of cyclic dinucleotide recognition by the immune system structural and functional analysis of ddx : a bispecific immune receptor for dna and cyclic dinucleotide the helicase ddx recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response the e ubiquitin ligase trim negatively regulates the innate immune response to intracellular double-stranded dna trim α is a negative-feedback regulator of the intracellular dna and dna virus-triggered response by targeting sting the ubiquitin ligase trim regulates innate immune responses to intracellular double-stranded dna trim protein modulates type i interferon induction and cellular antiviral response by targeting mita/sting protein for k -linked ubiquitination the critical role of toll-like receptors-from microbial recognition to autoimmunity: a comprehensive review the e ubiquitin ligase ro negatively regulates ifn-β production post-pathogen recognition by polyubiquitin-mediated degradation of irf self protection from anti-viral responses-ro promotes degradation of the transcription factor irf downstream of the viral toll-like receptors tripartite motif (trim ) differentially regulates the stability of interferon regulatory factor (irf ) isoforms e ubiquitin ligase tripartite motif negatively regulates tlr-mediated immune responses by proteasomal degradation of tnf receptor-associated factor in macrophages hung, t. trim negatively regulates tlr -mediated ifn-β signaling by targeting trif for degradation trim is an essential component of the tlr antiviral signaling pathway mechanisms and functions of inflammasomes activation and regulation of the inflammasomes activation of nucleotide oligomerization domain (nod ) by human cytomegalovirus initiates innate immune responses and restricts virus replication nod and nod signaling in infection and inflammation the e ubiquitin ligase tripartite motif is essential for cytosolic rna-induced nlrp inflammasome activation tripartite-motif protein negatively regulates nlrp inflammasome activation by modulating reactive oxygen species production the e ubiquitin ligase trim attenuates nlrp inflammasome activation by promoting proteasomal degradation of nlrp trim negatively regulates nod by ubiquitination and proteasomal degradation nucleo-cytoplasmic trafficking of trim , a novel oncogene, is involved in positive regulation of tnf induced nf-κb pathway trim modulates stat activity through negative regulation of pias trim inhibits tnfα-and il- β-triggered nf-κb activation by mediating lysosome-dependent degradation of tab / siglec suppresses antiviral innate immune response by inducing tbk degradation via the ubiquitin ligase trim tripartite motif-containing bridges endothelial inflammation and angiogenic activity by retaining expression of tnfr- and - and vegfr in endothelial cells interferons and viral infections tripartite motif (trim /tif α) tumor suppressor protein is a novel negative regulator of interferon (ifn)/signal transducers and activators of transcription (stat) signaling pathway acting through retinoic acid receptor α (rarα) inhibition trim /gerp ring finger protein interacts with socs- multiple functions of the ikk-related kinase ikkepsilon in interferon-mediated antiviral immunity iκb kinase ε (ikkε) regulates the balance between type i and type ii interferon responses control of foxo activity and cell survival by trim directs tlr -stimulated cells toward ifn type i gene induction or apoptosis the interferon-inducible staf gene is downregulated during t cell costimulation by cd and cd regulation of the interferon-inducible p target gene trim (staf ) in human t lymphocyte activation t-cell-intrinsic tif α/trim regulates il- r expression on th cells and th cell-mediated airway allergy tripartite motif containing protein negatively regulates cd t cells by ubiquitinating and inhibiting the class ii pi k-c β tripartite motif-containing protein modulates tcr-activated proliferation and effector functions in cd + t cells the cytoplasmic body component trim α restricts hiv- infection in old world monkeys trim α protein restricts both hiv- and murine leukemia virus a trim -cyclophilin a fusion protein found in owl monkey kidney cells can restrict hiv- cyclophilin a retrotransposition into trim explains owl monkey resistance to hiv- trim α requires ube w to anchor lys -linked ubiquitin chains and restrict reverse transcription dynamic conformational changes in the rhesus trim α dimer dictate the potency of hiv- restriction primate trim proteins form hexagonal nets on hiv- capsids mechanism of b-box domain-mediated higher-order assembly of the retroviral restriction factor trim α crystal structure of the trim α bbox domain from rhesus macaques describes a plastic oligomerisation interface recent insights into the mechanism and consequences of trim α retroviral restriction hexagonal assembly of a restricting trim αprotein trim is an innate immune sensor for the retrovirus capsid lattice proteasome inhibition reveals that a functional preintegration complex intermediate can be generated during restriction by diverse trim proteins proteasome inhibitors uncouple rhesus trim α restriction of hiv- reverse transcription and infection fates of retroviral core components during unrestricted and trim -restricted infection proteasomal degradation of trim α during retrovirus restriction nonhuman trim variants enhance recognition of hiv- -infected cells by cd + t cells capsid-binding retrovirus restriction factors: discovery, restriction specificity and implications for the development of novel therapeutics hiv- capsid recognition, and innate immune signaling trim α-mediated ubiquitin chain conjugation is required for inhibition of hiv- reverse transcription and capsid destabilization trim retroviral restriction activity correlates with the ability to induce innate immune signaling trim α restriction affects clinical outcome and disease progression in simian immunodeficiency virus-infected rhesus macaques preserved cd + central memory t cells and survival in vaccinated siv-challenged monkeys defective cd t cell memory following acute infection without cd t cell help requirement for cd t cell help in generating functional cd t cell memory a single amino acid change in the spry domain of human trim αleads to hiv- restriction evolution of a cytoplasmic tripartite motif (trim) protein in cows that restricts retroviral infection isolation of an active lv gene from cattle indicates that tripartite motif protein-mediated innate immunity to retroviral infection is widespread among mammals ovine trim α can restrict visna/maedi virus receptor usage dictates hiv- restriction by human trim α in dendritic cell subsets lack of endogenous trim α-mediated restriction in rhesus macaque dendritic cells endogenous trim α function is regulated by sumoylation and nuclear sequestration for efficient innate sensing in dendritic cells an hiv- capsid binding protein trim accelerates viral uncoating functional evidence for the involvement of microtubules and dynein motor complexes in trim α-mediated restriction of retroviruses influenza a virus uses the aggresome processing machinery for host cell entry hiv- uncoating is facilitated by dynein and kinesin cytoplasmic dynein promotes hiv- uncoating unanchored ubiquitin in virus uncoating mid and mid are required for xenopus neural tube closure through the regulation of microtubule organization hiv- transcriptional silencing caused by trim inhibition of sp binding to the viral promoter anti-hiv- activity of trim the interferon-induced antiviral protein pml (trim ) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion trim /pml restricts hiv infection in a cell type-dependent manner pml/trim -dependent inhibition of retroviral reverse-transcription by daxx translocalized iga mediates neutralization and stimulates innate immunity inside infected cells antibodies mediate intracellular immunity through tripartite motif-containing (trim ) trim is an igg receptor that is structurally, thermodynamically, and kinetically conserved intracellular antibody-bound pathogens stimulate immune signaling via the fc receptor trim simultaneous neutralization and innate immune detection of a replicating virus by trim trim promotes cgas and rig-i sensing of viral genomes during infection by antibody-opsonized virus trim immune signaling is more sensitive to antibody affinity than its neutralization activity intracellular antibody receptor trim prevents fatal viral infection swine trim restricts fmdv infection via an intracellular neutralization mechanism robust lys -linked ubiquitination of rig-i promotes cytokine eruption in early influenza b virus infection trim inhibits influenza a virus infection by targeting the viral nucleoprotein for degradation structure of influenza a polymerase bound to the viral rna promoter trim senses and restricts influenza a virus by ubiquitination of pb polymerase the c-terminal tail of trim dictates antiviral restriction of influenza a and b viruses by impeding viral rna synthesis historical perspectives on flavivirus research pathogenesis of flavivirus infections: using and abusing the host cell understanding the molecular mechanism(s) of hepatitis c virus (hcv) induced interferon resistance mutations in the nonstructural protein a gene and response to interferon in patients with chronic hepatitis c virus b infection trim inhibits hepatitis c virus infection by spry domain-dependent targeted degradation of the viral ns a protein interferon α(ifnα)-induced trim interrupts hcv replication by ubiquitinating ns a trim inhibits japanese encephalitis virus replication by degrading the viral ns a dengue virus inhibits α interferon signaling by reducing stat expression inhibition of interferon signaling by dengue virus blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein a overlapping and distinct molecular determinants dictating the antiviral activities of trim against flaviviruses and coronavirus identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus trim is a virus-and interferon-inducible e ubiquitin ligase that restricts pestivirus infection tick-borne flaviviruses antagonize both irf- and type i ifn signaling to inhibit dendritic cell function mechanisms of evasion of the type i interferon antiviral response by flaviviruses inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns as an interferon antagonist trim α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral rna polymerase trim enhances the antiviral action of zinc-finger antiviral protein (zap) promyelocytic leukemia isoform iv confers resistance to encephalomyocarditis virus via the sequestration of d polymerase in nuclear bodies identification and characterization of multiple trim proteins that inhibit hepatitis b virus transcription trim : a diverse and dynamic antiviral protein tripartite motif-containing inhibits the activity of hepatitis b virus core promoter, which is dependent on nuclear-located ring domain trim α promotes ubiquitination of rta from epstein-barr virus to attenuate lytic progression emerging role of pml nuclear bodies in innate immune signaling influenza a virus trims the type i interferon response the role of trim in development, disease and rna metabolism ubiquitin in influenza virus entry and innate immunity trim identification in the chinese goose: gene structure, tissue expression profiles, and antiviral immune responses in vivo and in vitro subcellular localizations of rig-i, trim , and mavs complexes the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination hijacking of rig-i signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type i interferon responses tripartite containing motif modulates proliferation of human neural precursor cells in hiv- neurodegeneration ns of dengue virus mediates stat binding and degradation dengue virus co-opts ubr to degrade stat and antagonize type i interferon signaling a family of mammalian e hendra and nipah viruses: different and dangerous evidence for ubiquitin-regulated nuclear and subnuclear trafficking among paramyxovirinae matrix proteins ubiquitin-regulated nuclear-cytoplasmic trafficking of the nipah virus matrix protein is important for viral budding hepatitis b virus x protein: trimming antiviral defences in hepatocytes suppression of interferon-mediated anti-hbv response by single cpg methylation in the -utr of trim chloroquine triggers epstein-barr virus replication through phosphorylation of kap /trim in burkitt lymphoma cells tripartite motif-containing gene- t/c polymorphism associated with hepatitis b virus infection in chinese han population identification of trim as a novel degradation target of herpes simplex virus icp the host e -ubiquitin ligase trim ubiquitinates the ebola virus vp protein and promotes virus replication ebola virus vp protein binds double-stranded rna and inhibits α/β interferon production induced by rig-i signaling mutual antagonism between the ebola virus vp protein and the rig-i activator pact determines infection outcome ebola virus protein vp impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk- filovirus replication and transcription. future virol eaten alive: a history of macroautophagy autophagy, immunity, and microbial adaptations autophagy fights disease through cellular self-digestion autophagy in leukocytes and other cells: mechanisms, subsystem organization, selectivity, and links to innate immunity trim-directed selective autophagy regulates immune activation trim proteins regulate autophagy and can target autophagic substrates by direct recognition trim proteins regulate autophagy: trim is a selective autophagy receptor mediating hiv- restriction specific recognition and accelerated uncoating of retroviral capsids by the trim α restriction factor functional interactions between ubiquitin e enzymes and trim proteins the authors declare no conflict of interest. key: cord- -sqxrwgif authors: paquin, ashley; onabajo, olusegun o.; tang, wei; prokunina-olsson, ludmila title: comparative functional analysis of mammalian ifn-λ orthologs date: - - journal: j interferon cytokine res doi: . /jir. . sha: doc_id: cord_uid: sqxrwgif ifn-λ is a novel type-iii interferon with strong clinical significance in humans. only a subset of individuals—up to % of asians, % of europeans, and % of africans—carry the Δg allele of a genetic variant rs -tt/Δg and are genetically able to produce ifn-λ protein. carriers of the Δg allele have impaired ability to clear infection with hepatitis c virus (hcv). ifn-λ is also predicted to exist and be functionally important in several nonhuman mammals. in this study, we present the first comparative analysis of mammalian ifn-λ orthologs in a human hepatic cell line, hepg , which supports signaling of the human ifn-λ . we show that despite differences in protein sequences, functional properties of the recombinant human and nonhuman ifn-λ proteins are comparable—they are all expressed as predominantly cytoplasmic proteins that are biologically active for induction of interferon signaling. we show that several ifn-λ orthologs can be detected by western blotting, flow cytometry, and confocal imaging using a monoclonal antibody developed for the human ifn-λ . studies of ifn-λ in animals should help improve our understanding of the biology of this novel clinically important interferon in normal and disease conditions. i nterferons (ifns) are an essential part of the innate immune response, which is the first line of defense against pathogens. the induction of ifns leads to activation of the jak/stat signaling pathway and expression of interferonstimulated genes (isgs) in the infected and the surrounding cells. this response can limit viral spread through mobilization of cellular defense mechanisms and elimination of infected cells (parkin and cohen ; levy and others ) . ifns are classified into three major groups-types i, ii, and iii-based on their receptor utilization. type-i ifns include a panel of ifn-a subtypes, ifn-b, ifn-e, ifn-k, and ifn-o, all of which signal through a ubiquitous ifnar receptor complex, consisting of the ifnar and ifnar receptors. ifn-g, the only known type-ii ifn, signals through its own receptor complex, ifngr, consisting of the ifngr and ifngr receptors. type-iii ifns (ifn-l - ) signal through the ifnlr receptor complex, which consists of the ifnlr receptor specific to type-iii ifns, and the il r receptor shared by all type-iii ifns and the il- family of cytokines. the signaling of type-iii ifns is restricted compared with other ifns because expression of ifnlr is largely limited to epithelial cells, such as of the respiratory and gastrointestinal tract. ifn activity in these cells, which are commonly exposed to exogenous pathogens, is important for prevention of viral entry and dissemination. a recently discovered type-iii ifn, ifn-l (prokunina-olsson and others ) reviewed in (o'brien and others ), can be created only in the presence of the dg allele of a dinucleotide genetic variant rs -tt/dg; this variant is polymorphic in humans, but only the ancestral dg allele seems to be present in nonhuman species. the human-specific tt allele, which eliminates ifn-l protein by a frameshift in the first exon, appeared only , years ago (key and others ) . apparently, this event was beneficial because it was favored by positive selection, which has resulted in a rapid increase of the tt allele frequency (or a rapid loss of the dg allele) in human populations (key and others ) . currently, up to % of asians, % of caucasians, and % of africans carry at least one copy of the dg allele and thus can generate ifn-l . carriers of the dg allele have impaired ability to clear hepatitis c virus (hcv) infection (prokunina-olsson and others ; aka and others ). chronic hcv infection eventually increases the risk of death due to liver failure and liver cancer, but this process takes decades, thus improvement of hcv clearance due to genetic inability to produce ifn-l could not be the reason for the positive selection observed for the rs -tt allele (key and others ) . the strength of this selection indicates that ifn-l might have interfered with clearance of other more deadly pathogens, suggesting that elucidation of ifn-l function could be important for understanding, prevention, and treatment of some existing and emerging infections. so far, beyond hcv, the dg allele was also found to be associated with increased risk of cytomegalovirus infection in patients receiving solid organ transplants without antiviral prophylaxis (manuel and others ) ; susceptibility to cytomegalovirus retinitis among hiv-infected individuals (bibert and others ) ; decreased resistance to hiv infection (real and others ) ; and unfavorable clinical and immunological status in hiv-infected individuals (machmach and others ). ifn-l has been reported to induce antiviral response against coronaviruses (hcov- e and mers-cov), yellow fever virus, and dengue virus (hamming and others ; lu and others ) ; this list is likely to grow with additional studies. previously, based on available genomic sequences, ifn-l was predicted to exist in a number of mammals. analysis of ifn-l protein sequences from mammalian species showed evidence of purifying selection, which is a process of eliminating genetic changes that cause amino acid substitutions while retaining neutral (synonymous) variations (key and others ) . this suggests that ifn-l is functionally important across species and evolutionary forces protect it from significant changes. although the function of human ifn-l is not completely understood, its ability to induce ifn signaling and antiviral response against several pathogens has been clearly demonstrated. it is possible that the human and animal ifn-l proteins have similar functions, which are relevant to antiviral response. in this study, we provide initial functional characterization of ifn-l orthologs from mammalian species, including those commonly used in biomedical research. studies of ifn-l across species should help improve our understanding of the biology of this clinically important ifn in normal and disease conditions. the human hepatoma cell line, hepg (atcc hb- ), was purchased from the american tissue culture collection (atcc) and maintained in dmem supplemented with % heat-inactivated fetal bovine serum (fbs). the custom isre-luc-hepg cell line stably expressing the luciferase reporter under control of the interferon-stimulated response element (isre) has previously been described (prokunina-olsson and others ); the cells were maintained in dmem supplemented with % fbs and mg/ml of puromycin (gold biotechnology). the mouse (mab-ifn-l ) and rabbit (rab-ifn-l ) monoclonal antibodies for ifn-l have previously been described (prokunina-olsson and others ). mab-ifn-l (mabf ; millipore) was raised against a synthetic peptide kalrdryeeealswgqrncsfrprrdsprps corresponding to amino acids - and rab-ifn-l was raised against a synthetic peptide pgssrkvpgaqkrr hkprradsprc corresponding to amino acids - of the human ifn-l protein (np_ ). the antibodies do not cross-react with the human ifn-l protein. to reduce unspecific background in flow cytometry experiments, mab-ifn-l was buffer exchanged to pbs using zeba k spin columns (thermo scientific), and for western blot and confocal imaging, the mab-ifn-l was used without buffer exchange. the expression constructs (human ifn-l -halo and control-halo) based on the pfc a vector (promega) have previously been described (prokunina-olsson and others ). the control-halo construct generates a halo-tag protein with a size of kda, and the ifn-l -halo construct generates the ifn-l -halo protein ( kda) consisting of the ifn-l protein ( kda) c-terminally fused with the halo-tag protein ( kda). for the animal expression constructs, fulllength open reading frames (orfs) were predicted from dna sequences available in the ucsc genome browser based on similarity to human ifn-l protein sequence. we used information based on our earlier sequencing of the genomic dna for chimpanzee ifn-l (genbank jx and nm_ ) and cynomolgus (crabeating macaque; genbank kc ). sequences for orangutan, marmoset, elephant, panda, megabat, cow, dog, and pig were only derived from the ucsc genome browser and not validated by sequencing. commercial genomic dna sample for rhesus was obtained from zyagen and used for sanger sequencing of the predicted ifn-l orf. complete orfs for ifn-l orthologs (supplementary table s ; supplementary data are available online at www.liebertpub.com/jir) were ordered as custom synthetic genes subcloned in pidtblue vector (idt). the sequences were then pcr amplified and recloned into halo-tag expression vector (promega) to create constructs similar to the human ifn-l -halo plasmid. the final constructs were fully sequenced for validation, propagated in e. coli, and purified with an endotoxin-free maxi plasmid kit (qiagen). hepg cells were reverse-transfected with corresponding constructs using lipofectamine and opti-mem (life technologies). after transfection for h in -well plates at a density of , cells/well, cells were lysed with ml of ripa buffer (sigma) supplemented with complete ultra protease inhibitor (roche). equal amounts of proteins were resolved on %- % bis-tris bolt gels and transferred using iblot (life technologies). detection was done using primary antibodies mab-ifn-l ( mg/ml), rab-ifn-l ( . mg/ ml), and mouse a-halo ( mg/ml, g ; promega). secondary hrp-tagged goat anti-rabbit (sc- ) or goat antimouse (sc- ) antibodies from santa cruz were used at a : , dilution. signals were detected with hyglo quick spray (denville scientific) and viewed on the chemidoc touch imager with imagelab . software (biorad). for initial screening, transfections were done for h in -well plates with , cells/well. constructs, which showed positive signals for mab-ifn-l , were further evaluated by transfections in -well plates with , cells/well, in triplicates. cells were harvested, fixed for min at room temperature with ml of cytofix (bd biosciences), and stained overnight in cytoperm (bd biosciences) with . mg/ml of a-halo, mg/ml rab-ifn-l , or mg/ml of mab-ifn-l . secondary antibodies were donkey anti-mouse and donkey anti-rabbit alexa fluor ( mg/ml; life technologies). cells were analyzed using multiparametric flow cytometry on a facs aria iii (bd biosciences) and flowjo. software (tree star). cells were transfected for h on nunc lab-tek ii chamber slides (thermo scientific) at a concentration of , cells/ chamber in ml reaction volumes. cells were fixed for min in cytofix (bd biosciences), and then coincubated for h with the mab-ifn-l ( mg/ml) in cytoperm (bd biosciences) and rabbit a-tubulin ab ( : dilution, ab- ; abcam). samples were then incubated for h at room temperature with donkey anti-mouse alexa fluor and donkey anti-rabbit alexa fluor secondary antibodies ( : dilutions; life technologies). alternatively, halo-tag was detected with cell-permeant halo-tag ligand (tmr red; promega), which was added to live cells ( : for min), then the cells were washed and fixed for min in cytofix (bd biosciences). slides were covered with mounting media (prolong gold antifade reagent with dapi). immmunofluorescent images were obtained with a confocal laser scanning microscope (lsm ; carl zeiss) and analyzed using zen software (carl zeiss). untransfected and transfected stable hepg -isre-luc cells were grown in -well plates at a concentration of , cells/well. the cells were lysed h after plating and assayed for luciferase expression of the isre-luc reporter using a glomax luminometer (promega). all transfections were done in biological replicates, except for untransfected cells ( biological replicates). ifn-l protein sequences were analyzed with free online tools-clustal w for sequence alignment and boxshade for annotating amino acid similarities. analysis of leader peptides was performed with online signalp . server (petersen and others ) , and psort ii analysis of nuclear localization signals and intracellular localization was performed with psort server (http://psort.hgc.jp/) (nakai and horton ) . statistical analysis and graphical presentation of results were done with graphpad prism (graphpad). ifn-l is a novel type-iii ifn with moderate similarity to other type-iii ifns; it has % amino acid identity with ifn-l , the most related member of this family (prokunina-olsson and others ). the three other type-iii ifns (ifn-l , ifn-l , and ifn-l ), which share high protein identity ( % between ifn-l and ifn-l and % between ifn-l and ifn-l ), were discovered in (kotenko and others ; sheppard and others ) . all four type-iii ifns are encoded by a kb genomic cluster on the human chromosome q . . due to low sequence similarity between ifnl and the genes for other type-iii ifns, ifnl could not be identified by a homology search. the reference human genome sequence has the rs -tt allele, which does not support the ifn-l orf, thus the existence of ifn-l was not predicted. ifn-l was identified by direct cloning and annotation of sequences discovered through rna sequencing of human hepatocytes treated with polyi:c to mimic viral infection (prokunina-olsson and others ). once the protein sequence of human ifn-l was known, it was used to search for ifn-l orthologs in all * vertebrate species with genomic information available through the ucsc genome browser. complete orfs encoding ifn-l proteins were found in several mammalian species, but not in nonmammals (key and others ) . the regions corresponding to ifn-l could not be found in the mouse and rat genomes, although orthologs for ifn-l and ifn-l are present in these species. now, we generated expression constructs for ifn-l orthologs ( fig. and supplementary table s ) and tested their functionality in a human hepatoma cell line, hepg , in comparison with human ifn-l . we used the human cell line, hepg , because it has all necessary components for type-iii ifn signaling and was used to characterize human ifn-l . we recognize that a human cell line may not be optimal for all ifn-l orthologs because the human receptors (ifnlr and il r ) and other signaling components may not work efficiently with nonhuman ifn-l s. however, our aim was to perform initial comparative characterization of ifn-l orthologs in the same established experimental system. additional studies on ifn-l orthologs should be performed in experimental systems relevant for species of interest. protein sequence alignment of ifn-l orthologs showed high similarity between primates, while more diversity was observed among nonprimate species (fig. ) . all orthologs were predicted to have a leader peptide, with a cleavage site located between amino acids and in most cases and between and or and in some cases. interestingly, the lowest leader peptide prediction score was for human ifn-l ( . ), followed by marmoset ( . ); all other orthologs had higher prediction scores ( . - . ) (table ) . however, a swap of leader peptides between the poorly secreted ifn-l and highly secreted ifn-l did not affect the secretion of ifn-l (hamming and others ), suggesting that the predicted strength of the leader peptide might not be relevant for ifn-l secretion. the most diverse area in ifn-l is the sequence immediately after the predicted leader peptide-the amino acid fragment was missing in all primates and this sequence was different in all nonprimates (fig. ) . this fragment did not seem to affect the leader peptide prediction scores and its functional significance is unclear. despite being only poorly secreted, human ifn-l can efficiently activate ifn signaling (prokunina-olsson and others ; onabajo and others ). we evaluated biological activity of the ifn-l orthologs after transient expression in hepg cells stably expressing an interferonstimulated response element (isre) coupled with luciferase reporter (isre-luc); this system was previously used to characterize human ifn-l . we observed differential biological activity of the ifn-l orthologs: compared with human ifn-l , proteins from chimpanzee, orangutan, marmoset, and cynomolgus showed similar levels of biological activity (isre-luc activation), while the dog protein showed significantly higher activity (by %). orthologs from rhesus, panda, and elephant were significantly less active ( % compared with the human ifn-l , fig. ). biological activities of the ifn-l orthologs measured in this experiment are likely to be affected by differential affinity of these proteins to human receptors and do not represent quantitative comparisons. however, the fact that all of the ifn-l orthologs were able to activate isre-luc reporter above the background level (control-halo) indicates that these proteins are biologically active as ifns, even in human cells. because all of the ifn-l orthologs are predicted to have leader peptides and showed biological activity for induction of ifn signaling, which is typical for exogenously acting ifns, it is likely that at least a portion of these proteins is secreted. at the same time, if these orthologs behave like the human ifn-l , they will also be retained in the cytoplasm. to test this, we evaluated protein expression and cellular localization of all orthologs transiently expressed in hepg cells. western blot analysis of protein lysates (fig. ) , flow cytometry analysis ( supplementary fig. s ), and confocal imaging ( supplementary fig. s ) confirmed that all ifn-l orthologs are detectable by an antibody or a fluorescent ligand for the halo-tag, which is present on all these proteins. confocal imaging showed that in most cases, ifn-l was expressed as a cytoplasmic protein, although, in some cases, it was also detected in the nucleus (such as in dog and panda, supplementary fig. s ). psort analysis (nakai and horton ) identified nuclear localization signals within ifn-l , and some of the orthologs were predicted to be nuclear proteins (supplementary table s ); however, there was no correlation between these predictions and intracellular localization observed by confocal imaging. it is possible that nuclear localization of ifn-l might be an alternative option utilized in some specific conditions. cytoplasmicnuclear shuttling has been reported for some factors involved in immune response ( jans and hassan ) , including ifn-g (subramaniam and others ) and irf (kumar and others ) , and the possible functional effects associated with nuclear translocation of ifn-l should be further explored in relevant experimental models. next, we tested the possibility of detection of ifn-l orthologs with two custom monoclonal antibodies generated against nonoverlapping peptides within the n and cterminal parts of the human ifn-l protein. in western blot analysis (fig. ) , flow cytometry ( fig. and supplementary figs s and s ), and confocal imaging (fig. ) , the mouse monoclonal antibody (mab-ifn-l ) recognized ifn-l from the human, chimpanzee, orangutan, rhesus, marmoset, cynomolgus (all the primates in the panel), and elephant. the results were similar for orthologs that were detectable both by halo-tag and mab-ifn-l . the rabbit monoclonal antibody (rab-ifn-l ) recognized only the human ifn-l . this is likely because the area that can be recognized by mab-ifn-l is more conserved between species compared with the area that can be recognized by the rab-ifn-l (fig. ) . further genomic studies may help to identify ifn-l orthologs in other relevant species. the success and accuracy of these predictions rely on the quality and completion of available genomic sequencing and additional targeted dna sequencing is often needed to validate the existing sequences and close the gaps. detailed analysis of functional properties of ifn-l orthologs in relation to differences in protein sequences could provide additional information on its biology. in conclusion, the importance of ifn-l in different aspects of antiviral response can be implied based on the conservation of its sequence and biological activity in mammals. however, it remains largely unknown what factors induce endogenous ifn-l expression in different conditions and why elimination of ifn-l was a beneficial event in humans strongly supported by positive selection. answers to these questions might be relevant for dealing with existing and emerging infections across species. viral infections have been directly responsible for some of the most dramatic and deadly disease pandemics in human history and many of these infections, including the recent ebola outbreak, are of zoonotic origin ( jones and others ). there are also indirect devastating effects of these infections through the loss of livestock and wildlife, causing food shortages and financial distress (frolich and others ; thumbi and others ) . some animals and corresponding derived cell lines are used for studies of human infections and vaccine development (meurens and others ; gerdts and others ) . thus, our findings and research tools might help to identify factors that trigger ifn-l expression and explore the spectrum of its biological effects in different conditions. association of the ifnl -deltag allele with impaired spontaneous clearance of hepatitis c virus deltag variant increases susceptibility to cytomegalovirus retinitis among hiv-infected patients a review of mutual transmission of important infectious diseases between livestock and wildlife in europe large animal models for vaccine development and testing interferon lambda signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses nuclear targeting by growth factors, cytokines, and their receptors: a role in signaling global trends in emerging infectious diseases selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda (ifnl ) ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex hepg cells were transiently transfected with expression constructs generating ifn-l orthologs and stained for ifn-l (mab-ifn-l , red), cytoskeleton (a-tubulin regulated nuclear-cytoplasmic localization of interferon regulatory factor , a subunit of double-stranded rnaactivated factor induction and function of type i and iii interferon in response to viral infection interferon-lambda is a cell-autonomous type iii interferon associated with pre-treatment hepatitis c virus burden ifnl ss polymorphism is associated with unfavourable clinical and immunological status in hiv-infected individuals influence of ifnl / polymorphisms on the incidence of cytomegalovirus infection after solid-organ transplantation the pig: a model for human infectious diseases psort: a program for detecting sorting signals in proteins and predicting their subcellular localization ifn-lambda : the paradoxical new member of the interferon lambda family expression of interferon lambda is associated with reduced proliferation and increased cell death in human hepatic cells an overview of the immune system signalp . : discriminating signal peptides from transmembrane regions a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus ifn-l rs polymorphism is associated with innate resistance to hiv- infection il- , il- and their class ii cytokine receptor il- r the carboxyl terminus of interferon-gamma contains a functional polybasic nuclear localization sequence linking human health and livestock health: a ''one-health'' platform for integrated analysis of human health, livestock health, and economic welfare in livestock dependent communities the work has been supported by the intramural research program (irp) of the division of cancer epidemiology and genetics, national cancer institute, national institutes of health, us. author disclosure statement l.p.-o. is a coinventor on a patent application related to ifn-l filed by the nci/nih. key: cord- -tpjxt w authors: mandl, judith n.; schneider, caitlin; schneider, david s.; baker, michelle l. title: going to bat(s) for studies of disease tolerance date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tpjxt w a majority of viruses that have caused recent epidemics with high lethality rates in people, are zoonoses originating from wildlife. among them are filoviruses (e.g., marburg, ebola), coronaviruses (e.g., sars, mers), henipaviruses (e.g., hendra, nipah) which share the common features that they are all rna viruses, and that a dysregulated immune response is an important contributor to the tissue damage and hence pathogenicity that results from infection in humans. intriguingly, these viruses also all originate from bat reservoirs. bats have been shown to have a greater mean viral richness than predicted by their phylogenetic distance from humans, their geographic range, or their presence in urban areas, suggesting other traits must explain why bats harbor a greater number of zoonotic viruses than other mammals. bats are highly unusual among mammals in other ways as well. not only are they the only mammals capable of powered flight, they have extraordinarily long life spans, with little detectable increases in mortality or senescence until high ages. their physiology likely impacted their history of pathogen exposure and necessitated adaptations that may have also affected immune signaling pathways. do our life history traits make us susceptible to generating damaging immune responses to rna viruses or does the physiology of bats make them particularly tolerant or resistant? understanding what immune mechanisms enable bats to coexist with rna viruses may provide critical fundamental insights into how to achieve greater resilience in humans. an estimated ∼ % of emerging infectious diseases are caused by pathogens which originate from a non-human animal source, referred to as zoonoses ( ) ( ) ( ) . moreover, the frequency of outbreaks caused by zoonotic pathogens has been increasing over time in the human population, with viruses being the most successful at crossing the species barrier ( ) ( ) ( ) . given the impact of viral zoonoses on global public health, considerable resources have been invested into better understanding patterns in their emergence to improve predictions of where they might arise. one key variable in such predictions is to determine the animal reservoir populations within which these novel viruses can be maintained indefinitely (with or without disease) and which therefore act as sources for transmission to humans ( ) . in some instances, epidemiological associations may provide clues to identifying a reservoir host species, and the detection of natural infection through seroconversion or the virus itself provides further evidence. recently, phylogenetic analyses have also been used to investigate viral origins-with a presence of greater diversity and of strains ancestral to those in humans being indicative of a virus circulating within a particular natural host population ( ) . once identified, viral reservoirs have historically been critical levers through which to reduce human cases ( ) . however, reservoir hosts may also provide us with fundamental insights into host-pathogen interactions and are a rich opportunity to examine the immunological processes that contribute to patterns governing which pathogens cross into humans, cause disease and why ( , ) . this can be particularly informative as in many instances, the zoonotic viruses that are so pathogenic in humans do not cause disease in the reservoirs with which they coexist. bats have been confirmed as reservoir hosts for many viruses, several of which are associated with fatality rates as high as % among diagnosed human cases. it has long been appreciated that rabies and other lyssaviruses causing lethal encephalitis can be transmitted from numerous bat species ( , ) . live marburg virus (marv) has been isolated from rousettus aegyptiacus fruit bats which, jointly with epidemiologic evidence and detection of viral rna, strongly suggests that r. aegyptiacus is a reservoir host of this filovirus ( ) . the related ebolavirus (ebov) likely also circulates in african fruit bats, with a few species having been implicated so far-the mobility of which accounts for the sudden appearance of ebola in west africa during the outbreak, a region where ebolavirus had not previously been detected ( , ) . the highly pathogenic henipaviruses, of which hendra virus emerged in australia and nipah virus in south-east asia via horse and pig intermediate hosts respectively, have been shown to be transmitted from pteropus bats ( , ) . in china, horseshoe rhinolophus bats have been identified as the reservoirs for sars coronavirus via palm civet intermediate hosts, the cause of a large outbreak of atypical pneumonia across several countries that began in in china ( ) ( ) ( ) . more recently, mers coronavirus that has caused lethal respiratory infections mostly in saudi arabia, likely transmitted via dromedary camels, was shown to be closely related to several bat coronaviruses, including those sequenced from neoromicia capensis, pipistrellus abramus, and vespertilio superans bats ( , ) . moreover, additional viruses may continue to emerge from bats, as in the single case of sosuga virus infection in a wildlife biologist collecting bats in south sudan ( ) . in addition to these emerging zoonotic viruses, bats may be the source of a number of viruses with which humans have older evolutionary associations. for instance, bats harbor viruses closely related to both mumps (rubula virus) and measles (morbilli virus) and have likely been donors of these viruses to other mammalian groups, possibly including humans ( , ) . furthermore, both old and new world bats carry diverse hepadnaviruses, some of which are related to hepatitis b virus and can infect human hepatocytes ( ) . hepaciviruses that are related to hepatitis c virus and pegiviruses that are related to human gb viruses were detected in the sera of many different bat species, and given the basal position of these bat viruses in phylogenetic trees, may also represent strains ancestral to those found in humans ( , ) . the preponderance of links between bat and human pathogens has led to a debate about whether bats disproportionately contribute to emerging viral infections crossing the species barrier into humans ( ) ( ) ( ) ( ) ( ) . given the diversity of the chiroptera order (figure ) , we may simply see more bat viruses because there are so many (> , ) species of bats ( ) . however, even when accounting for the fact that they make up ∼ % of extant terrestrial mammals, bats are overrepresented as reservoir hosts of pathogens with a high potential for spilling into human populations ( , ) . in fact, no known predictors that have been described to impact the likelihood of crossing the species barrier, including reservoir host ecology, phylogenetic relatedness to humans or frequency of reservoir-human contact, explain this pattern ( ) . thus, why bats are such a frequent source of pathogenic human viruses remains a tantalizing mystery. among viruses, those that have genomes encoded by rna generally jump across species boundaries more frequently, presumably due to their inherently greater mutation rates that facilitate the rapid adaptation to replicating within new hosts ( ) . interestingly, all pathogenic viruses that have made the jump to humans for which bat species may be reservoirs share the common feature that they have single-stranded rna genomes (with the exception of hepadnaviruses which have a dna genome but replicate via an rna intermediate). so far, available evidence suggests that bats remain disease-free when infected with the rna viruses they carry-even those highly pathogenic to humans-and are able to coexist with them without detectable fitness costs using measures such as changes in temperature, loss of body weight, or overt signs of inflammation ( ) . indeed, so far only one rna virus studied which circulates in a bat population has been shown to consistently cause significant morbidity and mortality: tacaribe virus in the jamaican fruit bat (artibeus jamaicensis), which recent evidence suggests is not a reservoir host for this virus ( ) . data from experimental rabies and lyssavirus infections suggests that rhabdoviruses may also cause disease in bats, although experimental infection outcome is very dependent on the infection route. intracerebral infection with different strains and in different bat species invariably led to death ( , ) . in contrast, intramuscular infection led to muscle weakness, paralysis and visible histological cns lesions in % of experimentally infected flying foxes (pteropus poliocephalus) ( ) . similarly, a subset of vampire bats (desmodus rotundus) experimentally infected intramuscularly with a high dose of rabies virus remained healthy despite viral shedding in the saliva and survived ( ) . naturally infected bats are thought to either die or remain healthy and seroconvert, but transmission in freeranging populations remains incompletely understood ( ) . while bats seem to be frequent hosts for rna viruses, current available data indicates that primates and humans disproportionately harbor dna viruses such as herpesviruses ( ) . interestingly, it is these dna viruses that can persist in an individual which can also be found in isolated, small indigenous groups-perhaps suggestive of humans having a more ancient relationship with such dna viruses ( ) . it may even be the case that persistent dna viruses in humans impact immune responses specifically to rna viruses, but this has not yet been examined. it is likely that differences in evolutionary history of pathogen exposure between bats and humans have led to distinct adaptations in anti-viral immune responses and the ability to tolerate certain infections without disease while being susceptible to others. importantly, bats differ in many aspects of their physiology and behavior from humans that may have direct or indirect effects on immune function. bats are a monophyletic mammalian group traditionally divided by morphological data into two suborders, the megabats and microbats, which more recent molecular data has revised into the yinpterochiroptera and yangochiroptera suborders (figure ). bats possess a suite of traits that make them distinct from other mammals in a number of ways. these unique life history traits may play a role in understanding which pathogens bats have evolved to coexist with and why. in particular, such traits may explain the ability of bat populations to maintain particular viral pathogens indefinitely, and may have effects on immune function through specific energetic or evolutionary trade-offs we have yet to better define. despite the diversity of viruses carried by bats, they are not typically known to cause mass bat die-offs or reduce bats' remarkable longevity. in this respect, bats represent a potential opportunity for long-term persistence of viruses within a population and across generations. bats live significantly longer than similarly-sized terrestrial mammals and, despite their small size, are characterized as "slow" mammals in the slow-fast continuum ( , ) . although their weights range from grams to kilograms, with respect to longevity bats group with large mammals such as humans and non-human primates ( ) . aerial living has an obvious advantage in avoiding predation, but bats outlive even birds. for example, the brandt's bat (myotis brandtii) lives up to years, compared to selasphorus platycercus, a bird species of similar size that lives for ∼ years ( , ) . thus, flight can only partially account for their extraordinarily long lives. initially, the longevity of some bats was attributed to seasonal hibernation, as temperate-zone species enter continuous torpor of up to days, with a dramatic drop in metabolic rate such that small fat reserves can sustain them throughout the entire hibernating season ( ) . however, even non-hibernating bat species live three times longer, on average, than predicted by their size, and heterothermy is not an accurate predictor of lifespan in other mammalian orders, suggesting that the driving force behind their surprising longevity is intrinsic to bats as a group ( ) ( ) ( ) . like other "slow" mammals, bat females typically only have one offspring per year, perhaps because the volant lifestyles of bats make it difficult to rear more than one offspring, as pregnant females and those with recent births must navigate and forage with added weight; on average, neonatal bat pups are ¼ of their mother's weight ( ) . the physical and energetic constraints of rearing multiple offspring may necessitate small litters, which would in turn require prolonged reproductive capability and enhanced longevity to ensure maintenance of the population over generations. thus, in bats, the dependence of colony survival as a whole may depend upon enhanced individual survival and delayed senescence ( ) . genetic analyses of several bat species have shown differences in the growth hormone (gh)/insulin-like growth factor (igf ) axis which in humans is associated with aging, resistance to diabetes and cancer ( ) . the determinants of adult survival in bats have been historically difficult to identify, as this requires tracking individuals over many years, and until recently longitudinal studies of bat mortality were conducted using tagged bats, of which only a fraction were recovered ( ) . recently, a year study of a colony of bechstein's bats demonstrated that unlike terrestrial mammals, survival could not be predicted by common indicators such as season, age, and body size. instead, the only accurate predictor of mortality was a single cataclysmic weather event that affected multiple countries in north-central europe. additionally, even the oldest female bats were reproductively capable, indicating that bat survival is primarily affected by catastrophic natural events rather than factors that normally dictate an individual's fitness ( ) . molecular phylogenetic studies of bats suggest that there are massive gaps in bat fossil records. as bats are the second most diverse order of mammals, outnumbered only by rodents, the number of species unrepresented in the fossil records is staggering. over half of microbat and nearly all of megabat fossil histories are missing ( , ) . the enormous incompleteness of the fossil records has made it difficult to identify when specific morphological traits of bats arose. as molecular phylogeny groups two echolocation-reliant microbat species with megabats (also called old world bats or pteropodids), which do not rely on echolocation, there is some debate as to whether echolocation first arose in the common ancestor of bats and was subsequently lost in megabats, or whether it arose twice, independently ( ) . pteropodids have adaptations that enhance visual acuity at night ( ), and they do not require echolocation for foraging ( ) . there are multiple types of echolocation that can be partially delineated by species, but are more clearly categorized by the type of environment. divergent species that inhabit the same type of environment, such as those that hunt in large, open spaces, often use the same form of echolocation, suggesting that habitat has a greater influence on echolocation than phylogeny ( ) . importantly, echolocation can result in the production of droplets or small-particle aerosols of oropharyngeal fluids, mucus, or saliva, thus facilitating transmission of viruses between individuals in close proximity ( , ) . the unique navigation tactic of many bat species may inadvertently facilitate virus transmission among bats in the same habitat. bats are the only mammal capable of powered flight, which likely evolved ∼ million years ago alongside birds following radical ecological changes that resulted in the extinction of the dinosaurs ( , ) . during flight, bats consume approximately four times as much oxygen, and they have a markedly higher concentration of red blood cells compared to small terrestrial mammals ( ). bat flight is markedly different from that of birds and insects, whose wing surfaces are typically composed of inflexible material, such as feathers or chitin. bat wings are constructed from live skin stretched across elongated arm and finger bones, making them extraordinarily malleable and sensitive to environmental cues ( ) . the plasticity of bats' wings allows them to navigate and inhabit diverse ecospheres, contributing to their extensive speciation. moreover, the capability of powered flight can allow the efficient spread of viruses and thus the introduction of pathogens to which colonies may otherwise have remained naïve. as flight is extremely metabolically demanding, in addition to evolving the physical mechanisms required for flight, bats have also evolved necessary underlying molecular mechanisms. the mitochondrial respiratory chain accounts for nearly all atp required for mobility in eukaryotes, and genetic analysis of both micro-and megabat species revealed an enrichment of genes specific to the oxidative phosphorylation (oxphos) pathway. specifically, . % of nuclear-encoded and % of mitochondrial oxphos genes have evidence of positive selection in bats, which is markedly higher than the expected % of orthologous genes in previous genome-wide studies that show evidence of positive selection ( ) . genomic analysis of pteropus alecto and m. davidii suggests positive selection for the dna damage checkpoint pathway and changes in overlapping aspects of this pathway with the innate immune system, indicating that evolutionary adaptations important for flight may have secondarily affected bat immunity ( ). as a group, bats exhibit the greatest diversity of social systems in mammals. tropical species are primarily responsible for this diversity, as temperate species are more restricted in their social behavior. generally, however, bats are extremely social creatures that tend to form dense roosting colonies ( ) , and almost all temperate-zone species live in closed societies with very little infiltration of foreign bats into established roosts ( , ) . in particular, female bats form maternity colonies in which males do not take part. as bats are capable of longdistance flight, dispersal barriers cannot explain the philopatry of females. instead, benefits such as knowledge of foraging areas and social thermoregulation likely selected for these colony types. additionally, there is evidence that forming closed societies limits the potential invasion of new pathogens, thereby protecting colony members that would otherwise be vulnerable to infection. for example, pseudogymnaoscus destructans has decimated north american bat populations that do not live in the type of closed societies observed elsewhere ( ) . dna analysis of a closed society of bechstein's bats revealed extraordinarily high conservation of mitochondrial dna and relatively low conservation of nuclear dna, suggesting stable maternal populations within colonies and gene flow between colonies via promiscuous mating with males. it is possible that the mating patterns of temperate-zone species may allow transmission of pathogens between colonies via traveling males while the frontiers in immunology | www.frontiersin.org more insular females may allow viruses to persist throughout generations within a colony. an important commonality among pathogenic rna viruses in humans presenting with disease is that the host response is an important contributor to the disease process, with dysregulated and excessive innate immune responses being particularly important drivers of tissue damage during infection ( ) . given the general absence of clinical signs of disease in bats infected with the same viruses that are so lethal in humans or other non-natural hosts infected experimentally, a critical question has been to understand whether bats might establish effective disease tolerance, thus maintaining fitness despite pathogen replication, or whether bats are more resistant to infection through more successful control of pathogen replication and what the contribution of the immune response is ( , ) . the lack of many fundamental immunological tools enabling the probing of bat immune responses has meant that truly mechanistic studies of bat immunity have been very limited, although recently there has been some progress in establishing approaches such as flow cytometry to identify distinct bat immune cell populations ( , ) . so far, studies of bat immunity have primarily taken one of three approaches, whereby each comes with important strengths and weaknesses that have to be kept in mind: (i) comparative genome studies, (ii) in vitro cell culture assays, and (iii) experimental infections. comparative genome studies have confirmed that the critical components of the innate and adaptive immune system are conserved in bats at the gene level and that bats have the machinery for innate responses to pathogen-associated molecular patterns (pamps), the production of anti-viral effector molecules such as type i interferons (ifn), t cell responses (variable t cell receptors, mhci and mhcii), and b cell responses [reviewed in ( ) ]. interestingly, based on the bat genomes sequenced so far, the only family of genes lost entirely in all of them are pyhin genes ( ) . members of the pyhin family are dna sensors capable of recognizing foreign dna, including dna viruses and damaged self dna which can be generated by rna viral infection. recognition of dna results in production of ifn through interaction with stimulator of interferon genes (sting). the pyhin family also encode the only identified class of dna sensors capable of activating the inflammasome. it has been hypothesized that the absence of the pyhin family may allow bats to limit activation of the innate immune response to damaged self-dna generated by rna viral infection, thus avoiding excessive inflammation ( , ) . genome comparisons highlighting contractions or expansions of specific gene families, specific genes under positive selection, or nonconserved sequence differences in critical protein domains can thus provide the basis for hypotheses worth testing further. however, it is important to note that much can be missed in absence of data on gene regulation, especially during infection when gene expression kinetics can make a critical difference to the infection outcome. moreover, the absence of a gene or gene family does not rule out that other proteins have evolved to compensate for their loss of function. thus, while whole genome analyses can provide a context for specific questions or be hypothesis-generating, on their own they cannot distinguish tolerance from resistance mechanisms. the repeated identification of signatures of positive selection in innate immune genes in particular, does however lend credence to the idea that bats have specific adaptations as a result of a long co-evolutionary history with viruses. cell culture assays with bat cell lines, or, in some instances, primary bat cells, have been used to assess whether bats are permissive for viral replication and to determine whether particular immune receptor signaling pathways are intact. as discussed below, such studies have probed the type i ifn pathway in particular, revealing some possible species-specific differences among bats ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . however, it is important to note that in some instances immortalized cells can behave differently from primary cells and that such cultures may miss additional differences imposed by changes in cell localization, cell recruitment or cell-cell interactions in a whole animal. careful experiments measuring the quality, magnitude, and kinetics of immune responses in bats during infection and upon administration with defined stimuli for which we have comparative information from humans remain to be done to provide additional evidence that specific innate immune pathways are wired differently. experimental infections come with the enormous challenge of having to house and/or breed colonies of bats and to have biosafety-level facilities in place to perform infections with viruses lethal to humans. moreover, some trial and error is involved in determining which route and dose leads to viral replication, establishing a source of the virus (humanadapted strains tend to replicate less well in bats than strains obtained from naturally infected bats), and amplifying this viral stock without extensive tissue culture passaging. studies to date have examined the kinetics of viral replication by quantifying the extent of viremia and dissemination to other tissues, and assessing changes in white blood cell counts, body mass, and temperature. given the generally low levels of viral shedding and short infectious periods observed so far it remains poorly understood how transmission occurs in the wild to sufficient levels that cross-species jumps occur. some infection experiments have also provided evidence that a particular bat species is unlikely to be a reservoir despite epidemiological evidence, for example for r. aegyptiacus and ebolavirus. certainly, once good experimental infection models are established, such studies have the potential to be hugely informative with regard to anti-viral immune responses elicited using, for instance, comparative transcriptome analyses. one drawback may be that experimental infections do not mimic the impact of chronic stress arising from the disruption of wildlife populations, which bats are particularly sensitive to jones et al. ( ) . comparison of either cave-roosting or foliage-roosting species in areas of malaysian borneo designated as actively logged forest, recovering forest, or fragmented forest revealed varying impacts of habitat disturbance on stress and circulating white blood cells ( ) . overall, the limited studies of bat immunity that have been done have focused largely on species: p. alecto and r. aegyptiacus. we summarize this work below, but comparisons of observations made across species suggest that although a number of species appear to be capable of avoiding the pathological effects of rna virus infection, each bat species may have achieved this through distinct pathways, possibly involving changes to both increase pathogen replication control and to mitigate any immunopathology through decreased inflammatory responses and hence increased disease tolerance. the most well studied bat species with regard to antiviral immune responses is the australian black flying fox (p. alecto). this interest has stemmed from the fact that pteropid bats have been identified as the natural reservoirs for the deadly hendra and nipah viruses ( ) , which continue to cause outbreaks [such as most recently in india in may ( )]. to date, several studies have examined the kinetics of viral infection in pteropus bats and the nature of transmission and replication in other susceptible species ( ) ( ) ( ) ( ) . in australia, all four species of pteropid bats (p. alecto, p. poliocephalus, p. scapulatus, and p. conspicillatus) have antibodies to hendra virus but only p. alecto and p. conspicillatus are considered to be the primary reservoir hosts ( , , ) . in south east asia, both pteropus spp. occurring in malaysia have been found to be seropositive for nipah virus neutralizing antibodies, and the virus has been isolated from p. hypomelanus and p. vampyrus ( , ) . experimental infections of pteroid bats with hendra or nipah virus result in sub-clinical infection with short periods of virus replication and shedding, and low antibody titres ( ) ( ) ( ) ( ) . upon subcutaneous infection of p. poliocephalus with hendra virus, viral antigen was detected by immunohistochemistry at dpi in blood vessels of spleen, kidney and placenta ( ) . similarly, oronasal hendra virus infection of p. alecto led to the presence of viral genome in lung, spleen, liver and kidney weeks later, but virus isolation was unsuccessful at this timepoint ( , ) . the malaysian flying fox, p. vampyrus and the australian species, p. poliocephalus demonstrate similarly short periods of viremia upon infection with nipah virus. in subcutaneously infected p. poliocephalus, virus was isolated from the kidney and uterus of bats euthanized at dpi, but no virus was isolated at any of the other timepoints examined ( , , , , or dpi) and there was no evidence of antigen in any tissue by immunohistochemistry, including tissues collected at dpi. in this study, low neutralizing antibodies were detected in all bats with the exception of one individual that developed a significant neutralizing antibody titre -possibly reflecting the fact that p. poliocephalus is not the natural host for nipah virus ( ) . in p. vampyrus challenged by oronasal nipah inoculation, viral genome was detected in a throat swab at dpi and a rectal swab of the same individual at dpi but virus was undetectable in tissues collected at postmortem from all individuals ( , , or dpi), consistent with a short period of viremia. similar to previous studies, antibody titres were low in all p. vampyrus bats ( ) . overall, these results are consistent with bats controlling replication rapidly, at least following experimental infections which involve higher doses of virus compared to what bats would likely be naturally exposed to in the wild. the absence of a robust antibody response also appears to be typical of all experimental hendra and nipah virus infections performed to date. since antibody responses are the only immune parameter that has been measured during experimental infections of bats so far, it is difficult to speculate on the mechanisms responsible for control of viral infections in vivo. pteropus alecto was among the first bat species to have its genome described in detail. genomic studies provided initial clues for possible differences in the innate immune system of bats, with evidence for selection of key innate immune genes and the expansion or contraction of specific immune gene families ( , , ) . the mhci region is contracted ( ) , as is the type i ifn locus, which in p. alecto contains fewer ifn genes than any other mammalian species sequenced, with only three functional ifn-α loci ( ) . in contrast, pteropid bats have the largest and most diverse family of apobec (apolipoprotein b mrna editing enzyme, catalytic polypeptide-like) proteins identified in any mammal ( ) . apobecs interfere with the replication of retroviruses by deaminating cytosine residues in nascent retroviral dna. this is notable, as bats are an important source of mammalian retroviruses, many of which have been transmitted to other mammals ( , ) . apobec diversification may therefore have occurred to counteract the effect of retroviruses and possibly other viruses, as apobecs have been shown to restrict the replication of other virus families including hepadnaviruses, and parvoviruses ( , ) . members of the apobeca protein family exhibit direct antiviral activity through dna cytosine deamination which results in hypermutation of the nascent retroviral dna which is then degraded or rendered non-functional ( ) . the mechanism of antiviral activity against non-retroviruses remains largely unknown. for parvovirus adeno-associated virus, apobec meditated inhibition has been speculated to involve direct interaction with the viral dna or the replication machinery ( ) . whether the expanded family of abobecs in bats have evolved other mechanisms to control dna and rna viruses remains to be determined. as apobecs can be induced by even low levels of type i ifn ( ) , one hypothesis to be tested is that bats, through their multiple apobecs, are able to restrict viral replication without causing inflammation. pteropus alecto is the only bat species to date in which apobec genes have been mapped, and whether the expansion of this gene family extends to other bat species remains to be determined. in addition to the identification of putative immune pathways distinct in p. alecto through genome studies, differences have been identified in the activation of innate immune effectors in p. alecto from studies performed in vitro, primarily using cell lines derived from tissues including the kidney and lung. ifns are the first line of defense following viral infection and unsurprisingly, because of this, they have been the most extensively studied group of genes in bats. both type i (ifna and ifnb) and iii (ifnl) ifns are detectable in bat cells. curiously, a unique characteristic of pteropid bats is the constitutive expression of mrna for ifna and the signaling molecule, ifn regulatory factor (irf ) in unstimulated tissues and cells [ , a] . constitutively expressed ifna and irf may allow bats to respond more rapidly to infection, thus avoiding the lag time between pathogen detection and response. furthermore, viral infection or stimulation with synthetic ligands result in little ifna induction in pteropid bat cells ( ) . the constitutive expression of ifna has been described in two species of pteropid bats (p. alecto and cynopterus brachyotis) and is a first for any species. ifnb and ifnl are activated following stimulation of cells from p. alecto and p. vampyrus with synthetic ligands such as polyic ( ) ( ) ( ) ( ) . moreover, bat ifns demonstrate antiviral activity ( , ( ) ( ) ( ) ( ) ) . however, viral infection of p. alecto splenocytes results in induction of ifnl but not ifnb, hinting at differences in the function of type i and iii ifns ( ) . in humans and mice, ifnl has recently been demonstrated to have a role not only in controlling virus replication, but also in dampening damage-inducing neutrophil functions and in modulating tissue-damaging, transcriptionindependent responses such as production of ros ( , ) . a hypothesis yet to be tested is whether upregulation of ifnl rather than ifnb has a similar function in bats. the endoplasmic reticulum (er) membrane protein, sting, is involved in induction of type i ifn by cytosolic dna ( ) . stimulation of bat splenocytes with gmp-amp, which is produced following sensing of cytosolic dna by cgas, results in little induction of ifn compared to responses observed in mouse splenocytes ( ) . bat sting contains an amino acid substitution of the highly conserved and functionally important serine residue s which may be responsible for dampening sting-dependent ifn activation in bat cells in response to dna. however, comparable levels of ifn induction in mouse and bat cells in response to the rna viral mimic polyic indicate that sting-associated inhibition of the ifn response does not extend to rna viruses ( ) , thus the relevance to rna viruses in bats remains unknown. downstream of the induction of ifns, novel subsets of ifn stimulated genes (isgs) have been detected in unstimulated and stimulated pteropid bat cells indicative of a response that is less damaging to the host. furthermore, the isg response is elevated for a shorter period of time in p. alecto compared to human cell lines which again may be a strategy to avoid tissue damage ( , ) . the less inflammatory profile of isgs may be the key to the ability of bats to tolerate higher ifn expression without adverse consequences. the balance between resistance and tolerance may therefore be achieved through careful selection of the pathways that are activated and shorter periods of activation or limited activation to prevent inflammation. in this regard, studies of the regulation of ifn signaling in bats is likely to provide important additional insights. a second bat species whose host responses to viral infections has been studied more recently is the egyptian fruit bat (r. aegyptiacus). marburg virus (marv) has been repeatedly isolated from this species with demonstrated seasonal pulses of active marv replication in juvenile bats living in caves in uganda ( , ) . moreover, r. aegyptiacus were a suspected reservoir for ebolavirus (ebov) based on epidemiological evidence and detected seroreactivity to ebov, but no infectious virus has been isolated thus far from wild rousettus bats ( ) . indeed, while cell lines from r. aegyptiacus are equally susceptible to marv and ebov ( , ) , experimental infections of r. aegyptiacus seem to confirm that it is a reservoir for marv, but is unlikely to be the source of ebov spillover to humans. subcutaneous ebov infection results in very low viral replication, no viremia, little dissemination to other tissues, and no viral shedding, although some animals seroconvert, suggesting that r. aegyptiacus are unlikely to perpetuate ebov in the wild ( , ) . in contrast, experimental marv infection of r. aegyptiacus resulted in acute viremia that peaked on days - post-infection (although generally at lower levels than in humans), oral shedding that peaked on days - postinfection, and dissemination to other tissues including spleen, liver, kidney and salivary glands ( , ( ) ( ) ( ) . interestingly, viral replication was not associated with increases in white blood cell counts, any clinical signs of infection such as changes in body temperature or body weight, and infected tissues showed little evidence of inflammatory infiltrates ( ) . in all experiments, viremia was cleared by day and oral shedding ceased by day . intriguingly, a cohousing experiment resulted in marv transmissions to uninfected bats - months after experimental infection, raising the question of whether persistent infection with intermittent shedding is possible or whether very long latent periods without detectable viral replication could follow exposure ( ) . upon secondary challenge of previously marv-infected bats, none showed any detectable viral replication or shedding, providing evidence that protective immunity is established ( ) . unlike for pteropus bats, no constitutive expression of type i ifns has been detected in r. aegyptiacus ( ) , but type i ifns are induced in r. aegyptiacus cell lines upon stimulation with sendai virus as seen in other mammals ( ) . furthermore, in r. aegyptiacus the type i ifn genes are expanded, again in contrast to p. alecto ( ), but like for p. alecto a number of genes in the type i ifn pathway or involved in innate immune recognition of pamps show signs of having been under positive selection ( ) . whether positive selection of genes in either bat species is associated with tolerance remains to be determined, especially given that innate immune genes in humans have also been under positive selection ( ) . a transcriptome study which generated rna sequencing libraries from tissues taken from female and male r. aegyptiacus found a reduced coverage of nk cell related genes compared to other mammals, but confirmed that in these bats the predominant t cells had an αβ t cell receptor, and showed that ige, igg, igm, and iga, as well as a number of pro-and anti-inflammatory cytokines, were all detectable ( ) . the recently sequenced r. aegyptiacus genome revealed substantial differences in the repertoire of nk cell receptors, with this bat species entirely lacking functional killer cell immunoglobulin receptors (kirs) and with all killer lectinlike receptors (klrs) encoding either activating and inhibitory interaction motifs, or inhibitory interaction motifs only ( ) . nk cells are important immune cell players in an antiviral response but without assessment of the consequences of these genomic differences it is difficult to draw any specific conclusions with regard to viral control or the magnitude of inflammation elicited upon infection with viruses like marv. nonetheless, these genomic data provide some interesting hypotheses to be tested in the future. some additional studies probing the induction of cytokines upon stimulation of bat cells with defined innate immune stimuli provides some evidence that innate immune recognition of viruses may be altered, leading to a reduction in proinflammatory responses. stimulation of kidney and myeloid cells from the big brown bat (eptesicus fuscus) with polyinosinicpolycytidylic acid (polyi:c) resulted in only limited activation of the inflammatory cytokine, tumor necrosis factor alpha (tnfα) compared to human cells which display a robust tnfα response. induction of tnfα is controlled by transcription factors, including the nf-kappa b (nf-κb) family which consists of five members, [rela (p ), relb, c-rel, nfκb- (p ), and nfκb- (p )] which form homo-or hetero-dimers that are bound by molecules of the inhibitor of nfκb (iκb) family and retained in the cytoplasm of the cell in an inactivated state ( ) . in e. fuscus, a potential repressor (c-rel) binding motif was identified in the tnfα promoter region which may explain the difference in induction of tnfα in e. fuscus cells. consistent with this hypothesis, partial knockdown of c-rel transcripts significantly increased basal levels of tnfα transcripts in e. fuscus cells ( ) . the transcription factor, c-rel has also undergone positive selection in the bat ancestor which may indicate that this mechanism is common to other species of bats ( ) . of note, low levels of tnfα induction have also been associated with tolerance in european bank voles which are a natural reservoir for puumala hantavirus (puuv) ( ) . stimulation of macrophages from the greater mouse eared bat (myotis myotis) suggested that this species may have also evolved mechanisms to avoid excessive inflammation caused by cytokines. while high levels of tnfα, il β, and ifnβ were produced in response to in vitro challenge with lipopolysaccharides (lps) and polyi:c, there was also a sustained, high-level transcription of the anti-inflammatory cytokine il- , which was not observed in mouse macrophages ( ) . furthermore, unlike in the mouse, m. myotis macrophages did not produce the proinflammatory and cytotoxic mediator, nitric oxide, in response to lps. the same study also showed evidence of bat specific adaptations in genes involved in antiviral and proinflammatory signaling pathways through comparison with other mammalian taxa, including rig-i, il b, il- , nlrp , sting, and casp , further supporting the evolution of adaptations associated with reducing inflammatory responses in bats ( ). even less is known about immune responses of bats to nonviral pathogens than to viral pathogens, but it is clear that while anti-inflammatory responses may be characteristic of antiviral responses in bats, they are susceptible to disease upon infection with particular pathogens-in some instances due to dysregulated and damaging immune responses. one particular example of this is the emerging infectious disease, white nose syndrome (wns), that has decimated north american bat populations beginning in , in what will likely rank as one of the most devastating wildlife diseases in history ( ) ( ) ( ) . for reasons that remain poorly understood, the psychrophilic fungus pseudogymnoascus destructans (formerly geomyces destructans) causes no mass mortality in european bats despite being abundantly detected ( , ) . indeed, evidence suggests that a single p. destructans genotype was introduced to north american bat species from europe ( ) . in north america, p. destructans infection is not specific to a particular bat genus, replicating in many different bat species during hibernation and targeting the furless skin of the wings, ears, and muzzle ( ) . distinct hypotheses have been proposed for why p. destructans is so deadly in north american bats, ascribing the impaired tolerance to infection compared to european bat counterparts to either physiological or immunological factors. on the one hand, more frequent arousal, electrolyte depletion, and dehydration are thought to contribute to mortality following infection ( , ) . the destruction of wing tissue in wns results in a marked electrolyte imbalance, as the wings play a critical role in maintaining water levels, especially during hibernation, during which bats are particularly vulnerable to dehydration ( , ) . dehydration catalyzes arousal in hibernating bats, which is extraordinarily metabolically costly and rapidly depletes the fat reserves necessary to survive until spring ( ) . an alternative hypothesis posits that the restoration of the immune system following emergence from hibernation induces the fatal pathology of wns. during hibernation, destruction of cutaneous tissue is limited and infiltrating immune cells are entirely absent, yet in the weeks following arousal, infected bats exhibit overt wing damage and corresponding neutrophilic and lymphocytic infiltration ( ) . hibernation does not preclude a localized immune response to p. destructans at the site of infection and transcriptomic analysis of infected tissue showed upregulation of some acute inflammatory genes in infected tissue ( , ) . however, the observed immune responses likely occur during arousal periods, which are more common in infected bats. ultimately, immunosuppression during torpor allows p. destructans to colonize infected bats relatively unchecked ( ) , and upon emergence from hibernation, the exuberant immune response may result in deadly immunopathology during wns ( ) . in addition to general studies of immune cell recruitment and transcriptional responses during wns, body mass and white blood cell counts were examined following lps administration in four bat species ( ) ( ) ( ) ( ) . subcutaneous lps challenge in of pallas's mastiff bats (molossus molossus) led to a loss of body mass of ∼ % within the first day, but did not result in changes in circulating white blood cell counts or body temperature ( ) . seba's short-tailed fruit bat (carollia perspicillata) also showed a decrease in body mass following lps challenge, but this was associated with increases in white blood cell counts as well as increases in derivatives of reactive oxidative metabolites (drom) ( ) . subdermal lps challenge of fish-eating myotis (myotis vivesi) led to body mass decreases, increased resting metabolic rate and skin temperature ( ) , while intraperitoneal lps challenge of wrinkle-lipped bats (chaerephon plicatus) caused an increase in circulating leukocytes, but did not result in a reduction in body mass compared to controls ( ) . the differential responses to lps challenge suggest that the immune response to bacterial infection varies across species. of note, postmortem examinations of ∼ dead bats comprising species from germany revealed inflammatory lesions, many of which had evidence of underlying bacterial or parasitic infections, particularly in the lung ( ) . bats have an array of unique life history characteristics that not only allow them to be particularly good reservoirs for viruses that are highly pathogenic in other species, but also appear to have shaped their immune systems. although research on bat antiviral immunity has focused on only a few species to date, at the genomic level, selection on genes is concentrated on the innate immune system across both suborders of bats. however, while these studies have provided a rich source of hypotheses, the majority remain to be tested at the functional level and many questions remain that cannot be answered from comparative genome studies. experimental studies to date have demonstrated some functional differences between bat species, with the common emerging theme that the overall antiviral response appears to converge on a lower inflammatory profile, with tight regulation of the cytokine and inflammatory response key to clearing viral infection without the pathological outcomes typically associated with infection. however, whether this is due to specific tolerance mechanisms that are at play or increased resistance to rna virus replication still remains unclear. fewer studies have examined the adaptive immune system than those probing innate immune pathways, but experimental infections with bat borne viruses have demonstrated that bats generate low or absent antibody responses which often wane rapidly. this is reminiscent of the response of another reservoir host, the sooty mangabey which is the natural reservoir for simian immunodeficiency virus (siv) and for yellow fever virus. sooty mangabeys given an attenuated yellow fever virus vaccine strain generate much lower, transient antibody responses as compared to humans or rhesus macaques. changes to innate immune responses are also evident in sooty mangabeys ( ) . thus, intriguingly, different reservoir hosts may have arrived at similar solutions to avoid the pathological 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circadian fluctuation of immune cells in wrinkle-lipped bats (chaerephon plicatus) diseases in free-ranging bats from germany distinctive tlr signaling, type i ifn production, and attenuated innate and adaptive immune responses to yellow fever virus in a primate reservoir host all authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the handling editor declared a shared affiliation, though no other collaboration, with the authors jm and cs.copyright © mandl, schneider, schneider and baker. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - mpz l e authors: mitchell, william m.; nicodemus, christopher f.; carter, william a.; horvath, joseph c.; strayer, david r. title: discordant biological and toxicological species responses to tlr activation date: - - journal: the american journal of pathology doi: . /j.ajpath. . . sha: doc_id: cord_uid: mpz l e toll-like receptors (tlrs) are highly conserved type membrane proteins that initiate a multiplicity of transient gene transcriptions, resulting in innate and adaptive immune responses. these essential immune responses are triggered by common tlr pattern recognition receptors of microbial products expressed through the cytoplasmic carboxy-terminal toll/il- domain. toll/il- adapter protein cascades are induced by an activated toll/il- to induce transient transcription responses. all tlrs, with the exception of tlr , use an myd adapter to toll/il- to initiate a proinflammatory cascade. tlr uses the toll receptor / induction factor adapter to initiate a different cytosolic adapter cascade with double-stranded rna agonists. this non-myd pathway induces both nf-κb and type interferon responses. by using a tlr -restricted double-stranded rna agonist, rintatolimod, we demonstrate significant unexpected differences in toxic responses between rats and primates. the mechanism of this differential response is consistent with a relative down-regulation of the nf-κb inflammatory cytokine induction pathway in the cynomolgus monkey and humans, but not observed systemically in rat. our findings suggest evaluation of tlr agonists in drug therapy. animal modeling is a time-tested standard for the development of novel therapeutics. the potential of such models to adequately translate findings for use in therapeutic development, however, has recently been questioned. a report by seok et al found a poor correlation between human and murine response to inflammatory stimuli, including responses to trauma, burn, and endotoxemia. discordant results at the level of gene expression array, temporal gene response patterns, and major signaling pathways, in response to these injuries, were demonstrated across species, but well conserved in humans, pointing toward inadequate and nonpredictive nature of available inflammation murine models. this may further explain the difficulty in translating promising pharmacological compounds into successful novel therapies. despite the heterogeneity of the patients and the causes of life-threatening trauma, the report found highly correlated genomic response profiles across the patient population, but not across species. burn, trauma, and endotoxemia yielded highly correlated genomic responses within species, but not across species, rodent to human. we have observed a similar dissociation of toxicities in a parallel proinflammatory system, the toll-like receptor (tlr ) induction of innate immune responses. the tlrs form a family of class transmembrane receptors originally discovered in drosophila, where they are essential elements in embryogenesis and an evolutionary ancient system of immune response to pathogen-associated molecular patterns (pamps). they were recognized further in plants, fish, , and mice, as well as primates. tlrs act as a first line of defense against microbial pathogens by the induction of innate immunity and further provide the initial cellular orchestration for the induction of adaptive immune responses to provide specific humoral and supported by hemispherx biopharma, inc. disclosures: w.m.m. is an independent member of the board of directors and a shareholder of hemispherx biopharma, inc., c.f.n. is a consultant for hemispherx biopharma, inc., w.a.c. is chief executive officer and member of the board of directors and shareholder of hemispherx biopharma, inc., j.c.h. is an employee of hemispherx biopharma, inc., and d.r.s. is the medical director and a shareholder of hemispherx biopharma, inc. rinitatolimod is an experimental drug whose clinical development and manufacture is funded by hemispherx biopharma, inc., under the trade name ampligen. cell-mediated immunity, mediated in part by inflammatory cytokines. they can be found especially in mature dendritic cells (dcs), central in the host adaptive immune response system. all of the tlrs use an myd -dependent signaling pathway, with the exception of tlr , which uses the myd -independent trif pathway. two other doublestranded rna (dsrna) inducers of gene expression that initiate innate immune responses are the cytosolic helicases, mda and retinoic acid inducible gene protein (rig- ), which are myd dependent. the pamp for tlr is dsrna detected in endosomes of antigen-presenting cells and on the cell surface of selected cells, including endothelial cells and airway epithelium. e the expression pattern is consistent with sentinel activity for the detection of replicating virus in the host organism. the cellular location of tlr is modulated by unc b , which promotes trafficking of differentially glycosylated tlr to the plasma membrane; unc b transcription is up-regulated by dsrna. to address the observed toxic heterogeneity of response to pamps across species, we have used a restricted tlr specific agonist, with much data reported in double-blind clinical trials , and unpublished open-label safety trials. rintatolimod (poly i:poly c u; ampligen) is a synthetic dsrna analogue composed of a single polypurine (inosine) strand and a single polypyrimidine (cytosine and uridine) strand. these are assembled in a dsrna structure that is maintained under physiological conditions by typical hydrogen bonding between purine and pyrimidine base pairs. the introduction of the pyrimidine base, uridine, at a : ratio into the polypyrimidine strand maintains the double-stranded structure but allows recurring sites of thermodynamic instability from nonhydrogen bonding of the mismatched base (uridine) with inosine of the polypurine strand. this small difference in structure, however, restricts induction of transitory gene activities to tlr , with its unique trif signaling pathways, with the exclusion of the mda and rig- dsrna proinflammatory dependence , (figure ). by using the restricted specificity of rintatolimod and the unique trif pathway of tlr signaling, we have been able to identify the apparent mechanism for the discordance in tlr -mediated toxicity between rodents and primates. patient serum samples from two clinical trials being conducted according to good clinical practice standards, under figure myd -independent and myd -dependent signaling pathways for the tlrs and helicases. a: the intracellular pathways for myd -independent tlr nuclear signal transduction initiated by trif binding to the tir of the tlr homodimer. tlr monomers dimerize with binding of the dsrna ligand. activated trif initiates two pathways. the first results in the transitory induction of the ifns. the second is a species-variable pathway (rodents >> primates) that operates through nf-kb (dashed line) that transiently induces the production of inflammatory cytokines. adapter protein cascade initiated by trif, tbk , traf / , nap , ikk, ikkε, p k, irf- / , tak , tab , and rip . the ectodomain of tlr consists of a horseshoe-shaped structure populated by leucine-rich b-sheets (orange disks) connected by nonordered chains containing rna-binding residues. the transmembrane a-helices (solid orange) connect the ectodomain to the cytoplasmic tir domain (dark green). the phosphorylated tir binds trif to initiate the adapter protein cascade. b: the intracellular pathways for myd -dependent signaling for tlr / and / heterodimers and tlr to homodimers. the diverse pamp ligands (green bar) are not necessarily accurate in placement, as is the dsrna ligand with tlr in a. tlr uses both the myd -dependent and myd -independent pathways. trif-tir domainecontaining adapter-inducing ifn, tbk -tankebinding kinase , traf-tnf receptoreassociated factor, nap -nckeassociated protein , ikk-ikb kinase, ikkε inhibitor of ikb kinase, p k-phosphoinositide -kinase, irf-ifn regulatory transcription factor, tab -tgf-beactivated kinase , and rip receptoreinteracting (tnfrsf) kinase . the american journal of pathologyajp.amjpathol.org the rintatolimod investigational new drug no. , , were analyzed for g-interferon (ifn), tumor necrosis factor (tnf)-a, il- p , and il- levels to assess acute and chronic changes that might be associated with rintatolimod dosing. all human protocols were conducted with institutional review board review and approval and patient informed consent. study subjects with a diagnosis of chronic fatigue syndrome qualified for participation if they met the protocol entrance criteria. patients received rintatolimod infusions at mg twice weekly. frozen serum samples from randomly selected patients in hemispherx biopharma's open-label treatment protocol, amp- , were obtained at pre-infusion, and again at , , and hours after infusion. serum samples in randomly selected patients from the double-blinded protocol, amp , were obtained at baseline pre-infusion and again at week or at early termination if the patient terminated participation before week . serum obtained from clinical testing sites was frozen and shipped to the hemispherx biopharma, inc., testing and manufacturing facility (new brunswick, nj), where it was assayed using commercially available enzyme-linked immunosorbent assay kits (human ifn-g, human tnf-a, human il- , and human il- p ) (supplemental table s ). animal toxicological analyses were conducted, as required, under investigational new drug guidelines. studies were conducted according to good laboratory practice procedures at licensed animal pharmacology facilities, according to institutional animal care committee review and approval. dose ranging was conducted at dupont (wilmington, de) and bioresearch (senneville, qc, canada), and -month chronic toxicological studies were conducted at chrysalis (cedex, france) and pharamakon (waverly, pa). maximum tolerated doses (mtds) were derived from acute and subacute dose-ranging studies and were defined as the maximum dose that did not cause mortality or moribund toxicity. repeat dosing studies were conducted with sprague-dawley rats, beagle dogs, cynomolgus monkeys, and new zealand white rabbits at doses ranging from to mg/kg, i.v. administered daily for to days (rats and rabbits) or twice weekly (monkey and dog) for and weeks, respectively. clinical observations were recorded during infusions, for hours after infusion, and otherwise twice daily. complete postmortem analysis was conducted, including gross and microscopic anatomical evaluations using standard animal pathological procedures. six-month chronic toxicological studies were conducted using twiceweekly doses of , , , and mg/kg rintatolimod under similar procedures. assessment of acute inflammatory cytokines in response to rintatolimod administration was conducted in collaboration with the lovelace respiratory research institute (albuquerque, nm) and hemispherx biopharma, inc., under good laboratory practice using an approved animal welfare protocol. thirty-two sprague-dawley rats were infused twice weekly for weeks at , , , or mg/kg and sampled for cytokines at , , , and hours after doses , , and , respectively. each time point after infusion required sacrifice of a male and a female rat. ten cynomolgus monkeys were dosed for weeks, twice weekly, at the same dosage levels. serum samples for cytokine measurement were obtained after doses , , , and . all serum samples were frozen and assayed for the corresponding patient cytokines using commercially available enzyme-linked immunosorbent assay kits (supplemental table s ). all results reported are on the basis of manufacturer-supplied standards yielding linear-dose responses. adverse event data in rintatolimod-and placebo-treated patients in controlled studies of chronic fatigue syndrome were assessed in the context of toxicological findings. adverse events were collected during serial patient evaluations of study subjects participating in prospective -to week studies of mg rintatolimod or placebo, infused twice weekly for the duration of the protocols. the x-ray crystallographic structure of the mouse tlr dimer/biol-dsrna complex and the human tlr monomer provided the coordinates for the model of human tlr , with rintatolimod bound to its active site. molecular modeling used accelrys' discovery suite software version . . (accelrys, inc., san diego, ca). the dsrna structure of the dimer/biol-dsrna complex was mutated in situ to the respective poly i (blue) and poly c u (magenta) chains maintaining the phosphate backbone linear translational coordinates of the x-ray crystallographic structure. the coordinates of the human tlr monomer were used to replace each homodimer. several unacceptable close van der waals contacts for the human tlr crystal coordinates were resolved by an alternate rotamer conformation of the individual amino acid r group. to demonstrate the relative equivalence of these two ligands as tlr agonists, we in silico measured the potential energies of the ligands, naked homodimers, and the tlr -ligand complex. the reduction in potential energy of the complex versus the sum of the potential energy of the components is the energy of binding. primary protein sequences for the human (genbank u ); monkey species, including macaca mulatta (gen-bank bag . and ay ), macaca fasicularis ajp.amjpathol.org -the american journal of pathology (genbank bag . ), papio anubis (xp_ ), callithrix jacchus (jab . ), and saimiri boliviensis (xp_ ); and rodents, including the house mouse (genbank af /mus musculus) and rat (genbank ab /rattus norvegicus), dog (genbank xp_ /canis lupus familiaris), and rabbit (genbank abb /oryctolagus cuniculus) were aligned using crystal w software version . . provided by dnastar (madison, wi). acute dose-ranging toxicological analysis was completed in the rabbit, dog, rat, and monkey ( table ). the mtd, defined as the highest acute dosage not associated with induction of a moribund state or mortality, was observed over a range of two orders of magnitude. the rabbit proved most sensitive to rintatolimod, with an mtd of . mg/kg per dose. the dog and rat were more tolerant, with mtds of and . mg/kg per dose, respectively. the cynomolgus monkey was substantially more tolerant of rintatolimod, with an mtd of mg/kg per dose, resulting in a surprisingly -fold differential in acute toxicities, with the nonhuman primate least susceptible to rintatolimod toxicity. because of the clinical development of the rintatolimodenvisioned chronic systemic dosing of the tlr agonist over prolonged periods in the treatment of chronic fatigue syndrome and to meet regulatory requirements [guidance for industry: nonclinical safety evaluation of drug or biologic combinations; u.s. department of health and human services, food and drug administration, center for drug evaluation and research (cder); http://www.fda.gov/ohrms/dockets/ fr/ d- -gdl .pdf, last accessed february , ], -month chronic toxicological studies were conducted in the sprague-dawley rat and cynomolgus monkey. animals were dosed similar to the human schedule, with twice-weekly infusions of rintatolimod at , , or mg/kg per dose, which were expected to be nonlethal on the basis of the acute toxicity studies. dose levels were determined by the human dose of mg per infusion, which is approximately mg/kg for an average-sized patient. results of these two chronic toxicological studies were consistent with the findings of the acute toxicological dosing (table ) , although lethality in the rat during the -month course of the study was commonly observed. the rat was extremely sensitive in multiple organs to both the -and -mg dosing regimens, and many animals in these groups did not survive the -month dosing period. in the rat, dose-dependent liver and renal toxicities were observed in all three dosage groups. additional findings in the rat included anemia and polychromasia, increased alkaline phosphatase, and profound elevation of hepatic transaminases. histopathological features were consistent with clinical analytes. dosedependent toxicities were observed in all dose groups, with hepatocellular degeneration, bile duct hyperplasia, and interstitial nephritis with renal tubular dilation. lymphoid depletion and hyperpigmentation (probably hemosiderin) were noted in the spleen, and extramedullary hematopoiesis and occasional miscellaneous organ inflammation were also noted in all dose groups. in contrast, monkeys tolerated the -month course without significant toxicity, except for occasional vomiting associated with infusion in the mg/kg group. there were transient elevations of hepatic transaminases only in the highdose group, associated with an unexplained decrease in alkaline phosphatase in the monkey. coagulation parameters were prolonged in the high-dose monkey group. there were no histopathological findings in the monkey, with the exception of increased myelopoiesis in the marrow and follicular thyroid hyperplasia in the and mg/kg dose groups. the thyroid morphological observation in the nonhuman primate was correlated with an increase in thyroid-stimulating hormone and thyroxine, suggesting a central effect on the pituitary causing thyroid hyperactivity and the observed hyperplasia. by using the clinical-grade formulation of poly i:poly c u, rintatolimod (ampligen), we have also found discordance between rodent and human inflammatory responses. as demonstrated with the nonhuman primate model (cynomolgus monkey), humans similarly have minor toxicities compared with the rodent model (sprague-dawley rat). as in the models of inflammation explored by seok et al, substantial between-species differences are apparent. in contrast to the rodent, in which sensitivity to endotoxin is magnitudes less than in the human, for the rintatolimodmediated tlr pathways, the rodent is highly sensitive, whereas primates are relatively tolerant. the data summarized in table demonstrate only minimal associations between rodent toxicological findings and the human experience. a low incidence of transient liver the american journal of pathologyajp.amjpathol.org function test abnormalities is seen in the human (ie, easily manageable, with dose reduction from to mg). some patients experience rigors and flu-like symptoms in association with infusion but not the vomiting noted in the high-dose monkey infusion or the general toxicities observed in the rat. no thyroid abnormalities observed in the monkey toxicity trial have been detected in the human clinical data set. thus, the human experience is similar to the monkey, although a major finding of pituitary-dependent thyroid hyperactivity noted in the monkey was not seen in the human. both human and monkey differ substantially in pathological response compared with the rodent. the unexpected differential toxicities observed between the rat and a nonhuman primate prompted an examination of inflammatory cytokines (g-ifn, tnf-a, and il- p ) associated with infusion of a tlr agonist, rintatolimod. preclinical studies in the rat and cynomolgus monkey assessed the induction of these three inflammatory cytokines, as well as il- , measured in the serum in response to three dosage levels ( , , and mg/kg) of rintatolimod administered by i.v. infusion. the -hour time point in response to initial infusion in rats gave the strongest systemic signals, which are illustrated in table , with the expression pattern consistent, although less intense, at later time points. the monkey exhibited minimal evidence of systemic cytokine production. a minimal ifn-g response was observed that was significantly different from that in the rat. il- p at the highest mg/kg dose level generated a small response in one monkey. in contrast, the rat showed a systemic cytokine response, with signals present for all three inflammatory cytokines and il- , even at the lowest mg/kg dose. the species difference for systemic inflammatory cytokine expression is statistically significant for each cytokine (p < . ). for comparison with humans, serum was obtained from patients participating in a concurrent open-label clinical study of chronic fatigue syndrome (amp- ), with a similar time course to the animal pharmacological characteristics and quantified for the same panel of cytokines. the human clinical trial patients were dosed with mg of rintatolimod i.v. over to minutes. similar to the monkey response, serum cytokine increases were minimally expressed with ifn-g, the highest in the aggregate, with a large sd (table ). finally, we assessed for evidence of chronic systemic cytokine changes at the week time point, obtained table illustrates results similar to those of the monkey, with one patient exhibiting a relatively high systemic tnf-a level. systemic inflammatory cytokines, observed in rats, are correlated with significant acute and chronic in vivo toxicities at doses that elicited no detectable systemic inflammatory cytokines and minimal toxicities in primates. moreover, the elevated thyroid activity in the cynomolgus monkey has not been detected in humans. again, systemic signals of cytokine change in association with weeks of rintatolimod at mg per dose i.v., twice weekly, were not evidenced. human dcs do show phenotypic changes in association with exposure to rintatolimod, including shift in maturation profile and local release of cytokines (il- p , ils , , and , ifn-g, and tnf-a) and the chemokines, monocyte chemoattractant protein- (ccl ), and macrophage inflammatory protein- a (ccl ) in the microenvironment of culture supernatants. , as demonstrated in this communication, systemic elevations of inflammatory cytokines are observed in rats. in monkeys and humans, any local elevations (microenvironment) induced by rintatolimod were rarely detected systemically. the difference is in the magnitude of the response. it is this difference in humans that appears to be responsible, at least in part, with the relative lack of toxicity in primates. toxicities associated with overwhelming infection are associated with systemic cytokine storms , of elevated measurable circulating cytokines. the lack of systemic cytokine detection is consistent with the observed minimal toxicity in primates, in contrast to the significant toxicities observed in nonprimates. rat toxicity correlates with systemic cytokine levels, and the lack of primate toxicity similarly is correlated with the lack of significant systemic inflammatory cytokine levels. in vitro data, however, demonstrate that cells expressing tlr respond to dsrna. , the difference between the systemic cytokine levels and the microenvironment is quantitative. inflammatory cytokines observed in the systemic circulation in rats are associated with extreme toxicity and are analogous to the toxicity seen in humans with lethal viral infections, such as highly pathogenic avian influenza (h n /h n ) and severe acute respiratory syndrome (sars coronavirus) with inflammatory cytokine storms. the standard human bioactive dose (approximately mg/ kg) is associated with substantial systemic toxicity in rodents, in which a dose only three times higher is often lethal. poly i:poly c is associated with significant human toxicity not observed with rintatolimod. we examined whether this dissociation of toxicities in humans is related to the affinities of these dsrna ligands to tlr . we modeled the predicted relative binding constants of poly i:poly c versus their mismatched poly c analogues. figure demonstrates an in silico molecular model of rintatolimod bound to the human tlr ectodomain, forming a homodimer structure, and its close Å amino acid contacts on the tlr variable nonhelical segments. these noncovalent contacts provide the collective energetics of binding to form the active homodimer. poly i:poly c demonstrates equivalent in silico energetics of binding with rintatolimod and several other structures with mismatched bases in the poly c strand ( table ). the insertion of a uridine in the poly c strand of poly i:poly c at various ratios of c/u has no significant effect on the binding affinity with tlr . thus, any biological differences observed between rintatolimod and poly i:poly c cannot be attributed to a differential affinity of tlr binding. adverse event data in rintatolimod and placebo-treated patients in well-controlled studies of chronic fatigue syndrome were assessed in the context of toxicological findings. adverse events were collected during serial patient evaluation of study subjects participating in prospective -to -week studies of rintatolimod ( mg) or placebo, infused twice weekly for the duration of the study. elevated lft results denotes alanine aminotransferase or aspartate aminotransferase greater than three times the upper limit of normal. no patients discontinued secondary to abnormal lft results. lft, liver function test; tsh, thyroid-stimulating hormone; t , thyroxine. the american journal of pathologyajp.amjpathol.org we evaluated the rabbit (o. cuniculus), dog (c. lupus familiaris), rat (r. norvegicus), and monkey tlr coding sequences relative to the human standard for differences in species' orthologs. the rabbit, dog, and rat show . % sequence nonidentities (supplemental figure s ). monkeys were highly homologous (> %) between the five species analyzed (supplemental figure s ). the major divergence was as anticipated between old world [m. mulatta, m. fasicularis (cynomolgus), and p. anubis (baboon)] and new world (c. jacchus and s. boliviensis) monkeys. the specific sequence differences between the cynomolgus monkey and rat, relative to humans, are listed in table . expressed tlr protein in the monkey and rat is similar in its total amino acid composition ( and residues, respectively) versus a z difference between rat and monkey cytokine levels across the three dosage levels using the jonckheere-terpstra test (two sided). the -mg infusions providing an approximately mg/kg average dosing. *high means and sd values were secondary to one patient with a value of . removal of this patient results in a mean value of . . ajp.amjpathol.org -the american journal of pathology shorter human tlr ( residues). as expected, the monkey tlr has greater homology to human ( %) than mouse/rat sequences ( %; note that the mouse and rat are % homologous). rodents have sequence differences versus humans in all of the leucine rich repeats. differences between humans and monkeys are present in leucine rich repears to and to ( of ). the endodomain is more highly conserved between the three species than the ectodomain, probably because of the critical cell signaling function of the cytoplasmic toll and il- receptor (tir), which suggests that the monkey and rodent signaling domains may have greater functional similarities than human, on the basis of the large disparity in residue length of human versus monkey and rodent tir endodomains. rintatolimod provided a unique tool that allowed us to dissect the differential effect of species on the systemic response to tlr activation. although poly i:poly c serves as a ligand for tlr , and is a prototypic tlr agonist for experimental purposes, it also activates the dsrna-sensing cytosolic helicases. the substitution of a mismatched base (uridine) in the poly c strand of rintatolimod inactivates its ligand activity for the cytosolic helicases (mda and rig- ) while maintaining its agonist activity for tlr . , the helicases act through the inflammatory myd pathway used by all mammalian tlrs, with the exception of tlr , which uses the trif adapter to initiate innate immune responses. figure illustrates the pattern recognition of the ectodomain of the tlrs and their cytoplasmic adapter pathways responsible for their pleiotrophic effects. figure a depicts the non-myd pathway used by tlr , and figure b depicts the myd -dependent pathways used by all tlrs and the helicases, with the exception of tlr . our toxicological analysis demonstrated systemic dose-dependent inflammatory cytokine responses in rats. although we did not examine inflammatory cytokine responses in rabbit or dog because of a lack of available test kits for the cytokines examined, we believe we would observe a similar quantitative cytokine response in the dog as observed in the rat. the rabbit was so the n-terminals of each tlr bind to opposite ends of the dsrna, with a minimum length of bp required for interaction with essential residues of tlr for activation of intracellular signaling. amino acids of tlr required for binding of rintatolimod are shown as cpk (van der waals' radii) associated with the phosphate backbone. b: the tlr homodimer complexed with rintatolimod, as seen down the long axis of the dsrna. the tlr homodimer is represented as structural elements, with the blue arrows signifying direction of the leucine repeat bsheets and the red cylinders signifying a-helices. poly i strand of rintatolimod (blue) and poly c u strand (magenta cytidines and green uridine). the american journal of pathologyajp.amjpathol.org sensitive to rintatolimod that the mechanism may be simply induced by systemic cytokine levels, although other toxicity mechanisms may be involved. there were, however, no significant detectable systemic inflammatory cytokines in either monkeys or humans. the data indicate that the parallel intracellular ifn and nf-kb transcriptional pathways are species dependent in magnitude of response because primate cells in culture respond to rintatolimod, with the biosynthesis of the ifns and inflammatory cytokines insufficient in vivo to provide a detectable systemic response. the lack of a systemic inflammatory response in primates is consistent with our observation of discordant in vivo toxicities between rats, monkeys, and humans and is consistent with the differences in gross analysis and histopathological characteristics observed between the species. in the case of myd -dependent tlr pathways, primates are more sensitive. in contrast, primates are much more resilient than rodents to inflammatory cytokine toxicity induced by nonemyd -dependent, trif-mediated activation of tlr . redundant helicase pathways may reduce the toxicological distinction between species for agonists that activate these pathways, such as poly i:poly c, in contrast to rintatolimod. potential mechanisms that may explain the speciesspecific differential toxicities observed with tlr activation by dsrna are apparent on analysis of gene structural differences between species. in addition to the primary sequence differences between rodents and primates illustrated in table , rodent and human tlr gene structures are remarkably dissimilar in the proximal promoter domains, as well as tlr isoforms. , an example of differential functional activity between mice and humans is the induction of tlr expression in murine macrophages by lipopolysaccharide (lps; ie, not seen in humans). expression in human blood cells (including monocytes, granulocytes, natural killer cells, t cells, and b cells) and tissue macrophages is undetectable at the transcript level after lps exposure and is only seen in myeloid dcs. , additional structural differences exist, resulting in possible differential tlr isoforms. tlr transcripts are initiated in either exon or exon of the mouse, indicating different promoters not present in humans. the cloning of a human tlr isoform suggests an alternate, but previously unrecognized, splicing pathway. the observed species-specific lps responsiveness in macrophages could be conferred by consensus motifs for nf-kb binding present in the murine tlr promoter, which are absent in human promoter. this has been confirmed by later studies that demonstrate that species-specific differences in tissue expression and responses to lps coincide with the presence of different evolutionary nonconserved promoter sequences in both species. however, despite the overall nonrelatedness of tlr promoter sequences, mrna expression of both tlr orthologs was induced by ifns, particularly by ifn-b. the basal and ifn-beinduced activation of promoters from both species largely depended on similar ifn regulatory factor (irf) elements, which constitutively total monkey-expressed tlr contains residues, versus residues for the human tlr . x total rodent (mouse and rat)eexpressed tlr contains residues, versus residues for the human tlr . *calculations with the accelrys discovery studio software version . . using a distance-dependent dielectric. net interaction energy is the energy of the complex minus energies of the components. y poly i:c u, rintatolimod. charmm, generalized force field; vdw, van der waals. ajp.amjpathol.org -the american journal of pathology bound irf- and recruited irf- after stimulation. in murine macrophages, tlr up-regulation induced by ifn-b required ifnar , stat , and, in part, irf- , but not the janus kinase family member, tyk . also, lps specifically up-regulates tlr expression through the induction of autocrine/paracrine ifn-b. in humans, however, ifn-be induced up-regulation of tlr was blocked by pretreatment with lps, despite the efficient induction of irf- . a recent publication provides additional evidence for differential cytokine responses between mice and humans. exposure of human primary dcs to agonist dsrna did not induce tnf-a, il- , or il- . in human macrophages, the anti-inflammatory cytokine, il- , was induced, but tnf-a and il- were not induced. this effect was specific for human cells because tnf-a or il- was induced by dsrna in murine dcs. moreover, there was differential regulation of nf-kb by murine versus human cells. as shown in numerous prior communications from other laboratories, it was demonstrated that nf-kb was activated by dsrna in murine cells but is absent in human dcs and macrophages, as was ikba degradation present in murine cells. differences between the ectodomain of humans and nonhuman primates ( of llrs) provide a potential mechanism to explain the small differential toxicity between the species. length disparities between the cytoplasmic signaling domain of rodents and nonhuman primates, compared with humans, may contribute to the small differences in toxicity observed between monkeys and humans. there have been numerous reports comparing species specificity of tlr responses, especially tlr , including mouse/ human, , , , monkey/human, and fish/drosophila/ human. , a comparison of tlr and tlr agonists demonstrated similar cytokine levels in humans and chimpanzees versus baboons, consistent with total dna sequence homologies and the small differences we observed between cynomolgus monkeys and humans. although universal tlr responses were largely conserved across primates, chimpanzee-specific immune signaling is enriched for hivinteracting genes that influence hiv disease progression. differences exist in the primate phenotypic responses to tlr agonists. nevertheless, they are relatively similar when compared with other species. we have observed significant species differences in the toxic responses to rintatolimod, a restricted tlr agonist. analysis of the dose-dependent toxicities between rats, monkeys, and humans was remarkably dissimilar. primates are far more resilient to tlr stimulation than rodents. the data are consistent with differential tlr nuclear transcriptional activities, with the nf-kb pathway in inflammatory cytokine production in primates playing a relatively minor role compared with the irf- /irf- nuclear ifn inducers. interestingly, rintatolimod has most recently been advanced clinically as a therapeutic modulator of chronic fatigue syndrome, a condition manifested by disordered energy metabolism and symptoms curiously reminiscent of the toxicity associated with inflammatory innate immune responses. it is hypothesized that the therapeutic effect may relate to feedback inhibition in these pathways, with rintatolimod being specific in its activity. discordance of toxicity in either direction is equally problematic for the successful clinical development of experimental therapeutics, either placing patients at risk for unexpected toxicity or, conversely, raising regulatory barriers for agents shown toxic in sensitive species. alternative methods of in vitro analysis, such as demonstrated by stebbings et al, need to be validated and adapted for regulatory evaluation for human safety. analogous issues may be encountered in modeling retinal disease across species in the context of rna-based therapeutics. the recognition of differential in vivo toxicities and understanding the mechanisms involved should be beneficial in the rational design and risk-benefit analysis of new pharmacological agents for human use. the fda critical path initiative and its influence on new drug development genomic responses in mouse models poorly mimic human inflammatory diseases a family of human receptors structurally related to drosophila toll toll and interleukin- receptor (tir) domain-containing proteins in plants: a genomic perspective expression analysis of the toll-like receptor and tir domain adaptor families of zebrafish characterization of toll-like receptor gene in rainbow trout (oncorhynchus mykiss) recognition of double-stranded rna and activation of nfkb by toll-like receptor toll-like receptor control of the adaptive immune responses dendritic cells and the control of immunity myd -dependent and myd -independent pathways in synergy, priming, and tolerance between tlr agonists discordant species responses to tlr pattern recognition receptors and the innate immune response to viral infection differential expression and regulation of toll-like receptors (tlr) in human leukocytes: selective expression of tlr in dendritic cells kleinman me: irna via tlr toll-like receptor, rig-i-like receptors and the nlrp inflammasome: key modulators of innate immune responses to doublestranded rna viruses the role of unc b protein in surface localization of tlr receptor and in cell priming to nucleic acid agonists chronic fatigue syndrome amp- study group, mitchell wm: a double-blind, placebo-controlled, randomized, clinical trial of the tlr- agonist rintatolimod in severe cases of chronic fatigue syndrome a controlled clinical trial in cfs (chronic fatigue syndrome) using a specifically configured rna drug, poly(i):poly(c u) structural requirements of the ri n -rc n complex for induction of human interferon sidwell rw: tlr is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c u), but not poly(i:c): differential recognition of synthetic dsrna molecules the microbial mimic poly ic induces durable and protective cd þ t cell immunity together with a dendritic cell targeted vaccine not all polyriboinosinicpolyribocytidylic acids (poly i: c) are equivalent for inducing maturation of dendritic cells: implication for alpha-type- polarized dcs toll-like receptor- as a target to enhance bioactivity of cancer immunotherapy fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice toxic properties of a synthetic doublestranded rna: endotoxin-like properties of poly i. poly c, an interferon stimulator of mice and men: species variations of toll-like receptor expression species-specific regulation of tolllike receptor genes in men and mice regulation of toll-like receptors in human monocytes and dendritic cells cloning of tlr isoform key differences in tlr /poly i:c signaling and cytokine induction by human primary cells: a phenomenon absent from murine cell systems use of murine embryonic fibroblasts to define toll-like receptor activation and specificity genetic analysis of toll/interleukin- receptor (tir) domain sequences from rhesus macaque toll-like receptors (tlrs) - reveals high homology to human tlr/tir sequences innate immune responses to tlr and tlr agonists differ between baboons, chimpanzees and humans functional comparison of innate immune signaling pathways in primates cytokine storm in a phase i trial of the anti-cd monoclonal antibody tgn cytokine storm" in the phase i trial of monoclonal antibody tgn : better understanding the causes to improve preclinical testing of immunotherapeutics short-interfering rnas induce retinal degeneration via tlr and irf we thank angus shieh (hemispherx biopharma, inc.) for extending his biostatistical expertise in comparative cytokine data analysis. supplemental material for this article can be found at http://dx.doi.org/ . /j.ajpath. . . . key: cord- -oyhzn kc authors: li, chenxi; di, di; wang, xin; xia, qiqi; wahaab, abdul; anwar, muhammad naveed; li, zongjie; liu, ke; shao, donghua; qiu, yafeng; wei, jianchao; li, beibei; ma, zhiyong title: duck karyopherin α (dukpna ) is involved in type i interferon expression and the antiviral response against japanese encephalitis virus date: - - journal: dev comp immunol doi: . /j.dci. . sha: doc_id: cord_uid: oyhzn kc karyopherin α (kpna ) is an adaptor molecule that mediates type i interferon (ifn) production by facilitating the nuclear translocation of ifn transcription factors. here, we cloned the duck kpna (dukpna ) gene and analyzed its involvement in type i ifn expression as well as antiviral response against japanese encephalitis virus (jev). the full-length dukpna gene encoded a -amino acid protein that shared . – . % sequence similarity with its orthologues in chickens, humans and mice. the dukpna was extensively expressed in various duck tissues at the mrna level. analysis of the subcellular localization of dukpna by immunofluorescence assays indicated that the dukpna was primarily distributed in both the cytoplasm and nucleus in primary duck embryonic fibroblasts (defs). however, it translocated from the cytoplasm to the nucleus in response to poly(i:c) stimulation or jev infection. the dukpna interacted with duck ifn regulatory factor and facilitated its nuclear translocation, thereby up-regulating the expression of ifn-α and ifn-β in defs in the presence of poly(i:c) stimulation. exogenous expression of dukpna significantly elevated the expression of ifn-α and ifn-β induced by jev infection and inhibited jev replication in defs. these data demonstrate the importance of dukpna in type i ifn signaling as well as the antiviral response against jev replication. karyopherin (kpna), also known as importin α, acts as an adaptor molecule between nuclear-localization-sequence (nls)-bearing cargo proteins and importin β (goldfarb et al., ) , and plays an essential role in the active transport of proteins from the cytoplasm to the nucleus (smith et al., ) . in eukaryotic cells, macromolecules (> nm in diameter or > kda) bearing an nls can be translocated from the cytoplasm into the nucleus by importin α/β heterodimers (fagerlund et al., ) . in eukaryotes, multiple subtypes of kpna have been identified. mice and humans have six (kpna , , , , and ) and seven (kpna , , , , , and ) subtypes, respectively, whereas budding yeast contains only a single kpna (srp ) (oka and yoneda, ) . on the basis of homology, kpna subtypes are classified into three subfamilies: α (kpna , kpna and kpna ), α (kpna and kpna ) and α (kpna and kpna ) (pumroy and cingolani, ; smith et al., ) . the three subfamilies of kpna show distinct nls-binding specificity (nadler et al., ) and exhibit multiple biological functions including cell development and differentiation, regulation of the immune response and control of tumorigenesis. among the identified kpnas, kpna in the α subfamily is involved in the innate immune response against viral infection ye et al., ) . several kpna orthologues from different species including humans (köhler et al., ) , mice (mihalas et al., ) , pigs (chen et al., ) and chickens (caldwell et al., ) have been identified. in mammals, human kpna , a specific adaptor molecule, mediates the nuclear import of interferon (ifn) transcriptional regulators, such as nf-κb p /p heterodimers and ifn regulatory factor (irf) , thereby regulating type i ifn-mediated immune responses (canton et al., ; shaw et al., ) . recently, pig kpna has been demonstrated to bind pig irf and initiate ifn-β production during porcine circovirus infection . however, the role of avian kpna , especially duck kpna (dukpna ), in type i ifn expression was unknown. the irf gene is absent in ducks, and the functions of duck irf (duirf ) complement most of irf 's functions in regulating type i ifn expression (magor et al., ) . therefore, we sought to explore whether dukpna interacts with duirf and induces type i li, et al. developmental and comparative immunology ( ) expression. domestic duck farming is a popular business in china. in addition, ducks, as an amplification host, are susceptible to japanese encephalitis virus (jev) infection (xiao et al., ) . jev is a single-stranded positive-sense rna virus of the flavivirus genus that is transmitted by mosquitoes and vertebrate-amplifying hosts including birds (dhanda et al., ) . jev infection causes stunted growth and death in ducklings (xiao et al., ) . in addition, jev ns protein interacts with human kpna and inhibits the nuclear translocation of irf and nf-κb in human cells (ye et al., ) . we therefore cloned the dukpna gene and analyzed its role in type i ifn expression as well as the antiviral response against jev in primary duck embryo fibroblasts (defs). specific pathogen free (spf) ducks, clinically healthy ducks and spf duck embryos were purchased from the harbin veterinary research institute of the chinese academy of agricultural sciences, china. all animal experiments were approved by the institutional animal care and use committee of the shanghai veterinary research institute (iacuc no: shvri-po- ) and were performed in compliance with the guidelines on the humane treatment of laboratory animals (ministry of science and technology of the people's republic of china, policy no. ) . defs were prepared from -to -day-old spf duck embryos and were grown in dulbecco's modified eagle's medium (gibco, grand island, ny, usa) supplemented with % fetal bovine serum (gibco). the jev sh strain (ncbi accession no. mh ) was maintained in our laboratory. the open reading frame (orf) encoding full-length dukpna was amplified by polymerase chain reaction (pcr) from the spleens of spf ducks. the forward primer dukpna -f and reverse primer dukpna -r (supplementary table ) for amplification of dukpna were designed according to the predicted sequence of dukpna (ncbi accession xm_ . ). the amplified sequence was confirmed by dna sequencing and cloned into the pcdna . vector to express dukpna tagged with ha (dukpna -ha). the multiple sequence alignment of dukpna with chicken (nm_ . ), helmeted guineafowl one hundred cells expressing dukpna -ha were randomly selected from different fields of microscope to calculate the cells with nuclear distribution of dukpna -ha. the percentages of cells with nuclear distribution of dukpna -ha in the presence and absence of poly(i:c) treatment were plotted. results are presented as the mean ± standard error from three independent experiments. **, p < . tested by student's t-test. (xm_ . ), human (nm_ . ) and mouse (nm_ . ) kpna was performed with geneious software. the functional domains of dukpna were analyzed with the smart website (http://smart. embl-heidelberg.de/). the phylogenetic tree of kpna was constructed by neighbor-joining in mega version . . samples of issues including the spleen, liver, ileum, jejunum, proventriculus, lung, kidney, gizzard, duodenum, brain, bursa, heart, appendix and blood were collected from three -day-old spf ducks and three -day-old clinically healthy ducks. total rnas from different tissues were extracted with trizol reagent according to the manufacturer's instructions (takara, dalian, china) and subsequently reverse transcribed to cdna with a primescript™ rt reagent kit with gdna eraser (takara). the relative expression levels of dukpna in different tissues were determined by quantitative real-time rt-pcr (qrt-pcr) with tb green™ premix ex taq™ ii (takara) and primers qdukpna -f and qdukpna -r (supplementary table ). the glyceraldehyde- phosphate dehydrogenase (gapdh) gene was used as an internal control. the subcellular localization of dukpna in defs was determined by immunofluorescence assays (ifas). defs were pre-cultured overnight to approximately % confluence and transfected with recombinant plasmids engineered to express dukpna -ha or empty vector, by using lipofectamine (invitrogen, carlsbad, ca, usa). after h of incubation, the transfectants were stimulated with poly(i:c) (invitrogen) fig. . up-regulation of expression of type i ifn by dukpna . defs were transfected with plasmid for expression of dukpna -ha or empty vector in the indicated amounts ( , and ng) for h and stimulated with poly(i:c) treatment (+poly(i:c)) or left untreated (-poly(i:c)) for an additional h. (a) the expression of dukpna -ha in the transfectants was confirmed by western blotting with anti-ha antibody. (b) the expression of ifn-α and ifn-β at the mrna level was detected by qrt-pcr and normalized to the expression of the gapdh gene. (c) the concentrations of ifn-α and ifn-β proteins in the supernatants were detected by elisa. (d, e and f) defs were transfected with three sirnas targeting different regions of dukpna mrna (sidukpna - , sidukpna - and sidukpna - ) or control sirna (sinegative) for silencing dukpna expression. the transfectants were incubated for h and subsequently stimulated with poly(i:c) treatment or left unstimulated for an additional h. the relative expression of dukpna mrna levels detected by qrt-pcr was normalized to gapdh mrna and is presented relative to the level in control sirna (sinegative) cells (set as ) (d). the relative expression of ifn-α and ifn-β mrna levels was normalized to gapdh mrna levels and plotted (e). the concentrations of ifn-α and ifn-β proteins in the supernatants were detected by elisa (f). results are presented as the mean ± standard error from three independent experiments. **, p < . ; *, p < . tested by student's t-test. c. li, et al. developmental and comparative immunology ( ) at a concentration of μg/ml for h. the defs were fixed in % paraformaldehyde for h, permeabilized with . % triton x- for min and blocked with % bovine serum albumin for h. subsequently, the cells were incubated with mouse anti-ha antibody (sigma, st louis, mo, usa) for h and treated with donkey anti-mouse igg(h + l) antibody conjugated with alexa fluor (invitrogen) for h. the nuclei were stained with ′, -diamidino- -phenylindole (dapi) (sigma). fluorescence images were taken with a fluorescence microscope (carl zeiss, zena, germany). defs at - % confluence were transfected with plasmids for expression of dukpna -ha or with empty vector for h and were stimulated with poly(i:c) ( μg/ml) for an additional h. the supernatants were collected to measure the protein levels of ifn-α and ifn-β with an elisa kit (lengton, shanghai, china), and the cells were used to extract total rnas for detection of the mrna expression of ifn-α and ifn-β by qrt-pcr. the primers used are listed in supplementary table . to further analyze the effects of dukpna on type i ifn expression, we synthesized three small interfering rnas (sirnas) targeting different regions of dukpna mrna (sidukpna - , sidukpna - , and sidukpna - ) in vitro to knock down the endogenous dukpna expression (supplementary table ). defs were transfected with the synthesized sirna or negative sirna with lipofectamine rnai max (invitrogen) and incubated for h. the transfectants were stimulated with poly(i:c) for an additional h and harvested for detection of the expression of ifn-α and ifn-β at both the mrna and protein levels, as described above. the orf of duirf was amplified from duck spleen by rt-pcr according to the sequence of duirf (genbank accession no. mg . ). the amplified sequence was confirmed by dna sequencing and cloned into the pcdna . vector to express duirf tagged with myc (duirf -myc) for co-immunoprecipitation assays. defs were co-transfected with recombinant plasmids for expression of dukpna -ha and duirf -myc and incubated for h. the transfectants were stimulated with poly(i:c) ( μg/ml) for h and lysed in icecold ripa lysis buffer (millipore, billerica, ma, usa) containing mm phenylmethyl sulfonylfluoride for min at °c. the supernatants of cell lysates were collected and incubated with anti-ha protein-g-sepharose beads for h at °c. the beads were washed three times with phosphate buffered saline with . % tween- , resuspended in × sds-page sample buffer and subjected to sds-page. the immunoprecipitated protein complexes were detected with western blot analysis with anti-myc antibody (millipore) and mouse anti-ha antibody (sigma), as described previously (zhao et al., ) . the protein levels of dukpna -ha and duirf -myc in the nuclear extracts were detected by western blotting. lamin b was detected as the nuclear marker (c). intensities of protein bands were determined by densitometric analysis. relative protein levels of dukpna -ha and duirf -myc in the nuclear extracts were normalized to those in the wcl and plotted (d). the significant difference was determined using one-way anova followed by tukey's multiple comparisons test. ***, p < . ; **, p < . ; *, p < . . c. li, et al. developmental and comparative immunology ( ) . . analysis of the effects of dukpna on the nuclear translocation of duirf defs were co-transfected with recombinant plasmids for expression of dukpna -ha and duirf -myc and incubated for h. the transfectants were stimulated with poly(i:c) ( μg/ml) for h and lysed with ne-per nuclear and cytoplasmic extraction reagents (invitrogen) to obtain cytoplasmic and nuclear extracts. the protein levels of duirf -myc in the nuclear extract were determined by western blot analysis. lamin b was detected as a nuclear marker with mouse anti-laminb antibody (cell signaling, danvers, ma, usa). defs were transfected with plasmid for expression of dukpna -ha and incubated at °c for h. the transfectants were infected with the jev sh strain at a multiplicity of infection (moi) of . and incubated at °c for the indicated times. the jev-infected cells were collected at , -and -h post-infection (hpi) for analysis of jev replication. the jev titers in the supernatants were determined with % tissue culture infective dose (tcid ) assays. the protein levels of jev ns in the cells were detected by western blotting with polyclonal antibody specific to jev ns (deng et al., ) . the relative number of rna copies of jev e in the cells was measured by qrt-pcr. the βactin gene was used as the internal control. the primers used are listed in supplementary table . the orf of the dukpna gene was cloned from duck spleen, which was bp in length and encoded a -amino acid protein (fig. a) . the nucleotide sequence of dukpna has been deposited in genbank under accession number mn . analysis of the deduced amino acid sequence revealed that dukpna had . %, . %, . % and . . % sequence similarity to the chicken, helmeted guineafowl, human and mouse kpna sequences, respectively (fig. a) , and showed a high degree of sequence similarity to orthologues in other species. structurally, dukpna contained three domains that were conserved in all orthologues of other species, including an n-terminal importin β-binding (ibb) domain, a series of armadillo (arm) repeats and a conserved short acidic cluster (fig. a) . the arm repeats are essential for nls-binding and are composed of eight regions (arm , , , , , , and ), which shared nearly % sequence identity with sequences from other species (fig. a) . phylogenetic analysis indicated that dukpna clustered together with chicken and helmeted guineafowl kpna in the bird group and was closest to chicken kpna (fig. b) . to examine the tissue distribution of dukpna expression, we collected different tissues from clinically healthy ducks and spf ducks and examined dukpna mrna expression by qrt-pcr. dukpna was ubiquitously expressed at varying levels in various tissues, including the spleen, liver, ileum, jejunum, proventriculus, lung, kidney, gizzard, duodenum, brain, bursa, heart, appendix and blood; the expression profiles were similar between clinically healthy ducks ( fig. a) and spf ducks (fig. b) . among the examined tissues, the blood showed the highest level of expression in both spf and clinically healthy ducks, followed by the kidney, lung, gizzard, spleen, and duodenum, whereas low levels of expression were observed in the ileum and jejunum (fig. ) . the subcellular location of dukpna was examined by ifa. because of a lack of a commercial antibody specific to dukpna , we exogenously expressed dukpna -ha in defs and detected the subcellular location of the expressed dukpna with anti-ha antibody. the dukpna -ha was distributed in both the cytoplasm and nucleus, with predominant expression in the cytoplasm (fig. a) . however, in response to poly(i:c) stimulation, dukpna -ha translocated from the cytoplasm to the nucleus, showing a predominant accumulation in the nucleus (fig. a-b) . these data indicated that poly(i:c) stimulation promoted the translocation of dukpna from the cytoplasm to the nucleus, thus implying a possible role of dukpna in type i ifn expression in ducks. to analyze whether dukpna might be involved in type i ifn expression, we transfected defs with plasmid for expression of dukpna -ha at different doses in the presence and absence of poly(i:c) stimulation, and detected the expression of interferon-α (ifn-α) and interferon-β (ifn-β) at the mrna level by qrt-pcr and at the protein level by elisa. the expression of dukpna -ha was confirmed by western blotting with anti-ha antibody (fig. a) . overexpression of dukpna -ha significantly promoted the expression of ifn-α and ifn-β at the mrna level in a dose-dependent manner in the presence of poly(i:c) stimulation, as compared with the expression in the control vector groups (fig. b) . the promotion of ifn-α and ifn-β expression by dukpna -ha was further confirmed at the protein level. the concentrations of ifn-α and ifn-β protein in the supernatants of dukpna -ha transfected cells were clearly higher than those in the control vector groups (fig. c) . to further confirm the involvement of dukpna in type i ifn expression, we knocked down the expression of endogenous dukpna in defs by rna interference with sirnas (sidukpna - , sidukpna - and sidukpna - ) targeting three different regions of dukpna mrna (supplementary table ) and analyzed the effects on the expression of ifn-α and ifn-β at the mrna and protein levels in the presence or absence of poly(i:c) stimulation. the down regulation of dukpna mrna expression in the sirna-treated cells was confirmed at the mrna level by qrt-pcr (fig. d) , because of a lack of a commercial antibody specific to dukpna . in response to poly(i:c) stimulation, the expression of ifn-α and ifn-β was significantly elevated at both the mrna and protein levels, as compared with the levels in cells without poly(i:c) stimulation. however, the elevated expression of ifn-α and ifn-β was significantly reduced at both the mrna and protein levels in cells treated with sirnas (sidukpna - , sidukpna - , and ) . results are presented as the mean ± standard error from three independent experiments. **, p < . ; *, p < . tested by student's t-test. c. li, et al. developmental and comparative immunology ( ) sidukpna - ) (fig. e-f) , thus suggesting that the silencing of endogenous dukpna expression eliminated the production of ifn-α and ifn-β in defs after poly(i:c) stimulation. together, these data indicated that dukpna is involved in type i ifn expression. irf and irf play critical roles in type i ifn-mediated innate immunity (takeuchi and akira, ). in mammalian cells, kpna interacts with irf and subsequently promotes its translocation from the cytoplasm to the nucleus, thus triggering the expression of type i ifn . the irf gene is absent in the duck genome; therefore duirf substitutes for most of irf 's functions in regulating type i ifn expression (chen et al., ) . given that dukpna upregulated type i ifn expression, we investigated whether dukpna might interact with duirf and promote the translocation of duirf from the cytoplasm to the nucleus. defs were co-transfected with plasmids for expression of dukpna -ha and duirf -myc, and their co-localization was determined by ifas in the presence or absence of poly(i:c) stimulation. the expressed dukpna -ha and duirf -myc mainly co-localized in the cytoplasm in the absence of poly(i:c) stimulation (fig. a) . however, in response to poly(i:c) stimulation, both the expressed dukpna -ha and duirf -myc translocated from the cytoplasm to the nucleus and co-localized in the nucleus (fig. a) , thus suggesting a possible interaction between dukpna and duirf . to confirm this interaction, we performed co-immunoprecipitation in defs co-transfected with plasmids for expression of dukpna -ha and duirf -myc, by using anti-ha antibody for immunoprecipitation and anti-myc antibody for detection of the immunoprecipitated proteins. the dukpna -ha co-immunoprecipitated with duirf -myc (fig. b) , thus revealing that dukpna interacted with duirf in defs. to further analyze whether dukpna promoted the nuclear translocation of duirf , we extracted the nuclear fractions from defs co-transfected with plasmids for expression of dukpna -ha and duirf -myc, in the presence of poly(i:c) stimulation, and performed western blotting analysis to detect the protein levels of dukpna -ha and duirf -myc. the expression of dukpna -ha and duirf -myc in the whole cell lysates (wcl) of defs was confirmed by western blotting (fig. c ). analysis of changes in the protein levels of duirf -myc in the nuclear fractions revealed that the nuclear accumulation of duirf -myc increased, an effect correlated with the increased nuclear accumulation of dukpna -ha ( fig. c and d) , thus indicating that dukpna facilitated the nuclear translocation of duirf , thereby upregulating type i ifn expression. given that jev infection induces type i ifn expression and that dukpna was involved in type i ifn expression in the presence of poly(i:c) stimulation (fig. ) , we investigated the involvement of dukpna in type i ifn expression during viral infection by using jev as a model. defs were infected with jev at different moi ( . , and ), and total rna was extracted from jev-infected cells to detect the endogenous mrna expression levels of dukpna . the qrt-pcr analysis indicated that jev infection did not significantly affect the mrna expression of endogenous dukpna in defs (fig. a) . however, the subcellular location of dukpna -ha shifted from the cytoplasm to the nucleus in defs in response to jev infection (fig. b) , similarly to the translocation induced by poly(i:c) stimulation (fig. ) . analysis of type i ifn expression in jev-infected defs with or without overexpression of dukpna -ha revealed that exogenous expression of dukpna -ha significantly elevated the mrna levels of ifn-α and ifnβ induced by jev infection, as compared with the levels in jev-infected defs without dukpna -ha expression (vector control) (fig. c) . the expression of dukpna -ha in jev-infected defs was confirmed by western blotting (fig. d) . together, these data indicated that dukpna resulted in nuclear translocation of duirf and facilitated type i ifn expression during jev infection, thereby implying its involvement in the type i ifn-mediated antiviral response. on the basis of the observation that dukpna facilitated type i ifn expression during jev infection (fig. ) , we investigated the effect of dukpna on jev replication. defs were transfected with plasmid expressing dukpna -ha or empty vector and subsequently infected with jev. jev replication was monitored by tcid assays and qrt-pcr. as shown in fig. a , the jev titers in defs transfected with plasmid expressing dukpna -ha were significantly lower than those in defs transfected with empty vector at , and hpi. the relative number of rna copies of jev e (fig. b ) and the protein levels of jev ns ( fig. c and d) in defs transfected with plasmid for expression of dukpna -ha were also significantly lower than those in defs transfected with empty vector, thus confirming the inhibitory effect of dukpna -ha on jev replication. mammalian kpna plays a role in regulating the nuclear transportation of type i ifn transcription factors, thereby triggering the expression of type i ifn and the subsequent immune response against virus infection (zhu et al., ; chen et al., ; li et al., ) . however, the role of avian kpna in type i ifn expression was unknown. the current study aimed to clone duck kpna and analyze its role in type i ifn expression as well as the antiviral response. the cloned dukpna comprised amino acid and shared . - . % sequence similarity with its orthologues from chicken, helmeted guineafowl, human and mouse, thus suggesting that kpna has been highly conserved through evolutionary history (goldfarb et al., ; miyamoto et al., ) . the ibb domain involved in binding importin β (kobe, ) , the arm repeats functioning as internal cargo nls-binding sites mediating the nuclear translocation of cargo protein (miyamoto et al., ) , and the short acidic cluster that acts as a binding region for the nuclear exporter of importin α (goldfarb et al., ) were highly conserved in dukpna . these observations suggested that dukpna might have biological roles similar to those of its orthologues from mammals. indeed, in response to poly(i:c) stimulation, dukpna facilitated the expression of type i ifn, showing a conserved role in type i ifn expression similar to that of human, mouse and pig kpna xinghui et al., ) . mechanistically, mammalian kpna acts as a specific adaptor molecule that mediates the nuclear translocation of nf-κb p /p heterodimers and irf , thereby inducing type i ifn expression (fagerlund et al., ; li et al., ) . knockdown of human kpna in mcf- cells significantly inhibits nf-κb activity and impairs ifn-α/β expression, thus leading to immunological dysfunction (zhu et al., ) , whereas up-regulation of kpna enhances nf-κb activation (chen et al., ) . porcine kpna has been found to be involved in the irf mediated type i ifn signaling pathway during porcine circovirus type infection . in ducks, the toll-like receptor signaling pathway and the retinoic-acid-inducible gene i signaling pathway largely resemble the human pathways (evseev and magor, ) and play essential roles in the regulation of type i ifn expression and host antiviral response (yu and levine, ) . notably, irf , as a downstream molecule in the toll-like receptor signaling pathway and the retinoicacid-inducible gene i signaling pathway, is absent in ducks, and duirf instead performs most functions of irf in inducing type i ifn expression (santhakumar et al., ) . in the current study, dukpna interacted with duirf and facilitated the nuclear translocation of duirf . mechanistically, dukpna promotes type i ifn expression, probably via the duirf -mediated type i ifn signaling pathway, in contrast to mammalian kpna . human kpna is detected in nearly all tissues at both the mrna and protein levels (nachury et al., ) . in this study, we detected dukpna expression only at the mrna level because of a lack of a commercial antibody specific to dukpna . the expression of dukpna was detectable in all most tissues tested, showing a ubiquitous expression pattern similar to that of human kpna (goldfarb et al., ; miyamoto et al., ) , thus suggesting the multiple biological functions of dukpna , such as involvement in antiviral immune response. indeed, in response to jev infection, dukpna translocated from the cytoplasm to the nucleus and elevated the type i ifn expression induced by jev infection. furthermore, exogenous expression of dukpna significantly inhibited jev replication in defs, thereby suggesting the involvement of dukpna in the host immune response against viral infection in ducks. this observation was consistent with a previous observation in humans, in which human kpna has been found to have a role in the inhibition of jev replication (ye et al., ) . given the highly conserved amino acid sequence similarity and the conserved role in type i ifn expression, kpna may be an important molecule in type i ifn signaling as well as type i ifn-mediated antiviral response, and may be targeted by viruses to antagonize the type i ifnmediated antiviral response. indeed, the b protein of middle east respiratory syndrome coronavirus binds kpna and impairs the interaction between kpna and the nf-κb-p subunit, thus interfering with the host innate immune response (canton et al., ) . jev ns protein competitively binds kpna and inhibits the nuclear translocation of ifn transcription factors, thus suppressing ifn expression (ye et al., ) . these previous observations together with our results emphasize the importance of kpna in the type i ifn-mediated antiviral response. however, these roles must be further mechanistically explored to gain more insight into the full biological functions of kpna . in summary, we cloned the dukpna gene, which encoded a amino acid protein with . - . % sequence similarity to its orthologues from other species. dukpna was extensively expressed in various tissues in ducks and was found to be involved in type i ifn expression in response to poly(i:c) stimulation. dukpna was found to interact with duirf and promote the nuclear translocation of duirf , thereby triggering type i ifn expression. in response to jev infection, dukpna translocated from the cytoplasm into the nucleus and elevated the expression of type i ifn expression. exogenous expression of dukpna significantly inhibited jev replication. together, these data suggested that dukpna is involved in type i ifn expression as well as the type i ifn-mediated antiviral response. full-length cdnas from chicken bursal lymphocytes to facilitate gene function analysis mers-cov b protein interferes with the nf-κb-dependent innate immune response during infection vitamin d deficiency 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virus ns inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor and nf-κb toll-like receptor , rig-i-like receptors and the nlrp inflammasome: key modulators of innate immune responses to double-stranded rna viruses differential antiviral immunity to japanese encephalitis virus in developing cortical organoids nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of trim by interfering with trim -mediated rig-i ubiquitination tdp- inhibits nf-κb activity by blocking p nuclear translocation supplementary data to this article can be found online at https:// doi.org/ . /j.dci. . . key: cord- -kmn e z authors: schultz, kimberly l.w.; griffin, diane e. title: immune responses to viruses in the cns date: - - journal: encyclopedia of immunobiology doi: . /b - - - - . - sha: doc_id: cord_uid: kmn e z for recovery from infection, the immune response in the central nervous system (cns) must eliminate or control virus replication without destroying nonrenewable, essential cells. thus, upon intracellular virus detection, the infected cell must initiate clearance pathways without triggering neuronal cell death. as a result, the inflammatory response must be tightly regulated and unique mechanisms contribute to the immune response in the cns. early restriction of virus replication is accomplished by the innate immune response upon activation of pattern recognition receptors in resident cells. infiltrating immune cells enter from the periphery to clear virus. antibodies and interferon-γ are primary contributors to noncytolytic clearance of virus in the cns. lymphocytes are retained in the cns after the acute phase of infection presumably to block reactivation of virus replication. for recovery after virus infection of the central nervous system (cns), the essential, nonrenewable nature of neurons requires a fine-tuned immune response that controls virus replication without damaging neuronal function. damage can result directly from virus replication or from the host immune response to infection. functional impairment or loss of neurons following infection can be fatal or leave survivors with neurological sequelae including cognitive deficits, seizures, or paralysis (hart et al., ; griffiths et al., ; silverman et al., ; ooi et al., ; sauder et al., ; finley et al., ) . thus, the immune responses required for successful clearance and control of virus infections in the cns are often distinct from those required for clearance from other organs and are characterized by noncytolytic, virus-specific processes. this strategy preserves cns function and minimizes the likelihood of autoimmunity. many viruses can infect the cns, including dna viruses, plus-and minus-strand rna viruses, and retroviruses, leading to varying outcomes from disease. dna viruses, such as herpesviruses (reviewed in koyuncu et al., ) , often establish a latent infection as opposed to rna viruses that generally lack a nuclear phase for their replication cycle and cause acute disease. in this article, we will focus on rna virus infections in the cns (table ) . much of our knowledge about the immune response to neurotropic viruses comes from studying well-characterized mouse models of infection. studies have investigated the course of disease and immune response both in immunocompetent mice and animals deficient in specific components of the immune response. these studies have provided detailed knowledge of the role of each arm of the immune response in control of virus replication and spread, virus clearance, and in immunopathology. in all infections, outcome of infection is dependent on the age and genetic background of the mouse and the strain of the virus used. for simplicity, we will focus on the most commonly studied strains of each virus family and infection of mature mice. detailed studies of immune responses to neurotropic viruses have included neuronal infections with rabies virus, flaviviruses, and alphaviruses, as well as infection of multiple cell types with natural mouse pathogens such as theiler's murine encephalomyelitis virus (tmev), mouse hepatitis virus (mhv), and lymphocytic choriomeningitis virus (lcmv). the immunological processes required for virus clearance from the cns are cell type and virus specific. experimental approaches to define these clearance mechanisms are dependent on the transient depletion of specific immune cell populations and on the use of mice that have selective deficiencies in various components of the immune system. because of the interdependent relationships of components of the immune system in the development of an immune response, deficiencies of one type of cell or molecule may affect several facets of the immune response, making it difficult to identify specific effectors that are crucial for in vivo clearance. infection is rarely initiated in the cns because viruses must invade the cns from initial sites of infection in the periphery with induction of the immune response in peripheral lymphoid tissues. entry of viruses, immunoglobulins, and immune cells from the blood is restricted by the blood-brain barrier (bbb), a selectively permeable barrier with tight junctions between cerebrovascular endothelial cells that are supported by astrocytes ( figure ). the bbb separates the parenchyma of the cns from the circulating blood and serves as a physical blockade to bloodborne infections of the cns. however, the endothelial barrier is more permeable at certain sites in the cns (e.g., choroid plexus) and inflammation increases permeability to allow immune cell infiltration along with opportunities for virus entry. historically, routes of cns infection have been deduced from data obtained by histological staining at early times after infection or disruption of a potential route of infection. entry routes are not mutually exclusive, as multiple routes have been described for some viruses. recently, new techniques such as intravital microscopy and clarity preparation of infected brains have been developed that may lead to new insights on the mechanisms of cns entry (yang et al., ; chung et al., ; mcgavern and kang, ) . in general, virus entry is either from the periphery by neuronal axonal transport or from the bloodstream across the vascular endothelium. sensory and motor neurons extend their processes into the periphery and provide a point of entry for some neurotropic viruses replicating in peripheral tissue. expression of viral receptors on neuromuscular junctions facilitates entry of poliovirus, adenovirus, and rabies virus into the cns (salinas et al., ) . olfactory neurons that project into the respiratory mucosal epithelium can provide a direct route to the brain for alphaviruses (phillips et al., ; powers and logue, ; charles et al., ) , flaviviruses (yamada et al., ; monath et al., ) , coronaviruses (barnett and perlman, ) , paramyxoviruses (munster et al., ) , bunyaviruses (bennett et al., ) , and occasionally influenza virus (van riel et al., ) . hematogenous entry occurs when a virus directly infects bbb endothelial cells or infects leukocytes that cross the bbb providing entry by a 'trojan horse' mechanism (neal, ; wilson, ; rhoades et al., ; kim, ; haase, ) . the cns is relatively protected from immunologic activity. in addition to the physical protection by the bbb, the brain parenchyma has no lymphatic vessels or professional antigenpresenting cells, low expression of major histocompatibility complex (mhc) molecules, and active maintenance of an immunologically quiescent state. however, the exclusion of immune cells and the role of active immune signaling in the cns has been redefined recently (schwartz et al., ; muldoon et al., ; elmer and mcallister, ; hernangómez et al., ) . resident cells in the nervous system, including neurons, play an active role in the immune response (schultz et al., ; o'donnell et al., ; chakraborty et al., ; daffis et al., a; castorena et al., ; daffis et al., ; commonly used in mouse models of viral encephalitis. jackson et al., ) . additionally, memory t cell and b cell are found in the cns long after infectious virus has been eliminated (phares et al., ; metcalf et al., ; wakim et al., ; wilson et al., ) . resident cells monitor the cns for infection and initiate and control inflammation when infection occurs. microglial cells, the resident macrophages of the cns, express the cd receptor (cd r), trem , cd a, and cd and are kept in a quiescent state through interactions with electrically active, healthy neurons expressing cd , hsp , cd , and cd and through the production of neurotrophins (chavarría and cárdenas, ; ransohoff and cardona, ; hoek et al., ) . local production of the antiinflammatory cytokines transforming growth factor (tgf)-b and il- by astrocytes, pericytes, and meningeal cells further inhibits cellular activation (schwartz et al., ; fabry et al., ; johnson et al., ) . activated t cells cross the bbb into the cns for immunological surveillance upon interactions with p-selectin on endothelial cells, but leave or die if antigen is not encountered (irani and griffin, ; wekerle et al., wekerle et al., , . the innate immune response initiated by resident cells in the cns upon virus infection is the first line of defense ( figure (a) ). detection of infection occurs through activation of cellular pattern recognition receptors that include the toll-like receptors (tlrs), rig-i-like receptors (rlrs), and nod-like receptors (nlrs). microglia, as the professional immune cells of the cns, express all of the known pattern recognition receptors. additionally, neurons, astrocytes, and to a lesser extent oligodendrocytes, express selected pattern recognition receptors and thus contribute to innate immune signaling (kigerl et al., ) . engagement of tlrs and rlrs activates the transcription factors irf- , irf- , and nfkb that control expression of type-i interferon (ifn)-a and ifn-b. for many cns infections, ifn production is critical for early control of infection. mice deficient in type-i ifn signaling, ifnar À/À , have increased virus replication and mortality upon infection by a variety of viruses including sindbis virus (sinv), west nile virus (wnv), lcmv, and vesicular stomatitis virus (samuel and diamond, ; byrnes et al., ; ryman et al., ; müller et al., ) . moreover, pretreatment with ifn is protective (frolov et al., ; lucas et al., ; grieder and vogel, ; després et al., a) . ifn signaling must be tightly regulated as excess ifn, particularly ifn-a, can be neurotoxic (nallar and kalvakolanu, ; reyes-vázquez et al., ) . in contrast, ifn-b coordinates the immune response and is generally neuroprotective (mclaurin et al., ) . autocrine and paracrine binding to the ubiquitously expressed ifna/b receptor initiates jak/stat signaling and directs ifn-stimulated gene (isg) expression. isgs restrict virus replication in infected cells and establish an antiviral state in neighboring cells to limit virus spread. although ifn signaling stimulates the expression of hundreds of isgs, antiviral effects oligodendrocyte: glial cell that produces the myelin sheath around neuronal axons and promotes conduction neuron: electrically active cell that transmits signals from the periphery and within the cns astrocyte: glial cell that secretes neuroprotective factors and is associated with maintenance of the bbb microglial cell: bone marrow-derived macrophage-lineage glial cell that is the resident 'macrophage' of the cns; constantly surveys the cns and becomes immunologically active upon pathogen detection blood brain barrier (bbb): endothelial cells connected by tight junctions that form a highly selective barrier between the circulating blood and the cns parenchyma of these proteins are both virus specific and tissue specific (cho et al., a; diamond and gale, ; schoggins et al., ; zhao et al., ) . for instance, the isg ifit restricts wnv in some regions of the brain, but did not affect replication in the cerebral cortex, spinal cord, or periphery (cho et al., b) . engagement of nlrs by infecting viruses can initiate inflammasome formation in the cns. the inflammasome activates caspase- to cleave precursors of the proinflammatory cytokines il- b and il- . secretion of mature il- b and il- helps to orchestrate the inflammatory response to infection. the magnitude and timing of the inflammatory response must be controlled to limit damage to bystander cells. inflammasome-mediated signaling has varying affects during cns infections (prow and irani, ; sergerie et al., ; liang et al., ) . inflammasome activation during wnv infection is protective, as mice deficient in inflammasome components have increased virus replication in the brain and decreased survival (kumar et al., ; ramos et al., ) . in contrast, inflammasome activation in microglia and astrocytes contributes to increased immunopathology and possibly bystander neuronal death during japanese encephalitis virus infection (kaushik et al., ; das et al., ) . neurons are active contributors to the innate immune response during virus infection as has been demonstrated in cultures of primary and immortalized neurons (schultz et al., ; farmer et al., ; cho et al., a; peltier et al., ; castorena et al., ; delhaye et al., ; préhaud et al., ) . in response to alphavirus, flavivirus, and bunyavirus infections, mature neurons rapidly activate irf- and irf- to induce expression of type-i ifn, limit virus replication, and preserve neuronal function (schultz et al., ; farmer et al., ; peltier et al., ; daffis et al., b; castorena et al., ; daffis et al., ) . additionally, il- b synergizes with ifn-b to control wnv replication in neurons (ramos et al., ) . the combination and importance of each innate immune signaling pathway in response to infection is likely cell type and virus specific. in addition to factors that control virus replication, infected cells produce factors that activate astrocytes and microglia, upregulate expression of mhc molecules on microglial cells, increase expression of adhesion molecules including intercellular adhesion molecule and vascular adhesion molecule (vcam ) on capillary endothelial cells to direct leukocyte infiltration to the site of infection, and modulate the inflammatory response. cytokines and chemokines important for these processes are induced in a virus-specific manner but often include ifn-g, il- , il- , il- , il- , tumor necrosis factor (tnf), ccl , ccl , ccl , cxcl , and cxcl (kulcsar et al., ; tun et al., ; lee et al., ; hayasaka et al., ; metcalf et al., ; ramos et al., ; stubblefield park et al., ; shrestha et al., ; klein et al., ; burdeinick-kerr and griffin, ; bergmann et al., ; chang et al., ; liang et al., ) . these factors facilitate recruitment of circulating leukocytes across the bbb and into the cns. in addition to controlling virus replication in cns cells, innate immune signaling initiates the virus-specific adaptive immune response. infiltration of mononuclear inflammatory cells into the cns typically begins - days after infection (figure (b) and (c)). t cell and b cell trafficking into the cns is promoted by neuronal expression of the chemokine cxcl that binds to cxcr on activated t cell and b cell (phares et al., ; zhang et al., ; klein et al., ) . additionally, proper trafficking of t cells to appropriate brain regions is promoted by signaling through ccr and cxcr (mccandless et al., ) . cells first accumulate in the perivascular areas and then infiltrate the parenchyma in the regions of virus infection. essentially, all components of the cellular immune response are detected in the infiltrate: natural killer (nk) cells, antigen-specific cd þ and cd þ t cells, b cells, and monocytes/macrophages (peña et al., ; zhao et al., ; lee et al., ; chang et al., ; rowell and griffin, ; parra et al., ; pearce et al., ; wesselingh et al., ) . the uninfected cns does not have professional antigen-presenting cells capable of activating naïve t cells, but dendritic cells are detected in the cns during inflammation after either entering from the circulation or developing from a subpopulation of activated microglia. presentation of viral peptide antigen in association with the appropriate mhc molecules, predominantly expressed on glial cells, retains activated t cells in the cns (kimura and griffin, ; irani and griffin, ; suzumura et al., ) . the continued presence of viral protein antigens promotes long-term retention of virus-specific b cells (phares et al., ; metcalf et al., ) . virus is cleared from the cns in a multistep process that must first stop cell-to-cell spread and eliminate cell-free infectious virus (figure (b) ). this phase of viral clearance can be assessed by measurement of infectious virus but, as neutralizing antibody is produced, virus clearance is best assessed by quantitative measurement of viral nucleic acid. initially, local production of type-i ifn reduces cell-to-cell spread through paracrine antiviral signaling. infectious virus is neutralized by antibody produced by b cells that enter the cns and interact with viral glycoproteins on the infected cell surface. additionally, ifn-g, interacting with ifn-g receptors expressed on the surfaces of infected cells, inhibits virus production (phares et al., ; metcalf et al., ; stewart et al., ; hooper et al., ; tschen et al., ; binder and griffin, ; ubol et al., ; levine et al., ) . for full recovery, virus-infected cells or viral genomes need to be cleared from the cns. in peripheral tissues, virus-infected cells are usually eliminated by virus-induced or immunemediated cytolysis. clearance of virus-infected cells in the cns becomes a more complicated process due to the nonrenewable and essential nature of neurons and the important role of glial cells in maintaining neuronal function. if the immune system destroys the infected cell, then the outcome of infection will be the same as if the virus caused cell death. however, if infected cells are allowed to survive, there must be a clearance mechanism that inhibits synthesis of viral nucleic acid and proteins and eliminates viral genomes. if clearance is not complete, mechanisms are needed to avoid progressive or relapsing disease. these processes must be tightly regulated to prevent immune-mediated damage to both infected and uninfected cells during the response to cns infection. for instance, the cd þ t cell response can be detrimental during wnv infection (szretter et al., ; wang et al., ) . during fatal encephalomyelitis due to infection with a neurovirulent strain of the alphavirus sinv, infiltration of th and th /th cd þ t cells is associated with a rapidly fatal paralytic disease. this response is modulated by il- produced by intrinsic cells of the cns and by infiltrating regulatory t cells (kulcsar et al., ) . il- also plays a protective role during coronavirus and flavivirus infections of the cns (tun et al., ; hayasaka et al., ; trandem et al., ) . generally, clearance of rna viruses from neurons occurs through noncytolytic antibody and cytokine-mediated mechanisms to preserve neuronal function. this process has been studied both in virus-infected mice and in cultured neurons. in mice, the clearance of sinv is a two-phase process (metcalf and griffin, ) . infectious virus is rapidly cleared during the first week after infection and then viral rna is cleared slowly over the next - days followed by persistence of a low level of rna. cd þ t cells followed by cd þ t cells and b cells enter the cns during the first phase when infectious virus is cleared. in the second phase, t cell and b cell are retained in the cns during viral rna clearance with overall larger numbers of cd þ t cells and b cells than cd þ t cells. numbers of immune cells in the cns gradually decrease with decreasing rna, but the resident populations are steadily enriched in those that are virus specific. within months after sinv infection, most antibody-secreting cells (ascs) produce sinvspecific igg (metcalf and griffin, ; tyor et al., ) . antibody that mediates virus clearance from neurons is often directed against viral structural proteins on the infected cell surface and has been most completely analyzed for cells infected with sinv (hooper et al., ; levine et al., ) . in addition to neutralizing free virus, antibody to the sinv e glycoprotein can bind to the surface of infected cells and may direct intracellular signaling to control virus replication (després et al., b; levine and griffin, ) . the antiviral effect of this antibody does not require complement or phagocytic cells, but is dependent on bivalent antibody, implying that cross-linking of viral proteins at the cell surface results in intracellular inhibition of virus production (ubol et al., ; levine et al., ) . antibody acts by unknown mechanisms to suppress virus replication and restore host protein synthesis, membrane potential, and type-i ifn responsiveness (després et al., a,b) . cd þ t cells can exert antiviral effector functions either through a noncytotoxic, cytokine-mediated, or a cytotoxic pathway. the most effective noncytotoxic cytokine identified is ifn-g and the cytotoxic effector pathways involve perforin and granzymes or cd (fas)-cd l interaction. cd þ t cells can be activated by interactions with mhc class i complexes on neurons (chevalier et al., ) or through cross-priming interactions with mhc class i molecules on surrounding glial cells. t cells recruited into the cns during sinv infection facilitate, but are not necessary, for rna clearance (rowell and griffin, ; kimura and griffin, ) . ifn-g alone can clear sinv from motor neurons, but not from cortical or hippocampal neurons which require antibody (burdeinick-kerr and griffin, ; binder and griffin, ) . ifn-g production by t cells is also important for clearance of mhv, borna disease virus, and measles virus infections from the cns (o'donnell et al., ; stubblefield park et al., ; richter et al., ; templeton and perlman, ; pearce et al., ) . clearance of wnv is dependent on cd þ t cells and monocytes, not antibody (sitati et al., ; shrestha et al., ; klein et al., ) . monocyte-derived cells likely signal to t cells, leading to optimal activation necessary for virus clearance (durrant et al., ) . t cells clear virus through ifn-g production or through cytotoxic methods, such as perforin (shrestha et al., ) . cytotoxic t cells are targeted to infected neurons upon upregulation of prodeath molecules (fas or trail ligand) and increased mhc class i expression (shrestha et al., ; chevalier et al., ; shrestha and diamond, ) . neurons infected with virulent strains of rabies virus upregulate fasl and b -h to inhibit t cell function and prevent virus clearance and the virus-induced inflammatory response (lafon et al., ; baloul et al., ) . additional upregulation of the nonclassical mhc class i molecule hla-g on infected neurons may promote tolerance . the best-studied examples of glial cell infections in mice are the picornavirus tmev and coronavirus mhv. failure to clear the acute infection by susceptible strains of mice leads to persistent production of infectious virus and immune-mediated demyelinating disease. thus, these infections have become models for the human demyelinating disease multiple sclerosis. tmev has an early encephalitic phase, which mainly involves infection of neurons, followed by persistent infection of glial cells. in resistant strains of mice, virus clearance is dependent on a rapid cd þ t cell response (lindsley and rodriguez, ) . in susceptible strains, infectious virus is cleared from the neurons, but not from microglia, astrocytes, or oligodendrocytes. establishment of a persistent infection involves failure of all stages of the immune response, beginning with innate immune signaling through tlr and tlr , proinflammatory molecule expression (il- ), and regulation of infiltrating t cells (jin et al., ; so and kim, ). il- inhibits cytotoxic t cell function and apoptotic death by preferential induction of il- -producing th cells (hou et al., ) . additionally, il- and il- synergistically promote expression of prosurvival bcl family members that facilitate survival of virus-infected cells (hou et al., ) . mhv infects a wide range of cell types including macrophages, microglia, astrocytes, and oligodendrocytes. the early adaptive response to mhv infection is characterized by expression of the chemokines cxcl , cxcl , ccl , ccl , and ccl and their receptors ccr , ccr , and cxcr by microglia and astrocytes (lane et al., ) . cxcl expression is important for recruitment of t cells (phares et al., ) . proinflammatory cytokine expression (i.e., decreases as the percentage of virus-specific cd þ t cells and expression of t cell support molecules (i.e., cxcl , ccl , and ifn-g) increases (lane et al., ; parra et al., ) . ifn-g plays a key role in dampening mhv replication and orchestrating t cell infiltration, along with maximal expression of mhc molecules on microglia and macrophages (whitman et al., ; bergmann et al., bergmann et al., , parra et al., ) . infiltrating cd þ t cells accumulate around blood vessels and provide supporting factors for infiltrating cd þ t cells that invade the parenchyma at the site of infection (phares et al., ; stohlman et al., ) . granzyme b-positive cd þ t cells target mhc class i-positive, infected cells for cytolysis, but effector function may be specific for the targeted cell type lin et al., ) . ifn-g is particularly important for clearance from oligodendrocytes, whereas perforin and cd þ t cell-mediated cytolysis is important for the clearance of virus from astrocytes and microglia (gonzález et al., ; bergmann et al., ; parra et al., ; lin et al., ; stohlman et al., ) . oligodendrocyte killing by cd þ t cells results in demyelination during the acute phase of infection (templeton and perlman, ) . cytolytic function declines concomitant with loss of viral antigen lin et al., ) . viral rna persists within the cns for over months, regardless of the presence of nonanergic, virus-specific cd þ and cd þ t cells retained in the cns (phares et al., (phares et al., , ramakrishna et al., ; bergmann et al., ) . additionally, persistent oligodendrocyte infection and the consequent immune response are associated with chronic demyelination. long-term control of virus infection (figure (c) ), regardless of infected cell type, is characterized by virus-specific antibody, a lack of infectious virus, and low levels of viral rna that are detected by sensitive methods such as qpcr (metcalf and griffin, ; stewart et al., ; appler et al., ; fragkoudis et al., ; tschen et al., ; tyor et al., ) . although control of the acute phase of mhv infection by the adaptive response is independent of antibody, long-term production of antibody is necessary to prevent reactivation of infection lin et al., ) . as the bbb does not allow antibody to efficiently enter the cns from the periphery, ascs must either be continuously recruited to the cns or maintained in the brain parenchyma to produce antibody locally (metcalf et al., ; stewart et al., ; hooper et al., ; diamond, ; diamond et al., ; ramakrishna et al., ; tyor et al., ; levine and griffin, ; parsons, ) . antibody controls persistent infection in the cns through a multifaceted defense strategy that preserves neuronal function following infection. the need for continued antibody production in the cns is highlighted in studies where passive transfer of antibody blocked recrudescence only during the time of treatment levine and griffin, ; levine et al., ) . molecular cues in the brain microenvironment orchestrate asc recruitment, retention, and maturation. ascs remain in the cns to block reactivation of virus replication. the mechanisms for recruitment and retention have been characterized during mhv and sinv infection (metcalf and griffin, ; marques et al., ; lin et al., ; tyor and griffin, ) . the chemokine receptor cxcr and its ligand cxcl are critical for recruitment of ascs to the cns during mhv infection (phares et al., ; gil-cruz and perez-shibayama, ; marques et al., ) . cxcl is also elevated during sinv, tmev, and rabies virus infections, although its role in asc recruitment has not been defined (rainey-barger et al., ; kuang et al., ; phares et al., ; hoffman et al., ) . infiltrating b cells early in infection are naïve/early activated but progress to a more differentiated, isotype-switched phenotype. consequently, asc that first enter the cns produce igm, likely contributing to neutralization of free virus. through the course of infection, igg predominates with iga also present and declining levels of igm (phares et al., ; metcalf and griffin, ; tschen et al., ; tyor and griffin, ) . long-term expression of b cell activating factor (baff) and a proliferating-inducing ligand (april) in the 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encephalitis interaction of t lymphocytes with cerebral endothelial cells in vitro cellular immune reactivity within the cns intracerebral cytokine mrna expression during fatal and nonfatal alphavirus encephalitis suggests a predominant type t cell response ifn-g-mediated suppression of coronavirus replication in glial-committed progenitor cells trafficking of immune cells in the central nervous system emerging viral infections brain lesions induced by experimental intranasal infection of japanese encephalitis virus in piglets single-cell phenotyping within transparent intact tissue through whole-body clearing cxcr mediates region-specific antiviral t cell trafficking within the central nervous system during west nile virus encephalitis global gene expression changes in bv microglial cell line during rabies virus infection innate immune response gene expression profiles in central nervous system of mice infected with rabies virus key: cord- - g h bz authors: idelsis, e.-m.; jesus, p.-e.; yaquelin, d.-r.; dania, v.-b.; monica, b.-r.; lisandra, b.-r.; jesus, c.-r.; lisbeth, c. c.; ernesto, p.-c.; saily, t.-p.; claudia, m.-s.; ivan, c.-l.; julio raul, f.-m.; hamlet, c.-r.; marisol, d.-g.; adriana, s.-m.; maura, g.-s.; sara maria, m.-m.; marel, a.-v.; francisco, h.-b.; hugo, n.-c.; dianela, b.-g.; abrahan, b.-c.; mary tania, v.-c.; gerardo, g.-n.; verena, m.-g.; iraldo, b.-r. title: effect and safety of combination of interferon alpha- b and gamma or interferon alpha- b for negativization of sars-cov- viral rna. preliminary results of a randomized controlled clinical trial. date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: g h bz abstract objectives: ifn-alpha b and ifn-gamma combination has demonstrated favorable pharmacodynamics for genes underlying antiviral activity which might be involved in the defense of the organism from a sars-cov- infection. considering this we conducted a randomized controlled clinical trial for efficacy and safety evaluation of subcutaneous ifn-alpha b and ifn-gamma administration in patients positive to sars-cov- . methods: we enrolled - years-old inpatients at the military central hospital luis diaz soto, havana, cuba. they were hospitalized after confirmed diagnosis for sars-cov- rna by real-time reverse transcription polymerase chain reaction. patients were randomly assigned in a : ratio to receive either, subcutaneous treatment with a co-lyophilized combination of . miu ifn-alpha b and . miu ifn-gamma (heberferon, cigb, havana, cuba), twice a week for two weeks, or thrice a week intramuscular injection of . miu ifn-alpha b (heberon alpha r, cigb, havana, cuba). additionally, all patients received lopinavir-ritonavir / mg every h and chloroquine mg every h (standard of care). the primary endpoints were the time to negativization of viral rna and the time to progression to severe covid- , from the start of treatment. the protocol was approved by the ethics committee on clinical investigation from the hospital and the center for the state control of medicines, equipment and medical devices in cuba. informed consent was obtained from each participant. results: a total of patients with laboratory-confirmed sars-cov- infection, including symptomatic or asymptomatic conditions, fulfilled the inclusion criteria and underwent randomization. thirty-three subjects were assigned to the heberferon group, and to the heberon alpha r group. sixty-three patients were analyzed for viral negativization, of them . % in the heberferon group negativized the virus after days of treatment versus . % of patients in the heberon alpha r groups (p= . ). time to reach the negativization of the sars-cov- measured by rt-pcr in real time was of . and . days for the heberferon and heberon alpha r groups, respectively. a significant improvement in the reduction of time for negativization was attributable to heberferon (p= . , log-rank test) with a hazard ratio of . and % ci of . to . , as compared to heberon alpha r treated group. worsening of respiratory symptoms was detected in two ( . %) and one ( . %) patients in heberferon and ifn-alpha b groups, respectively. none of the subjects transit to severe covid- during the study or the epidemiological follow-up for more days. rt-pcr on day after the start of the treatment was negative to sars-cov- in % and % of patients of the combination of ifns and ifn-alpha b, respectively. negativization for heberferon treated patients was related to a significant increase in lymphocytes counts and an also significant reduction in crp as early as days after commencing the therapeutic schedule. all the patients in both cohorts recover by day and were in asymptomatic condition and laboratory parameters return to normal values by day after treatment initiation. adverse events were identified in . % of patients, . % in the control group, and . % in the heberferon group, and the most frequent were headaches ( . %). conclusions: in a cohort of hospitalized patients between to years-old with positive sars-cov- , heberferon significantly negativized the virus on day of treatment when comparing with ifn-alpha b. heberon alpha r also showed efficacy for the treatment of the viral infection. both treatments were safe and positively impact on the resolution of the symptoms. none of the patients developed severe covid- . key words: covid- , treatment, drug, virus negativization, antiviral, interferon combination, sars cov- . properties of these molecules . in fact, severity of covid- disease correlates with the failure to implement an ifn response to sars-cov- infection . taking these into consideration and the fact that therapeutics that target the coronavirus alone, might not be able to reverse highly pathogenic infections, the cuban protocol for management of covid- includes heberon alpha r and other antiviral treatment since the symptomatic phase. cuban patients already showing symptoms or their near contacts are isolated in centers conditioned for that purpose and start receiving symptomatic treatments. as mentioned, this schedule incorporates heberon alpha r to lopinavir-ritonavir (kaletra) and chloroquine (cq). after confirmation of the positivity of sars-cov- , they are hospitalized and continue to, or start to receive heberon alpha r, kaletra, and cq as established by cuban health ministry guide-lines . this has resulted in a favorable evolution of the patients in a cohort of subjects confirmed for sars-cov- receiving heberon alpha r, where . % fully recovered from covid- , with only . % of case fatality rate . earlier studies of a combination of type i ifn and ifn-γ shown a synergistic inhibition of the sarscov virus replication in vitro , , , . ifn-γ is a key moderator in linking the innate immunity to adaptive immune responses , hence it is possible that a combinational therapy of ifns and other antiviral drugs could significantly inhibit virus replication and modulates clinical variables related to the immune response with a positive outcome in terms of viral infection resolution , , . in accordance to the previous comments we conducted this phase randomized trial to establish whether a combination of ifn-α b and gamma with the standard of care, can improve the viral load profile and clinical parameters in adults with covid- . hospitalized adult patients with rt-pcr confirmed sars-cov- were enrolled in this openlabeled, single center, prospective, randomized and controlled clinical trial at military central hospital "luis diaz soto" hospital, havana, cuba. patients were randomly assigned to receive the combination of ifn-α b and ifn-γ (heberferon, cigb, havana, cuba) or ifn-α b (heberon alpha r, cigb, havana, cuba) based on a power of %, and a level of confidence set at %, while also considering a dropout rate of %. patients were blocked randomized individually to one of two treatment arms by means of random computer-generated lists, with an allocation ratio of : , with block sizes of six patients. engineering and biotechnology (cigb), which has remained a product with proven antiviral efficacy and an adequate safety profile for years . heberferon (ifn-α b and ifn-γ, colyophilized in the same vial) is produced at cigb, and registered in cuba for the treatment of basal cell carcinoma. the study execution followed the ethical principles of the declaration of helsinki and the international council for harmonization-good clinical practice guidelines. no compensation was provided for enrollment in the trial. patient personal data were protected. (severe arterial hypertension, ischemic heart disease, diabetes mellitus, etc.), with a history of autoimmune diseases, presence of hyper inflammation syndrome, serious coagulation disorders, known hypersensitivity to any of the components of the formulation under evaluation, pregnancy or lactation, and obvious mental incapacity to issue consent and act accordingly with the study. the clinical trial protocol was approved by the ethics committee on clinical investigation of military central hospital "luis diaz soto", and the center for the state control of medicines, equipment and medical devices (cecmed) in cuba. patients were asked for written consent to participate after having been duly informed about the characteristics of the trial, objectives, benefits and possible risks. likewise, they were informed of their rights to participate or not and to withdraw their consent at any time, without exposing themselves to limitations for their medical care or other retaliation. the study was registered on april at: registroclinico.sld.cu/en/trials/rpcec . after a preliminary exploratory analysis of the outcomes of the first patients, the monitoring board considered a preliminary report and early publishing of the rt-pcr results from the available throat swabs in patients with available throat swabs, due to the significant effect of heberferon on the reduction of the time to viral clearance. the trial finally included patients that are now in the process of data collection for definitive processes and analysis. data collection: demographic, clinical, laboratory, treatments and outcome characteristics of patients were extracted from medical records and registered in to crf and then were entered in duplicate (independently by two operators) for the subsequent process of automatic comparison and correction of the databases, necessary for statistical analysis with accurate information from the trial. however the blinding was not feasible, it was maintained for laboratory sars-cov- rna detection by rt-pcr that is one of the endpoint of the study. the hospital received patients from several zones in havana city diagnosed in reference centers for sars-cov- infection following the cuban ministry of all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . health guidelines for diagnostic testing. patients were defined to have sars-cov- if they had two consecutive positive results, including the confirmatory test by rt-pcr targeting amplifications of e and /or rdrp genes. a cycle threshold up to value was defined as positive. specimens were obtained from throat swabs of patients at the hospital following standard procedures and transported to a bsl certified laboratory at the cigb for serial evaluation of sars-cov- viral nucleic acid detection by rt-pcr targeting after extraction by qiaamp® viral rna mini kit (qiagen, usa). a multiplexed detection by rt-pcr was carried out targeting e and/or rdpr genes plus eav internal extraction control (tib molbiol syntheselabor gmbh, berlin, germany) as described before using multiplex rna virus master (roche, usa). hospital "luis diaz soto" and included whole blood count, coagulation profile, serum biochemical tests (including renal and liver function, electrolytes, and coagulation), creactive protein (induced by various inflammatory mediators such as il- statistical analysis: quantitative variables were described with the arithmetic mean and its standard deviation and the median with its range. we used the absolute and relative frequency (%) for qualitative variables. the hypothesis test used was fisher's exact test. the viral negativization analysis was performed using the kaplan-meier plot representation and the comparison of factors was done with the mantel-cox log rank tests. the evolution of all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint laboratory parameters while under treatment was analyzed using a paired mixed model (which cannot handle missing values appearing due to patient release from hospital). correlations between virus negativization and laboratory parameters were studied using a two-tailed non-parametric spearman correlation with % confidence interval. p< . was considered statistically significant. statistical analysis was performed using the windows software package spss (version ) and graphpad prism v . . we have screened patients positive by rt-pcr to sars-cov- . fifty-seven patients did not fulfill the inclusion criteria, of them one with icterus, one with chronic decompensate renal insufficiency, two non-confirmed positive pcr for sars-cov- , and fifty-three patients with positive rt-pcr after more than days of persistent virus shedding, were excluded. patients with viral persistence were later treated with the heberferon out of the clinical trial (manuscript in preparation). eight patients that did not consent were also excluded. finally, seventy-night subjects met the inclusion criteria and were randomly assigned ( : ) to either the heberferon group ( patients) or the control group ( patients). twelve patients did not start the treatment, patients refused to start the treatment, although they have been signed the consent, and were excluded due to loss of inclusion criteria. seven patients withdrew by several causes: due to worsening of respiratory symptoms (two of them in the heberferon group, with asthma as underlying diseases) that changed to other non-permitted in the study drug treatment; patients from the control group with positive rt-pcr on day were switched to receive heberferon out of the clinical trial by medical decision; and with the appearance of an exclusion criterion (pregnancy) in the control group (see figure , flow chart of the study). four patients were not analyzed, three in the heberferon group, due to bad inclusions (were negative to viral rna before the beginning of treatment as identified by the board of monitors), and one in the control group because refused the swabs sampling. thirty and thirty-three patients were analyzed by intention to treat (itt) in the heberferon and control group, respectively. finally, swabs samples to test for viral negativization were obtained from sixty-three patients. in this cohort were symptomatic ( . %), with a median from the beginning of symptoms all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . (iqr: . symptomatic patients with more than days from the symptoms onset were more common in the heberferon arm ( . %), however, these numerically differences were not statistically significant (see table ). in the heberferon group . % of symptomatic were females and in the control symptomatic males were more frequent . % (p= . ). the more common symptoms were fever and unproductive cough ( . %), followed by headache ( . %), decay ( . %), odynophagia and nasal secretions ( . %), diarrhea, dyspnea, chills and general malaise ( . %), and others as sore throat and myalgia ( . %). fifty percent of patients had any comorbidity; the most frequent were hypertension ( %), asthma ( . %), diabetes and glaucoma ( . %). the vital signs at the time of hospital admission were not statistically different between groups. some imbalances existed at enrollment between the groups, including a higher median age in the heberferon than in the control group, as well as more patients with higher than days from onset of the symptoms in the heberferon group. no other major differences in symptoms, vital signs, laboratory results, disease severity, or treatments were observed between groups at baseline. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . in asymptomatic patients a lower rate of negativization was observed for both ifns. however, the heberferon showed a . % of negativization in comparison to . % for control group. the worsening of respiratory symptoms was detected in two ( . %) and one ( . %) patients in heberferon and control groups, respectively. none of the patients transit to severe covid- . the rt-pcr after treatment with ifns on day for hospital discharges was negative to sars-cov- in % and % of patients of heberferon and control cohorts, respectively. nevertheless, the kinetics for this recovery differ between treatments groups. earlier increase in lymphocytes percentage was observed only for heberferon treated patients (p= . ) with a marked trend for increment in lymphocytes concentrations. also a significant decrease in crp (p= . ) was notice for this group parallel to a trend in the reduction in cpk ( figure ). the correlation between laboratory data evolution and sars-cov- virus clearance data was analyzed using a two-tailed non-parametric spearman correlation with % confidence interval. table summarized the parameters identified with significant direct or indirect relation with the reduction in the time needed to achieve a negative pcr result. a particular assessment of the same parameters is also included for the symptomatic patients included for all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . nighty-four percent of adverse events were mild and none severe. there were no differences between the incidence of any of the adverse events or duration between the treatment groups. no serious adverse events were reported. no patients died during the study (table ) . asymptomatic incubation period with or without detectable viral rna, followed by nonsevere symptomatic step and viral presence, ending in a severe symptomatic stage with high viral load, characterizes the sars-cov- infection , that has been widely spread. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint beta- b . even % of negativization showed by herberon alpha- b is superior to the reported by other authors . time to reach the negativization of the sars-cov- measured by rt-pcr in real-time was . and . days from the start of treatment with heberferon and heberon alpha r, respectively, a difference statically significant. these results are in concordance with in vitro data about the greater sensitivity of sars-cov- to ifns with respect to sars-cov , and as compared to those treated with heberon alpha r. viral dissemination is determinant in the establishment of severe disease . therefore, the shortening of time to virus clearance as has been demonstrated for heberferon will impact very favorable in the disease outcome in covid- infected patients. the timing of initiation of antiviral therapy is another key factor in the treatment of viral infections. in the combat of sars-cov, no effect of several antiviral drugs was observed when the treatments were started - days after symptom onset the combination of kaletra with other antiviral agents, as has been done in sars , mers-cov, and our trial, might enhance antiviral effects and improve clinical outcomes. the confirmation of this therapeutic approach remains to be determined. however, it has been recently shown the combination of ifns with kaletra is associated with more favorable clinical outcomes than the use of kaletra alone in covid- patients . the presence of ifn-γ in the heberferon formulation additionally to its strong immune regulatory functions may restrict the angiotensin-converting enzyme (ace ) expression , a receptor for cell entry for sars-cov- . it has been reported that this cytokine can directly inhibit viral entry for several viral infections (hcv and hiv) by controlling the all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . in mers-cov infected mice delayed ifn treatment was associated with increased infiltration and activation of monocytes, macrophages, and neutrophils in the lungs; and enhanced pro-inflammatory cytokine expression . additionally, soon, after infection in human, application of antiviral therapy with rapid viral clearance can delay pro-inflammatory cell development, activation and their infiltration that will contribute to spar human life . delayed ifn response can also cause inflammation and tissue damage. the host may benefit from ifn presence early in the disease course, particularly when ifn system is antagonized by viral proteins or is of low competence in older aged patients . an important difference between treatments concerns their effects on lymphocytes percentages among leukocytes. only for heberferon treated patients a significant increase of lymphocytes percentages was observed by week and this fact, as well as lymphocytes concentrations, correlates significantly with the reduction in time to virus clearance (fig and table ). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . herein we detected a significant reduction in crp in patients after two administrations of heberferon (see figure ) . crp is correlated with the level of inflammation , and is an important index for the diagnosis and assessment of severe pulmonary infectious diseases , . in the early stage of covid- , crp levels could reflect lung lesions and disease severity. the downregulation of cpr levels by ifns in patients with covid- early in the diseases could avoid acute inflammatory pathogenesis and disease severity . then the administration of the heberferon in patients primed with the antivirals may result in a further boosted antiviral effect that contribute to shortening the time for viral clearance and to lower the probabilities to develop a more severe conditions of the diseases, that implies at the end, a lower lethality rate. although with significant more aged patients in the heberferon and cohort with % of symptomatic patients with median age of years-old, none of these patients became severe ill during the trial and all of them were discharged. however, two patients from heberferon group worsened the respiratory symptoms. these were asymptomatic men at admission, both years-old, with asthma as comorbidity that received and doses of heberferon. they were negative to sars-cov- rt-pcr since h and h since the first heberferon administration. during the days of symptoms worsening climate conditions were favorable to exacerbate asthma symptomatology. they recovered in hours after anti-inflammatory therapy. in the control group a symptomatic man of years-old, all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . with hypertension as comorbidity, that received only one dose of heberon alpha r also worsened the respiratory symptoms and was transfer to icu. his rt-pcr for viral rna was negative days after symptoms worsening and discharged. altogether these results indicate that with high probability the rapid viral elimination detected for heberferon treated patients is translated into the reduction of systemic inflammation markers while inducing a significant increase in circulating lymphocytes concentrations that may explain the symptomatic improvement observed in the more risky patients in the heberferon group. these results adds to the anti-inflammatory effect described for ifns in covid- patients , and are in correspondence with the finding of gene signature involved in type i and type ii response in mild-to-moderate covid- patients . about % of the confirmed covid- cases progress to the severe phase, with a higher risk for patients over years-old . using this estimate, in the patients included in our study approximately patients were expected to develop severe disease; however no patients became severely ill. no death was recorded in these mild or moderate patients. in similar cohort of patients, . % of mortality was described with the early use of ifns . in our trial several clinical parameters known to be related to covid- progression were significantly improved by the treatments or showed a trend to a favorable behavior. these results confirm the validity of early intervention with the treatment of ifns in patients with covid- , whereas demonstrated in the trial, the combination of type i and type ii ifns impacts strongly in the reduction of the risk for a severe disease likely through the efficient implementation of a timely controlled inflammatory antiviral response against the sars-cov- infection. before being approved recently by the fda and ema for severe covid- patients, remdesivir, was not successful in two randomized clinical trials , . its approval was sustained on the reduction of the illness duration in a few days . still the role of this antiviral in the inflammatory processes that drives the transit to severe and critical condition in covid- patients has not been described. heberferon formulation that combines in one vial ifn-α b and gamma results in an advantageous option for the treatment of covid- patients. first, due to the demonstrated better pharmacodynamics it is possible to administer less frequent and at lower doses than the other conventional ifns, (ifn-α b or ifn-β or ifn-λ) that need a thrice a week administration to have similar effect. ifn-β has been used at doses higher than fold (of miu/ml or miu/ml ) with respect to heberferon doses. second, the simultaneous all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint administration of both types of ifns will promote a faster and stronger innate and adaptive immune response. at least these two facts could be responsible for the quick clearance of sars-cov- detected in our trial. it has been proposed that interferon is efficient only in patients who lacked comorbidities , ; however we have obtained a high rate of negativitazation, resolution of symptoms, and hospital discharges for heberferon in a cohort of patients with % of coexisting comorbidities. moreover, it has been suggested that comorbidities like diabetes affect the response to ifn . two diabetic patients in our cohort negativized the virus on day from the beginning of the treatment and the other at least before day . our study had several limitations. this trial was open label, without a placebo group with unbalanced demographics (age years) between treatment arms. in addition, sampling methods were most likely suboptimal using the throat sampling, because of inability to do sampling of lower respiratory tract secretions. previous studies have shown that throat-swab specimens have lower viral loads . irrespective of these limitations the heberferon showed efficacy and was safe in shortening virus shedding, eliminating symptoms, and discharge of patients with covid- . perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint heberferon was a safe treatment, superior to heberon alpha r in shortening the time to sars-cov- viral rna negativization in a cohort of symptomatic or asymptomatic patients between and years-old, with more than % of patients negative to the sars-cov- in days of treatment. the rapid viral negativization contributes to implement an anti-inflammatory response that can protect the patients to enter in a more severe step of the disease. early isolation combined with early administration of antiviral treatments as ifns is an efficient approach that could contribute to save the life of patients in the covid- pandemic. the use of heberferon might be a distinctive element in the preventive and therapeutic strategy for current or future sars outbreaks. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . table . detection of sars-cov- in swabs by rt-pcr in the heberferon or control groups. throat swabs were taken from covid- positive patients at h, h, h and h after their treatment with heberferon ( patients) or standard of care ( patients). viral nucleic acid detection was carried out by rt-pcr resulting in positive or negative samples. for each analysis time, we represent the number of positive and negative patients, the percentage of negativization and the p value in a fisher test analysis in an overall analysis (all) and splitting patients in symptomatic (s) and asymptomatic (a). *: p< . ; **: p> . ; ns: p> . . all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . median of negativization for the two treatments were also calculated. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint leticia martínez hernández | internet@granma.cu. de junio de : : . que los problemas del país encuentren solución en la ciencia dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study therapeutic effectiveness 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treatment for covid- comparison of pre-and post-levothyroxine high-sensitivity c-reactive protein and fetuin-a levels in subclinical hypothyroidism diagnosis and treatment of acute pulmonary inflammation in critically ill patients: the role of inflammatory biomarkers association between c-reactive protein levels at hospital admission and long-termmortality in patients with acute decompensated heart failure the role of biomarkers in diagnosis of covid- -a systematic review diagnosis and treatment of acute pulmonary inflammation in critically ill patients: therole of inflammatory biomarkers association between c-reactive protein levels at hospital admission and long-termmortality in patients with acute decompensated heart failure clinical features of patients infected with novel coronavirus in wuhan, china impaired type i interferon activity and inflammatory responses in severe covid- patients clinical characteristics of coronavirus disease in china. the new england journal of medicine remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for or days in patients with severe covid- remdesivir for the treatment of covid- -preliminary report efficacy and safety of interferon β- a in treatment of severe covid- : a randomized clinical trial ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ifn-α a or ifn-β a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study baseline characteritic and outcomes of patients infectd with sars-cov- admitted to icu of the lombardy regii the authors were responsible for designing the trial and for collecting and analyzing the data.the authors assured the completeness and accuracy of the data collection and the adherence to the protocol. the details about the trial are provided in the protocol that has been posted in trials and is in processing by the editors of the journal.the primary endpoints were the time to viral rna negativization from the start of treatment and the time to progression to severe covid- . key: cord- -og k fc authors: konno, yoriyuki; kimura, izumi; uriu, keiya; fukushi, masaya; irie, takashi; koyanagi, yoshio; sauter, daniel; gifford, robert j.; nakagawa, so; sato, kei title: sars-cov- orf b is a potent interferon antagonist whose activity is increased by a naturally occurring elongation variant date: - - journal: cell rep doi: . /j.celrep. . sha: doc_id: cord_uid: og k fc one of the features distinguishing sars-cov- from its more pathogenic counterpart sars-cov is the presence of premature stop codons in its orf b gene. here, we show that sars-cov- orf b is a potent interferon antagonist, suppressing the induction of type i interferon more efficiently than its sars-cov ortholog. phylogenetic analyses and functional assays reveal that sars-cov- -related viruses from bats and pangolins also encode truncated orf b gene products with strong anti-interferon activity. furthermore, analyses of approximately , sars-cov- sequences identify a natural variant, in which a longer orf b reading frame was reconstituted. this variant was isolated from two patients with severe disease and further increased the ability of orf b to suppress interferon induction. thus, our findings not only help to explain the poor interferon response in covid- patients, but also describe the emergence of natural sars-cov- quasispecies with an extended orf b gene that may potentially affect covid- pathogenesis. the sars-cov- outbreak has originated from cross-species coronavirus transmission from these mammals to humans, the exact origin remains to be determined (andersen et al., ). one prominent feature that distinguishes covid- from sars in terms of immune responses is the poor induction of a type i interferon (ifn-i in this study, we therefore aimed to characterize the viral factor(s) determining immune activation upon sars-cov- infection. we particularly focused on differences in putative viral ifn-i antagonists and revealed that the orf b gene products of sars-cov- and sars-cov not only differ considerably in their length, but also in their ability to antagonize type i ifn. furthermore, we demonstrate that the potent anti-ifn-i activity of sars-cov- orf b is also found in related viruses from bats and pangolins. mutational analyses revealed that the length of the c-terminus determines the efficacy of ifn antagonism by orf b. finally, we describe a natural sars-cov- variant with further increased orf b-mediated anti-ifn-i activity that emerged during the current covid- pandemic. j o u r n a l p r e -p r o o f to determine virological differences between sars-cov- and sars-cov, we set out to compare the sequences of diverse sarbecoviruses. consistent with recent reports (lam et al., ; zhou et al., c), sarbecoviruses clustered into two groups, sars-cov- -related and sars-cov-related viruses (figures a and s ; the sequences used are listed in table s ( figure b) , we hypothesized that the antagonistic activity of orf b against ifn-i also differs between these two viruses. to test this hypothesis, we monitored human ifnb promoter activity in the presence of orf b of sars-cov- (wuhan-hu- ) and sars-cov (tor ) using a luciferase reporter assay. the influenza a virus (iav) non-structural protein (ns ) served as positive control (garcia-sastre et al., ; krug et al., ) . as shown in figure c , all three viral proteins dose-dependently suppressed the activation of the ifnb promoter upon sendai virus (sev) infection. notably, the antagonistic activity of sars-cov- orf b was slightly, but significantly higher than that of sars-cov orf b ( figure c, bottom (figures a and a) . since the lengths of orf b proteins in sars-cov- -related viruses including those from bats and pangolins were on average shorter than those from sars-cov and related viruses ( figure b), we next analyzed the variation of the orf b length in diverse sarbecoviruses. as shown in figure b , the vast majority of sars- the length of orf b is highly variable in sars-cov-related bat viruses ( figure b and table s ). only out of the orf b proteins of sars-cov-related bat viruses ( . %) are amino acids in length, % of them express a amino acid orf b ( figure b and table s ). similarly, only five out of the ten orf b proteins of sars-cov-related viruses from bats (rs , ynlf c, shaanxi , rm and f ) exhibited significant anti-ifn-i effects, and all these orf b proteins were shorter than amino acids ( figure c ). although three additional orf b proteins of sars-cov-related viruses from bats (hku - , gx and yunnan ) were shorter than amino acids in length, they did not exhibit anti-ifn-i activity, most likely because of their poor expression and/or stability (figures c and s b) . altogether, these findings suggest that the c-terminal region (residues - ) attenuate the anti-ifn-i activity of orf b. at its c-terminus ("l +nls") ( figure e) . furthermore, we also attached the c-myc nls to the c-terminus of rs orf b ( figure e ). as expected, sars-cov tor orf b l * as well as rs wild-type (wt) mainly localized to the cytosol, while the two mutants harboring the c-myc nls were localized to similar levels in both the cytosol and the nucleus ( figure f ). reporter assays showed that the sars-cov tor l * mutant exhibits significantly higher anti-ifn-i activity than wt sars-cov tor orf b although both are expressed at similar levels ( figure g) . moreover, the anti-ifn-i activity of both tor l * and rs orf b was attenuated by the addition of an nls (figure g ), suggesting that cytosolic localization of orf b is important to exhibit anti-ifn-i activity. consistent with the biochemical assays ( figures d and f) , immunofluorescence microscopy showed that wt sars-cov- orf b (wuhan-hu- ) as well as the sars-cov orf b l * mutant are mainly localized in the cytosol, while wt sars-cov orf b (tor ) and the tor l +nls mutant reside in both the cytosol and the nucleus ( figure h ). in parallel, we monitored the subcellular localization of irf , since this transcription factor is a key regulator of ifnb expression [reviewed in (park and iwasaki, )] that has previously been shown to be orf b and the sars-cov orf b l * mutant, but less so by wt sars-cov orf b and its l +nls mutant ( figure h ). collectively, these findings demonstrate that the c-terminal region of sars-cov orf b attenuates its anti-ifn-i activity by impairing its ability to prevent the translocation of irf into the nucleus. cov-glue webtool (http://cov-glue.cvr.gla.ac.uk) revealed that the orf b gene is highly conserved (table s and figure s d) . notably, however, we detected two viral sequences (gisaid accession ids: epi_isl_ and epi_isl_ ), in which the orf b gene was extended due to the loss of the first premature stop codon (* q) (figures b, bottom and s c and table s ). (figure b , bottom; see also figure s d ). ifnβ reporter assays revealed that the ecuador variant orf b exhibits significantly higher anti-ifn-i activity than the parental sars-cov- orf b ( figure d ). since we found that sars-cov- orf b hampers the nuclear translocation of irf (figure h) , we investigated the ability of wt sars-cov- orf b, the ecuador variant orf b, as well as sars-cov orf b and iav ns to suppress irf -driven gene expression. luciferase reporter assays revealed that the inhibitory activity of wt sars-cov- orf b was significantly higher than that of sars-cov orf b ( figure e) . importantly, the ecuador variant orf b was even more effective and suppressed irf -driven gene expression as efficiently as iav ns ( figure e) for recombination of orf b between the lineages of sars-cov- and sars-cov. notably, phenotypic differences in the ability of orf b to suppress ifn-i responses may also be associated with the likelihood of successful zoonotic transmission of sarbecoviruses to humans since many ifn-stimulated genes are antagonized in a species-specific manner. while more than sars-cov-related viruses were the full-length sequences (~ , bp) of sars-cov- (wuhan-hu- as a representative), sars-cov- -related viruses from bats (n= ) and pangolins (n= ), sars-cov (n= ), sars-cov-related viruses from civets (n= ) and bats (n= ), and outgroup viruses (n= ; bm - and btky ) were analyzed. accession number, strain name, and host of each virus are indicated for each branch. note that the branches including sars-cov (n= ) and sars-cov-related viruses from civets (n= ) were collapsed for better visualization. the uncollapsed tree is shown in figure s , and the sequences used are summarized in table s . independent experiments is shown. note that orf b and ns were run on separate blots for better visualization. figure s a shows them on the same blot with high and low exposure. for qrt-pcr, the expression levels of endogenous ifnb and gapdh were quantified. for the luciferase assay (c) and real-time quantification and statistical analysis data analyses were performed using prism (graphpad software). the data are presented as averages ± sem. statistically significant differences were determined by student's t test. statistical details can be found directly in the figures or in the corresponding figure legends. mean values of three independent experiments with sem are shown, and statistically significant differences (p < . ) compared to the sev-infected empty vector-transfected cells (#) and the same amount of the sars orf b-transfected cells (*) are shown. e, empty vector see also figures s and s and table s figure . c-terminal truncations increase the ifn-antagonistic activity of orf b (a) maximum likelihood phylogenetic tree of sarbecovirus orf b. the orf b sequences of sars-cov- (wuhan-hu- ) sars-cov (tor , gz , gz , urbani, bj sars-cov-related viruses from civets (civet ) and bats (rs , yn , rs , ynlf c, shaanxi , rm , f , hku - , gx and yunnan ), and two outgroup viruses the orf b sequences of all sars-cov-related viruses are summarized in table s , and the orf b sequences used in this study are summarized in table s bootstrap value; *, > %. (b) proportion of the orf b lengths in each sarbecovirus. the distribution of different lengths of orf b in each viral group is summarized in pie charts the number at the pie charts give the protein length indicated, and the numbers in bold indicate the most prevalent protein length for each viral group note that the amino acid sequences of zxc and zc are identical. an uncropped dot blot is shown in figure s b. (e) subcellular localization of sarbecovirus orf b. cell lysates of the hek cells transfected with a plasmid expressing ha-tagged sarbecovirus orf b were separated into cytosolic and nuclear fractions as described in the methods section. the percentage of orf b protein localized in the nucleus (top, n= ) and a representative western blot (bottom) are shown. tuba and lmna were used for as controls for cytosolic and nuclear proteins cell lysates of the hek cells transfected with a plasmid expressing ha-tagged orf b mutants were separated into cytosol or nuclear fractions as described in the methods section. the percentage of orf b protein localized in the nucleus (top, n= ) and a representative western blot (bottom) are shown. tuba and lmna were used for as controls for cytosolic and nuclear proteins. (g) hek cells were cotransfected with a plasmid expressing the sev was inoculated at moi . h post infection, cells were harvested for western blotting (top) and luciferase assay (bottom) subcellular localization of orf b and irf . hela cells were transfected with the indicated plasmids expressing ha-orf b and were infected with sev as described in the methods section. representative figures are shown. scale bar, µm. the white circles in the panels of one representative blot out of three independent experiments is shown. for the luciferase assay, the value of the sev-infected empty vector-transfected cells was set to %. the mean values of three independent experiments with sem are shown, and statistically significant differences sev-infected empty vector-transfected cells (#) are shown. in (d), red asterisks indicate statistically significant differences in (g), blue and green asterisks indicate statistically significant differences (p < . ) compared the same amount of either tor orf b l *-transfected cells or rs orf b wt see also figure s and tables s and s enhanced anti-ifn-i upon reconstitution of the cryptic sars-cov- orf b (a) schemes illustrating the genomic regions encoding orf , orf a, orf b and orf of sars-cov- and sars-cov. open squares with dotted red lines indicate a cryptic orf b reading frame in sars-cov- that is similar to sars-cov orf b (see also figure s a). asterisks indicate stop codons in the orf b frame. (b) sars-cov- * and *) are shown. asterisks indicate the stop codons in the original orf b frame. (bottom) a natural orf b variant detected in two sequences deposited in gisaid (gisai accession ids: epi_isl_ and epi_isl_ ; herein designated an "ecuador variant") are shown. the corresponding nucleotide and amino acid sequences are shown in figure s c hek cells were cotransfected with two different amounts of plasmids expressing the indicated ha-tagged sars-cov- orf b derivatives (wt, *, *, * and *; and ng) and p luc ( ng). h post transfection, sev was inoculated at moi enhanced anti-ifn-i activity of an ecuador variant orf b. hek cells were cotransfected with two different amounts of plasmids expressing ha-tagged "ecuador variant" orf b or parental sars-cov- orf b ( and ng) and p luc ( ng). h post transfection enhanced inhibition of the irf -mediated ifn-i activation by the ecuador variant orf b. hek cells were cotransfected with two different amounts of plasmids expressing the indicated ha-tagged viral proteins ( and ng) and p c b-luc ( ng). h post transfection for the luciferase assay, the value of the sev-infected empty vector-transfected cells was set to %. the mean values of three independent experiments with sem are shown, and statistically significant differences (p < . ) compared to the sev-infected empty vector-transfected cells (#) and the same amount of the sars-cov- orf b wt see also figures s and s and table s were transfected with using a fugene hd transfection reagent (promega) according to the manufacturer's protocol. for luciferase reporter assay, cells were cotransfected with ng of either p luc (expressing firefly luciferase driven by human ifnb promoter; kindly provided by dr ) and the pcaggs-based ha-tagged expression plasmid (the amounts are indicated in the figure legends). a cells ( , cells) were electroporated with ng of the pcaggs-based ha-tagged expression plasmid using a neon transfection system (thermo fisher scientific) according to the manufacturer's protocol ( v; ms; times pulse) ) was inoculated into the transfected cells at multiplicity of infection (moi) (for hek and a cells) or (for hela cells) briefly, µl of cell lysate was applied to a -well plate (nunc), and the firefly luciferase activity was measured using a picagene brillianstar-lt luciferase assay system (toyo-b-net), and the input for the luciferase assay was normalized by using a celltiter-glo . assay kit (promega) following the manufacturers' instructions subcellular fractionation was performed using nuclear/cytosol fractionation kit (biovision) according to the manufacturer's procedure transfected cells were lysed with x sds sample buffer ( . mm tris-hcl % -mercaptoethanol and . % bromophenol blue) ) using an hrp-conjugated rat anti-ha monoclonal antibody (clone f ; roche), a mouse anti-alpha-tubulin (tuba) monoclonal antibody (clone dm a; sigma-aldrich); a rabbit anti-lamin a/c (lmna) polyclonal antibody (cell signaling technology); an hrp-conjugated horse anti-mouse igg antibody (cell signaling technology); and an hrp-conjugated goat anti-rabbit igg antibody (cell signaling technology). dot blotting was performed using a bio-dot microfiltration apparatus -µm membranes (merck) were used for western blotting, while nitrocellulose . -µm membranes (bio-rad) were used for dot blotting thermo fisher scientific). cdna was synthesized using superscript iii reverse transcriptase (thermo fisher scientific) and oligo(dt) - primer (thermo fisher scientific). real-time rt-pcr was performed as previously described immunofluorescence staining twenty-four h post infection, cells were fixed with formaldehyde, permeabilized with triton x- , and then stained using an fitc-conjugated anti-ha antibody (clone f ; roche); a rabbit anti-irf polyclonal antibody (abcam); and an alexa -conjugated anti-rabbit igg antibody (thermo fisher scientific) antipode moutant with dapi (thermo fisher scientific) and observed using an fv- d confocal microscope cov-glue to survey the orf b derivatives in pandemic sars-cov- sequences, we used the viral sequences deposited in gisaid we constructed a phylogenetic tree using raxml-ng version . . (kozlov et al., ) with a tpm uf substitution model (figure s d). we also detected the two sars-cov- sequences (gisaid accession ids: epi_isl_ and epi_isl_ , collected in quito, ecuador) possessing the v t/t n substitutions in orf a all viral genome sequences used in this study and the respective genbank or gisaid (https://www.gisaid.org) accession numbers are summarized in table s . we first aligned the viral genomes using the l-ins-i program of mafft version . (katoh and standley, ). based on the multiple sequence alignment and the gene annotation of sars-cov, we extracted the region of the orf b gene. we then constructed phylogenetic trees using the full-length genomes (figures a and s ) and orf b genes (figure a ). we generated a maximum likelihood based phylogenetic tree using raxml-ng version . table s ) and the cryptic sars-cov orf b-like sequence in sars-cov- [wuhan-hu- (genbank accession no. nc_ . ), nucleotides - , see also figure s a ) was synthesized by a gene synthesis service (fasmac). the orf b derivatives were generated by pcr using primestar gxl dna polymerase (takara), the synthesized orfs as templates, and the primers listed in table s . the ha-tagged ecuador variant orf b (gisaid accession ids: epi_isl_ and epi_isl_ , which corresponds to the s q/l m mutant of sars-cov- wuhan-hu- orf b * ; see also figure s c ) was generated by overlap extension pcr by using primestar gxl dna polymerase (takara), the sars-cov- orf b * as the template, and the primers listed in key: cord- - lxq pi authors: jalkanen, juho; hollmén, maija; jalkanen, sirpa title: interferon beta- a for covid- : critical importance of the administration route date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: lxq pi nan interferon beta- a for covid- : critical importance of the administration route juho jalkanen , maija hollmén and sirpa jalkanen * type i interferons, especially ifn-beta, have been appointed as potential leading therapeutics to tackle severe covid- and are currently being evaluated in remap-cap and the who's solidarity trial. as a most recent example, combination treatments with ifn-beta, lopinavir-ritonavir, and ribavirin showed that the arm containing ifn-beta was superior in eliminating the virus from the nasopharyngeal swabs in phase ii clinical trial [ ] . recent papers on the matter unfortunately fall short of differentiating between subcutaneous (s.c.) and intravenous (i.v.) administration, which are completely different treatments concerning drug exposure and wanted effects in the lung endothelium, which is under attack in covid- [ ] . we wish to highlight the differences of these two treatment methods and also other crucial aspects of ifn-beta treatment for covid- and acute respiratory distress syndrome (ards). a recent report concluded that the pharmacological effects of s.c. vs. i.v. ifn-beta- a are the same, because they produce similar anti-viral responses [ ] . importantly, however, the pharmacokinetics of s.c. vs. i.v. ifn-beta are complete mirror images, "flip-flops" [ ] . maximum serum concentrations (cmax) and total exposure through serum concentrations are significantly higher after i.v. than s.c. injections (p = . ). prior corner stone pk studies investigating s.c. vs. i.v. administration of ifn-beta- a conclude that s.c. administration produces significantly lower drug concentrations and incomplete bioavailability compared to i.v. dosing. the bioavailability via the s.c. route is about one third of that obtained by i.v. injections [ ] . for critically ill patients on vasopressors and with very limited peripheral microcirculation, the bioavailability of s.c. dosed ifn-beta becomes even more questionable. ifn-beta is cleared almost solely through its receptor (ifnar). with s.c. dosing, ifn-beta is slowly taken up by the lymphatic system, from which it enters the blood during a number of hours with modest peak concentrations. in contrast, i.v. dosing achieves high serum concentration and efficiently reaches vast capillary beds of central organs, where it is taken up by its receptors without saturating the body and causing unwanted adverse events. this is an important aspect as endothelial dysfunction is connected to covid- infection [ , ] . nonetheless, the purpose of i.v. administered ifn-beta for the treatment of covid- and ards is to maximise bioavailability of the drug at the lung vasculature, as well as other vascular beds. this is hardly achieved with s.c. dosing in critically ill patients. ifn-beta increases cd in pulmonary capillaries. this is of utmost importance as cd is the key enzyme for vascular integrity under hypoxic conditions. the protective effect of ifn-beta on the lung is attributed to the clearance of pro-inflammatory atp and prothrombotic adp from circulation and converting them into highly anti-inflammatory adenosine via amp step by cd [ ] . it is well known that corticosteroids as immunosuppressors dampen our natural anti-viral responses, and the direct inhibitory effect of corticosteroids on ifn signalling has been reported [ ] . still corticosteroids are widely used to treat ards and severe viral respiratory infections even though several studies have shown that corticosteroid use is associated with harm in viral outbreaks such as h n and mers [ ] . in fact, reports on using type i ifns for the treatment of mers reveal that the majority of these patients received systemic corticosteroids with ifn. for example, % of mers patients received corticosteroids with type i ifn [ ] . in the recent interest study investigating the use of i.v. ifnbeta- a for ards, the primary analyses did not show any benefit for ifn-beta over placebo [ ] . however, nearly % of the patients received corticosteroids with ifn-beta. further studies revealed that corticosteroids block ifn signalling and the upregulation of cd expression in human pulmonary endothelial cells, and combining i.v. ifn-beta with systemic corticosteroids may be even more detrimental than corticosteroids alone [ ] . these findings suggest that the different anti-inflammatory pathways triggered by ifn-beta and corticosteroids should not be induced at the same time. immunomodulation is complex, and timing of the treatments is critical. there are a limited number of direct studies on the timing of immunomodulatory treatments such as ifn-beta, but given our basic understanding of human biology and viral defence, we suggest that ifn-beta should be given early to covid- patients. in mild cases such as in the recent clinical trial, even s.c. administered ifn-beta was effective [ ] , but in more severe cases, i.v. injections are needed to rapidly reach the endothelium. as ards rises together with a cytokine storm, corticosteroids may play a beneficial role during the later fibrotic phase or just by calming down the cytokine storm after ifns have had their impact. this is supported by villar et al. who showed that the use of dexamethasone was associated with better survival in ards [ ] . a notable feature of this study is that the enrolled patients were not on steroids when entering the trial. thus, initial endogenous ifn responses had not been tampered. sequential treatment strategy may be the future once we are able to reliably understand the time course of patients' immunological responses. severely ill covid- patients with increased levels of plasma cytokines (especially il- ) show signs of immune exhaustion and poor ifn responses [ ] . even in such cases, these patients would most likely benefit from ifnbeta, because it is the most potent anti-viral and antiinflammatory agent of all interferons. it can induce the desired immune boost, but simultaneously downregulate il- and il- [ ] and impair extravasation of neutrophils into lungs [ ] . ifn-beta is now among the leading candidates to treat covid- in various clinical trials, and i.v. and s.c. routes of administration are considered to be equal. this is not the case due to the different bioavailabilities of ifn-beta via i.v. and s.c. injections in target organs. this aspect needs to be taken seriously, when critically ill patients with compromised peripheral circulation are treated. gastrointestinal manifestations of sars-cov- infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- type interferons as a potential treatment against covid- receptor-mediated pharmacokinetic/ pharmacodynamic model of interferon-beta a in humans endothelial cell infection and endotheliitis in covid- purinergic signaling during inflammation the type i interferon signaling pathway is a target for glucocorticoid inhibition clinical evidence does not support corticosteroid treatment for -ncov lung injury ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study effect of intravenous interferon β- a on death and days free from mechanical ventilation among patients with moderate to severe acute respiratory distress syndrome: a randomized clinical trial glucocorticoids inhibit type i ifn beta signaling and the upregulation of cd in human lung dexamethasone treatment for the acute respiratory distress syndrome: a multicentre, randomised controlled trial imbalanced host response to sars-cov- drives development of covid- ifn-beta-mediated inhibition of il- expression requires the isgf components stat , stat , and irf- ifn-beta protects from vascular leakage via up-regulation of cd publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions all authors contributed to the writing of this comment; the funding agencies did not have any role. the authors read and approved the final manuscript. key: cord- -r rn yyz authors: omatsu, tsutomu; bak, eun-jung; ishii, yoshiyuki; kyuwa, shigeru; tohya, yukinobu; akashi, hiroomi; yoshikawa, yasuhiro title: induction and sequencing of rousette bat interferon α and β genes date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: r rn yyz bats are considered to be natural reservoirs for several viruses of clinical importance, including rabies virus, nipah virus, and hendra virus. type i interferons (ifns) is an important part of the immune system in the defense against viral infection. to investigate the function of type i ifns upon viral infection in bats, the nucleic acid, and amino acid sequences of egyptian rousette (rousettus aegyptiacus) ifn-α and -β were characterized. sequence data indicated that bat ifn-α consists of -bp encoded -aa, and ifn-β consisted of -bp encoded -aa. phylogenetic analysis of the overall identity of ifn-β shared the highest sequence homology with pig ifn-β in both nucleotide and amino acid level. stimulation of bat primary kidney cells (bpkcs) and bat lung cell lines, tb- lu, with polyinosinic–polycytidylic acid (poly(i:c)) or exogenous bat type i ifns resulted in increased type i ifns mrna expression in bpkcs, but not in tb- lu. characterization of the bat ifn-α and -β genes allows understanding of the immune responses upon stimulation in different tissues, thus providing practical strategies for control and treatment of clinically important diseases. these results are important especially for the virus infection, and suggest that future molecular studies on virus infection experiment of bats in vitro will require careful consideration of the differences of type i ifn expression patterns in different cell types. bats, chiropteras, are well-known vectors of rabies and some studies indicate that they may also naturally harbor some emerging viruses such as nipah virus, hendra virus, bat-sars-cov and ebola virus (chua et al., ; halpin et al., ; lau et al., ; leroy et al., ; mayen, ; mccoll et al., ; normile, ) . bat has two suborders, megachiroptera (flying fox) and microchiroptera (insectivorous bat). many emerging or re-emerging viruses, such as rabies, nipah virus, and hendra virus, were isolated form megachiroptera. in particular, european bat lyssavirus type i was also isolated from rousettus sp. (van der poel et al., ; wellenberg et al., ; wong et al., ) . bats were thought to have an important role for the infection cycle of these emerging and re-emerging viruses. in vivo experiment, www.elsevier.com/locate/vetimm available online at www.sciencedirect.com veterinary immunology and immunopathology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ebola virus inoculation studies showed that both flying foxes and insectivorous bats support viral replication and circulation with high viral titers without becoming ill (swanepoel et al., ) . studies in vitro have shown that ebola virus vp protein blocks the activation of interferon regulatory factor (irf- ) and ebola virus vp protein inhibits interferon (ifn) signaling (basler et al., ; reid et al., ) . these data suggested that ebola virus might evade the anti-viral activity of ifns in bat cells. therefore, it is crucial to investigate ifn regulation and function in bats because few immunological studies have been reported for this animal species. cells have many responses to viral infection. one of the responses is the secretion of type i ifns which are composed of multiple a subtypes and a single b subtype (sen, ) . type i ifns expression utilize two signal transduction pathways; the toll-like receptor (tlr)dependent pathway and tlr-independent pathway. in tlr-dependent pathway, cells recognize viral doublestrand rna, single-strand rna and cpg dna via tlr, and subsequently ifn-b is induced. in tlrindependent pathway, intracellular sensors such as retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated gene- (mda- ) detect viral components in the cytoplasm, and transactivate ifn-b mrna (hiscott et al., ) . expressed ifn-b binds to the type i ifn receptors and activates numerous ifnstimulated genes, such as the protein kinase r (pkr) gene, the - oligoa-denylate synthetases (oas) gene, and the myxovirus resistance (mx) gene. the product of these genes controls viral infection (samuel, ) . viral double-stranded rna (dsrna), a viral intermediate in the proliferation of many rna viruses, is known as an ifn-inducer through these sensors (gitlin et al., ) . polyinosinic-polycytidylic acid (poly(i:c)) is a synthetic mimetic of viral dsrna and a strong inducer of type i ifns in vivo and in vitro via these sensors (hertzog et al., ) . type i ifns stimulate anti-viral activity as mentioned above; however, such studies in bat have not been possible because the bat ifn related genes had not been previously identified. in this study, we determined the sequence of a subtype of ifn-a and ifn-b from rousettus aegyptiacus, including the full open reading frames (orfs), and analyzed phylogenetically based on ifns from other mammals. in addition, the upregulation of these mrnas in both bat primary kidney cells (bpkcs) and a bat lung cell line, tb- lu was examined using poly(i:c) or bat type i ifns derived from bpkcs. fresh liver sample and whole blood of r. aegyptiacus under anesthesia with ketamine ( mg/ml/kg) and medetomidine ( . mg/ml/kg) were collected by heart puncture. bat liver was fixed with % neutral buffer formalin. bat genomic dna was isolated from fixed liver with the wizard genomic dna purification kit (promega, madison, wi) and stored at À c until usage. bat genomic dna sample was used as a template of polymerase chain reaction (pcr) using takara ex taq (takara bio, ohtsu, shiga, japan). forward and reverse primers of ifn-a and ifn-b for pcr were designed from the sequence data of human, mouse, cat, pig, and horse ifn-a and ifn-b (table ). the accession numbers of these data in genbank are as follows: ifn-a table sequence of each pcr primers the pcr products were isolated by electrophoresis in an % agarose gel and purified using the wizard sv gel and pcr clean-up system (promega). the purified pcr products were cloned into pcr-topo vector using the topo ta cloning kit (invitrogen, carlsbad, ca) and the sequence was determined using the big dye tm terminator kit (abi, ca, usa) and abi prism tm dna sequencer (abi, ca, usa). we utilized the sequence data to design new primers for pcr amplification of the entire ifn-a and ifn-b cdnas. finally, we determined the orf for the ifn-a and ifn-b cdna sequences using ifn-a f , r and ifn-b cdsf, cdsr (table ) . the orfs and the deduced amino acid sequences were analyzed using the genetic information processing software genetyx-win version . . (software development, tokyo, japan). mammalian ifn nucleotide sequence information was obtained from genbank. species in the phylogenetic tree were limited because only several species have enough numbers of subtypes of known infs sequences. sequences were aligned using clustal w (version . ; http://www.cf.ac.uk/biosi/research/biosoft/downloads/clustalw.html), checked by eye, and all positions with gaps or ambiguous alignments were excluded from the analysis. a phylogenetic tree was constructed using phylip (version . . ; http://evolution.genetics.washington.edu/phylip.html) with the following full-length ifn orfs referred to genbank: human (ifn-a (np_ ) fresh kidney of rousettus leschenaulti under anesthesia with ketamine ( mg/ml/kg) and medetomidine ( . mg/ml/kg) were removed, sliced and treated with . % trypsin-edta in phosphate-buffered saline (pbs). whole blood of bat was collected by heart puncture under anesthesia. collected bat primary kidney cells were seeded on -cm plate in dulbecco's modified eagle's medium (dmem) (invitrogen) with % heat-inactivated fetal calf serum (fcs). bat lung epithelial cell line, tb- lu, was maintained in incubation of % co , at c in dmem containing % fcs. bpkcs and tb- lu cells were treated with % fcs-dmem including mg/ml poly(i:c) (sigma, st. louis, mo) and mg/ml diethylaminoethyl dextran (deae-dextran) (sigma) in % co at c for h. cells were then washed twice with pbs and total rna was isolated using isogen solution (nippon gene, toyama, japan). bpkcs were treated with % fcs-dmem including mg/ml poly(i:c) and mg/ml deae-dextran in % co at c for h. after treatment, the cells were washed twice and cultured in fresh % fcs-dmem for h. the whole supernatant, bat type i ifns-containing medium, was collected and stored at c until usage. bpkcs and tb- lu cells were prepared at  ml À in -well culture plate containing ml per well for days, and then treated with ml of bat type i ifns-containing medium for h. after that, cells were washed three times and then incubated in % co at c in dmem containing % fcs (primary kidney cells) or % fcs (tb- lu cells) for additional , , and h. after incubation, total rna was isolated from these cells with isogen solution (nippon gene). total rnas were treated with dnase i (takara bio) according to the manufacturer's instructions. rna samples were then reverse-transcribed using the oligo(dt) - primer and superscript tm ii (invitrogen) for synthetic first-strand cdna. cdnas were used as a template for semi-quantitative pcr with takara ex taq using gapdh f and r, ifn-a f and r and ifn-b cdsf and cdsr (table ) as primers. the pcr products were analyzed by % agarose gel electrophoresis and stained with ethidium bromide. in this study, the experiment was performed in accordance with the animal experimentation guideline, the university of tokyo, and was approved by the institutional animal care and use committee of the graduate school of agricultural and life sciences, the university of tokyo. the nucleic acid and amino acid sequences of one of the ifn-a genes and the ifn-b gene from r. aegyptiacus were determined. the full-length bat ifn-a orf was bp and encoded -aa polypep-tides. bat ifn-b orf was bp and encoded -aa polypeptides (fig. (a and b) ). direct comparison of bat ifn-a genes simultaneously against various animal species was complicated by the fact that there are various ifn-a subtypes and difficulties in the identification of subtype of the bat ifn-a. therefore, sequence comparisons were performed using ifn-b between bat and human, pig, cat, horse, and mouse. the identity of bat ifn-b with human, pig, cat, horse, and mouse ifn-b were . , . , . , . , and . % at the nucleotide level and . , . , . , . , and . % at the amino acid level, respectively. phylogenetic analysis using the amino acid sequences from several representative eutherian type i ifns and chicken type i ifn found that both bat ifn-a and bat ifn-b are homologous to the mammalian ifn group. further analysis showed that both bat ifn-a and ifn-b were most closely related to those of pig, and followed by horse (fig. ) . although phylogenetic relationship between bat and other animals remains inconclusive, molecular phylogenetics using mitochondrial dna or retro-transposon insertions indicated that chiroptera is included in fereuungrate or pegasoferae (perissodactyla, carnivora, pholidota, and chiroptera) (nikaido et al., ; nishihara et al., ) . comparison of the amino acid sequences of the cell surface molecule cd showed that bat is more closely related to cat and dog (omatsu et al., ) . our findings and these molecular phylogenetic analyses suggested that bat might have anti-viral mechanism similar to these animals. some investigators indicated that nipah virus spread from megachiroptera to pig and then from pig to human (tan and wong, ) , and pig might be more susceptible to the virus than other animals. in contrast, relatively low fig. . maximum likelihood phylogenetic tree constructed by the phylip . program using amino acid sequences from human, horse, pig, cat, dog, mouse, chicken, and bat type i ifns. the numbers at the nodes indicate bootstrap values. 'a' and 'b' reflect ifn-a and -b, respectively. homology of immune factors between bat and human suggested the presence of different anti-viral activity against some viral infections. these factors might be one of the key factors to control zoonoses from bat. to investigate whether bpkcs and tb- lu cells have the capacity of ifn production, we first examined the up-regulation of type i ifns in response to poly(i:c) treatment. in bpkcs, there is an increase in the expression of ifn-b mrna, but not ifn-a mrna, at h after poly(i:c) treatment. however, in tb- lu cells, poly(i:c) treatment induced ifn-a mrna production but production of inf-b mrna was not observed (fig. (a) ). to examine whether bpkcs or tb- lu expresses type i ifn mrna in response to the bat ifncontaining medium (exogenous ifn), the expression of type i ifns mrna was examined at , , and h after the exogenous ifn-treatment. in the case of bpkcs, ifn-a mrna was detected at each time point with a gradual increase, and ifn-b mrna which was not initially detected, peaked at h in response to exogenous ifn (fig. (b) ). in tb- lu, however, type i ifns mrna expression was not detected at all (data not shown). although bpkcs could induce type i ifns mrna in response to poly(i:c) via tlr , rig-i, and mda- , type i ifns-inducing signal was not sufficient for the stimulation of ifn-a mrna synthesis. in contrast, when type i ifns were supplied to bpkcs, ifn-b mrna was induced more rapidly than ifn-a. this indicated that ifn-b is involved in immediate response to invasion of viruses or microbes and ifn-a, which is responsible for anti-viral activity via stimulation of pkr, oas, and mx synthesis, is induced by ifnb and has more prolonged response in bpkcs (fig. (b) ). when tb- lu were treated with either poly(i:c) or bat ifns-containing medium, these cells did not express any ifn-a or ifn-b mrna. this suggests that in tb- lu the mechanism of dsrna recognition or the signaling pathway reacted to exogenous ifns is not utilized for up-regulation of type i ifns mrna. thus, the ifn signal responding to both poly(i:c) and exogenous type i ifns was different between bpkcs and tb- lu. bat is diversified into about a thousand species in the world. these results further indicated that extra considerations should be taken in the interpretation of experimental data of antiviral dynamics among various bat cell types and species. the bat immune system is of particular interest because of its ability to act as a reservoir for a variety of pathogens that pose serious health threats to humans. however, these studies are complicated because few studies on anti-viral mechanism of bat are available. thus, the nucleotide sequences of type i ifns of rousette bat were characterized for the first time. to investigate whether and how bats harbor clinically important pathogens, some basic information from inoculation studies performed in vivo and in vitro is very important. to determine how wild animals remain asymptomatic to pathogens, it will be necessary to understand their viral control mechanisms, such as ifn signaling. using bats as representative pathogenic carriers, this study provides some basic and important immunological information about bat. it is necessary for understanding zoonoses from bat, especially for megachiroptera. ifn-a mrna (lanes and ), ifn-b mrna (lanes and ), and gapdh control mrna (lanes and ) from bpkcs (lanes - ) and tb- lu cells (lanes - ) were analyzed using semi-quantitative reverse-transcription pcr of total rna followed by % agarose gel electrophoresis and ethidium bromide staining. (b) temporal change of type i ifn mrnas in bpkcs after treatment with bat ifns-containing medium. ifn-a mrna (upper panel), ifn-b mrna (middle panel) and gapdh mrna (control; lower panel) from bpkcs were analyzed following treatment with bat ifns-containing medium for the indicated period of time. the ebola virus vp protein inhibits activation of interferon regulatory factor isolation of nipah virus from malaysian island flying-foxes essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus the interferon in tlr signaling: more than just antiviral manipulation of the nuclear factor-kappab pathway and the innate immune response by viruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats fruit bats as reservoirs of ebola virus haematophagous bats in brazil, their role in rabies transmission, impact on public health, livestock industry and alternatives to an indiscriminate reduction of bat population bat lyssavirus infections monophyletic origin of the order chiroptera and its phylogenetic position among mammalia, as inferred from the complete sequence of the mitochondrial dna of a japanese megabat, the ryukyu flying fox (pteropus dasymallus) pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions virology. researchers tie deadly sars virus to bats molecular cloning and sequencing of the cdna encoding the bat cd ebola virus vp binds karyopherin alpha and blocks stat nuclear accumulation antiviral actions of interferons viruses and interferons experimental inoculation of plants and animals with ebola virus nipah encephalitis outbreak in malaysia characterisation of a recently isolated lyssavirus in frugivorous zoo bats presence of european bat lyssavirus rnas in apparently healthy rousettus aegyptiacus bats bats as a continuing source of emerging infections in humans r. aegyptiacus and r. leschenaulti were kindly supplied by ueno zoo, tokyo, japan and asa zoo, hiroshima, japan. this work was supported by research on emerging and re-emerging infectious diseases grant (h -emerging disease-ippan- ) from the ministry of health, labor and welfare of japan. key: cord- -m va er authors: raaben, matthijs; groot koerkamp, marian ja; rottier, peter jm; de haan, cornelis am title: type i interferon receptor-independent and -dependent host transcriptional responses to mouse hepatitis coronavirus infection in vivo date: - - journal: bmc genomics doi: . / - - - sha: doc_id: cord_uid: m va er background: the role of type i ifns in protecting against coronavirus (cov) infections is not fully understood. while covs are poor inducers of type i ifns in tissue culture, several studies have demonstrated the importance of the type i ifn response in controlling mhv infection in animals. the protective effectors against mhv infection are, however, still unknown. results: in order to get more insight into the antiviral gene expression induced in the brains of mhv-infected mice, we performed whole-genome expression profiling. three different mouse strains, differing in their susceptibility to infection with mhv, were used. in balb/c mice, which display high viral loads but are able to control the infection, and genes were significantly differentially expressed (≥ . fold change) upon infection at and days post infection, respectively. functional association network analyses demonstrated a strong type i ifn response, with irf and irf as the central players. at days post infection, a type ii ifn response also becomes apparent. both the type i and ii ifn response, which were more pronounced in mice with a higher viral load, were not observed in svev mice, which are much less susceptible to infection with mhv. svev mice lacking the type i interferon receptor (ifnar-/-), however, were not able to control the infection. gene expression profiling of these mice identified type i ifn-independent responses to infection, with ifn-γ as the central player. as the balb/c and the ifnar-/- svev mice demonstrated very similar viral loads in their brains, we also compared their gene expression profiles upon infection with mhv in order to identify type i ifn-dependent transcriptional responses. many known ifn-inducible genes were detected, several of which have previously been shown to play an important protective role against virus infections. we speculate that the additional type i ifn-dependent genes that we discovered may also be important for protection against mhv infection. conclusion: transcriptional profiling of mice infected with mhv demonstrated the induction of a robust ifn response, which correlated with the viral load. profiling of ifnar-/- mice allowed us to identify type i ifn-independent and -dependent responses. overall, this study broadens our present knowledge of the type i and ii ifn-mediated effector responses during cov infection in vivo. cytokines are key regulators that dictate many aspects of innate and adaptive immunity. induction of type i interferons (ifns), a well-known subset of cytokines with antiviral activity, is triggered by a selection of cellular pattern recognition receptors, including tlrs (toll-like receptors), rig-i (retinoic acid-inducible gene i), and mda (melanoma differentiation-associated protein ). these receptors are activated in response to a range of pathogenspecific factors, which includes double-stranded rna produced during virus infection [ , ] . secreted type i ifns (i.e. ifn-α and ifn-β), subsequently induce an antiviral transcription program in the infected cell as well as in adjacent cells, thereby magnifying the "danger" signal and protecting against the infection. the role of type i ifns in controlling coronavirus (cov) infections is not well understood. a number of studies has shown that covs, like the mouse hepatitis virus (mhv) and the severe acute respiratory syndrome (sars)-cov, are poor inducers of type i ifns in cell culture, and even escape from detection by cytoplasmic pattern recognition receptors [ ] [ ] [ ] [ ] [ ] [ ] . consistently, virus-encoded ifn antagonistic functions have been described for both mhv and sars-cov [ , ] . in vivo, however, mhv infection appeared to induce the production of ifn-α in plasmacytoid dendritic cells (pdcs) by a tlr -dependent mechanism [ ] . moreover, mhv infections of primary neuronal cultures and of the central nervous system (cns) induced ifn-β gene expression, indicating that the production of type i ifns in vivo is not limited to pdcs [ , ] . furthermore, neuronal cultures infected with mhv exhibited increased expression of several type i ifninduced transcription factors [ ] . more recently, roth-cross and co-workers reported that macrophages and macrophage-like microglia cells produce ifn-β in the cns of mhv-infected mice in a mda -dependent manner [ ] . several studies have demonstrated the importance of the type i ifn response in controlling mhv infection in vivo. the exogenous delivery of type i ifns was shown to inhibit mhv infection of and spread to the mouse brain [ , ] . consistently, infection of mice lacking the functional type i ifn receptor (ifnar-/-) with mhv resulted in increased viral replication and extended tissue tropism [ , , ] . although many type i ifn-responsive genes have been identified [ ] , the protective effectors against mhv infection are yet unknown [ ] . in order to get more insight into the antiviral gene expression induced in the brains of mhv-infected mice, we performed whole-genome expression profiling. three different mouse strains (balb/c, svev and ifnar-/- svev mice), differing in their susceptibility to infec-tion with mhv, were used. previously, we have observed that svev mice are significantly more resistant to infection via the intranasal route than balb/c mice [ ] . the reason for the significant difference in susceptibility is not known, but may be related to different antiviral immune responses in these two mouse strains. furthermore, gene expression profiling of svev mice lacking the type i ifn receptor, which are not able to control the mhv infection [ ] , allowed us to identify type i ifn-independent transcriptional responses. we started by comparing the whole-genome expression profiles in the brains of the balb/c and the svev mice upon infection with mhv. to this end, mice were inoculated intranasally with tcid of mhv strain a or with pbs (control). groups of mice (n = ) were sacrificed at and days post inoculation after which the brains were harvested and total rna was isolated. the extent of virus replication was determined by quantitative reverse transcriptase (rt)-pcr targeting mhv-specific rna sequences as described earlier [ ] . previously, we demonstrated that the viral rna load correlates well with viral infectivity in tissue homogenates [ ] . while no viral rna could be detected yet at days post inoculation (data not shown), viral rna was observed in the brain of both mouse strains at day ( figure a ). as expected, the balb/ c mice displayed a much higher viral rna load than the svev mice. next, the rna extracts were processed for microarray analysis using the pbs-inoculated groups as the reference. in total, and genes were significantly differentially expressed (≥ . fold change) in balb/c mice at and days post infection, respectively. in contrast, in the svev mice, no significant induction of gene expression was observed. the results are depicted in figure b as a gene tree that was built based on the genes with a significantly altered expression level in balb/c mice at days post infection (i.e. expression-based cluster analysis). from these data we were able to identify host genes, the increased expression (≥ . fold) of which could already be detected at day (i.e. early genes; figure c ) or only at day (i.e. late genes; figure d ). the group of earlyinduced transcripts contained many ifn-inducible genes, including the well-known interferon regulatory factor (irf ), signal transducer and activator of transcription (stat ), and '- ' oligoadenylate synthetase (oas) genes (additional file a). within the cluster of "late" genes (additional file b) several chemokines (i.e. ccl , ccl , ccl , cxcl , and cxcl ) could be identified. next, in order to construct a functional association network, we applied the string . software [ ] to the list of proteins encoded by the "early" and "late" genes. we also included known interactors of our hits in this analysis, while proteins that did not demonstrate any known interactions were excluded for clarity. the results are shown in figure a and b. functional association network analysis of the proteins encoded by the "early" genes revealed two main modules. one module contained several proteins involved in antigen presentation, while the other module contained numerous proteins involved in the type i ifn response. the key player in this latter module appeared to be irf , which is the master regulator of type i ifn-dependent responses [ ] . functional association network analysis of the proteins encoded by the "late" genes revealed a large network of proteins involved in host-pathogen interactions. although the microarray analyses did not reveal the induction of ifn-γ gene expression itself, ifn-γ appeared at a central position in the network. in addition, the induction of a type i ifn response was also evident from this network as demonstrated by the presence of the transcription factors irf and irf , both of which demonstrated elevated mrna levels upon mhv infection. in conclusion, these results demonstrate that mhv infection induces a robust ifn response both at and days post infection, in which the transcription factors irf , irf , and irf appear to be the key players. at days post infection, a type ii ifn response also becomes apparent. to confirm and extend these observations, we next analyzed the induction of type i and ii ifn gene expression (i.e. ifn-α and ifn-β , and ifn-γ, respectively) by using quantitative rt-pcr. in agreement with the microarray expression profiles, significant induction of these type i and ii ifns could only be detected in the mhv-infected balb/c animals ( figure e ). the observation that the balb/c mice, unlike the svev mice, exhibited abundant expression of ifn-responsive genes upon mhv infection appears counter intuitive as the svev mice are much more resistant to the infection than the balb/c mice. apparently, the resistance of svev mice to mhv infection is not controlled by a more robust ifn response. the reason for the observed difference in susceptibility between the different mouse strains after intranasal inoculation is not known. mhv-a was recently shown to replicate efficiently in the liver of svev mice after intraperitoneal inoculation [ ] . interestingly, the resistance of svev mice after intranasal inoculation is not restricted to infection with mhv, as it was also observed for vesicular stomatitis virus [ ] . the microarray expression profiles described above suggested that the induction of an ifn response correlates with the viral load within the brain. to confirm this, we examined the data of the individual balb/c mice at days post infection in more detail. clearly, the animals with the highest viral loads (mouse and ; figure a ), also dis-played significantly higher levels of induction of type i and ii ifn expression ( figure b ). likewise, the amplitude of the gene expression profiles ( figure c and additional file ) of the individual mice also correlated with the viral loads in the brain. these observations are in agreement with results obtained by the profiling of sars-covinfected macaques [ ] . also in that study a positive correlation between virus load and the induction of gene expression was observed. a few genes (n = ), including isg , showed an inverse correlation with the viral load. we currently have no explanation for this observation as expression of isg is known to be induced by type i ifns [ , ] . interestingly, isg has been shown to exhibit antiviral activity against other viruses [ , ] . to study the role of type i ifn-independent and -dependent gene expression in the control of mhv infection in vivo in more detail, we next made use of the ifnar-/-mice [ ] . these mice are highly susceptible to mhv infection as compared to the parental svev mice [ , ] . indeed, when these mice were inoculated intranasally with tcid of mhv-a , viral rna levels in their brains became much higher than in animals from the parental strain at days post infection ( figure a ). interestingly, at this time point the viral rna levels in the ifnar-/-mice were comparable to those in the brains of the balb/c mice. however, efficient dissemination of the infection, resulting in high viral loads in the liver as determined by quantitative rt-pcr, was only observed in the ifnar-/-mice and not in the wild-type mice, which displayed viral rna levels just above background ( figure b ). thus, in agreement with previous studies, a type i ifn-dependent response is required to inhibit virus dissemination [ , ] . whole-genome expression profiling of brains of the ifnar-/-mice revealed the significantly induced expression of genes (≥ . fold) at days post infection. in contrast, at day , hardly any alterations in gene expression could be detected in these knock-out mice (additional file ). figure c shows an expression-based cluster analysis of these genes for the wild-type and ifnar-/mice. comparison of the complete expression profiles of these mice revealed that the transcriptional profile at day in the ifnar-/-mice has a larger similarity with the profile at day of the parental svev mice than with that of the knock-out mice at day post infection ( figure c ). this observation may suggest the presence of an early host response to infection with mhv in the parental mice, even though no significant induction (≥ . fold) of gene expression could be detected ( figure b) . such a response, may not be evident in transcriptional profiles of whole organs, but might only be apparent at the cellular level. we speculate that early decisive events are happening in initial target cell populations such as dcs and macro-early and late transcriptional responses to infection with mhv as the knock-out mice lack a functional type i ifn receptor, the upregulation of gene expression observed in these mice apparently occurs independently of type i ifn signalling. not much is known yet about type i ifn-independent responses to infection. the observation that the transcriptional upregulation of irf was independent of type i ifn signalling is consistent with the notion that ifn-γ can also induce expression of this gene [ , ]. the type i ifn receptor-independent expression profile within the brains of ifnar-/-mice after mhv infection likewise, we also observed increased transcription of ifitm and ifitm independent of type i ifn signalling, again corresponding with the literature [ , ] . interestingly, the expression of various genes encoding proteins involved in antigen presentation (i.e. h , b m, psmb , psmb , and ctss) was also increased in the absence of type i ifn signalling. psmb and psmb encode immunoproteasome subunits which facilitate antigen presentation to cd + t cells after virus infection, a process that is primarily regulated by ifn-γ [ ] . furthermore, also the expression of the major histocompatibility complex class ii (mhc ii) invariant chain, also called cd [ ], was increased upon infection of the knock-out mice. these data are in agreement with the observation that the induction of genes involved in antigen processing is independent of stat activation by . we also observed the transcriptional upregulation of the isoforms of metallothionein (mt , mt , and mt ), which encode proteins known to scavenge toxic metals [ ] . the induction of these genes, which was not apparent in either wild-type mice, could reflect an acute-phase reaction in the brain of mhv-infected ifnar-/-mice, which likely contributes to pathogenesis as has been shown for other viruses [ - ]. we constructed a functional association network by applying the string . software [ ] to the list of proteins encoded by the type i ifn-independent genes (additional file ). we also included known interactors of our hits in this analysis, while proteins that did not demonstrate any interactions were again excluded for clarity. the result is shown in figure . the analysis revealed ifn-γ as the central player in the type i ifn-independent antiviral network as this protein appeared to link a number of smaller modules. the induction of ifn-γ gene expression could be confirmed using quantitative rt-pcr (data not shown). the finding that ifn-γ-mediated transcriptional responses are not dramatically affected in the absence of type i ifn signalling is in agreement with reports referred to above and with a recent publication by ireland et al. [ ] , which shows that ifn-γ expression is significantly induced in the cns of mhv-infected ifnar-/-mice. while the production of ifn-γ by nk cells plays a major role in the protection against infection with mhv [ - ], the ifn-γ-mediated transcriptional responses that we observed were not protective against acute mhv infection in the ifnar-/-mice. several studies have shown that mhv [ , , ] as well as several other viruses [ - ] replicate to much higher levels (up to fold difference) in ifnar-/-mice than in their wild-type counterparts. in this study we show that a strong correlation exists between the amplitude of type i and ii ifn host responses with the viral load. the huge differences in virus replication between wild-type and ifnar-/-mice therefore do not permit a fair comparison between gene expression profiles of these mice, with the aim of identifying type i ifn-dependent responses. indeed, as no significant gene expression is observed in the wild-type svev mice, a comparison with the expression profile of the ifnar-/-mice only provides information about type i ifn-independent and not ifndependent responses. we now observe, in agreement with our previous study, that the brain of balb/c and ifnar-/ - svev mice contain very similar mhv loads at day and post infection [ ] . since the type i ifn-responsive pathway is very well conserved among many different species [ ], we considered it acceptable to compare the gene expression profiles of these mice with the aim of identifying type-i ifn-dependent responses, although comparing transcriptional profiles of wild-type and ifnar-/-mice from a different genetic background should obviously be done very cautiously. ideally, a comparison between wildtype balb/c and ifnar-/-balb/c mice would have been more accurate. while the induced expression of a number of genes was similar for the two mouse strains (i.e. type i ifn signalling-independent gene-expression), that of other genes was only observed in the balb/c mice (i.e. tentative type i ifn signalling-dependent gene expression). the expression of yet other genes appeared to be partially dependent of type i ifn signalling: increased expression of these genes was observed in the ifnar-/mice, but much more so in the balb/c mice. genes, the expression of which was upregulated (≥ . fold) in the balb/c mice but not significantly changed in the ifnar-/-mice upon infection with mhv, were tentatively designated as type i ifn-dependent. genes, the transcriptional upregulation of which was at least times higher in the balb/c mice than in the ifnar-/-mice, were also added to the list of tentative type i ifn-dependent genes. as expected, this set of genes (n = ) contained many known ifn-responsive genes like isg , ifit , ifit , isgf g, mx and ube l (additional file ). functional association network analyses showed irf and irf to be the key players in the network (additional file ). several of the tentative type i ifn-dependent genes (including mx and ube l) have previously been shown to play an important protective role against virus infections [ - ]. we speculate that other genes present in this list may also be important for full protection against mhv infection. transcriptional profiling of mice infected with mhv demonstrated the induction of a robust ifn response, which correlated with the viral load. profiling of ifnar-/-mice allowed us to identify type i ifn-independent anddependent responses. overall, this study broadens our present knowledge of the type i ifn-mediated effector responses during cov infection in vivo. [ ] [ ] [ ] week old balb/c were obtained from charles river laboratories, while type i ifn receptor knock-out mice (ifnar-/-) [ ] and the parental svev mice were obtained from b&k universal ltd. mice were inoculated intranasally with tcid of mhv strain a and sacrificed at the indicated time-points for organ dissection. control animals were treated with pbs. the study proto-col was approved by the animal ethics committee of the utrecht university, and all experiments were performed in accordance with accepted institutional and governmental policies. whole brains and livers were dissected from the mhvinfected and control mice. the tissues were added to lysing matrix d tubes (mp biomedical), containing ml of the type i ifn-independent gene expression network figure the type i ifn-independent gene expression network. the genes listed in additional file (n = ) were subjected to functional association network analysis by using the string . database as described in the legend of figure . the key player in the network, ifn-γ, is indicated in red. rnapro™ solution (q-biogene), and processed using a fastprep instrument (mp biomedical). the tissues were homogenized at , rpm for sec and immediately placed on ice. subsequently, the homogenates were centrifuged at , rpm for minutes at °c and supernatants were harvested and stored at - °c. total rna was isolated from the homogenates using the trizol reagent (invitrogen) according to the manufacturer's protocol. rna was further purified using the rneasy mini-kit with subsequent dnasei treatment on the column (qiagen). rna integrity was determined by spectrometry and by a microfluidics-based platform using a uv-mini device (shimadzu) and a bioanalyzer (agilent technologies), respectively. ], respectively), were measured by quantitative pcr using assay-on-demand reagents and equipment (pe applied biosystems), according to the manufacturer's instructions. the quantitative pcr reactions were performed in a total reaction volume of μl containing μl taqman ® universal pcr master mix ( ×), μl cdna, μl taqman ® gene expression assay mix ( ×), and μl water using an abi prism sequence detection system under the following conditions: °c for mins, followed by cycles of °c for secs and °c for min. for all assays, we performed "no-rt" (reaction using total rna as the substrate) and "no template" (reaction using water as the substrate) controls. in both cases, omitting cdna from the reaction resulted in a lack of pcr product generation. all assays were analyzed with abi prism software v . . f (pe applied biosystems). the comparative ctmethod was used to determine the fold change for each gene (primer efficiencies were similar for both the endogenous control primer set and genes of interest primer sets [data not shown]). note that the ct values of all samples were within the limits of the standard curves (data not shown). the housekeeping gene gapdh (nm_ . ) was used as a reference in all experiments, since expression of this gene was found constant among samples. the amounts of viral rna were determined by quantitative rt-pcr as described before [ ] . the microarray experiments were performed as described previously [ ] . briefly, mrna was amplified from μg of total rna by cdna synthesis with oligo(dt) doubleanchored primers, followed by in vitro transcription using a t rna polymerase kit (ambion). during transcription, -( -aminoallyl)-utp was incorporated into the single stranded crna. cy and cy nhs-esters (amersham biosciences) were coupled to μg crna. rna quality was monitored after each successive step using the equipment described above. corning ultragaps slides, printed with a mouse array-ready oligo set (operon; , spots), were hybridized with μg of each alternatively labeled crna target at °c for - h. two independent dyeswap hybridizations ( arrays) were performed for each experimental group. after hybridization the slides were washed extensively and scanned using the agilent g aa dna microarray scanner. after data extraction using imagene . software (biodiscovery), lowess normalization [ ] was performed on mean spot-intensities in order to correct for dye and printtip biases [ ] . the microarray data was analysed using anova (r version . . /maanova version . - ) http://www.r-project.org [ ] . briefly, in a fixed effect analysis, sample, array and dye effects were modelled. pvalues were determined by a permutation f -test, in which residuals were shuffled , times globally. genes with p < . after family wise error correction were considered significantly changed. cluster analysis (standard correlation) was performed with genespring gx . software (silicon genetics). when indicated, the confidence level was increased by applying a fold change cut-off. the resulting genelists were subjected to genespring . software for further analysis. arrayexpress accession numbers miame-compliant data in mage-ml format as well as complete descriptions of protocols have been submitted to the public microarray database arrayexpress http:// www.ebi.ac.uk/arrayexpress/ with the following accession numbers: microarray layout, p-umcu- ; gene expression data of mhv-infected mice, e-mexp- ; protocols for total rna isolation and mrna amplification, p-mexp- ; crna labeling, p-mexp- and p-mexp- ; hybridization and washing of slides, p-mexp- ; scanning of slides, p-mexp- ; data normalization, p-mexp- . innate immune recognition of viral infection type i interferons in host defense group coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition transcriptional profiling of acute cytopathic murine hepatitis virus infection in fibroblast-like cells mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies preferential infection of mature dendritic cells by mouse hepatitis virus strain jhm mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor mouse hepatitis coronavirus a nucleocapsid protein is a type i interferon antagonist severe acute respiratory syndrome coronavirus nsp suppresses host gene expression, including that of type i interferon, in infected cells control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon differential regulation of innate and adaptive immune responses in viral encephalitis inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels viral induction of central nervous system innate immune responses murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia protective effect of recombinant murine interferon beta against mouse hepatitis virus infection non-invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells interferome: the database of interferon regulated genes interferon and cytokine responses to sarscoronavirus infection cyclooxygenase activity is important for efficient replication of mouse hepatitis virus at an early stage of infection this work was supported by grants from the m.w. beijerinck virology fund, royal netherlands academy of arts and sciences, and the netherlands organization for scientific research (nwo-vidi- . . ) to c.a.m. de haan. we thank connie bergmann for advice and monique oostra, marne hagemeijer, and mijke vogels for stimulating discussions. the authors declare that they have no competing interests. mr and mjagk conducted all the experiments. mr wrote the manuscript. pjmr and camdeh coordinated the research efforts and assisted with writing the manuscript. all authors read and approved the final manuscript. key: cord- -p p enk authors: schlee, martin title: master sensors of pathogenic rna – rig-i like receptors date: - - journal: immunobiology doi: . /j.imbio. . . sha: doc_id: cord_uid: p p enk initiating the immune response to invading pathogens, the innate immune system is constituted of immune receptors (pattern recognition receptors, prr) that sense microbe-associated molecular patterns (mamps). detection of pathogens triggers intracellular defense mechanisms, such as the secretion of cytokines or chemokines to alarm neighboring cells and attract or activate immune cells. the innate immune response to viruses is mostly based on prrs that detect the unusual structure, modification or location of viral nucleic acids. most of the highly pathogenic and emerging viruses are rna genome-based viruses, which can give rise to zoonotic and epidemic diseases or cause viral hemorrhagic fever. as viral rna is located in the same compartment as host rna, prrs in the cytosol have to discriminate between viral and endogenous rna by virtue of their structure or modification. this challenging task is taken on by the homologous cytosolic dexd/h-box family helicases rig-i and mda , which control the innate immune response to most rna viruses. this review focuses on the molecular basis for rig-i like receptor (rlr) activation by synthetic and natural ligands and will discuss controversial ligand definitions. receptors of the innate immune system sense foreign molecules and structures such as the highly conserved microbe-associated molecular patterns (mamps) like sugars, lipids, proteins, or nucleic acids of bacteria, fungi or viruses . innate immune receptor stimulation by mamps triggers intracellular defense mechanisms and the induction of innate immune responses, including secretion of cytokines and chemokines, which lead to alarming of neighboring cells and attracting immune cells. most of the known highly pathogenic and emerging viruses are rna genome-based; they give rise to epidemic and zoonotic diseases (flu, foot-and-mouth disease) or cause viral hemorrhagic fever including yellow fever, dengue, lassa fever and ebola (bray ) . the recognition of foreign pathogenic rna, resulting in induction of type i interferon (ifn), the most important antiviral cytokine, is therefore highly critical. innate immune cells express the endosomal toll-like receptors (tlr) , and , which sense gu-rich rna and cpg-containing dna. tlr stimulation leads to secretion of type-i ifn, il- and assorted chemokines (diebold et al. ; heil et al. ; hemmi et al. ; hornung et al. hornung et al. , judge et al. ; krieg et al. ) , reviewed in (barchet et al. ; schlee et al. schlee et al. , . in contrast to tlr , and , tlr is expressed in more cell types (e.g. endothelial cells, fibroblasts, astrocytes) (barchet et al. ; schlee et al. ) and was found to detect long double-stranded rna (alexopoulou et al. ) . unlike tlrs, the rig-i-like receptors (rlr) rig-i, mda and lgp are present in the cytosol of all cell types. similar to tlrs, rig-i and mda induce type i ifn and chemokines (but no il ) upon activation by viral but also bacterial rna. while the endosomal rna detecting tlrs do contribute to antiviral immunity, rlrs are essential for the immune recognition of and response to most rna viruses ( fig. ) (gitlin et al. ; hornung et al. ; kato et al. ; rothenfusser et al. ; venkataraman et al. ; yoneyama et al. ). this review summarizes the biological role of and ligand recognition by rlr with special focus on rig-i, which represents the most broadly studied and understood receptor to date. rig-i (retinoic acid-inducible gene i) and mda (melanoma differentiation-associated gene- ) are closely related dexd/h-box helicase family proteins. they consist of an n-terminal tandem caspase activation and recruitment domain (card) fused to a dexd/h-box helicase domain (composed of hel , hel and hel i) and the c-terminal domain (ctd; previously called rd = repressor domain) (luo et al. ; saito et al. ; yoneyama et al. but no recognition by rig-i or mda in bm-dc or fibroblasts (zhou and perlman ) , recognition by mda , not rig-i in macrophages and microglia (roth-cross et al. ) . **evasion of rig-i recognition by nuclease end cleavage leaving monophosphate at the end of the viral genome (garcin et al. ; habjan et al. ). ***evasion of rig-i recognition via substitution of triphosphate by vpg protein at the end of the viral genome (hruby and roberts ; lee et al. ; rohayem et al. ). ****evasion of rig-i recognition by overhang at the end of the viral genome (marq et al. b ). weber, : papers contributing to the characterization of the real ligand structure in vivo are underlined. ) (fig. ) . stimulation of rig-i or mda by viral rna release the associated cards, which aggregate with k polyubiquitin chains to card tetramers and then bind and activate the adaptor molecule mavs (jiang et al. ; zeng et al. zeng et al. , . mavs (also known as ips- , cardif or visa (kawai et al. ; meylan et al. ; seth et al. ; xu et al. ) ) recruits tbk- , which phosphorylates irf to induce transcription of type-i ifn genes (doyle et al. ; fitzgerald et al. ; sharma et al. ) . at present, the interaction of rig-i with its corresponding ligand rna is far better understood and analyzed than ligand-receptor interactions of mda or lgp . both the ctd and helicase domain (which is no active rna helicase) possess rna binding sites, whereas solely the ctd harbors the critical binding pocket for the rna ligand, the features of which will be discussed card card hel i ctd hel hel hel i ctd hel hel rig-i/mda lgp fig. . domain structure of rig-i, mda and lgp . below. as of now, high-resolution structure studies have been performed of the ctd alone (cui et al. ; takahasi et al. ) , the ctd with ligand (lu et al. b; wang et al. ) , mouse rig-i sf domain + non-hydrolysable atp (civril et al. ) , human rig-i( cards) + ligand (jiang et al. ; luo et al. ) , whole duck rig-i(ligand free) and helicase + ligand (kowalinski et al. ). the current model of rig-i ligand interaction resulting from the above-mentioned studies was extensively discussed (kolakofsky et al. ) . briefly, the card of non-stimulated rig-i binds to the so-called hel- i domain within the helicase domain, mediating an auto-inhibited state. upon stimulation the ctd-bound rna interacts with hel- i, leading to dislocation of the cards, which now become accessible for downstream interactions (card multimerization, mavs interaction, type i ifn induction) as described above. a similar activation mechanism for mda is thinkable. but as long as a mda recognition motif has not been clearly defined (discussed below) it remains unclear if the mda ctd mediates ligand specificity. a recently described crystal structure of mda ( cards) with a synthetic mer dsrna (which is not a mda activating ligand) revealed that the mda ctd does not cap the terminus of the blunt dsrna but rather binds the internal rna duplex structure (wu et al. ) . this would provide a prerequisite for a putative mda head-to-tail arrangement in a filament structure with exposed cards, which was suggested to be the mavs activating structure (wu et al. ) . the third rlr family member lgp lacks cards and does not induce type i ifn. its putative function will be discussed in the next section. the role of lgp in the immune response against viruses is not entirely understood. the lgp ctd resembles the rig-i ctd, albeit with different requirements for ligand binding, which will be discussed below (li et al. b; murali et al. ; pippig et al. ). while lgp structurally shares a helicase and ctd, it lacks cards, suggesting a putative ligand sequestering role. indeed, initial reports suggested a pure immune suppressive function for lgp (komuro and horvath ; rothenfusser et al. ; saito et al. ; yoneyama et al. ). confusingly, a rig-i-suppressing activity was found to be independent of dsrna binding (li et al. b ). the parainfluenzavirus type v protein was reported to interact with lgp , to stabilize a lgp /rig-i complex and in this way to cooperatively inhibit induction by rig-i ligands (childs et al. ) . further studies on lgp -deficient mice revealed that lgp absence impairs the immune response to viruses that are mainly detected by mda , and can both impair or enhance rig-i mediated antiviral responses (pippig et al. ; satoh et al. ; venkataraman et al. ). suthar et al. ( ) confirmed that lgp contributed to sustained rlr signaling of ifn-␤ expression in myeloid cells during west nile virus (wnv) or dengue virus infection. additionally, they discovered a role for lgp in cd (+) t cell survival: lgp modulated the sensitivity of cd (+) t cells to cd ligand-mediated cell death through the control of cd expression during wnv or lymphocytic choriomeningitis virus infection. although the authors excluded a mda /lgp interaction to be responsible for the observed cd modulation, it remained unclear if the effect in cd (+) t cells occurs independently of rig-i. in conclusion, lgp appears to have a modulatory role in fine-tuning the innate immune response to viruses. mda -biological role, target pathogens and ligand structure mda recognizes long double stranded rna and contributes to or even dominates the immune response to double strand (dsrna) and positive strand rna [(+)ssrna] viruses ( fig. ) (fredericksen et al. ; gitlin et al. ; kato et al. ; loo et al. ; mccartney et al. ; melchjorsen et al. ; roth-cross et al. ; saito et al. ) . it is crucial for raising innate immune responses against picornaviruses, like theiler's virus or encephalomyocarditis virus (emcv), enteroviruses, saffold virus , human parechovirus , equine rhinitis a virus or the caliciviridae family member norovirus, which escape rig-i recognition (feng et al. ; gitlin et al. ; kato et al. ; mccartney et al. ; triantafilou et al. ) (fig. ) . at first view, mda appears to target virus types which are known to produce considerable amounts of dsrna during their replication cycle, including (+)ssrna, dsrna or dna viruses (mccartney et al. ; melchjorsen et al. ; pichlmair et al. ; roth-cross et al. ; targett-adams et al. ; weber et al. ) . correspondingly, two independent groups identified the double stranded replicative intermediates of (+)ssrna enteroviruses as mda -stimulating rna species (feng et al. ; triantafilou et al. ) . a crystal with mda ( card) binding to dsrna could be obtained (wu et al. ) . however, the concept of dsrna recognition by mda seems incomplete. (−)ssrna paramyxoviruses express the immune suppressive v protein which binds to and inhibits mda directly, suggesting that also (−)ssrna viruses (which were shown not to generate long double stranded rna ) produce mda ligands (andrejeva et al. ; childs et al. ; luthra et al. ; motz et al. ) . in light of the above-mentioned studies, it appears unexpected that many double stranded rna species do not activate mda . thus far only one artificial, albeit enzymatically generated, mda -stimulating ligand (polyinosinepolycytidylic acid = poly i:c) has been described. it is composed of annealed strands of long (> nt) rna polymers of inosins (polyi) and cytidines (polyc) (gitlin et al. ; kato et al. kato et al. , . most studies investigating the recognition of "long double stranded rna" (dsrna) in fact have used poly i:c. of note, poly i:c is a very particular "dsrna", as it was reported as the only copolymer among many other artificial dsrnas which was capable of inducing high amounts of type i ifn in mammalian cells (field et al. ) . the absence of well-defined mda ligands impairs systematic investigations of the mda -ligand interaction. although the ctd of mda binds blunt-ended dsrna (li et al. a; wu et al. ), mda is not activated by short dsrna and no contribution of the ctd in discriminating mda stimulating rna has been demonstrated (saito et al. ) . even though poly i:c also binds and can stimulate rig-i in certain cell lines and experimental settings in vitro (kato et al. ; yoneyama et al. ) , it is important to note that poly i:c indeed fails to induce ifn-alpha when injected intravenously into mda -deficient mice or transfected in vitro into mda -deficient peritoneal macrophages, dendritic cells or mefs (gitlin et al. ; kato et al. ) . by using poly i:c fragments of different sizes from rnase-iii digestion, kato and colleagues observed that mda was only stimulated by long poly i:c fragments. an alternative interpretation of these results would be that rnase-iii destroys certain secondary structures, which are required for recognition by mda . pichlmair and colleagues concluded from testing of gelfractionated rnas of vaccina virus-infected cells that it was not the double-strandedness of rna that accounted for mda stimulating activity, but rather other higher order rna structures in large rna containing complexes (pichlmair et al. ). luthra et al. discovered a mrna fragment from the ss(−)rna parainfluenza virus (piv ) that activated type-i ifn expression in a mda -dependent manner (luthra et al. ). since type i ifn induction by this rna required rnase l, the authors concluded that rnase l recognizes and processes viral mrna into a mda activating structure. although a -nt-long region critical for mda stimulation was identified, no specific features of a minimal recognition motif were found. the observation by züst and colleagues that deficiency of the viral cap n - o-methyltransferase in a type of (+)ssrna corona virus (murine hepatitis virus; mhv) provoked recognition of this mhv by mda and tlr (zust et al. ) suggested a enddependent rna recognition by mda . this finding would contrast the findings by luthra et al., who expressed the mda stimulatory mrna from a promoter, which supports normal capping (including n - o-methylation). however, binding assays documenting the interaction/non-interaction of the end of viral transcripts with mda were not performed. indirect effects were therefore not excluded. later studies on a n - o-methyltransferase-lacking west nile virus did not reveal a role of mda in enhanced immune recognition of non-methylated cap structures (szretter et al. ) , suggesting that n - o-methylation does not generally impair mda engagement. despite increasing amounts of high resolution crystal data on the rig-i/ligand interaction, the ligand requirements for rig-i stimulation are still controversial in the literature. by giving an overview of the history of the rig-i ligand definition, the following section aims to help the reader to understand how different read-out systems and ligand preparation methods could lead to conflicting interpretation of data. when rig-i was discovered as antiviral sensor by the fujita group (yoneyama et al. ) , the requirements of its rna ligand were not explored. rig-i was shown to bind to and to be stimulated by poly i:c when overexpressed in cell lines. at the same time, the group around john rossi, while developing sirnas against hiv, observed that all rnas generated by phage-polymerase in vitro transcription (kim et al. ) strongly induced type-i ifn in several human cell lines (hela, k , hek , jurkat, cem). by contrast, synthetic sirnas did not show any immune stimulatory effect in the same cell lines. dna template-dependent rna transcription occurs primer-independently from the -to the -end of rna. for this reason, rna transcripts of all known rna polymerases, including phage polymerase, possess a triphosphate at the end (banerjee ) . by using phosphatase or rnase t (removes the end pppg), kim et al. could show that the triphosphate was the crucial type-i ifn-inducing structural element of in vitro transcribed rnas which was absent in synthetic sirnas (kim et al. ). this finding prompted us to analyze the ifn-alpha inducing capacity of in vitro transcribed triphosphorylated rna (ppprna) in human blood cells . at this time, plasmacytoid dendritic cells (pdc) were presumed to be the principal type i ifn producing cells (cella et al. ; siegal et al. ) . they express tlr and , and secrete large amounts of ifn-alpha upon tlr stimulation with single or double stranded rna (hornung et al. ) . human monocytes express the rna-sensing endosomal tlr . however, tlr stimulation does not induce ifn-alpha secretion (barchet et al. ) . unexpectedly, ppprna induced high levels of ifn-alpha not only in pdc but also in human monocytes. therefore, ppprna represented the first agent that induced ifn-alpha in human monocytes at comparable quantities to human pdc ). removal of the triphosphate abrogated ifn-alpha induction by in vitro transcribed rna in monocytes but not in pdc . integration of nucleotides with modified bases (pseudouridine, -thio-uridine) or backbone modifications ( o-methyl at uridines) abolished ifn-alpha induction by ppprna both in pdc and monocytes. using murine rig-i/tlr deficient primary cells, rig-i was identified to be crucial for ppprna mediated ifn-alpha induction in myeloid immune cells, while tlr was essential for ifn-alpha induction in pdc . at the same time, pichlmair and colleagues reported phosphate-dependent type i ifn induction by influenza virus vrna, which contained no dsrna detectable by a dsrna specific antibody (pichlmair et al. ) . saito and colleagues suggested that rig-i detects (+)rna viruses (hcv) in a sequence-dependent manner (saito et al. ) . they screened the hcv genome for rig-i activating motifs by in vitro transcription of small domains of the hcv genome and analyzed the results for rig-i binding and -activation. a transcript from an nt u-or a-rich region nt downstream of the end showed an exceptional rig-i inducing activity. the presence of triphosphate at the end was essential for rig-i stimulation. of note, a polyu sequence elicited a similar ifn response as the polya sequence, which could be explained by a phenomenon to be discussed below. while developing phage-polymerase transcription-generated shrna without rig-i stimulating activity, gondai and colleagues found that end extension by more than one g abolished type i ifn induction (gondai et al. ) . the results by saito and gondai suggested a sequence-dependent rna recognition by rig-i. however, later experiments with defined synthetic rig-i ligands indicated that the work of both groups needs to be re-interpreted (schlee and hartmann ; schlee et al. ; schmidt et al. ). before triphosphate was identified as the crucial rna modification to induce rig-i activation, marques and colleagues observed that synthetic blunt ended dsrna oligonucleotides can stimulate rig-i ( fig. ) (marques et al. ) . the read-out system used consisted of the glioblastoma cell line t g, which was transfected with blunt ended or overhang-possessing sirnas. type i ifn activity in these cells was monitored or h after transfection by western blot analysis of the type i ifn induced protein ifit (p ), a very sensitive assay. in contrast to overhangs possessing sirna, blunt-ended sirna induced substantial ifit upregulation. similar results were obtained with mrc- cells. sirna-mediated knockdown of rig-i in t g indicated involvement of rig-i in the blunt-ended sirna-induced type i ifn response. by contrast, ht cells and hela cells did not respond to blunt-ended dsrna, but exhibited ifit induction after transfection of in vitro transcribed rna. the response to blunt dsrna could be restored in ht cells by priming with type i ifn. according to the described results, the rig-i stimulation motif was defined as double bluntended dsrna longer than bp. single blunt-ended sirnas were less active. overhangs were reported to permit detectable activity after h of stimulation while overhangs abolished activity ( fig. ) (marques et al. ) . further studies analyzed the physical interaction of recombinant full-length rig-i or card-or ctd deficient mutants with synthetic blunt-ended dsrna in comparison to in vitro transcribed single stranded ppprna (ivtppp-ssrna) (cui et al. ) . while full length rig-i was highly activated by ivtppp-ssrna, synthetic non-phosphorylated dsrna induced rig-i to a much weaker degree. unexpectedly, for rig-i lacking the card domain, dsrna and ivtppprna showed comparable atpase activity. by contrast, interaction studies using fluorescence anisotropy with recombinant rig-i protein or the recombinant rig-i ctd domain confirmed the requirement of the triphosphate for substantial interaction with full-length rig-i (cui et al. ). takahasi and colleagues reported a rig-i-dependent type i ifn response to synthetic -monophosphorylated and -monophosphorylated dsrna oligonucleotides in an ifn-beta-primed murine cell line and in ifnbeta-treated mouse embryonic fibroblasts (mef). the type i ifn response was monitored by ifnbeta promoter reporter assays and irf- dimerization ( fig. ) (takahasi et al. ) . in this setting nonmodified synthetic dsrna did not induce type i ifn (takahasi et al. ). in accordance with earlier studies (marques et al. ), overhangs at the -monophosphorylated end abrogated the type i ifn response, while overhangs were not tested ( fig. ) (takahasi et al. ) . by contrast, nt overhangs in monophosphorylated dsrna induced a type-i ifn response (no other end structures, e.g. blunt, overhang, were analyzed). unexpectedly, the authors found that monophosphorylation did not enforce rig-i binding of dsrna but increased rna stability in the cells, suggesting that increased rna stability is responsible for the particular rig-i stimulating activity (takahasi et al. ) . since the triphosphorylated end sequence of rnas generated by phage polymerase is restricted to a conserved consensus starting nucleotide g (or a followed by g), in vitro transcription is not applicable for screening of sequence variations at the end of triphosphorylated oligonucleotides. therefore, our group (+++) = activity was not compared in one figure established a method to generate synthetic triphosphorylated rnas (schlee et al. ) which is based on the standard cyclotriphosphate protocol of triphosphate synthesis (ludwig and eckstein ) . unexpectedly, synthetic single stranded triphosphorylated rna (ppp-ssrna) did not induce type-i ifn in human monocytes, while the "same" rna sequence generated by in vitro transcription (ivtppp-ssrna) was a strong type-i ifn inducer. sequencing of products from the ivtppp-ssrna transcription mix revealed the presence of complementary sequences and double stranded hairpin species, which were obviously generated by template-dependent rna transcription, a side activity of phage polymerase that had been reported earlier (cazenave and uhlenbeck ; triana-alonso et al. ) . transcription reaction conditions that did not allow synthesis of complementary rna abrogated rig-i activation by ivtppp-ssrna completely, suggesting that rig-i was not stimulated by the intended ssrna transcript but rather by side products (schlee et al. ). hence, hybridization of a complementary ssrna strand reconstituted rig-i stimulation by synthetic ppp-ssrna. optimal rig-i agonists appeared to be blunt ended, while nt overhangs at the triphosphate end impaired rig-i activation by more than % (fig. ) . overhangs of the triphosphorylated end were not tolerated, thus demonstrating that base pairing of the nucleotide carrying the triphosphate is essential for rig-i activation. the non-phosphorylated end structure had no substantial impact on rig-i stimulation, as long as the dsrna encompassed at least base pairs. small ( nt) bulge loops in the center of the sequence were tolerated. all four nucleotides constituted active triphosphorylated ends of the rig-i ligand. activity of pppa, pppg and pppu differed only slightly (a = g > u), whereas pppc induced around % less type i ifn. of note, this sequence-dependency that was observed is based on only one dsrna sequence (nacacacacacacacacacacuuu), and remains to be verified in another sequence context. according to the public databases, no genomic viral rnas (vrna) start with pppc but most start vrna with pppa. at the same time, by testing synthetic ppp-ssrna, schmidt and colleagues (fig. ) confirmed the importance of dsrna ). in disparity with our results, they observed that rig-i tolerates a nt overhang at the ppp bearing end for one tested sequences. by using phage polymerase-generated hairpin ppprna structures with intended ppp overhangs, they concluded that longer (> nt) overhangs in hairpin ppprnas are tolerated. however, this interpretation may be misleading since in vitro transcribed ppprna hairpins with overhangs (accurate transcription/identity was not analyzed by mass spectrometry) are likely to be contaminated with completely double stranded material (cazenave and uhlenbeck ; triana-alonso et al. ) . in our experience, one-time size fractionation of hairpin rna is not sufficient to exclude contamination of transcripts with small size differences completely. on the other hand, it has to be considered that hairpins are in equilibrium with their self-complementary duplex (nakano et al. ) , which is supposed to be a more active ligand than the monomeric hairpin (binder et al. ) . thus, small contaminations can cause substantial effects. schmidt et al. reported that a double stranded region of minimum bp length is sufficient for rig-i activation. in their test they included three different sizes of ppp-dsrna ( , and bp, blunt at the pppend). curiously, a mer ppp-dsrna induced a stronger type-i ifn response than a mer. as a direct comparison of the full mer duplex ppprna to the mer and mer is missing, the interpretation of the result remains difficult ). altogether, it remains unclear whether just any mer duplex ppp-dsrna sequence can activate rig-i. it has to be considered that the possibility of non-canonical base pairings of rna (e.g. g-u wobbles) provides manifold alternatives to form double stranded structures, all of which have to be kept in mind when claiming that rna structures are single stranded. to date only a small number of synthetic ppp-dsrna sequences have been analyzed (seven in our work (schlee et al. ), one in the work of schmidt et al. ( ) ). it is possible that a stabilizing nucleotide sequence next to the ppp end enables a tolerance of nt -ppp-overhangs. using highly purified in vitro transcribed ppprna from arenavirus sequences marq et al. ( b) confirmed the requirement of a base paired -ppp end of dsrna for rig-i activation and suggested that some arenaviruses and bunyaviruses use a prime and realign mechanism for genome synthesis, leading to overhangs in order to evade rig-i recognition (marq et al. b) . the need of a base-paired -ppp end of dsrna was also validated by the assembly of the rig-i ligand within the rig-i ctd ppp-dsrna binding cleft (lu et al. b; wang et al. ) . pppterminal base pairing supports an essential stacking interaction with a conserved phenylalanine residue in the rna binding cleft of the ctd. in addition, in contrast to a single base pair, which allows free rotation of the following sequence, the double strand assembly stabilizes the helix in a fixed optimum position for interaction of the adjoining phosphodiester backbone with the ctd and the helicase domain (fig. ) (kolakofsky et al. ) . in disparity to previous studies using type-i ifn-primed murine cells as a readout (takahasi et al. ) , no considerable type i ifn induction was observed after h when human monocytes were transfected with monophosphorylated dsrnas (schlee et al. ; schmidt et al. ). of note, schmidt and colleagues tested the same sequences, which were previously reported to induce type i ifn in type i ifn-primed mefs takahasi et al. ) . by contrast, in both studies monophosphorylated and nonphosphorylated dsrna induced a substantial atpase activity of rig-i protein at higher rna doses (schlee et al. ; schmidt et al. ). this indicates that rig-i activation observed by marques et al. ( ) and takahasi et al. ( ) could occur because of relatively high local rna concentrations in the cytosol. further factors, sensitizing the readout and leading to contradictory results are most likely due to the use of highly rig-i responsive cell lines (t g) or murine cells combined with long incubation times ( - h (marques et al. ) ), pre-activation by incubation with type i ifn (marques et al. ; takahasi et al. ) and sensitive detection methods (ifit western blot, ifn-beta reporter assay, irf- dimerization (marques et al. ; takahasi et al. ) ). using gel shift experiments with radioactive labeled ligand, vela et al. could calculate that the triphosphate moiety of dsrna enhanced binding to the ctd fold (vela et al. ) . the crystallization of rig-i ctd with oh-dsrna by lu and colleagues revealed that binding of oh-dsrna in the ctd rna binding cleft is possible (lu et al. a) . although the assembly for oh-dsrna resembles that of ppp-dsrna, the crystal data reveal that oh-dsrna binds in a different angle, and with other amino acid positions. habjan and colleagues observed that crimean-congo hemorrhagic fever virus (cchtv), hantaan virus (htnv), and borna disease virus (bdv) can prevent rig-i mediated detection of their genomes by a prime and realign mechanism and cleavage of the terminal base of their genomic rna leaving monophosphorylated ends (habjan et al. ) (fig. ) . in contrast to ppp ended genomic rna, the genomic rnas of cchtv, htnv or bdv with monophosphorylated ends (prna) failed to bind or to activate rig-i when transfected into hek cells. this laborious procedure of (−)ssrna viruses to generate monophosphorylated genomes to prevent rig-i recognition does not support the concept that prna is a preferred target structure for rig-i during viral infection. the occurrence of aberrant dsrna during phage polymerase in vitro transcription questions the data interpretation from earlier studies, which intended to identify rig-i recognition sequences based on experiments with in vitro transcribed rna. the observations of gondai and colleagues can easily be explained by the non-acceptance of ppp-overhang structures (gondai et al. ) : shrnas consisting of a rna hairpin with base paired pppends and a uu -overhang induced rig-i. extra gs at the -ppp end generated single stranded or mismatched ppp ends ( pppoverhang), which fail to stimulate rig-i. the finding that ppprna composed of polya or poly u stretches are equally potent rig-i inducers can be explained by the possibility that both complementary rna species are generated in the phage-polymerase transcription reaction that was intended to produce only one ssrna species and form a duplex (saito et al. ) . by using the same template as used by saito and colleagues for phage polymerase-mediated generation of the rig-i stimulating "poly a" rich sequence (which in fact is composed of starting gs and a), schmidt and colleagues did not receive any rig-i stimulating activity when utp or ctp were omitted in the transcription mix (saito et al. ; schmidt et al. ). by contrast, addition of utp and ctp yielded rig-i-activating rna as reported (saito et al. ; schmidt et al. ), suggesting that a double stranded polya/polyu rich sequence constitutes the rig-i activating agent. potent rig-i stimulation by this structure can be explained by the fact that poly a and poly u represent sequences that are not able to form stable secondary structures. absence of secondary structures facilitates the hybridization of complementary rnas at low temperature to uniform dsrna structures in comparison to mixed high melting g/c containing sequences. it is important to note that subgenomic (single stranded) rnas of hcv were reported to have monophosphorylated ends . since rig-i activation by polyu strictly depended on the presence of triphosphate (saito et al. ) , triphosphate-dependent rig-i stimulation in vivo can happen by recognition of replicative ppprna intermediates, which are generated during replication and are, in fact, double stranded (targett-adams et al. ). in summary, for rig-i recognition structural features appear to be more important than the sequences of candidate ppp-dsrna. rig-i possesses two rna binding domains (dech domain and ctd). pioneering studies involving crystal structure analysis or nmr from the hopfner and the fuijita lab identified a basic binding cleft within the ctd of rig-i (amino acids - ) as the crucial ppprna binding structure that determines ligand specificity (cui et al. ; kolakofsky et al. ; takahasi et al. ). as described above, ppp-dsrna bound to the ctd displaces autoinactivated cards from binding to the helicase domain, leading to liberation and activation of cards and downstream events that culminate in the induction of type i ifn [reviewed in kolakofsky et al. ( ) ]. using synthetic or highly purified in vitro transcribed triphosphorylated rna led to the resolution of the crystal structure of the rig-i ctd bound to mer ppp-dsrna palindromic sequences (lu et al. b; wang et al. ). the crystal structures revealed an rna binding basic binding cleft with highly conserved amino acids involved in binding of the terminal base pair, the triphosphate structure itself, and backbone phosphate (fig. ) . k and k (amino acid numbers in the text always refer to human rig-i sequence) are in proximity to the gamma phosphate of the triphosphate. however, conservative k and k mutations to alanine (a) impaired rig-i activation only at very low ligand concentrations (wang et al. ) , suggesting a minor role of this interaction in rig-i mediated recognition of viral rna. k , h and k were identified to interact with the beta phosphate of the triphosphate. substitution of k to a, which is also in contact with alpha phosphate, or double substitution of h and k to a, abrogated rig-i activation by ppp-dsrna. likewise substitution of k , being in contact with the alpha phosphate group, inhibited recognition of ppp-dsrna by rig-i. the side chain of k is in contact with either the backbone phosphate between n and n (wang et al. ) or n and n (lu et al. b ). since substitution of k to a abolishes rig-i activation completely (wang et al. ) this backbone phosphate interaction appears to be crucial for the detection of the ribose backbone of a dsrna structure. the contact of k to the phosphate between n and n (wang et al. ) or n and n , as reported in another study (lu et al. b ), could either depend on the incorporated oligonucleotide sequence (ppp-gacgcuagcguc (wang et al. ) or pppggcgcgcgcgcgcc (lu et al. b )) or the crystal packaging. in summary, both studies exhibited very similar ppprna/ctd structures. of note, the amino acids mediating rna ligand binding (h , k , k , k and k ) are % conserved in the rig-i sequences of vertebrates, highlighting the importance of these positions for rig-i-mediated rna-virus recognition. f is also essential for rig-i activation (wang et al. ) . it conducts the crucial stacking interaction with the terminal base pair (fig. ) , mediating the rig-i selectivity for base paired triphosphorylated rna. the stacking interaction is stabilized by base pairing of n . in addition, base pairing of the interacting base pair prevents free rotation of the following rna strand and in this way can assure appropriate assembly in the ctd structure (kolakofsky et al. ) , another argument for the strict necessity of a base pairing at the triphosphate-bearing end of dsrna to stimulate rig-i. as suggested by the comparison of rig-i ctd of different vertebrate species, f can only be substituted by the functionally related tyrosine (y). h and c mediate possibly important backbone interactions with the oh groups of the ribose of n and n , suggesting a strict discrimination of rna versus dna at those positions. the conclusion by lu and colleagues that rig-i can bind and simultaneously be stimulated by single-stranded ppprna has to be questioned, since in vitro transcribed rna (which usually contains ppp-dsrna species) was used for stimulation of cells (lu et al. b ). although the rig-i ctd appears to interact only with the ppp-bearing rna strand, helix formation should be still crucial for appropriate interaction of the rna with the contacting amino acid residues of h , k , h and c . marq and colleagues found that non-stimulatory ppp-dsrna (ppp-dsrna with ppp-overhang) can bind rig-i with comparable affinity as active ligands (complete ppp-dsrna) indicating the possibility of stimulatory ("productive") and non-stimulatory ("non-productive") ligand binding modes to rig-i (marq et al. a) . these data suggest that binding to rig-i is essential but not sufficient for activation of rig-i. two independent studies found that the presence of cards strongly reduce the tolerance for binding of oh-dsrna, thus represent a considerable contribution to the selective recognition triphosphorylated rna (cui et al. ; vela et al. ) . additionally, based on energetic parameters of the rig-i dsrna interaction, vela et al. suggested that a relatively low affinity of full-length rig-i for dsrna and, therefore, enhanced target specificity is mediated through antagonistic domain binding between helicase and ctd (vela et al. ) . the ctd of the rig-i inhibiting helicase lgp is closely related to the rig-i ctd (li et al. b; pippig et al. ). similar to rig-i, lgp was reported to preferentially bind to blunt ended dsrna (li et al. b; murali et al. ; pippig et al. ), albeit in a triphosphate independent manner (pippig et al. ). amino acids mediating the interaction with the terminal base pair and the ribose backbone (h rig-i , f rig-i , k rig-i , k rig-i = h lgp , w lgp , k lgp , k lgp ) are conserved or at least functionally related between the rig-i and the lgp ctd, while triphosphate-interacting amino acids (h , k , k and k ) are missing in the lgp ctd. h lgp , w lgp k lgp and k lgp were found to be involved in dsrna binding of the lgp ctd (li et al. b; pippig et al. ). conversely, the binding mode of oh-dsrna to lgp differed considerably from the binding mode of ppp-dsrna. confusingly, mutation of the amino acids in the lgp ctd corresponding to k rig-i (k lgp → e) and k rig-i (k lgp → e) led to loss of rna binding but did not impair lgp -mediated inhibition of rig-i activation, suggesting a ligand-independent rig-i inhibiting mechanism by lgp (li et al. b) . the rig-i ligand requirements for short dsrna described above is in contrast to the initial finding that rig-i can be activated by poly i:c (yoneyama et al. ), a dsrna polymer with monophosphates at the end (grunberg-manago ) . in order to characterize ligand structure motifs differentiating rig-i and mda recognition, kato and colleagues fractionated rnase iii-digested poly i:c (resulting in monophosphates and nt overhangs). while kb poly i:c fragments (high molecular weight) were preferentially detected by mda , fractions equal to bp or smaller were exclusively recognized by rig-i (kato et al. ) . binder et al. ( ) found an inverse correlation: in their experiments, dsrnas of different length ( - bp) were assessed for rig-i stimulating activity in rig-i transgenic huh . cells. they observed that molecular weight of ppp-dsrna positively correlated with rig-i activation. of note, binder et al. compared rig-i activation at relatively low concentration with constant molarity while kato et al. analyzed rig-i activity transfecting high concentrations ( g/ml) at constant mass concentration (binder et al. ; kato et al. ). binder et al. could reproduce the results of kato et al. when transfecting high amounts ( . pmol/well) of rna. nevertheless, both studies observed triphosphate independent activation of rig-i by very long (≥ bp) dsrna. length-dependent, end-independent rig-i activation cannot be explained with the current model of ctd-mediated recognition. binder et al. proposed an alternative ctd-independent recognition mechanism (cooperative multimerization of rig-i) in which binding of one rig-i molecule facilitates the binding of a second, etc. (binder et al. ) . in this case, binding can only occur in a helicase-dependent manner and would enable displacement of the cards, leading to initiation of the signaling cascade. based on multi-angle light scattering and sizeexclusion chromatography-coupled small-angle x-ray scattering of rig-i/ mer dsrna complexes, beckham e al. suggested that binding of a first rig-i molecule changes the rna structure, thus constructively influencing the binding of a second rig-i molecule (beckham et al. ) . however, if rig-i can be activated by long dsrna in a end independent manner, the question still remains why long doublestranded replicative rna intermediates purified from (+)ssrna picornaviruses, are not recognized by rig-i (feng et al. ) . rnase cleavage products-ligands for rig-i and mda ? malathi et al. ( ) observed that activated antiviral endoribonuclease rnase l generates small rna cleavage products from self-rna that induce type i ifn production (malathi et al. ) . in this study both, mda and rig-i were reported to contribute to recognition of small (< nt) rnase l cleavage products of total cellular rna. importantly, the type i ifn-induced rnase l digests single-stranded rna into rna products with -oh and -monophosphate groups. by reverse sequencing in a follow-up study, malathi et al. discovered hcv genome sequence-derived rnase-l cleavage products that bind to rig-i (malathi et al. ) . one of rig-i binding rna sequences was able to significantly activate rig-i in a monophosphate-dependent but triphosphate-independent manner. the putative structure of rig-i activating sequence included long (> bp) dsrna regions but also long (> nt) single stranded and ends. as the sequence was generated by in vitro transcription and not validated by mass spectrometry, it remains unclear whether the intended structure or co-purified side products from in vitro transcription are responsible for rig-i stimulation. nevertheless, the study indicates that special rna structures exist, which can stimulate rig-i in a monophosphate-dependent manner and that these kinds of structures can be generated by rnase l cleavage of rna virus genomes. of note, monophosphate-dependent rig-i stimulation was also reported earlier by the fujita group (takahasi et al. ) . at the same time, the akira lab and the medzhitov lab reported a tlr -independent type i ifn induction when dsdna was transfected into the cytosol of cells stetson and medzhitov ) . knock-down of mavs in t cells significantly reduced dsdna-induced type i ifn, suggesting a mavs-dependent pathway of dsdna recognition in these cells ). unexpectedly, mavs-deficient murine cells still responded to dsdna (sun et al. ) . importantly, the akira lab used a special type of dsdna, the heteropolymer dadt (a polymer of the alternating sequence at). cheng et al. observed that human cell lines (huh , hek ) secrete type i ifn in a mavs and rig-idependent manner after transfection of dadt but not plasmid dna (cheng et al. ). the dadt enigma was later solved by two independent groups (ablasser et al. ; chiu et al. ): ablasser et al. and chiu et al. discovered that dadt functions as a template for the endogenous rna polymerase iii, which generates triphosphorylated au-polymers in the cytosol (chiu et al. ). at-rich dna is in two ways a special kind of dna: first, rna polymerase iii prefers transcription of at-rich sequences; second, the resulting pppau-polymers are self-complementary and can easily anneal to ppp-dsrna. they therefore represent excellent rig-i target structures (ablasser et al. ). thus, it has to be kept in mind that "rig-i stimulating dna" needs to provide two features: first, the dna needs to be able to serve as a template for rna polymerase iii, second, the transcript has to form an appropriate rig-i ligand structure (base paired ppp end + dsrna > bp). most natural dsdna structures do not provide both features. some dna virus (e.g. epstein barr virus and adenovirus) encode for small rnas (eber and vai) under the control of a polymerase iii-driven promoter; these were observed to stimulate rig-i (ablasser et al. ; minamitani et al. ; samanta et al. ) . unlike murine cells and human monocytic cells, most human cell lines are not stimulated by dsdnas (e.g. plasmid dna or pcr products) other than dadt. this indicates the absence of a receptor in the cytosol, which can recognize dna directly in those cell lines (ablasser et al. ; cheng et al. ). in those cells, the rna polymerase iii/rig-i pathway constitutes the only alternative to detect cytosolic dsdna. since some dna viruses and facultative intracellular bacteria induced a mavs or rig-i-dependent type i ifn response in non-immune cells, it was suggested that in these cells the innate immune response to intracellular dsdna-containing pathogens can occur via stimulation of rig-i by pathogen-dna templated polymerase iii transcripts (ablasser et al. ; chiu et al. ). as will be summarized in the next section, intracellular bacteria were actually shown to release rna into the cytosol of cells, which is then recognized by rig-i (abdullah et al. ; hagmann et al. ) . in general, all rna polymerase-dependent transcribed rnas initially possess a triphosphorylated start nucleotide. posttranscriptional processing or modification such as capping are key features of mrna translation regulation in eucaryotes. in contrast to eucaryotes, one third of escherichia coli (bacterial) mrna remains triphosphorylated (bieger and nierlich ) . regulation of the phosphorylation status of bacterial mrna, which determines mrna decay (celesnik et al. ) , occurs via the pyrophosphatase rpph (deana et al. ). the triphosphate moiety prevents degradation of bacterial mrna by rnase-e (celesnik et al. ). importantly, pyrophosphatase rpph preferentially targets single-stranded triphosphorylated nucleotides as a substrate, consequently leaving triphosphorylated dsrna (deana et al. ). this conclusion is supported by the finding that terminal stem-loops prolong the lifetime of bacterial rnas (emory et al. ; mackie ) . thus, base-paired triphosphorylated rnas (ppp-dsrna), which are an excellent target structure for rig-i, appear to represent a characteristic molecular pattern (mamp) for bacteria. in a sirna-based approach, opitz et al. observed that legionella pneumophilae, a facultative intracellular gram-negative bacterium with type iv secretion system, raised a mavs-dependent but rig-ior mda -independent type-i ifn response in a human endothelial cell line (a ) (opitz et al. ) . the involvement of mavs in the l. pneumophilae induced immune response was later affirmed in experiments with mavs-and mda -deficient, anti-rig-i shrnaexpressing bone marrow-derived macrophage cell lines (monroe et al. ). in contrast to findings by opitz et al. ( ) , contribution of mda and rig-i to the type i ifn response was reported (monroe et al. ). while knock-down of rig-i substantially reduced the response to transfected purified bacterial rna, it remained an open question whether legionella-derived rna actually gains access to the cytosol of the host cell during infection. as mda appeared to not directly sense bacterial rna, the authors speculated about an indirect mechanism leading to activation of rig-i and mda . importantly, dna from l. pneumophilae did not induce type i ifn in hek cells, thus excluding rig-i-mediated recognition of rna polymerase-iii transcripts in the host cell, as previously suggested (chiu et al. ; monroe et al. ). recognition of bacterial rna by rig-i was also observed for the bacterium helicobacter pylori (rad et al. ). abdullah et al. discovered that the facultative intracellular bacterium listeria monocytogenes actively secrete small rnas via its seca secretion system resulting in strong rig-i activating activity (abdullah et al. ) . recently, we visualized translocation of bacterial rna from l. monocytogenes into the cytosol of several human cell lines (hagmann et al. ) . previously, l. monocytogenes was reported to induce type i ifn exclusively via the sting pathway, an adaptor/receptor sensing second messenger molecules or cyclic nucleic acids either directly secreted by l. monocytogenes (cyclic di-amp) or generated downstream of cytosolic dna recognition (gmp-amp) in mammalian cells (ishikawa et al. ; sauer et al. ; sun et al. ; woodward et al. ; wu et al. ; zhong et al. ) . we found that the l. monocytogenes-triggered type i ifn response is dependent on rig-i recognition when the sting pathway is not present in cells, this being the case in tested non-immune cells (hagmann et al. ) . in immune cells such as the human cell line thp- the stingdependent recognition pathway dominates the immune response to l. monocytogenes while rig-i appears to not play any role in recognition (hagmann et al. ) . in this respect, data for murine cells are conflicting: while some studies excluded involvement of rig-i/mavs in l. monocytogenes recognition in macrophages (soulat et al. ; sun et al. ) another study observed a substantial contribution of the rig-i pathway (abdullah et al. ) . culturing conditions of bacteria and murine cells may influence the amount of transferred bacterial ppprna and the balance between rig-i and the sting pathway in murine macrophages, thus leading to controversial findings. interestingly, li et al. found that rna of commensal bacteria is recognized in a mavs-dependent manner and that mavs in cells of non-hematopoietic origin plays a dominant role in preventing dss-induced colitis (li et al. ) , supporting that rig-i-inducing bacterial rna indeed has access to non-immune cells in vivo and mediates important effects. this finding explained the observation by wang et al. that rig-i deficient mice easily develop colitis (wang et al. ) . considering the fact that mice with a defect in the type i ifn pathway exhibit a strong resistance to listeria-induced pathogenesis, it remains to be determined if the observed rig-i-dependent recognition of bacterial rna contributes more to the pathogenicity or to the clearance of listeria (auerbuch et al. ; o'connell et al. ). detection of viral rna -which structure stimulates rig-i? in addition to dsrna viruses positive single-strand rna [(+)ssrna] viruses also generate cytosolic dsrna species, such as replicative dsrna intermediates, during their replication (feng et al. ; targett-adams et al. ; triantafilou et al. ; weber et al. ) . together with triphosphate, such rna species represent ideal rig-i target structures. since picornaviruses were shown not to activate rig-i during infection (gitlin et al. ; kato et al. ) it was presumed that picornaviruses (and caliciviruses) (fig. ) are able to escape rig-i recognition because instead of a triphosphate their rna genomes possess a peptide (vpg) linked via a tyrosine residue to monophosphate (hruby and roberts ; lee et al. ; rohayem et al. ) . in line with these findings, feng et al. observed that purified picorna virus rna stimulated mda but not rig-i (feng et al. ) . additionally and rather unexpectedly, picornaviruses were observed to degrade rig-i during infection. the apparent need to degrade rig-i indicates the occurrence of some rig-i stimulating activity, the identity of which has not been clarified so far (barral et al. ; papon et al. ). rig-i dominates the immune response to many (−)ssrna viruses (cardenas et al. ; habjan et al. ; hornung et al. ; kato et al. kato et al. , loo et al. ; plumet et al. ; yoneyama et al. ) . however, three out of four so far analyzed (−)ssrna viruses were described not to generate double-stranded rna species during infection (no dsrna: influenza; sendai virus, sev; la crosse virus, lacv; dsrna detectable: new castle disease virus, ndv) (pichlmair et al. ; takeuchi et al. ; weber et al. ) . this finding may seem conflicting at first, but can be explained by the fact that the dsrna-visualizing antibody used in these studies is only able to bind dsrna longer than bases (bonin et al. ) but rig-i can recognize ppp-dsrna ≥ bp. in general, the complementary and terminal sequences of all (−)ssrna viruses genomes bear the potential to hybridize to so-called dsrna panhandle structures with blunt ended triphosphorylated ends (fig. , left bottom) . a certain degree of self-complementarity cannot be avoided by (−)ssrna viruses since the same viral polymerase which recognizes its start sequence needs to start mrna transcription or replication from the (−)ssrna or (+)ssrna intermediates. for bunyaviridae, including lacv, it was validated by psoralen-crosslinking and electron microscopy that the -and -ends of the viral genome indeed constituted a panhandle structure in vivo (hewlett et al. ; raju and kolakofsky ) . the lacv panhandle consists of a blunt-ended - bp dsrna stretch with only a few mismatched nucleotides (raju and kolakofsky ) , thereby achieving almost all rig-i ligand requirements (schlee et al. ) without being detected by the pb rna-specific antibody. marq et al. suggested that for this reason some arenaviruses and bunyaviruses circumvent rig-i recognition by conducting a prime and realign mechanism which enables generation of overhangs in their genomes (marq et al. b ). as mentioned above, habjan et al. identified viruses (bornaviridae, bunyaviridae) , which escape rig-i recognition by combination of a prime and realign mechanism to terminal cleavage, leaving a monophosphorylated end (fig. , "rlr escape"). by using replication and translation blocking agents, applying enzymatic probing and visualization by superresolution microscopy, weber et al. confirmed that rig-i indeed recognizes viral capsids of lacv upon entry into the cell by binding to the panhandle structure (weber et al. ) . similar to lacv, influenza virus genomic ppprna (comprising segments with conserved ends) forms triphosphorylated, panhandle structures, albeit with quite short double stranded stretches (about bp), including mismatches/bulge loop structures (desselberger et al. ; hsu et al. ). this panhandle structure represents the site of rna transcription-initiation for the viral rna polymerase complex in the nucleus of the host cell (portela and digard ) . dauber et al. observed that the influenza panhandle is accessible for the antiviral double-stranded rnadependent protein kinase (pkr) after export from the nucleus into the cytosol of the host cell (dauber et al. ). since pkr and rig-i recognize comparable dsrna structures-short triphosphorylated dsrna (nallagatla et al. ; schlee et al. ), it is likely that rig-i also has access to the influenza panhandle structure in the cytosol. if the influenza panhandle is indeed sufficient to activate rig-i still remains to be verified using well-defined rig-i ligands. rehwinkel and colleagues generated mutated influenza rna polymerases which either selectively produce viral mrna or replicative genomic rna (rehwinkel et al. ) . using this tool, they confirmed that rig-i activation occurs exclusively by the genomic rna and not mrna of influenza (rehwinkel et al. ) . additionally, analysis of rig-i-bound viral rna from influenza infected cells revealed that only triphosphorylated viral genomic rna coprecipitated with rig-i. in contrast to lacv and influenza, viral particles of sendai virus (sev) and measles virus (mev) which belong to the group of mononegavirales contain predominantly linear nucleocapsids because encapsidation with structural proteins prevents formation of double stranded or panhandle structures (bhella et al. ; gerlier and lyles ; loney et al. ). but sev and vsv were found to produce defective interfering (di) viral genomes during replication (kolakofsky ; lazzarini et al. ; perrault and leavitt ) . three kinds of di genomes were identified: di genomes with internal deletions, promoter duplications with completely complementary - ends, and hairpin di genomes "snap back", consisting of a dsrna hairpin of - bp (fig. ) . "panhandle" and "snap back" di rnas should result in excellent rig-i ligands. indeed, strahle et al. could correlate sendai virusinduced rig-i activation with the occurrence of snap back di genomes (di-h ) which are generated during infection without encapsidation and thus are able to form panhandle structures in infected cells (strahle et al. (strahle et al. , . in concordance with these data, by applying a deep sequencing approach after purification of rna attached to rig-i from sendai virus infected cells, baum et al. determined preferred binding of di genomes to rig-i (baum et al. ). it appears plausible that the genomic rna should be targeted by rig-i, primarily. actually, purified genomic rna from most examined (−)ssrna viruses like rabies virus , lassa virus, nipah virus, rift valley fever virus (habjan et al. ) activated rig-i. nevertheless, rna purification by denaturating agents removes rna-interacting viral nuclear proteins, allowing formation of secondary rig-i-activating rna structures which do not occur in vivo (e.g. hybridization of (−)ssrna and (+)ssrna). therefore it is uncertain if these viral genomes would activate rig-i during infection. however, the phenomenon that (−)ssrna viruses possess mechanisms to modify their genomic end, preventing rig-i recognition (habjan et al. ; marq et al. b) , implies that the dsrna structures that (can) form at the end of viral genomes are critical for the viruses with rig-i escape mechanisms (arenaviridae, bunyaviridae, bornaviridae). in fact, the principle of detecting panhandle structures represents an intelligent strategy to detect (−)ssrna viruses because replication of (−)ssrna viruses necessitates highly conserved promoters at both ends of the genome, consequently yielding self-complementary and ends. the intolerance of vsv for artificially introduced extra nucleotides at the and ends of its (−)ssrna genome suggests lack of flexibility of (−)ssrna viruses concerning promoter sequences (pattnaik et al. ) . therefore, a blunt-ended double-stranded rna structure with triphosphate, the consequence of two conserved promoters, constitutes a negative strand virus-associated molecular pattern recognized by rig-i. correctly processed viral mrna is unlikely to be targeted by rig-i, because rna viruses possess mechanisms to cap their mrna either by viral-encoded capping enzymes or cap-snatching mechanisms (fechter and brownlee ) leading to loss of, or masking of the triphosphate. nevertheless, other studies suggest that type i ifn induction by mononegavirales (e.g. sev, vsv, mev) is associated with mrna transcription rather than replication (reviewed in gerlier and lyles ( ) ). e.g., in contrast to influenza, replicationdisabled measles virus that was still capable of transcription was observed to still activate a type i ifn response (plumet et al. ). leader and trailer rnas (lerna and trrna) represent the only non-capped triphosphorylated transcripts occurring during the transcription of the genome. plumet et al. and bitko et al. observed (−) ssrna virus lerna dependent stimulation of rig-i (bitko et al. ; plumet et al. ). however, the viral rna which forms together with the lerna the rig-i activating dsrna species still needs to be determined. gerlier and lyles proposed that l-trrna read-through transcripts (viral l-mrna extended by the trrna template) hybridized to ppp-trrna could also reconstitute a source of perfect blunt-ended rig-i target structures during the nonreplicative transcription phase (fig. , left panel) (gerlier and lyles ) . dna genome-based viruses like adenovirus, vaccinia virus and herpesviridae (herpes simplex virus, hsv) generate copious amounts of dsrna during their life cycle ); these dsrnas are most probably generated by overlapping converging transcription (jacobs and langland ) . while melchjorsen et al. reported mda but not rig-i-dependent type i ifn induction by hsv (melchjorsen et al. ) , xing et al. reported that the hsv encoded us protein interacts with and inhibits both rig-i and mda , indicating that hsv -derived rna is also recognized by rig-i (xing et al. ). finally, ebv and adenovirus-encoded small rna polymerase iii transcripts (eber and vai) were described to activate rig-i (ablasser et al. ; minamitani et al. ; samanta et al. ) . despite the presence of crystal data, conflicting results on ligand motif definition still obscure the understanding of mda pathogen rna detection. as demonstrated for paramyxoviruses, conclusions from studies testing immune responses in rig-i/mda knock-out cells to whole viruses can be misleading as long as the function of viral proteins is unknown: viruses appear to be recognized by rig-i because they express proteins efficiently inhibiting mda , and the opposite is also thinkable. by contrast, recent progress in the synthesis and purification of defined synthetic ligands, native preparation and visualization of natural rig-i ligands (nucleocapsids) and crystallization of rig-i/ligand complexes has greatly improved the understanding of the molecular basis of rig-idependent virus recognition. most of the studies agree on terminal base pair recognition by rig-i, which is dependent on the presence of triphosphate in a physiological relevant ligand concentration range. completely end base pair-independent rig-i recognition as proposed for long (≥ bp) dsrna needs further investigation since it can currently not be explained by the model derived from recent crystal data. naturally occurring rig-i ligands are double stranded triphosphorylated replicative intermediates of (+)ssrna viruses (formal scientific proof is missing though) and the triphosphorylated panhandle structures of genomes of (−)ssrna viruses with the exception of mononegavirales, which are able to avoid forming panhandle structures in vivo. other viruses avoid rig-i recognition by laborious procedures leading to overhangs or monophosphorylation. interestingly, in analogy to phage polymerases in vitro, rna polymerases of mononegavirales generate rig-i ligands due to an error-prone transcription process, therefore counteracting their -in principle -perfect camouflage against rig-i recognition. on the other hand, a successful virus has to protect its host from detrimental consequences of infection. the best examples are herpesviruses, the infection of which usually proceeds asymptomatic: > % of adults are, for example, infected by ebv, which persists lifelong in its host without causing detrimental symptoms, making ebv a very successful virus (thorley-lawson ) . ebv is known for its close interaction with the immune system, limiting its own infection in the host (thorley-lawson ) . thus immune recognition of the virus at a certain stage of its spread in the host should be evolutionarily favored and complete immune evasion not the goal of virus adaption. the author is listed as inventor on a patent application covering structures described in a manuscript, which is cited in this review (schlee et al. ). rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate recognition of doublestranded rna and activation of nf-kappab by toll-like receptor the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter mice lacking the type i interferon receptor are resistant to listeria monocytogenes '-terminal cap structure in eucaryotic messenger ribonucleic acids accessing the therapeutic potential of immunostimulatory nucleic acids rig-i is cleaved during picornavirus infection preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing conformational rearrangements of rig-i receptor on formation of a multiprotein:dsrna assembly conformational flexibility in recombinant measles virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-space helical reconstruction distribution of -triphosphate termini on the mrna of escherichia coli molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) cellular la protein shields nonsegmented negative-strand rna viral leader rna from rig-i and enhances virus growth by diverse mechanisms determination of preferential binding sites for anti-dsrna antibodies on double-stranded rna by scanning force microscopy highly pathogenic rna viral infections: challenges for antiviral research ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling rna template-directed rna synthesis by t rna polymerase initiation of rna decay in escherichia coli by pyrophosphate removal plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon double-stranded dna and doublestranded rna induce a common antiviral signaling pathway in human cells paramyxovirus v proteins interact with the rna helicase lgp to inhibit rig-i-dependent interferon induction mda- , but not rig-i, is a common target for paramyxovirus v proteins rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway the rig-i atpase domain structure reveals insights into atp-dependent antiviral signalling the c-terminal regulatory domain is the rna -triphosphate sensor of rig-i influenza b virus ribonucleoprotein is a potent activator of the antiviral kinase pkr the bacterial enzyme rpph triggers messenger rna degradation by pyrophosphate removal the and -terminal sequences of influenza a, b and c virus rna segments are highly conserved and show partial inverted complementarity viral infection switches non-plasmacytoid dendritic cells into high interferon producers irf mediates a tlr /tlr -specific antiviral gene program a -terminal stem-loop structure can stabilize mrna in escherichia coli recognition of mrna cap structures by viral and cellular proteins mda detects the double-stranded rna replicative form in picornavirus-infected cells inducers of interferon and host resistance. ii. multistranded synthetic polynucleotide complexes lps-tlr signaling to irf- / and nf-kappab involves the toll adapters tram and trif establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda signaling through ips- the ends of hantaan virus (bunyaviridae) rnas suggest a prime-and-realign mechanism for the initiation of rna synthesis interplay between innate immunity and negative-strand rna viruses: towards a rational model essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus shorthairpin rnas synthesized by t phage polymerase do not induce interferon polynucleotide phosphorylase: structure and mechanism of action processing of genome termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction rig-i detects triphosphorylated rna of listeria monocytogenes during infection in non-immune cells species-specific recognition of single-stranded rna via toll-like receptor and a toll-like receptor recognizes bacterial dna circular forms of uukuniemi virion rna: an electron microscopic study -triphosphate rna is the ligand for rig-i sequencespecific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr quantitative expression of toll-like receptor - mrna in cellular subsets of human peripheral blood mononuclear cells and sensitivity to cpg oligodeoxynucleotides encephalomyocarditis virus rna. iii. presence of a genome-associated protein genomic rnas of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle a toll-like receptor-independent antiviral response induced by double-stranded b-form dna sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity when two strands are better than one: the mediators and modulators of the cellular responses to double-stranded rna structural basis of rna recognition and activation by innate immune receptor rig-i ubiquitin-induced oligomerization of the rna sensors rig-i and mda activates antiviral innate immune response sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna cell type-specific involvement of rig-i in antiviral response length-dependent recognition of doublestranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene differential roles of mda and rig-i helicases in the recognition of rna viruses ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction interferon induction by sirnas and ssrnas synthesized by phage polymerase isolation and characterization of sendai virus di-rnas a structure-based model of rig-i activation rna-and virus-independent inhibition of antiviral signaling by rna helicase lgp structural basis for the activation of innate immune patternrecognition receptor rig-i by viral rna cpg motifs in bacterial dna trigger direct b-cell activation the origins of defective interfering particles of the negative-strand rna viruses a protein covalently linked to poliovirus genome rna murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda structural basis of double-stranded rna recognition by the rig-i like receptor mda the rig-i-like receptor lgp recognizes the termini of double-stranded rna mitochondrial antiviral signaling protein (mavs) monitors commensal bacteria and induces an immune response that prevents experimental colitis paramyxovirus ultrastructure and genome packaging: cryo-electron tomography of sendai virus distinct rig-i and mda signaling by rna viruses in innate immunity crystal structure of rig-i c-terminal domain bound to blunt-ended double-strand rna without triphosphate the structural basis of triphosphate double-stranded rna recognition by rig-i c-terminal domain rapid and efficient synthesis of nucleoside - -( -thiotriphosphates), -triphosphates and , -cyclophosphorothioates using -chloro- h- , , -benzodioxaphosphorin- -one structural insights into rna recognition by rig-i duplex rna activated atpases (dras): platforms for rna sensing, signaling and processing activation of ifn-&# ; expression by a viral mrna through rnase l and mda stabilization of circular rpst mrna demonstrates the -end dependence of rnase e action in vivo small self-rna generated by rnase l amplifies antiviral innate immunity rnase l releases a small rna from hcv rna that refolds into a potent pamp short doublestranded rnas with an overhanging ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys unpaired ppp-nucleotides, as found in arenavirus double-stranded rna panhandles, are not recognized by rig-i a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells mda- recognition of a murine norovirus early innate recognition of herpes simplex virus in human primary macrophages is mediated via the mda /mavsdependent and mda /mavs/rna polymerase iii-independent pathways cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus adenovirus virus-associated rnas induce type i interferon expression through a rig-i-mediated pathway identification of host cytosolic sensors and bacterial factors regulating the type i interferon response to legionella pneumophila paramyxovirus v proteins disrupt the fold of the rna sensor mda to inhibit antiviral signaling structure and function of lgp , a dex(d/h) helicase that regulates the innate immunity response influence of cationic molecules on the hairpin to duplex equilibria of selfcomplementary dna and rna oligonucleotides -triphosphate-dependent activation of pkr by rnas with short stem-loops type i interferon production enhances susceptibility to listeria monocytogenes infection legionella pneumophila induces ifnbeta in lung epithelial cells via ips- and irf , which also control bacterial replication the viral rna recognition sensor rig-i is degraded during encephalomyocarditis virus (emcv) infection infectious defective interfering particles of vsv from transcripts of a cdna clone inverted complementary terminal sequences in single-stranded rnas and snap-back rnas from vesicular stomatitis defective interfering particles rig-i-mediated antiviral responses to single-stranded rna bearing -phosphates activation of mda requires higher order rna structures generated during virus infection the regulatory domain of the rig-i family atpase lgp senses double-stranded rna cytosolic -triphosphate ended viral leader transcript of measles virus as activator of the rig i-mediated interferon response the influenza virus nucleoprotein: a multifunctional rna-binding protein pivotal to virus replication extracellular and intracellular pattern recognition receptors cooperate in the recognition of helicobacter pylori the ends of la crosse virus genome and antigenome rnas within nucleocapsids are base paired rig-i detects viral genomic rna during negative-strand rna virus infection protein-primed and de novo initiation of rna synthesis by norovirus dpol murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia the rna helicase lgp inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna epstein-barr virus-encoded small rna induces il- through rig-i-mediated irf- signaling lgp is a positive regulator of rig-i-and mda -mediated antiviral responses the nethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides beyond double-stranded rna-type i ifn induction by prna and other viral nucleic acids the chase for the rig-i ligand-recent advances sirna and isrna: two edges of one sword recognition of triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus -triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf triggering the interferon antiviral response through an ikk-related pathway the nature of the principal type interferon-producing cells in human blood cytoplasmic listeria monocytogenes stimulates ifn-beta synthesis without requiring the adapter protein mavs recognition of cytosolic dna activates an irf -dependent innate immune response sendai virus defective-interfering genomes and the activation of interferon-beta activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway the specific and essential role of mavs in antiviral innate immune responses the rig-i-like receptor lgp controls cd (+) t cell survival and fitness -o methylation of the viral mrna cap by west nile virus evades ifit -dependent and -independent mechanisms of host restriction in vivo analysis of the end structure of hcv subgenomic rna replicated in a huh cell line nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna pattern recognition receptors and inflammation visualization of double-stranded rna in cells supporting hepatitis c virus rna replication ebv the prototypical human tumor virus -just how bad is it? self-coded -extension of run-off transcripts produces aberrant products during in vitro transcription with t rna polymerase visualisation of direct interaction of mda and the dsrna replicative intermediate form of positive strand rna viruses the thermodynamic basis for viral rna detection by the rig-i innate immune sensor loss of dexd/h box rna helicase lgp manifests disparate antiviral responses structural and functional insights into -ppp rna pattern recognition by the innate immune receptor rig-i rig-i −/− mice develop colitis associated with downregulation of g alpha i doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses incoming rna virus nucleocapsids containing a -triphosphorylated genome activate rig-i and antiviral signaling c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna herpes simplex virus tegument protein us downmodulates the rlr signaling pathway via direct interaction with rig-i and mda- visa is an adapter protein required for virus-triggered ifn-beta signaling shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity key role of ubc and lysine- polyubiquitination in viral activation of irf the adaptor protein mita links virus-sensing receptors to irf transcription factor activation mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna ribose -omethylation provides a molecular signature for the distinction of self and nonself mrna dependent on the rna sensor mda i thank janos ludwig for illuminating discussions and cristina amparo hagmann for critically reading the manuscript. present work in the laboratory was supported by grants from the deutsche forschungsgemeinschaft (sfb and dfg research grants program schl / - ). key: cord- -gr kk w authors: baxter, victoria k.; griffin, diane e. title: interferon-gamma modulation of the local t cell response to alphavirus encephalomyelitis date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gr kk w infection of mice with sindbis virus (sinv) provides a model for examining the role of the immune response to alphavirus infection of the central nervous system (cns). interferon-gamma (ifn-γ) is an important component of this response, and we show that sinv-infected differentiated neurons respond to ifn-γ in vitro by induction of antiviral genes and suppression of virus replication. to determine the in vivo effects of ifn-γ on sinv clearance and t cell responses, c bl/ mice lacking ifn-γ or ifn-γ receptor- were compared to wild-type (wt) mice after intracranial sinv infection. in wt mice, ifn-γ was first produced in the cns by natural killer cells and then by cd (+) and cd (+) t cells. mice with impaired ifn-γ signaling initiated clearance of viral rna earlier than wt mice associated with cns entry of more granzyme b-producing cd (+) t cells. however, these mice established fewer cd (+) tissue-resident memory t (t(rm)) cells and were more likely to experience reactivation of viral rna synthesis late after infection. therefore, ifn-γ suppresses the local development of granzyme b-expressing cd (+) t cells and slows viral rna clearance but promotes cd (+) t(rm) cell establishment. response to viral infections of the central nervous system (cns) poses a unique problem for the immune system. the restrictive nature of the blood-brain barrier limits the ability of proteins and immune cells to enter into the brain and spinal cord in response to a virus infection [ ] . resident cells of the cns, particularly neurons, have a limited capacity to express major histocompatibility complex (mhc) molecules [ , ] . because neurons are a valuable but finite and minimally renewable cell population, preservation of neuronal function requires that infected neurons be allowed to survive, necessitating noncytolytic immune mechanisms to control virus infection. sindbis virus (sinv) is the prototypic member of the alphaviruses, a genus of enveloped, positive-sense, single-stranded rna viruses belonging to the togaviridae family [ ] . sinv is neurotropic in mice, and when mice are infected with a strain of sinv that does not cause fatal encephalomyelitis (e.g., te), clearance of infection from the cns occurs in three phases [ ] . in phase , during the first to days post infection (dpi), both infectious virus and viral rna increase rapidly, followed by clearance of infectious virus that occurs primarily through cooperative effects of anti-sinv antibody and the cytokine interferon-gamma (ifn-γ) [ ] [ ] [ ] [ ] . in phase , from approximately to days, infectious four to six week-old male and female wild-type c bl/ (wt) mice, mice deficient in ifn-γ receptor (ifngr −/− , strain b . s -ifngr tm agt /j, jackson labs), and mice deficient in ifn-γ (ifng −/− , strain b . s -ifng tm ts /j, jackson labs) were intracranially (ic) inoculated with plaque forming units (pfu) of the te strain of sinv diluted in µl pbs or µl pbs vehicle while under light isoflurane anesthesia. for fresh tissue collection, mice were euthanized by an overdose of isoflurane anesthesia, perfused with ice-cold pbs, and cervical lymph nodes (clns), brains, and/or spinal cords were collected. the johns hopkins university institutional animal care and use committee approved protocols for all studies performed (mo h approved / / ; mo h approved / / ). for preparation of lysates, dap- cells pooled from three wells were incubated on ice for min in ripa buffer ( mm tris, mm nacl, % sds, % np- , . % na-deoxycholate, mm edta) and centrifuged at , rpm for min. total protein was quantified by dc protein assay (bio-rad, hercules, ca, usa) using a bsa standard curve, and µg was boiled in x sds loading buffer ( . m tris (ph . ), % glycerol, % sds, . % bromophenol blue, % β-mercaptoethanol) for min. samples were run on a % sds-polyacrylamide gel electrophoresis (page) gel and transferred to a nitrocellulose membrane (bio-rad). membranes were blocked in tbs- . % tween- (tbst) + % milk for h at room temperature on a rocker and incubated overnight at • c on a rocker with primary antibody diluted in tbst + % bsa ( : rabbit polyclonal anti-nsp ; : , rabbit polyclonal nsv anti-sera; : , mouse monoclonal anti-β-actin, (millipore, burlington, ma, usa) [ , ] . membranes were incubated with secondary antibody diluted in tbst + % nonfat milk ( : horseradish peroxidase (hrp)-conjugated donkey anti-rabbit igg for nsp and poly-nsv; : hrp-conjugated sheep anti-mouse igg for β-actin, ge healthcare) for h on a rocker and developed using amersham ecl western blotting detection reagent (ge healthcare) according to manufacturer's instructions. rna was isolated from dap- cells using the qiagen rneasy (germantown, md, usa) or rneasy plus mini kit following the manufacturer's directions. for mouse cns tissue, right brain halves or whole spinal cords were homogenized in one ml qiazol in lysing matrix d tubes (mp biomedicals, irvine, ca, usa) at . m/s for s using a fastprep- homogenizer (mp biomedicals). the qiagen rneasy lipid tissue mini kit was used to isolate rna, and cdna was synthesized using a high capacity cdna reverse transcription kit with random primers (life technologies, carlsbad, ca, usa), and quantitative real-time pcr (qrt-pcr) was performed using taqman universal pcr master mix (roche, indianapolis, in, usa) on a fast real-time pcr system. sinv rna copies were measured using taqman probe ( - -carboxyfluorescein (fam)-cgcatacagacttccgcccagt- -carboxytetra-methylrhodamine (tamra)- , applied biosystems, waltham, ma, usa) with primers to the sinv e gene (forward, -tgggacgaagcggacgataa- ; reverse, -ctgctccgctttggtcgtat- ). sinv e copies were quantified using a standard curve made of ten-fold dilutions of a plasmid containing the sinv subgenomic region genes and normalized to endogenous rodent gapdh. mrna was measured using commercially available taqman gene expression assays (applied biosystems or integrated dna technologies, coralville, ia, usa), and relative quantification was performed by the ∆∆ct method using endogenous rodent gapdh mrna for normalization. single cell suspensions were made from clns, brains, and spinal cords pooled from to mice per strain per time point as previously described [ ] . briefly, clns were dissociated using gentlemacs c tubes and dissociator (miltenyi biotech, auburn, ca, usa), and red blood cells were lysed with an ammonium chloride solution (sigma-aldrich, st. louis, mo, usa or ebioscience, san diego, ca, usa). brains and spinal cords were dissociated in a solution containing collagenase d (roche) or collagenase iv (worthington labs, worthington, oh, usa) and dnase i (roche), and mononuclear cells were separated on a supplemented percoll gradient. live mononuclear cells were quantified using trypan blue exclusion. for degranulation assessment, - × cells were stimulated for h at • c with ng/ml of phorbol- -myristate -acetate (pma, sigma), µg/ml ionomycin (sigma), and antibody against cd a (clone ebio d b, ebioscience) in rpmi + % fbs. after h, monensin (golgistop, : , bd pharmingen, franklin lakes, nj, usa) was added to block cellular protein transport. for intracytoplasmic cytokine staining (ics), - × cells were stimulated for h at • c with ng/ml pma and µg/ml ionomycin in the presence of brefeldin a (golgiplug, bd pharmingen) in rpmi + % fbs. following live/dead and surface antibody staining (see above), cells were fixed for min using fixation/permeabilization solution from the bd cytofix/cytoperm kit. cells were stained for min on ice with monoclonal antibodies against ifn-γ (clone xmg . ), il- (clone b ), il- a (clone ebio b ), granzyme b (clone ngzb), granzyme a (clone gza- g . ), gm-csf (clone mpi- e ), and tnf-α (clone mp -xt ) from ebioscience or bd pharmingen diluted in bd perm/wash buffer. cells were resuspended in µl facs buffer and run on a bd facscanto ii cytometer using bd facsdiva software, version , and analyses were carried out using flowjo software, version . cells were characterized as follows: cd t cells (cd hi cd + cd + ), cd t cells , and tissue resident memory t (t rm ) cells (cd hi cd l − cd + ). all flow cytometry data are presented as averages of to independent experiments. following euthanasia, mice were perfused with ice-cold % paraformaldehyde (pfa), and brains and spinal columns were collected. brains were cut into three coronal sections using an adult mouse brain slicer (zivic instruments, pittsburgh, pa, usa), fixed overnight in % pfa, and embedded in paraffin. spinal columns were fixed overnight in % pfa, decalcified for h in a % sodium citrate/ % formic acid solution, cut to isolate the l -l spinal cord regions, and embedded in paraffin. viruses , , of µm tissue sections from to mice per group were stained with hematoxylin and eosin (h&e). brain slides were coded and sections scored as previously described [ ] using a - ( ) scale: , no detectable inflammation; , one or two small inflammatory foci; , moderate inflammatory foci in up to % of × magnification fields per section; , moderate to large inflammatory foci in greater than % of × magnification fields. an additional point was given for excessive parenchymal cellularity, allowing for a maximum score of . spinal cord slides were coded and sections scored using a modified - ( ) scale as previously described [ ] : , no detectable inflammation; , one to two small inflammatory foci; , greater than two inflammatory foci per spinal cord or moderate to marked inflammatory foci. an additional point was given for excessive parenchymal cellularity, allowing for a maximum score of . statistical analyses were performed using graphpad prism software. time-course studies were analyzed by two-way anova with bonferroni's or tukey's multiple comparison post-test for two group and three group comparisons, respectively. comparisons between three groups at a single time point were made using one-way anova with tukey's multiple comparisons post-test. a p value of < . was considered significant for all analyses. to elucidate a potential role for ifn-γ in virus clearance from neurons, immature cycling cap- olfactory neuronal cells and mature bipolar differentiated dap- cells [ ] were infected with sinv (moi = ). both cap- and dap- cells supported virus replication, with cap- cells producing higher peak titers than dap- cells and dying by hpi ( figure a ). dap- cells continued to produce virus through hpi and were used for subsequent studies of the effect of ifn-γ on virus replication. dap- cells infected with sinv (moi = ) were treated with u/ml rat recombinant ifn-γ h before infection, hpi, or at hpi. virus replicated in all treatment groups, with titers peaking at about hpi ( figure b ). dap- cells treated with ifn-γ prior to infection and at hpi had significantly decreased virus production compared to untreated cells at , , and hpi, with the greatest effect seen in pretreated cells, demonstrating the ability of neurons to develop an ifn-γ-induced antiviral response. treatment with ifn-γ at hpi did not alter virus production. to assess the effect of ifn-γ on virus replication after infection was established, production of sinv proteins was examined by immunoblot in dap- cells infected with sinv alone or treated with ifn-γ at hpi. in untreated sinv-infected cells, production of the nonstructural nsp protein and structural capsid protein reached high levels by hpi ( figure c ). production of the e and e structural glycoproteins (along with precursor to e , pe ) was evident by hpi and diminished by treatment with ifn-γ. these results show that ifn-γ can decrease the production of sinv by neurons. we next sought to determine the effects of ifn-γ on production of viral rna. sinv-infected dap- cells were treated with ifn-γ at hpi, and cell pellets collected to quantify viral rna by qrt-pcr. viral rna copies were comparable at hpi. copy number in untreated cells continued to rise, peaking at hpi, while sinv rna levels plateaued in treated cells and by hpi had begun to decrease ( figure d ). overall, viral rna synthesis was significantly inhibited by ifn-γ treatment compared to untreated dap- cells from to hpi. these studies show that ifn-γ signaling affects production and clearance of both infectious virus and viral rna from neurons in vitro. because ifn-γ facilitates virus clearance from neurons, we examined induction of antiviral genes by ifn-γ. mrna expression of representative antiviral isgs was examined by qrt-pcr in dap- cells infected with sinv (moi = ) and treated with u/ml ifn-γ at hpi. gbp ( figure a ) and irgm ( figure b ), two genes associated with autophagy [ ] , were highly expressed by sinvinfected dap- cells treated with ifn-γ, as were oasl ( figure c ), a member of the '- 'oligoadenylate/rnasel system [ ] , and rsad ( figure d ), which encodes viperin, a protein that interferes with assembly and release of many viruses [ ] . zc hav ( figure e ), which encodes zap/parp , a protein involved in viral rna degradation and induction of the innate immune response that restricts alphavirus and flavivirus replication [ , ] , was less highly upregulated. all of these genes required ifn-γ for induction and generally were not induced by sinv infection alone. because ifn-γ facilitates virus clearance from neurons, we examined induction of antiviral genes by ifn-γ. mrna expression of representative antiviral isgs was examined by qrt-pcr in dap- cells infected with sinv (moi = ) and treated with u/ml ifn-γ at hpi. gbp ( figure a ) and irgm ( figure b ), two genes associated with autophagy [ ] , were highly expressed by sinv-infected dap- cells treated with ifn-γ, as were oasl ( figure c ), a member of the '- 'oligoadenylate/rnasel system [ ] , and rsad ( figure d ), which encodes viperin, a protein that interferes with assembly and release of many viruses [ ] . zc hav ( figure e ), which encodes zap/parp , a protein involved in viral rna degradation and induction of the innate immune response that restricts alphavirus and flavivirus replication [ , ] , was less highly upregulated. all of these genes required ifn-γ for induction and generally were not induced by sinv infection alone. to characterize the time course and source of ifn-γ throughout the course of sinv infection of the cns, flow cytometry was used to characterize cells from the clns, the draining lymph nodes of the brain, and the brains of wt mice ( figure ). cd + t cells, cd + t cells, and nk cells were examined for cytokine production during phase ( and dpi), phase ( and dpi), and phase ( dpi) of infection. in the clns, few cells produced ifn-γ at any time ( figure a ,c). in the brain, nk cells were the predominant source of ifn-γ at dpi, both as percentage of live cells and absolute numbers ( figure b ,d). numbers of ifn-γ-producing cells in the brain peaked at dpi and were predominantly cd + t cells. as cd + t cells decreased, cd + t cells became comparable contributors by dpi. in phase of infection, fewer t cells were present in the brains and cd + and cd + t cells produced the majority of ifn-γ. therefore, nk cells produce most of the local cns ifn-γ early in the course of infection, but at later times, cd + , and especially cd + , t cells become the predominant source. we next characterized the percentages of each cell population producing ifn-γ. little change was seen in the clns, with less than % of cd + t cells, less than % of cd + t cells, and - % of nk cells producing ifn-γ at any time after infection ( figure e ). in the brain, the percentage of cd + and cd + t cells producing ifn-γ increased over the course of infection, going from approximately to % at and dpi to over % at , , and dpi ( figure f ). in contrast, the percentage of brain nk cells producing ifn-γ remained between to %, similar to that in the clns. to compare the relative amounts of ifn-γ produced by each cell type, median fluorescence intensities (mfis) were determined. in the clns, the mfi for ifn-γ remained low and did not change for any cell population ( figure g ,i). however, in the brain, the ifn-γ mfis for cd + and cd + t to characterize the time course and source of ifn-γ throughout the course of sinv infection of the cns, flow cytometry was used to characterize cells from the clns, the draining lymph nodes of the brain, and the brains of wt mice ( figure ). cd + t cells, cd + t cells, and nk cells were examined for cytokine production during phase ( and dpi), phase ( and dpi), and phase ( dpi) of infection. in the clns, few cells produced ifn-γ at any time ( figure a ,c). in the brain, nk cells were the predominant source of ifn-γ at dpi, both as percentage of live cells and absolute numbers ( figure b ,d). numbers of ifn-γ-producing cells in the brain peaked at dpi and were predominantly cd + t cells. as cd + t cells decreased, cd + t cells became comparable contributors by dpi. in phase of infection, fewer t cells were present in the brains and cd + and cd + t cells produced the majority of ifn-γ. therefore, nk cells produce most of the local cns ifn-γ early in the course of infection, but at later times, cd + , and especially cd + , t cells become the predominant source. cells, but not nk cells, increased over time, with the greatest increase occurring between and dpi ( figure h ,j). . also evaluated were the percentage of each cell type producing ifn-γ (e, f) and the mfi of ifn-γ for each cell type presented in graph form (g,h) and as histograms (i,j) (n = - pooled mice per time point from three independent experiments, except for data from dpi clns, which were from two independent experiments; data are presented as the mean ± sem). to determine the in vivo role of ifn-γ during sinv encephalomyelitis, the responses of mice deficient in ifn-γ (ifng −/− ) or in the α-chain of the ifn-γ receptor (ifngr −/− ) were compared to those of wt mice. to assess expression of antiviral isg mrnas for the five antiviral isgs previously selected for in vitro analysis of neuronal responses plus oas a, a protein associated with the '- 'oligoadenylate/rnase l system that is more active than oasl in mice [ ] were examined by qrt-pcr ( figure ) . gbp , irgm , oasl , rsad , and zc hav were up regulated in the brains and spinal cords of all mice during sinv infection, likely reflecting the overlap with isgs induced by type i ifn. however, expression of gbp ( figure a ) and irgm ( figure b ) at - dpi in the brain and spinal . also evaluated were the percentage of each cell type producing ifn-γ (e, f) and the mfi of ifn-γ for each cell type presented in graph form (g,h) and as histograms (i,j) (n = - pooled mice per time point from three independent experiments, except for data from dpi clns, which were from two independent experiments; data are presented as the mean ± sem). we next characterized the percentages of each cell population producing ifn-γ. little change was seen in the clns, with less than % of cd + t cells, less than % of cd + t cells, and - % of nk cells producing ifn-γ at any time after infection ( figure e ). in the brain, the percentage of cd + and cd + t cells producing ifn-γ increased over the course of infection, going from approximately to % at and dpi to over % at , , and dpi ( figure f ). in contrast, the percentage of brain nk cells producing ifn-γ remained between to %, similar to that in the clns. to compare the relative amounts of ifn-γ produced by each cell type, median fluorescence intensities (mfis) were determined. in the clns, the mfi for ifn-γ remained low and did not change for any cell population ( figure g ,i). however, in the brain, the ifn-γ mfis for cd + and cd + t cells, but not nk cells, increased over time, with the greatest increase occurring between and dpi ( figure h ,j). to determine the in vivo role of ifn-γ during sinv encephalomyelitis, the responses of mice deficient in ifn-γ (ifng −/− ) or in the α-chain of the ifn-γ receptor (ifngr −/− ) were compared to those of wt mice. to assess expression of antiviral isg mrnas for the five antiviral isgs previously selected for in vitro analysis of neuronal responses plus oas a, a protein associated with the '- 'oligoadenylate/rnase l system that is more active than oasl in mice [ ] were examined by qrt-pcr ( figure ) . gbp , irgm , oasl , rsad , and zc hav were up regulated in the brains and spinal cords of all mice during sinv infection, likely reflecting the overlap with isgs induced by type i ifn. however, expression of gbp ( figure a ) and irgm ( figure b ) at - dpi in the brain and spinal cord were higher in wt mice than ifngr −/− and ifng −/− mice. smaller differences in expression levels in brain were seen at day and for oasl ( figure c ) and at dpi oas a ( figure d ) and at dpi for rsad ( figure e ) and zc hav ( figure f ). these results show that while isgs are induced during sinv infection in the cns of mice with impaired ifn-γ signaling, expression is diminished compared to that of mice with intact ifn-γ signaling. viruses , , x for peer review of cord were higher in wt mice than ifngr −/− and ifng −/− mice. smaller differences in expression levels in brain were seen at day and for oasl ( figure c ) and at dpi oas a ( figure d ) and at dpi for rsad ( figure e ) and zc hav ( figure f ). these results show that while isgs are induced during sinv infection in the cns of mice with impaired ifn-γ signaling, expression is diminished compared to that of mice with intact ifn-γ signaling. clearance of viral rna from the cns was examined by quantifying sinv rna in brains and spinal cords of sinv-infected wt, ifng −/− and ifnrg −/− mice by qrt-pcr using primers specific for the e gene ( figure ). viral rna peaked at to dpi in the brains ( figure a ) and spinal cords ( figure b ), with comparable amounts for all mice. however, at dpi, viral rna levels were higher in the brains and at and dpi in the spinal cords of wt mice compared to ifngr −/− and ifng −/− mice. viral rna levels were comparable throughout phase of infection; however, at occasional times during phase , when viral rna had reached a low-level steady state, viral rna increased, especially in the spinal cords of ifng −/− mice. the results indicate that while impaired ifn-γ signaling results in delayed infectious virus clearance [ , , ] , initiation of viral rna clearance was accelerated, but the likelihood of reactivation of viral rna synthesis late after infection was increased. clearance of viral rna from the cns was examined by quantifying sinv rna in brains and spinal cords of sinv-infected wt, ifng −/− and ifnrg −/− mice by qrt-pcr using primers specific for the e gene ( figure ). viral rna peaked at to dpi in the brains ( figure a ) and spinal cords ( figure b) , with comparable amounts for all mice. however, at dpi, viral rna levels were higher in the brains and at and dpi in the spinal cords of wt mice compared to ifngr −/− and ifng −/− mice. viral rna levels were comparable throughout phase of infection; however, at occasional times during phase , when viral rna had reached a low-level steady state, viral rna increased, especially in the spinal cords of ifng −/− mice. the results indicate that while impaired ifn-γ signaling results in delayed infectious virus clearance [ , , ] , initiation of viral rna clearance was accelerated, but the likelihood of reactivation of viral rna synthesis late after infection was increased. to examine the effect of ifn-γ signaling on the inflammatory response to sinv infection, we first examined brain and spinal cord pathology of sinv-infected wt, ifng −/− and ifngr −/− mice. most of the pathological changes seen with alphavirus encephalomyelitis are associated with infiltration of immune cells [ , , , [ ] [ ] [ ] . brains and spinal cords were examined for histopathological changes and inflammation at , , , and dpi ( figure ). sporadically at dpi and consistently at dpi, brains from wt mice had diffuse bilateral dilation of the lateral ventricles, extending along the dorsal aspects of the hippocampi. this change was rarely seen in brains from ifngr −/− or ifng −/− mice, and when present, was less severe. compared to mock-infected control mice ( figure a ), both perivascular cuffing and infiltration of mononuclear cells into the parenchyma in the brain were present at dpi in all sinv-infected mice ( figure b ). in the spinal cord, compared to mock-infected controls ( figure c ), sinv-infected wt and ifngr −/− mice had more inflammation than ifng −/− mice ( figure d ). for quantitative comparison of the inflammation in brain ( figure e ) and spinal cord ( figure f ) at , , and dpi, a scoring system was used to evaluate coded h&e-stained sections [ , ] . inflammation steadily increased during the first week of infection, peaking at dpi in wt and ifng −/− mice and at dpi in ifngr −/− mice. in both tissues, inflammation scores were lower in ifng −/− mice than in wt or ifngr −/− mice. minimal inflammation present in the brain but not spinal cord of mock-infected mice was likely due to trauma from the ic inoculation. to examine the effect of ifn-γ signaling on the inflammatory response to sinv infection, we first examined brain and spinal cord pathology of sinv-infected wt, ifng −/− and ifngr −/− mice. most of the pathological changes seen with alphavirus encephalomyelitis are associated with infiltration of immune cells [ , , , [ ] [ ] [ ] . brains and spinal cords were examined for histopathological changes and inflammation at , , , and dpi ( figure ). sporadically at dpi and consistently at dpi, brains from wt mice had diffuse bilateral dilation of the lateral ventricles, extending along the dorsal aspects of the hippocampi. this change was rarely seen in brains from ifngr −/− or ifng −/− mice, and when present, was less severe. compared to mock-infected control mice ( figure a ), both perivascular cuffing and infiltration of mononuclear cells into the parenchyma in the brain were present at dpi in all sinv-infected mice ( figure b ). in the spinal cord, compared to mock-infected controls ( figure c ), sinv-infected wt and ifngr −/− mice had more inflammation than ifng −/− mice ( figure d ). for quantitative comparison of the inflammation in brain ( figure e ) and spinal cord ( figure f ) at , , and dpi, a scoring system was used to evaluate coded h&e-stained sections [ , ] . inflammation steadily increased during the first week of infection, peaking at dpi in wt and ifng −/− mice and at dpi in ifngr −/− mice. in both tissues, inflammation scores were lower in ifng −/− mice than in wt or ifngr −/− mice. minimal inflammation present in the brain but not spinal cord of mock-infected mice was likely due to trauma from the ic inoculation. , and ifng −/− (white bars) mice either mock-infected or sinv-infected at , or dpi were scored for inflammation using a four-point (brain) or three-point (spinal cord) system (data are presented as the mean score ± sem for - mice per strain per group; * p < . and ** p < . , tukey's multiple comparisons test). to determine the effects of ifn-γ signaling on immune cell subsets after sinv infection, cells isolated from the clns and brains of wt, ifng −/− and ifngr −/− mice were examined by flow cytometry. in the clns at dpi, there were more total mononuclear cells in wt mice than ifng −/− mice ( figure a ), but similar numbers at and dpi. neither the percentage nor absolute number of cd + t cells ( figure c ) or cd + t cells ( figure d ) in clns were affected by impaired ifn-γ signaling, with a general overall decrease from dpi to dpi. , and ifng −/− (white bars) mice either mock-infected or sinv-infected at , or dpi were scored for inflammation using a four-point (brain) or three-point (spinal cord) system (data are presented as the mean score ± sem for - mice per strain per group; * p < . and ** p < . , tukey's multiple comparisons test). to determine the effects of ifn-γ signaling on immune cell subsets after sinv infection, cells isolated from the clns and brains of wt, ifng −/− and ifngr −/− mice were examined by flow cytometry. in the clns at dpi, there were more total mononuclear cells in wt mice than ifng −/− mice ( figure a ), but similar numbers at and dpi. neither the percentage nor absolute number of cd + t cells ( figure c ) or cd + t cells ( figure d ) in clns were affected by impaired ifn-γ signaling, with a general overall decrease from dpi to dpi. in contrast, wt mice had more total mononuclear cells in the brain at dpi than ifngr −/− and ifng −/− mice ( figure b ). numbers of infiltrating cd + t cells peaked at dpi, while infiltration of cd + t cells occurred later with peaks at dpi. wt mice had more cd + t cells than ifng −/− mice at dpi ( figure e) , while both the percentage of cells and absolute numbers of cd + t cells were lower in brains of wt mice than ifngr −/− and ifng −/− mice at dpi ( figure f ), despite the fact that wt mice had more overall brain mononuclear cells at this time ( figure b ). therefore, while the absence of ifn-γ signaling did not affect the proliferation of t cells in clns in response to sinv infection, it did affect recruitment of these cells to the site of infection in the brain. because wt mice have more inflammation ( figure ) and more mononuclear cells ( figure b ), but fewer cd + t cells ( figure f ) in brain compared to ifngr −/− and ifng −/− mice at dpi, non-t cell immune cell populations were assessed. macrophages ( figure g ) and nk cells ( figure i ), both as a percentage of the total live mononuclear cell population and absolute numbers, were higher in wt mouse brains compared to mice with impaired ifn-γ signaling, while neutrophils tended to be higher in ifngr −/− mouse brains ( figure h) . microglial cells as a percentage of the total brain mononuclear cell population were higher in ifng −/− compared to wt and ifngr −/− mice ( figure j ), but absolute numbers of microglia were comparable. therefore, the larger number of mononuclear cells in the brains of wt mice at dpi compared to mice with impaired ifn-γ signaling was due to infiltration of more macrophages and nk cells. because impaired ifn-γ signaling altered the numbers of cd + and cd + t cells infiltrating the brain during sinv infection, we sought to determine the effector function of these cells. brain cd + t cells at dpi were characterized further by measuring production of signature cytokines and transcription factors ( figure a -d) and expression of cytokine ( figure e -h) and transcription factor ( figure i -l) mrnas associated with different t helper (th) subsets. as expected, ifn-γ (th cells) was not produced by cd + t cells in ifng −/− mice, and the percentage of cd + t cells producing ifn-γ did not differ between wt and ifngr −/− mice ( figure a ). the percentage of cd + t cells producing il- (th cells) was lower in ifng −/− mice than wt or ifngr −/− mice ( figure b ). although not significant, more cd + t cells in ifngr −/− mice produced il- a (th cells) compared to wt and ifng −/− mice ( figure c) , and the percentage of cd + t cells expressing both cd and foxp (tregs) trended lower in ifng −/− mice ( figure d ). these results show that ifn-γ signaling affects cd + t cell function during sinv infection, but impaired signaling has only a modest effect on th subset profile. to further evaluate th subsets, mrnas for four cytokines associated with specific th profiles were examined: il for th cells ( figure e ), il for th cells ( figure f ), il a for th cells (figure g ), and il for tregs ( figure h ). at dpi, mrna expression of il and il was lower in ifng −/− mice, and expression of il a was higher in ifngr −/− mice. il expression did not significantly differ among strains. expression of mrnas for transcription factors tbx for th cells (figure i ), gata for th cells ( figure j) , rorc for th cells (figure k ), and foxp for tregs ( figure l ) was also measured. while significant differences were found between strains at various time points, no major trends were identified. therefore, cd + t cell differentiation mostly affected ifng −/− mice with fewer th and treg cells and ifngr −/− mice with more th cells. modulation of th profiles during sinv infection by ifn-γ signaling warrants further examination. to examine how ifn-γ signaling affects cd + t cell production of effector proteins at dpi, ifn-γ, tnf-α, gm-csf, and granzyme b in cells from the brains of wt, ifngr −/− , and ifng −/− mice were examined by flow cytometry. cd + t cells from ifng −/− mice did not produce ifn-γ, and the percentage of cd + t cells producing ifn-γ did not differ between wt and ifngr −/− mice ( figure a ). the percentage of cd + t cells producing tnf-α was not significantly different between strains ( figure b ), but more cd + t cells from wt mice than ifng −/− mice produced gm-csf ( figure c) , while more cd + t cells from both ifngr −/− and ifng −/− mice than wt mice produced granzyme b ( figure d) . furthermore, the granzyme b mfi of cd + t cells from ifngr −/− mice was also higher than wt mice ( figure e ), indicating that individual cd + t cells in the brains of mice with impaired ifn-γ signaling produced more granzyme b than cells with intact ifn-γ signaling. expression of granzyme a ( figure f ) and granzyme b ( figure g ) mrnas in brain was lower in ifng −/− mice compared to wt and ifngr −/− mice, but expression differences among strains for granzyme k (figure h ), granzyme m ( figure i ), and perforin ( figure j ) were less pronounced. effector function of cd + t cells in the spinal cords of infected mice were similar to the brain in that ifn-γ-producing cd + t cells were not detected in ifng −/− mice and were comparable between wt and ifngr −/− mice ( figure k ). tnf-α ( figure l ) and gm-csf ( figure m ) production by cd + to examine how ifn-γ signaling affects cd + t cell production of effector proteins at dpi, ifn-γ, tnf-α, gm-csf, and granzyme b in cells from the brains of wt, ifngr −/− , and ifng −/− mice were examined by flow cytometry. cd + t cells from ifng −/− mice did not produce ifn-γ, and the percentage of cd + t cells producing ifn-γ did not differ between wt and ifngr −/− mice ( figure a ). the percentage of cd + t cells producing tnf-α was not significantly different between strains ( figure b ), but more cd + t cells from wt mice than ifng −/− mice produced gm-csf ( figure c) , while more cd + t cells from both ifngr −/− and ifng −/− mice than wt mice produced granzyme b ( figure d) . furthermore, the granzyme b mfi of cd + t cells from ifngr −/− mice was also higher than wt mice ( figure e ), indicating that individual cd + t cells in the brains of mice with impaired ifn-γ signaling produced more granzyme b than cells with intact ifn-γ signaling. expression of granzyme a ( figure f ) and granzyme b ( figure g ) mrnas in brain was lower in ifng −/− mice compared to wt and ifngr −/− mice, but expression differences among strains for granzyme k (figure h ), granzyme m ( figure i ), and perforin ( figure j ) were less pronounced. t cells were not different, but the percentage of cd + t cells producing granzyme b ( figure n ) was lower in wt mice than mice with impaired ifn-γ signaling. these findings indicate that ifn-γ signaling not only inhibits infiltration of cd + t cells into the cns, but also inhibits cd + t cell synthesis of granzyme b. effector function of cd + t cells in the spinal cords of infected mice were similar to the brain in that ifn-γ-producing cd + t cells were not detected in ifng −/− mice and were comparable between wt and ifngr −/− mice ( figure k ). tnf-α ( figure l ) and gm-csf ( figure m ) production by cd + t cells were not different, but the percentage of cd + t cells producing granzyme b ( figure n ) was lower in wt mice than mice with impaired ifn-γ signaling. these findings indicate that ifn-γ signaling not only inhibits infiltration of cd + t cells into the cns, but also inhibits cd + t cell synthesis of granzyme b. . . effect of ifn-γ signaling on cd + t cell and nk cell degranulation and cytotoxic function during sinv infection cd + t cells and nk cells primarily exert their cytolytic effects through secretion of cytotoxic granzymes that kill target cells [ ] . because infiltration of cd + t cells and nk cells into the brain were differentially regulated by ifn-γ signaling, the extent of degranulation, as identified by cd a expression [ ] , and granzyme production were examined in cells from the clns and brains of wt, ifngr −/− , and ifng −/− mice at dpi. in clns, cd a expression by cd + t cells was minimally affected by ifn-γ signaling ( figure a ). in brain, the percentage of cd + t cells expressing cd a was higher in ifng −/− mice than wt mice, but absolute numbers were comparable ( figure c ). in contrast, wt mice had more degranulated nk cells in the clns ( figure b ) and the brain ( figure d ) than either ifngr −/− or ifng −/− mice. therefore, ifn-γ signaling affected the cytotoxic function of local cns nk cells and cd + t cells during sinv infection, but in opposite directions. because long-term control of viral rna is likely required to prevent reactivation of virus production and relapse of clinical disease, immune cells remain in the cns long term after sinv infection [ ] . tissue-resident memory (trm) cells are a subset of memory t cells that do not circulate, but instead permanently remain at sites of infection [ ] . cd + trm cells were assessed in clns and brains in wt, ifngr −/− , and ifng −/− mice at , , and dpi to determine a role for ifn-γ in their development, maintenance, and survival ( figure a ). very few cd + or cd + trm cells were present in clns of sinv-infected mice at any time after infection ( figure b ). higher percentages of cd + t cells in the brain were trm cells, but they did not change over time or differ among mouse strains granzymes a and b are the primary granzymes involved in cytotoxicity of cd + t cells and nk cells [ ] , so we next identified the granzymes produced in the brain at dpi during sinv infection and the effect of ifn-γ signaling using boolean gating ( figure e,f) . approximately to % of cd + t cells and to % of nk cells in the brain produced both granzyme a and b. however, if producing only one granzyme, cd + t cells preferentially produced granzyme b, while nk cells preferentially produced granzyme a ( figure f ). additionally, granzyme production was lower in cd + t cells but higher in nk cells of wt mice compared to mice with impaired ifn-γ signaling. taken together, these data suggest that ifn-γ promotes nk cell cytotoxicity but suppresses the cytotoxic potential of cd + t cells in the brain during sinv infection. because long-term control of viral rna is likely required to prevent reactivation of virus production and relapse of clinical disease, immune cells remain in the cns long term after sinv infection [ ] . tissue-resident memory (t rm ) cells are a subset of memory t cells that do not circulate, but instead permanently remain at sites of infection [ ] . cd + t rm cells were assessed in clns and brains in wt, ifngr −/− , and ifng −/− mice at , , and dpi to determine a role for ifn-γ in their development, maintenance, and survival ( figure a ). very few cd + or cd + t rm cells were present in clns of sinv-infected mice at any time after infection ( figure b ). higher percentages of cd + t cells in the brain were t rm cells, but they did not change over time or differ among mouse strains ( figure c , left panel). however, percentages of cd + t rm cells in the brain increased over time ( figure c , right panel) and were more abundant in the brains of wt mice than ifngr −/− mice at dpi and ifng −/− mice at and dpi. the results suggest that ifn-γ signaling promotes the development of cd + t rm cells in the brain during sinv infection and affects their presence following infectious virus clearance. ifn-γ is an important determinant of the outcome of virus infections of the cns, with both induction of antiviral genes and regulation of the immune response to infection. previous studies have shown that ifn-γ facilitates clearance of infectious virus from spinal cord neurons during sinv infection [ ] , and in vitro, ifn-γ treatment inhibits replication of sinv in neurons through the jak/stat signaling pathway [ , ] ; however, the antiviral genes induced were not identified. the current study showed that mrnas for gbp and irgm gtpase proteins associated with autophagy were figure . effect of ifn-γ signaling on t rm cell populations. flow cytometry was used to examine t rm cell populations by gating, denoted by blue frames, around cd + cells (a) at , , and dpi in the clns (b) and brains (c) of wt (black bars), ifngr −/− (gray bars), and ifng −/− (white bars) mice, and results are presented as a percentage of cd + (left graphs) and cd + t cells (right graphs) (n = - pooled mice per strain per time point from three to four independent experiments; data are presented as the mean ± sem; * p < . , *** p < . by tukey's multiple comparisons test). ifn-γ is an important determinant of the outcome of virus infections of the cns, with both induction of antiviral genes and regulation of the immune response to infection. previous studies have shown that ifn-γ facilitates clearance of infectious virus from spinal cord neurons during sinv infection [ ] , and in vitro, ifn-γ treatment inhibits replication of sinv in neurons through the jak/stat signaling pathway [ , ] ; however, the antiviral genes induced were not identified. the current study showed that mrnas for gbp and irgm gtpase proteins associated with autophagy were highly induced by ifn-γ signaling in sinv-infected neurons as well as in the cns of sinv-infected mice where multiple cells may be responding to secreted ifn-γ. autophagy can decrease virus replication by destroying virus or viral replication components, including alphaviruses, or by delivering them to endosomes for toll-like receptor (tlr) induction of the innate immune response [ ] [ ] [ ] . rna viruses, including the alphavirus chikungunya virus (chikv), target irgm to promote virus replication [ , ] . another antiviral protein system highly induced by ifn-γ signaling during sinv infection of neurons was the - -oligoadenylate synthetase (oas) family, a pathway that affects replication of several neurotropic viruses, including rabies virus, canine distemper virus, west nile virus (wnv), and jev [ , [ ] [ ] [ ] . two other antiviral genes usually more robustly induced by type i than type ii ifns, rsad and zc hav , were induced later and to lesser extents. rsad -encoded viperin can impair budding of enveloped viruses [ ] [ ] [ ] and alphavirus and flavivirus replication and assembly [ ] [ ] [ ] [ ] . zc hav encodes zinc-finger antiviral protein (zap) or poly(adp-ribose) polymerase- (parp ), an rna-binding protein that recruits rna decay factors to degrade viral rna and inhibit viral rna translation [ ] [ ] [ ] [ ] [ ] . however, the specific role for these isgs in ifn-γ-mediated virus clearance from neurons will require further study. the current studies show that the in vivo role of ifn-γ in pathogenesis of sinv-induced encephalomyelitis is complex. although ifn-γ is important for clearance of infectious virus, particularly from spinal cord neurons [ , ] , and was sufficient for viral rna clearance in neurons in vitro, ifn-γ delayed clearance initiation of viral rna from both the brain and spinal cord in mice ( figure ), suggesting an extra neuronal effect that secondarily affects viral rna clearance. ifn-γ signaling improved recruitment of nk cells but differentially affected the recruitment of t cells, with more cd + t cells and fewer cd + t cells infiltrating the brains of sinv-infected wt mice than ifngr −/− or ifng −/− mice. this modification of the local t cell response potentially explains the differential initiation of rna clearance among mice with intact and impaired ifn-γ signaling, leading us to further evaluate the composition and functionality of these t cells. cd + t cells primarily exert their effects via cytokine secretion, which identify th subsets that influence the differentiation and activation of other immune cells [ ] . treg cells, defined by expression of both foxp and cd , tended to be lower in ifng −/− mouse brains, and mrna expression of the regulatory cytokine il- was significantly lower compared to wt and ifngr −/− mice at dpi. th cells have been associated with fatal encephalomyelitis and virus persistence during infection with several viruses, such as sinv nsv, tmev, and jhmv, especially in the absence of ifn-γ [ , [ ] [ ] [ ] . ifngr −/− mice expressed more il a mrna and trended toward increased il- a production by cd + t cells compared to wt and ifng −/− mice. similar results were seen in ifngr −/− mice infected with sinv nsv and suggest preferential expansion of th cells [ ] . neutrophils, also associated with a th profile [ ] , were also increased in brains of ifngr −/− mice. these results show that ifn-γ signaling affects cd + t cell function, although major differences in th profile were not seen, and reiterates the differences between mice lacking ifn-γ production and mice with impaired receptor function in the immune response to sinv infection. in our mouse model of alphavirus encephalomyelitis, fewer cd + t cells infiltrated the cns in wt mice with intact ifn-γ signaling, an effect similar to the suppression of the cd + t cell response to friend virus infection observed in association with lactate dehydrogenase virus-induced ifn-γ [ ] . as with nk cells, cd + t cells primarily exert their effector function against virus infections through cytotoxic activity and produce granzymes and perforin for delivery through granule exocytosis to activate caspases and induce target cell apoptosis [ ] . a clear role for nk cells in pathogenesis of alphavirus encephalomyelitis has not been identified [ , ] and, in the current studies, greater numbers of nk cells and granzyme a expression in wt mice did not offset the diminished cd + t cell response. specific targeting of infected cells by cd + t cells is achieved through direct contact with an infected cell expressing mhc class i molecules. neurons have a limited capacity for mhc class i expression [ , [ ] [ ] [ ] [ ] , but cd + t cells can directly engage virus-infected neurons [ ] , and clearance of wnv, jev, and lcmv from the cns is dependent on the granzyme/perforin pathway [ ] [ ] [ ] . perforin production is decreased in the brains of wt mice compared to ifngr −/− and ifng −/− mice during sinv nsv infection [ ] , and perforin-mediated effector function is impaired during jhmv infection [ ] however, ifn-γ promotes granzyme b production during experimental coronavirus retinopathy [ ] . therefore, the effect of ifn-γ on the granule exocytosis pathway during cns infection appears to be virus-specific, and the mechanisms by which ifn-γ influences granzyme production during sinv infection remain to be understood. it is becoming increasingly understood that granzymes have non-cytotoxic as well as cytotoxic roles [ , [ ] [ ] [ ] . for instance, cd + t cells can inhibit reactivation of hsv- in neurons via a non-cytolytic mechanism [ ] [ ] [ ] . mouse granzyme k induces macrophages to release il- β during lcmv infection [ ] , and granzyme substrates with direct antiviral activity, either viral proteins or host cell proteins essential for virus replication, have also been identified. granzymes a, h, and m cleave viral proteins important for replication of moloney mouse leukemia virus, adenovirus, and human cytomegalovirus [ ] [ ] [ ] . granzymes b and h both cleave the rna-binding protein la, which is important in viral rna metabolism for several viruses [ ] [ ] [ ] [ ] . hnrnp k, which modulates viral rna replication of chikv, enterovirus , dengue virus, and hiv- [ ] [ ] [ ] [ ] and interacts with sinv nsp and subgenomic mrna [ , ] , is a substrate for granzyme b. silencing of hnrnp k or disruption of hnrnp-vrna binding decreases sinv rna replication in vitro [ , ] . therefore, we postulate that the increased granzyme b produced by cd + t cells of ifngr −/− and ifng −/− cleaves one or more cellular proteins important for sinv rna replication or stability to accelerate noncytotoxic viral rna clearance from the brain and spinal cord. further studies regarding viral rna clearance, the noncytotoxic roles of granzymes during sinv infection, and regulation by ifn-γ, are warranted. persistence of viral rna after cns infection is common [ , , [ ] [ ] [ ] [ ] [ ] and presents the potential for virus reactivation and relapse of disease. indeed, during phase of infection, transient increases in viral rna were seen in sinv-infected mice, particularly in ifng −/− mice. therefore, prevention of virus reactivation is likely achieved through continued presence of immune cells at the previous site of infection [ , ] . over the course of infection, the percentage of cd + , but not cd + , t cells expressing cd , a marker for t rm cells in the brain, increased. as permanent residents at sites of previous infection, t rm cells provide a rapid response to pathogen reactivation [ ] [ ] [ ] . ifn-γ produced by cd + t cells is required for generating cd + t rm cells in the lung after influenza virus infection [ ] , and rapid clearance of lcmv by brain t rm cells depends on ifn-γ signaling and cytotoxic granule release [ ] . brains of sinv-infected mice defective in ifn-γ signaling had both fewer cd + t cells and cd + t rm cells than wt mice, indicating a need for ifn-γ to promote the residence of t rm cells in the brain after infection. in conclusion, ifn-γ signaling has both positive and negative effects on sinv clearance and control. ifn-γ facilitates infectious virus clearance through direct antiviral effects on infected neurons and inducing production of b cell-attracting chemokines that fosters local production of anti-sinv antibody [ ] . however, ifn-γ impairs viral rna clearance, possibly by suppressing the cd + t cell response in the cns and granzyme b production. finally, ifn-γ promotes the development of cd + t rm cells in the brain, likely helping prevent reactivation of persistent virus. better understanding of the complicated interplay between the virus and host immune system in inducing pathology and promoting virus clearance is critical to developing effective therapies. the immune response in viral encephalitis induction of mhc class i genes in neurons the role of cd (+) t cells and major histocompatibility complex class i expression in the central nervous system of mice infected with neurovirulent sindbis virus fields virology alphavirus-induced encephalomyelitis: antibody-secreting cells and viral clearance from the nervous system antibody-mediated clearance of alphavirus infection from neurons interferon-gamma-mediated site-specific clearance of alphavirus from cns neurons synergistic roles of antibody and interferon in noncytolytic clearance of sindbis virus from different regions of the central nervous system interferon gamma modulation of disease manifestation and the local antibody response to alphavirus encephalomyelitis persistence of viral rna in mouse brains after recovery from acute alphavirus encephalitis virus specificity and isotype expression of intraparenchymal antibody-secreting cells during sindbis virus encephalitis in mice interleukin modulation of pathogenic th cells during fatal alphavirus encephalomyelitis contribution of t cells to mortality in neurovirulent sindbis virus encephalomyelitis interleukin- modulation of virus clearance and disease in mice with alphaviral encephalomyelitis biologic functions of the ifn-gamma receptors noncytolytic clearance of sindbis virus infection from neurons by gamma interferon is dependent on jak/stat signaling the molecular cell biology of interferon-gamma and its receptor antiviral actions of interferons an olfactory sensory neuron line, odora, properly targets olfactory proteins and responds to odorants molecular basis of sindbis virus neurovirulence in mice the nsp macrodomain is important for sindbis virus replication in neurons and neurovirulence in mice basis of neurovirulence in sindbis virus encephalomyelitis of mice the inflammatory response to nonfatal sindbis virus infection of the nervous system is more severe in sjl than in balb/c mice and is associated with low levels of il- mrna and high levels of il- -producing cd + t cells glutamine antagonist-mediated immune suppression decreases pathology but delays virus clearance in mice during nonfatal alphavirus encephalomyelitis immunity-related gtpase m (irgm) proteins influence the localization of guanylate-binding protein (gbp ) by modulating macroautophagy characterization of the "- -"oligoadenylate synthetase ubiquitin-like family the antiviral response. microbes infect multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity inhibition of japanese encephalitis virus infection by the host zinc-finger antiviral protein mice deficient in interferon-gamma or interferon-gamma receptor have distinct inflammatory responses to acute viral encephalomyelitis distinct immune responses in resistant and susceptible strains of mice during neurovirulent alphavirus encephalomyelitis immunopathogenesis and immune modulation of venezuelan equine encephalitis virus-induced disease in the mouse protective effects of glutamine antagonist -diazo- -oxo-l-norleucine in mice with alphavirus encephalomyelitis cytotoxic and non-cytotoxic roles of the ctl/nk protease granzyme b sensitive and viable identification of antigen-specific cd + t cells by a flow cytometric assay for degranulation granzyme a activates another way to die tissue-resident memory t cells gamma interferon-dependent, noncytolytic clearance of sindbis virus infection from neurons in vitro autophagy protects against sindbis virus infection of the central nervous system eating oneself and uninvited guests: autophagy-related pathways in cellular defense autophagy-dependent viral recognition by plasmacytoid dendritic cells irgm is a common target of rna viruses that subvert the autophagy network capsid, membrane and ns are the major viral proteins involved in autophagy induced by japanese encephalitis virus common host genes are activated in mouse brain by japanese encephalitis and rabies viruses interferon-stimulated genes-mediators of the innate immune response during canine distemper virus infection oas b-dependent immune transcriptional profiles of west nile virus infection in the collaborative cross the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts hiv- infection of human macrophages directly induces viperin which inhibits viral production in vivo and in vitro studies on the antiviral activities of viperin against influenza h n virus infection the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein a viperin restricts chikungunya virus replication and pathology cell-type-and region-specific restriction of neurotropic flavivirus infection by viperin viperin inhibits classical swine fever virus replication by interacting with viral nonstructural a protein expression of the zinc-finger antiviral protein inhibits alphavirus replication inhibition of filovirus replication by the zinc finger antiviral protein zinc-finger antiviral protein inhibits hiv- infection by selectively targeting multiply spliced viral mrnas for degradation inhibition of hepatitis b virus replication by the host zinc finger antiviral protein zap's stress granule localization is correlated with its antiviral activity and induced by virus replication cd t cells: fates, functions, and faults th cells enhance viral persistence and inhibit t cell cytotoxicity in a model of chronic virus infection il- signal affects both protection and pathogenesis of virus-induced chronic cns demyelinating disease ifn-γ protects from lethal il- mediated viral encephalomyelitis independent of neutrophils interleukin- : a novel inflammatory cytokine that bridges innate and adaptive immunity negative impact of ifn-γ on early host immune responses to retroviral infection natural killer cells appear to play no role in the recovery of mice from sindbis virus infection nk cell-mediated immunopathology during an acute viral infection of the cns viral persistence in neurons explained by lack of major histocompatibility class i expression consequences of cytotoxic t lymphocyte interaction with major histocompatibility complex class i-expressing neurons in vivo regulation of class i mhc gene expression in the developing and mature cns by neural activity detailed in vivo analysis of interferon-gamma induced major histocompatibility complex expression in the central nervous system: astrocytes fail to express major histocompatibility complex class i and ii molecules rapid formation of extended processes and engagement of theiler's virus-infected neurons by cns-infiltrating cd t cells control of lymphocytic choriomeningitis virus infection in granzyme b deficient mice cd + t cells require perforin to clear west nile virus from infected neurons cytolytic effector pathways and ifn-γ help protect against japanese encephalitis perforin-mediated effector function within the central nervous system requires ifn-γ mediated mhc up-regulation the critical role of ifn-γ in experimental coronavirus retinopathy are proteinases functional molecules of t lymphocytes? human and mouse granzyme a induce a proinflammatory cytokine response granzyme b-dependent proteolysis acts as a switch to enhance the proinflammatory activity of il- α gamma interferon can block herpes simplex virus type reactivation from latency, even in the presence of late gene expression selective retention of herpes simplex virus-specific t cells in latently infected human trigeminal ganglia noncytotoxic lytic granule-mediated cd + t cell inhibition of hsv- reactivation from neuronal latency mouse granzyme k has pro-inflammatory potential a secretable serine proteinase with highly restricted specificity from cytolytic t lymphocytes inactivates retrovirus-associated reverse transcriptase granzyme h destroys the function of critical adenoviral proteins required for viral dna replication and granzyme b inhibition granzyme m targets host cell hnrnp k that is essential for human cytomegalovirus replication functional characterization of the interaction between human la and hepatitis b virus rna cleavage of la protein by granzyme h induces cytoplasmic translocation and interferes with la-mediated hcv-ires translational activity la protein binds the predicted loop structures in the ' non-coding region of japanese encephalitis virus genome: role in virus replication la protein can simultaneously bind to both -and -noncoding regions of japanese encephalitis virus genome heterogeneous nuclear ribonuclear protein k interacts with the enterovirus ' untranslated region and participates in virus replication hiv nef enhances tat-mediated viral transcription through a hnrnp-k-nucleated signaling complex vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein and is important for viral replication and release mapping of chikungunya virus interactions with host proteins identified nsp as a highly connected viral component heterogeneous nuclear ribonuclear protein k interacts with sindbis virus nonstructural proteins and viral subgenomic mrna identification and characterization of sindbis virus rna-host protein interactions magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of semliki forest virus long term intraparenchymal ig secretion after acute viral encephalitis in mice persistence of japanese encephalitis virus in the human nervous system long-term effects of semliki forest virus infection in the mouse central nervous system persistence of west nile virus in the central nervous system and periphery of mice t cells facilitate recovery from venezuelan equine encephalitis virus-induced encephalomyelitis in the absence of antibody persistence of virus-specific immune responses in the central nervous system of mice after west nile virus infection recruitment and retention of b cells in the central nervous system in response to alphavirus encephalomyelitis dendritic cell-induced memory t cell activation in nonlymphoid tissues long-lived epithelial immunity by tissue-resident memory t (trm) cells in the absence of persisting local antigen presentation brain-resident memory t cells represent an autonomous cytotoxic barrier to viral infection cd + t cell help guides formation of cd + lung-resident memory cd + t cells during influenza viral infection this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank kimberly schultz, kirsten kulcsar, lisa mangus, and lauren peiffer for helpful discussions and elizabeth troisi and jane yeh for technical assistance. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. the authors declare no conflicts of interest. key: cord- - qt eth authors: cao, liyan; ge, xuying; gao, yu; herrler, georg; ren, yudong; ren, xiaofeng; li, guangxing title: porcine epidemic diarrhea virus inhibits dsrna-induced interferon-β production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: qt eth background: the lack of optimal porcine cell lines has severely impeded the study and progress in elucidation of porcine epidemic diarrhea virus (pedv) pathogenesis. vero cell, an african green monkey kidney cell line, was often used to isolate and propagate pedv. nonetheless, the target cells of pedv in vivo are intestinal epithelial cells, during infection, intestinal epithelia would be damaged and resulted in digestive disorders. the immune functions of porcine epithelial cells and interactions with other immune cell populations display a number of differences compared to other species. type i interferon (ifn) plays an important role in antiviral immune response. limited reports showed that pedv could inhibit type i interferon production. in this study, porcine small intestinal epithelial cells (iecs), the target cells of pedv, were used as the infection model in vitro to identify the possible molecular mechanisms of pedv-inhibition ifn-β production. results: pedv not only failed to induce ifn-β expression, but also inhibited dsrna-mediated ifn-β production in iecs. as the key ifn-β transcription factors, we found that dsrna-induced activation of ifn regulatory factor (irf- ) was inhibited after pedv infection, but not nuclear factor-kappab (nf-κb). to identify the mechanism of pedv intervention with dsrna-mediated ifn-β expression more accurately, the role of individual molecules of rig-i signaling pathway were investigated. in the upstream of irf- , tank-binding kinase (tbk )-or inhibitor of κb kinase-ε (ikkε)-mediated ifn-β production was not blocked by pedv, while rig-i-and its adapter molecule ifn-β promoter stimulator (ips- )-mediated ifn-β production were completely inhibited after pedv infection. conclusion: taken together, our data demonstrated for the first time that pedv infection of its target cell line, iecs, inhibited dsrna-mediated ifn-β production by blocking the activation of ips- in rig-i-mediated pathway. our studies offered new visions in understanding of the interaction between pedv and host innate immune system. porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded, rna virus of coronaviridae family, which is the main etiological agent of severe diarrhea in pigs of all ages and fatality in neonates [ ] . outbreaks of porcine epidemic diarrhea (ped) have received extensive attention for the considerable economic losses to the swine industry worldwide. great advances have been made in elucidation of the molecular epidemiology, diagnosis, prevention, and treatment of ped [ ] . recently, coronavirus interaction with host innate immune system has been a hot research field. previous studies indicated that transmissible gastroenteritis virus (tgev) infection enhanced type i interferon expression and its protein modulated type i ifn expression [ , ] . for mouse hepatitis virus (mhv), ifn production among different cell populations varied due to their diverse susceptibility to this virus [ ] [ ] [ ] [ ] [ ] . furthermore, both severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) do not induce type i ifn (ifn-α/ β) activation [ ] [ ] [ ] . so far, limited reports showed that pedv could inhibit type i interferon production [ , ] . during viral infection and replication, the host innate immune response is the first line of defense; therefore, the ability of viruses to suppress or avoid this response is crucial for their pathogenic potential. ifn-α/β is an essential element of the host innate immune response against viral infections. double-stranded rna (dsrna), the replicative intermediate of most viruses, is a potent inducer of ifn-β, which is recognized as a pathogen-associated molecular pattern (pamp) by host pattern recognition receptors (prrs). two of major prrs, retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated gene (mda ) detect dsrna in the cytoplasm [ ] . following dsrna binding, rig-i and mda recruit corresponding adapter protein ifn-β promoter stimulator (ips- ) that, in turn, activate downstream signaling of tank-binding kinase (tbk ) and inhibitor of κb kinase-ε (ikkε) transduction, leading to the activation of transcription factor ifn regulatory factor (irf- ) and nuclear factor-kappab (nf-κb). activated irf- , and nf-κb bind to ifn-β enhancer and initiate ifn-β transcription [ ] . vero cell, an african green monkey kidney cell line, was often used to isolate and propagate pedv [ ] . however, it was often considered that vero cells might lack genetic component necessary for ifn production [ ] [ ] [ ] . porcine intestinal epithelial cells (iecs) are thought to the target cells of pedv, which play an important role in the activation of host immune responses by induction of key signaling molecules, including cytokines, surface molecules, and chemokines during microoganism invasion [ , ] . in the present study, to determine if pedv infection suppresses ifn-β activation, we chose iecs as an infection model to research the molecular mechanisms of pedv infection and the host antiviral innate immune response. our results clearly suggested that pedv prevented dsrna-induced ifn-β synthesis by blocking rig-i-mediated pathways. pedv failed to induce ifn-β expression and inhibited poly (i:c)-mediated ifn-β production in iecs type i ifns (ifn-α/β) are critical to the host antiviral innate immune response. however, there is no evidence suggesting that iecs produce type i ifns in response to pedv infection. previous studies have showed that pedv could be propagated in iecs [ , ,] . to confirm whether pedv infection could induce ifn-β production in iecs or not, we transiently cotransfected the ifn-β/ luciferase reporter plasmid (ifn-β-luc) and the renilla luciferase construct phrl-tk and then infected with pedv (at an moi of or . , respectively) or mockinfected for h. the cells were retransfected with μg of poly (i:c) as a positive inducer. as shown in fig. a , ifn-β luciferase activity enhanced markedly in positive controls, while it was almost not detected in pedv-infected iecs. in addition, ifn-β mrna expression was hardly detected in pedv-infected iecs similar to mock-infected group, however, it had significant expression in poly (i:c)-transfected group at the indicated times ( h and h, p < . ) (fig. b) . this result was consistent with the luciferase reporter assay. taken together, pedv infection of iecs did not induce ifn-β activation. increasing evidence showed that viruses not only inhibit the induction of type i ifns, but also block dsrnainduced production of type i ifns to escape the innate immune surveillance of the host [ ] [ ] [ ] . to identify whether pedv was able to inhibit dsrna-induced ifn-β production, ifn-β-luc was transfected into pedv-infected and uninfected cells, respectively. the cells were retransfected with or without poly (i:c) h later. as a result, activation of the ifn-β promoter decreased significantly in poly (i:c)-transfected, pedvinfected cells compared with mock-infected cells transfected with poly (i:c) (fig. c) . it showed pedv also inhibited poly (i:c)-mediated ifn-β induction. pedv impeded poly (i:c)-mediated activation of irf- , but not nf-κb irf- and nf-κb are two essential ifn-β transcription factors. in unstimulated cells, irf- is ubiquitously present in the cytoplasm as an inactive monomer, whereas nf-κb is present as a homodimer or heterodimer bound to the inhibitory proteins iκb in the cytoplasm [ ] . phosphorylation, which is a key step during irf- and nf-κb activation, in turn, leads to nuclear translocation. therefore, we evaluated whether pedv infection induced irf- and p activation by western blot analysis. following pedv infection, the whole cell extracts were prepared for the indicative times, in fig. a , irf- still existed in the cytoplasm, and the levels of irf- protein was almost equal with mock-infected cells, while the phosphorylation of irf- (p-irf- ) did not detect in the pedv infected cells in comparision to a obviously signal in poly (i:c)-transfected cells. on the contrary, compared with the amount of nf-κb subunit p in the cytoplasm, the phosphorylation of p (p-p ) increased with progression of pedv infection in the nucleus. meanwhile, the concentration of poly (i:c)induced p nuclear translocation was clearly increased. pedv n protein was also detected in pedv-infected cells. these data suggested that pedv did not induce activation of irf- , but nf-κb. we then used luciferase reporter assay system to determine whether irf- and nf-κb are linked with the inhibition of ifn-β production after pedv infection. as shown in fig. b , irf- luciferase activity was sharply decreased in pedv-infected cells, and poly (i:c)-induced irf- activation was also inhibited by pedv in comparison to a remarkably signal in poly (i:c)-transfected cells. however, in fig. c , compared with mock-infected to detect the activation of irf- and p after pedv infection, the cell extracts were prepared at the indicated times and subjected to western blot analysis with antibodies specific for irf- , p , p-irf- , p-p and pedv n mcab. anti-β-actin was included as a control for sample loading. these experiments were performed in duplicate. b and c iecs were infected or mock-infected with pedv at an moi of for h, and then cells were cotransfected with (prdiii-i) -luc (b) or pnf-κb-luc (c) and phrl-tk for additional h. cells were retransfected with poly (i:c) for h, harvested, and then subjected to a dual-luciferase assay. all data are expressed as means ± sd of independent experiments. **p < . as compared with poly (i:c). d irf -gfp fusion protein transfected with iecs and then infected with pedv at an moi of and mock-infected cells served as negative controls. h later, cells were retransfected with poly (i:c) (positive control) (c and d) or untransfected (a and b) for h. cells were fixed with % paraformaldehyde, permeabilized with . % triton x- , and stained by dapi (blue). cells were incubated with anti-pedv rabbit polyclonal antibody (red) and tritc-labeled goat anti-rabbit secondary antibody, then analyzed for fluorescence by confocal microscopy. magnification, × (leica, wetzlar, germany) (see figure on previous page.) fig. pedv does not induce ifn-β production and inhibits poly (i:c)-mediated ifn-β induction. a iecs were cotransfected with ifn-β-luc and phrl-tk, then infected with pedv at an moi of and . for h. cells were retransfected with poly (i:c) as a positive control. after h, the cells were harvested and subjected to a dual-luciferase assay. b iecs were infected with pedv at an moi of , mock-infected as a negative control, or transfected with poly (i:c) as a positive control. at the indicated time points, total rna was extracted and ifn-β and β-actin mrna were subjected to real-time pcr. rna expression levels were normalized to β-actin. c in contrast to a, iecs were first mock-infected or infected with pedv at an moi of for h and then cotransfected with ifn-β-luc and phrl-tk for h. cells were retransfected with or without poly (i:c) for an addition h, harvested, and then subjected to a dual-luciferase assay. all data are expressed as means ± sd of independent experiments. *p < . ; **p < . as compared with poly (i:c) cells, nf-κb luciferase activity significantly enhanced both in pedv-infected and poly (i:c)-transfected cells. in addition, we found that poly (i:c)-induced activation of nf-κb was not blocked by pedv. to further identify pedv-inhibited poly (i:c)-mediated activation of irf- , confocal microscopy assay was used. as a result, irf -gfp remained in the cytoplasm of both mockinfected ( fig. d. a) and pedv-infected (fig. d . b) iecs compared with poly (i:c) controls, in which clear translocation to the nucleus was observed (fig. d. d) . furthermore, pedv could block poly (i:c)-mediated irf- nucleus migration (fig. d. c) . taken together, our date clearly implied that pedv impeded dsrna-mediated ifn-β transcription primary by interfering with irf- activation, but not nf-κb. pedv failed to block tbk /ikkε activity tbk and ikkε are essential kinases for the irf- activation [ ] . in order to ascertain whether pedv inhibited poly (i:c)-induced irf- activation by impeding tbk /ikkε kinase activity, we cotransfected plasmids expressing tbk /ikkε kinase and a plasmid encoding the ifn-β promoter of the luciferase reporter into infected or mock-infected iecs and retransfected the cells with or without poly (i:c) at h.p.i. as show in fig. , tbk /ikkε overexpression increased ifn-β promoter activity in both infected and mock-infected iecs, and obviously upregulation was detected in iecs transfected with poly (i:c), suggesting that pedv failed to block tbk /ikkε activity. however, poly (i:c)-induced ifn-β promoter activity in iecs overexpression of tbk / ikkε plasmids was significantly inhibited by pedv. it showed that pedv interrupting dsrna-induced ifn-β production should localize upstream from tbk /ikkε. it is possible that pedv blocks poly (i:c)-mediated ifnβ production by suppression of the individual molecules upstream of tbk /ikkε in rig-i signaling pathway. to explore this possibility, mock-and pedv-infected iecs were cotransfected with ips- expression plasmid and ifn-β promoter luciferase reporter plasmid, respectively. as shown in fig. a , overexpression of ips- in iecs could enhance ifn-β luciferase activity in mock-infected cells, but it was completely inhibited in pedv-infected cells. for the poly (i:c) transfection experiments, there appeared significant restriction of ifn-β luciferase expression in pedv-infected cells compared with that of mock-infected cells. these date indicated that pedv interacted with ips- to block poly (i:c)-mediated ifn-β transcription. ips- is an adapter molecule of rig-i, the data showed that pedv blocked ips- -induced ifn-β production in dsrna signaling pathway, thus, we speculated that rig-i-induced ifn-β production in this signaling pathway was also inhibited. to verify it, mock-and pedvinfected iecs were transfected with rig-i expression and ifn-β reporter plasmids. the results showed that ifn-β luciferase activity was markedly increased in iecs overexpressing rig-i, but was completely inhibited by pedv infection. and the ifn-β reporter signal could be observed in iecs stimulated with poly (i:c), while this signal could be sharply reduced in rig-itransfected, poly (i:c)-stimulated and pedv-infected iecs (fig. b) . in summary, the findings of the present study suggested that pedv-infection in iecs inhibits dsrna-induced ifn-β induction by interfering with irf- activity associated with rig-i-mediated signaling pathway. the target interaction molecules of pedv intervention of dsrnainduced ifn-β production primarily was ips- . however, as a limitation to this study, host cells may inhibit or activate multiple signaling pathways simultaneously in response to exogenous stimulus, and some other transcription factors may have been blocked or activated in this process. here, we only addressed the mechanisms of pedv-induced inhibition of ifn-β production in relation to the molecules of rig-i signaling pathways in vitro. further studies are needed. overall, elucidation of the cells were harvested and subjected to a dual-luciferase assay. data were analyzed and the ratio of firefly luciferase expression to renilla luciferase activity was shown. all data are expressed as means ± sd of independent experiments. **p < . compared with pedv-infected, expression plasmids or vector-transfected control influence of pedv evasion of the host innate immune response will aid in the development of antiviral agents to prevent the spread of pedv during the early infection phase. the african green monkey kidney cell line veroe and swine small intestine epithelial cells (iecs) [ , ] were respectively cultured in dulbecco's modified eagle's medium (dmem) and dulbecco's modified eagle's f ham medium (dmem-f ) supplemented with % fetal bovine serum at °c in a humidified atmosphere of % co . pedv strain cv was propagated in veroe cells in dmem containing . μg/ml of trypsin. poly (i:c) was purchased as a sodium salt (sigma-alorch, saint louis, mo, usa) and dissolved in water to obtain a stock solution of mg/ml. the dual-luciferase® reporter assay system was purchased from promega corporation (madison, wi, usa) and monoclonal anti-β-actin antibody was purchased from sigma-aldrich (st. louis, mo, usa). anti-irf- , anti-p anti-p-irf- and anti-p-p rabbit polyclonal antibodies and secondary horseradish peroxidase (hrp)-conjugated anti-rabbit igg were purchased from cell signaling technology, inc. (beverly, ma, usa). rhodamine isothiocyanate (tritc)-labeled goat anti-rabbit igg were purchased from the zhongshan company (beijing, china). anti-pedv rabbit polyclonal antibodies and anti-pedv n protein monoclonal antibody (mcab) were prepared in our laboratory, which could specifically react with pedv. the plasmids ifn-β-luc for ifn-β, prd (iii-i) -luc for irf- , and pnf-κb-luc for nf-κb were kindly donated by dr. shaobo xiao (huazhong agricultural university, wuhan, hubei province, china) [ ] . the pef-bos empty vector and pef-flag-rig-i recombinant expression plasmid were kindly provided by t. fujita (tokyo metropolitan institute of medical science, tokyo, japan) [ ] . the pef-bos-flag-trif, pcdna -flag-ikkε and pcdna -flag-tbk recombinant expression plasmids, and the pcdna empty vector, and the conjugate irf -green fluorescence protein (gfp) expression construct were kindly provided by k. fitzgerald (university of massachusetts medical school, worcester, ma, usa) [ ] . the porcine ips- (ncbi accession no: eu . ) gene was cloned from porcine kidney cells by reverse transcription polymerase chain reaction (rt-pcr) using the specific primer pair. the ips- primers were ′-ccgggtaccaccatgacgtttgccgaggacaa- ′ and ′-tttctcgagtcactggggcaggcgccgcc- ′. porcine ips- was inserted into pcdna . (+) using the restriction enzymes kpni and xhoi. cells were harvested and luciferase activity was analyzed using a dual-luciferase assay. all data are expressed as means ± sd of independent experiments. **p < . compared with pedv-infected, expression plasmids or vector-transfected control when the cells reached %- % confluence. the cells were then infected or mock-infected with pedv for h. cells were retransfected with or without poly (i:c) ( . μg) for an additional h. or iecs were infected or mock-infected with pedv for h prior to transfection the luciferase reporter plasmids alone or cotransfection the indicated expression plasmids ( . μg). the cell lysates were harvested and luciferase activity was analyzed using a dual-luciferase assay system and a luminometer (turner biosystems, inc. sunnyvale, ca, usa) according to the manufacturer's instructions. data represent relative firefly luciferase activity normalized to renilla luciferase activity. the resulting ratios were used to compare the expression of the firefly luciferase gene in pedvinfected cells to that present in mock-infected cells. total rna was extracted from the transfected cells using triquick reagent (beijing solarbio science & technology co., ltd., beijing, china) according to the manufacturer's instructions and then reverse-transcribed into complementary dna (cdna) using murine leukemia virus reverse transcriptase (gbi labs/golden bridge international, inc., mukilteo, wa, usa) with oligo dt random hexamers (haigene technology, harbin, china). the cdna was then subjected to real-time pcr with specific primer pairs targeting ifn-β (f: ′-gctaac aagtgcatcctccaaa- ′ and r: ′-ccaggagc ttctgacatgcca- ′) and β-actin (f: ′-ggctcag agcaagagaggtatcc- ′, and r: ′-ggtctcaaa catgatctgagtcatct- ′. β-actin mrna was used as an endogenous control. iecs were seeded in -well plates and then transfected with μg of irf -gfp fusion expression constructs per well using lipofectin transfection reagent (invitrogen corp.) when cells reached confluence. cells were then mock-infected or infected with pedv at a multiplicity of infection (moi) of . at h postinfection, cells were transfected with μg of poly (i:c) or left untransfected. after h, cells were removed from the culture medium and washed three times in cold phosphatebuffered saline (pbs). next, cells were fixed in % paraformaldehyde for min at room temperature, quenched with . m glycine for min, and then permeabilized with . % triton x- for min. afterward, the cells were incubated with anti-pedv antibody (dilution, : ) for h followed by tritclabeled goat anti-rabbit secondary antibody (dilution, : ) for min at °c. the nuclei were stained with ′, -diamidino- -phenylindole-dihydrochloride (dapi) (invitrogen corp.). cells were examined using a tcs sp aobs confocal microscope (leica camera ag, wetzlar, germany). iecs were infected with pedv at an moi of or treated with poly (i:c) for the indicative times, lysed in × sodium dodecyl sulfate (sds) sample buffer and boiled for min. whole cells extracts were separated by % sds-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, which was blocked with % (w/v) bovine serum albumin (bsa) in tris-buffered saline ( mm tris-cl at ph . and mm nacl) containing . % tween (tbst) at room temperature for h. the membranes were then incubated with a primary antibody (dilution, : ) at °c overnight and a secondary hrp-conjugated antibody (dilution, : ) for h at room temperature. protein blots were developed using an enhanced chemiluminescence (ecl) detection system and exposed to x-ray film (clinx science instruments co., ltd., shanghai, china). all data were expressed as means ± standard deviations (sd) of independent experiments. the statistical significance was tested by student 's t-test and p-values less than . were considered statistically significant. porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs 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double-stranded rna and virus infection: common and unique pathways ikkepsilon and tbk are essential components of the irf signaling pathway porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and upregulates interleukin- expression porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin- expression submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank shaobo xiao, takashi fujita, kate fitzgerald for kindly providing important constructs. this work was supported by the programme for new century excellent talents at the heilongjiang provincial university all authors declare that there are no financial or other relationships that might lead to a conflict of interests.authors' contributions lyc response for carrying out the experiments, date analysis and drafting the manuscript. xyg and yg construct the recombinant expression plasmids. ydr participated in date analysis. gh, xfr and gxl designed the experiments and reviewed manuscript. all authors have seen and approved the manuscript and have contributed significantly to the work. key: cord- -pw xqhwa authors: feeley, eric m.; sims, jennifer s.; john, sinu p.; chin, christopher r.; pertel, thomas; chen, li-mei; gaiha, gaurav d.; ryan, bethany j.; donis, ruben o.; elledge, stephen j.; brass, abraham l. title: ifitm inhibits influenza a virus infection by preventing cytosolic entry date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: pw xqhwa to replicate, viruses must gain access to the host cell's resources. interferon (ifn) regulates the actions of a large complement of interferon effector genes (iegs) that prevent viral replication. the interferon inducible transmembrane protein family members, ifitm , and , are iegs required for inhibition of influenza a virus, dengue virus, and west nile virus replication in vitro. here we report that ifn prevents emergence of viral genomes from the endosomal pathway, and that ifitm is both necessary and sufficient for this function. notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by ifn, and ifitm was required and sufficient for this action. we further demonstrate that ifn expands rab and lamp -containing structures, and that ifitm overexpression is sufficient for this phenotype. moreover, ifitm partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to ifitm 's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by ifn. therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats. the h n pandemic provided a strong reminder of the threat that influenza a virus poses to world health (http://www. cdc.gov/h n flu/cdcresponse.htm). the most effective means of protection against influenza is the seasonal vaccine. however, if the vaccine does not match the viral strains, its effectiveness can be reduced to % or less [ , ] . among small molecules, only two approved influenza drugs remain effective, zanamivir (relenza) and oseltamivir (tamiflu). although resistance to zanamivir is rare, there has been an increase in oseltamivir-resistant flu strains [ ] . of concern, both drugs target viral neuraminidase (na), precluding combinatorial therapy to minimize resistance [ , ] . thus, research to identify new anti-influenza strategies would be useful. the influenza a virus is - nm in size, encodes for up to proteins, and contains eight segments of negative single-stranded genomic rna ( ) . influenza a virus infection initiates with the cleavage and activation of the viral hemaglutinnin (ha) envelope receptor by host proteases [ , , , ] . ha then binds to sialylated proteins on the cell surface, eliciting endocytosis of the viral particle. endocytosed viruses are transported through the early and late endosomes, with late endosomal acidification triggering a conformational change in ha which results in viral-host membrane fusion [ , ] . fusion transitions from a hemifusion intermediate into a fusion pore through which the virus' eight viral ribonucleoproteins (vrnps) enter the cytosol. the vrnps are subsequently guided by the host cell's karyopherins into the nucleus [ , , ] , wherein the viral rna-dependent rna polymerase synthesizes viral genomes (vrna) and mrnas, both of which are exported to the cytosol, culminating in the production of viral progeny. genetic screens have identified multiple host factors and pathways which modulate influenza a virus infection in vitro [ , , , ] . using such a genetic screen, we identified the ifitm protein family members ifitm , and as antiviral factors capable of blocking influenza a viruses [ ] . we further tested the antiviral activity of ifitm protein using the seasonal influenza a strains, a/uruguay/ / (h n ) and a/brisbane/ / (h n ), and found similar levels of ifitm mediated viral inhibition [ ] . ifitm accounts for a significant portion ( - %) of ifn's (type i or ii) ability to decrease influenza a virus infection in vitro, and ifitm resides in vesicular compartments that are ifn-inducible [ ] . in addition, the ifitm family inhibits infection by the flaviviruses, dengue virus and west nile virus [ , ] , as well as the filoviruses, ebola and marburg, and the sars coronavirus [ ] . the ifitm proteins also block vesicular stomatitis virus-g protein (vsv-g)-mediated entry, but do not substantially alter the replication of moloney leukemia virus (mlv), several arena viruses, or hepatitis c virus (hcv, [ , ] ). the human ifitm proteins were identified years ago based on their expression after ifn stimulation [ , , ] . the ifitm , , and genes are clustered on chromosome , and all encode for proteins containing two transmembrane domains (tm and ), separated by a conserved intracellular loop (cil, [ ] ), with both termini extra-cellular or intra-vesicular [ , ] . tm and the cil are well conserved between the ifitm proteins and a large group of proteins representing the cd protein family. cd family members exist from bacteria ( members) to man ( members, with members in chordata), with no in depth functional data available for any member other than the ifitm proteins. ifitm , and are present across a wide range of species including amphibians, fish, fowl and mammals. the ifitm proteins have been described to have roles in immune cell signaling and adhesion, cancer, germ cell physiology, and bone mineralization [ , , , , , ] . ifitm expression can inhibit the growth of some ifn-responsive cancer cells [ ] . genetic evidence also points to ifitm /bril being required for early bone mineralization [ , ] . ifitmdel mice, which are null for all five of the murine ifitm genes, display a % perinatal mortality among null pups, but thereafter grow and develop normally in a controlled setting [ ] . however, cells derived from these ifitmdel mice are more susceptible to influenza a virus infection in vitro [ ] . ifitm inhibited infection by all influenza a virus strains tested including a pandemic isolate and two contemporary seasonal vaccine viruses [ ] . we have found ifitm to be the most potent of the ifitm protein family members in decreasing influenza a virus replication [ ] . viral pseudoparticles are differentially inhibited by the ifitm proteins based on the specific viral receptors expressed on their surfaces [ , ] . therefore, we have hypothesized that ifitm proteins inhibit susceptible virus families (orthomyxoviridae, flaviviridae, rhabdoviridae, filoviridae, and coronaviridae) during the envelope-dependent early phase of the infection cycle, which extends from viral binding to cell surface receptors through the creation of the fusion pore between viral and host membranes [ , , ] . in support of this notion, recent work demonstrated that ifitm protein overexpression did not prevent influenza a virions from accessing acidified compartments [ ] . consistent with its acting on endocytosed viruses, a portion of ifitm resides in structures that contain host cell endosomal and lysosomal proteins [ ] . furthermore, inhibition of influenza a virus infection depends on the palmitoylation of ifitm , a posttranslational modification that targets proteins to membranous compartments [ ] . here we directly test the idea that ifitm restricts influenza a viral infection during the envelope-dependent early phase of the viral lifecycle. consistent with previous studies, we find that ifitm inhibits influenza a viral infection after viral-host binding and endocytosis, but prior to primary viral transcription [ , ] . moreover, using a combination of assays, we find that either ifn or high levels of ifitm impede influenza a viruses from transferring their contents into the host cell cytosol, and that ifitm is necessary for this ifn-mediated action. therefore, we conclude that ifn is acting predominantly through ifitm to block viral fusion. we also find that ifn expands the late endosomal and lysosomal compartments, and that ifitm overexpression is sufficient for this phenotype. this study also presents data showing that ifitm overexpression leads to the expansion of enlarged acidified compartments consisting of lysosomes and autolysosomes. interestingly, we observe that viruses trapped in the endocytic pathway of ifitm -overexpressing cells are trafficked to these expanded acidified compartments. based on these results and those of others [ , ] , we present a model whereby ifn acts via ifitm to prevent viral fusion, thereby directing endocytosed viruses to lysosomes and autolysosomes, for subsequent destruction. collectively this study expands our understanding of how ifitm restricts a growing number of viruses by exploiting a shared viral vulnerability arising from their use of the host's endocytic pathway. the inhibition of ha-expressing pseudoparticles by the ifitm proteins pointed towards restriction occurring during the envelope-dependent phase of the viral lifecycle [ ] . therefore we tested ifitm 's impact on the most proximal phase of infection, viral binding, by incubating influenza a virus a/wsn/ h n (wsn/ , multiplicity of infection (moi) ) with a lung carcinoma cells either stably overexpressing ifitm (a -ifitm ) or an empty vector control cell line (a -vector, fig. a ). samples were incubated on ice to permit viral binding but prevent endocytosis. after incubation, cells were washed with cold media, fixed and stained for ha. when analyzed by flow cytometry, we observed no appreciable difference in surface bound ha between the vector and ifitm cells. there was also no difference in surface-bound virus over a series of ten-fold dilutions of viral supernatant (data not shown). we also determined that the stable expression of ifitm did not alter the surface levels of (a , ) or (a , ) sialylated cell-surface proteins (fig. s ) . to investigate ifitm 's impact on initial viral mrna production, we infected canine kidney cells, either expressing ifitm (mdck-ifitm ) or the empty vector (mdck-vector), with inf luenza a virus (a/puerto rico/ / h n (pr ), moi ). we used pr because of the purified high titer stocks available. next, the viral supernatant was removed and warm media was added ( min). at the indicated times, cells were processed and stained for the positive stranded np mrna of pr using a specific rna probe set (red, fig. b) , then imaged on a influenza epidemics exact a great toll on world health. thus research to identify new anti-influenza virus strategies would be useful. each of our cells contains antiviral factors that work to inhibit infection. a large component of this antiviral program is regulated by the interferon family of signaling molecules. here, we seek to better understand how one of these antiviral factors, ifitm , contributes to both baseline, as well as interferon-induced, antagonism of influenza a viral infection. we found that interferon prevents influenza a virus from entering our cells by blocking the virus' fusion with the cellular membrane. furthermore, we learned that ifitm is required for this antiviral action of interferon, and that high levels of ifitm alone can produce a similar viral inhibition. together, these results improve our understanding of how ifitm serves to defend us against viral invasion at a very early stage of infection. confocal microscope. based on np mrna staining, primary viral transcription begins by min. p.i. in the vector control, with the np mrna signal increasing through to min., when the export of viral mrnas to the cytosol can be observed. a decrease in primary viral transcription can be seen when comparing the ifitm cells to the vector control line. therefore, ifitm inhibits influenza a viral infection after viral-host binding but before primary viral mrna transcription. ifn interferes with vrnp nuclear entry and ifitm is necessary and sufficient for this antiviral defense we next used confocal imaging to track the nuclear translocation of vrnps ( fig. [ , ] ). at the start of infection, the np within infected cells is complexed with viral genomic rna forming vrnps. therefore, immunostaining for np permitted us to follow vrnp distribution intracellularly [ , , ] . normal diploid human lung fibroblasts (wi- cells) were stably transduced with empty vector (vector), ifitm cdna (ifitm ), or short hairpin rnas (shrna) either against ifitm (shifitm ) or a scrambled non-targeting control (shscramble, fig. , s ). wi- s were chosen because of their normal karyotype and relatively larger and flatter morphology. cells were first incubated on ice with pr (moi ). next, the viral supernatant was removed and warm media was added ( min). at the indicated times after warming, cells were fixed, permeabilized, stained for np and dna, and imaged on a confocal microscope. image analysis software was used to create an outline of each cell's periphery (white lines) and nucleus (blue lines). based on np staining, vrnps arrive in the nuclei by min in the vector control, shifitm , and in the shscramble cells, with the np signal increasing through to min ( fig. a, s a-d) . in contrast, we observed decreased nuclear and increased cytosolic np staining in the ifitm cells (fig. , s c) . moreover, in the ifitm cells greater than % of the cytosolic np colocalized with lysotracker red (ltred), a dye which marks acidic cellular compartments (late endosomes, lysosomes, ph# . ), and which was added to the warm media at time zero (fig. s a, d) . the increased np in the cytosol of the ifitm cells likely arises in part from an increase in the local concentration of viruses because a-np western blots (after trypsinizing the cells to remove adherent np) did not show substantial differences in internalized np levels between cell lines for up to min post infection (p.i., data not shown). because ifitm is required for the anti-viral actions of ifn in vitro [ ] , we performed a companion experiment with the wi- cells treated with ifn-a ( fig. b ). ifn-a treatment also decreased np nuclear staining in the wi- -vector cells, however this block was not as complete nor was it associated with similar levels of cytosolic np staining as those seen with high levels of ifitm . consistent with the gain-offunction data, the depletion of ifitm decreased ifn's ability to block vrnp trafficking to the nucleus ( fig. a and b , compare top and bottom rows). similar results were obtained either using a cells (fig. s ) or using mdck cells, with the latter experiments employing additional influenza a viral strains (x: , a/aichi/ (aichi h n ), fig. s a -c, wsn/ and a/victoria/ / h n , data not shown). it is important to note that the levels of ifitm protein in the a -ifitm cells are higher than those seen after treatment with ifn-a or -c (fig. s c) . however, we have not observed that other overexpressed proteins have either protected against viral infection or expanded the lysosome/autolysosome compartment (data not shown), arguing that this is a specific effect. to better assess the expanded ltred compartments observed with ifitm overexpression, we created mdck cells stably expressing the lysosomal protein, lamp , fused to a red fluorescence protein (lamp -rfp) and ifitm . as compared . ifn prevents vrnp nuclear entry, and ifitm is necessary and sufficient for this action. a) normal diploid human fibroblasts (wi- cells) were stably transduced with retroviruses containing either ifitm (ifitm ), a shrna against ifitm (shifitm ), an empty viral vector alone (vector), or a non-targeting control shrna (shscramble, fig. s ). cells were incubated with pr on ice, and then warm media was added at time zero. cells were fixed at the indicated times p.i. and stained for np (green) and dna and analyzed by confocal microscopy. image analysis software was used to define each cell's cytosolic (white lines) and nuclear peripheries (blue lines, based on dic images and dna staining, respectively). red arrows: cytosolic compartments containing np. images are representative of four independent experiments. (scale bar: mm). b) as in (a) except that cells were treated with ifn-a prior to infection. doi: . /journal.ppat. .g to control cells, the ifitm cells demonstrated extensive colocalization (. %) between the np and lamp -rfp signals, revealing that the entering viruses are trafficked to lysosomal compartments (fig. s ) . we extended this analysis by directly tracking the location of the vrna contained in the incoming vrnps. mdck cells stably expressing an empty vector or ifitm , were used in time-course experiments as above (fig. a-d) . at the indicated times, cells were processed and stained for the negative stranded np vrna of pr using a specific rna probe set (green). as seen with the wi- cells, we observed the nuclear translocation of vrna by min p.i. in the mdck-vector cells (fig. a) . the nuclear vrna signal was strongly decreased with ifitm overexpression based on the average number of vrna particles present per nucleus (fig. c) . consistent with the wi- results, the vrnas accumulated in the cytosol of the ifitm cells, with . % co-localizing with ltred-staining acidic structures (fig. d ). similar levels of retained cytosolic vrnps were observed in experiments without ltred (data not shown). interestingly, we observed the loss of the vrna signal in the acidic inclusions of the mdck-ifitm cells between and min. p.i. (fig. b ). by comparison, the vrnas in the control cells increased in number in both the nucleus and cytosol, as would be expected with the nuclear export of newly synthesized viral genomes [ ] . we next evaluated vrnp translocation in murine embryonic fibroblasts (mefs) derived from animals that have had all five ifitm genes deleted (ifitmdel / , [ , ] ). compared to wild-type (wt) matched litter mate controls, the ifitmdel / mefs displayed - fold more nuclear np staining, with or without ifn-c treatment (fig. , s c) . ifn-mediated viral restriction was restored when we transduced the null mefs with a retrovirus expressing ifitm (ifitmdel / ifitm , fig. s ). similar to what was observed with the ifitm overexpressing cell lines, the majority of the vrnp signal in the ifn-c-treated wt and ifitm rescued cells localized to acidic compartments (red, fig. s b ). an increase in acidic compartments occurred after ifn-c treatment with either the wt or the ifitmdel / ifitm mefs, but not in the ifitmdel / cells, suggesting that ifitm is required for this event (fig. , s ) . similar results were obtained with ifn-a (data not shown). we conclude from these experiments using orthologous reagents (cell lines and influenza a viruses) and methods, that ifn impedes vrnp nuclear entry, and ifitm is necessary and sufficient for this activity. viral pseudoparticle fusion mediated by either ha or vsv-g envelope proteins is decreased by ifn, and ifitm is necessary and sufficient for this activity to further characterize the mechanism of ifitm -mediated restriction, we used an established viral fusion assay [ , ] . lentiviral pseudoparticles containing the b-lactamase protein fused to the hiv- accessory protein vpr (blam-vpr) and expressing either ha and na (h n , wsn/ ), or vsv-g envelope proteins, were incubated for h with cells, which were then loaded with the b-lactamase flourogenic substrate, ccf . upon viral pseudoparticle fusion, blam-vpr enters the cytosol and cleaves ccf , producing a wave length shift in emitted light (from green to blue) when analyzed by flow cytometry (fig. a , [ ] ). in mdck-ifitm cells we observed a decrease in both ha-and vsv-g-directed fusion, which was comparable to the block produced by poisoning of the host vacuolar atpase (vatpases) with a low dose of bafilomycin a (baf, fig. b ). the inhibition of vatpases prevents the low-ph activation required by these two viral envelope proteins to produce membrane fusion. a block to fusion of pseudoparticles expressing h (pr ), h (a/udorn/ ), h (a/thai/ ) or h (a/fpv/ rostock/ ) subtypes of ha was also detected with mdck cells mefs, either a) wild type (wt) or b) ifitmdel / , which are missing all five of the mouse ifitm proteins, were either left untreated (left panels, buffer), or treated (right panels) with ifn-c. the following day cells were incubated with pr on ice. cells were next incubated in warm media containing ltred. cells were then fixed at the indicated times and immunostained with anti-np antibodies (green), stained for dna (blue), and imaged by confocal microscopy. image analysis software was used to define the nuclear boundaries (blue lines). images are representative of three independent experiments. (scale bar: mm). doi: . /journal.ppat. .g [ , ] comprised of lentiviral pseudoparticles (pps) containing the b-lactamase protein fused to the hiv- accessory protein vpr (blam-vpr, shown in orange/red) and expressing ha and na (wsn/ ) on their surfaces. the h n pps were incubated for h with cells, which were subsequently loaded with the b-lactamase flourogenic substrate, ccf . upon viral fusion, blam-vpr enters the cytosol and can cleave ccf , producing a wavelength shift from green to blue in emitted light when analyzed by flow cytometry ( [ ] ). b) mdck cells stably overexpressing ifitm (mdck-ifitm ) or empty vector control cells (mdck-vector) were exposed for h to viral pseudoparticles containing a blam-vpr and expressing either the ha and na envelope proteins of influenza a virus (wsn/ , h n pp) or the vsv-g envelope protein (vsv-gpp), then loaded with ccf . after incubation with the indicated pseudoparticles, the cells were fixed and assayed for cleavage of ccf by determining the conversion of the fluorescence emission from nm (uncleaved ccf ) to nm (cleaved ccf ) using flow cytometry. fusion of the pseudoparticles was inhibited by bafilomycin a (baf). these results are representative of six independent experiments. c) ifitm inhibits fusion of h n pps in normal diploid fibroblasts. wi- fibroblasts stably transduced with ifitm (wi- m ) or the empty vector (wi- v) were exposed for h to serial dilutions of h n pps containing blam-vpr, with or without baf. these results are representative of four independent experiments. d) fusion of h n pps increases after ifitm knockdown. wi- fibroblasts stably transduced with a shrna against ifitm (wi- shm ), a shrna control with a scrambled sequence (wi- shscr), or the ifitm cdna (wi- m ) were exposed to either no virus, h n pps or vsv-gpps containing blam-vpr. these results are representative of two independent experiments. e) fusion of h n pps is inhibited by ifn-c. wi- fibroblasts were treated with ifn-c for h or buffer alone prior to incubation with h n pps containing blam-vpr. these results are representative of three independent experiments. doi: . /journal.ppat. .g or with chicken embryonic fibroblasts (chefs), in which ifitm strongly inhibited viral replication (fig. s a , b, c). in the case of the mdck cells, the block to fusion closely paralleled the level of inhibition seen when the pseudoparticles were tested for productive infection using hiv- p expression as a readout (fig. s e) . consistent with earlier findings, pseudoparticles expressing an amphotropic mlv envelope protein were insensitive to ifitm , showing the specificity of these results (fig. s d) . similarly to its effect on h -expressing pseudoparticles, ifitm inhibited replication of infectious avian h n influenza a virus, a/vietnam/ / (vn/ ), isolated from a fatal human infection (fig. s f-h) . to enhance our analysis, we tested two additional cell lines, wi- and hela cells. a strong block to fusion in wi- -ifitm cells, similar to that of the baf and uninfected control samples, was seen at a range of serial dilutions of pseudoparticles, as well as an increase in fusion with ifitm depletion (shifitm , fig. c, d) . ifn treatment inhibited fusion of the h n pseudoparticles, albeit to a lesser extent than ifitm overexpression (fig. e) , and this effect was largely absent when ifitm was stably depleted in hela cells (fig. s ). similar results were obtained with ifn-a (data not shown). based on these experiments using multiple cell lines and ha, vsv-g, and mlv envelope-expressing pseudoparticles, we conclude that ifitm is required and sufficient for an ifn-mediated block of viral pseudoparticle fusion. importantly, the increase in pseudoparticle fusion seen when endogenous ifitm was depleted in either the hela or wi- shifitm cell lines argues that fusion inhibition underlies the first line defense provided by endogenous, as well as overexpressed, ifitm . mxa is an ifn-inducible large gtpase which interferes with secondary transcription during influenza a viral replication [ ] . a cells express mxa and have been used extensively in influenza a viral replication studies [ ] . therefore to clarify the antiviral roles of ifitm and mxa, we tested the levels of viral replication in a cells stably expressing one of three shrnas targeting ifitm (shifitm - , - , or - ). all three shifitm cell lines showed increased infection (wsn/ strain) and strong ifitm knockdown, when compared to the negative control cell line expressing a shrna against firefly luciferase (shluc), with or without ifn treatment (fig. s a, b) . the majority of the protective effect of either ifn-a or c was lost in the shifitm cell lines. we next confirmed both the baseline levels, as well as the ifn-inducibility of mxa in the a cells (fig. s c) . we also determined that mxa was both present and ifn-inducible in wi- normal fibroblasts, another cell line used in loss-of-function experiments in this work (fig. s d) . furthermore, if studies of wi- cells showed that mxa is expressed in an ifn-inducible vesicular pattern and that these structures did not appreciably colocalize with vesicles containing ifitm (fig. s e , [ ] ). we conclude that mxa is expressed in the a and wi- cell lines, but cannot fully compensate for loss of the antiviral actions of ifitm . ifitm is present in endosomes and lysosomes and these compartments are expanded with ifitm overexpression or ifn treatment our data demonstrate that ifn or ifitm inhibit viral fusion. influenza a virus fuses with the host membrane in late endosomes when the ph decreases to [ , , ] . rab is a late endosomal/lysosomal small gtpase that is required for the fusion of many ph-dependent viruses, including influenza a virus [ , ] . previous reports have shown that ifitm colocalizes with lamp and cd , components of lysosomes and multivesicular bodies, respectively [ ] . however, the relationship of ifitm and rab within the host cell infrastructure remains unknown. therefore we investigated the location of ifitm , by undertaking immunoflourescence (if) studies using antibodies that recognize ifitm , rab , or lamp [ ] . although the baseline level of ifitm in the a -vector cells was low, there was partial colocalization observed with either rab or lamp (fig. a-d, a,) . ifitm also partially colocalized with lamp and ltred-containing structures seen with ifitm overexpression (fig. a, b, a) . interestingly, either ifitm overexpression or ifn increased the staining intensity of rab and lamp (fig. a, b, s a ). partial colocalization of ifitm was also seen with either endogenous lamp , or an exogenously expressed rab -yellow fluorescence fusion protein (rab -yfp) in mdck cells (fig. e-i) . however, in all cases, co-localization was not complete because cells contained areas that uniquely labeled for each of the proteins. western blots indicated that ifitm overexpression led to modest increases in both lamp and rab proteins in the a -ifitm cells (fig. c) . however, these blots also showed that while ifn treatment of the a -vector cells increased ifitm protein levels as expected, the amount of rab and lamp remained unchanged. we conclude that ifitm partially resides in the late endosomal and lysosomal compartments along with rab and lamp , and that ifitm overexpression or ifn treatment expands these compartments through a mechanism that cannot be fully explained by increased protein expression alone. our assays showed that incoming influenza a viruses were retained in the expanded acidic compartments of both the ifitm overexpressing cell lines as well as the ifn-c-treated mefs, and that ifitm partially localized to these structures ( fig. - , s - , s ) . therefore, we extended our investigation of these compartments. an increase in acidic structures was seen in mdck and a cells overexpressing ifitm as compared to control cell lines, using either the vital acidophilic stain, acridine orange (ao), ltred, or a cathepsin-l substrate that fluoresces only after it is proteolyzed, when compared to the corresponding vector control cells (fig. a, b, a, b) . cathepsins are a family of lysosomal zymogens active in acidic environments (ph# . ) which are required for both the degradation of endocytic substrates and for the entry of several ifitm -susceptible viruses [ ] . flow cytometry revealed an increase in the total ltred fluorescent signal in both the mdck and a ifitm cell lines when compared to controls (fig. c ). this expanded compartment represents a heterogeneous population of lysosomes and autolysosomes, based on confocal imaging showing the colocalization of the autophagosome marker, microtubule-associated protein light chain (lc ), with either ltred or with cd , with the latter being a resident of multivesicular bodies, amphisomes and autolysosomes (fig. d, e) . furthermore, mdck-ifitm cells stably transduced with an lc protein fused to both a red fluorescent protein (mcherry) and an enhanced green fluorescence protein (egfp) showed a predominantly red signal, which occurs when the mcherry-egfp-lc protein resides inside the acidified interior of an autolysosome (fig. f, [ ] ). in keeping with previous reports that ifn-c induces autophagy [ , ] , we detected enhanced ltred staining in either ifn-c treated mefs or a cells (fig. a, s a) . we conclude that increases in ifitm levels expand the lysosomal/autolysosomal compartment. here we report several novel findings regarding the antiviral actions of ifn and the transmembrane ieg, ifitm . first, this study demonstrates that ifn inhibits the nuclear translocation of vrnps, and that ifitm is required for this ifn-mediated block, with both endogenous and overexpressed ifitm inhibiting vrnp nuclear entry. second, either endogenous or overexpressed ifitm , as well as ifn treatment, block the fusion of viral pseudoparticles expressing various influenza a virus envelope proteins (h , h , h and h subtypes of ha), or the vsv-g envelope protein; this block is specific because the fusion of pseudoparticles expressing mlv envelope is not inhibited by ifitm . third, our work reveals that ifitm partially resides with rab in late endosomes, thus placing it in position to block influenza a virus' cytosolic access. fourth, ifitm overexpression or ifn induce the expansion of late endosomal and lysosomal compartments containing rab and lamp . fifth, we show that similar to ifn-c treatment, ifitm overexpression expands the number and size of autolysosomes, and it is into these compartments that trapped viruses are trafficked and subsequently degraded. consistent with previous reports, our data show that high levels of ifitm do not prevent viral access to acidified compartments and that ifitm colocalizes with cd and lamp [ ] . this is in contrast to a report noting the exclusion of overexpressed ifitm from lamp -containing structures [ ] . therefore, this work adds substantially to our interpretation of previous reports by demonstrating that key downstream events in the viral lifecycle, fusion and vrnp nuclear translocation, are prevented by either ifn or ifitm . ifitm thus represents a previously unappreciated class of anti-viral effector that permits viral entry into the endosomal compartment, but prevents egress into the cytosol. these studies also raise new questions including i) how do ifn and ifitm prevent viral fusion? ii) how do ifn and ifitm alter the endosomal and autolysosomal compartments? and iii) is the latter action required for viral restriction, or alternatively does it arise as an outcome of ifitm 's potential cellular role? based on the substantial loss in ifn's potency observed when ifitm is depleted ( - % loss of viral inhibition, fig. s a , b, [ ] ) we conclude that inhibition of viral emergence from the endosomal pathway is a prominent component of ifn's antagonism of influenza a virus replication in vitro. our data also show that mxa cannot fully compensate for the loss of ifitm in ifn-treated cells challenged with influenza a virus. recent work by dittmann et al. [ ] and zimmermann et al. [ ] reveal that human influenza a viral strains have evolved a means to evade mxa, suggesting a possible explanation for the cellular reliance on ifitm for protection in vitro. similarly the ieg, ifit , prevents viral replication by targeting viral triphosphate-rnas (ppp-rna) for destruction [ , ] . given that ifitm is necessary for the majority of ifn-mediated restriction of influenza a virus in vitro, it may be that the virus has also evolved a means to at least partially nullify ifit , perhaps via the massive production of short ''decoy'' ppp-rnas, as previously postulated [ , ] . ifitm primarily resides in the endosomal compartment and partly colocalizes with rab and lamp . ifitm overexpression or ifn stimulation caused the endocytosed viruses to accumulate in acidic compartments that contained both ifitm and lamp . together with the blam-vpr fusion assay data, these results reveal that ifitm prevents viral-host membrane fusion within late endosomes, and likely within lysosomes as well, in light of studies showing ifitm-mediated restriction of filoviruses and coronaviruses, which depend on cathepsin-mediated activation prior to fusion [ ] . in doing so, ifitm traps the virus on a path which terminates in a degradative environment [ ] . in support of this, our experiments show the eventual loss of a detectable vrna signal in the ltred-positive compartments of the ifitm transduced cells, thus revealing the fate of viral fitness under those conditions. these studies also reveal that elevated levels of ifitm correlate with the expansion of host cell structures containing rab and lamp , and that ifitm was also present in these structures. in the mef and a experiments, ifn produced increased rab and lamp immunostaining, in addition to an increase in acidic structures. at present, we cannot explain the increased rab and lamp signals seen after ifn stimulation or ifitm overexpression solely on the slight elevations in the abundance of these proteins detected by immunoblotting. two possible explanations for the increased immunostaining observed, are that ifn stimulation induced these proteins to cluster together or alternatively unmasked sequestered epitopes; we find the latter possibility less likely since lamp and rab flourescent fusion proteins also showed larger and more intense signals under similar conditions. we envision that ifitm -mediated clustering of organelles and their protein cargoes might contribute to the host cell's antiviral state. earlier work reported no correlation between the size of the ifitm -induced acidified compartments and the level of viral restriction [ ] , however, we observe that increasing levels of ifitm result in both an expansion of lysosomes/ autolysosomes and increased viral inhibition. these observations might be explained by a common mechanism underlying the increase in these structures and viral inhibition, in addition to raising the possibility that they play a role in ifitm-mediated viral restriction. is there a common characteristic shared by ifitm -susceptible viruses? the late endosomal-and lysosomal-associated small gtpase, rab , is required for influenza a virus infection [ , ] . the ifitm -resistant viruses previously tested (mlv, the arena viruses and the hepacivirus, hcv) are all rab -independent, while the entry of the ifitm -susceptible viruses (influenza a, dengue, ebola, marburg, and sars) relies on rab [ , , , , , ] . standing against this hypothesis, is the lack of effect on vsv-g-mediated entry with expression of a dominant negative rab [ , , ] ). however, additional studies have shown that vsv-g-directed entry is dependent on transport to the late endosomes [ , ] ; these latter results, together with those of huang et al. and weidner et al. [ , ] , are consistent with the prediction that viruses that fuse in late endosomes or lysosomes are vulnerable to ifitm 's actions, while viruses whose genomes enter at the cell surface or in the early endosomes may avoid ifitm 's full effect. of note, we have been unable to demonstrate that ifitm blocks hiv- replication using tzm-bl hela cells and are working to address these differences with a published study ( [ ] , data not shown). this study, together with previous work, demonstrates that ifitm permits endocytosis of viruses, but prevents viral fusion and the subsequent entry of viral contents into the cytosol [ , ] . while the blam-vpr fusion assay demonstrates inhibition of fusion by ifn or by ifitm , we note that this assay uses an indirect readout to assess entry of viral contents. therefore several possibilities could explain the containment and neutralization of viruses within the endosomal pathway, including alterations in endosomal trafficking, acidification, or the host membrane's fusion characteristics (bending modulus, elasticity). while additional work is required to further define the mechanism, the lack of toxicity seen with cells stably overexpressing high levels of ifitm suggests that gross alterations in endogenous trafficking or ph control are unlikely (data not shown). therefore overexpressing or activating ifitm to produce an enhanced antiviral state may be an effective prevention strategy during high risk periods in vulnerable populations. we propose that ifn causes the degradation of endocytosed viruses by preventing their contents from entering the host cytosol, and that ifitm is necessary and sufficient for this defense ( figure g ). ifitm 's mode of defense could be envisioned as an effective means to neutralize pathogens during an organism-wide threat. such actions might confer an advantage to the host because if ifitm simply decreased viral attachment and/or entry, the repulsed viruses would be free to attack neighboring cells. of course while there are considerable differences between this simple scenario and the directed phagocytosis of pathogens by specialized immune cells, i.e. macrophages, the similarities none-the-less suggest an early prototype for a more evolved defense mechanism. cell lines and culture conditions u os, a , mdck, hela cells (all from atcc), and chicken embryonic fibroblasts (chefs, from charles river labs) were grown in complete media (dmem, invitrogen cat# ) with % fbs (invitrogen). wi- cells (atcc) were cultured in dmem (invitrogen cat# ), containing non-essential amino acids (invitrogen cat# ) and % fbs. wild type and matched ifitmdel / mefs were from adult ifitmdel+/ mice [ ] that were intercrossed and mefs derived from embryos at day . of gestation, as described previously [ ] . the mefs were genotyped by pcr and western blot, and the generation of the ifitmdel / ifitm cells have been previously described [ ] . the ifitm retroviral vector, pqcxip-ifitm and empty vector control (clontech) have been previously described [ ] . . dna = blue. e) mdck cells stably transduced with ifitm or with the vector alone were immunostained for confocal imaging of lc (endogenous, red) and cd (endogenous, green). dna = blue. f) confocal images of mdck cells overexpressing ifitm or the empty vector alone showing the distribution and fluorescence intensities of a stably expressed mcherry-egfp-lc b fusion protein using fluorescence channels that detect light emitted from the mcherry protein, egfp or both (merge). dna = blue. g) model of ifitm -mediated restriction of virus replication. endocytosed viruses enter late endosomes where ifitm is present. ifitm prevents viral fusion within the endosomes and likely lysosomes via an unknown mechanism, perhaps by altering ph, membrane characteristics, lipid composition, transport speed or destination. trapped viruses are trafficked to lysosomes and/or autolysosomes where they undergo degradation. doi: . /journal.ppat. .g # ) and was kindly deposited by jayanta debnath. plzs-rab -yfp and plvx-rfp-lamp were generously provided by walther mothes, section of microbial pathogenesis, yale university school of medicine. the following shrna sequences (sense strand sequence provided) were cloned into the papm shrna-expression lentiviral vector [ ] , to create the viruses used to generate the a ifitm knockdown cell lines in fig. s : ifitm - : -tcctcatgaccattctgctcat- ifitm - : -cccacgtactccaacttccatt- ifitm - : -tttctacaatggcattcaataa- influenza a virus a/puerto rico/ / (h n ) (pr , charles river labs) and a/wsn/ (h n ) (kind gift of dr. peter palese, microbiology dept., mt. sinai school of medicine, ny, ny) were propagated and assessed for viral infectivity as previously described [ ] . influenza a virus a/vietnam/ / (h n ) was propagated and characterized as previously described [ ] . human interferon (ifn)-c (invitrogen) was used at - ng/ml, human ifn-aa (pbl interferon source) was used at - u/ml. cells were incubated with cytokines for - h prior to if or viral infection experiments unless otherwise noted. murine ifn-c (pbl interferon source) was used at - ng/ml. whole-cell extracts were prepared by cell lysis, equivalent protein content boiled in sds sample buffer, resolved by sds/ page, transferred to immobilon-p membrane (millipore), and probed with the indicated antibodies. cells were seeded on glass coverslips for influenza a virus infection experiments. cells were incubated on ice with pr for min. at time zero, the viral supernatant was removed and uc media was added with or without lysotracker red dnd- (invitrogen). at the indicated time points post-warming, cells were washed twice with d-pbs (sigma) and incubated for seconds with room temperature . % trypsin (invitrogen). the cells were then washed with complete media twice and fixed with % formalin (pfa, sigma) in d-pbs. image analysis for quantitation of vrnp nuclear translocation was done using imaris . (bitplane scientific software). we generated a mask of the nucleus and applied this mask to the channel containing the viral signal (puncta) to determine vrna puncta contained in each nucleus. cells were incubated at uc and % co for min. with either lysotracker red dnd- or acridine orange (immuno-chemistry technologies). hoechst (dna stain, invitrogen) was incubated ( : , ) with the cells for the final min. the cathepsin l flourogenic substrate assay was performed as per the manufacturer's instructions (cathepsin l -magic red, immuno-chemistry technologies). cells were visualized live by confocal microscopy. cells were fixed in % pfa in d-pbs, and then incubated sequentially in . % tween (sigma), then % bsa with . m glycine (sigma), both in d-pbs. primary and secondary antibodies are listed below. slides were mounted in vectashield with dapi counterstain (vector labs). slides were imaged using a zeiss lsm , laser scanning inverted confocal microscope equipped with the following objectives: zeiss c-apochro-mat uv-vis-ir water, . na, zeiss plan-apochro-mat dic oil, . na, and zeiss plan-apochromat dic oil, . na. image analysis was performed using zen software (zeiss). laser intensity and detector sensitivity settings remained constant for all image acquisitions within a respective experiment. nuclear outlines were generated using metamorph software suite (molecular devices) using the kirsch/prewitt filter to define boundaries and then subtracting out the original binary images. the following antibodies were used in this study for either these experiments employ the quantigene viewrna slidebased assay kit from affymetrix (cat #qv ) with all components from that source unless noted. rna was visualized following a modified manufacturer protocol; changes made include the omission of the ethanol dehydration step, and use of vectashield mounting media. post-fixation with % pfa, cells adherent on coverslips were incubated with detergent solution or incubated in . % pbs-tween . cells were then incubated with proteinase k. next cells were incubated at uc in hybridization solution a containing a viewrna probe set designed against either the negative stranded rna np genome (vrna) of pr (affymetrix vx - - qg viewrna type probe set against np influenza a virus (a/puertorico/ / (h n )) at : ) or a probe set against the positive stranded np mrna. cells were then incubated in hybridization preamplifiers ( : in hybridization buffer b) at uc. finally cells were incubated with labeled probes ( : in hybridization buffer c), washed and imaged as above. all steps were followed by two d-pbs washes. sialic acid linkage expression studies a cells stably transduced to overexpress ifitm or with empty expression vector (pqcxip, clontech) were grown to , % confluency, dissociated with trypsin-free edta-based dissociation buffer (invitrogen) for min. at uc. cells were incubated at uc with fitc-conjugated sambucus nigra lectin (sna, vector labs #fl- ) to detect (a- , ) sialic acid linkages, and biotinylated maackia amurensis lectin ii (mal, vector labs #b- ) to detect (a- , ) sialic acid linkages, followed by streptavidin-pe-cy (invitrogen). cells were incubated with lectins individually and in combination, and the results of staining were indistinguishable. all cells were stained with violet cell-impermeable dye (invitrogen #l ), and cells were included in the analysis if viable by fsc/ssc and viability dye. binding assay a cells transduced with ifitm or the empty vector pqxcip were detached using enzyme free pbs-based dissociation buffer, and then washed in cold pbs extensively. cells and virus (wsn/ ) were pre-chilled on ice for min. and mixed at a moi of and incubated at uc for h with rotation. cells were washed extensively with ice cold pbs and then fixed using % pfa. the cells were then probed with anti-ha mouse monoclonal antibody (wistar collection, coriell institute, clone h -s , wc , if) for h at room temperature, followed by antimouse alexaflour- conjugated antibody (invitrogen) for h with pbs washes in between, then analyzed by flow cytometry. figure s ifitm overexpression does not alter the surface levels of (a- , ) or (a- , ) sialylated proteins. a cells stably transduced with ifitm or the empty vector were incubated with biotinylated maackia amurensis lectin ii (mal) to detect (a- , ) sialic acid linkages, followed by streptavidin-pe-cy , as well as fitc-conjugated sambucus nigra lectin (sna) to detect (a- , ) sialic acid linkages. a) the percentage of ifitm or vector cells staining positive for both sialic acid linkages (upper right hand quadrant), compared to unstained controls. b) ifitm overexpressing and vector cells are compared with regard to each sialic acid linkage in the double-stained populations. (pdf) figure s ifitm arrests influenza a virus in acidic cytosolic inclusions preventing vrnp nuclear translocation. a) normal diploid human fibroblasts (wi- cells) were stably transduced with retroviruses containing ifitm (wi- ifitm ) or (b) a non-targeting control shrna (wi- shscramble). cells were incubated with pr on ice, and then warm media containing ltred (red) was added at time zero. cells were fixed at min. p.i. and stained for np (green) and dna, then analyzed by confocal microscopy. image analysis software was used to define each cell's cytosolic (white lines) and nuclear peripheries (blue lines, based on dic images and dna staining, respectively). images are representative of four independent experiments. figure s ifitm expression rescues ifn-c-mediated inhibition of vrnp nuclear translocation in ifitmdel / mefs. a) ifitmdel / mefs stably overexpressing ifitm (ifitmdel / ifitm ), were left untreated (left panels, buffer), or treated (right panels) with ifn-c. the following day cells were incubated on ice with pr (moi ). cells were next incubated in warm media containing ltred ( min.). cells were then fixed at the indicated times p.i., immunostained with anti-np antibodies (green) and imaged by confocal microscopy. image analysis software was used to define the nuclear boundaries (blue lines). images are representative of three independent experiments. (scale bar cm) . b) percent colocalization of vrnp and ltred compartments in the indicated mef cell lines, with or without ifn-c treatment, are shown for the indicated times p.i. c) quantitation of nuclear vrnp particles. the number of vrnp particles per nucleus of the mef cell lines, with or without ifn-c treatment, at the indicated time points are shown. values represent the mean +/ the sd of three independent experiments. d) western blot of whole cell lysates from the indicated mefs probed with anti-mouse ifitm and using gapdh as a loading control. (pdf) figure s fusion of viral pseudoparticles expressing ha envelope subtypes, but not a mlv envelope, is decreased by ifitm . ifitm inhibits the replication of infectious h n virus. a) mdck cells stably transduced with ifitm or empty vector were incubated with pseudoparticles expressing n and ha subtypes (h n pp, h n pp, or h n pp). cells were then fixed and assayed for cleavage of ccf using flow cytometry. these results are representative of three independent experiments. b) chicken embryonic fibroblasts (chef) cells stably expressing the empty vector control or ifitm were incubated with pseudoparticles expressing n and either of the two avian influenza a viral ha subtypes, h or h , as in (a). these data are representative of three independent experiments. c) chef cells stably transduced with the empty vector control or overexpressing ifitm , were infected with wsn/ for h then stained for ha protein (red) and dna (blue). average percent infection is given for three independent experiments +/ sd. magnification. d) mdck-vector or mdck-ifitm cells were incubated with pseudoparticles expressing the amphotropic mlv envelope protein (mlvpp) and then assayed for cleavage of ccf using flow cytometry. these results are representative of two independent experiments. e) infectivity of ha-expressing pseudoparticles is decreased by ifitm . mdck-vector or mdck-ifitm cell lines were infected with the indicated pseudoparticles for h. cells were then immunostained for expression of hiv- p protein expressed from the integrated lentiviral genomes. percent infection is provided. these results are representative of three independent experiments. magnification. f) a cells were stably transduced with retroviruses containing ifitm or empty viral vector alone, then infected with a/vietnam/ / (h n ) influenza a virus (vn/ ). after h, the cells were fixed and stained for viral np expression (green) and for dna (blue). values given are percentage infected cells and are representative of two independent experiments. magnification. g) western blot of lysates from a -ifitm or a -vector cell lines probed with the indicated antibodies. h) a cell lines were infected with increasing amounts of h n vn/ . twelve hours after infection the cells were immunostained for np expression and scored for infection status. values are representative of two independent experiments. (pdf) figure s ifitm is required for ifn's inhibition of ha-mediated fusion. a) hela cells were stably transduced with retroviruses containing either ifitm , a shrna against ifitm (shifitm ), or a non-targeting control shrna (shscr). cells were left untreated (left panels), or treated with ifn-c (right panels), then exposed for h to h n pps (wsn/ ) containing blam-vpr. after incubation with the pseudoparticles, the cells were fixed and assayed for cleavage of ccf by flow cytometry. figure s ifn treatment both expands rab -and ifitm -containing structures, and increases the size and number of acidified organelles. a) confocal images of wi- cells treated with buffer, ifn-a, or ifn-c, and then immunostained for either ifitm (endogenous, red) or rab (endogenous, green), and dna (blue). arrows denote larger structures staining for rab and ifitm that were seen predominantly with ifn-c treatment. (scale bar: mm). b) a cells treated with either buffer or ifn-c, then incubated with ltred before fixation and dna staining (blue) followed by confocal imaging. images in this figure are representative of three independent experiments. 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s-palmitoylation-dependent antiviral activity of ifitm the ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells human host factors required for influenza virus replication orthomyxoviridae: the viruses and their replication nef does not affect the efficiency of human immunodeficiency virus type fusion with target cells an enzymatic virus-like particle assay for sensitive detection of virus entry human mxa protein: an interferon-induced dynamin-like gtpase with broad antiviral activity regulation of ifn-alpha/beta, mxa, , -oligoadenylate synthetase, and hla gene expression in influenza a-infected human lung epithelial cells differential requirements of rab and rab for endocytosis of influenza and other enveloped viruses sequential roles for phosphatidylinositol -phosphate and rab in tethering and fusion of early endosomes via their interaction with eea ) p / sqstm binds directly to atg /lc to facilitate degradation of ubiquitinated protein aggregates by autophagy traf and a regulate lysine -linked ubiquitination of beclin- to control tlr -induced autophagy autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages influenza a virus strains differ in sensitivity to the antiviral action of mx-gtpase the viral nucleoprotein determines mx sensitivity of influenza a viruses where, in antiviral defense, does ifit fit ifit is an antiviral protein that recognizes -triphosphate rna influenza a virus expresses high levels of an unusual class of small viral leader rnas in infected cells acid ribonuclease from hela cell lysosomes different mechanisms of cell entry by human-pathogenic old world and new world arenaviruses dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells endocytosis of chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments a spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating hepatitis c virus entry requires a critical postinternalization step and delivery to early endosomes via clathrincoated vesicles viral entry: a detour through multivesicular bodies endosome-tocytosol transport of viral nucleocapsids the ifitm proteins inhibit hiv- infection trim is an innate immune sensor for the retrovirus capsid lattice evolution of highly pathogenic h n avian influenza viruses in vietnam between we thank thomas murooka and thorsten mempel (center for immunology and inflammatory diseases, massachusetts general hospital) for the kind gift of the modified pbr ieg-nef+ plasmid. we thank marina boyarina, karen rogers, melissa hinely and cynthia hanes for their managerial support. we thank the university of iowa hybridoma bank and the nih aids reagent repository for the generous supplying of reagents. we thank richard sutton and walther mothes (yale university medical school) and gregory melikian (emory university medical school) for helpful discussions. we thank alex forrest-hay, kim crawford, quan nguyen and ankit patel of panomics/affymetrix for their expertise and guidance with the viewrna studies. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention or the agency for toxic substances and disease registry. key: cord- -elom nx authors: yip, tsz-fung; selim, aisha sami mohammed; lian, ida; lee, suki man-yan title: advancements in host-based interventions for influenza treatment date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: elom nx influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. keywords: host factors, influenza, cytokines, metabolism, immunomodulation introduction influenza remains a source of public health concern. influenza a virus (iav) has been the cause of historical noxious pandemics, such as the spanish flu h n , asian flu h n , hong kong h n flu , and more recently the pandemic of h n (swine flu). influenza also causes seasonal epidemics and outbreaks with high morbidity and mortality rates such as the h n outbreak in india ( , ) . the error-prone nature of the viral rna polymerase (rdrp) and virus' capacity for genetic re-assortment (antigenic drift and shift) result in the viral components' susceptibility to mutations, allowing the viruses to evade the immune system and increases their resistance to control strategies. currently, influenza vaccination and two classes of antiviral drugs-m ion channel blockers (amantadine and rimantadine) and neuraminidase (na) inhibitor (oseltamivir, zanamivir, and peramivir)-and the novel treatment option using polymerase inhibitor (favipiravir) are considered as mainstays in influenza infection treatment and control. the use of influenza vaccinations remains challenging due to antigenic drifts and shifts, with seasonal variation of new circulating species. production of vaccine is time consuming with efficacy concerns, especially in the case of pandemic. variations in vaccine efficacy caused by age should be aware, with studies suggesting that vaccineconferred protection may not be optimal in certain age groups ( ) . the disadvantages of using the conventional antiviral drugs have also been a concern. significant levels of resistance to both classes of drugs have been repeatedly reported ( , ) . high level of resistance (up to %) to m blockers has been reported in h n virus strain in american isolates ( ) . resistance has also been reported in h n virus ( ) . iav resistance to na inhibitors has also become an increasingly prevalent concern, with the recent highly fatal outbreak of influenza a(h n )pdm in india associated with oseltamivir drug resistance ( , ) . in addition, a large cluster of influenza a(h n )pdm viruses in japan was found to have increased oseltamivir and peramivir drug resistance ( ) . there is an urgent need to search for alternative targets to treat influenza virus infections, including non-viral targets such as host cellular factors; which are promising as viruses rely on the host machinery for replication. while host immune response is intended to confer a degree of protection against the infection, an impaired or exaggerated host immune response could be detrimental-iav h n and h n virus infection was reported to exaggerate aberrant cytokine release, resulting in a cytokine storm that caused accelerated host death ( ) ( ) ( ) . many recent studies have focused on the investigation of targeting host factors to control virus replication as well as modulate immune response, which we have previously evaluated ( ) . in this review, we will discuss the latest studies (in the past years) on the investigation of novel host-based approaches with potential for influenza treatment. the replication cycle of iav can be grossly divided into four different stages: ( ) entry, ( ) genome nuclear import, ( ) replication and protein synthesis, and ( ) genome nuclear export, apical transport, assembly, and budding. as an obligate intracellular pathogen, iavs are heavily dependent on host machinery for replication and propagation. to this extent, studies employing genome-wide rna interference (rnai) to screen for host factors involved in iav replication cycle have been performed ( , ) and an increasing number of approaches targeting these host factors to control iav replication have been investigated. entry of iav into the host cell is divided into several steps ( , ) . first, hemagglutinin (ha) on the surface of iav binds to the terminal α-sialic acid on the host cell receptor. this induces the internalization of the viral particle by clathrin-dependent, caveolin-, and clathrin-independent endocytosis ( ) . macropinocytosis was revealed as an alternative entry pathway for iav ( ) , which subsequently enters the canonical endocytic pathway ( , ) . the vesicle-containing viral particle forms an early endosome (also known as sorting endosome), which matures into a late endosome as the endocytic pathway progresses. a gradual decrease in intraluminal ph from ph . to . , mediated by v-atpase proton pump ( ) , takes place as the endosome matures ( , ) . this ph drop in the endosomal lumen induces a conformational change in ha, which is activated by proteolytic cleavage to generate ha and ha from precursor molecule ha ( , ) . this conformational change triggers the fusion of the viral envelope with the endosomal membrane, releasing the viral genome into the cytoplasm. acidification of the endosome causes the subsequent acidi fication of viral lumen via the iav m proton channel ( ) , which in turn promotes the dissociation of m layer from both the viral envelope ( ) and the viral ribonucleoprotein (vrnp) complex ( ) . interestingly, a sharp decrease in ph from neutral to an acidic ph of . as utilized by acid bypass has been observed to be sub-optimal for viral replication. it is hence proposed that a gradual decrease in endosomal ph is necessary for sequential reduction in viral stiffness, dissociation of m from the np in the vrnp complex, destabilization of m layer from the viral envelope, and the eventual conformational change of the ha for the release of viral genome and proteins to the cytoplasm from late endosome ( ) . proteolytic cleavage of ha to ha /ha is an important step in iav replication. this cleavage relocates ha , converting previously uncleaved ha to a metastable conformation that induces membrane fusion at acidic ph ( ) . inefficient cleavage and activation of ha leads to low infectivity ( ) . as identified proteins encoded by the viral genome do not possess proteolytic properties, the virus is dependent on host protease for the cleavage of ha. this provides a potential target to control iav infection. ha is commonly cleaved by trypsin-like proteases at the single arginine residue at position . human airway epithelium serine proteases hat and tmprss were identified as the host factors for cleavage at this residue ( ) . aprotinin, purified from bovine lung ( ) , is a protease inhibitor with a long history of clinical use as an antifibrinolytic agent in cardiac surgery ( ) . its potential as an anti-iav drug has been recognized for over a decade ( ) and has been shown to reduce the infectivity of a broad spectrum of iav strains ( , ) both in vitro ( ) and in vivo ( ) . once withdrawn from the western drug market due to its association with mortality ( ), aprotinin has been approved as a locally administered, small-particle aerosol drug for the treatment of iav infection in russia ( ) . however, side-effects associated with the systemic administration of aprotinin raises the need for an alternative protease inhibitor for use in treatment of iav infections. camostat, a serine protease inhibitor, was reported to demonstrate anti-iav potential in mice dating back to ( ) , but little to no research has been conducted to develop it into an anti-iav treatment. it was revisited and proven to be one of the most efficient serine protease inhibitors for the inhibition of iav replication in primary human tracheal epithelial cells in vitro when tested compounds were used at similar molarities ( ) . at present, camostat is widely administered for the treatment of liver fibrosis, chronic pancreatitis, and cancer ( , ) , making it a highly promising candidate for drug repurposing. despite the lack of association between camostat and increased mortality (as with aprotinin), reports of camostat potentially inducing acute eosinophilic pneumonia ( ) warrants the need for careful consideration and further research into the repositioning of drugs from the same class. highly pathogenic iav, such as the h and h subtypes, are reported to have ha cleavage sites rich in basic residues ( ) . the polybasic nature of the cleavage sites provides multiple targets for a broad spectrum of proteases, including the more ubiquitously expressed intracellular proteases such as furin ( ) . this increased protease spectrum could be utilized by these viruses for the activation of ha prior to viral budding, allowing for evasion of potential inhibition by exogenously administered serine protease inhibitors. furthermore, an in vivo study utilizing mice treated with a single protease inhibitor prior to infection with h virus bearing a polybasic cleavage site showed poor efficacy despite good results were obtained for infection with h n virus bearing single cleavage site ( ) , suggesting strain specificity in using serine protease inhibitors to treat iav infections. endosomal acidification is required for the release of iav genome (in the form of a vrnp complex) into the cytoplasm ( ) . research has shown that an increase in endosomal ph during the early phases of infection could inhibit iav infection in vitro ( ) , bringing to light the possibility of controlling iav infection through the prevention of endosomal acidification. the v-atpase inhibitor bafilomycin a , when used at high concentrations ( - nm) has been proven to inhibit iav replication through the efficient suppression of v-atpase ( , ) . however, prominent cytotoxicity to host cells was also observed at such concentrations ( ) . interestingly, lower concentrations ( . nm) of bafilomycin a lack inhibitory effects on v-atpase attenuated iav replication due to disruption of endosomal trafficking. thus, bafilomycin a is suggested to exert its antiviral function via distinct mechanisms at differing concentrations. diphyllin, isolated from the plant cleistanthus collinus, is a natural compound able to induce a v-atpase inhibitory effect ( ) . in contrast to bafilomycin a , diphyllin is well-tolerated in vitro without inducing obvious cytotoxic effects ( ) . most notably, diphyllin is found to effectively inhibit replication of viral strains resistant to amantadine and/or oseltamivir ( ) . since drug resistance to these widely administered antivirals is of major public health concern ( ), diphyllin is regarded as a promising antiviral against drug-resistant iav strains. the release of iav genomic material during replication requires the fusion of the endosomal membrane with the viral envelope. since cholesterol plays a major role in controlling the fluidity of the lipid bilayer in cells, it is hence suspected to have a role in the infection cycle of iav. interferon-induced transmembrane proteins (ifitms) are proteins expressed in many vertebrates (including humans) and are found on the plasma membrane, the membranes of early and late endosomes, as well as on lysosomes ( , ) . while humans express ifitm , ifitm , ifitm , ifitm , and ifitm , only ifitm , , and are both immune-related as well as interferon (ifn)-inducible ( ) , and have been observed to restrict the replication of different viruses, including iav ( ) . studies suggest that ifitms limit viral infection by reducing membrane fluidity and hence restrict the hemifusion (the mixing of lipid bilayer without the release of viral content) of viral and endosomal membranes ( ) , probably via the disruption of cholesterol homeostasis of late endosomes, where viral fusion and genome release conventionally take place ( ) . a recent study using rnai also demonstrated that cholesterol homeostasis can be regulated via acid phosphatase (acp )-mediated niemann-pick c activity and impaired the membrane fusion of iav and influenza b virus (ibv) ( ) , further suggesting the importance of controlling cholesterol homeostasis in the release of viral genome to cytoplasm. on the contrary, later studies suggest that ifitm exerts its antiviral activity in a cholesterol-independent manner, showing that an increase in cholesterol composition of late endosomal membranes fail to inhibit viral membrane fusion ( ) . in addition, studies suggested the accumulation of cholesterol level in the late endosome does not inhibit the iav genome release into cytoplasm ( , ) . with the modulation of cholesterol levels in host endosomal membrane as a mean to inhibit iav host cell entry is still under debate, further studies are required before clear conclusions can be drawn. by comparing the mirna profiles of the iav-permissive hek t cells and (less permissive) hela cells, mirna- a has been identified as a negative regulator for iav infection via the inhibition of archain (arcn , also known as δ-copi) ( ). arcn is a subunit of the copi complex that is required for intracellular trafficking and endosome function ( ) , depletion of which has been reported to inhibit iav infection ( ) . despite impaired iav internalization caused by arcn depletion via sirna ( , ), it was not able to recapitulate through acute inhibition of copi complex by pharmaceutical means ( ) . it is hypothesized that the long-term (lasting days) perturbation on arcn by rnai affected the general endosomal trafficking network, a phenomena which cannot be recapitulated by acute pharmaceutical inhibition to block iav infection ( ) . the potential of targeting arcn for iav treatment deserves further investigation, despite the favorable results from rnai studies. nuclear import of vrnp complexes from the cytoplasm following fusion of the viral and the endosomal membrane is required for replication to take place ( ) . an early study suggested that vrnp complexes could be transported to the periphery of the nucleus ( ), while recent studies report that vrnp complexes utilize the importin-α-importin-β (impα-impβ ) system for nuclear import ( , ) and lacking of importin-α , in an importin-α knockout mouse model were found to be resistant to iav infection ( ) . ivermectin has long been clinically administered for the treatment of parasitosis ( ) , but has recently come to attention as a potential inhibitor of impα/β ( ) . ivermectin inhibition of impα/β has shown to inhibit the replication of rna viruses such as dengue virus and hiv- ( ) . ivermectin was recently tested for the inhibition of iav in vitro, with nuclear import of vrnp complex (of both wild-type and antiviral mxa escape mutant) efficiently inhibited ( ) . given ivermectin's longstanding record of clinical applications and fda-approved status, repurposing of this drug for the treatment of iav should be considered, especially while under threat of pandemic iav outbreak. following the import of the vrnp complex into the nucleus of the host cell, rdrp uses the vrna as a template to synthesize mrna or crna. synthesized crna remains in the nucleus for new vrna generation, while mrna is exported out of the nucleus for translation. viral protein products are either transported to the cell surface via golgi (in case of ha and na) or imported back into the nucleus to bind with vrna, forming new vrnp complex ( ) . numerous host factors are involved in this process and hence could be possible targets for therapeutic intervention. out of the eight genome segments of iav, the m and ns segments are well known for undergoing splicing to generate at least two different mrnas per individual segment ( , ) . cdc -like kinase (clk ) is a kinase which regulates alternative splicing of pre-mrna ( ) . inhibition of clk by the chemical tg or knockdown of clk is shown to cause a decrease in m mrna generation and disrupt downstream m protein expression, prominently reduced iav propagation ( ) . clypearin and corilagin were both found to be potent anti-iav compounds, with a higher therapeutic index than tg in vitro ( ) . clypearin is isolated from herbs used by chinese medicine practitioners for treating respiratory tract diseases during replication, viral mrna is exported from the nucleus to cytoplasm, where protein synthesis takes place. human rna polymerase ii activity is found to be correlated with iav replication through the inhibition of nuclear export of certain viral mrnas, such as m mrna ( ) . cyclosporine a (csa) is a fda-approved drug with immunomodulatory functions ( ) that has been found to have an anti-iav effect in both cyclophilin a (cypa)-dependent and -independent manners ( ) . the cypa-dependent effect was found to correlate with nuclear export of vrnp complex (see targeting nuclear export complex). the cypa-independent effect caused inhibition of host rna polymerase ii. csa is a prospective drug candidate for treatment of iav infections with a relatively high barrier for development of intrinsic drug resistance, as opposed to commonly used antivirals ( ) . nuclear rna export factor (nxf ) is a host factor that has been identified to be involved in the nuclear export of iav mrna. the knockdown of nxf in hek t cells revealed prominent viral mrna nuclear retention in host cell nucleus ( ) . protectin d (pd ), an endogenously produced lipid in the respiratory tract, has been identified to have potent anti-inflammatory and antiviral effects ( ) . pd production was notably found to be reduced in the lungs of iav-infected mice. therapeutic administration of pd was shown to significantly reduce iav mrna expression, lower lung viral titer, as well as improve survival of iav-infected mice. mechanistic studies revealed attenuated cytoplasmic translocation of viral mrna with such treatment. a decrease in recruitment of viral transcripts to nxf was observed while nuclear export of host rna remained largely unaffected, suggesting a role of pd in regulating nxf in nuclear export of viral rna. natural pd expression in the human airway makes this an ideal candidate for novel therapeutics in the treatment of iav infection. the eukaryotic initiation factor- a (eif a) family plays an important role in protein translation ( , ) . eif a impairment has been proven to be related to antiviral activity in a broad spectrum of rna viruses in vitro ( ) , with inhibition of iav mrna translation ( ) . the eif a inhibitors, silvestrol and pateamine a were demonstrated to arrest viral protein synthesis, thus blocking viral genome replication in vitro ( ) . although both silvestrol and pateamine a caused high cytotoxicity at the concentration required effective for iav inhibition, drugs targeting mrna translation for various diseases have been approved by fda or are under active development ( ) . as such, inhibition of iav infections by disrupting mrna translation may well be a therapeutic approach in the future. post-translational modifications during protein maturation ensure proper function of proteins, with proteins of iav no exception. nitazoxanide, a fda-licensed drug used to treat enteritis, was found to be effective in controlling iav infection by interfering with ha n-glycosylation as well as intracellular trafficking in host cell and eventually led to a reduction in viral budding ( ) . despite the mechanism of nitazoxanide being presently unknown, its ability to inhibit replication of numerous viruses [iav, respiratory syncytial virus, coronavirus, hepatitis b virus, and many others ( ) ] suggests that it may act on host machinery. the drug has also been proven in vitro to inhibit the propagation of many circulating strains of human iav, including those resistant to oseltamivir or zanamivir ( ) . nitazoxanide has a high barrier of resistance to iav ( ) and other viral strains resistance to neuraminidase inhibitors ( ), making it a very promising therapeutic target for iav treatment. the drug is currently under phase iii clinical trials ( ). in the later stage of viral replication, viral rnas of iav packed with rdrp and np (known as vrnp complexes) are exported from the nucleus ( ), assembled ( ) , and transported to the plasma membrane [apical in polarized cells ( ) ] for budding. novel targets for influenza treatment frontiers in immunology | www.frontiersin.org july | volume | article exportin (xpo , also known as crm ) is well known for its function in the nuclear export of protein ( ) and rna, including viral rna ( ) . similar to hiv ( , ) , iav viral rna does not directly bind to xpo but is instead held together by several viral proteins. the viral nuclear export protein (nep, or previously known as ns ) and the vrnp complex have been proposed as the nuclear export complex ( ) . cellular xpo has been proven to be crucial in the nuclear export of the vrnp complex, with early studies using leptomycin b (lmb), a potent xpo inhibitor, revealing that in vitro inhibition of xpo led to nuclear retention of vrnp complex ( , ) . however, lmb was deemed unsuitable for development as a potential drug in the phase i clinical trial due to observed cytotoxic effects ( ) . verdinexor (also known as kpt- ) is a new bioavailable selective inhibitor of xpo . it has been shown to be effective against different strains of iavs both in vitro and in vivo as prophylactic and therapeutic treatments ( , ) . it is worth mentioning that delayed administration of verdinexor at day post-infection was still deemed beneficial, with reduced viral load in vivo ( ) . this suggests a prolonged therapeutic time window when compared to the mainstay antiviral drugs such as oseltamivir, where recommended administration is at the early stage of infection (within h of symptom onset) ( ) . currently, verdinexor has passed the phase i clinical study trials, suggesting that it does not pose severe cytotoxic effects as lmb does. in addition, a recent report demonstrated that a new drug, dp -e , which binds and inhibits the function of xpo , can suppress iav replication in vitro ( ) further strengthens the concept of iav intervention by targeting xpo . viral m protein is crucial in assisting the nuclear export of vrnp complex. it was commonly suggested that m protein links vrnp complex to viral nuclear export protein nep which interacts with xpo for nuclear export ( ) . thus, viral m protein may serve as a target to inhibit nuclear export of vrnp. as previously mentioned (see inhibition of mrna export), csa inhibits iav replication via both cypa-dependent and -independent mechanisms. a recent study using a transgenic mice overexpressing cypa showed greater resistance to iav challenge ( ) . in the cypa-dependent mechanism, csa enhances the binding of cypa to m protein ( ) , increases the self-association of m , and hinders m nuclear import ( ) . csa also promotes the cypa-dependent degradation of viral m protein ( , ) . csa seems to be a promising drug to inhibit the nuclear export of vrnp complex by inhibiting viral m protein stability and function. recently, cd , a tetraspanin (defined by four transmembrane domains with conserved residues) that is expressed abundantly in lungs and interacts with integrins has been implicated in the regulation of iav replication in vitro and in vivo ( ) . knockdown of cd in primary human nasal epithelial cells resulted in the nuclear retention of host xpo , viral np, nep, and m proteins, with an increased survival rate observed in iavinfected cd knockout mice. co-immunoprecipitation assays suggest that cd interacts with viral np, m , and nep proteins ( ) ; however, the exact domains involved in interaction and the mechanism of cd function in nuclear export remain unclear. given that a small molecule inhibitor for cd is now under development ( ) , more data revealing the role of cd in iav infection and subsequent use in targeting cd as anti-iav therapy is anticipated. during iav infection, raf/mek/erk signaling cascade is activated, while the inhibition of mek by u , probably mediated via myosin (light chain) ( ), a known motor protein, impairs the nuclear export of vrnp complexes ( ) . suppressing iav replication by inhibition of raf/mek/erk signaling cascade has been illustrated both in vivo ( ) and in vitro ( ) . the replication of ibv ( ) as well as borna disease virus ( ) was shown to be inhibited by u , suggesting the versatility of this approach in controlling infection by different viruses. despite being effective when administered locally to lungs via aerosol, u has little effect when administered orally ( ) . another mek inhibitor, ci- (also known as pd ) was shown to have high potency against iav in vitro ( ). ci- has completed phase ii clinical trials as an anti-tumor drug, with the application of ci- as a potential anti-iav drug candidate recently revisited. unlike u , ci- is orally bioavailable and oral administration of ci- at h post-infection protected % of the iav-infected mice, while the oseltamivir-treated group experienced a % death rate ( ) . oseltamivir is known to be effective only when administered in the early stages of iav infection. this suggests the potential use of ci- as an agent used in iav treatment due to its potentially longer therapeutic time window than mainstay antivirals. formyl peptide receptor (fpr ) located at the host cell surface was identified as an erk stimulator ( ) . antagonizing fpr promoted the survival of iav-infected mice ( ) . furthermore, fpr antagonists have been described to possess antiviral activity against not only iav but also ibv infection ( ) , promoting the idea that antagonizing fpr to suppress raf/mek/erk signaling cascade could potentially be a novel approach for the treatment of a broad spectrum of influenza viruses. after the nuclear export of the vrnp complexes, host cell's intracellular transport mechanism is required to deliver vrnp complexes to the host plasma membrane for the assembly of viral rnas and proteins at the final stage of viral replication. among the various vesicular compartments found in a cell, the rab a + endosomes are known to recycle endocytosed membrane proteins and lipids to the plasma membrane for membrane homeostasis ( ) , a property utilized by many rna viruses, including iav ( , ( ) ( ) ( ) . iav progeny virus production was found to be significantly reduced in rab a + knockdown human cell lines ( ) . furthermore, vrnp complex plasma membrane transport perturbation was observed in rab a knockdown cells ( , ) ; in cells expressing deletion mutant of rab family interacting proteins ( ) ; as well as cells treated with chemicals to interfere microtubule ( ) . direct interaction of vrnp complex with rab a has also been verified ( , ) , demonstrating the dependence of vrna complex transport on rab a + vesicles and the microtubule network during viral replication. since rab a proteins do not confer any mobile properties to the vesicle, molecular motors such as kinesins are required for the active transportation of vesicles through cytoskeletons. kif a, a kinesin- family member, was recently identified as a molecular motor for plasma membrane transportation of vrnp-loaded rab a + vesicles ( ) . kif a knockdown was found to reduce progeny virus production. overexpression of a mutant form of kif a lacking in motor capacity resulted in disruption of the plasma membrane distribution of vrnp complex during later stages of infection. this data suggest that the apical transport of viral components via rab a or kif a could potentially serve as therapeutic targets against iav infection. further examination is merited. tubulin acetylation and deacetylation affects microtubule stability ( ) . histone deacetylase (hdac ) was found to deacetylate α-tubulin, one of the subunits of microtubule ( ) . a study has demonstrated that hdac is involved in iav replication ( ) . inhibition of hdac by tubacin or knockdown of hdac gene resulted in an increase of progeny virus production with vrnp complex redistributed toward the periphery of infected cells. in addition, transportation of ha to the plasma membrane for viral budding was also found to be inhibited by hdac . this data suggests that activation of hdac by its stimulant could be a potential approach to anti-iav therapy, despite hdac stimulants still being under development. while several studies have suggested iav transmission between cells through apical membranes ( ) and intercellular connections ( ) , virus budding from cell membranes remains the major route for transmission of viruses to uninfected cells. na is responsible for the cleavage of sialic acid to prevent the interaction between ha and the host cell during viral budding. besides, viral na, viral ha, m as well as m , are also suggested to play an important role in the initiation of the budding process ( , ) . in section "controlling cholesterol homeostasis, " we discussed the involvement of host cholesterol in viral membrane fusion and viral genome release to cytoplasm. recent studies have demonstrated that host cholesterol may also play an important role in viral budding. it was demonstrated that overexpression of annexin a (anxa ), a phospholipid binding protein, could lead to a decrease in cholesterol levels within the golgi apparatus and plasma membrane ( ), ultimately causing a reduction in egression of progeny virion from infected cells ( ) . this reduction could be reversed by the addition of exogenous cholesterol ( ) . similar to anxa overexpression, addition of a hydrophobic polyamine, u a, could reduce cholesterol level in plasma membr ane, also inhibited viral replication ( ) . since iav is assumed to bud from lipid rafts (cholesterol-rich plasma membrane domains) ( ) , it was demonstrated that anxa overexpression or u a treatment could hinder progeny virus production by lowering the cholesterol content in the plasma membrane. this hypothesis was strengthened through recent studies resolving the cholesterol-binding site of viral m protein, suggesting that iav m clustering (which provides membrane curvature for scission) is mediated by cholesterol ( ) . a recent report utilizing two different fda-approved cholesterol-lowering drugs, gemfibrozil and lovastatin, stated that there was reduction in stability and infectivity of progeny virus compared to that replicating within cholesterol-sufficient host cells ( ) . taken together, this data suggests that controlling cellular cholesterol content would be an effective alternative with drugs available for repurposing iav treatment. further in vivo works are needed to confirm this hypothesis. the gi-type g-protein coupled receptor α -adrenergic receptors (α -ars) have been recently identified as a key host factor involved in iav replication ( ) . apical transport of the viral protein ha is inhibited by low intracellular camp level after stimulating the α -ar-mediated signaling. in vitro stimulation of α -ar by its agonist clonidine inhibits iav replication. therapeutic administration of clonidine reduced pulmonary edema and improved survival rate of iav-infected mice. development of a new antiviral targeting the α -ar-mediated signaling seems promising and deserves further investigation. although targeting host factors for viral interventions generally provides a better resistance barrier, emergence of resistance may still arise ( ) . therefore, combined use of interventions targeting both virus and host factors have been recommended to reduce opportunities for viral development of resistance. one such example would be the combined administration of na inhibitor (oseltamivir) alongside an anti-host factor [such as v-atpase inhibitor diphyllin ( ) , ha maturation inhibitor nitazoxanide ( ) , fpr antagonists ( ) , and xpo inhibitor verdinexor ( ) ]. while further direct assessment for the ease of emergence of escape mutants between single and combinatory use of drugs is required, the synergistic effects of a combined, multi-drug approach observed thus far highly suggest an increased effectiveness over a single-drug approach. table summarizes novel host targets regulating iav replication. compared to rnai, small molecular chemicals remain the best choice as drug candidates due to their fast acting and easy-todeliver properties. although small molecular chemicals targeting certain host factors aforementioned have yet to be developed, their rnai-identified involvement in the iav replication cycle provide leads for the development of new iav interventions. the immune system aims to protect the host from infection and clear the pathogen once an infection occurs. in addition, the complex networks formed between the host physiology and the immune system co-operatively shape the disease outcome; modulations on the networks could alleviate disease severity in iav infections. the immunological responses elicited by iav infection has been reviewed in detail ( ) ( ) ( ) . at the initial stage of iav infection, the respiratory epithelial cells are the primary target for infection. once the infection is initiated, the recognition of infection is accomplished via the detection of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) (see toll-like receptors), and lead to the expression and secretion of different cytokines and chemokines, such as il- , il- , tumor necrosis factor (tnf)-α, and ccl as well as type i and iii ifns. as sentinel cells, alveolar macrophages could also be infected, inducing cytokines and is the main source of type i ifns ( , ) . type i ifns are known inducer for the upregulation of death receptor , which is the receptor for tnfrelated apoptosis-inducing ligand (trail), in lung pneumocytes ( ) . il- and ccl produced by both epithelial cells and macrophages act as chemoattractants for neutrophils and monocytes, respectively. neutrophils are one of the earliest immune cells being recruited to the site of infection ( ) with transmigration of neutrophils carry out by adhesion molecules, such as cd a, cd b, and cd ( ) . in addition to the antiviral activity of neutrophil-released reactive oxygen species (ros), defensin and pentraxin ( ) , uptaking iav by neutrophils could also help in controlling viral propagation as these cells do not support replication of iav ( ) . besides controlling viral replication, neutrophils also play an important role in guiding the migration of iav-specific cd + t-cells in the infection site by secreting and leaving a trail of cxcl ( ) . infiltrated monocytes will, however, differentiate into macrophages or dendritic cells (dcs). the monocytes-derived macrophages are reported to be a permissive host for iav production ( ) , sustaining inflammation by producing cytokines in a magnitude larger than that of the resident alveolar macrophages. the monocyte-derived dc as well as the resident airway cd c low b + plasmacytoid dc (pdc) and two types of conventional dcs (cd + cd b low and cd − cd b hi ) acquire the antigen of the invading pathogen through either direct infection or up-taking infected dead cells ( ) . in the presence of type i ifns, dcs mature when encountering pamps from invading pathogen ( ) . depending on the sub-cellular localization of the antigen, cytosolic and endosomal antigen will be loaded onto major histocompatibility complex (mhc) class i and ii molecules respectively ( ) . once mature, dcs migrate from the infection site to the draining lymph nodes via the interaction of ccr and ccl /ccl ( , ) for antigen presentation via mhc class i and ii to naïve cd + and cd + t-cells, respectively ( ) ( ) ( ) ( ) . interestingly, monocytesderived dcs that engulfed the infected dead cells are poor antigen presenters for cd + t-cells and require the transfer of intact mhc class i/peptide complex to lymph node-resident cd α + dcs which are the most efficient antigen-presenting cells to cd + t-cells ( ) . in addition to antigen presentation, pdc are well known for their high ability in type i ifns production to limit viral propagation ( ) . within the lymph node, naïve cd + t-cells are activated by the dcs, differentiate and clonal expand into cytotoxic t-lymphocytes (ctls) with the aid of various cytokines, including ifn-γ, il- , type i ifns, and il ( , ) , and the help from activated cd + t helper cells ( ). differentiated ctls downregulate their lymph node homing receptor ccr and upregulate ccr and cxcr for the migration to the site of infection. within the site of infection, ctls control viral replication by targeting and inducing apoptosis of virus-infected cells via the secretion of perforin and granzymes as well as the ligation of death receptors on the infected cells by tnf, fas ligand, and trail. on the other hand, cd + t-cells are activated by the presentation of mhc class ii/ antigen complex by dcs, with co-stimulatory receptors such as cd expressed on the t-cells and the ligand for cd (cd and cd ) expressed on dcs playing an important role ( ). activation of cd + t-cells lead to differentiation into different effector cells subsets, including the classical th and th , and the more recently identified regulatory t cells, follicular t helper cells, th , and th subsets ( ). th cells regulate to the differentiation of ctls as mentioned whereas th cells contributes to the activation of b-cells through cd l. within the pregerminal center of the lymph node, the follicular t helper cells interact with antigen-primed b-cells and promote their proliferation. antigen-primed b-cells differentiates into plasmablast and undergo antibody class-switching in the germinal center ( ) . detailed functions of regulatory t cells, follicular t cells, th , and th cells are discussed elsewhere ( , ). plasmablasts enter the blood-stream, are recruited to the inflamed tissue, and terminally differentiate into plasma b cells which specialize in the production of antibody for pathogen neutralization, opsonization, and antibody-dependent cell-mediated cytotoxicity, etc. memory t-and b-cells are also developed during the maturation process, and has been discussed and reviewed elsewhere ( ) ( ) ( ) ( ) . a schematic diagram showing a summary of the immune response after iav infection has been illustrated in figure . the yin and yang theory is always used to describe the importance in balancing the host immune response. in the light of this theory, the treatment strategy aims to suppress the overwhelming activation of the host immune response and in reverse to compensate any unfavorable suppression. although adaptive immune responses are important in viral clearance, the immediate innate immunity play an important role in the early control of an infection, and conversely, is a major factor for disease severity due to immunopathology. dysregulated immune responses caused by viral infections have been implicated in severe disease development ( , ) , such as acute lung injury (ali). ali in its most severe form, known as acute respiratory distress syndrome (ards), is reported to be the most prevalent cause of mortality in iav-infected patients ( ) . studies suggested that iav strains could be associated with either over-activating (human infection by avian h n and h n ) ( , ) or suppressing (h n , h n ) ( ) immune response. recent history has seen the outbreak of iav pandemics of varying severity takes place at the cost of millions of lives. one such example would be the deadly spanish flu of , which claimed the lives of - million of the million people infected worldwide. the pathological examination of lung sections from mice infected with reconstituted iav virus revealed necrotizing bronchiolitis and severe alveolitis in tissue, with neutrophils observed as the predominant inflammatory cell type present ( ) , suggesting neutrophil involvement in the pathogenesis of iav infection. the majority of immune cells in blood circulation are neutrophils; of which they are among the first innate immune cells recruited to the site of infection ( ) . neutrophils characteristically control microbial infections by generating bactericidal ( ) neutrophil extracellular traps (nets), consisting of granule proteins, histones, and decondensed chromatin ( ) . both protective and destructive role of neutrophils in iav infections have been described. the contrasting role of neutrophils could be explained by factors such as viral strain and viral dose used in different experimental setup, etc. the protective role of neutrophils was observed when mice infected with a low, non-lethal dose of iav h n strain hkx displayed neutrophil-mediated viral clearance via phagocytosis ( , ) . depletion of neutrophils has found to enhance viral load in the iav-infected animals ( ) . on the contrary, this protective nature is disputed due to the association of neutrophil-generated nets. extensive net formation was observed in mice infected with pr , an iav strain highly pathogenic to mice ( ) . histones and myeloperoxidase within the net induce cell death of lung epithelium and endothelium ( ) , leading to the loss of integrity of the alveolarcapillary barrier, a characteristic of ali. yet, while histones have been shown to suppress iav replication in vitro ( ) , in vivo study demonstrated that there was increase in lung inflammation and damage in iav-infected mice treated with histones ( ) . interestingly, co-treatment of lethally infected mice with anti-histone antibody and oseltamivir resulted in an increase in animal survival when compared to infected mice groups treated solely with oseltamivir ( ) . in agreement with the in vitro and in vivo data, it has been reported that net produced by cultured neutrophils from patient with h n and severe h n infection increased alveolar epithelial cell permeability ( ) leading to ali. more importantly, plasma net level positively correlated with the disease severity index (including higher acute physiology and chronic health evaluation ii score) and multiple organ dysfunction syndrome ( ) , further demonstrating the detrimental role of net in the pathogenesis of severe iav infections. studies have demonstrated the involvement of superoxide dismutase and myeloperoxidase in netosis, the formation of net ( ) . the presence of anti-myeloperoxidase antibody as well as the superoxide dismutase inhibitor (detc) significantly reduced netosis. finally, tetrahydroisoquinolines ( ) and a panpeptidylarginine deiminase (pad) inhibitor, named cl-amidine ( ) have been suggested to inhibit netosis. despite it has been reported that during iav h n infection, pad knockout mice displayed only slight improvement in weight loss and a slight prolonged but no end-point survival advantage was observed compared to wt mice ( ) , based on the extensive findings presented above, targeting net to prevent ali in the severe case of iav infection, including the highly pathogenic avian iav, remain promising and may warrant further investigation. innate lymphoid cells are cells of lymphoid lineages that do not express antigen-specific b-or t-cell receptors ( ) . similar to t-helper cells, they are classified into subsets by their ability to produce type (th ), type (th ), and type (th and th ) cytokines. previous studies confirmed the involvement of ilcs of group linage (ilc ) in iav infection and airway inflammation ( , ) . on the positive side, during the recovery phase of iav infection, ilc expresses amphiregulin which promote airway epithelium repair ( , ) , thus facilitating the recovery of the infected lung. on the other hand, in response to il- produced by macrophages, dcs, and nkt cells, ilc secretes il- and il- and induce airway hyper-responsiveness. recruitment of eosinophils by il- to the lung also mediates airway inflammation ( ) . since eosinophilia is a characteristic of allergic asthma and influenza is a major cause for morbidity and mortality in asthma patients ( ) , it will be of particular interest to investigate the role of ilc in iav infection, particularly in asthma patients. ilc s have been initially described as immature nk cells residing in the liver and share many phenotypic similarities with nk cells ( ) . it was recently appreciated that tissue-resident ilc s other than the previously recognized nk cells are the major early source of the antiviral ifn-γ at the primary site of various viral infection, including iav ( ) . interestingly, ifn-γ was found to suppress ilc activity and reduce il production which exacerbates disease severity during influenza a(h n ) pdm infection ( ) . this data may highlight a link between ilc and ilc and suggesting ilc can suppress ilc activity via ifn-γ production during iav infection. with ilcs finally identified, functions of these cells and their role in immune response to tumors and pathogen infections have been massively investigated in recent years. type i ifns, prostaglandin i , corticosteroids, and testosterone have been reported to suppress ilc activity ( , ) . in addition to il- , the epithelial cytokines il- , thymic stromal lymphopoietin, as well as the lipid mediator prostaglandin d were found to activate ilc ( ) . the therapeutic potential of these ilc activators and suppressors is yet to be deduced. with more and more studies demonstrating the involvement of ilc in iav infection, the interplay between different ilc subtypes in iav infection would, therefore, be an interesting area to explore and modulate the ilc activity may be a future approach to combat iav infection. reactive oxygen species, generated by specialized enzymes such as nadph oxidases, are released during iav infection ( ) . the nadph oxidase family consists of enzymes containing different catalytic subunit named nox - and dual oxidase (duox) and . ros have been reported to display both beneficial (limiting viral replication) and detrimental (promoting ali) effects in the course of iav infection. interestingly, the protective or destructive effect of ros is dependent on the enzyme of which the ros is generated ( ) . dual oxidase and are found to be host-protective ( , ) . in vitro, ros generated by nuclear duox indirectly regulates the splicing of iav mrnas via the nuclear speckle-associated splicing complex ( ) . in addition to altering viral mrna splicing, ros generated by doux has been attributed to the production of ifn-λ, an important anti-iav ifn. in response to iav infection, increased viral mrna replication was observed when duox was silenced in vitro ( ) . increased viral replication was also observed in mice with doux silenced ( ) , further depicting the protective role of doux in iav infection. unlike doux, nox activation could be harmful to host. iav infection was reported to induce nox -dependent endosomal ros production ( ) . ros could target the conserved cys on toll-like receptor (tlr) , and inhibit tlr -mediated type i ifn expression during a mild iav h n infection in vivo ( ) . iav-infected mice treated with specific nox inhibitor, cholestanol-conjugated gp ds-tat, were found to have reduction in endosomal ros production, restored tlr activity, and displayed a decreased viral load ( ) . in addition to nox , nox dependent ros production has also been reported to activate mapk/erk signaling ( ) , enhancing the export of vrnp complex, thus increasing viral replication (see targeting the raf/ mek/erk pathway). nox knockdown resulted in a reduction of viral replication in vitro ( ) . targeting the different nadph oxidase isoforms, instead of scavenging ros should be considered as the therapeutic approach for iav infection, as doux-mediated ros production is beneficial ( , ) , while nox and nox are harmful during iav infections ( , ) . finally, ns (not to be confused with iav ns protein) has been demonstrated to be a nox inhibitor, which could inhibit the activity of nox , nox , and nox . a study demonstrated that ns suppresses iav-induced nox and significantly inhibits iav virus replication ( ) . besides cholestanolconjugated gp ds-tat and ns aforementioned, apocynin, a phagocytic nox inhibitor as well as ros scavenger ( ) ( ) ( ) , has been demonstrated to ameliorate hyper upregulation of cytokines induced by iav infection through socs and socs in vitro ( ) and reduce peri-bronchial inflammation and viral titer in vivo ( ) . interestingly, ebselen, another nox inhibitor and glutathione peroxidase mimetic, could reduce inflammatory status measured in bronchoalveolar lavage fluid (balf) of mice pre-exposed to cigarette smoke and subsequently infected with iav ( ) . taken together, these reports highlight the potential use of nadph oxidases inhibitors and ros scavengers to treat iav infections. dysregulated cytokine production has been associated with the elevated mortality rate observed in severe iav infections ( , ) . as such, the immunomodulation of cytokines are regarded as promising therapeutic tactics. recent advancements developed with this approach will be highlighted in the following section. tumor necrosis factor has two main functions during viral infection-it activates nf-κb, inducing the expression of cytokines responsible for the host immune response; and induces apoptosis through activation of a signaling cascade involving tradd, fadd, and caspase , , , and ( ) ( ) ( ) . tnf is known to be highly upregulated in iav-infected hosts, especially in hosts infected with highly pathogenic iav ( , ) . however, it is both protective and counter-protective functions associated with tnf that makes it a target in the treatment of iav. the protective role of tnf is observed during infection by low pathogenic iav, where extrinsically derived tnf is responsible for attenuating tissue-damaging cd + t-cell response ( ) . in addition to recruiting monocytic cells to the infection site, cd + t-cells response was observed to deteriorate lung pathology ( ) and damage healthy, non-infected lung epithelial cells ( ) upon iav infection. furthermore, tnf deficiency has been associated with an increased detection of il- and il- in balf ( ) , which promote the survival of and proliferation of cd + t-cells ( , ) and subsequent tissue damage. exacerbated lung pathology caused by the upregulation of the monocyte chemoattractant protein- was observed in tnf −/− mice infected with sub-lethal dose of iav ( ) . in addition, decreased cd + t-cell contraction due to enhanced expression of the anti-apoptotic protein bcl- was observed in sub-lethally iav-infected tnfdeficient mice when compared to wt mice ( ) . as a whole, there is substantial evidence supporting the protective role of tnf in iav infection. on the other hand, the correlation of tnf with pulmonary edema has been well-documented ( ) . tnf has been observed to stimulate the expression of cxcl in alveolar epithelial cells in a transgenic mice model resembling extensive iav infection in lung tissue, causing alveolar damage, lung edema, and hemorrhage ( ) . in addition to lung edema, tnf has also been reported to correlate with iav-associated encephalopathy ( , ) . however, it is notable that despite iav-associated encephalopathy, direct invasion of the central nervous system is rare ( ) , suggesting that iav-associated encephalopathy could instead be a result of peripheral infection. furthermore, tnf has been shown to increase the permeability of the blood-brain barrier (bbb) ( , ) , contributing to neural damage ( ) . these studies further support an anti-tnf approach as a potential therapy for severe iav infection. at present, etanercept, an anti-tnf drug administered in the treatment of rheumatoid arthritis, is the only tnf inhibitor (or even tnf directed treatment) tested for iav treatment. etanercept has been shown to protect against the in vivo lethal infection of mice with a highly virulent, mouse-adapted iav strain ( ) , with observations made of an increased survival rate with decreased morbidity, expression of the proinflammatory cytokine il- , lung injury, and edema ( ) . the protective role of il- was demonstrated in mice challenged with sub-lethal iav infection. il- -deficient mice displayed exacerbated pulmonary damage ( , ) and lung injury due to an observed decline in the survival of alveolar type ii cells and alveolar epithelial cells ( ) . iav suppresses the anti-apoptotic mcl- and bcl-xl expression, causing cell death of neutrophils which are critical in viral clearance ( ) . addition of il- restored the expression of mcl- and bcl-xl in vitro and is considered as the underlying mechanism for the observed survival advantage of wt mice over il- knockout mice during mild iav infection. il- has also been shown to induce the proliferation of lung il- + regulatory t cells and il- , which act to limit excessive proliferation of cd + t-cells and subsequent cd + -inflicted damage. this would hence prevent the tissue damage observed in lung immunopathology ( ) . despite the apparent protective role of il- , high levels of il- in serum or cerebrospinal fluid have been reported in severe neurologically complicated iav cases, with il- used as a marker for prognosis ( - , , ) . the role of il- in regulation of bbb permeability was reported ( ) , with potentially detrimental neurological complications. as such, the suppression of hyper-induced il- as a form of therapy in severe iav infection should be considered. one such option is the anti-il antibodybased drug tocilizumab, which is currently administered clinically for the treatment of rheumatoid arthritis. however, study on the usage of this drug to treat hyper upregulation of il- due to severe iav infection has yet to be conducted. on the other hand, in a case of h n virus-induced ards, the use of an extracorporeal cytokine hemoadsorption device to remove cytokines including tnf and il- from the bloodstream ( ) has showed beneficial to the patient ( ) . more research is required to confirm whether the removal or neutralization of il- could be a potential therapy for severe iav infections. the activation of cd + t-cell is crucial for viral clearance. it should, however, be tightly regulated to limit cd + t-cell inflicted host cell damage. il- mediates il- induction ( ) . il- acts to suppress cd + t-cells and reduce morbidity through il- and regulatory t-cells ( ) . much like other immunomodulatory approaches, the timing for applying il- should be carefully assessed. compared to placebo-treated iavinfected group, early administration of il- to iav-infected mice in fact led to poorer viral clearance, increased morbidity, and deteriorated lung histopathology, while il- administration during the recovery phase ( - days post-infection) accelerated recovery and improve lung immunopathology ( ) . notably, il- could also suppress th responses and increases susceptibility to secondary s. aureus infection ( ) . therefore, co-administration of antibiotics should be considered when utilizing il- as potential iav treatment. both type i and iii ifns have antiviral properties, with viruses counteract ifns to gain an advantage for their propagation. the iav viral protein ns inhibits the production of ifns by antagonizing irf- , a key transcriptional factor for ifns. this prevents the processing of cellular pre-mrnas (including those for ifns) and directly interacts with retinoic acid-inducible gene (rig)-i receptors, which are critical in innate sensing, to suppress ifn production during infection ( , ) . in addition to inhibiting ifn expression, the induction of socs inhibits ifns signaling by suppressing cytokine signaling has been documented ( ) . the recognition of ′ triphosphate on viral rna by rig-i receptor is shown to induce the expression of socs , which in turn represses type i ifns expression ( ) . due to ifns being a key contributor to antiviral immune response, an impairment of type i or iii ifn production may cause the escalation of otherwise mildly pathogenic iav infection into a life-threatening one ( ) . while type i ifn has been demonstrated to inhibit iav replication in vitro ( ) ; the in vivo administration of type i ifn in animal models only displayed effectiveness in a prophylactic capacity. a lowered viral titer was detected in the nasal wash of test animals. however, host susceptibility to iav infection remained unchanged ( ) . notably, this protective effect is only conferred by an optimal dose of type i ifn of low to moderate amounts ( - units per mice daily); with higher dosages ( , - , units per mice daily) shown to increase morbidity ( ) . in addition, clinical trials demonstrated that prophylac tic administration of type i ifn reduced disease severity and lowered susceptibility to iav in males and participants aged or above ( ) . despite relatively successful results seen in the prophylactic use of ifns, its therapeutic use is of greater clinical relevance. mice treated with type i ifn post-iav infection showed a successful reduction in lung iav titer but displayed increased morbidity and mortality in comparison to vehicle-treated mice ( ) . a possible explanation for this phenomenon is the induction of excessive inflammatory response and trail-dr -mediated epithelial cell death by type i ifn ( ) , which accounts for the observed lung pathology in iav-infected animals treated with type i ifn ( ) . in addition, downregulation of γδ t-cells by type i ifn has been correlated with increased susceptibility to secondary s. pneumoniae infection ( ) , further arguing against the potential use of type i ifns for the treatment of iav infection. in comparison to type i ifns, the administration of type iii ifns may provide advantages in the control of iav replication ( , , ) without the risk of previously reported type i ifns-mediated immunopathologic side-effects ( , , ) . however, a recent study aiming to stimulate ifns signaling through the systematic administration of rig-i ligand post-iav infection demonstrated that type i, but not type iii ifns signaling is important in conferring protection during fatal iav infection in vivo ( ) . though, this study did not measure the production of type i and iii ifns as well as any changes in viral load with respect to ifnar or ifnlr knockout. in addition, while human immune cells are not primary targets in iav infection, they could be susceptible to iav and become efficient host cells for virus replication. they are reported to possess a subpar response to type iii ifns ( ) ; leading to the preliminary conclusion that solely using type iii ifn as treatment may not be feasible. as such, reports suggesting the use of type iii ifns over type i ifns as a front-line therapeutic agent to counter iav infections may require further investigation. the inhibition of cox- by selective inhibitors, nimesulide and celecoxib, was previously demonstrated to suppress the hyper upregulation of pro-inflammatory cytokines induced by highly pathogenic avian iav ( ) ( ) ( ) . in addition, the use of zanamivir in tandem with a specific cox- inhibitor was shown to increase the survival rate of mice lethally infected with avian h n iav, when compared to mice treated solely with zanamivir ( ) . activated cox- regulates downstream prostaglandin production. one such example is pge , a major type of prostaglandin recently demonstrated to play an important role during iav infection. pge was significantly upregulated in response to iav infection, leading to the inhibition of antiviral type i ifn production in macrophages and the subsequent increase in virus replication ( ) . the use of chemicals ah and gw x to antagonize pge downstream signaling molecules ep and ep respectively, was shown to induce antiviral type i ifn production. the in vivo treatment of mice lethally challenged iav with both ep and ep antagonists significantly improved the survival rate. a recent study demonstrated the ability of a modified tcm decoction to reduce peg production and subsequent morbidity in mice lethally challenged with iav. improved lung pathology was observed ( ) . the long history of clinical tcm use supports the clinical feasibility of peg inhibition as an option to treat severe iav infections. pattern recognition receptors on host cells sense specific pamps present on the viral surface or generated during replication. prrs can be broadly divided into two classes by their function or location. when defined by location, prrs are classified into groups-membrane-bound (tlrs and c-type lectin receptors), cytosolic (rig-i-like and nod-like receptors), and secreted (collectins and pentraxins) ( ) . significant research has been conducted on prrs with regards to iav infection. tlrs and rig-i receptors have been extensively studied for their major roles in eliciting host immune responses (cytokine and ifn expression) during iav infection ( ) ( ) ( ) . rig-i receptors have been investigated for their functional relevance to iav infection and targeting these receptors as a form of iav treatment has been extensively reviewed ( ) ( ) ( ) . this section will cover recent research on tlrs and the targeting of different tlrs to treat iav infection. humans have been identified to express tlr - , while mice have been identified to express functional tlr - as well as tlr - ( ) . most tlrs-with the exception of tlr utilize myd as an adaptor protein during signal transduction. tlr utilizes trif as an adaptor. tlr is known for its ability to utilize either myd or trif, with the choice of adaptor dependent on its sub-cellular location ( ) . different tlrs, such as tlr , , and ( ) as well as tlr , tlr , and most recently tlr ( ) , have been revealed to play a role in the orchestration of host immune responses contributing to iav pathogenesis. with tlr being an exception ( ) ( ) ( ) , tlr activation largely causes the release of pro-inflammatory cytokines, with hypercytokinemia leading to ali as a major cause of mortality in severe iav infections. in addition to dysregulated cytokine release, excessive production of ros has been associated with ali development. in fact, lung injury during severe pulmonary infections, such as iav and sars, could be caused by oxidative stress ( ) . iav infection activates nadph oxidase that subsequently produces oxidized papc, an endogenous phospholipid. the oxidized papc serves as an agonist for tlr , activating a tlr -trif-traf -nf-κb signaling cascade to eventually trigger the release of il- , ultimately inducing the onset of ali. in addition to oxidized papc, the induction of endogenous protein s a upon intracellular prr ddx recognition of iav subsequently induces the activation of tlr , further contributing to iav-induced mortality ( ) . since tlr has been proven to be important in ali induction (and hence iav-related mortality), manipulating the stimulation and antagonism of tlr could potentially reduce the severity of iav infections. eritoran (e ) is a specific tlr antagonist initially purposed for the treatment of sepsis, but a failed a phase iii clinical trial due to improved patient care in the placebo group prevented its eventual use in sepsis treatment ( ) . in vivo administration of eritoran in mice lethally infected with iav resulted in improved clinical score, lung pathology results, and reduced viral titer. delayed administration of eritoran, at day after infection beyond the recommended therapeutic time window (within h after the first display of clinical symptom) for use of oseltamivir ( ), also demonstrated a significant benefit to infected mice compared to non-treated group, suggesting a prolonged therapeutic time window for iav treatment when compared to mainstay antiviral drug treatment. a newer and structurally simpler specific tlr antagonist, fp ( ), alongside a newly developed decoy peptide r that has been shown to disrupt tlr , , , and signaling via tirap, has been shown to protect mice from lethal iav infection ( ) . these results support the potential use of tlr antagonism as a means to treat severe iav infection. the suppression of other tlr signaling pathways-such as blocking tlr -mediated signaling through the use of an anti-tlr antibody, significantly protected against lethality when administered on day and post-iav infection ( ) . a study also demonstrated that h n -infected tlr knockout mice had better survival than h n -infected wild-type mice, which is evident through the significantly faster regaining of body weight post-infection, lower viral titer in the lung, and fewer pathological changes in the lung ( ) . an increasing number of tlr antagonists are now under development ( , ) , alongside several other agents also shown to have effects on tlrs. polysaccharides isolated from r. isatidis, a traditional chinese medicinal herb used to treat iav infection, have recently been shown to inhibit pro-inflammatory cytokines such as il- and ccl- in vitro by down-regulating upstream tlr expression ( ) . menk, an endogenous protein expressed in the adrenal medulla, was shown to both prophylactically and therapeutically increase the survival rate while reducing viral-caused lung pathology and viral titer in mice lethally challenged with iav ( ) . this was determined to be caused by the downregulation of tlr . these results suggest the potential of down-regulating tlr expression in the treatment of iav infection. the above-mentioned data suggest modulation of tlr signaling or expression as a promising approach in treating severe influenza disease and deserves immediate investigation. table summarizes new immunomodulatory approaches to combat iav infections. it is well documented that patients with diabetes mellitus have a greater tendency to develop severe iav infection than healthy patients ( ) . hyperglycemia increases susceptibility of the host to iav infection via viral uptake, through the promotion of v-atpase assembly ( ) and immunosuppression ( ) . in addition, viruses rely on host metabolism to perform essential functions during replication ( ) ( ) ( ) ( ) . these processes exert a large energy demand on the host within a very short period of time ( ) ; energy of which is supplied by and is dependent on host metabolism. iav viruses have been reported to modify the metabolic state of the host. for example, increased c-myc-dependent glycolysis and glutaminolysis has been demonstrated in infected cells ( ) . the changes in glucose and glutamine metabolism were reversed upon the addition of bez , which inhibited the iav-mediated c-myc induction. administration of bez days prior to infection and up to days post-infection was shown to decrease lung viral titer and improve the survival rate in iav-infected mice. small molecules such as clotrimazole and α-mangostin that target lipid metabolism have also been demonstrated to suppress iav replication in vitro ( ) . in addition to being important for generating energy and biosynthesis, recent research demonstrates that cellular metabolism affects immune cell function. dysregulated immune responses observed in many diseases are associated with specific metabolic configurations. viruses, influenza inclusive ( ) , were found to induce drastic alterations in metabolic levels and programs ( ) . macrophages in infected hosts were observed to have marked differences in the krebs cycle, a key metabolic pathway. this is of significance due to the role of macrophages, which are immune cells critical in the pathogenesis of many inflammatory diseases ( , , ) . in activated macrophages, succinate, a krebs cycle intermediate, was found to possess inflammatory signal. accumulation of succinate generates ros, leading to subsequent activation of hypoxia-inducible factor α and the induction of cytokines such as il- β ( ) . a recent study identified the ability of itaconate, another krebs cycle-derived metabolite, to block the production of inflammatory factors. this prevented inflammation, protecting mice from lethal levels of inflammation that can occur during infection ( ) . this data suggest the critical roles of krebs cycle intermediates in regulating cytokine profiles and inflammation. metabolites generated by innate immune cells in distinct configurations could have different roles beyond that of bioenergetics, with functions in signaling regulation, transcription, and orchestrating innate immune responses. despite the lack of research conducted thus far on the application of immunometabolic approaches to influenza treatment, the prospect of manipulating immune responses by modulating immune cell metabolic state is promising. further research should focus on the identification of metabolites for modulation of immune cell function with substantial improvement of therapeutic strategies to treat iav disease. latest advancements in high-throughput technologies, e.g., meta bolomics is a useful approach to systematically investigate the changes of metabolic mechanisms during iav infections. identification of important metabolites involved during iav infection should be a new approach by modulating the host metabolism for interventions. multiple host-based intervention strategies against influenza have been developed or are under development. while approaches targeting host machinery required for virus replication seem to be promising thus far, additional research is needed to determine the effect of modulating host immune response on influenza treatment. this is increasingly important, since targeted host factors may play distinct roles in response to infection by different influenza viral strains ( ) , making the management of influenza through solely targeting a single specific host factor is difficult. host-based interventions offer obvious advantages over conventional antivirals, such as a higher barrier to drug resistance ( , , ) due to greater genetic stability of host factors than the mutation-prone nature of viral components. in addition, administration feasibility is a key factor to consider the usage of drugs. the mainstays of antivirals for iav infections, the na inhibitors, and m blockers, are recommended to be administered within h of symptom onset for optimal antiviral activity. this short treatment window may not be fully fulfilled in a clinical setting. novel host-based interventions were reported to have therapeutic time windows longer than this conventional timeframe ( , , , ) , even up to days post-infection ( ) , providing a clear clinical advantage over na inhibitors and m blockers. in addition, hypercytokinemia and ards could contribute to disease severity and mortality in instances of severe influenza infection, with virustargeting antivirals providing little to no alleviation of such complications. since host immune response is indispensable in host defense against invading pathogens, the use of immune-modulators to suppress detrimental effects while retaining beneficial protection of the host remains challenging. the timing and dosage of medication administration would be critical in determining the drug effectiveness in influenza treatment. targeting virus-induced metabolic changes to restore host normal metabolism may be a new direction to combat influenza disease. further research in the immunometabolism field, along side studies on modulating immune response to infectious disease by altering host metabolic processes; would create a new direction for future research and is expected to yield significant discoveries that may provide new therapeutic options in the treatment of iav infections. smyl conceptualized the work. il and asms drafted some review sections and tfy and smyl wrote the manuscript. influenza a(h n )pdm outbreak detected in inter-seasonal months during the surveillance of influenza-like illness in pune avian influenza a (h n ) virus infections in humans across five epidemics in mainland china age and influenza-specific pre-vaccination antibodies strongly affect influenza vaccine responses in the icelandic population whereas disease and medication have small effects global transmission of oseltamivir-resistant influenza influenza a(h n )pdm virus exhibiting enhanced cross-resistance to oseltamivir and peramivir due to a dual h y/g r substitution high levels of adamantane resistance among influenza a (h n ) viruses and 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hif- alpha itaconate is an anti-inflammatory metabolite that activates nrf via alkylation of keap key: cord- -fgwf wy authors: wang, ben x.; fish, eleanor n. title: the yin and yang of viruses and interferons date: - - journal: trends immunol doi: . /j.it. . . sha: doc_id: cord_uid: fgwf wy interferons (ifns)-α/β are critical effectors of the innate immune response to virus infections. through activation of the ifn-α/β receptor (ifnar), they induce expression of ifn-stimulated genes (isgs) that encode antiviral proteins capable of suppressing viral replication and promoting viral clearance. many highly pathogenic viruses have evolved mechanisms to evade an ifn response and the balance between the robustness of the host immune response and viral antagonistic mechanisms determines whether or not the virus is cleared. here, we discuss ifns as broad-spectrum antivirals for treatment of acute virus infections. in particular, they are useful for treatment of re-emerging virus infections, where direct-acting antivirals (daas) have limited utility due to daa-resistant mutations, and for newly emerging virus strains in which the time to vaccine availability precludes vaccination at the onset of an outbreak. interferons (ifns)-a/b are critical effectors of the innate immune response to virus infections. through activation of the ifn-a/b receptor (ifnar), they induce expression of ifn-stimulated genes (isgs) that encode antiviral proteins capable of suppressing viral replication and promoting viral clearance. many highly pathogenic viruses have evolved mechanisms to evade an ifn response and the balance between the robustness of the host immune response and viral antagonistic mechanisms determines whether or not the virus is cleared. here, we discuss ifns as broad-spectrum antivirals for treatment of acute virus infections. in particular, they are useful for treatment of re-emerging virus infections, where direct-acting antivirals (daas) have limited utility due to daa-resistant mutations, and for newly emerging virus strains in which the time to vaccine availability precludes vaccination at the onset of an outbreak. ifns-a/b: host-derived broad spectrum antivirals virus infections range from mild and benign to highly virulent epidemics and pandemics, and significantly affect global health. daas, which target specific steps of virus replication, and vaccines, are currently the most effective therapeutic intervention strategies used against virus infections. newly emerging or re-emerging viruses that have undergone mutations may, however, be resistant to the effects of daas, whereas vaccines require that the virus strain be identified before vaccine production, precluding their use at the onset of any new virus infection outbreak. broad-spectrum antivirals, capable of modulating the innate immune response regardless of the infecting virus, present as ideal candidates as a first-line treatment for acute virus infections such as respiratory tract or sexually transmitted infections. ifns-a/b are produced by plasmacytoid dendritic cells (pdcs), macrophages, fibroblasts and endothelial cells, and are critical effectors in an innate immune response to virus infections [ ] . ifns-a/b are induced following pattern recognition receptor (prr) activation by viruses (box , as reviewed in [ ] ) and target many different stages of viral replication: for example, viral entry, envelope uncoating, genome replication, protein assembly, and release of viral progeny [ , ] . ifns-a/b also activate different cell types in the immune system to promote viral clearance and induce apoptosis of cells to prevent viral replication [ , ] . as host-derived innate immune response factors, ifns are, therefore, broad-spectrum antivirals, crucial for the primary host response to viral infection. ifns-a/b bind to and activate the ifnar complex, resulting in the rapid induction of transcription and translation of isgs ( figure , as reviewed in [ ] ). notably, ifnars are ubiquitously expressed on all cell lineages; probably an evolutionary consequence of different viruses being able to target and infect different cell types. ifnar expression on any and all cell types ensures that an ifn response to a virus may be induced upon infection. as an ifn response is central to a robust innate immune response, viruses have evolved a variety of mechanisms to interfere with ifn production and signaling, to disrupt innate host antiviral factors. successful viral clearance is determined by the balance between virus-encoded molecules that antagonize the host innate immune response and the robustness of the host innate immune response. here, we discuss how ifn therapy presents as a viable treatment option for a range of acute virus infections that target a variety of tissues, including respiratory tract infections by severe acute respiratory syndrome coronavirus (sars-cov) and influenza a viruses, infections of the liver by hepatitis c virus (hcv) and hepatitis b virus (hbv), and mucosal infections by herpes simplex virus (hsv). this may be particularly important given the paucity of broad-spectrum antivirals for treating newly emerging and re-emerging virus infections, which present a major threat to human health. yin: ifn activates the immune system in addition to the induction of isg expression in all cell types, ifns shape the landscape of the immune system in response to virus infection by promoting neutrophil survival [ , ] and the activation of macrophages [ ] , natural killer cells [ ] , dcs [ , ] , b cells [ ] and cd + t cells [ ] , and t helper (th) polarization of effector cd + t cells [ ] ( figure ). ifn therapy therefore has the advantage over daa treatments in that, in addition to stimulating genes that block viral replication in infected cells, ifns activate other innate and adaptive immune responses to combat the virus. polymorphisms in genes encoding factors involved in different stages of the ifn response can lead to marked differences in susceptibility to virus infection and severity of disease, and can also serve as predictive markers for the outcome of ifn treatment. for example, polymorphisms in host genes encoding proteins associated with regulation of an ifn response such as interferon receptor a-chain (ifnar ) [ ] , the ifn-inducible myxovirus resistance gtpase protein, mx [ ] , the ifn-inducible , -oligoadenylate synthetase (oas) [ ] and the suppressor of cytokine signaling (socs) associated with regulation of an ifn response [ ] , are predictive markers linked with the rate of sustained virological response (svr) to hcv infection following ifn-a treatment. this highlights the importance of an intact ifn response during viral infection and indicates that genetic variations among patients can present as a challenge for optimizing ifn therapy. yang: virulence factors antagonize the ifn response it is not surprising that many pathogenic viruses, including sars-cov, influenza a viruses, hcv and hsv, have developed mechanisms to disrupt and limit the ifn-a/b response (table ) . many viruses are able to evade the innate immune system by directly targeting pathways required for the induction of ifn-a/b production [ ] [ ] [ ] [ ] [ ] . these viruses are also able to inhibit an ifn response, by interfering with effectors in ifn-inducible signaling cascades [ ] [ ] [ ] . understanding the basis of these antagonistic mechanisms is essential for optimizing the timing and dosage of ifn treatments as a viable therapy for acute virus infections. box . ifn-a/b induction by viral pathogen-associated molecular patterns (pamps) prrs, including membrane-associated toll-like receptors (tlrs), and cytosolic rna and dna sensors such as rig-i, are able to detect both extracellular, endosomal or cytosolic viral pamps: viral genomic material. the primary outcome of this nonspecific surveillance system that detects any and all viruses, regardless of the target cell or tissue tropism of the virus, and independent of where the virus is located, is the transcriptional activation of ifn-a/b genes and the rapid production and secretion of these ifns. upon viral pamp recognition, prrs are able to trigger a phosphorylationdependent signaling cascade to activate irf and/or irf . for instance, endosomal rna/dna-sensing tlr and tlr activate irf via myeloid differentiation primary response gene (myd ), whereas endosomal dsrna-binding tlr and rig-i, are able to activate irf and irf through tbk and inhibitor of ikke. irf and irf activation results in their nuclear translocation where they act as transcription factors and up-regulate ifn-a/b gene expression. ifns-a/b bind with high affinity to the ifnar complex, composed of an a-chain, ifnar , which is structurally modified by cell membrane glycosphingolipids, galabiosylceramide (gb ) and globotriaosylceramide (gb ) to promote efficient ifn binding [ , ] , and a b-chain, ifnar . ifn binding to ifnar induces phosphorylation of the receptor-bound tyrosine kinases, tyrosine kinase (tyk ) and jak , leading to the subsequent regulation of: (a) protein synthesis via the activation of pi k and mammalian target of rapamycin (mtor); (b) histone modification, via the activation of p mapk; and (c) gene expression via the phosphorylation of stat proteins and mapk activation. tyk -and jak -mediated activation of insulin receptor substrate (irs) and irs is required for recruitment and activation of pi k. pi k phosphorylates akt, which inactivates inhibitors of mtor, tuberous sclerosis protein (tsc) and tsc [ ] . mtor activates the serine/ threonine kinase p s k, and inactivates eukaryotic translation initiation factor e-binding protein ( e-bp ), to upregulate cap-dependent mrna translation and protein synthesis. p is activated downstream of mapk kinases and (mkk / ) and regulates histone modification and gene expression through mitogen-and stress-activated protein kinase (msk) and msk . in addition, ifn signaling invokes promyelocytic leukemia zinc finger (plzf) protein-mediated histone modification to regulate isg expression [ ] . phosphorylation of stat proteins results in their dimerization and ifn-stimulated gene factor (isgf) formation. stat complexes translocate to the nucleus and bind to specific gene elements in the promoters of isgs, ifn-g activated sequence (gas) and ifn-sensitive response element (isre), to induce expression of antiviral genes. ifn therapy as a first-line treatment against newly emerging or re-emerging virus outbreaks: sars-cov the sars-cov outbreak originated in hong kong in late - and resulted in > cases of disease worldwide, with a . % mortality rate between november and july (http://www.who.int/csr/sars/country/ table _ _ /en/index.html). sars-cov is a singlestranded rna virus that encodes in its genome virulence factors that antagonize the ifn-a/b response. in infected host cells, sars-cov expresses the nonstructural protein (nsp) and nsp . nsp suppresses host gene expression by disrupting mrna translation and by upregulating mrna degradation [ , ] . immunoprecipitation and luciferase reporter studies have shown that nsp directly associates with the s ribosomal subunit to inhibit its translational activity [ ] . in addition, the nsp - s complex is able to modify mature -capped rnas to limit translation and promote degradation [ ] . in the context of an ifn response, these antagonistic mechanisms of nsp on host gene expression and protein synthesis inhibit ifn-a/b expression and production [ ] . in vitro, nsp also inhibits signal transducer and activator of transcription (stat) protein phosphorylation induced by ifn-a treatment [ ] . both nsp and nsp inhibit interferon regulatory factor (irf) and irf activation to downregulate ifn production in response to viral infection [ , ] . specifically, nsp inhibits irf in human bronchial epithelial cells via its papain-like protease (plp) domain, which [ , ] , tripartite motif-containing protein a (trim a) and trim , which are antiviral factors that limit hiv- infection [ ] , and transcription factor jun-d (jund) and claudin (cldn ) [ ] . ifn treatment primes cells for apoptosis by modulating the expression of proteasome subunits, major histocompatibility complex (mhc) class i, and fas receptor (cd ) [ ] [ ] [ ] [ ] . ifns-a/b also contribute to the activation and differentiation of cells involved in the (b) innate and (c) adaptive immune responses to virus infection. ifn-a/b induces production of interleukin (il)- , il- , and il- by dcs, and il- by macrophages to modulate b and t cell differentiation (th polarization) and activation [ ] . ifn-b signaling in pdcs leads to altered cd and sphingosine- -phosphate (s p ) receptor expression, thereby affecting pdc retention in lymph nodes [ ] . ifns-a/b increase mhc class ii, cd and cd expression on antigen presenting cells. ifn-a/b treatment induces macrophage and neutrophil phagocytosis [ , ] . moreover, ifns-a/b promote neutrophil survival by activating cellular inhibitor of apoptosis (ciap ) [ ] . natural killer (nk) cells respond to ifns-a/b with increased fas ligand (fasl) and perforin expression, and ifn-g production [ , ] . in response to ifns-a/b, b cells upregulate l-selectin and igg production [ , ] . trends in immunology april , vol. , no. interacts with irf to inhibit irf phosphorylation and nuclear translocation [ ] . in addition to nsp and nsp , the open reading frame (orf ) and matrix (m) proteins of the sars-cov also inhibit an ifn response [ , ] . orf localizes to the host endoplasmic reticulum (er) and blocks the transcription factor function of phosphorylated stat , by binding to nuclear import factors to prevent its translocation to the nucleus [ ] . the m protein interacts with rna sensor retinoic acid-inducible gene (rig-i), an rna helicase and key intracellular prr associated with induction of irf-dependent ifn production following detection of viral rnas. the m protein also interacts with the signaling effectors serine/threonine-protein kinase (tbk ), inhibitor of nuclear factor-kb kinase subunit e (ikke), and tumor necrosis factor (tnf)-associated factor (traf ), again associated with ifn gene induction [ ] . thus, there are multiple mechanisms by which sars-cov might inhibit the host ifn response. the implications are that an ifn response to sars-cov infection must be dramatically limited for virus replication to proceed, suggesting that the dominant immune response is the ifn response. different recombinant ifn-as and ifn-b are now approved for various clinical indications, including the treatment of chronic hcv infections [ , ] . through a comprehensive analysis of how structural features in the ifn-a/b moleculescrucial clusters of amino acidsaffect the sensitivity of target cells to ifn-induced biological responses, specific epitopes on the exposed surface of the ifn molecule have been identified that are associated with receptor recognition [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . accumulating evidence suggests that the affinity of a particular ifn-a/b subtype for ifnar determines the biopotency of the ifn, specifically in the context of antiviral and antiproliferative responses [ , ] . a direct consequence of this was the design and development of a synthetic ifn-a, ifn alfacon- , that exhibits optimized affinity for ifnar [ ] [ ] [ ] [ ] . initially, treatment for sars-cov infection focused on the use of a daa, ribavirin, in combination with corticosteroid therapy [ , ] . however, in a pilot clinical study, the therapeutic potential of ifn alfacon- was evaluated in individuals infected with sars-cov and hospitalized in toronto, canada [ ] . ifn alfacon- treatment together with corticosteroids is associated with reduced disease-associated impaired oxygen saturation, more rapid resolution of radiographic lung abnormalities and lower levels of disease-associated creatine kinase. in vitro studies to examine the mechanism of action of ifn against the sars-cov have revealed that ifn-inducible janus kinase (jak ), protein kinase c (pkc)-d and p mitogen-activated protein kinase (mapk) activation mediate ifn antiviral protection. target genes downstream of activation of these kinases are differentially expressed in the peripheral blood cells of sars patients treated with ifn alfacon- compared with patients not treated with ifn, and functionally these genes are associated with antimicrobial activity [ ] . treatment of a human bronchial epithelial cell line, calu- , with ifn alfacon- before infection with the sars-cov results in inhibition of virus infection and a reduction in overall virus yield, further supporting the idea that ifn alfacon- demonstrates antiviral activity against the sars-cov [ ] . these data demonstrate that despite the inherent ability of the sars-cov to inhibit ifn production and limit an ifn response, treatment with exogenous ifn-a overrides these inhibitory effects. these results support the further evaluation of ifn alfacon- as a first-line treatment for acute sars-cov infection and approved inhibits the function of pkr and , -oas via its dsrna-binding domain. [ ] review trends in immunology april , vol. , no. randomized clinical trial protocols are in place in the usa and canada should there be outbreaks of sars-cov. seasonal influenza a virus infections are a considerable health burden and vaccine programs are currently implemented in most developed countries. vaccines, however, are not relevant during an outbreak involving an emergent variant. the h n swine-origin influenza a virus is a prime example of how quickly a pandemic can develop given the potential for genetic shift and mutation of influenza a viruses among natural hosts. during the h n pandemic, daas such as the neuraminidase inhibitors oseltamivir and zanamivir were widely used before a vaccine became available [ ] . not surprisingly, however, daa-resistant variants of pandemic h n emerged [ , ] . avian h n influenza virus outbreaks, now affecting populations throughout asia and europe, are associated with mortality rates around % [ ] . notably, a number of h n strains are resistant to oseltamivir [ ] . to date, there have been no reported cases of human-to-human transmission of this lethal h n influenza virus infection, but if a newly emerging strain capable of human-to-human transmission appears, daa resistance will develop and until a vaccine becomes availableprobably - monthspopulations will be at risk in the absence of access to broad-spectrum antivirals. ns is the primary virulence factor encoded by influenza a viruses and it is expressed in host cells during the earliest stages of infection [ ] . in comparison with sars-cov nsp , influenza virus ns has both overlapping functions as well as unique mechanisms to inhibit the ifn response. ns acts both in the nucleus and cytoplasm of an infected cell, and is the primary antagonist of the host innate immune response. remarkably, ns has evolved to inhibit virtually all stages of the ifn response to virus infection, including inhibition of ifn production, interference with ifn signaling events, and inhibiting the function of antiviral factors induced by ifn signaling. ns inhibits the activity of rig-i (box ) where the ns dsrna-binding domain interacts directly with rig-i [ ] . within the nucleus of an infected cell, ns inhibits the processing and synthesis of host mrnas, including ifn-a/b mrnas, by binding to and inhibiting both cleavage and polyadenylation specific factor kda (cpsf ) and poly(a)-binding protein ii (pabpii), via its proteinbinding domain [ ] . the expression of avian h n ns disrupts ifn signaling events by downregulating the surface expression of one of the ifnar subunits, ifnar , and by upregulating socs protein expression, leading to a reduction in ifn-inducible stat phosphorylation and stat homo/heterodimer nuclear translocation [ ] . ns is able to block directly the antiviral activities of ifninducible antiviral proteins such as protein kinase rnaactivated (pkr) and , -oas/rnasel, via its proteinbinding domain and dsrna-binding domain, respectively [ ] . the src homology (sh )-binding domain within the protein-binding region of ns permits interaction with the internal sh domain of p b, the inhibitory subunit of phosphoinositide -kinase (pi k). this leads to activation of the pi k-akt pathway [ ] . activation of pi k, a downstream target of ifn-a/b signaling, by a specific ns promotes cell survival during the early stages of infection, illustrating the complex interplay between virus encoded factors and the ifn-a/b response [ ] . remarkably, distinct highly pathogenic respiratory viruses, namely influenza viruses and the sars-cov, encode nonstructural proteins in their genomes that function as virulence factors that specifically target the host innate ifn response, further emphasizing the importance of ifns as broad-spectrum antivirals. a recently completed randomized controlled trial has examined the safety and efficacy of recombinant ifn-a (rifn-a) treatment, administered in the form of a nasal spray, in military recruits, in the context of protection from respiratory virus infections [ ] . serum igm levels were measured as evidence of virus infection. subjects receiving rifn-a had lower concentrations of serum igm specific for h n influenza a virus, influenza b virus, adenovirus (species b), and parainfluenza virus types , and [ ] . specifically with regard to influenza a virus, only recruits treated with rifn-a had detectable levels of influenza a virus igm compared with recruits in the untreated control group [ ] . no adverse events were reported in the treatment group, the data demonstrating that ifn was well tolerated and was effective in preventing a variety of common viral respiratory infections. thus as for the sars-cov, the implications are that treatment with ifn-a can override the inhibitory effects of ns on an ifn response during influenza a virus infection. ifn therapy for highly pathogenic and oncogenic viral infections: hbv and hcv worldwide, > million people are infected with hcv, resulting in approximately deaths each year (http://www.who.int/mediacentre/factsheets/fs /en/). more than an estimated million people are chronically infected with hbv, resulting in approximately deaths each year (http://www.who.int/mediacentre/factsheets/fs /en/). hcv and hbv target the liver and cause both acute and chronic infections, resulting in liver cirrhosis and eventually, hepatocellular carcinoma [ , ] . the current approved standard-of-care treatment for hcv infection comprises daily ribavirin in combination with weekly pegylated ifn-a (peg-ifn-a) . the covalent linkage of polyethylene glycol to ifn-a increases the halflife of ifn-a in the circulation. common side effects associated with ifn therapy include a range of flu-like symptoms (fatigue, fever, myalgia), that often diminish spontaneously during the first few weeks of therapy. more severe neuropsychiatric disturbances including sleep disturbances and depressive mood changes have their onset within the first months of ifn therapy. hematological disturbances such as neutropenia or anemia may occur and are responsive to ifn dose reduction or treatment [granulocyte colony-stimulating factor (g-csf), erythropoietin (epo), respectively]. this combination ifn/ribavirin therapy has been very successful in patients infected with hcv genotypes or , and - % of patients go on to achieve an svr, characterized by undetectable hcv rna following weeks of treatment [ ] . the rate of svr falls to - % in patients infected with hcv genotypes or , trends in immunology april , vol. , no. following weeks of treatment with peg-ifn-a and ribavirin [ ] . the incomplete response to ifn treatment is partially attributable to virally encoded virulence factors that interfere with an ifn response: ns / a and ns a. ns / a is an hcv serine protease that targets mitochondrial antiviral signaling (mavs) proteins required for rig-i-mediated irf activation and subsequent ifn production [ ] . recently, the secondary structure of the hcv genotype b ns n-terminal region was identified as a predictive marker for the virological response in patients who had received ifn and ribavirin combination therapy for weeks. specifically, polymorphisms in the secondary structure of the ns amino-terminal region segregate hcv genotype b infected individuals into two groups and are predictive of the virological response to peg-ifn plus ribavirin therapy [ ] . ns a associates with intracellular membranes and its expression is vital for hcv genome replication. ns a is able to interact with ifn-inducible pkr to evade an ifn-induced antiviral response. polymorphisms in amino acid residues and in the hcv core and in ns a are also predictive markers of the virological response in patients receiving ifn and ribavirin therapy [ ] . notably, ns a is a target of ifn, because the ifn-activated gene (ifi )/interferon stimulated gene (isg ) encoding a -kda protein, promotes isgylation of ns a to enhance its degradation, thereby inhibiting hcv replication [ ] . in addition to hcv ns a, both the hcv envelope protein e and the internal ribosome entry site (ires) are able to inhibit ifn-inducible pkr activity [ ] . e contains a eukaryotic translation initiation factor (eif- a) phosphorylation homology domain through which it is able to interact with pkr, whereas the hcv ires binds to pkr, precluding dsrna binding, thereby preventing pkr activation [ ] . despite these potent inhibitory effects of hcv-encoded factors on an ifn response, clinical data provide direct evidence that peg-ifn-a treatment in combination with ribavirin is effective at limiting hcv infection and, dependent on the hcv genotype, may invoke a svr. the mechanisms by which hbv evades an innate immune response are less well understood. the hbv polymerase (pol) blocks irf signaling and subsequent ifn production by inhibiting tbk /ikke activity, associated with prr signaling [ ] . this inhibition is mediated by direct protein-protein interactions between pol and the host dead box (d-e-a-d amino acid sequence motif) rna helicase, ddx , that enhances tbk /ikke activity [ ] . peg-ifn-a is an effective treatment for hbv infection, again suggesting that ifn treatment can overcome virus-imposed inhibition of the innate immune response, specifically ifn production. peg-ifn-a is an effective treatment option for hepatitis b e core antigen (hbeag)positive disease, where detection of hbeag in the blood is indicative of viral replication. up to % of hbeag-positive patients treated with peg-ifn-a are able to develop hbeag-specific antibodies (seroconversion) by months after the end of treatment. this percentage rises to % at years after the end of treatment [ ] . in comparison to monotherapy with the daa lamivudine, which can lead to the emergence of mutant lamivudine-resistant hbv strains, peg-ifn-a alone or in combination with lamivudine is up to % more effective for inducing hbeag seroconversion, although more side effects are reported in patients receiving peg-ifn-a [ ] . in contrast to hbeag-positive disease, peg-ifn-a, alone or in combination with ribavirin, has limited effect in patients with late stage hbeag-negative disease, where the hbv mutation has resulted in loss of hbeag expression [ ] . in a randomized clinical trial, the percentage of hbeag-negative patients with hbv dna levels < copies/ml, receiving peg-ifn-a monotherapy, dropped from % to %, from the end of treatment to weeks later [ ] . the stage of viral disease can therefore affect the efficacy of ifn therapy and the timing of treatment contributes to the capacity to resolve an infection. moreover, as for influenza viruses and sars-cov, despite hbv and hcv encoding viral factors that antagonize an ifn response, exogenous ifn therapy has proven to be an effective treatment for establishing an svr. ifn therapy for highly transmissible viral infections: hsv- hsv- is a highly contagious virus, prevalent among sexually transmitted infections. hsv- is able to establish a latent infection in immunocompetent individuals by evading the immune system and is only reactivated when the host immune system is weakened [ , ] . the hsv- genome encodes a number of virulence factors, namely infected cell protein (icp) . , icp and icp , which are associated with immunoevasion and suppression of the innate immune response to virus infection [ , , , ] . specifically, icp . dephosphorylates eif- a to reverse pkr-mediated inactivation of eif- a [ ] , icp localizes to the cytoplasm and inhibits irf activity [ ] , and icp blunts the ifn-inducible jak-stat signaling pathway by inhibiting ifn-inducible stat phosphorylation and nuclear translocation [ ] . furthermore, hsv structural protein us , which has a dsrna-binding domain, disrupts the activation of the ifninducible antiviral proteins , -oas and pkr [ ] . for immunocompromised individuals infected with hsv- , viral pathogenesis can lead to serious life-threatening disease; more so in the context of emergent drug-resistant hsv- strains [ ] [ ] [ ] . different daas have been used to control hsv- infection, including acyclovir, penciclovir and foscarnet, resulting in the emergence of daa-resistant hsv- strains [ ] [ ] [ ] . ifn-g is able to exert antiviral activity by stimulating a t cell response. however, ifn-g alone may have limited efficacy in immunocompromised hsv- -infected individuals lacking a robust adaptive immune response. recent studies have shown that when immunocompromised nude mice are infected with a daa (acyclovir)-resistant hsv- variant and treated with ifn-b in combination with ifn-g, viral infection is reduced [ ] . these preliminary data are in further support of the broadspectrum antiviral activities of ifns-a/b. shifting the balance to favor the host innate immune response: the future of ifn antiviral therapy mechanisms for viral evasion of the host immune response include both the expression of many virulence factors by a review trends in immunology april , vol. , no. single virus to target different stages of the ifn response, or the expression of a single, highly specialized molecule that alone targets multiple facets of an ifn response. a priori, the widespread existence of these virally encoded virulence factors that target an ifn response highlights the critical role of a robust ifn response to limiting virus infection. the ability of ifns-a/b to target multiple types of viruses at different stages of viral replication, and the ubiquitous expression of ifn receptors on cells that are susceptible to different virus infections with different tissue tropisms, as well as the ability of ifns to activate innate immune cells and influence the adaptive immune response, emphasizes the relevance of ifns-a/b as broadspectrum antivirals. understanding the viral strategies for evasion of an ifn will permit the design of strategic ifn treatment regimens to both protect from and clear virus infections. the opportunity to limit virus infections even in the absence of characterizing the specific infecting virus, a reality during an outbreak of unknown etiology, or during a pandemic of a newly emerging or re-emerging virus strain, has profound implications for global health. indeed, early data indicate that ifn therapy may be effective in treating west nile virus [ ] , hemorrhagic yellow fever virus [ ] and ebola virus infections [ ] . moreover, short-term ifn therapy for an acute virus infection may not invoke the debilitating side effects associated with long-term ifn therapy for chronic infections such as hbv and hcv. preliminary data from pilot clinical trials of ifn treatment for sars-cov and for influenza a viruses showed this to be the case [ , ] . cognizance of the yin and yang of viruses and ifns opens the door to the widespread clinical application of these broad-spectrum antiviral ifns. type i interferons in host defense pathogen recognition by the innate immune system mechanisms of type-i-and type-ii-interferonmediated 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immunoproteasomes at the site of infection ifn-a secretion by type predendritic cells upregulates mhc class i in the hiv- -infected thymus involvement of fas-mediated apoptosis in the inhibitory effects of interferon-alpha in chronic myelogenous leukemia dynamic accumulation of plasmacytoid dendritic cells in lymph nodes is regulated by interferon-b effect of interferon-alpha( a) on neutrophil adhesion and phagocytosis in chronic myeloid leukemia and behçet's disease ifnalpha regulates nk cell cytotoxicity through stat pathway induction of interferon-gamma from natural killer cells by immunostimulatory cpg dna is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha inhibition of the interferon-inducible protein kinase pkr by hcv e protein key: cord- - aeu n v authors: honke, nadine; shaabani, namir; zhang, dong-er; hardt, cornelia; lang, karl s title: multiple functions of usp date: - - journal: cell death dis doi: . /cddis. . sha: doc_id: cord_uid: aeu n v since the discovery of the ubiquitin system and the description of its important role in the degradation of proteins, many studies have shown the importance of ubiquitin-specific peptidases (usps). one special member of this family is the usp protein (formerly ubp ). in the past two decades, several functions of usp have been discovered: this protein is not only an isopeptidase but also a potent inhibitor of interferon signaling. therefore, usp functions as 'a' maestro of many biological pathways in various cell types. this review outlines multiple functions of usp in the regulation of various immunological processes, including pathogen control, cancer development, and autoimmune diseases. ubiquitin-specific peptidase (usp ) is known as an isg isopeptidase and a negative regulator of type i and type iii interferon signaling. , the usp gene was originally called ubp because it encodes a -kda protein homologous with ubiquitin-specific proteases (ubps). it was first cloned by liu et al. , from mice expressing the leukemia fusion protein aml -eto and later by other groups from virus-infected porcine alveolar macrophages and human melanoma cell lines. the gene and protein were renamed usp and usp , respectively, according to the systematic nomenclature suggested by baker et al. for ubps and ubiquitin-like (ubl) proteins. on the basis of its amino-acid sequence, usp is a member of the usp family, which is responsible for removing ubiquitin or ubl proteins from their conjugated substrates. the usp gene spans . kb on mouse chromosome and includes exons. the transcript of bps is translated as a protein with amino acids (aa). the protein shares catalytic domains of ubps. in the mouse, a mutation of the usp protein within the cys box at position completely abolishes the isopeptidase activity of the protein by replacing the active site of cysteine c with codon specific for alanine c a. however, not only the cys box is responsible for isopeptidase function, but also the his box and the asn residue (figure ). another functional domain specific for usp has been mapped from exon - (aa - ); this domain facilitates binding to the intracellular domain of the ifnar subunit in order to regulate interferon signaling ( figure ). binding to ifnar is abolished by a mutation at position of the usp protein, a mutation that was induced by n-ethyl-n-nitrosourea mutagenesis, and mouse strains carrying this mutation are called usp ity . in humans, two isoforms of usp have been described. they differ in their n-terminal region because of translation from a non-canonical rare start codon cug; this translation produces a full-length protein or a canonical start codon, aug, and results in the n-terminal truncated isoform usp -sf. although usp is mainly located in the cytoplasm, usp -sf is evenly distributed in the cytoplasm and the nucleus. both proteins maintain their functional activity in term of enzymatic and ifnar binding ability, but the usp -sf isoform is the main delsgylation enzyme for nuclear proteins and therefore may serve cell-specific functions. in addition, because usp -sf is controlled by two independent mechanisms, its regulation is more flexible. additional studies are needed to characterize the role of each isoform and to determine whether the regulation pathway can influence the outcome of usp function. usp expression is detectable in several tissues but at different levels. for instance, high expression of usp has been measured in liver, spleen, and thymus. , however, a low but clearly detectable level of usp expression has been seen in bone marrow, adipose tissue, and lung tissue. the expression of usp has been studied in a wide spectrum of cells. for example, in our earlier studies we detected high levels of usp in cd + macrophages and bone marrow-derived dcs, but we found no usp in lung fibroblasts and bone marrow-derived macrophages. , a high level of usp expression has also been measured in peritoneal macrophages and monocyte-derived macrophages , and in two murine monocyte-related cell lines, raw . and m . usp is also expressed in various lymphatic and hematopoietic cell populations, including splenic tand b cells. in t cells, usp is highly expressed in naive, effector/memory, and natural regulatory t cells. microarray data indicate that a high level of usp expression is also maintained in t helper , th , and th cells but is diminished in th cells and inducible regulatory t cells. however, the expression level of usp is differently regulated during t cell activation, tolerance, and effector differentiation. usp is induced by ifn-β not only in lymphocytes but also in ho- human melanoma cells, in huh- . cells treated with irrelevant small-interfering rnas, in the choroid plexus and in ependymal cells. moreover, transfecting human hepatoblastoma hepg cells with hepatitis b virus (hbv) genome increased the expression of usp in those cells. the usp gene is rapidly and strongly upregulated after viral infection or by type i and type iii ifns, , , , , lipopolysaccharide (lps), , tumor necrosis factor alpha (tnf-α), or genotoxic stress , (figure a ). usp is degraded by proteasomes. , in most species, usp maps to a different chromosome ( table ) . the absence of usp strengthens the signaling of ifn-i and ifn-iii; , and is associated with prolonged janusactivated kinase/signal transducer and activator of in addition, absence of usp , which cleaves isg from its target protein, prolongs isg -mediated isgylation. , usp -deficient cells exhibit high sensitivity to treatment with ifn-i, poly i:c, and lps. treating usp -deficient hematopoietic cells with poly i:c decreases the number of white blood cells since apoptosis is not prevented by usp . moreover, knocking down usp markedly enhances the nf-κb signaling induced by various tlr ligands. a study using an oncogenic cell line (e a cells) found that usp activates the extrinsic tnf-related apoptosis-inducing ligand (trail) pathway after ifn-α challenge. in human promonocytic thp- cells, the expression of proinflammatory cytokines such as tnf-α, interleukin- (il- ), and il- β is significantly higher when usp is silenced with sirna. interestingly, in contrast to e a cells, usp deficient murine bone marrow cells and thp- cells that have been treated with ifnα/β do not experience apoptosis after treatment with trail or fasl. however, ifn-α/β still triggers apoptosis in these cells through the mitochondrial pathway and the reactive oxygen species pathway, a finding indicating that usp influences cell survival in various pathways depending on the cell type. the generation of usp -deficient mice gave researchers the opportunity to examine many immunological mechanisms and disorders. initially, usp −/− mice were generated on a mixed sv -c bl/ background. these mice are born at a normal ratio of wt to heterozygous littermates; however, homozygous knockout mice die of hydrocephalus - weeks after birth. further analysis showed that the difficulties in generating viable usp −/− mice on a c bl/ background occurred because the deletion of usp results in an increase in the concentration of free and conjugated isg at the fetomaternal interface and leads to fetal death. figure regulation and function of usp . (a) usp is upregulated through different signaling pathways (genotoxic stress, tnfr , tlr , ifnlr /il r , and ifnar / ifnar ). usp itself regulates signaling through a negative feedback mechanism either by inhibiting the binding of jak to the ifnar subunit or the ifnlr subunit, or by inhibiting the ubiquitination of the tak -tab complex of tnfr . (b) after isgylation of isg via a three enzymatic process, usp can deisgylate the ubiquitin-like protein isg from isg -conjugated substrate by cleaving the isopeptide bond between isg and the substrate. lacking of usp leads to an increase signaling of ifn-i, ifn-iii, tnf-α and high levels of conjugated isg interestingly, knockout mice generated on the fvb/n genetic background are more viable and do not exhibit the severe neurological symptoms that occur in mice generated on a c bl/ background, which develop brain injury because of necrosis of ependymal cells. this finding indicates that strain-specific modifiers may influence the neurological disorders and lethality induced by the absence of usp . although the role of isg in embryonic development has been demonstrated, the neurological disorders in usp −/− mice are not related to high expression of isg , because hydrocephalus also develops in mice with double knockout of usp and isg . homozygous usp -deficient mice are hypersensitive to ifn-i and die within h after treatment with synthetic double-stranded rna poly i:c. a recent study showed that strong signaling of ifn-i in usp −/− mice increases rankl-mediated osteoclast differentiation and elevates the induction of osteoclastogenic cytokines such as ip- and il- , which causes osteopenia. usp knockout mice are more resistant to intracerebral infection with lcmv and vsv but also display increased mortality to intravenous viral infection, as discussed below. to study specifically the enzymatic activity of usp , ketscher et al. generated a mutant mouse strain that lacks the isopeptidase activity of usp . using a knock-in targeting strategy, they replaced the enzymatic active site of cysteine (c ) with a codon specific for alanine (c a). in contrast to usp knockout mice, usp c a/c a mice do not exhibit brain abnormalities or increased lethality. they exhibit high levels of isg conjugates but normal ifn-i signaling. role of usp in cell development cd b + dcs: usp is involved in the development of conventional cd b + dcs. an in vitro study showed that the generation of bmdcs by usp -deficient mice is impaired. this defect is due to the suppressive role of usp on the ifn-i pathway, because dc development is also impaired after treatment with ifn-i. in vivo, the spleen and bone marrow of usp -deficient mice contain fewer cd b + dcs than wt mice. conventional cd b + dcs are developed via the ifn-i pathway independent of the role of usp in deconjugation of the ubl protein isg . th cells: during infection, cd + t cells have an important role. cd + t cells differentiate into separate subtypes that produce distinct effector cytokines. the main subtypes are th cells (which produce ifn-γ), th cells (il- ), and th cells (il- , il- f, il- , and il- ). th cells are involved in autoimmune diseases such as multiple sclerosis (ms). recently, liu et al. found that usp is necessary for th differentiation and autoimmune response. usp can inhibit the ubiquitination of the tak -tab complex, thereby inhibiting il- production and promoting il- production and synthesis. in mammalian cells, many proteins are modified by ubiquitination, a process which is important for different vital events. for instance, the ubiquitin pathway is essential for cell cycle, cell differentiation, and proliferation. this pathway mediates protein degradation, turnover of transport proteins, and transcription activation. at the immune level, this pathway is essential for antigen presentation. these mechanisms are controlled by a wide spectrum of enzyme family members called ubps, which remove ubiquitin from a large range of protein substrates. the family members vary in size and in amino acid sequences, but they all share six conserved regions. ubp (usp ) is one member of this ubiquitin protease family; [ ] [ ] [ ] , , it controls the protein binding of ubl protein isg . after stimulation with ifn-α/β, isg is upregulated and conjugated to various cellular substrates through a three-step enzymatic cascade: , e -activating enzyme (ube l), e -conjugating enzyme (ubch ), , and various e ligases , (figure b ). this conjugation mechanism is reversible and is controlled by the isopeptidase active site of ifn-inducible cysteine protease usp . this functional domain includes the cysteine box with a cysteine residue, a histidine box with a histidine residue as well as an adjacent asparagine. these sequences are necessary for the function of the enzymatic activity of usp . , cross-talk occurs between isg and usp . isg itself can stabilize usp ; this stabilization is important for preventing the undesirable autoinflammatory effect of sustained ifn-α/β. interestingly, this phenotype was observed only in human cells but not in murine cells. unlike humans, ifn-i signaling is downregulated in mice regardless of isg competency. in addition to isg , usp also specifically inhibits k -linked ubiquitination of nemo, leading to the negative regulation of nf-κb activation induced by the tak -tab complex. , a recent study found that usp can enhance the cellular transport rate in xenopus laevis oocytes by increasing the activity of pept and pept ; this activation was believed to be due to the reversal of ubiquitination and the subsequent degradation of carrier protein. interferon-i signaling is mastered through the binding of ifn-i to the heterodimeric interferon receptor, which consists of two subunits: ifnar and ifnar . both subunits are associated with kinase activity. ifn-i binding leads to phosphorylation of jak and tyk and in turn to phosphorylation of stats, which initiate the transcription of antiviral genes by binding to the interferon-stimulated response element. usp is considered to be a negative regulator of the ifn-i pathway by inhibiting jak-stat signaling , (figure a ). this function is independent of its activity as an isopeptidase. usp itself is upregulated in the presence of ifns through the activation of the jak-stat signaling pathway. , in turn, usp specifically binds to the ifnar subunit and thereby prevents the phosphorylation of jak by blocking the interaction of jak and the ifnar subunit and downstream signaling. this phenotype cannot be explained by the delsgylation activity of usp , because deleting isg or the isgylation-activating functions of usp n honke et al enzyme ube l in mice did not reverse the phenotype in usp -deficient mice. , , however, unlike mice, the presence of isg in human cells is essential to stabilize usp and guarantee the function of this protein as ifn-i suppressor. this means that, in humans, isg can also be considered as a good target protein by which we can strengthen ifn-i signaling and reduce viral replication. in the absence of usp , the phosphorylation of stat and stat is prolonged, and the expression of hundreds of antiviral genes, chemokines, cytokines, and antigenpresenting genes is enhanced, as confirmed by microarray data. additional in vitro studies found that the growth of prrsv is restricted in usp -overexpressed marc- cells. , this restriction is due to increased nuclear translocation of transcription factor p and decreased nuclear translocation of p . an in vivo study found that usp -deficient mice exhibit less replication of various viruses, including lcmv and vsv. the antiviral effect is not due to enhanced isgylation of isg in the absence of usp , because isg -deficient mice do not exhibit any antiviral impairment after infection with lcmv or vsv. in addition, usp knockout mice exhibit less replication of several other viruses, such as hbv, , sindbis virus, influenza b virus, hiv, and others in which the isgylation pathway is involved [ ] [ ] [ ] [ ] [ ] (table ) . not only viral infections but also sterile inflammation, which can be induced through different stimuli (chemical, physical, or metabolic noxious), can increase the usp expression through certain inflammatory cytokines (tnf-α and lps), which impairs ifn-i responses and viral control. for example, ischemic reperfusion injury upregulates usp expression in the liver and induces an ifn-α refractory state. that may raise the risk of an opportunistic viral infection. role of usp in enforced viral replication. the innate immune system reduces viral replication via ifn-i; this reduction is essential for inhibiting the spread of virus to other organs. simultaneously, it reduces the presentation of viral antigens to cells of the adaptive immune response. because the amount of antigen is positively correlated with the degree of activation of the adaptive immune system, , ifn-i can act as a double-edged sword. in our previous work using mice infected with vsv, we found that macrophages capture virus particles and suppress viral replication in the liver and the red pulp of the spleen in an interferon-dependent manner. however, the high expression of the gene encoding the inhibitory protein usp in cd + metallophilic macrophages reduces the responsiveness to ifn-i, which allows locally restricted replication of virus. this replication is essential to activate the adaptive immune system and to prevent the fatal outcome of infection (figure a ). an additional study showed that the same mechanism can lead to autoimmune diabetes as discussed below. as shown above, usp influences numerous cellular signaling and biological functions. consequently, many researchers have studied the role of usp during bacterial infection. the results show that strong ifn-i signaling resulting from the absence of usp leads to less growth of salmonella typhimurium in usp knockout mice than in control mice. however, survival rates are slightly higher for usp -deficient mice because of their hypersusceptibility to lps challenge. a more recent study by richer et al. found that a missense mutation (enu-induced mutation) in usp lty mice leads to lethal susceptibility to s. typhimurium and that the bacterial titer in liver and spleen is higher in these mice than in wt mice. moreover, infection with s. typhimurium enhances ifn-i signaling and inflammatory response in usp lty mice. this immunological response is explained by the impairment of stat phosphorylation and ifn-γ production as the result of stat hyperactivation. another group of researchers attributed the susceptibility of usp lty mice to s. typhimurium or mycobacterium tuberculosis infection to elevated levels of il- , il- β, or il- , in addition to the deregulation of autophagy markers. surprisingly, the contradictory nature of these two results obtained with usp knockout mice and usp lty mice can be explained by either the different backgrounds of the mice or the specific enu-induced mutation of usp . other studies demonstrated the upregulation of usp during infection with rickettsia conorii. as expected, this upregulation inhibits ifn-i-induced genes in human microvascular endothelial cells; however, whether usp plays a role in controlling bacterial infection has not yet been fully clarified. , role of usp in autoimmune diseases autoimmune diabetes. autoimmune diabetes, also called diabetes mellitus type , is characterized by the destruction of insulin-producing beta islet cells in the pancreas. the priming of autoreactive cd + t cells requires the presentation of autoantigen to cd + t cells. this presentation can occur during a viral infection that resembles cross-reactive epitopes of beta islet cells. dcs are considered to be mainly responsible for this process. a mouse model showed that the absence of dcs inhibits the viral replication that resembles autoantigen and, consequently, inhibits the onset of diabetes. because dcs express more usp than do other types of cells, the lack of a response to the antiviral effect of ifn-i leads to augmented viral replication and a sufficient amount of autoantigen. knocking down usp expression inhibits viral replication and consequently does not lead to autoimmune diabetes (figure b) . further studies show that the expression of usp by beta islet cells themselves is important for inhibiting diabetes. , on the one hand, the upregulation of usp expression by ifn-i in beta islet cells prevents the activity of proinflammatory chemokines such as ccl , cxcl , and il- and consequently inhibits insulitis. on the other hand, the upregulation of usp inhibits beta cell apoptosis. , ms. ifn-β is considered the first line of therapy against ms. , ifn-β can induce usp expression through ifnar. in turn, usp inhibits ifn-β signaling. it was recently shown that usp is found in microglia, tissueresident macrophages in the central nervous system. these macrophages have an essential role in controlling tissue homeostasis, and disorders in the function of microglia can lead to neuroinflammatory diseases. the presence of usp in microglia is essential for keeping them in a quiescent state. , when usp is absent, the microglia undergoes a prolonged period of activation of stats, leading to microgliopathy. usp is also involved in the differentiation of cd + t cells into th cells, as discussed above. th cells are widely figure role of usp -dependent enforced viral replication in activation of the adaptive immune system and onset of diabetes mellitus type i. (a) usp inhibits ifnar signaling, which leads to enforced viral replication in cd + macrophages. this replication guarantees sufficient amount of antigen, which is processed and presented via mhc complexes to activate the adaptive immune system. lack of usp strengthens the ifn-i signaling, inhibits viral replication and reduces the presentation of processed antigen and consequently diminished adaptive immune system activation. (b) in case of enforced viral replication in dendritic cells of a virus resembling an autoantigen, autoreactive cd + t cells will be primed, which leads to autoimmune diseases such as type i diabetes, whereas usp deficiency reduces the priming of autoreactive cd + t cells and onset of autoimmune diabetes owing to inhibition of enforced viral replication functions of usp n honke et al recognized as essential player for the development of ms. a study using usp -deficient mice found that the disease score of eae is significantly lower than in wt mice, a finding that demonstrates the important role of usp in the progression of ms. interestingly, in humans, usp expression is lower in ms patients than in healthy persons. a genetic study found that ms is associated with two snps, rs (c/t) is located in the promoter region of usp and rs (a/g) in the fourth intron of the usp gene. the usp haplotype, tg is associated with risk to ms, whereas the haplotype cg is protective. yan et al. found that usp affects tumor progression because of its effects on interferon signaling. using a wellestablished mouse model of bcr-abl-induced chronic myelogenous leukemia-like myeloproliferative disease, they showed that usp has an important role in regulating the latency and severity of leukemia development. the absence of usp in the breast cancer cell line mcf- results in an increase in the induction of apoptosis by chemotherapy and treatment with ifn-α. silencing usp in glioblastoma cells produces similar results, a finding suggesting that strengthening the ifn-i pathway by silencing usp elicits apoptosis in drug-treated cells with robust caspase- and caspase- activation independent of the mitochondrial pathway. the direct effect of usp on tumor progression has been studied with leukocytes. ubp can be considered as an antineoplastic target for the treatment of apl. because usp directly regulates the growth of apl cells, knocking down usp reduces cell growth and induces apoptosis. in addition, usp expression has been detected in several types of human malignant tissues, such as kidney and prostate tissues. , an increase in usp expression specifically stabilizes cyclin d , not only in a mouse model but also in clinical studies. , moreover, the role of ube l in repressing cyclin d has been described in diverse types of cancers. , usp is also directly involved in cell proliferation: higher expression of usp is associated with faster proliferation. in both murine and human cell lines, an increase expression of usp is associated with the activity of wt . wt binds directly to the usp promoter and suppresses its transcription. these findings illustrate the important role of usp in tumorigenesis of the kidney. another mechanism that rules out the importance of usp in tumor progression was found in a pyvmt model of mammary tumorigenesis. the absence of usp leads to the high production of t cell chemoattractant cxcl by mammary epithelial cells, and this increased production creates a tumor-suppressive microenvironment by recruiting cd + t cells. in addition, usp has been found to inhibit trail-induced apoptosis independently of the deisgylation pathway. furthermore, the depletion of usp leads to a strong increase in the levels and activity of mir- , and this activity in turn decreases the expression of egfr, leading to apoptosis and control of cancer cells. hong et al. found that ifn-γ can induce usp in tumor cells and that this protein plays an important role in inhibiting tumorigenesis and maintaining antitumor immunity. increasing the expression of usp in tumor cells suppresses tumorigenesis, whereas reducing its expression stimulates tumor development and decrease immunosurveillance. moreover, usp expression in tumor cells regulates the exogenous production of ifn-γ and the persistence of antigen-specific ctls in the tumor microenvironment. a recent study showed that usp can predict the survival of patients with mibc. longer cancer-specific survival is associated with decreased expression of usp with or without the expression of the dgcr gene. in summary, usp is a novel protein that can affect cell viability at various cellular levels. targeting this molecule may help to improve the immune system response and avoid many (auto)-immune diseases, either by up-or downregulation of usp expression (figure ). for instance, some vaccines should be weakened and not completely inactivated in order to generate memory b cells. according to this fact, upregulation of usp in macrophages and dcs will allow more sufficient amount of antigen and thereby may be useful during vaccine administration. vice versa, downregulation of usp expression in antigen-presenting cells can be helpful not only to increase the antiviral signaling of ifn-i but also to reduce the amount of autoantigen and induction of autoimmune diseases. in both cases, we should take into consideration the yin and yang effect of ifn-i. for tumor treatment, the role of usp is still controversial. on the one hand, usp has a negative role either by inhibiting the antitumor effect of ifn-i or by accelerating the cell proliferation. on the other hand, it regulates the exogenous production of ifn-γ and the persistence of antigen-specific ctls in the tumor microenvironment, which suppresses tumorigenesis. according to that, further studies are still needed in order to show whether up-or downregulation of usp in each specific tumor type is beneficial or not. in general, owing to the multiple functions of usp and its expression in various cell types, it would be complicated to generate one drug that can be used to treat all the diseases in which usp is involved. we believe that this difficulty can be resolved by using targeted drug delivery. such technology would be the best strategy to overcome the protein's wide expression and will allow recovery from various types of immunological dysfunctions. usp -based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response usp deficient mammary epithelial cells create an antitumour environment driven by hypersensitivity to ifn-lambda and elevated secretion of cxcl a novel ubiquitinspecific protease, ubp , cloned from leukemia fusion protein aml -eto-expressing mice, functions in hematopoietic cell differentiation cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine ubp (usp ) molecular responses of macrophages to porcine reproductive and respiratory syndrome virus infection cloning and characterization of human ubiquitin-processing protease- from terminally differentiated human melanoma cells using a rapid subtraction hybridization protocol rash identification, functional characterization, and chromosomal localization of usp , a novel human ubiquitin-specific protease related to the unp oncoprotein, and a systematic nomenclature for human ubiquitin-specific proteases dissection of usp catalytic domains reveals five common insertion points ubp is a novel regulator of interferon signaling independent of its isg isopeptidase activity contribution of increased isg , isgylation and deregulated type i ifn signaling in usp mutant mice during the course of bacterial infections n-ethyl-n-nitrosoureainduced mutation in ubiquitin-specific peptidase causes hyperactivation of ifn-alphass signaling and suppresses stat -induced ifn-gamma production, resulting in increased susceptibility to salmonella typhimurium two independent mechanisms promote expression of an n-terminal truncated usp isoform with higher deisgylation activity in the nucleus ubp (usp ) specifically removes isg from conjugated proteins enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus usp driven enforced viral replication in dendritic cells contributes to break of immunological tolerance in autoimmune diabetes usp inhibits nf-kappab and nfat activation during th differentiation by deubiquitinating the tak -tab complex silencing of usp potentiates the antiviral activity of interferon against hepatitis c virus infection dysregulation of protein modification by isg results in brain cell injury suppression of usp potentiates the anti-hbv activity of interferon alpha in hepg . . cells via jak/stat signaling rnase-l-dependent destabilization of interferon-induced mrnas. a role for the - a system in attenuation of the interferon response enhanced antibacterial potential in ubp -deficient mice against salmonella typhimurium infection by up-regulating type i ifn signaling lipopolysaccharide and tumor necrosis factor alpha inhibit interferon signaling in hepatocytes by increasing ubiquitin-like protease (usp ) expression usp negatively regulates nf-kappab signaling by targeting tak and nemo for deubiquitination through distinct mechanisms the isg isopeptidase ubp is regulated by proteolysis via the scfskp ubiquitin ligase deregulated proteolysis by the f-box proteins skp and beta-trcp: tipping the scales of cancer protein isgylation modulates the jak-stat signaling pathway identification of usp as an important regulator of the susceptibility to ifn-alpha and drug-induced apoptosis the mitochondrial pathway and reactive oxygen species are critical contributors to interferon-alpha/beta-mediated apoptosis in ubp -deficient hematopoietic cells ubp gene expression is required for normal isg expression and fetal development reexamination of the role of ubiquitin-like modifier isg in the phenotype of ubp -deficient mice elevated response to type i ifn enhances rankl-mediated osteoclastogenesis in usp -knockout mice role of isg protease ubp (usp ) in innate immunity to viral infection selective inactivation of usp isopeptidase activity in vivo enhances isg conjugation and viral resistance usp promotes conventional cd b+ dendritic cell development th cells in development: an updated view of their molecular identity and genetic programming how proteolysis drives the cell cycle dub- , a deubiquitinating enzyme with growth-suppressing activity three ubiquitin conjugation sites in the amino terminus of the dopamine transporter mediate protein kinase c-dependent endocytosis of the transporter rel/nf-kappa b/i kappa b family: intimate tales of association and dissociation inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on mhc class i molecules ubiquitin-dependent protein degradation regulation of ubiquitin-dependent processes by deubiquitinating enzymes conjugation of the -kda interferon-induced ubiquitin homolog is distinct from that of ubiquitin isg : the immunological kin of ubiquitin influenza b virus ns protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg protein interferon-inducible ubiquitin e , ubc , is a conjugating enzyme for protein isgylation the ubch ubiquitin e enzyme is also the e enzyme for isg , an ifn-alpha/beta-induced ubiquitinlike protein isg : a ubiquitin-like enigma the interferon-inducible ubiquitin-protein isopeptide ligase (e ) efp also functions as an isg e ligase functional analysis of the porcine usp and its role during porcine arterivirus replication human intracellular isg prevents interferon-alpha/beta over-amplification and auto-inflammation isg deficiency and increased viral resistance in humans but not mice usp sensitivity of peptide transporters pept and pept regulation of type i interferon responses isg , an interferon-stimulated ubiquitin-like protein, is not essential for stat signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus ube l and protein isgylation are not essential for alpha/beta interferon signaling usp establishes the transcriptional and anti-proliferative interferon alpha/beta differential microarray analysis reveals that type i interferon strongly increases the expression of immune-response related genes in ubp (usp ) deficient macrophages usp restricts prrsv growth through alteration of nuclear translocation of nf-kappab p and p in marc- cells the level of hepatitis b virus replication is not affected by protein isg modification but is reduced by inhibition of ubp (usp ) expression cell-type specific interferon stimulated gene staining in liver underlies response to interferon therapy in chronic hbv infected patients identification of interferon-stimulated gene as an antiviral molecule during sindbis virus infection in vivo innate antiviral response targets hiv- release by the induction of ubiquitin-like protein isg ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses protein interferon-stimulated gene conjugation delays but does not overcome coronavirus proliferation in a model of fulminant hepatitis potential relevance of cytoplasmic viral sensors and related regulators involving innate immunity in antiviral response isg , a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment activation of endogenous type i ifn signaling contributes to persistent hcv infection mechanisms of sterile inflammation t cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for t cell activation quantitative analysis of the contribution of tcr/pepmhc affinity and cd to t cell activation suppressor of cytokine signalling protein socs and ubp regulate the expression of type i interferon-stimulated genes in human microvascular endothelial cells infected with rickettsia conorii rickettsia conorii infection stimulates the expression of isg and isg protease ubp in human microvascular endothelial cells candidate genes for type diabetes modulate pancreatic islet inflammation and beta-cell apoptosis usp is a key regulator of the interferon-driven gene network modulating pancreatic beta cell inflammation and apoptosis critical review: assessment of interferon-beta immunogenicity in multiple sclerosis intramuscular interferon beta- a therapy initiated during a first demyelinating event in multiple sclerosis. champs study group usp lack in microglia causes destructive interferonopathy of the mouse brain poised for action: usp restrains microglial activation in the white matter search for specific biomarkers of ifnbeta bioactivity in patients with multiple sclerosis roles of the ubiquitin peptidase usp in multiple sclerosis and the response to interferon-beta treatment ubp regulates bcr-abl leukemogenesis via the type interferon receptor signaling ifns signaling and apoptosis resistance in glioblastoma cells blockade of the ubiquitin protease ubp destabilizes transcription factor pml/raralpha and inhibits the growth of acute promyelocytic leukemia evidence for the ubiquitin protease ubp as an antineoplastic target ubiquitin specific protease (usp ) is a wt transcriptional target ube l causes lung cancer growth suppression by targeting cyclin d ubiquitin and ubiquitin-like proteins in cancer pathogenesis microarray analyses uncover ube l as a candidate target gene for lung cancer chemoprevention the deisgylase usp limits trail-induced apoptosis through the regulation of trail levels: cellular levels of trail influences responsiveness to trail-induced apoptosis usp regulates epidermal growth factor (egf) receptor expression and cancer cell survival via microrna- usp is crucial for ifn-gammamediated inhibition of b melanoma tumorigenesis and antitumor immunity novel combination markers for predicting survival in patients with muscle invasive bladder cancer: usp and dgcr type i interferon: friend or foe? acknowledgements. we thank mouhammad wasseem shaabani for his contribution in designing figures. the authors declare no conflict of interest. key: cord- -gbwb fqi authors: christopher, mary e.; wong, jonathan p. title: broad-spectrum drugs against viral agents date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: gbwb fqi development of antivirals has focused primarily on vaccines and on treatments for specific viral agents. although effective, these approaches may be limited in situations where the etiologic agent is unknown or when the target virus has undergone mutation, recombination or reassortment. augmentation of the innate immune response may be an effective alternative for disease amelioration. nonspecific, broad-spectrum immune responses can be induced by double-stranded (ds)rnas such as poly (iclc), or oligonucleotides (odns) containing unmethylated deocycytidyl-deoxyguanosinyl (cpg) motifs. these may offer protection against various bacterial and viral pathogens regardless of their genetic makeup, zoonotic origin or drug resistance. influenza viruses are a major concern for public health officials due to their potential to cause pandemics. in recent years, influenza viruses a/h , a/h , a/h and a/h [ ] have resulted in human illness as the result of poultry-to-human transmission. at present, these viruses are not readily spread from human-to-human, but a mutated and/or reassorted virus with efficient human-to-human transmission could trigger an influenza pandemic. should the avian h n influenza virus currently circulating in asia assume the role of a pandemic agent, formidable technical difficulties relating to the properties of the virus itself, vaccine production, distribution and administration would ensure that open access vaccines would only become available after a significant lead time, with a low likelihood that a vaccine would be available during the first wave of the pandemic [ , ] . the use of existing antivirals, primarily oseltamivir (tamiflu) could be critical in the initial control of a pandemic [ ] [ ] [ ] , although there have been reports of oseltamivir resistance in influenza h n infected patients [ , , ] . drug resistance in influenza strains is increasing worldwide as evidenced by a survey of influenza h n , h n and h n field isolates. a significant increase in resistance to amantadine and rimantadine from . % in - to . % in - was observed with % of samples obtained in asia since being drug resistant [ ] . mucosal surfaces serve as the entry sites for the majority of infectious pathogens, including influenza viruses, and provide the first line of defence against infection [ ] . during the early stages of infection, the immune response is non-antigen specific, involving natural killer (nk) and natural killer t cells (nkt cells) which, through the activation of antigen presenting cells (apcs), indirectly respond to danger signals derived from invading pathogens. macrophages and dendritic cells (dcs) express numerous toll-like receptors (tlrs) [ ] and respond to microbial pathogens by producing type i interferons (ifn) and cytokines. there are ten different tlrs, each binding specific classes of compounds. unmethylated cpg dinucleotides present in bacterial dna are recognized by tlr [ ] [ ] [ ] , single-stranded (ss)rna viruses, such as vesicular stomatitis virus and influenza virus are recognized by tlr [ ] , and dsrna longer than base pairs, produced during the viral replicative cycle, is recognized by tlr [ , , , ] . in situations where a vaccine is unavailable and/or drug resistance is prevalent, it would be advantageous to stimulate the immune system to non-specifically respond to viral threats. poly (iclc), a dsrna, and cpg odns, molecular mimics for tlr and tlr , respectively, have been reported to non-specifically stimulate the innate immune system and to provide protection against various bacterial and viral pathogens, including influenza. in addressing whether these agents could potentially be used as therapeutic agents or vaccine adjuvants should an influenza h n pandemic arise, we need to understand the mechanism of action of poly (iclc) and cpg odns in relation to the pathogenic effects of influenza h n . cells are armed with various latent mechanisms that are able to sense viral components and initiate intracellular signal transduction to respond rapidly to viral infections. alveolar and bronchial epithelial cells, the primary target and principal host cells for influenza viruses, play a key role in the initiation of innate and adaptive immune responses to influenza virus [ , ] . double-stranded rna produced during viral replication is recognized by an intracellular protein, tlr , present in t, nk [ , , ] , mast [ ] and epithelial cells [ ] . in humans, tlr mrna is constitutively expressed in alveolar and bronchial epithelial cells, placenta, pancreas, liver, spleen, heart, brain and intestine, and is upregulated by influenza virus [ , ] . recognition by tlr results in enhanced production of ifn-α,β, -γ, and -λ, il- β, - , - , - , - and - , ip- , gm-csf, regulated on activation, normal t cells expressed and secreted (rantes), larc, mip- α, gro-α, ena- , icam- (which recruits inflammatory cells) and nitric oxide synthase [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , as well as stimulation of specific components of the cellular and humoral immune systems, including nk cell activation [ , ] and memory cd + t cell proliferation [ ] . additionally, dsrna-activated enzymes such as rna helicases (rig- and mda- ), protein kinases (pkr), '- '-oligoadenylate synthetase and rnaspecific adenosine deaminase which can directly inhibit viral replication are also induced. viruses evade these enzymes either by expressing dsrna-binding proteins or by inhibiting dsrna-induced pathways [ ] . synthetic dsrnas including polyriboinosinic-polyribocytidylic acid (poly (ic)) alone or stabilized with poly-l-lysine carboxymethyl cellulose (poly (iclc)), polyadenosinic-polyuridylic acid (poly (au)) and ampligen (polyi:polyc( )u) have been used as tlr molecular mimics [ , , , ] and tlr expression is up-regulated by these molecules [ ] . it has recently been demonstrated that tlr also binds polyriboinosinic acid, a ssrna [ ] . a comparative study in mice using poly (iclc), poly (ic) and poly (au) determined that ifn induction was highest with poly (iclc) and lowest with poly (au), with splenocyte cytotoxicity being significantly higher with poly (iclc) than with poly (au) [ ] . following intravenous (i.v.) infusion of poly (iclc), substantial increases in total ifn levels were observed to h later in humans and rhesus monkeys, with males of both species having consistently and significantly higher ifn responses [ , ] . the response to poly (ic) in the human tracheobronchial epithelial cell line beas- b was approximately h earlier than in the same cells infected with influenza a/scotland/ / (h n ), possibly reflecting the time required to generate dsrna within viral-infected cells [ ] . interestingly. although tlr is critical for ifn-γ production from cd + t cells, tlr has been implicated in the ifn-α response to poly (au) in murine plasmacytoid dcs (pdcs) [ ] . poly (iclc) has been shown to be effective in protecting rodents against influenza virus [ ] , rift valley fever virus [ ] , rabies virus [ ] , punta toro virus [ ] and western equine encephalitis virus [ ] . it also protects the marine crustacean litopenaeus vannamei from infections of white spot syndrome virus and taura syndrome virus [ ] . in primates, poly (iclc) is effective against yellow fever virus [ ] , venezuelan equine encephalomyelitis virus (veev) [ ] and rabies virus [ ] . poly(ic) has also been shown, through its effects on nk cells, to eliminate the histological lesions of graft-versus-host disease in mice [ ] . ampligen, a mismatched double-stranded rna currently under development by hemispherx biopharma in the u.s.a., acts by inducing ifn production (immunomodulator) and by activating an intracellular enzyme (rnase-l) against viral rna transcripts (antiviral). ampligen is indicated for the treatment of chronic fatigue syndrome and acquired immunodeficiency deficiency syndrome (aids) as part of the combination therapy. in may hemispherx announced that it had filed an expanded u.s.a. patent application covering its use for the potential treatment and prevention of severe acute respiratory syndrome (sars) and other emerging viruses [ ] . ampligen has been determined to be safe in phase iii human trials. in mice, two doses of mg/kg/dose poly (iclc) given intranasally (i.n.) provided complete protection against influenza a/pr/ / (h n ) or a/aichi/ / (h n ) viral challenge (figure ), whereas those pre-treated with a single dose had a slightly lower survival rate of % [ , ] . comparative studies demonstrated that two i.n. doses ( mg/kg/dose) of poly (iclc) was more effective than two i.n. doses ( , u/kg/dose) of either recombinant mouse ifn-α or -γ in protection of mice against influenza a/pr/ / infection, with survival rates of % and %, respectively [ ] . mice treated with two doses of poly (iclc), h apart, up to d prior to viral challenge were completely protected from infection, whereas survival rates decreased to , and %, when pre-treatment was given , or d prior to virus challenge, respectively [ ] . this increased survival, together with the observation that nk cell activity remained elevated for and d post-poly (iclc) treatment in liver and blood/spleen, respectively [ ] , suggests that poly (iclc) may provide short-term prophylaxis against influenza in an outbreak situation. poly (au) also increased nk cell activity in the liver but approximately a -fold higher dose was required than for poly (iclc) [ ] . poly (iclc) was able to provide protection to mice infected with rift valley fever virus provided that the mice were given two to three intraperitoneal (i.p.) doses with one dose being administered prior to infection [ , , ] . a single i.p. injection of ampligen administered either h or - h before infection with banzi virus provided significant improvements in survival. in comparison, ampligen administered prior to infection with semliki forest virus was able to significantly improve mortality when given - h, but not h, prior to infection [ ] . poly iclc treatment decreased the number of veev-infected monkeys that become detectably viremic and delayed the onset of viremia in the remaining monkeys [ ] . when mice were dosed i.p. with mg/kg ampligen h prior to sars-cov exposure, viral titres in the lungs were below detectable limits [ ] . poly (iclc) administered following influenza viral infection was less effective than when administered prophylactically. mice treated with two i.v. doses ( mg/kg/dose) of poly (iclc) and h post-infection (p.i.) showed a small increase in survival ( %) relative to untreated control mice and post-exposure treatment with a single dose was found to be almost completely ineffective [ ] . efficacy of post-exposure poly (iclc) treatment has primarily been observed when given in conjunction with pre-exposure treatment(s). efficacy has been reported for mice infected with punta toro virus [ ] and west nile virus when treatments were started d prior to infection, continuing every h until d post-infection. when treatment was delayed until - h before viral challenge, efficacy was greatly reduced [ ] . for treatment of rift valley fever at least four doses were required when treatment was started h p.i. [ ] . poly (ic) has also been used as an adjuvant with split-product influenza vaccines. coadministration of poly (ic) with the primary and booster doses of vaccine followed by challenge with influenza a/pr/ / resulted in cross-protection when administered with various h n virus vaccines (a/pr/ , a/beijing, a/yamagata), partial protection with heterologous influenza a vaccines (a/guizhou -h n ) and no protection with influenza b vaccines (b/ibaraki, b/yamagata, b/aichi) [ ] . t-cell activation and increased ifn-γ production was observed only in mice immunized with homologous antigens [ ] . treatment with vaccine plus poly (ic) rapidly up-regulated tlr expression in the nasal-associated lymphoid tissue, up-regulated il- and il- p , and induced ifnα, -β and -γ suggesting that route of administration of vaccine plus poly (ic) was important [ ] . a trivalent inactivated influenza vaccine co-administered with ampligen provided cross-protection against various strains of h n influenza virus (a/hongkong/ / , a/vietnam/ / and a/indonesia/ / ) when administered i.n. but not when given subcutaneously (s.c.), confirming the importance of the route of administration [ , ] . poly (iclc) adjuvant activity has been shown when co-administered with retinoic acid [ ] , veev vaccine [ , ] and the antimalarial drug chloroquine [ ] . synergism of poly (iclc) with various anti-hiv compounds including cytokines (rifn-α a, rifn-β ser , and rifn-γ), reverse transcriptase inhibitors (azidothymidine and phosphonoformate (foscarnet)), mrna capping inhibitors (ribavirin), lipophile (amphotericin b) and glucosidase inhibitor (castanospermine) was observed [ ] . similarly, priming with either ifn-α/β or poly (ic) completely blocked or transiently reduced west nile virus replication in macrophages from resistant mice or susceptible mice, respectively. combined pretreatment with ifn-α/β and poly (ic) elicited strong antiviral responses that completely prevented flavivirus replication in macrophages from susceptible mice [ ] . anti-semliki forest virus hyperimmune serum or poly (iclc) given i.p. were not protective when used alone following an intracranial semliki forest virus infection, but when given together survival rate increased to % and viremia and viral load in the brain became undetectable [ ] . a phase i study of poly (iclc), in combination with il- , in patients with a variety of cancers showed moderate toxicity of poly (iclc) at all doses tested. no increases in peripheral blood nk cell activity was observed after treatment with poly (iclc) alone but high doses of poly (iclc) (> . mg/m ) in combination with il- resulted in nk cell activity greater than that seen using the same dose of il- in combination with lower poly (iclc) doses [ ] . the potential of poly (iclc) as an anti-influenza agent is, however, limited by its intrinsic toxicity. toxicity of poly (iclc) is affected by the route of administration with s.c. administration being well tolerated in rabbits [ ] and mice (unpublished observations) and intratracheal administration being well tolerated in mice [ ] . intranasal, intramuscular (i.m.), i.v. or i.p. administration results in variable levels of toxicity depending on the animal species being used. in clinical trials, patients receiving multiple therapeutic doses of poly (iclc) i.v. or i.m. exhibited serious toxic reactions including hypotension, fever, anemia, leukopenia, thrombocytopenia, nausea, injection site inflammation and, in multiple sclerosis patients, neurological dysfunction [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . intra-articular administration induced arthritis, mediated by il- receptor (il- r) signalling, as early as d post-administration [ ] . attempt to improve safety and efficacy of poly (iclc) have focused on optimization of dosage and treatment regimes, encapsulation within liposomes, modification of poly (iclc), and coadministration of agents that mitigate cytokine-mediated adverse reactions. in patients with advanced cancer, poly (iclc) administered on an alternate-day schedule with gradual dose escalation was tolerated the best with the maximum tolerated dose varying over a several hundredfold dose range [ ] . in mice, poly (iclc) administration results in loss of up to % of the total body weight and hypothermia of up to o c [ ] . to mitigate the toxicity of poly (iclc) without adversely affecting its biological activities, poly (iclc) has been encapsulated within cationic liposomes composed of phosphatidylcholine, cholesterol (ch) and stearylamine. intranasal administration of μg free or liposome-encapsulated poly (iclc) at - and - d, followed by challenge with a lethal dose of influenza a/pr/ / at d resulted in no demonstrable reduction in weight loss following administration or influenza challenge, but the magnitude and duration of body temperature reduction was reduced [ ] . liposome-encapsulation completely mitigated the toxicity (as determined by absence of weight loss and changes in body temperature) seen with free poly (iclc) when administered i.v. [ ] . mice that received pre-treatment with liposome-encapsulated poly (iclc) d prior to virus challenge were fully protected whereas mice given free poly (iclc) were completely protected if treatment was within d of infection [ ] . liposomes have been shown to accumulate at sites of infection [ ] , possibly concentrating the encapsulated drug at the diseased site and thereby minimizing the exposures of healthy organs and tissues to the drugs. together, these results suggest that liposome encapsulation results in a gradual and sustained release of poly (iclc), thereby avoiding rapid systemic elevation of drug levels observed with some routes of administration. it is unclear whether the significant reductions in the toxicity of poly (iclc) provided by liposomes will result in corresponding decreases in clinical side effects seen in human patients. such attenuation of the toxic side effects in patients may result in increase drug tolerance and improve clinical outcomes. mitigation of cytokine-mediated adverse reactions has been attempted using il- r signalling pathway agonists and hydrocortisone treatment. the il- r signalling pathway, stimulated by poly (iclc), is implicated in increased plasma il- concentrations. male rats given il- receptor agonist (il- ra) prior to poly (ic) administration had elevated plasma tnf-α, but not il- , concentrations with a concomitant reduction in fever [ ] . hydrocortisone treatment prior to or following i.v. poly (iclc) administration reduced both the hypotensive responses and interferon induction in rabbits [ ] . rabbits mimic the human febrile and hypotensive response to poly (iclc) [ ] and thus would be useful experimental models to evaluate mechanisms / approaches to reduce poly (iclc)-mediated toxicity. a lower molecular weight ( s) poly (iclc) was able to generate high titers of ifn and ameliorated hypotensive responses in rabbits, however, it also induced high fevers [ ] . selective thiolation of the poly (c) strand at the five position of the cytosine base, generates a partially thiolated poly (c) (mpc) which, after annealing with a complentary unmodified poly (i), forms the thiolated dsrna, pi:mpc. optimal antiviral and antiproliferative activities were obtained when thiolation was at . % [ ] . these results suggest that further study into modification of poly (iclc) may be advantageous for the elimination / reduction of toxic side effects. cpg dinucleotides are under-represented in vertebrate dna ( in base pairs) and are generally methylated on the cytosine whereas bacterial dna contains unmethylated cpg dinucleotides at the expected frequency of in base pairs [ ] . cpg dinucleotide-containing dna or cpg-containing oligonucleotides (cpg odns) are rapidly internalized by immune cells through the endocytosis / phagocytosis pathway where they interact with the constitutively expressed receptor, tlr , present in endocytic vesicles, triggering swelling and acidification of the vesicle and generating reactive oxygen species [ , , ] . activation of the cpg odn/tlr signalling pathway culminates in the activation of several transcription factors [ , , ] which directly up-regulate production of proinflammatory cytokines (il- , - , - , - - , gm-csf, tnf-α, ifn-α, -β -γ, -λ, -ω), chemokines (mcp- , ip- , mip- α and β) and immunoglobulins [ , , , [ ] [ ] [ ] . cpg odns also stimulates maturation, differentiation, and proliferation of multiple immune cells, including b lymphocytes, monocytes, macrophages and dcs [ , [ ] [ ] [ ] [ ] and protects these cells from apoptosis [ ] . this, in turn, stimulates t cells to secrete additional cytokines and nk cells to secrete ifn-γ and have increased lytic function. as tlr molecules expressed by different species have diverged over evolutionary periods [ ] the precise sequence motif (cpg dinucleotide plus flanking sequences) optimal for stimulating immune cells varies between species (table ) . cell populations expressing tlr also differ between species. in mice, immune cells of the myeloid lineage (monocytes, macrophages, myeloid dcs) express tlr whereas, in humans, memory b (but not naïve b cells) [ ] and pdcs express tlr [ , , , , , ] . the activity of other immune cell subsets such as monocytes, nk cells, γδ t cells, and memory cd t cells is increased by cpg odn via pdc-derived cytokines, but due to the lack of tlr expression in these cell subsets, there is no direct effect of cpg odn on these cells [ , , ] . human peripheral blood mononuclear cells (pbmc) induce ifn-α and ifn-γ in response to cpg odn whereas mouse splenocytes induce ifn-γ, il- p and il- . this partial disparity in cytokine induction is due to the mouse splenocyte response being dominated by monocytes (a non-responder cell in humans) [ ] . cpg odns also upregulate cytokine and chemokines expression in cells of the central nervous system, promote angiogenesis and have an effect on bone formation [ , ] . [ , , ] cpg odns have been synthesized using either a phosphodiester, phosphorothioate or a mixed dna backbone with the phosphorothioate backbone rendering the cpg odn more resistant to nucleases [ ] , extending the plasma half-life from about min to - h, although a biphasic plasma elimination profile was observed [ ] , and lowering the dosage required for activity [ ] . liver and kidney have the highest uptake of phosphorothioate-containing cpg odns in mice and rats, with a significant amount being detected in the spleen [ ] . cpg odns accumulate intracellularly with little nuclear uptake [ ] except in hela cells where k-type cpg odns (table ) were observed to localize within the nucleus and mitochondria [ ] . dramatically different profiles and kinetics of immune activation have been observed with the various cpg odn backbones resulting in the classification of three distinct families of cpg odns: d-type (cpg-a), k-type (cpg-b) and c-type (cpg-c) ( table ) . studies of k-type cpg odns indicate that the sequence, number and location of cpg motifs influence the magnitude of the resultant response with cpg motifs at the ' end triggering significantly greater immune activation. addition of extra cpg motifs into d-or c-type cpg odns did not improve their activity, likely due to disruption of the palindromic sequence [ , ] . k-type cpg odns may preferentially trigger early type i ifn production, whereas d-type cpg odns may be able to support late type i ifn production via the ifn-αβ-mediated feedback loop [ ] . currently cpg odn-induced activation of innate immunity is being investigated in a wide range of models for protection against a variety of pathogens and for therapeutic activity against cancer and allergy [ , ] . as the cpg odn-stimulated immunomodulatory cascade peaks at - d and persists for several weeks, it has been suggested that cpg odns must be administered early (preferably before infection) to protect against rapidly lethal pathogens whereas treatment of slowly growing pathogens can be delayed until several weeks after challenge [ ] . in a study evaluating the efficacy of cpg odn against influenza, mice given μg of a k-type cpg odn d prior to infection with a lethal dose of influenza a/pr/ / (h n ) survived the viral challenge whereas pbs-treated mice did not (figure ), thus demonstrating that cpg odn pre-treatment is efficacious against influenza viral infection [ ] . cpg odns appear to enhance the immune response even when the response is impaired due to age or disease. cpg odn treatment of splenocytes from senescence-accelerated sam-p strain of mice increased ifn-γ and administration in vivo generated virus-specific cytotoxic t lymphocyte responses, nk cell activation, virus-specific ig isotype switch from igg to igg a, increased viral clearance and survival following influenza viral challenge. -active in mice [ ] , nonhuman primates [ ] -best activity in humans [ , , ] k-type -stimulate cytokine production [ ] -induce cell proliferation and il- production from human pbmcs [ ] -active in mice [ ] , human pbmc in vitro [ ] -poorly active in primates [ ] c-type -stimulate strong b-cell and nk cell activation [ ] -stimulate pdcs to produce ifn-α [ , ] -potent th adjuvant [ ] -stimulate b-cells to secrete il- , il- and igm [ , , ] this suggests that cpg odns could contribute to the development of a protective strategy in immunocompromised elderly persons [ ] . although siv-infected macaques have no detectable ifnγ production, treatment with d-type cpg odns increased ifn-α and reduced leishmania amazonensis-induced lesions when treated at - and + d, whereas those treated with k-type cpg odns had no reduction in infection although il- and cell proliferation was similar to healthy macaques [ ] . caution must be taken in extrapolating animal studies to humans because pbmc from healthy and hiv-infected donors had similar in vitro responses to k-and d-type cpg odns although the magnitude of the response to d-type cpg odns was reduced in hiv-infected donors relative to healthy donors [ ] . a clinical study on patients with various types of b cell non-hodgkin's lymphoma found that most b cell malignancies (with the exception of plasmacytoma) responded to cpg odns by up-regulating expression of co-stimulatory and antigen-presenting molecules, by increasing expression of cd and by proliferation, although the proliferative response was less than in normal b cells [ ] . these results suggest that it may be possible to use cpg odns as adjuvants to expand the potency and efficacy of antiviral vaccines in the population that is most at risk and yet has the least effective immune response to vaccination. cpg odns, when administered prior to pathogen challenge, have been shown to provide complete protection against listeria monocytogenes [ , [ ] [ ] [ ] [ ] , francisella tularensis [ ] and herpes simplex virus (hsv- ) [ ] in mice, escherichia coli [ ] in chickens and leishmania major [ , ] in rhesus macaques. when the cpg odn was administered following infection, complete protection was observed in mice challenged with leishmania major [ ] . complete protection was also observed against mycobacterium tuberculosis [ ] however, the cpg odn had to be given prior to and following infection. cpg odns provided partial protection to mice infected with plasmodium yoelii [ , ] , friend virus [ ] , hsv- [ ] , ebola virus [ ] or rml scrapie prion [ ] , to chickens infected with eimeria coccidiosis [ ] or to rhesus macaques infected with leishmania amazonensis [ ] . the disparate results observed with mice infected with hsv- can likely be attributed to the timing of cpg odn administration with treatment h prior to infection resulting in complete protection and treatment - h post-infection resulting in partial protection, although the strain of mouse used could also bias the results with balb/c and swiss webster mice being used, respectively. cpg odns increase influenza-specific antibody production when co-administered with inactivated influenza virus, influenza protein or plasmid-based vaccines. intranasal delivery of formalininactivated influenza virus vaccine/cpg odn mixture enhanced production of influenza-specific antibodies in the serum, saliva and genital tract with seven-fold higher antibody levels detected in mice administered vaccine with cpg odn [ , ] . intranasal delivery of plasmid dna encoding influenza a/pr/ / hemagglutinin (ha) administered with or without k-type cpg odn afforded protection against a lethal dose of influenza a/pr/ / with survivors having elevated anti-ha igg a titres. anti-ha igg b titres were also increased but only in cpg odn treated mice [ ] . covalent linkage of cpg odn to influenza virus ha resulted in secretion of high levels of ifn-and ha specific igg a antibodies [ ] suggesting that crosslinking cpg odns to peptides may be an efficient adjuvant method. encapsulation of cpg odn and influenza subunit vaccine within liposomes further enhances vaccine potency while reducing the number of administrations. three to twelve weeks post-vaccination, mice treated with liposome-encapsulated vaccine plus cpg odn had up to times higher serum and mucosal igg a and iga levels [ ] . in a double-blind study, mg of cpg odns were co-administered with a commercial trivalent killed split influenza vaccine (fluarix, smithkline beecham). inclusion of cpg odns did not increase the antibody response of naïve recipients when compared to fluarix alone but did significantly increase anti-ha titers among subjects with pre-existing anti-influenza antibodies. pbmcs from cpg odn-vaccinated subjects responded to in vitro re-stimulation by secreting significantly higher levels of ifn-γ than pbmcs from control vaccinees [ ] . vaccinees given cpg odn plus either a full or a reduced ( . ) dose of fluarix had similar hemagglutinin inhibition and anti-ha antibody titres, but antigen-specific ifn-γ secretion from pmbc was decreased in the reduced fluarix dose group [ ] . this suggests that cpg odn adjuvants can potentially expand the number of people that can be immunized when vaccine supply is limiting. human studies using cpg odn iss (immunostimulatory dna sequence), a -mer k-type cpg odn (dynavax technologies) [ , ] or cpg (promune, coley pharmaceuticals) [ , [ ] [ ] [ ] have been conducted. cpg odn co-administered with alum-absorbed hbsag increased the pool of high-avidity antibodies in an antigen and isotype-specific manner [ ] and co-administration with a commercial hepatitis vaccine (energix b, glaxosmith kline) generated protective levels of anti-hbs antibodies within two weeks of the priming vaccine, with subjects receiving higher doses of cpg odn having higher rates of positive cytotoxic t cell lymphocyte responses [ ] . in phase i clinical studies, seroprotective anti-hbsag antibody titers were observed after a single dose of rhbsag plus mg iss in . % of subjects whereas those not receiving iss did not produce protective antibodies [ ] . cpg odn iss has also been evaluated in phase i clinical studies as an adjuvant to the anti-cd chimeric monoclonal antibody rituximab (genetech, inc.) in patients with relapsed non-hodgkins lymphoma [ ] . in addition to influenza and hepatitis b, cpg odns have also been evaluated for their adjuvant potential with protein vaccines targeting hepatitis c virus [ ] , herpes simplex virus - (hsv- ) [ ] , sars-cov [ , ] , bovine herpesvirus [ ] , simian immunodeficiency virus [ ] , plasmodium yoelii [ ] and melanoma antigen [ ] and antibody therapy [ , ] . in contrast to mice, protocols for immunizing humans and livestock require higher doses of cpg odn to exert adjuvant activity [ ] . most adverse events associated with cpg odn co-administration with various vaccines were predominantly short-lived reactions (i.e. pain and erythema) at the injection site and flu-like symptoms [ , , , , , ] . however, concerns relating to cpg odn-induced production of il- and blockade of apoptotic death of activated lymphocytes, functions that predispose to the development of autoimmune disease by facilitating the persistence of self-reactive lymphocytes, have been raised [ , ] . some studies have found that repeated injection of immunostimulatory doses of cpg dna does not appear to induce or accelerate systemic autoimmune disease [ ] [ ] [ ] , however, allergic encephalomyelitis [ , ] , autoimmune myocarditis [ ] , joint inflammation [ ] and overproduction of tnf-α which can cause life-threatening toxic shock [ , [ ] [ ] [ ] have been demonstrated. immunosuppression was observed in mice given high ( μg) doses of cpg odn daily [ ] . since a single μg dose of cpg odn was able to afford protection against influenza [ ] it would be interesting to know whether daily treatment with a low dose of cpg odn also induced immunosuppression. as with poly (iclc), liposome-encapsulation of cpg odns has been used to reduce toxicity and prolong exposure due to gradual release of the cpg odn from liposomes [ , ] . mice treated with liposome-encapsulated cpg odn d prior to infection with a lethal dose of influenza a/pr/ / survived the influenza viral challenge with a lower weight loss and faster recovery than mice treated with free cpg odn, suggesting that liposome-encapsulation decreases cpg odn toxicity [ ] . cpg odn encapsulated within stabilized antisense-lipid particles increased plasma concentrations of il- , - , ifn-γ, monocyte chemoattractant protein- and tnf-α to a greater extent than unencapsulated cpg odn, with encapsulated phosphodiester cpg odns strongly stimulating cytokine induction at the early time points [ ] . this suggests that liposome-encapsulation can be used to decrease dosage required for activity. encapsulation of cpg odns within large ( . μm) multilamellar liposomes can change the type of response observed depending on what is co-administered. in mice, co-administration with either a subunit influenza vaccine or hbsag demonstrated a th -dominant or a mixed th /th response in the influenza and hepatitis b models, respectively [ ] . in cells lacking tlr , such as prostate cancer pc cells, treatment with phosphorothioate cpg odns had no effect unless co-administered with lipofection transfection agent suggesting that phosphorothioate cpg odns could also function in a tlr -independent manner and that liposome-encapsulation of cpg odns could potentially expand the variety of cell types responding to the cpg odn [ ] . co-delivery of cpg odn adjuvants and antigens in nanospheres has also been shown to be an efficient approach for immunization [ ] . conjugation of odns or ligands to cpg odns has been used in attempts to improve efficacy. in mice, a cpg odn-antigen conjugate inhibited influenza virus more efficiently than the coadministration of cpg odn and antigen [ ] . the location of ligand conjugation is important as cpg odns have reduced immunostimulatory activity when compounds were conjugated '- ', although conjugation of small molecules (i.e. phosphorothioate groups) had an insignificant effect. conjugation of an odn or a ligand through the ' end of cpg odn ( '- ' linkage) has no effect on immunostimulatory activity [ ] . two cpg odns linked '- ' are termed immunomers. a synthetic nucleoside "r" with a bicyclic heterobase [ -( '-deoxy-β-d -ribofuranosyl)- -oxo- -deaza- -methylpurine] has been used to replace the c in the cpg dinucleotide motif and has been evaluated in both a rpg odn and an immunomer. rpg odns activated nf-kВ and mitogen-activated protein kinase pathways and rpg immunomers induced high levels of il- and ifn-γ in a time-and concentrationdependent fashion in mouse splenocytes costimulated with il- . significantly, immunomers containing gtrgtt and gargtt were recognized to a similar extent by both mouse and human immune systems, stimulated proliferation of pbmcs and prevented conalbumin-and ragweed allergen-induced allergic inflammation in mice [ ] . tlr is an intracellular tlr, located on endosomal membranes, which recognizes ssrna in a sequence-independent manner as long as the rna contains several uridine residues in close proximity to each other [ , ] . nucleosides and nucleotides from intracellular pathogens, guanine nucleoside analogues, stabilized immunoregulatory rna, short dsrna oligonucleotides containing several uridine residues in close proximity (i.e. short interfering rna (sirna)) [ ] and imidoazoquinolinebased compounds such as a and imiquimod (imq) [ ] are also recognized by tlr . as with tlr , pdc are the primary ifn-α producing cells following exposure to a tlr agonist and stimulation of tlr results in induction of il- , il- , mip- α, mip- β, tnf-α and ifn-β among others [ ] [ ] [ ] [ ] [ ] . single-stranded rna induction of tlr also stimulates autophagy, a cellautonomous innate defence mechanism for elimination of intracellular pathogens, in macrophages. this induction of autophagy appears to be effective in eliminating intracellular microbes, even when the target pathogen is normally not associated with tlr signalling [ ] . tlr is essential for influenza viral recognition and inflammatory cytokine production by murine neutrophils [ , ] . mice pretreated with a tlr agonist ( -[ -amino- -hydroxy- -( -methoxyethoxy)purin- -ylmethyl]benzaldehyde) -mouse serum albumin conjugate prior to influenza a h n viral challenge had a significant delay in mortality relative to those not receiving the conjugate [ ] suggesting that stimulation of the influenza-binding receptor before infection can improve the outcome . intranasal administration of the synthetic tlr / agonist m- significantly inhibited h n influenza viral replication in the nasal cavity of rats when administered between h before and h after viral challenge. viral inhibition correlated with the ability of the tlr / agonist to stimulate type i ifn and other cytokines such as tnf-α, il- , and ifn-γ from rat pbmc. the activity of the tlr / agonist resulted in greater inhibition of viral titers compared to rat recombinant ifn-α administered in a comparable dosing regimen [ ] . there are, however, some serious concerns relating to stimulation of tlr receptors. in vivo studies in systemic lupus erythematosus (sle) mouse models demonstrate an essential role for tlr in the generation of rna-containing antinuclear antibodies and deposition of pathogenic immune complexes in the kidney, with tlr recognizing rna-and dna-containing autoimmune complexes and tlr amplifying the autoimmune response [ ] . transgenic analysis of tlr determined that a modest increase in tlr expression resulted in spontaneous development of autoimmunity and a substantial increase in tlr expression caused fatal acute inflammation and profound dc dysregulation, indicating that tlr must be tightly regulated in order to prevent spontaneous triggering of harmful autoreactive and inflammatory responses [ ] . inhibition of tlr and tlr with the immunoregulatory sequence inhibited the induction of ifn-α by human pdc in response to dna and rna viruses and immune complexes from sle patients and reduced sle disease severity [ ] . in humans, pbmcs isolated from females produced significantly higher ifn-α levels after tlr stimulation than did pbmcs isolated from males although there was no difference in tnfα production between cells isolated from both sexes. this sex-dependent activation of ifn-α by tlr may explain the higher prevalence of sle in females and the reported decrease in therapeutic efficacy of synthetic tlr ligands in males [ ] . influenza a is a highly contagious single-stranded rna virus that infects both the upper and lower respiratory tracts of humans. during a single-cycle infection, human viruses preferentially infect nonciliated cells, whereas avian viruses primarily infect ciliated cells. this correlates with the predominant localization of receptors for human (α- , -linked sialic acids) and avian (α- , -linked sialic acids) viruses on nonciliated and ciliated cells, respectively [ ] . sialic acid linked to galactose via α- , glycosidic bonds, is a cellular receptor located in the eye, which may account for the ocular tropism exhibited by zoonotic avian influenza a viruses such as h n in hong kong in , n n in the u.s.a. in , h n in the netherlands in and h n in canada in [ ] . infiltration of lymphocytes, neutrophils, and macrophages in the lungs and production of reactive oxygen species, which contributes to pulmonary tissue damage, is observed [ , ] . influenza virus infection induces expression of a variety of factors including il- β, - , - , tnf-α, fas ligand, ifn regulatory factor (irf)- , ifn-α, -β, eotaxin (eosinophil chemoattractant), rantes, tgf-β, dsrna dependent protein kinase (pkr), indolamine , -deoxygenase (ido) and '- 'oligoadenylate synthetase ( ) ( ) ( ) ( ) [ ] [ ] [ ] [ ] . inflammatory-mediated apoptosis is also induced [ ] [ ] [ ] . il- , produced by t cells specific for influenza a virus, is involved in t cell dependent ifn-γ production by nk cells, suggesting that at an early stage of recurrent viral infection, nk cellmediated innate immunity to the virus is enhanced by pre-existing virus specific t cells [ ] . a recent study showed that chickens infected with influenza h n viral strains were resistant to h n influenza viral challenge. this cross-protection was mediated by t cells bearing cd (+) and t-cell receptor (tcr) α/β vβ subset [ ] . protective immunity was closely related to the percentage of cd (+) t cells expressing ifn-γ in the lung, rather than in the spleen, suggesting that pulmonary cellular immunity may be very important in protecting naïve natural hosts against lethal influenza viruses [ ] . there are significant differences in cytokine responses between h n (a/hk/ / , a/vietnam/ / and a/vietnam/ / ) and h n influenza viruses, primarily in relation to ifn-β, il- , ip- and rantes which are elevated post-infection in human type ii pneumocytes, but significantly more so in h n infected cells, with more recent h n viruses from vietnam (h n / ) being more potent at inducing ip- [ ] . a comparative study using quantitative pcr and cdna arrays demonstrated that h n / viruses induced much higher gene transcription of proinflammatory cytokines, particularly tnf-α and ifn-β, than did h n or h n influenza viruses in human primary monocyte-derived macrophages in vitro [ ] . inactivation of the virus by uv irradiation prior to infection of alveolar epithelial cells abolished cytokine induction suggesting that virus replication was required for cytokine induction [ ] . transforming growth factor β (tgf-β), a potent proinflammatory cytokine that activates monocytes to induce the expression and release of various growth factors and inflammatory mediators, is induced by infection with non-hk-origin h n avian influenza viruses as early as -h p.i. and continues to increase for at least h. in contrast, hk-origin influenza virus infected mice showed no increase in tgf-β activity suggesting that hk viruses fail to activate latent tgf-β [ ] . influenza h n infection in birds is systemic, characterized by hemorrhage and edema resulting from virus replication in the endothelium [ ] . systemic infection has also been observed in cynomolgus macaques and humans [ , ] . vascular endothelial growth factor (vegf), produced primarily but not exclusively in alveolar epithelial cells, is induced in a time-and dose-dependent manner by ifn-γ, il- β and tnf-α, increasing microvascular permeability and contributing to pulmonary edema [ ] . in mice infected with influenza h n / , virus-infected cells initially appeared in the respiratory tract and later could be detected in neurons, glial and ependymal cells of the central nervous system [ ] . h n influenza viruses can be further classified into high and low pathogenicity viruses. the difference in host response to the lethal and nonlethal h n influenza virus is likely due to the nonstructural (ns) protein [ , , ] . mice infected with a lethal (a/hong kong/ / ) h n influenza virus had a significant decrease in the total number of circulating leukocytes (primarily lymphocytes) as early as d p.i. and a reduction in the number of cd (+) and cd (+) t cells [ ] . il- β, ifn-γ and mip- α in lung and lymphoid tissue were elevated in mice infected with either a lethal (a/hong kong/ / ) or nonlethal (a/hong kong/ / ) h n influenza virus although the degree of elevation was lower in mice infected with the lethal strain [ ] [ ] [ ] ; whereas, tnf-α and mip- levels were elevated in a similar manner by both strains [ , ] . it has been suggested that tnf-α may contribute to early disease severity whereas il- may play a role in viral clearance late in h n infection [ ] . mice infected with the lethal h n influenza virus hk also had increased concentrations of il- β, tnf-α, ifn-γ, mip- α and mip- in the brain, and apoptosis in the spleen and lung [ ] . although a/hk/ / infected mice showed no evidence of virus-induced encephalitis, the local synthesis of tnf-α or il- within the brain could contribute to anorexia, weight loss and death [ ] . the lethal h n influenza virus appears to possess the capacity to limit the induction of immune responses by targeting and destroying lymphocytes resulting in aberrant production of cytokines in serum and tissues ("cytokine storm"). in addition to the cytokine storm, systemic viral dissemination and alveolar flooding due to inhibition of cellular sodium channels contribute to the lethality of influenza h n disease [ , , , [ ] [ ] [ ] . in humans, influenza h n virus mediates a cytokine storm characterized by insensitivity to the antiviral effects of ifn (possibly due to insufficient production) and increased concentration of ifn-β, ip- , rantes, il- and tnf-α in the lung and macrophages. in mice, decreased cd (+) and cd (+) t cells, apoptosis of lymphocytes in the spleen and lung and detection of cytokines in the brain are associated with influenza h n pathology. theoretically, for non-specific immune stimulators to be effective in influenza h n viral infection, they should overcome the insensitivity to ifn and should prevent apoptosis of lymphocytes without contributing further to cytokine dysregulation. prevention of apoptosis of t cells could potentially increase tnf-β, ifn-γ, il- , - , - , - , - , - and - . preferably, tnf-α would not be induced by the non-specific immune stimulator as it is already highly induced by influenza a h n viral infection, although mice deficient for tnf-α or its receptors had no reduction in mortality when infected with a/vietnam/ / (h n ) suggesting that tnf-α alone is not responsible for the increased mortality of h n influenza viruses [ ] . inhibition of the cytokine response by corticosterone, the natural mouse glucocorticoid, was not sufficient to prevent death, regardless of when the drug was administered, [ ] suggesting that factors other than the cytokine storm are important for lethality. tlr activation by influenza virus stimulates cytokine production [ , ] , whereas a myd dependent pathway distinct from the tlr pathway appears to be involved in b cell responses to influenza virus [ , ] . the potent ifn-α induction by c-and d-type cpg odns in human pdcs and b cells is markedly reduced by stimulation of tlr , without affecting il- secretion or b cell proliferation [ ] [ ] [ ] . this suggests that a negative feedback mechanism has evolved which could act to prevent levels of ifn-α secretion that are detrimental to the host. experimental analysis of sequential activation of tlr , and in hek cells demonstrated that activation of tlr inhibited tlr activation but not vice versa [ ] . this suggests that cpg odns could be prophylactic agents against h n influenza viral infection. tlr agonist plus tlr / agonists, in the presence of the membrane permeability enhancer dotap, had an additive effect on ifn-α/β responses in human pbmcs [ ] . treatment of mice with liposome-encapsulated poly (iclc) has been evaluated for efficacy against influenza a/h n /chicken/henan (figure ). mice given one ld of the virus had a % survival rate whereas those given two doses of liposome-encapsulated poly (iclc) had a % survival rate. when the virus dose was increased to ld , survival of treated mice decreased to % whereas the untreated mice succumbed to infection. this demonstrates that non-specific immune stimulators may be an effective prophylaxis against a pandemic strain of influenza. similar to influenza h n virus, respiratory syncytial virus (rsv) is also a poor inducer of ifnα/β and is partially resistant to ifns antiviral activity. when poly (iclc), an ifn-α inducer, was given before rsv infection, mice had a milder disease and/or faster recovery with increased ifn production and reduced viral replication [ ] . however, when either poly (iclc) or cpg odn was administered h post-rsv infection, ifn-α production was almost completely inhibited [ ] . in human pdc, the tlr -dependent ifn-inducing pathways were abolished by infection with measles virus and rsv a with the rsv-mediated effects being attributed to the ns protein of rsv through interference with activation of the essential ifn transcription factor irf- [ ] . it has not yet been demonstrated whether influenza h n affects tlr /tlr signalling pathways. should an influenza h n pandemic occur, all age groups would be affected. influenza traditionally disproportionately affects the young and the elderly, with both the innate and adaptive immune systems being affected by aging. in the innate immune system, the functions of nk cells, macrophages (fewer number and less efficient antigen presentation) and neutrophils (impaired chemotaxis, degranulation and phagocytosis) are decreased with aging [ ] . age-related changes in the adaptive immune system include diminished/altered cytokine patterns (th bias), reduction in clonal expansion and function of antigen-specific t and b cells and a decline in antigen-presenting cell function [ ] . humoral immunity also exhibits changes albeit to a lesser extent, particularly the diminished ability to generate high-affinity protective antibodies against infectious agents. splenic and activated peritoneal macrophages from aged mice express significantly lower levels of all tlrs and macrophages from aged mice secrete significantly lower levels of il- and tnf-α when stimulated with known tlr ligands [ ] . poly (iclc) was able to effectively protect aged mice against lethal murine cytomegalovirus infection and effectively induced ifn [ ] . in immature and aging mice, treatment with cpg hastened maturation of dcs and recovery/enhancement of the th type response, respectively [ ] [ ] [ ] . similarly, immune compromised scid mice were almost completely protected against murine cytomegalovirus infection by poly (iclc) and poly (iclc) was able to induce ifn and nk cell cytotoxicity in these mice [ ] . 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polyribosinic-polycytidylic acid stabilized with l-lysine and carboxymethylcellulose, maleic anhydride divinyl ether and colony stimulating factor cpg oligodeoxynucleotides act as adjuvants that switch on t helper (th ) immunity cpg dna functions as an effective adjuvant for the induction of immune responses in aged mice cpg-dna stimulates cellular and humoral immunity and promotes th differentiation in aged balb/c mice nucleic acidbased antiviral drugs against seasonal and avian influenza viruses synergistic activation of innate immunity by double-stranded rna and cpg dna promotes enhanced antitumor activity differential production of cytokines, reactive oxygen and nitrogen by bovine macrophages and dendritic cells stimulated with tolllike receptor agonists key: cord- -gf bglm authors: scutigliani, enzo maxim; kikkert, marjolein title: interaction of the innate immune system with positive-strand rna virus replication organelles date: - - journal: cytokine growth factor rev doi: . /j.cytogfr. . . sha: doc_id: cord_uid: gf bglm the potential health risks associated with (re-)emerging positive-strand rna (+rna) viruses emphasizes the need for understanding host-pathogen interactions for these viruses. the innate immune system forms the first line of defense against pathogenic organisms like these and is responsible for detecting pathogen-associated molecular patterns (pamps). viral rna is a potent inducer of antiviral innate immune signaling, provoking an antiviral state by directing expression of interferons (ifns) and pro-inflammatory cytokines. however, +rna viruses developed various methods to avoid detection and downstream signaling, including isolation of viral rna replication in membranous viral replication organelles (ros). these structures therefore play a central role in infection, and consequently, loss of ro integrity might simultaneously result in impaired viral replication and enhanced antiviral signaling. this review summarizes the first indications that the innate immune system indeed has tools to disrupt viral ros and other non- or aberrant-self membrane structures, and may do this by marking these membranes with proteins such as microtubule-associated protein a/ b-light chain (lc ) and ubiquitin, resulting in the recruitment of ifn-inducible gtpases. further studies should evaluate whether this process forms a general effector mechanism in +rna virus infection, thereby creating the opportunity for development of novel antiviral therapies. the innate immune system forms the first line of defense against pathogens and its initial function is to recognize pathogenassociated molecular patterns (pamps) [ ] , which ultimately leads to induction of the antiviral state that effectively hampers spread of the infection. the adaptive immune system then kicks in to (in most cases) fully clear the virus and build up memory. viral rna is a very potent inducer of innate antiviral signaling [ , ] . therefore, detection of viral rna and the subsequent induction of antiviral effector mechanisms play an important part in the onset of an antiviral state in the context of rna virus infections. the study of pamp recognition and signaling by cytosolic and membrane bound sensors has intensified tremendously in the last decade. several comprehensive reviews on this subject have been published lately [ , ] . additionally, investigation of the involvement of intracellular organelle membranes of mitochondria, er, and peroxisomes and their mutual interactions indicated that these are important signaling platforms in innate immunity [ , ] . together, this has resulted in a better understanding of the details of innate immune responses that target rna viruses. in this review, we will focus on the interaction of the innate immune system with viruses that have a positive-strand rna (+rna) genome. in response to innate immune reactions, virtually all +rna viruses interfere to delay antiviral innate immune signaling in several ways [ , , ] . a key feature of immune evasion by +rna viruses, and simultaneously the hallmark of +rna virus infection, is rearrangement of host membranes into viral replication organelles (ros). as these structures are thought to shield viral rna from the host innate immune system and additionally seem to play a fundamental role in viral rna replication, ros in this sense seem to have a central and dual function in viral replication [ ] [ ] [ ] [ ] [ ] . therefore, disrupting integrity of ros might simultaneously result in impaired viral replication and enhanced antiviral immune signaling, which would be a beneficial effect from an antiviral immunity point-of-view. however, whether host cells possess effector mechanisms to disrupt +rna virus ros has hardly been investigated yet, and only quite recently some studies have shed more light on this, which will be the focus of this review together with the body of literature that surrounds it. positive-strand rna viruses have caused multiple outbreaks during the past decade: severe acute respiratory coronavirus (sars-cov) infected more than individuals in of which almost died [ ] . its close relative middle east respiratory syndrome coronavirus (mers-cov) emerged in saudi arabia in . while sars-cov infections have not been reported in humans after , mers-cov currently still regularly occurs in dromedary camels as well as in humans, and the virus displays a lethality rate of around % in the latter [ ] . zika virus, which received a lot of societal attention lately, is an example of a reemerged +rna virus with significant impact [ ] . these outbreaks, and the lack of tailored antiviral strategies against them, clearly illustrate the potential health risks associated with (re-)emerging +rna viruses, and emphasize the need for a precise understanding of host-pathogen interactions to facilitate development of novel antiviral therapies or vaccination methods. over the last decades, many +rna viruses have been extensively studied for their intriguing biology, including coronaviruses as those mentioned above, picornaviruses such as coxsackievirus and poliovirus, and flaviviruses such as west nile virus (wnv), hepatitis c virus (hcv), dengue virus (denv), zika virus, and japanese encephalitis virus (jev), as well as the family of togaviridae, including rubella virus and chikungunya virus. one of the intriguing features that is shared by all +rna viruses is the rearrangement of host membranes into viral ros, whereby particular cellular organelles (depending on the virus) are used as membrane donors. concerning their role in viral rna replication, it is thought that the structures form a scaffold that improves efficiency of enzymatic reactions and provides a spatiotemporal regulation of the different stages in the virus life cycle. besides this, it is thought that ros have an important role in shielding viral rna products from the rna sensors of the innate immune system. ros are therefore believed to have a dual role in +rna virus infection and innate immune evasion, which will be elaborated on further in this review. several comprehensive reviews have recently described the current knowledge of these structures [ , [ ] [ ] [ ] , therefore we will only briefly summarize this below. most studies until now focused on the architecture of structures induced by different viruses, and based on electron microscopy and electron tomography, ros were categorized into two different classes: the invaginated vesicle/spherule (inv) type, and the double membrane vesicle (dmv) type (fig. ) . inv ros are predominantly observed during infection with alphaviruses, such as semliki forest virus and sindbis virus, nodavirideae such as flock house virus, bromoviridae such as bromovirus, and flaviviridae such as rubella virus, denv, and wnv [ , ] . although the location and dynamics of the formation of inv ros varies per virus, the result is a single membrane invagination of which the content is connected to the cytosol. in addition, replicase proteins and newly synthesized viral rna were observed in these spherules, strongly suggesting that inv ros are sites of viral rna replication [ ] [ ] [ ] . furthermore, ribosomes are found in close proximity of some inv ros [ ] , suggesting that viral rna replication and translation are spatially separated. a similar organization of inv ros was found for flock house virus, rubella virus, denv, and wnv. moreover, a recent study visualized virus budding in close association with inv ros, suggesting a direct role for these structures in coordinating virus assembly [ ] . alternatively, dmv ros are known to be formed by enteroviruses such as coxsackievirus b and poliovirus, coronaviruses such as sars-cov and mers-cov, arteriviruses such as equine arteritis virus (eav), and the flavivirus hcv [ , ] . besides dmvs, for most of these viruses other kinds of structures are found during infection, mostly in close proximity to the dmvs themselves, such as single membrane vesicles, multi-membrane vesicles, vesicle packets, tubular structures or zippered er membrane. as for inv ros, replicases and viral rna of poliovirus, eav, and coxsackievirus b were demonstrated to localize to dmvs, suggesting that these structures might also support viral rna replication. likewise, for hcv replicase proteins and viral rna were predominantly found in dmvs, and the number of dmvs positively correlated to the amount of viral rna produced, suggesting that dmvs indeed serve as sites of rna replication. however, for coronaviruses, the number of dmvs is not necessarily proportional to replication capacity [ , ] . also, whereas sars-cov dsrna (a replication intermediate) was found inside dmvs, replicase proteins were more abundant in surrounding convoluted membranes, raising questions regarding the spatial organization of sars-cov rna replication. the lack of clear connections between the inside of corona-and arterivirus-induced dmvs and the cytosol (where the rna products have to go for translation and particle formation) further complicates our current understanding of coronavirus dmv functionality [ , ] . together, these data continue to cause debate on the exact function of dmvs and other features of dmv ros. the notion that expression of combinations of non-structural viral proteins (nsps) for some of these viruses mimics the formation of membrane alterations as observed during infection confirms the viral induction of these structures and opens possibilities for detailed study of this particular feature of the infection [ ] [ ] [ ] . . innate immune recognition and responses targeted towards viral rna replication, transcription, and translation rapid production of interferons (ifns) and pro-inflammatory cytokines is an important consequence of virus detection, as it contributes to an antiviral state in both the infected host cell and the (un)infected surrounding cells. in addition, ifns play an essential role in coordinating the antiviral adaptive immune response, which has been reviewed elsewhere [ ] . three types of ifns have been described. type i ifns consist of subtypes of ifna and a single subtype of ifn-b, ifn-d, ifn-e, ifn-k, ifn-t and ifnv. type ii ifn only contains one subtype of ifn-g, and type iii ifns comprise of ifn-l through Àl . whereas it is known that most cell types produce type i ifns in response to viral infection, type ii ifns are only produced after antigenic stimulation of an expanding group of certain immune cells, including t-cells, natural killer cells, dendritic cells, and macrophages [ , ] . in contrast, little is known about type iii ifn production in vitro and in vivo, although it is believed that most cell types that produce type i ifns are capable of producing type iii ifns as well [ ] . most cells are able to respond to ifn-i and Àii, whereas ifn-iii receptors are mainly found on epithelial cells [ ] . the protective role of ifns during viral infection is for example illustrated by inhibitory effects of ifna, ifn-b, and ifn-g on sars-cov replication in vitro and in vivo [ ] [ ] [ ] [ ] . importantly, correct timing and amount of ifn and subsequent pro-inflammatory cytokine expression is essential for an effective antiviral immune response, as delayed and/or elevated induction may stimulate immunopathological outcomes [ ] . below we will focus on the interactions of the innate immune system with the rna replication stages of +rna virus infection, including exposure of viral rna to the cytosol after the unpacking of virus particles, and formation of ros by the virus. we will also discuss examples of viral evasion surrounding these processes. ifn-i and -iii production is triggered after host cells detect viral rna, primarily using cytosolic rig-i like receptors (rlrs) and membrane-bound toll-like receptors (tlrs). rlrs retinoic acidinducible gene i (rig-i) and melanoma differentiation associated factor (mda ) have been studied extensively (reviewed in [ , ] ). besides their role in rna metabolism, rig-i and mda recognize viral rna by binding phosphorylated termini ( ppp-rna) in combination with dsrna or ssrna motifs. detection of viral rna activates rlrs and triggers downstream signaling through the mitochondrial antiviral signaling (mavs) adaptor, which is localized on the outer mitochondrial membrane [ ] [ ] [ ] [ ] . the importance of mavs is underscored by the observation that silencing of this protein by rna interference abolishes expression of type i ifns [ ] . subsequently, mavs recruits various adaptor molecules such as stimulator of interferon genes (sting) and tnf receptor-associated factors, resulting in the formation of large signaling complexes [ ] . ultimately, this leads to activation of kinase complexes ikke/tbk and ikka/ikkb/ikkg, resulting in activation of interferon regulating factor (irf ), (irf ) and nf-k b. these transcription factors then translocate to the nucleus and initiate the expression of ifns and pro-inflammatory cytokines. besides the role of rlrs, other rna sensing molecules have been shown to participate in antiviral responses. for example, of the types of tlrs described in humans, endosomal tlrs , , and are known to have a role in viral rna detection. a detailed overview on the role of tlrs in antiviral signaling can be found elsewhere [ ] . briefly, tlrs interact with various adaptor molecules after stimulation, and the combination of recruited adaptors influences downstream signaling events. tlr eventually recruits common adaptor molecule tir-domain-containing adapter-inducing interferon-b (trif), whereas tlrs and engage myeloid differentiation primary response gene (myd ). as in rlr signaling, this leads to activation of kinase complexes ikke/ tbk and ikka/ikkb/ikkg. furthermore, protein kinase r (pkr) and , -oligoadenylate synthetase (oas) have been demonstrated to induce antiviral activities upon binding of dsrna, as is described in detail elsewhere [ ] . well-established examples of antiviral activity provoked by pkr include inhibition of translation and inflammasome activation, and oas activates rnase l to initiate degradation of host and viral rna. in addition, pkr and oas have been suggested to amplify rlr-mediated antiviral immune signaling [ ] . the production and release of ifns and pro-inflammatory cytokines contributes to an antiviral state in both infected host cells and in (un)infected surrounding cells. despite the presence of multiple ifn and receptor types [ ] , the janus kinase-signal transducer and activator of transcription (jak/stat) pathway is utilized by all ifns to establish expression of interferon-stimulated genes (isgs). the observation that a deficiency in separate ifn receptors did not affect disease progression during murine sars-cov infection, in contrast to a deficiency in common signaling molecule stat , illustrates this high degree of redundancy [ ] . at the moment, hundreds of isgs have been identified. however, an exact function has only been clarified for a relatively low number of the corresponding proteins, a description of which can be found elsewhere [ ] [ ] [ ] . nonetheless, these studies suggest that isgs exhibit overlapping inhibitory activity towards most components of virus replication, including virus entry, uncoating, translation of viral proteins, rna replication, and egress [ ] . in summary, host cells detect viral rna by several detection mechanisms to establish an antiviral state via the induction of ifns and pro-inflammatory cytokines, leading to expression of isgs. as mentioned earlier, mavs localized on mitochondria is a key player in antiviral signaling. therefore, studies demonstrating that the mavs adaptor molecule sting is located on the er implied that earlier identified contacts between mitochondrial membranes and er membranes (mitochondrion-associated membranes: mams) were possibly involved in antiviral signaling [ ] . in support of this theory, expression of a mam-enriched marker protein followed by cell fractionation revealed that mavs localizes to mams, which was supported by immunofluorescence assays of mavs and mamenriched proteins [ ] . additionally, immunoprecipitation analysis of the mam fraction of sendai virus-infected hepatocytes confirmed that mam-localized mavs interacts with rig-i and signaling cofactor traf [ ] , indicating that mams are involved in the rlr-mediated antiviral immune signaling pathway. therefore, mams function as important signaling platforms that govern expression of type i and iii ifns. interestingly, mavs is also expressed on peroxisomal membranes, and interacts with stimulated rlrs during viral infection [ ] . however, in contrast to its mitochondrial counterpart, peroxisomal mavs was found to induce expression of isgs by an ifn-independent mechanism, and this relatively rapid process seems to provide short-term protection until the ifn-dependent expression of isgs mediated by other viral rna-sensing mechanisms is established [ ] . interestingly, recent studies found that direct contacts between peroxisomes and er membranes were involved in maintaining peroxisomal function [ ] [ ] [ ] , raising the question whether these contact sites might also regulate antiviral immunity-related processes. in summary, peroxisomal and mitochondrial membranes and their contactsites with the er are important regions for converting viral rna detection by rlrs to downstream events required for establishing an antiviral state. besides mams, increasing evidence suggest a role for stress granules (sgs) in antiviral immune signaling. sgs are nonmembranous compartments in the cell that contain aggregates of ribonucleoprotein (rnp) and mrna, and are formed as a physiological response to various stress stimuli that cause temporal stalling of mrna translation, suggestively preventing accumulation [ ] . consistent with the function of this structure, inhibition of translation through the inactivation of eukaryotic translation initiation factor (eif) a by phosphorylation is an important cue for sg formation [ ] . considering that various rna virus infections result in shutdown of host translation by inactivation of eif a by for example pkr, it is now known that formation of sgs occurs to different extends during infection with various rna viruses, including hcv, denv, and sfv [ , ] . interestingly, multiple lines of evidence have demonstrated that sgs in encephalomyocarditis virus-or influenza virus-infected cells contain multiple rna sensing molecules that were found to contribute to ifn production in vitro, including rig-i, mda , pkr, oas, rnasel, and dsrna [ , ] . however, it should be noted that the contribution of sgs to antiviral signaling differs per virus, as sg-localized mda , an rlr that greatly contributes to ifn induction upon encephalomyocarditis virus infection, was not found to support ifn expression upon infection with this virus [ ] . nonetheless, these findings suggest an important role for sgs as detection platforms for viral rna. taken together, increasing evidence suggests that the antiviral innate immune signaling cascade is spatially organized, and since different structures in the cell seem to be specialized in a particular aspect of this process, it can be hypothesized that interaction between these structures plays a fundamental role in orchestrating the antiviral innate immune response. positive-strand rna viruses developed multiple mechanisms to impair rna recognition and antiviral signaling (also reviewed in [ ] ). as mentioned above, the presence of viral rna in ros suggests a role for ros in shielding viral rna from the innate immune detection machinery. while there is no unequivocal evidence supporting this theory, the finding that isolated viral rnacontaining membrane alterations only become sensitive to nuclease treatment after membrane disruption by nonionic detergents supports this hypothesis [ ] [ ] [ ] [ ] . in addition, a positive correlation between cytosolic exposure of viral rna and ifn induction was recently found by an in vitro comparison of jev and denv infections [ ] . another important strategy by which rna viruses avoid recognition is their modification of viral rna molecules to mimic eukaryotic mrna. for example, the formation of a cap is catalyzed by viral phosphatases and methyltransferases in some viral families such as the coronaviruses. from work on mouse hepatitis virus (mhv) it became clear that coronaviruses need to take care of n-linked as well as o-linked methylation on the cap structure, in order to avoid recognition by innate immune sensors [ ] . mimicry of eukaryotic rna in this way was shown to be an important immune evasion strategy by +rna viruses such as sars-cov and jev [ , ] . a recent report indicated that coronaviruses also evade dsrna mediated recognition by pkr and oas by using their endonuclease (endou), which cleaves free viral (and cellular) rna that is somehow exposed to the cytosol, in order to prevent antiviral signaling [ ] . in addition to avoidance of recognition, +rna viruses actively interfere with antiviral signaling components to impair expression of ifns and pro-inflammatory cytokines. very often, the viral proteases that are expressed by +rna viruses, and which mostly have a primary function in cleavage of viral replicase polyproteins, seem to have a prominent role in this. for example, hcv ns / a and denv ns a cleave mavs [ , [ ] [ ] [ ] [ ] [ ] [ ] , and the denv protease complex and hcv ns b inhibit adaptor molecule sting by cleaving it [ ] [ ] [ ] [ ] [ ] . additionally, human cov nl and sars-cov nsp possess a papain-like protease (plp) domain that prevents dimerization of sting and its complex formation with mavs and ikke, and almost all nidovirus-encoded plps have been shown to display deubiquitinating activity [ ] . several have been suggested to facilitate deubiquitination of innate immune factors such as sting, rig-i, tbk and irf in order to halt innate immune signaling [ ] . moreover, in collaboration with others, our lab demonstrated that eav and mers-cov virus mutants that lack the dub activity of their plps while retaining polyprotein cleavage functions suppress ifn production less efficiently during infection ( [ , ] and our unpublished data). furthermore, viral proteins such as denv ns b/ and the sars-cov membrane glycoprotein interfere with complex formation of ikke/tbk and ikka/ikkb/ ikkg or downstream transcription factors, thereby effectively inhibiting both rlr and tlr signaling [ , ] . interestingly, accessory proteins encoded by open reading frames of covs have been shown to be involved in inhibition of irf , irf , and nf-kb by undefined mechanisms [ , ] . in addition, several methods are employed by +rna viruses to interfere with jak/stat signaling. for instance, jev ns and wnv ns b inhibit phosphorylation and activation of jak , and suppressors of jak are induced by viruses such as wnv, jev, and chikungunya virus [ ] [ ] [ ] [ ] [ ] . however, stat and stat are most heavily targeted. jev ns , wnv and denv ns b, and jev ns a have been shown to inhibit stat activation. in addition, denv ns promotes proteasomal degradation of stat . interestingly, sars-cov inhibits stat signaling in three different ways. firstly, sars-cov nsp binds stat to inhibit its phosphorylation [ ] . secondly, the accessory protein encoded by sars-cov open reading frame prevents nuclear transport of stat [ , ] . thirdly, sars-cov plp induces expression of e ubiquitin ligase e - k, which promotes proteasomal degradation of extracellular signal-regulated kinase , a protein responsible for activation of stat [ ] . to our knowledge, no studies have been published (yet) about specific interference of +rna viruses with ifn-ii production. interference of +rna viruses with antiviral signaling also has consequences for the spatial organization of the antiviral innate immune system. for instance, since sgs suggestively have an antiviral role, interfering with sg formation might constitute an opportunity for viruses to hamper the innate immune response. indeed, various proteins of poliovirus, coxsackievirus, encephalomyocarditis virus, denv, wnv, mers-cov, chikungunya virus, and jev have been shown to prevent the formation of sgs and thereby suppress ifn production [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . thus, dysregulation of sgs seems to be a common strategy for +rna viruses in order to dampen the innate antiviral immune response. in addition, the finding that mams are involved in the rlr-mediated antiviral immune signaling pathway shedded new light on the immunosuppressive role of viral proteases. by performing colocalization analysis with a mam-enriched marker protein and cell fractionation experiments in both uninfected and hcv-infected cells, it was found that ns / a also localizes to mams [ ] . intriguingly, despite the clear function for peroxisomal mavs in antiviral signaling, cleaved mavs was found solely in mam-enriched cell fractions. in addition, recent findings suggest that mams are also physically disrupted during denv infection [ ] . thus, these data indicate that mams are critical locations for antiviral signaling and have an important role in expression of type i and iii ifns. moreover, increasing evidence suggests that at least some +rna viruses in fact occupy or hijack mam-membranes during infection, as mams of hcv-infected cells were found to contain proteins involved in virus assembly and fully assembled virions [ ] . it remains to be investigated whether mams also serve as platforms for viral assembly of other +rna viruses. in addition, mam disruption as a consequence of denv infection was followed by the formation of convoluted membranes at the same location, which supported denv replication [ ] . moreover, promoting the formation of convoluted membranes further repressed the ifn response, underscoring the importance of mam disruption in both the replication and immune evasion of +rna viruses [ ] . taken together, these studies underscore the importance of mams as key antiviral signaling platforms, and disruption of mams constitutes an effective immune evasion strategy for +rna viruses. in conclusion, +rna viruses developed divergent methods to delay antiviral innate immune signaling at multiple levels. in addition, interference of +rna viruses with antiviral signaling leads to spatial disorganization of the antiviral innate immune system, and future studies will hopefully elucidate the consequences of this phenomenon in the context of other cellular compartments and viruses. assuming that ros impair antiviral signaling by shielding viral rna from the host, it is tempting to hypothesize the existence of host cell mechanisms aimed to disrupt ros, thereby exposing viral rna and promoting antiviral signaling. the fact that +rna viruses display such elaborate activities to inhibit viral rna recognition and subsequent signaling, as detailed in the former paragraphs, supports the view that disruption of ro integrity is a situation these viruses anticipate dealing with. however, evidence suggesting targeting of ros by the innate immune system is still scarce, although it was reported that hcv ros are attacked by the isg viperin [ ] . additionally, -hydroxycholesterol, a product produced by the isg cholesterol -hydroxylase also modifies hcv replication organelles [ , ] . recently, our lab published a study suggesting that ifn-b signaling also negatively influences arterivirus ros, since significantly less were formed and remaining structures showed drastically different morphology after ifn-b treatment. the results suggested that the treatment interrupted biogenesis of these membrane structures rather than breaking down structures that were already made [ ] . interestingly, neither viperin nor -hydroxycholesterol was involved in the observed effects, and the mechanistic details therefore have to be further investigated. other recent data suggest that there may indeed be specific mechanisms by which the innate immune system tags and disrupts so-called non-or aberrant-self membrane structures in the cell [ ] , which could include viral ros. most data originate from studies focusing on bacteria, fungi, or parasites that reside in rearranged membranes, known as pathogen-containing vacuoles (pvs), which prevent detection of cytosolic innate immune sensors in a similar way as viral ros are thought to do. multiple studies found that enzymes capable of disrupting pvs are gtpases, such as effector immunity-related p gtpases (irgs) and guanylatebinding proteins (gbps), which are part of a family which we will refer to as ifn-inducible gtpases [ ] . expression of irgs and gbps is induced upon ifn-g stimulation, and leads to their accumulation on pvs. subsequently, activation of these gtpases due to the exchange of gdp for gtp results in membrane disruption as a consequence of their dynamin-like activity [ ] . to prevent aspecific disruption, host cell membranes contain a set of proteins that inhibit gtpase activity, known as ''guard proteins'' [ ] . wellstudied pathogens in the context of this process are the protozoan parasite toxoplasma gondii (t. gondii), the bacterium chlamydia trachomatis and the microsporidian encephalitozoon cuniculi [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although it remains unclear for most of these pathogens how ifn-inducible gtpases are recruited towards pvs, recent studies on t. gondii infection demonstrated that host cells label these membrane structures, for example with various forms of microtubule-associated protein a/ b-light chain (lc ) to initiate this process [ ] [ ] [ ] [ ] (fig. ) . lc is a well-known factor in the autophagy pathway, and several forms of lc exist in the human proteome, which seem to have overlapping as well as distinct functions. the human genome encodes homologs lc a, lc b, and lc c and lc -like homologs gabarap, gabarapl , gabarapl , and gabarapl . we will refer to all of these as lc unless stated otherwise. lc was initially discovered as an essential protein for autophagosome formation, which requires a covalent interaction between cytosolic lc (lc -i) and the phospholipid phosphatidylethanolamine (pe), after which lc is referred to as lc -ii or lipidated lc [ ] . however, recent studies have now revealed that labeling of (foreign) membrane compartments by lc conjugation can also have various autophagyunrelated consequences, as has been reviewed elsewhere [ , ] . interestingly, multiple studies demonstrated that labeling of lc on pvs recruits ifn-inducible gtpases upon ifn-g expression during t. gondii infection, resulting in exposure of t. gondii to the host cell cytosol, thereby triggering anti-bacterial immune responses [ , , ] . considering the function of lc in autophagosome formation, jayoung choi and co-workers [ ] termed this process targeting by autophagy proteins (tag). in addition, a similar role was recently found for ubiquitin, the versatile regulator of numerous important cellular processes, and also a well-known autophagy-related protein [ ] . a study on t. gondii and chlamydia trachomatis infection demonstrated that pvs are also recognized and labeled by the ubiquitination pathway upon ifn-g expression, resulting in recruitment and activation of ifn-inducible gtpases [ ] . importantly, it was shown that virulent strains of t. gondii and chlamydia trachomatis interfere with the deposition of ifn-inducible gtpases on pvs, underscoring the importance of this mechanism in clearance of associated infections [ , , ] . collectively, these studies demonstrate the existence of various innate immune responses that result in tagging and subsequent disruption of non-or aberrant-self membrane structures in the cell, thereby exposing pamps and promoting immune signaling. importantly, the findings suggest that ifn-mediated membrane disruption might be a common principle in clearance of pathogens that use rearranged membranes to support replication and avoid innate immune detection. like pvs, ros induced by +rna viruses are membranous compartments that constitute an environment for efficient replication, simultaneously avoiding immune detection by the host. therefore, similar mechanisms might be aimed towards viral ros. the observation that ros induced by various +rna viruses consist of a double membrane initially suggested a role for the autophagy machinery in the formation of these structures. however, only nonlipidated lc , and not an intact autophagy pathway, was found to be essential for viral ro formation and viral replication upon infection with eav and mhv [ , ] , thereby contradicting this hypothesis. interestingly, lipidated lc was found to support ifn-g mediated protection against murine norovirus (mnv) infection by autophagy-unrelated means [ ] . furthermore, the induction of viral membranous structures by expression of mnv nsps led to a high colocalization between atg l , a protein involved in lc conjugation, and the mnv polymerase. since atg l determines the site for lc conjugation [ ] , and the mnv polymerase is associated with ros, this further supports the possibility that lc conjugation occurs on ros. other supporting in vitro experiments show that conditional ko of atg b inhibits replication of mnv and t. gondii more effectively compared to wildtype cells [ , ] . in contrast to the multiple autophagins involved in pre-processing of lc for conjugation, atg b is the only human homolog of yeast atg that efficiently deconjugates lc from membranes [ ] , and therefore plays a major role in negatively regulating the fraction of conjugated lc . several groups have reported enhanced conjugation of lc to membranes during knockdown of atg b in various cell types [ ] [ ] [ ] [ ] , including those used by aforementioned studies [ , ] . thus, it is likely that increased lipidated lc was present on ros and pvms during experiments in atg b-deficient recent studies demonstrate that lc is conjugated to the pvm by the lc conjugation system, leading to recruitment of ifn-g inducible gtpases to the pvm upon ifn-g stimulation. as a consequence, activation of ifn-g inducible gtpases leads to membrane disruption of the pvm, and exposure of t. gondii to the innate immune system. cells, thereby explaining the enhanced inhibition of replication. interestingly, findings in other studies are contradictory in the sense that only lc -i was observed on ros of mnv, eav, and mhvinfected cells [ ] [ ] [ ] . on this note, it is worth mentioning that lc -i does not support ifn-g mediated inhibition of viral replication, as ifn-g mediated inhibition of mnv-infected cells does not occur in the case of atg deficiency, one of the proteins responsible for lc conjugation [ ] . in conclusion, although there is no unequivocal evidence regarding the presence of conjugated lc on +rna virus induced ros, the studies described above provoke the hypothesis that membrane disruption of viral ros mediated by ifn-g inducible gtpases may be part of the innate immune response against +rna viruses (figure ). interestingly, current knowledge of ifn-inducible gtpases suggests that membrane disruption of ros by ifn-g inducible gtpases might be part of a common antiviral mechanism that utilizes ifn-inducible gtpases to combat viral infection. aside from irgs and gbps, other families of dynamin-like ifn-inducible gtpases exist, such as myxovirus resistance proteins (mx) and the very large ifn-inducible gtpases (vligs) [ ] . these enzymes are also known to have antiviral properties against a variety of viruses (reviewed in [ ] ). for example, the protective role of mx proteins has been demonstrated for orthomyxoviruses such as influenza, lentiviruses such as human immunodeficiency virus (hiv) , and, interestingly, several +rna viruses belonging to the picornaviridae and togaviridae [ ] . moreover, based on evolutionary studies, it was recently suggested that mx proteins mediate protection against even a wider variety of viruses [ ] . in contrast to ifn-g inducible irgs and gbps, expression of mx proteins is induced by ifns type i and iii, implying that all ifn types are capable of inducing ifn-inducible gtpases [ ] . therefore, all cell types known to respond to ifns could theoretically induce a variety of ifn-inducible gtpases in response to viral infection. in the case of +rna viruses, it is intriguing that multiple studies demonstrated how mxb suppresses replication of hiv- by targeting the viral capsid protein [ ] [ ] [ ] , given the parallels between retroviral capsids and +rna virus ros [ ] . in conclusion, these findings again suggest a general function for ifn-inducible gtpase effectors in the targeting of viral intracellular membrane structures that function to shield away viral components away from the cytosol. rearrangement of host membranes into ros is considered a hallmark of +rna virus infection. ros are believed to have at least a dual role in +rna virus infection and innate immune evasion, as they facilitate efficient rna replication while shielding viral rna from the host antiviral response machinery. as viral rna is a very potent inducer of antiviral signaling, we focused on how both the biochemical and spatial organization allows the innate immune system to convert detection of viral rna to an antiviral state. in addition, we described part of the divergent methods by which +rna viruses impair antiviral signaling surrounding their rna replication, transcription, and translation in order to establish infection. at last, based on recent studies that demonstrated how ifn-g inducible gtpases are capable of disrupting pvs, we discussed the possibility of a general function of ifn-inducible gtpases in the targeting of viral ros. in summary, upon infection, +rna viruses hamper ifn and isg induction at multiple levels to decelerate antiviral innate immune signaling. in this process, the formation of ros enables rapid viral rna replication while masking it from the host. however, in case of sufficient activation of ifn-inducible gtpases by ifn-i and -iii originating from various cell types, or ifn-ii produced by specialized immune cells, disruption of ros by ifn-inducible gtpases may result in enhanced exposure of viral rna, thereby amplifying the innate immune response and ensuring efficient clearance of the virus. however, considering the differences in morphology and origin of ros, the variety of infection dynamics within the category of +rna viruses, and a lack of knowledge regarding the exact mechanism(s) of disruption of non/aberrant-self membrane structures by ifninducible gtpases and possibly other factors, extensive further research is required to elucidate whether this process plays a general role in +rna virus infection, and might open possibilities for development of novel antiviral therapies. none funding this research did not receive any specific grant from funding 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formation human light chain /map lc b is cleaved at its carboxyl-terminal met to expose gly for lipidation and targeting to autophagosomal membranes hsatg b/hsapg b/autophagin- cleaves the carboxyl termini of three human atg homologues and delipidates microtubule-associated protein light chain -and gabaa receptor-associated protein-phospholipid conjugates autophagy proteins regulate erk phosphorylation autophagin- ) phosphorylation modulates autophagy evolutionary analyses suggest a function of mxb immunity proteins beyond lentivirus restriction mx is an interferon-induced inhibitor of hiv- infection human mx is an interferon-induced postentry inhibitor of hiv- infection the interferon-inducible mxb protein inhibits hiv- infection parallels among positive-strand rna viruses, reversetranscribing viruses and double-stranded rna viruses enzo scutigliani ( ) obtained a bsc in biomedical sciences with honor at the university of amsterdam (the netherlands) in , and is currently in the final process of becoming a msc with a specialization in cell biology and advanced microscopy. over the last years, he developed a particular interest for microbiology, and his ambition is to develop novel treatment or vaccination methods by understanding pathogens at the molecular level. to achieve this goal, he is currently specializing in combining microbiological research and advanced microscopy.a molecular sciences study at wageningen university and research center (the netherlands) motivated marjolein kikkert ( ) to pursue a ph.d. degree, which she achieved in at the department of virology of this university. her thesis was entitled "role of the envelope glycoproteins in the infection of tomato spotted wilt virus ". she then switched to mammalian virology, became a post-doc at the national institute of health and the environment in bilthoven, the netherlands with prof. emmanuel wiertz, and subsequently at leiden university medical center (lumc), working on immune evasion strategies of human cytomegalovirus. after this she started working with prof. eric snijder at the lumc as a post-doc, and developed into an assistant professor in the department of medical microbiology. her research currently focuses on the replication organelles and related virus-host interactions of nidoviruses and other +rna viruses, and the innate immune evasion mechanisms which these viruses employ. the knowledge gained from her research is being used for development of novel antiviral vaccines and Àdrugs. key: cord- -lvm o r authors: woo, bean; baek, kwang-hyun title: regulatory interplay between deubiquitinating enzymes and cytokines date: - - journal: cytokine growth factor rev doi: . /j.cytogfr. . . sha: doc_id: cord_uid: lvm o r deubiquitinating enzymes (dubs) are cysteine protease proteins that reverse the ubiquitination by removing ubiquitins from the target protein. with over dubs identified and categorized into at least families, many dubs interact with one or more cytokines, influencing cellular processes, such as antiviral responses, inflammatory responses, apoptosis, etc. while some dubs influence cytokine pathway or production, some dubs are cytokine-inducible. in this article, we summarize a list of dubs, their interaction with cytokines, target proteins and mechanisms of action. ubiquitination and deubiquitination are post-translational modifications for numerous proteins, which in turn affect many physiological processes. ubiquitination is defined as an attachment of one or more ubiquitin (ub) molecules onto the target protein through the function of a series of proteins: e , e and e ( fig. ) [ ] . seven lysine residues have been identified (k , k , k , k , k , k and k ) on the ubiquitin molecule [ ] . also, additional ub molecules can be attached onto one of the seven lysine residues or the n-terminal methionine to form polyubiquitin chains (polyub) [ , ] . perhaps, ub is most well-known as the crucial marker of the ubiquitin-proteasome system (ups), in which ubiquitinated proteins enter proteasomal degradation via s proteasome ( fig. ) [ ] . however, ub also affects many other aspects of tagged proteins, such as localization, protein interaction, function, etc. [ ] . on the other hand, deubiquitination refers to the process that reverses ubiquitination via deubiquitinating enzymes (dubs) (fig. ). little less than human dubs have been identified so far [ ] . dubs were categorized into five different families in the past [ ] , but at least seven different families are identified as of now, which include the ubiquitin-specific protease (usp), ubiquitin carboxyl-terminal hydrolase (uch), machado-josephin disease protein (mjd), ovarian tumor (otu), jab /mpn/mov (jamm), permutated papain fold peptidases of dsrna viruses and eukaryotes (pppde) and miu-containing novel dub family (mindy) [ , ] . cytokines are groups of small proteins that play a role in cell signaling and immune system by binding to their respective receptors. since dubs regulate diverse physiological processes, it was to be expected that dubs and cytokines affect one another. as anticipated, the more studies were performed regarding the functions of dubs, the more interaction between dubs and cytokines were revealed. recently, several reviews dealing with how dubs affect pathway of a specific cytokine were published [ ] [ ] [ ] , but none has yet introduced as a whole the interaction between dubs and cytokines. in this review, we wish to provide a brief overview of the dubs discovered to regulate cytokine signaling pathways and cytokine-inducible dubs. we will discuss the dubs that influence the pathways of interferons (ifn), tumor necrosis factors (tnf), tnf-related apoptosis-inducing ligand (trail), interleukins (il) and chemokines. ifn-α and ifn-β cytokines belong to type i ifn family that are essential for antiviral responses, cancer, inflammation, etc. [ ] . when a cell recognizes a viral infection through detecting ifn-stimulating signaling molecules or foreign double stranded dna in the cytosol, retinoic acid-inducible gene-i (rig-i) is activated, triggering the cascade of the second messenger system to activate and translate ifn-α and ifnβ signaling pathways (fig. ) [ ] . dubs interact with some of the key molecules in the ifn signaling pathway, which include, but are not limited to, rig-i, stimulator of interferon genes (sting), tumor necrosis factor receptor-associated factors (trafs), interferon regulatory factor are summarized in table . we will discuss the dubs in the order of ifn signaling pathway shown in fig. . the first group of dubs are those that deubiquitinate rig-i to inhibit the production of ifn. rig-i is a cytosolic protein that plays a significant role in ifn signaling by detecting viral dna and rna [ , ] , which is then ubiquitinated by tripartite motif (trim) to activate the signaling cascade to synthesize ifn [ ] . orf , usp , usp , usp , usp , cylindromatosis (cyld), porcine epidemic diarrhea virus papain-like protease (pedv plp ) and transmissible gastroenteritis virus papain-like protease (tgev pl ) are the dubs found to deubiquitinate rig-i. we will discuss them one by one. in a study using hek t cells and sendai virus (sev), knockdown of the gene transcribing usp resulted in upregulation of type i ifn, while overexpression of usp decreased type i interferon as usp showed dose-dependent inhibition of ifn-β [ ] . further experiment supported the idea that usp deubiquitinates k -polyub from rig-i [ ] . however, when the effects of usp with a mutated catalytic site and wild type usp were compared, the results were surprisingly similar, indicating that usp 's catalytic activity is not necessary for it to inhibit ifn synthesis [ ] . orf is a dub activity containing tegument protein, found within kaposi's sarcoma-associated herpesvirus (kshv) and murine gamma herpesvirus (mhv ) [ , ] . the expression of kshv orf in hek t cells led to suppression of both rig-i-induced and sev infection-induced ifn-β promoter activation [ ] . on the contrary, kshv orf -c g mutant, with defective deubiquitinating activity, resulted in lesser to no suppression, confirming the influence of orf on the ifn synthesis [ ] . also, the overexpression of trim , but not the mutant trim , reversed the orf 's effect on ifn production and reverted the ubiquitination of rig-i, further confirming the result [ ] . it is also noteworthy that orf was not capable of suppressing mavs-induced activation of ifn-β production [ ] . when mhv infected bone marrow derived dendritic cells (bmdc) were induced by mcmv and hsv- for type i ifn induction, no ifn was detected, but tnf-α, il- and il- β were expressed upon high dose stimulation [ ] . this provided evidence that mhv induced innate immunity of the host to a lesser extent [ ] . on the other hand, orf mutant mhv stimulated innate immune response [ ] . by utilizing dub activity of orf , mhv blocked viral dna induced, sting-mediated ifn production [ ] . in a study performed by zhong et el, usp , too, was found to deubiquitinate rig-i and reduce sev-induced ifn-β production in hek t cell line [ ] . knockdown of usp gene also led to augmentation of isre promoter upon sev induction [ ] . mutating the catalytic residue of usp was sufficient to block usp 's effect on ifnβ induction, supporting that ifn-β suppression via usp is dub activity dependent [ ] . usp targeted not only rig-i for deubiquitination, but also extended to traf [ ] , traf [ , ] and traf [ ] and to affect ifn signaling. usp also deubiquitinated traf and traf to regulate in il- signaling [ ] . however, some studies have given different results regarding usp 's ability to deubiquitinate traf . lin et al. stimulated usp knockout bmdc with sev or hsv- infection and added wild type (wt) usp or mutant usp , but k -ub of traf did not differ from one another [ ] . however, zhong et al.'s study using hek t cells supported deubiquitination of traf by usp [ ] . this variance in the result may be caused by the difference of the cell line used for the studies. furthermore, usp showed its ability to suppress phosphorylation of interferon regulatory factor (irf ) and p , also contributing to inhibition of ifn promoter activation [ ] . fan et al., knowing that usp inhibits rig-i-induced ifn-β production, searched for its mechanism [ ] . they unveiled that usp fig. . mechanism of action of ubiquitin proteasome system and deubiquitinating enzymes. ub attaches to the target protein by going through a series of reaction with e (ubiquitin activating), e (ubiquitin conjugating) and e (ubiquitin ligating) enzymes. a target protein could be ubiquitinated once or multiple times on lysine residues. s proteasome identifies target proteins with polyub chain and degrades them into amino acid segments and reusable ub. ubiquitinated proteins could also be deubiquitinated by dubs, resulting in a different fate. inhibited isre reporter activity induced by sev and rig-i-card, but not by tank-binding kinase (tbk ) in mouse embryonic fibroblasts (mef) cells [ ] . usp deubiquitinated rig-i in hek t cells [ ] . also, they found that usp 's function regarding antiviral response is compatible in mef and hek t cell lines by introducing each cell line's usp to the other cell line and observing the effect [ ] . usp 's specificity to rig-i was also confirmed in hela cells through coimmunoprecipitation (co-ip) of usp with rig-i, using rabbit polyclonal antibodies against usp [ ] . usp also deubiquitinated mda to inhibit antiviral response [ ] . usp is also a dub that deubiquitinates k -polyub chain of both rig-i and mda and suppresses ifn-β activation [ ] . usp 's effect was found viable in t, thp- , human peripheral blood mononuclear cells (pbmcs) and raw . cells, supporting that usp 's activity is viable in both human and murine cells [ ] . usp did not inhibit mavs, sting, tbk , irf and tirf, as demonstrated by isre-luc activity induction test [ ] . also co-ip demonstrated the interaction between usp and stimulated rig-i or mda , but not the unstimulated ones, supporting that ligand stimulation is required for usp to interact with rig-i or mda [ ] . more specifically, poly(i:c) (lmw) stimulation leads usp to have a strong interaction with rig-i, but a weak one with mda , while poly(i:c) (hmw) stimulation leads usp to have a strong interaction with mda , but a weak one with rig-i [ ] . cyld is another dub that removes k -ub chain from rig-i to decrease the ifn production [ , ] , but tbk and ikkε were also identified as the target of the deubiquitination of cyld in ebna cells [ ] , resulting in the same effect. cyld also interacted with ips- to negatively regulate it, but did not deubiquitinate it [ ] . schmid et al. found that in brain and peripheral blood of c bl/ , the mrna level of ifn-γ gene decreased with the knockdown of cyld, while the serum concentration of ifn-γ increased [ ] . a study conducted using human kidney mesangial cells (mc) showed slightly different results: silencing cyld in mc cells and stimulating them with poly ic increased the toll-like receptor (tlr )-induced activation of rig-i and mda [ ] ; however, the level of mrna of rig-i and mda actually decreased [ ] . the authors speculated this difference to be caused by the change in cell line used [ ] , but further study is necessary to determine the cause. cyld also decreased ifn promotor activation by deubiquitinating traf and traf in hek t cells, respectively [ , ] . cyld in u os/nod cells were found to deubiquitinate k -ub of ripk proteins, especially ripk , to suppress nod -induced nf-κb activation [ ] . when cyld was suppressed, ubiquitinated receptor interacting protein kinase (ripk ), also called rip , and ripk proteins accumulated within cells [ ] . plps, first discovered in coronavirus in [ ] , are multifunctional proteins with dub activity that are synthesized by many families of viruses that regulate ifn signaling pathway by interacting with rig-i [ ] [ ] [ ] [ ] . recently, the mechanism by which pedv plp suppresses ifn production in the host cell was identified. in hek t cells, pedv plp was found to deubiquitinate rig-i and sting, thereby affecting its downstream pathway, resulting in suppression of ifn production [ ] . tgev pl also was revealed to bind and deubiquitinate both rig-i and sting in hek t cells [ ] . studies on middle east respiratory syndrome coronavirus encoded papain-like protease (mers-cov pl pro ) showed that it also has a dub function [ ] . this was supported by a study by bailey-elkin et al., in which they obtained the crystal structure of pl pro -ub complex and showed that wt mers-cov pl pro , but not the dub mutant pl pro , suppresses ifn-β promotor activity [ ] . the targets of mers-cov pl pro were identified as rig-i, mda and mavs [ , ] . mers-cov pl pro and severe acute respiratory syndrome coronavirus (sars)-cov pl pro inhibited the proinflammatory signaling in hek t cells upon mda stimulation, which included decreased expression of ccl and ifn-β and decreased level of cxcl mrna [ ] . another study on sars-cov pl pro found that irf is ubiquitinated and that deubiquitinating activity of sars-cov pl pro was required for it to affect irf [ ] . sars-cov pl pro did not affect irf in other means, such as dimerization or nuclear translocation [ ] . dub domain mutated plp of equine arteritis virus (eav) also increased in expression of ifn-β and il- in equine long fibroblasts (elf) [ ] . plp domain was also found in nsp protein, which will be discussed in a later section. sting is a transmembrane protein found in mitochondria and endoplasmic reticulum that regulates ifn-promotor activation at the downstream of rig-i [ ] . zhang et al. studied the effect of usp (also known as ubp ) on sting and revealed that usp interacts with sting to affect ifn-promotor activity [ ] . however, when usp -/-mef cells with either wt usp or dub activity-mutated usp were induced with hsv- , hcmv or cytosolic dna, ifnb, ifna , tnf, il- or cxcl genes increased in expression, indicating that the deubiquitinating activity of usp is not responsible for this phenomenon [ ] . subsequently, they searched for dubs that interact with usp and found that knockdown of usp inhibited usp -induced deubiquitination of sting and knockdown of usp inhibited usp -induced deubiquitination of sting [ ] . immunoprecipitation revealed that sting, usp and usp are arranged as usp -usp -sting, but both usp and usp were associated with the n-terminus of sting [ ] . usp deubiquitinated k -or k -linked ubiquitin of sting [ ] . these results together supported that although usp does not deubiquitinate sting itself, usp recruits usp to deubiquitinate sting to suppress ifn synthesis [ ] . another way that usp inhibited nf-κb activation is by deubiquitinating k -ub of tak and nemo [ ] . usp strongly interacted with tak -tab and dub activity dependently deubiquitinated k -ub of tak in t cells [ ] and in th cells [ ] . usp also fig. . tlrs, ifnari and ifnarii induced ifn production pathway. pamps, ifn-α and ifn-β stimulate tlr and ifnar i & ii receptors respectively to induce ifn production as well as nf-κb activation. dubs that play a role in these pathways are indicated in the figure to show the mechanism of their action. decreased k -ub of nemo [ ] . in a study by malakhova et al., usp inhibited ifn-induced gene activation by affecting jak-stat signaling pathway in t cells [ ] . their study showed that usp does not interact with ifnar , but with box -box region of ifnar to disrupt its interaction with jak to inhibit jak's tyrosine kinase activity in a dub activity independent manner [ ] . consistent with this, usp knockout murine cells displayed hyperactivity towards type i ifn signaling, resulting in the increase of the level of phosphorylation of stat and stat [ ] . usp 's interaction with ifnar also interfered with ifnar 's ability to recruit ifnar , hindering ifn i signaling [ ] . herpes simplex virus (hsv- ) invades a host and escapes its ifn-mediated innate immunity by encoding a large tegument protein, ul , which has a motif with dub activity, named ul ubiquitin-specific protease (ul usp) [ ] . when hek t cells were transfected with markers of co-ip and ul usp or the c a (a dub motif mutant) and then infected with sev, the result showed reduction of ubiquitination of traf in cells with wt ul usp, while c a has no reduction of ul usp's ubiquitination [ ] . the result supported that ul usp deubiquitinates traf molecules to inhibit ifn-promotor activation [ ] . in a different study regarding the function of ul usp, it was found that ul usp inhibits cgas and sting dependent ifn-β production [ ] . nf-κb activation from overexpressing sting, tbk , ikkα and ikkβ was also inhibited, but not from overexpressing p [ ] . in this study, human foreskin fibroblast (hff) cells were infected with either hsv- or hsv- c a mutant and stimulated with ifn stimulatory dna [ ] . as a result, the level of endogenous iκbα in hff cells with mutant hsv- significantly decreased compared to hff cells with wt hsv- , supporting that ul usp decreases the degradation of iκbα in a dub activity-dependent manner [ ] . additionally, co-ip study in hek t cells showed decrease in ubiquitination of iκbα in those transfected with wt ul usp, but not in those transfected with c a mutant [ ] . taken together, ul usp deubiquitinates iκbα to inhibit its degradation, suppressing nf-κb activity [ ] . a study by lin et al. revealed that usp is required for both dna and rna virus-induced signaling [ ] . supporting this claim, silencing usp in mefs or mouse lymphatic fibroblasts (mlf) led to inhibition of expression of ifna , tnf, and il- upon triggering them with sev, vesicular stomatitis virus (vsv) or poly(i:c) [ ] . also, the level of ifnα and il- was reduced in mlfs, bmdcs or flt lpdc cells with usp knockdown [ ] . usp 's dub activity was also found to be necessary for virus-induced signaling, as usp knockdown mefs with wt usp reconstitution allowed expression of ifnb, ifna and il- upon sev or hsv- induction, while those with dub activity mutant usp did not [ ] . overexpressing usp in hek t cells resulted in reduction of irf phosphorylation when stimulated with sev, leading to inhibition of nf-κb activity [ ] . isre reporter activity was also inhibited by usp in a dose-dependent manner [ ] . taking it one step further, lin et al. uncovered that usp stabilizes traf in a dub activity dependent manner by deubiquitinating k -ub of traf in bone marrow-derived macrophages (bmdm) cells, inhibiting tlr signalinginduced innate immune responses [ ] . the nonstructural protein (nsp ) is a viral protein with deubiquitinating activity in its plp domain, which was found in scov and in mouse hepatitis virus a (mhv-a ) [ , ] . infecting mef cells with mhv-a did not result in detectable ifn-β induction, while infecting them with sev (the control) did result in ifn-β responses [ ] . when cells were given variants of nsp , the ifn-β induction only took place in cells that lacked wt plp domain [ ] . when the plp domain was present, ifn-β induction was suppressed upon viral infection [ ] . moreover, polyub of irf , which is necessary for ifn-β induction, was deubiquitinated in the presence of plp , which was further confirmed by co-ip indicated formation of a complex of plp and irf [ ] . this deubiquitination inhibited nuclear translocation of irf [ ] . in hek t cells and mef cells, k -polyub was also deubiquitinated by the plp domain of nsp , inactivating tbk -irf complex in the cytoplasm [ ] . monocyte chemotactic protein-inducing protein (mcpip ) is a protein, common to human and mouse, with dub activity toward traf , traf and traf , thereby inhibiting jnk and nf-κb signaling [ ] . a more recent study has uncovered through co-ip in sev infected hek t and hela cells that mcpip interacts with irf and through confocal microscopy that transfection with mcpip inhibited nuclear translocation of irf [ ] . additionally, the presence of mcpip inhibited traf and tbk activated ifn-β expression [ ] . co-ip also revealed possibility of mcpip to interact with ips- and ikkε as well [ ] . a (also known as tnfaip ) inhibited lps-induced nf-κb activity in mef cells in a study by boone et al. [ ] . upon further testing in hek t, they found that wt a , but not dub activity domain mutant a removed k -ub from traf [ ] . in raji cells, the n-terminal dub domain of a interacted with and deubiquitinated irf [ ] . however, in vitro study showed no interaction between a and irf , which is likely due to requirement of other intracellular proteins [ ] . irf has been known to be activated by epstein-barr virus (ebv)'s oncoprotein called latent membrane protein (lmp ) [ ] . a has been known for a long time as a negative regulator of nf-κb pathway mediated by rig-i. a does interact with rig-i, and suppresses rig-i-mediated nf-κb pathway [ ] , but whether a deubiquitinates rig-i or not still requires confirmation. similar to orf in mhv [ ] , bplf is an ebv encoded large tegument protein with dub activity that opposes tlr signaling in the host [ ] . gent et al.'s research revealed that bplf deubiquitinates traf and nemo in t cells [ ] . immunoprecipitation in t cells revealed that k -ub of traf and nemo was reduced when wt bplf was expressed, while mutant bplf did not [ ] . k -ub of iκbα was also identified as a target of bplf 's dub activity [ ] . the brcc isopeptidase complex (brisc) is a nuclear dub complex, composed of abraxas, brcc , brcc and merit , capable of deubiquitinating k -ub [ ] . in a study by zheng et al., serine hydroxymethyltransferase (shmt) formed a complex with brisc to form brisc-shmt complex [ ] . shmt allowed interaction of brisc with ifnar to deubiquitinate k -ub of ifnar , reducing ifnar 's internalization and degradation by lysosome [ ] . taken together, brisc is the first dub complex we discussed that works to actually increase responses to ifn. tnf is a cytokine that plays a significant role in inflammation and regulation of immune cells. since tnf shares some of its pathway with ifn, studies that focused on tnf rather than ifn are included in this section for the purpose of this review (fig. ) (table ) . usp was identified by studies to negatively regulate both tnf-αand il- β-induced nf-κb activation [ ] [ ] [ ] . jiang et al. observed that introducing small interfering usp (siusp ) to decrease usp level in microglia from the spinal cord of sprague-dawley rats led to an increase in p-p and traf expression as well as secretion of tnf-α and il- β, all of which decreased upon introduction of ha-usp plasmid [ ] . xiao et al.'s study added on to this by demonstrating usp 's interaction and deubiquitination of traf and traf , but not traf , both in vivo, in hek t cells, and in vitro [ ] . as a result, usp negatively influenced tnf-α-induced-nf-κb activation-mediated cytokine induction, including il- and il- in a and h cells [ ] . usp also deubiquitinates tak in hek t cells [ ] . usp also protected iκbα from degradation, [ ] , a necessary step for tnf-α-induced nf-κb activation [ ] . this was further supported by knockout of usp aiding iκbα degradation [ ] . l. infantum otubain (otuli) has been shown to induce inflammatory responses in peritoneal macrophages from c bl/ , shown by production of tnf-α and il- as well as lipid droplet synthesis [ ] . also otuli demonstrated strong dub activity on k -ub and weak activity on k -ub in vitro at ph . [ ] . we mentioned in a previous section that usp deubiquitinates traf and interacts with traf , increasing expression of tnf in hek t cells [ , ] , while in mef cells, usp negatively affects tnf-α-induced nf-κb activation [ ] . a also affects tumor necrosis factor receptor (tnfr ) signaling pathway. in bmdms and bmdc, a worked together with tax bp to interact with ubc , an e enzyme, resulting in the inhibition of e ligase activities of traf , traf and ciap [ ] . futhermore, a and tax bp participated in degrading ubc upon il- and tnf-α stimulation in mef cells [ ] . a 's zf motif was found to recruit a dimers to bind with ripk in the tnfr signaling complexes and inhibit ubiquitination of k -ub and k -ub chains of ripk , hindering tnf signaling [ ] . in intestinal epithelial cells (iec), a dimer interacted with the ripoptosome (also known as complex iia), which allowed ubiquitins on ripk to sustain, increasing caspase- activation to promote tnf-induced apoptosis [ ] . however, this effect was not dub activity dependent [ ] . cezanne is a dub that belongs to the a subgroup of otu family. similar to a , cezanne also was shown to suppress nf-κb signaling by deubiquitinating k -polyub from ripk [ , ] and traf [ ] . also, cezanne was recruited to the activated tnfr prior to deubiquitinating ripk , which was dependent on the ubiquitin-associated (uba) domain of cezanne [ , ] . consistnent with the findings, inhibiting cezanne production via sirna resulted in an increased production of il- upon tnf-α stimulation [ ] . cezanne's dub activity was required for inhibiting phosphorylation and degradation of iκbα [ ] . usp (also known as usp ) interacted with and deubiquitinated traf in beas b cells [ ] . a noteworthy fact is that traf in jnk pathway was targeted by usp , but not traf in nf-κb signaling [ ] . trail is a cytokine that binds to death receptors (dr) and induces apoptosis, especially in tumor cells. its specificity for tumor cells have made trail and its receptor as the targets for anti-cancer therapeutics. trail inducing dubs are also summarized in table . in a study of malignant mesothelioma, a loss of function mutation of brca associated protein (bap ) resulted in increased sensitivity fo trail induction [ ] . when testing for domains that play a role in trail sensitivity in h mm cells, only asxl / binding site-mutated bap and dub domain-mutated bap resulted reduction in rtrail sensitivity, indicating that asxl / binding sites play a role in trail sensitivity [ ] . this was in congruence with the fact that bap binds to asxl to form the polycomb repressive deubiquitinase complex (pr-dub) that deubiquitinates histone h a [ ] . also, flow cytometry analysis confirmed that the mutation of c a, or the deubiquitinating domain, of dub resulted in decreased expression of dr and dr in h cells [ ] . only dr expression increased in h cells upon bap knockout [ ] . on the same line with bap , usp knockout also increased trail sensitivity [ ] . a study by leznicki et al. introduced three isoforms of usp in hek cells: usp iso , usp iso and usp iso , although the focus was on the first two [ ] . usp iso was revealed as an integral membrane protein on endoplasmic reticulum, while usp iso was identified as a cytosolic protein [ ] . moreover, the proteins that they interacted with also varied [ ] . usp iso led to er stress, bap cleavage and activation of caspase- and caspase- , resulting in apoptosis [ ] . usp iso upregulated c/ebp homologous protein (chop) and dr in u osfipin and hela fipin cells [ ] . on the other hand, overexpressing usp iso exerted an opposite effect of delaying caspase- processing in trail-induced apoptosis [ ] . this effect was dependent on dub activity [ ] . b-ap is a small therapeutic molecule that inhibits usp and uchl [ ] . b-ap has been identified as an agent that increases trail receptors on many types of cancer cells, increasing their likelihood to enter apoptosis via nk cells [ ] . introduction of b-ap in a , hct and calu- cells increased the level of dr , but not the other death-inducing signaling complex (disc) components [ ] . the increase in the level of dr protein was due to reduction in the degradation of dr , leading to an increase in trail-induced apoptosis [ ] . in a different study, caspase-denependent apoptosis was increased in mantle cell lymphoma (mcl) cells when exposed to b-ap , which was confirmed with addition of pan-caspase inhibitor zvad-fmk, which resulted in an inhibition of apoptosis [ ] . this study indirectly demonstrated that usp and/or uchl partakes in decreasing dr expression. mcpip is another dub that decreases dr [ ] . exposing mda-mb- cells to doxycycline (dox) led to induction of mcpip , which then led to a decrease in dr [ ] . similarly, when a human lung cancer cells were exposed to mcp , mcpip level increased in a dosedependent manner, also resulting in a decrease in dr [ ] . likewise, dr level increased when mcpip was knocked down via short hairpin rna (sirna) [ ] . mcpip successfully achieved this by deubiquitinating dr , thereby stimulating lysosomal degradation of dr [ ] . also, the increase in dr level following mcpip knockdown catalized the formation of disc during the dr -induced apoptosis [ ] . we will now discuss dubs that induce interleukins, which are listed in table . usp has been shown to deubiquitinate traf and tlr and to interact with traf , increasing expression of il- in mef cells [ , ] . on top of decreasing production of ifn [ , ] , orf in mhv also induced the production of il- β [ ] . il- β production was dependent on nlrp and asc, rather than aim [ ] . usp negatively regulates il- β-induced nf-κb activation [ ] [ ] [ ] . increases (c bl/ lung homogenate) [ ] increase = inc. in production, + = induce positive effect. eeyarestatin i (esi), a small molecule that inhibits deubiquitination, has been found responsible for blocking il- β release [ ] . lopex-castejon et al., who reported this finding speculated uch or usp was responsible for this phenomenon, but futher study showed that they do not regulate il- β secretion individually or cooperatively [ ] . this result left possibility of an uncharacterized dubs or an additional dub(s) partaking in the process. usp knockout murine splenocytes and naïve t cells produced more il- compared to the wt splenocytes [ ] . under th polarizing condition, il- production was significantly higher in the usp knockout naïve cd + t cells compared to the wt naïve cd + t cells [ ] . also usp knockout naïve cd + t cells underwent hyperproliferation under th polarizing condition, which was reversed by adding il- neutralizing antibody [ ] . taken together, usp downregulates il- synthesis and tcr-induced t cell proliferation [ ] . additional mechanism of action has been discussed in the previous seciton. dufner et al. found that usp was essential in t cell maturation and homeostasis, although it was not required for negative selection [ ] . inhibiting usp leads to decrease in il- ra mrna as well as ccr [ ] . also, il- , il- p , ifn-γ and tnf levels were increased in the blood of usp f/f cd -cre mice than usp f/f [ ] . deleting otulin gene in mouse immune cells, t, b, natural killer cells (nk), dendritic cells (dc) and macrophage cells, resulted in production of cytokines specifically responsible for acute systemic inflammation, such as tnf, il- β, il- , mcp- , mip- α and g-csf [ ] . cytokines responsible for adaptive immunity were not affected by the deletion [ ] . it is noteworthy that this study suggests that although many cytokines partake in the inflammatory response in murine cells without otulin, the primarily responsible cytokine is tnf [ ] . unlike in immune cells, deleting otulin in myeloid cells resulted in activation of cytokines responsible for acute and chronic inflammation as well as autoimmunity, showing an increase in the level of out of cytokines tested [ ] . deficiency of otulin in macrophages resulted in nf-κb activation without an induction, which was due to the inability to manage polyub chains synthesized by linear ubiquitin chain assembly complex (lubac) [ ] . another study found that otulin overexpression inhibited tnf-α-induced nuclear translocation of p in hek t cells [ ] . interestingly, both wt and dub domain mutant otulin disabled lubac-induced nf-κb activation, indicating that otulin-mediated met -polyub is not the only factor influencing nf-κb activation [ ] . otulin has also been identified to deubiquitinate met -polyub of ripk and inhibited the binding of nemo and ripk , blocking the tnf-α-induced nf-κb response [ ] . ebv bplf suppresses ifn production, but il- production by cells upon malp- ligand stimulation was also abrogated upon bplf expression [ ] . ebv bplf suppressed production of proinflammatory cytokine il- in -tlr /cd cells [ ] . trabid is a dub from otu family, translated from the gene zranb [ ] . upon zranb knockout in mice, il- , tnf, il- a, il- b and il- a displayed a decrease in expression [ ] . trabid's deubiquitinating activity was necessary for recruiting c-rel and p to il- promoter by influencing histone modifications [ ] . knockout of trabid rendered bmdc incapable of producing il- and il- , leading to a decrease in the number of differentiation of cd + t cells to t h and t h cells, which was reversible with adding il- and il- [ ] . not many studies have focused their study objectives on discovering relationship between chemokines and dubs. some discovered interactions are listed in table . we have discussed how dubs induce cytokine production, signaling and effects. compared to dub's effects on cytokines, cytokine-inducible dubs are far less studied due to the difficult nature of planning such studies. however, this information can be as important as dub-induced cytokines. we will now discuss some known examples of cytokine-inducible dubs, as listed in table . dub- is one of the early identified cytokine-inducible dubs. studying the sequence of dub- gene, unveiled that it contained a il- inducible enhancer in ba/f murine lymphocyte cell line [ ] . the timing of il- induced dub- mrna increase was identified as early g phase [ ] . moreover, when dub- was constitutively expressed, majority of ba/f cells were arrested in g phase of the cell cycle, which was dub activity dependent [ ] . induction of dub- was dependent on viable jak and raf- , but not stat , suggesting that dub- expression is dependent on two pathways: jak and ras/raf- /mapk pathway [ ] . il- and granulocyte-macrophage colony-stimulating factor (gm-csf) also induced dub- transcription, which supported that β common (βc) subunit plays a part [ ] . dub- a decreased in expression when jak was suppressed, also suggesting dub- a to be affected by il- and jak pathway [ ] . dub- is similar to dub- in its sequence of amino acids and is also induced by jak /stat pathway [ ] . however, unlike dub- , dub- was induced only by il- in t cells, but not by il- [ ] . dub- also increase = inc. in production. + = induce positive effect. showed increase in jak/stat signaling pathway products by decreasing il- induced dephosphorylation of stat [ ] . also, dub- decreased apoptosis in ba/f cells upon withdrawal of cytokines [ ] . the mechanism by which dub- achieves these effects needs to be further studied. in myeloid d cells, dub- stabilized csf r and increased its signaling activity by decreasing lysosomal degradation of csf r by deubiquitinating it, leading to prolongation of stat phosphorylation in csf signaling pathway [ ] . on the other hand, dub- a is a dub expressed in hematopoietic cells, such as b and t cells [ ] . unlike dub- , which was more expressed by il- , dub- a was further expressed upon exposure to il- [ ] . although similar to dub- induction by il- [ ] , dub- a induction by colony-stimulating factor (csf ) in myeloid d cells did not require erk [ ] . dub- (also known as usp ) is a cytokine-inducible human dub that was found to deubiquitinate sds and block proliferation in hela cells [ ] . in mrna level, dub- expression increased in raji cells when treated with il- and in u cells when treated with il- [ ] . dub- also influenced the ras/mek/erk signaling pathway and deubiquitinated ras converting enzyme (rce ), decreasing proliferation of cells [ ] . otud- b, a dub from otu family, was upregulated in a mouse pro-b cells, ba/f cells, upon cytokine stimulation [ ] . stimulation with il- , il- , il- or gm-csf resulted in a dose-dependent increase of otud- b mrna in the first to h of stimulation, but quick decrease was observed from to h [ ] . overexpressing otud- b in ba/f cells resulted in downregulation of proliferation and increased the frequency of apoptosis [ ] . usp has been known to be induced by viral infection, genotoxic stress or interferon [ ] . consistent with this, usp 's mrna level increased in thp- cells and thp- -derived macrophages upon exposure to ifn-β [ ] . furthermore, tlr ligands, lps, pam csk and cl all gave the same result of increased mrna level of usp , supporting that tlr-induced signaling pathway induces usp expression [ ] . exposure to tnfα caused gsk β-mediated phosphorylation of usp , leading to deubiquitination of traf in beas b cells, increasing jnk signaling upon tnf-α-induction [ ] . a was identified since as a tnf-α-induced dub in human umbilical vein endothelial cells (huvec) [ ] . mrna of cezanne quickly increased in hek and huvec cells upon exposure to tnf-α, but not upon shear stress [ ] . we have discussed some known cases of dub-regulated cytokines and cytokine-inducible dubs. cytokines are intertwined in numerous cellular processes and dubs are closely related to cytokines. this review dealt with many dubs, but less than half of all known dubs are discussed. moreover, we cannot say for certain that all the functions of the dubs discussed here are discovered. this grants us to further investigate the molecular mechanism and their effects. studying dubs and their effects could enlighten us with a novel therapeutic approach to various diseases, including but not limited to immunological diseases and cancer. structural and functional insights to ubiquitin-like protein conjugation ubiquitin modifications the ubiquitin code in the ubiquitin-proteasome system and autophagy a genomic and functional inventory of deubiquitinating enzymes regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes an atlas of altered expression of deubiquitinating enzymes in human cancer mindy- is a member of an evolutionarily conserved and structurally distinct new family of deubiquitinating enzymes broad and diverse mechanisms used by deubiquitinase family members in regulating the type i interferon signaling pathway during antiviral responses the roles of ubiquitin modifying enzymes in neoplastic disease viral deubiquitinases: role in evasion of anti-viral innate immunity type i interferon in chronic virus infection and cancer triggering antiviral response by rig-i-related rna helicases double-stranded dna and doublestranded rna induce a common antiviral signaling pathway in human cells rna recognition and signal transduction by rig-i-like receptors trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-specific protease negatively regulates virus-induced type i interferon signaling via catalytically-dependent and -independent mechanisms a functional ubiquitin-specific protease embedded in the large tegument protein (orf ) of murine gammaherpesvirus is active during the course of infection inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirusencoded deubiquitinase orf evasion of innate cytosolic dna sensing by a gammaherpesvirus facilitates establishment of latent infection ubiquitin-specific proteases negatively regulates virus-induced type i interferon signaling induction of usp by viral infection promotes innate antiviral responses by mediating the stabilization of traf and traf ubiquitin-specific protease regulates tlr -dependent innate immune responses through deubiquitination of the adaptor protein traf negative regulation of il- -mediated signaling and inflammation by the ubiquitinspecific protease usp usp negatively regulates antiviral response by acting as a rig-i deubiquitinase usp inhibits type i interferon signaling by deubiquitinating rig-i-like receptors cylindromatosis (cyld), a deubiquitinase, attenuates inflammatory signaling pathways by activating toll-like receptor in human mesangial cells the tumour suppressor cyld is a negative regulator of rig-i-mediated antiviral response the deubiquitinating enzyme cylindromatosis dampens cd (+) t cell responses and is a critical factor for experimental cerebral malaria and blood-brain barrier damage loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating nf-kappab the ubiquitinmodifying enzyme a is required for termination of toll-like receptor responses cyld limits lys -and met -linked ubiquitin at receptor complexes to regulate innate immune signaling deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression mers-cov papain-like protease has deisgylating and deubiquitinating activities the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase transmissible gastroenteritis virus papain-like protease antagonizes production of interferon-beta through its deubiquitinase activity proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the sars coronavirus papain like protease can inhibit irf at a post activation step that requires deubiquitination activity deubiquitinase function of arterivirus papain-like protease suppresses the innate immune response in infected host cells modulation of stimulator of interferon genes (sting) expression by interferongamma in human keratinocytes usp recruits usp to promote innate antiviral response through deubiquitinating sting/mita usp negatively regulates nf-kappab signaling by targeting tak and nemo for deubiquitination through distinct mechanisms usp inhibits nf-kappab and nfat activation during th differentiation by deubiquitinating the tak -tab complex ubp is a novel regulator of interferon signaling independent of its isg isopeptidase activity microarray analysis reveals that type i interferon strongly increases the expression of immuneresponse related genes in ubp (usp ) deficient macrophages receptor dimerization dynamics as a regulatory valve for plasticity of type i interferon signaling a deubiquitinating activity is conserved in the large tegument protein of the herpesviridae herpes simplex virus ubiquitin-specific protease ul inhibits beta interferon production by deubiquitinating traf herpes simplex virus ubiquitin-specific protease ul abrogates nf-kappab activation in dna sensing signal pathway the papainlike protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production plp of mouse hepatitis virus a (mhv-a ) targets tbk to negatively regulate cellular type i interferon signaling pathway mcpinduced protein deubiquitinates traf proteins and negatively regulates jnk and nf-kappab signaling mcpip negatively regulate cellular antiviral innate immune responses through dub and disruption of traf -tbk -ikkepsilon complex the a deubiquitinase activity negatively regulates lmp activation of irf intracellular signaling molecules activated by epstein-barr virus for induction of interferon regulatory factor negative regulation of the retinoic acid-inducible gene i-induced antiviral state by the ubiquitin-editing protein a epstein-barr virus large tegument protein bplf contributes to innate immune evasion through interference with toll-like receptor signaling a brisc-shmt complex deubiquitinates ifnar and regulates interferon responses downregulation of usp promotes activation of microglia and subsequent neuronal inflammation in rat spinal cord after injury ubiquitin-specific protease (usp ) targets traf and traf for deubiquitination and inhibits tnfalpha-induced cancer cell migration usp targets tak to downregulate tnfalpha-induced nf-kappab activation ubiquitin signalling in the nf-kappab pathway revealing a novel otubain-like enzyme from leishmania infantum with deubiquitinating activity toward k -linked substrate inhibition of nf-kappab signaling by a through disruption of ubiquitin enzyme complexes dimerization and ubiquitin mediated recruitment of a , a complex deubiquitinating enzyme elevated a promotes tnf-induced and ripk -dependent intestinal epithelial cell death nf-kappab suppression by the deubiquitinating enzyme cezanne: a novel negative feedback loop in pro-inflammatory signaling the n-terminal ubiquitin-associated domain of cezanne is crucial for its function to suppress nf-kappab pathway cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting traf for deubiquitination the deubiquitinating enzyme usp stabilizes traf and reduces e-cadherin-mediated adherens junctions loss of functional bap augments sensitivity to trail in cancer cells histone h a deubiquitinase activity of the polycomb repressive complex pr-dub expansion of dub functionality generated by alternative isoforms -usp , a case study proteasome deubiquitinases as novel targets for cancer therapy a novel inhibitor of proteasome deubiquitinating activity renders tumor cells sensitive to trail-mediated apoptosis by natural killer cells and t cells the proteasome deubiquitinase inhibitor b-ap enhances dr activation-induced apoptosis through stabilizing dr the novel deubiquitinase inhibitor b-ap induces direct and nk cell-mediated antitumor effects in human mantle cell lymphoma monocyte chemotactic protein-induced protein- enhances dr degradation and negatively regulates dr activation-induced apoptosis through its deubiquitinase function deubiquitinases regulate the activity of caspase- and interleukin- beta secretion via assembly of the inflammasome the ubiquitin-specific protease usp is critical for the development and homeostasis of t cells the deubiquitinase otulin is an essential negative regulator of inflammation and autoimmunity otulin antagonizes lubac signaling by specifically hydrolyzing met -linked polyubiquitin epigenetic regulation of the expression of il and il and autoimmune inflammation by the deubiquitinase trabid dub- , a deubiquitinating enzyme with growth-suppressing activity jak is required for induction of the murine dub- gene the murine dub- gene is specifically induced by the betac subunit of interleukin- receptor dub- a, a novel deubiquitinating enzyme subfamily member, is polyubiquitinated and cytokine-inducible in blymphocytes the deubiquitinating enzyme dub- prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal dub- is a member of a novel family of cytokine-inducible deubiquitinating enzymes the deubiquitinating enzyme dub a enhances csf signalling by attenuating lysosomal routing of the csf receptor dub- a, a new member of the dub subfamily of hematopoietic deubiquitinating enzymes lys- -specific deubiquitination of sds by usp regulates hdac activity dub- , a cytokine-inducible deubiquitinating enzyme that blocks proliferation usp regulates ras activation and cell proliferation by blocking rce activity evidence for otud- b participation in b lymphocytes cell cycle after cytokine stimulation ubp (usp ) specifically removes isg from conjugated proteins the a cdna induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein we would like to thank members of baek's laboratory for their critical comments this study was funded by the korea ministry of environment (moe) as 'the environmental health action program ( )'. he has been serving as an editorial board member of more than a dozen of international journals. current research and clinical interests are in the molecular genetics of ubiquitination and deubiquitination systems relevant to various cancers. he first coined terms ubiquitomics and deubiquitomics in the field of the ubiquitin-proteasome system. in addition, he is also interested in genomics and proteomics during stem cell proliferation, differentiation, and reprogramming. key: cord- - ntdb yf authors: mair, kerstin h; müllebner, andrea; essler, sabine e; duvigneau, j catharina; storset, anne k; saalmüller, armin; gerner, wilhelm title: porcine cd α(dim/-)nkp (high) nk cells are in a highly activated state date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: ntdb yf natural killer (nk) cells play a crucial role in the early phase of immune responses against various pathogens. in swine so far only little information about this lymphocyte population exists. phenotypical analyses with newly developed monoclonal antibodies (mabs) against porcine nkp recently revealed that in blood nkp (-) and nkp (+) cells with nk phenotype exist with comparable cytotoxic properties. in spleen a third nkp -defined population with nk phenotype was observed that was characterised by a low to negative cd α and increased nkp expression. in the current study it is shown that this nkp (high) phenotype was correlated with an increased expression of cd and cd compared to the cd α(+)nkp (-) and nkp (+) nk-cell subsets in spleen and blood. additionally nkp (high) nk cells expressed elevated levels of the chemokine receptor cxcr on mrna level. functional analyses revealed that splenic nkp (high) nk cells produced much higher levels of interferon-γ and tumor necrosis factor-α upon stimulation with cytokines or phorbol- -myristate- -acetate/ionomycin compared to the other two subsets. furthermore, cross-linking of nkp by nkp -specific mabs led to a superior cd a expression in the nkp (high) nk cells, thus indicating a higher cytolytic capacity of this subset. therefore porcine splenic nkp (high) nk cells represent a highly activated subset of nk cells and may play a profound role in the immune surveillance of this organ. natural killer (nk) cells were initially characterised by their spontaneous lytic activity against certain tumor and virus-infected cells [ , ] . besides their role as cytotoxic cells through the production of perforin and granzymes, nk cells are potent producers of cytokines like interferon (ifn)-γ and tumor necrosis factor (tnf)-α [ ] and thus play important roles in immunomodulation and the defence against viral, parasitic and bacterial pathogens [ ] . a considerable number of phenotypically and functionally different nk-cell subsets have been identified up to date [ ] . for example, human nk cells can be divided into functionally and also developmentally distinct subsets according to their differing expression of cd in combination with cd [ , ] and more recently cd b and cd [ ] . in the mouse likewise cd and cd b (mac- ) are used to dissect nk cells into functionally and developmentally different subsets [ ] . additionally, the chemokine receptor cxcr is used in combination with cd to distinguish nk-cell subsets in the mouse [ ] . for porcine nk cells a perforin + cd + cd -cd -cd -cd -cd α + cd ß -cd b + cd + phenotype has been described and it was shown that these lymphocytes can perform immediate cytotoxicity against nk-susceptible targets [ ] [ ] [ ] . moreover, in parasitic as well as in viral infections increases in nk cell number and activity have been reported [ , ] , but also inhibitory effects on nkcell mediated cytotoxicity and cytokine production by viral infections are described [ ] [ ] [ ] [ ] . despite these hints on important functions of porcine nk cells in vivo, so far no investigations on the existence of functionally differing nk-cell subsets have been reported. nevertheless, a recent study from our group with newly developed monoclonal antibodies (mabs) against the activating receptor nkp enabled a more comprehensive insight into the phenotype of porcine nk cells and putative subsets [ ] . nkp (cd , ncr ) is a member of the natural cytotoxicity receptor (ncr) family, which is involved in the control of tumors and viral infections [ ] [ ] [ ] [ ] [ ] [ ] . moreover, it has been used as a marker for nk cell identification in different species like humans [ , ] , monkeys [ , ] , rodents [ ] [ ] [ ] , cattle [ ] and more recently in sheep [ ] and horses [ ] . in contrast, nkp in the pig was shown to divide porcine cd -cd α + nk cells into nkp and nkp + subsets in blood and all organs tested [ ] . cd -cd α + nkp -nk cells show phenotypic and functional properties of nk cells although they produce reduced levels of ifn-γ compared to the nkp + subset after in vitro stimulation. additionally, a third nk cell population with elevated nkp expression levels was identified in high frequencies in spleen and liver, pointing towards a special role of nk cells with this phenotype. therefore, in this study we focused on functional and phenotypical properties of cd α dim/-nkp high nkcells in the spleen. we observed that these cells differ in their expression of various nk-cell associated markers including cd , the tnf-receptor family member cd and the chemokine receptor cxcr compared to the cd α + nkp and nkp + nk-cell subsets. additionally, this nk-cell subset showed an increased cytokine production and cytolytic activity. thus, our data indicates that cd α dim/-nkp high nk cells in the pig are in a highly activated state. blood and spleens were obtained from - month-old healthy pigs from an abattoir. animals were subjected to electric high voltage anaesthesia followed by exsanguination. this procedure is in accordance to the austrian animal welfare slaughter regulation. peripheral blood mononuclear cells (pbmc) were isolated using density gradient centrifugation (lymphocyte separation medium, density: . g/ml, paa, pasching, austria) as described previously [ ] . dissected spleen was cut into small pieces and mechanically dissociated by forcing through a sieve. after a washing step in phosphate buffered saline (pbs, paa) cells were applied to density gradient centrifugation to isolate mononuclear cells. isolated lymphocytes were finally resuspended in culture medium or pbs containing % (v/v) porcine plasma for analysis by flow cytometry (fcm). isolated porcine pbmc and splenocytes were cultivated in rpmi (paa) with stable glutamine supplemented with % (v/v) heat inactivated foetal calf serum (fcs, paa), iu/ml penicillin and . mg/ml streptomycin (paa). medium for sorted nk cells was additionally supplemented with mm sodium pyruvate (paa), nonessential amino acids (paa) and μm -mercaptoethanol (sigma-aldrich, vienna, austria). where indicated, cells were additionally cultured in the presence of various cytokines as outlined below. freshly isolated pbmc or splenocytes were resuspendend in pbs containing % (v/v) porcine plasma and labelled for flow cytometric analysis. cultured cells were resuspended in pbs containing % (v/v) fcs for fcm staining. all incubation steps were performed for min on ice. the following primary antibodies were used for cell surface staining: unconjugated or alexa -conjugated anti-nkp (igg , clone viv-km , [ ] ), anti-cd (igg b, clone bb - e , southern biotech, birmingham, al, usa), percp-cy . -conjugated anti-cd (igg a, clone bb - e - c , bd biosciences, san jose, ca, usa), efluor -conjugated anti-cd (igg , clone ppt , custom-conjugation by ebioscience, san jose, ca, usa), unconjugated or fitc-conjugated anti-cd α (igg a, clone / / ), pe-conjugated anti-cd α (igg a, clone - - , bd biosciences), anti-cd (igg , clone g , serotec, raleigh, nc, usa), biotin-conjugated anti-cd (igg , clone b c , [ ] ). all non-commercial monoclonal antibodies were produced in-house [ ] . where indicated, these antibodies had been purified and covalently conjugated to fluorochromes or biotin. alexa fluor- protein labelling kit (life technologies, carlsbad, ca, usa) was used for conjugation of anti-nkp mabs according to manufacturer's instructions. fitc conjugation for anti-cd α mabs was performed as described elsewhere [ ] . anti-cd mabs were biotinylated using sulfo-nhs-lc-biotin (thermo scientific, pierce, vienna, austria) following manufacturer's instructions. unspecific binding was assessed by appropriate isotype-matched control antibodies. for indirect labelling, anti-mouse anti-igg -pe (southern biotech) and streptavidin-brilliant violet conjugate (biolegend, san jose, ca, usa) were used as second-step reagents. to discriminate between live and dead cells, fixable near-ir dead cell stain kit (life technologies) was used according manufacturer's protocol with . μl reactive dye per reaction. if unconjugated and conjugated antibodies with the same isotype were used in combination, a sequential staining was performed. unconjugated primary mab was used in a first step, followed by isotype-specific dye-conjugated antibodies. after secondary incubation, free binding sites of mouseisotype specific antibodies were blocked by whole mouse igg molecules ( μg per sample, jackson immunoresearch, suffolk, uk) followed by a further incubation step with fluorochrome-conjugated primary mabs. fcm analyses were performed on a facscanto ii or facsaria (bd biosciences). data of at least × lymphocytes per sample were recorded. data were analysed with facsdiva software (version . . , bd biosciences) and flowjo software (version . . ., tree star, ashland, or, usa). box plots were created by sigmaplot software (version . , systat software inc., erkrath, germany). for intracellular staining of ifn-γ, pbmc and splenocytes were stimulated in -well round-bottom plates at × cells per well in a final volume of μl. cells were either stimulated with iu/ml recombinant human interleukin (rhil)- (roche, vienna, austria) in combination with ng/ml recombinant porcine interleukin (rpil)- and ng/ml rpil- (both r&d systems, minneapolis, mn, usa) overnight, or left in medium alone as negative control. for ifn-γ labelling, brefeldin a (golgiplug, bd biosciences) was added to microcultures at a final concentration of μg/ml, h prior to harvest. cells were labelled with antibodies against cd , cd α and nkp as stated above. afterwards cells were fixed and permeabilized as described elsewhere [ ] and labelled with anti-ifn-γ-pe (igg , clone p g , bd biosciences) as well as corresponding isotype control mab (mouse-igg -pe, clone mopc- , bd biosciences). for sorting of cd -cd α + nkp and cd -cd α + nkp + nk cells of blood as well as cd -cd α + nkp -, cd -cd α + nkp + and cd -cd α dim/-nkp high nk cells from spleen, isolated mononuclear cells were labelled with primary antibodies against cd , cd α and nkp as described above. as secondary antibodies anti-igg -pe (southern biotech), anti-igg a-alexa and anti-igg b-alexa (both life technologies) were used. pbs containing % (v/v) fcs and mm edta was used for all washing steps. sorting was performed on a facsaria (bd biosciences). purity of sorted cell populations was at least . % or higher. sorted cells were either transferred directly into cell culture or resuspended in tri reagent (sigma-aldrich) and stored at − °c for subsequent mrna analysis. cd a degranulation assay nk cell receptor mediated degranulation was assessed by measuring the expression of cd a on the cell surface in combination with four-color flow cytometry to discriminate between the different nk-cell subsets. degranulation assays were performed according to a protocol for human nk cells [ ] and modified as follows. triggering of nkreceptors was performed by using monoclonal antibodies against nkp (igg , clone viv-km , [ ] ), cd (igg , clone g , serotec) or a combination of both. monoclonal antibodies were coated on -well round-bottom wells by incubation overnight at °c at a concentration of μg/ml each in pbs in a total volume of μl per well. isotypematched irrelevant antibodies served as control ( μg/ml in μl per well). plates were washed with pbs for three times before cells were added. freshly isolated pbmc and splenocytes were stimulated with rhil- ( iu/ml) and rpil- ( ng/ml, biosource, nivelles, belgium) overnight with × cells in a total volume of μl per well, using -well round-bottom plates. since nkp was rapidly internalised after receptor triggering, cells were labelled with alexa -conjugated anti-nkp mab prior to transfer into antibody-coated plates. the simultaneous use of viv-km for fluorescence-staining and viv-km for coating of plates was possible because the two mabs bind to different sites on nkp [ ] . after two washing steps to eliminate unbound anti-nkp -alexa antibodies, cells were used at a concentration of × cells in a total volume of μl per well (mab-coated -well round-bottom plate) in the degranulation assay. additionally, microcultures were supplemented with fitc-conjugated anti-cd a mab (igg , clone e / , serotec) at a final concentration of μg/ml and the two protein transport inhibitors brefeldin a (golgiplug, final concentration μg/ml) and monensin (golgistop, final concentration μg/ml) (both bd biosciences). after an incubation of one hour at °c, cells were re-labelled with alexa -conjugated anti-nkp in combination with pe-conjugated anti-cd α and efluor -conjugated anti-cd monoclonal antibodies for fcm as described above. analysis of ifn-γ and tnf-α production by elisa facs-sorted nk-cell subsets from blood and spleen were stimulated in -well round-bottom plates at × cells in a final volume of μl per well for cytokine production. for ifn-γ and tnf-α production cells were stimulated with a combination of rhil- ( iu/ml), rpil- ( ng/ml) and rpil- ( ng/ml). tnf-α production was also analysed after stimulation with ng/ml phorbol- -myristate- -acetate (pma) and ng/ml ionomycin (both sigma-aldrich). after h, supernatants were collected and tested for cytokine production with commercially available elisa kits for ifn-γ (mabtech, nacka strand, sweden) and tnf-α (r&d systems) according to manufacturers' protocols. optical densities (ods) were measured at / nm with an elisa reader (tecan, sunrise, crailsheim, germany). total rna from facs-sorted nk-cell subsets from blood and spleen was isolated using tri reagent (sigma-aldrich) according to manufacturer's protocol. rna quality control and cdna synthesis were performed as described elsewhere [ ] . expression of target genes was determined by real-time pcr, using an internal standard as calibrator. the internal standard (is) was generated by pooling equal aliquots of the cdna samples investigated in this study. primers for target genes were designed using either the public domain programmes primer [ ] or primer-blast [ ] for cxcr . all primers were synthesised commercially (eurofins mwg operon, ebersberg, germany). sequence information of used primers is listed in table . whenever possible, primers were forced to span over exon junctions in order to increase specificity. for amplification of target genes sybr w green i ( . ×, sigma-aldrich) was used as reporter dye. the qpcr reaction-mixes contained itaq w dna polymerase ( . u/reaction, bio-rad, hercules, ca, usa), gene specific primers ( nmol/l each), a final concentration of μmol/l dntp each and mmol/l mgcl (for cxcr . mmol/l were used) within provided reaction buffer ( ×, bio-rad). qpcr was performed on a cfx ™ (bio-rad), pcr conditions are listed in additional file . optimisation and validation of the qpcr assays with target gene-specific primers are likewise described in more detail in additional file . specificity of the generated pcr products using a cdna pool of samples was further verified by automated sequencing using the pgem-t easy vector system (promega, madison, wi, usa) and m standard sequencing primer (eurofins mwg operon). the multiplex qpcr assay for the reference genes (β-actin, cyclophilin a and gapdh) that were used to normalise each target-gene expression was performed as previously described [ ] . each plate contained corresponding randomly assigned rt-minus controls ( % of all samples investigated), the no-template controls (ntc), as well as the is. all samples were measured in duplicates. data were analysed using the cfx manager software (bio-rad) in the linear regression mode. for the quantification we applied the method described elsewhere [ ] . target gene expression was displayed as ^-ΔΔcq values representing the fold changes relative to is. data was analysed for statistical significance by spss w (spss statistics version . , ibm corp., armonk, ny, usa). datasets with two groups (pbmc) were analysed using paired two-tailed student's t-test. for datasets containing more than two groups (spleen) one-way variance analysis with bonferroni correction for paired sample means was applied. if sample size per group was < , no statistical evaluation was performed. three different levels of significance were defined: p < . (indicated by *), p < . (indicated by **) and p < . (indicated by ***). to expand the knowledge about the phenotype of previously described nkp -defined nk-cell populations in swine [ ] , we performed flow cytometric analyses of lymphocytes isolated from spleen and blood. a gating hierarchy was used throughout the experiments to exclude doublets, dead cells as well as cd + t cells. remaining cd lymphocytes were further analysed for cd α and nkp expression (see additional file ). as previously described [ ] , among cd lymphocytes two nk populations could be found in blood, namely nkp and nkp + cells that were both cd α + ( figure a , upper graph). the third nkp -defined subset that was found in spleen ( figure a , lower graph) was characterised by a low to negative cd α expression and increased expression of the activating receptor nkp . this applied to all animals analysed, resulting in a mean fluorescence intensity (mfi) for nkp that was - times higher than in the splenic nkp + subset ( ± to ± respectively, figure b , upper graph). cd α + nkp + cells in blood and spleen showed comparable expression levels of the activating receptor ( ± and ± respectively). we then investigated differences in the expression level of cd α in the respective nk-cell subsets. splenic nkp high nk cells showed a significantly reduced level of cd α expression compared to the other splenic nk-cell subsets in all animals analysed sequences of primers ( ´- ´) for target genes as well as primer positions on (+) strand, length of specific product in base pairs (bp) and product melting temperature in°c are indicated. (nkp high : ± , nkp + : ± , nkp -: ± , figure b , lower graph). of note, nkp and nkp + nk cells in blood as well as spleen also showed a differential expression of cd α, although it was not as obvious as for the nkp high subset. within each location nkp -nk cells showed the highest expression of cd α, thus indicating a correlation between an increase of nkp and a decrease of cd α expression on porcine nk-cell subsets. to get more insight into the phenotype of the nkp defined nk cells we further analysed the expression of cd and the tnf-receptor family member cd . the latter is an important marker to distinguish between nk-cell subsets in mouse and human [ ] [ ] [ ] ] and the porcine orthologue of cd was recently identified [ ] . no marked difference in cd or cd expression between blood nkp and nkp + nk-cell subsets could be observed, although nkp + cells showed a slightly increased cd expression ( figure c) . a clear difference between the three splenic nkp -defined nk-cell subsets could be observed for cd (nkp high : ± , nkp + : ± , nkp -: ± , figure c ) and was even more obvious for cd expression (nkp high : ± , nkp + : ± , nkp -: ± , figure c ). nkp -nk cells showed the lowest and nkp high nk cells the highest expression of both markers, thus indicating a positive correlation between nkp , cd and cd expression levels. in human and mice higher cd expression on nk cells is associated with an increased cytokine production [ , , , ] . if the same holds true for porcine nk cells, splenic nkp high cd high nk cells should be the most prominent cytokine producers compared to the other subsets. we therefore compared ifn-γ and tnf-α production between the different nk-cell populations in blood and spleen of several individuals (n = ). intracellular staining for ifn-γ in total pbmc and splenocytes was performed after in vitro stimulation with a combination of rhil- , rpil- and rpil- overnight. cells cultured in medium alone served as negative control and did not show any ifn-γ production. the percentage of ifn-γ + nk cells was significantly higher in the blood nkp + nk-cell subset compared to blood nkp cells after cytokine stimulation ( . % ± . versus . % ± . , respectively, figure a and b). nevertheless, the produced amount of ifn-γ per cell, investigated by the mfi, was similar for both blood nk-cell subsets ( ± for nkp + and ± for nkp -, figure b , right graph). splenic nkp high nk cells clearly showed the highest frequency of ifn-γ + cells compared to the other two splenic subsets whereas nkp cells had the lowest frequency (nkp high : . % ± . %, nkp + : . % ± , nkp -: . % ± . , figure a and b) . additionally, the amount of ifn-γ produced per cell was considerably higher in the nkp high subset (nkp high : ± , nkp + : ± , nkp -: ± , figure b , right graph). yet, stimulated splenic nk-cell subsets showed an overall lower frequency of ifn-γ producing cells compared to blood. results of intracellular cytokine staining were confirmed by elisa ( figure c ). facs-sorted cd -cd α + nkp and cd -cd α + nkp + nk cells from blood and cd -cd α + nkp -, cd -cd α + nkp + and cd -cd α dim/-nkp high nk cells from spleen were stimulated with rhil- , rpil- and rpil- overnight and supernatants were tested for ifn-γ production. blood nkp + nk cells produced higher levels of ifn-γ ( to -fold) compared to the blood nkp subset ( ± ng/ml versus ± ng/ml, respectively, figure c ), which is consistent with the data obtained by flow cytometry. in spleen differences between the nk-cell subsets were much more pronounced as nkp high nk cells showed a to -fold higher ifn-γ production compared to the nkp + ( ± ng/ml to ± ng/ml, figure c ), and to -fold higher production compared to the nkp -nk cells ( ± ng/ ml to ± ng/ml). in addition to ifn-γ, tnf-α production of the different facs-sorted nk-cell subsets was measured by elisa after cytokine or pma/ionomycin stimulation overnight (figure ). after stimulation with rhil- , rpil- and rpil- , splenic nkp high nk cells likewise showed the highest levels of tnf-α. thus tnf-α production was to -fold higher in the nkp high nk-cell subset compared to splenic nkp + nk cells ( ± pg/ml to ± pg/ml, figure a ) and to -fold compared to nkp -nk cells ( ± pg/ml to ± pg/ml, figure a ). blood nkp + nk cells showed a . to -fold higher tnf-α production compared to the blood nkp -nkcell subset ( ± pg/ml to ± pg/ml, figure a ). no obvious differences could be observed for tnf-α production between blood or spleen nkp and nkp + nk-cell subsets after pma/ionomycin stimulation ( figure b ) whereas splenic nkp high nk cells again showed an increased tnf-α production compared to splenic nkp + ( to -fold, ± pg/ml to ± pg/ml, figure b ) and splenic nkp -nk cells ( to -fold, ± pg/ml to ± pg/ml). data from both, ifn-γ as well as tnf-α production indicated that splenic nkp high nk cells, that also displayed an increased cd expression, are the most potent cytokine producing nk cell subset. it was already shown that blood nkp and nkp + nkcell subsets show comparable cytolytic activity against xenogeneic and allogeneic target cells in a nkp independent manner [ ] . to investigate whether the nkp high phenotype was also correlated with an increased cytolytic activity we performed cd a degranulation assays in combination with multi-colour flow cytometry. we used monoclonal antibodies against nkp or cd to mimic receptor-specific ligands to look at a possible correlation between receptor density and cytolytic activity of the different nk-cell subsets in blood ( figure ) and spleen ( figure ). background degranulation was determined by using irrelevant-isotype matched control antibodies. blood nkp + nk cells showed a clear cytolytic activity after triggering with anti-nkp mabs indicated by the induction of cd a ( figure ). as expected, nkp -nk cells showed no obvious increase in cd a expression after anti-nkp stimulation. although blood nkp as well as nkp + nk cells got activated by triggering of the fc receptor cd , this stimulation led to a higher cytolytic activation in the nkp + nk-cell subset ( . % ± . compared to . % ± . in the nkp -nk cells, figure b ). co-crosslinking of both receptors also led to a higher cd a expression in the blood nkp + nk-cell subset. interestingly, a comparison of data for the three different stimulations of the blood nkp + nk-cell subset revealed no co-stimulatory effect after co-crosslinking of nkp and cd ( figure a+b ). similar results were obtained for spleen nkp and nkp + nk-cell subsets ( figure ). splenic nkp + nk cells showed a higher cytolytic activity after triggering with anti-nkp and/or anti-cd mabs compared to nkp -nk cells ( % ± . to . % ± . for nkp and . % ± . to . % ± . for cd , figure a+b ). figure a+b) . a slight increase of both read-outs after co-receptor triggering with cd and nkp could be observed for this nk-cell subset compared to the nkp + cells where no obvious additive effect on degranulation could be found. the comparison of cytotoxic capability by degranulation assays demonstrated that blood nkp + nk cells showed an overall higher proportion of cd a + cells than splenic nkp + nk cells, regardless which receptor had been activated (mean of all stimulated fractions, blood nkp + cd a + : . % versus spleen nkp + cd a + : . %). however, cd a expression levels per cell, analysed by mfi, did not differ strongly (mean of all stimulated fractions, blood mfi nkp + cd a + : versus spleen mfi nkp + cd a + : ). instead, again regardless which receptor had been activated, the blood nkp + nk-cell subset showed more similar frequencies of cd a + cells to the spleen nkp high nk-cell subset (mean of all stimulated fractions, blood nkp + cd a + : . % versus spleen nkp high cd a + : . %). so far, our functional data indicated that splenic nkp high nk cells are in an elevated stage of activation. therefore we further analysed the expression of other nk-associated markers like the nk-receptors nkp and nkg d in this nk-cell subset. moreover, expression of the chemokine receptor cxcr was investigated since the expression of cxcr in combination with cd can be used for the identification of nk-cell subsets with different functional properties in the mouse [ ] . therefore, facs-sorted nkp -defined nk-cell subsets of blood and spleen were analysed for expression of these markers by quantitative rt-pcr ( figure ). additionally, nkp mrna levels in the different nk-cell subsets derived from blood and spleen were analysed. results confirmed data obtained from protein figure splenic nkp high nk cells produced the highest levels of tnf-α. analysis of tnf-α production of nkp -defined nk-cell subsets (cd α + nkp -: blue, cd α + nkp + : green, cd α dim/-nkp high : red) isolated from blood and spleen. facs-sorted cd -cd α + nkp and cd -cd α + nkp + nk cells from blood and cd -cd α + nkp -, cd -cd α + nkp + and cd -cd α dim/-nkp high nk cells from spleen were stimulated with rhil- , rpil- and rpil- (a) or pma and ionomycin (b) for h. supernatants were tested for tnf-α production in elisa. bar graphs on the left show data from one representative animal and are displayed as the mean of duplicates ± sd. tnf-α production of four animals analysed are shown on the right. mean values are represented by a black bar. significant differences between the subsets in blood or spleen are indicated (* = p < . , ** = p < . ). expression, showing the same differential expression of nkp mrna in the respective subsets ( figure ). interestingly, despite high nkp expression, for the ncrfamily member nkp a reduced expression was found for splenic nkp high nk cells compared to the other two splenic subsets. blood nkp -nk cells seemed to express slightly higher levels of nkp as blood nkp + nk cells. nkg d mrna levels were very homogeneous among the different nkp -defined subsets. also, this receptor showed the lowest variation in expression levels between different individuals. the most prominent difference in rna expression was observed for the chemokine receptor cxcr . splenic nkp high nk cells showed an overall higher expression of this receptor compared to nkp and nkp + nk-cell subsets from both spleen and blood. for nkp and nkp + nk cells from blood and spleen similar expression levels of cxcr were observed. recently, the phenotype of porcine nk cells was revisited in a study from our group using newly developed mabs against porcine nkp . interestingly, cd -cd α + nk cells in the blood were shown to be either nkp + or nkp - [ ] . additionally, a third nk-cell subset with elevated nkp expression was found in high frequencies in spleen and liver that was associated with a cd α dim/phenotype. in the current study we aimed to investigate this splenic nkp high nk-cell subset in more detail and elucidate possible functional as well as phenotypical differences to the nkp and nkp + nk-cell subsets in the pig. figure nkp high nk cells in spleen showed the highest cytolytic capacity. the cytolytic capacity of nkp -defined nk-cell subsets (cd α + nkp -: blue, cd α + nkp + : green, cd α dim/-nkp high : red) isolated from spleen was analysed after receptor-mediated degranulation. cells were stimulated and gated for flow cytometric analysis as outlined in figure . splenic nkp high nk cells showed highly elevated expression of this activating receptor compared to the other two nkp -defined subsets in spleen as well as in blood in all animals analysed, which could be demonstrated on protein as well as on mrna level. additionally, splenic nkp high nk cells displayed a strongly decreased expression of cd α, a receptor that was so far described to be expressed by all nk cells in the pig [ , ] . of note, also the nkp and nkp + nk cells in blood and spleen showed minor differences in their cd α expression. we observed a negative correlation, thus an increase of nkp expression was accompanied by decreased expression level of cd α. furthermore, expression levels of cd and the tnf-receptor family member cd differed between the three nkp defined nk-cell subsets. cd expression was clearly enhanced in splenic cd α dim/-nkp high nk cells and this subset also displayed slightly higher levels of cd compared to the other two splenic subsets. in contrast, nkp -nk cells showed the lowest expression of both receptors, thus indicating a positive correlation in the expression levels of nkp , cd and cd . only few reports highlight on differential expression levels of nkp on nk cells in other species, although the existence of nkp dull and nkp bright nk cells was already described in the late s on human nk cells [ ] . additionally, it was reported that cells within distinct human nk-cell subsets show different levels of nkp expression. thus, higher cd expression [ , , ] as well as higher cd expression [ , ] was associated with higher surface density of nkp on human nk cells. obviously, the latter is akin to the correlation of nkp and cd we observed in the pig. more recently a phenotype of human nk cells with elevated expression of nkp was reported, which show an activated phenotype and seem to play a profound role in hepatitis c virus infection [ ] . likewise to our findings, in that study nkp high nk cells showed a higher expression of cd . furthermore, the authors of this report used the different expression levels of nkp to separate nk cells into distinct subsets, similar to the approach of our study. cd is used to distinguish between different nk-cell subsets in mouse and human. in the mouse, cd in combination with cd b is used to differentiate functionally as well as developmentally different nk-cell subsets [ , ] . cd divides the mature cd b high murine nk cells into two distinct subsets. cd high nk cells show a higher proliferative capacity and additionally an increased cytokine production compared to the cd low nk-cell subset [ , ] . in regard to cytotoxicity, differing reports exist. it was shown that murine cd high nk cells show elevated cytolytic activity compared to the cd low nkcell subset [ ] . however, another report describes the cd low/-cxcr -nk cells to be the more cytolytic subset in the mouse [ ] . in human nk cells, high cd expression is also linked to higher cytokine production and the cd low/phenotype correlates with an overall higher cytolytic activity [ , , ] . in contrast, the recent study on human nk cells that used nkp to distinguish between different nk-cell subsets showed that nkp high cd high nk cells have an enhanced cytokine production and cytolytic activity [ ] . we likewise observed this bifunctionality in the nkp high cd high nk-cell subset in spleen, with an elevated ifn-γ and tnf-α production figure varying mrna expression of nk-associated markers on nkp -defined nk-cell subsets in blood and spleen. facs-sorted cd -cd α + nkp and cd -cd α + nkp + nk cells derived from blood and cd -cd α + nkp -, cd -cd α + nkp + and cd -cd α dim/-nkp high nk cells from spleen were analysed for their mrna expression of various nk-markers by quantitative rt-pcr. messenger-rna expression of the nk receptors nkp , nkp and nkg d as well as the chemokine receptor cxcr were analysed in the different nk-cell subsets (cd α + nkp -: blue symbols, cd α + nkp + : green symbols, cd α dim/-nkp high : red symbols). the ^-ΔΔct values for each individual animal (n = ) are shown as the fold differences relative to an internal standard (is = , black dotted line). geometric mean values of the three animals are represented by a black bar. after in vitro stimulation but also higher cytolytic capacity after triggering of the activating receptors cd or nkp compared to the other two splenic nk-cell subsets. consistent with this data, it was already suggested that higher density of the activating receptor nkp on the nk-cell surface is associated with higher cytolytic function [ , , ] . interestingly, we could not observe an obvious synergistic effect after co-crosslinking of both receptors in our study. nevertheless, similar results were shown for cd and nkp co-triggering in resting human nk cells [ ] . we observed an overall lower cytotoxic activity in the nkp subset in blood and spleen when stimulated with anti-cd antibody, although the expression level of cd was only slightly lower as in the nkp + nk cells. in our previous study we showed that blood nkp porcine nk cells had a cytotoxic capacity comparable to blood nkp + nk cells in killing assays using xenogeneic and allogeneic cell lines as targets [ ] . the lower killing capacity we observed in this study may be caused by the triggering of only a single activation pathway, whereas the killing of target cells is likely to result from the triggering of several activating receptors and/or lack of inhibitory signals. although splenic nkp high nk cells produced higher levels of ifn-γ compared to the other two subsets as shown by elisa, the overall proportion of ifn-γ producing cells was lower compared to blood nk cells. therefore, the higher cytokine production seems to result from a superior cytokine production on a single cell level as indicated by the higher mfi for ifn-γ in the splenic nkp high nk-cell subset. similar results could be observed for the cytolytic activity determined by cd a degranulation assays after receptor triggering. although the proportion of cd a + cells was not higher in the splenic nkp high subset compared to blood nkp + nk cells, nkp high nk cells showed the highest cd a expression levels on a per cell basis. thus, these data may suggest also a higher killing capacity on a single cell level within the splenic nkp high nk cells. taken the functional findings together, our data indicate that nkp high nk cells are in a highly activated state and can readily release high amounts of cytokines or cytolytic granules upon stimulation. to get further insight into the phenotype of the nkp defined nk-cells subsets in the pig we finally performed rt-qpcr analyses of distinct nk-cell associated markers. expression analyses of other activating receptors, namely nkp and nkg d, showed no marked differences between the nkp -defined nk cell subsets in blood as well as in spleen. splenic nkp high nk cells showed slightly lower levels of the ncr-family member nkp . nkg d that is described as important nk-cell receptor involved in target recognition in other species [ ] was very uniformly expressed between the different nk-cell subsets in the pig, which is consistent with data in mouse and human where nkg d shows an overall similar expression pattern between different nk-cell subsets [ , ] . the most prominent difference in expression between the nkp defined nk-cell subsets was observed for cxcr . splenic nkp high nk cells showed elevated levels of this chemokine receptor compared to the other two subsets. likewise, mouse cd high nk cells showed the highest levels of cxcr and recently a further sub-division of murine nk-cells by these two markers has been proposed [ ] . the chemokine receptor cxcr is associated with the recruitment of nk cells into the lymph node [ ] and accumulation in tumors [ ] . in the murine spleen, cxcr is important for intrasplenic trafficking of nk cells upon inflammatory signals [ ] as well as the mobilisation and migration of nk cells from the spleen into the periphery [ ] . thus, one can speculate that the elevated expression of cxcr on porcine splenic nkp high nk cells may indicate that this nk-cell subset is in a "ready-to-go" state for recruitment of nk cells into the periphery or within the spleen. in conclusion, our data shows that the splenic nkp high nk cells are in a highly activated state and can be readily activated upon in vitro stimulation. furthermore nkp high nk cells are characterised by a distinct receptor expression pattern compared to the nkp and nkp + porcine nk-cell subsets in blood as well as in spleen, including the tnf-receptor family member cd that can be used to separate functionally and developmentally different nk-cell subsets in other species. additional file : optimisation and validation of qpcr assays for nk-associated gene-specific primers. the suitability of the newly designed primers was verified in separate experiments by performing dilution series of pcr products in : or cdna pools in : steps in quadruplicates. the dilution series, in conjunction with the melt characteristics of the pcr product, were used to optimise the assays regarding the primer concentration, annealing and extension times and the efficiency for the pcr. the optimised pcr conditions including annealing and extension conditions as well as the reaction parameters (slope of the regression analysis corresponding to the efficiency of the qpcr) and the dynamic range for detecting % positive of the lowest dilution are indicated in the table. a product was detected in the rtminus control of some samples, nevertheless these showed at least . cqs or more difference to the respective rt-plus sample (Δct values are indicated in the table). calibration curve, melt curve and amplification blot for each target is illustrated. natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. ii. characterization of effector cells natural" killer cells in the mouse. ii. cytotoxic cells with specificity for mouse moloney leukemia cells. characteristics of the killer cell regulation of human nkcell cytokine and chemokine production by target cell recognition natural killer cells as an initial defense against pathogens natural killer cell subsets in man and rodents human natural killer cells: a unique innate immunoregulatory role for the cd (bright) subset cd bright cells differ in their kir repertoire and cytotoxic features from cd dim nk cells cd b and cd reflect distinct population and functional specialization in human natural killer cells cd dissects mature nk cells into two subsets with distinct responsiveness and migratory capacity murine cxcr +cd bright nk cells resemble the human cd bright nk-cell population expression of t-cell associated antigens by porcine natural killer cells discrimination between two subsets of porcine cd + cytolytic t lymphocytes by the expression of cd antigen perforin expression can define cd positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic t, natural killer t and mhc un-restricted cytotoxic t-cells host environment as a modulating factor of swine natural killer cell activity changes in lymphocyte populations in suckling piglets during primary infections with isospora suis infection with classical swine fever virus: effects on phenotype and immune responsiveness of porcine t lymphocytes natural killer cell dysfunction during acute infection with foot-and-mouth disease virus porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs porcine reproductive and respiratory syndrome virus induces pronounced immune modulatory responses at mucosal tissues in the parental vaccine strain vr infected pigs nkp expression discriminates porcine nk cells with different functional properties enhanced in vivo growth of lymphoma tumors in the absence of the nk-activating receptor nkp /ncr recognition and prevention of tumor metastasis by the nk receptor nkp /ncr natural killer p (high) expression defines a natural killer cell subset that is potentially involved in control of hepatitis c virus replication and modulation of liver fibrosis lethal influenza infection in the absence of the natural killer cell receptor gene ncr killing of avian and swine influenza virus by natural killer cells expansion of b + natural killer (nk) cells and decrease in nkp + nk cells in response to influenza p , a novel natural killer cell-specific surface molecule that mediates cell activation molecular cloning of nkp : a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity moretta l: identification, molecular cloning and functional characterization of nkp and nkp natural cytotoxicity receptors in macaca fascicularis nk cells identification, activation, and selective in vivo ablation of mouse nk cells via nkp the murine homologue of the human nkp , a triggering receptor involved in the induction of natural cytotoxicity rat nkp activates natural killer cell cytotoxicity and is associated with fcεriγ and cd ζ nkp defines a subset of bovine leukocytes with natural killer cell characteristics nkp defines ovine cells that have characteristics corresponding to nk cells generation and characterization of monoclonal antibodies to equine nkp monoclonal antibodies reactive with swine lymphocytes. ii. detection of an antigen on resting t cells down-regulated after activation porcine cd : identification, expression and functional aspects in lymphocyte subsets in swine characterization of swine leukocyte differentiation antigens preparation of cells and reagents for flow cytometry detection of intracellular antigens in porcine pbmc by flow cytometry: a comparison of fixation and permeabilisation reagents a rapid method for assessment of natural killer cell function after multiple receptor crosslinking porcine t-helper and regulatory t cells exhibit versatile mrna expression capabilities for cytokines and co-stimulatory molecules primer on the www for general users and for biologist programmers primer-blast: a tool to design target-specific primers for polymerase chain reaction quantitative simultaneous multiplex real-time pcr for the detection of porcine cytokines ra: cd defines phenotypically and functionally different human nk cell subsets application of cd as a marker for distinguishing human nk cell subsets nkp is the major triggering receptor involved in the natural cytotoxicity of fresh or cultured human nk cells. correlation between surface density of nkp and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells cd surface density identifies a functional intermediary between the cd bright and cd dim human nk-cell subsets analysis of natural killer cells in tap -deficient patients: expression of functional triggering receptors and evidence for the existence of inhibitory receptor(s) that prevent lysis of normal autologous cells maturation of mouse nk cells is a -stage developmental program synergy among receptors on resting nk cells for the activation of natural cytotoxicity and cytokine secretion roles of the nkg d immunoreceptor and its ligands induced recruitment of nk cells to lymph nodes provides ifnγ for t(h) priming natural killer cell accumulation in tumors is dependent on ifn-γ and cxcr ligands intrasplenic trafficking of natural killer cells is redirected by chemokines upon inflammation ifn-γ acts on t cells to induce nk cell mobilization and accumulation in target organs porcine cd α dim/-nkp high nk cells are in a highly activated state the authors thank maria stadler, katharina reutner, sandra groiß and sarah rosenthaler for their technical support. kerstin h. mair and part of this work were funded by the phd program "host pathogen interaction in the pig", university of veterinary medicine vienna, austria. the authors declare that they have no competing interests.authors' contributions khm carried out the laboratory work except qpcr analyses and participated in the design of the study. am performed practical work and data analyses for the qpcr assays. see and jcd performed qpcr assay design, validation and carried out qpcr data analyses. aks contributed to the design of experiments and drafting of the manuscript. as and wg were responsible for conception and design of the study. khm and wg wrote the manuscript and analysed the data. all authors read and approved the final manuscript. key: cord- -cb ntxs authors: nogales, aitor; l. dediego, marta title: host single nucleotide polymorphisms modulating influenza a virus disease in humans date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: cb ntxs a large number of human genes associated with viral infections contain single nucleotide polymorphisms (snps), which represent a genetic variation caused by the change of a single nucleotide in the dna sequence. snps are located in coding or non-coding genomic regions and can affect gene expression or protein function by different mechanisms. furthermore, they have been linked to multiple human diseases, highlighting their medical relevance. therefore, the identification and analysis of this kind of polymorphisms in the human genome has gained high importance in the research community, and an increasing number of studies have been published during the last years. as a consequence of this exhaustive exploration, an association between the presence of some specific snps and the susceptibility or severity of many infectious diseases in some risk population groups has been found. in this review, we discuss the relevance of snps that are important to understand the pathology derived from influenza a virus (iav) infections in humans and the susceptibility of some individuals to suffer more severe symptoms. we also discuss the importance of snps for iav vaccine effectiveness. influenza a viruses (iav) belong to the orthomyxoviridae family, and they contain a single-stranded (ss) negative-sense viral (v)rna genome formed by eight segments that are encapsidated into particles with an envelope ( figure a) . each of the vrna segments contains a long central coding region flanked at and termini by non-coding regions (ncrs), which work as promoters to initiate viral rna synthesis (transcription and replication). moreover, the packaging signals playing a role in the efficient encapsidation of the viral segments into nascent virions, are located at the and end of the coding regions ( figure b ) [ ] . structurally, vrnas form viral ribonucleoprotein complexes (vrnps), where vrnas are coated with multiple subunits of the viral nucleoprotein (np) and are associated with the heterotrimeric polymerase, which contains the polymerase basic and (pb and pb , respectively) and acidic (pa) proteins ( figure a ) [ ] [ ] [ ] . each vrnp acts as an independent transcription-replication unit using an uncommon mechanism among negative-sense rna viruses, given that viral rna synthesis occurs in the infected-cells nucleus. vrnas are used as templates by the viral polymerase to synthesize two positive-sense rna molecules, the complementary rnas (crnas), from which the same viral polymerase synthesizes more copies of genomic vrna, and the mrnas for viral protein synthesis [ ] [ ] [ ] [ ] [ ] [ ] . the small iav genome encodes for up to viral proteins through the viral envelope is decorated with the two viral glycoproteins hemagglutinin (ha) and neuraminidase (na) at a ratio of approximately four to one, respectively [ , ] . ha envelope protein mediates virus entry by binding to sialic acid-containing cell receptors, and then fusing endosomal and viral membranes during endocytosis [ , ] , while na is required for viral release from infected host cells, and it acts as a receptor destroying enzyme, cleaving terminal sialic acid residues from glycoproteins present at the cell surface [ ] [ ] [ ] . the matrix (m ) protein is also found in the viral membrane, although in much lower abundance than ha or na glycoproteins. m is a small transmembrane protein that forms a proton-selective ion channel in the viral envelope. m promotes uncoating of the vrnps after membrane fusion and the protein has also an essential role in viral assembly and release [ ] . under the viral envelop, there is an inner shell composed of the matrix (m ) protein, which interacts in the virion with the vrnp and the ha and na proteins. m apart from being a membrane-associated scaffold factor of the virion, acts as a crucial factor for different viral processes during infection, including virion assembly and budding [ ] [ ] [ ] . the nonstructural (ns) gene or segment of iav encodes an mrna transcript that is alternatively spliced to express two viral proteins, the nonstructural protein (ns ), produced from a continuous primary transcript, and the nuclear export protein (nep), which is produced by an alternatively processed transcript, using a weak splice site. nep is also located in the virion and may interact with m in the viral particle [ ] [ ] [ ] ( figure a) . during the infection, nep is responsible for the nuclear export of synthetized vrnp, ensuring that the vrnps are available for packaging [ ] . moreover, nep has also other functions during iav infection, contributing to viral budding and to regulate viral rna synthesis. ns is a multifunctional protein and a key viral factor that counteracts the host antiviral responses. ns has been shown to inhibit the production of interferon (ifn), the activity and expression of multiple interferon-induced genes (isg) and the processing and nuclear transport of host mrnas causing cellular shut-off [ , ] . segment of iav also encodes two proteins, the polymerase component pa and pa-x. pa is translated directly from the pa mrna, whereas pa-x is translated using a + frameshift mechanism from the same open reading frame (orf) [ ] . synergistically with ns , pa-x is also able to block the cellular antiviral responses by inhibiting host protein expression. moreover, the pa-x protein has been shown to modulate host inflammation, immune responses, apoptosis, and virus pathogenesis [ ] [ ] [ ] [ ] [ ] [ ] . human iav infections cause contagious respiratory diseases associated with mild to severe respiratory illness or even death, and they are considered as an important public health threat worldwide, which also results in significant economic losses [ ] [ ] [ ] . iav are divided into multiple subtypes, based on the ha and na glycoproteins. currently, there are ha (h to h ) and na (n to n ), but the growing iav surveillance programs and sequencing technologies could increase the number of subtypes in the following years. iav can infect a wide range of avian and mammalian species, although the natural reservoirs of iav are shorebirds and wild waterfowls [ ] [ ] [ ] [ ] . among all the ha and na subtypes, only h n and h n iav subtypes are circulating in human beings and they are responsible for annual recurrent epidemics that affect the entire world [ , ] . seasonal influenza infections are prevented and controlled through annual vaccination campaigns to decrease iav infections and viral transmission as well as to reduce their negative impact in the global economy. however, although vaccination remains the most effective approach to protect the population from seasonal infections, the effectiveness of current vaccination approaches is suboptimal [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . thus, the production of improved prophylactic approaches, including universal vaccines, are highly desired. concerns associated with iav are further aggravated by the adaptive capacity of the viruses to infect new hosts or escape to the immune system, as well as their ability to transmit efficiently in the population and the limited therapeutic options to treat viral infections [ , , , ] . because of the ability of iav to modify their genome using two main evolutionary mechanisms, antigenic drift and shift, viruses encoding novel antigenic proteins to which the population has limited or no preexisting immunity can be generated [ , , , ] . for that reason, seasonal vaccines have to be reformulated yearly to guarantee that the viral glycoproteins (ha and na) in the vaccine match seasonal viruses circulating worldwide [ , , ] . in addition, iav variability can lead to the generation of new virus strains with pandemic potential. for example, the first iav pandemic of this century occurred in and it is estimated that in approximately one year, the pandemic h n (ph n ) iav infected more than , human beings, causing near , deaths in over countries [ , ] . in addition, although only h n and h n are circulating in humans, the avian h , h , and h subtypes eventually cross the species barrier to infect humans, representing a new and serious public health problem [ , , [ ] [ ] [ ] . the cellular defense mechanisms provided by the innate immune system are a formidable barrier to inhibit virus infections [ ] and involve the recognition of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs). this recognition leads to the activation of signaling pathways and the production and secretion of ifns of type i (ifnα and ifnβ) and iii (ifnλ or il- a, ifnλ or il- b, and ifnλ or il- ) , and chemokines and cytokines involved in inflammatory processes [ ] . iav rnas are mainly recognized by the endosomal, membrane-associated prr toll-like receptors (tlrs) (double-stranded rnas, dsrnas) or / (ssrnas), respectively [ , ] , by the cytoplasmic prr retinoic acid-inducible gene i (rig-i), which detects dsrna and -triphosphates of the negative ssrna viral genome [ , ] , generated during replication of multiple viruses, by the nod-like receptor family member nod-, lrr-and pyrin domain-containing (nlrp ), which recognizes various stimuli (see below) [ ] and by the absent in melanoma (aim ) protein, recognizing not well-characterized influenza stimuli [ ] . the result of prr detection of viral pamps is the activation of multiple transcription factors, such as the nuclear factor kappa β (nf-κb), the activator protein (ap- ), and ifn regulatory factors (irf)- and irf- , which are responsible for the transcription of ifns [ , , ] and pro-inflammatory cytokines [ ] . secreted type i and iii ifns signal through different receptors in a paracrine or autocrine way to induce the transcription of ifn-stimulated genes (isgs), several of which counteract viral replication [ , , ] . just as an example mentioned below, ifitm is an isg playing antiviral roles against influenza virus infection and other viruses [ ] . type i and iii ifns signaling pathways lead to the post-translational phosphorylation of the signal transducer and activator of transcription (stat) and transcription factors [ ] , being the tyrosine kinase (tyk ) and janus protein tyrosine kinase (jak ) critical for the phosphorylation [ ] . moreover, stat is phosphorylated by ikkε during ifn signaling and this step is important for the ifn-inducible innate immune response [ , ] . upon phosphorylation, stat and stat associate with irf- forming the heterotrimeric isg factor (isgf ) complex [ ] . this heterotrimeric complex then translocates to the nucleus, and binds to ifn-stimulated response elements (isres) located in the promoters of isgs, up-regulating their expression [ , ] . inflammatory cytokines, such as interleukins (il)- a il- b and tumor necrosis factor (tnf)-α contribute to the proliferation and migration of different immune cells, such as monocytes, macrophages, neutrophils, and natural killer (nk) cells, to the infected tissue. nk cells have the ability to kill virus-infected cells, are important for the activation of a protective cytotoxic t lymphocyte (ctl) response [ ] , and nk-cell ifn-γ production is augmented by t-cell il- production in recall responses [ ] . neutrophils and resident alveolar macrophages are also important for virus clearance, due to their ability to destroy infected cells [ ] . in addition, cytokine signaling improves dendritic cells (dc) maturation, increasing the induction of adaptive immune responses by antigen presentation and co-stimulation [ , ] . these adaptive immune responses initiated upon innate immune activation are required for protection and viral clearance [ ] . nlrp is expressed by myeloid cells such as macrophages, monocytes, neutrophils, and dendritic cells [ ] or by human bronchial epithelial cells [ ] . upon stimulation, nlrp activates the inflammasome system, activating caspase- and leading to pro-inflammatory processes through the processing and activation of proil- b, proil- , and proil- [ ] . nlrp senses iav dsrna [ ] , and pb -f protein [ ] . furthermore, protein flux through the viral m ion channel activity in the trans-golgi network activates nlrp , leading to inflammasome activation [ ] . in addition to nlrp activation, iav activates the inflammasomes through aim , increasing iav-induced lung injury and mortality [ ] . the complement system is an important branch of innate immunity that plays an essential role in the clearance of pathogens. the complement system is triggered by three main pathways, the classical, the lectin, and the alternative pathways [ ] . the first two pathways are activated with the help of pattern recognition molecules, whereas the alternative pathway is activated spontaneously. interestingly, it is known that viruses are recognized by the three pathways. in the classical pathway, the c complex recognizes antigen-antibody complexes, which are formed on the pathogen surface. c qbp (complement c q binding protein) can bind to the globular heads of c q molecules, activating the classical pathway [ ] . on the other hand, in the lectin pathway, the mannan-binding lectin (mbl)/ficolin/mannan-binding lectin serin protease (map) complex recognizes specific carbohydrates on the pathogen surface. complexes activated after the classical and lectin pathways, cleave c and c , resulting in the generation of c bc a (c convertase). in the alternative pathway, spontaneous hydrolysis of native c results in the formation of c b-like c that binds factor b and after cleavage by factor d forms the initial c convertase [ ] . the three pathways converge at the cleavage of c into c a and c b by c convertases (c b, a and c b, bb). then, the c b molecules formed bind covalently to the c -convertases forming the c -convertases that cleave c into c a and c b. cd blocks c and c activation by preventing the formation of new c and c convertases [ ] . c b starts the formation of c b- or the membrane attack complex (mac). next, c binds to the membrane attached trimer and begins binding and polymerization of c that is inserted into the membrane, inducing virolysis [ ] . unregulated complement activation could play a central role in the acute lung injury (ali) pathology induced by highly pathogenic viruses, including severe acute respiratory syndrome (sars) coronavirus and avian iav h n , and h n [ ] . in virus-induced acute lung diseases, high levels of chemotactic, and anaphylatoxic c a can be generated as a result of excessive complement triggering and causing a "cytokine storm". accordingly, the blockade of c a signaling has been involved in treating the ali induced by highly pathogenic viruses [ ] . currently, particular attention is being paid to single nucleotide polymorphisms (snps) that are loci within the genome of an organism in which two or more alleles can exist. snps affect a single nucleotide or base pair and they are one of the most frequent types of genetic variations in the genome [ ] [ ] [ ] . snps need to be presented into the population with a frequency equal to or greater than % to be considered as polymorphisms. there are multiple types of snps, depending on their location that can be in different regions of the genes such as promoters, exons, introns or utrs ( figure ). snps in coding regions are classified as synonymous, when a nucleotide substitution does not change the amino acid sequence of the encoded protein, although other effects, such as changes in mrna structure or folding may account for variation in protein expression. on the other hand, non-synonymous snps are divided in missense or nonsense. in the first case, nucleotide substitution results in the change of one amino acid for another, affecting the protein sequence coded by a gene and therefore may lead to its dysfunction. in contrast, nonsense mutations are produced when instead of substituting one amino acid for another, the altered gene contains an early stop codon in the orf or a stop codon is abrogated, producing an elongated protein. this type of mutations results in shortened or elongated proteins leading typically to nonfunctional proteins. the functional role of snps in coding areas of the genome can be easily analyzed by studying the gene products. however, most snps fall within non-coding genome regions, therefore, predicting their effects is challenging. for example, snps in the promoter regions could affect their activity and regulation producing changes in gene expression levels. snps in utrs or intron regions have been related with an effect in protein translation or the production of splice variants of transcripts, leading to longer or shorter protein sequences, respectively. in summary, snps may influence gene regulation, the structure and stability of rna, the expression of rnas or proteins, the conformation and function of proteins, etc. thus, the identification of snps in genes and the analysis of their effects may lead us to better understand gene function or their impact on human health [ ] . in fact, snps that are or could be important for multiple human pathologies, such as cancer, diabetes, heart disease, schizophrenia, blood-pressure homeostasis, and autoimmune or metabolic diseases, have been identified [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, some described snps increase the human susceptibility to getting infected by viruses, bacteria or other pathogens [ , , [ ] [ ] [ ] [ ] [ ] [ ] . advanced sequencing and bioinformatics technologies have allowed the identification of a large number of human snps whose information is accessible in the databases. nevertheless, the biological significance and function for most of the snps found in the human genome remain unknown. currently, the scientific community recognizes the importance of this kind of genome variations that can act as biological markers and assist researchers in multiple aspects, such as: ( ) locate genes associated with multiple diseases, ( ) anticipate an individual's response to a specific infection, ( ) predict population responses to several treatments such as drugs or vaccines, ( ) design individualized therapies, ( ) identify markers for medical testing, ( ) perform pharmacogenetic studies, etc. this review focuses on the role of known snps on iav infection, as well as their impact on the effectiveness of vaccines against iav. instead of substituting one amino acid for another, the altered gene contains an early stop codon in the orf or a stop codon is abrogated, producing an elongated protein. this type of mutations results in shortened or elongated proteins leading typically to nonfunctional proteins. the functional role of snps in coding areas of the genome can be easily analyzed by studying the gene products. however, most snps fall within non-coding genome regions, therefore, predicting their effects is challenging. for example, snps in the promoter regions could affect their activity and regulation producing changes in gene expression levels. snps in utrs or intron regions have been related with an effect in protein translation or the production of splice variants of transcripts, leading to longer or shorter protein sequences, respectively. an snp is a variation on a single nucleotide which may occur at some specific point in the genome and that causes variations in dna sequences between members of the same species. (b) types of snps: dna variation can be located in non-coding or coding regions. snps within a coding sequence can be synonymous if they do not produce an amino acid change (silent mutation), or non-synonymous if they affect the protein sequence. nonsynonymous changes can be divided into missense (producing an amino acid change in the protein) or nonsense (producing a truncated or longer protein). an snp is a variation on a single nucleotide which may occur at some specific point in the genome and that causes variations in dna sequences between members of the same species. (b) types of snps: dna variation can be located in non-coding or coding regions. snps within a coding sequence can be synonymous if they do not produce an amino acid change (silent mutation), or non-synonymous if they affect the protein sequence. non-synonymous changes can be divided into missense (producing an amino acid change in the protein) or nonsense (producing a truncated or longer protein). risk factors, including underlying co-morbidities, age, and pregnancy, affect iav susceptibility, but do not explain all the conditions under which serious iav-associated disease can occur, making likely that snps in viral and host genes affect iav susceptibility and the outcome of the disease. in fact, there are some examples of the presence of snps in host genes affecting influenza severity (table ) , which will be discussed in this review. snps affecting iav disease have been found in genes recognizing viral components, in transcription factors important for ifn production and signaling, in isgs with antiviral activities, and in genes involved in inflammation. tlr recognizes dsrna, one of the iav replication intermediate products, and in turn activates ifn production, leading to an antiviral response. a missense mutation (f s) of the tlr gene was found in one out of three patients developing iav-associated encephalopathy (iae), a neurological consequence of severe viral infection [ ] . assays in tissue culture cells showed that a tlr receptor encoding the missense f s mutation was impaired in activating the transcription factor nf-κb, and in triggering downstream signaling via the ifnβ receptor, indicating that this genetic polymorphism could lead to increased iav replication [ ] . in a study of italian children diagnosed with iav h n infection, an additional tlr snp (rs , genotype c/t) was identified [ ] . this tlr snp was found in all the children developing iav-associated pneumonia ( cases). however, the snp was found in significantly less proportion in children with milder disease, suggesting a link between tlr and iav pathogenicity. furthermore, in a multicenter study involving adult cases of avian h n and ph n iav, in mainland china and hong kong, the tlr cc rs snp was associated with fatal cases [ ] . in addition to iav, there are other examples of snps in tlr or tlr signaling genes affecting viral infections. for instance, susceptibility to chikungunya virus (chikv) infection is highly increased in human and mouse cells with defective tlr molecules [ ] . furthermore, tlr snps, rs , and rs , leading to unknown functional consequences, were associated with an increased risk of chikv disease occurrence [ ] . patients with impaired tlr -mediated responses show an elevated susceptibility to herpes simplex- virus (hsv- )-mediated encephalitis by encoding tlr -deficient alleles [ , ] , or by encoding deficient traf , tbk and trif molecules, leading to impaired tlr- signaling [ ] [ ] [ ] . in a saudi arabian population, the tlr rs snp was strongly associated with hepatitis b (hbv) and hepatitis c (hcv) virus infections when compared to that in healthy control subjects [ , ] . the tlr rs c allele was also associated with hcv-related liver disease progression (cirrhosis and hepatocellular carcinoma) [ ] . however, the functional effects of these snps seem to be unknown. rig-i detects dsrna and -triphosphates of the negative ssrna iav genome, leading to innate immune responses activation [ ] . a caucasian male patient with severe iav h n infection during the swine flu pandemic showed two heterozygous variants (one in each chromosome): p.r h (snp rs ) and p.p s (snp rs ), located, respectively, in the caspase activation and recruitment domain (card) and rna binding domains of rig-i [ ] . these variants significantly decreased the recognition function of rig-i, and therefore, patient cells proved impaired antiviral responses to rig-i ligands and elevated proinflammatory responses to iav, providing evidence for dysregulation of the innate immune response and increased immunopathology [ ] . these results suggest that these rig-i polymorphisms may have contributed to severe iav outcome in this patient and reinforce that rig-i variants should be evaluated in future studies of host factors affecting ssrna virus infections. irf- is a transcription factor that increases interferon (ifn) production in response to viruses [ ] [ ] [ ] . a patient suffering from an unusual life-threatening disease after ph n infection encodes homozygous null mutations in the irf- factor. both irf- alleles from this patient encode mutations c. t>g/t (f v) and c. c>t/c (q x), which are mutations decreasing the ability of irf- to induce the transcription of ifn genes after iav infections [ ] . these findings suggest that irf- -dependent production of type i and iii ifns is required for controlling iav infections in humans. the rare allele a of two irf- snps, rs and rs , both located at exon/intron boundaries, were significantly associated with impaired levels of ifnα production by human plasmacytoid dendritic cells (pdcs) in response to human immunodeficiency virus (hiv- ) infection [ ] . therefore, these polymorphisms may affect the ability of human subjects to control hiv- infections, reinforcing the role of irf- in controlling viral infections. however, the effect of these snps should be further studied. irf- is a transcription factor essential for ifn signaling and the transcriptional induction of isgs [ ] . stat and stat , when phosphorylated, associate with irf- to form a heterotrimeric isg factor (isgf ) complex [ ] , which translocates to the nucleus, and binds isres present in the promoters of isgs, up-regulating their transcription [ , ] . a homozygous, loss-of-function mutation in irf- was described in a child born to first-cousin algerian parents and living in france affected by a severe pulmonary influenza infection [ ] . in particular, the homozygous mutation (c. g>a) occurred in the final nucleotide of exon and disrupted the essential splice site at the boundary of exon and intron , leading to deleted irf- proteins. the consequence of this mutation was an impaired activation of irf- , and therefore, an impaired transcription of isgs, many of which show antiviral activities [ ] . similarly, a family in which several members showed a surprising susceptibility to infection by different viruses, including iav, also showed to be irf deficient [ ] . the index patient, a boy with years born at term from healthy consanguineous parents (first cousins of portuguese origin and residing in venezuela) encoded a homozygous splicing mutation in the irf gene. the mutation, c. + g>t, was located in the donor splice site of introns and , leading to transcripts lacking exon . irf protein expression was undetectable in cells transfected with the c. + g>t irf construct, suggesting that either the protein was quickly degraded or the mrna was not translated. again, irf -deficient cells showed a profound defect in inducing the expression of multiple isgs [ ] . collectively, these findings show that human irf -and isgf -dependent type i and iii ifn responsive pathways are essential for controlling viral infections, including iav. the antiviral protein ifitm is an isg which abrogates the release of iav content from late endosomes into the cytoplasm [ ] . in addition, ifitm promotes the survival of mouse lung-resident cd + t cells following iav challenge, which may help clear the infection [ ] . furthermore, mice in which the expression of ifitm is abolished, showed severe disease after iav infection, compared to parental mice [ ] . one of the clearest associations of snps in genes affecting influenza severity is located in the isg ifitm . the human ifitm gene is encoded by two exons and is predicted to encode two splice variants that differ in the first amino-terminal amino acids. different studies have described the effect of ifitm snps in influenza disease severity. northern european patients infected with iav ph n virus requiring hospitalization showed over-representation of the snp rs in the ifitm gene, in which the majority t allele is replaced for a minority c allele [ ] . this leads to an alteration of the first splice acceptor site, originating an ifitm protein lacking the first amino acids (n∆ ) due to the protein starting from an alternative start codon. according to these results suggesting that this snp could affect influenza disease, the minority (cc) variant rendered homozygous cells more susceptible to iav infection, and this susceptibility correlated with decreased levels of ifitm protein expression in comparison to the majority (tt) variant cells [ ] . furthermore, cells expressing the n∆ protein showed an impaired ability to restrict viral replication when compared to wild-type ifitm cells [ ] . this data is consistent with previous results which show that the amino-terminal amino acids of ifitm are relevant for attenuating vesicular stomatitis virus (vsv) replication in vitro [ ] . moreover, the cc genotype was found in % of chinese patients showing mild disease after ph n virus infection compared to % in patients developing a severe ph n virus infection. in addition, the cc genotype was estimated to confer a six-fold increased risk for severe infection than the ct and tt genotypes [ ] , reinforcing the idea that ifitm is a factor affecting human iav disease [ ] . in another study, over-representation of the ifitm cc genotype was detected among fatal cases of chinese patients infected with iav ph n and h n viruses [ ] , and in a more general study, including twelve studies published before february with more than , subjects, revealed increased risk of severe influenza in both the east asian and white populations in the subjects encoding the ifitm cc genotype [ ] . another important snp (rs ) associated with risk of severe influenza in humans from the united states (us) infected with seasonal iavs is located in the -utr of the ifitm gene [ , ] . this snp affected ifitm expression being the risk allele associated with lower mrna expression. the mechanism for this lower mrna expression involves the decreased irf- binding and increased binding of the transcriptional repressor ccctc-binding factor (ctcf) in promoter-binding assays for the risk allele [ ] . moreover, the risk allele disrupted a cpg site that becomes differentially methylated in cd + t cell subsets, leading to less cd + t cells in the airways during natural influenza infection in the carriers of the risk allele, and suggesting that a critical role for ifitm may be to promote immune cell persistence at mucosal sites [ ] . interleukins a and b (il- a and il- b, respectively) are inflammatory cytokines that play critical roles in recruiting immune and inflammatory cells and developing adaptive immune responses. furthermore, accumulating evidence suggests that both cytokines play central roles in innate immunity against viral infections [ ] . the frequencies of snp (allele c) located base pairs upstream from the transcription start site (rs ), on the il- b promoter were associated with increased risk of influenza disease in chinese subjects [ ] . this nucleotide change is localized in a tata-box motif of il- b and modulates the transcription activity of il- b by binding to multiple transcription factors [ ] . the allele t of rs enhanced il- b protein expression, as indicated by several reports [ ] . people carrying allele t showed a higher il- b expression, which could lead to increased ifnγ production, which promotes virus clearance [ ] . in contrast, expression of il- b may be decreased in individuals who carry allele c, leading to a weaker immune response during viral infection. in addition, a t allele in il- a gene (snp rs ) increased the risk of iav ph n susceptibility, as observed in chinese subjects [ ] . the snp rs introduces a nonsynonymous mutation (a s) in il- a protein, suggesting that this genetic variant may lead to a functional variation in host susceptibility to ph n . nevertheless, the molecular mechanism needs to be evaluated and the real risk of these alleles should be analyzed in larger populations. tnf-α is a pro-inflammatory cytokine which orchestrates the host´s defense. a minor allele (a) at position - of tnf (snp rs ) was more frequent in greek patients infected with ph n virus compared to control subjects [ ] , and developing pneumonia was more uncommon in greek and mexican subjects with no copies of the minor allele compared to subjects with at least one copy of the minor allele [ , ] , leading to the hypothesis that this snp allele could be linked with an elevated susceptibility to infection with the ph n virus [ , ] . decreased tnf-α expression was observed in subjects encoding the minor allele at position - [ ] . this may explain how snps leading to lower production of tnf-α may predispose to more severe clinical symptoms following iav infections. however, the tnf-α rs minor a allele, associated with higher levels of tnf-α expression, was associated with susceptibility to japanese encephalitis virus infection in an indian population [ ] . the tnf-α rs minor a allele was a risk factor to develop liver cirrhosis and hepatocellular carcinoma following hbv infection in a han chinese population [ ] , suggesting that the protective or deleterious roles of tnf-α expression may vary depending on the infecting virus. chemokine receptor (ccr ) is expressed mainly on macrophages, t cells, and dendritic cells. ccr mediates leukocyte chemotaxis in response to its ligands, including mip- a, mip- b, and rantes. it can help direct multiple immune cell subsets, including regulatory t cells or th cells to sites of infection, supporting the antiviral immune response. evidence in humans support that homozygosity for the ccr -∆ allele, a naturally occurring polymorphism of ccr encoding a -bp deletion, prevents its expression on the cell surface, and is linked with an elevated susceptibility to west nile virus (wnv) [ ] and with increased severity of illness among patients infected with ph n [ ] , although this evidence is modest due to the limited number of subjects analyzed. in contrast, homozygous carriers of the ∆ mutation are resistant to hiv- infection because this molecule, absent in the cell surface in subjects encoding the deletion, is a molecule normally used by hiv- to enter cd + t cells [ ] . cd is an important complement regulatory protein which blocks c and c activation by preventing the formation of new c and c convertases, two proteases involved in inflammation and complement activation. consequently, cd protects cells from complement attack and decreases amplification of the complement cascade [ ] . the cd snp (rs , genotype t/t) was significantly associated with severe iav infection in chinese patients infected with ph n virus [ ] and was associated with increased death risk in greek patients [ ] . the rs snp of cd is located in the minimal promoter region [ ] and individuals with this genotype showed significantly lower levels of cd expression in comparison to those with the more frequent allele [ ] . therefore, patients who carry the t/t genotype may have more robust complement activation during iav infection, resulting in enhanced inflammation and disease severity [ , ] . according to these results, the polymorphism rs in gene cd was linked to disease severity in adult chinese cases of avian (h n ) and human ph n iav in another study [ ] . however, these findings need to be confirmed in bigger cohorts. c qbp can bind to the globular heads of c q molecules, activating the classical pathway of complement [ ] . an increased risk of severe disease after iav infection was found in patients homozygous for the minor allele of the snp rs in european and mexican populations [ , ] . however, the effect of this snps on gene expression and function is undescribed. soluble pattern-recognition molecules, forming part of the innate immune system, can neutralize iav infection. particularly, the serum mannose-binding lectin (mbl), several secreted human c-type lectins of the collectin family, collectin , and the pulmonary surfactant proteins (sp) -a , -a , and -d (sftpa , sftpa , and sftpd, respectively), may neutralize iav infectivity in vitro [ ] . mice lacking sp-a or sp-d were more susceptible to iav infection, indicating that sps exert relevant roles against iav infection [ ] [ ] [ ] . two frequent sp-a (sftpa ) missense alleles (rs -c, leading to the mutation q k and rs -a, leading to the mutation t n) were associated with acute respiratory failure, mechanical ventilation, and acute respiratory distress syndrome after infection with ph n virus in a spanish population [ ] . in addition to c-type lectins, s-type lectins have been described, such as galectins, which recognize galactose-containing oligosaccharides present in the cellular plasma membranes and in viruses, such as iav. importantly, intranasal treatment of galectin- enhanced survival of mice infected with iav by reducing viral load, apoptosis, and inflammation in the lung [ ] . moreover, galectin- knockout mice showed increased susceptibility to influenza virus infection than wild-type mice [ ] . to study human genetic susceptibility to avian iav h n infection, a genome-wide association study involving heavily-exposed healthy poultry chinese workers and iav h n patients was performed [ ] . functional variants of galectin- gene, including rs and rs , causing increased expression levels of galectin- expression, may confer more protection from iav h n infection to the carriers of these variants [ ] . the cleavage of the iav ha by host proteases is critical for viral infectivity. tmprss is a type ii transmembrane serine protease family member, which was shown to activate ha proteins of multiple human iavs in tissue culture cells. furthermore, deletion of tmprss in mice impairs the spread of h n influenza viruses, including the ph n swine iav [ ] . in addition, bodyweight loss and survival after h n iav infection were less severe in tmprss mutant mice compared to wild type mice [ ] . the genetic predisposition to severe ph n influenza virus was evaluated in chinese human subjects, finding that the gg genotype of rs , leading to increased expression of tmprss , was a risk variant to severe ph n influenza [ ] . furthermore, rs and rs , both of them associated with increased gene expression, were significantly associated with the susceptibility to iav h n [ ] . table . single nucleotide polymorphisms associated with susceptibility and severity of influenza infections. recognizes dsrna, triggering ifn production. rs not annotated; f s (nonsyn). rs (ncr). [ ] [ , ] rig-i detects dsrna and -triphosphates of the negative ssrna iav genome, leading to innate immune responses activation. rs ; r h (nonsyn). rs ; p s (nonsyn). [ ] irf- transcription factor that increases ifn production in response to viruses. rs ; f v (nonsyn) rs ; q x (nonsyn) [ ] irf- transcription factor essential for ifn signaling and the transcriptional induction of isgs. c. g>a occurred in the final nucleotide of exon and disrupted the essential splice site at the boundary of exon and intron (nonsyn). c. + g>t, was localized in the donor splice site of introns and and led to transcripts lacking exon (nonsyn). [ ] [ ] ifitm isg which abrogates the release of iav content from late endosomes into the cytoplasm. ifitm increases the survival of mouse lung-resident cd + t cells after iav infection, which can help clear the infection. rs , leading to an alteration of the first splice acceptor site, leading to an ifitm protein lacking the first amino acids (nonsyn). rs , is located in the -utr and affects ifitm expression with the risk allele showing lower mrna expression (ncr). [ , , , ] [ ] il- b inflammatory cytokine involved in the development of adaptive immune responses. furthermore, accumulating data has suggested that il- a and il- b have critical roles in innate immunity against viral infections. rs , located base pairs upstream from the transcription start site, on the il- b promoter. this nucleotide change is located in a tata-box motif of il- b, affecting the transcription activity of il- b (ncr). [ , ] galectin- recognizes galactose-containing oligosaccharides present in the cellular plasma membranes and in viruses, such as iav. -rs (ncr). -rs (ncr). [ ] tmprss type ii transmembrane serine protease family member which activates ha proteins of diverse human iav in tissue culture cells. deletion of tmprss in mice impairs the spread of h n influenza viruses, including the ph n . moreover, body weight loss and survival were less severe in tmprss mutant mice compared to wild type mice after infection with h n iav. -rs , localized in an intron (ncr). -rs , localized in an intron (ncr). [ ] syn-synonymous, nonsyn-nonsynonymous, ncr-non-coding region (intron, regulatory regions, promoter or utr). currently, iav vaccines are the main strategy to prevent iav infection, though their effectiveness is suboptimal in many cases. notably, the efficacy of vaccines against iav infections can fluctuate and there is a significant immune response variability across the population. factors such as previous exposure to iav infections or vaccines, age, and the closeness of the match between the vaccine and circulating strains are important to explain differences in vaccine effectiveness between seasons and group populations [ , , [ ] [ ] [ ] . however, multiple reports have demonstrated that the host genetic background and polymorphisms on key immune response genes modulate the immune response to infection or vaccination [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . therefore, new insights into iav-host interaction and immune response modulating factors could allow us to design better vaccination strategies. snps may modify the humoral immune response after iav vaccination. therefore, their impact on the immune responses induced after iav vaccination are being analyzed [ ] [ ] [ ] [ ] . the major histocompatibility complex (mhc) is localized in chromosome of the human genome, it includes multiple genes and exhibits considerable diversity between populations. moreover, in this genomic region, there is a higher presence of snps than in other sections of the genome. mhc class i and class ii molecules have an essential role in the adaptive immune system in response to infections. both classes of proteins bind peptide fragments derived from pathogens to be presented on the cell surface for recognition by appropriate t cells [ , , ] . in those genes, the human leukocyte antigens (hla) class i and ii are important because of their role in the immune system. gelder et al. studied whether hla class ii polymorphisms modulate anti-iav antibody responses to vaccination in a united kingdom population [ ] . for that, a cohort of hla-typed donors at risk was investigated, and hemagglutination-inhibition (hai) titers were evaluated before and days after the administration of seasonal trivalent influenza vaccine. a correlation between hla class ii alleles and iav hai titers in the influenza risk group was found. moreover, a positive association between non-responsiveness to influenza vaccine and hla-drb * and a negative association with hla-drb * and hla-dqb * - / [ ] was reported, suggesting that polymorphisms in hla class ii molecules affect antibody responses to iav vaccination. these findings are important because they could potentially identify individuals who may not be protected by current vaccination approaches. in another study, poland et al. analyzed the immunogenetic relationships between hla, cytokine and cytokine receptor gene polymorphisms in the induction of antibodies in response to inactivated seasonal vaccines [ ] . authors did not find statistically significant associations between hla class ii alleles and iav hai titers. however, they established a positive association of some hla class i alleles and iav h n hai titers, including hla-a* , a* , b* , b* , and c* . in contrast, they did not find associations between the hla-a, b or c alleles and hai antibody titers for iav h n . in addition, when authors evaluated a panel of cytokine and cytokine receptor snps, they identified several significant associations between snps, in regulatory or coding regions of cytokine (il- , il- b) or cytokine receptor (il- r, il- rb, tnfrsf a) genes and variations in hai antibody titers for iav h n [ ] (table ) . notably, snps from three genes, il- (rs ), il- b (rs ) and il- r (rs ) revealed links with iav h n -induced antibody responses in an allele dose-related way. the presence of snp allele c or g in the il- b or il- r genes, respectively resulted in reduced hai titers. however, high hai titers in the presence of minor snp allele g in the il- gene were observed [ ] . snps associations between cytokine or cytokine receptor genes and iav h n hai titers were also identified ( table ) . for example, a variant ga for non-synonymous snps within the il- receptor gene (rs ; d g) and tnf receptor gene (rs ; k e) displayed associations with lower hai titers, while a minor allele t variant (rs ) located in the region of the il- receptor gene was related with high antibody titers ( table ). these data suggest that host snps affect responses to influenza vaccine. mannose-binding lectin (mbl- ) is a protein that binds n-acetylglucosamine, mannose, and fucose on different microorganisms and activates the lectin complement pathway [ , ] . tang et al. studied the presence of snps in subjects who received an inactivated influenza vaccine. for that, authors classified the vaccine recipients in poor, normal or adverse responders. they observed that the g to a snp in the codon allele (rs ) in mbl- was associated with a decreased risk for the development of adverse or poor responses (table ) [ ] . in addition, they did not find a significant association between responses and either tnf-α or il- promoter snps among the response groups [ ] . cytokine expressed as a response to infections or tissue injuries. it plays an important role in host defense through the stimulation of acute-phase responses. -rs (ncr). -rs (ncr). iiv [ ] il- b cytokine that serves as a crucial inducer of th cell development. rs , located in ´utr (ncr). iiv [ ] ifn-b cytokine released as part of the innate immune response against infection by viruses or other pathogens. rs (ncr). iiv [ ] tnfrsf a cytokine receptor, its interaction with tnf-α control cell survival, apoptosis, and inflammation. rs (ncr). iiv [ ] il- r cytokine receptor involved in inflammatory and immune responses. rs , located in ´utr (ncr). iiv [ ] il- rb cytokine receptor that mediates the activation of the jak/stat signaling pathway leading to the expression of isg. rs , located in ´utr (ncr). iiv [ ] il- ra this cytokine receptor is important for the signaling pathway leading to immune cell differentiation and function. -rs (syn). -rs (ncr). iiv [ ] il- ra cytokine receptor that is involved in the inhibition of the synthesis of several proinflammatory cytokines. -rs (syn) -rs (ncr). iiv [ ] il- rb cytokine receptor that plays a role in th cell differentiation. rs ; d g (nonsyn). iiv [ ] il- rn cytokine receptor which modulates a variety of immune and inflammatory responses related with il- . -rs (syn). -rs located in ´utr (ncr). iiv [ ] tnfrsf b cytokine receptor involved in the recruitment of anti-apoptotic proteins. rs ; k e (nonsyn) iiv [ ] mbl- this calcium-dependent protein that plays an important role in innate immunity, and activates the lectin complement pathway. rs ; g d (nonsyn) iiv [ ] il- b (ifnl ) type iii ifn molecule, with brad functions in antiviral responses rs (ncr). iiv [ ] syn-synonymous, nonsyn-nonsynonymous, ncr-non-coding region (intron, regulatory regions, promoter or utr). iiv: inactivated seasonal vaccine. il- , interleukin . il- b, interleukin . ifn-b , interferon beta (ifnβ). tnfrsf a, tnf receptor superfamily member a. il- r , interleukin receptor type . il- rb, interleukin receptor subunit beta. il- ra, interleukin receptor subunit alpha. il- ra, interleukin receptor subunit alpha. il- rb , interleukin receptor subunit beta . il- rn, interleukin receptor antagonist. tnfrsf b, tnf receptor superfamily member b. mbl- , mannose binding lectin . il- b or ifnl , interferon lambda . other snps that are not related with immune responses have been also linked to vaccine effectiveness. egli et al. revealed that the presence of the t/g or g/g genotype (rs , minor-allele) in il- b (ifnλ ), a type iii ifn, was linked with increased seroconversion in recipients of an inactivated influenza vaccine (table ) [ ] . moreover, iav-stimulated b-and t-cells from the minor-allele carriers exhibited increased hla-dr and il- expression, respectively. in addition, the expression of il- b, but not il- a or il- , mrnas was significantly reduced in the rs , minor-allele carriers. authors also reported that the il- b rs polymorphism affected humoral responses to the iav vaccine, and had a strong outcome on cellular immune responses by modulating the th /th cytokine response [ ] . these findings are important because they will help to predict which individuals could not be protected by present vaccines and they can also be used to design personalized vaccine strategies to optimize the immune reaction. the sequencing of the human genome together with the development of novel bioinformatic tools have made possible the identification of multiple snps. more information is available for the scientific community in the databases. in addition, the identification and study of the human genome variability has opened the opportunity to investigate their association with the risk of developing multiple human diseases 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complex a journey through the lectin pathway of complement-mbl and beyond the role of mannose-binding lectin in health and disease host single-nucleotide polymorphisms and altered responses to inactivated influenza vaccine we apologize for those publications we could not refer due to space limitations. the authors declare no conflict of interest. key: cord- -x q t authors: davoudi-monfared, effat; rahmani, hamid; khalili, hossein; hajiabdolbaghi, mahboubeh; salehi, mohamadreza; abbasian, ladan; kazemzadeh, hossein; yekaninejad, mir saeed title: a randomized clinical trial of the efficacy and safety of interferon β- a in treatment of severe covid- date: - - journal: antimicrob agents chemother doi: . /aac. - sha: doc_id: cord_uid: x q t to the best of our knowledge, there is no published study on the use of interferon β- a (ifn β- a) in the treatment of severe covid- . in this randomized clinical trial, the efficacy and safety of ifn β- a were evaluated in patients with severe covid- . forty-two patients in the interferon group received ifn β- a in addition to the national protocol medications (hydroxychloroquine plus lopinavir-ritonavir or atazanavir-ritonavir). each -μg/ml ( million iu/ml) dose of interferon β- a was subcutaneously injected three times weekly for two consecutive weeks. the control group consisted of patients who received only the national protocol medications. the primary outcome of the study was time to reach clinical response. secondary outcomes were duration of hospital stay, length of intensive care unit stay, -day mortality, effect of early or late administration of ifn on mortality, adverse effects, and complications during the hospitalization. between february and april , patients were recruited, and a total of patients in the ifn group and patients in the control group completed the study. as the primary outcome, time to the clinical response was not significantly different between the ifn and the control groups ( . ± . versus . ± . days, respectively, p = . ). on day , . % versus . % of patients in the ifn group and the control group, respectively, were discharged (odds ratio [or], . ; % confidence interval [ci], . to . ). the -day overall mortality was significantly lower in the ifn than the control group ( % versus . %, respectively, p = . ). early administration significantly reduced mortality (or, . ; % ci, . to ). although ifn did not change the time to reach the clinical response, adding it to the national protocol significantly increased discharge rate on day and decreased -day mortality. (this study is in the iranian registry of clinical trials under identifier irct n .) the disease ( ) . hydroxychloroquine is another available choice that has been included in many national protocols ( ) . however, practitioners have been advised to restrict the administration of this drug for clinical trials due to its questionable efficacy and risk of adverse effects ( ) . the race is still on to find an effective treatment for covid- . most of our experience came from other coronavirus epidemics, severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) ( ) . interferon (ifn) subtypes were previously examined in the treatment of sars and mers. primary in vitro experience showed antiviral effects of ifns, especially ifn-␤ and ifn-␥, on sars-cov ( ) . the same results were reported for ifn-␤ against mers-cov ( , ) . later, in an animal model, higher antiviral activity of ifn-␤ compared to that of lopinavir-ritonavir was shown against mers-cov ( ) . the efficacy of ifn-␤ on mers is still being investigated ( ) . the antiviral effects of ifns are expressed through activating interferon-stimulated genes (isgs) ( ) . additionally, by decreasing the vascular leakage, ifn ␤- a improved ards complications, regardless of its antiviral properties ( ) . the higher expression of a protein named cd- also could lead to a better prognosis in ards. however, these data were not replicated in a later trial ( ) . to the best of our knowledge, there is no published study regarding the use of ifn ␤- a in the treatment of severe covid- . in this randomized clinical trial, the efficacy and safety of ifn ␤- a were evaluated in patients with severe covid- . patients and baseline features. during the study period, patients were screened, of whom subjects were eligible. considering dropouts, patients ( in the ifn and in the control group) completed the treatment for further analysis (fig. ). males were . % of patients. the mean age Ϯ standard deviations (sd) in the ifn and control groups was . Ϯ and . Ϯ years, respectively. fifty-two ( . %) patients had positive nasopharyngeal real-time pcr (rt-pcr) for sars-cov- , and ( . %) patients were diagnosed according to the clinical signs/symptoms along with the imaging findings. hypertension ( . %), cardiovascular diseases ( . %), diabetes mellitus ( . %), endocrine disorders ( . %), and malignancy ( . %) were common baseline diseases. endocrine disorders were dyslipidemia and hypothyroidism. there was no significant difference in terms of demographic data and baseline diseases between the groups. the most frequent chief complaints of patients were cough, fever, and dyspnea (table ) . apache ii score at the time of intensive care unit (icu) admission was not significantly different between the two groups ( . Ϯ in the ifn group versus . Ϯ in the control group, p ϭ . ). vital signs and laboratory data. median time from the onset of symptoms (according to patients' reports) to the administration of ifn was days (interquartile range [iqr], to ). the vital signs at the time of hospital admission were not statistically different, except respiratory rate was significantly higher in the ifn group ( versus , respectively, p ϭ . ). comparing the baseline laboratory data at the time of hospital admission revealed that the median blood urea nitrogen level was higher in the ifn than in the control group. the median inr value also was significantly higher in the control group than in the ifn group. other laboratory findings were comparable (table ) . treatment strategies. hydroxychloroquine is the main medication in iran's national protocol for the treatment of covid- . lopinavir-ritonavir or atazanavir-ritonavir may be added to hydroxychloroquine in severe cases. the antiviral regimens were not significantly different between the two groups. all antivirals were continued for days. deep-vein thrombosis prophylaxis and stress ulcer prophylaxis were considered for patients where indicated. based on patients' clinical conditions, azithromycin, intravenous ascorbic acid, antibiotics, intravenous immunoglobulin (ivig), or a corticosteroid was added to the antiviral regimens. a corticosteroid (methyl prednisolone, hydrocortisone, or dexamethasone) was administered for . % and . % of patients in the ifn and the control groups, respectively. the corticosteroid dose was equivalent to mg methylprednisolone daily for days. in addition, . % and . % of patients in the ifn and the control group, respectively, received ivig. the dose of ivig was g daily for days. supportive care modalities and administered medications are summarized in table . outcomes and complications. as a primary outcome, the time to clinical response was not significantly different between the ifn and control groups ( . Ϯ . versus . Ϯ . days, respectively, p ϭ . ), which is shown in the kaplan-meier plot (fig. ) . the log rank test also revealed that there was no statistically significant difference between the groups, considering time to clinical response (hazard ratio [hr], . ; % ci, . to . ; p ϭ . ). the six-category ordinal scale was assessed at days , , , and (table ) . on day , there was no significant difference between the groups in terms of the components interferon ␤- a in treatment of severe covid- antimicrobial agents and chemotherapy of this scale. on day of therapy, % of patients in the ifn group were discharged with no deaths. at this time, % of patients in the control group were discharged and % died. however, the difference was not statistically significant (odds ratio [or], . ; % ci, . to . ). on day , the results were statistically significant, and . % versus . % of patients in the ifn group and the control group, respectively, were discharged (or, . ; % ci, . to . ). when the administration of ivig and corticosteroid was considered a probable confounding factor, the adjusted odds ratio was . ( % ci, . to . ). regarding the time of ifn initiation, the analysis showed that early administration significantly reduced mortality (or, . ; % ci, . to ). however, late administration of ifn did not show significant effects (or, . ; % ci, . to . ). other secondary outcomes, such as duration of hospital stay, length of icu stay, and duration of mechanical ventilation, were not statistically different. however, more patients were extubated in the ifn group (p ϭ . ). additionally, the -day overall mortality was significantly lower in the ifn than the control group ( % versus . %, respectively, p ϭ . ). in multivariate analysis, the effect of ifn on the reduction of mortality was shown (or, . ; % ci, . to . ). when the model was adjusted for administration of ivig and corticosteroid as confounding factors, the effect not only remained but also became stronger (or, . ; % ci, . to . ) . kaplan-meier plot for assessment of survival was also determined (hr, . ; % ci, . to . ; p ϭ . ) (fig. ) . complications during the hospitalization course, incidence of organ failure, and adverse effects were not different between the groups. injection-related side effects (fever, chills, myalgia, and headache a few hours after injection of ifn) happened in ( %) patients (table ) . a hypersensitivity reaction occurred in one patient who received ifn. the reaction presented with maculopapular rash on the trunk and both upper and lower limbs. however, the patient was taking herbal medicine for cough consisting of thyme and honey and other medications, like hydroxychloroquine and lopinavirritonavir, concomitantly. interferon was discontinued after the fourth dose, and rashes began to disappear within days. according to the naranjo score, the reaction possibly was due to ifn. neuropsychiatric problems were detected in patients in the ifn group. two cases experienced severe agitation and two cases complained of mood swings (mostly depression). one of the patients with mood swings had a history of mild depressive disorder in past years. out of cases, two patients were in the hospital for nearly month. neuropsychiatric side effects of ifn are unlikely to happen in short-term use. all patients received a psychiatric consult. according to the naranjo score, ifn possibly and probably caused neuropsychiatric problems in three and one patients, respectively. the present study was the first randomized, open-label, controlled trial that assessed the efficacy and safety of ifn ␤- a in the treatment of patients diagnosed with severe covid- . time to reach the clinical response did not change following adding ifn to the national protocol medications. however, ifn significantly improved the discharge rate by day . the -day mortality also was significantly lower in the ifn group. patients who received ifn in the early phase of the disease experienced significantly more benefits from the treatment. some injection-related adverse effects of ifn occurred, and all were tolerable. as of now, no effective therapy has been introduced for covid- . some antiinflammatory agents and cytokine release inhibitors, like corticosteroids and tocilizumab (acting against il- ), have been proposed ( , ) . however, increasing risk of secondary infections, activation of latent tuberculosis, and other adverse effects are serious concerns ( ) . lopinavir-ritonavir did not improve time to the clinical improvement and mortality ( ) . the efficacy of other therapeutic modalities, like convalescent plasma, is not clear ( ) . among the coronavirus family, the efficacy of ifns at first was reported in sars ( ) . after the subsidence of the sars epidemic, ifn was again proposed for treatment of another coronavirus, mers. however, different subtypes of ifn (alpha and beta) in combination with ribavirin did not show significant efficacy in critically ill patients with mers ( ) . due to promising primary effects of ifn ␤- in mers, a trial for evaluating its efficacy is still running ( ) . thus, ifns, especially type i, are still interesting options for recent epidemics. one study evaluated the effects of ifn ␤- b in combination with lopinavir-ritonavir and ribavirin on mild to moderate covid- ( ) . in addition, nebulized ifn ␣- b in combination with oral arbidol was examined for the treatment of the disease ( ) . ifns may have multifunctional roles in the pathogenesis of sars-cov- . ifns are natural cytokines that are produced in response to viral infections. they can activate interferon-stimulated genes (isgs) and increase the expression of angiotensinconverting enzyme inhibitor (ace ). although the upregulation of ace might increase the risk of sars-cov- infection, by inactivating angiotensin ii, it might protect lung host cells from further injury ( ) . viral infections, including sars-cov- , can suppress the production of ifn types and but induce the production of il- and tnf. ifn type might remarkably reduce viral replication ( ) . interestingly, sars-cov showed the ability to act against the effects of ifns ( , ) . sars-cov encoded a family of proteins, the open reading frame (orf) family, that inhibits stat transporter from entering the nucleus and blocks interferon signaling ( ) . however, recently it has been shown that the function of some proteins in this family (orf and orf b) had changed in sars-cov- . this may have changed the pathogenesis of sars-cov- and its interaction with ifn ( ) . beside the antiviral effects of ifns, the potential role of ifn ␤- a in improving ards complications was proposed. expression of cd- proteins in lung cells and decrease in vascular leakage in ards and subsequent mortality were reported following treatment with intravenous ifn ␤- a ( ) . however, the results were not repeated in the next, larger trial ( ) . this may be related to the extensive use of glucocorticoids in the latter trial that can interfere with the effect of ifn. the antagonist effects of corticosteroids are considerable ( ) . the mean age of patients in the present study was Ϯ years. the mean or median ages were different in published studies, from to years ( , ) . the male gender was dominant in our study, resembling other studies of covid- ( , ) . although gender difference is evident in many studies of covid- , it does not affect outcomes. however, in one study, critically ill males had higher mortality ( ) . this difference was first explained by the increase in expression of ace (the receptor for entrance of sars-cov to the cell) in asian men ( ) . later, the issue was attributed to the higher rate of cigarette smoking in asian men than asian women. however, both hypotheses should be confirmed in future studies. at present, neither gender nor smoking is certainly correlated with severity of covid- ( ) . baseline vital signs and laboratory data were almost comparable between the groups, with some exceptions. the respiratory rate was significantly higher in the ifn group. the mean blood urea nitrogen level was higher in this group. this may be due to the higher rate of diarrhea as an initial symptom of covid- in this group compared to the control one ( % versus . %). diarrhea may cause dehydration and lead to higher blood urea nitrogen. two patients in the control group were under treatment with warfarin, which may explain the higher inr (international normalized ratio) mean value in this group. before interpretation of the laboratory results, it should be noted that serum levels of lactate dehydrogenase, creatine phosphokinase, and troponin were not measured for all included patients. as a primary outcome of the study, time to reach the clinical response was not significantly different between the groups. considering the dysregulated inflammatory response in the pathogenesis of the late phase of covid- , it is not surprising that antivirals do not have immediate effects in relieving the main symptoms at this stage. in the study of hung et al., as a secondary outcome clinical improvement occurred significantly faster in the ifn combination therapy group (lopinavir-ritonavir plus interferon ␤- b plus ribavirin) than in the control group, i.e., versus days ( ). however, it should be noted that most of the patients in this study had mild disease. remdesivir also shortened time to recovery in mild cases, i.e., days compared to days in the placebo group. the definition of time to recovery was somewhat different from that of our study ( ) . in severe cases, remdesivir showed better results in a -day than -day course. however, this arm of the study did not have a control group ( ) . the length of icu and hospital stays and duration of mechanical ventilation were not statistically different between the groups, like the other interferon trial ( ) . however, according to the six-category ordinal scale, more patients were discharged following ifn therapy at day . this scale was also used in the study of remdesivir, but the same results were not detected ( ) . one of the remarkable findings in our study was a decrease in -day mortality in the ifn group that was not achieved in other studies on covid- ( , ) . neither in early use (within days of the onset of the symptoms) nor in the late phase ( days after the onset of the symptoms) did remdesivir significantly change mortality in patients with severe covid- ( ) . although the effect of ifn on decreasing the mortality of patients with severe covid- is surprising, the results should be interpreted with caution and considering the limitations of the study. four patients in the ifn group died before receiving the fourth dose of ifn. according to the protocol of the study, these patients were excluded from the final analysis for assessing mortality. further studies are needed to confirm the results. as in previous reports, the mortality rate in our patients in the control group was high. in patients with severe covid- , mortality rate has been reported between % and % ( , ) . about half of the included patients had severe conditions and were transferred to the icu. most were intubated and were under mechanical ventilation. the clinical course of covid- is divided into early viral replication phase and late cytokine release phase ( ) . it was suggested that early administration of antiviral medications (within to days of the onset of the symptoms) would improve outcomes of patients with covid- ( ) . additionally, early administration of ifns was recommended in the treatment of mers ( ) . the early administration of antiviral agents in viral infections can accelerate viral clearance and postpone neutrophil infiltration. early administration of ifn ␤- a, even in severely ill, mechanically ventilated patients, led to a higher survival rate. late administration did not show more benefits. regarding the safety of ifn therapy in patients with covid- , injection-related reactions, including fever, chills, headache, and fatigue (early after injection), were antimicrobial agents and chemotherapy detected in % of the patients. all of these symptoms responded to the supportive therapy (acetaminophen) and change in the time of injection to late night. no erythema or injection site reaction, or any reaction that caused treatment interruption, was reported. considering the duration of the intervention, the incidence rate of ifn adverse reactions was lower than that reported in patients with multiple sclerosis ( ) . however, it should be accounted that some patients in our study were under mechanical ventilation, and the exact evaluation of these reactions was not feasible. as a component of the supportive care in covid- , most patients received analgesic and antipyretic concomitant with antiviral agents and ifn. these medications might mask the adverse reactions of ifn, too. nausea, vomiting, and abdominal pain were the most common gastrointestinal complications in our patients, and the incidence rates were not different between the groups. although two cases experienced slight elevation in serum amylase and lipase levels, in further evaluations, no pancreatitis was confirmed. covid- can cause several gastrointestinal symptoms. however, gastrointestinal symptoms that started after the hospital admission may be related to the medications. the incidence rates of aki and hepatic impairment were not significantly different between the ifn and the control groups. both renal and liver injuries can be covid- -associated organ dysfunction ( ) . the nephrotoxicity of medications like antibiotics and furosemide (which were frequently prescribed in our patients) and hepatotoxicity of antiviral agents also should be taken into account. no case of hepatotoxicity that led to the discontinuation of interferon was detected. indirect hyperbilirubinemia is one of the adverse effects of atazanavir-ritonavir ( ) . this study had some limitations. in this open-label, randomized clinical trial, patients in general and intermediate wards and intensive care units were recruited. most of the general wards actually were intermediate wards, but the accurate classification was not possible due to special and emergent conditions. due to restrictions in each pandemic event and low experience, the diagnosis of covid- was according to either positive ( ) . if discharged, the patient was followed up by phone. readmission was surveyed until may. outcomes assessment. the primary outcome of the study was time to reach clinical response. clinical response was defined according to the six-category ordinal scale ( ) . this scale classifies patients into six categories according to the severity of the viral pneumonia: ( ) discharge; ( ) hospital admission, not requiring oxygen; ( ) hospital admission, requiring oxygen; ( ) hospital admission, requiring noninvasive positive pressure ventilation; ( ) hospital admission, requiring invasive mechanical ventilation; ( ) death. time to clinical response was considered the number of days required to at least two scores of improvement on the scale or patient's discharge, whichever occurred sooner. secondary outcomes were duration of mechanical ventilation, duration of hospital stay, length of icu stay, -day mortality, effect of early or late (before or after days of the onset of symptoms) administration of ifn on mortality, adverse effects, and complications during the hospitalization. the following adverse effects of the antiviral regimen/ifn ␤- a and complications during the hospitalization course were assessed: gastrointestinal (nausea, vomiting, diarrhea, abdominal pain, and pancreatitis), anaphylaxis and allergic reactions (rash, urticaria, angioedema, bronchospasm, and dyspnea related to medication administration), ifn injection-related reaction (skin erythema and necrosis, chills, fever, and flu-like symptoms after injection), neuropsychiatric (sleep disorder, psychosis, agitation, depression, and mania), renal impairment (according to kdigo definition) ( ) , hepatic impairment (hepatic aminotransferase serum levels raised more than three times the upper limit of normal or serum total bilirubin above mg/dl) ( ), indirect hyperbilirubinemia (direct bilirubin level less than % of the total bilirubin) ( ), incidence of thromboembolism (deep-vein thrombosis or pulmonary thromboembolism), incidence of nosocomial infections, and diagnosis of septic shock (according to the surviving sepsis campaign guidelines) ( ) . the naranjo scale was used for evaluation of adverse effects of ifn. in this standard scale, several items, including previous reports, relationship with starting the agent, improvement after discontinuation, challenge result, alternative causes, data of drug assay, dose dependency, and patient's history of the same reaction were considered. total scores of Ͼ , to , and to were considered definite, probable, and possible correlations between the use of ifn and the adverse drug reaction, respectively ( ) . statistical analysis. the quantitative variables were reported as means Ϯ standard deviations (sd) if they had normal distributions or as median with iqr if they did not pass the normality test. the qualitative ones were reported as number (percent). for comparing the quantitative variables, t test or mann-whitney test was used. the qualitative variables were compared by chi-square test. the analysis was performed on a per-protocol basis, and patients who did not receive at least four doses of ifn were not included. the kaplan-meier plot and log rank test were used to compare the number of days to reach the clinical response and survival time between the groups. the hr and % ci for clinical improvement and death were estimated by cox proportional hazards model. the odds ratio was also calculated for patients who received ifn early versus late. the multiple logistic regression model was performed for the variables that were significantly different between the groups according to the univariate analysis. in the multiple logistic regression model, corticosteroid and 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surviving sepsis campaign: international guidelines for management of sepsis and septic shock a method for estimating the probability of adverse drug reactions we thank the nurses and other staff members of imam khomeini hospital complex for their kind support and ava khalili for proofreading the manuscript.we did not receive any funding for this work. recigen was a generous gift from cinnagen co.we have no conflict of interest to declare. key: cord- -z g fce authors: chu, yeonjeong; raja sekhara reddy, b.; pratap reddy gajulapalli, v; sudhakar babu, k.; kim, eunha; lee, sanghee title: design, synthesis, and biological evaluation of n-arylpiperazine derivatives as interferon inducers date: - - journal: bioorg med chem lett doi: . /j.bmcl. . sha: doc_id: cord_uid: z g fce type i interferon (ifn) signaling plays an important role in the immune defense system against virus infection and in the innate immune response, thus ifns are widely used as anti-viral agents and treatment for immune disorder or cancer. however, there is a growing demand for novel small-molecule ifn inducer due to tolerance, toxicity, or short duration of action following direct administration of ifns. in this study, we assessed arylpiperazine (arp) as a new core skeleton of ifn inducer. to investigate structure-activity relationship, we designed and synthesized a series of arp analogues and evaluated the ability to stimulate ifn response in thp- human monocyte cells. compound i was identified as a potent type i ifn inducer as it significantly increased cytokine secretion and increased expression of various ifn-stimulating genes which are representative biomarkers of type i ifn pathway. our results suggested a beneficial therapeutic potential of i as an anti-viral agent. type i interferon (ifn) signaling plays an important role in the immune defense system against virus infection and in the innate immune response, thus ifns are widely used as anti-viral agents and treatment for immune disorder or cancer. however, there is a growing demand for novel small-molecule ifn inducer due to tolerance, toxicity, or short duration of action following direct administration of ifns. in this study, we assessed arylpiperazine (arp) as a new core skeleton of ifn inducer. to investigate structure-activity relationship, we designed and synthesized a series of arp analogues and evaluated the ability to stimulate ifn response in thp- human monocyte cells. compound i was identified as a potent type i ifn inducer as it significantly increased cytokine secretion and increased expression of various ifn-stimulating genes which are representative biomarkers of type i ifn pathway. our results suggested a beneficial therapeutic potential of i as an anti-viral agent. keyword: interferon inducer, type i interferon, arylpiperazine, anti-viral agent, innate immunity we designed and synthesized a series of arylpiperazine derivatives as interferon inducers  we tested different linker system and carbonyl moiety resulted in increase of potency  i exhibited the activity of interferon inducer with ec of . μm and no cytotoxicity  i initiated immune responses by mediating type i ifn signaling interferons (ifns) are essential signaling peptides which act as the first line of surveillant in defense system and modulate innate and adaptive immune activation [ , ] . in response to virus infection, ifns are released by host cells, after which they bind to their specific receptors, thereby stimulating jak/stat signaling pathway and initiating transcription of various interferon-stimulated genes (isgs) such as cxcl , irf , ifit , and oas etc [ ] [ ] [ ] [ ] [ ] . activation of ifn signaling pathway regulates not only anti-viral response but also induces cellular immunity by stimulating macrophage or monocyte [ ] . based on the class of binding receptor and signaling cascade, ifns are categorized into three major group: type i, type ii and type iii. type i ifns including ifnα and ifnβ are considered as therapeutic targets for treatment of hepatitis b and c infections and multiple sclerosis [ , ] . in addition, ifns are used in cancer treatments of hematological malignancy such as leukemia and lymphomas [ , ] . despite their therapeutic potential, there are several limitations of ifns treatments including toleration, dose-related toxicity and side-effects such as dizziness, headache, muscle pain, and depression [ ] . moreover, ifns are typically administrated by intramuscular injection for therapeutic use. pegylated ifns with enhancing stability are generally used in clinical treatment to overcome the short duration of effect. however, pegylated ifns are not able to be used on patients with hyperbilirubinemia [ , ] . for these reasons, continuous efforts have been made to develop small-molecule modulators so as to stimulate ifns. for example, tilorone is the first synthetic small-molecule drugs used as orally active ifn inducer which showed efficient anti-viral activity against broad spectrum of viruses including ebola virus and middle east respiratory syndrome-related coronavirus (mers-cov), especially [ ] . anti-malarial agent anti-bacterial agent therefore, we aimed to develop new small-molecule regulators to stimulate type i ifn effect and identified that particular arylpiperazine (arp) derivatives exhibited the desired biological activity using phenotypic screening. remarkably, the arp core structure was reported to exert anti-malarial, antimicrobial, anti-cancer activity or selective -ht a antagonistic effect ( figure a ) [ ] [ ] [ ] [ ] . in addition, , -di-chloro substituent in arp pharmacophore plays important role in particular drugs such as cariprazine, aripiprazol or bioactive drd antagonist ( figure a ) [ ] [ ] [ ] . based on these important characteristics of the arp skeleton, we designed and synthesized a series of , -dichloro arp derivatives by further n-modification on piperazine part with different linker moiety such as thiourea, urea, and carbonyl functional group in order to assess structure-activity relationship (sar; figure b all the synthesized compounds were evaluated using isre reporter assay on thp- human monocyte cells for monitoring immune response. in this system, isg minimal promoter in conjunction with five isre elicits transcription of secreted luciferase reporter gene upon stimulation of the type i ifn pathway, and ifn-related immune activation was measured based on luminescence [ ] . as a result of the sar study, all thiourea derivatives revealed negligible or very weak potency, regardless of alkyl and aryl moiety at r ( a to f). no significant effects of p-nitro, fluoro, chloro substituents were observed on the aryl ring, however, the p-bromo substitution revealed undesired cellular toxicity ( c to e). in the aryl part, meta substitution lead to slightly increased activity compared to para substitution ( e and f). to enhance activity, we introduced a urea moiety as a linker with diverse substitution on the aryl ring, which increased potency of arp analogues for immune stimulation. compared with the thiourea linker, para substitution on the phenyl ring exhibited stronger potency than meta substitution in most urea linker cases ( a to h). in para-substituted derivatives, no significant activity was observed in the nitro moiety ( a) which corresponded to thiourea linker, whereas the fluoro and trifluoromethyl moieties showed increased activity ( b and c). however, compound c showed undesired cellular toxicity, thereby a trifluoromethyl substituent was replaced by a trifluoromethoxy moiety which substantially enhanced potency ( . -fold change) and generated no toxicity ( d). considering the interesting results of thiourea and urea linker, we next incorporated carbonyl moiety as a linker for arp derivatives. somewhat weaker or stronger potency was observed in case of trifluoromethyl and methoxy substituents, compared to individual functional group in urea linker ( c to e). for the further investigation, aliphatic chain or heteroaryl group were introduced at r position which produced no significant difference and only marginally increased activity ( a and b) . furthermore, disubstituted arp analogues were considered to increase activity ( f to h) that resulted in weak activity which was below that of p-methoxy analogue ( e). to increase potency of arp derivatives, we examined the effects on carbon element by incorporating a benzyl group with a trisubstituted functional group. for this purpose, trimethoxy benzyl ( i) or tri-fluoro benzyl ( j) were introduced at the phenyl ring. as a result, immune response was considerably improved due to tri-methoxy substitution on the aryl ring ( . -fold change), whereas tri-fluoro benzyl substitution showed no drastic change on isre result with a bit increase of activity ( . -fold change). in addition, we confirmed no effect on cell viability by benzyl group. for the further modification, we inserted a phenethyl moiety at r position ( k) to assess effects of carbon chain which showed activity similar to that of dichlorophenyl ( h) or trifluorobenzyl ( j) moiety ( . , . and . , respectively) based on these sar results, we identified the compound i as an effective type i ifn inducer and conducted further biological evaluation. corresponding to the cytokine level, mrna expression of ifnb and cxcl was clearly increased by i treatment (figure ). in addition to ifnb and cxcl , i significantly elicited transcription of various isgs such as irf , ifit , and oas which are considered biomarkers of type i ifn signaling pathway ( figure ). in conclusion, all these results indicated the potential ability of i as an efficient type i ifn inducer without undesired toxicity. although, it is hard to conclude that i showed superior potency compared to tilorone, an efficient small molecule anti-viral agent inducing type i ifn, due to the different evaluating system. considering mg/kg required to treat evola virus by tilorone [ ] , the fact that i induced significant increased ifnβ secretion at μm suggested structure insight for developing new anti-viral therapy. in conclusion, we confirmed arp motif as a new core skeleton of type i ifn regulation. moreover, we synthesized various arp analogues and investigated their biological activity for anti-viral state and innate immunity. based on the sar analysis, we verified the importance of the carbonyl linker and the benzyl moiety with a trimethoxy attachment on adjacent aryl ring in the arp pharmacophore. as a result, compound i was identified as a potent type i ifn inducer. further investigation about cytokine secretion and ifn-mediated isg expression by i indicated effective stimulation of type i ifn pathway. all these results suggest a beneficial therapeutic potential of i for anti-viral agent. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. type i interferons: crucial participants in disease amplification in autoimmunity cross-priming of cd + t cells stimulated by virus-induced type i interferon cxcl /ip- in infectious diseases pathogenesis and potential therapeutic implications type i inteferon gene induction by the interferon regulatory factor family of transcription factors ifn-induced tpr protein ifit potentiates antiviral signaling by bridging mavs and tbk interferon-inducible antiviral effectors interferon-stimulated genes and their antiviral effector functions a novel pathway of autocrine macrophage activation how type i interferons work in multiple sclerosis and other diseases: some unexpected mechanisms interferon beta a-induced severe autoimmune hepatitis in patients with multiple sclerosis: report of two cases and review of the literature recombinant interferon beta and gamma in the treatment of adult t-cell leukemia treatment of adult t-cell leukemia-lymphoma with a combination of interferon alfa and zidovudine antitumour actions of interferons: implications for cancer therapy impact of early elevation of serum bilirubin during treatment with pegylated interferon and ribavirin in patients with chronic hepatitis c mechanisms of hyperbilirubinemia during peginterferon lambda- a therapy for chronic hepatitis c infection: a retrospective investigation tilorone, a broad-spectrum antiviral for emerging viruses the silent and selective -ht( a) antagonist, way , produces via an indirect mechanism, a -ht( a) receptor-mediated behaviour in mice during the day but not at night synthesis and biological evaluation of arylpiperazine derivatives as potential anti-prostate cancer agents synthesis and antimicrobial activity of n-alkyl and n-arylpiperazine derivatives structure-activity relationship of new antimalarial -aryl- -susbtituted propanol derivatives: synthesis, preliminary toxicity profiling, parasite life cycle stage studies, target exploration, and targeted delivery dopamine d receptor antagonists as potential therapeutics for the treatment of neurological diseases discovery of cariprazine (rgh- ): a novel antipsychotic acting on dopamine d /d receptors interferon-induced nuclear factors that bind a shared promoter element correlate with positive and negative transcriptional control efficacy of tilorone dihydrochloride against ebola virus infection this study was financially supported by korea institute of science and technology (kist) institutional supplementary data to this article including detailed experimental procedures can be found online key: cord- -gs c fy authors: schreiber, gideon title: the role of type i interferons in the pathogenesis and treatment of covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: gs c fy type i interferons (ifn-i) were first discovered over years ago in a classical experiment by isaacs and lindenman, who showed that ifn-is possess antiviral activity. later, it became one of the first approved protein drugs using heterologous protein expression systems, which allowed its large-scale production. it has been approved, and widely used in a pleiotropy of diseases, including multiple-sclerosis, hepatitis b and c, and some forms of cancer. preliminary clinical data has supported its effectiveness against potential pandemic pathogens such as ebola and sars. still, more efficient and specific drugs have taken its place in treating such diseases. the covid- global pandemic has again lifted the status of ifn-is to become one of the more promising drug candidates, with initial clinical trials showing promising results in reducing the severity and duration of the disease. although sars-cov- inhibits the production of ifnβ and thus obstructs the innate immune response to this virus, it is sensitive to the antiviral activity of externally administrated ifn-is. in this review i discuss the diverse modes of biological actions of ifn-is and how these are related to biophysical parameters of ifn-i–receptor interaction and cell-type specificity in light of the large variety of binding affinities of the different ifn-i subtypes towards the common interferon receptor. furthermore, i discuss how these may guide the optimized use ifn-is in combatting covid- . type i interferons (ifn-i) are a family of cytokines that bind the type i interferon receptor, constituted of two transmembrane subunits, ifnar and ifnar ( figure ). the two receptors are constituted of an extracellular domain, which binds ifn-i, a transmembrane helix and an unstructured intracellular domain (icd) that binds jaks and stats ( , ) . jak is associated with ifnar and tyk with ifnar . stat and stat (and maybe also other stats) were found to be constitutively bound to the icd of ifnar ( ) ( ) ( ) . binding results in close proximity of the intracellularly associated jaks, jak and tyk , resulting in their activation through cross phosphorylation ( figure ) ( , ) . this also results in receptor phosphorylation, which role is still under debate ( , ( ) ( ) ( ) . the phosphorylated stats dissociate from the receptor and form homo and hetero dimers, which are transported to the nucleus, where they serve as transcription factors for a large number of genes. the most prominent effects are associated with stat /stat heterodimerization, which together with irf form the interferon-stimulated gene factor (isgf ), which bind a distinct group of target genes harboring the interferonstimulated response elements (isre). in addition to this, ifn-i drives stat /stat and stat /stat homodimerization, the formation of a stat /irf binary complex and more ( , ( ) ( ) ( ) (figure ). this leads to the transcription activation or suppression of over , genes, which drive a wide range of innate and adaptive immune functions. these, in turn respond against various pathogens, act as important regulators in tumor immunity and have a role in pathophysiology and autoimmune diseases ( , ( ) ( ) ( ) ( ) ( ) ( ) . stat knockout cells still activate a stat /stat response mediated by irf , while stat knockout cells activate a stat /irf -induced response ( ) . surprisingly, no change in the gene induction relative to wildtype cells was observed in stat knockout hela cells, despite the strong ifn-i-induced phosphorylation of stat . however, as ifn-i responses are cell-type specific, a stat /stat induced response may still be found in other cells than hela. due to this wide range of physiological responses, ifn-i has provided therapeutic benefits for multiple diseases, including multiple sclerosis, some cancers and viral diseases (hepatitis b and c) ( ) ( ) ( ) . due to the efficient activation of antiviral activities by ifn-is, most viruses have contemplated mechanisms to avoid its actions ( ) ( ) ( ) . for example, the ebola virus, which outbreak in central africa killed tens of thousands of people ( , ) , avoids ifn-i activity by producing the vp protein that binds the karyopherin alpha nuclear transporter. thereby, it inhibits the nuclear transport of phosphorylated stat , rendering cells refractory to ifn-is. another example of viral mechanisms that evolved to eliminate ifn-i functions in inducing innate immunity is given by the sars corona virus, where both the production of ifnb and the ifn-i induced signaling are attenuated. recently, a more infective version of sars has emerged, sars-cov- (which causes the covid- disease). covid- cases have been first reported by the end of in china, and rapidly became a world-wide epidemic with unprecedented consequences ( , ) . sars-cov- seems to have originated from horseshoe bats. similar virus strains that circulate in bats in hubei province in china may in the future cause further new zoonotic outbreaks ( ) . sars-cov- has % homology to the sars-cov virus that also spread from china in ( ) . sars-cov- proved to be much more infectious compared to the original sars virus, resulting in a global epidemic. as ifn-i drives strong antiviral activities, the mechanisms sars-cov and sars-cov- combat ifn-i activities has been a matter of intense research, with at least proteins being identified to counteract ifn-i functions in the sars-cov virus ( ) . in addition, ifn-is were implicated in contributing to the severity of the cytokine storm, which is a major complication of sars-cov and sars-cov- and can lead to respiratory distress syndrome (ards) and death ( , ) . in this review i will describe our current knowledge on the involvement of ifn-is in the development of the covid- disease, and how this relates to the different activities associated with type i interferons. type i interferon receptors are found on all cell types, and are a major component of the innate immune system. human type i interferons include similar ifnas with % homology between them and single ifnw, k, ϵ and b, with lower homology ( - %). all of them bind the receptor complex, composed of ifnar and ifnar at the same proximal location ( , , ) . despite structural similarities among the ternary ifn-i-ifnar -ifnar complexes, ifn-is drive a range of different activities, dependent on the cell type and the interferon subtype ( ) . this apparent paradox has major implications for understanding the role of ifn-i in health and disease and its varied applications as a drug against a pleiotropy of diseases. ifn-i signaling is initiated by binding of ifn-i to its receptor. it has been suggested that cytokine receptors are pre-associated, with ligand binding activating signaling through the induction of conformational changes ( ) . however, more recent singlemolecule receptor tracking on life cells has clearly shown that for many of the cytokines, its role is to bring the receptors into close proximity, which drives signaling ( ) . this seems to be the case also for ifn-i induction, as shown both using single receptor tracking and mutational analysis ( figure ) ( , ) . while structurally, the ternary ligand-receptor complex seems to be the same for all ifn-is, the binding affinity differs by many orders of magnitude. the tightest binding ifn-i is ifnb, which binds ifnar with nm affinity and ifnar with subnanomolar affinity. the different ifna subtypes bind ifnar with . to µm affinity and ifnar with to nm affinity, with ifna being the weakest binding ifna ( , ) . even weaker binding was measured for ifnϵ, with~ -fold reduced affinity relative to ifna proteins ( ) . interestingly, ifnϵ is constitutively expressed by the reproductive tract epithelium and is regulated by hormones during the estrus cycle, reproduction, menopause and by exogenous hormones. thus, its mode of action is different from other ifn-is ( ) . these large differences in binding affinity between ifn-i subtypes were suggested to result in major differences in biological activity. to obtain a better insight into the molecular mechanisms of their actions, ifna was engineered to cover the whole range of binding affinities of natural ifn-is to both the high affinity (ifnar ) and low affinity (ifnar ) receptor chains ( ) . these studies have shown that indeed, the binding affinity to both receptors is a major determinant of ifn-i activity ( ) . using both natural and engineered ifn-is has shown that even weak binding ifn-is activate the cellular antiviral program at very low (pm) concentrations ( ) . moreover, the antiviral program was activated in all cell-lines tested. despite the -fold higher affinity of ifnb over ifna towards binding ifnar receptors, its potency to elicit an antiviral response is similar. for example, in wish cells (originally thought to be of amniotic origin, but later found to be a hela (cervix cancer) contaminant) the ec for antiviral activity of ifna is . pm, while the ec for ifnb is . pm ( ) . wish cells have been extensively used to characterize ifn-i activity, including for definition of ifn-i unit activity. an upper limit for antiviral potency was further verified by engineering an ifna variant, yns-a -tail, with fold tighter binding to ifnar and -fold tighter binding to ifnar in comparison to ifna (thereby surpassing the receptor binding affinity of natural ifnb). still, the ec for antiviral activity is only -fold lower in comparison to ifna ( , ) . conversely to antiviral activity, ifnb is much more potent in activating the antiproliferative program relative to ifna , a result that was also verified using the ifna variant, yns-a tail ( ) . the ec for antiproliferative activity on wish cells is nm for ifna , pm for ifnb and pm for yns-a -tail. a similar increase in antiproliferative potency was observed also for ovcar and hela cells. interestingly, while antiviral activity was observed in all cell lines tested, some cell lines were not susceptible to ifn-i induced antiproliferative activity (for example t d and k ), independent on the concentration and subtype of ifn-i ( ) . to better understand the molecular basis for this finding, ifn-i induced gene expression was monitored using various ifn-i subtypes or engineered mutants on the background of different cell-lines. these experiments showed that low concentrations of weaker binding interferons activate the expression of mostly antiviral genes. higher concentrations of interferons activate also other genes, many of them related to immune-modulation ( ) . examples for such genes are chemokines such as cxcl and , which are involved in chemotaxis of t cells and natural killer cells, induction of apoptosis, regulation of cell growth and more. we gave the term of "robust" for the common ifn-i induced program (including its antiviral activity) and "tunable" for the other programs induced by ifn-is, which include between others antiproliferative and immunomodulatory activities ( ) . further investigations into these two programs has shown that cells with low receptor numbers activate only the robust program, and that not all cell types execute the tunable program, conversely to the robust program that is common to all cells ( ) . tighter binding ifn-is at higher concentrations are essential for the activation of the tunable program. genes upregulated by the robust program are mostly classical antiviral genes, such as mx and mx , oas and , pkr, ifit , and , isg , and many more. figure a according to string and go analysis, the commonly upregulated genes have a strong antiviral signature. the top go terms (fdr < − ) are response to type i interferon, innate immune response, response to virus, defense response and immune system process. it is interesting to note that antiviral genes constitute most of the upregulated genes common to all cell lines. antiviral genes are also the majority of upregulated genes in k and t d cells. conversely, ovcar and hela cells have many unique upregulated genes, many of them related to immunomodulatory functions, cell cycle, apoptosys and more. ifn-i. the diagram shows that genes are commonly upregulated by all cell-lines. figure b shows string protein interaction analysis of these common genes. clearly, these form a tightly interacting mesh of gene products. gene ontology analysis shows these genes to have an extremely high signature for antiviral activity and ifn-i activation. promoter analysis of common isgs has shown them to be driven by the classical isre promoter sequence ( ) . conversely, for tunable genes no clear promoter sequence was identified. the exact mechanism of how tunable genes are upregulated by ifn-i is thus not yet fully understood. from an immunological point of view, ifn-is have three major functions: . to activate an antiviral state in infected and neighboring cells that limits spread of infection. . modulate innate immune responses, including antigen presentation and natural killer cell functions while restraining pro-inflammatory pathways. . activating the adaptive immune system for the development of high-affinity antigen-specific t and b cell responses ( ) . as ifn-is are highly active molecules, their expression and signaling potency is highly regulated. opposing augmenting and suppressive signals are induced by host factors. suppressive pathways include ifn-i activation of usp , an isg that suppresses signal transduction by reducing the ability of ifn-is to form an active receptor complex ( , ) . a second inhibitory mechanism is the induction of socs and socs , which kir domain block the substrate binding groove on jak, thereby inhibiting stat phosphorylation ( ) . a third mechanism is by rapid endocytosis and subsequent lysosomal degradation of activated ifnar complexes ( , ) resulting in reduced receptor numbers ( figure ). it has been demonstrated that a mutant in ifnar (s a and s a in human and mouse respectively), which fails in ifnar endocytosis through blocking its ubiquitination result in high incidence of inflammation ( , ) . at the transcriptional level, ifn-i response can also be regulated by mir- , which is highly induced by pattern recognition receptors and inflammatory signaling, and suppresses the expression of over genes. between them genes related to the interferon pathway. it was shown that mir- -deficient cd (+) t cells had enhanced type i interferon signaling and were more susceptible to interferon's antiproliferative effect ( ) . high basal ifn-i levels are implicated in various immunological diseases, such as systemic lupus erythematosus and more ( , , ) . however, ifn-i has also anti-inflammatory effects, as best demonstrated by their ability to suppress multiple-sclerosis ( ) . it is important to note that beneficial results in treating multiplesclerosis were observed only for ifnb but not for ifna treatment ( ) . to see whether this relates to the higher receptor binding affinity of ifnb, we established a transgenic mouse harboring the human interferon-receptors extracellular domains fussed to the mouse intracellular domains and compared the severity of eae in a mice model upon treatment with ifna , ifnb and the high-affinity engineered ifn-yns-a -tail. we found that the ifn-yns-a -tail had the strongest suppressive effect on the development of eae ( ) . the effect was further enhanced by pasylation of ifn-yns-a -tail, which extends it plasma half-life by -fold. interestingly, we found a tight relation between the increased levels of expression of pd-l in mice and the severity of the disease. these data show that tight binding ifn-is induce preferential anti-inflammatory responses, at least in this ms mouse model. another example for the immunosuppressive activity of ifn-i was shown for lcmv infection, which induces consistent ifn-i production including the immunosuppressive factors il- and pd-l ( ) . in addition to the above, interferons contribute to inflammasome activation through several different mechanisms, including caspase- expression and the ifn-i inducible gbp protein expression, which was reported to have an important role in caspase- activation and pyroptotic cell death ( ) . ifn-is have important roles in protecting the lung from spread of respiratory viruses. in addition to their direct role, ifn-is have also been found to be critical in initiating lung inflammatory responses, by inducing recruitment and activation of immune responses, which have to be kept under control. ifn-is have been shown to result in the production of chemokines such as ccl and cxcl , which play important roles in the recruitment of monocytes/macrophages, t cells, nk cells, and dcs, therefore directly influencing inflammation in the lung ( ) . this varied effect of type i ifns on t cells is partly dependent on the different stats induced by type i ifns. in the absence of ifn-is, the detection of accumulating viral rna and downstream processing of the signal is compromised, leading to viral spread and also to reduced inflammation in the lung. interestingly, there is an age-related reduction of ifn-i production and isg induction after viral infection, which may be related to the higher susceptibility of elderly population to lung infections ( ). viruses have developed many strategies to interfere with the synthesis of ifn-is or the ifn-i induced responses. one of them, is the stimulation of turnover of the interferon receptors. among other viruses implicated in accelerating the turnover of ifnar are ebv, herpes simplex virus, hepatitis c and b viruses, vesicular stomatitis virus and the sars coronavirus ( , ) . sars-cov has been shown to suppress ifn-i responses in the host through multiple mechanisms. a subdued ifn-i response diminishes antigen presentation and reduces the antiviral adaptive th- immune response. ifn-is communicate between cells against pathogens and have a critical role in the immune system, such as activating natural killer (nk) cells and macrophages. in addition, ifn-is cause flu-like symptoms, which are observed in various diseases. these symptoms may have a role in alerting a person of his/her sickness, in order to limit disease-spread to other individuals. in sars-cov and mers-cov, the induction of ifnb is suppressed altogether. this dampening approach is highly associated with the disease severity and increased mortality ( ) . in the lethal cases of sars-cov or mers-cov infections, the increased influx of inflammatory cells is always observed. in a mouse model of sars-cov infection, imbalance in ifn-i and inflammatory cells were shown as the main cause of fatal pneumonia ( ) . in addition to these, sars-cov implements strategies to evade the immune response by antagonizing ifn-i induced signaling pathways. the orf protein blocks the expression of stat activated genes ( ) . sars-cov and mers-cov encode papainlike protease (plp) that is able to impede the immune response function ( ) . in addition, sars-cov interacts with isg and antagonizes the ifn-i-mediated antiviral response ( ) . the mers-cov orf b antagonizes the antiviral ifnb production by inhibiting irf and irf ( ) . also sars-cov inhibits activation of irf / , slowing ifnb production upon infection ( ) . while irf is expressed in many different cell types, plasmacytoid dendritic cells are the only cells constitutively expressing irf ( ) . ifn-i treatment has been studied against mers-cov and sars-cov in numerous experiments, both in vitro and in vivo, and in combination or not with lopinavir/ritonavir, ribavirin, remdesivir, corticosteroids, or ifng. while ifna and b were efficient in vitro and in certain animal models, their success in humans was less convincing [for review see, ( , ) ]. it should be noted that reduction in ards mortality (not related to sars) was also found to be at best marginal upon treatment with ifn-i ( ) . still, one has to consider that mice studies have shown the timing of ifn-i administration to be critical, with positive effects being observed if ifn-i was administered shortly after infection. conversely, ifn-i failed to inhibit viral replication and resulted in unwanted side-effects when administered later in the disease circle ( , ) . these include elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific t cell responses. it is interesting to note that a knockout of the ifn-i receptor in mice resulted in its protection from lethal sars-cov infection. these findings have major implications on how to treat humans against sars and mers, and could have affected the outcome of the clinical studies. the covid- pandemic started in december in wuhan, china. by the summer of , thirty million cases were reported worldwide, with over , fatalities. as covid- is closely related to the sars-cov virus, the interest in the effect of interferons on its disease progression, and its potential as a drug was immediate. disease progression of covid- goes through a number of stages. the initial stage, which last from to days (usually - days) from infection is asymptomatic. a certain proportion of patients never produce any symptoms (the percentage of those is under debate, but a range of - % is most likely). of those who develop symptoms, they are mostly mild ( % of those who develop symptoms). from the remaining %, about half will develop severe symptoms, which require hospitalization in intensive care units. the mortality rate, from those developing symptoms is % to %. the numbers given above are average, and change dramatically with age. at young age most of the infected people will be asymptomatic, while over the age of about % will have symptoms. moreover, as the age progresses, symptom severity increases ( ) . the major complication of severe infection is pneumonia, which can develop into acute respiratory distress syndrome (ards). in addition, covid- has been linked to cardiovascular sequelae, such as myocardial injury, arrhythmias, cardiomyopathy and heart failure, acute kidney injury, neurological complications, and acute ischemic stroke ( ) . developing severe symptoms and death is strongly related to background conditions. the strongest relation is to age, with the risk to people under being very small, while the risk peaks for people over the age of . in addition, chronic kidney disease, chronic obstructive pulmonary disease, immunocompromised state, obesity, heart conditions and type diabetes are linked to higher incidents of sever disease ( ) . cov- is presumed to infect people mostly though inhalation of viral particles, which can be airborne, in droplets or otherwise through infection through touching infected surfaces. the spike protein on the cov- surface binds to the human ace protein, which serves as its receptor ( figure ) . the homotrimeric spike glycoprotein is made from s and s subunits. its binding and subsequent cleavage by the host protease tmprss results in the fusion between cell and viral membranes and cell entry ( ) . blocking the ace receptors by specific antibodies voids viral entry ( ) ( ) ( ) . interestingly, cov- receptor-binding domain (rbd) exhibited significantly higher binding affinity to ace than the sars-cov rbd, which was speculated to relate to the higher infectivity of covid- in relation to sars. after membrane fusion, the virus enters through the endosomal pathway and the viral rna is released into the host cell. the viral rna is then translated into viral polyproteins, which are cleaved into small products by viral proteases (papain-like protease [plpro] and the main protease [mpro]). viral proteins and genome rna are subsequently assembled into virions in the er and golgi and then transported and released out of the cell. the exact mechanism of viral self-assembly is still under intense investigation ( , ) . investigating ace and the viral entry-associated protease tmprss expression levels in lung tissue and trachea has shown that tmprss is expressed in both tissues, while ace is predominantly expressed in a transient secretory cell type ( ) . in addition, ace and tmprss co-expressing cells were found within lung type ii alveolar cells (which also release pulmonary surfactant), enterocytes, and nasal goblet secretory cells ( ) . using single-cell rna-sequencing, ace and tmprss were found to be highly expressed also in the nasal goblet and ciliated cells ( ) . the inhaled virus likely binds to epithelial cells in the nasal cavity and starts replicating. the virus propagates and migrates down the respiratory tract along the conducting airways, and a more robust innate immune response is triggered. for about % of the infected patients, the disease will be mild and mostly restricted to the upper and conducting airways. unfortunately, about % of the infected patients will progress to more severe disease and will develop pulmonary infiltrates and some of them will develop ards ( ). like many other viruses, also sars-cov and sars-cov- have evolved mechanisms to reduce their exposure to ifn-i. in both viruses, mechanisms to block the production of ifnb were identified. while the antiviral potency of ifn-is on sars-cov is moderate, sars-cov- seems to be highly sensitive to ifn-i. this is evident by the significant reduction in viral replication observed following ifn-i treatment at both and h postinfection ( ) . in sars-cov- -infected cells, ifn-i results in elevated stat levels and isg production (in contrast to sars-cov infected cells). this raises the question of why the innate immune system fails to combat sars-cov- ? the apparent answer to this is in the inhibition of ifnb production by proteins of the sars-cov- virus. within cells, rna viruses are sensed by the innate immune system through three major classes of pattern recognition receptors (prrs): toll-like receptors (i.e. tlr- , - , - ), rig-i-like receptors (rlrs), and nod-like receptors (nlrs) ( ) . to identify the molecular mechanisms that block ifnb production through activation of irf / , several research groups transfected cells individually with all the cov- viral genes and with either rig i, mda , or mavs ( , ) . among the cov- proteins transfected to cells, they identified nsp and orf as competent suppressors of ifnb. yuen et al. also identified nsp and , while lei et al. identified nsp , nsp and the m protein as potent inhibitors of the mavs pathway, leading to inhibition of ifnb production ( figure ) . orf was between the strongest suppressors of ifnb production in both studies. orf was also the only sars-cov- gene suppressing the activity of an interferon-stimulated response element (isre) promoter in both studies. lei et al. also identified nsp and nsp as potent inhibitors of the induction of an isre promotor. in another study, li et al. showed that the viral orf , orf , and nucleocapsid proteins were strong inhibitors of ifnb production, and through this of the ifn-i innate immune response ( ) . in this study, orf and orf also inhibited induction of transcription an isre promotor driving a luciferase as reporter, following ifnb treatment. in addition to the above-mentioned sars-cov- genes, orf b was implicated by konno et al. as being a potent antagonist towards ifn-i production ( ) . an interesting civet in this study is the finding that a natural variant, with a longer orf b reading frame increased disease severity in two patients. in light of the much higher than expected coding capacity of the sars-cov- genome, where many more proteins than genes were identified ( ), we may find even more proteins and peptides being involved in eliminating the innate immune response, including through inhibition of ifn-i activities. another mechanism by which sars-cov- inhibit antiviral functions of the cell is thought the activity of the papain-like protease (plpro), which is essential for viral polyprotein processing. this gene was found to preferentially cleave the ubiquitin-like modifier interferon-stimulated gene (isg ), figure | sars-cov- has multiple effects on the immune system, including inhibition of ifnb production, which results in isgs not to be produced, cd + and cd + exhaustion and increased levels of pro-inflammatory proteins (tnfa, il , nf-kb). currently, the most promising drugs against covid- include ifn-is, antiinflammatory and antiviral drugs, protease inhibitors, antibodies, sars-cov -ace (receptor) binding inhibitors and more. which is an ifn-i induced gene with strong antiviral activity ( ) . this represents another layer of attenuation of ifn-i responses by sars-cov- and is similar to the mechanism previously identified for sars-cov ( ) . inhibition of ifnb production by cov- got further confirmation from measuring the levels of different cytokines in sars-cov- -infected patients. an integrated immune analysis, including immune cell analysis, whole-blood transcriptomics and cytokine quantification on covid- patients at to days after disease onset has shown an impaired ifn-i response that is a result of low ifn-i levels ( ) . this, in turn results in the low production of interferon-stimulated genes. conversely, high levels of il and tnfa were measured ( figure ) ( , ) . this is in contrast to what is seen in patients infected with highly pathogenic influenza viruses. the high production of pro-inflammatory cytokines and low production of ifn-is during sars-cov- infection suggests effective activation of nf-kb but not irf and irf ( ) . impaired ifn-i production during severe covid- may also lead to an imbalance in the pro-inflammatory versus pro-repair functions of airway macrophages. this was indeed seen in severely ill patients with covid- . other innate immune cells such as natural killer (nk) cells are also regulated by ifn-is during coronavirus infection. severe covid- is associated with exhaustion of cd + and cd + t cells ( ) , which may be a result of deficient ifn-i production, as ifn-is promote survival of t cells. an important issue to consider is that early production of ifn-is promote efficient t cell responses, while a delayed response may inhibit t cell proliferation or their exit from lymphoid organs and thus cause their functional exhaustion. indeed, t reg cell counts in covid- patients inversely correlate with disease severity ( , ) . interestingly, transcriptomic analysis of blood, lung, and airways of cov- -infected patients showed that while ifnb was indeed not highly expressed in either, a number of ifnas were highly upregulated in the lung and airways but not in blood ( ) . moreover, a clear ifn-i-induced gene expression profile was also detected for lung and airways, but not for blood (pbmcs). a similar finding of elevated ifna but not ifnb, during covid- infection was also found by wei et al. ( ) . in this study, the elevated ifn-i response was restricted to the stage in the disease were patients were in intensive care. in another study of patients, of whom did not produce ifn-i, those patients had higher viral load, required more aggressive medical intervention and their time of stay in the intensive care unit was longer that ifn-i producing patients ( ) . pdcs are the most rapid and abundant ifn-i producers. pdcs express tlr and tlr which are important in sensing viruses. the response of pdcs to viruses, particularly ifn-i production, is significantly impaired with ageing while secretion of all other pro-inflammatory cytokines was comparable to that of younger individuals ( ) . this may relate to the master regulator for ifn-i production, irf , which expression, phosphorylation and nuclear translocation decreases with age. in addition, local neutrophil-mediated inflammation is increased with age, while cytotoxicity of nk cells induced by type i ifn-is decreases in aged mice ( ) . in addition to age, other factors were also associated with reduced interferon responses. one of them is obesity, which is related to impaired ifna and ifnb responses, which may relate to inadequate response of obese people against viral infections ( ) . clinical trials of using ifn-i for treating corona viruses has a long history. already in , intranasal human ifna was given both before and after corona virus challenge, a strain that is causing common cold. the incidence of colds, the severity of symptoms and signs, and virus replication were all reduced in subjects receiving interferon as compared with those given placebo ( ) . for sars-cov, no randomized placebo-controlled trials have been performed to test the efficacy of ifn-is, however, comparing the clinical outcome of patients treated with ifn-a (infacon- ) with patients at different locations (not a control group) that were not treated, has suggested clinical benefits ( ) . these studies have raised the hope that ifn-i may be a potent drug also against covid- . this hope was further exuberated by the observation that externally administrated ifn-i induced a strong antiviral response, much more than that observed for sars-cov ( ) . while some of the sars-cov- proteins may affect isg production (most notably, orf and , see above), the main defense of sars-cov- against ifn-i innate immunity seems to be the prevention of ifnb production, which can be substituted by external administration. a major problem in assessing the efficiency of ifn-i against covid- is the lack of a good small animal model. while such models are now under development, they are still not perfect. in a recent study, mice were infected with a replication-deficient adenovirus containing human ace , and then infected with sars-cov- . these mice developed pneumonia, severe pulmonary pathology, and high-titer virus replication in lungs. to test the role of ifn-i in disease development, ifnar ko mice were infected with sars-cov- , showing higher viral titer over time. next, the mice were treated prior to infection with poly i:c, a strong inducer of ifn-i. this resulted in significantly diminished clinical disease and induced more rapid virus clearance ( ) . these results suggest that at least in a mice model, ifn-i may benefit disease recovery. due to the lack of a good animal model, and the availability of clinically approved ifn-i therapies, multiple clinical studies have been conducted administrating different subtypes of ifn-is using different routes of administration (for summary see table ). in a preventive study, nasal drops of ifna were given to , healthy medical staff in shiyan city hospital, hubei province for days to prevent sars-cov- infections. none of them developed serious side effects or was infected with cov- . while the study lacked a control group from the same city, overall in hubei province , medical staff were diagnosed with covid- ( ) . the study thus gives an indication that ifn-i may help in preventing infection for high risk medical personal. to test the benefit of subcutaneous injection of ifnb on early stage patients, an open clinical trial was conducted with patients, were assigned to the combination of lopinavir, ritonavir, ribavirin, and three doses of million international units of ifnb, while the control group of patients were given all the above except ifnb. the median number of days from symptom onset to start of study treatment was days. patients given also ifnb had a significantly shorter median time from the start of treatment to negative nasopharyngeal swab ( - days) in comparison to the control group ( - days) . moreover, ifnb reduced viral load and number of significantly ill patients relative to the control group, this without significant side-effects ( ) . in a medical study on the effects of treatment with ifna b in a cohort of confirmed covid- patients, some of the participants were given nebulized ifna b with or without arbidol while others were given only arbidol. treatment with ifna b with or without arbidol reduced the duration of detectable virus in the upper respiratory tract and reduced duration of elevated blood levels of il and c-reactive protein, which are inflammatory markers ( ) . while the study did not include a standard care group, and all patients recovered, it still provides an indication of ifn-i efficiency. the efficiency of ifnb a subcutaneously injected three times weekly for weeks for treatment of severe covid- was tested in a randomized clinical trial. all the patients (including the control group) received standard of care, including a range of other medicines (hydroxychloroquine, antibiotics, antiviral medicine and more). while the clinical response was not significantly different between the ifnb and the control groups, the -day overall mortality was significantly lower ( % vs. %) in the ifnb treated group ( ) . in a retrospective study of patients receiving ifna through inhalation, alone or in combination with other drugs at a relative early versus late stage of the infection, it was found that those receiving ifna at an early stage had a significantly lower rate of mortality. in contrast, late interferon therapy increased mortality and delayed recovery ( ) . the study suggests a relation between the time of ifn-i treatment and its efficiency. synairgen, a uk-based company, performed a controlled clinical trial of inhaled ifnb on patients and reported that compared with placebo the odds of developing severe disease during the treatment period decreased by % for hospitalized patients receiving sng , and that patients who received sng were more than twice as likely to recover from the virus during the treatment period versus those randomized to placebo. these are between the best results achieved so far in curing covid- . more clinical trials are now under way to evaluate ifn-i efficiency, but clearly the initial trials have been encouraging. moreover, due to the many years of experience in treating patients with ifn-is, the availability of the drug and its relatively modest cost make it an excellent candidate for mass treatment, once approved. however, critical questions remain concerning the use of ifn-is for covid- and other diseases ( figure ) . these questions relate to the optimal ifn-i subtype, drug-concentration, duration of treatment, mode of treatment and at which frequency should it be given. ample experience exists with subcutaneously administration, which is almost the only route ifn-is were used in the clinic. here, non-modified ifn-is are usually administrated two to three times weekly, while pegylated ifn-is are administrated once per week or less. injection of ifn-is will result in a systemic response, where ifn-is were shown to have antiviral functions as well as pro and anti-inflammatory functions. contrary, if given by inhalation, it will directly target the epithelial, and thus replace the ifnb, which production is inhibited by the virus. administration as nasal drops of ifna may be an excellent prophylactic method for people at high risk. ideally, these questions could be answered using animal models. the problem is that the disease in those is not equivalent to that observed in humans. due to the severity of the disease and the high proven safety of ifn-is, more clinical trials on humans, testing the many open questions related to its best mode of administration may be the fastest way forwards. the subtype to use is another important question. for multiple-sclerosis, ifnb has been used for many years ( ) , as it seems to provide a better anti-inflammatory response than ifnas. this may relate to its higher binding affinity to the interferon receptors, as has been demonstrated using a tight binding ifna mutant (yns-a tail), which binding affinity even surpasses that of ifnb [see above ( ) ]. for combating viral disease, most notable hepatitis c, ifna has been most commonly used ( ) , which was later replaced by pegylated (long plasma half-life) ifna ( ) . also, for cancers ifnas were mostly used ( ) . a good clinical explanation of why specific ifn-i subtypes were used is often missing, and decisions of which interferon to use may often relate to availability rather than to efficacy. moreover, due to the specie specificity of ifn-is, one cannot deduce from mouse experiments, which ifn-i to use in humans, as the data are not transferable ( , ) . the main difference between ifnas and ifnb is that the later has a stronger potency to induce antiproliferative and immunomodulatory responses (tunable), while ifna will provide a cleaner antiviral response (robust) without the additional responses associated with ifnb. the open question is which is desired for covid- treatment, where complications arise from the exuberated immune response. another, important parameter is the time of intervention by ifn-i, in early or late-stage covid- disease. in a recent study in mice it has been shown that prolonged ifn-i and iii signaling interferes with lung repair during influenza recovery, probably through p induction, which reduces epithelial proliferation and healing, while early treatment protects mice ( ) . in sars-cov- this is further complicated by the "cytokine-storm" symptoms of severe covid- , as indicated by elevated il and tnf-alpha levels. whether ifn-administration, particularly ifnb suppresses or exacerbate the sars-cov- cytokine storm needs to be urgently determined, as to provide a guide for future application of ifn-i therapy in sars-cov- treatment. the author confirms being the sole contributor of this work and has approved it for publication. structural linkage between ligand discrimination and receptor activation by type i interferons structural and dynamic determinants of type i interferon receptor assembly and their functional interpretation affinity of stat for the subunits of the interferon alpha receptor stat activation by type i interferons is dependent on specific 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recovery from viral infection the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © schreiber. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -qkjjxr p authors: li, liwei; wei, zuzhang; zhou, yanjun; gao, fei; jiang, yifeng; yu, lingxue; zheng, hao; tong, wu; yang, shen; zheng, haihong; shan, tongling; liu, fei; xia, tianqi; tong, guangzhi title: host mir- a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type i interferons date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: qkjjxr p micrornas (mirnas) play important roles in viral infections, especially by modulating the expression of cellular factors essential to viral replication or the host innate immune response to infection. to identify host mirnas important to controlling porcine reproductive and respiratory syndrome virus (prrsv) infection, we screened mirnas that were previously implicated in innate immunity or antiviral functions. over-expression of the mir- family strongly inhibited prrsv replication in vitro, as shown by virus titer assays, western blotting, and qrt-pcr assays. mir- a inhibited the replication of both type and type prrsv strains. mutating the seed region of mir- restored viral titers. luciferase reporters showed that mir- a does not target the prrsv genome directly but instead affects the expression of type i interferon and the ifn-stimulated genes mx and isg during prrsv infection. these results demonstrate the important role of mir- a in modulating prrsv infection and also support the possibility of using host mir- a to achieve rnai-mediated antiviral therapeutic strategies. micrornas (mirnas) are small (∼ nucleotides) non-coding rnas that bind to complementary sequences in the untranslated regions of target mrnas and contribute to gene regulation by reducing mrna translation or destabilizing transcripts (grassmann and jeang, ; skalsky and cullen, ) . the commonly accepted mechanism of mirna regulation is that the seed region ( ∼ nucleotides at the end) of an mirna is complementary to the or untranslated region ( -or -utr) of an mrna, leading to mrna degradation or translational inhibition (bartel, ; gottwein and cullen, ) . recent work has shown the importance of mirnas in regulating host-pathogen interactions and innate immunity (lodish et al., ; scaria et al., ; tenoever, ) . host mirnas can affect viral replication by binding directly to viral rna (lecellier et al., ) or by indirectly modulating host factors to provide a less permissive environment for virus replication (triboulet et al., ) . as mirnas are small molecules without antigenic properties, they are considered to have potential efficacy in antiviral therapeutic applications. for example, human mir- is an essential component of the biology of hepatitis c virus replication (jopling, ; jopling et al., ) and therapeutic blocking of mir- suppresses hepatitis c viremia in non-human primates (lanford et al., ) . porcine reproductive and respiratory syndrome virus (prrsv), a member of the arterivirus family, is the causative agent of porcine reproductive and respiratory syndrome [prrs; (chand et al., ) ]. based on their genetic and antigenic differences, prrsv strains are classified into two distinct genotypes, north american (type ) and european (type ), represented by the vr- (benfield et al., ) and lelystad virus (lv) (wensvoort et al., ) , respectively. these two genotypes on two different continents share only approximately % nucleotide sequence identity (forsberg, ; hanada et al., ) . many strategies for controlling prrsv transmission have been proposed but have generally shown little success, which has stimulated the search for new ways to control prrsv transmission. prrsv can escape from innate immunity and cause persistent infections (miller et al., ) . in mammalian cells, viral infection is a potent trigger of the interferon (ifn) response (sadler and williams, ; sen, ) . type i interferons can initiate the activation of jak/stat signaling to induce the expression of hundreds of ifn-stimulated genes (isgs), which play an important role in antiviral activities (albina et al., ; katze et al., ; overend et al., ) . however, in contrast to porcine respiratory coronavirus, prrsv is a poor ifn-inducer (buddaert et al., ) . many mirnas regulate ifn production pedersen et al., ) , maintain mrna stability (li et al., ) , and regulate signals downstream of ifn to modulate antiviral immunity (wang et al., ; yoshikawa et al., ) . while most mirnas characterized to date decrease the production of ifns (alam and o'neill, ) , a few mirnas that upregulate type i ifns have been reported. recent research has revealed that mir- may play a positive modulatory role in ifn production during prrsv infection (zhang et al., ) . given the breadth of mirna-mediated regulation of mammalian immunity (grassmann and jeang, ) , the role of host mirnas in prrsv infection is of significant interest. here, we found that mir- a is an anti-prrsv host factor. over-expression of mir- a inhibited infection by both of the major prrsv genotypes in a dosedependent manner. we found that mir- a does not target the prrsv genome directly, but rather affects the expression of type i interferon and the ifn-stimulated genes mx and isg during prrsv infection. our study reveals an example of a mirna that affects viral propagation and highlights a host factor that may be important for future control measures against prrs. marc- cells were grown in mem (invitrogen) with % fetal bovine serum (fbs, gibco-brl, gaithersburg, md, usa) and were maintained with % fbs at • c in a humidified % co atmosphere as described previously (yuan and wei, ) . baby hamster kidney (bhk- , atcc ccl ) cells were cultured in emem (atcc, manassas, va, usa) supplemented with % fbs. porcine alveolar macrophages (pams) were harvested from the lungs of -week-old prrsv-negative piglets as described previously (wensvoort et al., ) and maintained at • c in rpmi (gibco) supplemented with % fetal bovine serum (fbs). vaprrs (genbank accession no. gq ) (yuan and wei, ) and vshe (genbank accession no. gq ) (tian et al., ) were rescued from paprrs and pshe, respectively. vjx (at passage ) was isolated from the serum of a dying piglet displaying the clinical sings of porcine high fever disease (phfd) in . vjxm (genbank accession no. gq ) was obtained through serial passages of the highly pathogenic prrsv vjx strain (eu ) in marc- cells (wang et al., ) . the infectious cdna clone pjx was derived from vjx (lv et al., ) . high-titer virus stocks were obtained by infecting marc- cells at low multiplicities of infection (mois). infected cell supernatants were harvested after an % cytopathic effect (cpe) appeared, then the viruses were stored at − • c as stocks for further use. virus titer was determined by standard tcid assay using marc- cells. mirna mimics (table ) , which are double-stranded -omethyl-modified rna oligonucleotides with sequence complementarity to mature mirnas were synthesized by genepharma (shanghai, china). the sense sequences of the mir- mimics were: mir- a- -uucaaguaauccaggauaggcu- ; mir- b- -uucaaguaauucaggauaggu- ; corresponding non-seed-mutated mir- mimics ( - a, - u, and - a u) or seed-mutated mir- mimics ( a-m, b-m, c-m) are listed in table (underlined letters are mutated bases). the negative-control (nc) mimic sequence was -uucuccgaacgugucacgutt- . . . transfection of mirna mimic and viral multi-step growth kinetics mirna or nc mimics were transfected into pams or marc- cells at a concentration of nm (except for dosedependence experiments) using x-tremegene sirna transfection reagent (roche). twenty-four hours after transfection, cells were infected with prrsv. for analysis of prrsv growth, supernatants ( . ml/well) from cell cultures were collected at different time points post-infection and frozen at − • c. for virus quantification at each time point, a viral titer was measured in marc- cells by standard tcid assay using the method of reed and muench (reed and muench, ) . indirect immunofluorescence assays (ifa) were performed as described previously (tian et al., ) for the detection of nucleocapsid (n) protein in prrsv vjx infected marc- cells or pams pre-transfected with mir- family or mutant mimics. cells were fixed with cold methanol followed by blocking with % bovine serum albumin (bsa) and then incubated for h with a monoclonal antibody (sr a, rural technologies) that specifically recognizes type prrsv n proteins. after washing with phosphate-buffered saline (pbs), the cells were incubated for h with alexa fluor -labeled goat anti-mouse secondary antibody (invitrogen). cell nuclei were counterstained with g/ml of , -diamidino- phenylindole (dapi) for min. after a final pbs wash step, cells were visually analyzed using an olympus inverted fluorescence microscope. marc- cells were transfected with mir- a or nc mimics prior to prrsv infection. cells were washed twice with pbs at h post-infection and lysed with lysis buffer in the presence of mm n-ethylmaleimide (nem). after incubation for min on ice, cell lysates were centrifuged at , × g for min at • c and the supernatants were collected. protein samples were prepared in reducing buffer ( mm tris, ph . , % glycerol, % sds, . % [wt./vol.] bromophenol blue, mm dtt). samples then were heated at • c for min, resolved on % sds polyacrylamide gels, and transferred to hybond-pmembranes (amersham biosciences). membranes were blocked with % nonfat dry milk in tbst ( mm nacl, mm tris, ph . , . % tween ) for h at room temperature. membranes were incubated overnight at • c with primary antibody ( ag ) that specifically recognizes both type and type prrsv n proteins (kindly provided by ingenasa co., madrid, spain). after washing with tbst, blots were incubated with horseradish peroxidase (hrp)conjugated goat anti-mouse secondary antibody (santa cruz) for h at room temperature, washed again with tbst, and developed using supersignal west pico or femto chemiluminescent substrate according to the manufacturer's instructions (thermo fisher scientific). total rna and mirna were extracted with trizol (invitrogen) following the manufacturer's instructions. primescript tm st strand cdna synthesis kit (takara) was used for reverse transcription. quantitative rt-pcr (qpcr) analysis was performed using a step-one plus real-time pcr system (applied biosystems) and a sybr premix ex taq tm (takara). for detection of endogenous mirnas, a commercial mircute mirna first-strand cdna synthesis was purchased from tiangen biotech (beijing, china) and used for polyadenylation and reverse transcription. a commercial mircute mirna qpcr detection kit was purchased from tiangen biotech (beijing, china) for measuring mirna abundance. marc- cells infected with prrsv at a moi of . were collected at the indicated time points and total rna was extracted using trizol reagent (invitrogen). one g of this total rna was then used for reverse transcription with an rt-primer. the abundance of the mirna of interest in the resulting cdna was determined by qpcr using a universal reverse primer and a mirna-specific forward primer. the pcr procedure comprised pre-denaturation at • c for min, and cycles of • c for s, • c for s. the ubiquitously expressed u small nuclear rna (tiangen) was used for normalization purpose. all primers used for mirna qpcr were included in the commercial kit. the levels of orf rna, ifn-␣/␤, mx , isg mrna were quantified using a sybr premix ex taq tm (takara). relative expression levels were analyzed using the ct method (bookout et al., ) , and glyceraldehyde- -phosphate dehydrogenase (gapdh) mrna was used as an endogenous control. universal type i interferon was purchased from pbl interferonsource. all pcr experiments were performed in triplicate. other primers are listed in table . the pgl -control luciferase reporter vector (promega) was used as the cloning vector for luciferase assays to analyze potential mir- a target regions in the prrsv genome. twenty cdna fragments encompassing the prrsv genome were amplified by pcr from prrsv pjx and subcloned into the pgl -control vector downstream of the luciferase orf. the primers used are listed table sequence of oligonucleotide primers used in this study. sequence table . all cdna constructs were verified by dna sequencing. plasmids and mirna mimics were transfected into cells using lipofectamine (invitrogen) according to the manufacturer s protocol. for luciferase reporter assays, subconfluent bhk- cells cultured in -well plates were co-transfected with ng/well of the indicated reporter plasmid and ng/well of prl-cmv (as an internal control to normalize transfection efficiency, promega) along with the indicated amount of mir- a mimic. cells were lysed h later for determination of firefly luciferase activities using the luciferase assay system (promega). data are presented as the relative luciferase activities in mir- a mimic-transfected cells relative to nc mimic-transfected controls and are representative of three independent experiments. to screen potential mirnas for their ability to inhibit prrsv replication, mimics of mirnas that are well-conserved among different species and have been previously implicated in innate immunity and/or antiviral functions (banerjee et al., ; foley and o'neill, ; huang et al., ; yoo and liu, ; pauley and chan, ; schulte et al., ; selvamani et al., ) were synthesized (table ) . marc- cells were transfected with individual mirna mimics ( nm) and then infected with prrsv (vaprrs) at an moi of . . supernatants from infected cells were collected at and h post-infection to determine viral titers. among the mirnas tested, over-expression of the mir- a mimic strongly reduced prrsv titers (fig. a) . transfection of mir- a/ b inhibitors demonstrated the opposite effects (fig. b) , indicating that mir- has antiviral activity against prrsv replication and that mir- a is a more efficient suppressor than mir- b. all the other mirna mimics tested had no demonstrable impact on prrsv titers in marc- cells (fig. a) . furthermore, immunofluorescence assays using a fitc-conjugated monoclonal antibody against the prrsv n protein were consistent with viral titer data (fig. c) . to rule out the possibility that this antiviral effect of mir- a was specific to an individual prrsv strain, we analyzed the viral growth curves of two type prrsv strains (vjx , vjxm ) and a type prrsv strain (vshe) in marc- cells transfected with nc or mir- a mimics. over-expression of the mir- a mimic, but not the nc mimic, reduced prrsv replication in multiple prrsv strains of differing genotypes ( fig. a) . to corroborate our findings with mir- a further, marc- cells were transfected with increasing concentrations of mir- a mimic ( , , , nm) and then infected with vaprrs. both prrsv growth and the amount of orf mrna level were inhibited as a function of the dose of mir- a mimic ( fig. b and c) . consistent with this, transfecting the mir- a mimic also reduced the accumulation of the prrsv nucleocapsid (n) protein in a dose-dependent manner (fig. d) . to exclude the possibility that reduced prrsv replication was due to potential toxicity of the mir- a mimic, marc- cells were transfected with the mir- a mimic at different doses ( nm, nm, and nm). no appreciable effect of the mir- a mimic (at up to nm) on cellular viability and morphology was observed (data not shown). collectively, these data show that mir- a reduces prrsv replication in multiple prrsv genotypes in a dose-dependent manner. we next investigated the effect of other mir- mirna and mutants on prrsv replication. because mir- a is highly conserved between monkeys and pigs, we conducted the subsequent investigations in pams, which are the target cells of prrsv infection in vivo. as previously reported, mirna-mrna interactions may require seed-matched sites at nucleotides - (bartel, ). thus, we mutated mir- a mimic at non-seed nucleotides , , or and and mir- at seed nucleotides - ( a-m and bm). both mir- a and mir- b had anti-prrsv activity in pams. pams transfected with mir- a or mir- b mimics yielded significantly lower prrsv titers and orf gene expression compared with cells transfected with the nc mimic ( fig. a and b) . three mir- a mutants with non-seed mutations retained their ability to inhibit prrsv progeny production and gene expression ( fig. a and b). by contrast, seed mutations at nts - abrogated the ability of mir- family members to repress prrsv replication and gene expression (fig. a and b) , showing that the seed region was essential for inhibiting prrsv replication. to investigate further inhibition effect of mir- family and mutants on prrsv infection, we used an immunofluorescence assay to detect the prrsv n protein in pams. n protein expression in pams was suppressed by both mir- a and mir- b (fig. c , top) and by mir- non-seed mutants (fig. c, middle) , but was not affected by mir- seed mutants (fig. c, bottom) . we then analyzed the growth dynamics of hp-prrsv isolate vjx in pams transfected with mir- family or nc mimics. viral growth was suppressed about -fold in pams transfected with mir- a and about -fold in pams transfected with mir- b at h postinfection (fig. d) . notably, mir- a was more efficient suppressing viral growth than mir- b. these results indicated that mir- family members, especially mir- a, can inhibit vjx replication in pams. we then analyzed the kinetics of mir- expression in prrsv infected pam cells. the relative expression of mir- was upregulated as a function of prrsv infection time (fig. e) . targeting a specific viral sequence represents an efficient strategy by which mirnas can inhibit viral replication (jopling, ; lecellier et al., ) . in recent studies, mir- and mir- were confirmed to reduce viral gene expression and viral growth due to direct targeting of prrsv genomic rna (guo et al., ; zhang et al., ) . we determined whether mir- a specifically targets the prrsv genome to exert its antiviral effect by constructing a range of firefly luciferase reporter pgl -control based plasmids, which contained the cdna fragments representing the utr, nsp -nsp , orf -orf , and the utr of the prrsv genome statistical significance was analyzed using t-tests; *, p < . ; **, p < . ; ***, p < . . c. pams were transfected with the indicated mirna mimics and then infected with prrsv vjx (moi = . ) for h. cells were fixed and immunostained with the mouse monoclonal sr a antibody against the viral n protein and fitc-conjugated goat anti mouse igg. cellular nuclei were counterstained with dapi ( mg/ml). d. prrsv growth in pams transfected with mir- family mimics. pams were transfected with mir- family or nc mimics for h and then infected with prrsv vjx at an moi of . . culture supernatants were collected at the indicated times and titrated. e. time-course of mir- a/ b expression after prrsv infection. pam cells infected with vjx at a moi of . were collected at the indicated times and qrt-pcr analysis was performed to detect mir- a/ b expression. relative mir- a/b expression refers to the change in mir- a/b expression levels in prrsv-infected pams relative to mock pams. downstream of the firefly luciferase gene (fig. a ). if the prrsv cdna insert contains a mir- a target sequence, luciferase reporter expression is expected to be subjected to mir- a-regulation. mir- a or nc mimics were co-transfected with the individual reporter vectors into bhk- cells, along with an internal control vector prl-cmv. relative luciferase activities were quantified h post-transfection. the relative luciferase activities for different vectors containing various prrsv cdna fragments were not significantly different between cells transfected with mir- a mimic as compared with cells transfected with the nc mimic. thus, mir- a does not appear to target directly the prrsv genome. we found that over-expression of mir- a increased ifn-␣/␤ expression during vjx infection (moi = . ) at h in pams as compared with over-expression of nc (fig. a ). the ifn-stimulated genes mx and isg were also significantly upregulated (fig. b) . transfecting the mir- a mimic into pams in the absence of prrsv infection also enhanced type i ifn expression ( fig. a and b) . ifn-␣ and ifn-␤ were induced about . -fold in un-infected pams, and about . -and . -fold in prrsv infected pams, respectively. isg and mx were increased about . -and . -fold in un-infected pams, and about . -and . -fold in prrsv infected pams, respectively. in marc- cells, over-expression of mir- a up-regulated ifn-␣ and isg more strongly (fig. c ). ifn-␣ and isg were induced about . -and . -fold in un-infected marc- cells, and about . -and . -fold in prrsv infected marc- cells, respectively. transfection of mir- a inhibitors did not increase the expression of ifn-␣ or isg (fig. c) , confirming that the induction of the innate immune response is specifically mediated by mir- a. there is a growing body of evidence that cellular mirnas are important regulators of innate and adaptive immune responses and the intricate networks of host-pathogen interactions. herein, we identified mir- a as an inhibitor of prrsv replication that does not directly target the prrsv genome ( figs. and ) . overexpressed mir- a reduced prrsv replication and viral gene expression (fig. ) , in not only marc- cells, but also in pams (fig. ) , the main target cell for prrsv replication in vivo, confirming the biological relevance of this finding. mir- a belongs to a broadly conserved mirna family with perfectly identical sequences among vertebrates (griffiths-jones et al., ) . previous studies of mir- a have shown that this mirna is an important regulator of cell proliferation and differentiation that targets the smad transcription factor (ezh ), a suppressor of skeletal muscle cell differentiation (lu et al., ; luzi et al., ; sander et al., ; zhang et al., ) . by infecting pams with prrsv strain vr- , liu et al. generated small rna expression profiles at , and h post-infection to identify alterations in mirna expression associated with prrsv (yoo and liu, ) . overall, cellular mirnas were differentially expressed during at least one time point in prrsv-infected pams. however, in this study, mir- a was not mentioned (yoo and liu, ) . contrary to the previous study, we found that the expression of mir- a was up-regulated about -fold at h post-infection (fig. e) . one mechanism by which host mirnas regulate viral replication is the direct targeting of viral sequences (jopling, ; lecellier et al., ) . however, prrsv is a fast-evolving rna virus (prieto et al., ) and the relatively high mutation rate may limit the application of this kind of rnai-mediated antiviral therapeutic. cellular mirnas can also indirectly modulate cellular pathways that perturb the viral life cycle. in particular, the activation or enhancement of innate antiviral immune pathways has been suggested to be responsible for the antiviral effect of certain mirnas (lecellier et al., ; pedersen et al., ) . in the current study, the reduction of prrsv replication by mir- a did not appear to involve direct targeting of the prrsv genomic rna (fig. ) . moreover, this reduction occurred in both type and type prrsv strains ( fig. a) although these two genotypes share only approximately % nucleotide sequence identity. these data led us to hypothesize that mir- a might act on a cellular factor to reduce prrsv replication. the results presented here support a link between prrsv replication and the altered expression of mir- a in targeting host innate immune responses (fig. ) . type i interferons (ifns) are potent antiviral cytokines whose expression is triggered through recognition of viral components by pattern recognition receptors via a cascade of signaling molecules . pams are the main target cells for prrsv infection, and many gene expression studies have explored the immune response of pams to prrsv. such studies have shown that the expression levels of mx , usp, ifn-␤, il- , and tnf-␣ are affected by prrsv infection (albina et al., ; luo et al., ; van reeth et al., ) . overall, these analyses suggest that prrsv subverts host defenses by inhibiting the expression of pro-inflammatory cytokines (van reeth et al., ) and stimulating weak production of ifn-␣ . our results showed that over-expression of mir- a was capable of inducing expression of ifn-␣/␤ and the ifn-stimulated genes isg and mx , which might result in activation of the ifn response and further lead to the inhibition of virus infection. the restoration of innate immune responses to produce type i ifns in pams seems to be mirna specific, because another mirna (mir- b) had no such effect (data not shown). thus, it is possible that mir- a-induced type i ifn expression can overcome prrsv interference, contributing to viral clearance. this mechanism provides a higher genetic barrier to the emergence of viral escape mutants, so the identification and characterization of mir- a as an inhibitor of prrsv replication may open new ways to control fig. . mir- a increases type i ifn expression during prrsv infection. qrt-pcr analysis of (a. type i ifn ␣/␤ and b. mx /isg ) expression in pams transfected with nc or mir- a mimics or left untreated (mock) for h, and then infected with vjx for h at an moi of . , or left untreated. data were normalized to gapdh expression and are the mean ± standard deviation of three independent experiments. c. qrt-pcr analysis of ifn-␣ and isg expression in marc- cells transfected with nc, mir- a mimics or inhibitors, and then infected with vjx for h at an moi of . . data were normalized to ␤-actin expression. statistical significance was analyzed using t-tests; *, p < . ; **, p < . ; ***, p < . . future prrs outbreaks, for which effective control measures remain scant. our results showed that mir- a also can mediate the activation of ifns in the absence of prrsv infection (fig. ). the possible causes may relate to recent studies about a new function of mirnas, which is independent of their conventional role in post-transcriptional gene regulation fabbri et al., ; lehmann et al., ) . mir- , mir- a, and let- b have dual functions; on one hand, they bind to argonaute proteins and guide the silencing of target genes, and on the other hand, they act independently of argonaute proteins by interacting directly with tlrs. although there is no current evidence, mir- a may also serve as ligands for tlrs and activate ifns. future studies will be necessary to unravel the diverse functions of mir- a. overall, we demonstrated that over-expression of mir- a inhibits prrsv replication. although clearly defining the target and physiological role of mir- a remains an unfinished task, our study provided evidence that over-expression of mir- a enhances ifn-␣/␤ expression during prrsv infection, suggesting that mir- a could be used as a potential target for antiviral development. micrornas and the resolution phase of inflammation in macrophages immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) mir- a- p regulates differential activation of macrophages and inflammation micrornas: target recognition and regulatory functions characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc 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specialization of mir- and mir- in innate immune sensing chikungunya virus exploits mir- a to regulate nf-kappab pathway in human synovial fibroblasts viruses and interferons viruses, micrornas, and host interactions interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus rna viruses and the host microrna machinery chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype envelope proteins in the backbone of genotype suppression of microrna-silencing pathway by hiv- during virus replication differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity inducible microrna- feedback promotes type i ifn signaling in antiviral innate immunity by targeting suppressor of cytokine signaling development of a differentiable virus via a spontaneous deletion in the nsp region associated with cell adaptation of porcine reproductive and respiratory syndrome virus mystery swine disease in the netherlands: the isolation of lelystad virus characterization of the micrornaome in porcine reproductive and respiratory syndrome virus infected macrophages silencing of microrna- enhances interferon-alpha signaling in the liver through regulating socs promoter methylation construction of infectious cdna clones of prrsv: separation of coding regions for nonstructural and structural proteins pathologically decreased mir- a antagonizes apoptosis and facilitates carcinogenesis by targeting mtdh and ezh in breast cancer microrna- inhibits prrsv replication by directly targeting prrsv rna and possibly by upregulating type i interferons the study was supported by the grants from the national basic research program ( plan) (no. cb ), the national natural science foundation of china (no. , no. , no. ), and the natural science foundation of shanghai (no. jc ). key: cord- - x n cpt authors: lee, nelson; wong, chun k.; lam, wai y.; wong, ann; lim, wilina; lam, christopher w.k.; cockram, clive s.; sung, joseph j.y.; chan, paul k.s.; tang, julian w. title: chikungunya fever, hong kong date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: x n cpt nan nasal cultures in of the members of the families. in this niche, it was able to persist and cause a series of infections in a relatively large number of family members. even though the s. aureus isolated from active lesions were not available for testing, the recovery of identical pvl-positive organisms from nasal cultures strongly suggests the presence of a pathogenic clone that probably caused the recurrent infections in the affected family members. our investigation highlights the high transmissibility of this pvl-producing s. aureus clone, its high attack rate, and its virulence. the intervention in this outbreak might have prevented not only subsequent recurrences of cutaneous infections but also further spread of this clone and the manifestation of even more serious infections such as necrotizing pneumonia. increasing awareness among community-based healthcare providers of pvl-producing s. aureus infections is important to facilitate rapid and adequate response in similar clinical events in the future. ( , ) . no treatment or vaccine is available, and relatively little research has been conducted into its pathogenesis, compared with that of other arboviruses, such as dengue. recent reports have described a massive outbreak of chikungunya disease occurring on islands in the indian ocean, off the east coast of africa ( ). reemergence of chikungunya has also been reported from indonesia ( ). during march , a -year-old chinese man from hong kong visited mauritius where he was bitten by mosquitoes days before returning to hong kong. on the return trip, he experienced fever ( °c), severe finger joint and muscle pains, mild headache, and a skin rash, and he sought treatment at the prince of wales hospital (pwh) infectious diseases clinic on the second day of his illness. physical examination showed a generalized erythematous rash over the trunk and limbs and petechiae over the lower limbs. mild finger joint stiffness was observed, but no joint swelling. no lymphadenopathy or eschar was detected. level of c-reactive protein was elevated at . mg/l. results of screens for malaria and dengue were negative. results of other routine assessments were unremarkable. his symptoms subsided gradually within a week. serum specimens taken on days and were positive for chikungunya virus rna by in-house reverse transcription (rt)-pcr at the public health laboratory service (phls) (targeting the nonstructural protein- [nsp- ] gene) and pwh laboratory (targeting both nsp- and the envelope glycoprotein [e ] gene). an additional serum sample taken on day of illness, received by phls only, was also positive for chikungunya rna. both laboratories confirmed rt-pcr results by sequencing. at pwh, phylogenetic analysis was performed to determine the likely origin of the virus. in-house immunofluorescent slide serologic assays developed at phls found chikungunya immunoglobulin g (igg) titers < , , and in the serum samples taken on days , , and of illness, respectively, and detected chikungunya igm in the day serum. the acute cytokine immunologic response to this virus was also tested (online appendix available from http://www.cdc.gov/ ncidod/eid/vol no / - _ app.htm). sequencing and phylogenetic analysis was consistent with an imported infection, almost certainly originating from the current chikungunya outbreaks in the indian ocean. phylogenetic analyses of the nsp- and e regions, indicated that this virus is most closely related to previous african rather than south-east asian chikungunya viruses (see online appendix figures and , available from http://www.cdc.gov/ncidod/eid/ vol no / - _appg .htm and http://www.cdc.gov/ncidod/eid/ vol no / - _appg .htm). the persistence of viremia up to at least day of illness was unusual. standard texts state that viremia may be present during the first - days of illness, with neutralizing antibodies appearing by days - ( ) . the most striking finding from the cytokine analysis (table) is the high level of interferon-γ (ifn-γ)-inducible protein- (ip- /cxcl- ), up to and times the upper limit of the normal range at days and after disease onset, respectively. serum concentrations of interleukin- (il- ), monocyte chemoattractant protein (mcp) (mcp- ) and monokine induced by ifn-γ (mig/ cxcl ) are also elevated in both samples. notably, serum ifn-γ, tumor necrosis factor-α (tnfα), and il- β, , , and concentrations remain within normal limits in both samples, although the concentrations at local inflammatory sites (e.g., joints) are unknown. cxcl and mcp- /ccl concentrations decreased during clinical recovery. thus, the cytokine profile demonstrates that the levels of th chemokine cxcl was highly elevated and that the levels of chemokines il- /cxcl , ccl , and cxcl were moderately elevated. in contrast, ifn-γ and other inflammatory/th cytokines were not elevated during the illness. interpretation of the significance of these cytokine results is necessarily speculative. some comparison can be made with other viral infections. in severe acute respiratory syndrome-associated coronavirus (sars-cov) ( , ) and h n influenza ( ) infections, very high blood levels of cxcl and moderately high ccl , cxcl , and cxcl concentrations, or their enhanced expressions in vitro, have been reported. in dengue fever, which has similar clinical manifestations as chikungunya fever, only elevated cxcl , il- , il- , and tnfγ concentrations have been shown consistently ( , ) , although cxcl expression has not been studied. the function of cxcl is to act as a chemoattractant for th cells in the activation of cell-mediated immune response. its expression can be up-regulated by the th cytokine ifn-γ during acute inflammation. cxcl has been implicated in the pathogenesis of sars-cov and h n influenza infections, in which persistently high cxcl concentrations seem to correlate with disease severity and progression ( - ). ccl , cxcl , and cxcl , have also been found to have a pathogenic role in h n influenza, sars-cov, and dengue infections. notably, the level of antiviral cytokine ifn-γ was not elevated in our chikungunya case, though admittedly, this is only case. this finding may represent a way that the chikungunya virus evades host defenses and may provide a rationale for the use of ifn as a therapeutic option ( ) . such ifn therapy has been suggested and tried, experimentally, for sars-cov ( ) and dengue infections ( ). to the editor: in august , the laboratory response network (lrn) was established to better integrate and improve laboratory capacity for responding to public health threats ( ). however, while experts have focused on clinical indications for testing for agents of bioterrorism, laboratory methods for microbial identification, and needs for integrated communication networks ( ) ( ) ( ) , little attention has been given to how sentinel laboratories can effectively screen clinicians' requests for testing pathogens designated as global health threats. in times of crisis, clinicians often pressure laboratorians to perform testing for patients whose probability for disease is very low or for nonvalidated sample types. in , a few cases of anthrax triggered large numbers of nationwide requests to test nasal swabs for bacillus anthracis despite the absence of data to support this clinical practice outside epidemiologic investigations ( ) . similarly, a false-positive result for severe acute respiratory syndrome (sars) in from the national microbiology laboratory in canada created public alarm that sars was reemerging, when the virus was actually that of a common respiratory illness in a nursing home ( ) . the problem is further complicated when laboratories other than the lrn lack standardization, have greater access to nucleic acid amplification-based testing, and develop tests for global health threats outside a quality-regulated system. false-positive results caused by contamination or cross-reactivity with a microorganism of low virulence can disrupt a public health system, adversely affect patient care, and increase costs ( ) ( ) ( ) ; false-negative results may prompt clinicians to discontinue containment procedures and potentially risk transmitting a virulent microorganism. at our sentinel laboratory, we recognized these challenges and took steps to promote judicious use of testing for agents designated as global health threats. we report use of an algorithm to evaluate test requests for sars-associated coronavirus and highly pathogenic avian influenza h n ; however, the algorithm can be used to screen testing requests for any pathogen that has potential to threaten public health. during outbreaks of sars and h n , a laboratory protocol was established to notify the on-call laboratory professional when a sample was received for testing for of these pathogens (figure) . the protocol required the laboratorian to communicate directly with the clinician, using a script with questions based on criteria established by the centers for disease control and prevention, to determine the medical necessity for testing ( , ) . samples from patients not meeting these criteria were rejected. testing for sars used an inhouse real-time pcr assay with a standard laboratory protocol. samples accepted for h n testing were screened by a nonspecific hemagglutinin influenza pcr assay and, if results were positive, were to be forwarded to an lrn laboratory. positive results were to be reported only after confirmation by an lrn laboratory. laboratory professionals were specifically trained about the sensitivity, specificity, positive predictive value, and negative predictive value of test methods in relation to sample type, time between symptom onset and specimen collection, and disease prevalence. of samples ( sars and h n ) received for testing, ( %) samples were not tested because clinician responses failed to satisfy the screening criteria. the remaining ( %) samples met criteria for testing and all had negative results. in the genome microevolution of chikungunya viruses causing the indian ocean outbreak tracking the re-emergence of epidemic chikungunya virus in indonesia principles and practice of clinical virology plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome characterization of cytokine/ chemokine profiles of severe acute respiratory syndrome re-emergence of fatal human influenza a subtype h n disease systemic host inflammatory and coagulation response in the dengue virus primoinfection elevated plasma interleukin- levels in acute dengue correlate with disease severity in vitro inhibition of chikungunya and semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination randomized, placebo-controlled trial of nonpegylated and pegylated forms of recombinant human alpha interferon a for suppression of dengue virus viremia in rhesus monkeys key: cord- -v p rze authors: livonesi, márcia cristina; moro de sousa, ricardo luiz; badra, soraya jabur; figueiredo, luiz tadeu moraes title: in vitro and in vivo studies of the interferon-alpha action on distinct orthobunyavirus date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: v p rze oropouche, caraparu, guama, guaroa and tacaiuma viruses (orthobunyavirus genus) cause human febrile illnesses and/or encephalitis. to achieve a therapeutical agent to prevent and/or treat these diseases we evaluated the antiviral action of interferon-alpha (ifn-α) on these orthobunyaviruses. in vitro results showed that all the studied orthobunyaviruses are susceptible to antiviral action of ifn-α, but this susceptibility is limited and dependent on both concentration of drug and treatment period. in vivo results demonstrated that ifn-α present antiviral action on oropouche and guaroa viruses when used as a prophylactic treatment. moreover, a treatment initiated h after infection prevented the death of guaroa virus infected-mice. additionally, mortality of mice was related to the migration and replication of viruses in their brains. our results suggest that ifn-α could be potentially useful in the prevention of diseases caused by oropouche virus and in the prevention and/or treatment of diseases caused by guaroa virus. the oropouche (orov), caraparu (carv), guama (gmav), guaroa (grov) and tacaiuma (tcmv) viruses belong to distinct antigenic serogroups of the genus orthobunyavirus in the bunyaviridae family. these viruses are enveloped with trisegmented single-stranded rna genome of negative or ambisense polarity, replicate in the cytoplasm and bud into the golgi apparatus (elliot et al., ) or upon the plasma membrane (goldsmith et al., ) . orov (simbu serogroup) is transmitted mainly by the biting midge (culicoides paraensis) and has been associated with dengue-like acute febrile illness. orov fever has emerged over the past years as a serious public health problem in tropical and subtropical areas of central and south america, having caused at least reported outbreaks involving more * corresponding author. tel.: + ; fax: + . e-mail addresses: pink@rpm.fmrp.usp.br, livonesi@zipmail.com.br (m.c. livonesi). than half a million people (watts et al., ; pinheiro et al., ) . clinical features of orov fever include abrupt onset of fever, chills, severe headache, dizziness, myalgia, arthralgia, nausea, and vomiting. about half of the patients have a recurrence of symptoms within - days after they become afebrile. aseptic meningitis by orov has been reported during outbreaks. all ages and both sexes appear to be equally susceptible to infection (leduc and pinheiro, ) . similarly to orov, carv (group c serogroup), gmav (guama serogroup), grov (california encephalitis and bunyamwera serogroups) and tcmv (anopheles a serogroup) have also been associated with febrile illness in humans. these viruses are transmitted by mosquitoes and cause disease mainly in the south america countries (brinton et al., ; tavares-neto et al., ; jonkers et al., ; march and hetrick, ; travassos da rosa et al., ; iversson et al., ; iversson, ) . an antiviral therapy, if available, would be a very helpful intervention, reducing symptoms and disease period of orov fever as well as of those febrile illnesses caused by other orthobunyaviruses. however, until the moment, there is not treatment or vaccine for these viral diseases, and a recent study demonstrated that these viruses are resistant to antiviral action of the ribavirin, a broad-spectrum antiviral drug (livonesi et al., ) . the interferon (ifn) system is the first line of defense against viral infection in mammals. this system is able to block the spread of virus infection in the body, sometimes at the expense of accelerating the death of the infected cells. interferons are divided into two types, type i and type ii, both of which have antiviral activity (sen, ; galligan et al., ) . the type i interferons include the ifn-␣ and -␤ and they are secreted by virus-infected cells, and exhibit multiple biologic properties including antiproliferative, antiviral, and immunomodulatory effects (platanias et al., ; stark et al., ; goodbourn et al., ) . the type ii interferon present only one member, the ifn-␥, which is not virus-inducible, but it is secreted by activated t lymphocytes and nk cells and shows antiviral activity directly, through the induction of effector molecules (e.g. nitric oxide), and indirectly, through enhanced antigen presentation and the induction of apoptosis (boehm et al., ) . due to the antiviral properties of ifn-␣, it has been used clinically to treat chronic infections caused by hepatitis b and c viruses (davis et al., ; mchutchison et al., ) and in the treatment of infections caused by hpv (sen, ) . moreover, in vitro and/or in vivo experiments have demonstrated that ifn-␣ is able to inhibit the replication of many viruses as: sandfly fever sicilian (crance et al., ) , dengue (diamond et al., ) , severe acute respiratory syndrome-related coronavirus (sars cov) (tan et al., ; ströher et al., ; galligan et al., ) , vaccinia (liu et al., ) , and ebola (mahanty et al., ) . thus, in an effort to characterize antiviral agents that could attenuate infections caused by orov, carv, gmav, grov and tcmv, we tested the in vitro and in vivo actions of ifn-␣ on these viruses. oro (bean ), gma (bean ), gro (beh ), and tcm (bean ) viruses were kindly supplied by dr. pedro vasconcelos and dr. amélia travassos da rosa (evandro chagas institute, brazilian ministry of health, belém, brazil and university of texas medical branch, galveston, tx, usa). carv (span ) was kindly supplied by dr. terezinha lisieux coimbra (adolpho lutz institute, são paulo, brazil). viral stocks were obtained from the brains of intracerebrally infected newborn mice. brains were mixed with pbs (dilution : , w/v), macerated, and centrifuged at × g for min at • c. the supernatants were harvested and stored at − • c until use. african green monkey kidney (vero e ) cells were grown in minimum essential medium (mem, cultilab, brazil) supplemented with % inactivated, mycoplasma-free, fetal bovine serum (fbs, cultilab, brazil), % l-glutamine and . % sodium bicarbonate. interferon-alpha- a (ifn-␣- a) or roferon ® -a (hoffmann-la roche, usa) was used in the in vitro experiments. recombinant murine interferon-alphaa (ifn-␣a) (sigma-aldrich; st. louis, mo, usa) was prepared following the instructions of manufacturer and was used in the in vivo experiments. swiss newborn mice were obtained from the laboratory animal facility of the university of são paulo (ribeirão preto, brazil). the mice were maintained in microisolator cages in the animal housing facility of the center for research in virology (university of são paulo, ribeirão preto, brazil). the experiments were approved by the ethical committee on vertebrate animal experiments of the university of são paulo (no. / ). in vitro antiviral evaluation was done with a plaque assay (livonesi et al., ) . vero e cells were seeded in -well plates in mem with % fbs, for h at • c and % co . medium was removed, serial -fold dilutions of viral stocks diluted in mem with % fbs were added ( . ml/well) in quadruplicates, and the cells were incubated for h at • c. subsequently, the viral inoculum was removed, and . ml of a combination (v/v) of % low-melting-point agarose plus × mem ( % fbs) was added to each well; the plates were incubated at • c for days for orov and gmav, days for carv and grov, and days for tcmv. the plaques were visualized after staining with a naphtol blue black solution ( min) (morens et al., ) , preceded of removal of the agarose plug. the plaques were counted under an inverted microscope, and the virus titer was determined as log pfu ml − . ifn-␣- a was diluted in the medium and added to cells h before, or , , , h after viral infection. comparisons between the virus titers obtained from ifn-␣- a-treated and non-treated cell cultures were done and the results were plotted as percentage of inhibition on plaque formation (table ) . the ifn-␣- a was added to the cell cultures at the concentrations ≤ , iu ml − , because this concentration is not toxic to vero e cells as previously described (tan et al., ) . the most frequent adverse reactions caused by administration of ifn-␣ in humans include fatigue, myalgia, arthralgia, headache, fever, chills, anorexia, nausea, vomiting, diarrhea and abdominal pain, and usually occur in patients treated with high doses of ifn-␣ (the italian cooperative study group on cml, ; schmutz et al., ; márquez-peiró et al., ) . thus, the parameter chosen to evaluate the ifn-␣a toxicity in mice was a significant weight loss of ifn-␣a-treated mice in comparation with placebo-treated mice. the ifn-␣a doses used were , , , and , iu ml − ( l per mouse/day). the mice were treated intraperitoneally (i.p.) daily for days. the animal weights were determined daily and the concentration chosen of ifn-␣a was , iu ml − , because it was well tolerated by mice, which presented an increase of weight similar to the placebo-treated mice (fig. ) . furthermore, a dose of , iu ml − approximates to the highest levels of the recombinant ifn-␣ given to humans ( million units daily) (freireich et al., ) . three-day-old swiss mice were infected i.p. with orov ( ld ), carv ( ld ), gmav ( ld ), grov ( ld ), or tcmv ( ld ) in a volume of l per mouse. the mice were treated i.p. with ifn-␣a ( , iu ml − ) or placebo in a volume of l per mouse. the treatment was initiated h before, or or h after infection and maintained every day. the animals were monitored daily for mortality. suckling mice infected i.p. with orov, carv, gmav, grov and tcmv usually die after development of encephalitis, showing paralysis of forefoot, tremor and difficulty in eating, and presenting high viral titers in the brain some days before death . considering the brain as the principal target organ to viral replication, the virus titers obtained in this organ were used as parameter to demonstrate the efficacy of drug. thus, the brains of mice (two mice per group) were taken aseptically on days , , , , , and after infection. brains were mixed with pbs (dilution : , w/v), macerated and centrifuged at × g for min at • c. supernatants were harvested and stored at − • c before plaque assay on vero e cells. virus titers in brain were expressed as log pfu ml − . analysis of variance followed by the parametric tukey-kramer test was used in the in vitro experiments, while fisher's exact test was used in the in vivo experiments (instat soft-ware, graphpad, san diego, ca). a p value less than . was considered to indicate statistical significance. firstly, vero e cells were treated with ifn-␣- a ( , iu ml − ) at different periods of the cell infection by orov, carv, gmav, grov and tcmv. table shows that ifn-␣- a was able to inhibit the replication of all the studied orthobunyaviruses when treatment occurred either h before or h after infection (p < . ). moreover, ifn-␣- a presented a significant inhibitory activity on replication of gmav (p < . ) and tcmv (p < . ) when treatment was initiated h after infection. additionally, ifn-␣- a significantly inhibited the tcmv replication when cell treatment was initiated h after infection (p < . ). these results show that ifn-␣ present inhibitory effect on the orov, carv, gmav, grov, and tcmv replication, but this effect is dependent on timing of the treatment. next, we verified whether doses lower than , iu ml − would have inhibitory effect on the replication of distinct orthobunyavirus. thus, cells were treated h after viral infection with ifn-␣- a doses ≤ , iu ml − or medium. fig. shows that the concentration of , iu ml − is able to significantly inhibit carv, gmav, grov, and tcmv replication, but not of orov. furthermore, the concentration of iu ml − produces a significant inhibitory effect on carv, grov and tcmv replication (fig. ) , suggesting that antiviral effect of ifn-␣ on these orthobunyaviruses is also dependent on the concentration used. we firstly examined whether ifn-␣a treatment beginning day before viral infection would be effective on preventing lethal encephalitis caused by orov, carv, gmav, grov, and tcmv. intraperitoneal administration of the maximum nontoxic dose of ifn-␣a ( , iu ml − ) (fig. ) prevented the death of all mice infected by orov or grov (table ). these high survival rates were associated with the inhibition of viral migration and replication in brains of the mice (fig. ) . however, treatment of carv-, gmav-or tcmv-infected mice with ifn-␣a did not result in an increase in survival or prolongation of the mean time to death and did not prevent virus replication in the brain of animals (table and fig. , respectively) . next, the effect of administration of ifn-␣a initiated h after infection of animals by orov or grov was analysed. table shows that treatment with ifn-␣a was either unable to increase the survival time or inhibit the viral replication in the brain tissue of the mice infected by orov (fig. ) . on the other hand, ifn-␣a-treated-grov-infected mice had a survival rate of % (table ) , which was associated with inhibition of viral replication in the brain tissue of mice (fig. ) . treatment with ifn-␣a initiated h after mice infection by grov was unable to inhibit either the death of animals or the viral replication in the brain (table and fig. , respectively). fig. . ifn-␣-treated orov-or grov-infected mice did not show virus replication in the brain when treatment was initiated h before infection. groups of sixteen -day-old swiss mice were infected i.p. with orov, carv, gmav, grov, or tcmv and they were treated i.p. with ifn-␣ ( , iu ml − ) or placebo. mouse brains (two mice per group) were taken on days , , , , , and after infection. the virus titer in the brain was measured by plaque assay. the scale bars represent the mean of pfu ml − . similar results were obtained in a second experiment. h orov / ( %) / ( %) . ± . . ± . grov / / ( %) b . ± . > h grov / / ( %) . ± . . ± . a mean time to death. b p < . ; fisher's exact test. fig. . ifn-␣-treated grov-infected mice did not show virus replication in the brain when treatment was initiated h after infection. groups of sixteen -dayold swiss mice were infected i.p. with orov or grov and they were treated with ifn-␣ ( , iu ml − ) or placebo. mouse brains (two mice per group) were taken on days , , , , and after infection. the virus titer in the brain was measured by plaque assay. the scale bars represent the mean of pfu ml − . similar results were obtained in a second experiment. fig. . ifn-␣ treatment initiated h after infection did not inhibit replication of grov in the brain tissue of mice. groups of nine -day-old swiss mice were infected with grov and treated with ifn-␣ ( , iu ml − ) or placebo. the treatment was initiated h after infection. mouse brains (two mice per group) were taken on days , , , and after infection. the virus titer in the brain was measured by plaque assay. the scale bars represent the mean of pfu ml − . similar results were obtained in a second experiment. these results also show that the in vivo antiviral activity of ifn-␣ on orov and grov is dependent on timing of the treatment. in this study, we evaluated in vitro and in vivo antiviral activity of ifn-␣ on orov, carv, gmav, grov and tcmv. ifn-␣ had a significant antiviral effect in vitro on all studied orthobunyaviruses. the antiviral action observed was dependent on both administration timing ( table ) and concentration of the drug (fig. ) . in vivo experiments confirmed the antiviral activity of ifn-␣ on orov and grov, but not on carv, gmav and tcmv, being that this antiviral activity was dependent on the administration period of the drug (tables and ). the antiviral effect of ifn-␣ observed in vivo was related to its ability to inhibit viral replication in the brain tissue of infected mice . the antiviral action of exogenous ifn-␣ observed in vitro was probably due to its ability to induce an antiviral state on cells through the secretion of proteins that ultimately inhibit viral replication. for instance, the dsrna-dependent protein kinase (pkr) leads to phosphorylation of eif ␣ with a consequent blockade of translation of most cellular and viral mrnas; the , -oligoadenylate synthetases (oas), which are also activated by viral dsrna, produces , -oligoadenylates that in turn activate the rnase l, resulting in the degradation of viral and host rnas; and the mx proteins, gtpases of the dynamin family, whose mechanisms of action and functions await elucidation (stetson and medzhitov, ) . similarly, the antiviral effect observed in vivo after administration of exogenous ifn-␣ might have been either due to the above-mentioned cell-intrinsic mechanisms of this cytokine or the actions of type i ifns on the immune system. in fact, type i ifns are able to enhance the expression of mhc class i proteins on cells and thereby promote cd + t cell response (goodbourn et al., ) . moreover, type i ifns play an essential role in the differentiation and function of effector cd + t cells (stetson and medzhitov, ) . type i ifns are also able to enhance the cytotoxicity of nk cells by up-regulating the levels of perforins, and can stimulate the proliferation of nk cells to a limited degree (stetson and medzhitov, ) . ordinarily, cd + t and nk cells can eliminate infected cells, defending the host against intracellular infections (stetson and medzhitov, ) . however, the mechanisms through which the exogenous ifn-␣ performed its functions were not analysed in the present study. in vitro and in vivo results showed that ifn-␣ was able to prevent viral replication in a limited manner, exerting antiviral effect only when administrated early and in high doses, suggesting that orov, carv, gmav, grov and tcmv present some escape mechanism from antiviral actions of the ifn-␣. resistance to the ifn system was previously described for three members of bunyaviridae family, carv (brinton et al., ) , rift valey fever virus (bouloy et al., ) , and bunyamwera virus (weber et al., ; léonard et al., ) . brinton et al. ( ) , corroborating the results obtained by us, observed that carv resisted antiviral action of immmunomodulators, such as ifn-␣ and ifn-␤. however, the escape mechanism of carv was not studied by the authors. rift valey fever and bunyamwera viruses presented resistance to the ifn system associated with the non-structural protein nss which is encoded during viral replication. this protein showed to be able to block the production of ifn␣/␤ through the inhibition of transcription factors (bouloy et al., ; weber et al., ) . additionally, a recent study has demonstrated that the nss protein is also able to inhibit the ifn response through the interaction with the med component of mediator, a protein complex necessary for mrna production (léonard et al., ) . in our study, both cells and mice received high doses of exogenous ifn-␣ and a possible inhibition of the production of endogenous ifn-␣ by orthobunyaviruses would not be sufficient to block the antiviral effects derived from exogenous ifn-␣. moreover, it is known that vero e cells have an ifn i gene deficiency and thus they are unable to express endogenous type i ifn (mosca and pitha, ) . however, the ifn-dependent pathways are functional and can be activated by exogenously provided ifn (mosca and pitha, ). thus, it is possible that the studied orthobunyaviruses present a escape mechanism similar to that recently described for bunyamwera virus (léonard et al., ) , being able to inhibit the responses produced by ifn after association with the cell surface receptor. it would be interesting to further elucidate the escape mechanisms used by different bunyaviruses, especially those etiologically related to human diseases. it is important to mention that the cytokines tested in this study are part of a large group of existing ifns-␣ (foster and finter, ; yeow et al., ) . therefore, we cannot affirm whether other ifns-␣ would present a similar antiviral activity to that observed here. for instance, tan et al. ( ) evaluated the in vitro antiviral activity of four types of ifn-␣, roferon ® -a (ifn-␣- a), intron a (ifn-␣- b), wellferon (ifn-␣-n ) and alferon (ifn-␣-n ), on sars coronavirus (sars-cov) and observed that only two out of them, wellferon and alferon, were able to inhibit the cytophatic effect caused by this virus in vero e cell cultures. mechanisms for explaining these differences in activity of ifns-␣ are unknown. we may suppose that it could be related to distinct ifn-␣ sources because some preparations are derived from human lymphoblastoid or leukocyte cells, while other preparations are recombinantly produced in escherichia coli or mammalian cell culture (foster and finter, ) . in particular, the ifns-␣ used in our experiments were the roferon ® -a and the ifn-␣a, both produced in e. coli. in conclusion, our results demonstrated that the ifn-␣ presents in vitro antiviral activity on carv, gmav and tcmv, but this activity is limited and was not confirmed by in vivo experiments. therefore, ifn-␣- a could not be considered a suitable therapeutic drug for illnesses caused by these viruses. however, ifn-␣ presented in vitro and in vivo prophylactic antiviral activities on orov and grov and also showed therapeutic antiviral action on grov. thus, ifn-␣- a could be potentially useful in the prevention of diseases caused by oropouche virus and in the prevention and/or treatment of diseases caused by guaroa virus, mainly during epidemics or after occurrence of a laboratory accident. cellular responses to interferon-gamma genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein nss characterization of murine caraparu bunyavirus liver infection and immunomodulator-mediated antiviral protection inhibition of sandfly fever sicilian virus (phlebovirus) replication in vitro by antiviral compounds interferon alfa- b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis c modulation of dengue 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vaccinia infections in mice with interferon-␣ and interferon-␥ in vitro and in vivo studies of the ribavirin action on brazilian orthobunyavirus protection from lethal infection is determined by innate immune responses in a mouse model of ebola virus infection studies on guaroa virus identificación de oportunidades de mejora del tratamiento de la hepatitis c interferon alfa- b alone or in combination with ribavirin as initial treatment for chronic hepatitis c simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in bhk- cells: comparison of the bhk suspension test with standard plaque reduction neutralization transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis oropouche fever differences in interferon ␣ and ␤ signalling side effects of interferon alpha a and b viruses and interferons how cells respond to interferons type i interferons in host defense severe acute respiratory syndrome-related coronavirus is inhibit by interferon-␣ inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs serologic survey for yellow fever and other arboviruses among inhabitants of rio branco, brazil, before and three months after receiving the yellow fever d vaccine ifn-␣ a as compared with conventional chemotherapy for the treatment of chronic myeloid leukemia doenças infecciosas e parasitárias-enfoque amazônico oropouche virus transmission in amazon river basin of peru bunyamwera bunyavirus nonstructural protein nss counteracts the induction of alpha/beta interferon antiviral activities of individual murine ifn-alpha subtypes in vivo: intramuscular injection of ifn expression constructs reduces cytomegalovirus replication we thank dr. pedro vasconcelos, dr. amélia travassos da rosa, and dr. terezinha lisieux coimbra for kindly supply viruses used in this paper. this work was supported by a fapesp grant to luiz t.m. figueiredo (no. / - ) and by a fellowship from capes to márcia c. livonesi. key: cord- -glb y u authors: domingo, pere; mur, isabel; pomar, virginia; corominas, héctor; casademont, jordi; de benito, natividad title: the four horsemen of a viral apocalypse: the pathogenesis of sars-cov- infection (covid- ) date: - - journal: ebiomedicine doi: . /j.ebiom. . sha: doc_id: cord_uid: glb y u the pathogenesis of coronavirus disease (covid- ) may be envisaged as the dynamic interaction between four vicious feedback loops chained or happening at once. these are the viral loop, the hyperinflammatory loop, the non-canonical renin-angiotensin system (ras) axis loop, and the hypercoagulation loop. severe acute respiratory syndrome (sars)-coronavirus (cov)- lights the wick by infecting alveolar epithelial cells (aecs) and downregulating the angiotensin converting enzyme- (ace )/angiotensin (ang- – )/mas r axis. the viral feedback loop includes evading the host's innate response, uncontrolled viral replication, and turning on a hyperactive adaptative immune response. the inflammatory loop is composed of the exuberant inflammatory response feeding back until exploding in an actual cytokine storm. downregulation of the ace /ang-( – )/mas r axis leaves the lung without a critical defense mechanism and turns the scale to the inflammatory side of the ras. the coagulation loop is a hypercoagulable state caused by the interplay between inflammation and coagulation in an endless feedback loop. the result is a hyperinflammatory and hypercoagulable state producing acute immune-mediated lung injury and eventually, adult respiratory distress syndrome. in december , a new epidemic disease appeared in the huanan seafood wholesale market, wuhan, hubei province, china. it was characterized by an upper respiratory tract infection rapidly evolving to bilateral pneumonia and eventually respiratory failure [ ] . the etiologic agent was a new coronavirus which was named sars-cov- , whereas the disease was called covid- [ ] . the disease quickly expanded from its original nucleus in hubei and by march , the who declared it as a pandemic. as of june , , covid- has affected countries around the world, with . . confirmed cases worldwide and a death toll of . [ ] . early in the course of the pandemic, clinicians and researchers realized that full-blown covid- evolved in at least three phases: the first phase with cough, fever, wheezing, fatigue, headache, diarrhea, and dyspnea, reminiscent of an upper tract respiratory infection. the second phase, with the rapid appearance of bilateral pneumonia, infiltrates with variable degrees of hypoxemia, and omit in the third phase in which some patients developed respiratory failure leading to death [ ] . around % of people have sars-cov- infection asymptomatic or with mild to moderate illness, mostly restricted to the upper and conducting airways. the other % will develop symptomatic infection needing hospital admission, and % will require ventilatory support in the intensive care unit (icu) [ ] . the clinical phases of the infection reflect the pathogenic events starting with the virus gaining access to the lungs. the clinical manifestations and pathogenic events of any infectious disease, and covid- in particular, should be viewed in the light of the damageresponse framework in which several factors and forces may tip the scales to the host or pathogen side [ ] . therefore, sometimes the . the first horseman: a sneaky virus sars-cov- is a previously unknown b-coronavirus which shows % identity to the sequences of two bat-derived sars-like coronaviruses, . % identity to sars-cov, and about % identity to middle east respiratory syndrome (mers)-cov [ ] . the genome of sars-cov- is a positive-sense, single-stranded rna with a size of . kb, containing at least ten open reading frames (orfs) [ ] . recently, noncanonical orfs and at least rna modifications with an unknown function, were identified [ ] . the first orfs represent two-thirds of the viral rna. they are translated into two large polyproteins, which are later processed into non-structural proteins (nsp to nsp ) that form the viral complex replicase-transcriptase [ ] . these nsps rearrange endoplasmic reticulum into double-membrane vesicles, where viral replication and transcription take place [ , ] . the other third of the genome encodes four main structural proteins; spike (s), envelope (e), nucleocapsid (n), and membrane (m) proteins, and several accessory proteins whose functions are currently unknown but unrelated to viral replication [ ] . sars-cov- , like sars-cov, requires the ace as a receptor to enter the cells [ , ] . coronavirus s protein is a determinant of virus entry into host cells by binding the envelope spike glycoprotein to its cellular receptor ace [ , ] . although it was initially thought that sars-cov achieved entry by membrane fusion, a critical proteolytic cleavage at sars-cov s protein, mediated by type ii transmembrane serine protease (tmprss ), brings about membrane fusion and viral infectivity [ ] . after the virus entry, the rna genome is released into the cytoplasm and translated into two polyproteins and structural proteins [ ] . the survival of sars-cov in host cells is eased by strategies to evade the immune response. the evolutionarily conserved microbial structures called pathogen-associated molecular patterns (pamps) are recognized by pattern recognition receptors (prrs) such as tolllike receptor (tlrs), retinoic acid-inducible gene-i (rig-i)-like receptors, nucleotide-binding oligomerization domain (nod)-like receptors, and c-type lectin-like receptors [ ] . sars-cov induces the signal transducer and activator of transcription tace tnf-a converting enzyme tbk tank-binding kinase tlr toll-like receptor tmprss type ii transmembrane serine protease tnf-a tumor necrosis alpha traf tnf receptor-associated factor xcr xcl (chemokine [c motif] ligand ) and xcl (chemokine [c motif] ligand ) receptor production of double-membrane vesicles that lack prrs and can then replicate in these vesicles [ ] . furthermore, several structural and nsps encoded by sars-cov and mers-cov antagonize antiviral innate immune response. interferon (ifn) and interferon-stimulated genes (isgs) responses are counteracted by nsp , nsp macrodomain, nsps-deubiquitinase, and orf b, orf , and orf , thus overthrowing antiviral response [ ] [ ] [ ] [ ] [ ] [ ] . nsp inhibits ifn responses by three mechanisms, inactivation of host translational machinery, degradation of host mrnas, and inhibiting signal transducer and activator of transcription (stat ) phosphorylation [ , ] . part of the nsp is a papain-like protease that antagonizes ifn and cytokine production by blocking phosphorylation of ifn regulation factor (irf ) and disrupting nf-kb signaling [ ] . nsp and nsp are also ifn antagonists by an unknown mechanism [ ] . orf b exerts ifn antagonism through inhibition of ifnb induction by transcription factors irf and nf-kb, whereas orf antagonizes ifn by inhibiting signaling through the jak-stat pathway [ ] . m and n proteins flatten ifn signaling by inhibiting tank-binding kinase (tbk )/ikb kinase e (ikke), and the negative regulation of traf / -tbk -irf /nf-kb/ ap signals [ , ] . antagonism of ifn responses further promotes free virus replication resulting in increased viral pamps and damps that additionally dampen ifn signaling and stimulate prrs to induce an aberrant inflammatory response. the replicative capacity of sars-cov- is . folds more than that of sars cov in infected human lung tissue without significantly inducing types i, ii, and iii ifns [ ] . since innate immunity is the frontline defense against sars-cov- , a slow and poorly coordinated response may result in higher viral replication. this sequence of events, namely aecs infection, ifn signaling inhibition, and free viral replication depicts the viral vicious loop (fig. ). . the second horseman: a gathering storm sars-cov- infects primarily airway and alveolar aecs, especially type ii pneumocytes, the cells that produce alveolar surfactant and are predecessors of type i pneumocytes. however, it can infect any cell expressing the receptor ace , such as endothelial cells, pericytes, vascular smooth muscle cells, macrophages, fibroblasts, t-cells, cardiomyocytes, enterocytes, basal cell epidermal cells, and epithelial tubular distal cells [ ] [ ] [ ] . sars-cov- , in the face of unchecked replication because of dampened innate immunity, can replicate in high titres early after the infection [ , , ] . high viral replication in aecs induces cytopathic effects, as shown by the necropsy findings of multinucleated cells (syncytia), cytoplasmic viral inclusions, and apoptosis, an ultimate cellular response to stop virus replication [ , ] . these events are followed by the production of increased levels of proinflammatory cytokines and chemokines by aecs [ , ] . moreover, sars-cov nucleocapsid activates interleukin- (il- ) expression in lung epithelial cells via cellular transportation of nuclear factor kappa b (nf-kb) [ ] . massive infiltration of inflammatory cells into the lungs is, in turn, mounted by these cytokines and chemokines [ ] (fig. ) . although tissue-resident macrophages of the lungs localize to the airspace within alveoli, they do not seem to be the predominant subset in this response [ ] . accumulation of inflammatory monocyte-macrophages and neutrophils in the lungs following sars-cov- infection promotes the additional release of cytokines and chemokines [ ] (fig. ) . besides, the sars-cov spike promotes the upregulation of il- and tumor necrosis alpha (tnf-a) in macrophages [ ] . cytokines spill over from local inflammation to the systemic circulation. covid- patients have high serum levels of inflammatory cytokines, including interleukin (il)- , il- , il- , granulocyte-colony stimulating factor (g-csf), interferon gamma-induced protein (ip)- , monocyte chemoattractant protein (mcp)- , macrophage sars-cov- infects primarily type ii pneumocytes through binding to the ace receptor. the infected and surrounding pneumocytes secret cytokine and chemokines, which attract monocyte-macrophages and neutrophils to the alveolar space, which secrete additional cytokines and chemokines. ultimately the pneumocytes suffer apoptosis/pyroptosis releasing large amounts of proinflammatory factors. endothelial cells are infected, overexpress adhesion molecules, and release chemokines and cytokines. endothelial cells undergo apoptosis, which, together with alveolar cell apoptosis, increases vascular leakage and breaks the alveolar-capillary barrier. the hyperinflammatory milieu and endothelial dysfunction activate coagulation cascades through tissue factor expression, platelet activation, and netosis all of them promoting microcirculatory thrombi formation. the break of endothelial-alveolar barrier further promotes vascular leakage resulting in interstitial and alveolar space flooding. downregulation of the ace /ang-( À )/mas r axis contributes to increasing vasoconstriction, inflammatory signals, endothelial dysfunction, vascular leakage, and prothrombotic state. sars-cov- = severe acute respiratory syndrome coronavirus ; tnf-a = tumor necrosis factor alpha; il- = interleukin ; mcp- = macrophage chemoattractant protein ; mip- a = macrophage inhibitory protein a; il- = interleukin ; il- b = interleukin beta; nf-kb = nuclear factor kappa b. inflammatory protein (mip)- a, and tnf-a. these cytokine/chemokine levels correlate with disease severity [ , ] . covid- patients with severe disease have increased levels of il- more often than those with the mild or moderate disease [ ] . although viremia is not a prominent feature in covid- and is usually short-lived, the degree and duration of sars-cov- viremia relates to the severity of disease and the serum levels of il- [ ] . endothelial cells (ecs) are infected very early in the course of infection. because of speedy viral replication and exuberant proinflammatory cytokine/chemokine response they may suffer apoptosis [ , ] . this apoptotic phenomenon takes place via fas/fasl or trail-dr- -dependent mechanisms [ ] . besides, inflammatory monocyte-macrophages release tnf-a which also promotes apoptosis of both lung ecs and aecs [ ] . ecs and aecs apoptosis compromise lung microvascular bed and alveolar cell-capillary barrier integrity, thereby resulting in vascular leakage and alveolar edema [ ] (fig. ) . pericytes play an essential role in maintaining endothelial cell function in capillary vessels and are among the cells with the highest ace expression. their infection by sars-cov- may add to endothelial cell dysfunction leading to microcirculation disorders [ ] . a striking feature of full-blown covid- is severe lymphopenia. cov-specific t-cells are decisive for viral clearance and limitation of additional damage to host tissues since they can dampen hyperreactive innate immune response [ , ] . however, when exuberant inflammatory response induced by sars-cov- takes place, t-cell response is decreased because of tnf-a-mediated cell apoptosis, thus resulting in uncontrolled inflammatory responses [ ] (fig. ) . besides, normal t-cell activation can be suppressed by il- , further contributing to lymphopenia [ ] . in severe covid- patients with lymphocyte subsets examined, there is intense cd and particularly cd lymphopenia [ ] , both negatively correlating with tnf-a and il- serum levels [ ] . cd cells promote the production of virus-specific antibodies by activating t-dependent b cells, whereas cd cells are cytotoxic and can kill virus-infected cells. since cd cells account for about % of the total inflammatory lung interstitial infiltrate, highly cytotoxic cd lymphocytes can arbitrate immune-mediated tissue damage [ ] . also, in covid- patients, these cells exhibit markers of functionally exhausted t-cells such as programmed cell death protein . there is upregulation of apoptosis, autophagy, and p pathways in pbmc of covid- patients [ ] . mers-cov can induce t-cell apoptosis through activation of the intrinsic and extrinsic apoptosis pathways [ ] , and sars-cov e protein can also promote t-cell apoptosis mediated by cbl-xl binding [ ] . although sars-cov- can non-productively infect t lymphocytes, whether this infection induces t-cell apoptosis is not yet clear [ ] . alternatively, pyroptosis has been suggested as a cause of lymphopenia since covid- patients have increased serum il -b levels, which is the downstream indicator of cell pyroptosis [ ] . in sars-cov infection, viroporin a triggers the activation of nod-like receptor protein (nlrp ) inflammasome and the secretion of il- -b by macrophages [ ] . pyroptosis can release large amounts of proinflammatory factors [ ] . whatever the cause, during the late stages of infection, depletion of t-cells may promote viral survival and, consequently, may prolong the infection. essential to control the persistent phase of sars-cov- infection is the appearance of humoral immunity, in which antiviral neutralizing antibodies play a significant role. however, in animal models, anti-s protein-neutralizing antibodies (anti-s-igg) may cause severe lung injury by altering inflammatory responses [ ] . in sars-cov infection, the development of acute respiratory disease coincides with antiviral igg seroconversion in % of patients [ ] and patients who died developed anti-s-neutralizing antibodies faster [ ] . the presence of anti-s-igg promotes proinflammatory monocyte-macrophage lung accumulation and the production of mcp- and il- . such proinflammatory cytokine release would be mediated through the binding of the virus-anti-s-igg complex to the monocytes-macrophages fcgriia receptor since its blockade reduces the production of ifn-g, tnf-a, il- , and il- [ ] . it would also be possible that such complexes activate the classical pathway of the complement system or induce antibody-dependent cell-mediated cytotoxicity, thus leading to cellular damage. therefore, a possible underlying mechanism would be antibody-dependent enhancement (ade) of viral infection that occurs in some patients with early, sub-optimal antibody activity that cannot completely clear the virus, leading to persistent viral replication and inflammation [ ] . uncontrolled viral replication, because of a delayed innate immunity response, will cause cellular damage leading to an overexuberant and dysregulated immune kickback. this hyper reaction affecting the innate and adaptative immune responses will pave the way for immune-mediated damage of tissues and organs. this sequence of events conforms the inflammatory hurtful feedback loop (fig. ). the ras plays a critical role in the control of cardiovascular and renal functions by maintaining blood pressure homeostasis and hydro-electrolyte balance [ ] . initially, the ras was conceived as a linear hormonal system in which angiotensinogen synthesized in the liver is converted into the active peptide angiotensin i (ang i) through the action of renin [ ] . afterward, ang i is cleaved by the ace generating ang ii [ ] . two g protein-coupled receptors (gpcr) mediate the actions of ang ii, angiotensin ii receptor type (at r), and type (at r) [ ] . the primary role of this canonical or classical ras pathway (ace/ang ii/at r) is to increase the sympathetic nervous system tension, to cause vasoconstriction, increase blood pressure, and promote inflammation, fibrosis and myocardial hypertrophy [ ] . the ras also possesses a non-canonical, counter-regulatory branch composed of ace /ang-( À )/mas r. the activity of the system will depend on the balance between the two branches. ace is the main synthesizer of ang-( À ) by removing a single residue from ang i to generate ang-( À ) and by cleaving a single residue from ang ii to generate ang-( À ) [ , ] . the functional receptor for ang-( À ) is the gpcr mas r [ ] . the conformation of the negative or counter-regulatory axis is relevant not only because it downgrades the vasoconstrictive/ proliferative peptide ang ii to form the vasodilator heptapeptide ang-( À ), but also because it degrades ang i to ang-( À ), thereby limiting the availability of the substrate for ace. ang-( À ) binds to mas r, inducing vasodilation, inhibition of cell growth, anti-thrombotic, and anti-arrhythmogenic effects [ ] . ace activity is controlled by a disintegrin and metalloproteinase domaincontaining protein (adam- , also called tnf-a-converting enzyme, tace). adam- proteolytically cleaves ace causing the shedding of ace into the interstitium, which leads to decreased ace activity in the tissue and elevates circulating ace activity [ ] (fig. ) . since blood and urine measurement of ace levels is feasible, they could potentially be used as a prognostic biomarker in covid- [ ] . ras plays an essential role in the pathogenesis of inflammatory diseases in which most of the proinflammatory actions are caused by ang ii [ ] . ang ii activates several cellular functions and molecular signaling pathways related to tissue injury, inflammation, and fibrosis. they involve calcium mobilization, free radical generation, activation of protein kinases and nuclear transcription factors, recruitment of inflammatory cells, adhesion of monocyte and neutrophils to endothelial and mesangial cells, upregulation of adhesion molecules and stimulation of expression, synthesis, and release of cytokines and chemokines [ ] . at r mediates most of these actions [ ] . the counterregulatory ace /ang-( À )/mas r axis negatively modulates leukocyte migration, cytokine expression and release, and fibrogenesis pathways. hence, ace deficiency increases vascular inflammation by increasing the gene expression of vascular adhesion molecules, cytokines, chemokines, and matrix metalloproteases [ ] . the loss of ace results in higher increases in ang ii-induced expression of inflammatory factors, enhanced vascular permeability, increased lung edema, and neutrophil accumulation [ ] . the ace / ang-( À )/mas r axis also plays an essential role in haemostasis, since it stimulates prostacyclin (pgi ) production and nitric oxide (no) release by ecs and modulates platelet activity which is less adherent having, thus anti-thrombotic activity [ ] (fig. ) . ras exhibits high activity in lung tissue, which is the leading site of ang ii synthesis. ace is a zinc metallopeptidase, type i integral membrane glycoprotein orientated with the n-terminal, and the catalytic site facing the extracellular space [ ] . the union of ace with sars viral spike protein triggers enzyme internalization downregulating activity from the cell surface. once sars-cov binds to its receptor, the abundance on the cell surface, mrna expression, and the enzymatic activity of ace are significantly reduced [ ] . proteolytic shedding of its extracellular domain is a second mechanism for downregulating ace at the cell surface. s protein of sars, once it binds to ace , induces shedding by activating adam (tace) as do bacterial endotoxin and lipopolysaccharide (lps) [ ] (fig. ) . releasing ace from the cell membrane is a critical step in catalyzing substrates and implies that attenuation of ace activity might contribute to disease pathogenesis. the recently described induction of ace expression by type i ifn in human nasal epithelial cells, thus behaving as an isg, highlight an additional mechanism of ace downregulation by sars-cov- [ ] . since ifn-induced isgs are crucial for host antiviral response, the absence of ace induction due to hampered ifn responses will further cause tissue unprotection. therefore, in covid- , ace plays a pivotal role because of its multifaceted task as a facilitator of entry into aecs and its potential role in the pathogenesis of acute lung injury (ali) [ ] . in the mouse model of sars, downregulation of ace protein expression resulted in worse pneumonia, increased ang ii levels, increased vascular permeability, enhanced lung edema, neutrophil infiltration, and further worsened lung function [ , ] . catalytically active ace protein alleviated the symptoms, and active protein improved the outcome of respiratory failure [ ] . in covid- patients, plasma concentrations of ang ii were significantly higher than in healthy individuals and ang ii levels correlated with viral load and lung injury [ ] . owing to the widespread expression of ace , covid- is a disseminated infection. ace is highly expressed in the gut and sars-cov- can productively infect enterocytes [ , ] . despite ace the lung, and other organs, lose the protection of the non-canonical ras system as a result ace downregulation after sars-cov- -induced endocytosis. consequently, the canonical ace/ang ii/at r becomes dominant, levels of ang ii increase with the subsequent promotion of fibrosis, myocardial hypertrophy, increased ros, vasoconstriction, inflammation, and endothelial dysfunction. at r mediates most of these actions. endocytosed sars-cov- spike proteins mediates adam -mediated proteolytic cleavage of ace . adam- activity is enhanced through the activated at r by increased levels of ang ii. tnf-a is the primary substrate of adam . adam cleaves tnf-a releasing soluble tnf-a extracellularly where it has autocrine and paracrine functionalities. activation of tnf-a receptor by tnf-a also enhances adam activity. ace = angiotensin-converting enzyme ; sars-cov- = severe acute respiratory syndrome coronavirus ; ang ii = angiotensin ii; ros = reactive oxygen species; at r = angiotensin receptor; adam = a disintegrin and metalloproteinase domain ; tnf-a = tumor necrosis factor alpha; tmprss = transmembrane protease serine . expression in gut being higher than in the lung, only about À % of patients with covid- experience gastrointestinal symptoms [ ] . the contribution of the digestive system to the pathogenesis of covid- through impairment of mucous membrane barrier and increased inflammatory cytokine production has not been determined yet. similar to mers-cov and owing to ace expression in the brush borders of the proximal tubules and in podocytes, kidney injury in covid- is characterized by diffuse proximal tubule damage with virus-like particles in tubular epithelial cells and podocytes which is indicative of direct sars-cov- infection [ ] . these findings translate clinically into acute kidney injury and proteinuria which affect from . % to % of covid- patients [ ] . the consequence of impaired ace activity in the lung because of sars-cov- infection is a reduction of ang-( À ) production. ang-( À ) binding mas r promotes an array of biological responses to counteract ang ii-mediated processes such as apoptosis, angiogenesis, vasoconstriction, and inflammation in the lung [ , ] . consequently, the attenuation of ace catalytic function perturbs the pulmonary ras balance, resulting in enhanced inflammation and vascular permeability, leaving the lung defenceless in the face of the forthcoming raging cytokine storm. besides, infection of type ii pneumocytes will reduce the production of alveolar surfactant subsequently reducing pulmonary elasticity. moreover, the loss of type ii pneumocytes decreases restoration of type i pneumocytes which ultimately impacts on gas exchange and fibrosis [ ] . the above event sequence depicts the ras vicious feedback loop (fig. ). the association between covid- and coagulation disorders was beheld early during the pandemic when chinese physicians noticed that patients treated mainly with low-molecular-weight heparin had a decreased -day mortality [ ] . this mortality improvement was in patients with a sepsis score higher than four or a markedly elevated d-dimer [ ] . covid- is associated with coagulation disorders that include increases in procoagulant factors such as fibrinogen and d-dimers, both associated with poor prognosis [ , ] . patients admitted to the icu had an increased incidence of venous thromboembolic events ranging from % to % [ ] [ ] [ ] . moreover, standard prophylaxis for venous thromboembolism failed in . % of the patients [ ] . some found that most thrombotic complications were venous and primarily isolated pulmonary embolism, which suggests that it may be primary pulmonary thrombosis instead of embolic phenomena [ , ] . in line with that, microcirculatory thrombosis is a constant finding in lung pathologic studies [ , ] (fig. ) . infection of ecs, together with the derangements caused by cellular infiltration and high exposure to cytokines/chemokines, eventually leads to ecs dysfunction and apoptosis [ ] . all of them contribute to microvascular prothrombotic effects [ ] . there is an intense interplay between haemostasis and innate immunity, called thrombo-inflammation [ ] . both the intrinsic and extrinsic coagulation pathways can activate during inflammation. ecs and macrophages activate the extrinsic pathway through expression of tissue factor [ ] . the intrinsic pathway can be activated by neutrophil extracellular traps (nets) released by polymorphonuclear neutrophils (pmn) in a process called netosis. nets activate ecs, platelets, and the complement system and release proteases that inactivate endogenous anticoagulants [ ] . however, the role of nets in covid- is still a matter of discussion [ ] . platelets play a dual role. first, a proinflammatory role by secreting alpha granules that recruit pmn and macrophages, which are an essential source of il- b [ ] . besides, platelets stimulate pmn to undergo netosis which in turn activates platelets, creating a feedback loop. the second role of platelets is to activate the coagulation pathway by assembling enzyme-cofactor-substrate complexes on their exposed surface [ ] (fig. ) . complement activation, which has been seen in the mouse model of sars, contributes to immune-mediated pathology [ ] . activation of c and c promotes mast cell degranulation and recruitment of pmn and macrophages [ ] . the prothrombotic effects of activated c and c include platelet and ecs activation, together with increasing tissue factor and von willebrand factor expression [ ] . to close the loop, thrombin, and other components of the coagulation cascade can, in turn, activate c and c [ ] . the primary function of thrombin is to promote clot formation by activating platelets and by converting fibrinogen into fibrin [ ] . however, thrombin is a pleiotropic molecule and can increase inflammation via a proteinase-activated receptor (par), principally par- [ ] (fig. ) . the generation of thrombin is controlled by negative feedback loops and physiological anticoagulants such as antithrombin iii, tissue factor pathway inhibitor and the protein c system [ ] . il- b, il- , and tnf-a promote the release of ultra-large von willebrand multimers, and the production of tissue factor and factor vii/activated factor vii, leading to increased thrombin generation while decreasing the levels of endogenous anticoagulants [ ] . the ace /ang-( À )/mas r axis exerts antithrombotic effects through activation of mas r in platelets, which then release no and pgi and by protecting from endothelial dysfunction [ , ] . since this branch of the ras is not working properly in covid- , this protective mechanism is lost ( figs. and ) . in severe covid- , similar to other acute viral infections, a high prevalence od antiphospholipid antibodies was found, although the role of these antibodies in the prothrombotic state of sar-cov- infected patients is still a matter of debate [ ] . the progression of thrombo-inflammation may result in widespread thrombosis, which may be further enhanced by hypoxemia, hyperthermia, and hypovolemia [ ] . hypoxemia triggers increased expression of hypoxia-inducible factors, which may promote additional inflammation and may activate platelets and coagulation factors. they increase tissue factor expression, increase plasminogen activating inhibitor- , and inhibit the endogenous anticoagulant protein s [ ] . in the setting of a hyperinflammatory state and endothelial injury, activation of coagulation occurs whereas the counterregulatory force ace /ang-( À )/mas r axis is inactive, leaving the field to the full expression of a hypercoagulable state. this state may clinically translate into pulmonary thrombosis, venous thromboembolism, or other thrombotic events. if these events affect microvascular lung bed, they may further promote ali and impair gas exchange. whatever the location of the thrombotic event is, it worsens the patient's prognosis. hyperinflammatory state and defective ace /ang-( À )/mas r functioning activate the fourth hurtful feedback loop. hyperinflammation induces hypercoagulation and vice versa, while ace /ang-( À )/mas r axis avoidance maximizes both (fig. ) . the clinical spectrum of covid- is broad. not everyone who acquires sars-cov- becomes sick and the state that emerges after infection can vary among patients or within the same patient over time. consequently, it is envisaged that virus-dependent, hostdependent, and environment-dependent factors may modify the virus-host interaction explaining not only the individual susceptibility to infection but also the broad scale of damage seen in clinical disease. the initial viral titre in the airways could explain the different evolving patterns of covid- , since this will condition the intensity of cytopathic changes, which in turn will shape the strength of immune responses [ ] . sars-cov- replicates in high numbers very early after infection, and in turn, the magnitude of viral replication will impact on the extent of antiviral response [ ] . in humans, there is a strong correlation between sars-cov and mers-cov titres and disease severity [ ] . in animal models, the disease behaves differently if the virus infects airway epithelial cells or both airway and aecs (type i and type ii pneumocytes) predominantly. the viral antigen is mainly located in airway epithelial cells in mouse models permissible to infection, but which do not develop clinical disease. in contrast, in highly susceptible mice, the antigen is detected in both airways and alveolar type i and ii pneumocytes [ ] . consequently, infection of aecs seems critical for both host susceptibility and the development of lung pathology. an aspect that influences sars-cov- infection is the state of differentiation of human airway epithelia, which, in turn, correlates positively with the expression level of ace in these cells [ , ] . it is noteworthy that ace nasal gene expression is lower in children [ ] . this fact is connected to the striking age distribution of covid- in which children are often spared, affecting adults with enhanced severity and mortality as age increases [ , ] . however, the increasingly poor outcome with advancing age is influenced by the presence of common comorbidities, such as hypertension, cardiovascular disease, and diabetes, which bear a poor prognosis by themselves [ ] . besides, these comorbidities relate to a decreased activity of ace in elderly patients, a deficit further exacerbated by sars-cov- infection [ ] . ade is a potentially harmful, pro-inflammatory mechanism which occurs when suboptimal titres of neutralizing antibodies against sars-cov- are present. they are unable to control infection but instead facilitate viral entry into macrophages by a trojan horse mechanism. ade tends to happen when the time interval between coronavirus infections is long enough for antibody fall, which could be a possible mechanism for severe covid- in the aged [ ] . females with covid- usually present with milder disease than males. females exhibit higher ifn and ifn regulator factor and il- production from pbmc, lower production of tnf-a, lower expression of tlr in pmn, lower numbers of nk cells, and lower pmn phagocytic activity than males [ ] . oestrogens downregulate ang ii and upregulate ang-( À ) pathways, which makes apparent gender differences in expression, activity, and tissue responsiveness of ras components [ ] . besides, mas r expression was increased in female rats but not in males after the infusion of ang ii [ ] . in animal models of obesity, females appear to maintain circulating ang-( À ) levels and are protected from hypertension and metabolic complications induced by angiotensinogen, renin, angiotensin ii, and at r activation [ ] . stimulation of counterregulatory at r appears metabolically protective in female rodents, whilst there are inconsistent effects in males [ ] . in the uk, % of covid- patients in the icu were either overweight or obese [ ] , and % of dead north italians had obesity, hypertension, diabetes, heart disease, kidney damage, or cancer [ ] . the frequent occurrence of obesity as a factor of adverse outcome is frequently shadowed by other high prevalent comorbidities in obese people making the identification of the independent role of obesity steep [ ] . the association of covid- with obesity has been attributed to the chronic inflammatory status, the ineffective immune response or to interaction with the ras system. the immune pathways are all susceptible to genetic polymorphisms with functional consequences such as variability in cytokine expression, antigen-binding affinities, receptor ligation strength, and downstream signaling [ , ] . high interconnection is a prominent feature of immune pathways and thus functional resultant polymorphisms may hamper the growth of an optimal immune response to covid- . responses triggered by pamps recognition and its downstream molecules such as myeloid differentiation primary response (mdy ) may be altered by tlr polymorphisms [ , ] . hla genes present extreme allelic polymorphism. since they present viral peptides to host hla molecules to trigger an adaptative immune response, their polymorphisms may cause unevenness in antigen binding and presentation, and consequently in immune response. hla-b* : has been associated to the development and increased severity of sars-cov [ ] , and it has the fewest predicted binding affinity of sars-cov- peptides [ ] . il- plays a central role in the hyperactive immune response in covid- patients. since there are functional polymorphisms in the il- gene that modify its protein level expression, they may affect the severity of the disease [ ] . the role of ras in the pathogenesis of covid- is essential. single nucleotide polymorphisms and haplotypes in ace genes, such as polymorphism a/d in the ace gene, have been associated with circulating and tissue concentrations of ace levels and reduced expression of ace [ , ] . interestingly, the prevalence of covid- in europe correlates inversely with ace d allele frequency [ ] . a genetic variant of the ifn-induced transmembrane protein- gene is associated with covid- severity. ifn-induced transmembrane protein- is an immune effector protein that acts restricting membrane fusion [ ] . recently, a genomewide association study in covid- patients with respiratory failure identified an association signal at locus p . , which includes the genes slc a , lztfl , ccr , fyco , cxcr and xcr , while there was no association signals at the hla complex [ ] . lately, there has been a contention about the beneficial or detrimental role of ace inhibitors (acei) and angiotensin receptor blockers (arb) in the outcome of patients with covid- . currently, there is no evidence to support an advantageous or harmful effect of concomitant therapy with acei or arb in covid- patients [ ] . covid- is a systemic infection since it may impact any tissue or organ expressing ace . however, the most dreadful, often lifethreatening conditions, are ali and ards. therefore, the main challenge is to avoid their development to prevent icu admission and mechanical ventilatory support. we could envisage covid- as a tree in which aecs viral infection and ace downregulation represent the roots. the tree trunk would be the hyperinflammatory and hypercoagulable state. the branches would be an end-organ disease, such as ali, myocarditis, neurological disease, liver injury, gastrointestinal involvement, and skin disease. since the chain of events triggered by sars-cov- infection evolves quickly, any planned intervention must come as early as possible. besides, since the pathogenesis of covid- involves non-viral mechanisms, any intervention planned must also address the correction or modulation of these disbalances. hence, any therapeutic intervention must be early and combine antiviral and adjuvant therapies. however, the moment of diagnosis and eventual hospital admission will mark the timeframe of interventions. to tackle the roots of the disease, potential therapeutic interventions for covid- should first address the viral entry into aecs. the entry of sars-cov- into aecs takes place after binding of the spike to the receptor ace . specifically, the binding takes place in the receptor-binding domain of the s protein. thus, developing neutralizing monoclonal antibodies for this domain is a rational strategy to prevent the viral union and subsequent events [ ] . another possible way of targeting the interaction between ace and s protein may be the use of soluble recombinant ace , which may prevent the binding of the viral particle to the surface-bound, full-length ace [ ] . in the vero-e monkey cell line, a soluble form of ace blocks sars-cov replication and reduced sars-cov- recovery by a factor of À [ , ] . besides, since sars-cov- downregulates the ace /ang-( À )/mas r axis, recombinant human ace (rhace ) could prevent the development of ali in covid- . rhace attenuated arterial hypoxemia in a piglet model of lps-induced ali [ ] . in phase ii, open-label trial in humans with ards rhace was well tolerated, ang ii levels decreased, whereas ang-( À ) and surfactant protein d increased [ ] . however, the study was not powered to detect changes in acute physiological or clinical outcomes [ ] . there is a randomized controlled trial to assess rhace in patients with severe covid- (nct ). apart from ace , sars-cov- entry involves tmprss , whose inhibitor, camostat mesylate, significantly reduced lung cell line infection with sars-cov- [ ] . endocytosis is a crucial step in sars-cov- infection. ap- -associated protein kinase (aak ) regulates this process [ ] . baricitinib, a janus-kinase inhibitor, has been claimed as a candidate drug for covid- since it inhibits aak [ ] . arbidol inhibits viral entry by inhibiting the fusion between viral and cellular membranes [ ] . however, in a small retrospective study, arbidol did not meet noninferiority versus the combination of arbidol and lopinavir/ritonavir (lpv/r) [ ] . chloroquine and its safer derivative hydroxychloroquine are effective against sars-cov- in vitro [ ] . however, recent news from the large recovery trial showed that there is no beneficial effect of hydroxychloroquine in patients hospitalised with covid- ; therefore, that arm of the study was stopped [ ] . other planned large trials, such as solidarity, stopped enrolling patients to the hydroxychloroquine arm, and the national institutes of healthsponsored orchid study was also stopped [ , ] . numerous antivirals agents are being tested in clinical trials. lpv/r could not demonstrate enough efficacy when compared with placebo [ ] . the combination of ifn, lpv/r, and ribavirin showed a shorter time to negativize nasopharyngeal swabs and superiority versus lpv/ r in alleviating symptoms [ ] . in two double-blind, placebo-controlled trials, remdesivir was not associated with statistically significant clinical benefits in one, whereas in the other shortened the time to recovery in hospitalized adults [ , ] . as of now, there is no antiviral drug with proven efficacy for treating patients with covid- . another strategy tries to modulate the exuberant inflammatory response in covid- . the use of corticosteroids is controversial and not supported by previous experience in sars and mers [ ] . however, in the recovery trial, dexamethasone reduced deaths by one third in patients receiving invasive mechanical ventilation and by one fifth in patients receiving oxygen without invasive mechanical ventilation [ ] . tocilizumab, a specific il- receptor antagonist, is promoted to treat the hyperinflammatory state of covid- because of the pathogenic role il- plays. two observational studies have shown a clinical benefit of therapy with tocilizumab in covid- pneumonia with hyperinflammatory syndrome [ , ] . anakinra, a recombinant il- receptor antagonist, has proven useful in a small retrospective study of covid- patients with ards and hyper inflammation [ ] . there are additional trials in progress with tocilizumab, anakinra, and sarilumab. however, when trying to modify the cytokine response by targeting a single molecule or receptor, it should be recalled that the cytokine network is an intricate complex with a high degree of overlap, redundancy, and alternate pathways. this may explain therapy escape and eventually lack of response. therapeutic interventions for the consequences of hyperinflammatory and hypercoagulable states associated with covid- , such as ali, ards or thromboembolic events, are beyond this review's scope. knowledge of pathophysiology is the first step to address the management of a disease appropriately. it is familiar with the mechanism that the virus uses to evade host immune defense mechanisms or those that uses to harm will permit the design of appropriate strategies to neutralize the dysfunctions or disbalances generated either by the virus or by the consequences of the infection. from the knowledge gathered, it seems that most organ damage in severe covid- is done through an immune-mediated mechanism, although sars-cov- is the necessary initiator. the spectrum of disease is comprehensive, and since not all the patients will share the same evolving pattern, the search for predictive factors to promptly identify patients more prone to evolve to life-threatening disease is of the utmost importance. in severe cases, the quick evolving pattern of the disease makes early treatment imperative, at least until reliable predictive factors become widely available. the implication of viral and host-dependent mechanisms in covid- pathogenesis suggests that any therapeutic strategy must combine antiviral drugs and adjuvant therapy to modulate the host's responses. all these goals will be achieved through the broad effort of basic, translational, and clinical scientists and clinicians, and will demand a high degree of commitment from patients and their families, allied professionals, and everyone engaged in the fight against covid- . among them, politicians and health administration officers will play a unique role, since such a gigantic task will need the allocation of a vast amount of resources to overcome a health challenge to mankind like none other in recent times. while engrossed amid the pandemic, there was progress on the physiopathology of covid- . however, gaps regarding viral, environmental, and transmissibility aspects remain-the dynamic interplay between the host and the virus and how to modify it to improve disease prognosis not being the lesser. there is a big difference in transmissibility, which is highest for sars-cov- , among b-coronaviruses despite similar structure and functioning. asymptomatic viral shedding is the main factor. however, the role of newly described orfs and rna modifications and their functional correlations are not evident yet. although tmprss is involved in viral entry into the host cell, the involvement of other host proteins is still under discussion. the role of the different epithelial cells along the bronchial tree and the alveolar space needs to be ascertained. the virus's mechanisms to invade other organs beyond the lung are already poorly known. clinical disease progression is somewhat unpredictable. therefore, the identification of prognostic clinical and biological markers would optimize patients' care and resource consumption, which may be of utmost importance in pandemic times. this effort must include which role comorbidities and gender play. the definition and timing of the optimal therapeutic approach to covid- will represent a colossal effort, which can be accomplished only by randomized clinical trial performance. these should include concerted actions and a combination of diverse disciplines, resources, expertise, and techniques to contribute to advances in prevention, diagnosis, and therapy. this set makes up an almost flawless meaning of translational medicine defined by the european society for translational medicine (eustm) as "an interdisciplinary branch of the biomedical field supported by three main pillars: benchside, bedside, and community." for this review, our search strategy involved the review of original records, either journals or books, mainly from european and american sources, from to . from these sources, we hand searched reference lists of identified additional articles to retrieve additional studies. preference was given to most relevant research, but we were also keen to highlight the breadth of the topic and hence selected some publications that showcase particular areas of interest. we have searched pubmed and google scholar from database inception to may , , for records, journals, and books for the terms "sars-cov- , "covid- , "coronavirus", "raas system", "angiotensin-converting enzyme", "angiotensin-converting enzyme , "cytokine storm", "cytokine", "chemokine", "acute lung injury", "adult respiratory distress syndrome", "interferon", "interleukin ", "middle east respiratory syndrome", and "severe acute respiratory syndrome". references were examined in english. we declare no competing interests. the concept of the manuscript was devised by pd who also performed the overall literature searches. im, vp, hc, and jc designed the search strategy with inputs from pd and ndb. im, vp, hc, and jc carried out the literature searches and screening, and any discrepancies were discussed with pd and ndb. pd wrote the first draft of the review with inputs from all the authors. this work was partially 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arbidol combined with lpv/r versus lpv/r alone against corona virus disease : a retrospective cohort study remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro statement from the chief investigators of the randomised evaluation of covid- therapy (recovery) trial on hydroxychloroquine solidarity" clinical trial for covid- treatments study shows treatment does no harm, but provides no benefit a trial of lopinavir-ritonavir in adults hospitalized with severe covid- triple combination of interferon beta- b, lopina-virÀritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for the treatment of covid- -preliminary report clinical evidence does not support corticosteroid treatment for -ncov lung injury low-cost dexamethasone reduces death by up to one third in hospitalised patients with severe respiratory complications of covid- tocilizumab for the treatment of severe covid- pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of patients in pilot prospective open, single-arm multicentre study on off-label use of tocilizumab in patients with severe covid- interlekin- blockade with high-dose anakinra in patients with covid- , acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study we are indebted to jordi mancebo and m ant onia mangues for critical reading of the manuscript, and to richard pike for reviewing its writing. key: cord- - dsqbeg authors: mahlakoiv, t.; ritz, d.; enjuanes, l.; müller, m. a.; drosten, c.; staeheli, p. title: p combined action of type i and type iii ifn restricts initial replication of sars-coronavirus in the lung but fails to inhibit systemic virus spread date: - - journal: cytokine doi: . /j.cyto. . . sha: doc_id: cord_uid: dsqbeg introduction stat -deficient mice are more susceptible to infection with sars-coronavirus (sars-cov) than type i ifn receptor-deficient mice. the increased susceptibility of stat -deficient mice is potentially due to the lack of functional type iii ifn (ifn-λ) signalling. methods we used mice lacking functional receptors for both type i and type iii ifn (dko) to evaluate the possibility that type iii ifn plays a decisive role in sars-cov protection. results we found that viral peak titres in lungs of dko and stat -deficient mice were similar, although significantly higher than in wild-type mice. the kinetics of viral clearance from the lung was also comparable in dko and stat -deficient mice. surprisingly, however, infected dko mice remained healthy, whereas infected stat -deficient mice developed liver pathology and eventually succumbed to neurological disease. conclusion our data suggest that the failure of stat -deficient mice to efficiently control initial sars-cov replication in the lung is due to impaired type i and type iii ifn signaling, whereas the failure to control subsequent systemic viral spread is due to unrelated defects in stat -deficient mice. introduction. the single nucleotide polymorphisms (rs ), near the il b gene, is correlated with a sustained virological response (svr) in hepatitis c virus (hcv) infected patients treated with pegylated interferon-a combined with ribavirin ( , ) . however, the association of rs polymorphism with immune function of the liver, the site of hcv production, and the mechanism of svr remain still undefined. methods. patients chronically hcv-infected patients were genotyped for rs defining c/t polymorphism. c allele is associated to svr. liver samples were collected from the needle biopsy achieved for the diagnosis prior any treatment. single cell suspensions were prepared by mechanical disruption. liver lymphocytes (t, treg, nk and nkt) were identified by flow cytometry for the expression of cd , cd , cd , cd , cd and foxp as markers and cd a for degranulation activity. expression of cd b, foxp , il and hprt genes was measured by pcr. immunohistochemistry were performed on in paraffin sections for the detection of cd and foxp . statistical analysis wwas done with mann-whitney u test and wilcoxon matched-t test. results. lymphocytes from fresh liver biopsies displayed similar distributions of t (cd ), nkt (cd , cd ) and nk (cd ) among cd cells by flow cytometry multi parametric analysis, whatever the il b genotype of the patients. strikingly, higher degranulation activity, revealed by cd a surface expression, was observed in t (p = . ), nkt (p = . ) and nk cells (p = . ) of patients with cc genotype (n = ) compared to patients with ct or tt genotypes (n = ); patients with cc genotype displayed two fold higher degranulation activity than patients with ct genotype in t (p = . ), nkt (p = . ) and nk cells (p = . ); no significant difference was observed between patients with ct and tt genotypes. sections of liver from patients showed the frequency of cd -foxp lymphocytes two fold higher (p = . ) in patients with cc genotype (n = ) as compared to patients with ct genotype (n = ) supporting the presence of treg. previous study demonstrated that the ratio between the number of cd cells and foxp cells in parenchymatous necro-inflammatory areas is maintained in the early stage of the chronic hepatitis ( ). this is found only in patients with cc genotype, whereas this ratio is reduced in patients with ct genotype due to lower number of foxp cells. transcriptional analyses confirmed these data and further showed two strong correlations between: one between foxp and cd b, another between foxp and il- expressions in patients with cc genotype. conclusion. collectively these data provide new insights into the role of il b polymorphism related to svr in treatment of hcv infected patients. cc genotype, which is linked to good response, is associated to higher efficiency of effector lymphocytes (t, nk and nkt) of the liver. the liver immune response appears tightly regulated as suggested by the links between cd cells and cd -foxp cells, and between il and foxp gene expression. introduction: . innate immunity to rna virus infection is triggered when the cytosolic pathogen recognition receptor rig-i engages viral rna in infected cells. rig-i pathway signaling is transmitted by the rig-i adaptor protein mavs, which resides on mitochondria, peroxisomes, and the mitochondrial-associated membrane (mam), a distinct membrane that links er to mitochondria. during rna virus infection, rig-i is recruited into the mam where it binds mavs and drives the actions of a signalosome that mediates downstream induction of antiviral, proinflammatory, and immunomodulatory genes that impart control of infection and immunity. mamtethering to mitochondria and peroxisomes coordinates mavs localization to form a signaling synapse between membranes. the importance of the mam within this ''innate immune synapse" is highlighted by the fact that the hepatitis c virus (hcv) ns / a protease cleaves mavs on the mam, but not the mitochondria, to evade immunity. methods. to identify the components that regulate formation and function of the innate immune synapse and the mavs signalosome, we characterized the proteome of mam, er, and cytosol subcellular fractions from uninfected cells and from cells with either chronic (hcv) or acute (sendai) rna virus infections. results. comparative analysis of protein trafficking dynamics during both chronic and acute infection reveals differential protein profiles in the mam compartment under rig-i pathway activation. we also identified molecules recruited to the mam in both chronic and acute rna viral infections representing proteins that drive immunity and/or regulate viral replication. conclusion. our proteomic analysis reveals dynamic cross-talk between subcellular compartments during both acute and chronic rna virus infection, and demonstrates the importance of the mam as a central platform that coordinates innate immune signaling to initiate immunity against rna virus infection. methods. we used mice lacking functional receptors for both type i and type iii ifn (dko) to evaluate the possibility that type iii ifn plays a decisive role in sars-cov protection. results. we found that viral peak titres in lungs of dko and stat -deficient mice were similar, although significantly higher than in wild-type mice. the kinetics of viral clearance from the lung was also comparable in dko and stat -deficient mice. surprisingly, however, infected dko mice remained healthy, whereas infected stat deficient mice developed liver pathology and eventually succumbed to neurological disease. conclusion. our data suggest that the failure of stat -deficient mice to efficiently control initial sars-cov replication in the lung is due to impaired type i and type iii ifn signaling, whereas the failure to control subsequent systemic viral spread is due to unrelated defects in stat -deficient mice. introduction . the balance between pro and anti-inflammatory signaling in innate immune responses to bacterial infection is especially critical in the lung. airway epithelial cells, in addition to resident and recruited cells of immune origin, participate in what must be coordinated proinfammatory signaling in response to inhaled pathogens. the type iii interferons are especially important in pulmonary infection, produced in response to viral infection and bacterial pamps. ifn-k is activated by and induces nf-jb signaling and promotes expression of th cytokines. we postulated that common respiratory pathogens, staphylococcus aureus and pseudomonas aeruginosa, would stimulate an ifn-k response and that this would have an important effect on bacterial clearance from the airway. methods. to establish that bacterial components can stimulate ifn-k, we measured the induction of ifn-k by rt-pcr in murine bmdcs. biological significance of ifn-k induction was determined by comparing the ability of wt c bl/ and il- rÀ/À mice (lacking the ifn-k receptor) to handle an intranasal inoculation of cfu of either pak or usa . results. in response to either p. aeruginosa pak or usa mrsa on bmdcs there was a -fold increase in ifn-k transcript by h post infection which persisted for up to h; in contrast to the -fold induction of ifn-b by both organisms. the il- rÀ/À mice had significantly improved clearance of either pathogen from the airway and lung tissue (p < . for each). consistent with the expected participation of ifn-k in nf-jb signaling, we measured decreased expression of kc, tnf and gm-csf and increased il- in the il- rÀ/ -bal (p < . ), although there were no significant differences in the populations of immune cells (neutrophils, dc, macrophages) recruited to the airways of the wild type or il- rÀ/À mice. deleterious effects of ifn-k on bacterial clearance were confirmed by exogenous treatment of wt mice with ifn-k that significantly diminished clearance of both organisms. to address how ifn-k signaling interferes with bacterial clearance we examined the role of pdcd (programmed cell death protein ) in this model system. pdcd , which is negatively regulated by mir- , has been shown to divergently regulate nf-jb and il- signaling, consistent with the observed effects of ifn-k. at baseline there was significantly more pdcd expression in the wt murine lung as compared with the il- rÀ/ À mutant and levels of expression were not significantly different at h post usa or pak infection, in contrast to the significant increase in pdcd expression in the il- rÀ/À mice following exposure to either bacteria. to determine if pdcd in human airway epithelial cells is similarly regulated by ifn-k, we monitored the kinetics of pdcd expression in hbe cells noting a fold induction in response to ifn-kwhich was back to baseline by h. the expression of mir- was induced by -fold at h post ifn-k and decreased even more briskly back to baseline levels at h. conclusion. these results indicate that like ifn-b, bacterial pamps also activate ifnk signaling which may contribute to airway inflammation without augmenting pathogen clearance. further dissection of the components of ifn-k regulation may provide targets to diminish the pathology associated with airway inflammation without compromising the ability to clear pathogens. introduction. ligation of immunoreceptor tyrosine-based activation motif (itam)associated receptors in macrophages can initiate potent induction of negative regulators, including anti-inflammatory cytokine il- , signaling inhibitors socs , abin , a and transcriptional repressor hes [ ] . however under inflammatory conditions, the strong inhibitory pathway initiated by itam-bearing receptors was altered. in macrophages isolated from rheumatoid arthritis patients, as well as in blood monocytes/macrophages primed with ifn-c, itam-mediated induction of il- and other inhibitory molecules was markedly attenuated. here, we investigated mechanisms underlying the suppression of ifn-c on itam-mediated inhibitory pathway. methods. we utilized primary human macrophages in this study, and compared gene expression and signaling in ifn-c-primed versus non-primed cells, by q-pcr and western blotting respectively. fibrinogen (fb) was used to ligate itam-bearing b intergrin [ ] . a combination of biochemical and genetic approaches was conducted to investigate the role of gsk , including pharmacological inhibitors of gsk kinase and rna interference of gsk a/b genes. we further analyzed the subcellular localization of gsk , and its potential substrates, including b-catenin. microarray experiment was performed to determine the target genes of b-catenin. results. we found that ifn-c markedly increased itam-regulated gsk kinase activity and nuclear accumulation. inhibition of gsk using pharmacological inhibitors or rna interference reversed ifn-c suppression of il and hes , suggesting that gsk mediated the downregulation of inhibitory gene expression by ifn-c. b-catenin, a major substrate of gsk , is a transcription factor recently implicated in induction of anti-inflammatory mediator il- in murine dendritic cells [ ] . however, effective knockdown of b-catenin by sirnas had little effect on itam-mediated gene expression, as assessed by genome-wide microarray analysis. in contrast, we found that the expression of ap- , another target of gsk [ ] , as well as its nuclear accumulation was significantly suppressed by ifn-c. conclusion. we provided several lines of evidence that gsk was the major mediator in the crosstalk between itam and ifn-c signaling. ap- transcription factor, but not b-catenin, was the major target of gsk in this scenario. these findings yield insight into mechanisms of crosstalk between itam-associated receptors and ifn-c that are important for the orchestration of cytokine production and inflammation. disclosure of interest: none declared. indirect inhibition of toll-like receptor and type i interferon responses by itam-coupled receptors and integrins integrin signaling in neutrophils and macrophages uses adaptors containing immunoreceptor tyrosine-based activation motifs activation of beta-catenin in dendritic cells regulates immunity versus tolerance in the intestine ifn-gamma suppresses il- production and synergizes with tlr by regulating gsk and creb/ap- proteins ifn-a signals through the jak-stat pathway to induce expression of ifn-stimulated genes (isgs) with antiviral functions. usp is an ifn-inducible negative regulator of the jak-stat pathway. upregulation of usp results in a long-lasting desensitization of ifn-a signalling. as a result of this ifn-induced refractoriness, isg levels decrease back to baseline despite continuous presence of the cytokine. pegylated forms of ifn-a (pegifn-a) are currently in clinical use for treatment of chronic hepatitis c virus infection. pegifn-as show increased anti-hepatitis c virus efficacy compared to nonpegylated ifn-a. this has been attributed to the significantly longer plasma half-life of the pegylated form. however, the underlying assumption that persistently high plasma levels obtained with pegifn-a therapy key: cord- -hkyiy gm authors: nagata, noriyo; iwata, naoko; hasegawa, hideki; fukushi, shuetsu; harashima, ayako; sato, yuko; saijo, masayuki; taguchi, fumihiro; morikawa, shigeru; sata, tetsutaro title: mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice date: - - journal: the american journal of pathology doi: . /ajpath. . sha: doc_id: cord_uid: hkyiy gm advanced age is a risk factor of severe acute respiratory syndrome (sars) in humans. to understand its pathogenesis, we developed an animal model using balb/c mice and the mouse-passaged frankfurt isolate of sars coronavirus (sars-cov). we examined the immune responses to sars-cov in both young and adult mice. sars-cov induced severe respiratory illness in all adult, but not young, mice on day after inoculation with a mortality rate of to %. moribund adult mice showed severe pulmonary edema and diffuse alveolar damage accompanied by virus replication. adult murine lungs, which had significantly higher interleukin (il)- and lower il- and il- levels before infection than young murine lungs, rapidly produced high levels of proinflammatory chemokines and cytokines known to induce macrophage and neutrophil infiltration and activation (eg, tumor necrosis factor-α). on day after inoculation, young murine lungs produced not only proinflammatory cytokines but also il- , interferon-γ, il- , and il- . adult mice showed early and acute excessive proinflammatory responses (ie, cytokine storm) in the lungs after sars-cov infection, which led to severe pulmonary edema and diffuse alveolar damage. intravenous injection with anti-tumor necrosis factor-α antibody hours after infection had no effect on sars-cov infection. however, intraperitoneal interferon-γ injection protected adult mice from the lethal respiratory illness. the experimental model described here may be useful for elucidating the pathophysiology of sars and for evaluating therapies to treat sars-cov infection. in the severe acute respiratory syndrome-associated coronavirus (sars-cov) epidemic of winter to , ϳ people ( % of the Ͼ sars patients) suffered progressive respiratory failure and died. [ ] [ ] [ ] [ ] [ ] common symptoms of sars include fever, nonproductive cough, myalgia, and dyspnea. an age of years or older, co-morbid disease, male sex, high neutrophil counts, and several biochemical abnormalities are associated with poor outcomes. - the sars-cov spike (s) protein mediates the infection of cells bearing an appropriate receptor. one such receptor is angiotensin-converting enzyme (ace ), which binds sars-cov s protein with high affinity. [ ] [ ] [ ] [ ] that the binding of sars-cov to ace may contribute to sars-cov-associated pathology is suggested by several reports showing that angiotensin ii expression promotes severe lung failure on acute lung injury whereas ace expression protects from lung injury. , however, it is likely that the acute lung injury caused by sars-cov infection is also attributable to a complex pathophysiological process in which inflammatory cytokines released by activated alveoli macrophages induce immune system dysregulation. [ ] [ ] [ ] [ ] to understand the pathogenesis of sars-cov, the sars-cov susceptibility of experimental animals such as monkeys, cats, ferrets, mice, pigs, guinea pigs, ham-sters, chickens, and rats has been investigated. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] all of these animals are susceptible to sars-cov after intrarespiratory inoculation and exhibit virus excretion in pharyngeal or nasal swabs, histopathological pulmonary lesions, and seroconversion. however, the course of infection in these animals is shorter than that in humans. as in humans, an advanced age correlates positively and independently with adverse outcomes and is a predictor of mortality in animal models. - moreover, sars-cov isolates replicate better in aged balb/c mice than in younger mice. it is likely that the correlation between poor outcome and advanced age reflects the weakened immune responses of the elderly, in particular their impaired cytokine responses. this is significant because cytokines regulate the immune response to infection. indeed, analysis of the cytokine responses of elderly individuals to respiratory infections that lead to severe pulmonary diseases (eg, listeria monocytogenes, respiratory syncytial virus, influenza virus) - have revealed unbalanced th -type and th -type responses. we recently succeeded in establishing a rat model of sars using rat-passaged sars-cov. although the ratpassaged sars-cov was not lethal, it induced more severe pathological lesions in adult f rats than in young rats. we found that the severe inflammation in the adult rats was associated with high levels of inflammatory cytokines in the serum and lung homogenates, especially interleukin (il)- , along with low levels of the immunosuppressive cytokine il- . il- is an inflammatory cytokine that is produced by monocytes, leukocytes, endothelial cells, fibroblasts, and alveolar epithelial cells. sars patients have significantly elevated serum il- levels. il- is produced by macrophages, th lymphocytes, and b cells and inhibits tumor necrosis factor (tnf)-␣ production and neutrophil activation in lipopolysaccharide-induced acute lung injury, thereby suppressing lung tissue injury. it has been reported that serum il- levels increase in sars patients during the convalescence phase. in this study, we established a new and more useful experimental small animal model for sars by using balb/c mice and mouse-passaged sars-cov. this model allows us to better characterize the virus-host relationship and determine which immune responses are antiviral and which are pathogenic. here, we sought to determine why sars-cov infection is more frequently lethal in elderly patients by comparing sars-cov-infected adult and young mice in terms of their pulmonary pathology and immune responses. the frankfurt isolate of sars-cov used in this study was kindly supplied by dr. john ziebuhr, institute of virology and immunology, university of wü rzburg, wü rzburg, germany. the virus was propagated twice in vero e cells purchased from american type cell collection (manassas, va) that were cultured in eagle's minimal essential medium (mem) containing % fetal bovine serum, iu of penicillin g, and g of streptomycin per ml. titers of this stock virus were expressed as % of the tissue culture infectious dose (tcid )/ml on vero e cells, which was calculated according to the behrens-kä rber method. work with infectious sars-cov was performed under biosafety level conditions. compared to the original virus, the frankfurt isolate used in our laboratory has one amino acid change at position (his to tyr) in the s protein and another in open reading frame (orf) a (ala to ser). these changes presumably arose during the passage through vero e cells. female -week-old or -month-old balb/c mice were purchased from japan slc (shizuoka, japan) and maintained in specific pathogen-free facilities. on experimental infection, these animals were housed in biosafety level animal facilities. these animal experiments were approved by the animal care and use committee of the national institute of infectious diseases, tokyo, japan. the frankfurt isolate of sars-cov was serially passaged times in -week-old female balb/c mice, as follows. after intranasal inoculation, three mice were sacrificed on day after inoculation and their bronchoalveolar wash fluids were collected. these bronchoalveolar fluids were then used to inoculate three additional balb/c mice, whose bronchoalveolar fluids on day after inoculation were used to inoculate fresh mice. after such passages in mice, the lungs were removed under sterile conditions, washed three times, and homogenized in ml of phosphate buffer containing . % bovine serum albumin, iu of penicillin g, l of streptomycin, and g of amphotericin b per ml. the lung homogenates were centrifuged at ϫ g for minutes, and ml of the supernatants in ml of mem containing % fetal bovine serum were used to infect vero e cells. after hour of adsorption, the inoculum was removed and mem containing % fetal bovine serum was added. the cell cultures were incubated at °c with % co for days and then treated once with freezethawing. after centrifugation at ϫ g for minutes, the supernatants (referred to here as f-musx-veroe ) were used as the virus inoculum. compared to the original virus, f-musx-veroe has amino acid mutations in the s protein at positions (asp to glu) and (his to tyr); the latter change is identical to one of the mutations found in the frankfurt isolate. in the completely sequenced genome, f-musx-veroe also has two additional mutations in orf a (phe to leu) and orf ab (thr to ile). the mutation in orf a found in the frankfurt isolate was not present. mice were anesthetized by intraperitoneal injection with a . ml/ g body weight mixture of . mg ketamine and . mg xylazine. the animals were then inoculated intranasally in the left nostril with the frankfurt isolate or f-musx-veroe ( ϫ tcid in l) and observed for clinical signs. body weight was measured daily for or days. infected animals were also sacrificed at various time points after inoculation to analyze virus replication, hematology, cytokine expression, and pathology (n ϭ in each group). twenty percent (w/v) tissue homogenates of the lung, maxilla (including the nasal cavity), cervical lymph node, spleen, liver, and kidney were prepared in mem containing % fetal bovine serum, iu penicillin g, g streptomycin, and . g amphotericin b per ml (mem- fbs). bronchoalveolar and nasal wash fluids were also collected for analysis of virus replication. viral infectivity titers of respiratory tract and wash fluids were determined as described above. virus isolation from other tissues was performed by blind passage after freezing and thawing the first-round passage using vero e cells. total blood cell counts in peripheral blood collected in sodium-heparinized tubes were measured by an autoanalyzer (cell tuck; nihon koden, tokyo, japan). neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts were determined by microscopic analysis. antibodies used for flow cytometry were anti-cd -phycoerythrin-cy (ebioscience, san diego, ca), anti-cd ␤phycoerythrin (santa cruz biotechnology, santa cruz, ca), and anti-pan-nk cells-fluorescein isothiocyanate (ebioscience). cells incubated with these surface-binding antibodies were fixed in % paraformaldehyde in phosphate-buffered saline (pbs) and subjected to flow cytometry (epics elite; beckman coulter, fullerton, ca) using expo cytometer software (beckman coulter). homogenized lung tissue samples were diluted : with cell extraction buffer [ mmol/l tris, ph . , mmol/l nacl, mmol/l edta, mmol/l egta, mmol/l naf, mmol/l na p o , mmol/l na vo , % triton x- , % glycerol, . % sodium dodecyl sulfate, and . % deoxycholate (biosource international, inc., camarillo, ca)], incubated for minutes on ice with vortexing at minute intervals, and then centrifuged at , ϫ g for minutes at °c. supernatants were diluted : in assay diluent of the mouse cytokine -plex antibody bead kit (biosource international). sera and the % lung homogenate supernatants were subjected to ultraviolet irradiation for minutes to inactivated virus infectivity and stored at Ϫ °c until they were used to determine the presence of mouse cytokines, namely, basic fibroblast growth factor, gm-csf, interferon (ifn)-␥, il- ␣, il- ␤, il- , il- , il- , il- , il- , il- p /p , il- , il- , ip- , keratinocyte chemoattractant (kc), monocyte chemoattractant protein (mcp- ), mig, mip- ␣, tnf-␣, and vascular endothelial growth factor (vegf), by using the mouse cytokine -plex antibody bead kit and luminex tm (luminex co., austin, tx). animals (n ϭ in each group) were anesthetized and perfused with ml of % phosphate-buffered formalin. fixed lung, heart, kidney, liver, spleen, small and large intestine, brain, spinal cord, and maxilla (including nasal cavity) tissues were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin. maxilla samples were decalcified in phosphate-buffered saline (ph . ) plus % edta before being embedded. immunohistochemical detection of the sars-cov antigens was performed on paraffin-embedded sections, as follows. after deparaffinizing with xylene, sections were rehydrated in ethanol and immersed in pbs. antigens were retrieved by hydrolytic autoclaving for minutes at °c in mmol/l sodium citrate-sodium chloride buffer (ph . ). after cooling, the sections were immersed in pbs. endogenous peroxidase was blocked by incubation in % hydrogen peroxide in methanol for minutes. after washing in pbs, the sections were incubated with normal rabbit serum for minutes, and then with rabbit antibody against sars-cov , overnight at °c. after three washes in pbs, the sections were incubated with biotin-conjugated anti-rabbit igg for minutes at °c, followed by reaction with streptavidin-peroxidase for minutes at room temperature. peroxidase activity was detected by development with diaminobenzidine containing hydrogen peroxide. nuclei were counterstained by hematoxylin. sars-cov-and mock-infected adult and young mice were euthanized , , and days after inoculation by exsanguination under excess ether anesthesia, after which the lungs were harvested for pathological examination (three mice per group). mock infection was performed by using mem containing % fetal bovine serum. for staining with anti-mac- and anti-surfactant d (sp-d) antibodies and to detect sars-cov antigens, the lungs were fixed with % paraformaldehyde in pbs at °c for to hours and embedded in paraffin according to the manufacturer's instructions (bd biosciences pharmingen, san diego, ca). the paraffin-embedded sections were then subjected to a double-immunofluorescence staining method using a polyclonal rabbit antibody against sars-cov and the skot monoclonal mouse antibody against nucleocapsid protein or a monoclonal rat anti-mac- antibody against mouse mononuclear phagocytes (bd biosciences pharmingen), and a poly-clonal rabbit anti-sp-d antibody (chemicon international, inc., billerica, ma). briefly, after deparaffinization with xylene, the sections were rehydrated in ethanol and immersed in pbs. antigens were retrieved by hydrolytic autoclaving for minutes at °c in mmol/l sodium citrate-sodium chloride buffer (ph . ). after cooling, the sections were immersed in pbs, and then incubated with primary antibodies overnight at °c. to block background staining, normal donkey serum or the m.o.m. immunodetection kit for primary mouse monoclonal antibody (vector laboratories, burlingame, ca) were used according to the manufacturer's instructions. after three washes in pbs, the sections were incubated for minutes at °c with biotin-conjugated secondary antibodies, ie, a donkey anti-rat serum (jackson immunoresearch, west grove, pa) to detect the mac- antibody or a goat anti-rabbit serum (jackson immunoresearch) to detect the sp-d antibody. after three washes in pbs, the sections were incubated with streptavidin-alexa fluor (molecular probes, eugene, or) for minutes at room temperature. after three washes in pbs, to detect the sars-cov antibodies (the skot or the rabbit antibody), the sections were incubated with anti-rabbit or anti-mouse alexa fluor (molecular probes) for minutes at room temperature. the sections were counterstained with to-pro- nucleic acid staining (molecular probes) and images were captured and analyzed by confocal laser microscopy (fluoview, fv ; olympus, tokyo, japan). three hours after intranasal inoculation of f-musx-veroe ( ϫ tcid in l), adult ( -month-old) balb/c females mice were injected intravenously with l of anti-mouse tnf-␣ rat monoclonal antibody ( g/l, biosource), or isotype-matched control rat antibody ( g/l; mp biomedicals, solon, oh) in pbs, or injected intraperitoneally with l of recombinant mouse ifn-␥ ( . g/l; r&d systems, minneapolis, mn) or intraperitoneal injection with l of pbs/ . % bovine serum albumin was used as control. at least two independent experiments were performed (n ϭ or per group). sars-cov-and mock-infected mice were injected intravenously with l of % evans blue dye (tokyo kasei, tokyo, japan) hour before sacrifice (n ϭ in each group). mock infection was performed by using mem containing % fetal bovine serum. after perfusion with isotonic saline, the whole lung was removed and immersed in % phosphate-buffered formalin. the fixed lungs were immediately frozen in cold acetone with dry ice in % o.c.t. compound (sakura finetechnical co. ltd., tokyo, japan). cryosections ( m) (cm ; leica, wetzlar, germany) were mounted on mas-coated slides (matsunami, osaka, japan), air-dried, and examined with a fluorescence microscope. statistical significance was determined by student's ttest. p values Ͻ . were considered significant. the frankfurt isolate was passaged twice on veroe cells and then serially passaged times in young balb/c mice ( -week-old females) by intranasal inoculation of bronchoalveolar fluids from infected mice. the f-musx-veroe strain showed higher replication and pathogenicity in the respiratory tract of young balb/c mice than the original frankfurt isolate, as follows (figure , a-e). on day after inoculation, f-musx-veroe replication in the lung washes was higher than that of the original frankfurt isolate (p ϭ . ) but lower in the nasal washes (p Ͻ . ) ( figure a ). compared to frankfurt isolate-inoculated young mice, the f-musx-veroe inoculated young mice also evinced more lung inflammation, as shown by neutrophil, macrophage, and lymphocyte infiltration and virus antigen-positive cells in the alveolar spaces ( figure , b-e). however, the f-musx-veroe -inoculated young mice did not develop any obvious respiratory illnesses, although they did show transient weight loss for a few days after inoculation (data not shown). because advanced age is associated with higher mortality in human sars patients and sars-cov replicates better in aged mice, - , we experimentally infected -month-old (adult) female balb/c mice with f-musx-veroe or the frankfurt isolate. although none of the mice showed clinical signs of illness after intranasal inoculation with frankfurt isolate, all f-musx-veroe -inoculated mice became severely ill, as revealed by significant weight loss (ϳ % of their initial body weight), hunching, ruffled fur, and dyspnea on day after inoculation ( figure f ). three of the ten mice became moribund and died of severe respiratory illness on days , , and after inoculation ( % mortality rate). the surviving animals recovered their body weight during days to after inoculation. these results demonstrated that serial in vivo passage of sars-cov in mice increased the virulence of the virus, especially in adult mice. thus, we characterized the clinical and pathological features of f-musx-veroe -infected young and adult mice up until day after inoculation in more detail. the young mice again showed transient weight loss of up to . % (sd ϭ . %) during days to after inoculation but had recovered their weight by day after inoculation ( figure a ). in contrast, the adult mice showed continuous weight loss of up to . % (sd ϭ . %) of their initial body weight that continued until day after inoculation. all adult mice showed virtually identical clinical manifestations during days to after inoc-ulation (such as hunching and ruffled fur) that were not observed in the young mice. severe respiratory symptoms such as dyspnea were also observed in the adult mice from days after inoculation onwards. in this experiment, % of the adult mice had died by day after inoculation. the lungs of infected young and adult mice were weighed on days to after inoculation. the progressive increase in lung weight of the adult mice suggested the development of pulmonary edema (figure , b and c). by day after inoculation, the adults showed significantly greater lung weight changes than the young mice (p Ͻ . ). the lungs of infected young and adult mice were also subjected to histopathological analysis on days to after inoculation (figure , a-h) . on day after inoculation, both young and adult mice had antigen-positive epithelial cells in the bronchi and alveoli. the antigenpositive cells in the alveoli were considered on the bases of morphology and immunohistochemistry to be mainly type ii pneumocytes (figure , a and e; see supplemental figure s at http://ajp.amjpathol.org). on day after inoculation, antigen-positive atrophic and necrotic cells were seen in the alveolar area of both mice ( figure b ). in addition, antigen-positive activated alveolar macrophages associated with inflammatory infiltrations were seen in the alveolar area of adult mice ( figure f ). no antigen-positive cells were seen in the bronchi on day after inoculation or afterward in either young or adult mice. on day after inoculation, the young mice had diffuse inflammatory infiltrates consisting mainly of mononuclear cells (figure , c and d), and virus antigenpositive cells were seen in the alveolar area. activated macrophages, lymphocytes, and neutrophils were seen in the alveoli on days and after inoculation. in contrast, the adult mice evinced severe pulmonary edema, and congestion on day after inoculation ( figure , g and h). in these mice, the main inflammatory cells around the adult blood vessels and alveolar area on days to after inoculation were neutrophils and activated macrophages. fibrin deposition and hyaline membrane formation in the alveolar duct and alveoli were also observed ( figure h ), and microhemorrhages was seen in the alveolar area. the adult mice also had high virus titers in the lung and maxilla (including nasal cavity) and their fluid (figure , d and e) . after the infection, virus continued to be isolated from the cervical lymph nodes, spleen, liver, and kidneys of adult mice after day after inoculation whereas virus could no longer be isolated from any young mouse tissue (apart from the lung) after this time point (table ) . to analyze the immune responses of young and adult mice after infection with f-musx-veroe , we examined their peripheral blood white blood cell counts (figure ) , and measured the levels of different chemokines and cytokine levels in their plasma and lung homogenates (figures and ). before infection (day ), the adult mice had significantly lower white blood cells counts, especially with regard to lymphocytes (including cd ϩ and cd ␤ ϩ t cells), than the , lung wash fluid, and lung homogenates of young mice on days , , and after inoculation (n ϭ per group). the detection limit was . tcid /g of tissue. asterisks indicate statistically significant differences between f-musx-veroe and the frankfurt isolate (p Ͻ . ). b-e: histopathological features of the lungs of young mice on day after inoculation. b: after infection with the frankfurt isolate, inflammatory infiltrates in the lung were not detected. moreover, very few alveolar pneumocytes and alveolar duct and alveolus epithelial cells were sars-cov antigen-positive (c, arrowheads). in contrast, extensive cellular infiltration (d) and many virus antigen-positive cells (e) were seen in the alveolar area after f-musx-veroe infection. f: clinical illness in individual -month-old adult balb/c mice after frankfurt isolate or f-musx-veroe infection (n ϭ per group). shown are the changes in body weight (expressed as percentages of the body weight on day ). the mean initial body weight of the two mouse groups (on day ) were . Ϯ . g and . Ϯ . g, respectively. significant differences in body weight change were detected on days to after inoculation. for example, the average body weight f-musx-veroe -infected adult mice on day after inoculation was . Ϯ . % of the mean day body weight. this was significantly lower than the average body weight change of frankfurt isolate-infected adult mice on day after inoculation ( . Ϯ . %). three f-musx-veroe -infected adult mice died (crosses) of severe pulmonary edema on days , , and after inoculation. young mice (figure ). after infection, the neutrophil counts in the adult mice increased; however, this change was not observed in the young mice. in young mice, relative to counts on day , lymphocyte counts decreased signifi-cantly (p Ͻ . ) on days , , and after inoculation but then recovered, cd ␤ ϩ t-cell counts decreased significantly on day and then recovered, and cd ϩ t-cell counts decreased slightly and then showed a significant to assess the lungs for pulmonary edema, the lungs were weighed after mice were sacrificed on days to after inoculation by exsanguination under anesthesia (n ϭ per group). c: lungs from virus-and mock-infected young and adult mice obtained at the indicated time points after inoculation. arrowheads indicate focal congestion. on day increase on day . in contrast, although the lymphocyte counts of adult mice also dropped and were significantly lower than day counts on days and , they did not evidence an improvement on day after inoculation. moreover, the cd ␤ ϩ t-and cd ϩ t-cell counts of the adult mice also showed significant drops on days , , and after inoculation (p Ͻ . ) but had recovered poorly on day after inoculation, unlike the counts in young mice. with regard to the pannk ϩ cells counts, both the young and adult mice showed a marked drop on day after inoculation that was followed by a brief recovery and then another loss on day after inoculation cell count loss at day and days after inoculation compared with days after inoculation in adult mice (p Ͻ . ). with regard to the cytokine responses of the mice, the lung homogenates of adult mice on day after inoculation had significantly higher levels of monocyterelated chemokines [ie, mcp- , macrophage inflammatory protein (mip- ), and ifn-␥-inducible protein (ip- )] than those from young mice ( figure ). in contrast, on day after inoculation, the lung homogenates of young mice exhibited elevated levels of these three cytokines as well as kc, monokine induced by ifn-␥ (mig), and vascular endothelial growth factor (vegf) ( figure ). compared to young mice, the lung homogenates of adult mice on day after inoculation also had higher levels of il- ␣, il- ␤, and tnf-␣, and on day after inoculation, higher levels of il- were d a y days days observed ( figure ). in contrast, the lung homogenates of young mice had significantly higher levels of ifn-␥ (on day after inoculation), il- (on days to after inoculation), il- (on days , and to after inoculation), and il- (on days to , and and after inoculation). notably, the lung homogenates of preinfected adult mice (day ) had higher il- and lower il- and il- levels than young murine lungs. these observations indicate that the patterns of cytokine/chemokines responses are different between young and adult mice after sars-cov infection. adult mice showed early and acutely excessive proinflammatory responses in the lung after sars-cov infection. to determine whether the tnf-␣ response of the adult mice and the ifn-␥ produced by the young but not adult mice played significant roles in the development of sars-like illness by the f-musx-veroe -infected adult mice, we treated adult mice with an anti-tnf-␣ antibody or ifn-␥ hours after infection (figure , a and b). although the intravenous injection with anti-tnf-␣ antibody delayed the onset of this weight loss in the infected adult mice, as well as the onset of respiratory illness, both the anti-tnf-␣ antibody-treated and control adult mice showed significant body weight loss up until days after inoculation and there were no significant differences in mortality rates between treated and control adult mice (treated adult mice: . %, % mortality rate; control adult mice: . %, . % mortality rate in two separate experiments) ( figure a ). in contrast, the ifn-␥-treated mice rapidly recovered from the illness as evidenced by their body weight loss and severe acute respiratory symptoms and all animals survived after onset days after inoculation ( figure b ). in contrast, % of the control adult mice died. . asterisks indicate statistically significant higher or lower chemokine levels in adult mice (p Ͻ . ) compared to young balb/c mice. adult mice showed earlier induction of mcp- , mip- , and ip- in the lungs than young mice but these three chemokines and mig and vegf were at significantly higher levels in the lungs of young mice on day after inoculation. to determine whether the protective effect of ifn-␥ treatment is attributable to suppression of viral replication, the virus titers on the day after inoculation of nasal washes, maxilla homogenates, lung washes, and lung homogenates of ifn-␥-treated and pbs-treated adult mice after the infection were compared. however, the two groups did not differ significantly in terms of virus titers in the respiratory tracts ( figure c ). the ifn-␥-treated mice showed much milder histopathological changes than the untreated mice because only very mild edema with slight mononuclear cell infiltration was observed around the blood vessels after the infection ( figure d ). in contrast, the pbs-treated mice exhibited severe edema and infiltration of inflammatory cells, mainly neutrophils, around blood vessels ( figure e ). by examining evans blue dye extravasation, we found the ifn-␥treated mice also had lower blood vessel permeability than the pbs-treated mice (figure , a-g) . together, these results suggest that ifn-␥ treatment hours after inoculation protected the mice from severe sars-covinduced pulmonary edema that was responsible for the death of the untreated adult mice. to understand better the pathogenesis of sars after sars-cov infection, we developed a useful experimental mouse model of sars. when the frankfurt isolate of sars-cov was serially passaged in vivo in young balb/c mice, the passaged virus (f-musx-veroe ) exhibited in-creased infectivity in the murine lung. f-musx-veroe was also able to induce severe sars-like illness in adult ( -month-old) balb/c mice and several animals died of severe pulmonary edema and acute alveolar damage. however, young ( -week-old) mice were relatively resistant to f-musx-veroe and did not evince any obvious respiratory illness. when the immune responses of in- fected young and adult mice were compared, the adult animals had significantly lower pre-existing lymphocyte and cd ϩ and cd ϩ t-cell counts, their lungs expressed significantly higher il- and lower il- and il- levels before infection, and their lungs did not show the strong up-regulation of il- (a t-cell cytokine), il- , il- , and ifn-␥ that was exhibited by the young murine lungs after infection. during the infection, the lungs of adult mice also produced inflammatory chemokine-/cytokine-related macrophages (ie, mcp- , mip- and ip- , il- ␣, il- ␤, and tnf-␣) earlier than young mice (day after inoculation) and at higher levels. they also had higher il- levels on day after inoculation. in contrast, the young mice produced high levels of inflammatory chemokines and cytokines (ie, mcp- , mip- , ip- , kc, mig, vegf, and il- ␣) on day after inoculation and produced very little il- at any time point. these observations suggest that advanced-age balb/c mice have an early and acutely excessive proinflammatory cytokine reactions (ie, a cytokine storm) in response to f-musx-veroe . this cytokine storm results in severe pulmonary edema with macrophage and neutrophil infiltration, which causes severe acute lung injury and is likely to be the cause of death in infected adult mice. supporting this scenario are reports of age-related differences in pulmonary cytokine and chemokine responses to other pathogens, especially th and th cytokine imbalances. [ ] [ ] [ ] moreover, recent reports showed that unregulated ifn responses during acute sars prevented sars-cov-infected patients from switching from innate to adaptive immunity, , and some reports described that ifn-␥ may be responsible for lung immunopathology in sars patients. , interestingly, in our model, injection of ifn-␥ hours after inoculation significantly reduced the acute pulmonary edema induced by infection. notably, these protective effects of ifn-␥ injection were not associated with reductions in viral titers in the lung. this animal model and human results seem not to be consistent. however, these observations together suggest that the failure of the adult animals to produce ifn-␥, which is a prominent immunomodulator that plays a key role in host defense against intracellular pathogens, , was responsible for their inability to control the excessive and pathogenic innate immune response to the virus in the lung. supporting this is our previous study on the rat sars model, which showed that although adult rats developed severe inflammatory reactions that led to pulmonary edema, all of the infected animals survived; significantly, the infected adult rats had similar cytokine profiles as infected adult mice except that they also produced ifn-␥. thus, a strong and timely ifn-␥ response may be needed to prevent the immunopathology induced by sars-cov infection. with regard to the inflammatory reactions in the f-musx-veroe -infected adult lung, our histopathological and chemokine/cytokine analyses suggested that on day after inoculation, the affected respiratory epithelial cells and pulmonary macrophages released acute inflammatory chemokines (mcp- , mip- , ip- ) and cytokines (il- , tnf-␣, il- ). thus, pulmonary macrophage activation associated with the release of tnf-␣ and il- appears to predominate in the early phase of the inflammatory reaction in adults. tnf-␣ and il- are classic acute inflammatory cytokines that recruit neutrophils and monocytes to the area of infection along with increasing vascular permeability, where they activate these cells so that they can eradicate the pathogens. however, when tnf-␣ is overexpressed, it can induce systemic clinical and pathological abnormalities such as depressing cardiac dysfunction and cardiomyocyte death. , this double-edged aspect of tnf-␣ function may be why there were no effects of neutralizing tnf-␣ antibodies on sars-cov infection in this model. supporting the key role of tnf-␣ in f-musx-veroe -induced immunopathology is that the lungs of the adult mice on days and after inoculation were infiltrated with predominantly neutrophils; these mice also exhibited neutrophilia. young mice did not evince these changes. this suggested that infected adult mice produce tnf-␣, which then induces neutrophil-mediated inflammation in their lungs. although the significance of this is not clear, high neutrophil infiltration in the lung is known to be the cause or the result of lung injury characterized by hyaline membrane formation and pulmonary edema. high neutrophil counts in human sars cases have also been associated with poor outcomes. , we observed in the anti-tnf-␣ antibody and ifn-␥injection experiments that the control groups, which were injected hours after infection with rat igg serum intravenously or pbs intraperitoneally, showed body weight loss starting on day after inoculation. in contrast, untreated mice (such as those shown in figures f and a) only started to lose weight on day after inoculation. it may be that the injection with serum or pbs after the infection enhanced the pulmonary edema by hyperpermeability in the lung. we previously reported that serial in vivo passage of sars-cov in f rats increased the infectivity of the virus in rats, and that this correlated with a single amino acid change in the virus receptor-binding domain of the s protein. we detected the similar change in f-musx-veroe along with another amino acid change in the receptor-binding domain of the s protein in f-musx-veroe . the amino acid change may be responsible, at least in part, for the increased replication of f-musx-veroe in the pulmonary tissue of balb/c mice. [ ] [ ] [ ] [ ] , using another sars-cov isolate (urbani), roberts and colleagues have described the development and characterization of the mouse-adapted strain ma , which is lethal for young ( -to -week-old) female balb/c mice after intranasal inoculation. compared with the original urbani isolate, the ma strain had six coding mutations that may have been responsible for the mouse adaptation and increased virulence of this strain. the fewer amino acid mutations in our mouse-adapted f-musx-veroe strain may be responsible for the fact that it is less virulent than ma . in conclusion, we developed an experimental mouse animal model of sars by using in vivo-passaged sars-cov and balb/c mice. we found that the virus was lethal in adult mice because they generated an excessive innate immune response that induced lethal pulmonary edema and diffuse alveolar damage with infiltration of macrophages and neutrophils. ifn-␥ appears to play an important role in modulating the innate immune response because ifn-␥ treatment protected the animals from the lethal respiratory illness. this study, along with our previous study and studies of humans infected with sars-cov during the sars epidemic of winter of to , - improve our understanding of sars pathogenesis because they indicate that both advanced age and virus adaptation to a particular animal species may dictate the pathogenic capacity of sars-cov. the new experimental model described here may also be useful for elucidating the pathophysiology of sars and for evaluating anti-sars-cov vaccine candidates and antiviral agents. identification of a novel coronavirus in patients with severe acute respiratory syndrome koch's postulates fulfilled for sars virus anderson lj, the sars working group: a novel coronavirus associated with severe acute respiratory syndrome osterhaus adme: newly discovered coronavirus as the primary cause of severe acute respiratory syndrome yuen ky, members of the sars study group: coronavirus as a 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authors: selvaggi, carla; pierangeli, alessandra; fabiani, marco; spano, lucia; nicolai, ambra; papoff, paola; moretti, corrado; midulla, fabio; antonelli, guido; scagnolari, carolina title: interferon lambda – expression in infants hospitalized for rsv or hrv associated bronchiolitis date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: n jc jy objectives: the airway expression of type iii interferons (ifns) was evaluated in infants hospitalized for respiratory syncytial virus (rsv) or rhinovirus (hrv) bronchiolitis. as an additional objective we sought to determine whether a different expression of ifn lambda – was associated with different harboring viruses, the clinical course of bronchiolitis or with the levels of well established ifn stimulated genes (isgs), such as mixovirus resistance a (mxa) and isg . methods: the analysis was undertaken in infants with rsv or hrv bronchiolitis. nasopharyngeal washes were collected for virological studies and molecular analysis of type iii ifn responses. results: rsv elicited higher levels of ifn lambda subtypes when compared with hrv. a similar expression of type iii ifn was found in rsva or rsvb infected infants and in those infected with hrva or hrvc viruses. results also indicate that ifn lambda and ifn lambda – levels were correlated with each other and with mxa and isg -mrnas. in addition, a positive correlation exists between the ifn lambda levels and the clinical score index during rsv infection. in particular, higher ifn lambda levels are associated to an increase of respiratory rate. conclusions: these findings show that differences in the ifn lambda – levels in infants with rsv or hrv infections are present and that the expression of ifn lambda correlates with the severity of rsv bronchiolitis. keywords ifn lambda; il- ; il- ; rsv; hrv; mxa; isg ; viral load; bronchiolitis summary objectives: the airway expression of type iii interferons (ifns) was evaluated in infants hospitalized for respiratory syncytial virus (rsv) or rhinovirus (hrv) bronchiolitis. as an additional objective we sought to determine whether a different expression of ifn lambda e was associated with different harboring viruses, the clinical course of bronchiolitis or with the levels of well established ifn stimulated genes (isgs), such as mixovirus resistance a (mxa) and isg . methods: the analysis was undertaken in infants with rsv or hrv bronchiolitis. nasopharyngeal washes were collected for virological studies and molecular analysis of type iii ifn responses. results: rsv elicited higher levels of ifn lambda subtypes when compared with hrv. a similar expression of type iii ifn was found in rsva or rsvb infected infants and in those infected with hrva or hrvc viruses. results also indicate that ifn lambda and ifn lambda e levels were correlated with each other and with mxa and isg -mrnas. in addition, a positive correlation exists between the ifn lambda levels and the clinical score index during rsv infection. in particular, higher ifn lambda levels are associated to an increase of respiratory rate. conclusions: these findings show that differences in the ifn lambda e levels in infants with rsv or hrv infections are present and that the expression of ifn lambda correlates with the severity of rsv bronchiolitis. ª the british infection association. published by elsevier ltd. all rights reserved. bronchiolitis is a disorder most commonly caused in infants by viral lower respiratory tract infections; it is characterized by acute inflammation, edema and necrosis of epithelial cells lining small airways, increased mucus production, and bronchospasm. the most common virus causing bronchiolitis is the respiratory syncytial virus (rsv). , other viruses identified as causing bronchiolitis are rhinovirus (hrv), human metapneumovirus, bocavirus, and parainfluenza. in particular, hrv has been recently shown to infect the lower airway as well and confirmed to be the second most frequent cause of bronchiolitis. it has been demonstrated that the combination of both host and viral factors profoundly influence the severity of viral associated bronchiolitis. e however, it is not yet clear whether the different subtypes of rsv (a and b) or hrv species (a, b, and c) cause different grades of bronchiolitis severity. , furthermore, the role of the innate immune response, in the pathogenesis of severe rsv or hrv disease is still to be defined in detail. e among the main players of antiviral innate immune response, the type i interferons (ifns), ifn alpha and beta, are considered cytokines crucial for anti-viral resistance and represent an early antiviral host defense mechanism against viral infections. in , a novel class of antiviral cytokines was discovered, characterized and classified as type iii ifns: ifn lambda /il- , ifn lambda /il- a, and ifn lambda /il- b. at the amino acid level ifn lambda and lambda are highly similar having % sequence identity while ifn lambda shares approximately % sequence identity with ifn lambda and lambda . the type iii ifns possess antiviral properties similar to those of type i ifns but appear to be expressed especially by epithelial cells and consequently exert host protection primarily at epithelial surfaces. e despite the fact that it is known that ifn lambda contributes to the control of viral infections in epithelial cells of respiratory tract e and that the presence of single nucleotide polymorphism around ifn lambda (il- b) can increase the risk of hospitalization for bronchiolitis at early age, the ifn lambda e expression in the respiratory tracts of hospitalized infants with rsv or hrv infections has never been addressed. hence, considering the importance of the ifn lambda in protecting the airway tract from virus infections, e we hypothesized that the heterogeneity of ifn lambda e levels could, at least in part, explain the broad clinical spectrum of rsv or hrv bronchiolitis. therefore, we evaluated whether there was a difference in the gene expression of ifn lambda e subtypes between infants with a clinical diagnosis of rsv associated acute bronchiolitis and those with hrv infection. the same analysis was also performed between rsv or hrv subtypes. in addition, to characterize the activation of type iii ifns in the airway tract of infants with rsv or hrv infections, we evaluated whether there was a coordinate activation between ifns lambda and that of mxa and ifn-stimulated gene (isg) , which are well known markers of type i and iii ifn antiviral activity. finally, to further characterize the above issues, we also assessed whether a correlation between ifn lambda e levels and demographic, virological and clinical parameters in rsv and hrv infected infants actually exist. a total of infants with single rsv or hrv infection were retrospectively selected from a total of infants admitted with a clinical diagnosis of acute bronchiolitis during three epidemic seasons ( e ) to the paediatric department of policlinico umberto i hospital. the study was approved by the ethics committees and informed consent was obtained from the infant's parents. bronchiolitis was diagnosed from the presence of a history of upper respiratory tract infection followed by the acute onset of respiratory distress with cough, tachypnea, retraction, and diffuse crackles on auscultation (wheezing alone was not considered sufficient cause for inclusion in the study). the exclusion criteria were underlying chronic disease (such as cystic fibrosis, chronic pulmonary disease, congenital heart disease, and immunodeficiency) and recurrent (more than one) wheezing episodes. , the severity of the illness was assessed clinically on the following four indications, each of which was assigned a score within the range e . in particular, on admission to hospital, the clinical severity was assigned to each infant, based on respiratory rate (< breaths/min z , e breaths/min z , > breaths/min z ) arterial oxygen saturation in room air (> % z , e % z , < % z ), presence of retractions (none z , present z , present þ nasal fare z ), and ability to feed (normal z , reduced z , endovenous z ). nasopharyngeal washings were collected in the first h after admission to the hospital from infants suffering from acute bronchiolitis, and an aliquot was tested for viruses as previously described. in particular nasopharyngeal washings were obtained with ml of sterile saline physiological solution injected into each nostril and collected with a syringe. all samples were delivered on ice within e h to the virology laboratory and on arrival, if needed, they were vortexed with beads to solve mucus. they were divided into two aliquots: one was treated for nucleic acid extraction and viral detection; the second was centrifuged at rpm for min, and each cell pellet was resuspended in ml of phenol and guanidine isothiocyanate reagent (trizol, gibco-brl, ny) and frozen at À c for gene expression analysis. a panel of reverse transcription-pcr (rt-pcr) or nested pcr assays, some in a multiplex format, were used for the detection of respiratory viruses, including rsv; influenza viruses a and b; coronaviruses oc , e, nl , and hku ; metapneumovirus; adenovirus; hrv; and parainfluenza virus types e , human bocavirus as previously reported. , the evaluation of the sensitivity of the rt-pcr or pcr tests for the respiratory viruses was made as described in our previously published papers. , in particular, the integrity of the extracted nucleic acid was tested by means of the amplification of the cellular gene beta actin. rsv or hrv-positive samples were typed as rsva-b or as hrv a-c respectively. the rsv fragment to be sequenced was obtained by re-extracting rna from rsv-positive samples and directly amplifying it using superscript one-step rt-pcr for long templates kit (life technologies, monza, italy) with two expressly designed forward primers, a-fseq -aat gat ttt cac ttt gaa- ; b-fseq -gat gat tac cat ttt gaa- , corresponding to g gene position e of rsv-a and to position e of the rsv-ba reference strains, respectively, and with one reverse primer targeting the fusion protein gene end. hrv-positive samples were retrospectively amplified with primers widely used for genotyping targeting bases of the untranslated region ( utr) central portion. the mrna copy content of ifn lambda e , mxa and isg was measured by a real-time exonuclease rt-pcr assay using the light cycler sequence detector (roche, monza, italy). briefly, the total cellular rna was extracted from the cells collected from nasopharyngeal washings as described, using phenol and guanidine isothiocyanate reagent (trizol, gibco-brl, ny), by following the manufacturer's instructions, and was retro-transcribed as previously specified. primer pairs and probes for ifn lambda [forward primer, -ggacgccttggaagagtcact- ; reverse primer, -agaagcctcaggtcccaattc- ; probe, fam-agttgcagctctcctgtcttccccg- 'tamra ], ifn lambda e [forward primer, -ctgccacatagcccagttca- ; reverse primer, -agaagcgactcttctaaggcatctt- ; pro be, fam-tctccacaggagctgcaggccttta- 'tamra ], mxa (forward primer, -ctgcctggcagaaaaacttac- ; reverse primer, -ctctgttattctctggtgagtctcctt- ; probe, fam catcacacatatctgtaaatctctgcccctgtt- 'tamra); isg [forward primer, -tgaagaagctctagc-caacatgtc- ; reverse primer, -gagctttatccaca-gagccttttc- ; probe: 'fam -tatgtctttcgatatg cagccaagttttaccg- tamra ] were added to the universal pcr master mix (roche) at and nm, respectively, in a final volume of ml. the coamplification of the beta-glucuronidase gene (forward primer, -tctgtcaagggcagtaacctg- ; reverse primer, -gcccacgactttgttttctg- ; probe, fam-tcaagttggaagtgcgtcttttggatgc- 'tamra) was used to normalize the amount of total rna present using the threshold cycle relative quantification [the e(delta) ct] method according to the supplier's guidelines. all the determinations were performed in duplicate. a taqman-based real-time pcr technique for rsv or hrv rna quantification was performed on all nasopharyngeal washing specimens with positive rt-pcr results for rsv or hrv respectively. briefly, viral rna was extracted from nasopharyngeal washings npw that were positive for rsv or hrv, using a qiaamp viral rna mini kit (qiagen, milan, italy). the rna was dissolved in rnase-free water and the rsv quantification was performed by taqman assay after generation of cdna using a high capacity cdna archive kit (applied biosystems, monza, italy). type-specific primers and probes for n gene of both rsv a and b or utr region of hrv a-c strains were added to the universal pcr master mix (roche) at and nm, respectively, in a final volume of ml. the standards for rsv or hrv were obtained respectively by cloning the bp of rsv n gene or bp of the utr hrv region into the pcr . plasmid using a topo ta cloning kit (invitrogen corporation, san diego, ca, usa). a linear distribution (r z . ) was obtained between and copies of rsv or hrv-dna. viral load values were log transformed for analysis and data was expressed as the log number of rsv or hrv copies per ml of nasopharyngeal washings. all the determinations were performed in duplicate. all measurements are expressed median (range) or frequency (percentage), unless otherwise indicated. the demographic and clinical characteristics of infants suffering from rsv or hrv associated bronchiolits were compared using the mannewhitney test. differences in the clinical score index values were analyzed using student's t test. differences between infants with rsv or hrv infections and between rsv (a and b) or hrv (a and c) strains, in terms of the level of ifn lambda e measured in cells from nasopharyngeal washings, were compared using the man-newhitney test. spearman's rho coefficient was calculated in order to assess the correlation between the level of ifn lambda and ifn lambda e and between ifn lambda e and isgs, demographic, clinical and rsv or hrv viral load. differences in the ifn lambda levels in rsv infected infants divided into groups on the basis of the respiratory rate were evaluated by using kruskalewallis test. the significance was fixed at the % level. analysis was performed with spss v. . for windows. one hundred and eighteen infants, admitted over a period of years to the paediatric department of policlinico umberto i university hospital with a diagnosis of single rsv or hrv associated bronchiolitis were included ( table ) . as far as virological characteristics are concerned, a total of ( %) infants carried a single rsv infection the clinical severity was assigned to each infant with the range e , based on respiratory rate (< breaths/min z , e breaths/ min z , > breaths/min z ), arterial oxygen saturation in room air (> % z , e % z , < % z ), presence of retractions (none z , present z , present þ nasal fare z ), and ability to feed (normal z , reduced z , endovenous z ). whereas ( %) had an hrv single infection. in particular % ( / ) of the rsv positive infants had a rsva infection and the remaining had a rsvb infection. among hrv infected infants, % ( / ) had an infection with hrva and % ( / ) had an hrvc infection. interestingly, no infants had an hrvb infection. when we analyzed the demographic and clinical parameters of infants with rsv or hrv infection, there were no significant differences between infants with rsv or hrv infection and between infants with different rsv (a vs b) or hrv (a vs c) strains (table ) . on the contrary the differences between infants with rsv or hrv infection were statistical significant when the clinical score index, and the percentage of infants with fever were analyzed (table ) . moreover, the number of eosinophils was lower in infants with rsv infection compared to those with hrv infection ( table ) . the airway transcription levels of ifn lambda and ifn lambda e were evaluated using real-time rt-pcr in cells from nasopharyngeal washings collected from infants with rsv infection (n z ) or hrv infections (n z ). the gene expression level of ifn lambda and ifn lambda e showed high variability between infants with rsv or hrv infection [(coefficient of variation > %), fig. as reported in fig. (panel a) the transcript levels of ifn lambda and ifn lambda e in infants suffering from rsv infection were higher than in those with hrv infection [ifn lambda (rsv vs hrv): p z . ; ifn lambda e (rsv vs hrv): p z . ]. furthermore, we found no differences between mrna levels of ifn lambda and those of the ifn lambda e in rsv or hrv infections (fig. , panel a) . no significant differences in transcript levels of type iii ifn were observed between infants with different rsv (a vs b) or hrv (a vs c) strains (fig. , panel bec). in order to characterize the activation of type iii ifn response during bronchiolitis, we also evaluated whether there was a coordinate activation of ifn lambda subtypes in the airway tract of infants suffering from bronchiolitis. results indicate that in the respiratory tract of infants with rsv or hrv the transcript levels of ifn lambda were significantly correlated with those of ifn lambda e (table ) . furthermore, considering that ifn lambda induces the expression of ifn-stimulated genes (isgs), we evaluated whether there was a correlation between the expression of ifn lambda subtypes and that of well established isgs, namely mxa and isg . results indicate that type iii ifn mrna levels were significantly correlated with the transcript expression of mxa and isg in infants with rsv or hrv bronchiolitis ( table ) . the relationship between patient data as independent variables and ifn lambda e mrna levels measured in nasopharyngeal washings in infants with rsv or hrv bronchiolitis was analyzed (table ) . we found a significant positive correlation between the transcript level of ifn lambda and the clinical score index in infants with rsv infection (r z . , p z . ) but not in those hrv infected (table ). in particular, ifn lambda seems to be associated to an increase in the respiratory rate during rsv infection. indeed, as showed in fig. , when rsv infected infants were divided into groups on the basis of the respiratory rate (< breaths/min, e breaths/ min, and > breaths/min), there was a significant difference between the groups (p z . ). specifically, infants with > respiratory breaths per minute showed higher gene expression of ifn lambda compared with infants with < or e respiratory breaths per minute. in contrast, we failed to detect any correlation between the ifn lambda e gene expression levels and age, weight, number of days of hospitalization, and several immunological and biochemical parameters (numbers of neutrophils, lymphocytes, eosinophils or platelets, and levels of glycemia, sodium, c-reactive protein and hemoglobin). in addition, no differences were detected in ifn lambda e gene expression for male and female and between infants with fever or without fever (data not shown). in an attempt to determine whether rsv or hrv load influences the gene expression of type iii ifn, levels of rsv-or hrv-rna were analyzed respect to the expression of ifn lambda subtypes. no significant correlations were observed between rsv or hrv load and the levels of ifn lambda e (table ). it has been proposed that ifn lambdas are likely one of the main ifn produced during innate responses to respiratory viruses in the airway tract, , , , in line with the observations that the expression of the ifn lambda receptor is limited primarily to epithelial surfaces including that of the lung. , however the in vivo role of these cytokines in the host innate immune response to rsv or hrv infection is yet to be defined. our study gave some significant new insights into this complex issue. in particular, we found that in the respiratory tract of infants infected with rsv or hrv, there is a coordinate expression of different subtypes of ifn lambda: such an expression parallel also with those of mxa and isg , well established antiviral proteins induced by type i and iii ifns. this data could suggest that the airway tract is responsive to type iii ifns and that rsv or hrv caused a coordinate induction of ifn lambda subtypes that in turn can regulate antiviral pathways. however since both isgs analyzed are also regulated by ifn type i which is known to be produced during viral infections and to share the same pathways with ifn lambda, it remains unclear to which extent ifn lambda might specifically contribute to antiviral immunity against rsv or hrv in infants suffering from bronchiolitis. in this study we also compared the activation of ifn lambda e in the respiratory tract of infants suffering from rsv or hrv associated bronchiolitis. results demonstrated that in cells collected from nasopharyngeal washings of rsv positive infants there are higher mrna levels of type iii ifns compared to those observed in infants with figure gene expression of ifn lambda e during rsv or hrv bronchiolitis. the levels of type iii ifns were evaluated by using rt-taqman based real time pcr assays in cells from nasopharyngeal washings collected from infants suffering from rsv hrv infection. this is particularly interesting considering that it is known that the rsv ns and ns proteins can suppress in vitro the induction of ifn lambda. however, whether such response reflects host reactions for counteracting the ns /ns viral interference on ifn lambda induction is actually unknown. in our previous studies, we also observed a higher transcript level of isgs as well as of most pattern recognition receptors in infants with rsv compared to those with hrv infections. , all these findings may suggest that rsv infection are generally associate to a more robust innate immune response compared to those caused by hrv. in agreement, garcia et al. reported that concentrations of several cytokines in nasal wash tend to be higher in children with rsv than in those with hrv. in contrast, jartii et al. found that children with hrv-associated wheezing episodes show increased concentrations of th and th cytokines compared with those with rsv. however they measured cytokines concentrations in serum rather than respiratory tract secretions during wheezing episodes. as far as hrv is concerned, there are no studies on the evaluation of ifn lambda expression during hrv bronchiolitis: however wheezing during the first hrv infections is considered a risk factor for subsequent asthma development. interestingly, a deficient ifn lambda response to hrv infection has been reported in childhood in asthmatic subjects irrespective of their atopic status and in atopic patients without asthma. furthermore, contoli et al. reported a deficient induction of ifn lambda by hrv in asthmatic primary bronchial epithelial cells and alveolar macrophages, which was highly correlated with severity of hrv-induced asthma exacerbation and virus load in experimentally infected human volunteers. in apparent contrast, the presence of higher ifn lambda levels were recently associated with worsening illness of hrv associated asthmatic exacerbations in children. undoubtedly, the complex correlation between cytokine responses and viral infections deserves more studies to be performed carefully monitoring the various components of innate immunity during the natural course of respiratory viral infections. in this study, we also found no differences in the expression of type iii ifns between infants infected with rsva or rsvb subtypes and between those infected with hrva or hrvc strains, suggesting that the specific strain of rsv or hrv would not affect diversely the rate of activation of antiviral response. as far as the influence of specific rsv or hrv strains on the clinical course of bronchiolitis is concerned, no significant differences in the clinical characteristics between rsv (a vs b) or hrv (a vs c) infected infants were also found. however it must be considered that the samples size of infants analyzed in this study when divided according to the specific rsv or hrv strain was too small. conflicting results have been published on rsv (a or b) and hrv (a-c) association with recurrent gravity of respiratory diseases. , , e in particular, many, but not all, of the published studies , e found that hrv-c is associated with more severe lower respiratory disease and with wheezing, compared with hrv-a. nonetheless, because of the small size of hrv-positive specimens within each individual diagnosis category, most studies suffered from low power and were unable to detect significant differences among specific diagnoses, as discussed in one of the largest study ever. moreover, few data are available on the comparison of the ability of rsv a or b to modulate innate immune responses and no studies were performed on this issue for hrv a-c. therefore we retain that no definite conclusions can be drawn on such issues. interestingly, this study also demonstrated that levels of ifn lambda seem to be associated with severity of respiratory disease in rsv but not in hrv infected infants. this observation is not unusual for rsv pathogenesis. in fact, several studies have described severe rsv disease in children who have high levels of inflammatory cytokines and chemokines produced by innate immunity in respiratory secretions. , whether or not ifn lambda contributes to augmentation of inflammation in our rsv positive infants is currently unknown. however, in addition to their antiviral effects, type iii ifns have been shown to play a critical role in regulating the adaptive immune response by acting directly on th / polarization and cytokine production. , in particular, ifn lambda has been shown to increase production of il- , il- , and il- , with no concomitant increase in il- or tnf, suggesting it may not directly engender local tissue destruction, but could contribute to the inflammatory process during bronchiolitis. a reciprocal control of ifn lambda and th -associated cytokines seems also to exist. indeed, it has been shown that there is a reciprocal regulation between ifn lambda and il- or il- which are well established th cytokines associated with increased rsv disease severity. in addition, although type iii ifns seems to be activated during in vivo rsv infection, we observed that enhancement was not related to viral loads. indirectly the lack of a direct effect of ifn lambdas on viral replication may reflect a more relevant role of the cross-talk between inflammatory citokines and type iii ifn subtypes in determining the bronchiolitis clinical course. intriguingly, there are also indications that ifn lambda signaling may be resistant to feedback mechanisms targeting ifn alpha allowing it to be a more effective and durable antiviral and immunomodulator cytokine, at least under some circumstances, that could cause a prolonged, diffuse inflammatory response in the airway tract of rsv infected infants. the presence of this strong inflammatory response is consistent with the observation of the presence of string respiratory rates in rsv positive infants with increased ifn lambda levels. on the other hand, the reduced expression of ifn lambda subtypes observed in the airway tract of hrv infected infants compared to those with rsv infection, may not be enough to activate an excessive inflammatory response affecting the clinical course of bronchiolitis. in agreement with this hypothesis, wang q et al. found that mda deficient mice with reduced ifn lambda productions show less hrv induced airway inflammation. a greater number of eosinophils was also observed in hrv infected infants than in those with rsv accordingly to our previous study. in this regards it has been recently demonstrated that eosinophils may contribute to antiviral immunity and play a beneficial role in limiting viral respiratory lung dysfunction. however, it is also believed that eosinophilia is one of the atopic features that may contribute to the higher risk to develop recurrent wheezing and asthma. furthermore, in this study in line with previous studies , , a greater clinical severity in rsv infected infants than in those infected with hrv has been observed. therefore it is conceivable that the presence of reduced airway ifn lambda response in hrv infected infants than in those with rsv infections could reflect the presence of a milder bronchiolititis clinical course caused by hrv compared to that associated to rsv. indeed, miller et al. reported that ifn lambda levels were higher in wheezing children infected with hrv compared with no-wheezing and increased with worsening symptoms. alternatively, our data could indicate that an impaired ifn lambda production during hrv infection is present, not only in asthmatic subjects, , but also in infants with bronchiolitis which can be associated with the inability to control early virus replication and to mount an adequate th / th immune response which in turn may impact on recurrent wheezing predisposition and an exacerbation pathogenesis. these results on the presence of a positive correlation between ifn lambda levels and the clinical score of bronchiolitis in rsv infected infants does not seem to be in agreement with those obtained previously on the evaluation of isgs levels in infants with bronchiolitis. this discrepancy, although unexpected, might be explained considering that several host and viral factors independently from ifn lambda can regulate, directly or indirectly, the isgs production. , in addition it has been shown that the signaling pathways which lead to the activation of ifn regulatory factor can induce transcription of ifnstimulated response elements without the involvement of ifns. e thus it remains plausible that two different players of the same biological system might exert an opposite, complementary or simply additive effect on the clinical outcome of rsv bronchiolitis. all these findings would be greatly strengthened by comparing the level of expression of mrnas encoding type iii ifns with the level of expression of other cytokines or antiviral responses (e.g. type i ifns) in the cells from nasopharyngeal washings derived from infants with rsv or hrv associated bronchiolitis. this analysis could allow us to deep understand the complex picture of the airway ifn lambda response as well as the intensity and the dynamic nature of the antiviral and inflammatory pathways associated to type iii ifn response during pediatric lower respiratory tract infections. unfortunately the collected material was just enough to perform the experiments shown in the present study and the above issues could not be addressed. another important analysis which should be, but for the above reason have not been made, is the separate analysis of ifn lambda and ifn lambda subtypes expression in order to characterize the distinct contribute of these subtypes in rsv or hrv bronchiolitis. further studies specifically aimed to address these important issues are needed. in conclusion, we have shown for the first time to our knowledge that ifn lambda e are expressed in infants suffering from bronchiolitis although their levels may be different on the basis of which virus, rsv or hrv, has been detected in the respiratory tract. in addition, we have demonstrated that ifn lambda can influence the severity of bronchiolitis caused by rsv, but not by hrv, suggesting that the rate of activation of type iii ifn response may act as a double-edged sword in some circumstance during pediatric respiratory viral infections. however, it must be underlined that several factors associated or not with the ifn system may influence the clinical severity of bronchiolitis. the latter issue is exemplified by our observations about the opposite effect exerted by isgs and ifn lambda (this study) on the clinical outcome of bronchiolitis. all these findings highlight the importance of studying the interplay between the pathways of ifn lambda subtypes and those of other inflammatory cytokines or chemokines in order to deeply understand the influence of type iii ifn response on the clinical course of respiratory diseases. substantial variability in community 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work was supported by a grant from "sapienza" university of rome to carolina scagnolari ("ricerche universitarie", anno , prot c a x hp). key: cord- - kb b rq authors: koo, bonhan; jin, choong eun; lee, tae yoon; lee, jeong hoon; park, mi kyoung; sung, heungsup; park, se yoon; lee, hyun jung; kim, sun mi; kim, ji yeun; kim, sung-han; shin, yong title: an isothermal, label-free, and rapid one-step rna amplification/detection assay for diagnosis of respiratory viral infections date: - - journal: biosens bioelectron doi: . /j.bios. . . sha: doc_id: cord_uid: kb b rq recently, rna viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and middle east respiratory syndrome-coronavirus (mers-cov), and zika virus, are a major public health threats in the world. although myriads of diagnostic methods based on rna amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. a rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. here, we report an isothermal, label-free, one-step rna amplification and detection system, termed as iroad, for the diagnosis of respiratory diseases. it couples a one-step isothermal rna amplification method and a bio-optical sensor for simultaneous viral rna amplification/detection in a label-free and real-time manner. the iroad assay offers a one-step viral rna amplification/detection example to rapid analysis (< min). the detection limit of iroad assay was found to be -times more sensitive than that of real-time reverse transcription-pcr method. we confirmed the clinical utility of the iroad assay by detecting viral rnas obtained from human respiratory samples. we envision that the iroad assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases. emerging respiratory infections caused by viruses, such as influenza (ifn) type-a and b, respiratory syncytial virus (rsv) type-a and b, human coronavirus types oc and e, parainfluenza virus (piv) types - , adenovirus, and rhinovirus, present a major public health hazard (cox and subbarao, ; van der hoek et al., ; guy et al., ; loeffelholz and chonmaitree, ) . especially, acute respiratory tract infection is the leading cause of hospitalization, and a major reason of death in infants and children in developing countries (tregoning and schwarze, ; bourgeois et al., ) . in particular, the failure to rapidly and accurately diagnose contagious respiratory viruses results in an increased number of patients and duration of infection. hence, the testing methods for accurate pathogen identification have been explored to improve the patient's outcome, allow appropriate use of antibiotics, and facilitate cost-effective care. recently, the middle east respiratory syndrome-coronavirus (mers-cov) and zika virus outbreaks in has also heightened our awareness regarding the need for improved diagnostic methods (cho et al., ; kim et al., ; gourinat et al., ) . therefore, there is a great need for an affordable, robust, rapid, accurate, flexible, and simple point-of-care (poc) testing in order to control the unpredictable pandemics. although a myriad of techniques for rapid diagnosis of pathogens have been developed, the cell culture method remains the gold standard in many laboratories and hospitals owing to the lack of an alternative high sensitivity technique (moesker et al., ; ginocchio and mcadam ) . although cell culture is a time-consuming and laborious method, it can generally detect over % of the viruses within h (ginocchio and mcadam, ; pozzetto et al., ) . rapid antigen direct tests (radt) and direct fluorescent antibody testing (dfa) have been developed as alternative techniques, which are simple, cheap, with a rapid turnaround time of - min (mahony et al., ; leland and ginocchio, ; welch and ginocchio, ; landry, ). ). however, these methods are limited by the availability of antibodies against newly found viruses and sub-families of pathogens (guy et al., ; borg et al., ; renois et al., ) . in the absence of appropriate diagnostic methods, it is impossible to rapidly predict the type of pathogen from clinical signs and symptoms, due to an overlap in the clinical phenotype of various infections. alternatively, real-time reverse transcriptase (rt) polymerase chain reaction (pcr) is the current gold standard because of its superior sensitivity, rapid turnaround time ( - h), and ability to identify multiple types of pathogens in a single test (moesker et al., ; olofsson et al., ; pillet et al., ; mentel et al., ) . as a result, many techniques based on the real-time rt-pcr, such as respplex (qiagen), infiniti system (autogenomics), jaguar system (bd), filmarray system (biofire diagnostics), and plex-id (abbott molecular), have been commercialized for the detection of respiratory viruses in clinical use (caliendo, ; hammond et al., ) . however, these are not well implemented in all hospitals, as they require a molecular diagnostic laboratory with specialized personnel, equipment, and time to validate the products with large clinical samples. meanwhile, isothermal rna amplification techniques have emerged as an alternative to rt-pcr in order to bypass thermal constraints, as rna is easily degraded at high temperatures, and by materials and reagents (agrawal et al., ; compton, ; notomi et al., ; lizardi et al., ; vincent et al., ) . many isothermal amplification methods have been developed to allow exponential amplification at both constant and low temperatures. among the methods, recombinase polymerase amplification (rpa) does not require thermal cycling and operates at a single temperature (piepenburg et al., ; shin et al., a shin et al., , a . rpa forms a complex of a primer and a recombinase enzyme to extend the dna, which negates the need for a polymerase and cycle repetition. recently, reverse transcription-based rpa technique (rpa-rt) for rna has been developed and applied for the amplification of rna from bovine coronavirus (bcov), human immunodeficiency virus (hiv), influenza (ifn), dengue virus (denv), and ebola virus (evd) (yang et al., ; lillis et al., ; el wahed et al., ; teoh et al., ; el wahed et al., ; amer et al., ) . despite the advantages of the isothermal methods, these methods still require improved sensitivity, additional steps including gel electrophoresis, and labeling with a fluorescent dye for detection. together with this, biosensors that are label-free with real-time detection have been developed to overcome the limitations of isothermal methods, such as reduced reaction time and cost by eliminating the need for cycle completion, gel electrophoresis, and labeling baeumner et al., ; yan et al., ) . recently, silicon-based photonic biosensors have been developed as a sensitive bio-molecules detection technology that does not require additional materials for signal enhancement, such as chemical amplification or labeling of the analyte (iqbal et al., ; bogaerts et al., ) . particularly, silicon microring resonators (smrs) are refractive index-based optical sensors that provide highly sensitive, label-free, real-time multiplexed detection of biomolecules near the sensor surface. the silicon photonic sensors transduce the presence of target molecules based on binding-induced changes in the refractive index proximal to the waveguide surface (iqbal et al., ; bogaerts et al., ) . furthermore, the fabrication of the smrs by comple-mentary metal oxide-semiconductor (cmos) technology ensures the ability to minimize costs while providing large scale-up capabilities and makes the microring resonator a good candidate for a disposable sensor in poc diagnostics (park et al., ; shin et al., a; de vos et al., , . in this study, we report an isothermal and rapid one-step rna amplification/detection (iroad) assay to simultaneously amplify and detect the viral rna in a label-free and real-time manner. the assay is rapid, affordable, simple, and accurate. moreover, to the best of our knowledge, this is the first proof-of concept study that combines isothermal reverse transcriptase rna amplification with rpa-rt reagents as an asymmetric rna amplification method and a smr biophotonic sensor as a label-free and real-time detection in a single chamber. we demonstrated that the detection limit of iroad assay was -times higher than that of real-time rt-pcr method. furthermore, we demonstrated the clinical utility of the iroad assay by detecting respiratory viral rnas extracted from the nasopharyngeal samples with either ifn-a/b or hcov-oc / e or rsv-a/b. the iroad assay combines amplification and detection mechanism of rna, resulting in increased sensitivity and specificity of viral rna detection. using this strategy, respiratory virus nucleic acid from human specimens was simultaneously amplified and detected within min by a grafted complementary primer on the smr in a label-free and real-time manner. therefore, it is potentially adaptable for better diagnosis across various clinical applications involving rna. to use the iroad chip as a detection system, a previously described protocol was used with slight modifications for the detailed structure and fabrication of silicon microring resonators (smrs) (shin et al., a (shin et al., , b park et al., ) . the smr sensor device was provided from one biomed pte. ltd. briefly, the iroad chip [ . cm x cm x . cm] structures such as microring structures, waveguides, and gratings, were patterned on a commercially available mm silicon-on-insulator (soi) wafer with a nm thick top silicon layer, and µm thick buried oxide layer by nm deep ultraviolet (uv) lithography. the structures were then etched into the buried oxide layer by a reactive ion-etching process, followed by the deposition of . mm high-density plasma (hdp) oxide as a top cladding layer (park et al., ; shin et al., a) . we checked the layer thickness after every lay by layer deposition as a process control for the sensor uniformity during the chip fabrication. an array of microrings was designed to consist of four rings that are connected to a common input waveguide (through). each ring had a dedicated output waveguide (drop). one of the microrings, left under the sio cladding, is used as a reference sensor to monitor temperature-induced drift. the output signals of the remaining microrings are collected through a vertical grating coupler connected to a single-mode fiber optic probe (fig. s ). the tunable laser emits light from wavelength - nm, which corresponds to frequency from . × ghz to . × ghz. the ring resonance structure has multiple resonant wavelengths (frequencies) within the above wavelength range and the resonant peak we used for monitoring the wavelength shift is at~ nm ( . × ghz). the insertion loss (il) spectrum was measured using an exfo iqs- b dwdm passive component test system (park et al., ; shin et al., a) . for operation of the iroad, the chip was prepared in three steps. first, the surface of the chip was functionalized with an amine group for immobilization of the primer, as an asymmetric technique. the smr sensors were treated with oxygen plasma and immersed in a solution of % -aminopropyltriethoxysiane (aptes) in a mixture of ethanol-h o ( : , v/v) for h, followed by thorough rinsing with ethanol and diethylpyrocarbonate (depc)-treated deionized (di) water. the sensors were cured by drying under a nitrogen stream and heating to °c for min. the sensors were then incubated with . % glutaraldehyde (gad) in depc-di water containing mm sodium cyanoborohydride for h, rinsed with depc-di water, and dried under a nitrogen stream. second, for the immobilization of the target primers (table s ) , the pre-treated sensor was prepared by incubation with the primers of ifn, hcov, and rsv in pbs ( mm) containing mm sodium cyanoborohydride for h at room temperature. after the incubation, unbound dna probes were washed away with pbs and the sensors were dried using nitrogen. to streamline the assay procedure, an acrylic well [ mm x . mm x mm] was used to enclose the sensing area. at this time, the chips were considered ready for optical measurements. lastly, we prepared the rpa-rt solution for amplification and detection of the target rna using the iroad. for an optimized reaction, . ml of rehydration buffer, ml of rnase inhibitor and water, μm dtt, . ml of primers ( mm) were mixed. one dried enzyme pellet was added to each solution and vortexed. then, . ml of magnesium acetate solution was dispensed into the cap of each tube. a unidirectional shake mode mixing protocol guaranteed a homogeneous distribution of the molecules that are necessary for the reaction in the buffer. after mixing, the total volume of μl of reaction buffer was split into five μl aliquots. the viral rna samples obtained from the patient samples were used for the detection of viral rna. to start the reactions, μl of target rna (wildtype) were added to each μl reaction aliquot. finally, we added the rpa-rt solutions containing the viral rna targets or genomic rna (negative control) to the acrylic well at room temperature. before closing the acrylic well, we added mineral oil to protect the solution from evaporation during the amplification. the iroad assay was operated at a constant temperature ( °c). a thermo-electric cooler (tec), connected to a proportional integral derivative controller (alpha omega instruments, usa), was employed to maintain the constant temperature. the resonance spectrum of the device was immediately measured and used as a reference to obtain a baseline. the smrs allow target molecules to selectively bind to the immobilized primers in the evanescent field of the resonator waveguide, subsequently causing an increase in the proportion of each wavelength. during the amplification process, the wavelength shift was collected every min for up to min to monitor the amplification of target rna in a label-free and real-time manner. conventional assays, such as end-point reverse transcription (rt)-pcr and real-time rt-pcr, were compared with the iroad assay to test its utility. the forward and reverse primers were synthesized at the usual length of around bp (table s ). the target viral rna as a template for the conventional assays was obtained from the clinical samples. the human genomic rna was used as a non-target control (negative control). the end-point rt-pcr process consisted of an initial cdna synthesis step of min at °c, followed by min at °c and cycles of s at °c, s at °c, and s at °c, and a final elongation step at °c min. viral rna ( μl) was amplified in a total volume of μl, containing x one-step rt-pct buffer (qiagen one-step rt-pcr kit), . mm deoxynucleotide triphosphate, pmol of each primer, and unit of one-step rt-pcr enzyme mix (qiagen, germany). gel electrophoresis was used to separate pcr products on a % agarose gel containing ethidium bromide (etbr). the gel was visualized using a gel doc system (clinx science instruments). for real-time rt-pcr, the following procedure was modified in the ariamx (aligent) instrument protocol. briefly, μl of rna was amplified in a total volume of μl, containing x brilliant sybr green rt-qpcr master mix, pmol of each primer, and μl of rna template. an initial cdna synthesis step of min at °c, followed by min at °c, fifty cycles of s at °c, s at °c, and s at °c, and by cooling step of °c for s. the amplified products with sybr green signals were obtained using an ariamx real-time pcr system (agilent). to check the detection limit of the iroad assay, t in vitro transcribed rna was generated with either ifn-b or hcov-oc (megascript t kit, ambion, austin, tx, usa) (moll et al., ; bustin, ) . first, we performed end-point rt-pcr to obtain the pcr product of ifn-b or hcov-oc . then, a mixture including μl of x reaction buffer, mm ntp (atp, ctp, gtp, utp), enzyme mix, and . μg of pcr product in a volume of μl was incubated at °c overnight. to get rid of cdna in the mixture, μl of dnase was added to the mixture and incubated at °c for min. after the t in vitro transcription, we performed the purification of t in vitro transcribed rna (mega clear kit, ambion, austin, tx, usa). finally, we obtained the purified t in vitro transcribed rna with either ifn-b or hcov-oc . the purified t rna of ifn-b was stored at − °c until use. the nasopharyngeal samples from the patients infected with influenza (ifn)-a/b, human coronavirus (hcov)-oc / e, or respiratory syncytial virus (rsv)-a/b were obtained using protocols approved by the institutional review board of asan medical center (amc), republic of korea. institutional approval and written informed consent from the patients were obtained. the viral rna samples were extracted from the patient samples using qiaamp viral rna mini kit (qiagen, germany) according to the manufacturer's instructions (guy et al., ; leland and ginocchio, ; welch and ginocchio, ) . we used samples at a starting volume of μl each, and eluted around μl using viral elution buffer. the extracted rna was then aliquoted, and stored at − °c until use. . . iroad assay for viral rna detection fig. illustrates the iroad assay that was designed for the clinical detection of viral rnas, which were extracted from the nasopharyngeal swab samples with either influenza (ifn)-a/b or human coronavirus (hcov)-oc / e or respiratory syncytial virus (rsv)-a/b, by the qiaamp viral rna mini kit. following the extraction of viral rna, the target region was amplified by an isothermal-based asymmetric rna amplification method through recombinase polymerase amplificationreverse transcription (rpa-rt) reagents. for the reaction of iroad assay, one primer was grafted covalently to an optical sensor surface and the other was in solution, while the temperature was kept constant at °c. the amplified viral rna targets were simultaneously detected with the grafted primer on the sensor surface in a label-free and realtime manner (fig. ) . first, selection of primer pairs used for amplification and detection in both solid-and solution-phases was critical for the specific amplification of viral rna. the primer pair sequences from each viral infection were complementary to the target sequences (table s ). in order to avoid the formation of primerdimers, one primer was immobilized and possessed a ′-amine group as an asymmetric assay on the silicon microring resonators (smr), which could be used as unique refractive index sensitive sensors that allow target molecules to selectively bind to the immobilized receptors in the evanescent field of the resonator waveguide, subsequently causing an increase in the proportion of each wavelength during the reaction. second, during the amplification process, complementary dna (cdna) was obtained from the viral rna template after reverse transcription (rt) reaction. as a result, the cdna was hybridized with the immobilized primer on the amine-modified smr surface, and then the target amplification commenced from the rpa-rt mixture. this resulted in binding of the recombinase-primer complex to doublestranded cdna by components of the proteins (namely gp , uvsx, and uvsy) to facilitate strand exchange, leading to strand elongation and an exponential increase in the number of target cdna copies. this assay was placed on an in-house thermal pad with an optical instrument to enable the simultaneous amplification and detection of viral rna. finally, the exponentially amplified targets by repetition of the reaction on the sensor surface are monitored by the wavelength shift of the microring resonator without any labeling and in a real-time manner. subsequently, the iroad assay detected the respiratory viral targets from the clinical sample by measuring the resonance wavelength shift within - min (fig. ) . we first optimized the assay protocol to simultaneous amplify and detect the viral rnas extracted from the samples. the viral rnas were extracted from several types of viral infections, such as seasonal ifn-a/ b, hcov-oc / e, and rsv-a/b. in order to determine whether the iroad assay could be useful for detection of viral rnas for clinical use in a label-free and real-time manner, we examined the utility of the iroad assay compared to the one-step end-point reverse transcription (rt)-pcr method. as shown in fig. and fig. s , the resonance wavelength shift in min using the iroad assay was . pm ± . for ifn-a, . pm ± . for ifn-b, . pm ± . for hcov-oc , . pm ± . for hcov- e, . pm ± . for rsv-a, and . pm ± . for rsv-b in the presence of the target viral rnas. we also confirmed that the same target viral rna strands were strongly amplified using the one-step end-point rt-pcr (fig. c, f and fig. s c ). the human genomic rna from hct cells was used as a negative control to monitor non-specific interaction with the non-target rna. the wavelength shift in the absence of viral rnas was . pm ± . owing to the background caused by the binding of nonspecific components such as polymerase and other chemical compounds to the sensor surface ( fig. a-b , d-e and fig. s a-b) . the difference between the presence and absence of target rna in the wavelength shift is easily distinguishable after min. as a result, the data clearly indicate that the iroad assay is able to amplify and detect the target rna simultaneously on the optical sensor in a label-free and real-time manner, as compared to the non-target rna. the detection limit of the iroad assay was comprehensively characterized using the purified viral rna samples with either t in vitro transcribed ifn-b or hcov-oc . the serially diluted samples ranging from . × to copies/reaction were used as rna templates for determination of the absolute detection limit. we determined the relative detection limit of the iroad assay, as compared to the conventional methods (end-point rt-pcr and realtime rt-pcr), using the serially diluted samples (fig. ) . in case of the iroad assay with label-free and real-time capabilities, the wavelength shift was sequentially increased based on the concentrations ( . × to copies/reaction) of ifn-b within min, as compared to the negative control. the resonant wavelength shift from . × copies/ reaction of ifn-b sample was clearly distinguishable from that of human genomic rna (fig. a) . moreover, the difference between the target (ifn-b) and non-target (human genomic rna) was observed as early as min after simultaneous amplification and detection. fig. b shows good linearity (r = . ) for different concentrations of the target after min of amplification. in the case of the real-time rt-pcr assay, the fluorescent sybr green signal was observed in rna samples diluted up to . × copies/reaction and showed good linearity (r = . ) for different concentrations of the target (fig. c ). fig. shows that the limit of detection in the iroad assay fig. . schematic representation of the principle of an isothermal, rapid and label-free one-step rna amplification/detection (iroad) assay. first, preparation of the iroad chip through the primers (forward) grafting on the optical sensor would be needed for a ready-to-use viral rna detection assay (# ). then, the mixture containing recombinase polymerase amplification-reverse transcription (rpa-rt) reagents, reverse primers, and extracted rna is added into the reaction chip (# ). during the isothermal reaction, complementary dna (cdna) is synthesized from the rna template via rpa-rt kit (# ). thereafter, recombinase/primer complexes bind to double-stranded target cdna and facilitate strand exchange at a constant temperature. after the displaced strand forms a d-loop by gp (sky blue), the immobilized primers are extended by polymerase (light green) on the surface of the silicon microring resonator (# ). the formation of two duplexes is caused by the amplification of the solid and the solution. the exponential rna amplification after the reverse transcription based on the asymmetric assay is achieved by the repetition of the process (# ). the amplification and detection of the target is simultaneously monitored by measuring the wavelength shift on an optical sensor for min. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) was times more sensitive than that of the real-time rt-pcr method. in addition, we also determined the relative detection limit of the iroad assay with the serially diluted samples of hcov-oc (fig. s ). in the iroad assay, the resonant wavelength shift from . × copies/reaction of hcov-oc sample is clearly distinguishable from that of human grna within min (fig. s a ). fig. s b shows good linearity (r = . ) for different concentrations of the target in min of amplification. the fluorescent sybr green signal in the real-time rt-pcr method was observed in rna samples diluted up to . × copies/reaction and showed good linearity (r = . ) for the different concentration of the target (fig. s c ). in the hcov-oc sample, the detection limit of the iroad assay was superior to that of real-time rt-pcr method. therefore, this device would be useful as a rapid and highly sensitive method for respiratory viral rna detection, based on a molecular diagnostic method. to validate the clinical utility of the iroad assay, we analyzed clinical nasopharyngeal samples from ifn-a patients, ifn-b patients, hcov-oc patients, hcov- e patients, rsv-a patients, and rsv-b patients ( fig. and fig. s ). we also compared efficiency of the iroad assay with that of real-time rt-pcr assay using the same samples. the primers used for the detection of ifn-a/ b, hcov-oc / e, and rsv-a/b are shown in table s . fig. shows that the resonant wavelength shifts using the iroad assay were above pm within min when the target samples were amplified with the matched target primers (fig. a-b for ifn-a/b, fig. c -d for hcov-oc / e, and fig. e -f for rsv-a/b). in order to further verify whether the target primer was amplified specifically, we used human genomic rna obtained from hct cell line as a non-target sample. the wavelength shift was below pm by non-specific targeting. as a result, the viral rnas from the variety samples were amplified and detected as positive samples when the human genomic rna samples were used as negative controls ( fig. and fig. s ) . furthermore, the respiratory viruses subtypes may cause inaccurate detection due to cross-reactivity that should be distinguishable. hence, we performed the cross-reactivity testing of the iroad assay using the clinical samples ( fig. and fig. s ). for example, when we analyzed the ifn-a samples with the ifn-a primer for the sensitivity of the assay, the ifn-b samples with the ifn-a primer were used as negative controls for the specificity of the assay or vice versa (fig. a ). using the ifn-a primer, out of ifn-a samples were detected as true positives. one sample was observed as a false negative sample. on the other hand, out of ifn-b were detected as true negatives and one was observed as a false positive. in addition, when we analyzed the ifn-b samples with the ifn-b primer, ifn-a samples were used as negative controls (fig. b ). using the ifn-b primer, all ifn-b samples were detected as true positives. on the other hand, out of ifn-a samples were detected as true negatives. one sample was observed as a false positive sample (fig. b) . in case of hcov, when we analyzed the hcov-oc samples with the hcov-oc primer, hcov- e samples were used as negative controls or vice versa ( fig. c-d) . using the hcov-oc primer, out of hcov-oc samples were detected as true positives. one sample was observed as a false negative. on the other hand, out of hcov- e samples were detected as false positives. three samples were observed as true negatives. in addition, when we analyzed the hcov- e samples with the hcov- e primer, hcov-oc samples were used as negative controls (fig. c ). using the hcov- e primer, all hcov- e samples were detected as true positives. all hcov-oc samples were detected as true negatives (fig. d ). as shown in fig. s and table s , the iroad assay with ifn-a from min showed a value of - % and . % for sensitivity and specificity, respectively. the iroad assay with ifn-b from min showed a value of % and . % for sensitivity and specificity, respectively. furthermore, the iroad assay with hcov-oc from min showed a value of . % and - % for sensitivity and specificity, respectively. the iroad assay with hcov- e from min showed a value of % for both sensitivity and specificity. although the specificity of iroad for the hcov-oc was very low ( - %) due to the lack of the samples, the iroad was found to be a rapid ( < min), highly sensitive, and specific assay for viral rna detection. on the other hand, the sensitivity and specificity of the real-time rt-pcr assay were found to be insufficient using the same samples (table s ) . therefore, we showed that the sensitivity and specificity of the iroad assay was superior to that of real-time rt-pcr method. we have developed a new isothermal rna amplification and detection assay system for rapid, simple, and label-free detection of viral rnas. the combination of isothermal amplification and optical sensor-based detection not only simplifies the assay protocol considerably, but also enhances the sensitivity of detection. the iroad assay has many innovative features, presenting a new multidisciplinary approach to diagnosis of respiratory viral infection. first, the iroad assay enhances rna amplification and detection speed through realtime detection using an optical sensor. this is a significant progress from our previous approach that utilized dna oligonucleotides to target dna; a method limited to dna only. in contrast to dna, rna is easily degraded due to instability of the samples. hence, the system should be compatible with rna to avoid degradation. in addition, the cdna synthesis step from rna is a prerequisite for the amplification and detection of rna. the iroad assay is performed by the reverse transcription to cdna synthesis in a single chip, followed by simultaneous amplification and detection in a real-time manner. second, the iroad assay is adapting the rpa-rt isothermal method ( °c), which is widely used to avoid rna degradation at a high temperature. third, this assay is based on a label-free smr sensor system and it exhibits the following properties: simplicity, scalability, multiplexing capability, affordability, and rapid analysis time. although only single viral rna detection has been reported in this study, this assay can be used to simultaneously target multiple rna molecules. finally, the iroad is a versatile technology that could be readily applied to other rna-based studies and diseases. by changing the primer sequences, it could be used to detect emerging pathogens in hospitals. this prototype is being improved for more robust operation by using an array of microrings (fig. s ) for multiple detection in clinical use. moreover, we are developing a sample-processing device to construct a fully integrated device with iroad assay. based on low-cost thin film and nonchaotropic reagents for nucleic acid extraction (shin et al., b) , the platform enables robust nucleic acid extraction from a variety of sample sources such as blood, urine, and sputum. such a system will fig. . comparison of limit of detection with iroad assay and conventional method using t -in vitro transcribed ifn-b rna. (a) resonance wavelength shift in iroad assay. the colors represent the amount of the target: black ( . × copies/reaction), gray with darker % ( . × copies/reaction), gray with darker % ( . × copies/ml), gray with darker % ( . × copies/ml), gray with darker % ( . × copies/reaction), and black dot (negative). (b) linear relationship between wavelength by iroad assay and the concentration of target in min. error bars indicate standard deviation from the mean, based on at least independent experiments. (c) linear relationship between the concentration of target and ct value of fluorescence signal by real-time rt-pcr. : . × copies/reaction, : . × copies/reaction, : . × copies/reaction, : . × copies/reaction, : . × copies/ reaction, : . × copies/reaction, : . × copies/reaction, : . × copies/reaction and : negative control.. further optimize the protocol with a large clinical cohort for the improvement of the sensitivity and specificity in clinical applications. we envision the ultimate integration of sample processing and detection into a single device to enable technology for point-of-care testing. plos biol. , e supplementary data associated with this article can be found in the online version at http://dx.doi.org/ . /j.bios. . . . key: cord- - wj gr authors: katze, michael g.; fornek, jamie l.; palermo, robert e.; walters, kathie-anne; korth, marcus j. title: innate immune modulation by rna viruses: emerging insights from functional genomics date: journal: nat rev immunol doi: . /nri sha: doc_id: cord_uid: wj gr although often encoding fewer than a dozen genes, rna viruses can overcome host antiviral responses and wreak havoc on the cells they infect. some manage to evade host antiviral defences, whereas others elicit an aberrant or disproportional immune response. both scenarios can result in the disruption of intracellular signalling pathways and significant pathology in the host. systems-biology approaches are increasingly being used to study the processes of viral triggering and regulation of host immune responses. by providing a global and integrated view of cellular events, these approaches are beginning to unravel some of the complexities of virus–host interactions and provide new insights into how rna viruses cause disease. viruses can have a devastating effect despite their small genomes. all rna viruses encode proteins that are essential for structural components and replication, and most encode proteins that function to circumvent host antiviral responses [ ] [ ] [ ] . this limited number of proteins is sufficient to ensure the entry, replication and subsequent spread of the virus. however, viruses do not self-propagate and depend on various host-cell functions to complete their life cycle. the processes of viral entry, the triggering and regulation of the host antiviral response and subsequent viral replication together result in an intricate series of interactions between virus and host. much can be learnt about the nature and complexities of these interactions by global profiling of the transcriptional changes in host cells that occur during viral infection (box ) . in this review, we discuss how functional genomic and systems-biology approaches are contributing to our understanding of interactions between rna viruses and the host, of viral pathogenesis and of host immunity to infection. rather than providing a comprehensive literature review, we present examples of how these approaches are providing insight into the interaction of viruses with innate immune defence mechanisms, the evaluation of therapeutics that target these pathways and the crucial balance between protective immune responses and immunopathology. in addition, we describe how genomic approaches are being applied to vaccine evaluation and design, and how these approaches can be combined with other high-throughput technologies to provide an improved and integrated systems-biology view of virus infection. although genomic approaches are being used to study a wide variety of viruses, we highlight the current literature through discussion of a select few. among these is influenza virus, for which the looming threat of a new pandemic and concerns regarding therapeutic and vaccine preparedness have stimulated exciting new research efforts. we also review findings relating to hepatitis c virus (hcv) infection, for which genomic analyses are being used to shed light on the response of patients to treatment with type i interferons (ifns) and the relationship between hcv replication and liver disease. in addition, we highlight studies of west nile virus, severe acute respiratory syndrome-associated coronavirus (sars-cov) and ebola virus, all of which have revealed previously undescribed strategies used by these viruses to regulate innate immunity. finally, we discuss how genomic approaches are being applied to vaccine evaluation and how genomics is being combined with other high-throughput approaches to provide a systems-biology view of virus-host interactions. viruses and innate immunity a variety of cellular signalling networks have evolved in host cells to detect and respond to viral infection. one area in which genomics-based analyses are being put to abstract | although often encoding fewer than a dozen genes, rna viruses can overcome host antiviral responses and wreak havoc on the cells they infect. some manage to evade host antiviral defences, whereas others elicit an aberrant or disproportional immune response. both scenarios can result in the disruption of intracellular signalling pathways and significant pathology in the host. systems-biology approaches are increasingly being used to study the processes of viral triggering and regulation of host immune responses. by providing a global and integrated view of cellular events, these approaches are beginning to unravel some of the complexities of virus-host interactions and provide new insights into how rna viruses cause disease. these genes contain interferon (ifn)-responsive promoters and are responsible for the antiviral, antiproliferative and immunomodulatory properties of ifn. over such genes have been identified by microarray analysis. some, such as protein kinase r, ribonuclease l, mx (myxovirus resistance ) and isg (ifn-stimulated protein of kda), have well documented antiviral activities, but the precise biological function of the majority of these genes is unknown. particularly good use is in shedding new light on the components of innate antiviral defence mechanisms and the viral strategies used to overcome them. in this section, we review recent studies in which genomic approaches have been used to provide new information on how viruses trigger and regulate innate immune pathways, and to evaluate the use of type i ifn-based therapy as a means to enhance the innate immune response to hcv. mammalian cells have specialized proteins that are responsible for the recognition of virus infection, and other proteins that elicit responses to combat the invading virus. the antiviral response is triggered when host pathogen-recognition receptors (prrs) are engaged by pathogen-associated molecular patterns (pamps) in viral proteins and nucleic acids (reviewed in refs , ) . prrs that function in virus recognition include the cytosolic double-stranded rna helicases retinoic-acid-inducible gene i (rig-i) and mda (melanoma differentiation-associated gene ) and certain toll-like receptors (tlrs) that are present on the cell surface or in endosomal membranes. after binding to viral pamps, prrs initiate intracellular signalling cascades that result in the activation of transcription factors, including ifn-regulatory factors (irfs) and nuclear factor-κb (nf-κb). these transcription factors in turn regulate the expression of hundreds of genes, such as ifns and ifn-stimulated genes (isgs) , , and pro-inflammatory cytokines and chemokines that are involved in the orchestration of the adaptive immune response (fig. ) . one way in which gene-expression profiling has been used to examine this aspect of the antiviral response is through the use of mouse embryonic fibroblasts deficient in rig-i or mda . a recent study demonstrated that west nile virus infection of wild-type cells led to the induction of irf target genes and isgs, including several subtypes of ifnα (ref. ) . this was followed by a second phase of ifn-dependent antiviral gene expression that occurred at a later stage of infection. by contrast, cells lacking rig-i had delayed or inhibited initial and secondary gene-expression responses to the virus, indicating that rig-i has an essential but not exclusive role in initiating innate immune responses to west nile virus (fig. ) . the additional deletion of mda in these cells was found to further block their ability to respond to infection, indicating that the host immune response to west nile virus also involves mda . this is a noteworthy finding, as previous studies suggested that rig-i and mda recognized a specific subset of viruses, rather than acting cooperatively as found in the response to west nile virus . the role of rig-i in the response to influenza virus infection has also been assessed . similar to west nile virus, genomic analysis of influenza virus-infected wild-type and rig-i-deficient mouse embryonic fibroblasts revealed that rig-i is necessary for the type i ifn response to this virus (fig. ) . in rig-i-deficient cells, influenza virus fails to elicit the expression of ifnβ and of many isgs, including key antiviral mediators such as irf , stat (signal transducer and activator of transcription ), ifit (ifn-induced protein with tetratricopeptide repeats ; also known as isg ) and isg (also known as ifit ). this study also showed that, unlike during infection with west nile virus, mda does not function as a secondary mediator of the response to infection with influenza virus . important next steps in these studies will be to compare the profiles of genes induced by each of these viruses -and to determine whether some genes are specific for rig-i or mda signalling -and to begin to define the involvement of these genes in innate immunity. although this biological validation process will be necessary to follow-up genomic analyses, few studies so far have included such experiments. functional genomic analyses have also been helpful in elucidating the complex transcriptional events triggered following tlr signalling. tlrs are expressed by various immune cells, including macrophages, dendritic cells and lymphocytes, and a subset of these receptors are involved in viral recognition. so far, genomic studies have largely focused on the analysis of macrophages treated with tlr ligands, such as lipopolysaccharide (lps; a component of the cell wall of gram-negative bacteria) or polyinosinic-polycytidylic acid (a synthetic mimic of viral double-stranded rna, dsrna) [ ] [ ] [ ] . to obtain a comprehensive view of the transcriptional programmes that are induced by tlr activation, elkon et al. used a computational approach to analyse geneexpression data sets derived from four studies in which human or mouse macrophages were stimulated with pathogen-mimetic agents that engage various tlrs . this analysis identified one transcriptional profile that is universally activated by all tlrs and a second profile that is specific to both tlr (which specializes in the recognition of viral dsrna) and tlr (which recognizes genomics is broadly defined as the study of genomes. the term was first adopted nearly years ago to describe the emerging discipline of using nucleotide sequencing, gene mapping and computational biology to define the structure and organization of a genome . as ever increasing amounts of nucleotide sequence information have become available, the focus of genomics has expanded to include gene function . the human genome project was a driving force in advancing both structural and functional genomics, and the nucleotide sequence information generated by this project has fuelled tremendous advances in our understanding of human health and disease. one way in which this has occurred is through the convergence of comprehensive genome sequence information with advances in high-throughput technology. today, the standard technology in functional genomics is the oligonucleotide microarray [ ] [ ] [ ] . several alternative platforms are available, with the most common being microarrays for which thousands of oligonucleotide 'probes', each corresponding to an mrna transcript, are synthesized in situ directly on a glass slide. such microarrays enable researchers to simultaneously measure the expression of virtually all genes in a genome. for 'target' preparation, mrna is extracted from experimental samples and labelled with fluorescent dyes by reverse transcription. the labelled target is then hybridized with the microarray, and the fluorescence of the features is determined using an array scanner. following image analysis, the data are subjected to a variety of bioinformatic processes to identify statistically significant changes in gene expression between samples. because each comparison yields tens of thousands of data points, mining the data for biological meaning is a formidable challenge. a variety of sophisticated commercial and open-source analysis tools are therefore used to find relationships between differentially expressed genes, to identify networks or signalling pathways that are activated or repressed and to compare gene-expression profiles between experimental samples. envelope components of viruses and cell-surface components of bacteria (such as lps)). a computational analysis of promoter sequences identified nf-κb as the key regulator of the universal response, which occurs early after tlr stimulation, and the ifn-stimulated response element (isre) as the key component of the tlr and tlr response, which is induced after the nf-κb response. this computational approach provided additional knowledge regarding the kinetics of the tlr and tlr response, the regulatory circuitry involved and the identity of the genes figure | stimulation of interferon-stimulated gene expression and initiation of antiviral activity. pathogenassociated molecular patterns (pamps) in viral proteins and nucleic acids are recognized by cellular pathogen-recognition receptors (prrs) that include rig-i (retinoic-acid-inducible gene i), mda (melanoma differentiation-associated gene ) and certain toll-like receptors (tlrs). prr-pamp interactions trigger signalling cascades that result in the activation of transcription factors, including interferon (ifn)-regulatory factor (irf ) and nuclear factor-κb (nf-κb), which induce the production of type i ifns, ifn-stimulated genes (isgs) and pro-inflammatory cytokines and chemokines. the specific process differs between antigen-presenting cells, in which both the tlr pathway and the rig-i or mda pathway are operative, and other cell types, in which only the rig-i or mda pathway is present. activation of prr signalling induces an antiviral state in all cell types, and in antigen-presenting cells it can also induce the production of pro-inflammatory cytokines and chemokines. this normally results in an innate antiviral response that controls infection until it is resolved by the adaptive immune response. however, some viruses, such as the pandemic influenza virus, elicit an aberrant or disproportional response that results in immunopathology. alternatively, viruses that suppress the type i ifn response can subvert the mechanisms of innate surveillance and diminish the potential adaptive immune response, resulting in a chronic infection. for vaccine strategies, the best induction of a broad adaptive immune response might require some degree of type i ifn response in the initial stages of infection. dcs, dendritic cells; dsrna, double-stranded rna; ifnar, ifnα receptor; il, interleukin; ips , ifnb-promoter stimulator ; oas, ′, ′-oligoadenylate synthetase; pkr, protein kinase r; ssrna, single-stranded rna; stat, signal transducer and activator of transcription; tap , transporter associated with antigen processing ; tnf, tumour-necrosis factor. chimeric scid-alb/upa mouse model a chimeric mouse model of severe combined immunodeficient (scid) mice that contain a urokinase plasminogen activator transgene driven by an albumin promoter (alb/upa). these mice can be transplanted with human hepatocytes to generate chimeric mousehuman livers, providing the only small-animal infection model for hepatitis c virus infection. activated in both the universal and tlr -and tlr mediated responses. although these studies have provided considerable information regarding the genes activated downstream of tlr activation, it will be advantageous to extend genomic analyses in the context of viral infection using cells lacking the expression of specific tlrs. the ability of a virus to establish an infection depends, at least to some extent, on its ability to block the host innate immune response or to modulate the activity of antiviral effector proteins. hcv is one example of a virus that has devised a means to block the initial triggering of the host innate immune response. several studies have shown that the hcv ns -ns a serine protease blocks the tlr -dependent activation of irf (refs , ) . this is achieved by ns -ns a-mediated cleavage of trif (toll/interleukin- (il- ) receptor-domain-containing adaptor protein inducing ifnβ), an adaptor protein that links tlr to kinases that are responsible for activating irf and nf-κb , . hcv also inhibits the ability of rig-i to activate irf (refs , , , ), which is achieved through ns -ns a-mediated cleavage of ips (ifnb-promoter stimulator ; also known as visa, cardif, mavs), a recently identified rig-i adaptor protein [ ] [ ] [ ] [ ] [ ] . in light of these findings, it is both perplexing and paradoxical that virtually all gene-expression profiling carried out using hcv-infected tissue shows the induction of isg expression, including irf target genes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the induction of isg expression is observed in liver tissue from hcv-infected patients , , and during the initial host response in acutely infected chimpanzees , , and is a major part of the transcriptional response to hcv infection in the chimeric scid-alb/upa mouse model . this poses an interesting question about the source of both type i ifns and isg expression. it is possible that isgs are mainly expressed in uninfected hepatocytes and are induced in response to exogenous type i ifn released from adjacent hcv-infected cells. alternatively, it has been suggested that t cells and plasmacytoid dendritic cells that infiltrate the liver are a possible source of hepatic type i ifns . although this is possible, it is relevant to note that hcv infection in the scid-alb/upa mouse model is also associated with the induction of hepatic isg expression in the absence of these immune cell types . other genomic studies have revealed examples of highly virulent viruses that are relatively successful at inhibiting isg expression. perhaps the best example is a characterization of the host transcriptional response of human liver cells infected with filoviruses . this study demonstrated the marked suppression of genes in key innate antiviral pathways, including those mediated by irf . interestingly, this study also suggested a correlation between the antagonism of the type i ifn response and filovirus virulence. highly virulent viruses, such as zaire ebola virus and marburgvirus, inhibit the expression of most isgs that are induced in uninfected ifn-treated cells. by contrast, the relatively non-pathogenic reston ebola virus is less inhibitory and induces the expression of more than % of these genes. the suppression of the type i ifn response by the pathogenic viruses is associated with more rapid viral spread and higher rate of viral replication than that observed during reston ebola virus infection. a comparable trend was seen in a study evaluating the host transcriptional response and inflammation in the brains of mice infected with rabies virus . this study revealed that infection with an attenuated virus results in both inflammation and the induction of expression of key isgs. however, these events are either absent or diminished during infection with a highly pathogenic rabies virus. on the basis of results with filoviruses, it would follow that attenuation of the type i ifn response would be associated with higher viral replication and spread in the case of pathogenic infection with rabies virus; however, this was not measured in the study. similarly, infection with highly virulent pseudorabies virus suppressed the induction of a subset of isgs, even in type i ifn-treated cells . together, these data suggest that the virulence of acute, highly pathogenic viruses is at least partially related to their ability to suppress the host antiviral response, which seems to allow higher levels of viral replication. genomic analyses using cells that lack rig-i (retinoic-acid-inducible gene i) show the requirement for this pathogen-recognition receptor in the induction of interferonregulatory factor (irf ) target genes and interferon-stimulated genes (isgs) by west nile virus and influenza virus. a | the infection of rig-i-deficient cells by west nile virus results in the delay and partial inhibition of isg expression. deletion of mda (melanoma differentiation-associated gene ) further blocks the response to infection (not shown), indicating that the response to west nile virus also involves mda . b | by contrast, the infection of rig-i-deficient cells by influenza virus results in a near complete inhibition of isg expression that is not further blocked by the absence of mda , suggesting that mda does not mediate influenza virus-induced gene-expression changes. pamps, pathogen-associated molecular patterns. images generated from data in refs , . suppression of innate immunity and persistent infection. evidence discussed in this review suggests that suppression of elements of the innate immune response enables extensive viral replication and increased pathogenesis. does the converse hold true for a virus such as hcv, which typically establishes a persistent infection characterized by mild (or slowly progressing) disease? some evidence suggests that this might be the case; for example, studies using the chimeric scid-alb/upa mouse model indicate that an attenuated type i ifn response is associated with higher levels of intrahepatic hcv replication together with a greater induction of lipid metabolism and oxidative-stress genes, which have the potential to cause cytopathic effects . similarly, gene-expression profiling of serial liver biopsies obtained from patients that had received an hcv-infected liver transplant shows that rapid progression of fibrosis following transplantation is associated with the suppression of genes involved in the type i ifn response, antigen presentation and the cytotoxic t-cell response . although in these studies the apparent defect in the host antiviral response is probably related to host genetics rather than viral factors, the concept that a defective innate immune response correlates with enhanced pathogenesis is still evident. it is possible that the selective pressures on persistent viruses never resulted in a need for a complete subversion of host innate antiviral responses, so such viruses use these responses to limit their replication to a level that does not significantly affect the normal functions of the host cell. conversely, acute viruses, such as filoviruses, highly pathogenic influenza virus and rabies virus, seem to have evolved to antagonize these responses following cell entry to allow immediate, high levels of replication, which subsequently facilitate virus spread and transmission. given the importance of the innate immune response in regulating virus infection, there is considerable interest in enhancing or modulating this response for therapeutic benefit. one role for genomics in this area is assisting in the evaluation of type i ifn treatment of hcv infection. combination therapy with ifnα and the antiviral drug ribavirin results in virus clearance in only ~ % of individuals infected with hcv genotype and ~ % of individuals infected with hcv genotypes or (refs - ). as ifnα is the only approved treatment for chronic hcv, there is strong interest in improving this therapy, in understanding the molecular mechanisms that underlie treatment failure and in identifying markers to accurately predict a patient's response to treatment (that is, responders or non-responders). several groups that have used transcriptional profiling of patient hepatic tissue to address these issues have found that higher levels of expression of isgs before treatment are associated with treatment failure. for example, chen et al. carried out microarray experiments on pretreatment liver tissue obtained from a cohort of patients with chronic hcv infection who subsequently underwent ifnα and ribavirin therapy . this analysis identified a set of genes, many of which are known isgs; in general these genes were more highly induced in the livers of patients that did not respond to therapy. although the authors suggest that this set of genes could therefore be used to predict the response to therapy, it remains to be determined whether they can be used to accurately predict the response in other patient cohorts. similarly, feld et al. showed that non-responders have significantly higher intrahepatic pretreatment expression levels of isgs than patients who respond to type i ifn therapy . although these studies are intriguing, it is still unclear whether there is a causal relationship between higher pretreatment levels of isgs and therapy failure. other factors, such as viral quasispecies diversity, may also be important. owing to the technical and ethical issues of obtaining sufficient liver material for gene-expression studies, investigators have also used peripheral-blood mononuclear cells (pbmcs) to evaluate the response to treatment , . an example is virahepc, a multicentre study designed to define the differences in response rates among caucasian and african americans and to identify host and viral parameters associated with a lack of response to treatment . overall, this study showed that, during the first days of treatment, a lower level of induction of known isgs is associated with non-responsiveness to type i ifn treatment. however, in many cases, these differences are not strikingly dissimilar between responders and non-responders. the implication of such minor differences with respect to antiviral function is uncertain and the feasibility of using them for predicting a patient's response is questionable. in addition, analyses using pbmcs should be interpreted with caution, as a recent study showed that the transcriptional response to type i ifn treatment is significantly different in the blood and the liver of hcv-infected chimpanzees, presumably owing to the absence of hcv replication in pbmcs . although it has not yet been evaluated, this will almost certainly hold true for humans as well. an alternative mechanism of a failed response to type i ifn treatment could involve the induction of genes associated with ifn inhibitory pathways . walsh et al. found significantly increased intrahepatic expression of the gene encoding suppressor of cytokine signalling (socs ) in patients who did not respond to type i ifn treatment . enhanced intrahepatic socs expression is also thought to contribute to the non-responsiveness of hcv-infected chimpanzees to type i ifn therapy . however, a separate evaluation of patients for intrahepatic socs mrna expression before antiviral therapy actually found higher levels of expression in those patients who went on to respond successfully to type i ifn treatment . therefore, the relationship between treatment failure and induction of type i ifn inhibitory pathways is currently less clear than that between higher pretreatment levels of expression of isgs and treatment failure. there are still surprisingly few answers to the fundamental question of how virus infection results in disease pathology. although the mechanisms are certain to be different for each virus, a common theme is that there is abarrently high and sustained nature reviews | immunology infection resolves immunopathology a crucial balance between protective immune responses and immunopathology , . although the innate immune response is designed to target and eliminate invading pathogens, genomic analyses have indicated that some viruses, such as the highly virulent influenza virus that was responsible for the pandemic, elicit aberrant or disproportional innate immune responses that may also harm the host. the influenza virus pandemic (known as the spanish flu) killed as many as million people worldwide , and several studies have begun to provide clues to what made this virus so deadly (reviewed in . although genomic analyses have previously been carried out using engineered viruses containing one or more genes from the pandemic virus , , a major advance in the ability to study this virus came from its reconstruction based on nucleotide sequence information . genomic analyses of lung or bronchial tissue derived from mice or macaques that were infected with the reconstructed virus indicate how the beneficial role of the innate immune response can be tipped towards immunopathology. mice infected with the reconstituted influenza virus show severe pulmonary pathology and an increased and accelerated transcriptional activation of immuneresponse genes . this includes a marked activation of genes associated with pro-inflammatory and cell-death pathways by hours after infection (fig. ) , which remain unabated until the death of the animals. this response is in contrast to the less dramatic and delayed host immune responses (and less severe disease pathology) in mice that were infected with influenza viruses containing only subsets of genes from the virus, including the haemagglutinin (ha) and non-structural protein (ns) genes, or the ha, neuraminidase (na), matrix (m) and nucleoprotein (np) genes. these findings suggest that enhanced pro-inflammatory and cell-death responses can contribute to severe immunopathology. an additional study that evaluated the host response to the influenza virus using a cynomologus macaque (macaca fascicularis) infection model produced similar results . in macaques, the virus replicates to high levels and spreads rapidly throughout the respiratory tract of infected animals, causing severe lung damage and the massive infiltration of immune cells throughout the course of infection. functional genomic analyses of bronchial tissue revealed that the virus triggers the aberrantly high and sustained expression of numerous genes involved in the innate immune response, including pro-inflammatory cytokines and chemokines. although the timing of the response is somewhat different, the increased and sustained host response in macaques that were infected with the virus is similar to that observed in mice. these studies reveal similarities and differences in the host response to contemporary and pandemic influenza virus infection. first, contemporary and viruses each trigger an innate immune response that includes the expression of nf-κb and irf target genes, which is expected to occur if the virus triggers the rig-i pathway in infected respiratory cells. second, both viruses trigger a robust cytokine response that probably attracts immune-cell infiltration to infected tissues. unlike contemporary virus strains, in which the early response to infection is resolved, the innate immune response triggered by the virus is characterized by a strong and sustained induction that is associated with massive tissue damage and death of the infected animal. however, in preliminary genomic analyses carried out with lung tissue from macaques that were infected with avian h n viruses, we have found that there are significant differences in the regulation of antiviral responses by the pandemic and h n viruses (j. c. kash and m.g.k., unpublished observations). therefore, there may be differences in the ways in which highly pathogenic influenza viruses regulate the innate immune response and cause disease. the enhanced pathogenicity of the and h n influenza viruses might be attributed to distinct components of their genomes. although much emphasis has been placed on the ns protein of the virus acting as an inhibitor of the type i ifn response, recent evidence suggests that the viral proteins pb (a polymerase), ha and na contribute to its pathogenicity . likewise, the polymerases of h n viruses have been linked to increased viral pathogenesis , suggesting that the increased pathogenesis of these viruses may be related to their replicative fitness. another respiratory virus, sars-cov, has emerged recently and has caused great concern among the public health and research communities. it has been suggested that disease pathology associated with sars-cov is caused by a disproportional immune response, illustrated by increased levels of pro-inflammatory cytokines and chemokines [ ] [ ] [ ] . studies carried out in our laboratory have combined the use of functional genomics with a cynomologus macaque infection model to study the host response to this virus . we observed that sars-cov-infected macaques show a strong increase in the expression of innate immune response genes early after infection and that this response wanes after days. conversely, genes that are induced later in infection tend to be involved in the cell cycle and in cell repair. none of the animals used in this study succumbed to infection, and sars-cov-induced pathology in these macaques resembled the pathological changes seen in the majority of human patients with sars who recover from the disease . unlike the findings of the pandemic influenza virus study, these data suggest that early immune responses to sars-cov infection are productive and enable the host to properly fight the virus, allowing a return to cellular homeostasis. however, in the % of human infections in which sars-cov infection is fatal (mostly in the elderly), it is possible that the timing or magnitude of the response results in immunopathology. studies using aged macaques might help to address this possibility. viruses such as sars-cov, h n influenza virus and influenza virus are all zoonotic infections, in which a virus that was adapted to another host was transferred to humans. because the type i ifn response is somewhat different in different hosts, it is possible that these viruses, which have adapted to their normal animal hosts, elicit an aberrant response when infecting a human host in which adaptation has not occurred, resulting in immunopathology. this possibility also raises the question of how appropriate the various animal infection models (such as mice and macaques) are for the understanding of human pathogenesis. as reviewed elsewhere , there are both advantages and disadvantages associated with different animal models, and it is important to keep in mind that responses observed using an animal model may not always accurately reflect the response in humans. genomics in vaccine evaluation and design genomic information and high-throughput technologies are beginning to have an impact on the field of vaccine development, but the main focus has been directed towards identifying important conserved features of pathogens that could serve as immunogens and characterizing host genotypes associated with strong protective responses [ ] [ ] [ ] . in recent years, it has become evident that the type i ifn response has a significant role in the development of the adaptive immune response. this commences with the influence of type i ifns on the activation, maturation and migration of dendritic cells , . the development of the antibody response is also enhanced by type i ifns through the direct effect of ifn on b cells and on the priming or function of cd + t helper cells . there is now also evidence that type i ifns act directly on cd + t cells to promote clonal expansion and indirectly by stimulating cross-priming by antigen-presenting cells that have engulfed infected cells to acquire antigen [ ] [ ] [ ] . so, viruses that suppress the type i ifn response not only subvert the mechanisms of innate surveillance, but also diminish the potential adaptive immune response that could mediate viral clearance or establish a quiescent, non-pathogenic state. for vaccine strategies, the implication is then that the best induction of a broad adaptive immune response will require some degree of type i ifn response in the initial stages. just as dna microarray technology spurred the development of functional genomics, the development of immunomic microarray technology is driving the emerging field of functional immunomics (reviewed in ref. ) . the goal of immunomics is to provide a detailed understanding of host immunological responses to foreign antigens through the use of high-throughput technologies and computational methods. the technologies that are central to this effort include antibody microarrays (consisting of antibodies as probes and antigens as targets), peptide microarrays (consisting of antigen peptides as probes and serum antibodies as targets) and more recently peptide-mhc microarrays (consisting of recombinant peptide-mhc complexes and co-stimulatory molecules as probes and populations of t cells as targets). antibody microarrays are used to measure the concentration of specific antigens (such as cancer antigens), whereas peptide-mhc microarrays can map mhc-restricted t-cell epitopes which are involved in helper and regulatory functions of the immune system. peptide microarrays are used in various applications, including b-cell epitope mapping and detection and diagnostic assays. peptide microarrays are also being used in vaccine studies for mapping epitopes associated with effective immune responses and for testing the ability of experimental vaccines to generate specific antibody responses against those epitopes after immunization and challenge . studies of immune responses that are associated with different clinical outcomes, such as those of patients who are hiv positive and who rapidly progress to aids and those of infected long-term survivors, can also provide direction for the development of vaccines . it is probable that immunomics will become an increasingly integral part of a systems-biology approach to vaccine development and of obtaining a better understanding of host immunity to virus infection. animal models. we have used functional genomics to evaluate a live influenza virus vaccine in a macaque model, in which attenuation of the virus was accomplished by truncation of the gene encoding ns . this modification eliminates or reduces the ability of the ns protein to antagonize type i ifn production and, in mouse and swine models, such attenuated live viruses are immunogenic and protective , . gene-expression profiling of tracheal and bronchial epithelial cells from macaques immunized with the ns -truncated virus show clear evidence of a robust type i ifn response. compared with immunization with a traditional killedvirus vaccine, the attenuated live-virus-vaccine group had higher antibody titres before and after challenge and a broader range of influenza virus-specific t-cell responses. following challenge with infective virus, the protection afforded by the attenuated live-virus vaccine was evident by the limited viral replication and minor pathology observed in treated animals. in addition, gene-expression profiles of lung tissue from animals that received the attenuated live-virus vaccine show less upregulation of innate and pro-inflammatory response genes compared with animals immunized with the killed-virus vaccine or untreated animals. at the same time, the transcriptional profiles for the attenuated live-virus-vaccine animals showed a stronger induction of genes that are associated with b-cell and t-cell responses. the general picture overall is that the truncated-ns containing influenza virus vaccine undergoes minimal replication but induces sufficient type i ifns to galvanize the adaptive immune response, leaving the host in a state of adaptive preparedness after just one immunization. the early induction of type i ifns in response to the truncated-ns -containing vaccine might be especially important in the local b-cell response that is crucial for viral clearance. a relevant observation in this regard is that early stimulation of the respiratory-tract b cells (within hours of influenza virus infection) was shown to be strongly driven by virus-induced type i ifns , . human studies. at present, there are only limited examples in which gene-expression profiling applied to vaccine design supports a picture consistent with that described above for the influenza virus model. the standards for prevention of measles and yellow fever are immunizations with attenuated live-virus vaccines. to assess the impact of infection on primary target cells, gene-expression profiling was carried out in tissue-culture systems comparing wild-type and vaccine strains. for both measles and yellow fever, it was clear that the attenuated vaccine strains led to a greater induction of the type i ifn response than the pathogenic wild-type virus , . although in the case of measles virus this disparity in the ifn response has previously been shown by serological techniques , expression analysis indicated that the antagonism of the response by the wild-type virus originated at the level of transcription. this early induction of the type i ifn response was also evident in microarray studies examining chimeras of the yellow fever vaccine strain that were devised as attenuated live-virus vaccines against other flaviviruses such as dengue virus . this contrasted with the low-level induction of type i ifns by dengue virus infection as seen by expression profiling using infection of primary cells or macaque disease models , . it is interesting to note that the measles and yellow fever vaccine strains are attenuated by passage in cells from other species. therefore, with suitable molecular understanding, the ability of some viruses to induce type i ifns might be optimized by directed molecular techniques, as was done for the truncated-ns influenza virus strain. as an alternative, one might consider using recombinant type i ifns as vaccine adjuvants instead of inducing them with the vaccine constituents , but at our present level of understanding, these approaches have yet to prove clinically tenable . functional genomics for the evaluation of immunological memory. functional genomic studies have been more equivocal in assessing the significance of type i ifn production during the immunological memory response. in the aforementioned macaque influenza virus study, animals receiving the attenuated live-virus vaccine showed upregulation of type i ifn pathways in tracheobronchial cells days after challenge, and this coincided with the development of a strong memory response . this type i ifn induction seems to be weaker than that observed at the corresponding time after the primary exposure to the vaccine, but is far lower than the type i ifn induction observed after challenge of animals receiving the killed-virus vaccine or of naive animals. this would suggest some role of this innate pathway in stimulating immunological recall. in contrast to this, examination of transcriptional profiles observed shortly after rechallenge of human pbmcs from individuals previously immunized against influenza virus are more in accord with early production of ifnγ, possibly arising from antigenic stimulation of memory cells . dhiman et al. also did not see evidence of a type i ifn response in a microarray study of whole blood taken from individuals immunized with measles virus after rechallenge with an attenuated live-virus vaccine strain, although genes associated with lymphocyte activation and survival were upregulated . it could be considered that technical issues might hamper the relevance of these studies in assessing the role of type i ifns in the memory response. in the case of the first study , pbmcs are not a primary target of influenza virus, so virus internalization might have been inefficient and a type i ifn response might have been poor. in the measles study , the earliest time point examined was days after rechallenge rather than early, when the type i ifn response would be expected to be strongest. therefore, further functional genomic experiments, with appropriately designed models, are required to address whether an early innate immune response is a key stage in triggering immunological memory. functional genomics has proven to be a highly efficient method for providing broad views of the host response in studies of virus-host interactions. as we have discussed, these techniques have revealed the activation or nature reviews | immunology repression of innate immune signalling pathways, crosstalk between pathways, the timing and magnitude of the immune response and, depending on the experimental system, the degree to which the immune response varies among individuals. conversely, functional genomics has been less effective in pinpointing the role of specific host genes in the antiviral response or, somewhat surprisingly, in identifying previously undiscovered genes and pathways that are important in the infection process, despite this being one of its early goals . indeed, the early assumption that functional genomics would provide quick answers to the complexities of virus-host interactions has proved naive. how then can greater benefits be gained from using functional genomics to study virus-host interactions? rather than being used as a singular approach, the future of functional genomics in virology will be in the integration of genomic data with data derived from other high-throughput technologies (fig. ) . the obvious complementary approach to functional genomics is proteomics, which will provide much needed information regarding the correlation of gene expression with protein abundance [ ] [ ] [ ] . our group has begun to integrate genomic and proteomic data to better understand the host response to influenza virus infection . other possibilities for data integration are also beginning to unfold. for example, micrornas, which regulate both transcription and translation, might have an important role in mediating virus-host interactions . the discovery of micrornas in certain large dna viruses, such as herpesvirus, suggests that some viruses may encode micrornas to regulate cellular functions . in addition, immunomic strategies (box ) will provide additional opportunities to interrogate the host immune response; screens using small interfering rnas are currently being combined with genomic data to identify specific cellular proteins that are used by viruses during infection [ ] [ ] [ ] . together with virology, clinical and pathology data, this integrated set of information might provide the systems-biology view that will be needed to clearly understand the role of specific host genes and pathways involved in the development of immunity or disease after virus infection. another use for genomics that will no doubt expand is expression quantitative trait loci (eqtl) mapping . the combination of global gene-expression data with eqtl mapping provides greater power in elucidating complex genetic traits in addition to providing insights into specific genes or mutations that might be responsible for the trait in question. this approach is currently being used to better understand the genetic basis for various disease conditions in mice [ ] [ ] [ ] , and it is likely that it will also be useful in increasing our understanding of virus-host interactions. for example, using recombinant inbred strains of mice derived from parental strains that react differently to infection with a given virus, it should be possible to use eqtl mapping to determine chromosomal locations for potential traitcontributing factors and highlight genes of interest for the trait. with this increased level of complexity, however, it will be important to work closely with the bioinformatics and computational-modelling communities, and to make best use of the sophisticated bioinformatics tools, data-mining schemes and mathematical-modelling strategies that are continually being developed , . it might also be necessary to take a step back to simpler experimental systems (such as cell-culture models) to dissect cellular events before moving on to more complex in vivo models. the use of combined computational approaches that can account for gene-regulatory networks and cell-to-cell interactions will also facilitate the move to whole animal physiological modelling. functional genomics is clearly providing advances in our understanding of virus-host interactions, and the evolution to an integrated systems-biology approach holds even greater promise for the field. in addition to providing new insights into viral pathogenesis and host immunity, this approach provides a host-oriented antiviral discovery paradigm with the potential for discovering the benefits of functional genomics will be further enhanced by integrating genomic data with data derived from other high-throughput technologies. the potential information and biological insights provided by these technologies are shown. together, these approaches will help to provide a systems-biology view of virus-host interactions that spans the flow of biological information from dna (genetics) to mrna (genomics) to protein (proteomics) to protein function (immunomics). new targets for broad-spectrum antiviral therapies and for improving vaccine evaluation and design. we are optimistic about continuing advancements in the technologies and computational methods used to study virus-host interactions and in improved capabilities to identify, characterize and circumvent the strategies used by viruses to outsmart their long-suffering hosts. recent studies have shown that rig-i preferentially recognizes single-stranded rna (ssrna) with polyu motifs, whereas mda recognizes long dsrna molecules. these differences might help to explain the differential recognition and innate immune signalling induced by different rna viruses [ ] [ ] [ ] . pathogenic viruses: smart manipulators of the interferon system pathogen subversion of cell-intrinsic innate immunity viruses and interferon: a fight for supremacy principles of intracellular viral recognition toll-like receptors, rig-i-like rna helicases and the antiviral innate immune response functional classification of interferon-stimulated genes identified using microarrays identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda signaling through ips- in this study, genomic analyses show that rig-i and mda operate cooperatively to establish an antiviral state and mediate an ifn amplification loop that supports immune effector gene expression during west nile virus infection differential roles of mda and rig-i helicases in the recognition of rna viruses distinct rig-i and mda signaling by rna viruses in innate immunity systems biology approaches identify atf as a negative regulator of toll-like receptor transcriptional profiling of the lps induced nf-κb response in macrophages human macrophage activation programs induced by bacterial pathogens functional genomic delineation of tlrinduced transcriptional networks regulation of interferon regulatory factor- by the hepatitis c virus serine protease control of antiviral defenses through hepatitis c virus disruption of retinoic acid-inducible gene-i signaling immune evasion by hepatitis c virus ns / a protease-mediated cleavage of the toll-like receptor adaptor protein trif regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna 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methodologies for modelling, analysis and simulation of signalling networks the model organism as a system: integrating 'omics' data sets systems biology and the host response to viral infection a new discipline, a new name microarray analysis: basic strategies for successful experiments dna microarray technology for the microbiologist: an overview chips with everything: dna microarrays in infectious diseases from functional genomics to functional immunomics: new challenges, old problems, big rewards microarray profiling of antibody responses against simian-human immunodeficiency virus: postchallenge convergence of reactivities independent of host histocompatibility type and vaccine regimen microarray profiling of antiviral antibodies for the development of diagnostics, vaccines, and therapeutics nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses innate immunity induced by compositiondependent rig-i recognition of hepatitis c virus rna the length-dependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiationassociated gene we thank b. paeper and s. proll for discussions and assistance with preparation of the original figures. research in the authors' laboratory is supported by public health service grants (r ai , r hl , r ai , r r r , p a i , p a i , p da and p rr ) from the national institutes of health, usa. this study uses gene-expression profiling of serial liver-biopsy samples from patients that had received a liver transplant to demonstrate that rapidly progressive fibrosis is associated with an impaired immune response, as indicated by a lack of induction of genes associated with the ifnmediated antiviral response, antigen presentation and cytotoxic t-cell response. key: cord- - m e authors: boga, jose antonio; coto‐montes, ana; rosales‐corral, sergio a.; tan, dun‐xian; reiter, russel j. title: beneficial actions of melatonin in the management of viral infections: a new use for this “molecular handyman”? date: - - journal: rev med virol doi: . /rmv. sha: doc_id: cord_uid: m e melatonin (n‐acetyl‐ ‐methoxytryptamine) is a multifunctional signaling molecule that has a variety of important functions. numerous clinical trials have examined the therapeutic usefulness of melatonin in different fields of medicine. clinical trials have shown that melatonin is efficient in preventing cell damage under acute (sepsis, asphyxia in newborns) and chronic states (metabolic and neurodegenerative diseases, cancer, inflammation, aging). the beneficial effects of melatonin can be explained by its properties as a potent antioxidant and antioxidant enzyme inducer, a regulator of apoptosis and a stimulator of immune functions. these effects support the use of melatonin in viral infections, which are often associated with inflammatory injury and increases in oxidative stress. in fact, melatonin has been used recently to treat several viral infections, which are summarized in this review. the role of melatonin in infections is also discussed herein. copyright © john wiley & sons, ltd. the methoxyindole melatonin (n-acetyl- -methoxytryptamine) is a secretory product of the pineal gland. it was first reported as a skin lightening agent in amphibians [ , ] . further investigations showed that another function, supported by its direct effects in regions containing high densities of melatonin receptors, such as the circadian pacemaker (the suprachiasmatic nucleus) and the pars tuberalis, is to regulate and reset circadian rhythms as well as to be involved in the measurement of day length, an environmental variable used for seasonal timing of reproduction, metabolism and behavior in species responding to photoperiodic changes [ ] [ ] [ ] [ ] [ ] . in recent decades, melatonin has been reported to possess numerous additional functions and act in neural and non-neural tissues or cells that express melatonin receptors that are at lower densities than in the suprachiasmatic nucleus. thus, melatonin is involved in sleep initiation, vasomotor control, anti-excitatory actions, immunomodulation including possessing anti-inflammatory properties, antioxidant actions, and actions on energy metabolism, influences on mitochondrial electron flux, regulation of the mitochondrial permeability transition pore (mtptp), and mitochondrial protection against free radicals [ ] [ ] [ ] [ ] [ ] [ ] . deficiencies in melatonin production or melatonin receptor expression and decreases in melatonin levels (such as those that occur during aging) are likely to contribute to numerous dysfunctions [ ] [ ] [ ] . in fact, several clinical trials have shown that melatonin is efficient in preventing cell damage under acute (sepsis, asphyxia in newborns) and chronic states (metabolic and neurodegenerative diseases, cancer, inflammation, aging) [ ] [ ] [ ] [ ] [ ] [ ] . in humans, the efficacy of melatonin as a treatment of ocular diseases, cardiovascular diseases, sleep disturbances and several other pathologies, as well as a complementary treatment in anesthesia, haemodialysis, in vitro fertilization and neonatal care, has been assessed and reported to be beneficial [ ] . likewise, melatonin reduces the toxicity and increases the efficacy of a large number of drugs whose side effects are well documented [ ] . the beneficial effects of melatonin are explained by its properties as a potent antioxidant, a modulator of apoptosis and a positive regulator of immune functions [ ] [ ] [ ] [ ] [ ] . these actions suggest the potential to treat viral infections, which usually cause inflammatory injury and elevated oxidative stress [ , ] . a number of reports examining the ability of melatonin to protect against viral infections have been published, as summarized in the following section. encephalomyocarditis virus (emcv) is a highly pathogenic and aggressive virus that causes encephalitis and myocarditis in rodents. administration of melatonin prevented paralysis and death of mice infected with sublethal doses of emcv [ ] . melatonin also has a protective effect in mice infected with semliki forest virus (sfv), a classic encephalitis arbovirus, that invades the cns and whose replication in the mouse brain eventually leads to death. melatonin administration not only reduced the death rate but also significantly postponed the onset of the disease. furthermore, the level of virus in the blood in melatonin-treated mice was lower than in non-treated mice [ ] . although attenuated west nile virus (wnv) strain wn- is an encephalitis virus that does not invade the brain and does not normally cause encephalitis, exposure of mice to various stressful stimuli induces wn- encephalitis. melatonin counteracts the immunodepressive effect of stress exposure and prevents the stress-related encephalitis and death of wn- infected mice [ ] . venezuelan equine encephalomyelitis (vee) is an important human and equine disease caused by vee virus (veev), a mosquito-borne organism. outbreaks have occurred in northern south america from the s to the s with thousands of people and horses, donkeys and related species being infected. mice have been used as an animal model for this condition, because veev-infected mice show excitation and hypermotility followed by hypomotility, paralysis, coma and death. melatonin administration protects mice infected with veev by decreasing the virus load in brain and serum, reducing mortality rates, delaying the onset of the disease and deferring the time to death. furthermore, in surviving mice treated with melatonin, the veev-mediated igm antibody titres are highly elevated [ ] . aleutian mink disease is a natural condition caused by persistent infection with the aleutian mink disease virus (amdv). animals in the progressive state of the disease show a marked hypergammaglobulinemia, because of high titers of non-neutralizing admv antibodies. this is thought to cause lesions in the kidney, liver, lungs and arteries. melatonin implants reduced mortality in admv-infected mink [ ] . the findings in these reports document the ability of the melatonin to protect against viral infections [ table ]. the potential protective mechanisms include melatonin acting as a free radical scavenger, an antioxidant enzyme inducer, a positive regulator of immune functions and an inhibitor of inflammation, as well as a regulator of programmed cell death (pcd) [ table ]. free radicals are molecules formed naturally during many metabolic processes. they contain an unpaired electron in their valence orbital that makes them unstable and reactive. these reactive agents damage essential molecules in cells including lipids, proteins and dna [ , ] . among these reactants, the superoxide anion radical (o • À ), nitric oxide (no•) and especially their derivatives, the hydroxyl radical (•oh) and peroxynitrite (onoo À ), are highly biologically damaging elements produced in the host during microbial infections [ ] [ ] [ ] [ ] . phagocytes, such as neutrophils and macrophages are assumed to be the major generators of free radicals. elevated levels of o • À are although ifn-g is the major cytokine inducing inos and no• overproduction in the pathogenesis of these viral infections, inos expression is downregulated by il- , il- and transforming growth factor-b (tgf-b) [ ] [ ] [ ] . ifn-g is known to be associated with type helper t cell (th ) responses, and il- and il- are induced by type helper t cell (th ) responses; no• biosynthesis catalyzed by inos is precisely regulated by a polarized th -th balance. in other viral diseases, viral replication or viral components directly induce inos without mediation by pro-inflammatory cytokines. thus, the hiv envelope glycoprotein gp triggers inos expression in human astrocytes and murine cortical brain cells in culture [ , ] . rsv directly upregulates inos in human type alveolar epithelial cells (a cells) [ ] . free radicals are produced to eliminate the pathogenic agent or to kill the virus-infected cells by a non-specific response. thus, antiviral effects of no• have been described for some dna viruses such as murine poxvirus (ectromelia virus) and herpes viruses including hsv, ebv and some rna viruses such as coxsackie virus [ ] [ ] [ ] [ ] [ ] [ ] . the toxic oxygen and nitrogen-based reactants, unfortunately, cannot discriminate between exogenous invading pathogens and the host cells themselves, and therefore, they also damage the host. to minimize such self-damage during the elimination of pathogens, the host employs several primitive tactics; it uses recruited phagocytes for the physical containment of pathogens in infectious foci. most bacteria, for example, can be phagocytosed and confined to septic foci, which are typically abscesses or granulomas. under these conditions, free radicals can affect bacteria rather selectively with the surrounding normal tissue remaining mostly intact. in viral infections, in contrast, free radical mediators cause non-specific oxidative/nitrosative damage in virus-infected tissue and produce oxidative stress; this occurs when the virus cannot be confined to limited areas by the non-specific host defense [ , , ] . thus, no• has appreciable antiviral actions on several types of viruses including ortho-and paramyxovirus, murine vaccinia virus, coronavirus (mouse hepatitis virus), lymphocytic choriomeningitis virus, murine emcv, tickborn encephalitis virus (tbe-v) [ ] [ ] [ ] [ ] [ ] [ ] ; also, no• and its derivatives, especially onoo -, can be considered pathogenic in some viral infections. indeed, no• inhibition or lack of no• generation reduces the pathological consequences of viral pneumonia in mice caused by influenza virus, sev and hsv- , hsv- -induced encephalitis in rats, emcv-induced carditis and diabetes, and murine encephalitis induced by flavivirus (murray valley encephalitis virus, tbe-v) [ , , , [ ] [ ] [ ] [ ] [ ] . a similar pathogenicity with a lack of antiviral effects has been observed for o •in several experimental models of virus-induced pneumonia including those caused by influenza virus and cmv [ ] [ ] [ ] , , , ] . hcv-induced oxidative stress is emerging as a key step and a major initiator in the development and the progression of liver damage [ ] . ns , one of the non-structural proteins of hcv, was reported to induce reactive oxygen species by nadph oxidase in neutrophils [ ] . high-risk human papilloma virus (hpv), which causes cervical cancer, promotes inos-dependent dna damage, leading to dysplastic changes and carcinogenesis [ ] . epstein-barr virus is a herpes virus that infects the majority of the world population, generally during childhood; it has been linked to the genesis of a number of lymphoproliferative diseases and neoplasia such as the african burkitt lymphoma, nasopharyngeal carcinoma or gastric carcinoma. early stages of ebv infection generate oxidative stress either in b lymphocytes or in epithelial cells, so contributing to pathology [ ] . influenza a virus causes a respiratory disease, which ranges from mild upper respiratory tract illness with or without fever to severe complications such as pneumonia. the latter disease results in respiratory failure, acute respiratory distress syndrome, multi-organ failure and even death. an abrupt increase in o • À production occurs during phagocytosis, which induces injury in non-infected cells. these o • À -mediated pathways contribute to a portion of the extensive tissue injury observed during severe influenza-associated complications [ ] . to protect themselves against free radicalmediated damage, cells have developed an antioxidant defense that includes enzymatic and non-enzymatic mechanisms. free radical generation and a functionally efficient antioxidant defense system must be in equilibrium to avoid cellular damage caused by radicals and their derivatives. enzymes involved in the elimination of free radicals include the superoxide dismutases (sod), catalase (cat) and glutathione peroxidase (gpx). in addition to the enzymatic antioxidant system, organisms possess non-enzymatic free radical scavengers, which directly remove toxic reactants because of their electron donating ability. the best known nonenzymatic antioxidants are vitamin e (a-tocopherol), vitamin c (ascorbate), glutathione (gsh), b-carotene and, as recently described, melatonin [ ] . several radical scavengers have been efficacious in ameliorating the severity of viral diseases. n-acetylcysteine, a gsh precursor, inhibits hiv in vitro [ ] as did the natural thiol antioxidant, alpha-lipoic acid [ ] . glutathione administration to hiv seropositive individuals by aerosol treatment can correct the glutathione deficiency [ ] . the combination of several antioxidants with antiviral drugs synergistically reduces the lethal effects of influenza virus infections [ ] . thus, any agent that functions as a direct radical scavenger and also stimulates antioxidative enzymes could have utility in the treatment of patients with severe complications of viral infections. melatonin is a powerful and effective •oh scavenger, which provides protection against oxidative damage of cell components. it also scavenges the peroxyl radical to a lesser degree generated during lipid peroxidation with an activity that, in some situations, is reportedly greater than that of vitamin e [ , [ ] [ ] [ ] [ ] . also, melatonin directly detoxifies the onoo À and possibly peroxynitrous acid (onooh) [ ] . in vivo, melatonin stimulates several antioxidative enzymes including gpx, cat and sod, thereby potentiating its antioxidant properties [ ] [ ] [ ] [ ] . melatonin can cross anatomical barriers, including the placenta and the blood-brain barrier [ , ] , and easily enter cells [ ] . splenocytes infected with veev generated less of no•, when treated with melatonin; this finding suggests that the indoleamine protected mice infected with the veev by a mechanism involving a reduction in no• concentrations in tissue [ ] . elevated production of no• and lipid peroxidation products were also found in supernatants and cellular elements of veev-infected neuroblastoma cell cultures. both no• and lipid peroxidation were decreased by melatonin treatment in a timedependent manner with an associated reduction in inos expression [ ] . production of brain and serum nitrite, as well as neural lipid peroxidation products, was increased in veev-infected mice. melatonin treatment curtailed nitrite concentrations in the brain and serum of infected mice and lowered lipid peroxidation products [ ] . respiratory syncytial virus is a common cause of bronchiolitis, a severe lower respiratory tract affliction that infects nearly all infants by age three worldwide. mice inoculated intranasal with rsv showed elevated oxidative stress due to rises in no• and •oh. also elevated malondialdehyde (mda) and decreases in gsh and sod activities were observed. pre-administration of melatonin in vivo resulted in marked reduction of acute lung oxidative injury induced by rsv, suppressed mda, no• and •oh generation, and restored gsh and sod levels in the lungs of rsv-infected mice [ ] . rabbit hemorrhagic disease virus (rhdv) causes bleeding in the respiratory system, liver, spleen, cardiac muscle,and occasionally in the kidneys of infected rabbits with mortality over % in adults [ ] . the activity and mrna expression of the antioxidants enzymes gpx, glutathione-s-transferase (gst) and mn-sod were significantly reduced in the liver of rhdvinfected rabbits used as a model of fulminant hepatic failure; these changes were reduced by melatonin administration in a concentrationdependent manner. melatonin treatment also caused a rise in protein expression of the nuclear factor erythroid (nrf ), a transcription factor that plays a critical role by binding to the antioxidant response element in the promoter region of a number of genes encoding for antioxidant and detoxifying enzymes in several types of cells and tissues [ ] . the activation of nrf during prevention of oxidative liver injury by melatonin in rats treated with dimethylnitrosamine has been reported [ ] . during the early phase of infection and depending on the nature of the infected cells and the infecting virus, early innate defense mechanisms may be triggered to limit the extent of viral spread. the first mechanism to limit the extent of viral spread is the recognition of pathogenassociated molecular patterns (pamps), which are mostly viral nucleic acids, or their synthetic analogs produced during the viral infection, by a large repertoire of pattern recognition receptors (prrs), including toll-like receptors (tlrs), nodlike receptors (nlrs), rig-i-like receptors (rlrs) and aim -like receptors (alrs) [ ] [ ] [ ] [ ] . such recognition initiates signaling cascades that culminate in the activation of transcription factors including nuclear factor kappa b (nf-kb), activating transcription factor (atf- ), activating protein- (ap- ) and interferon regulatory factors (irf ) and (irf ). these stimulate the expression of type i ifn genes that are synthesized in most cell types and especially in plasmacytoid dendritic cells (pdc) [ ] . all ifns bind to specific ubiquitously expressed cell surface receptors and induce a large number of interferon-stimulated genes (isg), whose encoded proteins mediate the antiviral effects of interferons. among these isgs, dsrna-activated protein kinase (pkr) primarily inhibits replication of rna viruses such as vesicular stomatitis virus (vsv), emcv, wnv, hcv and dna viruses including hsv- [ ] . another group of isgs is the - -oligoadenylate synthetases (oas) that requires dsrna for its activation and is a major antiviral effector against picornaviruses (e.g. emcv) and influenza a virus, as well as other rna viruses [ ] . non-specific ssrna cleavage also occurs after induction of isg , a -exoribonuclease, which contributes to inhibition of rna viruses such as vsv [ ] . an additional, non-enzymatic mechanism of translation inhibition is pursued by the isg /ifit family proteins, which act against hcv [ ] [ ] [ ] . another ifn-induced protein is the human mxa, which is a key component in innate defense against orthomyxoviruses such as influenza virus as well as measles virus, vsv, hanta virus and sfv [ , ] , the viperin (cig ), which might interfere with viral budding of enveloped viruses, such as cmv, hcv, and influenza virus [ ] , and the nucleic acid-editing enzymes apobec g and À f, which inhibit retroviruses [ ] . a second mechanism is the triggering of effector functions of cellular components of the innate immune system, such as granulocytes, natural killer cells (nk) and natural killer t cells (nkt cells), macrophages, and dendritic cells, which are normally rapidly recruited and/or activated at the site of virus infection, causing a local inflammation [ ] . during this early phase, activated nk cells release ifn-g, which is not stimulated by viral pamps but by il- and il- released by activated macrophages [ ] . all of the cellular components of the innate immune system can participate in the antiviral response by killing infected cells, by producing chemokines (including eotaxin, rantes, mcp- , il- ) that recruit inflammatory cells into the infected tissue and by producing antiviral and immunoregulatory cytokines (including tnf-a, il- , il- , il- , il- , il- , il- , il- , gm-csf) that enable the adaptive immune response to recognize infected cells and perform antiviral effector functions [ ] [ ] [ ] [ ] . lymphocytes are cells of this adaptive immune system. among them, two subsets of cd + t cells, th and th , play a key role in antiviral immunity. after being stimulated by antigen presenting cells, th cells produce il- , tnf-a and ifn-g, which possess antiviral activities and regulate activation of cd + cytotoxic t cells, whereas th cells produce il- , il- , il- and il- , which stimulate b cells to produce antibodies [ ] . despite the fact that virus-specific th cells can be detected following primary infection by any virus, virus-specific th cells are usually much more abundant and reach very high numbers at the peak of the acute infection [ ] . moreover, their frequencies remain elevated following resolution of the infection. melatonin is synthesized in lymphoid organs, such as the bone marrow, thymus and lymphocytes [ ] [ ] [ ] , and there are high affinity membrane melatonin receptors as well as nuclear binding sites in circulating lymphocytes, spleen cells and thymocytes [ ] [ ] [ ] . melatonin is known to activate both innate and adaptive immune responses leading to an increase in immune responsiveness and regulation of several immune functions [ , , [ ] [ ] [ ] [ ] [ ] . melatonin has properties as an inflammatory regulator, because it differentially modulates pro-inflammatory enzymes, and controls the production of inflammatory mediators such as cytokines and leukotrienes. the timing of its pro-inflammatory and anti-inflammatory effects suggests that melatonin might promote early phases of inflammation, on the one hand, and contribute to its attenuation on the other hand, to avoid complications of chronic inflammation [ ] . melatonin enhances the production of il- , il- , tnf-a and il- from the monocytes [ ] and of il- , ifn-g and il- from cultured human peripheral blood mononuclear cells [ ] . it has been suggested that melatonin and ifn-g create an immunoregulatory circuit responsible for the antiviral, antiproliferative and immunomodulatory actions of . this cytokine increases serotonin and melatonin levels in lymphocytes and macrophages. the early stimulation in the production of ifn-g by melatonin suggests that earlier treatment with this indoleamine could increase the antiviral activity of ifn-g [ ] . in addition to stimulating the production of several cytokines that regulate immune function, melatonin enhances immune function by directly stimulating polymorphonuclear cells, macrophages, nk cells and lymphocytes [ ] . recently, considerable attention has been focused on the fact that melatonin treatment has been found to augment cd + t cells in lymph nodes of rats [ ] . consequently, melatonin is considered an immunoenhancing agent [ , ] . in retrovirus-infected people and mice, whereas th cytokine (il- and ifn-g) production declines, th cytokine (il- , il- , il- , and il- ) production increases [ ] [ ] [ ] . the excessive th cytokines suppress th cells, causing anergy of cell-mediated immunity, thus allowing the retrovirus as well as normal flora to reproduce and promote free radical generation by macrophages [ ] . female c bl/ mice infected with the lp-bm mlv develop murine aids. treatment with melatonin, alone or with dehydroepiandrosterone (dhea), prevented retrovirus-induced reduction in b-cell and t-cell proliferation and in th cytokine secretion, as well as overproduction of th cytokines and tnf-a [ ] . in fact, melatonin alters the balance of th and th cells mainly towards th responses increasing the production of th cytokines [ ] . a link between melatonin and the immune system has been also reported in patients infected with hiv- . although mean serum il- levels in hiv- -affected individuals did not significantly differ from healthy controls, the il- levels of hiv- patients with advanced disease (cdc stage c) were significantly lower than those of patients in less advanced cdc stages b and a. taking into account that serum il- levels run parallel with serum melatonin concentrations as the disease advances, a relationship between immune function and melatonin has been suggested; a reduction in serum melatonin could possibly affect il- production thereby contributing to the progress of hiv- infection [ ] . the protective effect of melatonin against veev by regulation of the immune system has been described by bonilla et al. [ ] . the endogenous production of ifn-g, il- b and tnf-a, but not of il- and il- , is stimulated in veev-infected mice treated with melatonin [ ] . nevertheless, the average mortality obtained during neutralization experiments with the corresponding anticytokine antibody suggests that although neither tnf-a nor ifn-g is essential for the protective effect of melatonin observed in murine veev infection, il- b induced by melatonin treatment is a target cytokine to promote the immune enhanced state. this in turn causes the viral clearance or helps generate an earlier immune response against the veev infection [ ] . in contrast, in the brain of veev-infected mice, melatonin stimulates the endogenous production of il- b but reduces the concentration of tnf-a [ ] . il- b is considered one of the earliest host mediators during infectious diseases of the cns and its role in infectious processes of the brain parallels its role in the peripheral immune system [ ] . although il- b deficiency is protective against fatal sindbis virus infection [ ] , mice deficient in il- b have increased susceptibility to influenza virus [ ] . in poxvirus animal models, the viral induction of this cytokine is also beneficial for the host [ ] . the increase in il- b levels detected in blood and in brain of veev-infected mice after melatonin treatment also plays a protective role, possibly by neuronal support and protection by inducing nerve growth factor secretion by astrocytes [ ] . this supplies a trophic factor for many neuronal cell types in times of stress such as that produced by veev infection. the significant reduction in the concentration of brain tnf-a induced by melatonin in veevinfected mice likely diminishes the inflammatory response caused by the migration of granulocytes and macrophages to inflammatory sites within the cns [ ] . these cells are recruited by colonystimulating factors produced by astrocytes stimulated by tnf-a and as a consequence of alterations in blood-brain barrier (bbb) permeability caused by the adhesive properties of astrocytes stimulated by tnf-a. tnf-a is known to induce intercellular adhesion molecules on neighboring endothelial cells [ ] , alter bbb permeability and promote inflammatory cell infiltration into the cns. by reducing adhesion molecule production, which melatonin is known to do [ ] , the indole would protect the brain infected with veev. respiratory syncytial virus bronchiolitis in infants is characterized by a massive infiltration of inflammatory cells into the airways. of the diverse intracellular signaling pathways, rsv is recognized by tlr , which initiates a signaling cascade that culminates in the activation of the transcription factor nf-kb; nf-kb is a central mediator of rsv-induced airway inflammation in vivo [ , , ] . rsv infection of raw . macrophages time-dependently stimulates the rapid activation of tlr and nf-kb, as well as subsequent nf-kb dependent genes, many of which encode for pro-inflammatory cytokines and chemokines including tnf-a and il- b. melatonin decreases tlr -mediated downstream gene expression in rsv-infected macrophages in a dose-dependent and time-dependent manner. such inhibition of nf-kb activity, as well as of tnf-a in serum, seems to be the key event required to explain the reduction in inflammatory gene expression caused by melatonin [ , ] . as obligate intracellular parasites, viruses are dependent on the host for each stage of replication and, therefore, constantly interface with multiple components of the host cell machinery, including cellular receptors and uptake pathways, gene expression mechanisms and the cell division apparatus. viral utilization of these systems likely causes cell stress and activates death-signaling pathways or alters expression of genes that control cell survival, evoking pcd [ , ] . apoptosis is one type of pcd, which is dependent on cleavage of important cellular factors by effector caspases such as caspase- and caspase- . two major pathways govern the activation of such effector caspases. in the intrinsic pathway, intracellular stresses sensed by the bh -only members of the bcl- family promote the formation of the apoptosome by activation of caspase- through release of proapoptotic molecules such as cytochrome c and smac/diablo from the mitochondria. the apoptosome directly activates effector caspases. in the extrinsic pathway, occupation of death receptors such as fas and tumor necrosis factor receptor (tnf-r) by death ligands including fasl and tnfa forms a death-inducing signaling complex (disc). this results in the activation of the initiator caspase, caspase- , which directly mediates effector caspase activation and causes cell death. the ability of melatonin to modulate apoptosis and to differentially regulate the expression of pro-apoptotic and anti-apoptotic mediators has been reported in many studies [ , [ ] [ ] [ ] [ ] [ ] . rhdv infection induces liver apoptosis with increased caspase- expression and activity [ , ] . these effects are attenuated by melatonin in a concentration-dependent manner. anti-apoptotic actions of melatonin on the intrinsic pathway were related to a reduced expression of bax and cytosolic cytochrome c release, increased expression of bcl- and bcl-xl, and inhibition of caspase- activity. melatonin treatment also has effects on extrinsic pathway resulting in a reduction in caspase- activity, tnf-r expression and phosphorylated janus kinase (jnk) expression, and increased expression of cellular fliceinhibitory protein (c-flip), an inhibitor of caspase- [ ] . these findings show that inhibition of apoptotic mechanisms contributes to the beneficial effects of melatonin in rabbits with experimental infection by rhdv and supports a potential hepatoprotective role of melatonin in fulminated hepatic failure. autophagy is a type of pcd characterized by the formation of autophagosomes to remove excessive proteins and thereby maintains homeostasis within the cell. autophagy is now recognized as a component of both innate and adaptive immune responses to bacterial and viral pathogens [ ] . varicella zoster virus infection provides an excellent example of autophagy in humans, because abundant autophagosomes are easily detected in the skin vesicles of both varicella and zoster [ ] . autophagy is also found during viral replication of hcv [ ] , rabbit calicivirus [ ] and poliovirus [ ] . given that melatonin modulates autophagy through redoxsensitive transcription factors [ ] , the role of melatonin in such viral infections involving autophagy should be examined. beneficial effects of melatonin when combined with several drugs, such as doxorubicin, cisplatin, epirubicin, cytarabine, bleomycin, gentamicin, cyclosporin, indometacin, acetylsalicylic acid, ranitidine, omeprazole, isoniazid, iron and erythropoietin, phenobarbital, carbamazepine, haloperidol, caposide- , morphine, cyclophosphamide and l-cysteine have been reported [ ] . recently, a single blind randomized study showed a higher percent of a complete regression of symptoms of hsv- infection after a treatment with melatonin plus sb- (an extract of aspergillus sp. with antiherpetic properties) compared with the treatment with acyclovir alone [ ] . effects of melatonin to increase the efficacy of other antivirals should be studied. melatonin is an endogenously produced and ubiquitously acting molecule [ ] [ ] [ ] . because of its highly diverse actions, this indoleamine has potential to combat a wide variety of pathophysiological conditions [ ] [ ] [ ] [ ] [ ] ; it has been tested in numerous clinical trials [ ] with the outcomes of the treatments always being beneficial. because of its essential and basic actions on cell physiology, melatonin qualifies for the moniker "molecular handyman," as indicated in the title of this review. in relation to viral infections, melatonin also seems to be beneficial as indicated in the experimental studies summarized herein. its favorable actions against viral infections likely relate to its ability to limit the negative molecular processes normally activated when viruses invade cells. melatonin's actions include an ability to promote immune surveillance, to scavenge free radicals thereby significantly reducing the associated molecular destruction and to modulate the processes related to apoptosis. these multiple actions suggest that melatonin should be evaluated in randomized controlled trials as a preventive agent or as a treatment of viral infections particularly in older individuals where endogenous levels of melatonin have declined. it is the hope of the authors that this summary will stimulate interest in experimental examination of melatonin's antiviral actions. isolation of melatonin, a pineal factor that lightens melanocytes structure of melatonin melatonin: a coordinating signal for mammalian reproduction synchronization of mammalian circadian rhythms by melatonin the melatonin rhythm: both a clock and a 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the rat hippocampus and cortex recognition of doublestranded rna and activation of nf-jb by toll-like receptor identification of nf-kappab-dependent gene networks in respiratory syncytial virusinfected cells melatonin decreases tlr -mediated inflammatory factor expression via inhibition of nfkappa b activation in respiratory syncytial virus-infected raw . macrophages role of virus-induced apoptosis in a host defense mechanism against virus infection enter the kill zone: initiation of death signaling during virus entry melatonin and cell death: differential actions on apoptosis in normal and cancer cells melatonin prevents capacitation and apoptotic-like changes in ram spermatozoa and increases fertility melatonin leucocyte apoptosis induced by intracellular calcium overload: relation with its antioxidant actions melatonin promotes puromycin-induced apoptosis with activation of caspase- and -adenosine monophosphate-activated kinase-alpha in human leukemia hl- cells programmed cell death in the pathogenesis of rabbit hemorrhagic disease melatonin attenuates apoptotic liver damage in fulminant hepatic failure induced by the rabbit hemorrhagic disease virus autophagy during common bacterial and viral infections of children varicella-zoster virus infection induces autophagy in both cultured cells and human skin vesicles autophagy proteins promote hepatitis c virus replication structural and functional analysis of virus factories purified from rabbit vesivirus-infected vero cells remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles melatonin modulates autophagy through a redox-mediated action in female syrian hamster harderian gland controlling cell types and gland activity regression of herpes viral infection symptoms using melatonin and sb- : comparison with pineal melatonin: cell biology of its synthesis and of its physiological interactions melatonin: a multitasking molecule a survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases sirtuins, melatonin and circadian rhythms: building a bridge between aging and cancer melatonin, cardiolipin and mitochondrial bioenergetics in health and disease melatonin as a therapeutic tool in ophthalmology: implications for glaucoma and uveitis drug-mediated ototoxicity and tinnitus: alleviation with melatonin matrix metalloproteinases in health and disease: regulation by melatonin the authors have no competing interest. jab is a researcher of isciii/ficyt. his stay at uthscsa has been subsidized by isciii (ba / ). key: cord- - cdqhrcw authors: seliger, barbara; ruiz‐cabello, francisco; garrido, federico title: chapter ifn inducibility of major histocompatibility antigens in tumors date: - - journal: adv cancer res doi: . /s - x( ) - sha: doc_id: cord_uid: cdqhrcw interferons represent a protein family with pleiotropic functions including immunomodulatory, cytostatic, and cytotoxic activities. based on these effects, interferons are involved in innate as well as adaptive immunity, thereby shaping the tumor host immune responses. these cytokines, alone or in combination, have been successfully implemented for the treatment of some malignancies. however, it has been recently demonstrated that tumor cells could be resistant to interferon treatment, which may be associated with an escape of tumor cells from immune surveillance. therefore, the aim of this chapter is to summarize the frequency of impaired interferon signal transduction, their underlying molecular mechanisms, and their clinical relevance. ag, antigen; apc, antigen presenting cells; apm, antigen-processing machinery; bh, bleomycin hydrolase; bp, base pairs; ciita, class ii transactivator protein; clip, class ii invariant chain peptide; ctl, cytotoxic t lymphocyte; dc, dendritic cell; er, endoplasmic reticulum; gas, gammainterferon-activated site; ifn, interferon; ifn-r , interferon-receptor- ; il, interleukin; irf, interferon regulatory factor; isg, interferon-stimulated genes; isgf , ifn-stimulated gene factor ; isre, interferon-stimulated response element; jak, janus kinase; lps, lipopolysaccharide; mapk, mitogenactivated protein kinase; mca, methylcholanthrene; mhc, major histocompatibility complex; nf, nuclear factor; nk, natural killer; pkc, protein kinase c; rcc, renal cell carcinoma; sclc, small-cell lung carcinoma; socs, suppressor of cytokine signaling; stat, signal transducer and activator of transcription; ta, tumor antigen; tap, transporter associated with antigen processing; tcr, t cell receptor; tfbs, transcription factor-binding sites; tnf, tumor necrosis factor; tpn, tapasin; tppii, tripeptidyl peptidase ii; tsa, trichostatin a; tyk, tyrosine kinase; uirr, upstream interferon response region; usf , upstream stimulatory factor ; wt, wild type. interferons (ifns) represent proteins that are secreted from cells in response to various stimuli and provide the basis for the understanding of the evolution, structure, and function as well as the pathways of other cytokines and their receptors (pestka, ; pestka et al., ) . they exert pleiotropic effects and are involved in host responses to bacterial and viral infection, in tumor surveillance mechanisms as well as in innate and adaptive immune responses (decker et al., ; pestka et al., ; stetson and medzhitov, ; takaoka and yanai, ) . in addition, ifns were the first cytokines used for the treatment of tumor patients. however, it has been suggested that tumor cells might develop either a transient or a permanent ifn insensitivity. this phenotype is linked to cytotoxicity resistance and might lead to escape of tumor cells from immune surveillance. we here summarize the current knowledge about (i) pleiotropic functions of ifns that mediate various biological responses, (ii) mechanisms of action and transduction pathways, (iii) the effect of type i and type ii ifns on the expression levels of molecules involved in proper major histocompatibility complex (mhc) class i and class ii antigen processing and presentation of tumor cells, (iv) the frequencies and the underlying molecular mechanisms of ifn resistance in tumors in association with alterations of the mhc class i and ii antigen-processing machinery, and (v) the clinical relevance of aberrant ifn signaling. the elucidation of the mechanisms leading to dysregulation of ifn signal transduction cascades triggering immune dysfunction and to tumor immune escape will benefit the design of strategies reversing these deficiencies, which could be of clinical relevance. interferons (ifns) are a family of multifunctional cytokines, which were originally described as antiviral cytokines, thereby protecting cells from viral infection (isaacs and lindenmann, ) . however, based on the current knowledge they exhibit a broad spectrum of activities including antiproliferative, immunomodulatory, anti-inflammatory, apoptosis-inducing, stress-mediated effects as well as regulation of cell differentiation steps and angiogenesis (amadori, ; baccala et al., ; theofilopoulos et al., ) . the ifn family is divided into type i, type ii, and type iii ifns. type i ifns consist of ifn-members and single members of ifn-, ifn-, ifn-, and ifn-e, respectively, which are all clustered on chromosome . in contrast, type ii ifn is represented only by a single gene, ifn-, encoded by chromosome (decker et al., ) . recently, type iii ifns have been discovered as a novel class of antiviral cytokines which are classified into ifn- , - , and - (oesterlund et al., ; sheppard et al., ; uze and monneron, ) . ifns bind to two distinct cell surface receptors. type i and ii ifn signal through a common -chain, thereby activating discrete, but related pathways leading to the transcriptional activation of the so-called interferonstimulated genes (isgs) ( table i; fig. ). isgs represent a functionally diverse group of genes involved in many cellular activities such as transcription, translation, regulation of cell cycle and apoptosis, intracellular communication as well as the processing and presentation of antigens. the transcriptional activity of isgs is necessary to mediate the effect of ifns. because of their diverse activities, ifns have been used for the treatment of various diseases such as chronic viral infections, like hepatitis c, multiple sclerosis, hematopoietic malignancies as well as solid tumors including renal cell carcinoma (rcc) and melanoma. the ifn therapy has been shown to reduce the rates of relapses and mortality by between and % in tumor patients (kirkwood et al., ) . however, during the last decade no further progress concerning the adjuvant therapy of tumor patients has been achieved. therefore, a better knowledge of the underlying molecular mechanisms of ifn action may lead to improved and more effective applications and the design of innovative, intelligent treatment strategies using ifns alone or in combination with other therapeutics. a wealth of information is available on the molecular processes underlying some of the ifn-induced signaling cascades. binding of ifns to their specific receptors lacking intrinsic kinase activity induces oligomerization of receptor subunits triggering diverse signaling pathways ( fig. ) , thereby leading to the transcriptional regulation of a plethora of target genes (kaur et al., ; li et al., ; schindler et al., ) . the physiologic relevance of ifn-dependent signal transduction cascades including the stat/jak pathway was established by generating and characterizing mice with targeted disruption of genes encoding stat /stat or jak , respectively (platanias, ; ramana et al., ) . both type i and type ii ifn receptors (ifn-r) initiate the activation of the jak/stat cascade, which consists of four janus kinases (jak , jak , jak , and jak ) and seven signal transducers and activators of transcription (stat , stat , stat , stat , stat a, stat b, stat c; fig. ifn signal transduction cascade and defects in this pathway. the type i and type ii receptors are transmembrane glycoproteins whose extracellular domains serve as ifn-binding sites, whereas their cytoplasmic domains associate with members of the jak kinase family and initiate signal transmission (dunn et al., ) . upon binding to their specific receptors both type i and type ii ifns induce a number of signal transduction cascades, which involve the phosphorylation of various components such as tyk , jaks, and stats. after recruitment to the receptor, stats become phosphorylated, form homo-or heterodimers, and migrate to the nucleus to bind to specific sequences in the promoter of target genes. type i ifn-induced signaling then induces homodimerization of stat and heterodimerization of stat and stat . stat and stat associate with the cytosolic transcription factor ifn-regulatory factor (irf ), forming a trimeric complex known as ifn-stimulated gene factor (isgf ) and activates transcription by binding to the isres. type ii ifn associates kinases, jak and jak phosphorylate stat , which then forms homodimers, translocates to the nucleus, and activates transcription by binding to the gas sequences. ifn-mediated signaling is controlled by several mechanisms including dephosphorylation of ifn-r , jak , and stat (mediated by sh -domain-containing protein tyrosine phosphatase , shp ), inhibition of the jaks (mediated by suppressor of cytokine signaling , socs ), proteasomal degradation of the jaks, and inhibition of stat (mediated by protein inhibitor of activated stat , pias ). shin-ya et al., ; yu and jove, ) . stats, sh -containing transcription factors, represent cytosolic proteins of - amino acids and are composed of (i) an extracellular domain that plays an important role in the association of stat with receptor molecules, (ii) a ligand-binding domain, and (iii) an intracellular domain that is responsible for the stat dimer formation. stat induces the expression of ifn-responsive genes through the activation of ifn-stimulated response element (isre)-containing promoters (yu and jove, ) . however, it has now become apparent that the activation of jak-stat pathways alone is not sufficient for the generation of all biological activities of ifns. there exists accumulating evidence that several other ifn-regulated signaling elements and cascades are required for the generation of many ifn responses. some of them operate independently of the jak-stat pathway, whereas others cooperate with stats to optimize the transcriptional regulation of target genes. these include in particular pathways linked to cellular stress and cell death like the mitogenactivated protein kinase (mapk), the stress-induced kinase p , and protein kinase c (pkc) signaling cascades. pkcs are known to be involved in both ifn-andsignaling pathways (kwon et al., ) . in this context, it is noteworthy that the ifn-, ifn-, and ifn-cascades exhibit overlapping activities, but also clearly different features ( fig. ; levy et al., ) . after the engagement with the type i ifn receptors (ifn-r), ifnbinding stimulates the cross-linking between the ifn-r chain (ifn-r ) and (ifn-r ), thereby bringing the receptor-associated kinases tyk and jak into close proximity. this triggers the activation of jak and tyk leading to the phosphorylation of tyr- of the ifn-r , which serves as a docking site for stat . the activated kinase subsequently phoshorylates stat and stat on tyr- and tyr- , respectively. both phosphorylated stats form a heterodimer and associate with the interferon regulatory factor (irf) , which does not undergo tyrosine phosphorylation to form the ifn-stimulated gene factor (isgf ), which, in turn, translocates to the nucleus and binds specific elements known as isres that are present in the promoters of certain isgs initiating the transcription of a broad variety of genes. in addition, phosphorylated stat , other stat complexes, and combinations of different stat-containing complexes can be formed which translocate to the nucleus and bind to the ifn--activated site (gas) leading to the transcription of further genes (caraglia et al., ) . it is noteworthy that ifn-can also activate stat and stat , but the role of stat in the ifn--mediated activity has still to be elucidated (uddin et al., ) . in contrast, ifn-mainly activates stat b. however, one can speculate that a fine balance between different stat complexes might account for specific responses and represent a key mechanism for ifn--induced activities. ifn-acts through a heterodimer consisting of the ifn-receptor- (ifn-r ) and ifn-r expressed on most cells, thereby upregulating specific genes. binding of ifn-initially leads to the formation of an ifn-r homodimer, which consecutively attracts the ifn-r chains. the ifn-r and -r homodimer is constitutively associated with jak and jak , which phosphorylate the tyrosine at the intracellular domain of the ifn-r serving as a docking site for the latent cytosolic transcription factor stat . stat is subsequently phosphorylated on tyrosine and serine leading to the homodimerization of phospho-stat molecules. these form a complex named the -activating factor (gaf) that translocates into the nucleus and upregulates the transcription of ifn--regulated genes including in particular the interferon-regulated factors (irf) and irf which represent transcriptional activators, whereas the constitutively expressed irf generally acts as a transcriptional repressor (harada et al., ) . irf subsequently activates the transcription of caspase genes involved in apoptosis next to genes encoded in the major histocompatibility complex (mhc) in particular components of the mhc class i and class ii antigen-processing machinery (apm) as well as -microglobulin ( -m) located on chromosome . the molecules of the antigen-processing pathway are required for the initiation and triggering of proper cd þ or cd þ t-cell responses, respectively. in addition, stat and irf cooperate with the ubiquitously expressed transactivating factor upstream stimulatory factor (usf) to activate the transcription of the class ii transactivator protein promoter iv (ciita-piv) that controls the expression of mhc class ii molecules (chen et al., ) . the expression of mhc class i and class ii molecules is critical for the presentation of antigens and essential for the generation of an adaptive immune response (cresswell et al., ; jensen, ) . in the last decades, cd þ cytotoxic t lymphocytes (ctl) have been implicated as main effector cells in antitumor responses. they recognize and attack tumor cells presenting intracellular antigens derived from different nonself peptides on their surface through the interaction of the t-cell receptor (tcr) with mhc class i peptide complexes. the generation and presentation of these antigens (ag) requires a coordinated expression of several genes ( fig. a) . briefly, endogenously synthesized proteins are cleaved by the multicatalytic proteasome complex, in particular the ifn--regulated proteasome subunits, such as the low molecular weight proteins (lmp) , lmp , and lmp . these peptides are further trimmed by cytosolic enzymes such as, for example, the tripeptidyl peptidase (tpp)ii and the bleomycin hydrolase (bh) generating the correct n-terminus (kloetzel, ; rock et al., ) . then the peptides are transported from the cytosol into the endoplasmic reticulum (er) via the transporter associated with antigen processing (tap), a heterodimer consisting of the tap and tap subunits. in the lumen of the er the mhc class i assembly occurs, which is assisted by various chaperones such as calnexin, calreticulin, the oxido thiol reductase erp , and tapasin (tpn). tpn facilitates the peptide loading onto mhc class i molecules. after successful peptide loading, mhc molecules are released from the peptide loading complex and the trimer consisting of mhc class i heavy chain (hc)/ -m/peptide is then transported through the trans-golgi apparatus to the cell surface and presented to cd þ ctl. thus, proper expression of the major components of the complex mhc class i apm components is obligatory for effective t-cell recognition of tumors (groettrup et al., ; jensen, ; seliger et al., ) . recently, it has been demonstrated that cd þ t cells are also important for proper antitumor immune responses (drozina et al., ; jensen, ) . these t cells recognize via their tcr antigens presented on mhc class ii molecules. in contrast to mhc class i antigens which are expressed on all nucleated adult cells, the expression of the heterodimeric mhc class ii molecules also representing transmembrane glycoproteins is highly restricted and preferentially found on the cell surface of professional antigen presenting cells (apcs). however, mhc class ii antigen expression can be induced in other cell types by various cytokines, in particular ifn-. mhc class ii expression is mainly controlled by the class ii transactivator protein (ciita), which acts as a master regulator for its coordinated constitutive and ifn--induced expression which also involves pkc delta (kwon et al., ; giroux et al., ) . ciita interacts with the transcription factors rfx, nfy, and creb (van den in the cytosol, endogenous peptides are generated by the proteasome, which were further trimmed by other peptidases and then transported into the er via the heterodimeric tap. erap is involved in the final aminoterminal trimming of peptides. the loading of mhc class i molecules with peptides is further assisted by the chaperone tapasin which is also a component of the plc. upon peptide loading, the plc dissociates and then transported via the trans golgi to the cell surface and there exposed to cd þ cytotoxic t lymphocytes. (b) mhc class ii pathway. mhc class ii molecules assemble in the er with the invariant chain (li), which contains an endosomal targeting signal. this complex is then transported to the endosomal compartment and there the ii is cleaved by a number of proteases leaving only the clip fragment, which occupies the peptide-binding groove. hla-dm and -do catalyze the release of clip, which is exchanged by antigenic peptides. hla-dm edit the repertoire of the mhc class ii-peptide complexes, which are then transported to the cell surface for recognition by cd þ t lymphocytes. exogenous proteins are internalized into the endosomal pathway by different mechanisms then unfolded and cleaved which is catalyzed by different proteases. in addition, the yielded peptides are further trimmed after binding to mhc class ii molecules. elsen et al., ) , thereby forming an enhanceosome governing the mhc class ii transcription. in addition, a coordinated expression of various mhc class ii apm components exists. mainly exogenous antigens are phagocytosed by apcs, directed then to lysosomes where they are cleaved into small peptide fragments (fig. b) . mhc class ii antigens are assembled in the er. the peptide-binding groove of these molecules is initially occupied by the invariant chain which is degraded into the class ii invariant chain peptide (clip) fragment by a series of key cleavage events, thereby protecting the mhc class ii-binding groove. the loading of mhc class ii molecules with exogenously derived peptides is assisted by the chaperone-like components hla-dm and -do, which results in an exchange of the clip fragment by these antigens. hla-dm is editing the peptides presented to cd þ t cells by catalyzing multiple rounds of peptide exchanges possibly favoring the most stable complexes. the peptide-loaded mhc class ii molecules are then transported to the cell surface and presented to cd þ t lymphocytes. in professional apc, exogenous antigens can gain access to the mhc class i pathway through distinct cross-presentation mechanisms. furthermore, the endosomal mhc class ii loading pathway could also receive peptides derived from endogenous antigens through autophagy and other mechanisms (dengjel et al., ; schmid et al., ) . the promoters of the mhc class i and class ii apm components have been intensely characterized and exert some similarities, but also unique properties. concerning the promoter of mhc class i apm components, some of them contain tata and caat boxes, whereas others completely lack these regulatory domains in the promoters). in addition, it is noteworthy that both tap and lmp are transcribed from a shared bidirectional promoter of only base pairs (bp) separating their atg translation initiation codon (wright et al., ) . the promoter of the major mhc class i apm components contain a combination of distinct transcription factor-binding sites (tfbs), like sp , creb, the nuclear factor (nf)-b, e f, and p , but all exhibit ifn-response elements, which hint toward their regulation by irfs chatterjee-kishore et al., , fig. ) . in terms of the mhc class ii pathway, the promoters of the invariant chain, hla-dm/-do and the mhc class ii hc, respectively, contain similar, but also distinct transcription factorbinding sites, whereas all of them contain an ifn-response element in their promoter. an exception is represented by ciita, which is regulated by multiple promoters differing in their tfbs composition. there exist three tissue-specific promoters for ciita, pi, pii, and piii. one promoter controls the constitutive ciita expression in dendritic cells (dc), whereas another is specific for the constitutive expression in b cells. the ciita-piv regulates the induction of ciita expression in different cell types. it contains several cis elements including a putative nf-b site overlapping with an ap site, the ifn-activating sequence (gas), the e box, and an irf element (dong et al., ; muhlethaler-mottet et al., ) . thus, the activity of the different mhc class i and ii apm component promoters can be induced, but to a different extent, by type i and type ii ifns, respectively. ifn-is a stronger inducer when compared to type i ifns, whereas a combination of both substances exerts additive or even synergistic effects on mhc class i and ii apm components. activation of adaptive immune responses by ifn, in particular ifn-, is partially due to transcriptional activation of genes encoding the mhc class i and class ii antigens and respective apm components such as the invariant chain, hla-dm/-do, ciita, tap, tpn, the lmps, and erap / . fig. promoter structure of major apm components. the structure of representative promoters of the major apm components is schematically illustrated, demonstrating a number of transcription factor-binding sites such as nf-b, ap , sp , and creb as well as interferon regulatory response elements (isre), which are involved in the inducibility by this cytokines. decrease in or absence of mhc class i molecules has been observed in a diversity of human tumor types (garrido and algarra, ; garrido et al., garrido et al., , ). an increasing proportion of tumors were found with total or selective hla allelic losses supporting the theory that altered hla expression phenotypes represent a major mechanism of tumor escape from t-cell recognition due to downmodulation of presentation of immunodominant tumor antigens. distinct hla class i abnormalities, including total loss or downregulation of hla class i antigens (paschen et al., ) , hla haplotype loss (ramal et al., ) , hla locus or allele loss (jimenez et al., ) has been described in tumors originating from different tissues and multiple molecular mechanisms have been identified as responsible for these changes (garrido and algarra ) . the mechanisms that underlie total or partial loss of hla class i antigens (table ii) include mutations of the -microglobulin ( -m) gene (perez et al., ) and loss of heterozygosity (loh) of mhc genes (maleno et al., ) . other causes of total hla class i downregulation comprise defects in the regulation of different components of the mhc class i antigen processing. structural defects of apm components cannot be corrected by cytokine treatment; therefore, it does not restore hla class i surface antigen expression. t-cell-based therapy may not be effective due to the irreversible loss of hla class i molecules. this is important when selecting the appropriate immunotherapy for a given cancer patient. abnormalities in the expression of various mhc class i apm components occur at a high frequency in human tumors of distinct origin like small-cell lung carcinoma (sclc), melanoma, colon carcinoma, breast carcinoma, renal cell carcinoma, and hematological malignancies and are frequently associated with malignant transformation (table ii) . this phenotype allows the tumor cells to evade recognition by mhc class i-restricted, tumor antigen (ta)-specific ctl. mutations in different apm components appear to be a rare event postulating that dysregulation rather than structural alterations is the major cause for aberrant apm component expression (fernandez et al., ; ramal et al., ; seliger et al., ; table ii) . this hypothesis is supported by experiments (i) identifying only few mutations in these molecules, (ii) characterizing the apm promoter activity in tumors, (iii) determining posttranscriptional regulatory mechanisms, and (iv) treating tumor cells with ifns to analyze whether deficiencies of apm component expression could be overcome by cytokines. indeed, impaired apm component expression of tumor cells could be often restored by ifn-/ and/or ifn-treatment. the ifn-mediated upregulation of apm components often results in enhanced mhc class i surface expression, which is required for the generation of an effective antitumor-specific immune response. indeed, the ifn-induced upregulation of apm components improves antitumor-specific ctl responses (seliger et al., ; tajima et al., ) and therefore represent a valuable strategy for the treatment of patients with apm component deficiencies. however, in some cases, tumors remain insensitive to ifn treatment despite the lack of structural alterations in apm components, rather suggesting an impaired ifn signal transduction. the unresponsiveness to ifn treatment was analyzed in a number of different tumor types and according to kaplan et al. ( ) can be frequently found in human cancers. approximately % of melanoma and non-adenocarcinoma lung tumor cell lines analyzed exhibit a quantitative reduction in ifn-sensitivity, while out of lung adenocarcinoma cell lines were totally unresponsive to ifn-. these data were extended in a recent study in which melanoma cell lines were analyzed for the ability to upregulate mhc class i surface antigens in response to stimulation with ifn-. a total unresponsiveness to ifn-was found in out of melanoma cell lines (rodriguez et al., b) . however, the number of tumor types and tumor samples analyzed for ifn resistance is still limited and requires further studies in order to determine the frequency, relevance, and molecular mechanisms of these deficiencies. it is noteworthy that an impaired ifnresponse despite a functional ifn-induction may exist. on the other hand, a lack of ifn-responsiveness can also be found in the presence of ifnsensitivity, suggesting that the ifn signal transduction cascades are not coordinately regulated in tumor cells. the importance and involvement of ifn signal transduction pathways in the transcriptional regulation of apm promoters have been established, but there exists only limited information about the underlying molecular mechanisms of defective ifn-inducible apm component expression. the impairment could occur at different steps along the ifn signal transduction pathways and might involve sequence abnormalities and/or different regulatory processes such as transcriptional, posttranscriptional, and epigenetic control ( fig. ; table iii ). the physiological relevance of the stat/jak and pi k pathway has been established in mice with a targeted disruption of these genes. the lack of jak activity was associated with a loss of ifn-to induce growth arrest and apoptosis as well as an increased tumorgenicity (sexl et al., ) . however, the observed ifn-response with respect to growth inhibition might also be attributable to the ifn-inducibility of lmp (hayashi et al., ) . so far, there exists only limited information regarding the molecular mechanisms of ifn resistance in tumors (huang et al., ; lesinski et al., ; wellbrock et al., ; wong et al., ) . based on the current knowledge that stat and irf are involved in the transcriptional regulation of the dual tap and lmp promoter, the loss of tap and lmp expression may be attributable to deficiencies of these regulatory factors. regarding the ifn-resistance of rcc cell lines, it is associated with a defective induction of stat that could be restored by the addition of a supernatant from pma-stimulated peripheral mononuclear cells (brinckmann et al., ) . this effect appears to be mediated by ifn-although other cytokines might also be involved in this process. in addition, the loss of the ifn--mediated upregulation of mhc class i apm components in some rcc cell lines appears to be due to the lack of irf -and stat -binding activities upon ifn-stimulation. the stat , jak , and jak proteins were expressed but not phosphorylated in the presence of ifn-. the ifn--mediated inducibility was not restored by gene transfer of jak and/or jak into rcc cells, whereas jak overexpression increased both tap and lmp expression independent of ifn-. therefore, the loss of tap and lmp induction was associated with a defect of an early step in the ifn-signal transduction pathway (dovhey et al., ) . furthermore, an association of impaired stat phosphorylation with the loss of ifn-mediated hla class i induction was also found in melanoma cell lines (rodriguez et al., b) . the absence of stat phosphorylation was at least partially due to the constitutive expression of the suppressor of cytokine signaling (socs)- protein, which could be mediated by the jak kinase inhibition via the socs phosphatase. socs- modulates the ifn-mediated signaling by binding to the autophosphorylation site of jak and by targeting bound jak to the proteasome for degradation (waiboci et al., ) . in addition, socs- expression correlates with melanoma progression and confers growth advantage (komyod et al., ; li et al., ) . in another study, the ifn-resistance was associated with socs expression. the resistant cell lines differed from the sensitive cells by a constitutive expression of socs , by the absence or a low degree of socs - activation following ifn-treatment, and by a short duration of the cytokine activatory signal (fojtova et al., ) . the expression of ifn--responsive genes is also reduced in the choriocarcinoma cells jeg and jar in comparison to the epithelial cell line hela (choi et al., ) . this is mediated by a compromised tyrosine phosphorylation of jak and stat at tyrosine and the reduced expression of irf . in addition, inhibition of the tyrosine phosphatases results in increased jak and stat phosphorylation and ifn--induced gene expression in these cells (choi et al., ) . the impaired expression of irf and deficient phosphorylation of stat were also observed in primary trophoblast cell lines suggesting that these defects are of clinical relevance. besides the posttranslational regulation of components of the ifn signal cascades, the absence of the ifn--mediated mhc class i expression can be controlled by epigenetic alterations in this pathway. indeed, methylation affects the binding of irf leading to an abrogation of the irf transactivation (rodriguez et al., b) . treatment with the demethylating agent deoxyazacytidine (dac) restored the irf expression and consecutively led to the reconstitution of the ifn--mediated mhc class i inducibility. other studies have identified that the ifn unresponsiveness is attributed to low expression of stat rather than to an absence of its phosphorylation (abril et al., ; xi et al., ) . the absence of stat expression has been correlated with the methylation of its promoter (xi et al., ) . finally, there exists evidence that genetic instability in tumor cells may lead to modulation of the expression of the ifn-r, which in some cases has been reported to be associated with cancer prognosis. for instance, the loss of ifn-r independently predicts poor prognosis in ovarian cancer and may be responsible for the limited success in the outcome of treatment of ovarian cancer with ifn- (duncan et al., ) . the multiple activities of ifns on tumor cells might coordinate the antitumor immune responses so that the early recognition and/or elimination of cancer cells by the innate immune system transitions to immune attack by the adaptive immune system (dunn et al., ) . the ifn-on the tumor cell immunogenicity mediate the immune response directed against tumor cells through distinct mechanisms. ifn-can downregulate the expression of the nkg d ligands and at the same time increase the expression of mhc class i molecules (bui et al., ) . in vitro treatment with ifn-decreased the death by nk cells independently from the expression of hla class i molecules, whereas an increased mhc class i expression increased the sensibility ctl-mediated lysis. besides these in vitro results, there also exist information that abnormalities in the ifn signaling occurs in vivo. lmp -/-mice exhibit an impaired proteasome function and % of female lmp -/-mice develop uterine leiomyosarcomas by months of age. thus, the development of spontaneous human uterine leiomyosarcomas might be probably due to defects in early steps of the ifn signal cascade. indeed, the defective tap and lmp expression in these tumors is associated with a g e mutation in the atp-binding region of the jak kinase domain, thereby affecting jak kinase activity, but neither jak expression and production nor its degradation (hayashi et al., ) . this allows the tumor cells to evade antitumor-specific immunity. in different tumor types, immunosuppression associated with stat activation and stat -mediated inhibition of dc function has been reported (yu and jove, ) . the biological function of stat and stat differs in terms of cell growth and induction of an antitumor immune response. whereas stat abrogates growth and mediates antitumor effects, stat promotes cell proliferation and tumorigenicity as it has been shown in melanoma and head neck squamous carcinoma. in both tumor entities, stat expression is associated with tumor progression and mediates immune suppression. in addition, unphosphorylated or phosphorylated stat and stat are coordinately upregulated by both ifn-and ifn-and may represent a marker for the dynamic mechanism of melanoma progression and host response. using methylcholanthrene (mca) and untreated ifn-r -/-, a significant tumor development was observed in the ifn-r control mice. the crossing of ifn-r and stat -/mice with p -/mice resulted in a spontaneous and more rapid tumor development in particular teratomas, hemangiomas, and chondrocytomas, whereas lymphoid tumors generally develop in ifn--sensitive p -/mice. interestingly, the ifn-sensitive tumor cells transfected with the dominant negative ifn-r mutant grew faster than untransfected tumors and were not rejected upon their treatment with lipopolysaccharide (lps) effectively eliminating control tumors . furthermore, downregulation of the ifn-r in association with loss of fas function is linked to tumor progression (yang et al., ) . thus, the ifn-responsiveness is an important mechanism in the control of tumor growth. an increased responsiveness to metastasespromoting agents might be induced by many mediators in the microenvironment of melanoma including type i and type ii ifns. both cytokines cooperate with tnf-, which involves a positive interplay between jak and pkc signal transduction (bianchini et al., ) . these data suggest that multiple signals were generated by the host inflammatory cells, which are accompanied by cooperate with the invasive properties of tumor cells. therefore, strategies targeting this cross-talk among tumor and host cells in the microenvironment are needed to prevent tumor growth. the chimeric ret/ptc (rearranged in transformation/papillary thyroid carcinoma) oncoproteins were constitutively expressed in papillary thyroid cancer and are able to phosphorylate the y of stat , which is accompanied by irf expression (hwang et al., ) . this is associated with an enhanced transcription of ciita and consequently with mhc class ii expression of papillary thyroid carcinoma cells and explain the immune cell infiltration of ret/ptc-positive cancers. furthermore, a synergistic activity of tnf-and ifn-on ciita was found in thyroid carcinoma (rahat et al., ) the ciita-independent mhc class i expression could be upregulated by histone deacetylases like trichostatin a (tsa) (chou et al., ; gialitakis et al., ) . ciita was refractory to ifn induction in many tumors. in colorectal and gastric carcinoma cells, ciita is silenced by epigenetic mechanisms resulting in the lack of ifn--induced mhc class ii expression (satoh et al., ) . in order to correlate the ifn unresponsiveness with the expression profile of isgs, cdna microarray analyses were employed using a customized microarray consisting of isg (holko and williams, ) . expression of genes associated with transcription precedes the expression of genes involved in signal transduction, whereas no differences in the stat induction were observed. however, subtle alterations in the expression profile might be responsible for the insensitivity to this cytokine. the maintenance of transcriptional activation following ifn treatment appeared to enhance ifn sensitivity. ifns have been used in various clinical settings, since they are potent negative regulators of cell growth either by modulating the cell cycle or by inducing pro-apoptotic genes. ifn-has been extensively studied in the treatment of various malignancies during the last two decades demonstrating improved clinical outcome of hematological malignancies (chronic myeloid leukemia, cutaneous t-cell lymphoma, hairy-cell leukemia, multiple myeloma), solid tumors including malignant melanoma, renal-cell carcinoma (rcc), aids-related kaposi's sarcoma, and viral syndromes (hepatitis c, hepatitis b, severe acute respiratory syndrome). ifn-has shown positive results in the treatment of chronic granulomatous disease, multiple sclerosis, and severe malignant osteopetrosis (parmar and platanias, , for review). however, the resistance to ifns has been described, which limits their anticancer activity. the impaired expression of ifn-responsive genes might have important implications not only in immunotherapy but also in transplantation, pregnancy, and the development of tumors such as choriocarcinoma. despite proven clinical efficacy in malignancies, viral infections, and multiple sclerosis, a substantial number of patients fail to develop positive clinical response to ifn therapy. although ifn- b is a clinically active therapeutic agent for malignant melanoma and rcc, only - % patients with metastatic melanoma respond to ifn therapy (marincola et al., ) . other reviews report even lower response rates of only % of treated melanoma patients (quesada et al., ; umeda and niijima, ) . in rcc, the best results of ifn treatment as determined by the response rate and the duration of the effect were obtained in patients with a previous nephrectomy without chemotherapy, in a good functional state, and with preferentially lung metastasis. in these patients the survival rate increased from to weeks upon ifn-administration (logothetis, ) . despite these positive results, there exist many aspects of these response factors which are not well understood. actually, none of these factors has been proved to be associated in an unambiguous way with the cytokine response and the patients' survival. the key aspect may be the right selection of patients, since currently all of them independent of previous nephrectomy and the presence of metastasis are enrolled into the treatment with poor clinical outcome. unlike type i ifns, ifn-has not been approved for cancer treatment by the fda. ifnproduces numerous antitumor effects and plays a central role in promoting natural immune responses directed against developing tumors. however, its practical application in immunotherapeutic protocols has been very limited. in clinical trials, an improved survival was observed in patients with ovarian cancer of stage ic-iiic treated with ifn- (windbichler et al., ) , when ifn was intravesically administered to patients with transitional-cell bladder carcinoma (giannopoulos et al., ) or when ifn was used in isolatedlimb perfusion of individuals with non-melanoma cancers of the extremities (lienard et al., ) . however, no effect was detected upon ifn-treatment of patients with metastatic rcc (gleave et al., ) , advanced colon cancer (wiesenfeld et al., ) , or small-cell lung cancer (jett et al., ) . the limited success of the therapeutic use of ifn-might reflect the inability to target ifns in the right place with an efficient concentration (dunn et al., ) . despite the proven pivotal role of endogenously produced antitumor immunity of ifn-in animal models, the limited success of this cytokine in cancer immunotherapy trials in humans might be explained by the resistance of tumor cells to ifn- (kaplan et al., ; rodriguez et al., a; wong et al., ) . in this context, it is important to note that unlike type i ifns, ifn-has a direct effect on tumor cells during the antitumor immune response supporting the relevance of ifn-in the cancer immunoediting process (dunn et al., ) . the targets of the immunologic unresponsiveness represent genes encoding components of the mhc apm components or the constituents of the ifn-r signaling pathway. in this context, in two recent studies from our laboratory, the physiological relevance of hla class i surface expression during the tumor rejection process in patients receiving different protocols of immunotherapy was assessed (cabrera et al., ; carretero et al., submitted) . in the first study, a significant difference in the immunotherapeutic response of patients exhibiting metastases with low levels of mhc class i surface antigens and those with high levels of mhc class i expression was detected. in a second trial, the impact of cytokine unresponsiveness was demonstrated by determination of hla class i antigen expression levels on metastatic melanoma lesions during the course of the disease in one patient undergoing ifn- b and autologous vaccination plus bcg (m-vax). bcg triggers the il- /ifn-axis and induces upregulation of genes associated with antigen presentation (feinberg et al., ; saban et al., ) . the level of the mhc class i antigen expression was dependent on the ifn response since neither of the progressor metastases increased the expression of hla class i antigens after vaccination. however, a significant increase in the hla class i surface expression was detected in the regressor metastases. therefore, the hla class i surface antigen on tumor cells significantly contributed to the therapeutic effect of bcg. in connection with these findings, downregulation of hla class i surface antigens in cancer cells has been considered a significant risk factor for recurrence in patients with intravesical bcg immunotherapy for bladder cancer (kitamura et al., ) . based on these results, a better understanding of the molecular mechanisms by which tumors modulate the cytokine signaling may be essential for the development of immunotherapeutic strategies with the aim to enhance mhc class i surface antigen expression in tumor cells. the balance of stat phosphorylation versus socs expression might be crucial in the activation of immunologic response through apm and mhc class i transactivation (wang et al., ) . for instance, the effects of high-dose ifn are associated with immunologic processes such as an upregulation of tap , tap , tpn, and lmp . the stat and stat pathways in melanoma cells are sensitized to ifn-by pretreatment of the cells with ifn-. thus, the biological response to ifn-might be mediated by a direct effect on melanoma cells and suggests also a potential role for ifn-in the treatment of this disease (carson, ) . in addition, it has recently been demonstrated that ifntreatment of patients with cutaneous melanoma significantly modulates the balance of stat /stat in tumor cells and host lymphocytes. this results in an upregulation of tap and an increased immune response (wang et al., ) . an increased knowledge of the factors responsible for the resistance to ifns might lead to an improved use of these cytokines in malignant diseases. the application of the molecular analysis of tumor tissues has now advanced to the point where better classification schemes and prognostic variables are used leading to an optimization of specific treatment programs and patients' selection. the identification of tumor lesions with the capacity to upregulate mhc gene expression will determine the ability to present new antigenic peptides to t lymphocytes favoring regression of primary or metastatic tumor lesions. in contrast, the identification of tumors with mhc irreversible genetic lesions will maintain an unaltered mhc expression, thereby not exposing new antigenic peptides to t cells, which subsequently favors tumor and/or metastases progression. we propose that suppression of ifn signaling in tumors contributes to tolerance by inhibiting expression of genes encoding subunits of hla class i/ii antigens and/or components of the mhc class i/ii apm that could be detrimental to successful antitumor responses. unresponsiveness to interferon associated with stat protein deficiency in a gastric adenocarcinoma cell line the role of ifn-alpha as homeostatic agent in the inflammatory response: a balance between danger and response? interferons as pathogenic effectors in autoimmunity expression of a metastatic phenotype in ifns-primed/tnfalpha-activated b murine melanoma cells: role of jak /pkcdelta signal transduction factors 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expression: transactivation of class ii transactivator promoter iv by ifn regulatory factor- is regulated by protein kinase c-alpha interferon gamma- b compared with placebo in metastatic renal-cell carcinoma peptide antigen production by the proteasome: complexity provides efficiency structurally similar but functionally distinct factors, irf- and irf- , bind to the same regulatory elements of ifn and ifn-inducible genes the mutation in the atp-binding region of jak , identified in human uterine leiomyosarcomas, results in defective interferongamma inducibility of tap and lmp functional annotation of ifn-alpha-stimulated gene expression profiles from sensitive and resistant renal cell carcinoma cell lines stat negatively regulates angiogenesis, tumorigenicity and metastasis of tumor cells regulation of signal transducer and activator of transcription (stat ) and stat -dependent genes by ret/ ptc (rearranged in transformation/papillary thyroid carcinoma) oncogenic tyrosine kinases 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carcinoma molecular mechanisms of hla class i antigen abnormalities following viral infection and transformation jak deficiency leads to enhanced abelson-induced b-cell tumor formation il- and their class ii cytokine receptor il- r intracellular interferon triggers jak/stat signaling cascade and induces p -dependent antiviral protection type i interferons in host defense interferon-gamma differentially regulates susceptibility of lung cancer cells to telomerasespecific cytotoxic t lymphocytes interferon signalling network in innate defence type i interferons (alpha/beta) in immunity and autoimmunity role of stat in type i interferon-signaling and transcriptional regulation phase ii study of alpha interferon on renal cell carcinoma. summary of three collaborative trials il- and il- : newcomers to the interferon family transcriptional regulation of antigen presentation both the suppressor of cytokine signaling (socs- ) kinase inhibitory region and socs- mimetic bind to jak autophosphorylation site: implications for the development of a socs- antagonist modulation of signal transducers and activators of transcription and signaling in melanoma by high-dose ifnalpha b stat contributes to interferon resistance of melanoma cells controlled clinical trial of interferon-gamma as postoperative surgical adjuvant therapy for colon cancer interferon-gamma in the first-line therapy of ovarian cancer: a randomized phase iii trial interferon-resistant human melanoma cells are deficient in isgf components, stat , stat , and p -isgf gamma coordinate regulation of the human tap and lmp genes from a shared bidirectional promoter decreased stat expression by promoter methylation in squamous cell carcinogenesis downregulation of ifn-gammar in association with loss of fas function is linked to tumor progression the stats of cancer -new molecular targets come of age this work was supported by grants from the fondo de investigaciones sanitarias (fis), red genomica del cancer (retic rd / ), plan andaluz de investigacion (group cts ), consejeria andaluz de salud (sas), proyecto de excelencia de consejeria de innovacion (cts ), proyecto de investigacion iþd (saf - ) in spain; and from the integrated european cancer immunotherapy project (oj /c , ) and by grants from the deutsche forschungsgemeinschaft dfg se - / and - (b.s). in addition, we thank tarish abbas for providing figure and anne wasilewski for excellent secretarial help. key: cord- -zr zx pv authors: hoo, regina; nakimuli, annettee; vento-tormo, roser title: innate immune mechanisms to protect against infection at the human decidual-placental interface date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: zr zx pv during pregnancy, the placenta forms the anatomical barrier between the mother and developing fetus. infectious agents can potentially breach the placental barrier resulting in pathogenic transmission from mother to fetus. innate immune responses, orchestrated by maternal and fetal cells at the decidual-placental interface, are the first line of defense to avoid vertical transmission. here, we outline the anatomy of the human placenta and uterine lining, the decidua, and discuss the potential capacity of pathogen pattern recognition and other host defense strategies present in the innate immune cells at the placental-decidual interface. we consider major congenital infections that access the placenta from hematogenous or decidual route. finally, we highlight the challenges in studying human placental responses to pathogens and vertical transmission using current experimental models and identify gaps in knowledge that need to be addressed. we further propose novel experimental strategies to address such limitations. the human placenta is the temporary extra-embryonic organ that is present only during pregnancy and is the anatomical boundary between the mother and fetus. it has a range of functions including transport of nutrients and gases, and hormonal production ( ) . the placenta forms a physical, selective barrier between the maternal and fetal circulations, preventing transfer of pathogens. the uterine mucosal lining, the endometrium, is transformed into the decidua during early pregnancy ( ) . a range of innate immune mechanisms can respond to pathogens in both the decidua and the placenta ( , ) . the maternal-fetal interface is a protective barrier against pathogens, but some pathogens can transfer from the mother to fetus by different routes and cause fetal infection ( , ) . vertical transmission during pregnancy can occur on distinct boundaries between the mother and the fetus: (i) the intervillous space (ivs), where placental villi is in direct contact with the maternal blood, (ii) the implantation site or decidua basalis, where maternal cells are in direct contact with the invading fetal trophoblast, and (iii) the fetal membranes, which are in direct contact with the uterine cavity ( ) . defense mechanisms in the cervix, such as the production of mucus and antimicrobial peptides (amp), limit ascending infection from pathogens present in the lower genital tract, that otherwise may access the uterine cavity ( ) . however, some pathogens can escape antimicrobial strategies at the cervix and ascend to the uterus, where they can bypass the fetal membranes and lead to the inflammation of the membranes-also known as chorioamnionitis-and infection of the amniotic fluid ( , ) . pathologic and immune features of chorioamnionitis and intra-amniotic infection are generally associated with bacterial invasion and inflammation [refer to ( , ) for a comprehensive review on these mechanisms]. here, we focus on infections and innate immune mechanisms at the uterine-placental interfacecases (i) and (ii) (figure ) . infections at the uterine-placental interface are commonly associated with viruses, parasites and few bacteria ( table ) . viral pathogens such as human cytomegalovirus (hcmv), zika (zikv), and rubella virus are the most common vertically transmitted pathogens through the decidual-placental interface ( table ) ( , ) . non-viral pathogens, such as toxoplasma gondii and listeria monocytogenes, can cross the placental barrier via cell-to-cell transmission ( table ) ( , ) . fetal infection can result in various forms of congenital anomalies in humans ( table ) . understanding the pathogenic mechanisms used by infectious agents is central to preventing vertical transmission and controlling infection during pregnancy. how the innate immune cells and mechanisms in the placenta and the uterus recognize and respond to protect both the fetus and mother remains controversial due to technical and ethical constraints. however, there are several different models currently used to interrogate the uterine-placental interface in pregnancy. firstly, mice are frequently used as a pregnancy model for infection. although the murine models have provided important insights into the pathogenesis of various infection agents in the context of pregnancy, there are still limitations with this approach. the anatomy of placentation, length of gestation, and use of inbred strains, make extrapolation to humans problematic ( , ) . secondly, a range of human trophoblast and choriocarcinoma cell lines are used as in vitro models for infection with pathogens. in contrast to the first trimester trophoblast in vivo, these cell lines do not recapitulate normal human trophoblast characteristics such as expression of the human leukocyte antigen (hla) class i and methylation of elf ( , ) . thirdly, human primary placental explants are frequently used. the syncytium dies rapidly in these cultures and it is virtually impossible to standardize the types of villi sampled ( ) . therefore, these in vitro experimental factors should be taken into careful consideration when interpreting studies of infection of trophoblast. in this review, we cover the innate immune features of the decidual-placental interface throughout gestation. we identify the gaps in knowledge and highlight the limitations of current studies and experimental models. finally, we discuss novel experimental strategies for understanding how infection affects pregnancy in humans. the trophoblasts of the placenta are the barrier between fetal and maternal tissues. they are derived from the trophectoderm, the outer layer of the blastocyst that forms an inner mononuclear layer with an outer primary syncytium following implantation ( ) . the trophoblast in contact with the maternal cells can be: (i) syncytiotrophoblast (sct), a single layer multinucleated, syncytial layer formed by fusion of the underlying villous cytotrophoblast (vct), and (ii) extravillous trophoblast (evt), that invade from the cytotrophoblast shell and anchoring villi into the transformed maternal endometrium, the decidua ( ) . the function of evt is to transform the uterine spiral arteries so that maternal blood is delivered to the intervillous space at low pressure. the arteries are surrounded by interstitial evt that destroys the smooth muscle cells of the arterial media, known as "fibrinoid" change ( , ) . subsequently, endovascular evt (eevt) moves down the spiral arteries from the placentadecidua boundary ( ) . these eevt form a plug of cells, limiting surges of arterial blood from damaging the delicate villi. evt invasion transforms the arteries to support optimal regulation of blood flow into the placenta during fetal development ( ) . the plugs dissipate between and weeks of gestation when the full hemochorial circulation is established ( ) . maternal blood then flows into the ivs, and establishes direct contact with the sct allowing for proper nutrient and gas exchange between the mother and the fetus. hofbauer (hb) cells are fetal macrophages of the human placenta ( ) . hb cells can be detected in the placental villous stroma as early as weeks post-conception and are present throughout pregnancy ( , ) . they are likely to have a variety of functions including control of villous remodeling and differentiation, hormonal secretion, and trophoblast turnover ( , ) . several lines of evidence have led to the postulation that hb cells may have a role in infection during pregnancy. hb cells with zikv viral particles detected intracellularly have been shown ( , ). human immunodeficiency virus (hiv- ) has also been detected in hb cells from first trimester infected placenta ( ) . whether the hb cells can serve as a reservoir or limit virus replication is still unknown. isolated hb cells from healthy term placenta show elevation of pro-inflammatory cytokines such as il- , mcp- , ip- , and ifn-α upon in vitro infection with zikv ( ) . hb cells from the first trimester placenta are also permissive for zikv infection and replication ( ) . however, this must be interpreted with caution because in vitro culture of hb cells do not entirely recapitulate the complexity of villous stromal microenvironment, such as presence of hormone and growth factors, all of which will influence the function and activity of hb cells ( ) . the sct is the barrier between maternal blood and the placental core as it separates the ivs from the underlying fetal villous stroma. blood-borne pathogens such as viruses and parasites can potentially be transmitted through the sct barrier (figure ) . how can pathogens cross the sct barrier and the vct to infect the villous stroma? although the sct is an efficient barrier due to its stiff, highly dense actin cytoskeleton network and continuous membrane ( ), the syncytium undergoes continuous breaks or gaps and dynamic repair processes ( ) . breaks in the syncytium could potentially lead to transmission of pathogens into the underlying vct. our recent work showed that a novel population of maternal macrophages (m ) is associated with the sct in early pregnancy and might be involved in repairing the breaks in the syncytium ( ). it is intriguing that m macrophages infected with intracellular pathogens could possibly gain access to the underlying vct via the syncytial breaks (figure ) . only a few viral entry receptors on the sct are described. notably, the sct lacks expression of zikv entry receptors, axl, and tyro ( ) and the hcmv entry co-receptor integrin α/β ( ) . this is further supported by the transcriptomic expression of viral receptors in placental cells ( , , ). expression of surface receptors commonly used by zikv such as axl and hcmv such as nrp and pdgfra are lowly expressed by the sct ( ) . in addition, there is minimal co-expression of ace , the receptor gene for human severe acute respiratory syndrome coronavirus (sars-cov- ), and tmprss , the viral spike protein serine protease gene ( , ) . in line with this, there is no conclusive and direct evidence of vertical transmission of sars-cov- in a placenta from a healthy individual. there are some reports showing sars-cov- is predominantly localized at the sct of the second trimester placenta ( , ) and can lead to severe inflammatory infiltrate in the ivs ( ) . however, these findings are presented in a very small number of patients with severe disease or pre-existing pregnancy complications ( , ) . alternative transplacental mechanisms have been postulated at the syncytial barrier. neonatal fc receptor (fcrn) is expressed on the apical surface of the sct and functions to selectively figure | toll-like receptors and potential inflammatory response at the sct-blood interface. predominant tlrs found in the human placenta from early and term pregnancies. tlr and tlr are expressed in human placenta sct, vct, and in hb cells. infiltration of infected maternal blood, infected immune cells, or release of pathogenic determinant such lipopolysaccharide (lps), peptidoglycan, or parasite materials such as hemozoin or gpi (glycosylphosphatidylinositol) into the ivs will activate tlr-mediated signaling, leading to the production of a wide range of cytokines and chemokines. severe infection is characterized by massive immune cell infiltration including monocytes and neutrophils from systemic circulation and overproduction of inflammatory cytokines upon tlr activation. this may lead to sct inflammation and damage. sct also secretes antimicrobial peptides as innate immune mechanisms. figure is created by biorender.com. frontiers in immunology | www.frontiersin.org transport maternal igg ( ) . fcrn could be exploited by certain viruses to enter the placenta including zikv, hiv- , and hcmv ( , , ) . transferrin receptor (tfr ) is expressed on the apical end of the sct, and functions as the primary iron transporter into the basal side of the sct to provide sufficient iron stores into fetal circulation ( ) . tfr has been associated with viral entry into a broad host cell range, including hepatitis c virus ( , ) suggesting a possible mechanism of viral transport across the sct barrier. some pathogens, although unable to cross the sct barrier, can still adhere to the syncytium and cause further pathology. for instance, plasmodium falciparum infected red blood cells can bind with high affinity to chondroitin sulfate a expressed on the sct, resulting in local inflammation, syncytial breaks, and damage ( ) ( ) ( ) . although the sct is an effective barrier to most pathogens, local inflammation, tissue damage, and fcrn or tfr -mediated viral entry at the sct can potentially allow pathogen to breach the syncytial barrier, giving opportunity for transmission from maternal blood into placental villi (figure ). during the first trimester of pregnancy, fetal evt invades deeply into the uterus. the decidua basalis, the region located at the implantation site, is populated at this time by a distinctive subset of innate lymphocytes, decidual natural killer cells (dnk), which constitute up to % of leukocytes. we have identified three major populations of dnk by single-cell rna-sequencing with unique phenotypes and functions in early pregnancy ( ). in addition, there are populations of decidual macrophages (dms) (∼ %), conventional dendritic cells (dcs) and small proportions of t cells (∼ - %), whereas b cells, plasma cells, mast cells, and granulocytes are virtually absent ( ) (figure ) . the proportion of immune cells will vary throughout pregnancy, with an increase in the proportion of t cells at term ( ) . systemic infections will reach all organs including the decidua. whether pathogens can also access the decidua via the cervix is still unclear. chlamydia trachomatis, a common sexuallytransmitted intracellular bacteria, was detected in glandular epithelial cells and unidentified decidual cells in decidual biopsies ( ) . this suggests the possibility of infections ascending and spreading from cell-to-cell from the lower genital tract into endometrial glands and vascular endothelium. the decidua basalis is in close contact with fetal cells and the maternal vasculature (figure ) . first trimester dms and decidual stromal cells are susceptible to zikv infection and replication ex vivo ( ) . hence, infection could possibly spread from infected maternal immune and non-immune cells at the decidua, into uninfected vct in the columns of the anchoring villi, and finally into the fetal compartment. however, this is likely to be limited to certain microorganisms which are capable of cell-to-cell spread, have an intracellular host niche, and are able to escape host innate defense mechanisms (table ) . hcmv, the most common cause of congenital infection, is mostly reported to infect from the decidua ( , ) . women with primary hcmv infection and first pregnancy are more likely to transmit the virus to their fetus, compared to multiparous women with previous infection and demonstrable antibodies ( ) ( ) ( ) . low affinity maternal antibodies against hcmv correlate with higher viral loads detected in the decidua, whereas patients with intermediate to high neutralizing antibodies have minimal viral replication ( ) , suggesting that maternal immunity against hcmv reduces risk of vertical transmission. hcmv protein was also detected in a range of cells within the decidua including endothelial, decidual stromal cells, dcs and macrophages ( , ) , suggesting that that infected maternal leukocytes could initiate transmission through contact and infection of endothelial cells that line decidual blood vessels. despite the evidence of decidual infection, the mechanism of vertical transmission for hcmv is still in debate. dnks have been proposed to play a protective role against hcmv infection through several mechanisms including modulation of their cytotoxic effector function ( ) and the interactions between the killer-cell immunoglobulin receptors (kirs) expressed by dnk and hla molecules expressed in the infected cells ( , ) . activating kir ds by dnks has been demonstrated to be more cytolytic against hla-c hcmv-infected maternal decidual stromal cells ( ) . similar cytotoxic response was also observed when peripheral blood nk cells expressing kir ds were exposed to hcmv-infected fibroblasts ( ) . hence, this implies that in the decidua, dnks are capable of eliminating harmful infection depending on the combination of kir/hla interactions between dnk and infected cells. dnks are also able to control hiv- infection in vitro through production of ifn-γ ( ) . the role of dnk in controlling viral infection may protect against potential risk of vertical transmission from the decidua. pattern recognition receptors (prr) are encoded in the germ-line and recognize specific, conserved pathogen-associated molecular patterns (pamps). these include gram-negative bacteria lipopolysaccharide (lps), gram-positive bacteria lipoteichoic acids, lipoprotein, dna, rna, glucans, and peptidoglycans ( , ). pathogen recognition is not only an essential component of the innate immune response against infection, but also plays an important role in bridging the innate and adaptive systems by toll-like receptors (tlr) activation of antigen presenting cells by up-regulation of major histocompatibility complex (mhc) and co-stimulatory molecules ( ) . tlrs, the most studied family of prr, are type i transmembrane proteins with large extracellular domains containing leucine-rich repeats that are expressed at the cell surface or intracellularly ( ) . each tlr recognizes distinct pamps, leading to the activation of the transcription factor nf-κb and/or the interferon-regulatory factor (irf) family, and the production of a wide range of cytokines and chemokines, including type i ifns ( , ) expression of tlrs is dynamic and changes in response to different pathogens and cytokines ( ) . tlr (which recognizes bacterial proteoglycan) and tlr (which recognizes bacterial lps) are the most well-studied, with immunohistochemical evidence of expression in healthy primary sct at term ( ) ( ) ( ) . in contrast, in the first trimester, tlr and tlr proteins are expressed in vct and evt, but minimally in sct ( , ) (figure ) . there is therefore variation in tlr and tlr expression in the different trophoblast lineages across pregnancy. why and how such dynamic regulation of tlr expression occurs during gestation requires further investigation in a broader range of human placental samples (different donors, gestation stages, genetic background, sampling regions). it is likely that alteration in cytokines profiles in the microenvironment as pregnancy progresses ( ) may result in the variation in the expression of tlrs in the placenta. current evidence is only limited to in vitro tlr / stimulation studies using placental explants and primary first trimester trophoblast cells, which drives the expression of figure | toll-like receptors and potential inflammatory response at the decidua. predominant tlrs found in the human placenta from early and term pregnancies. tlr and tlr are expressed in evt. dm and dnk also express a wide range of tlr families, where stimulation of tlr agonists lead to the production of a variety of cytokines and chemokines. infiltration of infected cells and release of pamps in the decidua, which will activate tlr-mediated signaling. overproduction of inflammatory cytokines at the decidua may lead to local inflammation. figure is created by biorender.com. pro-inflammatory cytokines il- , il- , tnf-α, and ifn-γ ( , , ) . tlr and tlr proteins are expressed in hb cells, confirmed by co-expression of cd in healthy term placentas ( ) . in early pregnancy, our findings indicate that only tlr but not tlr transcripts are expressed in steady-state hb cells ( ) (figure ) . enhancement of il- and il- secretion upon stimulation of isolated first trimester hb cells with tlr agonist, lps ( ), does suggest a role for tlrs on hb cells in bacterial recognition and placental inflammation during early pregnancy. hb cells are postulated to have a role in viral replication ( , ), however evidence on the expression and function of viral nucleic acid sensing receptors tlr , tlr , tlr , and tlr in hb cells is lacking. our findings show that tlr , which recognizes viral single-strand rna (ssrna) ( ) is expressed in steady-state hb cells (figure ) ( ) . other tlrs have also been shown to be expressed in decidua cells. dms and dnks isolated from first trimester pregnancies show steady state level expression of tlr - transcripts and respond to a broad range of pamps, including heat-killed bacteria, microbial membranes, and nucleic acids ( ) . stimulating primary dms with these pamps produces high levels of tnf-α, il- β, il- , il- , il- , il- , and il- ra, whereas dnks secrete il- , il- , and ifn-γ ( ) . this study suggests that, in addition to the physiological roles of dms and dnks in accommodating the uterus for placentation, dms and dnks may play a role in pathogen recognition and antimicrobial response via activation of tlr signaling (figure ) . the extent to which subsets of dms or dnks population ( ) are critical for tlr-mediated response at the decidua is currently unknown. in malaria endemic populations, single nucleotide polymorphisms (snps) within the tlr coding and tlr promoter regions are associated with variation in disease severity and parasitemia control ( , ) . in the case of pregnancy malaria, primiparous infected mothers with common tlr and tlr polymorphic variants are correlated with severe complications such as low birth weight and maternal anemia ( ) . this highlights the importance of studies involving large cohorts of individuals which include genotyping from pregnant mothers living in malaria endemic regions (see section on "challenges and future perspective"). animal models have also been used to study the functional role of tlr signaling, particularly for pathogens that are intracellular at some stage of their life cycle ( table ) . tlr and tlr are strongly activated by malaria parasite pamps such as glycosylphosphatidylinositol (gpi), dna, and hemozoin ( , ) (figure ) . in a mouse model of placental malaria, tlr , and myd signaling activation resulted in placental expression of pro-inflammatory markers, such as il- and tnf-α ( , ) . these studies also demonstrated that malaria parasite infection and inflammation in the mouse placenta lead to reduced fetus growth rate and disorganization of the vascular space in the placenta ( , ) . however, tlr-mediated inflammation and pathology in the human placenta upon malaria infection is unknown and remains to be further investigated. studies of congenital toxoplasmosis are also currently limited to animal models. tlr and tlr are associated with recognition of t. gondii's infection in mice ( ) . engagement of the t. gondii ligand by tlr and tlr at the sct-blood or in the evt-decidua compartments is plausible, although there is still no direct evidence for such host-parasite interaction in humans. tlr has a role in controlling t. gondii infection in mice ( , ) , however in humans tlr is a pseudogene and is not expressed ( ) . cytosolic prrs play an important role in fighting against viral infection by eliciting host type i interferons (ifn) antiviral response through recognition of single and double stranded rna (ssrna and dsrna) ( , ) . examples of prrs are the cytosolic retinoic acid-inducible gene-i-like (rig-i) and the melanoma differentiation-associated protein (mda ) receptors, both expressed in the sct and vct of term placenta ( ) . in the human placenta, there is limited information on the function of rig- and mda , but they may play a crucial role in recognizing a variety of rna viruses, including zikv and dengue virus ( ) . the nucleotide binding oligomerization domain-like receptors (nod-like receptors; nlr) recognizes intracellular pathogen products which have entered into the host cytoplasmic compartment ( ) . both nod- and nod- receptors, which are known to detect intracellular bacterial peptidoglycans ( ) , are expressed in the sct in the first trimester and term placentas ( , ) . the nlr pyrin-containing and proteins (nlrp and nlrp ) form the major inflammasome complexes, which contribute to activation of inflammatory caspases and pathogen clearance ( , ) . activation of nlrp and aim inflammasomes, together with high expression of il- r, il- β, and caspase- was recently shown in the placental tissue of mothers infected with p. falciparum with significant pathology ( ) . in a murine model of intra-amniotic inflammation induced by bacterial lps, tissue sections from the decidua basalis region expressed high levels of nlrp , but negligible caspase- activation suggesting a possible non-canonical activation of the nlrp inflammasome ( ) . our analysis shows that decidual dm expresses high levels of nlrp transcript at steady state compared to other cell types ( ) (figure ) , thus dm may play a role in nlrp -mediated pathogen recognition during early pregnancy. amp secreted by epithelial and immune cells are small peptides that bind and destroy most groups of pathogens-bacteria, yeasts, fungi, and viruses ( ) . in addition to direct killing of pathogens, amps can rapidly modulate innate host immune responses by recruiting myeloid cells and lymphocytes to the site of infection and mediating activation of tlr ( , ) . the human placenta expresses high levels of β-defensins, a family of broad spectrum antimicrobial peptides which participate in direct bactericidal and anti-viral activity ( ) . specific subtypes of β-defensins (hbd- , , and ) are expressed in scts ( ) , suggesting these amps can target potentially bacterial or viral infection from the maternal blood. recognition of pamps by prrs during infection leads to production of pro-inflammatory cytokines that can aid in clearing the pathogen ( ) . studies on the direct role of proinflammatory cytokines on the placenta in the case of infection is limited. inflammatory mediators can directly influence infection outcome and fetal development, but they can also cause damage to the placenta if produced in excess ( ). amongst the proinflammatory cytokines associated with uterine-placental infection during pregnancy, the antiviral ifn are the most well-characterized. ifns are secreted by a variety of cell types as the first line of defense against viral infection ( ) . type i ifns, including ifn-α and ifn-β, are potent antiviral cytokines. ifn-α and ifn-β bind to the ifnar / receptor and lead to expression of ifn stimulated genes (isgs), which control virus infection through a variety of mechanisms ( ) . loss of ifnar in the placenta leads to vertical transmission and fetal mortality in murine herpesvirus- (mhv ) infected mice ( ) . in the mouse model of zikv infection, type i ifn-mediated signaling is essential for the control of viral replication in the placenta, but can also lead to significant placental pathology and fetal mortality ( , ) . the mechanism of type i ifn-mediated placental pathology has been recently elucidated. ifn-induced transmembrane (ifitm) protein, which normally blocks viral entry into host cells, impairs syncytin-mediated fusion of vct to form sct, leading to aberrant placental development ( ) . type ii ifn, ifnγ, predominantly produced by nk and cd + t cells is crucial in controlling parasitic infection, such as t. gondii in mice ( , ) . however, elevated levels of ifnγ in response to t. gondii infection can lead to pathological effects during pregnancy including fetal demise ( , ) . severe placental pathology and fetal death have also been associated with elevation of ifnγ during pregnancy in a murine model of malaria ( ). hence, proper regulation of type i and ii ifn-mediated signaling at the uterine-placental interface is crucial in limiting pathogen replication, whilst preserving a balanced environment for normal placental development ( ) . type iii ifn, ifnλ, are constitutively secreted by the human sct, which presumably confers antiviral effects against zikv infection ( ) ( ) ( ) . tryptophan metabolism by ido indoleamine , -dioxygenase (ido) is a host intracellular enzyme which metabolizes the amino acid tryptophan ( ) . ido has been associated with maternal immunoregulation during pregnancy ( ) . it also plays a key role in the control of bacterial and viral replication, through limiting the bioavailability of tryptophan ( ) . ido also inhibits the replication of several parasitic pathogens including t. gondii in human fibroblasts ( ) and leishmania spp in human macrophages ( ) . mouse infection with l. monocytogenes showed that ido is elevated in an ifn-γ-dependent manner in stromal cells of the metrial gland and decidua basalis; a crucial process to resolve bacterial infection in the mouse placenta ( ) . our findings also show ido expression is enriched in epithelial glandular and dc cell type in the first trimester decidua ( ) (figure ) . the presence of ido in decidua suggests that the enzyme might have a central role in limiting parasitic, viral, and bacterial replication, thus preventing their spread to the fetus. research on how the human placenta safeguards itself against infections is challenging due to obvious logistical and ethical issues in obtaining tissue from early in gestation (box ). although animal experimental models have provided important insights relating to the immune responses to pathogenic infection, major differences between human and animal placentas must be considered ( , ) . likewise, differences between strains of pathogens adapted for mice compared with human clinical isolates should be taken into account as this may lead to variation in pathogenesis and cellular response. one such example is the use of mouse cmv, which is unable to cross the mouse placental barrier, unlike the hmvc counterpart which can be transmitted transplacentally in humans ( ) . therefore, all data obtained from studies of infection in pregnant animals needs careful interpretation and consideration prior to translation to clinical infection in humans. inherent properties of trophoblast cell lines, primary cultures or explants vary between donors, and are likely to be confounded by the area of the placenta that is sampled and as well as stage of gestation ( ) . for instance, villous placental explants will vary depending on the types of villi sampled and the presence of box | perspective of vertical transmission and innate immune function during pregnancy and infection. a variety of maternal infections can lead to vertical transmission ( table ) . the exact mechanisms these pathogens use to escape host defense and cross the placental barrier into the fetal compartment are not entirely known. experimental models that recapitulate infection of the human placenta and thus vertical transmission are challenging to set up. more data and representative experimental models are needed to answer these questions: (i) how do different pathogens escape or modulate the maternal-fetal host innate immune barrier (ii) why do some pathogens lead to congenital infection but not others? studying infected human placentas will be essential in understanding this but access to these samples is difficult especially in low and middle-income countries (lmic) where maternal infection is particularly prevalent (who, maternal mortality index ). despite evidence of expression in primary placental tissue, functional studies on important innate immune features such as tlrs, amps, rig-i, mda , nlrs, and ido during infection and pregnancy are lacking. understanding how different cell types at the uterine-placental interface (hb cells, dnks, and dms) respond to pathogen challenge is essential, but remains under-researched. a critical obstacle is to also extrapolate the protective and pathological mechanisms of cytokines from mouse to human infection. therefore, systematic comparison of the innate immune effector mechanisms across gestation, in the placenta and decidua from natural human infection vs. healthy pregnancy, will provide a more accurate representation in clinical settings. attached decidual tissue ( ) . caution is therefore needed when interpreting data using these experimental models. to overcome such limitations, population-based cohort studies of women with infection during pregnancy with extensive tissue sampling should be performed. these need to include and focus on lmic where infection is still a major cause of maternal and fetal mortality and morbidity. cohort studies and epidemiological surveillance on maternal infections can offer significant insights into disease pathogenesis and accelerate clinical interventions ( ) . collaborations between clinicians and researchers for population-based cohort collection and sample processing will be instrumental to achieving this goal. biological samples such as blood or placenta collected from controls and infected pregnant individuals could be stored and cryopreserved retrospectively. to capture the overall heterogeneity of infected and non-infected placenta samples, sampling, and biobanking criteria of different regions of placenta should be considered ( ) . protocols are now available to use frozen tissue processed for single-cell/nuclei and spatial genomics ( , ) . hence, application of single-cell "omics" on infected vs. healthy human placental and decidual samples will enable us to evaluate cellular heterogeneity in response to infection. the capacity to detect transcripts specific to host or pathogen mrna from the same tissue using in situ nucleic acid hybridization methods will provide direct quantification of infection burden and identification of potential target host cells within the same tissue ( ) . recent advances in spatial transcriptomics methods have also allowed gene expression signatures to be quantified and resolved from individual tissue sections ( ) . combination of these emerging technologies with new methods to integrate single-cell and spatial data computationally ( ) will provide an unbiased approach to characterize and profile the transcriptome of individual cells in situ from the placenta and decidua in response to infections. we anticipate that high-throughput datasets generated from cohort sampling studies will unravel novel cell states and tissue spatial localization associated with placental infections and inflammation. this will also allow us to better characterize not only the innate immune response or makers of infection, but also other adaptive immune states in the human placenta (box ). the use of in vitro models will also further define host responses to infection. the recent generation of human trophoblast stem cells (htscs) ( ) and three-dimensional ( d) trophoblast organoids ( , ) offer a great opportunity to study infections in early pregnancy where the access to first trimester placental samples is a concern. more importantly, the htscs and trophoblast organoids fulfill the criteria characteristic of human first trimester trophoblast in vivo ( ) . both htscs and trophoblast organoids can differentiate in vitro into sct and evt with appropriate media ( , ) allowing infection experiments on both the major trophoblast subpopulations present at the two major sites of contact between maternal and fetal cells. sequencing of both host and pathogen transcriptomes from infected trophoblast at single-cell resolution will also advance our understanding on host-pathogen interactions in placentas ( , ) . further refinement of the trophoblast organoid and htscs culture system is needed to address key biological questions unanswered by current models. these include studying the effect of infection on cellular crosstalk between trophoblast and other primary placental cells such as hb cells, or decidual cells in culture, such as dnk or decidual stromal cells. adaptation of crispr/cas genome editing technology for the trophoblast organoids or htscs will offer novel insights into essential host genes required for vertical transmission and placental defense mechanisms in humans. major maternal and fetal complications as a result of infection are still a concern, especially in lmic with highest prevalence reported in countries of sub-saharan africa (who, maternal mortality index ). profound limitations on current study models and ethical regulations on studying human placenta have significantly delayed the development of therapies and vaccines for maternal-fetal infection. how vertical transmission occurs and how the uterine-placental innate immune system reacts to infection remain as major unresolved questions. revolutionary advances in single-cell genomics, imaging, computational, and stem cell biology methods are currently underway to study the molecular and cellular mechanisms of human diseases. therefore, it is now an exciting time to apply these transformative technologies to comprehensively address fundamental questions on host-pathogen interaction at the human uterine-placental interface. rh, an, and rv-t wrote and edited the manuscript. all authors contributed to the article and approved the submitted version. rh and rv-t were supported by wellcome sanger core funding (wt ). an was supported by a sanger international fellowship, a nurture fellowship (d tw ) and a group leader award supported through the deltas africa initiative ( /z/ /z). the deltas africa initiative is an independent funding scheme of the african academy of science (aas), alliance for accelerating excellence in science in africa (aesa), supported by the new partnership for africa's development planning and coordinating agency (nepad agency) with funding from the wellcome trust (grant no. ), and the uk government. cellular basis of interaction between trophoblast and uterus at implantation immune mechanisms at the maternal-fetal interface: perspectives and challenges immune responses at the maternal-fetal interface zika virus -reigniting the torch antimicrobial peptides in the female reproductive tract: a critical component of the mucosal immune barrier with physiological and clinical implications evidence that intra-amniotic infections are 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analysis of gene expression in tissue sections by spatial transcriptomics spatial mapping of cell types by integration of transcriptomics data derivation of human trophoblast stem cells trophoblast organoids as a model for maternalfetal interactions during human placentation self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta scdual-seq: mapping the gene regulatory program of salmonella infection by host and pathogen single-cell rna-sequencing we would like to thank ashely moffett for the useful discussions and critical review of the manuscript. we are also very grateful to sarah aldridge, loren gibson, damiana alvarez, carlos talavera-lopez, and anna arutyunyan for their insightful comments and corrections. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © hoo, nakimuli and vento-tormo. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -n w f rr authors: lee, sang-myeong; schommer, susan k.; kleiboeker, steven b. title: porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: n w f rr type i interferons (ifn-α and -β) play an important role in the innate host defense against viral infection by inducing antiviral responses. in addition to direct antiviral activities, type i ifn serves as an important link between the innate and adaptive immune response through multiple mechanisms. therefore, the outcome of a viral infection can be affected by ifn induction and the ifn sensitivity of a virus. north american porcine reproductive and respiratory syndrome virus (prrsv) field isolates were studied with regard to ifn-α sensitivity and induction in order to understand the role of type i ifn in prrsv pathogenesis. prrsv isolates were differentially sensitive to porcine recombinant ifn-α (rifn-α) and varied in their ability to induce ifn-α in porcine alveolar macrophages (pam) cultures as measured by a porcine ifn-α specific elisa on cell culture supernatants. fifty-two plaques were purified from three prrsv isolates (numbers , , and ) and tested for ifn sensitivity and ifn induction. plaque-derived populations were composed of heterogeneous populations in terms of ifn-inducing capacity and sensitivity to rifn-α. when macrophages infected with isolates , , or were treated with polycytidylic acid (polyi:c), ifn-α production was enhanced. cells infected with isolate and treated with polyi:c showed the most consistent and strongest enhancement of ifn-α production. it was demonstrated that the relatively low concentrations of ifn-α produced by isolate contributed to the enhanced ifn-α synthesis in response to polyi:c. isolates and significantly suppressed the enhanced ifn-α production by isolate in polyi:c treated cells. to determine if suppression was at the level of ifn-α transcription, quantitative rt-pcr was performed for ifn-α mrna and compared to gapdh and cyclophilin mrna quantification. however, the relative number of ifn-α transcript copies did not correlate with ifn-α protein levels, suggesting a post-transcriptional mechanism of suppression. in summary, these results demonstrate that prrsv field isolates differ both in ifn-α sensitivity and induction. furthermore, a prrsv field isolate strongly enhance polyi:c-induced ifn-α production in pam cultures and this priming effect was suppressed by other prrsv isolates. porcine reproductive and respiratory syndrome (prrs) is one of the most economically important diseases of swine. this disease was first detected in the u.s. in (keffaber, ) and in europe in (wensvoort et al., ) . the etiologic agent for this disease, prrs virus (prrsv) is an enveloped, positive-stranded rna virus that is a member of the arteriviridae family, order nidovirales. the fulllength genomic sequence has been determined for prrsv isolates of both european and north american lineage (allende et al., ; meulenberg et al., ; nelsen et al., ; shen et al., ; wootton et al., ) . molecular analysis of the prototype prrsv vr- and lelystad strains (u.s. and european isolates, respectively) has suggested that divergently evolved strains emerged on two continents almost simultaneously, perhaps due to similar changes in swine management practices (murtaugh et al., ; nelsen et al., ) . analysis of partial genomic sequence data for hundreds of prrsv strains reveals extensive diversity but also well-conserved regions (andreyev et al., ; meng et al., ; reviewed by meng, ) . disease caused by prrsv is characterized by severe and sometimes fatal respiratory disease and reproductive failure. infection with prrsv also predisposes pigs to infection by bacterial pathogens as well as other viral pathogens (benfield et al., ) and prrsv is a key etiologic agent of the economically important porcine respiratory disease complex (prdc). the most consistent pathological lesions caused by prrsv during acute infection are interstitial pneumonia and mild lymphocytic encephalitis plagemann, ; rossow et al., rossow et al., , . tissue macrophages and monocytes are the major target cells during both acute and persistent infection (molitor et al., ) , although pneumocytes and epithelial germ cells of the testes have also been shown to be infected (sur et al., (sur et al., , . it is important to note that clinical disease caused by prrsv is highly variable, ranging from mild, subclinical infections to acute deaths of adult animals . the differences in virulence have been attributed to numerous factors including host genetics, management practices, and virus strain heterogeneity (halbur et al., , (halbur et al., , keffaber, ; wensvoort, ) . after the acute phase of prrsv infection, which is typically characterized by viremia and clinical disease, many pigs fully recover yet carry a low-level viral infection for an extended period of time. under experimental conditions, persistent infection with prrsv has been well documented (albina et al., ; allende et al., ; christopher-hennings et al., ; horter et al., ; sur et al., ; yoon et al., ; wills et al., ) . these ''carrier'' pigs are persistently infected with prrsv and shed the virus, either intermittently or continuously, and may infect naïve pigs following direct or indirect contact. most notably, infectious virus has been recovered for up to days postinfection . producers often vaccinate swine against prrsv with modified-live attenuated strains or killed virus vaccines. however, current vaccines do not provide satisfactory protection, possibly due both to strain variation and inadequate stimulation of the immune system. a protective immune response is possible since it has been demonstrated that previous exposure can provide protection when pigs are challenged with a homologous strain of prrsv (lager et al., ) . however, protective immunity has never been consistently demonstrated for challenge with heterologous strains (lager et al., ; mengeling et al., ) . the ability of prrsv to routinely establish a persistent infection in pigs coupled with experimental evidence of suboptimal humoral and cellular immunity to prrsv suggests that the adaptive immune response to prrsv is often ineffective (reviewed by murtaugh et al., ) . the innate immune response, of which type i ifn is a major component, plays a key role in establishment of an effective adaptive immune response. the type i ifn response serves as an important link between the innate and adaptive immune response through multiple mechanisms (reviewed by biron, ) . type i ifn: (i) promotes the development of cd + t cell response (reviewed by boehm et al., ) , (ii) regulates the expression of many proteins responsible for generating antigenic peptides to be displayed in association with mhc class i (reviewed by york and rock, ) , (iii) enhances differentiation of dendritic antigen-presenting cells (luft et al., ) , (iv) stimulates the division of memory t cells by inducing il- (tough et al., ) , (v) contributes to prolonging the lifespan of activated t cells (marrack et al., ) , and (vi) enhances igg production and down-regulates ige secretion in b cells (finkelman et al., (finkelman et al., , . in addition to a role in the establishment of an adaptive immune response, type i ifn also plays an important role in innate host defenses against viral infection. type i ifn has been shown to activate or induce the synthesis of several proteins including , oligoadenylate synthase (oas), double-stranded rna-dependent protein kinase (pkr), mx and ribonuclease l (rnase l) (vilcek and sen, ) , all of which result in induction of an antiviral state (reviewed by samuel, ) . not surprisingly, many viruses have evolved specific mechanisms to counteract the ifn response. for example, the hepatitis b virus orf-c product and terminal protein (whitten et al., ) , the human papillomavirus (hpv) e and e proteins (park et al., ; ronco et al., ) , and the influenza a virus ns protein (talon et al., ) are viral proteins that have been shown to inhibit ifn synthesis. some viral proteins, such as the adenoviral e a (zhang et al., ) , sendai virus c proteins (komatsu et al., ) and simian virus protein v (didcock et al., ) , block ifn signaling via the jak-stat pathways. additionally, herpes virus, sv and emcv are known to block ifn-induction of the , -oas/rnase l system (vilcek and sen, ) . better understanding of the type i ifn response following prrsv infection will provide an important basis for understanding the adaptive immune response to this pathogen. previous work (albina et al., a; buddaert et al., ; van reeth et al., ) has demonstrated that european prrsv strains do not induce a strong ifn-a response, yet they are sensitive to the effects of ifn and can suppress a viral-induced ifn response. in this study, north american field isolates of prrsv were evaluated with respect to induction, sensitivity and suppression of an in vitro type i ifn response. differences in ifn phenotype were observed among north american prrsv field isolates. the pam cultures were obtained by bronchoalveolar lavage of - week-old domestic piglets from a prrsv seronegative herd. the lungs were removed from the piglets immediately after death and rpmi- medium (life technologies, grand island, ny) was introduced through the main stem bronchi. bronchoalveolar lavage fluid was centrifuged at  g for min. when cell pellets contained obvious contamination with red blood cells (rbc), pam cultures were purified by histopaque- (sigma inc., st. louis, mo) according to manufacturer's directions to remove red blood cell contamination. after centrifugation, cell pellets were resuspended in rpmi- medium supplemented with % fetal bovine serum (fbs), mm lglutamine, . mg/ml fungizone, u/ml penicillin, mg/ml streptomycin sulfate and mg/ml gentamicin (biowhittaker, walkersville, md) and plated at a density of -  cells/well in a -well primaria plate (becton dickinson and company, franklin lakes, nj). the pam cultures were confirmed to be prrsv-negative by rt-pcr before use in subsequent experiments. pam cultures were incubated for h at c in a humidified % co incubator and washed once with complete rpmi- media before use. the marc- cell line is a clone of the african green monkey kidney cell line ma- which is highly permissive to prrsv infection (kim et al., ) . cells were cultured and maintained in dulbecco's modified eagle medium (dmem) supplemented with % fbs, . mg/ml fungizone, u/ml penicillin, mg/ml streptomycin sulfate and mg/ml gentamicin (biowhittaker inc., walkersville, md) and then held at c in a humidified % co incubator. swine testicular (st) cells were used to grow and titrate tgev. st cells were grown in dmem supplemented with % heat-inactivated fbs, mm l-glutamine, . mg/ml fungizone, u/ml penicillin and mg/ml streptomycin sulfate (bio-whittaker inc., walkersville, md). all cells were maintained at c in a humidified % co incubator. fifteen prrsv field isolates, numbered from to , were obtained from clinical cases submitted to the university of missouri's veterinary medicine diagnostic laboratory. genomic sequences of open read-ing frames - of prrsv isolates , , and were submitted to genbank and assigned accession numbers ay (prrsv b), ay (prrsv ), and ay (prrsv ), respectively. virus stocks of prrsv isolates were prepared in pam cultures. a low multiplicity of infection (moi < . ) was used to prepare viral stocks, and the third to fifth passages were used for all experiments. transmissible gastroenteritis virus (tgev) (purdue strain) was obtained from the university of missouri's veterinary medicine diagnostic laboratory serology section. to purify plaques from prrsv isolates, confluent marc- cells (kim et al., ) in -well plates were infected with prrsv at a very low moi (< . ). after h, cells were rinsed and overlayed with cell culture medium containing . % agarose and incubated for - days until plaques were visible. plaques were harvested by aspiration into a micropipette and diluted into a final volume of . ml of culture medium and used for experiments without further propagation. a total of plaques were harvested, from isolate , from isolate and from isolate . the titers of original stocks of prrsv plaques were analyzed by quantitative real-time rt-pcr using purified rna from a titered virus stock to establish the standard curve. titers from isolate , , and plaques were . ae . tcid /ml (mean ae s.e.m.), . ae . tcid /ml, and . ae . tcid /ml, respectively. the moi used in experiments with plaque-purified stocks was approximately . for plaques from isolates and , and . for isolate . pam cultures in -well plates were pre-incubated with or u/ml rifn-a (r&d systems inc., minneapolis, mn) in cell culture media for h before virus infection. cells were washed with cell culture media and infected with prrsv field isolates at moi = . . after h post-infection (p.i.), supernatants were harvested, frozen and thawed one time. infectious virus titers in cell culture supernatants were determined by serial dilution in -well plates and calculated by the method of reed and muench ( ) . for ifn-a sensitivity experiments of prrsv isolates and , rifn-a ( , or u/ml) was added h prior to prrsv infection (moi = ) and virus growth curve experiments were performed. cell culture supernatants were harvested at , , , , h p.i. infectious virus titers in cell culture supernatants were determined by serial -fold dilutions of viral stocks with % tissue culture infectious dose (tcid ) titers calculated by the method of reed and muench ( ) . ifn-a was measured with a porcine ifn-a specific elisa by using f monoclonal antibody (mab) and k mab (r&d systems inc., minneapolis, mn) as previously described (diaz de arce et al., ) . mab k was conjugated with horseradish peroxidase (hrp) using a peroxidase labeling kit (roche molecular biochemical, indianapolis, in). flat-bottomed -well plates (fisher scientific, houston, tx) were coated overnight at c with f at a concentration of mg/plate in coating buffer ( mm carbonate buffer, ph . , sigma inc., st. louis, mo). after blocking with % non-fat dried milk, . % tween in phosphate buffered saline (pbs) for h at c, the plates were washed five times with . % tween in pbs. samples ( ml) were added into each well containing ml of % non-fat dried milk, . % tween in pbs and incubated for h at c. following five washes, ml of peroxidase conjugated k was added to each well. after h incubation and five washes, ml of substrate solution, tetramethylbenzidine (sigma inc., st. louis, mo), was added to each well. after min, the reaction was stopped with n hcl and the optical density was measured at nm by an elisa plate reader. quantified recombinant porcine ifn-a (rifna, r&d systems inc., minneapolis, mn) was used as a standard, and ifn-a concentrations were calculated based upon a standard curve. one unit/ml of rifn-a is equivalent to pg/ml. pam cultures were infected with prrsv isolate , or at a moi = for h. after h p.i. the cell culture media was replaced with media containing polyi:c (sigma inc., st. louis, mo) at mg/ml. at h after the initial prrsv infection, supernatants were collected and the ifn-a concentration was measured by elisa. for suppression experiments, isolate was inoculated at h p.i. into pam cultures that were previously infected with either isolate , or . after h cell culture media was replaced with medium containing polyi:c at mg/ml. supernatants and cells were harvested at h p.i and stored at À c for subsequent analysis. prrsv isolates were uv-inactivated by exposing microcentrifuge tubes containing virus stocks to uv light (wavelength nm) for min. stocks of uvinactivated virus were demonstrated to be completely non-infectious for pam cultures. pam cultures were inoculated with either prrsv or uv-inactivated prrsv at moi = , incubated for h then washed with cell culture media. polyclonal antibody against porcine ifn-a ( neutralizing units/ml, r&d systems inc., minneapolis, mn) was added to cell culture media at the time of infection ( h) and again after infection ( h). at h p.i. cell culture medium was replaced with media containing polyi:c ( mg/ml). after an additional h incubation, cell culture media was collected and analyzed for ifn-a production by using porcine specific elisa. at h p.i. with isolate , or (moi = ), pam cells were harvested and resuspended in pbs at a concentration of cells/ml and assessed for cell viability. propidium iodide (sigma inc., st. louis, mo), which will positively stain dead cells with a disturbed cellular membrane, was added into . ml of cell suspension at mg/ml and incubated at room temperature. after min, cell viability was analyzed by flow cytometry. a second aliquot of infected cells with isolate , or was stained with . % trypan blue (bio-whittaker, walkersville, md), and then quantified microscopically with a hemocytometer to determine the total number of cells and the number of dead cells, i.e., those retaining trypan blue. the celltiter aq ueous one solution cell proliferation assay (promega corp., madison, wi) was performed as described by the manufacturer. briefly, ml of the celltiter solution reagent was added into each well of -well plate and the plate was incubated for h at c in a humidified, % co incubator. absorbance was measured at nm. the od measured at nm represents the amount of tetrazolium dye (mts)-toformazan conversion, which represents the number of viable cells. extraction of rna from samples of ifn suppression experiments was performed using trizol (invitrogen, carlsbad, ca) and the nucleospin rna ii kit (bd biosciences inc., palo alto, ca) with dnase i digestion performed directly on the spin column according to the manufacturer's instructions. extractions of the purified competitor rna were performed using the qiagen rnaeasy kit (qiagen inc., valencia, ca). heterologous competitor rna for quantification of swine ifn-a, cyclophilin or gadph was synthesized using the respective real-time rt-pcr primer sequences in a methodology previous described (kleiboeker, ) . the concentration of purified competitor rna was estimated by measuring the absorbance at nm and the purity was assessed by determining the ratio of absorbance at nm to the absorbance at nm. samples were considered to be relatively pure and suitable for use as quantification standards if the ratio was > . . following purification the rna was serially diluted in rnase-free dh o and stored as aliquots at À c. the number of molecules of competitor rna/ml was estimated based on the rna concentration and the molecular weight of the transcript. amplification of ml rna was performed using the qiagen quantitect probe rt-pcr kit (qiagen inc., valencia, ca) with thermocycling and detection performed in a stratagene mx (stratagene inc., la jolla, ca). samples were analyzed in triplicate. thermocycling conditions were: c ( min), c ( min), followed by cycles of denaturation ( c, s) and annealing/extension ( c, s). genbank accession numbers of sequences used for porcine ifn-a, porcine cyclophilin, and porcine gapdh real-time rt-pcr assays were nm , ay , and af , respectively. primers used for -exonuclease (taqman) amplification of swine ifn-a were (forward, position - ) -tctcatgcaccagagcca- , (reverse, position - ) -cctggaccacagaaggga- , and for amplification of swine cyclophilin were (forward, position - ) -atggcactggtggcaagt- , (reverse, position - ) -gatgccag-gacccgtatg- . primers used for -exonuclease (taqman) amplification of for swine gapdh were (forward, position - ) -tgcccagaacat-catccc- and (reverse, position - ) -ggatgaccttgcccacag- . all oligonucleotide primers were used at a final concentration of . mm. the dual-labeled probe used for detection of ifn-a transcript was - -fam-cttgagccttctggac-ctggttgc-bhq - (position - ), for detection of swine cyclophilin transcript was - -fam-catctatggagagaaatttgatgatgaga-bhq - (position - ), and for detection of swine gapdh transcript was -fam-cttct-accggcgctgccaag-bhq - (position - ). the dual-labeled probe used for detection of heterologous swine ifn-a, cyclophilin, or gapdh competitor rna was: -hex-tgtgctgcaaggc-gattaagttgggt-bhq - . each probe was used at a final concentration of . mm. all oligonucleotide primers and probes were synthesized by integrated dna technologies inc. (coralville, ia). the relative copy numbers of ifn-a mrna compared to cyclophilin or gapdh were determined as previously described (stordeur et al., ) . the student's t-test was used for the statistical analyses. p-values of less than . were considered statistically significant. to determine the sensitivity of north american prrsv field isolates to exogenous rifn-a, viral replication of field isolates was assessed in pam cultures that were pretreated with or u rifn-a/ ml cell culture media (fig. ) . for most isolates, viral titers were either unchanged or slightly decreased by the addition of u/ml of rifn-a. however, replication of one isolate (number ) was reduced by > log tcid /ml at this dose. all isolates demonstrated greater declines in replication following pretreatment with u/ml of rifn-a, with titers for of the isolates declining to the detectable limits of the assay. this experiment was repeated twice independently and results were consistent between experiments. to further characterize differences in sensitivity to rifn-a, two isolates, numbers and , were selected for use in a dose-response growth curve experiment. a representative experiment from independent experiments is shown (fig. ) . a dose-dependent reduction in viral titers was noted for both isolates. when pam cultures were pretreated with rifn-a at , and u/ml there was a significant (p < . ) reduction in viral titer from to h p.i. for isolate whereas for isolate only and u/ml of rifn-a resulted in a significant reduction in viral yield. for pam cultures that were pretreated with u/ml of rifn-a, titers of isolate were -fold lower than isolate at h p.i. compared to the untreated controls for each isolate. at , , and h p.i. the titers of isolate in u/ml rifn-a-treated cells were approximately . - . log % tissue culture infectious dose (tcid )/ml lower than in untreated cells. in contrast, the titers for isolate were reduced by about . - . log tcid /ml. when pam cultures were pretreated with u rifn-a/ml, virus yields of isolate were inhibited throughout the time course while virus yields of isolate increased through the time course and reached titers of the untreated control at h p.i. taken together, these results demonstrate that north american field isolates have different sensitivities to exogenous rifn-a in pam cultures. to investigate whether prrsv isolates are composed of heterogeneous populations with measurable differences in rifn-a sensitivity and ifn-a induction, plaque-derived populations of field isolates were prepared and tested. as shown in fig. , plaquepurified populations of all three isolates demonstrated a range in sensitivity to exogenous rifn-a. for isolates and , the range of reduction in viral titers was approximately . - . log tcid /ml. for isolate , the range of reduction in viral titers was - . log tcid /ml. in preliminary experiments, isolate was the most consistent ifn-a inducer compared to other field isolates tested. more than u/ ml of ifn-a was consistently synthesized in cells infected with isolate . in contrast ifn-a was typically undetectable in cultures infected with other isolates, although very low concentrations were detected in some experiments. plaque-derived populations of field isolates were also characterized with respect to ifn-a induction (fig. ) . while individual plaques from all three isolates induced relatively low concentrations of ifn-a compared to tgev, ifn-a production varied among plaques from each isolate. ifn-a induced by plaque-derived populations from isolates , and were . ae . , . ae . , and . ae . , respectively (mean ae s.d.). plaques from isolate induced a higher concentration of ifn-a than plaques of the other two isolates (p < . ). to investigate the ability of prrsv field isolates to suppress ifn-a induction by an ifn inducer, pam cultures were infected with prrsv then treated with polyi:c ( mg/ml) after h. in this experiment, no measurable amount of ifn-a was induced by prrsv infection alone with any of the three prrsv isolates (fig. a ). in the absence of prrsv, polyi:c failed to induce ifn-a at h, although ifn-a was induced at later time points. however, in the experiments shown samples from later time points were not used in order to avoid the influence of prrsv-induced cytopathic effect on ifn-a production. when prrsv-infected cells were treated with polyi:c, ifn-a production was consistently enhanced to the greatest extent for isolate . therefore it was tested whether isolates or could suppress ifn-a induced by a combination of isolate infection and polyi:c treatment of pam cultures. both isolates and significantly (p < . ) decreased ifn-a production by % and fig. . ifn-inducing capacity of prrsv plaque-derived populations. pam cultures were inoculated with ml/well of each plaque and supernatants were harvested after h and analyzed for ifn-a concentration using a porcine ifn-a-specific elisa. plaques from isolate induced a higher mean concentration of ifn-a than plaques of the other two isolates (p < . ). one unit/ml of rifn-a is equivalent to pg/ml. %, respectively. in contrast, ifn-a production was significantly (p < . ) increased by % in isolate superinfected cells. to demonstrate that suppression of ifn was not due to decreased cell viability, three cell viability assays (propidium iodide (pi) staining, trypan blue exclusion, and cell titer aq ueous cell proliferation and viability assay) were performed. when cell viability was analyzed by flow cytometry after pi staining, cell viability of prrsv-infected cells at h p.i. was . %, . %, and . % for isolates , , and , respectively. uninfected cells demonstrated . % viability. this result was in excellent agreement with the other two independent assays used to assess cell viability. to determine the effect of prrsvon ifn-a mrna synthesis, quantitative real-time rt-pcr was performed with samples from the suppression experiments. normalization of ifn-a mrna copy number was performed both with cyclophilin and gapdh transcript, with equivalent results obtained. as shown in fig. b , prrsv isolates induced variable quantities of ifn-a mrna in pam cultures. the quantity of ifn-a mrna did not correlate with the ifn-a protein level detected by elisa (fig. a) . consistently, ifna mrna synthesis was increased in prrsv-infected pam cultures (p < . ), despite a lack of detectable ifn-a protein. as a control, real-time pcr without the rt step was performed to determine if levels of contaminating genomic dna following dnase i treatment affected real-time rt-pcr quantification. relative amounts of dna in each sample were always less than % of the total signal, demonstrating that the signal analyzed was from rna rather than dna. uv-inactivated prrsv stocks were tested to determine whether virus binding to the cells is responsible for enhanced ifn-a production by polyi:c in cells infected with isolate (fig. ) . uvinactivation of virus reduced ifn-a in these cells to . % of control values obtained using fully infectious virus (p < . ). thus, virus binding to the cells alone was not sufficient to amplify ifn-a synthesis by polyi:c. to determine if ifn-a secreted after initial infection with isolate was responsible for enhanced ifn-a production in respond to polyi:c, neutralizing antibody against porcine ifn-a was used (fig. ) . neutralizing antibody against pig ifn-a reduced ifna production by polyi:c in cells infected with isolate - % of control values (p < . ) when added into cell cultures at a concentration of neutralizing units/ml prior to polyi:c treatment. pretreatment of macrophages with low concentration of rifn-a ( u/ fig. . effect of prrsv on ifn-a induced by polyi:c treated pam cultures. pam cultures infected with each isolate were infected with isolate at h p.i. (expressed as [# ]) and then treated with polyi:c at h p.i. supernatants were collected at h p.i. (a). ifn-a protein was measured by elisa. one unit/ml of rifn-a is equivalent to pg/ml. (b) quantitative real-time rt-pcr was performed by using total rna extracted from pam cultures. results are expressed as relative copy numbers of ifn-a mrna using cyclophilin as an internal control. values are shown as the means ae s.d. from duplicate wells and represent at least two independent experiments. *, significant difference compared with [# ] infected, polyi:c treated cells (p < . ). #, significant difference compared with polyi:c only treated cells (p < . ). no significant differences were detected in the copy numbers of ifn-a mrna among prrsv isolate infected cells and control cells. ml) also enhanced ifn-a production from u/ml to more than u/ml by polyi:c. thus these experiments suggest that even low concentrations of ifn-a induced by isolate are sufficient to strongly enhance the ifn response to polyi:c treatment. the type i ifn response plays an important role in host defenses against viral infection by performing immunoregulatory functions that link innate and adaptive immune responses by multiple mechanisms (reviewed by biron, ) as well as through direct antiviral effects. in this study, north american prrsv field isolates were evaluated with respect to induction, sensitivity and suppression of an in vitro ifn response in pam cultures. our results show that these isolates are sensitive to rifn-a in a dosedependent manner, but are generally poor inducers of ifn in vitro. notably the extent of the sensitivity and induction of ifn not only differed between prrsv isolates, but also showed variation among the plaquederived populations within each isolate. two of the field isolates studied were able to suppress the ifn-a production of cells dually-treated with polyi:c and a third north american prrsv isolate. while considerable variation was noted for the levels of ifn-a measured between replicates, most likely due to the variability of alveolar macrophage function among pigs (du manoir et al., ) , the trends observed were consistent between experiments. taken together, these results demonstrate that north american prrsv isolates differ in the ability to induce or suppress ifna, which may contribute to both virulence differences commonly observed among prrsv infections and the failure of prrsv infection to consistently induce a rapid and robust sterilizing immune response. the role of type i ifn in prrsv pathogenesis has been addressed by previous research (albina et al., a; buddaert et al., ; van reeth et al., ) , however this work was performed with two isolates (lelystad and sdrpi) of the european prrsv lineage, which is quite divergent ($ % sequence identity) compared to north american strains. in the present study, it was demonstrated that north american prrsv isolates were sensitive to rifn-a in a dose-dependent manner and were poor ifn-a inducers in vitro. these results are consistent with previous studies using european prrsv isolates (albina et al., a; buddaert et al., ; van reeth et al., ) . similar to results with type i ifn, north american prrsv replication was also blocked by a type ii ifn (ifn-g) and virus replication was restored by the addition of -aminopurine, an inhibitor of pkr (rowland et al., ) . biological, antigenic, pathogenic, and genetic variation among prrsv field isolates has been well documented (reviewed by meng, ) . the data presented herein demonstrated that north american prrsv field isolates were differentially sensitive to the in vitro antiviral effects of rifn-a and the use of plaque-derived populations from these field isolates demonstrated that closely related variants of prrsv may differ in their ifn responses. similarly, it has been shown that the sensitivity of reovirus, hepatitis c virus and lymphocytic choriomeningitis virus (lcmv) to type i ifn antiviral activity differs among viral strains (enomoto et al., ; moskophidis et al., ; reviewed by samuel, ) . a previous study suggested that significant differences of ifn-inducing capabilities could be a quasispecies marker of fig. . effect of uv-inactivated virus and anti-ifn-a antibodies on ifn-a production. pam cultures were infected with either prrsv or uv-inactivated prrsvat moi = . for h. polyclonal antibody against porcine ifn-a ( neutralizing units/ml) was added into cell cultures at the time of infection. at h p.i. cell culture media was replaced with media containing polyi:c ( mg/ml). after additional h incubation, cell culture media was collected and analyzed for ifn-a production by using porcine specific elisa. one unit/ml of rifn-a is equivalent to pg/ml. *, significant difference compared to polyi:c treated and isolate infected cells (p < . ). vesicular stomatitis virus (vsv) (marcus et al., ) . in contrast to the results presented herein, a study using the european prrsv strains sdrpi and sdrpii found no strain differences in ifn-a sensitivity or induction, despite differences in clinical virulence (albina et al., a) . previous experimental work has demonstrated evidence of a relationship between ifn phenotypes and virulence for other viruses. for example, virulent measles virus induces lower levels of ifn than attenuated strains and ifnresistant strains can establish persistent infections of the central nervous system (carrigan and knox, ). it has also been shown that the capability of noncytopathic bovine viral diarrhea virus to establish persistent infections in the early fetus is related to its ability to suppress type i ifn synthesis (charleston et al., ) . future in vivo studies will be needed to determine if the ifn phenotypes described in the present study are related to prrsv virulence or persistence. in the present study it was shown that polyi:c treatment of prrsv-infected cells resulted in greatly enhanced ifn-a production, especially in pam cultures infected with prrsv isolate . similar phenomenons have been reported by other studies. ifn-inducing listeria monocytogenes (havell, ) and type i ifn pretreatment (rosztoczy and megyeri, ) enhanced ifn production by polyi:c, sendai virus, or endotoxin. a plausible mechanism of this enhancement is by positive feedback regulation of type i ifn synthesis, in which a weak ifn-a/b signaling contributes to the enhancement of ifn-a/b synthesis due to the accumulation of transcription factor irf- (reviewed by taniguchi and takaoka, ) . this positive feedback loop of ifn synthesis could be efficiently induced by a small amount of ifna produced during the first h after isolate infection, resulting in amplification of the ifn-a response to polyi:c. consistent with this possibility was the observation that neutralizing antibody against ifn-a remarkably reduced ifn-a production by polyi:c in cells infected with isolate , and that u/ml of rifn-a added to poly i:c treated cells greatly increased the levels of ifn-a detected. tolllike receptor (tlr ) which recognizes polyi:c (alexopoulou et al., ) could be also involved in the enhancement of ifn production. measles virus strains with ifn-b inducing properties up-regulated the expression of tlr resulting in enhanced ifn-b production in response to polyi:c (tanabe et al., ) . therefore ifn produced by isolate may act through a similar mechanism to up-regulate tlr expression followed by increased ifn-a production after polyi:c treatment. for viruses such as sendai virus and vsv, low ifninducing strains have been shown to suppress ifn induction of high ifn-inducing strains of the same virus (marcus et al., ; mattana and viscomi, ) . in the present study, the levels of prrsv ifn induction were typically too low to use in a similarly designed suppression experiment. therefore the suppressive effect of prrsv isolates and on ifn-a production was evaluated in cells infected with isolate and treated with polyi:c, and significant suppression of ifn-a was demonstrated. european prrsv was also shown to suppress ifn-a production by tgev, both in vitro and in vivo (albina et al., a) . however, this result was not corroborated in vivo by another group using the closely related porcine respiratory coronavirus (prcv) (buddaert et al., ) . microarray experiments have indicated that polyi:c, ifn and viruses induced different subsets of the same cellular genes by activating diverse signaling pathways (geiss et al., ) . since ifn-a production was enhanced by the addition of polyi:c, it is likely that prrsv isolates and suppress an ifn-a synthesis pathway activated by isolate and polyi:c but not polyi:c alone. it has been shown that viruses which are resistant to the effects of ifn or can suppress ifn production have increased opportunities to spread before activation of the adaptive immune response (moskophidis et al., ; naniche et al., ) . mouse models of viral infection have clearly demonstrated that disrupting the ifn response leads to higher levels of viral replication. mice deficient in an ifn receptor exhibited increased susceptibility to lcmv (van den broek et al., ) and extensive spread of lcmv infection correlated with an isolate's relative resistance to ifn-a/b and ifn-g (moskophidis et al., ) . viruses can suppress ifn-a synthesis both at the transcriptional and post-transcriptional level. many viruses prevent mrna synthesis of ifn-a through various mechanisms but vsv inhibits ifn-a synthesis by shutting down the host protein synthesis (ahmed et al., ) . the present study showed that prrsv stimulated ifn-a mrna synthesis but inhibited ifna protein expression. this result suggests that the mechanism by which prrsv isolates suppress ifn-a production is mediated by generally inhibiting host protein expression or by specifically inhibiting ifn-a protein expression. half-life times of ifn mrna and protein could be different and that might be responsible for differences between mrna and protein levels of ifn. however, increases in ifn-a mrna expression for prrsv infected pam cultures were consistently detected at multiple times postinfection, yet for cells infected with isolate or very low to undetectable levels of ifn-a protein were typically observed at all times p.i. thus, although the mechanisms of ifn-a suppression by prrsv remain to be elucidated, data presented herein show that it is likely to be post-transcriptional in nature. while the immune response against prrsv is poorly understood, experimental work has demonstrated that the adaptive immune response of prrsvinfected pigs is generally ineffective (horter et al., ; mengeling et al., ; reviewed by murtaugh et al., ; wills et al., wills et al., , . specific evidence of this includes a slow neutralizing antibody response, which is typically not detected until weeks p.i. (albina et al., b) and does not reach maximum levels until - weeks p.i. (nelson et al., ; yoon et al., ) . wide variation has been shown in individual animals, with some studies demonstrating that many infected animals fail to develop a neutralizing antibody response (loemba et al., ; nelson et al., ) . while the importance of a cell-mediated response for protection against prrsv has not been debated, the effectiveness of this response during the early phases of disease also appears to be suboptimal (reviewed by murtaugh et al., ) . for example, the t-cell response to prrsv is weak and transient and cannot be restimulated for more than weeks post-challenge (meier et al., ; molitor et al., ) . additionally, ifn-g responses of prrsv-infected pigs were relatively weak and increased slowly in comparison to pseudorabies virus infected pigs (meier et al., ) . although the precise mechanisms for the ineffective nature of the adaptive immune response to prrsv is not known, prrsv evasion of the innate immune responses, such as the type i ifn response, may set the stage for subsequent subversion of the adaptive immune response. in conclusion, the data presented in this study demonstrates that north american prrsv field isolates are distinctively different in their in vitro ifn phenotypes, a difference that could contribute to the variable clinical signs and pathology observed in prrsv infections. moreover, the lack of ifn response following infection with prrsv could be involved in the establishment of persistent infections or secondary bacterial and viral infection, both of which are characteristic of prrsv infections in swine. however, in vivo studies are required to fully define the role of type i ifn in prrsv pathogenesis and virulence. the mechanisms involved in enhancement of ifn-a by prrsv infected and polyi:c treated cells remains to be elucidated. additionally, it will be important to determine whether prrsv generally inhibits host protein synthesis or specifically downregulates ifn expression and to identify the viral proteins that contribute to the different ifn response among prrsv isolates. knowledge about the role of ifn in prrsv pathogenesis will provide new hypotheses for improved vaccines or methods to modulate the immune response in order to more effectively control prrsv infection. ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host rna and protein synthesis immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units interferon-a response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) recognition of double-stranded rna and activation of nf-kappab by toll-like receptor north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions porcine 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unique insertion cytokine mrna quantification by real-time pcr in vivo detection of porcine reproductive and respiratory syndrome virus rna by in situ hybridization at different times postinfection porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis and induces germ cell death by apoptosis influenza a and b viruses expressing altered ns proteins: a vaccine approach mechanism of up-regulation of human toll-like receptor secondary to infection of measles virus-attenuated strains a weak signal for strong responses: interferon-alpha/beta revisited stimulation of naive and memory t cells by cytokines antiviral defense in mice lacking both alpha/beta and gamma interferon receptors differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity interferons and other cytokines mystery swine disease in the netherlands: the isolation of lelystad virus lelystad virus and the porcine epidemic abortion and respiratory syndrome identification of the hepatitis b virus factor that inhibits expression of the beta interferon gene prrs virus: a persistent infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate persistent and contact infection in nursery pigs experimentally infected with prrs virus characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection antigen processing and presentation by the class i major histocompatibility complex two contact regions between stat and cbp/p in interferon gamma signaling general overview of prrsv: a perspective from the united states key: cord- -mxt stat authors: saraya, takeshi; kurai, daisuke; ishii, haruyuki; ito, anri; sasaki, yoshiko; niwa, shoichi; kiyota, naoko; tsukagoshi, hiroyuki; kozawa, kunihisa; goto, hajime; takizawa, hajime title: epidemiology of virus-induced asthma exacerbations: with special reference to the role of human rhinovirus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: mxt stat viral respiratory infections may be associated with the virus-induced asthma in adults as well as children. particularly, human rhinovirus is strongly suggested a major candidate for the associations of the virus-induced asthma. thus, in this review, we reviewed and focused on the epidemiology, pathophysiology, and treatment of virus-induced asthma with special reference on human rhinovirus. furthermore, we added our preliminary data regarding the clinical and virological findings in the present review. more than different types of viruses, such as human rhinovirus (hrv), human metapneumovirus (hmpv), respiratory syncytial virus (rsv), and human parainfluenza virus (hpiv), are known to cause acute respiratory illness (ari; tsukagoshi et al., ) . we recently reported the issue of "virus-induced exacerbation in asthma and chronic obstructive pulmonary disease" (kurai et al., a) , however, among these causative viruses, hrv is now recognized to have a major impact on asthma pathogenesis (fujitsuka et al., ) . from this perspective, we reviewed the literature regarding the epidemiology of hrv-induced asthma in adults, together with preliminary epidemiological data obtained at our institution. hrv belongs to the genus enterovirus and family picornaviridae (turner and couch, ) . hrv possesses a single strand positive-sense rna (ssrna) genome of approximately . kb. the viral capsid is composed of four viral proteins (vp - ) which are assembled into protomers, resulting in a small icosahedral structure with a diameter of about - nm (turner and couch, ) . genetically, hrv is classified into three species; hrv-a, -b, and -c (simmonds et al., ) . furthermore, these species of hrv have more than genotypes (andries et al., ; arakawa et al., ; kiyota et al., kiyota et al., , . molecular epidemiological studies suggest that the dominant species are hrv-a and -c, while hrv-b is relatively rarely detected (arakawa et al., ; kiyota et al., ) . in particular, the vp and vp proteins have variations in their amino acid sequences, accounting for the large number of viral serotypes (turner and couch, ) . the host receptor for hrv in respiratory epithelial cells is the intracellular adhesion molecule (icam- , cd ) for the major hrv serotypes (hrv-a and -b), or low-density lipoprotein receptor (ldlr) for the other minor hrv serotypes. the receptor for hrv-c is not yet known. it has been suggested that the optimal temperature for replication of hrv is relatively cool ( - • c), which would limit infections to the upper airway; however, large or medium sized airways lower in the respiratory tract are now also considered cool enough for hrv replication, in spite of the higher temperature of the lung parenchyma ( • c; mcfadden et al., ) . therefore, hrv is potentially a causative agent of more severe ari such as bronchiolitis and pneumonia (turner and couch, ; watanabe et al., ; smuts et al., ; arakawa et al., ) , and may be associated with virus-induced asthma linsuwanon et al., ; fujitsuka et al., ; smuts et al., ) . hrv might therefore be involved in various aris and additional respiratory complications (kiyota et al., ) . lieberman et al. ( ) reported that the detection of any virus include hrv, the sensitivity rates for nasopharyngeal swab ( . %) was superior than that of oropharyngeal swab ( . %), respectively. the common cold is the third most common primary diagnosis in office visits (hsiao et al., ) , and this disease is generally selflimiting, usually lasting up to days (fashner et al., ) . among the general population, hrv infection causes common colds at a frequency of - % (makela et al., ; van gageldonk-lafeber et al., ) . tyrrell et al. ( ) reported that intranasal www.frontiersin.org inoculation with either hrv serotypes , , and , coronavirus type e, or rsv in healthy volunteers induced patterns of symptom development which were not substantially different from each other. however, individual signs or symptoms occurred earliest in hrv infections, then in coronavirus, and lastly in rsv, appearing up to days after inoculation, which demonstrated the long incubation periods of rsv in volunteers (tyrrell et al., ) . hrv has been implicated in patients with acute otitis media, exacerbation of chronic obstructive pulmonary disease, common cold, and lower respiratory tract infections in neonates, the elderly and immunocompromised. arruda et al. ( ) researching the frequency and natural history of hrv infections in adults during autumn, demonstrated that the first symptom noticed most often was sore throat ( %) in hrv culture-or pcr-positive patients, and stuffy nose in hrv-negative patients ( %), using nasal wash specimens. respiratory symptoms typically develop after - days after inoculation in studies, and uncomplicated hrv infections usually peak - days after inoculation. the median duration of hrv colds is week, but up to % last more than weeks (gwaltney et al., ; rotbart and hayden, ) . it should be noted that in illness caused by hrv, viral shedding occurs naturally for up to days, but predominantly over a - days period. hrv-a type (hrv- ), a major group virus commonly used for experimental human infection, and hrv-a type (hrv- ), which has been used in animal models of hrv infection, are closely related. grunberg et al. ( ) reported that experimental hrv- infection via nasal inhalation leads to a transient decrease of fev . in patients with asthma, and this decreased lung function was correlated with enhanced cold symptoms and / or airway hyperresponsiveness. contoli et al. ( ) demonstrated that type iii interferon (ifn-λ) production levels in ex vivo cell cultures derived from bronchial epithelial cells (becs) and macrophages obtained from asthmatic patients, were lower than in those derived from healthy controls. furthermore, deficient interferon-λ production was correlated with hrv viral load, severity of clinical symptoms and fev . . message et al. ( ) demonstrated that the severity of intranasally inoculated hrv-induced clinical illness in asthmatic subjects was correlated to virus load and lower airway virus-induced inflammation. on the other hand, demore et al. ( ) reported that no difference in clinical symptoms, and patterns of viral shedding, was noted between subjects with persistent allergic asthma and healthy subjects after experimental infection with hrv. these different results after experimental hrv infection in individual studies in asthmatic patients and healthy subjects might be dependent on the severity of the asthma of those subjects who enrolled in the studies. indeed, in several reports, neither defective ifn induction by hrv, nor increased hrv replication was observed in primary human becs derived from subjects with well controlled asthma (lopez-souza et al., ; bochkov et al., ; sykes et al., ) . a few animal models for rhinovirus infection have been showed because a major group of hrv (i.e., hrv- ) did not bind mouse icam- . only a minor group of hrv (i.e., hrv- b) infected the mouse. in this regard, bartlett et al. ( ) generated a transgenic balb/c mouse expressing a mouse-human icam- chimeric receptor for hrv- infection. this study also showed asthma exacerbation model by intraperitoneally sensitized with ovalbumin with aluminum hydroxide followed by intranasal inoculation of hrv- b or uv-inactivated hrv- b. although data regarding virus respiratory infections (vris) as precipitators of asthma attacks in adults are less clear, nicholson et al. ( ) reported that vris are as commonly linked to exacerbations in adults as they are in children (johnston et al., ; fujitsuka et al., ) . this study showed that viruses were detected in % of clinical exacerbative episodes with a decrease in peak expiratory flow rate (pefr) of ml/minute or more, and the most commonly identified virus was hrv, followed by coronaviruses and parainfluenza viruses (nicholson et al., ) . thus, the virus most commonly detected in asthma exacerbations appears to be hrv. although hrv is well known as the most frequent cause of the common cold, the implications of hrv infection vary according to respiratory diseases. table shows the frequency of hrv infection in various adult respiratory diseases such as exacerbation of asthma (nicholson et al., ; atmar et al., ; tan et al., ) , common cold (makela et al., ; van gageldonk-lafeber et al., ) , exacerbation of copd (seemungal et al., ; rohde et al., ; tan et al., ; beckham et al., ; papi et al., ; hutchinson et al., ; ko et al., ; mcmanus et al., ; kherad et al., ; dimopoulos et al., ; perotin et al., ) , community acquired pneumonia (jennings et al., ; johnstone et al., ; johansson et al., ; lieberman et al., ; fry et al., ; wootton et al., ; luchsinger et al., ; takahashi et al., ; huijskens et al., ) , exacerbation of idiopathic pulmonary fibrosis (wootton et al., ) , and asymptomatic infection (fry et al., ) . the risk of exacerbations of asthma in adults is elevated after children return to school, and around december th (the christmas holiday in westernized countries), and this is likely to be due to social interactions with children at these times. prospective monitoring studies using reverse transcription polymerase chain reaction (rt-pcr) indicate that as many as % of acute asthma exacerbations in children, and about % in adults, were associated with the presence of upper respiratory tract (urt) infections. corne et al. ( ) found that the detection rates of hrv in asthmatic ( . %) and healthy participants ( . %) were similar, but the lrt symptoms were significantly more severe and longer lasting in the asthmatic group than in the healthy group based on one definition of urt and lrt symptoms ( table ; johnston et al., ) . there is no common antigen across all strains of hrvs; therefore, no reliable diagnostic method for hrv infection has been established using hrv antigens or hrv-specific antibody. although viral culture is the conventional method for hrv detection, culture methods are not practical in clinical settings for the detection of hrv, because of its slow growing character and requirement for specific culture conditions. furthermore, the diagnostic capability of molecular amplification techniques frontiers in microbiology | virology exacerbation of asthma - nicholson et al. ( ) , tan et al. ( ) , atmar et al. ( ) common cold - makela et al. ( ) cited and adapted from johnston et al. ( ) . such as nucleic acid sequence-based amplification and rt-pcr is superior to those of culture methods (loens et al., ) . experimental hrv infections have been shown to lead to a longlasting excessive airway narrowing in volunteer subjects with asthma (cheung et al., ; grunberg et al., ) . of note, rhinovirus, unlike influenza and other viruses, causes minimal cytotoxicity (fraenkel et al., ) , and the amount of epithelial damage does not correlate with the severity of the symptoms. hrv infection can cause additive or synergistic effects in exacerbation of asthma via the influx of additional inflammatory cells in the airways with preexisted inflammation, resulting in airway cholinergic hyperresponsiveness (nagarkar et al., ) , as an allergic response. the effects of hrv infection such as enhanced contractility of airway smooth muscle (asm) cell and impaired relaxation to cholinergic or β-adrenaergic agonists are attributed solely to binding of the virus to its host receptor icam- on the asm cell surface. this proasthmatic-like effect was recognized even in the situation of complete inhibition of viral replication in vitro, but not in the setting of pretreatment of asm with neutralizing antibody directed against for icam- (grunstein et al., ) . thus, the hrv attachment to icam- itself can affects the contractility of asm cells in the absence of any cytopathic effects, and chun et al. ( ) reported that a cells infected with hrv in vitro produced a higher value of il- and rantes than those of rsv or adenovirus. in addition, only the combination of hrv with der f (house dust mites antigen) acted synergistically to induce il- production. these findings are the reason why the hrv can be a major pathogen for acute exacerbation of asthma. we present a schema for pathogenesis in hrv associated asthma exacerbations (figure ) , which requires the following steps, ( ) hrv attachment to airway epithelial cells, ( ) an innate immune response which leads to epithelial damage, ( ) infection-related airway remodeling. when rt-pcr is used to either supplement or replace conventional culture techniques, viruses have been found in approximately one half to three quarters of adults experiencing an acute wheezing episode (jackson and johnston, ) , and the majority ( %) of viruses identified were hrvs (nicholson et al., ) . however, the evidence is weak, and mechanisms are poorly understood. initially, hrv-a and -b attach to airway epithelial cells via icam- or ldlr (kennedy et al., ) . the receptor or receptors for the recently identified group hrv-c have yet to be clarified. hrv-infected becs secrete a wide range of cytokines and chemokines such as il- , il- , ccl /rantes (regulated on activation, normal t cell expressed and secreted), cxcl /il- , gm-csf, and cxcl /interferon-inducible protein (ip- ; jackson and johnston, ; proud, ) , which induce neutrophilic, lymphocytic, and eosinophilic inflammation together with airway hyperresponsiveness and airway remodeling (wark et al., ; proud, ) . clearance of viral pathogens begins with interferon secretion, and the underproduction of these factors has been postulated to lead to viral-induced exacerbations. there are three types of interferons, based on the receptors they bind: type i (ifn-α/β), type ii (ifn-γ), www.frontiersin.org and type iii (ifn-λ). hrv infection induced epithelial expression of mrna for both type i and type iii ifns, and it has been suggested that impaired epithelial production of ifn-β and ifnλ in asthmatic subjects may contribute to viral exacerbations of asthma (wark et al., ; contoli et al., ) . contoli et al. ( ) showed significant inverse correlations between ex vivo production of ifn-λ and severity of symptoms, bronchoalveolar lavage viral load and airway inflammation, and a strong positive correlation with reductions in lung function during in vivo infection. genome-wide association studies showed that single nucleotide polymorphisms involve in various diseases. interferon-λ polymorphisms may effect on the incidence of hrv infection (russell et al., ) . message et al. ( ) reported virus load in asthmatic subjects as being related to increased lower airway inflammation, and in turn increased lower airway inflammation being related to increased symptoms, reductions in lung function, and increases in bronchial hyperreactivity. these data suggest a causal role for hrv infection in the pathogenesis of asthma exacerbations. investigating virus-allergen interactions, durrani et al. ( ) demonstrated that another mechanism that increased expression and cross-linking of the high-affinity ige receptor, fcεri, on plasmacytoid dendritic cells is associated with reduced hrv-induced ifn-α and ifn-λ secretion, and allergic asthmatic children have significantly reduced hrv-induced ifn-α and ifn-λ production after cross-linking of fcεri. type , or inducible, nitric oxide synthase (inos) is the major nos isoform found in epithelial cells and can generate substantial amounts of nitric oxide (no). the no molecules both inhibit the replication of hrv in airway epithelial cells, and suppresses hrv-induced cytokine production (proud, ) . although the measurement of fractional no concentration in exhaled breath (feno) may be used to support the diagnosis of asthma (dweik et al., ) , however, increasing of feno seems to be not always correlated with viral load during the period of hrv infection (sanders et al., ) . other factors such as allergy, allergen exposure, tobacco smoke, particulates, ozone, stress, and infections such as sinusitis commonly contribute to exacerbations of asthma. grainge et al. ( ) reported that repeated bronchoconstriction in asthma promotes airway remodeling, and there is now clear evidence that airway remodeling begins in early childhood, and can be present even before clinical diagnosis of asthma is established (pohunek et al., ) . increasing evidence regarding hrv-induced wheezing or exacerbation of asthma raises the possibility that hrv infections could contribute to the initiation and subsequent progression of airway remodeling, which involves multiple factors such as increased epithelial release of mucin ac (mu ac), activin a, amphiregulin, matrix metalloproteinase (mmp ), epidermal growth factor (egf), fibroblast growth factor (fgf), and vascular endothelial growth factor (vegf). hrv infection upregulates production of muc ac from epithelial cells, which leads to airflow obstruction in asthma (hewson et al., ) . activin a is a member of the tgf-β superfamily and amphiregulin, a member of the egf family, alters repair processes (leigh et al., ) . both activin a and amphiregulin have been linked to subepithelial basement membrane thickening in asthma. mmp appears to have important roles in asthma exacerbation and airway remodeling (sampsonas et al., ) . expression of vegf and its receptors is increased in asthmatic subjects, and vegf is the major proangiogenic activator in asthmatic airways (feltis et al., ; simcock et al., ) . kuga et al. ( ) reported that . % of adult asthmatic patients with common cold suffered an asthma attack, and common cold was significantly associated with acute exacerbations of asthma. they also stated that hrv infection might be important as the virus was detected by rt-pcr in throat gargles (kuga et al., ) . virus-induced exacerbation of asthma is a critical issue for the general physician. however, among asthmatic patients with exacerbative status, distinguishing between those patients which have vris, and those who do not, is difficult. furthermore, epidemiological data regarding adult asthma exacerbations have been sparsely reported. to investigate the prevalence of vri in exacerbations of adult asthma in both hospitalized or not-hospitalized patients, characterization of clinical and radiological findings was performed. a prospective observational cohort study was conducted at kyorin university hospital, tokyo, japan from august to august (kurai et al., b) . all patients with respiratory symptoms associated with exacerbation of asthma were included, and samples were collected by nasopharyngeal or oropharyngeal swab, and subjected to a pcr method to detect common respiratory viruses. the patients who were enrolled consisted of hospitalized (n = ) or not-hospitalized patients (n = ; table ). in these two groups, the subject's backgrounds were similar for age, sex, smoking rates, and duration of illness, however, the measured value of spo was significantly lower in hospitalized patients ( ± . %) than in non-hospitalized patients ( . ± . %). the incidence of vri was significantly higher in the former group ( . %, n = ) than in the latter group ( . %, n = ; p = . ). in the latter group, influenza virus alone was detected in both patients. furthermore, all hospitalized patients ( %, n = ) had wheezing or severe exacerbation based on the ats (american thoracic society)/ers (european respiratory society) statement (reddel et al., ) , whereas, among nonhospitalized patients, only nine patients ( %) were considered as having a severe exacerbation (p < . ), and patients ( . %) had wheezing (p < . ). these findings suggested that virus infection was certainly associated with the hypoxemia and / or wheezing which resulted in a severe or serious asthma attack, based on the japanese guidelines (ohta et al., ) or the ats/ers statement (reddel et al., ) . previous studies using ohta et al. ( ) , † † defined by reddel et al. ( ) . ** p < . , *** p < . . all data are presented as (mean ± sd). pcr-based viral diagnostics found that viral respiratory infections were detected in up to % of exacerbations of asthma in children and about % of exacerbations in adults (nicholson et al., ; johnston et al., ) , which is similar to our results. serum inflammatory or allergic markers are not different between the hospitalized and non-hospitalized patients ( table ) . in hospitalized patients, the viruses identified were hrv (n = ), hmpv (n = ), and rsv (n = ). at the time of admission, the virus-positive group (n = ) had significant lower values of spo ( . ± . %) than those of the virus-negative group (n = , spo : . ± . %, p < . ), and for the patients whose data are available, the frequency of hypercapnea (paco torr) was significantly higher in the virus positive group ( . %, n = ) than in the virus negative group ( %; p = . ; table ). the mechanisms for hypercapnea in virus infected individuals have not been elucidated. however, cheung et al. ( ) reported that hrv infection causes long lasting excessive airway narrowing in response to methacholine in asthmatic subjects. we speculated that smooth muscle might have a role in exaggerated airway narrowing in virus positive asthmatic patients, as described by king et al. ( ) . interestingly, the incidence of ground glass opacities (ggo) on high resolution computed tomography seemed to be higher for virus-positive hospitalized patients than for virus-negative patients, but it did not reach statistical significance. for example, figure a shows a patchy ggo with thickening of interlobular septa in a -year-old woman who was admitted during an asthma attack induced by hrv-a. figure b also shows ggo in a -year-old man with an asthma attack caused by hrv-c infection. these ggo in both patients could only be detected in hrct, not in chest x-ray. these results suggested that hrv was the major cause of virusinduced asthma, and was possibly involved in lower airway or lung parenchyma features, appearing as ggo. viral infection significantly exaggerated the respiratory status (low spo and hypercapnea) when compared to that of virus-negative asthma exacerbative patients at the time of admission. indeed, in recent years, hrv has been recognized as a common cause of hospital admission, both as an agent of bronchopneumonia and through exacerbation of chronic pulmonary conditions, even in the elderly over years of age (pierangeli et al., ) . curiously, after initiation of treatment with intravenous steroid, both the virus-positive and -negative groups had no significant difference in duration of respiratory failure, wheezing, days in hospital, and even in the time required for steroid treatment. no established treatment for prevention of hrv-induced asthma is available, and we describe the exploratory interventions as follows. inhaled corticosteroid (ics) is the main drug for regular asthma therapy. ics treatment improved airway hyperresponsiveness in asthmatic patients experimentally challenged with hrv, however, ics treatment did not reduce accumulation of inflammatory cells, except for eosinophils in bronchial epithelium (grunberg et al., ) . double-stranded rna (dsrna), a viral product and a ligand for the toll-like receptor- (tlr ), upregulates the expression of inflammatory chemokines in airway epithelial cells. matsukura et al. ( ) reported that treatment of beas- b cells with fluticasone propionate significantly and dose-dependently inhibited dsrna-induced expression of ccl , cxcl , and cxcl protein and mrna. to confirm the effect on ssrna, such as that of hrv, would need further studies. leukotriene receptor antagonist was prescribed in asthmatic patients with or without ics. montelukast treatment did not improve asthma control or cold symptom scores when hrv were experimentally inoculated into mild asthmatics, or healthy subjects (kloepfer et al., ) . it is uncertain whether leukotriene receptor antagonist treatment is effective in the reduction of asthma symptoms associated with hrv infection. zambrano et al. ( ) reported that high serum ige levels in mildly asthmatic children with experimental hrv infection may be associated with enhanced lower respiratory symptoms and elevation of inflammatory markers, such as nasal eosinophil cationic protein and expired nitric oxide, than those of healthy subjects and/or low ige asthmatic patients. the prevalence of asthma was closely associated with the serum ige levels standardized for age and sex (burrows et al., ) , and airway hyperresponsiveness appears to be closely linked to the allergic diathesis, as reflected by the serum total ige level (sears et al., ) . omalizumab, an anti-ige monoclonal antibody, was indicated in inadequately controlled moderate-to-severe persistent allergic asthma patients who were treated with high dose ics. durrani et al. ( ) showed that the ige receptor fcεri is inversely associated with ifn-α and ifn-λ secretion when plasmacytoid dendritic cells derived from allergic asthmatic children were challenged with hrv. omalizumab downregulates fcεri expression on dendritic cells (prussin et al., ) , which may reduce exacerbation of asthma associated with increased production of ifns, through fcεri. no drugs are clinically used in hrv infection, although several drugs have been tried for treatment and prevention of hrv infection. these drugs are summarized in a review (jacobs et al., ) . ifns had a potential protective role in viral induced asthma (cakebread et al., ; gaajetaan et al., ) . becker et al. ( ) showed that exogenous ifn-α, ifn-β, ifn-λ , and ifn-λ inhibited hrv replication in becs from healthy donors. macrolides are known to possess anti-inflammatory and immunomodulatory actions extending beyond their antibacterial activity in pulmonary inflammatory disorders (takizawa et al., ; min and jang, ) . erythromycin inhibits hrv infection by reducing icam- expression on the surface of human tracheal epithelial cells, and modulates inflammation by suppressing the production of proinflammatory cytokines (suzuki et al., ) . yamaya et al. ( ) reported that the mucolytic drug ambroxol hydrochloride, antibiotic drug of levofloxacin (yamaya et al., b) , and bronchodilators (tiotropium, tulobuterol, and procaterol) for asthma or copd (yamaya et al., (yamaya et al., , a (yamaya et al., , may have a beneficial effect in hrv infection, by inhibiting hrv replication and partly reducing icam- expression and acidic endosome production, via the inhibition of nf-kappab activation (yamaya, ) . we reviewed the previous reports regarding hrv-induced asthma exacerbations, together with our results from an institutional prospective study. hrv is a major pathogen for asthma exacerbations, and certainly associated with more serious clinical conditions such as hypoxemia or hypercapnea in hospitalized patients. further accumulation of evidence of virus-induced asthma for multidisciplinary assessment would be helpful for physicians in recognizing the condition or understanding the pathogenic mechanisms. two groups of rhinoviruses revealed by 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risk groups of non-hospitalized patients viral infection in acute exacerbation of idiopathic pulmonary fibrosis virus infection-induced bronchial asthma exacerbation inhibitory effects of tiotropium on rhinovirus infection in human airway epithelial cells levofloxacin inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells procaterol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells tulobuterol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells ambroxol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells experimental rhinovirus challenges in adults with mild asthma: response to infection in relation to ige the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -jitao k authors: lei, yu; moore, chris b.; liesman, rachael m.; o'connor, brian p.; bergstralh, daniel t.; chen, zhijian j.; pickles, raymond j.; ting, jenny p.-y. title: mavs-mediated apoptosis and its inhibition by viral proteins date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jitao k background: host responses to viral infection include both immune activation and programmed cell death. the mitochondrial antiviral signaling adaptor, mavs (ips- , visa or cardif) is critical for host defenses to viral infection by inducing type- interferons (ifn-i), however its role in virus-induced apoptotic responses has not been elucidated. principal findings: we show that mavs causes apoptosis independent of its function in initiating ifn-i production. mavs-induced cell death requires mitochondrial localization, is caspase dependent, and displays hallmarks of apoptosis. furthermore, mavs(−/−) fibroblasts are resistant to sendai virus-induced apoptosis. a functional screen identifies the hepatitis c virus ns / a and the severe acute respiratory syndrome coronavirus (sars-cov) nonstructural protein (nsp ) as inhibitors of mavs-induced apoptosis, possibly as a method of immune evasion. significance: this study describes a novel role for mavs in controlling viral infections through the induction of apoptosis, and identifies viral proteins which inhibit this host response. in recent years, knowledge of host cell signaling responses to viral infection has progressed rapidly. it is known that cells of the immune system contain toll-like receptors (tlrs) capable of detecting extracellular or endosomal viral nucleic acid and activating appropriate signal transduction pathways leading to the up-regulation of immune and inflammatory cytokines. besides detecting extracellular viral products, somatic cells can also respond to intracellular viral rna by activating the recently identified mitochondrial antiviral signaling pathway. following cytoplasmic detection of viral nucleic acid by the rig-i-like helicases (rlh) family of receptors, these and other signaling proteins are recruited to the mitochondria where they interact with the mitochondrial antiviral signaling adaptor protein mavs (ips- , visa and cardif) [ , , , ] . in vitro and in vivo experiments have revealed a critical role for mavs and its mitochondrial localization in the activation of host antiviral responses [ , ] . although the role of mavs in type- interferon (ifn-i) responses is known, the localization of mavs to the mitochondria suggests other putative mitochondrial functions for mavs, prominent among these is apoptosis. however, to date, there are no comprehensive studies focused on testing this hypothesis. notably, host cell apoptosis is a successful strategy to impede viral replication and restrict virus spreading during a productive infection [ ] . multicellular organisms are equipped with at least two evolutionarily conserved defensive arms to eradicate viral infections: programmed cell death and innate immune responses. many proteins which function in both apoptotic and inflammatory signaling cascades contain a caspase recruitment domain (card), which functions as a homotypic interaction motif. in fact, the biological function of the card domain was initially described in a subset of caspases which activate mitochondria-dependent apoptotic signaling [ ] . for example, the card containing apaf- (apoptosis protease-activating factor- ) protein binds to cytochrome c and forms a ternary multimeric protein structure called the apoptosome which functions to activate caspase- via a proximity-induced mechanism [ ] . other card-containing proteins including some members of the nlr (nucleotide-binding domain and leucine-rich repeat containing) protein family have been linked with both apoptotic and inflammatory signaling [ ] . for example, the card-containing nlr, nod , has been shown to activate a caspase- dependent apoptosis and play a positive regulatory role in pathogen-induced nf-kb activation [ ] . similarly, nod , a protein linked with the etiology of the autoinflammatory crohn's disease, has been reported to augment caspase- -induced apoptosis when overexpressed [ ] . a third card-containing nlr, nlrc (ipaf), mediates cell death through a caspase- dependent fashion [ , , ] . similar to the aforementioned proteins, mavs contains an nterminal card-like domain, in addition to a central proline-rich region and a c-terminal transmembrane (tm) domain, which targets mavs to the mitochondrial outer membrane [ ] . recent crystal structure analysis reveals that the card-like domain of mavs is indeed a classical card fold with surface charge profiles of a typical card domain involved in homotypic associations [ ] . consequently, the presence of a card-like domain coupled with its mitochondrial localization suggests a putative role for mavs in both immune and cell death responses. in fact, both the n-terminal card-like and tm domains are indispensable for mavs-mediated activation of interferon regulatory factor- (irf- ) and subsequent transcription of the antiviral ifn-i, suggesting that these domains are critical to mavs function [ ] . as a survival mechanism, it is known that some viruses have evolved strategies to inhibit mavs function through selective targeting of these functional domains. for example, the genome of hepatitis c virus (hcv) has evolved to include a serine protease, ns / a, which cleaves the mavs tm domain and dislodges mavs from the mitochondria, thereby abrogating mavs mediated ifn-i production [ , ] . similar to hcv, hepatitis a virus (hav) encodes for the abc protein, which localizes to the mitochondria and inhibits mavs signaling via proteolytic cleavage [ , ] . currently, there are no reports of viral proteins targeting mavs for inhibition of virus-induced cell death responses. host cell apoptosis has been reported to suppress viral replication and the subsequent production of infectious progeny viruses [ ] . for example, adenoviruses and baculoviruses which are defective in anti-apoptotic genes are compromised in producing progeny viruses [ ] . in addition, several viruses infectious to humans, including the coronaviruses, are known to modulate host cell apoptotic responses [ , ] . in , researchers from several labs identified a unique coronavirus linked with the pathogenesis of severe acute respiratory syndrome (sars) in humans [ , , ] . the spread of sars-cov, reaching near pandemic levels, resulted in the death of over individuals with a case fatality rate of % [ ] . since that time, the sequencing of the complete sars-cov genome has allowed scientists to study the function of each sars-cov encoded protein in greater details. phylogenetic analyses revealed that sars-cov was not closely related to any other characterized coronaviruses [ , ] . the distinct nature of the sars-cov genome suggests possible unique strategies employed by this virus to subvert host defense mechanisms. notably, unlike other common human respiratory viral infections, such as influenza a virus, the viral loads in the upper airway of sars patients progressively increase, reaching a peak around days after the initial onset of symptoms [ ] . this indicates that at least during the initial stages of sars-cov infection, this virus might suppress host defensive responses. in fact, several sars-cov proteins have been shown to inhibit host ifn-i responses [ , , ] . although the fact that certain clinical manifestations of sars such as lymphopenia and cell death in the lung or liver are thought to be related to the ability of sars-cov to induce apoptosis in specific cell types and that several pro-apoptotic proteins have been found in sars-cov genome [ , , ] , the early pathogenesis of sars is poorly understood and how apoptosis contributes to the initial pathological changes is largely unknown. in addition, the replication of sars-cov seems to be restrained to the first two weeks upon symptom onset with little evidence of continued replication after this time window [ ] . clearly, the temporal and cell-type specific expression of sars-cov proteins could account for the dynamics of sars-cov infection and given that host cell fate decisions are a part of overall host responses to any pathogen, it is conceivable that like sars-cov inhibitory effects on ifn-i responses, this virus could employ proteins which function as inhibitors of host cell death. in this report, we describe a novel function of mavs in mediating virus-induced apoptosis, and identify viral proteins as inhibitors of this response. transient expression of mavs protein causes hek t cells to crenate and lose adherence, suggesting that mavs is cytotoxic (fig. a) . although some cell death was observed at hours posttransfection, it was most evident at hours post-transfection, which was quantitated by the trypan blue exclusion test of cell viability. in this assay, we observed a dose-dependent increase in the percentage of trypan blue positive cells at hours posttransfection with mavs plasmid (fig. b) . this result was confirmed by measurements of cell viability via xtt assay. mavs-induced cell death is potent as ng of mavs plasmid was sufficient to decrease cell viability by % (fig. c ). mavs contains a n-terminal card-like domain, which is well conserved from human to pufferfish [ ] ; and the card domain of other proteins has been shown to mediate the activation of caspases, facilitating apoptosis [ ] . therefore, we next sought to test the hypothesis that mavs induces apoptosis through a caspase-dependent mechanism. it is well known that blockade of caspase activity will inhibit the intrinsic apoptotic pathway [ ] . thus, we treated hek t cells with a pan-caspase inhibitor (z-vad-fmk) followed by transfection of increasing amounts of mavs plasmid. as expected, transient mavs expression in untreated cells resulted in a significant loss of cell viability as measured by xtt assay ( fig. a , black bars); and application of the pan-caspase inhibitor resulted in a complete reversal of mavs-induced cell death ( fig. a , grey bars). it is known that caspase-dependent apoptosis can cause poly (adp-ribose) polymerase (parp) cleavage, thus we investigated the effect of mavs expression on the triggering of parp cleavage. hek t cells were transfected with two different doses of mavs plasmid and both adherent and floating cells were harvested and lysed and hours post-transfection. immunoblotting followed by densitometry measurements revealed a dose-dependent increase in parp cleavage (fig. b, c ). in congruence with the parp cleavage pattern, caspases- and caspase- protein levels also showed dose-dependent increases following mavs expression (fig. b, c ). transmission electron microscopy examination of hek t cells transfected with mavs or empty vector was performed at hours post-transfection (fig. d ). unlike the otherwise healthy cells transfected with empty vector (fig. d , top panel), mavs-expressing cells exhibit the morphological hallmarks of apoptosis including an intact plasma membrane (fig. d , bottom panel arrow i), crenation, condensed and marginated chromatin (arrow ii), large vacuoles (arrow iii), cytoplasm shrinkage, membrane blebbing, an intact nuclear envelope, and swollen mitochondria (arrow iv) [ ] . a kinetic analysis shows that mavs expression reached a peak at h post-transfection (fig. s a) , while mavs-associated apoptosis lagged behind reaching a peak at h post-transfection (fig. s b ). thus the late onset of apoptosis does not appear to be due to the lack of mavs expression. it is possible that there are several molecular steps that have to occur before mavs could cause caspase- and caspase- activation, leading to apoptosis. the aforementioned studies analyzed the effect of transient expression of mavs on cell death. to explore the physiological role of endogenous mavs in mediating virus-induced apoptosis, we extended these findings to investigations of recombinant sendai virus expressing gfp (rsev-gfp) infected mouse embryonic fibroblasts (mefs) isolated from mavs knockout or wild type littermate control mice. mavs deficiency was confirmed by western blot (fig. s ). at hours post-infection, we infected mavs / and wildtype littermate control mefs with rsev-gfp, which is a known inducer of apoptosis [ ] . infection efficiency in each cell line was monitored by gfp positivity, which was similar between mavs wildtype and knockout mefs (fig. a , top graph). in contrast, the percentage of annexin v positive cells was . % among infected wildtype mefs compared to , % for the mavs / mefs (fig. a, bottom graph) . similarly, mavsdeficient mefs maintained a spindle-like fibroblastic morphology (fig. b , gfp bottom panel), while wildtype mefs crenated and became detached from the plate. in addition, rsev-gfp infected wildtype mefs have higher propidium iodide/hoechst staining ratio as compared to mavs / , further indicating an increase in cell death (fig. b , hoechst and pi panels). transmission electron micrographs taken for both cell types, with or without sev, demonstrated that sev infection resulted in the presence of typical apoptotic features in the wildtype mefs (fig. c , left panels). however, sev infected mavs / showed little signs of apoptosis and are indistinguishable from the uninfected controls (fig. c , right panels). previous reports have shown that the mavs c-terminal transmembrane domain (tm) is essential to mavs function as an adaptor in ifn-i signaling [ ] . therefore we sought to determine the essential domain that mediates mavs-induced apoptosis. we expressed full length mavs protein or three truncation mutants (fig. a ) in hek t cells and measured annexin v and -aad staining. the mavsdtm mutant, which lacks the mitochondrial transmembrane sequence, was completely incapable of inducing apoptosis, suggesting that similar to the role of mavs in interferon signaling, mitochondrial localization is essential to mavs activation of apoptosis ( (fig. s a, s b ), thus inability of these constructs to induce apoptosis is not due to ineffective protein expression. to solidify the mitochondrial dependency of mavs-induced cell death, we co-expressed mavs with the hepatitis c virus (hcv) protein ns / a. ns / a is a serine protease which has been shown to target the mavs tm domain for cleavage and subsequent inhibition of mavs antiviral signaling [ , ] . consistent with its role in inhibiting mavs mediated ifn-i signaling, ns / a inhibited mavs induced apoptosis (fig. b , bottom right panel). the inhibitory effects of mavsdtm mutant and ns / a were also verified by a measurement of cell viability via xtt assay (fig. c ). mitochondrial membrane potential collapse marks a point-of-no-return during apoptosis and occurs earlier than dna fragmentation [ , ] . only wildtype mavs resulted in compromised mitochondrial membrane potential as measured by tmre staining while the addition of ns / a abrogated this effect (fig. d ). it has been shown that exogenous mavs expression triggers ifn-i production [ , ] . type- ifns mediate their effects by binding to cell surface receptors, activating downstream interferon-stimulated genes or isgs, among which more than genes have pro-apoptotic functions [ ] . we sought to determine if mavs-induced apoptosis was the result of activation of a distinctive signaling pathway or as a consequence of ifn production. first, we induced ifn-i production in hek t cells by ectopic expression of upstream signaling molecules that activate mavs-mediated ifn production. as previously reported, a helicase domain truncation mutant of rig-i [ ] , full-length mavs [ ] , or mda- in conjunction with its ligand poly(i:c) [ ] zare all potent ifn-i inducers (fig. s ) . neither drig-i nor mda- plus poly(i:c) induced any observable cell death or annexin vpositive cells (fig. a, bottom panels) . only full-length mavs triggered apoptosis as quantified by annexin v and -aad staining (fig. a, top right panel) . to explore the role of ifn-b in mavs-induced apoptosis, hek t cells were treated with neutralization iu/ml ifn-b antibody prior to transient expression of mavs followed by xtt measurements. we found that blocking ifn-b from binding to its cell surface receptor had no effect on mavs-induced apoptosis ( fig. b ) even though the antibody efficiently blocked the function of secreted ifn-b (fig. s ). in congruence with these findings, rsev-gfp infections of the interferon-a/b receptor (ifnar) knockout and wildtype mefs demonstrated that this receptor is not required for sev-induced cell death as visualized by pi staining (fig. c ). mavs is also capable of inducing the transcription factor nf-kb, which is known to activate a wide array of genes linked to immune, inflammatory, and cell-fate processes [ ] . to test the involvement of nf-kb in mavs-induced apoptosis, we co-expressed mavs with the non-degradable superrepressor form of ikba. this super-repressor is a known potent inhibitor of nf-kb activation and as expected inhibited mavsinduced nf-kb activation (fig. s ) [ ] . however, transient expression of mavs in hek t cells induced apoptosis even in the presence of the nf-kb super-repressor (fig. d ). there are subtypes of type- ifns, and a plethora of other secreted soluble factors can be induced by mavs expression. therefore, we performed a transwell assay designed to definitively answer if mavs-induced cell death was intrinsic to the host cell or caused by some unknown secretory factor. hek t cells were plated in two different chambers (top and bottom) separated by an insert filter membrane, permissive to all secretory cytokines but impermeable to fugene :dna complexes. in this experiment, only the lower chamber was transfected with mavs. cells in the upper chamber were not transfected with mavs, but were exposed to the same cytokine milieu. as expected, mavs induced cell death in the lower chamber (transfected cells); however the cells in the upper chamber (untransfected cells) remained healthy and unchanged from controls (fig. e ). this indicates that the mavs-induced death is not caused by an undefined secretory product(s) which includes the interferon family members. irf is a critical transcription factor for ifn-i production but it has been associated with apoptosis, and furthermore can be activated by mavs [ , ] . we assessed whether mavs-induced apoptotic signaling depends on irf . we made numerous attempts to introduce mavs into irf / fibroblasts, however these cells were much more difficult to transfect than wildtype fibroblasts, hence the different efficiencies of transfection made the interpretation of data difficult. instead we used sirna to reduce irf expression. briefly, hek t cells were plated in -well plate and endogenous irf expression was reduced by transfecting a pool of four sirna into the cells. mavs was introduced hours after sirna transfection. cell viability was measured by xtt assay hours after cells were transfected with an expression plasmid containing mavs or a control empty vector. the reduction of irf did not prevent mavs-induced loss of cell viability ( figure a ). the same sirna was introduced into cells plated in a -well plate, and an immunoblot was used to verify the efficiency of sirna h post-transfection ( figure b ). as an alternate approach to measure cell death, the experiment was repeated in well plates and cells were harvested hours after plasmids transfection. half of the cells from each well were stained for annexin v and analyzed by flow cytometry, and the other half of the cells were lysed in ripa buffer for western blotting analyses of irf . as expected we were able to greatly reduce the endogenous irf expression, yet mavs-induced apoptosis was not abrogated (fig. c, d) . while host response that elicits apoptosis may function as an antiviral strategy, viruses have also evolved diverse mechanisms to evade these host antiviral responses. therefore we performed a functional screen of sars-cov-encoded proteins to identify any potential modulators of mavs-induced apoptosis. each of these sars-cov genes were cloned into an expression vector and co-expressed with mavs plasmid followed by xtt cell viability measurements. protein expression was verified by an immunoblot (fig. s ). of the tested proteins, only one sars-cov protein, nsp , significantly altered mavs-induced apoptosis of hek t cells (fig. a) . further examination showed that nsp significantly inhibited mavs-induced apoptosis in a dose-dependent manner (fig. b) . the anti-apoptotic function of nsp displayed specificity since it did not inhibit staurosporine-induced apoptosis (fig. c) . we sought to investigate if the anti-apoptotic function of nsp was specific for sars-cov or shared by other coronaviruses. when the nsp protein encoded by the sars-cov, hku or nl genome was coexpressed with mavs, only sars-cov nsp inhibited mavs-mediated apoptosis as measured by annexin v and -aad staining, while the others showed little effect on cell viability (fig. d ). host cellular response to virus infection involves the concomitant activation of parallel signaling pathways leading to the transcription of a plethora of cytokine genes, prominent among these are the genes encoding type- interferons (ifn-i). it is the autocrine and paracrine action of these and other cytokines which encompasses the comprehensive host immune response designed to defend against viral infection. this is accompanied by a reciprocal activation of programmed cell death in infected host cells, which is also known to reduce viral load. apoptosis has been suggested to play a protective role at the organismal level in preventing the virus from completing its replication and producing infectious progeny viruses [ ] . consequently, the exact mechanisms underlying both virus-induced apoptotic signaling in addition to viral strategies to subvert these responses is currently a topic of intensive research. while pathways that govern ifn-i have been extensively investigated, the molecular mediators that activate host cell apoptosis during viral infection are less known. in this study we have identified a role for mavs in the initiation of virus-induced apoptosis (fig. ) . furthermore, we have identified hepatitis c virus ns / a and sars-cov nsp proteins as inhibitors of mavs-mediated apoptosis. it is known that mavs is a potent inducer of ifn-i responses and that ifn-i can activate host apoptotic responses, therefore it was important to determine whether the cell death responses observed in the current study were a consequence of ifn-i secretion. in fact, ifn-i consist of several species including ifn-a, ifn-b, ifn-v and ifn-k and these cytokines bind to surface receptors leading to the activation of the jak-stat signal transduction pathway resulting in the transcriptional activation of virtually hundreds of ifn-stumulated genes (isgs), whose protein products play pivotal roles in a variety of biological events, including but not limited to immunomodulation, cell differentiation, anti-angiogenesis and programmed cell death. for example, type- ifn induction of apoptotic responses can be mediated by a multitude of isgs, illustrated by the finding that more than isgs have pro-apoptotic functions [ ] . in addition, the involvement of proteins on ifn axis in virusinduced host cell apoptosis has been implicated in another previous report, in which mavs has been shown to be critical for reovirus-triggered caspase- / activation in hek t cells [ ] , however, the study did not evaluate whether mavs mediates virus-induced apoptosis and what roles type ifns play in mavs-mediated apoptosis. using a multi-pronged approach we demonstrate that mavs-mediated apoptosis is not a consequence of ifn-i induction by mavs. we show that (a) the over-expression of the truncation mutant of rig-i that induce ifn-i does not lead to apoptosis; (b) anti-ifn-b antibody does not ablate mavs-induced apoptosis; (c) the targeted deletion of ifn receptor does not alter mavs-induced apoptosis; (d) the co-culture of mavs-transfected cells with nontransfected cells separated by an insert filter membrane does not induce apoptosis in the latter, indicating that a soluble secretory factor is not likely responsible for the apoptosisinducing activity. in the ifn-i induction pathway mediated by endogenous rig-i like helicases, irf lies downstream of mavs, and exogenous expression of mavs can lead to the phosphorylation and nuclear translocation of irf [ ] . in addition, others have shown that the expression of constitutively active form of irf mutant is toxic to cells and the transfection of wild type irf expression can augment sev-induced apoptosis [ , ] . hence it was important to evaluate if mavs-induced apoptosis depends on the presence of irf . our data shows that depletion of endogenous irf by means of rnai did not reverse mavs-induced cell viability loss. together with our findings that mavs-induced apoptosis is ifn- i-independent, we speculate that mavs-induced apoptotic signaling pathway is different from the classical mavs-mediated ifn-i response. furthermore, it is possible that the reported proapoptotic effects of the constitutively active form of irf might be independent of its ifn-i-inducing function. in fact our data suggests other ifn-i signaling molecules such as the constitutively active form of rig-i and mda do not lead to apoptosis despite of their roles in inducing ifn-i. the dual functions of mavs in virus-induced ifn-i and apoptosis highlight this molecule as a putative target for viral evasion strategies designed to escape host immunity. for example, it is already known that hepatitis c virus (hcv) produces a serine protease, ns / a, which disrupts ifn-i production through the targeted cleavage of the mavs transmembrane region and the subsequent dislodging of mavs from the mitochondria [ , ] . in fact, we found that loss of mavs mitochondrial localization through mutation of the transmembrane domain, also completely abolished its pro-apoptotic effects. consistent with these results, when hcv ns / a protein was co-expressed with mavs, this viral protein completely inhibited mavs-induced cell death. this result would suggest that the host immune evasion strategies of the hcv ns / a protein may be extended to inhibition of mavsmediated apoptosis. similarly hepatitis a virus (hav), a picornavirus, employs a cysteine protease abc to abrogate ifn-i production by targeting mavs [ ] . therefore, it is likely that other uncharacterized viral mechanisms target mavs for modulation of host cell death responses. one novel strategy described in this study is employed by the sars-cov. similar to sars-cov abilities to inhibit ifn-i responses, we have found that this virus is also capable of interfering with host cell death responses by targeting mavs. we showed that among the sars-cov proteins tested, nsp alone can completely abrogate mavs-induced apoptosis. the underlying mechanism is currently unclear since the function of nsp is not yet fully defined. however, an earlier report indicates that this protein is indeed important for sars pathogenesis in that it is essential for viral replication [ ] . however, until we understand the exact temporal expression patterns of each of the sars-cov proteins during the course of an infection, the relative contributions of each to the evasion of host immunity and apoptosis will not be fully understood. further studies are needed to determine the exact mechanism of action for nsp on inhibiting mavs-induced cell death. since sars-cov replication is limited to the first two weeks after symptom onset and nsp is critical for its replication [ , ] , this would support the theory that nsp may function as an inhibitor of host cell death during this time, which would possibly benefit viral replication. further studies are ongoing to determine how nsp and other sars-cov proteins contribute to overall viral evasion strategies. the finding that mavs mediates virus-induced apoptosis posits a new mechanism by which mitochondria serves as a bona fide intracellular sentinel for antiviral and apoptotic responses. it is well established that the permeabilization of the mitochondria outer membrane by the pro-apoptotic bcl- family member bak results in the activation of caspase- in a classical apaf- -dependent or an alternate apaf- -independent pattern [ ] . furthermore, it is known that proteins localized on the mitochondria are targeted by viruses to modulate host responses. for example, the cytomegalovirus rna can interact with the mitochondrial enzyme complex i (reduced nicotinamide adenine dinucleotide-ubiquinone oxido-reductase) to modulate the classical mitochondria-mediated apoptosis [ ] . recent discoveries also underscore the mitochondria as a platform orchestrating host antiviral type- ifns through the mitochondrial mavs protein. this study describes, for the first time, a duality of function for the mavs protein in regulating both ifn-i and apoptotic antiviral responses from within the mitochondria and suggests that mavs is a pivotal molecule in the bifurcation of host responses following viral challenge. furthermore, the identification of hcv ns / a and sars-cov nsp as inhibitors of mavs-mediated apoptotic responses reveals both novel host defense mechanisms as well as viral immune-evasion mechanisms that might serve as useful templates for the development of anti-viral drug strategies. hek t cells, mavs +/+ , mavs / , ifnar / mouse embryonic fibroblasts were maintained in dmem media supplemented with % fbs, % penicillin and mg/ml streptomycin. cells were passed every three days and grown at uc in % co . the mammalian expression plasmids of ns / a, wild type mavs and truncation mutants were kindly provided by dr. zhijian chen at the university of texas southwestern medical center. ha-tagged sars-cov proteins expression plasmids, hku nsp and nl nsp expression plasmids were provided by drs. ralph baric and matthew frieman at the university of north carolina. transfections and viral infections hek t cells were seeded in -well or -well plates, and the total dna transfected into these cells was ng/well and mg/ well respectively. standard transfection protocol was performed using fugene (roche applied science) according to the commercial protocol. cells were incubated for the indicated times prior to assay. rsev-gfp is a recombinant sendai virus expressing gfp and was originally made by dr. daniel kolakofsky [ ] . for viral infections, . mefs were plated into -well plate one day prior to sendai virus (sev) infection. viral infections were performed when cells reached % confluence. mefs were incubated with sev for h in serum-free dmem supplemented with . % tryple select at the moi of . . serum-free dmem supplemented with . % tryple select was used for mock infection. virus inoculum was removed and cells were replenished with complete media supplemented with . % tryple select following incubation with virus. cells were harvested and washed in cold facs buffer ( % fbs in pbs) twice and in annexin v binding buffer (bd bioscience, san diego, ca) once. cells were transferred into well plate and resuspended in ml annexin v binding buffer. cells were stained according to standard cell staining protocol [ ] using annexin v conjugated to fitc or apc (bd biosciences, san diego, ca). cells were washed in facs buffer and stained with -aad (invitrogen, carlsbad, ca) for min at room temperature. cells were washed twice and resuspended in facs buffer containing mg/ml actinomycin d. after min incubation, all samples were fixed in ml % em grade formaldehyde (polysciences, warrington, pa) for immediate flow cytometry analyses. for the rsev-gfp infected samples, gfp positive cells were gated; annexin v and -aad positive populations were assessed thereafter. mitochondrial membrane potential dy was assessed by flow cytometry analysis on a dysensitive fluorophore tetramethylrho- damine ethylester (tmre) (molecular probes, eugene, or). after treatment, hek t cells were incubated with tmre at the concentration of nm per cells for min at uc. immediate flow cytometry analysis was performed after staining. all flow cytometry data were collected on either a facscalibur (bd biosciences, san jose, ca) or cyan flow cytometer (dako north america, carpinteria, ca) and then analyzed by flowjo software (tree star, ashland, or). transmission electron microscopy examination was performed as described [ ] . mefs or hek t cells were harvested and fixed in glutaraldehyde and all samples were post-fixed in % oso for transmission electron microscopy examination. cells were embedded in london resin white and sections were analyzed using a leo em- transmission electron microscope (leo electron microscopy inc., thornwood, ny). hoechst and propidium iodide (pi) dual staining was used to evaluate sevinduced cell death. samples were photographed using a leica dmirb inverted fluorescence microscope (leica microsystems inc., bannockburn, il) with a digital camera (micropublisher, q-imaging, burnaby, bc, canada). cells were lysed in ripa lysis buffer ( % triton x- , . % doc, . % sds, mm tris ph . , mm nacl and mm naf) containing proteinase inhibitor cocktails (roche) for min at uc. whole cell lysates were suspended in laemmli's sample buffer and loaded onto nupage bis-tris - % gradient pre-cast gels (invitrogen, carlsbad, ca) for sds-page and subsequent immunoblotting for parp, caspase- and caspase- (abcam, cambridge, ma), irf (santa cruz biotechnology, santa cruz, ca), rodent specific mavs (cell signaling, danvers, ma). densitometry was performed using imagej analysis software. identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-beta signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus the specific and essential role of mavs in antiviral innate immune responses viral homologs of bcl- : role of apoptosis in the regulation of virus infection card games in apoptosis and immunity apaf- , a human protein homologous to c. elegans ced- , participates in cytochrome c-dependent activation of caspase- the nlr gene family: a standard nomenclature nod , an apaf- -like activator of caspase- and nuclear factor-kappab nod , a nod /apaf- family member that is restricted to monocytes and activates nf-kappab caspase- activator ipaf is a p -inducible gene involved in apoptosis differential activation of the inflammasome by caspase- adaptors asc and ipaf identification of ipaf, a human caspase- -activating protein related to apaf- crystal structure of human ips- / mavs/visa/cardif caspase activation recruitment domain hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor regulation of mitochondrial antiviral signaling pathways regulators of apoptosis on the road to persistent alphavirus infection open reading frame a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis induction of apoptosis by the severe acute respiratory syndrome coronavirus a protein is dependent on its interaction with the bcl-xl protein aetiology: koch's postulates fulfilled for sars virus identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study sars coronavirus and innate immunity severe acute respiratory syndrome coronavirus gene products contribute to virusinduced apoptosis a sars-cov protein, orf- , induces caspase- mediated, er stress and jnk-dependent apoptosis pathogenesis of severe acute respiratory syndrome time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars the mitochondrial apoptosome: a killer unleashed by the cytochrome seas caspases in cell survival, proliferation and differentiation the morphology of apoptosis the irf- transcription factor mediates sendai virus-induced apoptosis sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity nf-kappab signaling pathways in mammalian and insect innate immunity nf-kappab activation provides the potential link between inflammation and hyperplasia in the arthritic joint irf- activation by sendai virus infection is required for cellular apoptosis and avoidance of persistence retinoic acid-inducible gene-i and interferon-beta promoter stimulator- augment proapoptotic responses following mammalian reovirus infection via interferon regulatory factor- major genetic marker of nidoviruses encodes a replicative endoribonuclease apoptotic pathways: ten minutes to dead complex i binding by a virally encoded rna regulates mitochondria-induced cell death sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis c-c chemokine receptor on pulmonary fibrocytes facilitates migration and promotes metastasis via matrix metalloproteinase nlrx is a regulator of mitochondrial antiviral immunity we thank victoria madden for technical support with electron microscopy. we thank dr. mark heise and catherine cruz for the interferon protection assay. sars-cov proteins expression plasmids, hku and nl nsp expression plasmids were provided by drs. ralph baric and matthew frieman, university of north carolina at chapel hill. nf-kb super-repressor plasmid is a kind gift of dr. albert baldwin, university of north carolina at chapel hill. rsev-gfp was generously provided by dr.daniel kolakofsky, university of geneva. we also acknowledge visiscience inc. for scienceslides application in cartoon preparation. key: cord- -pp etmwq authors: baker, m. l.; schountz, t.; wang, l.‐f. title: antiviral immune responses of bats: a review date: - - journal: zoonoses public health doi: . /j. - . . .x sha: doc_id: cord_uid: pp etmwq despite being the second most species‐rich and abundant group of mammals, bats are also among the least studied, with a particular paucity of information in the area of bat immunology. although bats have a long history of association with rabies, the emergence and re‐emergence of a number of viruses from bats that impact human and animal health has resulted in a resurgence of interest in bat immunology. understanding how bats coexist with viruses in the absence of disease is essential if we are to begin to develop therapeutics to target viruses in humans and susceptible livestock and companion animals. here, we review the current status of knowledge in the field of bat antiviral immunology including both adaptive and innate mechanisms of immune defence and highlight the need for further investigations in this area. because data in this field are so limited, our discussion is based on both scientific discoveries and theoretical predictions. it is hoped that by provoking original, speculative or even controversial ideas or theories, this review may stimulate further research in this important field. efforts to understand the immune systems of bats have been greatly facilitated in recent years by the availability of partial genome sequences from two species of bats, a megabat, pteropus vampyrus, and a microbat, myotis lucifugus, allowing the rapid identification of immune genes. although bats appear to share most features of the immune system with other mammals, several studies have reported qualitative and quantitative differences in the immune responses of bats. these observations warrant further investigation to determine whether such differences are associated with the asymptomatic nature of viral infections in bats. despite being the second most species-rich and abundant group of mammals, bats are also among the least studied, with a particular paucity of information in the area of bat immunology. although bats have a long history of association with rabies, the emergence and re-emergence of a number of viruses from bats that impact human and animal health has resulted in a resurgence of interest in bat immunology. understanding how bats coexist with viruses in the absence of disease is essential if we are to begin to develop therapeutics to target viruses in humans and susceptible livestock and companion animals. here, we review the current status of knowledge in the field of bat antiviral immunology including both adaptive and innate mechanisms of immune defence and highlight the need for further investigations in this area. because data in this field are so limited, our discussion is based on both scientific discoveries and theoretical predictions. it is hoped that by provoking original, speculative or even controversial ideas or theories, this review may stimulate further research in this important field. efforts to understand the immune systems of bats have been greatly facilitated in recent years by the availability of partial genome sequences from two species of bats, a megabat, pteropus vampyrus, and a microbat, myotis lucifugus, allowing the rapid identification of immune genes. although bats appear to share most features of the immune system with other mammals, several studies have reported qualitative and quantitative differences in the immune responses of bats. these observations warrant further investigation to determine whether such differences are associated with the asymptomatic nature of viral infections in bats. little importance as vectors or reservoirs. much of our knowledge of bats and viruses is from studies of rabies virus and other lyssaviruses (calisher et al., ) . however, in recent years, many novel viruses of human and veterinary importance have been discovered that are hosted, or suspected to be hosted, by bats. in the s, novel paramyxoviruses, hendra and nipah viruses, caused outbreaks of fatal disease in australia and malaysia (murray et al., ; chua et al., ) . both viruses are hosted by species of pteropid bats. severe acute respiratory syndrome, caused by a coronavirus, was identified during an outbreak in china and hong kong in the early s. subsequent research has indicated its ancestor is a batborne virus (lau et al., ; li et al., ) . also in the s, marburg virus was demonstrated to be hosted by fruit bats, and there is compelling evidence that ebolaviruses are also hosted by fruit bats (leroy et al., ; towner et al., ). in addition, melaka and kampar viruses and related bat reoviruses from malaysia are associated with respiratory disease in humans (chua et al., (chua et al., , (chua et al., , . currently, more than viruses have been isolated from or detected in bats. the diversity of viruses found in bats is matched only by rodents that are the most abundant and diverse group of mammals and are reservoir hosts to a large number of viruses that also cause disease in humans and other species (meerburg et al., ) . it is now clear that bats have been substantially underappreciated as reservoirs of viruses important to human and veterinary health (calisher et al., ; wang et al., ) . the peridomestic nature of some bat species and the encroachment of humans upon bat habitats make it likely that new human pathogens from bats will be discovered after spillover events. bats have often been vilified by much of the public and have been frequently targeted for extermination, despite their critical roles in many ecosystems. because of this, many bat biologists, whose help laboratory scientists will need to answer many questions about bats, are often reluctant to engage or assist in biomedical research. in addition, obtaining bats for biomedical research is extremely challenging. most captive colonies are maintained by zoos, which are unwilling or unable to donate their excess animals for such research because of long-standing policies. the establishment of colonies from wild bats is also problematic because they require specialized housing and diets. moreover, the animals must be evaluated for possible pathogens, including rabies virus. therefore, most research is conducted on wild bats in their natural environments, or captured animals in laboratory settings, which introduces additional problems with housing, diet and occupational health (e.g. animal stress and rabies immunization for personnel). this practice severely limits experimental examination of viruses hosted by bats. although bats may be persistently infected with many viruses, evidence from experimental and naturally infected bats has demonstrated that they rarely display clinical symptoms (sulkin et al., ; swanepoel et al., ; williamson et al., williamson et al., , leroy et al., leroy et al., , middleton et al., ; towner et al., ) . experimental infection of bats has included viruses such as hendra and nipah viruses that are known to result in high disease mortality in other mammals including humans. these studies have confirmed the virulence of the viruses used for experimental infections using conventional laboratory mammals such as guinea pigs that succumb to the same dose of virus infection that bats respond to in the absence of disease (williamson et al., ; middleton et al., ) . the only viruses that have been demonstrated to cause clinical signs of disease in bats are rabies virus and the closely related australian bat lyssavirus (field et al., ; mccoll et al., ) . however, results of experimental infections are inconsistent, with only a small proportion of bats succumbing to infection (mccoll et al., ) . in addition, very few viruses have been shown to have negative impacts on natural bat populations. one exception is tacaribe virus, an arenavirus closely related to the south american haemorrhagic fever viruses, which caused the deaths of many artibeus bats in trinidad in the s (downs et al., ) and in experimental infections (cogswell-hawkinson et al., ) . it is unknown whether this virus is still circulating or what impact it may have on artibeus bats today. overall, these results demonstrate that for the most part, bats are able to coexist with viruses and may have evolved mechanisms to control viral replication more effectively than most other mammals. a similar situation exists in rodents that also show limited or no signs of disease in response to the viruses they harbour (fulhorst et al., ; botten et al., ) . all viruses must evade the immune response for a sufficient period of time to allow transmission to other susceptible hosts, and many viruses possess immunemodulating genes that provide a competitive advantage over the immune response. much of what has been learned about immune responses has been based upon pathology models; however, many zoonotic viruses have coadapted with their vertebrate hosts to cause persistent, apathogenic infections. because bats have not been examined in great detail, and because there are so many species of bats, virtually nothing is known about the role of their immune responses in control of viral infections, nor how viruses manipulate the immune responses of bats. fortunately, much of what is known about other natural zoonotic virus-reservoir relationships, particularly rodent hosts (fulhorst et al., ; easterbrook et al., ; schountz et al., ) , and the availability of novel and increasingly less-expensive deep sequencing methods (glenn, ) makes the study of specific bat reservoirs and viruses highly tractable. furthermore, the recent availability of partial genome sequences has provided important resources to study various aspects of bat biology, including genes associated with the immune system. two bat genomes have been sequenced as part of the us national institutes of health funded mammalian genome project, one from the megabat pteropus vampyrus and a second from the microbat myotis lucifugus. although both bat genomes are low coverage ( . · for p. vampyrus and . · for m. lucifugus), these projects have an important role to play in revealing the mechanisms that have evolved to allow bats to remain asymptomatic when infected by so many viruses. although few bat-specific reagents exist to identify specific cell types in bats, a variety of cells have been described based on morphological and physiochemical characteristics, demonstrating the presence of similar populations of cells in bats to other mammals. macrophages, b cells and t cells have been identified in the spleen and lymph nodes from the indian fruit bat (pteropus giganteus) using scanning electron microscopy and cellular adherence properties. these cells displayed similar characteristics to those from other mammals including humans and mice. in p. giganteus, the ratio of macrophages/b cells/ t cells was : : , similar to that of mice in which the ratio was approximately : : (sarkar and chakravarty, ) . a variety of immune cells including lymphocytes, neutrophils, eosinophils, basophils and macrophages have also been identified by morphology in histological sections from the brazilain free tailed bat (tadarida brasiliensis) following injection of the t-cell mitogen, phytohaemagglutinin (pha) (turmelle et al., a) . cells resembling follicular dendritic cells (fdcs) have also been described in p. giganteus (sarkar and chakravarty, ) . follicular dendritic cells are capable of capturing and retaining antigen in the form of immune complexes that can persist for months or even years and are important for the induction and maintenance of memory immune responses (mandels et al., ; . evidence for the ability of some viruses to retain infectivity when complexed within fdcs has been demonstrated (keele et al., ) . however, whether fdcs play a role in the persistence of viral infections in bats awaits further investigation. one hypothesis for the ability of bats to remain asymptomatic to viral infection is that they are able to control viral replication very early in the immune response through innate antiviral mechanisms. the recent description of a variety of innate immune genes in bats provides the first step in understanding the role of the innate immune system in antiviral immunity in bats. the recognition of pathogens by pattern recognition receptors (prrs) including toll-like receptors (tlrs) and retinoic acid inducible gene-like helicases (rlhs) provides the first line of defence against infection (xiao, ). toll-like receptors have been described in two species of fruit bats, pteropus alecto and rousettus leschenaultia (iha et al., ; cowled et al., ) . not surprisingly given the role of tlrs in the recognition of conserved molecular patterns, the bat tlrs were highly conserved between bats and other mammals cowled et al., ) . evidence from p. alecto for the presence of transcripts corresponding to tlrs - and provides evidence that bats are capable of recognizing a range of pathogens including viruses, bacteria and fungi. however, the p. alecto tlr transcript contained stop codons within its open reading frame and may represent a transcribed pseudogene (cowled et al., ) . to date, the only other mammals in which tlr has been identified are rodents. although the ligand for tlr is unknown, knockdown of tlr in mouse cells results in greater susceptibility to vesicular stomatitis virus (vsv), indicating it likely has a role in viral recognition (shi et al., ) . the transcription of a tlr pseudogene in pteropid bats may indicate that this gene has only recently undergone inactivation and at one time may have encoded a functional protein involved in viral sensing and may still be intact and functional in other bat species. the cytoplasmic rlhs, retinoic acid inducible gene i (rig-i), melanoma differentiation-associated protein (mda ) and laboratory of genetics and physiology (lgp ) have also been described in p. alecto, providing evidence for a similar repertoire of rlhs in megabats to other mammals (cowled et al., ) . overall, these reports provide evidence for the presence of the two major families of virus sensing prrs in bats consistent with recognition of a similar range of pathogens to other species of mammals. the interferon (ifn) response represents a potent first line of defence against viral infection conferring cells with an 'antiviral state' and preventing the spread of viral infection (randall and goodbourn, ) . the ifn signalling and production pathway is therefore a logical starting point in understanding the asymptomatic nature of viral infection in bats. three classes of ifn have been identified, designated types i, ii and iii, which differ in their amino acid sequences and the receptor complex they signal through (pestka et al., ; schroder et al., ) . type i (including a and b) and iii (k) ifns are induced directly in response to viral infection and thus play an important role in innate immunity. although they differ in the receptor complex they signal through, type i and iii ifns result in the induction of an overlapping set of ifn stimulated genes (isgs) that are responsible for the antiviral activity of ifns (sadler and williams, ) . type i ifns have been described in three species of fruit bats, rousettus aegyptiacus, the malaysian flying fox, p. vampyrus, and the greenish naked-backed fruit bat, dobsonia viridis, and from the microbat, m. lucifugus (omatsu et al., ; he et al., ; kepler et al., ) . in humans and mice, there are and ifna genes, respectively, but in bats, only seven ifna genes have been identified in the p. vampyrus genome and only ifna pseudogenes have been identified in the m. lucifugus genome sequence (van pesch et al., ; kepler et al., ) . however, as both currently available bat genomes are low coverage genome sequences, it is possible that some members of the type i ifn family are absent, despite the sequences being inferred from the unassembled trace archives that should contain a broader representation of the genome than the assembly. he et al. ( ) described the cloning of seven ifna subtypes and one pseudogene from d. viridis with evidence for positive selection among this gene family. both m. lucifugus and p. vampyrus also appear to have expanded the ifnw family of genes, with up to a dozen ifnw members in each species of bat. humans have only a single functional ifnw family member and at least two pseudogenes, and mice have a single ifnw pseudogene (hardy et al., ; kepler et al., ) however, this family has expanded in cats that have ifnw subtypes and in cattle that have potentially functional ifnw genes (yang et al., ; walker and roberts, ). furthermore, cat (felis catus) ifnw has been implicated in protection against parvovirus infection (paltrinieri et al., ) . thus, the expansion of the ifnw family in bats may also have implications for antiviral immunity. type iii ifns have also been identified in the m. lucifugus genome with the identification of a single full-length ifnl locus (fox et al., ). in the pteropid bat p. alecto, two ifnl genes (il a and il ) and the two chains of the type iii ifn receptor complex (il r and ifnkr ) have been characterized, and ifnkr has been demonstrated to act as a functional receptor (zhou et al., a,b) . furthermore, unlike the type iii ifn receptor of mammals such as mice and humans, in p. alecto, the type iii ifn receptor displays a wide tissue distribution consistent with a more significant role for the type iii ifns in antiviral immunity in bats (sommereyns et al., ; witte et al., ; zhou et al., a) . pteropid bat cells and cell lines readily secrete ifn in response to stimulation with synthetic tlr ligands including polyinosine-polycytidylic acid (polyic) and lipopolysaccharide (lps), demonstrating that ifn production pathways are functional in bat cells (stewart et al., a; crameri et al., ; kepler et al., ; zhou et al., b) . bats also demonstrate an ifn response following viral infection in vivo and in vitro. the earliest work on ifn production in bats described the detection of ifn in spleen and brain tissues from microbats (t. brasiliensis) infected with japanese b encephalitis (je) virus. although ifn was detected in both tissues during the first week of infection, it was detected only in brain tissue during the second week of infection despite the presence of virus in both tissues (stewart et al., a,b) . the persistence of the virus in certain populations of cells even in the presence of ifn has been speculated to reflect the presence of populations of cells that may be insensitive to the action of ifn (sulkin and allen, ) . in vitro studies have demonstrated the induction of ifnb in vsv-infected peripheral blood mononuclear cells (pbmcs) from p. vampyrus, demonstrating a delay in the ifn response in comparison with stimulation with the tlr ligands, polyic or lps, a result that is consistent with the mechanisms of ifn signalling of other mammals (kepler et al., ) . recently, evidence for differences in the ifn responses of bats and the ability of viruses to evade the ifn response of bat cells have also been described. in p. alecto splenocytes, type i and iii ifns appear to be differentially induced following infection with the bat-borne paramyxovirus tioman virus with type i ifns downregulated and type iii ifns upregulated following viral infection (zhou et al., b) . in contrast, henipavirus infection antagonized both type i and iii ifn production in human cell lines and ifn production and signalling in pteropid bat cell lines (virtue et al., a,b; zhou et al., b) . the ability of viruses to antagonize both the ifn signalling and production pathways in bat cells is intriguing and may indicate that factors other than ifn play a key role in antiviral immunity in bats. thus, these results demonstrate not only differences in the ifn response following infection with different viruses but also differences between bats and humans which may be significant in terms of the ability of bats to control viral replication. few studies have examined the ifn signalling pathway following ifn production to determine the ability of ifns to induce an 'antiviral state' in bat cells. ifns exert their antiviral actions through binding to cell surface receptors, which in turn activate the jak-stat pathway. activation of the receptor-associated janus family of tyrosine kinase enzymes results in the phosphorylation of latent cytoplasmic signal transduction and activator of transcription (stat) family of transcription factors. the phosphorylated stat and stat dimerize to interact with ifn regulatory factor and translocate to the nucleus resulting in isg production and the induction of an antiviral state (samuel, ) . the stat protein is the only component of this signalling pathway that has been characterized in bats. stat is present in the egyptian fruit bat, r. aegyptiacus, and is phosphorylated and translocated to the nucleus following stimulation with human ifna consistent with its activation in a similar manner to other mammals. rabies virus infection of human and bat cells antagonizes stat function, resulting in failure of stat to be translocated to the nucleus (brzó zka et al., ; fujii et al., ) . overall, this study demonstrated that the stat signalling pathway in r. aegyptiacus cells is similar to that of other mammals. further characterization of other signalling molecules involved in the ifn response will play an important role in understanding the nature of innate antiviral immunity in bats. the ability of ifn to induce an antiviral state through the induction of isgs is the hallmark of the ifn response (sadler and williams, ) . stewart et al. ( a) used ifn containing supernatant prepared from polyic stimulated t. brasiliensis embryo cells to compare the antiviral activity of bat ifn with ifns prepared from cells from other species. this study demonstrated each species of ifn has a characteristic spectrum of antiviral activity. although bat ifn displayed antiviral activity within a similar range to other species, this study did not examine the effect of bat ifn on any bat-borne viruses. more recently, recombinant p. alecto type iii ifn demonstrated antiviral activity against the bat-borne orthoreovirus, pulau virus (zhou et al., b) . the induction of isgs has also been demonstrated in bats with bat type iii ifn, resulting in the induction of isg and rig-i production in bat cell lines (zhou et al., a) . pteropid bat cell lines also produce isg and isg following stimulation with universal type i ifn that is an ifna hybrid constructed from recombinant human ifna a/d (virtue et al., a) . the induction of ¢, ¢-oligoadenylate-synthetase (oas ) has also been detected in p. vampyrus pbmcs following infection with vsv or stimulation with polyic or lps. the results of this study demonstrated a higher induction of oas by vsv compared with either polyic or lps (kepler et al., ) . these results provide evidence that the signalling molecules downstream of the ifn response are likely similar in bats to other mammals. the complement cascade kills foreign microbes by disrupting the microbial plasma membrane following activation through the binding of complement to antibodies that have attached to microbial surfaces (prodinger et al., ) . a variety of assays have been used to measure complement activity in bats to assess immunity under various environmental conditions. a comparison of complement activity in three microbats (eptesicus fuscus, m. lucifugus and t. brasiliensis) and one megabat (p. vampyrus) demonstrated higher levels of complement activity in the microbats by immune haemolysis but not by immune adherence. furthermore, complement activity in eptesicus microbats was relatively insensitive to changes in temperature above or below °c, whereas activities of guinea pig and pteropid bat complements decreased at temperatures above or below °c (hatten et al., ) . the ability to maintain complement activity may be a biological necessity in hibernating animals such as microbats where body temperatures can vary extensively and over prolonged periods of time. allen et al. ( ) used complement activity as a measure of bactericidal activity in t. brasiliensis bats, demonstrating variation in activity with roosting ecology, providing evidence for the influence of environmental factors on immune function. studies of bat adaptive immunity have provided evidence for the presence of both antibody and cell-mediated immunity in bats. however, several reports have demonstrated qualitative and quantitative differences in adaptive immune responses and in the generation and maintenance of immunological memory. these findings warrant further investigation to determine the relevance of these findings to the maintenance of viruses in bats. butler et al. ( ) demonstrated that four species of bats, including one megabat (cynopterus sphinx) and three microbats (carollia perspicillata, m. lucifugus and e. fuscus) transcribe igm, ige, iga and multiple igg classes, the latter of which appears to have diversified after speciation as in other mammals. serum fractionation using normal serum from the neotropical species, artibeus lituratus and from p. giganteus has confirmed that bat igm, igg and iga are homologous to corresponding human immunoglobulins (mcmurray et al., ; chakravarty and sarkar, ) . however, evidence so far indicates that igd may be unique to microbats, with igd present at the genomic and transcriptional level in m. lucifugus but not in the megabats. igd is an apparently ancient isotype and has a spotty distribution among vertebrates (ohta and flajnik, ) . among mammals, igd was once believed to be present only in humans and rodents until its more recent identification in various classes of animals including artiodactyls and monotremes (zhao et al., (zhao et al., , . although a broader survey of megabats will be required to rule out the presence of igd in this group, given that igd is not present in all mammals, it may not be surprising to find that megabats have lost this immunoglobulin isotype. immunoglobulin genes are assembled by recombination of germline-encoded gene segments: variable (v), diversity (d) and joining (j) for heavy (h) chains and v and j for light (l) chains. variation in the amino acid residues at the n terminal ends that are encoded by the v regions of both h and l chains contribute to antibody diversity and establishes antibody specificity (max, ) . to obtain insight into the antigen-binding capability and specificity of bat antibodies, several studies have examined the diversity of the vh regions of bat immunoglobulin genes. evidence from both megabats and microbats has demonstrated a highly diverse antibody repertoire, exceeding that of most of species and on par only with humans and mice (baker et al., ; bratsch et al., ) . in the pteropid bat, p. alecto, the amino acid sequence composition of the antigen-binding site of the expressed vh region is enriched in arginine and alanine residues and has a lower proportion of tyrosines compared to other mammals (baker et al., ) . tyrosines are directly involved in antigen binding and confer structural diversity, while arginines have been reported to be detrimental to antigen binding and may contribute to self-reactivity (radic et al., ; birtalan et al., ) . whether these characteristics are associated with differences in antigen-antibody interactions in bats awaits further functional characterization. however, differences in antigen binding may help to explain previous observations of the simultaneous presence of virus and antibody in bats (sulkin et al., ) . comparison of the germline and expressed vh repertoire of m. lucifugus has revealed a very low mutation rate consistent with the possibility that this species relies on combinatorial and junctional diversity rather than somatic hypermutation . all other mammals studied thus far use post-combinatorial mechanisms to fine tune their antibody repertoire resulting in antibodies that recognize fewer epitopes per antigen but do so with greater specificity and affinity. this result may provide evidence that bats rely solely on combinatorial mechanisms. however, as this study focused only on vh sequences expressed with igg in a single m. lucifugus, further work is required to confirm this result across multiple individuals and species using all of the immunoglobulin subclasses. the effector functions mediated by antibodies include neutralization, precipitation, agglutination, opsonization, antibody-dependent cellular cytotoxicity and the activation of the classical complement pathway. neutralizing antibodies to viruses including hendra virus, ebolaviruses and sars-like cov have been detected in wild-caught bats, demonstrating that bats are capable of mounting an antibody response (halpin et al., ; lau et al., ; leroy et al., ) . some of the earliest experiments performed on bat immune systems were measuring antibody responses. early studies of antibody responses in bats were consistent with differences in both the kinetics and magnitude of antibody responses compared with other mammals. several studies have used model antigens such as sheep red blood cells (srbcs), /x bacteriphage and , -dinitrophenylated bovine serum albumin (dnp-bsa) to compare the nature of the antibody response of bats with that of conventional laboratory animals (hatten et al., (hatten et al., , chakraborty and chakravarty, ; wellehan et al., ) . hatten et al. ( ) reported that the magnitude and duration of the neutralizing antibody response of big brown bats (eptesicus fuscus fuscus) maintained at and °c to immunization with /x bacteriophage was lower than that of guinea pigs and rabbits. a delay in attaining a peak in the primary antibody response was also reported in pteropid bats immunized with srbcs (chakraborty and chakravarty, ) . secondary responses also appeared to be slower or non-existent. a more pronounced igm response was observed in e. fuscus, and the appearance of igg appeared to be slower supporting poor isotype switching (hatten et al., ) . secondary immunization with / x bacteriophage has demonstrated an anamnestic response only in bats housed at °c but not at °c (hatten et al., (hatten et al., , . however, evidence for an increase in the affinity of antibodies for /x has been reported in e. fuscus (hatten et al., ) . clearly, further work is needed to understand the nature of antibody responses in bats. however, overall these studies demonstrate differences in both primary and secondary antibody responses in bats compared to conventional laboratory mammals. experimental infections and vaccinations have also been performed in bats to provide information on the kinetics and nature of antibody responses to viruses. consistent with the results obtained from bats immunized with /x or srbc antigens, vaccination and experimental viral infections have provided evidence for quantitative and qualitative differences in antibody responses in bats compared with other mammals. in addition, results from experimental infections appear to vary between species and viral infections. big brown bats (e. fuscus) experimentally infected with je virus generally demonstrate a neutralizing antibody response within days of infection. however, these studies have failed to detect evidence of complement fixation (cf) or haemagglutination (hi) by je virus antigen (sulkin et al., ; leonard et al., ) . as cf and hi responses were demonstrated in guinea pigs and rabbits during these experiments, the failure to detect a response in bats was considered to reflect a difference in the host antibody response rather than the assay. experimental infection of neotropical bats with venezuelan equine encephalitis virus resulted in a high hi and neutralizing antibody response in artibeus fruit bats but low or undetectable response in phyllostomus discolour (seymour et al., ) . furthermore, bats exposed to prolonged periods of cold ( °c) likely to be encountered during hibernation failed to develop an antibody response to je virus despite the persistence of the virus in various tissues but developed detectible antibody within week following transfer from to °c (sulkin et al., ) . these results are consistent with the ability of bats to maintain viruses, which are likely biochemically inert for long periods of time under states of immunosuppression. the ability of antibody to provide long-lasting protection is one of the hallmarks of the adaptive immune response. vaccination of bats against rabies virus appears to confer resistance to challenge compared to unvaccinated bats that succumb to disease. however, several studies have demonstrated that vaccinated bats are capable of clearing viral infection even in the absence of detectible neutralizing antibody (seymour et al., ; sétien et al., ; aguilar-setien et al., ; turmelle et al., b) . although these studies provide evidence that bats develop protective immunity following vaccination, the failure to detect an antibody response in some bats is striking and may indicate that the nature of protective immunity in bats differs from other mammals. evidence for viral recrudescence has also been reported in a captive p. vampyrus, which displayed changes in neutralizing antibody to nipah virus, providing evidence of the maintenance of virus in bats in a manner that does not sustain an antibody response. one individual was initially seropositive, became seronegative within - months and remained seronegative for months before displaying a gradual increase in neutralizing antibody and viral excretion (sohayati et al., ) . these results demonstrate that failure to detect specific antibodies may be insufficient evidence for excluding prior exposure. however, as this event was observed in only one individual, the significance of viral recrudescence in bat populations remains to be investigated and will require long-term studies of captive individuals of known history of viral exposure. differences in the numbers of cells expressing surface immunoglobulin (sig) have also been observed in p. giganteus with a higher number of sig-positive cells in peripheral blood ( %) compared to humans and mice ( - %) (chakravarty and sarkar, ) . as no batspecific reagents existed to further characterize the nature of this population of cells, the significance of this result remains unknown. further studies to characterize the nature of b cells in bats may assist in resolving whether differences in the numbers of b cells are a general characteristic of bats and whether this plays a role in the observed differences in antibody responses of bats. cell-mediated responses are controlled by t lymphocytes, which include cytotoxic and helper functions. the different populations of t cells in bats have not been characterized to date, and only one t-cell coreceptor, cd , has been characterized (omatsu et al., ) . however, a number of reports have described in vitro responses of lymphocytes to t-cell mitogens in pteropid bats and microbats (mcmurray and thomas, ; chakravarty and paul, ; paul and chakravarty, ) . these studies have indicated that bats display evidence for delayed responses to t-cell mitogens, pha and concanavalin a (cona) with a peak at h compared to h in mice (mcmurray and thomas, ; chakravarty, , ) . a delay in mixed lymphocyte responses (mlr) have also been observed with a peak at days for p. giganteus in comparison with days in mice, thus providing further evidence that cell-mediated immunity in bats is slower than that of other mammals (chakraborty and chakravarty, ) . the presence of suppresser t cells has also been implicated in the delay in mitogenic responses of b cells in bats . whether these cells are involved in the delay in t-cell-mediated immune responses observed in bats remains to be determined. more recently, an ifnc response was demonstrated following stimulation of pteropid bat lymphocytes with the t-cell mitogens pha and cona, demonstrating that bats are capable of a similar ifnc response to other mammals (janardhana et al., ) . in vivo cell-mediated responses in bats have been measured using delayed-type hypersensitivity (dth) tests using the pha skin test or skin sensitivity to - dinitrofluorobenzene (dnfb) (christe et al., ; allen et al., ; turmelle et al., a) . delayed-type hypersensitivity of p. giganteus to dnfb resulted in a characteristic dth response within h, similar to other mammals. however, only three of twelve bats tested in this study responded to treatment with dnfb, suggesting that bats may not be sensitive to dnfb to the same extent as other mammals (chakraborty and chakravarty, ) . a time series of histological skin sections taken from skin biopsies of t. brasiliensis following pha or saline injection has also demonstrated substantial individual variation but overall has provided evidence for a strong cell-mediated immune response (turmelle et al., a) . cell-mediated immunity has also provided evidence for changes in immunocompetence because of environmental and physiological factors. differences in immunocompetence because of roost ecology were observed in t. brasiliensis using subcutaneous pha injection as a measure of in vivo t-cell-mediated immunity, providing evidence for the effect of environment on immune responsiveness (allen et al., ) . christe et al. ( ) used pha skin tests to demonstrate that greater mouse-eared bats, myotis myotis, mount weaker cell-mediated responses during pregnancy compared with that of non-reproductive and lactating females. immunocompetence was also lower in early pregnancy than at later stages of gestation (christe et al., ) . this result is consistent with changes in immunocompetence reported in other mammals that undergo a shift in the immune response towards a humoral immune response and away from cell-mediated immunity during pregnancy (szekeres-bartho, ) . changes in immune function during pregnancy have been speculated to favour replication of viruses including zaire ebolavirus in bats. high titres of virus present in birthing fluids, blood and placental tissues may then be a source of infection to terrestrial mammals including apes (leroy et al., ) . further studies into the antiviral immune response during pregnancy may provide insights into whether changes in immune function influence viral infections in bats and/or correlate with spillover events from bats to other susceptible species. although cell-mediated responses both in vivo and in vitro have provided important information on the t-cellmediated responses of bats, no reagents currently exist to identify different populations of t cells in bats. the ability to identify and sort different populations of t cells would provide valuable insight into the role of t cells in antiviral immunity in bats. the mhc plays an important role in resistance to infectious diseases, autoimmunity, transplantation and reproductive success (kumánovics et al., ) . despite the importance of the mhc, no work has been reported on the mhc class i genes of bats and only a few studies have provided information on mhc class ii polymorphism in bats (mayer and brunner, ; richman et al., ; schad et al., ) . the earliest evidence for the degree of mhc polymorphism in bats came from mlr assays. mixed lymphocyte responses test the recognition and proliferation of t cells from different individuals, and this response is highly dependent on mhc class ii polymorphism (derks and burlingham, ) . pteropus giganteus lymphocytes undergo delayed and lower levels of proliferation in mlr tests compared to their responses to mitogens such as cona (chakraborty and chakravarty, ; chakravarty and paul, ) . as the proliferation of cells in mlrs correlates with the degree of genetic difference in mhc loci between individuals, delayed and weaker mlr responses in bats may be evidence for low mhc polymorphism. only recently has genetic evidence for the degree of mhc class ii polymorphism in bats been reported. the class ii dr beta (drb) locus is the most extensively studied of the mhc loci in mammals because of its high diversity and has been the focus of all of the mhc class ii analyses performed on bats to date (mayer and brunner, ; richman et al., ; schad et al., ) . richman et al. ( ) demonstrated extreme differences in polymorphism between bat species with extensive polymorphism at the mhc class ii drb locus in myotis velifer compared to the extremely limited polymorphism in myotis vivesi. m. velifer is a geographically widespread continental species compared to m. vivesi that is a narrowly distributed and endangered island endemic species. the lower population size of m. vivesi may have relaxed selection for the maintenance of many alternative alleles in the population, thus lowering mhc polymorphism. a single drb locus has been described in the bulldog bat, noctilio albiventris displaying moderate allelic variability within the range of other mammals. in addition, males displayed a significantly higher heterozygosity rate and genetic variability compared to female bats (schad et al., ) . the single drb locus of the sac-winged bat, saccopteryx bilineata, displayed low heterozygosity and evidence for diversifying selection. substantial nucleotide sequence variation between the drb alleles of s. bilineata was consistent with a history of balancing selection, but there was no evidence for ongoing balancing selection acting to maintain alternative alleles at intermediate frequency. in addition, unexpected homozygosity for a common allele was observed in this population of s. bilineata, consistent with pathogen-driven positive selection playing a role in the evolution of mhc genes in this species (mayer and brunner, ) . drb intron sequences from three species of bats (r. aegyptiacus, c. perspicillata and phyllostomus discolour) have also been used to infer phylogenetic relationships and demonstrate the monophyly of chiroptera (kupfermann et al., ) . overall, studies of drb polymorphism in bats provide evidence for the influence of factors such as population size and pathogen pressure on the diversification of class ii genes. the degree of variation in drb polymorphism described above may be consistent with wide variation in the mhc variability in bats that may in turn influence the ability of different populations of bats to respond to infections. a number of bat cytokine genes have now been characterized including cdnas corresponding to interleukin (il)- , il- , il- , il- , il- p and tumour necrosis factor (tnf) from rousettus leschenaultii . partial cdnas for il- , il- a, tnf and granulocyte macrophage colony-stimulating factor have been cloned from seba's fruit bat (c. perspicillata) (cogswell-hawkinson et al., ) . these cytokines appear to be highly conserved with those from other mammals. kepler et al. ( ) described the in silico identification of ifnc from p. vampyrus and m. lucifugus, confirming that both bats appear to have a single ifnc locus similar to other mammals. more recently, p. alecto ifnc has been described, including the characterization of its antiviral activity against semliki forest virus and hendra virus. this study included the generation of important bat-specific reagents for the detection of ifnc, an important step for future studies of the role of ifnc and t-cell-mediated immunity during viral infections in bats (janardhana et al., ) . although only limited work has been performed on bat cytokines, these studies pave the way for examining the role of cytokines in antiviral immunity in bats. as discussed above, functional and genome sequence analyses of bats have revealed some surprises, but overall, it appears that bats share many of the immunological features of other mammals. it is evident they have similar antibody and t-cell receptor genes, cytokines and chemokines, transcription factors, cluster of differentiation (cd) markers and activation pathways found in the immune responses of other mammalian species. bats have molecules involved in cell self-defence against viruses, innate response mechanisms and adaptive responses. defining these molecules will be relatively easy; understanding how the viruses and bat reservoirs have shaped one another, and the tempo and mode of immune responses will be more challenging. such functional studies will likely result in significant insights into host-virus relationships, the implications of which will have impacts on the development of novel therapeutics for other species and the ability to predict viral spillover events. the variability in the results obtained from studies involving wild-caught bats emphasizes the need for captive colonies of bats of known age and history of infections for focused studies of bat immunology. although challenging, information from such colonies would greatly assist in the interpretation of data obtained from wild-caught individuals. the development of cell lines also plays an important role in this regard. although cell lines have now been generated from a number of tissues from the pteropid bat, p. alecto (crameri et al., ) , the development of additional cell lines from immune relevant cells will also assist in developing assays for studying various aspects of bat immune function. future studies using expression tools, such as real-time pcr arrays, can be rapidly and inexpensively deployed to study particular species of bats and their viruses, and they can be highly informative regarding the genetic responses of bats during infection. however, transcription data are limited because many genes are post-transcriptionally regulated. furthermore, post-translational modification events such as phosphorylation cannot be assessed by transcriptional analysis. thus, it will be necessary to determine which antibodies currently available are crossreactive 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poliocephalus) il- a, il- b, and il- : promising cytokines with type i interferon-like properties innate immune recognition of nucleic acids cloning and characterization of a novel feline ifn-omega artiodactyl igd: the missing link ornithorhynchus anatinus (platypus) links the evolution of immunoglobulin genes in eutherian mammals and nonmammalian tetrapods type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity key: cord- - v pjqt authors: zhao, jun; zhu, ling; xu, lei; huang, jianbo; sun, xiangang; xu, zhiwen title: porcine interferon lambda (ifn-λ ) shows potent anti-prrsv activity in primary porcine alveolar macrophages (pams) date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: v pjqt background: porcine reproductive and respiratory syndrome virus (prrsv) is a serious viral disease of swine. at present, there are vaccines for the control of prrsv infection, but the effect is not satisfactory. the recombination of attenuated vaccines causes significant difficulties with the prevention and control of prrsv. type iii interferons (ifns), also called ifn-λs, were newly identified and showed potent antiviral activity within the mucosal surface and immune organs. results: therefore, primary porcine alveolar macrophages (pams) were used for this investigation. to this end, we found that the replication of prrsv in pams was significantly reduced after pre-treatment with ifn-λ , and such inhibition was dose- and time-dependent. the plaque formation of prrsv abrogated entirely, and virus yields were reduced by four orders of magnitude when the primary pams were treated with ifn-λ at ng/ml. in addition, ifn-λ in our study was able to induce the expression of interferon-stimulated genes (isg ), ′- ′-oligoadenylate synthase (oas ), ifn-inducible transmembrane (ifitm ), and myxoma resistance protein (mx ) in primary pams. conclusions: ifn-λ had antiviral activity against prrsv and can stimulate the expression of pivotal interferon-stimulated genes (isgs), i.e., isg , mx , oas , and ifitm . so, ifn-λ may serve as a useful antiviral agent. supplementary information: the online version contains supplementary material available at . /s - - - . type i interferons (ifn-α/β) and type iii ifns (ifn-λs), as the first line of defence in innate immunity, play a crucial role in the body's resistance to exogenous pathogens [ ] . type iii ifns, also called ifn-λs, were first described in [ , ] , consist of ifn-λ , ifn-λ , ifn-λ and ifn-λ in humans [ , ] , ifn-λ and ifn-λ in mice [ , ] , ifn-λ and ifn-λ in swine [ ] [ ] [ ] . both ifn-α/β and ifn-λs bind to unique receptors and induce the previous signalling pathway and expression of the ifn-stimulated genes (isgs) to mediate antiviral activity. type i ifn interacts with a receptor formed by interferon alpha/beta receptor (ifnar ) and interferon alpha/beta receptor (ifnar ). type iii ifns bind to the specific receptor chain ifn-λr and il- r [ ] . type i ifn receptor is ubiquitously expressed in various types of cells and organs; however, ifn-λr is widely expressed on epithelial cells, dendritic cells, human peripheral blood monocytes or macrophages [ ] , which means that ifn-λs may provide a focused antiviral response against mucosal and immune organ infections. interferon bind to its receptors on the cell surface and induce the production of a large number of isgs, including isg , myxoma resistance protein (mx) family, ′- ′-oligoadenylate synthase (oas) family and the ifninducible transmembrane (ifitm) family, through jak-stat signal transcription [ ] . isg is one of the most highly induced isgs, isg can inhibit viral translation, replication, or egress [ ] . mx is a broadly inhibitor and inhibits a wide range of viruses by blocking the endocytic traffic of incoming virus particles and the uncoating of ribonucleocapsids [ ] . oas can recognise the dsrna produced by the virus in the infected cells and play an antiviral role by activating the ribonuclease l (rnase l) to degrade the diseased mrna [ ] . the ifn-inducible transmembrane (ifitm) family has a role in blocking virus entry [ ] . ifitm has high potency against influenza a virus and severe acute respiratory syndrome (sars) coronavirus [ ] . prrsv is a member of the family arteriviridae in the order nidovirales, which causes severe reproductive failure in sows and respiratory distress in piglets and growing pigs [ ] . also, prrsv is an immunosuppressive virus that can infect the lymphatic system of the whole body and produce viraemia after infection [ ] . prrsv mainly infects and destroys porcine alveolar macrophages and leads to severe immunosuppression, which promotes the infection of mycoplasma pneumoniae, streptococcus, a. pleuropneumoniae, and other pathogens [ , ] . the primary pams derived from piglet alveoli is an appropriate model for studying the interaction of prrsv immune responses and host-pathogen in vitro. an in vivo antiviral test of type iii interferons from pigs has not been reported. in this study, the antiviral activity of porcine ifn-λ against prrsv in primary pams was evaluated, and the expressions of isgs genes induced by ifn-λ was also investigated in primary pams. previous study has confirmed that porcine ifn-λ possess the high specific activity against porcine epidemic diarrhoea virus (pedv), classical swine fever virus (csfv), hepatitis e virus (hev) and so on [ , , ] . in this study, we verified the antiviral effect of ifn-λ against prrsv in vitro on pams. as shown in fig. , treatment of primary pams with ifn-λ could reduce the multiplication of prrsv. the degree of cytopathic effect (cpe) decreased with the increase in ifn-λ concentration ( fig. a-e) . the number and size of viral plaques also decreased with the increase in ifn-λ concentration ( fig. f-j) . the virus titre was significantly reduced with the increase of ifn-λ treatment dose ( , fig. the cpe of primary pams treated with porcine ifn-λ and infected with prrsv. the primary pams were untreated or pre-treated with ifn-λ ( , , ng/ml). b the primary pams not treated. b the primary pams treated with ng/ml ifn-λ . c the primary pams treated with ng/ml ifn-λ . d the primary pams treated with ng/ml ifn-λ . e control primary pams. k the primary pams were treated or untreated with ng/ml of ifn-λ for h and then were infected with prrsv nj strain at . moi. infected cells were cultured for , , or h after infection. f, g, h, i, j corresponds to a, b, c, d, e with the same treatment. magnifications, × , ng/ml), and the maximum treatment dose could reduce the virus titre by four orders of magnitude compared with the control group ( fig. k, the raw data are shown in supplementary table s ). these results indicate that the ifn-λ could significantly inhibit the replication of prrsv in a dose-dependent manner in primary pams. to investigate the time-dependent manner of the ifn-λ inhibits the replication of prrsv, we used ifn-λ of ng/ml concentration to treat pams cells and infected with the prrsv. cell cultures were collected at specific time and determine the virus titre. as shown in fig. (the raw data are shown in supplementary file , table s ), the inhibition of ifn-λ on prrsv decreased with time in primary pams, but the inhibition still existes. prrsv proliferation slowed down within h to h in primary pams that were treated with ifn-λ . the above results showed that ifn-λ could maintain a potent anti-prrsv activity in the later stage and significantly inhibit the replication of prrsv in a timedependent manner in primary pams. isg , mx , oas and ifitm have well-known antiviral properties and may affect prrsv replication. therefore, we accessed the quantification of isgs induce by ifn-λ . as seen in fig. a to d, a dose-dependent induction of isg , mx , oas and ifitm has been observed in primary pams treated with ifn-λ . the expression of mrna for isg , mx , oas , and ifit m was up-regulated by , , , and times respectively at the concentration of ng/ml in primary pams. as shown in fig. e and f (the full-length blots are presented in supplementary file ), a dosedependent induction of the antiviral proteins isg , mx and oas has been observed in primary pams treated with ifn-λ . the expression concentration of three antiviral proteins increased with the increasing ifn-λ concentration. both isg and mx showed low expression in the untreated condition, and ifn-λ induced a large amount of expression. the expression of isg , mx and oas tended to be stable when the concentration of ifn-λ was higher than ng/ml (fig. f ). the results in our research confirm that the porcine ifn-λ shows potent anti-prrsv activity in primary pams. pams are the first line of defence against pathogenic microbe infections in the lung. prrsv replicates in monocytic lineage cell types, particularly in pams, and causes immunosuppression in swine [ ] . therefore, we selected the primary pams to carry out the ifn-λ in vitro anti-prrsv study. alveolar macrophages are resident phagocytes of the alveolar space [ ] . the expression of the ifn-λ receptor in alveolar macrophages has been confirmed and reported [ ] . macrophages express il- rβ and il- rα at both the mrna and protein levels [ ] . ifn-λ has the strongest antiviral function in ifn-λs [ ] . in our study, the prrsv proliferation reduced when primary pams were treated with ifn-λ ( ng/ml) (fig. i) . ifn-λ treatment could significantly reduce the virus titre of prrsv proliferation on pams, and the virus titre of the ng/ml treatment group was four orders of magnitude lower than that of the control group (fig. b) . consistent with these results, treatment with , or [ ] . the study of two other kinds of viruses targeted porcine intestinal epithelial, pedv and csfv, confirming that ifn-λ inhibits their infection in vitro [ , ] . all of these imply that porcine ifn-λ can inhibit the proliferation of porcine viruses such as csfv, pedv and prrsv. the antiviral activities of ifn-λ are due to isg induction and ifn-λ can induce the expression of isg. ifn-λ exerts its anti-hiv function by activating jak-stat pathway-mediated innate immunity in macrophages [ ] . ifn-λ can bind to cell surface receptors and induce the high expression of interferon-stimulating genes of the mx, oas and ifitm families [ , , ] . the gene transcription profile induced by ifn-λ , particularly the gene transcription profile induced by ifn-λ in primary pams has not been reported. in our study, we assessed whether the antiviral efficacy of ifn-λ was caused by the levels of isg expression induced by ifn-λ . consistent with the expecting result, the expression of isg , oas , mx , and ifitm was provoked in primary pam cells. the mrna transcription and protein translation of the isg , oas and mx showed dose-dependence. however, the rangeability of mrna and protein expression levels of isg , oas and mx were different. the expression levels of protein reached its peak when treated with ng/ml ifn-λ while the expression of mrna continuous increased (fig. ) . in summary, our data demonstrated that ifn-λ could inhibit the replication of prrsv in primary pams, and such inhibition is dose-and time-dependent. alveolar macrophages are one of the earliest immune defence cells in the lungs that contact pathogenic microorganisms. they are essential components of the innate and specific immunity of the host [ ] . pams are an essential host cell for prrsv natural infection. ifn-λ can stimulate the expression of pivotal isgs, i.e. isg , mx , oas , and ifitm . this study indicated that porcine ifn-λ might serve as a promising therapeutic agent against prrsv and other viruses in swine in the future. supplementary file (a to d) . the grey value of protein bands was measured by image j (f). data were presented as mean ± sem (n = ). *p < . ; **p < . ; ***p < . ; ****p < . by unpaired t-test to determine the anti-prrsv activity of ifn-λ in the primary pams, the e. coli-derived ifn-λ was prepared in our laboratory. to explore the dose-dependent of ifn-λ antiviral, primary pams were untreated or pretreated with ifn-λ ( , , ng/ml) for h. then, the cells were infected with prrsv nj strain at . moi for - h, washed and replenished with fresh medium containing the indicated ifn-λ . infected cells were cultured for h after infection. to explore the time-dependent effect of ifn-λ antiviral, primary pams were pre-treated with ng/ml ifn-λ for h. then, the cells were infected with prrsv nj strain at . moi for - h, washed and replenished with fresh medium containing the indicated ifn-λ . infected cells were cultured for , , , h after infection. all of the cells were submitted to two freeze-thaw cycles and titrated by % tissue culture infective dose (tcid ) in marc- cells. the cytopathic effect (cpe) units in culture plates were counted, and the viral titre analysis made use of the reed-muench method. to examine the level of isg expression in primary pams following ifn-λ stimulation, the cells were stimulated with the indicated concentrations ( , , ng/ml) of ifn-λ in -well plates for h. cells were then lysed, total rna was extracted for subsequent qpcr analysis and total protein was extracted for western blot analysis. every treatment group in this study had three duplicate samples (n = ). total rna was extracted from the cellular supernatant or cell lysates using the ez- spin column total rna isolation kit (sangon biotech (shanghai) co., ltd., china) according to the manufacturer's instructions and the rna concentration was measured using a nucleic acid concentration analyzer (scandrop , analytik jena, germany). reverse transcription was performed using the prime script™ ii st strand cdna synthesis kit (takara), and qpcr was performed in a light cycler (roche, switzerland) with tb green® premix ex taq™ ii (tli rnaseh plus) (takara). the thermal cycling conditions were °c for s, followed by cycles of °c for s, and °c for s. all acquired data were obtained using light cycler real-time pcr machines (roche) and analysed with light cycler software . based on the cycle threshold (ΔΔct) method. primers were designed using oligo . software and are shown in table . total protein was extracted from the cell lysates using the western and ip cell lysis buffer (sangon biotech (shanghai) co., ltd., china) according to the manufacturer's instructions and protein concentration was determined using the bca protein assay kit (sangon biotech (shanghai) co., ltd., china). after gel electrophoresis, the proteins were transferred to nitrocellulose membranes (bio-rad, usa), and blocked in % skim milk at °c overnight. after washing with pbst ( . % tween- in pbs), the membrane was incubated with primary antibodies for h at °c. after washing, the membrane was incubated with horseradish peroxidase (hrp)-conjugated igg antibody (abcam, no: ab ) for h at °c. the protein bands were detected using supersignal™ west pico plus chemiluminescent substrate (thermo scientific, usa) and chemiluminescence imaging system (bio-rad, chemidoc mp, california, usa). the primary antibodies of isg (no: ab ), oas (no: ab ), mx (no: ab ) and β-actin (no: ab ) was purchased from abcam. statistical analysis was performed and histogram were drawn using graphpad prism™ . (graphpad software, usa), paired student t-test, and one-way anova was used to test differences between different groups. p values< . were considered significant. the gray intensity of protein bolts was analyzed by image j (national institutes of health, usa). the layouts and cropping of the pictures were completed by adobe illustrator cs (adobe systems incorporated, california, usa). the online version contains supplementary material available at https://doi. org/ . /s - - - . additional file : table s . the viral titer at h after the pams stimulated with different dose of ifn-λ . table s . the viral titer at , , or h after the pams stimulated with ifn-λ ( ng/ml). abbreviations ifn-λ : interferon lambda ; pams: porcine alveolar macrophages prrsv: porcine reproductive and respiratory syndrome virus; ifns: interferons isg : interferon-stimulated genes ; oas : ′- ′-oligoadenylate synthase ifitm : ifn-inducible transmembrane ; mx : myxoma resistance protein isgs: interferon-stimulated genes; ifn-α: interferon alpha; ifn-β: interferon beta; ifn-λs: interferon lambdas; ifn-λ : interferon lambda ; ifn-λ : interferon lambda ; ifn-λ : interferon lambda ; ifnar : interferon alpha/beta receptor ; ifnar : interferon alpha/beta receptor ; ifn-λr : interferon lambda receptor ; il- r : interleukin receptor ; mrna: messenger rna pedv: porcine epidemic diarrhoea virus; csfv: classical swine fever virus hev: hepatitis e virus; cpe: cytopathic effect; il- rβ: interleukin receptor beta; il- rα: interleukin receptor alpha; hiv: human immunodeficiency virus african green monkey embryonic kidney epithelial cells dmem: dulbecco's modified eagle's medium; fbs: foetal bovine serum pcv : porcine circovirus type ; prv: pseudorabies virus; moi: multiplicity of infection; tcid : % tissue culture infective dose; qpcr: real-time polymerase chain reaction; pbst: . % tween- in pbs; hrp: horseradish peroxidase interferon-inducible antiviral effectors il- , il- and their class ii cytokine receptor il- r ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex ifn-λ : the paradoxical new member of the interferon lambda family interferon-lambda: a new addition to an old family murine interferon lambdas (type iii interferons) exhibit potent antiviral activity in vivo in a poxvirus infection model characterisation of the mouse ifn-lambda ligand-receptor system: ifnlambdas exhibit antitumor activity against b melanoma molecular cloning, expression and antiviral activity of porcine interleukin- (poil- ) antiviral activity of porcine ifn-λ against porcine epidemic diarrhea virus in vitro molecular characterisation and antiviral analyses of porcine type iii interferons contribution of type iii interferons to antiviral immunity: location, location, location interferon-stimulated genes: a complex web of host defences interferon-induced isg pathway: an ongoing virus-host battle structural basis of oligomerisation in the stalk region of dynamin-like mxa viral encounters with ′, ′-oligoadenylate synthetase and rnase l during the interferon antiviral response a diverse range of gene products are effectors of the type i interferon antiviral response distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus porcine reproductive and respiratory syndrome emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the mid-eastern region of china effects of porcine reproductive and respiratory syndrome virus (isolate tw ) on porcine alveolar macrophages in vitro alveolar macrophages: plasticity in a tissue-specific context emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark ifn-lambda mediates antiviral protection against porcine epidemic diarrhoea virus by inducing a distinct antiviral transcript profile in porcine intestinal epithelia immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and haemophilus parasuis in vitro lambda interferon inhibits human immunodeficiency virus type infection of macrophages comparison of antiviral activity of lambda-interferons against hiv replication in macrophages lambda interferon inhibits hepatitis b and c virus replication short communication: antiviral activity of porcine ifn-k against porcine epidemic diarrhoea virus in vitro ifn-λ inhibits hiv infection of macrophages through the jak-stat pathway antiviral activity of type i and type iii interferons against porcine reproductive and respiratory syndrome virus (prrsv) ifnlambda preferably inhibits pedv infection of porcine intestinal epithelial cells compared with ifn-alpha alveolar macrophages: plasticity in a tissue-specific context quantitative nitric oxide production by rat, bovine and porcine macrophages publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank prof. zhu for her insightful comments on the design of the study. all data generated during this study are included in supplementary file (table s , s ). however, the raw data is available from the corresponding author upon reasonable request. the sichuan provincial laboratory animal management committee (licence no: syxk (chuan) - ) approval has been received. the "guidelines for experimental animals" of the ministry of science and technology (beijing, china) were followed. not applicable. the authors declare that they have no competing interest.received: november accepted: october key: cord- -unrd bo authors: danesh, ali; cameron, cheryl m.; león, alberto j.; ran, longsi; xu, luoling; fang, yuan; kelvin, alyson a.; rowe, thomas; chen, honglin; guan, yi; jonsson, colleen b.; cameron, mark j.; kelvin, david j. title: early gene expression events in ferrets in response to sars coronavirus infection versus direct interferon-alpha b stimulation date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: unrd bo type i interferons (ifns) are essential to the clearance of viral diseases, however, a clear distinction between genes upregulated by direct virus–cell interactions and genes upregulated by secondary ifn production has not been made. here, we investigated differential gene regulation in ferrets upon subcutaneous administration of ifn-α b and during sars-cov infection. in vivo experiments revealed that ifn-α b causes stat phosphorylation and upregulation of abundant ifn response genes (irgs), chemokine receptors, and other genes that participate in phagocytosis and leukocyte transendothelial migration. during infection with sars-cov not only a variety of irgs were upregulated, but also a significantly broader range of genes involved in cell migration and inflammation. this work allowed dissection of several molecular signatures present during sars-cov which are part of a robust ifn antiviral response. these signatures can be useful markers to evaluate the status of ifn responses during a viral infection and specific features of different viruses. viral respiratory infections are a major worldwide cause of morbidity and mortality (kolling et al., ; thompson et al., ) . emerging viral threats, such as the severe acute respiratory syndrome coronavirus (sars-cov), avian influenza h n and pandemic influenza h n virus are well poised to cause epidemics or pandemics that could be socially and economically disastrous (dushoff et al., ; weiss and mcmichael, ; dawood et al., ) . for decades, ferrets have been used for the investigation of influenza infection since they are susceptible to influenza viruses (hull and loosli, ) . more recently, ferrets have also been shown to be a good model of human sars-cov infection (martina et al., ) . we have previously characterized ferret cytokine and chemokine genes as well as have developed immunological assays for evaluating the ferret immune system following sars and influenza infection danesh et al., ; ochi et al., ) . ligation of the interferon (ifn) alpha receptors and (ifnar and ifnar ) with ifn-α induces ifn signaling pathways and promotes ifn gene induction. formation of the signal transducer and activator of transcriptions and (stat -stat ) heterodimer occurs following the phosphorylation of janus kinase (jak ) and tyrosine kinase (tyk ) that are associated with ifnar and ifnar , respectively (marijanovic et al., ) . these two kinases phosphorylate stat and stat , which together form a complex with interferon regulatory factor (irf ) (takaoka and yanai, ) . the interferon stimulatory factor complex (isgf ) binds to interferon-stimulated response element (isre) and activates transcription of ifn-α inducible genes, including '- ' oligoadenylate synthase (oas ), myxovirus resistance (mx ), interferon stimulated gene (isg ) and many other ifn-response genes (irgs) (uddin and platanias, ) . ifn-α stimulation ultimately promotes a cellular antiviral state which is hallmarked by the upregulation of irgs (chevaliez and pawlotsky, ) . although ifn signaling gene upregulation during viral infection has been the subject of previous reports, there is little information regarding the host immune responses directly induced by viruses versus those that are upregulated due to secondary ifn stimulation (chelbi-alix and wietzerbin, ; haagmans et al., ; loutfy et al., ; cameron et al., ) . here we used our previously described ferret model (chu et al., ) to identify genes that were regulated by sars-cov infection compared to ifn-α b stimulation in the ferret model to elucidate immune responses during viral infection. we examined the phosphorylation status of signaling molecules in ifn-α b-stimulated peripheral blood mononuclear cells (pbmcs) . we also analyzed the in vivo gene expression profiles of ferret pbmcs and lung necropsies following ifn-α b injection during the time course. evaluation of gene expression patterns in pbmcs and lung necropsies of sars-cov-infected ferrets led us to the identification of upregulated irgs that also were upregulated in response to ifn-α b injection. our findings in ferrets suggested ifn-α b injection and sars-cov infection led to similar as well as unique gene expression signatures in a global point of view. increased knowledge of the interaction of these gene expression signatures may improve our understanding of the immune system of ferrets as a preferred animal model of severe respiratory viral illnesses. we first investigated the phosphorylation of the ifn-α receptor downstream signaling molecule stat to determine the signaling potential of ifn-α b in ferrets. the phosphorylation status of stat was evaluated using phosphorylated amino acid specific monoclonal antibody for flow cytometry analysis that cross-reacted with the phosphorylated ferret protein. pbmcs demonstrated a significant stat phosphorylation response min post-stimulation with ifn-α b in vitro compared to the control stimulated with pbs alone ( supplementary fig. ). since stat phosphorylation was observed in vitro, we then determined whether ifn-α b could activate stat in vivo. four ferrets were subcutaneously injected with μg/kg ifn-α b and peripheral blood samples were taken at , and h post-stimulation for flow cytometry examination. by h, samples extracted from all ferrets demonstrated significant stat phosphorylation in the pbmcs. control ferrets injected with pbs did not demonstrate marked stat phosphorylation at any time point. the stat average mean fluorescent intensity (mfi) of the ifn-α b-injected group was significantly increased compared to the average of its control group (p b . ) (fig. a) . these results indicated that stat was also inducible by ifn-α b in vivo. ifn signaling is critical to successful antiviral responses during infection (haller et al., ) . therefore, we next investigated the phosphorylation status of stat following sars-cov infection. we infected ferrets with sars-cov or pbs control and measured the phosphorylation of stat by flow cytometry. three ferrets infected with sars-cov demonstrated significant stat phosphorylation in pbmcs post-infection with a maximum peak at day (p b . ). control ferrets mock-infected with pbs did not demonstrate significant stat phosphorylation at any time point (fig. b) . since stat was phosphorylated following sars-cov infection and ifn-α b injection, we investigated select irg expression by qrt-pcr following in vitro stimulation of ferret peripheral whole blood with ifn-α b. in vitro stimulation with ifn-α b led to significant upregulation of stat and several irgs such as mx , oas , isg , isg , irf and interferon-induced protein (ifi ). as expected, activation of ifn-α receptor signaling therefore initiated transcriptional activation of interferon response genes ( supplementary fig. ). we then assessed genome-wide gene expression following in vivo ifn-α b administration in ferrets. ferrets were subcutaneously injected with pbs (control) or ifn-α b and blood samples were drawn for rna isolation and days after injection. without a commercially available ferret microarray, the rna was then used for microarray analysis on the affymetrix genechip canine genome . array (see materials and methods), because ferret genes show a high degree of homology with canine genes as we have previously established rowe et al., ; fang et al., table ). the peripheral blood gene expression data from ifn-α b-injected group were normalized to the control group. the t-test analyses showed the highest number of significant changes occurred at day , with a total of upregulated and downregulated genes in peripheral blood of the ifn-α b-injected ferrets (table ) . a threshold of at least . fold-change and a p value for the t-tests of less than . were chosen. the peak upregulation of a cluster of irgs, including mx , oas , oas , isg , ifi and ubiquitin specific protein (usp ), occurred at day , while peak upregulation of irgs such as jak , jak , protein inhibitor of activated stat (pias ) irf , interferon-γ receptor (ifngr ), and eukaryotic translation initiation factor -α kinase (eif ak ) occurred at day post-injection ( fig. a and supplementary table ) . after assessing the large scale gene expression profile following ferret in vivo ifn-α b stimulation, we validated the expression of selected irgs by qrt-pcr according to the availability of the ferret specific primers. we found stat and irgs such as mx , oas and isg were significantly upregulated in ferrets injected with ifn-α b compared to the controls (fig. b ). the lack of ferret sequences for other irgs prevented us from confirming the upregulation of these genes. to determine if stat phosphorylation was correlated with irg activation in our ferret animal model of sars-cov infection (chu et al., ) , we went on to analyze host gene expression following sars-cov infection. the gene expression data at day post-infection with sars-cov was normalized to the mock control dataset. unfortunately, blood samples from day post-infection did not meet minimal rna quality for microarray analysis, preventing us from performing a timecourse study on the peripheral blood. there were upregulated and downregulated genes (p b . , n . fold change) as ascertained by t-test analysis at day post-infection (table ) . irgs, including stat , mx , oas , oas , isg , ifi , suppressor of cytokine signaling (socs ), radical s-adenosyl methionine domain containing (rsad ), usp and oas ligand (oasl) were significantly upregulated ( fig. c and supplementary table ). the upregulation of stat , mx , oas and isg were validated with qrt-pcr (fig. d ). these gene expression and stat phosphorylation findings suggested that robust ifn responses were activated following sars-cov infection days post-infection. interferon canonical pathway analysis confirmed the similarities between the expression patterns of irgs at day . stat , mx , oas , usp , rsad , isg and ifi were upregulated in the peripheral blood of ifn-α b-injected and sars-cov-infected ferrets. in contrast, oasl, oas and socs were upregulated during sars-cov infection alone (fig. a) . since sars-cov infection causes severe lung pathology we went on to compare and contrast the genes upregulated by ifn-α b stimulation and sars-cov infection in the lungs of ferrets. microarray analysis was performed on lung necropsies of ifn-α b-injected ferrets compared to controls. the peak gene expression occurred at day with a total of upregulated and downregulated (p b . ) genes (table ) . interestingly, the strongest upregulation of several irgs, such as stat , mx , oas , oas , isg , ifi , ifi ligand (ifi l) and eif ak , occurred on day ( fig. a and supplementary table ). there was a marked increase in the total number of regulated genes from lung necropsies of sars-covinfected ferrets compared to lungs from ifn-α b-stimulated ferrets ( table ). the sars-cov infected ferrets had a peak in gene expression at day with upregulated versus downregulated genes (p b . ). both the number of upregulated irgs and the expression levels peaked at day , including stat , mx , oas , oas , isg , irf , interferon-induced protein with tetratricopeptide repeats ( ifit ), ifi , ifi , ifi l, proteasome subunit multifunctional beta (psmb ), eif ak and ifnrg . jak was the only irg that was downregulated at day (fig. c ). the upregulation of stat , mx , oas and isg was validated with qrt-pcr on lung necropsies of ferrets injected with ifn-α b or infected with sars-cov (fig. d ). the comparison of microarray results between the lung tissue of ifn-α b and sars-cov ferrets at day revealed commonalities in the expression patterns of most irgs. stat , mx , oas , oas , isg , ifi , ifi l and eif ak were among the overlapping genes (fig. b ). to further model the pathways involved in the host response to sars-cov and the direct effects of ifn-α b administration, functional analysis of the regulated genes was performed using ingenuity pathway analysis software. for each experimental group, genes showing changes in their expression levels were mapped into highlevel gene ontology categories: cellular process, metabolic process, intracellular signaling cascade, cell cycle and immune response ( table ). the number of genes present in each functional category is representative of the level of biological activity in each experimental group with respect to the controls. analysis of the ifn signaling canonical pathway showed the upregulation of stat , mx , oas , oas , isg and ifi in lung necropsies of ifn-α b injected and sars-cov infected ferrets (fig. ) . functional classification of upregulated genes showed that ifn-α b induces increased expression of phagocytosis-related genes, such as fc fragment of igg, high affinity ia, receptor cd (fcgr a) and dynamin -like (dnm l), leukocyte transendothelial migration genes, such as integrins beta and (itgb and itgb ), and upregulation of chemokine receptors, chemokine c-c motif receptors , and (ccr , ccr , ccr ) and chemokine c-x-c motif receptor (cxcr ) (supplementary table ). these results suggest that ifn-α b is able to activate specific functions of the leukocyte responses in blood samples after exposure. the lungs of ferrets infected with sars-cov showed broader immune responses than ifn-α b-injected ferrets, as demonstrated by the higher number of regulated genes in several functional categories related to the activation of the immune responses, including: complement and coagulation, cell adhesion molecules and leukocyte activation (fig. ). type i ifns are a critical component of the innate immune response during viral infections. the function of many downstream genes has been studied in-depth, however, it is likely that the presence of the virus and subsequent tlr-mediated signaling are required to deploy full irg-mediated antiviral activity (bosinger et al., ) . in this study we investigated the gene signatures induced following subcutaneous administration of ifn-α b in ferrets. we also analyzed the signaling pathways during an infection with sars-cov, and by means of comparative analysis we profiled ifn gene responses in the context of a respiratory infection. we used an experimental model of infection with sars-cov in ferrets, which causes mild symptoms without mortality. the pathological features of this model were previously published (chu et al., ) and a summary of the clinical information can be found in the supplementary information (supplementary table ). we assessed the capacity of subcutaneous administration of ifn-α b to activate antiviral responses in ferrets. the activation levels of several intracellular signaling proteins were studied by using phospho-specific antibodies and subsequent facs analysis. stat plays a key role downstream of ifn signaling while stat and stat are thought to be involved at a lesser extent, and/or weak participation of stat , mitogen activated protein kinase (p ) and extracellular signal-regulated kinase (erk) (li et al., ) . in vitro incubation of ferret pbmcs with ifn-α b led to strong phosphorylation of stat , weak phosphorylation of stat and stat and no phosphorylation of stat , p and erk. furthermore, the activation of the stat signaling pathway in vitro was confirmed at the mrna level with the presence of many downstream irgs, including mx , oas , oas , isg , and ifi . the in vivo effects of ifn-α b were also investigated. stat showed increased phosphorylation levels in the peripheral blood at early hours post-injection, while stat and stat remained unchanged. moreover, we did not observe mrna gene expression of interleukin (il- ) and suppressor of cytokine signaling (socs ) at the mrna level (data not shown), suggesting that stat (gharavi et al., ) and stat (barclay et al., ) , respectively, do not participate in vivo in response to ifn-α b. the global numbers of regulated genes found in the microarray results constitute good estimators of the intensity of the host response at different time-points. in vivo effects of ifn-α b can be observed h after the injection and their peak is reached h post-injection. irgs are markedly increased in both blood and lung tissue, however the responses in the blood show greater breadth and magnitude as compared with the responses observed in lung tissue (supplementary table ). this suggests that the administration protocol of ifn-α b used in this study is only capable of inducing a limited activation in lung tissue. therefore, alternative protocols including direct administration of ifn-α b into the respiratory tract or subcutaneous administration at higher doses should be explored in order to achieve stronger antiviral responses at the infection sites. gene expression during sars-cov infection, on the other hand, shows the presence of strong antiviral and inflammatory responses in the lungs h postinfection, fading on day post-infection in both blood and lung tissue. as expected, ifn-α b stimulates the increased expression of a variety of irgs that play a central role in the clearance of viral infections, including mx , oas , oas and isg . they exert their effects through different mechanisms of action, such as direct targeting of viral entry, inhibition of protein synthesis or degradation of viral rna. mx is a dynamin-like large guanosine triphosphatase (gtpase), which has antiviral activity against a wide range of rna viruses. the antiviral activity of mx is effective at the early stages of the viral cycle in the nucleus or cytoplasm (haller et al., ) . oas is an adenylate synthetase, which uses adenosine triphosphate to synthesize ', '-oligoadenylates. the latter activates latent rnase l that is involved in the degradation of viral rna (bonnevie-nielsen et al., ) . isg is a ubiquitin-like enzyme that covalently conjugates to a large number of cellular proteins; however this does not usually lead to protein degradation. in the case of hiv- , isg inhibits the release of virions (okumura et al., ) . upregulation of similar sets of irgs by sars-cov and ifn-α b were observed, including irgs (stat , isg , mx , oas , oas , ifi and ifi l) in the peripheral blood and lung tissue of both groups. in contrast, several irgs, including ifi , ifit and psmb , were only upregulated in the lungs during sars-cov infection. these results suggest that the expression of certain irgs lie beyond the direct control of ifn-α b, and additional signals such as activation of tlr-signaling by viral components are probably required to assemble a fully functional antiviral response. although the induction of irgs by ifn-α b is the hallmark feature of ifn responses, a full understanding of the biological effects of antiviral ifns requires a comprehensive study of the additional functional responses triggered by ifn-α b. in the blood of ferrets injected with ifn-α b, the upregulation of genes that participate in glycolysis-gluconeogenesis (e.g. acyl-coa synthetases and lactate dehydrogenases) (supplementary table ) are indicators of higher levels of metabolic activity. moreover, ifn also induces the expression of genes related with apoptosis (e.g. caspases and tnfsf ) and cell cycle (e.g. cyclins and smad family members). it is unclear whether ifn-α b alone is capable of inducing apoptosis and/or cell replication in vivo, however, the upregulation of these genes may indicate that pbmcs are now more responsive to signals capable of triggering cell cycle events. upregulation of chemokine receptors, such as ccr , ccr , ccr and cxcr may indicate that ifn-α b can increase the responsiveness of pbmcs to locally produced chemokines. likewise, increased levels of genes that are involved in leukocyte transendothelial migration and fc-gamma receptor-mediated phagocytosis (supplementary table ) suggest that ifn-α b enhances leukocyte responses (corssmit et al., ) . interestingly, a number of genes that are part to the wnt signaling pathway were found to be upregulated (supplementary table ). this indicates that in vivo administration of ifn-α b also has effects over lymphocyte maturation and differentiation (staal et al., ) . the lungs of ferrets infected with sars-cov show the upregulation of a broader variety of genes, as compared with ifn-α b administration, and depicts a more complex biological environment dominated by the antiviral responses, leukocyte infiltration and other inflammatory responses (fig. b) . a number of chemokine ligands, such as chemokine c-c motif ligand (ccl ), ccl , ccl , ccl and ccl , and cell adhesion molecules, such as activated leukocyte cell adhesion molecule (alcam) and intercellular adhesion molecule (icam ) are upregulated during sars-cov infection, but these were not induced by the administration of ifn-α b ( fig. a and supplementary table ) . sars-cov also induced the upregulation of genes of the complement system such as complement component (c ) and complement factor b (cfb) (fig. b) . taken together, these results depict how irgs and other arms of the innate immune responses are capable of resolving the respiratory infection caused by sars-cov infection. previously, gene regulation has been investigated using microarray analysis with the intent on revealing molecular pathways imperative to h n infection (shapira et al., ). here we investigated gene regulation of sars-cov infected and ifn-α b injected ferrets. microarray analysis was conducted on rna from lungs and blood on day and day . the number of upregulated genes was quantified and compared to the number of downregulated genes for each sample type. the number of downregulated genes was greater than upregulated genes in the day ifn-α b lungs and in the day sars-cov infected blood samples. to expose the molecular signature of this finding we then broke down the genes from each group into their respective functional pathways: cellular process, metabolic process, intracellular signaling cascade, cell cycle, and immune response. interestingly, we found that for every functional pathway the day ifn-α b-injected lungs had more downregulated genes than upregulated genes (except the immune response) where blood samples from the same animals had the opposite trend of more upregulated genes. furthermore, the sars-cov-infected animals had the opposite trend where day lungs had more upregulated genes and in the blood of day there were more downregulated genes. these findings may be indicative of the activity of the stimulant ifn-α b compared to sars-cov. moreover, the difference in the number of genes regulated shows that ifn-α b and sars-cov have different spatial stimulation which may be an important finding when determining the therapeutic efficacy of ifn-α b. it is possible that the increase in gene expression in the blood samples following ifn-α b injection is indicative of activation of systemic immunity where the sars-cov infection had an increase of lung gene expression signifying possible local inflammation. type i ifns play a critical role during antiviral responses, however their functions in vivo have not yet been fully resolved. additional research is required to define the optimal irgs profile that is present during successfully cleared viral infections. moreover, fine tuning of the irgs responses may achieve more prolonged and wider protection by therapeutic agents such as attenuated vaccines against respiratory viruses. male, kg, -month-old ferrets (mustela putorius furo) were purchased from marshall farms inc. (oak park, il) and housed at the toronto general research institute animal facility (toronto, canada) or at southern research institute (birmingham, al, usa). ferrets were quarantined and monitored days prior to tissue and blood collection. the ferrets' diet was based on a low fat/high protein regimen as recommended by marshal farms. animal protocols were approved and monitored by the animal care committee of the university health network or of the southern research institute. in vitro blood stimulation with ifn-α b whole blood was drawn from ferrets and diluted ¼ with cell culture media (invitrogen, ca). two ml of diluted blood from animals was stimulated with . μg/ml ifn-α b (pegylated ifn-α b, schering-plough, pointe-claire, canada) in separate wells and incubated at °c ( % co ) for and h. pbs was added to control wells. the cultured blood was then harvested and injected to paxgene tubes. rna was purified according to manufacturer's protocol (invitrogen, ca). one ml of blood stimulated with ifn-α b and or pbs was also added to ml lyse/fix buffer (bd biosciences, usa) for evaluation of phosphorylation status of signaling molecules at , , , , , and min, using phosflow antibodies (bd biosciences, usa) . subcutaneous injections of ml of pbs (control) or μg/ml of ifn-α b were performed on the back of each ferret. two ml of blood was collected directly into paxgene tubes. one gram of the lung necropsy was added to trizol® reagent (invitrogen ca). collected blood and lung tissues were used for rna isolation according to the manufacturer's protocol and used for microarray, and quantitative real-time pcr analysis. one ml blood was added to lyse/fix buffer (bd biosciences, san jose, ca) for analysis of the signaling molecule phosphorylation status. ferrets were infected with sars-cov in the animal biohazard safety level (absl ) facility at southern research institute (birmingham, al, usa), in accordance with the approved protocols. three male ferrets, weighing approximately - g, were infected intranasally with tcid sars-cov tor strain (isolated from a patient in toronto and sequenced at cdc, vancouver, bc) in ml pbs. an additional animals were mock-infected with ml pbs. animals were anesthetized and blood and lung necropsies were collected for rna purification. infection of ferrets with the above mentioned dose results in weight loss, decreased activity, temperature increase and histology lesions with no mortality during the disease course (chu et al., ) . a summary of natural history of ferrets infected with sars-cov has been provided as supplementary table . one ml of in vitro-stimulated blood with ifn-α b or pbs and/or ml of blood drawn from the ifn-α b or pbs injected ferrets and/or ml blood from infected ferrets with sars-cov or mock controls (in vivo) was added to ml lyse/fix buffer (bd biosciences, usa) and incubated in a °c water bath for min. tubes were then centrifuged at g for min and the cells were washed twice with cold pbs. one ml perm iii (bd biosciences, usa) was added to each tube and the tube was incubated on ice for min to permeabilize cells for intracellular staining. cells were washed with perm/wash (bd biosciences, usa) and cells were added to each tube for flow cytometry. twenty microliters of phosphorylated (p)-stat , p-stat , p-stat , p-stat , p-p and p-erk antibodies conjugated with alexa-fluor was added to separate tubes (bd biosciences, usa). matched isotype control was added to one tube as a negative control. tubes were incubated at room temperature in the dark for min. cells were washed with cold perm wash (bd biosciences, usa) and fixed with % paraformaldehyde in pbs. twenty thousand events were acquired with a bd facscalibur (bd biosciences, usa) and data were analyzed, using flowjo software (tree star inc., usa). cloning and sequencing was performed as described previously . briefly, purified rna was reverse transcribed to cdna using invitrogen rt-kits (invitrogen, carlsbad, ca). genespecific degenerate primers were designed based on multiple gene sequence alignment analysis of several species using clustalw ( . ) and then used to clone the cdnas for each gene. standard pcrs were performed and specific bands were gel-purified (qiagen, mississauga, canada) and cloned into the pcr . -topo vector (invitrogen, carlsbad, usa). sequences of positive clones were confirmed using an abi xl dna analyzer (applied biosystems, foster city, ca). the identification of genes was performed using basic local alignment search tool (blast) analyses against national centre for biotechnology information (ncbi) database. the following components were added to the reaction mixture plus cdna to a total volume of μl in distilled water: . μl cdna, nmol forward gene-specific primer, nmol reverse genespecific primer and μl cyber green (applied biosystems, foster city, ca). for every experiment, each reaction was performed in triplicate. an abi sequence detection system (applied biosystems, foster city, ca) was used for amplification. initial denaturation was min at °c, followed by cycles of amplification. each cycle consisted of a denaturation step ( seconds at °c) and an annealing/extension step ( min at °c). expression levels were normalized to β-actin and data were analyzed by sds . software (applied biosystems, foster city, ca). briefly, . μg of total rna was isolated using paxgene whole blood purification kits or trizol® reagent. oligonucleotide microarray analysis was performed using affymetrix two-cycle crna synthesis and ivt kits according to the manufacturer's protocols (affymetrix, santa clara, ca). crna samples ( μg) were labelled and hybridized to affymetrix genechip canine genome . arrays to monitor the gene expression of over , canis familiaris mrna/est-based transcripts and over , non-redundant predicted genes. as described earlier, canine arrays were used following the observation of high levels of homology between canine and ferret nucleotide sequences (average of % identity) rowe et al., ) . supplementary table demonstrates the amino acid identity of genes in this study compared to available orthologues of human and mouse. the arrays were scanned using an affymetrix gcs g system according to standard affymetrix protocols. probe-level analysis was performed using probe logarithmic error intensity estimate (plier). the raw intensity values for each individual target on the affymetrix chips were pre-processed with variance stabilization, log -transformation and were then normalized against the time zero datasets with arrayassist v . . (stratagene, usa). student's t-tests or edge time course differential expression analysis (storey et al., ) were performed with benjamini-hochberg false discovery rate (fdr) correction. genes with a significant difference were selected for agglomerative hierarchical clustering with pearson distance metrics and average linkage distance measurements between clusters using genelinker platinum v . . (improved outcomes software, kingston, canada). ingenuity pathway analysis . software (ingenuity systems inc., redwood city, ca) was used to annotate and organize the gene expression data into networks and pathways. pathways and functional categories were considered as over-represented when fisher's exact test p value was ≤ . . datasets are publicly available at the ncbi's gene expression omnibus (http:// www.ncbi.nlm.nih.gov/geo) accession number gse . t tests or edge analyses were used for statistical analysis considering a biological filter of . fold change compared to controls and a p value of ≤ . as significant. supplementary materials related to this article can be found online at doi: . /j.virol. . . . regulation of suppressor of cytokine signaling (soc ) by growth hormone in pro-b cells variation in antiviral ', '-oligoadenylate synthetase ( ' 'as) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the oas gene global genomic analysis reveals rapid control of a robust innate response in siv-infected sooty mangabeys interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome gene expression analysis of host innate immune responses during lethal h n infection in ferrets interferon, a growing cytokine family: years of interferon research interferon-based therapy of hepatitis c the sars-cov ferret model in an infectionchallenge study effects of interferon-alpha (ifn-alpha) administration on leucocytes in healthy humans cloning, expression and characterization of ferret cxcl emergence of a novel swine-origin influenza a (h n ) virus in humans mortality due to influenza in the united states-an annualized regression approach using multiplecause mortality data molecular characterization of in vivo adjuvant activity in influenza-vaccinated ferrets role of the jak/ stat pathway in the regulation of interleukin- transcription by oxidized phospholipids in vitro and in atherosclerosis in vivo pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques interferon-induced mx proteins in antiviral host defense adrenocorticotrophic hormone (acth) in the treatment of experimental air-borne influenza virus type a infection in the ferret leucocyte response and anti-inflammatory cytokines in community acquired pneumonia the interferon signaling network and transcription factor c/ebp-beta interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study comparable potency of ifnalpha and ifnbeta on immediate jak/stat activation but differential down-regulation of ifnar virology: sars virus infection of cats and ferrets cloning, expression and immunoassay detection of ferret ifn-gamma innate antiviral response targets hiv- release by the induction of ubiquitin-like protein isg modeling host responses in ferrets during a/california/ / influenza infection a physical and regulatory map of host-influenza interactions reveals pathways in h n infection wnt signalling in the immune system: wnt is spreading its wings significance analysis of time course microarray experiments interferon signalling network in innate defence mortality associated with influenza and respiratory syncytial virus in the united states mechanisms of type-i interferon signal transduction social and environmental risk factors in the emergence of infectious diseases we are indebted to nikki kelvin for her editing and critical review of this manuscript. we also would like to thank lixia guo and zujiang li for their assistance in cloning of the ferret genes. key: cord- -lgprrwee authors: bartok, eva; hartmann, gunther title: immune sensing mechanisms that discriminate self from altered self and foreign nucleic acids date: - - journal: immunity doi: . /j.immuni. . . sha: doc_id: cord_uid: lgprrwee all lifeforms have developed highly sophisticated systems equipped to detect altered self and non-self nucleic acids (na). in vertebrates, na-sensing receptors safeguard the integrity of the organism by detecting pathogens, dyshomeostasis and damage, and inducing appropriate responses to eliminate pathogens and reconstitute homeostasis. effector mechanisms include i) immune signaling, ii) restriction of na functions such as inhibition of mrna translation, and iii) cell death pathways. an appropriate effector response is necessary for host defense, but dysregulated na-sensing can lead to devastating autoimmune and autoinflammatory disease. their inherent biochemical similarity renders the reliable distinction between self na under homeostatic conditions and altered or exogenous na particularly challenging. in this review, we provide an overview of recent progress in our understanding of the closely coordinated and regulated network of innate immune receptors, restriction factors, and nucleases to effectively respond to pathogens and maintain host integrity. all lifeforms have developed highly sophisticated systems equipped to detect altered self and non-self nucleic acids (na). in vertebrates, na-sensing receptors safeguard the integrity of the organism by detecting pathogens, dyshomeostasis and damage, and inducing appropriate responses to eliminate pathogens and reconstitute homeostasis. effector mechanisms include i) immune signaling, ii) restriction of na functions such as inhibition of mrna translation, and iii) cell death pathways. an appropriate effector response is necessary for host defense, but dysregulated na-sensing can lead to devastating autoimmune and autoinflammatory disease. their inherent biochemical similarity renders the reliable distinction between self na under homeostatic conditions and altered or exogenous na particularly challenging. in this review, we provide an overview of recent progress in our understanding of the closely coordinated and regulated network of innate immune receptors, restriction factors, and nucleases to effectively respond to pathogens and maintain host integrity. nucleic acids (na) are a common building block of life, yet detection of exogenous genetic material is essential to host defense. the immune sensors employed by our cells to distinguish between self and non-self na are both effective and ancient, with recent publications revealing that even bacteria utilize sensors surprisingly similar to our own, such as cgamp synthase (cgas) and toll-interleukin receptor (tir) domain-containing proteins for anti-phage defense (cohen et al., ; doron et al., ) . although there are also sequence-based, adaptive forms of na sensing such as crispr/cas and rna interference (rnai), which are integral to host defense in other kingdoms and phyla (berkhout, ; hampton et al., ) , chordates principally rely on a discreet but powerful system of germline-encoded na sensors that are activated by molecular hallmarks of non-self or altered self na (schlee and hartmann, ) . sensor activation triggers a transcriptional form of host defense, including type-i interferon (ifn-i) release and the autocrine and paracrine induction of interferon-stimulated genes (isg), known as the antiviral state. in turn, to avoid immunodetection, pathogens and viruses in particular have engaged in a type of ''nucleic acid arms race'' to avoid sensing by the host. pathogens can sequester their na (e.g., in replication organelles), mask them with characteristics of self (e.g., viral cap-snatching), or even directly disable host signaling. indeed, recent publications indicate that, while rnai is present in vertebrate cells and active in embryonic cells (li et al., a; maillard et al., ) , it can be rendered ineffective by the anti-rnai mechanisms of many viruses (li et al., ; qiu et al., ) . thus, it is tempting to speculate that this disabling of rnai provided the evolutionary pressure leading to the dominance of the type-i interferon (ifn-i) signaling in vertebrate antiviral defense, a system which is both exceptionally potent and reciprocally antagonistic with rnai (maillard et al., ; seo et al., ) . it is interesting to note that, although many of the receptors activating type-i ifn signaling are highly conserved evolutionarily, their downstream signaling is unique to chordates. analogously, caspases and metacaspases are ancient participants in programmed cell death (bell and megeney, ), yet inflammatory caspase activation by inflammasome proteins, some of which can also be activated directly or indirectly by na, is unique to vertebrates (maltez and miao, ) . in particular, dna sensing by inflammasomes is subject to strong evolutionary pressure and divergence even among mammalian species (brunette et al., ; gaidt et al., ) . indeed, the functionalization of crispr/cas systems as a tool for genomic editing have revealed important differences in human and murine na sensing, including distinct cell subset expression patterns of na-sensing toll-like receptors (tlrs) or species differences in the structural requirements for the detection of cyclic dinucleotide cgamp by sting. in this review, we will focus on the specific molecular mechanisms employed by the nucleic acid sensors of the vertebrate innate immune system to distinguish between physiologically present and pathogen-derived or pathogenically altered na. we primarily focus on the distinction of self versus non self, and the molecular structure, availability, and localization of na ligands. our aim is to provide sufficient background to understand these findings within the broader context of the evolving field of nucleic acid immunity. special emphasis is placed on what we think is essential information for someone who is new to the field, such as the spectrum of antiviral responses elicited (see box , effector functions), the multiple issues around the first na ligand ''poly(i:c)'' (see box , polyi:c), or the advantages and disadvantages of using the most common enzymatic method for rna synthesis, in vitro transcription (ivt), and its implications for rig-i activation (see box , in-vitro transcription). principles of nucleic acid sensing: localization, structure, and availability unlike fundamentally exogenous, microbial substances, such as flagellin or lps, nucleic acids are common to all forms of life, whether pathogen or host. effective na sensing thus critically depends on specific and sensitive detection of pathogenic or altered na among abundant, physiological endogenous molecules. the central principles underlying this distinction are na ( ) structure, ( ) localization, and ( ) availability ( figure ). structural characteristics of na include their length, base pairing, secondary structure, -and -termini, and modifications. the localization of na refers to their cellular and subcellular compartmentalization, monitored by a system of strategically placed na sensors. endosomal na sensors, i.e., the na-sensing tlrs, are primarily expressed in phagocytic, professional im-mune cells and are ideally located to sense ligands released by the hydrolytic degradation of pathogens in the endophagosomal compartment (blasius and beutler, ; brubaker et al., ) . this compartmentalization is controlled by trafficking and proteolytic receptor activation in the endosomal compartment, and alterations of receptor localization can lead to fatal autoinflammation (mouchess et al., ) . cytosolic na sensors, including rig-i, mda , and cgas, are broadly expressed in nucleated cells where they sense cell-intrinsic infection. correspondingly, the type-i ifn receptor (ifnar) is also ubiquitously expressed (gonzá lez-navajas et al., ) , allowing, in theory, any nucleated cell to sense viral infection and enter an antiviral state via autocrine or paracrine ifn. moreover, recent research has also revealed the importance of nuclear na sensing (gentili et al., ; volkman et al., ) , revising the long-standing box . effector functions of nucleic acid sensing: signaling, restriction, and cell death upon na binding, immune sensing receptors activate an overwhelmingly transcriptional response. these signaling transduction cascades include nuclear factor-kb (nf-kb), interferon regulatory factors(irf) and and activator protein- (ap- ) (maniatis et al., ; marié et al., ) , which cooperatively transcribe chemokines and cytokines as well as type-i and, at mucosal barriers, type-iii interferons (onoguchi et al., ; ye et al., ) . this results in a complexly orchestrated response, including the induction of interferon-stimulated genes (isg) and the anti-viral state in infected and bystander cells, as well as the activation and the recruitment of innate and adaptive immune cells to the site of infection (gonzá lez-navajas et al., ) . in contrast, na restriction factors do not induce transcriptional signaling but rather serve to directly restrict the function of pathogen nucleic acids via their sequestration, destruction, and processing and/or presentation for na immune sensing receptors. these factors are generally isgs, and their presence is indicative of the anti-viral state. restriction mechanisms can be highly specific, such as the sequestration of o-unmethylated capped rna by ifit (daffis et al., ) or global, such as protein kinase r activation, which upon binding of long double-stranded(ds)rna induces the inhibition of cellular transcription and translation, halting the proliferation of cell and virus alike (levin and london, ) . the ultima ratio of global pathogen restriction in host defense is cell death, which simultaneously restricts pathogen replication, releases inflammatory mediators, and terminates transcriptional signaling (maelfait et al., ) . na-sensing can trigger a variety of forms of programmed cell death (pcd) from immunologically silent to proinflammatory. apoptosis can result from overwhelming activation of na sensors, such as sting tang et al., ) or mavs kaneda, ; matsushima-miyagi et al., ) , particularly in cancer cells. necroptosis, a lytic proinflammatory form of pcd, can be directly induced by the activation of the zbp- by viral or endogenous z-form rna (maelfait et al., ; thapa et al., ) but also indirectly by combined tnf and ifn exposure downstream of sting or mavs hyperactivation (brault et al., ) . pyroptosis, a rapid, lytic form of pcd accompanied by the release of pyrogenic cytokines, results from inflammasome activation and can be triggered directly by na-sensing inflammasome proteins or indirectly downstream of na sensing. in particular, the nlrp inflammasome is activated by cellular dyshomeostasis and thus induces pyroptosis secondary to other cell death pathways(de vasconcelos and lamkanfi, ), as has been described downstream of sting, mavs, and zbp- activation, as well as viral lytic cell death (da costa et al., ; franchi et al., ; gaidt et al., ; kuriakose et al., ) . since canonical inflammasome assembly induces caspase- -mediated proteolytic activation of both the pyroptotic effector gasdermin d as well as cytokines from the il- family, it sits at the crossroads of signaling and effector functions (de vasconcelos and lamkanfi, ) . however, it should be noted that this type of signaling is fundamentally different from the ifn and isg response: inflammasome activation is post-translational, not transcriptional and, theoretically, does not even requiring a living cell (franklin et al., ) . nonetheless, inflammasome activation is of critical importance to the immune response to many viral infections, since il- family cytokines support immune cell recruitment, nk-cell activation and the formation of anti-viral cd + t cell responses (reviewed in kanneganti, ) . since the type-i ifn response is inherently transcriptional, na-induced cell death also acts as an important limiter of antiviral responses. several studies have demonstrated that apoptotic caspases directly inhibit cgas/sting signaling (rongvaux et al., ; white et al., ) , with a recent report showing that activated caspase can cleave cgas, mavs, and irf (ning et al., ) . similar mechanisms have been demonstrated for pyroptosis. activated caspase has been reported to cleave cgas , and activation of gasdermin d-mediated pyroptosis can also resolve cgas/sting signaling through k+ efflux (banerjee et al., ) . although many recent studies to date have focused on the effect of pcd on cgas/sting, the reciprocal interplay between na sensing and cell death is undoubtedly important for all na-sensing pathways and continues to be the focus of intense research. paradigm that the nuclear compartment is ''immune privileged.'' indeed, given that the nucleus is the site the replication of most dna viruses, as well as retroviral genomic integration, these nuclear sensors are perhaps the last line of defense for our genomic integrity. na availability is the net result of the entry or generation of na structures in a specific compartment versus their sequestration (e.g., by pathogen vacuoles), shielding from receptor binding (e.g., by viral proteins), or degradation by nucleases. pathogen sequestration can be countered by host proteins, such as interferon-inducible gtpases (meunier and broz, ) , which release ligands into the cytosol. moreover, host and viral nucleases can act to limit or enhance the availability of potential na ligands. indeed, as we and others have shown, the same host nuclease (e.g., dnase , rnase t ) can potentially enhance the activity of some receptors while limiting the activity of others, thus acting as a rheostat for na sensor activation. although these principles are generally effective, the inherent risks of this strategy are clear. inappropriate or uncontrolled sensing of self na drives autoinflammatory disease, ranging from rare and devastating monogenetic forms of type-i interferonopathy (rodero and crow, ) to the inevitable senescence of our cells (gl€ uck et al., ; yang et al., ) . nonetheless, na sensing provides a clear evolutionary advantage in that it is essential for host survival, as demonstrated by numerous genetic models of pathogen infection. na sensors can be categorized into ( ) bona fide immune sensing receptors that act to detect pathogenic na and signal their presence to the host and ( ) anti-viral restriction factors that act directly upon viral na to limit viral replication but are not linked to the transcription of classical immune-related genes such as cytokines (schlee and hartmann, ) . the search for immune receptors detecting exogenous rna has led to the discovery of both endosomal and cytosolic rnasensing mechanisms (figure ). the transmembrane tlrs tlr , tlr , tlr , and, in lower vertebrates and rodents, tlr , are all genetically proven endosomal rna immune sensors (alexopoulou et al., ; diebold et al., ; heil et al., ; oldenburg et al., ) . tlr has been reported to act as an anti-inflammatory receptor of double-stranded (ds-)rna, but further studies will be needed to corroborate this finding . tlr was the first immune sensing receptor of polyi:c discovered (alexopoulou et al., ) and is a sequence-independent sensor of the ribose-phosphate backbone of dsrna > bp liu et al., ) and incomplete dsrna stem structures of sufficient length within ssrna molecules (tatematsu et al., ) . tlr signaling is distinct from other na-sensing tlrs in several respects: instead of myd , tlr signals via trif to induce ifn-b and nf-kb signaling (oshiumi et al., ; yamamoto et al., ) , with recent research indicating that the nutrient sensor mtorc is also critically required ; tlr is expressed in non-immune cells, including fibroblasts, endothelial cells, oligodendrocytes, astrocytes, and neurons (bsibsi et al., ; lafon et al., ; matsumoto et al., ; zimmer et al., ) , and it has been reported that, in several of these cell types, tlr can also be localized and signal from the cell surface, (jack et al., ; matsumoto et al., ; pohar et al., ) although this would presumably require an acidic environment for dsrna binding (liu et al., ) . despite the seeming redundancy of dsrna sensing (see box , polyi:c), humans expressing hypomorphic variants of tlr are susceptible to hsv- encephalitis (zhang et al., ) and severe influenza pneumonitis (lim et al., ) in box . poly i:c generated by the annealing of enzymatically generated inosine and cytosine homopolymers (michelson et al., ) , polyi:c is by far the most commonly used rna ligand in research. clinical applications for polyi:c were explored soon after the discovery that it could produce a robust ifn response (field et al., ; hilleman, ) , and clinical trials using polyi:c in tumor immunotherapy continue today (e.g., nct , nct ). moreover, over the last years, intense research has focused on characterizing the immune sensor(s) polyi:c activates. this popularity is remarkable given that polyi:c is neither a physiological ligand nor is its mode of action well defined. rather, its widespread use results from its amenability to enzymatic synthesis (michelson et al., ) and its unique ability to induce a robust type-i ifn response compared to other annealed homopolymers (field et al., ) , a feature which remains poorly understood. as a long dsrna with a diphosphate terminus, polyi:c is known to activate the sensing receptors tlr , mda , and rig-i (alexopoulou et al., ; gitlin et al., ; kato et al., ; yoneyama et al., ) , accessory proteins such including lgp and members of the ddx and dhx families (reviewed in oshiumi et al., ) as well as the restriction factors pkr and the oas family (farrell et al., ; hovanessian et al., ; zilberstein et al., ) . in theory, any receptor that binds dsrna could be activated by polyi:c, including further restriction factors, such as zbp- (z-rna), although characterizing the specific activity would clearly require multiple gene deletions. this plethora of potential receptors likely contributes to its high toxicity profile, which is reduced in the more selective derivative polyi:c u (junt and barchet, ) . despite over years of research, many open questions about the molecule's bioactivity remain: why does low-molecular weight polyi:c (< bp) preferentially activate rig-i given that rig-i senses the diphosphate terminus ? why is polyi:c a robust activator of mda while many other defined, long dsrnas such as poly(a:u) are not (colby and chamberlin, ; pichlmair et al., ) ? does the mesh-like structure of poly(i:c) contribute to its immune stimulatory activity, as opposed to just its length (pichlmair et al., ) ? why does the polyi:c derivative poly i:c u (ampligen) only activate tlr but not rig-i or mda- (gowen et al., ) ? childhood, and tlr À/À mice demonstrate a susceptibility to poliovirus , hsv- (davey et al., ) , and mcmv (tabeta et al., ) . moreover, perhaps due to its broader expression, tlr has a preeminent role in the cns , where it contributes to both protective and deleterious neuroinflammation during host defense (mé nager et al., ; perales-linares and navas-martin, ; sato et al., ; wang et al., ) . in contrast, tlr , tlr , and tlr signal via the adaptor myd and are restrictively expressed in immune cells (diebold et al., ; heil et al., ; hemmi et al., ; hornung et al., ; shi et al., ) . tlr and tlr result from a gene duplication (roach et al., ) and demonstrate numerous structural and functional similarities. both receptors can be activated by imidazoquinoline compounds and polyu and gu-rich ssrna (judge et al., ; jurk et al., ) . however, selective ligands have also been reported (forsbach et al., ; lu et al., ; ostendorf et al., ) . while tlr is functional in both mice and humans, the role of tlr in mice remains unclear (alexopoulou et al., ; heil et al., ) . in humans, tlr and tlr demonstrate a differential expression pattern (hornung et al., ) . tlr is highly expressed on monocytes, conventional dcs and neutrophils, whereas tlr is predominantly expressed in plasmacytoid dendritic cells(pdc) and b cells. in human monocytes, tlr activation leads to the release of ifn-b and proinflammatory cytokines, including il p , il , and tnf (bergstrøm et al., ; cushing et al., ) . very recently, the type-i-interferon-inducible tlr adaptor interacting with slc a on the lysosome (tasl) has been reported as an additional signaling component linking endolysosomal tlr and tlr to interferon regulatory factor- (irf ) and ifn-b induction (heinz et al., ) . tasl recruits and activates irf , and loss of tasl specifically impairs the activation of the irf pathway without affecting nf-kb and mapk signaling, revealing a mechanistic analogy with sting, mavs, and trif. in pdcs, tlr activation strongly induces ifn-a via the myd -traf -irf pathway (honda: jg. kawai et al., ) . tlr is also weakly expressed in primary monocytes, where it can also activate downstream signaling (de marcken et al., ; gantier et al., ) . the crystal structures of the ligand-bound ectodomains of tlr and tlr revealed that both bound rna degradation products (shibata et al., ; tanji et al., ; zhang et al., ) . in their first binding pocket, tlr binds guanosine and tlr , uridine, respectively, while the second binds short di-or trinucleotides. subsequent studies demonstrate that tlr activation by rna ligands critically requires upstream endosomal rnase activity (greulich et al., ; ostendorf et al., ) . since endosomal rnase expression varies substantially between cell types, their activity adds another layer to our understanding of tlr and tlr specific ligands. tlr is expressed in non-mammalian vertebrates, marsupials, and rodents but not in primates (hidmark et al., ; oldenburg et al., ) . two studies identify the sequence cggaaagacc and acggaaagacccc within s rrna of several species of bacteria as a tlr -activating motif oldenburg et al., ) . the crystal structure of the ectodomain of tlr reveals that it senses both rna sequence and stem-loop conformation (song et al., ) . to date, tlr is the only completely sequence-specific vertebrate rna immune sensor identified. the observation that the tlr À/À mouse still responds to polyi:c provided the first indication of intracellular rna immune sensing receptors (diebold et al., ) . the presence of cytosolic rna with hallmarks of non self can indicate viral replication , intracellular bacterial infection (hagmann et al., ; monroe et al., ) or the escape of rna from the endolysosome via transport or rupture (nguyen et al., ; watanabe et al., ) . two related dexd/h box rna helicases rig-i and mda- (yoneyama et al., ) , also known as rig-i like receptors (rlrs), sense cytosolic polyi:c and mount a phage-encoded dna-dependent rna polymerases are commonly used for in-vitro transcription (ivt) of dna into rna from linear dsdna or a plasmid dna template containing the appropriate promoter sequence (green and sambrook, ) . this method allows the rapid, cheap, and efficient generation of long rna molecules, many of which would be prohibitively expensive to produce synthetically. as such, ivt is a popular, often kit-based, method for producing mrna and crispr guide rna for use with recombinant cas proteins. however, since ivt utilizes promoter-based unprimed rna polymerization, it creates transcripts with a triphosphate terminus, much like other nascent viral rna transcripts. we and others used ivt-generated rna to demonstrate that triphosphate rna activated rig-i (hornung et al., ; pichlmair et al., ) . however, the rig-i activating species within the ivt is not its main product, triphosphate ssrna. end complementarity can prime a fold back reaction resulting in the production of complementary strands and rna hairpin structures (cazenave and uhlenbeck, ; triana-alonso et al., ) . as we and others subsequently showed, triphosphate base-paired, blunt-ended rna, a side product within an ivt, is in fact the true rna species activating rig-i schmidt et al., ) reviewed in (schlee, ) . unless modified nucleotides or specific sequences precluding the formation of the complementary strand are used, ivt can potentially generate rig-i ligands from a variety of templates (hornung et al., ; kim et al., ; schlee et al., ; wienert et al., ) . on the one hand, this can lead to unwanted immunostimulatory effects during the generation of mrna, grna, or sirna. on the other, it means that extreme caution should be used when inferring that a particular sequence or endogenous rna species activates rig-i if ivt rna was used to model its activity. most endogenous rna species are processed post-transcriptionally and thus lack accessible triphosphate moieties (gebhardt et al., ; schlee and hartmann, ) , thus using triphosphate ivt rna is prone to creating immunostimulatory artifacts. immunity , july , type-i ifn response via the mitochondrial adaptor protein mavs (kawai et al., ; seth et al., ) . while rig-i is activated by shorter (< bp, low-molecular weight, lmw) poly i:c molecules, mda requires longer dsrna (> bp, high-molecular weight, hmw) polyi:c (gitlin et al., ; kato et al., ; yoneyama et al., ) . this overlapping yet differential activity also applies to the sensing of viral replication (loo et al., ; schlee, ) . both sensors contribute to the immune response to dsrna viruses, such as reoviridae (loo et al., ) , but rig-i-mediated sensing dominates the response to many (-) ssrna viruses such as influenza, which form shorter dsrna panhandles in their genomes (rehwinkel et al., ; schlee et al., ) , whereas mda has a greater role in the host defense against (+) ssrna viruses, many of which are known to generate large amounts of dsrna during replication . whereas subsequent studies have made substantial advances in characterizing the precise molecular patterns activating rig-i (see rna motifs distinguishing self from non self), the motifs necessary to activate mda still remain ill defined (hartmann, ) . both rna sensors are expressed in almost all primary nucleated cells (hartmann, ; ida-hosonuma et al., ) . upon activation, rig-i and mda- oligomerize, thereby inducing the polymerization of mavs into fibrillar structures (hou et al., ; xu et al., ) and recruiting traf , traf , ikk, and tbk , which in turn activate nf-kb and irf and/or irf signaling (kawai et al., ; seth et al., ) and the transcription of proinflammatory cytokines and type-i and type-iii ifn. in addition to transcriptional signaling, mavs complexes can associate with fas-associated protein with death domain (fadd), receptor-interacting serine/threonine-protein kinase (rip ), and caspase (kawai et al., ; yang et al., ) . in this context, caspase has been reported to induce apoptosis downstream of mavs (el maadidi et al., ) but also to terminate signaling without cell death (rajput et al., ; sears et al., ; yang et al., ) . several studies have noted that malignantly transformed cells are more sensitive to rlr-induced cell death, although the precise molecular determinants for this difference remain unknown (hartmann, ; kumar et al., ) . while it is well established that inflammasome activation is important for antiviral defense (kanneganti, ) , the rna sensors involved are still the subject of intense research. to date, three inflammasome-building nlrs have been reported to participate in cytosolic rna sensing, with all three requiring accessory rna-binding proteins. via the adaptor protein dhx , nlrp is reported to sense viral dsrna, rnasel degradation products, and polyi:c, leading to inflammasome formation, caspase- cleavage and the release of il- b and il- from in human thp- cells and monocyte-derived macrophages (chakrabarti et al., ; mitoma et al., ) . via the rna figure . principles of self versus non-self or altered-self nucleic acid recognition unlike pathogen-specific molecules such as lps, nucleic acids in pathogens and the host are biochemically similar. for a reliable distinction of self versus nonself and altered-self nucleic acid recognition, information about the molecular structure, the availability, and the localization is integrated. the localization of nucleic acid receptors on different cell types, immune cells as well as non-immune cells, contribute as well. lists and cell types depicted are not comprehensive but just represent examples to better illustrate the principles. non-comprehensive overview of the most relevant rna sensing receptors at the relevant localizations, with their downstream signaling molecules and some of the functional outcomes and secondary consequences as applicable (e.g., rig-i sensing of rnasel degradation products). rna sensing receptors are in gray, signaling molecules in yellow, nucleases in green, inflammasome pathways is purple, and cell death pathways in orange. unlike other tlrs, tlr signals from the cell surface and the endolysosome. tlr is a sequence-independent sensor of the ribose-phosphate backbone of dsrna > bp and of incomplete dsrna stem structures of sufficient length within ssrna molecules. tlr signals via trif to induce ifn-b and nf-kb signaling. tlr and tlr are activated by rna degradation products, with the first pocket binding guanosine and uridine (tlr and tlr , respectively), and the second pocket binding short di-or trinucleotides. tlr activation requires upstream endosomal rnase activity (rnaset , rnase ) and, due to homology, rnase activity is likely required for tlr as well. tlr activation induces ifn-a via the myd -traf -irf pathway. tlr activation releases ifn-b and proinflammatory cytokines via a tak -ikkb-irf pathway. tlr adaptor interacting with slc a on the lysosome (tasl) was reported as a signaling component linking endolysosomal tlr and to irf . tlr recognizes bacterial s rrna in a sequence-specific manner. upon activation, the cytosolic immune sensors rig-i and mda- oligomerize thereby inducing polymerization of mavs into fibrillar structures leading to the recruitment of traf , traf , ikk, and tbk , which then activate nf-kb and irf and/or irf signaling. in addition, mavs complexes can associate with fadd, rip , and caspase and induce apoptosis downstream of mavs. ddx , dhx , dhx , and ddx enhance rig-i signaling. dhx acts as co-receptor for both rig-i and mda . lgp supports filament formation by mda but may compete with rig-i for ligand. cytosolic rna sensors with direct anti-viral activity include protein kinase r (pkr) and the '- -oligoadenylate synthetase system (oas) which both bind dsrna > bp including polyi:c. pkr phosphorylates elf a and inhibits cap-dependent translation of viral and host mrna. oas induces the formation of oligoadenylate which acts as a second messenger to activate ribonuclease l (rnase l), which in turn degrades cellular rna and viral rna to smaller rna molecules that can be sensed by rig-i and dhx . dhx activates the nlrp inflammasome, inducing pyroptotic cell death. ifn-induced proteins with tetratricopeptide repeats (ifit) sequester viral mrna and block their translation by sensing termini. ifit binds mrna with a cap structure ( mgpppnn), and ifit b binds cap , to a lesser extent cap ( mgpppnmn) structures but not cap ( mgpppnmnm) structures. b binds uncapped triphosphate rna, and ifit which binds au-rich rna. ddx can bind and sequester stem loop structures from some rna viruses. adenosine deaminase acting on rna (adar ) catalyzes the c deamination of adenosine to inosine in base-paired regions of rna, and the resulting non-synonymous coding causes amino acid substitutions and potentially renders viral proteins non-functional. the host rna decay machinery includes nonsense-mediated mrna decay (nmd), - rna degradation and the - rna exonuclease machinery (rna exosome). nmd targets mrna transcripts with a long utr but also senses viral rna. the - degradation machinery with the decapping enzymes dcp and dcp and the - exonuclease xrn (xrn-dcps) is involved in physiological cellular mrna turnover and exhibits antiviral activity. the superkiller viralicidic activity -like (skiv l) and zinc-finger antiviral protein (zap) support the binding and transport vrna to the rna exosome for degradation. the isg and viral restriction factor z-dna binding protein (zbp- ) is a death receptor downstream of dsrna sensing. zbp- (legend continued on next page) ll immunity , july , helicase dhx , the previously uncharacterized nlrp b (human homolog nlrp ) forms an inflammasome in intestinal epithelial cells in response to rotavirus, leading to the maturation of il- and the activation of gasdermin d-mediated pyroptosis (zhu et al., ) . via the rna-binding accessory protein dhx , nlrp has been reported to sense dsrna in intestinal epithelial cells . however, this does not result in caspase- activation but rather mavs-dependent type-i and iii ifn induction. this function of nlrp is reportedly restricted to the gastrointestinal tract, as nlrp À/À mice demonstrate increased mortality and viremia after oral but not systemic viral infection. since nlrp can form an inflammasome (elinav et al., ; shen et al., ) , how dhx induces nlrp -mavs interaction rather than oligomerization with the inflammasome adaptor asc remains unclear. other dexd/h-box proteins have been reported to act as accessory proteins to cytosolic rna signaling (oshiumi et al., ) . ddx , dhx , dhx , and ddx have been reported to enhance rig-i signaling (miyashita et al., ; oshiumi et al., ; pattabhi et al., ; yoo et al., ) , whereas dhx acts as a co-receptor for both rig-i (sugimoto et al., ) and mda . the individual contributions of these proteins to rna sensing remain unclear and occasionally contradictory. in one study, ddx has been found to induce mavs activation independently from rig-i and mda- (gringhuis et al., ) ; in another, it has been found to support rig-i signaling (oshiumi et al., ) , and, in another, it was even found to inhibit ifn-i release (loureiro et al., ) . likewise, the role of ddx is also controversial, with one group finding that it promotes rlr signaling and another reporting that it has no role (goubau et al., ; miyashita et al., ) . future studies will be needed to resolve these differences. lgp , a dexd/h-box protein from the rlr family, contains the rlr c-terminal domain (ctd) and helicase domains but lacks the card necessary for downstream signaling (schlee, ) . accumulating evidence indicates that lgp acts ( ) as a structural accessory protein that supports filament building and activation of mda (bruns et al., ; deddouche et al., ; satoh et al., ) and ( ) as an interferon-inducible inhibitor of rnai (takahashi et al., b; van der veen et al., ) . lgp has been reported to competitively bind dsrna and inhibit the rna silencing enhancer, tar-rna binding protein (trbp) (takahashi et al., b; , an accessory protein of dicer. this mechanism also highlights the inherent antagonism between dicer-mediated cleavage of dsrna during rnai and dsrna sensing by the innate immune system. in addition to the dexd/h-box protein, zink-finger protein zcchc has been reported to bind viral dsrna and act as a cofactor for rig-i and mda , and zcchc À/À mice are more susceptible to rna virus infection (lian et al., b) . the same group has also reported in parallel that zcchc acts as a cofactor for dsdna sensing and that zcchc À/À mice are also more susceptible to lethal herpes simplex virus type or vaccinia virus infection (lian et al., a) . here, further studies will be necessary to determine how zcchc fulfils such diverse roles in host sensing. protein kinase r (pkr) and the '- -oligoadenylate synthetase system (oas) are the first rna sensors discovered in the cytosol. both sense dsrna > bp and are activated by polyi:c (see box , polyi:c). pkr phosphorylates elf a and thus inhibits cap-dependent translation of viral and host mrna (levin and london, ) . oas binding of dsrna leads to the production of ' oligoadenylate (hovanessian et al., ; zilberstein et al., ) , a second messenger activating latent ribonuclease l (rnase l) (zhou et al., ) . rnasel, in turn, degrades cellular and viral rna. global rnase-l degradation does not only prevent viral replication but also generates smaller rna molecules sensed by rlrs (malathi et al., (malathi et al., , and dhx -mediated activation of nlrp , inducing pyroptotic cell death (mitoma et al., ) . in cells without nlrp , rnase l can also trigger apoptosis to prevent viral propagation (castelli et al., ) . the host rna decay machinery also contributes to antiviral defense, and the role of nonsense-mediated mrna decay (nmd), - rna degradation and the - rna exonuclease machinery (rna exosome) are all the subject of current, intense research. to guard the cell against aberrant self mrna, nmd targets mrna transcripts with a long utr, indicative of a mutation leading to a premature stop codon (molleston and cherry, ) . however, nmd also forms an important intrinsic barrier to viral infection (balistreri et al., ; fontaine et al., ; . conceivably, nmd senses viral rna via several complementary mechanisms, including the unusual translation dynamics of polycistronic viral transcripts, instability due to non-standard codon usage (hia et al., ) and targeting by nmd-associated proteins . the - degradation machinery, comprising the decapping enzymes dcp and dcp and the - exonuclease xrn (xrn-dcps), is primarily involved in physiological cellular mrna turnover. however, xrn is also specifically targeted by and inhibited by flaviviruses (molleston and cherry, ) , demonstrative of antiviral activity. one recent study demonstrates that xrn -dcps specifically colocalize with the rna of newcastle disease virus and encephalomyocarditis virus (emcv) and repress viral replication (ng et al., ) , although it is still unknown how xrn -dcps are recruited to viral rna. recruitment to the rna exosome is better understood. superkiller viralicidic activity -like (skiv l) (aly et al., ) and zincfinger antiviral protein (zap) (gao et al., ; guo et al., ) support the binding and transport viral rna to the rna exosome for degradation. how skiv l specifically targets viral rna is still unknown. however, recent studies have demonstrated that zap directly senses hiv- rna via its relative abundance in cg dinucleotides (ficarelli et al., ; takata et al., ) , thus revealing activates multiple programmed cell death pathways, including pyroptosis, apoptosis, and necroptosis, termed pan-optosis. upon dsrna binding, zbp- interacts with ripk supported by caspase- , resulting in mlkl activation and necroptosis, in caspase- -induced apoptosis and in nlrp dependent inflammasome activation and pyroptosis. three inflammasome-building nlrs participate in cytosolic rna sensing: dhx via nlrp , the rna helicase dhx via nlrp b (human homolog nlrp ), and the rna-binding accessory protein dhx , besides its supporting function for rig-i-like helicases, via nlrp . a sequence-dependent mechanism for self versus non-self discrimination. of note, the rna exosome is also involved in the degradation of immunostimulatory endogenous rna transcripts, and hypopmorphic variants of skiv l induce type-i interferonopathy that is dependent on rig-i (eckard et al., ) . we and others recently demonstrated that rnaset contributes to the activation of tlr (greulich et al., ; ostendorf et al., ) . however, hypomorphic variants of rnaset are associated with a rare form of proinflammatory cystic leukoencephalopathy (henneke et al., ) , likely resulting from cytosolic sensor activation by accumulating rna (haud et al., ) . our work also demonstrates that rnase , a member of the rnasea family only expressed in apes and old-world monkeys, synergistically participates in uridine release from ssrna to activate tlr in monocytes (ostendorf et al., ) . antimicrobial functions have also been attributed to other members of the rnasea family (lu et al., ) . members of the ifn-induced protein with tetratricopeptide repeats (ifit) family specifically sequester viral mrna and thus block their translation by sensing termini with markers on non self. ifit binds mrna with a cap structure ( mgpppnn), and ifit b binds cap , to a lesser extent cap ( mgpppnmn) structures but not cap ( mgpppnmnm) structures. ifit binds uncapped triphosphate rna (abbas et al., ; habjan et al., ; kumar et al., ) , and ifit binds au-rich rna . in addition to the ifits, ddx can bind and sequester stem loop structures from the rift valley fever virus in drosophila and humans (moy et al., ) , and it seems highly likely that other dexd/h box rna helicases can perform similar functions. adenosine deaminase acting on rna (adar ) catalyzes the c adenosine-to-inosine deamination of base-paired regions of rna (george et al., ) . due to their c carbonyl group, these new inosines are decoded non-synonymously as guanosines, causing amino acid substitutions and potentially rendering viral proteins non-functional (samuel, ) . in addition, the introduction of less stable i-u wobble pairs changes base-pairing dynamics ( spa cková and ré blová , ). these alterations of the secondary structure affect both viral regulation and host innate immune sensing, acting in a manner that can be pro-or antiviral (samuel, ; ) . in addition to the aforementioned cell death pathways, the isg and viral restriction factor z-dna binding protein (zbp- ) is a dedicated death receptor downstream of dsrna sensing. zbp- was originally described as a dsdna sensor capable of inducing type-ifn. however, subsequent studies have demonstrated that zbp- is a dsrna sensor that activates multiple pcd pathways, including pyroptosis, apoptosis and necroptosis, termed pan-optosis (malireddi et al., ) . upon dsrna binding, zbp- enters a homotypic rhim-rhim interaction with the kinase ripk supported by caspase- (zheng et al., ). this can result in mlkl activation and necroptosis (maelfait et al., ; thapa et al., ) , caspase- -induced apoptosis thapa et al., ) and, in nlrp expressing cells, inflammasome activation and pyroptosis (kuriakose et al., ) . how these normally hierarchical forms of pcd are coordinated after zbp- activation is not yet understood (maelfait et al., ) . nonetheless, zbp- -mediated pcd has been shown to be important to controlling murine influenza a virus (iav) infection. moreover, human h n pandemic iav but not seasonal iav has been demonstrated to suppress ripk -mediated necroptosis, indicating that inhibiting this cell death pathway also contributes to viral virulence in humans . rna motifs distinguishing self from non self cytosolic long dsrna is considered a hallmark molecular pattern of viral infection: it is generally absent from the cytosol of uninfected cells, yet it forms the genome of dsrna viruses and is generated during the replication of ssrna and dna viruses (son et al., ; weber et al., ) . however, recent research indicates that the absence of endogenous cytosolic dsrna is not coincidental. our genome contains a large number of complementarily inverted mobile elements, particularly alu elements, which could potentially form long dsrna (reich and bass, ) , and their overwhelming absence in the cytosol results from both strong purifying transcriptomic selection (barak et al., ) and a-to-i editing of the remaining transcripts by adar (ahmad et al., ; chung et al., ) . in addition, mitochondria utilize an rna helicase, suv , and polynucleotide phosphorylase (pnpase) to actively eliminate dsrna species resulting from bidirectional transcription thus preventing innate immune activation (dhir et al., ; pajak et al., ) . the importance of this sophisticated protective machinery becomes evident when it breaks down: hypomorphic variants of adar and hypermorphic variants of the dsrna sensor mda cause devastating forms of type-i interferonopathy (rodero and crow, ) resulting from the sensing of alu elements (ahmad et al., ; chung et al., ) . however, the contribution of adar to antiviral defense is less clear. a-to-i conversion destroys the coding potential of viral rna, thus inhibiting viral replication, yet it also destabilizes long dsrna structures indicative of replicating pathogens (samuel, ) . the same reciprocal antagonism in dsrna recognition occurs between the rnai system and rna sensing receptors. dicer-mediated processing of dsrna interferes with viral replication yet destroys the very structure which immune sensors recognize. the critical importance of dsrna as a molecular pattern is underscored by the convergent evolution of immune sensors and restriction factors it activates, making dsrna a ''promiscuous'' ligand with diverse downstream effects (hartmann, ) (see box , poly i:c). unfortunately, the precise requirements for the dsrna length of many of these sensors remains unknown. to date, we are unaware of any publications detailing a minimal ligand for the ddx and dhx proteins detailed in this review, adar , lgp , or zbp- . we hope future studies will characterize the precise nature of the dsrna activating these sensors. based on our knowledge to date, cytosolic dsrna sensing via its ribose-phosphate backbone requires dsrna > bp, including pkr, oas, and mda (> bp for polyi:c). detection of dsrna < bp requires amenable termini (see below), which contain further information on the origin of dsrna ( phosphorylation, capping, cap structure, etc.). this cutoff strongly suggests the presence of physiological endogenous dsrna < bp in our cytosol. nonetheless, mda ligands, while intensely researched, remain ill defined (hartmann, ). the length requirement of > bp has been determined for polyi:c (kato et al., ) but remains unknown for viral and endogenous ligands (schlee and hartmann, ) . indeed, while most inverted alu elements form dsrna of approximately bp, they are only weakly stimulatory for non-ags mda variants when compared to polyi:c (ahmad et al., ) . endosomal dsrna also represents an optimal recognition motif for viral infection. as it is more resistant to endosomal rnases than ssrna (ostendorf et al., ) , dsrna provides an important indication of viral infection in efferocytosed cells. here, it is interesting to note that the dsrna length required for tlr dimerization falls from > bp in the endosome to - bp in late endolysosome , at lower ph and after longer rnase exposure. thus, endosomal sensing seemingly results from a combined calculus of length and rnase resistance. the precise length requirement at the cell surface is unclear, but presumably > bp. of note, tlr can also sense dsrna structures within long > nt ssrna of poliovirus (tatematsu et al., ) , which would be indicative of a likely bulge tolerance between the two parts of the tlr dimer. however, reports of tlr activation with shorter rna, e.g., sirna < bp, are limited to mouse tlr (weber et al., ) , and one study on the activation of tlr by mrna is based on in vitro transcription (karikó et al., ) , which may have led to the generation of longer dsrna as well as rig-i ligands (see box , in-vitro transcription). nonetheless, certain endogenous rna species, such as unedited alu elements capable of activating mda , may also activate tlr . the terminus contains a wealth of information about rna origin. in higher eukaryotes, endogenous mrna receives a m gpppn cap, further modified by 'o-methylations to m gpppnm (cap ) and m gpppnmnm (cap ). the capping process controls gene expression by modulating nuclear export, splicing, protein translation and mrna turnover (dimitrova et al., ) . of note, 'o-methylation coevolved with the ifn system and is even used to specifically regulate isg expression (williams et al., ) . in addition to the extensive modification of mrna, small nuclear rnas (snrnas) receive methylguanosine caps which are hypermethylated into trimethylguanosine in the cytoplasm, and ribosomal rna (rrna) and transfer rna(trna) are both generated via processing of precursors which creates monophosphate ( p) termini and are extensively 'o-methylated. thus, capping, n and n 'o-methylation of capped rna, and the presence of p termini can all be viewed as hallmarks of self. the cytosolic dsrna sensor rig-i can be activated by dsrna > - bp but critically requires that the terminus is compatible with its c-terminal domain (ctd). strikingly, the ctd binding cleft accommodates rna bearing signs of ''non self'': it binds triphosphate ( ppp) (hornung et al., ; schlee et al., ) and diphosphate ( pp) ) and, to a lesser extent oh termini (binder et al., ; marques et al., ) , but not p (ren et al., ) . moreover, while a cap structure reduces rig-i activity, 'o-methylation (cap , cap ) abrogates it completely (schuberth-wagner et al., ) . detection of cap rna allows rig-i to sense viruses that code their own capping enzymes but do not have methyltransferase activity, such as sindbis virus (hefti et al., ) . moreover, the discrimination between p and oh for rig-i is of particular importance for the dsrna products of rnase ac-tivity. the rnases processing trna and rrna create p rna, as does dicer. in contrast, rnasel products have oh ends and can thus activate rig-i (malathi et al., ) . the rig-i response to ppp dsrna is strongly reduced by overhangs and abrogated by overhangs at the ppp end of the molecule . blunt dsrna occurs naturally in the panhandle genome of (-) ssrna viruses. however, rrna, trna, and dicer products all contain or overhangs, a further preclusion to rig-i activation. sensing of the terminus is also a strategy followed by members of the ifit family in their sensing of ssrna. ifit sequesters ssrna or dsrna with a overhang of > nt that have a ppp or a cap but not a cap structure. ifit b sequesters ppp, cap >> cap but not cap , and ifit senses ppp ssrna or dsrna with an overhang of at least nt (abbas et al., ) . in order to escape immunodetection, a number of viruses conceal the termini of their rna or disguise themselves with molecular markers of self. some (-) ssrna viruses, including hantavirus and bunyavirus, process their termini to p, avoiding detecting by rig-i and the ifits. arenaviruses use primeand-realign mechanisms to generate rna with overhangs, which allows for ifit binding but may even act as a competitive inhibitor of rig-i activity (marq et al., ) . others code for their own 'o-methyltransferases (daffis et al., ) , and some even perform ''cap snatching,'' removing the host g m cap along with - nt and integrating it into their own mrna. given the exquisite sensitivity of terminus sensing, it is hard to imagine the presence of many endogenous ligands capable of activating rig-i. however, hypomorphic variants of the skiv l subunit of the rna exosome drive type-i ifn mediated autoinflammation via rig-i (eckard et al., ) , which can recognize rna generated by inositol-requiring enzyme (ire- ) during the unfolded protein response. (see figure ) . however, to date the identity and structure of these transcripts remain unknown. another strategy to discriminate self from host is the sensing sequence-specific motifs. in the cytosol, ifit senses au-rich rna , although the precise function of this sensing is unclear, as au-rich elements also occur in our transcriptomes (elliott and ladomery, ) . zap, which ferries rna to the rna exosome, binds cg-rich motifs in rna (takata et al., ) , a strategy enabled by the cg-suppression of the vertebrate genome (karlin and mrá zek, ) that may explain why some viruses actively avoid cg motifs (schlee and hartmann, ) . in the endosome, tlr senses a genuinely sequencespecific motif: a(cggaaagacc)cc within s rrna. it is not clear why such as sequence-specific receptor evolved, but there are different possible explanations for its disappearance in higher mammals. oldenburg et al. ( ) has demonstrated that a known methylation coded by an erythromycin-resistance gene leading to cggmaaagacc could abrogate tlr activity. this gene is of ancient origin in bacteria and could explain a loss of utility for tlr . while li and chen ( ) demonstrate that this methylation has no effect, they find that this base within s rrna is exceptionally sequence-specific, i.e., that acggbaagacccc (b = not a) could no longer activate tlr . while it is remarkable that no further study has resolved this controversy, it seems clear ll that tlr is an exceptionally mutation-and/or modification-sensitive receptor, which may account for its evolutionary loss. human tlr has been suggested as an evolutionary replacement of murine tlr (kr€ uger et al., ) . while tlr is not active in mice, in humans, it is essential for recognition of bacterial rna (eigenbrod and dalpke, ) . however, this tlr -tlr equivalency only applies to pathogens containing the tlr consensus sequence. tlr also senses plasmodia rna in humans, (coch et al., ) , which activates tlr , not tlr in mice. indeed, how tlr senses non-self rna is an unresolved question. two studies have made clear that tlr senses rnase degradation products rather than ssrna (greulich et al., ; ostendorf et al., ) . in line with previous reports, it is clear that the degraded ssrna must contain uridine since free uridine is required for the first binding pocket (tanji et al., ) . one study has postulated that uuru is a minimal motif for tlr activation (greulich et al., ) , in line with its known stimulatory activity (forsbach et al., ) . however, under uridine supplementation, the di-or trinucleotide rna for the second binding pocket of tlr does not require uridine for activity (ostendorf et al., ; shibata et al., ) . thus, the precise requirements for the second binding pocket and the true nature of any minimal tlr ligand remain unclear. moreover, given the broad number of conceivable di-or trinucleotide ligands for the second binding pocket, it seems unlikely that such motifs are not contained in self rna, or that they could all be rendered inactive by modifications such as pseudouridine or 'o-methylation (freund et al., ) . in addition, it is unclear which inhibitory modifications affect tlr activation and which inhibit upstream rnases. 'o-methylation is a known inhibitor of tlr (and tlr ) activity (freund et al., ) but also of upstream rnaset and rnase (ostendorf et al., ). conceivably, self rna does not avoid immunorecognition in the classical sense but rather contains inhibitory motifs perhaps similar to those synthetically designed for tlr and tlr (schmitt et al., ) using 'o-methylation, but further studies will be necessary to determine if these sequences have natural counterparts in our self rna. endosomal sensing of dna tlr was the first immune na sensor identified (hemmi et al., ) and is the only endolysosomal dna receptor. despite extensive transient trafficking, the signaling competent form of tlr is exclusively localized to the endolysosome, and mislocalization of tlr to the cell surface induces strong autoinflammation (mouchess et al., ) . the availability of tlr ligands in the endolysosome is tightly regulated by dnases (see nucleases and the sensing of self versus non-self dna). tlr preferentially detects ssdna containing unmethylated cytosine-phosphate-guanine (cpg) motifs which are less frequent in eukaryotic self dna (methylation of the c carbon of cytosine) compared to bacterial dna hartmann and krieg, ; krieg et al., ) , with a recent study identifying highly conserved stimulatory cpg-dna fragments in multiple s and s rdna sequences in bacterial genomes (liu et al., ) . moreover, a recent structural study reports that tlr has two binding sites, one binding ssdna with an unmethylated cpg motif and the second binding short ssdna carrying a hydroxyl . the second site prefers hydroxyl ssdna with a cytosine at the second position from the end ( -xcx dna). combined cpg and -xcx dna binding cooperatively promotes tlr dimerization and activation. in line with this, previously established potent tlr ligands such as odn contain both structural elements, unmethylated cpg motifs and a cytosine at the second position from the end . previous work on stimulatory tlr ligands reveal important species-specific differences. in recent years, the sequence specificity of dna ligands for both humans and mice has been further investigated (pohar et al., a (pohar et al., , b . in these studies, the minimal dna sequence for human tlr has been defined as , and the -tcg was found to be essential for activation, consistent with the two binding sites identified by ohto et al. ( ) . interestingly, mouse tlr has a much lower requirement for the -xcx sequence than human tlr pohar et al., b) . in fact, mouse tlr can be dimerized by cpg dna alone due to tlr -cpg dna interactions unique to mice . not only ligand specificity but also the cellular expression pattern differs between murine and human tlr . whereas, in the mouse, tlr is widely expressed in immune cells, including myeloid cells, in humans, it is restricted to b cells and pdc (barchet et al., ) , and their downstream immune effects differ accordingly. notably, tasl was recently identified as an additional signaling protein linking tlr to irf (heinz et al., ) . the discovery that dsdna induces type-i ifn came years after dsrna stetson and medzhitov, ) . moreover, the first pathway identified involved rna but not dna sensing (ablasser et al., ; chiu et al., ). like polyi:c, poly(da:dt) was used as a standard dna stimulus due to its strong ifn-i activation and its amenability to enzymatic synthesis. however, poly(da:dt) provides a template for transcription of au-rich rna by the pol iii pathway. these polyua transcripts bear triphosphate and fold into dsrna, thus acting as rig-i ligands and inducing type-i ifn independently of a true dna sensor. at the end of , the cgamp synthase (cgas)-stimulator of interferon genes (sting) pathway was identified as the principle cytosolic dsdna sensor inducing type-i ifn wu et al., ) . a number of other candidate receptors have previously been proposed and may have accessory and/or cell-type-specific functions (see figure and box , cytosolic dna and type-i ifn). the carboxyl terminus of cgas binds to the ribose-phosphate backbone of dsdna in a sequence-independent manner, inducing dimerization and formation of the cyclic dinucleotide (cdn) c[g( - )pa( - ʹ)p]) ( ' cgamp). ' cgamp then acts as a second messenger (ablasser et al., a; gao et al., a) , activating sting, followed by tbk and irf phosphorylation. the downstream sensor sting itself is also a cytosolic (burdette et al., ; gao et al., b) . notably, murine sting was far more reactive to bacterial cdn than human sting (ablasser et al., a; gao et al., b) , with hsting h showing near-perfect selectivity for ' -cgamp (gao et al., b) . nevertheless, several natural polymorphisms in human sting influence the sensitivity to ' -cgamp and '-cdn (yi et al., ) , and the most common sting variant, hsting r shows a moderate selectivity for ' -cgamp (gao et al., b) . further studies will be needed to elucidate the relevance of human sting as a direct cdn sensor in host-pathogen interaction. the role of the pyhin proteins ifi and p in sting activation has been controversial (see box , cytosolic dna and type-i ifn). human ifi and its reported ortholog, murine p , are coded by the aim -like receptor (alr) locus, an evolutionary hotbed (see dna-induced inflammasome activation). an initial publication has demonstrated that rnai targeting human ifi and murine p reduces ifn-i induction from human thp- cells, murine embryonic fibroblasts, and raw . macrophages, respectively (unterholzner et al., ) . however, the importance of p has been challenged by subsequent studies (brunette et al., ; gray et al., ) , causing some confusion in the field. further studies using genome editing in human cells could provide clear evidence for the role of ifi in ifn-induction in keratinocytes and macrophages, both in supporting cgas signaling and cgas-independent dna sensing (almine et al., ; jønsson et al., ) . however, due to the genetic diversity of the alr locus, it is possible that this role in dna sensing is unique to primates. non-comprehensive overview of the most relevant dna-sensing receptors at the relevant localization, their downstream signaling molecules, and the functional outcome and secondary consequences as applicable (e.g., sting sensing of cgas product cgamp). dna-sensing receptors are in gray, signaling molecules in yellow, nucleases in green, and inflammasome pathways is purple. the signaling competent form of tlr is exclusively localized to the endolysosome and preferentially detects ssdna containing unmethylated cytosine-phosphate-guanine (cpg) motifs which are less frequent in eukaryotic self dna compared to bacterial dna. tlr has two binding sites, one site binding ssdna with an unmethylated cpg motif and the second site binding short ssdna carrying a hydroxyl, both cooperatively promoting tlr dimerization and signaling, including the recently identified signaling component tasl linking signaling to downstream irf . tlr ligands in the endolysosome are tightly regulated by endolysosomal dnase ii, pld , and pld which coordinately degrade single-and double-stranded dna. cytosolic dna sensing includes the rna sensor rig-i which detects rna transcribed from poly(da:dt) by pol iii. the cgamp synthase (cgas)/stimulator of interferon genes (sting) pathway is the principle cytosolic dsdna sensor pathway, activating a type-i ifn response, and via sting a nlrp dependent inflammasome response. availability of cytosolic dna for this pathway is regulated by dnase iii (trex ). accessory proteins for cgas are hmgb , the gtpase-activating protein sh domain-binding protein (g bp ), tfam, and cchc-type zinc-finger protein (zcchc ) which bind, bend, and stabilize dsdna in a manner amenable to the nucleation of cgas dimers along the dsdna strand. cgas is also localized in the nucleus where it senses foreign but not self dna. here, non-pou domain-containing octamer binding protein (nono), but also hmgb and tfam, assist in binding of dna to cgas. ifi is a nuclear restriction factor, binding, and silencing viral or transfected dna. the dna damage protein rad upon stimulation with dsdna induces proil b via the card -bcl pathway. in the human system, a sting-independent dna sensing pathway via the dna damage response protein dna-dependent protein kinase (dna-pk) senses linear dna, and signals via irf /irf and hspa /hsc . absent in melanoma (aim ) is the principle cytosolic dsdna sensor responsible for inflammasome activation. several family members of the apolipoprotein b editing complex (apobec) act as restriction factors by performing c-to-u editing on first(minus) strand cdna, resulting in g-to-a mutations in the plus strand. the sterile alpha-motif (sam) and histidine-aspartate (hd) domaincontaining protein (samhd ) is a deoxynucleoside triphosphate (dntp) triphosphohydrolase removing tripolyphosphate moieties from dntps thereby reducing the intracellular dntp concentration and inhibiting reverse transcription. in addition, a recent publication has identified a sting-independent dna sensing pathway (sidsp) in human but not murine cells via dna-dependent protein kinase (dna-pk) (burleigh et al., ) , a protein previously associated with sting-dependent responses (see box , cytosolic dna and type-i ifn). although dna-pk is important for sensing dna damage, sidsp does not occur downstream dna damage but requires the introduction of foreign dna. in contrast to cgas, dna-pk requires linear ligands, suggesting that it senses dsdna termini. its signaling pathway requires irf and/or irf but not tbk and possibly the dna-pk dependent phosphorylation of hspa and hsc . of note, a much earlier study has observed that dna-pk could directly phosphorylate irf (karpova et al., ) . to date, it remains unclear whether the sting-dependent dna-pk pathway in mice is related to sidsp in humans or what would be the basis for a species-specific requirement for sting activity. another dna damage protein that has been implicated in dna sensing is rad . upon stimulation with dsrna, rad has been reported to induce proil b mrna via the card -bcl pathway, thus providing proil b for concurrent dna-induced inflammasome activation (see dna-induced inflammasome activation) (roth et al., ) . cytosolic mislocalization of dsdna was long thought to be required for its sensing (schlee and hartmann, ) . however, many dna viruses replicate in the nucleus , and it is also the site of retroviral integration into genomic dna (volkman and stetson, ) . thus, despite the wealth of nuclear self dna, host defense in this compartment would be advanta-geous. several recent publications have demonstrated that cgas also localizes to the nucleus where it senses foreign but not self dna (gentili et al., ; volkman et al., ) as well as inhibiting homologous recombination and dna repair liu et al., ) , processes requiring direct interaction with nuclear self dna. one study has observed nuclear ''tethering'' of cgas via its ntase domain preventing activation by self dna, but how this tethering is regulated or relaxed to allow cgas to perform its various nuclear functions, such as the sensing of hiv- (lahaye et al., ) , remains unclear. ifi is also reported to participate in nuclear retroviral sensing as an inflammasome-forming protein (see next section). furthermore, several reports provide evidence that ifi works as a nuclear restriction factor, efficiently binding and silencing viral or transfected dna orzalli et al., ) . likewise, another alr pyhin (ifix) is reported to restrict herpesvirus in the nucleus (diner et al., a) . future studies will be necessary to characterize how these and other dna sensors act in the nuclear compartment. three dna-sensing inflammasomes have been described to date (see figure ): the absent in melanoma (aim ), ifi inflammasomes, and nlrp activation secondary to stingmediated cell lysis. aim and ifi are both alrs (brunette et al., ; unterholzner et al., ) , which can bind dna via their hin domain(s) and engage in homotypic pyrin-pyrin domain interaction with the inflammasome adaptor protein asc. the alr locus is genetically diverse: unique to mammals, it contains gene in marsupials and cows, in humans, in rats, and in mice, and is absent in monotremes and bats (ahn et al., a large number of receptors have been implicated in the immune sensing of cytosolic dna, including dhx , dhx , ddx , dai (zbp- ), mre , dna-pk/ku /ku , the alrs pyhin , ifi (p ), and the nucleotidyltransferase cgas (diner et al., b; unterholzner, ) . the type-i ifn response to cytosolic dna was described relatively late in the history of the field stetson and medzhitov, ) . here, the two principle ligands used were an annealed, synthetic, mixedsequence -mer designed to avoid cpg motifs, known as interferon-stimulatory dna (isd) (stetson and medzhitov, ) and the dsdna sequence poly(da-dt):poly(dt-da), known as poly(da:dt) synthesized in an enzymatic reaction . soon after, it was established that stimulator of interferon genes (sting) was involved dna-mediated activation of irf (ishikawa and barber, ; zhong et al., ) , and all of the putative receptors, except for dhx and dhx , were reported to act upstream of sting in some manner. while it is entirely possible that redundancies in function, species or cell-specific effects or other differences can account for the plethora of dna sensors postulated, these somewhat contradictory studies still pose a conundrum for the field. moreover, two groups also found that poly(da:dt) also activated rig-i after its transcription into rna by rnase polymerase iii (ablasser et al., ; chiu et al., ) , opening the question whether other dna ligands may be indirectly sensed by other pathways. with the discovery of cgas wu et al., ) , it became clear that cgas-sting was the principle pathway of cytosolic dna sensing in mammalian cells. however, the advent of crispr-cas genome editing in has also provided important genetic proof of the function of putative human dna receptors: human ifi has been shown to act as an important dna sensor upstream of sting (almine et al., ; jønsson et al., ) . however, one study removed the entire alr locus in mice (aim + paralogs), without affecting the ifn response to cytosolic dna (gray et al., ) , effectively dispelling any notion that an alr is required for dna-induced ifn in mice. of note, a very recent publication has proposed a further cytosolic dna sensing pathway which is active in human but not murine cells, dna-pk (burleigh et al., ) . however, in contrast to previous reports in murine cells, this study demonstrates that human dna-pk acts independently of sting. altogether, there seem to be important differences in mammalian dna signaling, and we hope that further studies will genetically investigate putative dna sensing pathways in humans (and other non-murine species) and clear up the controversies remaining in the field. immunity , july , ; brunette et al., ; cridland et al., ; khare et al., ) . aim is the best conserved gene in this locus and the principle cytosolic dsdna sensor for inflammasome formation in mice (b€ urckst€ ummer et al., ; fernandes-alnemri et al., ; . however, aim is a pseudogene in several mammalian species, including cows and dogs (cridland et al., ) . moreover, in human monocytes and the monocytic model cell line, transdifferentiated blaer , dna-induced inflammasome activation is dependent on nlrp , not aim (-gaidt et al., (-gaidt et al., , , and direct sting activation can also activate nlrp in monocytes. here, sting activation leads to a lytic form of cell death and k + efflux in turn activating nlrp . however, whether this form of sting-mediated cell death occurs in other cells is unknown. in cells connected by gap junctions, this seems implausible since bystander sting activation would theoretically induce a wave of cellular lysis and pyroptosis. moreover, aim acts as a dna-sensing inflammasome protein in other human myeloid cell types, including thp- cells (gaidt et al., ) and human macrophages (su et al., ) . human ifi forms a nuclear inflammasome in t cells, sensing hiv- cdna and inducing pyroptosis . ifi also senses kaposi sarcoma-associated herpesvirus (ksvh) in primary human endothelial cells (hmvec) and the monocytic cell line thp- (kerur et al., ; singh et al., ) . since pyroptosis is the predominant form of pcd ( %) of quiescent cd + t cell during hiv- infection, ifi -mediated pyroptosis may be of particular pathophysiological relevance for this disease . although p has been reported to the murine ortholog ifi , we are unaware of a report of p directly building an inflammasome, and the recent publication of the ifi À/À mouse indicates a distinct role in lps sensing (yi et al., ) . although it is conceivable that other alrs in the human and murine locus containing hin and pyrin domains could build inflammasomes, none have been reported to date. indeed, we are only beginning to understand the function of other alrs. the human alr pyrin-only protein does not contain a hin domain and is reported to act as an inflammasome inhibitor (khare et al., ) . human pyhin was reported to act as an ifn-i-inducing dna sensor (see box , cytosolic dna and type-i ifn) but has also recently been shown to control tnf and il- release downstream of sting activation (massa et al., ) . better understanding of the mammalian alrs is not only important to our own antiviral defense but also that of livestock and important mammalian viral reservoirs, such as bats for paramyxovirues and coronaviruses (drexler et al., ; li et al., ) . accessory proteins: cgas activation requires oligomerization (li et al., b) , even forming large protein foci through liquidphase separation (du and chen, ) , as previously described for p-bodies and stress granules (shin and brangwynne, ) . several accessory proteins have been reported that could contribute to lowering the activation energy for phase transition (see figure ). the dna structural proteins, hmgb and tfam, have been reported to bind, bend, and stabilize dsdna facilitating the nucleation of cgas dimers along the dsdna strand (andreeva et al., ) . other identified cofactors include cchc-type zinc-finger protein (zcchc ) (lian et al., a) , reported by the same group to act as an accessory protein to the rlrs (lian et al., b) , and gtpase-activating protein sh domain-binding protein (g bp ) (liu et al., ) . for nuclear cgas activation, the host protein non-pou domain-containing octamer binding protein (nono) supports the binding of hiv- dna to cgas (lahaye et al., ) . theoretically, these different cofactors could influence the activation state and multimeric structure of cgas. there are multiple reports of ifi acting as a viral restriction factor by suppressing viral promoters and interfering with replication (lo cigno et al., ; gariano et al., ; johnson et al., ) . nuclear ifi has been reported to bind and sequester unchromatinized dna and promote its integration into heterochromatin, thus restricting hsv- infection orzalli et al., ) , with one recent publication showing that it forms filamentous structures with viral dna, thus blocking the activity of hsv- rna pol ii (merkl and knipe, ) . the apolipoprotein b editing complex (apobec) family was discovered via a c-to-u base modification in apolipoprotein b (apob) mrna leading to a premature stop codon (arias et al., ) . the seven proteins(a-g) of the rapidly evolving primate apobec (or a ) subfamily are isgs countering retroviruses and endogenous retroelements (ito et al., ) . several apobec s act as restriction factors for hiv- , performing c-to-u editing on first(minus) strand cdna that result in plusstrand g-to-a mutations. apobecg mutates preferentially tgg motifs, converting them to a stop codon (tag) and is targeted by the hiv accessory-protein vif (along with apobec d, f, and h). apobec proteins are also active against other viruses, including parvovirus, hbv, htlv, and rsv (arias et al., ) . however, the drawbacks of this editing strategy include evolution of more virulent viral strains and genomic mutagenesis, which for apobec b has been linked to several forms of cancer (burns et al., ) . the sterile alpha-motif (sam) and histidine-aspartate (hd) domain-containing protein (samhd ) is a deoxynucleoside triphosphate (dntp) triphosphohydrolase that removes tripolyphosphate moieties from dntps (goldstone et al., ) . samhd was initially identified as a myeloid-cell specific hiv- restriction factor and target of the lentivirus auxiliary protein vpx (laguette et al., ) . samhd decreases the concentration of intracellular dntps in myeloid cells to inhibit reverse transcription (goldstone et al., ) , and also inhibits the replication of dna viruses, such as hsv- (hollenbaugh et al., ) . hypomorphic variants of samhd induce type-i interferonopathy in patients and mice, which is dependent on the cgas-sting pathway (maelfait et al., ) . similarly, hypomorphic variants of the principal cytosolic nuclease in mammalian cells, dnase three-prime repair exonuclease (trex or dnase iii), are linked to type-i interferonopathy as well as familial chilblain lupus (fcl) and systemic lupus erythematosus (sle) (rodero and crow, ) . trex degrades ss-and dsdna (grieves et al., ) , and trex -deficiency leads to cytosolic accumulation of dna ligands (stetson et al., ) and activation of the cgas-sting pathway (ablasser et al., ; gehrke et al., ) . trex À/À mice develop systemic inflammation (morita et al., ) ameliorated by simultaneous deficiency in sting or cgas (gray et al., ) . moreover, modifications such as uv-light exposure render dna trex -resistant and potentiate cgas activation (gehrke et al., ) . dnase ii is the predominant endolysosomal endonuclease and also degrades ss-and dsdna (drew, ) . dnase ii deficiency leads to dsdna accumulation which enters the cytoplasm, causing overwhelming inflammation (kawane et al., ; yoshida et al., ) . patients with expressing hypomorphic dnase ii present also present with type-i interferonopathy (rodero et al., ) , and dnase ii deficiency is embryonically lethal but can be rescued by ifnar deficiency (kawane et al., ) . of note, cgas-sting and aim both contribute to the phenotype of dnase ii deficiency (ahn et al., ; baum et al., ; jakobs et al., ) . recently, two further endosomal nucleases have been discovered, phospholipase d (pld ) and d pld . unlike dnase ii, pld and pld are exonucleases which degrade ssdna from the terminus (gavin et al., ) . this discovery has its origins in genome-wide association studies linking pld to alzheimer disease and pld to rheumatoid arthritis and systemic sclerosis. pld -deficient mice demonstrate elevations in ifn-g and splenomegaly. pld and pld activity requires a oh terminus and the absence of secondary structure, both features of dnase ii cleavage products, enabling it to act cooperatively with dnase ii (see next section). nucleases and the sensing of self versus non-self dna initially, sensing of dsdna was thought to rely entirely on subcellular localization (see figures and ) . while dna does not undergo the same exquisite processing as rna, nuclear dna sensing clearly depends on hallmarks of self and non self, and dna modifications can strongly affect the availability of cytosolic and endosomal dna. dna sensing is tightly controlled by its availability. cytosolic dsdna must contend with the high-affinity -exonuclease trex . similarly, activation of tlr is antagonized by the -exonuclease activity of pld / in the endosome. exonuclease activity is an elegant strategy for controlled sensor activity: ( ) the na ligand is degraded base by base, allowing for a gradual removal of ligands, ( ) whereas inhibiting endonucleases requires extensive modification to a substrate, exonucleases can be blocked by a simple modification on the or terminus, which can, in turn, be removed by an endonuclease. cytosolic dsdna becomes potently stimulatory with modifications that inhibit trex degradation, e.g., 'oh guanosine after uv irradiation (gehrke et al., ) . endosomal pld and only degrades ssdna and is inhibited by a p-terminus (gavin et al., ) . dnase i, trex , and caspase-activated dnase perform p- oh cleavage of phosphodiester bonds. thus, incoming endosomal dna will be predominantly ( ) double-stranded and ( ) have a p terminus, rendering it inaccessible to pld and . in contrast, dnase ii is a oh- p endonuclease with activity on ss-and dsdna and is thus critically required to render dna accessible to pld and pld . furthermore, phosphorothioate (pto) modification also inhibits pld and activity -to- -fold. pto-linkage has been employed for decades to potentiate stimulatory oligonucleotides and also occurs naturally in bacterial dna, rendering it a better stimulus for tlr . however, single pto modifications do not inhibit the endonuclease dnase ii. in parallel to the role of rnase t and rnase for tlr , dnase ii does not inhibit tlr activity. rather, it is critically required for the generation of tlr ligands from complex natural ligands (chan et al., ; pawaria et al., ) by releasing ssdna from genomic dsdna and generating oh termini for the second binding pocket . as with dsrna, length is a key determinant of whether dsdna is stimulatory. trex is non-processive, so longer dsdna will have a longer half-life in the cytosol (hö ss et al., ) . longer dsdna is more easily bent into structures amenable to dimer formation and cgas-dependent phase transformation (andreeva et al., ; du and chen, ; hooy and sohn, ) . for ifi and aim , longer naked dsdna stretches are more amenable to the oligomerization of adjacent pyrin domains, a process which supports hin -dsdna binding and stability (morrone et al., (morrone et al., , , provides a platform for the pyrinpyrin interaction with asc during inflammasome activity (matyszewski et al., ) , and effectively sequesters and neutralizes long dsdna molecules. the influence of dsdna length on receptor oligomerization, signal strength, and duration may account for the distinct signaling outcomes, e.g., restriction, cytokine induction, cell death, observed with different stimuli and cell types. for humans, cgas require dsdna of at least bp and aim requires bp, whereas murine cgas sensing can occur with shorter sequences (r bp) (fernandes-alnemri et al., ; jin et al., ; luecke et al., ) . at the structural level, bp was shown to be the minimal length for the assembly of two human cgas dimers (andreeva et al., ) . whereas ifi is reported to require at least - bp for ifn activation (unterholzner et al., ) , we are not aware of a study determining the minimal length necessary for inflammasome activation and other ifi effector functions. another important aspect of dsdna sensing is packaging. an essential distinction between self and non self in the nucleus is the tight winding of chromosomal dna (gentili et al., ; volkman et al., ) , which allows cgas binding but not activation. this seems difficult to reconcile a priori with the activation of cytosolic cgas by micronuclei (harding et al., ; mackenzie et al., ) . possibly, micronuclei contain unwound regions or other features amenable to cgas sensing, or cgas activation has different requirements in the cytosol. understanding this process is of great importance to studies of cgas-mediated cellular senescence. a notable exception to the length requirement is cgas activation by g-rich y-form dna, base-paired dna r bp containing an unpaired g in adjacent ssdna . this mechanism could explain various forms of ssdna sensing attributed to cgas, including secondary-structured ssdna originating from retroviral genomes (coquel et al., ; herzner et al., ) . although direct sensing of y-dna was demonstrated in a cellfree system , an accessory protein may contribute in cellulo as has been observed for many other ligands. regardless, y-dna-mediated cgas activation provides evidence of -and -terminal sensing (here, of guanosines) of cytosolic dsdna. another recent example is the sting-independent dna-pk pathway, which fitting its function as a sensor of dna damage and due to its requirement for linear dsdna, seems interacts with dsdna termini, although the precise structural features required remain unknown (burleigh et al., ) . immunity , july , sting is can be activated cell intrinsically and in bystander cells by endogenous ' cgamp (ablasser et al., b; gao et al., a) . ' cgamp is targeted by hydrolysing poxins expressed by members of the poxviridae, such as vaccinia virus (eaglesham et al., ) . clearly, inhibiting cgas-sting signaling is of advantage to dna viruses. in contrast, certain human sting alleles have evolved to tolerate bacterial cdn while retaining sensitivity for endogenous cgamp. this intentional desensitization to non self is highly unusual and thus likely indicative of a strong evolutionary advantage for not avoiding ifn induction in response to certain bacteria. here, it should be noted that the role of ifn in bacterial defense remains rather unclear and differs starkly between bacterial species (boxx and cheng, ) . there is abundant self ss-and dsdna in the endolysosome of phagocytes, requiring a strategy beyond sensing of the ribosephosphate backbone. to avoid unnecessary activation: ( ) tlr signaling is strictly contained within the endolysosomal compartment via an intricate system of trafficking and proteolytic activation, ( ) tlr activation is regulated by the activity of endosomal dnases (see previous section), and ( ) tlr senses unmethylated cpg dinucleotides within ssrna. cpg motifs are suppressed in eukaryotic dna (krieg, ) , since their methylation can drive their hydrolytic deamination (shen et al., ) . however, unmethylated cpg-rich dna can be found in bacteria, viruses, and fungal pathogens. the tremendous progress outlined above suggests a tight functional integration of innate immune responses and cell-autonomous defense mechanisms in response to exogenous na. in this context, na-related research to date has followed three, almost completely separate, principle research directions: immune sensing of na, antiviral restriction factors, and na metabolism. we now understand that those three principles are all integral parts of a tightly controlled na defense system. impairment or failure of this system caused by dysregulation, genetic alterations, or pathogen challenge causes erroneous detection of self na resulting in autoinflammation. therefore, an improved understanding of the intricate mechanisms that govern the distinction of endogenous and exogenous na as summarized in this review will guide better diagnostic procedures and treatments for inflammatory and infectious diseases. in particular, this insight allows for targeted activation of functionally distinct na-sensing pathways in order to elicit immune response pathways that have not been previously accessible for treatment. gunther hartmann is a founder of rigontec gmbh which was acquired by msd in . gunther hartmann is an inventor of the following patents: au b , us b , us a , us a , us b , us b , us b , us b , us a , hrp t , us a , au a , za b, ep a , ep a , us a , ep b , hk a , au a , ma b , au a , jp a, ep a . structural basis for viral -ppp-rna recognition by human ifit proteins rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate cgas produces a - -linked cyclic dinucleotide second messenger that activates sting cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp trex deficiency triggers cell-autonomous immunity in a cgas-dependent manner breaching self-tolerance to alu duplex rna underlies mda -mediated inflammation sting manifests self dna-dependent inflammatory disease unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing recognition of double-stranded rna and activation of nf-kappab by toll-like receptor ifi and cgas cooperate in the activation of sting during dna sensing in human keratinocytes rna exosome complex regulates stability of the hepatitis b virus x-mrna transcript in a non-stop-mediated (nsd) rna quality control mechanism accessing the therapeutic potential of immunostimulatory nucleic acids sting contributes to abnormal bone formation induced by deficiency of dnase ii in mice evolution of caspase-mediated cell death and differentiation: twins separated at birth tlr senses staphylococcus aureus rna in human primary monocytes and macrophages and induces ifn-b production via a tak -ikkb-irf signaling pathway rnai-mediated antiviral immunity in mammals proapoptotic signaling induced by rig-i and mda- results in type i interferon-independent apoptosis in human melanoma cells molecular mechanism of signal perception and integration by the innate immune sensor retinoic acidinducible gene-i (rig-i) intracellular toll-like receptors the roles of type i interferon in bacterial infection intracellular nucleic acid sensing triggers necroptosis through synergistic type i ifn and tnf signaling innate immune pattern recognition: a cell biological perspective extensive evolutionary and functional diversity among mammalian aim -like receptors the innate immune sensor lgp activates antiviral signaling by regulating mda -rna interaction and filament assembly broad expression of toll-like receptors in the human central nervous system an orthogonal proteomic-genomic screen identifies aim as a cytoplasmic dna sensor for the inflammasome sting is a direct innate immune sensor of cyclic di-gmp human dna-pk activates a sting-independent dna sensing pathway evidence for apobec b mutagenesis in multiple human cancers a study of the interferon antiviral mechanism: apoptosis activation by the - a system rna template-directed rna synthesis by t rna polymerase rnase l activates the nlrp inflammasome during viral infections dnase ii-dependent dna digestion is required for dna sensing by tlr rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway human adar prevents endogenous rna from triggering translational shutdown the nuclear dna sensor ifi acts as a restriction factor for human papillomavirus replication through epigenetic modifications of the viral promoters higher activation of tlr in plasmacytoid dendritic cells by microbial dna compared with self-dna based on cpg-specific recognition of phosphodiester dna human tlr senses rna from plasmodium falciparum-infected red blood cells which is uniquely required for the ifn-g response in nk cells cyclic gmp-amp signalling protects bacteria against viral infection the specificity of interferon induction in chick embryo cells by helical rna samhd acts at stalled replication forks to prevent interferon induction the mammalian pyhin gene family: phylogeny, evolution and expression irak kinase activity controls tolllike receptor-induced inflammation through the transcription factor irf in primary human monocytes rna viruses promote activation of the nlrp inflammasome through cytopathogenic effect-induced potassium efflux -o methylation of the viral mrna cap evades host restriction by ifit family members cutting edge: priming of cd t cell immunity to herpes simplex virus type requires cognate tlr expression in vivo tlr and tlr activate distinct pathways in monocytes during rna virus infection recent insights on inflammasomes, gasdermin pores, and pyroptosis identification of an lgp -associated mda agonist in picornavirus-infected cells mitochondrial double-stranded rna triggers antiviral signalling in humans viral infection switches non-plasmacytoid dendritic cells into high interferon producers innate antiviral responses by means of tlr -mediated recognition of singlestranded rna rna -o-methylation (nm) modification in human diseases the functional interactome of pyhin immune regulators reveals ifix is a sensor of viral dna the emerging role of nuclear viral dna sensors cell death by pyroptosis drives cd t-cell depletion in hiv- infection systematic discovery of antiphage defense systems in the microbial pangenome structural specificities of five commonly used dna nucleases bats host major mammalian paramyxoviruses dna-induced liquid phase condensation of cgas activates innate immune signaling viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling the skiv l rna exosome limits activation of the rig-i-like receptors bacterial rna: an underestimated stimulus for innate immune responses nlrp inflammasome regulates colonic microbial ecology and risk for colitis molecular biology of rna interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded rna aim activates the inflammasome and cell death in response to cytoplasmic dna cpg dinucleotides inhibit hiv- replication through zinc finger antiviral protein (zap)-dependent and -independent mechanisms inducers of interferon and host resistance. ii. multistranded synthetic polynucleotide complexes the cellular nmd pathway restricts zika virus infection and is targeted by the viral capsid protein identification of rna sequence motifs stimulating sequence-specific tlr -dependent immune responses cytosolic double-stranded rna activates the nlrp inflammasome via mavs-induced membrane permeabilization and k+ efflux the intra-and extracellular functions of asc specks rna modifications modulate activation of innate toll-like receptors the dna inflammasome in human myeloid cells is initiated by a sting-cell death program upstream of nlrp modeling primary human monocytes with the trans-differentiation cell line blaer tlr is involved in sequence-specific sensing of singlestranded rnas in human macrophages inhibition of retroviral rna production by zap, a ccch-type zinc finger protein ] is the metazoan second messenger produced by dna-activated cyclic gmp-amp synthase structure-function analysis of sting activation by c the intracellular dna sensor ifi gene acts as restriction factor for human cytomegalovirus replication pld and pld are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing discrimination of self and non-self ribonucleic acids oxidative damage of dna confers resistance to cytosolic nuclease trex degradation and potentiates stingdependent immune sensing the n-terminal domain of cgas determines preferential association with centromeric dna and innate immune activation in the nucleus an rna editor, adenosine deaminase acting on double-stranded rna (adar ) essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence hiv- restriction factor samhd is a deoxynucleoside triphosphate triphosphohydrolase immunomodulatory functions of type i interferons antiviral immunity via rig-i-mediated recognition of rna bearing -diphosphates mouse superkiller- -like helicase ddx is dispensable for type i ifn induction and immunity to multiple viruses tlr is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c u), but not poly(i:c): differential recognition of synthetic dsrna molecules cutting edge: cgas is required for lethal autoimmune disease in the trex -deficient mouse model of aicardi-goutiè res syndrome the aim -like receptors are dispensable for the interferon response to intracellular dna vitro transcription systems. cold spring harb. protoc. . published online tlr is a sensor of rnase t degradation products exonuclease trex degrades double-stranded dna to prevent spontaneous lupus-like inflammatory disease hiv- blocks the signaling adaptor mavs to evade antiviral host defense after sensing of abortive hiv- rna by the host helicase ddx signalling strength determines proapoptotic functions of sting the zinc-finger antiviral protein recruits the rna processing exosome to degrade the target mrna sequestration by ifit impairs translation of 'o-unmethylated capped rna rig-i detects triphosphorylated rna of listeria monocytogenes during infection in non-immune cells the arms race between bacteria and their phage foes mitotic progression following dna damage enables pattern recognition within micronuclei nucleic acid immunity mechanism and function of a newly identified cpg dna motif in human primary b cells delineation of a cpg phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo pandemic h n influenza a viruses suppress immunogenic ripk -driven dendritic cell death rnaset mutant zebrafish model familial cystic leukoencephalopathy and reveal a role for rnase t in degrading ribosomal rna nucleotide sequence of sindbis viral rna species-specific recognition of single-stranded rna via toll-like receptor and tasl is the slc a -associated adaptor for irf activation by tlr - a toll-like receptor recognizes bacterial dna small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway rnaset -deficient cystic leukoencephalopathy resembles congenital cytomegalovirus brain infection sequence-specific activation of the dna sensor cgas by y-form dna structures as found in primary hiv- cdna codon bias confers stability to human mrnas a human dna editing enzyme homologous to the escherichia coli dnaq/mutd protein mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells the alpha/beta interferon response controls tissue tropism and pathogenicity of poliovirus structural basis for species-specific activation of mouse toll-like receptor a toll-like receptor-independent antiviral response induced by double-stranded b-form dna sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling retroviruses drive the rapid evolution of mammalian apobec genes tlr signaling tailors innate immune responses in human microglia and astrocytes aim drives joint inflammation in a self-dna triggered model of chronic polyarthritis chromatin-bound cgas is an inhibitor of dna repair and hence accelerates genome destabilization and cell death structures of the hin domain:dna complexes reveal ligand binding and activation mechanisms of the aim inflammasome and ifi receptor ifi restricts hsv- replication by accumulating on the hsv- genome, repressing hsv- gene expression, and directly or indirectly modulating histone modifications ifi is required for dna sensing in human macrophages by promoting production and function of cgamp sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna translating nucleic acid-sensing pathways into therapies human tlr or tlr independently confer responsiveness to the antiviral compound r- the rig-i/mavs signaling pathway in cancer cell-selective apoptosis central roles of nlrs and inflammasomes in viral infection mrna is an endogenous ligand for toll-like receptor compositional differences within and between eukaryotic genomes interferon regulatory factor- is an in vivo target of dna-pk cell type-specific involvement of rig-i in antiviral response differential roles of mda and rig-i helicases in the recognition of rna viruses length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene interferon-alpha induction through toll-like receptors involves a direct interaction of irf with myd and traf ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction chronic polyarthritis caused by mammalian dna that escapes from degradation in macrophages samhd is the dendritic-and myeloid-cell-specific hiv- restriction factor counteracted by vpx nono detects the nuclear hiv capsid to promote cgas-mediated innate immune activation recognition of double-stranded rna and regulation of interferon pathway by toll-like receptor the tlr signaling complex forms by cooperative receptor dimerization regulation of protein synthesis: activation by double-stranded rna of a protein kinase that phosphorylates eukaryotic initiation factor sequence specific detection of bacterial s ribosomal rna by tlr bats are natural reservoirs of sars-like coronaviruses rna interference functions as an antiviral immunity mechanism in mammals cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization induction and suppression of antiviral rna interference by influenza a virus in mammalian cells identification of antiviral roles for the exon-junction complex and nonsense-mediated decay in flaviviral infection zcchc is a co-sensor of cgas for dsdna recognition in innate immune response the zinc-finger protein zcchc binds rna and facilitates viral rna sensing and activation of the rig-i-like receptors severe influenza pneumonitis in children with inherited tlr deficiency structural basis of toll-like receptor signaling with double-stranded rna nuclear cgas suppresses dna repair and promotes tumorigenesis g bp promotes dna binding and activation of cgas the discovery of potent immunostimulatory cpg-odns widely distributed in bacterial genomes distinct rig-i and mda signaling by rna viruses in innate immunity ddx suppresses type i interferons and favors viral replication during arenavirus infection vtx- is a novel tlr agonist that activates nk cells and augments adcc immune modulation by human secreted rnases at the extracellular space cgas is activated by dna in a length-dependent manner a novel mitochondrial mavs/caspase- platform links rna virus-induced innate antiviral signaling to bax/bak-independent apoptosis cgas surveillance of micronuclei links genome instability to innate immunity restriction by samhd limits cgas/sting-dependent innate and adaptive immune responses to hiv- sensing of viral and endogenous rna by zbp /dai induces necroptosis nucleic acid sensors and programmed cell death antiviral rna interference in mammalian cells inactivation of the type i interferon pathway reveals long double-stranded rna-mediated rna interference in mammalian cells small self-rna generated by rnase l amplifies antiviral innate immunity rnase l releases a small rna from hcv rna that refolds into a potent pamp zbp and tak : master regulators of nlrp inflammasome/pyroptosis, apoptosis, and necroptosis (pan-optosis) reassessing the evolutionary importance of inflammasomes structure and function of the interferon-beta enhanceosome differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor- short double-stranded rnas with an overhanging ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells pyhin regulates pro-inflammatory cytokine induction rather than innate immune dna sensing in airway epithelial cells establishment of a monoclonal antibody against human toll-like receptor that blocks double-stranded rna-mediated signaling trail and noxa are selectively upregulated in prostate cancer cells downstream of the rig-i/mavs signaling pathway by nonreplicating sendai virus particles digital signaling network drives the assembly of the aim -asc inflammasome toll-like receptor (tlr ) plays a major role in the formation of rabies virus negri bodies role for a filamentous nuclear assembly of ifi , dna, and host factors in restriction of herpesviral infection interferon-inducible gtpases in cell autonomous and innate immunity synthetic polynucleotides the dhx rna helicase senses cytosolic rna and activates the nlrp inflammasome ddx , a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling attacked from all sides: rna decay in antiviral defense identification of host cytosolic sensors and bacterial factors regulating the type i interferon response to legionella pneumophila ifi dna sensor is required for death of lymphoid cd t cells abortively infected with hiv gene-targeted mice lacking the trex (dnase iii) -> dna exonuclease develop inflammatory myocarditis cooperative assembly of ifi filaments on dsdna provides insights into host defense strategy assembly-driven activation of the aim foreign-dsdna sensor provides a polymerization template for downstream asc transmembrane mutations in toll-like receptor bypass the requirement for ectodomain proteolysis and induce fatal inflammation stem-loop recognition by ddx facilitates mirna processing and antiviral defense spatio-temporal characterization of the antiviral activity of the xrn -dcp / aggregation against cytoplasmic rna viruses to prevent cell death sidt transports extracellular dsrna into the cytoplasm for innate immune recognition apoptotic caspases suppress type i interferon production via the cleavage of cgas, mavs, and irf ripk activates parallel pathways of mlkl-driven necroptosis and fadd-mediated apoptosis to protect against influenza a virus toll-like receptor contains two dna binding sites that function cooperatively to promote receptor dimerization and activation tlr recognizes bacterial s rrna devoid of erythromycin resistance-forming modification viral infections activate types i and iii interferon genes through a common mechanism nuclear interferon-inducible protein promotes silencing of herpesviral and transfected dna ti-cam- , an adaptor molecule that participates in toll-like receptor -mediated interferon-beta induction dead/h box (ddx ) helicase binds the rig-i adaptor ips- to up-regulate ifn-betainducing potential the tlr /ticam- pathway is mandatory for innate immune responses to poliovirus infection accessory factors of cytoplasmic viral rna sensors required for antiviral innate immune response immune sensing of synthetic, bacterial, and protozoan rna by toll-like receptor requires coordinated processing by rnase t and rnase defects of mitochondrial rna turnover lead to the accumulation of double-stranded rna in vivo dhx is a coreceptor for rlr signaling that promotes antiviral defense against rna virus infection cutting edge: dnase ii deficiency prevents activation of autoreactive b cells by double-stranded dna endogenous ligands toll-like receptor in viral pathogenesis: friend or foe? rig-i-mediated antiviral responses to single-stranded rna bearing -phosphates activation of mda requires higher-order rna structures generated during virus infection the role of unc b protein in surface localization of tlr receptor and in cell priming to nucleic acid agonists short single-stranded dna degradation products augment the activation of toll-like receptor selectivity of human tlr for double cpg motifs and implications for the recognition of genomic dna human virus-derived small rnas can confer antiviral immunity in mammals rig-i rna helicase activation of irf transcription factor is negatively regulated by caspase- -mediated cleavage of the rip protein rig-i detects viral genomic rna during negative-strand rna virus infection mapping the dsrna world rig-i selectively discriminates against -monophosphate rna the evolution of vertebrate toll-like receptors type i interferon-mediated monogenic autoinflammation: the type i interferonopathies, a conceptual overview type i interferon-mediated autoinflammation due to dnase ii deficiency apoptotic caspases prevent the induction of type i interferons by mitochondrial dna rad -card interactions link cytosolic dna sensing to il- b production adenosine deaminases acting on rna (adars) are both antiviral and proviral adenosine deaminase acting on rna (adar ), a suppressor of double-stranded rna-triggered innate immune responses combating herpesvirus encephalitis by potentiating a tlr -mtorc axis lgp is a positive regulator of rig-i-and mda -mediated antiviral responses master sensors of pathogenic rna -rig-i like receptors discriminating self from non-self in nucleic acid sensing recognition of triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus dna virus replication compartments -triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i identification of an optimized -o-methylated trinucleotide rna motif inhibiting toll-like receptors and a conserved histidine in the rna sensor rig-i controls immune tolerance to n - 'o-methylated self rna caspase- -mediated cleavage inhibits irf- protein by facilitating its proteasome-mediated degradation reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf the rate of hydrolytic deamination of -methylcytosine in double-stranded dna molecular mechanism for nlrp inflammasome assembly and activation a novel toll-like receptor that recognizes vesicular stomatitis virus guanosine and its modified derivatives are endogenous ligands for tlr liquid phase condensation in cell physiology and disease kaposi's sarcoma-associated herpesvirus latency in endothelial and b cells activates gamma interferoninducible protein -mediated inflammasomes double-stranded rna is detected by immunofluorescence analysis in rna and dna virus infections, including those by negative-stranded rna viruses structural basis for specific recognition of single-stranded rna by toll-like receptor role of inosine À uracil base pairs in the canonical rna duplexes recognition of cytosolic dna activates an irf -dependent innate immune response trex prevents cell-intrinsic initiation of autoimmunity immune checkpoint inhibition overcomes adcp-induced immunosuppression by macrophages helicase proteins dhx and rig-i cosense cytosolic nucleic acids in the human airway system cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway toll-like receptors and as essential components of innate immune defense against mouse cytomegalovirus infection lgp virus sensor regulates gene expression network mediated by trbp-bound micrornas virus sensor rig-i represses rna interference by interacting with trbp through lgp in mammalian cells cg dinucleotide suppression enables antiviral defence targeting non-self rna agonist-mediated activation of sting induces apoptosis in malignant b cells toll-like receptor senses degradation products of single-stranded rna toll-like receptor recognizes incomplete stem structures in single-stranded viral rna dai senses influenza a virus genomic rna and activates ripk -dependent cell death self-coded -extension of run-off transcripts produces aberrant products during in vitro transcription with t rna polymerase the interferon response to intracellular dna: why so many receptors? ifi is an innate immune sensor for intracellular dna the rig-i-like receptor lgp inhibits dicer-dependent processing of long double-stranded rna and blocks rna interference in mammalian cells the enemy within: endogenous retroelements and autoimmune disease tight nuclear tethering of cgas is essential for preventing autoreactivity toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis nlrp regulates intestinal antiviral innate immunity inflammasome activation triggers caspase- -mediated cleavage of cgas to regulate responses to dna virus infection raftlin is involved in the nucleocapture complex to induce poly(i:c)-mediated tlr activation double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses toll-like receptor (tlr) immune modulation by unformulated small interfering rna or dna and the role of cd (in tlr-mediated effects) apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production in vitro-transcribed guide rnas trigger an innate immune response via the rig-i pathway the mrna cap -o-methyltransferase cmtr regulates the expression of cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna structural basis for the prion-like mavs filaments in antiviral innate immunity role of adaptor trif in the myd -independent toll-like receptor signaling pathway crystal structure of isg reveals a novel rna binding structure and potential functional mechanisms cgas is essential for cellular senescence control of antiviral innate immune response by protein geranylgeranylation interferon-l orchestrates innate and adaptive mucosal immune responses single nucleotide polymorphisms of human sting can affect innate immune response to cyclic dinucleotides p is required for canonical lipopolysaccharide-induced tlr signaling in mice the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity dhx enhances rig-i signaling by facilitating pkr-mediated antiviral stress granule formation lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested dna tlr deficiency in patients with herpes simplex encephalitis tlr immunity to infection in mice and humans the adaptor protein mita links virussensing receptors to irf transcription factor activation expression cloning of - a-dependent rnaase: a uniquely regulated mediator of interferon action nlrp b inflammasome restricts rotavirus infection in intestinal epithelial cells dhx functions as an rna co-sensor for mda -mediated emcv-specific antiviral immunity isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase activation of endothelial toll-like receptor impairs endothelial function key: cord- - uwlmjx authors: kuchipudi, suresh v. title: the complex role of stat in viral infections date: - - journal: j immunol res doi: . / / sha: doc_id: cord_uid: uwlmjx signal transducer and activators of transcription- (stat ) regulates diverse biological functions including cell growth, differentiation, and apoptosis. in addition, stat plays a key role in regulating host immune and inflammatory responses and in the pathogenesis of many cancers. several studies reported differential regulation of stat in a range of viral infections. interestingly, stat appears to direct seemingly contradictory responses and both pro- and antiviral roles of stat have been described. this review summarized the currently known functions of stat in the regulation of viral replication and pathogenesis of viral infections. some of the key unanswered questions and the gap in our current understanding of the role of stat in viral pathogenesis are discussed. signal transducers and activators of transcription (stats) are a family of transcription factors that play crucial roles in regulating a number of diverse biological functions including cell proliferation, differentiation, apoptosis, inflammatory response, immunity, and angiogenesis [ ] . there are seven stat proteins (stats , , , , a, b, and ), which are activated by the signals from cytokine and growth factor receptors in the plasma membrane and regulate gene transcription [ ] . a unique feature of stat proteins is their dual roles, which include signal transduction through the cytoplasm and functioning as transcription factors in the nucleus [ ] [ ] [ ] . stats were first discovered through their capacity to mediate signalling from interferon (ifn) and interleukin- (il- ) receptors following binding of cytokines [ , , [ ] [ ] [ ] . each stat family protein responds to a defined set of cytokines (table ) , and certain cytokines can activate more than one stat protein [ ] . cytokine receptors on plasma membrane do not usually possess intrinsic tyrosine kinase activity and their engagement activates receptor-associated tyrosine kinases, prominent of which are janus kinase (jak) family kinases (jak , jak , jak , and tyk ) [ , , [ ] [ ] [ ] . stat proteins are activated by phosphorylation of specific tyrosine residues, following which they form stable homodimers or heterodimers with other stat proteins through reciprocal phosphotyrosine-src homology (sh ) domain interactions [ ] . stat dimers then translocate to nucleus where they regulate the transcription of a set of specific genes. a number of the downstream target genes of stats encode cytokines and growth factors, which in turn mediate autocrine and paracrine stat activation [ ] . in addition to the canonical model of jak/stat signalling, stats also form dimers in the absence of the activating tyrosine phosphorylation [ ] . in the noncanonical model, unphosphorylated stats are consistently found as a result of constant nuclear import and export. these unphosphorylated nuclear stat molecules might also contribute to gene regulation [ ] . of all the stat family proteins, stat is unique as it is known to direct seemingly contradictory responses [ ] and is essential for early embryonic development in mice [ ] . stat regulates cell-cycle progression and apoptosis, plays a key role in oncogenesis [ ] , and is aberrantly expressed in cancer cells [ ] . stat function has been extensively journal of immunology research table : activators of stat family proteins (adapted from yu et al., [ ] ). key activators stat ifn , ifn , and ifn stat ifn and ifn stat il- , il- , il- , il- , il- , lif, and osm stat il- stat a and stat b il- , gm-csf, il- , il- , il- , il- , growth hormones, and prolactin stat il- and il- studied in cell culture systems. stat is known to regulate a number of distinct responses in different cells, including induction of an acute-phase response in hepatoma cells, stimulation of proliferation in b lymphocytes, activation of terminal differentiation and growth arrest in monocytes [ ] , and maintenance of the pluripotency of embryonic stem cells [ , [ ] [ ] [ ] [ ] . the seemingly contradictory responses of stat could be explained in part by the activation of distinct sets of target gene by stat in different cells [ ] . nd ccd dbd linker sh tad the structure of stat is similar to the other stat family members comprising six structural regions, namely, nterminal domain (nd), coiled-coil domain (ccd), dnabinding domain (dbd), linker domain, sh domain, and a c-terminal transcriptional activation domain (tad) (figure ). the core fragment of stat comprising ccd, dbd, linker, and sh domains is monomeric and the dimer interface observed in the unphosphorylated stat core fragment structure is absent in the stat structure [ ] . stat is activated by phosphorylation at tyrosine in the c-terminal domain [ ] and more than different polypeptide ligands are known to cause stat phosphorylation [ ] . stat was initially described as a dna-binding factor that is capable of selectively interacting with an enhancer element in the promoter region of acute-phase genes in interleukin- (il- ) stimulated hepatocytes [ ] . subsequently, it became evident that stat can be activated by the entire il- family and other cytokines including leukemia inhibitory factor (lif), cardiotrophin- , ciliary neurotrophic factor (cntf), il- , il- , il- , il- , il- , il- , il- , il- , ifn-, tnf-, light, a member of the tnf superfamily, monocyte chemotactic protein- (mcp- ), macrophage inflammatory protein- (mip- ), ccl- /rantes, stem cell factor (scf), and oncostatin m (osm) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . various growth factor receptors also activate stat , which include epidermal growth factor receptors (egfrs), hepatocyte growth factor receptors (hgfrs), fibroblast growth factor receptors (fgfrs), platelet-derived growth factor receptors (pdgfrs), insulin-like growth factor receptors (igfrs), and vascular endothelial growth factor receptors (vegfrs) [ ] . in addition, many carcinogenic agents such as nicotine in cigarette smoke, diesel exhaust particles, bacterial lipopolysaccharide (lps), environmental stress including ultraviolet light, osmotic shock, heat shock, and oxidative stress, ca + /calmodulin-dependent protein kinase ii (camkii ), bile acids, leptin and low ph, black soy peptides, diazoxide, isoliquiritigenin, and olanzapine have been found to activate stat (reviewed by siveen et al., [ ] ). growth factor or cytokine receptor-ligand interaction results in dimerization of gp , a signal transducer protein in cytoplasm [ ] . this is followed by phosphorylation of jak family of tyrosine kinases especially jak which in turn mediates stat phosphorylation [ ] . stat has two important phosphorylation sites at tyr and ser . ser phosphorylation has been considered to be a secondary event after tyr phosphorylation. however, recent evidence suggests that ser phosphorylation on stat is not necessarily a secondary event after tyr phosphorylation but has a role in the regulation of cell survival activity and nuclear translocation of stat in melanocytic cells [ ] . further, stat phosphorylation at serine in the c-terminal transactivation domain promotes maximal transcriptional activation of a subset of target genes [ ] . furthermore, ser phosphorylation of stat appears to be important in tumorigenicity. for example, in glioma, reduced stat ser phosphorylation enhances tumorigenicity which may be regulated in part by ck -pp a pathway [ ] . the phosphorylation of stat promotes homodimerization, wherein the sh domain of each stat monomer interacts with the y residue on another monomeric stat [ ] . stat homodimers translocate into the nucleus which is mediated by importin /npi- [ ] and bind to specific dna response elements such as the ifn-stimulated response element (isre) in the promoter regions of responsive target genes and regulate their transcription [ ] [ ] [ ] (figure ). stat dimers recognize an -to -base pair inverted repeat dna element with a consensus sequence of -tt(n)aa- [ ] . further, nonphosphorylated stat monomers are also capable of dimerization and induction of transcription through binding to nfkappab [ ] . activation of jak/stat pathway begins with the binding of extracellular signalling proteins (esps) to specific receptors associated with jaks. activated jaks then activate stat proteins by phosphorylation which otherwise remain latent in the cytoplasmic [ , ] . a range of endogenous protein regulators tightly control the receptor-induced stat activation [ , [ ] [ ] [ ] . several protein tyrosine phosphatases (ptps) including shp- , ptp b, ptp c, tc , and shp- have been implicated in the termination of stat signaling. because only stat dimers bind to dna, the nuclear ptp tc may be important in the termination of stat -mediated transcriptional activation [ , ] . protein inhibitors of activated stat (pias) family members are involved in blocking the dna-binding ability of stat proteins thereby inhibiting their function as transcription factors. pias family of proteins shares a highly conserved domain structure comprising the n terminus sap domain that can bind to at-rich dna sequences and a pro-ile-asn-ile-the (pinit) motif, which is involved in the nuclear retention of pias proteins. pias has been shown to specifically bind to stat and block its dna-binding activity and transcriptional activation [ , , ] . the family of suppressors of cytokine signalling (socs) acts as classical feedback inhibitors of the jak/stat pathway. stat activates the transcription of socs which can block stat signalling either by direct binding and inhibition of jaks, by competing with stat for py-binding sites on activated receptor chains, or by binding signalling proteins and targeting them for proteasomal degradation [ , ] ( figure ). cytokine response stat is a signaling mediator of il- and il- family members and other cytokines such as leptin and g-csf [ , ] . n-terminal domain of stat negatively regulates type interferon (ifn) response in mice, which is independent of its function as a transcriptional factor [ ] . while stat and stat mediate antiviral and inflammatory effects of type i ifns, stat has been shown to negatively regulate the inflammatory properties of type i ifns possibly through suppression of stat function [ ] . stat activation results in either activation or suppression of inflammatory response depending on the physiological status of the cells. stat is found to be constitutively activated in cancer cells and the persistent activation of stat in cancer cells mediates tumour-promoting inflammation [ ] . stat also promotes a potent anti-inflammatory response (air); for example, toxoplasma gondii exploits host stat to prevent lps-triggered proinflammatory cytokine production in infected mouse macrophages [ ] . il- regulates both acute and chronic inflammation [ ] and stat is essential for all known aspects of the il- -regulated anti-inflammatory effect both in vivo and in vitro [ ] [ ] [ ] . while it is not clear how the pro-and anti-inflammatory functions of stat are regulated, possible factors could be the physiological status of the cells and the length of stat activation during a response. several studies described the involvement of stat in the replication and pathogenesis of viruses in humans and animals. notably, both pro-and antiviral functions of stat have been documented and its precise role in the pathogenesis of viral infections is not yet fully established. a number of dna and rna viruses are known to regulate stat , which is summarized in table . stat is either positively or negatively regulated in a range of viral infections depending on the type of virus [ ] , and varicellazoster virus (vzv) [ ] promote stat phosphorylation in infected cells. in contrast, influenza a virus (iav) nonstructural protein (ns- ) [ ] and human metapneumovirus [ ] impede stat phosphorylation in infected cells. however, human cytomegalovirus (hcmv) infection inhibits stat phosphorylation but rapidly promotes nuclear localization of unphosphorylated stat to the nucleus and disrupts il- -induced gene expression [ ] . severe acute respiratory syndrome coronavirus (sars-cov) infection of vero e cells results in stat dephosphorylation at tyr [ ] . in addition hcv promotes stat ubiquitination and degradation via the proteasome [ ] . stat plays an important role in adaptive immune response, in particular in the regulation of t lymphocyte function. stat mediates il- -dependent t cell proliferation by preventing apoptosis [ ] . further, stat regulates proliferation, survival, and differentiation of cd + [ ] and cd + t cells [ ] . furthermore, stat is essential in upregulating cd + t cell-mediated responses to viruses. for example, stat plays an important role in the activation of cd + t cells effective response during herpes simplex virus (hsv- ) infection [ ] . it is evident that viruses either promote or disrupt stat mediated gene transcription and host immune responses against viruses. further, activation or inhibition of stat mediated signalling appears to be dependent on virus and host cell type involved. inhibition of stat activation could be a strategy of viruses to subvert host immune responses. on the other hand stat -mediated gene transcription of proviral factors could be essential for certain other viruses. it is possible that the timing of stat regulation during virus infection may be critical and hence require further in depth studies to profile stat regulation during different stages of viral infection. the role of stat in virus replication appears to be complex, as it appears to function as a proviral factor in some viral infections and antiviral factor in others. stat cooperatively interacts with hepatocyte nuclear factor (hnf- ) and activates hepatitis b virus (hbv) gene expression [ ] . there is a stat binding site within the core domain of hepatitis b virus (hbv) enhancer . il- and epidermal growth factor stimulates the interaction of hbv enhancer dna-stat protein resulting in overall stimulation of hbv enhancer function and viral gene expression [ ] . hcv constitutively activates stat in liver cells which plays an important role in hcv rna replication [ ] . hcv replicon-expressing cells showed constitutive activation of stat which is mediated by oxidative stress and influenced by the activation of cellular kinases, including p mitogenactivated protein kinase, jnk, jak- , and src [ ] . hcmv primarily utilizes unphosphorylated stat to promote, either directly or indirectly, the initiation of hcmv dna replication [ ] . hcmv infection disrupts il- induced phosphorylation of stat and expression of a subset of il- -induced stat -regulated genes including socs . hcmv -kda immediate-early (ie ) protein associates with stat and rapidly promotes nuclear localization of stat in the absence of robust phosphorylation at y and inhibition of stat nuclear localization or stat expression during infection results in diminished hcmv genome replication [ ] . stat activation was shown to be critical for replication of vzv that causes varicella (chickenpox) during primary infection and zoster (shingles) upon reactivation [ ] . vzv triggers stat phosphorylation in cells infected in vitro and also in human skin xenografts in scid mice in vivo. stat activation induces the antiapoptotic protein survivin and both stat and surviving are essential for vzv replication as inhibition of stat phosphorylation and survivin results in restricted vzv replication [ ] . mumps virus v protein functions as a ubiquitin ligase that targets stat for degradation and stat evasion has been proposed to be beneficial to the replication of paramyxoviruses [ ] . further, mumps virus v protein prevents responses to interleukin- and v-src signals and can induce apoptosis in stat -dependent multiple myeloma cells and transformed murine fibroblasts [ , ] . in a similar fashion, measles virus also interferes with stat activation and it was proposed that this could provide several general or tissuespecific replication advantages to the virus [ ] . influenza a virus (iav) ns protein interferes with ifn production [ ] which correlates to reduced phosphorylation of stat . transfection of ns from a highly pathogenic avian influenza (hpai) h n virus in human lung epithelial a cells resulted in a notable reduction in ifn-inducible stat phosphorylation [ ] . viruses such as hbv, hcv, and hcmv appear to exploit host genes transcriptionally regulated by stat for their gene replication which could be independent of the regulatory effects of stat on type ifn response. whereas stat inhibition appears to be a strategy of viruses such as paramyxo-and orthomyxoviruses to evade host innate and adaptive immune responses, it is plausible that inhibition of stat signalling could provide a much broader spectrum of cytokine and growth factor suppression to allow replication and spread of these viruses in vivo. stat role in viral pathogenesis appears to be complex with reports suggesting both promotion of innate antiviral response and contribution to the detrimental effects of viral infection. stat is constitutively phosphorylated in neoplastic cells [ ] and many viruses exploit the oncogenic effects of phosphorylated stat (pstat ). stat plays a central role in the pathogenesis of oncogenic viruses such journal of immunology research as the -herpesviruses, kshv, ebv, and herpesvirus saimiri [ , , ] . stat role has been well characterized in the pathogenesis of viral infections resulting in liver disease in humans. hcv infection in liver cells causes constitutive activation of stat , which plays a central role in chronic hepatitis and often results in liver cirrhosis and hepatocellular carcinoma [ ] . hcv core protein directly interacts with and activates stat through phosphorylation of the critical tyrosine and is responsible for the virus-induced transformation. activation of stat by the hcv core results in rapid proliferation and upregulation of bcl-xl and cyclin-d and additional expression of stat in hcv core-expressing cells results in anchorage-independent growth and tumorigenesis [ ] . cytokine stimulation of hbv gene expression represents an important regulatory scheme of direct relevance to the pathogenesis of liver disease associated with hbv infection [ ] . hcmv infection of phh and hepg cells results in activation of the il- -jak-stat pathway which results in the transformation of phh cells and enhanced hepg tumorsphere formation raising the possibility that hcmv infection might be involved in the genesis of hepatocellular carcinoma [ ] . stat activation and upregulation of antiapoptotic protein survivin play an important role in the pathogenesis of lytic as well as tumorigenic herpesviruses [ ] . stat and survivin have been shown to play a major role in the malignant transformation of cells infected by -herpesviruses, such as kshv [ ] . stat activation is essential for the skin infections caused by vzv which is necessary for viral transmission and persistence in the human population [ ] . stat is also known to promote host defense against virus infections and play a protective role in regulating virus mediated proinflammation. gp -stat signalling plays an important role in the innate immune response in cardiac myocytes against coxsackievirus b infection [ ] . dysregulation of host proinflammatory response is a key contributing factor to the morbidity and mortality of virulent influenza virus infections such as the highly pathogenic avian influenza (hpai) h n viruses [ ] [ ] [ ] [ ] . we recently showed that elevated proinflammatory response in chickens is a major pathogenicity factor of hpai-h n virus infection possibly mediated by inhibition of stat phosphorylation [ ] . hpai-h n virus infection results in stat inhibition and elevated proinflammatory response in chicken cells. in contrast stat inhibition was not found in hpai-h n virus infected duck cells which show a moderate proinflammatory response. in summary, stat plays a significant role in the complex interplay between viruses and their hosts and functions as either a pro-or antiviral factor depending on the virus and host cell type involved. while stat role has been reasonably well characterized in the pathogenesis of oncogenic viruses and viruses causing liver pathology, its role in many other viral infections is less well understood. stat regulates antiviral and proinflammatory responses, either through transcriptional regulation of other cellular factors or through pathways independent of its role as a transcription factor. the seemingly contradictory roles of stat in viral infections raise a number of interesting questions: "what factors determine the switch between pro-and anti-inflammatory functions of stat ?," "how are viruses able to exploit stat signalling for their gene replication?," and "does stat either negatively or positively regulate type ifn response depending on the virus type involved?" further in depth studies to dissect the role of stat in viral infections could provide valuable insights into viral pathogenesis and development of novel antiviral therapies. in vitro studies using cell culture systems are a valuable tool to dissect the molecular basis of stat role in innate response to viruses. however, in vivo studies are essential to elucidate stat role in adaptive immune response to viruses. complementary in vitro and in vivo experiments are therefore essential to better understand the seemingly contradictory role of stat in viral infections. the author declares that there is no conflict of interests regarding the publication of this paper. stat signaling is active during early mammalian development stat signaling: anticancer strategies and challenges jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins interleukin- -type cytokine signalling through the gp /jak/stat pathway how cells respond to interferons stat : a stat family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin- molecular cloning of aprf, a novel ifn-stimulated gene factor p -related transcription factor involved in the gp -mediated signaling pathway jak-stat signaling: from interferons to cytokines stats in cancer inflammation and immunity: a leading role for stat stats: transcriptional control and biological impact dynamics and noncanonical aspects of jak/stat signalling canonical and non-canonical jak-stat signaling what does stat do? targeted disruption of the mouse stat gene leads to early embryonic lethality potential role of signal transducer and activator of transcription (stat) signaling pathway in inflammation, survival, proliferation and invasion of hepatocellular carcinoma targeting the stat signaling pathway in cancer: role of synthetic and natural inhibitors leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells self-renewal of pluripotent embryonic stem cells is mediated via activation of stat essential role of stat for embryonic stem cell pluripotency stat activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells roles of stat in mediating the cell growth, differentiation and survival signals relayed through the il- family of cytokine receptors crystal structure of unphosphorylated stat core fragment ciliary neurotrophic factor and stress stimuli activate the jak-stat pathway in retinal neurons and glia il- and granulocyte-macrophage colony-stimulating factor activate stat and stat and promote pim- and cyclin d protein expression in human eosinophils il- induces ccl expression via stat signalling in human airway smooth muscle cells activation of stat by il- and il- in primary human macrophages is differentially modulated by suppressor of cytokine signaling stat and stat mediate il- -dependent and inflammation-associated gastric tumorigenesis in gp receptor mutant mice interleukin signaling in t helper type (th ) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (stat) and stat il- contributes to jak /stat activation and promotes cell growth in alkpositive anaplastic large cell lymphoma il- is related to development of human colon cancer by activation of stat a novel role for interleukin- (il- ) as mediator of intestinal epithelial barrier protection mediated via differential signal transducer and activator of transcription (stat) protein signaling and induction of antibacterial and anti-inflammatory proteins activation of signal transducer and activator of transcription- (stat ) expression by interferon-gamma and interleukin- in hepatoma cells tumor necrosis factor alpha (tnf-alpha) activates jak /stat -stat b signaling through tnfr- in human b cells light, a member of the tnf superfamily, activates stat mediated by nik pathway the chemokine monocyte chemotactic protein triggers janus kinase activation and tyrosine phosphorylation of the ccr b receptor rantes and mip- alpha activate stats in t cells steel factor induces serine phosphorylation of stat in human growth factor-dependent myeloid cell lines oncostatin m promotes stat activation, vegf production, and invasion in osteosarcoma cell lines small molecule inhibitors of signal transducer and activator of transcription (stat ) protein signal transducer and activator of transcription in liver diseases: a novel therapeutic journal of immunology research target role and regulation of stat phosphorylation at ser in melanocytes and melanoma cells stat as a central mediator of neoplastic cellular transformation reduced phosphorylation of stat at ser- mediated by casein kinase -protein phosphatase a enhances stat tyr- induced tumorigenic potential of glioma cells regulation of stat nuclear import by importin and importin via two different functional sequence elements t /pei complex: a potent therapeutics for prostate cancer that targets stat signaling stat expression in salivary gland tumours activated stat is a mediator and biomarker of vegf endothelial activation unphosphorylated stat accumulates in response to il- and activates transcription by binding to nf b polypeptide signalling to the nucleus through tyrosine phosphorylation of jak and stat proteins interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor principles of interleukin (il)- -type cytokine signalling and its regulation inhibitors of cytokine signal transduction nucleocytoplasmic shuttling of persistently activated stat regulation of cytokine signaling pathways by pias proteins identification of recurrent nab -stat gene fusions in solitary fibrous tumor by integrative sequencing somatic stat mutations in large granular lymphocytic leukemia signal transducer and activator of transcription- , inflammation, and cancer: how intimate is the relationship? stat negatively regulates type i ifn-mediated antiviral response role of stat in type i interferon responses: negative regulation of stat -dependent inflammatory gene activation cutting edge: il- -independent stat activation by toxoplasma gondii mediates suppression of il- and tnf-in host macrophages interleukin- suppression of myeloid cell activation -a continuing puzzle toll-like receptor-dependent production of il- p causes chronic enterocolitis in myeloid cell-specific stat -deficient mice aberrant inflammation and lethality to septic peritonitis in mice lacking stat in macrophages and neutrophils general nature of the stat -activated anti-inflammatory response epstein-barr virus regulates stat through latent membrane protein dengue virus inhibits alpha interferon signaling by reducing stat expression epstein-barr virus lmp activates egfr, stat , and erk through effects on pkc human immunodeficiency virus type (hiv- ) nef activates stat in primary human monocyte/macrophages through the release of soluble factors: involvement of nef domains interacting with the cell endocytotic machinery extracellular hepatitis c virus core protein activates stat in human monocytes/macrophages/dendritic cells via an il- autocrine pathway human hepatitis c virus ns a protein alters intracellular calcium levels, induces oxidative stress, and activates stat- and nf-b mitochondrially associated hepatitis b virus x protein constitutively activates transcription factors stat- and nf-kappa b via oxidative stress persistent activation of stat by latent kaposi's sarcoma-associated herpesvirus infection of endothelial cells activation of stat transcription factor by herpesvirus saimiri stp-a oncoprotein signal transducer and activator of transcription (stat ) and survivin induction by varicella-zoster virus promote replication and skin pathogenesis influenza virus nonstructural protein (ns ) disrupts interferon signaling human metapneumovirus inhibits the il- -induced jak/stat signalling cascade in airway epithelium human cytomegalovirus ie protein disrupts interleukin- signaling by sequestering stat in the nucleus tyrosine dephosphorylation of stat in sars coronavirus-infected vero e cells hepatitis c virus targets the interferon-jak/stat pathway by promoting proteasomal degradation in immune cells and hepatocytes stat activation is responsible for il- -dependent t cell proliferation through preventing apoptosis: generation and characterization of t cell-specific stat -deficient mice stat protein promotes t-cell survival and inhibits interleukin- production through up-regulation of class o forkhead transcription factors stat regulates proliferation and survival of cd + t cells: enhances effector responses to hsv- infection, and inhibits il- + regulatory cd + t cells in autoimmune uveitis interaction between stat- and hnf- leads to the activation of liver-specific hepatitis b virus enhancer function hepatitis c virus (hcv) constitutively activates stat- via oxidative stress: role of stat- in hcv replication stat ubiquitylation and degradation by mumps virus suppress cytokine and oncogene signaling stat protein interference and suppression of cytokine signal transduction by measles virus v protein the multifunctional ns protein of influenza a viruses dangerous liaisons: stat and nf-kappab collaboration and crosstalk in cancer stat activation induced by epstein-barr virus latent membrane protein causes vascular endothelial growth factor expression and cellular invasiveness via jak and erk signaling activation of stat by the hepatitis c virus core protein leads to cellular transformation hcmv activates the il- -jak-stat axis in hepg cells and primary human hepatocytes innate defense mechanism against virus infection within the cardiac myocyte requiring gp -stat signaling innate immunity to h n influenza viruses in humans induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? pathogenesis of hong kong h n influenza virus ns gene reassortants in mice: the role of cytokines and b-and t-cell responses highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and proinflammatory responses key: cord- - p scli authors: majzoub, karim; wrensch, florian; baumert, thomas f. title: the innate antiviral response in animals: an evolutionary perspective from flagellates to humans date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: p scli animal cells have evolved dedicated molecular systems for sensing and delivering a coordinated response to viral threats. our understanding of these pathways is almost entirely defined by studies in humans or model organisms like mice, fruit flies and worms. however, new genomic and functional data from organisms such as sponges, anemones and mollusks are helping redefine our understanding of these immune systems and their evolution. in this review, we will discuss our current knowledge of the innate immune pathways involved in sensing, signaling and inducing genes to counter viral infections in vertebrate animals. we will then focus on some central conserved players of this response including toll-like receptors (tlrs), rig-i-like receptors (rlrs) and cgas-sting, attempting to put their evolution into perspective. to conclude, we will reflect on the arms race that exists between viruses and their animal hosts, illustrated by the dynamic evolution and diversification of innate immune pathways. these concepts are not only important to understand virus-host interactions in general but may also be relevant for the development of novel curative approaches against human disease. the animal kingdom, including humans, has evolved while facing constant threats from viral elements. viruses can be, in some cases, beneficial for a given animal species and drive its evolution [ ] . however, their uncontrolled replication may cause disease and prove fatal to their hosts. consequently, animal cells have evolved devoted pathways which ( ) sense and recognize pathogen-associated molecular patterns (pamps) and, more particularly, virus-associated molecular signatures; ( ) initiate signaling cascades stemming from the site of detection, translocating the information to the nucleus; and ( ) induce a transcriptional program that confers an antiviral state to the host ( figure ). interestingly, a closer examination of individual factors constituting these pathways shows a different conservation status between different animal species. while genes encoding sensors and signaling platforms are generally well conserved amongst animals, virus-stimulated genes (vsgs) are clearly less so and are subject to faster evolution [ , ] . in vertebrates, one such vsg is the secreted interferon (ifn) cytokine, that signals in an autocrine and paracrine fashion. secreted ifn molecules bind to cell-surface receptors and initiate signal transduction involving the janus kinase/signal transducer and activator of transcription (jak-stat) pathway. this pathway induces the transcription of a major antiviral program composed of hundreds of so-called ifn-stimulated genes (isgs) that comprise effectors of the cell-autonomous antiviral defense [ ] . a lot of our understanding of the innate antiviral immune system in animals is a result of studies conducted in vertebrates and more particularly in mammalian species. therefore, the ifn system has been heavily studied over the last years. however, during the last two decades, we came to appreciate that the ifn system, as we know it, is a vertebrate particularity. indeed, while some animal species like insects or nematodes are devoid of ifns and rely on rna interference (rnai) as the major antiviral pathway, some others, like mollusks, have conserved all the components that lead to ifn production but have no obvious homologs of type i ifn cytokines. nevertheless, these are predicted to use an ifn-like antiviral cytokine [ ] . in fact, the ifn cytokine itself seems to be an evolutionary novelty, however, the pathways dictating its production existed early in metazoan evolution [ , ] (figure ). interestingly, ifn is not the only vsg induced upon viral detection in mammals. certain isgs that directly interfere with the viral life cycle like viperin are also immediately induced after viral infection in an ifn-independent fashion [ ] [ ] [ ] . in this review, we will focus on the conserved pathways that are responsible for sensing viral pamps, signaling and inducing antiviral genes upon infection in animals. we will start by describing our current view on the immediate activation of the ifn and nf-kb pathways in vertebrate species. we will then zoom in on two important viral nucleic acid receptor families, toll-like receptors (tlrs) and rig-i-like receptors (rlrs), describe their function in viral rna detection and their conservation across animal species. next, we will focus on a central hub in the signaling pathways induced by dna viruses, the stimulator of ifn genes (sting). we will then examine how the evolutionary conflicts between viruses and host immune factors are shaping antiviral immunity in animals. viruses and transposable elements are very powerful drivers of evolution [ , ] . however, their uncontrolled replication and spread can be catastrophic to host cells. it is therefore not surprising that every known living species on the planet has evolved measures to recognize and counteract parasitic genetic elements. it is suggested that anti-sense mediated targeting of viral nucleic acids was the most primordial strategy protocells used to fend off viral threats [ ] . this is illustrated by argonaute and crispr-based defenses in bacteria, archaea and rnai systems in plants, all of which rely on anti-sense nucleic acids that program a nuclease to target and degrade the complementary invading viral genome [ ] [ ] [ ] . because many of the core rnai machinery components can be found in all eukaryotic superkingdoms, it is thought that this antiviral defense mechanism predated the emergence of pattern-recognition receptor (prr)-based immunity ( figure ) [ ] . interestingly, antiviral defense in eukaryotes has diversified greatly during evolution, with some species maintaining rnai-based defenses [ ] and others innovating and adopting completely novel antiviral strategies. in chordates and more particularly in vertebrate animals, the emergence of ifn-i and a recombinational adaptive immune system seems to coincide with the loss of rnai as the main antiviral mechanism in somatic cells. there is strong evidence of an intrinsic incompatibility between an antiviral rnai and the prr-ifn system. for instance, while long dsrnas can produce functional small interfering rnas (sirnas) in stem cells in the absence of ifn, the same dsrna molecule is not processed onto sirna and is sensed as a pamp in vertebrate somatic cells [ , ] . interestingly, experimental evidence of antiviral rnai in mammals seems to be limited to specialized pluripotent cells in which the prr-ifn system is not fully deployed yet. the emergence of both the ifn-i (innate) and somatic dna recombination systems (adaptive) in vertebrates constituted a major evolutionary event that dispensed them from using rnai for antiviral purposes. interestingly, retrotransposition and selfish transposable elements were determinants in the acquisition of these two systems [ , [ ] [ ] [ ] . the emergence of somatic dna recombination in vertebrate animals was considered an "immunological big bang" [ , ] . indeed, somatic dna recombination in specialized b and t cell lineages provided jawed vertebrates with large repertoires of major histocompatibility complexes (mhc), t-cell receptors (tcrs) and immunoglobulins (igs). until relatively recently, adaptive immunity was believed to be exclusive to gnathostomes (jawed vertebrates). we now know that agnathans (jawless vertebrates), including lampreys and hagfish, have also evolved an equivalent adaptive immune system with specialized lymphocytes termed, vlra, vlrb and vlrc cells, in which specific variable lymphocyte receptors (vlrs) are produced through somatic leucine-rich repeat (lrr) rearrangements [ ] . in both gnathostomes and agnathans, somatic recombination events in specialized cells permitted a pathogen-tailored response and endowed vertebrate species with an immune memory. until the end of the last century, most vertebrate immunologists concentrated their efforts on studying the adaptive arm of the immune system. nearly years ago, charles janeway predicted the presence of an evolutionary ancient immune system that detects conserved microbial and danger signals, termed the pathogen-associated molecular patterns (pamps) and danger-associated molecular patterns (damps), respectively. janeway predicted that this innate immune system precedes and instructs the adaptive system [ , ] and posited that pamps and damps must be sensed by germ-line encoded prrs. at that time, the innate immune components were severely understudied and tlrs, rlrs and sting's respective functions in immunity were completely unknown. this illustrates the immense leap forward the innate immunity field has experienced in the last three decades. today, we know that vertebrates share with other invertebrate animals specialized phagocytic cells that are able to discriminate between self and non-self. by recognizing general pathogen molecular patterns (pamps, e.g., viral double-stranded rna) and danger signals (damps), these cells establish an immediate and general inflammatory response translated into an antimicrobial and/or antiviral state. although mainly studied in vertebrates, the machineries responsible for these responses transcend this group and are fairly conserved in all animals. we will describe a particular arm of the innate immune pathways, the innate antiviral system that is best studied in mammals. unlike bacteria, viruses represent a unique challenge for prrs because they possess few unique signatures that could serve as pamps [ ] . however, viral nucleic acids (dna or rna) could have peculiar biochemical features that differentiate them from endogenous host rna [ ] . in rna molecules, for example, the lack of a -methylguanosine cap structure, double strandedness or the trior bi-phosphorylation at their ends are often used by prrs for self/non-self-discrimination. prrs that detect viral infection can be classified into four families: tlrs, rlrs, aim -like receptors (alrs) and the cgas-sting sensors [ ] . after viral detection, prr-mediated signaling directly or indirectly induces transcription factors, including ifn-regulatory factors (irfs) and nuclear factor k-b (nf-kb) to upregulate expression of vsgs including pro-inflammatory cytokines. another class of prrs such as double-stranded rna (dsrna) activated protein kinase r (pkr; also known as eif ak ), adenosine deaminase acting on rna (adar ) and - -oligoadenylate synthetase (oas ) also contribute to innate immunity [ ] . these also recognize viral signatures; however, their main function is not necessarily to induce a transcriptional immune response, but rather to directly attack viral rna by degrading it or inhibiting its translation. for this reason, these are not usually considered receptors. viruses are obligatory intracellular parasites, therefore their detection by prrs most often occurs in the intracellular milieu. endosomal transmembrane tlrs, including tlr , tlr and tlr recognize dsrna in the endosome lumen [ ] [ ] [ ] . rlrs including rig-i [ ] , melanoma differentiation associated gene (mda ) [ ] , and laboratory of genetics and physiology (lgp ) [ , ] detect viral rnas in the cytosol, whereas cytosolic viral dna is mainly recognized by cyclic-gmp-amp (cgamp) synthase (cgas) [ ] . therefore, the nature of the viral particle (e.g., enveloped vs. non-enveloped) and the viral genome (e.g., dna vs. rna) dictates which of these receptors recognizes the infection first ( figure single-stranded rna (ssrna) is a potent tlr and tlr ligand, while tlr is specific for dsrna. tlr , for example, recognizes dsrna viruses from reoviruses [ ] , but can probably also recognize dsrna intermediates from (+) strand rna viruses like coxsackievirus and west nile virus (wnv) and (-) strand rna viruses like the human respiratory syncytial virus (hrsv) [ ] . indeed, all rna viruses are thought to produce dsrna intermediates as part of their replication cycle, so both ssrna and dsrna viruses have the potential to be sensed by tlr . tlr and , on the other hand, have been shown to prefer ssrna ligands from (-) strand rna viruses such as vesicular stomatitis virus (vsv) and influenza a virus (iav) [ , ] . when it comes to rlrs, most rna viruses have been shown to be detected by rig-i or mda , as these receptors have a high affinity to dsrnas. while rig-i prefers viral rnas bearing di-or tri-phosphate groups at their while the cytosolic recognition of viral rna is almost exclusively mediated by rlrs, several proteins have been proposed to play a role in dna sensing and triggering innate immune responses, such as the dna-dependent activator of ifn-regulatory factors (dai), ddx , rna polymerase iii, ifi and dna-pk [ ] [ ] [ ] [ ] [ ] [ ] . however, among all the proposed sensors, only cgas knock-outs can completely shut down ifn production in response to cytosolic dna [ ] . the cgas protein is now thought to be the major viral dna sensor and has been shown to detect adenovirus, human papillomavirus (hpv), herpes simplex virus- (hsv- ) and cytomegalovirus (cmv) [ , [ ] [ ] [ ] . aim has also been shown to activate the inflammasome upon dna stimulation [ ] but will not be discussed in this review. rna ligands cause the endosomal transmembrane tlr , and to dimerize and then to oligomerize through their cytoplasmic tir (toll/il- receptor) domains. this allows tlrs to recruit signaling adaptors via tir-tir interactions [ ] . tlr recruits the adaptor protein trif (tir-domain-containing adapter-inducing ifn-β) [ , ] . trif is able to play a dual role by inducing the ifn or the nf-kb pathways. when it comes to ifn, after activation, trif recruits the ubiquitin ligase traf (tumor necrosis factor receptor-associated factor ) through an ubiquitination mechanism which in turn recruits tank-binding kinase (tbk ) [ , ] . the trif/tbk complex is then able to phosphorylate the transcription factor irf , triggering its dimerization and nuclear translocation. phosphorylated irf dimers specifically bind to ifn-stimulated response elements (isres) present in the ifn-β gene promoter which leads to the transcription of this cytokine [ ] . trif can also recruit ripk (receptor-interacting serine/threonine-protein kinase ) that leads to the activation of the ikk complex, releasing the nf-kb transcription factor from its ikb inhibitory subunit and resulting in its translocation to the nucleus to induce the transcription of pro-inflammatory cytokines [ ] (figure ). unlike tlr , the activation of tlr and tlr recruits the adaptor protein myd (myeloid differentiation primary response ) through tir-tir domain interaction. myd death domains oligomerize which triggers the formation of the myddosome signaling complex consisting of myd and the irak family of kinases (il- receptor-associated kinases), irak , and . through a series of phosphorylations and the help of the e ubiquitin ligase traf , the myddosome is able to recruit and activate the transcription factors irf , irf and nf-kb that translocate to the nucleus to induce the transcription of ifn-α genes and other proinflammatory cytokines [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure ). rlrs (rig-i, mda and lgp ) are characterized by a central dead-box helicase/atpase domain and a c-terminal regulatory domain (ctd) essential for rna recognition and autorepression in the absence of rna ligands. with the exception of lgp , rlrs also possess two n-terminal caspase activation and recruitment domains (cards). upon rna binding rig-i is remodeled into an active conformation in which the ctd and helicase domains organize into a ring around the rna ligand and the card domains are exposed [ , ] which facilitate their interactions with other card domains resulting in rig-i tetramers. although rig-i and mda share similar domain architectures, mda seems to prefer longer dsrna, assembling along these molecules to form helical, filamentous oligomers [ , ] . a poly-ubiquitination reaction by ubiquitin ligases like riplet and trim (tripartite motif-containing ), is thought to enhance rig-i and mda oligomerization and activation [ ] [ ] [ ] [ ] . rig-i and mda oligomers then serve as a scaffold for binding to the adaptor protein mavs (mitochondrial antiviral signaling protein, also known as ips- , visa, and cardif) [ ] [ ] [ ] . mavs has been shown to be critical for mounting an efficient immune response to infection by several rna viruses [ ] . its c-terminal transmembrane domain is inserted into the outer mitochondrial membrane [ ] , whereas its n-terminal card domain mediates its aggregation on the mitochondrial surface by interacting with the tandem cards of rig-i or mda oligomers [ , ] . mavs aggregates then recruit several e ubiquitin ligases including traf , traf and traf . although traf-mediated ubiquitination is essential to activate mavs downstream signaling, the ubiquitination targets of traf remain unknown [ ] . subsequently, the ubiquitin sensor nemo (nf-κb essential modulator, also known as ikkγ) [ , ] is then recruited to the mavs/trafs complex, which in turn recruits ikk and tbk to the mavs complex leading to activation of nf-kb and irf and their translocation to the nucleus to induce the transcription of antiviral genes [ ] [ ] [ ] [ ] (figure ). after trif and mavs were discovered, sting was identified as a third adaptor protein that is also able to activate irf and ifn production [ , ] . sting is an endoplasmic reticulum (er) resident membrane protein with cytoplasmic c-and n-termini. sting has been shown to be essential for dna-mediated ifn production in different tissues, for example, it is crucial for host defense against the dna virus hsv- [ ] . sting has also been shown to sense cyclic dinucleotides (cdns), which are the second messengers known to be produced by bacteria such as listeria monocytogenes [ ] [ ] [ ] [ ] . although it can bind bacterial cdns, sting is unable to bind dna and relies on an upstream sensor, cgas [ ] . cgas is an enzyme that contains a nucleotidyltransferase (ntase) domain and can synthesize the second messenger -cyclic gmp-amp (cgamp) from atp and gtp upon dna recognition ( figure ). loss of cgas in various cell lines and also in vivo results in a complete loss of type i ifn induction upon dna delivery or viral infections [ , ] . cgas preferentially binds longer dna (> bp) as a dimer to form stable protein-dna ladder networks responsible for strong cgamp production [ , ] . a unique cgamp isomer termed -cgamp with particular phosphodiester linkages is produced by cgas [ , ] . -cgamp is a potent sting ligand and has a higher affinity to this protein than other cgamp molecules containing different phosphodiester linkages such as -cgamp, -cgamp or bacterial cdns [ , ] . apart from activating sting in the cell where cgas initially detects viral dna, cgamp second messengers can also travel to neighboring cells, through gap-junctions [ ] or after being packaged in newly formed virions [ , ] . this intercellular transfer of free or packaged cgamp permits uninfected cells to mount a preventive ifn response, protecting them from infection or providing a faster response to dna viruses that encode cgas antagonists. upon cgamp binding, sting undergoes a conformational change that results in the release of its c-terminal tail (ctt) from its autoinhibitory state and in the formation of sting homodimers that translocate to perinuclear regions to colocalize with tbk [ , , ] . tbk recruitment results in the phosphorylation of sting and the phosphorylated site serves as a platform for irf dimerization and activation which ultimately results in ifn-β induction [ ] (figure ). sting has also been shown to induce nf-kb, map kinase and stat activation, as well as the stimulation of lc puncta formation, a hallmark associated with autophagosome formation [ , [ ] [ ] [ ] . however, the molecular mechanisms by which sting induces these non-ifn responses remain poorly understood. tlrs comprise an ancient family of membrane-spanning receptors that recognize ligands through their extracellular domains and initiate an intracellular response upon stimulation (see above). the toll gene was first identified as a developmentally important gene in drosophila in [ ] . in the mid- s the discovery that this gene also plays an essential role in the ability of drosophila to resist fungal infections connected for the first time toll receptors to innate immunity [ , ] . although in flies toll functions as a cytokine receptor, a human toll receptor (tlr ) was rapidly identified [ , ] and shown to induce an immune response in mice after induction by lps [ ] . we now know that there are ten tlrs in humans that can respond to many bacterial and viral pamps [ ] . prototypical tlrs contain three structural elements, a hydrophobic ectodomain containing a variable number of lrrs, a transmembrane domain and a tir domain, which mediates downstream signaling through adaptor proteins [ ] . tlrs are likely very ancient immune sentinels since two of their characteristic building blocks (lrr and tir domains) are observed in placozoans (e.g., trichoplax animals) [ ] and porifera (e.g., sponges) [ ] . full tlrs were detected in cnidarian species, like the starlet sea anemone (nematostella vectensis; one single tlr) [ , ] and the acroporid corals (acropora digitifera; four tlrs) [ ] (figure ) . interestingly, both developmental and immunological roles of tlrs have been described in cnidarians. tlrs from both the sea anemone (nematostella vectensis) and the mountainous star coral (orbicella faveolata) have been shown to signal via myd leading to nf-kb activation [ , ] . in the bilateria phylum, tlrs can be found in most studied species, however, their numbers vary greatly among species, ranging from a single tlr in nematodes like caenorhabditis elegans, to over two hundred in echinoderms like the pacific purple sea urchin strongylocentrotus purpuratus (figure ). the expansion of the tlr repertoire in some animals like the sea urchin, reflects the adaptation of their immune arsenal to rapidly changing environmental stressors [ ] . amongst a multitude of other innate immune factors in this species, such as nacht domain-lrrs and scavenger receptors, sea urchin genomes encode for tlrs. among those, tlrs belong to a greatly expanded set of genes with vertebrate like features, many of which seem to have duplicated recently. the high prevalence of pseudogenes ( % to %) among those might reflect a history of strong positive selective pressures. another phylum where tlrs have undergone a significant expansion is in mollusca [ ] , like the pacific oyster crassotrea gigas [ ] (figure ). the pacific oyster encodes for tlrs in total, potentially reflecting a highly specialized response to environmental challenges and response to pathogens. the spread of pathogens in c. gigas natural habitats occurs very quickly, which is highlighted by the mass mortality events the ostreid herpesvirus (oshv ) has caused in many oyster nurseries. tlr sensing of oshv results in the differential regulation of more than a thousand genes, many of which are related to viral infection (e.g., cytosolic dna sensing and dna replication) [ , ] . in contrast to the very diverse set of tlr repertoires found in other bilateria species (e.g., nematodes, sea urchins and oysters), chordates and more particularly vertebrates contain roughly equal numbers of tlrs, reflecting the reduced need for highly diversified pattern recognition due to the acquisition of adaptive immune components (figure ). in general, vertebrate tlrs can be grouped into six major families [ ] . the families responsible for sensing of viral pamps are the tlr family, which recognizes dsrna, the tlr family (including tlrs , and ) which recognizes nucleic acid motifs and the large tlr family (tlr , , , , , , , and ) . the reduced number of tlrs in vertebrates does not necessarily mean that the tlr-response in those species cannot be tailored to a particular environment. a peculiar example is tlr , one of two virus sensing tlrs present in the pufferfish takifugu rubripes. tlr is widely conserved among teleosts and amphibians but does not seem to be present in avian or mammalian animals, which indicates that tlr might be required only in vertebrates living in water [ ] . in mammals, one last case of tlr adaptation and rapid evolution that is worth mentioning comes from bat species. analyses of tlr evolution in bats reveal adaptations acquired by tlrs , , and , with unique mutations fixed in ligand-binding sites [ , ] . these adaptations are thought to stem from the unique lifestyle of bat species, that are the only known flying mammals, and that represent important viral reservoirs [ ] . evolutionary studies paint a complex and dynamic picture of the emergence and functional diversification of rlrs across the animal kingdom. initially, several studies proposed that rig-i and mda /lgp evolved in animals independently through gene fusion and domain grafting events [ , ] . for instance, it has been proposed that the two card domains have been acquired by rig-i and mda in two separate events: the first domain being gained by the ancestor of rig-i and mda before their duplication and the second acquired after their divergence [ ] . these studies suggested that full-length rlrs are a vertebrate-specific evolutionary novelty, although their building blocks may have been present in closely related invertebrate animals [ , ] . a more recent study challenges this view and finds that the rlr-based immunity is not vertebrate-specific but originated in the earliest multicellular animals [ ] (figure ). in this study, the authors show that rlrs functionally diversified through a series of gene duplication events, followed by protein-coding changes that modulated their rna-binding properties. using homology-based gene prediction based on confirmed human rlrs the authors were able to identify full-length rlrs in early-branching animal genomes, including porifera (e.g., sponges) and cnidaria (e.g., jellyfish). however, they were unable to identify rlrs in non-metazoan eukaryotes, including fungi and choanoflagellates [ ] (figure ). it is therefore proposed that the ancestral rlr (rig-i/mda /lgp anc) duplicated in bilateria to give rise to rig-i and mda /lgp lineages, followed by a more recent duplication of the mda /lgp ancestor, giving rise to mda and lgp lineages in jawed vertebrates after their split from jawless vertebrates [ ] . the emergence of rlrs early in animal evolution is a very plausible scenario, since other components of the signaling pathways downstream of rlrs, like the irf genes, are also found in early metazoans [ ] ( figure ). another recent evidence suggesting that rlrs predated vertebrate evolution comes from studies performed in mollusks (pacific oyster; c. gigas). the invertebrate c. gigas not only encodes up to rlrs, but also mavs, traf , tbk and irf family proteins, which have been shown to have functional antiviral roles [ , , ] (figure ) . even though there is no consensus on the exact evolutionary history of rlrs, it is clear that these receptors (and/or their building blocks) existed very early in metazoan evolution and most importantly, they are subject to a very dynamic evolution. this is illustrated by the lineage-specific loss of rlr genes in many species. for example, although mda and lgp homologs were found in many teleost fish, rig-i homologs have only been identified in some fish species like salmon and carp [ ] . rig-i is absent in the chicken genome although mda and lgp are both present [ , ] . interestingly, chickens suffer severely from avian influenza virus (aiv) infection compared to ducks (that do possess the rig-i gene) which could be due to the loss of rig-i affecting their first line of defense in epithelial cells [ ] . most studied mammals possess rig-i, however, it has been lost in at least one mammalian species; the chinese tree shrew [ ] . interestingly, with the loss of rig-i, both mda and lgp have undergone strong positive selection in chinese tree shrews, and positively selected sites in mda endowed the substitute function for the lost rig-i [ ] . another eloquent example illustrating the dynamic evolution of these receptors is the loss of all rlr genes (rig-i, mda and lgp ) in insects (figure ). in drosophila, for example, although the nf-kb and jak/stat pathways are present and contribute to antiviral defenses [ , ] , all components of the rlr-mavs-irf-axis have been lost. instead, drosophila like other insects and relies on the rnai mechanism as the major antiviral system protecting it from viral infections [ , , ] . interestingly the rnase iii dicer- , a central player in insect antiviral immunity, responsible for generating small interfering rnas (sirnas), also contains an n-terminal dexd/h-box helicase domain that is highly homologous to the helicase domains of vertebrate rlrs [ , ] . moreover, dicer- has been shown to be responsible for the transcriptional upregulation of an antiviral gene (vago) that could function as a cytokine by activating the jak/stat pathway and triggering systemic antiviral immunity in various mosquito tissues [ , ] . although the pathway leading to the transcriptional activation of vago is still poorly understood in insects, these studies established that dexd/h-box helicase containing proteins, like dicer and rlrs, may represent an evolutionarily conserved set of viral nucleic acid sensors that direct antiviral responses in animals [ ] . one last observation exemplifying the dynamic and rapid evolution of these receptors comes from mammalian species. indeed, rlrs seem to be experiencing very recent adaptive changes in some mammals. for example, rig-i seems to have accumulated adaptive changes altering its rna-binding properties throughout mammalian evolution [ ] . moreover, in humans, for example, a number of protein-coding polymorphisms have been identified in rig-i which may contribute to differences in viral susceptibility and risk of autoimmune diseases [ , , ] . sting presence in animal genomes is probably more ancient than that of rlrs, since sting homologs can be found in most animal phyla including unicellular choanoflagellates ( figure ) [ , ] . furthermore, the ability of sting to bind cdns seems to be an ancient property. in an elegant study, kranzusch and colleagues show that a sting homolog in the starlet sea anemone n. vectensis (nvsting) is not only structurally very similar to that of human sting but is also able to bind cgamp with very high affinity [ ] . however, sting's ctt domain, which is crucial for tbk recruitment and downstream ifn induction, appeared only in vertebrate species [ ] . consequently, nvsting lacking the ctt is unable to induce ifn-β production in response to cdns when transfected in mammalian cells [ ] . the lack of a ctt domain in invertebrates does not mean that sting could not have an immune function in these animals. a first indication comes from invertebrate species like the lophotrochozoa phylum that includes the pacific oyster c. gigas and the annelid worm capitella teleta. in these animals, an unusual sting architecture can be found, where a sting domain is fused to a tir domain, known to be involved in innate immune signaling [ , ] . the second indication that sting lacking a ctt could function in immunity comes from arthropods. recent studies in drosophila, that lack an ifn system, clearly show that sting is important for antimicrobial and antiviral nf-kb activation in this model [ , ] (figure ) . interestingly, the emergence of the ctt domain of sting in vertebrate species seems to coincide with the development of the ifn system. nevertheless, sting ctt domain function, which dictates downstream signaling, seems to be plastic amongst vertebrate species. in a recent study, authors show that sting ctt-dependent activation of irf and nf-kb varies between vertebrate species [ ] . while sting ctt from mammalian species is able to induce a strong ifn-β and a weaker nf-kb response, an extension of this domain in ray-finned fish species elicits a dramatic enhancement of nf-kb activation and weaker irf -ifn signaling [ ] . another indication of sting ctt structure-function plasticity comes from bat species. a highly conserved and functionally important serine residue (s ) in sting's ctt domain is lost in bats [ ] . the replacement of this critical residue in this mammalian species significantly dampens sting-dependent ifn activation. the authors of this study suggest that the lifestyle of bat species (e.g., flight induced cytosolic dna, high viral titers) imposes a strong selective pressure on sting. this results in functionally dampened sensing and signaling mechanisms to avoid ifn overactivation and to cope with high cytosolic dna content. taken together, present studies suggest an evolutionarily ancient role of sting in antiviral immunity and modulation of its structure and function to accommodate species-specific pathogen burdens. the picture is less clear for the cgas enzyme when it comes to antiviral immunity. although cgas homologs have been identified in a variety of ancient metazoan lineages [ , ] , it is believed that the ability of cgas to bind and detect dsdna emerged in vertebrates. indeed, cgas' zinc-ribbon domain, required for dna binding and cgamp synthesis in response to dna in the cytosol, seems to be a vertebrate innovation [ ] [ ] [ ] . interestingly, primate cgas seems to have undergone rapid evolution in this lineage, as observed by the positive selection at its nucleic acid binding interfaces [ ] . these studies argue that although the cgas enzyme existed early in metazoans, its function has been repurposed for dna sensing only recently in vertebrates. clearly, cgas and sting seem to have acquired novel features throughout evolution. specifically in vertebrates cgas evolved the zinc ribbon motif to detect dna and sting evolved the ctt domain that expanded its signaling potential. as obligate intracellular parasites, viruses have evolved an array of evasion mechanisms to escape their elimination by the host's immune system. interestingly, viral antagonism is a general strategy and is not a peculiarity of animal viruses. many bacteriophages, for instance, encode crispr-cas inhibitors, termed anti-crisprs, to counter prokaryotic antiviral systems [ ] . plant viruses also encode viral suppressors of rnai (vsrs) the main antiviral system in plant cells [ ] . likewise, several evasion strategies and immune antagonisms by animal viruses have been described [ ] [ ] [ ] [ ] [ ] . these include hiding the viral genome from immune detection, shutting off host translation or transcription machineries, inhibiting host rna processing and trafficking and interfering directly with either proteins that sense viral presence, or factors that signal the information to the nucleus. since interfering with the innate immune system is less damaging for the host than targeting vital cellular machineries (e.g., translation), many studied viruses seem to have opted for this strategy. several studies describe viral evasion mechanisms at both the recognition and sensing step (tlrs, rlrs and cgas-sting) or at the downstream signaling steps through the targeting of proteins such as mavs, tbk , irf , irf and nf-κb. evasion strategies and immune antagonisms by animal viruses are a very active area of research, that have yielded a rich literature in the past few years. we will here just give some select examples of viral strategies that curb sensing and signaling by tlrs, rlrs and cgas-sting in animals, with an obvious bias towards viruses infecting humans. for a more complete picture on the subject, readers can refer to excellent reviews, published recently, describing those strategies [ ] [ ] [ ] [ ] [ ] . tlr signaling has been shown to be inhibited by the vaccinia virus (vacv) protein a r, that targets specific tir-domain-containing adaptor proteins. a r itself contains a tir domain which allows it to competitively interact with tir-domain-containing complexes such as myd , trif or tram, thereby inhibiting the activation of both nfkb and irfs [ , ] . human t-cell leukemia virus type- (htlv- ) is also able to interfere with tlr -dependent signaling. the htlv- encoded viral protein p binds and disables a transcription factor, pu. , required for tlr surface expression [ ] . trif, an important player in the tlr signaling cascade, is a target of choice of many viruses. the ns / a protease of hcv and the c proteases of several picornaviruses such as coxsackievirus b, ev and hepatitis a virus (hav), can all recognize and proteolytically cleave trif, producing trif fragments that are unable to signal [ ] [ ] [ ] [ ] [ ] . when it comes to rlrs, one basic strategy used by cytosolic viruses to escape surveillance is to simply prevent these receptors from accessing viral genomes. denv, for example, replicates in convoluted membranes of the er concealing its dsrna intermediates from the cytosol and thereby prevents the activation of rlrs [ ] . other viruses like ebola virus (ebov) and marburg viruses encode viral protein (vp ) that tightly binds and 'shields' the viral genome from detection by rig-i [ , ] . another strategy used by viruses to 'hide' from rlrs consists of modifying the very molecular features these receptors rely on to recognize viral genomes. for example, both, hantaan viruses from the bunyaviridae family and borna disease virus (bdv) from the bornaviridae family, encode phosphatases that process the triphosphate group at their genome termini, to a -monophosphate to escape rig-i surveillance [ , ] . lassa virus (lasv) from the arenaviridae family evolved a unique strategy in which its nucleoprotein (np) acquired a - exonuclease activity, that enables it to digest free dsrna, preventing the activation of rig-i [ ] . however, the most direct way of interfering with rlr function and their signaling partners is to either directly target them for cleavage and degradation or to manipulate their phosphorylation and ubiquitination statuses, which are crucial for their activation. indeed, many viruses encode proteases that directly cleave rlrs. while the cpro proteases of both poliovirus and ev cleave rig-i, the apro of ev cleaves mda [ , ] . mavs, a crucial hub for both rig-i and mda -mediated signaling is also frequently targeted and cleaved by numerous viral proteases, such as cpro from hav, apro from ev , ns -ns a from hcv, apro and cpro from rhinovirus and cpro from coxsackievirus b (cvb ) [ , , [ ] [ ] [ ] . mavs can also be indirectly degraded by particular viruses. for instance, measles virus (mev) can trigger a selective form of autophagy, called mitophagy, responsible for the degradation of mitochondria, which leads to a decrease of mavs abundance [ ] . another example of indirect mavs degradation comes from studies with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov). this virus has evolved a strategy in which its b protein localizes to mitochondria and subverts the cellular e ubiquitin ligase atrophin- -interacting protein (aip ) to degrade mavs [ ] . post-translational modifications of both mavs and rlrs have also been shown to be subverted by viruses to inhibit their downstream signaling. ns proteins from many influenza a virus strains (iav) interact with the host ubiquitin ligase trim and inhibit its oligomerization, a crucial step for its enzymatic activity of attaching lys -linked polyubiquitin to the card domains of rig-i [ , ] . other viruses encode deubiquitinating enzymes (dubs) to remove the lys -linked ubiquitination off rig-i. orf from kaposi's sarcoma herpesvirus (kshv), papain-like protease (plp) from sars-cov, leader proteinase (lpro) from foot-and-mouth disease virus (fmdv) and the ovarian tumor (otu)-type proteins of arteriviruses and nairoviruses have all been shown to possess a deubiquitination activity and interfere with rig-i mediated signaling [ , [ ] [ ] [ ] . rig-i and mda phosphorylation status can also be subverted by viruses. in normal conditions, phosphorylation of serine or threonine residues keeps rig-i and mda in an inactive state. upon viral infection, pp phosphatases are recruited to dephosphorylate specific marks on those receptors and activate them. v proteins from measles and nipah viruses (mev and niv) act as decoys and have been shown to bind pp -α and pp -γ, sequestering them away from mda and rig-i [ , ] . similar to evasion strategies that counter the rna sensing machinery described earlier, dna viruses use numerous strategies to escape cgas-sting-dependent detection and signaling. they could either hide their viral genomes or cleave, degrade, post-translationally modify or even relocalize dna sensing and signaling factors [ ] . hepatitis b virus (hbv), that causes chronic hepatitis and increases the risk of developing liver cirrhosis and hepatocellular carcinoma, has developed an array of mechanisms to inhibit the host's immune systems (reviewed in [ ] ). notably, the hbv polymerase can bind to sting to block its lys -linked ubiquitination, inhibiting the production of ifn-β [ ] . moreover, even though cgas is expressed in human hepatocytes and is able to sense and signal upon transfection of naked relaxed-circular hbv dna; during a natural infection, hbv dna seems to escape cgas detection, likely due to packaging of the genome into the viral capsid [ ] . kshv, another dna virus has been shown to act on both cgas and sting. several kshv proteins (e.g., orf and lana) can either sequestrate stimulatory dna or directly bind to cgas inhibiting its enzymatic activity [ , ] . kshv has been also shown to encode a viral interferon regulatory factor (virf ) that interacts with sting thereby preventing tbk binding and sting activation by tbk -dependent phosphorylation [ ] . the ns protease of denv, together with its ns b co-factor, has been shown to target the residues - (lrrg) of human sting, leading to its cleavage and degradation [ , ] . interestingly, mouse sting lacks these lrrg residues, and ns b/ns of denv is neither able to cleave the murine sting, nor to block murine ifn-β production. therefore, it has been proposed that the inability of denv to cleave mouse sting might explain its host tropism, as murine cells are not very susceptible to denv infection [ , ] . in animals, prrs and their associated signaling pathways are early and potent cellular sensors of viral elements, that mobilize the organism's defenses by inducing an antiviral state. major advances have been made in the last two decades in the understanding of their function in mammalian immunity. new genomics data and gene editing tools can now let us interrogate prr-like pathways in poorly studied animal species and define their evolutionary trajectories. studying the evolution of immune components and their interplay with viral pathogens is extremely important since our immune responses to contemporary viruses have been shaped by our evolutionary responses to previous infections. the modern innate immune system is generally not yet optimized against modern viruses but rather was selected for by previous rounds of co-evolution with ancient viruses [ ] . studying the biological arms race between host and virus, referred to as the "red queen hypothesis" [ ] , in which each entity maintains a relatively constant fitness cost, will be instrumental in the fight against future infections. such studies will help us understand many aspects of viral infections including viral zoonoses, tropism, global epidemics and disease progression. furthermore, exploring these pathways and mechanisms for therapeutic purposes may offer novel strategies to cure human disease. indeed, modulating the action of the aforementioned immune sensors is proving to be an effective strategy to develop vaccines and vaccine adjuvants [ ] [ ] [ ] [ ] [ ] or to treat viral infections [ ] [ ] [ ] [ ] [ ] [ ] . finally, the use of tlr, rlr and sting modulators, to treat inflammation, auto-immune disease [ , ] and also in cancer immunotherapy [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] provides an eloquent incentive to continue studying these pathways and to look ahead with great optimism. a virocentric perspective on the evolution of life evolution of innate immunity: clues from invertebrates via fish to mammals evolution of interferons and interferon receptors transcriptional regulation of antiviral interferon-stimulated genes antiviral defense and innate immune memory in the oyster dynamic 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nf-kappab signaling pathways in mammalian and insect innate immunity the evolution of antiviral defense systems the frustrated gene: origins of eukaryotic gene expression antiviral immunity directed by small rnas crispr-cas immunity in prokaryotes the evolutionary journey of argonaute proteins on the origin and functions of rna-mediated silencing: from protists to man rna interference to treat virus infections specific interference with gene expression induced by long, double-stranded rna in mouse embryonal teratocarcinoma cell lines antiviral rna interference in mammalian cells transposition mediated by rag and rag and its implications for the evolution of the immune system primordial emergence of the recombination activating gene (rag ): sequence of the complete shark gene indicates homology to microbial integrases evolution of immune systems from viruses and transposable elements re-evaluation of the immunological big bang pattern recognition receptors and control of adaptive immunity approaching the asymptote? evolution and revolution in immunology crosstalk between cytoplasmic rig-i and sting sensing pathways discriminating self from non-self in nucleic acid sensing innate immune pattern recognition: a cell biological perspective recognition of double-stranded rna and activation of nf-kappab by toll-like receptor recognition of single-stranded rna viruses by toll-like receptor human tlr- -, - -, and - -mediated induction of ifn-alpha/beta and -lambda is irak- dependent and redundant for protective immunity to viruses the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses inhibition of the rna polymerase iii-mediated dsdna-sensing pathway of innate immunity by vaccinia virus protein e rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate dna-pk is a dna sensor for irf- -dependent innate immunity dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response the interferon response to intracellular dna: why so many receptors? immunobiology ifi is an innate immune sensor for intracellular dna the helicase ddx senses intracellular dna mediated by the adaptor sting in dendritic cells adenovirus detection by the cgas/sting/tbk dna sensing cascade pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc ticam- , an adaptor molecule that participates in toll-like receptor -mediated interferon-beta induction role of adaptor trif in the myd -independent toll-like receptor signaling pathway specificity in toll-like receptor signalling through distinct effector functions of traf and traf critical role of traf in the toll-like receptor-dependent and -independent antiviral response type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors cutting edge: tnfr-associated factor (traf) is essential for myd -dependent pathway but not toll/il- receptor domain-containing adaptor-inducing ifn-beta (trif)-dependent pathway in tlr signaling interferon-alpha induction through toll-like receptors involves a direct interaction of irf with myd and traf helical assembly in the myd -irak -irak complex in tlr/il- r signalling protein kinase ikkbeta-catalyzed phosphorylation of irf at ser induces its dimerization and nuclear translocation in myeloid cells an oligomeric signaling platform formed by the toll-like receptor signal transducers myd and irak- ikkbeta is an irf kinase that instigates inflammation interleukin- receptor-associated kinase- plays an essential role for toll-like receptor (tlr) -and tlr -mediated interferon-{alpha} induction structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna mda assembles into a polar helical filament on dsrna cooperative assembly and dynamic disassembly of mda filaments for viral dsrna recognition trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-induced oligomerization of the rna sensors rig-i and mda activates antiviral innate immune response riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf essential role of ips- in innate immune responses against rna viruses an autoinhibitory mechanism modulates mavs activity in antiviral innate immune response mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response structural basis for the prion-like mavs filaments in antiviral innate immunity mavs recruits multiple ubiquitin e ligases to activate antiviral signaling cascades activation of ikk by tnfalpha requires site-specific ubiquitination of rip and polyubiquitin binding by nemo sensing of lys -linked polyubiquitination by nemo is a key event in nf-kappab activation key role of ubc and lysine- polyubiquitination in viral activation of irf sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf transcription factor activation sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity sting is a direct innate immune sensor of cyclic di-gmp coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for v. cholerae virulence mpys is required for ifn response factor activation and type i ifn production in the response of cultured phagocytes to bacterial second messengers cyclic-di-amp and cyclic-di-gmp the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization cgas produces a - -linked cyclic dinucleotide second messenger that activates sting cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting viruses transfer the antiviral second messenger cgamp between cells transmission of innate immune signaling by packaging of cgamp in viral particles structure-function analysis of sting activation by c[g( , )pa( , )p] and targeting by antiviral dmxaa atg a controls dsdna-driven dynamic translocation of sting and the innate immune response phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation activation of stat by sting is critical for antiviral innate immunity cytosolic-dna-mediated, sting-dependent proinflammatory gene induction necessitates canonical nf-kappab activation through tbk activation of autophagy by alpha-herpesviruses in myeloid cells is mediated by cytoplasmic viral dna through a mechanism dependent on stimulator of ifn genes establishment of dorsal-ventral polarity in the drosophila embryo: the induction of polarity by the toll gene product signals from the il- receptor homolog, toll, can activate an immune response in a drosophila hemocyte cell line the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults chromosomal localization of til, a gene encoding a protein related to the drosophila transmembrane receptor toll, to human chromosome p prediction of the coding sequences of unidentified human genes. i. the coding sequences of new genes (kiaa -kiaa ) deduced by analysis of randomly sampled cdna clones from human immature myeloid cell line kg- defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene the role of pattern-recognition receptors in innate immunity: update on toll-like receptors evolutionary origins of toll-like receptor signaling innate immunity in the simplest animals-placozoans sea anemone model has a single toll-like receptor that can function in pathogen detection, nf-kappab signal transduction, and development the innate immune repertoire in cnidaria-ancestral complexity and stochastic gene loss differential and convergent utilization of autophagy components by positive-strand rna viruses a conserved toll-like receptor-to-nf-kappab signaling pathway in the endangered coral orbicella faveolata genomic insights into the immune system of the sea urchin massively parallel rna sequencing identifies a complex immune gene repertoire in the lophotrochozoan mytilus edulis massive expansion and functional divergence of innate immune genes in a protostome teleost tlr recognizes rna duplex to induce ifn and protect cells from birnaviruses adaptive evolution of virus-sensing toll-like receptor in bats the evolution of bat nucleic acid-sensing toll-like receptors immune system modulation and viral persistence in bats: understanding viral spillover origin and evolution of the rig-i like rna helicase gene family characterization of the mollusc rig-i/mavs pathway reveals an archaic antiviral signalling framework in invertebrates retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) in fish: current knowledge and future perspectives chicken cells sense influenza a virus infection through mda and cardif signaling involving lgp association of rig-i with innate immunity of ducks to influenza genome of the chinese tree shrew loss of rig-i leads to a functional replacement with mda in the chinese tree shrew the kinase ikkbeta regulates a sting-and nf-kappab-dependent antiviral response pathway in drosophila the jak-stat signaling pathway is required but not sufficient for the antiviral response of drosophila the rna silencing endonuclease argonaute mediates specific antiviral immunity in drosophila melanogaster essential function in vivo for dicer- in host defense against rna viruses in drosophila sensing viral rnas by dicer/rig-i like atpases across species the dexd/h-box helicase dicer- mediates the induction of antiviral activity in drosophila secreted vago restricts west nile virus infection in culex mosquito cells by activating the jak-stat pathway dicer- -dependent activation of culex vago occurs via the traf-rel signaling pathway nucleic acid sensing in invertebrate antiviral immunity the rig-i atpase core has evolved a functional requirement for allosteric stabilization by the pincer domain the selective footprints of viral pressures at the human rig-i-like receptor family evolution and functional impact of rare coding variation from deep sequencing of human exomes cyclic di-nucleotide signaling enters the eukaryote domain evolutionary origins of cgas-sting signaling toll signaling: the tireless quest for specificity analysis of drosophila sting reveals an evolutionarily conserved antimicrobial function modular architecture of the sting c-terminal tail allows interferon and nf-kappab signaling adaptation dampened sting-dependent interferon activation in bats structure of human cgas reveals a conserved family of second-messenger enzymes in innate immunity ] is the metazoan second messenger produced by dna-activated cyclic gmp-amp synthase structural mechanism of cytosolic dna sensing by cgas overlapping patterns of rapid evolution in the nucleic acid sensors cgas and oas suggest a common mechanism of pathogen antagonism and escape the discovery, mechanisms, and evolutionary impact of anti-crisprs decoding type i and iii interferon signalling during viral infection viral evasion of dna-stimulated innate immune responses ten strategies of interferon evasion by viruses viral evasion of intracellular dna and rna sensing viral evasion and subversion of pattern-recognition receptor signalling a r and a r from vaccinia virus are antagonists of host il- and toll-like receptor signaling vaccinia virus protein a r targets multiple toll-like-interleukin- receptor adaptors and contributes to virulence the htlv-i p interferes with tlr signaling and modulates the release of pro-and anti-inflammatory cytokines from human macrophages innate immunity evasion by enteroviruses: insights into virus-host interaction toll-like receptors in antiviral innate immunity hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity the coxsackievirus b c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling disruption of tlr signaling due to cleavage of trif by the hepatitis a virus protease-polymerase processing intermediate the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling structural basis for marburg virus vp -mediated immune evasion mechanisms sequestration by ifit impairs translation of o-unmethylated capped rna old world hantaviruses do not produce detectable amounts of dsrna in infected cells and the termini of their genomic rnas are monophosphorylated structure of the lassa virus nucleoprotein reveals a dsrna-specific to exonuclease activity essential for immune suppression rig-i is cleaved during picornavirus infection enterovirus apro targets mda and mavs in infected cells cleavage of ips- in cells infected with human rhinovirus disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor mitophagy enhances oncolytic measles virus replication by mitigating ddx /rig-i-like receptor signaling sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf deubiquitinase function of arterivirus papain-like protease suppresses the innate immune response in infected host cells antagonism of the phosphatase pp by the measles virus v protein is required for innate immune escape of mda measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp phosphatases the cgas-sting defense pathway and its counteraction by viruses immune evasion strategies during chronic hepatitis b and c virus infection. vaccines (basel) , , hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dna-sensing pathways hepatitis b virus evasion from cyclic guanosine monophosphate-adenosine monophosphate synthase sensing in human hepatocytes inhibition of cgas dna sensing by a herpesvirus virion protein cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas modulation of the cgas-sting dna sensing pathway by gammaherpesviruses denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity evolutionary conflicts between viruses and restriction factors shape immunity the evolutionary conundrum of pathogen mimicry enhanced influenza virus-like particle vaccination with a structurally optimized rig-i agonist as adjuvant pika as an adjuvant enhances specific humoral and cellular immune responses following the vaccination of mice with hbsag plus pika a tlr ligand that exhibits potent inhibition of influenza virus replication and has strong adjuvant activity has the potential for dual applications in an influenza pandemic as , an aluminum salt-and tlr agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity the tlr agonist, monophosphoryl lipid a, attenuates the cytokine storm associated with respiratory syncytial virus vaccine-enhanced disease sting agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice the tlr antagonist eritoran protects mice from lethal influenza infection targeting innate immunity for antiviral therapy through small molecule agonists of the rlr pathway direct antiviral properties of tlr ligands against hbv replication in immune-competent hepatocytes safety, efficacy and pharmacodynamics of vesatolimod (gs- ) in virally suppressed patients with chronic hepatitis b antibody and tlr agonist delay viral rebound in shiv-infected monkeys therapeutic effects of the artemisinin analog sm on lupus-prone mrl/lpr mice via inhibition of tlr-triggered b-cell activation and plasma cell formation tak- (resatorvid), a small-molecule inhibitor of toll-like receptor (tlr) signaling, binds selectively to tlr and interferes with interactions between tlr and its adaptor molecules essential for the antitumor effect of immune checkpoint blockade sa- - bbl and monophosphoryl lipid a constitute an efficacious combination adjuvant for cancer vaccines magnitude of therapeutic sting activation determines cd (+) t cell-mediated anti-tumor immunity immunogene therapy using immunomodulating hvj-e vector augments anti-tumor effects in murine malignant glioma promising targets for cancer immunotherapy: tlrs, rlrs, and sting-mediated innate immune pathways immunogenic cell death of human ovarian cancer cells induced by cytosolic poly(i:c) leads to myeloid cell maturation and activates nk cells sting-mediated dna sensing promotes antitumor and autoimmune responses to dying cells sting agonist formulated cancer vaccines can cure established tumors resistant to pd- blockade this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this work has been supported by the h marie-curie actions msca-if- -hipshot (km). this work was also supported in part by the national institutes of health grants u -ai (tfb) by arc, paris and institut hospitalo-universitaire, strasbourg (therahcc and therahcc . ihuarc ihu and ihuarc to t. acknowledgments: the authors would like to thank jean-luc imler and joao t. marques for their critical reading of the manuscript. the authors apologize to colleagues whose work could not be cited due to space limitations. the authors declare no conflict of interest. key: cord- -ynoxec k authors: matsuyama, toshifumi; kubli, shawn p.; yoshinaga, steven k.; pfeffer, klaus; mak, tak w. title: an aberrant stat pathway is central to covid- date: - - journal: cell death differ doi: . /s - - - sha: doc_id: cord_uid: ynoxec k covid- is caused by sars-cov- infection and characterized by diverse clinical symptoms. type i interferon (ifn-i) production is impaired and severe cases lead to ards and widespread coagulopathy. we propose that covid- pathophysiology is initiated by sars-cov- gene products, the nsp and orf proteins, leading to a catastrophic cascade of failures. these viral components induce signal transducer and activator of transcription (stat ) dysfunction and compensatory hyperactivation of stat . in sars-cov- -infected cells, a positive feedback loop established between stat and plasminogen activator inhibitor- (pai- ) may lead to an escalating cycle of activation in common with the interdependent signaling networks affected in covid- . specifically, pai- upregulation leads to coagulopathy characterized by intravascular thrombi. overproduced pai- binds to tlr on macrophages, inducing the secretion of proinflammatory cytokines and chemokines. the recruitment and subsequent activation of innate immune cells within an infected lung drives the destruction of lung architecture, which leads to the infection of regional endothelial cells and produces a hypoxic environment that further stimulates pai- production. acute lung injury also activates egfr and leads to the phosphorylation of stat . covid- patients’ autopsies frequently exhibit diffuse alveolar damage (dad) and increased hyaluronan (ha) production which also leads to higher levels of pai- . covid- risk factors are consistent with this scenario, as pai- levels are increased in hypertension, obesity, diabetes, cardiovascular diseases, and old age. we discuss the possibility of using various approved drugs, or drugs currently in clinical development, to treat covid- . this perspective suggests to enhance stat activity and/or inhibit stat functions for covid- treatment. this might derail the escalating stat /pai- cycle central to covid- . the etiology of severe acute respiratory syndrome coronavirus (sars-cov- ) infection and the progression of the resulting coronavirus disease of (covid- ) disease are novel. a person may be contagious - h before clinical symptoms appear, and % of transmissions are presymptomatic [ ] . viral loads are high at symptom onset but decrease within days. in contrast, in the severe acute respiratory syndrome (sars) disease caused by sars-cov- , the highest viral shedding occurs days after symptom onset [ ] . sars-cov- also binds more tightly than sars-cov- to their common cell entry receptor, human angiotensin-converting enzyme (ace ) [ ] [ ] [ ] . sars-cov- chiefly infects the lower airways, while sars-cov- is initially present in the upper respiratory tract and later moves to the lower airways. for sars-cov- , the highest viral load in the upper respiratory tract occurred during the first days, and viral load is higher and persists for longer in the lower respiratory tract of patients who are severely ill with covid- . for sars, upper edited by g. melino respiratory tract infection rarely occurred, and as a consequence, the transmission of sars-cov- was rare during the first days of illness [ ] . these differences accentuate the greater infectivity of sars-cov- . a strong correlation exists between sars-cov- viral load and disease severity and progression [ ] . pathological examinations have established that covid- causes widespread thrombosis with microangiopathy in pulmonary vessels [ ] . among covid- patients in the intensive care unit (icu) with pneumonia, the cumulative incidence of thrombotic complications was % [ ] . levels of ddimer, a coagulopathy indicator, correlated well with covid- illness severity [ ] . accordingly, coagulopathy and thrombosis are pervasive pathological features of covid- . hospitalized patients also commonly exhibit lymphopenia [ ] . pronounced laboratory abnormalities are also present in severe covid- disease. immunopathologic phenomena include the temporal downregulation of the type i and type iii interferon responses, with concomitant increases in proinflammatory cytokine and chemokine production [ ] . in contrast to other viral infections [ ] , crp levels are significantly elevated and positively correlated with disease severity in covid- [ , ] . these high crp levels have never been observed in any other infectious viral disease [ ] . clinical studies of hospitalized patients revealed that the most common comorbidities were hypertension ( . %), obesity ( . %), and diabetes ( . %) [ ] . sars-cov- is a sarbecovirus with an overall structure similar to that of sars-cov- . the sars-cov- genome contains the large ′ open reading frame (orf) ab encoding two polyproteins, including nonstructural proteins (nsps), namely nsp -nsp . the ′ end of the genome encodes the structural proteins spike (s, composed of two subunits s and s ), envelope (e), membrane (m), and nucleocapsid (n). interspersed among these genes are orfs encoding the nonstructural accessory proteins orf a, orf b, orf , orf a, orf b, and orf [ ] [ ] [ ] . several sars-cov- proteins antagonize the antiviral activities of ifns and the downstream jak (janus kinase)-stat signaling pathways they activate. jak family kinases (jak , jak , jak , tyk ) display a wide range of functions during ontogeny, in immunity as well as in chronic inflammation, fibrosis, and cancer [ ] . comparative genetic structural studies have suggested that sars-cov- has similar ifn antagonist activity [ , ] . after a careful review of the scientific literature, we realized that the sars-cov- -mediated inhibition of ifn and stat , and the subsequent shift to a stat dominant signaling network (see below), could result in almost all of the clinical features of covid- . here, we discuss the pathophysiology of sars-cov- , with a specific focus on sars-cov- 's effects on ifn and jak/ stat signaling. we propose that covid- is a disease caused by a catastrophic cascade of failures stemming from the sars-cov- -mediated dysregulation of stats. specifically, the dysfunctions of stat and stat induced by sars-cov- proteins may be the foundation of severe covid- pathophysiology. target cell entry sars-cov- cell entry depends on the binding of the spike protein's s subunit to ace on the target cell surface [ ] [ ] [ ] . host proteases, furin [ ] , as well as tmprss (transmembrane serine protease ) [ ] , then processes the s protein to facilitate membrane fusion. cleavage at the s / s junction and s ' site mediates the fusion of the viral and cellular membranes, in a process driven by the s subunit [ , ] . furin is ubiquitously expressed and its cleavage site at the boundary between the s /s subunits is not present in sars-cov- and sars-related viruses [ , ] , allowing sars-cov- to have enhanced proteolytic activation in a wider range of tissues [ ] . potential host cells for sars-cov- express high levels of both ace and tmprss , and include type alveolar cells, nasal goblet cells, nasal ciliated cells, corneal cells, and intestinal epithelial cells [ ] . sars-cov- can also directly infect endothelial cells [ , ] and engineered human blood vessels in kidney organoids in vitro [ ] . among immune cells, sars-cov- appears to infect mononuclear phagocytes, but not lymphocytes [ , ] . cells detect viral attack through membrane-bound or intracellular pattern recognition receptors (prrs). among these, coronaviruses activate the toll-like receptors (tlrs), retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein ( fig. ) [ ] . virusinduced oligomerization of prrs leads to the activation of downstream interferon regulatory factors (irfs) and nuclear factor-kappa b (nf-κb) transcription factors that induce the production of ifn-i, ifn-ii, and ifn-iii [ ] . the ifn released from infected cells binds to ifn receptors on neighboring cells, alerting them to a viral attack (fig. ) . the ifn-i and ifn-ii receptors are almost ubiquitously expressed, while ifn-iii receptors are only expressed on cells lining the epithelial barrier [ ] . the engagement of ifn-i and ifn-iii receptors activates various members of the jak ( , fig. ) and stat families ( and , fig. ), and specific transcription factor complexes are formed. for example, stat interacts with stat and irf- to constitute the transcription factor complex "ifn-stimulated gene factor " (isgf ) [ ] . in contrast, ifn-ii activates jak and jak , producing a phosphorylated stat homodimer known as "γ-interferon activation factor" (gaf) [ ] . interestingly, both isgf and gaf can be evoked by all ifns [ , ] . in any case, before they can exert their transcription factor activity, the isgf and gaf complexes must be transported to the nucleus and the subsequent upregulation of the interferon-stimulated gene products (isgs) [ ] . karyopherin-α (kpna ) is essential for the nuclear transport of stat [ ] , and the interaction between stat and kpna (stat /kpna ) involves a nonclassical nuclear localization signal (nls). in addition to ifn signaling, stat proteins are involved in signal transduction for other families of cytokines, including il- [ , ] . the sars coronaviruses use various mechanisms to hamper ifn production and response [ ] . consequently, target cells proximal to the site of the initial infection fail to receive critical and protective ifn signals, allowing the virus to spread and replicate without hindrance. a hallmark of sar-cov- infection is impaired ifn-i and iii production and responses, which masks the ifn-related fever symptoms [ ] and leads to naive spreading of the virus [ , , ] . the sars-cov- nsp protein impedes stat phosphorylation [ ] , and nsp , nsp , and nsp are involved in the establishment of double-membrane vesicles (dmvs) [ ] . dmvs create a platform by assembling the replicase proteins, virus genomes, and host proteins required for replication, while physically separating the replication sites from the cytoplasmic sensors of the innate immune response [ , ] . nsp and nsp prevent detection by the host's rig-i, which recognizes unmethylated rnas as non-self [ ] . sars-cov- structural proteins also quench the ifn response. the n protein binds to the e ubiquitin ligase trim (tripartite motif protein ) and interferes with the association between trim and rig-i [ ] . the m protein impairs the formation of the traf /tank/tbk /ikk ϵ complex, which is important in irf /irf signaling [ ] . the orf a protein induces serine phosphorylation within the degradation motif of ifn alpha-receptor subunit (ifnar ) and enhances ifnar ubiquitination [ ] . finally, the sars-cov- orf b, orf , and n proteins block irf phosphorylation [ ] . sars-cov- proteins structurally resemble those of sars-cov- and thus are likely to have the similar effects on ifn production and response (figs. and ), but there are some important differences. the sars-cov- orf b gene contains a premature stop codon, resulting in a truncated protein ( aa) as compared to the sars-cov- orf b protein ( aa) [ ] . although this sars-cov- orf b aa peptide lacks the c-terminal nls, it retains the ability to efficiently inhibit ifn-i signaling [ ] . orf b proteins in bat coronaviruses (sl-covs) [ ] also inhibit fig. possible differential effects of sars-cov- rna/proteins on ifn-i and proinflammatory cytokine/chemokine production. molecular patterns derived from sars-cov- -associated molecules, such as ssrna, dsrna, and viral proteins, bind to host prrs and trigger the activation of signal transducers and transcription factors that drive the production of ifn-i and proinflammatory cytokines and chemokines. soon after infection, the engagement of rig-i and mda- by these molecular patterns induces the activation of irf , or irf , through mavs. in addition, viral ssrna, dsrna, and proteins can engage tlrs to trigger the myd -and trif-dependent pathways, primarily leading to the activation of the nf-κb (p /p ) transcriptional complex. sars-cov- proteins that inhibit ifn-i production are indicated in black boxes, and the associated blocked pathways are indicated as dashed lines. note that only the ifn-i production pathway, and not the secretion of proinflammatory cytokines/chemokines, is inhibited by the viral proteins. proinflammatory cytokine/chemokine production is further activated by the engagement of tlrs by a high viral load. ifn-i and konno et al. have described a larger, natural sars-cov- orf b variant ( aa) with increased anti-ifn-i activity [ ] . yang et al. found that the sars-cov- inhibition of stat phosphorylation leads to the attenuation of the interferon-stimulated genes transcription in monocyte-derived dendritic cells and macrophages [ ] . perhaps sars-cov- nsp blocks stat phosphorylation in a similar manner as sars-cov- nsp [ ] , contributing to the inhibition of interferon response. the sars-cov- orf protein is a particularly vital viral factor that uses multiple measures to inhibit ifn production and responses. the sars-cov- orf protein appears to have similar functions. several lines of evidence demonstrate the effect of sars-cov- orf on the ifn response. ( ) frieman et al. showed that sars-cov- orf is an er/golgi membrane protein that binds kpna , together with kpnb . this recruitment of kpnb into the membrane complex limits the bioavailability of kpnb needed for the nuclear import of stat / kpna complexes. as a result, orf blocks the nuclear import of stat [ ] . virions [ ] , and thus the cell is exposed to orf immediately upon viral infection. moreover, when coexpressed with the sars-cov- s, m, and e proteins, orf is incorporated into virus-like particles [ ] . therefore, the orf protein may interfere with intracellular signaling prior to authentic viral replication. ( ) sars-cov- orf modifies membranes and produces perinuclear vesicles resembling dmvs [ ] . orf co-immunoprecipitated with viral rnas and co-localized on cytoplasmic vesicles with replicating viral rnas [ ] . thus, orf is also involved in establishing dmvs for viral rna replication. ( ) the introduction of sars-cov- orf into a ( ) are activated after ifn-i stimulation. stat is normally activated in ifn-i signaling to induce isgs (stat -isgs) by isgf (stat /stat /irf ). stat is also activated and becomes a homodimer, but the response is small. b ifn-i signaling with sars-cov- infection. after the infection, stat activity is inhibited by the sars-cov- proteins, nsp , and orf ( ). with stat activity restricted, stat ( ) then becomes dominant and induces stat -isgs. both stat and stat induce socs and socs ( ) that inhibit the kinase activity of jaks for the negative feedback of ifn-i signaling. pias and pias ( ) inhibit the binding of stat and stat to dna, respectively, to regulate ifn-i signaling. the role of pias becomes critical when stat is aberrantly activated and uncoupled from socss regulation. protein tyrosine phosphatases (ptps, ) have regulatory activities on activated jaks and stats, but their role in the viral infection needs further clarification. egfr ( ) is upregulated by acute lung injury or by reduced stat activity in the sars-cov- -infected lung. stat is activated through directly binding to egfr, through egfr-activated src ( ), or through jak (data not shown). pias normally limits the activity of stat but pai- produced during infection blocks pias activity ( ) and an escalating cascade in the stat /pai- axis is established. sublethal mouse hepatitis virus (mhv) strain promoted viral proliferation and caused fatal encephalitis in infected mice [ , ] . the expression of orf before infection with wild-type mhv facilitated the production of significantly more progeny. sars-cov- -encoded proteins and found that the orf protein was the most potent antagonist. although sars-cov- orf shares only % sequence homology with sars-cov- orf , the sars-cov- orf protein reduced the ifn-beta promoter activity by -fold and suppressed ifn signaling to levels comparable to those imposed by sars-cov- orf . the sars-cov- and sars-cov- orf proteins inhibit ifn-i and ifn-iii secretion induced by sendai virus infection, a method often used to elicit ifn-i production, and have profound effects on irf phosphorylation, thus accounting for their potent inhibition of ifn-i production [ , ] . once stat function is impaired by the nsp or orf protein of sars-cov- ( , fig. ), a concomitant and compensatory shift to signaling via stat -independent pathways may occur, and a stat -dependent transcriptional profile becomes dominant in many situations ( , fig. ) [ ] [ ] [ ] . notably, ifn-i signaling not only activates stat but also stat , and stat has a fine-tuning role in stat -mediated ifn-i responses [ ] . several potential mechanisms have been proposed as to how stat inhibits the stat -mediated ifn-i response: ( ) stat prevents the formation of the stat homodimer by making heterodimers with stat , ( ) stat inhibits the binding of isgf (stat /stat /irf ) to dna in cooperation with repressors, ( ) stat directly and indirectly reduces the expression of isgf components [ ] . stat may also compete with stat for a nuclear translocation factor, kpna [ ] . if competition between stat and stat for these nuclear translocation factors occurs in sars-cov- -infected cells, then stat would have to outcompete stat for binding to kpna [ ] and orf for binding to kpnb [ ] to achieve proper nuclear localization. these situations would create a higher functional stat :stat ratio immediately after virus infection, shifting the dominant transcriptional network to that governed by stat , the stat -stimulated genes (stat -isgs; , fig. ) [ ] . both stat and stat transcriptionally induce both suppressor of cytokine signaling (socs ) and socs that inhibit activity of jaks ( , fig. ). in the nucleus, protein inhibitor of activated stat (pias ) and pias can bind activated stat and stat dimers ( , fig. ), respectively, and block them from binding to dna, thus inhibiting stat mediated transcription. tyrosine-phosphorylated stats or jaks are targets of tyrosine phosphatases, such as shp , shp , ptp b, and tc-ptp [ ] ( , fig. ). however, their roles in covid- remains to be clarified. sometimes the negative regulation of stat by socs / socs becomes dysfunctional. ramana et al. reported that the ifn-ii induction of socs in stat -deficient mouse embryonic fibroblasts was derived from activated stat [ ] . in wild-type fibroblasts, stat is normally dominant, but in stat -deficient fibroblasts stat is activated strongly and in a sustained manner. the src kinase inhibitor su suppressed ifn-ii activation of stat significantly in stat -deficient fibroblasts but not in wild-type fibroblasts indicating that stat is activated through src kinase activity in the absence of stat . stat can be tyrosine-phosphorylated by the dimerized epidermal growth factor receptor (egfr), egfr-activated src ( , fig. ), or by egfr-activated jak [ ] , but only the latter pathway is regulated by socs . recently, egfr signaling was shown to inhibit ifn-i through activated stat [ ] . egfr is upregulated ( , fig. ) during acute lung injury [ ] , or when stat is deficient [ ] . this scenario is consistent with a study with an alpha coronavirus, porcine epidemic diarrhea virus. stat was virologically suppressed by the infection [ ] and ifn-i signaling was inhibited by upregulated egfr and activated stat [ ] . therefore, in covid- , egfr signaling may become an alternative pathway that activates stat specifically when the lung is damaged while the production of ifn-i is severely impaired by sars-cov- infection [ ] . this aberrant transcriptional rewiring toward stat may lead to the symptoms most commonly observed in hospitalized covid- patients: rapid coagulopathy/ thrombosis, proinflammatory conditions, profibrotic status, and t cell lymphopenia. the next sections describe how stat hyperactivation can be linked to each of these abnormalities. a summary of these events, starting with the downregulation of stat ( , fig. ) and compensatory upregulation of stat ( , fig. ) induced by nsp and orf of sars-cov- , is presented in fig. . pervasive hemostasis disorders are a life-threatening feature of covid- [ ] . inflammation-induced coagulation is initiated by the expression of the transmembrane protein "tissue factor" (tf) [ ] . in the lung, tf is expressed in alveolar epithelial cells [ ] , myeloid cells [ ] , and endothelial cells [ ] in response to proinflammatory cytokines [ ] or crp [ ] [ ] [ ] [ ] [ ] . tf promotes the transformation of prothrombin into thrombin, which converts circulating fibrinogen into fibrin, leading to fibrin-based blood clots [ ] . recombinant human crp, administered in concentrations commonly seen in patients with inflammation, induced a -fold increase in the tf procoagulant activity of human pbmcs [ ] . because tf is induced by crp, and crp is a downstream target of stat [ ] , aberrantly activated stat may prime the initial phase of coagulation ( , fig. ). in addition, the full transcriptional induction of crp requires the synergistic cooperation of stat , c-fos, and t cell factor (tcf- , or hnf- α) [ ] . c-fos is activated by sars-cov- orf b [ ] and tcf- is expressed in lung cells [ ] . thus stat , c-fos, and tcf- could participate in sars-cov- -infected cells and act to raise plasma crp levels. it then follows that the positive correlation between crp level and disease severity in covid- patients [ , ] also reflects the number of virus-infected cells. another critical factor in coagulopathy and thrombosis is pai- (serpin e ), a serine protease inhibitor secreted by vascular endothelial cells, hepatocytes, adipocytes, and epithelial cells [ , ] . several lines of evidence suggest that pai- and stat interact to promote coagulopathy and thrombosis in covid- ( , fig. ) . ( ) pai- is upregulated by stat . pai- is expressed in damaged type alveolar cells [ ] and indirectly upregulated by stat through five pathways. first, microrna- a (mir- a) targets the pai- gene in non-small cell lung carcinoma (nsclc), and mir- a transcription is suppressed by stat [ ] . this negative regulation of pai- by mir- a [ ] , and of mir- a by stat [ ] , has also been documented in cancer cell lines. therefore, stat indirectly upregulates pai- through mir- a ( , fig. ) . second, stat (plus c-fos and tcf- ) induces crp [ ] , and crp induces pai- in human aortic endothelial cells [ ] . third, the tumor suppressor p fig. a dysregulated stat -pai- signaling node is common to covid- pathophysiology. proposed role of stat -pai- signaling node in catastrophic cascades underlying covid- pathophysiology. infection by sars-cov- intracellularly delivers nsp and orf , which efficiently inhibit stat function ( ). repression of stat increases stat ( ) activity. stat upregulates pai- ( ) by repressing mir- a, a pai- inhibitor ( ). this increased pai- reciprocally activates stat by blocking pias , a stat inhibitor ( ) . stat can activate has , a hyaluronic acid synthase, which produces hyaluronan (ha) and leads to diffuse alveolar damage (dad) characterized by hyaline membrane formation ( ) . fragments of ha (lmw-ha) activate pai- ( ). an escalating cycle of stimulation between stat and pai- begins a catastrophic cascade of events, resulting in combinations of coagulopathy/thrombosis ( ), macrophage production of cytokines and chemokines ( ), and profibrotic changes ( ) . hypoxia eventually results, which further induces pai- transcription through hif- α ( ). this elevated pai- activity then drives il- production via tlr , which in turn stimulates even more stat activity (no. ). elevated stat also activates pd-l in endothelial cells, leading to t cell lymphopenia (no. ). details of these events are described in the main text. italicized outside labels are cell types, and non-italicized outside labels are locations. activates pai- production [ ] , and activated stat induces p transcription [ ] . fourth, as detailed below, stat indirectly activates tgf-β in the extracellular matrix (ecm), and tgf-β upregulates pai- production [ ] . fifth, severe ards cases of covid- result in diffuse alveolar damage (dad) and the formation of hyaline membranes [ ] . hyaluronan (ha) is essential for many biological functions, but it is a causative agent in ards when it becomes a low-molecular-weight ha (lmw-ha) [ ] . both stat and pai- are associated with the production of ha. stat activates the transcription of has , the gene for ha synthase [ ] , and lmw-ha stimulate the production of pai- ( , fig. ) [ , ] . furthermore, variations in genes involved in ha synthesis control the levels of plasma pai- .( , fig. ) [ ] indeed, the stat /pai- axis is intimately connected to the production of ha. ( ) pai- may activate stat . pai- can interact with pias ( , fig. ), an endogenous stat inhibitor ( , fig. ), to regulate stat -dependent gene expression in nsclc [ ] . other studies have found a more indirect effect. for example, pai- engages tlr [ ] and activates il- , which could then activate stat [ ] . in addition, pai- can promote monocyte migration through its interaction with lipoprotein receptor protein- (lrp- ). pai- /lrp- binding influences monocyte/macrophage polarization toward the m type, which features transcriptional upregulation of il- and activation of stat [ ] . ( ) pai- is highly expressed in plasma and lungs in covid- cases. sars patients were characterized by increased plasma levels of pai- [ ] and high pai- protein expression in alveolar cells [ ] compared to controls. consistently, pai- is significantly elevated in the plasma of hospitalized covid- patients [ ] . furthermore, pai- mrna levels are higher in the lungs of covid- patients, as compared to those of uninfected or influenza patients [ ] . ( ) pai- is closely linked with covid- risk factors. network pathway analyses have identified pai- as a key protein in obesity, diabetes [ ] , and cardiovascular disease [ ] . furthermore, higher levels of plasma pai- are present in hypertensive patients [ , ] and older adults [ , ] . all of these conditions are high-risk factors for severe covid- pathology. the above studies suggest that the increase in pai- levels due to stat activation may efficiently inhibit both tissue-type plasminogen activator (tpa) and urokinase-type plasminogen activator, leading to coagulopathy/thrombosis ( , fig. ). in addition, ifn-i has been shown to upregulate tpa expression through stat [ ] . therefore, the early repression of stat and ifn activities imposed by viral proteins could synergistically inhibit tpa and promote coagulopathy/thrombosis. in addition to the coagulopathic changes within the pulmonary vasculature, copin et al. observed a peculiar pattern in the lungs of six covid- patients that was different from the classical dad pattern of ards [ ] . the pattern, acute fibrinous and organizing pneumonia, consisted of a fibrin component and was characterized by extensive deposition of intra-alveolar fibrin ("fibrin balls") in the lungs. mcgonagle et al. suggested that the occurrence of enhanced thrombin generation and fibrin deposition within the bronchoalveolar system might be primarily driven by the upregulation of tf expression within the alveoli, coupled with a pai- -induced reduction in fibrinolysis [ ] . because tf is a downstream target of activated stat [ ] and produced in type alveolar cells [ ] , the secretion of both tf and pai- into the alveolar space may lead to the enhanced deposition of intra-alveolar fibrin. significant efforts are presently underway to define the proinflammatory cytokines and chemokines active in covid- . huang et al. studied in-patients ( icu patients and non-icu patients) and reported that the plasma levels of inflammatory cytokines, including il- , il- , g-csf, ip- , mcp- , mip a, and tnf-α, were all elevated in critically ill patients [ ] . in critically ill covid- patients, serum il- is significantly greater than those with moderate or severe disease [ ] . the excessive production of these inflammatory cytokines in the lungs of covid- patients appears to be due to increased macrophage activation [ ] [ ] [ ] . gupta et al. reported that pai- activates macrophages and increases their proinflammatory cytokine production by binding to tlr [ ] ( , fig. ). pai- binding to tlr leads to the activation of nf-κb, which induces the production of chemokines and il- . il- stimulates stat ( , fig. ) to produce tgf-β [ ] in a positively regulated reciprocal feedback loop involving pai- [ ] , thereby amplifying the tlr -mediated inflammatory response. pai- thus appears to be partly responsible for the excessive production of inflammatory cytokines and chemokines by macrophages in the lungs of severely ill covid- patients. in addition, accumulation of lmw-ha molecules in the small airways not only stimulates macrophages to release chemokines, cytokines, and growth factors, but also promotes fluid retention in the extracellular space, contributing to interstitial and alveolar edema [ ] . interstitial and alveolar edema severely impairs physiological gas exchange, causing hypoxia, which further stimulates pai- expression through hypoxia-inducible factor α (hif- α) [ , ] . thus, the central node involving stat and pai- may favor hypoxia through multiple routes ( , fig. ). tgf-β plays a key role in covid- . xiong et al. performed transcriptome sequencing of rnas isolated from bronchoalveolar lavage fluid and pbmc specimens of covid- patients, and found that sars-cov- infection induced il- and tgf-β production [ ] . many viral infections modulate tgf-β signaling to block cell apoptosis and promote fibroblast proliferation and myofibroblast differentiation. thus, the increased expression of tgf-β in covid- patients may drive the observed pulmonary fibrosis [ ] . the possible sources of tgf-β in covid- are the ecm, blood-circulating platelets, recruited neutrophils, macrophages, and infected type alveolar cells [ , ] . the latent form of tgf-β in the ecm requires activation by integrin αvβ and thrombospondin, which are induced by stat [ ] [ ] [ ] [ ] [ ] . therefore, aberrantly activated stat in type alveolar cells may contribute to tgf-β activation and subsequent fibrosis ( , fig. ) . experiments in a murine model of systemic sclerosis support the indirect upregulation of pai- ( , fig. ) by stat through tgf-β [ ] . activated tgf-β induces pai- transcription [ ] , establishing another positive feedback loop involving stat and pai- . the transcription of profibrotic genes encoding collagens, proteoglycans, integrins, connective tissue growth factor, and matrix metalloproteinases (mmps) is also actuated by activated tgf-β [ ] , establishing a profibrotic environment. in fact, lessons from sars-cov- suggest that the sars-cov- n protein may enhance tgf-β-induced expression of pai- and collagen i, and thus promote lung fibrosis [ ] . lymphopenia is a common characteristic of covid- patients [ , , ] and correlates with disease progression, but its etiology remains unclear. diao et al. reported that, in hospitalized cases, % of the patients had remarkably reduced total t cell counts, which were significantly lower in icu patients than in non-icu cases. they also observed that pd expression on t cells increased as patients progressed from the prodromal to the overtly symptomatic stage. because pd is a marker of t cell exhaustion, this group proposed that the elevated proinflammatory cytokines in covid- patients are responsible for their t cell lymphopenia [ ] . moreover, chen et al. analyzed spleens and lymph nodes from six autopsy cases, and suggested that sars-cov- could directly infect tissue-resident cd + macrophages in secondary lymphoid organs and induce lymphocyte apoptosis [ ] . alternatively, t cell lymphopenia could be caused directly by sars-cov- entering t cells via cd [ ] . although sars-cov- cannot replicate in t cells, the apoptosis-inducing activity of the sars-cov- orf a protein [ ] could effectuate t cell lymphopenia. the effects of sars-cov- on pd and its binding partner pd-l differ by disease phase; i.e., the early/mild phase vs. the late/critical phase of covid- . the early/mild phase of sars-cov- infection is limited to type alveolar cells and alveolar macrophages. once infected, these cells secrete chemokines, including ip- (cxcl ), in a process that requires stat activation [ ] . as a result, plasma ip- is increased in severely ill covid- patients [ ] . ip- recruits activated cxcr + t cells from the regional capillaries [ ] [ ] [ ] [ ] , and lymphocytes infiltrate the diseased lung tissues of these patients [ , , ] . because type alveolar cells [ ] and alveolar macrophages [ ] express pd-l together with virus antigens, virus-specific t cells migrate to the area and then upregulate pd upon recognition of the antigens [ ] . pd-l engagement with pd induces the apoptosis of the activated t cells [ , ] , resulting in moderate t cell lymphopenia ( , fig. ). in the severe phase of sars-cov- infection, activated stat promotes the secretion of mmp- by type alveolar cells [ ] and alveolar macrophages [ ] . mmp degrades collagen iv, the major component of the basement membrane, and compromises its integrity. the virus may then migrate into the interstitium and enter capillaries, where it could infect endothelial cells. the infection causes aberrant stat regulation in these endothelial cells and may also induce pd-l activity, as demonstrated for some cancer cells with constitutively active stat [ , ] . in the critical stages of covid- , the pd /pd-l -induced death of t cells becomes systemic, resulting in severe t cell lymphopenia ( , fig. ). we hypothesize that covid- disease is due in large part to the actions of the sars-cov- nsp and orf proteins, which cripple stat function and predominantly promote stat activation. stat in turn upregulates pai- , and together these molecules serve as a central hub of reactions that perpetuate a catastrophic cascade. our understanding of immune responses, coupled with lessons from sars-cov- and recent research on sars-cov- , point to stat and stat as enticing drug targets because they function upstream of the cytokine storm and thrombosis. developing vaccines will take some time, and attacking the downstream cytokine storm is difficult due to its many targets. hence, in the short term, the manipulation of stat and/or stat may be the most practical strategy for treating covid- . fortunately, many therapeutic regulators of these stats have been, or are being, developed. the first consideration in treating sars-cov- infection should be to preserve stat function in its earliest stages. however, patients may be asymptomatic or presymptomatic for days while the virus replicates, spreads, and evades the ifn response. if the virus has already compromised stat when symptoms arise, then treatment should focus on preventing the excessive activation of stat that drives the release of proinflammatory cytokines and chemokines. care must be taken to apply these therapeutic strategies in a stageappropriate manner, because some approaches that may help in the earlier stages of the disease could be detrimental if used in its later stages. in the following sections, we discuss the potential use for covid- treatment of existing drugs that enhance stat or inhibit stat functions. problems could arise from this stat-approach to covid- therapy. chronic mucocutaneous candidiasis (cmc) was present in % of patients with gain of function (gof) mutations in stat in one study of individuals, and the few that did not have cmc, had invasive fungal or bacterial infections. generally, the immune profile was relatively normal, but % had decreased th cells [ ] . stat and stat reciprocally regulate each other, and consistently, stat gof patients have reduced levels of stat [ ] . stat ige syndrome (stat -hies) is a rare autosomal dominant condition caused by loss of function mutations in the stat gene. patients have eczema, recurrent skin and respiratory tract infections, and usually high levels of ige [ ] . these examples of genetic defects are likely the most extreme consequences of increased stat or decreased stat activities. it is unknown if temporary treatment with stat activators and stat inhibitors would be detrimental, but it is promising that a high stat to stat ratio is advantageous in cancer treatment [ ] . at the very onset of infection, sars-cov- infects a minority of cells, making its invasion hard to detect. the virus acts to delay antiviral responses while it hijacks the cell's functions for viral replication. if the infection is detected early, then an effective strategy might be to use ifn-i to activate stat in neighboring uninfected cells. it is proposed that the timing of ifn treatment is critical [ ] . early induction of ifn-i signaling protects the patients, but delay in ifn administration not only fail to inhibit viral replication, but also increase proinflammatory cytokine production, leading to fatal pneumonia [ ] . since the upper respiratory tract is the primary entry site for sars-cov- , mucosal treatments with ifn-i for prevention of covid- is an ideal strategy. meng et al. used ifn-i nasal drops prophylactically applied to more than high-risk medical staff who were in direct contact with sars-cov- -infected patients. remarkably, the use of the ifn-i nasal drops, with thymosin-α , an immune-stimulator, protected all of the high-risk staff from covid- pneumonia [ ] . the use of agents that induce ifn production is another option for activating stat in uninfected cells. ampligen, poly(i:c( )u), is a synthetic dsrna polymer that stimulates ifn production [ ] . mice treated with ampligen h prior to lethal infection with sars-cov- were protected against death, showed reduced virus titers in the lungs, and exhibited significantly reduced lung disease scores and weight loss [ ] . notably, because ifn-i inducers can be administered prophylactically, the innate immune system can theoretically be primed to respond to a sars-cov- attack by immediately activating stat . however, such use of ifn inducers must be carefully monitored, because these agents can exacerbate the disease in the later stages of infection. administration of the h- blocker famotidine to patients hospitalized with covid- , but not initially in the icu, was associated with a twofold reduction in clinical deterioration leading to intubation or death [ ] . computational modeling indicated that famotidine directly binds and inhibits the sars-cov- processing enzyme nsp [ ] , but this drug lacked a direct anti-sars-cov- effect in vitro in vero cells, and h- -mediated antiviral effect is suggested [ ] . in vitro, histamine pretreatment of c bl/ mouse splenocytes enhances stat phosphorylation, and an h- antagonist (but not an h- antagonist) can augment stat phosphorylation to a similar extent [ ] . famotidine may function as an h- receptor antagonist that promotes stat activation and ifn responses. therefore, early stage treatment with famotidine might significantly decrease the mortality rate of covid- patients. ivermectin displays broad-spectrum antiparasitic and antiviral activities. caly et al. reported that a single dose of ivermectin reduced the amount of sars-cov- rna by -fold. they speculated that ivermectin inhibits the kpna/kpnb mediated nuclear import of viral proteins [ ] . apart from its possible role in blocking nuclear transport, ivermectin may promote a positive clinical outcome by inhibiting stat and il- production. ivermectin inhibits p activated kinase (pak ), a serine/threonine kinase with oncogenic activity, which then compromises stat activity. in this case, ivermectin suppresses akt/mtor signaling by promoting the ubiquitination-mediated degradation of pak [ ] . in addition, pak physically binds to both jak and stat , and the resultant pak /stat complex activates il- gene transcription [ ] . when ivermectin inhibits jak/ stat signaling by promoting pak degradation, stat activity is compromised and il- production is decreased. there are a number of approved drugs, or drugs in cancer development, that inhibit stat directly. stat antagonists have been described comprehensively in recent reviews by qin et al. [ ] and bharadwaj et al. [ ] . a brief description of some prominent candidates follows. napabucasin was initially identified by its ability to inhibit the properties of cancer cell stemness and stat activity in gel retardation assays [ ] . a phase iii clinical trial involving napabucasin in combination with other standard chemotherapeutic agents is currently underway for several advanced malignancies [ ] . in rectal cancer, napabucasin reduced not only pstat levels but also angiogenesis through a ros-mediated effect [ ] . another promising anti-stat agent is the -mer antisense oligonucleotide azd (danvatirsen), which targets the ′utr of the stat gene and inhibits its transcription [ ] . a phase ii trial of danvatirsen plus anti-pd-l monoclonal antibody in patients with head-and-neck squamous cell carcinoma is currently underway, with encouraging results thus far [ ] . metformin is a first-line oral antidiabetic drug that has been used to treat type diabetic patients for over years. metformin inhibits stat by specifically reducing its phosphorylation at tyr and ser [ ] . in addition, metformin prevents both venous and arterial thrombosis by inhibiting platelet activation and extracellular mitochondrial dna (mtdna) release. mtdna induces platelet activation through a dc-sign-dependent pathway [ ] . despite metformin's apparent utility in reducing stat activation and thrombosis, a major concern is that the dose that elicits these activities is very high ( - mg/kg/day). the regulation of endogenous inhibitors is another way to control aberrantly activated stat . pias is an ideal target because of its potency to inhibit the activated stat [ ] . specifically, curcumin and resveratrol have been shown to suppress constitutive activation of stat , through upregulation of pias [ , ] although widely used for many indications, curcumin and resveratrol have not been shown to be conclusively effective in any randomized, placebo-controlled, clinical trial. the occurrence of a cytokine storm in covid- (il- , il- , il- , il- , g-csf, ifnγ, mip α, and tnf-α [ , ] , which triggers cytokine receptors coupled to the jak-stat pathway, suggests that inhibition of the jak pathway may be an appropriate therapeutic strategy for the management of covid- . although the jak pathway affects many stats, perhaps the inhibition of the jak pathway may lead to the therapeutically favorable effect of quenching cytokine storm via stat inhibition. the available jak / inhibitors [ ] could be studied for their effects in covid- . however, as jak inhibitors also inhibit stat activation, the application of jak inhibitors in covid- cases needs careful consideration. as discussed earlier and shown in fig. , acute lung injury and/or the loss of functional stat can lead to the upregulation of egfr that may cause the constitutive activation of stat . therefore, it is reasonable to assume that targeting egfr signaling is an attractive strategy for covid- treatment. a promising candidate is erlotinib. lupberger et al. reported the combination of erlotinib and ifn-i resulted in a highly synergistic antiviral response against hepatitis c virus. furthermore, erlotinib reduced ifn-iinduced stat activity by induction of socs expression [ ] . very recently, the remarkable inhibition of sars-cov- replication by several growth factor signaling inhibitors was reported [ ] . these include lonafarnib (ras inhibitor), omipalisib (pi k inhibitor), pictilisib (pi k inhibitor), ro (raf and mek inhibitor), sorafenib (raf inhibitor, stat inhibitor [ ] ), because ras/raf/ mapk pathway [ ] and pi k pathway [ ] are also involved in egfr-mediated stat activation, these inhibitors can possibly synergistically potentiate ifn-i's anti-sars-cov- activity. as noted above, pai- is intimately involved in the pathogenesis of covid- , and its inhibition may be a key point at which to treat the disease once the escalating cycle between pai- and stat has been established. unfortunately, no fda-approved pai- inhibitor is currently available. pai- 's labile structure appears to make it a difficult target for the development of small-molecule inhibitors [ ] . alternatively, therapeutically targeting tlr , pai- 's binding partner, may be equally effective. tlr has already attracted keen interest as a therapeutic target for sepsis cases. although the molecular mechanisms have yet to be clarified, it is worth investigating whether pai- /tlr binding can be inhibited by the several tlr antagonists in development, or by approved drugs with anti-tlr activity. one tlr inhibitor, eritoran, is now in clinical trials to treat ards in covid- [ ] . the sars-cov- virus has evolved multiple tools to escape immune detection and destruction, and thus has become the most formidable virus in over years. our survey of the current literature reveals that severe cases of covid- are commonly dependent on the over-stimulation of the stat / pai- signaling network. we believe that this shared node may be the achilles' heel of covid- and a vulnerable point of the disease. we therefore urge the immediate investigation and application of stat therapy as a treatment for this perplexing disorder, and hope that our article provides a sound foundation for doing so. a final note: during our analysis of the literature on covid- and related topics, we noticed many similarities in the pathogenesis of covid- and cancers. in fact, both stat and pai- have been separately implicated in cancer development and are the subjects of extensive clinical investigations. a shared node of stat and pai- activities may also function in some pstat -positive cancers, creating a cascade of harmful responses comparable to those of covid- . any stat-related agents developed to treat covid- may therefore eventually enjoy much wider clinical use. • specific sars-cov proteins inhibit the functions of stat and ifns. in the absence of stat , stat is activated in a compensatory manner. • stat upregulates pai- through five signaling pathways mediated by mir- a, crp, p , transforming growth factor-β (tgf-), or hyaluronan fragments. • pai- is upregulated in aged individuals and in those suffering from hypertension, obesity, or diabetes, which are risk factors for covid- . • in severe cases of covid- , there is a common escalating cycle of stat and pai- activation that is shared among diverse disease manifestations and leads to catastrophic consequences. • is ifn production different between asymptomatic carriers, and patients with less or more severe cases of covid- ? • is ifn production different between people infected with sars-cov- , sars-cov- , mers coronavirus, or other coronaviruses? • is stat activated in sars-cov- -infected tissues? • will prophylactic activation of stat protect against the severe symptoms of covid- ? • will inhibition of the stat /pai- axis decrease the severities of the cytokine storm and thrombosis/ coagulopathy in covid- ? • will inhibition of egfr signaling potentiate the antiviral activity of 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analyses of sar-cov genomes from different geographical locations and other coronavirus family genomes reveals unique features potentially consequential to host-virus interaction and pathogenesis structure, function, and antigenicity of the sars-cov- spike glycoprotein sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor sars-cov- entry factors are highly expressed in nasal epithelial cells together with innate immune genes endothelial cell infection and endotheliitis in covid- inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace the novel severe acute respiratory syndrome coronavirus (sars-cov- ) directly decimates human spleens and lymph nodes comparative in vitro transcriptomic analyses of covid- candidate therapy hydroxychloroquine suggest limited immunomodulatory evidence of sars-cov- host response genes type i and type iii interferons-induction, signaling, evasion, and application to combat covid- stats and infection. landes biosci cross talk between interferon-γ and -α/β signaling components in caveolar membrane domains functional crosstalk between type i and ii interferon through the regulated expression of stat extracellular signal-dependent nuclear import of stat is mediated by nuclear pore-targeting complex formation with npi- , but not rch the role of interleukin during viral infections current clinical applications of interferon asymptomatic transmission, the achilles' heel of current strategies to control covid- imbalanced host response to sars-cov- drives development of covid- severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp and rational design of an attenuated strain severe acute respiratory syndrome coronavirus nonstructural proteins , , and induce double-membrane vesicles virus-induced double-membrane vesicles crystal structure and functional analysis of the sarscoronavirus rna cap '-o-methyltransferase nsp /nsp complex the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk /ikkepsilon complex the sars coronavirus a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type interferon receptor severe acute respiratory syndrome coronavirus open reading frame (orf) b, orf , and nucleocapsid proteins function as interferon antagonists sars-cov- orf b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant bat severe acute respiratory syndrome-like coronavirus orf b homologues display different interferon antagonist activities attenuated interferon and pro-inflammatory response in sars-cov- -infected human dendritic cells is associated with viral antagonism of stat phosphorylation severe acute respiratory syndrome coronavirus orf antagonizes stat function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane severe acute respiratory syndrome coronavirus accessory protein is a virion-associated protein and is released from protein-expressing cells the n-terminal region of severe acute respiratory syndrome coronavirus protein induces membrane rearrangement and enhances virus replication severe acute respiratory syndrome coronavirus protein accelerates murine coronavirus infections a severe acute respiratory syndrome-associated coronavirusspecific protein enhances virulence of an attenuated murine coronavirus stat deficiency unexpectedly and markedly exacerbates the pathophysiological actions of ifn-alpha in the central nervous system stat regulates lupus-like chronic graftversus-host disease severity via interactions with stat stat and stat in tumorigenesis: a matter of balance fine-tuning of type i interferon response by stat stat regulates the type i ifn-mediated antiviral response by interfering with the nuclear entry of stat negative regulation of type i ifn signaling stat -independent induction of socs- by interferon-gamma is mediated by sustained activation of stat in mouse embryonic fibroblasts targeting stat in hepatocellular carcinoma: sorafenib again epidermal growth factor receptor signaling impairs the antiviral activity of interferon-alpha human epidermal growth factor receptor signaling in acute lung injury overactive epidermal growth factor receptor signaling leads to increased fibrosis after severe acute respiratory syndrome coronavirus infection porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat porcine epidemic diarrhea virus-induced epidermal growth factor receptor activation impairs the antiviral activity of type i interferon understanding pathophysiology of hemostasis disorders in critically ill patients with covid- . intens care med tissue factor: an essential mediator of hemostasis and trigger of thrombosis the alveolar epithelium can initiate the extrinsic coagulation cascade through expression of tissue factor myeloid tissue factor does not modulate lung inflammation or permeability during experimental acute lung injury endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. effects of thrombin inhibitors th and th t-helper cells exert opposite regulatory effects on procoagulant activity and tissue factor production by human monocytes c-reactive protein induces human peripheral blood monocytes to synthesize tissue factor c-reactive protein induces expression of tissue factor and plasminogen activator inhibitor- and promotes fibrin accumulation in vein grafts c-reactive protein induces tissue factor expression and promotes smooth muscle and endothelial cell proliferation c-reactive protein enhances tissue factor expression by vascular smooth muscle cells jialal i. c-reactive protein stimulates superoxide anion release and tissue factor activity in vivo tissue factor: newer concepts in thrombosis and its role beyond thrombosis and hemostasis stat participates in transcriptional activation of the c-reactive protein gene by interleukin- transcriptional complex formation of c-fos, stat , and hepatocyte nf- alpha is essential for cytokinedriven c-reactive protein gene expression sars-cov accessory protein b induces ap- transcriptional activity through activation of jnk and erk pathways betacatenin in the fibroproliferative response to acute lung injury aging and plasminogen activator inhibitor- (pai- ) regulation: implication in the pathogenesis of thrombotic disorders in the elderly pai- promotes the accumulation of exudate macrophages and worsens pulmonary fibrosis following type ii alveolar epithelial cell injury pai- /pias /stat /mir- a forms a positive feedback loop to promote emt-mediated metastasis through stat signaling in non-small cell lung cancer mir- a exerts as a key regulator in the dedifferentiation of osteosarcoma via pai- -sox axis il- r/stat /mir- a feedback loop promotes emtmediated colorectal cancer invasion and metastasis c-reactive protein increases plasminogen activator inhibitor- expression and activity in human aortic endothelial cells: implications for the metabolic syndrome and atherothrombosis regulation of airway and alveolar epithelial cell apoptosis by p -induced plasminogen activator inhibitor- during cigarette smoke exposure injury stat /p pathway activation disrupts ifn-β-induced dormancy in tumorrepopulating cells activation of stat integrates common profibrotic pathways to promote fibroblast activation and tissue fibrosis accelerated hyaluronan concentration as the primary driver of morbidity and mortality in high-risk covid- patients: with therapeutic introduction of an oral hyaluronan inhibitor in the prevention of induced hyaluronan storm syndrome. public and global health hyaluronan in acute respiratory distress syndrome (ards): simply a biomarker or a deeper insight into ards mechanisms? extracellular udp-glucose activates p y receptor and induces signal transducer and activator of transcription (stat ) tyr phosphorylation and binding to hyaluronan synthase (has ) promoter, stimulating hyaluronan synthesis of keratinocytes regulation of plasminogen activator inhibitor- and urokinase by hyaluronan fragments in mouse macrophages effects of different molecular weight hyaluronan products on the expression of urokinase plasminogen activator and inhibitor and gelatinases during the early stage of osteoarthritis genetic variation in hyaluronan metabolism loci is associated with plasma plasminogen activator inhibitor- concentration plasminogen activator inhibitor- stimulates macrophage activation through tolllike receptor- plasminogen activator inhibitor promotes immunosuppression in human non-small cell lung cancers by enhancing tgf-β expression in macrophage plasminogen activator inhibitor- promotes the recruitment and polarization of macrophages in cancer analysis of thrombotic factors in severe acute respiratory syndrome (sars) patients severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with smad and modulates transforming growth factor-beta signaling endotheliopathy in covid- -associated coagulopathy: evidence from a single-centre, cross-sectional study serpine links obesity and diabetes: a pilot study plasminogen activator inhibitor and the risk of cardiovascular disease: the framingham heart study relationship between plasma plasminogen activator inhibitor- and hypertension in american indians: findings from the strong heart study plasminogen activator inhibitor- activity and the g/ g polymorphism are prospectively associated with blood pressure and hypertension status stress/inflammation and pai- as stellar processes in the aging and associated pathologies plasminogen activator inhibitor- (pai- ): a key factor linking fibrinolysis and age-related subclinical and clinical conditions interferons induce stat -dependent expression of tissue plasminogen activator, a pathogenicity factor in puumala hantavirus disease time to consider histologic pattern of lung injury to treat critically ill patients with covid- infection. intens care med immune mechanisms of pulmonary intravascular coagulopathy in covid- pneumonia upregulation of tissue factor by activated stat contributes to malignant pleural effusion generation via enhancing tumor metastasis and vascular permeability in lung adenocarcinoma clinical features of patients infected with novel coronavirus in wuhan profiling serum cytokines in covid- patients reveals il- and il- are disease severity predictors alveolar macrophages regulate neutrophil recruitment in endotoxin-induced lung injury monocyte-derived alveolar macrophages: the dark side of lung repair? pulmonary macrophage subpopulations in the induction and resolution of acute lung injury tgf-beta -induced pai- gene expression requires mek activity and cell-to-substrate adhesion induction of the plasminogen activator inhibitor- gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor- in rat hepatocytes hypoxia-inducible factor- α mediates tgf-β-induced pai- production in alveolar macrophages in pulmonary fibrosis transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients a potential treatment of covid- with tgf-β blockade tgf-β -a truly transforming growth factor in fibrosis and immunity cloning and characterization of the human integrin β gene promoter constitutively activated stat induces tumorigenesis and enhances cell motility of prostate epithelial cells through integrin beta intermittent hypoxia mediated by tsp dependent on stat induces cardiac fibroblast activation and cardiac fibrosis what the lung has taught us about latent tgf-beta activation targeting tgf-β mediated smad signaling for the prevention of fibrosis sars coronavirus and lung fibrosis lymphopenia is associated with severe coronavirus disease (covid- ) infections: a systemic review and meta-analysis clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury cd as a target for covid- treatment: suggested effects of azithromycin and stem cell engagement the orf a protein of sars-cov- induces apoptosis in cells macrophages induce differentiation of plasma cells through cxcl /ip- plasma ip- and mcp- levels are highly associated with disease severity and predict the progression of covid- differentiated human alveolar type ii cells secrete antiviral il- (ifn-lambda ) in response to influenza a infection ip- is critical for effector t cell trafficking and host survival in toxoplasma gondii infection ifn-gamma-inducible protein (ip- ; cxcl )-deficient mice reveal a role for ip- in effector t cell generation and trafficking identification a novel subset of alveolar type cells expanding following pneumonectomy and enriched in pd-l scoring of pd-l expression intensity on pulmonary adenocarcinomas and the correlations with clinicopathological factors the pd- /pd-l axis and virus infections: a delicate balance tumor-associated b -h promotes t-cell apoptosis: a potential mechanism of immune evasion pd- and its ligands in tolerance and immunity gelatinases a and b are up-regulated in rat lungs by subacute hyperoxia: pathogenetic implications overexpression of alveolar macrophage gelatinase b (mmp- ) in patients with idiopathic pulmonary fibrosis: effects of steroid and immunosuppressive treatment stat inhibition reduced pd-l expression and enhanced antitumor immune responses stat activity promotes programmed-death ligand expression and suppresses immune responses in breast cancer heterozygous stat gain-of-function mutations underlie an unexpectedly broad clinical phenotype gain-of-function stat mutations impair stat activity in patients with chronic mucocutaneous candidiasis (cmc) hyper ige syndrome associated with novel and recurrent stat mutations: two case reports steering of carcinoma progression by the yin/yang interaction of stat / stat an experimental trial of recombinant human interferon alpha nasal drops to prevent coronavirus disease in medical staff in an epidemic area. infectious diseases (except hiv/aids) interferon and its inducers-a never-ending story: 'old' and 'new' data in a new perspective a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo famotidine use is associated with improved clinical outcomes in hospitalized covid- patients: a propensity score matched retrospective cohort study. gastroenterology covid- : famotidine, histamine, mast cells, and mechanisms involvement of histamine h and h receptors in the regulation of stat- phosphorylation: inverse agonism exhibited by the receptor antagonists the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro ivermectin induces cytostatic autophagy by blocking the pak /akt axis in breast cancer the pak -stat signaling pathway activates il- gene transcription and human breast cancer stem cell formation stat as a potential therapeutic target in triple negative breast cancer: a systematic review napabucasin (bbi ), a potent chemoradiosensitizer in rectal cancer stat antisense oligonucleotide azd in a subset of patients with heavily pretreated lymphoma: results of a phase b trial metformin targets stat to inhibit cell growth and induce apoptosis in triple-negative breast cancers metformin uniquely prevents thrombosis by inhibiting platelet activation and mtdna release specific inhibition of stat signal transduction by pias curcumin suppresses constitutive activation of stat- by up-regulating protein inhibitor of activated stat- (pias- ) in ovarian and endometrial cancer cells shp and socs expression patterns in cervical cancers: relevance with activation and resveratrol-caused inactivation of stat signaling growth factor receptor signaling inhibition prevents sars-cov- replication stat serine phosphorylation by erk-dependent and -independent pathways negatively modulates its tyrosine phosphorylation akt-stat pathway as a downstream target of egfr signaling to regulate pd-l expression on nsclc cells mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor- key: cord- - g ukml authors: clementi, nicola; ferrarese, roberto; criscuolo, elena; diotti, roberta antonia; castelli, matteo; scagnolari, carolina; burioni, roberto; antonelli, guido; clementi, massimo; mancini, nicasio title: interferon-β- a inhibition of severe acute respiratory syndrome–coronavirus in vitro when administered after virus infection date: - - journal: j infect dis doi: . /infdis/jiaa sha: doc_id: cord_uid: g ukml the ongoing coronavirus disease pandemic has forced the clinical and scientific community to try drug repurposing of existing antiviral agents as a quick option against severe acute respiratory syndrome–coronavirus (sars-cov- ). under this scenario, interferon (ifn) β- a, whose antiviral potential is already known, and which is a drug currently used in the clinical management of multiple sclerosis, may represent as a potential candidate. in this report, we demonstrate that ifn-β- a was highly effective in inhibiting in vitro sars-cov- replication at clinically achievable concentration when administered after virus infection. the ongoing coronavirus disease pandemic has forced the clinical and scientific community to try drug repurposing of existing antiviral agents as a quick option against severe acute respiratory syndrome-coronavirus (sars-cov- ). under this scenario, interferon (ifn) β- a, whose antiviral potential is already known, and which is a drug currently used in the clinical management of multiple sclerosis, may represent as a potential candidate. in this report, we demonstrate that ifn-β- a was highly effective in inhibiting in vitro sars-cov- replication at clinically achievable concentration when administered after virus infection. keywords. sars-cov- ; ifn-β- a; covid- clinical trial. the current severe acute respiratory syndrome-coronavirus (sars-cov- ) pandemic is severely affecting global health, putting an unprecedented strain on health facilities worldwide. the lack of effective direct-acting antiviral drugs and of immune modulatory therapies, validated through large population studies, worsens the scenario. while waiting for specific antivirals and vaccines to be developed, the biomedical community are also focused on drug repurposing: this is the case with hydroxychloroquine, viral protease inhibitors, and several immunomodulatory drugs already in clinical use for other indications [ ] . in this scenario, interferons (ifns) may also be considered, including ifn-β- a, which has been widely used, and is still applied in some settings, for the management of relapsingremitting multiple sclerosis [ ] . at this time, several articles have already suggested that type i ifns can interfere with coronavirus infections [ , ] . in particular, the activity of ifnβ- a has been described against sars-cov- both in vitro and in vivo, showing a protective effect on acute lung injury in a macaque model of infection [ , ] . in the current study, we assessed its anti-sars-cov- activity in vitro to give a preclinical background to clinical trials evaluating the possible therapeutic role of ifn-β- a in patients with coronavirus disease (covid- ). vero e cells (vero c ; clone e -crl- ; american type culture collection) were cultured in dulbecco's modified eagle medium supplemented with nonessential amino acids, penicillin/streptomycin, hepes buffer, and % (vol/vol) fetal bovine serum (fbs). a clinical isolate of sars-cov- (hcov- / italy/unisr / ; gisaid accession no. epi_isl_ ) was obtained and propagated in vero e cells. virus stocks were titrated using both plaque reduction (plaqueforming units per milliliter) and end-point dilution ( median tissue culture infective dose per milliliter) assays. in plaque reduction assays, confluent monolayers of vero e cells were infected with eight -fold dilutions of virus stock. after hour of adsorption at °c, the cell-free virus was removed. cells were then incubated for hours in dulbecco's modified eagle medium containing % fbs and . % agarose. cells were fixed and stained, and viral plaques were counted. in end-point dilution assays, vero e cells ( × per well) were seeded into -well plates and infected with base dilutions of virus stock. after hour of adsorption at °c, the cell-free virus was removed, and complete medium was added to cells. after hours, cells were observed to evaluate the cytopathic effect (cpe). vero e cells were seeded into -well plates hours before the experiment, and when at % confluency for each well, infected for hour with sars-cov- at a multiplicity of infection (moi) of . [ , ] . cells were washed with phosphate-buffered saline × to remove cell-free virus particles, and μl of fbs-free medium containing different concentrations ( to . iu/ml) of ifn-β- a (avonex; biogen idec) was added to cells. the experiment ended hours after infection. the possible drug toxicity of ifn-β- a at a concentration of iu/ml was also tested on uninfected cells. two experiments were performed in quadruplicate; live images were acquired (with an olympus ckx inverted phase-contrast microscope) at , , and hours after infection, and cell supernatants were collected for real-time quantitative reverse-transcription polymerase chain reaction (qrt-pcr) analysis at and hours after infection. the sars-cov- rna relative amounts detected in each experimental condition as a cycle threshold (ct) value were compared, with a mean ct value determined for the positive infection control. the viral rna was purified from μl of all cell-free culture supernatant, using the qiaamp viral rna mini kit (qiagen) and following the manufacturer's instructions. the purified rna was subsequently used to perform the synthesis of first-strand complementary dna, using the superscript first-strand synthesis system for rt-pcr (thermo fisher scientific), following the manufacturer's instructions. real-time pcr, using the sybr green dye-based pcr amplification and detection method, was performed to detect the complementary dna. we used the sybr green pcr master mix (thermo fisher scientific), with the forward primer n f (tta caa aca ttg gcc gca aa), the reverse primer n r (gcg cga cat tcc gaa gaa), and the following pcr conditions: °c for minutes, cycles of °c for seconds, annealing at °c for seconds and elongation at °c for seconds, followed by a final elongation at °c for minutes. rt-pcr was performed using the abi-prism ht fast real time instrument (applied biosystems) and opticalgrade -well plates. samples were run in duplicate, with a total volume of μl. cpe cells observed were normalized to corresponding virus infection control and used to fit a curve with nonlinear regression for half-maximal effective concentration (ec ) interpolation. the qrt-pcr results were analyzed, calculating the difference in ct (Δct) as the difference between ct values obtained for tested drug concentrations and for the infection control. then -way analysis of variance and dunnett multiple comparisons tests were performed to evaluate differences in Δct means evaluated for each group. vero e cells were treated with concentrations ranging from to . iu/ml of ifn-β- a hour after inoculation with sars-cov- and monitored for cytopathic effect and real-time-pcr quantitative evaluation at , , and hours after infection. inhibition of the sars-cov- by ifn-β- a was dependent on both time and drug concentration. no morphological alterations related to drug toxicity was observed in uninfected cells treated with ifn-β- a at iu/ml. in particular, cpe was assessed at , , and hours after infection ( figure a ). first signs of cpe were already observed at the first time point, when cells were treated with low drug concentrations. marked cpe was evident at hours, showing that iu/ml of the drug gave full protection from virus infection, while it was inhibited only partially with lower concentrations ( to . iu/ml). as expected, -hour images showed that only higher concentrations of ifn-β- a ( to iu/ml) completely protected cells from sars-cov- infection. lower tested concentrations ( . and . iu/ml) had no effect on hindering virus replication. data were used for ec calculations at different time points, resulting in . iu/ml ( % confidence interval, . - . iu/ml) at hours, . iu/ ml (. - . iu/ml) at hours, and . iu/ml ( . - . iu/ml) at hours after infection ( figure b) . cell supernatants collected and hours after infection from different cells treated with all drug concentrations were analyzed using rt-pcr. the results were fully comparable with cpe data ( figure c) for both time points, as ct levels detected were inversely proportional to the amount of target nucleic acid in the sample. the Δct values were reported as the differences between ct values for treated and untreated cells. significant Δct values were observed down to the ifn-β- a concentration of iu/ml, at hours (p < . ) and especially at hours (p < . ). the ct for iu/ml was higher at hours than at hours (both p < . ), and results obtained with both and iu/ml concentrations were significantly different from the infection control at both time points (p < . ). several clinical trials on the administration of ifn to patients with covid- are currently ongoing, even without experimental preclinical evidence of anti-sars-cov- potential [ ] (https://www.hra.nhs.uk/covid- -research/approved-covid- -research/ /). among the ifns currently available for clinical use, ifn-β- a represents an interesting option, because its pharmacological features are well known. a very recent article, just released as a preprint during the submission of the current manuscript, describes the effect of ifn-β- a when used before infection of cells with sars-cov- [ ] . our in vitro observations shed light for the first time on that antiviral activity of ifn-β- a against sars-cov- when administered after the infection of cells, highlighting its possible efficacy in an early therapeutic setting. to this point, we detected that ifn-β- a effectively inhibits both infectious virus particles and viral rna on treated cells, when compared to viruspositive infection control without toxicity at its highest tested concentration. moreover, the drug ec evaluated at , , and hours after infection can be easily accessed in the clinical setting and could therefore help in addressing drug administration regimens in vivo [ ] . from this perspective, it is important to note that, in our experimental setting, ifn-β- a activity is retained up to hours after its use on the infected cells. we are aware of the limitations of this preliminary study, such as the lack of a parallel evaluation of the activity ifnβ- a on other viruses, such as vesicular stomatitis virus, whose clinical sensitivity to the drug is well known, to establish the level of susceptibility of sars-cov- to type i ifn. moreover, owing to the lack of standardized phenotypic tests for this novel coronavirus, we have preferred to set the virus amount used for all assays on the cpe observed at the time points ( , , and hours after infection), rather than using a predetermined moi. hence, the antiviral activity of ifn-β- a against sars-cov- was evaluated only at a single low moi in a multiple-cycle replication condition, as previously reported for sars-cov- [ , ] . it would also be interesting to test the activity of ifn-β- a on other sars-cov- isolates featuring different phenotypic behaviors and possibly on animal models of covid- to further assess, and dissect, the clinical potential of this therapeutic approach [ , ] . this would have certainly have allowed a more complete evaluation of the clinical potential of ifn-β- a activity in the clinical setting of covid- . moreover, further in vitro testing on other cells of different ifns, such as ifn-λ, may complement our preliminary results, it being of extreme importance to continue supporting ifn-based clinical trials [ ] . finally, we are fully aware that the preclinical evaluation of the antiviral activity of a drug, such as ifn-β- a, is only a partial assessment of its possible clinical role in a disease such as covid- , in which the beneficial or detrimental effect of type i ifn is still to be established and in which immune-mediated damage is probably extremely important in determining the development of the worst outcomes of the infection [ ] . nonetheless, while we are surprised by the current lack of data on ifn-β- a against sars-cov- in the literature, it is both urgent and clinically important to deliver data indicating whether type i may display direct antiviral activity against this virus. obviously, its antiviral potential deserves further investigation in such an atypical setting. targeting sars-cov- : a systematic drug repurposing approach to identify promising inhibitors against c-like proteinase and '-o-ribose methyltransferase the interferon beta therapies for treatment of relapsing-remitting multiple sclerosis: are they equally efficacious? a comparative review of open-label studies evaluating the efficacy, safety, or dosing of different interferon beta formulations alone or in combination treatment of sars with human interferons type interferons as a potential treatment against covid- interferon-β a and sars coronavirus replication exacerbated innate host response to sars-cov in aged non-human primates severe acute respiratory syndrome coronavirus replication is severely impaired by mg due to proteasome-independent inhibition of m-calpain severe acute respiratory syndrome-related coronavirus is inhibited by interferon-α interferon-a b treatment for antiviral activities of type i interferons to sars-cov- infection recombinant leukocyte a interferon: pharmacokinetics, single-dose tolerance, and biologic effects in cancer patients sars-coronavirus- replication in vero e cells: replication kinetics, rapid adaptation and cytopathology animal models for emerging coronavirus: progress and new insights the effectiveness of antiviral agents with broad-spectrum activity against chikungunya virus varies between host cell lines type i interferon and hiv: subtle balance between antiviral activity, immunopathogenesis and the microbiome key: cord- -asbt mcj authors: schulz, katharina s.; mossman, karen l. title: viral evasion strategies in type i ifn signaling – a summary of recent developments date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: asbt mcj the immune system protects the organism against infections and the damage associated with them. the first line of defense against pathogens is the innate immune response. in the case of a viral infection, it induces the interferon (ifn) signaling cascade and eventually the expression of type i ifn, which then causes an antiviral state in the cells. however, many viruses have developed strategies to counteract this mechanism and prevent the production of ifn. in order to modulate or inhibit the ifn signaling cascade in their favor, viruses have found ways to interfere at every single step of the cascade, for example, by inducing protein degradation or cleavage, or by mediate protein polyubiquitination. in this article, we will review examples of viruses that modulate the ifn response and describe the mechanisms they use. the mammalian immune system evolved to detect and fight viral infections effectively. the induction of type i interferon (ifn), predominantly ifn-α and ifn-β, forms the first line of defense. the type i ifn response consists of two parts. first, the cell produces type i ifn, when triggered by a viral stimulus. the ifn is then secreted and, in the second part of the response, it is sensed by the producing, as well as neighboring cells, resulting in the production of ifn-stimulated genes (isgs) [reviewed in ref. ( ) ]. viruses, which have coevolved with their host, develop strategies to counteract the signaling cascades of the innate immune system and ensure their replication. recently, several reviews were published, describing the innate immune evasion strategies of individual viruses or virus families, such as influenza virus ( , ) , phleboviruses ( ), herpes viruses ( - ), coronaviruses severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) ( ) , human immunodeficiency virus (hiv) ( , ) , as well as multiple rna viruses ( , ) . moreover, there are recent articles that review how viruses prevent detection by pathogen recognition receptors (prrs) ( , ) and how viruses modulate innate immune signaling by use of viral deubiquitinases ( ) . in this review, we will compare the different strategies viruses have developed to suppress innate immune signaling of individual components of the innate immune signaling cascade. due to the tremendous amount of data in this field, we will focus on recent discoveries. older studies were summarized in ref. ( , ) . ( ) ]. the most important viral markers for the innate immune system are viral nucleic acids. the detection of viral dna through the cgas-sting pathway and the counter measurements taken by viruses have been reviewed recently ( ) and are not part of this review. viral rnas, which are mostly double-stranded (ds-)rna, are recognized by three prrs: the endosomal toll-like receptor (tlr ), the cytoplasmic retinoic acid-inducible gene i (rig-i)like receptors (rlrs), and the nucleotide-oligomerization domain (nod)-like receptors (nlrs) ( ) . tlr and the rlrs are important for inducing the type i ifn response, whereas nlrs have been shown to regulate interleukin- β (il- β) maturation through activation of caspase- ( ) . the group of rlrs consists of rig-i, melanoma differentiation-associated gene (mda ), and laboratory of genetics and physiology (lgp ). the three receptors have a similar structure, all containing a caboxyterminal domain, which functions as a repressor domain (rd) in rig-i and lgp ( ) and a central helicase domain, but lgp lacks the caspase activation and recruitment domains (cards) that function in signaling [reviewed in ref. ( , ) ]. both the helicase and the carboxy-terminal domain are required for rna binding. rig- and mda- detect specific viral rna pamps, while lgp negatively regulates rig-i signaling and promotes rna binding to mda [reviewed in detail in ref. ( ) ]. in unstimulated cells, rig-i and mda- are kept in a repressed state due to phosphorylations on serine and threonine residues in the cards and carboxy-terminal domains ( , ) . upon binding of rna, both rig-i and mda- undergo conformational changes, resulting in release of their cards ( , ) . recruited phosphatases remove the phosphate residues, and e ubiquitin ligases attach lys -linked ubiquitin polymers onto the cards and c-terminal domain of rig-i, which are important for rig-i tetramerization ( ) ( ) ( ) ( ) ( ) . rna-bound rig- then interacts with - - ε, a mitochondrial trafficking protein, and the trim ubiquitin ligase, which together transport rig-i to the mitochondria ( ) . there the cards of rig-i or mda- interact with the card of the mitochondrial activator of virus signaling (mavs, also known as ips- , visa, and cardif), which is an essential signaling adaptor protein. the activation of mavs has recently been reviewed in detail in ref. ( ) . tlr interacts with trif, which serves as a molecular platform and forms physical interactions with several adaptor molecules ( ) . by interacting with upstream adaptors, trif undergoes conformational changes and recruits the downstream tnf receptor-associated factor (traf) and traf [reviewed in ref. ( ) ]. the kinase receptor-interacting protein- (rip- ) is part of both the signaling pathways downstream of tlr and rig-i. it can interact with trif to induce nfκb activation ( ) . moreover, the dsrna-activated tlr can recruit trif, rip- , and caspase- and induce apoptosis ( ) . also, rip- and its adaptor protein fas-associated protein with death domain (fadd) are part of the signaling cascade downstream of rig-i and mda- and involved in the activation of the transcription factors interferon regulatory factor (irf) and irf ( ) . traf serves as a linker between the upstream adaptor proteins (trif or myd for tlrs and mavs for rlrs) and the downstream signaling kinases tbk /ikkε or irak /ikkα. the recruitment of traf to the tlr or rlr signaling complexes activates the e ligase activity of traf , which then catalyzes its own k -linked ubiquitinylation. subsequent traf activates tbk /ikkε or irak/ikkα [reviewed in ref. ( ) ] (figure ) . viruses target rig-i directly or indirectly to block the type i ifn response. the phlebovirus toscana virus expresses a non-structural protein, which directly interacts with rig-i and induces its proteasomal degradation ( , ) . foot-and-mouth disease virus (fmdv) proteins l pro , c pro , and b increase the rig-i mrna expression but decrease the protein expression of rig-i. l pro and c pro both induce rig-i degradation, whereas the mechanism of how b reduces rig-i protein levels has not been solved yet ( ) . other viruses target rig-i indirectly. hepatitis b virus (hbv) induces mir a, which then posttranscriptionally inhibits the expression of rig-i and suppresses the production of type i ifn ( ) . the dengue virus ns protein binds to - - ε and prevents the translocation of rig-i to mavs. the binding site on ns is a highly conserved phosphomimetic motif, which was verified by generation of a virus containing a mutation in this motif ( ) . it has been proposed that in certain cell types rig-i requires sentinels, such as the protein ddx , which associates with rig-i and promotes the rig-i rna-binding activity ( , ) . other studies question ddx acting as a broadly active enhancer of antiviral responses ( , ) and instead suggest that ddx only functions in the antiviral response to specific viruses, such as hepatitis c virus ( ) . however, there are data indicating that influenza a virus and hepatitis c virus attenuate ifnβ-promoter activation by targeting the sentinel ddx . both viruses activate the epidermal growth factor (egf) receptor, which in turn phosphorylates ddx on tyr- and tyr- . this results in the attenuation of ddx -dependent rig-i activation. in addition, independent of its role as sentinel for rig-i viral rna recognition, ddx plays a role in viral rna degradation ( ) (figure ). mitochondrial activator of virus signaling is blocked by different viruses in various ways. the dengue virus protein ns a targets mavs, and the interaction prevents the binding of mavs to rig-i ( ) . the porcine reproductive and respiratory syndrome virus (prrsv) c-like protease ( clsp), by contrast, cleaves mavs in a proteasome-and caspase-independent manner at glu (e /g ). both cleavage products fail to activate the type i ifn response ( ) . likewise, the hepatitis c virus protein ns - a ( , ) , as well as the highly pathogenic porcine reproductive and respiratory syndrome virus (hp-prrsv) protein nsp ( ) have been shown to cleave mavs and block rlr signaling. the porcine epidemic diarrhea virus (pedv) also targets mavs in small intestinal epithelial cells (iecs). however, the exact mechanism has not been solved yet ( ) (figure ) . the sars coronavirus protein orf b not only influences antiviral signaling but also alters host cell mitochondria morphology by inducing degradation of the dynamin-like protein (drp ). mavs becomes concentrated into small puncta in the presence ( ) . in addition to mavs, also the levels of traf and traf are reduced by orf b. however, it is unlikely that traf and traf are targeted directly. more likely, they are degraded due to their interaction with mavs ( ) (figure ) . human t-cell lymphotropic virus type i (htlv- ) protein tax disrupts innate immune signaling in multiple ways: it binds to the rip homotypic interaction motif (rhim) domains of rip- and disrupts the interaction between rip- and rig-i or mda- and the activation of the type i ifn promoter. tax also binds to trif and thereby interrupts the tlr signaling cascade. finally, tax blocks the association between rip- and irf , which resulted in repression of the irf activity ( ) (figure ) . middle east respiratory syndrome coronavirus m protein interacts with traf and disrupts the interaction between traf and tbk , which ultimately leads to a reduced irf activation. for the interaction with traf , the n-terminal transmembrane domain of the mers-cov m protein is sufficient ( ) , similar to what has been shown for sars-cov before ( ) (figure ). triggering of the tlr -and rlr-signaling cascade results in the activation of the transcription factors nfκb and irf /irf . in its inactive state, the transcription factor nfκb is complexed with its inhibitor iκb ( ) . upon stimulation, iκb is phosphorylated by the iκb kinase (ikk) complex, which is composed of two catalytic subunits, such as ikkα and ikkβ, and a regulatory subunit, such as nfκb essential modulator (nemo) ( ) . the phosphorylation of iκbα induces its polyubiquitination through the e ubiquitin ligase β-transducin repeat-containing protein (β-trcp) and subsequent proteasomal degradation ( ) , allowing nfκb to translocate into the nucleus and induce the expression of target genes ( ) (figure ) . encephalomyocarditis virus (emcv) protein c cleaves traf family member-associated nfκb activator (tank), which inhibits traf -mediated nfκb activation, on gln . as a result, nfκb is activated and the unstable c-terminal fragment of tank is subjected to proteasomal degradation ( ) . also, other viruses express proteases that cleave tank, although on other residues, such as porcine reproductive and respiratory syndrome virus (prrsv) (tank is cleaved by nsp ), fmdv (protease c cleaves tank), and equine arteritis virus (eav) (tank is cleaved by nsp ). thus, tank seems to be a common target of several positive rna viral proteases ( ) (figure ) . several viruses have been shown to disrupt ifn signaling by cleaving nemo. pedv c-like protease, nsp , cleaves nemo at gln ( ), whereas the hepatitis a virus c protease ( c pro ) cleaves nemo at gln ( ) and the picornavirus fmdv protease c pro at gln , removing the c-terminal zinc finger domain from the protein ( ) . the human rotavirus has developed another way. its non-structural protein (nsp ) has been shown to inhibit the nfκb pathway by degrading β-trcp and consequently stabilizing iκb ( ) (figure ) . tank-binding kinase (tbk ) and inhibitor of κb kinase ε (ikkε) are classified as non-canonical serine/threonine kinases and are both able to induce irf and irf phosphorylation and subsequent dimerization ( ) ( ) ( ) ( ) . however, while tbk is constitutively expressed in most cell types, the expression of ikkε is more restricted ( ) . upon stimulation, tbk and ikkε are recruited by adaptor proteins to signaling complexes to be activated by phosphorylation on ser and both have been shown to be subjected to k -linked polyubiquitination [reviewed in ref. ( , ) ]. for tbk , k -linked polyubiquitination seems to be important for tlr-and rlr-induced ifn production, as ubiquitin chains might serve as a platform for the assembly of tbk signaling complexes. moreover, deubiquitinases are able to terminate the tbk -mediated pathway by cleaving the k linked ubiquitin chains [reviewed in ref. ( , ) ]. activated tbk /ikkε phosphorylates irf and/or irf in the cytosol at specific serine residues. this phosphorylation results in homo-or heterodimerization of irf and irf and nuclear translocation ( , ) . interestingly, while irf is constitutively expressed, irf is expressed at low levels in most cell types and expression is induced upon ifn signaling. therefore, in most cells, irf strongly enhances the production of ifn [reviewed in ref. ( ) ]. once phosphorylated irf and/or irf dimers have translocated into the nucleus, they bind to the transcription coactivator creb-binding protein (cpb)/p ( , ) . together with other factors, such as nfκb, they form the enhanceosome on the ifnβ promoter and induce the expression of type i ifn [reviewed in ref. ( ) ]. the viral proteins that target tbk act by either blocking activation of tbk by mavs or by inhibiting activation of irf by tbk . the mers-cov protein orf b blocks ifnβ production by binding to tbk and ikkε and suppressing the formation of a mavs/ikkε complex ( ) . in addition to inhibiting tbk /ikkε activation, orf b can also inhibit the production of ifnβ in the nucleus; however, the mechanism has not been solved yet ( ) . recently, two herpes simplex virus proteins have been shown to target tbk /ikkε and inhibit the phosphorylation of irf : icp ( ) and vp ( ) . also, dengue virus serotype non-structural proteins ns a and ns b, as well as the ns a and ns b proteins of other dengue viruses, inhibit the phosphorylation of tbk ( ) and pedv n protein has been shown to interact with tbk , hampering the association of tbk with irf and preventing the activation of irf activation ( ) . the human t-cell leukemia virus type oncoprotein tax has been shown to also interact with tbk . however, studies came to contradicting results on how that influences the production of ifnβ. while one group showed that tax activates tbk and the production of ifnβ ( ), another group showed that tax suppresses the ifn production by interaction with tbk ( ) . interestingly, when a recent study tested how the rabies virus p protein of street strains behaves compared to laboratory-adapted strains with regard to the induction of type i ifn, they found that both street strains and laboratory strains inhibit tbk -mediated signaling, but only the p protein of street strains also interacts with and inhibits ikkε-inducible irf dependent ifnβ expression ( ) (figure ) . interferon regulatory factor is targeted by many viruses to impair innate immune signaling. most viruses inhibit the phosphorylation and thereby also the dimerization and translocation of irf , such as the porcine deltacoronavirus ( ) or poliovirus ( ) . hepatitis e virus protein orf also suppresses irf phosphorylation, but in an indirect way. it activates the signal regulator protein α (sirp-α), which negatively regulates type i ifn induction ( ) . in contrast, porcine bocavirus (pbov) np protein does not affect irf expression, phosphorylation, or nuclear translocation. instead, it interacts with the dna-binding domain of irf and inhibits the dna-binding activity ( ) . a very interesting way of how to circumvent the host innate immune response was found when studying gammaherpesviruses kaposi's sarcoma-associated herpesvirus (kshv) and rhesus macaque rhadinovirus (rrv). they express several viral homologs to the irfs, called viral irfs (virfs). these virfs have found multiple ways to suppress type i ifn production. for kshv, different strategies have been reviewed in ref. ( ) . recently, the rrv virf r has been shown to interact with the transcriptional coactivator cbp in the nucleus, similar to the kshv virf . as a result, cbp cannot form a complex with the phosphorylated irf , and the ifn expression is not induced ( ) ( ) ( ) . interestingly, rrv r is the first virf for which an association with the viron could be shown. therefore, virf v can shut down the type i ifn response shortly after the cell was infected, rendering the cell more susceptible to infection ( ) . the pedv protein nsp also targets cbp. nsp induces cbp degradation in a proteasome-dependent manner and thereby interrupts enhanceosome assembly and the production of type i ifn ( ) (figure ) . for most of these interactions, the molecular mechanisms have not been unraveled yet. a protein that has been shown to interact with and induce proteasomal degradation of irf some time ago is classical swine fever virus (csfv) npro ( , ) . recently, the molecular mechanism has been published. irf and npro interact direct and form a soluble : complex. moreover, it was shown that npro interacts with the full-length irf , not with individual domains, and that npro binds the constitutively active form of irf in the presence of cpb. thus, npro interacts with both the monomer and the active irf dimer and likely targets both species for ubiquitinylation and proteasomal degradation ( ) . interferon regulatory factor is targeted by two human enteroviruses, such as enterovirus and enterovirus . they downregulate irf by cleaving it with their protease c, leaving the cleavage products unable to induce ifn expression. while enterovirus cleaves irf once at gln -ser ( ), enterovirus cleaves it twice, the cleavage sites being gln and gln ( ) . moreover, megalocytivirus, a dna virus that infects marine and freshwater fish, induces the expression of the host microrna pol-mir- , which then specifically suppresses the expression of irf ( ) (figure ) . the type i ifns act in an autocrine, paracrine, or systemic manner to stimulate antiviral responses. they are recognized by the ifnα/β receptor (ifnar), which consists of the subunits ifnar and ifnar expressed on virtually all cell types ( ) . the interaction of type i ifn with the receptor results in the phosphorylation and activation of the ifnar -and ifnar -associated tyrosine kinases tyrosine kinase (tyk ) and janus kinase (jak ), which then phosphorylate ifnar tyrosine residues, resulting in the recruitment and activation of signaling molecules, such as the signal transducer and activator of transcription (stat) family of transcription factors ( , ) . upon activation, stat and stat , together with irf , form the ifn-stimulated gene factor (isgf ), which then translocates into the nucleus to induce transcription of isgs [reviewed in detail in ref. ( ) ( ) ( ) ]. several viruses target ifnar to prohibit ifn binding and signaling. influenza virus induces the degradation of ifnar . hemagglutinin (ha) triggers the phosphorylation and ubiquitinylation of ifnar , thus promoting protein degradation ( ) . encephalitic flaviviruses, such as tick-borne encephalitis virus or west nile virus, inhibit ifnar surface expression. their protein ns binds the cellular dipeptidase prolidase (pepd), which is involved in ifnar maturation and accumulation, activation of ifnβ-stimulated gene induction, and ifn-dependent viral control. this interaction inhibits ifnar intracellular trafficking and glycosylation but does not promote ifnar degradation ( ) (figure ) . both stat and stat are targeted by many viruses to suppress isg induction. pedv induces stat ubiquitinylation and targets it for degradation in the proteasomes ( ) . ( ) . similarly, human metapneumovirus (hmpv) protein sh impairs stat expression, phosphorylation, and activation ( ) . simian varicella virus not only inhibits stat phosphorylation but also promotes degradation of irf in a proteasome-dependent manner through its protein orf ( ) . also, infectious bronchitis virus (ibv) inhibits phosphorylation and nuclear translocation of stat . however, despite detailed analyses, it is unclear which viral protein is responsible. it was, however, shown that the accessory protein a contributes to ibv resistance to type i ifn, although the target is unknown as well ( ) . in case of the human parvovirus b , it becomes evidently clear that both the virus and the immune system constantly evolve to prevail. while its protein ns suppresses stat phosphorylation, the immune system senses the protein and triggers the production of type i ifn ( ) . sftsv, an emerging tick-borne pathogen, developed multiple ways to prevent isg induction. the viral non-structural protein ns impairs stat expression, phosphorylation, and activation ( ) and interacts with stat and sequesters stat and stat into viral inclusion bodies, where they are trapped ( ) (figure ) . the jak-stat signal transduction pathway is negatively regulated by the suppressor of cytokine signaling (socs) family of proteins in form of a classical feedback loop ( , ) . some viruses induce the expression of socs to take advantage of this mechanism to minimize the induction of isgs. japanese encephalitic virus (jev) downregulates the expression of micro-rna mir- , which then results in upregulated socs levels ( ) . varicella-zoster virus (vzv) infection induces the expression of socs ( ) and respiratory syncytial virus (rsv) nonstructural proteins ns and ns induce upregulation of socs and socs , which also inhibited the induction of chemokines ( ) (figure ). viruses fully depend on the translation machinery of the host cell for replication. accordingly, they have evolved multiple ways to hamper host protein synthesis [reviewed in ref. ( ) ]. one way is to shut off host protein synthesis. for some time, it was thought that gamma-and deltacoronaviruses do not induce host shutoff, such as alpha-and betacoronaviruses do. however, a recent study showed that the infectious bronchitis gammacoronavirus induces host shutoff using its protein b. it seems like b is a functional equivalent of nsp , the host shutoff protein of alpha-and betacoronaviruses ( ) . viruses evolved to have various strategies to circumvent the innate immune response by blocking the production of type i ifn or the expression of isgs. while these diverse strategies may appear contradictory between viruses, several factors require consideration. for example, the use of clinical isolates versus viral evasion of interferon pathways frontiers in immunology | www.frontiersin.org november | volume | article laboratory-passaged strains could yield different results, particularly with rna viruses that rapidly accumulate mutations due to error-prone rna-dependent rna polymerases. moreover, the choice of cell line can greatly influence experimental outcomes, as many immortalized or transformed continual cell lines harbor mutations in critical innate immune signaling ( ) . likewise, the use of genetic knockout versus knockdown cell lines or organisms can influence experimental outcomes, as can the experimental procedures themselves, particularly when endogenous interactions are disrupted with the use of overexpression approaches. studying the mechanisms used by viruses to prevent an immune response is of great importance for the development of new strategies to limit the sequelae of viral infections. identification of key immune evasion proteins allows development of antivirals to target these proteins. alternatively, identification of key cellular antiviral pathways allows development of strategies to enhance these pathways to overwhelm incoming viruses. information on key immune evasion factors further facilitates the engineering of safe and effective vaccine strains and designing strategies to target new emerging viruses from the same or closely related family. ks and km conceptualized the scope of the review article. ks wrote the review with input from km. this work was supported by a postdoctoral fellowship from the deutsche forschungsgemeinschaft (schu / - ). work in the mossman laboratory on innate antiviral signaling is supported by the canadian institutes for health research. induction and function of ifnbeta during viral and bacterial infection functions of the influenza a virus ns protein in antiviral defense to conquer the host, influenza virus is packing it in: interferon-antagonistic strategies beyond ns phleboviruses and the type i interferon response the tiers and dimensions of evasion of the type i interferon response by human cytomegalovirus herpesviruses: interfering innate immunity by targeting viral sensing and interferon pathways evasion of host antiviral innate immunity by hsv- , an update middle east 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phosphorylationmediated negative regulation of rig-i antiviral activity phosphorylation of rig-i by casein kinase ii inhibits its antiviral response structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna a structure-based model of rig-i activation trim ringfinger e ubiquitin ligase is essential for rig-i-mediated antiviral activity reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity conventional protein kinase c-alpha (pkc-alpha) and pkc-beta negatively regulate rig-i antiviral signal transduction a distinct role of riplet-mediated k -linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses dephosphorylation of the rna sensors rig-i and mda by the phosphatase pp is essential for innate immune signaling the mitochondrial targeting chaperone - - epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity how rig-i like receptors activate mavs antiviral responses induced by the tlr pathway a unique host defense pathway: trif mediates both antiviral and antibacterial immune responses rip is an essential mediator of toll-like receptor -induced nf-kappa b activation dsrna induces apoptosis through an atypical death complex associating tlr to caspase- a fadd-dependent innate immune mechanism in mammalian cells traf : a novel tumor suppressor gene in macrophages. macrophage (houst) ( ) :e toscana virus nss protein inhibits the induction of type i interferon by interacting with rig-i truncation of the c-terminal region of toscana virus nss protein is critical for interferon-beta antagonism and protein stability foot-and-mouth disease virus viroporin b antagonizes rig-i mediated antiviral effects by inhibition of its protein expression hepatitis b virus inhibits intrinsic rig-i and rig-g immune signaling via inducing mir a a phosphomimetic-based mechanism of dengue virus to antagonize innate immunity ddx , a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling ddx is involved in rig-i-dependent and independent antiviral responses, and its function is attenuated by virus-induced egfr activation a diverse range of gene products are effectors of the type i interferon antiviral response mouse superkiller- -like helicase ddx is dispensable for type i ifn induction and immunity to multiple viruses dengue virus subverts host innate immunity by targeting adaptor protein mavs porcine reproductive and respiratory syndrome virus c protease cleaves the mitochondrial antiviral signalling complex to antagonize ifn-beta expression hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity hepatitis c virus ns - a inhibits the peroxisomal mavs-dependent antiviral signalling response highly pathogenic porcine reproductive and respiratory syndrome virus nsp cleaves visa to impair antiviral responses mediated by rig-i-like receptors porcine epidemic diarrhea virus inhibits dsrna-induced interferon-beta production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome pcbp mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip oncogenic human t-cell lymphotropic virus type tax suppression of primary innate immune signaling pathways middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk -dependent phosphorylation of irf suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain a firm hand on nfkappab: structures of the ikappabalpha-nfkappab complex ikappab kinases: key regulators of the nf-kappab pathway inducible degradation of ikappabalpha by the proteasome requires interaction with the f-box protein h-betatrcp shaping the nuclear action of nf-kappab encephalomyocarditis virus c protease relieves traf family member-associated nf-kappab activator (tank) inhibitory effect on traf -mediated nf-kappab signaling through cleavage of tank porcine epidemic diarrhea virus c-like protease regulates its interferon antagonism by cleaving nemo hepatitis a virus c protease cleaves nemo to impair induction of beta interferon foot-and-mouth disease virus c protease cleaves nemo to impair innate immune signaling nsp of human rotaviruses commonly inhibits nf-kappab signalling by inducing beta-trcp degradation ikkepsilon and tbk are essential components of the irf signaling pathway triggering the interferon antiviral response through an ikk-related pathway regulation and function of ikk and ikk-related kinases involvement of the ubiquitin-like domain of tbk /ikk-i kinases in regulation of ifn-inducible genes ikappab kinase epsilon (ikkepsilon): a therapeutic target in inflammation and cancer regulation of tbk activity by optineurin contributes to cell cycle-dependent expression of the interferon pathway negative regulation of tbk -mediated antiviral immunity irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors the irf family transcription factors in immunity and oncogenesis the irf family transcription factors at the interface of innate and adaptive immune responses direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf- and cbp/p mechanism for transcriptional synergy between interferon regulatory factor (irf)- and irf- in activation of the interferon-β gene promoter middle east respiratory syndrome coronavirus orf b protein inhibits type i interferon production through both cytoplasmic and nuclear targets hsv- icp targets the tbk -activated sting signalsome to inhibit virus-induced type i ifn expression herpes simplex virus serine protease vp blocks the dna-sensing signal pathway by abrogating activation of interferon regulatory factor dengue virus ns proteins inhibit rig-i/mavs signaling by blocking tbk /irf phosphorylation: dengue virus serotype ns a is a unique interferon-regulating virulence determinant porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf and tbk htlv- tax protein recruitment into ikkepsilon and tbk kinase complexes enhances ifn-i expression suppression of type i interferon production by human t-cell leukemia virus type oncoprotein tax through inhibition of irf phosphorylation contribution of the interaction between the rabies virus p protein and i-kappa b kinase to the inhibition of type i ifn induction signalling porcine deltacoronavirus (pdcov) infection suppresses rig-i-mediated interferon-beta production proteolysis of mda and ips- is not required for inhibition of the type i ifn response by poliovirus hepatitis e virus infection activates signal regulator protein alpha to down-regulate type i interferon porcine bocavirus np protein suppresses type i ifn production by interfering with irf dnabinding activity functional analysis of human herpesvirus -encoded viral interferon regulatory factor and its association with cellular interferon regulatory factors and p viral interferon regulatory factor of kaposi's sarcoma-associated herpesvirus (human herpesvirus ) binds to, and inhibits transactivation of, creb-binding protein a rhesus rhadinovirus viral interferon (ifn) regulatory factor is virion associated and inhibits the early ifn antiviral response suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp classical swine fever virus npro interacts with interferon regulatory factor and induces its proteasomal degradation the npro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target interferon regulatory factor- pestivirus npro directly interacts with interferon regulatory factor (irf ) monomer and dimer cleavage of interferon regulatory factor by enterovirus c suppresses cellular responses c protease of enterovirus d inhibits cellular defense mediated by interferon regulatory factor pol-mir- , a teleost mirna upregulated by megalocytivirus, negatively regulates virus-induced type i interferon response, apoptosis, and cell cycle arrest the interferons and their receptors -distribution and regulation jak-stat signaling: from interferons to cytokines stats get their move on transcriptional regulation by stat and stat in the interferon jak-stat pathway stat and irf : beyond isgf . jakstat ( ) dynamic control of type i ifn signalling by an integrated network of negative regulators hemagglutinin of influenza a virus antagonizes type i interferon (ifn) responses by inducing degradation of type i ifn receptor flavivirus antagonism of type i interferon signaling reveals prolidase as a regulator of ifnar surface expression porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat la piedad michoacan mexico virus v protein antagonizes type i interferon response by binding stat protein and preventing stats nuclear translocation human metapneumovirus small hydrophobic (sh) protein downregulates type i ifn pathway signaling by affecting stat expression and phosphorylation varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms infectious bronchitis coronavirus inhibits stat signaling and requires accessory proteins for resistance to type i interferon activity nonstructural protein (ns ) of human parvovirus b stimulates host innate immunity and blunts the exogenous type i interferon signaling in vitro suppression of type i and type iii ifn signalling by nss protein of severe fever with thrombocytopenia syndrome virus through inhibition of stat phosphorylation and activation disruption of type i interferon signaling by the nonstructural protein of severe fever with thrombocytopenia syndrome virus via the hijacking of stat and stat into inclusion bodies the role of suppressors of cytokine signaling (socs) proteins in regulation of the immune response regulation of the immune system by socs family adaptor proteins japanese encephalitis virus exploits the microrna- to regulate the expression of suppressor of cytokine signaling (socs) suppressor of cytokine signaling expression induced by varicella-zoster virus infection results in the modulation of virus replication respiratory syncytial virus nonstructural proteins upregulate socs and socs in the different manner from endogenous ifn signaling viral subversion of the host protein synthesis machinery infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein b deregulation of interferon signaling in malignant cells the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -hviv zar authors: masucci, maria grazia title: viral ubiquitin and ubiquitin-like deconjugases—swiss army knives for infection date: - - journal: biomolecules doi: . /biom sha: doc_id: cord_uid: hviv zar posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubiquitin-like polypeptides regulate numerous cellular processes that are captured by viruses to promote infection, replication, and spreading. the importance of these protein modifications for the viral life cycle is underscored by the discovery that many viruses encode deconjugases that reverse their functions. the structural and functional characterization of these viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infections and the host’s antiviral defense. given the growing body of evidence demonstrating their key contribution to pathogenesis, the viral deconjugases are now recognized as attractive targets for the design of novel antiviral therapeutics. viruses have shaped the fate of human societies throughout history. understanding how these potentially life-threatening pathogens establish infection and how they interact with their hosts is our best strategy for acquiring the means to control the diseases they cause. being obligatory intracellular parasites, viruses face a double challenge. on one side, they need to commandeer the molecular machinery of the host cell to support the production of new virus particles, while on the other side, they must hold back the multifaceted cellular and organismal defenses that are triggered by infection. these challenges are met by the expression of specialized viral products that hijack or manipulate critical cellular functions. many of these viral pathogenicity factors are multifunctional proteins that mimic the activity of cellular counterparts whose activity controls key aspects of normal cell physiology. virtually all cellular processes are regulated by posttranslational modifications that dictate the function, subcellular localization, and interactions of effector proteins. among those, the covalent attachment of small polypeptides of the ubiquitin family (henceforth collectively referred to as ubiquitin-like polypeptides, ubls) provides a flexible means to control the activity and fate of the modified substrate. cell functions that orchestrate the outcome of infection such as the cell cycle, cell survival and programmed cell death, gene expression, protein trafficking and degradation, autophagy, and the immune response are all dependent on ubl modifications [ ] . it is therefore not surprising that viruses have evolved means to interfere with the ubl signaling networks in order to secure a cellular environment conducive to their own replication and spread. the ubls are a family of structurally related small polypeptides that share a β-grasp fold organization consisting of a mixed β-sheet structure with a central α-helix [ ] . to date, seventeen human ubls have been reported to be conjugated to other molecules. in addition to ubiquitin (ub), the ubls include the small ubiquitin-related modifier (sumo)- , - , and - ; nedd (neural precursor cell expressed, developmentally downreagulated- ); isg (interferon stimulated gene- ); ufm (ubiquitin-fold modifier- ); urm (ubiquitin-related modifier- ); fat (hla-f adjacent transcript cell expressed, developmentally downreagulated- ); isg (interferon stimulated gene- ); ufm (ubiquitin-fold modifier- ); urm (ubiquitin-related modifier- ); fat (hla-f adjacent transcript ); mnsfβ (monoclonal nonspecific suppressor factor β); and the lc (microtubule-associated light chain- ) and gabarap (γ-aminobutyric acid receptor associated protein) family of modifiers [ ] . the attachment of ubls to their protein or, in the case of the lc /gabarap family, lipid substrates is mediated by an enzymatic cascade that starts with processing of the ubl precursor by a specific protease, which generates a c-terminal gly residue required for conjugation. the mature ubl becomes the substrate of an activating enzyme (e ) that forms a high-energy thiolester bond with the c-terminal gly and then loads the activated ubl on the catalytic cys residue of a conjugating enzyme (e ). the e transfers the ubl to the substrate with the help of a ligase (e ) that promotes the transfer [ , ] (figure ). as a rule, each ubl conjugation system involves distinct sets of dedicated e , e , and e , enzymes but enzyme sharing is not uncommon and e ligases with mixed specificity for ub and isg , e.g., trim (tripartite motif- ) [ ] ; ubiquitin and nedd , e.g., mdm (mouse double minie- ) [ ] ; or ub and sumo, e.g., topors (top binding arginine/serine rich protein), traf (tnf receptor associated factor- ), uhrf (ubiquitin like with phd and ring finger domain- ), and trim [ ] [ ] [ ] [ ] have been described. figure . schematic illustration of the ubl activation, conjugation and deconjugation cycle. the covalent attachment of ubls to their substrates involves sequential catalytic reactions that initiate with processing of the ubl precursor by a specific ubl protease. the mature ubl is activated by an activating enzyme (e ) and then transferred to a conjugating enzyme (e ) that, with the help of a substrate-specific ligase (e ), transfer the activated ubl to the ε-amino residue of a lys on the target protein via a covalent isopeptide bond. additional ubls can be linked to the previous one to form chains. ubl-specific proteases can reverse the modification, supplementing the cellular pools of free ubls. the attachment of a ub moiety to the n-terminal met or to an internal lys residue of the previous ub (k , k , k ,k ,k ,k or k ) results in the formation of topologically different poly-ub chains that, upon recognition by signal transducers contain dedicated binding domains, target the substrates various fates and cellular functions ubiquitin is the first recognized and best-known member of the family. the covalent attachment of ub, ubiquitination, is mediated by specific combinations of e s and e s that promote the formation of a peptide bond between the c-terminal gly and the n-terminal met or the ε-amino group of a lys residue in the substrate. in addition to the attachment of a single ub to one (mono-ub) or several figure . schematic illustration of the ubl activation, conjugation and deconjugation cycle. the covalent attachment of ubls to their substrates involves sequential catalytic reactions that initiate with processing of the ubl precursor by a specific ubl protease. the mature ubl is activated by an activating enzyme (e ) and then transferred to a conjugating enzyme (e ) that, with the help of a substrate-specific ligase (e ), transfer the activated ubl to the ε-amino residue of a lys on the target protein via a covalent isopeptide bond. additional ubls can be linked to the previous one to form chains. ubl-specific proteases can reverse the modification, supplementing the cellular pools of free ubls. the attachment of a ub moiety to the n-terminal met or to an internal lys residue of the previous ub (k , k , k ,k ,k ,k or k ) results in the formation of topologically different poly-ub chains that, upon recognition by signal transducers contain dedicated binding domains, target the substrates various fates and cellular functions ubiquitin is the first recognized and best-known member of the family. the covalent attachment of ub, ubiquitination, is mediated by specific combinations of e s and e s that promote the formation of a peptide bond between the c-terminal gly and the n-terminal met or the ε-amino group of a lys residue in the substrate. in addition to the attachment of a single ub to one (mono-ub) or several (multi-ub) lys residues, poly-ub chains can be formed upon attachment of a new ub moiety to met or lys , , , , , , or of the first conjugated ub. in the poly-ub chain, ub is usually attached to the same lys residue on each ub in the chain but mixed-linkage and branched poly-ub chains may be more common than originally thought [ ] (figure ). the attachment of ub or poly-ub chains generates new interaction surfaces in the modified substrate that are recognized by signal transducers via dedicated ubiquitin binding domains [ ] . signal transducers that recognize other ubls have their own specific binding motifs resulting in a broad spectrum of distinct signals that engage the modified substrate in specialized functions ( figure ). for example, lys -linked poly-ub chains (k poly-ub) usually target the substrate for degradation by the proteasome whereas k poly-ub have non-proteolytic functions, often related to protein localization and protein-protein interactions [ ] . mono-or poly-sumoylation regulates protein localization and the formation of protein complexes involved in dna replication and stress responses [ ] . poly-sumo chains may also serve as a signal for ub-dependent proteasomal degradation following recognition by specialized e that carry multiple sumo-interacting motifs [ ] . the activation of cullin-ring ligases (crls) by neddylation of the cullin scaffold provides another example of cross-talk between different types of ubl modification [ ] . other ubls play important roles in different types of stress responses as illustrated by involvement of urm and ufm in the regulation of oxidative stress [ , ] and er stress [ , ] , respectively, and by the involvement of fat- [ ] and isg [ , ] in the cellular and immune response to infection. the conjugation of ubls of the lc /gabarap family to the membrane lipid phosphatidylethanolamine underlies their involvement in the expansion and fusion of autophagic membranes [ ] . biomolecules , , of (multi-ub) lys residues, poly-ub chains can be formed upon attachment of a new ub moiety to met or lys , , , , , , or of the first conjugated ub. in the poly-ub chain, ub is usually attached to the same lys residue on each ub in the chain but mixed-linkage and branched poly-ub chains may be more common than originally thought [ ] ( figure ). the attachment of ub or poly-ub chains generates new interaction surfaces in the modified substrate that are recognized by signal transducers via dedicated ubiquitin binding domains [ ] . signal transducers that recognize other ubls have their own specific binding motifs resulting in a broad spectrum of distinct signals that engage the modified substrate in specialized functions ( figure ). for example, lys -linked poly-ub chains (k poly-ub) usually target the substrate for degradation by the proteasome whereas k poly-ub have non-proteolytic functions, often related to protein localization and protein-protein interactions [ ] . mono-or poly-sumoylation regulates protein localization and the formation of protein complexes involved in dna replication and stress responses [ ] . poly-sumo chains may also serve as a signal for ub-dependent proteasomal degradation following recognition by specialized e that carry multiple sumo-interacting motifs [ ] . the activation of cullin-ring ligases (crls) by neddylation of the cullin scaffold provides another example of cross-talk between different types of ubl modification [ ] . other ubls play important roles in different types of stress responses as illustrated by involvement of urm and ufm in the regulation of oxidative stress [ , ] and er stress [ , ] , respectively, and by the involvement of fat- [ ] and isg [ , ] in the cellular and immune response to infection. the conjugation of ubls of the lc /gabarap family to the membrane lipid phosphatidylethanolamine underlies their involvement in the expansion and fusion of autophagic membranes [ ] . the best-known function of ubiquitin is the marking of substrates for degradation by the proteasome, but different types of ubiquitination regulate endocytosis protein trafficking, transcription and translation, cell signaling, histone modification and dna repair. other ubl have similar but usually more restricted roles in the regulation of cellular functions. sumoylation is involved in the formation of protein complexes that regulate transcription, dna repair different stress responses and can also mark proteins for ubiquitin-dependent degradation. nedd is best known for its role in regulating the activity of cullin-ring ligases, which in turn regulates substrate degradation. ubls, of the lc /gabarap family are involved in the process of autophagy. the best-known function of ubiquitin is the marking of substrates for degradation by the proteasome, but different types of ubiquitination regulate endocytosis protein trafficking, transcription and translation, cell signaling, histone modification and dna repair. other ubl have similar but usually more restricted roles in the regulation of cellular functions. sumoylation is involved in the formation of protein complexes that regulate transcription, dna repair different stress responses and can also mark proteins for ubiquitin-dependent degradation. nedd is best known for its role in regulating the activity of cullin-ring ligases, which in turn regulates substrate degradation. ubls, of the lc /gabarap family are involved in the process of autophagy. the conjugation of ubls is highly dynamic and reversible, allowing for the fine-tuning and rapid remodeling of signal transduction pathways in response to different stimuli. deconjugating enzymes catalyze the removal of the ubls from their substrates, resulting in either complete loss or editing/trimming of the ubl chain. in humans, around hundred ub-specific deconjugases (also called deubiquitinases, dubs), belong to families that differ in structure and catalytic mechanisms [ , ] . the majority are cysteine proteases containing an active-site catalytic triad composed of a cys nucleophile and closely situated his and asp/asn residues. the cys protease families include ub-specific proteases (usps), ubiquitin c-terminal hydrolases (uch), ovarian tumor domain proteases (otus), machado-josephin disease proteases (mjd), motif interacting with ubiquitin novel dub family (mindy), and zinc finger with ufm -specific peptidase domain protein (zufsp), whereas the jab /mpn/mov (jamm) deconjugases are zinc-dependent metalloproteases. members of the usp family generally cleave all ub linkage types without a clear preference [ ] , while the members of the otu enzymes are often linkage specific [ ] . as a rule, different ubls have their own sets of specific deconjugases. however, several ub deconjugases exhibit promiscuous activity against isg or nedd [ , ] , probably due to the closely similar c-terminal tail region of these ubls. in view of their limited genome size, viruses need to co-opt the host-cell machineries for virtually all aspects of their life cycle. hence, ubl-regulated cellular functions are exploited for viral entry, transcription and replication of the viral genomes, synthesis of viral proteins, and assembly of new virions and for the maturation and exit of viral particles from the infected cell [ ] [ ] [ ] [ ] [ ] . in addition, ubls play key roles in the regulation of the innate and adaptive immune defense that counteract infection. a large number of ubl ligases and deconjugases participate in different aspects of the antiviral immune response, ranging from the signaling of viral nucleic acid sensors in innate immunity and inflammation to the maturation of antigen presenting cells and the activation of antigen-specific t-cell responses [ , ] . the pleiotropic role of ubls in orchestrating the antiviral defense is well illustrated by their contribution to the activation and fine-tuning of the early response to infection [ ] (figure ). the recognition of incoming viral genomes by dna or rna sensors located in endosomes (toll-like receptors, tlrs) or in the cytosol of the infected cells (including the retinoic acid inducible gene, rig-i-like receptors, rlrs, and cytoplasmic dna sensors such as cyclic gmp-amp synthase, cgas) leads via a cascade of signal transduction events to the activation of executor transcription factors that drive the synthesis of antiviral molecules such as type i and ii interferons (ifns) and pro-inflammatory cytokines [ , ] . in turn, binding of ifns to their specific receptors activates a new signaling cascade that leads to transcription of ifn-sensitive genes for which the products mediate establishment of an antiviral state [ ] . e ligases regulate these signaling pathways via the attachment of different types of polyubiquitin chains and ubl polypeptides [ , ] . for example, the viral nucleic acid sensor rig-i is activated by k -polyubiquitination mediated by the ligases trim [ ] , riplet [ ] , and trim [ ] , while signaling is terminated by k -polyubiquitination mediated by the ring finger protein (rnf ) ligase [ ] , which promotes proteasomal degradation. the attachment of m -or k -polyubiquitin chains to signaling mediators such as irak (interleukin associated kinase- ) [ , ] , traf [ ] , rip (receptor interacting protein- ) [ ] , traf (tnf receptor associated factor- ) [ ] , mavs (mitochondrial antiviral signaling protein) [ ] , nemo (nf-κb essential modulator) [ ] , and sting (stimulator of ifn genes) [ ] promotes activation of the kinases ikk (iκb kinase), tak (transforming growth factor beta activated kinase- ), and tbk (tank binding kinase- ) [ , ] that phosphorylate the executor transcription factors nf-κb (nuclear factor-κb, irf (interferon regulatory factor- ), and irf , leading to their activation and nuclear translocation. phosphorylation may also serve as a signal for ubiquitination as illustrated by the phosphorylation-dependent k -polyubiquitination of iκbα by the βtrcp e ligase, which leads to degradation of the inhibitor and activation of nf-κb [ ] . other types of ubl modifications exert biomolecules , , of similar regulatory functions. thus, isgylation targets rig-i for degradation by autophagy, reducing the levels of both basal and virus-induced ifn promoter activity [ ] , while isgylation of irf by the herc (hect and rld domain containing- ) ligase was shown to promote sustained signaling by protecting irf for ubiquitination and proteasomal degradation [ ] . fat was shown to form an inhibitory complex with rig-i, leading to the formation of insoluble rig-i aggregates, which prevented the translocation of rig-i to mavs and halted signaling [ ] . neddylation of myd was shown to negatively regulate nf-κb signaling by antagonizing its ubiquitination [ ] , while sumoylation of rig-i, irf , and irf was shown to affect both their stability and signaling properties [ ] . biomolecules , , of containing- ) ligase was shown to promote sustained signaling by protecting irf for ubiquitination and proteasomal degradation [ ] . fat was shown to form an inhibitory complex with rig-i, leading to the formation of insoluble rig-i aggregates, which prevented the translocation of rig-i to mavs and halted signaling [ ] . neddylation of myd was shown to negatively regulate nf-κb signaling by antagonizing its ubiquitination [ ] , while sumoylation of rig-i, irf , and irf was shown to affect both their stability and signaling properties [ ] . the production of type i ifn leads to transcriptional activation of numerous ifn stimulated genes (isgs) whose products cooperate in the establishment of an antiviral state in both the infected and adjacent cells [ ] . isg and its conjugation enzymes are strongly upregulated by ifn, and hundreds of putative targets of isgylation have been identified by mass spectrometry analysis although only a few have been experimentally validated [ ] . the conjugation of isg to both viral and cellular proteins was shown to impair virus replication and spread [ , ] . thus, the isgylation of de novo synthesized viral proteins may hinder their interaction with host proteins that are required for replication, may disrupt their catalytic function, or may alter the oligomerization of capsid proteins leading to a decrease in the number and infectivity of virus particles [ ] [ ] [ ] . in addition, isgylation inhibits the function of cellular proteins that regulate vesicular trafficking and are required for virus budding and release, including components of the endosomal sorting complex required for transport (escrt) [ , ] . recent studies suggest that isg may participate in the regulation of herpesvirus latency. numerous isgs were strongly upregulated in primary human oral fibroblasts latently infected with kaposi's sarcoma-associated herpesvirus (kshv) and in kshvpositive primary effusion lymphoma cells, while knockdown of isg or the isg ligase herc induced virus reactivation and the release of infectious virus [ , ] . it should be noted that conjugation-independent functions of isg may also contribute to the control of infection. high serum levels of unconjugated isg have been detected in patients treated with interferon and in mice infected with different viruses [ ] . furthermore, extracellular isg was shown to function as a cytokine with immune modulatory activity [ ] and as a chemotactic factor for neutrophils [ ] , pointing to a possible function of soluble isg in the modulation of inflammatory responses. the production of type i ifn leads to transcriptional activation of numerous ifn stimulated genes (isgs) whose products cooperate in the establishment of an antiviral state in both the infected and adjacent cells [ ] . isg and its conjugation enzymes are strongly upregulated by ifn, and hundreds of putative targets of isgylation have been identified by mass spectrometry analysis although only a few have been experimentally validated [ ] . the conjugation of isg to both viral and cellular proteins was shown to impair virus replication and spread [ , ] . thus, the isgylation of de novo synthesized viral proteins may hinder their interaction with host proteins that are required for replication, may disrupt their catalytic function, or may alter the oligomerization of capsid proteins leading to a decrease in the number and infectivity of virus particles [ ] [ ] [ ] . in addition, isgylation inhibits the function of cellular proteins that regulate vesicular trafficking and are required for virus budding and release, including components of the endosomal sorting complex required for transport (escrt) [ , ] . recent studies suggest that isg may participate in the regulation of herpesvirus latency. numerous isgs were strongly upregulated in primary human oral fibroblasts latently infected with kaposi's sarcoma-associated herpesvirus (kshv) and in kshv-positive primary effusion lymphoma cells, while knockdown of isg or the isg ligase herc induced virus reactivation and the release of infectious virus [ , ] . it should be noted that conjugation-independent functions of isg may also contribute to the control of infection. high serum levels of unconjugated isg have been detected in patients treated with interferon and in mice infected with different viruses [ ] . furthermore, extracellular isg was shown to function as a cytokine with immune modulatory activity [ ] and as a chemotactic factor for neutrophils [ ] , pointing to a possible function of soluble isg in the modulation of inflammatory responses. interfering with ubl-dependent processes through deconjugation is a powerful strategy used by viruses to regulate many cellular functions that contribute to or counteract infection. common means of regulation involve altering the expression of host deconjugases or redirecting the activity of the cellular enzymes towards new cellular or viral substrates [ , ] . in addition, many viruses encode their own deconjugases and increasing evidence supports the involvement of these viral enzymes in the control of infection [ ] [ ] [ ] . significant effort has been devoted to uncovering the substrates and cellular functions targeted by viral deconjugases. however, caution should be used in the interpretation of the increasing body of data since many experiments have relied on the overexpression of recombinant viral proteins or isolated enzymatic domains that, outside of the physiological context of infection, often exhibit very potent and broad deconjugase activity. indeed, temporal and spatial constraints operating in the infected cells are likely to determine the accessibility of a given substrate, while other viral factors expressed during infection may influence substrate specificity. further complications arise when the viral enzyme exerts both deconjugase and protease activity, as observed for the enzymes encoded by rna viruses. while some of these caveats can be addressed by sophisticated technologies, including structure determination and powerful mass spectrometry, the use of recombinant viruses expressing catalytically dead mutants of the enzymes has in several cases provided conclusive evidence on the cellular functions targeted during infection and reliable information on the putative substrates. both dna and rna viruses were shown to encode proteins with ubl deconjugase activity (table ) , and bioinformatics analysis coupled with in vitro enzymatic assays suggest that the largest viruses may even contain more than one deconjugase, as exemplified by the identification of three bona-fide dubs in the genome of epstein-barr virus (ebv) [ , ] . sequence-and structure-based comparisons with eukaryotic ubl-specific protease families have provided interesting clues on the origin and biology of the viral enzymes. in contrast to the eukaryotic enzymes, the deconjugases encoded by viruses usually target more than one ubl. for example, the adenovirus encoded adenain cleaves ub and isg conjugates but shares structural similarities with the ubiquitin c-terminal hydrolase uchl and with ulp/senp-like proteases that cleave sumo and nedd [ ] ; the papain-like proteases encoded by coronaviruses (cov) that are structurally related to mammalian dubs such as usp and usp show specificity for ub and isg and possibly nedd [ ] [ ] [ ] . ub and isg conjugates are also recognized by the otu-like proteases encoded by several animal rna viruses although this double specificity is not observed in their mammalian counterparts [ ] . while the structural similarities point to common ancestry, adaptation mechanisms operating in the context of infection may have selected for variants with broader specificity, which could counterbalance the limited coding capacity of the viral genomes. based on tertiary fold and architecture of the catalytic triad, the deconjugases encoded by herpesviruses constitute a unique family of enzymes that belong to the papain protease superfamily but are only distantly related to known cellular dubs [ ] . these enzymes cleave with comparable efficiency ub and nedd conjugates but fail to recognize isg [ , ] , pointing to a distinct set of substrates and targeted cellular functions. a common feature of the viral deconjugases is the embedding of catalytic domains in large multidomain proteins that play pleiotropic roles in infection. for example, the cov-encoded deconjugases are contained within a relatively well-conserved approximately kd region of the membrane anchored nonstructural protein- (nsp ) [ ] . the multidomain nsp is the largest protein encoded by the cov genome, with an average molecular mass of about kd and an essential component of the replication/transcription complex. although the domain organization differs between cov genera due to duplication or absence of some domains, eight domains are found in all known nsp . these include, in addition to the deconjugase domain, two ubiquitin-like domains, a catalytically active adp-ribose- -phosphatase domain that may play a role during synthesis of viral sub-genomic rnas, a nucleic acid binding domain with chaperone function, and other less characterized domains including an er luminal zn finger domain [ ] . via interaction with other nsps, nsp scaffolds the assembly of the replicase complex that utilizes er membranes to organize a microenvironment where the genome replication and transcription machinery is localized. in a similar fashion, the deconjugases encoded by different herpesviruses are located in an approximately kd n-terminal domain of the - kd large tegument proteins [ ] . the function of the large tegument proteins is only partially understood. studies on the herpes simplex virus (hsv) encoded member of the family, ul /vp - , show that, via binding to the viral capsid protein ul [ ] and inner tegument protein ul [ ] , ul promotes the transport of a viral dna loaded capsid along microtubules to the sites of secondary envelopment in the trans-golgi network [ ] . deletion of ul results in failure to fully assemble infectious virus particles [ ] . this role in virus assembly is shared by all members of this protein family [ , ] and is independent on the deconjugase function [ ] . in addition, upon de novo infection, ul guides the transport of incoming semi-uncoated capsids to the nuclear pore, where the viral genome is discharged into the nucleoplasm for viral transcription and replication [ ] . interestingly, cleavage of the n-terminal domain of ul that contains the deconjugase activity is required for the release of viral dna into the nucleus [ ] . a feature that distinguishes viral enzymes from their eukaryotic counterparts is the double function as ubl deconjugases and endopeptidases that play key roles in the virus cycle by processing viral proteins. the adenovirus protease (avp), adenain, is incorporated into immature virus particles, where it becomes activated by forming a thiol bond with an eleven-residue cleavage product of the capsid protein pvi (pvic) [ ] . the activated enzyme cleaves several viral capsid precursor proteins via recognition of the (m/i/l)xgx-g and (m/i/l)xgg-x sequence motifs [ , ] ; proteolytic maturation promotes the assembly of entry-competent viruses and primes the incoming virus particles for uncoating, which is essential for infectivity [ ] . interestingly, binding to the activating peptide induces preferential cleavage at the gx-g site that is overrepresented in the viral proteins [ ] , suggesting that the substrate repertoire of adenain may change during different phases of infection depending on availability of the activating peptide. in a similar fashion, the cov papain-like protease (plpro) contained in nsp cooperates with the major chymotrypsin-like protease ( clpro) in processing of the viral polyprotein to give rise to the sixteen nsps that form the viral replicase complex [ ] . plpro recognizes the sequence lxgg at the nsp / , nsp / , and nsp / boundaries that is identical to the c-terminal sequence of ub, isg , and nedd [ , ] . processing of the polyprotein by plpro is required for virus replication, which highlights the essential role of the enzyme in the virus life cycle. analysis of the crystal structure of plpro bound to ub-aldehyde and models of the interaction with ub-chains and isg revealed a likely mechanism for discrimination between the ubl-conjugates and viral substrates. the recognition of poly-ub chains and isg was shown to be dependent on simultaneous engagement of two binding domains, s and s , on the surface of plpro [ ] . mutation of the distal s domain significantly impaired the processing of isg and poly-ub conjugates but did not affect the activity of the protease against the viral polyprotein [ ] . given the importance of coronaviruses, adenoviruses, and herpesviruses for human diseases, the next sections will be focused on the deconjugases encoded by these viruses, with particular emphasis on the affected cellular functions and validated substrates. comprehensive reviews on the ubl deconjugases of animal viruses, including the very interesting family of out-domain containing proteases encoded by nairoviruses, were recently published [ ] . coronaviruses are enveloped viruses with positive-sense, single-stranded rna (ssrna) genomes [ ] . the first human cov was identified in the s and was recognized as a causative agent of the common cold [ ] . since then, highly pathogenic covs causing severe acute respiratory syndrome (sars-cov) [ , ] , middle east respiratory syndrome (mers-cov) [ ] , and the ongoing cov-disease- pandemics (sars-cov- ) [ ] have emerged in humans via zoonotic transmission. the three highly pathogenic human viruses cause similar forms of atypical pneumonia with bilateral parenchymal "ground-glass" consolidative lesions that may progress to acute respiratory distress syndrome (ards) [ ] . the frequency of such complication varies, being highest for mers-cov and lowest for sars-cov- , suggesting important differences in disease pathogenesis. the severe cases show progressive lymphopenia with loss of both cd + and cd + t lymphocytes; massive infiltration of the alveolar walls by neutrophil and eosinophil granulocytes; and significantly elevated levels of il- , il- , il- , and ifn-γ, pointing to a direct correlation between the magnitude of the inflammatory response and the severity of the disease [ ] . importantly, all three viruses induce very little, if any, type i ifn [ ] [ ] [ ] . work in a mouse model of sars suggests that the impaired ifn production is responsible for the recruitment of monocyte-macrophages and production of proinflammatory cytokines in the lung, resulting in vascular leakage and impairment of the immune response [ ] . based on available crystal structures, the plpro encoded by sars-cov, mers-cov, and sars-cov- share a similar domain organization with "thumb", "palm", and "fingers" subdomains arranged together to resemble an extended right hand [ ] [ ] [ ] , ] . the cys-his-asp catalytic triad is located at the interface of the thumb and palm domain, with a topology similar to that found in papain. in addition to the core catalytic domain, the enzymes contain an n-terminal ub-fold domain that is not required for catalysis but may play a role in the immunomodulatory activity of plpro [ ] . the three enzymes cleave ub-and isg -reporter substrates and synthetic poly-ub chains, and both sars-cov and sars-cov- exhibit weak deneddylase activity [ ] . differences in specificity and efficiency of catalysis are likely explained by subtle changes in the surface binding pockets that mediate interaction with the substrates. as a general rule, dubs that disassemble ub chains have surface pockets that bind the ub moiety preceding (s ) and following (s ) the scissile bond, whereas dubs that recognize mono-or-polyubiquitinated substrates lack the s pocket but contain one (s ) or more (s , s , and s ) additional pockets that can accommodate distal ub moieties. based on structure and mutation analysis, the preferential recognition of k poly-ub by sars-cov plpro relies on interaction with both s and s binding pockets, where solvent-exposed hydrophobic residues within the thumb domain interact with the ile patch of the distal ub [ ] . the s site is also involved in the recognition of the distal ub-fold domain of isg [ ] , although interaction with the proximal s pocket was shown to play a dominant role [ ] . important differences in the architecture of both the catalytic core and binding pockets of mers-cov plpro correlate with overall decreased catalytic activity and capacity to recognize all types of poly-ub chain linkages [ ] . in line with the high degree of homology ( % identity), the plpro encoded by sars-cov- resembles the sars-cov enzyme in the capacity to target both k poly-ub and isg conjugates. however, changes in the palm domain were shown to improve the interaction with isg [ ] , while mutation of a key leu residue that engages ub in the s pocket of sars-cov to thr diminishes the ability to process k poly-ub chains [ ] [ ] [ ] , which results in the preference of sars-cov- for isg conjugates. the deconjugase activity of cov plpro has been implicated in the downregulation of innate immune responses [ , ] . type i ifns and pro-inflammatory cytokines are hardly expressed or appear late in cell-culture based models of cov infection [ ] , and dysregulation of the ifn response is associated with severe lung immunopathology, influx of inflammatory monocyte-macrophages, and elevated levels of cytokines and chemokines in mouse models of sars-cov [ ] and mers-cov [ ] infection. a similar imbalance of the host response was recently observed in sars-cov- -infected patients [ ] . two lines of evidence support a key role of the viral deconjugase in these effects. first, ectopic expression of both sars-cov [ , ] and mers-cov [ , ] and more recently sars-cov- plpro [ ] was shown to inhibit innate immune signaling pathways. second, direct evidence for the contribution of the deconjugase activity to immune evasion was obtained using recombinant mers-cov viruses encoding for plpro mutants with selective loss of the deconjugase activity but preserved polyprotein cleavage. the transcription of type i ifn and ifn-stimulated genes was markedly increased in cells infected with the mutant viruses, and infected mice showed significantly increased survival rates and faster virus clearance in spite of comparable virus replication rates in the lungs [ ] . although the capacity of plpro to regulate innate immune responses appears to be firmly established, the mechanism and cellular substrates involved in this effect are not well understood. the inhibition of the type i ifn by sars-cov plpro was shown to correlate with impaired phosphorylation and nuclear translocation of irf , suggesting that signaling through tlr or rig-i may be affected [ ] . surprisingly, while plpro interacted with irf both in transfected cells and in cells infected with sars-cov, this was dependent on the presence of the nsp transmembrane domain (plpro-tm) and neither binding nor inhibition of irf phosphorylation were affected by mutation of the catalytic cys to ala. a possible explanation may be found in the demonstration that plpro-tm interacts with the sting-traf -tbk complex via binding to the sting transmembrane domain, which promotes disruption of the complex and is associated with reduced ubiquitination of rig-i, traf- , sting, tbk , and irf [ ] . in addition, expression of a plpro construct lacking the tm domain but containing the n-terminal ubiquitin-fold domain was shown to block nf-κb signaling by stabilizing phosphorylated iκbα [ ] . stabilization of iκbα may be due to deubiquitination and the consequent inhibition of proteasomal degradation, but this was not formally proven. a recent study comparing the plpro of sars-cov and sars-cov- suggests that their different activity towards k poly-ub and isg parallels the extent of inhibition of nf-kb versus ifn signaling, with the dominant de-isgylase activity of sars-cov- plpro being associated with a stronger decrease of isgylated irf and stronger inhibition of the ifn response [ ] . however, the multiple roles of isgylation in the inhibition or enhancement of the ifn response via targeting of rig-i [ ] or rnf [ ] call for some caution in assessing the significance of this finding in the context of infection. it is noteworthy that silencing of isg did not affect or even enhance the type i ifn response in mice infected with different rna viruses [ , ] . it is also important to remember that isg is itself an ifn target gene and that secreted isg regulates the activity of various types of immune cells, including natural killer (nk) cells, dendritic cells (dc), and neutrophils [ , ] . thus, much remains to be done to achieve a precise molecular understanding of the mechanisms by which the plpro encoded by different coronaviruses modulate innate immunity and how this reflects in the severity of the disease. adenoviruses are double-stranded dna, non-enveloped viruses with a ≈ kd genome that codes for at least different proteins expressed during the early and late phases of infection. more than forty adenovirus serotypes infect humans, causing a range of pathologies including respiratory, ocular, and gastrointestinal infections [ ] . the adenovirus protease (avp) adenain is a kd protein encoded by the conserved late gene l [ ] . the protease activity plays an essential role in the maturation of virion-associated precursor proteins, which is required for assembly of infectious virus particles and is also involved in the uncoating of incoming virions during primary infection [ ] . adenain exhibits a papain-like fold and is structurally related to the ub-specific protease uch-l and sumo deconjugase ulp , with topology of the active site cys and his residues resembling that of ulp and organization of s -s substrate binding pocket similar to that of ubiquitin hydrolases [ ] . recombinant adenain was shown to cleave k tetra-ub and isg precursor peptides in vitro, and expression of the active enzymes correlated with a global decrease of poly-ubiquitinated proteins in adenovirus-infected cells. however, the cellular substrates of these activities have not been characterized. interestingly, overexpression was associated with decreased levels of mono-ubiquitinated histone h a in transfected hela cells, suggesting a possible role of the protease in chromatin-related events or in the regulation of the dna damage response. herpesviruses are large dna viruses with double-stranded dna genomes ranging from to kb. a characteristic property of herpesviruses is their capacity to establish latent infections in certain cell types, which allows life-long persistence in the infected hosts [ ] . seven herpesviruses are important human pathogens. herpes simplex virus- and (hsv- and - ; hhv- and - ) and varicella zoster virus (vzv; hhv- ) infect epithelial cells, causing cold sores or shingles, and establish latency in sensory neurons [ , ] . human cytomegalovirus (hcmv; hhv- ) and human herpesvirus- and - (hhv- and - ) infect myelomonocytic and lymphoid cells and cause mononucleosis-like syndromes in immunosuppressed patients and roseola in children [ , ] ; epstein-barr virus (ebv; hhv- ) and kaposi sarcoma herpesvirus (kshv; hhv- ) establish latency in b-lymphocytes and are associated with the pathogenesis of lymphoid, endothelial, and epithelial cell malignancies [ , ] . the establishment of latency poses a particular challenge to these viruses since it entails adaptation to different cellular environments and the consequent establishment of cell type-dependent programs of viral gene expression. in addition, transmission to new hosts is dependent on the reactivation of virus production in the face of specific and highly effective immune responses, which requires sophisticated immune evasion strategies [ ] . the development of activity-based ubiquitin probes capable of forming covalent adducts with the catalytic cys of dubs [ ] was instrumental for the discovery of deconjugase activity in the n-terminal fragment of the hsv- large tegument protein ul [ ] . the activity is conserved in all ul homologs encoded by human and animal herpesviruses investigated to date [ , [ ] [ ] [ ] [ ] [ ] , supporting the notion that the enzymes play important roles in the biology of these viruses. in spite of very low amino acid sequence similarity, sequence alignment identified relatively well-conserved cys and his boxes, and crystal structure of the homolog encoded by the mouse cytomegalovirus (mcmv) m revealed a unique organization of the catalytic core, suggesting that the viral enzymes may represent a new family of deconjugases [ ] . the in vitro cleavage of fluorogenic substrates and in vivo assays in cells transfected with tagged ubls showed that the enzymes recognize comparable efficiency of k and k poly-ub [ ] . in addition, a bacterial screen based on co-expression of the ebv encoded homolog, bplf , with ubl-gfp (green fluorescent protein) reporters revealed specificity for both ub and nedd [ ] .the deneddylase activity was shown to be conserved in the homologs encoded by hsv- , hcmv, kshv, and mouse herpesvirus mhv- [ ] , and sirna knockdown confirmed the involvement of bplf in the progressive decrease of neddylated substrates in ebv-positive cells entering the productive virus cycle [ ] , corroborating the notion that the deneddylase operates in infected cells under physiological levels of expression. the strict host-specificity of herpesviruses hampers direct testing of the contribution of the deconjugases to viral pathogenesis in humans, but compelling evidence from cell culture and animal models of herpesvirus infection supports an important role of the enzymes in virus replication and pathogenesis. while deletion of the entire or large fragments of the large tegument proteins severely impaired the release of infectious virus [ ] , as may be expected given their essential role in the architecture of the mature virions, decreased virus yields were also observed upon infection with recombinant viruses carrying mutation of the active site cys residue [ , , ] . ultrastructural analysis of cells infected with a mouse pseudorabies virus (prv) carrying an inactivating mutation of the pul deconjugase revealed accumulation of naked nucleocapsids in the cytoplasm [ ] , suggesting that enzymatic activity is required for virus assembly and egress. the contribution of the deconjugase to viral pathogenesis in vivo is clearly illustrated by the strongly reduced formation of t cell lymphomas in chicken infected with mutant marek's disease virus (mdv) [ ] and decreased neuro-invasion and longer survival of mice infected with mutant prv [ ] . interestingly, in line with the known role of the large tegument protein in the early phases of herpesvirus infection, abrogation of the deconjugase activity was shown to cause the accumulation of incoming viral genomes in the cytoplasm of mhv- -infected cells, which correlated with strongly enhanced activation of the type i ifn response and hampered the establishment of latent infection [ ] . the capacity to interfere with the ifn response is conserved in the deconjugase encoded by human herpesviruses, as confirmed by comparing type i ifn production in cells infected with wild type and mutant viruses [ , , , ] . collectively, the findings illustrate a pleiotropic role of the herpesvirus deconjugases in the regulation of multiple steps of the virus life cycle from virus entry, uncoating, and viral genome replication to the assembly and release of infectious virus particles. in addition, by halting the innate immune response, the deconjugase may promote establishment of a cellular and host environment conducive to latency and permissive for virus reactivation. in line with the broad effect of the herpesvirus deconjugases on different cellular functions, several putative substrates have been identified, often based on candidate approaches where the capacity of the isolated enzymatic domains to deconjugate known ubl substrates was tested in co-transfection assays. while the very potent and broad deconjugase activity of the overexpressed enzymes calls for some caution in the interpretation of the data, at least some of the candidate substrates could be validated by mapping the sites of interaction and by comparing their fate in cells infected with wild type and mutant viruses. cullins are the main cellular targets of neddylation and an obvious candidate substrate for the deneddylase activity of the ebv-encoded bplf in productively infected cells [ ] . bplf was shown to interact with a conserved region in the c-terminal domain of the cullins scaffolds, close to the site of neddylation, and to promote cullin deneddylation and their degradation by the proteasome [ , ] . the phenotype of cells expressing catalytically active bplf is similar to that induced by chemical blockade of the neddylation cascade, with accumulation of several substrates of nuclear cullin ligases and arrest in the s/g phase of the cell cycle [ ] , pointing to a role of the deconjugase in the induction of a pseudo s-phase environment that is required for efficient replication of the herpesvirus genomes [ ] . in line with this possibility, viral dna replication was strongly decreased upon sirna-mediated knockdown of bplf in cells entering the productive virus cycles, which correlated with failure to accumulate several substrates of cullin ligases, including the cellular dna polymerase licensing factor cdt (chromatin licensing and dna replication factor- ) [ ] . viral dna replication was restored following reconstitution of cdt expression in bplf knockdown cell, supporting the involvement of the cellular licensing factors in viral dna replication. the dub activity of the viral enzymes is likely to synergize with the deneddylase activity in promoting the efficiency of virus replication. reactivation of the productive virus cycle triggers the dna damage response [ ] . in response to dna damage, pcna (proliferating cell nuclear antigen) is monoubiquitinated by rad (ring type ubiquitin ligase- ) to activate the translesion synthesis pathway of post-replication repair. pcna accumulates at the replication sites of many dna viruses, although its function in viral replication is not fully understood. the ebv-encoded bplf [ ] and hsv- -encoded ul [ ] were shown to de-ubiquitinate pcna and to prevent the formation of pcna foci. in addition, bplf was shown to interact with and to promote the accumulation of the pcna ligase rad [ ] and translesion synthesis polymerase polη [ ] . taken together, these findings point to an important role of the viral deconjugase in regulating the stability, localization and activity of a variety of cellular factors that are recruited at the site of viral replication to assist or counteract the production of infectious virus. several members of the type i ifn and nf-κb signaling pathways have been proposed as putative targets of the inhibitory effect of the herpesvirus deconjugases on the innate immune responses. in different experimental setups, expression of the catalytically active enzymes was accompanied by impaired ubiquitination of rig-i [ , ] traf [ , , ] , traf [ , ] , irak [ ] , irf [ ] , sting [ , ] and iκbα [ ] . however, since the activity of these signaling molecules is interconnected via different ubiquitination and deubiquitination events, the identity of the viral substrates and the molecular interactions leading to failure to activate the key executor transcription factors remain in many cases unknown. in addition, since the poor amino acid sequence conservation of the n-terminal domains is likely to influence the binding properties of the viral enzymes, it is unclear whether the same or different ubiquitination events are targeted by the various member of the family. an alternative unbiased approach to the identification of putative substrates and targeted signaling pathways relies on the identification of binding partners by co-immunoprecipitation and mass spectrometry. while the huge size renders this approach technically challenging for the entire large tegument proteins, the physiological relevance of the much shorter n-terminal domain is supported by the finding that processing by caspase- [ ] , or by a yet unidentified protease [ ] , releases the catalytic domain of bplf and ul to promote localization of the enzymatic activity to the nucleus. gene ontology analysis of protein interacting with the catalytic domain of bplf showed enrichment in proteins involved in numerous cellular functions including rna transcription and metabolism, nuclear transport, intracellular trafficking, cell cycle, and apoptosis, with major interacting hubs centering around proteasome subunits, nuclear transport proteins, and several members of the - - family of adaptor proteins [ ] . the - - proteins are conserved molecular scaffolds expressed in all eukaryotic cells that bind as homo-or heterodimers to a multitude of functionally diverse proteins, including kinases, phosphatases, and transmembrane receptors [ , ] . network analysis revealed that bplf and - - share a high number of interacting partners, in addition to cullins, the trim ligase that regulates the ifn response via ubiquitination of rig-i [ ] . binding of - - was shown to stabilize the interaction of trim with rig-i [ ] , which is essential for targeting the ubiquitination rig-i to mavs for downstream signaling [ ] . the recruitment of bplf to the - - :trim complex was shown to promote activation and autoubiquitination of the ligase and was associated with failure to ubiquitinate rig-i. interestingly, while catalytically inactive bplf induced the k -linked autoubiquitination and degradation of trim , the active viral enzyme trimmed the polyubiquitin chain to mono-or di-ubiquitin and promoted the formation of trim cytosolic aggregates decorated by the autophagy receptor p /sqstm [ ] . aggregate formation and the inhibition of ifn response were abolished by mutation of solvent exposed residues in helix- of bplf that are also involved in the interaction with cullins [ , ] , pointing to a critical role of the - - :bplf interaction in the assembly of the inactivating trimolecular complex. mapping of the interacting domains provided interesting insights on the possible mechanism of inhibition. the formation of - - homo-or heterodimers builds a groove with two symmetrically oriented binding pockets [ ] . in vitro binding assays using isolated trim domains and bacterially expressed wild type and mutant - - and bplf suggest that dimeric - - could stabilize the trimolecular complex by simultaneously accommodating in each of the two binding pockets acidic residues located in bplf helix- and on the tip of the trim coiled-coil domain. docking models predict that bplf would not prevent the recruitment of rig-i but that the presence of one or two conjugated ubiquitins may hinder correct positioning of the substrate towards the e and may contribute to functional inactivation of the ligase [ ] . the finding that the bplf -mediated inhibition of ifn signaling is dependent on the formation of a tri-molecular complex with - - and trim points to trim as the primary target of the viral deconjugase. while the capacity to inhibit the ubiquitination of rig-i and other components of the signaling cascade was previously reported [ , , , ] , the homologs encoded by hcmv and kshv shared with bplf the capacity to bind to - - and trim and to promote the functional inactivation of trim , suggesting that this early step of the signaling cascade is a common target of the viral enzymes. interestingly, this property was not shared by the hsv ul homolog where changes in the exposed residues of helix- prevent efficient binding to - - [ ] . it remains to be seen whether and how the targeting of different steps of the ifn and nf-κb signaling pathways impacts the life cycle of these viruses. given the growing body of evidence demonstrating the contribution of viral deconjugases to virus replication and pathogenesis, the enzymes are now recognized as attractive targets for the design of antiviral therapeutics. high-throughput screening of small-molecule libraries and rational structure-guided design have led to the identification and development of several lead compounds capable of inhibiting the activities of the plpro of coronaviruses [ ] . among those, naphthalene inhibitors are particularly interesting due to their capacity to act as competitive, non-covalent inhibitors of sars-cov plpro via interaction with a mobile loop in the fingers domain that, upon binding of the inhibitor, closes towards the catalytic cleft and shuts down the active site [ ] . the compounds were shown to inhibit the deconjugase activity against model substrates and to block ifn production and virus replication in infected cells. while structural difference in the mobile loop are likely to explain the failure of the compounds to inhibit the plpro of mers-cov [ , ] , recent findings demonstrate potent inhibitory activity against the plpro of sars-cov- [ ] . of note, the naphthalene compounds did not inhibit the activity of human deconjugases, caspases- , and cathepsin-k in biochemical assays [ ] , which may explain their lack of toxicity in cell cultures and makes them promising candidates for the development of specific antiviral drugs. both covalent and non-covalent inhibitors of the adenovirus protease working at nanomolar concentrations in biochemical assays have been described [ ] . however, the compounds have either weak antiviral activity, suggesting poor cell permeability, or are highly cytotoxic for cultured cells. thus, additional medicinal chemistry optimization will be required to harness their therapeutic potential. the involvement of the herpesvirus deconjugases in the regulation of both virus production and antiviral responses makes them interesting candidates for the development of much needed drugs for the treatment of many diseases caused by herpesvirus infection and reactivation. unfortunately, research in this area is still scarce. recently, the antiparasitic drug suramin was identified as a possible candidate in a high-throughput screen for small molecule inhibitors of bplf [ ] . suramin inhibited the cleavage of k poly-ub at sub-micromolar concentrations and decreased in a dose-dependent manner the production of infectious virus without apparent cell toxicity. however, comparable levels of inhibition were observed against a panel of ten human dubs, indicating that the compound has relatively poor selectivity and may be acting via a nonspecific mechanism. as regulators of both the virus life cycle and the host innate immunity, viral ubl deconjugases serve as multifunctional swiss army knives to facilitate viral infection and pathogenesis (figure ). herpesviruses provide a particularly enticing example of the involvement of ubl deconjugases in the regulation of a multitude of nuclear and cytoplasmic events that are critical for the establishment of both latent and productive infection in different cell types. since the first report on the ubiquitin deconjugase activity of adenain some twenty years ago, significant progress has been made in the identification, functional characterization, and structure determination of this fascinating class of viral enzymes. however, a precise understanding of the mechanism of action and the identification of viral and cellular substrates remain in many cases a challenge. the huge societal impact of the coronavirus pandemics is providing a strong stimulus towards the characterization of the deconjugases encoded by these viruses and the development of therapeutic inhibitors. regrettably, work on the adenovirus and herpesvirus deconjugases is lagging behind. while the similarity to cellular enzymes is likely to be a major hinder towards the development of specific inhibitors targeting the catalytic core, the mapping of binding domains involved in substrate interaction may offer new opportunities for regulating the function of the viral deconjugases. funding: the work of the author was funded by grants awarded by the swedish research council, the swedishcancer foundation, and the karolinska institutet. acknowledgments: i sincerely thank t frisan, umeå university, n dantuma, karolinska institutet and all members of the masucci group for their critical reading of the manuscript and apologize to many contributors to the field whose work was not cited due to space limitations. the author declares no 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environment virus dna replication and the host dna damage response the translesion polymerase pol eta is required for efficient epstein-barr virus infectivity and is regulated by the viral deubiquitinating enzyme bplf epstein-barr virus deubiquitinase downregulates traf -mediated nf-kappab signaling during productive replication essential role of hcmv deubiquitinase in promoting oncogenesis by targeting anti-viral innate immune signaling pathways hsv vp - deubiquitinates sting to block type i interferon expression and promote brain infection the - - proteins: integrators of diverse signaling cues that impact cell fate and cancer development - - proteins: structure, function, and regulation the mitochondrial targeting chaperone - - epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity roles of rig-i n-terminal tandem card and splice variant in trim -mediated antiviral signal transduction - - scaffold proteins mediate the inactivation of trim and inhibition of the type i interferon response by herpesvirus deconjugases interaction with - - correlates with inactivation of the rig-i signalosome by herpesvirus ubiquitin deconjugases catalytic function and substrate specificity of the papain-like protease domain of nsp from the middle east respiratory syndrome coronavirus x-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases structure-based design and optimization of potent inhibitors of the adenoviral protease small molecule screening identifies inhibitors of the epstein-barr virus deubiquitinating enzyme, bplf this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: i sincerely thank t frisan, umeå university, n dantuma, karolinska institutet and all members of the masucci group for their critical reading of the manuscript and apologize to many contributors to the field whose work was not cited due to space limitations. the author declares no conflict of interest. the funders had no role in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the manuscript. key: cord- -wp wr b authors: peng, hui; yang, li-tao; li, jian; lu, zhi-qiang; wang, ling-yun; koup, richard a.; bailer, robert t.; wu, chang-you title: human memory t cell responses to sars-cov e protein date: - - journal: microbes infect doi: . /j.micinf. . . sha: doc_id: cord_uid: wp wr b e protein is a membrane component of severe acute respiratory syndrome coronavirus (sars-cov). disruption of e protein may reduce viral infectivity. thus, the sars-cov e protein is considered a potential target for the development of antiviral drugs. however, the cellular immune responses to e protein remain unclear in humans. in this study, we found that peripheral blood mononuclear cells (pbmcs) from fully recovered sars individuals rapidly produced ifn-γ and il- following stimulation with a pool of peptides overlapping the entire e protein sequence. analysis of the immune responses by flow cytometry showed that both cd (+) and cd (+)t cells were involved in the sars-cov e-specific immune responses after stimulation with sars-cov e peptides. moreover, the majority of ifn-γ(+)cd (+)t cells were central memory cells expressing cd ro(+)ccr (+)cd l(−); whereas ifn-γ(+)cd (+) memory t cells were mostly effector memory cells expressing cd ro(−)ccr (−)cd l(−). the results of t-cell responses to individual peptides indicated that the e protein contained at least two major t cell epitopes (e amino acid [aa] – and e – : aa – ) which were important in eliciting cellular immune response to sars-cov e protein in humans. severe acute respiratory syndrome (sars) is a newly emerged infectious disease caused by a novel type of coronavirus, designated sars coronavirus (sars-cov) [ e ] . during the period january to may , sars spread rapidly among large human populations and led to thousands of infected patients and hundreds of deaths in over countries [ e ] . as reported, substantial efforts have been made to isolate the sars-cov and study its genomic sequence [ , ] , and its ace- binding receptors have been identified on target cells [ ] . increasing evidence in both animal and human models indicates that sars-cov infection results in multiple organ dysfunction [ , ] . moreover, it has been also reported that sars-cov infection induces specific antibody production that is considered both an effective method of diagnosis, as well as potential for a passive and active immunization for the prevention and treatment of sars [ e ] . in addition to the induction of humoral immune responses, cellmediated immune responses to sars-cov were detected in both sars patients and animal models [ e ] . clinical data showed that pronounced lymphopenia was observed in most sars patients [ ] . all of those reports show that t cell responses may not only participate in the clearance of virus in recovered sars patients but also contribute to immunopathology in early stages of the disease. therefore, it is very important to understand the role of t lymphocytes in human sars-cov infection. sars-cov has four structural proteins (s, n, m, and e proteins) that have various functions [ , ] . many reports concerning the immune responses to sars-cov s and n proteins have been published [ e ], the immune responses against e protein in human and animal models have not yet been studied extensively [ ] . e protein is a small, e kda integral membrane protein. a previous study demonstrated that sars-cov e protein could form ion channels [ ] . e protein also plays a role in viral assembly and morphogenesis [ ] . disruption of the e protein may possibly abrogate viral infectivity [ ] . recently, yang et al. [ ] showed that sars-cov e protein induced apoptosis in transfected jurkat t cells, which possibly contributes to the sars-cov-induced lymphopenia observed in most sars patients. thus sars-cov e protein may be a potential target for the development of antiviral drugs. here we analyzed memory t cell responses against the e protein in a group of individuals who had clinical sars-cov infection between january and may . our results demonstrated that sars-cov e protein specific memory t cell responses including central and effector memory cd þ and cd þ t cells could be persistent for two years. we also showed that sars-cov e protein might contain at least two different major epitopes, aa e and aa e , which could be important in eliciting cellular immune response against sars-cov e protein in humans. eleven recovered sars individuals ( males and females, aged to ) were recruited from the second afflıiated hospital of sun yat-sen university and guangdong provincial hospital of traditional chinese medicine, guangzhou, guangdong, china. all participants had been diagnosed as sars patients based on clinical examination during the period january to may . the diagnostic criteria for sars-cov infection followed the world health organization definition of sars [ ] . the diagnosis of sars-cov infection was further confirmed by serological detection of sars-cov-specific antibodies [ ] . normal subjects without any contact history with sars patients were used as negative controls. nine synthetic peptides that spanned the entire sequence of the sars-cov e protein were kindly provided by drs. koup and bailer at the vaccine research center of the national institute of allergy and infectious diseases (niaid), national institutes of health (nih), usa. the peptides used in the subsequent experiments were e mers, overlapped by amino acids and named e ee . peripheral blood mononuclear cells (pbmcs) were isolated from heparinized venous blood by ficoll-hypaque density gradient centrifugation. cells were washed twice with rpmi- (gibco) and suspended in complete culture medium (rpmi- containing % fetal calf serum, u of penicillin per ml and mg of streptomycin per ml). pbmcs were seeded into the wells of -well culture plates (becton dickinson) (  cells/well) in triplicate. peptides and costimulatory mabs to cd (bd pharmingen) and cd d (bd pharmingen) were added, each at mg/ml, to the wells. in addition, pbmcs stimulated with mabs to cd and cd were used as a positive control; as a negative control, pbmcs were not stimulated. the plates were incubated for h at c in a humidified atmosphere containing % co and % air. supernatants were collected and the level of ifn-g was measured by elisa kit (r&d) according to the manufacture's instructions. the detection limit of the ifn-g assay kit was pg/ml. elispot assay kit for ifn-g was purchased from bd biosciences and assays performed as described [ ] . briefly, -well plates (millipore) were coated with anti-ifn-g mab and incubated at c overnight. the plates were washed three times before blocking with complete culture medium. fresh pbmcs were plated at  cells per well in triplicate. peptides and costimulatory mabs to cd and cd d were added at mg/ml each. pbmcs stimulated with cd and cd ( mg/ ml) mabs were used as a positive control, and as a negative control, pbmcs were not stimulated. after incubation for h at c, the cells were removed and incubated with biotinylated anti-human ifn-g detection antibody for h at room temperature. after washing, wells were developed for h with streptavidin-hrp, followed by incubation with substrate reagent according to the manufacturer's protocol. spot-forming cells (sfc) were detected with the elispot image analysis system (sage creation). the frequency of ifn-g-producing cells was calculated as the number of spots/number of total pbmcs per well and was adjusted as the number of ifn-g-producing cells/ pbmcs. the number of spots in negative control wells was in a range of e spots. intracellular cytokines ifn-g and il- were assessed as previously described [ ] . pbmcs (  cells) were placed into polystyrene tissue culture tubes (becton dickinson) with ml of complete medium (cm) and were stimulated with peptides plus costimulatory mabs as described above. culture tubes were incubated at c in a humidified % co atmosphere for h. brefeldin a (sigma-aldrich) was added to cells at a final concentration of mg/ml to prevent secretion of cytokines. after incubation, the cells were harvested and washed twice with pbs containing . % bsa plus . % sodium azide. cell phenotype was determined by cell surface staining with percp-labeled anti-cd , percp-labeled anti-cd , fitclabeled anti-cd ro, pe-labeled anti-ccr and pe-labeled anti-cd l mabs (bd pharmingen) at c for min. for intracellular ifn-g or il- detection, apc-labeled anti-ifn-g and pe-labeled anti-il- (bd pharmingen) antibodies were used. briefly, cells were fixed with % pfa for min at room temperature, washed in pbs-bsa buffer and incubated for h with in pbs-bsa buffer containing . % saponin. cells were washed in pbs-bsa- . % saponin buffer and stained with the labeled abs at c for min. data acquisition was performed on a flow cytometer (facs calibur). the data were analyzed using cellquest . software (becton dickinson). to assess memory t cell response specific for e protein after sars-cov infection in humans, pbmcs from individuals who had fully recovered from sars two years after infection were stimulated with a pool of peptides spanning the entire amino acid sequence of the sars-cov e protein, or the cells were stimulated with anti-cd and anti-cd antibodies under the same culture conditions as positive controls. after incubation for h, the culture supernatants were collected and assessed for the production of ifn-g by elisa. as shown in fig. a , stimulation of pbmcs from all sars-recovered donors with a pool of sars-cov peptides resulted in significantly higher levels of ifn-g production compared with the cells from normal individuals. similarly, the frequency of sars-cov e antigen-specific ifn-g-producing cells determined by ifn-g elispot assay in pbmcs from fully recovered sars individuals was significantly higher than that of the cells from the normal donor controls in response to e peptides (fig. b) . the median frequency of ifn-g-producing cells from sars-recovered individuals was sfc/million pbmcs and the range was e sfc/million pbmcs. in contrast, there was no obvious difference in the frequencies of ifn-g-producing cells between sars donors and normal controls when pbmcs were stimulated with anti-cd and anti-cd mab (data not shown). these results demonstrated that the t cell response specific to e protein could persist for two years after sars-cov infection. to analyze the cell populations involved in the e proteinspecific t cell response, multiple-color flow cytometry was used to characterize the phenotype and determine the frequency of cytokine-producing cells. pbmcs from sarsrecovered donors were incubated with a pool of e peptides and stained for cell surface expression of cd and cd and for intracellular ifn-g expression (fig. a) . the results showed (fig. b ) that ifn-g-producing cd þ t cells in response to e protein were detected in all of the sarsrecovered donors with a mean frequency of . % (range: . e . %). the frequency of ifn-g-producing cd þ t cells specific for e protein was similar to that of the cd þ t cells (mean: . %, range: . e . %). together, these data demonstrated that both cd þ and cd þ t cells were involved in sars-cov e-specific immune responses. the culture supernatants were collected and assessed for the production of ifn-g by elisa. all assays were performed in triplicate. (b) pbmcs from eight sars-recovered donors were stimulated with or without a pool of e peptides. ifn-g-producing cells were determined by elispot assay. the number of spots in the medium control wells ranged from to . all assays were performed in triplicate. normal subjects without any contact history with sars patients were used as negative controls. statistical analysis was performed using the student's t-test, and a statistically significant difference was set at p < . (**). bars indicate mean values. in addition to detection of ifn-g expression, the t cell responses to sars-cov e peptides based on of their ability to express il- were also evaluated by flow cytometry. pbmcs from sars-recovered donors were stimulated with a pool of e peptides and were stained for cell surface expression of cd and cd and also for intracellular il- expression (fig. ) . the results showed that sars-cov e protein specific cd þ memory t cells were also capable of producing il- (fig. a) , and a very low number of specific cd þ memory t cells could produce il- (data not shown). to further analyze the subsets of il- -and ifn-g-producing cd þ t cells, co-staining for ifn-g and il- expression was performed and analyzed by flow cytometry. the results, indicated in fig. b , show that t cells specific for sars-cov e peptides could be divided into three subsets based on il- and ifn-g expression: ifn-g secreting cells; co-expression of il- and ifn-g secreting cells; and il- secreting cells. the majority of t cells specific for sars-cov e peptides were single ifn-g-secreting cells, and the frequency of cells secreting both il- and ifn-g was very low in specific cd þ t cells (fig. c) . the phenotypic characteristics of sars-cov e protein specific cd þ and cd þ memory t cells were further assessed. following stimulation ex vivo with e peptides, pbmcs from sars-recovered donors were stained for surface expression of cd , cd , cd ro, cr , cd l and for intracellular ifn-g and subsequently analyzed by flow cytometry (fig. ) . the results showed that % of ifn-g þ cd þ t cells were ccr þ , . % of them were cd l À , and % of them expressed cd ro. compared to ifn-g þ cd þ t cells, % of ifn-g þ cd þ t cells did not express ccr , % of them were cd l À , and % of them were cd ro À . overall, these data indicated that ifn-g þ cd þ t cells were mostly contained within the cd ro þ ccr þ cd l À cell population, whereas most ifn-g þ cd þ t cells were contained within the cd ro À ccr À cd l À cell population, further suggesting that cd þ and cd þ memory t cells are different in phenotype and also demonstrating the heterogeneity of cd þ and cd þ memory t cells. pbmcs from sars-recovered donors were tested in a single peptide screening from overlapping peptides of sars-cov e protein by using ifn-g elispot assay. although responses were obtained with all nine peptides in one or more donors, the highest responses were obtained with peptides e (aa to ), e (aa to ) and e (aa to ), whereas the rest of the peptides induced lower responses (fig. ) . therefore, peptide e (aa to ) and peptides e , e (aa to ) were the major dominant antigen sites of e protein and contained at least two different epitopes. peptides e , e and e could effectively stimulate t cell responses when detected with elispot assay. to assess the populations of t cells for the response to individual peptides, pbmcs from sars-cov-recovered donors were stimulated for h ex vivo in the presence or absence of peptides e , e or e . then the cells were stained for surface cd and cd and for intracellular ifn-g and analyzed by flow cytometry (fig. ) . the frequency of sars-cov e-peptide-specific t cells induced by the selected peptides ranged from . to . % of cd þ t cells and from . to . % of cd þ t cells. these data indicated that the individual peptides e , e and e were able to induce the immune responses of both cd þ and cd þ memory t cells. our study provides the first evidence that cd þ and cd þ memory t-cell responses to sars-cov e protein were generated and persistent in the individuals who had had sars caused by sars-cov infection two years previously. in line with our observation, it has recently been shown that immunization of mice with dna vaccine of the e, m, and n genes can induce high levels of specific antibodies, t cell proliferation, ifn-g, dth responses, and in vivo cytotoxic t cell activities in response to sars-cov antigens [ ] . however, compared with pcd d/n vaccine, a lower level of immune responses is generated by the pcd d/e vaccine. in addition, it has been reported that bhpiv-based sars-cov dna vaccines encoding n, m, e or me protein are not able to generate neutralizing antibody and detectable resistance to sars-cov challenge, gated on cd + t cells fig. . functional characterization of e protein-specific ifn-g-producing memory t cells. pbmcs from sars-recovered donors were stimulated with a pool of e peptides for h. the expression of ccr , cd l and cd ro in ifn-g þ cd þ or ifn-g þ cd þ t cells was assessed by flow cytometry. cd þ cd À or cd À cd þ cells were gated and analyzed. results shown are from one experiment performed on pbmcs from a sars patient. while sars-cov dna vaccine encoding s is capable of inducing neutralizing antibody, and its protective efficacy can increase slightly by coexpression of m and e [ ] . therefore, the e protein can a induce lower level of specific immune response compared with other structural proteins of sars-cov. our results demonstrated that e protein-specific memory t cell response was persistent two years after sars-cov infection in humans. in another study by our group, we found that n protein-specific memory t cell responses were readily observed two years after sars-cov infection. however, memory t cell responses specific to e protein are much lower, compared to those of n protein (wu et al., authors' unpublished data), probably due to its small size and low abundance in the virions, and also possibly due to the sars-cov-e protein-induced lymphopenia via apoptosis. previously, it was believed that memory t cells could be divided into two functionally distinct subsets based on expression of ccr [ , ] . ccr À effector memory t cells (t em cells) were present in the blood, spleen and nonlymphoid tissues, whereas ccr þ central memory t cells (t cm cells) were found in lymph nodes, spleen and blood but not in non-lymphoid tissues. recent studies in both mice and humans have demonstrated that these two cell subsets were able to rapidly produce ifn-g and tnf-a. the il- production remains a property of t em cells following stimulation with antigen [ e ]. our results showed that sars-cov-specific ifn-g þ cd þ t cells were mostly contained within the cd ro þ ccr þ cd l À cell population, and most ifn-g þ cd þ t cells were contained within the cd ro À ccr À cd l À cell population. moreover, these two cell subsets from sars-recovered donors' subsets were able to rapidly produce ifn-g in response to sars-cov e peptides. while the frequency of il- -secreting cd þ memory t cells was much higher than that of cd þ memory t cells, consistently with other studies on cd þ memory t cells [ ] , our observations also demonstrated that cd þ and cd þ memory t cells were different in phenotype and function. as the t-cell epitopes are usually to aa long, the e peptides used in our study overlapped by aa in each peptide to minimize the possibility of missing the t-cell epitopes of sars-cov e protein. the clear difference in protein recognition among sars-cov immune donors suggests that the nature and composition of the immune response against the virus varies between individuals possibly due to the difference in hla types. although the results showed that the t-cell fig. . memory t cell responses specific to peptides e , e and e . pbmcs from sars-recovered donors were incubated with the indicated peptides (e , e or e ) for h. intracellular staining for ifn-g was performed. cd þ and cd þ t cells were gated and subsequently analyzed for ifn-g expression by flow cytometry. results shown are representative of independent experiments from sars patients. similar results were obtained in other experiments. epitopes were scattered throughout the sequence of e protein, some peptides were recognized more frequently than others. the peptides that were frequently recognized by t cells were e (aa to ), e (aa to ) and e (aa to ). therefore, at least two different epitopes, corresponding to aa to and aa to , were the major dominant antigen site on e protein for t cell responses. as e protein with amino acids has limited immunogenicity, very few studies have focused on identification of epitopes on e protein. liu et al. [ ] selected specific recombinant scfv antibodies against e and n protein of sars-cov from the phage display library and found that the two scfv antibodies b and c could recognize the non-overlapping epitopes of e protein of sars-cov. in conclusion, the current study demonstrates that both cd þ and cd þ t cells participate in sars-cov e-specific immune responses and that the memory t cell immune responses specific for sars-cov e antigen are persistent for a long period of time after recovery. peptides e (aa to ), e (aa to ) and e (aa to ) are important epitopes of sars-cov e antigen. persistence of e-specific memory t cell responses in sars patients may play an important role in protection from sars-cov re-infection. coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a cluster of cases of severe acute 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coronavirus vaccine in monkeys a dna vaccine induces sars coronavirus neutralization and protective immunity in mice mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus induction of th type response by dna vaccinations with n, m, and e genes against sars-cov in mice sars coronavirus e protein forms cation-selective ion channels assembly of human severe acute respiratory syndrome coronavirus-like particles expression of sars-coronavirus envelope protein in escherichia coli cells alters membrane permeability bcl-xl inhibits t-cell apoptosis induced by expression of sars coronavirus e protein in the absence of growth factors updated sars case definition using laboratory criteria th predominance and cd þ memory t cell depletion in patients with severe acute respiratory syndrome rapid effector function in cd þ memory t cells mapping t cell epitopes by flow cytometry contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity two subsets of memory t lymphocytes with distinct homing potentials and effector functions similarities and differences in cd þ and cd þ effector and memory t cell generation functional heterogeneity of memory cd t cell responses in different conditions of antigen exposure and persistence human cd þ t-cell differentiation in response to viruses identification of naive or antigen-experienced human cd þ t cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific cd þ t cell response recombinant scfv antibodies against e protein and n protein of severe acute respiratory syndrome virus this study was supported by grants from national nature we thank the individuals who donated their blood for this study. we also thank z. peng for performing flow cytometry assays. key: cord- -lk ao m authors: annamalai, thavamathi; saif, linda j.; lu, zhongyan; jung, kwonil title: age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: lk ao m porcine epidemic diarrhea (ped) is an enteric coronaviral infection that causes severe morbidity and mortality in suckling pigs, but less severe disease in older pigs. consequently, it causes significant economic losses to the pork industry. there are limited studies on the innate immune responses to ped virus (pedv) in pigs. the aims of our study were to investigate differences in innate immune responses to pedv infection in suckling and weaned pigs and to examine if disease severity coincides with reduced innate immune responses. weaned -day-old pigs (n = ) and -day-old nursing pigs (n = ) were assigned to pedv inoculated or uninoculated control groups. the pigs were observed daily for clinical signs, virus shedding and were euthanized at post-inoculation days (pids) and to assay immune responses. blood samples were collected at pids , and . the natural killer (nk) cell frequencies, nk cell activities (lysis of target k tumor cells in vitro), cd +cd + t cell and cd +cd + t cell frequencies were measured in blood and ileum at pids and . the pedv infected suckling pigs showed severe diarrhea and vomiting at pid , whereas the pedv infected weaned pigs showed milder clinical signs starting at pid . pedv infected suckling pigs had significantly higher diarrhea scores, earlier fecal pedv rna shedding and significantly higher viremia (viral rna in serum) compared to weaned pigs. there was no mortality in either infected suckling or infected weaned pigs. the control pigs not inoculated with pedv did not show any clinical signs and no detectable fecal or serum pedv rna. strikingly, pedv infected suckling pigs had significantly lower nk cell frequencies, undetectable nk cell activity and lower ifnγ producing nk cells in blood and ileum compared to pedv infected weaned pigs. pro-inflammatory cytokine profiles of pedv infected suckling pigs differed from those of pedv infected weaned pigs and coincided with onset of fecal pedv rna shedding and serum pedv rna titers. the infected suckling pigs have higher and earlier increases in serum ifnα, but lower serum il- and tnfα levels compared to infected weaned pigs. cd +cd + t cell frequencies were significantly higher in ileum of suckling pigs than in weaned pigs, whereas there was no difference in cd +cd + t cell frequencies. in conclusion, the observations of impaired lytic activity and ifn-γ production by nk cells in suckling pigs coincided with the increased severity of pedv infection in the suckling pigs compared with the weaned pigs. porcine epidemic diarrhea virus (pedv) is an enteric coronavirus (genus alphacoronavirus, family coronaviridae, order nidovirales) causing significant morbidity and mortality in suckling pigs. pedv was first diagnosed in the usa in may, (stevenson et al., ) and has spread throughout the usa and was also reported in mexico and canada (vlasova et al., ) . the estimated annual economic losses in the us from pedv is $ million to $ . billion (paarlberg, ) . pedv causes severe enteric disease in suckling pigs (chen et al., ; stevenson et al., ) , but milder disease in older weaned pigs . this is similar to earlier observations for another enteric coronavirus infection of pigs, transmissible gastroenteritis (tge) (saif et al., ) . therefore, similar to tge virus (tgev) infection, biofeedback of intestinal contents of affected pigs to older pigs to build herd immunity is considered as an important method to reduce losses from pedv (jung and saif, ) . viral infections induce both innate and adaptive immunity. innate immunity involves production of cytokines and interferons as well as recruitment of innate immune cells such as http://dx.doi.org/ . /j.vetimm. . . - /© elsevier b.v. all rights reserved. dendritic cells, macrophages and natural killer (nk) cells (rouse and sehrawat, ) . the innate immune response plays a significant role in controlling primary viral infections and in development of adaptive immune responses (aoshi et al., ; janeway and medzhitov, ) . nk cells are innate immune cells that display cytotoxic action against virus infected host cells and tumor cells (campbell and hasegawa, ; herberman et al., ; trinchieri, ) and thus play an important initial role in containing the viral infection. nk cells are also a major source of certain cytokines such as ifn␥ and tnf␣ (fauriat et al., ; vivier et al., ) . they play a key role in initial clearance of infection in viral diseases (brandstadter and yang, ) . cytokines are important in viral infections in that they are necessary for cell to cell communication for inflammation and immune responses (akira and kishimoto, ) . the early cytokines secreted during a viral infection help to modulate immune responses. the cytokines examined in this study are early cytokines that have mainly proinflammatory and antiviral action. interferons are a group of cytokines whose major function is antiviral activity (isaacs and lindenmann, ; wheelock, ) . ifn␣ is a type i interferon produced by most cells in response to viral infection, with the major source being innate immune cells such as monocytes and dendritic cells (trinchieri et al., ) . ifn␥ is a type ii interferon produced initially by innate immune cells such as macrophages, dendritic cells and nk cells, and later on by activated t cells (sen, ) . ifn␥ is important in enhancing the activities of phagocytic cells such as macrophages and nk cells (carnaud et al., ) . il- is produced by various cell types and is a proinflammatory cytokine due to its chemoattractive properties for inflammatory cells (arndt et al., ; huber et al., ) . il- is a proinflammatory cytokine secreted by th cells, as well as ␥␦t cells (innate immune cells in mucosa) (jin and dong, ) . it stimulates the inflammatory response to viral infections (ryzhakov et al., ) . il- is a proinflammatory cytokine produced mainly by phagocytic cells and is involved in activation of nk cell activity including ifn␥ production by nk cells (trinchieri, ) . tnf␣ is a proinflammatory cytokine secreted mainly by macrophages that regulates cell death, differentiation and inflammation (bradley, ) . studies of rotavirus infection of children showed that the cytokine responses varied depending on severity of clinical signs in individuals (jiang et al., ) . studies of human rotavirus infected gnotobiotic pigs showed similarly that the proinflammatory cytokine responses were more marked with virulent virus compared with attenuated virus infection (azevedo et al., ) . the above cytokines were examined in the present study to understand if differences in proinflammatory cytokine responses between suckling and weaned pigs may be involved in susceptibility of suckling pigs to severe disease by pedv infection. there is a lack of information on innate immune responses of young and older pigs to pedv infection that might explain some of the differences in disease severity between young and older pigs. in the present study, we investigated the innate immune responses such as cytokine and nk cell activity as well as changes in frequencies of t cells to examine if differences coincide with the higher disease severity of suckling versus weaned pigs. the virus inoculum used in this study was the wild-type virulent us pedv strain pc a which was from the intestinal contents of a pedv positive field piglet, then serially passaged two times in gnotobiotic pigs (jung et al., ) . the original sample was negative by pcr for other enteric viruses such as tgev/porcine respiratory coronavirus (prcv), porcine deltacoronavirus, rotavirus groups a, b, and c, porcine enteric caliciviruses, st-valerien-like viruses, porcine astroviruses, enterovirus, kobuvirus, and bocavirus (amimo et al., a,b; chung et al., ; jung et al., b; kim et al., ; sisay et al., ; wang et al., ) . immune electron microscopy of the original sample using gnotobiotic pig hyperimmune serum to pedv showed only pedv particles. the gnotobiotic pig passaged pc a intestinal contents were diluted in minimum essential medium (mem) and used as inoculum in this study as noted below. seronegative pregnant sows and -day-old, pedvseronegative weaned, large white × duroc crossbred pigs were obtained from a pedv-free specific pathogen free (spf) (confirmed by history, lack of qrt-pcr-pedv positive fecal samples and pedv antibodies) swine herd of the ohio state university. the spf osu herd was also seronegative for antibodies to porcine respiratory and reproductive syndrome virus, prcv, tgev and porcine circovirus type . the sows farrowed naturally and nursed their piglets until the end of the study. the four experimental groups in the study were as follows. group : pedv inoculated -day-old suckling pigs (n = ); group : mem only inoculated -day-old suckling pigs (n = ); group : pedv inoculated day-old weaned pigs (n = ); group : mem only inoculated -day-old weaned pigs (n = ). all experimental procedures on animals were approved by the institutional animal care and use committee of the ohio state university. pigs in pedv groups were inoculated orally with pedv inoculum [ . log ge (genomic equivalents) (≈ . log plaque forming unit)/pig] and pigs in mem only inoculated groups received mem. the inoculation dose was based on a previous pathogenicity study in our lab (jung et al., ) . following pedv inoculation, pigs were monitored for clinical signs daily until necropsy. diarrhea was assessed by scoring fecal consistency. fecal consistency was scored as, = solid; = pasty; = semi-liquid; = liquid, with scores of or more considered diarrheic. inoculated and mock pigs (n = - /group at each time-point) were euthanized for immunological studies at an acute stage on post inoculation day (pid) and at a later stage (pid ) of infection. blood samples were taken at pid (n = - pigs per group), pid (n = - pigs per group) and pid (from euthanized pigs, n = - per group) and separate serum aliquots were prepared for cytokine analysis and viral rna quantification. rectal swabs were collected from all pigs on the designated pids to determine fecal ped viral shedding (pedv rna quantified by rt-qpcr). two rectal swabs were suspended in ml mem (jung et al., ) . the rna was extracted from l of serum or supernatants following centrifugation of the fecal suspensions ( × g for min at • c), using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. pedv rna titers in rectal swab supernatants and sera were determined by rt-qpcr as described previously (jung et al., ) . blood and ileum were collected on the day of euthanasia and processed for isolation of mnc as previously described (yuan et al., ) . the isolated cells were resuspended in rpmi medium (roswell park memorial institute medium) containing % fetal bovine serum, mm l-glutamine, mm sodium pyruvate, . mm suckling pigs had more severe clinical signs, earlier fecal pedv rna shedding and higher serum pedv rna compared to weaned piglets. following pedv inoculation, pigs were monitored every day and rectal swabs and blood for serum were collected at pid , and . fecal consistency scores of or more considered diarrheic. fecal shedding and serum pedv rna were determined by rtqpcr. the detection limit of rt-qpcr was genomic equivalents (ge) per reaction, corresponding to . log ge/ml of rectal swab fluid or . ge/ml of serum. therefore, viral rna titers more than . log ge/ml in rectal swab fluid or . ge/ml in serum are considered positive. statistical analysis was done to compare suckling and weaned pigs (across rows) for different parameters measured. values with different alphabetical superscript within a parameter and time point are considered different at p < . . the uninoculated pigs did not show any clinical signs. the rectal swab fluids and serum of uninoculated pigs were tested at similar time points and no pedv rna was detected. nonessential amino acids, mm hepes and antibiotics (e-rpmi) and used for assays. k (human erythroleukemia cell line) tumor cells were used as target cells and the assay was done as described previously with a few modifications (cao et al., ; park et al., ) . the k cells were initially stained with carboxy fluorescein succinimidyl ester (cfse) (ebioscience, usa), washed and used for the assay. mncs from blood and ileum were used as effector cells. effector: target cell ratios of : , . : and . : were used. the cells were mixed at the specified ratios and incubated overnight in e-rpmi at • c. the cells were then incubated with -aminoactinomycin d ( -aad) (life technologies, usa) for min at • c to stain dead cells. the cells were examined by flow cytometry and the percentage of cfse positive cells that were also stained with -aad were assessed as dead k cells. cfse labeled k cells incubated without mncs and stained similarly with -aad were used as controls for spontaneous death of k cells. the procedure was followed as described previously (chattha et al., ; yuan et al., ) with a few modifications. mononuclear cells from blood and ileum were cultured for h at • c in e-rpmi. the protein transport inhibitor, brefeldin a ( mg/ml; sigma-aldrich, usa), was added for the last h to prevent secretion of ifn␥ produced by the cells. the cells were stained with cd -fitc (fluorescein isothiocyanate) (clone ppt ; southern biotech, birmingham, al, usa), cd -sprd (spectral red) (clone - - ; bd biosciences, usa), and cd -biotin followed by streptavidin apc (allophycocyanin) (bd biosciences, usa) as secondary antibody. samples were stained intracellularly with anti-porcine ifn-␥-pe (phycoerythrin) (clone p g ; bd biosciences, usa). cd -cd -cd + ifn-␥+ cells were expressed as percentage of cd -cd -cd + nk cells. isotype antibody-labeled cells were used as controls. to determine the frequencies of t helper cells (cd +cd +), cytotoxic t cells (cd +cd +) and nk cells (cd -cd -cd +), cell samples were stained with anti-porcine cd -fitc, cd -pe (clone - - ; bd biosciences), and cd -sprd for min at • c. the frequencies of t cells or nk cells were expressed as percentage of lymphocytes expressing the respective markers. cells stained with isotype antibodies were used as controls. serum was separated by centrifuging blood at × g for min, and the collected serum was stored at − • c until tested. il- , il- , il- and ifn␣ were measured as previously described (azevedo et al., ; chattha et al., ) . for tnf␣, a porcine tnf␣ elisa kit was used per manufacturer's recommendations (kingfisher biotechnologies, st. paul, mn). all values are expressed as the means ± standard error of the means (sem). fecal consistency scores and viral rna titers in rectal fluids were analyzed and compared by a student's t-test using graphpad prism software (graphpad prism inc.). nk cell activity, nk cell numbers, t cell numbers and cytokine amounts were analyzed by one-way anova using graphpad prism software. a value of p < . was considered statistically significant. . . suckling pigs had more severe clinical signs, earlier fecal pedv rna shedding and higher serum pedv rna titers compared to weaned pigs the suckling pigs showed severe diarrhea and vomiting at pid , whereas the weaned pigs showed milder clinical signs starting only at pid ( table ). the fecal consistency scores were significantly higher in suckling pigs compared to weaned pigs at all time points examined (p < . ) ( table ). the fecal shedding pedv rna titer was high in suckling pigs starting from pid , whereas the weaned pigs started shedding pedv rna at pid and shed at significantly higher titers at pid compared to the suckling pigs (p < . ) ( table ). the serum pedv rna titers were significantly higher in suckling pigs at all time-points sampled ( table ). the higher severity of disease in suckling pigs coincided with the higher serum pedv rna titers in suckling pigs compared to weaned pigs. the mem only inoculated control pigs had no detectable pedv rna in either serum or feces. the suckling pigs had no detectable nk cell activity in blood and ileal mncs regardless of pedv infection, whereas the pedv infected and uninfected weaned pigs had low, but detectable nk cell activity (percentage of target lysis: . - . ) at pids and (fig. a-d) . suckling piglets had lower nk cell activity (% lysis of k cells) compared to weaned piglets in blood and ileal mononuclear cells at pid (a and b) and pid (c and d). blood and ileal mononuclear cells were co-cultured with cfse stained k cells at indicated ratios overnight. the dead cells were stained by incubating with -aad. the cells were observed by flow cytometry to obtain % of dead k cells. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the bar graphs labeled with different alphabetical letters are significantly different (p < . ). the infected weaned pigs had significantly increased nk cell activity in ileum at pid compared to uninfected weaned pigs (p = . ) (fig. d ). the nk cell activity was similar for infected versus uninfected weaned pigs at pid ( fig. a and b ). this coincides with the delayed onset of virus shedding in the infected weaned pigs. the nk cell activity in ileum of uninfected weaned pigs was about -fold higher than in the blood of uninfected weaned pigs (p < . ). the nk cell activity in ileum of infected weaned pigs was about -fold higher than in the blood of infected weaned pigs at pid (p < . ) ( fig. a and b) and about -fold higher at pid (p < . ) ( fig. c and d). there was no significant difference in the nk cell activity of blood mncs of infected and uninfected weaned pigs at pids and ( fig. a and c). the increase in nk cell activity was not correlated to the increase in nk cell frequencies in the ileal mncs. . . weaned infected pigs had significantly higher nk cell frequencies in blood and ileum at pid compared to suckling infected pigs the uninfected weaned pigs had significantly higher blood nk cell frequencies compared to uninfected suckling pigs (p < . ) ( fig. a) . the infected weaned pigs also had significantly higher nk cell frequencies in blood and ileum at pid (p < . ) compared to infected suckling pigs ( fig. a and b) . at pid , the infected weaned pigs had similar nk cell frequencies in blood to uninfected weaned pigs. at pid , the infected suckling pigs showed a significant increase in nk cell frequencies in blood compared to uninfected suckling pigs (p < . ) (fig. a) . the ileal nk cell frequencies were similar across groups at pid (fig. b) . however, at pid , the infected weaned pigs had significantly higher nk cell frequencies in ileum compared to infected suckling pigs, although there was an increase in nk cell frequencies in ileum of infected suckling and weaned pigs compared to uninfected suckling and weaned pigs, respectively (p < . ) (fig. b ). the nk cell frequencies of suckling and weaned pigs were higher in blood mncs than in ileal mncs at all time-points. . . ifn producing cd -cd -cd + nk cell frequencies were significantly higher in blood of weaned pigs compared to suckling pigs at pid and , regardless of pedv infection status and in ileum at pid for pedv-infected pigs like for the nk cell activity ( fig. a and c) , the ifn␥ producing nk cells were undetectable in blood of suckling piglets (fig. a) . the infected weaned pigs had a significantly higher ifn␥ producing nk cell frequency in blood than uninfected weaned pigs at pid (p < . ), although there was no difference between the two groups at pid . the ifn␥ producing nk cell frequency in ileum was similar in uninfected/infected suckling (pid ) and weaned pigs (pid , ) (fig. b) . at pid , the infected weaned pigs had similar ileal ifn␥ producing nk cell frequencies compared to infected suckling pigs. the infected suckling pigs had significantly lower ileal ifn␥ producing nk cell frequencies than the infected weaned pigs at pid (p < . ). at pid , the ifn␥ producing nk cell frequency was significantly lower in the ileum of infected suckling pigs compared to uninfected suckling pigs (p < . ). however, in the weaned fig. . weaned infected piglets had higher nk cell frequencies compared to suckling infected piglets in blood (a) and ileum (b) at pid . blood and ileal mncs were stained with anti-porcine cd -fitc, cd -pe and cd -sprd. the nk cell (cd -cd -cd +) frequency was expressed as percentage of lymphocytes. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the different time points were also compared within an age group and tissue type. bar graphs labeled with no common alphabetical letter are significantly different (p < . ). infected pigs there was no significant reduction in the ileal ifn␥ producing nk cell frequency compared to uninfected weaned pigs. . . pro-inflammatory cytokine profiles of pedv infected suckling pigs differed from pedv infected weaned pigs and coincided with fecal and serum pedv rna titers there was a marked induction of serum ifn␣ in infected suckling pigs at pid which declined significantly thereafter. for infected weaned pigs, the response was highest at pid (fig. a ). infected suckling pigs had significantly higher ifn␣ levels compared to infected weaned pigs at pid (p < . ). at pids and , there was no difference in the ifn␣ induction levels in infected weaned and suckling pigs. overall, the peak induction of serum ifn␣ in suckling pigs was at pid and was much higher than the peak induction of ifn-␣ in weaned pigs which was at pid (p < . ); both peaks coincided with the peaks of pedv rna shedding in feces and serum (suckling pigs) or serum (weaned pigs) and onset of diarrhea. the uninfected suckling and weaned pigs had similar low serum ifn␣ levels. serum il- levels followed a similar trend. il- levels of infected suckling pigs were highest at pid , whereas for infected weaned pigs, the induction was highest at pid (fig. b) . there was no statistical difference in the peak il- levels between the infected suckling pigs at pid and infected weaned pigs at pid . however, serum il- levels were significantly higher in infected suckling pigs compared to infected weaned pigs (p < . ) at pid . the infected weaned pigs had significantly higher serum il- fig. . ifn␥ producing cd -cd -cd + nk cell frequencies were higher in weaned pigs compared to suckling pigs at pid and in blood (a) and at pid in ileum (b). blood and ileal mncs were cultured in e-rpmi with protein transporter inhibitor, brefeldin a added. the cells were stained with cd -fitc, cd -sprd, and cd -biotin followed by streptavidin apc as secondary antibody. samples were stained intracellularly with anti-porcine ifn␥-pe. flow cytometric analysis was done and the percentage of cd -cd -cd + nk cells that were also ifn␥ positive was calculated. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the different time points were also compared within an age group and tissue type. bar graphs labeled with no common alphabetical letter are significantly different (p < . ). levels at pid compared to infected suckling pigs (p < . ). the uninfected suckling and weaned pigs had similar serum il- levels at all days. overall, serum il- levels were much lower than for all other cytokines measured. serum il- levels were significantly higher only in infected weaned pigs compared to infected suckling pigs at pid (p < . ) (data not shown). otherwise, there were no significant differences in the serum il- between weaned and suckling infected pigs at pids and . the uninfected suckling and weaned pigs had similar serum il- levels. serum il- levels were significantly higher only in infected weaned pigs compared to infected suckling pigs at pid (p < . ) (fig. c) . there was no difference in the serum il- between weaned and suckling infected pigs at pids and . the uninfected suckling and weaned pigs had similar serum il- levels. serum tnf␣ levels also followed similar trends to ifn␣ including the pids of peak levels in infected weaned and suckling pigs. infected suckling pigs had highest tnf␣ levels at pid , whereas for infected weaned pigs, the response was highest at pids and (fig. d) . serum tnf␣ was significantly higher in weaned infected pigs than in infected suckling pigs at pids and (p < . ), whereas there was no significant difference in the serum tnf␣ between infected suckling and infected weaned pigs at pid . the peak tnf␣ level in weaned pigs at pid was significantly higher than the peak tnf␣ level in suckling pigs at pid (p < . ). in suckling and weaned pigs at pids , and . blood samples were collected from the pigs at the specified time points and sera separated by centrifugation and frozen at − • c until use. the cytokine assays were done using standard assays. the groups that were compared were, infected suckling versus infected weaned at different time points, uninfected suckling or weaned versus infected suckling or weaned at different time points. the different time points within infected groups of suckling or weaned pigs were also compared. bar graphs labeled with no common alphabetical letter are significantly different (p < . ). the serum ifn␥, il- and tgf␤ levels were measured and there were no differences in their levels between the treatment groups (data not shown). . . cd +cd + t cell frequencies were higher in blood and ileum of suckling pigs than in weaned pigs, whereas there was no difference in cd +cd + t cell frequencies in ileum of suckling and weaned pigs the cd +cd + t cell frequencies in blood and ileum of uninfected suckling pigs were significantly higher than those in uninfected weaned pigs at pids and (p < . ) (fig. a and b) . however, the infected suckling pigs had statistically similar cd +cd + t cell frequencies in blood at pid compared to those of infected weaned pigs, whereas blood (fig. a) and ileum (fig. b ) of infected suckling pigs had higher cd +cd + t cell frequencies compared to infected weaned pigs at pid (p < . ). the cd +cd + t cell frequency in blood did not differ between pids and within the infected or uninfected groups of suckling or weaned pigs. the cd +cd + t cell frequencies in blood and ileal mncs were lower in infected suckling pigs compared to uninfected suckling pigs at pid (p < . ) (fig. a and b) . the frequency fig. . cd +cd + cells were higher in ileum of suckling pigs than weaned pigs whereas there was no difference in cd +cd + cells in ileum of suckling and weaned pigs. the graphs show the percentage of cd +cd + cells in blood (a) and ileum (b) of suckling and infected pigs at pids and , and the cd +cd + cells in blood (c) and ileum (d) of suckling and infected pigs at pids and . blood and ileal mononuclear cells were stained with cd -fitc, cd -pe and cd -sprd for min at • c. the frequency of t cells or nk cells was expressed as percentage of lymphocytes. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the different time points were also compared within an age group and tissue type. bar graphs labeled with no common alphabetical letter are significantly different (p < . ). of ileal cd +cd + t cells was higher at pid compared to pid for suckling infected pigs (p < . ). the infected suckling pigs had transient relative leukopenia of cd +cd + and cd +cd + t cells at pid compared to uninfected suckling pigs. there was no difference in the cd +cd + t cell frequency in blood and ileal mncs in weaned infected and uninfected pigs at either time point. the cd +cd + t cell frequency in blood mncs was significantly lower in infected suckling pigs compared to uninfected suckling pigs at pid (p = . ). the infected weaned pigs had significantly higher frequency of cd +cd + t cells in blood at pid compared to pid . there was no difference in the cd +cd + t cell frequency in blood mncs in infected and uninfected weaned pigs at either time point. there was no difference between the suckling and weaned uninfected pigs in cd +cd + t cell frequencies in blood (fig. c ) and ileal (fig. d ) mncs. suckling infected pigs had significantly lower cd +cd + t cell frequency in blood mncs compared to weaned infected pigs at pid . the cd +cd + t cell frequency in ileal mncs was similar in uninfected suckling and weaned pigs. there was also no difference in the ileal cd +cd + t cell frequency between the infected weaned and suckling pigs. the cd +cd + t cell frequency in ileal mncs of infected weaned pigs was significantly higher compared to uninfected weaned pigs (p < . ) and the same was true for infected and uninfected suckling pigs (p < . ). the infected suckling pigs had significantly higher frequency of cd +cd + t cells in ileum at pid compared to pid . similarly, infected weaned pigs had significantly higher frequency of cd +cd + t cells in ileal mncs at pid compared to pid (p < . ). in the present study, we investigated the differences between suckling and weaned pigs in innate immune responses to pedv infection and attempted to assess if these differences coincide with the greater severity of ped in suckling pigs. the major findings of this study were: ( ) suckling pigs had earlier onset and more severe diarrhea and earlier fecal and higher serum pedv rna titers compared to weaned pigs; ( ) pedv infected and uninfected suckling pigs had lower nk cell frequencies and no activity and lower ifn␥ producing nk cell frequencies compared to weaned pigs; ( ) ifn␣ and pro-inflammatory cytokine profiles of pedv infected suckling pigs differed from pedv infected weaned pigs. in infected suckling and weaned groups, peak ifn␣, il- and tnf␣ levels coincided with onset of diarrhea and fecal pedv rna shedding and peak serum pedv rna titers (pids and , respectively); and ( ) frequencies of cd +cd + t cells were significantly higher in ileum of suckling pigs than in weaned pigs, whereas there was no difference in frequency of cd +cd + t cells. there was a transient relative leukopenia in cd +cd + and cd +cd + t cells in blood of suckling, but not weaned pigs at pid . the nursing pigs showed severe clinical signs and fecal and serum pedv rna as early as pid as reported previously (jung et al., ; stevenson et al., ) , but the weaned pigs showed mild and delayed clinical signs and delayed shedding of pedv rna in feces, in agreement with previous findings . in general, neonates of various species are more susceptible to severe disease than adults (camacho-gonzalez et al., ; rose, ; rosenberg et al., ; tregoning and schwarze, ; wohlfender et al., ). one of the reasons for the high susceptibility of neonates to severe disease from infections is deficient innate and adaptive immune responses/memory (levy, ; levy et al., ) . in the absence of previous exposure to pathogens, and therefore no adaptive immune responses, the newborn depends on innate immune responses to clear or reduce viral infection. in instances when the virus infection overwhelms the already weak innate immunity of the neonate, severe disease results (firth et al., ) . the virus levels in blood were directly related to severity of certain enteroviral diseases in infants (dagan et al., ; yen et al., ) as well as in other viral infections (maggi et al., ; vaughn et al., ) . interestingly in the present study, the moderate titer of pedv rna in serum of suckling pigs and -fold lower titers in weaned pigs coincided with the greater severity of disease in suckling pigs compared to weaned pigs. the severity of disease in suckling pigs also coincided with the early high titer pedv rna fecal shedding. porcine nk cells are identified as cd -cd + cells (gerner et al., ) . suckling pigs had no detectable nk cell activity in blood or ileal mncs and the activity did not change with infection, although the suckling pigs had comparable nk cell frequencies in most instances (except pids of infected pigs) to weaned pigs. this is in confirmation of previous studies of nk cell frequency and cytotoxic activity of lymphocytes of healthy suckling pigs against k cells (onizuka et al., ; yang et al., ) . newborn pigs also did not have nk cell mediated cytotoxic activity against pk- cells persistently infected with tgev (cepica and derbyshire, ) . studies of tge showed that vaccination with a modified live tgev vaccine did not increase nk cell activity in newborn pigs (raymond and wilkie, ) , but in vitro stimulation of adult monocytes increased nk cell activity (charley et al., ) . in the present study, pedv infected weaned pigs showed increased frequencies as well as activity of nk cells in the ileum. although uninfected suckling pigs had comparable nk cell frequencies to weaned pigs, their frequencies were reduced in blood and slightly increased in ileum following infection, but nk cell activity remained undetectable. studies of human infants showed that neonates had significantly lower nk cell lytic activity and the activity was further reduced by infection or sepsis (georgeson et al., ; uksila et al., ) . nk cells of infants also had reduced antiviral activity such as defective killing of virus infected cells and reduced cytokine secretion (jacobson et al., ) . collectively, reduced nk cell activity and nk cell frequencies may have contributed to the early onset of virus shedding and the severe clinical signs in the suckling versus weaned pigs. coinciding with nk cell cytotoxic activity, the weaned pigs also had higher frequency of ifn␥ positive nk cells compared to suckling pigs. further, pedv infection reduced the frequencies of ifn␥ producing nk cells in ileum of suckling pigs. neonatal mncs are deficient in ifn␥ production in response to antigenic stimuli (wilson et al., ) . nk cells are the major source of ifn␥ and activation of nk cells by ifn␥ is essential for their lytic activity (wang et al., ) . studies have shown that the increase in frequency, activity of nk cells and ifn␥ production by nk cells is essential for reduction of viral load in mice infected with lymphocytic choriomeningitis virus or influenza virus (biron et al., ; mack et al., ; stein-streilein et al., ) . further, production of ifn␤ and ifn␥ in intestine of mice was essential and sufficient to induce innate immune cells to eliminate certain bacterial pathogens in mice (sotolongo et al., ) . since the suckling pigs showed reduced ifn␥ positive nk cell frequencies, this could result in reduced viral clearance contributing to the enhanced disease. similar to our observations, foot and mouth disease virus decreased the lytic activity of nk cells in swine (toka et al., ) . studies showed that il- production stimulates ifn␥ production and il- production is less in intestinal epithelium of neonatal pigs compared to older pigs (muneta et al., ) . thus, it appears that the failure to induce sufficient ifn␥ production by nk cells might be one reason for increased severity of disease and higher systemic load of pedv in suckling pigs. the weaned pigs had a delayed pro-inflammatory cytokine induction compared with suckling pigs which coincided with delayed infection, disease and shedding of pedv rna in feces of weaned pigs. ifn␣ is an antiviral cytokine produced in response to viral infection by monocytes and dendritic cells (hansmann et al., ; siegal et al., ) . consistent with fecal virus shedding and serum viral rna titers, the suckling pigs had the highest serum ifn␣ induction at pid . the higher systemic ifn␣ response in suckling pigs at the early stage of infection coincides with the serum pedv rna titer and very severe infection. collectively, lack of nk cell activity, reduced nk cell frequency and ifn␥ production might have contributed to the early onset of viral shedding, viremia (viral rna in serum) and severity of clinical signs. this is similar to the ifn␣ induction by tgev infection in suckling pigs (la bonnardiere and laude, ) . notably, treatment of newborn pigs with the interferon inducer, polyinosinic: polycytidylic acid complexed with poly-l-lysine, resulted in a delayed onset of clinical signs when pigs were infected with tgev (lesnick and derbyshire, ) and the ifn␣ treatment enhanced nk cell activity (charley et al., ) . also, treatment of tgev infected suckling pigs with oral human ifn␣ increased their survival rates (cummins et al., ) . in vitro studies of human dendritic cells stimulated with viruses show that neonatal cells are capable of producing ifn␣ responses similar to those of adults (renneson et al., ). the increased ifn␣ may be the result of infection in suckling pigs in the early stages and may have led to the reduced pedv rna shedding in feces at a later stage compared to the weaned pigs. the present studies showed that weaned infected pigs had higher il- at pid and higher tnf␣ induction at pids and compared to suckling pigs. tnf␣ is a proinflammatory cytokine secreted by activated macrophages, nk cells, t cells and other cells (bradley, ) . serum levels of tnf␣ are often associated with severity of disease (jiang et al., ; waage et al., ) . studies of rotavirus infected children show that higher tnf␣ and lower ifn␥ are associated with more severe symptoms of infection (jiang et al., ) . similarly, studies of human rotavirus infection in a gnotobiotic pig model show that levels of cytokines like ifn␥ and tnf␣ are higher when pigs are infected with virulent virus compared to attenuated virus (azevedo et al., ) . human neonatal monocytes/macrophages stimulated in vitro with bacterial lipopolysaccharides or virus showed reduced tnf␣ responses when compared to monocytes/macrophages from adults (levy et al., ; valero et al., ) . in the present study, although the suckling pigs had more severe disease, their tnf␣ levels were similar to weaned pigs at pid and less than weaned pigs at pids and . il- is a chemoattractant for neutrophils and is secreted by various cell types (bickel, ) . pigs infected with porcine respiratory and reproductive syndrome virus that subsequently cleared the infection had higher il- than pigs that did not clear the infection (kim and chae, ) . therefore, the higher il- in weaned pigs may have contributed to lower severity of disease. similar to the pattern of ifn␣, the serum levels of cytokines il- and il- were high in suckling pigs at pid and in weaned pigs at pids or and coincided with the delay in onset of clinical signs and fecal pedv rna shedding in weaned pigs. studies of human neonates show that they are capable of producing certain pro-inflammatory cytokines in comparable levels to adults (schnurr et al., ) . therefore, suckling piglets could have a similar ability to produce these cytokines as the weaned pigs. thus, pro-inflammatory cytokine profiles of suckling and weaned pigs mirrored the severity of infection and viremia. the suckling pigs lacked innate immune activity to delay onset of viral shedding and viremia that resulted in more cells infected and higher ifn␣ (jung et al., a) . in contrast, the robust innate immune activity in weaned pigs may have delayed the onset of viral shedding and viremia, resulting in a lower ifn␣ response. however, the innate immune response was not enough to prevent the higher fecal viral shedding at pid in weaned pigs although there was reduced diarrhea compared to suckling pigs. the results also indicated the ability of suckling pigs to produce proinflammatory cytokines comparable to weaned pigs. cd + t cells represent t helper cells that aid in the humoral immune response and are responsible for th cytokine production. cd + t cells represent cytotoxic t cells that are important in killing virus infected cells (germain, ; gerner et al., ) . the cd +cd + t cell frequency was higher in the uninfected suckling pigs compared to uninfected weaned pigs, whereas infected suckling pigs had transient relative leukopenia at pid and then had increased cd +cd + t cell frequencies at pid compared to uninfected suckling pigs. neonates have a polarity toward th responses dominated by cd +cd + t cells and this changes when they are given viral vaccines and it also changes with age (dowling and levy, ; kelly et al., ; kovarik and siegrist, ; siegrist et al., ) . the present study confirmed that the neonatal swine intestine has increased cd +cd + t cells that can be altered by viral infection. the cd +cd + t cell frequency was comparable in suckling and weaned uninfected pigs and infection increased the intestinal cd +cd + t cell frequency in both groups. thus, the observed differences in pedv infectivity between suckling and weaned pigs might not be influenced by early cd +cd + t cell frequency. since cd +cd + t cells are involved in early antiviral adaptive immune responses, these cells are expected to play important roles in pedv infection of the intestine. longer observations are required to determine if there are differences in adaptive immune responses between the suckling and weaned pigs. in summary, deficiency in innate immune function of nk cells may have contributed to the higher severity of pedv infection in suckling versus weaned piglets. interventions to enhance the nk cell activity and innate immunity may reduce the morbidity and mortality associated with pedv in suckling piglets. further 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disease severity is possible in severe neonatal enterovirus infection systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease virus-specific intestinal ifn-gamma producing t cell responses induced by human rotavirus infection and vaccines are correlated with protection against rotavirus diarrhea in gnotobiotic pigs we thank dr. juliette hanson, andrew wright, megan strother, and ronna wood for assistance with care of experimental animals. we also thank xiaohong wang, kyle scheuer, dr sukumar kandasamy, bryan eyerly and john blakenship for technical assistance. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work was supported by a grant from the oardc seeds, grant # oaoh (jung k, pi). key: cord- -j w vsw authors: stockman, lauren j; bellamy, richard; garner, paul title: sars: systematic review of treatment effects date: - - journal: plos med doi: . /journal.pmed. sha: doc_id: cord_uid: j w vsw background: the sars outbreak of – presented clinicians with a new, life-threatening disease for which they had no experience in treating and no research on the effectiveness of treatment options. the world health organization (who) expert panel on sars treatment requested a systematic review and comprehensive summary of treatments used for sars-infected patients in order to guide future treatment and identify priorities for research. methods and findings: in response to the who request we conducted a systematic review of the published literature on ribavirin, corticosteroids, lopinavir and ritonavir (lpv/r), type i interferon (ifn), intravenous immunoglobulin (ivig), and sars convalescent plasma from both in vitro studies and in sars patients. we also searched for clinical trial evidence of treatment for acute respiratory distress syndrome. sources of data were the literature databases medline, embase, biosis, and the cochrane central register of controlled trials (central) up to february . data from publications were extracted and evidence within studies was classified using predefined criteria. in total, sars treatment studies, in vitro studies, and three acute respiratory distress syndrome studies met our inclusion criteria. within in vitro studies, ribavirin, lopinavir, and type i ifn showed inhibition of sars-cov in tissue culture. in sars-infected patient reports on ribavirin, studies were classified as inconclusive, and four showed possible harm. seven studies of convalescent plasma or ivig, three of ifn type i, and two of lpv/r were inconclusive. in studies of steroid use, were inconclusive and four were classified as causing possible harm. conclusions: despite an extensive literature reporting on sars treatments, it was not possible to determine whether treatments benefited patients during the sars outbreak. some may have been harmful. clinical trials should be designed to validate a standard protocol for dosage and timing, and to accrue data in real time during future outbreaks to monitor specific adverse effects and help inform treatment. the sars outbreak of - presented clinicians with a new, life-threatening disease for which they had no experience in treating and no research on the effectiveness of treatment options. the world health organization (who) expert panel on sars treatment requested a systematic review and comprehensive summary of treatments used for sars-infected patients in order to guide future treatment and identify priorities for research. in response to the who request we conducted a systematic review of the published literature on ribavirin, corticosteroids, lopinavir and ritonavir (lpv/r), type i interferon (ifn), intravenous immunoglobulin (ivig), and sars convalescent plasma from both in vitro studies and in sars patients. we also searched for clinical trial evidence of treatment for acute respiratory distress syndrome. sources of data were the literature databases medline, embase, biosis, and the cochrane central register of controlled trials (central) up to february . data from publications were extracted and evidence within studies was classified using predefined criteria. in total, sars treatment studies, in vitro studies, and three acute respiratory distress syndrome studies met our inclusion criteria. within in vitro studies, ribavirin, lopinavir, and type i ifn showed inhibition of sars-cov in tissue culture. in sars-infected patient reports on ribavirin, studies were classified as inconclusive, and four showed possible harm. seven studies of convalescent plasma or ivig, three of ifn type i, and two of lpv/r were inconclusive. in studies of steroid use, were inconclusive and four were classified as causing possible harm. despite an extensive literature reporting on sars treatments, it was not possible to determine whether treatments benefited patients during the sars outbreak. some may have been harmful. clinical trials should be designed to validate a standard protocol for dosage and timing, and to accrue data in real time during future outbreaks to monitor specific adverse effects and help inform treatment. the severe acute respiratory syndrome (sars) is a febrile respiratory illness primarily transmitted by respiratory droplets or close personal contact. a global outbreak of sars between march and july caused over , probable or confirmed cases and deaths [ ] . the causative organism has been identified as a novel coronavirus (sars-cov) [ ] [ ] [ ] . the overall mortality during the outbreak was estimated at . % [ , ] . the overriding clinical feature of sars is the rapidity with which many patients develop symptoms of acute respiratory distress syndrome (ards). this complication occurred in approximately % of all patients with sars, and when it occurred was associated with a mortality rate of % [ , ] . at the time of the sars epidemic it was not known what treatments would reduce sars-related illness and deaths. because the urgency of the international outbreak did not allow time for efficacy studies, physicians in canada and hong kong treated the earliest patients with intravenous ribavirin, based on its broad-spectrum antiviral activity [ , ] . corticosteroids and immune-modulating agents were often prescribed empirically. soon after sars-cov was identified as the causative agent, antiviral screening programs were initiated; these programs reported several antiviral agents that inhibited sars-cov replication in vitro. these results led to the experimental use of protease inhibitors and interferon alpha (ifn-a) in the treatment of patients. the most commonly used treatments for sars are associated with adverse effects when used for other conditions (table s ). in october , the who established an international sars treatment study group, consisting of experts experienced in managing sars. the group recommended a systematic review of potential treatment options to identify the targets for proper evaluation in trials should the disease recur [ ] . this paper reports on this systematic review designed to summarise available evidence on the effects of ribavirin, lopinavir and ritonavir (lpv/r), corticosteroids, type i ifn, intravenous immunoglobulin (ivig), or convalescent plasma in relation to ( ) sars-cov replication inhibition in vitro; ( ) mortality or morbidity in sars patients; and ( ) effects on ards in adult patients. we prepared a protocol that defined our scope, inclusion criteria, and outcomes to be assessed. the interventions we included were defined by the who: ribavirin, lpv/r, corticosteroids, type i ifn, convalescent plasma, or ivig. the types of study we included were: ( ) in vitro studies, in which the authors examined inhibition of sars-cov viral replication, and data from an assay in human or animal cell line; ( ) in vivo studies, which included randomised controlled trial (rct), or prospective uncontrolled study design, or retrospective cohort design, or case-control design, or a case series, and patients treated for sars, and ten or more patients; and ( ) studies of ards that included rct, or systematic review, and treatment for ards or acute lung injury, and or more patients. in february , we systematically searched the literature databases medline, embase, biosis, and the cochrane central register of controlled trials (central) for articles that included the selected treatments (table s ) . the full text of each identified study was retrieved and each was independently reviewed by two authors (ls and rb). publications in chinese were selected after review of the english abstract. unpublished data were not sought, as the task of summarising existing published data was extensive and the international sars treatment group indicated that much of the clinical data had already been published. we used the quorom checklist to help ensure the quality of this review (table s ) . data from the full text of studies in english were extracted independently by two authors (ls and rb). data from the chinese literature were extracted with the assistance of a translator. because the chinese articles were reviewed by only one author, the consistency of the translated information with that from english articles was maintained by subsequent discussion with the translator to verify the extracted data. we established explicit criteria to assess the level of evidence for each human treatment study (box ). since the treatments chosen for evaluation were often given in combination, evidence was classified by the treatment that was given to all patients in the cohort or given to some with the author's intention of studying its effects. if putative effects within a study included several drugs, then we extracted data for each intervention. the level of evidence was independently classified by two authors (ls and rb). chinese studies were appraised and classified in the same way using translated information extracted from each report. discrepancies were resolved by consensus. in vitro evidence was available in studies. clinical evidence of sars treatment in humans was reported in studies ( in english, in chinese). three studies addressed treatment of ards ( figure ). in vitro. we found six studies that described the antiviral effect of ribavirin in vitro (table s ) ; four showed an antiviral effect (table s ) . a synergistic antiviral effect between ribavirin and type i ifn (ifn-b a or leukocytic ifn-a) was described in two studies performed in human cell lines and vero cell lines [ , ] . in sars patients. we found studies that described ribavirin treatment in cohorts larger than ten patients (table s ). our formal assessment classified studies as ''inconclusive,'' due to study design or because the effect of ribavirin could not be distinguished from the effects of other treatments (such as steroids and antiviral drugs). four publications presented evidence of possible harm ( ) ( ) ( ) ( ) . three of these studies, each of which included over patients, documented a fall in haemoglobin levels after ribavirin treatment when compared to levels in patients before treatment [ ] [ ] [ ] . of patients treated with ribavirin, / to / ( %- %) developed haemolytic anaemia, a recognised complication with this drug, although it is not possible to rule out the possibility that sars-cov infection caused the haemolytic anaemia, as there is no control group. one study noted that over % of sars patients had some degree of liver dysfunction indicated by alt levels higher than normal, and the number of patients with this complication increased to over % after ribavirin treatment (table s ) [ ] . in the chinese literature six additional reports described patients with sars treated with ribavirin (often with steroids). these six reports were determined to be inconclusive in the evaluation of treatment for sars (tables s and s ). in vitro. of three studies, two demonstrated that lopinavir inhibits cytopathic effects of sars-cov in fetal rhesus monkey kidney cells (table s ). one study showed detectable but reduced activity in vero-e cells [ ] , and one study concluded that neither lopinavir nor ritonavir had an effect [ ] . a synergistic effect of lopinavir with ribavirin has been reported (table s ) . in sars patients. we found two studies of lpv/r (lopinavir mg with ritonavir mg orally every h) in cohorts larger than ten patients (table s ) . patients also received ribavirin and corticosteroids. lpv/r use was compared among three groups of patients: those who received it as an early sars treatment, those who received it as a late treatment, and those who did not receive it at all. when lpv/r was added as an initial treatment to ribavirin and corticosteroid therapy, the death rate was lower than among those who received ribavirin and corticosteroids ( / [ ] . both studies were determined to be inconclusive due to possible bias in the selection of control group or treatment allocation. no additional studies were identified from the chinese literature. in vitro. no studies were found on the cytopathic effect of corticosteroids alone against sars-cov. corticosteroids act as immunomodulatory agents, and therefore studies to measure direct antiviral effects in vitro were not expected. in sars patients. fifteen articles examined corticosteroid treatment in ten or more patients. of these cohorts were also treated with ribavirin (table s ) . we determined that of the studies were inconclusive. of these, in an uncontrolled and nonrandomised study, / ( %) of patients treated with high-dose methylprednisolone ( . - mg/kg prednisolone on day of illness, followed by hydrocortisolone mg every h, and pulse-doses of methylprednisolone . g iv for d) after the first week of illness recovered from progressive lung disease (table s ) [ ] . two studies contained evidence of possible harm from corticosteroids [ , ] . one measured sars-cov plasma viral load across time after fever onset in a randomized, double-blind, placebo-controlled trial; corticosteroid use within the first week of illness was associated with delayed viral clearance. the other study, which was case-controlled, found that patients with psychosis received higher cumulative doses of steroids than patients without psychosis ( , mg versus , mg; p ¼ . ) [ ] . in the chinese literature, we found reports in which steroids were used (table s and table s ). twelve studies were inconclusive and two showed possible harm. one study reported diabetes onset associated with methylprednisolone treatment [ ] . another study (an uncontrolled, retrospective study of sars patients) reported avascular necrosis and osteoporosis among corticosteroid-treated sars patients [ ] . in ards patients. three clinical trials examined the effect of corticosteroids on mortality in patients with established ards (table s ). in two trials, high-dose methylprednisolone given for approximately d was not effective for early ards [ , ] . one small rct that used a regimen of lower dose methylprednisolone ( mg/kg per day), tapered after wk, showed possible evidence of ards improvement (table s ) [ ] . in vitro. twelve in vitro studies with data on the antiviral effect of ifn type i have been reported, and all demonstrated an antiviral effect against sars-cov (six for ifn-a and ten for ifn-b) (tables s and s ) . antiviral effects have been demonstrated in monkey (vero; vero-e ), fetal rhesus monkey kidney (frhk- ), and human (caco , cl , and hpek) cell lines. three reports presented evidence that ifn-b was superior against sars-cov compared to ifn-a and found rifn-a virtually ineffective against sars-cov compared to other ifns [ ] . synergistic effects were reported for leukocytic ifn-a with ribavirin [ ] , ifn-b with ribavirin [ , ] and ifn-b with ifn-c [ , ] . ''inconclusive'' if a study could not be used to inform a decision about treatment efficacy due to having either outcomes which were not reported consistently, an inconsistent treatment regimen, no control group or a control group which was a likely source of bias. a control group was considered a likely source of bias if there were differences in co-morbidities, sex, age and markers of severe disease compared to the treatment group. ''possible harm'' if a study reported adverse effects of treatment that were consistent with adverse effects reported with the use of the drug in the treatment of other conditions. evidence of direct causality was not required. a study could be classified as suggesting possible harm from the drug even if the study had methodological weaknesses. ''possible benefit'' if a study had evidence of benefit for an important outcome measure which was recorded consistently (e.g., case fatality, need for mechanical ventilation, duration of hospitalization, frequency of ards) in patients treated in a defined way compared to a valid control group. a control group was considered valid if randomized, or if patient characteristics and illness severity were comparable to the treatment group. evidence of direct causality was not required. ''definite harm'' if a study contained statistically significant evidence of harm demonstrated in a double-blind randomized trial, which did not contain serious methodological weaknesses. ''definite benefit'' if a study contained statistically significant evidence of harm demonstrated in a double-blind randomized trial, which did not contain serious methodological weaknesses. in sars patients. two studies of ifn-a given with steroids and/or ribavirin were reported (table s ). no significant difference was seen in outcome between ifn-a treatment group and those treated with other regimens. results of both studies were inconclusive due to a lack of a consistent treatment regimen or suitable control group (table s ). in the chinese literature, one additional study reported the use of ifn-a as part of a regimen that included ribavirin and steroids [ ] . we determined this study to be inconclusive because a variety of treatments given masked the effect of ifn-a alone (table s and table s ). in vitro. no studies were found on the cytopathic effect of this treatment on sars-cov. convalescent plasma and ivig act as immunomodulatory agents and therefore studies to measure direct antiviral effects in vitro were not expected. in sars patients. five studies of either ivig or convalescent plasma treatment given in addition to steroids and ribavirin were reported for treatment of sars (table s ) . these studies were inconclusive, because the effect of convalescent plasma or ivig could not be discerned from effects of patient comorbidities, stage of illness, or effect of other treatments (table s ). in the chinese literature, two additional studies reported evidence on the effect of convalescent plasma as a treatment for sars [ , ] . these studies were inconclusive (table s and table s ). evidence collected on the benefit or harm of drugs used to treat sars is summarized in table . the rapid spread and subsequent control of sars precluded controlled clinical treatment trials during the in this report we summarize the results of a systematic evaluation of the findings from published reports of treatments used for sars during the epidemic. publications from the chinese literature were included to capture as much evidence as possible. we developed specific criteria (box ) to look for large, obvious effects of benefit, adverse or poor outcomes, or evidence of potential benefit that could be used to prioritise future research of sars treatments. a summary of this evidence in sars patients is shown in table . despite thirty reports of sars-infected patients treated with ribavirin, there is no convincing evidence that it led to recovery. haemolytic anaemia, a recognized side effect of this treatment, was observed in three studies. we would infer from these findings that any future use of ribavirin for sars should be within the context of a controlled trial with close attention given to adverse effects. corticosteroids were commonly prescribed to sars patients with worsening pulmonary disease or progressing abnormalities on chest x-rays. treatment regimens varied widely but can be classified into two groups, early treatment and rescue treatment given at a later stage of illness. it is difficult to make a clear recommendation about whether corticosteroids should be used to treat sars-associated lung injury in any stage of illness, particularly as the drug is immunosuppressive and may delay viral clearance if given before viral replication is controlled [ ] . of added concern are infectious complications, avascular necrosis, and steroidinduced psychosis-recognized adverse effects of corticosteroid use. fungal superinfection and aspergillosis have been noted in case reports and autopsy findings of sars patients given corticosteroids at high doses or for prolonged periods [ , ] . this review has found evidence of avascular necrosis and steroid-induced psychosis in sars patients. seven studies of treatment with convalescent plasma or ivig, three with ifn type i, and two with lpv/r were inconclusive by the criteria used in our analyses. authors of four of the ivig studies commented that patients seemed to improve upon treatment, but that more controlled trials of this approach are needed to provide evidence of an effect for sars. important caveats should be considered in this review. most of the studies of sars patients were descriptions of the natural course of the disease and had not been designed to reliably assess the effects of the treatments used. patient characteristics such as age and presence of diabetes mellitus have been associated with severe disease and can confound treatment effects. a diagnostic test for early sars illness was not validated or widely available, and in general, treatment was initiated once patients fulfilled a clinical and epidemiological case definition. it is possible that the inclusion of patients without laboratory confirmation of sars-cov infection in this review could cause an underestimate of any true effect of antiviral treatment on sars. the variation in treatment regimens-particularly the wide range in doses, duration of therapy, and route of administration of ribavirin and corticosteroids-is a major obstacle to a clear interpretation of the data in this review. the nonstandardised collection of clinical information limits the conclusions that can be drawn from a retrospective analysis. we suggest that, in the event of a future outbreak of sars-cov or another novel agent, attempts be made to develop treatment protocols and to collect and contribute information for a standardized minimum dataset that could facilitate analysis of treatment outcomes among different settings. as observational studies pose problems of interpretation, the need is great for good-quality randomised trials, despite the difficulties in organising such trials. the group recommended a systematic review of potential treatments for sars. in particular, it was considered important to summarise the available evidence on the use of certain antiviral drugs (ribavirin, lopinavir, and ritonavir), steroids, and proteins called immunoglobulins, which are found naturally in human blood. the who group wanted to know how these treatments affected the virus outside the body (''in vitro'') and whether it helped the condition of patients and reduced the death rate, particularly in those patients who developed the dangerous complication called acute respiratory distress syndrome (ards). this study is a systematic review conducted in response to the who request. what did the researchers do and find? they did no new work with patients or in the laboratory. instead they conducted a comprehensive search of the scientific and medical literature for published studies that fitted their carefully predefined selection criteria. they found sars treatment studies, in vitro studies, and three ards studies that met these criteria. some of the in vitro studies with the antiviral drugs found that a particular drug reduced the reproduction rate of the viruses, but most of the studies of these drugs in patients were inconclusive. of studies on steroid use, were inconclusive and four found that the treatment caused possible harm. what do these findings mean? from the published studies, it is not possible to say whether any of the treatments used against sars were effective. no cases of sars have been reported since but it is always possible that the same or a similar virus might cause outbreaks in the future. it is disappointing that none of the research on sars is likely to be useful in helping to decide on the best treatments to use in such an outbreak. the authors discuss the weaknesses of the studies they found and urge that more effective methods of research be applied, in a timely fashion, in any similar outbreaks in the future. while the systematic review suggests that we do not know which if any of the potential treatments against sars are effective, its recommendations mean that researchers should at least be better prepared to learn from potential future outbreaks. additional information. please access these web sites via the online version of this summary at http://dx.doi.org/ . /journal.pmed. . wikipedia entry on sars (wikipedia is a free online encyclopedia that anyone can edit) medlineplus pages on sars wikipedia entry on systematic reviews, which includes links to other web sites where more detailed information may be found summary of probable sars cases with onset of illness from a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the severe acute respiratory syndrome the epidemiology of severe acute respiratory syndrome in the hong kong epidemic: an analysis of all patients critically ill patients with severe acute respiratory syndrome acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada a major outbreak of severe acute respiratory syndrome in hong kong future clinical trials for sars, informal meeting and workshop ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines in vitro susceptibility of clinical isolates of sars coronavirus to selected antiviral compounds common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada clinical features and short-term outcomes of patients with sars in the greater toronto area severe acute respiratory syndrome: report of treatment and outcome after a major outbreak temporal patterns of hepatic dysfunction and disease severity in patients with sars ( ) hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients factors associated with psychosis among patients with severe acute respiratory syndrome: a case-control study glucocorticoidinduced diabetes in severe acute respiratory syndrome: the impact of high dosage and duration of methylprednisolone therapy highdose corticosteroids in patients with the adult respiratory distress syndrome early steroid therapy for respiratory failure effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial increased sensitivity of sars-coronavirus to a combination of human type i and type ii interferons interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) pulmonary pathology of severe acute respiratory syndrome in toronto fatal aspergillosis in a patient with sars who was treated with corticosteroids we acknowledge dr. larry j. anderson for his review of manuscript drafts and consultation on in vitro data for sars treatment, dr. haoqiang zheng for extracting and translating data from the articles published in chinese, ms. vittoria lutje for helping with the literature search strategy, and the who international sars treatment study group for prioritising this research project and identifying treatments for review.the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention.author contributions. ls, rb, and pg drafted the protocol for this review. ls and rb reviewed all abstracts and extracted data. all authors appraised included studies, interpreted results, commented critically on the manuscript, and contributed text to the final version. key: cord- - hkogdh authors: samaddar, arghadip; grover, malika; nag, vijaya lakshmi title: pathophysiology and potential therapeutic candidates for covid- : a poorly understood arena date: - - journal: front pharmacol doi: . /fphar. . sha: doc_id: cord_uid: hkogdh coronavirus disease (covid- ), an acute onset pneumonia caused by a novel betacoronavirus, severe acute respiratory syndrome coronavirus (sars-cov- ), emerged in the wuhan city of china in december and evolved into a global pandemic. to date, there are no proven drugs or vaccines against this virus. hence, the situation demands an urgent need to explore all potential therapeutic strategies that can be made available to prevent the disease progression and improve patient outcomes. in absence of clinically proven treatment guidelines, several repurposed drugs and investigational agents are currently being evaluated in clinical trials for their probable benefits in the treatment of covid- . these include antivirals (remdesivir, lopinavir/ritonavir, umifenovir, and favipiravir), interferon, antimalarials (chloroquine/hydroxychloroquine), antiparasitic drugs (ivermectin and nitazoxanide), biologics (monoclonal antibodies and interleukin receptor antagonist), cellular therapies (mesenchymal stem cells and natural killer cells), convalescent plasma, and cytokine adsorber. though several observational studies have claimed many of these agents to be effective based on their in vitro activities and extrapolated evidence from sars and middle east respiratory syndrome (mers) epidemics, the currently available data remains inconclusive because of ill-defined patient selection criteria, small sample size, lack of concurrent controls, and use of intermediary outcomes instead of patient-relevant outcomes. moreover, there is a need to clearly define the patient populations who warrant therapy and also the timing of initiation of treatment. understanding the disease pathology responsible for the clinical manifestations of covid- is imperative to identify the potential targets for drug development. this review explains the pathophysiology of covid- and summarizes the potential treatment candidates, which can provide guidance in developing effective therapeutic strategies. in december , a cluster of cases of unexplained acute pneumonia was reported from the wuhan city of china's hubei province. as the causative agent could not be identified, these initial cases were classified as "pneumonia of unknown etiology." later on, the cause of this illness was attributed to a novel betacoronavirus, which was designated as -novel coronavirus ( -ncov) by the world health organization (who) (cascella et al., ) . on january , , as per the international health regulations (ihr, ) , the outbreak was declared a public health emergency of international concern (pheic) by the who. on february , , the disease was renamed as coronavirus disease , and on the same day, the coronavirus study group (csg) of the international committee on taxonomy of viruses designated -ncov as severe acute respiratory syndrome coronavirus (sars-cov- ) due to its phylogenetic similarity with severe acute respiratory syndrome coronavirus (sars-cov) (cascella et al., ) . considering its potential to evolve into a pandemic, the who raised the threat to the epidemic to the "very high" level on february , . with the alarming increase in the number of covid- cases outside china, affecting thousands of people across several countries, the who declared covid- a pandemic on march , . as of august , , covid- has affected more than million people across countries and territories, with , deaths. usa accounts for the maximum number of cases, followed by brazil, india, and russia. coronaviruses (covs) are a group of enveloped viruses with positive sense single-stranded rna genome and having a crownlike appearance under an electron microscope. they belong to the order nidovirales, family coronaviridae, and subfamily orthocoronavirinae. based on genetic and antigenic criteria, covs are classified into four genera: alphacoronavirus (a-cov), betacoronavirus (b-cov), deltacoronavirus (d-cov), and gammacoronavirus (g-cov). to date, seven covs capable of infecting humans (hcovs) have been identified (cascella et al., ) . according to an estimate, % of the population are healthy carriers of covs and they account for to % of acute respiratory infections . the common hcovs, hcov-oc and hcov-hku (b-covs) and hcov- e and hcov-nl (a-covs), cause mild self-limiting respiratory tract infections. other human covs, sars-cov, sars-cov- , and middle east respiratory syndrome coronavirus (mers-cov) (b-covs), cause epidemics with variable clinical severity featuring respiratory and extrarespiratory manifestations. sars-cov and mers-cov infections possess pandemic potential and can cause life-threatening disease with mortality rates up to % and %, respectively (cascella et al., ) . the g-covs infect avian species, while d-covs tend to infect both mammals and birds (zumla et al., ; de wilde et al., ) . phylogenetic analysis has placed sars-cov- under the subgenus sarbecovirus of the genus betacoronavirus. next generation sequencing data has revealed that the genome of sars-cov- bears . % sequence homology with a bat coronavirus ratg and shares . % identity with sars-cov (zheng, ) . based on phylogenetic and evolutionary analyses, it has been proposed that both bat-cov ratg and sars-cov- might have evolved from a common ancestor, and sars-cov- might have jumped from bats to humans via some unknown intermediate hosts (perrotta et al., ) . covid- is an acute respiratory disease with a clinical spectrum ranging from mild and moderate disease ( %) to severe ( %) and critical illness ( %), with an overall case fatality rate (cfr) of . - . %. the severe and critical illness categories (nearly % of all infections) are of special concern in elderly population and those with underlying comorbidities, as the severity and cfr are particularly high in these groups (perrotta et al., ) . several risk factors related to disease severity have been outlined by the united states centers for disease control and prevention (cdc). advanced age, male sex, and smoking have been reported as independent risk factors for disease progression, severity, and mortality. it was observed that % of the patients in italy over years of age succumbed to the disease (livingston and bucher, ) , and as per cdc reports, - % of the patients above the age of in the united states required hospitalization (vishnevetsky and levy, ) . a weekly surveillance report by the who regional office for europe reported that over % of all deaths due to covid- were people aged years or above, and more than % were people aged years or older. it has been hypothesized that there occurs an age-related decline in the clearance of inhaled particles in small airways, possibly due to decrease in the number of cilia and ciliated epithelial cells in the airways (svartengren et al., ) . an age-dependent increase in nasal-cavity volume coupled with decreased nasal resistance and upper airway size are other contributory factors (levitzky, ) . as age advances, there occurs a disruption of the innate and adaptive arms of the immune system with an impairment of both effector memory t cell and competent b cell functions, along with continuous production of inflammatory mediators and cytokines (inflammaging) (aw et al., ) . in healthy state, angiotensin converting enzyme (ace ) catalyzes the conversion of angiotensin to angiotensin − and thus, maintains a homeostasis between inflammatory and anti-inflammatory pathways. ace levels have been found to decrease in old age causing elevated angiotensin- , which increases pulmonary vascular permeability and inflammation, thereby worsening lung injury due to covid- in such patients (dhochak et al., ) . moreover, in the elderly, there occurs an age-related decrease in the vital capacity of lungs and perfusion of vital organs, such as, heart, lungs, and kidneys. sars-cov- causes a much more severe pneumonia in the aged than younger individuals. it has been observed that the incidence of acute respiratory distress syndrome (ards) is higher in the elderly and those with heart, liver and kidney ailments (perrotta et al., ; wang l. et al., ) . also, older age is a surrogate for comorbid illnesses, such as, respiratory and cardiovascular disorders, morbid obesity (i.e., body mass index of ≥ ), hypertension, diabetes mellitus, and significant renal and hepatic impairment (preskorn, ) . all these risk factors have been linked to higher rates of intensive care unit (icu) admission, greater disease severity, and poor prognosis. it has been observed that % of the patients who require hospitalization, icu admission, or succumb to the disease have one or more comorbid conditions, irrespective of age (garg et al., ) . therefore, in the elderly, immunosenescence and underlying comorbidities are likely to be the major contributory factors for life-threatening respiratory failure and multisystemic involvement associated with covid- . on the contrary, children develop milder symptoms, rarely require hospitalization, and have an overall better prognosis when compared to adults (ludvigsson, ) . a systematic review and meta-analysis including children with covid- from countries reported milder self-limiting symptoms in majority of the cases, with . % being critical and seven deaths. unlike adults, children rarely progressed to severe disease requiring icu admission (hoang et al., ) . this can be attributed to the immature immune system in pediatric population, cross-protection from related coronaviruses and other rna viruses to which they get exposed early in life, competitive inhibition of sars-cov- by other respiratory viruses simultaneously invading the airways and the lungs, trained non-specific immunity due to childhood immunization (e.g., bacillus calmette-guerin vaccine and mumps measles rubella vaccine), good regenerative capacity of lungs in children, absence of immunosenescence and age-related comorbidities, and high ace expression causing increased metabolism of angiotensin (dhochak et al., ) . despite overwhelming global efforts, covid- remains a poorly understood disease with limited success in the field of drug development. understanding the disease pathogenesis is crucial for choosing effective drug targets. this review explains the pathophysiology of covid- and summarizes the potential treatment candidates, which can provide guidance in developing efficient therapeutic strategies. sars-cov- is a positive sense, single-stranded rna virus belonging to the genus betacoronavirus (subgenus sarbecovirus, subfamily orthocoronavirinae). the genomic mrna has a ´-cap and a ´-poly (a) tail and can act as an mrna for translation of the viral polyproteins. in addition, both ´and ´ends of the genomic rna contain a highly structured untranslated region (utr) that plays an important role in the regulation of rna replication and transcription. the sars-cov- genome contains open reading frames (orfs), preceded by transcriptional regulatory sequences (trss). the two main transcriptional units, orf a and orf ab, comprise two-thirds of the viral genome and encode two major polyproteins: pp a (~ kda) and pp ab (~ kda), respectively. the synthesis of pp ab involves programmed ribosomal frame shifting during translation of orf a. these polypeptides are cleaved by virally encoded chymotrypsin-like protease ( clpro), main protease (mpro), and papain-like protease (plpro) into non-structural proteins (nsp -nsp ), which assemble to form the replicationtranscription complex (rtc) involved in genome transcription and replication (naqvi et al., ; romano et al., ) . pp a is cleaved into non-structural proteins (nsp -nsp ) and pp ab into five (nsp -nsp ). the non-structural proteins play an important role in the pathogenesis of covid- . nsp and nsp encode plpro and clpro, respectively, which help in peptide cleaving and host innate immune antagonism. nsp and nsp encode rna-dependent rna polymerase (rdrp) and rna helicase, respectively. other orfs at the ´end of the viral genome encode four structural proteins: the spike surface glycoprotein (s), membrane (m), envelope (e), and the nucleocapsid (n) proteins, which are the major components of the virus playing a crucial role in structural integrity and pathogenesis (romano et al., ; . the s protein is a homotrimeric transmembrane glycoprotein that determines diversity to coronaviruses and host tropism. it has two functional subunits: s , responsible for binding to the host ace receptors and s , for the fusion of the virion and cellular membranes. the m protein helps in transport of nutrients across the cell membrane, bud release, and the formation of viral envelope. the e protein plays a significant role in viral morphogenesis and assembly. the n protein plays an important role in packaging of viral rna into ribonucleocapsid and also helps in immune evasion by attenuating host immune responses (astuti, ; naqvi et al., ) . besides these structural proteins, the ´end also contains eight putative orfs for accessory proteins: a, b, p , a, b, b, b, and orf . the structural and accessory proteins are translated from a set of nested sub-genomic rnas (sgrnas) . the life cycle of sars-cov- consists of five steps: attachment, penetration, biosynthesis, maturation, and release. entry of the virus into the host cells is facilitated by interactions between the s protein and its receptors, ace , which are found in various organs such as heart, lungs, kidneys, and gastrointestinal tract. the s protein binds to ace through the receptor binding domain (rbd) region of the s subunit, which consists of a core and a receptor binding motif (rbm). rbm specifically recognises human ace as its receptor (yuki et al., ) . the s protein/ace interaction (attachment) is the primary determinant to infect a host species and also controls tissue tropism. ace mediates human-to-human transmission, and also acts as a receptor for sars-cov and respiratory coronavirus nl (astuti, ) . following binding of the virus to the host ace receptors, the s protein undergoes a two-step sequential proteolytic cleavage, one at s /s cleavage site for priming and another at s ˊsite for activation. the latter acts as a viral fusion peptide that inserts into the membrane, followed by the joining of two heptad repeats in s forming a six-helix bundle. the formation of this bundle results in fusion and entry of virus into the host cell (penetration) (tang et al., ) . another receptor, which has found to be of importance in viral invasion, is cluster of differentiation (cd ), also known as extracellular matrix metalloproteinase inducer (emmprin) or basigin . a characteristic unique to sars-cov- is the existence of a novel furin cleavage site (prrars) at s / s , which confers the ability to infect organs and tissues where furin is ubiquitously expressed such as the brain, lung, liver, gastrointestinal tract, and pancreas . other proteases that may play a role in virus entry are transmembrane protease serine (tmprss ) and cathepsin l. following internalization, there is uncoating and release of viral ssrna in the host cell cytoplasm, which then gets attached to the ribosomes and is translated into two large polyproteins, pp a and pp ab. these polyproteins are cleaved by virus-encoded proteinases into nsps. many of these non-structural proteins congregate to form the rtc in double-membrane vesicles (dmvs), which are mainly an assembly of rdrp-and helicase-containing subunits (astuti, ; . synthesis of genomic rna follows the translation and assembly of viral replicase complexes. rtc is responsible for rna replication and transcription of the sgrnas. the latter serve as mrnas for the translation of structural and accessory proteins (biosynthesis). following translation, the s, e, and m proteins are transported to the endoplasmic reticulum where they move along the secretory pathway into the endoplasmic reticulum-golgi intermediate compartment (ergic). in the compartment, the viral genomes are encapsidated by the n protein, which then bud into the membrane resulting in formation of the mature virus (maturation) (fehr and perlman, ) . the m protein regulates most of the protein-protein interactions required for virus assembly. however, virus-like particles (vlps) can only be formed when m protein is co-expressed with e protein, suggesting the role of these two proteins for production of viral envelope. following assembly, the virions are transported to the cell surface in vesicles and released by exocytosis (release) (fehr and perlman, ; astuti, ) . host immune response to sars-cov- the entry of virus into the host cells triggers stimulation of innate immune response via antigen presenting cells (apcs) like dendritic cells and macrophages, which represent the first line of defence against viruses. apcs have pattern recognition receptors (prrs), such as, toll-like receptors (tlrs), nod-like receptors (nlrs), rig-i-like receptors (rlrs), and melanoma differentiationassociated protein (mda ) present at various locations like plasma membrane, endosomal membrane, lysosomes, and cytosol . they recognize various structural components of the virus, such as, nucleic acids, carbohydrate moieties, glycoproteins, lipoproteins, and dsrna and induce a signaling cascade to produce the immune system effectors. the apcs present the viral antigenic peptides to the cd + t cells in association with major histocompatibility complex (mhc) class i. the cd + t cells get activated, undergo clonal expansion and develop into virus-specific effector and memory t cells. with their perforin and granzymes, cd + t cells lyse the virusinfected cells and induce apoptosis. in addition, there occurs an upregulation of natural killer (nk) cell activation and production of pro-inflammatory cytokines via the nuclear factor kappa b (nf-kb) and interferon regulatory factor (irf ) signaling pathways. this leads to further recruitment of neutrophils and monocytes to the site of infection and activation of several other pro-inflammatory cytokines (astuti, ; li et al., ) . during an infection, activation and priming of innate and adaptive immune responses result in pathogen clearance and recovery. however, sars-cov- causes suppression of host's innate immune response by inhibiting certain signaling pathways and thus, evades detection by the immune system, leading to a more severe disease and fatal outcomes (felsenstein et al., ) . it has been postulated that sars-cov- , like sars-cov, alters the ubiquitination and degradation of rna sensors (rig-i and mda ) and inhibits the activation of mitochondrial antiviral-signaling protein (mavs), thereby preventing the activation and nuclear translocation of irf in response to activated rna sensors. moreover, sars-cov inhibits tumor necrosis factor (tnf) receptor-associated factors (traf) and , which are crucial for the induction of irf- / in response to tlr / and nfkb signaling pathways. it also inhibits the phosphorylation of janus kinase/signal transducers and activators of transcription (jak/stat) transcription factor and blocks type i/iii interferon (ifn) signaling pathways (kindler et al., ) . these mechanisms allow the virus to replicate evading the innate antiviral responses and induce the production of cytokines required for recruitment of adaptive immune cells. the transition between innate and adaptive immune responses is critical for the clinical course of covid- . this phase determines whether the immune regulatory events will culminate in protective immunity or an exacerbated immune response. the protective immunity is t cell mediated, with cd + t cells eliminating the virus-infected cells and cd + t cells helping the b cells to produce neutralizing antibodies and orchestrating the response of other immune cells. the t cells account for % of the infiltrating cells in sars-cov- infection. however, a dysregulated t cell response can result in immunopathology leading to exaggerated cytokine release and a cytokine storm (cao, ; tay et al., ) . this condition is characterized by increased secretion of pro-inflammatory cytokines, such as interleukin (il)- b, il- , il- , il- , il- , il- , il- , and il- ; granulocyte-macrophage colony stimulating factor (gm-csf); tnf-a, ifn-g and ifn-g inducible protein (ip ); monocyte chemoattractant protein (mcp- ); macrophage inflammatory protein- alpha and - beta (mip- a and - b); chemokines like cc chemokine ligand (ccl ), ccl , and ccl ; and c-x-c motif chemokine ligand (cxcl ), cxcl , and cxcl . the cytokine storm induces a hyperinflammatory state causing acute lung injury and various complications like ards, respiratory failure, shock, disseminated intravascular coagulation, multiorgan failure and death (qin et al., ; xu z. et al., ) . this complex cascade of inflammatory response triggers platelet activation, endothelial dysfunction, and vascular stasis. recent studies suggest that covid- induces a hypercoagulable state that may predispose to venous and arterial thromboembolic events and worsened outcomes (abou-ismail et al., ) . the humoral immune response is critical for virus clearance and preventing reinfection. sars-cov- elicits a robust b cell response, as evidenced by detection of virus-specific neutralizing antibodies in most cases following infection. seroconversion occurs between and days after symptomonset, and antibody titers persist in the weeks following virus clearance (vabret et al., ) . the rbd of s protein is highly immunogenic, and antibodies against this domain can block virus interaction with the host ace receptors and thus, prevent virus entry (ju et al., ) . the subepithelial dendritic cells and macrophages recognize the viral proteins and present them to cd + t cells in association with mhc class ii, which induces differentiation of these t cells into th , th , and memory t follicular helper (t fh ) subsets. the t fh cells induce the conversion of b cells to plasma cells and promote the production of virus-specific igm, iga, and igg antibodies (cao, ) . like other viral infections, the initial antibody response in covid- is predominantly igm, which is transient and shortlived and soon gets replaced by igg antibodies. the latter has a longer half-life and lower molecular weight, which enable it to confer long-term protection and effective tissue penetration. however, different patterns of igm and igg seroconversion have been observed, such as synchronous seroconversion, igm seroconversion preceding that of igg, and igm seroconversion later than that of igg. secretory iga plays a crucial role in mucosal immunity by neutralizing the virus and preventing its attachment to the mucosal epithelium (long et al., ) . the proposed host immune response to sars-cov- has been shown in figure . currently, there are no clinically proven antiviral drugs or biologics for the treatment of covid- patients. a protocol issued by national health commission of the people's republic of china states that optimized symptomatic management, together with respiratory support should be the mainstay of treatment . most existing data on antiviral therapy for covid- are derived from related coronaviruses, such as, sars-cov ( ) , and mers-cov ( ) and noncoronaviruses such as ebola virus. how well these data can be extrapolated to sars-cov- remains unclear. moreover, a lack of pharmacokinetic/pharmacodynamic or clinical data comparing achievable exposures with treatment outcomes further questions the clinical relevance of in vitro activity of antiviral drugs, which may vary widely and therefore, should be compared cautiously. since the onset of this pandemic, several studies emphasizing the therapeutic benefits of a wide range of antiviral drugs and biologics have been published in medical literature. however, a thorough analysis of these drugs is warranted to ascertain whether the existing evidence supports the currently proposed management strategies. an overview of various repurposed and investigational drugs undergoing clinical trials against covid- has been depicted in figure (tu et al., ) . there are more than ongoing clinical trials evaluating the safety and efficacy of these drugs. the major proposed therapeutic candidates that seem promising for the treatment of covid- are summarized in table . remdesivir (veklury; gilead sciences, inc.) is an analog of adenosine triphosphate, which incorporates into the nascent viral rna chains and results in delayed chain termination during replication of viral rna. it has broad-spectrum antiviral activity against several rna viruses including ebola, marburg, mers-cov, sars-cov, respiratory syncytial virus (rsv), nipah virus, and hendra virus and has demonstrated prophylactic and therapeutic efficacy against coronaviruses (gordon et al., ) . use of remdesivir in sars-covinfected mice resulted in reduced viral loads and improved disease outcomes. recently, the drug has been shown to possess in vitro activity against sars-cov- . remdesivir seems to possess a favorable safety profile, as evidenced in participants, including healthy volunteers and patients who received remdesivir for ebola virus disease (mulangu et al., ) . its prophylactic and therapeutic efficacy was demonstrated in a rhesus macaque model of mers-cov infection, in which prophylactic administration of remdesivir hours prior to mers-cov inoculation completely prevented clinical disease, inhibited viral replication, and prevented the development of pulmonary lesions. therapeutic administration of the drug hours post-inoculation reduced the severity of clinical symptoms, attenuated viral replication, and decreased the pulmonary lesions (de wit et al., ) . gilead sciences, in a recent case series, considered compassionate-use of remdesivir in covid- patients with severe disease and reported that % of the cases showed clinical improvement after a median followup of days, with mortality of % and a favorable safety profile (grein et al., ) . the findings were, however, not compared with a control group that received only standard care. at present, there are six ongoing clinical trials evaluating the safety and efficacy of remdesivir in adult patients diagnosed with covid- (moderate/severe disease): two initiated by gilead sciences, one by national institute of allergy and infectious diseases (niaid), one by inserm (france), and two by china-japan friendship hospital. all these clinical trials are currently in phase iii. formal recommendations regarding the use of remdesivir can be made once these trials come up with some conclusive evidence. lopinavir/ritonavir (lpv/r; kaletra) is a combination of protease inhibitors used for the treatment of hiv infection. ritonavir is also a potent inhibitor of cytochrome p , a class of enzymes responsible for metabolism of lopinavir, and the co-administration augments the plasma levels of lopinavir, improving its antiviral activity (molla et al., ) . lpv/r has demonstrated in-vitro antiviral activity against sars-cov and mers-cov. since this combination was not specifically formulated for treatment of coronavirus infections, this alone may not demonstrate a significant advantage over placebo in reducing viral load (yao et al., ) . a clinical trial involving patients with laboratory-confirmed sars-cov- infection reported that lpv/r combination did not offer any clinical benefit over the standard management . there are several ongoing clinical trials comparing the efficacy of lpv/r alone and in combination with other drugs like umifenovir, carrimycin, danoprevir/ritonavir, interferon, xiyanping, and traditional chinese medicines. lpv/r in combination with ifn-b b reduced mers-cov viral load and improved lung pathology in a marmoset model (yao et al., ) . however, sheahan et al. ( ) reported that combining lpv/r with ifn-b did not significantly augment the antiviral activity of the latter against mers-cov. in an open label clinical trial involving hospitalized sars patients, lpv/r in combination with ribavirin was found to decrease the mortality rate and requirement of ventilator support compared to the control group (median, days versus days; % ci, − - ) . thus, considering the therapeutic benefits in the treatment of sars and mers, the safety and efficacy of lpv/r based combination regimen in the treatment of covid- needs to be evaluated. figure | host response to sars-cov- . the virus attaches to ace receptors and enters the target cell by membrane fusion. upon entry, the virus is recognized by innate immune receptors tlr / , cytosolic rna sensors rig-i/mda- , and the inflammasome sensor nlr family pyrin domain-containing- (nlrp ). this leads to the activation of nf-кb and irf / and the subsequent production of pro-inflammatory cytokines (e.g., il- b, il- , and tnf-a) and type i ifns, respectively. cytokines released by infected cells modulate the adaptive immune response by causing recruitment and activation of macrophages, b cells, and t cells which facilitate elimination of the virus. however, an unbalanced immune response can cause massive release of pro-inflammatory cytokines, leading to a cytokine storm which is responsible for the severe clinical manifestations of covid- . umifenovir (arbidol, pharmstandard ltd.) is a fusion inhibitor that interacts with viral hemagglutinin and prevents the fusion of viral envelope with host cell membrane. the drug is currently licensed for use only in russia and china for the treatment and prophylaxis of influenza and other respiratory viral infections. umifenovir has a broad-spectrum antiviral activity due to its dual action as direct-acting antiviral and host-targeting agent. it has been found to be active against several enveloped and nonenveloped rna and dna viruses, including chikungunya virus, zika virus, foot-and-mouth disease virus, lassa virus, ebola virus, hsv, hbv, hcv, chikungunya virus, reovirus, hantaan virus, and coxsackie virus b (blaising et al., ; kadam and wilson, ) . it also inhibits clathrin-mediated exocytosis and intracellular trafficking by interacting with the cell membrane (blaising et al., ) . considering its unique mechanism of action, umifenovir alone and in combination with antiretroviral drugs is currently being investigated for treatment and prophylaxis of covid- . however, a retrospective study by lian et al., patients showed that umifenovir did not shorten the sars-cov- negativity time or improve the prognosis in non-icu patients compared to the supportive treatment . there are currently four ongoing clinical trials of umifenovir for covid- treatment: one in comparison with the basic treatment , and the other three comparing the effects in combination with oseltamivir , lopinavir/ ritonavir , and carrimycin. , ) . besides influenza a and b, it has been found to be effective against avian influenza. it has also been investigated for the treatment of infections caused by ebola virus, lassa virus, and now, sars-cov- . favipiravir is a prodrug that gets metabolized to an active form favipiravirribofuranosyl- ′-triphosphate (favipiravir-rtp), which selectively binds to rdrp and inhibits viral replication. in contrast to the existing antivirals against influenza that primarily block the entry and exit of the virus from cells, favipiravir's novel mechanism of action allows its active form to get incorporated into the nascent rna strand, thus preventing strand elongation and viral proliferation. the drug has an oral bioavailability of . and is % plasma protein-bound with an elimination half-life of - hours . the rdrp gene of sars-cov- is structurally similar to that of sars-cov and mers-cov, as revealed by genome sequencing (cascella et al., ) . a clinical trial (chictr ) conducted in shenzhen, china reported that covid- patients who received favipiravir demonstrated significantly shorter viral clearance time and higher improvement in chest imaging, compared to the control group ( days, . % versus days, %) (cai et al., ) . in another multi-centre randomized trial (chictr ), treatment with favipiravir was found to be beneficial for covid- patients with diabetes and/or hypertension as evidenced by decreased time-to-relief for fever and cough. also, seven days clinical recovery rate increased from . to . % . these studies indicate that favipiravir can be a safe and effective treatment option for covid- . the drug is currently undergoing phase iii clinical trial, which is expected to be completed by july . ifns are a family of inducible cytokines produced by various cell types in response to viral infections. ifns exert their actions through pattern recognition receptors (prrs), which are largely species specific. of particular interest are the type ifns (viral ifns), which are secreted by the plasmacytoid dendritic cells and are among the first cytokines produced during a viral infection. ifn-i comprises of several subtypes (a, b, ϵ, w, and k) (samuel, ) , which exert their actions after binding with interferon-a/b receptor (ifnar). ligand binding induces phosphorylation of the receptor and activation of signal transducers and several transcriptional factors such as stat and stat . these form complexes that are translocated to the nucleus, where they activate interferon-stimulated genes (isg). isgs include prrs, irfs, and members of the jak-stat signaling pathway, which sensitize the cell to pathogens, and play a prominent role in inflammation, antiviral innate signaling, immunomodulation, and interfere with several steps of viral replication (schneider et al., ) . thus, ifn-i plays a vital role in antiviral immunity. because of their immunomodulatory and antiviral properties, they are often evaluated for the treatment of several emerging viral infections. sars-cov- bears a close resemblance with other members of the coronaviridae family such as mers-cov and sars-cov and exhibits similar properties, despite differences in their epidemiology, pathology, and several of their structural proteins. numerous in vivo and in vitro studies have evaluated the role of ifn-i in the treatment of mers-cov and sars-cov, either alone or in combination with lopinavir/ritonavir (chan et al., ) , ribavirin (omrani et al., ) , remdesivir, corticosteroids, and ifn-g (sainz et al., ) . though both ifn-a and-b have demonstrated efficacy in vitro and succeeded in certain animal models, they failed to improve the disease in humans. such difference in therapeutic responses could be attributed to ifn signaling pathway used by the viruses, limited number of study subjects, varied experimental settings or clinical conditions, and ifn-subtype diversity. studies have shown that ifnb, particularly the b subtype (ifnb b or ifnb a), is a more potent inhibitor of coronaviruses than ifna and thus appears to be more relevant in the treatment coronavirus infections (stockman et al., ) . in the lungs, ifnb stimulates the secretion of anti-inflammatory adenosine and promotes maintenance of endothelial barrier function by up-regulating cd in pulmonary endothelial cells. this can be a possible explanation to the reduction of vascular leakage in ards with ifnb a treatment (bellingan et al., ) . the timing of ifn-i administration plays a critical role, with positive effects being observed early in the course of infection while delayed administration failed to inhibit viral replication (channappanavar et al., ) . based on previous knowledge, it has been hypothesized that sars-cov and mers-cov are able to disrupt the interferon signaling pathway probably through involvement of orf and orf b (kopecky-bromberg et al., ) . however, due to the truncated nature of orf and orf b proteins in sars-cov- , they may have lost their antiinterferon activities. this could be a possible explanation for sars-cov- displaying substantial in vitro sensitivity to ifn-i. thus, ifn-i is expected to be more promising for the treatment of covid- than for sars (lokugamage et al., ) . the assumption is further supported by the fact that ifna b sprays minimise the infection rate of sars-cov- and can be used prophylactically against the virus administration of million units of ifna by vapor inhalation twice a day, in combination with ribavirin (dong et al., ) . vapor inhalation offers the advantage of specifically targeting the respiratory tract. the efficacy of ifn-i can be further improved if given in combination with lopinavir/ritonavir, ribavirin, or remdesivir because of the efficacy of such combinations observed in vitro against other coronaviruses (sheahan et al., ) . further research on ifn-based treatment is expected in near future, which should give more accurate information on the efficacy of this therapy and possible outcomes. ivermectin (stromectol; merck & co., inc.) is a broad spectrum anthelmintic agent belonging to class of avermectins and is derived from the soil bacterium streptomyces avermitilis. it's selective and high affinity binding with glutamate-gated chloride channels in nerve and muscle cells of nematode, increases the permeability of the cell membrane to chloride ions, resulting in hyperpolarization of cells and paralysis and death of the parasite. it is . % plasma protein-bound and has a half-life of hours following oral administration. the drug was originally launched by merck laboratories in for use against onchocerciasis (river blindness) as a part of the onchocerciasis control programme in west africa. subsequently, the drug was approved for the treatment of a number of human parasitic infections including strongyloidiasis, ascariasis, trichuriasis, enterobiasis, lymphatic filariasis, and scabies in several countries (australia, france, japan, the netherlands, usa, etc) (ikeda, ) . besides its anti-parasitic action, several studies have demonstrated the potent antiviral activity of ivermectin against a broad range of viruses in vitro (caly et al., ) . it has been shown to inhibit the interaction between the hiv- integrase protein (in) and the importin (imp) a/b heterodimer, causing inhibition of hiv- replication (wagstaff, ) . ivermectin has also been reported to limit infections caused by several rna viruses (dengue viruses - , west nile virus, venezuelan equine encephalitis virus, and influenza virus) and dna virus (pseudorabies virus) (wagstaff, ; caly et al., ) . studies have found that host cell division might be affected during sars-cov infection, due to a signal-dependent nucleocytoplasmic shutting of the viral nucleocapsid protein involving impa/b (timani, ; wulan, ) . the antiviral activity of the stat transcription factor is blocked by sars-cov accessory protein orf , which causes sequestration of impa/b on the rough endoplasmic reticulum/golgi membrane (frieman, ) . considering ivermectin's inhibitory action on impa/b -mediated nuclear import, it is presumed to be effective against sars-cov- . caly et al. ( ) studied the antiviral activity of ivermectin against sars-cov- and observed that a single treatment with ivermectin was able to cause ∼ -fold reduction of virus titre at h in vero/hslam cell culture. ivermectin has a favorable safety profile in humans with high dose therapy considered as safe as the standard low-dose regimen. however, the therapeutic benefits from multiple drug dosing need to be evaluated in covid- patients. an effective antiviral drug given early in the course of infection can help reduce the viral load and prevent disease progression while limiting person-person transmission. ivermectin's unique antiviral action combined with a favorable safety profile allows it for further consideration as a possible treatment option in covid- . hydroxychloroquine (hcq) (plaquenil; sanofi-synthelabo inc.) is an aminoquinoline like chloroquine and is indicated for the treatment of uncomplicated malaria, prophylaxis of malaria in places without chloroquine resistance, chronic discoid lupus erythematosus, systemic lupus erythematosus, and rheumatoid arthritis. in addition, hcq has been found to be effective against intracellular bacteria such as coxiella burnetii (raoult et al., ) and tropheryma whipplei (boulos et al., ) . hcq has also been shown to possess antiviral properties and is already being used in clinical trials for the treatment of hiv infection. it increases endosomal ph which prevents viral fusion and entry into the host cells, inhibits antigen processing and presentation, blocks dimerization of major histocompatibility complex (mhc) class ii, and reduces host inflammatory response by decreasing the release of cytokines like il- and tnf-a. hcq inhibits terminal glycosylation of ace receptor, the main portal of entry for sars-cov and sars-cov- . non-glycosylated ace interacts less efficiently with the viral spike protein, thus preventing viral entry (colson et al., ) . several studies have proposed that repurposing of approved drugs such as chloroquine, hcq, azithromycin, metformin, losartan, and simvastatin could be useful in the treatment of covid- . clinical trials from china have shown the efficacy of chloroquine in the treatment of covid- patients, as evidenced by subsidence of fever, improvement of radiological findings, and delay in disease progression. azithromycin (az) is a macrolide antibiotic that has demonstrated in vitro activity against zika and ebola viruses (bosseboeuf et al., ) . several authors have mentioned a synergistic effect of hcq/az combination in the treatment of covid- . an open label non-randomized clinical trial from france showed that covid- patients treated with mg of hcq daily had a significant reduction in viral carriage at day post-inclusion, with % of the patients having a negative pcr test result compared to only . % in the untreated control group. moreover, patients who were treated with a combination of hcq and az ( mg on day , followed by mg daily for the next four days) showed complete virological cure at day postinclusion compared to . % in the group that received hcq alone (gautret et al., a) . another study from france claimed that patients who received a combination of hcq and az had a significant clinical improvement as evidenced by a rapid fall in viral load, with % tested negative by quantitative pcr on day and % on day . virus cultures of respiratory samples were negative in . % patients on day (gautret et al., b) . however, the apparent beneficial effects of hcq in the treatment of covid- have been completely negated by a pilot study from china, where no significant differences in outcomes were observed between hcq-treated group and the control group . a large observational study in hospitalized covid- patients in the us also showed that treatment with hcq was not associated with significant clinical benefits and has no influence on intubation or death (geleris et al., ) . furthermore, the use of hcq alone or in combination with az is not free from hazards. both these drugs are associated with an increased risk of qt c prolongation, torsades de pointes, ventricular tachycardias, and gastrointestinal side effects. it has been observed that patients receiving a five-day course of az had an increased risk of sudden cardiac death with a hazard ratio of . (ray et al., ) . considering the cumulative adverse effects of hcq and az on cardiac conduction, it is advised to have baseline and follow-up ecg monitoring, along with careful consideration for other concomitant medications known to prolong the qt c interval, if this combination has to be used. guidelines published by the infectious disease society of america mentioned that despite a higher proportion of clinical improvement in the hcq group, the beneficial effect of hcq on viral clearance or disease progression cannot be judged by the currently available evidence due to certain drawbacks such as small sample sizes, ill-defined patient selection criteria, cointerventions, and methodological limitations (bhimraj et al., ) . moreover, none of the studies have addressed patientrelevant outcomes like mortality, rate of disease progression to ards, and need for mechanical ventilation. also, the mortality rate among patients receiving hcq/az combination was not compared with an untreated cohort. though studies have claimed that patients receiving hcq and az experienced less virologic failure ( % pooled virologic failure) as compared to historical controls ( % virologic failure) (gautret et al., b; molina et al., ) , such comparison lacks certainty because of unmeasured confounding and selection bias. furthermore, these studies have relied mainly on intermediary outcomes such as reduction in development of pneumonia, and less hospital or icu admission to ascertain therapeutic benefits, which raise question on their precision and feasibility. therefore, a rct should be the ideal approach for determining the therapeutic effects of hcq in covid- patients. the leading cause of mortality in covid- is respiratory failure from ards. a cytokine profile resembling secondary hemophagocytic lymphohistiocytosis (hlh), characterized by a fulminant and fatal hypercytokinemia with multiorgan failure is associated with covid- . there is a massive and uncontrolled release of pro-inflammatory cytokines like il- , il- , g-csf, ip , mcp- , mip- -a and tnf-a (mehta et al., ; xu z. et al., ) . a recent retrospective study involving confirmed covid- cases from wuhan, china, revealed that elevated levels of serum ferritin and il- were independent predictors of fatality, probably due to virally driven hyperinflammation (ruan et al., ) . tocilizumab (actemra, roche) is a humanized monoclonal antibody against the interleukin- receptor (il- r) approved for the treatment of seriously ill covid- patients with elevated il- by the national health commission of china. xu x. l. et al. ( ) observed the effects of tocilizumab in covid- patients with severe disease, in addition to routine therapy, and reported significant therapeutic benefits as evidenced by subsidence of fever and other symptoms within a few days and improvement of oxygen saturation in % of patients. there were no obvious treatment-related adverse reactions. in another report from china, a case of covid- with pre-existing multiple myeloma was successfully treated with tocilizumab, highlighting its potential therapeutic benefits in the treatment of covid- patients . on march , , the drug entered phase iii clinical trial for the treatment of covid- pneumonia. the main contributory factors for increased mortality in covid- patients are acute lung injury (ali) and ards, brought about by a cytokine-mediated hyperinflammatory response. pulmonary edema is the key detrimental feature of ali/ards. covid- is associated with more exaggerated pulmonary mucus exudation than sars as revealed by autopsy . pulmonary imaging and histopathological examination also support similar findings. however, specific pharmacotherapy to combat this pathology is lacking. vascular endothelial growth factor (vegf) is one of the most potent inducers of increased vascular permeability in covid- affected lungs, causing fluid extravasation and pulmonary edema. expression of vegf is induced by hypoxia through activation of prolyl hydrolases (phd)-hypoxia inducible factor (hif)- pathway, which upregulates transcription of vegf. therefore, blockade of vegf signaling pathway might help in reducing inflammation and improving tissue perfusion in patients with severe covid- . bevacizumab (avastin; genentech ltd.) is a recombinant humanized monoclonal antibody targeted against vegf and is currently recommended for the treatment of malignancies (colorectal, lung, breast, renal, brain, and ovarian), age-related macular degeneration, and diabetic retinopathy. it acts by reducing the elevated vegf levels secondary to hypoxia and severe inflammation, thereby improving tissue perfusion. (wang et al., ) . this might help in subsidence of pulmonary edema in covid- patients. qilu hospital of shandong university, china is conducting two clinical trials of bevacizumab, both of which are expected to be over by may . thus, bevacizumab holds promise as a potential therapeutic option in the treatment of severe covid- patients. studies till date recognize angiotensin converting enzyme (ace ) as the major entry portal for sars-cov- . however, a novel route of viral invasion through direct interaction between the sars-cov- spike protein and cd , also known as emmprin, expressed on epithelial cells has been recently described by wang k. et al. ( ) meplazumab (ketantin, jiangsu pacific meinuoke biopharmaceutical co. ltd.) is a humanized igg monoclonal antibody against cd that has demonstrated dose-dependent inhibitory action on sars-cov- replication and virus-induced cytopathic effect in vitro (bian et al., ) . cd binds to cyclophilin a (cypa), a proinflammatory cytokine up-regulated in viral infection, and regulates cytokine secretion and leukocyte chemotaxis. meplazumab is a monoclonal anti-cd antibody that inhibits cypa-induced t cell chemotaxis and thus reduces local inflammation. bian et al. ( ) studied the effects of meplazumab in hospitalized patients with covid- at tangdu hospital, china, and reported that meplazumab treatment significantly improved the clinical outcomes in severely ill patients. also, the time to virus negativity in the meplazumab group was shortened compared to the control group. these evidences suggest that meplazumab therapy improves the recovery of patients with sars-cov- pneumonia and has a favorable safety profile. the drug is currently in phase ii clinical trial, which is expected to be completed by december . itolizumab (alzumab, biocon ltd.) is a humanized anti-cd igg monoclonal antibody that was introduced in india in for the treatment of chronic plaque psoriasis. it binds specifically to domain of cd and modulates the activation and proliferation of t cells by cd co-stimulation, without interfering with the interaction between cd and activated leukocyte-cell adhesion molecule. it inhibits intracellular phosphoproteins like mitogen-activated protein kinase (mapk) and stat and interferes with cd mediated intracellular signaling pathways and th development. itolizumab downregulates the transcription of pro-inflammatory cytokine genes and thus leads to decreased levels of ifn-g, il- , and tnf-a, causing attenuation of cytokine storm and t cell infiltration (menon and david, ) . considering its unique mechanism of action, the drug has been repurposed for the treatment of crs, which is the leading cause of death in covid- . a prospective, multi-centric, randomized phase ii study conducted on severely ill covid- patients ( cases and controls) in india showed significant improvement in blood oxygen levels with reduced levels of proinflammatory cytokines and reduced mortality rate in patients who received itolizumab. a similar trial conducted in cuba also indicated positive results with . % of the patients discharged from icu after weeks of treatment. itolizumab has been approved by drugs controller general of india for the treatment of crs in moderate to severe ards patients with covid- . anakinra (kineret; amgen inc.) is a recombinant human il- receptor antagonist that competitively inhibits the binding of il- a and il- b to the high-affinity il- receptor. it is the first biological agent approved for the treatment of rheumatoid arthritis. it is administered through subcutaneous route and has an absolute bioavailability of % (cvetkovic and keating, ) . in covid- patients, halting the disease progression from manageable hypoxia to frank respiratory failure and ards can have a significant impact on patient management and outcomes. therefore, a therapy directed at intercepting the cytokine storm may be beneficial in this regard. there is an ongoing prospective, randomized, interventional trial comparing the therapeutic effects of individual and simultaneous blockade of il- and il- versus standard care in covid- patients. the trial will include participants whose clinical status after days of treatment will be assessed to measure the effectiveness of anakinra alone and in combination with tocilizumab and siltuximab in restoring lung homeostasis. the study is estimated to be completed in december . considering the role of il- in the pathogenesis of acute lung injury in covid- , anakinra seems to be a promising therapeutic option in the management of such patients. several studies have recognized the potential benefits of cellbased therapies in a number of disease processes including pulmonary, cardiovascular, hepatic, renal, metabolic, and mulculoskeletal disorders. a guideline published by the italian college of anesthesia, analgesia, resuscitation and intensive care has mentioned that stem cells have the potential to decrease icu admission and curtail the number of icu days in covid- (vergano et al., ) . currently, usfda recommends autologous bone marrow stem cells as the only candidate for stem cell therapy. mesenchymal stem cells (mscs) have shown benefit in the treatment of musculoskeletal disorders such as lowback pain and spinal injuries. the other stem cells that can be considered for clinical use include adipose, amniotic, and umbilical cord stem cells. among these, umbilical cord stem cells seem to be the more attractive as unlike bone marrow, umbilical cord (wharton jelly) has a high concentration of mscs that can be extracted noninvasively (arutyunyan et al., ) . moreover, they have fast doubling times, more plasticity, greater potency, and can be efficiently be expanded in the laboratory to cater the large number of expected coronavirus patients (nagamura-inoue and he, ). despite being allogenic, mscs can evade the host immune system as they express low levels of mhc i, mhc ii, and t cell co-stimulatory molecules, cd and cd , on their surface. at a cellular level, mscs demonstrate powerful immunomodulatory activity through secretion of anti-inflammatory molecules by paracrine effect and direct interaction with t and b lymphocytes, dendritic cells, macrophages, and nk cells. all these may help in attenuating the cytokine storm (tipnis et al., ) . they suppress the hyperactive immune system and promote endogenous repair by improving the cellular microenvironment. multiple studies have demonstrated the beneficial effects of mscs in the settings of ali and ards. when given intravenously, mscs accumulate in the lungs and improve lung function by decreasing inflammation, reducing pulmonary endothelial permeability, facilitating alveolar fluid transport, preventing pulmonary fibrosis, and promoting tissue repair. several clinical trials have documented the safety and efficacy of mscs in immune-mediated inflammatory diseases, such as graft versushost disease (gvhd) and autoimmune disorders (li et al., ; atluri et al., ; behnke et al., ) . mscs secrete antimicrobial peptides and proteins (amps) such as cathelicidin ll- , human beta-defensin- (hbd- ), hepcidin, and lipocalin- (lcn ) and anti-inflammatory molecules such as indoleamine , dioxygenase (ido) and interleukin (il)- . amps cause disruption of membrane integrity, inhibition of protein and nucleic acid synthesis, and blockade of interaction with intracellular targets (alcayaga-miranda et al., ) . mscs regulate the host immune response by maintaining a dynamic equilibrium between pro-and anti-inflammatory cytokines. there was a concern that sars-cov- can infect the stem cells and render them ineffective. however, a study of seven covid- patients (one critically ill, four serious and two mild) in beijing revealed that sars-cov- was not able to infect the injected umbilical cord mscs. all patients who received single dose of stem cell therapy recovered during the days follow-up period, while two out of three patients (with serious disease) who did not receive stem cell therapy (control group) had unfavorable outcomes (one died and one developed ards). there was gradual normalization of oxygen saturation and levels of inflammatory biomarkers like crp, aspartic aminotransferase, creatine kinase, and myoglobin in the treated group with no treatment-related adverse events. follow-up ct scan of lungs showed significant radiological improvement (leng et al., ) . thus, mscs can be a safe and effective treatment option for patients with covid- pneumonia. natural killer (nk) cells (large granular lymphocytes) are innate lymphocyte subsets that constitute the frontline defence system against virus infected and tumor cells. they originate in the bone marrow and represent up to % of peripheral blood mononuclear cells. nk cells are phenotypically defined by expression of cd and absence of cd and do not require prior stimulation to perform their effector functions. nk cells display a diverse range of biological activities that are controlled by several inhibitory and activating receptors. the inhibitory receptors recognize self-mhc class i and prevent nk cell activation. in viral infections, there is upregulation of activating receptors and downregulation of mhc class i expression, which causes activation of nk cells. the major activating receptors include cytotoxicity receptors (nkp and nkp ), c-type lectin receptors, and immunoglobulinlike receptors. among the inhibitory receptors, the killer immunoglobulin-like receptors and leukocyte inhibitory receptors have prominent role in defence against viral infections. nk cells lack antigen-specific receptors and kill virus-infected cells through the production of cytokines (tnf-a, gm-csf, ccl /rantes, and ifn-g), perforin-granzyme-mediated cellular destruction, and death receptor-mediated cytolysis (cooper et al., ) . perforin, a pore forming protein, increases the cell permeability, which allows granzymes, a family of serine proteases, to enter into the cell and disrupt cell cycle progression, inflict dna damage, and promote karyolysis (vivier et al., ) . they also cause recruitment and activation of other effector cells, including cd + t cells and cd + th cells. patients with deficient nk cell response are predisposed to recurrent viral infections (jost and altfeld, ) . currently, the role of nk cells for immunotherapy in infectious diseases is being explored and results seem to be promising. as hunt for new therapeutic options in the treatment of covid- continue to expand, focus has been on the potential benefits of nk cell-based therapy. on april , , usfda approved the use of cynk- , the only cryo-preserved allogeneic nk cell therapy, derived from placental hematopoietic stem cells, in adults with covid- . the agent's manufacturer celularity, a new jersey-based therapeutic company, in collaboration with sorrento therapeutics is about to launch a phase i/ii clinical trial on cynk- , involving covid- patients. the therapy is already being tested in patients with acute myeloid leukemia, multiple myeloma, and various solid tumors. in january , celularity's cynk- was approved by usfda for treatment of glioblastoma multiforme. thus, considering the potent antiviral and immunomodulatory properties of nk cells, their efficacy in the treatment of covid- seems promising and needs to be evaluated in clinical trials. convalescent plasma therapy (cpt) is a passive immunization strategy that has been used for the prevention and treatment of several infectious diseases for more than a century. cpt has been successfully used in the treatment of sars (cheng et al., ) , mers (ko et al., ) , and influenza a h n (hung et al., ) , with satisfactory efficacy and safety profile. a protocol for the use of convalescent plasma (cp) in the treatment of mers was established in . cpt is associated with a significant reduction in viral load and pooled mortality as revealed in a large meta-analysis on sars and severe influenza (mair-jenkins et al., ) . patients with a high titer of nab, after having recovered from covid- may be a valuable donor for cp. it has been observed that the nabs titers in covid- patients remain low for the first days following disease-onset and tends to increase thereafter, reaching a peak in to days after the onset . usfda has laid down eligibility criteria for covid- cp donors which include: i) evidence of confirmed covid- documented by a positive nasopharyngeal pcr at the time of illness or a positive sars-cov- antibody test after recovery, ii) complete resolution of symptoms at least days prior to donation or at least days prior to donation and negative results for covid- , either from a nasopharyngeal swab specimen or by a molecular diagnostic test from blood, iii) male/female donors tested negative for hla antibodies, and iv) sars-cov- neutralizing antibody titers of ≥ : . in a study from china, cpt supplemented with supportive care and antiviral agents was associated with significant clinical and radiological improvement with a rise in neutralizing antibody titers and a fall in c-reactive protein levels within days of initiation of treatment. no treatment-related adverse effects were observed (duan et al., ) . similar findings were reported by . a systematic review on cpt for the treatment of covid- revealed that cpt is safe, effective, and reduces mortality in critically ill patients (rajendran et al., ) . a clinical trial evaluating the benefits of cp in the treatment of covid- is being conducted by universidad del rosario, colombia (nct ), the results of which are expected to be declared by december . cytosorb (cytosorbents corp.) is an extracorporeal cytokine adsorber that acts by removing the circulating cytokines and redirecting the activated neutrophils to the site of infection. this may help in ameliorating cytokine storm that can otherwise trigger uncontrolled systemic inflammatory response, organ failure, and death. cytosorb offers significant survival benefits in septic shock as observed in several studies. it has been safely used in over , cases worldwide, primarily in the treatment of several immune-mediated life-threatening conditions such as septic shock, influenza, ards, secondary hlh, liver failure, and pancreatitis. cytosorb helps in protecting endothelial tight junctions, thus reducing capillary leak syndrome. it also modulates pulmonary metabolism, edema formation, and cellmediated infiltration and injury to the lungs. on april , , the usfda approved emergency use of cytosorb for the treatment of adult covid- patients admitted to icu with features of respiratory failure. sars-cov- can induce a sepsis-like syndrome, and in such cases, since pharmacological approaches fail to give promising results, removal of proinflammatory cytokines by hemoadsorption through cytosorb should be considered. to date, more than critically ill patients with covid- infection have been treated with cytosorb across various centers in italy, china, and germany. based on positive results in italy, the brescia renal covid task force has formally recommended the use of cytosorb in severe covid- patients with stage acute kidney injury, receiving continuous renal replacement therapy (crrt). cytosorb therapy has also been recommended by the national guidelines for the care of adult patients covid- , panama. in addition, the handbook of covid- prevention and treatment, issued by zhejiang university school of medicine, china is also recommending cytosorb therapy for the management of cytokine storm in critically ill covid- patients. currently, an ongoing clinical trial (nct ) is investigating the efficacy of cytosorb in the treatment of patients with severe covid- disease. it is expected to be completed by november . formulating appropriate treatment strategies for covid- poses a considerable challenge. during pandemics, in absence of clinically proven treatment guidelines, the tendency is to repurpose drugs based on their antiviral and immunomodulatory activities, as evidenced through observational studies. however, such studies have certain drawbacks like lack of concurrent controls, ill-defined patient selection criteria, small sample size without randomization, and use of intermediary outcomes like viral clearance rather than patient-relevant outcomes. though several repurposed drugs have shown promising results, and their potential clinical benefits appear to outweigh the relatively minor risk of adverse events, conclusive evidence is lacking. there is a need to clearly define the patient populations who warrant therapy and the timing of initiation of treatment. since viral loads are highest early in the course of infection and the disease progression can occur rapidly in stable patients, it is rational to consider rapid initiation of therapy in highrisk populations (old age, hospitalized patients, those with underlying diseases and comorbidities), ideally in the context of a well-controlled, randomized clinical trial. moreover, the demand for unproven therapies can cause shortages of medications that are otherwise indicated for more prevalent diseases like hiv, malaria, hypertension, and diabetes mellitus. the idsa guidelines for treatment of patients with covid- raise concern upon these aspects. in an attempt to generate and disseminate clinical data on an urgent basis, a phenomenal increase in fast-track publications related to covid- has been observed. however, caution should be exercised because the bulk of the available clinical data are often uncontrolled, not peer reviewed, and subject to publication bias (with an intention to publish outstanding results, there may be a tendency to publish positive outcomes and disregard the negative findings). there are several ongoing clinical trials, some with versatile designs that can reasonably explain the therapeutic benefits offered by these drugs in the management of covid- . given the plethora of uncertainties concerning the reliability of existing data and the safety and efficacy of the proposed treatments, it would be wise to wait for the results of clinical trials than to adopt clinically unproven therapies. this article has been released as a 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findings of covid- associated with acute respiratory distress syndrome effective treatment of severe covid- patients with tocilizumab a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease- treatment option covid- pathophysiology: a review first case of covid- in a patient with multiple myeloma successfully treated with tocilizumab sars-cov- : an emerging coronavirus that causes a global threat coronaviruses-drug discovery and therapeutic options the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © samaddar, grover and nag. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -fzgxuak authors: penman, sophie l.; kiy, robyn t.; jensen, rebecca l.; beoku‐betts, christopher; alfirevic, ana; back, david; khoo, saye h.; owen, andrew; pirmohamed, munir; park, b. kevin; meng, xiaoli; goldring, christopher e.; chadwick, amy e. title: safety perspectives on presently considered drugs for the treatment of covid‐ date: - - journal: br j pharmacol doi: . /bph. sha: doc_id: cord_uid: fzgxuak intense effort is underway to evaluate potential therapeutic agents for the treatment of covid‐ . in order to respond quickly to the crisis, the repurposing of existing drugs is the primary pharmacological strategy. despite the urgent clinical need for these therapies, it is imperative to consider potential safety issues. this is important due to the harm‐benefit ratios that may be encountered when treating covid‐ , which can depend on the stage of the disease, when therapy is administered and underlying clinical factors in individual patients. treatments are currently being trialled for a range of scenarios from prophylaxis (where benefit must greatly exceed risk) to severe life‐threatening disease (where a degree of potential risk may be tolerated if it is exceeded by the potential benefit). in this perspective, we have reviewed some of the most widely‐researched repurposed agents in order to identify potential safety considerations using existing information in the context of covid‐ . taleb-gassabi, & dayer, ) . a standard dose of lopinavir-ritonavir is mg/ mg twice a day for hiv- treatment, and this has also been used for sars-cov- treatment . the most frequent reported aes for lopinavir-ritonavir treatment are gastrointestinal disturbances including diarrhoea, nausea and vomiting (chandwani & shuter, ) . dose-related diarrhoea have been reported in up to % of patients and are thought to occur through a number of mechanisms including decreased proliferation of intestinal epithelial cells, disruption of intestinal barrier function, inducing endoplasmic reticulum stress and activating the unfolded protein response (x. wu, li, peng, & zhou, ) . diarrhoea is also a symptom in some covid- patients and so lopinavir-ritonavir has the potential to exacerbate this. pancreatitis has been reported in a small number of patients following lopinavir-ritonavir treatment although this was more frequent in those with a pre-existing history of pancreatitis (chandwani & shuter, ; oldfield & plosker, ) . additionally, patients with underlying liver diseases should have regular monitoring of hepatic function (palacios et al., ) . caution should be exerted for those patients taking concomitant medication as lopinavir-ritonavir inhibits p-glycoprotein (p-gp) and cytochrome p (cyp) - a , which therefore may alter the pk of other compounds (l. zhang, zhang, & huang, ) . a covid- drug interaction website has been developed by the liverpool drug interaction group which details ddis with lopinavir-ritonavir and a number of drugs, which in some cases can lead to potentially serious and/or life-threatening reactions (group, ; abbvie inc., ) . since the sars-cov- outbreak, clinical trials have been registered (up to th july ) to test lopinavir-ritonavir as a potential treatment for sars-cov- with variable outcomes in terms of efficacy. in one trial of patients with confirmed sars-cov- , patients on the lopinavir-ritonavir arm were withdrawn due to aes . in a different trial, patients who were administered lopinavir-ritonavir ( mg/ mg) also experienced gastrointestinal aes (y. li et al., ) . chloroquine and its derivative, hydroxychloroquine, are widely used as inexpensive and safe antimalarial drugs. in particular, the established good tolerability of chloroquine/hydroxychloroquine has made them safe to use even in pregnancy (villegas et al., ) . in addition to anti-malarial activity, both drugs have immunomodulating effects and are used for the treatment of autoimmune diseases including systemic and discoid lupus erythematosus, psoriatic arthritis, and rheumatoid arthritis. chloroquine/hydroxychloroquine concentrate extensively in acidic vesicles including the endosomes, golgi vesicles, and the lysosomes (ohkuma & poole, ) . this leads to lysosomal membrane permeabilisation or dysfunction of several enzymes including acid hydrolases and palmitoyl-protein thioesterase (rebecca et al., ; savarino, boelaert, cassone, majori, & cauda, ; schrezenmeier & dorner, ) . although the precise mechanisms of the anti-viral effects are not fully understood, it has been proposed that chloroquine/hydroxychloroquine can prevent virus infection (pre-infection) by interfering with the glycosylation of cellular receptors and impair viral replication by increasing endosomal ph (post-infection) (savarino et al., ; savarino et al., ; vincent et al., ) . owing to their efficacy against viruses (mostly demonstrated in vitro) including influenza, hiv, coronavirus oc , and sars-cov, a large number of clinical trials (> ) have been registered worldwide using chloroquine/hydroxychloroquine alone, or in combination with other drugs (e.g. azithromycin) for the treatment of covid- . despite promising in vitro antiviral results for hydroxychloroquine/chloroquine, there is no convincing evidence of efficacy at present (gao, tian, & yang, ; gautret, lagier, parola, hoang, meddeb, mailhe, et al., ; gautret, lagier, parola, hoang, meddeb, sevestre, et al., ; magagnoli, ; mathian et al., ; million et al., ; tang, ; yao et al., ) . a post-exposure prophylaxis randomised controlled trial of participants failed to show any benefit of hydroxychloroquine (n= ) compared with placebo (n= ) (boulware et al., ) . at the time of writing, the recovery trial (clinical trial identifier nct ) which is the largest randomised control trial so far conducted for the treatment of covid, has stopped recruiting to the hydroxychloroquine arm ( patients compared with on standard care) because of no beneficial effect either in terms of mortality or hospital stay (p. . there are still many other trials on-going testing the efficacy of hydroxychloroquine for either prophylaxis or treatment. both chloroquine and hydroxychloroquine have been in clinical use for many years for rheumatoid diseases, and thus their safety profile is well established. dose-dependent retinal toxicity has long been recognized as the major ae with long-term use of chloroquine/hydroxychloroquine (marmor et al., ) . besides retinal toxicity, gastrointestinal, liver and renal toxicity have also been reported (giner galvan, oltra, rueda, esteban, & redon, ; michaelides, stover, francis, & weleber, ; mittal, zhang, feng, & werth, ) . as both drugs are mainly metabolised in the liver and excreted by renal clearance, their use in patients with liver or renal impairment may worsen the function of these organs. for chloroquine treatment, prescribing information recommends the full dose at all degrees of renal impairment but suggests that monitoring of renal function may be useful . for hydroxychloroquine, reductions in dosage are advised for patients with impaired renal function, as well as those taking concomitant medications with known risks of kidney damage (concordia pharmaceuticals inc, ) . this article is protected by copyright. all rights reserved. a serious ae associated with chloroquine/hydroxychloroquine is cardiotoxicity, which can take many forms including cardiomyopathy in rare instances. prolonged treatment or high dosage of chloroquine/hydroxychloroquine has been shown to increase of the risk of qt interval prolongation, polymorphic ventricular tachycardia, and sudden cardiac death (chatre, roubille, vernhet, jorgensen, & pers, ) . a large epidemiological analysis in patients with rheumatoid arthritis has recently shown that -day cardiovascular mortality was increased by more than -fold when hydroxychloroquine was combined with azithromycin. the lethal ventricular arrhythmias are primarily due to inhibition of a potassium channel (the inward rectifier kir . channel) and may occur at low µm concentrations (ic = . m) (rodriguez-menchaca et al., ) . while therapeutic doses of chloroquine typically result in plasma concentrations of - µm, much higher concentrations in the heart are expected based on a -fold increase observed in rat pk studies (mcchesney, banks, & fabian, ; walker, dawodu, adeyokunnu, salako, & alvan, ) . both drugs act on various potassium channels including the inward rectifier currents (kir . and kir . ) and rapid delayed rectifier currents (kv . /herg) (ponce-balbuena et al., ; rodriguez-menchaca et al., ; sánchez-chapula, navarro-polanco, culberson, chen, & sanguinetti, ) . the binding of chloroquine to the inward rectifier kir . channel can be stabilized by negatively charged and aromatic amino acids (rodriguez-menchaca et al., ) . to a lesser extent, chloroquine also blocks the rapid delayed rectifier ikr, possibly through cation-π and π-stacking interactions with tyrosine and phenylalanine in the s domain of herg (sánchez-chapula et al., ) . the effect of inhibition of these potassium channels on the heart rate appears to be complex. however, blocking the herg channel has proven to be the most common mechanisms by which drugs cause qt interval prolongation (traebert & dumotier, ) . the binding of chloroquine/hydroxychloroquine to proteins is also stereoselective, but whether one of the chloroquine/hydroxychloroquine enantiomers has a stronger interaction with the kir . channel is not known. caution is needed when hydroxychloroquine is used in combination with other drugs (including azithromycin), which increase the qt interval because of a pharmacodynamic synergistic interaction. given the comorbidities in many patients with covid- , especially those with underlying cardiovascular disease, and the fact that covid- itself is associated with cardiac manifestations, this may increase the risk of cardiotoxicity associated with the use of chloroquine/hydroxychloroquine. indeed, excessive qtc prolongation was observed in % of patients as reported by bessiere at al. and greater qtc prolongation was also seen in patients taking the combination of hydroxychloroquine and azithromycin than those taking hydroxychloroquine alone, highlighting the importance of pharmacodynamic interactions (bessiere et al., ; mercuro et al., ) . furthermore, a phase iib trial in brazil showed that a higher dose of chloroquine ( mg twice daily) in patients hospitalised with covid- had a higher fatality rate ( %) compared with % in the lower dose ( mg twice daily) group (borba et al., ) . qtc interval prolongation > msec was observed in % of the high dose group compared with % of the low dose group. the us prophylaxis randomised control trial however did not show any increase in cardiovascular aes (boulware et al., ) . we await the publication of the recovery trial to determine whether there was an excess of cardiovascular events. however, it is important to note that despite the size of the recovery trial (n = patients), it may still be under-powered to identify an excess number of cardiovascular events when compared with standard of care. remdesivir is an investigational compound that was developed for the treatment of ebola (mullard, ; tchesnokov, feng, porter, & gotte, ) . remdesivir is a monophosphoramidate prodrug and acts as a broad-spectrum antiviral that can be incorporated into viral rna (agostini et al., ; sheahan et al., ; warren et al., ) . many anti-virals are proving to be ineffective against covid- due to the presence of a proofreading exoribonuclease (exon) specific to coronaviruses, encoded in non-structural protein (nsp ) (agostini et al., ) . remdesivir is able to evade this viral proofreading, meaning its incorporation into viral rna results in the inhibition of rna-dependent rna polymerases (rdrps), thereby preventing subsequent viral replication (warren et al., ) . furthermore, arshad et al. suggest that the maximum serum concentration (cmax) of remdesivir is sufficient to inhibit % of sars-cov- replication, a parameter which is suspected to be of vital importance in the treatment of covid- (arshad et al., ) . remdesivir is administered intravenously, with single doses ranging between to mg being well patients receiving remdesivir via the uk early access to medicines scheme (eams) is similar to that which was evaluated for ebola treatment: a loading dose of mg on day , followed by mg daily for - days depending on symptom severity (medicines and healthcare products regulatory agency, b). as such, it is likely that many of the aes observed in the ebola study will translate to covid- patients treated with remdesivir. this article is protected by copyright. all rights reserved. mild to moderate alt and aspartate transaminase (ast) elevations were observed in several ebola patients during the multiple-dose study, thus reflecting observations made in human hepatocytes in vitro (clinical trials.gov, ; world health organisation, ) . this is likely to be due to the high cell permeability of hepatocytes, in combination with the effective intracellular metabolism of remdesivir to its active form within the liver (world health organisation, ) . emerging data has suggested that sars-cov- may target ace on hepatocytes leading to liver injury as evidenced by a significant increase in alt and bilirubin in severe cases of covid- (guan et al., ) . therefore, it is likely that differentiating between covid- -induced transaminase elevations and those induced by remdesivir presents challenges (bangash, patel, & parekh, ; c. zhang, shi, & wang, ) . however, a recent study found that only . % of covid- patients receiving remdesivir treatment suffered serious (grade or ) transaminase elevations, with there being no significant difference between the remdesivir-and placebo-treated groups (beigel et al., ) . this data implies that remdesivir is relatively well-tolerated in sars-cov- -positive patients. regardless, as advised by the drug manufacturer, daily liver function tests are essential in any patients receiving remdesivir, with suggested discontinuation of the drug in patients whose alt levels reach ≥ times the upper limit of normal (uln) (gilead, ) . adhering to these guidelines is of particular importance in patients with pre-existing liver disease, or in those taking other medications which can also induce transient alt and ast elevation (world health organisation, ) . the reported differences between preclinical and clinical data regarding the safety of remdesivir highlight the inadequacies of preclinical models in some contexts. for example, with regards to covid- , a concerning element of theoretical toxicity is that which affects the respiratory system. a study using mice models of middle east respiratory syndrome coronavirus (mers-cov) found remdesivir improved pulmonary pathology in infected mice and rhesus monkeys, and no respiratory toxicity was observed (gilead, ; sheahan et al., ) . in contrast, a respiratory safety study in rats showed that remdesivir had no impact on tidal volume or minute volume, but did increase respiratory rate, which returned to baseline by hours post-dose (world health organisation, ). clearly, increased respiratory rate is a manifestation of covid- , and there would be problems in assessing causality if remdesivir was also likely to cause of respiratory problems in a clinical setting. fortunately, a recent double-blind, randomized, placebo-controlled trial showed there to be no significant differences in adverse respiratory events between the remdesivir-treated and control arms (beigel et al., ) . in addition to this, preclinical safety studies performed in rats and cynomolgus monkeys suggested that the kidney was the target organ for remdesivir-induced toxicity (gilead, ) . this was a significant concern before the initial covid- clinical trials, as it is known that sars-cov- can cause acute kidney failure in severe cases (ronco, reis, & husain-syed, ) . however, this has not this article is protected by copyright. all rights reserved. been reflected in covid- clinical trials, where the presence of biomarkers indicative of renal injury have not differed in patients treated with remdesivir compared to those on placebo (beigel et al., ; gilead, ) . however, due to the inclusion of the solubility enhancer sulfobutylether βcyclodextrin sodium (sbecd) within remdesivir formulations, remdesivir is contraindicated in patients with severe renal impairment (egfr < ml/min) (european medicines agency, ). finally, remdesivir is not exempt from ddis. co-administration of remdesivir with several antibiotics including rifampicin is contraindicated, which could cause problems for any patients being treated concomitantly for tuberculosis (group, ) . this occurs because of enzyme induction which reduces systemic exposure to remdesivir. a similar interaction has also been seen with enzyme-inducing anticonvulsants, including carbamazepine, phenytoin, and phenobarbital (group, ) , where reduction in remdesivir exposure may lead to inadequate treatment of covid- . favipiravir is another broad-spectrum anti-viral prodrug which undergoes intracellular phosphoribosylation to produce its active form, favipiravir-ribofuranosyl- ′-triphosphate (favipiravir-rtp) (yousuke furuta, komeno, & nakamura, ) . it is thought that this anti-viral primarily acts by inducing lethal mutagenesis of rna viruses, although it also selectively and potently inhibits viral rdrp by acting as a pseudo purine nucleotide (dawes et al., ; sangawa et al., ) . favipiravir is currently licensed in japan for the treatment of novel and re-emerging influenza (yousuke y. furuta et al., ) . its extensive spectrum of activity against various rna virus polymerases led to favipiravir being cited as a potentially 'crucial pandemic tool', even before the outbreak of the novel coronavirus, covid- (adalja & inglesby, ) . the pk of favipiravir was initially characterised in healthy japanese volunteers (madelain et al., ) . a cmax of . µg/ml was found to occur hours post-administration, but plasma concentrations decreased rapidly due to the relatively short half-life of favipiravir (between and . hours) (madelain et al., ) . however, both cmax and half-life increase slightly after multiple doses and it has been suggested that favipiravir is capable of reaching a cmax in humans sufficient to inhibit % of sars-cov- replication, thus establishing it as an important compound in the ongoing search for covid- therapies (arshad et al., ) . marked differences in cmax have been observed between japanese and american patients with cmax values in japanese subjects being on average . µg/ml greater than those in american subjects (pmda, ) . this highlights the need for relevant covid- clinical trials to include a diverse range of subjects so that factors such as weight and ethnicity can be considered to optimise dose. the bioavailability of favipiravir is high at . % and only % of the drug is plasma protein-bound, suggesting high tissue penetration would be likely (madelain et al., ; pmda, ) . in vivo work in mice showed that the half-life of favipiravir in the lungs is double that of favipiravir in plasma, indicating slower elimination from the lungs (pmda, ) . this is thought to be of high importance in covid- , where viral load is particularly high in the lungs. for influenza treatment in adults, mg favipiravir is given twice on day of treatment, followed by mg twice daily from days to (pmda, ). however, the dosing period has been extended in ongoing covid- clinical trials: up to days in chictr and days in chictr (guan et al., ) . it is therefore essential that all pk parameters are monitored in these trials as differences, including increased cmax and decreased clearance, are expected during this prolonged dosing regimen which may impact upon safety. favipiravir has been linked to teratogenicity and embryotoxicity, and is therefore contraindicated in pregnancy (yousuke furuta et al., ) . overall, favipiravir is generally thought to have a good safety profile (asrani, devarbhavi, eaton, & kamath, ; group, ; nhs, ) . this is likely to be due to the fact that unlike other antiviral drugs such as ribavirin, favipiravir does not appear to disrupt non-viral rna or dna synthesis. however, very little is known about the long-term safety of favipiravir, as in previous clinical trials patient follow-up has been as little as days . this is perhaps less of a concern in covid- as treatment is time-limited. drug-drug interactions have been reported with favipiravir. for example, coadministration with favipiravir can increase exposure to paracetamol by around %, which may be a concern for patients with pre-existing liver disease as paracetamol is the leading cause of acute drug-induced liver injury (dili) in the uk and usa (asrani et al., ; group, ) . favipiravir can also increase patient exposure to many contraceptives, including progesterone-only pills, combined pills, and several contraceptive implants, which may cause discomfort, prolonged vaginal bleeding, and nausea (group, ; nhs, ) . whether the increased exposure to oestrogens caused by concomitant treatment with favipiravir can enhance the risk of thrombosis is not known but should be monitored, given the overwhelming evidence that covid- increases the risk of blood clots (atallah, mallah, & almahmeed, ; di micco et al., ; spiezia et al.) . interestingly , large clots are most common in patients under the age of ; almost % of women aged between - in the usa currently use either oral or long-acting contraceptives, and thus represent a particular risk group (hurley, ; prevention, ). sars-cov- virus is capable of eliciting an immune reaction in the infected individual. laboratory examinations have revealed that inflammatory factors such as interleukin (il)- , il- , il- and tumour necrosis factor-α (tnfα) are upregulated during infection and can instigate an inflammatory response in the lower airways leading to lung injury in some instances guo et al., ) . additionally, in patients with severe symptoms of covid- , there may be activation of a cytokine storm, which can cause significant tissue damage (mehta, mcauley, et al., ; shi et al., ) . a smaller proportion of patients can progress to a hyper-inflammatory state which in covid- has been suggested to resemble secondary haemophagocytic lymphohistiocytosis (shlh), a rare syndrome characterised by uncontrollable fever, cytopenia, raised ferritin levels and acute respiratory distress (seguin, galicier, boutboul, lemiale, & azoulay, ) . interleukin and tnf-α levels show the greatest increase in those who require admission to the intensive care unit (icu), suggesting that the cytokine storm is instrumental in severe covid- cases (huang et al., ) . therefore, there has been a logical progression towards the use of immunosuppressive agents as potential therapies to alleviate inflammation and hyperinflammation associated with covid- (mehta, mcauley, et al., ) . dexamethasone is a glucocorticoid that can be administered both orally and intravenously. it acts as a glucocorticoid receptor agonist and is over times more potent than endogenous cortisol, thus resulting in dose-dependent suppression of pro-inflammatory genes through a number of pathways in common with other steroids (papich, ; whelan & apfel, ; yasir & sonthalia, ) . low doses of glucocorticoids have an anti-inflammatory effect while higher doses are immunosuppressive (buttgereit et al., ) . dexamethasone can be used for inflammatory diseases such as rheumatoid arthritis (crohn's & colitis foundation, ; freeman, ) , but is recommended for short-term treatment (spanning from one to days) because of the major adverse effects which can occur with long-term treatment. one of the commonest uses of dexamethasone is for reducing cerebral oedema. as of th july , dexamethasone was undergoing evaluation in clinical trials. on th june, the results of the dexamethasone arm of the recovery trial were announced. the trial results, which are available in preprint form, showed that patients had received either oral or intravenous lowdose ( mg) dexamethasone daily for ten days (peter horby et al., ) . when compared to control patients receiving usual care only, it was shown that dexamethasone reduced deaths by one third in sars-cov- positive patients requiring ventilation, and by one fifth in patients receiving this article is protected by copyright. all rights reserved. oxygen. no benefit was observed for patients with milder covid- symptoms who did not require respiratory support (peter horby et al., ) . recent work has found that tissue inflammation and organ dysfunction seen in fatal cases of covid- are not consistent with sars-cov- distribution in tissues and cells (dorward et al., ) . tissuespecific tolerance to the virus may therefore important, and suggests that fatalities arising from covid- may be mainly due to host-mediated immune response rather than pathogen-mediated end-organ inflammation. this is consistent with the dexamethasone result in the recovery trial. dexamethasone has a bioavailability of - % and is % protein bound in plasma (spoorenberg et al., ) . it is -hydroxylated by hepatic cyp a to α-and β-hydroxy-dexamethasone, and can also be reversibly metabolised to -dehydroxymethasone and back to dexamethasone by renal corticosteroid -beta-hydrogenase isozyme (diederich et al., ; diederich, hanke, oelkers, & bähr, ; tomlinson, maggs, park, & back, ) . unlike many glucocorticoids which are predominantly excreted in urine, only about % of dexamethasone is excreted in urine (dexcel pharma technologies ltd.). glucocorticoids are generally safe drugs when given at low doses and for short periods of time (< weeks), with the risk of adverse events increasing with dose and therapy duration (yasir & sonthalia, ). short-term use of dexamethasone can result in increased appetite, mood changes, and insomnia, but most of the adverse reactions are self-limiting (nhs, ). dexamethasone can lead to b and t cell depletion, and hence lymphopenia (marinella, ) , which interestingly is also found in up to % of patients with covid- (liu, blet, smyth, & li, ) . however, despite this, the recovery trial was able to show a mortality benefit in the most severely affected covid- patients. a critical issue may be the dose that is administered -in recovery, mg/day was administered over days, which is a relatively low dose. a recent systematic review and meta-analysis of corticosteroid treatment in patients with coronavirus infection suggested that corticosteroids were associated with higher rates of bacterial infections, longer time spent in hospital and higher rates of mortality (z. yang et al., ) . however, most of the studies analysed in this meta-analysis were retrospective observational studies, generally of poor quality and did not analyse the effects according to steroid dose. other studies which have used low-to-moderate-dose corticosteroids as treatment for diseases such as viral and bacterial pneumonia reflect the results of the recovery trial, with low dose corticosteroids resulting in decreased mortality and morbidity in patients with severe pneumonia (h. li et al., ; stern et al., ) . in these studies, low-to-moderate dose corticosteroids ( - mg prednisolone, which equates to - . mg dexamethasone) were given to patients for between and days (stern et al., ) (national institute for health and care excellence, b). in keeping with the known adverse effects of corticosteroids, the systematic review showed that hyperglycaemia was significantly more frequent in the corticosteroid-treated group (stern et al., ) . dexamethasone can be involved in both pharmacokinetic and pharmacodynamic interactions. combining it with other immunosuppressants may increase the risk of serious infection (national institute for health and care excellence, c). co-treatment with ibuprofen or other nsaids increases the risk of gastrointestinal bleeding (national institute for health and care excellence, c), while its gluconeogenic effects can lead to hyperglycaemia, which in diabetic patients can lead to increased insulin doses being required (consilient health ltd., ). dexamethasone is a cyp a inducer, and may therefore interact with remdesivir, a cyp a substrate, potentially reducing its plasma exposure. although clinicians should be aware of this interaction, the risk is small given that both drugs are indicated for days or less. both tocilizumab and sarilumab are humanised anti-il- receptor monoclonal antibodies used for the treatment of moderate -severe rheumatoid arthritis, whereas siltuximab is a chimeric, human-mouse anti-il- receptor monoclonal antibody used for treatment of multicentric castleman's disease (mcd) (deisseroth et al., ; national institute for health and care excellence, e). due to their long half-life, il- inhibitors do not need to be taken daily; however, given that they are currently indicated for chronic diseases, patients receive il- inhibitor treatments for life or until treatment failure (janssen biotech inc; roche pharma; sanofi-aventis). clinical trials to assess the efficacy and safety of tocilizumab, sarilumab and siltuximab for the treatment of the inflammatory phase of covid- are ongoing. whilst the exact dosing regimens vary between trials, covid- patients will be receiving a single or short course intravenous infusion or subcutaneous injection of the il- inhibitor (clinical trial identifiers nct , nct , nct , nct , nct ). due to their similarity, it is not surprising that tocilizumab, sarilumab and siltuximab have comparable safety profiles. thus far, evidence from clinical trials in patients with rheumatoid arthritis and mcd or post-marketing have revealed that il- inhibitors are generally well-tolerated. participants were enrolled on these trials for a minimum of months and in some cases up to months. individuals with diabetes, a history of recurrent infection, age ≥ and corticosteroid use have been shown to be at an increased risk of developing a more serious infection following il- inhibitor use (jones et al., this article is protected by copyright. all rights reserved. ). whilst adverse reactions were typically seen following chronic il- inhibitor treatment, the potential for covid- patients to develop an adverse drug reaction (adr) following a single or small number of doses should not be ignored. the most common infections reported in patients receiving anti-il therapy include skin infections, respiratory infections, urinary tract infections and in some cases, opportunistic infections ranging from tuberculosis to herpes (emery et al., ; smolen et al., ) . liver injury has also been reported with a liver biopsy from a female patient who had taken tocilizumab for a month revealing focal this article is protected by copyright. all rights reserved. necrosis of hepatocytes with steatosis and early fibrosis (mahamid et al., ) . covid- also has effects on the liver, and again causality assessment may be difficult (guan et al., ) . the prescribing instructions for tocilizumab and sarilumab indicate that liver function tests are required every - weeks following treatment commencement and then every months thereafter (roche pharma; sanofi-aventis). if liver enzymes are - x uln, the dose of tocilizumab and sarilumab can be reduced until alt or ast have normalised and then treatment resumed at the therapeutic dose. where laboratory findings are > - x uln, treatment with il- inhibitors must be paused and then recommendations for - x uln followed. if elevations persist or are > x uln, tocilizumab and sarilumab treatment must be discontinued immediately (roche pharma; sanofi-aventis). whilst sarilumab and siltuximab are associated with abnormalities in liver function tests, they are typically short-lived and asymptomatic (livertox, (livertox, , b . pre-existing liver disease can worsen symptoms of dili, and in some cases increase susceptibility (david & hamilton, ) . tocilizumab, sarilumab and siltuximab are expected to undergo metabolism via catabolic pathways and not cyp processes (mccarty & robinson, ) . therefore, due to the lack of hepatic metabolism, it is assumed that the pk of the il- inhibitors will not be altered in patients with preexisting liver disease (abou-auda & sakr, ). however, tocilizumab, sarilumab and siltuximab have been shown to restore and improve cyp levels (janssen biotech inc, ; roche pharma, ; sanofi-aventis, ). this is of particular importance as cyp levels may remain elevated following treatment discontinuation due to the long half-life of the compounds. therefore, this may be a consideration for further evaluation for any dosing adjustment requirements if patients are taking medication that are metabolised by cyp enzymes. anakinra is a kd, recombinant human il- receptor antagonist that blocks the activity of proinflammatory cytokines il- α and il- β (cawthorne et al., ; dinarello, simon, & van der meer, ) . anakinra is primarily used in combination with methotrexate for reducing the symptoms and slowing the progression of joint damage in rheumatoid arthritis (national institute for health and care excellence, a). it is also used for rare inflammatory conditions such as cryopyrin-associated periodic syndromes and still's disease (national institute for health and care excellence, a). it is administered via subcutaneous injection and is supplied as a single-use, pre-filled syringe containing mg/ . ml (swedish orphan biovitrum ltd, ) . rheumatoid arthritis patients and those with still's disease and a body weight > kg must be administered mg anakinra, while patients with still's disease with a body weight < kg should have weight-based dosing starting at - mg/kg this article is protected by copyright. all rights reserved. (swedish orphan biovitrum ltd, ). the recommended starting dose for patients with cryopyrinassociated periodic syndromes is - mg/kg. if tolerated, the dose can be increased to - mg/kg to a maximum of mg/kg (swedish orphan biovitrum ltd, ) . anakinra has a short terminal half-life of approximately - hours and so must be administered daily, preferably at the same time each day (amgen inc., ) . anakinra is currently not licensed for intravenous administration or treatment of shlh but its use is endorsed by clinicians, where intravenous infusion, as opposed to subcutaneous injection, can achieve quicker and greater maximal plasma concentrations (carter, tattersall, & ramanan, ; la rosée et al., ; mehta, cron, hartwell, manson, & tattersall, ) . thus far, clinical trials have been registered to assess the use of anakinra in patients with severe covid- . additionally, two recent studies have reported positive outcomes with anakinra in covid- induced acute respiratory distress syndrome (cavalli et al., ; clinical trials.gov, c; huet et al., ) . participants were dosed mg twice daily subcutaneously for hours followed by mg daily for days in addition to standard of care (huet et al., ) . this retrospective study found that anakinra reduced rates of mortality and the need for mechanical ventilation in icu patients (huet et al., ) . anakinra was administered either subcutaneously or intravenously in the covid- biobank study (huet et al., ) . participants received subcutaneous injections at a dose of mg twice daily or via slow intravenous infusion at mg/kg per day until there was a % reduction in serum c-reactive protein levels and sustained respiratory improvements (cavalli et al., ) . whilst no safety concerns emerged with anakinra administered subcutaneously, it was discontinued due to a lack of clinical improvement and limited reduction in c-reactive protein (cavalli et al., ) . by contrast, intravenous anakinra was well-tolerated and improved clinical outcomes. notably, % of patients had improved respiratory function in comparison to % within the standard treatment group (cavalli et al., ) . in both studies, cases of alt ≥ x uln were observed in both the anakinra and the standard treatment arms. four cases of bacteraemia following intravenous anakinra were reported in the covid- biobank study, but there were no cases of bacterial infection in the ana-covid study (cavalli et al., ; huet et al., ) . whilst both studies are encouraging, they should be considered proof-of-concept trials and larger randomised trials are still needed (cavalli et al., ; huet et al., ) . subcutaneous administration of anakinra is associated with injection site reactions (kaiser et al., ) . in a review of five rheumatoid arthritis clinical trials, % of participants receiving anakinra therapy reported injection site reactions in comparison to % of participants on placebo (mertens & singh, ) . injection site reactions can range from immediate to delayed. in immediate cases, the reaction manifests as a burning sensation whereas delayed reactions present as a rash, pruritus or swelling (kaiser et al., ) . anakinra has also been reported to lead to infection, neutropenia, this article is protected by copyright. all rights reserved. thrombocytopenia, headache, and blood cholesterol increase when administered subcutaneously (swedish orphan biovitrum ltd, ) . injection site reactions that arise immediately can be eased by placing an ice pack on the injection site before and after anakinra administration and delayed reactions can be treated with topical corticosteroids or anti-histamines (kaiser et al., ) . increases in serious infection rate are common following anakinra use and frequently include upper respiratory infections, sinusitis, urinary tract infection and bronchitis (bresnihan et al., ; cohen et al., ; r. m. fleischmann et al., ) . whilst rare, cases of opportunistic infection have been reported in anakinra monotherapy or in those receiving anakinra in combination with immunosuppressive agents (salvana & salata, ; swedish orphan biovitrum ltd, ) . neutrophil counts must be monitored during the first six months of anakinra treatment and quarterly henceforth (swedish orphan biovitrum ltd, ) . in patients where the anc is < . x /l, treatment must be discontinued immediately (swedish orphan biovitrum ltd, ) . the higher doses being used in covid- trials and the potential for a greater cmax due to intravenous administration potentially raise additional safety concerns. however, earlier detection of aes should be possible since the duration of treatment will be shorter than that used in rheumatoid arthritis, coupled with the fact that patients will already be hospitalised. anakinra is catabolised and eliminated via glomerular filtration (swedish orphan biovitrum ltd, ; b.-b. yang, baughman, & sullivan, ) . caution should be exercised and dose-adjustments may be required in moderate to severe renal impairment (swedish orphan biovitrum ltd, ; b.-b. yang et al., ) . during general infections and inflammatory diseases, cyp enzymes are primarily downregulated (mallick, taneja, moorthy, & ghose, ) . similar to il- inhibitors, it may be possible that anakinra treatment restores cyp levels in infected patients (swedish orphan biovitrum ltd, ) . therefore, caution should be exerted in covid- patients receiving concomitant medications with a narrow therapeutic window drug. mild interactions can occur between anakinra and warfarin, clopidogrel, clozapine and phenytoin (group, ) . baricitinib is an oral disease-modifying anti-rheumatic drug (dmard), traditionally used in the treatment of moderate to severe active rheumatoid arthritis (al-salama & scott, ). by acting as an atp-competitive kinase inhibitor, baricitinib can selectively and potently inhibit janus kinases (jaks) - and - in a reversible manner. jaks are essential in the transduction of intracellular signals for various cytokines involved in the inflammatory and immune responses, and so by inhibiting these kinases, baricitinib is able to relieve symptoms of rheumatoid arthritis for many patients (fridman et al., ) . as described previously, a common characteristic of covid- , much like another beta-coronavirus disease sars, is a profuse inflammatory response (huang et al., ; stebbing et al., ) . increased levels of pro-inflammatory cytokines, such as interferon (ifn) -γ and il- β, have been observed in confirmed covid- cases (huang et al., ; mehta, mcauley, et al., ; russell et al., ) . furthermore, the levels of some specific cytokines appear to be related to disease severity; patients requiring admission to intensive care units show increased levels of tnfα and monocyte chemoattractant protein (mcp ). the rationale behind repurposing baricitinib as a treatment for covid- is centred on this potential for severely ill patients to present with a cytokine storm (mehta, mcauley, et al., ; russell et al., ) . by dampening the inflammatory response, it is postulated that baricitinib will be able to relieve covid- symptoms. data modelled using artificial intelligence techniques suggests baricitinib may work by inhibiting virus entry into cells via an endocytic regulator known to be involved in coronavirus internalisation, ap -associated protein kinase (aak ) (burkard et al., ; richardson et al., ) . baricitinib, as well as being capable of jak and jak inhibition, is a high-affinity inhibitor of aak . patients tend to tolerate baricitinib well, and it has a relatively good safety profile (keystone et al., ) . however, as with tocilizumab and sarilumab treatment, a very common (≥ / ) ae observed in patients taking baricitinib, but not in the placebo arm, is upper respiratory tract infection, which may be related to its ability to suppress the immune system (eli lilly, ) . patients taking baricitinib have the potential to develop respiratory tract infections which may make it difficult to distinguish whether any deterioration is due to covid- or a secondary infection. other opportunistic infections including herpes zoster and urinary tract infections were also more common in the treated arm compared to placebo, and dose reduction is recommended for patients with a history of chronic infections (eli lilly, ; josef s. smolen et al., ) . secondary infections are not uncommon in severe covid- patients and so the use of a drug that may make patients increasingly prone to infections will depend on the harm-benefit ratio for severe cases of covid- (world health organisation, a). baricitinib is currently still being trialled in patients with covid- with a therapeutic dose of - mg once daily which is the same as the recommended dosage for the treatment of rheumatoid arthritis (cantini et al., ; richardson et al., ) . there have been a small number of reports from patients taking this recommended dosage for the treatment of rheumatoid arthritis presenting with deep vein thrombosis (dvt), which was severe in some of these cases (taylor et al., ) . this is a cause for this article is protected by copyright. all rights reserved. concern as there are increasing reports of covid- patients, especially those who are critically ill and in the icu, with thrombotic complications including pulmonary embolism and other venous and arterial thrombotic events (klok et al., ; middeldorp et al.) . as baricitinib has been reported to cause dvt, there is the potential for disease-drug interactions with covid- patients taking baricitinib potentially more likely to develop thrombotic complications. in order to mitigate this risk, alternative jak inhibitors, which have a lower risk of thrombotic events, such as ruxolitinib, may be considered in the context of covid- (alvarez-larran et al., ) . however, unlike baricitinib, ruxolitinib is primarily metabolised by cyp a (l. p. h. yang & keating, ) . this means that prescribing ruxolitinib instead of baricitinib may increase the risk of cyp a -related ddis (ogu & maxa, ) . baricitinib is not predicted to be involved in any problematic ddis. coadministration with both cyp a inhibitors (fluconazole) and inducers (rifampicin) failed to result in any clinically relevant changes to baricitinib exposure (eli lilly, ). emerging reports have revealed that patients with covid- experience renal impairment, which could be attributed ace receptor expression on kidney endothelial cells (varga et al., ) . baricitinib should not be given to patients with renal impairment as the majority of the drug is cleared through the kidneys, and monitoring of renal function will be important to prevent aes related to over-exposure to baricitinib in those with deteriorating renal function (eli lilly, ) . type ifns are a group of cytokines produced during viral infection. notably, ifn-β- a has a leading role in activating genes involved in immunomodulation, suppressing the inflammatory response and anti-viral effects (sallard, lescure, yazdanpanah, mentre, & peiffer-smadja, ) . whilst a variety of type ifns exist, in vitro evidence has shown that ifn-β- a and ifn-β- b are the most potent in the inhibition of sars-cov and mers-cov (chan et al., ; hensley et al., ) . within the lungs, ifnβ- has been shown to upregulate levels of the enzyme cluster of differentiation (cd ), which inhibits vascular leakage, increases the secretion of anti-inflammatory adenosine and preserves pulmonary endothelial barrier function (kiss et al., ; sallard et al., ) . however, in vivo research has revealed that timing of administration of ifn-β- is imperative for positive effects. when administered shortly after mers-cov infection, ifn-β- protected mice from lethal infection, whereas delayed administration failed to effectively inhibit viral replication or pro-inflammatory cytokines, leading to fatal pneumonia (channappanavar et al., ) . interestingly, in vitro evidence has revealed this article is protected by copyright. all rights reserved. that sars-cov- is more sensitive to ifn-β- treatment than mers-cov and sars-cov, and thus supports the tenet that treatment with ifn-β- may be beneficial for covid- patients (lokugamage, schindewolf, & menachery, ; sheahan et al., ; thiel & weber, ) . it is assumed that treatment of covid- patients with ifn-β- will strengthen the host immune response and prevent the worsening of severe respiratory tract manifestations. ifn-β- therapy has been used for the long-term management of multiple sclerosis (ms) and has been associated with a number of aes. when administered subcutaneously in ms patients, the most common aes were flu-like symptoms, injection site reactions, worsening of ms symptoms, menstrual disorders, mood alterations and laboratory abnormalities (walther & hohlfeld, ) . the most common laboratory abnormalities were neutropenia, leukopenia, lymphopenia and raised aminotransferases (walther & hohlfeld, ) . a genome-wide association study of patients with ifnβ induced liver injury showed that rs which has been linked to differential expression of interferon regulatory factor (irf)- is a predisposing factor (kowalec et al., ) . this may be related to the fact that irf leads to apoptosis in the presence of ifn-β. depression is a common ae reported in patients receiving subcutaneous ifn-β- therapy, and thus caution is needed when administering to those with a previous or current history of depressive disorder (biogen) . whilst rare, careful monitoring of clinical manifestations such as new onset hypertension, thrombocytopenia, impaired renal function and fever are required in order to identify cases of thrombotic microangiopathy (tma) (biogen) . tma is rare and has been reported at different time points of ifn-β- therapy (biogen; nishio et al., ; yam, fok, mclean, butler, & kempster, ) . laboratory findings of a decreased platelet count, increased serum lactate dehydrogenase (ldh) and red blood cell fragmentation are suggestive of tma (biogen) . if diagnosed, patients must discontinue ifn-β- therapy and will require plasma exchange (biogen) . sng is an inhaled form of ifn-β- a produced by synairgen. the company have tested the efficacy and safety of the drug for the prevention and treatment of symptoms associated with respiratory viral infection in asthma and chronic obstructive pulmonary disease (copd) (synairgen plc, ). a randomised, placebo-controlled phase trial is currently ongoing to assess the safety and efficacy of inhaled sng for the treatment of patients with covid- (nct ). data from the asthma trials have revealed that when administered via inhalation, high levels of ifn-β- a are achieved within the lungs with lower levels within the circulation leading to improvements in lung function, antiviral responses and better asthma control (djukanović et al., ) . inhaled sng seems to have a good safety profile; patients within the sng arm reported cardiac palpitations whereas no cases were reported in the placebo arm, but symptoms were mild and not considered clinically significant (djukanović et al., ) . this article is protected by copyright. all rights reserved. a clinical trial has been undertaken in hospitalised covid- patients where the triple combination of ifn-β, lopinavir-ritonavir and ribavirin was compared to lopinavir-ritonavir and ribavirin (hung et al., ; shalhoub, ) . patients in the triple combination therapy arm achieved negative tests results faster than those in the control arm, with improved patient symptoms, decreased viral shedding and decreased overall length of stay in the hospital compared to those in the control group (hung et al., ) . aes reported in both groups included nausea and diarrhoea. however, due to polypharmacy in this trial, it was difficult to determine the effect of ifn-β on sars-cov- alone. ifn-β has reported ddis with other covid- therapies including chloroquine and hyrdroxychloroquine, and with anakinra, sarilumab and tocilizumab (group, ) . ddis have also been reported with metamizole (analgesic), linezolid (antibacterial), clozapine (antipsychotic), zidovudine (hiv antiretroviral therapy) and some immunosuppressants (adalimumab, azathioprine and pirfenidone) (group, ) . reviewing the safety of potential covid- treatments (table ) is complex due to the fast-moving pace of research in this field. for example, chloroquine and hydroxychloroquine with or without an accompanying macrolide antibiotic, have consistently been at the forefront of covid- research efforts since the outbreak began. however, the astonishing developments over a week or so have led to retraction of a highly publicised paper, and results from a post-exposure prophylaxis trial and a treatment trial (recovery), both of which have shown no beneficial effect of hydroxychloroquine (boulware et al., ; mehra, ruschitzka, & patel, ) . this highlights that the rapid rate of discoveries surrounding covid- therapies generates the need to update this perspective frequently, in order to ensure that the safety of any newly repositioned therapies, novel developmental compounds, or new therapeutic combinations are investigated. for example, the potential use of heparin in novel forms, including nebulised therapy (clinical trial identifier nct ), as an antiviral agent is currently the subject of several investigational trials. in addition, the potential utility of nitazoxanide is currently the subject of several clinical trials (clinical trials.gov, a; pepperrell, pilkington, owen, wang, & hill, ; rajoli et al., ) . it is clearly essential that the harm:benefit ratio of any pharmaceuticals being considered for use in the treatment of covid- are thoroughly considered. this ratio changes dependent upon the disease stage and is correlated to potential mortality. for example, a higher risk may be accepted for patients in the later stage of severe disease than the same therapeutic agent administered in mild disease. this difference in harm-benefit analysis becomes even more striking when considering the use of such agents to prevent infection. as is the case for many highly contagious viruses, prevention by prophylaxis would be incredibly valuable. some of the agents described in this review, including chloroquine and ritonavir have been suggested as potential prophylactic agents, but to date, data on efficacy have been disappointing (rathi, ish, kalantri, & kalantri, ; spinelli, ceccarelli, di franco, & conti, ) . clearly, treatment duration for prophylaxis is expected to be longer than for treatment of covid- , and this may further alter the harm-benefit ratio, reinforcing the need for safety considerations at the outset of any clinical trials. similarly, the evaluation of therapy risk also applies to long-term recovery. as the current pandemic progresses, it is becoming apparent that being discharged from hospital does not necessarily mean that patients are free from covid- symptoms. large numbers of patients who have survived severe sars-cov- infection may have incurred long-term health problems, including some permanent loss of lung and kidney function (foundation, ; su et al., ; summers, ) . consequently, it is probable that long-term therapies will be required for many patients to maintain, or ideally restore, normal physiological organ function. it is vital that therapies which will be used to treat patients during their long-term recovery are also undergoing evaluation for their safety, particularly as many of these agents may need to be administered over much longer periods of time than initial covid- treatments. the identification and characterisation of biomarkers of disease and safety will be invaluable in the further development and deployment of therapies for covid- . disease biomarkers, for example of lung injury or the hyperinflammatory reponse, may allow the stratification of therapy in order to select the agent best suited to the stage of disease. moreover, biomarkers should be considered to monitor patient safety in cases of known aes. for example, the manufacturer's guidelines for remdesivir recommend daily liver function tests due to the risk of transaminase elevations (gilead, ) . these tests are essential, particularly with regards to covid- where increased alt levels are reported to be common amongst hospitalised patients (bangash et al., ; l. zhang et al., ). looking to the future, improvements in the specificity, predictivity and reliability of drug-induced organ damage, through academic-industry partnerships such as the biomarker qualification program in the critical path institute in the us, and the european innovative medicines initiative consortium transbioline, will help improve clinical assessment of covid- drug safety issues. continued enhancements in the speed, predictivity, and human translation of safety assessment for toxicity of anti-viral compounds is clearly warranted, and this may include animal models of sars-cov- as well as in vitro models, in order to assess efficacy alongside safety. such a full understanding for individual therapies will indicate the combinations that can have the potential to provide the best this article is protected by copyright. all rights reserved. synergy for benefit, while forewarning of the potential for increased risk/harm through pharmacokinetic or toxicodynamic interaction. although outside the scope of this review, a vaccine for covid- remains the greatest hope to end the pandemic and protect the population. as of th july , according to who there are vaccines in clinical trial stages and in preclinical stages of evaluation (world health organisation, b). currently, potential vaccines are only just beginning to be tested for efficacy in humans in early phase studies, and therefore safety data will begin to emerge as larger numbers of individuals are administered the vaccine. safety data regarding preliminary vaccinations against sars and mers are limited, but the available information may be useful during the development of covid- vaccines due to the similarities between the coronavirus strains (padron-regalado, ). one safety concern relevant to coronaviruses is the potential for the induction of antibody-dependent enhancement (ade), a phenomenon which was observed in cats vaccinated against feline infectious peritonitis coronavirus, and has also been seen in patients vaccinated against zika virus and dengue virus (khandia et al., ; padron-regalado, ; vennema et al., ) . ade can occur when nonneutralising antibodies bind to virus particles and increase their uptake into host cells, instead of rendering them non-infectious (padron-regalado, ; tirado & yoon, ) . this caused concern in initial sars vaccine development, but can reportedly be avoided by using truncated versions of the viral s glycoproteins (he et al., ) . acknowledging safety concerns such as this, as well as the ways they can be attenuated, may be paramount in the timely development of a vaccine against covid- . in conclusion, although expanding extremely rapidly, the field of therapies to treat covid- remains in its infancy. safety will continue to play a major role in therapeutic success, as apparent with recent reports of increased cardiac toxicity associated with the use of chloroquine/hydroxychloroquine in the treatment of covid- , despite its long history of use as an antimalarial. above all, this perspective has exemplified the need to view safety concerns in the context of the individual and specific phase of disease in order to formulate a comprehensive harm-benefit balance. importantly, an awareness of potential safety concerns will support the development of the next stage of therapy targeting prophylaxis and recovery post-covid infection. it is imperative that safety scientists look to rise to the challenge of covid- by utilising their expertise in mechanistic understanding, biomarker development and toxicokinetic modelling in order to support the development of covid- therapies that can be used effectively and safely. aa, bkp, cbb, ceg, rlj, rtk, shk, slp and xm declare that that they have no conflicts of interest. ao declares no direct conflict of interest but is director and cso for tandem nano ltd and a co-inventor of patents relating to drug delivery of infectious disease medicines. aec reports no direct conflict of interest but receives research funding for the support of sp and rlj from servier pharmaceuticals and astrazeneca, these are unrelated to the published work. aec receives additional unrelated research funding from janssen pharmaceuticals. ao has received consultancy and /or research funding from viiv healthcare, merck, astrazeneca, gilead, and janssen unrelated to the current paper. db received educational grants and/or consultancy from abbvie, novartis, merck, gilead and viiv healthcare outside the submitted work. mp receives research funding from various organisations including the mrc, nihr, eu commission and health education england. he has also received partnership funding for the following: mrc clinical pharmacology training scheme (co-funded by mrc and roche, ucb, eli lilly and novartis); and a phd studentship jointly funded by epsrc and astra zeneca. he has also unrestricted educational grant support for the uk pharmacogenetics and stratified medicine network from bristol-myers squibb and ucb. none of the funding received is related to the current paper. figure : overview of the mechanisms of action of the repurposed drugs undergoing clinical trials for the treatment of covid- that will be reviewed in this perspective. compounds in red represent those that are viral entry inhibitors, compounds in green represent disruptors of cellular viral processing, compounds in blue are modulators of the hyperinflammatory phase of infection and compounds in yellow stimulate host immunomodulatory and anti-viral activity. abbreviations: ace , angiotensin converting enzyme ; il- , interleukin- ; il- , interleukin- ; jak, janus kinase; rdrp, rna-dependent rna polymerases; 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title: efficacy and adverse events during janus kinase inhibitor treatment of savi syndrome date: - - journal: j clin immunol doi: . /s - - - sha: doc_id: cord_uid: bc vths objectives: mutations affecting the tmem gene cause sting-associated vasculopathy with onset in infancy (savi). no standard immunosuppressive treatment approach is able to control disease progression in patients with savi. we studied the efficacy and safety of targeting type i ifn signaling with the janus kinase inhibitor, ruxolitinib. methods: we used dna sequencing to identify mutations in tmem in patients with peripheral blood type i ifn signature. the jak / inhibitor ruxolitinib was administered on an off-label basis. results: we identified three patients with savi presenting with skin involvement and progressive severe interstitial lung disease. indirect echocardiographic signs of pulmonary hypertension were present in one case. following treatment with ruxolitinib, we observed improvements of respiratory function including increased forced vital capacity in two patients, with discontinuation of oxygen therapy and resolution of echocardiographic abnormalities in one case. efficacy was persistent in one patient and only transitory in the other two patients. clinical control of skin complications was obtained, and one patient discontinued steroid treatment. one patient, who presented with kidney involvement, showed resolution of hematuria. one patient experienced increased recurrence of severe viral respiratory infections. monitoring of peripheral blood type i interferon signature during ruxolitinib treatment did not show a stable decrease. conclusions: we conclude that targeting type i ifn receptor signaling may represent a promising therapeutic option for a subset of patients with savi syndrome and severe lung involvement. however, the occurrence of viral respiratory infection might represent an important cautionary note for the application of such form of treatment. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. type i interferonopathies are a new class of disorders linked to the upregulation of type i interferon (ifn) [ , ] . stimulator of ifn genes (sting)-associated vasculopathy with onset in infancy (savi) is caused by gain of function mutations in tmem [ ] , which lead to a constitutive production of high levels of type i ifns without infectious triggers [ ] [ ] [ ] . savi is characterized by systemic inflammation, chronic anemia, growth failure, cutaneous necrotic lesions, and interstitial lung disease [ , ] . low-titer autoantibodies (i.e., anti-nuclear antibody, anti-cardiolipin antibodies) can also be present. therapeutic management is challenging: steroids are partially effective; patients respond poorly to disease-modifying therapies, such as methotrexate, mycophenolate mofetil, antimalarials, infliximab, and rituximab [ , , ] . prognosis is poor especially in patients with a severe lung involvement, with a high mortality in the first two decades of life [ ] . in light of the encouraging results obtained with the use of jak inhibitors in other interferonopathies [ ] , and the ability of blocking type i ifn pathway activation in savi pbmc in vitro [ ] , a few savi patients have been treated with jak / inhibitors with good responses [ , ] . we describe three savi patients with severe pulmonary involvement in which treatment with the jak / inhibitor, ruxolitinib, led to marked amelioration of disease manifestations that was sustained in one patient, but only transient in the other two. the local ethics committees of giannina gaslini institute and ospedale pediatrico bambin gesù approved the study. ifn signature was performed as described [ ] with minor modifications. briefly, rna was extracted from whole blood from peripheral venipuncture drawn in paxgene tubes using paxgene blood rna kit (qiagen, hilden, germany). cdna was retrotranscribed using superscript® vilo™ cdna synthesis kit (invitrogen, carlsbad, california, usa). selected ifn-stimulated gene (ifi , ifi l, ifit , isg , rsad , siglec ) expression was quantified by real-time pcr using gene-specific primers and probes (roche) with the ddct method relatively to a healthy donor calibrator using hprt and g pd as reference genes. in case samples were run in different assays, the same healthy donor calibrator was used and a positive control was added to assess inter-assay variability. mutations in the tmem gene were assessed by standard sanger sequencing (p and p ) or targeted resequencing using a customized panel and analyzed with the nextseq sequencing platform (illumina, san diego, california, usa) (p ). sequences are analyzed using a bioinformatics pipeline integrated in the basespace illumina system, and variants are called by variantstudio software. variants identified by nextgeneration sequencing have been validated by sanger sequencing. the dyspnea score was defined as follows: : absence of dyspnea; : dyspnea only during intense physical activity, such as gymnastics at school; : dyspnea on moderate physical activity, such as climbing steps; : dyspnea walking; and : dyspnea at rest. patient (current age years old), born from healthy unrelated parents, presented at the age of eight months with isolated erythematosus-infiltrated skin lesions with pustular evolution followed by scarring, chilblains, and severe nail dystrophy by three years of age (fig. a) . skin biopsy of an active nummular violaceous lesion and a scar resolution of a preexisting lesion revealed granulomatous nodular dermatitis, with deep granulomatous folliculitis and secondary fibrosis. the child presented recurrent episodes of bronchospasm, recurrent fever episodes, and worsening of skin lesions. at the age of eight years, focal thickening of the interlobular septa with areas of ground glass opacities with predominant sub-pleural distribution were evident at chest ct (fig. a) . a lung biopsy revealed lymphocytic aggregates in the peribronchial interstitial areas with aspects of capillaritis, and contiguous focal sub-atelectasis with macrophages infiltrating the alveoli (table ) . immunological studies revealed increased igg and ige, normal lymphocyte subset distribution, and normal lymphocyte proliferation in response to mitogens. tetanus toxoid-specific igg were below the protective level when tested at nine years of age (table ) . spirometry revealed mixed restrictive and obstructive features (fig. a , table ). differently from the cases described so far, mild renal involvement (microscopic hematuria, mild proteinuria, no biopsy was performed) was also present, associated with hypertension. steroids (prednisone mg/kg/day) ameliorated the clinical manifestations with normalization of inflammatory markers. however, disease relapses were observed during steroid tapering. azathioprine and etanercept were ineffective. peripheral blood type i ifn signature revealed the activation of type ifn-induced gene expression ( supplementary fig. a ). sanger sequencing of the tmem gene identified a de novo c. g>a p.val met mutation [ ] . patient (current age ten years old) was recently described [ ] . she presented at the age of three months with growth failure and respiratory distress (table ) . livedo reticularis was present on the lower limbs (fig. b) ana anti-nuclear antibodies, anca anti-neutrophil cytoplasm antibodies, pr anti-proteinase antibodies, b glg anti-beta glycoprotein, igg subtype, pl anti-phospholipid antibodies, pt antiprothrombin antibodies, aza azathioprine, mtx methotrexate nonprotective to tetanus toxoid and diphtheria ( ) protective to tetanus toxoid ( ) not tested numbers in italics means abnormal for age-specific reference ranges [ , ] dtp diphtheria, tetanus toxoid, and pertussis, hib haemophilus influenzae type b, mmr measles, mumps, and rubella, pha phytohemagglutinin infections (bronchiolitis and pneumonia). ct scan showed extensive ground glass abnormalities (fig. b) . chronic pseudomonas aeruginosa infection was documented. lung biopsy showed interstitial fibrosis without signs of vasculitis. echocardiographic imaging showed indirect signs of pulmonary hypertension and mild dilation of the pulmonary artery. steroids were only partially effective, and association of methotrexate and infliximab were ineffective, resulting in steroid dependency. daily oxygen support and non-invasive ventilation at night were required. cheeks and nose numbers in italics means abnormal according to age-specific reference ranges * interrupted after min for respiratory distress. p did not perform respiratory function tests due to his young age ** restarted oral prednisone at mg/kg/day at months because of worsening of the radiological signs of disease at chest ct np did not perform, mwt min walking test, fvc forced vital capacity, crp c-reactive protein, esr erythrocyte sedimentation rate, hb hemoglobin telangiectatic skin lesions and unilateral vocal cord paresis were also observed (fig. b , table and table ). except for the elevation of total iga and igg and a mild lymphopenia, immunological studies did not reveal major alterations (tables and ) . peripheral blood type i ifn signature was positive ( supplementary fig. a) . targeted sequencing revealed a c. g>a p.arg gln mutation in tmem [ ] . patient (current age three years old), the only child of healthy, unrelated parents, presented at three days of life, with an erythematosus vesicular rash on the nose and cheeks, later spreading to her hands and feet with evolution to pustules and scars ( fig. c and table ). she was admitted at the age of three months with recurrent low-grade fever, cough, diarrhea, and dermatitis variably responsive to glucocorticoids and antibiotics in the suspect of a combined immunodeficiency. skin biopsy was consistent with neutrophilic dermatosis with karyorrhexis throughout the vessel wall (fig. d) . chest ct revealed focal thickening of the interlobular septa with areas of ground glass opacities. blood investigations revealed microcytic anemia, increased acute-phase reactants, hypergammaglobulinemia, mild reduction of c levels, and low-titer autoantibodies (anti-cardiolipin), with a normal distribution of lymphocyte subsets. lymphocyte proliferation was normal in response to pha and low but present in response to okt (tables and ) . peripheral blood type i ifn signature was positive (supplementary fig. a) . the patient was started on glucocorticoids ( mg/kg/day prednisone) with improvement of clinical and laboratory findings, but recurrence of symptoms at tapering. targeted genome sequencing revealed a de novo heterozygous mutation in tmem c. a>g (p.asn ser). treatment with ruxolitinib was started initially at . mg/kg/ day in two doses, and subsequently progressively increased until clinical efficacy was obtained, namely, to . mg/kg/ day in p , . mg/kg/day in p , and . mg/kg/day in p . all patients showed an initial clinical response starting in the first weeks of treatment and evident after three months (figs. a and a, table ), with amelioration of lung disease (assessed by pulmonary functional tests and ct scans) and resolution of cutaneous lesions that was stable in p and only transient in p and p . furthermore, p showed resolution of the microhematuria and was able to progressively taper steroids and stop them after two years. p also showed amelioration of the skin phenotype, reduction of the dyspnea that correlated with an improvement in the -min walking test (o saturation at the end of the test improved from to %), and increase in total forced vital capacity (figs. b and b, table ). furthermore, echocardiographic improvement of the indirect signs of pulmonary hypertension was documented, with normalization of pulmonary artery size. p initially showed a good clinical response with amelioration of skin lesions and radiological lung findings (fig. c , table ) . notably, the clinical responses did not correlate with decreased type i ifn signatures, which improved only transiently in p during concomitant treatment with high dose steroids and ruxolitinib (fig. b) . during three years of ruxolitinib treatment, p presented three flares of the skin lesions requiring brief cycles of steroids. no severe infections occurred. bk virus in the urines was intermittently detected (viral copies from thousands to million/ml), without bk viremia, nor alteration of kidney function. p (follow-up of months) starting from seven months of therapy, experienced an increase in hospitalization because of respiratory infections, caused by pseudomonas aeruginosa exacerbation, several episodes of rhinovirus infection, influenza a, varicella, and coronavirus (the last requiring mechanical ventilation and extracorporeal membrane oxygenation for days), prompting the decision of reducing ruxolitinib dosage (fig. c) . at months of therapy, ct scan revealed a worsening of the interstitial disease with a ground glass appearance and interlobular thickening (fig. b) . p (follow-up of months) after ten months on treatment on ruxolitinib presented clinical and radiological relapse of lung disease requiring glucocorticoid therapy ( mg/kg/day of prednisone) with a prompt response (fig. a) . she never presented viral or bacterial infections since the beginning of therapy. cell blood counts and ruxolitinib serum levels were regularly monitored ( supplementary fig. b our report confirms that jak inhibition is a therapeutic resource worth considering for patients with savi, including very young children, as in the case of p , also taking into account that the disease poorly responds to any other available immune-modifying agents. however, contrary to published reports, we observed several episodes of severe viral infections in one patient, suggesting the possibility that jak inhibitors might significantly increase the risk of infections possibly in cases of more severe lung involvement or in the presence of genetic modifiers, not investigated in our patient, with consequent deterioration of t he lung disease. interestingly, the most frequent virus isolated was rhinovirus, which may be a direct consequence of effective type i ifn inhibition by ruxolitinib at least in respiratory epithelial cells, where ifn-β is required to control rhinovirus [ , ] . on the other end, these infections might result from a cumulative effect of the drug with the reported developmental and in vitro proliferative defects of sting mutant t lymphocytes [ , ] . considering the severity of lung disease and the lymphopenia (table ) present before ruxolitinib treatment, p was started on antibiotic prophylaxis with bactrim and azithromycin; that, however, did not seem to prevent the febrile episodes, mostly if not always caused by viral pathogens. of note, the patient is now on ivig prophylaxis and no severe infections have been reported so far. whether this is due to ruxolitinib reduction and/ or ivig treatment, it is difficult to understand. we observed a variable medium-and long-term clinical response to the therapy. only p seemed to reach a stable control of the disease, while p and p required at some point the introduction (p ) or the increase (p ) in steroids to control flares. several factors might have played a role, including variability of disease severity and inadequate plasma drug levels. indeed, while all patients were steroid dependent and required several hospital admissions to treat flares, p and p appear to have a more severe phenotype, with p presenting several life-threatening exacerbations requiring admission to icu and ventilator support during infection episodes and p presenting a very early disease onset (three days of life). additionally, despite dose adjustment and plasma level monitoring, drug dosage appeared to be stably adequate only in p (supplementary fig. and supplementary table ) . notably, we reduced ruxolitinib dosage in p after one year of therapy, in the attempt to prevent infections (fig. c) . overexpression of a set of interferon-stimulated genes (isgs) in peripheral blood has consistently been found in all reported patients with savi syndrome [ , , ] . modulation of ifn signature following treatment may represent a surrogate biomarker. however, we did not observe a consistent decrease in ifn signature during the treatment. in previous reports of ruxolitinib treatment in patients with savi [ , , ] , only a partial or no decrease in ifn signature was observed. the lack of downregulation of ifn signature might be explained by the fast kinetic of jak inhibition by ruxolitinib as shown by the transient decrease in pstat following drug intake [ ] which seems not to be coupled with a clear impact on isg expression ( supplementary fig. ) . otherwise, it might suggest that the six gene-based test is not appropriate to assess type i interferon activation at least in patients with savi treated with a jak inhibitor. finally, it is important to consider that ruxolitinib inhibits not only the signaling of type i interferon receptor but also that of other receptors involved in important inflammatory pathways such as il , il / , and interferon γ. it is possible that the inhibition of some or all of these pathways is responsible for the therapeutic efficacy (and side effects) we see in savi. in conclusion, savi disease still appears to be orphan of ideal therapeutic targets. despite the encouraging results obtained in this and previous reports [ , ] , prospective studies with a greater number of patients are warranted to address all the crucial issues linked to jak inhibitor use in patients with savi. type i interferonopathies: a novel set of inborn errors of immunity type i interferonopathies in pediatric rheumatology activated sting in a vascular and pulmonary syndrome inherited sting-activating mutation underlies a familial inflammatory syndrome with lupus-like 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necker-enfants malades, paris, france) for the help in patient diagnosis and advice in the use of ruxolitinib, dr. francesca conti (irccs ospedale bambin gesù, rome, italy) for patient care, and dr. nihel khoudour (laboratory of pharmacology, université paris-est créteil, france) for serum drug dosage authorship contributions sv designed and performed experiments, acquired and analyzed the data, followed the patients, and wrote the manuscript. ai acquired and analyzed the data, followed a patient, and participated in the writing of the manuscript. rc, as, fc, vm, os, stl, fcar, mr, dg, md, cc, ar, pp, ad, and mec acquired the data and followed the patients. cp, es, gm, pb, gc, and cp conducted the experiments and acquired the data. pt and cg performed and analyzed the radiological exams. fcan supervised the project and participated in the writing of the manuscript. fdb followed a patient and supervised the project. mg followed the patients, supervised the project, and participated in the writing of the manuscript. conflict of interest the authors declare that they have no conflict of interest. key: cord- -l dm pp authors: santhakumar, diwakar; rohaim, mohammed abdel mohsen shahaat; hussein, hussein a.; hawes, pippa; ferreira, helena lage; behboudi, shahriar; iqbal, munir; nair, venugopal; arns, clarice w.; munir, muhammad title: chicken interferon-induced protein with tetratricopeptide repeats antagonizes replication of rna viruses date: - - journal: sci rep doi: . /s - - -y sha: doc_id: cord_uid: l dm pp the intracellular actions of interferon (ifn)-regulated proteins, including ifn-induced proteins with tetratricopeptide repeats (ifits), attribute a major component of the protective antiviral host defense. here we applied genomics approaches to annotate the chicken ifit locus and currently identified a single ifit (chifit ) gene. the profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. this highly virus- and ifn-responsive chifit protein interacted with negative sense viral rna structures that carried a triphosphate group on its ′ terminus (ppp-rna). this interaction reduced the replication of rna viruses in lentivirus-mediated ifit -stable chicken fibroblasts whereas crispr/cas -edited chifit gene knockout fibroblasts supported the replication of rna viruses. finally, we generated mosaic transgenic chicken embryos stably expressing chifit protein or knocked-down for endogenous chifit gene. replication kinetics of rna viruses in these transgenic chicken embryos demonstrated the antiviral potential of chifit in ovo. taken together, these findings propose that ifit specifically antagonize rna viruses by sequestering viral nucleic acids in chickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health significance. innate immune responses, primarily triggered by interferons (ifns) and their antiviral effectors, can establish an extremely potent antiviral state to efficiently restrict virus replication and virus-induced pathologies in the susceptible host . to initiate these innate responses, viruses are detected by the host pathogen recognition receptors (prrs) through recognition of viral molecular signatures known as pathogen associated molecular patterns (pamps) . pamps associated with most viruses include viral double-stranded rna (dsrna), which is produced as replicative intermediate and single-stranded adenosine/uridine (au)-rich regions . these pamps effectively activate cascades of signalling events that culminate in the production of ifns in virus-infected cells. the released ifns transcriptionally activate hundreds of ifn-stimulated genes (isgs) in uninfected neighbouring cells that instigate direct or indirect antiviral activities , . well-studied examples of these antiviral effectors are dsrna-activated protein kinase r and ′- ′ oligoadenylate synthetase which bind to dsrna, and interferon induced proteins with tetratricopeptide repeats (ifit)- and - that bind to ′-triphosphate containing rna , . ifit genes are evolutionary conserved and are originated possibly by gene duplication . all members of the ifit family consist of multiple tetratricopeptide repeats (tpr) throughout the length of the protein that are mainly responsible for the protein-protein interaction and assembly of larger protein complexes , . most mammals encode several ifit genes including ifit /isg , ifit /isg , ifit /isg and ifit /isg , however, mice and rats lack ifit and horses lack ifit . amongst all known members of the family, ifit and ifit are highly responsive both at the transcription and translation levels to diverse cellular stresses including those induced by dsrna, virus infections and lipopolysaccharides , . the ifit proteins family is responsible for diverse array of cellular activities including nucleic acid sensing and direct antiviral effects . specifically, ifit is proposed to be a negative-feedback regulator of virus-triggered induction of type i ifns and cellular antiviral responses whereas ifit potentiate antiviral signalling . additionally, mammalian ifit can sense and bind to numerous short cellular rnas such as initiator trna, and these interactions are mapped across the protein surface . the most prominent features of ifit proteins are attributed to their involvements in the inhibition of virus replication through nucleic acid sensing and leading to possible inhibition of translation , . ifit protein, in addition to ifit , discriminates the cellular and viral mrna for initiating downstream antiviral activities by recognition of discrete features at the ′ termini , . most eukaryotic cellular ribosomal and transfer rnas (rrnas and trnas) carry monophosphate at ′-termini whereas messenger rnas (mrnas) bear n -methlguanosine cap (cap ) attached to the first base through ′- ′ triphosphate bridge that recruits cellular factors involved in rna processing and translation initiation , . additionally, in most higher eukaryotes methylation occurs at the ′-o position of the first or second base yielding the cap (m gpppnmn) or cap (m gpppnmnm) structures, respectively . although cap and cap are not crucial for mrna translation, human and murine ifit protein can inhibit translation of cap -lacking mrna , . these features of cellular rnas are also mimicked by several viruses as countermeasure strategies . however, viral genomic and subgenomic rna of negative sense single-stranded viruses such as influenza a viruses (iav) and newcastle disease viruses (ndv) bear triphosphate at the ′-termini . these pamps are sensed by cellular prrs and initiate innate immune responses, which ultimately restrict virus growth , . significant genetic, functional and structural features have been recently attributed to the mammalian ifit genes and proteins [ ] [ ] [ ] [ ] . there is limited information available on the repertoires of cellular proteins that recognizes different populations of viral nucleic acid in avian species especially in chickens which differ significantly in mounting innate immune responses and are infected by pathogens that continuously pose zoonotic threats to public health . here, we have genetically characterized chicken ifit and revealed that chickens, in contrast to other vertebrates, encode only one ifit gene (chifit ). the chifit gene was transcriptionally highly responsive to both type i ifns and rna viruses (iav and ndv) and interestingly this responsiveness was mapped to isre motif in the cis-acting elements. we also demonstrated that chifit specifically interacts with ssrna carrying ′-ppp moiety. through this interaction, chifit applies strong antiviral activities in lentivirus-transduced stable cell lines whereas crispr/cas -mediated chifit knockout promoted virus replication. finally, employing the rcas-based retroviral gene transfer vector system , we generated transgenic chicken embryos expressing chi-fit and demonstrated its antiviral potential in ovo. these findings were further evaluated by rcas-mediated gene silencing in developing transgenic chicken embryos by assessing the replication kinetics of rna viruses. these analyses provide evidence of the presence of a functional homologue of ifit and expand our understanding on the breaths and dynamics of nucleic acid sensing in chicken. genomic annotation of ifit locus revealed that chicken genome encodes ifit gene. the genome of all major mammalian species (including human, mouse, dog and horse) encodes for ifit genes, however, these genes are only genetically and functionally characterized in a limited number of species , . the human genome encodes four ifit genes (ifit , ifit , ifit and ifit ) whereas rats and mice lack ifit and horses lack ifit (fig. a ). in addition, several pseudogenes have been identified in different animal species including ifit b (in human, mice and rabbits), ifit c (in mice), ifit -like (in dogs and mice) and ifit -like gene (in dogs and rabbits). to identify corresponding ifit homologues in chicken, initially several ifit genes from human and mouse were used in the blast algorithm in the ensembl database. based on high genetic similarity with huifit , only a single gene (ensgalt ) was identified in the chicken genome (fig. a) . the ifit locus, encoding all ifit genes, is primarily mapped in between the lysosomal acid lipase/cholesteryl ester hydrolase (lipa) and pantothenate kinase (pank ) genes. since only a single ifit gene (ifit ) was identified in the chicken genome, we next examined an approximately . kb genomic sequence spanning aadn . contigs in the chicken chromosome (chromosome : , , - , , ). immediately adjacent to the lipa gene, a sequence gap was identified whose estimated length was bps in the ensembl chicken genome build (ensembl release -july ) ( supplementary fig. a ). long primers (supplementary table ) flanking each end of the gap were used to amplify genomic fragments and subsequent sequencing of the products was used to cover the genomic gap. we used the sequenced fragments as input in webaugustus and predicted any possible genes in the entire ifit locus. only one gene (ifit ) was predicted in any strand of the input dna using chicken as species parameters ( supplementary fig. a ). the genomic gap has now been filled with the latest ensembl chicken genome (ensembl release -dec ) and no gene other than ifit has been identified (until april ). to confirm the sequence of the cdna and to identify the genomic structure of the identified ifit gene, the transcript was amplified from rna extracted from ndv-infected cefs. complete sequence analysis of the gene revealed an open reading frame (orf) of bps ( amino acids excluding stop codon) with high sequence identity ( % and %) with the human and duck ifit proteins, respectively ( supplementary fig. b) . the identified gene showed a characteristic exon/intron organization where two exons were separated by a few kilobases (kb) long intron (fig. b) . the first exon encodes barely a kozak sequence (catg) and ′ untranslated region (utr). the second exon codes the rest of the orf ( bps) and ′ untranslated regions (utr) for chicken ifit gene. based on the transcriptomic data from marek's disease virus (mdv, strain rb b)-infected cefs (fig. b) and cdna sequencing data, a complete ifit gene was annotated. phylogenetic analysis of characterized chicken (ch) ifit gene with all known human, mouse and duck ifit genes clustered the chicken gene with scientific reports | ( ) : | doi: . /s - - -y duck and human ifit (fig. c) . one of the structural hallmarks of all ifit proteins is the presence of multiple trp motifs dispersed throughout the length of the protein , . the consensus chicken, duck and human ifit sequences were used to predict trps using ncbi's conserved domain database. both duck and human ifit proteins structurally carried ten trp motifs and two multi-domains whereas chicken ifit encoded eight predicted and ten structure-based tprs (fig. d) . taken together, the gene synteny, genetic similarity, genomic architecture and annotation indicate that chickens encode only one ifit gene compared to at least four in mammals. based on genetic clustering, it is highly similar to ifit genes of other avian (chickens and ducks) and mammalian species. moreover, no ortholog for ifit , ifit , ifit and no pseudogenes were identified in the current ensembl chicken genome build (ensembl release -dec ). mouse ifit protein is predominantly expressed in the cytoplasm and upon stimulation with the ifn, it accumulates in the cytoplasm (> × molecules per cell), and this abundance is identified to be crucial for its antiviral function . beside the distribution pattern of mouse ifit , no other ifit proteins have been investigated for their subcellular distributions. to delineate the expression dynamics and sub-cellular distribution, df- cells were transfected with v -tagged chifit and transient expression of chifit was compared in ndv-stimulated or mock-treated cells. confocal microscopy figure . genomic architecture along with relative loci around ifit genes in human, mouse, dog, horse and chicken, and gene annotation in chicken. (a) the ifit locus in compared species is flanked upstream with lipa gene and downstream with pank gene. direct syntenic analysis identified a single ifit gene in chicken compared to four in other species aslong with other pseudogenes. (b) transcriptomics profiling of chicken primary fibroblasts which were mock-infected or infected with rb b strain of mdv. blue bar represents the transcript in the current chicken genome assembly whereas the red bar represents the mappability of the transcript to the chicken genome. a final transcript from mdv-infected data and gene characterization is shown at the bottom. (c) phylogenetic analysis of ifit genes in different species. based on the clustering patterns and sequence homologies, the single identified gene clustered closer to ifit of duck and human. (d) putative tetratricopeptide repeats (tpr) showing characteristic features of ifit proteins. (e) expression and subcellular distribution of chifit in chicken embryo fibroblasts. chicken cells were transfected with ng of mammalian expression vectors encoding v -tagged chifit for hours and were left untreated (ndv-) or were treated with moi of ndv-gfp (ndv+) for another hours before fixation, staining for nucleus (blue), chifit (red) and gfp marker (ndv). scientific reports | ( ) : | doi: . /s - - -y using anti-v antibodies showed that chifit was exclusively cytoplasmic and was expressed throughout the cytoplasm; however, this expression was concentrated at the cell surface (fig. e, upper panel) . we were unable to demonstrate the distribution pattern of chifit under ndv infection (fig. e , lower panel) probably due to the profound antiviral state induced by chifit so that infection of ndv in the chifit -expressing cells could not be achieved. it has previously been demonstrated that human ifit enhances the innate immune responses by interacting with rig-i (not identified in chicken) and mavs and this interaction occurs at the mitochondria . since mavs localizes on both mitochondria and endoplasmic reticulum (er)-derived membranes (mem), we labelled mitochondria and er in presence of chifit to assess the distribution and localization of chifit with mavs. in df- cells, chifit localized in close proximity to the mitochondria and no co-localization was observed between er and chifit (data not shown). owing to lack of cross-reactivity of human ifit antibodies and specificity of anti-sera which we have raised against chifit , these experiments were performed using tagged-chifit . therefore, future studies are required to assess the expression patterns of the endogenous chi-fit under virus or non-virus stimuli. transcriptional profiling of chifit in vitro and ex vivo. we next assessed the nature of ligands that can transcriptionally induce the expression of chifit . different ligands that can either directly induce the transcription of ifit such as ifn-β or stimuli that result in the expression of ifns, which in turn induce the ifit transcription, were assessed ( fig. a) . tlr (lipopolysaccharide, lps) and tlr (poly i:c, synthetic dsrna) ligands significantly induced the expression of chifit gene by and folds, respectively (fig. b) . the induction is likely through the activation of ifns since the chifn-β stimulated cells profoundly increased the transcription of chifit by folds compared to untreated or mock-treated cells. there are evidences that negative sense single stranded rna viruses (e.g. influenza and ndv) produce dsrna as intermediate by-product during the virus replication cycle , - . therefore, it is plausible that induction of chifit expression in chicken cells infected with ndv is mediated by virus-generated dsrna (fig. b ). to further assess the temporal effect of ndv infection on the induction of chifit , a time course profiling ifn and ifn-regulated genes was evaluated. the virus-induced expression of chifit was profoundly observed as early as hours post-infection (fig. c ) and the expression was maintained for hours. a slight reduction in ifit expression was observed at hours post-infection (hpi) was reconstituted at hpi (fig. c ). this biphasic expression of ifit after virus infection was repeatedly observed in chicken cells. however, the pattern of expression of myxovirus-resistance protein (mx) gene, which is another well-characterized isg , shows a steady up-regulation and peak expression was observed at the latest time post-infection (fig. e ). the levels of chifn-β gene induction was proportional to the ndv replication (fig. d) , therefore, it can be inferred that the virus-induced expression of chicken ifit is ifn-dependent and that chifit is an early-isg with capacity to modulate initial steps of virus life cycle. to investigate transcriptional activation of ift following virus infection in chicken, we analysed a panel of rna transcripts derived from selected tissues (liver, kidney, spleen, beak, trachea, lungs and duodenum) of chickens ( -weeks-old rhode island red) which were mock-infected or infected with h n avian influenza virus strain a/chicken/pakistan/udl / (udl /h n ). all tissues from infected birds contained relatively higher levels of chifit compared to the corresponding tissue samples derived from mock-infected chickens (fig. g) . these results conclude that virus infection positively regulates the transcriptional dynamics of chifit in chickens. promoter structures contributing to the ifn, dsrna or virus-mediated transcriptional activation of chifit gene. most studied-ifit genes respond to ifn or ifn stimuli through one to four ifn-stimulated response elements (isre), which are mainly present within base pair (bp) of the transcriptional start site in the orientation of the encoded gene or in the reverse complement order . on the other hand, several ifit genes lack isre motifs in their promoters including ifit from chimpanzee, ifit b from human, chimpanzee and dog, and ifit from horse, chimpanzee and dog . since our data and previously published transcriptomics studies support the profound expression of ifit gene against a wide range of stimuli, we analysed the ′ flanking region of chifit gene for motifs that regulate the expression of chifit . in addition to the gene-encoding sequences, approximately . kb sequence including the putative promoter (supplementary fig. a ) was isolated and sequenced. inspection of the promoter region revealed the presence of two consecutive isre motifs within bps from the transcriptional start site ( fig. a and supplementary fig. b ). these elements were preceded by tata element, and the binding motif for specificity protein (sp ) transcription factor. a putative and weak ifn-gamma-activated site (gas) was identified at the distal end of the promoter and six consecutive gaaann elements were predicted between gas and sp motifs (fig. a) . gas is essential for ifn-gamma-mediated transcription of the genes whereas gaaann elements have been demonstrated to regulate the virus-induced and type i ifn-regulated genes via the binding of interferon regulatory factors , . to determine the role of these cis-acting regulatory elements in the responsiveness of different ifn ligands and stimuli, the entire promoter region ( bp) was cloned upstream to the luciferase gene in a promoter-less pgl . -basic vector (fig. a) . additionally, five reporter constructs were generated each containing either all of the gaaann elements, sp , tata box, double or single isre promoter sequences (fig. a,b) . luciferase reporter assays were performed to assess whether the upstream region of the chifit gene can mediate responses to stimuli from chifn-β, dsrna or virus infection. df- cells were transiently transfected with these reporter plasmids and subsequent luciferase activity was monitored after stimulation with either chifn-β (fig. c) , dsrna ( fig. d ) or with ndv (fig. e ). similar to ifn-induced promoter activation of chmx gene , the chifn-β induced -fold higher luciferase activities in full-length promoter (fig. c ). however, further deletion of gas, gaaann, sp and tata box resulted in an approximately -fold reduction in basal transcription activity. additional deletion of the distal isre reduced the ifn-responsiveness of the promoter. however, repetitive luciferase assays demonstrated that isre motif just proximal to the transcriptional start site was exceedingly responsive to the ifn-treatment with a highest of -fold induction in luciferase activity as compared to the control vector without a promoter sequence (fig. c ). in the dsrna-dependent promoter induction (fig. e ), results demonstrated that the above elements and motifs (gaaann, spi and tata box) are least important in controlling the transcriptional activation of luciferase gene and a non-significant difference was observed in the absence or presence of these motifs. however, promoter sequence containing dual isre motifs severely compromised the responsiveness of dsrna on the ifit promoter and the dsrna-dependent promoter induction was restored when distal-isre was deleted, further suggesting the importance of this isre motif (fig. e ). similar to dsrna induction, the construct containing only one isre motif was highly inducible by ndv compared to the construct carrying both isre motifs (fig. e) . as was observed in the ndv-induced chmx promoter activation, all other constructs with variable lengths and motifs were also responsive to virus infections. comparison of these three stimuli indicated lower luciferase activities in ndv and dsrna-treated chicken cells compared to chifn-β induced promoter activation, presumably due to indirect activation of ifit promoter by inducing endogenous ifns. following the demonstration of robust reporter function of the chifit promoter-containing proximal isre motif by the chifn-β, dsrna and ndv, we compared the sequence of cis-acting regulatory elements with the previously characterized chmx promoter . in silico analysis of highly responsive bp sequence indicated that the isre motif ( ′-gctttcactttct- ′ at position − to − in pchifit − /+ construct) was identical to the consensus isre (g/a/t)g/ctttcn - tttc(a/t/c) found in most of the ifn-inducible genes including chmx promoter sequence (fig. a ). for further absolute delineation of importance of this motif, we mutated both triplet thymidines (ttt), the core residues for the ifn-inducible activation of genes ( fig. b and supplementary fig. c ). responsiveness of the wild type (wt) and mutated isre motifs was assessed in chicken df- cells with or without stimulation by chifn-β, dsrna or ndv using luciferase assays. while both pchifit - /+ and pchifit - /+ -mut promoters were not auto-stimulatory, stimulation with either of the stimuli positively regulated the luciferase genes in pchifit - /+ . interestingly, stimulation of mutated promoter with chifn-β ( . . chicken and human ifit restricts the replication of rna virus in stably expressing chicken fibroblasts. to assess the antiviral potential of chifit protein, we generated a lentivirus construct which bicistronically expresses chifit and a red fluorescent protein tagrfp under the control of emcv (encephalomyocarditis virus) internal ribosome entry sequence (ires) . cef cells were transduced with the appropriate vsv-g pseudotyped lentiviral particles and infected with gfp expressing ndv. after hours, the virus infectivity (gfp+) was quantified in lentivirus transduced (rfp+) cell population using fluorescence-activated cell sorting (facs) (fig. a ). lentivirus expressing firefly luciferase (ffluc), not expected to affect the virus replication, was used as a negative control. human irf (huirf ), a potent virus restriction factor , was used as a positive control ( fig. b and supplementary fig. a ). compared to ffluc control, both huirf and chifit significantly inhibited ndv replication (fig. b,c) , while the antiviral effect of chifit was greater than that of huirf in cefs. since two populations of gfp+ cells were observed, further gating of low gfp+ and high gfp+ indicated a profound and cumulative antiviral effect compared to ffluc control ( supplementary fig. b ). these results showed that the chifit -mediated antiviral effect is not profound at individual cell levels but it attenuates replication of the virus after initial infection. to compare anti-viral effects of chifit (only ifit gene identified in chickens so far) with its orthologous and homologous human proteins, huifit , huifit , huifit and huifit were expressed in chicken cells and antiviral affect was monitored (fig. a ). similar to chifit , both huirf and huifit showed significant inhibition of ndv replication whereas no antiviral activities were observed by other huifit proteins in chickens (fig. d ). to further demonstrate the effect of ifit -induced inhibition of ndv replication, we observed an exclusive replication of either ndv (gfp+) or transduction of lentivirus expressing ifit (rfp+) in % cells compared to ffluc expressing cells (n = ) (fig. e, upper panel) . it is possible that transduction of lentivirus particles may interfere with the virus entry and/or ndv replication. to examine the lentivirus-mediated restriction of ndv replication, we used ffluc-transduced cells in the control group. the analysis of several microscopic fields showed simultaneous expression of lentiviruses-transduced ffluc protein and ndv infection (fig. e , lower panel and fig. b ), demonstrating that the transduction of lentivirus particles doesn't interfere with the ndv replication. collectively, these results demonstrate that ifit proteins of both human and chicken are potent cellular restriction factors against rna virus infection in chicken primary cells. to further evaluate the antiviral effect of chifit on virus replication, we applied a loss-of-function approach using crispr/cas genome editing technology. using synthetic grna and cas expression plasmid (fig. a) , the chifit was targeted for editing. df- cells were co-transfected with hybridized crrna and tracr-rna as well as a vector expressing hspcas and puromycin resistance marker (puro r ) gene. for fast and efficient enrichment of genetic modification, a population of stably-integrated cells was selected with puromycin, and was further enriched using facs (fig. a) . the relative frequency of gene editing in the puromycin-resistant and facs-enriched cell was estimated from a dna mismatch detection assay using t endonuclease i (t e ) (fig. b) . t e assay showed a mutation frequency of % within the chifit gene, however, t e assays are likely to underestimate the fold enrichment . we subsequently sequenced the mutated sites and confirmed the in-frame or out-of-frame gene editing (fig. c) . results of the t e assay and sequencing showed that sgrna sufficiently edited the gene and puromycin selection greatly improved the enrichment of the cells. additionally, most of the mutations in the chifit gene appeared to be deletions, which introduce a pre-mature stop codon to the beginning of exon of the chifit gene. the chifit deletion-confirmed df- cells (chifit ko df- ) were isolated, expanded and assessed for virus replication. both wt df- and chifit ko df- were infected with ndv and vsv, and replication of viruses was monitored for hours. both ndv (fig. d) and vsv (fig. e ) replicated efficiently in wt df- cells over the time course of infection ( to hours). however, deletion of chifit by crispr/cas further supported the replication of ndv (fig. d) and vsv (fig. e) , confirming that chifit is a crucial antiviral effector and elimination of such factors weakens the hostile barriers of the host. these results also highlight the possible exploitation of innate immune genes in promoting virus replication for vaccine production. additionally, we applied facs to quantify the percentage infectivity of the wt df- and chifit ko df- cells for gfp-expressing recombinant ndv. data demonstrated that the wtdf- cells infectivity by ndv ( . %) could further be enhanced ( . %) in the absence of chifit (supplementary fig. ) . cumulative quantitative measurement of ndv-infectivity was significantly enhanced in chifit ko df- cells compared to wt df- cells (fig. f ). next, in order to counter-confirm the antiviral potential of chifit , the level of vsv replication was assessed in conventional plague assay in df cells either overexpressing or knocked out with chifit (fig. g) . the vsv replicated effectively in wt df- cells and overexpression of chifit suppressed the vsv whereas crispr/cas knockout df- cells substantially supported the virus replication (fig. g) . together, these results confirm the potential of chifit as an important host restriction factor, at least against evaluated rna viruses. chicken ifit interacts with ′ppp-containing rna. different human and mouse ifit proteins (ifit , ifit , ifit and ifit ) interact with rna carrying multiple genetic modifications at their ′ ends , , - . since ifit was the only identified ifit protein in chickens, we aimed to explore whether chifit interacts with rna in a similar mechanism reported for huifit or chifit -rna interaction is redundant to other members of ifit family. in order to explore the molecular mechanisms of chifit recognition of rna, we used bp rna without any known similarity to viral rna sequences and structure, and generated (i) rna bearing terminal ′ hydroxyl group ( ′ohrna), and (ii) rna bearing ′ triphosphate ( ′ppprna) (fig. a) . these populations of rna, mimicking viral rna ends, were biotinylated and coupled with agarose beads. the beads were then incubated with chicken df- cells expressing v -tagged chifit , the ribonucleoproteins were purified (fig. b ) and the interaction of chifit was determined by staining chifit . while neither huifit nor chifit interacted with the ′ohrna, both proteins recognized rna carrying ppp at the ′ end (fig. c) . overexpression of chicken chifit in transgenic chicken embryos restricts the replication of rna virus. we next asked whether the in vitro demonstrated antiviral activities of chifit are translatable in ovo in developing chicken embryos. for this purpose, we constructed rcasbp(a)-chifit recombinant viruses to generate mosaic-transgenic chicken embryos that are constitutively expressing chifit , and monitored its impact on the replication of ndv (fig. a) . in addition, a reporter virus, rcasbp(a)-egfp, was generated to monitor the rescue of the virus and to investigate the effect of retroviruses on host responses. rcasbp(a)-chifn-β, expressing chifn-β which is a known antiviral cytokine , was generated as a positive control (fig. a) . expression of this transgene did not compromise the replication of retroviruses, and the induction of innate immune responses was not significant (data not shown), confirming previous reports that rcasbp is a safe in ovo gene transferring system , . stable cells lines expressing retrovirus-mediated chifit and chifn-β were used to monitor virus replication. while mock-infected or rcasa(bp)-wt infected df- cells did not interfere with the replication of ndv (fig. b) and vsv (fig. c) , stable expression of chifit and chifn-β established a profound antiviral state against both viruses over the time course of infection. these functionally validated infectious df- cells were used to generate transgenic chicken embryos. three-day-old embryonated chicken eggs were inoculated with wt retroviruses or viruses that were expressing chifit or chifn-β to generate mosaic transgenic chicken embryos (fig. d) . additionally, embryos were either mock-inoculated with pbs or with non-infectious df- cells to exclude the possibility of antiviral effects by the retrovirus infected df- cells. these five groups of embryonated eggs were either mock-challenged with pbs or virus-challenged with ndv, and were incubated for another five days (fig. d ). compared to controls, retroviral expression of ifit negatively affected the development of healthy embryos until day post-embryonation before challenge with ndv (fig. e) . quantitative analysis of ndv in allantoic fluids showed that the wt retroviruses or non-infectious df- cells alone were unable to interfere with the replication of ndv, however, transgenic embryos stably expressing chifit or chifn-β significantly restricted the virus replication compared to the mock-treated control (fig. f) , indicating that chifit can restrict virus replication in developing transgenic chicken embryos. collectively, these results further confirm the antiviral activity of chifit in ovo. ablation of chicken chifit in transgenic chicken embryos ameliorates the replication of rna viruses. next, we asked whether silencing of endogenous chifit in developing embryos would facilitate the replication of ndv. for this purpose, we streamlined two individual gene delivery protocols for specific and optimal gene silencing in chicken cells and embryos. first, a total of three short hairpin rnas (shrna) targeting the chifit and a scrambled shrna with a highly confident predicted score were cloned in the prf-prnaic as described before (fig. a) . transfection of chicken df- cells with these vectors expressing shrna under a chicken u promoter showed a reliable level of expression of the rfp marker gene indicating the functional integrity of the system (fig. b) . although all shrna were effective in silencing, quantitative analysis of ndv-induced chifit mrna showed that shrna# was the most effective (> %) (fig. d) . next, the validated shrna cassette that included chicken u promoter, shrna, leader and trailer sequences was transferred to the compatible rcasbp(a) vectors for silencing of chifit in ovo (fig. c) . additionally, we used shrna silencing-resistant rcasbp(a)-chifit retroviruses ( supplementary fig. ) to stably express the chifit in developing embryos in the presence of shrna against endogenous chifit gene. the replication competency of shrna expressing retroviruses was compared to the wt retroviruses (fig. e ) before using them to generate transgenic chicken embryos. using the experimental strategy depicted in fig. f , we monitored the replication of ndv in transgenic embryos that were either stably expressing chifit or were expressing chifit in the endogenously silenced ifit gene. the retroviral overexpression of chifit or silencing of chifit were normalized to non-infectious df- cells or wt rcaspb(a) that did not express the transgene. quantitative analysis of ndv showed an expected reduction in replication of the virus in ifit -overexpressing embryos, whereas silencing of endogenous chifit favoured the replication of ndv compared to the control df- cells (fig. g) . a moderate reduction in the virus replication was observed in transgenic embryos that were simultaneously silenced for endogenous chifit and were stably expressing silence-resistant chifit (fig. g) . taken together, these results not only confirm the antiviral activities of chifit in different experimental settings but also highlight the potential use of rcaspb(a) in enhancing the replication of avian viruses in embryonated eggs. innate immune responses are key to the success of host resistance to virus infection and isgs provide a front-line role in these defences by acting at multiple stages of the virus replication cycle [ ] [ ] [ ] [ ] . among the myriad of isgs, members of the ifit family have attracted recent attention for both immunological as well as virological reasons wild type and chifit ko -confirmed df- cells were infected with ndv or were left uninfected for hours before processing them for facs. cumulative mfi of gfp positivity in wild type and chifit ko df- cells based on at least independent experiments. (g) df- cells were either mock transfected (wt df- cells) or were transfected with ug of chifit expression plasmid (chifit oe df- cells). these cells and crispr/cas knockout df (chifit ko ) were either left un-infected or were infected with vsv for hours before staining with crystal violet. the plagues developments were imaged using hand-held camera. scientific reports | ( ) : | doi: . /s - - -y due to their abundant and profound transcription and translation responses to diverse stimuli such as ifns and viruses , . significant advances have been made during studies of the mechanisms of both human and mouse ifit proteins . however, the knowledge on the breadth and plasticity of the antiviral properties of ifit proteins are currently inadequate for non-mammalian hosts. characterizing the repertoire of ifit proteins and investigating their functions in chicken are of special interest because of the unique features of the chicken immune system including the absence of essential components of innate immune induction and signalling such as rig-i, irf , and irf in chickens. intriguingly, while chickens are lacking these components, they still mount profound innate responses against virus infections. understanding the alternative means of innate immune regulation and antiviral defences in chicken could establish the foundation to control chicken-mediated emergence of zoonotic infections such as influenza viruses . in order to explore the ifit genes in chicken, we began with genetics and functional genomic approaches and revealed that chicken encodes only a single ifit gene compared to genes in mammals (human and mice). based on its sequence and structure similarities, and phylogenetic associations, this single chicken ifit gene is classified as ifit (chifit ). currently the chicken genome is about % annotated and all chromosomes are correctly characterized , however, the possibility of orthlogous and pseudogenes in the non-annotated part ( %) of the chicken genome cannot be excluded at this stage. with regard to the complex ifn pathways and a number of major missing genes in chicken, the innate immune genes, as general sensors of self and non-self, are under continues evolutionary selection pressure compared to the pathogen-specific adaptive immune responses. therefore, it is plausible that ifit proteins are generated by paralogue expansions and/or gene deletions in chicken . ifit is the only gene that is missing in the mice and rat genomes whereas it is the only gene identified in chicken, as this study shows. it remains to be determined in future if chifit also plays additional antiviral functions of the redundant ifit and ifit . we observed a profound transcription of chifit gene by diverse stimuli that acted upon the ifn induction or ifn signalling pathways, which was further confirmed by the transcriptomics profiling of the mdv-infected chicken fibroblasts. interestingly, structural and functional characterization of the chifit promoter, required for effective transcriptional regulation of chifit , was mapped within bp of the transcriptional start site and carried a single isre motif. this minimum essential requirement of the promoter justifies several folds induction of chifit gene as early as hours of post ndv-infection. although transcription of ifit was generally proportional to the virus replication and ifn induction, an ifn-independent regulation of chifit is also possible, especially when the chifit gene up-regulation was observed in earlier time points of virus-infection when the ifn gene was barely detectable. it is also plausible that transcriptional activation of chifit is directly regulated by irf or related transcription factors and thus warrants future investigations. nevertheless, these transcriptional patterns augment essential roles of the chifit protein during the early stages of virus replication such as interaction with viral genetic material (viral rna/dna) and viral protein translation. previous rna-protein analysis revealed that all ifit proteins assemble into multimeric complexes (except ifit ) and that these interactions are crucial for co-functionalities of these proteins . while all ifit proteins can make multimers, ifit exists as a poorly characterized monomer . probably due to these structural flexibilities, reports on ifit functionalities are inconsistent , - . however, ifit protein can arguably sense a broad range of rna structures including single stranded rna with mono-(p) and tri-phosphates (ppp) at the ′ ends, double stranded dna and rna with cap modifications , , . in order to understand the binding potential of chifit to modified rna that either interacts specifically with human ifit or with human ifit , we coupled modified rna-coated beads with quantitative binding assays for chifit . our results indicated that chifit specifically interacted with rna that carried ′-ppp modifications and failed to interact with rna in which ′-ppp was replaced with oh. these rna-protein interaction studies highlighted the principal roles of chifit for direct recognition of foreign ppp-rna and to subsequently exert downstream antiviral activities. since ppp-rna is found within the genome of most viruses carrying negative sense single stranded rna genomes such as influenza, ndv, vsv , and ppp-rna is produced as an intermediate product during the replication of viruses with positive sense rna genomes such as coronaviruses , it is plausible that chifit sense foreign rna (bearing ppp-rna) while ignoring self rna (bearing cap in the case of mrna and monophosphate in the case of rrna and trna). compared to four essential ssrna cellular sensors including rig-i, tlr , tlr and ifit in mammals , , rig- is missing and tlr is disrupted due to insertion of retroviruses in the tlr locus in chickens , , . because of these fundamental differences in innate immune genes between avian (e.g. chicken) and mammals, future studies are needed to investigate if the interaction of ifit with ′-ppp-ssrna can induce downstream ifn signalling , and if so, does this interaction compensate for the deficiency of rig-i and tlr in chickens. this specific interaction with ppp-rna could lead to attenuation of virus replication by sequestering viral rna for transcription and translation. to investigate this possibility, we applied both gain-in-function and loss-in-function methodologies and evaluated the antiviral potential of ifit against negative sense single stranded rna viruses, including a poultry specific (ndv) and model rna virus (vsv). lentivirus-delivered stable expression of chifit or huifit compromised the replication of viruses, whereas crispr/cas mediated knocking-out of the chifit gene supported virus replication in chicken fibroblasts. intriguingly, overexpression of human ifit , ifit and ifit failed to establish an antiviral state in chicken fibroblasts, suggesting that chickens have opted exclusively for the antiviral activities of ifit . our attempts to monitor virus replication in mosaic transgenic chickens overexpressing chifit , or silenced for endogenous chifit yielded strong evidence that this cytokine possesses antiviral activities in developing embryos. thus rcas-mediated gene delivering and silencing approaches can be exploited to study gene functionalities in ovo at the early embryonic developmental stage and may establish the basis for evaluation of genetic resistance against pathogens. taken together, we characterized the ifit locus in chicken and systemically analysed the functional rationale for antiviral activities of chifit against rna viruses using both functional genomics and molecular biological approaches. the foundations built in this study warrant future investigations to assess the potential of chifit in sensing the nucleic acid of many diverse viruses and bacteria (which also generate ppprna), and the impact of these interactions on host innate immunity. data mining and sequence analysis. chicken genome (ensembl) and expressed sequence tags (est) databases were screened for the homologues of ifit family gene members using the basic local alignment search tool (blast) algorithm. a single transcript showing high sequence-similarity to human ifit was identified in the putative ifit locus. using sequences from public databases and transcriptomics data from marek's disease virus (mdv)-infected chicken embryo fibroblasts (cef), an open reading frame (orf) was revealed and extracted. the chicken ifit (chifit ) coding region was amplified from ndv-infected primary cefs, whereas sequence-covering gap in the ifit locus was amplified from genomic dna using primers mentioned in supplementary table . the genomic nucleotide sequence of chifit promoter region was amplified using primers provided in supplementary table . the orf and homology searches for chifit were carried out in the orf finder programme (http://www.ncbi.nlm.nih.gov/projects/gorf) and blast tool (http://www.ncbi.nlm.nih.gov/ blast/) integrated in the ncbi database. possible gene transcription start sites were identified using promoter predictor programme (http://www.fruitfly.org/seq_tools/promoter.html) whereas potential transcription factor binding sites were identified using the matinspector server (http://www.genomatix.de). gene synteny and tetratricopeptide repeats (tpr) were predicted using ensemble as well as conserved domain databases, respectively. the ifit sequences from aves and non-aves were acquired from ncbi and aligned using clustalw programme. the phylogenetic analysis was performed using neighbour-joining method with bootstrap value of n = , in the mega programme version . cells culture, media and antibodies. cefs were prepared from -day-old embryonated eggs at the pirbright institute as described previously . immortalized chicken fibroblasts (df- ), human embryonic kidney cells t (hek- t) and madin-darby canine kidney (mdck) cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with % foetal bovine serum (fbs), % penicillin and streptomycin (p/s) at °c in % co incubator. amv- c -s (gag) antibodies were purchased from hybridoma bank of iowa, university of iowa. α-v and j antibodies for the detection of v tag and dsrna were from genetex, and scicons, respectively. alexa-flour secondary antibodies were purchased from invitrogen carlsbad, ca, usa and irdye cw α-mouse secondary antibodies were acquired from li-cor, nebraska usa. poly i:c scientific reports | ( ) : | doi: . /s - - -y (a synthetic analogoue of dsrna), dimethyl sulfoxide (dmso) and lipopolysaccharide (lps), were purchased from invivogen and sigma whereas chicken ifn-β was produced in hek- t cells . (ndv-gfp) was generated using reverse genetics system as described before and rescued virus particles were propagated in embryonated chicken eggs . the ndv-gfp strain was quantified using immunostaining and expressed as focus-forming units (ffu). vesicular stomatitis virus (vsv) expressing gfp (vsv-gfp) was kindly provided by dennis rubbenstroth (institute for virology, medical centre -university of freiburg, germany). vsv-gfp was propagated and quantified in df- cells and was represented in ffu or images showing plaques. allantoic fluids and infectious viruses from cell culture supernatants were titrated by plaque assays on mdck cells. briefly, mdck cells were inoculated with -fold serially diluted samples and overlaid with . % agarose (oxoid, hampshire, uk) in overlay dmem ( × mem, . % bsa v, mm l-glutamate, . % sodium bicarbonate, mm hepes, × penicillin/streptomycin (gibco, carlsbad, ca, usa) and . % dextran deae, with µg ml − tpck trypsin (sigma-aldrich, dorset, uk). plates were incubated at °c for h and plaques were developed using crystal violet stain containing methanol. plasmids construction and mutagenesis. the full-length chicken and human ifit was pcr-amplified using primers that were tailed with ′ bamhi site, ′ ecori/spei site and a consensus kozak translation sequence (ccaccatg) (supplementary table ). the bamhi and ecori/spei digested amplicons were sub-cloned in the mammalian expression vector, pefplink-v (kindly provided by steve goodbourn, st. george's university of london), which contains an n-terminal v tag. for identification of cis-acting elements in the chifit promoter, a . kb genomic sequence was amplified and cloned into the kpni and xhol sites in the promoter-less vector, pgl . basic (promega) and named as pchifit - /+ . subsequent five truncated versions of the promoter were amplified from full-length pchifit - /+ using primers mentioned in the supplementary table and cloned between kpni and xhol sites in the pgl . basic vector. for production of the chifn-β, the orf for chifn-β (accession number, nm_ ) was cloned in pcdna . + and final constructs were labelled as pcdna . -chifn-β . the reporter plasmid pgl -p-chmx-luc was kindly provided by nicolas ruggli, switzerland and renilla luciferase plasmid (phrl-sv ) was purchased from promega, madison, wi, usa. mitochondrial (dsred -mito- , # )) and er (mcherry-er- , # ) markers were obtained from addgene. triple thymidine duplex (tttnnnttt) in pchifit - /+ -wt construct was mutated into tatnnntat using quikchange lightning site-directed mutagenesis kit (agilent technologies) and was named as pchifit - /+ -mut. all mutagenesis oligonucleotides were designed in the quikchange primer design tool and these primers are provided in the supplementary table . all clones were sequenced from both ends for correct frame and orientation or were digested with unique cleavage sites to confirm the gene inserts. confocal microscopy. chicken cells were transfected with individual or combined plasmids for indicated time points using lipofectamine (invitrogen) at a ratio of : or were infected with lentiviruses, retroviruses or ndv-gfp for indicated time points. these cells were then fixed for h in % paraformaldehyde and permeabilized using . % triton-x before incubation with primary antibodies raised against v tag, dsrna (j ), or gag protein of retroviruses. additionally, depending upon the experimental needs, different fluorescent markers (rfp, gfp) were used. afterwards, cells were incubated with corresponding secondary antibodies at °c for h. after brief staining with ′, -diamidino- -phenylindole (dap ) (nuclear), slides were visualized using a leica sp confocal laser scanning microscope. western blotting. all the transfections for subsequent western blot analysis were performed following the same protocol as described for immunofluorescence unless otherwise indicated. cells were lysed in a hypotonic buffer and protease inhibitor cocktail (sigma). proteins were separated by sds-page under reducing conditions and analysed by western blotting using anti-v (gentek) and irdye-labelled secondary antibodies (li-cor biosciences). the signals were acquired and quantified using the odyssey infrared imaging system (li-cor biosciences). ifn bioassay. ifn-induced protection against vsv-gfp was used to identify ifn-producing stable clones and to quantify ifn preparations, as described before . briefly, df- cells were seeded in -well plates until they are % confluent and treated with serial dilutions of supernatants containing interferons for hours. these interferon stimulated cells were inoculated with vsv-gfp (moi of ). at hours post-infection, vsv-gfp replication was correlated with the change in gfp fluorescence signal intensities using luminometer (promega, madison, wi, usa). the percentage antiviral activity of ifns were determined by comparing the percentage reduction of corrected gfp signal intensity (gfp signal intensity of ifn treated and virus infected wells minus background fluorescence signal intensity of uninfected control) with the mock treated and vsv-gfp-infected control wells. one unit (u) of ifn in the tested ifn preparations was defined as the volume containing % inhibitory activity against vsv-gfp. a total of us of ifns were used for stimulation of cef or df- cells. tronic expression and gateway-compatible destination vector (ptrip.cmv.ivsb.gene.ires.tagrfp) for lentiviruses was kindly provided by charles rice, the rockefeller university, usa. to generate chifit entry clone, gene encoding chifit was pcr amplified with oligonucleotides (supplementary table ) containing attb sites flanking gene-specific sequences. pcr products were purified over qiagen columns (qiagen) and cloned into pdonr (invitrogen) with bp clonase. bp clonase reactions were transformed into escherichia coli (invitrogen), and colonies were screened by restriction digestion and sequencing. the gene sequences from pentr clones scientific reports | ( ) : | doi: . /s - - -y were moved into ptrip.cmv.ivsb.gene.ires.tagrfp using lr clonase ii (invitrogen) according to the manufacturer's instructions. after lr reaction products transformation, one or two colonies for each construct were grown in ml luria-bertani (lb) broth with ampicillin, and transfection-quality plasmid dna was purified over anion-exchange columns (qiagen). lentivirus constructs expressing human ifit , ifit , ifit , ifit , irf (positive control) and ffluc (negative control) were kindly provided by charles rice, the rockefeller university, usa. all constructs were sequenced using primers provided in supplementary table to confirm the gene insertion before rescuing lentiviruses. poly-lysine pre-coated plates with seeding density of × per well of -wells plates and were co-transfected with gene expressing proviral dna (huifit , huifit , huifit , huifit , chifit , huirf , or ffluc), hiv-i gag-pol and vsv-g in a ratio of : . : . using lipofectamine (invitrogen). supernatants collected at h and h post-transfection were cleared by centrifugation ( rpm for min) and were pooled and supplemented with mm hepes and μg/ul polybrene (sigma). for titration of lentiviral pseudoparticles, cef cells ( × ) were transduced with serial dilutions of individually rescued pseudoparticles for hours. trypsinized cells were fixed with % paraformaldehyde and processed for facs for quantification of percentage rfp+ cells. the volume of the lentiviral pseudoparticles that infected % of cef cells was used to transduce cefs. titrated lentiviral pseudoparticles were stored at − °c before use and the same stock of the virus was used for all experimentation. duced with moi of lentivirus-expressing specific gene in dmem media containing % fbs, mm hepes and μg/ml polybrene. transduction was facilitated by centrifugation ( g for h at °c) and cells were incubated at °c. a day later, cells were infected with gfp-tagged virus (ndv-gfp) at . moi and the infection was stopped after hr and replaced with fresh dmem containing % fbs. after hours of infections, cells were trypsinised and the cell suspensions were incubated with live dead marker (near ir cat no: l ) according to the manufacturer's protocol. the cells were then fixed with % paraformaldehyde (pfa) for min before analysing the cells by flow cytometry. as described before , live and singlet cells were gated based on forward and side scatter, and four-quadrant plots were generated using the untransduced and uninfected (rfp negative and gfp negative), uninfected (rfp positive and gfp negative), and untransduced (rfp negative and gfp positive) cells. analysis was carried out using flowjo software applying the same gating and analysis strategies for all samples. construction of ifit knockout cell line using crispr/cas . a synthetic gene-targeting approach was applied to specifically knockout the chicken ifit from the chicken embryo fibroblasts. for this purpose, two components (crrna and tracrrna) of single guide rna (sgrna) which are crucial for targeting specificity and scaffolding/binding ability for crispr associated protein (cas ) nuclease were synthesized by dharmacon. targeting the beginning of the second exon of chicken ifit , two individual crispr rna (crrna) were designed with the highest-predicted score and lowest off-target affects; sgrna acaggagaagtctcgttacc and sgrna gcttggatctactaccacat. using dharmafect duo transfection reagents, df- cells were co-transfected with individual crrna and a common trans-activating crrna (tracrrna) as well as plasmid expressing cas nuclease and puromycin resistance gene (puro r ) separated by self-cleavage t a sequence. cells were split after hours transfection and selected for puromycin antibiotics ( μg/ml) for one week or until complete eradication of non-transfected control cells. for fast and efficient enrichment of genetic modification, a population of cells with stable integration was enriched using facs. for this purpose, puromycin-selected cells were transfected with gfp-expression plasmid and individual cells were sorted by facs before being seeded in -well plates. at least clones were expanded and the relative frequency of gene editing in the puromycin-resistant and facs-enriched cells was estimated from a dna mismatch detection assay using t endonuclease i (t e ) (neb). the dna fragments flanking the target editing sites were amplified from genomic dna extracted by dneasy kits (qiagen) using primers mentioned in supplementary table . a total of ng of the pcr products were denatured at °c and allowed to anneal gradually at room temperature to form heteroduplex dna. the re-hybridized dna was digested with t ei and resolved in a . % agarose gel to determine the gene editing efficiency. additionally, the pcr products were sequenced using pcr-amplification primers and aligned with the corresponding wild-type genomic sequence to identify mutations, deletions and insertions. t ei and sequence verified clones were used to monitor virus replication. rna was extracted from ifn-β ( u), lps ( μg/ml), dsrna ( μg/ml), or ndv-stimulated (moi of ) df or cefs using trizol reagents (invitrogen, carlsbad, ca, usa). additionally, organs were collected from specific pathogen free (spf) chickens, which were infected or mock-treated (intranasally) for days with pfu of a/ chicken/pakistan/udl- / (h n ). a total of ng of rna was used in pcr reactions using superscript ® iii platinum ® sybr ® green one-step qrt-pcr kit (invitrogen, carlsbad, ca, usa). the abundance of specific mrna was compared to the s rrna (supplementary table ) in the applied biosystems prism system. the reaction was carried out in abi light cycler using the following thermo profile; °c for minutes hold, °c for minutes hold, followed by cycles of °c for seconds and °c for seconds. melting curve was determined at °c for seconds, °c for minute, °c for seconds and °c for seconds. primers for isgs including chifit are provided in supplementary table . primers specific for a conserved region of the influenza a and ndv matrix genes were used as described previously , . short hairpin design and expression systems. to silence endogenous chifit gene in developing embryos, a total of three -nucleotides-long rna interference (rnai) short hairpin rnas (shrna) were designed using the genscript rnai target finder (https://www.genscript.com/ssl-bin/app/rnai). double stranded dna products for each of three chifit specific and a control scrambled target were generated by pcr using random and gene-specific oligonucleotides together with hp-l and hp-r (supplementary table ) as described before . the amplified pcr products were cloned between nhei and mlui into microrna (mirna) cloning sites of prfprnaic (kind gift of stuart wilson, the university of sheffield, uk). all shrna coding plasmids were sequenced to confirm the inserts and orientations. to evaluate the silencing potential of individual shrna, df- cells were transfected with ng plasmid using lipofectamine according to the manufacturer's protocol and the knockdown effects on the chicken ifit was monitored and compared with the scrambled rna transfected control. next, the validated shrna cassette that included chicken u promoter, individual shrna, leader and trailer sequences was transferred to the rcasbp(a) retrovirus vector between noti and clai sites. rescue of rcasbp(a)-sh ifit retroviruses and generation of mosaic transgenic chickens are detailed below. to determine responsiveness of chicken ifit promoter to chicken ifn-β, dsrna, and virus-stimulation, chicken fibroblasts were grown in -well plate format at × to × cells in addition pgl -p-chmx-luc was used as a positive control whereas pgl . basic vector was used as a negative control. correspondingly, df- cells were co-transfected with phrl-sv and pchifit - /+ -wt or pchifit - /+ -mut constructs. all transfections were performed using lipofectamine sk-as) was linearized with unique bamhi restriction digestion and the purified dna was used for in vitro transcription in the presence of bioin- -utp using ribomax ™ large scale rna production system-sp (promega, cat# p ) as recommended by the manufacturer and reported previously with a few modifications. briefly, a reaction of μl was established containing μl × sp buffer, μl ntp-bioutp mixtures, μg linearized plasmid, and μl enzyme mix. the reaction mixture was first incubated at °c for hours followed by digestion of the dna remnant with u rnase-free dnase (thermo scientific) for another minutes at °c. the biotinylated uracil triphosphate (bioin- -utp) was incorporated during in vitro transcription for purification of ribonucleoproteins and due to nascent nature of polymerase a ′-ppprna was over-hanged as a signature for ifit protein interaction. after the completion of in vitro transcription, the quality of in vitro transcription rna was assessed by agarose gel electrophoresis and rna was purified with rneasy minelute cleanup kit (qiagen) according to the manufacturer's recommendations. a total of μg of purified in vitro transcribed and biotinylated ppp-rna was either mock treated or rna samples were purified with rneasy minelute cleanup kit and eluted with μl nuclease-free water for use in the rna-protein interactions to prepare chifit protein, chicken df- cells ( × ) were transfected with μg v -tagged chifit plasmid for hours and lysed with tap buffer in the presence of protease and rnase inhibitors. the rna-coated beads were incubated with mg chifit protein lysate on a rotary wheel for hours at °c, and washed three times to remove unbound proteins the gfp/gag expression-confirmed cell cultures were split into cm flasks and were passaged again into cm flasks after days. finally cells were expanded into cm flasks until the desired number ( cells/egg) was achieved. mosaic-transgenic chicken embryos were generated by inoculation of one million rcasbp(a)-chifn-β, rcasbp(a)-chifit , rcasbp(a)-shrna and rcasbp(a)-wt infected df- cells at day post-embryonation or were left un-infected. at day post-embryonation ( days post-infection), each egg was either left unchallenged or was challenged with pfu ndv-gfp. embryo mortality was monitored daily and allantoic fluids were harvested at days of embryonation and were subjected to the plaque assay for virus quantification. statistical analysis. pairwise comparisons of treated and control groups were performed using student's t-test interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures cytosolic sensing of viruses interferon-stimulated genes and their antiviral effector functions interferons and their actions lineage-specific expansion of ifit gene family: an insight into coevolution with ifn gene family ifit is an antiviral protein that recognizes ′-triphosphate rna structural basis for viral ′-ppp-rna recognition by human ifit proteins interferon-induced ifit proteins: their role in viral pathogenesis isg is a negative-feedback regulator of virus-triggered signaling and cellular antiviral response ifit potentiates anti-viral response through enhancing innate immune signaling pathways trna binding, structure, and localization of the human interferon-induced protein ifit broad and adaptable rna structure recognition by the human interferon-induced tetratricopeptide repeat protein ifit conventional and unconventional mechanisms for capping viral mrna eukaryotic ribonuclease p: a plurality of ribonucleoprotein enzymes when your cap matters: structural insights into self vs non-self recognition of ′ rna by immunomodulatory host proteins sequestration by ifit impairs translation of ′ o-unmethylated capped rna inhibition of translation by ifit family members is determined by their ability to interact selectively with the ′-terminal regions of cap -, cap -and ′ ppp-mrnas the broad-spectrum antiviral functions of ifit and ifitm proteins the rcas vector system the glucocorticoid attenuated response genes garg- , garg- , and garg- /irg encode inducible proteins containing multiple tetratricopeptide repeat domains when two strands are better than one: the mediators and modulators of the cellular responses to double-stranded rna double-stranded rna is detected by immunofluorescence analysis in rna and dna virus infections, including those by negative-stranded rna viruses activation of the pkr/eif α signaling cascade inhibits replication of newcastle disease virus the chicken mx-promoter contains an isre motif and confers interferon inducibility to a reporter gene in chick and monkey cells differential expression profile of chicken embryo fibroblast df- cells infected with cell-adapted infectious bursal disease virus triggering the innate antiviral response through irf- activation the rna polymerase ii core promoter genotyping with crispr-cas-derived rna-guided endonucleases distinct induction patterns and functions of two closely related interferoninducible human genes, isg and isg crystal structure and nucleotide selectivity of human ifit /isg coordinated regulation and widespread cellular expression of interferon-stimulated genes (isg) isg- , isg- , and isg- in the central nervous system after infection with distinct viruses prolonged effect of baff on chicken b cell development revealed by rcas retroviral gene transfer in vivo antiviral activity of lambda interferon in chickens a robust system for rna interference in the chicken using a modified microrna operon association of rig-i with innate immunity of ducks to influenza rig-i in rna virus recognition a virological view of innate immune recognition identification and characterization of a functional, alternatively spliced toll-like receptor (tlr ) and genomic disruption of tlr in chickens molecular evolutionary genetics analysis version . cell-culture methods a novel cytokine with antiviral activities tissue tropism in the chicken embryo of non-virulent and virulent newcastle diseases strains that express green fluorescence protein biological characterization and phylogenetic analysis of a novel genetic group of newcastle disease virus isolated from outbreaks in commercial poultry and from backyard poultry flocks in pakistan a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity development of a real-time reverse-transcription pcr for detection of newcastle disease virus rna in clinical samples development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h and h hemagglutinin subtypes construction and rescue of ifit expressing rcas system, and generation of transgenic embryos. the orf of chicken ifit and coding regions (signal and mature peptide sequence) of the chifn-β were amplified from rna extracted from the ndv-infected primary cefs. the amplified products were sub-cloned into an improved version of rcasbp(a)-Δf (kindly provided by stephen h. hughes, national cancer institute, md, usa) via the clai and muli restriction sites which replace the src gene while maintaining the splice accepter signals. the resultant constructs were named as rcasbp(a)-chifit and rcasbp(a)-chifn-β. similarly, a gfp encoding rcasbp(a), referred as rcasbp(a)-egfp, was generated by introducing the coding sequence of the gfp in between the clai and muli sites. additionally, a codon optimized and shrna-silencing resistant chifit gene was chemically synthesized (geneart, invitrogen) and cloned in the corresponding sites and labelled as rcasbp(a)-shrna . the inserted gene orientation and sequence validity were confirmed by dna sequencing.to rescue recombinant rcasbp(a) viruses, a total of . × df- cells were seeded in cm flasks and maintained at °c, % (vol/vol) co for hours (~ % confluent). cells were washed with pbs and transfected ethics statement. all animal studies and procedures were carried out in strict accordance with the guidance and regulations of european and united kingdom home office regulations under animal risk assessment numbers ar and ar . as part of this process the work has undergone scrutiny and approval by the ethics committee at the pirbright institute. supplementary information accompanies this paper at https://doi.org/ . /s - - -y. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - bmdzgla authors: sun, xinjuan; wang, tianyuan; cai, dayong; hu, zhiwei; chen, jin’an; liao, hui; zhi, liming; wei, hongxia; zhang, zhihong; qiu, yuying; wang, jing; wang, aiping title: cytokine storm intervention in the early stages of covid- pneumonia date: - - journal: cytokine growth factor rev doi: . /j.cytogfr. . . sha: doc_id: cord_uid: bmdzgla clinical intervention in patients with corona virus disease (covid- ) has demonstrated a strong upregulation of cytokine production in patients who are critically ill with sars-cov -induced pneumonia. in a retrospective study of patients with covid- , most patients with sars-cov- infection developed mild symptoms, whereas some patients later developed aggravated disease symptoms, and eventually passed away because of multiple organ dysfunction syndrome (mods), as a consequence of a severe cytokine storm. guidelines for the diagnosis and treatment of sars-cov- infected pneumonia were first published january (th), ; these guidelines recommended for the first time that cytokine monitoring should be applied in severely ill patients to reduce pneumonia related mortality. the cytokine storm observed in covid- illness is also an important component of mortality in other viral diseases, including sars, mers and influenza. in view of the severe morbidity and mortality of covid- pneumonia, we review the current understanding of treatment of human coronavirus infections from the perspective of a dysregulated cytokine and immune response. of global disease burden (johns hopkins university -https://coronavirus.jhu.edu/map.html) is over . million infections and , deaths worldwide. social distancing in cities and countries around the world remains the only means available to limit the impact of virus transmission. at the same time, an unprecedented response from the world's biomedical research community seeks to identify treatments for covid- that include antiviral drug and vaccine development. current clinical observations indicate that sars-cov infection can range from an inapparent non-symptomatic infection, to a respiratory illness presenting with spiking fever and dry cough, accompanied by a high rate of human-human transmission. one of the most serious complications of corona virus disease (covid- ) is the development of an atypical upper respiratory tract pneumonia that poses a major challenge to clinicians in terms of disease management. an abnormal and uncontrolled production of cytokines has been observed in critically ill patients with covid- pneumonia and the ensuing uncontrolled cytokine storm in covid- patients is centrally involved in the exacerbation of symptoms and disease development, and represents a major factor contributing to covid- mortality. in this sense, covid- disease shares similarities with other viral diseases such as sars, mers and influenza, where the development of a cytokine storm is a warning sign of disease escalation. in support of the above observations, a retrospective study of patients with covid- showed that most sars-cov- infected patients present clinically with mild symptoms, while a minority of patients progressively declined from the infection and eventually died of acute respiratory distress syndrome (ards) and multiple organ dysfunction syndrome (mod). guidelines for the diagnosis and treatment of sars-cov- infected pneumonia were first published on january th , and recommended for the first time that cytokine monitoring be applied to improve the curative rate and reduce mortality . in view of the severe morbidity and mortality of covid- pneumonia, here we review the current understanding of treatment of human coronavirus infections from the perspective of a dysregulated immune response. the emerging and re-emerging viruses (ebola, zika, chikungunya, h n , dengue, sars, mers) have led to numerous global public health crises in recent years and continue to threaten public health and security. unfortunately, these viruses are often difficult to control due to the lack of approved antiviral drugs and vaccines. in addition to sars-cov , two other novel coronaviruses have emerged as global health threats since , namely severe acute respiratory syndrome coronavirus (sars-cov in ) that was transmitted to countries, and the middle east respiratory syndrome coronavirus (mers-cov in ) that was transmitted to countries. severe pneumonia caused by pathogenic human coronaviruses (hcov) are often associated with induced hypercytokinemia, also termed cytokine storm, in immunocompetent individuals; uncontrolled overproduction of inflammatory cytokines contributes to acute lung injury and acute respiratory distress syndrome (ards). the secretion of multiple cytokines, also termed cytokine release syndrome (crs), is closely j o u r n a l p r e -p r o o f related to development of clinical symptoms; for example, ifn-γ can cause fever, chills, headaches, dizziness, and fatigue; tnf-α can cause flu-like symptoms similar to ifn-γ, with fever, general malaise, and fatigue, but can also cause vascular leakage, cardiomyopathy, lung injury, and acute-phase protein synthesis . il- , which is an important target in crs induced by adoptive cell therapy, can lead to vascular leakage, activation of complement and the coagulation cascade, leading to the characteristic symptoms of severe crs, such as diffuse intravascular coagulation (dic) , . it is noteworthy that il- is likely to cause cardiomyopathy by promoting myocardial dysfunction, which is often observed in patients with crs . in addition, activation of endothelial cells may also be one of the hallmarks of severe crs. endothelial dysfunction can lead to capillary leakage, hypotension, and coagulopathy ( figure ). taken together, these studies argue that the virus-induced immunopathological events play a crucial role in the fatal pneumonia observed after hcov infections . the development of a cytokine storm is a potentially fatal immune condition characterized by rapid proliferation and hyperactivation of t cells, macrophages, natural killer cells and the overproduction of more than inflammatory cytokines and chemical mediators released by immune or nonimmune cells , . in viral infections, the aberrant release of pro-inflammatory factors leads to lung epithelial and endothelial cell apoptosis which damages the lung microvascular and alveolar epithelial cell barrier, leading to vascular leakage, alveolar edema and hypoxia. the uncontrolled production of pro-inflammatory factors, containing il- , il- , il- β, and gm-csf, and chemokines such as ccl , ccl- , ip- , and ccl , together with reactive oxygen species cause ards leading to pulmonary fibrosis and death . in sars-cov infected patients, high levels of serum pro-inflammatory cytokines (ifn-γ, il- , il- , il- , and tgfβ) and chemokines (ccl , cxcl , cxcl , and il- ) were detected in cases of severe disease compared to patients with uncomplicated sars . in mers infection, high levels of serum pro-inflammatory cytokines (il- and ifn-α) and chemokines (il- , cxcl- , and ccl ) were likewise observed in severe disease, compared to mild or moderate disease . in contrast to sars and mers, doctors in wuhan central south hospital found that the levels of il , il , il , gscf, ip , mcp , mip a and tnf α were significantly elevated in the blood of severely ill patients, compared to patients with mild illness . the level of il- in the blood of the severe group was % higher than that of the mild group ( %) . furthermore, histological examination and biopsy samples obtained from a patient who died from severe sars-cov- infection showed an increased concentration of highly proinflammatory ccr +ccr + th + cd t cells, suggesting that t cell hyperactivation contributesdin part to the severe immune injury in this patient . pulmonary examination in other patients with early phase sars-cov- pneumonia also revealed patchy inflammatory cell infiltration; however, the pathological results in early stage of sars-cov pneumonia require further confirmation . in short, aberrant release of multiple cytokines appears to trigger a cytokine storm that produces immunopathogenic damage to tissues and organs, even while the immune response seeks to suppress and eradicate the virus (figure ). j o u r n a l p r e -p r o o f in view of the above observations, therapeutic strategies to treat the cytokine storm in the pathogenesis of severe covid- pneumonia deserve special attention. in accordance with current who guidance , supportive therapy remains the most important management strategy for this pneumonia, including supplemental oxygen therapy, conservative fluid management and empiric antimicrobial application in the time of need. it is noteworthy that the use of glucocorticoids remains controversial. current who guidance recommends that corticosteroids should not be used in sars-cov -induced lung injury or shock, except in the setting of a clinical trial. however, in clinical settings, front-line physicians in china tend to use corticosteroids prudently at a low to moderate dosage in patients with sars-cov infection . in the period of sars epidemic ( ) ( ) , corticosteroids were generally used by clinicians for immunomodulatory treatment, and according to clinical feedback, could bring early beneficial changes including a decline in fever, resolution of radiographic lung infiltrates, and the improvement of oxygenation , . however, further studies indicated opposite clinical outcomes and a systematic review in sars, mers and influenza infections indicated no survival benefit and possible harm (avascular necrosis, psychosis, diabetes, delayed viral clearance and secondary infections) with corticosteroids . in our opinion, the use of corticosteroids is not recommended for patients with hcov infections, although corticosteroids may be used prudently in critically ill patients. in the treatment of patients with sars-cov- infection, clinicians should pay close attention to the impact immune inflammatory factor release, and several effective cytokine storm blockers and therapeutic methods have been reported. in the clinical process of covid- pneumonia, there is window period between the diagnosis and the occurrence of mods (about - days). after this window, the majority of patients tend to improve (~ %), whereas ~ % of the patients progress to severe pneumonia, with ~ % mortality , , . to improve the prognosis, we suggest that patients with covid- pneumonia be given immunotherapy treatment at the time of diagnosis, in order to block the possibility of a subsequent cytokine storm. the early use of immunological intervention in the evaluation of patients with mods may reduce mortality in the most severe patients. ( figure , position ). one strategy that has received considerable attention in the face of covid- is the use of convalescent plasma, also called passive antibody therapy, to treat patients with advanced disease. the treatment uses plasma from a patient who has survived covid- infection to provide neutralizing antibodies against the virus; the antibodies are available and active immediately, but only for a limited time. as an example of one study, five patients critically ill with covid- and on mechanical ventilation received convalescent plasma to days after being admitted to a hospital in shenzhen, china. all patients improved; three were discharged after days in hospital, while the other two patients were in stable condition. well controlled clinical evaluation of this strategy is currently ongoing in light of such positive anecdotal responses. another potential treatment involves the use of anti-il- monoclonal antibody (siltuximab) (johnson & johnson) which was used previously to treat the consequences of cytokine storm following by car-t cell therapy for cancer. interleukin- (il- ) has become a key point in some crs. originally described as b-cell differentiation factor- (bsf- ) and macrophage and granulocyte induction factor- (mgi- ), il- has significant pro-inflammatory and pyrogenic properties, and given chronic overproduction in covid- patients, anti-il- monoclonal antibody may be beneficial in controlling cytokine release. also il- inhibitor (secukinumab) (novartis) was used as specific treatment for severe patients with covid- pneumonia to control th cell activation. an additional observation indicated that the expression levels of cd + t and cd + t were low, while il- levels were high in patients with severe pneumonia, suggesting that t cell subsets and cytokine levels could be used as one predictor of the transition from mild to severe pneumonia . pegylated and non-pegylated interferons (ifn) have been under intensive study for some time. however, in the case of hcov infection, the results of ifn therapy were mixed, as predicted by animal and human hcov infection models. early application of ifn was slightly beneficial to reduce viral load and improve clinical outcome. however, delayed ifn administration was of no benefitscompared with the placebo group. early application of ifn and ribavirin moderately improved disease severity without affecting mortality . the use of sifalimumab monoclonal antibody, produced against multiple ifn subtypes (medimmune) has not been examined clinically but could hold promise in situations of constitutive ifn production. the early use of ifn had some beneficial effects in sars-cov-infected animal model, although the timing of ifn treatment was crucial in determining the course of disease. the use of ifn-αβ receptor inhibitors or antagonists could be considered as an approach to avoid excessive inflammatory reactions in late stage severe hcov infection . since sars-cov and mers-cov infect mainly airway epithelial cells and ifn-λ stimulates antiviral gene expression in these cells without over-stimulating the immune system, ifn-λ might be a option in the therapy of hcov infection. oxidized phospholipids (oxpl) have been demonstrated to promote acute lung injury by increasing the production of cytokines/chemokines from lung macrophages via tlr -trif signaling in influenza a virus (iav)-infected mice . in a recent study, therapeutic administration of the tlr antagonist eritoran protected mice from lethal iav infection by decreasing the levels of oxpl and inflammatory cytokines/chemokines . because pathogenic human coronaviruses can cause acute lung injury and promote the production of oxpl in the lungs, the strategies of oxpl suppression by using eritoran (boc sciences) or other similar compounds may have value in controlling hcov induced inflammation. signal transduction by the sphingosine- -phosphate receptor (s p ) in mouse endothelial cells j o u r n a l p r e -p r o o f nfected with influenza a virus has been shown to contribute to the pathogenesis of inflammation responses . targeted s p antagonism inhibited the recruitment of inflammatory cells, limited proinflammatory cytokine/chemokine release, and reduced mortality and morbidity induced by influenza a virus . s p antagonism may be considered a potential therapy in hcov-infected individuals to limit cytokine responses. the results of studies in animal models have borne out claims that imms paly pathogenic roles in the process of fatal hcov infections. in the mice cardiac inflammation model, the systemic administration of optimized lipid nanoparticles including ccr -silenced small interfering rna (sirna) can effectively degrade ccr mrna and destroy the imm recruitment in the inflammatory site, thus improving the outcome of the disease . because hcovs are single-chain rna viruses and tlr agonist r (a synthetic single-chain rna analog) stimulates imms which causes exuberant inflammatory response. therefore, imm specific tlr- signal may promote the excessive inflammatory response caused by hcov infection. therefore, a tlr antagonist targeted approach to alleviate inflammation reaction could be useful. continuous renal replacement therapy (crrt) may benefit severe patients by removing potentially harmful components and maintaining haemodynamic and metabolic status. in addition to the conventional aim of maintaining renal function, crrt can be used to regulate the immune response of patients with sepsis, with the goal to regulate circulating levels of inflammatory cytokine mediators, as shown in studies demonstrating removal of inflammatory mediators containing cytokines and complement (tnfα, il- β, il- , il- , complement factors c a, c a, and d) by crrt . at present, there is a need for well-designed clinical trial to evaluate the efficacy of such a treatment regimen. after covid- infection, some patients develop systemic inflammatory response syndrome (sirs) and mods characterized by the uncontrolled release of inflammatory mediators, giving rise to a cytokine storm that contributes to increased mortality in ards. in summary, further experimentations is required to understand the changes in the immune response of patients with covid- infection and the mechanisms of abnormal cytokine expression in covid- pneumonia. accurate prediction and targeted intervention during the course of covid- pneumonia will be essential to improve patient survival (figure ). a summary of the process of onset sars-cov pathogenesis with potential treatment options against the virus-induced cytokine storm. nowcasting and forecasting the potential domestic and spread of the -ncov outbreak originating in wuhan, china: a modelling study clinical features of patients infected with novel coronavirus in wuhan, china a rapid advice guideline for the diagnosis and treatment of novel coronavirus ( -ncov) infected pneumonia (standard version) cytokine release syndrome immunotherapeutic implications of 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pneumonia in sars-cov-infected mice identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury the tlr antagonist eritoran protects mice from lethal influenza infection endothelial cells are central orchestrators of j o u r n a l p r e -p r o o f cytokine amplification during influenza virus infection interaction of angiotensin ii and atrial natriuretic peptide in the human fetoplacental unit therapeutic sirna silencing in inflammatory monocytes in mice immunomodulatory therapy for severe influenza the effect of inhibition of pp and tnfalpha signaling on pathogenesis of sars coronavirus mediator removal with crrt: complement and cytokines schematic representation of clinical features versus pathogenic inflammatory cytokine response in sars-cov- infections there are no financial or other interests with regard to the paper that represent a conflict of interest. key: cord- - n jafsk authors: feng, qian; langereis, martijn a.; van kuppeveld, frank j.m. title: induction and suppression of innate antiviral responses by picornaviruses date: - - journal: cytokine growth factor rev doi: . /j.cytogfr. . . sha: doc_id: cord_uid: n jafsk the family picornaviridae comprises of small, non-enveloped, positive-strand rna viruses and contains many human and animal pathogens including enteroviruses (e.g. poliovirus, coxsackievirus, enterovirus and rhinovirus), cardioviruses (e.g. encephalomyocarditis virus), hepatitis a virus and foot-and-mouth disease virus. picornavirus infections activate a cytosolic rna sensor, mda , which in turn, induces a type i interferon response, a crucial component of antiviral immunity. moreover, picornaviruses activate the formation of stress granules (sgs), large aggregates of preassembled mrnps (messenger ribonucleoprotein particles) to temporarily store these molecules upon cellular stress. meanwhile, picornaviruses actively suppress these antiviral responses to ensure efficient replication. in this review we provide an overview of the induction and suppression of the mda -mediated ifn-α/β response and the cellular stress pathway by picornaviruses. on the front line of antiviral immunity is the production of type i interferons (ifn-a/b) and other antiviral cytokines at the site of infection. ifn-a/b can be produced by virtually all nucleated cell types and act in autocrine and paracrine manners (i.e. on both the infected cells and neighboring non-infected cells). ifn-a/breceptor engagement induces the expression of large numbers of interferon-stimulated genes (isgs), which together establish a so-called antiviral state in the recipient cells. to recognize the invading pathogens timely and correctly, cells employ specialized pattern recognition receptors (prrs) to detect specific pathogenassociated molecular patterns (pamps). rig-i-like receptors (rlrs) are a family of ubiquitously expressed prrs that detect ''non-self'' viral rnas in the cytoplasm. two rlrs, rig-i and mda , mediate ifn-a/b production upon infection of various rna viruses [ , ] . while rig-i is required for sensing, among others, paramyxoviruses, influenza virus, japanese encephalitis virus and hepatitis c virus, mda recognizes picornaviruses, mouse norovirus, mouse hepatitis virus and defective interfering particles of paramyxoviruses [ , ] . another rlr, lgp , lacks functional domains required for downstream signaling, and therefore has been long suspected to play regulatory roles on rig-i and mda . besides the ifn-a/b and inflammatory responses, cells also engage various other mechanisms to cope with undesirable conditions. one of such mechanisms is the formation of stress granules (sgs) in the presence of stress such as oxidative, heat, or nutrient stress, uv radiation and viral infections. although the stress pathway was initially thought to function independently of classical innate antiviral responses such as ifn-a/b, it was recently suggested that the stress response may also directly or indirectly play an antiviral role [ ] . during the long co-evolution with their hosts, viruses have acquired strategies to actively counteract host antiviral responses. both the rlr-mediated ifn-a/b induction pathway as well as the stress pathway are targeted by various viruses [ , ] . this review summarizes our current knowledge on the recognition and suppression of host antiviral pathways by picornaviruses, a large family of human and animal pathogens. we focus on two important and well-studied genera of picornaviruses, namely enterovirus and cardiovirus, and discuss how their rnas are recognized by rlrs, and how they antagonize the ifn-a/b induction pathway and sg formation in infected cells. the family picornaviridae comprises of small, non-enveloped, positive-strand rna viruses and contains many human and animal pathogens including enteroviruses (e.g. poliovirus, coxsackievirus, enterovirus and rhinovirus), cardioviruses (e.g. encephalomyocarditis virus), hepatitis a virus and foot-andmouth disease virus. picornavirus infections activate a cytosolic rna sensor, mda , which in turn, induces a type i interferon response, a crucial component of antiviral immunity. moreover, picornaviruses activate the formation of stress granules (sgs), large aggregates of preassembled mrnps (messenger ribonucleoprotein particles) to temporarily store these molecules upon cellular stress. meanwhile, picornaviruses actively suppress these antiviral responses to ensure efficient replication. in this review we provide an overview of the induction and suppression of the mda -mediated ifn-a/b response and the cellular stress pathway by picornaviruses. ß elsevier ltd. all rights reserved. (sero)types of important human pathogens [ ] [ ] [ ] . poliovirus (pv) is the causative agent of poliomyelitis. various types of coxsackieviruses (cvs), echoviruses, and other enteroviruses (evs) can cause viral meningitis, encephalitis, myocarditis and pancreatitis, and have also been implicated in the development of type i diabetes [ , ] . enterovirus (ev ) is an emerging virus that can cause severe neurological symptoms in young children, and have caused many outbreaks in the past decade, mostly in southeast asia [ ] . human rhinoviruses (hrvs) are responsible for approximately one third of common colds in adults, and are also associated with asthma exacerbations and chronic obstructive pulmonary disease (copd) [ ] . the cardiovirus genus contains mostly animal pathogens such as encephalomyocarditis virus (emcv) and theiler's murine encephalomyelitis virus (tmev). both emcv and tmev are primarily rodent pathogens, but emcv also causes severe, sometimes lethal, infections in other animals such as pigs, elephants, lions and primates, posing problems in zoos and national parks [ , ] . recently, a new cardiovirus that infects humans has been discovered, namely the saffold virus, which has been associated with gastroenteritis, and respiratory and neurological infections [ ] . members of the picornaviridae family are small, non-enveloped, positive-strand rna viruses. the viral genome, a single-stranded (ss) rna molecule of . - . kb, encodes a single open reading frame (orf), an untranslated region (utr) at either terminus, and a poly(a) tail at the extreme end. the terminus of the viral rna is coupled to a small viral peptide vpg (also known as b) via a phosphodiester bond as a result of vpg-primed viral rna replication. the genomic rna also contains several structured rna elements that are crucial for virus replication. the internal ribosomal entry site (ires) in the utr contains several stemloop structures and drives viral cap-independent translation. stem-loop structures in the utrs and a cis-acting rna element (cre) -the position of which varies across different genera -are crucial for viral rna replication [ ] . picornaviruses share a similar life cycle (fig. ) , with some details varying across genera [ ] . infection is initiated via receptor-mediated endocytosis, followed by uncoating to release the genomic rna in the cytoplasm. a cellular enzyme then releases the vpg peptide from the genomic rna [ ] , for reasons not yet understood, generating a single-stranded viral rna carrying a monophosphate group. the viral genome is then immediately translated by the host translation machinery to generate a large polyprotein, which undergoes proteolytic processing by the virally encoded proteinases. all picornaviruses carry a c pro , which mediates most of the proteolytic processing, and members of some genera carry an additional proteinase that also participates (e.g. a pro for enteroviruses and l pro for aphthoviruses). additionally, these viral proteinases also cleave host factors to aid virus rna replication and/or to evade host antiviral responses. next, several viral non-structural proteins hijack regulatory mechanisms of host membrane metabolism to induce extensive remodeling of the intracellular membranous structures to form the so-called replication organelles (ros) where viral rna replication takes place. the process of rna replication is carried out by the virally encoded rna-dependent rna polymerase d pol (fig. ) . first, d pol uridylylyates vpg, and uses the resulting vpg-pu-pu as a primer to transcribe the positive-strand rna into a complementary, negative-strand rna molecule. during this process, a long dsrna intermediate product is produced, which is referred to as the replicative form (rf). next, d pol uses the negative-strand rna as a template, and again vpg-pu-pu as a primer, to produce a large number of nascent positive strands. this step leads to the production of another intermediate product, namely the replicative intermediate (ri), which comprises of a single negative-strand rna and multiple incomplete positive-strand rnas that are undergoing active transcription. the completed nascent positive-strand rnas either enter a new round of translation and rna replication, or are encapsidated to form new virions. at the end of the replication cycle, progeny virus particles are released by cell lysis [ ] . a and l are the two most divergent picornaviral proteins. both can act as proteinases in some genera but not in others [ ] . because of their activities in inducing host shutoff, modulating cell death and counter-acting immune responses, these proteins have been classified as viral security proteins [ ] . in fact, the l proteins rig-i-like receptors (rlrs) are cytoplasmic rna sensors that initiate a type i interferon response upon rna virus infections. currently, the rlr family contains three dexh/d box helicases, namely rig-i, mda and lgp . upon ligand recognition, rig-i and mda interact with a mitochondrial adaptor molecule mavs (also known as visa, cardif and ips-i), which in turn, activates the tbk / ikk-e and ikk-a/b/g pathways (fig. ). tbk then phosphorylates and activates irf , leading to transcription activation of ifn-a/b genes, whereas the ikk-a/b/g complex leads to nf-kb activation and the transcription of many proinflammatory cytokine genes. lgp also binds rnas but lacks the signaling domains, thereby the ability to initiate downstream signaling cascades. instead, it has been shown to play regulatory roles on rig-i and mda [ , ] . lots of effort have been invested into characterizing rig-i and mda ligands [ , ] . in vitro studies using synthetic ligands showed that rig-i requires relatively short dsrnas, or triphosphate ( ppp)-containing ssrnas with base-paired regions for activation. in agreement with these findings, the ppp-containing panhandle rna structures of many negative-strand rna viruses (e.g. influenza virus and sendai virus) have been shown to potently activate rig-i. mda is activated by long dsrnas, as evidenced by transfection studies using poly(i:c). to date, there is no evidence that specific terminal groups are required on mda ligands. it is well established that picornavirus infections induce mda mediated ifn-a/b response in both cultured cells as well as mice [ ] [ ] [ ] [ ] [ ] [ ] . mda -stimulatory activity was observed in total rna extracts from emcv-or tmev-infected cells [ ] . further analysis in this study suggested that the mda -stimulating rna was a high molecular weight rna complex that consisted of both ssrnas and dsrnas, hinting that cardiovirus infections may lead to production of various pamps that can activate mda . as mentioned above, several viral rna species are produced during picornavirus rna replication, all bearing ''non-self'' features that could potentially be recognized by cellular sensors including mda . picornavirus ssrnas lack a cap structure but, instead, contain either a covalently linked vpg peptide or a monophosphate at the terminus. the rf is a dsrna molecule of . - . kbp, and the ri is a complex, partially double-stranded rna. recently, triantafilou et al. and our group both examined the abilities of purified picornaviral rna species in stimulating mda . together, the results clearly show that the two viral ssrna species are in fact poor ifn-a/b inducers upon transfection, despite of the highly structured regions and the ''non-self'' features they carry [ , ] . in contrast, both viral rna replication intermediate products, namely the rf and the ri, induced potent ifn-a/b response upon transfection, which was completely dependent on mda [ , ] . additionally, the rf was shown to activate the atpase activity of recombinant mda in the absence of any additional proteins [ ] , demonstrating that this rna can serve as a direct ligand of mda . while these recent data from transfection and in vitro experiments provided significant insights into picornavirus rna recognition by mda , they cannot be taken for granted to reflect mda activation in infected cells. during infection, the viral rnas may be less, if at all, accessible because they are bound by various viral and host factors that participate in viral rna replication, and may also be (partially) shielded by the virus-induced membranous ros. recently, this question was tackled by using compounds that inhibited viral rna replication at different steps in infected cells. inhibition of negative-strand rna synthesis, and therefore rf formation, caused a complete abrogation of ifn-a/b response in infected cells. in contrast, a significant ifn-a/b response was observed when rf formation was allowed to proceed but the subsequent step, nascent positive-strand rna synthesis, thereby the formation of ri, was inhibited [ ] . these results strongly suggest that picornavirus rf is a physiological mda ligand in virus-infected cells. the role of ri in stimulating mda under physiological conditions is less clear. in contrast to rf, the ri is a primarily ssrna molecule containing double-stranded regions and many protruding single-stranded ends [ ] . mda activation, however, requires long dsrnas [ , , ] . the average length of the double-stranded regions in ri molecules is unknown. yet, ri induced high levels of mda -mediated ifn-a/b response upon transfection [ ] , suggesting that there is sufficient length of basepaired regions in purified ri to induce mda activation. whether this is also the case for the dynamic structure of ri during active viral rna replication remains to be established. besides the classically recognized viral rna species of picornaviruses, deddouche and colleagues suggested in a recent study that an additional mda -stimulating viral pamp might be produced in emcv-infected cells. lgp -associated rnas from emcv-infected cells were found to exert mda -stimulatory activity upon transfection of naive cells [ ] . subsequent deepsequencing analysis of the rna pool that co-immunoprecipitated with lgp revealed a clear enrichment of a -nucleotide fragment that is complementary to the coding region of the viral protein l. while this is a remarkable and unexpected finding, it also raises a number of questions. firstly, it is not yet clear whether this l antisense rna is produced as a specific entity during emcv infection since northern blotting analysis failed to detect this rna species in total rna preparation from emcv-infected cells [ ] . therefore this nucleotide fragment might represent the most stable fragment of a larger trunk of, or even the full-length, negative-strand rna during the isolation procedure. alternatively, small rlr-stimulating rnas can be generated by rnase l from host and viral rnas during virus infections [ ] [ ] [ ] , and perhaps the l antisense rna fragment is such a product. however, there was no evidence of the involvement of rnase l, which is also in line with a previous observation that emcv-induced ifn-a/b response was comparable in wt and rnase l knockout cells [ ] . it remains to be clarified if and how this emcv l antisense rna is produced during infection. secondly, it is unclear how such a small ssrna could activate mda in an lgp -dependent manner. involvement of lgp in emcv-induced ifn-a/b response has been previously reported [ ] . the newly discovered l antisense rna was recovered from lgp but not mda immunoprecipitations [ ] , suggesting that lgp directly binds to this small rna and then facilitate mda activation. both mda and lgp have been shown to bind short ssrnas [ ] [ ] [ ] [ ] , although mda can only be functionally activated by long dsrnas [ , ] . lgp has also been shown to interact with mda , though this strictly depended on the presence of dsrnas [ ] . it remains to be demonstrated whether the base-paired regions in emcv l antisense rna are sufficient to facilitate such lgp /mda complex formation, or that lgp induces mda activation via a yet unknown mechanism. in any case, the l antisense rna seems to contain some immune-stimulatory signature since in vitro transcripts (without ppp to exclude rig-imediated recognition) of this fragment, but not that of the complementary sequence (l sense), induced an mda -mediated ifn-a/b response upon transfection [ ] . lastly, it is important to realize that the l antisense rna is not the sole mda ligand produced during emcv infection (see section . ). a recombinant emcv carrying deletions in the l-encoding region (dl), which thereby could not produce the l antisense rna (or the l protein, the known ifn-a/b antagonist), induced a potent ifn-a/b response. yet, this response was less strong as compared to another recombinant emcv that lacked a functional l protein due to mutations (l mut ) but could still produce the l antisense rna [ ] . whether picornaviruses from other genera also produce small mda -stimulating ssrnas remains to be further investigated. many viruses have evolved to actively suppress the ifn-a/b system (often at multiple steps) [ , , , ] , and picornaviruses are no exception. it has long been suspected that picornavirus-induced host gene expression shutoff and secretory pathway blockade (the latter of which has only been reported for some genera such as enterovirus) contribute to ifn-a/b suppression [ ] . however, we and others have shown that little irf activation can be detected during enterovirus and cardiovirus infections [ ] [ ] [ ] , implying that the primary blockade of this pathway lies upstream of ifn production. enterovirus infections trigger little irf phosphorylation [ ] [ ] [ ] . a recent report showed that tbk phosphorylation, a prerequisite to irf phosphorylation, is inhibited in cvb -infected cells, suggesting that inhibition of the rlr-mediated ifn-a/b induction pathway lies upstream of tbk [ ] . in line with this observation, several enteroviruses have been reported to directly interfere with upstream factors. mda was shown to be degraded during infections of pv and ev in a caspase-dependent manner, and in the case of pv, also via proteasome activities [ , ] . also the downstream adaptor molecule mavs has been shown to be targeted by several enteroviruses including hrv a [ ] , cvb [ ] and ev [ ] , via various proposed mechanisms. a pro of ev and c pro of cvb were suggested to be responsible for mavs cleavage during infection of these viruses, whereas a pro , c pro as well as caspase were all implicated in hrv a-induced mavs cleavage. it was somewhat surprising that enteroviruses seemed to employ a variety of means to shut down the rlr pathway, since these viruses often utilize the same strategies to target a particular host factor or pathway. recently, a systematic examination of signaling components of the rlr pathway revealed that both mda and mavs are cleaved during cvb infection, and could be reproduced by recombinant viral proteinase a pro . a pro 's of three enteroviruses belonging to different species, namely ev (enterovirus a), cvb (enterovirus b) and pv (enterovirus c), also targeted mda and mavs for cleavage when inserted into the genome of a emcv (which does not induce these cleavages by itself) [ ] . these data suggest that enteroviruses very likely employ a unified mechanism to interfere with the rlr-mediated ifn-a/b induction pathway. recently, we also observed that insertion of enterovirus a pro in emcv l mut not only resulted in cleavage of mda and mavs, but also almost a complete inhibition of ifn-a/b induction (data not shown). these data show that a pro is indeed the enterovirus ifn-a/b antagonist. that mda is specifically targeted by the viral a pro [ ] seems to contradict earlier reports that mda is degraded by caspases and the proteasome during enterovirus infections [ , ] . however, it is important to point out that the caspase-and proteasome-mediated mda cleavage events were observed under conditions where mda expression level was artificially upregulated prior to infection (either by poly(i:c) or viral rna transfections) [ , ] , which may sensitize cells for virus-induced apoptosis, thereby promote caspase-and proteasome-mediated protein degradations. in contrast, the a pro -mediated mda cleavage was observed without manipulations of its expression levels [ ] . the importance of studying cleavage of endogenous proteins is also illustrated by the observation that c pro can cleave overexpressed mavs [ , ] but not endogenous mavs. cleavage products observed in infected cells are only observed upon expression of a pro [ , ] , suggesting that a pro -mediated mavs cleavage is likely a more prominent event under physiological conditions. interestingly, rig-i is also cleaved by various enteroviruses via their c pro activity [ , ] . however, it remains to be elucidated why these viruses target a rna sensor that does not participate in their recognition [ , ] . it has been proposed that rig-i may directly turn on isg transcription via stat activation in an ifn-a/bindependent fashion [ ] . whether enteroviruses cleave rig-i to prevent an augmentation of isg expression via the stat pathway remains to be investigated. as mentioned above, enterovirus a pro is an effective ifn-a/b antagonist. while it is tempting to attribute this ifn-a/b-suppressing effect to the cleavage of mda and mavs, it is known that a pro also targets many other host proteins, thereby affecting many cellular processes, which may possibly influence ifn-a/b response against these viruses. one group of cellular substrates of a pro are the nucleoporins (nups), proteins that form the nuclear pore complex and regulate protein and mrna trafficking through the nuclear pore. nup cleavage during enterovirus infection results in a bidirectional loss of selective macromolecule trafficking across the pore [ ] [ ] [ ] [ ] , a phenomenon often referred to as the nucleocytoplasmic trafficking (nct) disorder. this virus-induced nct disorder may interfere with ifn-a/b transcription activation by affecting shuttling and/or activation of irf , which must translocate to the nucleus to activate target gene transcription [ ] . alternatively localization of other (regulatory) factors necessary for ifn-a/b transcription may be deregulated by the nct disorder. the potential blockade at the level of irf activation may seem unnecessary since upstream tbk activation is already inhibited in infected cells [ ] . however, it is common for viruses to employ redundant strategies to interfere with the ifn system to ensure an effective inhibition. furthermore, these two mechanisms may also be important at different stages of infection. the nct disorder can be observed extremely rapidly upon enterovirus infections -hours before the cleavages of mda and mavs could be detected. thus, it is possible that enteroviruses rely on the nct disorder to keep ifna/b under control before mda and mavs could be inactivated. of note, it cannot be excluded that mda and mavs are already cleaved earlier during infection, locally at the site of viral protein synthesis and rna replication, but are undetectable when analyzing these factors in whole cytoplasmic lysates [ ] . the relative contributions of the nct disorder and mda /mavs cleavage to ifn-a/b antagonization remain to be demonstrated. cardioviruses do not target mda or mavs, however, still effectively prevent irf phosphorylation [ ] . it was also shown that tbk is phosphorylated -believed to reflect activationduring emcv infection [ ] , pinpointing the viral antagonization to a step between tbk activation and irf phosphorylation. the cardiovirus ifn-a/b antagonist has long been established, namely the l protein, a small peptide ( amino acids in the case of emcv) produced at the extreme end of the viral polyprotein. the mature l protein contains an n-terminal chcc zn-finger domain, a hinge region and a c-terminal highly acidic domain [ , ] . this protein is non-essential for viral rna replication, but very important for counteracting host antiviral responses. l mut viruses replicate to similar levels as wt viruses in ifn-a/b-deficient cells/mice, but are attenuated in ifn-a/b-competent systems [ , , ] , suggesting that it is an important player in ifn-a/b antagonization. l has neither enzymatic activity, nor known homologs [ , ] . it is thus far unknown how l inhibits irf activation and subsequently ifn-a/b production. this effect of l may be linked to its role in the nct disorder that is induced by cardioviruses, which like enteroviruses also disrupt macromolecule trafficking across nuclear pores [ , ] . l has been shown to form a tight complex with the small gtpase ran, which is currently the only reported interaction partner of l [ , ] . hereby, l disrupts the rangdp/gtp gradient across the nuclear pore, a crucial regulatory mechanism for nuclear import and export [ , ] . l also induces hyperphosphorylation of several nups (e.g. nup , , & ) within the domains that form the physical barriers of the nuclear pore and provide important docking sites for transport receptors [ ] [ ] [ ] . hereby l may physically interfere with interactions of cargo transporters with nups [ ] . mutant l proteins harboring substitutions in the zn-finger domain or acidic domain that fail to induce the nct disorder [ , , , ] -due to impaired ran binding and nup hyperphosphorylation [ , , ] -are also deficient in suppressing ifn-a/b [ , , , , , ] . these results suggest that ifn-a/b suppression by l may be in one way or another caused by its activity to deregulate nct. as mentioned in section . . , nct disorder may interfere with irf activation by preventing its shuttling and/or nuclear translocation upon activation by tbk . future research is necessary to demonstrate a clear cause-effect relationship between the nct disorder and ifn-a/b suppression, as well as the exact underlying mechanism. it has been reported that emcv also proteolytically targets rig-i in infected cells, either via the viral c pro [ ] , or caspases [ ] . however, rig-i cleavage was not observed throughout infection of either wt or l mut emcv in another study [ ] . it is currently unclear what causes this disagreement in literature. nonetheless, assuming rig-i is targeted by emcv, how could this benefit virus replication? as discussed above (see section . . ), rig-i does not participate in detecting picornaviruses [ , ] . it has been suggested that rig-i may directly activate stat , thereby activating isg expression independently of ifn-a/b production [ ] . thus, it is possible that cardioviruses inactivate rig-i to prevent isg transcription activation, though experimental proof remains to be provided. picornaviruses from other genera than enterovirus and cardiovirus are also known to actively interfere with ifn-a/b induction. hav, a member of the hepatovirus genus, proteolytically targets mavs. in this case, a precursor of the viral c pro proteinase, namely the abc, is responsible, and both the proteinase activity of c pro and a transmembrane domain in a, which targets abc to mitochondria where mavs is localized, are required [ ] . another picornavirus, fmdv from the aphthovirus genus, also targets the rlr pathway. the fmdv viral proteinase l pro (which is unrelated to the cardiovirus l) exerts deubiquitinating activity on k as well as k ubiquitin chains, and has been shown to reduce ubiquitination of, among others, rig-i and tbk [ ] , which is known to modulate the activity of these factors. in addition, the c pro of fmdv has been reported to cleave nf-kb essential modulator (nemo, also known as ikk-g), a factor required for nf-kb activation [ ] . these results clearly demonstrate that picornaviruses employ a diverse range of mechanisms to suppress host antiviral responses, and knowledge obtained from one genus cannot be directly applied to other genera. of note, another important genus of picornaviruses, parechovirus, which can cause several neonatal infections in humans [ ] , do not encode for an l protein, and neither do their a proteins exert any protease activity [ , ] . how these viruses interfere with host antiviral responses remains to be elucidated. stress granule (sg) formation is part of the cellular stress response and is important for cell survival during stress conditions. these granules are dynamic and serve to temporarily store pre-initiation mrna complexes (fig. ) [ , ] . they contain ras gtpase-activating protein-binding proteins (g bp / ), t-cell intracellular antigen (tia- ), tia-related protein (tiar), many translation initiation factors, s ribosomes, mrnas, and numerous other rna-binding proteins. many more proteins have recently been reported to associate with sgs formed under specific types of stress [ ] , indicating that there is some level of specificity of the cellular stress response. in most cases, sg formation is triggered by a halt in cap-dependent translation, which is often caused by phosphorylation of eukaryotic translation initiation factor a (eif a) by one of the four kinases -protein kinase r (pkr), pkr-like endoplasmic reticulum kinase (perk), heme-regulated kinase (hri) and general control non-depressible kinase (gcn ). however, eif a phosphorylation-independent sg formation after oxidative stress or direct inhibition of eif g or eif a has also been reported [ ] . growing evidence suggests that sgs exert an antiviral function, though the exact mechanism is largely unknown. since the primary function of the stress pathway is to halt cap-dependent translation and temporarily store preformed translation initiation complexes in sgs [ , ] , it has been suggested that also viral translation is inhibited. indeed, in the case of influenza virus, which relies solely on cap-dependent translation for viral protein synthesis, sg formation was associated with reduced viral protein accumulation levels and thus virus replication [ ] . also for picornaviruses, which use ires-dependent translation, sg formation appears to be disadvantageous for virus replication, albeit only to moderate levels [ ] [ ] [ ] . how sg formation acts on picornavirus replication is incompletely understood. hypothetically, viral mrnas can get trapped in the sgs, thereby precluding them from translation and/or replication. however, it is still under debate whether picornavirus rnas localize to sgs (see section . . ). alternatively, it has been suggested that host and/or viral proteins, which play a role in virus replication, are scavenged in sgs, thereby limiting virus replication [ ] , but this has not been investigated. recent observations suggest that there is a link between the stress pathway and the ifn-a/b pathway -which is discussed here below -providing novel insights into the possible antiviral role of sgs. recently, sgs were suggested to function as sites for viral rna recognition by rlrs. the group of fujita showed that both rig-i and influenza viral ssrna (ligand of rig-i) translocate to sgs during influenza virus dns infection. inhibition of sg formation, either by g bp knockdown or pkr knockout, resulted in a -to -fold reduction in ifn-b response [ ] . these results led the authors to propose that sgs may serve as a platform for viral rna recognition by rig-i. also mda , the sensor for picornavirus rna [ ] , localizes to sgs [ ] . this observation suggests that mda may also use sgs as sites for viral rna recognition. however, it is thus far unclear whether picornaviral dsrna (ligand of mda ) also localizes to sgs. on the basis of propidium iodide staining it was concluded that dsrna localized to sgs in emcv-infected cells [ ] . however, detection of emcv or tmev viral rnas using either a dsrna-specific antibody or in situ hybridization, respectively, failed to show any viral rna in virus-induced sgs [ , ] . these observations suggest that it is unlikely that mda utilizes sgs as (primary) site for viral rna recognition during picornavirus infections. accumulating evidence suggest that there is cross-talk between the stress pathway and rlr-mediated ifn-a/b response. it has been reported that pkr and g bp enhance activation of the nf-kb pathway [ , ] and consequently also ifn-a/b production [ ] . vice versa, dhx , a novel activator of rig-i [ ] , as well as mavs [ ] were recently shown to be involved in pkr activation. clearly, activation of the one pathway does not necessarily lead to fullblown activation of the other. langereis et al. showed that activation of the stress pathway and subsequent sg formation did not induce ifn-a/b production, and conversely, activation of the rlr signaling pathway or ifn-a/b treatment of cells did not lead to sg formation [ ] . notwithstanding this, ifn-a/b treatment did sensitize cells for stress pathway activation [ , ] , possibly due to ifn-induced increase in pkr expression. it remains to be established to what extent, and under what circumstances, the cross-talk between these two pathways occurs. since stress pathway activation acts as an antiviral response, viruses have found ways to inhibit this pathway. enteroviruses (e.g. pv and cvb ) induce sg formation early during infection, which may partly result from virus-induced cap-dependent translation shutoff via eif g cleavage [ ] . these granules disappear as infection proceeds due to cleavage of g bp by the viral proteinase c pro [ , , ] . overexpression of a g bp mutant that can no longer be cleaved by the viral c pro (g bp -q e) led to stable sg formation and stronger repression of pv replication than wt g bp overexpression. this observation underscores the important role of c pro -mediated cleavage of g bp on enhancing enterovirus replication [ ] . besides enteroviruses, also alphaviruses like semliki forest virus [ ] and chickungunya virus [ ] prevent sg formation by interacting with g bp , suggesting that targeting this protein is an effective way to inhibit the antiviral role of sgs. cardioviruses also suppress sg formation. as influenza a virus, which utilizes its ns protein to suppress both ifn-a/b activation and sg formation [ , ] , cardioviruses also employ a single viral protein, namely l, to antagonize these two antiviral pathways [ , ] . while no sgs are observed during wt cardiovirus infections, l mut emcv and tmev induced pkr-dependent activation of the stress pathway [ ] and persistent sgs throughout infection [ , ] . moreover, overexpression of l alone was sufficient to repress de novo sg formation induced via hri and perk activation [ ] , suggesting that l expression inhibits sg formation at a common step downstream of pkr, hri and perk, possibly at the level of eif a phosphorylation or a yet unknown downstream step. as mentioned in section . . , cardiovirus l also induces nct disorder. although there is no proof that coordinated trafficking of proteins between the nucleus and the cytoplasm is essential for stress pathway activation and sg formation, tia- is known to migrate from the nucleus to the cytoplasm upon stress pathway activation. since tia- is an essential structural component of sgs, it is possible that the l-induced nct disorder affects tia- migration and thus sg formation. of course, it cannot be excluded that l acts on a central host factor or process that controls nct, ifn-a/b response as well as sg formation, though no plausible candidate has been suggested, to date. recently, it was reported that emcv induced sg formation early in infection, and the sgs disappeared later in infection as a result of g bp cleavage via viral c pro , similar to what is described for enteroviruses [ ] . while this is a remarkable observation, it provides no explanation as to how l mut cardioviruses, which all express active c pro , potently induce sg formation [ , ] . in summary, both enteroviruses and cardioviruses are able to inactivate the stress pathway. however, the mechanism how this is achieved, especially for cardioviruses, remains to be investigated in more detail. studying how picornaviruses target the stress pathway and how sg formation acts as an antiviral response may also help reveal novel insights into the interplay between stress and innate immune responses. the interaction between pathogens and the innate antiviral responses is extremely intricate. recent studies provided a first glimpse on mda activation during the course of a normal picornavirus infection, however, much is still to be learned about mda activation during virus infections in general. what rna(s) are directly bound by mda ? what is the minimum length of mda filaments that can activate mavs in cells? is lgp a positive regulator of mda in general, or is it only needed for specific (types of) rna ligands? in addition, the immune evasion strategies of many important picornaviruses are still incompletely understood. furthermore, research on the antiviral activities of the stress response, and the underlying mechanisms, are also still in its infancy. while addressing these questions in future research, it is important to realize that viruses replicate at specific subcellular microenvironments that can largely differ from in vitro experimental conditions. efforts should be made to study events during natural infections. in addition, the spatial and temporal aspects of viral rna recognition and viral evasion strategies are important issues that need to be clarified. at what stage during a picornavirus infection does mda become activated? where does mda gain access to viral rna pamps that are produced in restricted subcellular microenvironments? where and when do viral proteinases encounter and target crucial factors of immune pathways? these important questions await future investigation and warrant an exciting research area of the interaction between picornaviruses and innate antiviral responses. rig-i-like receptors: cytoplasmic sensors for nonself rna cytosolic sensing of viruses diversion of stress granules and p-bodies during viral infection enter at your own risk: how enteroviruses navigate the dangerous world of pattern recognition receptor signaling viral subversion of host functions for picornavirus translation and rna replication picornavirus and enterovirus diversity with associated human diseases enterovirus infection and type diabetes mellitus: systematic review and meta-analysis of observational molecular studies clinical features, diagnosis, and management of enterovirus virus-induced exacerbations in asthma and copd encephalomyocarditis virus infection in an italian zoo encephalomyocarditis virus infections in an australian zoo saffold virus, a novel human cardiovirus with unknown pathogenicity the picornaviruses an rna virus hijacks an incognito function of a dna repair enzyme viral security proteins: counteracting host defences the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor the leader protein of theiler's virus inhibits immediate-early alpha/beta interferon production differential roles of mda and rig-i helicases in the recognition of rna viruses the toll-like receptor -mediated antiviral response is important for protection against poliovirus infection in poliovirus receptor transgenic mice essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus melanoma differentiationassociated gene is critical for protection against theiler's virus-induced demyelinating disease mda and mavs mediate type i interferon responses to coxsackie b virus activation of mda requires higher-order rna structures generated during virus infection mda detects the double-stranded rna replicative form in picornavirus-infected cells visualisation of direct interaction of mda and the dsrna replicative intermediate form of positive strand rna viruses structure of poliovirus replicative intermediate rna electron microscope analysis of rna cross-linked in vivo with psoralen derivative length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene identification of an lgp -associated mda agonist in picornavirusinfected cells activation of ifn-beta; expression by a viral mrna through rnase l and mda rnase l releases a small rna from hcv rna that refolds into a potent pamp small self-rna generated by rnase l amplifies antiviral innate immunity lgp is a positive regulator of rig-i-and mda -mediated antiviral responses the rig-i-like receptor lgp recognizes the termini of double-stranded rna regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp solution structures of cytosolic rna sensor mda and lgp c-terminal domains: identification of the rna recognition loop in rig-i-like receptors cooperative assembly and dynamic disassembly of mda filaments for viral dsrna recognition lgp plays a critical role in sensitizing mda- to activation by double-stranded rna the coxsackievirus b c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling mda plays a crucial role in enterovirus rna-mediated irf activation cleavage of ips- in cells infected with human rhinovirus enterovirus a pro targets mda and mavs in infected cells mda- is cleaved in poliovirus-infected cells enterovirus protease a pro targets mavs to inhibit anti-viral type i interferon responses rig-i is cleaved during picornavirus infection ra-inducible gene-i induction augments stat activation to inhibit leukemia cell proliferation bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores differential targeting of nuclear pore complex proteins in poliovirus-infected cells differential processing of nuclear pore complex proteins by rhinovirus a proteases from different species and serotypes rna nuclear export is blocked by poliovirus a protease and is concomitant with nucleoporin cleavage nuclear/cytoplasmic localization of irfs in response to viral infection or interferon stimulation a picornavirus protein interacts with ran-gtpase and disrupts nucleocytoplasmic transport encephalomyocarditis virus leader is phosphorylated by ck and syk as a requirement for subsequent phosphorylation of cellular nucleoporins mengovirus leader is involved in the inhibition of host cell protein synthesis inhibition of mrna export and dimerization of interferon regulatory factor by theiler's virus leader protein nucleocytoplasmic traffic disorder induced by cardioviruses the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins encephalomyocarditis virus leader protein hinge domain is responsible for interactions with ran gtpase nucleoporin phosphorylation triggered by the encephalomyocarditis virus leader protein is mediated by mitogenactivated protein kinases mengovirus-induced rearrangement of the nuclear pore complex: hijacking cellular phosphorylation machinery large cargo transport by nuclear pores: implications for the spatial organization of fg-nucleoporins mda localizes to stress granules but this localization is not required for the induction of type i interferon the mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ ferritin-mediated activation of nf-kappa b the viral rna recognition sensor rig-i is degraded during encephalomyocarditis virus (emcv) infection disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor the leader proteinase of footand-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase foot-and-mouth disease virus c protease cleaves nemo to impair innate immune signaling regulation of stress granules in virus systems eukaryotic stress granules: the ins and outs of translation influenza a virus inhibits cytoplasmic stress granule formation production of a dominantnegative fragment due to g bp cleavage contributes to the disruption of mitochondria-associated protective stress granules during cvb infection inhibition of cytoplasmic mrna stress granule formation by a viral proteinase encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity the leader protein of cardioviruses inhibits stress granule assembly the n-terminus of pkr is responsible for the activation of the nf-kappab signaling pathway by interacting with the ikk complex ikappabalpha and ikappabalpha/nfkappa b complexes are retained in the cytoplasm through interaction with a novel partner, rasgap sh -binding protein jnk and ikkbeta are required for activating the innate response to viral infection dhx enhances rig-i signaling by facilitating pkr-mediated antiviral stress granule formation ips- plays an essential role in stress granule formation induced by dsrna through interacting with pkr and mediating its activation dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection induction of stress granules by interferon and downregulation by the cellular rna adenosine deaminase adar sequestration of g bp coupled with efficient translation inhibits stress granules in semliki forest virus infection chikungunya virus nsp blocks stress granule assembly by recruitment of g bp into cytoplasmic foci we would like to thank the netherlands organisation for scientific research (nwo) for the following funding: q. key: cord- -wkrj n d authors: zhang, pengfei; yu, linyang; dong, jianguo; liu, yanling; zhang, leyi; liang, pengshuai; wang, lei; chen, bin; huang, li; song, changxu title: cellular poly(c) binding protein interacts with porcine epidemic diarrhea virus papain-like protease and supports viral replication date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: wkrj n d porcine epidemic diarrhea virus (pedv) belongs to the alphacoronavirus genus in the coronaviridae family. similar to other coronaviruses, pedv encodes two papain-like proteases. papain-like protease (plp) has been proposed to play a key role in antagonizing host innate immunity. however, the function of plp remains unclear. in this study, we found that overexpression of plp significantly promoted pedv replication and inhibited production of interferon-β. immunoprecipitation and mass spectrometry were used to identify cellular interaction partners of plp . host cell poly(c) binding protein (pcbp ) was determined to bind and interact with plp . both endogenous and overexpressed pcbp co-localized with plp in the cytoplasm. overexpression of plp upregulated expression of pcbp . furthermore, overexpression of pcbp promoted pedv replication. silencing of endogenous pcbp using small interfering rnas attenuated pedv replication. taken together, these data demonstrated that plp negatively regulated the production of type interferon by interacting with pcbp and promoted pedv replication. porcine epidemic diarrhea virus (pedv) was identified as the causative agent of porcine epidemic diarrhea (ped) in (pensaert and de bouck, ) and has had catastrophic impacts on the global pig industry. the clinical signs and symptoms of ped include severe enteritis, vomiting, watery diarrhea, and high mortality. in china experienced and outbreak of a mutant pedv, leading to huge economic losses (sun et al., a) . pedv has a positive-sense single stranded rna genome that is approximately kb in size and contains six open reading frames (orfs). orf ab encodes polyprotein (pp) a and pp ab, which are further cleaved into non-structural protein (nsp) - . the structural spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins are encoded by orf , orf , orf and orf , respectively. orf encodes the accessory protein orf (kocherhans et al., ) . nsp is the largest nsp, and comprises papain-like protease (plp) and plp domains (lei et al., ) . the innate immune response is the first line of host defense against viral infection (o'neill and bowie, ) . type interferons (ifns) play a key role in host resistance to viral infections. rna viruses induce the production of ifns through toll-like receptor (tlr ) and retinoic acid-inducible gene (rig)-i-like receptor-dependent pathways (kawai and akira, ) . the virus can evade host innate immune response in two major ways: by modifying or hiding pathogen-associated molecular patterns (pamps), and by encoding specific proteins to block immune responses. coronaviruses have evolved specific mechanisms to evade or inhibit host antiviral innate immune j o u r n a l p r e -p r o o f responses (g devaraj et al., ; zhou and perlman, ) . many pedv proteins are involved in escaping the innate immune response. nsp , nsp , nsp , nsp , nsp , and nsp , as well as the structural e, m and n proteins, demonstrated ifn antagonism . the mechanisms underlying the ifn antagonism of nsp , plp , nsp , and n protein have been elucidated (wang et al., ; xing et al., ; zhang et al., ) . pedv nsp inhibits production of type i ifns by degrading cyclic adenosine monophosphate responsive element-binding protein-binding protein and inhibiting immune stress granule expression (dragan et al., ; zhang et al., ) . pedv nsp is a c-like protease that proteolytically cleaves the nuclear transcription factor kappa b (nf-κb) essential modulator (nemo) at glutamine , impairing the ability of nemo to activate ifn production (wang et al., ) . the pedv n protein interacts with the tank-binding kinase, blocking its association with interferon regulatory factor (irf ) and thus inhibiting irf activation and type i ifn production (ding et al., ; hu et al., ) . pedv can also resist immune responses by co-opting host cell proteins. poly(c) binding protein (pcbp ) belongs to a class of proteins that bind poly(c) sequences in both rna and dna and are involved in maintaining mrna stability, regulating translation and cellular antiviral responses (makeyev and liebhaber, ) . pcbp expression is induced following viral infection and acts as a negative regulator of mitochondrial antiviral signaling protein (mavs), triggering its degradation (you et al., ) . it was previously reported that pcbp interacted with porcine reproductive and respiratory syndrome virus (prrsv) nsp β and supported viral replication (beura et j o u r n a l p r e -p r o o f wang et al., ) . pcbp also antagonized vesicular stomatitis virus growth by affecting viral gene expression . it is unclear whether pcbp is also involved in pedv replication. although significant progress has been made in understanding pedv evasion of innate immune responses, the mechanism of interaction between viral and host cell proteins remains unclear. in the present study, we demonstrated that plp interacted with pcbp to inhibit ifn-β production and promote pedv replication. human embryonic kidney (hek) t cells, porcine intestinal epithelial ipec-j cells and african green monkey vero e kidney cells were maintained in dulbecco's modified eagle's medium supplemented with % heat-inactivated fetal bovine serum (fbs), units/ml penicillin and μg/ml streptomycin. pedv strain gdgh (genbank accession number: mg ) was isolated and stored in our laboratory. a dna sequence encoding pedv plp protein was cloned into pcmv-ha as previously described to yield the pcmv-plp -ha expression vector (yu et al., ) . pcbp and pcbp were amplified by pcr and cloned into the pecmv- ×flag-n vector using the primers listed in table . all plasmids were verified by dna sequencing. hek t cells were seeded on coverslips in -well plates and cotransfected with j o u r n a l p r e -p r o o f ng of ifn-luc, ng of tk-luc and ng of pcmv-plp -ha or empty pcmv-ha vector. twenty-four hours post-transfection (hpt), cells were stimulated with ng/ml tumor necrosis factor (tnf)-α for h. cell lysates were prepared for analysis of luciferase activity using a luciferase enzyme assay system in accordance with the manufacturer's instructions (promega, beijing, china). total cellular rna was extracted using the minibest universal rna extraction kit (takara, japan) in accordance with the manufacturer's protocol. total rna was reverse transcribed to cdna using prime script™ rt master mix (takara, japan) and relative gene expression levels were quantified by quantitative rt-pcr using sybr® green real-time pcr master mix (toyobo, japan) using the cycle threshold method. βactin was used as the internal control. all primers were designed using primer premier . software and are listed in table . hek t cells were grown to % confluence in a mm dish and transfected with empty pcmv-ha vector or pcmv-plp -ha using lipofectamine ltx reagent (thermo scientific, china) and following the manufacturer's instructions. at hpt, cells were washed with phosphate-buffered saline (pbs) and lysed with lysis buffer ( mm tris, ph . , containing mm nacl, % triton x- , sodium pyrophosphate, β-glycerophosphate, ethylenediaminetetraacetic acid, na vo , and leupeptin) supplemented with mm phenylmethylsulfonyl fluoride. lysates were centrifuged at j o u r n a l p r e -p r o o f , g for min at °c. the supernatant was collected and incubated with µl of anti-ha agarose (pierce® ha tag ip/co-ip kit, thermo scientific) at °c with shaking overnight. immunoprecipitates were washed with tbst buffer ( mm tris-hcl, ph . , containing . m nacl and . % tween ) and eluted in non-reducing sample buffer. the samples were separated by sds-page and either transferred to a nitrocellulose membrane for western blotting or silver stained for mass spectrometry (pierce tm, thermo scientific). the silver stained gel was analyzed, and one differentially expressed protein spot was selected for liquid chromatography-mass spectrometry (lc-ms/ms) analysis. briefly, the excised band was cut into small cubes approximately . - mm in size, decolorized in a °c water bath, further decolorized in decolorizing solution ( mm potassium ferricyanide and mm sodium thiosulfate) in a °c water bath, reduced with mm dithiothreitol at °c for h, then alkylated with mm iodoacetamide for min at room temperature in the dark. the samples were digested with trypsin overnight. tryptic peptides were analyzed using easy-nl c and q-exactive (thermo scientific, waltham, ma). trapped peptides were separated on an analytical c column ( μm × cm, thermo scientific). the mobile phases consisted of % acetonitrile (acn) a and % acn b, both containing . % formic acid. a gradient of %- % solvent b was used to elute the peptides at a constant flow rate of nl/min for min. data were acquired using a ms scan range (m/z) of - , j o u r n a l p r e -p r o o f acquisition mode dda and a resolution of , . thermo xcalibur . software (thermo) was used for data acquisition. protein identifications were assigned using the ncbi nr databank and swissprot/uniprot databank. cells were harvested and lysed with lysis buffer. protein samples were mixed with sds sample loading buffer, electrophoresed on % or % polyacrylamide gels and transferred to a nitrocellulose membrane. the membrane was incubated with primary antibodies for h at °c. plp was detected using a primary anti-ha-tag rabbit monoclonal antibody (cell signaling technology, danvers, ma). an anti-flag mouse antibody (sigma-aldrich, st. louis, mo) was used to detect flag-tagged proteins and an anti-tubulin antibody (cell signaling technology) was used to detect tubulin. rabbit polyclonal pcbp antibody (proteintech, chicago, il) was used to detect endogenous pcbp protein. horseradish peroxidase-conjugated anti-mouse igg and goat anti-rabbit igg antibodies were used as secondary antibodies. chemiluminescence was detected using the gel imaging system tanon- multi (tanon, shanghai, china). vero e cells were grown in glass slides and cotransfected with pcmv-plp -ha, pecmv- ×flag-pcbp or empty vector (pecmv- ×flag). at hpt, the cells were washed three times with pbs, fixed with . % paraformaldehyde for h at room temperature, and blocked with % bovine serum albumin for h. subsequently, the cells were permeabilized with . % triton x- for min, and then incubated at °c primers for pcbp quantitative rt-pcr are shown in table . anti-tubulin antibody and rabbit polyclonal pcbp antibody (proteintech) were used to assess expression levels of tubulin and pcbp proteins. all results were presented as the means ± standard errors of the means of three independent experiments. data were analyzed using graphpad prism . (graphpad software, inc., la jolla, ca). differences between and among groups were assessed j o u r n a l p r e -p r o o f using the student's t-test and two-way analysis of variance, respectively. values of p < . were considered statistically significant and were indicated as follows: *p< . , **p< . , and ***p< . . ifn-α/β is a key component of the host innate immune response to viral infection. pedv plp has been proposed to play a key role in antagonizing innate immunity (xing et al., ) . to investigate the effect of plp on pedv replication, vero e to investigate the effect of plp on type i ifn promoter activation, hek t cells were cotransfected with ifn-luc and pcmv-plp -ha or empty pcmv-ha vector. tk-luc was cotransfected as an internal control. at hpt, the cells were stimulated with ng/ml tnf-α for h and prepared for luciferase analysis. as shown in fig. b , tnf-α significantly activated the ifn promoter and plp overexpression significantly inhibited tnf-α induced ifn-luc activity. to investigate the effects of plp on type i ifn production, hek t cells were stimulated with tnf-α ( ng/ml), then cells were cotransfected with pcmv-plp -ha or empty pcmv-ha vector. at , and hpt, cells were harvested for total j o u r n a l p r e -p r o o f rna extraction and ifn-β mrna abundance was assessed by quantitative rt-pcr. as shown in fig. c , tnf-α stimulated ifn-β mrna transcription, whereas overexpression of plp significantly inhibited tnf-α-induced transcription of ifn-β mrna. these results indicated that plp acted as a negative regulator of ifn-β, thus augmenting pedv infection. to further investigate the mechanism through which pedv plp inhibited expression of ifn-β, we identified cellular interaction partners of plp using immunoprecipitation and mass spectrometry. hek t cells were transfected with pcmv-plp -ha or pcmv-ha empty vector. at hpt, the cells were lysed and proteins were immunoprecipitated using the pierce® ha tag ip/co-ip kit (thermo scientific, china). the eluent was collected and analyzed by sds-page. silver staining showed multiple and distinct protein bands immunoprecipitated in plp overexpressing cells but not in cells transfected with empty vector. we chose the band with the largest expression difference for mass spectrometry analysis (fig. b ). gene ontology (go) and kyoto encyclopedia of genes and genomes (kegg) analysis was performed to analyze the functions of target proteins (fig. ) . the identified host proteins were classified based on molecular function (mf), cellular component (cc) and biological process (bc) (fig. a) . the proteins were involved in cellular processes, metabolic processes and biological regulation. the majority of proteins pulled down with plp are listed in table . analysis of the top pathways showed that the majority of proteins played a role in organismal systems, human diseases and viral j o u r n a l p r e -p r o o f infection (fig. b) . identification of cellular interaction partners showed that pcbps were involved in the immune response against pedv infection. on the basis of the mass spectrometry results, we initially identified two proteins (pcbp and pcbp ) related to immune responses for further study. the genes encoding these two proteins were amplified and cloned into pecmv- ×flag, and hek t cells were cotransfected with pcmv-plp -ha and pecmv- ×flag-pcbp or pecmv- ×flag-pcbp . at hpt, cells were lysed and immunoprecipitated with mouse anti-flag antibody. the results showed that pcbp and pcbp were both pulled down, and that pcbp interacted most strongly with plp ( fig. ) . therefore, pcbp was selected for further studies. to examine the co-localization of pcbp with plp , vero e cells were cotransfected with pcmv-plp -ha and pecmv- ×flag-pcbp . at hpt, the cells were stained with anti-ha and anti-flag antibodies and analyzed by confocal microscopy. overexpressed plp and pcbp were mainly co-located in the cytoplasm (fig. a) . we further analyzed the co-localization of plp and endogenous pcbp in vero e cells. the cells were transfected with pcmv-plp -ha or pcmv-ha, then analyzed by confocal microscopy following staining with anti-ha and anti-pcbp antibodies. as shown in fig. b , plp was mainly co-localized with endogenous pcbp in the cytoplasm. to study whether plp promoted pcbp upregulation, vero e cells were transfected with different concentrations of pcmv-plp -ha or empty pcmv-ha vector and western blotting was performed using anti-ha, anti-pcbp , and anti-tubulin antibodies. plp overexpression enhanced endogenous accumulation of pcbp protein in a dose-dependent manner (fig. ) . to investigate the effect of endogenous pcbp on viral replication, expression of pcbp was silenced using sirnas. three sirnas ( to ) were designed. following transfection of ipec-j cells, the effect of knockdown was assessed by western blotting. levels of pcbp protein were significantly decreased in all sirna-treated groups compared with the control group, and sirna showed the most efficient pcbp silencing (fig. a) . next, ipec-j cells were treated with sirna or a negative control (nc) sirna for h and then infected with pedv at a moi of . . levels of pcbp mrna and pedv rna were determined at and hpi by quantitative rt-pcr. as shown in fig. b and c, pcbp mrna levels in sirna-treated ipec-j cells were significantly reduced compared with nc-sirna-treated cells, and pedv replication was significantly suppressed (fig. b and c ). to further investigate whether overexpression of pcbp would affect pedv replication, ipec-j cells were transfected with pecmv- ×flag-pcbp or empty vector. at hpt, the cells were infected with pedv at a moi of . and pedv viral loads were determined at , and hpi by quantitative rt-pcr. we found that pcbp overexpression enhanced pedv replication (fig. d) . these results indicated j o u r n a l p r e -p r o o f that plp acted as a negative regulator of ifn-β, and plp functions to augment pedv infection by interacting with pcbp . this work was supported by the national key technologies r&d program nsp is the largest protein encoded in the coronavirus genome and comprises two subdomains (plp and plp ), which are mainly responsible for cleavage of nsp /nsp and nsp /nsp , respectively (barretto et al., ; harcourt et al., ) . not all coronaviruses have a plp domain, while the plp domain is conserved in all coronaviruses. plp proteins encoded by various viruses have been shown to inhibit innate immunity (zheng et al., ) . severe acute respiratory syndrome coronavirus plp inhibits irf activation by blocking ubiquitination of rig-i, tnf-receptor associated factor , and stimulator of interferon genes (a lindner et al., ) . transmissible gastroenteritis virus plp and prrsv plp rely on deubiquitination activity to antagonize ifn production hu et al., ; j o u r n a l p r e -p r o o f b). similar to other coronaviruses, pedv can also inhibit the production of type ifns (zheng et al., ) . pedv plp has been shown to have deubiquitination activity and is an ifn antagonist, but plp has no deubiquitination activity (xing et al., ) . our results showed that plp is also an ifn antagonist. overexpression of plp enhanced pedv infection (fig. a) . using a dual fluorescent reporter, we found that plp could inhibit tnf-α-induced production of ifn-β. in hek t cells transfected with plp expression vectors, induction of ifn-β by tnf-α was inhibited (fig. c) . these results suggested that plp could enhance pedv infection by inhibiting ifn-β production. studies have shown that the plp of pedv cv strain has no ifn antagonism (zheng et al., ) . there are amino acid substitutions distinguishing the plp s of pedv gdgh and cv strains (data not shown). we suspect that these substitutions may lead to differences in plp function. future studies should investigate the effects of amino acid mutations at these sites on plp function. the innate immune response is an important line of defense, and type ifns are essential for host defense against viral infection. the double-stranded rna (dsrna) molecules produced by viral rna replication viral are pamps and can be recognized by pattern recognition receptors (prrs) (matzinger, ) . tlrs and rig-i-like receptors are two crucial prrs that recognize pathogens and stimulate downstream signaling to activate immune responses (huang et al., ; kawai and akira, ) . mavs is a downstream adaptor protein of rig- . rig- mediates the activation of nf-κb and irf by interacting with the caspase-recruiting-like domain of mavs to inhibit the production of type i ifns (biacchesi et al., ; kawai et al., ; meylan et al., j o u r n a l p r e -p r o o f seth et al., ) . pcbp is a negative regulator of mavs whose expression is induced following viral infection and results in mavs degradation (you et al., ) . we found that overexpression of pcbp promoted pedv replication (fig. d) . pedv replication could be inhibited by silencing pcbp expression (fig. c ). pcbp has been reported to play a role in a variety of viral infections luo et al., ; wang et al., ) . we found that plp interacted with pcbp ( fig. and fig. ), and that overexpression of plp induces the expression of pcbp (fig. ) . these results indicated that plp plays an important role in the antagonism of ifn responses. therefore, the study of plp proteins is important for understanding viral escape from host immune responses. compared with plp , the function of plp has received less attention. the plp s of many coronaviruses have deubiquitination activity, and can interfere with host antiviral responses (barretto et al., ) . we found that pedv plp also has ifn antagonism and contributes to viral replication. our results indicate that plp interacted with pcbp , thereby helping pedv escape from host immune responses. our findings reveal a new mechanism evolved by pedv to circumvent the host antiviral response, and contribute to our understanding of the role of coronavirus selectivity in isg and ubiquitin recognition 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respiratory syndrome virus replication the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase pcbp mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip porcine epidemic diarrhea virus nsp induces pro-inflammatory cytokine and chemokine expression inhibiting viral replication in vitro suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by key: cord- - txogxek authors: zhang, xiao-nan; liu, jiang-xia; hu, yun-wen; chen, hui; yuan, zheng-hong title: hyper-activated irf- and stat contribute to enhanced interferon stimulated gene (isg) expression by interferon α and γ co-treatment in human hepatoma cells date: - - journal: biochimica et biophysica acta (bba) - gene structure and expression doi: . /j.bbaexp. . . sha: doc_id: cord_uid: txogxek abstract previous reports suggest that type i and type ii interferon can co-operatively inhibit some virus replication, e.g. hcv, sars-cov, hsv- . to find out the molecular mechanism underlying this phenomenon, we analyzed the transcription profile stimulated by ifn-α and ifn-γ in huh- cells and found that the transcription of a subset of ifn stimulated genes (isgs) including bclg, xaf , trail and tap was enhanced when ifn-α and γ were both present. promoter analysis of bclg revealed that irf- and stat were both required in this process. enhanced irf- /dna complex formation was observed in interferon co-treatment group by gel shift analysis. furthermore, irf- activation was found to be generally required in this cluster of isgs. stat tyrosine phosphorylation was elevated by ifn combination treatment, however, only the hyper-transactivation of gas but not isre was observed. in conclusion, hyper-activation of irf- and elevated stat dimer formation may be two general switches which contribute to a much more robust antiviral symphony against virus replication when type i and type ii ifns are co-administered. hyper-activated irf- and stat contribute to enhanced interferon stimulated gene (isg) expression by interferon α and γ co-treatment in human hepatoma cells xiao-nan zhang a,b, , jiang-xia liu a, , yun-wen hu a,b , hui chen a , zheng-hong yuan a,b, ⁎ interferons (ifns) are pleiotropic cytokines that mediate antiviral, anti-proliferative, anti-tumor and immuno-modulatory activities [ ] . interferons are categorized into two major classes, i.e. type i (predominantly ifn-α and β) and type ii (ifn-γ) interferon. these two classes of interferon bind to different pairs of receptors on the cell membrane but have overlapping route of signaling downstream [ ] . type i interferon preferentially triggers the formation of the isg factor (isgf ) complex composed of phosphorylated stat and stat together with irf- which binds to ifn-stimulated response elements (isre) within promoters of certain isgs. type ii interferon induces the tyrosine phosphorylation of stat and results in the formation of stat homodimers which bind to ifn-γ activated site (gas) elements [ ] . in addition, type ii interferon can potently induce the expression of irf- , which has been shown to stimulate various genes with anti-viral, anti-proliferative or immuno-modulatory activities [ ] . human type i ifns have long been recognized clinically for their antiviral and immuno-modulatory effects. ifn-α has been widely used to treat hepatitis b and hepatitis c virus infection although with limited percentage of sustained response [ , ] . similar to ifn-α, ifn-γ has potent antiviral activity in many in vitro and in vivo virus replication models [ , ] , however its application in clinical viral infection turned out to be unsuccessful and toxicity was also frequently observed [ ] . interestingly, in recent years, a growing body of evidence has shown that when type i and type ii ifn are coadministered, the replication of many viruses is inhibited even more strikingly. sainz et al. demonstrated in a series of articles that type i and type ii ifn could synergize to inhibit herpes simplex virus type (hsv- ), human cytomegalovirus and severe acute respiratory syndrome coronavirus(sars-cov) replication [ ] [ ] [ ] , fuchizaki et al. also reported that combination of mouse ifn-α and ifn-γ can prolong the survival period of mice infected with mouse hepatitis virus type . this is consistent with the lower levels of heptocellular necrosis and serum alanine aminotransferase (alt) and decreased titers of mhv- virus load [ ] . it was also the case in an in vitro hcv replicon system in which ifn-α or ifn-β is co-administered with ifn-γ [ , ] . furthermore, an ifn-γ priming ifn-α boost protocol in a clinical trial successfully invoked the response against hcv in six of the nineteen patients who all failed in a previous -month ifn-α mono-therapy [ ] . in the light of all the experimental and clinical evidence, it is timely to probe the molecular process underlying these phenomena. a small cluster of genes were identified whose expression was enhanced after co-treatment including bclg (bcl- family protein g), xaf (x-linked inhibitor of apoptosis associated factor- ), trail (tnf-related apoptosis inducing ligand) and tap (transporter ). subsequent promoter analysis of bclg showed that irf- (interferon regulatory factor ) and stat (signal transducer and activator of transcription ) were both essential for full enhancement of gene expression. moreover, enhanced irf- /dna complex formation was also observed in emsa analysis. knock down of irf- expression by specific small interfering rna (sirna) not only suppressed the enhancement of bclg but also other members of this cluster suggesting the general role of irf- in this process. stat tyrosine phosphorylation was enhanced, while only the activation of gas (ifn-gamma-activated site) but not isre (interferon-stimulated response element) was observed. taken together, we postulate that irf- hyper-activation and elevated stat dimer formation associate with enhanced expression of a subset of interferon stimulated genes (isgs) which may lead to even greater antiviral activity. huh- cells were maintained in dmem supplemented with % fetal calf serum, mmol/ml l-glutamine, penicillin and streptomycin (gibco, brl). plasmid dna was transfected using fugene (roche), sirna was transfected with oligofectamine (invitrogen). the antibodies to irf- (sc- ), irf- (sc- ) and irf- (sc- ) were obtained from santa cruz, antibodies to stat (# ) and phospho-stat (tyr ) (# ) were from cell signaling, bclg antibody (pab ) was obtained from orbigen. antibody to β-actin was from sigma. human ifn-α and ifn-γ were purchased from calbiochem and peprotech respectively. total rna of huh- cells after various stimulations was extracted using trizol reagent (gibco brl) followed by min of dnasei digestion of remaining genomic dna. pure rna was reverse transcribed using superscript ii (invitrogen) and random hexamer primer according to the manufacturer's instructions. real-time pcr (icyler, bio-rad) was performed as instructed in icycler resource guide. briefly, reactions were carried out in μl volume containing μl cdna template, nm of each forward and reverse primer, sybr greeni and . unit hot-start extaq (takara). a linearized plasmid containing bp gapdh cdna was quantified and diluted to × - × copy/μl as standard. cycle numbers of the logarithmic linear phase were plotted against the logarithm of the concentration of template dna. the primers used are listed in table (upper panel). dual luciferase assays were performed according to the manufacturer's protocols (promega). huh- cells were extracted by using sds sample buffer ( mm tris-hcl, ph . , % w/v sds, % glycerol mm dtt and . % w/v bromophenol blue). equal amount of protein extracts were loaded on a . or % sds-page gel. after electrophoresis, protein was transferred onto a . μm nitrocellulose membrane (protran™, perkin elmer) in transfer buffer ( mm tris, mm glycine and % methanol, ph . ). the membranes were incubated with various antibodies followed by the addition of horseradish peroxidaseconjugated goat anti-rabbit or goat anti-mouse igg (santa cruz). proteins were visualized by an ecl western blotting system (western lightning™, perkin elmer). bclg promoter (− to + ) was amplified from human genomic dna of a healthy donor by pcr and inserted into pgl -basic (promega). subsequent truncation or deletion mutants were done by further subcloning or fusion pcr. the sequences of primers used for cloning and table primers, dna probes and sirnas used in this study sequence ( ′- ′) the huh- nuclear extracts were prepared using a nuclear extraction kit (active motif). p labeling of dna probe and binding reaction was done using a gel shift kit from promega (e ) according to the instructions. briefly, μg/reaction nuclear extract was incubated at room temperature with p labeled irf-e in bclg promoter in × binding buffer. the sequences of the probes are listed in table (middle panel). the antibodies to irf- , irf- , irf- , cold irf-e or mutant irf-e were added min before p labeled probe was included in supershift or competition reactions. after min of incubation with probe at room temperature, the complex was loaded on a % polyacrylamide gel in . × tbe and subjected to electrophoresis. the gel was dried and analyzed using a phosphor-imaging instrument (fuji medical systems). the sequences of sirnas are listed in table (lower panel). all the sirnas were synthesized by genechem co. ltd. transfection of sirna was performed as described in the oligofectamine™ user manual (invitrogen). in a microarray analysis, a class of genes was shown to be hyper-activated when ifn-α/γ was co-administered (unpublished data). subsequent real-time pcr confirmed that, at least, the transcription of bclg long form, tap , xaf and trail was indeed enhanced ( table ) . bclg is a novel member of bcl- family with bcl- homology domains and is pro-apoptotic when over-expressed. bclg encodes two proteins by alternative splicing; the long form of bclg is expressed widely in various tissues whereas the short form is restricted in testis [ ] . since this gene had not been reported to be an isg at the time of analysis, it was selected as a prototype for further examination. we cloned − to + region of bclg promoter and inserted it into pgl -basic. in silico analysis of this region identified an ifn-gamma-activated site (gas) and an interferon regulatory factor element (irf-e) close together between − and − (http: //mbs.cbrc.jp/research/db/tfsearch. html). a camp responsive element (cre) was also identified downstream (fig. a) . the expression of full-length reporter construct showed clear synergism in ifn-α/γ co-treatment (fig. b) . the activation was totally dependent upon tyrosine phosphorylation of stat as over-expression of a y f mutant made bclg unresponsive to interferon co-treatment (fig. c) . truncation of ′ proximal sequence did not alleviate but increased the synergism until the gas element was deleted (bclg , fig. b) . however, we could still detect about three fold induction in co-treatment suggesting that another factor is involved in − to + region. further internal deletions on the downstream sequence until − did not alter the expression pattern (fig. d) , deletion of cre also had no effect. we therefore focused on the gas and irf-e elements. deletions of gas or irf-e both significantly altered the responsiveness to ifn-α/γ treatment. double deletion led to complete unresponsiveness to either cytokine (fig. e ). since the gas and irf-e are very close with only bp in between, we changed the intervening sequence into other two random sequences, however, no significant difference was observed (fig. f) . thus, we conclude that irf-e and gas are both important for synergistic activation of bclg transcription. since previous results (fig. ) demonstrated indispensable role of irf-e, we sought to find out which irf binds to this element. neither irf- nor irf- seemed to participate in bclg transcription as co-transfection of their dominant negative forms did not significantly alter its expression pattern (data not shown). however, when irf- was co-expressed, the basal expression was greatly increased. besides, a dose dependent phenomenon was also observed ( fig. a) . gel shift analysis suggested that over-expressed irf- could bind to the irf-e of bclg promoter (arrow a), addition of irf- specific antibody effectively eliminated this complex (fig. b) . to investigate whether irf- was indeed responsible for the synergistic activation of bclg in physiological settings, we cotransfected bclg and irf- and then treated the cells with ifn-α/γ (fig. c) . ifn-α did not significantly alter the reporter expression, whereas ifn-γ could slightly increase luciferase activity which supported the idea that other factors (e.g. stat ) may be involved in the activation of bclg transcription in ifnγ treatment. co-expression of irf- also abolished the enhanced reporter expression after ifn co-treatment which confirmed the critical role of irf- in this process. to further verify that irf- is the nuclear factor associated with irf-e, we performed emsa analysis by using irf-e corresponding to − to − of bclg promoter as the probe. as shown in fig. d , ifn-α/γ co-treatment led to an enhanced band shift (arrow a) compared with ifn-γ treatment. while addition of a specific irf- antibody super-shifted the complex (arrow b), addition of antibody to irf- and irf- had no effect. the shift band was effectively inhibited by adding fold cold probe but not by mutant oligo. western blot analysis of bclg confirmed the synergistic effect although slightly different compared with the transcription level (fig. ) . when ifn-α was added, a marked decrease of bclg was observed and it is possible that bclg degradation was activated in ifn-α treatment since bclg was found to be linked to mnsf (monoclonal nonspecific suppressor factor), a ubiquitin homology protein [ ] . although ifn-γ treatment did not result in a significant increase of bclg protein, co-treatment clearly up-regulated its expression (fig. ) . the different induction profiles in protein and mrna level are presumably caused by the half-life of bclg and possibly by the different degradation efficiency in various stimulation contexts. as to irf- level, minimal expression was observed in untreated or ifn-α treated cells, but after ifn-γ treatment, an obvious induction was seen as described in previous reports [ ] . in contrast to the enhanced binding activity in emsa experiment, no significant increase of irf- was observed in ifn-α/γ cotreatment which is still not well understood. irf- level remained constant in all treatment groups. to further elucidate the role of irf- in altered transcription profile and synergistic antiviral activity after ifn co-treatment, synthetic sirna directed against irf- was used to abrogate its activation. irf- induction after ifn-γ treatment was sig-nificantly inhibited when irf- sirna was transfected (fig. ) while irf- expression unchanged. the effect of irf- sirna on synergistically enhanced genes was further assessed by quantitative rt-pcr. as expected, enhanced mrna level of bclg was significantly reduced ( . × copy/ gapdh versus . × after ifn-α/γ treatment, p < . ). similar expression profile changes were observed in tap ( . × versus . × , p < . ), xaf ( . × versus . × , p < . ) and trail ( . × versus . × , p < . ) (fig. a) . in silico transcription factor binding site analysis on the promoter of xaf and trail also identified putative irf- binding sites (fig. b) . to ascertain the specificity of our irf- sirna, the expression of mxa, a well-known irf- independent isg, was also quantified. indeed, the transfection of irf- sirna did not affect the mrna level of mxa ( . × versus . × , p > . ) after ifn combination treatment (fig. a) . these data strongly support the idea that irf- activation is a general switch leading to enhanced gene expression in a subset of isgs. interestingly, a previous report has shown that irf- is required for trail induction by both retinoic acid and ifn-γ [ ] . as to tap , a gas and irf-e arrangement in its promoter similar to that of bclg has been indicated and that both elements were crucial for its induction after ifn-γ treatment [ ] . since there has been evidence showing that ifn-γ priming could amplify ifn-α induced stat activation in human macrophage [ ] , we checked stat phosphorylation in huh- cells after min of ifn-α/γ treatment. tyrosine phosphorylation of stat could be detected in either ifn-α or ifn-γ mono-treatment although with differing intensities, in accordance with published data, stat hyper-phosphorylation was readily observed after co-treatment (fig. a) . as stat plays a pivotal role in transcription activation in both ifn-α and ifn-γ signaling, we monitored the induction of downstream interferon-stimulated response element (isre) and gas reporter expression. ifn-γ treatment led to a lagged isre activation compared with ifn-α, however, the combined cytokines did not enhance isre activity (fig. b) suggesting that interferon stimulated gene factor (isgf ) complex formation was not elevated. in the gas assay, ifn-α/γ co-treatment resulted in about two fold activation compared with ifn-γ treatment which suggested increased stat homo-dimer formation (fig. c ). basic research and clinical trials have repeatedly demonstrated that type i and type ii ifn are inter-dependent. signaling by ifn-γ is shown to be dependent on the ifn-α/β receptor components in knock-out mice models [ ] . a recent clinical trial using ifn-alfacon and ifn-γ b to treat hcv patients unresponsive to peg-ifn and ribavirin treatment has shown about % sustained virological response which was remarkably higher than that in prolonged standard treatment [ ] . the underlying mechanism is believed to be two fold: ( ) the innate antiviral effects of ifn-α may be enhanced by addition of type ii ifn, ( ) the adaptive immunity is re-tuned to th response thus facilitating virus eradication by specific ctls (cytotoxic t lymphocyte). in this study, we focused on the immediate antiviral activity after ifn combination treatment in a host cell-line which supports hcv replication. a cluster of genes (bclg, trail, xaf and tap ) was shown to be transcriptionally enhanced after ifn-α/γ co-treatment, subsequent promoter analysis of bclg revealed irf- and stat as crucial regulators. interestingly, a similar gas and irf-e arrangement in the promoter of gp (phox) has also been documented, which allowed rapid induction of gp expression by ifn-γ treatment in u cells [ ] . nevertheless, due to its strict tissue specific (myeloid) expression pattern [ ] , gp (phox) could not be detected in huh- cells (unpublished data). although increased stat phosphorylation was demonstrated, we found that only the gas but not isre mediated transcription was enhanced suggesting that stat homo- dimer rather than isgf formation was preferably induced. as to irf- , it is dramatically upregulated upon some virus infection or ifn stimulation [ ] . other observations suggest that irf- functions as a regulator of cellular response to ifns by affecting a set of isgs [ ] . interestingly, in this study, the dna binding activity of irf- was shown to be enhanced after ifn-α/γ co-treatment. further analysis supports the general role of irf- in regulating this cluster of genes. indeed, basal expression level of irf- was found to be significantly lower in cells harboring the replicon [ ] indicating that irf- could greatly hamper the replication of hcv, and a variant clone of hcv replicon could block the dna binding activity of irf- and suppress the expression of some irf- dependent isgs [ ] . intriguingly, based on previous reports, bclg, xaf and trail are all thought to be associated with apoptosis [ , [ ] [ ] [ ] . bclg can be conjugated with mnsf, a . kda protein with % identity with ubiquitin, the post-translational modification of bclg might be implicated in t-cell activation [ ] . in our preliminary study, bclg long form did not show significant pro-apoptotic effect, nor was there any direct anti-hcv activity at least in huh- cells (data not shown). given that its molecular function is still not well defined, bclg merits further investigations in anti-viral or pro-apoptotic activities. trail, a member of the tnf family, has selective apoptosis activity in cancer cells while normal cells are largely insensitive [ ] . trail−/− mice display no overt phenotype but an increased susceptibility to tumor initiation and metastasis [ ] . x-linked inhibitor of apoptosis associated factor- (xaf ) was shown to be associated with xiap; expression of xaf triggers a redistribution of xiap from cytosol to the nucleus and antagonizes xiap activities [ ] . xaf protein induction is observed predominantly in melanoma cell lines sensitive to pro-apoptotic effects of ifnβ. cells constitutively expressing xaf were more sensitive to trail induced apoptosis [ ] . the similar expression kinetics and co-operative proapoptotic activity indicated that this cluster of enhanced genes is finely regulated to fulfill a specific task, e.g. immunosurveillance of cancer cells as reported [ ] . however, their roles in control of virus replication cannot be excluded. indeed, it has been proposed that a novel antiviral function of type i ifn is the selective induction of apoptosis in virally infected cells [ ] [ ] [ ] . on the other hand, viruses also encode various genes that can antagonize the pro-apoptotic signaling. for example, ns a protein of hcv is well known for its anti-apoptotic activity although the exact mechanism is still controversial [ , ] ; ebv (epstein-barr virus)-encoded poly(a) rnas (ebers) exhibit striking resistance of ifn-α induced apoptosis in burkitt's lymphoma cell lines [ ] . overall, our data provide the evidence that hyper-induction of subclass of isgs is mainly controlled by hyper-activated irf- and elevated stat dimer formation when ifn-α and ifn-γ are co-administered. although only four enhanced genes were unequivocally identified in this study, we believe that many other genes of this cluster are yet to be found. further investigations are needed to expand this cluster and elucidate the functions of its members as some of them may play important roles in the innate antiviral activity of interferon. we are also aware of the fact that the enhanced irf- trans-activation capacity, as shown in emsa, does not correlate well with its protein expression level. intensive analysis is being done to elucidate the mechanism underlying this phenomenon. how cells respond to interferons mechanisms of type-i-and type-ii-interferon-mediated signalling activities of irf- mechanism of action of interferon and ribavirin in treatment of hepatitis c hepatitis b virus infection noncytolytic control of viral infections by the innate and adaptive immune response the role of ifn-gamma in immune responses to viral infections of the central nervous system treatment with human gamma interferon of chronic hepatitis b: comparative study with alpha interferon alpha/beta interferon and gamma interferon synergize to inhibit the replication of herpes simplex virus type interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) synergistic inhibition of human cytomegalovirus replication by interferon-alpha/beta and interferon-gamma huh- cells were treated with ifn-α ( u/ml) and/or ifn-γ ( u/ml), cell extracts were prepared min after. western blotting of tyrosine phosphorylated stat , total stat and β-actin was shown (a). (b) and (c) huh- cells were co-transfected with isre or gas reporter and prl-tk, h later, ifn-α ( u/ml) and/or ifn-γ ( u/ml) was added synergistic antiviral effect of a combination of mouse interferon-alpha and interferon-gamma on mouse hepatitis virus synergistic antiviral activity of human interferon combinations in the hepatitis c virus replicon system enhancement of antiviral activity against hepatitis c virus in vitro by interferon combination therapy immunological response to interferon-gamma priming prior to interferon-alpha treatment in refractory chronic hepatitis c in relation to viral clearance bcl-g, a novel pro-apoptotic member of the bcl- family characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the bcl family irf family of transcription factors as regulators of host defense tumor suppressor irf- mediates retinoid and interferon anticancer signaling to death ligand trail regulation of murine tap and lmp genes in macrophages by interferon gamma is mediated by stat and irf- amplification of ifn-alpha-induced stat activation and inflammatory function by syk and itam-containing adaptors cross talk between interferon-gamma and -alpha/ beta signaling components in caveolar membrane domains combination interferon (type i and type ii) therapy for chronic hepatitis c cooperation of stat- and irf- in interferon-gamma-induced transcription of the gp (phox) gene cloning the gene for an inherited human disorder-chronic granulomatous disease-on the basis of its chromosomal location regulation of hepatitis c virus replication by interferon regulatory factor regulation of pkr and irf- during hepatitis c virus rna replication increased susceptibility to tumor initiation and metastasis in tnf-related apoptosis-inducing ligand-deficient mice identification of xaf as an antagonist of xiap anti-caspase activity identification of x-linked inhibitor of apoptosisassociated factor- as an interferon-stimulated gene that augments trail apo l-induced apoptosis apoptosis and interferons: role of interferonstimulated genes as mediators of apoptosis type i interferons are essential mediators of apoptotic death in virally infected cells alpha/beta interferons potentiate virus-induced apoptosis through activation of the fadd/caspase- death signaling pathway host defense, viruses and apoptosis antiapoptotic and oncogenic potentials of hepatitis c virus are linked to interferon resistance by viral repression of the pkr protein kinase the hepatitis c virus ns a protein activates a phosphoinositide -kinase-dependent survival signaling cascade epstein-barr virus rna confers resistance to interferon-alpha-induced apoptosis in burkitt's lymphoma we thank dr. reena (shanghai public health center) for helpful discussions on our manuscript. this work was supported by chinese state basic research foundation grant ( cb ), national natural science fund for distinguished scholars ( ) and shanghai sci & tech research program ( dz , xd ). key: cord- -a x hvkk authors: wong, lok-yin roy; lui, pak-yin; jin, dong-yan title: a molecular arms race between host innate antiviral response and emerging human coronaviruses date: - - journal: virol sin doi: . /s - - - sha: doc_id: cord_uid: a x hvkk coronaviruses have been closely related with mankind for thousands of years. communityacquired human coronaviruses have long been recognized to cause common cold. however, zoonotic coronaviruses are now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses causing severe respiratory diseases. infections by these emerging human coronaviruses are characterized by less robust interferon production. treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. the mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. this review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against sars and mers coronavirus infection are discussed. on the other hand, the counter-measures evolved by sars and mers coronaviruses to circumvent host defense are also dissected. with a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived. [image: see text] coronaviruses (covs) are classified into four genera, namely alpha-, beta-, gamma-and deltacoronavirus, under the family of coronaviridae and the order of nidovirales (woo et al., ) . the first three genera were previously known as groups i, ii and iii, respectively (lau et al., ; zhong et al., ) . covs have been shown to infect many different hosts including bats, birds, dogs, mice and human (woo et al., ; de groot et al., ) . the infections are commonly zoonotic in nature (chan et al., ) . in the past years, several human covs (hcovs) were identified. hcov- e and hcov-oc , belonging to alpha-and betacoronavirus respectively, were the first two hcovs identified in the mid- s (tyrrell and bynoe, ; hamre and procknow, ; mclntosh et al., ) . healthy individuals infected with either hcov-oc or hcov- e develop illnesses within the range of typical common colds with good prognosis (bradburne et al., ) . since the identification of these two hcovs, extensive studies were conducted to understand their pathogenicity. however, almost all studies showed that hcov-oc and hcov- e caused mild illnesses with high titers of neutralizing antibodies (bradburne et al., ) . the idea of hcov being a relatively weak respiratory diseasecausing agent was therefore presented to the field. this idea was generally accepted until the outbreak of sars in . sars-cov was the first hcov identified to cause acute respiratory distress syndrome (ar-ds) (cheng et al., ; graham et al., ) . according to world health organization (who), a total of cases from countries were reported with a case mortality rate of . %. the sars outbreak changed the landscape of cov studies entirely and marked the new era of combating infectious diseases. tremendous efforts have been put into understanding sars-cov pathogenicity, opening a new page of cov biology. despite advances in infection control and quarantine measures in the past decade, another hcov causing ards was identified in saudi arabia as a novel lineage c betacoronavirus in september (zaki et al., ) . the newly identified hcov was later named mers-cov. up to october , laboratory-confirmed cases were reported to who with related deaths in countries, including a recent outbreak involving cases and deaths in south korea. mers-cov is closely related phylogenetically to two bat covs, hku and hku , shedding light on the possible zoonotic reservoir of mers-cov (zaki et al., ; . together with hcov-hku identified in (woo et al., ) and hcov-nl discovered in (fouchier et al., ; van der hoek et al., ) , six hcovs have been documented up to date. these hcovs present diseases with a range of clinical severity from typical common cold in hcov-oc , hcov- e, hcov-hku and hcov-nl to ards in sars-cov and mers-cov. why these covs show dramatically different pathogenicity in human is an important but unanswered question in the field. one model to explain this difference is based on adaptation and host immunity. according to this model, bats are reservoir of various covs. bat covs constantly emerge in human via intermediate hosts such as civets and dromedaries. exposure of immunologically naïve human populations to these covs commonly causes severe diseases plausibly due to aberrant activation of innate immunity and lack of immune memory. when some covs become better adapted in human by acquiring the ability to transmit from human to human readily, pandemics could arise. meanwhile, as they become fully adapted, the covs might only cause mild diseases in human. existing evidence supports the origin of hcov-oc , hcov- e, hcov-hku and hcov-nl from bats and other animals (woo et al., ; huynh et al., ; corman et al., ) . adaptation and virus-host interaction are also known to be major determinants in cov pathogenesis (pepin et al., ; chan et al., ) . it will therefore be of great interest to see whether emerging human covs might be particularly capable of evading innate antiviral response while activating pathological inflammation. in other words, we need to determine whether the more severe clinical presentations might be accounted for by the specific interaction between host and emerging human covs, namely sars-cov and mers-cov. in this review, the host innate antiviral response to cov infection is particularly focused. in addition, the viral strategies adopted by sars-cov and mers-cov to subvert innate immunity are also summarized to provide inspiring insights that may explain the discrepancies in virulence (figure ). covs are polycistronic positive-sense single-stranded rna (ssrna) viruses with genomes of about kb in size. the ′ most two-thirds of cov genome encodes polyprotein a (pp a) and pp ab replicase polyproteins, which are further cleaved by viral proteases to yield nonstructural proteins (nsps), while the ′ end of the genome encodes structural and lineage-specific proteins (durai et al., ) . the cov life cycle begins with the binding to cellular receptor followed by membrane fusion as well as viral rna and protein synthesis in the cytoplasm. the pp a and pp ab polyproteins are co-translationally processed resulting in the formation of the replicase complex. a set of nested subgenomic mrnas and genomic rna, which possess both the same ′ end and a common ′ leader sequence derived from the ′ end of the genome, is then transcribed. normally, only the ′ end of each mrna is translated. virion assembly is achieved by budding into intracellular membranes and virion release is accomplished through the secretory pathway (cheng et al., ; durai et al., ) . the coronaviral spike (s) protein is responsible for binding to specific host receptor on cell surface and fusing viral envelope with lipid membrane of host upon infection (bosch et al., ; rota et al., ; chen et al., ) . hcov-nl and sars-cov from α-and β-genera respectively recognize angiotensin-converting enzyme (ace ) (li et al., ; pyrc et al., ; frieman et al., ; chen et al., ) while mers-cov infects cells through another cell surface enzyme dipetidyl peptidase (dpp ) (chen et al., ; raj et al., ) . aminopeptidase n (apn) has also been found to be recognized by some α-genus covs like hcov- e (yeager et al., ) . cell surface receptor binding dictates species-specific viral entry as well as tropism. this also confines the direction of cellular antiviral response. we and others have shown the ability of cov s proteins to activate unfolded protein response and endoplasmic reticulum stress fung et al., ; siu et al., b) . the activity of s might also be functionally related to coronaviral perturbation of innate antivir-al response including ifn and cytokine production. pattern recognition receptors (prrs) constitute an indispensable part of the host innate immune defense mechan-ism by the detection of foreign, non-self patterns from invading microbes distinct from host. these pathogenassociated molecular patterns (pamps) are usually biomolecules derived from the surface or generated during the life cycle of the microbes. the detection of pamps by host prrs activates innate immune response including the expression of type i ifns and cytokines for clear- cycle are exposed to host innate immune sensors, rig-i/mda in cytoplasm or toll-like receptors tlr / / in endosome. activation of these immune sensors initiates a downstream signaling cascade that leads to ifn-β gene expression. rig-i/mda conveys signal through a mitochondrial adaptor mavs while tlr signals through trif/myd . both pathways share the common traf adaptor to activate transcription factors. traf serves as an adaptor which activates tank × tbk /ikkε complex for irf phosphorylation and subsequent dimerization, while traf is responsible for the activation of ikk complex which phosphorylates the canonical inhibitor of nf-κb (iκb). activated transcription factors are translocated into the nucleus to drive ifn-β expression. (right) ifn-β are secreted into extracellular space and bound to its cognate receptors ifnar to activate downstream jak-stat signaling. receptor-associated tyrosine kinases jak and tyk are brought to juxtaposition for self-phosphorylation and activation. stats are recruited to and phosphorylated by the tyrosine kinases. phosphorylated stat / with irf forms a ternary complex isgf which translocates into the nucleus and binds to isre in the promoter region upstream of isg genes. isg genes are expressed consequently to establish an antiviral state in cells. oas is an example of isg which produces ′, ′-oligoadenylate ( ′, ′-a) upon detection of dsrna and activates rnase l to cleave viral rna to yield more rlr ligand as a positive-feedback mechanism of ifn production. the cov-encoded proteins shown in red are known to intervene the host innate immune signaling at various action points as evasion mechanisms to sustain viral replication and propagation. the action points at which viral proteins function marked with a question mark (?) represent controversial and inconclusive findings in the field or molecular mechanisms not well studied. mhv: mouse hepatitis virus. arms race between innate immunity and human coronaviruses ance of invading microbes. during cov infection, retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) and toll-like receptors (tlrs) are believed to bear pivotal importance in stimulating host type i ifn induction. it is therefore essential to review the sensing mechanism of the prrs to understand viral evasion mechanisms and provide insights on the development of potential viral antagonists. after viral entry, cov genomes are exposed in the cytoplasm for expression of viral proteins, providing an opportunity for viral rna sensing by host. rlrs are ubiquitously expressed cytoplasmic rna helicases of dexd/h box family responsible for sensing doublestranded rna (dsrna) (yoneyama et al., ) . three types of rlrs have been identified up to now, including rig-i, melanoma differentiation-associated gene (mda ) and laboratory of genetics and physiology (lgp ) (loo and gale, ) . rig-i and mda consist of n-terminal caspase activation and recruitment domain (card) in two tandem copies, a central dexd/h box helicase domain and a c-terminal domain (ctd) (yoneyama et al., (yoneyama et al., , . the n-terminal cards are the effector domain of rlrs to mediate downstream transduction, which is held by the ctd when unstimulated (jiang et al., ; kowalinski et al., ; luo et al., ) . however, in the presence of residual amount of cytoplasmic dsrna, rlrs bind to dsrna through the central dexd/h box helicase domain and ctd with atp, causing a conformational change that exposes the n-terminal cards for signal transduction (yoneyama et al., ; jiang et al., ). lgp lacking the n-terminal cards is thought to act as co-factor that augments the function of rig-i and mda (satoh et al., ; bruns et al., ) . exposure of cards leads to oligomerization of rig-i or mda to form filamentous structure (berke et al., ; peisley et al., ; wu et al., ) . the card filament recruits and further initiates similar filamentous structure formation of card on mavs, an adaptor protein which further recruits downstream effectors tumor necrosis factor receptor-associated factor (traf ), tank-binding kinase (tbk ) and iκb kinase ε (ikkε) (loo and gale, ; wu et al., ) . tbk and ikkε form a complex of activated protein kinase for phosphorylation and activation of not only mavs adaptor , but also irf transcription factor (loo and gale, ) . activated irf are phosphorylated, dimerized and eventually translocated to the nucleus. on the other hand, traf / is also recruited to mavs for nf-κb activation. specifically, canonical nf-κb inhibitor iκb is phosphorylated and then degraded through proteasomes in a ubiquitinationdependent fashion (loo and gale, ) . iκb degrada-tion exposes nuclear localization signal on nf-κb dimer for nuclear translocation. activated irf and nf-κb together with other transcription factors including c-jun assemble the enhanceosome that binds to ifn-β promoter for ifn-β expression (ford et al., ; loo and gale, ) . infection with mouse hepatitis virus induces rig-i expression. in addition, the activation of type i ifn production by this cov in oligodendrocytes requires both rig-i and mda . thus, rlrs might play an important role in the sensing of cov infection. several critical questions concerning rlr recognition of covs merit further investigations. first, the role of rlrs in cov sensing should be studied in rlr-null and cov-susceptible cells and animals. when necessary cr-ispr/cas technology might be used to disrupt rlr genes in target cells (hsu et al., ; yuen et al., ) . second, the cov pamps recognized by rlrs should be identified and characterized. particularly, it will be of interest to see whether and how common and highly structured regions in coronaviral genome, such as the aforementioned ′ leader sequence, might be recognized by rlrs. for example, a polyuridine motif in the ′ untranslated region of hepatitis c virus genome and the panhandle structure in rna viruses such as influenza a virus have previously been shown to be rig-i agonists (saito et al., ; weber et al., ; kell et al., ; liu et al., b) . in addition, possible involvement of viral proteins such as nucleocapsid (n) in this recognition as in the case of other rna viruses (saito et al., ; weber et al., ) should also be clarified. finally, comparative analysis of sars-cov, mers-cov and other hcovs for their ability to activate rlrs will shed light on whether rlr activation would be a critical determinant in cov virulence. covs have been observed to infect host cells through more than one pathway. while cov entry by the fusion of viral envelope and host membrane has been described, the endosomal pathway is still considered the classical entry pathway for covs. in this pathway the activation of s protein cleavage by cathepsin l and transmembrane serine protease tmprss occurs in the absence of cell surface proteases in certain cell types (shirato et al., ; burkard et al., ) . in this regard, tlr family may play an essential role in sensing cov infection through the endosomal pathway. tlr family was identified as another prr homologous to drosophila toll receptor (boehme and compton, ) , sensing various pamps within the endosome which leads to induction of cytokines and ifns. in human, each of the tlrs is known to specifically recognize a particular pamp and preferentially resides in either plasma or endosomal membrane. the cellular localization of tlrs defines their functions in detecting different pamps. for example, tlrs critically involved in viral nucleic acid sensing, including tlr for dsrna, tlr and tlr for ssrna, and tlr for unmethylated cpg island of dsdna viruses, are mainly localized in endosomal membrane while other members having a role in sensing other biomolecules derived from microbial surface components localized to plasma membrane of infected cells (xagorari and chlichlia, ; kawai and akira, ) . tlr family members being type transmembrane proteins share a similar structure with a single transmembrane domain. tlr specificity is determined by the ectodomain made up of various number of leucine-rich repeats (lrrs) that bind the corresponding pamp directly (boehme and compton, ) . signal transduction begins with ligand binding to lrrs in the ectodomain, thus recruiting cytosolic adaptor protein myd with cytoplasmic toll/il- receptor (tir) domain by homotypic tir-tir domain interaction (xagorari and chlichlia, ) . the tlr-myd complex then recruits and activates interleukin r-associated kinase (irak) by phosphorylation. the activated irak then in turn associates with traf and activates a series of downstream effectors leading to the activation of a range of cytokines and ifn-stimulated genes (isgs), while activation of type i ifn expression by tlr is independent of myd but dependent on trif (boehme and compton, ; xagorari and chlichlia, ) . tlr pathway is significantly involved in the suppression of cov replication and induction of type i ifn expression. mice deficient of either tlr or tlr were more prone to sars-cov pathogenesis (mazaleuskaya et al., ; totura et al., ) . notably, disruption of either myd or trif arm of the tlr signaling pathway causes lethal sars-cov disease, indicating the importance of both arms in host innate immunity against sars-cov (totura et al., ) . full characterization of the role of tlrs in host innate antiviral response against sars-cov and mers-cov versus other hcovs will not only provide new knowledge about how tlr activation might impact cov pathogenesis, but might also identify new strategies for antiviral and vaccine development. for example, synthetic tlr agonists could potentially serve as antivirals and vaccine adjuvants in the prevention and control of covs. innate antiviral response is the first line of defense against cov infection. type i ifns are important antiviral and immunomodulatory agents. type i ifns function by binding to ifn-α receptor- (ifnar- ) and ifnar- receptor complex, thus activating janus family tyrosine kinase (jak), leading to the phosphorylation of signal transducer and activator of transcription (stat), a family of transcription factors regulating the expression of isgs. activated stat and irf form ifn-stimulated gene factor (isgf ), stimulating expression of isgs by binding to ifn-stimulated response element (isre) in promoters of isgs (levy et al., ; samuel, ) . viral induction of isgs was abrogated in stat -/mice infected with sars-cov. the viral infection could not be cleared resulting in severe disease, extensive lung injury and % mortality zornetzer et al., ) . this indicates the importance of stat in sars-cov pathogenesis. isgs are the workhorses of the innate antiviral response with diverse functions including direct antiviral activities and regulation of adaptive immune system (schneider et al., ) . for example, ifn-inducible gene p evokes apoptosis in virus-infected cells (takaoka et al., ) . ifn-inducible protein kinase pkr, ′, ′-oligoadenylate synthetase (oas) and rnase l are important modulators involved in dsrna sensing, viral gene expression and replication. they act sequentially to trigger viral rna degradation and suppression of viral activities (samuel, ) . other isgs encoding antiviral effectors such as mx proteins, cholesterol- -hydrolse, ifitm proteins, trim proteins, viperin, tetherin, cgamp synthase and sting could also be highly relevant to cov infection (schneider et al., ; schoggins et al., ; ma et al., a; ma et al., b) . inflammatory responses triggered by inflammatory cytokines like tumor necrosis factor α (tnf-α) and ifn-γ are also found to be ifn-dependent (samuel, ) . ifns do not only exert antiviral effects through activation of innate immunity but also act as modulators of adaptive immunity. adaptive immune response is activated by increased level of ifns. the levels of major histocompatibility complex (mhc) proteins class i and ii are found up-regulated by ifns. this facilitates efficient antigen presentation and hence cellular immune response to cov infection (samuel, (samuel, , ivashkiv and donlin, ) . in addition, the roles of non-conventional isgs including micrornas, long non-coding rnas and alternatively spliced isoforms have been increasingly recognized in recent years (schneider et al., ) . it will be of importance to determine whether sars-cov and mers-cov might be unique in isg activation as suggested in a recent study, which demonstrated that mers-cov induces repressive histone modifications to down-regulate specific subsets of isgs (menanchery et al., b) . in relation to this, two areas concerning isg activation by covs might require more attention and research efforts. first, unbiased and large-scale screening of antiviral isgs using rna interference or crispr/cas technology might be carried out to identify key cellular factors that restrict sars-cov and mers-cov replication and infection. second, small-molecule compounds that activate antiviral isgs could be identified and tested for inhibition of sars-cov and mers-cov replication and infection. for example, establishing the significance of cgas and sting in cov infection might lead to the development of cyclic dinucleotides such as c-di-gmp and cgamp as novel anti-cov agents. covs have been reported to directly or indirectly suppress ifn production and signaling pathways by a subset of viral proteins via various mechanisms. in many cases, infected patients have shown diminished levels of type i ifns. this is especially true for sars and mers patients with severe diseases (faure et al., ) . it was also shown that sars-cov and mers-cov were capable of evading type i ifn production and signaling to different extents in cultured cells (kindler et al., ) . when the deficiency in type i ifn production in cov-infected cells was remedied by ifn-α treatment, cov replication was inhibited (falzarano et al., ) . combination of ifn-α with other antiviral drugs further improves the survival of infected patients (omrani et al., ) . this evidence suggests an essential role of type i ifns in the antiviral effect against cov infection. covs have evolved strategies to counter host antiviral response by antagonizing type i ifn production and signaling. cov proteins have been characterized to exhibit innate immunosuppressive effects in cellular models. below we will discuss them in three categories: structural, lineagespecific and non-structural proteins (nsps) (de groot et al., ) . nsps of covs are involved in the assembly of the replicase complex for viral rna synthesis (sevajol et al., ) . certain nsps have also been reported to possess innate immunosuppressive effect that facilitates viral replication and propagation, although these proteins per se are not required for viral life cycle (narayanan et al., b; lokugamage et al., ) . nsps of different covs are more or less evolutionarily conserved suggesting their functional significance, with the exception of nsp and nsp , which are thought to contribute to virulence of certain covs (neuman et al., ) . four structural proteins are found in covs, namely s, membrane (m), envelope (e) and n proteins. structural proteins contribute the architecture for virion assembly. accessory proteins are lineage-specific with diverse behaviors in different covs but are not essential for viral replication and propagation (de groot et al., ) . cov nsps have shown suppressive effects in various immune pathways including type i ifn production and signaling. sars-cov and mers-cov nsp proteins have been shown to selectively induce degradation of host mrna by inducing endonucleolytic cleavage while leaving viral rnas intact (huang et al., ; lokugamage et al., ) . in addition to the induction of endonucleolytic cleavage of host mrna, general inhibition of host mrna translation is achieved by binding of s subunit of ribosome with sars-cov nsp (huang et al., ) . particularly, sars-cov nsp inhibits innate immune response by translational repression of ifn mrna transcripts, hence altering ifn production and signaling (narayanan et al., a; tanaka et al., ) . mers-cov nsp has also been characterized to specifically induce endonucleolytic cleavage of nuclear transcribed mrna while sparing cytoplasmic host mrna and viral rna (lokugamage et al., ) . this suggests a novel mechanism for evading host immune response. cov nsp protein has been characterized with a papain-like protease (plpro) domain for enzymatic cleavage of pp a and pp ab as well as a plp domain with deubiquitinating and deisgylating activity (clementz et al., ; mielech et al., ) . mers-cov plpro is able to antagonize ifn production induced by rig-i and mda as well as nf-κb activation . mers-cov plpro is catalytically more efficient (báez-santos et al., ) and its catalytic activity is indispensable for the suppressive effect on rig-i, mda and nf-κb (mielesh et al., ) . in contrast, sars-cov plpro does not require enzymatic activity for ifn antagonism (clementz et al., ) . hcov-nl and sars-cov plp transmembrane domain can also act as potent ifn antagonists to suppress ifn production induced by rig-in, a dominant active form of rig-i (clementz et al., ) . in another view of direct inhibition of ifn induction, nsp with deubiquitinating and deisgylating activity may also influence the ubiquitination and isgylation pattern and dynamics thus indirectly hindering innate immune response against cov infections (clementz et al., ) . for example, isgylation and ubiquitination of irf required for optimal activation is probably altered by plp domain of nsp . apart from directly manipulating the signaling pathway involved in ifn production, several cov nsps were identified to act on viral rna to minimize ifn stimulation. n -methylguanosine is the fundamental moiety of eukaryotic mrna cap structure and ′-o-methylation on this moiety is a representative host signature to avoid prr activation as well as isg action. particularly, viral rna with this modification evades recognition by mda or ifit family antiviral factors (züst et al., ; daffis et al., ) . this is a common immunoevasive mechanism adopted by not only different covs but also other rna viruses. functional screening in yeasts suggested a novel function of sars-cov nsp as a guanine-n -methyltransferase, the activity of which is required for viral replication and transcription (chen et al., ) . another nsp of sars-cov, nsp , also possesses ′-o-methyltransferase activity (menachery et al., a; menachery et al., c) . structural modeling suggested that sars-cov nsp associates with nsp in : ratio to form a complex of mature ′-o-methyltransferase for viral cap methylation (chen et al., ; decroly et al., ) . a short peptide derived from nsp conserved region has been shown to be a promising nsp antagonist which outcompetes native nsp to blunt ′-o-methyltransferase activity and restrict viral replication . plausibly, cov nsps might execute their innate immunosuppressive roles by targeting type i ifn production and signaling. further investigations are required to clarify whether and how far the sensing of cov rna and the induction of innate antiviral response are involved in the inhibitory activity of the nsp antagonists on cov replication. cov structural proteins have been shown to inhibit ifn production and signaling at multiple levels. sars-cov n protein showed inhibitory effects on ifn production induced by sendai virus and dsrna analogue poly(i:c) but no inhibition could be observed when downstream signaling molecules of tlr and rlr pathway were overexpressed. truncation mutant of n protein shows that the c-terminal domain is critical for rna-binding and ifn-antagonizing effect (lu et al., ) . this suggests sars-cov n may interfere with rna recognition by host immune sensors such as rig-i and mda thus achieving suppressive role in ifn production. other than n protein, sars-cov m protein has been characterized to potently down-regulate ifn production by impeding the formation of traf ·tank· tbk /ikkε complex through the first transmembrane domain (siu et al., (siu et al., , a . sars-cov m protein inhibits ifn production possibly through a sequestration model in which components of traf ·tank·tbk /ikkε complex, an active complex for irf phosphorylation, are sequestered to specific locations in the cell (siu et al., ) . sars-cov m protein therefore exerts its inhibitory effects by impeding the formation of traf ·tank· tbk /ikkε complex but not by modulating the catalytic activity of the complex. mers-cov m protein also exhibits ifn-antagonizing effects similar to its counterpart in sars-cov. in a previous study, mers-cov m is shown to impede ifn production by preventing irf translocation into the nucleus (yang et al., ) . however, the detailed mech-anism of inhibition remains unknown. recently, our group has characterized the mode of inhibition of ifn production by mers-cov m. consistently with previous report, we show that mers-cov m suppresses ifn production by preventing irf activation. we showed that mers-cov m interacts with traf which impedes the recruitment of tbk to traf complex. irf activation and dimerization have also been hampered as a result. the inhibitory effect is at least in part accounted for by the n-terminal transmembrane domains. despite of the similar behaviors, mers-cov m can only moderately suppress ifn expression when compared to sars-cov m. interestingly, hcov-hku m protein does not exert any inhibitory effects on ifn production (siu et al., a) , suggesting that the ifn-antagonizing activity of structural proteins is unique to each cov but not universal. it will be of great interest to see whether this may correlate with the pathogenicity of different hcovs. eight accessory proteins have been identified in sars-cov and five are found in mers-cov (narayanan et al., b) . sars-cov genome encodes orf a, orf b, orf , orf a, orf b, orf a, orf b and orf b as accessory proteins (narayanan et al., b) . sars-cov orf b and orf have been found to antagonize type i ifn production and signaling. particularly, sars-cov orf b and orf suppress ifn-β production by perturbing irf activation induced by sendai virus infection. sars-cov orf b and orf also suppress ifn-β-induced activation of isre in isg promoters (kopecky-bromberg et al., ) , although they are not able to reduce the level of phosphorylation of stat , a transcription factor that activates isre activity once phosphorylated. however, sars-cov orf has been shown to inhibit stat translocation for isre activation (kopecky-bromberg et al., ) . the findings suggest a mode of inhibition of ifn-β signaling by sars-cov. ifn antagonism of accessory proteins has also been observed in another deadly hcov. mers-cov genome encodes orf , orf a, orf b, orf and orf b (de groot et al., ) . among the five accessory proteins, orf a, orf b and orf show the ability to dampen ifn production (yang et al., ) . suppression of ifnβ promoter-driven luciferase activity has been observed in cells transfected with orf a, orf b and orf plasmids. all these accessory proteins are able to block irf translocation to the nucleus to activate ifn promoter (yang et al., ) . mers-cov orf a shows an additional level of inhibition of innate immunity by intervening nf-κb activation. in another study, orf a has been shown as an antagonist of ifn production by inhib-iting irf translocation but has no effect on ifn signaling (niemeyer et al., ) . our group demonstrated that mers-cov orf a interacts with pact, a cellular dsrna-binding protein that optimally activates rig-iand mda -induced type i ifn production, in an rnadependent manner (siu et al., c) . this suggests that orf a may compete with rig-i and mda for rna, rendering the inactivation of rig-i and mda . direct interaction of orf a with pact may also prevent interaction of pact with rig-i and mda , thus compromising pact-dependent activation of rig-i and mda required for optimal induction of ifn production. although we and others have observed the ifn-antagonizing activity of mers-cov orf b, different activity profiles and mechanisms have been suggested (yang et al., ; matthews et al., ) . one recent report suggested that orf b directly interacts with and inhibits tbk /ikke in the cytoplasm but might also perturb type i ifn production in the nucleus through an unknown mechanism (yang et al., ) . mouse hepatitis virus, another betacoronavirus closely related to hcov-oc and hcov-hku , encodes a lineage-specific accessory protein named ns with innate immunosuppressive property (zhao et al., ) . biochemical assays indicate that ns protein has phosphodiesterase activity against ′, ′-a, the product of oas . thus, ns is a potent inhibitor of an ifn effector molecule and it might represent a new family of viral and cellular proteins with innate immunosuppressive activity gusho et al., ) . whether distantly related proteins in hcov-oc and hcov-hku might have similar activity remains to be determined. more importantly, it will be of interest to see whether sars-cov and mers-cov might encode proteins with similar enzymatic activity. multiple ifn antagonists have been identified and characterized in sars-cov and mers-cov. some differences between these ifn-antagonizing viral proteins and their counterparts in other covs such as the parental bat viruses of mers-cov have also been noticed (siu et al., c) . existing evidence supports several important notions. first, although sars-cov and mers-cov share some features in common, they are distinct and use unique mechanisms for innate immune evasion (perlman and zhao, ) . second, both sars-cov and mers-cov are bat-origin covs that are well adapted in bats but newly emerge in human. this provides a golden opportunity for the study of cov-host interaction, cov adaptation as well as the arms race between host innate antiviral immunity and covs. observing how the arms race between the host and sars-cov or mers-cov might evolve when the viruses become adapted to human will be most revealing and could provide important clues as to how a balance of power in this arms race might result in attenuation with increased transmissibility. finally, studies on sars-cov and mers-cov have overturned existing concepts and derived new principles and thoughts to cov biology. particularly, mechanisms by which sars-cov and mers-cov evade innate immunity have attracted increasing attention. however, many key issues remain obscure. particularly, better in vivo evidence should be obtained to clarify whether more potent inhibition of innate ifn production and signaling by sars-cov and mers-cov is a key determinant in virulence and disease severity. covs have drawn a lot of interests in the light of the recent emergence of mers-cov. it remains to be understood whether the emerging deadly covs causing ards might ultimately be established and adapted in human resulting in significant attenuation of virulence. from the identification of the first two hcovs, hcov- e and hcov-oc in the mid- s, we learned that hcov was able to cause only common cold. however, the outbreaks of sars and mers that have claimed hundreds of lives revealed the other extreme of cov pathogenicity and raised new questions in cov biology. so far no vaccines have been developed against sars-cov and mers-cov. infection with sars-cov and mers-cov has been accompanied with suppression of innate immune response, most notably with the suppression of type i ifn production and signaling pathways. as the first-line defense in the immune system, suppression of innate immune response by these covs has impeded the host ability to restrict infection, causing significant casualties. although many reports have shed light on the molecular mechanism by which various cov proteins antagonize type i ifn production and signaling, most of the studies were performed with overexpression experiments in cellular models. future emphasis should be put on the characterization of knock-out viruses with which the function of a particular viral gene could be studied in a more physiologically relevant context. infectious clones and replicons for sars-cov and mers-cov have been generated for this reverse genetic approach (yount et al., ; almazán et al., almazán et al., , almazán et al., , scobey et al., ) . ifn and cytokine profiles of deadly hcovs such as sars-cov and mers-cov can be compared with hcov- e and hcov-oc causing mild diseases. the pivotal significance of type i ifns in innate immune activation and modulation has been discussed in this review. suppression pattern of ifn may provide insights on the high pathogenicity of deadly hcovs. the arms race between host innate antiviral response and emerging human covs might evolve after their 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isolation of a novel coronavirus from a man with pneumonia in saudi arabia homologous ′, ′-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology recent progress in studies of arterivirus-and coronavirus-host interactions transcriptomic analysis reveals a mechanism for a prefibrotic phenotype in stat knockout mice during severe acute respiratory syndrome coronavirus infection ribose ′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda we thank hinson cheung, kitty fung, edwin kong and sam yuen for reading the manuscript critically. coronavirus research in our laboratory was supported by hong kong health and medical research fund ( , and hkm- -m ) and hong kong research grants council (hku /crf/ g, c - r and t - / -r). the authors declare that they have no conflict of interest. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord- -xypg evo authors: market, marisa; angka, leonard; martel, andre b.; bastin, donald; olanubi, oladunni; tennakoon, gayashan; boucher, dominique m.; ng, juliana; ardolino, michele; auer, rebecca c. title: flattening the covid- curve with natural killer cell based immunotherapies date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xypg evo natural killer (nk) cells are innate immune responders critical for viral clearance and immunomodulation. despite their vital role in viral infection, the contribution of nk cells in fighting sars-cov- has not yet been directly investigated. insights into pathophysiology and therapeutic opportunities can therefore be inferred from studies assessing nk cell phenotype and function during sars, mers, and covid- . these studies suggest a reduction in circulating nk cell numbers and/or an exhausted phenotype following infection and hint toward the dampening of nk cell responses by coronaviruses. reduced circulating nk cell levels and exhaustion may be directly responsible for the progression and severity of covid- . conversely, in light of data linking inflammation with coronavirus disease severity, it is necessary to examine nk cell potential in mediating immunopathology. a common feature of coronavirus infections is that significant morbidity and mortality is associated with lung injury and acute respiratory distress syndrome resulting from an exaggerated immune response, of which nk cells are an important component. in this review, we summarize the current understanding of how nk cells respond in both early and late coronavirus infections, and the implication for ongoing covid- clinical trials. using this immunological lens, we outline recommendations for therapeutic strategies against covid- in clearing the virus while preventing the harm of immunopathological responses. natural killer (nk) cells are a key component of the innate immune system and are critical in the response to many viral infections in humans and animal models ( ) ( ) ( ) . in addition to their beneficial antiviral role, nk cells have also been associated with immunopathology in infections such as respiratory syncytial virus (rsv) ( ), influenza a virus ( ) ( ) ( ) ( ) , and hepatitis b ( ) . additionally, in the context of non-respiratory viral infections by hiv and hcv, nk cells appear to act as a rheostat by eliminating activated cd + and cd + t cells, thus preventing t cell-mediated autoimmunity ( ) . the etiologic agent of the outbreak of pneumonia in wuhan, china, was identified as belonging to the coronaviridae family and named severe acute respiratory syndrome coronavirus (sars-cov- ). this virus causes the coronavirus disease (covid- ) which was declared a pandemic by the world health organization (who) on march th, ( , ) . with the paucity of information currently available, there is a lack of consensus on the role played by nk cells in the response to coronavirus (cov) infection. in this review, we will explore evidence for both the protective and pathological role that nk cells may play in cov infection. based on this knowledge we will comment on immune modulating treatment options that are being developed for the current covid- crisis. first discovered in the s, covs are part of the coronaviridae family of enveloped positive single-strand rna viruses ( , ) . the subfamily orthocoronaviridae includes four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus ( ) . alpha-and betacoronaviruses circulate in mammals, including bats, gammacoronaviruses infect mostly avian species, and deltacoronaviruses infect birds and mammals ( ) . low pathogenic human covs (hcovs), such as hcov- e ( ) , infect upper airways and etiological studies suggest they account for - % of common colds ( , ) . on the other hand, highly pathogenic covs infect the lower respiratory tract and can cause severe pneumonia ( ) . these highly pathogenic covs include sars-cov- , the virus responsible for the - severe acute respiratory syndrome (sars) epidemic, and mers-cov, the virus responsible for the outbreak of middle eastern respiratory syndrome (mers) in ( ) ( ) ( ) . while highly pathogenic covs have become a relatively recent issue for humans; feline, canine, and bovine covs have long been recognized as significant pathogens with implications in veterinary medicine and agriculture ( , ) . all covs have a roughly kb genome packed into an enveloped helical capsid ranging from to nm ( ) . at minimum, coronaviridae members encode structural and non-structural proteins ( ) with the family owing its name to the crown-like appearance produced by their spike (s) proteins ( ) . mutations in the s protein have allowed sars-cov / to co-opt ace or mers-cov to co-opt dipeptidyl peptidase (dpp ) receptor/cd as viral entry receptors, thus facilitating the zoonosis of nonhuman covs ( , ( ) ( ) ( ) . in addition, another mechanism that may have allowed these viruses to adapt to human hosts is through s protein cleavage by host cell proteases to expose the s domain fusion peptide, which induces viral and cellular membrane fusion and results in the release of viral genome into the cytoplasm ( ) . genetic sequencing revealed sars-cov- to be a betacoronavirus that shares . % nucleotide identity with sars-cov- and . % identity to mers-cov ( ) . the epidemic of sars in - caused by sars-cov- illustrated the devastating potential of coronaviruses to cause serious disease in humans ( ) . sars ultimately reached countries and continents causing over , infections and over deaths. the basic reproductive rate (r ) or the number of expected cases arising from one infected individual, ranges from to ( , , ) . with its reservoir in bats, sars-cov- is a zoonosis that was transmitted to humans by palm civets ( , , ) . sars-cov- infects lung pneumocytes ( ) and enterocytes in the digestive tract ( ) most often producing flulike symptoms ( , ) . more severe presentations including pneumonia, pronounced lymphopenia, liver abnormalities, and acute respiratory distress syndrome (ards) were also reported, with most fatalities due to respiratory failure ( , ( ) ( ) ( ) ( ) . the subsequent mers-cov outbreak in also originated in bats, with dromedary camels being the intermediary host ( , , ) . the r for mers-cov is estimated to be under ( ) . the extent of mers-cov transmission was more limited than sars-cov- , but its case fatality rate was greater with , cases over countries and deaths being reported at the end of ( ) . common presentations for mers-cov include fever, dyspnea, muscle pain, and digestive tract symptoms and disease progression is more likely in those with comorbidities ( ) . like sars-cov- and mers-cov, sars-cov- is thought to have originated in bats through an unknown intermediary host ( ) . at the time of writing, the number of global infections is estimated to be over , , with over , deaths ( ) and the r is roughly . ( ) . like other diseases caused by infectious covs, most patients present with flu-like symptoms including fever, cough, and lethargy, with the development of pneumonia and ards often proving fatal ( ) . furthermore, patients with underlying conditions are at risk for further complications if infected with covid- , such as those with cardiovascular disease ( ) . sars-cov- has been posthumously detected in not only the lungs, but the pharynx, heart, liver, brain, and kidneys ( ) . transmission of sars-cov- is thought to mainly occur through direct contact/inhalation of respiratory droplets and aerosols from infected carriers, but indirect transmission by fomites has also been reported, although less efficient ( , ) . sars-cov- viral entrance is thought to be mediated by binding of the s protein to the ace receptor ( , ) , although this is still under debate ( ) . while direct cytopathic effects are thought to play a major role in cov pathology, studies have suggested that a dysregulated immune response resulting in pathological inflammation is also partly responsible ( ) . with the current pandemic already surpassing the previous cov outbreaks ( ) , rapid deployment of novel approaches to understanding and treating coronavirus infections are needed. innate immunity is essential in disease prevention and viral clearance. among the first responders to viral infections, tissueresident macrophages and dendritic cells (dcs) ( ) recognize evolutionarily conserved microbial structures termed pathogenassociated molecular patterns (pamps) via germline-encoded pattern recognition receptors (prrs) ( ). in the context of respiratory rna viruses, airway epithelial cells, that also express some prrs ( ) , are often infected and have a major role in the first line of defense. tlr , tlr , tlr , mda- , and rig-i are prr expressed by immune and non-immune cells that are especially relevant in fighting respiratory rna viruses, such as coronaviruses ( ) . sensing through prrs results in the transcription of genes involved in the inflammatory response, with type i interferons (ifns) (ifn-α/β) production being a critical part of the antiviral response ( ) . type i ifns are produced by many immune and non-immune cells ( , , ) and in addition to eliciting intrinsic antiviral responses ( ), they are also essential to prime innate and adaptive lymphocytes, including nk cells ( ) . nk cells are cytotoxic lymphocytes that directly target infected, stressed, or transformed cells and play a critical role in bridging the innate and the adaptive immune responses ( ) . in humans, mature nk cells comprise - % of total peripheral blood leukocytes and are described phenotypically as cd − cd − cd − cd + cd +/− ( ). nk cells do not undergo clonal selection but instead express several germline-encoded receptors that regulate their activity ( , , ) . upon viral infection, host cells become more susceptible to nk cell killing through: (i) upregulation of self-encoded molecules induced by infection/cellular stress ( , ) that bind activating nk cell receptors such as natural cytotoxicity receptors (ncrs) (nkp , nkp , and nkp ) ( ), c-type lectin-like receptors nkg d and nkp ( ) , and co-activating receptors such as dnam- ( ); (ii) downregulation of ligands for inhibitory receptors such as killer immunoglobulin-like receptors (kirs) ( ) ( ) ( ) and the c-type lectin-like receptor cd -nkg a ( , ) which suppress nk cell activation, and; (iii) direct recognition of viral moieties, via engagement of pamps ( ) or transmembrane activating receptors such as mouse ly h ( ) or human nkg c ( ) . moreover, nk cells can eliminate virus-infected cells via cd -mediated antibody-dependent cellmediated cytotoxicity (adcc), which has been shown to be particularly important for herpesvirus clearance ( ) . finally, nk cell activity is modulated by cytokines, including, but not limited to, the activating cytokines interleukin (il)- / / / ( ) and type i ifn, which can be produced by virally infected cells or activated antigen presenting cells ( , ) . il- / / / , alone or in combination, promotes nk cell survival, proliferation, cytotoxicity, and cytokine production, including ifn-γ ( ) . therefore, nk cells are uniquely equipped to sense and quickly respond to viral infections. nk cells are found in circulation and in peripheral tissues ( ) and can be quickly recruited to sites of infection where they facilitate and accelerate viral clearance. in fact, nk cells are not thought to have permanent tissue residency but instead move dynamically between the blood and tissues, such as the lungs ( ) . nk recruitment is regulated by chemokine gradients that are sensed via chemokine receptors ( , ) . activated nk cells induce the apoptosis of target cells through the engagement of death receptors, such as trail and fas ( ) or via direct cytotoxicity through ca + -dependent exocytosis of cytolytic granules (perforin and granzymes) ( ) . moreover, nk cells secrete cytokines, including ifn-γ, which have key anti-viral properties ( ) . in addition to being essential first responders to viral infection, nk cells can elicit a stronger secondary response resembling the memory features of adaptive lymphocytes ( , ) . nk cell memory has been initially described in mice infected by mcmv, where ly h + nk cells quickly expand and have stronger responses after a secondary encounter with the virus ( ) . interestingly, a similar nk cell subset has been identified in humans, where nk cells expressing nkg c are expanded and persist in cmv infected patients ( ) . both ly h and nkg c bind viral determinants, highlighting how nk cell memory is linked with the ability of nk cells to directly recognize viruses ( , ) . in addition to direct recognition of viral molecules, longlasting changes in nk cells are induced by the cytokine milieu ( , ) , which can be elicited by viral infection. the relevance of nk cells in fighting viral infections has been highlighted by several studies where nk cells, in mice and humans, were not present or had compromised functions ( ) . for example, individuals with nk cell deficiencies (nkd), a subset of primary immunodeficiency diseases, are highly susceptible to viral infection, particularly by herpesvirus and papillomavirus families ( ) . the seminal case of nkd in an adolescent female with severe herpesvirus infections (varicella pneumonia, disseminated cmv, and disseminated hsv) revealed how functional nk cell deficiencies have clinical consequences in terms of viral infections ( ) . cancer patients are also at risk of viral infections ( ) , which may be explained, at least in part, by an impairment of nk cell responses often observed in humans and in murine tumor models ( ) ( ) ( ) ( ) ( ) ( ) . unsurprisingly, cancer patients are at a significantly increased risk of severe covid- ( , ) . elderly patients are also more susceptible to viral infections ( ) . mouse studies highlighted how a decreased number of circulating mature nk cells in aged animals paralleled with increased susceptibility to viral infections ( ) . studies in humans suggest that although nk cell numbers can actually increase with aging, nk cell activity declines significantly ( , ) . przemska-kosicka et al. investigated nk cell function in response to seasonal influenza vaccination in young and old populations and observed quantitative and qualitative changes associated with impaired responses in the nk cell population and this was associated with poor seroconversion in the older population ( ) . additionally, obesity, which has been shown to cause systemic nk cell dysfunction ( , ) , has also been linked to increased covid- severity and could be the reason behind the high prevalence of severe covid- in younger people ( ) . in short, nkd and individuals with reduced nk cell numbers or function are more susceptible to viral infections. unsurprisingly, the cdc has already highlighted a higher risk of infection and severity of covid- in older individuals and individuals with comorbidities such as obesity and cancer ( ) . however, this point is still controversial as a systematic review showed that primary immunodeficiencies are not linked with increased covid- severity ( ) , but these data have to be interpreted keeping in mind that a large part of covid- pathology is caused by excessive immune activation, which is arguably harder to reach in immunocompromised individuals. given the paradoxical role of the immune response in covid- patients, it would be extremely useful to be able to rely on immunological functional biomarkers that could predict the outcome of disease severity. such assays are readily available for determining nk cell activity, e.g., nkvue tm , and there is therefore an opportunity to conduct studies that would link nk cell functions to disease severity. evasion of host immune responses is necessary for the successful propagation of a virus. mechanisms employed by covs to evade the immune response could provide insights into how the immune system, and nk cells in particular, responds to sars-cov- . covs have been shown to target components of the innate ifn response, employing non-structural proteins (nsps), structural proteins, and accessory proteins to achieve this goal. nsp methylates viral rna therefore preventing recognition by mda and dampening type i ifn production ( ) . nsps also suppress type i ifn responses via the inhibition of the transcription factor stat mrna transcription (nsp ) and deubiquitination of transcription factors like interferon regulatory transcription factor (irf) (nsp ) ( ) . moreover, viral-encoded accessory proteins from sars-cov- open reading frame (orf) b and mers-cov orf a/ b also block ifn production and signaling ( ) . in addition, the mers-cov orf -encoded protein blocks p-stat import, thus blocking ifn signaling ( ) . finally, the structural m protein of mers-cov ( ) physically sequesters kinase proteins rig-i, tbk , ikke, and traf and the sars-cov- n protein inhibits activator protein (ap)- signaling, protein kinase r function, and nfκb activation, all of which act to impede ifn responses ( ) . in vivo murine studies report young mice rapidly clear sars-cov- infection, while old mice do not and that this discrepancy is due to a delay in type i ifn. furthermore, early administration of ifn-β induces a stronger immune response and reduces mortality in old mice ( ) . since type i ifns are critical for nk cell activation and effector functions, it is possible that nk cell-mediated clearance of sars-cov- is being subverted by these mechanisms. further research into the role of nk cells in cov clearance and potential immune evasion mechanisms are necessary to inform therapeutic development and use. there is currently a paucity of studies into the role of nk cells not only in covid- pathophysiology, but also in other coronavirus infections. an in vivo study reported that beige mice on a b background cleared sars-cov- normally, indicating that functional lymphocytes, including nk cells, may not be required to eliminate sars-cov- in murine models ( ) . however, in a more recent study characterizing the cellular immune response to sars-cov- in - -month old balb/c mice, t cell depletion did not prevent control of sars-cov- replication ( ), suggesting a role for the innate immune system, and nk cells, in viral clearance. importantly, in this study cd -depletion resulted in enhanced lung immunopathology and delayed viral clearance, while cd -depletion did not affect viral replication or clearance, thus highlighting an important role for cd + t cells in coronavirus infection. these conflicting results may be due to the inherent limitations of cov murine models. in - week-old mice, sars-cov- is associated only with mild pneumonitis and cytokines are not detectable in the lungs ( , , ) . a sars-cov- isolate (ma- ) replicates to a high titer and is associated with viremia and mortality, however the model lacks significant inflammatory cell infiltration into the lungs ( ) . thus, mouse models developed for the study of sars fell short in terms of reproducing the clinical and histopathological signs of disease ( , ( ) ( ) ( ) . it is therefore necessary to develop a usable animal model that is capable of reproducing the clinical and histopathological signs on covid- . israelow et al. recently described a sars-cov- murine model based on adeno associated virus (aav) -mediated expression of human (h)ace , which replicated the pathologic findings found in covid- patients ( ) . this model, which overcame the inability of murine (m)ace to support sars-cov- infection, was used to show the inability of type i ifn to control sars-cov- replication ( ) . in a similar attempt to overcome the lack of infectability through mace , dinnon et al. recently described a recombinant virus (sars-cov- ma) with a remodeled s protein mace interface, which replicated in upper and lower airways in young and aged mice with disease being more severe in aged mice. the authors used this model to screen therapeutics from vaccine challenge studies and assessed pegylated ifn-λ- as a promising therapeutic. the authors suggested that this model has greater ease of use, cost, and utility over transgenic hace models ( ) to evaluate vaccine and therapeutic efficacy in mice ( ) . a preliminary analysis of nk cell function and phenotype has been performed by zheng et al. using peripheral blood from covid- patients ( ). on admission, nk cell levels in the peripheral blood inversely correlated with disease severity. furthermore, covid- patients with severe disease had significantly lower numbers of circulating nk cells, as compared to mild disease (p < . ) ( ) . additionally, circulating nk cells in severe disease displayed increased expression of the inhibitory receptor nkg a and had an hyporesponsive phenotype with lower levels of ifn-γ, tumor necrosis factor (tnf)-α, il- , and granzyme b, although degranulation was maintained ( ) . finally, as compared to patients with active disease, patients recovering from covid- had higher numbers of nk cells and lower nkg a expression ( ) . liao et al. performed singlecell rnaseq on the cells obtained from bronchoalveolar lavage fluid of severe and mild covid- patients and found that covid- patients had significantly more nk cell infiltrates into the lungs, however patients with severe disease had reduced proportions of nk cells ( ) . in addition, klrc (nkg a) and klrd (cd ) were highly expressed by nk cells ( ) . carvelli et al. analyzed myeloid and lymphoid populations by immunophenotyping from blood and bronchoalveolar lavage fluid (balf) in healthy controls, paucisymptomatic covid- patients, pneumonia patients, and patients with ards due to sars-cov- and found that absolute numbers of peripheral blood lymphocytes, including nk cells, were significantly reduced in the pneumonia and ards groups compared to healthy controls. furthermore, the proportion of mature nk cells was reduced in patients with ards and nk cells showed increased nkg a, pd- , and cd ( ) . finally, wilk et al. performed single-cell rna-sequencing on covid- patients and healthy controls and found that the cd bright population was depleted in all covid- patients but the cd dim population was depleted only in patients with severe covid- . furthermore, nk cells had increased expression of the exhaustion markers lag and havcr ( ) . nk cell cytopenia seems to be a consistent characteristic among sars-cov- infected patients ( ). altogether, these data indicate alterations in the nk cell phenotype and functional profile that are consistent with the hypothesis that to establish a productive and lasting infection, sars-cov- needs to dampen the nk cell response. nk cell dysfunctions were also observed in patients from the previous cov outbreaks. wang et al. assessed nk cell number and phenotype using peripheral blood from sars patients admitted to hospitals in beijing, china ( ) . nk cell proportion and absolute number were significantly reduced in sars patients as compared to healthy donors and patients infected with the bacterium mycoplasma pneumoniae ( ). nk cell number correlated inversely with disease severity and patients with anti-sars cov-specific igg or igm antibodies had significantly fewer nk cells ( ) . the patients assessed had varied disease duration from to days (mean . days) and this allowed for patient stratification by disease duration. within the first days of sars-cov- infection, nk cell numbers remained high but this period was followed by the development of lymphopenia with levels recovering only around day ( ) . dong et al. also observed a reduction of nk cell numbers in sars patients, and these levels were lower in patients with severe, as compared to mild, sars ( ) . in addition, mers infection is strongly associated with leuko-and lymphopenia ( , ( ) ( ) ( ) . the mechanisms underlying the reduction of circulating nk cells in patients infected with covs are still unclear. as most studies have focused on peripheral blood nk cells, it is possible that the reduced number of circulating nk cells is due to redistribution of blood nk cells into the infected tissues ( ) . while it is hard to assess nk cell migration to infected tissues in covid- patients, this hypothesis was corroborated by mouse studies, where nk cells have been shown to migrate to the lungs in cov infected animals ( ) . an abundance of inhibitory factors, such as tgf-β, may be partially responsible for the nk cell hyporesponsiveness observed in covid- patients. in support of this hypothesis, huang et al. found significantly higher tgf-β levels in sars patients compared to healthy controls and this positively correlated with length of stay ( ) . given the importance of tgf-β in suppressing nk cell functions, it is possible that the higher levels of tgf-β (as well as other inhibitory cytokines) in cov patients leads to suppression of nk cell antiviral activity ( ) . early studies of covid- patients report secondary (super-) infections, including nosocomial pneumonia or bacteremia as a complication of sars-cov- infection ( ) . since nk cells are critical first responders that play a role in preventing and clearing infections ( ) , a poor nk cell count or exhausted phenotype, in addition to negatively influencing covid- patient outcomes, could facilitate the development of secondary infections and have a significant negative impact on patient outcomes. one of the main barriers in studying the role of nk cell activation in the early clearance of cov infection in asymptomatic or mildly symptomatic patients is the fact that these individuals are rarely diagnosed in the clinic and therefore an opportunity to collect samples for research does not exist. thus, while there is currently no direct evidence to support a role for nk cells in the clearance of sars-cov- , evidence showing that viral infection has a negative effect on the nk cell compartment is accumulating. given the importance of nk cell activity in early viral clearance and late immunopathology, having a rapid and reliable test to predict nk cell function, such as nkvue tm (atgen canada/nkmax), whereby whole blood is stimulated by an nk cell-specific activating cytokine mix and activity is measured via ifn-γ production, might allow researchers to predict who will mount an adequate response with asymptomatic or minimally symptomatic viral clearance and who will need icu admission, as has been shown with cancer patients ( ) . further research will be required into the innate immune response to cov infection to more fully understand nk cell contributions to viral clearance. in the context of covs, the significant morbidity and mortality associated with severe disease is due to acute lung injury (ali) and the development of ards ( , ) . pathological analysis of tissues obtained from sars and mers patients showed edematous lungs with areas of consolidation, bronchial epithelial denudation, loss of cilia, squamous metaplasia, pneumocyte hyperplasia, and bronchial submucosal gland necrosis ( , ) . histological features include diffuse alveolar damage and acute fibrinous and organizing pneumonia ( ) . a heightened inflammatory response in the lungs resulting in tissue damage has been hypothesized to explain the development of ali. there are several key factors that may be responsible for the induction of this dangerous inflammation ( ) . both sars-cov- and mers-cov replicate to high titers early in infection, which could lead to enhanced cytopathic effects and increased production of pro-inflammatory cytokines/chemokines by infected cells. chen et al. developed a pneumonia model where pulmonary replication of sars-cov- was associated with histopathological evidence of disease, including bronchiolitis, interstitial pneumonitis, diffuse alveolar damage, and fibrotic scarring ( ) . they identified a biphasic cellular immune response in which cytokines (tnf-α and il- ) and chemokines [interferon gamma-induced protein (ip)- , monocyte chemoattractant protein (mcp)- , macrophage inflammatory protein (mip)- a, rantes] were produced early, likely by infected airway epithelial cells, alveolar macrophages, and recruited inflammatory monocyte-macrophages and neutrophils, which have been shown to replace resident alveolar macrophages ( , ) . sars-cov- and mers-cov encode structural and non-structural proteins that antagonize the interferon response, which may initially delay the innate immune response but eventually potentiate inflammatory monocyte-macrophage responses ( ) . in covid- patients, liao et al. reported increased lung infiltration by macrophages identified via rnaseq analysis of bronchoalveolar lavage fluid. patients with mild cases exhibited infiltration by alveolar macrophages [fatty acid binding protein (fabp) + ] while patients with severe ards exhibited infiltration by highly inflammatory [ficolin (fcn ) + ] monocyte-derived macrophages ( ) . in the sars-cov- pneumonia model, the first wave of cytokines and chemokines induced an accumulation of nk cells, as well as plasmacytoid (p)dcs, macrophages, cd + t cells and nkt cells in the lungs. a second wave of inflammatory mediators was detected later on day post-infection [cytokines tnf-α, il- , ifn-γ, il- , il- , and chemokines mcp- , mip- a, rantes, monokine induced by gamma interferon (mig), ip- ] and correlated with lung infiltration of t cells and neutrophils ( ) . these findings are consistent with studies that have shown increased levels of activating and inhibitory cytokines and chemokines in the blood and lungs of sars patients, as well as histological studies of sars and mersinfected lungs which show extensive cell infiltrates ( , , ( ) ( ) ( ) . when huang et al. investigated the cytokine/chemokine profile in the acute phase of sars infection in a cohort of taiwanese patients, they observed an ifn-γ-led cytokine storm ( ) . they assessed sera from hospitalized patients prior to the administration of immunomodulators and found significantly increased levels of ifn-γ, il- , ip- , mcp- , mig, and il- ( ) , which returned to basal levels in convalescent sera. ip- , mig, mcp- , and il- levels were all significantly increased in death vs. survival groups. interestingly, they found an inverse relationship between ifn-γ levels and lymphocyte numbers and suggested this could either be due to ifn-γ-induced lymphocyte apoptosis or sequestration of chemokine-recruited lymphocytes in the lungs ( ) . indeed, this hyper-cytokinemia has been consistently observed in sars-infected patients ( ) . however, a recent study found that levels of six pro-inflammatory cytokines (il- b, il- ra, il- , il- , il- , and tnf-α) implicated in the cytokine storm in covid- patients did not differ significantly from levels in cytokine storms caused by other conditions. they suggest that it is therefore possible that increased levels of proinflammatory cytokines in the context of severe covid- may simply reflect an increased viral burden rather than an exuberant immune response and suggest that immunotherapies should therefore be used with caution ( ) . altogether these studies show that during acute cov infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce cytokines and chemokines that induce the activation and migration of lymphocytes, including nk cells, to the lungs, where they could be one of the main producers of ifn-γ ( ). under normal conditions, human lung nk cells are typically hyporesponsive but dynamically migrate in and out of pulmonary tissues ( ) . this supports the hypothesis that during infectious respiratory diseases, an increased recruitment of hyperresponsive nk cells would worsen the festering immunopathology ( ) . in fact, through viral-track scanning of unmapped single-cell rnasequencing data, bost et al. showed that patients with severe covid- exhibited a hyperinflammatory response with an enriched and highly proliferative nk cell compartment ( ) . high levels of ifn-γ leads to epithelial and endothelial cell apoptosis and vascular leakage, suboptimal t cell response, accumulation of alternatively activated macrophages and altered tissue homeostasis, and ards ( ) , all of which may contribute to covid- disease severity. in summary, the evidence is consistent with the hypothesis that nk cells are involved in the cytokine storm associated with cov infection and that this hyper-cytokinemia contributes significantly to disease severity via inflammation-mediated lung damage (figure ) . interestingly, this duality of nk cell roles mirrors what is seen in critically ill patients with sepsis. studies suggest that while early nk cell stimulation and ifn-γ production is beneficial to combat infections, excessive and prolonged stimulation of nk cells leads to reduced nk cell numbers and an exhausted phenotype and was associated with increased systemic inflammation in systemic inflammatory response syndrome (sirs)/sepsis and increased mortality ( ) ( ) ( ) ( ) . this review of the literature suggests that nk cells may play an important role in both cov clearance and immunopathology. the continued probing of nk cell involvement is essential for a more complete understanding of cov pathophysiology and for the deployment of immunotherapeutics. depending on the patient, the stage of disease, and other still poorly understood factors, it may be necessary to either boost nk cell activity to ensure viral clearance, e.g., at exposure or during early infection, or to finely tune nk cell effector functions in late stage infections to prevent hyper-cytokinemia and inflammatory lung damage. indeed, all covs that infect humans are zoonoses and there is an extensive reservoir of covs that could serve as a source for future pandemics ( , ) . therefore, a broader understanding of the immune response to coronaviruses and insights into therapeutic implications will be of significant value not only for the current covid- pandemic, but also for potential future pandemics. the race to vaccinate and find a cure for covid- has resulted in a spectacular effort from researchers and medical practitioners around the world. early attempts at creating targeted therapeutics have mostly relied on historical evidence from related, but not identical, coronaviruses and on the paucity of studies investigating sars-cov- . these strategies have attempted to combat the virus by targeting various stages of its life cycle starting with neutralizing sars-cov- virions using monoclonal antibodies or plasma from convalescent patients ( ) . the entry mechanism of covs has been shown to rely on binding the ace receptor and using proteases such as tmprss for s protein priming ( ) . thus, preventing ace receptor binding through blocking antibodies or competitive binding with soluble ace and tmprss protease inhibitors (camostat mesylate) are being tested ( ) . upon viral entry, the viral proteolysis or replication cycle can be targeted with protease inhibitors (lopinovir and ritonavir) ( ) or rna-dependent rna polymerase inhibitors (remdesivir and ribavirin) ( ) . at the time of writing this review, the results of these trials have not been released or are still preliminary and will require further evaluation to assess their clinical efficacy in larger cohort studies. as nk cell activity is critical for viral clearance and may be involved in disease immunopathology, a rapid and reliable predictor of nk cell function may allow for the prediction of clinical progression and the stratification of patients to receive therapeutic intervention. the remainder of this review will discuss the various ways immunotherapies are being deployed to tackle covid- , with a focus on therapies that use nk cells (table ) . lastly, while nk cells play an important role in combating viral infections, we also need to be fully cognizant of the potential damage immunotherapies could have in severe cases of covid- , and how these adverse effects may need to be attenuated ( table ) . in the absence of a clinically approved vaccine against sars-cov- , scientists have begun developing therapeutics to halt the spread of covid- by alternative strategies. studies have reported that patients infected with sars-cov- have lower levels of circulating nk cells and these express a greater level of inhibitory receptors (e.g., nkg a) while producing less ifn-γ ( , , ) . these findings provide a rationale for pursuing nk cell-based therapies as a tool to fight covid- . although nk cell-based therapies have mostly been developed for use against cancer, similar concepts and mechanisms could provide guidance in the fight against viruses. therapeutic nk cell products can be thought of as "living drugs" as they generally use either primary nk cells isolated from peripheral blood mononuclear cells (pbmcs) or are generated from stem cell precursors or genetically engineered immortalized human nk cell lines ( ) . primary nk cell products are often pre-treated and expanded in vitro with cytokines or via co-culture with target cells before being infused into patients. patients can also receive immune stimulants [e.g. recombinant il- ( ) or il- ( ) ] with the goal of improving the in vivo activity and persistence of the nk cell products ( ) as is being tested in this covid- trial (nct ). the first cell-based investigational drug to be approved by the fda for clinical testing in covid- patients is an allogeneic, off-the-shelf, cryopreserved nk cell therapy made by celularity (cynk- ), originally developed for cancer immunotherapy ( ) . the trial (nct ) is split into two phases. phase i will assess the frequency and severity of adverse events in mild, non-icu covid- patients (n = ) following infusion of nk cells derived from placental cd + cells. the subsequent phase ii trial will recruit up to patients and include a standard of care comparator at a : allocation. genetically modified nk cells are also being investigated for efficacy against covid- . chimeric antigen receptor nk cells (car-nk cells) are engineered to express virtually any receptor(s) of interest and were originally designed to enhance the ability of nk cells to eliminate cancer cells via receptors targeting egfr ( ) or cd ( ) , which are present on many cancer types and b cell hematological malignancies, respectively ( ) . although the efficacy of car-nk cells to control viral infections has yet to be rigorously tested in large scale clinical trials, the promising safety profile of car-nk cells in cancer patients, who are often immunocompromised, suggests that car-nk therapy can be well-tolerated in early phase/mild covid- patients. notably, car-nk cells are considered "safe" largely because they are less likely to lead to cytokine release syndrome (crs), a severe adverse event of car-t cell therapy ( ) . but as these are unchartered waters, it is critical that car-nk cells are used cautiously and not given to late/severe covid- patients. a phase i/ii study in early stage covid- patients (within days of illness) employing car-nk cell therapy is currently being tested using off-the-shelf nk cells derived from human umbilical cord blood expressing nkg d and ace cars (nct ). this complex five-arm study will compare the efficacy of different car-nk constructs: (i) nk cells, (ii) nk cells secreting il- , (iii) nkg d car-nk cells, (iv) ace car-nk cells, and (v) nkg d-ace car-nk cells. nkg d car-nk cells have shown promising preclinical results in cancer studies ( ) , and although not proven for sars-cov- , the rationale for expressing nkg d derives from work showing that nkg d-ligands (nkg dl) are upregulated on virally infected cells ( ) . similarly, the investigators hypothesize that expressing ace on nk cells will facilitate the elimination of sars-cov- virions and infected cells by binding the viral spike proteins-but it is unknown whether or not car-nk cells can eliminate virions or if infected cells display sufficient levels of spike protein to be recognized by ace -nk cells upon viral infection. the investigators also suggest that expressing ace on nk cells may also have a secondary benefit as a decoy cell that will be infected by the virus thereby indirectly protecting lung epithelial cells. as described previously, it is unclear whether this strategy will work to stop viral spread to healthy epithelial cells or if it will serve to perpetuate viral spread if the virus can replicate in nk cells. in arms ii-v of this trial, the car-nk cells have been engineered to secrete il- based on studies showing improved in vivo persistence of car-nk cells in cancer patients ( ) . however, the addition of the proinflammatory cytokine il- to this treatment strategy should be monitored closely for life-threatening toxicities, as elevated il- has been previously reported to accompany chronic pulmonary inflammatory diseases ( ) and mers-cov infection ( ) even if no correlation has been reported for sars-cov- . interestingly, a study compared il- levels from lung tissue homogenates following sars-cov infection in aged vs. juvenile monkeys and showed that il- concentrations were only elevated in juvenile monkeys days post-infection ( ) . this study would suggest that il- therapy may be tolerated and effective in older covid- patients that may not be able to produce il- , however this has not been confirmed. lastly, all the car-nk cells in this trial secrete gm-csf neutralizing scfv antibodies, since this cytokine has a known role in crs in cancer patients treated with car-t cells ( ) , and has been shown to be correlated with covid- disease severity in association with pathogenic cd + th cells ( ) . although nk cell based therapies are versatile, have shown safety and efficacy in cancer patients, and can be utilized in immunocompromised individuals, their potential has yet to be fully realized as an antiviral therapy. furthermore, the logistics of manufacturing nk cell products (cost and time) may pose limitations and barriers to access. for this reason, therapies focused on stimulating a patient's own nk cells offer many advantages over adoptive transfer of nk cells. the importance of the interferon pathway is underscored by the fact that many viruses actively interfere with host interferon responses, for which coronaviruses are a prime example. as described above, covs utilize numerous tactics to avoid elimination by disrupting the host type i ifn response ( ) . therefore, since the majority of covs fail to induce any detectable type i ifn response, eliciting a type i ifn response is a very attractive therapeutic strategy ( , ) . given the robust immunomodulatory nature of type i ifns, uninfected or early symptomatic patients would benefit the most from this therapy to prevent exacerbating immunopathology at later stages of disease. numerous clinical trials have been initiated investigating type i ifns ( table ) . a large study (nct ) of ∼ , medical staff allocated participants to two trial arms: (i) low-risk (non-isolated wards or laboratories) or (ii) highrisk (isolated wards in direct contact with covid- patients). in addition to the ifn-α- b nasal drops, high-risk medical staff will also receive the immune-modulating tlr activator, thymosin α , which indirectly activates nk cells through pdcs ( , ) . interestingly, reports in sars-cov- studies showed that ifn-β therapy had a -fold greater anti-viral activity in vero cells than ifn-α treatment ( ) . promising results have been published from a phase ii study (nct ) ( ) , showing that complementing lopinavir-ritonavir and ribavirin with subcutaneous ifn-β- b in mild-to-moderate covid- patients is safe with no serious adverse events reported in the triple combination therapy group, and highly effective, with significant and clinically meaningful reductions in time to complete alleviation of symptoms, hospital length of stay, and time to negative viral load ( ) . despite our best efforts in timing type i ifn therapy to mitigate immunopathology, these treatments still increase the risk of excessive activation of proinflammatory signals, which could damage host tissues and perpetuate immunopathology ( , ) . for this reason, alternative therapeutic avenues to direct type i ifn administration are being explored. type iii ifns can be a valid alternative to type i ifns, because they maintain antiviral functions yet are less toxic and less prone to mediate immunopathology ( ) . the type iii ifn, ifn-λ, activates nk cells indirectly (compared to type i ifns which directly act on nk cells), resulting in a less potent and slower immune response ( , ) . ifn-λ activates nk cells by stimulating macrophages to produce il- which in turn induce nk cells to produce ifn-γ ( ) . pegylated ifnλ is being tested in covid- positive patients with mild symptoms in the absence of respiratory distress (nct ). while ifn-λ can lead to the eventual activation of nk cells, its primary utility is in preventing the tissue damaging potential of neutrophils at mucosal surfaces, such as the lungs. however, ifn-λ also has been shown to reduce the rate of tissue repair, which in the context of covid- which has a long disease course, could mean greater risk of secondary infections. since exogenous administration of any ifn therapy poses the risk of tipping the balance toward severe covid- immunopathology, broggi et al. assessed the levels of ifns in upper and lower respiratory samples from healthy and covid- patients. in this preprint, they report that while the upper airway swabs showed similar mrna expression levels of type i and iii ifn compared to healthy controls, the balf samples of severe covid- patients had significantly elevated type i and iii ifn levels ( ) . therefore, as with all of the therapies discussed in this review, careful consideration about safe and effective timing should guide our design of clinical trials. in addition to ifn cytokine therapy, interleukin cytokine therapy can enhance the effector functions of nk cells ( ) . the use of whole, unmodified recombinant cytokines as a monotherapy has resulted in minimal success in humans in cancer immunotherapy. the earliest cytokine therapies to gain fda-approval were ifn-α and recombinant il- , approved for renal cell carcinoma and metastatic melanoma ( ) . although approved, they were limited by their in vivo half-life, marginal anti-tumor activity, and associated toxicities. the next generation of cytokine therapies were created to address these issues by first improving their biological stability through pegylation and fusion to chaperone molecules and secondly improving their specificity by fusing cytokines with antibodies or intratumoral administration. these advances in the field have allowed for the reassessment of the therapeutic potential of specific cytokines ( ) . given the importance of il- signaling and nk cell function, researchers have developed il- "superagonists" which are il- :il- r heterodimers that have better in vivo stability and bioactivity compared to monomeric il- ( ) . although at the time of writing il- superagonists are not being studied for their efficacy in covid- patients, il- superagonists, such as alt- , are safe in humans ( ) and have been used in conjunction with many of the therapies being discussed in this review including: car-nk cell therapy, adoptive nk cell transfers, checkpoint inhibitors, and the bcg vaccine in cancer ( ). it should be noted that although the therapeutic potential of cytokine therapy to specifically stimulate nk cells is enticing, exogenous cytokine therapy has a high risk for exacerbating crs if given at the incorrect time. some viruses are known to induce a state of functional hyporesponsiveness in t cells that is essential for the productive establishment of chronic viral infections ( ) . a vast body of literature has identified inhibitory checkpoint receptors, including ctla and pd- , as key regulators of this process ( ) . interestingly, cancer exploits similar mechanisms to escape the immune response, which provided the rationale for the introduction of antibodies targeting checkpoint receptors for cancer immunotherapy ( ) . ctla and pd- /pd-l blockade have revolutionized cancer immunotherapy, and their success provides a strong rationale for the use of these drugs in covid- patients, where emerging evidence suggests that the immune response is also subverted. a clinical trial (nct ) is currently assessing the efficacy of pd- blocking antibodies in severe covid- patients within h of reported respiratory distress. pd- has also been shown to play a role in regulating nk cell responses, in addition to modulating t cell functions ( ) ( ) ( ) ( ) ( ) , and has been reportedly increased in covid- patients ( ) . inhibitory receptors on the surface of nk cells regulate nk cell activation and can be targeted by antibody therapy. one of the most promising is certainly the inhibitory receptor nkg a, which binds to hla-e ( , , ) . nkg a expression is increased in circulating ( ) and balf nk cells from covid- patients, in contrast to nkg c, an activating receptor closely related to nkg a, which remains unchanged ( ) . however, it is unclear whether the observed increase in nkg a + nk cells is due selective proliferation of nkg a + cells or if it is the result of nkg a negative cells migrating out of circulation to infected tissues. circulating nk cells from patients with active hepatitis b disease had higher levels of nkg a compared to patients without active disease, however antiviral administration was associated with a reduction in nkg a expression. additionally, blocking nkg a in vitro with nkg a monoclonal antibodies led to improved nk cytotoxicity ( ) . given the association between nkg a expression in patients with severe covid- ( , ) , a promising avenue of investigation would be anti-nkg a therapy, even in light of results showing that nkg a + nk cells are tuned to present a higher level of responsiveness to stimulation ( ) . while nk cells can be stimulated directly by cytokines such as interferons and interleukins, their activity can also be enhanced through a by-stander effect following stimulation of other innate immune cells, such as macrophages and pdcs ( table ). this type of coordinated innate immune response may be more effective at cov viral clearance and mitigation of severe covid- . trained immunity has been recently described as an epigenetic re-wiring occurring in myeloid cells and progenitors upon stimulation that primes for a stronger response to subsequent stimuli, even of a different nature ( , , ) . whereas, the consensus is that myeloid cells are primarily responsible for trained immunity ( ) , it is likely that the resulting alteration in the cytokine milieu also has an effect on nk cells ( , ) . this is the case for the bcg vaccine, which has been shown to provide non-specific protection against yellow fever viral infection ( , , ) . the bcg vaccine is composed of a live attenuated strain of mycobacterium bovis originally given to young children to protect against tuberculosis (m. tuberculosis) ( ) . this vaccine provides an initial boost to innate immunity, but more importantly, results in the secretion of il- β from monocytes/macrophages, which feeds back to further stimulate the innate response ( ) . the use of a heterologous vaccine to provide enhanced protection against non-specific/new pathogens makes this a compelling strategy against covid- that warrants thorough investigation in randomized controlled trials ( , ) . the bcg vaccine is undergoing clinical trials in healthcare workers in the netherlands (nct ), australia (nct ), egypt (nct ), and the usa (nct ) to enhance overall innate immunity and provide heterologous protection against sars-cov- . interestingly, an association was found that linked lower covid- -attributable mortality rates in countries using bcg in their national immunization schedules ( ) . on the contrary, a study that assessed the association of childhood bcg vaccination in adults living in israel did not show a beneficial difference in covid- infection rates. the discrepancy between these two reports likely stem from the fact that the latter study only included adults who were previously vaccinated during childhood, supporting the fact that heterologous vaccination may not result in long-term protection ( ) . childhood bcg immunization has a limited window of opportunity to protect younger individuals from infection ( ) , but it is hypothesized that reducing the number of infected children can have a meaningful impact on curbing the spread of covid- to the rest of the population ( , ) . another heterologous vaccine in the process of clinical trial development for covid- studies is imm- (cctg id# ic ). created by immodulon therapeutics ltd, imm- is composed of heatkilled mycobacterium obuense and may have an improved safety profile over the bcg vaccine ( ) . imm- has been studied in multiple clinical trials for its non-specific immune stimulating properties as a cancer immunotherapy in pancreatic ( ) and melanoma patients ( , ) . agonists of toll-like receptors (tlrs) have been shown to broadly activate different immune populations and have had both preclinical and clinical success as adjuvants in vaccination and in the treatment of a variety of viral pathogens ( ) . for example, cpg oligodeoxynucleotides (cpg odns) are short dna sequences that contain unmethylated cpg dinucleotides which activate tlr particularly on dcs and b cells ( ). bao et al. showed that their cpg odn construct, bw , had protective effects against sars-cov- in a mechanism that relied on nk cell activation likely through a dc intermediate ( ) . amidst the ongoing sars-cov- pandemic, two clinical trials (nct , nct ) have opened using the tlr / / agonist, pul- , in order to prevent infection. ascorbic acid, more commonly known as vitamin c, has been shown to exhibit potent immunomodulatory, antioxidant, and antimicrobial effects ( ) . vitamin c has been shown to restore nk cell cytotoxicity in individuals exposed to toxic chemicals through protein kinase c expression, a critical component in lymphocyte metabolism ( ) . additional reports have shown that vitamin c also enhances the expression of nkp , cd , cd and ifn-γ production by nk cells ( ) and can increase the expression of irf in lung tissues of influenza infected, pneumonia-induced mice ( ) . vitamin c also harbors potent antioxidant attributes which can scavenge reactive oxygen species (ros) and prevent lung injury ( , ) . although ros production is an important component in the host defense response to viruses, they can be harmful to cells and lead to the pathogenesis of viral-induced host injury ( ) . the underlying rationale to investigate the therapeutic potential of vitamin c has been based on two key observations: (i) critically ill patients have lower levels of vitamin c ( ) ( ) ( ) and (ii) vitamin c has pleiotropic immunomodulatory, antioxidant, and antiviral effects ( ) . it is important to underscore that reports on the clinical outcomes of vitamin c treatment in humans are mixed and context dependent. a thorough metaanalysis on vitamin c supplementation for the common cold has been reported by hemilä and chalker ( ) . briefly, they concluded that while the incidence of colds was not reduced, the duration and severity of colds was reduced when assessing studies of regular vitamin c intake ( ). interestingly, a separate metaanalysis on vitamin c and cardiac surgery showed a reduction in the length of icu stay and shortened the need for mechanical ventilation ( ) . this is an important correlation as clinical trials are currently investigating the efficacy of vitamin c to reduce mortality and hospital burden in covid- patients ( table ) . a phase ii clinical trial (nct ) was initiated in wuhan where covid- patients will be given a high dose intravenous infusion of vitamin c. lastly, whether oral dosing of vitamin c can achieve therapeutically relevant concentrations, as described in the above studies, is currently unknown, thus caution should be taken as exceeding the recommended dietary allowance of - mg/day may lead to mild toxicities including abdominal discomfort and diarrhea ( , ). the main cause of death for covid- patients has been pulmonary complications and respiratory failure often as a result of an unregulated cytokine storm ( ) . it is unclear whether the hyperinflammation seen in severe cases of covid- is the result of the viral replication within pulmonary epithelial cells or an overactive/avalanching immune response. however, studies in sars-cov- reported hyperinflammation in later stages of disease progression, despite reduced viral titers, suggesting that the damage was immune-mediated ( ) . the most appropriate course of therapy can only be determined by elucidating the pathophysiology of disease progression. scientists and physicians, however, have had to respond quickly to the growing number of severe covid- cases and this has resulted in therapy mainly through a combination of anti-inflammatory and anti-viral interventions ( table ) . as described above, there is a potential for nk cells to contribute to the cytokine storm and therefore the development of ali. a possible explanation for the observed lymphopenia in covid- patients is that nk cells and other lymphocytes migrate out of the circulation and into pulmonary tissues to aid in the elimination of infected epithelial cells ( ) . this could be the premise for the large, unintended, amount of tissue damage that worsen the respiratory distress ( ). for this reason, therapeutics that dampen the immune response have been effective in mitigating immunopathology in severe covid- patients. the following review papers have thoroughly discussed many of these immunotherapies already ( ) ( ) ( ) ( ) ( ) ( ) , therefore, this section will focus on immunotherapies and their potential implications on nk cells. the main cytokines responsible for the life threatening respiratory distress seen in reported cases of severe covid- are il- , il- , il- , il- , g-csf, ip- , mcp- , mip a, and tnf-α ( ) . many clinical trials have focused on targeting il- signaling with anti-il- r monoclonal antibodies (e.g., tocilizumab, sarilumab, siltuximab) because of the important role il- has in propagating crs ( ) . tocilizumab, in particular, is being used as the primary therapy in the majority of these trials, likely owing to its fda approved status as a therapeutic for crs in car-t cell therapy ( ) . a case report demonstrated the potential for tocilizumab therapy in treating severe covid- illness, where a single dose on day of symptoms led to progressive reduction in il- levels and resolution of symptoms ( ) . a phase iii study (nct ) led by hoffman-la roche is recruiting patients to study the safety and efficacy of tocilizumab therapy in a randomized, double-blind, placebocontrolled, multicenter study in over patients with severe covid- pneumonia ( table ) . targeting the il- axis in severe covid- patients may also serve to improve nk cell functions as cifaldi et al. showed that increased il- negatively impacts nk cell function ( ) . they also showed that tocilizumab treatment improved nk cell function in vitro ( ) . mazzoni et al. recently reported that serum il- levels were inversely correlated (p = . ) with nk cell function in covid- icu patients. additionally, in a small subset of covid- icu patients (n = ), nk cells displayed improved markers of activation (granzyme a and perforin) after tocilizumab treatment ( ) . similar therapies have emerged in the fight against covid- including an il- r antagonist (anakinra; nct ) ( ) and cytosorb (nct ) ( ) . high dose anakinra therapy has shown promising safety and efficacy in a small retrospective study, as part of the covid- biobank study (nct ) ( ) . cytosorb therapy is used in conjunction with conventional dialysis through a whole blood cartridgebased filtration system designed to remove middle molecular weight molecules (which include inflammatory cytokines < kda) through extracorporeal cytokine adsorption ( ) . it is reported to be effective at removing ferritin and il- in a case study of a -year-old with severe crs following car-t cell therapy ( ) . jak / inhibitors (jaki) are also undergoing clinical trials in moderate-severe covid- patients, such as baricitinib (nct ). in addition to their ability to impede the production of il- , thus curb the excessive inflammation, they may also block clathrin mediated endocytosis-indicating a dual role for jaki ( ) . however, jaki can also lead to the transient increase in nk cells as shown in baricitinib treated rheumatoid arthritis patients ( ) , which could be detrimental for severe covid- patients. corticosteroids have played a key role in the treatment of auto-immune diseases over the past years ( , ) . whether endogenous or exogenous, corticosteroids decrease the number of circulating monocytes and lymphocytes and decrease synthesis of pro-inflammatory cytokines (il- , il- , tnf-α) ( ) . their strong anti-inflammatory and immunosuppressive effects make them good candidates for rapidly suppressing inflammation during early auto-immune disease or viral infections. corticosteroids have been shown to inhibit nk cells in ex vivo experiments ( , ) . while corticosteroids may delay clearance of infections, their major benefit lies in suppressing excessive innate immune responses, thus preventing lung damage and ards commonly present in severe viral infections ( ) ( ) ( ) . in fact, this was the main rationale for the widespread use of corticosteroids during mers and sars infections ( , ) . specific to covid- , some groups have advocated for the use of low-dose corticosteroids in a specific subset of critically-ill patients with refractory ards, sepsis, or septic shock ( table ) ( ) . there is one known ongoing randomized clinical trial examining the effect of the corticosteroid ciclesonide in adults with mild covid- infections (nct ). this trial is based on preclinical studies showing in vitro antiviral activity of ciclesonide against sars-cov- . while there may be a benefit to using corticosteroids in a subset of critically-ill patients with refractory ards or sepsis ( ) , their routine use in covid- is not recommended outside of clinical trials, based on expert opinion and who recommendations ( ) ( ) ( ) . corticosteroids also cause a multitude of side effects, most notably diabetes mellitus, osteoporosis, and increased risk of infections ( ) . controversially, a systematic review of over , influenza patients showed that corticosteroids actually led to increased mortality, length of icu stay, and secondary infections ( ) . additionally, one retrospective observational study examined the use of corticosteroids in covid- patients, and reported no significant association between corticosteroids and viral clearance time, hospital length of stay, or duration of symptoms ( ) . these studies highlight the need to be vigilant in our attempts to fight covid- . healthy, uninfected individuals, who are at a high risk of becoming infected (through situational circumstances such as healthcare workers) would be most fit and suitable to receive investigational prophylactic therapies such as exogenous ifns and heterologous vaccines. (b) individuals who have tested positive for covid- that are asymptomatic or have mild to moderate disease progression may benefit from receiving investigational immune stimulating therapies, including nk cell-based therapies. it is critical that investigators must be vigilant to assess the safety profile and potential immunopathologies associated with these immunotherapies. (c) in severe covid- patients, the most appropriate therapies to investigate would be those that mitigate immunopathologies, such as anti-inflammatory and immunosuppressive therapies. given the relatively low chance of toxicity and the wide range of beneficial immune effects, natural health products such as vitamin c and vitamin d can be suitable for investigation at all categories of covid- patients. non-steroidal anti-inflammatory drugs, or nsaids, are one of the most commonly prescribed drugs for treating fever, pain, and inflammation. nsaids include over-the-counter household names such as ibuprofen, naproxen, and aspirin. given the widespread use of these medications it is appropriate that researchers have investigated the potential benefits and harms of nsaids in patients diagnosed with covid- . thus far, the evidence for using nsaids in the context of covs are mixed and might not be generalizable to all nsaids as reports tended to focus on specific nsaids. these studies also focused on the potential for nsaids to act as an antiviral, with a potential added benefit of being able to treat inflammatory symptoms. one report showed that the nsaid indomethacin could directly inhibit sars-cov replication in vero cell monolayers in a dosedependent manner ( ) . the antiviral properties of naproxen have been described in the context of influenza virus ( , ) and has prompted the initiation of a clinical trial investigating the efficacy of naproxen as a treatment for critically ill covid- infected patients (nct ). nsaid therapy should be used with caution as they have been shown to interfere with immune responses and ability to produce antibodies, with ibuprofen having the greatest suppressive effect ( ) . furthermore, ibuprofen has been reported to increase the expression of the ace receptor ( ) which could facilitate sars-cov- viral entry. this finding should be considered for any current (nct ) and potential covid- clinical trial assessing ibuprofen therapy. nsaids also have been shown to have a direct suppressive effect on nk cell ifn-γ and tnf-α production ( ) which may be beneficial for late stage covid- patients. the relevance of nk cells as antiviral first responders is highlighted in patients with nkd and immunocompromised individuals who show increased susceptibility to viral infections. while there is currently little direct evidence to support a role for nk cells in the clearance of sars-cov- there is a paucity of research in this field. however, studies in admitted covid- patients with mild and severe disease reported a reduction in circulating nk cell levels and function as compared to healthy individuals. furthermore, reduced nk cell levels and function were inversely correlated with disease severity, suggesting that nk cells may be involved in some capacity. one of the potential mechanisms by which nk cells may become hyporesponsive is via sars-cov- interference with type i ifn pathways. in investigating the pathogenesis of other cov infections, namely sars and mers, studies suggest that during acute cov infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce chemokines and cytokines that induce nk cell migration and activation. as nk cells are one of the main producers of ifn-γ, they may be involved in the ifn-γ-led cytokine storm that is responsible for the induction of inflammation-mediated ali, ards, and subsequent mortality associated with covid- . inarguably, more research into the role of nk cells in covid- is required. despite the knowledge gaps in covid- pathophysiology, there has been a surge of clinical trials as the fda continues to fast-track the approval of investigational therapeutics ( ). here we have outlined potential therapeutics with a focus on mediating nk cell activity, including prophylactic treatments that could boost innate immunity in addition to therapeutics that could mitigate the immunopathological consequences of covid- , thereby relieving the 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administration the authors would like to thank dr. doug gray for his thorough editing, proofreading, and thoughtful suggestions. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © market, angka, martel, bastin, olanubi, tennakoon, boucher, ng, ardolino and auer. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -b yikvj authors: giotis, efstathios s.; robey, rebecca c.; skinner, natalie g.; tomlinson, christopher d.; goodbourn, stephen; skinner, michael a. title: chicken interferome: avian interferon-stimulated genes identified by microarray and rna-seq of primary chick embryo fibroblasts treated with a chicken type i interferon (ifn-α) date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: b yikvj viruses that infect birds pose major threats—to the global supply of chicken, the major, universally-acceptable meat, and as zoonotic agents (e.g. avian influenza viruses h n and h n ). controlling these viruses in birds as well as understanding their emergence into, and transmission amongst, humans will require considerable ingenuity and understanding of how different species defend themselves. the type i interferon-coordinated response constitutes the major antiviral innate defence. although interferon was discovered in chicken cells, details of the response, particularly the identity of hundreds of stimulated genes, are far better described in mammals. viruses induce interferon-stimulated genes but they also regulate the expression of many hundreds of cellular metabolic and structural genes to facilitate their replication. this study focusses on the potentially anti-viral genes by identifying those induced just by interferon in primary chick embryo fibroblasts. three transcriptomic technologies were exploited: rna-seq, a classical ′-biased chicken microarray and a high density, “sense target”, whole transcriptome chicken microarray, with each recognising – regulated genes (curated for duplication and incorrect assignment of some microarray probesets). overall, the results are considered robust because of the compiled, curated list of regulated genes were detected by two, or more, of the technologies. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. the interferon (ifn) response is one of the most important arms of host innate immunity against virus infection [ , ] . infected cells are able to recognise foreign nucleic acids and induce the synthesis and secretion of type i ifn (ifn-α and ifn-β) and type iii ifn (ifn-λ), which bind to receptors on the surface of neighbouring cells and trigger the transcriptional regulation of genes involved in the antiviral state. studies in mammals have demonstrated that there are several hundred such ifn-regulated genes (irgs). because the vast majority are up-regulated they are overwhelmingly referred to as ifn-stimulated genes (isgs) so, hereafter, they will be referred to generically as isgs (or specifically as chicken isgs, chisgs), except where the more generic term avoids confusion. induction of isgs involves the jak/stat signalling pathway: stat is either recruited directly to target promoters for a relatively weak activation or, more commonly, is recruited in a complex called isgf in association with stat and irf [ , ] . isgs are the focus of considerable current attention with regard to: (i) their antiviral activity, (ii) an increasing appreciation of the complexity of their regulation and (iii) their targeting by virus-encoded modulators of ifn-induced responses [ , , ] . these studies require comprehensive catalogues of the isgs, especially where system-wide approaches are undertaken. even though many key mammalian isgs have been known for some time, it is with the relatively recent advent of transcriptomic technologies that the full complement has been catalogued (mainly using microarrays [ ] ; see also schoggins et al. [ ] ). in contrast to the mammalian ifn system our equivalent knowledge of the avian system has lagged behind. although ifn was discovered in chickens in [ ] the first chicken ifn gene was characterised in [ ] and the key chicken isg, pkr, was identified in [ ] . the derivation of the chicken genome sequence, first drafted in [ ] , did not greatly advance our understanding of chicken isgs because of the incomplete nature of the gallus gallus genome assembly, even at v (galgal ), which might be partly due to the fact that the chicken karyotype has six pairs of macrochromosomes (but pairs of microchromosomes), and the difficulties in annotating immunity genes, which are some of the most divergent between mammals and birds [ ] . however, it has become apparent that key genes of the innate immune system, such as the transcription factors irf and one member of the irf /irf dyad [ , ; unpublished] , are absent from avian species, indicative of significant functional differences between them and mammals. moreover, for reasons that are not understood, the cytosolic pattern recognition receptor, rig-i, appears to have been lost from chicken as well as other galliformes [ , ] . to generate a chicken isg database we have compared data from three transcriptomic technology platforms: (i) the classical ′-biased genechip chicken genome array ( k; affymetrix, high wycombe, uk), (ii) the chicken gene . sense target (st) whole transcriptome array (affymetrix) and (iii) illumina (little chesterford, uk) rna-seq. this three-way comparison allowed a high level of cross-validation of data from each technology, beyond what would normally be achieved by qrt-pcr. it also allows subsequent studies, constrained to use any particular technology, to be more broadly compared. we monitored irg expression in chicken embryo fibroblast (cef) induced for h with units recombinant chicken ifn-α (rchifn ; hereafter routinely referred to as ifn), a time chosen to reflect predominantly primary signalling targets. the expression data for selected genes were also validated by pcr and qrt-pcr. overlapping data show generally high degrees of concordance in the identity of the irgs and their relative levels of regulation by ifn, with disparity mainly where multiple microarray probes exist for single genes. the study was presented in a preliminary form as a poster at the international cytokine and interferon society (icis) meeting ("cytokines "; october - , ) in bamberg, germany [ ] . freshly isolated cef were provided by the former institute for animal health (compton, uk, now the pirbright institute, pirbright, uk). cells were seeded in t flasks (greiner bio one, kremsmünster, austria; . × cells/flask) and cultured overnight in . ml media (gibco thermo fisher scientific, paisley, uk) supplemented with % heat-inactivated newborn bovine serum (nbcs; gibco), % tryptose phosphate broth (tpb; sigma-aldrich, gillingham, uk), % nystatin (sigma-aldrich) and . % penicillin streptomycin (gibco). recombinant chicken ifn-α (rchifn ) was prepared as previously reported [ ] and was added in culture media to a final concentration of units/ml. confluent cells were treated with ifn or mock-treated and incubated for six hours before harvesting. cells were stored at − °c in rnalater (sigma-aldrich) until rna extraction. the experiment was repeated in triplicate with three different batches of cef. total rna was extracted from cells using an rneasy kit (qiagen, crawley, uk) according to the manufacturer's instructions. on-column dna digestion was performed using rnase-free dnase (qiagen) to remove contaminating genomic dna. rna samples were quantified using a nanodrop spectrophotometer (thermo fisher scientific, paisley, uk) and checked for quality using a bioanalyzer (agilent technologies, wokingham, uk). all rna samples had an rna integrity number (rin) ≥ . . rna samples were processed for microarray with the genechip ® chicken genome array (affymetrix) using the genechip ® ′ ivt express kit (affymetrix) or for microarray with the chicken gene . st array (affymetrix) using the ambion (paisley, uk) wt expression kit for affymetrix genechip ® whole transcript (wt) expression arrays (ambion) and the genechip wt terminal labelling and controls kit (ambion), following the manufacturers' instructions, as described previously [ ] . total rna ( ng) was used as input and quality checks were performed using the bioanalyzer at all stages suggested by the manufacturer. rna samples were processed in two batches of but batch mixing was used at every stage to avoid creating experimental bias. hybridisation of rna to chips and scanning of arrays was performed by the medical research council's clinical sciences centre (csc) genomics laboratory (hammersmith hospital, london, uk). rna was hybridised to genechip chicken genome array chips (affymetrix) in a genechip hybridization oven (affymetrix), the chips were stained and washed on a genechip fluidics station (affymetrix), and the arrays were scanned in a gene-chip scanner g with autoloader (affymetrix). cdna was synthesised from rna samples from untreated and ifn-treated cef using the quantitect ® reverse transcriptase system (qiagen) according to the manufacturer's instructions. the glyceraldehyde -phosphate dehydrogenase (gapdh) was used as a reference gene. all target gene expression levels were calculated relative to gapdh expression levels and the target gene expression level in − h uninfected cef using the comparative c t method (also referred to as the −ΔΔct method). triplicate untreated (control) and ifn-treated cef were processed for transcriptome analysis by rna-seq. the cell samples used were identical to those used for the microarray analyses. total rna was extracted as for microarrays (above) and rna libraries were prepared for deep sequencing using the truseq rna sample preparation kit (illumina) according to the manufacturer's instructions. total rna ( . μg) was used as an input for each library. a total of six rna adapter indices were randomly assigned to the samples to allow multiplexing of libraries. at the end of the protocol, libraries were quantified using a nanodrop spectrophotometer and checked for quality using a bioanalyzer high sensitivity dna chip (agilent technologies). rna library qpcr quantification, multiplexing and sequencing was performed by the medical research council's clinical sciences centre (csc) genomics laboratory, hammersmith hospital, london, uk. libraries were quantified using the kapa biosystems (london, uk) library quantification kit (kk ) on an abi fast qpcr machine (applied biosystems). libraries were then diluted to a nm stock solution, pooled for multiplexing, denatured and diluted to a final molarity of pm. libraries were loaded on to the flow cell ( - pm per lane) for clustering and cluster generation was performed by the illumina cbot using version kits. sequencing of the flow cell was then carried out on the illumina hiseq using the version kits. data were processed using microarray data were processed using workflows in genespring ™ (agilent) and partek ™ (partek inc., st louis, mo, usa) commercial software suites. data (.cel files) were analysed and statistically filtered using either partek genomic suite . (partek gs) or genespring version . (agilent technologies) software. input files were normalized with either gcrma or genespring algorithms for gene array on core metaprobesets. a one-way anova was performed using either software across all samples. statistically significant genes were identified using mixed model analysis of variance with a false discovery rate (benjamini-hochberg test) of p < . . fold-change values <± . were removed. rna-seq data were imported into clc bio's genomics workbench (clc bio, aarhus, denmark; now qiagen), quality-controlled and thereafter processed using that package (versions and ). after quality control, the reads were subjected to quality trimming then mapped against ensembl galgal annotated genes (release [ ] ) for quantitative analysis of expression. fold change and false discovery rates (fdr) were calculated using kal's z test [ ] , with pooled data, or baggerly's test [ ] , using separate triplicates. initially, we used the k genechip ® chicken genome array (affymetrix) because, as well as displaying probes for chicken transcripts, it displays probes for transcripts from different viral pathogens of chickens, which offers advantages to those studying virus infections in a chicken background. subsequently, we used the more refined chicken gene . st array (affymetrix) because it offers a higher probe density against chicken genes and should allow detection of transcript isoforms, including non-polyadenylated and alternatively polyadenylated, though it does not include probes for viral genes. separate weekly batches of cef, produced from pools of eggs from the same flock (rhode island red) held in spf-like conditions at the former compton laboratory of the institute for animal health (now the pirbright laboratory) served as biological replicates. principal component analysis of the microarray data (data not shown) indicated limited variation between batches so, thereafter, biological triplicates were used routinely. irgs were identified from expression analysis data determined using the k genechip following ifn treatment ( units, h) of cef. after quantile normalization, significant hits were identified with genespring using an unpaired t test with asymptotic p-value computation and benjamini-hochberg multiple testing correction to generate false discovery rates (fdr). a matrix of fdr (from < . to ) plotted against fold change (fc; from . to > ) is shown in table . a relatively conservative fdr of < . returned differentially expressed probesets. overlaying this with a value for fc for which changes in expression might reasonably be expected to be readily and reliably assayed using other technologies, namely > , reduced the number of selected, significant probesets to a manageable ( up, down). these settings were therefore chosen for further analysis. for of these probe sets, no currently recognised genes were automatically assigned. of the remaining probe sets, were assigned to genes recognised in duplicate by other probe sets. consequently recognised genes were identified as differentially expressed (the down-regulated transcript was not, at that time, assigned to a recognised gene). with the chicken gene . st array, probe sets demonstrated differential expression ( up, down) at the same settings (fc > , fdr < . ). amongst these, there were five duplicated probe sets and that were not automatically assigned to recognised genes therefore recognised genes were uniquely identified as differentially regulated. illumina rna-seq yielded a total of million reads ( bases; paired) for the mock-treated cef triplicate samples and million for the ifn-treated samples. upon quality trimming and mapping to ensembl galgal annotated genes (release ), using clc bio's genomic workbench, recognised genes were identified as differentially regulated ( up, down) using kal's proportion-based z test [ ; as implemented in the clc bio package] at the same settings (fc > , fdr < . ). kal's is performed on the pooled reads from ifn-treated and untreated samples. it is perhaps, therefore, more widely applicable; it also returned a number of irgs comparable to those returned by the microarrays. triplicate-based analysis using baggerly's proportionbased beta-binomial test [ ; as implemented in the clc bio package] at the same settings (fc > , fdr < . ) returned an additional up-regulated genes. comparison of the complete raw gene lists from the three technologies using the most compatible identifier (essentially the gene symbol) with an online venn diagram tool (venn diagram generator; [ ] ) demonstrated that recognised genes were identified as differentially regulated. of these, were identified in common by all three technologies and a further were identified by two out of three technologies, meaning that were identified by at least two technologies. a total of were therefore each identified only by individual technologies ( figure a) . as well as comparing the identities of the differentially regulated genes, the correlation of expression of the genes identified by the different platforms was examined in terms of both level and rank of fc (figures a and b) . for instance, comparing rna-seq data with the k genechip data, spearman correlation values were . for fc level and rank. considering the current state of assembly and annotation of the chicken genome, the correlation of isgs in terms of gene identity as well as the level and rank of induction as indicated by all three technology platforms is reassuring. nevertheless the platform transcriptomic data were validated for selected genes by rt-pcr (data not shown) and by qrt-pcr ( figure a) . a h time point was chosen for microarray and rnaseq analysis of ifn treatment as it has been widely used and is known to result in significant levels of a broad range of isgs in mammals, making it suitable for defining the chicken interferome. use of this single time point does not, however, provide unequivocal insight into mechanistic interpretation of isg induction; for instance, it does not discriminate between strictly isre-dependent induction of isgs and isre-independent induction of isgs by mechanisms that might include immediate high-level induction of irf , which has been observed in mammalian systems [ ] [ ] [ ] . kinetic analysis of the induction of expression of a subset of isgs was therefore conducted at , , and min post application of ifn (see figure b ). even among highly-induced isgs, different temporal profiles were observed, from the rapid accumulation of ifit ( -fold by min) and rsad (which remain at steady levels to min) to the steadier, sustained accumulation of mx and the more modestly induced stat ; with lgp and trim peaking at min. although differences in mrna stability and turnover will influence the profiles, this identification of the isgs will allow detailed analysis of their promoters to investigate elements (and the factors that bind them) that contribute to the complexity of the observed induction patterns. of the irgs initially identified by all three technologies, ). this suggests either that the mammalian equivalents are isgs but that they are not included as such in interferome or that they are not isgs in mammals. the raw lists were refined by manual "curation", allowing for synonyms of recognised genes (for instance isg - versus isg ( )) and, after bioinformatic analysis using blast, etc., assigning recognised gene identifiers to probe sets that previously lacked them. at the end of this process ( figure b ; additional files , ), it was apparent that some (n = ) differentially regulated genes identified by the microarrays were also identified as differentially regulated by rna-seq but that they fell outside of the strict fc > and fdr < . parameters, reflecting unsurprising disparity in the sensitivity of the three technologies. those genes that were expressed down to fc > . or with an fdr up to < . were, therefore, also incorporated to produce a final list ( figure c ; additional files , ). it is obvious that this manual curation of the data, to allow for alternative gene id nomenclature used by the three technologies and for differences in sensitivity, introduced minor changes to the figures from the automatic comparisons cited above (figure ; additional files , ). curation, therefore, reduced the number of irgs from to . it also increased the number of differentially expressed genes detected by two out of three technologies from to (compare figures a and b) . relaxing the criteria for detection of differentially regulated genes by rna-seq (to fc > . and/or fdr < . ) further increased the number of genes detected by all three technologies from to (representing %) or by at least two of the technologies from to ( %), leaving genes detected by single technologies (compare figures b and c) , with of those detected by rna-seq alone (using the kal's test, at fc > . and fdr < . ; additional files , ). of the additional isgs identified by rna-seq as significant (fc > fdr < . ) by the more sensitive baggerly's test but not by kal's (table ) , two were also identified as significant by kal's using the relaxed criteria (fdr < . ). baggerly's, therefore, identified isgs additional to those described in the above analyses using rna-seq (kal's analysis) and the microarrays (table ) analysis of rna-seq data depends directly on the extant annotated genome sequence. perhaps not surprisingly therefore, rna-seq identified the largest proportion of genes amongst the set of unique irgs that we compiled ( ; %). nevertheless, the microarrays each identified % of the genes ( and ) . congruence was highest, and almost identical, between rna-seq and each microarray ( and ; ± %; all percentages referring to the total of unique irgs). between microarrays it fell to % ( ). for two-way-only comparisons, the distribution of unique genes between the microarrays was symmetrical ( and ; %). between rna-seq and each microarray, unique genes were biased > -fold towards rna-seq: ( %) versus ( %) against the genechip and ( %) versus ( %) against the st array. clearly in simple terms of numbers of irgs identified, rna-seq outperforms the microarrays. this is probably attributable to the historic nature of the array design based on earlier genome assemblies and annotations, with consequent effects on overall coverage (which might disproportionately affect conditionally expressed genes such as those of the innate immune responses). nevertheless, the ability of microarrays to quantify expression of % (about ) of such a large pool of important genes will often prove sufficient for the experimental objectives where other considerations might affect the choice of technology (see below). moving away from actual numbers of genes, it is worth noting that deeper analysis (in the form of validation by alternative approaches) will, by definition, be required to determine which of the genes identified uniquely as irgs by individual technologies are actually irgs. genomic loci for each of the predicted isgs were visually inspected using genomic workbench's genome browser, displaying tracks showing: gene, transcript, exon and orf annotations for the current chicken genome build as well as read-mapping for control and ifn-treated reads [ ] . on occasions, such inspection revealed the presence of non-annotated, inducibly-transcribed regions, representing exons, whole genes or even gene families. examples include those previously described at the chicken ifitm locus [ ; data not shown], at the herc locus (described below) or downstream of ccl (loc ; "c-c motif chemokine -like"; figure ). systematic analysis of these isgs is outside the scope of this manuscript but the data deposited from this study (european nucleotide archive (ena) study number prjeb [ ] ) will facilitate ongoing study and improved annotation. in some cases, although not currently annotated on the ensembl chicken genome, the genes have ids in ncbi and were identified as isgs by one of the microarrays. examples of these include loc , loc ("guanylate-binding protein -like") and loc ("hect domain and rld -like", a member of the herc family, discussed below). about % of the reads from cefs did not map to the current chicken genome. the unmapped reads combined from the control and ifn-treated samples were assembled into contigs using the de novo assembly function of genomic workbench. the rna-seq function of genomic workbench was then used to quantitate expression of the contigs in control and ifn-treated samples. one of the most highly-expressed contigs was one which, when analysed by blast, proved to represent a homologue of stat , which is missing from the current ensembl annotated reference chicken genome in (b) pyurf shows -fold suppression by ifn but the sequence surrounding pyurf shows -fold induction from the right-hand end of the unannotated, antisense loc and considerably higher upregulation from the left-hand end (due to its lower uninduced levels), consistent with these representing homologues of ifn-inducible human genes herc and herc . assembly (galgal ; release ), though at ncbi it has recently been placed as a refseq gene on chromosome in the new assembly galgal (an annotated form of which has not yet been released and is currently not scheduled for release). the de novo assembled contig sequence was used to derive primers for rt-pcr; characterisation of chicken stat will be reported elsewhere. the data on differential expression showed an overwhelming over-representation of genes up-regulated by ifn. for each of the technologies, only one gene was detected as down-regulated. corresponding geneids were pyurf (pigy upstream reading frame; ensgalg ) for rna-seq and pigy (phosphatidylinositol glycan anchor biosynthesis, class y; ncbi geneid: ) for the st array. the down-regulated k genechip probe (gga. . .s _at), though not mapped to a known gene at the time of initial processing, according to the affymetrix netaffx ™ analysis center [ ] is now also assigned as pyurf. in humans, pigy and pyurf represent different open reading frames on the same spliced transcript of a gene on hs chromosome located downstream of herc then herc . the pyurf/pigy gene is overlapped on the opposite strand by herc , which extends downstream to be followed by fam a. similarly, the chicken pigy (ncbi) and pyurf (ensembl) genes map to a locus lying upstream of herc then fam a on gg chromosome (see figure ) , with herc-like loc ("hect domain and rld -like") starting upstream but spanning and extending downstream of the chicken pyurf. our rna-seq data ( figure ) indicate that this locus is poorly annotated and demonstrates complex regulation of the component genes by ifn. thus, although the pigy/ pyurf transcript is down-regulated by ifn, as recorded by all three technologies, it appears to be closely flanked upstream and downstream by still unannotated multiple exons that are clearly strongly induced by ifn (figure ). sequences within these upstream and downstream regions (which are represented by the single ncbi refseq (gal-gal ) gene, loc , but appear as though they may represent two separate genes, figure ) bear homology with genes of the herc family, consistent with the fact that herc neighbours the human pigy/pyurf gene and that herc neighbours the chicken pigy/pyurf gene. the chicken herc gene shows no evidence of induction by ifn. description of the interferon-inducibility of the chisgs serves as the first step in understanding the regulation of their expression and their role in anti-viral (and potentially broader anti-microbial) activities. there is considerable current interest in the antiviral responses of particular cell types, particularly those of the lymphoid, myeloid and dendritic lineages. however, the definition of a wide variety of these cell types is not so advanced in avian species so we felt it best to produce baseline data for readily available, primary cells, namely chick embryo fibroblasts (cef) as they are highly responsive to ifn. they also remain important for commercial production of vaccine viruses (including human vaccines) as well as for the routine isolation and diagnosis of avian pathogens. given the currently incomplete nature of the chicken genome assembly (even at galgal ) and of its annotation (as currently available for galgal and even as awaited for galgal ) it is inevitable that updates will continue to be released but the primary data reported here, and publicly-available, for microarrays and rna-seq, can always be applied to updated microarray assignments as well as to subsequent genome assemblies and annotations. all things being equal, rna-seq would seem to be the method of choice for transcriptomic analysis of chicken ifn responses, particularly given its ability to produce high-resolution quantitative and qualitative data. moreover the data are readily portable and can be easily mined by others with different research focus. they can also be applied immediately to newly released genome assemblies and annotations (whether global or local), whereas microarray analysis must await the generation of annotation updates for each technology. however, although the cost of sequencing has fallen, and will probably continue to do so, there remain considerable overheads to handling large data sets from extensive, complicated experiments, especially in terms of computing and data storage capacity, as well as speed of processing and archiving. for such experiments, microarrays continue to offer a tractable approach, capable of quickly quantifying and comparing the expression of the central core of irgs producing relatively compact data for rapid analysis and easy archiving. induction of innate responses with pamps will trigger different or broader ranges of responses by virtue of the fact that they will trigger other or more pathways than just the ifn-pathway. for instance we (giotis et al. unpublished) and others [ ] have begun to analyse the responses induced by the dsrna analogue poly[i:c]. regulation of isg expression might affect the innate responses observed in different cell lines or tissues so it will be important to understand the mechanisms involved. additionally, we have observed suppression of isg induction in the spontaneously immortalized chicken fibroblast cell line, df- [ ] , due to their enhanced basal expression of the regulatory isg, socs (giotis et al., unpublished) . identification of the isgs means that their promoters, enhancers and other regulatory elements can be systematically analysed to help understand the complex kinetics of expression of their expression (figure ). several studies have investigated changes in host gene expression in response to infection in vivo or in culture with particular avian viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although many of these genes will represent innate (and potentially antiviral) host responses, the majority will be involved in the metabolic, cell cycle and ultrastructural changes that the virus has to induce to facilitate replication. furthermore, it is not unusual for viruses to modulate the expression of signalling molecules key to the antiviral responses or of antiviral effectors themselves. for instance, we have shown that even an attenuated strain of fowlpox virus blocks induction of ifn-β (chifn ) and is highly resistant to the antiviral activity induced by ifn [ , ] . the results of existing and future studies of infection in vivo or in culture with particular avian viruses can now be compared with data presented here for isg induction by ifn to look for evidence of modulation of isg expression by viruses, whether that be modulation of individual isgs, subsets [ ] or the complete set. for instance, fowlpox virus blocks essentially all isg expression but a mutant defective in the fpv ankyrin repeat/f-box protein identified by laidlaw et al. [ ] induces modest levels of a subset of the isgs (giotis et al., unpublished) . such analyses can be extended to important avian zoonotic viruses and pathogens with huge impact on the global poultry industry. although this study relates to type i ifn, extensive comparison with the effects of type iii ifn could now be conducted, extending on the qrt-pcr comparison made by masuda et al., who looked at induction of mx and oas by ifn-β, ifn-γ and ifn-λ [ ] . interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures inborn errors of anti-viral interferon immunity in humans interferon-stimulated genes: a complex web of host defenses pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses functional classification of interferon-stimulated genes identified using microarrays a diverse range of gene products are effectors of the type i interferon antiviral response virus interference. i. the interferon chicken interferon gene: cloning, expression, and analysis characterization of the chicken pkr: polymorphism of the gene and antiviral activity against vesicular stomatitis virus international chicken genome sequencing c ( ) sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution evidence of the adaptive evolution of immune genes in chicken functional analysis of chicken irf in response to dsrna analog poly(i:c) by integrating overexpression and knockdown defense genes missing from the flight division innate sensing of viruses by pattern recognition receptors in birds id: : transcriptomic analysis of the chicken interferome genetic screen of a library of chimeric poxviruses identifies an ankyrin repeat protein involved in resistance to the avian type i interferon response species difference in anp a underlies influenza a virus polymerase host restriction dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources differential expression in sage: accounting for normal between-library variation involvement of the irf- transcription factor in antiviral responses to interferons constitutive expression of an isgf /irf transgene leads to interferon-independent activation of interferon-inducible genes and resistance to virus infection ifn regulatory factor- bypasses ifn-mediated antiviral effects through viperin gene induction interferome v . : an updated database of annotated interferon-regulated genes chicken interferon-inducible transmembrane protein restricts influenza viruses and lyssaviruses in vitro the df- chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses transcriptomic profiling of virus-host cell interactions following chicken anaemia virus (cav) infection in an in vivo model molecular responses to the influenza a virus in chicken trachea-derived cells early host responses to avian influenza a virus are prolonged and enhanced at transcriptional level depending on maturation of the immune system transcriptional analysis of host responses to marek's disease viral infection analysis of the early immune response to infection by infectious bursal disease virus in chickens differing in their resistance to the disease a comparative analysis of host responses to avian influenza infection in ducks and chickens highlights a role for the interferon-induced transmembrane proteins in viral resistance analysis of the crow lung transcriptome in response to infection with highly pathogenic h n avian influenza virus integrated analysis of microrna expression and mrna transcriptome in lungs of avian influenza virus infected broilers differential expression of micrornas in marek's disease virus-transformed t-lymphoma cell lines genetic screen of a mutant poxvirus library identifies an ankyrin repeat protein involved in blocking induction of avian type i interferon biological effects of chicken type iii interferon on expression of interferon-stimulated genes in chickens: comparison with type i and type ii interferons we are grateful for the skilled support of laurence game, nathalie lambie and adam giess of the medical research council's (mrc) clinical sciences centre's (csc) genomics facility in conducting microarray analysis and illumina sequencing. we gratefully acknowledge sarah butcher and geraint barton of the bioinformatics support service at imperial college london for their advice. the datasets supporting the conclusions of this article are available from the following repositories: european bioinformatics institute (ebi) arrayexpress accession numbers e-mtab- (for the k genechip; [ ] ) and e-mtab- (for the st array; [ ] ). european nucleotide archive (ena) study number prjeb (for illumina rna-seq; [ ] ). additional file . table of additional file . detailed information on chisgs identified by rna-seq, and microarray technologies ( ). technologies identifying significant irgs are listed as " " rna-seq (using kal's z test); " " affymetrix k genechip chicken genome array and " " chicken gene . st array' . chisgs significant by one or both microarrays and rna-seq using kal's z test under relaxed criteria (fc > . or fdr < . ) are indicated by "( )". "+" after the technology identifier indicates that ifn-induced rna-seq read density was observed at the location of the unannotated gene. ( ) interferome status [ ] . ( ) human homologue data (hugo) [ ] . ( ) mouse orthologue data (mgi) [ ] . ifn: interferon; irgs: ifn-regulated genes; isgs: ifn-stimulated genes; cef: chicken embryo fibroblasts; rchifn : recombinant chicken ifn-α; rin: rna integrity number; qrt-pcr: quantitative real-time pcr; gapdh: glyceraldehyde -phosphate dehydrogenase; fc: fold change; fdr: false discovery rate. the authors declare that they have no competing interests. esg and rcc design of the study, data acquisition and analysis, drafting the manuscript. ngs data compilation and analysis, drafting the manuscript. cdt design, production, curation and maintenance of chisg browser website. sg design of the study, critically reviewing the manuscript. mas design of the study, data analysis, finalizing manuscript. all authors read and approved the final manuscript. key: cord- -f wvh la authors: zhou, peng; cowled, chris; mansell, ashley; monaghan, paul; green, diane; wu, lijun; shi, zhengli; wang, lin-fa; baker, michelle l. title: irf in the australian black flying fox, pteropus alecto: evidence for a unique expression pattern and functional conservation date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: f wvh la as the only flying mammal, bats harbor a number of emerging and re-emerging viruses, many of which cause severe diseases in humans and other mammals yet result in no clinical symptoms in bats. as the master regulator of the interferon (ifn)-dependent immune response, ifn regulatory factor (irf ) plays a central role in innate antiviral immunity. to explore the role of bat irf in the regulation of the ifn response, we performed sequence and functional analysis of irf from the pteropid bat, pteropus alecto. our results demonstrate that bat irf retains the ability to bind to myd and activate the ifn response despite unique changes in the myd binding domain. we also demonstrate that bat irf has a unique expression pattern across both immune and non-immune related tissues and is inducible by double-strand rna. the broad tissue distribution of irf may provide bats with an enhanced ability to rapidly activate the ifn response in a wider range of tissues compared to other mammals. the importance of irf in antiviral activity against the bat reovirus, pulau virus was confirmed by sirna knockdown of irf in bat cells resulting in enhanced viral replication. our results highlight the importance of irf in innate antiviral immunity in bats. bats have been implicated in the spillover of many deadly viruses including rabies, henipaviruses (hendra and nipah), ebola virus, and the coronaviruses (cov): severe acute respiratory syndrome (sars-cov) and the recently emerged middle eastern respiratory syndrome virus (mers-cov), all of which impose a significant threat to human health [ , , , , , ] . as natural hosts, bats rarely show clinical signs of disease during infection [ ] . how bats co-exist with viruses and the role of the bat innate immune system in controlling viral replication remain poorly understood [ ] . identifying the mechanisms responsible for controlling viral replication in bats has profound implications for the development of therapeutic strategies targeting viral infections in humans and other species. one of the most important early anti-viral defenses in mammals is the ifn system, which not only provides pivotal protection immediately following infection but also shapes the adaptive immune response [ ] . of the three ifn families discovered, type i (including a and b) and type iii (l) ifns respond directly to viral infection. due to the importance of ifns in controlling viral replication, the regulation of the ifn response has been extensively studied in humans and other mammals. key to the regulation of ifn production and signaling is the ifn regulatory factor (irf) transcription factor family. the irf family consists of nine members which share functional and structural characteristics. however, only irf , irf , irf and irf have been implicated as positive regulators of type i ifn transcription, and only irf and irf are designated as antiviral irfs [ , ] . since their first discovery within the biological context of epstein-barr virus latency, irf was identified as the master regulator of the type i ifn-dependent immune response, and perhaps that of type iii ifn as well [ , , ] . irf is expressed only at low levels in most cells but is constitutively expressed in certain immune cells such as plasmacytoid dendritic cells (pdc) which specialize in ifn production. correspondingly, the tissue distribution of human irf is restricted to immune tissues which contain large numbers of specialized immune cells including spleen, thymus and peripheral blood lymphocytes whereas non-immune tissues including the intestine and colon express almost undetectable levels of irf [ ] . although irf is expressed at low levels in most cell types, it is induced strongly by type i ifn mediated signaling in all cells [ ] . interestingly, multiple fish species (japanese flounder, crucian carp, mandarin fish, snakehead fish and atlantic salmon) have been demonstrated to express irf constitutively in all tissues including both immune and non-immune tissues [ , , , , ] . viral sensing either by toll like receptors (tlrs) or retinoic acid-inducible gene (rig-i)-like receptors can result in the activation of irf and subsequent induction of ifns [ , ] . all tlrs with the exception of tlr activate irf through the adaptor protein, myd (myeloid differentiation primary response gene ) through the myd -dependent pathway. myd forms a complex with the kinases irak- (interleukin receptor associated kinase ), irak- and traf- (tnf receptor associated factor). this complex binds directly to irf leading to ubiquitination by traf- and phosphorylation by irak or ikk- (ikb kinase- ) and translocation from the cytosol to the nucleus where irf binds to promoter elements inducing ifn production [ , ] . tlr and tlr activate irf through the myd -independent pathway through the adaptor molecule trif (tir-domain-containing adapter-inducing ifn-b) which forms a complex with tbk (tank binding kinase ), ikk-e (inhibitor of nuclear factor-kb kinase e) and irf . in this case the phosphorylated irf forms a homodimer or heterodimer with irf and translocates to the nucleus where it binds to the ifn promoter via its dna-binding domain to induce type i or type iii ifn [ ] . in irf knockout mice, viral induced ifn production through the tlr (myd -independent) pathway is greatly impaired. as a result, mice become more susceptible to viral infection [ ] . although irf has been reported to preferentially activate ifn-b over ifn-a genes, irf is believed to efficiently activate both ifn-a and ifn-b [ ] . the human ifn-b promoter region contains four positive regulatory domains (prds to iv) that serve as binding sites for irfs. in the human ifn-a promoter region there may be two or three prd modules depending on the ifn-a subtype [ ] . due to the importance of irf in the innate immune response, it is an active target for viruses to evade the host immune response [ ] . a role for irf in immunosurveillance has also been identified in breast cancer [ ] . using the australian black flying fox (p. alecto) as a model species we have begun to explore the role of the ifn system in the control of viral replication in bats. we have demonstrated that tlrs, rig-ilike receptors, and some ifn stimulated genes (pkr, mx and oas ) appear to be conserved in sequence compared to other mammals [ , , ] . however, bats appear to have relatively higher expression of type iii ifn and wider distribution of type iii ifn receptors consistent with a role for type iii ifns in antiviral immunity [ , ] . bat genome analysis has also provided evidence for positive selection of genes within the ifn pathway, including tlr , c-rel, tbk- , ifn-c, isg and rig-i [ ] . these changes may have occurred in response to the co-evolution of bats with viruses and may have consequences for the clearance of viral infections and the ability of bats to coexist with viruses. due to the central role of irf in the regulation of the ifn response, we performed sequence and functional analysis of p. alecto irf . our results provide the first description of irf in any species of bat and evidence for conserved irf functional activity despite variation at the sequence level in the bat irf gene. all animal experiments were approved by the australian animal health laboratory (aahl) animal ethics committee (protocol number ). immortalized and cloned p. alecto kidney (pakit ) and lung (palut ) cells established previously [ ] were cultured in dmem/f -hams (sigma), supplemented with % foetal calf serum (fcs, hyclone), units/ml penicillin, mg/ml streptomycin and mg/ml gentamycin (sigma). human embryonic kidney hek t cells were cultured in dmem supplemented with % fcs (hyclone), mm lglutamine, mg/ml penicillin, neaa/na-py/fungizone. all cells were maintained in a humidified atmosphere of % co at uc. sendai virus (sev, cantell strain) was prepared in chicken embryos as described previously [ ] . pulau virus (pulv) was prepared and titered as described previously [ ] . for infection of cells, virus was incubated with cells for one hour at uc, then replaced with normal cell culture medium for the indicated time. full-length irf , irf and myd open reading frames (orfs) were identified in the p. alecto genome (ncbi id p. alecto asm v ) [ ] using blastx. for comparative purposes, sequences were obtained from the current genome assemblies from the ensembl database for the following species: ensp , homo sapiens (human); ensmusp , mus musculus (mouse); ensecap , equus caballus (horse); enssscp , sus scrofa (pig); ensbtap , bos taurus (cow). the microbat myotis davidii irf amino acid sequence was deduced from irf orf annotated from the published m. davidii genome (ncbi id m. davidii asm v ) [ ] . the p. alecto irf sequence has been submitted to genbank under accession number kj . sequence alignment was performed using clustalx and visualized using genedoc (http://www.nrbsc.org/gfx/genedoc/ index.html). alignment files were visualized using emboss plotcon to determine the conservation of irf proteins among different species. genomic intron-exon maps of the genes were drawn using fancy gene v . by comparing individual irf orfs of p. alecto, horse and human (http://host .bioinfo .ifomieocampus.it/fancygene/). phylogenetic trees were constructed using the neighbour joining method and mega . program with bootstrap replicates [ ] . primers listed in table were designed based on the p. alecto genomic sequences and used in rt-pcr to amplify irf , irf and myd from rna extracted from freshly isolated bat splenocytes. to construct expression plasmids, pcr products corresponding to full-length irf and irf were ligated directly to vivid colors pcdna . /emgfp topo vector (life technologies) with an n-terminal gfp tag. the myd orf was ligated to the pflag-cmv expression vector (sigma) using restriction enzymes noti and sali with an n-terminal flag tag for detection. to generate a truncated bat irf (tirf ) that lacked the myd binding region at amino acids (aa) - , overlapping pcr was performed [ ] . the resulting tirf pcr product was ligated to the pcdna . /emgfp topo vector. the human irf (hu-irf ) and hu-myd plasmids have been described previously [ ] . hu-irf is in the pegfp-n vector and hu-myd is in the pef-bos vector with an n-terminal flag tag. mouse ifn-a , ifn-a and human ifn-b promoter plasmids, abbreviated as mu_ifn-a p, -a p and hu_ifn-bp respectively, have been described previously [ ] . the bat ifn-b promoter plasmid was constructed using sequence bp upstream from the start codon of ifn-b orf from the p. alecto genome. promoter prediction was performed using the online transcriptional start site prediction tool, matinspector in the genomatix software suite (http://www.genomatix.de/cgi-bin//matinspector_prof). regions containing putative irf or irf binding sites were identified from to bp from the atg of the bat ifn-b gene by comparison with human ifn promoters and cloned into the pgl . expression vector (promega). a transfection control prl-tk plasmid containing renilla luciferase was obtained from promega. details of primers used during plasmid construction can be found in table . hek t cells were transfected using fugene (promega) according to the manufacturer's instructions. approximately cells per well in a -well plate were co-transfected with ng of the relative ifn promoter plasmids, ng of prl-tk (promega) served as an internal control. where indicated, expression plasmids for bat irf , human irf or bat myd were included in the transfection mix. cells were harvested h post-transfection and lysed using passive lysis buffer provided in the following kit. luciferase activities were determined using the dual-luciferase assay system (promega) using a thermo fluoroskan ascent fl machine. for promoter experiments in pakit cells, similar transfections were performed using lipofectamine (life technologies). a smartpool consisting of four small interfering rnas (sirnas) targeting bat irf (siirf ) was designed based on the full-length irf orf sequence using dharmacon custom services (thermo). information of the four siirf can be found in table . transfection of siirf was performed in pakit cells using the neon transfection system (life technologies) according to the manufacturer's instructions. briefly, nm of each of the four siirf was used for every cells. cells were harvested into rlt lysis buffer (rneasy kit, qiagen) h post-transfection and stored at uc prior to rna extraction. rna was extracted from cell lysate from the sirna knockdown experiment using a qiagen rneasy kit and converted to cdna using the quantitect reverse transcription kit for real-time pcr (qiagen). all experiments were performed according to manufacturer's protocols. preparation of cdna from p. alecto tissues including brain, kidney, liver, lung, lymph nodes, spleen, heart, small intestine, wing, salivary gland, thymus and testis from three individual bats and from polyi:c stimulated palut cells has been described previously [ ] . for each sample, mg rna was applied to reverse transcription using the quantitech reverse transcription kit (qiagen). irf qrt-pcr primers were designed using primer express . (applied biosystems) with default parameter settings and are listed in table . primers for ifn-b and s rrna have been described previously [ ] . reactions were carried out using express sybr greener qpcr supermix universal (life technologies) in an applied biosystems fast real-time qrt-pcr instrument. for each reaction from cdna, ml of : diluted cdna were used with a final concentration of nmol of each primer. the cycling profile for cdna samples consisted of an initial denaturation at uc for minutes followed by cycles of uc for seconds, uc for minute, followed by melt curve analysis. expression levels of target genes were calculated using the standard curve method after normalisation to the housekeeping gene s rrna. pakit cells were seeded onto glass coverslips in -well plates at cells per well one day before transfection. they were transfected with ng each of gfp-irf and flag-tagged myd (human or bat) using lipofectamine (life technologies). at h post-transfection, cells were fixed in % paraformaldehyde in pbsa at room temperature for minutes. after removal of fixative, cells were washed three times with pbsa, followed by treatment with . % triton x- for minutes and blocked with . % bsa in pbsa for minutes. mouse anti-human myd antibody (santa cruz, cat. sc ) was diluted : in . % bsa and applied to cells and incubated at uc for h. cells were then incubated with alexa conjugated goat anti-mouse secondary antibody at uc for h and washed three times in pbsa. nuclei were labelled with dapi and coverslips were mounted on glass slides for analysis. all slides were examined under a leica confocal microscope (leica, germany). the immunoprecipitation method for analysis of irf and myd has been described previously [ ] . in brief, hek t cells were seeded h prior to co-transfection with . mg of both flag-myd and gfp-irf (human or bat) using fugene (promega). cells were then lysed h later with lysis buffer ( mm tris (ph . ), . % triton x- , mm nacl, mm edta, mm na vo , mm naf, mm pmsf and protease cocktail inhibitor mixture; promega). cell lysates were cleared by centrifugation ( g, min, uc), and then pre-cleared with protein g-sepharose beads for min at uc and flag-myd immune complexes were immune-precipitated from supernatant using anti-flag m -agarose (sigma) conjugated beads for h at uc. beads were washed with x lysis buffer and eluted by boiling beads in volumes of sds page sample buffer. lastly, sds page and western blot analysis were performed using the samples obtained. for the blotting, antihuman myd rabbit antibody and anti-human irf goat antibody (both from santa cruz) were used at : dilution during primary antibody incubation and ap-conjugated goat antirabbit or rabbit anti-goat secondary antibody (both from life technologies) were used at : dilution. to explore possible differences in the bat antiviral immune system which may influence the association between bats and viruses, irf family members were chosen as primary targets due to their importance in ifn induction and signaling [ ] . all irf members from irf to irf were identified in the bat genome, indicating their relative conservation at the family level. we next performed sequence analysis on the four known positive regulators of type i ifn transcription in humans; irf , irf , irf and irf . comparison of the sequence similarity of the deduced protein sequence of human and bat irfs demonstrated significant conservation of irf ( %), irf ( %) and irf ( %). in contrast, bat irf shares only % and % amino acid similarity to human and mouse respectively. to determine whether the relatively low sequence conservation of the bat irf gene compared to human and mouse affects the functional activity of bat irf , this gene was chosen for further functional analysis. the recently published p. alecto whole genome sequence and transcriptome data were used to identify the irf gene [ , ] . primers based on the genomic irf sequence were used to amplify full-length irf by pcr from bat spleen cdna resulting in the identification of a single full length irf transcript. by aligning the bat irf cdna sequence with the corresponding region in the p. alecto genome, we were able to determine the intron and exon structure of this gene. as shown in figure , bat and horse irf have nine exons compared to the ten exon structure of human irf . horse was included in this comparison due to the close phylogenetic relationship between bats and horses (figure s and [ ] ). nine exons were also identified in irf genes from other laurasiatheria species, including pig, cow and dog. of the sequences available in the ensembl database, the ten exon structure appears to be typical only among primate irf genes (data not shown). analysis of the putative bat irf promoter region around bp upstream of the start site of the orf resulted in the identification of two ifn stimulated response elements (isres) and one nuclear factor kappa b (nf-kb) binding site. to determine whether the presence of two isre sites was unique to bats, the irf promoters of other species (human, mouse, horse, cow, dog, cat and rat) were also examined using the publicly available databases. all species examined contain two isre sites (one isre and one irf binding site) with the exception of human which has a single isre (data not shown). based on the deduced protein sequence, p. alecto irf was aligned with six other species: human, mouse, pig, cow, horse and the microbat, david's myotis (m. davidii) available from the recently completed genome sequence [ ] . these species were chosen because human and mouse have been well studied, while all other species are phylogenetically close to p. alecto [ ] . fulllength irf contained multiple functional domains including an n-terminal dna-binding domain (dbd), followed by a constitutive activation domain (cad), virus-activated domain (vad), inhibitory domain (id), and c-terminal serine-rich region ( figure ; [ , ] . the vad is responsible for binding to the upstream activators of irf : myd , traf or tbk- , while the serine-rich region is the target for virus-inducible phosphorylation [ , ] . from the alignment, bat irf appears to have a conserved dbd ( figure s ), c-terminal serine-rich domain which is the target of phosphorylation, and auto-inhibitory domain [ ] . however, the region between amino acids - , which corresponds to the vad and contains the myd binding site is not conserved in sequence among all species ( figure b) . furthermore, in the putative myd binding region, both bats are less conserved not only to human but also to other laurasiatheria species shown here (pig, cow and horse). as the myd -irf pathway is critical to ssrna-induced human ifn production, alignment was performed between bat myd and myd from human, mouse, pig, cow and horse. no significant change that would potentially alter the functionality of bat myd was identified based on sequence analysis (figure s ). to determine the tissue distribution of bat irf , its transcription was examined in a range of immune and non-immune associated bat tissues. the tissues were from three apparently healthy bats caught from the wild in which the irf level should represent the normal expression pattern in wild bats. as shown in figure a , bat irf is widely expressed among all bat organs at the mrna level, with spleen, small intestine and lung having the highest irf expression and wing and salivary gland the lowest. with the exception of the wing, all other tissues showed similar expression levels of irf with around -fold difference between spleen which had the highest expression and salivary gland with the lowest. this pattern differs from the transcription pattern of bat tlr , and , which appear to be predominantly expressed in immune tissues [ ] . next, the inducibility of irf by a known virus mimic was explored by testing irf transcription using our cloned and immortalized bat lung cell line (palut cells) following stimulation with the double stranded rna (dsrna) ligand, alecto irf with irf from human, mouse, cow, pig, horse and m. davidii using a amino acid sliding window and created using emboss plotcon (http://emboss.bioinformatics.nl/cgi-bin/emboss/plotcon). the similarity across the whole orf between these species is shown by scores. the lower the score the lower the sequence conservation. the least conserved region is boxed. doi: . /journal.pone. .g polyi:c. the palu cell line has previously been demonstrated to produce ifn in response to polyi:c in a dose dependent manner [ ] . as shown in figure b , stimulation by either treatment or transfection with polyi:c, which mimics ifn production through tlr or rlh pathways respectively resulted in strong induction of irf . notably, irf mrna was induced at the earliest time point of h following stimulation, to a peak level of around times higher than mock treated cells, highlighting the importance of irf in early antiviral defense in bats. these data are consistent with bat irf being inducible by both tlr and rlh pathways. in summary, bat irf is constitutively transcribed in all tissues and cell lines tested (palut cells and pakit cells, below) and can be strongly induced by dsrna. due to the limitations of the human irf antibody used in this study, the expression of irf at the protein level awaits the development of a suitable bat specific reagent. to test the ability of irf to induce ifn-b production a bat ifn-b promoter assay was used. the putative promoter region of the bat ifn-b gene was examined and predicted to contain irf and irf binding modules ( figure s ). this region was ligated to the pgl . luciferase reporter vector. interestingly, the bat ifn-b promoter contains one residue difference in its prdi which may abolish its ability to bind to irf or irf [ ] . in contrast, the second prd (prdiii) is almost identical to the corresponding domain of the human ifn-b promoter region and was therefore predicted to be functional [ ] . comparison of the prdi domain of other closely related species available in the ensembl database and the microbat, m. davidii, revealed the disruption of prdi is unique to p. alecto ( figure s ). in the bat immortalized kidney cell line (pakit cells), a plasmid encoding bat irf was co- transfected with the bat ifn-b promoter plasmid for h and cells were then infected with sev for another h before performing the luciferase test. the pakit cells were chosen for these experiments due to their ability to be successfully transfected and sev was chosen due to its potent ability to induce ifns and other cytokines in the absence of viral products that block the ifn response [ ] . results clearly indicate that irf alone can induce ifn-b activation and sev induces enhanced activation of irf in a dose dependent manner ( figure a) . to examine whether the poorly conserved myd -binding region in bat irf influenced its transactivation potential, bat irf was compared with that of human irf using ifn promoter assays. this experiment was designed to test the hypothesis that the sequence differences identified in the bat irf region do not affect its ability to activate ifn transcription. this response was tested using mouse ifn-a , mouse ifn-a or human ifn-b promoter plasmids co-transfected with increasing doses of bat or human irf expression plasmids in hek t cells. as shown in figures b-d , bat irf activates all three promoters in a dosedependent manner. together with the results described above using the bat ifn promoter, these results demonstrate that bat irf is capable of activating ifn in bat cells following stimulation with sev or in human cells co-transfected with human or mouse ifn promoters. cells transfected with mock or empty vector failed to activate the ifn promoters significantly. although identical doses of human and bat irf plasmid were used in these experiments, the results are not statistically comparable due to the difference in irf protein expression from the two plasmids. of note, we used human hek t cells and mouse ifn-a and human ifn-b promoters in this assay because they are a well established system for testing the activation of ifn [ ] . given the high conservation in the dna-binding domain of bat irf , it was predicted to be capable of binding to the human and mouse ifn promoters followed by activation by downstream factors. to explore the importance of bat irf in the ifn production pathway, a knockdown approach was used in our p. alecto pakit cells. transfection of sirna targeting bat irf (siirf ) for h resulted in a reduction of native irf mrna expression to approximately % compared to mock transfected cells. the statistical analysis of the knock down effects was calculated by comparing mrna expression in the knock down samples to mock transfected cells. to exclude the possibility of off-target effects of sirna transfection, the expression of a closely related gene, irf was examined in transfected cells. as shown in figure a , there was a decrease in irf transcription in siirf transfected cells due to possible toxic effects and/or off target effects of the sirna smartpool but this change was not statistically significant. having confirmed the knockdown effect of siirf , we then explored the downstream effect of reduced irf on ifn-b production and viral replication. two experiments were performed; firstly, h after siirf transfection, cells were stimulated with sev for h and ifn-b mrna was detected by qpcr. knockdown of irf impaired the induction of ifn-b by sev by . fold relative to untransfected cells ( figure b) . notably, bat cells maintained some ifn-b induction in siirf cells, a result which likely reflects insufficient knockdown of irf or ifn-b induction through alternative (irf or nf-kb) pathways. to examine the effect of ifn knockdown on the replication of a bat-borne virus, pulv, a dsrna reovirus originating from pteropid bats, was used to infect siirf -transfected bat cells [ ] . a dose of moi was used to infect pakit cells one day after siirf transfection (or mock transfection). cell supernatant containing virus was collected h after infection and applied to a tcid test. figure c shows that when bat irf was knocked down, pulv replicated to a titer more than four-fold higher than in mock-transfected cells. these data demonstrate that bat irf is functionally important in sev induced ifn-b production and antiviral defense against pulv infection of bat cells. we next wanted to determine whether bat irf is involved in the production of ifn-a and ifn-b by the myd despite the divergent nature of its myd binding domain. the transactivation activity of bat irf was compared to that of human irf using expression plasmids containing bat or human myd and irf co-transfected with mouse ifn-a or ifn-a promoter plasmids. in mice, ifn-a is the earliest ifn-a induced by viral infection, while ifn-a is induced later in the response. a dose of ng or ng of irf was co-transfected with myd for the ifn-a p and ifn-a p promoter assay respectively in hek t cells. these doses of irf were chosen as they do not result in huge induction of the native ifn promoter. as shown in figure a , the activation of ifn-a p by both human and bat irf was increased by co-transfection with myd . co-transfection of cells with bat myd and irf resulted in a higher response compared to co-transfection with the corresponding human plasmids. our results demonstrate that even with a significant difference in its myd binding region, bat irf is still capable of inducing ifn-a transcription via myd ( figure ). some differences were observed between ifn-a and ifn-a inducibility which may be due to differences in their irf or nf-kb binding motifs. no ifn-a activation occurred following cotransfection of bat myd with bat irf confirming that irf is myd independent ( figure s a) . a similar experiment was performed to confirm that bat irf is capable of activating the human ifn-b promoter confirming the activity of bat irf ( figure s b ) [ ] . thus, only ifna production by irf is dependent on myd . to confirm that bat irf interacts with bat myd , experiments were performed to examine the interaction between the two proteins. firstly, hek t cells were co-transfected with plasmids encoding flag-tagged myd (either human or bat, as indicated) and gfp-tagged irf (human or bat). cells were lysed and protein immunoprecipitated with anti-flag antibody conjugated beads followed by immunoblotting with anti-flag or anti-human irf antibody. human irf protein was successfully captured by human myd which was detected in ip samples demonstrating protein interaction between human myd and human irf ( figure a, panel ) . the detection of only a faint band may be due to the relatively low expression of the input human irf and myd proteins from these expression plasmids. clear signals were detected for irf and myd following co-ip of bat myd with bat or human irf ( figure a ). there is a slight difference in the molecular weights of human and bat myd and irf which are reflected on the blot (human and bat myd are . and . kd respectively and human and bat irf have a molecular weight of . and . kd respectively). these results clearly demonstrate that bat myd protein is capable of binding both bat and human irf proteins. confocal microscopy was used to determine the colocalisation of the two proteins, to further confirm protein interaction. bat kidney pakit cells or human kidney hek t cells were used to examine colocalisation of irf with myd . a dose of ng/ well of either human or bat myd and irf plasmids were used to transfect cells grown overnight on coverslips in -well plates. sixteen hours later, cells were fixed and stained with anti-human myd antibody and examined under the confocal microscope. myd transfection alone resulted in the formation of very large condensed aggregates in the cytoplasm of both human and bat cells. human myd and human irf colocalised in a manner similar to previous studies ( figure b ) [ , ] . similarly, bat myd and irf proteins also demonstrated clear co-localisation. as shown in figure b , bat irf appeared to be surrounded by myd in an aggregated form, which is the typical myd structure [ , ] . in addition, bat myd also colocalised with human irf , which is consistent with our ip results ( figure a ). as expected, no such aggregated structure was observed following co-expression of bat myd with bat irf , ruling out the possibility of interaction between these two proteins. having confirmed the functional reliance and binding capability of bat irf to bat myd , it appeared that the poorly conserved myd -binding region (between aa - ) retains a similar function to the corresponding human domain. to further confirm this was the case, an internal deletion mutant of bat irf , tirf was constructed ( figure a ). this form of bat tirf contained a deletion of aa - which has been shown to abolish the ability of human irf to transactivate the ifn-a promoter [ ] . the mouse ifn-a promoter transactivation activity by tirf was compared to the full-length irf protein using a luciferase assay. having confirmed successful expression, figure b clearly demonstrates that tirf , was unable to activate the mouse ifn promoter, even in the presence of myd ( figure b ). these data are consistent with functional conservation of the region corresponding to aa - in bats with that of human irf despite significant sequence variation. irf is a master regulator of ifn expression in mammals and is therefore central to the innate antiviral immune response. in humans, irf acts predominately in pdcs via activation of tlr / and the myd dependent signaling pathway [ ] . regulation of the ifn response may play an important role in the ability of bats to coexist with viruses in the absence of clinical signs of disease. this report describes the analysis of irf from our model bat species, the australian black flying fox, p. alecto, an important reservoir for viruses including hendra virus, which has resulted in the deaths of numerous horses and humans since its discovery in [ , ] . our results support the conservation of functional activity of irf in p. alecto but provide evidence of a wider tissue distribution which has implications for broader activation of the ifn response in bats. our results provide the first functional characterization of irf in any species of bat and contribute to our understanding of the function and evolution of irf in mammals. bat irf was identified from the bat genome and bat transcriptome data [ , ] , together with rt-pcr results from spleen cdna resulting in the identification of a single full length variant of irf . in humans and mice, irf expression is very low in most tissues and cells with the exception of pdcs and cells that have been activated by ifn [ , ] . in contrast, the transcription of p. alecto irf was detected not only in immune-related tissues but comparable expression was observed in many other organs as well. although there is a lack of data on the tissue distribution of irf in mammals besides human and mouse, there have been several studies on fish irf . interestingly, at least five species of fish including crucian carp, mandarin fish, snakehead fish, atlantic salmon and japanese flounder express irf constitutively in a wide variety of tissue types although different irf transcripts were expressed in each species. these tissues were neither primarily immune-related nor serve as portals for microbial infection, where the immune response is easily initiated. since these fish irf s can also be induced by dsrna, they were hypothesised to play an important role in fish immunity [ , , , , ] . although further analysis of the cell types responsible for constitutive irf expression in bats is required, a constitutively expressed irf in a broad range of cells and organs may result in faster and stronger ifn production upon viral infection [ ] . this observation is similar to the pattern of type iii ifn receptor expression which has a wide distribution in bats but only limited distribution in other mammals [ ] . thus, bats may maintain the potential to rapidly activate the innate immune response in a broader subset of tissues and cells than other mammals. induction of irf by treatment or transfection of our bat kidney cell line with the dsrna ligand, polyi:c resulted in a peak in the induction of irf at h post-treatment, which is h later than the peak in bat type i and type iii ifns but similar to that of isgs mx , oas and pkr described previously in bat cells [ , ] . this result is consistent with the induction of irf through type i ifn feedback similar to other species. in humans, irf is generated through multiple pathways following ifn induction. following the production of ifn and binding to the . bat irf interacts with bat myd . (a) binding of bat irf to bat myd . hek t cells were transfected with gfp-tagged irf and flag-tagged myd (human or bat, as indicated). at h post-transfection, whole cell lysate was prepared and immunoprecipitated with anti-flag antibody m . immunoprecipitated complexes (ip) were analysed by immunoblot for irf and myd expression using an anti-human irf antibody (top panel), and an anti-flag antibody m for detection of flag tagged myd (middle panel). whole cell lysate ( ul) was also run on an sds-page gel and subsequently analysed for irf expression using anti-human irf antibody (bottom panel). (b) bat irf co-localizes with bat myd . pakit cells were transfected with bat gfp-irf /irf and bat flag-myd and hek t cells were transfected with human gfp-irf and human or bat flag-myd . hours post-transfection (or h), cells were fixed for indirect immunofluorescence assay using an anti-human myd antibody and red fluorescence-conjugated secondary antibody. pictures show co-localisation of bat irf or bat irf with bat myd in pakit cells and human irf or human irf with human myd in hek t cells. doi: . /journal.pone. .g ifn-ar, a complex consisting of activated stat , stat and irf , called the ifn stimulated gene factor (isgf ) is formed, which in turn binds to the isre on the irf promoter and induces irf transcription. the human irf promoter region contains an nf-kb binding site and a single functional isre approximately . -kb upstream from the atg start site, both of which are important in the induction of irf [ , ] . analysis of the putative bat irf promoter region resulted in the identification of two isres and one nf-kb binding site indicating that multiple mechanisms for irf activation may also exist in bats. however, two isre sites were also identified in the irf promoters of other species examined (mouse, horse, cow, dog, cat and rat). thus, whether the broad distribution of constitutively expressed irf is the result of the presence of a more efficient irf promoter region driven by transcription factors other than irfs, or simply due to enriched immune-related cells in all tissues will require further study. sequence differences in the myd binding domain of bat and human irf led to the hypothesis that there may be functional differences in the activation of bat irf and the regulation of the ifn response that may contribute to the ability of bats to resist the clinical outcomes of viral infection. our results demonstrate that these sequences differences do not appear to affect irf function either in ifn transactivation activity or activation by myd . bat irf was capable of activating both ifn-a and ifn-b promoters and the levels of transactivation were equivalent to or higher than that of human irf . similarly, bat myd and bat irf maintained binding capability similar to their human counterparts. deletion of the myd -binding region of bat irf impaired its ability to activate ifn, demonstrating functional conservation of the myd binding domain with that of human irf . collectively, these data demonstrate that bat irf is capable of inducing ifn and myd binding in a similar manner to human irf . although the myd binding domain of bat irf has low sequence conservation with the equivalent region in human irf , experimental data demonstrate a fully functionally irf exists in pteropid bats. our results describing the experimental knockdown of irf using sirna is to our knowledge the first description of the use of sirnas in bat cells. the successful knockdown of irf is consistent with the presence of an rna-silencing mechanism in bats similar to that in other mammals. irf knockdown resulted in impaired ifn-b induction in sev infected cells and enhanced pulv replication. although further work will be required to determine whether irf is the master regulator of the bat ifn response, these results confirm that irf plays an important role in anti-viral defense and the early innate immune response in bats [ ] . analysis of the bat ifn-b promoter also revealed one residue difference in the prdi module, known to be associated with activation by irf or irf [ ] . a similar change in the human ifn-ap impairs its inducibility by irfs [ ] . although bat ifn-b is strongly inducible in bat cells following either stimulation or viral infection, further work will be necessary to determine whether this mutation affects the induction of ifn-b under conditions other than those described here [ ] . the presence of this mutation may also indicate that transcription factors other than irf and irf are involved in the regulation of ifns in bats. therefore, future work focusing on ifn promoters (including ifn-a and ifn-l) will be necessary to explore whether the bat irf-ifn induction pathway is as critical to ifn induction as it is in other species. in humans, there are around transcription factors that have been recognized [ ] . whether these factors play similar roles in bats or whether they perform different functions resulting in differences in the expression of downstream genes remains to be determined. understanding the mechanisms responsible for the regulation of the ifn response will assist in discovering how bats successfully coexist with viruses. in conclusion, our data clearly demonstrate that bat irf exhibits a constitutive expression pattern across a broad range of immune and non-immune related organs, intact ifn transactivation function, and binds to and is activated by bat myd . these findings not only provide the first information on the myd -irf dependent ifn production pathway in bats, but also explore the functionality of bat irf and identify critical point mutations in the bat ifn-b promoter. ultimately, we hope these advances may help uncover the mechanisms underlying the ability of bats to co-exist with deadly viruses, and that this will in turn lead to the development of potential therapeutic strategies targeting viral infections in other mammals. severe respiratory illness caused by a novel coronavirus nipah virus: a recently emergent deadly paramyxovirus isolation and characterization of a bat sars-like coronavirus that uses the ace receptor isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus fruit bats as reservoirs of ebola virus public health awareness of emerging zoonotic viruses of bats: a european perspective bats: important reservoir hosts of emerging viruses antiviral immune responses of bats: a review interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures the irf family, revisited type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors irf- , a new interferon regulatory factor associated with epstein-barr virus latency irf- is the master regulator of type-i interferon-dependent immune responses ifn regulatory factor family members differentially regulate the expression of type iii ifn (ifn-lambda) genes positive feedback regulation of type i ifn genes by the ifn-inducible transcription factor irf- molecular cloning and characterization of interferon regulatory factor molecular cloning and characterization of crucian carp (carassius auratus l.) interferon regulatory factor gene structure and transcription of irf- and irf- in the mandarin fish siniperca chuatsi gene structures and promoter characteristics of interferon regulatory factor (irf- ), irf- and irf- from snakehead channa argus regulation and function of interferon regulatory factors of atlantic salmon interferon-alpha induction through toll-like receptors involves a direct interaction of irf with myd and traf multiple regulatory domains control irf- activity in response to virus infection ikappab kinase-alpha is critical for interferon-alpha production induced by tolllike receptors and involvement of the ubiquitin-like domain of tbk /ikk-i kinases in regulation of ifn-inducible genes differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor- the role of differential expression of human interferon-a genes in antiviral immunity interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures silencing of irf pathways in breast cancer cells promotes bone metastasis through immune escape bat mx and oas , but not pkr are highly induced by bat interferon and viral infection molecular characterisation of toll-like receptors in the black flying fox pteropus alecto molecular characterisation of rig-i-like helicases in the black flying fox, pteropus alecto type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity comparative analysis of bat genomes provides insight into the evolution of flight and immunity establishment, immortalisation and characterisation of pteropid bat cell lines mega : molecular evolutionary genetics analysis (mega) software version . phosphorylation-induced dimerization of interferon regulatory factor unmasks dna binding and a bipartite transactivation domain suppressor of cytokine signaling negatively regulates toll-like receptor signaling by mediating mal degradation the immune gene repertoire of an important viral reservoir, the australian black flying fox comparative analysis of bat genomes provides insight into the evolution of flight and immunity role of a transductional-transcriptional processor complex involving myd and irf- in toll-like receptor signaling type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity regulation of virusinduced interferon-a genes evidence for a nuclear factor(s), irf- , mediating induction and silencing properties to human ifn-beta gene regulatory elements sendai virus defective-interfering genomes and the activation of interferon-b pulau virus; a new member of the nelson bay orthoreovirus species isolated from fruit bats in malaysia regulation of myd aggregation and the myd -dependent signaling pathway by sequestosome and histone deacetylase distinct roles of tir and non-tir regions in the subcellular localization and signaling properties of myd irf : activation, regulation, modification and function type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto regulation of the promoter activity of interferon regulatory factor- gene. activation by interferon snd silencing by hypermethylation a census of human transcription factors: function, expression and evolution we thank drs prasad paradkar and glenn marsh for critical review of the manuscript. key: cord- - qh dsew authors: stegelmeier, ashley a.; van vloten, jacob p.; mould, robert c.; klafuric, elaine m.; minott, jessica a.; wootton, sarah k.; bridle, byram w.; karimi, khalil title: myeloid cells during viral infections and inflammation date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qh dsew myeloid cells represent a diverse range of innate leukocytes that are crucial for mounting successful immune responses against viruses. these cells are responsible for detecting pathogen-associated molecular patterns, thereby initiating a signaling cascade that results in the production of cytokines such as interferons to mitigate infections. the aim of this review is to outline recent advances in our knowledge of the roles that neutrophils and inflammatory monocytes play in initiating and coordinating host responses against viral infections. a focus is placed on myeloid cell development, trafficking and antiviral mechanisms. although known for promoting inflammation, there is a growing body of literature which demonstrates that myeloid cells can also play critical regulatory or immunosuppressive roles, especially following the elimination of viruses. additionally, the ability of myeloid cells to control other innate and adaptive leukocytes during viral infections situates these cells as key, yet under-appreciated mediators of pathogenic inflammation that can sometimes trigger cytokine storms. the information presented here should assist researchers in integrating myeloid cell biology into the design of novel and more effective virus-targeted therapies. the ability of the immune system to recognize invading pathogens and tissue damage, and subsequently respond in a targeted and reproducible manner bestows longevity to our existence. within the diverse cellular network of the immune system, recent research has shown that myeloid cells deserve new-found attention due to their ability to detect and mitigate viral infections and promote inflammation. upon viral infection, there are a number of myeloid cell subsets that play various roles in the subsequent inflammatory, cellular, and humoral responses. myeloid cells are granulocytic and phagocytic leukocytes that traverse blood and solid tissues. when they recognize virus-infected cells or tissues damaged by viruses, these sentinels rapidly initiate an innate immune response [ ] . this multifaceted response involves cellular activation [ ] , signaling cascades [ ] , and the release of cytokines [ ] to guide leukocytes to mount an effective response. evidence is accumulating that two myeloid cell subsets, in particular, are playing a larger role in recognizing and halting viral infections than was previously thought. researchers are discovering that both neutrophils and inflammatory monocytes are intertwined in the immune system's anti-viral response. moreover, they play unique immuno-regulatory roles post-infection, and are critical for restoring homeostasis. neutrophils are the most abundant leukocyte subset in mammals, ranging from - % of white blood cell counts [ ] . they are responsible for both pro-inflammatory and anti-viral responses, and, therefore, constitute a first line of defense against invading pathogens and cell damage [ ] . neutrophils are effector innate cells that live for a relatively brief five days [ ] and exist in one of three states: quiescent, primed, or active. although they are predominately considered cells that target extracellular organisms such as bacteria via phagocytic uptake, their control of other cell subsets enables them to play important indirect roles in clearing viral infections and modulating inflammation. monocytes are large mononuclear leukocytes that are involved with the inflammation and clearance of pathogens. these non-dividing cells are able to further differentiate into other myeloid subsets such as dendritic cells (dcs) and macrophages. monocytes constitute a heterogeneous population that is endowed with a high degree of plasticity, allowing them to respond to environmental cues in tissues. current research is uncovering the role that inflammatory monocytes play during inflammation and viral infections. this subset preferentially traffics to inflamed regions, where they secrete inflammatory cytokines [ ] . however, they can also function as regulatory cells [ ] . for example, alveolar macrophages have been shown to recruit inflammatory monocytes through a type i interferon (ifn)-mediated mechanism [ ] . these monocytes can then provide protection against virus-induced pathology. evidence exists that both neutrophils and monocytes can contribute to viral clearance or exacerbate pathological damage depending on the context of the infection ( figure ). in terms of myeloid cells contributing to virus-induced pathologies, a linkage can be made between the induction of cytokine storms and dysregulated type i ifn responses. in cases where these cells are beneficial, they can be therapeutically boosted, whereas they can also be depleted when viruses have commandeered them towards destructive fates. exploring the pronounced involvement that myeloid subsets have in mitigating viral replication and pathology, therefore, has the potential to create novel therapeutics that are more efficacious against viral infections. the aim of this review is to explore recent advances in our understanding of the roles that neutrophils and inflammatory monocytes play during viral infections. although previous reviews have provided comprehensive coverage on the impact that these myeloid subsets have during bacterial infections [ , , ] , there is no current review with an extensive focus on their contributions to mitigating viral infections. further, this review has a novel focus on the expanding literature discussing the regulatory roles of these cell types during viral infections, as well as a possible link between the virus-mediated blockade of type i interferon signaling and virus-induced cytokine storms. figure . schematic of myeloid cells highlighting their ability to respond to pulmonary viral infections via the initiation and modulation of anti-viral inflammatory activity. lung-resident myeloid cells, such as alveolar macrophages, utilize a complex sensory system to integrate disturbances of pulmonary tissues by viruses such as respiratory syncytial virus (rsv) into the activation of local effector leukocytes. (a) rsv enters and infects the lungs. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna or danger associated molecular patterns (damps), are detected by pattern recognition receptors (prrs) in or on sentinel cells in the lungs, such as tlr in the endosomes of lung-resident macrophages. tlr stimulation activates the nf-κß signaling cascade, resulting in the release of chemokines and inflammatory cytokines. a chemokine gradient forms between the lungs and bone marrow. (b) homeostatic bone marrow tends to retain cxcr + neutrophils and monocytes through endogenous expression of high levels of cxcl . however, the release of pamps, as well as the secretion of cytokines and chemokines as a consequence of pulmonary rsv infections, is sensed by cells in the bone marrow, which in turn allow recruitment of new neutrophils and monocytes from the bone marrow into the lungs. specifically, g-csf downregulates cxcr on neutrophils, triggering their release. similarly, ccl is produced in the bone marrow by endothelial cells following tlr signaling in infected lungs, which is crucial for inflammatory monocyte release into the bloodstream. once in the bloodstream, these cells sense disrupted endothelium from the viral infection, which triggers a complex adhesion cascade. activated ly c hi inflammatory monocytes are recruited to the site of infection by a variety of chemokine receptors including ccr , and , as well as cxcr binding to their respective ligands. (c) once at the site of infection, they differentiate into dendritic cells and macrophages that initiate an inflammatory cascade that includes copious amounts of inflammatory cytokines, in particular il- and ifn-γ, which are potent inducers of th -biased immune responses. once these dendritic cells and macrophages acquire viral antigens, they home to lymph nodes via chemokine receptors, including ccr . monocyte-derived dendritic cells that home to lymph nodes present viral antigens to naïve cd + and cd + t-cells that are required to kill infected cells. (d) the basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement. neutrophils are also capable of promoting tissue repair and increased angiogenesis. further, monocytes can suppress lymphocytes in various clinical scenarios. in lungs, myeloid cells are able to inhibit pro-inflammatory tissue-resident leukocytes through direct cell-to-cell contact through galectin /tim and the effect of tgf-β on nkp in order to regulate tcells and nk cells, respectively. myeloid cells can also exert suppressive functions through secretion of soluble factors such as il- , arginase- and indoleamine , -dioxygenase. (e) we speculate that disruption of the cellular sensing of type i ifn responses can result in excessive production of proinflammatory cytokines, including ifn-γ, il- , il- , and tnf-α, leading to a toxic cytokine storm. figure . schematic of myeloid cells highlighting their ability to respond to pulmonary viral infections via the initiation and modulation of anti-viral inflammatory activity. lung-resident myeloid cells, such as alveolar macrophages, utilize a complex sensory system to integrate disturbances of pulmonary tissues by viruses such as respiratory syncytial virus (rsv) into the activation of local effector leukocytes. (a) rsv enters and infects the lungs. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna or danger associated molecular patterns (damps), are detected by pattern recognition receptors (prrs) in or on sentinel cells in the lungs, such as tlr in the endosomes of lung-resident macrophages. tlr stimulation activates the nf-κß signaling cascade, resulting in the release of chemokines and inflammatory cytokines. a chemokine gradient forms between the lungs and bone marrow. (b) homeostatic bone marrow tends to retain cxcr + neutrophils and monocytes through endogenous expression of high levels of cxcl . however, the release of pamps, as well as the secretion of cytokines and chemokines as a consequence of pulmonary rsv infections, is sensed by cells in the bone marrow, which in turn allow recruitment of new neutrophils and monocytes from the bone marrow into the lungs. specifically, g-csf downregulates cxcr on neutrophils, triggering their release. similarly, ccl is produced in the bone marrow by endothelial cells following tlr signaling in infected lungs, which is crucial for inflammatory monocyte release into the bloodstream. once in the bloodstream, these cells sense disrupted endothelium from the viral infection, which triggers a complex adhesion cascade. activated ly c hi inflammatory monocytes are recruited to the site of infection by a variety of chemokine receptors including ccr , and , as well as cxcr binding to their respective ligands. (c) once at the site of infection, they differentiate into dendritic cells and macrophages that initiate an inflammatory cascade that includes copious amounts of inflammatory cytokines, in particular il- and ifn-γ, which are potent inducers of th -biased immune responses. once these dendritic cells and macrophages acquire viral antigens, they home to lymph nodes via chemokine receptors, including ccr . monocyte-derived dendritic cells that home to lymph nodes present viral antigens to naïve cd + and cd + t-cells that are required to kill infected cells. (d) the basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement. neutrophils are also capable of promoting tissue repair and increased angiogenesis. further, monocytes can suppress lymphocytes in various clinical scenarios. in lungs, myeloid cells are able to inhibit pro-inflammatory tissue-resident leukocytes through direct cell-to-cell contact through galectin /tim and the effect of tgf-β on nkp in order to regulate t-cells and nk cells, respectively. myeloid cells can also exert suppressive functions through secretion of soluble factors such as il- , arginase- and indoleamine , -dioxygenase. (e) we speculate that disruption of the cellular sensing of type i ifn responses can result in excessive production of pro-inflammatory cytokines, including ifn-γ, il- , il- , and tnf-α, leading to a toxic cytokine storm. the fatal outcome of severe lung infections is shown to be correlated with the early persistent production of inflammatory cytokines and chemokines that recruit neutrophils and monocytes. while inflammatory cytokines and chemokines are essential for effective control of viral infections, they can also contribute to the severity of disease and tissue damage. multiple progenitor cell types arise from self-renewing multi-potent hematopoietic stem cells that become committed in the bone marrow to lineage-specific myeloid cells of the immune system [ ] . clonogenic myeloid-primed precursors (cmps) give rise to myeloid cells, which then further differentiate into granulocyte/monocyte progenitors (gmps) [ ] . subsequently, gmps undergo multiple stages of differentiation before they are terminally differentiated into neutrophils or monocytes in the bone marrow [ ] . monocytes require the growth factor colony-stimulating factor- to develop [ ] and high levels of the transcription factor pu. to steer gmps to commit to a monocyte lineage [ ] . monocyte-dc progenitors (mdps) create descendants that are destined to become either dcs or monocytes [ ] . recent discoveries have expanded our understanding of neutrophil development. advances in isolation techniques have elucidated that neutrophils are derived from unique cd b + ly g lo ly b int cd − precursors that possess proliferation capabilities [ ] . indeed, transcriptional profiling coupled with mass cytometry has provided additional information on the process required for gmps to differentiate into neutrophils [ ] . researchers determined the bone marrow possesses three distinct subsets: the aforementioned proliferative precursor cells, as well as non-proliferative immature and non-proliferative mature neutrophils. precursors required the transcription factor c/ebpε to differentiate from gmps. as precursors further shift into non-proliferative populations, they exchange proliferation capacity for increases in effector function and migration [ ] . further experiments on neutrophil precursors demonstrated their ability to expand in the presence of cancers such as melanoma and suppress regulatory t cells [ ] . the role of precursor neutrophils during viral infections has not been determined and represents a novel avenue of research. once viruses such as influenza virus or respiratory syncytial virus (rsv) manage to infect a tissue ( figure a ), type i ifns are released from the infected cells and stimulate the expression of hundreds of genes [ ] , appropriately known as ifn-stimulated genes (isgs), in neighboring cells. this induces an antiviral state within minutes to hours that is characterized by reduced transcription and translation [ ] , the induction of enzymes that degrade viral rnas and proteins, and even the sensitization of cells to apoptosis [ ] . products of isgs, including cytokines and chemokines, also recruit leukocytes, including neutrophils and monocytes to the virally infected tissue [ ] . the induction of the ifn response following viral infections fundamentally changes the bone marrow microenvironment ( figure b) , leading to the enhanced differentiation of myeloid cells [ ] and emigration of neutrophils and monocytes to the site of infection, which is facilitated by chemokine gradients interacting with their cognate receptors ( figure a ) [ ] . murine ly c hi monocytes originate from the bone marrow and travel to sites such as skin, lungs, and lymph nodes [ ] , whereas ly c low monocytes typically scan vasculature and the endothelial cells lining the lumen for damage. the human counterparts to these subsets are cd + cd − and cd low cd + monocytes, respectively [ ] . monocytes require kruppel-like factor- to differentiate into inflammatory monocytes in vivo [ ] . a recent advance by yáñez and colleagues demonstrated that gmps and mdps can independently generate functionally distinct monocytes [ ] . gmps and mdps are both derived from cmp-flt + progenitors but differentiate into the above subsets when either the toll-like receptor (tlr)- agonist lipopolysaccharide (lps) or the tlr agonist cpg dna, respectively, are injected into mice. ly c hi monocytes can be derived from either subset [ ] . therefore, the innate pathogen-associated molecular patterns (pamps) and their cognate receptors, known as pattern recognition receptors (prrs), will dictate which monocyte subset is preferentially generated. infections can affect hematopoiesis and influence the proportions of cell subsets. human immunodeficiency virus (hiv) is capable of infecting bone marrow microvascular endothelial cells and provoking hematopoietic dysfunction [ ] . a plethora of regulatory signals required to differentiate and release myeloid cells, including granulocyte-colony-stimulating factor and interleukin (il)- , can be suppressed by hiv. human t-cell leukemia virus (htlv)- has recently been shown to infect several lineages of hematopoietic stem cells in addition to t-cells [ ] . both neutrophil and monocyte lineages were permissive to infection, as evidenced by the viral tax protein in neutrophils and the ability of monocyte progenitors to become infected. indeed, these infected monocytes were capable of differentiating into dcs and spreading the infection to t-cells. moreover, four subsets of neutrophils have been characterized in infants with viral respiratory infections [ ] . these subsets include suppressive, progenitor, mature, and immature neutrophils, which are present in the blood of infected individuals. however, cd high cd l low suppressive neutrophils were only observed in patients with bacterial co-infections. strikingly, the viral dysregulation of hematopoiesis can lead to numerous diseases [ ] . for example, the epstein-barr virus (ebv) causes infectious mononucleosis, characterized by a dramatic increase in white blood cells in the bloodstream. in rare instances, this virus can cause pancytopenia, which is a severe reduction in the number of platelets, red, and white blood cells [ ] . pancytopenia has also been found in patients who have contracted hepatitis c virus (hcv) [ ] . reducing the number of progenitor cells available to differentiate into lymphoid and myeloid cells may be a reasonably common viral strategy to avoid clearance by the immune system. the functional capacity of myeloid cells to respond to viral particles is influenced by the origin of their precursors. defining the molecular and cellular mechanisms underlying myeloid cell precursor development in viral illnesses will provide a better understanding of the susceptibility of patients to different viruses and the immunological events that may ultimately be exploited for therapeutic benefit. indeed, progenitor cells constitute a promising gene therapy target to treat hiv infections because they can differentiate into multiple cell lineages, all possessing a therapeutic transgene such as an anti-hiv ribozyme [ ] . other viral infections that, in theory, may be successfully treated by targeting hscs with gene therapies include viruses that dysregulate hematopoiesis, such as hcv or ebv. the human body relies on a robust innate sensory system to quickly eliminate many viruses. prrs are present in and on a variety of cells including neutrophils and monocytes to recognize pamps ( figure a ). tlrs are a subset of prrs that recognize pamps. there are multiple tlrs in and on neutrophils and monocytes that specifically recognize viral pamps or danger associated molecular patterns (damps) released from virus-damaged cells. the nucleic acid from rna and dna viruses constitutes a predominant source of viral pamps that can be recognized either via phagocytosis of cellular debris such as epithelial cells, or in cases where viruses infect myeloid cells. within endosomes, tlr recognizes dsrna from viruses (dsrna constitutes the genome of one family of viruses, but is also generated during the life cycle of many viruses) [ ] , ssrna is recognized by tlr and tlr [ ] , whereas tlr recognizes dna viruses while distinguishing from host dna [ ] . monocytes are activated via signaling through surface-bound tlr during varicella-zoster virus [ ] , measles [ ] , and type and herpes simplex virus (hsv) infections [ ] . tlr can recognize a wide range of viral pamps including the glycoproteins gb and gh/gl from hsv [ ] and hemagglutinin from measles [ ] . tlr stimulation after phagocytosis activates the nf-κb signaling cascade, resulting in the release of inflammatory cytokines such as tnf-α, il- , and il- from monocytes [ ] to control virus infections by direct antiviral mechanisms and the recruitment of other leukocytes. direct antiviral mechanisms of monocytes and neutrophils, including phagocytosis and oxidative burst, were reduced in patients who had contracted hcv and were taking ifn-based therapies [ ] . neutrophils also use tlrs to conduct anti-viral surveillance, and express ten out of eleven known human tlrs (they lack tlr ) [ ] . the endosomal tlr is essential for recognition of influenza viruses by neutrophils via sensing viral single-stranded rna when they phagocytose cell debris [ ] . lack of tlrs is associated with increased mortality during viral infections. for example, blocking tlr leads to increased mortalities associated with influenza virus infections by disrupting phagocytosis of infected cells [ ] . although influenza viruses do not contain lps, tlr activation is also involved with delaying fusion between lysosomes and phagosomes, thereby preventing virus entry, and thus has an additional role in innate immunity besides recognition of pamps [ ] . the multifaceted functions of tlrs should, therefore, be studied in greater detail to determine whether additional tlrs have unappreciated mechanisms to mitigate viral infections. another method for host recognition of viruses involves retinoic acid inducible gene-i (rig-i) and melanoma differentiation factor (mda ) [ ] . to clear viral infections, rig-i-like receptors and mda recognize cytosolic viral rnas via the helicase domain [ ] . in contrast to tlrs that are predominately present in leukocyte subsets, these receptors are ubiquitous in human cells. neutrophils and monocytes themselves can become infected by viruses [ ] and therefore possess cytoplasmic and endosomal mechanisms to recognize them, including rig-i and mda signaling cascades in the cytoplasm and endosomal tlrs. in fact, the double-stranded rna mimetic poly(i:c) stimulates neutrophils to increase many antiviral genes, including type i ifn mrna transcripts, ifn-responsive genes, tnf-α, and ifn regulatory factor (irf) [ ] . when infected with encephalomyocarditis virus (emcv), mda -deficient mice mount significantly reduced tnf-α and ifn-β responses [ ] . similar results were also observed after infections with coxsackie b virus (cvb) [ ] and west nile virus (wnv) [ ] . notably, tnf-α and ifn-β have the capacity to upregulate the expression of major histocompatibility complex molecules on antigen-presenting cells, which would make viruses more susceptible to t-cell-mediated clearance. damps are endogenous molecules that are released in response to tissue damage from trauma, including cells killed by viruses, and, like pamps, trigger an immune response ( figure a ). damps can be derived from a variety of cellular components, including the nucleus, cytoplasm, exosomes, plasma, or the extracellular matrix [ ] . damps that promote inflammation and immunogenic cell death [ ] include the chromatin protein high mobility group box (hmgb ) and mitochondrial damps such as mitochondrial dna and formyl peptides [ ] . hmgb interacts with neutrophils and monocytes [ ] by binding to the inflammatory receptor for advanced glycation end-products (rage). this damp causes monocytes to secrete pro-inflammatory cytokines, including il- and tnf-α, reorganize their cytoskeleton, and increases migration across epithelial barriers. monocytes are also capable of secreting hmgb themselves when lysosome exocytosis is induced by the inflammatory lipid lysophosphatidylcholine [ ] . neutrophils, in turn, have upregulated transcription of genes for pro-inflammatory molecules involving the nf-κb, p mapk, and erk / pathways in response to recognition of hmgb [ ] . cell damage from viral infections leads to a release of damps and subsequent detection by myeloid cells. for example, infection of epithelial cells with dengue viruses results in the release of hmgb from necrotic cells [ ] . the interaction between viral pamps and prrs in or on myeloid cells can play an essential survival role in the response to viral infections but may, simultaneously, be responsible for tissue injury associated with severe virus-induced inflammation. in theory, mechanisms involved in the recognition of danger signals by neutrophils and monocytes could be targeted selectively to enhance protection against detrimental viral infections while, simultaneously, preventing exaggerated, pathological innate immune responses. chemokines and their receptors play a critical role in dictating the migration and positioning of myeloid cells. an extensive list of chemokines, their receptors, and their various functions has been described [ ] . neutrophils and monocytes begin their journey to a site of infection by first leaving the bone marrow ( figure b ). neutrophil and monocyte retention in the bone marrow is dictated by steady signaling between the chemokine (c-x-c motif) receptor (cxcr ) and its ligand cxcl expressed on bone marrow stromal cells. during maturation, these cells downregulate cxcr and become less sensitive to cxcl , causing their release into the bloodstream [ ] . cxcr , and mainly cxcr expression, on neutrophils grants an additional form of chemotaxis away from the bone marrow via their respective ligands, cxcl and cxcl [ ] , which are produced by macrophages and mast cells at the site of infection [ ] . however, retention is typically favored in the steady state, as cxcl appears to be constitutively expressed in the bone marrow. inflammation mediated by viral infections that induce g-csf enhances cxcl release and decreases cxcr expression on bone marrow-resident neutrophils, tipping the balance in favor of neutrophil release [ ] . ly c hi inflammatory monocytes appear to require chemokine receptor (ccr ) signaling to efficiently exit the bone marrow and travel to sites of inflammation, whereas ccr signaling appears to be contextually dependent for monocyte emigration from circulation into virus-infected tissues [ ] . more research needs to be conducted to ascertain if ccr signaling is required to respond to viral infections of various tissues. ccr signaling, via ccl binding to the receptors on monocytes, causes the downregulation of cxcr and renders the monocytes less sensitive to cxc , causing their release from the bone marrow [ ] . interestingly, low concentrations of circulating tlrs cause rapid ccl release by mesenchymal stem cells and their progeny in the bone marrow, which triggers the release of monocytes [ ] . the dissemination of neutrophils and monocytes to virally infected tissues involves many complex processes ( figure b) [ , , ] . generally speaking, myeloid cell migration to infected tissues relies on transmigration through vascular endothelium from the blood. this transmigration is dictated by a milieu of cytokines, and chemokines produced by tissue injury and resident sentinel cells in response to damps and viral pamps. the disruption of homeostasis confers a change to the vascular endothelium near sites of infection [ ] . the multitude of changes to the endothelium can happen rapidly, and have been reviewed extensively elsewhere [ ] . in brief, endothelial changes that start and subside within minutes are known as type i activation and can be mediated by factors such as histamine [ ] ). alternatively, type ii activation can last hours to days with substantial changes in gene expression profiles mediated by tumor necrosis factor (tnf)-α [ ] . both forms of activation cause increased blood flow, vascular leakage of plasma proteins, and the recruitment of leukocytes [ ] . these disruptions in endothelium homeostasis can trigger a leukocyte adhesion cascade [ ] that, in harmony with various cytokines released by inflamed endothelium, such as il- and monocyte chemoattractant protein (mcp)- [ ] , initiates the selectin-mediated rolling of leukocytes along the surface of endothelial cells. trafficking of neutrophils and monocytes through the endothelium towards the site of infection is then facilitated by crawling via macrophage-antigen- (mac- /cd b) expressed on monocytes and the intercellular adhesion molecule- (icam- /cd ) expressed on endothelial cells [ ] . crawling appears to facilitate the paracellular (between cells) transmigration of neutrophils and monocytes, which is generally the preferred method of trafficking (occurring - % of the time), as opposed to transcellular (through cells) transmigration [ ] . the dissemination of neutrophils and monocytes from the vasculature into infected tissues is critical for viral clearance. neutrophils are initially recruited to sites of infection by their ability to recognize tissue damage via sensing of h o , dna, n-formyl peptides, adenosine triphosphate, uric acid, and other damps [ ] . further guidance to sites of infection is provided by a family of cxcl chemokines originating from concentrated sites of pamps and damps, including cxcl , cxcl , cxcl , cxcl , cxcl , cxcl , and cxcl (il- ), which are sensed by cxcr and cxcr on neutrophils and monocytes [ ] . within the context of viral infections, experimental data from mice infected with theiler murine encephalomyelitis virus have demonstrated that cxcl released from epithelial cells, macrophages, and neutrophils recruits both neutrophils and monocytes to sites of infection [ ] . macrophages infected with rotaviruses release cxcl to recruit neutrophils [ ] and nipah virus c protein is capable of inducing the release of numerous chemokines, including cxcl , cxcl , and cxcl , from endothelial cells [ ] . pamps from viruses tend to amplify neutrophil recruitment. the inflammatory ly c hi subset and the "patrolling" ly c low cx cr hi subset migrate along luminal and endothelial cell surfaces, with the latter being able to respond rapidly to infections in a cx cr -dependent fashion [ ] . the migration of inflammatory monocytes to tissues is ccr -dependent. however, as mentioned above, this tends to only be required to exit the bone marrow. nonetheless, ccr signaling appears to be critical for inflammatory monocyte recruitment in cases of west nile virus-induced encephalopathies, and influenza virus infections [ , ] . in summary, a range of trafficking signals and endothelial barrier regulatory molecules shape myeloid cell recruitment to virally infected and inflamed tissues. neutrophils are able to lyse and phagocytose virus-infected cells [ ] , and are one of the first leukocyte subsets to enter inflamed tissues ( figure c ). the magnitude of the neutrophil response is a predictor of the host's ability to clear an influenza virus infection with minimal damage [ ] . depleting neutrophils causes greater viral spread and host mortality [ ] , and neutrophils are also crucial to mitigate hsv type- corneal infections in a murine model [ ] . moreover, tate and colleagues demonstrated that neutrophils are critical for limiting the replication of influenza viruses [ ] and that a loss of neutrophils increases disease severity. thus, the antiviral response of neutrophils contributes to clearing viral infections. neutrophils can directly mediate innate immune responses, activate adaptive immunity and recruit lymphoid cells to sites of viral infections [ , ] . a key mechanism of action that enables neutrophils to neutralize invading viruses is the production of neutrophil extracellular traps (nets) [ ] . nets are strands of dna and granule proteins secreted by neutrophils that form around viral particles, preventing their spread [ ] . poxvirus infections in mice were mitigated in liver microvasculature via this mechanism [ ] . in addition to the physical containment of infections, nets are coated with antiviral enzymes that enable neutrophils to concentrate lethal antimicrobial proteins such as histones at sites of infection [ ] . neutrophils are also capable of mediating antibody-dependent cellular cytotoxicity (adcc) or antibody-dependent phagocytosis, which involve the release of cytolytic granules or phagocytosis, respectively, after binding antibodies via fc receptors [ ] . these antibody-dependent processes are critical in the clearance and neutralization of certain viruses such as hiv [ ] . adcc responses peak quickly (i.e., within four hours) and are controlled by the fcγr family of receptors and can also utilize the extracellular release of reactive oxygen intermediates [ ] . reactive oxygen intermediates are also involved in other pathological responses, including exocytosis. exocytosis is a cellular active transport process whereby membrane-bound vesicles transport molecules to the cell surface. neutrophils emit an array of compounds including myeloperoxidase to control sepsis [ ] , antiviral lysozyme with anti-hiv properties [ ] , and n-formyl-methionyl-leucyl phenylalanine (fmlf)-stimulated superoxide release in the presence of periodontitis pathogens [ ] . exocytosis, therefore, expands the neutrophil arsenal to neutralize the array of pathogens they encounter. neutrophils are incredibly diverse in their functions. in addition to trafficking to sites of infection to phagocytize viruses and form nets, they also stimulate virus-specific adaptive immune responses [ ] . neutrophils that have detected viral antigens can home to draining lymph nodes dependent on il- r, where they can act as antigen-presenting cells [ , ] . neutrophils present processed viral antigens to naïve cd + t-cells via the major histocompatibility complex i and t-cell receptor interactions, along with the expression of cd and cd to provide co-stimulation, thereby providing the two signals required to activate t-cells [ ] . furthermore, neutrophils are responsible for the recruitment of effector cd + t-cells to sites of viral infections. the mechanism by which they recruit t-cells during influenza virus infections has been linked to cxcl deposits left behind like a "trail of breadcrumbs". cd + t-cells follow this chemoattractant trail left behind by neutrophil uropods to the sites of influenza virus infections [ ] . rsv causes lung infections that are characterized by neutrophils contributing to host damage [ ] . rsv is capable of delaying the apoptosis of neutrophils and eosinophils, which is hypothesized to delay antigen presentation and increase tissue damage. il- and tlr / binding was determined to contribute to this delay and depended on nf-κb and pi k activation. the authors of this study did not directly examine whether this delay resulted in an increase in host tissue damage in their model, but hypothesized this was the case, constituting an area of future study. during an rsv infection, neutrophils migrate through infected airway epithelial cells [ ] . these neutrophils are characterized by the increased expression of myeloperoxidase and cd b, and their migration promotes epithelial shedding and airway tissue damage. aside from delaying apoptosis, rsv infection has also been shown to increase eosinophil recruitment and degranulation based on the macrophage inflammatory protein (mip) -α and eosinophil cationic protein concentrations measured in lower respiratory airway secretions [ ] . ly c hi monocytes migrate to injured sites, induce inflammation, and eliminate the cause of tissue injury ( figure c ) [ ] . for instance, type i ifns amplify the production of mcp- , the primary chemokine responsible for recruiting inflammatory monocytes to the lungs during influenza virus infections [ ] . these monocytes have been implicated in influenza virus-induced lung injury [ ] . importantly, elevated mcp- levels have been associated with severity of illness in pediatric influenza virus infections [ ] . in mice, the recruitment of monocytes to lungs was shown to be accompanied with an increase in type i ifn production, nlrp inflammasome activation, and alveolar epithelial barrier dysfunction [ ] . it has been identified that increased pro-inflammatory monocytes are a major immunological determinant of severity of disease in previously healthy adults with life-threatening influenza virus infections [ ] . this provides a possible mechanistic cause for disease severity in these patients, a potential early identifier and a modifiable immune pathway for therapeutic targeting. however, there is no role for recruited monocytes in the lungs of mice infected with the natural rodent pathogen, pneumonia virus of mice [ ] , indicating the pathogen-specific functions of these cells. interestingly, monocytes have been proposed to be educated in the bone marrow to promote their tissue-specific functions at sites of persistent challenge [ ] . long-lasting epigenetic alterations in monocyte precursors may account for the "trained immunity" phenomena [ ] . indeed, monocytes have an immunological memory of past insults. thus, this evidence shows that neutrophils and inflammatory monocytes participate in inflammation that is needed for an effective immune response against viruses. a shared feature of neutrophils and monocytes is their ability to synthesize pro-inflammatory cytokines that help the host overcome viral diseases. however, these responses can also be overly robust, thereby contributing to virus-induced tissue damage. future research directions should include a focus on furthering our understanding of the diverse antiviral arsenal of myeloid cells. robust immune responses are critical for protecting hosts against lethal viral infections. it is equally important that immune responses are of adequate magnitude and duration. the capacity for a host to resolve inflammation and return to homeostasis has important consequences for health ( figure d ). the induction of an immune response that is too severe or the failure to return to homeostasis can result in immunopathology [ ] , including tissue and organ damage [ ] , cytokine storms ( figure d ) [ ] , chronic inflammation [ ] , and autoimmune diseases [ ] . as innate immune responders, myeloid cells are key players in orchestrating appropriate inflammatory responses and the return to homeostasis following virus infections. the role of myeloid cells in the regulation of immune responses is complex and involves specialized cellular subsets, suppressive receptors, and cytokines. in addition, much of what we know about the regulatory and immunosuppressive effects of myeloid cells originates from research investigating bacterial, fungal, and sterile inflammation models, but has implications for virus infections. neutrophils possess multiple mechanisms to control inflammation, despite their predominately pro-inflammatory role ( figure d ) [ ] . one mechanism involves the formation of aforementioned nets [ ] . these nets function via serine proteases to degrade excess cytokines and chemokines in areas with high densities of neutrophils [ ] . neutrophils are also capable of reducing lung injury during influenza virus infections [ ] . a neutrophil depletion study in a h n murine model demonstrated that their absence led to weight loss, viremic spread, and increased inflammation. the basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement [ ] . they are also capable of healing mucosal regions of the intestine [ ] , and increasing angiogenesis [ ] . a recent advance in our knowledge of neutrophils concerns their ability to de-prime [ ] . originally considered an irreversible process, neutrophils are capable of returning to quiescence. neutrophils can be spontaneously de-primed in the circulatory system via the degradation of a superoxide anion response [ ] , with a de-priming half-life of approximately forty minutes [ ] , or retained in the bone marrow [ ] to limit the number of primed cells that can traverse the body and cause damaging effects such as lung injury [ ] . recent experimental data have demonstrated that inflammatory monocytes are capable of exhibiting suppressive properties. inflammatory monocytes are recruited to sites of vaccine-mediated inflammation via mcp- [ ] . within the vaccine draining lymph node, monocytes sequester cysteine, resulting in t-cell suppression [ ] . blocking monocyte suppression in this context may prove to be an effective mechanism to improve vaccine effectiveness. monocytes are also capable of suppressing b cells. in vitro studies have demonstrated that monocytes suppress b cell differentiation, proliferation, and ig class distribution [ ] . monocytes, therefore, represent a prime example of a cell type that can be both pro-inflammatory and suppressive, depending on the context. the resolution of immune response is an active regulatory process, which is initiated via the release of soluble mediators such as cytokines and chemokines, as well as through cell-to-cell interactions mediated by surface-expressed ligands and receptors [ ] . evidence has revealed that monocytes that are part of inflammation also can be reprogrammed to cells that are highly anti-inflammatory and contribute to resolution of inflammation [ ] . moreover, during sepsis, human monocytes have been shown to undergo a transition from a pro-inflammatory to an anti-inflammatory status [ ] , although it remains unclear whether the conversion of monocytes from pro-inflammatory to a regulatory phenotype occurs in viral diseases. further studies are needed to understand the mechanisms to explain how monocytes can be switched into suppressor/anti-inflammatory cells during a viral infection, which in turn would allow intervention with targeted therapeutics to control and down-modulate excessive inflammation in viral diseases. an additional myeloid subset of interest is the myeloid-derived suppressor cells (mdscs). mdscs can suppress immune responses in numerous anatomical locations, including tumor microenvironments, virally infected tissues, and sites of inflammation. the subset of mdscs with neutrophil-like properties have been designated polymorphonuclear (pmn)-mdscs or granulocytic (g)-mdscs, while their myeloid counterparts have the nomenclature m-mdscs. viral infections can induce mdscs, as is the case with hcv [ ] . cd + mdscs were upregulated upon co-culture with hcv infected hepatocytes, resulting in t cell suppression mediated by reactive oxygen species. moreover, nk cells are also suppressed by mdscs during hcv infection [ ] . the production of mdsc-derived arginase- resulted in a decrease in ifn-γ production by nk cells. the suppression of key effector cells contributes to viral persistence. hcv is not the only virus to control mdscs to evade the immune system. patients with hiv- have m-mdsc populations that suppress helper t cells [ ] , and elevated levels of these myeloid cells were correlated with increased viral loads. future research should focus on determining whether other viruses engage mdscs to prolong infections. additionally, more research is required to fully determine the position these subclasses have in myeloid cell differentiation. although a recent review concluded that mdscs constitute bona fide alternate lineages [ ] , future studies will be required to cement their status within the field of immunology. myeloid cells are able to translate micro-environmental cues into an effector profile that initiates lymphocyte responses [ ] . innate lymphoid cells (ilcs) react to pathogens indirectly through myeloid or epithelial cell-derived cytokines and other inflammatory mediators including il- , il- , and il- [ ] . ilcs are derived from a lymphoid progenitor but do not contain either a b or t-cell receptor due to the absence of the recombination-activating gene [ ] . there are three major subsets of ilcs: groups , , and . group includes cells that produce ifn-γ and tnf-α and is predominately composed of classical natural killer (nk) cells. ilcs that require gata and rorα to develop and express the cytokines il- and il- are denoted as group , while intestinal ilcs that express nkp and depend on rorγ comprise group [ ] . since evidence shows that ilcs are tissue-resident cell types with limited capacity to directly recognize pamps [ ] , myeloid cells may play a crucial role in controlling ilc homeostasis and function [ ] . in the steady state, monocytes enter tissues and replenish macrophages and dcs [ ] . however, during viral infections they are recruited to infected tissues and mediate direct antiviral activities [ ] . for instance, in mice infected with murine cytomegalovirus, inflammatory monocytes are recruited to the liver and produce mip- a, which recruits nk cells [ ] . nk cells are relevant to viral infections because they target infected cells for destruction. nk cells are cytotoxic ilcs that require il- to develop, differentiate, and survive [ ] . il- is secreted by several cell types, including monocytes after viral recognition [ ] , which therefore places nk cells under the control of myeloid cells. expression of the activating receptor nkg d is upregulated on nk cells in response to il- . il- -activated nk cells show preferential expression of the tnf-related apoptosis-inducing ligand (trail) as well as activation and phosphorylation of erk and , and increases in perforin production [ ] . the increased expression of these activating receptors and effector compounds increases the killing potential of nk cells. many viruses down-regulate the expression of mhc on infected cells to escape detection by cd + t-cells [ ] . therefore, il- secretion by monocytes constitutes a mechanism to upregulate multiple cell receptors. changes in granzyme regulation were not documented in these studies, but represent an area of future investigation due to the role of this compound in the apoptosis of virus-infected cells. human monocytes express membrane-bound il- constitutively, with its expression increased in the presence of ifn-γ [ ] . the monocyte-mediated production of il- was increased in the presence of the anti-inflammatory cytokine il- , but was unaffected by il- or il- [ ] . il- also influences monocytes and can transform them into dcs in airway epithelia [ ] , which has implications for improving the presentation of viral antigens, suggesting a cross-talk between nk cells and myeloid cells under viral inflammatory conditions. recently, ashkar and colleagues [ ] showed that type i ifns produced during a viral infection stimulated vaginal mcp- production, which is a chemoattractant that is responsible for inflammatory monocyte migration to inflamed sites. once recruited, type i ifns stimulate inflammatory monocytes to produce il- , which then signals through the il- receptor expressed by nk cells to induce their production of ifn-γ. interestingly, cytokine il- also promotes the secretion of ifn-γ by nk cells [ ] and neutrophils [ ] . neutrophils can also increase ifn-γ production by nk cells using multiple pathways. the first method is to interact with dcs via icam- to further upregulate il- p [ ] , creating a positive feedback loop. the direct co-stimulation of nk cells also occurs with cd and icam- binding on neutrophils and nk cells, respectively [ ] . our unpublished data (personal observation by karimi k and bridle b) have demonstrated that the induction of viremia in mice, which induces the release of high concentrations of inflammatory cytokines into the circulation, is accompanied by increased numbers of pulmonary ilc subsets and the accumulation of multiple myeloid cell subsets that, interestingly, were type i ifn-dependent (data not shown). additionally, we demonstrated that the induction of inflammation by concanavalin a in mice, which occurs due to macrophage activation downstream of the rapid stimulation of t-cells, led to increased numbers of ilc populations in all organs examined, including the bone marrow, spleen, and liver [ ] (unpublished data). recently, mortha and burrows [ ] discussed how the feedback communication between ilcs and myeloid cells contributes to stabilize immunological homeostasis. further studies are needed to dissect cell-to-cell interactions between myeloid cells and ilcs other than nk cells in viral inflammatory conditions. the concept that neutrophils can initiate, amplify and/or suppress adaptive immune effector responses by establishing direct bidirectional cross-talk with t-cells has garnered attention in the past few years [ ] . a th response can be induced by neutrophils in a murine model [ ] , which increases the number of cd + cytotoxic t-cells available to lyse virally infected cells. indeed, in vivo murine studies have demonstrated that neutrophils can cross-present ovalbumin to cd + t-cells in a tap-and proteasome-dependent manner [ ] . neutrophils can further impact the adaptive immune response by inducing dc maturation, which in turn increases antigen presentation to adaptive cells [ ] . neutrophils have been observed to cluster with immature dcs and bind their mac- to dc-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign). dc-sign is also referred to as cd and is a prr that recognizes and binds to mannose residues, a conserved pamp associated with a variety of viral infections. however, neutrophil depletion studies have demonstrated an increase in antigen presentation to cd + t-cells. the mechanism by which this phenomenon occurs is thought to be a reduction in competition for viral antigens between neutrophils and dcs [ ] . there are extensive demonstrations that neutrophils in humans and mice can also suppress t-cell responses ( figure d ). suppressive neutrophils that express low levels of cd l are induced after acute inflammation arising from either viral infections or tissue injury [ ] . they have been shown to impair t-cells by releasing hydrogen peroxide into an immunological synapse, which impairs t-cell migration via the cxcl chemokine gradient. ball and colleagues have shown that cxcl -induced migration to sites of infection decreases as the concentration of hydrogen peroxide released into the immunological synapse is increased. results demonstrate the impaired recruitment of th and cd + t-cells to the periphery. ultimately, the mechanistic consequence pertains to defective migration mechanisms rather than tcr:mhc signal transduction. it is also important to note that this interaction required mac- (cd b). additional research has demonstrated that mac- -expressing neutrophils are crucial in limiting pathology caused by t-cells in a murine model of infection with influenza virus, presumably by suppressing t-cell proliferation [ ] . we have demonstrated that a subset of neutrophils function as negative regulators of excessive cytokine production in a mouse model of viremia, in which type i ifn signaling has been disrupted (karimi k and bridle b, unpublished data). altogether, these findings allow us to envision the therapeutic potential of subsets of neutrophils. however, one of the major challenges would be the heterogeneity of immunosuppressive or regulatory neutrophils. future studies taking advantage of flow cytometry technology and next-generation sequencing to phenotypically and functionally define neutrophil subsets will extend our knowledge about the immunoregulatory role neutrophils play in viral infections and inflammation. neutrophils also have an indirect mechanism to modulate t cells during a viral infection. the bacteria mycobacterium tuberculosis is capable of delaying neutrophil apoptosis, which delays an adaptive cd + t-cell response [ ] . although this has not been demonstrated via a viral infection, it nonetheless demonstrates a key effect neutrophils have on controlling a cd + t helper cell response. this response may be delayed because dcs ingest whole infected neutrophils [ ] to acquire antigens and present them to t-cells. additionally, dcs that ingest neutrophils possessing pathogen-derived antigens can migrate to lymph nodes more efficiently [ ] . the differentiation of inflammatory monocytes into cd b + pulmonary dcs is triggered by the presence of respiratory viruses such as influenza virus [ ] . defects in this differentiation delay the clearance of influenza viruses and significantly reduce the activation of cd + t-cells [ ] . while inflammatory monocytes are key regulatory cells in maintaining macrophage and dc populations in healthy tissues, a function of homeostasis, they are quintessential in the clearance of infections due to their ability to induce adaptive immunity and prime a variety of lymphocytes, including t-cells ( figure c ) [ ] . upon viral infection, inflammatory monocytes in the blood are recruited to the primary site of infection or the draining lymph node. cells that traffic to the primary site of infection play a critical role in the recruitment of t-cells and, thereby, the activation of inflammatory responses and cellular immunity [ ] . however, inflammatory monocytes that traffic to draining lymph nodes acquire a dc phenotype that enables them to present viral antigens to naïve t-cells [ ] . in particular, studies have shown that inflammatory monocytes stimulate a th -biased immune response via production of il- that promotes production of ifn-γ by t cells primed in lymph nodes [ ] . this th immunity is critical in the defense against intracellular pathogens, such as viruses [ ] . although memory is traditionally considered a hallmark of the adaptive immune response, recent advances have shed light on the contributions of innate memory. innate memory, also referred to as trained immunity, is a multifaceted response. a recent component of trained immunity involves its modulation of hematopoiesis [ ] . although myeloid cells have a short lifespan in circulation, the administration of the agonist ß-glucan resulted in myeloid progenitor expansion and subsequent improved responses to a secondary challenge with the agonist lps. trained immunity was able to reduce myelosuppression from chemotherapy, and was associated with metabolic shifts in cholesterol biosynthesis and glucose metabolism [ ] . other benefits of innate myeloid memory have been elegantly reviewed by netea and colleagues [ ] . in brief, monocytes are influenced by vaccination and viral infections, and are more responsive upon re-challenge. this innate memory response helps mitigate pathogens via upregulated cytokine production and enhanced pathogen elimination response times. this exciting new field may allow vaccines to be optimized for viruses by targeting the innate memory response. clearly, the cross-talk that is occurring between monocytes, neutrophils, and t-cells constitutes a crucial bridge between innate and adaptive immunity. future investigations are encouraged to examine the full extent of communication between these cells, further elucidate the mechanisms, and the anatomical locations of these interactions. depletion assays will be beneficial to determine which cell subsets can mount effective anti-viral responses, not just by t-cell and apc interactions, but also by direct interactions with neutrophils and monocytes. extensive studies have highlighted the role type i ifns play in initiating an anti-viral state in cells through the inhibition of viral replication [ ] . in some cases, the disruption of this response results in the excessive production of cytokines, leading to a so-called cytokine storm that can be very toxic ( figure e ) [ ] . this is a cause of mortality in cases of severe acute respiratory syndrome (sars) [ ] , infection with some strains of influenza viruses [ ] , ebola virus [ ] , and dengue virus [ ] . during viral infections, the regulation of cytokine networks and the mechanisms by which the cytokines may interact with neutrophils and monocytes are poorly documented. the fatal outcome of severe influenza infections is shown to be correlated with the early persistent production of inflammatory cytokines and chemokines that recruit neutrophils and monocytes [ , ] . lethal outcomes of h n influenza infections in humans correlated with early excessive innate immune response, involving type i ifns followed by prolonged inflammatory responses, and were associated with high viral loads and hypercytokinemia [ , ] . while inflammatory cytokines and chemokines are absolutely essential for the effective control of viral infections, they can also contribute to the severity of disease [ , ] . other fatal viral infections that are hallmarked by dysregulated type i ifn responses and cytokine storms are hantaviruses [ ] and wnv [ , ] . given the dynamic nature of cytokines, the complexity of signaling pathways they interact with, and the fact that their excessive production is often associated with some of the worst clinical outcomes of viral infections, there is a need for much more research into the mechanisms by which virus-induced cytokine storms are triggered or controlled. investigation into the mechanisms involved in host responses to viral infections demonstrates a complex and carefully balanced interaction between type i ifns and inflammatory neutrophils and monocytes. recent analysis of mrnas in the blood of humans responding to infections with influenza viruses revealed that early gene expression patterns of anti-viral molecules, such as the genes encoding for myxovirus resistance protein- (mx ) and isg- , are correlated with the heightened production and activation of type i ifns after viral infections [ ] . late gene expression patterns were also induced by type i ifns, but in contrast to patterns of antiviral molecules being observed, the transcriptional profiles of patients in the late stages of infections were highly reflective of neutrophil and inflammatory molecule activation [ ] , suggesting an important interplay between the secretion of type i ifns and the activation of neutrophils and inflammatory monocytes. it is important to study the receptors mediating the neutrophil antiviral response to reduce aberrant host responses and damage. nlrp is a nucleotide-binding domain leucine-rich repeat protein that is expressed on blood-derived leukocytes, including monocytes, and modulates neutrophil recruitment by increasing the chemokine cxcl through the il- -nlrp axis and increasing vascular permeability [ ] . another activator and recruiter of neutrophils is produced by liver cells and is entitled serum amyloid a (saa) [ ] . injections of saa increased phagocytosis of influenza viruses by neutrophils, resulting in the release of il- . modulating these protein concentrations might represent a promising therapeutic strategy to achieve ideal neutrophil responses to promote elimination of influenza viruses without excessive bystander damage to tissues. neutrophil-mediated antiviral responses have varying effects on the outcome of influenza virus infections, depending on the strain of virus [ ] . neutrophils contributed to terminating infections with h n influenza virus strains of intermediate virulence and h n strains that were highly virulent, while they did not limit the severity of disease during infection with an h n strain of low virulence. the early production of virus-induced type i ifns has been observed to upregulate genes in neutrophils that encode pro-apoptotic molecules, such as ifn-induced dsrna-activated protein kinase, and the oligoadenylate synthase-like proteins and the rnase l system [ ] . experiments with irf- -/x irf- -/double-knockout mice and wnv [ ] concluded that the viral induction of cellular ifn-β secretion depends on interferon-β promoter stimulator- -mediated signaling without requiring the ifn transcription factors irf / , suggesting the essentiality of the immediate and optimal activation of the type i ifn response. sars-coronaviruses are highly pathogenic and cause alveolar damage, fibrin deposition, and tissue necrosis [ ] . the delayed expression of the type i ifn response in mice infected with sars-coronaviruses was implicated in the promotion of inappropriate and chronic inflammatory responses, such as excessive inflammatory monocyte, neutrophil and cytokine accumulation, and impaired virus-specific t-cell responses due to augmented t-cell apoptosis, leading to lung damage [ ] . in contrast, an early type i ifn response reduced the immunopathological damage observed, linking the early activation of the type i ifn response to the control of overly robust inflammation. additionally, type i ifns have been implicated in the regulation of myeloid cell migration during initial exposure to viral infections, heightening inflammatory and virus-specific b and t-cell responses [ , ] . the production of type i ifns by sentinel leukocytes, in particular that of plasmacytoid dcs that serve as a potent source of ifns, upon viral infection initiates a type i ifn-dependent secretion of neutrophil and inflammatory monocyte chemoattractants such as il- α, cxcl and cxcl [ , ] , highlighting the role of virus-induced type i ifns in the regulation of neutrophil and monocyte trafficking. pollara et al. [ ] demonstrated that the secretion of type i ifns by hsv- -infected myeloid dcs results in the activation of uninfected dcs. this process enables the adaptive immune system to become activated even during a viral infection that targets myeloid cells and prevents their maturation, such as in the case of hsv. the protective functions of type i ifns have been associated not only with the recruitment of neutrophils and inflammatory ly c hi monocytes to sites of viral infections, but also with the prevention of excessive monocyte and neutrophil activation, thereby controlling inflammation caused by type ii ifns, such as ifn-γ [ ] . the interplay between type i and ii ifns was crucial for mitigating damage stemming from influenza a virus-induced inflammation in rag -/-, ifnar -/-, ifngr -/and stat -/-c bl/ mice [ ] . both ifns were required to prevent excessive numbers of neutrophils trafficking into lungs. stat was experimentally determined to coordinate inflammation via type i and ii ifn receptors. when type i ifns were absent, ly c lo monocytes transitioned to being more inflammatory than ly c hi monocytes. in the absence of type i ifn signaling, ly c lo monocytes traditionally associated with tissue re-modeling became phenotypically and functionally more pro-inflammatory during infection with influenza a viruses [ ] . notably, infection of trophoblasts with zika virus induced a lower secretion of type i ifns, and higher immunopathological inflammatory immune responses when compared to trophoblasts infected with yellow fever virus and dengue virus [ ] . measurement of immune mediators in nasal fluids from rsv-infected infants indicated that severe disease caused by heightened inflammatory responses was also associated with diminished type i ifn responses [ ] , furthering the idea that a link between type i ifns and the promotion versus suppression of virus-induced inflammation exists. taken together, these findings suggest that type i ifn signaling drives a balance of pro-and anti-inflammatory effects on the functions of monocytes and neutrophils in response to viral infections; providing protective immunity while simultaneously limiting immunopathology. these results suggest that the administration of type i ifns at optimized time points and doses could prove beneficial in the limitation of toxic cytokine storm onset and the control of excessive immunopathological damage. indeed, in vitro evidence suggests that the administration of exogenous type i ifns can mitigate excessive cytokine production induced by sars-coronaviruses [ ] . determining the means by which type i ifns control excessive inflammation while ensuring effective anti-viral responses is required. neutrophils, inflammatory monocytes, and their roles in mitigating bacterial infections have been extensively studied and well characterized. exciting new research in immunology and virology has demonstrated that these first responders of the innate immune system are also crucial in limiting viral infections, replication, and associated off-target pathological damage. a multifaceted range of tactics is utilized to combat an equally diverse range of viruses, including phagocytosis, the formation of extracellular traps, the production of cytokines such as ifns, and modulation of ilcs and lymphocytes. despite rapid advances in the field, many exciting unknown aspects of the involvement of neutrophils and inflammatory monocytes in combating viral infections remain to be clarified. current research has documented the impact of neutrophil/monocyte retention in the bone marrow as it pertains to viral infections, but we still do not completely understand all mechanisms by which myeloid cells are recruited from the blood stream to the primary sites of infection. future studies should aim to elucidate the specific signaling cascades that recruit myeloid cells into infected tissues and the mechanistic consequences of disruptions in these cascades via the chemokine gradient as well as depletions of specific ligands. if the scientific community can determine how different cell subsets can influence the production of chemokine populations and hone in on the essential ligands required for migration into the primary sites of infection, drugs could potentially be developed to exploit this localized production of chemokines. the discovery of pharmaceuticals that could fine-tune myeloid cell trafficking could prove beneficial to inducing rapid antiviral responses. differential ligation versus the blockade of prrs associated with protective versus pathological inflammation constitutes another strategy to balance rapid viral clearance and minimize host damage. current knowledge from myeloid cell studies in bacterial diseases demonstrated that neutrophils are essential for monocyte recruitment and function. additionally, it has been shown that the ratio of neutrophils to lymphocytes is higher in bacterial than viral infections among patients hospitalized for fevers [ ] . it is clear that neutrophils and monocytes work in concert to enhance immune responses against bacterial pathogens. however, future studies are needed to explore the mechanisms by which these myeloid cells collaborate with each other to control viral infections, with the aim of gaining new insights into how they function in virus-infected microenvironments to regulate cell-to-cell communication within the innate and adaptive arms of the immune system. gaining a better understanding of the role of myeloid cells in the pathogenesis of viral diseases will facilitate the design of better therapies. importantly, viruses and virus-mediated tissue damage stimulate both neutrophils and monocytes, triggering a cascade of cytokine/chemokine-mediated innate immune responses. this antiviral activity is not always beneficial for a host and, when improperly regulated, may contribute to immunopathologies such as cytokine storms that have been observed in many severe viral infections and could 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interferons is essential to limit influenza a virus-induced tissue inflammation herpes simplex virus type- -induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type i ifn secretion dengue and yellow fever viruses induce differential anti-viral immune responses in human monocytic and first trimester trophoblast cells differential ability of pandemic and seasonal h n influenzaa viruses to alter the function of human neutrophils characterization of the antiviral effects of interferon-alpha against a sars-like coronoavirus infection in vitro role of neutrophil to lymphocyte and monocyte to lymphocyte ratios in the diagnosis of bacterial infection in patients with fever key: cord- -yabt jf authors: tuttle, kathryn d.; minter, ross; waugh, katherine a.; araya, paula; ludwig, michael; sempeck, colin; smith, keith; andrysik, zdenek; burchill, matthew a.; tamburini, beth a.j.; orlicky, david j.; sullivan, kelly d.; espinosa, joaquin m. title: jak inhibition blocks lethal sterile immune responses: implications for covid- therapy date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: yabt jf cytokine storms are drivers of pathology and mortality in myriad viral infections affecting the human population. in sars-cov- -infected patients, the strength of the cytokine storm has been associated with increased risk of acute respiratory distress syndrome, myocardial damage, and death. however, the therapeutic value of attenuating the cytokine storm in covid- remains to be defined. here, we report results obtained using a novel mouse model of lethal sterile anti-viral immune responses. using a mouse model of down syndrome (ds) with a segmental duplication of a genomic region encoding four of the six interferon receptor genes (ifnrs), we demonstrate that these animals overexpress ifnrs and are hypersensitive to ifn stimulation. when challenged with viral mimetics that activate toll-like receptor signaling and ifn anti-viral responses, these animals overproduce key cytokines, show exacerbated liver pathology, rapidly lose weight, and die. importantly, the lethal immune hypersensitivity, accompanying cytokine storm, and liver hyperinflammation are blocked by treatment with a jak -specific inhibitor. therefore, these results point to jak inhibition as a potential strategy for attenuating the cytokine storm and consequent organ failure during overdrive immune responses. additionally, these results indicate that people with ds, who carry an extra copy of the ifnr gene cluster encoded on chromosome , should be considered at high risk during the covid- pandemic. one sentence summary inhibition of the jak kinase prevents pathology and mortality caused by a rampant innate immune response in mice. cytokine release syndrome (crs) or hypercytokinemia, often referred to as a 'cytokine storm', has been postulated to drive mortality during severe respiratory viral infections, such as influenza ( ) -including the spanish flu epidemic ( ) and the h n bird flu ( ) , as well as during the severe acute respiratory syndrome coronavirus epidemic of (sars-cov- ) ( ). increasing evidence supports the notion that mortality during infections with sars-cov- , which causes coronavirus disease of , is driven by the exacerbated immune response to the virus, leading to a cascade of events involving a cytokine storm and acute respiratory distress syndrome (ards), often accompanied by myocardial damage and multiple organ failure ( , ) . this pathological cascade resembles what is observed in other lethal viral lung infections, where the immune response to the virus triggers a primary wave of cytokines, including type i and type iii interferons (ifns), followed by infiltration and activation of diverse immune cells and production of secondary cytokines and chemokines, including type ii ifn (ifn-g), accelerated immune activation, and progressive decline of respiratory function ( ) . importantly, many of the cytokines and chemokines involved in the cytokine storm employ janus protein kinases (jaks) for signal transduction. cytokine analysis of covid- patients indicate an important role for the magnitude of the cytokine storm in prognosis and clinical outcome. levels of c-reactive protein (crp) and il- at the time of hospitalization were reported to be significantly higher in patients who eventually died versus those that survived ( ) . in a different study, patients admitted to intensive care units (icus) showed significantly higher levels of il- , il- , il- , ip- , mcp- , mip- α, g-csf, and tnf-α relative to non-icu patients ( ) . using artificial intelligence methods, high levels of circulating alanine aminotransferase (alt), a marker of liver inflammation, was associated with progression toward ards ( ) . altogether, these findings support the rationale for combining antiviral treatment and targeted immunosuppression as a therapeutic approach in covid- ( ) . accordingly, several clinical trials are currently testing the safety and efficacy of inhibitors of il- signaling and jak/stat signaling. furthermore, hydroxychloroquine, a molecule shown to attenuate toll-like receptor signaling and cytokine production that is approved for the treatment of rheumatoid arthritis and systemic lupus erythematosus ( , ) , is also being testing in many clinical trials for covid- . within this context, we report here relevant results obtained during studies of the hyperactive immune response in a mouse model of down syndrome (ds). we recently discovered that trisomy (t ), the genetic cause of ds, causes consistent activation of the ifn transcriptional response in multiple cell types ( ) ( ) ( ) ( ) , which is explained by the fact that four of the six ifn receptors are encoded on chromosome (ifnar , ifnar , ifngr , il rb). additional investigations of the proteome ( ), metabolome ( ) , and immune cell lineages ( , ) of people with ds demonstrated that t causes: ) changes in the circulating proteome indicative of chronic autoinflammation with clear dysregulation of ifn-inducible cytokines ( ) , ) activation of the ifn-inducible kynurenine pathway, leading to elevated production of neurotoxic tryptophan catabolites ( ) , and ) widespread hypersensitivity to ifn stimulation across the human immune system ( , ) . altogether, these results support the notion that ds can be understood in good measure as an interferonopathy, whereby increased ifn signaling could account for many of the developmental and clinical impacts of t . now, we report results generated during our investigation of immune responses in the dp ( )/yey mouse strain (referred hereto as dp ) ( ) , a widely used mouse model of ds. the dp strain carries a segmental duplication of murine chromosome that is syntenic to human chromosome , encoding ~ genes, including the four ifnrs. we found that dp mice overexpress ifnrs in all immune cell types tested, display hypersensitivity to type i and type ii ifn stimulation, overproduce key cytokines, and display increased liver inflammation. remarkably, dp mice display lethal immune responses upon challenge with viral mimetic molecules, such as the tlr agonist poly-i:c [p(i:c)] or the tlr / agonist imiquimod, a phenotype that is not observed in their wild type (wt) littermates or mouse strains carrying triplications of other genomic regions syntenic to chromosome . strikingly, both the lethal immune phenotype and associated cytokine storm are blocked by treatment with a small molecule inhibitor of the jak kinase, which also decreases liver pathology. overall, these results have potential far-reaching significance for the treatment of covid- , justifying a deeper study of jak inhibitors as a therapeutic strategy to attenuate the cytokine storm and downstream organ failure in this condition, while also indicating that individuals with ds should be considered a high-risk population during the covid- pandemic. in order to model the impact of t on chronic innate immune responses in mice, we employed the dp mouse strain, which harbors triplication of a region syntenic to human chromosome including four ifnrs ( figure a ) ( ) . to define the relationship between gene copy number and ifnr protein expression, we evaluated surface expression of ifnar in peripheral immune cells from dp mice by flow cytometry. dp( ) yey/+ and dp( )yey/+ mice (hereafter referred to as dp and dp ), which have three copies of genes that are syntenic to chromosome , but are disomic for ifnrs ( ) , were included as controls ( figure a) . expression of ifnar on the surface of cd + white blood cells was significantly higher in dp mice, but not dp or dp animals, relative to wt littermates ( figure b) . when different immune cell types were analyzed by flow cytometry, ifnar expression was significantly higher in all cell types tested ( figure c) . these results are consistent with previous reports of mrna overexpression for all ifnrs in this gene cluster in various tissues of dp mice ( , ) . to test whether upregulation of ifnrs conferred stronger responses to ifn ligands, we stimulated wt and dp blood samples with ifn-a and evaluated downstream phosphorylation of stat (pstat ) by flow cytometry. dp cells responded more strongly to ifn-a as defined by significantly higher levels of pstat relative to wt cells, in all immune lineages examined ( figure d ). this widespread heightened response to ifn-a was not observed in dp and dp cells; however, t cell subsets from the dp strain exhibited more robust pstat ( figure e-f) . although dp mice have only two copies of the ifnr genes, this result suggests that some aspect of triplication of the region of chromosome syntenic to human chromosome leads to differential regulation of type i ifn signaling in t cells. given that one of the type ii ifn receptor subunits (ifngr ) is also encoded on murine chromosome and triplicated in dp mice, we next investigated the response to ifn-g stimulation. indeed, dp cells responded more strongly to ifn-g stimulation in most cell types examined ( figure g) . overall, these results suggest that elevated expression of the ifnrs in the dp mouse strain confers increased sensitivity to type i and type ii ifns. since dp cells mount enhanced responses to ifn ligands, we next investigated the response of dp mice to innate immune stimuli known to trigger an ifn response. chronic systemic induction of ifn signaling was elicited by repeated administration of the tlr agonist polyinosinic:polycytidylic acid [p(i:c)], which is known to produce an acute spike of type i ifn production ( - days), followed by low but persistent expression of type i ifn ligands and a robust cytokine response in the chronic phase ( - days) ( ) . wt and dp mice were given intraperitoneal injections of mg/kg of p(i:c) or an equivalent volume of vehicle (sham) every other day, for up to days, with the experiment completed at day . remarkably, dp mice were profoundly sensitive to the treatment, which was largely tolerated by wt mice (figure a ). body weight measurements during the course of the experiment revealed rapid weight loss in dp mice upon treatment ( figure b) . eventually, all but one of the twelve dp mice had to be removed from the study for excessive weight loss (> %). in contrast, wt animals did not lose as much weight, and all but three out of nine survived to the end of the experiment (figure a ,b). dp and dp mice also tolerated chronic immune stimulation with p(i:c) (figure c-f). although p(i:c) treatment caused some weight loss in both dp and dp mice, the percentage lost was comparable to wt levels. this p(i:c)-induced weight loss was clearly dosedependent, as mice receiving half the lethal dose ( mg/kg) displayed only minor weight loss, with no observable differences between dp and wt mice ( figure s ). in order to determine whether hypersensitivity was restricted to tlr agonists or more broadly observed across activation of other pattern recognition receptors, we treated the mice with the tlr agonist imiquimod. topical administration of imiquimod causes ifn production, acute skin inflammation, systemic inflammation, and dehydration, and is commonly employed as a model of psoriasis ( ) . imiquimod treatment caused significant weight loss in dp mice despite daily administration of supportive fluids, which led to rapid termination of the study in observation of the humane endpoint ( figure g,h) . in contrast, wt animals receiving identical treatment maintained their weight (figure g,h) . altogether, these results indicate that the hypersensitivity to ifn stimulation observed at the cellular level in dp mice associates with increased sensitivity and lethality to ifn-inducing viral mimetic agents at the organismal level. we next compared the immune response in dp mice relative to their wild type littermates during the acute and chronic responses to p(i:c). first, we measured levels of circulating ifn-a hours after the first injection of p(i:c) (or sham). ifn-a levels were induced by p(i:c), with a stronger response in dp mice ( figure a) . elevated ifn ligand production in dp mice is consistent with a predicted fast-forward loop driven by increased ifnr expression in ds ( ) . we then measured mrna expression for several ifn-stimulated genes (isgs) in the spleen of wt and dp mice at hours after a single injection of p(i:c) (or sham). this experiment revealed strong induction of isg expression upon p(i:c) treatment, with several isgs being more significantly elevated in the dp mice than in wt animals ( figure b ). next, we measured the levels of several cytokines and chemokines in the bloodstream hours after a single p(i:c) injection. expectedly, p(i:c)-treated animals showed increased levels of many pro-inflammatory factors, regardless of genotype ( figure c and figure s a ). in this acute phase, the most significantly elevated inflammatory markers were ip- (cxcl ), mcp- , rantes (ccl ), and kc/gro (cxcl ) (figure c ). on average, dp mice did not show significant differences in expression of cytokines relative to their wt littermates. then, we measured circulating cytokines and chemokines during the chronic response. we compared serum levels between dp mice taken at the time of harvest due to reaching the humane endpoint of % weight loss to the levels in the wt littermates that survived to the end of the -day experiment. at these later stages of chronic inflammation, several of these inflammatory markers were significantly elevated in the dp animals, such as mcp- and mip- a, but not in the wt littermates ( figure d) . altogether, these results indicate that activation of tlr signaling leads to strong induction of key cytokine and chemokines in our experimental paradigm, with several inflammatory markers being more elevated in the dp mice. although not a single cytokine or chemokine stands out as the sole driver of phenotypes in the dp mice, the mild over-production of several cytokines could potentially contribute to their exacerbated immune sensitivity. next, we investigated markers of liver inflammation and injury. the serum levels of the enzymes alanine transaminase (alt) and aspartate transaminase (ast), two commonly used markers of hepatocyte injury, were significantly elevated upon p(i:c) treatment in the dp mice, reaching concentrations nearly an order of magnitude higher than those observed in their wt littermates ( figure a) . prompted by these results, we investigated liver pathology. liver tissue sections were stained with hematoxylin and eosin (h&e) and evaluated by a trained histologist, blinded against treatment group and genotype. scoring of liver pathology was done with procedures adapted for mice ( ) using a validated histological scoring system ( ) , that included parameters such as cell injury, inflammation, and reactive changes, leading to an overall liver pathology score. this analysis revealed that dp mice have increased liver pathology even before p(i:c) treatment, as demonstrated by significantly higher levels of cell injury, inflammation, and reactive changes relative to their wt littermates (figure b,c) . this basal level of liver pathology was not observed in the dp and dp mice (figure d,e) . importantly, upon treatment with p(i:c), the livers of wt animals developed pathology scores comparable or greater than those observed at baseline in dp mice, including signs of strong recruitment and/or expansion of inflammatory cells. when p(i:c) was administered to dp mice, all metrics of liver pathology increased, leading to significantly higher overall pathology scores relative to both dp sham treatment and wt upon p(i:c) treatment (figure b-c) . livers from dp and dp mice that received p(i:c) also experienced increases in liver pathology, but these changes did not differ from wt mice (figure d,e) . overall, these results indicate that dp mice display increased liver inflammation and pathology, both at baseline and upon immune activation, which could potentially contribute to their heightened sensitivity to ifn-inducing agents. all three types of ifn signaling employ the jak kinase for signal transduction, in combination with either jak or tyk (figure a) . therefore, we sought to determine whether inhibiting jak specifically would block the lethality and pathology caused by p(i:c) treatment in dp mice. to this end, we used the jak -specific inhibitor incb . animals received a dose of mg/kg of incb (or an equivalent volume of methylcellulose used as the vehicle) twice daily via oral gavage, beginning hours prior to the first p(i:c) injection, and every day during the course of the experiment. remarkably, dp mice that received the jak inhibitor all survived the p(i:c) treatment, while dp animals that received the vehicle and p(i:c) experienced weight loss and had to be euthanized ( figure a) . we then analyzed the impact of jak inhibition on cytokine production by comparing the dp mice treated with p(i:c) with and without co-treatment with the jak inhibitor. importantly, cytokine production was strongly abrogated by jak inhibition (figure b and figure s ) . lastly, we evaluated the impact of jak inhibition on markers of liver inflammation and injury. indeed, the jak inhibitor led to a reduction of circulating levels of alt and ast, as well as a decrease in overall liver pathology scores ( figure c) . altogether, these results indicate that blocking the catalytic activity of the jak kinase prevents the lethality associated with a rampant innate immune response in the dp mouse model. the efficacy of this treatment likely relies on a combination of inter-related effects, such as reduced cytokine production and improved liver function. as the covid- pandemic rapidly progresses worldwide, there is a dire need to identify new prophylactic and therapeutic strategies. based on recent data from analyses of inflammatory markers in covid- ( , ) and prior knowledge about inflammatory responses in other lethal viral lung infections, targeted immune-suppression is now appreciated as a potential strategy to prevent ards, fulminant myocarditis, organ failure, and mortality at advanced stages of the condition ( ). accordingly, clinical trials for diverse immune-suppressants have been started around the world, including inhibitors of il- and jak/stat signaling. here, we demonstrate a key role for the jak kinase in driving cytokine storms and accompanying mortality in a mouse model of lethal anti-viral immune responses. jak is one of several jak kinases required for mediating inflammatory signaling downstream of a large number of cytokines, including all three types of ifn signaling and il- ( ). we show that eliciting the innate anti-viral immune response with tlr agonists is lethal in a sensitized mouse model carrying triplication of four of the six ifn receptors. within days, the dp mice rapidly lose weight and die if unattended, concurrent with induction of many prominent cytokines linked to the severity of covid- -driven pathology, such as il- , tnf-α, il- , ip- , mcp- , and mip- α ( ) . furthermore, dp mice display exacerbated liver inflammation and pathology, which, together with stronger production of some cytokines, may converge into a pathological cascade leading to wasting and death. importantly, these phenotypes are blocked by jak inhibition. based on these results, we propose here that jak inhibitors are a valid therapeutic approach for attenuating the cytokine storm in covid- . four jak inhibitors are currently fda-approved for the treatment of diverse medical conditions: jakafi/ruxolitinib, a jak / inhibitor approved for myelofibrosis, polycythemia vera, and graft-versus-host-disease; xeljanz/tofacitinib, a jak / inhibitor approved for rheumatoid arthritis, psoriatic arthritis and ulcerative colitis; olumiant/baricitinib, a jak / inhibitor approved for rheumatoid arthritis; and rinvoq/upadacitinib, a jak inhibitor also approved for rheumatoid arthritis. many jak specific inhibitors are currently being developed and tested in clinical trials for diverse inflammatory and autoimmune conditions, including the molecule used in this study, we hypothesize that jak inhibitors could be superior agents in terms of attenuating the cytokine storm caused by covid- relative to existing anti-il agents, which consist of injectable monoclonal antibodies that inhibit the interaction between il- and its receptor il- r ( ) . in contrast, jak inhibitors are available as drugs administered orally, with very well characterized pharmacodynamics and pharmacokinetics, and may provide a more appropriate strategy to transiently tone down the cytokine storm to prevent ards and fulminant myocarditis. furthermore, jak inhibitors not only block il- signaling, but also other inflammatory pathways involved in the cytokine storm ( ) . depending on the degree of inhibition of various jak kinases, it is predicted that available jak inhibitors will block various aspects of the cytokine storm, with potential for varying degrees of therapeutic benefit. with regards to its relevance to therapeutic strategies for covid- and other conditions associated with cytokine storms, this study has several limitations. first, our mouse model does not fully reproduce the human conditions associated with cytokine storms. although pandemic. our studies of the dp mice were originally driven by our interest in studying immune dysregulation and ifn hyperactivity in ds. of note, our results are consistent with studies of sars-cov- demonstrating that type i ifn signaling contributes to sars-driven pathology and mortality in mice ( ) . we predict that, as has been observed during rsv ( ) and h n ( ) infections, people with ds will develop more severe complications upon sars-cov- infection, including higher rates of hospitalization and mortality. we hope these results will encourage special attention to individuals with ds, including closer monitoring of hyperinflammation during covid- , and inclusion in clinical trials for targeted immunesuppressants. table s . q-rt-pcr. q-rt-pcr was performed using the applied biosystems viia ™ -well block real time pcr system. q-rt-pcr master mix was prepared with applied biosystems sybr select master mix for cfx. standard curves were run for every primer pair in each q-rt-pcr experiment to ensure efficient amplification of target transcripts within all experimental tissues. all samples were run in triplicate, averaged and normalized to s rrna. primer sequences at provided in table s . cytokine measurements. blood cytokine levels were measured using mesoscale discovery assays and/or biolegend legend plex assays per manufacturer's instructions. all samples were analyzed in duplicate and the average used for statistical analysis. missing values were set to the lower limit of detection. liver histopathology. formalin-fixed paraffin-embedded pieces of liver were sectioned at microns and stained with hematoxylin and eosin (h&e). scoring of liver pathology used procedures adapted for mice as described ( ) table s . primers for q-rt-pcr. primers used for q-rt-pcr analysis of interferonstimulated genes and s rrna, including gene, refseq id, forward primer sequence, reverse primer sequence. the cytokine storm of severe influenza and development of immunomodulatory therapy preparing for the next pandemic confronting potential influenza a (h n ) pandemic with better vaccines an interferon-gamma-related cytokine storm in sars patients clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china clinical features of patients infected with novel coronavirus in wuhan into the eye of the cytokine storm towards an artificial intelligence framework for data-driven prediction of coronavirus clinical severity covid- : consider cytokine storm syndromes and immunosuppression mode of action of hydroxychloroquine in ra-evidence of an inhibitory effect on toll-like receptor signaling chloroquine and hydroxychloroquine equally affect tumor necrosis factor-alpha, interleukin , and interferon-gamma production by peripheral blood mononuclear cells trisomy consistently activates the interferon response trisomy dysregulates t cell lineages toward an autoimmunity-prone state associated with interferon hyperactivity trisomy activates the kynurenine pathway via increased dosage of interferon receptors mass cytometry reveals global immune remodeling with multilineage hypersensitivity to type i interferon in down syndrome trisomy causes changes in the circulating proteome indicative of chronic autoinflammation duplication of the entire . mb human chromosome syntenic region on mouse chromosome causes cardiovascular and gastrointestinal abnormalities rodent models in down syndrome research: impact and future opportunities lifespan analysis of brain development, gene expression and behavioral phenotypes in the ts cje, ts dn and dp( ) /yey mouse models of down syndrome re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type i interferons imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the il- /il- axis signaling a link between interferon and the traits of down syndrome ketohexokinase c blockade ameliorates fructose-induced metabolic dysfunction in fructose-sensitive mice design and validation of a histological scoring system for nonalcoholic fatty liver disease jak inhibition as a therapeutic strategy for immune and inflammatory diseases targeting interleukin- signaling in clinic interaction of sars and mers coronaviruses with the the establishment of reference sequence for sars-cov- and variation analysis dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice down syndrome and the risk of severe rsv infection: a meta-analysis pandemic (h n ) virus and down syndrome patients we are grateful to all staff at the linda crnic institute for down syndrome, specially hannah dougherty, jessica baxter, dayna tracy, and ross granrath. we also thank staff at the university of colorado cancer center flow cytometry shared resource, the human immune monitoring shared resource (himsr), and the barbara davis center flow cytometry core who assisted with various aspects of this work. we are grateful to incyte corporation for providing incb for these studies and for expedited review of these results prior to publication. key: cord- -jv j wx authors: lee, kyungjin; kim, dong-eun; jang, kyoung-soon; kim, seong-jun; cho, sungchan; kim, chonsaeng title: gemcitabine, a broad-spectrum antiviral drug, suppresses enterovirus infections through innate immunity induced by the inhibition of pyrimidine biosynthesis and nucleotide depletion date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: jv j wx gemcitabine, an anti-cancer chemotherapy drug, has additionally shown the antiviral activity against a broad range of viruses and we also have previously reported its synergistic antiviral activity with ribavirin against enteroviruses. as a cytidine analog, gemcitabine has been reported to have an inhibitory activity on the pyrimidine biosynthesis. in addition, a few inhibitors of the pyrimidine biosynthesis have shown to induce the innate immunity in a yet-to-be-determined manner and inhibit the virus infection. thus, we also investigated whether the anti-enteroviral activity of gemcitabine is mediated by innate immunity, induction of which is related with the inhibition of the pyrimidine synthesis. in this study, we found that the addition of exogenous cytidine, uridine and uridine mono-phosphate (ump) effectively reversed the antiviral activity of gemcitabine in enterovirus-infected as well as enteroviral replicon-harboring cells, demonstrating gemcitabine’s targeting of the salvage pathway. moreover, the expression of several interferon (ifn)-stimulated genes (isgs) was significantly induced by the treatment of gemcitabine, which was also suppressed by the co-treatment with cytidine. these results suggest that the antiviral activity of gemcitabine involves isgs induced by the inhibition of the pyrimidine biosynthesis. enteroviruses belong to the picornaviridae family, which is characterized by a single stranded positive-sense rna genome with about - nucleotides, and have been emerged as the major causative agents of various human diseases. coxsackievirus b (cvb ), one of the most well-studied enteroviruses, causes viral meningitis, myocarditis and pancreatitis [ , ] . in addition, enterovirus (ev ) is a causative agent of hand-foot-mouth disease and also of severe neurological symptoms, which can lead to even death [ ] [ ] [ ] . however, despite the increasing public threat, no effective therapy is currently available for the treatment of these infections. enteroviruses have hundreds of distinct viruses, and newly emerging enteroviruses have been increasingly reported in recent years. moreover, many rna viruses including influenza, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and zika virus (zikv) have become an enormous threat for public health. therefore, broad-spectrum antiviral drugs are necessary to efficiently control various viral infections. in another aspect ineffectiveness of conventional enzymetargeting drugs due to the rapid development of resistant mutants is another hurdle we need to tackle. in order to achieve the development of broad-spectrum antiviral drug with a low rate of mutation, two strategies have been generally considered. one is targeting host cellular factor that is essentially required for the viral life cycle. this strategy would have a low potential of producing resistant viruses, but undesirable side effects could be accompanied. the other is activating innate immune response such as interferon (ifn) signaling so as to boost host antiviral defense system [ ] [ ] [ ] [ ] . actually, ifn itself or in combination with other antiviral drugs such as ribavirin has been primarily used for the treatment of various rna virus infections. more recently, a few inhibitors of nucleoside biosynthesis have been shown to induce the innate immunity and suppress a broad range of virus infections [ ] [ ] [ ] [ ] [ ] . for instance, wang et al identified a broad-spectrum antiviral compound (brequinar) targeting dhodh, a key enzyme of the pyrimidine biosynthetic pathway, and subsequently inducing innate immune response [ ] . previously, we identified gemcitabine, a drug currently being used for anti-cancer chemotherapy, as an effective inhibitor of enteroviruses including cvb , ev and human rhinoviruses (hrvs) [ ] . its antiviral activity has been also shown against various rna viruses including hepatitis c virus (hcv), human immunodeficiency virus (hiv), influenza virus, poliovirus, mers-cov and zikv [ ] [ ] [ ] [ ] [ ] [ ] . gemcitabine, as a cytidine analog, was reported to interfere with the pyrimidine biosynthesis [ ] . however, the role of pyrimidine inhibition and the involvement of subsequent innate immunity in the antiviral action of gemcitabine have not been explored yet. in this study, we examined the role of pyrimidine inhibition in the antiviral activity of gemcitabine by adding the exogenous nucleosides to cvb -infected or cvb replicon-harboring hela cells. as a result, the antiviral effect of gemcitabine was remarkably suppressed by the pyrimidine nucleosides. further analysis demonstrated that gemcitabine inhibited the salvage pathway of pyrimidine biosynthetic pathway most probably by targeting cytidine and/or uridine synthesis. moreover, the treatment with gemcitabine activated the expression of several ifn-stimulated genes (isgs), the major effectors in the innate immunity, which was also suppressed by the supplemented cytidine. previously, we identified a new indication of gemcitabine as an effective anti-enteroviral inhibitor [ ] . as a cytidine analog, gemcitabine is known to have an inhibitory activity on the pyrimidine biosynthesis. besides, a few inhibitors of the pyrimidine biosynthesis have been reported to show the antiviral activity, at least partly, through activating the innate immune response [ , , ] . thus, in this study we sought to examine if the anti-enteroviral activity of gemcitabine is also associated with the modulation of pyrimidine biosynthesis and innate immunity. at first, in order to test if the anti-enteroviral activity of gemcitabine is related with the inhibition of pyrimidine biosynthesis, hela cells were infected with cvb and simultaneously treated with excessive nucleosides (adenosine, guanosine, uridine and cytidine) in the presence of various doses of gemcitabine for hours. antiviral activity was measured by staining infected cells with an anti-cvb cpro antibody and secondary antibody conjugated with af fluorescent dye and counting cells with a fluorescent signal. as previously reported, gemcitabine itself exhibited a strong antiviral activity on cvb infection, with an estimated ic of ~ µm and maximal efficacy of > % ( figure a ). when gemcitabine was co-treated with excessive nucleosides each, only two pyrimidine nucleosides (cytidine and uridine) could significantly suppress the antiviral activity of gemcitabine, resulting in the sustained infection. especially, cytidine had the strongest effect, which can be explained by the fact that gemcitabine is a cytidine analog. the effect of cytidine was evidently demonstrated by a dose-dependent suppression of gemcitabine's antiviral activity (supplementary figure a) . in contrast, purine nucleosides (adenosine and guanosine) had little effect. as a control experiment, cell viability was analyzed in the same condition by using mtt assay ( figure b ). there were little changes except that the treatment with adenosine without gemcitabine decreased the cell viability by about %, indicating the cytotoxic effect of adenosine. this phenomenon was further confirmed in an independent experiment using increasing concentrations of each nucleosides without cvb infection (supplementary figure ) . to further confirm the effect of pyrimidine nucleosides on the antiviral activity of gemcitabine, cvb subgenomic replicon system was additionally used. this system contains the firefly luciferase gene in place of structural genes (vp -vp ) of cvb viral genome and allows the quantitative measurement of viral replication [ , ] . hela cells were transfected with in vitrotranscribed cvb replicon rnas and simultaneously treated with nucleosides each in the presence of various doses of gemcitabine for hours. as similar to the results from cvb infection experiment ( figure a) , addition of excessive pyrimidine nucleoside (cytidine or uridine) remarkably suppressed the antiviral activity of gemcitabine, thereby protecting the replication of cvb replicon (supplementary figure a) . according to our previous study, gemcitabine has a strong antiviral effect on ev as well as cvb [ ] . therefore, we also tested pyrimidine nucleosides in ev replicon system. as a result, only cytidine and uridine suppressed the antiviral activity of gemcitabine on the replication of ev replicon, which is similar to those observed from cvb based assays (supplementary figure b) . collectively, these results strongly indicate the involvement of pyrimidine biosynthesis in the antiviral activity of gemcitabine. the observation that the antiviral effect of gemcitabine is suppressed by the supplemented pyrimidine nucleosides directed us to further define the effect of gemcitabine on the pyrimidine biosynthetic pathway. pyrimidine biosynthesis occurs by two separate pathways: de novo synthesis and the salvage ( figure c ). to determine which pathway and step were affected by gemcitabine, a few intermediates of both pathways were tested. dihydroorotate (dho) and orotic acid were used as intermediates of de novo biosynthetic pathway, and nucleobases (adenine, guanine, uracil and cytosine) were additionally used as intermediates of the salvage pathway. uridine mono-phosphate (ump) acting downstream of both pathways was also included. ump is synthesized from uridine by uridine kinase in the salvage pathway and separately from orotidine mono-phosphate (omp) by omp decarboxylase in de novo biosynthetic pathway ( figure c ). gemcitabine, as a nucleoside analog, may directly affect the activity of enzyme(s) that are involved in the pyrimidine biosynthesis, resulting in the decrease of intermediates acting downstream of its target. thus, excessive addition of any intermediate acting downstream of gemcitabine's target can rescue the deficit and supposedly shows a suppressive effect on the antiviral activity of gemcitabine. when selected intermediates were co-treated with gemcitabine in cvb -infected hela cells, only ump significantly showed a suppressive effect with an extent similar to that of cytidine ( figure a ). other intermediates for de novo pathway (dho and orotic acid) and for the salvage pathway ( nucleobases) had marginal effects. under the same condition, little changes in cell viability were shown ( figure b ). moreover, similar effect of ump was also observed in cvb replicon-harboring cells (supplementary figure ) , even though it was slightly weaker than that of cytidine. taken together the results from figures and , it seems that gemcitabine majorly targets the salvage pathway rather than de novo pathway, particularly uridine and/or cytidine synthesis from uracil and/or cytosine ( figure c ). there are a few antiviral compounds interfering with the nucleoside biosynthesis, which subsequently induce innate immunity involving the upregulation of isgs [ ] [ ] [ ] ] . in those studies antiviral compounds induced the expression of ifn-stimulated response element (isre)-luciferase reporter even without ifn stimulation. thus, we also examined if gemcitabine itself has a stimulatory effect on isre-luciferase reporter. hela cells were transfected with the reporter plasmid and then treated with gemcitabine ( . to μm) and ifn-α ( . to ng/ml) for or hours. at hours posttreatment gemcitabine increased the luciferase activity up to ~ % of the control ( figure a ). the increase of luciferase activity was much more evident at hours post-treatment, maximally reaching ~ % of the control ( figure c ). in contrast, ifn-α induced the luciferase expression up to ~ and % of the control at and hours post-treatment, respectively, indicating a rapid stimulation ( figure b and d) . both gemcitabine and ifn-α significantly stimulated the isre promoter but with apparently different time-kinetics. isre promoter more slowly responded to gemcitabine compared to ifn-α. differential mechanisms of isre activation by gemcitabine and ifn-α will be proposed in the discussion section. to further confirm that isre activation by gemcitabine is correlated with its antiviral activity, we tested if the antiviral activity is enhanced by the longer treatment of gemcitabine. hela cells were treated with gemcitabine for hours prior to cvb infection and maintained for another hours, achieving hours of treatment ( h exposure). antiviral activity was assessed by quantifying the virus-infected cells showing a fluorescent signal of cpro protein at hours after treatment with gemcitabine. as a result, a considerably stronger antiviral effect was exhibited by a longer time treatment ( h exposure) than a shorter time treatment ( h exposure) (supplementary figure ) . the estimated ic of gemcitabine in ' h exposure' cells (~ . μm) was much lower than that in ' h exposure' cells (~ μm). moreover, the maximum efficacy of gemcitabine in ' h exposure' cells (~ %) was apparently higher than that in ' h exposure' cells (~ %). these results indicate that the enhanced antiviral activity by a longer exposure of cells with gemcitabine is likely correlated with stronger activation of isre promoter attained at hours of treatment. ifns trigger an intracellular signal through the jak/ stat pathway and transcriptionally induce numerous isgs under the control of isre [ ] . according to our aforementioned results, gemcitabine itself has a stimulatory effect on isre promoter like ifns. however, their modes of activation seem to be quite different. thus, in order to further confirm the activation of isrecontaining promoters by gemcitabine and to know how differently these promoters respond to gemcitabine and ifn-α, the transcriptional expression of isgs (cxcl , irf , irf , ifit , and ddx ) were analyzed. hela cells were treated with increasing doses of gemcitabine or ifn-α for hours and then total rnas were prepared for quantitative real-time pcrs. as a result, gemcitabine strongly induced two genes (ifit and ddx ) up to and ump) ( μm) of pyrimidine biosynthetic pathway were treated as described in figure a (a) and in figure b (b) . cytidine was used as a positive control. the average and standard deviation were obtained from three independent experiments. *** indicates p < . . (c) pyrimidine biosynthetic pathway. tested metabolites were indicated as boxes and metabolites with the significant effect were shown as orange boxes. the salvage pathway affected by gemcitabine was depicted by blue arrows. more than -fold of the control and moderately three genes (cxcl , irf and irf ) up to less than -fold of the control ( figure a ). on the other hand, ifn-α strongly induced isgs (irf , irf , ifit and ddx ) up to more than -fold of the control ( figure b ). these results indicate that gemcitabine has a different mode of isg activation from ifn-α. in order to further confirm whether the isg activation by gemcitabine is mediated by the modulation of pyrimidine biosynthesis, we tested the effect of cytidine on the activation of ifit and ddx genes, the two strongest responders to gemcitabine. as expected, the supplemented cytidine considerably suppressed the gemcitabine-induced expression of ifit and ddx ( figure ). in contrast, cytidine had no significant effect on the ifn-induced ifit and ddx expression. to further clarify whether the stimulation of innate immune response by gemcitabine is independent of ifn-induced jak/stat pathway, we first examined the phosphorylation of stat at tyr , a major event occurred at the early step of ifn signaling. hela cells were treated with various doses of gemcitabine or ifn-α for hours or hours and then cell lysates were analyzed by western blotting. as a result, the levels of phosphorylated stat (ptyr ) proteins was not changed at all by gemcitabine at both and hours of treatment (supplementary figure a and c) . on the contrary, treatment with ifn-α remarkably increased the phosphorylation of stat protein at both time conditions (supplementary figure b and d) , as expected. in addition, we further examined whether the gemcitabineinduced isg expression involved irf , which forms ifnstimulated gene factor (isgf ) complex by interacting with the phosphorylated stat /stat and functions as a transcriptional activator of isgs [ ] . sirna-mediated knockdown of irf did not affect the gemcitabineinduced expression of ddx , while it had a significant reducing effect on that induced by ifn-α (decreased by ~ % of the control) (supplementary figure ) . collectively, these results demonstrate that the antiviral effect of gemcitabine involves the activation of isgs, the mode of which is different from ifn-dependent conventional way but which is mediated, at least partly, by the inhibition of pyrimidine biosynthesis discussion gemcitabine, currently in use for cancer therapy, has the antiviral activity against a broad range of rna viruses, including poliovirus [ ] , hcv [ ] , influenza [ ] , mers-cov [ ] and zikv [ ] . in the previous study, we also identified gemcitabine as an effective and potent anti-enteroviral agent [ ] . herein, we further defined the underlying mechanism of gemcitabine's antiviral action that involved the modulation of pyrimidine biosynthesis and the subsequent activation of isgs. there have been accumulating reports that nucleoside analogs act as the antiviral agents through the modulation of nucleotide biosynthesis [ , [ ] [ ] [ ] . as a cytidine analog, gemcitabine was also expected to work in this way. indeed, the antiviral activity of gemcitabine in both cvb -infected and cvb repliconharboring hela cells was significantly suppressed by the addition of exogenous pyrimidine nucleosides (cytidine and uridine) but not by purine nucleosides (figure and supplementary figure ) . further delineation demonstrated that gemcitabine acted through the salvage pathway rather than de novo pathway and targeted most probably uridine and/or cytidine synthesis from uracil and/or cytosine (figure and supplementary figure ) . therefore, it is conceivable that gemcitabine induces the decrease of pyrimidine nucleosides and their subsequent metabolites including ump. there have been a few reports that the inhibition of pyrimidine biosynthesis induces the innate immune response, especially the activation of isgs, which is the major antiviral mechanism in the early stage of virus infection [ , , ] . these previous findings were confirmed in our test by observing the activation of isre promoter by leflunomide, an inhibitor of dihydroorotate dehydrogenase (dhodh) on the pyrimidine biosynthetic pathway (supplementary figure ) . more importantly, we newly revealed that gemcitabine also activated the isre promoter ( figure a and c) . the activation of isre promoter by gemcitabine was further confirmed by the significant induction of all endogenous isgs (cxcl , irf , irf , ifit , and ddx ) tested, which was evaluated by quantitative real-time pcr analysis ( figure a) . intriguingly, the increase of ifit and ddx mrnas was particularly outstanding, and the increase of those mrnas was definitely suppressed by the additional treatment with cytidine, suggesting the role of ifit and ddx in the antiviral effect of gemcitabine. ifit family members are among the most highly induced isgs in response to ifn [ ] and there are many reports concerning their antiviral functions on various viruses. for instance, ifit proteins exhibit the antiviral effect generally by sequestrating viral genomes having '-triphosphates [ ] . they also inhibit hcv translation initiation by binding to eif , which is required for the efficient ribosome recruitment on hcv ires [ , ] . in addition, ifit's targeting of hpv e helicase, which is essential for viral dna replication [ ] , was reported as anti-human papillomavirus (hpv) effect of ifit protein. ddx , the other isg strongly stimulated by gemcitabine, encodes retinoic acid-inducible gene i (rig-i). it is part of the rig-i-like receptor family, which functions as a pattern recognition receptor that is a sensor for single-or double-strand rna of viruses such as flavivirus, influenza a and reovirus, and is involved in triggering an antiviral response [ ] [ ] [ ] . also, ifit and ddx proteins may majorly contribute to the antiviral action of gemcitabine in a way similar to the aforementioned mechanisms. still, we would not exclude the contribution of other isgs such as cxcl , irf , and irf , and other untested isgs as effectors to the full antiviral activity of gemcitabine. one of the most interesting observations from our study is that gemcitabine seems to activate the expression of isgs in a way different from ifn-α. first, isre promoter more slowly responded to gemcitabine, compared to ifn-α ( figure ). gemcitabine weakly activated the isre promoter at hours and almost fully at hours after treatment, which is quite contrary to ifn-α that achieved the full activation even at hours after treatment. second, the pattern of isg activation was quite different between gemcitabine and ifn-α (figure ). gemcitabine exhibited a relatively strong induction of ifit and ddx mrnas over irf and irf mrnas, while ifn-α had a similarly strong stimulatory effect on all of irf , irf , ifit and ddx mrnas. considering that all isgs contain stat / -binding element in their promoters, the mode of gemcitabine seems to be weakly dependent on stat / or independent of them, which is contrary to ifn-α that highly depends on them. third, the phosphorylation of stat (tyr ) was not induced by gemcitabine, while a remarkable induction of stat phosphorylation was observed by ifn-α (supplementary figure ) . fourth, the expression of ddx mrnas induced by gemcitabine was not affected by sirna-mediated irf knockdown, which is contrary to the result that the ifn-α-induced expression of ddx mrnas was significantly reduced at the same condition (supplementary figure ) . fifth, the gemcitabine-induced expression of ifit and ddx mrnas was significantly suppressed by the additional treatment with cytidine, while that by ifn-α was not affected ( figure ). this observation is of particular significance in that it strongly suggests a crosstalk between the pyrimidine biosynthesis and the innate immune response, which is induced by gemcitabine but not by ifn-α. decreased pyrimidine pool or inactivation of metabolic enzyme(s) might trigger a signal, which is delivered to certain cis-acting element on a subset of isgs possibly through the relay of some kinase(s). as mentioned above, this signal is less likely to be dependent on stat / -irf (ifn-stimulated gene factor ; isgf ) that is the major transcriptional complex in the ifn-induced jak/stat pathway [ ] . rather, the possible role of irf or irf needs to be alternatively evaluated. further investigation in the following study will clarify the underlying molecular mechanism of gemcitabine's antiviral action. our results, particularly the suppression of the gemcitabine-induced isg activation by cytidine ( figure ), indicate that the gemcitabine-induced innate immune response is mediated, at least partly, by its pyrimidinedepleting effect. moreover, according to the results in figure a and a, the gemcitabine's pyrimidine-depleting effect seems to occur mainly through its targeting of the salvage pathway. however, considering that neither excessive cytidine, uridine nor ump could achieve the full suppression of gemcitabine's antiviral effect but by up to - % of dmso control ( figure a and a) , there seems to be the other way of contribution that accounts for the residual portion of antiviral effect (~ - %). besides the salvage pathway of pyrimidine biosynthesis, gemcitabine is known to inhibit ribonucleotide reductase catalyzing the formation of dntps from ntps [ ] , which is another cause of nucleotide depletion. thus, the residual antiviral activity might be explained by these additional inhibition of nucleotide biosynthesis, seemingly resulting in the severe nucleotide depletion and the subsequent stronger isg activation. alternatively, gemcitabine could be incorporated into newly synthesized viral genome or directly inhibit rdrp activity, resulting in the reduced viral replication. in this study, we demonstrated the involvement of pyrimidine inhibition-induced innate immune response in the antiviral activity of gemcitabine, particularly by targeting the salvage pathway. the antiviral action of gemcitabine through innate immunity supported the feasibility of its therapeutic application as a broadspectrum antiviral drug. hela cells were purchased from atcc and cultured as previously described [ ] . the stock of cvb (nancy; atcc vr- ) was also purchased from atcc and amplified in hela cells. gemcitabine, nucleosides (adenosine, guanosine, cytidine and uridine), nucleobases (adenine, guanine, cytosine and uracil), pyrimidine synthetic intermediates [dihydroorotate (dho), orotic acid and uridine mono-phosphate (ump)], ifn-α- a and leflunomide were purchased from sigma-aldrich. rabbit polyclonal anti-cvb cpro antibody was generated in house by immunizing with cvb cpro recombinant protein. anti-rabbit secondary antibody conjugated with alexa fluor (af ) was purchased from life technologies. to examine the involvement of pyrimidine biosynthetic pathway in the antiviral activity of gemcitabine, hela cells were infected with cvb ( moi) and simultaneously treated with nucleosides, nucleobases or pyrimidine biosynthetic intermediates ( μm). at hours post-infection cells were fixed with icecold mixture of methanol-acetone ( : , v/v). after washing twice with pbs, infected cells were stained with rabbit polyclonal anti-cvb cpro antibody and anti-rabbit secondary antibody conjugated with af . nuclei were counterstained with hoechst (life technologies). images were captured and viral infection was quantified as previously described [ ] . infection was calculated as a percentage relative to dmso control. mtt assay was used for measuring cell viability as described previously [ ] . cell viability was calculated as a percentage relative to the control. plasmids p cb -luc and pribfluc-ev , which contain the firefly luciferase gene in place of the p capsid coding region of cvb and ev viral genomes, were kindly provided by frank j. m. van kuppeveld (utrecht university, the netherlands) [ , ] . synthesis of cvb and ev replicon rnas and replicon assays were performed as previously described [ ] . briefly, hela cells ( × cells/well) on a -well plate were transfected with . μg of replicon rna, split into well plates ( × cells/well), and simultaneously treated with nucleosides, nucleobases or pyrimidine biosynthetic intermediates in the presence of gemcitabine. eight hours after compound treatment, cells were assayed for the firefly luciferase activity using one-glo luciferase assay system (promega). hela cells ( × cells/well) in a -well plate were transfected with an isre-luciferase reporter plasmid using lipofectamine (invitrogen) and split into -well plates ( × cells/well). the ifn-stimulated response elements (isre)-luciferase reporter plasmid was described previously [ ] . forty-eight hours after transfection, cells were treated with gemcitabine or ifn-α for another or hours. thereafter, cells were assayed for the firefly luciferase activity using one-glo luciferase assay system (promega). total rnas from cells treated with compounds in a -well plate were prepared using rneasy mini kit (qiagen) and then cdnas were generated using superscript iv (life technologies) and random hexamers. for the quantification of isg expression, we performed a real-time pcr using ssofast evagreen supermix (bio-rad) and primers described in the previous report [ ] . the β-actin mrna was also quantified and used as the endogenous control [ ] . hela cells ( × cells/well) in a -well plate were treated with gemcitabine or ifn-α for or hours. cell lysates were prepared and subjected to western blot analysis with anti-stat and -phospho-stat (tyr ) antibodies. gapdh proteins were also analyzed as a loading control. more detailed procedures were described previously [ ] . anti-stat and -phospho-stat (tyr ) antibodies were purchased from cell signaling technology, and gapdh antibody was purchased from santa cruz biotechnology. hela cells ( × cells/well) in a -well plate were transfected with irf or negative control sirnas by using lipofectamine rnaimax reagent (invitrogen) for hours and then treated with gemcitabine or ifn-α for another hours. thereafter, total rnas were prepared and analyzed for the quantitative real-time pcr of ddx mrnas. the sirnas were synthesized by bioneer (daejeon, south korea). in figures, data are presented as the means ± standard deviations obtained from or more independent experiments. * , ** , and *** represent p< . , p< . , and p< . , respectively, which were analyzed by student's t-test. the authors declare no conflicts of interest. enterovirus infections: diagnosis and treatment host and virus determinants of picornavirus pathogenesis and tropism enterovirus isolated from cases of epidemic poliomyelitis-like disease in bulgaria an overview of the evolution of enterovirus and its clinical and public health significance antiviral activity of hederasaponin b from hedera helix against enterovirus subgenotypes c and c a pharmacologic activation of the innate immune system to prevent respiratory viral infections the anti-tumor agent, , -dimethylxanthenone- -acetic acid (dmxaa), induces ifn-beta-mediated antiviral activity in vitro and in vivo ro - enhances tlr and rlr agonist induced antiviral response regulators of innate immunity as novel targets for panviral therapeutics cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis e virus replication discovery of a broad-spectrum antiviral compound that inhibits pyrimidine biosynthesis and establishes a type interferon-independent antiviral state ribavirin enhances interferon-stimulated gene transcription by activation of the interferon-stimulated response element mycophenolic acid augments interferon-stimulated gene expression and inhibits hepatitis c virus infection in vitro and in vivo synergistic suppression of dengue virus replication using a combination of nucleoside analogs and nucleoside synthesis inhibitors synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses cellular growth kinetics distinguish a cyclophilin inhibitor from an hsp inhibitor as a selective inhibitor of hepatitis c virus activity of a novel combined antiretroviral therapy of gemcitabine and decitabine in a mouse model for hiv- obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection cell-based high-throughput screening assay identifies ', '-difluoro- '-deoxycytidine gemcitabine as a potential antipoliovirus agent repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism preclinical characteristics of gemcitabine genetic analysis of a hydrophobic domain of coxsackie b virus protein b: a moderate degree of hydrophobicity is required for a cis-acting function in viral rna synthesis structural and functional characterization of the coxsackievirus b cre( c): role of cre( c) in negative-and positive-strand rna synthesis inhibition of dengue virus through suppression of host pyrimidine biosynthesis interferon-stimulated genes and their antiviral effector functions mechanisms of type-i-and type-ii-interferonmediated signalling the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase ribavirin inhibits in vitro hepatitis e virus replication through depletion of cellular gtp pools and is moderately synergistic with alpha interferon -ethynyl- -beta-dribofuranosylimidazole- -carboxamide). a novel potent inhibitor of inosinate dehydrogenase activity and guanylate biosynthesis the isg /ifit gene family ifit is an antiviral protein that recognizes '-triphosphate rna structure and function of hcv ires domains alpha interferon induces distinct translational control programs to suppress hepatitis c virus rna replication interferon-inducible protein, p , inhibits hpv dna replication by binding to the viral protein e retinoic acidinducible gene-i-like receptors rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates the rna helicase rig-i has an essential function in doublestranded rna-induced innate antiviral responses induction of apoptosis by gemcitabine antiviral activity of micafungin against enterovirus hepatitis b virus-induced parkin-dependent recruitment of linear www.impactjournals.com/oncotarget ubiquitin assembly complex (lubac) to mitochondria and attenuation of innate immunity antiviral activity of kr- targeting nuclear export of influenza b virus ribonucleoproteins inactivation of human dgat by oxidative stress on cysteine residues key: cord- -atnrw ew authors: vabret, nicolas; blander, j. magarian title: sensing microbial rna in the cytosol date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: atnrw ew the innate immune system faces the difficult task of keeping a fine balance between sensitive detection of microbial presence and avoidance of autoimmunity. to this aim, key mechanisms of innate responses rely on isolation of pathogens in specialized subcellular compartments, or sensing of specific microbial patterns absent from the host. efficient detection of foreign rna in the cytosol requires an additional layer of complexity from the immune system. in this particular case, innate sensors should be able to distinguish self and non-self molecules that share several similar properties. in this review, we discuss this interplay between cytosolic pattern recognition receptors and the microbial rna they detect. we describe how microbial rnas gain access to the cytosol, which receptors they activate and counter-strategies developed by microorganisms to avoid this response. when janeway formulated the theory of pattern recognition in , he proposed that host cells could sense microbial infection owing to receptors able to recognize invariant molecular structures defined as pathogen-associated molecular patterns (pamps). these patterns would be present in groups of pathogens, but absent in the host ( ) . years later, janeway and medzhitov described the activity of the first mammalian member of the toll-like receptor (tlr) family, toll-like receptor ( ) . tlrs comprise a family of transmembrane proteins able to recognize conserved microbial features and activate the immune response ( ) . once activated, tlrs and others pattern recognition receptors (prrs) initiate several intracellular pathways, including those mediated by nuclear factor-κb (nf-κb), mitogen-activated protein kinases (mapks), and interferon regulatory factors (irfs). another outcome of activation of distinct members of cytosolic prrs is their oligomerization into multimeric cytosolic structures called inflammasomes, which activate the cysteine protease caspase- , subsequently leading to the production of biologically active forms of pro-inflammatory cytokines ( ) . initially thought to detect exclusively microbial derived ligands, prrs were later shown to recognize host derived danger signals, which are released in response to stress conditions such as cellular damage or tissue injury ( ) . under normal physiological conditions, these ligands are not accessible to their respective prrs and do not activate the immune system. conversely, it was first suggested that self-dna artificially introduced into the cytoplasm by transfection could activate nf-κb and the mapk pathway ( ) . evidence that any dna, regardless of its origin, can engage innate immune receptors when localized outside of the nucleus was further confirmed by the identification of several endosomal and cytosolic dna sensors [reviewed in ref. ( ) ]. in contrast to cytosolic dna, rna sensing in the cytoplasm raises many questions on the mechanisms used by the innate system to specifically distinguish non-self-rna from self-rna. during infection, microbial rnas share the cytosolic cellular compartment with several host rna species, including messenger rna (mrna), transfer rna (trna), ribosomal rna (rrna), microrna, and other small regulatory rnas. as a consequence, cytosolic sensors must display a high affinity for specific microbial features to avoid activation by host molecules that would otherwise elicit autoimmune responses. despite this apparent challenge, efficient detection of foreign rna in the cytosol is essential for innate immunity. during certain viral infections, rna may be the only microbial pamp produced throughout most of the replication cycle. additionally, our laboratory previously showed that recognition of bacterial mrna in the cytosol was critical to elicit a robust innate response against bacterial infection ( ) . finally, cytosolic sensing of pathogen invasion by non-immune infected cells provides the very first steps of innate response against infection, before phagocytosis-competent immune cells are recruited to the site of infection. in this review, we summarize the current understanding of cytosolic rna sensing. we describe instances in which microbial rnas gain access to the cytosol, the prrs they activate, their corresponding ligands and strategies developed by microorganisms to conceal their rnas. rna entry into host cells generally takes place during the first steps of a microbial infection. we distinguish four processes leading to the presence of microbial rna in the cytosol of eukaryotic cells, where it can engage host prrs (figure ). figure | cytosolic recognition of microbial rna. genomic rna from rna viruses access the cytosol immediately after the cell entry step of the replication cycle, where it may be amplified by viral rna-dependent rna polymerase (rdrp). genomic dna from dna viruses is transcribed by viral or cellular rna polymerase, including the cytosolic rna polymerase iii. bacterial rna can access the cytosol through the activity of auxiliary secretion systems or during passive leakage of phagosomal products. once in the cytosol, microbial rna binds different families of prrs classified as rlrs, non-rlr helicases, and other receptors. downstream signaling pathways include activation of mavs, trif, and the nlrp inflammasome. black arrows, rna entry; red arrows, signaling pathways. step of the replication cycle. viruses can directly release their genome at the plasma membrane after binding to a receptor. alternatively, they can be first internalized through endocytosis or macropinocytosis. endocytosed virus particles will typically traffic through endosomal vesicles by actin-dependent and/or microtubule-dependent transport ( ) . specific environmental triggers like endosomal ph acidification induce either fusion of enveloped virus with the endosome, or membrane penetration of viral proteins, allowing viral genetic material to be released into the cytoplasm ( ) . alternatively, viruses can spread by direct cell-cell contact ( ) . cell-to-cell transmission of viral material can activate cytoplasmic innate pathways, as exemplified with hepatitis c virus ( ), lymphocytic choriomeningitis virus ( ) , or human immunodeficiency virus transmission ( ) . other viral rna pamps can be produced during viral replication. david baltimore has defined a classification of viruses based on the mechanism of mrna production ( ) . viruses are clustered in seven groups depending on their genomes (dna, rna), strandedness (single or double), sense or antisense, and method of replication. the type of rna ligands produced during viral replication will depend on the type of viral genome and the strategy used to generate mrna. rna ligands can be generated by dna viruses and retroviruses via genome transcription, or by synthesis of mrna and replication intermediates by rna-dependent rna polymerases (rdrps) of rna viruses ( ) . it has been shown that ligands generated in phagolysosomes after phagocytosis of bacteria by immune cells can engage cytosolic innate immune receptors ( ) . similarly, we showed that rna from escherichia coli could activate receptors in the cytosol after phagocytosis by macrophages ( ) . we demonstrated that phagosomes carrying e. coli exhibit intrinsic leakiness, suggesting a mechanism by which bacterial rna, irrespective of the activity of virulence factors, can gain access to the cytoplasm ( ) . alternatively, bacteria express secretion systems to translocate products outside of the bacterial cell wall. in the case of intracellular bacteria, auxiliary secretion systems like seca in listeria monocytogenes have been shown to actively translocate listeria rna into the cytoplasm, resulting in activation of cytosolic sensors ( , ) . similarly, another study proved that cytosolic rna sensors participate in the type interferon (ifn-i) response to legionella pneumophila. although the authors did not demonstrate the translocation of legionella rna into the cytosol of infected cells, they discuss their data through a model where it would be the case ( ) . future studies looking for additional secreted rna will likely provide additional insights on their interaction with the innate immune system. two independent groups have demonstrated that cytoplasmic dsdna triggers ifn-i production via rna polymerase iii, which transcribes dna into '-triphosphate ( -ppp) rna, subsequently recognized by cytosolic rna receptors ( , ) . this pathway has been involved in the sensing of dna viruses, like epstein-barr virus, or intracellular bacteria, like l. pneumophila ( , ) . although the rna intermediate produced is not sensu stricto microbial, its generation is due to the activity of a microbial invader. the best-studied cytosolic rna sensors are the three members of rig-i-like receptors (rlrs), a subfamily of the dexd/hbox family of helicases. they consist of retinoic acid-inducible gene i (rig-i), melanoma differentiation factor (mda ), and laboratory of genetics and physiology (lgp ). they share a similar organization with three distinct domains: (i) a c-terminal repressor domain (rd) embedded within the c-terminal domain (ctd); (ii) a central atpase containing dexd/h-box helicase domain able to bind rna; and (iii) a n-terminal tandem card domain that mediates downstream signaling, and which is present in rig-i and mda but absent in lgp . upon activation by rna ligands, rig-i and mda are subsequently recruited to the adaptor protein mitochondrial antiviral signaling (mavs) via a card-card interaction and lead to activation of nf-κb and irfs ( ) ( ) ( ) ( ) . in contrast to tlr expression that is predominantly expressed in specialized immune cells such as macrophages and dendritic cells (dcs), rlrs are found in the cytosol of most cell types and are strongly induced in response to ifn-i ( , ) . the rig-i ligand has been characterized as an rna molecule with two distinct features: (i) a -ppp moiety ( , ) and (ii) bluntend base pair at the '-end ( , ) . blunt-end base pairs can be found in double-stranded rna (dsrna) and secondary rna structures such as hairpin or panhandle conformations ( , ) . recent structural studies have contributed toward a better understanding of ligand binding and activation of rig-i. specificity of -ppp binding is conferred by the ctd, and the helicase domain binds the double-stranded part of the rna. rig-i is normally held in an auto-repressed conformation, and ligand binding results in a conformational change, releasing the card domain which can subsequently initiate signaling by association with mavs ( ) ( ) ( ) . despite the increasing amount of high-resolution crystal data, the consensus definition of rig-i ligand remains controversial. other rig-i ligands have been indeed described in the literature including long ( ) or short dsrna ( ) ( ) ( ) lacking the -ppp. however, thermodynamic analysis have shown that the full-length human rig-i protein binds '-ppp dsrna with -fold higher affinity than '-oh dsrna, and dsrna with a -fold higher affinity than short single stranded rna (ssrna) lacking secondary structure ( ) . many viral families display blunt-end base-paired rna with a -ppp, directly in their genomic rna or in replication intermediates. consistent with this notion, rig-i has been shown to be involved in the recognition of many viruses, either antisense (−)ssrna viruses ( , ) or sense (+)ssrna/dsrna viruses ( , ) . notably rig-i can detect panhandle structures found in lacrosse viral particles ( ) or in influenza genomic rna ( , ) . sendai virus (sev) and other mononegavirales produce defective interfering (di) viral genomes containing panhandle structures that activate rig-i in infected cells ( ) . retinoic acid-inducible gene i recognition has not been limited to rna virus since rig-i is involved in recognition of dna viruses, such as epstein-barr virus or adenovirus through the rna polymerase iii pathway ( , , ) . moreover, rig-i is also able to detect bacterial infections. bacterial mrna are not capped and it has been estimated that approximately % of rna oligonucleotides in e. coli have a -ppp ( ) . reports in the literature describe sensing of l. monocytogenes secreted rna ( , ) or purified legionella ( ) and helicobacter pylori rna ( ) by rig-i. finally, rig-i can also sense shigella flexneri infection in macrophages through the rna polymerase iii pathway ( ) . melanoma differentiation factor ligand is less characterized than rig-i. using poly(i:c) as a synthetic dsrna mimic, studies have shown that mda binds long, but not short dsrna ( , , ) . structural analyses have demonstrated that mda specifically recognizes the internal duplex structure of dsrna and uses it as a platform to stack along dsrna in a head-to-tail arrangement. this mechanism promotes stochastic assembly of the tandem card oligomers that activates the signaling adaptor mavs ( ) . melanoma differentiation factor detects infection by viral families known to produce long dsrna structures during their replication cycle, including (+)ssrna viruses like picornaviruses, dsrna viruses like reoviruses, or dna viruses like poxviruses ( , ( ) ( ) ( ) ( ) . in the case of (+)ssrna virus infection, fluorescent imaging studies have confirmed that mda recognizes preferentially the dsrna generated during the replication of these viruses, but not the genomic ssrna ( ) . prior to the structural study mentioned above, multiple observations raised the possibility that there may exist additional mda ligands, different from the consensus long dsrna. thus, a study has shown that mda cooperates with the ribonuclease rnase l to induce ifn-i in response to a viral mrna from parainfluenza virus ( ) . interestingly, rnase l converts rna into small rna products, with shorter length than the current mda www.frontiersin.org ligand definition ( ) . another work published the same year has shown that mrna lacking '-o-methylation at their ' cap structure induces production of ifn-i through mda activation ( ) . however, the data published, which focus on coronavirus infection, did not elucidate whether the absence of methylation was directly recognized by mda or via another intermediate ( ) . after binding to their specific ligands, both rig-i and mda activate mavs to trigger a common signaling pathway. the majority of mavs is localized on the mitochondrial membrane and its engagement by rlrs causes a conformational change that propagates to adjacent un-activated mavs in a prion-like behavior ( ) . the formation of these very large mavs aggregates results in a largescale amplification of the signaling cascade. this cascade involves the recruitment of cytosolic adaptor molecules, followed by the activation of the canonical ikks, ikk-α, ikk-β, and ikk-γ, the mapk and the non-canonical ikk-related kinase, tbk and ikki/ε. ultimately, specific transcription factors, such as irf , nf-κb, and depending on the cell type irf and irf , are translocated to the nucleus where they promote the expression of ifn-i genes and pro-inflammatory cytokines [reviewed in ref. ( ) ]. finally, mavs has been recently shown to interact with nodlike receptor family, pyrin domain containing (nlrp ) and promote its recruitment to the mitochondria. the authors emphasize the central role of mavs in innate immune signaling events by showing its importance in the functioning of nlrp inflammasome and the production of il- β ( ) . of note, mavs independent activation of the nlrp inflammasome by rig-i has also been reported ( , ) . the third member of rlrs, lgp , is able to bind dsrna ( , ) , however, its role in immune activation is poorly understood. lgp was proposed to be a modulator of rlr signaling. studies showed that lgp was required for rig-i and mda activity, in particular during picornaviral infection ( ) ( ) ( ) . another work proposed that lgp would inhibit rig-i through competition with its ligand ( ) . it is however unclear whether lgp binds microbial rna in an infectious context, and what specific features of the rna it would recognize. further studies will be required to clarify the precise role of lgp . apart from rlr, several recent studies have highlighted the importance of other dexd/h-box helicases in microbial rna sensing. rna helicases of the dead box family are involved in various different steps of rna metabolism [reviewed in ref. ( ) ]. they share eight conserved motifs that are involved in atp binding, atp hydrolysis, nucleic acid binding, and rna unwinding activity. additionally, most dexd/h-box helicases contain auxiliary nand c-terminal domains that confer on them functional specificities, such as an ability to induce downstream signaling or to bind specific rna targets ( ) . ddx (ddx x) can bind poly(i:c) or vesicular stomatitis virus (vsv) rna and was shown to enhance the ifn-i response to vsv infection by interaction with the rlr-mavs complex. overexpression assays suggest that ddx precipitates with rig-i and mda ( ) . since ddx is easily detected in resting cells, the authors propose a sentinel role for this helicase, the activity of which would be required during the initial steps of viral infection. another study showed that upon sev infection, ddx interacts with ikkε, an essential component of the irf signaling pathway, increasing the induction of the ifn-β promoter ( ) . moreover, ddx is targeted by vaccinia virus protein k ( ) , an inhibitor of ifn-β production, and by hcv core protein, which can disrupt its interaction with mavs ( ) . these observations highlight the importance of ddx in efficient viral sensing. using overexpression and knock-down experiments, dhx was shown to be required for the production of ifn-i and proinflammatory cytokines in response to poly(i:c), influenza virus, and reovirus by a murine splenic dc line and bone-marrow derived dcs. dhx can bind dsrna via its dsrna-binding motif and interact with mavs through both its helicase c-terminal domain and ha -duf ( ). myeloid dcs have also been shown to express a complex composed of ddx , ddx , and dhx that triggers an antiviral program in response to poly(i:c), in a pathway dependent of the adapter molecule tir-domain containing adapter-inducing interferon-β (trif). ddx binds to poly(i:c) via its helicase a domain, while dhx and ddx bind the tir domain of trif via their ha -duf and prk domains, respectively. this complex seems to be required for the innate response against influenza or reovirus infection ( ) . notably, a separate study also characterized dhx and dhx as a sensor for the dsdna species cpg-a and -b, respectively. in this case, both dhx and dhx activate the cytosolic adapter protein myeloid differentiation primary response gene (myd ) by binding to its tir domain ( ) . another recent study by yong-jun liu's group identified another helicase, dhx , as a cytosolic rna receptor able to activate the nlrp inflammasome ( ) . dhx is involved in inflammasome activation after sensing cytosolic rna such as poly(i:c) or reoviral rna when directly delivered by lipofection to the cytoplasm of a macrophage cell line or human monocyte-derived macrophages. additional experiments suggested that dhx could also possibly be involved in detection of cytosolic bacterial rna. the authors showed that dhx can bind to dsrna through its helicase c domain and to nlpr through its dead domain ( ) . a few months later, another study performed on myeloid dcs confirmed the role of dhx in the sensing of cytosolic poly(i:c) and reoviral rna. surprisingly, in this case, poly(i:c)-induced activation of mapk, nf-κb, and irf was mediated by mavs, which binds the helicase c domain of dhx ( ) . ddx has also been shown to enhance the ifn-i response to rna and dna stimulation through formation of complexes with frontiers in immunology | molecular innate immunity rig-i, mda , and lgp but not with mavs. this complex formation has been deciphered with overexpression assays in the case of mda and lgp , and with endogenous rig-i during vsv infection. ddx expression is induced by viral infection and its helicase domain can bind ds-or ss-vsv rna generated in vitro, independently of the -ppp ( ) . interestingly, ddx can also bind dsdna, and was shown to play role in ifn-i expression after infection with herpes simplex virus- , a dna virus. this ability to bind both dsrna and dna raises the question of the feature ddx recognizes. it should be finally noted that the role of ddx in the ifn-i pathway has been questioned ( ) . several other cytoplasmic receptors have been shown to play a role in microbial rna recognition. this is the case for the cytoplasmic protein kinase r (pkr), which is important for antiviral activity. pkr is activated by dsrna from viruses and is a component of mapk and nf-κb signaling pathways [reviewed in ref. ( ) ]. activation of pkr can also be mediated by short -ppp rnas containing limited secondary structures ( ) . proteins from the interferon-induced protein with tetratricopeptide repeats (ifits) family, such as ifit and , bind -ppp of viral rna ( ) . using short in vitro transcribed oligonucleotides, crystal structure studies have demonstrated that ifit proteins contain a positively charged cavity designed to engage, without any particular sequence specificity, ssrna with a -ppp end. contrary to rig-i, ifit proteins cannot bind blunt-ended -ppp dsrna, and owing to the limitations imposed by their rna-binding pockets, ifit and ifit require '-overhangs of at least or nt, respectively ( ) . using a -o-methyltransferase mutant of japanese encephalitis virus, another study showed that ifit preferentially binds to capped -o-unmethylated mrna ( ) , confirming previous findings showing that -o-methylation of viral mrna caps promotes ifit evasion ( , ) . the mechanism of ifit antiviral action is not completely understood, and it has been proposed that ifit might sequester viral rnas ( ) or inhibit viral mrna translation ( ) . the crystal structure of ifit (known as isg ) was also described. ifit specifically binds adenylate uridylate (au)rich rnas in vitro, independently of the presence of a -ppp ( ) . the authors showed that rna-binding capacity of this protein mediates its antiviral properties, using a model of hek t cells infected by newcastle disease virus or sev ( ) . nucleotide-binding oligomerization domain containing protein (nod ) is a member of the nod /apaf- family and encodes a protein with two card domains and six leucinerich repeats (lrrs). nod is primarily known for its ability to recognize bacterial peptidoglycan, but it also plays a role in the antiviral response. nod has been shown to activate mavs after stimulation with viral ssrna or human respiratory syncytial virus infection ( ) . nlrp is involved in cytosolic rna sensing. caspase- cleavage triggered by influenza virus, sev, or bacterial mrna is dependent on nlrp inflammasome activation ( , , ) . however, direct binding of nod or nlrp to microbial rna has not been established. leucine-rich repeat flightless-interacting protein (lrrfip ) contributes to the production of ifn-β induced by vsv and l. monocytogenes in macrophages ( ) . mostly located in the cytosol, lrrfip can also be found in rna-containing lysosomes ( ) . lrrfip can bind both dsrna and dsdna and subsequently induce ifn-i expression through β-catenin phosphorylation. activated β-catenin is translocated to the nucleus and increases ifn-β expression by binding to the c-terminal domain of the transcription factor irf and promoting the recruitment of the acetyltransferase p to the ifnb promoter. several other microbial rna features have been suspected or proposed to act as potential signals for cytosolic sensing, suggesting the existence of receptors detecting these characteristics. a computational analysis identified cpg motifs in an au-rich rna as an immunostimulatory feature. this sequence motif is underrepresented in both ssrna viruses and host innate immune gene mrna, and its frequency in influenza virus genomes has decreased throughout evolution ( ) . since this evolutionary pressure seems to also be applied on host mrna, the implication of a cytosolic receptor is possible, although experimental studies identified endosomal tlr as a potential prr ( ) . another study identified the nucleotide bias of a-rich hiv- genome as a strong inducer of ifn-i and potent mediator of lentiviral pathogenicity. the authors showed that the ability of rna sequences derived from the hiv- genome to induce an interferon response correlated with their nucleotide bias and that codon-optimized sequences lost their stimulatory activity ( ) . the experimental procedure used in this study consisted of direct delivery via lipofection of in vitro transcribed rna sequences into the cytosol of a reporter cell line, suggesting a potential role for a cytoplasmic rna sensor ( ) . recently, our group identified bacterial mrnas as an activator of the nlrp inflammasome. polyadenylation of these rnas abrogated their immunostimulatory activities, suggesting that features at the end of mrna, rather than the end, could engage cytoplasmic cellular sensors ( ) . philip bevilacqua's group has shown that different nucleoside modifications on rna, such as base or sugar internal modifications, suppress their intrinsic ability to activate immune sensors, notably pkr. the authors propose that self-rna editing could be a mechanism used by the innate immune system to discriminate self-transcripts from "unmodified" microbial rnas ( , ) . conversely, microbial rna editing by cellular deaminase enzymes such as dsrna-specific adenosine deaminase (adar) have been shown to enhance its recognition by cytosolic sensors ( ) . other host transcript specificities, like association to cellular components that prevent prr binding, or specific tertiary structure such as the eukaryotic mrna closed loop conformation ( ) , could be determinants for the differentiation of host mrnas from microbial rnas. identification of receptors able to recognize such features are lacking so far. infectious microorganisms have developed several strategies to evade cytosolic sensing. one of these strategies, which we only mention briefly here, is the direct targeting by microbial proteins www.frontiersin.org some (−)ssrna viruses edit the -ppp moieties in their genomes as well as replication intermediates into mono-phosphates to avoid recognition by rlrs ( ) . arenaviruses produce rna panhandle structures with a -ppp containing a gtp overhanging nucleotide. this viral structure is suggested to act as a rig-i ligand decoy, by trapping rig-i but not activating it ( ) . we are beginning to understand how eukaryotic cells use nucleoside modifications in order to protect self-rnas from innate sensing. for example, higher eukaryotes have acquired the ability to -omethylate their mrnas, allowing cellular receptors to distinguish self from unmethylated non-self mrna through specific types of antiviral sensors such as mda and ifits ( , ) . consistent with the red queen hypothesis ( ) , which postulates that parasites have to constantly evolve in order to adapt to their host species, the same immune escape strategy has been mimicked by several pathogens, like flaviviruses ( , ) . similarly, -o-methylation of g (gm ) on bacterial trna suppresses activation of the immune response in plasmacytoid dcs ( , ) . flaviviruses and other viruses are also known to induce cellular membrane reorganization that allows them to replicate in subcellular compartments, creating new replication-dependent organelles ( ) . thus, tick-borne encephalitis virus or japanese encephalitis virus have been shown to rearrange endoplasmic reticulum membranes to provide a compartment where viral dsrna is concealed from prr recognition. this hijacking of internal cell membrane induces a delayed cytosolic exposure of viral rna to innate receptors and accordingly, ifn-i responses are only measured late in the replication cycle ( ) ( ) ( ) . the ns protein from influenza virus can prevent rna sensing through the formation of a chain of ns molecules along the influenza dsrna backbone ( ) . picornaviruses mask their ppp with a viral encoded protein, vpg, which functions as a cap and as a primer during rna synthesis. interestingly, studies have shown that vpg could be used to evade rig-i recognition ( ) . similarly, ebola virus vp assembles into dimmers to cap the ends of viral dsrna and hide the specific rig-i recognition site ( ) . while one vp monomer binds the terminus and backbone of dsrna, the other vp monomer binds only the phosphate backbone of the dsrna, displaying a unique mode of dsrna concealing from prr ( ) . another hemorrhagic fever virus, lassa fever virus, uses the '- ' exonuclease activity of its nucleoprotein (np) to degrade stimulatory dsrna ( ) . this activity seems to be shared by other arenaviruses ( ) . finally, the protein c from human parainfluenza virus type (hpiv ), a paramyxoviridae, has been shown to limit the accumulation of dsrna. cell infection by a virus mutant defective for the c protein displays higher accumulation of several viral rnas, including viral genome, antigenome, and mrna, eventually leading to the accumulation of dsrna. thus, by limiting intracytosolic quantities of viral dsrna, the c protein of hpiv avoids dsrna triggering of mda and pkr in infected cells ( ) . the multiplicity of prr pathways is an essential determinant of the immune system's ability to sense with precision the level of frontiers in immunology | molecular innate immunity microbial threat and to respond accordingly ( ) . however, as far as cytosolic rna sensors are concerned, it is striking to observe the contrast between the high number of prrs that have been isolated and the similarities of the pamps they recognize ( table ) . while -ppp and dsrna are undoubtedly powerful triggers of the innate immunity, they cannot account for the diversity of responses that the organism is able to elicit against a wide range of pathogens. our understanding of how the immune system distinguishes between foreign and self-nucleic acids will continue to improve over time. this will help us better define the precise role played by cytosolic rna sensors in the global immune response against pathogens. approaching the asymptote? evolution and revolution in immunology a human homologue of the drosophila toll protein signals activation of adaptive immunity approaching the asymptote: years later beyond pattern recognition: five immune checkpoints for scaling the microbial threat activation of target-tissue immune-recognition molecules by double-stranded polynucleotides immune sensing of dna detection of prokaryotic mrna signifies microbial viability and promotes immunity principles of virology cell-to-cell transmission of viruses plasmacytoid dendritic cells sense hepatitis c virus-infected cells, produce interferon, and inhibit infection human pdcs sense lcmv infected cells in vitro innate sensing of hiv-infected cells expression of animal virus genomes bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids rig-i detects triphosphorylated rna of listeria monocytogenes during infection in non-immune cells identification of host cytosolic sensors and bacterial factors regulating the type i interferon response to legionella pneumophila rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf visa is an adapter protein required for virus-triggered ifn-beta signaling expression analysis and genomic characterization of human melanoma differentiation associated gene- , mda- : a novel type i interferon-responsive apoptosis-inducing gene the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses '-triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates recognition of ' triphosphate by rig-i helicase requires short blunt doublestranded rna as contained in panhandle of negative-strand virus '-triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna structural insights into rna recognition by rig-i molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses the thermodynamic basis for viral rna detection by the rig-i innate immune sensor rig-i detects viral genomic rna during negative-strand rna virus infection incoming rna virus nucleocapsids containing a '-triphosphorylated genome activate rig-i and antiviral signaling differential roles of mda and rig-i helicases in the recognition of rna viruses innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing epstein-barr virus-encoded small rna induces il- through rig-i-mediated irf- signaling adenovirus virus-associated rnas induce type i interferon expression through a rig-i-mediated pathway the ' ends of rna oligonucleotides in escherichia coli and mrna degradation extracellular and intracellular pattern recognition receptors cooperate in the recognition of helicobacter pylori ifngamma inhibits the cytosolic replication of shigella flexneri via the cytoplasmic rna sensor rig-i essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses activation of mda requires higher-order rna structures generated during virus infection mda detects the double-stranded rna replicative form in picornavirus-infected cells innate immune response after adenoviral gene delivery into skin is mediated by aim , nalp , dai and mda visualisation of direct interaction of mda and the dsrna replicative intermediate form of positive strand rna viruses activation of ifn-&# ; expression by a viral mrna through rnase l and mda new insights into the role of rnase l in innate immunity ribose '-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response intracellular pathogen detection by rig-i-like receptors the adaptor mavs promotes nlrp mitochondrial localization and inflammasome activation recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production type i ifn triggers rig-i/tlr /nlrp -dependent inflammasome activation in influenza a virus infected cells the rig-i-like receptor lgp recognizes the termini of double-stranded rna the regulatory domain of the rig-i family atpase lgp senses doublestranded rna lgp is a positive regulator of rig-i-and mda -mediated antiviral responses atp hydrolysis enhances rna recognition and antiviral signal transduction by the innate immune sensor, laboratory of genetics and physiology (lgp ) lgp plays a critical role in sensitizing mda- to activation by double-stranded rna from unwinding to clamping -the dead box rna helicase family dexd/h-box rna helicases as mediators of anti-viral innate immunity and essential host factors for viral replication dead/h box (ddx ) helicase binds the rig-i adaptor ips- to up-regulate ifn-beta-inducing potential viral targeting of dead box protein reveals its role in tbk /ikkepsilon-mediated irf activation hepatitis c virus core protein abrogates the ddx function that enhances ips- -mediated ifn-beta induction dhx pairs with ips- to sense double-stranded rna in myeloid dendritic cells ddx , ddx , and dhx helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells aspartateglutamate-alanine-histidine box motif (deah)/rna helicase a helicases sense microbial dna in human plasmacytoid dendritic cells the dhx rna helicase senses cytosolic rna and activates the nlrp inflammasome the interaction between the helicase dhx and ips- as a novel pathway to sense double-stranded rna and rna viruses in myeloid dendritic cells ddx , a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling cytosolic sensing of viruses interferon-inducible antiviral effectors '-triphosphate-dependent activation of pkr by rnas with short stem-loops ifit is an antiviral protein that recognizes '-triphosphate rna structural basis for viral '-ppp-rna recognition by human ifit proteins ifit inhibits japanese encephalitis virus replication through binding to ' capped '-o unmethylated rna '-o methylation of the viral mrna cap evades host restriction by ifit family members '-o methylation of the viral mrna cap by west nile virus evades ifit -dependent and -independent mechanisms of host restriction in vivo crystal structure of isg reveals a novel rna binding structure and potential functional mechanisms activation of innate immune antiviral responses by nod critical role for cryopyrin/nalp in activation of caspase- in response to viral infection and double-stranded rna the nlrp inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the cytosolic nucleic acid sensor lrrfip mediates the production of type i interferon via a betacatenin-dependent pathway characterization of lrrfip patterns of oligonucleotide sequences in viral and host cell rna identify mediators of the host innate immune system oligonucleotide motifs that disappear during the evolution of influenza virus in humans increase alpha interferon secretion by plasmacytoid dendritic cells the biased nucleotide composition of hiv- triggers type i interferon response and correlates with subtype d increased pathogenicity nucleoside modifications modulate activation of the protein kinase pkr in an rna structure-specific manner native tertiary structure and nucleoside modifications suppress trna's intrinsic ability to activate the innate immune sensor pkr inosine-containing rna is a novel innate immune recognition element and reduces rsv infection the mechanism of eukaryotic translation initiation and principles of its regulation targeting of immune signalling networks by bacterial pathogens processing of genome ' termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction short doublestranded rnas with an overhanging ' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys a new evolutionary law identification of modifications in microbial, native trna that suppress immunostimulatory activity the '-o-methylation status of a single guanosine controls transfer rna-mediated toll-like receptor activation or inhibition viral reorganization of the secretory pathway generates distinct organelles for rna replication tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles delayed cytosolic exposure of japanese encephalitis virus double-stranded rna impedes interferon activation and enhances viral dissemination in porcine cells formation of membrane-defined compartments by tick-borne encephalitis virus contributes to the early delay in interferon signaling x-ray structure of ns from a highly pathogenic h n influenza virus the genome-linked protein vpg of vertebrate viruses -a multifaceted protein ebolavirus vp uses a bimodal strategy to bind dsrna for innate immune suppression structure of the lassa virus nucleoprotein reveals a dsrna-specific ' to ' exonuclease activity essential for immune suppression structures of arenaviral nucleoproteins with triphosphate dsrna reveal a unique mechanism of immune suppression the c proteins of human parainfluenza virus type limit double-stranded rna accumulation that would otherwise trigger activation of mda and protein kinase r the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -lr e w authors: lehtoranta, liisa; latvala, sinikka; lehtinen, markus j. title: role of probiotics in stimulating the immune system in viral respiratory tract infections: a narrative review date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: lr e w viral respiratory tract infection (rti) is the most frequent cause of infectious illnesses including the common cold. pharmacological solutions for treating or preventing viral rtis are so far limited and thus several self-care products are available in the market. some dietary supplements such as probiotics have been shown to modulate immune system function and their role in reducing the risk and the course of rtis has been investigated extensively within the past decade. however, the mechanism of action and the efficacy of probiotics against viral rtis remains unclear. we searched pubmed, google scholar, and web of knowledge for pre-clinical and clinical studies investigating the effect of probiotics on respiratory virus infections, immune response, and the course of upper and lower respiratory tract illness. the literature summarized in this narrative review points out that specific probiotic strains seem effective in pre-clinical models, through stimulating the immune system and inhibiting viral replication. clinical studies indicate variable efficacy on upper respiratory illnesses and lack proof of diagnosed viral infections. however, meta-analyses of clinical studies indicate that probiotics could be beneficial in upper respiratory illnesses without specific etiology. further studies aiming at discovering the mechanisms of action of probiotics and clinical efficacy are warranted. respiratory viruses cause the most common infectious illnesses in humans-acute rtis that can be divided into upper rtis (urti), e.g., the common cold, and lower rtis (lrti), e.g., bronchitis and pneumonia. these illnesses affect all age groups annually and cause a high burden on health care systems and global economics due to absenteeism from daycares, school, and work. over virus types have been identified as causative agents for respiratory illnesses [ , ] . in most cases, especially illnesses of the upper respiratory tract are mild to moderate and often self-limiting. on the other hand, lrtis leading to pneumonia can be especially fatal among children and the elderly, or immunocompromised subjects [ ] . in the past decade, studies linking the microbiota and immune system function have laid the foundation for the opportunities in microbiota modulation and bacterial therapeutics in health and disease. further, studies have associated the gut and airway microbiota with upper and lower respiratory tract health and immunity [ , ] . thus, modulation of the gut microbiota and immunity by dietary supplements or pharmaceuticals is of increasing interest in finding novel solutions to manage rtis. among the promising candidates are probiotics that have been studied for immune function modulation and viral infections. meta-analyses suggest that probiotics could be beneficial in the context of acute urti typically caused by viruses [ , ] . viruses that cause rtis are found in various virus families, which differ in virulence and utilize variable strategies to infect the host cells and to evade the host immune system [ , ] . respiratory viruses spread via nasal secretions that can be transmitted through the air or by hand-to hand and surface-to-hand contact [ ] . infection requires penetration of the virus through the host mucus layer, including the microbiota, and antiviral molecules in the mucus, such as antibodies and collectins. once on the mucosal epithelial cells, respiratory viruses attach to specific receptors, such as intercellular adhesion molecule (icam)- (rhinoviruses), peptidases (coronaviruses), or sialic acids (influenza viruses), that mediate the internalization of the virus by endocytosis [ , ] . the viral receptors are differentially expressed on host cells resulting in virus-specific host cell tropism that is one key factor in viral pathogenesis. for example, influenza viruses typically infect bronchial cells, whereas rhinoviruses infect the epithelial cells of the upper airways, resulting in differences in illness presentation. viral genomic structure can be, for example, positive-or negative-sense single-stranded (ss) or double-stranded (ds) rna or dna [ ] . most rti causing viruses, picornaviruses, influenza viruses, and coronaviruses are ssrna viruses, whereas adenoviruses are dsdna viruses. the genomic structures are recognized by different receptors in the host and activate different types of immune responses. some respiratory viruses, e.g., coronaviruses, are surrounded by a viral envelope which confers additional protection from the host immune system. respiratory viruses have further developed molecules that help in evading the immune response, for example, by disrupting the interferon (ifn) response and hijacking the host's cellular machinery for the production of virus copies [ ] . once the viruses have penetrated into the host cells, the epithelial and immune cells detect the viral structures by pattern recognition receptors (prr) of which toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) play a central role. tlr , tlr , tlr , and tlr are located in the endosomes and can identify viral ss (tlr and ) and ds (tlr ) rna structures, and dna (tlr ) [ ] . the recognition by tlrs leads to activation of transcription factor nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) and ifn regulatory factors (irf) , , and [ , , ] , resulting in expression of pro-inflammatory cytokines and type i ifns, ifn-α and ifn-β. type i ifns are broadly secreted by cells, but epithelial cells further secrete type iii lambda ifns in response to viral infections. cytoplasmic rnas, on the other hand, are recognized by rlrs, of which rig-i recognizes ssrna and melanoma differentiation associated (mda- ) dsrna. the activation of rlrs leads to type i (and type iii in epithelial cells) ifn production via mitochondrial antiviral signaling protein (mavs). type i and iii ifns induce an antiviral state in the surrounding cells which is not, however, necessarily sufficient to resist the infection, but delays the spreading of the infection [ , ] . the activation of rlrs, and tlrs by viral infection and cellular stress, leads to formation of nucleotide-binding oligomerization domain (nod)-, leucine-rich repeat (lrr)-, and pyrin domain-containing protein (nlrp ) inflammasome [ ] . although the role of nlrp is still somewhat unclear in viral rtis, it seems to play a role at least in rhinovirus, influenza, adenovirus, and rsv infections. nlrp inflammasome activation drives caspase -dependent il- β and il- cytokine response and inflammatory programmed cell death (pyroptosis) [ ] . epithelium-derived pro-inflammatory cytokines tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , chemokine (c-c motif) ligand (ccl) , ccl , chemokine (c-x-c motif) ligand (cxcl) , and cxcl induce innate cellular responses by attracting and activating natural killer (nk) cells, macrophages, and neutrophils that further amplify the innate cytokine and chemokine response [ ] . the role of other innate cells like, for example, intraepithelial lymphocytes, γδt cells, mucosa associated invariant t cells (mait), and innate lymphoid cells (ilc) is less well described in viral infections, however, they are likely to contribute to innate and adaptive responses against viral infections, as exemplified by the nk cells [ , ] and by the role of ilc cells in overcoming an influenza infection [ ] . if the innate immune or memory responses cannot clear the pathogen effectively and the adaptive immune system is unexperienced with the virus, an adaptive immune response is initiated and required. key are dendritic cells (dcs) that present the viral antigens and induce b and t cell responses against the pathogen in the secondary lymph nodes. b and t cell responses initiate within four-six days post-infection and peak later at days - depending on the respiratory virus [ ] [ ] [ ] . typically, common respiratory viruses, such as rhinovirus and influenza virus, are cleared before adaptive immune responses are activated [ , ] indicating that memory responses and innate immunity are essential in viral eradication. however, the induction of cytotoxic cd t cells, cd t cells, and antibody responses is key for virus eradication by adaptive immunity and for establishing protective immunity for secondary infections. the activation of the epithelium, innate immune cells, and adaptive responses is important for defense against respiratory viruses, but on the other hand, the host inflammatory response is the major cause of symptoms and more severe pathologies [ , , ] . chronic activation of cd t cell responses and adaptive immunity may lead to pulmonary damage and acute respiratory distress syndrome, like in severe cases of coronavirus infections (e.g., sars-cov or sars cov- ) and pandemic influenza virus infections [ , , ] . in milder colds, rhinoviruses are not cytolytic and do not actually cause considerable damage to host cells and may pass asymptomatically. presentation of cold symptom severity seems to correlate with host inflammatory response. specifically, the early expression of pro-inflammatory il- [ ] and high levels of neutrophils in nasal aspirates [ ] have been shown to correlate with symptom severity of rhinovirus and influenza infection [ , ] . production of anti-inflammatory il- , resolvins, and regulatory t cell responses acts as a natural mechanism to control lung inflammation during acute influenza virus (and others) infection [ ] [ ] [ ] . virus-host immune interactions are key to viral pathogenesis and to ultimately determine the outcome of the infection. probiotics, by definition, are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [ ] . most probiotics are lactic acid bacteria, belonging to lactobacillus spp., (now with new taxonomy including lacticaseibacillus spp., lactiplantibacillus spp., levilactobacillus spp., ligilactobacillus spp., limosilactibacillus spp. [ ] ) or bifidobacterium spp. furthermore, some strains of other microbial genera, such as propionibacterium spp., and bacillus spp., have been reported to have probiotic properties. traditional lactic acid bacteria have long been considered safe and suitable for human consumption as very few instances of infection have been associated with these bacteria, and several published studies have specifically addressed their safety (reviewed, e.g., by [ ] [ ] [ ] ). regarding probiotics safety in rtis, meta-analyses conclude that the reported side effects related to the consumption of probiotics were minor [ , , ] . probiotics are mostly consumed orally in the form of dietary supplements and food (e.g., yoghurt). therefore, their primary site of action is in the gastrointestinal (gi) tract [ ] . however, probiotics have been detected with pcr-based methods from nasopharyngeal mucosa, adenoids, and tonsils after oral consumption [ , , ] , but it is unclear what the contribution to upper respiratory tract immune stimulation against viral rti by probiotics is, as oral ingestion results in the stimulation of the intestinal immune system as well. the small intestine, that is naturally exposed to microbes and nutrients due to the thin mucosal layer, seems to play an important role in immune stimulation by probiotics [ , ] . however, dissecting the relative contributions of the small and large intestine and upper gi tract on immune stimulation against rti is challenging with available research data. independent of the actual mucosal inductor site of the probiotic, it has been shown that lymphocytes circulate between mucosal tissues [ ] . thus, local mucosal stimulatory effects may influence immune responses at other mucosal tissues and contribute to antiviral immunity. even very closely related bacteria have differences in their antigenic structures and thus influence the immune system uniquely. probiotics are thought to influence immune function primarily in a strain-specific manner [ ] . although these effects in general are strain-specific, probiotics also share common mechanisms of immune stimulation, such as the secretion of metabolites. for instance, short chain fatty acids are known to have immunomodulatory effects [ ] . direct effects of the probiotics on immune function are driven by interactions of bacterial structures or metabolites with receptors, like tlrs, on the host epithelial and immune cells. on the other hand, probiotics may influence immune function indirectly by changing the composition and/or activity of the host microbiota [ ] . for example, in the small intestine where the number of endogenous bacteria is lower than in the large intestine, ingestion of probiotics temporally changes the microbiota composition and influences the host immune response [ , ] . in rtis, orally consumed probiotics may elicit systemic effects from the gi tract via the "gut-lung axis" by modulating mucosal immune function [ , ] . probiotics are taken up by m cells or by cx c chemokine receptor (cx cr )+ macrophages located in the gut epithelium and then transferred to dcs in the subepithelial tissue. probiotics are able to modulate dc polarization and function [ ] that influence the subsequent t and b cell responses [ ] in the inductive sites (peyer's patch, and mesenteric lymph nodes). t and b cells can also enter the circulation and migrate to extraintestinal sites, such as the respiratory tract [ , ] . however, the exact mechanisms of probiotics (and their metabolites) in respiratory infections has not been clearly established and may be influenced by the investigated probiotic strain, microbiota composition, and immunological status of an individual. in the following chapters, we review the current pre-clinical evidence of immunomodulatory and antiviral mechanisms of probiotics against respiratory virus infections with a focus on the direct stimulatory effects of probiotics on virus-infected immune cells and animal models. probiotic bacteria can engage and activate tlrs leading to the activation of nf-κb and irfs in immune cells that are essential in antiviral defense. for example, it has been demonstrated in murine bone marrow-derived dcs that lactobacillus acidophilus ncfm and l. acidophilus x induce the upregulation of tlr , il- , and ifn-β in a tlr -dependent manner [ ] . in macrophage-derived raw . cells, lactobacillus gasseri sbt upregulated ifn-β and myxovirus resistance (mx) mrna expression [ ] and in human monocyte-derived macrophages, two lacticaseibacillus rhamnosus strains, gg and lc , induced type i ifn-dependent gene activation [ ] . pro-inflammatory and ifn-regulated genes including il- , il- , il- β, il- , ccl , cxcl , mx , and mx were induced by both lacticaseibacillus strains. in addition, the gene expression of tlr and tlr , receptors recognizing viral dsrna and ssrna, respectively, was upregulated by these strains. tlr gene expression was upregulated only by l. rhamnosus lc , the strain with higher antiviral potential, while tlr was moderately upregulated by both strains. in vitro studies with immune cells have also shown the ability of probiotics and their components to restrict viral replication. in human monocyte-derived macrophages, lacticaseibacillus strains showed the ability to prevent influenza a virus replication which correlated with the ability to activate type i ifn-dependent antiviral genes [ ] . similarly, mouse adapted influenza a virus (pr ) titer was reduced in raw . cells by l. gasseri sbt [ ] . in mouse bone marrow dcs, the inhibition of viral replication by l. acidophilus atcc s-layer protein was demonstrated [ , ] . priming of cells with s-layer protein prior to h n avian influenza virus infection inhibited the invasion and replication of the virus, stimulated the type i ifn signaling pathway, increased il- mrna, and decreased tnf-α mrna expression [ ] . polyinosinic/polycytidylic acid (poly i/c), a synthetic mimic of dsrna, is widely used in in vitro studies to stimulate tlr . it induces characteristic inflammatory responses associated with virus infections such as increased production of inflammatory cytokines. stimulation of airway epithelial cells with poly i/c has been found to closely mimic inflammatory responses associated with respiratory virus infections [ ] . poly i/c challenge in peripheral blood mononuclear cells (pbmcs) induced changes in the gene expression of tlr , ifn, and nf-kb-dependent pathways similar to acute viral infections [ ] . moreover, poly i/c was able to induce a pulmonary dysfunction similar to rsv in a mouse model [ ] . probiotic bacteria have shown the ability to modulate poly i/c-induced responses. studies with heat-killed lacticaseibacillus casei crl showed that the strain reduced tnf-α, ifn-γ, and il- levels when it was introduced before or simultaneously with poly i/c challenge in pbmcs alone and in a co-culture system with alveolar epithelial cell line a [ ] . il- and il- (also known as ifn-λ ) were induced in response to poly i/c together with heat-killed l. casei crl , indicating a boost in pro-inflammatory responses and the activation of anti-inflammatory and antiviral mechanisms. in the intestinal human colon cell line (hct ), regulation of poly i/c response by lactiplantibacillus plantarum subsp. plantarum du , latilactobacillus sakei du , and weissella cibaria du was examined [ ] . these strains modified poly i/c-induced expression of cytokines and antiviral genes by upregulating ifn-β, tlr , and rig-i while dampening the inflammatory response. moreover, the probiotic strains induced ifn-α, ifn-β, and il- and reduced the expression of inflammatory cytokines il- β and tnf-α in human monocytic thp- macrophages [ ] . in addition to the pro-inflammatory and antiviral gene activation described above, also direct interactions of viruses and probiotic bacteria have been demonstrated between porcine influenza a virus and enterococcus faecium in vitro [ ] . similar upregulation of ifn response by probiotics has been shown in several studies in intestinal epithelial cells [ ] [ ] [ ] and macrophages [ , ] . overall, the in vitro studies indicate that probiotics may stimulate similar innate immune pathways to respiratory viruses and potentially modulate virus-induced immune responses. several mouse studies have shown that the administration of probiotics can help to fight against viral rtis. the beneficial effects of oral probiotic supplementation on mouse survival and health status is well demonstrated [ , [ ] [ ] [ ] [ ] [ ] . for example, oral administration of lacticaseibacillus paracasei subsp. paracasei cncm-i- [ ] , l. gasseri lg [ ] , and bifidobacterium longum mm- [ ] reduced mortality and improved immune control in influenza-infected mice. probiotic administration resulted in better health status of the mice and lower virus loads in the lungs after influenza infection. in addition, the immune response to viruses was modulated by probiotic administration. for instance, l. paracasei cncm-i- modified the pro-and anti-inflammatory cytokine release in the lungs before and after influenza infection and affected total cell counts in the lungs after probiotic treatment [ ] . similarly, b. longum mm- suppressed inflammation in the lower respiratory tract through the decrease in influenza virus proliferation and pulmonary il- and tnf-α cytokine production [ ] . activation of host defense systems by increased ifn-γ, il- , il- , and il- gene expression and nk cell activation in lungs was also demonstrated. in non-infected mice, b. longum mm- significantly enhanced ifn-γ production by peyer's patch cells and splenic nk cell activity. in the infected mice, nk cell activity was significantly enhanced both in the spleen and lungs by the probiotic strain. another bifidobacterium (b. bifidum) improved anti-influenza immune responses by inducing both humoral and cellular immunities [ ] . decreased il- levels were detected in the lung and higher igg and igg levels in the sera of probiotic-treated mice compared with control mice. furthermore, l. gasseri sbt has been found effective in preventing both influenza a virus [ ] and rsv [ ] infections in mice. pre-treatment with l. gasseri sbt induced the expression of antiviral genes mx and - oligoadenylate synthase (oas) a in lung tissues before viral infection and reduced lung inflammatory responses after viral infection [ ] . the same l. gasseri strain was found effective in preventing rsv infection in mice by reducing lung viral loads and pro-inflammatory cytokines and by stimulating ifns and ifn responsive gene expression such as ifn-β , ifn-γ, interferon-inducible transmembrane protein (ifitm) , oas a, and interferon-stimulated gene (isg) [ ] . in addition to live probiotics, also orally administered heat-killed bacteria seem to confer protection against viral rtis in mice [ ] . heat-killed l. paracasei mcc reduced symptom scores and lung virus titers in influenza-infected mice and induced antigen-specific iga production in the small intestine, serum, and lungs. the proportion of iga+ b cells and follicular helper t cells (tfh) in peyer's patches was increased as was the gene expression of il- p , il- , il- , signal transducer and activator of transcription (stat) , and b cell lymphoma protein (bcl)- , which are associated with tfh cell differentiation. to conclude, different probiotic strains have been shown effective in inhibiting the replication of various respiratory viruses including influenza viruses and rsv in vitro. similar effects have been demonstrated in several mouse studies with the ability to reduce virus titers in lung tissues and modulation of antiviral and pro-inflammatory gene expression before and after viral infection. accumulating clinical evidence suggests that probiotics in general may have favorable effects against rtis. for instance, several systematic reviews and/or meta-analyses have evaluated the effects of prophylactic ingestion of probiotics on the rti-associated outcomes, e.g., either only in children [ , ] , or both in children and adults [ , , ] (table ) . of note, the majority of the outcomes in these analyses are related to urti, and data on lrti outcomes are either not available or are very limited. therefore, in the below chapter, we primarily focus on clinical trials on probiotics' effects on urti symptoms/episodes/duration. probiotic consumption had no effect on the duration of rtis ( rcts, n = , md - . in children (below years), the meta-analysis by wang et al., , reported that probiotic use compared with placebo significantly decreased the number of subjects having at least one rti episode, had fewer numbers of days of rtis per person, and had fewer numbers of days absent from daycare or school [ ] . however, the meta-analysis did not find a statistically significant difference on the illness episode duration between the probiotic and the placebo. laursen and hojsak [ ] limited the analysis to children up to years old and reported that probiotic use was associated with reduced risk of at least one urti and reduced the risk of antibiotic use, but the use was not associated with a reduction in rti duration or missed days of daycare due to rti [ ] . this meta-analysis also discussed the effects of the individual probiotic strains on rti outcomes. the results of the analysis showed that the most effective probiotic strains on rti-related outcomes were l. rhamnosus gg (rti duration) and l. acidophilus ncfm as a single supplement and in combination with b. lactis bi- (rti duration and antibiotic use). interestingly, these strains have shown in vitro the ability to induce antiviral ifn signaling pathways (see section . ) which may potentially explain their beneficial effects observed in rtis. however, as multiple studies with probiotic strains other than l. rhamnosus gg are limited or lacking, comparison and interpretation of the strain specific results should be made carefully. meta-analyses that pool data from clinical trials conducted with children, adults, and the elderly show that probiotic use is more beneficial over placebo in reducing the number of participants experiencing episodes of acute urti [ , ] , reducing antibiotic prescription rates for acute urtis [ , ] , and reducing the mean duration of an episode of an acute urti as well as cold-related school absences [ , ] . when the literature search was conducted, meta-analyses were not found in the databases searched on probiotic effects on respiratory infections restricted to the elderly population, potentially due to the fact that data are fairly limited regarding this age group. while there is consensus that probiotics could have potential in reducing the risk for rtis, it should be noted that clinical trials in the meta-analyses have been conducted in populations of different ages and genetic backgrounds, with various strains and/or their combinations, supplementation matrices, and doses. moreover, the measured outcomes and data collection procedures between the trials (i.e., infection episode definition) are not harmonized and therefore may vary considerably. consequently, pooling all the data creates a bias, as the probiotic effect is generally dependent on the dose, population, and strain. moreover, as discussed above, the probiotics effects on the immune system are strain-specific which affects the interpretation of the results. with regard to probiotics effects to specific respiratory viruses in clinical settings, several trials have characterized the respiratory infection etiology in infants [ ] , in children [ ] [ ] [ ] , in adults [ ] , and in the elderly [ ] . in addition, two clinical trials have investigated the efficacy of probiotics in an experimental rhinovirus challenge model [ ] [ ] [ ] (table ) . children had less days with respiratory symptoms per month ( . vs. . , p < . ). no effect on the occurrence of respiratory viruses during the study or respiratory symptoms associated with viral findings. l. rhamnosus gg cfu of live or heat-inactivated (by spray-drying) in ml of fruit juice or control juice daily for weeks. in the clinical trials conducted in free-living subjects in the community, no consistent data exist that show that specific probiotics would reduce the incidence of laboratory-confirmed respiratory virus infections as such. in preterm infants, the use of l. rhamnosus gg for days was associated with lower incidence of rhinovirus-induced episodes (comprising % of all rti episodes) compared with the placebo. however, l. rhamnosus gg had no effect on rhinovirus rna load during infections, duration of rhinovirus rna shedding, duration or severity of rhinovirus infection, or the occurrence of rhinovirus rna in asymptomatic infants. in children attending daycare, l. rhamnosus gg [ ] consumption for weeks did not reduce the occurrence of any of the common respiratory viruses either. in otitis-prone children, supplementation of a combination of l. rhamnosus gg, l. rhamnosus lc , b. breve , and propionibacterium jensenii js, for six months, reduced the number of human bocavirus-positive nasopharyngeal samples when compared with placebo, but not the number of rhino/enterovirus-positive samples [ ] . furthermore, in schoolchildren, the consumption of levilactobacillus brevis kb during influenza season was associated with lower incidence of physician-diagnosed influenza virus cases [ ] . in adults attending military service, the use of a combination of l. rhamnosus gg and b. lactis bb- for either or days was not overall associated with lower occurrence of common respiratory viruses upon presentation of cold symptoms [ ] . however, in a subgroup, there was a lower occurrence of rhino/enteroviruses after three months in the probiotic group when compared with the placebo. in nursing home residents, wang et al., , reported that the use of l. rhamnosus gg for six months was not associated with the reduction in occurrence of confirmed viral respiratory infections [ ] . the differences between the findings in these trials may be explained by the fact that these studies were conducted in various age groups with different immune system statuses (infants vs. children vs. healthy adults vs. the elderly), different seasons, as well as variable probiotic strains, strain combinations, doses, and variable lengths of intervention. furthermore, most of the studies were not designed for analyzing the viral infection etiology as the primary outcome and the diagnosis for the identification of the viral agent was not applied. since over respiratory virus types can cause respiratory infections and, in many cases, the infections and symptoms overlap, or the etiology is undiagnosed, the potential antiviral effects of probiotics against specific viruses can be difficult to determine in clinical trials targeting free-living subjects within the community. to overcome this caveat, two probiotics have been investigated in an experimental rhinovirus challenge model that allows investigation of the effect of a probiotic strain to a specific viral pathogen. in a rhinovirus (type ) challenge model, b. lactis bl- was administered for days prior and during five days of experimental rhinovirus infection to healthy volunteers [ ] . b. lactis bl- supplementation resulted in significantly lower rhinovirus titers in nasal washes during the infection as well as in a lower number of infected participants shedding the virus compared with the placebo. moreover, b. lactis bl- induced a significantly higher concentration of il- in nasal washes after days of supplementation and prior to infection. given the reduced viral titer, an increase in il- could indicate priming of the mucosal immune system prior to infection. this hypothesis is in line with a clinical study conducted in healthy active adults, where supplementation of b. lactis bl- reduced the risk of urti episodes compared with placebo [ ] . in another similar experimental rhinovirus type challenge pilot trial, no significant antiviral effect was seen with live or inactivated l. rhamnosus gg supplementation compared with placebo [ , ] , suggesting potential strain-specific differences on the efficacy of probiotics in respiratory virus infections. nevertheless, further adequately powered trials with harmonized study designs are necessary to draw conclusions on the efficacy of probiotics against specific respiratory viruses. viral rtis are the most common infections of mankind and the health and financial impact of seasonal epidemics and global pandemics on society is high. due to the large number of various respiratory viruses, the development of efficient therapies, such as vaccines, is challenging. when preventative measures are scarce or lacking, the role of a well-functioning immune system becomes crucial for providing resistance to an infection. within the past decade, research highlighting the importance of the microbiota on immune system function has raised interest in understanding the role of microbiota modulation and bacterial therapeutics by dietary and pharmaceutical solutions in health and disease. of the available solutions, probiotic bacteria have been studied for immune function modulation in the context of respiratory viral infections. in this review, we have summarized the current evidence on the effects of probiotics on antiviral immune function in vitro and in vivo, and clinical evidence on the effect of probiotics on viral rtis and on the course of rti (figure ). importance of the microbiota on immune system function has raised interest in understanding the role of microbiota modulation and bacterial therapeutics by dietary and pharmaceutical solutions in health and disease. of the available solutions, probiotic bacteria have been studied for immune function modulation in the context of respiratory viral infections. in this review, we have summarized the current evidence on the effects of probiotics on antiviral immune function in vitro and in vivo, and clinical evidence on the effect of probiotics on viral rtis and on the course of rti (figure ). in vitro data indicate that probiotics have strain-specific immunomodulatory effects on the host and immune cells by engaging tlrs that stimulate ifn pathways. the upregulation of ifn response seems to prime cells for better resistance against virus infection as probiotics were shown effective in inhibiting the replication of various respiratory viruses, including influenza viruses and rsv. similar effects have been demonstrated in mice with the ability of the probiotics to reduce virus titers in lung tissues and to modulate antiviral and pro-inflammatory gene expression before and after viral infection. interestingly, some studies in mice show an increase in il- response, suggesting control of the pro-inflammatory response that typically drives lung pathology in severe infections. most likely probiotics' effects in the gut are transferred into the respiratory tract via the gut-respiratory tract axis, however, this mechanism of action remains to be studied in more detail. the pre-clinical studies further show improvement in the symptom scores of mice, suggesting potential clinical benefits. indeed, some evidence exists for specific probiotic strains, e.g., from the species of l. rhamnosus, l. acidophilus, and b. lactis for their ability to induce antiviral immune responses in preclinical models, which is in agreement with their effects observed in clinical trials in reducing the risk of rti-associated outcomes. however, translation of probiotic effects from cell culture and animal studies to humans can be challenging and variable confounding factors, e.g., age, diet, microbiome, genetic and epigenetic immune status of an individual, study season, and variable viral epidemiology, all have an impact on the study outcome and are difficult to standardize. the clinical studies that have diagnosed and characterized viral etiology are limited, nevertheless, the metaanalyses investigating probiotic clinical interventions on rtis show that probiotic use is associated with lower incidence and duration of mild rtis, both in children and in adults. further studies aiming at discovering the mechanism of action of probiotics and establishing the association of immune system function stimulation and clinical efficacy are warranted. in vitro data indicate that probiotics have strain-specific immunomodulatory effects on the host and immune cells by engaging tlrs that stimulate ifn pathways. the upregulation of ifn response seems to prime cells for better resistance against virus infection as probiotics were shown effective in inhibiting the replication of various respiratory viruses, including influenza viruses and rsv. similar effects have been demonstrated in mice with the ability of the probiotics to reduce virus titers in lung tissues and to modulate antiviral and pro-inflammatory gene expression before and after viral infection. interestingly, some studies in mice show an increase in il- response, suggesting control of the pro-inflammatory response that typically drives lung pathology in severe infections. most likely probiotics' effects in the gut are transferred into the respiratory tract via the gut-respiratory tract axis, however, this mechanism of action remains to be studied in more detail. the pre-clinical studies further show improvement in the symptom scores of mice, suggesting potential clinical benefits. indeed, some evidence exists for specific probiotic strains, e.g., from the species of l. rhamnosus, l. acidophilus, and b. lactis for their ability to induce antiviral immune responses in pre-clinical models, which is in agreement with their effects observed in clinical trials in reducing the risk of rti-associated outcomes. however, translation of probiotic effects from cell culture and animal studies to humans can be challenging and variable confounding factors, e.g., age, diet, microbiome, genetic and epigenetic immune status of an individual, study season, and variable viral epidemiology, all have an impact on the study outcome and are difficult to standardize. the clinical studies that have diagnosed and characterized viral etiology are limited, nevertheless, the meta-analyses investigating probiotic clinical interventions on rtis show that probiotic use is associated with lower incidence and duration of mild rtis, both in children and in adults. further studies aiming at discovering the mechanism of action of probiotics and establishing the association of immune system function stimulation and clinical efficacy are warranted. the common cold epidemiology of viral pneumonia. clin microbiome and disease in the upper airway the influence of the microbiome on respiratory health probiotics for preventing acute upper respiratory tract infections probiotics for preventing acute upper respiratory tract infections respiratory virus infections emerging respiratory viruses other than influenza sars-cov- viral load in upper respiratory specimens of infected patients epidemiology 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infection-a randomized controlled trial probiotic supplementation for respiratory and gastrointestinal illness symptoms in healthy physically active individuals key: cord- -fymfqvp authors: channappanavar, rudragouda; perlman, stanley title: pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology date: - - journal: semin immunopathol doi: . /s - - -x sha: doc_id: cord_uid: fymfqvp human coronaviruses (hcovs) can be divided into low pathogenic and highly pathogenic coronaviruses. the low pathogenic covs infect the upper respiratory tract and cause mild, cold-like respiratory illness. in contrast, highly pathogenic hcovs such as severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov) predominantly infect lower airways and cause fatal pneumonia. severe pneumonia caused by pathogenic hcovs is often associated with rapid virus replication, massive inflammatory cell infiltration and elevated pro-inflammatory cytokine/chemokine responses resulting in acute lung injury (ali), and acute respiratory distress syndrome (ards). recent studies in experimentally infected animal strongly suggest a crucial role for virus-induced immunopathological events in causing fatal pneumonia after hcov infections. here we review the current understanding of how a dysregulated immune response may cause lung immunopathology leading to deleterious clinical manifestations after pathogenic hcov infections. coronaviruses belong to the virus family coronaviridae and are enveloped, positive-sense rna viruses. the coronavirus genome is approximately kb, making these viruses the largest known rna viruses [ , ] . coronaviruses infect a variety of host species, including humans and several other vertebrates. these viruses predominantly cause respiratory and intestinal tract infections and induce a wide range of clinical manifestations [ , ] . coronaviruses infecting the respiratory tract have long been recognized as significant pathogens in domestic and companion animals and as the cause of mild and severe respiratory illness in humans [ , ] . in general, coronaviruses infecting humans can be classified into low pathogenic hcovs, which include hcov- e, hcov-oc , hcov-nl , and hcov-hku and highly pathogenic covs such as severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov) [ , ] . low pathogenic hcov infect upper airways and cause seasonal mild to moderate cold-like respiratory illnesses in healthy individuals. in contrast, the highly pathogenic hcovs (pathogenic hcov or hcov hereafter) infect the lower respiratory tract and cause severe pneumonia, which sometimes leads to fatal acute lung injury (ali) and acute respiratory distress syndrome (ards), resulting in high morbidity and mortality [ ] [ ] [ ] [ ] [ ] . highly pathogenic hcovs pose a substantial threat to public health. during the - epidemic, sars-cov infected approximately individuals with a . % overall mortality rate [ , ] . more recently, mers-cov crossed species to infect individuals resulting in deaths (∼ % mortality rate) as of april , [ , ] . recent identification of sars-like coronaviruses in bats and mers-cov in domesticated camels makes it likely that these viruses will continue to cross species barriers and cause additional outbreaks in human populations [ ] [ ] [ ] [ ] . these highly pathogenic hcovs cause a wide spectrum of clinical manifestations in humans, with a large fraction of patients developing short period of moderate clinical illness and a small but a substantial number of patients experiencing severe disease characterized by ali and ards [ ] [ ] [ ] ] . thus, there are basically two groups of patients, those developing milder disease, which resolved and those with severe disease, which was commonly fatal. the disease severity in pathogenic hcov infections was also influenced by several factors such as initial viral titers in the airways and age and comorbid conditions of the infected individual. while younger individuals below years experience mild-moderate clinical illness, elderly individuals exhibit worse outcomes after infection with sars-cov or mers-cov [ , , ] . additionally, individuals with comorbid conditions such as diabetes, obesity, heart failure, and renal failure among others experience severe disease, particularly after mers-cov infection [ , ] . despite several years of research, specific factors causing the unusually high morbidity and mortality following pathogenic hcovs are incompletely understood. virus-induced direct cytopathic effects and viral evasion of host immune responses are believed to play major roles in disease severity. however, studies from humans who died of sars and more recent studies in animal models suggested that a dysregulated immune response occurred, resulting in an exuberant inflammation and lethal disease. in this review, we discuss recent advances in our understanding of hcov pathogenesis, with a special emphasis on cytokine storm and immunopathology as causes for deleterious consequences during hcov infections. sars-cov infection in humans resulted in an acute respiratory illness that varied from mild febrile illness to ali and in some cases ards and death [ , ] . the clinical course of sars presents in three distinct phases. the initial phase was characterized by robust virus replication accompanied by fever, cough, and other symptoms, all of which subsided in a few days. the second clinical phase was associated with high fever, hypoxemia, and progression to pneumonia-like symptoms, despite a progressive decline in virus titers towards the end of this phase [ ] . during the third phase, ∼ % of patients progressed to ards, which often resulted in death [ , ] . because of a progressive decline in virus titers, the third phase is thought to have resulted from exuberant host inflammatory responses. the most common clinical manifestations of mers include flu-like symptoms such as fever, sore throat, nonproductive cough, myalgia, shortness of breath, and dyspnea, which rapidly progress to pneumonia [ , ] . other atypical presentations include mild respiratory illness without fever, chills, wheezing, and palpitations. mers-cov in humans also causes gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea. the majority of mers patients with dyspnea progress to develop severe pneumonia and require admission to an intensive care unit (icu). although most healthy individuals present with mild-moderate respiratory illness, immunocompromised and individuals with comorbid conditions experience severe respiratory illness, which often progressed to ards [ ] . overall, mers-cov caused severe disease in primary index cases, immunocompromised individuals and in patients with comorbid conditions, but secondary cases of household contacts or healthcare workers were mostly asymptomatic or showed mild respiratory illness. gross and microscopic pathology of sars typically, analyses of lungs from patients who succumbed to sars showed lung consolidation and edema with pleural effusions, focal hemorrhages, and mucopurulent material in the tracheobronchial tree. diffuse alveolar damage (dad) was a prominent histological feature in sars lungs [ , ] . other changes included hyaline membrane formation, alveolar hemorrhage, and fibrin exudation in alveolar spaces with septal and alveolar fibrosis observed during later stages [ , ] . staining for viral antigen revealed infection of airway and alveolar epithelial cells, vascular endothelial cells, and macrophages [ , ] . furthermore, sars-cov viral particles and viral genome were also detected in monocytes and lymphocytes [ ] . in addition to these changes, histological examination of lungs from patients who died of sars revealed extensive cellular infiltrates in the interstitium and alveoli. these cellular infiltrates included neutrophils and macrophages with macrophages being the predominant cell type [ , ] . these results correlated with increased numbers of neutrophils and monocytes and lower cd and cd t cell counts in the peripheral blood samples of patients with fatal sars [ - ]. despite numerous laboratory-confirmed cases and deaths due to mers-cov infection in several countries, only one autopsy report of mers in humans is available. analysis of lung tissue from this patient showed pleural, pericardial, and abdominal effusions associated with generalized congestion, edema, and consolidation of lungs [ ] . similar to sars-cov infection, dad was a prominent feature in the lungs. additionally, epithelial cell necrosis, sloughing of bronchiolar epithelium, alveolar edema, and thickening of alveolar septa were also noted. immunohistochemical examination showed that mers-cov predominantly infected airways and alveolar epithelial cells, and endothelial cells and macrophages. the severity of lung lesions correlated with extensive infiltration of neutrophils and macrophages in the lungs and higher numbers of these cells in the peripheral blood of mers patients [ ] . cytokines and chemokines have long been thought to play an important role in immunity and immunopathology during virus infections. a rapid and well-coordinated innate immune response is the first line of defense against viral infections, but dysregulated and excessive immune responses may cause immunopathology [ ] [ ] [ ] . although there is no direct evidence for the involvement of pro-inflammatory cytokines and chemokines in lung pathology during sars and mers, correlative evidence from patients with severe disease suggests a role for hyper-inflammatory responses in hcov pathogenesis. additionally, sars-cov-infected airway epithelial cells (aecs) also produce large amounts of ccl , ccl , ccl , and cxcl [ ]. the delayed but excessive production of these cytokines and chemokines is thought to induce a dysregulated innate immune response to sars-cov infection. high serum levels of pro-inflammatory cytokines (ifn-γ, il- , il- , il- , and tgfβ) and chemokines (ccl , cxcl , cxcl , and il- ) were found in sars patients with severe disease compared to individuals with uncomplicated sars [ ] [ ] [ ] [ ] . conversely, sars patients with severe disease had very low levels of the anti-inflammatory cytokine, il- [ ] . in addition to pro-inflammatory cytokines and chemokines, individuals with lethal sars showed elevated levels of ifn (ifn-α and ifn-γ) and ifn-stimulated genes (isgs) (cxcl and ccl- ) compared to healthy controls or individuals with mild-moderate disease [ - ]. these results were the first to suggest a possible role for ifns and isgs in the immunopathogenesis of sars in humans. thus, it appears from these studies that dysregulated and/or exaggerated cytokine and chemokine responses by sars-cov-infected aecs, dcs, and macrophages could play an important role in sars pathogenesis. similar to sars, mers-cov infection of human airway epithelial cells induces significant but delayed ifn and proinflammatory cytokine (il- β, il- , and il- ) responses [ ] . while mers-cov replicates both in naïve and activated human monocyte-macrophages and dcs, only activated t cells support mers-cov replication [ ] [ ] [ ] . this is in contrast to sars-cov, which abortively infected monocyte-macrophages, dcs, and t cells. mers-cov infection of thp- cells, a monocyte cell line, and human peripheral blood monocyte-derived macrophages and dendritic cells induced delayed but elevated levels of pro-inflammatory cytokines and chemokines such as ccl- , ccl- , ccl- , il- , and il- [ , ]. however, induction of ifn-α/β by monocytemacrophages and dcs was not substantial except for plasmacytoid dendritic cells, which produced copious amounts of ifns upon mers-cov infection [ ] . recent studies showed elevated levels of serum pro-inflammatory cytokines (il- and ifn-α) and chemokines (il- , cxcl- , and ccl ) in individuals with severe mers compared to those with mild to moderate disease [ , ] . high serum cytokine and chemokine levels in mers patients correlated with increased neutrophil and monocyte numbers in lungs and in the peripheral blood, suggesting a possible role for these cells in lung pathology [ , , ]. several inbred mouse strains have been evaluated to study sars-cov pathogenesis. mice infected with the human strain of sars-cov (sars-cov-urbani) were permissive to virus replication but developed only mild lung pathology and clinical illness [ ] . subsequently, isolation of mouseadapted strains of sars-cov (e.g., sars-cov-ma ) allowed studies of lethal sars [ ] [ ] [ ] . ma infects airway and alveolar epithelial cells and epithelial cells of other organs [ ] . young mice of many strains (e.g., c bl/ , ) support ma replication in the lungs but are resistant to developing significant clinical disease [ , ] . in contrast, young balb/c mice infected with ma develop lethal disease characterized by diffuse alveolar damage, enhanced monocyte/macrophage and neutrophil accumulation, pulmonary edema, and hyaline membrane formation [ ] . furthermore, aged mice of all strains develop lethal clinical disease and succumb to infection [ , , ] . in addition to mouse models, sars-cov infection of aged rhesus macaques resulted in significantly more pathology than young adult animals [ ] . these animal models replicated several key features of sars-cov infection in humans and were thus useful for investigating sars pathogenesis. studies in animal models have been particularly useful in elucidating the role of cytokines and chemokines in mediating lung immunopathology following hcov infections. infection of non-human primates (nhps) with sars-cov induced a dysregulated immune response resulting in increased disease severity in aged but not young nhps, despite similar viral titers in the airways [ ] . since enhanced expression of genes regulating inflammation but not virus titers correlated with disease severity, an exaggerated immune response is thought to induce lethal disease in aged nhps [ ] . similarly, in sars-cov-infected balb/c mice, disease severity in aged mice correlated with early and disproportionately strong upregulation of ards-associated inflammatory gene signatures [ ] . in a recent study, we identified a pathogenic role for ifn-i in mice infected with ma . our results showed that rapid sars-cov replication in balb/c mice induced a delayed ifn-α/β response accompanied by an excessive influx of pathogenic inflammatory monocyte-macrophages (imms) [ ] . the accumulating imms themselves produced additional levels of monocyte chemo-attractants such as ccl , ccl , and ccl (through ifn-α/β receptor stimulation), resulting in further accumulation of pathogenic imms, which in turn enhanced disease severity. these infiltrating imms produced elevated levels of pro-inflammatory cytokines such as tnf, il- , il -β, and inos. blocking ifn signaling, depleting imms, or neutralizing a single inflammatory cytokine, tnf, protected mice from lethal sars-cov infection. additionally, ifn-α/β or imm-derived pro-inflammatory cytokines sensitized t cells to undergo apoptosis, further impeding virus clearance [ ] . in another study of sars-cov infection, loss of tir-domain-containing adapter-inducing interferon-β (trif), an adapter molecule for tlr and tlr signaling, resulted in a distinct inflammatory signature characterized by neutrophil and other inflammatory cell infiltration [ ] . a dysregulated immune response to sars-cov in trif-deficient mice was associated with aberrant antiviral ifn (ifn-α and ifnβ), pro-inflammatory cytokine and chemokine (il- , tnf, ifn-γ, and ccl ), and interferonstimulated gene (rsad , ifit , and cxcl ) responses. notably, virus titers were significantly higher in tlr −/− and trif −/− mice compared to their wt controls [ ] . although the viral factors regulating the pro-inflammatory response of neutrophils and monocyte-macrophages remain to be identified, the e protein of sars-cov has been shown to enhance pro-inflammatory cytokine and chemokine and inflammasome activity via its ion channel activity [ ] [ ] [ ] . these results support the notion that higher virus titers and dysregulated cytokine/chemokine responses cause a bcytokine storm^with lung immunopathological changes following sars-cov infection. animal models employed to study mers include rhesus macaques, rabbits, marmosets, and mice among others. mers-cov challenged rhesus macaques developed mild to moderate disease [ ] . similarly, mers-cov-infected rabbits displayed mild clinical disease with mild-moderate perivascular, peribronchiolar infiltration, and to a lesser extent lung interstitial inflammation [ , ] . in contrast, marmosets displayed moderate-severe respiratory disease characterized by broncho-interstitial pneumonia, alveolar edema, and fibrin deposition [ ] . marmosets with severe disease showed increased neutrophil and macrophage infiltration in alveoli and interstitial septa, although whether marmosets develop severe disease remains controversial [ , ] . although gross and histological lesions and inflammatory cell infiltration in mers-cov infected marmosets resemble human disease, there are no data available describing cytokine and chemokine responses in these animals. small laboratory animals, particularly rodents, do not support mers-cov replication due to inability of mers-cov-spike protein to bind to human dpp (hdpp ) orthologs in these animals [ ] . the first mouse model to study mers was generated by intranasal transduction of adenovirus encoding hdpp . these mice developed mild to moderate pneumonia, especially in immunodeficient mice [ ] . several hdpp transgenic mouse models developed thereafter exhibited variable organ tropism and disease severity, depending on the promoter driving the hdpp expression [ , ] . more recently, hdpp knock-in mice in which hdpp is expressed under the mouse hdpp promoter have also been described. these mice also developed moderate clinical disease after infection with human isolates of mers-cov [ ] . we and others recently developed a similar mouse model and showed that serial passage of human isolate of mers-cov resulted in mouse adaptation. mice infected with this adapted virus caused lethal respiratory illness and will be useful for studies of pathogenesis [ , ] . overall, delayed and aberrant antiviral and proinflammatory cytokine production in mers-cov-infected human macrophages and dendritic cells and high serum proinflammatory cytokine levels in patients with severe disease compared to mild-moderate clinical disease suggesting that possible dysregulated and enhanced cytokine responses promote lung pathology following mers-cov infection. to counter innate antiviral cytokine responses, sars-cov and mers-cov encode several structural and non-structural proteins (nsps) that antagonize antiviral immune response. sars-cov encoded nsp , nsp -macrodomain, nsp deubiquitinase (dub), and orf b, orf , and orf b subvert antiviral response by antagonizing ifn and isg responses [ ] [ ] [ ] [ ] [ ] [ ] . while nsp impairs ifn responses by unknown mechanism, nsp inhibits ifn responses by blocking stat phosphorylation [ , ] . additionally, structural proteins such as the membrane (m) and nucleocapsid (n) proteins dampen ifn signaling by inhibiting tbk /ikke and by unknown mechanisms, respectively [ ] [ ] [ ] [ ] . similarly, mers-cov structural proteins m and n and accessory proteins orf , orf a, and orf b antagonize ifn responses [ , , ] . it should be noted that most if not all of these putative antiviral mechanisms were demonstrated in transient expression assays and whether they are actually important in the context of infectious virus remains to be determined. structural and nonstructural protein antagonism of ifn responses further amplifies inflammatory responses by promoting unrestrained virus replication resulting in increased viral pamps that further dampen ifn signaling and stimulate prrs to induce an aberrant inflammatory response. lack of ifn signaling also leads to an excessive accumulation of ly c low monocytes and neutrophils. despite several years of research studying sars and mers pathogenesis, specific host factors that drive lung pathology following hcov infections are relatively unknown. however, a careful review of the literature related to sars-cov and mers-cov pathogenesis in humans and animal models highlights several key factors that may play a crucial role in the initiation and progression of an exacerbated inflammatory responses. studies from animal models, especially mouse models, provide correlative evidence for differential disease outcome if the viruses predominantly infect airway epithelial cells versus both airway and alveolar epithelial (type i and type ii pneumocytes) cells. in b and strains, both of which are permissive to virus replication but resistant to developing clinical disease, viral antigen is predominantly located in airway epithelial cells early after infection. in contrast, in highly susceptible balb/c mice, virus antigen is detected in the lung airways and in alveolar type i and ii pneumocytes (fig. ) . these results suggest a critical role for hcov-infected type i and ii pneumocytes in mediating lung pathology and host susceptibility. cov-specific t cells are crucial for virus clearance and limit further damage to host [ , ] . additionally, t cell responses also dampen overactive innate immune responses [ , ] . exuberant inflammatory responses caused by pathogenic hcov diminish the t cell response, in the case of sars-cov infection via tnf-mediated t cell apoptosis, thus resulting in uncontrolled inflammatory response. . accumulation of alternatively activated macrophages and altered tissue homeostasis: in some sars patients with extended duration of disease, dad was accompanied by fibrosis of interstitial and alveolar spaces and hyperplasia of pneumocytes. similar histological features were noticed in lungs of sars-cov-challenged stat −/− mice on b and backgrounds. lungs from these mice revealed an enhanced perivascular infiltration of alternatively activated macrophages, neutrophils, and fibroblasts accompanied by extensive fibrin deposition and alveolar collapse, features observed during end stage ali and ards in humans [ , ] . further studies revealed that abrogation of stat signaling, specifically in myeloid cells, resulted in alternative activation of macrophages [ ] . in addition, a delicate balance between host coagulation and fibrinolysis processes regulates tissue remodeling and ali [ ] . . ards: inflammatory mediators play a key role in the pathogenesis of ards, a primary cause of death in patients infected with sars-cov or mers-cov [ , ] . several pro-inflammatory cytokines, including il- , il- , il- β, and gm-csf, reactive oxygen species, and chemokines such as ccl , ccl- , ip- , and ccl contribute to ards [ , , ] . additionally, uncontrolled epithelial cell proliferation and impaired tissue remodeling during later stages induce ards leading to pulmonary fibrosis and death. a summary of causes and consequences of cytokine storm and immunopathology to hcov pathogenesis is demonstrated in fig. . high virus titers and subsequent exuberant inflammatory cytokine and chemokine responses correlate with high morbidity and mortality observed during pathogenic hcov infections. a systematic review of therapeutic effects of several commonly used antiviral and immunomodulatory agents used during sars outbreak showed inconclusive results [ ] . similarly, therapeutic interventions aimed towards reducing viral load were somewhat beneficial when administered early but not during later stages of mers-cov infection [ ] [ ] [ ] . these results suggest that besides controlling viral load, novel strategies directed at attenuating inflammatory responses will likely improve clinical outcomes. here, we describe agents that have the potential to mitigate hcov-induced inflammation. corticosteroid therapy corticosteroids are a class of steroidal hormones that exert anti-inflammatory functions and are generally used to suppress inflammatory conditions. during the sars epidemic, corticosteroids were the mainstay of immunomodulatory therapy. the timely administration of corticosteroids often leads to early improvement in terms of reduced fever, resolution of radiographic lung infiltrates, and better oxygenation [ ] [ ] [ ] . however, while some studies showed no beneficial effect, other demonstrated adverse outcomes following corticosteroid therapy during sars-cov infection in humans. early treatment of corticosteroids in sars patients enhanced plasma viral load in non-icu patients, thus leading to exacerbated disease [ ] . overall, these results show that the timing, dosage, and duration of corticosteroid therapy are critical if this intervention is to be beneficial in hcov infections. in general, corticosteroid therapy is not recommended for treatment of hcov respiratory infections. interferons pegylated and non-pegylated interferons have been under investigation for therapeutic purposes in hcovinfected individuals. however, therapeutic use of these agents produced mixed results both in humans and animal models of hcov infections. early administration of ifn was marginally beneficial in reducing viral load and resulted in moderate improvement in clinical manifestations. in contrast, delayed administration of ifn did not have any advantage compared to placebo controls. similarly, early administration of combination of ifn and ribavirin modestly ameliorated disease severity but did not affect mortality [ , , , ] . other possible therapeutics ifn-αβ inhibitors and ifn-λ ifn-αβ restrict virus replication through induction of isgs. however, ifn-αβ can also exacerbate disease by enhancing recruitment and function of imms and other innate immune cells. while an early interferon response was protective in sars-cov-infected mice, delayed ifn-αβ signaling dysregulated the anti-sars-cov immune response suggesting that timing of ifn therapy is critical in determining the disease outcome. based on these results, the administration of ifn-αβ receptor blockers or antagonists should be considered as an option to prevent exuberant inflammatory responses during later stages of severe disease, particularly during sars [ ] . in contrast to ifn-αβ, ifn-λ mainly activates epithelial cells and lacks monocytemacrophage-mediated pro-inflammatory activity of ifn-αβ [ ] . additionally, ifn-λ suppresses neutrophil recruitment to the site of inflammation [ ] . since sars-cov and mers-cov predominantly infect aecs and ifn-λ stimulates antiviral gene in epithelial cells without over-stimulating the immune system, use of ifn-λ may be an ideal therapeutic option. suppression of oxidized phospholipids oxidized phospholipids (oxpl) have been shown to promote ali by increasing lung macrophage cytokine/chemokine production via tlr -trif signaling in influenza a virus (iav)-infected mice [ ] . in a recent study, therapeutic administration of the tlr antagonist, eritoran, protected mice from lethal iav infection by reducing the levels of oxpl and inflammatory cytokines and chemokines [ ] . despite potent immunomodulatory functions, eritoran has no direct antiviral activity, suggesting its use in the amelioration of inflammatory responses. since pathogenic human coronaviruses cause acute lung injury and promote oxpl production in the lungs [ ] , strategies to suppress oxpl either by using eritoran or other similar compounds could be of value in dampening hcovinduced inflammation. sphingosine- -phosphate receptor agonist therapy in mice infected with iav, sphingosine- -phosphate receptor fig. schematic representation of protective versus pathogenic inflammatory responses to pathogenic hcov infections (s p ) signaling in endothelial cells was shown to orchestrate pathogenic inflammatory responses [ ] . targeted s p agonism restrained excessive inflammatory cell recruitment, suppressed pro-inflammatory cytokines and chemokines, and reduced iav induced morbidity and mortality [ , ] . , so that sars-cov infection of endothelial cells may drive s p -mediated inflammatory cytokine/chemokine responses and neutrophil and macrophage accumulation. therefore, s p agonism could be a potential therapeutic agent in hcov patients to dampen pathogenic cytokine and chemokine responses, if a role for an excessive immune response by these cells is demonstrated. inhibitors of monocyte recruitment and function studies in animal models demonstrate pathogenic roles for imms during lethal hcov infections. in a mouse model of cardiac inflammation, systemic delivery of optimized lipid nanoparticles containing a ccr -silencing short interfering rna (sirna) efficiently degraded ccr mrna and impaired imm recruitment to sites of inflammation thus resulting in improved disease outcome [ , ] . since hcovs are single-stranded rna (ssrna) viruses and stimulation of imms with the tlr agonist, r (a synthetic ssrna mimic), induces strong inflammatory responses, it is possible that immspecific tlr- signaling promotes excessive inflammation in response to hcov infection. thus, a tlr antagonisttargeted approach to mitigate inflammation could prove beneficial. other immunomodulatory agents several other immunomodulatory agents that could ameliorate inflammatory responses following pathogenic hcov infections include cytokine/chemokine inhibitors and danger-associated molecular pattern (damp) antagonists [ ] . studies from animal models show a significant contribution of tnf to acute lung injury and impaired t cell responses in sars-covchallenged mice. in vivo neutralization of tnf activity or infection of mice lacking tnfr provides protection against sars-cov-induced morbidity and mortality [ , ] . however, it is to be noted that tnf was not detected in the serum of sars patients at least during later stages of infection. inflammation is an indispensable part of an effective immune response, without which successful elimination of an infectious agent is difficult. the inflammatory response begins with the initial recognition of a pathogen, which then mediates immune cell recruitment, eliminates pathogens, and ultimately results in tissue repair and return to homeostasis. however, certain viruses such as highly pathogenic covs, iav, and ebola viruses induce excessive and prolonged cytokine/ chemokine response known as bcytokine storms,^which results in high morbidity and mortality due to immunopathology. although studies reviewed in this manuscript provide evidence that bcytokine storms^and immunopathology can occur during pathogenic hcov infections, we do not yet have a sufficient understanding of the specific factor/s responsible for exuberant inflammatory responses. studies from human autopsies and animal models strongly suggest a pathogenic role for inflammatory cytokines/chemokines derived from imm and neutrophils. therefore, therapeutic interventions targeting these pro-inflammatory cytokines and chemokines could prove beneficial in ameliorating undesirable inflammatory responses. additionally, since high virus titers at early and later stages of infection strongly correlate with disease severity in humans, strategies directed at controlling viral load as well as attenuating the inflammatory response might prove beneficial. therefore, future studies should focus on identification of specific signaling pathways that mediate inflammatory responses in hcov-infected patients and animals. 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sphingosine analog provides protection against pathogenic influenza virus silencing of ccr in myocarditis therapeutic sirna silencing in inflammatory monocytes in mice immunomodulatory therapy for severe influenza the effect of inhibition of pp and tnfalpha signaling on pathogenesis of sars coronavirus acknowledgements we thank dr. anthony fehr for careful review of this manuscript. this work was supported in part by grants from the n.i.h. (po ai , ro ai ). key: cord- -qek jy c authors: lee, su jeen; park, hyo‐jung; ko, hae li; lee, jung eun; lee, hyun joo; kim, hun; nam, jae‐hwan title: evaluation of glycoprotein e subunit and live attenuated varicella‐zoster virus vaccines formulated with a single‐strand rna‐based adjuvant date: - - journal: immun inflamm dis doi: . /iid . sha: doc_id: cord_uid: qek jy c introduction: varicella‐zoster virus (vzv), a human alphaherpesvirus , elicits both chickenpox and shingles and/or postherpetic neuralgia. a live attenuated vaccine (lav) and glycoprotein e (ge) subunit vaccine were developed to prevent vzv‐induced diseases. we recently reported that single‐strand rna (ssrna) based on the intergenic region of the internal ribosome entry site of cricket paralysis virus (crpv) is an effective adjuvant for protein‐based and virus‐like particle‐based vaccines. here, chinese hamster ovary expression system and an lav from oka/sk strains. methods: we appraised the adjuvant effect of the same crpv ssrna encoding the ge gene formulated in the two vaccines using vzv‐primed c bl/ mice and guinea pigs. humoral immunity and cell‐mediated immunity were assessed by enzyme‐linked immunosorbent assay (elisa) and elispot in ge subunit vaccine and by elisa and fluorescent antibody to membrane antigen in lav. results: the ge subunit vaccine‐induced ge‐specific antibodies and cd (+) t‐cell responses (indicated by interferon‐γ [ifn‐γ] and interleukin‐ secretion) in the ssrna‐based adjuvant containing the vzv ge gene. therefore, an ssrna adjuvant combined with ge antigen can trigger the innate immune response and induce an adaptive immune response to ultimately activate humoral and cell‐mediated responses. vzv lav could also induce vzv‐specific antibodies and ifn‐γ stimulated by lav, whereas the effect of ssrna as a vaccine adjuvant could not be confirmed. however, the ssrna adjuvant increased vzv‐specific neutralizing antibody response. conclusions: taken together, these results highlight that the ge subunit vaccine and lav developed in this study can be functional vzv vaccines, and ssrnas appear to function better as adjuvants in a subunit vaccine than in an lav. varicella-zoster virus (vzv) induces chickenpox (varicella), shingles (herpes zoster), and/or postherpetic neuralgia. varicella is the primary vzv infection and it occurs most frequently in children. herpes zoster occurs mainly in adults or immunocompromised hosts as a consequence of latent vzv reactivation. vzv is a member of the human herpesvirus family encoding five major glycoproteins designated gpi-gpv. glycoproteins are critical factors for vzv entry and replication. thus, they are attractive targets for antiviral drug development. vzv ge among vzv glycoproteins is the most abundant and immunogenic. it participates in viral replication and cell-cell transmission. moreover, it contains b-cell and cd + t-cell epitopes and elicits complementdependent neutralizing antibodies and cell-mediated immunity. vzv-specific cd + t cells synthesize th -like cytokines such as interleukin- (il- ) and interferon-γ (ifn-γ). they induce major histocompatibility complex class ii-restricted cytotoxicity. , therefore, cd + t cells expressing il- and ifn-γ were selected as immune markers to evaluate cell-mediated immune responses to vzv vaccines. , - vzv ge is an attractive candidate for the development of vzv subunit vaccines because the vzv ge antigen, also known as cd + t-cell antigen, is capable of inducing both humoral and cell-mediated immune responses. [ ] [ ] [ ] [ ] vaccines currently used to prevent vzv include live attenuated vaccine (lav) developed by takahashi and colleagues in and several other varicella vaccines licensed in several countries. the herpes zoster lavs, zostavax (merck & co., inc., darmstadt, germany) and skyzoster (sk bioscience co ltd, andong, korea), have been licensed. lav has preventive efficacy against varicella in the range of % to %. in contrast, its preventive efficacy against herpes zoster is only~ %. lav promotes relatively lower vzv-specific cellular immune responses against herpes zoster in older patients as immunosenescence accompanies the aging process. immunosenescence is characterized by decreased t-cell numbers and impaired t-cell function. a subunit vaccine comprising vzv ge and the liposome-based adjuvant shingrix (glaxosmithkline biologicals, rixensart, belgium) was licensed in . its reported efficacy against herpes zoster was %. therefore, the subunit vaccine may have greater efficacy against herpes zoster than lav. a recombinant subunit vaccine is a potential alternative to live attenuated herpes zoster vaccine. however, the low immunogenicity of individual viral proteins may have to be enhanced with adjuvants. adjuvants are immunomodulating substances that may be combined in formulations to increase their immunostimulatory efficacy. certain adjuvants have been approved for clinical use. all new adjuvants should be compared with the gold-standard aluminum-based adjuvants (alum). alum has been used to increase vaccine formulation efficacy for > years. however, there are still limitations of alum that require supplementation to boost vaccine efficacy. for example, alum typically induces a th response, which mediates the differentiation of b cells that secrete th -cellassociated antibody isotypes, as opposed to inducing a very low th response that is required to activate innate immune response, , as a prerequisite to guarantee a more effective and/or long-term immune response. the ideal adjuvant induces innate immune responses, thereby activating adaptive immune responses. , toll-like receptor (tlr) agonists and oil-in-water emulsions have been developed to complement alum adjuvants. , our new candidate adjuvant is a single-strand rna (ssrna) derived from the cricket paralysis virus (crpv) intergenic region (igr) of the internal ribosome entry site (ires). it induces balanced th /th responses, enhances innate immune response, and increases vaccine efficacy. here, we modified this novel ssrna adjuvant to encode the vzv ge gene. the latter was then tested in a ge subunit vaccine expressed in a chinese hamster ovary (cho) cell culture platform and lav bearing the oka/sk strain. in vzv-primed mice, we assessed whether this ssrna adjuvant induces humoral-and cell-mediated immunity in the vzv ge subunit vaccine. adjuvants are not usually included in lav formulations. however, we used guinea pigs to evaluate the ability of the ssrna adjuvant to enhance neutralizing antibody production and cell-mediated immune response in vzv lav. based on all results, we showed the potential of ssrna adjuvants to compensate for the limitations of protein-based vaccines with respect to low t-cell activity and short-term responses, as well as to increase the neutralizing antibody in lav for preventing vzv-induced disease. a truncated form of the vzv ge antigen with a deleted transmembrane (tm) domain was used in this study. gene cloning for the ge antigen was performed with a primer and a vector (pbudce . ) ( table ) after isolating the dna from oka/sk wvss (working virus seed stock). the vzv ge antigen was expressed in a cho-k (atcc ccl- ) cell line via a cho transfection kit including lipofectamine ltx-plus (no. - ; invitrogen, carlsbad, ca). single-cell colonies were obtained according to ge expression in isolated single cells. an enzyme-linked immunosorbent assay (elisa) identified four stable cell clone candidates with high antigen expression that were adapted in serum-free media. after serial passages, only one clone was selected. cells with confirmed ge antigen expression were cultured to l. the antigen was purified via serial ultrafiltration/diafiltration (uf/df) steps and anion exchange chromatography and concentrated to kda for immunization. uf/df was performed on a -kda pellicon membrane (no. p b a ; pellicon mini ultrafiltration module biomax; emd millipore, billerica, ma). samples were loaded onto a tmae(m) column (minichrom column fractogel® tmae (m); merck, darmstadt, germany) and eluted with mm nacl in mm piperazine buffer. the eluted samples were concentrated in a -kda centricon® plus- centrifugal filter (emd millipore). the final mass of the total antigen after concentration and purification was mg. the rna platform was designed with crpv igr ires and sv late-polyadenylation signal sequences. the rna platform consisted of four restriction enzyme sequences and a multicloning site between the untranslated regions to permit the insertion of the vzv orf (ge) gene. the dna platform was designed with crpv igr ires and sv late-polyadenylation signal sequences. dna templates were linearized with noti. in vitro transcription was performed with an ez t high-yield in vitro transcription kit (enzynomics, seoul, korea). three micrograms of linearized dna template were incubated with t transcription buffer, mgcl , mm dithiothreitol, enhancer solution, mm rntp, nuclease-free water, and units t enzyme mix for hour at °c. the transcripts were incubated with rnase-free dnase i (promega, madison, wi) for minutes at °c. the reaction was terminated by incubation at °c for minutes. rna was purified by the licl method. rna purity and concentration were evaluated with a nano-drop spectrophotometer (thermo fisher scientific, waltham, ma). the rna integrity was evaluated by denaturing gel electrophoresis. a cells were cultured in a six-well plate for hours at a density of × per well in a medium free of % (v/v) fetal bovine serum. the cells were then stimulated with μg ssrna adjuvant for hours, lysed by vortexing at -min intervals for hour in radioimmunoprecipitation assay buffer containing halt protease and phosphatase inhibitor cocktail, and centrifuged at g for minutes. the resulting supernatant was used as a whole-cell lysate. fifty-microgram protein was loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and electrophoretically transferred to a polyvinylidene fluoride membrane. the membrane was incubated for hours with the indicated vzv-antibody (cha biotech, seoul, korea) and then incubated for hours with horseradish peroxidaseconjugated goat anti-mouse antibody. the protein band of interest was visualized with a chemidoc imaging system (bio-rad laboratories, hercules, ca). equal protein six-week-old c bl/ mice were primed with vzv bulk (oka/sk; sk bioscience co ltd) at a dose of~ pfu mouse − . thirty-five days after priming, vzv ge protein ( μg vzv antigen mouse − ) formulated with μg ssrna adjuvant was injected twice into the upper thigh muscles at -wk intervals between inoculations. the mice were immunized in the same way with addavax (cat. no. vac-adx- ; μg; invivogen, san diego, ca) as a reference control. five groups were designated as follows: negative control (g ); lav priming (g ); ge antigen (g ); addavax (g ); and ssrna adjuvant (g ). six-week-old dunkin-hartley guinea pigs were primed with vzv bulk (oka/sk; sk bioscience co ltd) at a dose of~ pfu guinea pig − . thirty-five days after priming, the guinea pigs were subcutaneously injected twice with a human dose ( . ml) of live attenuated herpes zoster vaccine (skyzoster) with or without ssrna adjuvant ( μg) at -week intervals between inoculations. three groups were designated as follows: negative control (g ); lav (g ); and ssrna adjuvant (g ). vzv-specific total immunoglobulin g (igg), igg , and igg a in mouse serum and total igg, igg , and igg in guinea pig serum were measured by eelisa. the -well plates (nunc maxisorp tm ; thermo fisher scientific) were coated with ng well − vzv ge for mice and pfu well − vzv for guinea pigs and incubated overnight at °c. the wells were then blocked with μl of % (v/v) skim milk for hour at room temperature (rt). diluted serum samples and vzv ge ab (no. - ; raybiotech, inc, peachtree corners, ga) were added to the plates and incubated for hours at rt. the wells were then washed three times with μl phosphate-buffered saline (pbs) mixed with . % (v/v) tween (pbst). the following antibodies were then added: anti-mouse igg (ab ; abcam, cambridge, uk), igg (ab ; abcam), and igg a ab (ab ; abcam) or anti-guinea pig igg (ab ; abcam), igg (abin ; antibodies, cambridge, uk), and igg ab (gagp/igg /po; nordic mubio, susteren, the netherlands). the mixtures were then incubated for hour at °c. after washing, , ′, ′ ′-tetramethylbenzidine (tmb) substrate was added to the wells and the mixtures were incubated for minutes. a stop solution was then added to halt the reaction. optical densities were measured at nm in a microplate reader. the spleen from a mouse immunized with vzv ge antigen was washed with rpmi media and lysed with ammonium-chloride-potassium lysing buffer. complete media were then added to the sample, the suspension was centrifuged for minutes at g, and the pellet was washed with pbs and recentrifuged to obtain the splenocytes. one hundred microliters of the cells was loaded in each well ( cells per well). the stimulation factor phytohemagglutinin (pha; l ; μg ml − ; sigma-aldrich corp, st louis, mo) served as a positive control. then ge protein ( μg ml − ; sk bioscience, co ltd, andong, korea) and pepmix vzv ge ( . μg ml − ; pm-vzv-ge; jpt peptide technologies, berlin, germany) were added and the suspension was incubated for hours at °c. the cells were washed with pbst and anti-murine ifn-γ and il- biotin detection ab (immunospot® murine ifn-γ and il- single-color enzymatic enzyme-linked immune absorbent spot (elispot) kit; cellular technology ltd., shaker heights, oh) was added to each well. the plate was incubated for hours at rt and washed three times with μl pbst. strep-ab solution from the aforementioned kit was diluted to : . then μl of this dilution was added to each well and the suspensions were incubated for minutes at rt. eighty microliters of diluted substrate solution was then added to each well. the wells were rinsed with distilled water to stop the reaction, the plates were allowed to air-dry, and the spots were counted. cultured guinea pig splenocytes ( ) harvested at days after the second immunization with live vzv bulk containing oka/sk strains and ssrna adjuvant were mixed with a live virus ( plaque-forming unit, pfu) and pha ( μg ml − ). the cells were incubated for hours at °c and plated in a -well plate coated with antibodies against ifn-γ (guinea pig interferon-γ elisa kit; abx ; abbexa ltd, cambridge, uk) or il- (guinea pig il- elisa kit; abx ; abbexa ltd). the standards and samples were added to the wells and the suspensions were incubated and washed with wash buffer in the kit. one hundred microliters of biotin-conjugated antibody against ifn-γ was used for detection. after washing, μl tmb substrate was added to the wells. the suspensions were incubated for minutes at °c to visualize horseradish peroxidase activity. to stop the reaction, μl stop solution was added to each well. optical densities were measured in a microplate reader at nm to calculate the ifn-γ or il- concentration. fluorescent antibody to membrane antigen (fama) was used to measure the production of neutralizing antibodies to vzv. to determine the anti-vzv igg level, μl dulbecco's phosphate-buffered saline (dpbs) was added to u-bottom -well plates. guinea pig serum was serially diluted from : to : . cell-associated virus ( μl) from infected cells was added to the wells and the suspensions were incubated for minutes at °c. after centrifugation at rpm for minutes, the supernatant was removed and the cells were washed with % (v/v) gelatin-dpbs ( : ) buffer. then μl of a : dilution of anti-gp igg-fluorescein isothiocyanate conjugate was added to each well and the suspensions were incubated for minutes at °c. after washing with % (v/v) gelatin-dpbs buffer, μl glycerol-dpbs ( : ) was added to each well and the suspensions were visualized by fluorescence microscopy. all histomorphometry data are expressed as means ± standard deviations (sd). one-way analysis of variance was run to compare group means. a levene test was performed to evaluate variance homogeneity. if no significant deviations were found, the data were then analyzed by the least significant difference test. if the levene test detected significant deviations from variance homogeneity, the data were then assessed by a bonferroni test. statistical analyses were conducted in spss v. . for windows (release . k; ibm corp, armonk, ny). student t test was run to identify differences between groups with large errors. differences were considered significant at p < . . to prepare the vzv ge antigen for mouse immunization, we isolated the orf gene (ge + gpi) from oka/sk wvss (sk bioscience co ltd). the full-length ge gene was bp and it encoded amino acids. however, the truncated form used for ge antigen expression here was bp and it encoded amino acids. in the latter case, the transmembrane and the cytoplasmic tail (ct) of the c-terminal were deleted ( figure a ). the cloned truncated ge genes ( figures b and s ) were expressed in cho-k cells. a western blot analysis of the purified vzv ge proteins confirmed the expression of clones , , , and in the first single cells (figures a and s ) and in clones derived from clone ( figure b) . elisa revealed that clones , , , , and had high antigen expression ( figure c,d) . thus, they were selected for suspension culture adaptation in a serum-free medium. the lav used to immunize the guinea pigs was manufactured by sk bioscience co ltd. it was approved in under the name skyzoster and prescribed as a prophylactic against shingles (zoster) for adults aged ≥ years. its active ingredient is oka/sk strain and the other constituents include stabilizers such as gelatin, sucrose, and urea. the master-and working virus seeds (oka/sk) were established with the attenuated oka vaccine strain. vaccine safety and immunogenicity were validated in clinical phase ii/iii trials and compared against zostavax (merck & co inc, whitehouse station, nj). one lot from the commercial products the ssrna adjuvant was prepared using the rna platform derived from crpv igr ires ( figure a ) and the vzv orf gene (ge). paci-saci was used in the gene of interest ( figure b ). western blot with ge antibody (cha biotech, seoul, korea) confirmed that the ssrna adjuvant with vzv orf gene expressed -kda ge protein in transfected a cells ( figure c) . to confirm the immune responses in mice subjected to the ssrna adjuvant with the vzv ge gene, mouse blood was sampled for igg detection at days before the first immunization and again at and days after the first immunization ( figure a ). total igg, igg , and igg a titers were all similar at days and for nearly all mouse groups. the vzv ge total igg antibody titers were higher in g (with ssrna adjuvant) than g (without ssrna adjuvant) ( figure b ). the igg titers indicating a th response were similar for g and g ( figure c ) whereas the igg a titer indicating a th response for g was about -fold higher than that for g ( figure d ). g (with addavax) presented with the highest igg and igg a titers ( figure c,d) . vzv-specific cytokines (ifn-γ and il- ; also known as cd + t-cell cytokines) were measured by elispot assay to confirm enhancement of the vzv-specific cellular immune response , with splenocytes from immunized mice at day after the first immunization. the numbers of ifn-γ-and il- -secreting cells in the cultured splenocytes had increased in g (with ssrna adjuvant) compared with those in g (without ssrna adjuvant) after stimulation with ge protein and ge-specific peptide mixture (pepmix) (figure a-d) . moreover, the numbers of cells secreting ifn-γ and il- were similar in g (with ssrna adjuvant) and g (with addavax) used as the reference control ( figure a-d) . the numbers of ifn-γand il- -secreting cells after pha stimulation (as a positive control) were dramatically increased in all groups ( figure s (a) and (b) ). we tested the effects of ssrna adjuvant in lav skyzoster (sk bioscience co ltd) which was approved for administration as a shingles vaccine. blood samples were obtained at days before the first immunization and again at and days after the first immunization ( figure a ). total igg, igg , and igg titers were not significantly increased in g (with ssrna adjuvant) relative to those in g (without ssrna adjuvant) ( figure b-d) . thus, the ssrna adjuvant had no influence on the live vaccine. igg (th immune response) showed a weak titer at days after the first immunization but was dramatically increased after the second immunization at day in all groups ( figure c ). cd + t-cell-mediated immune response was assessed by measuring ifn-γ and il- in cultured splenocyte supernatants after vzv stimulation. the vzv-induced cytokines ifn-γ and il- were slightly increased in g (without ssrna adjuvant) and g (with ssrna adjuvant) compared to that in g (negative control). however, the cytokine levels were not significantly different between g and g ( figure e,f) . as a positive control, the numbers of ifn-γ-secreting cells after pha stimulation were increased in all groups ( figure s a ). but the numbers of il- -secreting cells were not increased significantly after pha stimulation ( figure s b) . therefore, the ssrna adjuvant was ineffective in lav. . | neutralizing antibodies against vzv in guinea pigs vzv-specific neutralizing antibodies were measured by fama. the titers in g (with ssrna adjuvant) were about -fold higher than those in g (without ssrna adjuvant) ( figure a,b) . therefore, the ssrna adjuvantinduced neutralizing antibodies and, by extension, a humoral immune response in the lav to the same level as the protein subunit vaccine even without corresponding increases in elisa antibodies or cytokines. ssrna is recognized by tlr and/or tlr , which are highly expressed in antigen-presenting cells (apcs) to stimulate an adaptive immune response for antigen-specific t cells, accompanied by high-affinity antibody production. despite this potential adjuvant effect, to date, rna has not been applied in conventional vaccines. here, we produced a vzv ge subunit vaccine and a lav containing the oka/sk strain and integrated our recently developed crpv igr ires-derived ssrna adjuvant encoding the vzv ge gene into them. mice immunized with the subunit vaccine containing the ssrna adjuvant responded with igg (th response) and igg a (th response) titers that were higher than those for the groups without ssrna adjuvant. the numbers of ifn-γ-and il- -secreting cells were to -fold higher in the ssrna adjuvant-immunized groups than in those immunized with vzv ge antigen alone. ifn-γ and il- activate th cells that enhance proinflammatory, cell-mediated immunity. increases of the il- -secreting immune cells indicates elevated t-cell activation, expansion, differentiation, and maintenance. it also indicates the differentiation of cd + t cells into terminal effector-and memory cells. , induction of vzv-specific t-cell activation with ssrna adjuvant is essential for the development of vzv vaccines for administration to older patients because t-cell activity declines with age. thus, ssrna adjuvant may play an important role in vzv-specific t-cell immunity and enhance cell-mediated immune response to vaccination. our data demonstrated that the ssrna adjuvant boosts ge antigen immunogenicity and augments ge-specific antibody and cd + t-cell responses. therefore, it enhances cell-mediated-and humoral immune responses. these findings corroborate those of a previous study in which the ssrna adjuvant was formulated with middle east respiratory syndrome coronavirus spike protein. protein-based vaccines are relatively less immunogenic because the potency of t-cell activation is low. thus, the ssrna adjuvants must compensate for these deficiencies. the ge protein produced from the cho-k cell expression system-induced vzv ge-specific antibody. thus, it is a good candidate for vzv protein vaccine. the ssrna adjuvant in lav (skyzoster) did not increase humoral-or cellular immune responses in guinea pigs. lavs can generally initiate innate immunity in dendritic cells (dcs). dcs induce innate inflammatory responses to pathogens in a manner similar to responses to viral infections. these innate immunity responses drive subsequent adaptive responses such as memory t-and b-cell activation. [ ] [ ] [ ] adjuvants may function as pathogen-associated molecular patterns that trigger innate immune responses via different mechanisms including apc activation and maturation and the initiation of downstream adaptive immune activity. in an earlier study, we showed that ssrna adjuvant-induced immune response-related genes similar to those induced by lav. f i g u r e cytokine-secreting cell frequencies in immunized mouse splenocytes. ifn-γ-secreting cell frequency in immunized mouse splenocytes after stimulation with ge protein (a) and pepmix (b). il- -secreting cell frequency in immunized mouse splenocytes after stimulation with ge protein (c) and pepmix (d). data are means ± sd. ge, glycoprotein e; ifn-γ, interferon-γ; il- , interleukin- ; n.s., no significance. *p < . therefore, elicitation of the innate and adaptive immune responses by ssrna adjuvant may be offset by the effects of vzv lav. the neutralizing antibodies measured by fama were about two-fold higher in the groups receiving the ssrna adjuvant in vzv lav than in those that did not receive it despite the lack of difference in elisa antibody (igg and igg a) titers or th -related cytokine induction. the ssrna adjuvant must recruit comparatively more apcs (especially dcs) at the injection site and the resultant increase in apcs may present specific epitopes to b cells. however, further research is needed to validate this hypothesis. addavax was the reference control vaccine used in this study. it resembles mf and is a squalene-based oil-inwater nanoemulsion that enhances cellular-and humoral immune responses. in contrast, alhydrogel (aluminum hydroxide gel) drives a th response. addavax significantly increases antibody titers with more balanced th /th responses than those obtained with alum. here, the ssrna adjuvant-induced humoral and balanced th /th immune responses even though its antibody and cytokine induction levels in the vzv ge protein subunit vaccine were lower than those for addavax. nevertheless, the mode of action of addavax has not yet been elucidated. , a previous study established that ssrna adjuvants are relatively safe. taken together, these results verify that the ge protein produced in this study can be a new vzv protein vaccine candidate. moreover, the ssrna derived from crpv igr ires encoding the ge gene, which could express ge in transfected cells, can effectively function as a vaccine adjuvant in a protein-based subunit vaccine by activating humoral and cell-mediated immune responses. these findings are in accordance with our previous report. in addition, we showed that the ssrna adjuvant could boost the production of neutralizing antibodies even in an lav. f i g u r e humoral immune responses and ifn-γ and il- levels in guinea pigs immunized with lav. a, study design for vzv lav immunization and schedules. all groups were inoculated twice at -week intervals. sera at pre-injection (day - ), days, and days after the first immunization were analyzed for total igg (b), igg (c), and igg (d). ifn-γ (e) and il- (f) levels in supernatants of cultured splenocytes after stimulation with lav were measured by cytokine elisa. data are means ± sd. elisa, enzyme-linked immunosorbent assay; ifn-γ, interferon-γ; il- , interleukin- ; igg, immunoglobulin g; lav, live attenuated vaccine.** p < . , *p < . compared with pbs immunization group g thus, it is expected that this previously developed ssrna adjuvant will be useful as an immune stimulator for various vaccine types, including protein-based and even lavs. f i g u r e neutralizing antibodies analyzed by fama to determine boosting effect of ssrna adjuvants on vzv lav. study design for vzv lav immunization is shown in figure a . guinea pig serum was serially diluted from : to : . anti-gp igg-fitc conjugate was used with : . a, average fama titer was for g (lav alone) and for g (lav + ssrna). b, cells were observed under fluorescence microscope. fama, fluorescent antibody to membrane antigen; fitc, fluorescein isothiocyanate; igg, immunoglobulin g; lav, live attenuated vaccine; ssrna, single-strand rna; vzv, varicella-zoster virus purification, characterization and immunogenicity of recombinant varicellazoster virus glycoprotein ge secreted by chinese hamster ovary cells antibody-binding sites on truncated forms of varicellazoster virus gpi(ge) glycoprotein. vaccine varicella-zoster virus glycoproteins: entry, replication, and pathogenesis cell-mediated immune responses to a varicella-zoster virus glycoprotein e vaccine using both a tlr agonist and qs in mice identification of cd + t cell epitopes in the immediate early protein (ie ) of varicella-zoster virus, and evaluation of frequency of cd + t cell response to ie , by use of ie peptides after varicella vaccination persistent high frequencies of varicella-zoster virus orf protein-specific cd + t cells after primary infection measurement of varicella-zoster virus (vzv)-specific cell-mediated immunity: comparison between vzv skin test and interferon-γ enzymelinked immunospot assay effector and central memory poly-functional cd + and cd + t cells are boosted upon zostavax vaccination comparative preclinical evaluation of as versus other adjuvant systems in a candidate herpes zoster glycoprotein e subunit vaccine a phase / clinical trial evaluating safety and immunogenicity of a varicella zoster glycoprotein e subunit vaccine candidate in young and older adults varicella-zoster virus glycoprotein ge: endocytosis and trafficking of the fc receptor recombinant varicellazoster virus glycoprotein e and i: immunologic responses and clearance of virus in a guinea pig model of chronic uveitis zoster vaccination increases the breadth of cd + t cells responsive to varicella zoster virus live vaccine used to prevent the spread of varicella in children in hospital a new era in cytomegalovirus vaccinology: considerations for rational design of nextgeneration vaccines to prevent congenital cytomegalovirus infection safety and immunogenicity of three different formulations of an adjuvanted varicella-zoster virus subunit candidate vaccine in older adults: a phase ii, randomized, controlled study safety and immunogenicity of an as -adjuvanted varicella-zoster virus subunit candidate vaccine against herpes zoster in adults ≥ years of age vaccine adjuvant systems containing monophosphoryl lipid a and qs induce strong and persistent humoral and t cell responses against hepatitis b surface antigen in healthy adult volunteers optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want towards an understanding of the adjuvant action of aluminium advances in aluminum hydroxide-based adjuvant research and its mechanism length of dsrna (poly i:c) drives distinct innate immune responses, depending on the cell type unmet needs in modern vaccinology: adjuvants to improve the immune response mechanisms of action of adjuvants tlr-based immune adjuvants vaccine adjuvants: putting innate immunity to work cricket paralysis virus internal ribosome entry site-derived rna promotes conventional vaccine efficacy by enhancing a balanced th /th response development of an rna expression platform controlled by viral internal ribosome entry sites recombinant glycoprotein e produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology immunogenicity and safety of a new live attenuated herpes zoster vaccine (nbp ) compared to zostavax® in healthy adults aged years and older varicella-zoster virusspecific immune responses to herpes zoster in elderly participants in a trial of a clinically effective zoster vaccine measurement of cellmediated immunity with a varicella-zoster virus-specific interferon-γ elispot assay: responses in an elderly population receiving a booster immunization t cell subsets and t cell-mediated immunity the dual role of il- in the generation and maintenance of cd + memory t cells the role of interleukin- in memory cd cell differentiation adjuvant-enhanced cd t cell responses are critical to durable vaccine immunity role of inflammatory dendritic cells in innate and adaptive immunity the role of dendritic cell subsets and innate immunity in the pathogenesis of type a diabetes and other autoimmune diseases varicella-zoster virus infection of human dendritic cells and transmission to t cells: implications for virus dissemination in the host vaccine adjuvants: from to and beyond the adjuvant alhydrogel elicits higher antibody titres than addavax when combined with hiv- subtype c gp from cap a novel multi-peptide subunit vaccine admixed with addavax adjuvant produces significant immunogenicity and protection against proteus mirabilis urinary tract infection in mice model adjuvants and the vaccine response to the ds-cav -stabilized fusion glycoprotein of respiratory syncytial virus comprehensive analysis of the safety profile of a single-stranded rna nano-structure adjuvant the authors thank the personnel of the laboratory of viral immunology for their excellent technical assistance with the experimental animals. the authors declare that there are no conflict of interests. all data are shown within the manuscript and figures. the raw data and the analysis details that support the findings of this study are available from the corresponding author upon reasonable request. key: cord- -bu pzbnv authors: miller, craig s. title: pleiotropic mechanisms of virus survival and persistence date: - - journal: oral surg oral med oral pathol oral radiol endod doi: . /j.tripleo. . . sha: doc_id: cord_uid: bu pzbnv viruses are enormously efficient infectious agents that have been implicated in causing human disease for centuries. transmission of these pathogens continues to be from one life form to another in the form of isolated cases, epidemics, and pandemics. each infection requires entry into a susceptible host, replication, and evasion of the immune system. viruses are successful pathogens because they target specific cells for their attack, exploit the cellular machinery, and are efficient in circumventing and/or inhibiting key cellular events required of survival. this article reviews some of the advances that have taken place in human virology in the past years, emphasizing mechanisms that contribute to, and are involved with, virus survival and persistence. the field of virology was barely half a century old in when dr thomas francis wrote articles, ''viruses as agents of disease'' and ''the prevention of virus disease,'' that were published in this journal. early leadership by mayer, ivanovsky, loeffler, frosch, walter reed and others, allowed progress from ''contagium vivum fluidum'' and a simple understanding of the existence and predatory nature of viruses to the characterization of viruses with regard to size, resistance to chemical and physical agents, host and tissue selectivity, and pathogenic and immunologic effects. these investigations made it clear that viruses were a very diverse group of pathogens. however, our knowledge of viral-cell interactions and the effect of viruses on the immune system was rudimentary. we held the understanding that a recovered individual is not susceptible to reinfection with the same virus, and that serum contained components that when mixed with virus and injected into a susceptible animal that animal was protected. these basic concepts served as the basis for classic studies of active and passive immunization. but several important advances that occurred between and jump-started the field of modern virology. these included the development of cultures of single animal cells, , watson and crick's identification of dna and the genetic code, the development of optimal medium for growing cells, and the development of the viral plaque assay. by the early s, max theiler and jonas salk (killed virus) had developed vaccines for yellow fever and polio, respectively, and through the benefits of growing the viruses in cell culture, shortly thereafter sabin developed the oral (live attenuated virus) vaccine. introduction of these vaccines into the human masses remains to this day one of the greatest accomplishments of preventive medicine. in the s, the transition from basic virology to molecular biology began. viruses and components of viral infections were analyzed using gel electrophoresis, protein-antibody interactions, and biochemical assays to answer basic biologic questions. as a result, greater knowledge of virus replication, viral and cellular receptors, and immunologic interactions was achieved. specifically, research during this time led to an understanding of the regulation of gene expression including transcription factors, enhancer elements, promoters, aspects of rna polymerase, and reverse transcriptase, as well as the discovery of proto-oncogenes and tumor suppressor proteins. scientists tagged viruses to identify intracellular locations of viral proteins, understand nuclear and cytoplasmic shuttling, and map neural circuitry. complete genomic sequences of viruses have been recorded and entered into public databases. the benefits of databases such as fasta and blast have led to searches in homology between motifs characteristic for specific gene products and the identification of novel viral genes and their functions. our knowledge also has advanced from the use of positive and negative selection procedures. positive selection being the method whereby genomic fragments or single candidate genes are expressed in a suitable cell system and tested for functionality (ie, infection phenotype). in contrast, negative selection is based on the construction of viral mutants that lack specific genes and the implementation of studies that identify a change in phenotype when the viral mutant infects a particular this work was supported in part by nih grant de . cell or animal. use of these techniques has led to the identification of virulence factors, novel mechanisms of regulation of cell surface receptors, and signal transduction pathways. within the last decade, the emerging fields of genomics and proteomics have allowed for the functional analysis of a large number of transcripts and protein sequences that are expressed during viral infection that provide new clues as to the regulation of acute, chronic, and persistent viral infections, as well as reactivation and malignant transformation. excitingly, the last decade has demonstrated that scientists have the knowledge and skill to harness unique features of viruses (eg, adenoviruses, retroviruses, herpesviruses) in implementing gene therapy and targeting processes important in chronic disease and cancer. however, mastery of this field remains to be seen. despite the exponential growth in virology during the last years, mankind still suffers from transmission and disease when humans serve as hosts to viruses. moreover, the outcomes are often severe when humans serve as novel hosts to emerging virus infection (eg, avian flu virus, ebola virus, equine hemorrhagic fever viruses, hanta virus, human immunodeficiency virus (hiv), hendra virus, nipah virus, sudden acute respiratory syndrome (sars) coronavirus, and west nile virus). of great importance is the fact that the oral cavity continues to be the source of transmission of many viruses, the site of replication and asymptomatic shedding of viruses, and a site where persistent viral infections exist, the latter being a prerequisite for virally induced malignant transformation. clearly, the field of virology has grown to the extent that a ''state-of-the-art'' paper would be exhaustive in length. accordingly, this review focuses on specific viral cell interactions that allow the virus to survive the cellular attack and evade the immune system, establish persistent infections, and cause chronic disease. additional topics will be covered in future reviews. viruses have developed numerous strategies for subverting the host defenses that are launched during infection. the first innate defense encountered is cellular selectivity during the entry process. viral attachment proteins bind to specific cell receptors (proteins, carbohydrates, or glycolipids) and coreceptors. the absence of a specific receptor shields the cell from attack. if this level of defense is foiled, upon binding receptors can sense microbial infection and trigger a multitude of antimicrobial and inflammatory responses. the toll-like receptor (tlr) family which consists of to members are well characterized in their ability to detect bacterial components (ie, lipoproteins and lipoteichoic acids, flagellin) as well as unmethylated cpg motif dna of bacteria and viruses (detected by tlr ), double-stranded rna (detected by tlr ) and single-stranded viral rna (detected by tlr ). in particular, tlrs , , , and specialize in viral detection and recognition of nucleic acids within the intracellular compartments which results in defensive signaling. after receptor binding, entry is modulated by either direct fusion with the plasma membrane or clathrin-or nonclathrin-mediated endocytosis. viruses that gain entry uncoat and deliver their genetic material and undergo a permissive or nonpermissive infection. a permissive cell permits virus replication and ultimate lysis of the host cell. in contrast, a nonpermissive cell downregulates virus replication and lytic gene expression resulting in little to no viral progeny. nonpermissive infections can be abortive or persistent, and persistent infections can be active or latent. latent infections are characterized by silencing of gene transcription, intermittent reactivation, or rarely oncogenic transformation. at the onset of infection, for a virus to survive within a cell, the virus must balance its own growth with death of the host and circumvention of the immune response. strategies for survival involve regulating apoptosis, inhibiting interferon production, modulating the major histocompatibility complex (mhc) class i function that ultimately affects the cytotoxic lymphocyte (ctl) and natural killer (nk) response, and limiting cytokine and chemokine production/function. long-term survival (ie, latency) requires downregulation of lytic gene expression, inhibition of apoptosis, and minimizing the inflammatory response. apoptosis, or programmed cell death, is a highly regulated and conserved series of sequential cellular events that results from receptor-or mitochondrialmediated pathways in response to a variety of stimuli, including viral infection and the appearance of doublestranded rna. the process is regulated (fig ) by a family of aspartate-specific cysteinyl proteases, or caspases, that converge at a number of downstream points resulting in proteolytic cleavage and enzyme activation. caspases are segregated into distinct subfamilies. the ''apoptotic'' caspases ( , , , , , , and ) are involved in the cascade that results in protease production, chromatin condensation, and cellular degradation. the ''inflammatory'' caspases ( , , and ) provide a second round of defense against viral infection. the inflammatory caspases are involved in the proteolytic maturation of key cytokines (ie, interleukin (il)- b and il- ). cytokine il- , also known as interferon (ifn)-inducing factor, directs the production of ifn-c. in turn, ifn-c induces expression of proteolytic active subunits that lead to proteolysis and antigenic processing by tap proteins. tap proteins are critical for displaying viral antigens on the cell surface (see cellular immunity, below). clearly, apoptosis is an important target of virus defense, because early destruction of an infected cell could greatly reduce replication and the number of viral progeny produced. interestingly, viruses have evolved several methods for suppressing or delaying apoptosis as well as encoding proteins that function as inducers of apoptosis. this apparent yin-yang relationship with apoptosis is important to prolong the life of the cell yet facilitate the release and spread of viral progeny at the appropriate time. , viruses regulate apoptosis by several mechanisms including the targeting of the tumor suppressor gene product p , the fas death receptor, and by producing caspase inhibitors and viral bcl- homologs. adenovirus, for example, encodes several gene products that influence apoptosis. the e a gene product stabilizes p and induces p -dependent apoptosis. , in contrast, the adenovirus e gene product promotes degradation of fas, and the adenovirus e b proteins antagonize p function. viral homologs of bcl- , an apoptosis suppressor that binds with bax, are produced by adenovirus, epstein-barr virus (ebv, bhrf protein), and other viruses. , there are several classes of caspase inhibitors encoded by viruses. these include the serine proteinase inhibitors (serpins: crma/spi- ), viral inhibitors of apoptosis (viaps), p , and inhibitors of procaspase protease (also known as flice). crma and p block caspase , previously termed il- beconverting enzyme (ice). caspase functions primarily as an activator of proinflammatory cytokines, but also has apoptosis-inducing ability in select mammalian cells, such as neurons. the viaps appear to inhibit bax-mediated apoptosis in human cells rather than directly inhibiting caspases. several human herpesviruses encode fliceinhibitory proteins (flips) that block trail-mediated cell death by interfering with procaspase protease (flice) activation. for example, the b-herpesviruses (cytomegalovirus (cmv)) encode a viral inhibitor of caspase activation (vica) which inhibits caspase (flice) activation, and c-herpesviruses encode vflips (eg, k ) which inhibit activation of caspases by molecular mimicry. cmv also encodes a viral mitochondrial inhibitor of apoptosis (vmia) (encoded by the u l gene) which inhibits activation of mitochondrial pores in a manner similar to members of the antiapoptotic bcl family. , the alpha herpesvirus hsv- encodes several antiapoptotic gene products (ie, icp , icp , c . , u s , gj) [ ] [ ] [ ] [ ] [ ] that modulate apoptosis at several levels, including antagonism of double-stranded rna-activated protein kinase (pkr), a downstream induction molecule of the interferon signaling pathway , of note, all c-herpesviruses express viral homologues of cellular antiapoptotic genes, including or bcl- homologues. interferon, discovered in the late s when scientists observed that virus-infected cells secrete a factor that mediates the transfer of a viral-resistant state, is a family of regulatory glycoprotein cytokines that modulate both innate and adaptive antimicrobial immunity. they are products of an infected cell genome and one of the key factors in the host response against viral infection. ifns serve as an early defense system that precedes the onset of the immune response and are triggered by envelope glycoproteins, cpg dna, or double-stranded rna. in recombinant formulations, they have been used in medicine and dentistry to combat various viral infections. , human ifns are classified based on the sequence of amino acids into main groups -a, b, and c -and that are less extensively studied (x, j, and s, not discussed further in this review). ifn-a and -b are produced rapidly when viral factors interact with cellular patternrecognition receptors such as tlrs and cytosolic receptors. historically, synthesis of ifn-a has been attributed to macrophages and b cells, and ifn-b has been considered to be produced by fibroblasts. more recently, plasmacytoid dendritic cells have been shown to produce ifn-a preferentially to ifn-b. both ifn-a and -b prevent the replication of viruses by inducing formation of secondary messengers which include ifn regulatory factor (irf) , irf- , irf- , c-jun/atf- , and nf-jb. ifn-c is synthesized by activated t lymphocytes and natural killer (nk) cells following receptor-mediated stimulation or in response to cytokines produced by macrophages or antigen-presenting cells (ie, primarily il- , il- , and ifn-a/b) or by stimulation through t cell receptors (tcrs) or nk cell receptors. it is a powerful activator of mononuclear phagocytes, thus enhancing their ability to destroy intracellular microorganisms and tumor cells. ifns mediate their antiviral action through ifn-stimulated genes (isg), which number in hundreds. ifns also regulate the cell cycle and have antiproliferative effects. viral evasion of ifn occurs by several strategies. in the majority of infections, viruses encode products that antagonize either the ifn signal transduction pathway or cellular proteins induced by ifn that are responsible for inhibiting virus replication (fig ) . adenovirus, ebv, papillomavirus, and members of the paramyxovirinae subfamily encode proteins that inhibit the jak-stat (janus kinaseesignal transducer and activator of transcription) signaling pathways that are required for ifn production. specifically, adenovirus encodes the oncoprotein e a which inhibits the activation of isg factor (isgf ). , paramyxovirinae reduce the effectiveness of the ifn response by targeting stat for degradation or by interference with stat phosphorylation or stability. , kaposi's sarcomaeassociated herpesvirus (kshv) encodes the pleiotropic gene product latency-associated nuclear antigen (lana) that acts downstream of isgf and inhibits p . , in an alternate approach, hpv encodes proteins, e and e , that bind to irf- and irf- , respectively, both of which inhibit the transactivation functions of the bound irf. , viruses also encode proteins that mimic cellular components of the ifn signal transduction pathway, including homologs of the ifn receptors, a viral isrelike promoter element, and viral homolog of irf (virf). for example, poxviruses antagonize ifn signals by encoding soluble ifn receptor homologs. , ebv encodes a viral isre and hhv encodes virf from the k orf that functions as a repressor of transcriptional activation induced by ifn-a, -b, and -c. in addition, several viruses have developed strategies to inhibit ifn-inducible, rna-dependent protein kinase (pkr). pkr, when antagonized, leads to phosphorylation of eif- a which results in inhibition of the ifn-induced antiviral response of the host. adenovirus, herpesviruses, influenza, and sv antagonize pkr by different mechanisms involving degradation of pkr, prevention of pkr activation, and resistance to downstream kinase activation. [ ] [ ] [ ] the mhc class ierestricted t cell response can result in a lethal hit before virus replication. thus, many viruses have developed strategies for interfering with antigen presentation to mhc class i molecules and intracellular trafficking of mhc molecules. viruses target the mhc-i at almost all steps of its trafficking: in the endoplasmic reticulum (er), in the cytoplasm on its way to the surface, and after the mhc reaches the cell surface (fig ) . one key target in the viral defense against the cellular arm of the immune system is attack of the transporter protein associated with antigen processing (tap). tap loads short antigenic peptides to the mhc which stabilize the class i complexes and allows their migration to the cell surface. without the peptide cargo, mhc class i molecules are unstable and dissociate. hsv- and hsv- encode infected cell polypeptide (icp)- , an immediate early gene product, that interacts with the tap protein in the cytosol to prevent peptide binding to tap. human cytomegalovirus (hcmv) encodes u s , a eamino acid glycoprotein, that blocks peptide transport by binding to tap in the endoplasmic reticulum. although efficient at both retaining mhc-i molecules and preventing ctl recognition, hcmv also uses additional viral proteins (u s , u s , u s , and u s ) to evade the immune system. a different approach is taken by ebv. this human c-herpesvirus encodes a glycine-alanine repeat (gar) domain on ebv-encoded nuclear antigen (ebna) that inhibits ubiquitin/proteasome-dependent proteolysis of ebv antigens. thus, processing (ie, degradation) of viral proteins into antigenic peptides is restricted. kshv, a lymphotropic c-herpesvirus, interferes with mhc-i antigen presentation by ubiquitinating the cytosolic domain of the mhc-i. herpesviruses also produce proteins that target mhc class i molecules for degradation in lysosomal compartments and downregulate expression of major histocompatibility complex molecules by shutting off host cell protein synthesis by the gene known as virion host shutoff (vhs, u l ). in contrast, hiv with its simple genome encodes fewer proteins but accomplishes similar immune evasion by pluripotent accessory proteins. for example, nef, of the regulatory proteins encoded by hiv, has multiple functions. in addition to enhancing virion infectivity, nef binds to and inhibits the surface expression of the major mhc-i, downregulates the cell surface expression of cd (the main hiv receptor), and facilitates cd receptor endocytosis. through the function of nef in redirecting the trafficking of immune receptors, infected t lymphocytes are able to hide from the immune system allowing for viral spread. hpv utilizes early proteins (e and e ) to persist undetected within epithelial cells. the early gene product e of the oncogenic strains hpv- and - downregulates mhc-i expression at the transcriptional level by inhibiting the promoters of the mhc-i heavy chain, tap- , and lmp- . e decreases mhc-i expression at the transcriptional level and causes retention of mhc-i in the golgi apparatus. within the golgi, hpv e inactivates the atpase proton pump system. as a result, acidification is blocked, local ph rises, and mhc-i trafficking is perturbed. thus, it is clear that viruses have achieved ingenious methods for interference with mhc-i antigen presentation and inhibition of the cellular immune response. viruses persist in cells because they are able to downregulate key processes that if left unattended would result in cell death. originally attributed in part to the immune response, increasing evidence suggests regulation of key genes plays an important role in the process. specifically, regulation of viral transcription and genomic replication allows for long-term viral stability and survival. many viruses, including those that cause persistent infections and chronic disease (ie, hepatitis c virus, hepatitis b virus, hiv, human herpesviruses, hpv, and jc virus), are successful because of their cell tropism and ability to autoregulate their replication efficiently within specific cells. common features of autoregulation include sensors to the external environment, negative feedback loops, transcriptional enhancers specific for cells that host the persistent infection, and transcriptional silencers. in some cases autoregulation results in steady-state levels of virus replication; in other infections, the virus enters latency only to reactivate intermittently. the importance of autoregulation is apparent from both in vivo and in vitro studies. for example, during lentivirus (hiv, simian immunodeficiency virus, and feline immunodeficiency virus) infection, viremia peaks early after infection then declines to a steady-state level. the effect is not altered by the presence of steroidinduced immunosuppression, and clearance of infected white blood cells is not associated with an earlier presence of antibody, cytokine response, or cytotoxic lymphocyte activity. in another common clinical example, successful antiviral therapy results in dramatic drops in viremia, often to undetectable levels. however, replication of virus often rebounds rapidly to pretreatment levels upon drug withdrawal, and in vitro studies indicate that cellular and immune functions are not contributory to the observed outcome. even when antiviral therapy achieves a sustained virologic response (ie, absence of viremia months after the end of treatment), highly sensitive assays (ie, polymerase chain reaction) detect residual viral genomes in most patients, indicating the ability of viruses to persist and autoregulate based on their environment. , herpesviruses are well known to establish latency in a variety of cell types, and this family of viruses has the ability to autoregulate. this is illustrated by hsv- and hsv- , which can undergo dichotomous life cycles: a lytic infection in epithelium and a latent infection in neurons. in fact, when neuronal cells are infected with hsv- or hsv- at low multiplicity of infection in vitro, the majority of cells can survive for many days even in the absence of immune cells and without the addition of antiviral drugs to the culture medium. similarly, if the rare neuronal cells supporting replication are eliminated using acycloguanosine in the above mentioned system, over % of the remaining population harbors a quiescent infection for weeks after the antiviral drug is removed, again in the absence of immune cells. , viruses regulate replication of their genome in a complex manner, but achieve the outcome through use of viral sensors, repressors, and effectors. viral sensors ''sense'' perturbations in the viral equilibrium within the cell and signal change at the appropriate time. with many viruses (ie, hiv, hepatitis c virus, hepatitis b virus), envelope proteins play the role of sensors, because envelope proteins can influence virus replication in both a positive and a negative manner. [ ] [ ] [ ] [ ] for hpv, regulation is through cellular factors that bind to the promoters of e and e . for hsv- , transcription of the immediate-early (ie) genes during the lytic infection is regulated by the binding of a tegument protein (vp ) with the cellular protein host cell factor (hcf) and oct- . thus, vp would seem to be a logical choice for a sensor. however, latency can be established in the presence of vp and reactivation occurs without the transactivating domain of vp . thus, downstream factors of vp (eg, icp , icp , or other unknown factors) serve as sensors of the environment and regulate the balance between latency and reactivation. the ''effectors'' (transactivators and replicative enzymes such as rna polymerase) modulate virus replication and are the targets of the sensors. effectors are tightly regulated (ie, repressed at certain times) but dynamically modifiable, typically by proteins bound to critical regions of the genome. these proteins afford protection by limiting changes in conformation of, or enzymatic action on, the restricted gene. histones are the most notable guardians of the effectors. histones permit access of dna to specific activators or repressors, general transcription factors, and rna polymerase by posttranslational modification (acetylation, methylation and phosphorylation) of their amino terminal tails. , for example, hyperacetylation of histones is associated with an ''open chromatin'' conformation and transcriptional activation, whereas hypoacetylation of the histone complex is associated with condensed (hetero-) chromatin and gene silencing. several human herpesviruses - utilize these mechanisms for regulating latency and reactivation. in addition, the active regions of the latent a-herpesvirus genome appear to be segregated from the repressed gene regions by boundary or insulator elements, similar to that found on cellular chromosomes (d bloom, personal communication). these chromatin insulators appear to be able to protect genes in one region from the regulatory influence of adjacent regions through conserved ctcf motifs. herpesviruses also encode proteins such as lana and latency-associated transcripts (lat) that appear to regulate viral transcription during latency. , integration, and the site of integration, into the host chromatin is another mechanism that can regulate viral gene transcription. for example, viruses that integrate (ie, hiv) preferentially select chromosomal sites where high-level transcription of key transactivators is maintained. this is accomplished by viruses preferentially integrating in chromatin regions characterized by an open structure (a hallmark of actively transcribed genes). the process by which this is regulated is not completely clear, but it has been suggested that cellular proteins may interact with integrase, the viral protein that catalyzes the integration reaction, in a manner that is site specific. viral persistence increases the likelihood of chronic infection and replication, but under certain circumstances also contributes to increased risk of oncogenic transformation. this can occur through chromosomal instability and virus integration, and the ability of several specific viral proteins to bind and inactivate p or less frequently prb (fig ) . p is a checkpoint protein that interacts with cdk/cyclin inhibitors and p , p , and p to arrest the cell cycle in the g phase and can send signals for apoptosis through the regulatory proteins bax, bcl- , and c-myc. the clinical importance of p inactivation is exemplified in that persistent hpv infection is associated with an increased risk of developing cervical cancer in young women, and recent findings suggest that the persistence of hpv dna in treated tissue after cancer therapy is highly predictive of local recurrence. in this brief review, examples of mechanisms that contribute to survival and persistence of viruses within their host were presented. emphasis was placed on mechanisms that permit survival of host defenses, evasion of the immune system, and establishment of chronic infections. detailed knowledge of these processes has led to many therapeutic successes. however, additional knowledge is required for us to make strides in eliminating human suffering caused by these intracellular pathogens. it is hoped that years from now when another review may appear in this journal on this topic, we will have a better understanding of how to eliminate persistent viral infections and identify patients at risk for virally induced complications of acute and chronic infections and will have harnessed the power of viruses to undergo selective lytic replication in tumor cells and modulate chronic disease. the origins of virology further studies on the proliferation in vitro of single isolated tissue cells the infection of cells in tissue culture with rous sarcoma virus the structure of dna a plaque assay for foot-and-mouth disease virus and kinetics of virus reproduction 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within the context of viral genomes expression of the herpes simplex virus alpha transinducing factor (vp ) does not induce reactivation of latent virus or prevent the establishment of latency in mice a herpes simplex virus type mutant containing a nontransinducing vmw protein establishes latent infection in vivo in the absence of viral replication and reactivates efficiently from explanted trigeminal ganglia functional consequences of histone modifications heterochromatin and epigenetic control of gene expression herpes simplex virus type virion host shutoff protein regulates alpha/beta interferon but not adaptive immune responses during primary infection in vivo histone acetylation and reactivation of epstein-barr virus from latency potential role for luman, the cellular homologue of herpes simplex virus vp (alpha gene trans-inducing factor), in herpesvirus latency identification of a novel multifunctional structural domain in the herpes simplex virus type genome: implications for virus latency insulators: many functions, many mechanisms a lat-associated function reduces productive-cycle gene expression during acute infection of murine sensory neurons with herpes simplex virus type integration site selection by retroviruses viral and host factors in human papillomavirus persistence and progression perspectives in studies of human tumor viruses human tumor suppressor p and dna viruses persistence of human papillomavirus infection as a predictor for recurrence in carcinoma of the cervix after radiotherapy key: cord- -nf ouaq authors: es-saad, salwa; tremblay, nicolas; baril, martin; lamarre, daniel title: regulators of innate immunity as novel targets for panviral therapeutics date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: nf ouaq interferons (ifns) have long been used as an immunomodulatory therapy for a large array of acute and chronic viral infections. however, ifn therapies have been plagued by severe side effects. the discovery of pathogen recognition receptors (prr) rejuvenated the interest for immunomodulatory therapies. the successes obtained with toll-like receptor (tlr) agonists in activating immune cells and as adjuvant for prophylactic vaccines against different viruses paved the way to targeted immunomodulatory therapy. better characterization of pathogen-induced immune disorders and newly discovered regulators of innate immunity have now the potential to specifically withdraw prevailing subversion mechanisms and to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than ifn therapies. salwa es-saad , , nicolas tremblay , , martin baril and daniel lamarre , interferons (ifns) have long been used as an immunomodulatory therapy for a large array of acute and chronic viral infections. however, ifn therapies have been plagued by severe side effects. the discovery of pathogen recognition receptors (prr) rejuvenated the interest for immunomodulatory therapies. the successes obtained with toll-like receptor (tlr) agonists in activating immune cells and as adjuvant for prophylactic vaccines against different viruses paved the way to targeted immunomodulatory therapy. better characterization of pathogen-induced immune disorders and newly discovered regulators of innate immunity have now the potential to specifically withdraw prevailing subversion mechanisms and to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than ifn therapies. the innate immune system is the first line of defense for organisms that possess an adaptive immune system. it relies on the presence of specific receptors able to recognize recurring pattern in molecules associated with pathogens but not with host cells, allowing discrimination between self and non-self. these receptors are named pattern recognition receptors (prr) and recognized pathogen-associated molecular patterns (pamp) to induce the expression of cytokines and chemokines that restrict dissemination, eliminate pathogens and instruct pathogenspecific adaptive immune responses. in the recent years, tremendous advances in the characterization of prr families, nucleic acid sensing, downstream signaling pathways and effector responses have revealed essential role of novel proteins and dynamic protein interactions network in the triggering of immune responses to intracellular pathogen such as viruses. in the near future, targeting specific regulators of prr-mediated innate response to withdraw viral subversion mechanisms, and access to novel surrogate measurable effector markers, hold the promise of new panviral therapeutics that will minimize adverse effects associated with type i ifn therapy. this review briefly summarizes strategies and challenges of present and future targeted immunomodulatory therapies according to our increasing knowledge in regulation of innate immunity and of virus-induced immune host dysfunction. signaling pprs include the major families of toll-like receptors (tlrs), retinoic acid-inducible gene i (rig-i)like receptors (rlrs) and nucleotide-binding oligomerization domain (nod)-like receptors (nlrs). pathogen sensing takes place in all nucleated cells to generate cellintrinsic innate immunity and in professional antigen presenting cells (apcs) to promote specific adaptive immune responses. while tlrs sense pamps in the extracellular space and endosomes, rlrs and nlrs function as pathogen sensors in intracellular compartments [ ] . interestingly, only a few of the known tlrs have the ability to recognize viral molecules: tlr for viral dsrna, tlr / for viral ssrna and tlr for viral unmethylated cpg dna. three cytosolic sensors of viral rna have been characterized thus far: rig-i for the sensing of triphosphate structure and blunt-end base paring, mda for the sensing of long dsrna and lgp a cardless regulator of its counterparts [ ] . following their activation, the card domain of rig-i and mda interacts with the card domain of the signaling adaptor mavs (mitochondrial antiviral signaling protein) [ ] . both tlr and rlr viral sensing pathways converge to activate ifn regulatory factor irf -mediated and irf -mediated type i ifn (a/b) antiviral response and nf-kb-mediated inflammatory pathway [ ] (figure ). recent studies aims at better defining innate immune responses have identified several novel signaling and regulatory molecules [ ] . global proteomic analysis has further revealed signaling modules with high interconnectivity and adaptor proteins regulating signalosome assembly upon antiviral response and type i ifn production [ ] . cytokines and type i ifns increase expression of mhc class ii antigens, cd and cd on apcs [ ] . cytokines produced at sites of infection play a key role in the activation and differentiation of dendritic cells (dc), macrophages, neutrophils and nk cells, all major players of the innate immune response [ ] (figure ). when mature dcs detect virus derived antigens, they migrate to the lymph nodes to present antigens to cd + and cd + t cells and b cells, inducing their activation [ ] . thus, modulation of prr-mediated antiviral responses can have important ripple effects on both qualitative and quantitative aspects of the specific adaptive immune responses to maximize the therapeutic potential of immunomodulatory drugs [ ] . negative regulation of innate immune response and pathological consequences antiviral innate response must be tightly regulated in order to prevent uncontrolled production of cytokines that might have deleterious effects on the host. type i ifn signature induced by prr activation has been observed in diverse autoimmune disorders including diabetes, and is believed to play a role in the induction of chronic inflammatory disorders such as asthma and rheumatoid arthritis. in the recent years, a better picture has emerged in the biology of regulators illustrating the existence of numerous negative regulators that often play a nonredundant role and target the same positive regulator [ ] . many negative regulators have been characterized that are either involved in direct interaction with prrs, dissociation of adaptors complexes, degradation of signal proteins or transcriptional regulation [ ] . posttranslational modifications (phosphorylation and ubiquitination) have emerged as key mechanisms to regulate innate immune responses. degradation of signal proteins mediated by the ubiquitin-proteasome and autophagy systems plays crucial roles in negative regulation of tlr signaling, and unlike disruption of adaptors regulators of innate immunity as novel targets for panviral therapeutics es-saad et al. contributes to termination of signaling as these degradations are irreversible [ ] . examples include proteins socs and pin that promote polyubiquitination and proteasomal degradation of mal adaptor and irf / respectively, to suppress type i ifn and antiviral responses. recently, mirnas have also emerged as fine tuners of innate immune responses, which target mrnas encoding tlrs, intracellular signaling proteins and cytokines. examples include mir- that targets irak and traf , and mir- that targets myd , tab and ikke [ ] . thus, targeting specific negative regulator of the innate immune response may offer a new immunotherapeutic strategy to treat a range of infectious and inflammatory diseases [ ] . cellular defence has evolutionarily challenged viruses that in turn have developed strategies to counteract innate immune response. indeed tlr and rlr sensing pathways are fundamental targets for virus-encoded immune suppression. these viral subversion mechanisms include recruitment of ubiquitin proteasome system, mimicry of the host cell components and sequestration and cleavage of key components of the immune system. one notable example is mavs adaptor that is targeted by numerous viruses through proteolytic cleavage by hepatitis c virus (hcv), hepatitis a virus (hav), coxsackievirus b (cvb ), human rhinovirus a (hrv a) and gb virus b (gbv-b), through decrease of the mitochondrial membrane potential by influenza a virus (flu) or through inhibition of its interaction with rig-i by hepatitis b virus (hbv). processes of viral evasion are varied and are beyond the scope of this review, but are recapitulated in figure (reviewed in [ ] ). importantly, host proteins targeted by multiples viruses highlight key players of innate immunity, which represent potential therapeutic targets to restore antiviral response and eventually cure cells from viruses. however, these specific viral evasion strategies must also be taken into account when developing immunomodulatory therapeutics to provide the greatest clinical benefits. type i ifns were rapidly used as a therapeutic agent against hbv and hcv, and demonstrated antiviral activity against infection with sars-cov [ ] , flu [ ] , west nile virus (wnv) [ ] , yellow fever virus (yfv) [ ] and ebola virus [ ] . refinement of therapies was explored with the development of improved ifn molecules like consensus interferon (cifn: a completely synthetic interferon) [ ] , albinterferon (a fusion protein between ifna a and human albumin) [ ] and y shape interferon [ ] . recently, virus-induced type iii ifns (ifn-l - : il- , il a, il b) have gained a lot of interest to treat viral infections since naturally occurring variants of the il b gene have been a major prediction factor in spontaneous and treatment-induced clearance of hcv [ , ] . early clinical trials of recombinant pegylated-ifn-l in hcv-infected patients showed reduced adverse effects compared to ifn-a, likely linked to minimal expression of ifn-l receptors in hematopoietic cells [ , ] . tlr targeted therapies (table ) the discovery of tlrs heralded the rebirth of interest in innate immunity. their specificity in recognizing most classes of pathogens, as well as their role in the pathogenesis of multiple diseases represent the strongest evidences that tlrs are valuable therapeutic targets. tlr targeted drugs have been approved and small-molecule compounds are being investigated in the treatment of viral infections as stand-alone treatment or adjunct to direct acting antivirals (daas). the most advanced examples of tlr agonists are imiquimod (aldara, m) and resiquimod (r- , m), which are members of the imidazoquinolinamines [ ] . imiquimod is the only approved tlr agonist and is used for topical treatment of external genital and perianal warts resulting from human papillomavirus (hpv) infection [ ] . resiquimod is a mixed tlr / agonist that reached phase iii trial for the treatment of genital herpes before being suspended due to a lack of efficacy [ ] . isatoribine ana- (anadys pharmaceuticals) is a second generation of orally bioavailable prodrug of isatoribine that signals through tlr , which is expressed in b cells and dcs [ ] . in hcv infected patients, ana- was generally well tolerated and resulted in a significant À . log decrease in hcv rna levels following days of treatments [ ] . ana- is now assessed in phase iia, and its efficacy will be evaluated in combination with ribavirin and daas as an ifn replacement. synthetic cytosine-phosphate-guanine containing oligodeoxynucleotides (cpg-odns) are potent tlr agonists, which interact directly with dcs to stimulate cytokine release and induce adaptive immune responses [ ] . in phase i clinical trials, subcutaneously administration of imo- (idera pharmaceuticals) as monotherapy resulted in a more than À log decrease in hcv rna levels in prior nonresponders to peg-ifn/ribavirin after weeks [ ] , and in combination with ribavirin to a À . log decrease in hcv rna in treatment-naïve patients at day [ , ] . on the basis of its efficacy, imo- could provide an alternative to ifns for hcv therapy. however, idera pharmaceuticals delayed a phase ii study after the observation of atypical lymphocytic proliferation in preclinical toxicology study. vaccine adjuvants using tlr agonists tlr agonists have been an extensively explored area in the development of vaccine adjuvants for prophylactic and therapeutic applications by linking innate and adaptive immune systems. the proof-of-concept of this approach was made with the as adjuvant system that combines monophosphoryl lipid a (mpla), an agonist of the tlr receptor and aluminium salt [ ] [ ] [ ] . as has been approved in prophylactic vaccine against hbv (fendrix, glaxosmithkline) [ ] and hpv and (cervarix, glaxosmithkline) [ ] . the mechanism of action of as is mediated by a transient and local activation of nf-kb activity and cytokine production, thus providing an innate immune signal for optimal activation of apcs [ ] . other notable examples of adjuvants in clinical development are heplisav and vaxinnate. heplisav is a hbv vaccine comprised of an immunostimulatory sequence (iss- , dynavax technologies) that targets tlr receptor and hbv surface antigen. in phase iii clinical trials, heplisav demonstrated earlier and higher protection with fewer doses than currently licensed vaccines [ ] . vax-innate corporation is developing vaccines using highly conserved influenza immunogens fused to tlr agonist salmonella typhimurium flagellin type as an adjuvant to potentially protect against all strains of seasonal and pandemic flu strains (vax , vax , vax and vax ) [ ] [ ] [ ] . future immunomodulatory targeted therapy and panviral approaches (table ) in the past decade, many newly emerging or re-emerging virus infections and fear of future pandemics have accentuated the need for novel antiviral therapy. panviral therapeutics with a targeted therapy approach would be an ideal treatment for acute and chronic viral infections, either as a standalone treatment or in combination with daas. the major challenge in developing future immunomodulatory therapy will be to minimize adverse effects. the aggravation of psoriatic plaques in hpv-infected patients treated with imiquimod illustrates that triggering innate immune responses can lead to uncontrolled activation of the inflammatory response. furthermore, immunomodulatory molecules, such as peptidoglycans, that bind to multiple prrs (tlr , nod proteins and peptidoglycan recognition proteins) increase the risk of undesired side effects. development of therapeutics will require more extensive structural information of receptor-ligand interaction to maximize the specificity and avoid undesired interactions. the selection of specific targets will require a comprehensive knowledge of innate immunity signaling pathways and regulators that are induced by and common to numerous viral infections. the mapping of an innate regulators of innate immunity as novel targets for panviral therapeutics es-saad et al. table development status of tlr-targeting molecules for treatment of viral infections. immune protein interaction network regulating ifnb has revealed signaling modules with high interconnectivity including mavs, tbk and irak [ ] . each module interacts with many signaling proteins of the pathway offering multiple drug targets with specific immune effector function. using a genome-wide rnai screen assessing virus-induced ifnb transcription in human cells, we identified novel proteins and pathways capable of negatively and positively regulating innate immune responses (unpublished data). comprehensive epistasis analysis of the various regulators acting at different steps of the antiviral responses from virus sensing, signal propagation/amplification up to feedback regulation, offers valuable information for selection of drug targets. in principle, strategies of targeted therapy could include small molecule-mediated activation of positive regulators or inhibition of negative regulators. an example of targeting a negative regulator could be the immuno-mirna mir- , which is induced by virus infection and downregulate myd , irak , tab and ikke gene expression to suppress tlr signaling [ ] . silencing mir- function using antagomirs or locked nucleic acid (lna) in infected cells could potentially restore tlr signaling. a better knowledge of surrogate end point measurable makers of immune effector function (correlating with pan antiviral efficacy) in relevant infected biological material will undoubtedly enhance selection process and therapeutic value of drug targets. indeed, microarray analysis of infected primary cells can be used to identify early and late response innate immune genes, as well as virus-mediated inhibition of these genes [ ] [ ] [ ] . finally, the knowledge of virus-induced immune host dysfunction and of immune proteins targeted by multiples viruses will validate key viral host interfaces, leading to hypothesis-driven selection of therapeutic targets intended to restore innate immune responses. tlrs agonists reflect substantial promise as therapeutic targets and demonstrate the huge potential of targeting innate immunity in fighting viral infections. in the future, integration of structural, proteomics and functional genomics data will pave the way to the identification of key regulators of innate immunity. targeting immune regulators that promote prr signaling to maintain transient activation of innate immune responses upon viral infection should pioneer the discovery of panviral therapeutics. such targeted immunomodulatory therapy approach could change the way we treat infectious diseases by allowing a single treatment to be effective against numerous viruses, with minimal viral breakthrough. in the near future, the increasing availability and potency of new targeted immunomodulatory panviral therapeutics could allow the re-thinking of temporal aspects of treatments that, in combination with available daas, could achieve viral eradication. the ultimate goal is to shape tlr-dependent and rlr-dependent innate immune responses to restore antiviral effects and to generate an optimal global immune response, while controlling inflammation. table current and future development of immunomodulatory targeted therapy [ , - ] . activation leads to pro-inflammatory cytokines and type i ifns production set the pace for an adaptive immune response via t and b cells. activation leads to pro-inflammatory cytokines and type i ifns production modulation of the adaptive immunity through dc and nk cells extensive structural and functional studies available [ ] tlr agonists marketed or in phase i-iii clinical trials structure recently identified [ , ] ssrna rig-i ligand reduces infection with flu [ ] rig-i agonist as adjuvant in flu vaccine [ ] restore immunity by counteracting viral evasion processes papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest pattern recognition receptors and inflammation immune signaling by rig-i-like receptors mavs forms functional prion-like aggregates to activate and propagate antiviral innate 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c virus-infected patients the structural biology of toll-like receptors highlights the variation in the nature of the interactions of tlr paralog extracellular domains following ligand-induced dimerization rig-i like receptors in antiviral immunity and therapeutic applications structural basis of rna recognition and activation by innate immune receptor rig-i structural insights into rna recognition by rig-i gold nanorod delivery of an ssrna immune activator inhibits pandemic h n influenza viral replication coexpressed rig-i agonist enhances humoral immune response to influenza virus dna vaccine a signaling polypeptide derived from an innate immune adaptor molecule can be harnessed as a new class of vaccine adjuvant viral and cellular micrornas as determinants of viral pathogenesis and immunity this work was supported by grants from the canadian institutes for health research (cihr-ci - ) and by the novartis/canadian liver foundation hepatology research chair to dl. nt is a recipient of a ph.d. scholarship from the fonds de recherche en santé du qué bec (frsq). key: cord- -hmsidrc authors: grunwell, jocelyn r.; stephenson, susan t.; mohammad, ahmad f.; jones, kaitlin; mason, carrie; opolka, cydney; fitzpatrick, anne m. title: differential type i interferon response and primary airway neutrophil extracellular trap release in children with acute respiratory distress syndrome date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: hmsidrc acute respiratory distress syndrome (ards) is a heterogeneous condition characterized by the recruitment of large numbers of neutrophils into the lungs. neutrophils isolated from the blood of adults with ards have elevated expression of interferon (ifn) stimulated genes (isgs) associated with decreased capacity of neutrophils to kill staphylococcus aureus and worse clinical outcomes. neutrophil extracellular traps (nets) are elevated in adults with ards. whether pediatric ards (pards) is similarly associated with altered neutrophil expression of isgs and neutrophil extracellular trap release is not known. tracheal aspirate fluid and cells were collected within h from seventy-seven intubated children. primary airway neutrophils were analyzed for differential isg expression by pcr, stat phosphorylation and markers of degranulation and activation by flow cytometry. airway fluid was analyzed for the release of nets by myeloperoxidase-dna complexes using an elisa. higher stat phosphorylation, markers of neutrophil degranulation, activation and net release were found in children with versus without pards. higher nets were detected in the airways of children with ventilator-free days less than days. increased airway cell ifn signaling, neutrophil activation, and net production is associated with pards. higher levels of airway nets are associated with fewer ventilator-free days. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ we have previously demonstrated that neutrophils exposed to airway fluid from mechanically ventilated children with virally-induced respiratory failure and bacterial coinfection have decreased respiratory burst and bacterial killing capacity . however, the mechanisms underlying these observations are still unclear. respiratory viruses have been shown to induce type i interferon (ifn) (ifnα/β) responses that then upregulate expression of interferon-stimulated genes (isgs) such as mx , isg , and ifit , , which influence neutrophil behavior and function , . since viral lower respiratory tract infections are a primary trigger for pards, we questioned whether differential isg expression was associated with neutrophil responses in intubated children at risk or with pards. we hypothesized that children with pards would have a greater type i ifn response resulting in more neutrophil extracellular trap (net) release. this prospective, observational study was performed in the thirty-six-bed academic medical/surgical pediatric intensive care unit (picu) at emory university/children's healthcare of atlanta at egleston from january through february . the study was approved by the institutional review board at emory university (irb and irb ) and all methods were carried out in accordance with relevant guidelines and regulations in the declaration of helsinki. informed consent was obtained from the parents of all subjects prior to collection and use of their samples. all patients admitted to the picu who were greater than days, with a corrected gestational age of at least weeks, and were younger than years old that met criteria for being at risk or having pards, as defined by the pediatric acute lung injury consensus conference (palicc), were screened for eligibility . children had to have lung injury within days of a known clinical insult, new infiltrate(s) consistent with acute pulmonary parenchymal disease on chest imaging and be receiving oxygen delivered either noninvasively or invasively to maintain an oxygen saturation between and %. children were excluded if they had any peri-natal related lung disease, respiratory failure fully explained by cardiac failure or fluid overload, chronic respiratory failure with mechanical ventilation via a tracheostomy or ram cannula, confirmed immunodeficiency disorder, immunosuppression from chemotherapy for an oncologic process, chronic immunosuppression in a bone marrow transplant or solid organ transplant recipient, no parent or legal guardian present to provide written informed consent, or the attending physician did not wish the patient to participate in the study. children were enrolled as controls if they were endotracheally intubated for airway protection and without lung pathology or signs of systemic infection or inflammation. for example, children selected as controls were electively intubated to facilitate radiologic imaging, for non-airway or cardiothoracic surgeries, and for airway protection following an acute ingestion or altered mental status without suspicion for infection or systemic inflammation. clinical data were abstracted from the medical record onto a standardized form. variables included demographics; fraction inspired oxygen, mean airway pressure, arterial oxygen saturation or arterial oxygen pressure used to calculate an oxygen saturation index (osi) or oxygenation index (oi), respectively; laboratory and microbiology results; length of mechanical ventilation and need for reintubation; length of picu stay, use of high frequency oscillatory ventilation (hfov) or extracorporeal life support (ecls), and vital status. severity of illness was determined by the pediatric risk of mortality (prism)-iii and pediatric logistic organ dysfunction (pelod) scores were calculated within h of intubation , . need for mechanical ventilation to -days was monitored to calculate ventilator-free days . lung injury severity was categorized according to palicc criteria . tracheal aspirate collection. tracheal aspirates were obtained from patients on conventional mechanical ventilation by instilling - ml of sterile saline through the inline ballard suction catheter and into a sterile luken's trap as part of routine suctioning per published protocols . children who were mechanically ventilated with hfov were suctioned only if clinically indicated and approved by the attending physician. tracheal aspirate samples were immediately placed on ice for transport to the laboratory for processing. airway sample processing. tracheal aspirate was gently dissociated using repeated passage through an g needle after the addition of ml of pbs-edta. dissociated tracheal aspirate was then centrifuged at ×g to generate a cell pellet and a fluid fraction. the fluid fraction was spun at ×g to generate cell-free airway supernatant (asn), aliquoted, and stored at − °c . airway cells were resuspended in pbs-edta and cell density was quantified using a countess hemocytometer using trypan blue exclusion to determine cell viability. cell purity was also assessed by cytospin preparations and diff-quik staining of airway cells. cell viability was also assessed using a live/dead aqua stain in the surface staining flow cytometry panel. gene expression assays. rna was isolated from airway cells stored at − c in rna later using the nucleospin rna ii kit with on-column genomic dna digestion according to the manufacturer's protocol (takara, mountain view, ca). rna was quantified using a nanodrop fluorospectrometer (therma scientific). rna integrity (rin) was measured at the emory integrated genomics core on an agilent bioanalyzer (see supplementary table s statistical analysis. statistical analyses were performed using jmp pro (sas institute, cary, nc) and graphpad prism for windows. unless otherwise stated, comparisons between samples of children with versus without pards were made using a two-tailed mann-whitney u test for nonparametric data. for pcr studies, a rout outlier test using a % false discovery rate was performed prior to a two-tailed mann-whitney u test. statistical significance was defined as a p value less than . . subject characteristics. seventy-seven patients were enrolled into the study within h of intubation and mechanical ventilation. of these children, ( . %) had pards and ( . %) did not have pards. table shows the demographics and clinical characteristics of the enrolled patients. the respiratory viral pcr panel and respiratory culture results from the clinical microbiology lab are reported in supplementary table s online. children with pards were more likely to be supported by extracorporeal life support (ecls), had a longer duration of mechanical ventilation, and spent more days in the hospital and the picu (table ) . there were no significant differences in severity of illness (prism iii) or organ dysfunction (pelod) scores, the absolute number or percentage of alive neutrophils from children based on pards status (table ) . this study was a survey of the activation status of neutrophils is pards. we assessed markers of airway neutrophil activation by flow cytometry. the gating strategy for selecting airway neutrophils is shown (fig. a-c) . there was no difference in total number (fig. d ) or percent cd b + neutrophils (fig. e ) in intubated children with or without pards. children with pards also had increased neutrophil surface expression of cd , a marker of primary granule exocytosis (fig. f) , and sphingosine -phosphate receptor (s pr ), a protein that forms a heterodimer with the neutrophil il -induced chemotaxis receptor, cxcl , and is detected with increased abundance in blood neutrophils from adults with bacterial pneumonia (fig. g ) . there was no significant difference in surface expression of the type iii fcγ receptor, cd or arginase i (arg ), an enzyme stored in the primary and tertiary granules of human neutro- www.nature.com/scientificreports/ phils ( fig. h-i) . examples of cytospin preparations stained with diff-quik from four unique patient tracheal aspirates show the predominance of neutrophils in the airway samples (fig. j) . we next determined whether the type i interferon (ifnα/β) signaling pathway was activated by measuring the phosphorylation of signal transducer and activator of transcription (stat ) by intracellular staining of fixed airway samples. airway cells from children with pards had increased expression of phosphorylated-y stat (p-stat ) and stat compared with children who did not meet pards criteria (fig. ) . cells were gated on forward and side scatter and representative histograms for both p-stat and stat are shown ( fig. a) . quantification of both the mean fluorescence intensity (mfi) and percent positive cells are summarized in fig. b -e. children with pards have higher levels of stat and p-stat compared with children without pards. activation of the type i interferon signaling pathway results in increased expression of many www.nature.com/scientificreports/ www.nature.com/scientificreports/ ifn-stimulated genes (isgs) . three isgs, ifit , isg , and mx , were used to identify activation the type i ifn signaling pathway , . isg expression of children with versus without pards were compared. isg expression was variable; however, children with pards did not show a significant difference in expression level of isg , ifit or mx compared to children without pards (fig. ) . our results show that markers of neutrophil degranulation and activation are elevated in children with versus without pards. in addition, activation of the type i ifn signaling pathway, as indicated by increases in p-stat , and isg expression occurs in some children with pards. (fig. a) . higher airway net levels were associated with fewer ventilator-free days (f = . , p value < . ), a lower proportion of children with ventilator-free days over days (fig. b) acute lrti are the trigger for the majority of pards . viral and bacterial infections activate type i ifn signaling pathways resulting in an increase in antiviral isg expression and proinflammatory responses critical for host defense , . a finely tuned type i ifn response is crucial to a host as an excessive or prolonged type i ifn response may lead to impaired gas exchange in the lung due to cellular and tissue destruction . ifns prime mature neutrophils for net release upon stimulation with a second stimulatory signal . although antimicrobial proteins expressed on nets can inactivate pathogens and prevent viral spread to neighboring cells, the lung is www.nature.com/scientificreports/ vulnerable to the damage that histones and proteolytic enzymes contained within nets can inflict to host tissue. therefore, excessive and prolonged type i ifn signaling may contribute to the production of nets which fill the alveolar spaces, lead to increased inflammation, and result in impaired lung function, which are the hallmarks of pards. our study assessed neutrophil activation, the differential expression of isgs and activation of the stat signaling pathway, and netosis in the airways of intubated children with acute respiratory failure due to lower respiratory tract infections. we found increased neutrophil degranulation markers, phosphorylation of stat (y ), and nets, as measured by mpo-dna complexes, in the airways of children with pards compared with children without pards. higher levels of airway nets were associated with fewer ventilator-free days. our findings are summarized in fig. . the timing and magnitude of the type i ifn response are important in modulating the immune response to viral infection , . at higher levels, isgs contribute to dysregulated lung inflammation, disease progression, and are linked to worse clinical outcomes . for example, in a mouse model of sars-cov infection, the delayed expression of high levels of type i ifn in the presence of high viral titers resulted in lethal pneumonia ; however, early treatment with type i ifn within the first six hours of sars-cov infection was protective. in influenza a virus, disease severity and progression are associated with overshooting the ifn-driven inflammatory response whereby exogenous supplementation with type i ifn correlated with increased morbidity and mortality , . rsv also induces high levels of pro-inflammatory cytokines directly related to type i ifn, and mice that lack ifnar have less proinflammatory cytokine release resulting in a less severe disease course following rsv infection . by contrast, there was no effect of ifnar deletion on pathogenicity of mice infected with sars-cov; however, stat deficient mice showed increased susceptibility, prolonged viral shedding and mortality . differential type i ifn and isg expression are associated with neutrophil dysfunction and worse clinical outcomes , . for example, high expression of a panel of isgs (mx , ifit , isg ) from circulating neutrophils from a subgroup of adults with ards compared with normal isg expressing neutrophils had reduced migration toward the neutrophil chemokine interleukin- (il- ), decreased p map kinase phosphorylation, superoxide anion release, il- release, and a shift from necrotic to apoptotic cell death that was associated with a diminished capacity to kill staphylococcus aureus, but not pseudomonas . subsequent hierarchal clustering analysis of the aforementioned isg panel from circulating neutrophils of ards patients within h of initiation of mechanical surface expression of the primary granule exocytosis marker, cd , and the lipid signaling g protein-coupled receptor, sphingosine- -phosphate receptor (s pr ), are higher in children with versus without pards. the type i interferon (ifn) signaling pathway transcription factor, stat , is upregulated and phosphorylated, and transcript levels of isg is increased in children with versus without pards. neutrophil extracellular trap (net) release is regulated by a nadph oxidase (nox) respiratory burst (reactive oxygen species (ros) triggered mechanism and an intracellular calcium-dependent trigger. it is not known which trigger dominates in the airways of children with pards. children with pards have elevated levels of nets in their airways as detected by myeloperoxidase (mpo)-dna complexes in our study. elevated net levels are associated with a higher number of ventilator-free days (vfd) over days in a -day period (i.e. if the child survived, then they were more likely to spend ≥ days endotracheally intubated and mechanically ventilated). created with biorender.com using the web version, which may be accessed at https ://biore nder.com/, with a paid individual subscription granting permission to publish in journals. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ ventilation showed that both high-and low-range expression had fewer -day ventilator-free and icu-free days and higher -day mortality compared with mid-range isg expressing patients . these data suggest that a targeted middle ground for ifn levels exists to benefit the host. an ifn signal below a lower threshold would result in an ineffective antiviral host response, while an ifn signal above an upper threshold would trigger a detrimental inflammatory host response. ifn signaling outside the target zone would increase lung inflammation and ards severity , , . by contrast, our findings are from airway cells with a neutrophil predominance rather than from circulating neutrophils for adults with ards , . additionally, we assessed each isg, ifit , isg , and mx , individually rather than in aggregate, and we note that isg expression is highly variable in children with versus without pards. finally, the release of nets was not assessed in the aforementioned studies. excessive net formation has been reported in the serum and bronchoalveolar lavage fluid from adults with ards; however conclusions with respect to clinically relevant outcomes are difficult to draw due to the heterogeneity in study design , , . nets play prominent roles in bacterial pneumonia [ ] [ ] [ ] , rsv bronchiolitis , , influenza pneumonia , , , sepsis [ ] [ ] [ ] [ ] , sars-cov , small-vessel vasculitis , systemic lupus erythematosus , , and transfusion-related acute lung injury . nets induce dose-dependent cytotoxic effects on human alveolar epithelial cells due to histone and myeloperoxidase induced damage to alveolar epithelial and endothelial cells , . nets also influence the macrophage function , t cell proliferation , and amplify ifn production by plasmacytoid dendritic cells . conversely, interferons influence the process of netosis in mature neutrophils , . high ifn/isg expression polarizes neutrophils to an activated "n " phenotype that can lead to increased net production, degranulation, and influence neutrophil interactions with other immune and airway epithelial cells . interferonopathies, autoimmune diseases, and tumor-associated neutrophils are all examples where high ifn/isg levels influence neutrophil activation and function , . priming of neutrophils with ifn-α and subsequent stimulation with c a resulted in increased stat phosphorylation at tyrosine and net production in mature neutrophils . activation of neutrophils by type i ifns led to increased netosis that triggered biofilm formation by pseudomonas aeruginosa and persistence in the lung . net production, like ifn signaling, exists in a balance. excessive net release can lead to the systemic spread of inflammation through platelet interactions and result in multiple organ dysfunction , , ; however, depletion or defective net production can lead to the spread of infection early in the course of disease . several mechanisms regulating the formation of nets are known and include nadph oxidase (nox ) dependent and calcium channel nox-independent netosis - . netosis is driven by net-specific kinases that regulate transcription initiation in neutrophils. for example, erk was shown to differentially regulate noxdependent netosis . interestingly, khan and colleagues also noted that stat was a transcription factor that was upregulated in the nox-dependent netosis pathway . we were not able to study the relative importance of nox-dependent versus calcium influx (nox-independent) mechanisms on net formation given that netosis has already occurred by the time of tracheal aspirate sampling in our patients. additionally, we did not quantify the relative amounts of neutrophils undergoing netosis versus apoptosis. quantifying neutrophil apoptosis should be part of future studies as nox-dependent netosis is dependent on the activation of the kinase akt to suppress apoptosis and switch to netosis . while we detected higher type i ifn signaling and net production in the airways of children with versus without pards, due to the clinical nature of our study, we are not able to attribute causation of neutrophil dysfunction to high isg expression in study participants. there are several additional limitations to our study. first, this is a single-center study with a limited sample size; however, despite the limited number of children studied, we were able to detect differences in neutrophil type i ifn signaling pathway phosphorylation and function in intubated children with versus without pards. the majority of children in this study were at risk for developing pards, with the remaining children being equally distributed amongst mild, moderate and severe pards categories; however, the degree of pards severity was not associated with neutrophil dysfunction due to heterogeneity in neutrophil function and a limited sample size. we also excluded immunocompromised patients from our study which is a major risk factor associated with pards-related mortality [ ] [ ] [ ] . second, there is heterogeneity in the identity of viral and bacterial pathogens infecting study participants precluding any conclusions regarding the influence of organism on study results. we did not stratify the patients based on bacterial growth in an endotracheal respiratory culture as there is controversy regarding whether this is a true bacterial coinfection versus merely codetection of bacterial and viral organisms . thirdly, we are not powered to detect differences in mortality associated with higher isg gene expression as seen in the adult ards cohort; however, we did detect a significant difference in ventilator-free days in children with a higher airway net burden . we are limited in the ability to study the mechanisms regulating airway net release as netosis has already occurred in patients at the time of sample collection' however, network analysis of transcription factor signaling pathways from recovered airway neutrophils could be performed as previously described . additionally, future experiments using blood-derived neutrophils from healthy donors could be incubated in patient airway fluid to simulate the airway environment with pma and calcium-ionophore stimulation, and appropriate nox inhibition, to study mechanistic regulation of net formation. finally, we did not explore the role of other signaling pathways, such as that initiated through the sphingolipid receptor or other kinase signaling cascades, as mechanisms involved in the pathogenesis of nets or pards severity. in summary, we describe the differential type i ifn signaling based on the presence or absence of pards and show that airway levels of nets are higher in children with versus without pards. higher levels of airway nets are associated with fewer ventilator-free days. future work will explore the influence of type i ifn signaling, nets and the pards airway environment on macrophage and t-cell activation and function. 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species generation and citrullination and cleavage of histone. front. immunol. , akt is essential to induce nadph-dependent netosis and to switch the neutrophil death to apoptosis a simple and robust bedside model for mortality risk in pediatric patients with acute respiratory distress syndrome predicting mortality in children with pediatric acute respiratory distress syndrome: a pediatric acute respiratory distress syndrome incidence and epidemiology study subtypes of pediatric acute respiratory distress syndrome have different predictors of mortality pediatric ventilator-associated infections: the ventilator-associated infection study we acknowledge the emory + children's flow cytometry core for flow cytometry instrumentation. this study was supported in part by the emory integrated genomics core (eigc), which is subsidized by the emory university school of medicine and is one of the emory integrated core facilities. additional support was provided by the georgia clinical and translational science alliance of the national institutes of health under award number ul tr . the content is solely the responsibility of the authors and does not necessarily reflect the official views of the national institutes of health. the authors thank the bedside caregivers of the patients involved in this study for their skilled and compassionate care. j.g. and a.f. conceived and developed the study, supervised the acquisition of the biological data, analyzed and interpreted the data. j.g. drafted and edited the manuscript. a.f. assisted with drafting and editing the manuscript. s.s. and a.m. helped with patient sample processing, performed experiments and helped to interpret the data. k.j. and c.o. assisted in identifying, consenting, acquiring patient samples. k.j., c.o., and c.m. assisted in collecting clinical information about the patients. all authors edited and approved the final version of this manuscript. drs. grunwell and fitzpatrick received support for research from the national institutes of health. funding was provided by nih grants k hd (atlanta pediatric scholars program), k hl - , and an emory university pediatrics research alliance junior faculty focused pilot award to jg. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.r.g.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - anybxnk authors: ireland, derek d. c.; stohlman, stephen a.; hinton, david r.; kapil, parul; silverman, robert h.; atkinson, roscoe a.; bergmann, cornelia c. title: rnase l mediated protection from virus induced demyelination date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: anybxnk ifn-α/β plays a critical role in limiting viral spread, restricting viral tropism and protecting mice from neurotropic coronavirus infection. however, the ifn-α/β dependent mechanisms underlying innate anti-viral functions within the cns are poorly understood. the role of rnase l in viral encephalomyelitis was explored based on its functions in inhibiting translation, inducing apoptosis, and propagating the ifn-α/β pathway through rna degradation intermediates. infection of rnase l deficient (rl(−/−)) mice with a sub-lethal, demyelinating mouse hepatitis virus variant revealed that the majority of mice succumbed to infection by day p.i. however, rnase l deficiency did not affect overall control of infectious virus, or diminish ifn-α/β expression in the cns. furthermore, increased morbidity and mortality could not be attributed to altered proinflammatory signals or composition of cells infiltrating the cns. the unique phenotype of infected rl(−/−) mice was rather manifested in earlier onset and increased severity of demyelination and axonal damage in brain stem and spinal cord without evidence for enhanced neuronal infection. increased tissue damage coincided with sustained brain stem infection, foci of microglia infection in grey matter, and increased apoptotic cells. these data demonstrate a novel protective role for rnase l in viral induced cns encephalomyelitis, which is not reflected in overall viral control or propagation of ifn-α/β mediated signals. protective function is rather associated with cell type specific and regional restriction of viral replication in grey matter and ameliorated neurodegeneration and demyelination. type i interferon (ifn-a/b) induced anti-viral responses are critical for the initial control of most virus infections. although anti-viral responses are initiated in infected cells, they act in an autocrine and paracrine fashion, antagonizing virus replication in host cells and inducing a protective anti-viral state in neighboring cells. the anti-viral state is distinguished by the expression of numerous ifn stimulated genes (isg), which directly or indirectly interfere with both viral and host rna transcription and translation [ ] . many of these pathways can also result in the apoptosis of infected cells [ ] . nevertheless, the relative contributions of these various anti-viral effector mechanisms to overall virus control differ with the virus as well as the tissue and cell type infected. the best characterized anti-viral mechanisms are mediated by activation of double-stranded rna-dependent protein kinase (pkr) and the - -oligoadenylatesynthase (oas)/ribonuclease l (rnase l) pathways [ ] [ ] [ ] . upon recognition of double-stranded rna, the oas family of proteins convert atp into unique, unstable - -a oligomers, which are bound by latent rnase l monomers, driving the dimerization and activation of rnase l. activated rnase l cleaves single-stranded rna, of u-a and u-u sites found in both viral and cellular rna. rnase l endoribonuclease activity is thus not limited to viral rna, but also degrades host cell rna, including ribosomal rna. as oas genes are stimulated by ifn-a/b, elevated oas levels enhance the antiviral state in adjacent, non-infected cells responding to ifn-a/b. nevertheless, inappropriate activation of rnase l is counterbalanced by the unstable nature of - a oligomers and the endogenous rnase l inhibitor, rl [ ] . a novel aspect of the oas/rnase l pathway is the cleavage of host rna releasing free -monophosphates that can activate the cytoplasmic rna helicases rig-i and mda- [ ] . recognition by these pattern recognition receptors initiates ifn-b transcription, similar to the activation mediated by recognition of specific viral rna structures. rnase l thus not only exerts anti-viral effects via rna degradation and apoptosis, but also has the capacity to amplify and prolong expression of anti-viral genes and other isgs. the anti-viral activity of the oas/rnase l system has been studied in several virus infections in vitro and in vivo [ , ] . protective mechanisms of the oas/rnase l pathway are generally more evident for rna relative to dna virus infections. however, the contribution of rnase l to disease severity and viral control varies significantly depending on the virus and even virus strain studied, presumably reflecting the diverse mediators and targets of these anti-viral mediators. for example, encephalomyocarditis virus (emcv) replication was only modestly increased in rnase l deficient (rl / ) mouse embryonic fibroblasts, consistent with enhanced susceptibility to infection in vivo. although rnase l prolonged survival, mortality rates of infected rl / and control mice were similar. furthermore, ifn-b treatment increased the survival time of infected wild-type (wt) and to a lesser extent rl / mice, indicating that alternate ifn-dependent anti-viral mechanisms act in the absence of rnase l [ ] . in contrast, the requirement for rnase l activity to protect from another picornavirus, coxsackievirus b , was evident by significantly increased mortality rates of infected rl / compared to wt mice. however, increased mortality was not the result of uncontrolled virus replication, as virus titers were only slightly elevated within pancreatic islet cells in vivo. by contrast, pancreatic islet cells derived from rl / mice, treated with ifn-a and infected in vitro exhibited increased virus replication and loss of cellular integrity, compared to ifn-a treated rnase l sufficient cells [ ] . these results revealed distinct differences in direct antiviral functions of rnase l in vitro and in vivo. cell type specific effects of anti-viral rnase l activity are also clearly evident from studies with west nile virus (wnv). while mouse embryonic fibroblasts display rnase l dependent anti-viral activity [ ] , ifn-b treatment revealed no affect of rnase l in reducing wnv replication in either cortical or peripheral motor neurons [ ] . nevertheless, a modest effect was observed in bone marrow derived macrophages, consistent with increased viral burden in cd b + splenocytes derived from rl / compared to wt mice [ ] . furthermore, increased mortality of rl / mice in response to peripheral wnv infection correlated with increased replication in lymphoid tissue, but not enhanced viral dissemination into the cns. a minor role for rnase l in control of wnv in the cns was supported by similar viral titers in brains as well as kinetics and rates of mortality following intracranial infection of rl / and wt mice. although viral replication was only transiently increased in spinal cords of intracranially infected rl / mice, the most prominent effect was increased spread to the spleen and liver [ ] . the more critical role of rnase l in limiting wnv spread in peripheral tissues rather than the cns highlights the complexities of oas/rnase l mediated anti-viral mechanisms in vivo. ifn-a/b mediated innate responses are also essential to control virus replication and survival following coronavirus infections [ ] [ ] [ ] [ ] . infections in vitro and in vivo induce upregulation of ifn-a/b and anti-viral mediators, including pkr and oas [ , , ] ; however, the participation of these pathways in the anti-viral response to cns infection has not been elucidated. in vitro, the dual liver and neurotropic mhv-a strain does not elicit degradation of s and s ribosomal rna, suggesting the absence of rnase l activation in hela cells [ ] . however, administration of an rnase l agonist inhibited replication of the hepatotropic mhv- strain in peritoneal macrophages in vitro and in liver in vivo [ ] . nevertheless, reduced viral replication via rnase l function in vivo was insufficient to prevent liver necrosis and death. the nonlethal gliotropic jhm strain of mouse hepatitis virus (mhv-jhm) replicates exclusively in the cns following intracranial infection and is controlled by both innate and adaptive responses [ , ] . cns infection results in a non-lethal encephalitis with immune-mediated demyelination resulting in hind limb paralysis [ ] . based on expanded tropism to hippocampal neurons and widespread dissemination within the cns of mice deficient in ifn-a/b signalling (ifnar / ) [ ] , the gliotropic mhv-jhm variant was used to probe an anti-viral role of rnase l within the cns. while the vast majority of wt mice survived, rl / mice succumbed to infection, albeit delayed compared to ifnar / mice. although enhanced morbidity suggested a prominent role of rnase l in anti-viral protection, the absence of rnase l neither diminished overall virus control in the cns, nor enhanced neuronal infection. furthermore, rl / mice did not reveal any impairment in ifn-a/b induction, alterations in proinflammatory cytokines or inflammation compared to wt mice. these data rather reveal a novel role of rnase l in specifically counteracting focal microglia/macrophage infection, thus potentially sustaining microglia mediated neuroprotective effects and ameliorating demyelination. mice deficient in ifn-a/b signalling succumb to an otherwise sub-lethal gliotropic mhv-jhm within days of infection despite functional cd t cell responses [ ] . uncontrolled viral replication in the absence of ifn-a/b implicated a crucial role of anti-viral innate mechanisms in stemming viral spread within the cns. to elucidate the role of rnase l in cns pathogenesis, gliotropic mhv-jhm infection was examined in rl / mice [ ] . increased susceptibility of rl / mice was evident by more severe clinical symptoms of acute encephalitis at - days post infection (p.i.) (fig. , top panel) . with the exception of increased severity, onset and progression of clinical symptoms in infected rl / were similar to those exhibited by infected wt mice. these included initial symptoms of acute viral encephalitis, including: lethargy, dehydration and weight loss, which progressed to diminished hind limb function. furthermore, mortality rates were significantly higher in rl / mice, with over % succumbing to infection by days p.i. (fig. , middle panel) , approximately - days delayed relative to ifnar / mice [ ] . assessment of direct anti-viral functions of rnase l [ , ] revealed no initial spread of viruses is controlled by type i interferon induced antiviral molecules. early intervention with viral replication is especially critical in central nervous system infections to reduce loss of non-renewable cells and mitigate immune pathology. one of the best characterized anti-viral mechanisms is mediated by ribonuclease l (rnase l). rnase l exerts activity at multiple levels, including degradation of viral and host rna, induction of apoptosis, and propagation of the ifn-a/b pathway. recent studies suggest that rnase l antiviral activity is dependent on the virus, as well as the cell type and tissue infected. this study demonstrates that rnase l protects mice infected with a sub-lethal, demyelinating neurotropic coronavirus by ameliorating encephalitis and morbidity, albeit without affecting control of infectious virus or ifn-a/ b expression. rnase l specifically protected the brain stem from sustained infection and prevented spread of virus to microglia/macrophages located in spinal cord grey matter. the subtle regional alteration in tropism in the absence of rnase l coincided with increased apoptotic cells and earlier onset as well as increased severity of axonal damage and demyelination. the results demonstrate how subtle regional alterations in viral tropism within the cns may severely affect the balance between neuroprotection and neurotoxicity mediated by microglia/macrophages. significant changes of infectious virus in brains of rl / , compared to wt mice. virus titers were increased less than . log in rl / mice (fig. , bottom panel) . very modest anti-viral rnase l activity in the cns was confirmed by analysis of viral rna from cns tissue. levels of viral mrna encoding the viral nucleocapsid protein were increased less than . -fold in spinal cords of rl / compared to wt mice at any time point (table ) . as liver may constitute an extraneural infection site in immunocompromised mice, viral replication in this tissue was also assessed by real-time pcr. the abundant viral mrna encoding the nucleocapsid protein was detected in liver at very low levels compared to the cns in both groups, albeit only at early time points (table ) . although viral rna was elevated in livers of rl / mice at day p.i., clearance by day p.i. suggested hepatitis did not contribute to mortality. necrotic foci were not evident in either mouse group by gross examination. a minor role of rnase l in viral control in vivo was reminiscent of the previously described picornavirus models [ , , ] . in addition to rna degradation, rnase l also induces a multitude of genes in the ifn-a/b pathways during infections with sendai virus and emcv [ ] . thus, both self and virus small rna products released by rnase l activate cytoplasmic rna helicases rig-i and mda- to optimize and perpetuate anti-viral responses. however, ifn-a and ifn-b expression levels were slightly increased, rather than decreased in brains of mhv-jhm infected rl / mice relative to wt mice (fig. ) . furthermore, oas- , ifit- , ifit- , and irf- , representing prominently activated isgs, were induced to similar, or modestly increased levels in the cns of infected rl / mice. slightly enhanced ifn-a/b and isg expression was also observed in spinal cords from infected rl / relative to wt mice (data not shown). consistent with similar virus replication and control in the cns, neither ifn-a/b nor the expression of downstream isgs were impaired at the tissue level. these data suggest that a positive rnase l-dependent feedback loop in propagating ifn-a/b signalling is not activated during mhv-jhm infection in the cns. an unanticipated feature of rl / mice is delayed tissue rejection in transplant studies [ ] , suggesting a role of rnase l in modifying antigen presentation, lymphocyte trafficking or function. to assure that altered pathogenesis was not due to differential inflammatory responses, the cns was examined for extent, composition and localization of infiltrating cells. characterization of cells recruited to the cns using flow cytometry revealed similar total numbers of infiltrating cells and no alterations in their composition (fig. ). unlike ifnar / mice [ ] , neutrophil infiltration was not affected in rl / mice and macrophages comprised the most prominent population early p.i. in both strains. cd + and cd + t cells peaked to nearly identical numbers at day p.i. in both groups. furthermore, cns infiltrating cd + t cells in both groups were comprised of % and % virus specific cells at day and pi, respectively, as monitored by d b /s class i tetramer staining [ ] . similarly, no significant differences in the extent of inflammatory cells were observed by histochemical analysis (data not shown). these data confirm that priming and trafficking of virus specific t cells was unaffected by the loss of rnase l activity, consistent with effective clearance of infectious virus. rnase l did not overtly affect overall cns viral replication; however, it may act in a tissue or cell type specific manner [ ] . mhv-jhm initially infects ependymal cells and spreads to astrocytes, microglia/macrophages and predominantly oligodendrocytes, but rarely infects neurons [ ] . however, mhv-jhm induced mortality in the absence of ifn-a/b signalling is associated with a dramatically expanded virus tropism to hippocampal neurons [ ] . to assess the possibility that rnase l exerts cell type dependent anti-viral effects not apparent from whole tissue homogenates, the distribution of virus infected cells was analysed by immunohistochemistry. sequential analysis demonstrated a limited infection of neurons within the brain with a predominance in the brain stem at day p.i. in both rl / and wt mice (fig. ) . glia tropism prevailed in both groups and overall distribution of virus infected cells was similar with occasional neurons still infected at day p.i. (data not shown). by day p.i. virus infected cells declined in cortex and brain stem of both wt and rl / mice. however, in contrast to wt mice which had cleared virus from brain stem, virus infected cells were sustained in the brain stem of rl / mice and were predominantly microglial in appearance (fig. ) . the overall extent and distribution of viral infected cells was thus similar during peak virus replication in brains of rl / and wt mice, with the exception of sustained infection in brain stem in the absence of rnase l. preliminary experiments indicate that these cells are microglia or macrophage/ monocytes (see below). these data contrast with the infection of ifnar / mice [ ] which show predominant neuronal infection. therefore, preferential infection of neurons could not account for the increased mortality in rl / mice. the data also indicated that protective rnase l functions are not overtly reflected in early anti-viral activity but are manifested in region specific protection. rnase l activation leads to apoptosis and elimination of infected cells, a process requiring activated caspase [ , ] . furthermore, ifna/b mediated activation of rnase l also induces apoptosis in non-infected cells [ ] . during mhv-jhm infection of wt mice the majority of apoptotic cells are lymphocytes [ ] , which are not targets of infection. although oligodendrocyte apoptosis has been linked to the demyelinating process, the demonstration of apoptotic oligodendrocytes during mhv-jhm infection has been elusive [ , ] . the retention of virus infected cells in brain stem may result in enhanced local t cell activation and apoptosis of either t cells, infected cells, or both. therefore, the frequency of apoptotic cells was examined at day p.i. in contrast to the brain stems of wt mice which contained few apoptotic cells, apoptotic cells were prominent in brain stems of rl / mice (fig. ) coincident with retention of virus infected cells. although increased focal virus infection associated with substantially increased apoptotic cells presumably dysregulates neuronal function and potentiates tissue damage, there was no evidence that apoptotic cells are preferentially located adjacent to neurons. sustained brain stem infection and apoptosis in rl / mice coincided with increased morbidity and mortality, despite similar overall control of viral replication. the morbidity in mhv-jhm infected rl / mice also coincided temporally with the onset of acute primary demyelination in wt mice. rl / mice were thus examined for enhanced tissue pathology and demyelination. brain stems of infected wt mice revealed minimal demyelination by day p.i. (fig. ). by contrast, demyelination was more severe in brain stems of rl / mice, correlating with increased numbers of virus infected as well as apoptotic cells. demyelination is associated with a variable degree of axonal damage [ , ] . primary immune mediated demyelination in the spinal cord during mhv-jhm infection coincides with early axonal degeneration [ , ] . while axons were largely intact in brain stems of wt mice, axonal integrity within demyelinated lesions was severely compromised in rl / mice as indicated by a striking decrease in neurofilament and an increase in dystrophic axons within lesions (fig. ) . in spinal cords of wt mice, demyelination was also minimal at day p.i. and increased by day p.i. (fig. ) , when control of virus replication is clearly evident (table ) . by contrast, large focal areas of severe demyelination were already apparent in rl / mice at day p.i., resembling those in wt mice at day p.i. in addition to the accelerated kinetics of demyelination, the severity of myelin loss was more pronounced in the absence of rnase l at both days and p.i. consistent with the more rapid loss of myelin in infected rl / mice, quantification of demyelinated areas at day p.i. revealed . . % demyelination in spinal cords of wt mice and . . % in rl / mice. increased demyelination in both brain stem and spinal cord are thus a hallmark of infected rl / mice, irrespective of overall viral control. apoptotic cell numbers and axonal damage in the spinal cords of rl / mice were also evaluated to determine an association with increased demyelination. in wt mice, apoptotic cells were undetectable in spinal cord at day p.i. (data not shown), but were prominent in white matter by day p.i. (fig. ) . by contrast, spinal cords from rl / mice already exhibited small areas of apoptotic cells at days p.i., albeit only in white matter. however, by day p.i. numerous apoptotic cells were evident in both white matter and grey matter of spinal cords from infected rl / mice. furthermore, in contrast to brain stems, apoptotic cells were often detected in close proximity to neurons (inset fig. ) ; however, there . rnase l does not alter the extent of neuronal infection but delays viral clearance from brain stem. viral nucleocapsid antigen detected by immunoperoxidase staining using mab j. . in brains from infected wt and rl / mice at days and p.i. (top panels: brown chromogen; hematoxylin counterstain). note neuronal infection (arrows) and similar foci of viral antigen in glial cells in brain stem at day p.i. by day p.i. infection is controlled in wt mice, but foci of infected non neuronal cells with glia morphology are sustained in rl / mice. scale bar = mm. immunohistochemical staining for activated caspase- (bottom panels) at day p.i. indicates increased numbers of cells undergoing apoptosis in brain stem of rl / relative to wt mice. scale bar = mm. doi: . /journal.ppat. .g was no evidence for neuronal apoptosis. these data suggest that the absence, rather than presence, of rnase l contributes to enhanced apoptosis in white matter, and subsequently also in grey matter. analysis of axonal integrity revealed that all axons were intact in spinal cords of wt mice consistent with minimal demyelination at day p.i. (fig. ) . by contrast, a striking decrease in neurofilament and an increase in dystrophic axons within demyelinated lesions demonstrated axonal integrity was already compromised in white matter of rl / mice by day p.i. at day p.i. the amount of axonal degeneration in wt mice resembled that of rl / mice at day p.i., consistent with the severity of demyelination. however, by day p.i. spinal cords of rl / mice showed marked axonal loss located in areas of myelin loss (fig. ) , similar to results in brain stem. the increased pathological changes in both brain stem and spinal cord thus correlated with the high mortality rates of rl / mice starting at day p.i. the inability to directly link increased axonal damage with enhanced neuronal infection remains puzzling and supports dysregulation of oligodendrocyte function and/or neuroprotective functions of microglia as contributing factors to the severe pathological phenotype and clinical outcome. increased demyelination in spinal cords has been correlated with the extent of white and grey matter infection in mice infected with heterologous mhv strains [ ] . furthermore increased apoptosis, specifically in spinal cord grey matter of rl / mice supported grey matter infection. increased demyelination as well as apoptosis in grey matter in rl / mice thus prompted a more detailed investigation of viral antigen distribution in spinal cords, where the demarcation between grey and white matter is linear as opposed to the more complex organization of brain stem. neuronal infection was not observed in spinal cords of either rl / or wt mice. furthermore, the number of infected cells with oligodendrocyte morphology in the white matter of rl / mice was similar to wt mice at day p.i. (fig. ) . however, a prominent difference in viral antigen positive cells was noted in spinal cord grey matter. rl / mice harboured several foci of infected cells with microglia morphology, whereas viral antigen positive cells were rarely observed in spinal cord grey matter of wt mice (fig. ) . furthermore, a large number of infected cells in the grey matter of rl / mice were identified as microglia and/or infiltrating monocytes based on co-localization with iba- positive cells (fig. ). foci of iba- positive cells co-expressing viral antigen in the grey matter were only observed in rl / but not wt mice, confirming results obtained by immunohistochemistry (fig. ) . nevertheless, infected iba- positive cells were dispersed throughout the white matter of wt and rl / mice. similar characterization of the brain stem at day p.i. also identified iba - positive cells as prominent cells harbouring sustained viral infection (data not shown). co-stains with the astrocyte specific marker gfap showed no evidence of infected astrocytes in spinal cord grey matter; astrocytes were also only rarely infected in white matter of wt and rl / mice (data not shown). these data indicate that rnase l mediated antiviral function acts specifically in microglia and/or monocytes. moreover the focal nature of infection in grey matter suggests microglia localization is associated with enhanced susceptibility to infection in the absence of rnase l. to determine whether infection of iba- positive cells was biased to microglia or infiltrating monocytes, both cell populations were purified from spinal cords of infected mice by fluorescence activated cell sorting. measurement of viral mrna encoding the nucleocapsid protein revealed that microglia derived from rl / mice harbored . -fold higher levels of viral rna at day p.i., while the relative levels in monocytes was only increased . -fold relative to infected wt mice (table ) . however, viral mrna levels decreased in both cell types reaching comparable levels by day p.i. while rna levels for tnf were elevated in rnase l deficient microglia and monocytes by -fold and . fold, respectively, inos levels remained similar relative to wt derived cells (data not shown). ifnc relative to gapdh mrna levels in spinal cord were also modestly increased from . . in wt mice to . . in rl / mice at the peak (day p.i.) and declined to . . and . . , respectively, by day p.i., confirming a modest contribution of proinflammatory cytokines to pathogenesis. moderately increased viral rna levels in microglia are thus consistent with foci of microglia infection in spinal cord grey matter not present in wt mice. overall these data demonstrate that accelerated and enhanced demyelination in the absence of rnase l coincided with significant neuronal damage in the absence of expanded neuronal infection. furthermore, the limited extent of increased focal glial infection appeared insufficient to mediate the pathological effect. to assess potential differences in the overall activation state of microglia, the distribution and morphology of iba- positive cells was assessed in spinal cord grey matter. microglia in wt mice were activated as demonstrated by their intense staining and ramified phenotype and localized in close proximity to neuronal cell bodies (fig. ) . by contrast, in infected rl / mice, fewer microglia were activated based on their iba- staining and morphology and did not show preferential association with neurons. whether the absence of rnase l itself or infection of microglia mitigates a neuroprotective function of microglia remains to be explored. the notion that inflammatory insults disrupt neuroprotective functions by microglia is based on microglia mediated neuroprotection in models of injury and lps preconditioning [ ] [ ] [ ] and loss of protection following viral infection [ ] . ifn-a/b dependent innate immunity is essential to contain viral spread during most viral infections prior to control by adaptive responses. nevertheless, the in vivo anti-viral effector mechanisms contributing to innate viral control remain poorly understood. the best characterized pathways are mediated by activation of pkr and rnase l [ ] [ ] [ ] . however, in contrast to the conclusive effects of pkr and rnase l deficiency on viral replication in vitro, in vivo studies demonstrate more subtle anti-viral effects [ , , ] . during wnv infection, rnase l deficiency is manifested in profoundly altered morbidity, despite similar viral control after footpad inoculation [ ] . ifn-a/b-mediated anti-viral responses are also critical for controlling spread of neurotropic coronavirus within glial cell populations and preventing infection of neurons [ ] . based on its activation by numerous rna viruses, rnase l was investigated as a prototypical anti-viral effector molecule in controlling mhv-jhm virus replication in the cns. rnase l deficiency was not sufficient to duplicate uncontrolled replication and the rapid uniform lethality observed in ifnar / mice [ ] . nevertheless, a critical contribution to virus susceptibility was demonstrated by a significant increase in both clinical disease and mortality. mortality was delayed by - days in rl / relative to ifnar / mice, but could not be attributed to uncontrolled viral replication or spread to neurons. in fact, overall virus replication in brains and spinal cords of rl / mice was only modestly increased and declined with kinetics similar to wt mice. these latter findings were reminiscent of transiently increased viral replication in the cns of rl / mice following intracerebral wnv inoculation [ ] . in addition to direct anti-viral activities, activated rnase l degrades viral and host rna [ , ] . the cleavage products may in turn activate rig-i/mda- cytoplasmic pattern recognition receptors, propagating the expression of ifn-a/b and isgs [ ] . while this function of rnase l is indeed evident by reduced ifn-b production in emcv infected rl / mice [ ] , mhv-jhm infection of the cns presented no evidence for this pathway. the negligible affects of rnase l deficiency on overall viral replication [ ] . nevertheless, the preferential susceptibility of rnase l deficient microglia/ monocytes to mhv-jhm infection demonstrates that viral rna does activate cellular rna sensors, albeit in a cell type specific manner. this is supported by mda- triggered activation of the ifn-a/b pathway in microglia and macrophages infected with mhv-a in vitro [ ] . whether the apparent inability of gliotropic mhv-jhm to activate rnase l in other cell types in vivo resides in distinct basal oas/rnase l expression levels, activation of distinct oas enzymes, or other rna sensing receptors such as mda- has not been elucidated. numerous functions of rnase l, not directly associated with anti-viral activity, may contribute to the increased susceptibility of rl / mice to mhv-jhm infection. rnase l plays a role in translational inhibition, regulation of mrna stability, apoptosis, proliferation and tumor suppression [ , ] . for example, rnase l contributes to ifn-a/b mediated apoptosis, as well as homeostasis of thymocytes and splenocytes in young naïve mice [ ] . rnase l deficiency also delays tissue graft rejection [ ] implicating a defect in t cell function or trafficking. nevertheless, both histochemical and flow cytometric analysis revealed a similar extent and composition of inflammatory cells in the cns of infected rl / and wt mice. the lack of rnase l mediated alterations in t cells was supported by similar peripheral expansion of virus-specific cd t cells and kinetics of viral control. increased morbidity could thus not readily be attributed to altered inflammation or pro-inflammatory signals at the tissue level. neuronal infection by gliotropic mhv-jhm is sparse and only evident early p.i. in wt mice. no evidence for enhanced neuronal infection in rl / mice thus suggested that the ifn-a/b mediated anti-viral mechanisms in neurons are rnase l independent. this contrasts with a protective role for rnase l in inhibiting hsv- replication in neurons of ifn-b treated trigeminal ganglia [ ] , and supports virus specific susceptibilities to innate anti-viral immunity. distinguishing features in mhv-jhm infected rl / mice are the sustained areas of microglia infection in brain stem and focal areas of infected microglia within the spinal cord grey matter. infected cells in spinal cord grey matter are also evident in scid [ ] and ifnar / mice (unpublished observation) following infection with the gliotropic mhv-jhm. in rl / mice the prominent infected cells in brain stem and spinal cord grey matter were identified as iba- positive microglia or infiltrating monocytes, which cannot readily be distinguished by immunohistochemistry. nevertheless the morphology of infected cells in the grey matter was consistent with activated microglia, rather than the monocyte/macrophages with dense cytoplasm found prominently in demyelinating lesions. furthermore, preferential microglia infection was supported by a relatively greater increase of viral rna in microglia relative to monocytes, when comparing rl / versus wt mice. the prominent location of iba- positive cells in grey matter of rl / mice could not be attributed to the absence of activated microglia in grey matter of wt mice. in fact, microglia in wt mice exhibited a more ramified phenotype surrounding neurons compared to microglia in rl / infected mice. it remains unclear whether abrogation of the proximal localization of microglia to neurons in rl / spinal cords reflects an unknown function of an rnase l enhanced neuroprotective effect, or altered function due to infection. surprisingly, both prolonged brain stem infection and enhanced spinal cord grey matter infection was associated with more severe demyelination and axonal damage. overall, the cns pathology characteristic of mhv-jhm infection was accelerated by - days in rl / relative to wt mice. although infection of microglia correlated with a subsequent increase in apoptotic cells, apoptotic cells surrounding neurons were only evident in spinal cord, not in brain stem. the inability to detect increased numbers of infected or apoptotic cells in spinal cord white matter is consistent with both an apparent lack of rnase l activation in oligodendrocytes during mhv-jhm infection and paucity of apoptotic oligodendrocytes in wt mice [ ] . whether apoptotic cells originated from infected myeloid cells themselves or from lymphocytes migrating to the infected areas and exerting effector function is unclear. preliminary analysis showed no co-localization of activated caspase and iba- (data not shown). overall, the neurologic disability and morbidity of the infected rl / mice appears to result from axonal or neural degeneration, independent of neuronal infection in both brain stem and spinal cord. the enhanced demyelination phenotype is thus distinct from demyelination attributed to enhanced white matter infection by recombinant neuronotropic mhv variants [ ] . rnase l deficiency and infection of microglia may contribute to this pathology in several ways. infection in the absence of rnase l may impair neuroprotective effects exerted by microglia under inflammatory conditions [ ] [ ] [ ] . alternatively, enhanced infection of microglia may increase proinflammatory responses resulting not only in enhanced recruitment of t cells and monocytes, but also increased local production of neurotoxic factors such as tnf, nitric oxide, oxidative radicals, and matrix metalloproteases. however, only modest increases in ifnc mrna in spinal cord, as well as minor differences in tnf and inos mrna in microglia/monocytes suggest these effects may only be apparent at a focal level. increased localized t cell effector function, manifested in release of perforin and granzymes, has also been shown to contribute to axonal injury without affecting demyelination [ ] . lastly, rnase l deficient and/or infected microglia may perturb normal microglia functions in maintaining neuronal health as indicated by disruption of neuronal autophagy by siv infected microglia [ ] . it thus remains to be determined to what extent accelerated and more severe pathology is due to disruption of the neuroprotective functions of microglia and/or from overt activation due to infected cells [ ] [ ] [ ] [ ] [ ] . independent of a pathogenic effect, it is interesting to note that the absence of rnase l also enhanced macrophage susceptibility to wnv infection [ ] . whether this reflects differential activation of oas enzymes or participation of other pattern recognition receptors such as mda- in susceptible cell types in vivo remains to be elucidated. in summary, these data highlight a novel role of rnase l in protection from virus induced demyelination. although rnase l did not play an overt anti-viral role as measured by viral replication, it did provide specific protection from focal infection of microglia/macrophages in the cns. rnase l was not crucial in protecting neurons from infection and played no obvious role in protecting oligodendrocytes or astrocytes. furthermore, rnase l deficiency did not alter proinflammatory responses, diminish ifna/b mediated signals, or dampen adaptive immune mediators. the results rather uncover how subtle local alterations in viral tropism may affect the balance between neuroprotection and neurotoxicity mediated by microglia/macrophages. animals, viruses, and clinical scores c bl/ mice were purchased from the national cancer institute (nci, fredrick, md). c bl/ -rl / mice were bred and housed under pathogen-free conditions in the biological resources unit of the cleveland clinic. all procedures were performed in compliance with protocols approved by the institutional animal care and use committee. mice were infected at weeks of age by intracranial injection with pfu of the gliotropic mhv-jhm variant v . - [ ] in ml endotoxinfree dulbecco's phosphate buffered saline (dpbs). the severity of clinical disease was graded as previously described [ ] : = ruffled fur, = slow righting reflex, = loss of righting reflex, = moribund. following intraperitoneal administration of ketamin/xylaxine ( mg/kg/ mg/kg), mice were perfused intracardially with ml dpbs. brains, spinal cords, spleens, cervical lymph nodes (cln) and livers were collected and processed as described below. brains were bisected sagittally. one half-brain and whole spinal cord from each mouse were homogenized in ice cold tenbroeck glass grinders in ml or ml of dpbs, respectively. homogenates were clarified by centrifugation for min at g. supernatants were stored at uc. virus in supernatants was measured by plaque assay on monolayers of delayed brain tumor (dbt) astrocytoma cells as previously described [ ] . cns cells from homogenate pellets were resuspended in rpmi containing mm hepes, adjusted to % percoll (pharmacia, upsalla, sweden), underlayed with ml % percoll, centrifuged at g for minutes and collected from the %/ % percoll interface as previous described [ ] . purified cns cells were washed and resuspended in rpmi. cell populations isolated from the brain and spinal cord were phenotyped using four-color flow cytometry. prior to staining, cells were incubated with % mouse serum and % rat anti-mouse fcciii/iir monoclonal antibody (mab) in fluorescent antibody cell sorting (facs) buffer ( . % bovine serum albumin in dpbs) for minutes at uc to block non-specific binding. cell types were identified using fluorescein isothiocyanate-, phycoerythrin-, peridinin-chlorophyll-protein complex-or allophycocyanin-conjugated anti-mouse mab: ly- g ( a ), cd (gk . ), cd ( . ) (all from bd biosciences, san diego, ca) and f / (ci:a - ; serotec, raleigh, nc). virus specific cd t cells were identified using h- d b /s mhc class i tetramers as described previously [ ] . cells were incubated with antibodies for minutes on ice, washed twice with facs buffer and fixed with % paraformaldehyde for minutes on ice. at least , events were acquired on a facscalibur flow cytometer (bd biosciences, san jose, ca) for subsequent data analysis using flow-jo software (tree star, inc. ashland, or). for fluorescence activated cell sorting of microglia and monocyte populations, spinal cords from eight mice per group were finely minced using a razor blade and dissociated in a . % trypsin solution as described [ , ] at uc for minutes with periodic tituration. trypsin was quenched by addition of rpmi supplemented with mm hepes and % new born calf serum. the dissociated cells were washed in rpmi containing mm hepes, % fcs, then isolated from the interphase of a %/ % percoll gradient as described above. cells were incubated with % mouse serum and cd / prior to staining with allophycocyanin-conjugated mab specific for cd ( -f ), peridinin-chlorophyll protein-conjugated cd b (m / ) (bd biosciences, san diego, ca) and phycoerythrin-conjugated mab specific for f / (ci:a - ; serotec, raleigh, nc). monocyte/ macrophages and microglia were purified on a facsaria cell sorter (bd biosciences, san diego, ca) based on their respective cd hi cd b + f / + and cd lo cd b + f / + phenotypes. one half-brain and whole spinal cords were fixed with formalin and embedded in paraffin. sections were stained with either hematoxylin and eosin or luxol fast blue as described [ ] . distribution of viral antigen was determined by immunoperoxidase staining (vectastain-abc kit; vector laboratory, burlingame, ca) using mab j. . , specific for the carboxyl terminus of the viral nucleocapsid protein as primary antibody, biotinylated horse antimouse as secondary antibody, streptavidin-conjugated horse radish peroxidise and , -diaminobenzidine substrate (vector laboratory) [ ] . similarly, immunoperoxidase staining for neurofilament used mouse anti-phosphorylated and anti-non-phosphorylated neurofilament mab (smi- and smi- , covance, princeton, nj) and immunoperoxidase staining for microglia/macrophages used anti-ionized calcium-binding adapter molecule- antibody (iba- , abcam, cambridge, ma). apoptotic cells were detected by rabbit anti-activated caspase- ab (asp , cell signalling technology, beverly, ma). sections were scored for inflammation, viral antigen, apoptotic cells, axonal damage and demyelination in a blinded fashion. representative fields-of-view were identified based on average scores of all sections in each experimental group. for calculation of the percentage area of demyelination, sections were digitized and analysed as previously described [ ] . to identify infected cell types, spinal cords were embedded in tissuetek o.c.t. compound (andwin scientific, tryon, nc), flash frozen in liquid nitrogen and stored at uc. blocks were warmed to uc prior to cutting mm sections by cryostat at this temperature. following fixation with % paraformaldehyde for minutes at room temperature, non-specific antibody binding was blocked using % bovine serum albumin, % normal goat serum. infected cells were identified using anti-mhv-jhm j . mab, rabbit anti-glial fibrillary acidic protein antibody (gfap, abcam, cambridge, ma) and rabbit anti-ionized calcium-binding adapter molecule- antibody (iba- , wako, richmond, va) in combination with goat anti-mouse alexa-fluor and goat anti-rabbit alexa-fluor -conjugated igg (invitrogen, carlsbad, ca) as secondary antibodies, respectively. sections were mounted with prolong gold antifade mounting media containing , -diamidino- -phenylindole (invitrogen, carlsbad, ca). imaging of immunofluorescent sections was performed with a leica sp- confocal microscope (leica microsystems, bannockburn, il). z-projections of sections were processed by imagej software (national institutes of health, bethesda, md) one-half brains and whole spinal cords were snap frozen in liquid nitrogen and stored at uc. frozen brain or spinal cord tissue was homogenized in ml and ml trizol reagent (invitrogen, carlsbad, ca), respectively, in rnase-free tenbroeck glass grinders. rna was purified according to the manufacturer's protocol (invitrogen, carlsbad, ca). briefly, . ml chloroform/ ml trizol (sigma-aldrich, st. louis, mo) was added to homogenate, mixed and centrifuged at , g for minutes at uc. rna was precipitated from the aqueous phase by addition of isopropyl alcohol and centrifugation at , g for min at uc washed in rnase-free % ethanol and resuspended in ultrapure dnase/rnase-free water (invitrogen, carlsbad, ca). cells isolated by facs were immediately resuspended in ml of trizol reagent (invitrogen, carlsbad, ca) and treated as above. isolated total rna was treated with dnase , using the dna-free kit (applied biosystems, foster city, ca) following the manufacturer's protocol. the concentration and purity of rna was measured by spectrophotometry at / nm. rna integrity was confirmed by . % formaldehyde-agarose gel electrophoresis. reverse transcription was performed on mg total rna isolated from brain and spinal cord or all total rna isolated from facs sorted cells, primed with ng random hexamers, (invitrogen, carlsbad, ca) using mmlv reverse transcriptase (invitrogen, carlsbad, ca) for minutes at uc. real-time pcr was performed using sybr green master mix (applied biosystems, foster city, ca) for the following primer sets: interferon-induced protein with tetratricopeptide repeats ( the reaction conditions were: uc for minutes, followed by cycles of: denaturation at uc for seconds, elongation at uc for seconds and annealing at uc for seconds. ifn-b , ifn-a and ifn-c levels were determined by real time pcr using abi gene expression assays with universal taqman fast master mix (applied biosystems, foster city, ca), using manufacturer default cycling conditions and gapdh expression as an endogenous control. all real-time reactions were run using an abi fast real-time cycler and analysed with fast software (applied biosystems, foster city, ca). data presented are expressed as fold-induction relative to gapdh based on the following formula: ( (ct(gapdh)-ct(target)) )* . students' t-test with equal variance was used to compare rl / and wt c bl/ mice. significant differences between groups are noted by: *, = p# . . interferons at age : past, current and future impact on biomedicine type interferons and the virus-host relationship: a lesson in detente a scientific journey through the - a/rnase l system viral encounters with , -oligoadenylate synthetase and rnase l during the interferon antiviral response the dsrna protein kinase pkr: virus and cell control cloning and characterization of a rnase l inhibitor. a new component of the interferonregulated - a pathway small self-rna generated by rnase l amplifies antiviral innate immunity interferon action and apoptosis are defective in mice devoid of , -oligoadenylate-dependent rnase l rnase l and double-stranded rna-dependent protein kinase exert complementary roles in islet cell defense during coxsackievirus infection rnase l plays a role in the antiviral response to west nile virus pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells interferon and cytokine responses to sars-coronavirus infection control of coronavirus infection through plasmacytoid dendritic cell-derived type i interferon type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection viral induction of central nervous system innate immune responses murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia mouse hepatitis coronavirus a nucleocapsid protein is a type i interferon antagonist a , -oligoadenylate analogue inhibits murine hepatitis virus strain (mhv- ) replication in vitro but does not reduce mhv- -related mortality or induction of procoagulant activity in susceptible mice coronavirus infection of the central nervous system: host-virus stand-off murine coronavirus infection: a paradigm for virus-induced demyelinating disease skin allograft rejection is suppressed in mice lacking the antiviral enzyme, , -oligoadenylate-dependent rnase l inverted immunodominance and impaired cytolytic function of cd + t cells during viral persistence in the central nervous system sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv- ) leads to a characteristic distribution of demyelination diverse functions of rnase l and implications in pathology ctl effector function within the central nervous system requires cd + t cells inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination differential induction of apoptosis in demyelinating and nondemyelinating infection by mouse hepatitis 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neurodegeneration interferon action: rna cleavage pattern of a ( - )oligoadenylate-dependent endonuclease interferon actionsequence specificity of the ppp(a p)na-dependent ribonuclease rnase l: its biological roles and regulation interferon-beta suppresses herpes simplex virus type replication in trigeminal ganglion cells through an rnase l-dependent pathway memory cd + t-cell-mediated protection from lethal coronavirus encephalomyelitis microglia as neuroprotective, immunocompetent cells of the cns molecular and cellular immune mediators of neuroprotection multiple sclerosis: novel perspectives on newly forming lesions experimental models of neuroprotection relevant to multiple sclerosis the role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: hiv-associated dementia, alzheimer disease, and multiple sclerosis pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies induction of class i antigen processing components in oligodendroglia and microglia during viral encephalomyelitis perforin-mediated effector function within the central nervous system requires ifn-gamma-mediated mhc up-regulation the authors thank dr. bruce trapp for helpful discussions and comments on the manuscript. we also thank wen wei and ernesto barron for excellent assistance with the histopathology and jennifer powers for cell purification by facs. conceived and designed the experiments: ddci sas ccb. performed the experiments: ddci pk. analyzed the data: ddci sas drh pk rhs raa ccb. contributed reagents/materials/analysis tools: ccb. wrote the paper: ddci sas drh rhs ccb. key: cord- - ugzkf authors: weeratunga, prasanna; uddin, md bashir; kim, myun soo; lee, byeong-hoon; kim, tae-hwan; yoon, ji-eun; ma, jin yeul; kim, hongik; lee, jong-soo title: retracted article: interferon-mediated antiviral activities of angelica tenuissima nakai and its active components date: - - journal: j microbiol doi: . /s - - - sha: doc_id: cord_uid: ugzkf angelica tenuissima nakai is a widely used commodity in traditional medicine. nevertheless, no study has been conducted on the antiviral and immune-modulatory properties of an aqueous extract of angelica tenuissima nakai. in the present study, we evaluated the antiviral activities and the mechanism of action of an aqueous extract of angelica tenuissima nakai both in vitro and in vivo. in vitro, an effective dose of angelica tenuissima nakai markedly inhibited the replication of influenza a virus (pr ), vesicular stomatitis virus (vsv), herpes simplex virus (hsv), coxsackie virus, and enterovirus (ev- ) on epithelial (hek t/hela) and immune (raw . ) cells. such inhibition can be described by the induction of the antiviral state in cells by antiviral, ifnrelated gene induction and secretion of ifns and pro-inflammatory cytokines. in vivo, angelica tenuissima nakai treated balb/c mice displayed higher survivability and lower lung viral titers when challenged with lethal doses of highly pathogenic influenza a subtypes (h n , h n , h n , and h n ). we also found that angelica tenuissima nakai can induce the secretion of il- , ifn-λ, and local iga in bronchoalveolar lavage fluid (balf) of angelica tenuissima nakai treated mice, which correlating with the observed prophylactic effects. in hplc analysis, we found the presence of several compounds in the aqueous fraction and among them; we evaluated antiviral properties of ferulic acid. therefore, an extract of angelica tenuissima nakai and its components, including ferulic acid, play roles as immunomodulators and may be potential candidates for novel anti-viral/anti-influenza agents. viral diseases range from trivial infections to plagues that alter the course of history. because of the enormous variations in viruses and in their epidemiology and pathogenesis, there is no single, magic-bullet approach to control. each virus presents its own set of problems. as an example, influenza viruses are highly infectious and constitute a major causative agent for recurrent epidemics and pandemics. on average, approximately % of the world's population is infected by the virus annually, resulting in an estimated , deaths, hence posing a serious health threat (rajasekaran et al., ) . moreover, new and re-emerging infectious viral diseases are a rising global health threat, and the risk of spreading these viruses between continents and countries is even greater. hiv/aids, severe acute respiratory syndrome (sars) and the h n influenza pandemic and mers epidemic are only a few of many examples of emerging infectious diseases in the modern world (morens and fauci, ; who, ) . several preventive and therapeutic measures, including biosecurity, vaccination and antiviral drugs, are routinely used or tried to prevent and treat viral diseases. vaccines are the basis of prevention of many viral infections; however, there are considerable drawbacks. because vaccination requires regular monitoring to confirm matching between the vaccines and the circulating virus strains, time-consuming generation processes limit its availability. failures of influenza vaccines have been widely documented, and in the elderly populationin which most mortality occurs-influenza vaccines are only approximately % effective (reichert et al., ) . in the eventuality of a pandemic infection with a new strain, antiviral drugs also represent the first line of defense. owing to their metabolic properties, viruses are difficult to control, and the limited availability, associated side effects, and rapid development of antiviral resistance have limited the usefulness of these drugs. therefore, within this evolving environment, groundbreaking strategies and responses are required to reduce the economic and human health risks associated with viral diseases. the development of safe, effective and inexpensive antiviral drugs is among the top global priorities in drug development. currently, there is a large and ever-expanding global population base that prefers the use of natural products for preventing and treating medical conditions (gandhiraja et al., ) and many pharmaceutical companies are attempting to produce new antimicrobial formulations extracted from plants or herbs. plants are an important sources of lead compounds; up to % of modern drugs are derived from plant material (pan et al., ) . for the viral diseases, empirical evidence of the ethno-medical benefits of plants, coupled with bioassay-guided fractionation and isolation, has the potential to identify novel antiviral drugs. in our study, approximately natural oriental herbal extracts were screened for antiviral effects. of these, extracts from angelica tenuissima nakai (atn) showed considerable therapeutic promise because of its broad spectrum of antiviral activities and immune-enhancing properties. angelica tenuissima nakai belongs to the family apiaceae and grows in certain areas in china and rocky slopes in the korean peninsula (ka et al., ) . it is widely used to treat headache, diarrhea, epilepsy and rheumatic arthralgias in traditional oriental medicine (nam et al., ) . however, the antiviral activity or immune-modulatory potential of the crude plant extract of angelica tenuissima nakai have not been reported in detail. hence, we evaluated the antiviral activities of total aqueous extracts from this herb against a wide array of viruses in vitro. we also examined the effects of angelica tenuissima nakai on innate immune responses. in addition, we used highperformance liquid chromatography (hplc) analysis to determine the active molecules present in the aqueous fraction. commercial dried bark of the angelica tenuissima nakai was obtained and verified by professor ki hwan bae at the college of pharmacy, chungnam national university. the water extract of angelica tenuissima nakai was prepared by vitabio corporation. further, the extract quality was assured by herbal medicine improvement research center, korea institute of oriental medicine, daejeon, republic of korea. hundred gram ( g) of the dried bark was mixed with l of distilled water and extracted by heating for . h at °c using a medical heating plate (gyeongseo extractor cosmos- ). then, angelica tenuissima nakai was filtered using a filter paper ( . μm) (millex ® ). the extract was next centrifuged at , rpm for min and the supernatant was collected, and the ph was adjusted to . . the total aqueous extract was then filtered through a syringe filter ( . μm) and lyophilized. the final concentration was adjusted to . mg/ml with phosphate buffered saline (pbs) and kept at °c until utilization. raw . (atcc ® tib- tm ), hek t (atcc ® crl- tm ), hela (atcc ® ccl- tm ), mdck (atcc ® ccl- , nbl- ), and a (atcc ® ccl- tm ) cells were cultured in dulbecco's modified eagle's medium (dmem) (invitrogen) supplemented with % fetal bovine serum (fbs; gibco) and % antibiotic-antimycotic solution (gibco) in a humidified incubator at °c and % co . green fluorescent protein (gfp)-fused viruses such as pr -gfp, vsv-gfp, h -gfp, and ev- were kindly provided by dr. jae u. jung, department of molecular microbiology and immunology, university of southern california, usa. challenge viruses were provided by dr. y. k. choi, chungbuk national university, cheongju, republic of korea. antiviral assays in angelica tenuissima nakai pretreated raw . , hek t, and hela cells raw . cells were cultured in -well tissue culture plates ( × cells/well) and incubated at °c for h. hek t and hela cells were cultured in six-well tissue culture plates ( × cells/well) under similar conditions. antiviral assays were performed according to moon et al. ( ) , with some modifications. dmem alone (untreated and virus-only groups), dmem with , u/ml of recombinant mouse/human interferon (ifn)-β (positive control, sigma) and dmem with . μg/ml ( μl/ml or % v/v) of angelica tenuissima nakai were incubated in different wells. at hpt (hour post treatment), all the wells were gently washed with pbs once and raw . cells were infected with either vsv-gfp (moi = . ) or pr -gfp (moi = . ), hek t cells were infected with vsv-gfp (moi = . ) or hsv-gfp (moi = . ) and hela cells were infected with h -gfp (moi = . ) or ev- (moi = . ) viruses using dmem containing % fbs. two hours post-infection (hpi), the culture medium was renewed, and incubated for an additional h. gfp expression was observed was quantified with the glomax multi-detection system (promega). gfp expression, virus titrations and cell viabilities were determined at and/or hpi. in vitro pro-inflammatory cytokine inducing effect of angelica tenuissima nakai was tested using commercial elisa kits. in raw . cells, murine interleukin (il)- and ifn-β were measured, as previously described (wadsworth and koop, ) . raw . cells were treated with recombinant murine ifn-β (sigma-aldrich) and . μg/ml ( μl/ml or % v/v) angelica tenuissima nakai and cultured supernatants were collected at , , and hpt, clarified by centrifugation at , × g for min at °c and dispensed into murine ifn-β (pbl interferon source) elisa plates or murine il- or murine tnf-α (bd bioscience) capture antibody-coated elisa plates. in the case of hek t cells, recombinant human ifn-β (sigma-aldrich) was used as the positive control and the clarified supernatant was dispensed into human ifnβ (tfb, inc.) and human il- (invitrogen) elisa plates. murine ifn-β, human ifn-β, and human il- elisa were performed in duplicate, and other elisa's was performed in triplicate. interferon-mediated antiviral activities of angelica tenuissima nakai determination of the effect of angelica tenuissima nakai on type i ifn-related protein phosphorylation in raw . cells by immunoblot analysis raw . cells were grown in -well tissue culture plates and incubated at °c. after h the cells were treated with dmem containing % fbs alone (negative control), dmem with ng/ml lps (positive control), or dmem with . μg/ml ( μl/ml or % v/v) angelica tenuissima nakai, and the cells were harvested at , , , and hpt. the cell pellets were lysed in lysis buffer for immunoblot analysis; loaded on sds-page and then transferred onto a polyvinylidene difluoride membrane (bio-rad) for h. the membranes were blocked for h in tris-buffered saline containing . % tween- (tbst) and % bovine serum albumin. then incubations were performed at °c overnight with indicated primary antibody such as anti-irf (abcam, #ab ), antiphopho-irf (ser ), anti-p , anti-phopho-p , anti-stat , anti-phospho-stat , anti-tbk , or anti-phospho-tbk , anti-p , anti-phopho-p (cell signaling technology), or anti-β-actin (santa cruz sc# ) antibodies. horseradish peroxidase-conjugated secondary antibody (sigma) was used to visualize the respective proteins by means of an enhanced chemiluminescence detection system (ecl-ge healthcare, uk) using a las- mini lumino image analyzer. raw . cells were grown in -well tissue culture plates ( × cells/well) and incubated at °c. the cells were treated with dmem containing % fbs alone (negative control), dmem with u/ml of recombinant murine/ human ifn-β, dmem with . μg/ml ( μl/ml or % v/v) angelica tenuissima nakai, and the cells were harvested at , , , , and hpt. the total rna from the cells was isolated using the rneasy mini kit (qiagen), and cdna was synthesized using reverse transcriptase (toyobo). the different levels of cdna were quantified by real-time polymerase chain reaction (pcr) using a quantitect sybr green pcr kit (qiagen) on a mygenie thermal block (bioneer). the pcr primers are listed in tables and . fifty two female, five-week-old balb/c mice were separated into four experimental sets, with two groups per set. of the four sets, one had two groups with mice each (six for lung virus titration at and day's post-infection [dpi]). the other three sets had two groups containing mice each. the mice were orally inoculated . mg/ml angelica tenuissima nakai at a total volume of μl ( μg/head) , , and days before infection. the control groups were orally inoculated μl of pbs. all mice were intra-nasally infected ( μl) with five times the % mouse lethal dose (mld ) of h n , h n , h n or h n . mice showing a more than % body weight loss were regarded to have reached the experimental end point and were humanely killed. the body weight and survival were recorded up to dpi. at and dpi, three mice from each of the two groups from the h n infected set were randomly sacrificed to measure the lung virus titers. lung tissues were collected from euthanized mice aseptically, and virus titers were measured by % tissue culture infectious dose (tcid ) (quan et al., ) . first, lung tissues were mechanically homogenized in ml of pbs solution added with % antibiotic/antimycotic solution, centrifuged ( min, , × g and °c) and stored at - °c. madin-darby canine kidney (mdck) cells cultured in -well microtiter plates were infected with -fold serial dilutions (in dmem with % fbs) of lung homogenate ( μl/well) in quadruplicate and incubated at °c. at hpi, the media was removed and renewed with medium containing l- -tosylamido- -phenylethyl chloromethyl ketone (tpck) trypsin (thermo scientific) and incubated for h. viral cytopathic effects (cpe) were monitored daily and the titers were determined by the hemagglutination assay (ha). the virus titer was calculated by the reed and muench method (zhao et al., ) and expressed as log tcid /lung. twenty four, five-week-old balb/c mice were divided into two groups with mice each. in one group, mice were orally administered . mg/ml angelica tenuissima nakai at a total volume of μl ( μg/head) , , and days before the collection of samples. the control group was orally administered μl of pbs. three mice from each group were randomly selected at , , , and hpt of angelica tenuissima-nakai or pbs, and their bronchoalveolar lavage fluid (balf) and small-intestinal fluids (sif) were collected as described below. balf: balf was collected as previously described with some modifications (viana et al., ) . briefly, lungs of mice were lavaged four times with ml of hank's balanced salt solution (hbss), collected and immediately stored at - °c and later subjected to elisa (murine iga, il- , ifn-λ). sif: small intestines of mice were collected as previously described (lefrançois and lycke, ) . briefly, mice were euthanized by cervical dislocation; midline incision was performed and retracted the skin. small intestine was cut ~ cm above cecum to separate it from others. then, the intestine was flushed very carefully with μl of hbss (sigma-aldrich), centrifuged and fluids were immediately stored at - °c. the sif supernatants were further assayed by the elisa (murine iga). a reversed-phase high performance liquid chromatography (hplc) system was an agilent technologies infinity (agilent technologies co.) having g c quaternary pump, g b auto-sampler, g a column oven, g c multiple wavelength detector a pump l- . the column was carried out on a zorbax eclipse xdb-c column ( × . mm i.d. s- um) and the column oven temperature was kept at °c. the mobile phase consisted of % formic acid (solvent a) and acetonitrile (solvent b) in the gradient mode as: - min - % b; - min - % b; - min - % b; - min - % b; - min - % b. the flow rate was . ml/min, and the elution was monitored at nm. fraction (fr)- (later identified and confirmed as ferulic acid) was purified by a semi prep-hplc agilent technologies infinity (equipped with a fraction collector, fc b, gilson) using ymc-pack ods-a ( × mm i.d. s- μm, nm) (ymc) at a flow rate of ml/min, with constant temperature of °c. purified fractions were further subjected to antiviral assays and cytokine inducing ability in immune (raw . ) cells using an effective dose of . μg/ml. antiviral activity against pr -gfp in raw . cells, virus titration and induction of cytokine secretion were determined according to the protocols described in 'materials and methods.' the viral titers were determined by standard plaque assays using vero cells (coil and miller, ) . to assay the viral replication, culture supernatants were collected from vsv-gfp and h -gfp infected cells both at and hpi and viral titer was determined in vero cells. in the case of pr -gfp titration, cells were collected at hpi and subjected to five cycles of freezing at - °c and thawing at rt and titer was determined in vero cells. viral titers for ev- , brv, and hrv were measured by the median tissue culture infectious doses (tcid ) using hela cells (wilden et al., ) with some modifications. briefly, supernatants collected from the infected cells were used to infect hela cells cultured in -well microtiter plates with -fold serial dilutions ( μl/well). after hpi, dmem ( % fbs) containing tpck trypsin (thermo scientific) was added to the infected wells and incubated for additional days. viral cpe were observed daily and titers were determined by cpe-tcid . raw . , hek t and hela cells were cultured in well plates ( . × cells/well, × cells/well and × cells/well, respectively) and incubated at °c in a % co atmosphere. after h, two-fold serially diluted angelica tenuissima nakai ( μl/well) was added. at hpt, the cells were infected using dmem containing % fbs. raw . cells were infected with pr -gfp (moi = . ) or vsv-gfp (moi = . ), hek t cells were infected with vsv-gfp (moi = . ) or hsv-gfp (moi = . ) and hela cells were infected with coxsackie-gfp (moi = . ) or ev- (moi = . ) viruses. at hpi, the culture medium was renewed. gfp expression was measured at hpi with the glomax multi-detection system (promega). the ec values were then calculated as the angelica tenuissima nakai concentration yielding % gfp expression. the experiments were performed in triplicate. the cc was assessed in a cell viability assay through the trypan blue exclusion (strober, ) . the assay was performed using -well tissue culture plates. increasing concentrations ( - μl/ml or . - μg/ml) of the water extract were added to confluent raw . , hek t and hela cell monolayers. clarified cells were stained with . % trypan blue stain (invitrogen) (ratio: : ) at hpt and mounted to a hemocytometer to get the percentages of viable cells. cc was calculated as the concentration of the extract resulting in % cell viability. the experiment was performed in triplicate. all the experiments were repeated in triplicate and the data were assessed as the means ± standard deviation (sd). statistical significance was evaluated using a student's t-test or one-way analysis of variance (anova), and was considered significant with p< . (*p< . and **p< . ). results for percent initial body weight were also compared by student's t-test. comparison of survival was done by log-rank test using graphpad prism version. to determine the antiviral effects of angelica tenuissima nakai in epithelial cells, we checked the antiviral activity in hek t and hela cells. in the hek t cells, the antiviral activity was determined with gfp-fused vsv or hsv upon pre-treatment with the extract ( . μg/ml [ μl/ml or % v/v]). upon pre-treatment ( fig. a and b), gfp expression was significantly reduced in hek t cells in comparison with the untreated groups and showed reduced viral titers by nearly -fold or -fold against vsv-gfp and hsv-gfp, respectively at hpi. moreover, a significant reduction in cell death was observed in angelica tenuissima nakai extract-treated hek t cells compared with the untreated cells. also, we examined the antiviral activity of angelica tenuissima nakai in the hela cells against gfp-fused coxsackie virus (h -gfp) and enterovirus- (ev- ). pre-treated cells with angelica tenuissima nakai exhibited markedly reduced h -gfp expression and reduced viral titers by nearly . fold at hpi, together with a significant reduction in cell death (fig. c ). in addition, ev- -induced cpes were markedly inhibited upon pre-treatment with angelica tenuissima nakai (fig. d ), resulting in a low level of viral replication. collectively, these results indicate that the total aqueous extract of angelica tenuissima nakai reduces the replication of rna and dna viruses in epithelial cell lines. to investigate the antiviral effects in immune cells, we first assessed the replication of divergent gfp-expressing viruses that were treated or untreated with cytotoxic-free (data not shown) angelica tenuissima nakai in raw . cells. a total aqueous extract of angelica tenuissima nakai-treated ( . μg/ml [ μl/ml or % v/v]) raw . cells exhibited markedly reduced gfp expression; however, the untreated groups had high levels of gfp expression for vsv ( fig. a) and pr (fig. c) . when quantitated, a significant reduction in the gfp expression was observed in the extract-treated cells compared to untreated groups ( fig. b and d middle panel). these findings further correlated with the observed viral titers from vsv-gfp or pr -gfp infected cells (fig. b and d right panel) . interestingly, the angelica tenuissima nakai-treated cells had a ≥ % cell viability within hpi for all the tested viruses compared with the untreated cells, which were shown to have significantly higher cell death following virus infection ( fig. b and d left panel) . consequently, these results also suggest that the aqueous extract of angelica tenuissima nakai could significantly inhibit the replication of vsv and pr viruses in immune cells. we then developed an improved gfp assay to determine the ec values of angelica tenuissima nakai against divergent viruses in vitro using hek t and raw . cells (magadula and suleimania, ; lin et al., ) . the ec is the extract concentration that results in a % reduction in virus, and the extract concentration that results in % cell viability is the cc . because we primarily used gfp-fused viruses, a % reduction in gfp expression was considered equivalent to a % reduction in virus titer. in the case of ev- , a % reduction in cpe was considered equivalent to a % reduction in virus titer. the aqueous extract of angelica tenuissima nakai inhibited the replication of vsv-gfp (moi= . ) and hsv-gfp (moi= . ) by % at an ec of . ± . μg/ml and . ± . μg/ml, respectively in hek t cells (table ). in raw . cells, vsv-gfp (moi= . ) and pr -gfp (moi = . ) by % at an ec of . ± . μg/ml and . ± . μg/ml, respectively (table ). in addition, the observed ec values against coxsackie-gfp (moi= . ) and ev- (moi = . ) were . ± . μg/ml and . ± . μg/ml, respectively, in hela cells. given its effectiveness and convenience during the experiments, . μg/ml has been chosen as the optimal dose of angelica tenuissima nakai for further in vitro antiviral assays, observed from ec values. following treatment with various concentrations, a cell viability test was performed to assess the cytotoxicity of angelica tenuissima nakai extract. the extract had a cc of . ± . μg/ml, . ± . μg/ml, and . ± . μg/ml in hek t, raw . and hela cells, respectively (table ) . the selection indexes (sis) of angelica tenuissima nakai for vsv and hsv on hek t cells were . and . , respectively; those for vsv and pr on raw . cells were . and . , respectively; and those for coxsackie and ev- on hela cells were . and . , respectively. notably, the sis of angelica tenuissima nakai were several magnitudes higher for the tested viruses in their respective cell lines, a clear indication of the extract's broad prophylactic and therapeutic potential. because angelica tenuissima nakai possesses antiviral activity, we hypothesized that angelica tenuissima nakai might be involved in the type i ifn signaling pathway. to test this hypothesis, we first evaluated the levels of interferon-β (ifnβ) and the pro-inflammatory cytokines in the extract-treated cell supernatant of hek t or raw . cells (fig. ) . angelica tenuissima nakai ( . μg/ml [ μl/ml or % v/v]) induced high levels of secreted ifn-β and il- in hek t cells (fig. a) and tnf-α, il- , and ifn-β in raw . cells at both hpt and hpt (fig. b) . importantly, the secreted cytokine amounts were significantly higher in hek- t cells, although the secreted level was not as high as the levels observed in the raw . cells in comparison with the non-treated groups. these results suggest that the aqueous extract of angelica tenuissima nakai can induce the secretion of ifns and pro-inflammatory cytokines, which can stimulate the cellular antiviral state for inhibition of viral replication. further, we checked the phosphorylation of interferon-related signal molecules or p molecule related to nf-kb activation to observe correlation of above results with the ifninducing signaling pathway. for this study, immunoblot analyses were performed using whole-cell lysates of the angelica tenuissima nakai-treated ( . μg/ml [ μl/ml or % v/v]) raw . cells. as shown in fig. c , we found that the significant upregulation of type i interferon or nf-kb related signal molecules (irf , stat , tbk , p , and p ) in angelica tenuissima nakai-treated cells such as lps-treated cells. taken together, these results clearly indicate that the aqueous extract of angelica tenuissima nakai can induce the secretion of ifns and pro-inflammatory cytokines through the activation of signal molecules in the type i ifn and nf-kb signaling pathway. given that the angelica tenuissima nakai extracts were able to secrete ifn-β and pro-inflammatory cytokines, we further confirmed an interaction between angelica tenuissima nakai and the transcriptional levels of antiviral genes or interferonstimulatory genes (isgs) in vitro. as confirmed by real-time pcr assay, the mrna expression levels of various antiviral and interferon stimulatory genes in the cells treated with angelica tenuissima nakai ( . μg/ml [ μl/ml or % v/v]) were up-regulated to levels similar to those found with the ifn-β-treated positive controls (fig. ) . also, to determine the transcription levels of various antiviral genes in the angelica tenuissima nakai-treated hek t cells (fig. a) , an ifn-β real-time pcr assay was performed from to hpt to monitor the time-dependent mrna changes. interestingly, the highest fold inductions of cellular transcriptional levels were observed at hpt for all the tested primers (the primers are listed in table ). in hek t cells, upon treatment of angelica tenuissima nakai extract, transcriptional levels for ifn-β, mx- , gbp- , tnf-α, il- , and il- were up-regulated by . -fold, . -fold, . -fold, . -fold, . -fold, and . -fold, respectively (fig. a) . similarly, transcriptional activation patterns were also observed in the angelica tenuissima nakai-treated raw . cells. the results showed that the extract-treated cells displayed a nine-fold increase in the level of ifn-β mrna at hpt and a thirteen-fold induction at hpt compared with the untreated cells (fig. b) . hence, we performed a pcr assay for other genes of interest at , , and hpt using specific primers (the primers are listed in table ) in raw . cells. we found that various antiviral gene tran-scriptional levels of were to be up-regulated by angelica tenuissima nakai at hpt, including mx , oas- , pml, pkr, il- , isg- , and isg- , to levels that were . -fold, . fold, . -fold, . -fold, . -fold, . -fold, and . -fold, respectively, higher than those of the control (fig. b) . thus, overall results suggests that angelica tenuissima nakai extracts were able to up-regulate the transcriptional levels of ifn-β, isgs, and various antiviral related genes in hek t and raw . cells which can induce antiviral state. to study the angelica tenuissima nakai induced prophylactic interferon-mediated antiviral activities of angelica tenuissima nakai after challenge, the control (pbs) mice groups were suffered severe illnesses and the body weights loss significantly. by approximately - dpi, most of the infected mice in the con-trol groups displayed severe clinical signs of respiratory disease, including labored respirations and respiratory distress. moreover, the control groups succumbed to death by dpi for all of the viruses tested. however, the angelica tenuissima nakai-inoculated mice showed a ≤ % body weight loss between and dpi and had begun to regain by dpi, returning to their normal state by - dpi (fig. ) . besides, all the angelica tenuissima nakai-inoculated groups had higher survival rates: % survival for the groups infected with h n (fig. b) , h n (fig. c) , and h n (fig. a) ; and % survival infected with h n (fig. d ). the surviving mice in these groups did not show obvious clinical signs, except for negligible weight loss. because influenza virus is transmitted primarily by aerosols via the respiratory system of mice and replicated most . . induction of immune state by angelica tenuissima nakai in balb/c mice. five-week-old female balb/c mice were orally administered . mg/ml angelica tenuissima nakai (atn) at a total volume of μl ( μg/head) at , , and days before the collection of samples. control groups were orally administered with μl of pbs. three mice from each group were randomly selected at , , , and hpt of angelica tenuissima nakai or pbs, and their bronchoalveolar lavage fluid (balf) and small-intestinal fluid (sif) were collected and analyzed for the presence of secreted murine (a) il- or ifn-λ, (b) immunoglobulin a (iga). the test was performed in duplicate for iga and ifn-λ and triplicate for il- . the data shows representative means ± sd of each cytokine measured over time (*p< . indicates a significant difference between groups compared by student's t-test). efficiently in lungs (bouvier and lowen, ) , it is important to examine the ability of angelica tenuissima nakai extracts to inhibit viral replication in lungs. to evaluate the ability of angelica tenuissima nakai to inhibit viral replication in the h n infected lung tissues, three mice from each group were sacrificed, and their lungs were collected at and days post-infection for viral titration. in comparison of control groups, h n virus replicated efficiently in the lungs with a viral titer of . log tcid /lung and . log tcid /lung on and dpi, respectively (fig. e) . comparatively, the viral loads in the angelica tenuissima nakai-treated groups were significantly lower: . log tcid /lung and . log tcid /lung, at dpi and dpi, respectively (fig. e ). consequently, inoculation of angelica tenuissima nakai extracts induce the sufficiently strong inhibition of viral replication and endorsed the survival of mice against diverse influenza a subtypes lethal infections. to observe the immune status in balf of angelica tenuissima nakai inoculated balb/c mice, we measured the level of the cytokines and iga in the balf and sif. after inoculation with angelica tenuissima nakai extracts into mice, we detected significant level of il- and ifn-λ which has antiviral function such as type i ifns, at each time point starting from to hpt in the balf. and also, we detected the presence of iga in balf and sif in all the tested samples. however, minor levels of iga secretion were detected in all the samples from the pbs-treated mice (fig. b) . these results indicate that oral inoculation of angelica tenuissima nakai extracts stimulate mucosal immune cells of small intestine and ultimately induce the strong inhibition of influenza virus replication in bronchoalveolar through the mucosal immune response system. for the main component profile of the water extract of angelica tenuissima nakai, we used a hplc system. a reversed-phase hplc method was employed with a mobile phase of % formic acid (solvent a) and acetonitrile (solvent b) in the gradient mode as follows: - min, - % b; - min, - % b; - min, - % b; - min, - % b; and - min, - % b. the flow rate was kept constant at . ml/min for the total run time, and fractions were purified successfully. we then evaluated the antiviral effect of the identified fractions (fr- to fr- ) in raw . cells. viral replication was monitored with pr -gfp in response to pre-treatment of various fractions. an effective dose of . μg/ml was chosen on the basis of our preliminary experiments on the efficacy of purified fractions (data not shown). interestingly, treatment with fr- markedly inhibited the virus replication (fig. c) . of fractions, fr- was detected at a wavelength and retention time of nm and . min, and the concentration of fr- in . mg/ml (w/v) of angelica tenuissima nakai extract was found to be . μg/ml ( fig. a and b) . ultimately, fr- was confirmed the ferulic acid by lc-mass. the ferulic acidtreated cells displayed reduced gfp expression and reduced viral titers compared with the untreated cells. ferulic acid treatment reduced the viral titers by nearly . -fold against pr -gfp at hpi (fig. c) . furthermore, treatment with ferulic acid ( . μg/ml) markedly increased cytokine secretion in the raw . cells (fig. d) . therefore, our results strongly suggest that ferulic acid, a major constituent of angelica tenuissima nakai, might be able to induce the antiviral state in cells and subsequent inhibition of virus replication. historically, natural products and their derivatives have been invaluable sources of therapeutic agents. recent technological advances, coupled with unrealized expectations of lead-generation strategies, have led to renewed interest in natural products in drug discovery (koehn and carter, ) . plants have a long evolutionary history of developing resistance against viruses and have increasingly drawn attention as potential sources of antiviral drugs. for instance, many plant extracts and compounds of plant origin have been shown to have activity against influenza viruses (park, ; koehn and carter, ) . at present, plant and herb resources are unlimited, have provided mankind with remedies for many infectious diseases and continue to play a major role in primary health care as therapeutic remedies in developing countries. here, we evaluated the antiviral activity of angelica tenuissima nakai in vitro and in vivo. despite the known biological properties of the plant, no study has been conducted on the antiviral activity and the underlying mechanism of action. first, we found that the total aqueous extract of angelica tenuissima nakai displayed a broad spectrum of antiviral properties in vitro. this herb has been used for a variety of purposes for a long time, and no adverse effects of its use have been reported. notably, angelica tenuissima nakai did not show any significant cytotoxic effect on the tested cell lines. its cell cytotoxic concentration (cc ) was several magnitudes higher than the effective concentration (ec ) of the tested viruses, and the sis of the herb for various viruses indicate a higher safety margin of the extract for therapeutic or prophylactic purposes (table ). in examining the in vitro antiviral activity, we determined that angelica tenuissima nakai inhibited the replication of influenza (fig. c) , vsv (figs. a and a) , hsv (fig. b) , coxsackie (fig. c) and ev- (fig. d ) viruses in immune (raw . ) and epithelial (hek t and hela) cells. upon viral infection, host cells initially recognize an infection and quickly evoke antiviral innate immune responses, including secretion of type i ifns and pro-inflammatory cytokines (takeuchi and akira, ) . secreted ifns and cytokines induce an antiviral state, which is important to protect the host cells against invading viruses (tenoever et al., ) . induction of an antiviral state at an early stage of virus infection is critical to control the spread and pathogenesis of viruses (boasso, ) by specific agents could be effective approaches for limiting viral infection (jackson, ; perlman, ) . likewise, we hypothesized that angelica tenuissima nakai induces an antiviral state via the induction of type i interferons and proinflammatory cytokines, and we determined the induction of antiviral, ifn-stimulated genes (fig. ) and the secretion of ifn-β and il- ( fig. ) by angelica tenuissima nakai in vitro. to elucidate the features in antiviral signaling, we also evaluated the effect of angelica tenuissima nakai on the phosphorylation of irf- , p , tbk , stat , erk, and p , which are key signaling molecules in the type i ifn and nf-κb signaling pathways. upon stimulation of the pattern recognition receptors (prrs) or unknown receptors of the host cell by foreign materials containing pathogens or diverse ligands, downstream signal transduction is activated, including the activation of adaptor signal molecules or transcriptional factors, and can initiate the induction of type i ifns and pro-inflammatory cytokines to up-regulate the antiviral status of the host cell (mogensen, ) . in this study, we found that angelica tenuissima nakai extracts treatment can induce the phosphorylation of irf- , stat , and tbk in a time-dependent manner, providing evidence of downstream signal transduction in the type i ifn signaling pathway (fig. c) . additionally, the activation of nf-κb (p , p ), which leads to a strong secretion of pro-inflammatory cytokines, was also observed (fig. c ). this phosphorylation could lead to the rapid production of type i ifns and various inflammatory cytokines that play a crucial role in stimulating the antiviral state and the subsequent clearance of viruses (price et al., ) . oral administration of angelica tenuissima nakai extracts not only increased the survival rate of mice subjected to lethal challenges with divergent influenza a subtypes, including h n , h n , h n , and h n , but also led to rapid weight recovery (fig. ). mice treated with the abstract initially displayed little weight reduction; ultimately, the majority did not lose more than % of their body weight. by contrast, all of the mice in the control groups displayed weight loss of more than % within dpi and were humanely killed. influenza virus causes a rapid reduction in the body weight of infected mice; hence, % body weight loss is considered the humane end point for sacrificing influenza virus-infected mice . in addition, lung virus titration (fig. e ) correlated with survival, and the extract-treated mice displayed reduced lung viral titers, suggesting that angelica tenuissima nakai is sufficiently potent to inhibit viral replication and promote the survival of mice against lethal infections of diverse influenza a viruses. to support the antiviral effects of angelica tenuissima nakai in vivo, we observed the immune status in balf of angelica tenuissima nakai inoculated balb/c mice. as shown in fig. , after inoculation with angelica tenuissima nakai extracts into mice, we could detect significant level of il- and ifn-λ in balf. previous literature has indicated that elevated levels of serum il- correlate with induction of the antiviral state and therefore play an important role in the inhibition of virus replication (spellberg and edwards, ; melchjorsen et al., ) . and ifn-λ which is an exciting new chapter in the field of ifn research, is also able to activate the same intracellular signaling pathway and many of the same biological activities as those displayed by type i ifns, including antiviral activity, in a wide variety of target cells (lazear et al., ) . moreover, we also found significant level of secreted iga (siga) in balf and sif, after inoculation with angelica tenuissima nakai extracts. secretory iga (siga) is the most abundant immunoglobulin in body secretions, including saliva, tears, colostrum and gastrointestinal secretions, and is the main effector of the mucosal immune system, providing an important first line of defense against most pathogens that invade the body at the mucosal surfaces (woof and mestecky, ; mantis et al., ) . thus, significant level of il- , ifn-λ or siga which induced in balf by angelica tenuissima nakai extracts, maybe correlate with the higher survival rates observed in the balb/c mice against divergent influenza a infection. macrophages have been widely used in drug development because of their significant role in the immune system, especially in antiviral responses (wilden et al., ; kim et al., ) . here, we used murine macrophages (raw . ) to evaluate the antiviral effect of angelica tenuissima nakai in vitro. in addition, we used epithelial cells such as hek t and hela, which are very susceptible to viral replication. it was recently reported that hek t (graham et al., ) and hela (kolli et al., ) cells have fewer prrs than tolllike receptors (tlrs). this finding means that an aqueous extract of angelica tenuissima nakai may contain active components that can bind or penetrate the cell membrane. those active components may stimulate the cell surface prrs, cytoplasmic prrs, or both, and ultimately induce antiviral immune responses in immune or epithelial cells. surely, we tested angelica tenuissima nakai for endotoxin contamination using a limulus amebocyte lysate (lal) assay and found that it was not contaminated with endotoxin (data not shown). there have been phytochemical reports on various types of compounds from angelica tenuissima nakai, including phthalides, coumarins, terpenoids, and phenylpropanoids (islam et al., ; nam et al., ) . some of these compounds were found to have diverse biological activities, such as antioxidative (ka et al., ) , anticancer (park et al., ) , angiotensin converting enzyme (ace) inhibitory (liang et al., ) and antifungal effects . to confirm these reports, we conducted hplc analysis to identify the active compounds present in the total aqueous fraction. we observed major peaks (fig. a) , and fractions (fr- to fr- ) were successfully purified. of the fractions, fr- was identified as having significant antiviral properties, like those displayed by angelica tenuissima nakai; fr- was later confirmed to be ferulic acid (fig. a) . here, we newly defined the antiviral effects of ferulic acid via type i ifn stimulation. thus, the antiviral and immunomodulatory effects of an aqueous extract of angelica tenuissima nakai might be due to a cumulative effect of ferulic acid including with other fractions or other unknown active compounds present in the extract. the relationship between the mechanisms of the antiviral effects and active compounds, including ferulic acid, should be studied further. interferon-mediated antiviral activities of angelica tenuissima nakai in summary, we demonstrated that the aqueous extract of angelica tenuissima nakai can be a major alternative antiviral agent. the aqueous extract of angelica tenuissima nakai inhibit the diverse viral infection through the induction of type i ifn signaling and pro-inflammatory cytokines, leading to an antiviral state in epithelial and immune cells. the aqueous extract of angelica tenuissima nakai can reduce influenza-induced mortality by disrupting viral replication or preventing viral infection by creating an antiviral state in in vivo (lungs). given the antiviral activities, angelica tenuissima nakai extracts or its components, including ferulic acid may be used as a preventive or therapeutic agent to limit viral replication which have been caused a serious damage to human or livestocks. furthermore, angelica tenuissima nakai may also be used as a target for drug design to prevent the viral infection through the innate immune responses. prasanna weeratunga, md bashir uddin designed and executed all cell biological experiments; myun-soo kim, byeong-hoon lee, ji-eun yoon performed all virus infection experiments, analyzed the data; jin yeul ma, hongik kim analyzed the data. jong-soo lee designed the overall study and wrote the paper. the animal study was conducted under appropriate conditions with the approval of the institutional animal care and use committee of bioleaders corporation, daejeon, korea. protocol number: bsl-abls- - . none of the authors have any financial or personal relationships with other people or organizations that could inappropriately influence or bias this study. type i interferon at the interface of antiviral immunity and immune regulation: the curious case of hiv- animal models for influenza virus pathogenesis and transmission phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus phytochemical screening and antimicrobial activity of the plant extracts of mimosapudica l. against selected microbes treatment with a toll-like receptor inhibitory gpg oligonucleotide delays and attenuates lupus nephritis in nzb/w mice characterization of the anti-influenza activity of the chinese herbal plant paeonia lactiflora a validated lc method for simultaneous determination of phenolic, coumarin and phthalide compounds in the ethanolic extract of angelica tenuissima intranasal administration of 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susceptibility of cells to infection by newcatle disease virus important role of interferon regulatory factor (irf)- in the interferon response of mouse macrophages upon infection by newcastle disease virus mucosal immunoglobulins in vitro and in vivo antifungal activities of decursin and decursinol angelate isolated from angelica gigas against magnaporthe oryzae, the causal agent of rice blast an m e-based multiple antigenic peptide vaccine protects mice from lethal challenge with divergent h n influenza viruses the authors thank dr. j. u. jung of the university of southern california, usa for providing green fluorescence key: cord- - lyt gfq authors: griffiths, samantha j.; koegl, manfred; boutell, chris; zenner, helen l.; crump, colin m.; pica, francesca; gonzalez, orland; friedel, caroline c.; barry, gerald; martin, kim; craigon, marie h.; chen, rui; kaza, lakshmi n.; fossum, even; fazakerley, john k.; efstathiou, stacey; volpi, antonio; zimmer, ralf; ghazal, peter; haas, jürgen title: a systematic analysis of host factors reveals a med -interferon-λ regulatory axis against herpes simplex virus type replication date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: lyt gfq herpes simplex virus type (hsv- ) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. to comprehensively investigate the complex interaction between hsv- and its host we combined two genome-scale screens for host factors (hfs) involved in virus replication. a yeast two-hybrid screen for protein interactions and a rna interference (rnai) screen with a druggable genome small interfering rna (sirna) library confirmed existing and identified novel hfs which functionally influence hsv- infection. bioinformatic analyses found the hfs were enriched for several pathways and multi-protein complexes. of particular interest was the identification of med as a strongly anti-viral component of the largely pro-viral mediator complex, which links specific transcription factors to rna polymerase ii. the anti-viral effect of med on hsv- replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to hsv- , as a range of other viruses including vaccinia virus and semliki forest virus were unaffected by med depletion. we found med significantly upregulated expression of the type iii interferon family (ifn-λ) at the mrna and protein level by directly interacting with the transcription factor irf . the synergistic effect of med and irf on ifn-λ induction suggests this is the major transcription factor for ifn-λ expression. genotypic analysis of patients suffering recurrent orofacial hsv- outbreaks, previously shown to be deficient in ifn-λ secretion, found a significant correlation with a single nucleotide polymorphism in the ifn-λ (il b) promoter strongly linked to hepatitis c disease and treatment outcome. this paper describes a link between med and ifn-λ, provides evidence for the crucial role of ifn-λ in hsv- immune control, and highlights the power of integrative genome-scale approaches to identify hfs critical for disease progression and outcome. up to % of the global population is infected with the aherpesvirus herpes simplex virus type i (hsv- ). whilst hsv- is largely responsible for outbreaks of vesicular oral skin lesions (fever blisters, or cold sores), it can also cause a variety of more severe diseases including encephalitis, meningitis and keratitis [ , ] . furthermore, the frequency of association with genital lesions (previously associated mainly with hsv- infection) is increasing. as co-infection with hsv is a significant contributing factor to transmission of the human immunodeficiency virus (hiv), our understanding of hsv disease, and herpesviruses in general, has wide implications for global healthcare. like all herpesviruses, hsv- establishes lytic (epithelial cells) and asymptomatic latent infection (sensory neurons in trigeminal and sacral ganglia) which undergoes periodic reactivation [ ] . the equilibrium between these two infection states requires a fine balance between innate and adaptive immune responses, and viral immune evasion mechanisms [ ] . whilst aspects of the hsv- replication cycle have been intensively investigated, there remain gaps in our understanding of the complexity of virus:host interactions. for example, a proteomics study identified over changes in the cellular proteome within the first h of infection with hsv- [ ] , and a recent analysis of virionincorporated cellular proteins found that about % of these directly affected virus growth [ ] . to systematically identify host factors (hfs) required for viral replication, rnai screens have been performed with a range of different rna and dna viruses including hiv- [ , , ] , influenza a virus [ , , ] , hepatitis c virus [ ] , west nile virus [ ] , dengue virus [ ] , enterovirus [ ] and vaccinia virus [ , ] . the overlap between the results of these studies is generally very low [ ] , reflecting either differences in biology, or different experimental set-ups, cutoff and selection criteria. in addition, microenvironmental effects might also play a role for the differences of the results [ ] . whilst loss-of-function sirna screens provide functional information on specific genes, protein interaction studies can provide insight into the mechanism of action by identifying physical interaction partners between pathogen and host. genome-scale virus-host protein interaction screens using the yeast-two-hybrid system have been performed for hcv [ ] , influenza a virus [ ] , epstein barr virus (ebv) [ ] , vaccinia virus [ , ] , sars coronavirus [ ] and several non-human viruses [ ] . based on these genome-scale studies and individual interactions found by literature curation, several virus-host interaction databases have been created including the hiv- , human protein interaction database at ncbi [ ] , virhostnet [ ] , virusmint [ ] , pig [ ] and hpidb [ ] . although there is little overlap between individual cellular interactors of different viruses, targeting of a number of cellular processes such as cell cycle regulation, nuclear transport and immune response appears to be conserved [ ] . understanding the complex interplay between viral and host components is critical to the definition of herpesvirus infection and pathogenesis. as herpesviruses encode a large number of proteins, in contrast to small rna viruses such as hiv and influenza, many cellular processes may be directly affected by viral proteins, and whilst there exists a wealth of information on individual viral proteins, there remain large gaps in our understanding of the hsv- life cycle and its interaction with its host. here, we present data from the first integrative and systematic screening approach to characterise the role of cellular proteins in the hsv- life cycle. a genome-scale rnai knockdown screen to identify hfs functionally influencing hsv- replication was performed in parallel with a yeast two-hybrid (y h) protein interaction screen to simultaneously gain insight into potential mechanisms of action. combined analyses confirmed the importance of known cellular proteins involved in processes such as cell cycle, proteins transport and gene expression important for virus replication. furthermore, we identified a subunit of the mediator multi-protein complex, med , as a key regulator of ifn-l induction, which appears to be of crucial significance for the control of hsv- both in vitro and in vivo. these data demonstrate the power of a combined screening strategy to investigate pathogen:host interactions and identify novel host factors and cellular pathway targets for the development of essential clinical interventions. host factors (hfs) which positively or negatively regulate hsv- replication were identified by screening a druggable genome sirna library ( sirnas per gene) targeting , human genes against a hsv- reporter virus expressing the enhanced green fluorescent protein (egfp; hsv- strain c ) in the epithelial hela cell line, due to their ease of transfection and susceptibility to hsv- infection [ ] . to generate a robust and reliable dataset the screen was carried out three times in triplicate, with one replicate used in a cell viability assay to determine any cytotoxic effects of gene depletion and duplicates infected for the virus infection assay. the sirna library was reversetransfected into hela cells before infecting with hsv- and monitoring virus growth kinetics as a measure of gfp-fluorescence (figure b). by following virus growth over multiple rounds of replication, host proteins involved in all stages of the virus life cycle can be identified. replication slopes during linear growth were normalized to controls (mock-transfected cells, and cells transfected with a sirna unable to be processed by the rna silencing complex, rscf) and the mean of six replicates was calculated. sirnas found to be cytotoxic ( in total) were excluded from further analyses, and a hitlist of containing the top . % inhibitory and the top . % enhancing hfs was generated ( table s in text s ). the identified hsv- hfs were compared to datasets from published sirna depletion screens aimed at identifying cellular factors affecting hiv- [ , , ] , west nile virus (wnv) [ ] , hepatitis c virus (hcv) [ ] , dengue virus [ ] and influenza a virus [ , , ] . of our hfs, cellular proteins ( . %) overlapped with these other virus screens (influenza a, ; hiv- , ; hcv, ; wnv, ; dengue virus, ) ( figure c ; table s in text s ). a genome-scale yeast two-hybrid protein interaction screen identifies novel viral protein interaction partners hsv- is currently known to encode at least proteins, expressed sequentially under strict temporal regulation during herpes simplex virus type (hsv- ) infects the vast majority of the global population. whilst most people experience the relatively mild symptoms of cold sores, some individuals suffer more serious diseases like viral meningitis and encephalitis. hsv- is also becoming more common as a cause of genital herpes, traditionally associated with hsv- infection. co-infection with hsv- is a major contributor to hiv transmission, so a better understanding of hsv- /hsv- disease has wide implications for global healthcare. after initial infection, all herpesviruses have the ability to remain dormant, and can awaken to cause a symptomatic infection at any stage. whether the virus remains dormant or active is the result of a finely tuned balance between our immune system and evasion techniques developed by the virus. in this study we have found a new method by which the replication of the virus is counteracted. the cellular protein med was found to actively induce an innate anti-viral immune response in the form of the type iii interferons (ifnlambda), by binding irf , a key regulator of interferons, and modulating its activity. interferon lambda is well known to be important in the control of hepatitis c infection, and a genetic mutation correlating to an increase in interferon lambda levels is strongly linked to clearance of infection. here we find the same association between this genetic mutation and the clinical severity of recurrent cases of hsv- infection (coldsores). these data identify a med -interferon lambda regulatory axis of innate immunity, show that interferon lambda plays a significant role in hsv- infection, and contribute to the expanding evidence for interferon lambda in disease control. infection. to gain further mechanistic insight into host factors involved in hsv- infection, in parallel to the sirna depletion screen we carried out a yeast two-hybrid protein interaction screen to identify cellular interaction partners of viral proteins. we generated a collection of partial and full-length hsv- cdna constructs and tested them for interactions with proteins encoded by a library of , human cdna clones [ ] . hsv- human protein interactions were detected once (low confidence), and more than once (high-confidence)( table s in text s ) . using these high-confidence interactions, the previously reported hsv- interactome [ ] was connected into a human interactome ( , published protein interactions) to generate a combined pathogen-host interactome ( figure s a ). both degree centrality (which indicates the number of interactions a protein has, where high values represent highly interactive 'hubs') and betweenness centrality (which indicates the number of shortest paths between ( replicates) or the capacity to influence replication of the hsv- gfp reporter virus c ( replicates) from to h post-infection. virus replication slopes during the linear phase were calculated and normalized to mock-transfected cells. replication slopes were then compared to replication upon knockdown of essential (icp , vp ) or non-essential (vp / ) viral genes, a cellular receptor for hsv- (hvem) or control risc-free sirna (rscf). (c) overlap between the hsv- hfs identified in this study with those published in hiv- [ , , ] , hepatitis c virus (hcv) [ ] and influenza a virus [ , , ] . doi: . /journal.ppat. .g any pair of proteins passing through the protein considered) were significantly increased for hsv- interactors, particularly in the high-confidence network (figure s b-e). these data suggest hsv- proteins preferentially target highly connected central human proteins in the cellular interaction network, similar to other viruses [ ] . analysis of this interactome for hfs identified by rnai found they were enriched in the fraction of cellular proteins that directly interact with viral proteins or that interact via one intermediate, in comparison to proteins that only interact via or more intermediates (p = . , fisher's exact test)( figure s f) . a direct comparison of hsv- protein interaction partners and the sirna screen hfs found genes in common. of those, ten ( . %) were identified as a hit in both screens ( table s in text s ), suggesting that these technologies identify complimentary yet not necessarily overlapping hfs. an extended literature and database search identified cellular proteins that interact with or are involved in infection with human herpesviruses. the overlap between the high-confidence y h cellular interactors ( ) and hfs ( ) with this set was statistically significant (p = . ) (figure a; figure s h ; table s in text s ). from this combined analysis, a subset of hfs was chosen for further validation. protein interactions were tested in a mammalian cell system by lumier pull-down assay [ ] . of the interactions tested, ( . %) were confirmed, with strongly positive (z-score . ) and weakly positive (zscore - ) ( figure s g ). sirna deconvolution ( sirnas per gene tested individually) was used to further validate hfs (figure b ; figure s ). the replication phenotype could be confirmed ($ or more sirnas gave the same or better replication slope than observed in the primary screen) in a high proportion ( . %) of candidates, highlighting the reliability of the primary screen dataset. quantitative rt-pcr analysis of mrna expression levels found a minimum depletion of % (mean %) in a subset of genes (data not shown; table s in text s ) confirming the observed effects on hsv- replication are genuine and not due to 'off-target' effects or insufficient gene knockdown. to further investigate the virus-specificity of our identified hfs, we tested this subset for their effect on the replication of an additional a-herpesvirus (varicella-zoster virus, vzv), the bherpesvirus cytomegalovirus (cmv), and a completely unrelated rna virus, semliki forest virus (sfv). none of the three proteins which enhanced hsv- replication upon knockdown had an effect on either vzv or cmv, and one (nr c ) was even inhibitory for sfv ( figure c ). of the sirnas which inhibited hsv- , ( . %) were also inhibitory for vzv, for cmv ( . %) and ( . %) for sfv replication ( table s in text s ) . some functional groups (transcriptional regulators) were required by most viruses, but there were notable differences between other proteins. for example ifitm- , previously identified as an inhibitor of influenza a, dengue virus and wnv [ ] , inhibited vzv yet had a positive effect on hsv- replication. these data suggest that whilst there are some hfs which are broad in their effects on virus replication, a large proportion are species-specific. table s in text s ). pathways included those involved in gene expression, transcription, splicing and translational regulation (rnai screen), and protein transport, cell cycle, and transcriptional repressor activity (y h screen). a combined analysis of hfs from both screens found dominant functional categories centred on the regulation of transcription (rna polymerase ii-associated genes, splicing factors, transcription activation and the mediator complex) ( figure s c, d) . the physiological relevance of some hfs and pathways was confirmed by further biological validation. protein transport pathways (in the form of dynein microtubule networks) are exploited by hsv- early after infection to shuttle viral capsids to the nucleus. these screens confirmed known interactions between dynein subunits and viral proteins, and identified additional previously unknown interactions (text s and figure s a ). several dynein chain subunits were found to be essential for virus replication, whilst the moderate effect of depletion of other subunits demonstrated a level of functional redundancy in hsv- capsid transport ( figure s b -e) [ , ] . intrinsic anti-viral host defense mechanisms, in the context of cellular e ubiquitin ligases, were also investigated. the immediate-early viral protein icp , an e ubiquitin ligase, is crucial for blocking anti-viral defense mechanisms by degrading promyeloctic leukemia (pml) nuclear bodies (nd domains) in the presence of cellular e -ubiquitin-conjugating enzymes (e s). our sirna screen found multiple e s were required for this, and suggests that hsv- icp is promiscuous in its exploitation of e s to mediate pml degradation and ensure successful infection (text s and figure s ). combined bioinformatic analyses of protein interaction and sirna depletion screens found a significant functional enrichment for proteins involved in transcription, and identified multi-protein complexes enriched for pro-viral hfs which strongly inhibited hsv- upon depletion, including the rna-polymerase ii, eif and mediator complexes ( figure a) . the mediator complex links the cellular transcription machinery (rna polymerase ii) to specific transcription factors, and the identification of many mediator subunits as hfs in other viral sirna depletion screens highlights its significant role in viral genome transcription [ , , , ] (table s in text s ) . further, several mediator subunits (med , , and ) are known to interact with the hsv- transactivator vp (ul ) and other herpesviral proteins [ ] ( figure s a) . consistently, the mediator complex was found to be strongly required for hsv- replication, with depletion of the majority of subunits (med , , , , , , , , , , and ) leading to a severe reduction in virus replication in the primary screen ( figure s b ) or in confirmatory deconvolution assays ( figure b) . however, depletion of the med subunit was striking in that it led to a significant enhancement of virus growth ( figure b ). flow cytometry quantification found that removal of med not only increased the total number of infected cells (combination of gfp lo and gfp hi cells; . % in comparison to . % in mock-transfected cells) but also the copy number of virus genomes (gfp hi cells; . % in comparison to . % in mocktransfected cells) (figure c med could exert anti-viral effects either by having an inhibitory effect on viral transactivators or by interacting with and having a positive effect on an existing anti-viral factor. we first tested whether med directly affects viral gene expression using luciferase reporters with hsv- promoters, however observed no inhibitory effect (data not shown). since the mediator complex and med in particular is known to be involved in jak/statmediated interferon signaling [ ] , we used the lung epithelial cell line a and its stat- -deficient derivative a -v [ ] to determine if med influences hsv- replication by modulating innate immunity. in the parental a cells the phenotype of hsv- replication was the same as that observed in hela cells, where depletion of med enhanced replication and overexpression inhibited virus growth. however, in the stat -deficient a -v cells hsv- replication was unaffected by both depletion and over-expression of med (fig. a) , indicating that med requires an intact jak/stat signalling pathway to exert its anti-viral effects. to determine which interferon may be responsible for the antiviral effects of med , a cells were depleted for med and infected with hsv- following pre-stimulation with type i (ifn-a or ifn-b), type ii (ifn-c) or type iii (the distinct ifn-l or the almost identical ifn-l and -l , termed ifn-l / ) interferons. whilst treatment with ifn-a, -b and -c significantly decreased hsv- replication levels, the observed , -fold enhancement of hsv- replication following med depletion was still seen. however, pre-treatment with the both ifn-l and ifn-l / blocked the enhancing effect of med depletion (figure b) . investigation into the effect of med on interferon induction by qrt-pcr found that whilst med over-expression induced ifnb (, -fold increase), induction of ifn-l and l / was considerably and statistically significantly higher (, -fold induction; p = . and . , respectively) ( figure c ). this induction was specific, as levels of other cytokines and interferon-regulatory factors (irfs) were unaffected by med overexpression ( figure s a ). secretion of ifn-l / protein was also increased in all cell lines tested, but most significantly to , -fold in a cells (figure d) , which is consistent with a recent report showing that type iii interferons are the dominant type of ifns expressed by primary airway epithelial cells [ ] . furthermore, qpcr analysis found depletion of med inhibited the induction of ifn-l expression following hsv- infection of a cells in comparison to cells transfected with the rscf sirna control ( figure e ). together, these data suggest that ifn-l is responsible for the observed inhibitory effect of med on hsv- replication. as ifn-l expression is induced following activation of pathogen recognition receptors (prrs) by virus infection [ , , , ] , we tested whether med induced ifn-l by directly interacting with an interferon-responsive transcription factor (irf). y h and confirmatory co-immunoprecipitation experiments in mammalian cells with a panel of irfs found that med interacted with irf and irf (figure a ; figure s b ). we also observed a weak interaction with irf , which may explain the previously observed effect of med on jak/stat signalling [ ] . to determine if this interaction had a functional effect, we looked at whether med influenced irf-mediated induction of ifn-l. in a luciferase reporter assay, neither irf nor irf led to a significant induction of the ifn-l promoter, either alone or in conjunction with med (data not shown). irf induced expression from the ifn-b and ifn-l promoters to similar levels (, -fold and -fold higher than background, respectively), whilst the isre, induced by irf and also present in the irf promoter, was induced , -fold ( figure b ). whilst co-expression of med with irf had no further effect on ifn-b expression, a synergistic induction of the ifn-l promoter and, to a lesser extent, the isre, was observed (ifn-l doubled to , -fold, p = . ) ( figure b ). interestingly, a med mutant unable to induce immediate early gene expression via jun/fos (r q, or r q in med transcript variant used here) synergistically induced isre expression with irf , yet was unable to further enhance irf mediated induction of ifn-l (data not shown). a similar synergistic effect of med and irf was seen at the protein level, where co-expression increased supernatant levels of ifn-l more than -fold those seen with med or irf alone ( figure s c , d). successful disease and treatment outcome in hepatitis c virus infection (demonstration of a sustained virologic response) is strongly associated with a single nucleotide polymorphism (snp) in the ifn-l promoter (rs ; cc genotype over ct or tt) and higher plasma levels of ifn-l [ ] , [ ] . furthermore, ifn-l expression is impaired in a cohort of ethnically italian individuals suffering recurrent hsv- -related herpes labialis reactivation [ ] . to determine if the clinical severity of hsv- disease is due to the observed deficiency in ifn-l expression, we screened a subset of the recurrent herpes labialis (hl) cohort and additional subjects for the ifn-l promoter polymorphism. genotypic analysis found the presence of a t (ct or tt genotype) had a dose-dependent association with clinical severity, with the homozygous tt genotype being more prevalent as disease severity increases ( figure ). in spite of the relatively small sample numbers in some clinical categories (table ) , the association of a ct or tt genotype with the most severe recurrence of herpes labialis (h+) was statistically significant (p = . ; fishers's exact t-test). as the cc genotype is directly associated with increased ifn-l levels [ ] , these data highlight a previously unknown association between the frequency/severity of recurrence of herpes labialis, the ct/tt genotype and subsequent reduction in secretion of ifn-l . it is of importance to investigate this genotype association with a larger cohort of hl patients, as well as those suffering with other hsv- -related disease, in order to determine the role of ifn-l in the full spectrum of hsv- pathogenesis. was selected for validation with deconvoluted sirnas to confirm the phenotype observed in the primary screen. the effect of the four individual sirnas ( - ) and a reconstituted smartpool (sp) were tested by reverse-transfecting into hela cells before infecting after h with hsv- -egfp (c ) and monitoring replication. replication slopes were calculated and normalized as described, and compared to the primary screen slope ( u). a heat map of replication slopes was generated where red represents inhibition (replication slope , . ) and green represents enhancement (slope . ). the phenotype was considered validated if $ sirnas produced the same or better phenotype as the primary screen. (c) virus specificity of hsv- hfs. the effect of hf sirna smartpools on the replication of vzv (a-herpesvirus), hcmv (b-herpesvirus) or semliki forest virus (sfv; rna virus) was determined and compared to hsv- . normalized replication stdev of the controls was considered inhibiting/enhancing. doi: . /journal.ppat. .g taken together, these data identify med as a novel anti-viral factor which acts as a key regulator of ifn-l expression by interacting with and enhancing the activity of irf , a major transcription factor involved in innate immunity. our observation of a link between the clinical severity of hsv- disease and, a ct/ tt genotype at a snp known to regulate ifn-l secretion demonstrates the significance of ifn-l in the control of hsv- replication in vivo. whilst this study provides no direct link between the ifn-l promoter polymorphism and med , these associations of ifn-l with hsv- disease, combined with our observations that med is required for the induction of ifn-l following hsv- infection, identifies for the first time a link between med and ifn-l, provides a clinical context for med regulation of ifn-l expression and underscores the potential biological significance of these data. the use of hsv- in a combined genome-scale screening approach has led to the identification of a regulatory axis in antiviral innate immunity, and this important finding not only highlights the power of such combined genome-scale screening approaches to identify novel host candidates for anti-herpesvirus drug discovery, but provides an invaluable dataset to the herpesvirus and scientific community at large. by nature of their scale, high-throughput screening technologies have limitations. rnai technology is limited by technical issues such as off-target effects, where an alternative gene to the intended target is degraded, and insufficient gene knockdown. similarly, y h protein interaction screens can generate both false-positive interactions, due to 'sticky' proteins and auto-activation of the reporter gene used, and false-negative interactions. whereas the number of false positives can be considerably reduced by stringent screening and selection criteria, the low sensitivity of the y h assay, which detects - % of known interactions, is inherent to the system and can only be marginally improved. this poor sensitivity is caused by factors such as structural restraints of the y h bait and prey fusion proteins, a lack of or existence of distinct protein modification in yeast cells, and cellular localization signals in bait and prey proteins preventing nuclear import [ ] . however, as all other high-throughput methods for measuring binary protein interactions possess a similarly low sensitivity, but are considerably more laborious and expensive, the y h system is still the most commonly used technology [ ] . in this study we have exploited a combined genome-wide screening approach to investigate hsv- replication and interaction with its host. this identified functional hfs modulating hsv- replication, and cellular interaction partners. in validation experiments, . % of the interactions were confirmed by co-immunoprecipitation assays in mammalian cells, and of the functional hfs identified in the sirna screen, the phenotype of . % was confirmed in deconvoluted sirna experiments. this, combined with qpcr data demonstrating a minimum gene depletion of %, suggests that the functional phenotypes on virus replication are genuine, and not due to 'off-target' effects. the confirmation of such a high proportion of selected validation candidates, in spite of the potential technical drawbacks, highlights the reliability of our primary screen datasets, and thus provides an invaluable resource for the herpesvirology research community. one interesting outcome of this study was the surprisingly low overlap between hits identified using these different technologies. of the genes in common between the sirna and cdna libraries, only ( . %) were classified as a hit by both methods. this, however, is not unexpected, as even the overlap between studies using the same technology has been reported to be low. for example, the overlap between the three previously published hiv screens was only % [ ] . furthermore, the degree of functional redundancy within the sirna library, and cellular pathways in general, the potential situation-specificity of virus-host interactions, and the possibility of indirect interactions between viral and host proteins, suggest that these methodologies detecting functional outcomes or physical interactions are linked, but complementary rather than confirmatory. the identified hfs were enriched for a range of cellular processes, such as transcription, gene expression, protein transport and cell cycle ( figure s and s ) , and involved at different stages of viral infection. we investigated hfs involved in capsid transport and ubiquitination of antiviral intrinsic host defence factors in more detail. incoming hsv- capsids are transported to nuclear pores via the microtubule-organizing centre (mtoc), mediated by capsid proteins vp (ul ) and ul binding to the dynein light chains dynlt (tctex ) and dynlt (rp ) [ , ] . our y h screen confirmed the known interaction between the capsid protein vp and the dynein light chain dynlt (text s and figure s ). combined with the sirna screen data, which found depletion of multiple light chain subunits had moderate anti-viral effects on hsv- , these data confirm propositions of redundancy in the capsid transport process which ensures successful infection in the event of viral mutations [ , ] , and provide further evidence that hsv- has evolved to be highly promiscuous in its exploitation of cellular pathways to its advantage. to overcome the intrinsic host defence, hsv- induces a proteasome-dependent degradation of anti-viral promyelocytic leukemia (pml) nuclear bodies (nd domains) by the ringfinger ubiquitin ligase icp expressed during early infection [ ] . in vitro, icp is a biochemically active e ubiquitin ligase in the presence of e ubiquitin conjugating enzymes (e s) [ube d (ubch a) and ube e (ubch )] [ ] , but which e s are used during infection has remained unclear. we identified cellular e s that are able to influence hsv- replication (text s and figure s ). depletion of ube d - , ube e - and ube n significantly increased the number of pml-positive cells postinfection, in an icp -dependent manner, indicating that icp can use multiple e s to degrade pml [ , ] . one of the multi-protein complexes affecting hsv- replication was the mediator complex, a large (. subunits) complex which links specific transcription factors to the rna polymerase ii transcription machinery [ ] . as the requirement of mediator subunits in the replication of herpes and other viruses is already well-known [ , , , ] , it was striking that depletion of the med subunit exerted the opposite phenotype and led to a strong increase in virus growth. the mediator is composed of four distinct modules termed the head, middle, tail and kinase domains, which provide the mediator with some degree of active control over transcription [ ] . as individual subunits of this large complex interact with and exert functional effects via specific transcription factors, it is not unexpected that the observed antiviral effects were specific to med [ ] . within the mediator, med forms a tight sub-complex with med and med [ , ] . the increase in virus replication observed upon depletion of med may be caused by the destabilisation of the structure of this sub-complex ( figure s b) . investigations into the mechanism of action revealed med inhibits hsv- replication by preferentially inducing a type iii interferon response (ifn-l) at the mrna and protein level. this induction was mediated via a direct interaction with the transcription factor irf , which resulted in a synergistic increase in ifn-l expression. med was unable, however, to further enhance irf -induced levels of ifn-b, suggesting an additional level of complexity to the regulation of interferon signalling. interestingly, the inhibitory effect of med was specific to hsv- , with replication of a range of other viruses including vaccinia virus and semliki forest virus being unaffected by med depletion. as vaccinia virus is resistant to ifn-l anti-viral activity [ ] , this observation further highlights the importance of ifn-l, as opposed to ifn-b, in the anti-viral effect of med . the r q mutation in med (r q in med transcript variant , used here) was unable to enhance irf -induced ifn-l expression. this mutation causes hereditary dementia [ ] , and the failure to induce ifn-l and thereby control hsv- in the brain may be a potential cofactor for the development of dementia, similar to alzheimer's disease [ ] . there is mounting evidence for a role of the ifn-l family in the regulation of virus pathogenesis [ ] , particularly in the case of hepatitis c infection where a polymorphism in the promoter region of ifn-l (il- b; polymorphism rs ), which correlates with plasma levels of ifn-l [ ] , is associated with disease and treatment outcome [ ] . individuals with recurrent hsv- reactivation have been shown to be deficient in ifn-l expression [ ] , and here we found the similar association between the ifn-l promoter polymorphism and ethnically italian patients suffering recurrent and severe reactivations of hsv- -related oral herpes outbreaks, albeit with a small sample group (n = ). furthermore, sporadic mutations and genetic polymorphisms in innate immune receptor and signalling molecules that lead to the induction of type i and iii ifns have also been shown to be associated with herpes encephalitis [ ] , as well as oral and genital herpes [ , ] . hsv infection controlled by a complex, interconnected and highly regulated network of cytokines expressed by innate immune cells. type i ifns mainly produced by hsv-infected keratinocytes [ ] and pdcs [ ] inhibit the spread from neurons to epithelial cells and between epithelial cells [ ] , similar to ifn-c. type iii ifns are also able to directly inhibit hsv- infection in primary neurons, astrocytes, macrophages and dendritic cells [ , ] . ifn-c levels produced by peripheral blood cd + t-cells correlate with the frequency of hsv- reactivation [ ] . ifn-l is able to induce expression of both itself and the type i ifns, and a similar effect has also been observed for type i ifns which induce both type i and iii ifns [ , ] . type iii ifns are mainly expressed by myeloid dendritic cells (mdc) and monocyte-derived macrophages [ ] , and signal through the heterodimeric il rb/il ra receptor complex whose expression is largely restricted to cells of epithelial origin and plasmacytoid dendritic cells (pdc), in contrast to the broadly expressed type i ifn receptor (ifn-ar / ) [ , ] . since primary hsv- infection and reactivation affects skin and mucosa in the majority of cases, ifn-l may play a much greater role in the control of hsv- pathogenesis, likely in a complex network of coregulated type i and ii ifns, than previously thought. we hypothesize that hsv-infected dcs at the site of the lesion (such as skin langerhans dcs whose role in ifn-l production is currently unknown, or intruding myeloid dcs) in individuals with the rs t/t or c/t haplotype express reduced levels of type iii ifns, and, in consequence, of type i ifns, which leads to a reduced inhibition of local hsv- replication and the occurrence of fresh skin lesions. however, the relative contribution of ifn-l and l / to the interferonmediated control of hsv- replication in vivo, and indeed the role of med in this, remains to be seen. in summary, this study provides a comprehensive and robust analysis of hfs that influence hsv- replication in vitro, which will benefit many future studies on hsv- . the identification of med as a crucial cellular component for ifn-l expression, and evidence for the significant role of type iii ifn in the innate immune control of hsv- in vitro and in vivo, demonstrates the power of combined, genome-scale studies to identify physiologically important hfs for virus pathogenesis. future studies will clarify the role of genetic variations in both med and ifn-l in hsv- -related diseases, such as meningitis, keratitis and orolabial/genital reactivations. sirna smartpools ( sirnas per gene) at . mm were dispensed in ml volumes using a rapidplate liquid handler (qiagen) into triplicate black -well plates (corning), sealed with adhesive seals (thermofisher) and plastic lids. plates were stored at uc until needed (minimum h, maximum h). on the day of transfection, assay plates were thawed at room temperature and ml transfection reagent (dharmafect , dharmacon), diluted in hank's buffered saline solution (hbss, thermofisher) to give a final concentration of . %, was added using a multidrop (thermofisher). plates were incubated for min at room temperature to allow formation of transfection complexes. during complex formation, low-passage (p - ) hela cells (ecacc) from , % confluent flasks were washed in pbs and trypsinised in trypsin-edta (lonza) before diluting in phenol red-free, antibiotic-free transfection medium (dmem/f- : / % fcs with mm hepes and l-glu; gibco). cells were counted and cells in ml were added to each well using the multidrop . plates were incubated for h at uc in a humidified incubator with % co . to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillin-streptomycin; lonza) or virus (hsv- -egfp strain c , diluted to moi . in infection media) [ ] was added using the multidrop . plates were incubated at uc for h before ml infection media was added and plates returned to the incubator. replication was monitored as a function of egfp fluorescence from h to h post-infection using the polarstar optima plate reader (bmg labtech). virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. cells were transfected as described above, and the cytotoxicity of sirnas was determined using the celltiter blue (ctb, promega) reagent, which gives a fluorescent or absorbance signal relative to the number of live cells. briefly, ml ctb was added per well using the multidrop . plates were incubated at uc in a humidified incubator with % co for h before measuring fluorescence (polarstar optima plate reader). readings were normalized to viability of mock-transfected cells, per plate, and mean cell viability over three replicates was calculated. distribution analysis of cell viability values identified median viability as %, and values , % were considered cytotoxic. the hsv- clone collection was cloned by recombinatorial (gateway tm , invitrogen) and conventional cloning into the bait vector pgbkt , and screened against a library pooled from , mgc clones [ ] in the pgadt prey vector using a semiautomated y h assay [ ] . interacting prey cdnas were identified by sequential blasting of refseq, ensembl and unigene databases. blast hits with identical parameters (score, expectation value, length of alignment) were considered indistinguishable and counted separately. a high-confidence dataset was generated from interaction pairs isolated at least twice, or where the bait interacted with two highly related, non-promiscuous preys. interactions between hsv- and human proteins were connected to a network of human protein-protein interactions (a total of , ) taken from the databases hprd [ ] (release ), biogrid [ ] , dip [ ] , mint [ ] and intact (downloaded may th ). a high-confidence interaction set ( , interactions) was compiled from interactions identified in at least two studies. betweenness centrality (g(v) of a protein v was calculated as g(v) = gs {v{t (s st (v)/s st ), where s st is the total number of shortest paths from protein s to protein t, and s st (v) is the number of those shortest paths that contain v. betweenness centrality was normalized by dividing by the total number of protein pairs in the network. enrichment for functional annotations from gene ontology (go) [ ] , kegg [ , ] , reac-tome [ , ] , and biocarta was performed using david [ ] . data on known human protein complexes was retrieved from the corum database, and complexes with subunits showing consistently stronger effects (inhibiting or enhancing) than expected by chance were detected using wilcoxon's rank-sum test. genes included in the rnai screen were ranked by their distance from the median knockdown, with the most inhibiting and enhancing genes being ranked highest. fdr was used for multiple testing correction. the hsv- replication phenotype observed in the primary screen was validated for a subset of candidates by deconvoluting the assay smartpools. the four individual sirnas targeting different regions of each gene, as well as a reconstituted smartpool, were diluted to . mm in sirna buffer and dispensed to black well plates. transfection and infection was carried out as described above. replication slopes were calculated and normalized as described, and a phenotype was considered validated if two or more of the four sirnas resulted in the same, or better, phenotype. for inter-viral comparison, sirnas were considered inhibitory or enhancing if normalized replication was stdev of the controls a) hsv- replication assays. selected sirna smartpools were diluted to . mm in sirna buffer and dispensed in black -well plates (corning). to this ml dharmafect , diluted in hbss to a final concentration of . %, was added using the multidrop . following a min incubation to enable complex formation, hela cells in ml transfection media were seeded on to the complexes bringing the final volume of transfection to ml in each well. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillinstreptomycin; lonza) or virus (strain c , diluted to moi . in infection media) was added using the multidrop . plates were incubated at uc for h before virus was removed by plate inversion and ml infection media was added. plates were returned to the incubator before replication was monitored as a function of egfp fluorescence from h to h post-infection. virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. b) vzv replication assays. selected sirna smartpools were diluted to . mm in sirna buffer and dispensed in black -well plates (corning). to this, ml dharmafect diluted in hbss to a final concentration of . % was added using the multidrop . following a min incubation to enable complex formation, . mewo cells (atcc, htb- tm ) in ml media (emem/ % fcs/ % non-essential amino acids) were seeded on to the complexes bringing the final volume of transfection to ml in each well. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion and , colony forming units of vzv-egfp-infected mewo cells (vaccine strain oka) [ ] diluted in mewo growth media were seeded on to the complexes using the multidrop . virus growth was measured in h intervals as a function of egfp fluorescence from to h post-infection. virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. c) hcmv replication assays. selected sirna smartpools were diluted to . mm in sirna buffer and dispensed in ml volumes in black -well plates (corning). to this, ml dharmafect, diluted in hbss to a final concentration of . %, was added using the multidrop . following a min incubation to enable complex formation, mrc- (atcc, ccl- tm ) in ml growth medium (phenol red-free dmem/ %fbs/l-glutamine/ % non-essential amino acids were seeded on to the complexes bringing the final volume of transfection to ml in each well. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillin-streptomycin) or virus hcmv-gfp (strain ad ) [ ] , diluted to moi . in infection media, was added using the multidrop . plates were incubated at uc for h before ml infection media was added and plates returned to the incubator prior to monitoring virus replication. replication was monitored as a function of egfp fluorescence from h to h post-infection using the polarstar optima plate reader (bmg labtech). virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. d) sfv replication assays. selected sirna smartpools were diluted to . mm in sirna buffer and dispensed in ml volumes in black -well plates (corning). to this, ml dharmafect diluted in hbss to a final concentration of . % was manually added. following a min incubation to enable complex formation, hela cells in ml transfection media (dmem/ % fcs/l-glu) were seeded on to the complexes bringing the final volume of transfection to ml in each well. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillin-streptomycin; lonza) or virus (sfv ( h)-rluc [ ] diluted in phosphate buffered saline (pbs)/ . % bovine serum albumin to an moi . ) was manually added. plates were incubated at uc for h before media (as for transfection media) was added manually to increase the volume to ml per well. plates were incubated at uc for h before cells were lysed using a passive lysis buffer and renilla luciferase levels measured with a microplate reader (promega) using a dual luciferase reporter assay kit (promega). luciferase activity, which is representative of virus genome replication, was normalized to mock-transfected cells and mean luciferase activity from six replicates used for subsequent data analyses. e) vaccinia virus replication assays. hela cells were transfected as described in primary sirna screen. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillin-streptomycin) or ml media containing vaccinia virus strain wr with egfp-tagged a protein [ ] , diluted to moi . , was added using the multidrop . plates were incubated at uc for h before ml of media was added to each well, the plates inverted to remove the media and virus, and a final volume of ml of media added to the plates before they were returned to the incubator. replication was calculated as a function of egfp fluorescence at h post-infection using the polarstar opti-ma plate reader (bmg labtech). virus replication was normalized to mock transfected wells on individual assay plates, and the mean replication from eight replicates used for subsequent data analyses. hela cells were transfected with selected smartpool sirnas in -well plates, in triplicate, as described. after h transfection, medium was removed, cells rinsed in pbs and lysed in ml trizol (invitrogen). triplicate wells were combined, and rna extracted by standard phenol:chloroform extraction methods. mrna levels were determined by taqman qpcr, using the one-step rt-qpcr kit (thermofisher), with gene-specific primers ( table s in text s ), and probes from the universal probe library (roche). expression levels normalized to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase (hprt) and calibrated to mock-transfected cells. qpcr was carried out in duplicate for each sample, and the mean of normalized expression levels calculated. proteins were transiently expressed in hek cells as hybrid proteins with the staphylococcus aureus protein a tag or renilla reniformis luciferase fused to their amino termini. ng of each expression construct were transfected into hek cells using . ml of lipofectamine (invitrogen) in -well plates. after h, medium was removed and cells were lysed on ice in ml of ice-cold lysis buffer ( mm tris ph . , mm nacl, % tritonx- , mm edta, mm dtt, protease inhibitor cocktail (roche), phosphatase inhibitor cocktail (roche), benzonase (novagen) units per ml final concentration) containing sheep-anti-rabbit iggcoated magnetic beads (invitrogen, dynabeads m , mg/ml final concentration). lysates were incubated on ice for minutes. ml of wash buffer (pbs, mm dtt) were added per well, and % of the diluted lysate was removed to determine the luciferase activity present in each sample before washing. the remaining sample was washed times in wash buffer in a tecan hydroflex plate washer. luciferase activity was measured in the lysate as well as in washed beads. negative controls were wells transfected with the plasmid expressing the luciferase fusion protein and a vector expressing two copies of protein a. for each sample, four values were measured: the luciferase present in % of the sample before washing (''input''), the luciferase activity present on the beads after washing (''bound''), and the same values for the negative controls (''input nc'', and ''bound nc''). normalized interaction signals were calculated as follows: log(bound)/log(input) -log(bound nc)/ log(input nc). normalized interaction signals were z-transformed by subtracting the mean and dividing by the standard deviation. the mean and standard deviation were calculated from large datasets of protein pairs which were not expected to interact, i.e. from negative reference sets. selected sirnas smartpools were diluted to nm in hbss and ml was incubated with ml dharmafect diluted in hbss to a final concentration of . %. after min incubation, hela cells in ml transfection medium were added, mixed with the transfection complexes and transferred to -well glass bottomed chamber slides (becton dickinson). plates were incubated for h at uc in a humidified incubator with % co before infection by removing medium and adding ml hsv- -egfp at a moi of . after incubation for h at uc, virus was removed and replaced with ml growth medium. images were acquired h post-infection. select sirnas smartpools were diluted to nm in hbss and ml was incubated with ml dharmafect diluted in hbss to a final concentration of . % in individual wells of a well plate. after min incubation, hela cells in ml transfection medium were added. plates were incubated for h at uc in a humidified incubator with % co before infection by removing medium and adding ml hsv- -egfp at a moi of . after incubation for h at uc, virus was removed and replaced with ml growth medium. after h, medium was removed, cells rinsed in pbs and dislodged by trypsinisation. cells were washed in pbs and pelleted by centrifugation for min at g. supernatant was removed and cells fixed in % paraformaldehyde before analysing for egfp expression by flow cytometry (facs diva, bd biosciences) using the cellquest software package. for transient over-expression, . hek cells were seeded in black -well plates. the following day, cells were transfected with ng pcr -med using lipofectamine tm ltx (invitrogen) and incubated for h before infection with the recombinant hsv- reporter viruses c and vp -yfp at moi . . replication growth curves were monitored, and endpoint replication (as determined by fluorescence) was normalized to untransfected cells. for stable expression, hela cells were transduced with plenti-med , generated using the virapower tm lentiviral expression system (invitrogen), as per manufacturers' instructions. stable cells were infected, and replication monitored, as above. for confirmation of overexpression, rna was extracted (qiagen rna-easy kit) and expression quantified by one-step rt-qpcr (thermofisher), with gene-specific primers (table s in text s ), and probes from the universal probe library (roche). expression levels were normalized to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase (hprt) and calibrated to mock-transfected cells. qpcr was carried out in duplicate for each sample, and normalized expression levels averaged. the vp -yfp reporter virus was generated from a kos bac kindly provided by david leib using the red-mediated recombination system, where the first amino acids of vp were replaced with yfp [ , , ] . the effect of med over-expression and depletion was investigated in interferon-deficient cells. the human alveolar epithelial cell line a , and a -v, a stat -deficient derivative cell line stably expressing the v protein from simian virus [ ] , were seeded at cells per well in a -well plate, and transfected with med sirna or pcr -med and infected as described for hela cells. qrt-pcr analysis of interferon and cytokine induction a cells were transfected with ng pcr or med overexpression plasmids, or rscf or med smartpool sirna ( nm) in duplicates, in -well plates as described. rna was harvested h post-transfection, and mrna expression levels quantified by qrt-pcr, as described. induction of ifn-l by med was determined in a range of cell types by seeding cells in -well plates to be , % confluent the next day. cells were transfected in duplicates with ng pcr or pcr -med using lipofectamine ltx with plus reagent (invitrogen), in antibiotic-free medium. ifn-l levels quantified - h post-transfection. the synergistic effect of med and irfs on ifn-l induction was determined by co-transfection of pcr or pcr -med ( ng) with pcr -irf ( ng) in a cells, with lipofectamine ltx with plus reagent. the effect of med depletion on ifn-l induction was determined by transfection of a cells in -well plates with nm rscf or med sirna. ifn-l was quantified h post-transfection. ifn-l protein expression was quantified in supernatants using a commercial ifn-l duoset elisa kit (r and d systems). a point mutation (r q) was introduced into med (transcript variant ) by pcr with specific primers (see table s in text s ) and the clone verified by sequence analysis. a cells were co-transfected with ng of pcr , pcr -med or pcr -r q, ng of pcr -irf , ng of ifn-b-, ifn-l -or isre-responsive luciferase reporter constructs and ng prl-tk, a renilla luciferase transfection control, in antibiotic-free low serum ( %) medium. after h cells were lysed, and firefly (promoter reporter construct) and renilla (transfection control) luciferase activity was measured (dual-luciferase reporter kit, promega). relative luminescence activity was normalized to renilla (as a transfection control). a cells were transfected in triplicate in black -well plates, as described. after incubation at uc for h, cells were serumstarved for h by growing in low serum ( %) medium, before cells were left untreated or stimulated with ng/ml ifn-a, ifnb or ifn-c, or ng/ml ifn-l or ifn-l / in serum-reduced medium. after h cells were infected with hsv- c diluted to moi . in ifn-containing serum-reduced medium. after h incubation, virus was removed and media replaced with ml serum-reduced growth medium containing no interferon, ifn-a/b/-c at ng/ml, or ifn-l /-l / at ng/ml. replication was monitored as described and normalized to replication in mock-transfected, unstimulated cells. potential interactions between med and the irfs were determined by yeast two-hybrid analysis and confirmed by coimmunoprecipitation (co-ip) in mammalian cells. a) co-immunoprecipitation. co-immunoprecipitation was performed using the epitope-tagged plasmids pgbkt (myc epitope) and pgadt (ha epitope) containing the t promoter, and recombinant vaccinia virus vtf- expressing the t rna polymerase (nih aids repository). hek cells were seeded in cm dishes and the following day infected with vtf- (moi ) for h before transfecting mg each of empty bait (pgbkt ) or med -bait, and irf-prey (pgadt ) vectors with lipofectamine (invitrogen). after h cells were lysed on ice for min in np buffer, containing protease and phosphatase inhibitors. debris was removed by centrifugation, and protein quantified with a bca protein assay kit (thermo scientific, uk) as per manufacturers' instructions. equal protein quantities ( mg per sample) were pre-cleared by continuous mixing with ml preequilibrated protein g sepharose beads for h at uc. samples were centrifuged and supernatants halved before overnight mixing incubation with beads pre-coated with mg a-ha (roche, uk) or a-c-myc (santa cruz, uk) at uc. beads with precipitated proteins were washed three times with ice-cold np buffer, resuspended in sds protein sample buffer and boiled for min before sds-page separation on two % polyacrylamide gels. proteins were transferred onto nitrocellulose membranes overnight at uc before blocking with % milk in tbs-tween then incubating with either a-ha or a-myc antibody (diluted : in % milk/tbs-tween). membranes were washed in tbs-tween before incubation with hrp-conjugated secondary antibody (diluted : in % milk/tbs-tween). after further washes, proteins were detected by incubation with ecl western blotting detection system, exposing to x-ray film and developing films in an optimax x-ray film processor. b) yeast two-hybrid assays. haploid yeast strains ah and y were transformed with mg prey (pgadt or pgadt -irf , -irf , -irf , -irf , -irf , -irf or -irf ) or bait (pgbkt or pgbkt -med ) plasmid dna, respectively, and grown overnight in synthetic defined (sd) medium lacking either leucine (-l; prey) or tryptophan (-w; bait) before prey-and bait-expressing haploid yeast cells were mated overnight in sd-lw/ % ypda medium. haploids were selected in sd-lw for h before transferring to triple-knockout, histidine-deficient sd-lwh liquid medium containing -aminotriazol ( -at) and methylumbelliferyl-b-d-galactopyranoside ( -mux) for - days. interactions were tested in quadruplicates, detected by growth of colonies on sd-lwh agar with -at, and quantified by measurement of fluorescence released from a-galactosidase cleavage of -mux upon protein interaction. relative fluorescence (rfu) was normalized to negative control interaction (empty pgadt mated with empty pgbkt ). ethnically italian subjects with or without a history of recurrent herpes labialis (hl) gave written voluntary informed consent and were enrolled in this study at the university of rome tor vergata with the approval of the ethical committee at the university of rome tor vergata. all subjects were interviewed by medically trained investigators using an appropriate questionnaire and agreed to provide saliva and/or blood samples. data and blood sample collection was carried out as previously described [ ] . a total of healthy immunocompetent individuals ( men and women) age - years (overall median age . yr; male median age yr, range - ; female median age , range - ) participated in the study. none of the patients presented with an active lesion at the time of or in the weeks preceding saliva sample collection. for the purpose of this study, patients were characterized into no recurrence of hl (nr), low recurrence (l; - hl episodes/yr, with a maximum extension of cm, mild symptoms and healing time , days), high recurrence (h; or more hl episodes/yr, extension of lesions more than cm), very high recurrence (h+; more than hl episodes/yr, extension of lesions . cm and/or involving nose or cheek beyond the lip, more severe and long lasting associated symptoms including itch, burning, paresthesias and/or neuralgia, with healing times . days and who required antiviral therapy ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ]). saliva samples (, ml) were obtained from each subject after an overnight fast and after rinsing the mouth twice with water, split into two aliquots and frozen at uc. samples were anonymised and stored with dual code labels before shipping on dry ice to the university of edinburgh for dna extraction. saliva and pbmc samples were thawed and dna extracted using a qiaamp dna blood mini kit (qiagen) as per manufacturer's instructions, quantified using a nanodrop and il b genotype determined by melt-curve analysis pcr on a lightcycler (roche) using the lightmixh kit il b (tib molbiol) as per manufacturer's instructions. significance of genotype association was determined by fisher's exact test, comparing the frequency of the cc, ct or tt genotype in the nr group versus the l (pvalue = ), h (p-value = . ) or h+ (p-value = . ) clinical groups. below lists the geneid numbers for genes and proteins mentioned within the text of this manuscript: ifitm ( ) text s text for supporting figures. descriptive text for figures s and s validation of hsv- -host y h interactors. a subset of protein interactions identified in the hsv- -host y h screen were validated using the lumier pull-down assay in a mammalian cell system. strength of interaction was determined by z-score, where a score to represents a weak interaction and score . represents a strong interaction. (h) distribution of direct hsv- targets in the rnai screen. the proteins directly targeted by hsv- were taken from the y h data set and from the literature curation. an enrichment of literature-derived targets could be observed in the top % most inhibiting knockdowns ( . -fold enrichment; p = . ), but the same could not be observed for the y h-detected direct targets. (tif) figure s validation of hf identified by rnai. hfs identified in the hsv- perturbation screen were validated with deconvoluted sirnas and qpcr: (a) chromoboxes, (b) homeoboxes, (c) general transcription factors, (d) nuclear receptors, (e) proteasome family members, (f) topoisomerases, (g) mediator complex subunits, (h) proteins involved in vesicle transport, (i) integrins, (j) y h interactors, (k) ubiquitin e ligases, (l) vesicle transport -further candidates, (m) interferon-stimulated membrane proteins and (n) others. hsv replication is presented as normalized replication slope, and is the mean of six individual assay points. error bars represent standard deviation of the six data points. deconvoluted sirnas which had a sequence different to that in the original screen are highlighted in red. genes for which the primary screen phenotype was not confirmed in the deconvolution assay are shown in bold text. (tif) figure s hsv- hfs are involved in diverse cellular pathways and at multiple stages of the hsv life cycle. enrichment of protein functions among the hsv- hfs. the enrichment for gene ontology (go) terms and kegg, bio-carta or reactome pathway annotations among the hsv- hfs identified by (a) rnai and (b) y h assay was performed using david bioinformatics software. (c) direct interactions between human and hsv- proteins. the protein-protein interactions (ppis) depicted are from the high confidence y h data set and from literature curation. circles and diamonds correspond to human and hsv- proteins, respectively. human proteins detected in the y h screen are drawn with red borders. human genes that showed the strongest effects in the rnai screen are colored yellow (extreme %) and orange (extreme %). (d) highly interconnected regions in the human interaction network composed of hfs. highly interconnected regions in the human interaction network composed of hfs. we assembled a human interaction network using data from the major ppi databases. a subnetwork consisting of hfs was then defined by limiting the network to the hfs detected in the y h screen, the rnai (extreme %) screen, and the literature curation. highly interconnected regions in the subnetwork were sought out using the mcode algorithm. the top six scoring regions are shown. proteins displayed in red correspond to hfs that are known only from the literature, and those in green are those that were detected in either of the screens performed in this study. the three boolean values beside each gene symbol represent whether the hf is present in the y h screen, the rnai screen (extreme %), or the literature curated set, in that order. dominant functional categories could be observed in each of the regions, including transcription (e.g., mediator complex, rna polymerase ii and associated genes), translation initiation, splicing, and intracellular transport. (tif) figure s hfs involved in viral entry and capsid transport. (a) diagrammatic summary of the role of dynein chains in hsv- infection. (b) microtubule transport is required for hsv- infection. the role of dynein microtubule transport components in hsv- replication was analysed by comparing the replication slope of hsv- -egfp (c )-infected cells depleted for a range of dynein chains from the primary sirna perturbation screen. error bars represent the mean of three independent experiments done in duplicate. (c) depletion of dyneins inhibits virus particle release. the effect of dynein chain depletion on hsv- particle release was determined by quantifying virus titer in supernatants of hela cells depleted of dync h (heavy chain), dync i (intermediate chain) and dync li (light intermediate chains) in a high multiplicity (moi ; hsv- kos) growth assay. titers were compared to control transfected cells (nt, nontargeting sirna). (d) depletion of dynein chains prevents immediate-early gene expression. immediate-early (icp ) and late (vp ) viral protein expression in cells depleted of dync h , dync i or dync li was analysed and quantified by western blot. levels were compared to control transfected cells (nt, non-targeting sirna). (e) quantification of icp and vp protein expression. protein levels of icp and vp in cells depleted of dync h , dync i or dync li were quantified with an odyssey imager and normalized to protein levels in control transfected cells (non-targeting sirna). (tif) figure s e ubiquitin conjugating enzymes in hsv- immune evasion. (a) depletion of e ubiquitin ligases inhibits pml degradation following hsv- infection. hela cells mounted on coverslips were depleted for a range of e ubiquitin ligases for h before infecting with hsv- +. cells were fixed and stained for icp (green) and pml (red), analysed by confocal microscopy and pml-positive cells were counted ( fields of view per coverslip) and expressed as a mean percentage of pmlpositive cells remaining ( independent experiments). error bars represent the standard deviation over independent experiments. (b) inhibition of pml degradation by e s is icp -dependent. hela cells were seeded on coverslips, transfected as above and infected with an icp ring-finger deletion mutant (fxe). remaining pml-positive cells were quantified as above. (c) immunofluorescence staining for pml bodies in cells transfected with control sirna not incorporated into the risc complex (rscf) or cells depleted of the e ubiquitin conjugating enzymes e d or e l . the arrows highlight cells that have wt pml levels remaining in them whilst containing wt icp . (tif) figure s med is an anti-viral component of the largely pro-viral multi-protein mediator complex. (a) diagrammatic summary of the role of mediator complex subunits in virus replication. subunits are coloured according to whether hsv- replication was unchanged (grey), inhibited (top %, red; top %, orange) or enhanced upon gene knockdown (top %, light green; top %, dark green). subunits not included are white; herpesvirus proteins reported to target mediator subunits are violet; mediator subunits detected in other viral rnai screens are highlighted by coloured diamonds. (b) mediator complex subunits influence hsv- replication. individual subunits of the mediator complex and associated proteins were depleted by sirna knockdown and infected with hsv- c (moi . ). replication was monitored over multiple rounds and the slope of replication over the linear phase was calculated and normalized to controls (mock-transfected cells). grey, no significant effect; red, strongly pro-viral; green, strongly anti-viral. error bars represent the mean of six replicates. herpes simplex virus type infection: overview on relevant clinico-pathological features herpes simplex virus infects most cell types in vitro: clues to its success the family herpesviridae: a brief introduction herpesviruses and immunity: the art of evasion proteomic analysis of cells in the early stages of herpes simplex virus type- infection reveals widespread changes in the host cell proteome analysis of virion-incorporated host proteins required for herpes simplex virus type infection through a rna 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experimentally derived confidence score for binary protein-protein interactions herpes simplex virus type capsid protein vp interacts with dynein light chains rp and tctex and plays a role in retrograde cellular transport walking the walk: how kinesin and dynein coordinate their steps replication of icp -null mutant herpes simplex virus type is restricted by both pml and sp herpes simplex virus type immediateearly protein icp and is isolated ring finger domain act as ubiquitin e ligases in vitro the degradation of promyelocytic leukemia and sp proteins by herpes simplex virus is mediated by the ubiquitinconjugating enzyme ubch a the metazoan mediator co-activator complex as an integrative hub for transcriptional regulation transcription control by e a and map kinase pathway via sur mediator subunit the trap component of the trap/mediator complex is essential in broad transcriptional events and development inhibition of type iii interferon activity by orthopoxvirus immunomodulatory 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in human monocyte-derived macrophages and dendritic cells is dependent on virus replication and is counteracted by icp targeting nf-kappab and irf- the cycle of human herpes simplex virus infection: virus transport and immune control expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand-and cell-dependent interferon-lambda: a new addition to an old family ifn-lambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo automated yeast two-hybrid screening for nuclear receptor-interacting proteins human protein reference database- update the biogrid interaction database: update the database of interacting proteins: update gene ontology: tool for the unification of biology. the gene ontology consortium kegg: kyoto encyclopedia of genes and genomes the kegg resource for deciphering the genome reactome: a knowledgebase of biological pathways reactome: a knowledge base of biologic pathways and processes systematic and integrative analysis of large gene lists using david bioinformatics resources varicella-zoster virus infection of a human cd -positive t-cell line regulation of the transcription and replication cycle of human cytomegalovirus is insensitive to genetic elimination of the cognate nf-kappab binding sites in the enhancer properties of non-structural protein of semliki forest virus and its interference with virus replication vaccinia virus cores are transported on microtubules live visualization of herpes simplex virus type compartment dynamics construction and characterization of bacterial artificial chromosomes containing hsv- strains and kos en passant mutagenesis: a two step markerless red recombination system we are grateful to tomozumi imamichi (national cancer institute, bethesda), rick randall (university of st andrews) and geoffrey l. smith (university of cambridge) for providing reagents, as well as george sorensen, tobias bergmann, gabriella siszler, frank schwarz, melanie ott, verena raschbichler, hannah striebinger, philippa beard and susanne bailer for assistance. key: cord- -t jtjmi authors: chen, weiye; cao, wenyan; zhao, huijun; hu, qianqian; qu, linmao; hu, sen; ge, jinying; wen, zhiyuan; wang, xijun; li, haobo; huang, kehe; bu, zhigao title: establishment of a stable cho cell line with high level expression of recombinant porcine ifn-β date: - - journal: cytokine doi: . /j.cyto. . . sha: doc_id: cord_uid: t jtjmi abstract a cho cell clone (cho-poifn-β) with stable porcine ifn-β expression under control of cmv promoter was selected under g pressure. in a cm cell culture flask ( ml culture medium), the cumulative protein yield of recombinant poifn-β reached . × iu/ml. this cells clone maintained stable expression for at least generations even in the absence of g selection pressure. the expressed recombinant poifn-β could induce the expression of porcine mx protein in pk cells, and activate the chicken mx promoter-controlled luciferase reporter gene expression, confirming that the recombinant poifn-β has the biological activity of natural porcine type-i interferon. in addition, the recombinant poifn-β fully protected pk cells against tcid of porcine transmissible gastroenteritis virus and pseudo-rabies virus infection, demonstrating its high potential in therapeutic applications. this is the first report of establishing a mammalian cell line with stable expression of porcine ifn-β. type-i interferon, an important component of the innate immune system in vertebrates, has gained much attention in modern medical research. currently, one of the research focuses is the mechanism employed by viruses to evade the innate immune defense such as interferons when infecting the host [ ] [ ] [ ] , this in theory provides strong support for effective viral disease prevention and treatment. viruses such as porcine reproductive and respiratory syndrome virus (prrsv) [ , ] , pseudo-rabies virus (prv) [ , ] , porcine arteritis virus (poav) [ ] , swine fever virus [ ] [ ] [ ] and transmissible gastroenteritis virus (tgev) [ ] , employ the strategy to evade the host immune system by destroying type-i interferon system when infecting the host. type-i interferon has played an important role in the treatment of chronic hepatitis b [ , ] , chronic hepatitis c [ ] , multiple sclerosis [ ] , tumor [ , ] and other diseases [ , ] . in veterinary medicine, porcine type-i interferon has good prospect in the treatment of common viral diseases such as tgev [ ] , swine fever virus [ ] , prv [ , ] , etc. therefore, type-i interferon with high activity is needed whether it is for basic research or clinical application. in addition, pure and stable interferon with high activity per unit mass is needed as the standard for accurate and convenient determination of the interferon biological activity. recombinant interferon from mammalian cell expression displays correct folding and glycosylation in comparison to that from other expression systems, best suited for use in therapeutics [ ] and as standards [ ] . however, currently, the production of porcine interferon is mainly from the prokaryotic [ , , ] , yeast [ , [ ] [ ] [ ] [ ] and baculovirus [ ] expression systems. it is therefore important for basic and applied research to establish a high level expression system for porcine type-i interferon in mammalian cells. the purpose of this study was to establish highly efficient and stable expression of recombinant porcine ifn-b in cho-k cell line, and further characterize the biological activity of the product. porcine kidney (pk ) cells and madin-darby bovine kidney (mdbk) cells preserved in our laboratory were cultured in the dmem (gibco) culture medium containing % fbs (gibco). chicken embryo fibroblasts (cefs) were prepared from -day-old spf chicken embryos according to routine method. mdbk-mxp-luc cell line with stably integrated luciferase reporter gene under chicken mx promoter was established in this laboratory (unpublished (prv) were preserved in the harbin veterinary research institute for swine diseases of the chinese academy of agricultural sciences, and determination of tcid was performed on pk cells. the lasota strain of newcastle disease virus (ndv) was preserved in this laboratory, and titrated on primary cefs. vesicular stomatitis virus (vsv) was preserved in this laboratory, titrated on mdbk cells. porcine ifn-a standard were from pestka biomedical laboratories (piscataway, nj, usa). plasmid pet (a+) was purchased from novagen corporation. poifn-b orf sequence was obtained from genbank (genbank accession no. s ). primers were designed as: the upstream primer -tgccaccatggctaacaagtgcatc- , the downstream primer -agccacaggggggagatgttcagt- . kozak sequence was added to the upstream primer before the initiation codon atg to facilitate expression in the eukaryotic cells. pk cells with about % confluence were infected with ndv at a moi (multiplicity of infection) of . and harvested after h. total rna was extracted using trizol (invitrogen) and subjected to rt-pcr using the mentioned primers above. poifn-b pcr product was then subcloned into pmd -t vector (takara) to generate plasmid pmd -poifn-b and sequenced. pmd -poifn-b was double-digested with ecor i/sal i to obtain the poifn-b orf fragment, which was then subcloned into xho i/nhe i double-digested pcaggs plasmid to generate plasmid pca-poifn-b, or sma i digested pci-neo plasmid (promega) after end-blunted with t dna polymerase (mbi) to generate plasmid pcn-poifn-b. antiviral activity of interferon was titrated as described [ ] with modifications. in brief, mdbk cells in -well plate were grown to - % confluence and the supernatant was discard, ll of -fold serially diluted ifn sample was added to each well ( - -fold dilution) with two parallel wells set up for each dilution, and the cells were treated at °c and % co culture conditions for h. after the supernatant was removed, ll of vsv virus diluted to , pfu/ ll in dmem containing % fbs was applied to each well. three wells of virus control (vc) with virus added but no interferon treatment, and three wells of blank control (bc) with neither interferon nor virus were set up. when cytopathic effect (cpe) in vc-wells reached %, the cells were stained with naphthol blue-black and the absorbance at nm was read using a microplate reader (biorad). antiviral activity units (iu) were calculated using porcine ifn-a standards as reference [ , ] . pca-poifn-b and pcaggs were used to transfect bhk- cells with fugene (roche) according to the manufacturer's instructions. medium was changed h after transfection and culture continued for another h. culture supernatants were collected, named poifn-b pca and mock pca respectively, and stored in aliquots at À °c before use. cho-k cells were transfected with pcn-poifn-b using fugene . twenty-four hours after transfection, the cells were passaged : and cultured in the pressure selection medium containing lg/ml g . after - days, the neomycin-resistant cell colonies were isolated using cloning rings, trypsin digested and culture expanded in -, -and -well plates sequentially. the se-lected cell clones were seeded into a -well plate with .  cells per well and grown to a density of % for h, and then the cell culture supernatants were collected for titration of antiviral activity. the cell clone with the highest level of expression was subjected to single cell cloning using the limited dilution method. the steps above were repeated and the cell clone with the highest expression was selected and freeze preserved according to conventional methods. the cell clone was maintained in the culture medium containing lg/ml g . prokaryotic expression of the chicken mx protein and purification steps were as follows: ndv (moi = . ) was used to infect cefs of about % confluence. when cpe reached - %, the cells were harvested and total rna were extracted with trizol. using the primers in parentheses (upstream primer -ggggatatcag-caatcagatggctttc- , introducing restriction site ecor v; downstream primer -tttgtcgactgggatgacctcgttttg- , introducing sal i restriction site), rt-pcr was performed to amplify the first half of the orf gene fragment of chicken mx protein. the pcr product was double-digested with ecor v/sal i and cloned into pet (a+) (novagen) underwent the same double-digestion. the chicken mx protein produced from this prokaryotic expression plasmid was a fusion protein with the his tag. the plasmid was transformed into bl and induced with . mm iptg at °c for h. the fusion protein was purified with ni-nta agarose affinity resin (invitrogen) according to the manufacturer's instructions and further dialysed to remove urea. then the recombinant chicken mx protein was used to immunize balb/c mice according to conventional method [ ] , and serum was collected and stored at À °c before use. pk cells grown in -well plates to confluent monolayers were incubated with serially diluted recombinant poifn-b in % co at °c for h. the cells were digested with trypsin and collected by centrifugation at rpm/min for min. the cell pellets were then mixed with ll each  sds sample buffer, boiled in water for min, and loaded for sds-page. the proteins were transferred to a nitrocellulose membrane, blocked with % fish skin protein (prepared in pbst) overnight, and then incubated with mouse polyclonal anti-chicken mx protein antibody : diluted in pbst, or mouse polyclonal anti-porcine beta-actin protein antibody (sigma) : diluted in pbst as internal reference, followed by horseradish peroxidase-anti-mouse igg (sigma) : diluted in pbst and color developed in dab for - min before termination with deionized water. mdbk-mxp-luc cells in -well plate were grown overnight to approximately % confluence, ll of ifn samples -fold serially diluted in dmem containing % fbs was added to each well, and culture continued at °c and % co . after h, intracellular luciferase expression was determined using the bright-glo luciferase assay system (promega) according to the manufacturer's instructions. two parallel wells were set up for each dilution, and wells with no ifn treatment were set as blank control (bc). pk cells were grown to % confluence in -well plates; the medium was replaced with ll of fresh culture medium with -fold serially diluted poifn-b cho , and incubated in % co at °c for h. after removal of the culture medium, tcid (in ll dmem) of tgev or prv was added to each well and incubated for h. the virus solution was then removed and dmem containing % fbs was added and culture continued. wells receiving virus in the absence of ifn treatment were set up as the virus control (vc), and wells not treated with either virus or ifn were the blank control (bc). when the cpe of the vc-wells reached - %, the inhibitory effect of interferon on the replication of tgev and prv was observed under an inverted microscope, and the culture medium in each well were collected to titer tgev or prv on pk cells in -well plates. in order to construct a stable expression plasmid for poifn-b, it was first examined whether the acquired sequence was right. poifn-b fragment amplified by pcr was bp in size by electrophoresis as expected and further confirmed by sequencing to be the complete poifn-b gene orf after cloning into the pmd -t cloning vector, completely matching the poifn-b gene sequence from the genebank (genbank accession no. s ). to examine whether the protein coded by this cloned poifn-b gene has antiviral activity, the poifn-b gene orf was subcloned into the eukaryotic expression plasmid pcaggs to obtain pca-poifn-b. the pca-poifn-b and pcaggs plasmids were used to transiently transfect bhk- cells. the cell culture supernatants harvested were named poifn-b pca and mock pca , respectively, and antiviral activity was assayed in mdbk cells using vsv. the result showed that the antiviral activity of poifn-b pca was .  iu/ ml, while -fold diluted mock pca had no antiviral activity. together with the sequencing results, it is confirmed that the correct full orf of poifn-b gene was obtained, and subcloned to pcn to generate a stable recombinant expression plasmid pcn-poifn-b. after pcn-poifn-b transfection, cho cells were selected in the selection medium containing lg/ml g . the obtained neomycin-resistant cell colonies were harvested with cloning rings and expanded. based on the antiviral activity of the collected culture supernatant, three cell clones with the highest expressions were selected. the clones were further screened through single cell cloning, once again their expression levels were tested, and the one with the highest level of expression was selected and freeze preserved, named cho-poifn-b. afterwards cho-poifn-b cells were maintained and passaged in the selection medium containing lg/ml g . although the bioactivity of poifn-b was detected in the culture medium of cho-poifn-b cells, the rt pcr was carried out to ensure the poifn-b gene was transcribed. total rna from cho-poifn-b cells was extracted with trizol, and subjected to rt-pcr to amplify the poifn-b gene fragment. the expected size of bp was confirmed in cho-poifn-b cells (fig. a, lane ) but not in cho cells (fig. a, lane ) . the result showed that poi- cho-poifn-b cells in cm flasks were passaged in culture media with or without g every - days; samples were taken from the th, th, th, th and th generations, respectively. cell culture was repeated three times under each condition. prior to sampling, cells were seeded at cells/ml in cell culture flask with ml of culture medium. when cells formed a contiguous layer, the culture was continued for additional h before sampling. the samples were stored at À °c, and antiviral activity titration was performed when all the samples were ready. as shown in fig. poifn-b cho sample ( .  iu/ml) expressed from cho-poifn-b cells was -fold serially diluted, and was used to stimulate pk cells for h. cells were harvested, and then subjected to immunoblotting for detection of porcine mx protein expression using mouse polyclonal antibody against chicken mx protein and sheep anti-mouse igg antibody. as shown in fig. a , using beta-actin protein as internal reference, poifn-b cho induced clearly detectable mx protein (molecular weight of about kda) at a threshold of . iu/ml, and the extent of mx protein expression is positively correlated with the poifn-b cho unit activity in the range of - .  iu/ml. there was no mx protein expression in cells not stimulated with poifn-b cho (fig. a, lane ) . in parallel experiment, poifn-b cho was -fold serially diluted as above and used to stimulate mdbk-mxp-luc cells (fig. b) . the results showed that poifn-b cho as low as . iu/ml ( -fold dilution) could induce luciferase expression; and the expression level of luciferase was linear to the antiviral activity of poifn-b cho in the range of about . - iu/ml ( - -fold dilution). in order to study the antiviral capacity of recombinant poifn-b against porcine viruses in vitro, pk cells were treated with -fold serially diluted poifn-b cho (start from .  iu/ml) for h, and then infected with tcid of tgev (fig. a-c) or prv (fig. f-h) . when cpe formed in the virus control wells reached - % (fig. d and i) , virus suppression by poifn-b cho was observed and compared with the blank control (fig. e and j) . the results showed that: iu/ml of the poifn-b cho completely inhibited tgev infection in pk cells (fig. a) ; iu/ml of the poifn-b cho completely inhibited prv infection in pk cells (fig. f) . and the tittering results of tgev or prv in supernatant in each well were concordant with the cpe results above (fig. k) , no tgev were detect in iu/ml poifn-b cho treated wells, and no prv were detect in iu/ml poifn-b cho treated wells, while virus could be detected in mock wells and lower diluted-poifn-b cho treated wells. even if a few more days were allowed, tgev and prv still could not replicate to generate cpe (data not shown), indicating that poifn-b cho could adequately protect pk cells against tcid of tgev and prv infection. however, if more than tcid of virus were used for infection, even .  iu/ml of poifn-b cho could not provide complete protection (data not shown). since there is no established standard from world health organization (who) for porcine ifn-b [ ] , commercial ifn-a from prokaryotic expression system was used in this study as the standard to determine interferon activity. the cho cell-derived recombinant ifn-b is very suitable as a standard [ ] , so that the recombinant poifn-b in the present study has potential application as the standard for poifn-b products. to acquire a cell line with stable gene integration and stable expression, it is necessary to perform one to two rounds of single cell cloning after the cell clones with high expression were selected after transfection. in this study, after single cell cloning, the expression of cho-poifnb cell line remained stable after passages regardless of g presence in the culture medium, indicating that poifn-b expression framework has been integrated into the cho-k cell genome. in order to verify the biological activity of expressed interferon in this study, both conventional antiviral activity titration and mx promoter activation were adopted. chicken mx promoter is not only suitable for the detection of chicken type i ifn, but also suitable for the detection of biological activity of mammalian type i ifn [ ] . that the antiviral activity of poifn-b needed was times lower against the tgev (coronavirus branch, single-stranded rna virus) than the prv (herpesviridae, double-stranded dna virus) indicates that poifn-b is more effective against rna viruses. interferon from prokaryotic expression has lower activity per unit mass [ ] and more pyrogens in addition to being more antigenic, thus having more side effects in clinical applications and reducing the therapeutic effect of interferon. in contrast, ifn expressed from mammalian cells has higher activity per unit mass and less pyrogens, is less antigenic, and thus having less side effects and so on. it is worthy to note that ifn from yeast and baculovirus expressions differs significantly with natural ifn in glycosylation, while recombinant ifn-b expressed by mammalian cells displays almost the same properties in terms of glycosylation and folding as its natural counterpart, and correct glycosylation of the ifn-b is essential for its activity and stability [ , , ] . porcine leucocyte-derived interferon is used as quality standards for veterinary biological products promulgated by the ministry of agriculture of china. it is obtained by inducing swine leukocytes using newcastle disease virus. however, the production process is complicated, the product has a complex composition, and the activity of the final product is about , iu/ml. in com-parison, recombinant poifn-b production from cho cells has great advantages in that the process is simple, the product composition is easy to control, and the protein yield of poifn-b in this study is times higher. human ifn-a expression in mammalian cells could reach the level of .  iu/ml [ ] , times the expression of ifn-b in this study. the difference in the expression levels may be due to the differences in screening tags, promoters, cells, ifn activity detection systems and units defined for activity, etc. by optimizing the culture medium and cultivation techniques, it is expected that expression levels can be greatly increased to meet future demand for industrial production. currently, enrichment and purification of recombinant poifn-b are in progress. in conclusion, research of animal interferon expression in mammalian cells lags far behind that of human interferon. this study reported for the first time internationally to have established a mammalian cell line with stable expression of poifn-b, in an exploration of using mammalian cells to express interferon for veterinary use. the cho-poifn-b cell line established in this study expresses the recombinant poifn-b that has the same biological function as natural porcine type i ifn. the recombinant poifn-b displays a high level of antiviral activity of up to .  iu/ml, and has a very high activity per unit mass and stability. the recombinant poifn-b can be used as a standard for the detection of biological activities of porcine ifn-b, and has good prospect in clinical applications. viruses and interferons inverse interference. how viruses fight the interferon system type i interferons in host defense vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes induction of porcine cytokine mrna expression after dna immunization and pseudorabies virus infection suppression of the interferon-mediated innate immune response by pseudorabies 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interferon beta cloning, high level expression and purification of porcine ifn gamma gene modification and high prokaryotic expression of porcine interferon alpha- expression of porcine interferon-gamma gene in pichia pastoris and its effect of inhibiting porcine reproductive and respiratory syndrome virus secreted expression of porcine interferon-gamma gene in pichia pastoris high level secretion expression of poifnalpha in pichia pastoris secretory expression of porcine interferon-gamma in baculovirus using hbm signal peptide and its inhibition activity on the replication of porcine reproductive and respiratory syndrome virus expression of porcine gamma-interferon in recombinant baculovirus and determination of its antiviral activity assays for antiviral activity automated, quantitative cytopathic effect reduction assay for interferon cytopathic effect inhibition assay for interferon: microculture plate assay get effective polyclonal antisera in one month biological assays for interferons inducible expression of amplified human beta interferon genes in cho cells human interferons alpha, beta and omega expression and purification of recombinant, glycosylated human interferon alpha b in murine myeloma nso cells we thank dr. changming liu for providing the transmissible gastroenteritis virus and pseudo-rabies virus, and dr. yanwu wei for the help in the part of antiviral experiment in vitro using transmissible gastroenteritis virus and pseudo-rabies virus. key: cord- -dk w z authors: kikkert, marjolein title: innate immune evasion by human respiratory rna viruses date: - - journal: journal of innate immunity doi: . / sha: doc_id: cord_uid: dk w z the impact of respiratory virus infections on the health of children and adults can be very significant. yet, in contrast to most other childhood infections as well as other viral and bacterial diseases, prophylactic vaccines or effective antiviral treatments against viral respiratory infections are either still not available, or provide only limited protection. given the widespread prevalence, a general lack of natural sterilizing immunity, and/or high morbidity and lethality rates of diseases caused by influenza, respiratory syncytial virus, coronaviruses, and rhinoviruses, this difficult situation is a genuine societal challenge. a thorough understanding of the virus-host interactions during these respiratory infections will most probably be pivotal to ultimately meet these challenges. this review attempts to provide a comparative overview of the knowledge about an important part of the interaction between respiratory viruses and their host: the arms race between host innate immunity and viral innate immune evasion. many, if not all, viruses, including the respiratory viruses listed above, suppress innate immune responses to gain a window of opportunity for efficient virus replication and setting-up of the infection. the consequences for the host's immune response are that it is often incomplete, delayed or diminished, or displays overly strong induction (after the delay) that may cause tissue damage. the affected innate immune response also impacts subsequent adaptive responses, and therefore viral innate immune evasion often undermines fully protective immunity. in this review, innate immune responses relevant for respiratory viruses with an rna genome will briefly be summarized, and viral innate immune evasion based on shielding viral rna species away from cellular innate immune sensors will be discussed from different angles. subsequently, viral enzymatic activities that suppress innate immune responses will be discussed, including activities causing host shut-off and manipulation of stress granule formation. furthermore, viral protease-mediated immune evasion and viral manipulation of the ubiquitin system will be addressed. finally, perspectives for use of the reviewed knowledge for the development of novel antiviral strategies will be sketched. sion. many, if not all, viruses, including the respiratory viruses listed above, suppress innate immune responses to gain a window of opportunity for efficient virus replication and setting-up of the infection. the consequences for the host's immune response are that it is often incomplete, delayed or diminished, or displays overly strong induction (after the delay) that may cause tissue damage. the affected innate immune response also impacts subsequent adaptive responses, and therefore viral innate immune evasion often undermines fully protective immunity. in this review, innate immune responses relevant for respiratory viruses with an rna genome will briefly be summarized, and viral innate immune evasion based on shielding viral rna species away from cellular innate immune sensors will be discussed from different angles. subsequently, viral enzymatic activities that suppress innate immune responses will be discussed, including activities causing host shut-off and manipulation of stress granule formation. furthermore, viral protease-mediated immune evasion and viral manipulation of the ubiquitin system will be addressed. finally, perspectives for use of the reviewed knowledge for the development of novel antiviral strategies will be sketched. the epithelium of the lungs is the largest surface in the human body that is in contact with our environment. huge amounts of air and aerosols pass these cells each day, whereby the lung tissue, as well as the rest of the respiratory tract is probably almost constantly exposed to viruses and bacteria present in the inhaled air. an elaborate machinery is therefore present at this large surface to defend this tissue against invading pathogens, including mechanical barriers such as a mucus layer. the first line of defense at the entire length of the tract from the nasopharynx to the alveolar membrane is formed by the innate immune system [ , ] . in this review, the focus will be on the selection of common viruses that invade the lungs: coronaviruses (covs), rhinoviruses, respiratory syncytial virus (rsv), and influenza, which all have an rna genome. this latter feature is of importance to the set of cellular innate immune sensors that recognize these viruses when they enter the cells of the respiratory tract, and the subsequent downstream signaling cascades that are triggered as a result. a myriad of different cell types such as alveolar macrophages, airway epithelial cells, innate lymphoid cells, and dendritic cells (dcs) have a major role in this first defense, while in these and other cells of the respiratory tract the sensing, and several subsequent specific molecular intra-and intercellular signaling cascades ensure the establishment of the so-called antiviral state in the lungs. this state can inhibit the development of a productive infection with each of these invading viruses, thereby preventing or at least mitigating illness, before adaptive immunity kicks in to completely clear these viruses from the lungs. importantly, as a countermeasure against these elaborate defense mechanisms, invading respiratory viruses evolve activities that either circumvent or suppress the innate immune responses to create a window of opportunity for efficient virus replication, thereby often causing disease. ultimately, the balance between the efficacy of the combined innate and adaptive responses on the host's side, and the virulence and its capacity to evade the host's immune responses on the virus' side, together dictate the disease outcome. this review will focus on the evasion of the innate immune system by the array of respiratory viruses as introduced above, to highlight this important aspect of the virus-host interaction that may provide us with possible opportunities for exploration of novel antiviral strategies against these important viruses. particular viral activities will be highlighted and different viruses compared, but the information discussed will not be complete. i therefore apologize to any authors who miss discussion of their interesting work in this review. to facilitate comparison between the respiratory viruses described here, known and arguably important innate immune evasion strategies are listed, and for each strategy it is discussed how each virus group exploits its own mechanism. innate immune evasion obviously links to the innate immune responses that are known to be elicited by respiratory and other (rna) viruses, and while this will be elaborated to a limited extent below, they have also been reviewed comprehensively in recent reviews by others [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . arguably, the innate immune system is more important in early life, when the adaptive functions are still underdeveloped [ ] . yet, the young infant is probably exposed to as many incoming pathogens as older children and adults are, so the innate immune system plays a very important role in the protection from respiratory infection in young children. the fact that respiratory infections are one of the leading causes of mortality in children under years of age [ , ] suggests that the interactions of the (innate) immune responses in the infant respiratory tract with incoming pathogens is indeed a delicate one, and the balance between severe illness and overcoming an infection may be relatively easily tipping towards the dangerous side. that the innate immune response plays an important role in defense against respiratory infections in early life may be further illustrated by the fact that severe rsv infections in children are linked with polymorphisms in genes encoding innate immune factors (reviewed in [ , ] ). also later in life, the innate immune system plays an important role in the response against respiratory viruses (reviewed in [ ] ), and in the lungs these first responses against incoming viruses are governed primarily by alveolar and interstitial macrophages, dcs, airway epithelial cells, innate lymphocytes, and neutrophils. the innate immune response signaling cascade starts with the recognition of pathogen-associated molecular patterns by pattern recognition receptors (prrs). for rna viruses in the lungs, the toll-like receptors (tlrs) , and , which are expressed on several of the mentioned cell types, are important prrs. also, intracellular cytosolic prrs such as mda and rig-i, which are present in virtually any cell type including those of the lung, have been shown to be relevant for respiratory infections, as will be elaborated below. each of these mentioned receptors, or sensors, recognize forms of rna (e.g., ′ triphosphate rna, double-stranded rna [dsrna]) that are produced by (respiratory) rna viruses during their infection process, and which are distinguishable from the rna species that are normally present in the cells (such as capped mrna in the cytosol). in this way, the innate immune system senses foreign material that is possibly pathogenic, and this triggers downstream signaling to ultimately induce transcription factors in the nucleus which in turn stimulate expression of types i and iii interferons (ifns) and other proinflammatory cytokines. a second round of autocrine and paracrine signaling subsequently ensures that infected, and the surrounding uninfected cells, express a myriad of interferon stimulated genes (isgs) that establish a so-called antiviral state. this state quite efficiently inhibits further spread of the infection, and simultaneously triggers further adaptive responses that in most cases eventually will clear the virus from the infected individual. during all these signal transduction pathways, regulation of activation and inhibition of signal transduction in the cascades is governed in a strict manner by phosphorylation events as well as ubiquitination of different linkage types (k , k , k , etc.) on numerous factors in the pathways (reviewed in [ ] ). these events critically regulate the downstream signaling to ensure a sufficiently strong, but not overly explosive triggering of innate immune responses, and a timely downregulation of these responses to protect the individual from damaging immunopathology. recently, it has become clear that particular type iii ifns (il- / ), or ifn lambdas, which were discovered in [ , ] , play a prominent role in defense of epithelial surfaces such as that in the lung (reviewed in [ , , ] ). they bind to a distinct heterodimeric receptor consisting of ifnlr and il rb (as opposed to type i ifn that binds to ifnar / ), but seem to trigger downstream signaling that is very similar to the type i ifn-induced pathways, and are also induced by the same prrs as those triggering type i ifns. however, whereas type i ifns are made by many different cell types, ifn lambdas are primarily expressed by epithelial cells and dcs. recent literature suggests that despite the clear similarities between the types i and iii ifn signaling pathways, the type iii ifn machinery seems especially equipped to protect epithelial surfaces from pathogenic attacks, and forms the primary local defense upon invasion of low doses of viruses and bacteria. when this first activation of the type iii ifn machinery is insufficient due to higher doses of pathogens coming in, the more systemic type i ifn machinery forms the second line of defense over broader areas of the tissue (reviewed in [ ] ). additionally, it seems that type iii ifn does not trigger inflammation as much as type i ifn, and this probably indicates an important unique aspect of the type iii ifn induction, which may have a role in the protection of, for example, the lung epithelial tissue from immunopathology [ ] . recently, it has become clear that the strict distinction between innate and adaptive responses that has been the general view for a long time is probably not accurate. in the respiratory tract, several of the newly identified cell types and mechanisms that integrate aspects from both branches of human immunity are now thought to be very important for the defense against respiratory infections. natural killer t cells, mucosal-associated invariant t cells, and neutrophils, for example, each form a bridge between the innate and adaptive machineries and play very important roles during the clearance of respiratory viruses (reviewed in [ , , ] ). aspects of immunological memory, which were thought to be only present in the adaptive immune system, have now clearly been shown to play a role in the innate immune response as well, also that induced by viruses, and was named "trained innate immunity" [ ] . the general idea about the mechanism governing this is that epigenetic changes on innate immune factor genes in specialized immune cells such as macrophages are made after the activation of the innate immune response. this then positively influences the response upon a subsequent pathogen encounter, just as in the adaptive immune system [ ] . recently, it also became clear that after respiratory (bacterial) infections this mechanism indeed has a role, and strikingly, signaling from adaptive (cd + t cell responses) "back" to innate immune systems (alveolar macrophages) via ifn-gamma plays a role in generating epigenetically triggered innate immune memory to protect from re-infection [ , ] . besides these different responses, most of which are ifn-mediated, small non-coding (micro, circular, ...) rnas, rnai, and ifn-independent antiviral responses can be regarded as part of the innate immune response package as well [ ] [ ] [ ] . an emerging hot topic is also the interplay of innate immune response with cellular metabolism, so-called immunometabolism, which likely is quite relevant for respiratory viral infections [ , , ] . the general idea is that immune cells such as macrophages and dcs adapt the choice for the use of their metabolic systems to an immune-activated situation that requires increased amounts of energy. this resembles "the warburg effect", as described in tumor cells, and after pathogen sensing innate immune response thus triggers changes in the cell's metabolism from oxidative phosphorylation to glycolysis, thereby optimizing the cell's metabolism for the new situation [ ] . since the new insights mentioned above have generally not yet, or only to a limited extent, been investigated in the context of viral evasion, this will not be further elaborated in the subsequent sections for the selected respiratory viruses. viruses with an rna genome, such as the respiratory viruses highlighted in this review, produce several rna species during viral replication, which are normally absent in uninfected cells. for example, dsrna and rna with a ′-triphosphate are commonly produced by rna viruses during replication, but since the host cells do not normally copy rna from rna templates, these intermediate rna species are recognized by the innate immune sensors discussed above as foreign, resulting in antiviral effector activation. to be able to set up a productive infection in the cell, these viruses therefore need to circumvent and/or suppress these intracellular innate antiviral responses. an obvious primary strategy would be to shield away the replication intermediates with their dangerous, recognizable features, from the innate immune sensors roaming the cytosol. indeed, the viruses that have a +rna genome, which replicate exclusively in the cytosol such as the covs and rhinoviruses that invade the lungs, generally modify intracellular membranes elaborately to form headquarters of viral rna replication, also called "replication organelles" (ros; covs), "replication factories," "double membrane vesicles" (dmvs; covs, enteroviruses), "invaginations," or other (reviewed in [ ] [ ] [ ] ). also, the negative-stranded rsv genome and its replication enzymes are found associated with cytosolic occluded structures, in that case named inclusion bodies [ , ] . expression of a selection of specific hydrophobic viral proteins can usually mimic the formation of these structures, for example, nsp and nsp of covs [ ] , the n and p proteins of rsv [ ] , and b, c and a proteins of enterovirus (polio; [ ] ). all these structures, while divers in morphology and contents, seem to concentrate the viral replication machinery, intermediates and products inside membrane-bound vesicles or invaginations, seemingly unreachable for the innate immune sensors of the cytosol. it is interesting to note that very little is known about the details of interaction of viral replication organelles with the innate immune system. while the protective function of such organelles in the context of innate immune sensing is assumed by many researchers, hardly any reports present investigation, let alone proof, of this concept. a report by al-mulla and co-workers showed that in cov mutants that produced only half the number of ros during infection or in which the structures were smaller, replication as well as fitness of these viruses was in fact unaffected or even higher than for wt viruses. this was also true in cultures of primary host cells, which presumably have a fully functional intracellular innate immune system [ ] . their results indicated that there is no strict correlation between the number of replication organelles and the replication rate of these viruses. it is not clear, however, whether (part of) the viral replication takes place outside the replication organelles in these mutant virus infections, and whether replication organelles do, or do not, protect viral replication from innate immune attack therefore remains elusive after all. importantly, virtually all research investigating the role and structure of viral ros was performed in cell cultures, and little is known about their presence or numbers during infections in animal models or real hosts. investigation of the latter will, therefore, be pivotal for the true understanding of viral ros and their role in protection from innate immune responses. besides the question whether the replication organelles protect from innate immune sensors that recognize viral rna, it is also largely unclear whether the innate immune system possesses sensors or effectors that target viral replication organelles themselves. after all, all +rna viruses produce membranous replication organelles, and since they are probably indeed supporting viral replication, recognizing and attacking them would provide an efficient way for the innate immune system to inhibit viral infection. our recent research revealed that the type i ifn signaling cascade, which is utterly relevant for defense against +rna viruses, indeed includes effectors that influence the integrity of ros induced by equine arteritis virus, a +rna arterivirus and a distant relative of the covs [ ] . however, it is not yet clear which type i ifn-inducible factors are responsible. some recent reports (reviewed in [ ] [ ] [ ] ) suggest that intracellular membrane modifications such as viral ros can be recognized and targeted by guanylate-binding proteins (gbps), a family of dynamin-related large gtpases, of which mxa is a member. mxa is a well-known human type i and iii interferon-inducible factor that inhibits influenza virus infections [ ] . although the exact mechanism of inhibition is still not clear for several of the viruses inhibited by mx proteins, mx gtpase family members bind to intracellular membranes, and in cytosolic +rna virus infections mx proteins could target the ros [ ] . since influenza replicates in the nucleus (see also below), the idea is that mxa attacks influenza while its products are in the cytosol. several reports indicate that gbps other than the mx proteins act against human +rna viruses such as hepatitis c virus, classical swine fever virus, and dengue virus, which are all members of the flavivirus family, possibly by attacking their ros. in pigs, gbps inhibit porcine reproductive and respiratory syndrome virus (an arterivirus, distantly related to the covs). in mice, encephalomyocarditis virus and murine norovirus, which are both +rna viruses, are suppressed by interferon (-gamma)induced gbps [ ] . for murine norovirus, it has now become clear that gbps are indeed targeted to viral ros and that this depends on part of the autophagy machinery, namely the lc conjugation system [ ] . lipidated lc associates with viral ros, and while this does not depend on ifn-gamma induction it is clearly stimulated by it. the authors of this paper also mention in their discussion that similar mechanisms can be shown for encephalomyocarditis virus, suggesting that at least several +rna virus induced ros can be targeted by the innate immune system via gbps [ ] . ultimately, the idea is that once gbps associate with the viral ro membranes, they cause disruption and/or modification of these structures, resulting in less efficient viral replication [ , ] . mechanistically, this effect on viral replication could link to the viral rna species and intermediates becoming exposed upon disruption of ro membranes by gbps to the cytosolic innate immune rna sensors such as rig-i and mda , which subsequently triggers antiviral innate and adaptive immune responses to suppress further replication. further research is needed to confirm such a hypothesis. interestingly, while covs, rhinoviruses, and rsv replicate in the cytosol of respiratory epithelial cells and shield their replicating rnas as discussed above, influenza virus apparently takes another route, and as the only known exception to the rule this rna virus replicates in the nucleus. rna sensors like the rig-i-like sensors or tlrs were thought to be absent there, and therefore replication inside the nucleus may have been an alternative solution to avoid innate immune recognition of viral rna intermediates during replication. however, recent data indicated that rig-i can be active in the nucleus against influenza rna [ ] . the viral genome, packaged in nucleocapsid proteins and bearing a panhandle-and ′-triphosphate structure is recognized by rig-i, presumably in the cytosol while on its way to the nucleus, or when being incorporated into new virus particles [ ] [ ] [ ] . the recognition by rig-i is the major trigger to the production of type i ifn during influenza infection, while also tlr plays a role [ ] . additionally, the cell has evolved multiple ways to attack influenza replication, for example, by gbps that are localized in the nucleus and the cytosol [ ] . in summary, the formation of membranous headquarters may be a major strategy for respiratory viruses to avoid innate immune recognition of viral nucleic acid products in the cytosol. whether the cell can in turn recognize and attack these structures is still relatively unknown, along with viral countermeasures against these attacks. this kind of interactions illustrates the arms race between the cellular immune responses and viral evasion, which due to continuous evolution often has multiple levels. protection of the ′ terminus of viral rnas apparently, the shielding of viral replication products by ros is not a watertight system, and to further avoid recognition of their foreign rna species, respiratory viruses have evolved several means of directly modifying these rnas to avoid recognition by the innate immune rna sensors. adding a cap-structure or a mimic of this structure to the ′-end is an effective way, since in this way the cell's own mrnas are protected from recognition by the innate immune sensors. the respiratory viruses discussed here use quite diverse methods to achieve this kind of protection from recognition, concomitantly making sure their mrnas can be properly recognized by the translation machinery of the cell, which they "chose" to utilize. the rhinoviruses are members of the picornavirus family, and these use a specialized, virally encoded capmimicking peptide, called vpg, and attach this to the viral rna ′ end to protect it from recognition by the innate rna sensors [ , ] . these viruses indeed do not need a cap structure for translation of their rnas, since they use cap-independent internal ribosomal entry site-mediated translation [ , ] . influenza viruses steal mrna cap-structures from host mrnas in the nucleus during transcription in a process called "cap-snatching," in j innate immun ; : - doi: . / which the viral nucleoprotein plays a major role [ ] . rsv and covs provide their mrnas with cap-structures themselves, using enzymatic functions in their polymerase complexes. interestingly, rsv rnas have capstructures that contain a -methyl guanosine; however, these caps are devoid of '-o-methylation [ ] . both methylations are part of the canonical cap-structures on cellular mrnas, but why this is necessary was actually unknown. a more recent report in which covs and their cap-structures were studied indicated that the latter viruses make sure to add '-o methylation to their capstructures using a dedicated viral enzyme called nsp . this turned out to be important to avoid recognition by the mda sensor and subsequent triggering of innate immune responses [ , ] . rsv apparently does not need this '-o-methylation on its caps, and this may be explained by the observation that this virus is able to sequester mda (and innate immune adapter mavs) into its inclusion bodies (the rsv replication headquarters as discussed above) using association with its n protein, to avoid mda -dependent recognition of viral rna species and subsequent innate immune response [ ] . yet another activity provides additional means of avoiding recognition, and that is viral endoribonuclease activity. covs encode endonuclease activity in one of their non-structural proteins, and recent reports indicated that this is instrumental to avoid recognition by the mda , protein kinase r (pkr), and oas/rnase l machineries [ , ] . the latter systems recognize and destroy foreign rna in the cytosol independently of the rig-i-like sensors to remove microbial products. though it may be counter-intuitive for an rna virus to express an rnase, the virus apparently destroys its own rna at certain locations or in certain stages of the infection to avoid the triggering of the rna sensing and virus-destroying machineries. influenza also encodes one or more endoribonucleases, the primary one in the pa protein, which is part of the viral polymerase complex together with the pb- and pb- subunits. the pa endonuclease is responsible for cleaving the host mrnas for cap-snatching during transcription of the influenza rna [ , ] , another mechanism of innate immune evasion that was discussed above. additionally, many influenza strains express shorter forms of this protein encoded by the same gene, overlapping with pa at the n-terminal region, but with an alternative or truncated c-terminal region, added through a ribosomal frame shift or by natural truncation, respec-tively [ ] . these alternative products of the pa gene from segment of the influenza genome are called pa-x or paxdeltac , which were discovered recently to also have an endonuclease activity. these were shown to have a role in innate immune evasion, although the truncated paxdeltac seems to have very low endonuclease activity [ ] . the immune modulation by these alternative pa proteins is thought to be achieved by stimulating host shut-off, another innate immune evasion strategy further discussed below, whereby host cell mrnas are destroyed to suppress the expression of host proteins, including those involved in the activation of the innate antiviral state. interestingly though, pa-x was shown to cleave dsrna quite efficiently [ ] , which may not be very relevant for host shut-off, as the cell does not really produce dsrna. whether pa-x also degrades viral dsrna species to prevent recognition by cytosolic rna sensors is not entirely clear, but mutant viruses in which this pa-x protein was expressed in significantly lower amounts elicited higher levels of innate immune response; for example, ifn-beta production was much higher in these infections [ ] . this indeed suggests that pa-x, besides having a role in the degradation of cellular mrnas, may also degrade viral rna to prevent recognition by innate immune sensors and activation of innate immune responses, similar to what was shown for the covs. to my knowledge, an endoribonuclease has not been identified in the rsv genome, so this virus may use alternative innate immune evasion strategies, as discussed elsewhere in this review. the same counts for the rhinoviruses. besides the replication organelles, the viral ′ end rna capping/protection mechanisms, and the viral endonucleases, other ways of shielding rna from innate immune sensors or protecting it from degradation are exploited by respiratory viruses. influenza non-structural protein ns , for which many different innate immune evasion strategies have been described, binds and sequesters viral rna to protect it from being sensed by rig-i, and this also protects from the activation of pkr and oas/rnase l-mediated viral rna degradation [ ] [ ] [ ] . recent data hint at the importance of protecting the ′ ends of viral rnas as well, besides the ′ ends, as it was shown that tut and tut , cellular terminal uridylyltransferases, can add one or uridines to the ′ ends of polyadenylated influenza mrnas, as well as rnas of several other viruses, to target these rnas for degradation by cellular machineries [ ] [ ] [ ] . additionally, a recent report indicated that cytosolic coronaviral mrnas are targeted by the cellular nonsense-mediated decay pathway, a pathway that detects aberrant translation termination doi: . / features such as premature termination codons in mrna, resulting in the degradation of these mrnas [ ] . in the case of cov, the viral n protein plays a role in counteracting this latter effect [ ] , presumably by packaging the viral rnas, thereby protecting them from degradation. all these data suggest that viruses likely evolved escape mechanisms to avoid all these different cellular mechanisms for rna degradation to be able to set-up a productive infection in this hostile environment, however, the details of several of these mechanisms for the respiratory viruses discussed here are still unknown. host shut-off general host shut-off, that is viruses halting cellular protein expression, is an effective way to actively suppress all cellular innate immune responses against the virus, and simultaneously provide the virus with the full capacity of the cellular translation machinery for their own use. besides using viral endoribonucleases pa-x and derivatives to attack cellular mrnas, as has been briefly discussed for influenza viruses above, the viral polymerase complex and the viral "immune evasion" ns each also contribute importantly to host shut-off during influenza infection. since the polymerase complex takes care of cap-snatching, it will leave considerable amounts of cellular mrnas without a cap, and this in fact triggers the degradation of these molecules by cellular machineries such as xrn exonuclease, diminishing the general cellular mrnas available for translation. additionally, the interactions of viral polymerase complex with the cellular translation machinery cause degradation of pol ii, thereby inhibiting cellular mrna production and translation [ ] . in , nemerof et al. discovered the role of influenza encoded ns in host shut-off [ ] . ns interacts with an essential component of the ′ end processing machinery of cellular pre-mrnas, cpsf , whereby ′-end cleavage and polyadenylation of cellular mrnas is inhibited, thereby contributing to host shut-off. in the past decades, details of the molecular mechanism in which ns influences host shut-off have been investigated, and it is also clear that these mechanisms can be strain-specific [ , ] . like influenza viruses, covs such as sars-cov and mers-cov also use a combination of ways to achieve host shut-off both at the transcriptional and the translational levels. nsp , the most ′-terminal subunit of the replicate polyprotein of these viruses, was shown to cause host shutoff by binding to cellular factors of the translation machin-ery thereby preventing translation of host mrnas. sars-cov nsp binds the s subunit of ribosomes to halt translation [ ] [ ] [ ] [ ] , however, for the mers-cov encoded nsp the mechanism of halting translation of cellular mrna seems a bit different [ ] . one of the differences is that mers-cov encoded nsp distinguishes between cellular mrnas produced in the nucleus, and viral mrnas in the cytosol, and the translation of the latter is not inhibited by mers-cov nsp . in this way, specificity towards disrupting cellular mrna translation is achieved [ ] . this is different from sars-cov nsp , which inhibits all mrna translations. additionally, the nsp protein of both viruses causes host mrna degradation, however, not through intrinsic endoribonuclease activity of nsp itself but by activating the cellular mrna degradation machinery and its exonuclease xrn [ , , , ] . rhinoviruses, like poliovirus and other enteroviruses, cleave translation initiation factor elf g to shut down cap-dependent translation of cellular mrnas. this does not interfere with the translation of viral mrnas since these viruses depend on internal ribosomal entry site-mediated translation (see above). the a protease of these viruses is responsible for this, by directly cleaving this factor [ , ] . recent work indicated that interaction of rhinovirus a encoded a protease with elf e, another subunit of the cellular translation initiation complex, is required for the cleavage of elf g during infection [ ] . finally, for rsv little is known about possible host shut-off mechanisms. a report by bruce et al. [ ] suggested that rsv specifically targets mrna encoding surfactant protein a, an innate immune factor with an important role in the epithelial tissue of the lung, which directly binds to virus particles to cause their destruction by host defense mechanisms. during rsv infection, surfactant protein a mrna translation efficiency seems inhibited, however, the mechanism for this effect has not been elucidated to date. an indirect way for viruses to manipulate host mrna expression besides the classical host shut-off mechanisms discussed for the other respiratory viruses above, may be the induction of stress granules. in these structures, cellular mrnas are accumulated upon induction of cellular stress responses that lead to inhibition of cellular translation. rsv, for example, seems to induce stress granules and this benefits its replication, as will be discussed in the next section. stress granules are structures in which, upon stress responses such as resulting from virus infections, the cell concentrates mrnas that are produced but can no longer j innate immun ; : - doi: . / be translated. the triggering of pkr, a viral rna sensor, for example, causes phosphorylation of the eif alpha translation factor, which halts cellular translation thereby also affecting viral translation. the accumulation of untranslated mrnas and stalled translation and pre-initiation complexes trigger the formation of stress granules. recent insights suggest that stress granules may form a platform for innate immune responses, since the accumulation of (viral) rna species there provides a pool of substrates for cellular sensors such as rig-i and mda [ ] [ ] [ ] . indeed, these sensors have been shown to be recruited to stress granules, supporting this view [ ] [ ] [ ] . in the last decade, it has become clear that many viruses manipulate stress granule formation to benefit their replication, for example, rsv, as was also mentioned above. in the later stages of rsv infection in epithelial cells, stress granules are formed, and if expression of g bp, a factor that is essential for stress granule formation, is knocked-down, replication of rsv is inhibited, suggesting a beneficial role for stress granules [ ] . a subsequent report showed that pkr activation is required for the induction of stress granules by rsv, however, this is dispensable for viral replication [ ] . several other reports also show how rsv counteracts the formation of stress granules [ ] , suggesting a negative effect of stress granule formation on rsv infection. up till today, it therefore remains unclear what the role of stress granules during rsv infection is exactly. covs also manipulate stress granule formation. in collaboration with the group of frank van kuppeveld, our lab showed that mers-cov encoded a protein (translated from orf in the virus) impedes dsrna-mediated pkr activation, thereby preventing stress granule formation [ ] . protein a binds viral dsrna, which is essential for its antagonistic function in pkr activation and stress granule formation, suggesting that a prevents recognition of viral rna by pkr. recombinant mers-cov in which orf (encoding a and b proteins) was removed, however, still suppressed stress granule formation in vero cells, suggesting that a's activity is not the only way in which the virus inhibits stress granule formation [ ] . indeed, cov nsp with its host-shut-off activities (see above) is a likely candidate viral protein that could play a role. a later report by nakagawa et al. [ ] however showed that the orf mers-cov mutant virus did induce stress granules in another cell line (hela/ cd ), and also a virus mutant in which a alone was removed was not able to suppress sg formation in these cells. this suggests that the activity of a, and possibly other stress granule-inhibiting mers-cov proteins, may differ per cell line, or that cell lines differ in the activity of their antiviral pathways. influenza virus infection is also negatively influenced by the triggers that induce stress granule formation [ , ] . indeed, this virus also inhibits the formation of stress granules, and influenza virus encoded ns seems to play a major role in this [ ] . this is not surprising given the role of ns in host-shut-off as well as in protecting the viral rna from recognition by rna sensors in the cell (see above), thereby preventing the activation of pkr and concomitant eif alpha phosphorylation and stress granule formation. interestingly, this innate immune evasion activity of ns is counteracted by cellular protein nf , which partly prevents the suppression of pkr triggered stress granule formation by ns by binding both pkr and ns [ ] . besides ns , the influenza nucleoprotein np and polymerase subunit pa-x help to prevent stress granule formation, due to their rna protection and host-shut off functions, respectively [ ] . for rhinoviruses, nothing is known about their capacity to manipulate stress granule formation, however, for other picornaviruses the a and l proteases have recently been shown to interfere by cleaving stress granule factors such as g bp and g bp [ ] [ ] [ ] [ ] . a recent report showed that binding of eif gi translation factor to stress granule-inducing protein g bp is essential for antiviral stress granule formation, and this interaction is disrupted by the a or l proteases of picornaviruses [ ] . given these data, it may be likely that rhinoviruses also affect stress granule formation using their proteases, which is further supported by data described in the next paragraph, but this needs to be investigated. most, if not all, positive strand rna viruses encode proteases, which they generally use to cleave their viral polyproteins into functional subunits during the viral life cycle. it has lately become apparent that these proteases often have side-functions that support immune evasion by these viruses. among the viruses discussed here, rhinoviruses and covs carry a positive strand rna genome, and each of the members of these virus families encode at least proteases. rhinoviruses use their a papain-like protease (plpro) to effectively disable cap-dependent translation by cleaving eif g to induce host-shut off. this may well also prevent stress granule formation, however as mentioned, this has not been investigated for rhinoviruses yet. addition-doi: . / ally, rhinovirus a protease cleaves nuclear pore proteins nup and nup , while c protease seems to cleave nup [ , ] . these activities are thought to influence host immune response signaling for which cytosolnucleus communication and trafficking is essential. it recently became clear that rhinovirus a protease activity also plays a role in targeting rhinovirus c protein to the nucleus [ , ] , however, it is not clear what c protease is doing there exactly [ , ] . as discussed in the beginning of this review, the type i ifn antiviral pathway is very relevant for rna virus infections, and an essential adaptor that enables downstream signaling in this pathway is ips- (also called mavs). this factor is cleaved by both a and c proteases of rhinovirus to halt type i ifn signal transduction [ ] . rhinovirus c protease can inhibit apoptotic cell death and activation of antiviral protein complexes by cleaving cellular apoptosis factor ripk [ , ] . cov proteases also cleave cellular substrates to benefit the infection. the functions of plpro of covs in manipulating the ubiquitin regulation of the innate immune system will be discussed later. the main, or c-like, protease of covs may have side-functions in cleaving innate immune factors as it was shown for porcine covs that their main proteases cleave nemo [ , ] , however, nothing is known yet in this respect for the human respiratory covs. the ubiquitin system is essential for the correct functioning of virtually all important cellular processes. the central molecule is ubiquitin, a small amino acid protein that can be conjugated with its c-terminus to lysine residues in substrate proteins. three classes of enzymes are needed for the conjugation: activating e enzyme, conjugating e enzyme, and an e ligase. additional ubiquitins can be added to the first via one of lysines in ubiquitin itself, yielding poly-ubiquitin chains. the signal that the ubiquitin chain gives depends on the linkage type(s) of the chain. k and k -linked ubiquitin chains are best studied and are generally the cause of degradation or activation of the substrate, respectively. in antiviral innate immune signaling, ubiquitin is an important regulating factor, and isg , an interferon-induced ubiquitin-like molecule, is also an important factor in antiviral innate immunity. it is therefore not a surprise that a lot of viruses have evolved ways to manipulate the ubiquitin system and ubiquitin-like molecules such as isg , which they do in very diverse ways [ ] . after the discovery of a structural resemblance between sars-cov expressed plpro and the cellular deubiquitinase hausp/usp [ ] , it soon became clear that cov plpros had intrinsic deubiquitinating activity and could potentially deconjugate cellular (or viral) substrates to disrupt ubiquitin-mediated signaling, as well as deconjugate isg off its substrates [ ] . it is still not clear which cellular and viral factors are deconjugated by plpro during infection, but mutant mers-cov in which the deubiquitinating/de-isg ylating function of plpro was removed clearly showed increased type i ifn innate immune responses (knaap et al., unpublished results) , indicating that plpro's dub activity has an important role in the suppression of innate immunity during infection. for plpro from human common cold virus hcov-nl it was shown, although only by over-expression experiments, that it can deubiquitinate mdm , the e ligase that mediates p ubiquitination and subsequent degradation, thereby possibly inhibiting apoptosis and innate immune signaling [ ] . similarly, sars-cov plpro can deubiquitinate e ligase rchy to stimulate ubiquitination of p by this ligase, and thus also potentially inhibit apoptosis [ ] . for influenza, several different interactions with the ubiquitin system have been identified that critically influence the outcome of the infection [ ] . generally, the activation of the rig-i -mavs -irf signaling axis in type i ifn signaling, which is important for all viruses discussed in this review, is governed by ubiquitin linked through its lysine at position (forming k -linked chains). about a decade ago, influenza virus ns was shown to bind e ligase trim , thereby interfering with k -linked ubiquitination of rig-i, and therefore uniquely inhibiting innate immune signaling in the type-i ifn pathway [ ] . there has been a recent debate as to whether these chains are actually conjugated to rig-i or other factors within the cascade or whether they are free ubiquitin chains that provide a scaffold for activating the aggregation of rig-i and mavs, which in turn enables downstream signaling [ ] . influenza b virus-encoded ns additionally inhibits isg antiviral activity by binding the n-terminus of human isg (and not mouse isg ) [ ] . furthermore, and similar to what some of the cov plpro's may do (see above in this section), influenza ns was recently shown to destabilize mdm e ligase which somehow benefits the iav infection. according to the authors, this is because mdm seems to have a p -independent antiviral function which is then alleviated [ ] . this is, however, in contrast to what was mentioned for nl cov, where plpro seems to stabilize innate immune evasion by respiratory viruses mdm to also benefit infection [ ] . further research is needed to conclude whether these opposite effects indeed benefit the respective infections, or whether either of the results is incorrect. finally, influenza ns was shown to mediate the upregulation of a , a deubiquitinase with a role in the downregulation of rig-i activation, to suppress the activation of rig-i [ ] . rsv also manipulates ubiquitin-mediated signaling, mainly directed by its non-structural proteins ns and ns . quite recently it was shown that rsv ns targets trim to suppress rig-i ubiquitination, very similar to influenza's ns 's strategy [ ] . this probably corroborates the importance of trim -mediated ubiquitination in the innate immune signaling cascade. earlier reports suggested that ns of rsv can direct proteasomal degradation of signal transducer and activator of transcription (stat ) in lung epithelial cells [ , ] . stat and stat are transcription factors in the second round of innate immune signaling after binding of ifn to its receptor on the original, or surrounding cells. however, the mechanistic details of ns 's action has not become completely clear yet, although it has been claimed that ns somehow stimulates (k -linked) ubiquitination of proteins, which can be alleviated again by a com-bination of mutations in ns . these mutations when introduced into the virus prevent stat from being degraded during infection, providing possibilities for novel vaccines [ ] . although it was reported that rsv infection in cell culture and in patients causes induction of isg and that isg conjugation to proteins has an antiviral effect [ ] , it is not clear whether rsv inhibits or evades isg antiviral effects or not. for rhinoviruses, it is unclear how it interacts with the cell's ubiquitin system. while picornavirus family member foot-and-mouth disease virus leader protease was shown to have deubiquitinating activity [ ] , neither a nor c protease from rhinovirus has been implicated in ubiquitin-regulated processes to date, and no other reports hinting at manipulation of the ubiquitin system by rhinoviruses have been published to my knowledge. the data summarized and discussed above illustrate that innate immune evasion is a major function of respiratory and other rna viruses (fig. ) , which probably takes a significant volume of the genetic capacity of these viruses. this also implies that, given the restricted genetic space available to these viruses, the evasive functions must be pivotal for viruses to survive, otherwise they would likely not have evolved. since each virus employs multiple different activities to suppress immune responses, and often evolved multifunctional proteins to do so, it remains difficult to acquire a complete picture of the immune evasive arsenal of a virus and how this is balanced with symptoms and disease outcome in different cell types or situations. nevertheless, in-depth knowledge of this virus-host interaction creates important avenues for novel antiviral strategies, some of which have already been mentioned in the text above, and some more examples will be discussed in the next section. besides the major strategies for innate immune suppression by respiratory viruses discussed, several other mechanisms of innate immune evasion have been described for the respiratory viruses discussed here, and/ or members of their families, of which many may be unique to only one or of the respiratory viruses discussed here. one example is the virus-encoded macrodomain. these domains have been identified in covs (and several other non-respiratory +rna viruses) and have been shown to counteract ifn signaling with a yet unknown mechanism [ ] . they are absent in influenza virus, rhinoviruses, and rsv, and therefore has not been discussed in this review. many of these additional evasive activities have been comprehensively reviewed recently by others [ , , , , , , , . undoubtedly, yet other evasive activities are additionally still to be identified. the newly discovered aspects of human innate immunity, such as trained innate immunity and the integrated innate/adaptive cell types, as well as the links between innate immune responses and cellular metabolic changes, as discussed in the first part of this review, due to their recent discovery have not yet been studied extensively in the context of possible viral evasion strategies. this direction of course forms an obvious avenue for new research that should be undertaken, since it is likely that viruses also target these newly discovered mechanisms. an important question is how exactly the viral innate immune evasive functions of respiratory viruses influence disease outcome and ultimate immune responses. it is noticeable that many of the viruses discussed here do not elicit a long-lasting immune protection after infection, and indeed rhino, corona, and rsv can re-infect individuals sometime after earlier infection, again causing symptoms (reviewed in [ , ] ), which is in sharp contrast to several other childhood-associated viral infections, where lifelong protection is achieved after generally experiencing only one episode of disease. it may well be that, besides their strong genetic variation, the innate immune evasive activities of the mentioned respiratory viruses play a role in this lack of eliciting protective immunity [ ] , and to possibly improve our options for effective antiviral strategies, it seems pivotal to further investigate this. for influenza the situation is slightly different, since this virus elicits protective immunity [ ] ; however, its genetic drift and shift causes new strains that are not, or inefficiently, recognized by existing influenza immunity which generally means that individuals will experience multiple influenza infections in the course of their lives. besides contributing to the problem of limited immunological protection, viral innate immune evasion may also contribute to often reported immune over-reactions associated with respiratory infections, including cytokine storms, damaging inflammation, and other severe complications [ ] [ ] [ ] [ ] . some studies on sars-cov and mers-cov infections in patients suggest that the delayed innate immune response that is the result of temporary suppression by innate immune evasion, contributes to an exacerbated response [ ] . how this works exactly is unclear to date. respiratory pathogens are associated with asthma. the exacerbation of asthma symptoms upon infection with rhinoviruses have been associated with defective types i and iii ifn responses [ , ] . in lung tissue, antiviral defenses may be further compromised by other mechanisms that impair these defenses such as th cytokines il- and il- [ ] , and possibly high affinity ige receptor expression and crosslinking [ ] . however, suppressed antiviral innate immune response during virus-induced asthma exacerbations is likely also influenced by the innate immune evasive functions of respiratory viruses, as these activities contribute to more severe pathogenicity and slower virus clearance, likely stimulating asthmatic manifestations [ , ] . understanding (innate) immune evasion by respiratory viruses could, therefore, shed light on the possibilities for the prevention and cure of asthmatic complications associated with respiratory infections. for particularly rsv and influenza, efforts to develop effective and long-lasting vaccines and antivirals have been relatively unsuccessful for decades [ , ] link to the viruses' capability to manipulate the host's immune responses, thereby breaking through pre-existing natural or vaccine inflicted immunity. detailed knowledge on the mechanisms whereby these viruses deal with and modify the immune responses they encounter is therefore pivotal to genuinely advance this field. some studies have focused on mapping the interactions of respiratory viruses, and their immune evasion proteins, with their host cells to find promising cell-based drug targets [ , ] , and this could be an effective way to developing novel vaccines and antiviral drugs. effective rhinovirus and cov antivirals and vaccines have also been lacking, and for these viruses causing common colds, an additional hurdle is the cost-effectiveness of these medicines. the general symptoms of these virus infections are mild, and before the public is willing to buy specific and effective medicines against these infections, these should be relatively cheap. given that these viruses are generally difficult to control due to factors of viral immune modulation, the more knowledge we gain on the link between virus infection and (innate) immune responses in the host, the higher the chance that we may be able to develop successful and cost-effective remedies. although the impact of a common cold may not be high, the fact that these infections are extremely widespread in the human population makes controlling these viruses a desirable goal. the cost-effectiveness balance is also a factor for the covs causing severe infections, that is, sars-cov and mers-cov, since infections with these viruses are either not being reported any more (sars-cov), or are quite localized and relatively scarce (mers-cov). still, since % of mers-cov-infected patients succumb to the infection, and the lingering threat of larger outbreaks is felt as long as the virus replicates in humans, who has been recommending the development of specific vaccines for both viruses. recently, a number of efforts for mers-cov vaccines have reached the stage of clinical trials, and having these vaccines "on the shelf" will at least ease societal concerns of dangerous outbreaks with this lethal virus [ ] . a more or less obvious way of exploiting a virus' innate immune evasive functions for the development of new vaccines is to remove one or more of these from the virus using reverse genetic technology. in this way, the 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advances in the treatment of virus-induced asthma. expert rev respir med challenges in developing a cross-serotype rhinovirus vaccine identification of common biological pathways and drug targets across multiple respiratory viruses based on human host gene expression analysis viral immune modulators perturb the human molecular network by common and unique strategies middle east respiratory syndrome vaccine candidates: cautious optimism. viruses influenza virus: a master tactician in innate immune evasion and novel therapeutic interventions. front immunol crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants the author wishes to thank prof. pieter hiemstra (lumc, leiden, the netherlands) for critically reading the manuscript and for helpful comments and discussions. the author declares no conflicts of interest. the author was funded by a primary institutional appointment as associate professor at leiden university medical center, leiden, the netherlands. key: cord- -yd idp authors: zhang, chengfei; yan, yan; he, hongwang; wang, li; zhang, na; zhang, jie; huang, hongjun; wu, nannan; ren, hua; qian, min; liu, mingyao; du, bing title: ifn-stimulated p y( ) protects mice from viral infection by suppressing the camp/epac signaling pathway date: - - journal: j mol cell biol doi: . /jmcb/mjy sha: doc_id: cord_uid: yd idp among the most important sensors of extracellular danger signals, purinergic receptors have been demonstrated to play crucial roles in host defense against infection. however, the function of p receptors in viral infection has been little explored. here we demonstrated that p y( ) and its ligand adp play an important role in protecting hosts from viral infections. first, we demonstrate that p y( ), as a typical interferon-stimulated gene, is induced together with extracellular adp during viral infection. most importantly, extracellular adp restricts the replication of different kinds of viruses, including vesicular stomatitis virus, newcastle disease virus, herpes simplex virus , and murine leukemia virus. this kind of protection is dependent on p y( ) but not p y( ) or p y( ), which are also considered as receptors for adp. furthermore, cyclic adenosine monophosphate and epac are downregulated by extracellular adp through the p y( )-coupled gi alpha subunit. accordingly, inhibition or deletion of epac significantly eliminates adp/p y( )-mediated antiviral activities. taken together, our results show that p y( ) and adp play pivotal roles in the clearance of invaded virus and have the potential as antiviral targets. upon viral infection, the host can sense the virus through the recognition of pathogen-associated molecular patterns by pattern recognition receptors, especially toll-like receptors (tlrs) and rig-i-like receptors (rlrs) (kawai and akira, ; chen et al., ) . when activated by viral nucleic acids, tlrs and rlrs will activate the tbk -irf axis, leading to type i interferon (ifn) expression and secretion (garcia-sastre and biron, ; he et al., ) . then, the released ifns induce hundreds of interferon-stimulated genes (isgs) via the janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway to clear the invaded virus (platanias, ; garcia-sastre and biron, ; shu et al., ) . among them, the double-stranded rna-dependent protein kinase pkr, the myxovirus resistance gene mx, the ′- ′ oligoadenylate synthetases, and interferon induced transmembrane protein (ifitm ) have been shown to disrupt viral infection in various manners rafique et al., ; liu et al., ) . more than genes have been characterized as isgs. however, their roles and mechanisms in fighting against multiple distinct classes of viruses remain largely unexplored. purinergic signaling and purinergic receptors are important in both innate and adaptive immune responses. purinergic signaling was first reported in and, after several decades of study, many of the biologic effects of atp, utp, adp, udp, and adenosine signaling were discovered (eltzschig et al., ; idzko et al., ) . in tissue injury or pathogen infection, nucleotides can be released from stressed cells as a danger signal, alerting the cells by interaction with purinergic receptors in an autocrine or paracrine manner . nineteen different purinergic receptor subtypes have been identified, which can be classified into p and p receptors. four p receptor subtypes can be activated by adenosine. p receptors can be further divided into p y receptors (p yrs) and p x receptors (p xrs) based on their structures and distinct signaltransduction mechanisms. p yrs are g-protein-coupled receptors for atp, adp, utp, and udp, while p xrs are nucleotide-gated ion channels and can be activated by atp (ralevic and burnstock, ; junger, ; idzko et al., ) . many studies revealed crucial roles for p receptors during inflammatory and infectious diseases. for example, atp-triggered p x activation is crucial for the activation of nlrp inflammasome and the subsequent release of interleukin- β (di virgilio et al., ) . atp/utp receptor p y signaling has been identified as a 'find-me' signal to recruit motile phagocytes, thereby promoting clearance of apoptotic cells or bacteria (elliott et al., ; chen et al., ) . our previous study demonstrated that the adp-mediated p y /p y signal pathway protects the host defense against bacterial infection via erk signaling (zhang et al., ) . however, whether adp-(or its receptors-) mediated immune regulation is also involved in viral infection remains unclear. p y , p y , and p y are all receptors for adp. p y binds to gtp-binding protein subunit alpha q (gq), which can evoke calcium signaling. while p y and p y interact with gtpbinding protein subunit alpha i (gi), which can suppress the activity of adenylate cyclase (adcy) and the production of cyclic adenosine monophosphate (camp) (zhang et al., ; shinozaki et al., ) . in this study, we demonstrate that p y , as an isg, and its ligand, adp, is released from infected cells as a danger signal during viral infection, which restricts the replication of both dna and rna viruses. adp/p y -mediated protection against viral infection operates by suppressing the expression of exchange protein activated by camp (epac ), which is an alternative key intracellular sensor for camp. therefore, we demonstrated that adp/p y restricts viral infection through suppressing the camp/epac signaling pathway, which has potential therapeutic significance in the prevention and control of viral diseases. the expression of p y is increased by ifn through jak/stat pathway to identify p y family members potentially involved in antiviral immune responses, we treated mouse bone marrowderived macrophages (bmdms) with ifn-α, ifn-β, and ifn-γ to appraise the rna expression of all p y family members by quantitative real-time pcr (q-pcr). as shown in figure a , the rna expression of p y was upregulated obviously by all three ifns. the protein level of p y also upregulated by ifn-β ( figure b) . besides, when blocked ifn receptor (ifnar) with the anti-ifnar antibody, the upregulated expression of p y was decreased ( figure c ), suggesting that p y upregulation is dependent on signals downstream of ifn. the jak/stat pathway is crucial for ifn-induced cellular antiviral signaling. to determine whether ifn-induced p y upregulation via the jak/stat pathway, we pretreated mouse peritoneal macrophages (pems) with ruxolitinib (a jak inhibitor) and fludarabine (a stat inhibitor) and found that ifnβ-induced p y expression was blocked by both inhibitors ( figure d and e). inhibition of stat also reduced vsv-induced p y expression ( figure f ). in addition, knockout of tlr to block tlr -mediated activation of ifn also suppressed poly(i:c)-induced p y expression ( figure g ). taken together, these data demonstrated that p y has the typical characteristics of isgs. as an endogenous ligand to purinergic receptors, adp can be released from injured cells to activate p y , p y , and p y receptors. to investigate the potential role of adp as a danger signal in viral infection, the release of endogenous adp in viralinfected cells was measured. as shown in figure a and b, extracellular adp in vsv-infected cell supernatant was clearly increased in a time-dependent manner. besides, ndv-infected bmdms and hsv- -infected hek t cells also release large amounts of adp ( figure c and supplementary figure s a ), suggesting adp can be released as a danger signal during viral infection. to investigate the biological significance of adp, we pretreated the cells with adp before vsv infection. to our surprise, the rna replication of vsv in adp-treated raw . cells was reduced significantly in a time-( figure d ) and concentration-( figure e ) dependent manner. accordingly, the viability of vsv-infected raw . cells was increased dramatically by adp in a concentration-dependent manner while have little influence on uninfected cells ( figure f and supplementary figure s b ), indicating that extracellular adp protects cells from vsv infection. to confirm whether adp has broad-spectrum antiviral activities, raw . cells and bmdms were pretreated with adp before vsv, ndv, and hsv- infection. as shown in figure g -j and supplementary figure s c , the rna expression of vsv, ndv, hsv- and the titers of vsv were all strikingly decreased by adp. similar results were observed in hek t cells, where cell cytopathic effects were induced by vsv and hsv- , as determined using a crystal violet staining assay (supplementary figure s d and e). we further infected hek t and hela cells with vsv-gfp and mlv-gfp and found that the number of gfppositive cells was decreased in adp-treated cells, indicating that vsv-gfp and mlv-gfp were restricted by adp (supplementary figure s f-i). to rule out the possibility that adp degradation products mediate the suppression of viral replication, we treated cells with non-hydrolyzable adp analog adp-β-s. as shown in supplementary figure s j , adp-β-s has a similar role as adp in restricting vsv replication. taken together, our data suggested that extracellular adp has broad-spectrum antiviral activities, which have great potential in protecting hosts from a viral infection. to explore the key receptors involved in adp-mediated antiviral activities, we detected the expression of p y , p y , and p y after vsv infection. as shown in figure a , only the expression of p y was increased significantly, whereas the expression of p y and p y was little changed in vsv-infected raw . cells. similar data were observed in poly(i:c)-treated bmdms ( figure b ). we then used mrs and mrs to inhibit p y and p y , respectively, to verify whether p y and p y contributed to adp-mediated antiviral activities. we found that adp-mediated antiviral activities were little influenced by mrs but significantly decreased by mrs as shown in figure c -f, and both of mrs and mrs have little influence on cell viability (supplementary figure s a and b). consistently, when infected wild-type, p y -, p y -, and p y deficient bmdms with vsv, the vsv rna replication was increased only in p y -deficient bmdms, not in p y -or p y -knockout bmdms ( figure g ). meanwhile, adp-reduced vsv rna replication was compensated in p y -deficient pems ( figure h) . furthermore, the protein expression of vesicular stomatitis virus glycoprotein (vsv-g) was significantly increased in p y -deficient pems and overexpression of p y in hela cells significantly repressed vsv replication ( figure i ; supplementary figure ifn signal pathway increases p y expression. (a) mouse bmdms were treated with ifn-α, ifn-β, or ifn-γ ( ng/ml) for h, and mrna expression of p y family was detected by q-pcr. (b) mouse pems were treated with ifn-β ( ng/ml) for or h, and the expression of p y protein was detected by western blotting. (c) mouse bmdms were pretreated or not with anti-ifn-α/β receptor igg ( μg/ml) for h before being treated with ifn-β ( ng/ml) for h, and the expression of p y mrna was detected by q-pcr. (d) mouse pems were pretreated or not with ruxolitinib ( μg/ml) for h and then stimulated with ifn-β ( ng/ml) for h. the expression of isg and p y mrna was detected by q-pcr. (e) mouse pems were pretreated with or without fludarabine ( μg/ml) for min and then stimulated with ifn-β ( ng/ml) for h. the expression of isg and p y mrna was detected by q-pcr. (f) mouse pems were pretreated or not with fludarabine ( μg/ml) for min and then infected with vsv (moi = ) for h. the expression of p y mrna was detected by q-pcr. (g) tlr +/+ and tlr −/− mouse pems were treated with poly(i:c) ( μg/ml) for h. the expression of ifn-β, isg , and p y mrna was detected by q-pcr. data are shown as mean ± sd. **p < . ; ***p < . . figure s c and d). taken together, our data suggested that p y deficiency facilitates vsv infection and adp-mediated antiviral activities primarily through the p y receptor. to further confirm the in vivo antiviral activities of adp/p y , we challenged wild-type and p y -deficient mice with a lethal and then infected with vsv (moi = ) for h. vsv titers in supernatants were measured by plaque assay. (i and j) mouse bmdms were treated with adp ( μm) for h and then infected with ndv (moi = . ) and hsv- (moi = . ) for h virus rna replicates were detected by q-pcr. data are shown as mean ± sd. *p < . ; **p < . ; ***p < . . dose of vsv. as shown in figure a , the survival of adp-treated wild-type mice was increased obviously, but there was little change in that of p y -deficient mice. p y -deficient mice were more susceptible to vsv infection. on the other hand, if we intraperitoneally injected mice with adp to activate p y , vsv distributions in the liver, spleen, and lung were all significantly decreased ( figure b ). similar data were observed during hsv- infection ( figure c ). besides, more vsv rna replicates were found in p y -deficient mice liver ( figure d ). as a neurotropic virus, vsv can invade the nervous system and is transmitted directly via the transcutaneous or transmucosal route. thus, to mimic the natural infection of vsv, we treated mice with adp through nasal dripping and then intranasally infected with vsv. as shown in figure e and f, vsv rna replication and vsv-g in olfactory bulbs were both significantly reduced by adp. taken together, these data demonstrated that adp/p y plays an important role in protecting mice from vsv infection, suggesting that adp/p y has potential as a novel antiviral drug target. adp/p y restricts viral replication by inhibiting camp signaling type i ifn plays pivotal roles in fighting against the invaded virus, so we tested whether it was involved in adp/p y mediated antiviral activities. however, when we treated bmdms with adp, the rna expression of ifn-α and ifn-β was little changed (supplementary figure s a and b) . p y signaling can suppress the activity of adcy and the production of camp. to confirm that, raw . cells were pretreated or not with mrs before being treated with adp. as shown in figure a , adp significantly suppressed the production of camp, while p y antagonist mrs rescued this phenomenon. adp ( mg/kg) for h and then intraperitoneally injected with vsv ( × pfu/g). mice were executed for days and mouse survival was recorded for days. (b) six-week-old mice were intraperitoneally treated with adp ( mg/kg) for h and then intraperitoneally injected with vsv ( × pfu/g) for h. vsv rna replicates in organs were detected by q-pcr. (c) six-week-old mice were intraperitoneally treated with adp ( mg/kg) for h and then intraperitoneally injected with hsv- ( × pfu/g) for h. hsv- rna replicates in livers were detected by q-pcr. (d) eight-week-old p y +/+ or p y −/− mice were intraperitoneally injected with vsv ( × pfu/g) for h and vsv rna replicates in livers were detected by q-pcr. (e) six-week-old mice were treated with adp ( mg/kg) through nasal dropping for h and then intranasally infected with vsv ( × pfu/g) for h. vsv rna replicates in olfactory tissues were detected by q-pcr. (f) six-week-old mice were treated as in e, and vsv invaded in olfactory tissues were detected by immunofluorescence. scale bar, μm. data are shown as mean ± sd. **p < . ; ***p < . . however, to our surprise, vsv infection caused an obvious increase in cellular camp levels ( figure b) . besides, p y -deficient pems showed higher cellular camp levels than wild-type pems during vsv infection ( figure c ). so, we examined the role of camp in adp/p y -mediated antiviral activities. we first found that adcy activator forskolin blocked adp-mediated the cells were then infected with vsv (moi = . ) for h. vsv rna replicates were detected by q-pcr and vsv titers in supernatants were measured by plaque assay. (i) raw . cells were pretreated or not with kh ( μm) for h before being treated with adp ( μm) for h. the cells were then infected with hsv- (moi = . ) for h, and hsv- rna replicates were detected by q-pcr. data are shown as mean ± sd. *p < . ; **p < . ; ***p < . ; ns, not significant. antiviral activity completely ( figure d and e). furthermore, camp treatment promoted viral replication obviously and also rescued the adp-reduced cell cytopathic effects and vsv replication ( figure f and g; supplementary figure s c and d). consistent with this, kh (a selective inhibitor of adcy) treatment significantly inhibited cell cytopathic effects and vsv replication (supplementary figure s e and f). adp-mediated antiviral activities were also significantly blocked by kh ( figure h and i). the cell viability assays revealed that these phenomena were not caused by the toxicity of forskolin, camp, and kh (supplementary figure s g-i) . taken together, these results suggested that camp plays a key role in adp/p y -mediated antiviral activities. adp/p y restricts viral replication in an epac -dependent manner when intracellular camp is increased by upstream signaling, epac and protein kinase a (pka) could be activated as classical sensors for camp. thus, we treated hela cells with esi- (epac / inhibitor), hjc (epac selective inhibitor), and h (pka inhibitor) at a safety concentration (supplementary figure s a -c). as shown in supplementary figure s d , the replication of vsv is dramatically suppressed by both esi- and h , but not hjc , suggesting that epac and pka may be involved in adp/p y -mediated restriction to vsv. however, when we inhibited pka with h , the replication of vsv ( figure a ) and hsv- ( figure b ) in hela cells are still suppressed by adp, whereas adp-reduced vsv, hsv- , and ndv replication was significantly blocked by esi- , but not hjc ( figure c -f and supplementary figure s e) . besides, the adpreduced vsv-g expression was also blocked by esi- ( figure g) . these data show that epac is crucial in adp/p y mediated antiviral activities. in addition, we generated an epac knockout cell line using the crispr/cas system ( figure h ). as shown in figure i and j, when epac is deficient, adp-mediated suppression of vsv rna replication and vsv-g expression were eliminated. taken together, these results demonstrated that adp/p y -mediated antiviral activities are dependent on epac . inhibiting epac with esi- in raw . cells significantly suppressed vsv and hsv- replication, and epac knockout also inhibited hsv- replication, demonstrating that either inhibition or deletion of epac has broad-spectrum antiviral activities (supplementary figure s f-h) . to further confirm the key role of epac in viral infection, we detected the expression of epac in viral-infected cells. as shown in figure a , when infected raw . cells with vsv, ndv, and hsv- , rna expression of epac was increased significantly. deficiency in p y maintained higher expression of epac in pems after infection with vsv ( figure b ). in addition, the expression of epac protein also upregulated after virus infection as shown in figure c and supplementary figure s a , b. however, to our surprise, the increased expression of epac induced by vsv was reduced by adp in a dose-dependent manner (supplementary figure s c and d) . to exclude that the downregulation of epac may be due to the attenuation of vsv replication by adp, cell lines were only treated with adp. as shown in figure d -f and supplementary figure s e , the endogenous rna expression of epac was directly inhibited by adp. the protein expression of epac was also inhibited by adp ( figure g and h). taken together, these data demonstrated that the virusincreased epac expression is suppressed by adp, which restricts the viral infection in host cells. p y and p y , as membrane receptors for adp, are both gi-coupled receptors that inhibit the activity of adcy and reduce the production of camp (vitiello et al., ) . p y is highly expressed in platelets and plays a central role in platelet activation, which has become a drug target for atherothrombotic diseases, whereas p y is mainly expressed in the bone marrow, spleen, liver, and brain (savi et al., ; vitiello et al., ) . previous studies have shown that p y and p y are both involved in the development of pain behavior during nerve injury and the expression of pro-inflammatory cytokines (kobayashi et al., ; liu et al., ) . however, the role of p y and p y receptors in viral infection remains unclear. here we demonstrate that p y is upregulated by type i ifn (p y is little changed) and its endogenous ligand, adp, is released as danger signals from virusinfected cells. consequently, extracellular adp-activated p y protects the host against viral infection in a camp-dependent manner. thus, our study reveals an important role of adp/p y in fighting against viral infection, which demonstrates the potential of purinergic receptors as novel antiviral targets. upon viral infection, the host initiates a series of signaling cascades to induce the production of type i ifn, which results in the induction of isgs to exclude invaded virus (ooi et al., ; zhan et al., ) . previous studies reported that isgs could further promote the production of type i ifn to eliminate the virus, such as isg and ifit (lu et al., ; liu et al., ) . therefore, we tested whether adp/p y influences the expression of type i ifn. our results show that adp enhanced ifn expression by only a little. because adp clearly inhibited virus replication, we hypothesized that there must be another way for adp/p y to restrict viral infection. thus, the classic gi-coupled signaling pathway came to mind. interestingly, we found that adp-inhibited vsv replication and cell cytopathic effects were rescued by both adcy activator forskolin and camp. moreover, similar to adp, the adcy inhibitor kh also inhibited vsv replication, suggesting that camp plays a key role in adp/p y mediated antiviral activities. as the first identified second messenger, camp plays a crucial role in microbial pathogenesis (mcdonough and rodriguez, ) . pka and epac are the two classical sensors for intracellular camp, which are essential for many biological processes (gloerich and bos, ) . pka plays important roles in metabolism, cell cycle, cell migration, differentiation, and apoptosis (yan et al., ) . previous studies have reported that pka can inhibit ifn induction of the jak/stat pathway (david et al., ) . recently, a new study reported that pka catalytic (pkac) figure adp-mediated antiviral activities are dependent on epac . (a) hela cells were treated or not with h ( μm) for h before being treated with adp ( μm) for h and then infected with vsv (moi = . ) for h. vsv rna replicates were detected by q-pcr. (b) hela cells were treated or not with h ( μm) for h before being treated with adp ( μm) for h and then infected with hsv- (moi = . ) for h. hsv- rna replicates were detected by q-pcr. (c) hela cells were pretreated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with vsv (moi = . ) for h and vsv rna replicates were detected by q-pcr. (d and e) hela cells were pretreated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with hsv- (moi = . ) or ndv (moi = . ) for h and viral rna replicates were detected by q-pcr. (f) raw . cells were treated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with hsv- (moi = . ) for h, and hsv- rna replicates were detected by q-pcr. (g) hela cells were pretreated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with vsv (moi = . ) for h and vsv-g protein levels were detected by western blotting. (h) epac +/+ and epac −/− hela cells were exposed to vsv (moi = . ) for h, and the expression of epac was detected by western blotting. (i) epac +/+ and epac −/− hela cells were treated with adp ( μm) for h and then infected with vsv (moi = . ) for h. vsv rna replicates were detected by q-pcr. (j) epac +/+ and epac −/− hela cells were treated with adp ( μm) for h and infected with vsv (moi = . ) for h. vsv-g protein levels were detected by western blotting. data are shown as mean ± sd. *p < . ; **p < . ; ***p < . ; ns, not significant. subunits α and β could negatively regulate rna virus-triggered signaling and ultimately attenuate the innate antiviral response (yan et al., ) . epac has two isoforms, epac and epac . the camp/epac signal pathway is important for controlling various cellular functions, including cell adhesion, exocytosis, and microtubule dynamics. previous studies have reported that epac is a potential target for the treatment of fatal rickettsioses and that the silencing of epac gene expression renders cells resistant to mers-cov infection (gong et al., ; tao et al., ) . in our study, we found that both pka and epac inhibitors suppress viral replication. however, only epac inhibition could block adp/p y -mediated antiviral activities. also, we found that the epac inhibitor could not block adp/p y -mediated antiviral activities. these findings suggest that adp/p y -mediated antiviral activities are mainly dependent on epac . knockout of epac in hela cells showed similar results and further proved this point. taken together, our results showed that, upon viral infection, the virus can trigger an upregulation of intracellular camp levels to activate epac and thereby facilitating virus replication. however, the virus also triggers adp released from these cells, which will activate p y and suppress the camp/ epac signal pathway to restrain virus replication. this phenomenon revealed a negative feedback regulation in animals during viral infection, and that activation of p y and inhibition of the camp/epac signal pathway are potential antiviral strategies. p y , p y , and p y knockout mice were described previously (zhang et al., ) . tlr knockout mice were a gift from professor yuping lai (east china normal university) and were described previously (wu et al., ) . c bl/ wild-type mice were purchased from the shanghai laboratory animal company. all of the mice were maintained in specific pathogen-free (spf) facilities at the animal center of east china normal university. all animal experiments were done following institutional guidelines. reagents adp, adp-β-s, mrs , forskolin, camp, kh , esi- , h , anti-β-actin antibody, and anti-vsv-g antibody were purchased from sigma-aldrich. ruxolitinib was from targetmol. fludarabine was obtained from calbiochem. polyinosinic-polycytidylic acid [poly(i:c)] was purchased from invivogen. adp-glo™ kinase assay kit was purchased from promega. mrs and anti-gpr (p y ) antibodies were purchased from abcam. hjc was purchased from selleck. the anti-epac antibody was from bioss. primescript rt master mix and sybr premix ex taq were from takara. ifn-α, ifn-β, and ifn-γ were purchased from sino biological inc. the camp elisa kit, mouse ifn-α/β receptor block/neutralize polyclonal goat igg (af ), and normal goat igg control were from r&d systems. p y plasmid was obtained from biogot technology, co., ltd. the bmdms were prepared as follows: bone marrow cells were collected from tibias and femurs by flushing with dmem, and the cell suspension was filtered through a -μm cell strainer to remove any cell clumps. bone marrow cells were then cultured in dmem containing % fbs, % penicillin/streptomycin, and % l culture supernatants. the pems were prepared as follows: mice were intraperitoneally injected with ml of % sterile thioglycollate medium and, days later, mice were sacrificed and the peritoneal cavities washed with ml pbs. the cell suspension was filtered through a -μm cell strainer to remove any cell clumps. pems were then cultured in dmem containing % fbs and % penicillin/streptomycin. raw . , hek t, and hela cells were obtained from the american type culture collection and cultured in dmem containing % fbs and % penicillin/streptomycin. after stimulation, total rna was extracted with rnaiso plus (takara) from the cells. cdna was synthesized from extracted total rna using the primescript rt master mix perfect real time kit (takara) according to the manufacturer's protocol. quantitative real-time pcr was performed using a sybr-green premix (takara). the sequence-specific primers are listed in supplementary table s . adp release assay raw . cells, mouse bmdms, and hek t cells were plated in -well plates ( × cells per well) overnight and then infected with viruses for various periods. adp release into the cell culture supernatants was detected using adp-glo™ kinase assay kit according to the manufacturer's protocol. raw . cells were plated in -well plates ( × cells per well) overnight. the cells were treated with adp for h and then infected with vsv for h. mts ( μl, promega) was added to each well. after incubated for h at °c, the absorption was measured at nm. cell viability of the untreated group was normalized to %. hela and hek t cells were seeded into -well plates ( . × cells per well) overnight. the cells were treated with adp for h and then infected with vsv-gfp or mlv-gfp for h. cells containing virus were determined after gating on gfp-positive cells. hek t cells were plated in -well plates ( × cells per well) overnight. twelve hours after vsv (moi = . ) or hsv- (moi = . ) infection, cells were fixed with % paraformaldehyde in pbs for min at room temperature. after incubation with crystal violet for min, the cells were then washed with pbs five times before the photographs were taken. cells were grown in -well plates. after stimulation, cells were lysed with radio immunoprecipitation assay (ripa) buffer supplemented with protease and phosphatase inhibitors. whole-cell lysates were separated by % sds-page, transferred to nitrocellulose membranes, and blocked with % bsa. immunoblots were hybridized with the respective primary antibodies. protein bands were visualized with the appropriate fluorescent secondary antibodies and the odyssey laser digital imaging system (gene company). cells were infected with viruses for the indicated time and moi. for mouse survival assay, -week-old p y +/+ or p y −/− mice were intraperitoneally treated with adp ( mg/kg) or not treated for h and then intraperitoneally injected with vsv ( × pfu/g). executed these for days and mouse survival was recorded for days. for in vivo studies of viral replication, to -week-old mice were intraperitoneally treated with adp ( mg/kg) or not treated for h and then intraperitoneally injected with vsv ( × pfu/g) or hsv- ( × pfu/g) for the indicated time and viral rna replicates in organs were detected by q-pcr. vero cells were plated in -well plates ( × cells per well) overnight. the supernatants from vsv-infected cells were serially diluted and infected vero cells for h. then the supernatant was removed and the cells were covered with dmem containing % low-melting point agarose. plaques were counted after h. for immunofluorescence assay, -week-old mice were anesthetized with ketamine/rompun and treated with mg/kg adp through nasal dropping for h before being intranasally infected with vsv ( × pfu/g). after h of infection, mice were sacrificed, and the olfactories were fixed with % paraformaldehyde in pbs overnight. slices ( -μm) from olfactories were permeabilized with . % triton x- and blocked with % bsa. tissues were then incubated with anti-vsv-g antibody overnight and alexa-fluor- -conjugated secondary antibody for h. finally, the tissues were stained with dapi and visualized with a fluorescence microscope. for camp detection, raw . cells or pems were plated in -well plates ( × or × cells per well, respectively) overnight. after stimulation, the cells were lysed with μl cell lysis buffer and the amounts of camp were measured by elisa (r&d systems) according to the manufacturer's instructions. to generate an epac -knockout cell line, a crispr/cas system was used. the epac -knockout target sequence used was ′-aaatgcactccggaccacg- ′. a lenticrisprv plasmid containing the epac -knockout target sequence was transduced into hela cells. three days later, puromycin-resistant single clones were selected, and the epac -knockout clones were identified by western blotting with the anti-epac antibody. all data were analyzed using the graphpad prism software and shown as mean ± sd. the student t-test (two-tailed) was used to analyze the significance of data and p-value < . was considered statistically significant. supplementary material is available at journal of molecular cell biology online. induction of siglec-g by rna viruses inhibits the innate immune response by promoting rig-i degradation purinergic signaling: a fundamental mechanism in neutrophil activation purinergic p y receptor modulates stress-induced hematopoietic stem/progenitor cell senescence activation of protein kinase a inhibits interferon induction of the jak/stat pathway in u cells the p x receptor in infection and inflammation nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance purinergic signaling during inflammation impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action. microbiol type interferons and the virushost relationship: a lesson in detente epac: defining a new mechanism for camp action exchange protein directly activated by camp plays a critical role in bacterial invasion during fatal rickettsioses errα negatively regulates type i interferon induction by inhibiting tbk -irf interaction nucleotide signalling during inflammation immune cell regulation by autocrine purinergic signalling the role of pattern-recognition receptors in innate immunity: update on toll-like receptors multiple p y subtypes in spinal microglia are involved in neuropathic pain after peripheral nerve injury interferon-inducible cholesterol- -hydroxylase broadly inhibits viral entry by production of -hydroxycholesterol ifn-induced tpr protein ifit potentiates antiviral signaling by bridging mavs and tbk p y and p y receptors involved in adpβs induced the release of il- β, il- and tnf-α from cultured dorsal horn microglia isg enhances the innate antiviral response by inhibition of irf- degradation the myriad roles of cyclic amp in microbial pathogens: from signal to sword novel antiviral host factor, tnk , regulates ifn signaling through serine phosphorylation of stat mechanisms of type-i-and type-ii-interferon-mediated signalling positional effect of phosphorylation sites and in the cytoplasmic domain of the e protein of hepatitis c virus a genotype: interferon resistance analysis via sequence alignment receptors for purines and pyrimidines the active metabolite of clopidogrel disrupts p y receptor oligomers and partitions them out of lipid rafts transformation of astrocytes to a neuroprotective phenotype by microglia via p y receptor downregulation adap is an interferon stimulated gene that restricts rna virus entry blocking of exchange proteins directly activated by camp leads to reduced replication of middle east respiratory syndrome coronavirus immunoregulation through extracellular nucleotides hyperglycaemia inhibits reg a expression to exacerbate tlr -mediated skin inflammation in diabetes pkacs attenuate innate antiviral response by phosphorylating visa and priming it for march -mediated degradation phosphatase pp negatively regulates type i ifn production and antiviral innate immunity by dephosphorylating and deactivating tbk two disparate ligand-binding sites in the human p y receptor extracellular adp facilitates monocyte recruitment in bacterial infection via erk signaling this work was supported by the national key r&d program of china ( yfa to b.d.), the national natural science conflict of interest: none declared. key: cord- -co fgz authors: lobo‐silva, diogo; carriche, guilhermina m.; castro, a. gil; roque, susana; saraiva, margarida title: interferon‐β regulates the production of il‐ by toll‐like receptor‐activated microglia date: - - journal: glia doi: . /glia. sha: doc_id: cord_uid: co fgz pattern recognition receptors, such as toll‐like receptors (tlrs), perceive tissue alterations and initiate local innate immune responses. microglia, the resident macrophages of the brain, encode tlrs which primary role is to protect the tissue integrity. however, deregulated activation of tlrs in microglia may lead to chronic neurodegeneration. this double role of microglial responses is often reported in immune‐driven neurologic diseases, as in multiple sclerosis (ms). consequently, strategies to manipulate microglia inflammatory responses may help to ameliorate disease progression. in this context, the anti‐inflammatory cytokine interleukin (il)‐ appears as an attractive target. in this study, we investigated how activation of microglia by tlrs with distinct roles in ms impacts on il‐ production. we found that activation of tlr , tlr , and tlr induced the production of il‐ to a greater extent than activation of tlr . this was surprising as both tlr and il‐ play protective roles in animal models of ms. interestingly, combination of tlr triggering with the other tlrs, enhanced il‐ through the modulation of its transcription, via interferon (ifn)‐β, but independently of il‐ . thus, in addition to the modulation of inflammatory responses of the periphery described for the axis tlr /ifn‐β, we now report a direct modulation of microglial responses. we further show that the presence of ifn‐γ in the microenvironment abrogated the modulation of il‐ by tlr , whereas that of il‐ had no effect. considering the therapeutic application of ifn‐β in ms, our study bears important implications for the understanding of the cytokine network regulating microglia responses in this setting. indeed, pathologic neuroinflammation is reported in many cns diseases, including multiple sclerosis (ms), alzheimer's disease, parkinson's disease, stroke, and traumatic brain injury (czirr & wyss-coray, ; shastri, bonifati, & kishore, ) . the critical balance between neuroprotection and neurodegeneration is largely dependent on microglia immune responses (ransohoff & brown, ) . microglia, the resident macrophages of the cns (hanisch & kettenmann, ) , play an important role in patrolling the parenchymal tissue, contributing to the maintenance of neuronal homeostasis and the initiation of innate immune responses in the brain (kettenmann, hanisch, noda, & verkhratsky, ; perry & teeling, ; ransohoff & brown, ) . in line with their myeloid origin, microglia express numerous pattern recognition receptors (prrs), among which toll-like receptors (tlrs) (eggen, raj, hanisch, & boddeke, ; shastri et al., ) , involved in the recognition of pathogen-or damage-associated molecular patterns (pamps and damps, respectively). upregulation and activation of several tlrs in microglia has been described as a protective mechanism during infection by viruses, bacteria and parasites (sochocka et al., ) . in the context of neurodegeneration, the initial activation of microglia (and other glial cells) by damps plays a pivotal role in repairing injury and maintaining the cns homeostasis, but with the increase in neuronal death, this very same activation further potentiates cell death, thus enhancing neurodegeneration (hanke & kielian, ; hayward & lee, ; kigerl, de rivero vaccari, dietrich, popovich, & keane, ; shastri et al., ; su, bai, zhou, & zhang, ) . consequently, it is conceived that regulating the activation of microglia, for example, through tlrs, may allow the regulation of several neurological diseases (pedras-vasconcelos, puig, & verthelyi, ). tlr responses are mediated by the intracellular adaptor molecules myeloid differentiation primary response gene (myd ) and tirdomain-containing adapter-inducing interferon-b (trif) (kawai & akira, ; pandey, kawai, & akira, ) . most tlrs signal uniquely via myd , the exceptions being tlr , which signals via both myd and trif, and tlr , which signals uniquely through trif (kawai & akira, ) . both myd and trif initiate a series of intracellular signaling cascades that culminate with the production of several immune mediators, as cytokines, chemokines, reactive oxygen species, and nitric oxide (kawai & akira, ) . myd -dependent signals efficiently activate the nf-kb family of transcription factors, leading to the production of many cytokines, as tumor necrosis factor (tnf), interleukin (il)- , il- b, and also il- (pandey et al., ) . trif-mediated signals induce interferon regulatory factor (irf)- , leading to the production of type i interferons (ifns) and other ifn-responsive factors (pandey et al., ) . of all these cytokines, two have been associated with protection during neurodegeneration: type i ifns and il- . type i ifns comprise a family of cytokines with prominent roles during viral resistance (mcnab, mayer-barber, sher, wack, & o'garra, ) . in the context of cns infections, protective type i ifn responses are also observed, for example, during intracerebral infection with lymphatic choriomeningitis virus (merigan, oldstone, & welsh, ; nayak et al., ) or with la cross virus or coronavirus mouse hepatitis virus, where microglial cells were identified as type i ifn sources (kallfass et al., ; roth-cross, bender, & weiss, ) . a role for microglial type i ifn signaling was also described during the resolution of sterile injury (khorooshi & owens, ) . furthermore, the induction of type i ifns is critical for limiting experimental autoimmune encephalomyelitis (eae) as mice deficient for type i ifn receptor (ifnar) suffer from a higher disease course, increased macrophage, tcell and b-cell infiltration, and greater demyelination (prinz et al., ) . importantly, administration of ifn-b is the first-line drug for the treatment of ms (inoue & shinohara, ; marrie & rudick, ) . despite all these protective effects, chronically increased levels of ifn are linked to various diseases, including encephalitis or other type i interferonopathies (goldmann, blank, & prinz, ) . il- is a powerful anti-inflammatory cytokine produced by most immune cells that plays the important role of preventing exacerbated immune responses and subsequent tissue immunopathology (o'garra & vieira, ) . the production of il- by tlr-activated microglia is well documented (butchi, du, & peterson, ; jack et al., ; ledeboer et al., ; mizuno, sawada, marunouchi, & suzumura, ; olson & miller, ) . the potential of il- in controlling inflammatory responses in the brain has gained recent importance (kwilasz, grace, serbedzija, maier, & watkins, ) . il- has been tested as a therapeutic approach to a series of neurologic pathologies, where excessive and persistent neuroinflammation acts as a driver of neurodegeneration. examples of this include the administration of il- in animal models of brain ischemia (perez-de puig et al., ) , parkinson's disease (joniec-maciejak et al., ) , and ms (cua, hutchins, laface, stohlman, & coffman, ) . there are, however situations, such as in alzheimer s disease, where a detrimental role for il- has been shown (chakrabarty et al., ; guillot-sestier et al., ) . in either case, modulating il- in the cns appears as an attractive approach to rewire otherwise detrimental immune responses. this calls for a deep understanding on the molecular mechanisms regulating il- production in the brain, something that remains poorly studied (lobo-silva, carriche, castro, roque, & saraiva, ) . owing to the potential of il- in restoring the immune balance in the brain, targeting the molecular mechanisms that regulate il- production by brain resident cells is an interesting area that deserves attention. in this study, we investigated the induction of il- upon tlr activation in microglia and some of the operating mechanisms that can enhance it. we also probed how microenvironmental cues further modulate il- . all animal experiments were performed in strict accordance with the recommendations of the european union directive / /eu. animals were kept and bred with water and food ad libitum, according with the portuguese national authority for animal experimentation guidelines. newborn mice were humanely euthanized by decapitation and every effort was made to minimize suffering. adult wild-type, il- -deficient, and type i interferon receptor (ifnar)-deficient mice, all in c bl/ background, were kept and bred at icvs. the ifnar deficient mice were a kind gift of dr anne o'garra (francis crick institute, london, uk). postnatal day (p )-p mice were used to perform primary microglia cell cultures. adult mice ( - weeks) were used to generate bone marrow-derived macrophages. dulbecco's modified eagle medium (dmem), fetal bovine serum (fbs), hepes solution, sodium piruvate, l-glutamine, and penicillin-streptomycin were purchased from invitrogen. cells were cultured in dmem supplemented with % fbs, % hepes solution, % sodium piruvate, % l-glutamine, and % penicillin-streptomycin (cdmem). pam csk (tlr /tlr ligand), polyi:c (tlr ligand), and cpg (tlr ligand) were obtained from invivogen. lipopolysaccharide (lps) from escherichia coli (tlr ligand) was bought from sigma. mouse recombinant ifn-b, il- , ifn-g, and il- were bought from r&d systems. neutralizing ifn-g antibody (clone xmg . ) was a kind gift of dr rui appelberg (ibmc, porto). all media was prepared using endotoxin-free plastics and all stimuli were suspended in endotoxin-free media or water. primary microglia cultures were established from p -p c bl/ mice brains. brains were aseptically removed, washed with cdmem to remove blood leftovers, and homogenized using a lm cell strainer. . cells were plated in cdmem onto cm flasks and cultured for weeks in a humidified atmosphere at c with % co . the media was changed on days , , , and . on day , flasks were shaken for h at rpm to detach microglial cells from the mixed cultures. on day , around % of the collected cells were cd b , as detected by flow cytometry. cells were collected and cells plated in ll of cdmem per well in -well plates for further stimulation. at different times poststimulation, the cells were harvested for rna analysis and the supernatants for cytokine detection. bone marrow-derived macrophages were differentiated from bone marrow precursors cultured in cdmem, supplemented with % l cell conditioned media (lccm), as previously described (teixeira-coelho et al., ) . briefly, total bone marrow cells were cultured in microbiological petri dishes (sterilin) and kept at c and % co . cells were fed on day with equal volume of cdmem containing % lccm. on day , macrophages were harvested, counted and seeded into -well tissue culture plates at cells in ml per well in culture medium. cells were stimulated as indicated below and the culture supernatants harvested hr later for il- detection. purified microglial cells recovered on day or bone marrow-derived macrophages recovered on day were stimulated for different time points as appropriate. unless specified in the figure legends, tlr stimuli were used at a concentration of mg/ml for pam csk , ng/ml for polyi:c, ng/ml for lps, and mm for cpg. recombinant cytokines were supplemented at the following concentrations: ng/ml for ifn-b; ng/ml for il- ; - - u/ml for ifn-g; and - - ng/ml for il- . . | mrna stability determination mrna stability was determined as described before (teixeira-coelho et al., ) . microglia cultures were tlr-stimulated for hr and at that time-point actinomycin d added to the cultures. after , , or min, the cells were lysed and the expression of il- was analyzed by rt-pcr as indicated above. data are expressed as mean sd and analyzed by one or two-way analysis-of-variance (anova) tests or student's t test, as indicated in the figure legends. the p values considered as statistically significant were *p . , **p . , ***p . , and ****p . . to obtain an overview of the inflammatory landscape of microglia trig- with the aforementioned tlr agonists, the culture supernatants were assayed for a panel of cytokines by multiplex or elisa ( figure ) . of the cytokines tested, il- and il- were consistently below detection level for all the tlr agonists studied. also below detection level were all the tested cytokines in nonstimulated cells. we observed that whereas certain cytokines (il- , il- , il- , ifn-b, ifn-g, il- , and il- ; figure a -g) were greatly dependent on the tlr triggered, others (tnf, il- , il- , and il- ; figure h -k) were produced in equivalent amounts, independently of the stimulus. within the first group, it was interesting to note that tlr was the poorest inducer of cytokine production ( figure a -g), with the exception of ifn-b (figure d ), which was induced at higher levels upon tlr stimulation of microglia. furthermore, the production of anti-inflammatory il- ( figure a ) by microglia was among the most affected by the type of tlr stimulated. indeed, il- production varied from the highest amounts induced downstream of tlr triggering, to barely detectable ones downstream of tlr ( figure a ). these differential responses were not dependent on the dose of agonist used to stimulate microglia, as increasing the doses of tlr ligands did not result in enhanced secretion of il- , nor of tnf (supporting information, figure ). in particular, increasing the doses of tlr agonist did not improve il- secretion, which was for most of the cases below detection level (data not shown). we then tested whereas non-tlr stimuli would also induce il- production by microglia. we found that il- production was also triggered by fungal ligands for the prr dectin- (supporting information, figure a ). the combined stimulation dectin- /tlr was a more potent stimulus than either independent one (supporting information, figure a ), being the same pattern was observed for tnf production (supporting information, figure b ). finally, we compared the production of il- upon tlr stimulation of microglia to that of myeloid cells of the periphery, namely primary mouse bone marrow-derived macrophages. the pattern of il- secretion upon tlr stimulation of macrophages for hr (supporting information, figure ) was similar to that observed for microglia. interestingly, for both cell types, tlr stimulation remained the poorer inducer of il- (supporting information, figure ). taken together, we show that microglia respond to different tlr stimulation with the production of an array of cytokines. we highlight il- as a molecule which production is highly dependent on the stimuli and tlr as a poor inducer of il- . nevertheless, the pattern of il- production is similar for myeloid cells of distinct embryonic origins. because both the activation of tlr and the presence of il- have been linked to neuroprotection in ms and eae (gooshe, abdolghaffari, gambuzza, & rezaei, ; kwilasz et al., ) , we were surprised to see that activation of tlr in microglia was uncoupled from il- secretion. we next questioned if tlr signaling could potentially interfere with other tlrs, in what respects the induction of il- in microglia. it was interesting to observe that co-stimulation of tlr -, tlr -, or tlr -activated microglia with tlr significantly enhanced il- production (figure a-c) . so, although tlr triggering of microglia does not lead to productive il- secretion, it is an enhancing signal for il- production in these cells. to further understand the molecular events underlying the modulation of il- upon tlr co-stimulation of microglia, we focused on the combination tlr /tlr . we measured the transcription of the il gene over time in microglia stimulated via tlr alone, tlr alone or the combination tlr /tlr . consistent with the decreased amounts of il- protein, the transcription of the il gene upon tlr stimulation was significantly reduced as compared to that induced by tlr activation ( figure d ). this suggested that the signaling downstream tlr is not productive in what concerns il- transcription. co-stimulating microglia with tlr and tlr did not enhance the amount of il mrna early poststimulation, when compared with tlr stimulation alone ( figure d ). however, whereas upon tlr activation a peak of il gene expression was detected at hr poststimulation and progressively decreased during the analyzed hr, upon tlr /tlr activation, the level of il- mrna peaked at hr and then again at hr poststimulation ( figure d ). this suggested that in the combined stimulation a second wave of il transcription may be occurring, which would justify the higher amounts of il- protein detected in this case. alternatively, maintenance of the il mrna could result from increased il mrna stability, as previously shown for macrophages (teixeira-coelho et al., ) . to investigate this hypothesis, microglia were stimulated with tlr or tlr /tlr and at hr poststimulation actinomycin d was added to the cultures and the il- transcription followed for an extra min. a similar decline in the detection of il- either in single or combined stimulation was observed (figure e) . therefore, the combination of tlr with tlr triggering led to enhanced il- transcription, rather than enhanced mrna stability, thus explaining the higher levels of il- produced by microglia upon tlr /tlr co-stimulation. as the effect of tlr on il- occurred after the initial wave of gene transcription mediated by tlr , we questioned whether a feedback loop resulting from cytokines downstream of tlr could contribute to the higher il- production observed upon tlr /tlr co-stimulation of microglia. considering the cytokine landscape obtained upon tlr triggering of microglia (figure ), if such mechanism was operating, the best candidate molecule would be ifn-b, as it is strongly induced by tlr ( figure g ) and it has been described to potentiate il- in studies performed with bone marrow-derived macrophages and dendritic cells (iyer, ghaffari, & cheng, ; f. w. mcnab et al., ; wang et al., ) . furthermore, we observed an early transcription of the ifnb gene downstream of tlr signaling in microglia (figure a) , which suggested that ifn-b would be produced early enough to modulate the second il transcriptional wave. also, in line with the protein data ( figure g ), no ifnb transcription was observed upon tlr stimulation of microglia (figure a) . to test the role of ifn-b in inducing il- in microglia, we generated microglia from wt or ifnar deficient mice, which do not respond to type i ifns (ifn-b or ifn-a) . although il- production in response to single tlr or tlr stimulation was not affected by the absence of ifnar, microglia deficient for this receptor failed to upregulate il- production hr poststimulation with tlr fig ure inflammatory landscape of tlr-stimulated microglia. (a-k). primary microglial cell cultures were left unstimulated or stimulated for hr with chemical tlr agonists for tlr , tlr , tlr , or tlr , as described in section . cell culture supernatants were collected and cytokine production was measured by multiplex assay (il- , tnf, il- p , il- , il- , il- , ifn-g, il- , and il- ) or elisa (il- and ifn-b). unstimulated cells did not produce detectable amounts of cytokines. the detection limit for each cytokine is represented as a dotted line in each graph. represented are the mean sd for triplicate wells per condition set after mixed cultures generated from independent mice. statistical differences were assessed by one-way anova or student's t test. significant statistical differences relative to tlr are represented by *; to tlr by #; to tlr by $. one symbol, p < . ; two, p < . ; three, p < . ; and four, p < . . bdl, below detection level and tlr (figure b ). thus, our findings strongly suggest that the production of type i ifn downstream of tlr triggering is a key event for the modulation of il- production by this receptor. to further validate these findings, we compared the amount of il- secreted by microglia co-stimulated with tlr /tlr or with tlr in combination with recombinant ifn-b. as shown in figure c , treatment of microglia with recombinant ifn-b did not induce il- on its own, but enhanced it when combined with tlr stimulation. furthermore, the transcriptional profile of the il gene upon costimulation of microglia with tlr and ifn-b was similar to that observed for tlr /tlr (figure d ) and the il mrna stability was not altered by ifn-b (figure e ). co-stimulation of microglia with tlr enhanced il- production via ifn-b, thus opening a novel mechanism underlying its beneficial role in ms and being a possible tool to locally regulate inflammatory responses. for this, it is however important that tlr co-activation does not promote an overall cytokine storm. to investigate if this was the case, we run the multiplex panel in supernatants from microglia stimulated with tlr in combination with tlr ( figure ). costimulation of microglia via tlr and tlr did not lead to an overall deregulation of microglia responses, with many cytokines remaining at a level identical to that observed for tlr single stimulation (figure ) . it was interesting to note that il- b secretion, which was undetectable upon single stimulation of tlr or tlr , became detectable upon costimulation of microglia (figure k ). in addition to il- , another molecule found to be markedly upregulated in the case of tlr /tlr , as compared to tlr alone, was il- ( figure e ). this cytokine has previously been implicated in the regulation of il- production by macrophages in some reports (iyer et al., ) . we thus questioned whether il- could play a role in inducing il- by tlr -activated microglia. because il- enhancement upon tlr triggering depended on ifnar (figure b ), we first measured il- production in wt or ifnar-deficient microglia activated via tlr / fig ure tlr potentiates il- production by tlr-stimulated microglia, by modulating the transcription of the il gene. (a-c) wt microglial cells were left unstimulated or stimulated for hr with chemical agonists for tlr , tlr , or tlr alone or in combination with a tlr agonist. cell culture supernatants were collected and il- production measured by elisa. il- production by unstimulated cells was undetectable. (d) wt microglial cells were left unstimulated or stimulated with tlr (open circle) and tlr (close square) agonists alone or in combination (close circle). rna samples were collected at the indicated time-points and rt-pcr was performed to evaluate il gene expression normalized to that of hprt. (e) wt microglial cells were stimulated for hr with the tlr agonist alone (open circle) or in combination (close circle) with the tlr agonist and then actinomycin d was added to cells. rna samples were collected at , , and min after actinomycin d addition and rt-pcr was performed to evaluate il gene expression as before. the dotted line represents % of the rna detected at hr poststimulation. represented are the mean sd for triplicate wells per condition for two independent experiments. statistical differences were assessed by student's t test or two-way anova. significant statistical differences are represented by ** to p < . ; *** to p < . and **** to p < . . actd, actinomycin d; bdl, below detection level tlr . il- production by tlr /tlr stimulated microglia was diminished in the absence of ifnar (figure l ). we then assessed whether il- would augment il- production by tlr -stimulated microglia. as before, stimulation of microglia with tlr and ifn-b augmented the production of il- ( figure m ). however, this was not observed when ifn-b was replaced by il- , nor did the addition of both to the cultures further increased il- production ( figure m ). of note, stimulation of microglia with either recombinant cytokine in the absence of tlr did not result in il- production (figure m) . therefore, the increase in il- secretion by tlr /tlr -stimulated microglia seems to be directly potentiated by ifn-b, with no major role for il- . altogether, our data show that tlr potentiates il- production in microglia stimulated with a variety of prr agonists and that this involves the production of ifn-b and the activation of the ifnar. furthermore, this il- enhancement is not accompanied by an overshoot cells generated from wt and ifnar / mice were stimulated with tlr and tlr agonists alone or in combination. cell culture supernatants were collected hr poststimulation and il- production was measured by multiplex. (c) wt microglial cells were stimulated with tlr and tlr agonists in the presence or absence of recombinant ifn-b. cell culture supernatants were collected and il- production was measured by elisa. (d) wt microglial cells were stimulated for hr with the tlr agonist (open circle), recombinant ifn-b (close square) or with their combination (close circle). rna samples were collected at the indicated time-points poststimulation and rt-pcr was performed to evaluate il gene expression. (e) wt microglial cells were stimulated for hr with the tlr agonist alone (open circle) or in combination with recombinant ifn-b (close circle) and then actinomycin d was added to cells. the il mrna stability was measured as before. the dotted line represents % of the rna detected at hr after stimulation. represented are the mean sd for triplicate wells per condition for two independent experiments. statistical differences were assessed by one-or two-way anova. significant statistical differences are represented by ** to p < . ; *** to p < . ; and **** to p < . . actd, actinomycin d; bdl, below detection level lobo-silva et al. fig ure tlr co-stimulation of microglia does not lead to overshooting responses. (a-k) wt microglial cells were stimulated for hr with tlr or tlr agonists alone or in combination. cell culture supernatants were collected and cytokine production was measured by multiplex assay or elisa (tnf, il- , ifn-b, and il- b). significant statistical differences relative to tlr are represented by *; to tlr by #. (l) wt and ifnar / microglial cells were stimulated for hr with the tlr or tlr agonists alone or in combination. cell culture supernatants were collected and il- production was measured by multiplex. (m) wt microglial cells were stimulated for hr with the tlr agonist, ifn-b or il- alone or in combination. cell culture supernatants were collected and il- production was measured by elisa. significant statistical differences relative to tlr are represented by *; to tlr ifnb by #; to tlr il- by $. the detection limit for each cytokine is represented as a dotted line in each graph. represented are the mean sd for triplicate wells per condition set after mixed cultures generated from independent mice. statistical differences were assessed by student's t test or one-way anova. significant statistical differences are represented by one symbol, p < . ; two, p < . ; three, p < . ; and four, p < . . bdl, below detection level of the microglia response. these findings are interesting because il- expression in the brain has been proposed as a therapeutic approach for a series of neurological diseases with an immune component, such as ms, parkinson's disease, and brain injury (kwilasz et al., ) . furthermore, because ifn-b is currently used in the clinics to treat ms patients, it is tempting to speculate that it may exert its local action by promoting il- secretion. however, in the case of complex diseases such as ms, the immune environment generated in the cns also comprises t-cell-derived cytokines, notably ifn-g and il- . to understand whether the presence of these cytokines would impact on the modulation of il- by tlr signaling, we stimulated microglia via tlr and tlr and combined this with increasing doses of recombinant il- or ifn-g. interestingly, whereas the il- -enriched environment did not alter the modulation of il- secretion by tlr (figure a) , the ifng-enriched milieu totally abrogated it (figure b) . therefore, the successful modulation of il- production in microglia by tlr or ifn-b will clearly depend on the immune composition of the tissue microenvironment, a factor that needs to be taken into account in il- -based therapies. surveillance of the cns by innate immune cells, such as resident microglia, occurs in both physiological conditions and pathological states (ousman & kubes, ) and is critical for the maintenance of the cns homeostasis. pathways for initiating inflammation include an expanding number of cellular sensors, namely the tlr family. members of this family are expressed on innate immune cells, including microglia cells and astrocytes (holley et al., ; kawai & akira, ) . activation of microglia through tlrs, and other prrs, plays an important role in initiating innate immune responses that protect the cns from aggressions, such as the presence of pathogens, and that promote tissue regeneration after injury (okun et al., ) . however, cumulative evidence show that persistently activated microglia, and reactive astrocytes, can also contribute to pathogenesis of several types of cns diseases, such as ms (goverman, ; okun et al., ) . therefore, microglial cells may contribute to either neuroprotection or neurodegeneration, depending on the setting and the context they are in. consequently, switching microglial responses toward neuroprotection may be beneficial in different diseases, for example, in ms (weiner, ) . in this setting, the modulation of il- production by activated microglia might prove of interest. several studies addressed the importance of tlrs in ms pathology, showing that the expression of these receptors is increased in brain lesions of both ms and eae (bsibsi, ravid, gveric, & van noort, ) . interestingly, whereas activation of tlr , tlr , and tlr is detrimental in ms and eae (prinz et al., ; visser et al., ) , that of tlr protects from disease (gooshe et al., ; touil, fitzgerald, zhang, rostami, & gran, ) . we started this study by looking at the inflammatory landscape of microglia triggered by these tlrs. we found that, overall, tlr was the least inflammatory tlr, as only low cytokine levels were detected upon activation of microglia with polyi: c, a tlr agonist. this poor reactivity of tlr may underlie its protective nature in eae (gooshe et al., ) . furthermore, tlr triggering led to pronounced ifn-b secretion, a molecule that is currently used to treat ms patients (plosker, ; trojano et al., ) and which presence is protective in eae (goldmann et al., ; touil et al., ) . however, in apparent contrast with these data, we found that another protective cytokine in eae (bettelli et al., ) , the anti-inflammatory cytokine il- , was poorly induced downstream of tlr . in the context of ms or eae, tissue inflammation is accompanied by cellular death, which releases several damps and propagates microglia reactivity. it is therefore most likely that several tlrs, and indeed other prrs, get activated. when tlr activation was combined with that of the other tested tlrs, a significant increase on the production of il- fig ure the cytokine milieu impacts the tlr -driven enhancement of il- . wt microglial cells were stimulated for hr with tlr or tlr agonists alone or in combination in the absence or presence of increasing doses of (a) il- or (b) ifn-g. cell culture supernatants were collected and il- production was measured by elisa. represented are the mean sd for triplicate wells per condition for two independent experiments. statistical differences were assessed by student's t test or one-way anova. significant statistical differences are represented by *** p < . . bdl, below detection level lobo-silva et al. | was observed. importantly, this increase of il- production as a result of combined tlr /tlr stimulation of microglia has a functional impact. indeed, whereas the production of il- downstream of tlr or tlr stimulation is only mildly increased in il- -deficient microglia, it is markedly enhanced in co-stimulated microglia, in the absence of il- (supporting information, figure ) . thus, the enhancement of il- in tlr /tlr co-stimulated microglia appears to hamper proinflammatory responses. we narrowed the regulation of il- by tlr down to a positive feedback loop involving ifn-b that mechanistically induced a second wave of il gene transcription, but did not affect the stability of the il mrna. this observation mirrors the effect of ifn-b in innate immune cells of the periphery (iyer et al., ; mcnab et al., ; wang et al., ) , supporting the existence of transversal mechanisms operating in circulating versus resident macrophages, such as microglia. we herein also show that the production of il- by tlractivated microglia follows a pattern similar to that observed in conventional macrophages derived from the bone marrow. tlr is the sole tlr signaling only via the trif pathway (kawai & akira, ) , which has been previously involved in sustaining the stability of the il mrna in bone marrow-derived macrophages (teixeira-coelho et al., ) . thus, it is tempting to speculate that cell-specific mechanisms are also in place, to ensure specificity in the regulation of il- . pinpointing the common, and the cell-specific, mechanisms regulating this cytokine is critical if tailor-made interventions are to be envisaged. the protective role for tlr in ms and eae appears to be associated with the triggering of neuroprotective responses in astrocytes (bsibsi et al., ) , but also with the release of ifn-b (touil et al., ) and of il- (fitzgerald et al., ) by innate immune cells of the periphery. in eae, lack of endogenous ifn-b in the cns leads to augmented microglia activation, resulting in a sustained inflammation, cytokine production, and tissue damage with consequent chronic neurological deficits (teige et al., ) in support of an anti-inflammatory role for this molecule. furthermore, expression of ifn-b within the cns (khorooshi et al., ) , including in microglia (kocur et al., ) , plays a protective role in eae. other studies further link ifn-b therapy with an enhancement of il- in ms and an accompanying inhibition of t helper (th) cell responses (krakauer, sorensen, khademi, olsson, & sellebjerg, ; kvarnstrom, ydrefors, ekerfelt, vrethem, & ernerudh, ; ramgolam, sha, jin, zhang, & markovic-plese, ; sweeney et al., ) , but all do so in immune cells of the periphery. we now reveal another protective action of ifn-b, by directly targeting microglia leading to enhanced il- and il- , both protective cytokines in ms. at least in microglia, these events seem to be unrelated, with the ifn-b potentiation of il- being independent of il- . this is in line with other studies performed in macrophages (mcnab et al., ) and contrasts with a possible role of il- in promoting il- production by myeloid cells (iyer et al., ) and effector th cells (freitas do rosario et al., ) . as mentioned above, the response to ifn-b therapy in ms has been correlated with an inhibition of th cells (krakauer et al., ; kvarnstrom et al., ; ramgolam et al., ; sweeney et al., ) . as herein shown, the direct effect of ifnb in potentiating il- secretion by tlr-activated microglia would still occur in the context of il- , but would be largely compromised in the presence of ifn-g. the role of ifn-g in modulating il- production by microglia is still not fully understood, but it is likely dependent on the activation of glycogen synthase kinase (gsk)- (green & nolan, ) . it is interesting to note that low amounts of ifn-g were detected upon stimulation of the microglia cultures with tlr , , and agonists, but not upon tlr triggering. yet, this pattern of endogenous ifn-g production was not related to that of il- expression. the fact that absence of ifn-g under tlr stimulation did not license il- production fits with the mechanism we propose, where low il- triggering in response to tlr stimulation is related to deficient basic transcriptional activity. in further support of an independent endogenous ifn-g vs il- production, blocking of ifn-g with a neutralizing antibody did not alter il- secretion by tlr -stimulated microglia (supporting information, figure ). in sum, by using an in vitro model of neonate microglia activation, our study illustrates the complexity of the events regulating microglial responses, providing hints to possible mechanisms operating in the adult brain in the context of eae. it will be important to now probe these mechanisms in vivo, by resorting to various genetically modified mice and to also address the effects of microglia modulation on adjacent cells, notably on neurons. our data showing that ifn-b is a direct modulator of il- in tlractivated microglia also has implications in the study of animal models of ms. in a recent study, the most common mouse backgrounds used in laboratory research (balb/c and c bl/ ) were shown to bear important differences in the ability of their macrophages to produce il- in response to tlr triggering (howes et al., ) . these differences were related to a differential induction of ifn-b in either background (howes et al., ) . if the same differences apply to microglia, the use of either mouse strain to induce eae may lead to distinct results. in line with a possible impact of the mouse genetic background in the response of microglia to tlr activation, previous studies reported ifn-b production downstream of tlr activation in microglia generated from irw mice (butchi et al., ) . this was not the case in our study, where c bl/ mice were used (figure d ). the interaction between the brain and the immune system is now accepted to play pivotal roles in health and disease, particularly in pathologies involving a strong neuroimmune component. microglia are key pieces in this interaction. as such, it is critical to understand the network of events that drive microglia responses. our study contributes to this understanding in what regards the intrinsic orchestration of il- expression and the impact of the cytokine microenvironment in this regulation. understanding these processes is chief to the targeted manipulation of il- in the brain. we declare that we have no conflict of interest. ds and gmc performed the experiments. ds, agc, sr, and ms designed the study. ds, agc, sr, and ms analyzed and interpreted the data. all authors read and approved the final manuscript. il- is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of il- -and il- -deficient and transgenic mice the microtubule regulator stathmin is an endogenous protein agonist for tlr broad expression of toll-like receptors in the human central nervous system interactions between tlr and tlr agonists and receptors regulate innate immune responses by astrocytes and microglia il- alters immunoproteostasis in app mice, increasing plaque burden and worsening cognitive behavior central nervous system expression of il- inhibits autoimmune encephalomyelitis the immunology of neurodegeneration microglial phenotype and adaptation suppression of autoimmune inflammation of the central nervous system by interleukin secreted by interleukin -stimulated t cells il- promotes il- production by effector th cd t cells: a critical mechanism for protection from severe 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a review of its use in multiple sclerosis innate immunity mediated by tlr modulates pathogenicity in an animal model of multiple sclerosis distinct and nonredundant in vivo functions of ifnar on myeloid cells limit autoimmunity in the central nervous system ifn-beta inhibits human th cell differentiation innate immunity in the central nervous system murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia innate immunity and neuroinflammation inflammatory response in the cns: friend or foe microglial toll-like receptors and alzheimer's disease il- mediates the response to ifn-beta therapy in multiple sclerosis patients by inhibiting th cells ifn-beta gene deletion leads to augmented and chronic demyelinating experimental autoimmune encephalomyelitis differential post-transcriptional regulation of il- by tlr and tlr -activated macrophages cutting edge: tlr stimulation suppresses experimental autoimmune encephalomyelitis by inducing endogenous ifn-beta italian multiple sclerosis database network real-life impact of early interferon beta therapy in relapsing multiple sclerosis proinflammatory bacterial peptidoglycan as a cofactor for the development of central nervous system autoimmune disease the role of glycogen synthase kinase in regulating ifn-beta-mediated il- production a shift from adaptive to innate immunity: a potential mechanism of disease progression in multiple sclerosis the authors thank drs joão relvas and ana cordeiro gomes for critically reading the manuscript. additional supporting information may be found online in the supporting information tab for this article.how to cite this article: lobo-silva d, carriche gm, castro ag, roque s, saraiva m. interferon-b regulates the production of il- by toll-like receptor-activated microglia.glia. key: cord- -f etlk i authors: olofsson, peter; nerstedt, annika; hultqvist, malin; nilsson, elisabeth c; andersson, sofia; bergelin, anna; holmdahl, rikard title: arthritis suppression by nadph activation operates through an interferon-β pathway date: - - journal: bmc biol doi: . / - - - sha: doc_id: cord_uid: f etlk i background: a polymorphism in the activating component of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex, neutrophil cytosolic factor (ncf ), has previously been identified as a regulator of arthritis severity in mice and rats. this discovery resulted in a search for nadph oxidase-activating substances as a potential new approach to treat autoimmune disorders such as rheumatoid arthritis (ra). we have recently shown that compounds inducing ncf -dependent oxidative burst, e.g. phytol, have a strong ameliorating effect on arthritis in rats. however, the underlying molecular mechanism is still not clearly understood. the aim of this study was to use gene-expression profiling to understand the protective effect against arthritis of activation of nadph oxidase in the immune system. results: subcutaneous administration of phytol leads to an accumulation of the compound in the inguinal lymph nodes, with peak levels being reached approximately days after administration. hence, global gene-expression profiling on inguinal lymph nodes was performed days after the induction of pristane-induced arthritis (pia) and phytol administration. the differentially expressed genes could be divided into two pathways, consisting of genes regulated by different interferons. ifn-γ regulated the pathway associated with arthritis development, whereas ifn-β regulated the pathway associated with disease protection through phytol. importantly, these two molecular pathways were also confirmed to differentiate between the arthritis-susceptible dark agouti (da) rat, (with an ncf- (da )allele that allows only low oxidative burst), and the arthritis-protected da.ncf- (e )rat (with an ncf (e )allele that allows a stronger oxidative burst). conclusion: naturally occurring genetic polymorphisms in the ncf- gene modulate the activity of the nadph oxidase complex, which strongly regulates the severity of arthritis. we now show that the ncf- allele that enhances oxidative burst and protects against arthritis is operating through an ifn-β-associated pathway, whereas the arthritis-driving allele operates through an ifn-γ-associated pathway. treatment of arthritis-susceptible rats with an nadph oxidase-activating substance, phytol, protects against arthritis. interestingly, the treatment led to a restoration of the oxidative-burst effect and induction of a strikingly similar ifn-β-dependent pathway, as seen with the disease-protective ncf polymorphism. rheumatoid arthritis (ra) is one of the commonest autoimmune diseases, with a prevalence of . - % [ , ] . ra is a chronic and severely disabling disease of unknown etiology, although both environmental [ ] and genetic factors [ ] are believed to play roles in its cause. there is presently no cure for ra, although a variety of different drugs is used to treat the symptoms. the most common treatments include disease-modifying antirheumatic drugs such as,methotrexate [ , ] . other efficient antirheumatic drugs are the recently developed biologicalresponse modifiers, such as tumor necrosis factor (tnf)α blockers, which reduce both the established inflammation and joint destruction [ ] . the drawback of these treatments is an increased risk of infections because the body's defense system is impaired. other biological-response modifiers targeting other cytokines or costimulatory molecules are currently being clinically tested and evaluated [ ] . despite this range of antirheumatic drugs, there is still a large number of ra patients for whom none of these treatments is effective [ , ], thus making the development of new therapies essential. identification of new targets for development of antirheumatic drugs is hampered by the heterogeneity of ra and the complexity of its molecular pathology. however, recent efforts with linkage-association studies, both in human populations and in animal models, are now giving results in the form of successful identification of autoimmunity-regulating genes [ ] [ ] [ ] [ ] [ ] . the combined results from such studies will eventually lead to a greater understanding of the molecular regulation of this complex disease and they present potential new targets for drug development. neutrophil cytosolic factor (ncf ), also known as p phox, was one of the first single genes identified to regulate arthritis severity. this finding was a result of linkage analysis and positional cloning in an arthritis model in rats using a cross between the arthritis-susceptible dark agouti (da) rat and the arthritis-resistant e strains da.ncf e ([ , ] . ncf- is the activating component of the nadph oxidase complex, which, upon activation, produces reactive oxygen species (ros) [ ] . we found that the dramatic increase in arthritis severity was caused by a decreased capacity of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex to produce ros. interestingly, the lower oxidative burst led to a reduction in cell membrane proteins and activation of autoreactive and arthritogenic t cells [ ] . thus, the identified ncf polymorphism and its effect on the immune system by decreased ros production was concluded to be associated with the regulation of arthritogenic cd t cells in the immune priming phase [ , ] . nadph oxidase-activating substances such as phytol ( , , , -tetramethyl- -hexadecene- -ol), were identified from studies performed on a human neutrophil cell line, and have subsequently been shown to be very efficacious in the treatment of arthritis in rats [ ] . however, the mechanism through which these compounds act is still not completely understood. the intention of this study was to obtain a molecular insight into the anti-inflammatory mechanism of nadph oxidase activation by phytol in an experimental arthritis model in rats, the pristane-induced arthritis (pia) model [ ] . to analyze the in vivo effects of the nadph oxidase activator phytol versus the effects of the arthritis-inducing compound pristane, global gene-expression profiling was performed. a biodistribution analysis of phytol was performed to determine which tissue and time point would be most relevant to investigate. phytol was observed to accumulate slowly in inguinal lymph nodes, with peak levels being reached approximately days after subcutaneous administration, which coincides with the onset period of arthritis. in the global gene-expression profiling, inguinal lymph nodes obtained days after administration were analyzed. this analysis revealed several genes that were differentially expressed in rats with pia compared with rats injected with phytol. a group of interesting genes was selected and verified by quantitative real-time pcr analysis in three further separate biological experiments, including a comparative study between the da and da.ncf e congenic rats. the comparison between the two strains was aimed at determining whether the selected genes were differentially expressed in the arthritis-susceptible da rat versus the arthritis-resistant congenic da.ncf e rat. such data would indicate whether the nadph oxidase-activating compound phytol is activating a pathway that is normally inactive in the absence of a fully functional ncf . by studying gene-expression profiles and performing pathway analysis, we have identified the importance of an ifn-β-dependent pathway as one molecular mechanism for the arthritis-ameliorating efficacy of nadph oxidaseactivating compounds. the discovery that nadph oxidase-derived ros have disease-protecting effects in arthritis opens up new possibilities for drug development against autoimmune and inflammatory diseases. to obtain a more detailed understanding of the molecular mechanism of this regulation, gene-expression profiling experiments were performed on inguinal lymph nodes from rats with pharmacologically (phytol) and genetically (ncf ) modified nadph oxidase activity. phytol has previously been identified as a potent nadph oxidase activator [ , ] . the in vivo distribution of phytol was analyzed and examined by subcutaneous injection of tritium-labeled phytol into the base of the tail in da rats. the rats were killed and dissected , , , and days after administration. various organs (inguinal lymph nodes, spleen, heart, thymus, kidney, liver, lung, fat, muscle and peripheral blood) were collected, homogenized and analyzed in a beta counter to determine tissue-incorporated tritiated phytol. most of the administered phytol remained as a depot at the injection site (> %, data not shown), whereas phytol distributed to tissues was recovered mainly from the inguinal lymph nodes. furthermore, the accumulation of phytol in the lymph nodes reached its maximum level days after administration ( figure ). no phytol was detected in peripheral blood at the time points studied or in blood collected at earlier time points ( , , or hours after administration, data not shown). further analysis of the biodistribution of phytol at the microscopic level in the inguinal lymph nodes was performed by microautoradiography. it was shown that phytol was distributed primarily to the cortical regions and also in the sinusoidal space of the inguinal lymph nodes (data not shown). moreover, the staining seemed to appear between cells or in the cell membrane, but not intracellularly. previous results have shown that the nadph oxidase activator phytol has ameliorating properties on arthritis when administrated prior to arthritis induction and when given as therapeutic treatment [ ] . to obtain a molecular understanding of the protective effects of phytol on pia, four groups of animals were subjected to different conditions; (i) induced arthritis by injection with pristane, (ii) injection with phytol, (iii) injection with both pristane and phytol, and (iv) no treatment. all animals were killed close to disease onset, i.e. days after administration. global gene-expression profiling using chip arrays (genechip ® ;affymetrix, santa clara, ca, usa) was performed on the collected inguinal lymph nodes from the described animals. the gene-expression pattern was quite different between treated rats and naïve controls. comparing any of the group of treated animals and untreated animals, > differentially expressed genes were detected (at significance level p < . and absolute fold change > . ; data not shown). however, as the most interesting differences were to be found between the different treatments, the analysis focused on these groups. when comparing phytol-treated versus pristane-treated animals, the expression of genes was significantly changed (p < . , with absolute fold change > . ). of these genes, showed expression in phytol-treated rats, whereas genes showed higher expression in pristane-treated rats ( figure ). because more complex gene induction was observed in animals given phytol plus pristane, further studies concentrated on analyses of single-compound administrations. to minimize erroneous conclusions due to technical variability and multiple testing effects inherent to the microarray technology, as well as biological variation, quantitative real-time pcr analysis was used to validate expression profiles of nine differentially expressed genes (table ) in separate biological material. this verification confirmed all examined transcripts to be differentially expressed (data not shown). in addition, there was strong correlation between the fold changes detected using global gene expression and the quantitative real-time pcr analysis for the identified genes ( the genes upregulated by phytol were best , irf , ifit , oas , mx and s a best (bone-expressed sequence tag ) is mainly expressed in bone marrow and spleen and has been proposed to be involved in bone formation. the cytokines ifn-α and ifn-γ have been shown to induce best expression in osteoblasts [ ] .interferon regulatory factor (irf)- belongs to the irf family of transcription factors involved in cell growth, antiviral defense and immune activation in lymphoid cells in spleen, thymus, and peripheral blood [ , ] the protein ', '-oligoadenylate synthetase (oas)- belongs to the oas family, which was one of the first groups of ifn-induced antiviral proteins to be characterized. the activation of this mechanism is induced by the ifn type i pathway, which is activated upon pathogenic invasion as part of an antiviral response [ ] . myxovirus resistance (mx)- has a functional role in the defense against viral infections and its expression is partly tissue distribution of tritium-labeled phytol in rats. biodistribution was estimated as relative counts per minute per gram of tis-sue figure tissue distribution of tritium-labeled phytol in rats. biodistribution was estimated as relative counts per minute per gram of tissue. a large fraction of phytol remained as a depot in the injection site (> %, not shown). besides that, the inguinal lymph nodes were the primary tissue for accumulation of phytol. the distribution of phytol to the inguinal lymph nodes showed the highest accumulation > week after injection and showed a reduction after weeks. values are means from groups of four animals. controlled by type i ifns [ ] . mx is induced by type i ifns, and to a less extent by ifn-γ and lipopolysaccharide [ ] . s a is a small calcium-binding protein that belongs to the s family. it is primarily expressed by neutrophils but also by activated monocytes and macrophages [ ] . s a has been shown to have a chemoattractant function in inflamed tissue, attracting neutrophils and inducing the adhesion of the attracted cells [ ] . elevated levels of s a have been found in the synovial fluid [ ] and plasma [ ] of patients with ra. to find a connection between the differentially expressed genes bioinformatic tools (ingenuity pathway analysis; ingenuity systems inc., ca, usa, and pathwayassist; stratagene, ca, usa) and literature studies were used to assign pathways for these genes. using these analysis tools, large schematic diagrams, covering all published interactions hierarchical clustering of differentially expressed genes in phytol-treated compared with pristane-treated rats figure hierarchical clustering of differentially expressed genes in phytol-treated compared with pristane-treated rats. euclidean distance was used as a similarity measure. each column represents one individual rat, and each horizontal stripe represents a gene transcript. the affymetrix probe set identification, the gene symbol, the average fold change (fc) for phytol versus pristane together with the p value, and the average fold change for phytol/pristane versus pristane and its p value are given to the right. the colors in the clustering represent the gene-expression level in each individual rat compared with the average expression in all arrays, where green indicates low expression and red represents high expression. low high ccl s a s a between the identified genes and common denominators, are produced. however, in this study we chose to focus on and validate one common denominator for the regulation of the identified differentially expressed genes, i.e.ifn types i and type ii (ifnγ) [ , ] . in addition, this stratification of the bioinformatic information was performed to make it possible to verify these findings using quantitative real-time pcr and to validate the biological importance of the identified pathway in further biological experiments. hence, the identified central pathways for further studies included the genes increased in expression due to treatment with pristane (ass, cxcl and mmp ), which are regulated by ifn-γ and genes with increased expression due to phytol (best , irf , ifit , oas , mx and s a ), which are induced by ifn-α/β. both ifn-α/β and ifn-γ have an important role in the immunological response to pathogens and viruses. however, type i ifns are mainly produced by plasmacytoid dendritic cells [ ] , whereas ifn-γ is produced by macrophages, natural killer (nk) cells and activated t helper (th) cells. consequently, the ifn genes were included in the panel of genes to be further studied. to analyze how the expression of the selected genes (table ) varies over time, a third biological experiment was performed. in this experiment, only pristane-treated or phyto-treated animals were studied, and inguinal lymph nodes were isolated at , , , , , , and days after injection. this time study enabled us to follow the expression of these genes during the development of arthritis induced by pristane ( figure ). the three genes associated with increased expression after pristane administration (ass, cxcl and mmp ) showed significantly increased expression (p < . ) after disease onset (i.e. days - after pristane administration) compared with the phytol-treated group. phytol induced a small increase in expression of these genes around the day of disease onset (day - ) and then decreased to the same low expression as seen at day ( figure ). the fact that the highest expression of these genes occurs after arthritis onset makes it plausible that the increased expression of these genes is associated with the inflammatory response. however, as seen for the expression of cxcl ( figure b ), which also increased significantly (p < . ) at days - after pristane injection, the expression of such genes also plays a role in the early response after injection. all genes associated with increased expression after phytol administration (best , irf , ifit , oas , mx and s a ) showed significantly increased expression (p < . ) compared with pristane ( figure ) in the early disease-onset period (day - ). the expression levels then decreased to the same low level as for the pristane-treated rats. besides the genes described above, s a and the mx related gene mx were also analyzed in this time study and shown to be expressed in a similar pattern to that of s a and mx , respectively (data not shown). as these genes are closely related, only one of each was followed for further expression characterization. the expression of ncf was also included in this analysis as a control. however, no marked difference in ncf expression was detected between the groups of animals (data not shown). the expressions of the ifn genes were also analyzed over time ( figure ). the expression of ifnγ was significantly increased in the rats injected with pristane (p < . ) and expression of ifnα and ifnβ was increased after injection with phytol. to further validate the significance of the increased expression of ifnα and ifnβ in da rats at day after treatment with phytol, rna samples produced from all four separate experiments was analyzed for ifnα and ifnβ expression levels in da rats treated with either pristane (n = ) or phytol (n = ). this combined dataset showed an upregulation of ifn-α (p < . ) and an upreg- rn. chemokine (c-x-c motif) ligand - . . the genes were chosen based on the difference in expression levels between phytol and pristane treatment in the affymetrix genechip ® analysis. *phytol versus pristane. ulation of ifn-β (p < . ) in phytol-treated compared with pristane-treated animals. the increased expression of ifnα/β coincided with the most pronounced increase in expression of best , irf , ifit , oas , mx and s a , i.e. day - after treatment ( figure and figure a , b). hence, ifnα and ifnβ could be postulated to be common regulatory genes for this molecular pathway of genes that were all induced by phytol treatment. however, it should be noted that the expression levels of the studied type i interferons were very low and that the difference in expression levels could only be detected using quantitative real-time pcr (figures and ). tnf-α and il- are other cytokines identified as important immunological regulators in the same molecular pathways as the interferons. transcription of tnfα and il- is increased in macrophages activated by ifn-α to act synergistically with ifn-γ in initiating a chronic inflammatory response. the expression of tnfα and il- was also analyzed, but only a small decrease in the expression of tnfα could be observed after phytol administration, and the expression of il- was below the detection limit (data not shown). the selected genes that were identified as being associated with either the disease state (pristane) or treatment state (phytol) of da rats, were further analyzed in another biological experiment including a comparison between da and congenic da.ncf e rats. these rats carry different alleles of the ncf gene, and differ dramatically in arthritis severity; the da rat is highly susceptible and the congenic da.ncf e is almost completely resistant to arthritis [ ] . this analysis was included to study the importance of the selected genes for the difference in arthritis susceptibility caused by different ncf alleles, and thus also nadph oxidase functionality in pia compared with phytol treatment. administration with either pristane or phytol leads to increased expression of ass, cxcl and mmp- in both strains, compared with the untreated controls (p < . ) (figure ) . further, a clear and significant difference (p < . ) between the pristane-treated and phytol-treated animals was observed, although no significant difference was found between the two rat strains. expression of best , irf , ifit , mx , oas and s a were all induced by phytol, compared with untreated controls (p < . ). interestingly, after pristane injection, there was significantly higher expression (p < . ) of best , irf , ifit and mx in da.ncf e rats than in da rats. no difference in expression levels between the strains was observed for s a . overall, the gene expression of best , irf , ifit , mx and oas showed the highest expression levels in rats that had been injected with a combination of pristane and phytol (figure ). ifnα was expressed at a lower level in both the pristanetreated and phytol-treated animals, and in the rats with combined treatment, compared with the untreated control rats (p < . ) ( figure a ). the expression of ifnβ was significantly higher (p < . ) in the pristane-injected or phytol-injected groups than in the untreated control group, while the combined treatment induced even higher expression ( figure b ). the expression of ifnγ was significantly higher (p < . ) for the rats injected with pristane than for both control and phytol-treated rats (figure c) . interestingly, only ifnβ showed different expression in the da.ncf e rats compared with da rats after pristane injection (p < . ). the expression level of ifnβ in da rats after pristane injection was comparable with that of untreated control rats; however, after phytol administration, the level of ifnβ was increased to a level equal to that of da.ncf e rats ( figure b ). the levels of different cell populations in the studied tissue may affect the outcome of gene-expression profiling. therefore, fluorescence-activated cell-sorting (flow cytometry, bd biosciences) analyses were performed on arthritis development in rats after subcutaneous administra-tion of pristane (circles) or phytol (squares) in the time study experiment figure arthritis development in rats after subcutaneous administration of pristane (circles) or phytol (squares) in the time study experiment. only animals injected with pristane developed arthritis. values are means ± sem from groups of four animals. levels of significance were calculated using student's t-test (***p < . ). isolated inguinal lymph nodes to determine whether the observed differentially expressed genes could be explained by a skewed cell population. in both the pristane-treated and phytol-treated rats, the number of b and nk cells were raised compared with untreated controls, whereas the relative number of t cells were lower. significantly more b and nk cells were observed after phytol treatment than after pristane treatment (p < . , da and da.ncf e combined). no significant alterations in the subset of cells containing macrophages and dendritic cell populations (i.e. cd +tcr-cells) could be detected (data not shown). no difference between the strains was observed for all of the studied groups of animals ( figure ). these effects on cell populations could have a major impact on the gene-expression profiles, but as no difference between the strains was observed, the differentially expressed genes assigned to the ifnβ pathway are unlikely to be the result of differences in these cell populations. ncf is a gene encoding the activating component of the nadph oxidase complex. by positional cloning in rat models of arthritis, a polymorphism of ncf was found to regulate arthritis severity [ ] . in fact, it was shown that a less functional ncf , and the resulting decrease in nadph oxidase capacity to produce ros, is a major cause of increased arthritis severity in both rat and mouse models of arthritis, an observation that challenges the general dogma of the inflammatory role of ros. furthermore, nadph-activating substances have both preventive and therapeutic effects on arthritis, which opens up new approaches for disease treatment [ ]. however, from the results of the positional cloning of ncf and the data showing strong ameliorating effects of ros-inducing compounds in animal models, we are still a long way from fully understanding the underlying mechanism of action. one approach to obtaining a molecular insight into a complex biological system is to use global gene-expression profiling [ ] . an advantage of this method is the large set of genes that can be analyzed in the same experimental setup, making it possible to use clustering and pathway analysis of large sets of genes [ , ] . the assembled biological information can then be stratified into molecular pathways that can be studied and thoroughly validated. however, the intricate choice of time point and tissue/cell type to be selected for analysis presents a significant hurdle [ ] . a further difficulty is the need for biological replicates and the genetic heterogeneity that is involved in human studies. this has naturally led to some skepticism regarding the utility of gene-expression profiling as a method to achieve an understanding of rheumatic dis- values are means ± sem from groups of four animals. levels of significance were calculated using student's t-test (*p < . ; **p < . ; ***p < . ). eases [ ] . however, recent analysis of gene-expression fingerprints of individual patients with ra might give hope for this still technically evolving method for understanding complex disorders [ ] [ ] [ ] [ ] . in this study, we used global gene-expression analysis and quantitative real-time pcr techniques in four separate arthritis experiments in rats to investigate the downstream effects of preventive arthritis treatment with the nadph oxidase activator phytol. based on biodistribution analyses of phytol after sc administration, global gene-expression analysis was performed on inguinal lymph nodes at a time point just before the estimated day of disease onset. we observed that the expression of our set of differentially expressed genes in our material varies dramatically during the disease progress (figures , , ) . therefore, it is necessary to have information about which time point and tissue to analyze before initiating gene-expression profiling. furthermore, we also analyzed, in other tissues (i.e. thymus, blood and spleen) the expression of the genes identified as differentially expressed in inguinal lymph nodes, and observed that the expression levels could be quite different or even undetectable in these tissues (data not shown). as a result, it is not possible to extrapolate the information between tissues and time points. in addition, the number of biological replicates used in the compared groups should be high enough to enable statistical analysis. altogether, these issues make these studies extremely cumbersome to perform, especially when analyzing human samples. by using animal models, a more optimized study design may be provided [ ] , as a number of identical (inbred) individuals under the same treatment and environmental conditions are compared and tissue collected at the same time point by the same researcher. in this study, we identified a molecular mechanism that links the protective effects observed from treatment with nadph oxidase activators with a relevant inflammatory mechanism. however, it is not the differentially expressed genes per se that are most interesting, but the molecular pathways that can be extracted (figure ). from our comparison between pristane (arthritis inducer [ ]) and the in lymph nodes at , , , , , , and days after injection with pristane (circles) or phytol (squares). values are means ± sem from groups of four animals. levels of significance were calculated using student's t-test (**p < . ). phytol (nadph oxidase-activating and arthritis-ameliorating compound [ ]), two main pathways were identified. the pristane-induced pathway was marked by increased expression of ass, cxcl and mmp (table ) . these genes all share ifn-γ as a common regulator [ , , ] , which was also shown to be upregulated in the inguinal lymph nodes, in a similar pattern, after induction of arthritis with pristane (figures , c, and c). ifn-γ is known to effect the balance between th and th cells, and increased expression of this pro-inflammatory cytokine plays an important role in the progression of ra [ ] . although pristane stimulates the expression of these cytokine-regulated genes, no direct conclusions about whether these genes are involved in the cause of arthritis can be drawn, as no difference in gene expression could be seen between the arthritis-susceptible strain (da) and the arthritis-protected strain (da.ncf e ) (figures and c) . as the da.ncf e congenic rats carry the arthritis-protecting variant of ncf , one might speculate that the effect caused by the functional ncf is to generate a resistance to the effect from induction of ass, cxcl and mmp genes, which might otherwise contribute to the development of arthritis. further investigation of the effect on disease regulation is needed to be able to propose a more exact role for these genes as potential markers of arthritis [ ] . six genes (best , irf , ifit , mx , oas and s a ) were identified as genes induced by phytol ( table ) interestingly, a clear tendency in the expression of ifnβ between da and da.ncf e strains was observed after injection with pristane, with the da.ncf e rats having higher expression of ifnβ than the da rats ( figure b ). this difference in expression level clearly resembles the differences observed for the phytol-induced genes best , irf , ifit , mx and oas (figure ). it is therefore likely that the downstream effect of the polymorphism in ncf , as well as the therapeutic effect of phytol, involves regulation of an ifn-β pathway. this ifn-β-regulated gene profile is interesting with respect to similarities to the patterns that have been identified in patients with sle and in patients with juvenile arthritis treated with anti-tnf-α [ ] [ ] [ ] [ ] , and in mouse models of sle [ ] . subpopulations with active ra have also been shown to express increased levels of ifn type i signature genes in peripheral blood [ ] . these reports show an ifnα/β expression signature, which is very similar to that identified in the rats treated with phytol. however, in our study, the ifn type i expression profile was linked to disease protection while the sle ifn type i profile was linked to disease progression. this may indicate that the interferon balance could be an important threshold denominator of autoimmune dis-eases, functioning as an essential balance for immune regulation, with an imbalance resulting in either sle or arthritis. this is also exemplified by a recent study in which pristane was used to induce sle in mice. in that study, chronic peritoneal administration of pristane elicited increased expression of the type i interferon-inducible genes mx , irf , ip- and isg- as a consequence of sle [ ] . however, despite the similarities in expression pattern, it must be noted that the sle studies were performed on tissues other than inguinal lymph nodes and on established disease, so that differences in gene profiles could be caused by tissue differences or time-dependent regulation. also interesting is the fact that the present study points towards a disease-ameliorating pathway that is regulated by ifn-β. therefore, as the present observation of increased ifn type i signature genes was observed to be a signal for prevention of disease onset, in contrast to the studies in ongoing sle [ ] and arthritis [ ] , one might speculate that increased ifn type i regulation is a way to downregulate an ongoing immune response. such an attempt to limit the inflammatory response has been suggested previously [ ] , and has also been indicated by experiments in our laboratory using ifnβ-deficient mice, schematic representation of the differentially expressed genes and how they are induced upon injection by (a) pristane or (b) phytol figure schematic representation of the differentially expressed genes and how they are induced upon injection by (a) pristane or (b) phytol. the molecular pathway that is shared between the arthritis resistant da.ncf e rat strain and the arthritis treatment achieved with phytol is the ifn-β-dependent pathway, which results in the upregulation of best , irf , ifit , oas and mx . the inflammatory arthritis that is induced by an injection with pristane is characterized by increased expression of the ifnγ-connected signature genes ass, cxcl and mmp , which potentially could be used as molecular markers for an initiating inflammatory response. as this pathway is also induced by pristane in the da.ncf e strain this upregulation per se does not cause arthritis. most important in this mechanism is how administration of phytol induces arthritis protection via the ifn-βregulated pathway in the arthritis-susceptible da rats. where a prolonged arthritis severity was observed due to ifnβ deficiency [ ] . in fact, treatment with recombinant ifn-β significantly reduces cartilage destruction and bone destruction in collagen-induced arthritis in mice, which suggested a beneficial effect in patients also [ ] . however, to date no positive outcome of clinical trials using recombinant ifn-β in ra has been presented [ , ] . as we have shown that the effect of phytol on ifnβ-related genes is time-dependent and also correlates with the biodistribution of phytol to the inguinal lymph nodes, it is possible that tissue distribution as well as the dose and frequency of administration are crucial for the efficacy of arthritis treatment via this pathway. the data presented here, together with gene-expression profiles of sle, [ ] [ ] [ ] [ ] and arthritis [ ] strongly suggests the importance of the ifn-α/β pathway as a key mechanism in autoimmune conditions [ ] . the fact that no difference in cell populations was observed to explain the differential levels of mrna for ifn-β-regulated genes does not prove that alterations in cell populations in the inguinal lymph nodes are not crucial. as ifn-β is mainly produced by plasmacytoid dendritic cells [ ] , which were not specifically addressed in this study, more careful analysis of this cell population might provide further insight into the disease-protecting effects of phytol in arthritis. increased expression of s a was observed in rats treated with phytol ( figure f ). however, the expression of s a did not show any difference between da and da.ncf e rats ( figure f ), and the expression profile was not similar to that of ifnβ ( figure b ). as a result, we consider s a not to be regulated by ifn-β. it is more likely that the increased levels of s a expression is a direct effect of increased concentrations of intracellular ca + induced by the nadph oxidase-produced ros [ , ] . increased expression of s a is mostly reported to be proinflammatory. however, this knowledge is based on analyses from tissues and blood during ongoing inflammation, while these studies analyzed inguinal lymph nodes prior to disease onset. hence, the expression levels for different timepoints of the disease process could differ. even so, s a may be a good biomarker for the in vivo efficacy of administered phytol as an activator of nadph oxidase specifically in lymph node tissues. by targeting the nadph oxidase complex with activating compounds such as phytol, we highlight a new mechanism to treat autoimmune conditions such as arthritis. by extracting and verifying a relevant biological molecular mechanism from gene-expression profiling data, we also indicate a plausible relation between increased levels of ros and an anti-inflammatory response regulated by an ifn-β pathway. the use of gene-expression profiling to compare treatments in animal models of complex diseases also points to a useful pharmacogenomic approach to extract relevant information about the mechanism of action and to identify potential molecular biomarkers to be used in future animal experiments and clinical trials. da rats used in the microarray analysis, the first verifying quantitative real-time pcr study, the time study and the biodistribution study were purchased from harlan netherlands, and the da.ncf e rats with background origin from zentralinstitut für versuchstierzucht, hannover, germany [ , ] . all animals were kept in a climate-controlled environment with -hour light/dark cycles, housed in polystyrene cages containing wood shavings and fed standard rodent chow and water ad libitum. pristane (sigma-aldrich, st. louis, mo, usa) and/or phytol ( , , , -tetramethyl- -hexadecene- -ol) (sigma-aldrich) were injected into the rats (age - weeks) by a single subcutaneous injection of μl at the base of the tail. arthritis development was monitored with a macroscopic scoring system of the four limbs ranging from to ( point for each swollen or red toe, point for midfoot digit or knuckle, points for a swollen ankle). the scores of the four paws were added, yielding a maximum total score of for each rat [ ] . in the global gene-expression profiling and the first verifying quantitative real-time pcr experiment, five rats per group were used. in the comparative experiment between the da and the da.ncf e strain, - rats per group were used. in all three experiments, the analyzed groups were; naïve controls, pristane-treated, phytol-treated and pris-tane plus phytol-treated animals. all rats were killed days after injection. in the time-resolution study for comparison between pristane and phytol, four animals from each group were killed at , , , , , , and days after injection. the inguinal lymph nodes were immediately surgically removed and stored in a tissue-storage reagent (rna later; qiagen, germany). da rats were injected with μl phytol (sigma-aldrich) mixed with tritiated phytol (moravek biochemicals, ca, usa) to a final dose of μci/rat. the rats, four each day, were killed at , , , or days after injection, and the inguinal lymph nodes, spleen, heart, thymus, kidney, liver, lung, adipose tissue, muscle, injection-site tissue and blood were collected in equal amounts of saline solution (blood samples had heparin added to prevent coagulation) in pre-weighed tubes. the tissues were weighed, homogenized, and mixed with ready-safe scintillation liquid (beckman coulter, ca, usa). tissue distribution of phytol was determined as counts per minute (cpm) of tritium using a beta counter (lkb wallac, turku, finland) and cpm/g tissue was determined as the relative distribution of phytol. microautoradiography was performed on three individual rats days after subcutaneous administration with tritiated phytol ( μci/rat). the inguinal lymph nodes were snap-frozen in liquid nitrogen and shipped on dry ice to quest pharmaceutical services (newark, de, usa). the frozen lymph nodes were embedded in optimum cutting temperature (oct) embedding media (vwr international, bristol, ct, usa) for cryosectioning. sections of μm thickness were heated at °c for minutes, coated with photographic emulsion (kodak ntb; kodak, new haven, ct, usa) and dried. the coated slides were exposed at °c in lightproof boxes. the exposed slides were stained with hematoxylin and eosin and developed for tritium labeling. single-cell suspensions were made from inguinal lymph nodes and cells were stained with the anti-rat antibodies ox- (anti-lca, lymphocytes), ox- (anti-cd ra, bcells), and r (anti-αβtcr, t cells) (all bd pharmingen, san diego, ca, usa), and with . . (anti-nkrp , nk cells, produced from an in-house hybridoma) for minutes at °c. after washing with phosphate-buffered saline (pbs), cells were resuspended in pbs and analyzed in a facsorter (becton dickinson, san jose, ca, usa). gates were set for the relevant cell type and analysed as percentage of total lymphocytes. total rna from inguinal lymph nodes was isolated using a commercial kit (rneasy ® mini kit; qiagen, germany). the protocol for animal tissues was followed with the addition of the optional dnase digestion (rnase-free dnase set; qiagen). the rna yield was quantified spectrophotometrically (rna nano assay kit;agilent technologies, palo alto, ca, usa) and the quality analyzed ( bioanalyzer; agilent). the average ratio between s/ s rrna was . , indicating high rna quality. all rna and cdna samples were stored at - °c. in total, μg of total rna spiked with poly-a controls (pgibs-trp, pgibs-thr, and pgibs-lys; american type culture collection) was converted to cdna, using a t promoter-polyt primer (affymetrix, santa clara, ca, usa) and the reverse transcriptase superscript ii (invitrogen, paisley, uk), followed by a second-strand cdna synthesis (invitrogen). double-stranded cdna was in vitro transcribed to biotinylated crna (ivt labelling kit; affymetrix) and then fragmented. the fragmented crna was mixed with hybridization spike controls (oligonucleotide b and a crna cocktail: biob, bioc, biod, and cre; affymetrix,). aliquots of each sample were hybridized ( hours at °c) to an array (genechip ® rat expression set a arrays; affymetrix). the arrays were subsequently washed, stained and scanned according the manufacturer's instructions (genechip ® expression analysis technical manual; affymetrix). the data were analyzed using specific sofware (robust multi-chip analysis in genetraffic ® uno version . - ; stratagene, la jolla, ca, usa, and spotfire decisionsite for functional genomics, version . ;spotfire inc., göteborg, sweden). the intensities were log transformed and the mean log intensity for each group calculated. the mean log fold change was calculated for the phytol-treated animals versus the pristanetreated animals by subtracting the mean log intensity for the pristane-treated rats from that for the phytol-treated rats. the total number of probe sets in the used rat affymetrix chips was , and the average present call was %. statistical significance of the difference in gene expression was determined using the two-sided student's t-test. a transcript was considered differentially expressed if the mean absolute fold change was > . and the p value < . . in addition, the mean intensity in the group showing the highest expression should be > . the average log fold change between the animals treated with phytol plus pristane versus the animals treated with pristane alone and the corresponding statistical analysis were also calculated, although these data were not used to identify differentially expressed genes. the global gene-expression profiling data is deposited online [arrayexpress: e-mexp- ]. the primers used in the quantitative real-time pcr were designed using software (primer express . ; applied biosystems, foster city, ca, usa) and the sequences are listed in table . total rna ( μg) was transcribed to cdna using a commercial system (superscript™ first strand synthesis system; invitrogen). the pcr reaction was performed in a μl volume including × sybr ® green (applied biosystems) and nm of each primer, except for infβ, for which nm of each primer was used. all reactions were performed in duplicate, amplified and quantified (abi sequence detection system; applied biosystems). the relative quantities of mrna were calculated according to the standard curve method [ ] and arbp (alias b ), was used as endogenous control [ ] . quantitative data is expressed as mean ± sem and significance analysis was performed using two-sided student's ttest. * represents a significance value of * p < . , ** p < . and 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clinical laboratory markers of inflammation identification and isolation of dominant susceptibility loci for pristaneinduced arthritis positional cloning of ncf -a piece in the puzzle of arthritis genetics genetic analysis of mouse models for rheumatois arthritis appliedbiosystems: user bulletin # abi prism sequence detection system b cdna used as an estradiol-independent mrna control is the cdna for human acidic ribosomal phosphoprotein po po, an, mh and en contributed to the design and performed the experiments, collected and interpreted the data, and wrote the manuscript. sa and ab performed the real-time pcr under supervision of an and po. rh contributed to the study design, interpretation of collected data and the writing of the paper. the global gene-expression profiling data is deposited at arrayexpress (accession number e-mexp- ). key: cord- -xuszdrsa authors: hackbart, matthew; deng, xufang; baker, susan c. title: coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors date: - - journal: proc natl acad sci u s a doi: . /pnas. sha: doc_id: cord_uid: xuszdrsa coronaviruses (covs) are positive-sense rna viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. covs encode an endoribonuclease designated endou that facilitates evasion of host pattern recognition receptor mda , but the target of endou activity was not known. here, we report that endou cleaves the ′-polyuridines from negative-sense viral rna, termed pun rna, which is the product of polya-templated rna synthesis. using a virus containing an endou catalytic-inactive mutation, we detected a higher abundance of pun rna in the cytoplasm compared to wild-type−infected cells. furthermore, we found that transfecting pun rna into cells stimulates a robust, mda -dependent interferon response, and that removal of the polyuridine extension on the rna dampens the response. overall, the results of this study reveal the pun rna to be a cov mda -dependent pathogen-associated molecular pattern (pamp). we also establish a mechanism for endou activity to cleave and limit the accumulation of this pamp. since endou activity is highly conserved in all covs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging cov infections. coronaviruses (covs) are positive-sense rna viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. covs encode an endoribonuclease designated endou that facilitates evasion of host pattern recognition receptor mda , but the target of endou activity was not known. here, we report that endou cleaves the ′-polyuridines from negative-sense viral rna, termed pun rna, which is the product of polya-templated rna synthesis. using a virus containing an endou catalytic-inactive mutation, we detected a higher abundance of pun rna in the cytoplasm compared to wildtype−infected cells. furthermore, we found that transfecting pun rna into cells stimulates a robust, mda -dependent interferon response, and that removal of the polyuridine extension on the rna dampens the response. overall, the results of this study reveal the pun rna to be a cov mda -dependent pathogen-associated molecular pattern (pamp). we also establish a mechanism for endou activity to cleave and limit the accumulation of this pamp. since endou activity is highly conserved in all covs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging cov infections. coronavirus | endoribonuclease | endou | nsp | interferon c oronaviruses (covs) are positive-sense rna viruses that replicate in the cytoplasm of infected cells. the positivesense virion rna is translated to generate the viral replication machinery, which then replicates the positive-sense rna into negative-sense, genomic rna and subgenomic rnas (sgrnas). the negative-sense rnas then function as templates for synthesis of positive-sense genomic rna and sgrna ( , ) . this replication strategy can generate long double-stranded rna (dsrna) intermediates ( ) , that may act as pathogen-associated molecular patterns (pamps) recognized by cytoplasmic pattern recognition receptors (prrs) ( , ) . the specific prr that recognizes cov rna is mda , which can activate the type i interferon (ifn) response in macrophages ( ) . other host dsrna prrs, such as pkr and oas, are also activated and operate to limit cov replication ( ) ( ) ( ) ( ) ( ) . covs encode multiple proteins that antagonize these innate immune responses, particularly the activation of the ifn response ( , ( ) ( ) ( ) ( ) ( ) , ultimately leading to a dysregulated immune response and increased immunopathogenesis ( , ) . understanding the mechanisms used by covs to delay ifn signaling may provide opportunities for the development of antivirals and live-attenuated vaccines to limit cov infections. here, we investigate the mechanism used by one cov ifn antagonist, the nonstructural protein (nsp ), which is an endoribonuclease designated endou. endou is highly conserved in all known covs ( , ) . endou is similar to the cellular endoribonuclease xendou, as revealed by bioinformatic analysis of the amino acid sequence ( ) . x-ray structures of endou revealed conserved endoribonuclease folds with catalytic histidine residues required for endoribonuclease activity ( ) ( ) ( ) ( ) ( ) . purified endou was shown to cleave single-stranded rna and dsrna at uridine residues in in vitro assays ( , , ( ) ( ) ( ) ( ) . however, the target of endou activity during viral infection was unknown. initial studies revealed that endou colocalizes with the viral replication complex ( , ) , and it was suggested that endou was necessary for efficient virus rna replication in cell culture ( , ) . more recent findings, however, revealed that endou catalytic mutant (endoumut) viruses replicate as well as wild-type virus in ifn-nonresponsive cells, but are severely impaired for replication in ifn-responsive macrophages ( , ) . these recent results revealed that endou activity is important for limiting the sensing of viral rna by host dsrna sensors such as mda , pkr, and oas/rnasel. limiting viral rna recognition contributes to delayed type i ifn responses; thus viruses with intact endou activity are more virulent than their endou-mutant counterparts ( , , ) . in this study, we show that cov endou activity limits the abundance and length of the polyuridine (polyu) extension on ′-polyu-containing, negative-sense (pun) rnas for both the beta-cov mouse hepatitis virus strain a (mhv-a ) and the alpha-cov pedv. importantly, we found that the pun rnas can act as pamps recognized by mda . overall, we propose a mechanism for endou, which is to cleave polyu sequences from pun rnas, thus limiting the formation of a pamp and impeding the ability of mda to activate the innate immune response to infection. endou activity reduces the accumulation of an epitope recognized by an anti-dsrna antibody in cov-infected hepatocytes. previously, we reported that endou activity delays the accumulation of an epitope recognized by the k antibody in the cytoplasm of ifnar −/− bone marrow-derived macrophages (bmdms) as measured by immunofluorescence ( ) . the k antibody was shown to recognize significance cells carry sensors that are primed to detect invading viruses. to avoid being recognized, coronaviruses express factors that interfere with host immune sensing pathways. previous studies revealed that a coronavirus endoribonuclease (endou) delays activation of the host sensor system, but the mechanism was not known. here, we report that endou cleaves a viral polyuridine sequence that would otherwise activate host immune sensors. this information may be used in developing inhibitors that target endou activity and prevent diseases caused by coronaviruses. dsrna; therefore, we hypothesized that the cov epitope was dsrna. to determine whether this phenotype is present in a stable cell line, we infected ifn-responsive aml hepatocytes with wild-type or endoumut mhv and measured accumulation of replication complexes (anti-nsp / ) and dsrna foci (anti-dsrna, k ) at h postinfection (hpi) (fig. a) expression during infection (si appendix, fig. s ). we quantified the number of nsp / foci and dsrna foci from individual cells. we found that, while the numbers of nsp / -labeled replication complexes were not significantly different (fig. b, right) , the total number of dsrna foci per cell was elevated in endoumutinfected cells (fig. b, left) . median fluorescent intensity of the individual dsrna foci was also brighter in endoumut-infected cells (fig. c) . these results indicate that endoumut infection results in increased abundance of an epitope recognized by the k anti-dsrna antibody. the viral rna recognized by the k antibody during cov infection is negative-sense rna. since the rna bound by the k antibody accumulates in the absence of endou activity, we sought to identify this rna. to this end, we sequenced the rna precipitated with the k anti-dsrna antibody. we obtained ∼ million reads for each total rna sample and ∼ million reads for immunoprecipitated samples. upon mapping the reads to the mouse genome, we found similar read counts to host genes from both wild-type− and endoumut-infected cells (data available at ncbi geo database, accession no. gse ) ( ) . we then mapped the reads to the mhv-a genome (genbank accession no. ay ) ( ) , and separated the viral reads by strand specificity, expecting to identify complementary sequences from positive-and negative-sense rna. surprisingly, we found that the majority of reads from the immunoprecipitated rna sample mapped to negative-sense rna ( fig. a) . we discovered that . % of the reads from the input rna sample mapped to positive-sense rna. in contrast, . % of the reads from the immunoprecipitated rna mapped to negative-sense rna. we found that the reads from the input rna sample mapped across the entire mhv genome, as expected ( fig. b and d). similarly, the reads from the immunoprecipitated rna sample also mapped across the entire genome ( fig. c and e). we concluded that the k antibody immunoprecipitated fulllength, negative-sense rnas. when comparing the read counts between wild-type virus-and endoumut virus-infected samples, we found an eightfold increase ( × read counts versus × read counts) in the abundance of the reads from the endoumut virus-infected samples ( fig. a) . these results are consistent with the increase in dsrna foci observed in endoumut-infected cells by immunofluorescence staining (fig. ) . to determine the abundance of the dsrna signal in other cell types, we infected ifnar −/− bmdms, c bl/ bmdms, and aml cells with either wild-type or endoumut virus, and performed the anti-dsrna immunoprecipitation experiment. we used random hexamers as primers for complementary dna (cdna) synthesis, which allows for generation of cdna from both positive-and negative-sense rna, and then evaluated the abundance of cdna by qpcr. we consistently detected elevated levels of viral rna immunoprecipitated by the dsrna antibody from endoumut virus-infected cells as compared to the levels detected in wild-type virus-infected cells (fig. a) . the total input viral rna was similar between wild-type− and endoumutinfected cells (fig. b) . overall, our sequencing and qpcr results suggest that endou reduces the accumulation of a negative-sense viral rna epitope that can be recognized by the anti-dsrna antibody. endou activity limits abundance and length of pun rnas. previous studies showed that the ′ end of the cov negative-sense rna contains polyu extensions ( ) , and that endou cleaves at uridine residues ( , , ( ) ( ) ( ) ( ) . therefore, we considered the pun rna as a potential target for endou activity. we hypothesized that pun rnas accumulate in the absence of endou activity. to quantitate the pun rnas, we generated cdna from the negative-sense rna using a strand-specific primer and performed a series of qpcrs with primers shown in fig. a . primer set flanks a taqman probe and provides a measurement of total negative-sense rna. primer set measures the pun rna. by normalizing set to set , we can compare relative proportions of the negative-sense rna that contain polyu sequences. to control for potential "self-priming" of the viral rna during cdna synthesis, we performed cdna synthesis in the presence or absence of the negative-sense cdna primer and quantified rna expression by qpcr (fig. b ). for both set and set qpcrs, we detected a significantly higher signal with the negative-sense primer compared to no primer. when comparing wildtype− and endoumut-infected cells, we detected a -fold increase in pun rnas from endoumut-infected cells as compared to wild-type virus-infected aml cells (fig. c , left) and detected a -fold increase in ifnar −/− bmdms (fig. d, left) . to determine whether the polya tail on the positive-sense rna was similarly reduced by endou activity, we used either random hexamers or oligo-dt primers for reverse transcription and determined that the abundance of polya tails on positive-sense rna does not differ between wild-type and endoumut infections ( fig. c and d, middle and right). we concluded that endou activity reduces the abundance of negative-sense rna that contains polyu extensions. to determine whether endou reduces the lengths of the polyu extensions on the pun rna, we completed a nested pcr to obtain polyu-containing pcr products with a minimum predicted size of ∼ base pairs (bp) (fig. a ). we detected pcr species of ∼ bp from both wild-type− and endoumutinfected cells, and detected a smear of larger pcr species unique to endoumut virus-infected cells (fig. b ). to determine whether the length of polya tails on the positive-sense rna was affected by endou activity, we generated cdna with oligo-dt primers to select for polya-containing rnas and performed the nested pcr reactions. we found that the products generated from positive-sense rna were similar between wild-type and endoumut viruses, consistent with our previous results indicating that the polya tail is not cleaved by endou activity (fig. c ). to determine whether the smear of pcr amplicons represents extended polyu sequences, we sequenced the amplicons with next-generation sequencing and found that endoumut pcr amplicons had an increase in the number of reads and proportion of products with extended polyu sequences (fig. d ). the most striking feature of the sequencing results is the bimodal distribution of the polyu extensions present in the endoumut-infected cell samples. we found that the majority ( %) of the reads from wild-type virus infection contained uridine residues. in contrast, only % of the reads from the endoumut virus-infected sample contained uridine residues. this was not due to a difference in the number of reads with uridines but to an increase in longer polyu extensions detected in endoumut-infected cells. we detected variability in the polyu extensions in the endoumut virus-infected sample, with % of the reads containing from to uridine residues. we note that, while pun rnas in endoumut-infected cells are only a few uridines longer than the pun rnas from wild-type virusinfected cells, the pun rnas are -fold more abundant in endoumut virus-infected cells (fig. ) . overall, these experiments revealed that endou activity reduces the abundance and length of polyu extensions on pun rnas, consistent with our hypothesis that endou cleaves the pun rnas during virus replication. since endou is conserved among covs, we sought to determine whether endou reduces the abundance and length of the pun rnas in the alpha-cov porcine epidemic diarrhea virus (pedv). although the endou domains of mhv and pedv exhibit only about % overall amino acid similarity, the catalytic histidines are % conserved ( ) . we showed that inactivation of endou in pedv results in an increased type i and type iii ifn response during infection ( ) . to determine whether endou limits the accumulation of pun rnas during pedv infection, we infected cells with either wild-type or endoumut pedv, isolated rna, and evaluated the levels of pun rnas. we found that, relative to wild-type virus-infected cells, endoumut virus-infected cells contained abundant pun rnas in pk (fig. a ) and vero cells (fig. b) . sequences of pcr products templated by pun rna revealed that the length of the polyu extensions on the pun rnas was increased during endoumut virus infection (fig. c and e) , with a similar bimodal distribution of polyu extensions shown in fig. d . during pedv infection, we did not observe a difference in polya tail length (fig. d) . taken together, these results indicate that pun rnas are generated during alpha-and beta-cov replication, and that the highly conserved endou activity targets the polyu extensions in the pun rna. pun rna is a pamp. since endou both reduces pun rna abundance and suppresses host mda activation, we hypothesized that cov pun rna is a pamp. to directly test this hypothesis, we measured ifn stimulation following introduction of pun rnas derived from mhv-a into aml cells. pun rna was synthesized by t in vitro transcription of digested plasmids that contained sequences representing the ′ end or ′ end of the viral genome (fig. a) . the pun rna, designated n , and other cov positive-and negative-sense rna termini (p , p , n ) were transfected into aml cells. total cellular rna was harvested at h posttransfection (hpt) and subjected to qpcr for ifnβ messenger rna (mrna) expression. we found that the presence of pun rnas increased ifnβ expression by , -fold (fig. b) , which was fourfold higher than any other in vitro transcribed viral rna, indicating that pun rna is a pamp. to determine whether the polyu sequence contributed to the robust ifn stimulation of the pun rna, we transcribed pun rna containing either uridines (n ) or no uridines (n .nou) at the ′ end. we found that removing the uridines from the pun rna significantly decreased the ability of that rna to induce ifnβ expression (fig. c) . also, removing sections of the ′ end of the pun viral sequence (n . and n . ) resulted in a decrease in ifnβ expression, suggesting the polyu sequence alone is not sufficient to induce the ifn response (fig. c) . shortening the polyu extension to eight uridines (n . u) or four uridines (n . u) also diminished the ifn activation by the pun rna (fig. d) . these results suggest that a polyu sequence of uridines can enhance the ifn response to pun rna. previous studies documented that mhv-a infection induces ifn through mda signaling ( , ) . to determine whether pun rna activates mda , we generated mda knockdown (mda -kd) aml cells by crispr-cas transduction (fig. e) ifnβ expression in an mda -dependent manner (fig. f and g) . during viral infection of mda -kd cells, both wild-type and endoumut virus infections had a significant reduction of ifnβ expression (fig. f) . ifnβ induction by in vitro transcribed pun rna also was significantly reduced in mda -kd cells (fig. g) . importantly, we found that a single-stranded, in vitro-transcribed rna activated mda , which was previously known to be activated by long complementary dsrna. taken together, these data suggest that the pun rna can act as an mda -dependent, viral pamp. endou can degrade pun rna and dampen ifn activation. to determine whether endou activity can cleave the pun rna pamp, we performed a series of in vitro cleavage assays ( ) . we incubated endou with ′ negative-sense rna containing a -uridine extension (rna ) or without the -uridine extension (rna ) (fig. a) . when either rna or rna is mixed with endou in the presence of mncl , the rna is degraded over time (fig. b ). this degradation is most likely due to the presence of multiple uridines throughout rna and rna , which is consistent with previous studies ( ) . we observed that endou cleaves rna more slowly than rna in this assay. we speculate that the polyu extension on rna may promote the formation of rna secondary structures, which could contribute to the relative stability of rna versus rna . to determine whether the polyu extension can be cleaved, we substituted the viral sequence uridines with adenosines and generated rna and rna (fig. a ). when mixed with endou and mncl , the polyu extension of rna is cleaved, producing a cleavage product the size of rna (fig. c) . rna was not cleaved, consistent with the requirement of uridine residues for endou recognition and cleavage. to determine whether endou cleavage can decrease the ability of pun rna to stimulate ifn, we cleaved the pun rna with endou (fig. d ). in the presence of endou and mncl , the pun rna was degraded into smaller rna fragments. after endou treatment, we transfected the pun rnas into aml cells and measured ifn stimulation (fig. e) . we found that transfecting the rna treated with endou decreased the ifn stimulation activity. we note that the pun rna with mncl migrated faster in the agarose gel, likely due to the addition of the mn + cation ( ), but we do not observe a difference in ifn stimulation in the presence of mncl alone. overall, endou is capable of cleaving and degrading pun rna, which then reduces the ability of pun rna to stimulate ifn. our study reveals that cov endoribonuclease activity degrades pun rna, which acts as a viral pamp. endou cleaves the polyu sequence on the pun rna, limiting the length and abundance of the polyu extension. this reduces the ifn-stimulating effect of pun rna, which, without endou digestion, activates host sensor mda . the fact that endou is highly conserved in all covs suggests that endou activity is important for sustained replication in the host ( , ) . our study reveals that the pun rna is a pamp and that endou activity is essential for limiting the accumulation of pun rna. we developed a model consistent with our findings (fig. ). we hypothesize that, during the synthesis of negative-sense rna, the cov rna-dependent rna polymerase uses the polya tail as a template to generate negative-sense rnas with variable lengths of polyu extensions. endou can recognize and cleave the polyu extensions, which limits the ability of the negative-sense rna to form a viral pamp. in the absence of endou activity, the polyu extension on the pun rna enhances the interactions of the pun rna with a complementary region of the viral genome to form an epitope recognized by mda and the k anti-dsrna antibody. one of the surprising findings from our study is that antibody k , which was developed as an anti-dsrna antibody ( ) , recognizes cov negative-sense rna (figs. and ). our immunofluorescence studies showed that the epitope recognized by k accumulates in endoumut-infected cells. using rna sequencing (rna-seq), we determined that the rna bound by k was negative-sense rna. we speculate that cov negative-sense rna forms a higher-order rna structure recognized by the k antibody, and that this rna is also recognized by host sensors. supporting this idea, a previous study showed that the viral rna recognized by the k antibody during encephalomyocarditis virus infection formed a higher-order rna structure and could activate mda ( ) . our approach using rna-seq analysis of immunoprecipitated rna could be widely used to determine whether other unique dsrna epitopes are generated during viral infections. schönborn et al. developed four anti-dsrna antibodies: j , j , k , and k ( ) . these antibodies were generated against the l species of dsrna from saccharomyces cerevisiae, and each antibody has unique binding specificities to different dsrna species. for example, the k antibody was reported to be highly specific to poly i:c, whereas the j antibody is specific to the l species of dsrna ( , ) . the differing specificities suggest that each anti-dsrna antibody recognizes unique dsrna structures or sequences. ultimately, structural studies are needed to fully elucidate the higher-order rna structures that these dsrna antibodies are recognizing during cov infection. identifying viral pamps and the host prrs that are activated by the pamps is critical for developing strategies for treating viral infections. previous studies implicated mda as the prr important for macrophages to respond to cov infection ( , ) . consistent with these studies, we report that the pun rna acts as a pamp, recognized by mda (fig. ) . we show that knocking down mda by crispr-cas limits the ifn stimulation by pun rna. canonically, mda binds to long dsrna species, such as poly i:c, to induce ifn signaling ( ). our study suggests that the negative-sense rna may form a higher-order rna structure that can bind and activate mda . interestingly, a longer polyu extension (> uridines) on the rna drives a heightened ifn response. currently, many studies utilize nucleic acid as an adjuvant to stimulate innate immune responses ( ) . with the pun rna forming an mda -recognized structure, it rna treated by endou cleavage was transfected in aml cells, and, at hpt, rna was purified from cell lysates, and ifnβ gene expression was measured by qpcr. ifnβ gene expression is normalized to s rrna and set relative to mock. values were analyzed by student t tests. data are representative of three independent experiments and presented as mean ± sd. n.s., not significant. would be interesting to determine whether the pun rna could act as an mda adjuvant to elicit robust ifn responses during immunizations. our study raises the question of whether other viruses have mechanisms to limit polyu-containing rna from activating host prrs. the hepatitis c virus (hcv) genome encodes a long polyu stretch on the positive-sense rna, but the polyu region is flanked by highly structured rna, which may limit immune stimulation ( ) . furthermore, the hcv replication complexes may hide the viral rna from recognition by host sensors ( ) . polioviruses prime replication of the negative-sense rna with a polyu sequence attached to vpg ( ) . the vpg linkage may prevent the exposure of a polyu structure to host sensors, thus preventing the polyu sequence from acting as a pamp. for influenza viruses, the polyu sequence on negative-sense rna is essential for polyadenylation, because it is part of a unique stem− loop structure ( ) . however, this rna structure is localized to the nucleus and not exposed to cytoplasmic dsrna sensors. these examples illustrate that many viruses have polyu sequences that may act as pamps, and that each virus may have evolved unique mechanisms or structures that limit their detection. our study also raises interesting questions about how the cov polya tail is generated during positive-sense rna synthesis. studies from influenza virus revealed that a short polyu sequence within a unique rna stem−loop mediates a stuttering mechanism used to polyadenylate the positive-sense rna ( ) . in contrast, peng et al. ( ) implicate that covs utilize a noncanonical cytoplasmic polyadenylation site to synthesize the positive-sense polya tail. they identified a conserved viral sequence on the positive-sense rna that could be bound by host proteins, including cytoplasmic polyadenylation element binding protein (cpeb ) ( ), a protein that mediates polyadenylation. in addition, cov nsp has been demonstrated to contain ′ terminal adenylyltransferase (tat) activity ( ) . nsp can synthesize the polya tail on the positive-sense rna, and having a complement negative-sense rna with a polyu extension greater than five uridines enhances the tat activity. since we observe endou controlling the abundance of longer polyu sequences which stimulate ifn, it would be interesting to determine whether there is an "ideal length" of polyu sequences that lack immune stimulation by mda , but promote tat activity during addition of the polya tail. one caveat of these in vitro studies is that the assays are performed in the absence of other viral replication complex proteins that may alter the binding, recognition, and activity of the rna processing proteins. studies have shown protein−protein interactions between nsp , nsp , nsp , and nsp , which may alter the activities of these proteins ( , ) . while endou can fully degrade pun rnas in vitro, the cleavage activity may be more specific during viral infection, due to interactions with other viral proteins in the membrane-associated replicase complex ( , ) . while endou can cleave pun rna, there may also be other endou cleavage sites during cov infection that were not detected in this study. further studies are needed to fully elucidate the mechanisms covs use to alter and process viral rnas. our study reveals that the cov endoribonuclease activity is distinctly different from three other previously documented viral ribonucleases. influenza pa-x is an endoribonuclease that selectively degrades host mrna by hijacking host rna splicing machinery ( ) . pa-x inhibits the translation of host proteins to perturb the cell functions. pestivirus rnase e(rns) is an endoribonuclease that is secreted outside infected cells and degrades extracellular viral rnas to block innate immune activation ( ) . lassa virus encodes an exonuclease that will specifically degrade intracellular dsrna ( ) . both rnase e(rns) and lassa virus exonuclease cleave viral rnas thought to be pamps. our study reveals an additional mechanism for a viral endoribonuclease to degrade a viral pamp. we found that pun rnas with variable lengths of polyu sequences are generated in the absence of endou activity. we predict that these pun rnas can fold back and generate stem−loop structures by hybridizing with an a/g-rich domain located within the pun rna or on adjacent rnas. this stem−loop structure may be recognized as dsrna by host prrs, thus stimulating the host innate immune response. the function of endou during replication is to reduce the length of polyu sequences, thus limiting the potential for generating pamps. the current outbreak of -ncov and the associated morbidity and % mortality highlight the importance of developing effective vaccines against covs ( ) ( ) ( ) . one vaccine strategy is to generate attenuated viruses that can efficiently be produced and signal for robust, protective, antiviral immune responses. endou is not necessary for viral replication, and endoumut covs are attenuated in vivo while still eliciting a protective immune response ( , ) . therefore, endou may be one of several immune antagonists targeted for generating an attenuated and recombinationresistant cov vaccine ( , ) . ideally the ifn antagonist mutations would be conserved so they can be applied to any current or emergent cov, including the -ncov. in addition, the enzymatic activity of endou could be targeted for the development of an antiviral therapeutic. in summary, this study provides evidence for a mechanism used by the cov endou to cleave a viral rna pamp, which would otherwise be recognized by mda . endou activity delays recognition by the host innate immune sensors, and thus is a highly conserved virulence factor and a potential target for antiviral and vaccine strategies. cells, viruses, and reagents. aml hepatocytes (crl- ; atcc) were cultured in dulbecco's modified eagle's medium (dmem)/f- ( - , invitrogen) supplemented with % penicillin/streptomycin, % fetal bovine serum (fbs), insulin, transferrin, and selenium ( ; life technologies), and dexamethasone ( ng/ml, d ; sigma). l cell line was gifted from francis alonzo, loyola university of chicago, maywood, il, and maintained in dmem ( - -cv; corning) supplemented with % fbs, % l-glutamine, % sodium pyruvate, % nonessential amino acids, and % penicillin/streptomycin. differentiated bmdms were maintained in bone marrow macrophage media containing dmem ( - -cv; corning) supplemented with % l cell supernatant, % fbs, % l-glutamine, % sodium pyruvate, and % penicillin/streptomycin. methods for generation of bmdms are described in deng et al. ( ) . porcine kidney epithelial cells, pk (cl ; atcc), were grown in growth medium containing modified eagle medium (mem) ( - -cv; corning) supplemented with % fbs, and % penicillin/streptomycin. vero cells were grown in growth media containing mem ( - ; gibco) supplemented with % fbs, . % lactalbumin enzymatic hydrolysate ( - - ; sigma), and % penicillin/streptomycin. wild-type mhv strain a (genbank accession no. ay ) and endoumut (h a) were previously generated by reverse genetics and full-genome sequenced ( ) . infectious clones of wildtype pedv or endoumut pedv (h a) were previously generated by reverse genetics and full-genome sequenced ( ) . dsrna immunoprecipitation pcrs. ifnar −/− bmdms, c bl/ bmdms, or aml cells were infected with wild-type or endoumut virus at a multiplicity of infection (moi) of . at indicated times postinfection, rna was isolated with rneasy kit ( ; qiagen). one microgram of rna was mixed with dsrna antibody (k ; scicons) or mouse anti-beta actin (a - ; genscript), rna binding buffer ( mm tris ph . , mm nacl, % nonidet p- , and u/ml ribolock [eo ; thermofisher]). after overnight incubation, protein g beads (lskmagg ; millipore) were added and incubated rotating for h at °c. protein g beads were precipitated with magnets, then washed with cold binding buffer. rna was purified off beads with rneasy kit and reverse-transcribed into cdna with rt first strand kit ( ; qiagen). the qpcr was performed as described below. for rna-seq, rna was processed by university of chicago genomics facility. the cdna sequencing libraries were generated with truseq stranded total rna with ribo-zero extraction (illumina), then sequenced on illumina hiseq . rna-seq reads were analyzed with galaxy's online platform (https://usegalaxy.org/). reads were groomed, clipped, and mapped with hisat to the wild-type mhv strain a (genbank accession no. ay ) or host genome (grcm ensembl build of the c bl/ j). the number of reads at individual nucleotides was calculated by plotcoverage. rna-seq data have been deposited and are available in the national center for biotechnology information gene expression omnibus (ncbi geo) database (accession no. gse ). qpcr. rna was isolated with rneasy kit from cells at stated times postinfection. isolated rna was reverse-transcribed with rt first strand kit. the qpcr reactions were performed using rt kit ( ; qiagen). the qpcr reaction is ) °c for min, ) °c for s, ) °c for min, and a repeat of steps and for cycles. polyu extension pcrs. cells were infected with wild-type or endoumut virus at an moi of . at indicated times postinfection, rna was isolated with rneasy kit. for strand-specific cdna, ng of rna was reverse-transcribed with omniscript ( ; qiagen) with a reaction temperature of °c to reduce self-primed cdna synthesis ( ) . for negative-sense rna, the cdna primer was ′-gaattctggtggtgctgatgaac- ′ for mhv and ′-gcag-cattgctctttggtg- ′ for pedv. for total rna, the cdna primers were random hexamers, and positive-sense rna primer was oligo-dt. the qpcr was performed with ssoadvanced universal probes supermix ( ; bio-rad). the qpcr reaction was performed with an annealing temperature of °c with either primer set polyu qpcr set or polyu qpcr set . primers are listed in si appendix, table s . polyu length nested pcrs were performed with pfu ultra polymerase ( ; agilent) with an annealing temperature of °c. the cdna was generated as described above. for negative-sense rna, the cdna primer was ′-gaattctggtggtgctgatgaac- ′ for mhv and ′-gcagcattg-ctctttggtg- ′ for pedv. for positive-sense rna, the cdna primer was ′-ggggatccgcggtttttttttt- ′. a pcr was performed with set primers for polyu length. then the product was diluted : , and utilized in a pcr with set primers for polyu length. pcr products were separated on a % polyacrylamide gel and stained with sybr green ii dye (s ; thermofisher). primers for cdna synthesis and pcrs are listed in si appendix, table s . for polyu length sequencing, sequencing libraries were generated with custom amplicon primers with nextera xt indexes, and the amplicons were sequenced on an illumina miseq v with paired-end -bp reads. reads were analyzed and mapped to viral genomes with galaxy's online platform. rna transfections. the pcaggs constructs were generated containing a t promoter, the viral genome segment, and a hindiii cleavage site. viral sequences are listed in si appendix, table s . plasmids were digested with hindiii-hf (r ; neb), then purified with wizard gel and pcr purification kit (a ; promega). rna was in vitro-transcribed using a t rna polymerase (ep ; thermofisher) and purified by licl precipitation. five picomoles of rna or poly i:c (p ; sigma) was transfected into aml cells using lipofectamine ( ; thermofisher). at hpt, rna was isolated using rneasy kit and qpcr for ifnβ (ppm c; qiagen), and s ribosomal rna (rrna) (ppm e; qiagen) was performed as described above. generation of mda -knockdown cells. a modified crispr/cas protocol, based on the gecko system ( ) , was used to knock down the function of mda in aml cells. single-guide rna was identified with benchling (benchling, inc.) to target the ifih gene. sequence used for targeting ifih was ′-atggacgcagatgttcgtgg- ′. the cdna versions of guide rna were annealed and inserted into a plenticrisprv -puro (addgene ) cassette between flanking bsmbi sites. transducing particles (tps) were generated by transfecting hek- t/ cells with plenticrisprv -puro, ppax , and phef-vsv-g and collecting supernatant. tps were centrifuged at , × g for min at °c then filtered through a . -μm filter (millipore sigma). aml cells were transduced with tps, then incubated for h at °c in % co . transduced aml cells were then selected with μg/μl puromycin (invivogen) for h. puromycin-selected cells were then grown and cloned into a monoclonal population. knockdown of mda was determined by western blot using rabbit anti-mda (sab ; sigma) and mouse anti-actin (a - ; genscript). rna cleavage assay. cleavage of rna substrates was performed according to kang et al. ( ) . purified, wild-type endou was kindly gifted by c. kao, formerly of indiana university, bloomington, in, currently at aligos therapeutics, san francisco, ca. briefly, μm rna was mixed with endou in cleavage buffer ( mm tris ph . , mm kcl, mm dithiothreitol) with or without mm mncl . reactions were incubated at °c for indicated time, and reactions were stopped by addition of rna gel loading buffer (b s; neb) and incubation at °c for min. reaction products were immediately loaded into a % polyacrylamide gel with tris-borate-ethylenediaminetetraacetic acid (edta) buffer or a % agarose gel with tris-acetate-edta buffer, and bands were separated by electrophoresis. gels were then stained with sybr green ii dye and visualized with a chemidoc xrs+ imager (bio-rad), and processed with image lab software (bio-rad). data availability statement. rna-seq data have been deposited and are available in the ncbi geo database (accession number: gse ). the molecular biology of coronaviruses 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evasion of dsrna sensors and limits apoptosis in macrophages murine coronavirus delays expression of a subset of interferon-stimulated genes pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses middle east respiratory syndrome coronavirus ns b protein inhibits host rnase l activation antagonism of dsrna-induced innate immune pathways by ns a and ns b accessory proteins during mers coronavirus infection interaction of sars and mers coronaviruses with the antiviral interferon response ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice an "old" protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group lineage rna recognition and cleavage by the sars coronavirus endoribonuclease crystal structure and mechanistic determinants of sars coronavirus nonstructural protein define an endoribonuclease family new antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease nsp crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp suggests a role for hexamerization as an allosteric switch the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor major genetic marker of nidoviruses encodes a replicative endoribonuclease biochemical and genetic analyses of murine hepatitis virus nsp endoribonuclease mutational analysis of the sars virus nsp endoribonuclease: identification of residues affecting hexamer formation in situ tagged nsp reveals interactions with coronavirus replication/ transcription complex associated proteins colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells dsrna ip of coronavirus infected ifnar −/− bmdms systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a the ′ end of coronavirus minus-strand rnas contains a short poly(u) tract a guide to ions and rna structure biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease monoclonal antibodies to double-stranded rna as probes of rna structure in crude nucleic acid extracts activation of mda requires higher-order rna structures generated during virus infection a study of the transmission and structure of double stranded rnas associated with the killer phenomenon in saccharomyces cerevisiae combination and inducible adjuvants targeting nucleic acid sensors uridine composition of the poly-u/ uc tract of hcv rna defines non-self recognition by rig-i the hepatitis c virus-induced membranous web and associated nuclear transport machinery limit access of pattern recognition receptors to viral replication sites poly(a) at the ′ end of positive-strand rna and vpg-linked poly(u) at the ′ end of negative-strand rna are reciprocal templates during replication of poliovirus rna direct evidence that the poly(a) tail of influenza a virus mrna is synthesized by reiterative copying of a u track in the virion rna template characterization of the role of hexamer aguaaa and poly(a) tail in coronavirus polyadenylation cpeb: a life in translation identification and characterization of a human coronavirus e nonstructural protein -associated rna ′-terminal adenylyltransferase activity structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors rna replication of mouse hepatitis virus takes place at double-membrane vesicles sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum timing is everything: coordinated control of host shutoff by influenza a virus ns and pa-x proteins the viral rnase e(rns) prevents ifn type-i triggering by pestiviral single-and double-stranded rnas structure of the lassa virus nucleoprotein reveals a dsrna-specific ′ to ′ exonuclease activity essential for immune suppression clinical features of patients infected with novel coronavirus in wuhan a novel coronavirus emerging in china-key questions for impact assessment a pneumonia outbreak associated with a new coronavirus of probable bat origin evaluation of a recombination-resistant coronavirus as a broadly applicable, rapidly implementable vaccine platform middle east respiratory syndrome vaccine candidates: cautious optimism potential pitfalls in the accuracy of analysis of natural sense-antisense rna pairs by reverse transcription-pcr genome-scale crispr-cas knockout screening in human cells acknowledgments. we would like to acknowledge the members of the s.c.b. laboratory for helpful discussions, especially dr. amornrat o'brien for help with figure design. we would also like to acknowledge roberto limiero at loyola university chicago and the staff at the university of chicago genomics facility for their help with the sequencing experiments. we would also like to thank dr. cheng c. kao (formerly of indiana university, currently at aligos therapeutics) for his help with the endou cleavage assays. this work was supported by nih grants ro ai (to s.c.b.) and t ai - (for support of m.h.). key: cord- -xqt aw authors: jalkanen, juho; pettilä, ville; huttunen, teppo; hollmén, maija; jalkanen, sirpa title: glucocorticoids inhibit type i ifn beta signaling and the upregulation of cd in human lung date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: xqt aw nan dear editor, glucocorticoids are widely used to treat acute respiratory distress syndrome (ards) despite their use being highly controversial based on randomized controlled trials and meta-analyses [ ] . as type i interferons (ifns) are our first line of defense against severe viral respiratory infections, we explored whether glucocorticoids interfere with ifn signaling and whether their use associates with outcome of ifn beta treatment of ards. methods are described in the esupplement. we performed a propensity-matched post hoc analysis using data from the recent randomized interest trial comparing ifn beta- a to placebo in ards patients [ ] . seventy-eight out of patients ( %) included in the ifn beta- a treatment arm of the inter-est trial received glucocorticoids during the -day study period, % ( / ) at randomization (d ), % ( / ) during the treatment (d - ) and % ( / ) after the treatment (d onward). day- mortality for patients receiving glucocorticoids with ifn beta- a was . % compared to . % for patients receiving ifn beta- a alone. the kaplan-meier curves of the ifn beta- a treatment arm adjusted by ards severity and divided according to the overlapping (d -d ) use of glucocorticoids with ifn beta- a treatment demonstrate significantly increased mortality by glucocorticoid use (p = . , see supplement). in the post hoc propensity-matched analysis of the ifn beta- a arm (n = ), baseline systemic glucocorticoid treatment was independently associated with d mortality (or . , % ci . - . , p < . ) according to logistic regression. when the propensity-matched analysis was performed using an exact matching (a precision of . propensity units), there were pairs of patients who received or did not receive glucocorticoids (n = ). among these patients, or for increased mortality was . ( % ci . - . ) for those who had baseline systemic glucocorticoid treatment and . ( % ci - ) for those who initiated glucocorticoid treatment while receiving ifn beta- a (fig. a) . based on the results of these analyses, we utilized human lung tissue and pulmonary endothelial cell cultures to investigate the effect of hydrocortisone on ifn nuclear signaling and the protein transcription of cd , a molecule responsible for vascular integrity and leukocyte infiltration to sites of inflammation [ ] . when ifn beta- a was applied to the lung organ cultures, cd expression was upregulated. however, in the presence of hydrocortisone, cd upregulation was inhibited (fig. b, c) . ifn beta- a signaling via its receptor leads to the formation of a heterotrimeric transcription complex isgf containing stat -stat and irf , which then enters the nucleus and binds to the ifn beta responsive elements of several genes or assembly on dna [ ] . in pulmonary endothelial cell cultures, ifn beta- a induced the translocation of irf *correspondence: sirjal@utu.fi into the nucleus, which was reduced by hydrocortisone treatment (fig. d, e) . in addition, hydrocortisone treatment decreased irf and stat mrna synthesis when measured by qpcr (fig. f ) , which was also seen at the protein level in fig. d . we conclude that glucocorticoids inhibit type i ifn beta signaling and the upregulation of cd in human lung. this provides the mechanistic basis for the harmful association of glucocorticoids in ifn beta-treated patients in the interest trial. this study takes the earlier preclinical evidence that steroids block endogenous ifn signaling [ ] to the critical care setting. our findings give mechanistic support to the icm recommendation not to use glucocorticoids at the early stages of severe covid- or viral-induced ards in general and highly recommends not to use systemic glucocorticoids together with type i interferons because of the harmful effects of this combination. the online version of this article (https ://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. this article is licensed under a creative commons attribution-noncommercial . international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by-nc/ . /. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. fig. glucocorticoid use associates with mortality and blunts ifn beta-induced cd upregulation in lung vasculature and ifn beta- a dependent signaling pathways in pulmonary endothelial cells. a (i) independent factors associated with day mortality in all ifn beta- a-treated patients (n = ) by logistic regression adjusted for propensity score and ards severity (odds ratios, or with % confidence intervals, ci). (ii) association of glucocorticoid treatment with d mortality in only propensity-matched ifn beta- a-treated patients (n = ) by logistic regression (or with % ci) b immunohistochemical staining of cd (brown) in lung tissue incubated in the presence of ifn beta with or without hydrocortisone (hc) for and days (d). d indicates baseline expression of cd in fresh lung tissue. c quantification of cd positive vessels/mm . the samples were incubated in the presence of two different ifn beta- a formulations (fp- , circles n = (day ) and n = (day ); ds, triangles n = ). each data point marks an individual. the dotted line represents the placebo-induced level of cd expression. statistical significance was analyzed using the mann-whitney test. d immunofluorescence staining of irf in primary human pulmonary endothelial cells after ifn beta treatment with or without hc showing marked inhibition of nuclear translocation of irf by hc, the representative results of three independent experiments with the similar results. e immunoblotting of the cytosolic and nuclear fractions of pulmonary endothelial cells after ifn beta- a ± hc demonstrating both decreased irf signal and its nuclear translocation. specific irf band is indicated by an arrow, a representative blot of five independent analyses with the comparable results. gapdh and h (histone) are loading controls for the cytoplasmic and nuclear fractions, respectively. f qpcr results of two independent experiments demonstrating the decrease in ifn beta- a-triggered mrna synthesis of irf and stat by hc. scale bars μm group is ( ) effect of intravenous interferon β- a on death and days free from mechanical ventilation among patients with moderate to severe acute respiratory distress syndrome: a randomized clinical trial crucial role for ecto- ′-nucleotidase (cd ) in vascular leakage during hypoxia a molecular switch from stat -irf to isgf underlies interferon-induced gene transcription the type i interferon signaling pathway is a target for glucocorticoid inhibition we thank riikka sjöroos and sari mäki for expert technical assistance and the study group of the interest trial for participating to the study at the trial sites. key: cord- -txryqil authors: kulka, m.; chen, a.; ngo, d.; bhattacharya, s. s.; cebula, t. a.; goswami, b. b. title: the cytopathic f strain of hepatitis a virus induces rna degradation in frhk cells date: journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: txryqil the mechanism responsible for the induction of apoptosis by the rapidly replicating hm / f strain of hepatitis a virus (hav) was investigated. full length hav rna and viral capsid protein vp were detected in f infected cells at earlier times post-infection than in hm /clone infected cells. analysis of total cellular rna from hm / f infected frhk cells by denaturing agarose gel electrophoresis and northern blot hybridization revealed extensive degradation of both the s and s ribosomal rna (rrna) molecules. similar degradation was observed when these cells were infected with human coxsackievirus b , a fast replicating enterovirus. in contrast, the parental strain of f, hm /clone did not induce rna degradation. inhibition of rna degradation correlated with inhibition of virus replication. the pattern of rrna degradation resembled degradation of rrnas by rnase l, an enzyme activated in interferon-treated cells following infection with certain viruses. ribosomal rna degradation was accompanied by the reduction in the levels of several cellular rnas including those for β-actin and glyceraldehyde- -phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. interferon mrnas could not be detected in either infected or mock-infected control cells, and stat , a key regulator of interferon action was not phosphorylated following virus infection. these results reveal a heretofore-undescribed pathway that involves the regulation of rna degradation and apoptosis following hav/ f replication in frhk cells. hepatitis a virus (hav), the only member of the genus hepatovirus within the picornaviridae family, is a major cause of infectious hepatitis worldwide [ ] . the virus is characterized by its resistance to heat, acid ph, polar solvents, and common disinfectants [ ] . it is primarily transmitted by the fecal-oral route and is a principal agent of localized food-borne outbreaks of infectious hepatitis in developed countries [ ] . the virus contains a single (+) stranded rna genome that is approximately . kb long and contains a poly (a) tail. the end is uncapped but covalently linked to the viral coded protein vpg [ ] . the viral genome has been cloned and sequenced from a number of strains. it codes for a single polyprotein, which is subsequently cleaved by the viral protease c and possibly cellular proteases into mature viral proteins [ , , ] . strains of hav can be grouped into three categories based on their distinctive growth characteristics in permissive cells: i) wild-type (wt) strains, defined by a long incubation period between infection and detection of replication and a cytopathic effect (cpe) is not observed; ii) tissue culture adapted (cc) strains, defined by shorter incubation periods than for wt strains and cpe is not observed; and iii) tissue culture adapted (cp) strains, defined by shorter incubation periods than for cc strains and cpe is observed. the cp strains arise during repeated passage of cc strains in permissive cells [ , , , , , , ] . although the molecular basis for the development of the cp phenotype is poorly understood, it is well documented that both viral genome replication and viral protein expression can be detected much earlier in permissive cells infected with cp strains such as hm / f, hm / a and hm /hb . , than in cells infected with the parental cc strains from which they were derived [ , , , , ] . the rapid replication and viral gene expression observed for cp strains has been attributed to a more efficient initiation of translation from the ires (internal ribosomal entry site) of cp strains due to mutations in this region [ , , ] , though some have disputed this interpretation [ , ] . apoptosis is observed following infection by a number of picornaviruses, especially when virus replication is restricted [ , , , ] . while cell killing by apoptosis may aid in spreading progeny virus or escape cellular defense mechanisms [ ] , premature induction of apoptosis may limit virus growth and spread [ , , ] . the cytopathic effect that is observed in cells following infection with cp strains of hav has been attributed to the induction of apoptosis [ , ] . apart from the demonstration of a link between rapid replication and cytopathogenicity of the cp strains of hav, the actual molecular events leading to apoptosis following infection with cp strains are unknown. expression of p region proteins b and c from either cp or cc strains in frhk cells result in the accumulation of a tubularvesicular network [ , ] . expression of the p region protein ab has been shown to result in membrane alterations in bacterial cells with resultant changes in membrane permeability [ ] . the lack of correlation between apoptosis and expression of either p or p region proteins prompted us to investigate alternative mechanisms of apoptosis induction by cp strains of hav. in this study, we report that infection of frhk cells with the hav cp strain hm / f results in the degradation of ribosomal rna (rrna) and the reduction of several cellular mrnas including β-actin and gapdh. ribosomal rna degradation was specific for the f strain and the fast replicating enterovirus coxsackievirus b (cbv ), while the parental cc strain of f did not induce rrna cleavage. the effect of f or cbv infection on rrna integrity was observed in the absence of interferon (ifn) induction. ribosomal rna degradation was detected early after virus infection and prior to induction of cpe. this degradation was blocked by inhibitors of virus replication. the pattern of rrna degradation suggests the involvement of the dsrna activated rnase l; the regulation of this enzyme could provide an alternative mechanism by which f infection induces apoptosis. frhk monkey kidney cell line was a kind gift of dr. g. kaplan (fda, center for biologics evaluation and research, bethesda, md). the cells were grown in eagles minimal essential medium (emem, life technologies, gaithersburg, md) containing % heat inactivated fetal bovine serum (fbs), mem non-essential amino acids and sodium pyruvate (all from life technologies), with routine weekly sub-culturing. under these conditions the cells increase in number about -fold in to days. the cell line is contact inhibited and can be kept in growth medium or maintenance medium ( % fbs) for several weeks without degeneration of the monolayer. hav strains hm / f and hm /clone [ , ] , and human coxsackievirus b (cbv ) were obtained from atcc. all three viruses were grown in frhk cells. virus stocks were prepared essentially as described [ , ] . f and cbv were titered by plaque assay, while clone virus was titered by an enzyme immunoassay (eia) in well plates as described previously [ ] using the havab eia diagnostic kit (abbott laboratories, abbott park, il). confluent cultures of frhk cells in cm flasks ( × cells) were infected with - pfu/cell of f or cbv, or tcid of clone in . ml of mem containing % heat inactivated fbs or mock infected. after h of adsorption, . ml of the same medium was added to each flask and incubation continued until the indicated times of cell harvest. cells were harvested by scraping, centrifuged to remove medium, and the pellets washed once with pbs (ca ++ and mg ++ free) and either stored frozen at − • c or used immediately. for rna isolation, cell pellets were dissolved in trizol (life technologies) containing mm β-mercaptoethanol (sigma), following the protocol suggested by the manufacturer. all rnas were routinely digested with rnase-free dnase (promega, madison, wi) in the presence of ribonuclease inhibitor (rnasin, promega) for min at • c, followed by phenol/chloroform extraction and alcohol precipitation. rnas were quantitated by uv absorption at nm. infected or mock infected cells were treated with µg/ml cycloheximide, . mm guanidine hydrochloride, or µg/ml of brefeldin a in mem containing % fbs immediately following the virus absorption period. for measurement of virus replication, well plates containing approximately . × cells were used. cells were fixed in methanol/pbs at hpi and viral antigens detected by eia [ ] . for rna isolation, cm flasks, containing approximately × cells at the time of infection were harvested at or hpi, and rnas isolated as described above. a cm flask was seeded with frhk cells and infected at approximately % confluence with clone virus as described above. the virus was allowed to replicate for days and thereafter the cells were trypsinized and seeded into a cm flask. subsequently, the flask was sub-cultured at weekly intervals by trypsinization and seeding a new flask with one tenth of the cell suspension. to monitor virus replication, periodically cells were seeded into well plates and cultured for to h until the plates were confluent. cells were fixed in % methanol and viral antigens were measured by the havab eia procedure [ ] . uninfected cells were used as a negative control. samples of rna ( µg unless otherwise indicated) were denatured by heating at • c for min in µl of buffer containing × mops buffer, % formamide and . % formaldehyde. samples were separated in a % agarose gel containing × mops buffer, . m formaldehyde, and µg/ml etbr [ ] . the gel was photographed under uv light and transferred overnight to nytran n membranes (schleicher & schuell) in × ssc. the membrane was washed briefly in × ssc, baked under vacuum and stored at • c prior to hybridization. probes for β-actin and gapdh were labeled by random priming of decatemplate tm -βactin-mouse and decatemplate tm -gapdh-mouse respectively (ambion, austin, tx), using α- p datp ( ci/mmol, icn, costa mesa, ca). probe for s rrna was decatemplate tm - s-mouse, also obtained from ambion and was similarly labeled. the anti-sense rna probe for the s rrna (ptri rna s, ambion) was labeled using sp polymerase and α- p utp. the probe for hav genomic rna was obtained by xba digestion of the plasmid phav/ (kindly provided by dr. s. emerson nih, bethesda, md). following gel purification, the large xba fragment, representing almost all of the genomic rna (except the ires), was labeled by nick translation using α- p datp. blots were prehybridized at • c in hyb- (gentra) supplemented with denatured salmon sperm dna or yeast trna (for the rna probe) for - h with continuous agitation and hybridized with probe ( - × cpm/ml) overnight. filters were washed once in × ssc/ % sds for min and then times in . × ssc/ . % sds for min each time at • c, prior to exposure to film or phosphorimager screen (molecular dynamics). anti-vp antiserum was obtained from rabbits immunized with a synthetic peptide cvpetf pelkpgesrht, corresponding to amino acids - of hav viral capsid protein vp , covalently linked to klh. synthetic peptide, free and klh-conjugated, was purchased from new england peptide, inc (fitchburg, ma). pre-immune serum was collected from new zealand white rabbits - days prior to immunization and stored at − • c. rabbits were immunized with klh-conjugated peptide emulsified in freund's complete adjuvant and challenged at regular intervals, over a to week period, with klh-conjugated peptide emulsified in incomplete freund's adjuvant. the animals were hyperimmunized using unconjugated peptide resuspended in sterile pbs. the serum was collected and stored at − • c. all animal studies were conducted in compliance with the guide for the care and use of laboratory animals under an approved iacuc protocol. culture media was removed from mock or virus infected cell cultures and the monolayer were scraped into ca ++ and mg ++ free phosphate-buffered saline (pbs). cells were pelleted ( × g, • c) and washed twice with cold pbs. to obtain a total cell lysate, the cell pellet (while on ice) was resuspended in cold ripa buffer ( mm tris-cl, ph . , mm nacl, % np- , . % sodium deoxycholate, . % sds) containing mm pmsf, and : dilution of protease inhibitor cocktail (sigma st. louis, mo) and phosphatase inhibitor cocktails i and ii (sigma). additional pmsf was added ( mm) to the lysate prior to its passage through a gauge needle ( - times). following min of incubation on ice, the sample was centrifuged at , × g for min • c to pellet insoluble material. the extracts were stored as aliquots at − • c. protein concentrations of lysates were estimated using the bca- protein assay (pierce chemical co., rockford, il) according to the manufacturer's instructions. sixty five µg of each protein extract were adjusted to equivalent volumes in denaturing sample buffer and heated at • c for min and subjected to electrophoresis in sds- % polyacrylamide gels followed by electrophoretic transfer onto supported nitrocellulose membranes (optitran, ba-s , schleicher and schuell inc.) in transfer buffer ( mm tris base, mm glycine) containing % methanol. the membranes were blocked with tbs-t (tris buffered saline/ . % tween ) containing % nonfat dried milk (nfdm) for min at room temperature and washed four times with tbs-t. using a mini-blotter manifold, membranes were incubated either overnight at • c with anti-stat and anti-p-stat antibodies (cell signaling technologies, beverly, ma) diluted : in tbs-t containing % bsa or h at room temperature with anti-actin antibody (sigma) and anti-vp antibody diluted : in tbs-t containing % nfdm. after four washes in tbs-t, the blots were incubated with goat anti-rabbit igg-hrp antibody conjugate (cell signaling) diluted : , in tbs-t containing % nfdm. following four washes at room temperature in tbs-t, the supersignal west pico chemiluminescent substrate kit (pierce chemical co. rockford, il) was used according to manufacturer's instructions followed by exposure to x-omat-xar (kodak, inc.) for the detection of bound antibody-hrp conjugates. cells were grown in cm flasks and infected when confluent with f as above and labeled at hpi for h with µci/ml of a mixture of s-methionine and s cysteine (icn) in methionine and cysteine-free medium supplemented with % dialyzed fbs (icn) following a min pre-incubation in methionine and cysteine-free medium. cbv infected cells and clone persistently infected cells were labeled similarly at h or days post-infection (pi), respectively. cells were collected by scraping, washed once with pbs and the pellets lysed in µl ripa buffer containing protease inhibitors cocktail (sigma). tca precipitable incorporation was measured using µl of clarified lysate and protein concentration was measured with µl lysate. labeled proteins were separated in a - % novex pre-cast gel (invitrogen, carlsbad, ca), after heating the samples at • c for min in × sample buffer containing dithiothreitol. following electrophoresis, the gel was fixed and stained with brilliant blue g (sigma) in % methanol, % acetic acid and destained in % methanol, % acetic acid. the gel was then treated with amplify (amersham biosciences, piscataway, nj) for min, dried and exposed to a phosphorimager screen, which was scanned in a typhoon scanner (molecular dynamics). total rna preparations from mock infected, f infected ( hpi), cbv infected ( hpi), and hm infected ( dpi) frhk cells were labeled with α- p datp using the cds primers for human . array (clontech, palo alto, ca), clontech enzyme and reagents provided with the array. the labeled cdna preparations were purified using the nucleospin extraction kit (clontech). four identical arrays were pre-hybridized at • c in hybridization bottles in a hybaid oven (national labnet, woodbridge, nj) in ml of expresshyb (clontech) containing µg/ml denatured sheared salmon sperm dna (eppendorf scientific, westbury, ny). hybridization was performed overnight at • c by the addition of denatured probe ( . × cpm/ml) and µg human cot- dna (clontech) directly to the pre-hybridization solution. the membranes were washed twice ( min each) with × ssc- % sds and twice ( min each) with . × ssc− . % sds at • c, followed by a final wash at room temperature with × ssc for min. the membranes were wrapped in saran wrap and exposed to phosphorimager screens at room temperature for to h. the screens were scanned in a molecular dynamics typhoon scanner at -micron resolution and analyzed using the atlasimage . software (clontech). µgrna samples were reverse transcribed in µl reactions using oligo (dt) as the primer and amv reverse transcriptase and µl of each cdna pool were pcr amplified using primers bg and bg as described [ ] . ten µl samples were withdrawn after and cycles and analyzed by agarose gel electrophoresis. bands were quantitated by the imagequant program from molecular dynamics following scanning of the etbr stained gel by a typhoon phosphorimager. at a moi of - pfu/cell, the rapidly replicating cp strain hm / f of hav ( f) induced cell rounding in frhk cells within h post-infection (pi) in approximately to % cells. the percentage of cells exhibiting cpe increased for the next h, so that by h, to % of the cells were rounded and detached from the surface of the culture flask. thus, even with the f virus, infection is characterized by a slow and asynchronous recruitment of cells into supporting viral replication. similar effects have been reported for other cp strains of hav, namely hm / a, and hb . [ , ] . in agreement with these results, we confirmed the induction of apoptosis in f infected frhk cells by a colorimetric tunel assay (data not shown). the parental hm /clone virus (clone ), also used in the present study, produced no changes in cell morphology after several months of continuous culture of infected cells, although viral antigens could be detected by eia after four weeks of weekly subculture following the initial infection. the coxsackie virus strain b (cbv ) used in this study replicated very rapidly in frhk cells, producing marked cpe within hpi and all cells were floating by to hpi. we observed the appearance of rna bands in the region between the s and s species, as well as below the s rrna band, upon denaturing agarose gel electrophoresis of total rna preparations isolated from f-infected cells (fig. ) . the apparent degradation products were easily detectable after ethidium bromide (etbr) staining of formaldehyde-agarose gels (fig. a) . rna degradation was observed only in f infected cells at and hpi (lanes and ), while no degradation was observed at hpi in clone infected cells (lane ) or mockinfected cells (lane ). degradation of rrna has not been reported previously in hav infected cells, therefore, the rrna origin of the observed degradation the probe for s rna was a . kb fragment of the mouse s rna gene (nucleotides to ), labeled by random priming with α- p datp. the probe for hav genomic rna was the large xba i fragment from a genomic clone phav/ , labeled by nick translation with α- p datp. an rna ladder ( . to . kb) was included in the analysis and the position of the . to . kb fragments identified in panel a. an arrow is used to identify the putative s rrna degradation product in a and the position of the s rna and its degradation products in b. full length hav rna is . kb (c) products was confirmed by northern blot analysis. duplicate rna samples were electrophoresed and transferred to nytran n membranes. one half of the blot was hybridized to a probe for s rna (fig. b) , and the other half to a probe for hav rna (fig. c) . the s probe hybridized to intact s rna and to two additional species of rna (indicated by arrows) from f infected cells, while these species were not detected in rna preparations from either mock or clone infected cells. apparently full length hav rna ( . kb) was detected in f infected cells at both and hpi, while no viral rna was detected in clone infected cells at hpi, confirming that the f virus indeed was a faster replicating strain. to further investigate whether rna degradation was specific for f infection, and to determine the temporal relationship between rna degradation and onset of cpe, we isolated total rna from cells infected with cbv , hm /clone , and hm / f at different times, and analyzed them by formaldehyde-agarose gel electrophoresis (fig. ) . in cbv infected cells, degradation of both species of rrna was observed after cbv infection at - hpi, when almost all the cells in the monolayer were rounded but still attached to the surface ( fig (fig. b, lane ) , though small amounts of degradation products may be detected at days following infection (fig. b, lane ) . the levels of viral rna and protein in clone infected cells at and days pi were comparable to those observed in f infected cells, as measured by rt-pcr and western immunoblotting (fig. ) and eia (data not shown). we have continuously cultured the persistently clone infected cells for over five months without significant changes in cell morphology or rrna integrity. when rna was obtained from f-infected monolayers following separation of the rounded cells from cells with a normal morphology by vigorous agitation, rrna degradation was apparent in both populations of cells (fig. b, lanes and ) . in lane , only µg of rna was applied, since the percentage of floating cells at hpi was low and the yield of rna much less compared to the cells that remained attached (fig. b, lane ) . in lane , no intact s or s bands were visible, indicating that almost complete degradation of the rrna species has occurred in these cells. it should be noted that cbv infection proceeds much faster and synchronously in frhk cells. since we did not examine rrnas from cbv infected cells between and hpi, it is quite possible that rna degradation occurs in cbv infection prior to the onset of morphological changes associated with cpe. to confirm that s rrna degradation was occurring after infection with f or cbv , a northern blot of the rna gel shown in fig. (panel a) was hybridized to a probe specific for s rrna (fig. ) . the short probe ( bases from the end of the s rna) hybridized strongly to a fragment migrating faster than the . kb marker, as well as to intact s rrna, but did not hybridize to apparent fig. a were subjected to denaturing agarose gel electrophoresis. the gel was northern blotted and hybridized to a labeled anti-sense rna probe specific for s rrna (nucleotides to ). the probe was synthesized from ptrirna- s by transcription with the sp polymerase. the arrow indicates the position of a s rna degradation product. b an identical northern blot was hybridized to a nick translated dna probe for hav as described in fig. . arrows indicate position of full-length hav rna ( . kb) and putative degradation products degradation products seen in etbr stained gels (figs. and ) that migrated to the region between the s and s species. it is likely that these latter products were derived from the end of the s rrna and could not be detected by a probe targeted to the end. nevertheless, it is clear that both the large and small rrna molecules were degraded in cbv and f infected cells, and that this degradation coincided with the appearance of cpe in cbv infected cells but preceded the appearance of cpe in f-infected cells. in contrast, infection of permissive cells with mouse hepatitis virus (a coronavirus) resulted in the degradation of only the s species [ ] . apparently intact hav rna was detected in f-infected cells at hpi and hpi, and, albeit at lower levels in clone infected cells at dpi (fig. b ). this blot was exposed longer to reveal the existence of hav rna in clone infected cells. while overexposure of the blot showed that some degradation of viral rna might also be taking place, the bulk of the viral rna remains intact, possibly protected from degradation by its association with capsid proteins. on the other hand, the smaller virus specific rna molecules could have resulted from premature termination of transcription [ ] . to investigate whether the degradation of rrnas observed in f infected cells required replication of the virus, we studied the effects of several inhibitors of virus replication on rna degradation. cyclohexamide (ch) is a strong inhibitor of protein synthesis in many cultured cell lines and produces a marked cpe in treated cells that is attributed to induction of apoptosis [ , ] . in agreement with the results of brack et al. [ ] , we observed f-like cpe after treatment of frhk cells with ch at concentrations ranging from to µg/ml. despite the induction of cpe/apoptosis, ch treatment failed to elicit rna degradation in uninfected frhk cells (fig. b, lane ) , and inhibited both virus replication (fig. a) , and degradation of rna in f infected cells (fig. b, lane ). the inhibition of virus replication was not unexpected since ch inhibits picornavirus genome replication by preventing translation of input genome to produce viral rna dependent rna polymerase [ ] . brefeldin a (bfa) has been shown to induce apoptosis in other cell lines through the activation of caspase and disruption of golgi complex function, and to inhibit replication of hav and some picornaviruses, as well as viral rna synthesis [ ] . treatment of frhk cells with µg/ml bfa produced marked apoptosis within h while suppressing f replication and f induced rna degradation (fig. , panels a and b respectively). thus, rna degradation in f-infected cells was prevented by both ch and bfa. ch and bfa induced apoptosis in frhk cells was synchronous and was complete by h as evidenced by cell rounding and eventual detachment from the growth surface. however, these changes were not accompanied by rna degradation following treatment with either ch (fig. b) or brefeldin a (data not shown). treatment of f-infected cells with . mm guanidine hydrochloride (guhcl), on the other hand, had a marginal effect on f replication at high moi, and a somewhat larger effect at low moi (fig. a) . however, guhcl treatment did not prevent rna degradation in f-infected cells (fig. b ). it has been reported that guhcl, an inhibitor of poliovirus replication, is ineffective against hav due to a mutation in the genome [ ] , but guhcl was found to be a strong inhibitor of hm /p virus [ ] . these experiments clearly show that in frhk cells, replication of the f virus is required for rna degradation, and that degradation of rna is not a consequence of apoptosis. degradation of rrna is a feature of virus infection in interferon (ifn) treated cells and is believed to be due to the availability of double stranded rna (dsrna) during replication or transcription of the viral genome, resulting in the activation of the rnase l pathway [ , ] . however, in the absence of ifn pretreatment, dsrna can activate the rnase l pathway by activating pre-existing - a synthetase, which ultimately results in cell death [ , , ] . since viral rna replication and expression occurs much earlier post infection in f-infected cells compared to clone infected cells, the degradation of rrnas could result from an early dsrna dependent activation of rnase l in f-infected cells. we further investigated whether the lack of rna degradation and cpe in clone infected cells observed at later times pi may correlate with diminished levels of viral rna or protein. frhk cells were infected with hm /clone , and the cells were serially passaged at weekly intervals until viral antigens were detectable by eia (data not shown). after days, the level of viral antigens was comparable to that observed in f infected cells after an acute infection. total cellular rna, and proteins were isolated from the persistently infected cells. the level of viral rna in the persistently infected cells was measured at and days of continuous culture by rt-pcr (fig. a) . the level of viral rna was somewhat lower in clone infected cells after days, but at days the levels were almost the same as in f infected cells at hpi. pcr amplification was carried out for and cycles to ensure that the signal strengths of the pcr products were obtained before the reactions reached saturation ( cycles) . western blot analysis with anti-vp antibody also showed lesser but easily detectable accumulation of the viral capsid protein vp in persistently clone -infected cells (fig. b) . however, the cells did not show any discernable morphological change after more than days of weekly subculture and produced viral antigens and rna as determined by eia and rt-pcr. the data indicate that, unlike f infection, clone replication and expression does not induce rrna degradation. a reduction in cellular rna and protein levels during virus infection may aid in the replication of viruses possessing a cp phenotype (e.g., hav hm / f), or a rapid growth/cpe characteristic (e.g., cbv) in cell lines permissive for virus growth. for example, translation of hav rna in infected cells requires interaction of trans-acting cellular protein factors with cis-acting elements present at the [ , ] . indeed, a competition between gapdh and polypyrimidine tract-binding protein (ptb) for stem-loop iiia of hav ires has been reported by yi et al. [ ] . while ptb functions as a positive effector of hav rna translation, the binding of gapdh to stem-loop iiia of hav ires has been shown to destabilize rna secondary structure and inhibit translation [ , ] . these results prompted us to investigate the levels of gapdh mrna in f-infected cells since decreased levels of this mrna may indicate a functional relationship between f replication and regulation of effector proteins such as gapdh. as a control, we also investigated whether virus infection affects the level of another cellular housekeeping gene β-actin. northern blot hybridizations to β-actin and gapdh specific probes were used to assess the levels of these two cellular mrnas (fig. ) . the levels of both mrnas were reduced in a time dependent manner in cbv and f infected cells while there was little effect of clone infection on these molecules. however, unlike the rrnas, we did not find any degradation products of these rnas. two reasons for this could be that these mrnas are transcriptionally repressed and/or the degradation products are too small to detect by this analysis. the degradation of rrna into discrete products is a hallmark of the antiviral effect of interferon. since the cell line used for these experiments was not treated with interferon prior to virus infection, it was important to determine whether the frhk cell line is a natural producer of interferon, or if interferon is induced as a result of virus infection. to investigate if interferon mrnas were present in frhk cells prior to or following virus infection, nylon arrays, each containing human genes including those for interferons α, β, and γ were hybridized to cdna probes synthesized using total rna from uninfected and virus infected cells as templates. surprisingly, we did not find significant hybridization signals table . induced and down-regulated genes during f infection of frhk cells. array hybridizations were carried out in duplicate with cdna probes prepared using two independent rna preparations each from mock and virus infected frhk cells. independent phosphorimager scans for each duplicate sample (control or infected) were averaged using atlasimage program to produce composite arrays for control and infected cells. the composite arrays were then compared to detect differences in signal levels using atlasimage software. gene signal values that were less than two-fold over background values were considered insignificant. ratios of less than one represent down-regulation in the infected cells. ratios of higher than one represent up-regulation in the infected cells. a less than two-fold difference of signal intensity between control and infected samples are considered insignificant. nd means the pixel value for that gene was equivalent to background corresponding to the interferon genes in either uninfected or hav infected cells (table ) . these results are supported by the data from pietiäinen et al. [ ] suggesting that ifn mrnas are neither present nor induced during pv , ev and cbv infection of hos and hela cells. however, low levels of ifn rnas, and any comparative differences between uninfected and infected cells, may not be detectable by array hybridization. rt-pcr analysis of total rna from virus infected frhk cells did not show increased level of β-ifn mrna compared to uninfected cells. however, treatment of these cells with dsrna resulted in β-ifn mrna induction (data not shown). we investigated the possibility that low levels of ifn may be present in control or infected frhk cells by looking at the phosphorylation status of stat by western immunoblotting (fig. ) . signal transducer and activator of transcription (stat) and are regulators of ifninduced gene expression. in cells treated with ifn, both stat and stat are phosphorylated by janus kinase (jak), which is a prerequisite for dimerization and translocation to the nucleus [ , ] . the antibody to stat readily detected the two forms of this protein in both control and virus infected cells, as well volumes of extracts containing equal number of cpm or protein were heated in sds sample buffer containing reducing agent prior to loading on a - % polyacrylamide gel that was run in mops-sds buffer along with molecular weight markers. the gel was stained, destained and, and treated with amplify (amersham) prior to drying according to standard protocol and exposed to phosphorimager screen, which was scanned by a typhoon scanner as in control and interferon treated hela cell extracts. both stat proteins in hela migrate as slightly larger molecules. using an antibody specific for the phosphorylated stat , stat phosphorylation was not detected in either control or f-infected frhk cells (fig. ) , confirming the results obtained by array hybridization. in addition, stat phosphorylation was not observed in cbv infected frhk cells while phosphorylated stat was readily detected in ifn treated hela cells (fig. , panel a) , and frhk cells (fig. , panel b) . these results are in agreement with those obtained by array hybridization, indicating that ifn is not expressed and, therefore, is not mediating rna degradation in f-infected cells. unlike most enteroviruses, even cp strains of hav lack the ability to shut off host protein synthesis. this inability to cause host shut-off is most likely due to a non-functional a protease, and is probably a major reason for the slow growth phenotype of hav [ ] . while it would contradict published reports, it seemed possible that f induced rna degradation could result in host shut-off triggered by events peculiar to frhk cells. therefore, we investigated the effect of f infection on overall protein synthesis by pulse labeling of cells for h and analysis of the products by sds-polyacrylamide gel electrophoresis (sds-page). as shown in fig. , cbv infection resulted in a dramatic reduction of cellular protein synthesis at hpi, and viral proteins could be clearly identified. in contrast, the effect of f infection was much less pronounced, and comparable to the effect of the parental cc (clone ) strain. thus no positive identification of hav specific proteins could be made from such an analysis. it should be noted that the clone infected cells used for protein labeling in this experiment have been routinely sub-cultured for more than two months, and were actively producing viral antigens as detected by a colorimetric eia procedure (data not shown) as well as viral rna and vp capsid protein (fig. ) . however, rna degradation was not evident in these cells. in conclusion, rrna degradation alone is not sufficient for host shut-off. cytopathic (cp) strains of hav have been isolated in several laboratories during the past few years. the cp strains arise during repeated passage of cc strains in permissive cells and the genetic basis for the development of the cp phenotype is poorly understood [ , , , , , , ] . there is no evidence to indicate conclusively which nucleotide changes arising in the cp genotype are responsible for both the rapid growth and the cp phenotype [ , , , , , ] . these characteristics may be the result of different mutational events that have occurred during cc strain replication. the data presented here identify a previously unreported event that occurs during infection with a cp strain of hav, namely the degradation of rrnas and the reduction in the level of cellular rnas. similar effects on rrna, and β-actin and gapdh mrnas were also observed after cbv infection. however, the non-cpe inducing parent strain of f, hm /clone had only a marginal effect on rrna integrity during long-term persistent infection. similar to the effects of enteroviruses, such as pv , cbv and ev , on the expression of certain cellular genes involved in apoptotic pathways, c-myc and c-jun levels were moderately increased in f-infected cells, while c-jun level was markedly reduced in clone infected cells (table ) . full length viral rna was detectable much earlier in f-infected cells compared to cells acutely infected with the clone virus (figs. and ) . significantly, viral rna and viral capsid protein vp were present at comparable levels following extended culture of clone -infected cells in the absence of significant rna degradation (fig. ) . degradation of rrna following infection of interferon treated cells has been reported for a number of dna and rna viruses [ , ] . moreover, dsrna, a likely intermediate of rna virus replication or transcription, has been shown to promote rrna degradation without interferon treatment in some cell lines [ , , , , , , , , ] . the function of dsrna as an effector of the interferon response is dependent on both the cell type and the nature of the virus [ , ] . for example, vaccinia virus is known to evade the effects of ifn treatment, but in suspension cultures of mouse l cells treated with ifn, rapid degradation of rrna was observed [ ] . activation of the - a/rnase l pathway has been reported during estrogen withdrawal from chick oviducts [ ] . the enzyme responsible for the synthesis of - a, - -oligoadenylate synthetase can apparently be also induced by hepatitis c virus coat protein [ ] . while the role of a viral protein in the activation of - a/rnase l pathway in f or cbv infected frhk cells cannot be ruled out, previous reports with emc virus and the studies involving dsrna clearly suggests a similar mechanism of rrna degradation reported here. it remains unclear why rrna degradation and apoptosis are absent in clone infected cells despite the presence of viral rna and protein. perhaps cellular inhibitors of rnase l play a role in blocking rna degradation in clone infected cells [ ] . it is also possible that a mechanism, analogous to the adenovirus vai rna mediated inhibition of pkr activity is used by the clone virus to disarm the rnase l pathway [ , ] . clearly, accumulation of sufficient viral rna or proteins cannot account for either rrna degradation or the cp phenotype, since cc strains accumulate enough viral rna and proteins after an initial lag (fig. ) . it is interesting to note that while the precursor of mature vp protein vp - a is present in equal amounts in both f and clone infected cells, vp itself accumulates to higher levels in f infected cells, suggesting a slower processing of the precursor in clone infection. surprisingly, we did not see a general inhibition of protein synthesis in frhk cells infected with the cp ( f) strain of hav despite massive rna degradation, although an almost total shutoff of host protein synthesis was observed in cbv infected frhk cells (fig. ) . this is in contrast to the general inhibition of protein synthesis observed upon activation of the rnase l pathway in some cell lines [ , , , , , , ] . the effect of cbv infection on protein synthesis is most likely a combination of rrna cleavage (this study) and cleavage of eif g, poly (a)-binding protein (pbp), and other translation factors mediated by an active a protease [ , , ] . it has been shown that both caspase activation and apoptosis induction can occur when - a synthetase is activated and rrna degradation is induced [ , , , , ] . while we have not directly assessed the level of - a following virus infection, no other rnase currently known produces the characteristic degradation pattern of rrna that we have observed. it is apparent from our data, however, that rna degradation is dependent upon cp virus strain replication, since compounds, such as cycloheximide and brefeldin a, that inhibited f replication also abolished rrna degradation in f infected cells (figs. and ) . treatment in the absence of infection did not induce rna degradation. additionally, the treatment of uninfected cells with these compounds induced apoptosis in the absence of rna degradation (figs. and ) . in view of the temporal relationship between onset of rrna degradation and induction of cpe/apoptosis, these results suggest that rna degradation following f (or cbv ) infection is the cause, rather than the effect, of apoptosis. on the other hand, an apoptotic reaction may also be induced by the preferential synthesis of apoptosis inducing proteins such as c-jun [ ] or c-myc [ ] . as shown in table , several gene transcripts linked to apoptosis are present in higher levels in f-infected cells compared to clone infected cells. particularly intriguing are lack of induction of c-myc and downregulation of c-jun in clone infected cells. we cannot rule out the possibility, therefore, that apoptosis in f-infected cells may be induced via regulation of relevant proteins such as c-jun or c-myc. another interesting feature of the cp strains of hav is that all apoptosis inducing cp strains are derived from persistently infected monkey kidney cells and induction of apoptosis (or cpe) is restricted to monkey kidney cells. therefore, the nature of the host cell is clearly as important as the genetic makeup of the virus. it is interesting to note that a base insertion is present in the ires of cp strains hm / f as well as hm / a. it would be tempting to speculate that conformational changes brought about by this or other mutations result in a better inducer of - a synthetase, a possibility that is currently under investigation. activation of rnase l resulting in rrna cleavage is usually accompanied by inhibition of protein synthesis [ , , , , ] and picornavirus growth [ , , ] . we believe that the apparent lack of inhibition of host protein synthesis in f-infected cells is a result of asynchronous rna degradation, which masks the effect of rna degradation on protein synthesis. the other possibility is the absence of activation of dsrna stimulated pkr, which may be lacking in these cells. however, several different mechanisms exist, through which viruses can disarm the pkr response [ , ] . further studies are required to assess the level of activity of this enzyme in virus-infected cells. recent studies using dna arrays have revealed that genes involved in the interferon pathway (interferon response genes, isgs) are the major targets of a diverse group of viruses [ ] . a direct effect of interferon in inducing - a synthetase during f or cbv infection is unlikely because we failed to detect ifn mrna or stat phosphorylation in either uninfected or virus infected cells. these results are supported by previous findings that cell culture adapted hav does not induce ifn in cultured cells [ ] . moreover, recent studies also have shown that a non-cytopathic cc strain of hav down regulates dsrna-induced transcription of β-ifn [ ] . however, there are now several studies to show that isgs can be regulated in the absence of ifn by viral dsrna or viral proteins [ , , ] . while many viruses have evolved mechanisms to disarm various facets of the ifn inducible antiviral response, the usurpation of the ifn response by the cp strain f to facilitate it's spread is unique. the fact that only a few cell lines of primate kidney origin are susceptible to apoptosis in response to rapidly replicating hav variants suggests that both cellular processes and genotypic changes in the viral genome are responsible for the establishment of a cp phenotype. in order to define the contributions of these two processes, it will be necessary to examine strain differences at the genetic level as they relate to substantive changes in viral protein expression/function, and to investigate the connection between viral and cellular events at the level of cellular protein expression. in conclusion, rrna degradation in combination with lower levels of cellular mrnas such as β-actin, gapdh and others could confer on the f virus a replicative advantage over the parental cc strain. clearly, reduction in the levels of cellular mrnas is advantageous to the virus, even if it leads to a small reduction rather than a dramatic shut-off of host protein synthesis. competing death programs in poliovirus-infected cells: 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-oligoadenylate synthetase cdna results in increased antiviral activity and growth suppression proteinase c of hepatitis a virus (hav) cleaves the hav polyprotein p -p at all sites including vp / a and a/ b specific interaction of glyceraldehydes -phosphate dehydrogenase with the nontranslated rna of hepatitis a virus the structures of picornaviral proteinases stability of hepatitis a virus viruses and interferons how cells respond to interferons apoptosis-inducing and apoptosis-preventing functions of poliovirus apoptosis in acute and chronic central nervous system disease induced by theiler's murine encephalomyelitis virus isolation and molecular cloning of a fast growing strain of human hepatitis a virus from its double stranded replicative form hepatitis a virus infection and the interferon system hav f induces rna degradation regulation of two interferon inducible human genes by interferon, poly(ri):poly(rc) and viruses detection of a genome-linked protein (vpg) of hepatitis a virus and its comparison with other picornaviral vpgs low efficiency of the nontranslated region of hepatitis a rna in directing cap-independent translation in permissive monkey kidney cells functional significance of the interaction of hepatitis a virus rna with glyceraldehydes -phosphate dehydrogenase (gapdh): opposing effects of gapdh and polypyrimidine tract binding protein on internal ribosome entry site function an infectious cdna clone of a cytopathic hepatitis a virus: genomic regions associated with rapid replication and cytopathic effect impact of rnase l overexpression on viral and cellular growth and death division of molecular biology we thank dr. darcy hanes for help in preparing the anti-vp antiserum, dr. joseph e. leclerc and dr. dan levy for critically reading the manuscript, dr. c.d. atreya and dr.stephen feinstone for helpful discussions, and marcia meltzer for help with the graphics. key: cord- -kv n qj authors: rabaan, ali a.; alahmed, shamsah h.; bazzi, ali m.; alhani, hatem m. title: a review of candidate therapies for middle east respiratory syndrome from a molecular perspective date: - - journal: journal of medical microbiology doi: . /jmm. . sha: doc_id: cord_uid: kv n qj there have been laboratory-confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) in countries, with a mortality rate of . %. there is no specific therapy. the current therapies have mainly been adapted from severe acute respiratory syndrome (sars-cov) treatments, including broad-spectrum antibiotics, corticosteroids, interferons, ribavirin, lopinavir–ritonavir or mycophenolate mofetil, and have not been subject to well-organized clinical trials. the development of specific therapies and vaccines is therefore urgently required. we examine existing and potential therapies and vaccines from a molecular perspective. these include viral s protein targeting; inhibitors of host proteases, including tmprss , cathepsin l and furin protease, and of viral m(pro) and the pl(pro) proteases; convalescent plasma; and vaccine candidates. the medline database was searched using combinations and variations of terms, including ‘middle east respiratory syndrome coronavirus’, ‘mers-cov’, ‘sars’, ‘therapy’, ‘molecular’, ‘vaccine’, ‘prophylactic’, ‘s protein’, ‘dpp ’, ‘heptad repeat’, ‘protease’, ‘inhibitor’, ‘anti-viral’, ‘broad-spectrum’, ‘interferon’, ‘convalescent plasma’, ‘lopinavir ritonavir’, ‘antibodies’, ‘antiviral peptides’ and ‘live attenuated viruses’. there are many options for the development of mers-cov-specific therapies. currently, mers-cov is not considered to have pandemic potential. however, the high mortality rate and potential for mutations that could increase transmissibility give urgency to the search for direct, effective therapies. well-designed and controlled clinical trials are needed, both for existing therapies and for prospective direct therapies. middle east respiratory syndrome coronavirus overview middle east respiratory syndrome coronavirus (mers-cov) was first isolated in jeddah in the kingdom of saudi arabia (ksa) from a -year-old male hospital patient, who died june , days after presenting with acute pneumonia and subsequent renal failure [ ] . since then, the who have been notified of laboratory-confirmed cases, including deaths [ ] . while most cases have arisen in the middle east, cases have also emerged in countries worldwide in travellers from the middle east and/ or in their contacts [ ] . most human mers-cov infections are considered to be the result of multiple zoonotic transfers. bats are the most likely mers-cov natural reservoir, as with other mammalian coronaviruses (covs), while camels are likely to be the major zoonotic source for human infections [ ] [ ] [ ] . secondary human-to-human transmission is considered to be limited, occurring mainly within family and healthcare settings. the first cluster of cases in humans was retrospectively identified to have occurred in a public hospital in jordan in april [ ] . multiple healthcare facility-associated outbreaks have since occurred in the middle east, most notably in ksa, often linked to deficiencies in infection control procedures [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although cases outside the middle east have mainly been isolated, a large outbreak occurred in korea in june , in which human-human transmission resulted in cases and deaths [ ] . increased vulnerability to either cross-species or trans-human transmission could result from viral adaptations [ ] . mers-cov infection is often accompanied by acute viral pneumonia, and sometimes gastrointestinal symptoms. clinical severity varies from asymptomatic to death, and the extent of asymptomatic spread is unclear. the high mortality rate is mainly accounted for by acute respiratory distress syndrome (ards) [ , , ] . higher mortality is observed among vulnerable patients, such as older individuals and those suffering from comorbid illness, and is also associated with high viral load [ , , , ] . in one study in a ksa hospital, intensive care unit (icu) admission among mers-cov patients was associated with a mortality rate of . % [ ] . while mers-cov is not currently considered to have pandemic potential, it is clear that human-human transmission does occur. the exact mechanisms by which mers-cov is transmitted from animals to humans have not been fully elucidated. in the south korean outbreak, the virus emerged in second-and third-generation contacts, resulting in the first human case to be imported into china [ ] . this raised concern that viral mutations were contributing to humanhuman transmission. given its high mortality and poor outcomes for vulnerable patients, and the potential for viral mutations, there is no room for complacency in the search for therapeutic options for mers-cov. there is currently no specific therapy. many of the therapeutic options used have been adapted from approaches used to treat severe acute respiratory syndrome (sars-cov) during the outbreak of , and/or the h n influenza virus during the outbreak of [ ] . however, while mers-cov and sars-cov are phylogenetically related betacoronaviruses, they differ in many important respects. mers-cov utilizes human dipeptidyl peptidase (dpp ; cd ) receptors, with binding mediated by the viral spike (s) protein, not the angiotensin-converting enzyme (ace- ) receptors used by sars [ ] [ ] [ ] [ ] [ ] . mers-cov also has a wider cellular tropism [ ] [ ] [ ] . therapies currently used include broad-spectrum antibiotics, corticosteroids and anti-viral treatments, such as interferons (ifn), ribavirin, lopinavir-ritonavir, or mycophenolate mofetil [ , , [ ] [ ] [ ] [ ] [ ] . however, the efficacy and/or safety of many of these therapies is unclear, and none are specific to mers-cov. ribavirin monotherapy, for example, is associated with multiple side-effects in the treatment of other viral illnesses, including sars-cov, has uncertain efficacy, and has not been tested in animal studies or randomized control trials for mers-cov [ , , ] . corticosteroids have not been successful in the treatment of respiratory distress or lung fibrosis in mers-cov [ , ] . meanwhile, studies in sars-cov and h n patients suggest that corticosteroid use may in fact increase viral replication in airways, and sars patient and animal studies indicate that it contributes to immunosuppression [ ] [ ] [ ] . mycophenolate mofetil has been associated with fatal disease and high viral loads in a marmoset model of mers-cov infection [ ] . ifn therapy, alone or in combination with ribavirin or lopinavir-ritonavir, has shown greater promise in in vitro, animal and human studies [ ] [ ] [ ] [ ] . however, clinical studies on ifns vary with respect to factors such as time of administration and type of patient [ , , ] . overall, there is a lack of randomized control trials (rcts) designed to test the safety and efficacy of any potential therapies specific to mers-cov, and much of the information available for existing therapies is based on in vitro and/or animal studies [ , , ] . a position paper on the evidence base for specific mers-cov therapies, published by public health england (phe) and the world health organization-international severe acute respiratory and emerging infection consortium (isaric-who), suggested that benefit was likely to exceed risk for convalescent plasma, lopinavir-ritonavir, ifns and monoclonal/polyclonal antibodies, while, by contrast, for ribavirin monotherapy and corticosteroids it was considered that the risks would outweigh the benefits [ ] . for interferon/ribavirin combination therapy, nitazoxanide and chloroquine, the available data were considered to be inadequate for assessment [ ] . in this review, we consider potentially effective mers-cov therapies, including ifns, lopinavir-ritonavir and inhibitors of proteases, including tmprss and cathepsin l, as well as mers-cov-specific potential therapies, including convalescent plasma, monoclonal antibodies (mabs), antiviral peptides and candidate vaccines. these therapies will be considered from a molecular perspective, in the context of the infection and replication mechanisms of mers-cov. the therapies are summarized in table . mers-cov lineage and structure mers-cov is a betacoronavirus belonging to clade c (lineage ) of the betacoronaviruses [ ] . its closest known coronavirus relatives are the prototypic clade c betacoronaviruses, tylonycteris bat virus hku , pipistrellus bat hku virus and neoromicia zuluensis bat pml/ (neocov) virus [ , [ ] [ ] [ ] [ ] [ ] . in common with other coronaviruses, the genome of mers-cov is a single, positive-stranded rna of over nucleotides. it encodes predicted open reading frames (orfs) and genes for structural proteins, namely the spike (s), nucleocapsid (n), membrane (m) and envelope (e) proteins (figs and ) [ ] [ ] [ ] . orf a and b encode virus replication-related proteins (pp a, pp ab), which are cleaved to give non-structural proteins (nsps) involved in synthesis of viral rna and recombination ( fig. ) [ ] [ ] [ ] . these include nsp- , which contains a -to- exoribonuclease (exon) domain that is important in viral proofreading and in determining the sensitivity of rna viruses to mutagens. thus small-molecule inhibitors references s /dpp binding antibody (mouse): s rbd in vitro [ ] antibody (human): s rbd m , m , m in vitro/in vivo (mouse, rabbit -m ) [ ] [ ] [ ] antibody (human): s rbd in vitro [ , ] antibody (mouse-humanized): s rbd in vitro/in vivo (mouse) prophylactic and therapeutic [ ] antibody (mouse-humanized): s rbd hms- in vitro/in vivo (mouse) [ ] antibody (human): s rbd in vitro/in vivo (mouse) potential for more mers-specific agents [ ] of exon activity could be candidates for mers-cov and other coronavirus therapies [ ] . as with other coronaviruses, the mers-cov s protein is critical to host cell receptor binding and cell entry, and is considered to have been under strong positive selection pressure when the virus was transmitted to humans [ , ] . hence the s protein is a major target for potential anti-mers-cov therapies [ ] . the s protein of mers-cov is composed of s and s subunits ( fig. ) [ ] . in common with other coronaviruses, entry into host cells depends on the s subunit, which contains a receptor-binding domain (rbd) comprising a core subdomain and a receptor-binding motif (rbm). the mers-cov rbm differs from that of sars-cov and dictates that mers-cov uses the dpp receptor, as opposed to the ace- receptor [ , ] . the infection process is shown in fig. . dpp , which is widely expressed in tissues, including the lung and kidneys, is critical in the species tropism of mers-cov infection; bat, human, camel, non-human primate and swine cells, for example, are permissive for mers-cov infection, whereas mouse, hamster and ferret are not [ , ] . host species restriction has been attributed to differences in five amino acids involved in dpp -rbd binding, with glycosylation of the mouse dpp also identified as being important in the inhibition of mers-cov infection [ ] [ ] [ ] . the human dpp receptor is therefore a potential target for mers-covspecific therapeutics, in particular anti-dpp mabs (fig. , table ) [ ] [ ] [ ] [ ] . adenosine deaminase (ada), which is a dpp -binding protein, competes with mers-cov for dpp binding and hence is a natural mers-cov antagonist; this gives potential insights for the development of therapeutic antagonists [ ] . mers-cov binds to the permissive host cell dpp via the rbd of the s domain, one of the major targets for potential mers-cov therapies [ ] . similarly to other coronaviruses, mers-cov then uses the s subunit for virus-host membrane fusion (fig. ) . fusion results in cleavage of the s protein at the s /s boundary by host proteases [ ] . the s subunit contains the fusion peptide, two heptad repeat domains termed hr and hr , and a transmembrane (tm) domain ( fig. ) [ ] . membrane fusion requires conformational rearrangement of s , the formation of a sixhelix bundle ( hb) fusion core, of which hr and hr are essential elements, and exposure of the fusion peptide, which inserts itself into the host cell membrane [ , , ] . hr -derived peptides have been identified as potentially effective anti-viral agents for treatment of mers-cov [ ] ( fig. ; table ). the availability of host cell proteases is essential for mers-cov entry into cells [ , ] . the host proteases responsible for s protein cleavage at the s /s boundary include the serine protease tmprss , endosomal cathepsins such as cathepsin l, and furin protease [ , , [ ] [ ] [ ] . in vitro studies suggest that uncleaved mers pseudovirus can enter host cells by cathepsin l-dependent endocytosis, but that cleavage of virus during maturation by host proteases such as tmprss results in viral entry at neutral ph and the formation of massive syncytia [ ] . host cell proteases are therefore potential molecular therapeutic targets for mers-cov prophylaxis and/or treatment (fig. , table ). the tmprss inhibitor camostat, for example, has been identified as a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [ ] . following host cell entry, mers-cov pp a and pp ab are synthesized and then cleaved by two viral proteases, the main protease (mpro/ clpro) and the papain-like protease (plpro) (fig. ) [ , ] . thus viral proteases represent further potential molecular targets for therapy ( table ) . the recently described mers-cov mpro crystal structure resembles other coronavirus mpro proteases [ ] . the sars-cov pl(pro) inhibitors, -mercaptopurine ( mp) and -thioguanine ( tg), can inhibit mers cov protease activity in vitro, as can the immunosuppressant drug mycophenolic acid [ ] . however, caution is required, as the results of studies on marmosets have associated use of mycophenolate mofetil with fatal disease and high viral loads [ ] . other mers-cov proteins involved in helping the virus to circumvent the immune system also present potential molecular targets (fig. ) . for example, the accessory protein products of orf a, b and are interferon (ifn) antagonists [ , ] . the orf a protein both inhibits type i ifn production via cytoplasmic and nuclear mechanisms, and interferes with the ifn-mediated interferon-stimulated response element (isre) promoter element signalling pathways [ , ] . this gives a molecular level rationale for the use of ifn as a therapeutic option in mers-cov treatment. mers-cov can also infect dendritic cells and macrophages [ , , ] . endosomal uptake of mers-cov by dendritic cells following binding via dpp prompts these cells to produce abundant amounts of type i and iii ifns [ ] . this gives context to the ifn antagonism exhibited by mers-cov accessory proteins. recently, mers-cov has also been shown to infect t cells, which are rich in dpp , both in vitro and in marmoset spleen [ ] . this results in t cell apoptosis and could contribute significantly to viral pathogenesis and further emphasizes the potential therapeutic utility of molecular targeting of dpp and/or the mers-cov s protein. both convalescent plasma containing virus-specific antibodies and the use of specific mabs provide options for targeting mers-cov infection at a molecular level. the use of convalescent plasma [or hyperimmune iv immunoglobulin (hvig) from the plasma of convalescent donors] for infectious disease treatments has a long history, including in the treatment of respiratory diseases [ ] [ ] [ ] [ ] . for influenza and sars-cov infection, early convalescent plasma treatment within - days of symptoms is associated with decreased viral load and reduction in mortality [ ] [ ] [ ] [ ] . however, for sars-cov the quality of studies has been inconsistent and the results have been inconclusive, with a lack of adequate clinical trials [ , ] . according to the phe and isaric-who position paper, convalescent plasma (or high neutralizing antibody titre products) is indicated for the treatment of serious mers-cov infection [ ] . one rct on critically ill patients with h n infection identified a significant reduction in viral load and mortality in patients who received hvig within days of the onset of symptoms [ ] . to date, no rcts have been completed on the use of convalescent plasma/hvig in mers-cov patients. in the light of results from sars and influenza patients, an ongoing clinical trial on the safety and efficacy of convalescent plasma treatment for critically ill mers-cov patients was initiated in may and is due to report in june [ ; clinicaltrials.gov identifier: nct ]. this trial is being carried out in ksa. however, as is common for convalescent plasma therapies, the trial has been affected by logistical and technical issues, including the availability of sufficient donors [ , ] . issues can also arise in the collection of convalescent plasma that has sufficient levels of mers-cov antibodies, particularly outside the middle east [ , ] . while there are two case reports in which intravenous immunoglobulin (ivig) was used in treatment of mers-cov, it is uncertain as to whether this contributed to patient recovery [ , ] . thus, while convalescent plasma is a promising potential therapy for mers-cov, the available clinical evidence is very limited and the results of the ongoing clinical trial will be vital in guiding any future use [ ] . focused development of neutralizing monoclonal antibodies targeted against specific mers-cov proteins has meanwhile yielded promising in vitro and/or in vivo results. monoclonal antibodies: s -dpp binding a number of mouse and human neutralizing mabs against the s region of mers-cov have been developed and tested in vitro and/or in animal models [ ] . targeting of s protein for therapeutic purposes was recently comprehensively reviewed by du et al. [ ] [ ] . in particular, the s rbd is a popular target, as mabs directed against this region have the most potent neutralizing capacity. however, in terms of vaccine development, neutralizing antibodies raised by immunization with full-length s or s protein expression vectors may produce a more effective immunogenicity through the targeting of multiple epitopes and the reduction of the possibility of escape mutations [ ] . nevertheless, many mouse and human mabs targeting the s rbd have given promising results in vitro and in mouse models [ ] . (table ) . a mouse monoclonal antibody, mersmab , blocks mers pseudovirus cell entry in vitro by binding to the rbd and preventing s binding to dpp [ ] . meanwhile, the human monoclonal antibodies m , m and m , which target overlapping epitopes in the rbd, all potently neutralize pseudovirus and live mers-cov in vitro [ ] . significantly, intraperitoneal injection of m has also been shown to have both prophylactic and therapeutic protective effects against mers-cov infection in a wellestablished human dpp (hdpp )-expressing transgenic mouse model, and in rabbits [ , ] . other anti-rbd human antibodies, mers- and mers- , which recognize distinct rbd regions and block binding to dpp , likewise have potent in vitro neutralizing activity against pseudovirus and live virus infection, and they also act synergistically [ , ] . mers- has anti-syncytia formation activity [ ] . the crystal structure of mers- bound to the dpp receptor revealed two critical rbd residues [ ] . the crystal structure of another anti-rbd antibody c , which was raised in mice, has also been elucidated. this has allowed the identification of an epitope that partially overlaps the rbd receptor binding unit [ ] . c was consequently humanized to give an antibody with prophylactic and therapeutic properties, shown by a reduction of mers-cov lung viral titres in an ad -hcd (hdpp ) transgenic mouse model [ ] . another humanized anti-rbd antibody, hms- , similarly has potent in vivo protective properties against fatal mers-cov infection in a transgenic hdpp mouse model [ ] . the human antibody lca targets both the n-terminal domain (ntd) and the rbd of the s region, and was isolated from b cells of a mers-cov-infected human donor before being used to rapidly establish a stable cho cell line that can be used to reliably produce clinical grade antibody [ ] . this is a promising candidate for clinical development, given the antibody's potent prophylactic and therapeutic activities against mers-cov infection in ad /hdpp transgenic mice and type i interferon receptor (ifnar)-ko mice [ ] . the human anti-rbd antibody b -n is another promising candidate that prophylactically reduces lung pathology in rhesus monkeys infected with mers-cov [ ] . targeting the s -dpp interaction from the host side through the development of anti-dpp (cd ) antibodies is another possible therapeutic option. the anti-cd antibodies f , f and ys target the mers-cov entry into cells in vitro [ ] . the f epitope maps close to the binding site of ada, a natural dpp binding protein and mers-cov antagonist, while the f and ys epitopes lie outside this region [ , ] . thus, targeting of the s -dpp interaction by use of mabs is a promising strategy for the clinical development of molecular therapeutics against mers-cov. another molecular approach involves targeting of the s -mediated mers-cov-host cell fusion element of the mers-cov infection cycle by use of antiviral peptides. the role of hr and hr in viral fusion makes them potentially effective molecular therapeutic targets. this has been borne out by in vitro and in vivo results obtained using hr peptides (table ). hr peptides are ineffective antivirals as they aggregate in physiological solutions [ ] [ ] [ ] . a peptide named hr p, which spans residues - of hr , effectively inhibits viral replication and s proteinmediated cell fusion in vitro [ ] . a hr p analogue named hr p-m is an even more potent fusion blocker in vitro, and inhibits mers cov-expressing pseudovirus infection [ ] . hr p-m interacts with an hr peptide to effectively block hb bundle formation. in vivo hr p-m intranasal administration to ad /hdpp transgenic mice protected them from mers-cov infection, as evidenced by the reduction of the lung viral titres by more than -fold [ ] . the addition of ifn-b along with hr p-m enhanced the protective effect [ ] . thus, s hr peptides have potential as mers-cov intranasal antiviral treatments. the s protein is also the focus of a number of candidate vaccines (table ) [ , [ ] [ ] [ ] . a fusion product combining truncated rbd and the fc portion of human igg can bind human dpp and inhibit mers-cov infection in an in vitro cell culture model [ ] . importantly, this rbd-igg fusion product can induce a humoral response in mice vaccinated by subcutaneous injection, hence blocking rbd-dpp binding and inhibiting mers-cov infection [ ] . further in vivo studies have indicated that intranasal administration to mice induces similar long-term igg humoral responses to those achieved with subcutaneous injection, but superior cellular immune responses and local mucosal responses in lungs [ , ] . this suggests that this type of construct is both potentially effective and readily deliverable by intranasal means. use of an adjuvant, particularly mf , significantly improves the humoral and t cell immunogenicity of the rbd s - -fc igg fusion construct in subcutaneously immunized mice [ ] . the possibility of using the s rbd as a vaccine molecular target for a range of divergent mers-cov strains and escape mutants has also been explored recently [ ] . the use of five recombinant rbds with mutations observed in different mers-cov outbreaks or in camel strains induced potent neutralizing antibody responses against several mers-cov pseudoviruses [ ] . however, while the rbd of the s subunit is a logical and promising target for mers-cov vaccine development, the epitope scope is relatively limited and full-length s protein may be a preferable option [ ] . technical difficulties in stably expressing abundant quantities of full-length s protein have presented a barrier. however, studies on delivery options, including the use of adjuvants and nanoparticles, may help in overcoming such issues. one study undertaken by novavax (gaithersburg, maryland, usa) showed that the inoculation of mice by intramuscular injection with fulllength s protein proprietary nanoparticles produced a relatively low neutralizing antibody response after days [ ] . however, the addition of the adjuvants alum or matrix m resulted in a robust and sustained anti-mers-cov neutralizing antibody response [ ] . [ ] . these viruses are expected to enter clinical trials as a proposed prophylactic mers-cov vaccine. likewise, intramuscular injection of ad or ad expressing full-length s protein induces both antigen-specific t cell and neutralizing antibody responses in mice [ ] . finally, intraperitoneal injection of measles virus expressing either membraneanchored, full-length s protein or soluble s protein lacking the tm domain induces robust mers-cov antigen-specific neutralizing antibody and cytotoxic t lymphocyte in interferon-a/b receptor (ifnar)-deficient mice [ ] . recently, an mva-based vaccine expressing s protein has been shown to induce mucosal immunity in mers-cov-infected dromedary camels, with a reduction in excreted virus and viral transcripts [ ] . this has potential for veterinary use and the reduction of cross-species infection of humans by camels [ ] . dna plasmids gls- is a dna-plasmid vaccine that encodes mers-cov s protein (table ) [ , ] . it was co-developed by inovio, geneone life science, inc. and the walter reed army institute of research, and has become the first potential mers-cov vaccine to enter human testing [ , ] . a phase i clinical trial in healthy volunteers commenced in for the evaluation of its safety and ability to generate antibody and cellular immune responses over a -year period, using one of three dosages in a three-injection regimen [ ] . the vaccine has already undergone pre-clinical trials in mice, camels and macaques [ ] . it induced robust and antigen-specific cytotoxic t lymphocyte and neutralizing antibody responses, which effectively protected animals against viral infection [ ] . gls- and other potential vaccine candidates provide an opportunity to develop a prophylactic mers-cov vaccine. however, the barriers to development of a prophylactic vaccine include the current relatively low mers-cov incidence in humans, as well as sourcing suitable small animal models [ , , ] . these factors complicate the definition of a target population for mass prophylactic vaccination and pre-clinical demonstration of vaccine efficacy [ ] . in this context, the monoclonal antibodies described above may be invaluable resources in an outbreak situation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in vitro and animal studies while mers-cov-specific therapies are offering promising pre-clinical results, and gls- has entered clinical trials, there is as yet no specific evidence-based therapy or vaccine clinically available for mers-cov. as described in the mers-cov infection and replication section, mers-cov accessory protein products are ifn antagonists [ , ] . attenuation of the ifn response is an important mers-cov immune response circumvention mechanism [ ] . the orf a in particular inhibits ifn-b production via the inhibition of interferon regulatory transcription factor (irf)- and nuclear factor (nf)-kb actions, and thus irf- -activating small molecules, for example, may be potential therapeutic agents for restoring ifn responses [ , ] . toll-like receptor- (tlr- ) is also involved in the immune response of mice to sars-cov and mers-cov, recognizing viral molecular patterns and initiating the innate response that leads to ifn production (fig. ) [ ] . thus, tlr- agonists are another possible candidate for mers-cov-specific anti-viral agents [ ] . therapeutically, ifn itself is particularly useful prophylactically or during the early days of viral exposure, including for coronaviruses [ , ] . in vitro and animal studies have confirmed the potential efficacy of ifns in mers-cov therapy, in particular in combination with other therapeutic agents such as ribavirin and/or lopinavir. in vitro, mers-cov was substantially more susceptible to ifn-a than sars-cov [ ] . while mers-cov in vero or llc-mk cells was sensitive to both ifn-a b and ribavirin separately, relatively high concentrations were required to reduce viral replication [ ] . however, combination therapy allowed the concentrations of each to be substantially reduced [ ] . combination therapy of ifn-a b and ribavirin in macaques administered hours after mers-cov infection reduced systemic and local viral effects, and reduced viral genome copy number and gene expression levels [ ] . bioinformatics data from microarray analysis recently showed that ifn-a b and ribavirin treatment impacts on mers-cov gene expression in different pathways, including genes involved in recognition of pathogens, immune responses and release of cytokines [ ] . both ifn-b b and lopinavir treatment, alone or in combination, also protected marmosets from the adverse clinical, radiological and pathological effects of mers-cov infection [ ] . clinically, the use of ifn monotherapy, or ifn therapy in combination with ribavirin and/or lopinavir/ritonavir, has shown some promise (table ) [ ] [ ] [ ] [ ] . however, the interpretation of clinical studies has been complicated by variability in factors such as the stage of infection at which therapy was administered. the available data are limited to case studies and retrospective cohort studies [ , ] . in one case study on a patient who died in a greek hospital, pegylated ifn along with ribavirin and lopinavir was administered as part of the treatment regime, but not until the thirteenth day of the illness [ ] . by contrast, in another preliminary study on two patients, the first patient was treated with ifn-a b and ribavirin within a day of admission prior to mers-cov diagnosis, but he was also being treated with antibiotics, steroids and non-invasive ventilation [ ] . patient , the wife of patient , was treated prophylactically after developing a low-grade fever and poorly defined lung infiltrates, but a diagnosis of mers-cov was not formally made [ ] . thus, while patient survived and patient had only a mild course of illness, it is difficult to draw any firm conclusions regarding the efficacy of the treatment. in another case study on a patient in korea, administration of pegylated ifn-a a along with ribavirin and lopinavir days after hospital admission was deemed to have been effective in viral clearance and patient survival [ ] . these case studies do not overall provide firm evidence for the efficacy or otherwise of ifn combination therapy for mers-cov. in one case involving a series of five patients who were critically ill with mers-cov infection and on mechanical ventilation and corticosteroids, ifn-a b and ribavirin was administered on average days after admission [ ] . all five patients died, but they may not have benefited, as they were treated late in their illness and were already critically ill [ ] . the benefit of earlier treatment in less vulnerable patients was suggested in another series of six patients in which three who received ifn-a b and ribavirin early in the illness survived, while three other patients who were older and had comorbid conditions received the combination treatment later and all died [ ] . however, in another study in which mechanically ventilated patients with severe mers-cov infection who received pegylated ifn-a a and ribavirin early in treatment were compared to patients who did not receive the combination therapy, the -day survival rate was significantly higher in the treatment group, but the -day survival rate was equivalently low in the two groups [ ] . in another retrospective analysis of results from a series of patients who received either ifn-a a or ifn-b a in combination with ribavirin, no significant difference in outcome between the two types of ifn was shown, and there was no survival benefit due to use of either ifn [ ] . however, most of the patients in this study were aged more than years and some had comorbid conditions, including end-stage renal disease [ ] . thus the retrospective studies that have been carried out are heterogeneous in terms of type of patient, stage of disease and type of ifn used, including whether or not it was pegylated or short-acting. there is an urgent need for well-controlled clinical trials for ifn combination therapy in mers-cov, preferably early in the illness, as ifns are routinely available agents whose safety and efficacy is established for other viral illnesses and whose use has a sound molecular basis for mers-cov treatment. another type of therapy with a logical molecular basis for mers-cov treatment is the targeting of proteases, both host and viral (table ; fig. ) [ , , , , , [ ] [ ] [ ] [ ] . camostat, an inhibitor of tmprss , is a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [ ] . in a pathogenic mouse model of sars-cov infection, viral spread and pathogenesis was effectively blocked by camostat, and it is likely that it would have a similar impact on mers-cov [ ] . as camostat is already in clinical use for the treatment of chronic pancreatitis, it represents a potentially safe and effective therapeutic option. recently, another tmprss inhibitor, nafamostat, was identified in a split protein-based cell-cell fusion assay as a potent inhibitor of mers-cov s protein-mediated hostviral membrane fusion in vitro [ ] . nafamostat is already clinically approved for use by the us food and drug administration (fda) and is used as an anticoagulant [ ] . the cathepsin l inhibitor teicoplanin, a glycopeptide antibiotic, was recently shown, via high throughput screening of fda-approved drugs, to block entry of mers-cov, sars-cov and ebola pseudoviruses into the cytoplasm [ ] . teicoplanin is currently used clinically as an antibiotic in both prophylaxis and the treatment of serious grampositive bacterial infections. it also has derivatives, including dalbavancin, oritavancin and telavancin, all of which also block viral entry. the viral proteases, mpro ( clpro) and pl(pro), also represent potential molecular therapeutic targets [ , ] . as well as its role in viral maturation, the mers-cov pl(pro) causes deubiquitination of ifn regulatory factor (irf- ), and hence suppression of ifn b production, which contributes to viral suppression of the innate immune response (fig. ) [ , ] . the x-ray d crystal structure of mers-cov pl(pro) is similar to that of sars-cov, and includes ubiquitin-like and catalytic core domains [ ] . thus the sars-cov pl(pro) inhibitors, -mercaptopurine ( mp) and -thioguanine ( tg), can inhibit mers cov protease activity in vitro [ ] . however, the mers-cov pl pro crystal structure also has unique aspects, including the oxyanion hole, and s and s subsites, which may be viable molecular targets for antivirals specifically designed against mers-cov [ ] . a commercial compound termed compound (commercial code f - ,life chemicals) has been identified as an inhibitor of mers-cov and sars-cov plpro activity [ , ] . the critical binding interactions and mode of inhibition differ between the two viral proteases, with the compound acting as a competitive inhibitor against mers-cov pl(pro), but an allosteric inhibitor of sars-cov pl[pro) [ ] . however, f - may lose potency in physiological reducing environments [ ] . lopinavir is a protease inhibitor with activity against the sars-cov main protease m pro [ ] . in a screen of a library of fda-approved drugs to identify anti-mers-cov activity in cell culture, lopinavir emerged as one of four compounds that inhibited viral activity in a low micromolar range [ ] . however, the clinical efficacy of lopinavir in mers-cov treatment has not yet been fully established. as mentioned above, it has usually been used clinically in combination with ifn and data are only available from case studies and series. however, notably, lopinavir-ritonavir treatment resulted in better clinical, radiological and pathological outcomes and reduced mortality in marmosets infected with mers-cov [ ] . lopinavir has also been identified in a position paper from phe and isaric-who as a potential mers-cov therapy whose benefits are likely to exceed its risks [ ] . thus far, mers-cov has not been considered to have pandemic potential. most cases have occurred in the middle east, particularly in ksa. outbreaks have been primarily linked to healthcare institutions, and shortcomings in infection control and prevention procedures [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, potential viral mutations could facilitate expanded viral host range and enhance cross-species and human-human transmission [ , , ] . the outbreak in korea resulted in mers-cov emergence in second-and third-generation contacts, highlighting the potential for mutational changes that could increase the likelihood of human-human transmission [ , ] . mers-cov also exacts a high mortality rate, mainly due to the development of ards [ ] [ ] [ ] . these factors emphasize the importance of developing targeted therapies and/or vaccines. the most promising advances in the development of specific molecular mers-cov therapies relate to targeting of the viral s protein by means of anti-s monoclonal antibodies, hr-targeted antiviral peptides and viruses or plasmids bearing s protein as potential vaccine candidates [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the use of ifns, usually in combination with other therapies such as ribavirin or lopinavir, also has a logical molecular basis given that ifn antagonism is an important mechanism by which the virus circumvents the innate immune system [ , , , ] . targeting of host and viral proteases is also a sound molecular approach, as host proteases are important in viral-host membrane fusion, while viral proteases are key to viral maturation and are also involved in targeting ifns [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the therapies currently used for mers-cov have mainly been extrapolated from those used for sars-cov treatment, regardless of the important differences in receptor usage and cellular tropism between the viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] . none of these therapies have been subject to well-controlled trials, and in some cases the risks are likely to outweigh any poorly defined benefits [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in general, the clinical research response to mers-cov may have been too slow [ ] . thus, while there are many promising lines of research in terms of specific molecular targeting of mers-cov, no potential therapies have yet been subject to welldesigned clinical trials, and none have been approved for clinical use, apart from the gls- dna-plasmid vaccine [ , ] . continuing outbreaks of mers-cov, with possible increases in human-human transmission, are likely to galvanize the research community to push ahead with the design and performance of clinical trials for some of the available monoclonal antibodies and/or antiviral peptides for use in outbreak situations. there are various challenges inherent in the development of specific mers-cov therapies. these include the difficulty of identifying a target population for potential prophylactic vaccines, limited small animal model availability and dependence on transgenic mouse models, and the current relatively low incidence of infection, which complicates the performance of adequate clinical trials [ , , , ] . for example, one currently ongoing trial on convalescent plasma therapy has been affected by logistical and technical issues, including insufficient available donors and difficulty in collecting convalescent plasma containing sufficient mers-cov antibody levels [ , ] . thus, while numerous monoclonal antibodies have been raised with anti-mers activity, in particular against the s protein [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and promising antiviral hr peptides have been synthesized [ , ] , the available data are thus far limited to in vitro and animal studies. despite these issues, there is cause for optimism, given the many candidate antibody and peptide therapies. there is also some promising in vitro and animal model evidence suggesting that use of ifns, which are well-established therapies in other viral illnesses, may be of benefit if used sufficiently early in mers-cov treatment, or as a prophylactic, especially in combination with other therapies, including ribavirin or lopinavir-ritonavir [ ] [ ] [ ] [ ] . likewise, other drugs that are currently in clinical use for other conditions have been shown to be potentially useful for mers-cov treatment, including camostat and nafamostat; teicoplanin and its derivatives dalbavancin, oritavancin and telavancin; and the sars-cov pl(pro) inhibitors, -mercaptopurine ( mp) and -thioguanine ( tg) [ , , [ ] [ ] [ ] [ ] . these drugs have already been shown to be safe and well-tolerated by humans. repurposing of existing drugs may therefore prove to be the most viable option in mers-cov therapy. for example, screen of approved drugs uncovered candidates with in vitro activity against both mers-cov and sars-cov, including oestrogen receptor inhibitors and dopamine receptor inhibitors [ ] . thus, there are 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respiratory syndrome coronavirus infection repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection mechanisms of coronavirus cell entry mediated by the viral spike protein the authors received no specific grant from any funding agency. the authors declare that there are no conflicts of interest. this is a review article and no experimental work with humans was performed. five reasons to publish your next article with a microbiology society journal . the microbiology society is a not-for-profit organization. . we offer fast and rigorous peer reviewaverage time to first decision is - weeks. . our journals have a global readership with subscriptions held in research institutions around the world. . % of our authors rate our submission process as 'excellent' or 'very good'. . your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord- - l ydspz authors: webb, l. g.; veloz, j.; pintado-silva, j.; zhu, t.; rangel, m. v.; mutetwa, t.; zhang, l.; bernal-rubio, d.; figueroa, d.; carrau, l.; fenutria, r.; potla, u.; reid, st. p.; yount, j. s.; stapleford, k. a.; aguirre, s.; fernandez-sesma, a. title: chikungunya virus antagonizes cgas-sting mediated type-i interferon responses by degrading cgas date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: l ydspz chikungunya virus (chikv) is a mosquito-borne alphavirus known to cause epidemics resulting in predominantly symptomatic infections, which in rare cases cause long term debilitating arthritis and arthralgia. significant progress has been made in understanding the roles of canonical rna sensing pathways in the host recognition of chikv; however, less is known regarding antagonism of chikv by cytosolic dna sensing pathways like that of cyclic gmp-amp synthase (cgas) and stimulator of interferon genes (sting). with the use of cgas or sting null cells we demonstrate that the pathway restricts chikv replication in fibroblasts and immune cells. we show that dna accumulates in the cytoplasm of infected cells and that chikv blocks dna dependent ifn-β transcription. this antagonism of dna sensing is via an early autophagy-mediated degradation of cgas and expression of the chikv capsid protein is sufficient to induce cgas degradation. furthermore, we identify an interaction of chikv nsp with sting and map the interaction to residues in the cytosolic loop of the adaptor protein. this interaction stabilizes the viral protein and increases the level of palmitoylated nsp in cells. together, this work supports previous publications highlighting the relevance of the cgas-sting pathway in the early detection of (+)ssrna viruses and provides direct evidence that chikv interacts with and antagonizes cgas-sting signaling. introduction chikv is an arbovirus belonging to the genus alphavirus (family: togaviridae) which is transmitted primarily by mosquitos of the aedes spp [ , ] . approximately - % of chikv infections are symptomatic and the hallmark of infection is an acute febrile illness with a sudden high-grade fever as well as debilitating arthritis and arthralgia which can persist, in some infected individuals, for months to years after clearing the infection [ ] [ ] [ ] [ ] . since its first characterization in , chikv has been noted to cause sporadic and explosive outbreaks resulting in a potentially debilitating range of inflammatory diseases [ ] [ ] [ ] . between - , chikv was responsible for an outbreak on the island of la reunion where more than one third of the island's population was infected in the span of a few months [ ] . this outbreak then spread to india, where an estimated . million infections occurred, southeast asia, and ultimately to italy where the first subtropical autochthonous transmission was established [ , ] . the chikv genome is a positive-sense single stranded rna (+ssrna) with a type , ' methyl-guanosine cap and poly(a) tail, which encodes six structural and four non-structural proteins. after entry and uncoating of the viral nucleic acid, translation of the non-structural proteins occurs. two polyproteins are produced: either p or, if read-through of an opal stop codon takes place, p [ ] [ ] [ ] . non-structural protein (nsp ) of chikv has been shown to anchor viral replication complexes to cytosolic membranes through both an amphipathic helix as well as through palmitoylation of three cystine (cys) residues - [ , ] . mutations of these residues have been shown for both chikv and other alphaviruses to severely hamper viral replication kinetics and decrease pathogenicity in mouse models [ , ] . viral structural proteins are then translated from the s rna and processed threaded into the endoplasmic reticulum (er) with exception of the capsid protein which remains in the cytosol prior to encapsidation of the viral genomic rna [ ] . innate immune defenses are at the forefront of restricting viral pathogens. successful replication and subsequent release of viral progeny is largely dependent upon the ability of a virus to evade or inhibit both detection and activation of cellular antiviral responses. chikv is no exception, the type-i interferon (ifn) response has been demonstrated to be key in controlling chikv infection [ ] [ ] [ ] [ ] [ ] [ ] and primary sensing of the virus is via the pattern recognition receptor (prr) retinoic acid-inducible gene i (rig-i)-mitochondrial antiviral signaling protein (mavs) axis [ , ] . alternatively to pathogen associated molecular patterns (pamps), cell intrinsic molecules released as a result of cellular stress, termed danger associated molecular patterns (damps), are also potent inducers of innate immune responses [ , ] which lead to the induction of type-i ifns. type-i ifns are a family of cytokines, composed of both ifn alpha (ifna) and ifnß, which are induced though prr signaling [ ] . these cytokines signal in an autocrine and paracrine manner, and are critical in inducing an antiviral state in uninfected cells proximal to infected cells [ ] . antiviral states are induced by transcription and translation of hundreds of interferon stimulated genes (isgs) as a result of type-i ifn signaling [ ] . previous work has shown that chikv nsp inhibits ifn mediated signaling [ ] [ ] [ ] downstream of type-i ifn production and can inhibit rna pol ii dependent transcription [ ] . meshram et al. identified that mutations within nsp alter the production of ifn beta (ifnß) [ ] . to date, however, no function has been identified for any chikv proteins, in restricting the induction of type-i ifn due to dna stimuli. while the canonical view of prr mediated antiviral responses associates rna sensing prrs with rna based pathogens and dna sensing prrs with dna based pathogens, these systems do not exist in isolation. recent work has demonstrated a role for cytosolic dna innate immune sensors in initiating and responding to rna viral infections and it has been shown that there is a crosstalk present between rig-i like receptor (rlr) signaling and dna signaling through the adaptor protein, stimulator of interferon genes (sting) [ , ] . specifically, the cyclic gmp-amp synthase (cgas)-sting pathway has been shown to restrict both human flaviviruses and coronaviruses and the viruses in-turn have mechanisms to inhibit the pathway [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . antiviral responses are activated when cgas binds to double stranded dna or dna-rna hybrids and synthesizes the secondary messenger molecule, '- ' cyclic-guanosine monophosphate (gmp)-adenosine monophosphate (amp), (cyclic gmp-amp or cgamp) which then bind to sting [ , ] . upon cgamp binding, sting dimerizes, translocates to the trans-golgi-network (tgn), where it associates with tank binding kinase (tbk ), which ultimately results in phosphorylation of interferon regulatory factor (irf ) and induction of type-i ifn transcription [ , , , ] . interestingly, activation of the cgas-sting pathway with chemical activators or via overexpression has been shown to restrict alphaviruses, including chikv [ , , ] ; however, no studies have identified direct antagonism of the cgas-sting pathway by chikv. given the importance of type-i ifn in restricting chikv, as well as recent work highlighting the relevance of the cgas-sting pathway in restricting rna viruses, we asked whether chikv was able to antagonize the cgas-sting pathway. chikv has been shown to replicate well in primary human foreskin fibroblast (hff- ) cells and fibroblasts are thought to be primary sites of viral amplification upon infection [ ] . to better understand the innate immune response induced in primary human cells by chikv / , hff- s were infected either with chikv / or the paramyxovirus newcastle disease virus (ndv). ndv, like chikv, is sensed via rlrs [ ] ; however, ndv is an avian pathogen with limited ability to antagonize the induction of the innate immune response in human cells [ , ] . chikv / replicated to high titers in hff- s (fig a) but had a notably muted induction of ifn-ß, isg , and tnfa transcripts at hpi when compared to ndv ( fig b- d ). ndv replication was measured in hff- s and peaked by hpi (fig e) . to better understand if chikv / had similar replication in hff- s to other chikv isolates, it's replication and transcriptional profile were compared to a chikv isolate of the indian ocean lineage (chikv-iol). chikv / replicated better in hff- s when compared to chik-v-iol but did not induce largely different innate immune responses at the transcriptional level (s fig). and supernatants were plaqued on bhks at indicated timepoints. (b-d) rt-qpcr from chikv growth curve in (a) for specified genes (ifnb, isg , and tnfa) relative to rps at hpi. data representative of three independent experiments. data represented as means ± sd (n = ). statistical analysis was performed with by a one-way anova with tukey's multiple comparisons. (e) ndv viral rna relative to rps from infected hff- s (moi = . ) at indicated timepoints. data represented as means ± sd (n = ). (f) diagram of coinfection experiments. briefly, hff- s were infected with either mock, uv-inactivated chikv / (uvc), or chikv / at an moi of . . infections were allowed to proceed for hrs in order to allow for viral protein expression. (g & i) hpi, cells were then treated with mock, mva at an moi of . , or e. coli dna ( ug/ . x cells). and hrs postsecondary treatment, cell lysates were collected. ifnß transcripts were quantified via rt-qpcr for cells stimulated with mva or e. coli dna, respectively, while quantification of isg transcripts was performed as previously stated (g & i). transcripts are represented as "fold over respective mock" e.g. uvc ➔ mva condition was normalized to uvc ➔ mock condition to determine the relative gene induction resultant from secondary treatment. to investigate the ability of chikv to block dna sensing, hff- cells were infected first with infectious or uv-inactivated chikv (uvc) and subsequently infected with either modified vaccinia ankara virus (mva), a dna virus known to be sensed via cgas [ ] , or as an alternative source of cgas ligand, transfected with e. coli dna, respectively ( fig f) . surprisingly, chikv drastically reduced ifn-ß transcription induced by both a dna virus (mva) and a direct cgas agonist (e. coli dna) (fig g & i) . a slight reduction in ifn-ß transcripts was observed in the uvc condition (fig g & i) . this minor inhibition could be mediated by an intrinsic component of the viral nucleocapsid, playing a role in reducing cgas-sting signaling. importantly, the initial infections did not alter the ability of mva to replicate in these cells at hpi, demonstrating that the reduction in innate immune transcripts observed in fig g & i is a result of chikv-mediated inhibition and not due to a reduction in mva replication ( fig h) . the role of individual chikv non-structural proteins in potential inhibition of type-i ifn production was tested using a previously established type-i ifn reporter system [ ] . these reporter cells are hek- t cells that are stably transduced to express firefly luciferase under the control of an interferon beta (ifnβ) promoter and are deficient for both cgas and sting ( t-ifnβ-ffluc) (s a fig). the ability of chikv nsps to inhibit dna mediated induction of the ifnβ promotor was assessed by co-expressing the non-structural proteins of chikv, ross thailand isolate (chikv-rt), with cgas and sting. hrs post transfection, firefly luciferase activity was quantified as a proxy for activity of the ifnβ promoter. inhibition of dna dependent ifnβ promoter activity was observed for nsps , to test for chikv nsp mediated degradation of sting, nsps of chikv-rt were co-expressed with sting. when sting was co-expressed with the four individual chikv-rt nsps no degradation or cleavage of sting was observed, though a clear cleavage of sting was seen in the positive control, sting co-expressed with denv ns b [ ] (s d fig). additionally, nsps - of chikv-rt were individually expressed with cgas to look for potential interactions or degradation of the protein. immunoprecipitation of cgas pulled down nsp of chikv-rt; however, no degradation of cgas was observed in any of the co-expression conditions (s e fig). next, we validated the interaction of chikv / nsp with cgas in the context of infection by over-expression of cgas in hek- t for hrs followed by infection with chikv / and subsequent cgas immunoprecipitation. interestingly, no cgas was observed in the input sample of the infected condition with chikv / (s f fig, lane ) . however, when the immunoprecipitation for cgas was performed, we detected a reduced presence of cgas, even less than in the positive control, denv ns b , which has already been shown to interact with and degrade cgas[ ] (s f fig). to further confirm the interaction between nsp and cgas, hek- ts were transfected with chikv-rt nsp and seeded on glass-bottom plates for imaging by immunofluorescence. a clear co-localization of cgas with nsp was observed in cells which expressed both proteins (s g fig). the lack of cgas degradation seen in the exogenous expression system when compared to the infected cells shows that although there is a clear interaction of cgas with nsp , the degradation of cgas observed is not mediated by the non-structural proteins of chikv, but must require other viral or host factors. representative of two independent experiments (n = ). data are represented by means ± sd (n = ). statistical analysis was done with student's t tests. statistical significance represented as follows: ns = not significant, � = p< . , �� = p< . , ��� = p< . , ���� = p< . . https://doi.org/ . /journal.ppat. .g chikungunya virus inhibits dna sensing by degrading cgas during viral infection, stresses placed upon cells can lead to the release of dna into the cytoplasm of infected cells, thus leading to the activation of cytosolic dna sensors, such as cgas [ , ] . we previously detected such cytosolic dna in denv infected cells via immunofluorescence [ ] . to test if chikv infection results in the appearance of cytoplasmic dna in infected cells, hff- s were infected with chikv / at low or high multiplicity of infection (moi) for hrs. the cells were then probed with antibodies against nsp (viral infection), an anti-dna antibody, clone [ ] [ ] [ ] [ ] , which binds both double stranded and single stranded dna (dsdna/ssdna), and dapi which binds dsdna. during chikv infection we observed a distinct accumulation pattern of dna puncta which were absent in the mock infected condition (fig a) . furthermore, the presence of dna puncta increased in conjunction with the increase in moi (fig a) . high resolution confocal microscopy of infected cells illustrated the presence of this dna to be in the cytoplasm (fig b ( d view inset) ). wild type murine embryonic fibroblasts (mefs) have been shown to be permissive to chikv replication [ ] . we obtained wt and sting null, goldenticket (gt) mefs [ ] (fig a) and infected them with chikv / in order to understand the restriction imposed by sting on chikv. by hpi there was a log increase in infectious particle release from the gt mefs when compared to wt (fig b) . viral transcripts were also significantly lower in wt mefs when compared to their sting null counterparts ( fig c) indicating that murine sting serves as a potent restriction factor for chikv replication and infectious particle release. next, commercially available raw . cells, a murine macrophage cell line, deficient for cgas or sting, were infected with chikv / . infectious virus release was quantified from the supernatant of infected cells and revealed a sharp increase in chikv replication from both the cgas and sting null cells when compared to their wt counterpart ( fig d) . taken together, these data support previous reports that the cgas-sting pathway restricts the replication of chikv [ , , ] and provides direct evidence that the pathway poses a significant restriction upon viral replication. to determine the integrity of the cgas during chikv infection, whole cell lysates were collected from hff- s at , , and hpi. lysates were analyzed via sds-page and endogenous cgas and sting were detected via western blotting (wb). we observed a drastic decrease in cgas expression as early as hpi, before even viral non-structural proteins were detectable via wb in infected cells, while sting expression was not altered (fig a) . because chikv can inhibit cellular transcription, the reduction in cgas expression over time in chikv infected cells could also be due to reduced cgas transcripts, ultimately leading to reduced newly translated cgas. mrna levels of cgas were quantified and there were no differences in chikv infected cells when compared to mock or ndv at hpi (fig b) indicating that a decrease in cgas transcripts is not responsible for the rapid loss of cgas expression following chikv infection. alternatively, cgas levels could be reduced via global translational inhibition by the viral protein nsp [ ], resulting in a reduction in cgas protein levels through normal homeostatic protein recycling. to assess if the half-life of cgas was shorter than the time in which it takes chikungunya virus inhibits dna sensing by degrading cgas chikungunya virus inhibits dna sensing by degrading cgas chikv infection to result in a loss of cgas expression, a puromycin pulse-chase was performed. this technique has been shown to be highly sensitive in detecting a snapshot of active translation in cells [ ] . hff- s were pretreated with mock or cycloheximide (chx) for hrs to block cellular translation and then "pulsed" for min with puromycin to label newly synthesized proteins after which cells were washed and re-fed with complete media for min. we observed a clear reduction of puromycin incorporation in cells which were treated with chx for hrs, demonstrating an inhibition of new protein synthesis in treated cells. although translation had been halted in chx treated cells for hrs, there was no change in cgas expression, demonstrating that the half-life of cgas in hff- s is longer than hrs ( fig c) . in order to assess whether viral induced translational shutoff coincides with loss of cgas expression, chikv-mediated translational inhibition was determined by the previously described puromycin pulse-chase method ( fig d) . in accordance with a previous publication [ ] , chikv mediated translational shutoff was not detectable at or hpi while there was a significant reduction in cgas expression as early as hpi, when compared to mock (fig d) , demonstrating that the loss of cgas expression observed in infected cells is not due to chikv mediated translational inhibition. interestingly, there is a noticeable increase in translation in chikungunya virus inhibits dna sensing by degrading cgas both mock and infected cells by hpi suggesting that the stress produced during the process of infection (mock or chikv) in hff- s stimulates increased translation at early timepoints ( fig d, lanes and ) . as we observed a slight decrease in dna stimuli-dependent type-i ifn transcripts in the uvc conditions (fig g & i ) and because the decrease in cgas occurs very early during the infection, we hypothesized that the viral factor responsible for cgas degradation is a component of the virion. upon viral membrane fusion in endosomes, the viral nucleocapsid is exposed in the cytoplasm which is followed by disassembly of the nucleocapsid resulting in the release of the viral genome along with capsid into the cytoplasm of infected cells. to determine if chikv capsid was sufficient to result in a degradation of cgas, the viral protein was coexpressed with cgas in increasing amounts. a dose dependent cgas degradation was observed when co-expressed with capsid and this reduction was specific, as no reduction in cgas expression was observed when the innate immune sensor was co-expressed with another viral protein, nsp ( fig e) . capsid co-expression was also sufficient to inhibit cgas-sting mediated induction of a type-i ifn reporter, indicating a functional inhibition of the innate immune sensing pathway (fig f & g) . these data indicate an immediate restriction of the cgas-sting pathway by chikv via degradation of cgas. this degradation is independent of viral transcriptional and translational shutoff and is mediated by the viral capsid protein. during infection, chikv is known to induce autophagy which has a proviral effect on replication [ ] . previous work from our group demonstrated an autophagy-mediated degradation of cgas during denv infection, in order to prevent the activation of the cgas-sting pathway by mis-localized mtdna [ ] . we hypothesized that the cgas degradation observed in chikv infection might also occur via autophagy. to test this, hek- ts were transfected with cgas before treatment with a commonly used chemical inhibitor of autophagy, -methyladenine ( -ma)[ , [ ] [ ] [ ] and later infected with chikv / . treatment with -ma was able to rescue expression of cgas in infected cells, indicating a role for autophagy in cgas degradation during chikv infection (fig a) . confirmation that autophagy participates in cgas degradation during chikv infection was assayed via knockdown of autophagy related protein (atg ), a critical component in the formation of phagophores [ ] . knockdown of atg was able to rescue cgas expression in infected cells, but did not have an appreciable effect in non-infected cells (fig b) . importantly, knockdown of atg did not result in an accumulation of the lower molecular weight band of lc , demonstrating a functional inhibition of the process of autophagy in the siatg conditions ( fig b) . upon quantitative analysis of protein expression, atg knockdown did increase expression of cgas in mock-infected cells by . fold, when compared to a non-targeting control, indicating that atg participates in regulating homeostatic levels of cgas expression (fig c) . in chikv infected conditions, however, there was a striking . fold representative of two independent experiments). (d) hff- s were infected with mock or chikv at an moi of . . min prior to indicated timepoints, cells were pulsed with puromycin as described in (c). lysates were collected and proteins were detected as previously stated (data representative of two independent experiments). (e) hek- ts were transfected with indicated constructs at indicated plasmid amounts. cells were allowed to rest for hr post transfection before lysis and sds-page/immunoblotting analysis (data are representative of three independent experiments). (f) t-ifnb-ffluc cells were transfected with empty vector (vec), cgas and sting in conjunction with vec, or cgas and sting with the capsid of chikv / . cells were allowed to rest for hrs before lysis for collection of protein or quantification of luminescence. data are representative of six independent experiments. data represented as fold induction over vector alone. data are represented by means ± sd (n = ). statistical analysis was done with student's t tests ( �� = p< . )). (g) protein input for ifn reporter assay in (f). https://doi.org/ . /journal.ppat. .g chikungunya virus inhibits dna sensing by degrading cgas increase in cgas expression due to atg knockdown, confirming that autophagy modulates cgas expression during chikv infection (fig c) . reduction in atg expression when compared to respective non-targeting controls were . % (mock) and . % (chikv), respectively ( fig d) . no difference in viral infectious particle release was observed between siatg or sictl, indicating that the increase in cgas expression was not due to reduced viral replication ( fig e) . these data demonstrate a critical role of autophagy in chikv dependent cgas degradation and expand our previous work with denv, suggesting a conserved virushost interplay across different class iv rna viruses. interactions of chikv nsps with sting were tested by immunoprecipitation against the flag tag from total cell lysates and visualized via wb. a clear interaction of chikv nsp with sting was noted in an overexpression system ( fig a) . next, the nsp interaction was tested in the context of chikv / infection. once again, the interaction of nsp with sting was observed, indicating that this interaction occurs in the context of viral infection (fig b) . to better characterize the interaction between nsp and sting, deletion mutants of sting were generated (fig c) . sequential deletions of sting were made from the c-terminus of the protein to delete functional domains, but so as not to disrupt the insertion of the protein in the er membrane or affect subcellular localization. sting Δ eliminates the c-terminal domain (ctd) of sting, which includes a critical interaction region for tank binding kinase interaction [ , ] . the Δ mutant deletes the functional cyclic gmp-amp (cgamp) binding ability of the protein and the dimerization domain (dd), while the Δ mutant leaves only the transmembrane spanning portion of sting [ ] . chikv nsp was then coexpressed with the deletion mutants of sting and an immunoprecipitation was performed ( fig d) . each mutant co-precipitated nsp , indicating that interaction of nsp with sting only requires the transmembrane spanning domains of the innate immune signaling protein. given the predicted structure of sting as a four-pass transmembrane protein there are only two regions of sting exposed to the cytosol with which nsp could be interacting, the first aa at the n-term or a cytosolic loop region spanning approximately amino acids - [ , ] . deletion mutants of sting were generated lacking the first aa at the n-term or which had small deletions in the cytosolic loop of the protein, either aa - or aa - , respectively (fig e) . immunofluorescence of hek- ts transfected with sting internal deletion variants represented in e showed no significant changes in subcellular localization of the proteins (s fig). co-expression of nsp with a Δ mutant of sting illustrated that the n-terminal tail of sting is not critical for interaction, but rather that the nsp -sting interaction is mediated by the aa cytosolic loop of sting (fig f) . interestingly, a loss of nsp s interaction with sting resulted in decreased expression of the viral protein (fig f, lane ) . alternatively, co-expression of nsp with full length sting significantly and specifically increased nsp expression (s fig). recently it has been demonstrated that murine sting is palmitoylated at cysteine residues and and that this modification is critical in activation of the antiviral response against dna viruses [ ] . these residues are conserved between human and murine sting and are in the cytosolic loop region which nsp of chikv is interacting with. additionally, it has been represented as means ± sd. (b-e) data representative of two independent experiments. statistical analysis was done with student's t test. https://doi.org/ . /journal.ppat. .g chikungunya virus inhibits dna sensing by degrading cgas chikungunya virus inhibits dna sensing by degrading cgas demonstrated that nsp of chikv is palmitoylated and inhibition of this post-translational modification has myriad effects ranging from altering replication kinetics to pathogenesis in mice [ ] [ ] [ ] [ ] ] . because of this association we hypothesized that nsp s interaction with sting could result in increased levels of palmitoylated nsp and this could serve as a mechanism by which sting stabilize nsp expression. interestingly, sting mediated nsp stabilization was independent of palmitoylation as demonstrated by transfecting nsp with sting both in the presence and absence of a global palmitoylation inhibitor, -bromopalmitate ( fig g) , although there was an increase in the levels of palmitoylated nsp when the protein was co-expressed with sting ( fig h) . the described sting-nsp interaction, also resulted in a significant inhibition of the ifnβ promotor activation by cgas-sting overexpression (s b fig) . these data provide the first description of chikv interaction with sting. furthermore, we mapped the interaction to a aa cytosolic loop of sting and demonstrates that this interaction could have a pro-viral role with respect to increasing the amount of nsp in infected cells in a sting dependent manner, while downregulating the ifnβ production pathway. cgas and sting have been previously implicated in restricting the replication of alphaviruses [ , , ] . in mef and raw . cells deficient for cgas or sting, we observed that the individual proteins in this dna-sensing pathway serve to functionally restrict chikv replication and infectious particle release. these data are in accordance with previous publications which have implicated cgas and sting as restriction factors for chikv replication [ , , ] and provide further evidence that this pathway is important in inhibiting not only dna viruses but also different class iv rna viruses. similar to what our group has described during denv infection[ ], we also observed the production of distinct puncta of extranuclear dna upon chikv infection. as defined subcellular compartmentalization of dna is fundamental to the normal lifecycle of eukaryotic cells the presence of dna in the cellular cytoplasm results in a strong type-i ifn response mainly mediated by cgas-sting [ , ] . the identity of the mis-localized dna in chikv infected cells has yet to be understood, but its presence in the cytoplasm by definition serves as a danger associated molecular pattern (damp)[ , , ] . when the cgas-sting pathway was stimulated with either a dna virus or e. coli dna, we observed a replication dependent inhibition of ifnß transcripts. interestingly, there was a slight reduction in ifnß transcripts when the cells were treated with a uv-inactivated chikv (uvc). this could be because uvc treatment resulted in the cells being refractory to infection with mva; however, we did not observe a reduction in mva replication measured by quantitative pcr. another possibility was that a viral factor intrinsic to the nucleocapsid was responsible for the reduction in cgas-sting dependent signaling observed. indeed, exogenous expression of chikv capsid was sufficient to reduce cgas expression and was able to significantly inhibit cgas-sting mediated induction of a type-i ifn reporter. immunoblotting of infected primary human chikv-rt nsp and the different sting mutants indicated in (c) and cells were lysed hrs post transfection. an immunoprecipitation was performed against the flag epitope and protein samples were then analyzed via sds-page and immunoblotting performed as described previously (data representative of two independent experiments). (e) schematic of sting inserted in the er membrane highlighting regions deleted which are located in cytosolic facing domains (schematic representative of poor artistic skill). (f) t cells were transfected with internal deletion sting constructs and nsp . cells were lysed hpt and an anti-flag ip was performed followed by sds-page and immunoblotting (data representative of three independent experiments). (g) hek- t cells were transfected cells with indicated constructs overnight followed by treatment with um -bp for h. cells were lysed and analyzed via western blotting. (h) hek- t cells were transfected with indicated constructs overnight and treated for h with um alk- palmitoylation chemical reporter reagent prior to cell lysis. immunoprecipitation was performed against the ha epitope followed by click chemistry reaction with azido-rhodamine for visualization of protein palmitoylation via fluorescence gel scanning. (g & h) data representative of two independent experiments. https://doi.org/ . /journal.ppat. .g chikungunya virus inhibits dna sensing by degrading cgas fibroblasts revealed that endogenous cgas is degraded as early as hrs post infection and that this degradation is independent of global transcriptional or translational repression induced by chikv. the ability of a viral structural protein to reduce cgas expression explains the rapid loss of cgas protein levels at time-points prior to a full replication cycle of the virus with de novo production of viral proteins. furthermore, capsid mediated degradation of cgas explains the slight reduction in cgas-sting signaling observed when cells were treated with uvc prior to stimulation with mva or e. coli dna. here, no data is presented identifying antagonism of cgas-sting signaling at the level of sting, however, given the current view of cgas dependent sting signaling, the degradation of cgas by chikv, by definition, leads to an inhibition of cgas-sting signaling. further studies must be performed to understand if during chikv infection, hallmarks of sting activation are present including phosphorylation of serine , interactions with tbk , or a subcellular re-localization of sting to the trans-golgi-network (tgn). the role of autophagy during chikv infection is cell type specific and has been demonstrated to have both pro and antiviral effects [ , , , ] . in addition to canonical antiviral signaling, other antiviral functions of sting have been documented including sensing of viral fusion [ , ] and translational repression [ ] . research regarding the function of these alternative sting-dependent pathways during chikv infection is critical in developing a more complete understanding of the interplay of chikv with the cgas-sting pathway. interestingly, nsps - of chikv were also able to inhibit cgas-sting mediated induction of a type-i ifn promotor, suggesting that multiple chikv proteins may work individually or in concert to antagonize cgas-sting signaling during viral infection. antagonism mediated by the non-structural proteins was not due to any direct effects on the levels of cgas or sting expression indicating that they do not modulate the pathway via alteration of cgas or sting protein levels. with these data, no direct mechanistic conclusions can currently be drawn as to inhibition of cgas-sting signaling by nsps - of chikv. they do however provide insight that degradation of cgas may only be part of a repertoire of methods by which chikv antagonizes cytosolic dna sensing. we observed an interaction of nsp with cgas, however, nsp did not significantly alter the activity of a type-i ifn reporter system. interestingly, for sindbis virus, another alphavirus, nsp has been shown to be degraded during infection via the proteasome by the n-end rule pathway during infection [ ] . thus, degradation of cgas could be mediated partially by nsp , but only when the host protein is degraded by the proteasome in the context of infection. this potential alternative protein degradation mechanism would explain why there is no total cgas recovery during autophagy inhibition in chikv infection. additionally, for other alphaviruses it has been demonstrated that nsp interacts with all three of the other non-structural proteins [ ] [ ] [ ] and the viral rdrp, nsp is the first protein to be cleaved from the polyprotein nsp - upon chikv infection. this early cleavage event in conjunction with replicative intermediates produced immediately upon infection could synergistically result in antagonism of cgas dependent signaling, but only in the context of infection. by using two complementary methods to inhibit autophagic flux, a chemical inhibitor, -ma, and sirna knockdown of atg , a critical factor in initiation of autophagosomes, we observed a significant recovery of cgas expression in chikv infected cells. specifically, when cgas levels were normalized to their respective non-targeting controls and atg expression was taken into account, there was an . fold increase in cgas expression in chikv-infected conditions versus a . fold increase in mock-infected cells. importantly, a reduction in the amount of lc ii was observed in knockdowns of atg when compared to the non-targeting controls, indicating the knockdowns were functionally reducing autophagy. interestingly, it has been reported that there is an atg /atg independent form of autophagy in murine embryonic fibroblasts (mefs) [ ] . this atg /atg independent macro-autophagy did not result in the accumulation of lc ii in cells. because lc ii was observed in the atg knockdowns, atg dependent autophagy was reduced but we cannot rule out whether or not atg independent autophagy, plays a role in cgas degradation during chikv infection. furthermore, it has been demonstrated that during infection, chikv capsid interacts with selective autophagy mediator p and this interaction results in capsid degradation via autophagy [ ] . another study demonstrated that p can interact with and modulate cgas expression via autophagic degradation [ ] . it is thus possible that the degradation of cgas by chikv capsid is mediated via an intermediary interaction between cgas-p -and -capsid which results in both being degraded during infection. chikv nsp was found to interact with sting. nsp is the only non-structural protein of chikv which is anchored to cellular membranes [ ] [ ] [ ] and sting is a transmembrane protein which has been shown to localize to the endoplasmic reticulum (er), mitochondrial associated membranes (mam), and cytoplasmic vesicles [ , ] . it is possible that the interaction between nsp and sting is enhanced because of their proximity due to membrane association. the interaction between nsp and sting was mapped to a amino acid cytosolic loop region of the protein. it is possible that through this interaction, nsp is disrupting dimerization of sting and thus dampening antiviral responses. however, the cytosolic loop domain of sting has not been shown to function in dimerization, so that possibility is unlikely. further work must be done to understand the residues required for sting interaction from the molecular perspective of nsp as well as if there are differences between human and murine sting with respect to the nsp interaction as previously reported for denv ns b [ ] . it has been reported that sting interacts with both rig-i and mavs and that sting null cells have reduced type-i ifn production when stimulated with vesicular stomatitis virus (vsv) or sendai virus (sev) [ , , ] . the nsp -sting interaction, could thus be affecting rig-i-like receptor (rlr) signaling by disrupting the innate immune signaling complex of rig-i-mavs-sting, resulting in an inhibition of rlr mediated type-i ifn induction during chikv infection. alternatively, this interaction could serve to inhibit another reported antiviral role of sting: translational repression during rna virus infection [ ] . furthermore, no research has directly addressed whether or not chikv is able to inhibit rlr mediated antiviral sensing or signaling. for a complete understanding of chikv mediated antagonism of innate immunity, it will be critical to study potential viral antagonism of rlr sensing as well as the role of sting's crosstalk with these sensors. interestingly, the nsp -sting interaction serves to increase protein levels of nsp in cells specifically when compared to a gfp control. we found that the increase in nsp levels resulted in increased palmitoylated nsp in cells, although an inhibitor of palmitoylation, -bp, did not alter the increased nsp expression indicating that the process of palmitoylation was not required for the increase in nsp expression. palmitoylated nsp has been previously shown to enhance replication kinetics and pathogenesis of alphaviruses in mice [ , ] while mutant chikv viruses with non-palmitoylatable nsp s showed severely altered replication kinetics and nsp membrane association was disrupted [ ] . this provides a potential dual role for the nsp -sting interaction: simultaneously inhibiting sting mediated antiviral signaling while being stabilized, thus increasing the total levels of nsp . furthermore, chikv nsp has been identified as a druggable target [ ] . currently, there are no complete protein models of either sting or chikv nsp , hindering in silico modeling of protein-protein interactions. by determining the cytosolic loop domain of sting as being critical for the nsp -sting interaction and identifying that sting increases nsp expression, our data provide valuable information for understanding the molecular basis of interfacing between these two proteins which can aid in small molecule inhibitor screening and design. significant evidence supports that the dna sensing pathway of cgas and sting plays an important role in restricting rna virus infections and viral antagonists of cgas and sting have been identified for both flaviviruses and human coronaviruses [ , , , , , , ] . in this work we sought to understand if chikv interacted with or restricted the cgas-sting innate immune signaling pathway. taken together, these data have strong potential implications for the rational design of attenuated chikv viruses, provide information regarding nsp for small molecule inhibitor design, and identify, for the first time, direct antagonism of a cytosolic dna sensing pathway by chikv. human foreskin fibroblast (hff- ) cells were obtained through atcc (atcc scrc- ) and cultured in dulbecco's modified essential medium (dmem) supplemented with % fetal bovine serum (fbs). human embryonic kidney- t (hek- t) cells were also obtained from atcc (atcc crl- ) and were grown in dmem supplemented with % fbs, u ml - l-glutamine, u ml - penicillin/streptomycin. mosquito cells from aedes albopictus mosquitos, clone c / (obtained originally from j. munoz-jordan, cdc, puerto-rico) were maintained in rpmi medium with % fbs at ˚c. baby hamster kidney cells (bhk) were passaged in minimum essential medium (mem) alpha + glutamax-l purchased from gibco and supplemented with % fbs, u ml - penicillin/streptomycin, and mm -( -hydroxyethyl)- -piperazineethanesulfonic acid (hepes). u os cells were a gift from dr. carolyn coyne's laboratory and were maintained in media used for the hek- ts. hek- t-ifnß reporter cells, previously described [ ] were grown as stated for hek- ts. vero cells were purchased from atcc (atcc ccl- ) and were maintained in media described for hek- ts. all tissue culture reagents were purchased from invitrogen. mefs, wt and gt, were a gift from dr. jonathan miner (wustl) and were maintained in (dmem) supplemented with % fetal bovine serum (fbs). raw . cells were purchased from invitrogen and cultured according to the manufacturer's instructions. catalog numbers: wt: rawl-isg, cgas ko: rawl-kocgas, and sting ko: rawl-kostg. the chikungunya / (chikv / ) strain, originally derived from a patient isolate in thailand [ ] , used in this study was kindly provided by dr. st. patrick reid at the university of nebraska, omaha. the virus was passaged one time in vero cells and supernatants were collected, clarified, and stored at - ˚c. viral titers were determined by limiting dilution plaque assay on bhk- s as previously described for dengue virus (denv) [ ] . newcastle disease (ndvb -gfp), originally obtained from dr. adolfo garcia-sastre, were grown in nine day old embryonated chicken eggs and tittered via tcid on chicken embryonic fibroblasts (cefs) as previously described [ , ] . vaccinia virus aliquots were a kind gift from dr. nacho mena, of the gracia-sastre laboratory. dengue virus serotype (denv- ) strain was grown for six days as previously described in c / cells [ ] . chikv indian ocean lineage (chikv iol) was generated from the la reunion (strain - ) infectious clone (coffey et al, ). to generate infectious virus, the infectious clone plasmids were linearized overnight with noti, purified, and used for in vitro transcription with the sp mmachine kit (ambion). in vitro transcribed rna was phenol:chloroform extracted, ethanol precipitated, aliquoted at μg/μl and stored at - ˚c. μg of in vitro transcribed rna was electroporated into bhk cells and virus was harvested at hours post electroporation. working virus stocks chikungunya virus inhibits dna sensing by degrading cgas were generated by passaging the virus in bhk cells for hours. viral titers were determined by plaque assay on vero cells. uv-c bulb was placed inches from the ml viral aliquots in -well culture dishes (corning) with the lid removed. plates were placed on a magnetic stir plate and sterile magnetic stir bars were placed in each well. uv-inactivation was allowed to proceed for min at room temperature. viral aliquots were then stored at ˚c for future use. uv-c bulb used: g t . w. the wavelength of light was nm. the ability of different chikv nsps to inhibit the induction of the ifnß reporter was assessed in hek- t cells stably expressing firefly luciferase under the control of an ifn-ß promotor ( t-ifnß), previously described [ ] . , t-ifnß cells were transiently reverse transfected using lipofectamine (thermo) with ng total dna per well of different constructs expressing either human cgas-pcmv (origene), human sting-(pcdna), denv ns b -(pcaggs), chikv gfp-nsps plasmids, (kindly provided by dr. subhash g vasudevan (duke-nus graduate medical school, singapore), chikv capsid (ptr- ) or pcaggs with no coding insert (empty vector (ev)), in -well plates, using lipofectamine reagent (invitrogen) per the manufacturer's protocol. hours post transfection, ifn-ß promotor induction was measured using the neolite luminescence reporter gene assay system (perkinelmer) per manufacturer's protocol. western blots were performed as described in "immunoblot analysis" section. primary human foreskin fibroblasts (hff- ) were seeded in -well plates at a density of . x cells/well. hours after seeding, cells were treated with either mock (dmem), ndvb -gfp at an moi of . , or chikv, either chikv iol or / at an moi of . . infections were allowed to proceed for hr in a total volume of μl of sera-free dmem. after hr, infection media was removed and cells were re-fed with ml of hff- media (dmem with % fbs). , , , and hours post infection supernatants were collected and quick-frozen in dry ice/ethanol then stored at - ˚c. at the selected time-points, rna from cells was collected according to the manufacturers' protocol using the quick-rna mini-prep (zymogen) and stored at - ˚c. protein lysates were collected by re-suspending cells in ripa lysis buffer (sigma aldrich) and were subsequently stored at - ˚c. prior to infection, . x hff- s were seeded in -well culture dishes (corning). cells were then treated with mock, chikv / or uv-inactivated chikv / (uvc) at an moi of . . after primary treatment, cells were allowed to rest for hrs. the secondary treatments were administered hrs post primary treatment. cells were infected with either mock or modified vaccinia ankara (mva) at an moi of . to induce the rlr or cgas-sting pathways and rna was collected and hrs post-secondary treatment. alternatively, cgas-sting induction was performed via mock or e. coli dna ( ug/well) (invivogen) transfection using lipofectamine according to manufacturer's instructions. rna was collected at and hrs post transfection. all rna collections were performed according to the manufacturer's protocol using the quick-rna mini-prep (zymogen). determination of induction of innate chikungunya virus inhibits dna sensing by degrading cgas immune signaling gene transcripts was determined by normalizing all conditions to rps and then determining fold induction over respective mock e.g. induction of isg transcripts as a result of mva infection was determined by comparing uvc ➔ mva over uvc➔mock (primary ➔ secondary) conditions. rna from cells was extracted using quick-rna mini-prep (zymogen) according to the manufacturers protocol (including the in-column dnase treatment). concentration of ribonucleic acid was determined via spectrophotometer at nm. rna was then stored at - ˚c until rt reaction. rt reaction was done with the iscript cdna synthesis kit (bio-rad) utilizing random hexamer priming, according to the manufacturer's instructions with - ng of total rna. rt-qpcr was used to quantify relative levels of gene expression in infected and uninfected cells and was performed using the iq sybr green supermix (biorad) according to the manufacturer's instructions. the biorad c thermal cycler was used with the following pcr profile: ˚c for min followed by cycles of ˚c for s then ˚c for s. quantification of gene expression was performed based on ct values of a given gene normalized to the housekeeping gene, rps , gapdh, or both where indicated. cellular lysates were obtained by incubating cells in ripa lysis buffer (sigma aldrich) supplemented with edta-free, complete ultra tablets, mini (roche) for min on ice. quantification of protein in cellular lysates was performed via colorimetric bradford assay (bio-rad) utilizing bovine serum albumin (bsa) for generation of a standard curve. cellular lysates were re-suspended, in a : ratio, in x laemmli sample buffer (bio-rad) supplemented with -mercaptoethanol and boiled at ˚c for min in a heating block (fisher scientific). all samples were then loaded on polyacrylamide-sds gels and the denatured proteins were separated by electrophoresis via conventional methods. protein was then transferred to nitrocellulose membranes (bio-rad). blots were blocked with phosphate buffered saline (pbs) with % milk for one hour at room temperature. antibodies used: cgas (d d g), sting (d p f), rig-i (d h ), atg (d b ) (cell signaling technology) at a : dilution, anti-flag (f ), anti-ß-actin (a ), and anti haemagglutinin (ha) (h ) (sigma aldrich) at a : dilution, anti-gfp (ma - ) (invitrogen), anti-chikungunya virus clone a (mabf ), anti-puromycin clone d (mabe ) (emd millipore). antibodies against chikv / were kind gifts from drs. stapleford and reid. secondary antibodies against mouse (na v) and rabbit (na v) (ge healthcare). detection of immunocomplexes were performed using supersignal chemilumisescence system (thermo). densitometry analysis was performed using imagej software. t cells were seeded on -well glass-bottomed plates (mattek) which had been pre-coated with . % polylysine for hr at rt or hff- s were seeded directly on the glass-bottomed plates without pretreatment. hrs post transfection or infection, cells were fixed at rt with . % formaldehyde then permeabilized with . % triton-x before blocking with % bsa in pbs for hr at rt. cells were incubated overnight at ˚c with primary antibodies: (h : sigma chikungunya virus inhibits dna sensing by degrading cgas aldrich), anti-ssdna (mab : emd millipore), anti-nsp (provided by dr. stapleford), anti-flag (f : sigma), or anti-calnexin (ma - : invitrogen). cells were then incubated with for hr at rt with alexa fluor-conjugated anti-mouse , anti-rabbit (life technologies), μg ml - dapi (invitrogen) or phalloidin (a : invitrogen) as indicated. confocal imaging was performed using a zeiss lsm with airyscan. images were collected at bits and a resolution of x pixels. d images were created via reconstruction of z-stacks in zen blue software. deletions of functional regions of sting were generated and cloned into the ptr- mammalian expression vector using ecori and bamhi restriction sites utilizing the in-fusion hd cloning kit (clontech). immunoprecipitation was performed using the ezview red anti-flag m affinity gel (sigma aldrich). briefly, affinity gel was washed x in lysis buffer (ripa lysis buffer (thermo) supplemented with complete mini edta-free protease inhibitor (roche). whole cell lysates were mixed with the affinity gel and incubated for hr at ˚c, rotating. after incubation, affinity gel was washed x for min each in lysis buffer. following washes, the affinity gel was resuspended in μl x laemmli buffer (bio-rad) with -mercaptoethanol (sigma aldrich) and boiled for min at ˚c. samples were then stored at - ˚c until immunoblot analysis. chikungunya virus inhibits dna sensing by degrading cgas studies of protein palmitoylation were performed according to detailed published protocols [ ] [ ] [ ] . in brief, transfected cells were treated for h with the alk- ( um) chemical reporter of protein palmitoylation. proteins of interest were immunoprecipitated from cell lysates and reacted via the copper(i)-catalized azide alkyne cycloaddition reaction ("click chemistry") with azido-rhodamine (kindly provided by dr. howard hang of the rockefeller university) for visualization of protein acylation via fluorescence gel scanning on a typhoon (amersham) fluorescence imager. western blotting of the samples provided controls for loading and sample inputs. for inhibition of protein palmitoylation, transfected cells were treated for h with um -bromopalmitate (sigma). unpaired, two-tailed, student's t-test was used for direct comparisons while one way or two way anovas with tukey's multiple comparisons we used for viral growth curves or multiple comparisons. specific analysis used for respective figures are listed in the figure legends. p-values were determined to be significant when p < . . relevant p-value cutoffs used are listed in figure legends. no samples were excluded when analyzing these data. cells were allowed to rest for hrs before lysis for collection of protein or quantification of luminescence. (c) input protein expression for reporter experiment (b) was visualized via sds-page followed by immunoblotting. data representative of four independent experiments. data are represented by means ± sd (n = ), fold induction over mock. statistical analysis was done with student's t tests ( � = p< . , �� = p< . , ��� = p< . ). (d) hek- t cells were transfected with indicated constructs and cells were lysed hpt. denv- ns b served as a positive control for sting cleavage/degradation while the catalytically inactive ns b s a was used as a negative control. gfp tagged chikv-rt nsp constructs were used to test for degradation or cleavage of sting. protein lysates were analyzed via sds-page and subsequent immunoblotting. data representative of one independent experiment. (e) hek- t cells were transfected with indicated constructs (nsps - -ha chikv-rt) and cells were allowed to rest for hrs before lysis. lysates were subjected to immunoprecipitation against a flag epitope and proteins were visualized via sds-page and immunoblotting. data representative of three independent experiments. (f) indicated constructs were expressed in t cells and cells were allowed to rest for hrs. after resting, cells were infected with either mock or chikv / (moi = . ). hpi cells were lysed and an immunoprecipitation preformed against a flag epitope. protein interaction was analyzed via sds-page followed by immunoblotting. data representative of two independent experiments. chikungunya virus inhibits dna sensing by degrading cgas togaviridae: the viruses and their replication. fields virology high level of vector competence of aedes aegypti and aedes albopictus from ten american countries as a crucial factor in the spread of chikungunya virus - pmid: chikungunya fever: epidemiology, clinical syndrome, pathogenesis and therapy persistent arthralgia associated with chikungunya virus: a study of adult patients on reunion island a report of cases of rheumatoid arthritis following chikungunya fever. a mean follow-up of two years chikungunya virus and arthritic disease an epidemic of virus disease in southern province reemergence of chikungunya virus an epidemic of virus disease in southern province, tanganyika territory, in - . i. clinical features a major epidemic of chikungunya virus infection on reunion island the signal for translational readthrough of a uga codon in sindbis virus rna involves a single cytidine residue immediately downstream of the termination codon fatty acid synthase promotes the palmitoylation of chikungunya virus nsp role of the amphipathic peptide of semliki forest virus replicase protein nsp in membrane association and virus replication effects of palmitoylation of replicase protein nsp on alphavirus infection mutations at the palmitoylation site of non-structural protein nsp of semliki forest virus attenuate virus replication and cause accumulation of compensatory mutations replication cycle of chikungunya: a re-emerging arbovirus emerging alphaviruses are sensitive to cellular states induced by a novel small-molecule agonist of the sting pathway inhibition of dengue and chikungunya virus infections by rig-i-mediated type i interferon-independent stimulation of the innate antiviral response characterization of a novel human-specific sting agonist that elicits antiviral activity against emerging alphaviruses type i ifn controls chikungunya virus via its action on nonhematopoietic cells pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity chikungunya virus induces ips- -dependent innate immune activation and protein kinase r-independent translational shutoff cytosolic sensing of viruses sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion type i interferons in infectious disease interferon-stimulated genes: a complex web of host defenses chikungunya virus nonstructural protein inhibits type i/ii interferon-stimulated jak-stat signaling activation of sting requires palmitoylation at the golgi characterization of reemerging chikungunya virus influenza virus evades innate and adaptive immunity via the ns protein newcastle disease virus v protein is a determinant of host range restriction modified vaccinia virus ankara triggers type-i ifn production in murine conventional dendritic cells via a cgas/sting-mediated cytosolic dna-sensing pathway collateral damage during dengue virus infection: making sense of dna by cgas chikungunya virus-induced autophagy delays caspase-dependent cell death the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides sunset, a nonradioactive method to monitor protein synthesis chikungunya triggers an autophagic process which promotes viral replication the autophagic inhibitor -methyladenine potently stimulates pka-dependent lipolysis in adipocytes systemic application of -methyladenine markedly inhibited atherosclerotic lesion in apoe(-/-) mice by modulating autophagy, foam cell formation and immune-negative molecules dual role of -methyladenine in modulation of autophagy via different temporal patterns of inhibition on class i and iii phosphoinositide -kinase apg p/cvt p: a novel proteinactivating enzyme essential for autophagy structural analysis of the sting adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-gmp binding molecular evolutionary and structural analysis of the cytosolic dna sensor cgas and sting plos pathogens chikungunya virus inhibits dna sensing by degrading cgas the effects of palmitoylation on membrane association of semliki forest virus rna capping enzyme inhibition of virus multiplication by foreign nucleic acid recognition of cytosolic dna activates an irf -dependent innate immune response structural mechanism of cytosolic dna sensing by cgas the cgas-cgamp-sting pathway of cytosolic dna sensing and signaling species-specific impact of the autophagy machinery on chikungunya virus infection identification of a candidate therapeutic autophagy-inducing peptide virus-cell fusion as a trigger of innate immunity dependent on the adaptor sting influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses sindbis virus rna polymerase is degraded by the n-end rule pathway modification of asn of nsp suppresses a sindbis virus nsp minus-strand polymerase mutant requirement for the amino-terminal domain of sindbis virus nsp during virus infection suppressor mutations that allow sindbis virus rna polymerase to function with nonaromatic amino acids at the n-terminus: evidence for interaction between nsp and nsp in minus-strand rna synthesis discovery of atg /atg -independent alternative macroautophagy trim inhibits cgas degradation mediated by selective autophagy receptor p to promote innate immune responses semliki forest virus mrna capping enzyme requires association with anionic membrane phospholipids for activity chikungunya virus nsp interacts directly with nsp and modulates its atpase activity membrane binding mechanism of an rna virus-capping enzyme plos pathogens chikungunya virus inhibits dna sensing by degrading cgas the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita the adaptor protein mita links virus-sensing receptors to irf transcription factor activation the viral capping enzyme nsp : a novel target for the inhibition of chikungunya virus infection sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity inhibition of the type i interferon response in human dendritic cells by dengue virus infection requires a catalytically active ns b complex development of an attenuated strain of chikungunya virus for use in vaccine production infection of human cells by dengue virus is modulated by different cell types and viral strains dengue virus inhibits the production of type i interferon in primary human dendritic cells newcastle disease virus (ndv)-based assay demonstrates interferon-antagonist activity for the ndv v protein and the nipah virus v, w, and c proteins modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm chemoproteomics reveals toll-like receptor fatty acylation the palmitoyltransferase zdhhc enhances interferon-induced transmembrane protein (ifitm ) palmitoylation and antiviral activity the authors would like to thank dusan bogunovic, florian krammer, and jean lim for their valuable input on the direction of the project as part of lgw phd advisory committee. we thank paula lopez-monteagudo for her invaluable technical advice and camaraderie as a lab mate. we also thank jonathan miner, carolyn coyne, subhash g vasudevan, adolfo garcia-sastre and nacho mena for providing reagents. key: cord- - c flue authors: chinnaswamy, s title: gene–disease association with human ifnl locus polymorphisms extends beyond hepatitis c virus infections date: - - journal: genes immun doi: . /gene. . sha: doc_id: cord_uid: c flue interferon (ifn) lambda (ifn-λ or type iii ifn) gene polymorphisms were discovered in the year to have a strong association with spontaneous and treatment-induced clearance of hepatitis c virus (hcv) infection in human hosts. this landmark discovery also brought renewed interest in type iii ifn biology. after more than half a decade since this discovery, we now have reports that show that genetic association of ifnl gene polymorphisms in humans is not limited only to hcv infections but extends beyond, to include varied diseases such as non-alcoholic fatty liver disease, allergy and several other viral diseases including that caused by the human immunodeficiency virus. notably, all these conditions have strong involvement of host innate immune responses. after the discovery of a deletion polymorphism that leads to the expression of a functional ifn-λ as the prime ‘functional’ variant, the relevance of other polymorphisms regulating the expression of ifn-λ is in doubt. herein, i seek to critically address these issues and review the current literature to provide a framework to help further understanding of ifn-λ biology. supplementary information: the online version of this article (doi: . /gene. . ) contains supplementary material, which is available to authorized users. in the year , two different groups reported on the presence of three novel genes closely placed to each other, on human chromosome that coded for interferons (ifns) with potent antiviral properties. , these genes due to their relatedness to the interleukin (il- ) family were initially christened il- a, il- b and il- , and subsequently changed to ifnl , ifnl and ifnl . the genes encode ifnλ- , ifnl-λ and ifnl-λ , respectively; together with the newly discovered ifnl-λ (or ifnl ) they constitute the type iii ifns or the lambda ifns (ifnl-λs). ifnl-λ , and activate antiviral responses through the jak-stat (janus kinase-signal transducer and activator of transcription) pathway by utilizing a distinct receptor complex made of a heterodimer formed between ifn-λr and il- r . , subsequent studies showed that unlike the receptors that bind to type i ifns, the ifn-λ receptors were expressed on selective cell types mainly of epithelial origin, hepatocytes and some immune cells. [ ] [ ] [ ] [ ] the discovery of ifn-λs was seminal in the sense that it showed the presence of an alternate system to the well-known type i ifns (ifn-α and ifn-β) that the different nucleated cells in the body, especially the ones on epithelial surfaces, can utilize to combat viral infections. later studies in mice have shown that type iii ifns form a strong barrier at the host-environment interface, which encompasses large regions of epithelial lining to the respiratory, gastrointestinal and urogenital tracts of mammals. [ ] [ ] [ ] [ ] a major boost in the area of ifn-λ research came after another discovery in the year . three independent groups conducted genome-wide association studies (gwass) involving treatment response to chronic hepatitis c virus (hcv) infections, in three different geographical regions of the world, and reported that single-nucleotide polymorphisms (snps) in the ifnl locus (figure ), had strong association with treatment-induced hcv clearance irrespective of ethnicity and geographical location of the hosts. [ ] [ ] [ ] the search for a 'causative' or a 'functional' snp at the ifnl locus that could give a biological explanation for the hcv-gwas results was taken up rigorously by several groups, but none seem to have given a better explanation to the hcv-ifn-λ 'puzzle' than the group from the national institutes of health, usa, that discovered the presence of another ifnl upstream to ifnl , named as ifnl (refs , ; figure ; in figure b , the alleles for the respective snps are shown as beneficial (b) or non-beneficial (nb) with respect to the studies on hcv (reviewed in ref. ; the major allele for each of the snps depicted in figure b has been shown to be the beneficial one in hcv infections). functional ifn-λ is expressed only in a subset of individuals, due to a frameshiftcausing deletion polymorphism (ifnl -Δg; rs ) in the first exon of ifnl (figure ). the presence of the alternate allele (ifnl -tt) renders ifnl a pseudogene and this allele is seen iñ % of the european population and in most of the east asian population, but ifnl is a functional gene (ifnl -Δg allele) in majority (~ %) of the african population, suggesting that human evolution has played an active role in elimination of a functional ifn-λ in the human species. in fact, the pseudogene shows strong positive selection in human evolution, whereas the functional gene is conserved in other mammalian species except in mice and rats where the gene is completely absent. in this article, i review the literature on genetic association studies that have shown the involvement of the hcv-gwas snps in non-hcv disorders that involve both viral diseases and some non-infectious conditions. i also update the progress on transcriptional studies of the ifnl gene and examine whether the functional ifn-λ -generating snp is sufficient to explain the molecular mechanism of causality in the diseases it is associated with, and whether the other ifnl locus snps (mainly the ones regulating ifn-λ expression) may have any functional roles to play in the observed phenotypes. ifnl-λs and innate immunity against viruses innate and adaptive immunity are the two indispensable arms of the mammalian immune system. although we had a clearer understanding of the principles of functioning of the adaptive immunity arm, a lack of advanced molecular techniques and incomplete understanding of molecular mechanisms made us remain unaware of the intricacies of functioning of the innate immunity arm, for a long time. with the advent of superior molecular biology techniques and the discovery of the pathogenassociated molecular pattern (pamp) or pattern recognition receptors, we now have better understanding of how nucleated cells can differentially recognize different classes of pathogens and propagate signals to their surroundings, in the process raising the immediate alarm in the host. large strides were made in the area of molecular recognition of viral pamps and signal transduction that leads to raising of antiviral states within virusinfected cells. , the epithelial cells, being at the interface between the host and the environment in the respiratory, gastrointestinal and the urogenital tract, are not only prone to a variety of viral infections but are strategically located to respond and propagate alarm signals to the underlying immune cells ( figure ) . however, the primary function of the epithelium is to provide a physical barrier between the underlying lamina propria and the lumen of the cavity or the exterior. even though they can sense and respond to pamps and damage-associated molecular patterns, the epithelial cells are not professional immune cells and due to their high level of differentiation, may lack the plasticity required to send out amplified and prolonged signals to the lamina propria. therefore, a crucial link still remained missing about how an adaptive immune response is shaped within distantly located lymph nodes that have obligatory 'immune-rich environments', by taking cues from signals generated by viral infections at the epithelium ( figure ). the discovery of the new class of effector immune cells called the innate lymphoid cells (ilcs) may seem to have provided the answer to this puzzle ( figure ). ilcs are derivatives of common lymphoid precursors along with t and b cells. these cells are stationed near the epithelial surfaces in larger numbers and respond to signals from the surrounding cells by secreting cytokines and chemokines in large quantities, thereby acting as signal amplifiers in both health and disease. the ilcs are considered the innate immunity counterparts to the various th (t helper) cell (cd + (cluster of differentiation)) subsets of the adaptive immune system (figure ; ref. ) . for example, ilc s respond to il- generated from influenza virus-infected epithelial cells by secreting il- and il- , both known inducers of th immunity. the natural killer cells, now classified as ilc cells, are considered the tc (t cytotoxic) cell (cd +) counterparts. even though most studies so far on ilcs have been on mice, ilcs have also been characterized in a variety of human tissues and are deregulated in many human diseases. with emerging roles of type iii ifns at the epithelium, , , ilcs and ifn-λs now seem to be the major players in innate immunity at barrier surfaces. ). the minor allele (mi) is the non-beneficial allele (that leads to expression of a functional ifn-λ and also lowers expression of ifn-λ ) and the major allele (ma) is the beneficial allele (that does not produce a functional ifn-λ and is associated with higher levels of expression of ifn-λ ) for all snps, again with respect to hcv studies. the information on ld values has been obtained from genomes project reference panel (http://www. genomes.org), october release; some ld (r ) values for yri (marked with *) were obtained from ref. . the maf values were obtained from dbsnp (national center for biotechnology information). in asian population, mafs of rs , rs and rs are from jpt population; and mafs of rs , rs and rs are from chb-jpt populations. yri, yoruba in ibadan, nigeria; ceu, residents with ancestry from northern and western europe; chb/jpt, han chinese in beijing, china/japanese in tokyo, japan; -, maf information not available for the ta repeat polymorphism rs . how ilcs respond and interact with the epithelial cells during viral infections and how they cross-talk with other immune cells at the barrier surfaces in shaping and maintaining the optimal th responses, will form the next exciting wave in innate immunity research. similarly, research on the production, regulation and functions of ifn-λs in viral infections has also been exciting in the last decade. human ifn-λs are secreted (except ifn-λ that is poorly secreted ) in response to the detection of viral rna intermediates from the cytoplasm of epithelial cells via the toll-like receptor and retinoic acid inducible gene i-like receptor) pathways. , , ifn-λs are also known to be secreted by macrophages, plasmacytoid dendritic cells, monocyte-derived dendritic cells and hepatocytes. , , [ ] [ ] [ ] the ifn-λr receptor is expressed on limited cell types including epithelial cells, hepatocytes, b cells and monocytes. , there is currently no information on whether the newly discovered ilcs secrete any of the ifn-λs and whether they express ifn-λr . ifn-λs act in a paracrine and/or autocrine manner to raise an antiviral state in the infected and to-be-infected cells by reprogramming the target cell gene expression patterns. , the ifnl snps would have functional roles if they can affect the diversification of innate and adaptive immune cell subsets. diversification of ilc subsets will lead to the polarization of dendritic cells and macrophages and will eventually influence the th /th balance by favoring either a th or a th response ( figure ). although there is no evidence for this belief, it is known that the ifnl snps do affect the expression of ifn-λ (ref. ) and that they give rise to a new ifn (ifn-λ ). ifn-λ , and are known to deflect the th /th balance to a th predominant mode in vitro and also in vivo in humans and mice , (reviewed in ref. ) . no such studies are reported for ifn-λ . one hypothesis is that different levels of ifn-λ expression dictated by the underlying genetic polymorphisms are responsible for eliciting a th or a th response. the role played by ifn-λ in this process is only speculative at this stage ( figure ). how ifn-λs interact with the newly discovered ilcs is also unknown. a recent report showed that rotavirus infection in mice is controlled by il- produced by ilc s for which the presence of an intact ifn-λ signaling pathway is required. an even more important question in humans will be on what role does the ifnl snps, and therefore ifn-λ , play in the diversification and functioning of ilcs. ifn-λ apart from being antiviral to hcv is also known to inhibit other flaviviruses such as dengue virus, yellow fever virus and human corona viruses. some of the other ifn-λs are known to be induced by several human pathogens including m. tuberculosis, human papilloma virus, influenza virus and human metapneumovirus (also induces ifn-λ ), and have clear antiviral activities against some but not other viruses in mice (reviewed in ref. ) . in a recent finding, murine ifn-λ but not ifn-α/ifn-β was responsible for protecting mice against norovirus persistence in mice colon, strikingly, even in the absence of t and b lymphocytes. similar results were seen in reovirus infections of mice colon. in conclusion, ifn-λs are potent antiviral molecules and their cross-talk with innate immune cells can potentially figure . immune responses to viral infections of the epithelia. resident macrophages and dendritic cells (dcs) form the first line of cellular resistance to invading viruses. the newly discovered innate lymphoid cells (ilcs) may have important roles due to their ability to respond to signaling molecules/alarmins secreted by the epithelial cells. ilcs diversify and respond by secreting large amounts of effector cytokines and chemokines locally. these effector molecules can potentially lead to the polarization of dcs and macrophages, and therefore may be critical in shaping the adaptive immunity mediated by t-helper (th) and t-cytotoxic (tc) cells that develop in the nearby lymphatic tissue (green lobed structure). mhc, major histocompatability complex. orchestrate innate immunity against several viruses and they may be particularly important at the barrier surfaces. ifn-λs also modulate adaptive immunity by affecting th /th balance, tilting it to a th -favoring response that is required for clearing viral infections. the function of the newly discovered ifn-λ in these immune processes remains to be determined. in cognizance of the potential that ifn-λs may hold in innate immunity, researchers across the world have got intrigued with the ifnl snps and have started to test them in candidate gene case-control studies in both infectious and non-infectious diseases. so far, association has been reported with nonalcoholic fatty liver disease (nafld), allergy and infections with several viruses. causing chronic infections and is especially a problem in immunosuppresed individuals. egli et al. tested the association of the ifnl snp rs with cmv replication in a small number of solid organ transplant patients (n = ) who were seronegative for cmv (but received an organ transplant from a cmvseropositive donor) and who had stopped receiving antiviral prophylaxis. they found that the minor allele at rs (g) was associated with decreased cmv replication (p = . ) in a dominant model of inheritance. further, their in vitro studies provided evidence for a beneficial effect of the minor allele against cmv replication. a study in cmv-seropositive kidney transplant patients also found that the minor allele (t) at rs had a dominant beneficial effect against cmv replication. although in another study in allogenic stem cell transplant recipients, the minor allele t at rs protected recipients against cmv infection in a recessive model of inheritance. two more studies have also reported on the association of ifnl snps with cmv. , the first report by bibert et al. tested for the occurrence of cmv retinitis among those human immunodeficiency virus (hiv)-infected patients who were at risk of developing cmv retinitis due to low cd counts and cmv seropositivity. they found that carriers of two copies of the minor allele (Δg/Δg), increased the risk of getting cmv retinitis (p = . ) in multivariate regression models. in the second report by manuel et al., solid organ transplant patients were tested for the association of rs with cumulative incidence of cmv replication. the results showed that the minor allele homozygotes (Δg/Δg), only in the pre-emptive antiviral therapy group but not among those receiving antiviral prophylaxis, had higher incidence of cmv replication. these results are indeed very interesting and sound, but the paradoxical findings of the five groups in terms of the model of inheritance of ifnl snps raise some questions. although two groups showed that their results fit best with a recessive model of inheritance of ifnl snps , in affecting cmv replication/retinitis wherein minor homozygosity was non-beneficial, the other three studies show an opposite trend where the minor allele had a beneficial effect against cmv replication in both patients and cell culture experiments, involving either dominant , or recessive models of inheritance. in stark contrast, a dominant model of inheritance (of the non-beneficial ifnl snp minor allele) has consistently given the best explanation on the observed phenotypes in association studies with both spontaneous clearance and ifn-based treatment response in chronic hcv infections. [ ] [ ] [ ] this discrepancy within the cmv studies may be partly due to statistical fluctuations owing to low sample sizes, , and high heterogeneity among patient groups, end points and antiviral regimens that are followed in the different studies. the discrepancy needs be resolved by well-designed replication studies to gain a proper understanding of the role of ifnl snps in cmv replication and disease. human t-lymphotrophic virus. human t-lymphotrophic virus (htlv)- is an ancient retrovirus causing chronic infections in humans and is associated with adult t-cell leukemia/lymphoma. it is also associated with inflammatory disorders such as htlv- associated myelopathy/tropical spastic papaparesis (ham/tsp), htlv-associated arthropathy and several other related disorders including rheumatoid arthritis. a spanish study reported for the first time that an ifnl snp (rs ) was associated with ham/ tsp in a small number of patients (n = ) who had htlv- proviral dna in their blood cells. they showed that the presence of the minor allele made / patients to have a sixfold higher risk (p = . ) of having symptoms of ham/tsp compared with asymptomatic carriers (n = ) in a dominant model of inheritance. however, proviral dna load was a confounder in this association; covariate analysis suggested that both the snp and proviral dna load were linked in their association with ham/tsp. the minor allele-carrying genotypes (ct and tt) were indeed having higher proviral dna in their blood cells compared with the major homozygotes (p = . ). the major drawback of this study seems to be the low sample number; although the strength of the study was that there was significant effect of the minor allele on proviral dna copy number, a functional association that could be directly linked with the antiviral role of ifn-λ . it is known that the ifnl snp minor alleles are associated with decreased expression of ifn-λ (reviewed in ref. , although an opposite effect is evident in chronic hcv infections, where the minor allele carriers have low baseline virus levels ). in a later study from brazil, both rs and rs were tested in a cohort consisting of htlv-positive subjects ( ham/tsp patient and asymptomatic carriers). the minor allele g of rs was significantly associated with ham/tsp in both univariate and multivariate analysis with a recessive model of inheritance (p o . ). in this study, the proviral dna load as a covariate did not seem to interfere with the association of rs with ham/tsp unlike the previous spanish study. with respect to rs , the minor allele t was associated with ham/tsp only as a heterozygote in univariate analysis (p = . ) and weakly in multivariate analysis (p = . ). however, a series of studies have also reported conflicting results to the above two reports on the association of ifnl snps with htlv- -associated diseases. first, sanabani et al. show clearly a lack of association of ifnl snp rs with ham/tsp and/or adult t-cell leukemia/lymphoma. this brazilian study had more number of samples (n = ) than the spanish study of trevino et al. (n = ). they also did not find any correlation of proviral dna load with rs genotypes. another report from brazil with a sample size of also reflected similar findings, where they found no association of rs with ham/tsp and proviral dna load. this study also compared healthy controls with htlv- -positive subjects and found no association with rs and htlv- infection. yet another recent study from brazil analyzed the genotypes at rs , rs and rs in healthy controls and htlv- -infected individuals, and found no association with htlv-associated arthropathy and any of the three snps when tested individually. when they carried out haplotype analysis, they found some association (p = . ) with htlv- infection and one of the seven haplotypes (cct) involving the three snps (rs , rs and rs ); with htlv-associated arthropathy and another haplotype (ttg; p = . ). they also found an association of the three snps individually with levels of some cytokines (such as ifn-γ) and proviral dna load (p o . ). however, no multiple testing corrections seem to have been carried out in their analysis, thus severely undermining the results. further, a japanese study also failed to see any association with adult t-cell leukemia/lymphoma and rs and also with htlv- and hcv mono-or co-infections. last, a study from france on htlv- -positive subjects of afro-carribean lineage compared the distribution of genotypes of rs and the ifn-λ -generating snp rs , and found no association with ham/tsp. in summary, the results so far are not entirely convincing on a true association of the ifnl snps with htlv infection-related diseases. further, in the two reports , that did see an association, the models of inheritance used to fit the phenotype data do not agree with each other, raising doubts on the underlying functionality of the observed genetic associations. therefore, more functional characterization of the observed association may be needed to rule out false positivity. if at all there is another human disease where ifnl snps were expected to have associations as strong as that of hcv infections, it was the case of hepatitis b virus (hbv). this expectation is because both viruses are hepatotropic and cause chronic infections; both diseases can be effectively treated using ifn-α even though they differ in their pamp ligands recognized by innate immune receptors. , mixed results have been obtained about the role of ifnl snps in both spontaneous clearance and ifn-α-induced clearance of hbv and the literature until the year - has been reviewed elsewhere. [ ] [ ] [ ] more studies have also been conducted since then (results from of them are summarized in supplementary table ), but have largely failed to resolve the conflict. two studies have also reported on a lack of association of ifnl snps on spontaneous as well as ifn-induced clearance of hepatitis d virus, a co-infecting satellite virus that requires hbv for replication and assembly. , although consistent results were obtained across numerous studies with hcv-ifnl snp association mainly because they had only virological end point phenotypes that correlated well with serological and biochemical parameters of the infection, several drawbacks in case of hbv studies may be responsible for the inconsistent results. some of them are: presence of different hbv genotypes as mixed infections, with some genotypes (genotype d ) showing better association than others; variation in treatment regimens (ifn alone or with nucleotide analogs); end point phenotypes varying from serology to viral load, to biochemical parameters without a common quantitative parameter to assess the phenotype; prolonged clinical course of the disease with fluctuating virological, serological and biochemical markers; and prevalence rates of disease differing in different ethnicities/populations, to name a few. the reports that have shown an association of ifnl snps with hbv spontaneous or treatment-induced clearance are largely in agreement with the results from hcv studies in terms of the model of inheritance (supplementary table ). although the conflicting data so far on hbv-ifnl snp association do lead to doubts about its true positive nature, the data are also not fully supportive of the notion that the association may be false positive. in fact it has been argued that if properly assessed, the association between hbv disease progression and ifnl snps could have clinical value. but it appears that the effect of the ifnl snps on hbv persistence and/or progression within the human host is highly variable; it may involve more complex interactions with other variables and genes than was observed in chronic hcv infections. human immunodeficiency virus. another important viral disease that has been tested for the influence of ifnl snps is acquired immunodeficiency syndrome (aids) caused by hiv. particularly, it was of interest to see how the ifnl snp beneficial alleles are distributed in a unique group of hiv-infected individuals that can suppress the virus from replicating to high levels (defined as elite controllers/suppressors or natural viral suppressors) and defer the progression to aids (called as long-term non-progressors) without any antiretroviral therapy; and another unique group that remains hiv-seronegative despite being at high risk for infection due to intravenous drug use (highly exposed seronegative or exposed seronegative)). more interestingly, the natural viral suppressor patients have also been known to efficiently clear hcv in hcv-hiv co-infections, compared with controls in both african americans and caucasians. a few reports have come up in this area, some showing no correlation of hiv infection/disease progression to the ifnl snps, whereas others see clear association. [ ] [ ] [ ] [ ] [ ] [ ] [ ] one of the earlier reports tested the association of rs in high-risk seronegative and hiv-positive subjects comprising both white and black subjects in the usa. no association was evident for either hiv positivity or for disease progression within the infected subjects. in another study reported by rallon et al., the association of rs was tested in two different cohorts. the first comprised of long-term non-progressors and typical progressors to aids; the second included exposed seronegative and hiv-positive partners. thus, the study tested both hiv progression and protection, although in a small number of patients. no significant difference in distribution of genotypes between cases and controls was evident in both cohorts, although the beneficial cc genotype was more frequent in the exposed seronegative patients compared with hiv-positive patients ( % vs %), suggesting that there may be a protective role for the cc genotype against hiv that was not detected in the study, likely due to inadequate power. another study from the usa tested whether the beneficial cc genotype at rs was overrepresented in african-american elite controllers/suppressors compared with hiv-infected patients with high viral loads, and found no statistically significant difference. confirming this report, sajadi et al. also found that cc genotype (for rs ) was not significantly over-represented in natural viral suppressors of african-american origin compared with hivpositive (n = ) and -negative controls (n = ). three reports published subsequently have seen association with rs and rs , and spontaneous control of hiv and/or aids progression. [ ] [ ] [ ] interestingly, all three studies were carried out on whites/caucasians, whereas the reports that failed to see an association described above were mostly carried out on african americans (except that by rallon et al that used white subjects) or mixed group of blacks and whites. first, machmach et al. found an association of rs with spontaneous hiv control when tested on white natural viral suppressors and matched non-controllers at p = . after correcting for occurrence of hla-b (human leukocyte antigen) protective alleles (which also have independent association with hiv and hcv spontaneous clearance) and gender, in multivariate analysis. second, machmach et al. confirmed and extended their results in another report, where they found the association of rs with aids progression. last, a recent study carried out on a well-characterized spanish white cohort of hcvseropositive men exposed to hiv infection through shared needles shows a clear association of ifn-λ -generating rs with hiv positivity. the study had men who were hiv seropositive and were highly exposed seronegative. they found that the protective tt/tt genotype was overrepresented in the highly exposed seronegative group ( . vs . ; p = . ). further, this association had no interaction with the hiv-protective ccr (c-c chemokine receptor type ) deletion (p = . ). interestingly, all three studies that found an association show data that the non-beneficial minor alleles are following a recessive model of inheritance, - suggesting a common underlying functionality in the observed genetic association between the three studies. this is however different from the dominant model of inheritance seen in hcv studies, [ ] [ ] [ ] suggesting that the two viruses may have different interactions with ifn-λ-driven immune responses. however, unlike in the case of cmv studies discussed above, [ ] [ ] [ ] [ ] [ ] all three hiv studies [ ] [ ] [ ] show that the minor alleles are non-beneficial, similar to the observations in chronic hcv infections. in summary, it is evident that ifnl snps do associate with hiv replication and disease progression, even though in an ethnicity-specific manner. it appears that the beneficial alleles of ifnl snps have protective roles in whites/caucasians. in african americans, more studies with the functional ifn-λ -generating snp rs rather than rs will reveal any true association. this is especially true as the two snps are not in strong linkage disequilibrium (ld) in this population (figure b) . herpes simplex virus. herpes simplex virus resides inside the human host latently in sensory neurons, but gets reactivated due to the altered host immunity and replicates efficiently in epithelial cells causing genital or oral lesions (the latter is referred to as 'cold sores'). an association of rs with the recurrence and severity of herpes simplex virus- -induced 'cold sores' was reported from a study on a small number of individuals (n = ). a recent report carried out on a large number of individuals (n = for genital herpes; n = for oral herpes) clearly found no association of rs with recurrence of genital or oral herpes episodes. among other phenotypes, the latter report ruled out an effect of the snp on 'frequency of recurrence' of oral herpes, it however did not rule out an effect of the snp on severity of infection. non-infectious diseases and miscellaneous conditions. ifnl snps have also been tried as candidate gene snps in some noninfectious inflammatory diseases. the association of ifnl snps with nafld was earlier reported by petta et al. this study with subjects identified an independent association with the cc genotype at rs and the severity of lobular inflammation (cc genotype positively correlated with severity of inflammation) in a cohort of patients with nafld (p = o . ). the study was conducted taking lead from previous reports of increased hepatic necroinflammation in hcv patients with the cc genotype at rs . another report showed no such association in caucasian biopsy-confirmed nafld patients. however, all the patients in this cohort were obese (with body mass index ), whereas in the previous study only % were obese. after the latter report, petta et al. revisited their data and indeed found an association with the ifnl snp only with their non-obese patients (n = ; p = o . ) but not with the obese patients (n = ; p = . ). these initial doubts seem to have been resolved with a recent report by eslam et al. who worked on a relatively large number of nafld patients (n = ) and confirmed the association of the cc genotype at rs with increased severity of liver inflammation and fibrosis (p = o . ). they also found a similar strong association with hepatic inflammation and fibrosis in separate cohorts of viral hepatitis c (n = ) and b (n = ), strongly suggesting that even though the major allele genotype cc is beneficial against hcv and hbv, it is also responsible for excess inflammation of the liver in viral and non-viral hepatitis. all four studies - report on dominant models of inheritance for the minor alleles; but unlike in the context of chronic hcv infections, [ ] [ ] [ ] the minor alleles in nafld are beneficial rather than being non-beneficial, as they are responsible for less severe hepatic inflammation. extending the potential role for ifnl snps in inflammatory diseases, there is also a recent report that showed association of rs and allergy in children aged o years. although the study involved a small sample size (non-allergic, n = ; allergic cohort , n = ; food allergy cohort , n = ), a large effect size (odds ratio = . , p = . , for cohort ) was seen wherein the non-beneficial t allele at rs was over-represented in the allergy group in a dominant model of inheritance. another interesting finding in this study was that the effect of the ifnl snp was more pronounced in females than males. this report suggests that the non-beneficial ifnl snp minor allele-carrying genotypes may predispose children to an allergic phenotype by skewing the th /th balance to a th predominant one. this extrapolation is also based on the findings from a recent study on mice model of allergic asthma, which showed that ifn-λ was able to rescue mice from allergy by suppressing th cytokines. these conclusions would also suggest a role for ifnl snps in immune response to respiratory viral infections, which account for disease exacerbations in conditions such as asthma. a study carried out on infants with respiratory syncytial virus-induced bronchiolitis shows no association of r and rs with viral load or other clinical features. interestingly, the non-beneficial tt genotype of rs was associated with early age of hospitalization in the infants (p = . ). more studies are awaited on the association of ifnl snps with allergy and allergy-related diseases. in the autoimmune disorder multiple sclerosis, rs and rs did not show any association with ifn-β treatment. as evidence for a potential role of ifn-λs and ifnl snps in adaptive immune responses, a study has linked ifnl snps with development of effective vaccine response against human influenza virus in immunosuppressed individuals. the study shows that minor allele (g)-carrying individuals at rs show better vaccine responses (odds ratio = . ; p = . ) in a dominant model of inheritance, and that t cells from minor allele-carrying individuals produce more il- (a th cytokine). this study also showed that recombinant ifn-λ increased th responses from human peripheral blood mononuclear cells while inhibiting th responses (th cytokines are needed for effective seroconversion). studies have accumulated evidence on the different transcription factors (tfs) that bind and drive transcription from all four ifnl genes. roles for several virus-inducible tfs such as nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells), ifn regulatory factor- and ifn regulatory factor- have been defined for ifnl , and genes, and have been reviewed elsewhere. , , we recently showed that apart from these three tfs, specificity protein ( a gc-rich dna-binding tf) also has a role in driving transcription from the ifnl promoter in a cells (figure a) . a cpg island of~ . kb is located upstream of the ifnl gene and overlapping the ifnl gene, and also includes two of the most important ifnl snps rs and rs . epigenetic regulation of the transcriptional activity at the ifnl locus is likely to involve this cpg island and needs further exploration. , even though ifnl messenger rna (mrna) was shown to be highly expressed from stimulated primary hepatocytes in the original report that described the discovery of ifnl , subsequent studies have not seen robust expression levels of ifnl gene in human samples. for example, amanzada and others found ifnl transcripts expressed at . - . -fold lower levels than ifnl / mrna in liver biopsies of hcv-infected patients. in another report, ifnl mrna was detectable in only in / and / patient-derived peripheral blood mononuclear cells that were stimulated or not with ifn-poly(i:c), respectively. all patients carried at least one copy of the functional ifn-λ -generating allele, Δg. in the report by liang and colleagues also, ifnl transcripts were seen in only / liver biopsies from hcv-infected patients. furthermore, lu et al. found that only % of the transcripts generated from poly(i:c)-stimulated primary hepatocytes derived from two heterozygous (tt/Δg at rs ) individuals, represented functional ifn-λ transcripts that originated from the Δg allele. similarly, they also found no expression of the full-length functional ifn-λ mrna transcripts from poly(i: c)-stimulated a cells. it is possible that the qpcr assays utilized in these studies may be suffering from technical difficulties in dealing with several different mrna isoforms of ifnl gene. all the four studies described above have used primer sets where at least one of the primers binds to the exon/intron/exon-intron junctions (figure b) . using a primer set that uniquely binds to the ′-untranslated region of ifnl mrna, ifn-λ induction was seen at levels similar to that of ifn-λ / , upon human metapneumovirus infection of a cells by banos-lara et al. we used the same primer set and found similar high expression levels upon poly(i:c)/hcv rna transfection in a cells. two other studies have reported no such problems in amplifying ifnl transcripts from liver biopsies. , although honda et al. used the allelespecific taqman assay described earlier, konishi et al. do not provide the primer sequence information that they used in their taqman assays. the inconsistency in the above reports in detection of ifnl transcripts (either the full-length functional ifn-λ isoform or the other isoforms) suggests that rna-sequencing may be the optimal approach to measure ifnl transcripts. furthermore, the pre-mrna splicing mechanism that leads to the expression of different ifn-λ isoforms under different stimulation and cell-type conditions needs to be examined. the relevance of other 'ifn-λ functional snps' after the discovery of functional ifn-λ -generating snp till date, three functional snps have been identified that regulate ifnl gene transcription/translation (referred to as 'ifn-λ functional snps' in this section). although rs is a snp present at~ nucleotides upstream of the start codon of ifnl and is known to affect downstream gene expression by differentially binding to nf-κb, rs was identified in the ′-untranslated region region of ifnl (figure a ) that affects stability of the mrna by interfering with au-rich element decay (amd). both these snps are in high ld with each other and with rs and were predicted to be two of the four potential causal snps from among the hcv-gwas hits. , a recent study defines another mechanism by which rs affects the stability of rna by remodeling its secondary structure. furthermore, a third functional snp is the ta repeat polymorphism rs , originally reported by the mizokami group. this snp located within the proximal promoter affects transcription of ifnl , depending on the number of repeats present. recent reports have confirmed the significance of this snp in association studies involving chronic hcv infections. [ ] [ ] [ ] apart from several reports that showed genotype-dependent differences in expression levels of ifn-λ (reviewed in ref. ) recent reports also have confirmed this finding in ex vivo and in vivo conditions. , these results suggest that ifn-λ expression levels dictated by the alleles present at the three functional snps may have a role in the observed phenotype in health and disease. however, the discovery of the ifn-λ -generating snp (rs , referred to as 'ifn-λ functional snp' in this section) has overshadowed the importance of the 'ifn-λ functional snps', as most of the new reports have chosen to test for the 'ifn-λ functional snp' rather than rs , which is the tag-snp for the 'ifn-λ functional snps'. another snp within the coding region of ifnl that leads to a non-synonymous mutation in the functional ifn-λ protein is thought to be a better marker of association along with rs , in chronic hcv infections. so the question arises: is the ifn-λ -generating snp the sole functional snp or the other snps regulating the expression of ifn-λ also have independent roles? the answer to this question will be difficult to obtain under in vivo conditions due to high ld between these snps in most populations that precludes any attempts to assess their independent effects (figure b) . a recent report compared the strength of association of rs and rs in ifn treatment response in hcv-infected african americans, who have the lowest reported ld values among all ethnicities between rs and rs snps (figure b) . they found that rs was more strongly associated with the outcome than rs . in another independent report, lu et al. applied multivariate regression to test the independence of rs from rs in predicting response to ifn treatment in hcv patients of african-american decent. they found that correcting for the effect of s on rs abolished the latter snp's association with treatment response, whereas correcting for the effect of the latter snp reduced the strength of association of the former snp (from p = . to . ). this could have resulted due to the low sample size (n = ); nevertheless, more results are awaited to conclude that ifn-λ -generating snp is the sole functional snp and that the observations giving credence to the functionality of the other 'ifn-λ functional snps may only be artifacts. studies so far point to the functional ifn-λ -generating snp rs as the prime causal variant at the human ifnl locus. therefore, a detailed investigation is needed on the function of ifn-λ as both an antiviral cytokine in different viral diseases and as a potential player in diversification and maintenance of innate and adaptive immune cell subsets. in case of ifn treatment response in chronic hcv infections, even though the ifn-λ -generating snp can explain a large amount of variance in the observed phenotypes, questions still remain on its role in spontaneous clearance of hcv. it is known that expression of functional ifn-λ is associated with high levels of ifn-stimulated gene (isg) expression in non-responders who further fail to upregulate their isg expression upon ifn-α treatment. however, such an elegant explanation is lacking in the case of spontaneous clearance of hcv ( figure ). although the majority of studies on spontaneous hcv clearance are with rs and not rs , the fact that these two snps are in high ld in most populations allows us to extrapolate the results. so, how does the expression of a functional ifn-λ in patients who get an acute hcv infection will lead them to become chronically infected? one explanation could be that similar to the ifn treatment nonresponders group, the subjects expressing functional ifn-λ also have higher baseline levels of isgs, which do not get further upregulated upon acute hcv infection. having a higher basal innate immune response may have helped those populations with higher frequency of the functional ifn-λ -generating allele in dealing with prevalent viral infections. higher baseline levels of isgs in people who can express a functional ifn-λ may be offering protection against excess inflammation of the liver in hepatitis of non-viral origin such as nafld. [ ] [ ] [ ] [ ] however, as is evident from studies on spontaneous and treatment-induced clearance of hcv, such a pre-emptive state may become nonbeneficial in dealing with hepatitis of viral origin. further, there may be other conditions that we do not yet know in which expression of a functional ifn-λ may be beneficial. therefore, an epidemiological investigation to assess basal isg expression levels, in humans both in health and disease, and their correlation with expression of ifn-λ , is needed. further, studies are also needed to examine why hepatic ifn-λ levels are lower in the beneficial allele/genotype-carrying individuals during chronic hcv infection and how this phenomenon is reversed once treatment is initiated (figure c ; ref. ) . although ifn-λ , and are known to associate with a th response, , , no information is available in this regard for ifn-λ . if the hypothesis that higher levels of ifn-λ expression (due to presence of major alleles) will lead to a th predominant response is to be believed, then by extrapolation, ifn-λ should promote th responses (figure ; as 'ifn-λ functional snps' and 'ifn-λ functional snp' are in strong ld and the minor allele will give rise to ifn-λ ). this explanation is difficult to reconcile with the observations that ifn-λ , except for being a poorly secreted ifn, requires the same dimeric receptor for signaling and can stimulate not only similar set of isgs but also at similar levels when compared with ifn-λ . , , besides, both ifn-λ and ifn-λ have similar antiviral potencies in vitro. , therefore, expression of ifn-λ and its associated isgs in a subset of individuals carrying minor alleles is less likely to favor a completely opposite phenotype to that seen in those carrying major alleles that induce higher levels of ifn-λ expression. human ifnl snps came to the limelight after genetic studies showed their relevance in chronic hcv infections in the year . since then, case-control studies in humans have been reported involving ifnl snps and several other viral diseases, nafld, allergy and even in vaccine responses. although it appears that the associations are well replicated (such as in nafld and aids) and strong in some diseases (such as in pediatric allergy), conflicting reports weaken the association in some (such as those involving htlv and hbv), whereas further studies are needed in others (such as those involving cmv and herpes simplex virus) to clear discrepancies. the ifnl-λs may have critical roles in shaping innate and adaptive immunity in general and in viral infections in particular. however, unlike their effect in hcv infections, the ifnl snps may involve interactions with variables other than just standard end point phenotypes in many of the reported diseases/ conditions. therefore, future studies should aim at dissecting these interactions to arrive at meaningful results. well-designed replication studies and functional studies to strengthen the genetic association results are required to arrive at firm conclusions. importantly, ifn-λ has emerged as the key causal link to the genetic associations, but many questions remain on its functional role in the diversification and shaping of innate and adaptive immune responses in viral infections and other inflammatory conditions. ) and therefore are expected to express ifn-λ . a recent study has documented that type iii ifns including ifn-λ / and ifn-λ expression levels correlate with isg expression in the liver of patients undergoing anti-hcv therapy. the ifn-λ / and ifn-λ levels and their associated isg levels are higher in the liver of patients who will eventually not respond to the therapy compared with those who will respond, suggesting that an unknown effect inhibits the expression of ifn-λ in the latter group of patients (shown as an ellipse in c). it seems that ifn-α-ribavirin treatment induces the expression of ifn-λ once treatment begins (depicted as a circle in c) preferably in those patients who will eventually respond to the treatment (these patients have lower frequency of the functional ifn-λ -generating alleles and hence are shown without ifn-λ expression (c)). this induction is further dependent on whether the patients carry beneficial alleles at rs . the patients who have the beneficial alleles show higher increase in their hepatic ifn-λ levels than those who do not, once treatment is initiated. a previous report also had seen similar changes in serum ifn-λ levels. the isg levels are shown in correlation with ifn-λ expression. ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il- , il- and their class ii cytokine receptor il- r il- a, il- b, and il- : promising cytokines with type i interferon-like properties viral infection and toll like receptor agonists induce a differential expression of type i andλ interferons in human plasmacytoid and monocyte-derived dendritic cells ifn-lambda (ifn-λ) is expressed in a tissue-dependent 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innate immunity and incidence of cmv infection in seropositive kidney transplant patients effect of the il b rs c/t polymorphism on the incidence and features of active cytomegalovirus infection in allogeneic stem cell transplant patients the ifnl / Δg variant increases susceptibility to cytomegalovirus retinitis among hiv-infected patients influence of ifnl / polymorphisms on the incidence of cytomegalovirus infection after solid-organ transplantation development of tropical spastic paraparesis in human t-lymphotropic virus type carriers is influenced by interleukin b gene polymorphisms il b gene polymorphism snp rs genotype gg is associated with htlv- carriers lack of evidence to support the association of a single il b genotype snp rs with the htlv- clinical outcomes and proviral load htlv- -associated myelopathy/ tropical spastic paraparesis is not associated with snp rs of the il- b gene il b gene polymorphisms and th /th cytokine levels might be associated with htlv-associated arthropathy paradoxical expression of il- b mrna in peripheral blood in human t-cell leukemia virus type- mono-infection and co-infection with hepatitis c virus absence of association of ifnl /il b rs and ifnl ss polymorphisms with the neurological status of htlv- afro-caribbean subjects in martinique nucleic acid sensors involved in the recognition of hbv in the liver-specific in vivo transfection mouse models-pattern recognition receptors and sensors for hbv regulation of hepatic innate immunity by hepatitis c virus systematic review with meta-analysis: do interferon lambda polymorphisms predict the outcome of interferon-therapy in hepatitis b infection? one more piece in the interleukin b gene puzzle? the case of hepatitis b interleukin b genetic polymorphism and hepatitis b virus infection no impact of interleukin- b polymorphisms on spontaneous or drug induced hepatitis delta virus clearance effects of polymorphisms in interferon λ (interleukin b) on sustained virologic response to therapy in patients with chronic hepatitis d virus infection hepatitis c infection in hiv- viral suppressors hepatitis c virus replication in caucasian hiv controllers il b polymorphism does not determine outcomes of hepatitis b virus or hiv infection interleukin- b gene polymorphisms do not influence the susceptibility to hiv-infection or cd cell decline protective interleukin- b genotype affects hepatitis c virus clearance, but does not contribute to hiv- control in a cohort of african-american elite controllers/ suppressors il b genotype does not correlate with hiv control in african americans il b single-nucleotide polymorphism rs is associated with spontaneous hiv control in white subjects ifnl ss polymorphism is associated with unfavourable clinical and immunological status inhiv-infected individuals ifnl rs polymorphism is associated with innate resistance to hiv- infection a systematic analysis of host factors reveals a med -interferon-λ regulatory axis against herpes simplex virus type replication interferon lambda genotype is not associated with recurrence of oral or genital herpes il b and pnpla polymorphisms affect histological liver damage in patients with nonalcoholic fatty liver disease genome wideassociation study identifies il b polymorphism to be associated with baseline alt and hepatic necro-inflammatory activity in chronic hepatitis c patients enrolled in the ideal study il b rs is not associated with histologic features of nafld in a cohort of caucasian north american patients reply to: "il b rs is not associated with histologic features of nafld in a cohort of caucasian north american patients interferon-λ rs genotype and liver fibrosis in viral and non-viral chronic liver disease genetic variations in il b and allergic disease in children anti-viral agents: potential utility in exacerbations of asthma evaluation of interleukin b single nucleotide polymorphisms in infants suffering from bronchiolitis il b polymorphisms are not associated with the response to interferon-β in multiple sclerosis il- b is a regulator of b-and t-cell vaccine responses against influenza mechanisms of type iii interferon expression interferon induction and function at the mucosal surface roles for transcription factors sp , nf-ĸb, irf , and irf in expression of the human ifnl gene il b expression depends on a novel tt/-g polymorphism which improves hcv clearance prediction identification of improved il b snps and haplotypes for prediction of drug response inn treatment of hepatitis c using massively parallel sequencing in a crosssectional european cohort interferon-λ (ifnl ) transcript expression in human liver tissue samples impaired induction of interleukin b and expression of interferon λ associated with nonresponse to interferon-based therapy in chronic hepatitis c hepatic expression levels of interferons and interferon-stimulated genes in patients with chronic hepatitis c: a phenotype-genotype correlation study interferon-lambda genetic polymorphism is associated with the therapy response for hepatitis c virus recurrence after a living donor liver transplant hepatic interferon-stimulated genes are differentially regulated in the liver of chronic hepatitis c patients with different interleukin- b genotypes a single nucleotide polymorphism associated with hepatitis c virus infections located in the distal region of the il b promoter influences nf-ĸb mediated gene transcription the favourable ifnl genotype escapes mrna decay mediated by au-rich elements and hepatitis c virus-induced micrornas estimating the net contribution of interleukin- b variation to spontaneous hepatitis c virus clearance ifnl mrna structure is remodelled by a functional non-coding polymorphism associated with hepatitis c virus clearance genetic variation of the il- b promoter affecting gene expression prevalence of thymine-adenine dinucleotide repeat, il b and ifnl in thai population and correlation with spontaneous clearance and treatment outcome of hepatitis c infection pretreatment prediction of the outcome of response-guided peginterferon-α and ribavirin therapy for chronic hepatitis c a thymineadenine dinucleotide repeat polymorphism near il b is associated with spontaneous clearance of hepatitis c virus ifnl genotype is associated with differential induction of ifnl in primary human hepatocytes reduced ifnλ activity is associated with improved hcv clearance and reduced expression of interferon-stimulated genes comparison of functional variants in ifnl and ifnl for association with hcv clearance il- b genetic variation is associated with spontaneous clearance of hepatitis c virus, treatment response, serum il- b levels in chinese population impact of il b gene polymorphisms on interferon-λ plasma levels during pegylated interferon-α/ribavirin therapy for chronic hepatitis c in patients coinfected with hiv the author declare no conflict of interest. key: cord- - gej h d authors: meier, william a.; husmann, robert j.; schnitzlein, william m.; osorio, fernando a.; lunney, joan k.; zuckermann, federico a. title: cytokines and synthetic double-stranded rna augment the t helper immune response of swine to porcine reproductive and respiratory syndrome virus date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: gej h d immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine initially elicits a weak interferon (ifn)-γ response. to improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (il)- or ifn-α was co-administered during vaccination. in the presence of either adjuvant, at least a three-fold increase in the primary virus-specific ifn-γ response was observed. while this enhancement was only transient ( week) when the il- expressing plasmid was used, the effect was not only still apparent at weeks after vaccination in the presence of the ifn-α expressing plasmid but even after challenge with a virulent genetically divergent prrsv. in contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. despite the apparent augmentation of a t helper (th) type response by the inclusion of ifn-α or il- during vaccination, this modulation did not necessarily correlate with a reduction in viremia. since a similar increase in the degree of the ifn-γ response to the prrsv vaccine could be achieved by substituting polyinosinic–polycytidylic acid in lieu of either cytokine, exposure to prrsv in the presence of a variety of th polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. conceivably, such intervention could be applied to improve the formulation of anti-prrsv vaccines. porcine reproductive and respiratory syndrome (prrs) is an economically important disease of swine characterized by abortion, stillbirth and weak-born pigs. in its non-reproductive form, this syndrome affects younger pigs more severely than older animals, causing reduced growth and pneumonia that can be made more severe by co-infection with other pathogens (thacker, ) . the etiologic agent for this disease is an rna virus (prrsv) that belongs to the family arteriviridae, which targets macrophages for infection (wensvoort et al., ; collins et al., ) . prrsv exhibits a significant degree of genetic diversification (murtaugh et al., ; key et al., ; goldberg et al., ) and this characteristic probably contributes to the difficulty in controlling infectious outbreaks caused by it (meng, ) . under experimental conditions, the currently available modified live virus (mlv) vaccine against this pathogen has been shown to provide immunized pigs adequate protection from subsequent challenge with the homologous strain (lager et al., a (lager et al., , b . however, despite the demonstrated ability of this vaccine to afford pigs partial protection against infection by a genetically divergent prrsv strain (osorio et al., ; lager et al., ; mengeling et al., a mengeling et al., , b , the overall protective immunity provided to swine in commercial settings is generally inadequate (halbur, ) . infection of pigs with wild type prrsv (nelson et al., ; loemba et al., ; vezina et al., ; yoon et al., ; albina et al., b; gonin et al., ) or their vaccination with a live attenuated form of this virus (labarque et al., ; ostrowski et al., ) elicits an exuberant production of nonneutralizing antibodies. in contrast, a transient t cell mediated prrsv-specific lymphoproliferative response is detected at weeks post-infection and lasts an additional (bautista and molitor, ) to weeks (lopez-fuertes et al., ) . moreover, during this time interval, limited quantities of ifn-g secreting cells (sc) are also generated (meier et al., ; xiao et al., ) . interestingly, in the absence of additional antigenic stimulation this polarity reverses within the ensuing months, as manifested by a decreasing antibody response and a gradual increase in the intensity of the ifn-g response (meier et al., ) . the initial antibody-dominated immune response is not the result of insufficient antigenic stimulation, since neither the inclusion of a commercial adjuvant during primary vaccination (meier et al., ) nor booster immunizations of previously heavily vaccinated pigs (bassaganya-riera et al., ) enhances virus-specific cell-mediated immunity (cmi). thus, prrsv seems to inherently stimulate an imbalanced immune response characterized by an abundance of humoral immunity and a limited, but potentially protective, th -like ifn-g response . one characteristic of prrsv infection that probably contributes to the retarded development of a specific cell-mediated immune response is the lack of induced ifn-a production (albina et al., a; buddaert et al., ; van reeth et al., ) . usually, virus-infected cells secrete type i ifn and the released cytokine interacts with a subset of naïve t cells to promote their conversion into virus-specific ifn-g sc (cella et al., ; cousens et al., ; kadowaki et al., ; biron, ; levy et al., ) . however, to counteract a virus that is a poor inducer of type i ifn, the development of a th response can be generated by a pathway that utilizes interleukin (il)- (cousens et al., ; biron, ) . indeed, the injection of recombinant at the time of vaccination and three additional times during the following week enhanced the host cellular immune response to prrsv (foss et al., ) . based on the success of this intervention and the noted absence of ifn-a generation during prrsv infection, the present studies were undertaken to determine if the antiviral adaptive immunity induced by vaccination with prrs mlv could be modified simply by the co-administration of ril- protein directly, or of either porcine ifn-a or il- indirectly via plasmids expressing these cytokines. in addition to these two elements that can deliver th polarizing signals (biron, ; kadowaki and liu, ; kapsenberg, ) , the effect of stimulation with a known inducer of ifn-a production, namely double-stranded rna, was examined. although all three adjuvants positively influenced the intensity and rate of development of the virus-specific ifn-g response, the results indicate that a stronger immunomodulator will be needed to overcome the tendency of prrsv not to elicit this type of th response. prior to use for immunization, the ingelvac prrs mlv vaccine (boehringer ingelheim vetmedica inc., st. joseph, mo) was reconstituted from lyophilized vials according to manufacturer's instructions. the highly virulent, ''atypical'' prrsv strain ia- - - ( - , genbank accession number af ) was originally isolated from a severe case of reproductive failure in an iowa swine herd in and was used as the challenge virus in the animal studies. the extent of nucleotide sequence identity in orf between this wild-type strain and the parental strain of the vaccine (vr ) used in this study is approximately %. for elispot assays of prrsv-specific ifn-g sc, prrsv strains vr- (american type culture collection, manassas, va) and ia- - - were used as sources of antigen. when required, prrsv was propagated and titrated in monolayers of marc- cells as previously described (meier et al., ) . porcine peripheral blood mononuclear cells (pbmc) were isolated from venous blood that had been anti-coagulated with mm heparin and the cells were subsequently maintained as described by meier et al. ( ) . for generating a plasmid capable of expressing ifn-a in mammalian hosts, an intact cdna encoding porcine ifn-a was first prepared by rt-pcr using rna isolated from pig lymphocytes previously infected with pseudorabies virus (to stimulate ifna production). forward (tctgcaaggttcccaa-tg) and reverse (gtctgtcactccttcttcctg) primers were designed based on the nucleotide sequence of porcine ifn-a cdna (lefevre and la bonnardiere, ) . products of the anticipated size ( bp) were cloned into the pcr . plasmid (invitrogen corp., carlsbad, ca), and an insert having the predicted restriction enzyme sites was sequenced. a comparison of the coding region within the selected amplicon to the previously reported ifn-a cdna revealed three nucleotide differences. these variations resulted in two amino acid changes of a tyrosine to a cysteine at position and of an arginine to a leucine at position of the intact protein. the ifn-a cdna was then excised from the recombinant pcr . plasmid and placed under the transcriptional regulation of the cytomegalovirus promoter in pcdna (invitrogen) to generate pina. to verify that an active cytokine was encoded by the amplified cdna, chinese hamster ovary (cho) cells were transfected with pina and single cell clones resistant to . mg geneticin per ml growth medium were prepared. supernatant medium from the clones were tested for the ability to inhibit the replication of an interferoninducer negative strain of vesicular stomatitis virus (sekellick and marcus, ) in madin derby bovine kidney (mdbk) cells as previously described (rubinstein et al., ) . clones producing from to greater than , units ( unit inhibits % of vsv replication in mdbk cell monolayers) of ifn-a were detected. for generating a plasmid capable of expressing a composite il- moiety consisting of the p and p subunits joined in tandem by an inert amino acid stretch in mammalian cells, cdna encoding the amino acid long leader region and amino acids of the mature form of the il- p subunit was first derived from plasmid p - race # (provided by dr. m.p. murtaugh, university of minnesota) that contains approximately the half of il- p subunit cdna. this fragment was then inserted into pcdna and ligated to a segment that was excised from the yeast expression plasmid ppic scil- (foss et al., ) and consisted of the remainder of the il- p subunit cdna linked by a nucleotide stretch to cdna encoding the mature form of the il- p subunit. the resultant plasmid, pcpil , utilizes the cytomegalovirus promoter to regulate expression of a complete il- complex consisting of one of each of the two subunits. to verify that a biologically active, secreted cytokine could be produced by pcpil , cho cells were either mock-transfected or transfected with the plasmid and after h the overlaying medium was subjected to a bioassay designed to demonstrate the presence of porcine il- based on its ability to increase the ifn-g response of memory t cells to recall viral antigen (meier et al., ) . by comparison to a yeast-derived porcine ril- standard (provided by dr. m. moody, endogen, woburn, ma), > ng/ml bioactive cytokine was found in the transfected cell medium whereas no activity was found to be associated with the control supernatant. escherichia coli strain dh a (invitrogen) was transfected with either pina or pcpil and grown in l of lb medium supplemented with mg/ml ampicillin (sigma, st. louis, mo) for h at c with constant shaking ($ rpm). plasmid purification was conducted using a qiagen plasmid maxi kit (qiagen inc., valencia, ca) according to manufacturer's instructions. plasmid pina or pcpil was precipitated onto the surface of gold particles (average diameter of mm; bio-rad laboratories, inc., hercules, ca) at a concentration of . mg dna/mg gold. plastic tubing was then coated with . mg of the dna-bound gold particles using a tubing prep station (bio-rad) following the manufacturer's instructions and cut to yield cartridges containing . mg dna. immediately prior to use as a standard in the il- bioassay or as an adjuvant in the animal studies, lyophilized yeast-derived porcine ril- (endogen) was reconstituted in low endotoxin-tested pbs (mediatech, herndon, va) to a concentration of mg/ml. stabilized polyinosinic:polycytidylic acid (poly i:c) was prepared by the method of levy et al. ( ) with minor modifications. briefly, poly i:c (sigma) at mg/ml in pyrogen-free . % nacl was denatured at c for h and allowed to re-anneal while cooling slowly to ambient temperature. the annealed poly i:c solution was then mixed with equal volumes of . mg poly-l-lysine/ml pyrogen-free . % nacl and % carboxymethylcellulose in pyrogen-free . % nacl. the final preparation was stored at c until needed. in the first study, -week-old yorkshire x landrace cross-bred pigs were obtained from a prrsv-free herd and randomly segregated into five groups (n = ) and a sixth group of only two individuals. the latter group was kept in a prrsv-free environment and was not vaccinated or challenged. all other animals were immunized in their adductor muscles (inner thigh) with . ml of ingelvac prrs mlv vaccine. at the same time, some of the pigs were inoculated intramuscularly (i.m.) by needle with either ml of saline (group ), mg pina (group ) or pcpil (group ) per animal or intradermally (i.d.) with mg of pcpil /animal (group ) via biolistic delivery with a gene gun (bio-rad) at locations adjacent to the site of vaccination. twenty micrograms of porcine ril- in a ml volume were co-administered to the members of group , which also received a second i.m. injection of the cytokine h later. at weeks postimmunization, all pigs receiving only the vaccine except one (group ), or the vaccine in conjunction with an i.m. application of either plasmid pina (group ) or pcpil (group ) were transferred to a biocontainment facility together with five additional prrsv-naïve pigs (group ) obtained from the same herd mentioned above. at this time all of the transferred animals were challenged with . tcid / . ml ( . ml/nostril) of prrsv strain ia- - - . fourteen days later all pigs were euthanized. for the second study, twelve -week-old yorkshire  landrace cross-bred pigs were obtained from the same prrsv-free herd described above and were randomly assigned to one of two groups (n = ). while all pigs were immunized i.m. with . ml of ingelvac prrs mlv vaccine, . mg poly i:c/kg of body weight was co-administered to the animals of one group only. this dose of poly i:c was selected based on its demonstrated ability to induce the maximum presence of ifn-a in the sera of -week-old pigs (loewen and derbyshire, ) . pigs were bled at , , and h after poly i:c administration and differential white blood cell numbers were determined with a cell-dyne (abbott laboratories, abbott park, il) and confirmed by microscopy by an experienced technician. eight weeks later, members of both groups were given a secondary ''booster'' immunization identical in composition to that of the primary vaccination. the extent of the host cell-mediated immune response to prrsv was measured by using a single cell elispot assay meier et al., ) to enumerate virus-specific ifn-g-sc in the pbmc population. the presence of prrs virus-neutralizing (vn) antibodies in the sera was determined as previously described by benfield et al. ( ) . briefly, twofold serial dilutions of serum ( ml) in modified eagle's minimal essential medium (mem) were mixed with an equal volume of mem containing tcid of the selected prrsv strain. after incubation at c for h, the mixture was added to monolayers of marc- cells in -well tissue culture plates. the cells were then examined daily during a -day interval, and the end point titer was expressed as the reciprocal of the highest serum dilution that neutralized the development of a prrsv-induced cytopathic effect. quantities of non-neutralizing prrsv-specific antibodies in the sera were measured by using a commercial elisa assay (herd-chek prrs, idexx, westbrook, me). sample to positive (s/p) ratios greater than . were considered positive and were calculated based on the manufacturer's instructions utilizing the formula: s/p = (test sample a( ) against prrsv antigen À test sample a( ) against normal host cell antigen)/(positive control against prrsv antigen À positive control against normal host cell antigen). virus was detected in serum samples collected on days , and post-prrsv challenge and from tonsil biopsies obtained at the termination of the experiment. prior to use, tonsils ( . g) were first homogenized by grinding the tissue with a pestle in a . ml microfuge tube, adding ml of mem supplemented with mg of gentamicin per ml, and vigorously shaking the sample with the pestle in the tube. after removal of the pestle, an additional ml of mem was added, and the homogenate was again mixed vigorously. the suspension was then passed through a . mm pore-size filter and stored at À c. to detect infectious prrsv, quadruplicate sets of marc- cell monolayers in -or -well plates were incubated with either ml of serial -fold dilutions of serum or tonsil homogenate (starting with : dilution) in mem supplemented with mg of gentamicin per ml for h at c in % co . afterwards, the inocula were removed and replaced with ml mem supplemented with % fetal calf serum and mg of gentamicin per ml. the cells were then incubated for days at c and checked daily for signs of cytopathic effect. results are expressed as the mean ae the standard error of the mean (s.e.). statistical significance was determined by analysis of variance. when significant differences at the . % confidence level were present, individual differences between treatment groups were determined with fisher's protected least significant difference (plsd). the comparison between the quantity of virus in sera and tonsils was done by using the fisher's exact test. the analyses were performed with the statview program v . (abacus concepts, berkeley, ca). immunization of pigs with only prrs mlv vaccine induced a relatively mild cmi response during the ensuing weeks as evidenced by the low frequency of prrsv-specific ifn-g sc in the vaccinated pigs' pbmc populations (fig. ) . although a similar response was evident during this time period for vaccinated pigs also i.m. injected with exogenous il- either directly as protein or indirectly via il- cdna, immunized animals that were i.d. inoculated with il- cdna exhibited an overall . -fold transient increase in the frequency of prrsv-specific ifn-g sc at weeks post-vaccination. likewise, a slightly greater enhancement of three-fold was found in those vaccinated animals that had received ifn-a cdna. however, in this case, the ifn-g response remained elevated at about this extent throughout the following weeks. since the pigs immunized with prrsv mlv in the presence of pina exhibited a significantly enhanced and sustained ifn-g response as compared to those in the other vaccinated groups, these animals as well as those receiving the vaccine only or the vaccine plus an i.m. injection of pcpil were transferred to a biocontainment facility and challenged with prrsv strain ia- - - . as controls, an additional group of age matched prrsv-naïve pigs were included and also infected with the virulent virus. during the weeks after challenge, the animals originally immunized in the presence of ifn-a cdna continued to display a statistically significant, greater ifn-g response relative to that measured in the other pigs. all mlv-vaccinated pigs exhibited strongly positive anti-prrsv antibody titers (s/p ratios ranged from . to . ) at weeks following immunization and their levels of humoral immunity had increased when measured weeks later (s/p ratios ranged from . to . ) (fig. ) . however, there were no significant differences between the mean s/p ratios of the cohort receiving the vaccine alone and of any of those groups whose vaccination was supplemented with a cytokine adjuvant. in contrast, vn antibodies were not detected until weeks after immunization and then only at low titers ( : ; v:v) and in at most one or two pigs per group (table ) . differences between the various cohorts in regards to the proportion of pigs that contained vn antibodies were not found to be significant. immediately prior to challenge with the virulent prrsv ia- - - strain at weeks postvaccination, vn antibodies were not detected in serum collected from any member of the three cohorts being challenged (mlv only, mlv + pcpil i.m., and mlv + pina i.m.; fig. ) . although subsequently by weeks post-challenge the majority of the pigs had developed a detectable vn antibody response, the three groups could still not be statistically differentiated based on their average vn titers. despite use of the challenge strain in lieu of prrsv isolate vr- as an antigen source appearing to result in a slightly more sensitive assay for the presence of vn antibody, this visual difference was determined not to be statistically significant. as expected due to the short interval between exposure to virus and performance of the assay, prrsv-neutralizing antibodies were not injections of porcine ril- at a daily interval (group ). an additional group served as unvaccinated controls (group ). at weeks post-immunization, vaccinated animals in groups - as well as five, newly acquired prrsv-naïve pigs (challenge control, group ) were challenged with prrsv strain ia- - - . pbmc were isolated from the pigs at the indicated times post-vaccination and the presence of virus-specific ifn-g sc was determined by using an elispot assay. asterisks indicate significant differences (p < . ) between the frequencies of ifn-g sc in the blood of the animals immunized with the mlvalone as compared to the other immunized groups. each value represents the mean response of five animals ae s.e.m. except for group that was downsized to four pigs immediately prior to challenge and group that consisted of two animals. unvaccinated / / / / nd a serum was drawn from all members of groups - described in the legend for fig. at the indicated times after vaccination and assayed for the presence of virus neutralizing (vn) antibodies against prrsv isolates vr and ia- - - . b the numerator values represent the number of vn positive pigs, and the denominator values represent the total number of pigs in each respective group. the vn titer in positive samples was : against either virus isolate. c not done. fig. . comparison of the presence of prrsv-neutralizing antibodies in the serum of vaccinated pigs after a challenge with wild type prrsv. immediately prior to and weeks after challenge with prrsv strain ia- - - , serum was drawn from four members of group and all pigs in groups , and described in the legend to fig. and assayed for the presence of prrsv-neutralizing antibodies. results are presented as the reciprocal of the lowest two-fold dilution of serum that inhibited the replication of either prrsv strain vr- or ia- - - in marc- cell monolayers. each value represents the mean prrsv neutralizing antibody titer in the blood of five animals ae s.e.m. except for group that was downsized to four pigs immediately prior to challenge. detected in the sera of the challenged, non-vaccinated pigs. at days post-prrsv challenge, one of four pigs immunized with mlv vaccine alone (group ) and three of five pigs immunized in conjunction with an i.m. injection of pcpil (group ) had detectable viremia (level of sensitivity . tcid /ml serum) (fig. ) . in contrast, at this time all of the unvaccinated pigs (group ) and all of those that received pina in combination with the vaccine were viremic (group ). three days later, only three of the five animals injected with pina (group ) as well as all of the nonimmunized pigs (group ) remained viremic (level of sensitivity . tcid /ml serum). by days post-challenge, virus could be found in the serum of only one of the unvaccinated pigs and none of the vaccinated pigs (level of sensitivity . tcid /ml serum). prrsv was also detected in the tonsils of three of the five non-immunized animals, but not in biopsies removed from any of the vaccinated pigs, when the experiment was terminated at days after challenge (level of sensitivity . tcid /g tissue). the lower sensitivity of the virus detection assay at days after challenge was due to the toxicity of the serum at a : dilution. this toxic effect was not present in the serum samples collected or days after challenge. to evaluate the impact of poly i:c on the host response to prrsv, the immune status of pigs receiving the mlv vaccine alone or in conjunction with this synthetic double-stranded rna and then revaccinated with the same formulations weeks later was monitored for a total of weeks. a nearly immediate effect of poly i:c on the pigs was observed as evidenced by an approximately % reduction in the quantity of white blood cells in their peripheral blood as compared to the other animals at h after the fig. . comparison of the virus titers in the sera and tonsils of vaccinated pigs after a challenge with wild type prrsv. serum was drawn from four members of group and all pigs in groups , and described in the legend to fig. at , and days post-challenge with prrsv strain ia- - - , while tonsil biopsies were obtained at days after challenge for all virus-challenged pigs. virus titers (log tcid per ml of serum or per gram of tonsil) are presented for each pig (circle). the limit of detection for each assay is indicated by the dotted line and values at this level have been placed on the line. bars indicate the mean of four or five measurable titers within a group. statistical significance (p < . , p < . ) is based on differences on the percent of viremic pigs between the indicated treatment groups as determined by fisher's exact test. administration of this compound. this leukopenia was transient as it was no longer apparent h later (data not shown). at week post-immunization, the poly i:c-treated pigs also exhibited a significant . -fold increase in the frequency of their prrsv-specific ifn-g sc as compared to the untreated pigs (fig. ). this enhancement tapered off but was still > -fold when measured and weeks later. from then until the time of the second immunization, significant differences between the frequencies of the ifn-g sc in the two groups were not detected. however, week after the booster immunization, once again the frequency of virus-specific ifn-g sc in the poly i:c-treated animals increased in comparison to the untreated animals-in this case a statistically significant . -fold. during the ensuing -week interval until the termination of the experiment, no differences between the two groups in regards to this parameter were observed. although prrsv-specific antibodies were readily detected in all of the pigs' sera when collected at and weeks post-primary immunization, no significant differences were found to exist between the average antibody titers of the poly i:ctreated and untreated groups (data not shown). the data presented here demonstrate the adjuvant effects of ifn-a when provided exogenously in the form of an expressible cdna (pina) or circuitously via induction of ifn-a synthesis (poly i:c) on the vaccine-induced ifn-g response to prrsv. however, despite the positive modulation of th immunity, no significant alteration in the development of the humoral immune response was observed. thus, even with such intervention at the initiation of prrsv immunization, the usual rapid onset of anti-prrsv antibody production and delayed appearance of vn antibodies (labarque et al., ; ostrowski et al., ; meier et al., ) still occurred. although in previous studies a similar increased presence of prrsv-specific ifn-g sc was afforded to pigs receiving multiple injections of il- at the time of vaccination and during the ensuing week (foss et al., ) , the use of a conventional oil-in-water adjuvant was found to be ineffective in this regard (meier et al., ) . moreover, in that study, even though this compound demonstrably enhanced the genesis of vn antibodies recognizing pseudorabies virus in addition to specific ifn-g sc, the intensity of either type of immune response to prrsv was not affected. apparently prrsv possesses inherent structural elements that prevent the timely development of protective innate or adaptive immunity capable of inhibiting its infectious process. thus, the ultimate outcome of the interaction between this virus and its host will be determined by the elicited host response which is highly variable, as evidenced by the inconsistency of the clinical outcomes seen upon challenge of naïve or immune pigs with prrsv (labarque et al., ; mengeling et al., a mengeling et al., , b . this is likely due to the significant variability within the swine population in regards to their innate and adaptive immune responses to prrsv (xiao et al., ; royaee et al., ) . fig. . comparison of the intensities of the virus-specific ifn-g sc response of pigs to immunization with a prrs mlv vaccine in the absence/presence of poly i:c. groups of pigs were vaccinated with prrs mlv alone or in conjunction with poly i:c at an -week interval. pbmc were isolated from the pigs at the indicated times post-vaccination and the presence of virus-specific ifn-g sc was determined by using an elispot assay. significant differences (p < . ) between the frequencies of ifn-g sc in the blood of the two groups are indicated by an asterisk. each value represents the mean response of six animals ae s.e.m. the ability of prrsv to not initially elicit protective immunity in an infected host is relatively novel among viruses that even after being inactivated can usually still promote a th -like response (de wit et al., ) . in this regard it is notable that the ifn-a response to exposure to prrsv is nearly non-existent. for example, ifn-a production in the lungs of pigs acutely infected with prrsv was either almost undetectable, or -fold lower than that induced by another pathogen, porcine respiratory coronavirus (prcv) (buddaert et al., ; van reeth et al., ) . such lack of efficient stimulation of ifn-a production by a pathogen has a significant impact on the nature of the host's adaptive immune response, since ifn-a up-regulates ifn-g gene expression, and thus controls the dominant pathway that promotes the development of adaptive immunity, namely, t cellmediated ifn-g responses and peak antiviral immune defenses (cousens et al., ; levy et al., ) . in this regard, it has become evident that the link between innate and adaptive immunity in viral infections occurs through the interaction of dendritic cells with type i interferon (montoya et al., ; tough, ) and the dendritic-cell controlled polarization of t-cell function (kapsenberg, ) . the production of ifna by plasmacytoid dendritic cells/natural ifn-a/bproducing cells (nipc) has an autocrine effect that promotes their functional and phenotypic activation, which is necessary for their optimal expression of costimulatory molecules and subsequent ability to cause naïve t cells to differentiate into ifn-g-sc (cella et al., ; kadowaki et al., ; fitzgerald-bocarsly, ; montoya et al., ; honda et al., ) . presumably, prrsv is a poor inducer of ifn-a production by nipcs since unlike transmissible gastroenteritis virus (charley and lavenant, ; nowacki et al., ) and type-a cpg oligonucleotides (guzylack-piriou et al., ) it fails to stimulate the secretion of ifn-a from cultured porcine pbmc (albina et al., a; zuckermann et al., unpublished observations) . thus, direct examination of the outcome of the interaction of prrsv with porcine nipcs will likely reveal important information on the immunobiology of this virus, especially since this virus is susceptible to the antiviral effects of ifn-a (albina et al., a) . the application of molecular tools such as real-time pcr assays to measure the expression of key immune mediators that regulate the development of th responses in swine will be particularly useful in this endeavor. it should be noted that in the absence of ifn-a/b production, the cytokine il- whose synthesis is not induced by most viral infections (orange and biron, ) can increase ifn-g production by t cells (cousens et al., ) . thus, two alternative routes (il- -or type i ifn-dependent) can lead to an adaptive th cell-mediated immune response with potent antiviral effects (biron, ) . according to a scenario involving the presence of less than a requisite amount of ifn-a, il- could provide the necessary impetus for the development of an anti-viral ifn-g response. in this regard, il- mrna has been detected in porcine macrophages infected with prrsv (thanawongnuwech et al., ) , and transiently in the lungs of prrsv-infected pigs (chung and chae, ) . however, this pathogen is also apparently a poor stimulator of il- production, since a negligible quantity of il- mrna or protein was produced by porcine pbmc exposed in vitro to prrsv zuckermann et al., unpublished observations) . the observation that the inclusion of either il- and ifn-a during immunization increased the intensity of the ifn-g response to prrsv validates the proposed role of these two innate cytokines in directing the in vivo differentiation of swine th cells, and helps explain the poor virusspecific ifn-g response that normally develops as a result of the exposure of pigs to prrsv (meier et al., ; xiao et al., ) . to compensate for the lack of stimulation of innate cytokine expression by prrsv, novel adjuvants have been used during immunization. the administration of il- in combination with a live or killed prrsv vaccine resulted in an increased lymphoproliferative response to this virus (wee et al., ) . inclusion of either a combination of il- and il- or cholera toxin correlated with an enhanced response to prrsv but only at weeks after the initial exposure (foss et al., ) . moreover, in that study, the frequency of virusspecific ifn-g sc did transiently increase between and weeks post-vaccination of pigs also injected with porcine ril- . although similar results were obtained in the current study, albeit after biolistic injection of expressible il- cdna in lieu of protein, the provision of ifn-a cdna had a more pronounced and sustained effect on the intensity of the cell-mediated immune response. it was notable that the i.d. administration of il- cdna with a gene gun was more successful at enhancing the vaccine-induced ifn-g response than the i.m. injection of the same plasmid. this observation is in agreement with the reported higher efficiency of in vivo dna transfection by biolistic delivery (fynan et al., ; colosimo et al., ) . the greater effectiveness of the plasmid encoding ifn-a rather than il- at enticing an ifn-g response could be attributed to the relatively low amounts of complete il- receptor on swine lymphocytes and the limited up-regulation of expression of the il- receptor ß subunit gene as compared to other species (solano-aguilar et al., ) . in this regard, it should be noted that the injection of bioactive il- into naïve pigs did not stimulate a strong t cell response (solano-aguilar et al., ) . likewise, the introduction of a known inducer of ifn-a production in pigs, poly i:c (derbyshire and lesnick, ) , during vaccination temporarily amplified the quantities of prrsvspecific ifn-g sc, but was not as efficient as the ifn-a encoding plasmid at enhancing the ifn-g response to the vaccine. this difference could be attributed to the presence of immunostimulatory cpg motifs in the eukaryotic expression vector pcdna , which was used in this study to express the cytokine genes, and has been shown to induce ifn-g expression by porcine leukocytes (magnusson et al., ) . it is plausible that the combination of direct stimulation of nipc by the cpg motifs in the ifn-a-encoding plasmid, presumably through their toll-like receptor (shimosato et al., ) , in combination with the plasmid-driven production of ifn-a would provide the necessary stimulatory signals to promote the maturation of dendritic cells and the creation of a microenvironment conducive for a sustained ifn-g response to prrsv. although poly i:c induces ifn-a production, it does not entice porcine nipc to differentiate (guzylack-piriou et al., ) , and thus might not provide enough impetus to promote a sustained t-cell-mediated ifn-g response to this virus. this notion is in agreement with the role attributed to cpg-containing oligonucleotides in promoting the maturation of nipc in humans (krug et al., ) and swine (guzylack-piriou et al., ) . the inability of ifn-a cdna to enhance the vn antibody response to prrsv is in contrast to the positive effect exerted by the co-administration of adenovirus expressing ifn-a in combination with a foot-and-mouth disease virus (fmdv) subunit vaccine into pigs . the notion of an structural inherent property unique to prrsv that deters the elicitation of a strong vn antibody response is in accord with the observation that in the case of prrsv-neutralizing antibodies, the close association of a nearby n-glycan with a recognized epitope in the virus's envelope glycoprotein may impede neutralization of this virus (plagemann et al., ) . in addition, decoy epitopes that delay the development of vn antibodies also exist (ostrowski et al., ) . remarkably, as we have shown here, the titer of vn antibodies increased equally in all vaccinated groups as a result of the pigs being challenged with a genetically divergent, virulent virus. similarly, augmented titers of vn antibodies against prrsv have been observed in pigs previously vaccinated with prrs mlv after they received a booster immunization with an inactivated genetically divergent virus but not when exposed to the same attenuated virus (osorio et al., ; bassaganya-riera et al., ) . as compared to the control (prrsv naïve) pigs, a lower proportion of those vaccinated with prrs mlv exhibited viremia at and days after challenge. however, no further reduction was observed in animals also receiving either il- or ifn-a. rather, injection of pina increased the percentage of viremic pigs, although the mean virus titer in their sera was intermediate between those values determined for the vaccinated and un-vaccinated groups at days after challenge. the significance of this observation with regards to protective immunity is unclear. this is due to the fact that while vaccination can decrease the duration and magnitude of viremia following an experimental challenge (van woensel et al., ; verheije et al., ) , the reduction in viremia is not necessarily associated with commensurate amelioration of the severity of other clinical parameters associated with prrs such as a lessened rate of weight gain, fever, respiratory distress or virus transmission to sentinel pigs (nodelijk et al., ; labarque et al., ; mengeling et al., a) . a similar lack of correlation between viremia and clinical signs was also noted when two different age groups of non-immune pigs were infected with prrsv. whereas a greater frequency of viremia with an accompanying higher virus titer was found in the younger animals ( months of age), the older pigs ( months of age) exhibited more severe clinical signs . the difficulty of deciphering prrsv biology is further revealed by the marked degree of variability and irreproducibility of consecutive trials conducted by the same investigators (labarque et al., ; mengeling et al., a mengeling et al., , b . moreover, although a positive association between protection from disease and the intensity of the ifn-g response in swine has been observed in regard to pseudorabies virus van rooij et al., ) , a similar relationship concerning prrsv was not apparent from the results of this study. thus, the identification of an immunologic mechanism responsible for mediating protective immunity against prrsv poses a significant challenge and will perhaps require monitoring the extent of reproductive failure caused by this virus as a measurement of the degree of protection (lager et al., ) . notably, in recent, related field studies we have noticed a positive correlation between the reduction of abortion/still births in sows and the relative frequency of prrsv-specific ifn-g sc in their blood (lowe et al., ) . in any event, perhaps a more marked enhancement of the vaccine-induced ifn-g response will be needed in order to improve protective immunity against prrsv as we found to be the case when il- was used as an adjuvant for an inactivated pseudorabies virus vaccine, which like the prrs mlv vaccine stimulates a weak ifn-g response . clearly, further studies will be required to clarify how the outcome of a prrsv infection is influenced by the intensity of the ifn-g response by memory t cells. nevertheless, the potential importance of eliciting a strong ifn-g response against this pathogen might not be limited to preventing virus replication (bautista and molitor, ; rowland et al., ) . since ifn-g can restrain b cell differentiation and even polyclonal b cell activation (cowdery and fleming, ) , it is reasonable to propose that a strong ifn-g response could mediate protective immunity by thwarting the polyclonal b cell activation that is associated with prrsv infection and results in immunopathology (lemke et al., ) . the uncontrolled b cell activation induced by prrsv could also contribute to the observed weak cell-mediated immune response, since activated b cells can release il- (burdin et al., ) , a cytokine that inhibits both ifn-g production by porcine t cells (waters et al., ) , and ifn-a synthesis by human pbmc in response to viral stimulation (payvandi et al., ) . in the accompanying manuscript we demonstrate that pbmc obtained from pigs at weeks after immunization with prrs mlv spontaneously secrete il- , il- , and il- and that the production of these cytokines are indeed affected by the administration, at the time of vaccination, of the same ifn-a expressing plasmid utilized in the present study. such repression may be critical since unabated prrsv infection will actually stimulate synthesis of il- (asai et al., ) , a known inhibitor of th cell development (diehl and rincon, ) and promoter of polyclonal immunoglobulin production following b cell activation by the murine arterivirus, lactate dehydrogenase-elevating virus (markine-goriaynoff et al., ) . strategies designed to shift the bias of the initial reaction to prrsv from the strong elicitation of nonneutralizing antibody production towards a greater th -like immune response are worth exploring since they could conceivably lead to the development of an improved vaccine against this pathogen. the need to develop such methodology is made palpable by the unusual kinetics of the immune response to this virus, as evidenced by the lack of a marked cmi response upon vaccination and subsequent challenge with virulent virus. the ifn-g response to prrsv therefore appears to be determined at the time of the first exposure to this pathogen and is only minimally affected by re-exposure. although it is also possible that we did not monitor the immune response long enough post-challenge to detect a significant increase in the number of virus-specific ifn-g sc, this is unlikely since in the accompanying manuscript a similar minimal enhancement was observed when the immune response was measured for up to weeks after a booster immunization . in addition, similar limited changes in the ifn-g response have been observed upon challenge of vaccinated pigs with wild-type virus (foss et al., ) or booster immunization with mlv vaccine (meier et al., ) . remarkably, the t-cell proliferative response to prrsv was also not increased by a booster immunization in pigs that had previously been repeatedly exposed to a mlv vaccine, but rather appeared to be suppressed as compared to that elicited by the same vaccine in naïve pigs (bassaganya-riera et al., ) . the mechanism responsible for this unusual effect is currently unknown, but might be related to the persistence of the virus in lymphoid tissues associated with the site of infection (xiao et al., ) . such sustained presence of virus could adversely affect a subsequent response due to an inherent and yet unknown property of prrsv. in summary, it is apparent that prrsv elicits in swine a polarized immune response characterized by an abundance of non-neutralizing antibodies and a paucity of ifn-g sc. the molecular pathway responsible for generating this type of immunity is unknown at this time, but based on the results presented it likely involves the limited induction of ifn-a and il- production and/or inherent structural elements of the virus that promote such a response. we propose that the strong humoral immunity bias of the host response to prrsv is mostly responsible for the difficulties in 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parenteral immunization with a pepsin-digested serpulina hyodysenteriae bacterin efficacy of porcine reproductive and respiratory syndrome virus vaccine and porcine interleukin- mystery swine disease in the netherlands: the isolation of lelystad virus the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection use of interleukin to enhance the cellular immune response of swine to an inactivated herpesvirus vaccine interleukin- enhances the virus-specific interferon gamma response of pigs to an inactivated pseudorabies virus vaccine the research work was supported by illinois agricultural experimental station swine research funds to fz and usda ars cris funds to jl. we gratefully acknowledge drs. tony goldberg and elizabeth greeley for helpful discussions and suggestions in the preparation of this manuscript. key: cord- -tibtngy authors: muñoz-moreno, raquel; cuesta-geijo, miguel Ángel; martínez-romero, carles; barrado-gil, lucía; galindo, inmaculada; garcía-sastre, adolfo; alonso, covadonga title: antiviral role of ifitm proteins in african swine fever virus infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: tibtngy the interferon-induced transmembrane (ifitm) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. while ifitms are widely known to inhibit several single-stranded rna viruses, there are limited reports available regarding their effect in double-stranded dna viruses. in this work, we have analyzed a possible antiviral function of ifitms against a double stranded dna virus, the african swine fever virus (asfv). infection with cell-adapted asfv isolate ba v is ifn sensitive and it induces ifitms expression. interestingly, high levels of ifitms caused a collapse of the endosomal pathway to the perinuclear area. given that asfv entry is strongly dependent on endocytosis, we investigated whether ifitm expression could impair viral infection. expression of ifitm , and reduced virus infectivity in vero cells, with ifitm and ifitm having an impact on viral entry/uncoating. the role of ifitm in the inhibition of asfv in vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that ifitm is acting at the late endosome, preventing the decapsidation stage of asfv. upon infection with pathogens such as bacteria or viruses, the host cell activates the innate immune response as a first line of defense. the group of cytokines known as interferons (ifn) plays a major role in the cell immunity by inducing a cascade of interferon-stimulated genes (isgs) that encode for several antiviral innate immune effectors. among isgs, the interferoninduced transmembrane proteins (ifitms) are known to inhibit entry of a wide variety of enveloped rna viruses [ ] . this group of proteins is present across a wide range of species: from amphibians, fish and birds to mammals. ifitms in humans were identified years ago as interferon-stimulated genes upon induction of type-i and type-ii ifn [ , ] . human ifitm , ifitm and ifitm are expressed in almost every cellular type, whereas ifitm is expressed primarily in osteoblasts, as it is required for bone mineralization [ ] . ifitms are found mainly distributed at the plasma membrane and/or at endosomal membranes. the ifitm , , and genes are clustered on chromosome , and they encode for relatively small proteins (about amino acids) with both extra-cytoplasmic termini separated by two transmembrane domains (tm and tm ) and a cytoplasmic loop (cil) [ ] [ ] . tm and the cil are well conserved between the ifitm proteins and a large group of members of the cd protein family. ifitm , , and are currently known to inhibit the replication of multiple rna viruses that enter the host cell via endocytosis, including influenza a virus (iav), west nile virus (wnv), dengue virus (denv) [ ] , severe acute respiratory syndrome coronavirus (sars cov) and hepatitis c virus (hcv) [ ] . in contrast, ifitms do not inhibit the entry process of mouse leukemia virus (mlv), machupo virus (mach), lassa virus (lasv) or lymphocytic choriomeningitis virus (lcmv) [ ] . little is known about the ifitm-mediated antiviral activity against dna viruses. only ifitm has been recently described to inhibit rana grylio virus (rgv), a frog/fish iridovirus, at the entry stage [ ] . on the other hand, ifitm , and have been reported not to affect the replication of other dna viruses, such as human papillomavirus (hpv), human cytomegalovirus (hcmv) and adenovirus (ad ) [ ] . the antiviral effect of ifitms is mainly exerted through their effects on the endocytic pathway and would affect viruses entering the cell through a late endosomal compartment [ ] . to further expand our understanding on the antiviral activity of ifitms against dna viruses, we investigated the role of these proteins in the replication cycle of the african swine fever virus (asfv), belonging to the nucleocytoplasmic large dna virus (ncldv) superfamily [ ] . asfv infection is strongly dependent on the endocytic pathway [ , ] , thus a possible ifitm-mediated inhibition of the virus could likely occur in the endosomal compartments. asfv is the only member of the asfarviridae family and is responsible of a highly lethal and hemorrhagic disease affecting domestic swine, which often results in important economic losses in many countries due to the high rate of mortality associated with the illness and the lack of an effective vaccine [ ] . an epidemic outbreak is currently affecting east europe and is slowly spreading between neighboring countries [ ] [ ] [ ] . we previously reported that asfv enters into the host cell by dynamin-dependent and clathrin-mediated endocytosis [ , ] . thus, our goal in the current work was to test whether the ifitm family of proteins affected early entry steps of asfv infection in vero cell cultures using the cell-adapted ba v isolate. vero cells were obtained from the american type culture collection (atcc, richmond, va, usa) and maintained in dulbecco modified eagle medium supplemented with % fetal bovine serum (fbs), lu/ml penicillin, ug/ml streptomycin and mm l-glutamine at °c at % co . cells were pretreated with , or , u/ml of universal type-i ifn (pbl assay science) for h, as indicated. the tissue culture-adapted asfv isolate ba v was used in most experiments [ ] . for flow cytometry analyses, a recombinant virus expressing the viral protein p fused to the green fluorescent protein (gfp) was used (b gfp) [ ] . preparation of viral stocks, titrations and infection procedures were carried out in vero cells as previously described [ ] . when synchronization of viral infection was required, the adsorption phase took place at °c to allow viral attachment to the cell surface but impeding its internalization. when indicated, asfv was semi-purified by sucrose cushion ( %) in pbs at , xg for min at °c. to generate stable cell lines expressing different proteins, the commercially available lentiviral expression vector plvx-puro (clontech) was used to clone the proteins of interest. t cells were transfected at % confluency using lipofectamine (life technologies) with opti-mem (life technologies) in -cm plates. plates were previously pretreated with poly-l-lysine (sigma-aldrich) at a final concentration of . mg/ml to avoid cell detachment. co-transfection of plvx-puro expression vector together with vesicular stomatitis virus glycoprotein (vsv-g) and the human immunodeficiency virus (hiv) gag-pol expressing plasmids was performed to produce pseudotyped lentiviral vectors. supernatants containing the pseudotyped lentiviruses were collected twice at h and h postransfection. cell debris was removed by brief centrifugation at , rpm for min and cleared supernatants were . μm-filtered and stored at - °c until use. sub-confluent vero cells were transduced with the pseudotyped lentiviruses expressing the gene of interest and supplemented with μg/ml of polybrene (emd millipore). h later, transduced cells were selected by adding μg/ml of puromycin (life technologies). optimal puromycin working concentration was previously titrated in non-transduced cells. finally, protein expression levels of vero stable-cell lines were determined by western blot (wb). cells were seeded and grown on glass coverslips. mock-infected and infected cells were fixed with % paraformaldehyde in pbs for min at room temperature (rt). following cell fixation, aldehyde fluorescence was quenched by incubation of cells with mm nh cl in pbs for min. then, cells were permeabilized with pbs- . % triton x- or saponin (sigma) for min at rt. after blocking with bovine serum albumin (bsa; sigma) or normal goat serum (sigma), cells were stained with primary and secondary antibodies and then incubated with topro- (molecular probes) in pbs at a : , ratio for dna staining. after washing, coverslips were finally mounted on glass plates and cells were observed under a leica tcs sp -aobs confocal microscope (leica-microsystems, wetzlar, germany) using a x immersion oil objective. to detect cholesterol distribution we used filipin staining (sigma), as previously described [ ] . cholesterol was visualized in a conventional leica dm rb microscope by combining a x immersion oil objective and a uv filter set. images were captured with leica application suite advanced fluorescence software (las af) and imagej software. finally, digital images were processed with adobe photoshop . . the primary antibodies used for immunofluorescence assays included the following: rabbit polyclonal antibodies to ifitm , ifitm and ifitm (proteintech), : ; anti-asfv p mouse monoclonal antibody, : (kindly given by dr. j.m. escribano, inia); asfv mouse monoclonal antibodies anti-p (clone bc for immunofluorescence : , or clone bg for wb : , ) and anti-p (clone ah , ingenasa), : , ; mouse monoclonal to cd (developmental studies hybridoma bank, university of iowa, clone h c ), : ; rabbit polyclonal to lamp (abcam), : ; rabbit polyclonal to eea and rab (cell signalling), : . secondary antibodies were purchased from molecular probes and diluted : . specificity of labeling and absence of signal crossover were determined by examination of single labeled control samples. cells were harvested in sample buffer laemmli x concentrate (sigma). then, the samples were incubated for min at °c and resolved by sds-page in % or % polyacrylamidebisacrylamide gels. afterwards, separated proteins were transferred to a nitrocellulose membrane (bio-rad) and the non-specific antibody binding sites were blocked with skimmed milk diluted in pbs and then incubated with the specific primary and hrp (horseradish peroxidase)-conjugated secondary antibodies. antibodies used for western blotting included: rabbit polyclonal to ifitm , ifitm and ifitm (proteintech), : , ; anti-asfv p mouse monoclonal antibody, : ; anti-asfv p mouse monoclonal antibody (clone bc , ingenasa), : , , and mouse monoclonal to tubulin (sigma-aldrich), : , . as secondary antibody, anti-mouse igg (ge healthcare) or anti-rabbit igg (bio-rad) conjugated to horseradish peroxidase was used at a : , dilution. precision protein streptactin-hrp conjugate (bio-rad) was used to reveal the ladder precision plus protein westernc (bio-rad). as a loading control an anti-mouse antibody against β-tubulin (sigma) was used, : , . finally, bands obtained after development with ecl reagent (ge healthcare, piscataway, nj, usa) were detected using a chemidoc xrsplus imaging system (bio-rad). band densitometry was performed with image lab software (bio-rad) and normalized to control values. the quantitation of the number of copies of asfv genome was achieved by quantitative realtime pcr (qpcr) using specific oligonucleotides and a premix extaq (tm) ( x; takara) probe. fluorescent hybridization probes were used to amplify a region of the p viral gene, as described previously [ ] . dna from cells mock-infected or infected with asfv ba v at moi of pfu/cell was extracted at hpi and purified with a dnaeasy blood and tissue kit (qiagen). dna concentration was measured using nanodrop. the amplification mixture was prepared on ice as follows: ng template dna diluted in mq h o to a total volume of μl, μl oligonucleotide oe f ( pmol), μl oligonucleotide oe r ( pmol), μl pcr premix ex taq(tm) ( x; takara), μl taqman probe se ( pmol) [ ] . positive amplification controls included dna purified from asfv purified virions at different concentrations as standards. negative amplification controls consisted in dna from mock-infected cells. each sample was included in triplicates and values were normalized to standard positive controls. reactions were performed using the abi fast real-time pcr system (applied biosystems) with the following parameters: cycle at °c for min, cycles at °c for s, and cycles at °c for min. to study virion decapsidation in asfv, a protocol was adapted from a previous publication [ ] . briefly, stable cell lines vero-ifitm , ifitm , ifitm or control cells containing the empty vector were infected at moi of pfu/cell after viral synchronization at °c for minutes to enable virus attachment to the cell but restricting viral entry. infection was allowed to proceed for minutes at °c, % co . cells were then washed with cold pbs x and treated with . % trypsin-edta (gibco) for minutes at °c to remove the membrane-bound virus. finally, cells were placed in media containing fcs to quench trypsin activity and washed with pbs. after this treatment, only internalized virions were observed in an immunofluorescence assay as described above. we used specific antibodies to detect the major viral capsid protein p and the viral core protein p , and staining was analyzed by confocal microscopy. decapsidated virions were single labeled for p and were counted for each condition and normalized to the total number of fully encapsidated virions which were double labeled for p and p . stable cell lines vero-ifitm , ifitm , ifitm or control cells containing the empty vector were infected with ba v or b gfp at the indicated moi. recombinant b gfp is a recombinant asfv expressing green fluorescent protein as a fusion protein of viral p [ ] . samples infected with b gfp at hpi were just fixed and washed with facs buffer three times before analysis. vero cells infected with ba v at hpi or hpi (early or late postinfection times respectively), were harvested by trypsinization, washed with facs buffer (containing pbs, , % sodium azide, and , % bovine serum albumin), fixed and permeabilized with perm (bd science) for min at rt and finally incubated with specific primary antibodies against p and p for min at °c. the secondary antibody used was phycoerythrin (pe) conjugated (dako) : diluted in facs buffer for min at °c. after repeated washes, , cells/ tube were analyzed in the facscalibur flow cytometer (bd science) in triplicates. the obtained infection rates were always normalized to the corresponding control. bonferroni's multiple-comparison test was used to compare different experimental groups. prism software (graphpad software, inc.) and instat software were used for the statistical analysis. values were expressed in graph bars as mean ±sd of at least three independent experiments unless otherwise noted. metrics were normalized to control values and represented in graphics. asterisks denote statistically significant differences ( ÃÃà p< . , Ãà p< . and à p< . ). to determine the effect of interferon on asfv infection, vero cells were pretreated with , or , u/ml of universal type-i ifn (pbl assay science) and h later, they were infected with recombinant asfv b gfp at a moi of pfu/cell (fig ) . viral infection was quantified by analyzing the number of gfp-positive cells by flow cytometry at hpi (fig a) . pretreatment of vero cells with universal type-i ifn at both concentrations completely abrogated asfv infectivity when compared to untreated control cells. a sample flow cytometry profile is shown (fig a) . ifn treatment induces expression of ifitm proteins ifitm proteins are located in different cellular compartments and their antiviral properties strongly correlate with their capacity to alter the fluidity and fusion ability of the membranes in which they reside [ , ] . then, we analyzed ifitms expression and distribution in vero cells upon ifn treatment. to this end, cells were incubated with either , or , u/ml of universal type-i ifn for h and ifitms , and distribution was analyzed by confocal microscopy ( fig b) . although basal levels of ifitm and were detected prior to treatment with to further analyze the expression of ifitm , and upon ifn treatment, vero and t cells were incubated with universal type-i ifn for h and analyzed by wb (fig ) . while ifitm and ifitm were induced by ifn in both cell lines (fig a and b) , ifitm showed the highest induction (fig c) . in order to analyze the possible impact of ifitms in asfv infection, we generated vero cells stably expressing the human ifitm , , (hereinafter referred to as vero-ifitm , or cells respectively) or control cells containing the empty vector. to generate the stable cell lines we used a lentiviral transduction system. our proteins of interest were cloned into the plvx vector (see material and methods section for detailed experimental procedures). positively transduced vero stable cells were selected with μg/ml of puromycin. once these cells were established, the expression of different ifitm proteins was confirmed by wb analysis ( fig a) . as shown in the corresponding wb densitometry (fig b) , the highest expression levels within the ifitm family members corresponded to ifitm , followed by ifitm and ifitm . after assessing the expression levels of the vero-ifitm cells, we next wanted to ascertain the subcellular distribution of each ifitm. expression of ifitm , and in vero-ifitm cells was compared with the distribution of ifitms in vero cells with the empty vector. confocal microscopy experiments revealed that, ifitm was mainly distributed at the plasma membrane and to a lesser extent in perinuclear compartments, resembling endosomal structures (fig c, lower left panel) , while endogenous ifitm was barely detected in vero cells containing the empty vector (fig c, upper left panel) . in vero-ifitm cells, overexpression led to a high accumulation of the protein in the perinuclear region, colocalizing with vesicular structures that resembled endosomes (fig c, medium lower panel). consistent with fig b, there was a significant expression of endogenous ifitm in control vero cells containing the empty vector, which displayed a mitochondrialike pattern together with vesicular-like structures. finally, overexpressed ifitm was found predominantly accumulated in the perinuclear region, showing a pattern consistent with localization at clustered endosomal structures ( fig c, lower right panel) . endogenous ifitm was barely detected in vero cells containing the empty vector (fig c, upper right panel) . analysis of endogenous ifitm expression in control vero cells suggested a mixed mitochondrial and vesicular pattern. to confirm this observation, we analyzed subcellular localization of ifitm using mitotracker red as a specific marker for mitochondria. confocal images revealed a distribution of ifitm to mitochondrial structures in cells containing the empty vector (fig ) . however, in vero-ifitm cells, ifitm labelling localized to endosomal-like structures and we found few areas of colocalization between ifitm and mitochondria (fig b) . then, we investigated the possible mechanism underlying the inhibition caused by ifitm in the viral infection. to further analyze the vesicular localization of ifitms, we studied the cell. data are expressed as mean±sd of three independent experiments. statistical significance was evaluated by one-way anova followed by bonferroni's multiple comparison test. differences are marked with asterisks as indicated (***p< . ). an example of a typical facs profile is shown. (b). confocal fluorescence images of ifitm , and subcellular distribution in untreated vero cells or upon increasing universal ifn concentrations ( , or , u/ml) for h. bar = μm. (fig a) . endosomes are normally distributed through the cytoplasm and this dispersed distribution was found for the different maturation stages of endosomes in controls and in vero-ifitm cells. however, in vero-ifitm and ifitm cells dispersed distribution changed and endosomes aggregated around the nucleus (fig a) . this endosome redistribution was analyzed by confocal microcopy in x, y, z planes by measuring the mean distance between each endosomal marker and the cell nucleus, using the "distance measure" imagej plug-in (fig b) . a total of cells were analyzed for each condition. we concluded that overexpression of ifitm and ifitm altered endosome distribution by accumulating these vesicles to the perinuclear region, similar to the pattern previously found after ifn treatment of vero cells (fig b) and this redistribution might reflect alterations in endo-lysosomal maturation and function. next, we analyzed the colocalization rate between ifitms and endosomal structures. cd remains mainly associated with intracellular vesicular membranes and it is particularly abundant in endosomal structures called multivesicular bodies (mvbs), which constitute a late and acidic endosomal compartment filled with intraluminal vesicles (ilvs). these ilvs are filled with cholesterol and are crucial for endosomal membrane backfusion and necessary for late endosome maturation. co-staining of ifitms and cd by immunofluorescence assay revealed a clear colocalization of ifitms and cd in vero-ifitm cells, with % colocalization for vero-ifitm cells and % in vero-ifitm and in ifitm cells (fig a) . then, ifitm , and to a lesser extent ifitm and , were located primarily in endosomal compartments as indicated by higher colocalization with endosomal marker cd (fig c) when compared to the empty vector ( fig b) . ifitm proteins could reduce curvature of cell membranes for fusion pore formation [ , ] at the outer endosomal membrane, also called endosomal-limiting membrane, to differentiate it from the membranes of intraluminal vesicles inside multivesicular bodies (mvb/le). this would lead to an alteration of membrane fusion and impaired cholesterol endosomal efflux. changes in endosomal distribution such as those found in ifitm stably expressing cells (fig ) is a characteristic phenotype of an altered cholesterol endosomal efflux [ , ] . conversely, a conserved endosomal cholesterol efflux is required for a correct endosomal function. therefore, we analyzed intracellular and intra-endosomal cholesterol levels by using the cholesterol marker filipin. vero-ifitm and ifitm cells showed intense intracellular cholesterol accumulation at the perinuclear area ( fig a) that was absent in control cells containing the empty vector (fig b) . this cholesterol accumulation also colocalized with the ifitm-labeled endosomal vesicles. in contrast, in vero-ifitm cells, only discrete colocalization areas between ifitm and cholesterol were found at the plasma membrane. collectively, these findings indicate that ifitm and ifitm overexpression in vero cells results in cholesterol accumulation in endosomal compartments, and as a result it might be responsible of an altered endosomal function possibly altering viral entry through this pathway. next, we investigated whether overexpression of ifitms could restrict asfv infection or not. asfv is known to require acidic endosomal compartments for entry into host cells. successful asfv entry and egress from endosomes depends on the acidic ph of late endosomes [ ] . previous experiments in the laboratory revealed that the inhibition of acidification impaired viral decapsidation and viral particles were retained in rab -positive late endosomes, thus blocking viral infection progression [ ] . given that ifitm restriction could be mediated at the endocytic pathway [ , ] , we hypothesized that ifitm overexpression may affect virus entry process and subsequent asfv infection. acidic ph of late endosomal compartments is required for viral decapsidation, which is the first step required for uncoating previous to endosomal escape of the virus and the start of replication [ ] . the asf virion is composed of several concentric domains. the viral capsid is formed by major capsid protein p organized in capsomers. hence, after decapsidation, p staining of virions is lost. the internal core is composed by the nucleoid containing genome coated by the core shell, a thick protein layer that can be labeled with antibodies against p , a core shell protein derived from processed core shell polypeptides [ ] . . (b) . the change in distribution was quantified by measuring the mean distance to the nucleus of the different endosomal markers in x, y and z planes as described in materials and methods section. as shown in graphics, distance was reduced in vero-ifitm and cells. graphics depict mean±sd of n = cells per condition. statistical significance was evaluated by a one-way anova followed by bonferroni's multiple comparison test. differences are marked with asterisks as indicated (*p< . ; **p< . ). doi: . /journal.pone. .g we used antibodies against the major viral capsid protein p to detect viral capsids and against viral core protein p to detect viral cores (fig ) . we analyzed the number of encapsidated viral particles, double labeled for p and p , and compared with the number of successfully decapsidated viral cores positive for p at minutes postinfection (mpi). at this time point, most virions undergo uncoating and progress to replication in normal conditions, otherwise, encapsidated virions would accumulate. confocal microscopy revealed that the number of viral cores was severely decreased in vero-ifitm cells when compared to control cells containing the empty vector (fig b) . double-labeled encapsidated viral particles were counted and the ratio of decapsidated viral cores compared to the total number of virions per individual cell was calculated and expressed in percentages (fig a) . the percentage of decapsidated virus severely decreased under ifitm expression. however, ifitm expression produced an accumulation of virions leading to higher numbers of total virions (fig c) . this increase in the number of total virions might be the result of an impaired progression of the asfv replication cycle. these virions would neither proceed with infection nor be degraded and this would be consistent with an inhibition of the membrane fusion capacity at the endosomal level. altogether, these results indicate that viral entry was the rate-limiting step in vero-ifitm and ifitm cells for asfv infectivity. finally, we analyzed the impact of ifitm expression on other infection parameters to find reduced number of copies of asfv genome at hpi (fig a) . ifitm overexpression induced a consistent reduction in infectivity as measured by early protein p expression by flow cytometry (fig b) . however, these reductions were even more marked when analyzing infected cell percentages by late p protein expression (fig c) . finally, we also analyzed viral protein expression by wb (fig d- f) . the expression of protein p at hpi ( fig d) and p at hpi (fig e) resulted significantly reduced as other infection parameters. in the present work, we have studied the antiviral effect of the ifitm family of proteins in the context of cell-adapted asfv infection in vero cells. while different ifitm proteins have been repeatedly described as inhibitors of a broad spectrum of rna viruses [ ] , their antiviral role involving dna viruses is poorly studied and only reported in the rana grylio virus (rgv), blocking the virions at the entry stage into the host cell [ ] . asfv ba v infection in vero cells is significantly affected upon ifn treatment [ , ] . in general, the sensitivity towards the induction of the innate immune response of the host cell has led viruses to acquire different strategies to regulate the ifn pathway for its own benefit. such is the case of asfv viral gene a r, which negatively regulates the induction of ifn through targeting irf in a nfκb-independent manner [ ] . another example is the asfv gene i l, which codifies for a viral tlr homolog with inhibitory activity against ifn [ ] . finally, the myxovirus resistance gene a (mxa), which is another interferon stimulated gene (isg), also inhibits the replication and the late gene expression of asfv [ ] . we report here that upon ifn treatment of vero cells, the distribution of ifitm proteins changes into a perinuclear vesicular pattern resembling endosomes. our analysis of endogenous ifitm expression in the absence of ifn induction showed colocalization with mitochondrial structures. interestingly, ifitm underwent vesicular pattern redistribution around the cell nucleus when overexpressed and upon ifn treatment as well. our characterization of the cellular distribution of ifitm , and also unveiled specific localization patterns linked to the endosomal pathway upon overexpression, particularly in the late endosomal compartments. this distribution is coincident with previously reported data of other groups, which described localization of endogenous ifitm at the plasma membrane and early endosomes, and of ifitm and ifitm in late endosomes and lysosomes [ , ] . interestingly, overexpression of ifitm has been recently described to delay the proteolytic degradation of human papilloma virus (hpv) capsids in keratinocytes [ ] . this, however, did not affect the replication of the virus. our analysis of asfv ba v uncoating correlated the expression of ifitm with a decrease in the numbers of decapsidated asf virions in vero cells. this suggests a possible role for ifitm in inhibiting asfv exit from late endosomes. in contrast, ifitm did not modify the ratio between encapsidated and decapsidated virions. instead, ifitm apparently increased the accumulation of virions that are probably retained and do not proceed to degradation and/ or to a productive infection. these results may also suggest that the presence of ifitm affects the release of the virions from the late endosomal compartments. asfv enters into the host cell by dynamin-dependent and clathrin-mediated endocytosis [ , ] and macropinocytosis [ ] . in fact, endosomal pathway integrity is known to be important for asfv infection, both for culture-adapted isolates in cell lines [ ] or for virulent and attenuated isolates in primary macrophages [ ] . hence, it is not surprising that infection could be impaired by these restriction factors acting at the endosomal membrane. the aforementioned ifitm -and ifitm -mediated inhibition of asfv entry has been previously reported in other viruses, including iav, flaviviruses (denv and wnv) [ ] , filoviruses (marv and ebov) and coronaviruses (such as sars) [ ] . in contrast, infection with alphaviruses, arenaviruses and mlv (a retrovirus) seems to be unaffected by ifitm protein expression [ ] . in general, ifitm-mediated viral inhibition has been related to impaired viralhost membrane fusion subsequent to viral binding and endocytosis [ , ] . ifitm has also been reported to modulate the fluidity and the bending modulus of the cell membrane, thus making it resistant to viral fusion machinery [ ] . we also studied whether the inhibition of asfv entry could be due to an alteration of the endosomal compartments. analysis of endosome distribution upon ifitm overexpression revealed that ifitm and ifitm altered the normal distribution of early and late endosomes and lysosomes. however, this alteration was not found in the presence of ifitm . a collapse of endosomes to the perinuclear area is also a characteristic phenotype of an alteration of endosomal cholesterol efflux [ ] . also, recent publications from our laboratory demonstrated that accumulation of cholesterol in endosomes caused by a chemical inhibition of cholesterol flux, causes virion retention inside endosomes and inhibition of infection progression [ ] . there are currently two proposed models trying to explain the ifitm-mediated inhibition of viral entry. the first one, known as the "tough membrane" model, postulates that intramembranous interactions between adjacent ifitms alter the fluidity and bending of the host cellular membrane, making it resistant to the viral fusion machinery [ ] . the second model suggests that ifitms can induce the accumulation of cholesterol in the endosomal membrane and membrane fusion defects [ ] disturbing intracellular cholesterol homeostasis that finally blocks the viral release into cytosol [ ] . ifitm and ifitm have been previously reported to alter the cholesterol homeostasis at the late endosome, leading to cholesterol accumulation and blocking the viral release [ ] . in the present study, we have described accumulation of cholesterol upon overexpression of ifitm and ifitm . we have recently reported that inhibition of cholesterol exit from endosomes using chemical inhibitors causes retention of virions inside these vesicles, thus impairing progression of the infection [ ] . altogether these data could suggest that the antiviral action of ifitms may affect to a higher extent to those viruses that require the endosomal pathway during the early stages of infection. collectively, our results illustrate a close relationship between the ifitm protein family and the endosomal pathway, leading to the inhibition of asfv infection. the antiviral action of ifitms could rely on alterations of the endosomal physiology and ongoing studies in our laboratory will be focused on antiviral targets at the molecular regulation of late endosomes. also, a role in cell-to-cell viral transmission has been postulated for ifitms, which are incorporated into hiv- virions [ ] . we have performed this study in a cellular system using the cell-adapted ba v isolate in vero cells but these results will be next extended to other asfv isolates with porcine macrophages. further studies will be required for better understanding the relevance of ifitms in the context of asfv infection. in summary, ifitms represent a broad and previously unappreciated class of restriction factors that degrade invading enveloped viruses and may therefore be considered as potential antiviral components to protect the host cell. ifitms restrict the replication of multiple pathogenic viruses transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells molecular analysis of a human interferon-inducible gene family the broad-spectrum antiviral functions of ifit and ifitm proteins the cd /cd signal transducing complex of human b lymphocytes includes the target of antiproliferative antibody- and leu- molecules the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus ifitm is a tight junction protein that inhibits hepatitis c virus entry evidence for paralichthys olivaceus ifitm antiviral effect by impeding viral entry into target cells the antiviral restriction factors ifitm , and do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus common origin of four diverse families of large eukaryotic dna viruses endosomal maturation, rab gtpase and phosphoinositides in african swine fever virus entry african swine fever virus infects macrophages, the natural host cells, via clathrin-and cholesterol-dependent endocytosis african swine fever virus-cell interactions: from virus entry to cell survival european comission, . . cases of asf confirmed in latvia. the veterinary record epidemiology of african swine fever in poland since the detection of the first case. polish journal of veterinary sciences dynamin-and clathrin-dependent endocytosis in african swine fever virus entry titration of african swine fever (asf) virus. the journal of general virology visualization of the african swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p membrane protein chimera attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation development of a taq-man pcr assay with internal amplification control for the detection of african swine fever virus the major cellular sterol regulatory pathway is required for andes virus infection the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry ifitm restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion alteration of dynein function affects alpha-synuclein degradation via the autophagosome-lysosome pathway phosphatidylinositol -kinases: hostages harnessed to build panviral replication platforms african swine fever virus morphogenesis. virus research effect of interferon-alpha, interferon-gamma and tumour necrosis factor on african swine fever virus replication in porcine monocytes and macrophages. the journal of general virology interferon cures cells lytically and persistently infected with african swine fever virus in vitro identification and utility of innate immune system evasion mechanisms of asfv. virus research a novel tlr inhibitor encoded by african swine fever virus (asfv) inhibition of a large double-stranded dna virus by mxa protein - pmid: ifitm inhibits influenza a virus infection by preventing cytosolic entry distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus african swine fever virus uses macropinocytosis to enter host cells the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication cholesterol flux is required for endosomal progression of african swine fever virions during the initial establishment of infection the diverse functions of oxysterol-binding proteins ifitm proteins incorporated into hiv- virions impair viral fusion and spread distance measure software plug-inn of imagej was kindly provided by dr. esteban veiga and giulia morlino from institute for health research "hospital universitario la princesa", cnb, madrid.funding sources for ca: ministerio de economía y competitividad (es) http://www. mineco.gob.es/portal/site/mineco/idi agl - and agl - -r and for ag-s national institute for allergy and infectious diseases (niaid); http://www.niaid.nih.gov/; u ai . key: cord- -qzm wgde authors: ellermann-eriksen, svend title: macrophages and cytokines in the early defence against herpes simplex virus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: qzm wgde herpes simplex virus (hsv) type and are old viruses, with a history of evolution shared with humans. thus, it is generally well-adapted viruses, infecting many of us without doing much harm, and with the capacity to hide in our neurons for life. in rare situations, however, the primary infection becomes generalized or involves the brain. normally, the primary hsv infection is asymptomatic, and a crucial element in the early restriction of virus replication and thus avoidance of symptoms from the infection is the concerted action of different arms of the innate immune response. an early and light struggle inhibiting some hsv replication will spare the host from the real war against huge amounts of virus later in infection. as far as such a war will jeopardize the life of the host, it will be in both interests, including the virus, to settle the conflict amicably. some important weapons of the unspecific defence and the early strikes and beginning battle during the first days of a hsv infection are discussed in this review. generally, macrophages are orchestrating a multitude of anti-herpetic actions during the first hours of the attack. in a first wave of responses, cytokines, primarily type i interferons (ifn) and tumour necrosis factor are produced and exert a direct antiviral effect and activate the macrophages themselves. in the next wave, interleukin (il)- together with the above and other cytokines induce production of ifn-γ in mainly nk cells. many positive feed-back mechanisms and synergistic interactions intensify these systems and give rise to heavy antiviral weapons such as reactive oxygen species and nitric oxide. this results in the generation of an alliance against the viral enemy. however, these heavy weapons have to be controlled to avoid too much harm to the host. by il- and others, these reactions are hampered, but they are still allowed in foci of hsv replication, thus focusing the activity to only relevant sites. so, no hero does it alone. rather, an alliance of cytokines, macrophages and other cells seems to play a central role. implications of this for future treatment modalities are shortly considered. virus-host interactions are crucial for the outcome of infections. several strategies have been utilized by viruses to overcome the host defence. for the virus to be successful, these evasive strategies have to be balanced with the pathology induced and the possibilities of transmission to new susceptible individuals. the mammalian host utilizes ubiquitous and redundant antiviral defence mechanisms. in different viral infections, different parts of the host defence seem to be crucial. however, the redundancy ensures that other systems are ready to take over, if one of them fails. the final outcome of a viral infection depends on a delicate regulation and timing of these antiviral effector mechanisms in response to the invading virus. a viral infection of an individual thus involves a conflict between the virus and the host, which could conceptually be viewed upon as a human controversy escalating to invasion and armed struggle. to understand the resulting course of events it is important to know each party of the conflict and to conduct an analysis of the powerful weapons held by each of the combatants. the present review analyzes the early non-specific events in the conflict upon herpes simplex virus (hsv) infection. initially, each participant of the conflict, the infecting hsv and the non-specific antiviral weapons of the host, are described. subsequently, the early events of the conflict, the armament, early strikes and the opening battle between hsv and the host are discussed. insight into the early non-specific defence mechanisms are important for our understanding of the conflict and may indicate how to intervene during serious systemic infections. herpesviruses are ubiquitous viruses generally infecting humans early in life. the majority of humans has had a primary infection with one or more herpesviruses and harbour these viruses in a latent state for the rest of their lives. the initial infection is most often asymptomatic, but can be symptomatic depending on the herpesvirus in question and the age and immune status of the host. the viruses are phylogenetically old and humans and herpesviruses have evolved together [ ] . this co-evolution has created viruses which are well adapted to the human host and environment. thus, herpesviruses are capable of coping with the human immune defence in a balanced manner generally without serious threads to the life of the host. infection with a foreign herpesvirus, normally hosted by another species, does not always hold this balance, and the pathology is unpredictable. this is seen when humans are infected with the simian b virus, which often shows serious clinical outcome [ ] . the human herpes simplex viruses were initially identified by lowenstein, who passed it onto rabbits in , and found it to be sensitive to alcohol and higher temperatures [ ] . the viruses were classified into two serologically different types by schneweiss in [ ] , and these are now known to belong to the subfamily of alphaherpes-virinae together with varicella-zoster virus. these alphaherpesviruses all show neurotropic latency, and mucosal or skin lesions are frequently seen as a result of viral reactivation from sensory nerves. the two types of herpes simplex virus confer the genera simplexvirus and - , which were formally designated by the international committee on taxonomy of viruses as human herpesvirus (hhv) and [ ] . herpes simplex virus (hsv) type and type are very closely related, showing a homology at the dna level of % in protein coding regions and less in noncoding regions [ ] . the genetic map of the two herpes simplex viruses is colinear [ ] , and the genomes are of approximately the same size, hsv- of kbp [ ] and hsv- of kbp, and code for corresponding genes [ ] . the minor sequence variations give different cleavage sites for restriction endonucleases, which has been used intensively as an important epidemiological tool [ ] [ ] [ ] . as all other herpesviruses the herpes simplex viruses are enveloped, icosahedral dna viruses with a capsid of approximately nm ( fig. ) [ ] . the envelope holds at least different glycoproteins protruding from the outer side (gb, gc, gd, ge, gg, gh, gi, gk, gl, and gm). the glycoproteins are primarily responsible for attachment to cellular receptors and fusion of membranes (especially gb and gd) [ ] [ ] [ ] [ ] . in addition, there are two unglycosylated proteins in the viral envelope. the glycoproteins of the envelope have several immunoregulatory effects besides their primary more mechanical functions in viral attachment and entry [ ] [ ] [ ] [ ] [ ] . in the space between the envelope and the capsid, the complete viral particles posses an almost amorphous structure which was termed the tegument by roizman and furlong [ ] . the tegument consists of several viral proteins involved in the initial phases of viral infection and replication such as transport of the viral dna out of the capsid [ ] , early shutoff of cellular protein synthesis (vhs) [ ] , and initiation of transcription of viral genes (α-trans-inducing factors) [ ] . besides the tegument seen in complete viral particles, tegument-like structures are seen in enveloped particles lacking a capsid and dna, the so called light particles [ , ] . the capsid is composed of a complex icosahedral structure of capsomeres, each with a central channel running from the outside to the interior of the capsid. inside the capsid the double stranded linear dna is packed as a spool with the ends in close proximity [ , , ] . the genome consists of a long (l) and a short (s) segment which are covalently linked [ ] , and contains a high density of genetic information with about open reading frames (orf) and encodes approximately polypeptides [ , ] , of which only are required for replication of the virus in cultured cells [ , ] . the viral genes are expressed in a cascade in groups classified as immediate early (ie, α), early (β), early late (γ ) and late (γ ) genes, each with a certain characteristic group of promoters regulating the sequential expression [ , ] . generally, the α-gene products are transcription inducers, the β-gene products are viral enzymes such as the thymidine kinase and the viral dna polymerase, and the products of γgenes are the structural proteins of the viral particle [ ] . the viral transcriptional chain is closed by some of the tegument proteins (e.g. vp /vmw ) which are γ-gene products with structural properties in the tegument of the viral particle and besides this harbour transcriptioninducing capacity upon α-gene promoters crucial in the induction of the next replication cycle of the virus [ , ] . the hsv infection is initiated by adsorption of the viral particle via gb or gc to a cellular receptor, which is a heparan sulphate chain on cellular proteoglycans [ ] . thus hsv adsorption can be inhibited by heparin and soluble heparan sulfates [ , ] . this initial binding, in which gc is important but dispensable, is of greater significance for hsv- than for hsv- , a divergence which could have implications for the different pathogenic patterns of the two strains [ , ] . furthermore, trapping of hsv to heparan sulfate motives in the tissues, e.g. basal laminas, may be of importance for containment of the infection at a specific site [ ] . binding to the heparan sulfate-containing cellular receptors, which are in size with the hsv particle itself, is reversible, and serves to concentrate the viral particle in near proximity to the cell ( fig. ) [ , , ] . a crucial step is then conducted by gd binding to an entry receptor, of which three classes has been described [ ] . these include herpes virus entry mediator (hvem), later designated as herpes virus entry protein a (hvea), which is a member of the tumour necrosis factor receptor family, nectin- (hvec) and nectin- (hveb), both members of the immunoglobulin superfamily, and heparan sulfate sites modified by -o-sulfotransferases [ ] [ ] [ ] [ ] . the differential use of these receptors is of importance for hsv entry of different cell types and infection of polarized cells [ ] [ ] [ ] [ ] [ ] , exemplified by nectin- , which is of importance in infection of the vaginal mucosa [ ] . upon binding to one of these entry receptors, conformational changes in gd lead to interaction with gb or gh-gl dimer, which results in membrane fusion by a mechanism not known in detail ( fig. ) [ , ] . the membrane fusion can take place both with the plasma membrane on the surface of the target cell and with an endosomal membrane after intraluminal ph-reduction, as it is seen for some other enveloped viruses [ , - ]. following these initial steps of infection several immunomodulatory cellular events are induced, but the potential importance of signalling through receptors involved in adsorption and membrane fusion is only scarcely analysed [ ] . the receptor molecule hvem is by its normal ligand capable of inducing activation of nuclear factor κb (nf-κb) and activation of t cells. by interaction with hsv-gd these receptor responses are inhibited. thus, the hsv interaction with at least one of its receptors has multiple potentials for modulation of the host response to the infection [ , ] . upon fusion, the hsv nucleocapsid is transported by microtubules to a nuclear membrane pore where the viral dna is released into the nucleus [ , ]. both viral tegument products and cellular kinases are responsible for the initiation of α-gene transcription [ ] . in these initial events the determination of whether it will lead to a lytic infection cycle or a latent infection seems to be directed largely by the infected cell type in question [ , ] . a key event in this seems to be early induction of latency-associated transcripts (lats) with sequences antisense to the infected cell protein null (icp ) and icp [ ] [ ] [ ] . in the hsv composition and entry figure hsv composition and entry. electron micrograph of negatively stained hsv particle with indications of major structural elements. important mediators of adsorption to cells ( ), receptor binding ( ) and fusion of membranes ( ) during the process of infection are drawn stylistically. initial phase of the lytic replication cycle, the ie-gene products, besides being transcription factors for the next wave of viral proteins, intimately regulate cellular functions in favour of viral replication and immune evasion [ , ] . of these, the icp , a promiscuous transactivator without much dna-binding capacity, forces the cell to a pre-dividing state optimal for viral protein synthesis [ , ] . furthermore, icp is active in inhibiting immune mechanisms such as interferon production and antiviral effects of interferons [ - ] and induces degradation of cellular proteins, involving the proteasome [ , ]. very early in infection, the first transcriptional activity is seen just inside the nuclear membrane at the site were the viral dna enters the nucleus [ ] . the produced icp colocalizes with the promyelocytic leukaemia (pml) nuclear bodies and initiates degradation of these, an event which seems to be important for productive replication of the virus [ , ] . icp binds to parental viral dna which is juxta-localized to the pml bodies, and later, when the bodies are degraded, replication compartments are formed, in which also icp can be found [ , , ] . icp affects the posttranscriptional polyadenylation and splicing of rna, and it is thus an element of the delayed host protein shutoff [ ] . immune evasion is additionally induced by the ie protein icp which binds to transporter associated with antigen processing, tap /tap and blocks the presentation of viral peptides by the major histocompatibility complex (mhc)-i [ ]. the hsv progeny is formed in the nucleus of the infected cell, where the viral dna is packed into preformed capsids. these are assembled with the tegument proteins and bud through the inner nuclear membrane to the perinuclear space [ ] . the route of virus from here to the external side of the cell is controversial. apparently two routes of viral egress are possible [ ] . one way is by continuous passage through vesicles and the golgi apparatus, where the membrane proteins are modified. the other route is by fusion of the newly acquired envelope with the outer nuclear membrane or the membrane of a vesicle, generating naked nucleocapsids in the cytoplasm. from here a new budding event should take place, for instance into the golgi apparatus. the progeny virus thus acquires the envelope from other membranes, than the inner nuclear lamella, as it is indicated by analysis of membrane lipids [ ] . increasing evidence is pointing at this latter possibility of de-envelopment and re-envelopment as the dominating route of hsv egress [ - ]. the progress of hsv infection in tissues is influenced by the capacity of hsv to infect adjacent cells directly through cell junctions. the virus is thus avoiding exposure to extracellular substances such as antibodies and complement. the glycoproteins ge and gi are crucial for this kind of polarised transmission which primarily takes place in epithelial infections [ , ] . as it is the case at the molecular level, the two herpes simplex viruses show similarities in their clinical appearance, both giving rise to primary infections of mucosal membranes and showing latency in sensory nerve ganglia [ ] . the primary infections with hsv are often asymptomatic, especially at young age, but in a minority of cases vesicular or ulcerative lesions are seen. although hsv- and - can give rise to indistinguishable clinical infections, there are differences in the anatomical distribution of these infections, as described in by dowdle et al. [ ] . hsv- is predominantly giving rise to infections above the waist, and hsv- to infections below the waist. this pattern is, however, not as straightforward as primarily described. in the last decades changes in both prevalence and distribution of hsv infections have been seen. the overall prevalence of hsv infection is very different in different countries and ethnic and social populations [ ] [ ] [ ] . a decline in hsv prevalence has been observed in the western countries, probably because of improved socioeconomic conditions [ ] [ ] [ ] . in parallel to the decline in prevalence, the aetiology of herpes genitalis has changed in several countries, presumably because of altered human habits and conditions of life [ ] . in some areas of the world the proportion of genital infections caused by hsv- is still low ( - %) the aetiology of a genital infection is not insignificant, in that the frequency of recurrence is higher in hsv type infected individuals than in those infected with type [ , ] . the frequency of primary and recurrent infections with both hsv- and - has been reported to be higher among women than men [ , , ] . overall, these epidemiological changes could have implications for the risk of neonatal infection from vaginal delivery, in that more women are seronegative at delivery and thus a higher number have the risk of caching a primary hsv infection. on the other hand, less hsv is circulating, reducing the risk of those who are susceptible. clinical appearance and pathogenesis as described above, primary infection with hsv is most often asymptomatic, especially in younger children [ ] . however, some individuals experience a symptomatic primary infection with vesicular herpetic gingivostomatitis or in adolescence more often a pharyngitis [ ] . as it is the case with orofacial infections, a primary genital hsv infection can be both asymptomatic and symptomatic with ulcerative lesions and with or without generalized symptoms such as fever, headache etc. [ , ] . rarely, the infection disseminates to one or several organs giving rise to infections such as necrotising hepatitis, meningitis, encephalitis or to disseminated intravascular coagulopathy [ ] [ ] [ ] [ ] . such a clinical course, although uncommon, is most often seen in immunosuppressed patients e.g. transplant patients, neonates or pregnant women [ ] [ ] [ ] . in pregnancy, primary infection with hsv without previous seroconversion at the time of delivery seems to be the main risk factor for infection of the newborn [ , ] . genital hsv reactivations at labour only seem to posses a minor risk for neonatal infection of the baby [ , ] , but in spite of this, approximately % of neonates infected are born by asymptomatic women [ ] . the amount of virus in vaginal secretions during reactivations is much lower than the amount of virus in primary infections, and in reactivated cases maternal antibodies furthermore seems to be protective for the neonate [ , [ ] [ ] [ ] [ ] . when transmitted, the course of hsv infection in the newborn varies. in the pre-acyclovir era about one third of cases were mucocutaneus infections only involving the skin, mouth and eyes, one third were infections of the central nervous system (cns) with or without mucocutaneus involvement, and the last third were disseminated infections involving multiple organs, including the liver, lungs, adrenals, and often the cns [ ] . of these, neonates with a generalized infection had a one-year mortality of approximately %, those with cns-infections had intermediary mortality, and nearly no mortality was seen in the group of patients with only mucocutaneus involvement [ ] . in infected with multi-organ involvement the deaths are often set off by infection of liver or lungs or by coagulopathy. sequelae, such as mental or neurological disabilities are seen in some of those with cns involvement [ ] . now a day, after initiation of high-dose acyclovir treatment, the mortality and sequela rates have dropped [ ] . the clinical pattern of neonatal hsv infections has changed in that less of the mucocutaneus infections disseminate to generalized infections when treated [ ] . even with high-dose acyclovir, improvements in treatment protocols are still needed, because the mortality is still as high as % in disseminated infections. reduction in the time from debut of symptoms to initiation of therapy is vital and passive immunotherapy with hsv-specific antibodies could posses a potential as adjuvant to the antiviral treatment [ , , ] . other adjuvant treatment modalities are still needed in both neonatal infections and in generalized infections at later ages. the pathology of hsv infections is mainly caused by a direct cytopathic effect of the virus, resulting in cellular lysis and focal necrosis of the infected area [ , , ] . in tissues capable of regeneration, this is not devastating, provided that the lesions do not totally destroy the organ or result in functional disability during the infection. in the brain, however, the capacity for regeneration is small, and larger necroses induced by viral infection will result in life-long sequelae [ , ] . a delicate balance exists between the direct hsv-induced pathology and the immunopathology induced by immune reactions to the virus and the toxic and functional side effects of these reactions [ ] . immunopathogenesis seems to be the main aspect of hsv stromal keratitis, which often leads to blindness [ , ] . the scarification from this infection has even been attributed to autoimmunity by molecular mimicry [ ] . weak immune response to the virus leads to severe infections because of massive viral replication and dissemination. an immense immune reaction, especially with high amounts of virus to trigger a response, can bring about increased symptoms of infection, local symptoms such as high intra-cerebral pressure or pulmonary complications, as well as generalized or septic symptoms [ , [ ] [ ] [ ] [ ] . it is thus clear that early control of hsv replication in the initial phases of infection is crucial for the host. early containment or at least inhibition of viral replication can prevent dissemination of the infection, and the early nonspecific immune reactions thus have the potential to inhibit development of a symptomatic infection. obviously the host will benefit from an attenuated or asymptomatic course of infection, but hsv -with the potential of subsequent reactivation from a latent site -could also benefit from such a course of infection, in that the host will survive and the activity of the host in society will not be hampered by symptoms from infection. thus, the hsv has excellent chances to reach new susceptible hosts which bring the virus and the host in a situation of mutual benefit [ ] . with phagocytic activity. in metchnikoff named them macrophages (large eaters) in contrast to microphages (the polymorphonuclear leukocytes) [ ] , and in aschoff defined the reticuloendothelial system by the criteria of uptake of vital dye [ ] . the macrophages are now more precisely defined as an important member of the mononuclear phagocyte system, defined in by van furth and colleagues [ ] . in the tissues they constitute a dynamic pool of cells with many functional capabilities, among which the capacity of phagocytosis, microbial killing, motility, and adherence to surfaces are classic [ ] . the macrophages originate from the bone marrow, where proliferating promonocytes give rise to monocytes which enter the blood stream [ ] . after a mean circulation time of approximately / day, the blood monocytes migrate to the tissues [ ] . in the tissues the monocytes differentiate into macrophages with characteristics determined by the environment of the tissue in question [ ] . the tissue macrophages in the major organs are represented by kupffer cells in the liver, alveolar and interstitial lung macrophages, spleenic and sinusoidal lymph node macrophages, microglia in the brain, osteoclasts in bone, and langerhans cells of the skin. thus, macrophages are strategically situated all over the body taking care of debris from the organism itself and foreign material, among others invading microorganisms, including viruses [ , ] . macrophages in different organs have different characteristics and functional capabilities and can not totally substitute one another in studies on macrophages [ , [ ] [ ] [ ] [ ] . likewise, macrophages from different species can possess differences in their functional capability, e.g. the capacity for nitric oxide (no) production [ , ] . macrophages in tissues are, as described above, in part originating directly from monocytes, but they are also in part originating from local proliferation. this local proliferation in the tissues is performed by newly recruited monocytes, and in the steady state situation they only constitute a small fraction of the mononuclear phagocytes present [ ] . of the monocytes produced in the bone marrow of mice and passing through the blood, approximately half are targeting the liver, % are going to the lungs, % to the spleen and % to the peritoneal cavity [ ] [ ] [ ] . in the lungs, % of tissue macrophages in the steady-state originate from monocyte influx and % from local proliferation [ ] . this proportion might vary between different tissues, as the lifespan of tissue macrophages in different organs also varies from around days in mouse spleen to approximately one month for alveolar macrophages [ , ] . in the skin, langerhans cells are a very stable and long-lived population of cells staying there for at least month in the steady-state situation. however, in inflammation the langerhans cells are within weeks replaced and supplemented by circulating mononuclear cells [ ] . when an inflammatory process is initiated, the dynamics of monocytes and macrophages are changed. monocytes and other white blood cells are produced and recruited from the bone marrow, and the white blood cell count in the circulation is increased. the monocytes are mainly passing through the blood to become tissue macrophages, and the number of macrophages in the inflamed tissue can be increased by more than ten times [ ] . in inflamed tissue the local proliferation of macrophages does not seem to increase, although the number of newly recruited cells is high, indicating that the differentiation of monocytes in the tissues is accelerated [ ] . the differentiation of monocytes and activation of macrophages have been a focus of interest for many years because of the observation that macrophage activation is crucial in the defence against many intracellular pathogens [ ] [ ] [ ] [ ] . it became clear relatively early that lymphocytes and soluble factors secreted by these (lymphokines) are important in activation of macrophages for killing of intracellular bacteria, e.g. listeria [ ] . in the killing of bacteria, interferon (ifn)-γ was shown to be an important stimulator of macrophage activation [ ] . as mechanisms in performance of the killing simple toxic substances of reactive oxygen species (ros) and nitric oxide were identified and seem to conduct their action in synergy [ ] [ ] [ ] . the toxic substances are chemically simple, but their production and regulation in macrophages are very complex and still a matter of intense studies [ ] . the state of the activated macrophage has changed conceptually from being viewed as one specific condition of the cell towards a more dynamic picture, provoked by the fact that macrophages activated by different means show different phenotypical characteristics [ , ] . the activated macrophage is now viewed as a cell with floating characteristics of many functional capacities regulated by a multitude of stimulating substances, such as the cytokine environment, hormones, and pathogenic and foreign substances [ , ] . among variables, controlling macrophage activity in infected individuals, are the genetic constitutions of the host. the genetic background has been shown to be of importance for the regulation of both basic proliferation and function of macrophages and for the more specific antimicrobial responses [ , ] . soluble mediators of lymphocyte activities were described as early as , but the first lymphokines/cytokines found and characterized were the type i interferons. soon after, many other soluble mediators of lymphocyte and monocyte/macrophage activities were found [ ] [ ] [ ] . the term lymphokine was introduced by dumonde et al. in , to describe lymphocyte derived factors, and the term monokine was used as a description of factors coming from the mononuclear phagocyte system, both acting on many cells, primarily leukocytes [ ] . because of a broader view on origin and function of these factors, the term cytokine is now more often used. each cytokine was originally named according to biological activity in a functional assay, which often gave several different names to one cytokine, and thus confusion at the molecular level. to straighten this out, a numerical nomenclature of interleukins (between leukocytes) was introduced in [ ] . this numbering system has clarified the field, but since it has no mnemonic functional anchorage it has drawn critique since then [ ] [ ] [ ] . the cytokines are generally smaller proteins, some composed of two subunits, utilizing specific receptors on target cells for induction of their functional effects. they are structurally related in three families, with the prototypes being il- , il- and il- [ ] . functionally, cytokines are highly potent regulatory proteins acting in a paracrine or autocrine manner at picomolar concentrations [ ] . the cytokine receptors are also structurally clustered in families, and functionally utilize a battery of overlapping kinases and nuclear binding proteins in their signalling pathway and thus have overlapping functions [ ] . the final functional capacity of the effector cell thus reflects the cytokine environment experienced by the cell [ ] . thus the cytokines comprise a network of factors inducing or inhibiting each others secretion and function in different cells, giving rise to a constantly floating landscape of a large array of functional capacities [ ] . in the early hours of a viral infection, the cytokines produced by cells infected or coming into contact with viral products are vital in conduction of the innate immune response to the infection [ , ] . the interferons (ifns) were described and named in by isaacs and lindenmann [ ] , who characterized the substances involved in the previously described interference of one virus with the replication of another unrelated virus, and the interfering activity of inactivated influenza virus with the subsequent infection of chorioallantoic membranes [ ] [ ] [ ] . the ifns were the first cytokines described in detail, and thus provided the fundamental basis for the understanding of the cytokine concept [ ] . the ifns are divided into three major groups. the two original groups of ifns are designated type i and type ii, type i being the so called non-immune ifn, and type ii the immune ifn. type ii (ifn-γ) is produced in high amounts as part of a specific immune reaction, whereas the type i ifns can be produced by many cell types in response to, in immunological terms, non-spe-cific stimulation. the many functions of ifns and the growing understanding of signalling and regulation indicate that ifn analogues may play a major role in the next generation of new antiviral compounds [ ] . the type i ifns are a diverse group of cytokines, consisting of ifn-α, ifn-β, ifn-ε, ifn-κ, ifn-ω, ifn-δ, ifn-τ, and ifn-ξ/limitin [ , ] . the first five of these are expressed in humans, and their relative production depends on the stimulus and the cell type in question. the ifn-α family consists of multiple species and some of these in different allelic forms in both humans and mice. in humans ifn-α genes and one pseudogene and in mice ifn-α genes and pseudogenes have been identified, clustering on chromosome in mouse and chromosome in man [ ] . the functional importance of such a diversity is largely unknown. the subtypes differ in potency and have previously been shown to vary in their profile of activities [ , ] , but new studies show correlation between antiproliferative and antiviral effects of various ifn-α species [ ] . thus, it seems that the importance of the diversity could come from varying expression patterns of the different ifn-α species. most of the α ifns are n-glycosylated, but glycosylation does not correlate with activity of the molecule, but rather with in vivo stability, and recombinant ifns are shown to have activity comparable with that of the naturally produced molecules [ , ] . only one ifn-β species exists, coded by a gene situated in the ifn type i cluster on chromosome in mouse and chromosome in man, as described above [ ] . the natural ifn-α and -β have a molecular weight of - kda and most species retain stability at ph [ ] . all type i ifns bind to one common receptor composed of two subunits, ifn-α-receptor(r) and ifn-αr . the ifnα/β receptor (ifnar) signal through the jak/stat-pathway by phosphorylation of the janus kinase (jak) , tyrosine kinase (tyk) , signal transducer and activator of transcription (stat) and stat , and induces genes with an ifn-stimulated response element (isre) in their promoter [ , ] . generally the type i ifns exhibit a huge range of biological effects, such as antiviral and antiproliferative effects, stimulation of immune cells such as t cells, natural killer (nk) cells, monocytes, macrophages, and dendritic cells, increased expression of mhc-i, activation of pro-apoptotic genes and inhibition of anti-apoptotic mechanisms, modulation of cellular differentiation, and inhibition of angiogenesis [ ] . the newly discovered ifn-ξ/limitin also interacts with the ifn-α/β receptor, and is regarded as a type i ifn [ , ] . antiviral activity of ifn-ξ has been shown against many viruses including hsv, and it exhibits both immunomodulatory and anti-tumour effects, but the lymphosuppressive activity is less than that of ifn-α [ , ] . a human homolog of ifn-ξ could thus have interesting potential in the therapy of tumours and viral infections. the type ii ifn is represented by only one member, the ifn-γ [ ] . structurally, ifn-γ is distinct from the type i ifns, and it signals through a different receptor. for many years ifn-γ was thought only to be expressed by t cells. later the large granular lymphocytes (nk cells) were recognised as important producers by the fact that ia-antigen (mhc-ii) expression on mouse macrophages could be induced by listeria monocytogenes infection in scid mice lacking t cells [ ] [ ] [ ] . in recent years it has, however, been clear that other cell types, originally thought not to be producers of ifn-γ, are in fact capable of ifn-γ expression. so now macrophages, b cells, nkt cells and professional antigen-presenting cells are also recognized as ifnγ producers in certain situations [ ] [ ] [ ] [ ] [ ] [ ] . induction and production of ifn-γ in antigen-presenting cells and nk cells seem to be vital in the early non-specific response to infections and of importance in the linkage to the adaptive specific responses coming up later [ ] [ ] [ ] . the induction of ifn-γ production in non-t cells (e.g. nk cells) is conducted by cytokines, especially il- in synergy with other proinflammatory cytokines, largely produced by mononuclear phagocytes [ , ] . ifn-γ exerts its effects through a distinct class ii cytokine receptor, the ifn-γ receptor (ifngr), composed of two subunits, ifn-γr and ifn-γr . upon binding of a homodimer of ifn-γ to the receptor complex, jak autophosphorylates and then transphosphorylates jak . activated jak in turn phosphorylates ifn-γr , which allows binding of the stat homodimer to the receptor and subsequent phosphorylation of stat [ ] . the ifngr and stat are preformed as hetero-and homo-dimers, and upon receptor binding, the ifn-γ-ifn-γr -stat complex seems to be internalized and translocated to the nucleus, where the activated stat homodimer binds to dna at gas elements and induces the first wave of responses [ , [ ] [ ] [ ] [ ] [ ] . many of these initial ifn-γ induced products are transcription factors participating in further regulation of the many ifn-induced cellular response. among these products are the ifn regulatory factors (irfs) which stimulate or inhibit transcription of genes possessing an isre in the promoter region [ , ] . for many years the key mediator of macrophage activation during antigen-induced processes was recognised as macrophage activating factor (maf) [ ] . only later, the crucial importance of these effects was attributed to ifn-γ [ , ] . ifn-γ has antiviral activity, but the most important effects of ifn-γ seem to be activation of macro-phages, antigen-presenting cells, and nk cells and inhibition of t-helper type (th ) cells, resulting in a th driven cell-mediated response to infection [ ] . experiments in knock out (ko) mice with deficient ifn-γ, ifngr, or stat expression have shown that this system is of major importance, but not vital, in the host response to viral infections [ ] [ ] [ ] [ ] . besides the two traditional groups of ifns, a new group of ifn-like cytokines has been described in various species and named il- a (ifn-λ ), il- b (ifn-λ ), and il- (ifn-λ ) [ , ] . these cytokines are antiviral proteins interacting with a distinct heterodimeric class ii cytokine receptor composed of ifn-λr and il- r , but sharing with the type i ifns some intracellular signalling pathways through the isre [ ] . thus, they have a largely similar antiviral effect as the type i ifns [ ] . tumour necrosis factor (tnf, former designated tnf-α) and lymphotoxin (lt; former tnf-β) were for many years also known as cachectin from their involvement in cachexia of cancer patients [ ] . tnf is a prototype and the second member of the tnf ligand superfamily (tnfsf ), now encompassing over known signalling molecules, among which the ltα, ltβ, and light (ltlike, exhibits inducible expression, and competes with hsv glycoprotein d for hvem, a receptor expressed by t lymphocytes) are some of the more prominent ligands [ , ] . each member is the ligand of one or two distinct receptors of the tnf receptor family sharing a high degree of homology. the current nomenclature of these ligands and receptors has now been gathered on the internet [ ] . tnf is a type ii transmembrane glycoprotein coded from the human chromosome and from chromosome in mice [ ] . it is synthesized as a kda transmembrane pro-tnf, primarily located in the membranes of the golgi apparatus [ ] . the pro-tnf is cleaved by a metalloprotease releasing the kda extracellular portion of the molecule [ , ] . production and release of tnf from the cell is regulated at both the transcriptional and translational level and by post translational modification as described above [ ] . during hsv infection both preand post-transcriptional regulatory mechanisms are involved in tnf production [ ] . tnf is produced by many cell types of immune origin, primarily mononuclear phagocytes, neutrophils, nk cells and t cells, and has diverse effects on different cells [ ] . both membrane bound and soluble tnf interact as homotrimers with two different receptors, the p tnfr (tnfrsf a) and the p tnfr (tnfrsf b) [ ] . as most other receptors of this family, tnfr holds a death domain important in the pro-apoptotic pathway. tnfr is expressed virtually on every cell type except erythrocytes, whereas tnfr is mostly expressed on endothelial and bone marrow derived cells [ ] . the tnfr activates nf-κb (p , p /rela, and p /relb) by ubiquitin-mediated degradation of inhibitor-κb (iκb) after phosphorylation by an iκb kinase (ikk). besides inducing apoptosis, tnfr also activates nf-κb (p / p ) [ , ] . furthermore, the activator protein (ap- ) is activated by mitogen-activated protein kinases (mapks) and together with nf-κb primarily acts in the proinflammatory pathways. thus, signalling from the tnf receptor family induces a delicate balance between life and death (apoptosis) of the cell. both of the tnf receptors can by proteolytic cleavage be converted to soluble receptors with the capacity to compete with their signalling ancestors, but also act to stabilize the trimeric tnf and thus maintain its activity [ , ] . the tnf superfamily seems to have evolved with the adaptive immune system in vertebrates and is crucial for the embryonic development of lymphoid tissue [ ] . furthermore, tnf is, as a proinflammatory cytokine, involved in activation of many immune cells and is thus an important factor of both the early non-specific and the specific immune response [ ] . the importance of the tnf superfamily in antiviral defence is illustrated by the fact that different viruses have developed mechanisms for interference with nearly every step of activity of this system [ , ] . il- is the prime member of a small group of heterodimeric cytokines, all with the capacity to induce production of ifn-γ in a variety of cells. il- was first described as an nk cell stimulatory factor (nksf) and identified as a heterodimeric molecule composed of a p and a p subunit, which are covalently linked [ ] . the p subunit has homologies to il- , and p is homologous to the extracellular domain of the haematopoietin receptor family, particularly the il- rα chain [ ] . the two il- subunits are coded from different chromosomes, i.e. the human chromosomes and and the mouse chromosomes and , respectively [ ] . these genes are regulated separately, and coordinated induction in the same cell is required for secretion of the biologically active il- p heterodimer [ ] . il- is produced by monocytes, macrophages, dendritic cells, neutrophils and b cells [ , ] . in the initial response of spleen cells in mice injected in vivo with extracts of toxoplasma gondii or with lipopolysaccharide (lps), the cellular source was found to be dendritic cells, but cultured macrophages have by themselves also been shown to produce il- p upon hsv- infection [ , ] . such differences could depend on variations in the signalling mechanisms involved, which is also illustrated by the observation that the production in dendritic cells and macrophages has dif-ferent kinetics. this difference could be brought about by differences in the requirement for co-stimulation with ifn-γ [ ] . a collaborative action of dendritic cells and macrophages could be important, as indicated for il- induction by influenza virus and other inducers [ ] . the receptor for il- is found on nk cells, t cells and dendritic cells and consists of two subunits (β and β ), which signal by the β subunit through the jak/stat pathway, primarily by activated stat [ ] . the primary effect of il- is induction of ifn-γ production in nk cells and t cells, and il- activates the cytotoxic potential of these cells. the ifn-γ locus in nk cells is constitutively demethylated and is thus ready for transcription of the gene, which is in contrast to that of t cells, [ ] . macrophages and nk cells are then stimulated by ifn-γ, resulting in activation for enhanced antimicrobial capacity [ , ] . il- and ifn-γ in conjunction are the main responsible factors for activation of a th -driven adaptive cellular immune response, important for the long-term control of intracellular pathogens [ ] . il- stimulates proliferation of naïve t cells, and in conjunction with ifnγ inhibits th cell differentiation and the production of th cytokines (e.g. il- , il- , and il- ) [ ] . thus il- holds a key position in induction and control of the th response. the il- -induced ifn-γ production is synergistically enhanced by other cytokines such as tnf and il- [ ] , and ifn-γ production can even be induced in macrophages by co-stimulation with il- [ , , ], a cytokine which by itself does not possess major ifn-γinducing capacity [ ] . a positive feed-back loop is initiated by the il- -induced production of ifn-γ, in that ifn-γ is an important primer of il- production, thus accelerating the system [ ]. furthermore, t cells enhance il- production through signals of the proinflammatory tnf family [ ] . in virus-infected macrophages a similar autocrine feed-back loop involving il- , il- , ifn-α/β, and ifn-γ could be speculated [ ] . this potentially harmful situation, with accelerating ifnγ production, regulated in a positive feed-back loop by il- , is inhibited by cytokines possessing anti-inflammatory properties. among these il- holds a crucial position as an inhibitor of il- production, an effect which is also conducted by transforming growth factor-β (tgf-β) [ ] [ ] [ ] .the th cytokines of the other side of the adaptive response, il- and il- , inhibit il- induction in the early phases of stimulation, but later they can be potent inducers of il- production, although they still inhibit many of the ifn-γ-induced activities [ , , ] . phagocytosis of apoptotic cells by macrophages inhibits production of il- , a regulatory mechanism which seems to be important in restriction of the damages induced by uncontrolled defence mechanisms [ ] . injection of high doses of il- to virus-infected mice is toxic, and leads to death with the pathology of tnf-related toxic shock, an effect which was explained by increased sensitivity to the toxic effects of tnf, and found to be dependent on the genetic constitution of the host [ , ] . the small il- cytokine family also includes two other heterodimeric cytokines, il- and il- , and a homodimer of il- p . the latter is found in vivo in mice and functions as an antagonist of il- , but it is debated whether it exists in humans [ , ] . il- is composed of the il- p and a p subunit and likewise binds to a receptor with one of the il- receptor subunits (il- rβ ) and a distinct il- r subunit [ , ] . the production and function of il- is quite similar to that of il- , but il- has a unique capacity to induce proliferation of memory t cells [ ] , and it has been found in nervous ganglia of hsv-infected mice on day of infection [ ] . il- drives il- production of nk cells, which mobilizes neutrophils and promotes production of the proinflammatory cytokines il- , il- , and tnf [ ] . il- is the newest recognized member of the family, constructed of two distinct subunits (ebi and p ), but still with functional capacities alike those of il- [ ] . the functional implications of these later discovered members of the il- family is not yet clear, but it seems as if they are contributors to the overall effects of the il- family and fine-tune the system [ , [ ] [ ] [ ] [ ] . the induction of ifn-γ and activation of nk cells is not only mastered by members of the il- cytokine family. other cytokines, like il- , are also implicated in development, function, and activation of these cells [ , ] . generally, the il- cytokine family has shown itself of importance in early defence against several viral infections, and as a vital inducer and regulator of the adaptive immune response against viruses and other intracellular pathogens [ , , , ] . upon an accelerating pro-inflammatory response induced by initial viral replication the organism has to embank the ifn-γ-activated potentially harmful actions of macrophages and nk cells. important mediators of this embankment are il- and il- , which as described above repress the induction of il- , and thus put a brake on the positive feed-back loop of ifn-γ production [ , ] . furthermore, il- suppresses the production of other proinflammatory cytokines such as tnf and il- [ ] . most importantly, il- and il- are potent inhibitors of the efferent arm of the pro-inflammatory system, and thus inhibit production of reactive oxygen species and nitric oxide. the production of these two potentially harmful effector mechanisms of activated macrophages is hampered by inhibition of production of the responsible enzymes in these reactions, the nadph oxidase and the inducible nitric oxide synthase (inos) [ ] [ ] [ ] . the primary producer cells of il- and il- are the th cells, but these cytokines are also produced by basophils and mast cells [ ] [ ] [ ] . the receptors for il- and il- are expressed on most cells and are composed as dimers of four different chains. il- is the ligand of two receptors: a high-affinity heterodimer of il- rα and the il- r common γ-chain and another heterodimeric receptor composed of il- rα and il- rα . il- binds to three complexes: a high-affinity heterodimer of il- rα and il- rα and two homodimers composed of either il- rα or il- rα , which are both coded from genes on the human x-chromosome [ ] . the immunomodulatory signalling is conducted through the jak/stat-pathway utilizing jak , jak and stat . phosphorylated and homodimerized stat binds to stat binding elements (sbe), which includes gas, and either trans-activates or inhibits transcription of the adjacent genes [ ] . the functions of il- and il- are nearly overlapping with only discreet discrepancies [ , ] . il- was discovered in on the basis of another important effect of the cytokine, namely the ability to induce proliferation of b cells, and it was from this effect in the early years called b cell growth factor [ ] . as this, some other effects of il- are stimulating, in that it furthermore activates other th -like effects such as b cell class-switching and expression of mannose receptor and fc receptor for ige on macrophages [ ] . despite the anti-inflammatory profile il- has in vivo been shown to confer some resistance to hsv infection [ , ] . il- is thus not only an inhibiting cytokine but essentially an immunomodulatory cytokine with regulatory effects on macrophages as well. the early innate defence mechanisms have for many years been regarded as important for the course of many viral infections, including infections with hsv [ ] . the control of viral replication and dissemination during the first days of an hsv infection seems to be vital for the final outcome. if the viral replication is not halted by natural defence mechanisms during induction and maturation of the antigen-specific immune response, the adaptive immune system can be overwhelmed by massive viral infection at the dawn of activity of the specific reactions. the mechanisms of the anti-herpetic natural defence have been analysed extensively. it became relatively early clear that antiviral activity of macrophages [ ] and nk cells [ ] and early activity of the ifn-system [ ] were important mediators of innate resistance to hsv. the relative contribution of each of these players in the early defence has been much debated, and as more interactions and molecular mechanisms are now elucidated, it seems clear that all of these players each hold a crucial position in an integrated antiviral natural defence system. an important model used in the study of resistance mechanisms in defence against generalized infection with hsv is a mouse model, where mice infected intra-peritoneally or intra-venously experience a generalized infection with hsv replication in most organs, including the liver, spleen, and eventually the brain [ ] . the dissemination of infection to the brain and the severity of infection of the peripheral organs depend in part on the age of the mice, as is the case in humans, where neonates have difficulties in controlling a hsv infection [ , [ ] [ ] [ ] . the course of infection in mice also depends on the type of hsv in question. furthermore, in lopez described a differential susceptibility of inbred mice to generalized infection with hsv, and this genetic difference in sensitivity has since been used for analysis of resistance factors of importance for the anti-herpetic defence [ ] . in generalized infections, the genetics of the relative resistance to hsv- was shown to segregate with the x-chromosome [ ] . this pattern of resistance to the generalized infection was for both hsv- and - attributed to a genetically determined difference in the capacity for ifn-α/β production [ , , ] , and it was shown that the x-linked pattern of resistance segregated with the hsv- -induced production of ifn-α/β in macrophages during the first hours of infection [ ] . furthermore, macrophages from female mice respond to hsv with higher ifn-α/β production than macrophages from male mice [ ] . this observation is in line with female mice being more resistant to hsv infection in vivo [ ] . early production of ifn-α/β has been correlated to resistance of hsv infections in several other studies. treatment of mice with antibodies to ifn-α/β increases and accelerates mortality of a generalized hsv- infection and with higher doses of virus, mice are dying already after three to four days, a period where antigen-specific mechanisms are still in the induction and proliferation phase [ ] . furthermore, mice treated with mercuric chloride showed higher titres of hsv- in the first days of infection, an effect which could be correlated to impaired production of ifn-α/β [ , ] . in studies on peripheral hsv infections, such as cutaneous or corneal infections, ifn-α/β has been shown to be produced locally and to restrict the local replication of hsv and infection of nervous ganglia cells of the area, an effect which has also been correlated to the genetic constitution of the host [ ] [ ] [ ] [ ] . the genetic background for the x-linked trait of hsv resistance and ifn-α/β production of macrophages remains unravelled. induction of ifn-α/β upon hsv infection seems to be governed by different mechanisms in different cells [ ] . ifn-α/β can be induced early by both infectious and uv-inactivated hsv in various cells, with the infectious virus being the more potent inducer in mouse peritoneal macrophages, whereas the uv-inactivated virus showed most potency in human peripheral blood mononuclear cells (pbmc) [ , [ ] [ ] [ ] [ ] . production of ifn-α/β was induced by gd of hsv- in pbmc, but not in murine macrophages [ , , ] . in pbmcderived dendritic cells, however, the cellular mannose receptor was shown to be involved [ , ] . furthermore, different toll-like receptors (tlrs) have been shown to react with hsv [ ] . tlrs are transmembrane pattern recognition receptors (prrs) that detect redundant microbial molecular motives and induce antiviral and proinflammatory cytokines in response to alerting signals. in dendritic cells, tlr -signalling, induced by the gc-rich hsv genome, has been shown to govern the induction of ifn-α/β, but tlr -ko mice are still capable of controlling hsv infections in vivo [ ] [ ] [ ] . however, in mouse macrophages the tlrs do not seem to be crucial for ifn-α/β induction upon hsv infection [ ] . this is in agreement with the observation that the majority of ifn-α/β produced by spleen cells and dendritic cells and the total production from bone marrow macrophages was independent of tlr or myd , which is necessary for signalling by most tlrs [ ] . in this study, heat inactivated virus was shown still to induce ifn-α/β in cells utilizing tlr . as resident peritoneal macrophages do not produce ifn-α/β in response to even high doses of heat inactivated hsv, this gives an additional indication of independency from tlr of ifn-α/β production in macrophages [ ] . moreover, efficient induction of ifn-α/β by hsv in macrophages required dsrna-activated protein kinase (pkr) activity and infectivity of the virus [ ] . this is in agreement with the observation that dsrna, which is produced by most viruses during replication, induces ifn through pkr, and not through tlr , which also binds dsrna [ ] . furthermore, another mechanism of ifn induction by dsrna through a rna helicase has been proposed [ ] . the different induction patterns in different cells types, and the fact that ifn-α/β seems largely to be induced by other mechanisms than tlrs, explain the fact that knocking out tlr-signalling by myd did not influence the in vivo infection with hsv in mice [ ] . other tlrs have also been shown to mediate signals in hsv infections. in hsv encephalitis in tlr -ko mice, viral replication seemed unchanged or slightly increased during the first days of infection, and the production of il- and monocyte chemoattractant protein were impaired, but interestingly pathological changes and mortality were reduced [ ] . in relation to the x-linked resistance pattern of hsv infection and ifn production upon hsv infection, it is interesting that some of the tlrs are coded from the xchromosome [ ] . these are the tlr and tlr , which are triggered by guanosine-or uridine-rich ssrna in the endosomal compartment of cells [ , ] . there are, however, no indications that this pathway is implicated in ifn induction in cells during hsv infection, but the question has still not been directly addressed. regulation of the ifn-α/β gene induction is in part governed by activation of the transcription factors irf- and - , which are induced by ifn-α/β itself, resulting in a positive feed back loop, an effect which has been known for years without knowledge of the signalling mechanisms [ ] [ ] [ ] . thus, one possible explanation for the genetic differences in hsv-induced ifn-α/β production could be an elevated physiological level of this ifn self-stimulating system [ ] [ ] [ ] [ ] . an analysis of the levels of irf- and - in normal macrophages from these mice could be of interest. analysis of the levels of the ifn-induced enzyme '- '-oligoadenylate synthetase (oas) in uninfected cells showed low but slightly higher levels in cells from relatively resistant mice [ ] . with lps, cells from the relatively resistant (c bl/ ) mice show an early induction pattern of ifn-α/β, peaking within hours, whereas cells from the susceptible balb/c mice demonstrate a delayed response, peaking hours after induction [ ] . among other transcription factors involved in induction of the various ifn-α/β genes are the heterodimeric nf-κb family, which is activated by tlrs, il- r, and tnfr [ ] . during a hsv infection nf-κb is activated and translocated to the nucleus [ ] . many regulatory mechanisms of nf-κb activation exist, one of them exerted through tnf, which is produced by macrophages very early during hsv infection ( fig. ) [ , , , ] . thus, the responsible mechanisms might be exerted by other regulatory signals, influencing the magnitude of the hsv-triggered ifn-α/β induction pathway, and perhaps not by this pathway in itself [ ] . a number of x-linked immunodeficiencies have been described, one of them being the wiskott-aldrich syndrome with defects in a protein expressed in haematopoietic cells, facilitating reorganization of the actin cytoskeleton, and thus influencing the mobility of immune cells and chemotaxis of macrophages. patients with this x-linked immunodeficiency show aggravated herpetic infections, and cells from some patients seem to produce lower amounts of ifn in response to hsv [ ] [ ] [ ] . cells from patients with another x-linked immunodeficiency with mutations in the cd -ligand, a member of the tnf family, showed decreased ifn-α/β production when infected with hsv- , but these patients apparently show a normal response to viral infections [ , ] . this supports the notion above that other regulatory signals might be involved. the overall effects of the ifn-α/β system, besides the production as described above, are determined by the sensitivity of cells to the secreted ifn-α/β. the effector mechanisms of ifn-α/β on hsv replication are not fully elucidated. several ifn-α/β-activated systems are involved, including the dsrna-activated pkr, which phosphorylates, and thereby inhibits, the elongation initiation factor (eif)- α, resulting in inhibition of translation [ ] . another important mediator of the antiviral activity is the oas system, which activates '- 'oligoadenylate-dependent rnase l with the capacity to degrade single-stranded rna [ ] . lately, the pml bodies have been described as crucial for the anti-hsv effect of ifn-α/ β [ ] . in mice exhibiting a relatively hsv-resistant phenotype, the direct antiviral effect of ifn-α/β in embryonic cells was found to be approximately three-fold higher than in cells from susceptible mice [ ] . data from another study showed comparable results on ifn-α/β sensitivity concerning the replication of encephalomyocarditis virus (emcv) in cells from the same mouse strains [ ] . this phenomenon was inherited as a co-dominant autosomal trait without any apparent influence of x-linked genes [ , ] . further studies in mouse fibroblasts have revealed that tnf intensify the antiviral effect of ifn-α/β and, thus, the in vivo situation seems more complicated ( fig. ) [ , ] . in the original publication on genetics of hsv susceptibility in inbred mice, lopez reported fibroblasts from the different mice to replicate hsv equally, and the same was found in the cells showing differential sensitivity to ifn-α/β [ , ] . in line with these results, the ifn-activated oas, an inhibitor of hsv replication, was induced to a higher degree in cells from the resistant mice upon ifn-α/β treatment [ , , ] . furthermore, the level of stimulated and unstimulated oas was generally found to vary between different inbred mouse strains [ ] . thus, the genetic difference in antiviral action of type i ifns seems to affect the replication of several different viruses and to correlate with resistance to hsv. the viral host protein synthesis shutoff, exerted by the hsv vhs-protein of the tegument, has major effects on the cytokine production of infected cells and reduces the effect of ifn-α/β on hsv replication [ ] . furthermore, the tegument proteins have been shown to induce cellular inhibitors of the jak/stat pathway, resulting in inhibition of both ifn signalling and production [ , ] . the ie protein icp inhibits activation of irf- and thereby also restricts ifn-induced pathways [ - ], and icp , icp and icp induce late shutoff of protein synthesis with decreased mrna stability and thus reduced cytokine production [ , ]. as outlined, it thus seems hsv has evolved several mechanisms to evade the consequences of the ifn-α/β system, which underline the importance of these cytokines in the antiviral defence. during hsv infection macrophages are activated and possess an increased antiviral potential [ , ] . classically, the macrophage antiviral activity has been described as intrinsic or extrinsic [ ] . resting macrophages possess a high degree of intrinsic activity against hsv, generally being non-permissive to viral replication. the macrophages are thus a blind end for the hsv infection, and they can in that way protect other cells from infection, for example as a barrier lining the liver sinusoids [ ] . the extrinsic antiviral activity refers to the ability of macrophages to inactivate virus outside the macrophage itself or to inhibit viral replication in other cells [ ] . the intrinsic antiviral activity depends among other factors on macrophage differentiation and has been correlated to ifn activity, either physiological levels of "spontaneous" preinfection-synthesized or rapidly acting autocrine ifn-α/β [ ] . in that respect, macrophages from mice of the resistant phenotype showed higher intrinsic activity by being less permissive to hsv replication [ , ] . one potential antiviral mechanism of macrophages may be the production of ros. these were originally assigned to bacterial killing, but the effect of ros has also been correlated to antiviral functions, although they might not be of major importance [ ] . the ros are mainly produced by nadph-oxidases (nox), which are membrane-bound multi-component enzymes primarily situated in the phagolysosome [ ] . activation of the nahpd-oxidase, by phosphorylation and fusion of the enzyme subunits, primarily results in production of superoxide anion (o -), which by superoxide dismutase can be converted to hydrogen peroxide (h o ). the h o in turn is then by fe + (fenton reaction) or by fe + and o -(haber-weiss reaction) converted to hydroxyl radical (·oh), hydroxyl anion (oh -) and singlet oxygen ( o ), or by the myeloperoxidase to hypochlorous acid (hocl) [ , ] . small amounts of ros are also produced by the mitochondria and may be of importance as signalling molecules from tnf [ , ] . during hsv infection in vivo, macrophages are activated and achieve an increased capacity to react with a respiratory burst of ros when appropriately triggered, i.e. by phorbol esters (fig. ) [ ] . this macrophage activation is induced early in response to hsv infection, reaching a plateau within the first hours of i.p. infection [ ] . in vitro, macrophages were shown to be the cell type responding with an oxidative burst, and this capacity peaked after only hours of infection with hsv [ ] . this hsv-induced capacity for an increased respiratory burst was shown to be governed by autocrine ifn-α/β as a sine qua non phenomenon [ , ] . nevertheless, tnf was also found to influence the macrophage activation. by itself, tnf reduced the macrophage capacity for a respiratory burst, but in combination with ifn-α/β it synergistically enhanced the ifn-induced activation [ , ] . interestingly, a secreted portion of the hsv-gg acts as a phagocyte chemoattractant and induces production of ros by signalling through the receptor activated by the phorbol esters [ ] . the hsv-induced activation of macrophages in vivo is influenced by the genetic constitution of the host, with the most pronounced activation of macrophages originating from resistant mice, as expected on the basis of the genetics of ifn-α/β production in response to the infection. furthermore, the genetics of the efferent part of the ifn-α/β-mediated hsv-induced activation of macrophages, displayed a co-dominant autosomal trait, as was the case with the antiviral effect of ifn-α/β in fibroblasts [ ] . thus, the genetically-determined sensitivity to ifnα/β seems to be expressed in different cell-types. the influence of tnf on the genetics of this phenomenon has not been addressed. in contrast to these observations, the genetics concerning the antiproliferative effect of ifn-α/β in bone marrow cells seems to be reversed [ , ] . this might, however, be linked, in that ros are shown to activate various signalling molecules, mediate apoptosis, and exhibit antiproliferative effects depending on the dose and time of exposure [ ] . little is known on the potential antiviral effect of ros. by examining peroxidized lipids, which is an oxidative product from ros in tissues, it has been documented that these are produced during the acute hsv infection in vivo, and speculations on antiviral mechanisms have focused on induction of apoptosis [ , , ] . hsv triggers apoptosis of infected cells by several pathways, and the importance of this phenomenon is indicated by the fact that the virus has evolved mechanisms to counteract each of these pathways [ ] . macrophages generally suppress apoptosis in hsv infections, as seen by increased apoptosis in macrophage-depleted mice [ ] . several studies on the mechanisms involved in the early battle against hsv, performed in in vivo animal models, have pointed to ifn-α/β as a crucial player. in adoptive transfer experiments, the effect of adult mouse spleen cells on the initial phase of a generalized hsv infection in suckling mice was conducted by ifn-α/β [ ] . furthermore, administration of a hematopoetic growth factor to neonatal mice increases the number of dendritic cells, b cells and nk cells, and confers resistance in a cutaneous model of hsv infection. the effect in this model could also largely be attributed to the actions of ifn-α/β, with some additional contributions by ifn-γ [ , ] . in ko-mice ifn-α/β was able to control the initial phase of a generalized hsv infection without contributions from nk, t-or b cells, but these latter players were necessary for survival and long term control of the infection [ ] . the importance of an early, local ifn-response in models including in vivo progression and evaluation of final outcome of infection is more unclear, in that many other viral and host factors are of importance in these more complicated models with several stages of infection and involvement of different organs. such models are, however, more close to the normal human hsv infection, starting at an epithelial surface, but to expect that one resistance factor in such a complicated system will come out clear as the responsible factor for the outcome downstream the sequence of events, is too simplistic. nevertheless, induced expression of ifn-α/β in the eye by plasmid dna or an adenovirus vector was shown to inhibit early local replication of hsv and the concomitant spread of virus to the brain and death from encephalitis [ , ] , and in ifn-α/βr ko-mice hsv replicated to much higher titres than in normal mice [ ] . this tells us that the innate and adaptive immune systems exhibit much redundancy, and that ifn-α/β is of vital importance in local inhibition of hsv replication. the multitude of antiviral mechanisms, be it innate or adaptive, have varying effects and importance in the different phases of infection, such as initial local infection, dissemination to other organs, establishment of latency and reactivation, and conclusions can not be drawn from one situation to another. the reactions discussed above, involving production of ifn-α/β and tnf, take place within the first to hours of a hsv infection, and thus are reactions, which can execute an effect within the first replication cycle of the virus. a little later, other cytokines such as il- , il- and ifn-γ are produced and give rise to other weapons in the battle against the virus. they will, in turn, within the next replication cycle execute their actions, with potential harmful consequences for either parts of the conflict. a few hours after the type i ifn and tnf response, macrophages react upon hsv infection with production of il- , which is seen from to hours after infection and on [ , ] . the same was found with other viruses to hours after infection [ ] . in these and other studies, the producers of il- p during viral infection seem to be inflammatory cells, including macrophages, and not the infected stromal cells [ , ] . the il- induction during hsv infection requires infectious virus, and it was shown to be regulated at the transcriptional level [ ] , as it is also the case when it is induced by lps [ , ] . the dependence on infectivity is, however, in conflict with results from in vivo production of il- p and ifn-γ in draining lymph nodes from sites injected with uv-inactivated hsv [ ] . high doses of uv-inactivated virus were used, and some minimal transcription of viral genes could have taken place, although the virus was not replication competent. transcription of the il- p gene in macrophages requires de novo protein synthesis during the inducing hsv infection, which could explain the relatively late appearance of il- production [ , ] . the κb-sequence of the il- p promoter binds nf-κb in hsv-infected cells, and the production of il- p was found to be repressed by an inhibitor of nf-κb activation [ ] . both these observations indicate that signalling through nf-κb is of significance in hsv-induced il- production. in human macrophages, tnf has been shown to inhibit il- p production, but not p production, by a mechanism not involving nf-κb [ ] . furthermore, il- has in a mouse model been shown to stimulate tnf expression [ ] , indicating that tnf can participate in a negative feed-back loop in the regulation of the il- system [ ] . likewise, ifn-α/β has been shown to inhibit il- production in both humans and mice [ ] [ ] [ ] . the implication of such inhibition by ifn-α/β and tnf, which are secreted very early in hsv infections, well before the production of il- , has so far not been elucidated. as described earlier, the il- p induction is influenced by ifn-γ in a positive feed back loop. ifn-γ could activate il- transcription through binding of irf- , - , and - to an isre site in the promoter-region of il- [ , ] . upon hsv infection, ifn-γ is produced as part of the nonspecific response to the virus. a marked synergism between hsv and ifn-γ in il- induction has been demonstrated [ ] , indicating that the il- / ifn-γ autoaccelerating system is of importance during hsv infections. the ifn-γ-inducing activity of the produced il- is pronounced in mouse peritoneal cells after hours of infection with hsv [ ] . in a study by kirchner et al. ifn-γ was detected as early as on day of in vivo hsv infection, and the ifn-γ production was correlated to the genetics of hsv resistance [ ] . during hsv infection, the production of ifn-γ is mainly induced as a concerted action of several factors and not by il- alone. ifn-α/β by itself was shown to be a weak inducer of ifn-γ production by nk cells, but in synergy with il- the production of ifnγ was markedly enhanced [ ] . in elicited peritoneal macrophages, hsv induced efficient ifn-γ production through cooperation of il- , ifn-α/β and il- [ ] . in such a proinflammatory environment even other cells than nk and t cells, e.g. macrophages, might produce lower levels of ifn-γ [ , , ] . il- signals through stat , but stat translocation to the nucleus of nk cells has also been seen after ifn-α/β stimulation [ , ] . likewise, ifn-α/β induces stat phosphorylation in t cells [ ] , indicating that il- and ifn-α/β at this point act through a shared signalling pathway. furthermore, the synergistic action of il- and il- in ifn-γ production by macrophages was shown to be dependent on stat [ ] . in addition to these factors, tnf and il- have also been shown to act in synergy with il- in ifn-γ induction [ , , ] and vice versa, ifn-γ has been shown to synergize with hsv in induction of tnf production [ ] . this further emphasizes the concept of positive feed-back mechanisms in the regulation of early ifn-γ production. the important direct effect of ifn-α/β on hsv replication was found to be enhanced synergistically by ifn-γ in both cell culture and in vivo in mice [ , [ ] [ ] [ ] . this is, however, in conflict with an early study, which could not reveal any synergism between ifn-α/β and ifn-γ on the replication of hsv in human blood mononuclear cells [ ] . synergistic action of the two types of ifn is further supported by the observation of synergism between ifn-γ and tnf on hsv replication in corneal cells, and the fact that this was exerted through production of ifn-β [ ] [ ] [ ] . the effect was, however, greatly dependent on the cell type examined, which could explain the above-mentioned inconsistency. synergism between tnf and ifn-γ in inhibition of hsv replication has now been shown to be mediated by activation of a tryptophan-depleting enzyme [ ] . thus, relatively small amounts of early ifn-γ produced by nk cells in response to il- , ifn-α/β, tnf, and il- could in collaboration with the already present ifn-α/β and tnf have important local effect on hsv replication in permissive cells ( fig. ). this conclusion is further supported by observations in ko mice, indicating that collaborated action of ifn-α/β and ifn-γ is of importance in control of subcutaneous hsv infections [ ] . in vivo studies on hsv infections in immunodeficient, ko, and antibody-treated mice have shown that the il- , second early wave of response figure second early wave of response. regulatory pathways controlling production and action of ifn-γ during early hsv infection. when infected with hsv macrophages (mφ) produce several cytokines, including il- , which stimulate production of ifn-γ, primarily in nk cells. ifn-γ then induces no production in macrophages and stimulate the direct antiviral activity of ifn-α/β in other cells. stimulatory pathways are indicated by green arrows (→), and inhibitory pathways are drawn in red. - / ifn-γ system is able to control the infection, affecting both the survival rate and the hsv titres early in infection [ , ] . the effect of il- in hsv infections seems to be conducted in synergy with il- [ ] , as it has also been shown for vaccinia virus [ ] . in hsv corneal infections in ko mice, il- was shown to participate in the immune pathogenesis [ ] , but in another study utilizing il- encoding plasmid dna, corneal expression of il- reduced the angiogenesis, and thus the pathology of the infection [ ] . however, both studies agreed that il- does not affect the local titres of hsv in the eye. after a thermal injury, wide-spread hsv infections are an important risk, and treatment of injured mice with il- combined with soluble il- r results in augmentation of the ifn-γ production and decreased viral replication and mortality [ ] . in mice infected with murine cytomegalovirus (mcmv) production of il- -induced ifn-γ by nk cells has been demonstrated in vivo, and the system was further shown to lower the viral titres [ , ] . the il- / ifn-γ system seems, however, not to be of importance in all viral infections, in that the latter study could not detect any production of early il- or ifn-γ in a model of infection with the arenavirus lymphocytic choriomeningitis virus. analyses of the il- , - / ifn-γ system in humans with genetic defects and in ko-mice reveal more redundancy in man than in mouse and indicate that the system is of more importance in dna-than in rna-virus infections [ ] . the producers of early ifn-γ, the nk and nkt cells, and the cytokine il- and the transcription factor t-bet, which are both crucial for the differentiation and function of these cells, have all been shown to be decisive for the early control of hsv infection in vivo [ , [ ] [ ] [ ] . although nk cells but not ifn-γ was shown to be decisive for survival from ocular infections [ ] , such an effect of ifn-γ has been seen by others [ ] . furthermore, a review of genetic functional nk cell defects found nk cells and their innate ifn-γ production to be of central importance in herpesvirus infections [ ] . overall, it can be concluded that the il- / ifn-γ system is active in hsv infections and possesses an important antiviral potential, capable of controlling viral replication during the early phases of infection. in macrophages exposed to ifn-γ, the enzyme inducible nitric oxide synthase is induced, which eventually results in production of no from molecular oxygen and a guanidino nitrogen by conversion of l-arginine to l-citrulline [ ] . upon hsv infection, the inos gene is induced, as shown by detection of inos-mrna in infected mouse peritoneal cells and corneal neutrophils [ , ] . the production of no in hsv-infected cultures of resting mouse peritoneal cells, which comprise a mixed population of macrophages, lymphocytes, nk cells etc., is dependent on the virus being infectious [ ] . this is in line with the requirement of infectious hsv for il- production and thus for production of ifn-γ as described previously [ ] . no could itself be involved in a positive feed-back, in that signalling of il- utilizing tyk requires the activity of no [ ] . when exogenous ifn-γ is added to virus-infected cells, a marked synergism is seen. this synergistic effect of hsv on the ifn-γ-induced no production in macrophages was shown to be mediated by autocrine secretion of tnf [ , ] . in line with this, mice with a targeted disruption of the tnf gene showed impaired resistance to hsv and increased viral replication within the first days of infection [ ] , and antibodies to tnf and an inhibitor of no production impaired early control of hsv infection in peripheral nervous tissue [ ] . the induction of inos and the following production of no in response to ifn-γ and hsv is a relatively slow reaction, coming up after about hours of infection [ ] . in in vivo vaginal hsv infections inos mrna could be detected after hours of infection [ ] . thus, the production of this relatively toxic substance is part of the second wave of innate defence mechanisms. the retarded production of no and the requirement for two or more signals for induction of inos are logic considering the in hsv-infected macrophages exposed to ifn-γ, inos is induced synergistically though tnf-induced nf-κb activation and translocation to the nucleus, as shown by binding of a heterodimeric complex of p /p and a homodimer of p to the κb-site of the inos promoter during infection [ ] . the crucial position of nf-κb in the induction of inos and production of no is also indicated by experiments showing that antibodies to tnf inhibit activation of nf-κb and production of no in hsvinfected cells and abolish the synergism between the virus and ifn-γ, an observation which was also seen with inhibitors of nf-κb activation [ ] . further analysis of the signalling mechanism has revealed that the synergism upon hsv infection is influenced by physical interaction of irf- and the nf-κb subunit p and controlled by the isresite and the distal κb-site of the inos promoter ( fig. ) [ ] . a further support for this notion comes from the observation that the dna-binding capacity of nf-κb and the nuclear translocation of irf- have similar kinetics upon hsv infection [ ] and the fact that irf- is essential for inos induction [ ] . induction of other genes such as ifn-β and vascular cell adhesion molecule also involve physical interaction of irf- and nf-κb [ ] , and both irf- and irf- have in other cells types been shown to form complexes with nf-κb [ , ] . another potential mechanism in the synergistic induction of inos could involve complex formation of ifn consensus sequence-binding protein (icsbp or irf- ) and irf- , which is also important for high-output no production but has still not been studied in hsv infections [ ] . thus, high-output no production from activated macrophages is controlled by a "double-lock" signalling mechanism restricting the production of this antiviral toxic substance to sites of active viral replication, and sparing uninfected tissue from the detrimental effects ( fig. ). the antiviral effects of no have been documented in several viral infections, although there clearly exist viruses and conditions where no does not exhibit major antiviral properties. no is thus not a magic bullet against virus infections [ ] . in hsv infections, no has been shown to confer a substantial part of the antiviral activity induced by ifn-γ in a macrophage cell line and to participate in the extrinsic anti-hsv effect of macrophages [ ] [ ] [ ] [ ] . an exogenously added donor of no has in several cell lines been shown to reduce the replication of hsv [ ] . in vivo, analysis of mice treated with an inhibitor of no production showed higher titres of hsv in the lungs but increased survival rates due to reduced inflammation [ ] . recently, a study using another inhibitor of no production has confirmed the anti-herpetic effect of no during a hsv respiratory infection, but in this study mice with inhibited no production showed increased inflammatory responses, symptoms of infection, and mortality ifn-γ il- (at high ifn-γ / stat ) [ ] . replication of hsv during vaginal infection was increased in the presence of an inhibitor of no production, and this enhanced viral replication was most prominent during the first hours of infection [ ] . in inos-ko mice, the herpes virus mcmv replicates to higher titres in various organs and in macrophages, and this results in impaired survival of the animals [ ] . weanling mice with a targeted disruption of the inos gene showed increased hsv replication, but apparently without differences in hsv titres during the first days of infection [ ] , and in adult ko mice, we could not detect any significant effect of no during the early days of a generalised hsv infection (ellermann-eriksen, unpublished results). probably, these in vivo results are due to redundancy of the antiviral system. [ ] . the final effects of no on hsv infections therefore appear to be balanced between antiviral versus toxic effects, and the final outcome seems to depend on the timing, infectious dose, and tissues involved. thus no production in the early phases of hsv infection is one of the effector mechanisms of the innate immune response inhibiting hsv replication, but when overproduced, no might itself result in pathology, as discussed in the following section. as outlined above, positive feed-back mechanisms exist at the afferent side of the early cytokine response, involving especially the production of ifn-γ, il- , ifn-α/β and tnf, and synergisms at the efferent side, resulting in highoutput no production. as a result of coordinated induction of the inos gene by several transcription factors, activated by especially ifn-γ and tnf, a potent early antiviral system is activated. however, no causes damage to dna, proteins and lipids in cells and tissues and could thus be deleterious for the host [ ] [ ] [ ] [ ] . a study in ko-mice indicates that no can be responsible for inflammation and life-threatening symptoms to hsv infection of the lungs [ ] . this effect of no on pulmonary symptoms is also observed in influenza virus infections [ ] , although no inhibits replication of both influenza virus and severe acute respiratory syndrome coronavirus [ , ] . consequently, when this system is activated, it has to be controlled and eventually closed down, as it would otherwise induce unnecessary harm to the host. such negative regulations of the inos gene induction in ifn-γ activated macrophages is conducted by il- and il- [ , , ] . furthermore, tgf-β can exhibit downregulation of no production through several post-transcriptional regulatory mechanisms, but the contribution of these pathways have not been analysed in hsv infections [ , ] . il- production during hsv infection has in vaginal and cns infections been demonstrated on day of infection and to increase for the next days [ , ] . in peritoneal cells from mice infected i.p. pro-duction of il- could be detected at day of infection [ ] . at low ifn-γ concentrations, il- has been shown to inhibit inos induction through stat competition with stat binding to the gas element of the irf- promoter region. this results in reduced expression of the transcription factor irf- , which is crucial for induction of inos [ ] . generally, stat was shown to be a key factor in il- -and il- -induced inhibition of inos gene transcription induced by ifn-γ (fig. ) [ ] . at higher ifn-γ concentrations, activated stat is no longer able to compete with the high amounts of activated stat dimer [ ] . however, in this situation il- is still able to inhibit the production of no from ifn-γ-stimulated macrophages [ , , ] . in the presence of high levels of ifn-γ, il- is not able to alter the induction of irf- , but the production of irf- is increased [ ] . the human promoter region of irf- contains a sbe, and the induction of irf- could thus potentially be mediated by stat binding to this element [ ] . this is in agreement with the fact that irf- is known to compete with the binding of irf- to isre sites and to antagonize the transactivating activity of irf- in the regulation of other ifninduced genes [ ] [ ] [ ] . inhibition of inos expression by high concentrations of irf- relative to irf- has thus been proposed as a controlling mechanism in situations with high levels of ifn-γ ( fig. ) [ ] . furthermore, another mechanism could evolve from the observation that il- signalling can result in disruption of the complex formation of icsbp and irf- and thereby inhibit inos induction [ ] . other mediators of il- -induced repression of inos induction might exist, in that another dnabinding transcriptional repressor competing with irf- has been described [ ] . in ifn-γ activated macrophages the il- -and il- induced inhibition of inos induction can thus be overruled by hsv infection, leading to a sustained no production ( fig. ) [ , ] . this effect of hsv infection is mediated through tnf production and nf-κb activation [ , ] . however, pre-treatment with il- has in a theiler's murine encephalomyelitis virus model showed inhibition of nf-κb activation [ ] . in thioglycollateinduced peritoneal cells, lps and tnf could only overcome the inhibiting effect of il- in situations, where il- was added simultaneously or after the stimulators [ ] , a sequence of events which, however, is in agreement with the sequence of cytokine production in hsv infections. when activated, the nf-κb p physically interacts with irf- and trans-activate inos transcription in hsvinfected and tnf-treated cells [ , ] . it is thus tempting to speculate that the nf-κb-irf- complex has higher affinity for the combined dna-binding site and thus is able to obstruct the binding of irf- to the isre site of the inos promoter and in that way turn the competition towards transcriptional activity ( fig. ) [ ] . this will block the inhibiting effect of il- in foci of hsv replication and open up for no production at sites where the antiviral effect is of more importance than the potential toxicity. in treatment of hsv infections, we have for many years had a very powerful tool in the antiherpetic drug acyclovir and related compounds. but there are still therapeutic problems in the group of patients with generalized or cns infections, and therefore it is tempting and timely to hypothesize on possible future treatment strategies. as described, it is clear that relatively discrete but early actions of the non-specific defence systems are crucial for the long term outcome of the infection. the same holds for the antiviral therapy, and early presumptive therapy and rapid diagnostics could thus potentially improve the final outcome. in the seeking for improved antiviral treatment, adjuvant therapy with anti-hsv antibodies could potentially accelerate the clearance of viral particles, and block viremic dissemination in patients, who are still seronegative at the time of treatment. immunomodulatory treatment modalities imitating the early non-specific antiviral defence, working as described in this review, could be considered. the key players exhibiting the least toxicity by themselves could be used, taking advantage of potential synergy with other cytokines in the foci of hsv infection. in the future, molecules with affinity for various receptors are expected to be produced, and when we know the signalling mechanisms in detail and all the potential interactions, molecular signalling could be addressed directly by pharmaceuticals. in consequence of the crucial position of the type i ifns in innate response to hsv, future analogues of ifn-α/β seem obvious as candidates for adjuvant treatment of severe hsv infections. this could be supplemented with il- , which would give the highest ifn-γ production in foci of hsv infection because of other cytokines such as ifn-α/β, tnf and il- being present there. with focussed production of ifn-γ at sites of active viral replication and treatment with ifn type i analogues the focal antiviral activity could be increased markedly, without too much activity in areas without infection. to hamper systemic consequences of the enhanced proinflammatory reactions, such pro-inflammatory treatment could perhaps benefit from concomitant treatment with il- or other stat -activating therapeutics in the future. this would further focus the activity to sites of active hsv replication. in situations with massive viral replication in nearly all organs, high-dose aciclovir should perhaps only be supplemented with anti-inflammatory medications and inhibitors of tnf, since many of these individuals risk to die from septic reactions. the author(s) declare that they have no competing interests. the family herpesviridae: a brief introduction cercopithecine herpesvirus (b virus) aetiologische untersuchungen über der fieberhaften, herpes serologische untersuchungen zur 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are important in innate defense against genital herpes simplex virus type infection in mice but are not required for the development of acquired gamma interferon-mediated protective immunity interleukin- -and gamma interferon-dependent innate immunity are essential and sufficient for long-term survival of passively immunized mice infected with herpes simplex virus type il- and il- act in synergy to clear vaccinia virus infection: involvement of innate and adaptive components of the immune system reduced severity of hsv- -induced corneal scarring in il- -deficient mice il- suppresses the expression of ocular immunoinflammatory lesions by effects on angiogenesis therapeutic protective effects of il- combined with soluble il- receptor against established infections of herpes simplex virus type in thermally injured mice requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin administration an absolute and restricted requirement for il- in natural killer cell ifn-gamma production and antiviral defense. studies of natural killer and t cell responses in contrasting viral infections interleukin- and natural killer and nkt cells play a critical role in innate protection against genital herpes simplex virus type infection in the absence of t cells, natural killer cells protect from mortality due to hsv- encephalitis protective immunity to genital herpes simpex virus type infection is mediated by t-bet the role of natural killer cells in protection of mice against death and corneal scarring following ocular hsv- infection kinetics of cytokine production in the cornea and trigeminal ganglion of c bl/ mice after corneal hsv- infection human natural killer cell deficiencies and susceptibility to infection nitric oxide as a secretory product of mammalian cells herpes simplex virus type synergizes with interferon-gamma in the induction of nitric oxide production in 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synergistically induce major histocompatibility class i gene expression interferon regulatory factor- physically interacts with nf-kappa b in vitro and inhibits nf-kappa b induction of major histocompatibility class i and beta -microglobulin gene expression in transfected human neuroblastoma cells complex formation of the interferon (ifn) consensus sequence-binding protein with irf- is essential for murine macrophage ifn-gamma-induced inos gene expression does nitric oxide play a critical role in viral infections? inhibition of viral replication by interferon-gammainduced nitric oxide synthase interferon-gamma induced type i nitric oxide synthase activity inhibits viral replication in neurons inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites nitric oxide and macrophage antiviral extrinsic activity evidence for antiviral effect of nitric oxide. inhibition of herpes simplex virus type replication early inhibition of nitric oxide production increases hsv- intranasal infection role of nitric oxide synthase type in acute infection with murine cytomegalovirus mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced th cell responses phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase epr characterization of molecular targets for no in mammalian cells and organelles dna strand breakage, activation of poly (adp-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. formation of novel nitrogen-containing oxidized lipid derivatives altered immune responses in mice lacking inducible nitric oxide synthase rapid interferon gamma-dependent clearance of influenza a virus and protection from consolidating pneumonitis in nitric oxide synthase -deficient mice osterhaus ad: inhibition of influenza virus replication by nitric oxide nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus interleukin- -mediated inhibition of nitric oxide production in interferon-gamma-treated and virus-infected macrophages mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta spontaneously increased production of nitric oxide and aberrant expression of the inducible nitric oxide synthase in vivo in the transforming growth factor beta null mouse effect of the deletion of us and us from herpes simplex virus type on immune responses in the murine vagina following intravaginal infection small amounts of exogenous il- increase the severity of encephalitis induced in mice by the intranasal infection of herpes simplex virus type herpes simplex virus type infection of macrophages impairs il- -mediated inhibition of no production through tnfalpha-induced activation of nf-kappab il- -induced stat suppresses ifngamma-stimulated stat -dependent transcription in mouse macrophages impaired il- -mediated functions of macrophages in stat -deficient mice structure and regulation of the human interferon regulatory factor (irf- ) and irf- genes: implications for a gene network in the interferon system structurally similar but functionally distinct factors, irf- and irf- , bind to the same regulatory elements of ifn and ifn-inducible genes absence of the type i ifn system in ec cells: transcriptional activator (irf- ) and repressor (irf- ) genes are developmentally regulated recognition dna sequences of interferon regulatory factor (irf- ) and irf- , regulators of cell growth and the interferon system b lymphocyte-induced maturation protein (blimp)- , ifn regulatory factor (irf)- , and irf- can bind to the same regulatory sites inhibition of no production in macrophages by il- is counteracted by herpes simplex virus infection through tumor necrosis factor-alpha-induced activation of nk-kappa b interleukin- and interleukin- modulate nuclear factor kappab activity and nitric oxide synthase- expression in theiler's virus-infected brain astrocytes interaction of interferon regulatory factor- and nuclear factor kap-pab during activation of inducible nitric oxide synthase transcription i wish to thank all members of the group for highly constructive discussions and especially søren c. mogensen for critical review of the manuscript. for excellent technical help with the electron microscopy i thank ruth nielsen. furthermore, i wish to thank the department of clinical microbiology, aarhus university hospital, skejby and department of medical microbiology and immunology, university of aarhus for their hospitality and support. key: cord- - towah t authors: malmo, jostein; moe, nina; krokstad, sidsel; ryan, liv; loevenich, simon; johnsen, ingvild b.; espevik, terje; nordbø, svein arne; døllner, henrik; anthonsen, marit w. title: cytokine profiles in human metapneumovirus infected children: identification of genes involved in the antiviral response and pathogenesis date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: towah t human metapneumovirus (hmpv) causes severe airway infection in children that may be caused by an unfavorable immune response. the nature of the innate immune response to hmpv in naturally occurring infections in children is largely undescribed, and it is unknown if inflammasome activation is implicated in disease pathogenesis. we examined nasopharynx aspirates and blood samples from hmpv-infected children without detectable co-infections. the expression of inflammatory and antiviral genes were measured in nasal airway secretions by relative mrna quantification while blood plasma proteins were determined by a multiplex immunoassay. several genes were significantly up-regulated at mrna and protein level in the hmpv infected children. most apparent was the expression of the chemokine ip- , the pro-inflammatory cytokine il- in addition to the interferon inducible gene isg . interestingly, children experiencing more severe disease, as indicated by a severity index, had significantly more often up-regulation of the inflammasome-associated genes il- β and nlrp . overall, our data point to cytokines, particularly inflammasome-associated, that might be important in hmpv mediated lung disease and the antiviral response in children with severe infection. our study is the first to demonstrate that inflammasome components are associated with increased illness severity in hmpv-infected children. the negative sense single strand rna virus human metapneumovirus (hmpv) has since its discovery in emerged as a commonly detected respiratory tract pathogen involved in the npas were tested using pcr for hmpv (a , a a/b, b , b ), adenovirus, human bocavirus- , coronavirus (oc , e and nl ), enterovirus, influenza a and b virus, parainfluenza virus type - , parechovirus, rsv, rhinovirus, bordetella pertussis, chlamydophila pneumoniae and mycoplasma pneumoniae. nucleic acid was extracted using nuclisens easy-mag according to the manufacturer's protocol (biomerieux). all pcrs were in-house realtime assays based on taqman probes [ ] . for hmpv detection, primers targeting the n-gene allowing detection of all four hmpv genotypes were used as described elsewhere [ ] . genotyping was performed by sequencing an amplified f-gene pcr product [ ] using a genetic analyzer (applied biosystems) and comparing sequenced data with the nucleotide blast database (www.ncbi.nlm.nih.gov/blast/). a total of children tested positive for hmpv ( . %) in npa. for the purpose of the present study, children satisfying all of these criteria were recruited: an npa was sampled less than one week after onset of symptoms and within one day after admittance, either hmpv genotypes a or b were detected in npa, no viral co-infections detected in npa, and serum levels of crp < mg/l. clinical data were recorded prospectively or retracted from medical records. upper respiratory tract infection (urti) was diagnosed when rhinitis, pharyngitis, tonsillitis and/or otitis media (serous, simplex or purulent) was found without signs of lower respiratory tract infection (lrti). lrti was diagnosed in children with signs and symptoms of respiratory difficulty such as tachypnea, retractions and nasal flaring, signs of lower airway obstruction (wheezing, prolonged expiration, rhonchi), focal findings (crepitations) and/or a positive chest x-ray (infiltrates, atelectasis, air trapping). lrti was divided in bronchiolitis (age < years, tachypnea, retractions, wheezing, crepitations), obstructive bronchitis (age > years, signs of lower airway obstruction), asthma exacerbation with signs of lower airway obstruction, and pneumonia (cough, tachypnea, localized crepitations, x-ray infiltrates). a clinical severity score previously used to determine hmpv/rsv disease severity in children [ , ] was calculated for each infected child as the sum of these variables ) length of hospital stay days ( point), ) oxygen demand ( point), ) ventilatory support by continuous positive airway pressure ventilation ( point), and ) ventilatory support by endotracheal intubation and overpressure ventilation ( points). ten children admitted for elective surgery with no symptoms of rti in the last two weeks were randomly selected and included as controls. the clinical and virological findings are summarized in table , and a detailed overview for the individual patients is presented in the s table. mrna quantification cdna was synthesized from rna isolated from the collected npas using the qscript kit according to the manufacturer's protocol (quanta). quantitative pcr (qpcr) was performed using perfecta sybr green reaction mix (quanta) and a steponeplus instrument (life technologies) with the temperature profile °c for s, cycles at °c for s and °c for s. fold-change in ifn-β, ifn-γ, il- , iκbα, il- β, il- , nlrp , ip- , tnf-α, il- and isg gene expression relative to the control with highest levels of target gene mrna was calculated using the ddc t -method with gapdh as housekeeping gene. data was plotted as boxplots where the box represents % of the values, the horizontal line indicates the median, whiskers show the maximum and minimum values, and the closed circles indicate outliers. primer sequences used in this study are shown in the s table. protein quantification blood samples were collected in vacuette edta tubes (greiner bio-one) and the fractions separated according to the manufacturer's protocol. the concentrations of proteins in the plasma fraction were measured using a bio-plex multiplex system (bio-rad) and cytokine assays for ifn-β, ifn-γ, il- a, il- β, il- , ip- , tnf-α, il- , gro-α and gro-β (bio-rad). categorical variables were analyzed with the pearson chi-square test or the -tailed fisher's exact test. continuous and nearly normal distributed variables were analyzed with the student's t-test or anova-test, and non-parametric variables were compared by mann-whitney u tests. a two-sided p-value less than . was considered statistically significant. normally distributed and continuous variables are presented as average±standard deviation and nonnormally distributed variables as median (range). analyses were performed using the statistical package of social science (spss, version . ) and sigmaplot (version . ). the study was approved by the regional committee for medical and health research ethics (rek, mid-norway). caregivers to all patients and controls received written information about the study. written consent was obtained at the hospital from the caregivers of all controls and the majority of the patients included in the cohort. due to practical challenges by enrolling patients hours a day, days a week, some patients were enrolled after discharge from the hospital. their caregivers received written information after the hospital stay and children were included if the caregivers did not resist enrollment by taking contact to the hospital within weeks. this study was based on a cohort involving children hospitalized with respiratory tract infection (rti) where tested positive for hmpv. from the hmpv positive children we selected samples without detectable co-infections (n = ) as described in the methods section. further, children admitted for elective surgery without recent symptoms of rti were randomly selected and included as controls (n = ). table shows that the majority of the children were diagnosed with lower respiratory tract infection (bronchiolitis or pneumonia), and the disease severity ranged from - . one half of the patients had more severe disease with a score higher than zero (s table) . during the period of this study, a ( % of the samples) and b ( %) were the dominant genotypes found in the cohort and therefore our focus. the length of hospitalization was average . ± . days for a and . ± . days for b infected children (p> . , student's t-test). the maximum c-reactive protein (crp) values in the patients ranged from approximately to mg/l with the majority below mg/l, indicating that severe bacterial infections were unlikely. to determine the presence of antiviral cytokines in children infected with hmpv and controls, we initially investigated the expression of type i, ii and iii ifns. fig a shows that only a infected children had slightly elevated mrna levels of the type i ifn-β compared to the controls. as shown in fig b, the mrna levels of the type ii ifn-γ was not elevated in any of the patient groups relative to the controls. in correspondence with this, ifn-γ protein was not detected in the blood samples from any of the investigated children (data not shown). finally we evaluated the mrna expression of the type iii ifn il- . fig c shows that both the a and b positive patients had significantly increased levels of il- expression. next, we wanted to investigate the possible involvement of nf-κb and inflammasome-associated genes in response to hmpv infection. fig shows the mrna expression of a) iκbα, a repressor gene induced by nf-κb activation [ ] , b) il- β, c) il- and d) nlrp in hmpv infected children and controls. the expression of il- was significantly increased for both a and b infected children. however, for the other genes investigated their expression was not significantly increased compared to the controls. it should be noted that the standard deviations for the iκbα, il- β and nlrp expression were high. this was caused by a considerable fraction of the hospitalized children having undetectable expression while others had high mrna levels relative to control subjects. in contrast, children from the control group exhibited similar levels of iκbα, il- β and nlrp mrna levels. to illustrate this individual variation, differences in gene expression for individual a and b positive patients are outlined in fig . from this figure, it can be seen that a) iκbα, b) il- β and c) nlrp were significantly upregulated in several patients. interestingly, most of the patients with upregulation of iκbα, il- β and nlrp had increased expression of all three genes. to evaluate the involvement of other relevant immunomodulator genes, we measured the mrna expression of ip- , tnf-α, il- and isg . as shown in fig a, the expression of ip- was increased for both the a and b infected patients. the tnf-α expression (fig b) was significantly higher in patients infected with b compared to the controls. further, the expression of il- ( fig c) was not increased at mrna level in any of the groups with hmpv infected children. finally, the expression of isg (fig d) was markedly increased for both a and b infected patients. as mentioned earlier, the expression of inflammatory genes can possibly promote pathogenic events during rti. consequently, we wanted to investigate if the gene expression profiles differed in children with a severity index of zero compared to those with more serious disease and a higher severity score. as summarized in table , the mrna expression of il- β and nlrp were more frequently upregulated in the children with a severity score of - . finally, the concentration of several secreted cytokines in blood plasma was measured for a random subset of the patients (n = ) and the controls (n = ) as shown in fig . herein, we also included the chemokines gro-α and gro-β to evaluate implications of neutrophil recruitment to the lungs. fig shows that all of the cytokines included in the analysis, except for il- , were detected at significantly higher concentrations in patients compared to the controls. the percentage with up-regulated genes is given in the brackets. statistical significance is indicated with the p-value from a fisher's exact test comparing the two groups. further, as mentioned earlier, ifn-γ was not detected above the threshold value in the controls or patient samples. this study aimed to identify inflammatory genes associated with hmpv mediated disease. we performed an analysis of npas and blood samples collected from children hospitalized with rti. herein, we identify specific cytokines, such as ip- , il- , tnf-α, and isg , that are elevated in hmpv mediated lung disease. of noteworthy interest is the up-regulation of il- β and nlrp in samples from children with the most severe respiratory tract infection. this suggests that the inflammasome is involved in the innate immune response to hmpv in these patients, either as a protective mechanism or as a response to lung damage. type i and iii ifns are responsible for the induction of an antiviral state in cells immediately after infection [ ] . while type i ifns are produced by many different cell types, type iii ifns seems to be mostly produced by plasmacytoid dendritic and epithelial cells where the epithelial cells are the main targets for activity [ ] . due to their powerful and potentially harmful effects, the production of these cytokines is tightly regulated [ ] . our results showed that the type i ifn-β mrna in npa was marginally up-regulated for the a infected patients, but the protein concentrations in plasma were markedly increased for both a and b positive patients. this inconsistency could be explained by the protein being produced by tissue resident cells in e.g. the lung or lymph nodes. another explanation could be a lag in the induction of protein synthesis in the epithelial cells after the elevation of mrna transcription, since it is assumed that cytokines are able to enter the bloodstream from lung tissue during inflammation. for type iii ifn il- we observed increased mrna expression both for a and b infected children. however, il- protein was not significantly elevated in blood plasma. il- is prominently expressed in lung epithelial cells, and suggested to prevent viral invasion through skin and mucosal surfaces [ ] . hence, the lack of increased il- protein in blood could, similar as suggested for ifn-β, be explained by a lag in the induction of protein synthesis. the expression of il- has been shown to be triggered by viral infection, and it displays immunological properties similar to the type i ifn-α/β [ ] . recently, it has been shown that hmpv induces expression of il- in mice and the cytokine was suggested to have an important protective role [ ] . for rsv, which is phylogenetically and clinically closely related to hmpv, an in vitro study has shown that il- , in contrast to ifn-α/β, is induced during infection of primary respiratory tract cells [ ] . further, a study on infants hospitalized with rsv infection and bronchiolitis showed induced expression of il- in the airways [ ] . overall, our results adds to existing data suggesting that ifn-β and il- are induced to some extent during hmpv infection. chemoattractants are induced during acute inflammation events to recruit effector cells of the immune system. in our study, elevated mrna expression of the chemoattractant ip- was observed for a and b infected patients. further, elevated levels of ip- protein, and also of the chemokines gro-α and gro-β were found in blood plasma. ip- has previously been shown to be significantly elevated seven days post infection with hmpv a in mice [ ] . the expression of ip- may be induced by ifn-γ [ ] , ifn-α/β [ ] or tnf-α [ ] secreted by different immune-cells. in our study, despite elevated ip- , we did not measure any increased expression of the type ii interferon ifn-γ at mrna or plasma protein levels. the lack of ifn-γ detection in npas from hmpv infected children has also recently been reported elsewhere [ ] . our data suggests that ifn-β and tnf-α, which was significantly detectable in plasma, might be responsible for induction of ip- . in addition, ifn-γ-independent induction of ip- has been demonstrated in human peripheral blood mononuclear cells [ ] . in this setting the type iii interferon il- , which is closely related to il- that was shown to be upregulated herein, was correlated to ip- induction. of note, when infecting human epithelial cells with hmpv in vitro, we observed that il- mrna was induced and that il- showed a similar trend (data not shown), as also suggested elsewhere [ ] . hence, il- / could mediate ifn-γ independent ip- induction in hmpv infection. ifn-γ-independent ip- expression has also previously been reported in bal samples from patients during respiratory tract virus infection [ ] . we found that mrna expression of the pro-inflammatory cytokine tnf-α was elevated only in the b -infected children. however, the level of tnf-α protein in plasma was significantly increased both for a and b infected patients. an explanation for this could be different expression kinetics for mrna and protein. tnf-α induces nf-κb [ ] and, among other functions, it is involved in the recruitment of neutrophilic cells to the lungs [ ] . tnf-α production has previously been shown to be at high levels in nasopharyngeal secretions from rsor influenza-virus infected children [ ] . when comparing the patients with a severity index of and those with an index of - , tnf-α had significantly higher levels of expression (p< . , student's t-test). this could indicate that the presence of tnf-α in the respiratory tract directly or indirectly affects the severity of disease in hmpv infected children. it is worth mentioning that the hmpv c t value was similar for the patients with severity index and higher (s table) , suggesting similar extent of viral replication in these patients. we did not detect any increase in mrna levels of the pro-inflammatory cytokine il- . on the other hand, the levels of il- in blood plasma were significantly increased for the hmpv infected patients. similar as for ifn-β, this observation could be explained by il- protein being produced by tissue-resident cells or, alternatively, downregulation of gene expression after a peaked response. a previous study comparing the expression of several inflammatory cytokines in hmpv, rsv and influenza virus, detected elevated levels of tnf-α, il- and il- β protein in nasal washes from infants with rti [ ] . together with our data showing increased detection of these proteins in blood from patients with rti compared to the controls, this could suggest that at least a part of the protein detected in blood might have been secreted from cells in the respiratory tract. the antiviral cytokine isg was expressed at the highest relative levels of any of the genes included in this study. the mrna expression was up-regulated for both hmpv a -and b -positive npa samples. isg is known to be induced by viruses downstream of pattern recognition receptors and ifns [ ] . the expression of isgs is initiated by type i and type iii interferons such as ifn-β and il- . we did indeed find that these ifns were up-regulated at mrna and/or protein level in hmpv infected children. the functional effects and clinical involvement of isg are largely unknown. nevertheless, isg has been shown to inhibit viral replication in vitro and in vivo [ ] . interestingly, mice lacking isg have recently been shown to be subject to high mortality when infected with the mouse respiratory tract pathogen sendai virus-a negative sense, single-stranded rna paramyxovirus [ ] . this suggests that isg has a protective function and limits pathogenesis in mice. however, in our study, we were not able to identify any differences with respect to length of hospitalization or severity index when comparing the groups of patients expressing isg or not (p> . , student's ttest), suggesting a general protective function of this cytokine in children. our study reveal that most of the hmpv-infected children exhibited low levels of mrna coding for inflammasome-associated proteins, except for il- which was induced in both a and b infected patients, both at mrna and protein levels. interestingly, some patients showed distinct expression profiles with elevated levels of several genes involved in the inflammasome mediated immune-response. more specific, approximately / of the patients showed increased expression of iκbα, il- β and nlrp . as mentioned earlier, the repressor gene iκbα expression is induced by nf-κb activation [ ] . nf-κb induces mrna expression of the potent proinflammatory cytokines pro-il- β and pro-il- [ ] . further, increased level of nlrp mrna expression is the initial essential step in activating the nlrp -inflammasome. taken together, the increased levels of nlrp mrna in this group of children along with enhanced il- β and il- mrna expression and protein secretion could suggest that the nlrp -inflammasome is activated to produce il- β and il- in the respiratory tract of these patients. previously, it has been shown that influenza virus is able to activate the nlrp -inflammasome in mice [ ] . this was found to be critical for survival and reduction of viral titers in mice lung. further, rsv was recently shown to activate the nlrp -inflammasome in vitro using primary human lung epithelial cells [ ] . our results show that the hmpv infected children with a severity score exceeding zero more frequently exhibited elevated levels of il- β and nlrp than children with severity score zero. this suggests a possible involvement of these genes in hmpv infected children with severe disease. as mentioned earlier, previous studies on rsv have shown a variation in disease severity for different genotypes, but similar studies on hmpv have been inconclusive [ ] [ ] [ ] [ ] . consequently, we wanted to see if our data suggested any differences in gene expression or disease severity for the hmpv a and b genotypes. however, we did not detect any differences between a and b with respect to expression profiles except for ifn-β and tnf-α which was significantly higher expressed in a and b positive patients, respectively, at mrna level. further, we did not detect any differences in the length of hospital stay or disease severity in the a and b infected children. our study population includes infants and up to years old children. consequently there is a risk that children with both primary and secondary hmpv infections are included. these might experience different immune responses. to address this we compared children less than one year of age with older children. we found no differences in gene expression profiles (data not shown). in addition, the average age of children with severity score - was not significantly different from those with severity score zero (p = . , student's t-test), indicating that the youngest children where not more disposed to severe disease compared to the older. of note, there was a difference in age distribution between hmpv-infected and control children. the controls were symptom-free and screened for the same airway pathogens as the patient samples, and no pathogens were detected. hence, we would not anticipate that the inflammatory genes are expressed above a basal level in the controls. further, to our knowledge, no studies so far have shown that basal expression is significantly different in children less than one year and older. our use of clinical samples from selected, well-classified patients is a strength of the study. for studies aimed to examine immune responses to a particular viral infection in vivo it is a challenge, especially in children, to obtain sufficient number of samples collected at similar conditions. in particular, interfering viral and bacterial co-infections are common. in our study population extensive viral analyses of each sample revealed that nearly half of the population had viral co-infections and were excluded from further analysis. in addition, bacterial infections were unlikely because most children had received one or more conjugated pneumococcal vaccinations, and only patients with maximal crp levels less than mg/l were included. furthermore, we only included samples that were collected within the first week after onset of symptoms. finally, separating the samples based on the two most frequent hmpv genotypes enabled us to see if there was a genotype-specific cytokine gene expression profile or differences in disease severity. this allowed us to study the initial host immune response towards particular hmpv genotypes, which extends current literature. however, our selection criteria limited the total number of patients that were eligible for our study. consequently, it will be necessary to confirm our findings in other cohorts, in particular with respect to inflammasome-associated genes in children with severe rti. in summary, our study has identified inflammasome components that may be key players in the innate immune response against hmpv in children. the recent discovery of nlrp inhibitors [ ] that may prevent or even treat rti-mediated inflammation, emphasizes the need to establish the role of inflammasomes in the response to rti in children. our study provide an important contribution in this regard. supporting 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implications posttranscriptional control of type i interferon genes by ksrp in the innate immune response against viral infection ifn-lambda (ifn-λ) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo impact and regulation of lambda interferon response in human metapneumovirus infection type-iii interferon, not type-i, is the predominant interferon induced by respiratory viruses in nasal epithelial cells interferon lambda - expression in infants hospitalized for rsv or hrv associated bronchiolitis pulmonary infection of mice with human metapneumovirus induces local cytotoxic t-cell and immunoregulatory cytokine responses similar to those seen with human respiratory syncytial virus biochemical characterization of a gamma interferon-inducible cytokine (ip- ) interferon-independent, human immunodeficiency virus type gp -mediated induction of cxcl /ip- gene expression by astrocytes in vivo and in vitro essential involvement of cross-talk between ifn-γ and tnf-α in cxcl production in human thp- monocytes premature infants have impaired airway antiviral ifn[gamma] responses to human metapneumovirus compared to respiratory syncytial virus interferon lambda- (ifn-[lambda] //il- ) induces elr-cxc chemokine mrna in human peripheral blood mononuclear cells, in an ifn-[gamma]-independent manner detection of respiratory viruses and the associated chemokine responses in serious acute respiratory illness tnf activates nf-κb by phosphatidylcholine-specific phospholipase c-induced "acidic" sphingomyelin breakdown role of p mitogen-activated protein kinase in a murine model of pulmonary inflammation severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses the broad-spectrum antiviral functions of ifit and ifitm proteins ifit is an antiviral protein that recognizes [prime]-triphosphate rna sendai virus pathogenesis in mice is prevented by ifit and exacerbated by interferon tlrs, nlrs and rlrs: a trinity of pathogen sensors that co-operate in innate immunity inflammasome inhibition: putting out the fire we would like to thank the research nurses at the children's clinic (st. olav's hospital) and the technicians at the department of medical microbiology (st. olav's hospital). key: cord- -bnzbppof authors: biesold, susanne e.; ritz, daniel; gloza-rausch, florian; wollny, robert; drexler, jan felix; corman, victor m.; kalko, elisabeth k. v.; oppong, samuel; drosten, christian; müller, marcel a. title: type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: bnzbppof bats harbor several highly pathogenic zoonotic viruses including rabies, marburg, and henipaviruses, without overt clinical symptoms in the animals. it has been suspected that bats might have evolved particularly effective mechanisms to suppress viral replication. here, we investigated interferon (ifn) response, -induction, -secretion and -signaling in epithelial-like cells of the relevant and abundant african fruit bat species, eidolon helvum (e. helvum). immortalized cell lines were generated; their potential to induce and react on ifn was confirmed, and biological assays were adapted to application in bat cell cultures, enabling comparison of landmark ifn properties with that of common mammalian cell lines. e. helvum cells were fully capable of reacting to viral and artificial ifn stimuli. e. helvum cells showed highest ifn mrna induction, highly productive ifn protein secretion, and evidence of efficient ifn stimulated gene induction. in an alphavirus infection model, o'nyong-nyong virus exhibited strong ifn induction but evaded the ifn response by translational rather than transcriptional shutoff, similar to other alphavirus infections. these novel ifn-competent cell lines will allow comparative research on zoonotic, bat-borne viruses in order to model mechanisms of viral maintenance and emergence in bat reservoirs. the order chiroptera (bats) is one of the most diverse and geographically wide-spread orders within the mammals constituting % of all mammalian species [ ] . chiroptera are subdivided into two suborders yango-and yinpterochiroptera. the latter includes frugivorous/nectarivorous bats (flying foxes) with species like rousettus aegyptiacus (r. aegyptiacus), pteropus alecto (p. alecto) and eidolon helvum (e. helvum) as well as the insectivorous bat rhinolophus cf. landeri (r. cf. landeri, rhinolophidae). yangochiroptera comprise bats from the three superfamilies emballonuroidea, noctilionoidea, vespertilionoidea including the insectivorous species myotis daubentonii (m. daubentonii), pipistrellus spec. (both vespertilionidae), tadarida brasiliensis (t. brasiliensis, molossidae), hipposideros cf. caffer/ ruber (h. cf. caffer/ruber, hipposideridae) [ , ] . bats have been shown to host relevant human pathogens like rabies-, ebola-, marburg-, henipaviruses (hendra-, nipah-) and severe acute respiratory syndrome (sars)-like coronaviruses [ ] . the ability to control such highly pathogenic viruses raises the question whether bats might have evolved particularly effective mechanisms of immune control [ ] [ ] [ ] . little is known about the immune system of bats. it has been shown that bats develop immunoglobulins after infection and have lymphoid development similar to that in other mammals [ ] [ ] [ ] . however, information on the innate immune response of bats is particularly scarce. toll-like receptor genes have been identified in r. leschenaulti and p. alecto [ , ] . cells from pteropus species have been shown to produce high amounts of interferon (ifn)-l after stimulation with the double-strand (ds)rna analogue poly ic, and after infection with the bat-associated paramyxovirus, tioman [ ] . conversely, infection with the highly pathogenic paramyxovirus hendra virus resulted in no induction of ifn expression and concomitant inhibition of ifn signaling, suggesting the presence of specific viral ifn antagonists [ ] . a conserved functionality of ifn signaling in different mammalian cell cultures including epithelial lung cells from tadarida brasiliensis (tb -lu) was already described earlier [ , ] . however, there remains a fundamental lack of knowledge on the ways type i ifns are induced and ifn signals are processed in bat cells. because type i ifn is a major barrier towards virus infection, quantitative comparisons between different mammalian systems are of particular interest. currently there are hardly any bat cell lines available whose fundamental properties in ifn induction and -response have been characterized in a comparative manner. here we present a set of essential tools to characterize ifn induction and -response in bat cells, and introduce a novel group of highly ifn-competent, immortalized bat cell lines from the species e. helvum that hosts relevant zoonotic viruses including henipa-and lyssaviruses [ , ] . we compare paramount patterns of ifn induction and response in these e. helvum cells with that in prototype murine and primate cell lines. for all capturing and sampling, permission was obtained from the wildlife division, forestry commission, accra, ghana. samples were exported under a state contract between the republic of ghana and the federal republic of germany, and under an additional export permission from the veterinary services of the ghana ministry of food and agriculture (permit no. chrpe / ; a ). all cells were cultivated in dmem (dulbecco's modified eagles medium) (paa, cölbe, germany) with . g/l glucose (paa), supplemented with % fetal bovine serum (paa), % penicillin/streptomycin concentrate (penicillin units/ml, streptomycin mg/ml) (life technology), % l-glutamine mm, % sodium pyruvate mm (paa), % mem nonessential amino acids (neaa) concentrate (paa). cells were generally incubated at uc and % co . as prototype mammalian cells we applied simian virus (sv) large t antigen immortalized mouse embryonic fibroblasts (mef) generated inhouse from /svj mice [ ] , african green monkey kidney cells (ma , kindly provided by friedemann weber, university of marburg) and human lung adenocarcinoma epithelial cell line (a , ccl- ). for titration of o'nyong nyong virus (onnv) vero e cells (atcc crl- ) were used. under the auspices of ghana authorities bats were caught with mist nets, anaesthetized with a ketamine/xylazine mixture and euthanized to perform organ preparations (permit no. chrpe / ; a ). organs from e. helvum (embryo kidney and lung), r. aegyptiacus (kidney), m. daubentonii (lung), pipistrellus spec. (kidney), h. cf. caffer/ruber (embryo) and r. cf. landeri (kidney) were minced, trypsinized, and cultured in dmem medium by titration and seeding at : dilution in cell culture flasks as described previously [ ] . imipenem (zienam, msd, haar, germany) and amphotericin b (paa) were added to minimize contamination risks. immortalization was done by lentiviral transduction of the large t antigen of sv . immortalized cells were expanded and stock frozen or processed further for subcloning. all cell cultures were genotyped by amplification of mitochondrial cytochrome b as previously described using primers l and h (table s ) [ , ] and were controlled for mycoplasma [ ] , sv (in-house assay, table s ), lyssaviruses [ ] and filoviruses [ ] by rt-pcr. nucleic acid extraction and real-time rt-pcr viral rna was extracted from cell culture supernatant with the qiaamp viral rna mini kit (qiagen, hilden, germany). total rna from % confluent cells was isolated using the rneasy mini kit (qiagen) and reverse-transcribed with random hexamer primers (life technologies, karlsruhe, germany). fragments of target genes were amplified from cdna by lowstringency pcr. after initial denaturation for min at uc, touchdown pcr was done for cycles ( uc/ s, - uc, delta uc/ s, uc/ s). the remaining pcr cycles included uc/ s, uc/ s, uc/ s. fragments were sequenced and used to design real-time rt-pcr assays targeting genes relevant for the ifn system as well as reference genes, in domains conserved among bat species (table s ) . probes were tagged with -carboxyfluorescein and -black hole quencher (biomers, ulm, germany). real-time rt-pcr was processed using the lightcyclerh real-time pcr system (roche, basel, switzerland). for quantification of onnv genome equivalents (ge) the dilution end-point was defined as one pcr unit. log pcr units per ml for each experimental sample were calculated from the linear equations of the dilution series [ ] . to determine the foldinduction of the different target genes (ifn, mxa, isg ) the ddct method was applied with tata-box binding protein (tbp) as housekeeping gene [ ] . onnv infections were performed at multiplicity of infection (moi) of . and . respectively. virus was diluted in serumfree medium optipro (life technologies). cells were inoculated for h at uc and washed twice with pbs after infection. samples were taken at time points , and h post infection (hpi). titration of onnv was performed by plaque assay as previously described [ , ] . vero e cells were seeded in -well plates at a density of cells per ml. virus supernatant was added and after h incubation the inoculum was removed and cells were washed with pbs (paa). mem (biochrom, berlin, germany), supplemented with . % (w/v) nahco (roth, karlsruhe, germany), % fcs (paa) and % penicillin/ streptomycin, was mixed in a : ratio with . % avicel (fmc, biopolymers, brussels, belgium) and . ml per well was added. after days the overlay was discarded and cells were stained with a . % crystal violet and % ethanol (roth) containing solution. rift valley fever virus clone (rvfv ) infection for ifn induction was done as described above by infecting cells at an moi of . after h, all supernatants were harvested and virus was inactivated by b-propiolactone (b-pl, ferak berlin, germany) treatment as described below. ifn stimulation by poly ic was performed by transfection of cells per -well with mg poly ic using nanofectin (paa) according to the manual instructions. rvfv -and onnv-containing supernatants were inactivated with b-pl as previously described [ ] [ ] [ ] . briefly, the supernatants were collected and rvfv was inactivated by incubating with . % of b-pl at uc for h. the hydrolysis of b-pl was achieved by incubation at uc for h. for onnv a final concentration of . % of b-pl was needed for total inactivation. all b-pl treated samples were subjected to a virus plaque assay to control complete inactivation. to ensure equal treatment of the samples, supernatants of negative controls and cells stimulated with poly ic were also treated with b-pl. a classical vsv bioassay was performed as described previously [ ] . eidni/ . , mef and ma cells were seeded in -well plates at a density of cells per ml. after h the inactivated supernatants or a pan-species ifn-a (pan-ifn, a universal recombinant type i human ifn-a a/d hybrid, pbl biomedical laboratories/axxora, lörrach, germany) standard dilution series were added to the respective cell lines. an internal standard curve comprising five concentrations ( . , , , and units/ml) of pan-ifn diluted in medium of mock treated cells was applied in every experiment. the external pan-ifn standard curves for the ec calculation were performed in quadruplicates and comprised pan-ifn concentrations between . and units/ml diluted in dmem. h posttreatment, ifn containing supernatants were removed and cells were washed with pbs. the cells were then infected with vsv at an moi of . . after h of virus adsorption cells were washed again and overlayed with avicel as described above. after days the overlay was discarded, the cells were fixed and stained as before. the amount of plaques of the applied pan-species ifn standard curves or of the test samples was calculated in percentage (maximal plaque count from the negative control was set to %). the equation of the internal standard was used to calculate ifn amounts in the samples. ec values were defined as ifn concentrations which reduced the number of plaques to %. afterwards the values were multiplied by the dilution factors and figures were normalized to the amount of ifn per ml. to define comparable, species-independent ifn concentrations the calculated amount of ifn was divided by the ec of each internal standard curve. thus, the normalized amount of ifn could directly be compared. protein analysis was essentially done as described elsewhere [ ] . generally, cells were lysed in ripa lysis buffer ( mm nacl, % igepal ca- , . % sodium deoxycholat, . % sds, mm tris (ph . ), protease inhibitor cocktail iii, benzonase units/ml, mm dithiothreitol (merck, darmstadt, germany)) and separated on a % sds-page gel. western blotting was performed by using mouse-anti-mx / / (c- , santa cruz, heidelberg, germany), goat-anti-ifit/isg (l- , santa cruz), mouse-anti-actin (sigma) immunoglobulins at dilutions : . secondary detection was done with the help of horseradish peroxidase labeled goat-anti-mouse and rabbit-anti-goat antibody ( : , dianova, hamburg, germany) and supersignalh west femto chemiluminescence substrate (thermo fisher scientific, bonn, germany). old world frugivorous/nectarivorous bats or flying foxes (pteropodidae; formerly classified as ''megachiroptera'') comprise species like r. aegyptiacus and e. helvum. whereas r. aegyptiacus is a known reservoir for marburg virus [ ] [ ] [ ] , e. helvum was shown to carry henipa-like viruses [ ] and lagos bat virus [ ] . only for r. aegyptiacus [ ] and tadarida brasiliensis (atcc: ccl- ) cell cultures are commercially available. e. helvum bats roosting in kumasi, ghana, were investigated shortly before the parturition season in . one pregnant female was euthanized by injection of ketamine/xylazine according to approved protocols, under a license from the veterinary services and the ministry of food and agriculture, accra, ghana. primary cell cultures from embryonic kidney tissue of e. helvum were generated at the kumasi collaborative centre for research in tropical medicine, kumasi ( figure a) . after expansion of primary cells to confluent monolayers, cells were immortalized by lentiviral transduction of the sv large t antigen gene [ ] . primary cells could be passaged only twice, while transduced cells could be passaged continuously (. passages). out of the second passage, clonal cell lines were prepared by end point dilution plating. a clonal cell line named eidni/ . ( figure a ) was chosen for in-depth characterization. for comparison, two clonal cell lines from the same preparation (eidni/ . and eidni/ . ), as well as the st passage of a mixed culture (eidni/ ), were also preserved ( figure a) . additionally, a kidney cell culture ( th passage) from an adult r. aegyptiacus bat was prepared (data not shown) to investigate putative differences between the two related frugivorous bats. all bat cell cultures were genotyped by amplifying mitochondrial cytochrome b fragments [ ] followed by a blast analysis. eidni and roni cells showed % identity with cytochrome b (genbank accession no. ab . (e. helvum) and jf . (r. aegyptiacus), data not shown). as already shown for primary cells of the pteropus species, bat cells can readily secrete ifn upon virus infection or poly ic transfection [ , , , ] . however, immortalization of cells can damage cytokine genes and signaling pathways. [ , ] . second, cells were transfected with poly ic [ ] . to measure the induction of ifn transcription, a real-time rt-pcr assay for the ifn-b gene was designed. to this end the ifn-b gene was amplified by nested rt-pcr from cultures of a phylogenetically representative range of bats differing in main diet and hunting strategies including the insectivorous m. daubentonii, pipistrellus spec. (vespertilionidae), h. cf. caffer/ruber (hipposideridae),and r. cf. landeri (rhinolophidae) as well as the frugivorous/nectarivorous r. aegyptiacus and e. helvum (pteropodidae) ( figure b) , using primers designed upon alignments of available ifn-b genes from horses, swine, cattle, humans, mice and rats. real-time rt-pcr primers (see table s for genbank no. and primer sequences) were placed in sites conserved among bats ( figure c) . as shown in figure d , eidni/ . cells were readily infected by rvfv , and luciferase expression representing virus replication levels was comparable with that in several reference cell cultures including mef, ma and vero e cells. nevertheless, the induction of the ifn-b gene in a single cycle infection experiment, at an moi = , was approximately -fold stronger in eidni/ . cells than in mef and ma cells ( figure e ). the induction in vero e cells was not examined because of their intrinsic ifn-b gene defect [ , ] . interestingly, excessive ifn induction as compared to reference cells was only observed upon virus infection, but not upon poly ic transfection. it was concluded that the capability of eidni/ . cells to react on natural and artificial ifn stimuli had not been affected by immortalization. since cellular mrna levels are prone to differential expression over time ifn protein secretion was analyzed next. it was tested whether eidni/ . cells were able to produce and secrete effective doses of type i ifn. to this end it was necessary to compare and calibrate species-specific ifn secretion between primate, rodent, and bat cells, using an appropriate experimental model. to achieve this, a vsv-based ifn bioassay was established. vsv growth and plaque morphology was compared in all cell lines (figure a) . countable plaques were visible after days post infection (dpi). to obtain a calibration of ifn secretion across species, all cell lines were incubated with gradients of equivalent concentrations of recombinant pan-species ifn and subjected to vsv plaque reduction assays (exemplified for eidni/ . in figure b ). the antiviral responses in eidni/ . , mef, and ma cells upon equivalent doses of ifn are compared in figure c . ec values (amount of pan-species ifn to achieve a % vsv plaque reduction) were calculated for all cell lines ( figure c ). using this calibration, it was possible to compare the equivalent activity levels of species-specific ifn secreted from each cell line upon stimulation. in accordance with the ifn mrna induction, the highest equivalent amount of bioactive secreted ifn upon rvfv virus infection and poly ic transfection was measured in eidni/ . cells, followed by mef and ma (figure ). the observed difference in ifn mrna induction between rvfv virus and poly ic transfection ( figure e) was not seen in this case favoring the idea of differential mrna regulation in e. helvum cells upon poly ic treatment. in conclusion, eidni/ . cells were capable of inducing ifn upon natural and artificial stimuli, to secrete active type i ifn, and to induce an antiviral state upon ifn stimulation. since e. helvum cells produced considerable amounts of ifn upon infection with rvfv we investigated in addition the effects of infection with a non-attenuated wild-type rna virus. the old world alphavirus onnv was chosen because alphaviruses have been detected previously in bats [ , ] and exhibit strong induction of ifn [ ] . interestingly, alphaviruses utilize a rather general mechanism to evade ifn, by causing a translational shutoff that affects cellular translation more than viral translation [ ] [ ] [ ] . to establish a synchronized infection of onnv, a high moi = . was used. virus growth was measured by an onnv-specific real-time rt-pcr and titration of virus in supernatants hpi. virus rna and infectious virus were detected in all cell lines (figures a and b) . increases of infectious virus formation were about -fold within hpi, and specific infectivities, expressed as pfu per genome equivalent (pcr units), were highly comparable between cell cultures ( figure c) . to investigate if onnv infection caused ifn induction, the amount of ifn mrna (y-axis; for absolute values refer to figure s ) was directly compared to viral rna (x-axis) from supernatant at equivalent time points in each cell line ( figure a ). whereas no induction of ifn mrna was detectable at hpi, onnv replication clearly induced ifn mrna expression by hpi. comparison of secreted ifn with infectious virus production at hpi revealed that increased viral titers corresponded to lower amounts of secreted ifn protein ( figure b and s ). eidni/ . cells showed highest levels of secreted ifn but the lowest level of infectious particles, suggesting that virus replication might be limited in response to ifn. to determine whether virus control correlated with levels of secreted ifn or merely with the induction of genes under control of the ifn promoter, the ratios of secreted ifn to ifn mrna were determined in all cell lines. in all cells stimulated by rvfv or poly ic, the ratios of secreted ifn versus ifn mrna induction was comparable. in contrast, in all cells infected with onnv the relative levels of secreted ifn were clearly reduced, while overall ifn mrna induction was less affected ( figure c ). this effect was independent of the cell species. in order to exclude that the onnv-mediated reduction of ifn protein production was cell-clone specific, another cell line named eidni/ . as well as the original mixed cell culture eidni/ was analyzed, essentially showing the same reduction of relative ifn secretion upon onnv infection ( figure d ). similar effects were also seen in a mixed cell culture generated from the related flying fox r. aegyptiacus (roni/ ). a human lung adenocarcinoma epithelial cell line (a ) showed an even higher decrease of ifn secretion than ma and mef ( figure d) . alphaviral induction of a transcriptional and/or translational shutoff affecting mainly the cellular but not the viral protein production has been described in other cells [ ] . while our results suggested similar effects to be in place in e. helvum and r. aegyptiacus cells, control of onnv replication seemed to be relatively more efficient in both applied bat cells. one reason might be differences in the capability of bat cells to react on ifn signaling. to investigate this, the mrna abundance of two different isgs (mxa and isg ) as well as two reference genes (actin-b and tbp) was analyzed and compared to the amounts of secreted ifn at and hpi. whereas the expression of mxa is strictly ifn signalingdependent, isg can also be directly activated in the absence of secreted ifn and without involvement of the jak/stat pathway [ , ] . as shown in figure e , the ratios of mxa and isg mrna to secreted ifn showed an approximately fold increase hpi. thus, transcription did not seem to be generally blocked after onnv infection in all cells. in bat cells (eidni/ . ) but not in murine or primate cells, the level of mxa mrna induction in relation to ifn in supernatant was more than -fold higher compared to the same ratio for isg . this was also the case in different clonal and mixed e. helvum kidney cells (eidni/ . and eidni/ ), as well as in r. aegyptiacus kidney (roni/ ) cells ( figure f ), suggesting that ifn signaling-dependent induction of isgs may be highly efficient in e. helvum and r. aegyptiacus bat cells. in our experiments this explained an apparently superior control of onnv replication in both applied bat cells. to confirm alphaviral translational shutoff in bat cells, protein expression of mxa, p and actin was determined by western blot analysis hpi. as depicted in figure , rvfv infection clearly caused higher or similar (ma cells) expression of mxa in cells from all species while expression of p was hardly increased over levels seen in uninfected cells. in contrast, onnv infection resulted in a reduced expression of mxa and p proteins. this effect was generally more pronounced for mxa than for p , suggesting that ifndependent signaling rather than jak/stat-independent induction were affected ( figure ). interestingly, this effect was observed in all applied bat cell lines but not in mef ( figure a ). in summary, while e. helvum and r. aegyptiacus bat cells showed particularly efficient induction of mxa mrna, the production of this highly active antiviral protein was antagonized by onnv in those bat cells to a larger extent than in cells from rodents or primates. finally, it had to be excluded that the observed reduction of ifn and isg protein levels might be due to attenuation of virus replication. alphaviruses have been shown to acquire mutations in the utr [ , ] , nsp [ ] , or nsp genes [ ] , which resulted in low level replication and enabled virus persistence without ifndependent elimination. the observed increase of ifn mrna upon onnv infection ( figure a ) and viral titers of up to pfu per ml ( figure b ) would in fact speak against this hypothesis. however, to exclude attenuated replication in bat cells we analyzed virus growth at a very low moi of . . virus replication was monitored by real-time rt-pcr at time point , and hpi ( figure a ) and viral supernatants were titrated after h ( figure b) . additionally, the specific infectivity of virus supernatants was compared hpi ( figure c ). all cell cultures supported virus growth efficiently, despite -fold lower mois applied in this experiment. the specific infectivity was higher compared to the infection at moi = . , suggesting an efficient infectious particle formation in all cells at low moi. whereas ma primate cells yielded a to -fold higher specific infectivity compared to the high moi experiment, eidni/ . and mef had similar ratios at both doses. taken together the highly productive virus formation at a low moi suggested that decreased ifn and isg protein levels were generally not due to attenuation of replication. in conclusion, this study is the first to directly compare ifn induction, secretion and signaling in african fruit bat cell lines with that in other mammalian cell lines. we show that these bat cells were fully capable of reacting to viral and artificial ifn stimuli. our bat cells showed strikingly high ifn mrna induction, efficient ifn protein secretion, and evidence of highly efficient isg induction. onnv exhibited typical strong ifn induction but evaded the ifn response by translational rather than transcriptional shutoff, as typical in alphavirus infection of mammalian cells. these well-characterized, highly interferoncompetent cell lines will enable comparative research on zoonotic, bat-borne viruses in order to model mechanisms of viral maintenance and emergence in bat reservoirs. we thank ute winke and stephan kallies for excellent technical assistance, antje seebens (noctalis) and peter vallo (academy of sciences of the czech republic) for field work in ghana, and beate kümmerer for critical review of the data. we are 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high-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing roles of nonstructural protein nsp and alpha/beta interferons in determining the outcome of sindbis virus infection university of bonn, for mouse embryonic kidney cells and to friedemann weber, university of marburg, for the donation of rvfv . key: cord- -oyg oyx authors: li, shasha; yang, jinping; zhu, zixiang; zheng, haixue title: porcine epidemic diarrhea virus and the host innate immune response date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: oyg oyx porcine epidemic diarrhea virus (pedv), a swine enteropathogenic coronavirus (cov), is the causative agent of porcine epidemic diarrhea (ped). ped causes lethal watery diarrhea in piglets, which has led to substantial economic losses in many countries and is a great threat to the global swine industry. interferons (ifns) are major cytokines involved in host innate immune defense, which induce the expression of a broad range of antiviral effectors that help host to control and antagonize viral infections. pedv infection does not elicit a robust ifn response, and some of the mechanisms used by the virus to counteract the host innate immune response have been unraveled. pedv evades the host innate immune response by two main strategies including: ) encoding ifn antagonists to disrupt innate immune pathway, and ) hiding its viral rna to avoid the exposure of viral rna to immune sensors. this review highlights the immune evasion mechanisms employed by pedv, which provides insights for the better understanding of pedv-host interactions and developing effective vaccines and antivirals against covs. porcine epidemic diarrhea virus (pedv) is the etiological agent of porcine epidemic diarrhea (ped) that causes an acute and highly contagious enteric disease of swine characterized by vomiting, diarrhea, dehydration, and anorexia in pigs of all ages, especially resulting in severe diarrhea and high mortality rate in piglets. in serious cases, outbreak of ped even leads to a mortality rate of % in pigs [ ] [ ] [ ] . the causative agent of ped was first described in the s in england [ ] . in , an unrecognized enteric disease was reported in several european countries [ ] . the viral diarrhea was collectively designated "ped" in [ ] . endemic ped had been described in both developed and developing countries, but with a low impact on the swine industry until . in , outbreaks of ped caused by highly pathogenic variant pedv strains occurred in china, and this was immediately reported in other asian counties, causing up to % mortality in suckling piglets [ , [ ] [ ] [ ] . in april , pedv entered the united states (us) for the first time and the virulent strains spread rapidly across the us [ ]. apart from almost % mortality rate in suckling piglets, pedv infection also damaged the growth performance of finishing pigs [ ] . the highly pathogenic strains of pedv spread worldwide, resulting in serious problems to the swine industry and substantial economic losses [ , , , ] . vaccination used to be the main strategy to prevent and control the rate of pedv infection [ ] , however, the current available pedv vaccines cannot provide complete protection for the pigs affected by the highly pathogenic strains. the optimal vaccines should induce efficient maternal antibodies in sows that could be transmitted to the offspring and protect neonatal suckling piglets from pedv. the vaccination (s ) protein [ ] . apart from apn, pedv is able to bind to sialic acids [ ] . it remains unknown whether pedv uses sugar coreceptors during viral infection [ , ] . pedv infects multiple cell lines from different species including bat and primate (human and non-human) in vitro. the ability of pedv to infect the cells of different species indicates that the virus utilizes the evolutionarily conserved cell components as receptors, thereby enhancing the potential for cross-species and potentially, zoonotic transmission [ , ] . the highly pathogenic variant strains of pedv were identified in and caused a high morbidity of up to % in piglets, and since then, these strains become dominant, leading to most of the acute outbreaks of ped worldwide [ , , ] . the high virulence of these strains is critically associated with the immune evasion mechanisms employed by the virus. pedv has evolved different strategies to delicately manipulate and damage the host innate immune system for their multiplication. clarification of these mechanisms is critical for understanding the host range, tropisms, pathogenesis, and for developing effective vaccines and antiviral drugs to curb the spread of pedv in pigs. in this review, we provide an overview of different mechanisms used by pedv to evade host innate immune responses. pedv is an enveloped, positive-sense, single-stranded rna virus. the pedv genome constitution represents a standard cov arrangement. the viral genome is approximately kb in length, containing a terminal cap, a poly (a) tail, as well as seven open reading frames (orfs), including the orf a, orf b, s, orf , e, m, and n genes ( figure ) [ ] [ ] [ ] . the n terminal orf a and orf b encode two large replicase polyprotein precursors (pp a and pp ab), which are subsequently processed into nonstructural proteins (nsp to ). orf a encodes pp a which is cleaved by viral proteases into nonstructural proteins (nsp -nsp ). the orf b generates five additional nonstructural proteins (nsp - ) that are proteolytically cleaved by the viral proteases from pp ab [ ] . nsp contains two papain-like protease domains (plp and plp or pl pro ) that cleave the nsp - region of the replicase polyprotein at three sites. nsp , a chymotrypsin-like enzyme also known as c-like protease, cleaves the polyprotein at remaining cleavage sites [ ] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf [ ] [ ] [ ] . the nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [ ] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf [ ] [ ] [ ] . the nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [ ] . the viral proteins of pedv perform different biological functions during viral entry, replication cycle and propagation (table ) . pedv s protein, a type i membrane glycoprotein protein located on the envelope of the virus, consists of an n-terminal signal peptide, a large extracellular region, a single transmembrane domain, as well as a short cytoplasmic tail [ , ] . the ectodomain of s protein comprises s and s subunits. the n-terminal s region, containing n-and c-terminal domains (s -ntd and s -ctd), is mainly responsible for receptor binding [ ] . the c-terminal membrane-anchored s region is mainly involved in triggering the fusion of the viral envelope with host cell membranes [ , ] . the interaction of the cov s protein with its host cell surface receptor is a key determinant for host tropism. the s -ctd of most known members of α-cov genus, including pedv, interacts with aminopeptidase n (apn) to entry into the target cell [ , , [ ] [ ] [ ] [ ] . in addition, s protein contains the epitopes that are the major targets of the neutralizing antibody. n protein is the most abundant viral protein during the early phase of infection in cov-infected cells [ ] . similar to the n proteins of other covs, the pedv n protein has multiple functions, such as acting as a structural protein that forms nucleocapsid with viral genomic rna, playing important roles in viral replication, transcription, and assembly [ , ] . the expression of the n protein in intestinal epithelial cells extends the s-phase of cell cycle, causes endoplasmic reticulum (er) stress, and upregulates interleukin- expression [ ] . moreover, the n proteins of several α-covs and β-covs, including pedv, pdcov, sars-cov, and mouse hepatitis virus (mhv), have been identified as innate immunity antagonists [ ] [ ] [ ] [ ] . however, the involved antagonistic mechanisms are particularly different. pedv m protein participates in virion assembly and virus budding through collaboration with other viral proteins, and engages in the induction of neutralizing antibodies against pedv [ , ] . pedv m protein is distributed throughout the cytoplasm. it induces the cell growth retardation in intestinal epithelial cells (iec) and arrests the cells in s-phase [ ] . in addition, pedv m protein is identified as an interferon (ifn) antagonist with an unrecognized mechanism [ ] . pedv e protein is important for the virus packaging and budding [ ] . it is predominantly localized in the er, having no effect on cell growth, cell cycle and cyclin a expression in iec. however, it causes er stress and activates the nuclear factor-κb pathogens , , of (nf-κb) pathway which is responsible for the up-regulation of il- and the anti-apoptotic protein bcl- expression [ ] . orf has been predicted to possess multiple transmembrane domains [ ] , while it is predominantly distributed in the cytoplasm [ ] . orf also detains cells at s-phase, facilitating vesicle formation, and thus promoting pedv multiplication [ ] . a recent study suggests that orf interacts with s protein during pedv assembly and consequently benefits viral replication [ ] . cov nsps play multiple roles in the synthesis or processing of viral rna, or in virus-host interactions aiming to create an optimal environment for virus replication, such as facilitating viral entry, viral gene expression, rna synthesis, and virion release. nsp is a n-terminal cleavage product of orf a polyprotein [ ] , a -kda protein, that exists only in α-covs and β-covs [ ] . the nsp of α-covs is not very similar to β-covs nsp with regard to sequence homology and size [ , ] . based on the sequence alignment analysis of the genomes of different covs, the viral nsp can be regarded as a genus-specific marker [ ] . moreover, β-covs nsp has been widely reported to inhibit host protein expression. however, the biological functions of α-covs nsp remain largely unknown. despite the lack of overall sequence similarity, the nsp of different covs shares a similar function to interfere with host protein expression [ ] . these studies suggest the importance of nsp in the life cycle of different lineages of covs. it is shown that tgev nsp inhibits host gene expression and is critical for viral virulence [ ] . pedv nsp induces the degradation of cbp and nf-κb to abate ifn response [ ] , but the detailed mechanisms remain unclear. the sizes and amino acid sequence identity of nsp are variable among different covs [ ] . nsp of mhv and sars-cov are involved in viral rna synthesis [ ] . pedv nsp has unknown functions in replication, and may implicate the virus-host interactions and virulence. nsp is the largest nsp protein, containing two papain-like protease (plp and plp ) domains, of which pedv plp acts as a viral deubiquitinase (dub), to negatively regulate type i ifn signaling [ ] . covs plps domains exhibit multiple functions, serving as a viral protease, dub, as well as an ifn antagonist [ ] . covs, like other positive-stranded rna viruses, induce membranous rearrangements of varying morphologies that are essential for rtcs anchoring [ , ] . the cov-induced replicative structures consist of double-membrane vesicles (dmvs) and convoluted membranes (cms), which form a large reticulovesicular network that are critical for viral replication and transcription [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . among the cov nsps, nsp , nsp , and nsp include the hydrophobic transmembrane domains engaging in anchoring the viral rna synthesis components to the membranes [ ] . for mers-cov and sars-cov, co-expression of nsp and nsp is required to induce dmvs [ ] . sars-cov nsp has membrane proliferation ability as well, which also contributes to dmvs formation [ ] . the structure and functions of α-cov nsp are largely unknown [ ] . nsp is also a marker for cov-induced membrane structures; some results indicate that the nsp - of pp a act as a large complex through multidomain structure or scaffold during viral rna replication progress, before its cleavage into individual products [ ] . covs nsp encodes a c-like proteinase ( cl pro ). the polyproteins pp a and pp ab are processed into individual elements of replicase by c-like protease and plps [ ] . moreover, pedv nsp plays a crucial role in virus replication and also blocks host innate immune responses [ ] . crystallographic or nuclear magnetic resonance structures have shown that nsp , nsp , nsp , nsp , nsp , and nsp have the plprob and the adp-ribose -phosphatase (adrp) activity [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the crystal structure of sars-cov nsp suggests that nsp is dimeric and it is able to bind to single-stranded rna [ ] . the crystal structure of sars cov nsp protein suggests that nsp is a zinc-finger protein, which is existent exclusively in covs so far [ ] . moreover, nsp - have rna binding activity and nsp encodes a single rna-dependent rna polymerase (rdrp). the biochemical characterization and crystallization of sars cov nsp and nsp manifests that eight copies of nsp and eight copies of nsp form a supercomplex [ ] . the complex is supportive for nucleic acid binding and may be associated with the processivity of viral rdrp [ , ] . recently, structural studies have described that the sars-cov nsp polymerase binds to the nsp and nsp complex [ ] that may increase the polymerase activity of nsp rdrp [ ] . cov nsp , a ntpase/helicase, is also determined to play essential roles in viral replication [ ] . cov nsp is a multifunctional protein with - pathogens , , of exoribonuclease activity and n- -methyltransferase [mtase] activity [ , ] . nsp catalyzes the n -methylation of gppp-rna to form a cap- structure. cov nsp encodes an endoribonuclease (endou), performing functions through a hexamer in many covs [ ] [ ] [ ] . the endoribonuclease activity of nsp is not essential for cov replication [ , ] . for covs, the end of the viral genomic rna and subgenomic mrna (sgmrna) is supposed to have cap structures: an n- methylated guanosine nucleoside (m gpppn) (cap ) and a methyl group at the -o-ribose position (cap ) of the first nucleotide [ ] . these cap structures enhance the initiation of translation of viral proteins, protect viral mrnas against cellular - -exoribonuclease and limit the recognition of viral rna by host innate system [ , ] . nsp is proposed to catalyze the first step of the -capping reaction of viral rnas [ ] . the methylation of the two sites in the cap are catalyzed by three nsps; nsp (the n- -mtase), nsp (the -o-methyltransferase), and nsp [ , [ ] [ ] [ ] [ ] . in addition, the - exoribonuclease activity of nsp is involved in a replicative mismatch repair system during rna synthesis, which improves the replication fidelity of cov [ ] . although these nsps have been demonstrated to play essential roles in viral replication, transcription and/or post-translational polyprotein processing [ ] , the nsp - of pedv and other covs are poorly characterized to date, except for sars-cov. nsp is an ifn antagonist; nsp inhibits type iii ifn response; nsp is involved in nucleic acid binding; nsp enhances the inhibitory effect of nsp on ifn-β production. [ , [ ] [ ] [ ] nsp encoding a rdrp; viral replication [ ] nsp a ntpase/helicase that is essential for viral replication [ ] nsp n- -mtase; catalyzing n -methylation of gppp-rna to form a cap- structure; - exoribonuclease activity involves in a replicative mismatch repair system during rna synthesis; ifn antagonist [ , , , host cells generally defend against virus infection by mounting an innate antiviral immune response to prevent the spread of the infection and aid in initiating an adaptive immune response which eventually removes the viruses from host. therefore, the first barrier to restrain viral infections is the host innate immune system, which is related to multiple proteins and mechanisms, including ifns, inflammatory cytokine, apoptosis, autophagy, and so on. the activation of type i ifn responses is composed of three stages: ( ) recognition of pathogen-associated molecular pattern (pamp) by prrs; ( ) secretion of type i ifns through paracrine or autocrine pathways; and ( ) expression of numerous antiviral ifn-stimulated genes (isgs) which bring the host into the antiviral state [ ] . at least three important prrs have been identified in recognition of viral nucleic acids, including retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) (detection of viral rna in the cytoplasm) [ ] , the membrane-bound toll-like receptors (tlrs) (recognition of viral rna or dna in the endosome) [ ] , as well as a structurally unrelated group of viral dna sensors (e.g., cgas (cyclic gmp-amp synthase) and ifi ) localized in the host cytoplasm and/or nucleus [ ] . in the cytosol, the formation of specific secondary structure of viral rna is closely related to viral rna delivery and replication. these molecular signatures are detected by rlrs including rig-i (also known as ddx ), melanoma differentiation-associated gene (mda ), and laboratory of genetics and physiology- (lgp ) [ , ] . prrs recognize pamps, inducing an intracellular signaling cascade, thus leading to the activation of transcription factors such as irfs and nf-κb, which in turn induce the production of ifns [ ] . both rig-i and mda have two n-terminal card domains to interact with the card domains of the downstream adaptor proteins, and a dead/h-box rna helicase domain for rna binding. however, lgp does not have the n-terminalcard domains, and the involved functions remain unclear [ ] [ ] [ ] . dsrna, as a specific secondary structure of viral rna, can be sensed by rig-i/mda to induce ifn-α/β production through the cascade activation of the rlr pathway [ ] [ ] [ ] . activated rig-i/mda forms homo-oligomers and recruits the adaptor mitochondrial antiviral signaling (mavs) to induce the formation of signaling complex of mavs with other proteins on mitochondria. tnf receptor-associated factor (traf ) associates with tnfr -associated death domain protein (tradd), tripartite motif (trim ), and pyruvate carboxylase (pc), resulting in the activation of iκb kinases (ikks). ikks (ikkα, ikkβ and ikkγ) phosphorylate nf-κb inhibitor (iκb), leading to the ubiquitination of iκb and its subsequent degradation. nf-κb is then activated and translocated into the nucleus to turn on the expression of proinflammatory and type i ifn. mavs also recruits and activates tbk /ikkε by trafs that are pre-associated with tbk /ikkε, via direct interaction between the sdd domain of tbk /ikkε and the coiled-coil domain of trafs [ ] . activated tbk /ikkε phosphorylates ifn regulatory factors (irf /irf ), that further dimerize and import into the nucleus to promote type i ifn production. on the other hand, mavs interacts with mita (also known as sting), that is located in mitochondria and the endoplasmic reticulum (er) membrane. mita interacts with the trap complex, which may be involved in recruiting tbk and ikkε to phosphorylate irf . ubiquitination and deubiquitination are decisive in the regulation of rlr pathways activation [ ] . upon binding to viral dsrna, rig-i and mda undergo conformational changes and release the n-terminal tandem card domains [ ] [ ] [ ] . the card domains of rig-i are modified by lysine (k ) polyubiquitin chains through the ubiquitin ligases trim , rnf , and riplet. this modification is crucial for rig-i to recruit mavs [ , ] . in addition to the ubiquitination of rlrs, the polyubiquitylation of traf and traf also play an important role in the regulation of innate immune signaling by activation of tbk and ikks, respectively. the k polyubiquitin chains can be removed by dubs such as the tumor suppressor protein cyld, duba and a , providing a mechanism to downregulate immune responses [ ] . ubiquitination and deubiquitination are in a dynamic equilibrium to maintain immune homeostasis. type i ifns are secreted by secretory cells and peripheral cells through self-secretion or paracrine secretion manners. extracellular type i ifns bind to a heterodimeric complex composed of subunits of ifn-α receptors (ifnar ) and (ifnar ) located on the cell surface, which activates the tyrosine kinase (tyk )/janus kinase (jak ) signal transducer. tyk /jak subsequently induces the phosphorylation of transcription factors stat and stat , which dimerize and in turn recruit ifn-regulatory factor (irf ), to form stat -stat -irf trimerized complex (isgf ). this complex then translocates to the nucleus, where it binds to the ifn-stimulated response elements (isre motif; conserved sequence is tttcnntttc) [ ] . the binding of isgf to isre finally triggers expression of ifn-stimulated genes (isgs) that directly or indirectly exert antiviral effects in host cells [ ] . three types of ifns (types i, ii and iii) have been identified. type i and type ii ifns have been widely reported. type ii ifns only contain ifn-γ. ifn-γ is produced by natural killer (nk) cells and activated cd + and cd + t cells in response to the cytokines such as interleukin- (il- ) and il- [ ] . ifn-γ binds to the type ii ifn receptor composed of two subunits, ifngr and ifngr . ifngr and ifngr induce the formation of stat -stat homodimers. stat -stat homodimers translocate to the nucleus and bind to the promoter of the ifn-γ-activation site (gas) elements, to initiate the transcription of ifn-γ-regulated genes [ ] . type iii ifns have been explored in recent years, to unravel the underlying mechanisms that manipulate host innate immune responses. type iii ifns in humans contains ifn-λ (interleukin ), ifn-λ (il- a), ifn-λ (il- b), and ifn-λ [ ] [ ] [ ] . their expression profiles, signaling pathways, and gene expression programs resemble those of type i ifns. the production of type i and iii ifns are both initiated through the recognition of pamps or damage associated molecular patterns (damps) by prrs [ ] . despite the fact that the same transcriptional factors are required for the activation of promoters of type i and iii ifns, the nf-κb pathway is a pivotal regulator in ifn-λ production, whereas the irfs pathway dominates type i ifns expression. the promoter of ifn-λ includes more nf-κb binding sites compared with in the ifn-β promoter [ ] . the signal transduction of type iii ifns depends on the ifn-λ-specific receptor, il- ra chain and il- r chain [ , ] . synthetic ifn-λ binds to il- rα and induces a conformational change within the receptor subunits, that triggers the activation of the receptor-associated tyrosine kinases (tyk and jak ), which then phosphorylate stat and stat . stat and stat are heterodimerized and interact with irf to form the isgf transcription complex that binds to isre in the promoters of isgs, to induce the expression of hundreds of proteins with antiviral functions [ ] . induction of ifn-α/β is the most rapid and effective mechanism for hosts to initiate antiviral innate immune responses. sars-cov, mhv and many other covs are sensitive to ifns. a great number of viral dsrnas intermediates are generated during covs infection that contribute to ifn production, but these covs remain highly pathogenic. as a matter of fact, covs have developed a set of elaborate mechanisms to evade or inhibit the host antiviral innate immune response during virus evolution [ , ] . the evasive strategies utilized by pedv are classified into four major types: ( ) inhibition of rlrs-mediated ifn production pathways, ( ) inhibition of the activation of transcription factors responsible for ifn induction, ( ) disruption of the signal cascades induced by ifn, and ( ) hiding its viral rna to avoid the exposure of viral rna to immune sensors. in the past decade, accumulating evidence demonstrates that pedv n protein, nsp , plp , nsp , nsp , and nsp antagonize type i ifn or type iii ifns production [ , , , , , , , ] . this explains why only weak ifns' and cytokines' expression is detected in pedv-infected cells [ , ] . n protein, as an abundantly produced structural protein within cov-infected cells, has multiple functions, including virus replication, transcription, and assembly [ , ] . pedv n protein has been identified as an ifn antagonist that blocks the expression of ifn-β and isgs by suppression of the pathogens , , of irf and nf-κb activities [ ] . pedv n protein inhibits the activation of the ifn-β promoter induced by tbk and its upstream rig-i, mda- , visa, and traf , while not affecting the activation of the ifn-β promoter driven by irf . further experiments confirm that n directly interacts with tbk to obstruct the association between tbk and irf , which inhibits tbk -induced irf phosphorylation and ifn-β production [ ] . moreover, the effect of pedv n protein on type iii ifn production has also been evaluated [ ] . n protein inhibits polyinosinic-polycytidylic acid (poly(i:c))-induced ifn-λ production by blocking the nuclear translocation of nf-κb, but does not antagonize the type i or type ii ifn expression induced by poly(i:c) in ipec-j cells [ ] . recent studies show that sars-cov n protein inhibits type i ifn production through suppressing trim -mediated rig-i ubiquitination [ ] . the mers-cov n protein also blocks ifn production by interacting with trim [ ] . in addition, both mhv and sars-cov n proteins perturb the function of cellular protein activator of protein kinase r (pact), which can bind to rig-i and mda to activate ifn production, and thus antagonize type-i ifn signaling [ ] . these results indicate the important function of the covs n protein in modulating host innate immune response. whether pedv n protein targets trim or pact should be investigated. although several studies have been performed to understand the pathogenicity of pedv, there remains limited information about the interaction between viral proteins and host cell factors during viral infection. cov n protein is a vital viral protein involved in virus replication. current researches have indicated that n protein interacts with many host proteins, such as hcypa [ ] , proteasome subunit p [ ] , smad [ ] , hnrnp-a [ ] , and the chemokine cxcl [ ] . in the host cells, a large number of host proteins reveal various functions. however, for the virus, the genome only encodes several limited viral proteins. therefore, these viral proteins have to be multifunctional, which is pivotal to virus replication and existence. pedv nsp is the n-terminal cleavage product from polyproteins pp a and pp a/b processed by nsp and nsp [ ] and is about amino acids in length [ , ] . nsp of many α-cov and β-cov exhibits both functional conservation and mechanistic diversity in suppressing host gene expression and ifn signaling. for sars-cov, nsp triggers the decay and cleavage of host mrna and inhibits host protein translation, subsequently inhibiting type i ifn production [ , ] . sars-cov nsp also blocks the expression of ifn-inducible genes, by restraining the signal transduction during virus infection [ , ] . the tgev nsp considerably suppresses host protein expression during viral infection [ ] . structural studies show that the core structure of pedv nsp is highly similar to those of sars-cov nsp and tgev nsp [ ] . pedv nsp inhibits host gene expression and three motifs (amino acids to , to , and to ) form a stable functional region for inhibition of host protein synthesis, differing considerably from sars-cov nsp [ ] . pedv nsp has been identified as an ifn antagonist, which constrains poly (i:c)-induced ifn-β promoter activity [ ] . nsp significantly inhibits the activation of ifn-β promoter triggered by irf , whereas it does not inhibit irf phosphorylation and its nuclear translocation. nsp interrupts the association of irf with creb-binding protein (cbp), by promoting cbp degradation in the nucleus via the proteasome-dependent pathway. cbp/p , the transcription co-activator camp responsive element binding protein (creb), forms a complex with the activated irf in nucleus. the irf -cbp/p complex binds to the positive regulatory domain (prd) regions of the ifn-β promoter, to assemble the enhanceosome with nf-κb and other factors, which ultimately turn on the transcription of type i ifn genes [ ] [ ] [ ] . therefore, pedv nsp blocks type i ifn production in the nucleus. activated nf-κb induces the production of type i ifns and proinflammatory cytokines and is important for inhibiting viral infection. pedv nsp has been shown to interfere with the nf-κb activity [ ] and is the most potent suppressor of proinflammatory cytokines at early infection. it inhibits the phosphorylation and degradation of iκbα, and blocks p nuclear translocation, leading to the suppression of both ifn and the early production of pro-inflammatory cytokines [ ] . moreover, pedv inhibits type iii ifn production and nsp , nsp , nsp , nsp , nsp , nsp , nsp , orf , e, m, and n are identified as type iii ifn antagonists. among these antagonists, nsp is the most potent suppressor [ ] . pedv nsp blocks the nuclear translocation of irf and decreases the amounts of peroxisomes and then suppresses irf -mediated type iii ifns. the conserved residues of pedv nsp protein are crucial for ifn suppression [ ] . multiple effects of nsp on modulating innate immune response during pedv infection suggest the vital role of nsp in the pedv replication cycle. the antiviral innate immune signaling pathways are regulated by several posttranslational modifications (ptms), such as phosphorylation, ubiquitination, glycosylation, neddylation and sumoylation [ ] , of which ubiquitination is a critical modification to modulate the stability and activity of prrs and other components of innate immune signaling pathways. during viral infection, a reciprocatory action (occurrence of ubiquitination and deubiquitination) helps maintain the homeostasis of host immune responses. hence, deubiquitinases (dubs) are indispensable in the regulation of virus-induced type i ifn signaling [ ] . many host dubs have been reported engaging in the regulation of innate immune signaling pathways [ ] [ ] [ ] . in recent years, a variety of viral dubs have been discovered to target key components of type i ifn pathway during various rna virus infections. for example, foot-and-mouth disease virus leader proteinase (fmdv lb pro ) [ ] , and porcine reproductive and respiratory syndrome virus nsp (prrsv nsp ) possess ubiquitin-deconjugating activity to deubiquitinate key host components [ , ] . to counteract host antiviral response, covs likely take advantage of dub activity to break host innate immunity. indeed, the plps of mouse hepatitis virus a (mhv-a ) [ ] , sars [ ] , and human cov nl have dub activity and antagonize ifn induction [ ] . pedv plp has been reported as having a deubiquitinase activity as well, and it can be co-immunoprecipitated by rig-i and sting. as mentioned above, fmdv lb pro , mhv plp and sars plps all counteract host innate immune response through blocking the ubiquitination of the components of rlrs pathways. similarly, pedv plp removes the ubiquitinated conjugates from rig-i and sting by its dub activity, to negatively regulate type i ifn production. pedv plp probably interacts with rig-i and sting, which prevents the activation of rig-i and sting by hindering the recruitment of downstream signaling molecules. as expected, the interference with the ubiquitination of rig-i and sting by plp clearly benefits pedv replication [ ] . pedv nsp contains two core domains of plps (plpl and plp ). it is determined that pedv plp , but not plp , inhibits the ifn-β promoter activation in hek t cells. the dub activity of plp is highly dependent on its catalytic activity. three catalytically inactive mutants of pedv plp (c a, h a and d a) are defective in the deubiquitination of its targets and fail to impair virus-induced ifn-β production. sars-cov plp interacts with mdm (mouse double minute homolog) to deubiquitinate and stabilize mdm , approving the degradation of p and the suppression of ifn signaling [ ] . pedv infection degrades p by upregulation of mdm expression [ ] . pedv plp may be responsible for targeting the p pathway and inhibiting p -dependent apoptosis, leading to immune evasion. a recent study determined that tgev pl inhibits the ifn-β expression and interferes with the rig- and sting-mediated signaling pathway through a viral dub activity [ ] . it suggests that different viral proteins are involved in the deubiquitination of host proteins for different covs. however, these studies offer a probability to design a common therapeutic against different viral dubs to reduce the replication and pathogenesis of covs. therefore, further studies are required to understand more about the substrate specificity of these viral dubs and clarify the precise functions of cov protease/dub activity. notably, c pro is a critical ifn antagonistic protein identified in multiple different families of viruses. the c pro of picornaviruses, including fmdv [ ] , hepatitis a virus (hav) [ ] , enteroviruses (ev , ev-d ) and coxsackieviruses (cvb , cv-a , cv-a ) [ ] [ ] [ ] [ ] , antagonize innate immune signaling by targeting the critical components of the ifn pathways for proteolysis. a newly emerged picornavirus, seneca valley virus (svv), has also evolved an effective mechanism to escape host antiviral innate immune using its c pro . moreover, c pro cleaves the signaling components (mavs, trif, and tank) of type i ifn pathway and induces the degradation of the transcription factors irf and irf to constrain host antiviral response [ , ] . cov nsp is called c-like protease ( cl pro ), that resembles the c pro of other rna viruses. for cov, the polyprotein precursors (pp a and pp b) are mainly processed to generate mature nonstructural proteins by cl pro . to date, the cl pro of covs, including pedv and pdcov, have been confirmed to antagonize type i ifn production by the cleavage of nf-κb essential modulator (nemo) and stat [ , , ] . nemo is essential for rna virus-induced activation of nf-κb, irf , and irf [ ] . nemo is required for mavs-induced ikkα/β activation and is also crucial for the activation of tbk /ikkε [ ] . to establish successful infections, pedv targets nemo to subvert host innate immune responses. pedv nsp significantly inhibits sendai virus (sev)-induced ifn-β synthesis and the process depends on its protease activity [ ] . further experiments show that pedv nsp inhibits rig-i/mda signaling and targets the upstream of tbk . the cleavage of nemo by nsp is identified as responsible for this inhibitive effect. the pedv nsp -mediated cleavage of nemo efficiently blocks nemo-mediated downstream signaling. the cleavage site within nemo that is grasped by nsp has been determined. of these reported immune evasion strategies employed by covs, the cleavage of innate immune adaptors is a particularly effective manner to disrupt antiviral responses. nsp is essential for the life cycle of pedv and other covs [ , ] . it is a potential target for the development of anti-coronaviral therapeutics. although pedv nsp does not target stat mediated type i ifn signaling pathway, pedv nsp has been reported to inhibit the stat and stat induced activation of isre [ ] . nsp competes with karyopherin α (kpna ), which is an adaptor mediating nuclear translocation of isgf , in combination with stat , to block isgf nuclear transport. however, the expression and phosphorylation of stat and stat are not affected by pedv nsp . in fact, pedv infection degrades stat , leading to the inhibition of ifn signaling [ ] . therefore, other pedv encoded proteins likely target ifns mediated signaling. covs belong to rna viruses, which produce several rna species, such as dsrna intermediates and rna with a -triphosphate during replication. these rna intermediates are potent stimulators of prrs and are associated with the organelles of viral rna replication, dmvs [ , ] . dmvs formed from membranous rearrangements seem to sequester the replication intermediates using membrane-bound vesicles or invaginations to keep away from prrs. therefore, the form of dmvs may be a strategy for pedv to escape innate immune recognition in the cytosol (figure ). however, whether dmvs alone are sufficient to shield rna from prrs remains unknown. besides, the replication organelles, the endoribonuclease activity and viral end rna capping/protection mechanisms are also the critical ways of avoiding rna recognition or protecting it from degradation [ , ] . ( ) pedv nsp cov nsp has endou catalytic activity that was initially thought to play a vital role in virus replication. however, the catalytic-defective endou of mhv shows only a subtle defect in viral replication compared to wt virus in fibroblasts [ ] . similar results are found for the nsp mutants of sars-cov and hcov- e [ ] . these findings suggest that the endou activity of nsp is not required for rna synthesis. recently, nsp has been demonstrated to act as a new ifn antagonist of covs [ , , ] . recent reports indicate that covs' endou activity is essential for prevention of rna recognition by mda , protein kinase r (pkr), and oas/rnase l system [ ] . pkr and oas/rnase l recognize and destroy foreign rna in the cytosol to defend viral infections. to counteract the function of pkr and oas/rnase l, the virus hides or modifies its viral rna, to avoid the exposure of viral rna to these molecules. in all covs, the endou catalytic domain in nsp is highly conserved. pedv endou activity has been indicated as having an antagonistic effect on ifn signaling [ ] . the endou activity of pedv nsp not only inhibits the type i ifn response in porcine macrophages, but also antagonizes the type iii ifn response in porcine epithelial cells. the replication of endou-mutant pedv (icpedv-enumt) is considerably impaired in porcine epithelial cells compared to the wild type pedv (icpedv-wt). the icpedv-enumt clearly induces early and robust type i and type iii ifns production, as well as isgs' expression compared with that induced by icpedv-wt. the endou-deficient pedv infected animals also show reduced viral shedding and mortality. these results indicate that the endou activity of pedv nsp plays a vital role in evading host antiviral innate immunity (figure ) [ ] . ( ) pedv nsp cov nsp is a -o-methyltransferase ( -o-mtase). to evade recognition by the host immune sensors, many covs encode methyltransferases involved in the capping of viral rna. this modification makes the viral rna indistinguishable with host cell mrna, which is important to avoid the recognition of viral rna by mda ( figure ) [ , ] . methylation of the two sites in the cap is catalyzed by three nsps, including nsp (the n- -mtase), nsp (the -o-mtase), and nsp [ , [ ] [ ] [ ] [ ] . for example, sars-cov nsp acts as a -o-mtase to prevent innate immune recognition and promote viral proliferation [ , , ] . pedv nsp and nsp have been identified as the viral ifn antagonists [ , ] . the overexpression of nsp or nsp remarkably inhibits ifn-β production, but nsp appears to play a more important role in innate immunity regulation than nsp [ ] . nsp is a highly conserved methyltransferase which contains an invariant kdke motif within the methyltransferase core [ ] . this kdke motif is required to mediate its activity. notably, the mutation of any of the kdke active sit has been shown to abolish the -o-mtase activity [ ] . pedv nsp kdke motif plays a critical role in the inhibition of type i ifn production, suggesting the important role of the -o-mtase in pedv-mediated immune evasion. pedv nsp negatively regulates rlr-mediated signal pathway activation, and inhibits the expression of the ifn-stimulated ifit family members (ifit , ifit , ifit ), which in turn promotes pedv replication. taken together, these results demonstrate that pedv nsp negatively regulates cellular antiviral response to promote viral replication [ ] . screening inhibitors targeting the -o-mtase of nsp might be a prominent strategy to inhibit cov infections and develop antivirals for treatment of the diseases caused by covs. additionally, covs nsp also includes the -to- exoribonuclease (exon) activity [ ] . a mutation of tgev nsp exon generates lower levels of dsrna than wildtype tgev and thus triggers a reduced antiviral response [ ] . nsp exon activity is also critical for the resistance of host innate immune response in mhv-infected cells [ ] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. activity is also critical for the resistance of host innate immune response in mhv-infected cells [ ] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. heat shock protein (hsp ) belongs to the small heat shock proteins family, which has been identified as a multifunctional protein involved in cytoskeletal stability, proinflammatory processes, and the inhibition of apoptosis [ , ] . several hsps have been reported to be implicated in pedv infection in vitro and in vivo [ , ] . indeed, the infection of many viruses up-regulates hsp expression by different mechanisms to delay cellular apoptosis, then supplies sufficient time for viral replication [ , ] . however, pedv infection significantly induces the decreased expression of hsp in vero cells [ ] and marc- cells [ ] . hsp activates the phosphorylation of nf-κb, and thus promotes the mrna expression of ifn-β in marc- cells. as hsp is an upstream regulator of antiviral immune signaling, overexpression of hsp significantly inhibits the pedv replication. pedv has developed a strategy via mediating the suppression of hsp production to escape from host innate immune response [ ] . hsp , the most conserved hsp, is also important for the multiplication of several covs. the recruitment of hsp is thought to be a viral survival strategy for several viruses in their hosts [ ] . the relationship between hsp and pedv should be exploited further in future. viral infection triggers host immune response to induce ifns' and inflammatory cytokines' production. released ifns elicit the expression of numerous isgs which limit viral replication in the infected cells. however, the release of excessive amounts of ifns and inflammatory cytokines will lead to autoimmune and auto-inflammatory diseases. the concomitant uncontrolled apoptosis is also one outcome that is harmful to the host. to maintain the reaction in a proper balance, hosts have evolved a series of effective mechanisms to control the antiviral innate immune response [ ] . in contrast, viruses often break this balance, causing improper apoptosis reaction, which benefits viral replication. pedv infects various host cells including vero, pk- and marc- and cause obvious cytopathic effects. pedv-induced apoptosis of the infected cell has been demonstrated both in vitro and in vivo [ ] . apoptosis is induced through the activation of apoptotic caspases, including caspase- , - , - , - , - , - , and - [ ] . pedv infection results in obvious caspase- and caspase- activation, as well as the cleavage of apoptosis-inducing factor mitochondria-associated (aifm ) and poly adp-ribose polymerase (parp), which leads to apoptotic nuclear fragmentation. pedv spike protein s significantly elicits host cell apoptosis, while the nsp - and other structural proteins (m, n, e, s , and orf ) have none or few effects on cell apoptosis. therefore, s protein is probably the critical protein mediating the apoptosis induced by pedv [ ] . the multiple stages of cov replication cycle are closely associated with cellular membrane compartments, especially the endoplasmic reticulum (er). the shape and functions of er can be influenced by different physiological states and environmental conditions. when protein synthesis amounts surpass the folding capacity, the accumulation of a large amount of unfolded proteins in the er leads to er stress. consequently, cells manifest a corresponding biological reaction that is widely known as the unfolded protein response (upr) [ ] . once the upr is induced, it alleviates the problems by host protein translation inhibition (by the transducer pkr-like er protein kinase (perk)-induced phosphorylation of eif a), stimulating protein folding. if homeostasis cannot be re-established, apoptosis eventually is triggered. indeed, the activation of upr regulates a wide variety of signaling pathways, such as apoptosis, autophagy, mitogen-activated protein (map) kinase activation, and innate immune response [ ] . furthermore, α-cov and β-cov may induce er stress in the infected cells [ ] . pedv orf , as the only accessory protein encoded by pedv, is thought to be related to virus production and virulence of pedv [ ] . a series of studies suggest that orf plays multiple roles, in addition to acting as an ion channel during pedv replication. recent studies show that pedv orf consists of four transmembrane domains (tmds) and localizes in the cytoplasm in the aggregation manner [ ] . orf is a transmembrane protein, and the confocal microscopy analysis indicates that the aggregated orf localizes in the er to induce the er stress associated with either apoptosis or autophagy. however, pedv orf induces the autophagy via driving conversion of lc -i to lc -ii, but not influencing the apoptosis. orf -induced autophagy is dependent on er stress response. pedv orf triggers er stress response via the up-regulation of grp protein expression and the activation of the perk-eif α signaling pathway. moreover, orf protein is identified as an ifn antagonist to block ifn response by an unknown mechanism in pedv-infected cells [ ] . the functions of pedv orf should be further exploited. pedv has caused epidemic and endemic infections in pig populations in many countries and has become a major economic threat to the swine industry. previous studies have identified viral factors that target key signaling molecules in the rlrs' pathways, as well as viral factors that target the downstream signaling pathways responsible for isgs induction. of the pedv-encoded proteins, at least viral proteins have been identified as type i ifn antagonists [ , ] . the mechanisms utilized by pedv nsp , plp , nsp , and n protein to antagonize type i ifn production have been clarified (figure , [ , , , , ] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp , as well as nsp , has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. have been clarified (figure , [ , , , , ] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp , as well as nsp , has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. removes ubiquitinated conjugates from rig-i; pedv nsp induces cleavage of nemo; pedv n protein directly interacts with tbk to obstruct the association between tbk and irf ; pedv nsp causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p activation. pedv nsp inhibits type i ifn production and nsp enhances the inhibitory effect of nsp on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp blocks the nuclear translocation of irf and reduces the amounts of peroxisomes. pedv nsp inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp interacts with stat and stat to block nuclear translocation of isgf . removes ubiquitinated conjugates from rig-i; pedv nsp induces cleavage of nemo; pedv n protein directly interacts with tbk to obstruct the association between tbk and irf ; pedv nsp causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p activation. pedv nsp inhibits type i ifn production and nsp enhances the inhibitory effect of nsp on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp blocks the nuclear translocation of irf and reduces the amounts of peroxisomes. pedv nsp inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp interacts with stat and stat to block nuclear translocation of isgf . author contributions: the manuscript was prepared by s.l., j.y., z.z., and h.z., and reviewed by the co-authors. all authors have read and agreed to the published version of the manuscript. outbreak of porcine epidemic diarrhea in suckling piglets porcine epidemic diarrhea in china experimental infection of pigs with a new porcine enteric coronavirus, cv an apparently new syndrome of porcine epidemic diarrhoea virus-like particles associated with 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orf protein causes endoplasmic reticulum stress to facilitate autophagy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are thankful to everybody who participated in the studies. the authors declare no conflict of interest. key: cord- - g zxne authors: jartti, t.; palomares, o.; waris, m.; tastan, o.; nieminen, r.; puhakka, t.; rückert, b.; aab, a.; vuorinen, t.; allander, t.; vahlberg, t.; ruuskanen, o.; akdis, m.; akdis, c. a. title: distinct regulation of tonsillar immune response in virus infection date: - - journal: allergy doi: . /all. sha: doc_id: cord_uid: g zxne background: the relationships between tonsillar immune responses, and viral infection and allergy are incompletely known. objective: to study intratonsillar/nasopharyngeal virus detections and in vivo expressions of t‐cell‐ and innate immune response‐specific cytokines, transcription factors, and type i/ii/iii interferons in human tonsils. methods: palatine tonsil samples were obtained from elective tonsillectomy patients. adenovirus, bocavirus‐ , coronavirus, enteroviruses, influenza virus, metapneumovirus, parainfluenza virus, rhinovirus, and respiratory syncytial virus were detected using pcr. the mrna expression levels of ifn‐α, ifn‐β, ifn‐γ, il‐ , il‐ , il‐ , il‐ , il‐ , il‐ , tgf‐β, foxp , gata , rorc , and tbet were directly analyzed by quantitative rt‐pcr. results: fifty percentage of subjects reported allergy, % had ≥ nasopharyngeal viruses, and % had ≥ intratonsillar viruses. tonsillar virus detection showed a strong negative association with age; especially rhinovirus or parainfluenza virus detection showed positive association with ifn‐γ and tbet expressions. il‐ expression was positively associated with atopic dermatitis, whereas ifn‐α, il‐ , il‐ , and tbet expressions were negatively associated with allergic diseases. network analyses demonstrated strongly polarized clusters of immune regulatory (il‐ , il‐ , tgf‐β, foxp , gata , rorc , tbet) and antiviral (ifn‐α, ifn‐β, il‐ , il‐ ) genes. these two clusters became more distinctive in the presence of viral infection or allergy. a negative correlation between antiviral cytokines and il‐ , il‐ , il‐ , foxp , and rorc was observed only in the presence of viruses, and interestingly, il‐ strongly correlated with antiviral cytokines. conclusions: tonsillar cytokine expression is closely related to existing viral infections, age, and allergic illnesses and shows distinct clusters between antiviral and immune regulatory genes. results: fifty percentage of subjects reported allergy, % had ≥ nasopharyngeal viruses, and % had ≥ intratonsillar viruses. tonsillar virus detection showed a strong negative association with age; especially rhinovirus or parainfluenza virus detection showed positive association with ifn-c and tbet expressions. il- expression was positively associated with atopic dermatitis, whereas ifn-a, il- , il- , and tbet expressions were negatively associated with allergic diseases. network analyses demonstrated strongly polarized clusters of immune regulatory (il- , il- , tgf-b, foxp , gata , rorc , tbet) and antiviral (ifn-a, ifn-b, il- , il- ) genes. these two clusters became more distinctive in the presence of viral infection or allergy. a negative correlation between antiviral cytokines and il- , il- , il- , foxp , and rorc was observed only in the presence of viruses, and interestingly, il- strongly correlated with antiviral cytokines. conclusions: tonsillar cytokine expression is closely related to existing viral infections, age, and allergic illnesses and shows distinct clusters between antiviral and immune regulatory genes. exacerbations of childhood asthma, adult asthma, and chronic obstructive pulmonary disease have been linked to positive virus detections in %, %, and % of the cases, respectively ( ) ( ) ( ) . susceptibility to certain viral infections and defects in viral clearance could play a role in pulmonary inflammatory processes. pediatric studies have shown a strong link between susceptibility to rhinovirus-associated wheezing and the development of asthma ( , ) . in addition, respiratory syncytial virus (rsv)-induced bronchiolitis has been linked to asthma and atopy development ( , ) . studies in adults have shown an association between persistent/latent abbreviations adv, adenovirus; bov, bocavirus- ; ct, cycle threshold; cv, coronavirus; ef -a, elongation factor- a; ev, enteroviruses; foxp , forkhead box protein ; flu, influenza a or b virus; ifn, interferon; il, interleukin; mpv, metapneumovirus; pbmc, peripheral blood mononuclear cells; pcr, polymerase chain reaction; piv, parainfluenza virus types - ; rorc , retinoic acid receptor-related orphan receptor c ; rsv, respiratory syncytial virus; rv, rhinovirus; suppl., supplementary; th, t helper cell; tgf-b, transforming growth factor-b. adenovirus infections and chronic lung diseases ( ) . moreover, the persistence of respiratory viruses, virus load, and virus coinfections have been related to more severe respiratory illnesses ( , ) . the susceptibility to rhinovirus infections has been associated with low interferon (ifn)-a/-b/-c/-k and interleukin (il)- responses and increased t helper cell (th) -type (il- , il- , il- ) cytokine responses in peripheral blood mononuclear cells (pbmc) ( , ) . similarly, low ifn-c response, increased il- /ifn-c ratio in pbmc, and low foxp + natural regulatory t-cell numbers in the circulation have been suggested as risk factors for rsv-induced bronchiolitis ( , ) . factors linked to the persistence of adenovirus infection are not well known ( ) . it is generally accepted that low or deficient innate and adaptive immune responses may contribute to the morbidity and mortality of viral infections. tonsils represent the primary nasopharyngeal lymphoid tissue and constitute the first contact point of the immune system to food and aeroallergens and infectious agents. a peripheral t-cell repertoire exists in tonsils, and tonsils have an active role in inducing and maintaining peripheral tolerance to allergens ( , ) . peripheral t-cell tolerance to allergens mediated by regulatory t (treg) cells is an essential mechanism in allergen tolerance developed by allergen-specific immunotherapy or natural high-dose allergen exposure ( , ) . considering tonsils as organs that have a role in immune tolerance induction, intensive research has been needed on the associations between the features of cytokine response and viral infections to better understand the link between respiratory viral infections and chronic respiratory diseases. therefore, we studied the associations between tonsillar cytokine and t-cell subset-related transcription factor expressions and respiratory viral infections. we investigated the in vivo intratonsillar mrna expressions of th (ifn-c, tbet), th (il- , gata ), th (il- , rorc ), and treg type (il- , tgf-b, foxp ) and type i/iii interferons (ifn-a, ifn-b, il- , il- ) and the recently discovered il- family cytokine il- in connection with intratonsillar virus expressions in elective tonsillectomy patients. human tonsil samples were obtained from consecutive routine tonsillectomy patients of all ages with or without reported allergy from satakunta central hospital, pori, finland. the inclusion criteria were elective tonsillectomy according to clinical indication and written informed consent from the study subject or his/her parents. the study protocol was approved by the ethics committee of satakunta central hospital, pori, finland. a standard questionnaire was used to obtain information on allergic diseases and respiratory symptoms within days before the operation (table s ). tonsillectomy was performed according to routine clinical procedure, and tonsils were stored in rnalater rna stabilization reagent (qiagen, hilden, germany) ( ) . during anesthesia, nasopharyngeal aspirate was also obtained using a standardized procedure and stored at À °c ( ) . more details are shown in supplementary methods. viral diagnostics of naive nasopharyngeal aspirates and intratonsilar samples were performed according to clinical routine using pcrs for adenovirus, bocavirus- , coronaviruses ( e, oc , nl , and hku ), enteroviruses, influenza a and b viruses, metapneumovirus, parainfluenza virus types - , respiratory syncytial virus and rhinovirus for all samples, and polyomaviruses ki and wu for samples ( , ) . gene expressions of ifn-a, ifn-b, ifn-c, il- , il- , il- , il- , il- , il- , tgf-b, foxp , gata , rorc , and tbet were analyzed by quantitative real-time pcr (applied biosystems, foster city, ca, usa) ( ) . for more details, see suppl. methods and table s . in addition to conventional statistics, linear and logistic regressions were used to analyze the associations between virus detection, age, and atopic characteristics (version . . , sas institute inc. cary, nc, usa). agreement statistics between intratonsillar and nasopharyngeal virus diagnostics were calculated with kappa coefficients. in heatmaps, partial spearman correlations between gene expressions were computed over different disease groups, controlling for age. the differences between gene-to-gene correlations according to disease groups were analyzed using the procedure developed by fisher ( ) . the statistically significant correlations are also displayed in network representations (cytoscape software, www.cytoscape.org) ( ). statistical significance was established at the level of p < . . more details are in suppl. methods. tonsil and nasopharyngeal aspirate samples were obtained from routine tonsillectomy patients. patient characteristics are shown in table . briefly, the median age of the study subjects was years and . % reported allergy. all operations were carried out during the 'cold phase' of chronic tonsil condition. main indications for tonsillectomy were hypertrophic tonsils and/or recurrent tonsillitis ( table ). the median for the last day of respiratory symptoms was days before the operation, excluding sore throat in subjects due to operation indication. medication data are shown in suppl. results. fifty-nine percent of the nasopharyngeal aspirates were positive for ≥ viruses and % for ≥ viruses (fig. a , table s ). rhinovirus ( %) was the most prevalent virus followed by bocavirus- ( %), adenovirus ( %), enteroviruses ( %), coronavirus ( %), and other viruses (< % each). twentyfour percentage of the tonsillar samples were positive for ≥ viruses and % for ≥ viruses. bocavirus- , adenovirus, and enteroviruses were found % each, followed by parainfluenza virus ( %) and other viruses (< % each). both nasopharyngeal and intratonsillar virus detection rates decreased by age (both p < . ). nasopharyngeal samples were clear of viruses by the age and tonsillar samples by the age , excluding one case (fig. b) . in nasopharyngeal samples, rhinovirus, bocavirus- , adenovirus, and more than one virus detection rates decreased by age (all p < . ). in tonsillar samples, bocavirus- , enterovirus, adenovirus, parainfluenza virus, and virus co-detection rates decreased by age (all p < . ). enterovirus, but not any other virus, detection showed agreement between nasopha-ryngeal and intratonsillar samples (kappa coefficient . , % confidence interval . , . ; table s ). the intratonsillar mrna expression levels of the analyzed cytokines and transcription factors are listed in table s . increasing age showed a significant negative correlation with il- , il- , il- , and tbet mrna expressions in tonsils (all p < . , fig. ), but did not show any correlation with the other investigated cytokines and transcription factors (fig. s ). when allergic diseases were considered, atopic dermatitis was associated with higher il- expression in tonsils, and this association remained significant after adjustment for age (p = . , fig. a ). the inverse correlation between il- and age was seen in patients without atopic dermatitis (r = À . , p < . ), but not in those with atopic dermatitis (p > . ). cytokine and transcription factor data and their associations with clinical factors are shown in tables s and s . in brief, allergy was associated with lower tbet values are shown as medians (interquartile range) or n (%). *range , years. †chronic white patches in tonsils (n = ), recurrent fever (n = ), throat abscess (n = ), teeth braces (n = ), and food remnants in tonsils (n = ). ‡seven subjects had cough, had rhinitis, had temperature > °c, and ≤ had other symptoms. forty subjects had sore throat, but it was excluded from the count due to operation indication. §if > days, days was used in the calculation. sore throat was excluded from the calculation. expression and lower tbet/gata ratio, which both remained significant after adjustment for age (both p < . ). allergic rhinitis was associated with lower il- and il- expressions, and asthma was associated with lower ifn-a and il- expressions (all p < . , adjusted for age). in addition, respiratory symptoms within the last weeks before tonsillectomy correlated negatively with foxp expression (mean difference À . , % confidence interval À . , À . , p = . ). rhinovirus detection was independently associated with higher ifn-c in tonsils (p = . , fig. b ). intratonsillar virus detection overall, number of detected viruses, rhinovirus detection, and parainfluenza virus detection were independently associated with high tbet expression (p < . for all, adjusted for age and allergy, fig. c ). the significant univariable associations between intratonsillar virus detection and il- or il- expressions did not remain significant in multivariable models, while age and/or atopic dermatitis remained significant as adjustments (fig. d) . rorc expression was independently and negatively associated with nasopharyngeal and/or tonsillar virus detection (p < . ). the association between cytokine and transcription factor data and virology are fully detailed in tables s and s and fig. s a and b. this study enabled us to investigate the correlations between the expressions of cytokines and transcription factors, because all of them were directly analyzed in the tonsils without any further cell culture, representing the in vivo situation. il- expression most closely and positively correlated with il- , il- , foxp , gata , rorc , and tbet expressions (all p < . , fig. ) . in contrast, a negative correlation of il- was observed with ifn-a, ifn-b, and ifn-c when all of the tonsils were considered (all, p < . ). age-adjusted gene-to-gene correlations are illustrated in heatmaps in fig . overall, two clusters, which showed high positive intracluster correlations, were observed as 'antiviral' cluster (ifn-a, ifn-b, ifn-c, il- , il- ) and 'immune activation/regulatory' cluster (il- , il- , il- , tgf-b, foxp , gata , rorc , tbet) (see overall age-adjusted data in fig. s ). these two clusters showed strong negative correlations between each other excluding ifn-c, which appeared as an intermediary link between antiviral immune response and general immune response. interestingly, correlations between ifn-c and other antiviral genes were less prominent in the presence of a virus. the four key th cell lineage-specific transcription factors, namely foxp , gata , rorc , and tbet, showed very strong positive correlations with each other. the immune regulatory and treg cell-related factors il- , il- , tgf-b, and foxp also closely and positively correlated with each other. the presence of a tonsillar and/or nasopharyngeal viral infection induced a very strong negative correlation between the antiviral cluster cytokines and the regulatory cluster, which mostly disappeared in the absence of a virus (fig. a) . the negative correlation of foxp , il- , and particularly il- with antiviral cluster was most prominent only in the the existence of allergy also modestly deepened the negative relationship between the immune regulatory and antiviral clusters when compared to individuals without allergy (fig. b) . interestingly, allergy was associated with strong positive correlation between tgf-b and tbet, whereas the positive correlation between il- and il- in nonallergic patients disappeared in the presence of allergy. gene-to-gene correlations are shown in more detail in tables s and s . gene network graphs in fig. illustrate the antiviral and regulatory clusters in disease states of virus infection and allergy and their negative interrelations, excluding the only positive connection through ifn-c (p-values are shown in fig. ) . overall, the distinction between the two clusters significantly increased in the presence of a tonsillar virus or clinical allergy. this study provides new insights into the immune regulation and antiviral response in human tonsils in relation to age, infection, and allergic illnesses. interestingly, intratonsillar virus detection strongly decreased by age, and only one virus-positive case was found above the age of . the network analyses showed two main clusters, antiviral and immune activation/regulation-related clusters. these two clusters had very strong negative intercluster correlation, which became more distinctive in the presence of viral infection or reported allergy. it was remarkable that having allergy influenced many gene-to-gene interactions by markedly deepening the negative relationships between the antiviral and immune activation/ regulatory clusters. a fine balance between allergen-specific t-cell activation vs allergen-specific tolerance lies in the expression intensity of th cells vs il- -secreting treg cells in human tonsils ( ) . indeed, changes in gene-to-gene correlations were found between il- and il- and between tgf-b and tbet in relation to allergy. interestingly, the newly discovered anti-inflammatory cytokine il- was positively associated with atopic dermatitis, and subjects with allergy showed lower th expression and lower innate immune responses than those without allergy. the presence of asthma was negatively associated with type i/iii interferons, which is in agreement with previous in vivo data ( ) . the role of il- (formerly il- family member ) in respiratory viral infections or allergy development has not been previously studied. it has previously been demonstrated as a natural suppressor of innate inflammatory and immune responses in vitro and in animal studies ( ) ( ) ( ) . our data may suggest that il- may inhibit ifn-c expression as shown previously with t regulatory cells ( ) . in agreement, we showed low-to-moderate positive correlation between il- and il- , il- and foxp . our findings on il- expression, the independent positive association with atopic dermatitis and positive association with virus infection, could imply that an inflammatory state in general, not just a viral infection, is likely to increase the expression of il- similar to il- expression ( ) . several isoforms of il- exist, but our primer set covered them all. moreover, il- is produced as a precursor that needs to be activated by caspase ( ) ; therefore, the relationship between mrna expression of il- and biological activity is not necessarily direct. one of the key findings of the present study was that il- fell apart from typical t-cell activation response and unexpectedly showed very strong correlation and clustered with antiviral cytokines that became particularly significant in the presence of a tonsillar virus or allergy status. this finding may help to explain the role of il- in virus-induced exacerbation of asthma. interestingly, our data suggest a different way of regulation in contrast to gata ( ) . in virus infection, il- released together with antiviral interferons may reduce the extent of inflammation and tissue injury, because il- inhibits monocyte and macrophage production of proinflammatory factors ( ) . the data are scarce on the disconnection between gata and il- in viral infection, but specific viral infections may either decrease or increase gata -mediated expression of th cytokines ( , ) . moreover, ifn-b may induce the expression of il- recep-tor, leading to a consequential suppression of responsiveness to il- ( ) . our network analyses revealed that virus infection is also associated with many alterations in gene-to-gene interactions similar to allergy. in light of the recent demonstration of breaking allergen-specific t-cell tolerance in human tonsils by triggering of toll-like receptor (tlr) or tlr and the proinflammatory cytokines il- b or il- ( ), we speculate that viral infections may have an important role in breaking peripheral tolerance and thereby promoting the development or exacerbation of allergic diseases. rhinovirus is particularly interesting in this regard because it is the most important trigger of wheeze and asthma, especially in atopic individuals ( , , , ) . we demonstrate as a new finding that the intratonsillar virus, especially rhinovirus and parainfluenzavirus, detection was independently and positively associated with th type expression. ifn-c, which is mainly controlled by tbet, has an important antiviral activity and capacity to downregulate th type response ( , , , ) . the existence of virus infection and allergy increased the complexity of the gene networks, and interestingly, both disease conditions had similar polarizing influence on the overall gene-to-gene correlation profiles. a negative correlation between antiviral and regulatory genes, particularly il- , was observed only in the presence of a virus. il- is a proinflammatory cytokine, and its downregulation most likely limits virus-induced inflammation ( ) . it was remarkable that the only positive interaction between the antiviral and regulatory clusters was through ifn-c. in the search for covariates, we found that age showed low negative correlation with the expressions of several genes and viruses in tonsils. with high age, each component of immunity is affected, t cells being most susceptible ( ) . virology, follicular helper t cells as a possible cellular source of investigated genes, and potential limitations of the study have been discussed in more detail in suppl. discussion. taken together, the present study provides several new and potentially important findings considering tonsils as a new in vivo model for the understanding of immune response development and immune tolerance induction. intranodal immunotherapy has been demonstrated highly effective ( ) , but more data are needed on the basic immune mechanisms in lymphnoids ( ) . t.j. and his laboratory are supported by the academy of finland (grants and drs jartti and akdis c participated sufficiently in the work to take public responsibility for the whole content; jartti, palomares, and akdis c contributed to study concept and design; jartti, palomares, waris, nieminen, puhakka, r€ uckert, aab, vuorinen, allander, akdis m, and akdis c involved in the acquisition of data; jartti, palomares, tastan, vahlberg, akdis m, and akdis c involved in analysis and interpretation of data; jartti, palomares, tastan, vahlberg, akdis m, and akdis c drafted the manuscript; jartti, palomares, waris, tastan, nieminen, puhakka, r€ uckert, aab, vuorinen, allander, vahlberg, ruuskanen, akdis m, and akdis c involved in critical revision of the manuscript for important intellectual content; jartti, palomares, r€ uckert, vuorinen, akdis m, and akdis c provided administrative, technical, or material support; akdis c supervised the study. the authors declare that they have no conflicts of interest. additional supporting information may be found in the online version of this article: figure s . the correlations between age and the expressions of ifn-a, ifn-b, ifn-c, il- , il- , il- , foxp , gata , rorc , and tgf-b. figure s . intratonsillar cytokine and transcription factor expression according intratonsillar virus detection. figure s . age-adjusted heatmap of intratonsillar gene-togene correlations in all study patients. table s . health questionnaire. table s . primer sequences for cytokines or transcription factors. table s . virus etiology of nasopharyngeal aspirate and intratonsillar samples. table s . the comparison of tonsil vs. nasopharyngeal virus detections. table s . intratonsillar transcription factor and cytokine expressions. table s . the univariable associations between atopic characteristics and intratonsillar cytokine expressions. table s . the univariable association between atopic characteristics and intratonsillar transcription factor expressions. table s . the association between nasopharyngeal and intratonsillar virus detections and intratonsillar cytokine expressions. table s . the association between nasopharyngeal and intratonsillar virus detection and transcription factor expressions. table s . comparison of gene-to-gene correlations between virus-positive (intratonsillar and/or nps positive) and virus-negative (intratonsillar and nps negative) subjects. table s . the comparison of gene-to-gene correlations between allergic and nonallergic subjects. epidemiology of respiratory viruses in patients hospitalized with nearfatal asthma, acute exacerbations of asthma, or chronic obstructive pulmonary disease interleukin- gene expression in acute virus-induced asthma human bocavirus and acute wheezing in children wheezing rhinovirus illnesses in early life predict asthma development in high-risk children prednisolone reduces recurrent wheezing after first rhinovirus wheeze: a -year follow-up asthma and allergy patterns over years after severe rsv bronchiolitis in the first year of life early infection with respiratory syncytial virus impairs regulatory t cell function and increases susceptibility to allergic asthma molecular mechanisms of decreased steroid responsiveness induced by latent adenoviral infection in allergic lung inflammation new molecular virus detection methods and their clinical value in lower respiratory tract infections in children rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production deficient antiviral immune responses in childhood: distinct roles of atopy and asthma type and type cytokine imbalance in acute respiratory syncytial virus bronchiolitis cd + natural regulatory t cells are critical in limiting innate and adaptive immunity and resolving disease following respiratory syncytial virus infection induction and maintenance of allergen-specific foxp + treg cells in human tonsils as potential first-line organs of oral tolerance evidence for a stepwise program of extrathymic t cell development within the human tonsil immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific t regulatory and t helper cells in vivo switch to il- -secreting t regulatory cells in high dose allergen exposure simultaneous detection and differentiation of human rhino-and enteroviruses in clinical specimens by real-time pcr with locked nucleic acid probes on the probable error of a coefficient of correlation deduced from a small sample cytoscape . : new features for data integration and network visualization il- is a fundamental inhibitor of innate immunity interleukin expression protects mice from colitis interleukin- reduces liver inflammatory injury via effects on hepatocytes and nonparenchymal cells regulatory t cells inhibit acute ifn-c synthesis without blocking t-helper cell type (th ) differentiation via a compartmentalized requirement for il- interleukins, from to , and interferon-c: receptors, functions, and roles in diseases bidirectional interactions between viral respiratory illnesses and cytokine responses in the first year of life lentiviralmediated gata- rnai decreases allergic airway inflammation and hyperresponsiveness the innate antiviral response upregulates il- receptor a in bronchial fibroblasts triggering of specific toll-like receptors and proinflammatory cytokines break allergen-specific t cell tolerance in human tonsils and peripheral blood allergic sensitization is associated with rhinovirus-, but not other virus-, induced wheezing in children the role of viruses in acute exacerbations of asthma development of spontaneous airway changes consistent with human asthma in mice lacking t-bet t-box transcription factor t-bet, a key player in a unique type of b-cell activation essential for effective viral clearance dysregulation of t-cell function in the elderly: scientific basis and clinical implications intralymphatic immunotherapy for cat allergy induces tolerance after only injections mechanisms of peripheral tolerance to allergens key: cord- -u je d o authors: diaz-salazar, carlos; sun, joseph c title: natural killer cell responses to emerging viruses of zoonotic origin date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: u je d o emerging viral diseases pose a major threat to public health worldwide. nearly all emerging viruses, including ebola, dengue, nipah, west nile, zika, and coronaviruses (including sars-cov , the causative agent of the current covid- pandemic), have zoonotic origins, indicating that animal-to-human transmission constitutes a primary mode of acquisition of novel infectious diseases. why these viruses can cause profound pathologies in humans, while natural reservoir hosts often show little evidence of disease is not completely understood. differences in the host immune response, especially within the innate compartment, have been suggested to be involved in this divergence. natural killer (nk) cells are innate lymphocytes that play a critical role in the early antiviral response, secreting effector cytokines and clearing infected cells. in this review, we will discuss the mechanisms through which nk cells interact with viruses, their contribution towards maintaining equilibrium between the virus and its natural host, and their role in disease progression in humans and other non-natural hosts. emerging viral diseases pose an ongoing threat to mankind, especially in a globalized and highly interconnected world. a prime example of such a threat is the current covid- pandemic, with more than eighteen million confirmed cases and over deaths worldwide at the time of this publication [ ] . all of the diseases with the greatest potential to cause a public health emergency, as identified by the world health organization, are driven by viruses of zoonotic origin [ ] . these include viruses that cause haemorrhagic fever (e.g. ebola, marburg, dengue, and lassa viruses), highly pathogenic respiratory coronaviruses (e.g. those causing mers and sars), and other viruses (e.g. nipah, zika, and chikungunya). these zoonotic viruses are extremely diverse in nature, as some are transmitted through vectors such as mosquitoes or ticks (e.g. dengue, tick-borne encephalitis), whereas human-to-human is the main mode of transmission for others (e.g. ebola, sars-cov ). some zoonotic viruses have a wide range of natural hosts (e.g. huaiyangshan, west nile), whereas others are restricted to specific species such as bats (e.g. marburg, nipah) (table ) . however, most zoonotic viruses have shared characteristics, such as being single-stranded rna (ssrna) viruses, and causing mild or asymptomatic infections in its natural host animal, while provoking profound pathologies in humans [ ] . understanding the immune mechanisms that allow animal reservoirs to tolerate these viruses will shed light into how viral zoonotic infections progress to severe illness in humans. this review describes the role of natural killer (nk) cells, a critical component of early antiviral immunity, in the establishment of tolerance to viral infections in natural hosts, as well as their role in the development of disease in nonnatural hosts. nk cells are lymphocytes of the innate immune system with the unique ability to rapidly destroy virally infected cells and tumors without previous sensitization [ , ] . they play a critical role in the control of viral infections, as deficiencies in the nk cell compartment are associated with increased susceptibility to certain viral infections in humans [ ] [ ] [ ] . unlike t and b cells, nk cells do not express a rearranged antigen receptor, but instead express a wide array of germline-encoded receptors, which can be activating or inhibitory [ , ] . nk cells are 'educated' through their inhibitory receptors to recognize 'self' (e.g. ligands normally present on healthy cells, such as mhc class i molecules) [ , ] . in contrast, activating receptors can recognize 'non-self' pathogen components (e.g. viral ligands, proteins, or peptides expressed during infection) or stress signals (e.g. nkg d ligands expressed during injury, infection, or tumorigenesis) [ , ] . nk cells also express activating fc receptors (e.g. cd ), which helps them recognize antibody-coated target cells and clear them via antibodydependent cell-mediated cytotoxicity (adcc) [ ] . moreover, during viral infection, pro-inflammatory table overview of nk cell response to emerging viruses of zoonotic origin, as identified by the who [ , ] . general upregulation of mhc class i, downregulation of nkg d ligands [ , ] likely. associations of inhibitory kirs with case fatality [ ] . association of nk cell expansion with recovery [ ] . unknown. unlikely inhibition of type i ifn responses [ , ] probable. reduced nk cell numbers in mouse models [ ] unknown. unclear. elevated ifn-a levels but reduced nk cell killing [ ] . dampened responsiveness to type i ifn? [ ] likely. nk cells strongly activated and proliferative during acute phase. high levels of ifn-g in human patients [ ] possible. nk cells increased in csf of tbe patients [ , ] very likely. strong nk cell activation and killing to wninfected cells [ ] . increased ifng production and mature phenotype of nk cells from patients with previous wn infection [ , ] . unclear. nk cells increase in cns but depletion in mice does not change disease outcome [ ] yellow fever flaviviridae monkeys (lemurs?) extremely rare % asymptomatic. fever, malaise, vomit. in severe cases ( %), jaundice, hemorrhage, shock, organ failure. % fatality rate. inhibition of type i ifn responses [ ] very likely. strong nk cell expansion, increased ifn-g production and cytokine responsiveness [ ] [ ] [ ] . correlates with better protection in humanized mouse model [ ] . [ , ] . upregulation of mhc class i? [ ] conflicting reports. nk cells are activated early [ ] , proliferate [ ] , and activation correlates with viral clearance [ ] . others report nk cells do not proliferate nor respond during zika infection [ , ] possible. increased nk cell infiltration in cns in fatal zika cases and mouse models [ , ] [ , ] , accumulation of nk cells in sites of viral replication [ ] . unclear. higher nk cell number in severe cases in humans [ ] , but others report no correlation [ ] . orthomyxoviridae poultry, (other birds) extremely rare fever, cough, shortness of breath. fatality rate ranges from % (h n strain) to % (h n strain). unclear. induction of type i ifn responses [ , ] and downregulation of mhc class i [ ] . strain specific? lower nk cell activation and killing in infection with highly pathogenic h n strain, compared to low pathogenic strains [ , ] . h n strain induces hyperresponsiveness in human lung nk cells [ ] . strain specific? nk cell depletion improves disease outcome in mice infected with h n strain [ , ] . inhibition of type i ifn responses [ ] . however, ifn-b produced in vitro [ ] . unclear. increased cytotoxic cell markers and nk cell markers in survivors monkeys [ ] , and increase in nk cell numbers and proliferation in one survivor [ ] possible. nk cell signature predicted to be contribute to fatal outcomes [ ] . phenuiviridae rodents, (other mammals) extremely rare severe fever, vomiting diarrhea. in severe cases, thrombocytopenia, multiple organ failure. fatality rate %. dampening of type i ifn responses [ , ] . induction of anti-inflammatory cytokines (il- ) [ ] possible. increase in nk cell percentage during early disease stages in recovery patients [ ] . no difference in nk cell percentage between fatal and recovered cases [ ] cytokines (e.g. ifn-a, il- , and il- / ) can directly activate nk cells [ ] [ ] [ ] . when engaging a potential target, all these signals are integrated to determine whether the nk cell will kill, make cytokines, and proliferate. upon activation, nk cells can release granzymes and perforin to lyse infected target cells, produce effector cytokines such as ifn-g to alert the surrounding tissue of infection, and in some cases undergo robust proliferation [ , , likely. kirs with case incidence [ ] . strong nk cell activation and expansion during acute phase [ ] very likely. nk cell depletion ameliorates joint pathology [ ] the striking diversity of nkrs suggests an intense selective pressure from the pathogens they encounter, especially viruses. this co-evolution of the nkr repertoire with the viral interface is arguably best studied in the mouse and human nk cell response to cytomegalovirus (cmv) [ ] [ ] [ ] . cmv is a species-specific herpesvirus that persists through latency in healthy hosts, and thought to intimately interact with the host immune system. to avoid cd + t cell recognition, human and mouse cmv (hcmv and mcmv, respectively) possess multiple genes encoding immunoevasins that interfere with antigen presentation by degrading, retaining, or preventing the assembly of mhc class i on the cell surface of infected cells [ ] . however, because downregulation of mhc class i causes cmv-infected cells to be susceptible to nk cell recognition of "missing self", cmv has evolved mhc class i-like proteins as a decoy to engage inhibitory nkrs and tune down nk cell reactivity [ ] . as perhaps an evolutionary countermeasure, activating nkrs recognizing the same decoy ligands have appeared in mouse and human genomes that allow for nk cell recognition of cmv-infected cells [ ] . examples of this host-pathogen evolutionary adaptation include the inhibitory ly i and activating ly h receptor pair in mice, which both recognize the mcmv m glycoprotein [ ] , and the inhibitory nkg a and activating nkg c receptors in humans, which both recognize the viral ul peptides presented on the nonclassical mhc class i molecule hla-e [ , ] . in further evidence of this evolutionary 'tug-of-war' between host and virus, mcmv strains isolated from wild mice have been shown to exhibit high polymorphism in its m gene, and hcmv isolates that promote strong nkg c engagement are far rarer than those inducing weak activation in humans [ , ] . thus, both mouse and human immune systems have evolved remarkably similar molecular mechanisms of viral control of cmv using different families of nkrs, representing a strong example of convergent evolution. animals with the greatest potential to transmit zoonotic diseases to humans include mammals such as bats, primates, and rodents [ ] . while ecological and biological factors certainly play a role in the transmission of zoonotic diseases, a growing body of evidence suggests that transmission across species may occur because these animals possess a more permissive innate immune response allowing them to carry a high viral burden (figure ). bats are reservoirs to ebola and marburg viruses, hendra and nipah viruses, and sars coronaviruses, among others [ , ] . their roosting behaviour, ability to fly, widespread distribution, and consumption as bushmeat makes them especially well-positioned to transmit zoonotic viruses to humans [ ] . despite having a high metabolic rate associated with the energetic cost of flight, bats have unusually long lifespans [ ] . consistent with this energetic demand, genes involved in oxidative phosphorylation and dna damage response are under positive selection in bats [ ] . in turn, bats may have fine-tuned their response to high cellular stress in order to avoid overt inflammatory responses, which has been hypothesized to provide a unique niche for certain viruses. indeed, bat cells show dampened activation of the nlrp inflammasome during viral infection, and the production of interferons (ifns) following viral entry is rapid but more transient and lower in magnitude compared to other mammals [ , ] . moreover, interferonstimulated genes (isg) such as isg may have been positively selected for in certain bat species, and some ifn gene families are greatly expanded [ , , ] . altogether, it is believed that quick control of viral infections and reduced induction of pro-inflammatory cytokines have allowed bat viruses to rapidly co-evolve with their host without provoking major immune- the careful study of the immune system in reservoirs of zoonotic diseases will certainly offer insights into how these animals carry high viral loads while remaining asymptomatic. a growing body of evidence suggests that differences in the innate immune system, particularly in viral recognition and induction of pro-inflammatory cytokines, may explain such phenomenon (figure ). since nk cells are key players in the integration and amplification of these signals, some animal reservoirs may have adapted their nkr repertoire and functionality to avoid excessive immune activation leading to immunopathology. as such, a deeper understanding of the nk cell response to zoonotic viruses in its natural host may shed light into their role in disease pathology in humans. nk cells have been proposed to contribute to immunopathology in some zoonotic diseases, while having protective effects against other viral infections. here, we will discuss their role in the progression of diseases caused by sars coronaviruses and some haemorrhagic fever viruses (hfvs). this data is summarized in table and figure , alongside with other relevant viral diseases of zoonotic origin, including zika, west nile, and nipah. finally, we will examine their contribution to the protection conferred by vaccines against zoonotic viruses. the coronaviruses mers-cov, sars-cov, and sars-cov have caused three global outbreaks in the past two decades, with the latter being the causative agent behind the current covid- pandemic. negative outcomes have been associated with aberrant immune responses and subsequent lung pathology. mers-cov induced high levels of inflammatory cytokines associated with strong myeloid responses (e.g. il-b, il- , and il- ), but failed to induce early type i ifns [ ] [ ] [ ] . common human coronaviruses induce strong type i ifn responses compared to sars and mers, which may account for their lower fatality rate [ , ] . interestingly, ifn-a production anecdotally correlated with survival of mers patients, as did serum levels of ifn-g [ ] . although definitive data is lacking, these studies suggest that early ifn production (e.g. ifn-g production by nk cells) may contribute to viral control in these patients. conversely, delayed recognition and responses may result in virusmediated cytopathy and aberrant production of highly inflammatory cytokines leading to lung pathology. all three coronaviruses mentioned above have been documented to provoke severe lymphopenia and lower circulating nk cell numbers in some patients, although the exact role of nk cells in disease pathology is not clear [ ] [ ] [ ] . although mouse models of sars infection suggest that lymphocytes do not play a major role in pathology, these models do not accurately replicate disease progression in humans [ , ] . given our understanding of viral infections in hamsters and macaques, we would argue that developing more relevant animal models is key to studying virus-immune interactions, and immunemediated pathobiology. currently, one us-based company has obtained approval from the fda to use nk cellbased immunotherapy as a potential treatment for covid- , with others following suit [ , ] . although the available data indicates that nk cell-targeted therapy may have beneficial effects on virus control and disease progression early on, these data also suggest that nk cells can exacerbate the severity of the disease at later time points. thus, extreme caution should be exercised when designing such treatment strategies, and there should not be a rush to haphazardly implement such cellular treatments without careful design. hfvs are a diverse group of viruses with the common denominator of causing fever and severe bleeding, although disease severity is highly variable. hfvs include arenaviruses (e.g. lassa virus), filoviruses (e.g. ebola virus), flaviviruses (e.g. dengue virus) and hantaviruses. here we attempt to summarize the nk cell response to representative examples of these viruses. ebola virus-infected cells have been shown to be resistant to nk cell-mediated killing [ , ] . ebola is known to potently inhibit type i ifn induction in infected myeloid cells, and ifn-a and il- are difficult to detect in infected individuals [ , [ ] [ ] [ ] . in contrast, viral-like particles (vlps) of ebola, which contain no rna or inhibitory proteins, are able to readily activate dcs, and promote nk cell cytotoxicity and ifn-g production [ , ] . these findings suggest that nk cells can more efficiently target ebola-infected cells when they receive the proper cytokine signals. indeed, transfer of vlpprimed nk cells provided almost complete protection against lethal ebola challenge in mice, where protection was dependent on cytotoxicity and not on ifn-g production by nk cells [ ] . ebola can evade nk cell recognition through additional mechanisms. vlp-treated nk cells recognize ebolainfected cells through nkp and possibly nkg d; however, ebola uses its glycoprotein to conceal nkp and nkg d ligands expressed on the surface of infected mechanisms of nk cell evasion or activation by zoonotic viruses. left: zoonotic viruses can avoid interferon responses by blocking prr sensing, inhibiting interferon production, and dampening interferon receptor signalling. these viruses also evade nk cell recognition by upregulating ligands for inhibitory nkr, downregulating or shielding activating nkr ligands, and inducing anti-inflammatory cytokines. right: when activating signals (green arrows) outweigh inhibitory signals (red flat-end arrows), nk cells become activated and secrete pro-inflammatory cytokines (e.g. ifn-g), release cytotoxic granules to kill target cells, and undergo proliferation. see also table for a complete list of references. prr, pattern recognition receptors. nkr, nk cell recognition receptors. isg, interferon stimulated genes. cells, thereby shielding them from nk cell killing [ ] . interestingly, while the glycoprotein also impairs mhc class i presentation and thus recognition by the t cells, engagement of inhibitory nkrs can still occur. altogether, these vlp studies suggest that nk cells can recognize viral proteins present in the vlp, but additional evasion proteins in the ebola genome can impair nk cell activation. the direct mechanisms and relevance of these ebola immunoevasins in human infections remain to be determined. ebola infections are also characterized by profound lymphopenia, lymphocyte apoptosis, and extensive cellular damage. do nk cells directly (via cytotoxicity) or indirectly (via ifn-g secretion) contribute to these phenotypes? one study suggested that nk cells can contribute to pathology by migrating to infected organs and killing infiltrating t cells [ ] . furthermore, ifn-g was markedly elevated in serum samples from patients that succumbed to haemorrhagic shock, but not in those who survived [ , ] . ifn-g mrna levels in pbmcs were surprisingly similar in both patient groups, suggesting that translation into ifn-g protein may be exacerbated in those that led to fatality, a step-wise mechanism for ifn-g production that has been recently characterized in nk cells [ ] . dengue nk cells and ifn-g production appear to be important for dengue virus control, as nk cell depletion and ifn-g blockade led to high viremia and virus-mediated pathology in humanized mouse models [ ] . furthermore, nk cells have been suggested to recognize and lyse dengue-infected cells in vitro through activating receptors such kir ds (proposed to recognize ns peptides in the context of hla-c ) and nkp (proposed to recognize dengue envelope protein) [ , ] . however, despite a strong ifn response and potent nk cell activation, viral replication can reach high levels in patients and in vitro [ ] [ ] [ ] [ ] . rather than directly impeding the engagement of nk cell activating receptors, like ebola, dengue instead can evade nk cell targeting by upregulating hla molecules on infected cells [ ] . indeed, blocking hla in vitro restores nk cell degranulation against dengue-infected cells, and nk cell cytotoxicity is diminished at early disease stages when viral titers are highest [ , ] . the upregulation of hla molecules by dengue suggests that inhibitory nkrs are engaged during nk cell surveillance of infected cells, which is supported by the observation that expression of inhibitory receptors kir dl , kir dl , and kir dl are more prevalent in dengue-infected patients than healthy controls [ ] . furthermore, kir dl has been suggested to directly recognize dengue ns -derived peptides presented on hla-b [ ] . however, given that kir dl can already directly recognize endogenous peptides presented on hla-bw , whether ns -derived peptides can directly alter nk cell function in vivo is unknown. after an initial febrile phase, dengue viral titers dwindle and a latter critical phase ensues which can progress into serious complications, including haemorrhage and fatal shock [ , ] . this latter critical phase is likely immune-mediated. nk cell activation and ifn-a titers were higher in haemorrhagic patients compared to milder symptomatic patients, suggesting that these cellular and soluble immune components may directly contribute to pathology at these later stages [ , , ] . by comparison, infection with chikungunya virus elicits similar initial symptoms, but very rarely results in fatal haemorrhage and shock. furthermore, whereas nk cells only increased in number late during dengue infection, their numbers increased early during chikungunya infection [ ] . lastly, nk cells from dengue-infected patients produced more ifn-g but were less cytotoxic than those from chikungunya patients [ ] . many other factors beyond the specific immune components herein presented distinguish the pathologies driven by these viral infections. nonetheless, the available data suggest that nk cells may play a role in the onset of haemorrhagic complications. lassa virus has previously been described to evade nk cell killing by maintaining high levels of mhc class i on infected cells while inhibiting expression of nkg d ligands [ , ] . indeed, expression of the inhibitory nkr kir dl has been associated with increased fatality rate [ ] . although nk cells can be activated by lassa virus-infected cells, they do not produce ifn-g [ , ] . the reduced inflammatory profile may partly explain the lower occurrence of haemorrhage and mortality in comparison with other hfvs. interestingly, nk cells will proliferate to a greater degree in lassa-infected monkeys that recover from the infection, but are reduced in number in those that succumb to the virus [ ] . whether nk cell proliferation provides protection against lassa virus has not been formally addressed. altogether, it is tempting to speculate that although nk cells provide critical protection in the early phases of hfv infection, the exacerbated nk cell response observed in the later stages contributes to the onset of haemorrhagic complications. among the biggest challenges we face with zoonotic viruses is the lack of effective vaccines. to date, only vaccines against rabies, tick-borne encephalitis, japanese encephalitis, yellow fever, and recently ebola, are available. rabies infection leads to delirium and certain death if untreated, but vaccination after suspected exposure effectively controls the infection [ , ] . interestingly, in prime-boost studies (where three doses are recommended for full protection), nk cells are among the first and strongest responders during each re-vaccination dose [ ] . ifn-g production and degranulation predominantly occurred through an adcc mechanism, suggesting that protective antiviral nk cell responses may depend on rabies-specific antibodies generated following vaccination [ ] . an effective vaccine against ebola developed in canada was recently approved for use in the us and europe [ ] [ ] [ ] . a systems vaccinology approach revealed a correlation between a strong nk cell activation signature and development of an antibody response to this ebola vaccine [ ] . furthermore, and similar to the rabies vaccine, nk cells from ebola-vaccinated individuals mounted an antibodydependent response against ebola glycoprotein, where ifn-g production and degranulation correlated in magnitude with neutralizing antibody titers [ ] . currently, two recombinant rabies-based vaccines are being developed against marburg and lassa viruses [ , ] . although these vaccines provided almost complete protection from viral challenge, the antibodies generated were non-neutralizing. instead, these virusspecific antibodies elicited strong nk cell-mediated adcc against infected cells expressing the viral glycoprotein used for vaccination. blocking fc receptors on nk cells or directly depleting nk cells decreased killing of target cells, and immunized mice lacking the fc receptor became susceptible to viral challenge [ , ] . altogether, these studies highlight the critical role of nk cells in conferring protection through vaccination against zoonotic diseases. nk cells are critical for the control of viral infections. the receptors they use to recognize virally infected cells have rapidly evolved to adapt to the viral niche in each species. indeed, some animal reservoirs of emerging viruses (e.g. bats) have exquisitely complex and expanded nk cell receptor repertoires. emerging viruses may persist in their natural hosts by avoiding the induction of strong pro-inflammatory responses. in turn, natural hosts may have evolved enhanced interferon responses to prevent pathology. when this equilibrium is disturbed, or transmission occurs, the virus can cause severe disease in closely related or unrelated species (figure ). although some emerging viruses can evade nk cell recognition by dampening type i ifn signalling, others increase the expression of inhibitory ligands while downregulating activating ligands for nk cell receptors in the infected cells. furthermore, while nk cells contribute to viral clearance early during infection, exacerbated or prolonged nk cell activation at later stages of infection may contribute to severe immunopathology. the complex interaction of viral ligands with nk cells is the result of a frenetic arms-race between host and pathogen ( figure ). while this delicate equilibrium has developed over millennia and will likely continue for many more, we hope that new insights into the biology of antiviral nk cells will tilt the balance in our favour. nothing declared. papers of particular interest, published within the period of review, have been highlighted as: of outstanding interest blueprint for r&d preparedness and response to public health emergencies due to highly infectious pathogens reservoir host immune responses to emerging zoonotic viruses in this review, the authors provide a detailed analysis of immune mechanisms in natural hosts of emerging zoonotic viruses that allows for viral tolerance and persistance the dynamic life of natural killer cells natural" killer cells in the mouse. ii. cytotoxic cells with specificity for mouse moloney leukemia cells. characteristics of the killer cell orange js: natural killer cell deficiency keeping nk cells in highly regulated antiviral warfare control of human viral infections by natural killer cells natural killer cell education and tolerance up on the tightrope: natural killer cell activation and inhibition sondel pm: nk cellmediated antibody-dependent cellular cytotoxicity in cancer immunotherapy type i ifn promotes nk cell expansion during viral infection by protecting nk cells against fratricide proinflammatory cytokine signaling required for the generation of natural killer cell memory characterization of early il- , ifnalphabeta, and tnf effects on antiviral state and nk cell responses during murine cytomegalovirus infection evolutionary struggles between nk cells and viruses viral evasion of natural killer cells human cytomegalovirus encodes a glycoprotein homologous to mhc class-i antigens structural elucidation of the m mouse cytomegalovirus ligand for ly natural killer cell receptors hla-e binds to natural killer cell receptors cd /nkg a, b and c surface expression of hla-e, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpul murine cytomegalovirus m mutation and variation leads to immune evasion of natural killer cells peptidespecific recognition of human cytomegalovirus strains controls adaptive natural killer cells the authors show that adaptive nkg c+ nk cell responsiveness is dictated by the affinity of nkg c to the variable hcmv ul peptides from clinical isolates bats as' special' reservoirs for emerging zoonotic pathogens going to bat(s) for studies of disease tolerance novel insights into immune systems of bats bat tolerance to viral infections comparative analysis of bat genomes provides insight into the evolution of flight and immunity dampened nlrp -mediated inflammation in bats and implications for a special viral reservoir host active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment mers-cov infection is associated with downregulation of genes encoding th and th cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis national research project for sars beijing group: the involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study increasing the translation of mouse models of mers coronavirus pathogenesis through kinetic hematological analysis phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected sars coronavirus mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice fda accepts ind for nk cell therapy cynk- to treat patients with covid- cytovia therapeutics and macromoltek to develop dual-acting natural killer immunotherapy against sars cov (covid- the ebola interferon inhibiting domains attenuate and dysregulate cell-mediated immune responses role of natural killer cells in innate protection against lethal ebola virus infection in this article, the authors show that nk cells primed with ebola virus like particles completely protect mice from lethal ebola infection human asymptomatic ebola infection and strong inflammatory response inflammatory responses in ebola virus-infected patients human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis nkp -dependent cytolysis of filovirusinfected human dendritic cells the ebola-glycoprotein modulates the function of natural killer cells nk cells accumulate in infected tissues and contribute to pathogenicity of ebola virus in mice markedly elevated levels of interferon (ifn)-gamma, ifnalpha, interleukin (il)- , il- , and tumor necrosis factoralpha associated with fatal ebola virus infection defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in ebola virusinfected patients activation receptor-dependent ifn-g production by nk cells is controlled by transcription, translation, and the proteasome dengue virus-infected dendritic cells, but not monocytes, activate natural killer cells through a contact-dependent mechanism involving adhesion molecules show that nk cells provide early control against dengue infection, and that nk cell activation by dengue virus kir ds recognizes conserved peptides derived from viral helicases in the context of hla-c nkp receptor mediates interaction of the envelope glycoproteins from the west nile and dengue viruses with nk cells production of interferon alpha by dengue virus-infected human monocytes high levels of interferon alpha in the sera of children with dengue virus infection analysis of plasma viral rna levels during acute dengue virus infection using quantitative competitor reverse transcriptionpolymerase chain reaction longitudinal analysis of natural killer cells in dengue virus-infected patients in comparison to chikungunya and chikungunya/dengue virus-infected patients defining the role of nk cells during dengue virus infection ló pez-vergè s s: hla upregulation during dengue virus infection suppresses the natural killer cell response influence of kir genes and their hla ligands in susceptibility to dengue in a population from southern brazil interaction of a dengue virus ns -derived peptide with the inhibitory receptor kir dl on natural killer cells immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms early cd expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever lassa virus infection of human dendritic cells and macrophages is productive but fails to activate cells nk cells are strongly activated by lassa and mopeia virus-infected human macrophages in vitro but do not mediate virus suppression hla-crestricted viral epitopes are associated with an escape mechanism from kir dl (+) nk cells in lassa virus infection early and strong immune responses are associated with control of viral 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two conserved amino acids within the nss of severe fever with thrombocytopenia syndrome phlebovirus are essential for anti-interferon activity severe fever with thrombocytopenia syndrome phlebovirus non-structural protein activates tpl signalling pathway for viral immunopathogenesis dynamic changes of laboratory parameters and peripheral blood lymphocyte subsets in severe fever with thrombocytopenia syndrome patients genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein nss rift valley fever virus inhibits a proinflammatory response in experimentally infected human monocyte derived macrophages and a pro-inflammatory cytokine response may be associated with patient survival during natural infection a sap complex inhibits ifn-beta expression in rift valley fever virus infected cells innate immune basis for rift valley fever susceptibility in mouse models cd t cells, cd t cells, and monocytes coordinate to prevent rift valley fever virus encephalitis subversion of the immune response by rabies virus. viruses control of acute arboviral infection by natural killer cells association of hla class-i and inhibitory kir genotypes in gabonese patients infected by chikungunya or dengue type- viruses phenotypic and functional analyses of nk and nkt-like populations during the early stages of chikungunya infection caribbean and la reunion chikungunya virus isolates differ in their capacity to induce proinflammatory th and nk cell responses and acute joint pathology world health organization methodology to prioritize emerging infectious diseases in need of research and development we thank members of the sun lab, dr. lewis lanier, dr. daniel calabrese, and regina bou puerto for the useful discussion and comments on this review. the sun lab was supported by grants from the burroughs wellcome fund, the american cancer society, and the national institutes of health (ai , ai , and p ca ). key: cord- -u ud vmq authors: lussi, carmela; schweizer, matthias title: what can pestiviral endonucleases teach us about innate immunotolerance? date: - - journal: cytokine growth factor rev doi: . /j.cytogfr. . . sha: doc_id: cord_uid: u ud vmq pestiviruses including bovine viral diarrhea virus (bvdv), border disease virus (bdv) and classical swine fever virus (csfv), occur worldwide and are important pathogens of livestock. a large part of their success can be attributed to the induction of central immunotolerance including b- and t-cells upon fetal infection leading to the generation of persistently infected (pi) animals. in the past few years, it became evident that evasion of innate immunity is a central element to induce and maintain persistent infection. hence, the viral non-structural protease n(pro) heads the transcription factor irf- for proteasomal degradation, whereas an extracellularly secreted, soluble form of the envelope glycoprotein e(rns) degrades immunostimulatory viral single- and double-stranded rna, which makes this rnase unique among viral endoribonucleases. we propose that these pestiviral interferon (ifn) antagonists maintain a state of innate immunotolerance mainly pertaining its viral nucleic acids, in contrast to the well-established immunotolerance of the adaptive immune system, which is mainly targeted at proteins. in particular, the unique extension of ‘self’ to include the viral genome by degrading immunostimulatory viral rna by e(rns) is reminiscent of various host nucleases that are important to prevent inappropriate ifn activation by the host’s own nucleic acids in autoimmune diseases such as aicardi-goutières syndrome or systemic lupus erythematosus. this mechanism of “innate tolerance” might thus provide a new facet to the role of extracellular rnases in the sustained prevention of the body’s own immunostimulatory rna to act as a danger-associated molecular pattern that is relevant across various species. viruses are never able to propagate and survive on their own, i.e., they are totally dependent on a host that they can infect. consequentially, the field of virology is intimately linked to immunology as, during the long time of co-evolution, the hosts have acquired a vast array of antiviral defense mechanisms and vice versa. bovine viral diarrhea virus (bvdv) is probably one of the most wide-spread viruses, at least among the "terrestrial viruses". in this mini review, we will focus on the interplay of bvdv, especially its viral rna, with the innate immune defense of the host animals, because this is the key element to explain the long-term survival of this virus in its host population. finally, we hypothesize that the survival strategy of bvdv to induce adaptive and, likewise, innate immunotolerance might provide a new viewpoint for the way the host handle its own, potentially immunostimulatory, self nucleic acids to prevent them from inappropriate chronic activation of the interferon (ifn) system. the pestiviral mechanisms to avoid detection and, thus, to behave identical as the body's own rna, might well be analogous to the mechanisms of its host not to recognize own structures, the latter being of paramount importance to prevent autoimmune reactions. bovine viral diarrhea virus (bvdv), including the two species (also called 'genotypes') bvdv-i and bvdv-ii, classical swine fever virus (csfv), border disease virus (bdv) of sheep, and several tentative species belong to the genus pestivirus in the family flaviviridae [ ] . bvdv is a cattle pathogen of major importance with a worldwide distribution. its viral life cycle was recently described in a number of excellent reviews [ ] [ ] [ ] [ ] [ ] and, thus, is only briefly summarized here. pestiviruses are single-stranded (ss) rna viruses with an envelope containing three viral glycoproteins, i.e., e rns , e , and e . the genome with positive polarity encodes for a single large open reading frame (orf). by virtue of a large variety of possible mutations, bvdv exists as a cytopathic (cp) and a noncytopathic (ncp) biotype, defined by their effect on cultured cells. upon attachment of the virus particle to the cell surface, the virus enters its host cell via clathrin-mediated endocytosis. fusion of the viral with the endosomal membrane is then initiated by acidification of the organelle. cap-independent translation is mediated by an internal ribosomal entry site (ires) and the polyprotein is then further processed by cellular and viral proteases into at least structural and non-structural viral proteins. virus assembly most likely occurs in intracellular vesicles and exocytosis of mature particles occurs in a non-lytic way, at least for the ncp biotype. infection of cattle with either biotype results in transient viremia and infected animals show no disease signs, mild diarrhea, fever, and coughing, but severe thrombocytopenia and hemorrhages have also been reported [ ] . upon resolution of infection, the animals will be protected from re-infections. by contrast, infection of pregnant cows within the first days of gestation with an ncp, but not cp, biotype of bvdv may result in the birth of persistently infected (pi) calves. the clinical symptoms of such pi animals vary considerably and range from unapparent infection to severe growth retardation. gastrointestinal and/or lung diseases are frequently described, with lung-centered pathology observed mainly in young calves and mucosal pathology predominantly in older animals ( [ ] , and references therein). these pi calves are highly susceptible to secondary infections with other pathogens, and are at risk of developing fatal mucosal disease (md). the latter can occur at any time during the life of the pi animal if mutations or recombination with viral or cellular rna with the persisting ncp strain lead to the generation of an antigenically homologous cp biotype (for reviews, see refs. [ , , ] ). however, the development of such a cp biotype in these pi animals is rather an evolutionary misfortune as the cp strain will be eliminated with the death of its host animal [ ] . it is exclusively the ncp biotype of pestiviruses that is transmitted in the long term from pi animals to naïve pregnant host's in order to produce new pi calves [ ] . hence, persistent infection at the level of the single animal is responsible for viral persistence in the host population. consequently, it's the pi animals that are specifically searched for and eliminated in order to eradicate bvdv in regional and national control programs [ , [ ] [ ] [ ] . as positive-sense rna viruses, pestivirus replication occurs via a semi-conservative model [ ] using a double-stranded (ds) rna template to synthesize plus-strand, genomic viral rna. accordingly, replicative forms (rf) and replicative intermediates (ri) were detected in bvdv-infected cells, and it was estimated that the ri contain - nascent strands per template [ ] [ ] [ ] . thus, pestiviral replication involves the formation of dsrna intermediates in the cytosol of infected cells as seen with a variety of rna and dna viruses [ ] . this could be confirmed in bvdv-and csfv-infected cultured cells by immunofluorescence or immunoelectron microscopy and flow cytometry using a dsrna-specific monoclonal antibody [ ] [ ] [ ] . in line with the rather unrestrained replication of the cp biotype of pestiviruses [ ] , the amount of plus-and minus-strand viral rna and, thus, also of dsrna is up to two order of magnitude higher in cells infected with cp than with ncp viruses ( [ , , ] , and references therein). as dsrna is a potent pathogen-associated molecular pattern (pamp) [ ] , it comes of no surprise that pestiviruses are able to induce a type-i ifn response. however, ifn induction strongly depends on the virus strain, its biotype, virulence, and the cell type being infected. thus, infection of many cell types by pestiviruses of the cp, but not the ncp, biotype induces ifn type-i synthesis in vitro (for review, see ref. [ ] ), whereas in calf testicle cells [ ] and porcine plasmacytoid dendritic cells (pdc) [ ] , ifn type-i was induced by ncp bvdv and ncp csfv, respectively. notwithstanding, the amount of dsrna present in cells infected with pestiviruses of the ncp biotype is basically able to induce ifn expression in most cell types, which become obvious as mutant ncp strains lacking the ifn antagonist n pro (see below) replicate to similar or only partially reduced levels as its wt parent strains but readily induce ifn synthesis [ ] [ ] [ ] . thus, it can be conceived that the threshold for ifn induction, e.g., the amount of trigger required to induce an innate immune response, and the effectiveness of the pestiviral ifn antagonists varies between different cell types. the pattern recognition receptors (prr) that sense the pestiviral infection are much less well characterized. viruses are mostly recognized by virtue of their genome (for reviews, see e.g., refs. [ ] [ ] [ ] ), and thus, the cytosolic rlrs (rig-i-like receptors, such as rig-i, mda- and lgp ) and the toll-like receptors (tlrs) in the endolysosomal compartments were obvious candidates. transfection of total rna extracts isolated from cp bvdv-infected cells into uninfected mdbk cells induced ifn synthesis in a triphosphate-independent manner [ ] , which points to mda- as possible prr. however, as rig-i is not strictly dependent on a triphosphate moiety [ ] , other receptors in addition to mda- could not be excluded. accordingly, hüsser et al., using lentivirusmediated transduction of short hairpin rna, nicely demonstrated that csfv is sensed by mda- , rig-i and tlr- in porcine pk- cells [ ] . in addition, ifn-a secretion in pdcs induced by csfv infection or by cell-cell contact with csfv-infected cells was severely reduced by an oligodeoxynucleotide inhibitor of tlr [ ] . activation of pdcs by csfv infection required replication of the virus, as uv inactivation or neutralization of the virus suspension with neutralizing antibody completely abrogated ifn-a release, whereas ifn expression upon cell-dependent rna transfer was independent on infectious virus particles [ , ] . these results show that cytosolic and endolysosomally localized prrs are able to detect the presence of pestiviral rna. rlrs located in the cytosol most probably detect replicative double-stranded intermediates of various length formed during viral replication in productively infected cells. however, it remains unknown how this dsrna gets access to tlr- containing compartments. on the one hand, dsrna might be liberated from infected cells that undergo spontaneous or virus-induced apoptosis and then becomes endocytosed by neighboring cells [ ] . on the other hand, viral rna present in the cytosol might be shuttled to endosomes by the formation of autophagosomes as shown for vsv and activation of tlr- in mouse pdcs [ ] . but the fact that pestiviral replication was purported to be even enhanced by autophagy in pk- or mdbk cells [ , ] argues against the latter possibility. finally, virus replication complexes might be already formed in membranous vesicles as described, e. g., for corona-or hepatitis c viruses [ , ] , but such large membrane arrangement were not observed in pestivirus infected cells [ ] . notwithstanding, dsrna was localized inside the lumen and outer membranes of multivesicular bodies (mvbs) in mdbk cells infected with the pestivirus strain giraffe- , but whether these vesicles represent autophagosomes that hide the dsrna from detection by the innate immune system followed by disposal in lysosomes, or whether they are true sites for viral replication is currently unknown [ ] . finally, viral ssrna represents a pamp on its own as shown by the activation of tlr- in porcine pdcs [ ] . this indicates that viral replication is not required to generate a danger signal, but viral rna synthesis might be required in order to produce a sufficient amount of trigger molecules. the exact structure of the ssrna molecule required to activate tlr- / is not yet known, but au-or gu-rich regions, or inosine-containing immunostimulatory ssrna were reported to effectively activate tlr- [ , ] . interestingly, inosine incorporation seems to increase the rna's secondary structure that enables its recognition by tlr- [ ] , further demonstrating that highly structured ssrna in addition to dsrna is a tlr- agonist [ ] . this is confirmed by the fact that in vitro transcribed ssrna of the bvd viral genome possess highly structured regions resistant to serum rnases and to rnase a (preferentially ssrnases) that are able to induce activation of tlr- [ , ] . the -und -utrs and long-range interactions between these two regions might be especially immunostimulatory [ , ] , but most regions within the bvd viral genome seem to possess high-ordered structures able to induce tlr activation [ ] . in summary, various single-and double-stranded intermediates of viral rna replication and viral genomic ssrna or fragments thereof that were released by premature decay of extracellular or endosomal virus particles represent immunostimulatory nucleic acids. based on their different localizations, a variety of prrs in different compartments, e.g., rlrs in the cytosol and tlrs in endolysosomes, might become activated by pestiviruses. infection of the fetus at an early stage of development as described above effectively bypasses the adaptive immune system by establishing self-tolerance including b-as well as t-cells. in addition, maternal neutralizing antibodies cannot cross the ruminant epitheliochorial placenta, further protecting the virus from a humoral immune response within the fetus. however, in order to succeed in establishing persistent infection, bvdv still requires to cope with the innate immune system already active from the outset. thus, the interaction of ncp bvdv with its host bypasses the adaptive immunity by inducing central immunotolerance, as well as evades innate immunity, with the ifn system as one of the most important antiviral defense systems of the host. the n-terminal non-structural autoprotease n pro and the envelope glycoprotein e rns are two ifn antagonists expressed by pestiviruses that are unique to this genus within the flavivirus family. in the past few years, it became more and more evident that both antagonists are required in a non-redundant way to successfully establish persistent fetal infection [ ] . n pro expressed in virus infected cells is responsible for polyubiquitinylation and proteasomal degradation of the transcription factor irf- in an as yet unknown manner but without requiring its proteolytic activity. by contrast, secreted e rns prevents the activation of the host's tlrs also in non-infected cells by endonucleolytic degradation of viral rna prior to their activation of the corresponding prrs (for reviews, see refs. [ , , , , ] ). collectively, it appears that n pro and e rns effectively reduce or at least delay ifn induction. the fact that the highly replicating cp biotype of pestiviruses similarly express these two ifn antagonistic proteins indicate that there exists a delicate balance between the level of pamps and the capacity to limit their effects on the innate immune response of the host. with pestiviruses exhibiting a rather broad cell tropism, expression of n pro as the very first protein enables an efficient and fast inhibition of dsrna-induced responses in all cells containing replicating virus. in addition, the rather unspecific cell and host tropism of soluble e rns (see below), which is even active, e.g., in canine or human cells [ ] , prevents tlr activation in a large variety of uninfected cells within the host animal. nevertheless, despite the availability of such effective inhibitory mechanisms, both biotypes of bvdv induce the expression of ifn in vivo after infection of adult animals [ ] [ ] [ ] . by contrast, only ncp bvdv strains are immunologically silent in fetuses and pi animals [ , ] . the latter fact might still be debated as chronic up-regulation of type-i and type-ii interferon was reported in bovine fetuses infected early in utero [ ] . indeed, % of pi animals but only % of non-pi control animals expressed mx protein in pbmcs ex vivo. however, there was no correlation with the amount of viral rna or e rns protein and only a week positive correlation with the infectious virus titer in the plasma of the pi animals (t.t.h. pham blume and m. schweizer, unpublished observation). this indicates that it is rather the increased susceptibility of pi animals to secondary infections [ ] than the persisting virus itself that is the cause for the increased activation of the ifn system in these animals. whether this increased susceptibility is related to the selective immunosuppression elicited by the expression of the ifn antagonists and whether it is more distinctive for pathogens exploiting these pathways remains to be shown. thus, the precise adjustment of inhibitory and stimulatory triggers of the innate immune system and their spatiotemporal control in vivo are not yet sufficiently characterized. notwithstanding, it appears that the evasion of the ifn response is the central element for ruminant pestiviruses to induce persistent infections. in the next paragraphs, we will specifically describe the mechanisms of e rns contributing to the establishment and maintenance of innate immunotolerance in pi animals, and put it into relation to other host and viral nucleases that are involved in the depletion of immunostimulatory self and nonself nucleic acids. e rns , initially termed e , was first detected at the surface of pestiviral particles and shortly thereafter also in the supernatant of virus infected cells [ , ] . accordingly, e rns was detected in vivo with concentrations up to ng/ml in the serum of pi animals [ ] . both, secreted and structural e rns are mostly found as disulfidelinked homodimers of around kda [ ] , with carbohydrates contributing approximately half of the apparent molecular weight [ ] . e rns contains nine highly conserved cysteine residues that form four intramolecular disulfide bonds [ ] . the c-terminal cysteine at the position (c ) forms an intermolecular disulfide bond between two e rns monomers and a substitution of this residue results in a loss of the dimeric status [ ] . to that effect, viruses encoding monomeric e rns are not restricted in their replication in vitro but are attenuated in vivo [ , ] . as envelope glycoprotein, e rns plays a role in virus attachment through interactions with the cell surface glycosaminoglycans (gags) [ , ] . binding of e rns to gags involves a cluster of basic residues ( -kklenksk- ) near the c-terminus, with the lysine residues at positions and being critical for binding [ ] . nonetheless, pseudotyped particles containing only e and e of csfv were still able to mediate virus entry [ ] . in addition to its contribution to virus attachment, the c-terminus is folded into an amphipathic helix that anchors e rns in plane into the membrane of the viral envelope [ , ] . detailed analyses depicted that the amphipathic helix lies with a slight tilt within the membrane just underneath the lipid head domain [ , ] . to ensure a sufficient number of protein molecules for the production of new virus particles, a large number of the e rns proteins remains within the infected cells in a not yet defined part of the endoplasmic reticulum (er) by a specific retention signal located within the cterminal amphipathic helix [ ] . in summary, the c-terminus of e rns fulfills different tasks: it anchors the protein into the viral envelope, it attaches soluble e rns to the cell surface via its gagbinding sites and it helps to maintain an appropriate ratio of cellassociated and soluble e rns . in , it was reported that, in addition to its function as envelope glycoprotein, e rns resembles ribonucleases of the rnase t family and indeed degrades preferentially ssrna ( [ ] ; for review, see refs. [ , , ] ). thereby, e rns , but not rnase-inactive mutants, potently inhibit ifn expression induced by the addition of extracellular synthetic or viral ss-or dsrna [ , , ] . according to x-ray structure analyses, however, e rns is only able to bind ssbut not dsrna in the active site [ ] . hence, the mechanism of e rns to degrade dsrna remains to be investigated, but we propose that it might act as a nicking endoribonuclease targeting the two strains of dsrna individually ( [ ] , and lussi et al., in preparation). these in vitro results are also applicable in vivo, as bvdv and csfv mutant viruses encoding for an e rns protein lacking its rnase activity are severely attenuated [ , ] . owing to the gag-binding site within the amphipathic helix, e rns rather unspecifically binds to cell surfaces, and is thus able to act as ifn antagonist in cells of various species, e. g., caprine, ovine, canine, or human cells. mutant proteins lacking amino acid residues of the c-terminus, including the gag-binding site, were severely limited in their inhibition of dsrna-induced ifn expression [ ] . furthermore, binding to the cell surface was followed by an energy-dependent uptake via clathrin-mediated endocytosis, which strongly indicates that e rns cleaves its substrate in an intracellular compartment [ ] (fig. b) . this is corroborated by the fact that rnase-active e rns effectively inhibited activation of tlr- by extracellularly added dsrna or viral ssrna that is resistant to serum rnases of the host (fig. a) , and of tlr- by virus-infected cells in contact with pdcs [ , ] . by contrast, activation of tlr- by r was not inhibited by e rns [ ] further indicating that it does not inhibit any downstream signaling but rather degrades the viral pamp prior to activation of tlrs (fig. ) . as extracellular dsrna was reported to be delivered to endosomal andby an unknown wayto cytosolic prrs, it cannot formally be excluded that rlrs are activated as well [ ] . but e rns potently inhibits ifn induction by extracellular dsrna, and it can thus be postulated that the dsrna might well be degraded within an endolysosomal compartment prior to any transfer to the cytosol. accordingly, ifn induction was unimpeded in e rnsexpressing cells upon lipofectin-mediated transfection of poly (ic), whereas the effect of dsrna added to the medium was completely blocked [ ] . an endosomal location of e rns is also in agreement with its preference for a slightly acidic milieu. e rns is active in a broad ph range from about - . with an optimum around a ph value of , measured in a mm sodium-or tris-acetate buffer [ ] . by contrast, e rns degraded poly(ic) but not in vitro transcribed dsrna in cell culture medium (ph . - . ) with the latter being cut at a ph value of . [ ] . however, by comparing the activity of e rns at ph . and . in tris-acetate buffer versus cell culture medium (mem), we demonstrated that the activity of e rns is much lower in hepesbuffered mem irrespective of the ph tested ( table ), implying that there might be an unknown inhibitory factor in the cell culture medium different from the buffer substance itself. based on the fact that gag-binding of e rns is required prior to its uptake by endocytosis, we were able to effectively prevent or reverse binding of e rns to cell membranes by heparin treatment (fig. a) . furthermore, even after removal of all extracellular e rns by heparin after one hour of incubation, the viral rnase was still able to inhibit dsrna-induced ifn expression in bovine turbinate cells up to - days later [ ] . finally, complexation of dsrna with the human cathelicidin ll- completely protected the nucleic acid from degradation by e rns in vitro. nonetheless, ifn induction by these dsrna-ll- complexes was inhibited in e rnstreated cells, which suggests that the rna dissociates from ll- in order to the nucleic acid being accessible to degradation by the viral rnase within the corresponding compartment (fig. b ). this is in complete accordance with e rns being active at ph values as low as . and with the fact that poly(ic) dissociates from ll- at low ph values, e.g., upon endosomal acidification [ ] , which is a further indication for the endolysosomal localization of e rns . the exact location of e rns inside the various cell types, and the mechanism of how it specifically encounters its targets, i.e., ss-and dsrna, remain to be established. fig. . pestiviral e rns inhibits ss-and dsrna-induced type-i interferon (ifn) synthesis. viral ss-and dsrna, e.g., from dying infected cells, are potent pathogen-associated molecular pattern (pamp) that are sensed by the corresponding pattern-recognition receptor (prr) such as tlr- and tlr- . both toll-like receptors are located in endolysosomal compartments and, once bound by their substrates, they induce downstream signaling that leads to ifn expression. as many regions of the bvd viral genome are resistant to degradation by extracellular serum rnases, they effectively induce the cell's innate immune response (a). by contrast, soluble e rns is taken up by clathrinmediated endocytosis that enables this rnase to effectively degrade the viral pamps prior to tlr activation (b). the annotation of the elements in the figure is depicted in the panel on the right. a number of viruses express ribonucleases, which play crucial roles in viral replication and in virus-host interactions. these viruses comprise negative-strand rna virus of the orthomyxo-, arena-, or bunyavirus families, plus-strand rna viruses such as nidoviruses, or dna viruses such as herpesviruses. the role of these virus-encoded exo-and endoribonucleases, e.g., in proof-reading, mrna cap snatching, protein synthesis shutoff, or rna interference were described in a number of excellent reviews [ , , [ ] [ ] [ ] . in the following, we therefore only describe few examples for the role of viral rnases in evasion of the host's innate immune response. viruses in the order nidovirales, including corona, arteri-and roniviruses, encode for a large number of ifn antagonists within their extraordinary large rna genome [ ] . among them, the exonuclease exon within non-structural protein (nsp) , the endoribonuclease endou encoded by nsp , and possibly the nsp b of the arterivirus porcine reproductive and respiratory syndrome virus (prrsv) possess rnase activity. the latter seems to be conserved only in prrs viruses, whereas nsp in coronaviruses was reported to lead to host translational shut off by degrading host mrnas through a yet unknown host endonuclease, but its precise role and conservation among the nidoviruses remain to be table degradation of in vitro transcribed pestiviral ss-and dsrna [ ] by e rns in various buffer systems at ph . or . , with rating from very strong degradation (+++; green) to no degradation (red). trisac: tris-acetate buffer; mem: minimum essential medium; n.d.: not done. fig. . pestiviral e rns inhibit viral rna-induced ifn synthesis in endosomal compartments. blocking e rns from entering the cell by heparin treatment prevents this viral rnase to inhibit ss-and dsrna-induced ifn expression (a). by contrast, despite protection from rnase degradation by complexation of nucleic acids, e.g., by ll- , ifn expression induced by complexed viral rna is nevertheless inhibited by e rns , as endosomal acidification leads to separation of ll- from the rna that makes it immediately amenable for degradation by e rns also at low ph values (b). the annotation of the elements in the figure is depicted in the panel on the right. clarified [ , , ] . the exoribonuclease exon within the nterminal part of nsp in coronaviruses (that also harbors an n methyltransferase activity at the c-terminus) is responsible for 'proof-reading' during replication of these large nidoviruses, but additionally, a role for exon in degrading rna to avoid their recognition by the host's prrs was suggested [ ] . finally, the endonuclease nsp (nsp in arteriviruses) cleaves ss-and dsrna preferably at uridine residues [ , , ] . the substrate of the rnase activity in nsp is not yet known, but it might be conceivable that nsp degrades viral rna to avoid its recognition as pamp by cellular prrs [ ] . alternatively, nsp of the arterivirus prrsv was reported to degrade mavs mrna [ ] , but this is still debatable as this conclusion was presumably drawn from experiments using single protein overexpression and the original data were not yet published. akin to pesti-and coronaviruses, arenavirus infections are linked to suppression of the host innate immune system. the nucleoprotein (np) of the prototypic arenavirus lymphocytic choriomeningitis virus (lcmv) was shown to be able to inhibit an ifn type i response by blocking irf- nuclear translocation [ ] . this observation was later extended to other arenaviruses, including lassa virus, and it was demonstrated that a - exoribonuclease activity in the c-term of np is essential for the ifn suppression. determination of the crystal structure of np of lassa and tacaribe virus and biochemical analyses confirmed that the structure of np contains an exonuclease domain of the dedd family that is able to cleave -triphosphate dsrna templates in vitro. collectively, there is strong evidence that the exonuclease activity of np is conserved in all arenaviruses and is required to prevent activation of rig-i by the degradation of viral pamps ( [ , ] , and references therein). almost every virus encodes for at least one mechanism to prevent its nucleic acid being recognized by the host and thereby activating the host's ifn response [ ] . overall, however, there is only few experimental evidence that viral nucleases indeed degrade their viral pamps to evade the host's innate immune defense. by contrast, there are several instances of host nucleases that use a similar strategy to degrade own danger molecules that might participate in autoimmune pathologies as exemplified in the following section. inappropriate activation of type-i interferon goes along with many autoinflammatory and autoimmune diseases, and there is strong evidence that dysregulation of various host pathways that are required to contain immunostimulatory self nucleic acids play a causative role (for recent reviews, see e.g., refs. [ ] [ ] [ ] [ ] [ ] [ ] ). in the following, a few examples are briefly described to illustrate the role of ifns and of nucleases regulating ifn synthesis in autoimmune diseases. in systemic lupus erythematosus (sle), circulating ifn-a levels correlate with disease severity. the recognition of self-dna or self-rna by tlrs followed by an ifn-dependent auto-amplification loop seems to be a major mechanism of disease pathogenesis. thus, in sle, pdcs are continuously activated by immune complexes (ic) comprising self-nucleic acids (e.g., from apoptotic or necrotic cell material) and autoantibodies to self-rna, -dna or nucleoproteins followed by fc receptor mediated uptake, which ultimately leads to the secretion of large amounts of ifn type-i in a tlr- / or tlr- -dependent manner. the constant ifn production, the ifn-dependent maturation of myeloid dendritic cells followed by stimulation of autoreactive t-cells and the differentiation of bcells into autoantibody-secreting plasma cells further aggravate the disease symptoms [ , [ ] [ ] [ ] . anti-inflammatory treatment of sle by glucocorticoids, which are thought to act via inhibition of nf-kb, shows only limited success in relieving sle disease symptoms, as the continuous activation of tlrs in pdcs by selfnucleic acids also activate nf-kb that enhances pdc survival and, thus, continued ifn secretion is not abrogated [ ] . in addition to autoantibodies, the antimicrobial cathelicidin peptide ll- and high-mobility group box protein (hmgb , a nuclear dna-binding protein released from necrotic or cytokine-stimulated cells) might protect the self-nucleic acids from degradation by host nucleases, and facilitate their uptake by pdcs and b-cells. interestingly, ll- which is produced by keratinocytes and neutrophils after skin injuryis similarly overexpressed in psoriatic skin lesions, and its ability to complex self-rna and self-dna, to enhance its retention in the endosomes of plasmacytoid and of myeloid dendritic cells, and to trigger tlr-dependent ifn synthesis might be responsible for the observed breaking of self-tolerance [ , , ] . finally, there is genetic evidence obtained from studies with various knockout mice and genome-wide association studies that support the role of innate and adaptive immune responses in the development of sle, such as pathways involved in removal of apoptotic bodies and ics (including dnase i and trex- (dnase iii)), prr activation and ifn expression, and interference with t-and bcell signaling [ ] . remarkably, mice with the y-linked autoimmune accelerating (yaa) locus show enhanced sensitivity to develop lupus depending on the background of the mice, which was attributed to a x to y chromosomal translocation resulting in the duplication of the gene encoding for tlr- . the latter observation might also relate to the fact that women are around times more often affected by sle than men, which might be caused by incomplete x chromosome inactivation leading to insufficient tlr- dosage compensation [ ] [ ] [ ] . aicardi-goutières syndrome (ags) is a genetically determined, autosomal recessive disease of progressive encephalopathy of early childhood, very similar to congenital viral infections. some children develop early-onset sle or a cutaneous form thereof, familial chilblain lupus. ags is caused by mutations in trex- , various components of the rnase h complex, samhd , adar- and mda- (for review, see e.g., [ , ] ). trex- is the main - dna exonuclease in mammalian cells and might be involved in the disposal of dna from endogenous retroelements. recently, it was reported that trex- exerts in addition rna exonuclease activity on ssrna and on rna/dna hybrids and both, dna and rna exonuclease activity, are lost by the mutations found in ags patients [ ] , which in the end leads to spontaneous activation of the cytosolic dna sensor cyclic gmp-amp synthase (cgas) followed by ifn expression [ ] . rnase h is composed of three different subunits performing endonuclease activity cleaving rna in rna/dna hybrids or it cleaves the phosphodiester bond of individual ribonucleotides in dna duplexes. the precise role of samhd in ags is not yet known. the wild-type enzyme is a triphosphohydrolase, which reduces the cellular dntp pool, and is an rnase, whose endogenous rna substrate still needs to be identified. in any case, spontaneous ifn expression in samhd knockout mice was reported to involve intracellular rna and dna sensors, as additional knockout of the adapter proteins mavs or sting abolished ifn production [ ] . finally, adar- deaminates adenosine to inosine in dsrna and was reported to suppress ifn signaling, possibly by marking endogenous dsrna as self avoiding recognition by mda- [ ] . analogously, the mutations found in mda- in ags lead to a gain-of-function phenotype with increased affinity to dsrna and enhanced ifn expression. combined, mutations that cause an aberrant nucleic aciddependent signaling and increased ifn expression were described in all of these autoimmune disorders that finally lead to disease pathology. the "ifn signature" is a hallmark of all these "type i interferonopathies" [ ] , and retroelements, which make up half of the human genome, seem to be an important source of endogenous ligands for the host prrs. finally, there is a strong overlap between the control of self-nucleic acids and the antiviral innate immune response, further highlighting the need for a tight regulation. during viral infections, the adaptive immune system is primarily responsible for the detection of nonself proteins. as viruses lack the elements of metabolism required for independent multiplication and, therefore, depend on the enzymes of their host cells to synthesize their proteins, viruses are largely recognized by their dna and rna genomes. thus, it is the innate immune system that is in charge of detecting viral nucleic acids, which makes it a formidable task for the host to separate foreign from body's own as nucleic acids are not pathogen-specific by default. the difficulty of differentiation of self from infectious nonself nucleic acids becomes especially apparent when considering that around % of our own genome consists of retroviral sequences [ , ] . to specifically detect nonself nucleic acids by the corresponding prrs, a number of strict controls are required in order to avoid autoimmune reactions. for instance, nucleic acid-sensing tlrs are mostly confined to the endolysosomal compartment and their activation is ph dependent; modification of host nucleic acids, e.g., methylation of nucleosides or incorporation of pseudouridine, prevents their recognition by prrs or even negatively regulates tlr activation; or sequestration might render rna or dna invisible to the host. finally, if all else fails, improper activation of the innate defense is prevented by degradation of immunostimulatory nucleic acids by host rnases or dnases [ , ] . thus, quite some knowledge was gained on the role of host nucleases in preventing autoimmune diseases (compare section . ), but most of these are localized intracellularly. by contrast, despite extracellular rnases were mentioned many times in the literature to play a role in eliminating free extracellular rna (for instance in ref. [ ] ), a specific role of these rnases in the elimination of immunostimulatory self rna has not unequivocally been demonstrated. one reason for this might be the large variety of endogenous rnases found in the serum, e.g., rnases of the rnase a and t family [ ] [ ] [ ] , which might prevent the establishment of single-gene knockout mice with a clear phenotype. in order to survive in the host population, we propose the hypothesis that ruminant pestiviruses induce persistence in its host animals by completely pretending to be part of the body's own, which is a clear advantage for the survival of the host and for successful virus transmission. as a result of the early fetal infection, bvd viral proteins already become part of the host's own with regard to the adaptive immune system by inducing central immunotolerance. as there is no or only limited long-term innate immune memory [ ] , maintaining tolerance to self nucleic acids is an enduring challenge for any host. as the pestiviral genome appears to be at least partially resistant to the host's extracellular rnases, the host's safeguard mechanism as described above fails to prevent tlr activation by misdirected viral ss-and dsrna. thus, the extracellularly secreted viral endonuclease e rns might be regarded as an extension of the host's rnase substrate specificity to avoid inappropriate activation of the innate immune system by immunostimulatory viral rna (fig. ) . with its capability of being endocytosed into endolysosomal compartments and with its rnase being active over a broad ph range, e rns is able to efficiently degrade viral pamps at all relevant compartments. even in the case of extracellularly sequestered immunogenic rnas that are protected from degradation by rnases, e rns effectively prevents them from stimulating tlrs as they are required to dissociate prior to activation of tlrs [ ] , which immediately exposes them to the pestiviral rnase (fig. ) . consequently, the virus in persistently infected animals is entirely tolerated by the host similarly to its own immunostimulatory nucleic acids without inducing overt disease [ ] . similarly, overexpression of tlr- in transgenic mice lead to a lupus-like disease phenotype, which could be partially reversed by additionally overexpressing secreted bovine rnase a [ ] . the host's ifn response is the prime antiviral defense system by inducing direct innate immune reactions and by shaping adaptive immunity. thus, the survival strategy of bvdv consists of being non-cytopathogenic and producing less dsrna than its cp counterpart, and expressing the ifn antagonists n pro as the first protein in order to reduce or even avoid ifn production in infected cells and e rns to degrade immunostimulatory viral rna before they might activate the host's prrs. notably, both pestiviral ifn antagonists are not only required to constantly maintain innate immunotolerance during persistent infections, but they also play an important role in acute infections [ ] . thus, rnase-inactive mutants of pestiviruses are attenuated upon acute infections [ , ] , and the evasion of the host's ifn response by n pro and e rns upon acute infection with csfv is also important for the virulence of the virus and the severity of immunopathology caused by the infection [ ] . thus, the pestiviral ifn antagonist, on the one hand, extend the host's specificity to tolerate self nucleic acids to its own viral rna during persistence and, on the other hand, participate in the evasion of the host's ifn response during acute, transient infections, further illustrating the dichotomy of immunotolerance and the antiviral immune response. this model might well shed new lights on fundamental questions on the innate tolerance to self nucleic acids and the specific detection of viral nonself rna. these aspects are highly relevant also for the prevention of chronic ifn induction and autoimmunity induced by "self-rnas" that might be fundamental beyond the mechanism of an animal disease [ ] . finally, the mechanism of the soluble pestiviral endoribonuclease e rns during persistent infection to support the virus in its strategy to pretend to be part of the body's own might be analogous to the role of the host's own extracellular nucleases to continuously maintain innate immunotolerance to its own immunostimulatory nucleic acids. matthias schweizer received his m.sc. in biochemistry in from the department of biochemistry at the university of zurich, switzerland and his phd in at the institute of biochemistry i at the swiss federal institute of technology (eth) in zurich, switzerland. the doctoral studies were on the oxidative regulation of mitochondrial ca + homeostasis and included a stay at tcom, university of north texas, fort worth (tx). after postdoctoral work at the eth and the institute of veterinary virology (ivv) at the university of bern, he received his "habilitation" (postdoctoral lecture qualification) in virology at the vetsuisse faculty of the university of bern, switzerland. since , he is head of the research group "virus infections of ruminants" and deputy head of the virology section of the institute of virology and immunology (ivi) of the federal food safety and veterinary office in cooperation with the vetsuisse faculty of the university of bern. his main interests are the interaction of ruminant pestiviruses with their host cells to induce persistence, the evolution of bvdv, and in applied research projects to further understand transmission and pathogenesis of pestiviral infections. carmela lussi is a phd student in the group of dr. matthias schweizer at the institute of virology and immunology (ivi) of the federal food safety and veterinary office in cooperation with the vetsuisse faculty of the university of bern. after receiving the bachelor of science in biology, she obtained her master of science in molecular life sciences with further specification in microbiology and immunology in at the faculty of science of the university of bern. within her phd studies, carmela lussi focusses on the role of pestiviral protein e rns in innate immunotolerance. virus taxonomy molecular biology of bovine viral diarrhea virus bovine viral diarrhea virus: global status molecular biology of pestiviruses the molecular biology of pestiviruses bovine viral diarrhea virus infections: manifestations of infection and recent advances in understanding pathogenesis and control clinical appearance and pathology of cattle persistently infected with bovine viral diarrhoea virus of different genetic subgroups rna recombination in pestiviruses-cellular rna sequences in viral genomes highlight the role of host factors for viral persistence and lethal disease cytopathic bovine viral diarrhea viruses (bvdv): emerging pestiviruses doomed to extinction bovine virusdiarrhöe (bvd): von der biologie zur bekämpfung pestivirus control programs: how far have we come and where are we going? anim bvdv control and eradication in europe-an update parallels among positive-strand rna viruses, reversetranscribing viruses and double-stranded rna viruses the replicative intermediate molecule of bovine viral diarrhoea virus contains multiple nascent strands characterization of rna synthesis during a one-step growth curve and of the replication mechanism of bovine viral diarrhoea virus properties of the bovine viral diarrhoea virus replicase in extracts of infected mdbk cells doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses role of doublestranded rna and n pro of classical swine fever virus in the activation of monocyte-derived dendritic cells the viral rnase e rns prevents ifn type-i triggering by pestiviral single-and doublestranded rnas morphogenesis of pestiviruses: new insights from ultrastructural studies of strain giraffe- a cellular jdomain protein modulates polyprotein processing and cytopathogenicity of a pestivirus the double-stranded rna-induced apoptosis pathway is involved in the cytopathogenicity of cytopathogenic bovine viral diarrhea virus intracellular detection of viral nucleic acids bvdv: a pestivirus inducing tolerance of the innate immune response self" and "nonself" manipulation of interferon defense during persistent infection: bovine viral diarrhea virus resists alpha/beta interferon without blocking antiviral activity against unrelated viruses replicating in its host cells classical swine fever virus n pro limits type i interferon induction in plasmacytoid dendritic cells by interacting with interferon regulatory factor the amino-terminal domain of bovine viral diarrhea virus n pro protein is necessary for alpha/beta interferon antagonism rnasedependent inhibition of extra-but not intracellular, dsrna-induced ifn synthesis by e rns of pestiviruses classical swine fever virus interferes with cellular antiviral defense: evidence for a novel function of n pro no love lost between viruses and interferons interferons and viruses: an evolutionary arms race of molecular interactions microbial sensing by toll-like receptors and intracellular nucleic acid sensors, cold spring harbor perspect viral rna detection by rig-i-like receptors identification of the role of rig-i, mda- and tlr in sensing rna viruses in porcine epithelial cells using lentivirus-driven rna interference efficient sensing of infected cells in absence of virus particles by plasmacytoid dendritic cells is blocked by the viral ribonuclease e rns fc gamma rii-dependent sensitisation of natural interferonproducing cells for viral infection and interferon-alpha responses extracellular dsrna: its function and mechanism of cellular uptake autophagydependent viral recognition by plasmacytoid dendritic cells autophagy during early stages contributes to bovine viral diarrhea virus replication in mdbk cells autophagy enhances the replication of classical swine fever virus in vitro to sense or not to sense viral rna-essentials of coronavirus innate immune evasion flaviviridae replication organelles: oh what a tangled web we weave identification of rna sequence motifs stimulating sequence-specific tlr -dependent immune responses inosine-mediated modulation of rna sensing by toll-like receptor (tlr ) and tlr inosine-containing rna is a novel innate immune recognition element and reduces rsv infection beyond dsrna: toll-like receptor signalling in rna-induced immune responses pestiviral e rns blocks tlr- -dependent ifn synthesis by ll complexed rna conserved rna secondary structures and long-range interactions in hepatitis c viruses the untranslated regions of classic swine fever virus rna trigger apoptosis bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting e rns rnase and n pro protease pestiviruses: how to outmaneuver your hosts charleston, type i and iii interferon production in response to rna viruses prolonged activity of the pestiviral rnase e rns as an interferon antagonist after uptake by clathrinmediated endocytosis alpha/beta and gamma interferons are induced by infection with noncytopathic bovine viral diarrhea virus in vivo in vitro and in vivo detection of mx gene products in bovine cells following stimulation with alpha/beta interferon and viruses acute non-cytopathic bovine viral diarrhea virus infection induces pronounced type i interferon response in pregnant cows and fetuses establishment of persistent infection with non-cytopathic bovine viral diarrhoea virus in cattle is associated with a failure to induce type i interferon innate and adaptive immune responses to in utero infection with bovine viral diarrhea virus processing of the envelope glycoproteins of pestiviruses a second envelope glycoprotein mediates neutralization of a pestivirus, hog cholera virus hog cholera virus: molecular composition of virions from a pestivirus a structural model of pestivirus e rns based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide mutation of cysteine of pestivirus e rns rnase prevents homodimer formation and leads to attenuation of classical swine fever virus dimerisation of glycoprotein e rns of classical swine fever virus is not essential for viral replication and infection passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein e-rns interactions of bovine viral diarrhoea virus glycoprotein e rns with cell surface glycosaminoglycans identification of the glycosaminoglycan-binding site on the glycoprotein e rns of bovine viral diarrhoea virus by site-directed mutagenesis characterization of classical swine fever virus entry by using pseudotyped viruses: e and e are sufficient to mediate viral entry the carboxy-terminal sequence of the pestivirus glycoprotein e rns represents an unusual type of membrane anchor the pestivirus glycoprotein e rns is anchored in plane in the membrane via an amphipathic helix structure of the membrane anchor of pestivirus glycoprotein e rns , a long tilted amphipathic helix lipid binding of the amphipathic helix serving as membrane anchor of pestivirus glycoprotein e rns a new type of intracellular retention signal identified in a pestivirus structural glycoprotein identification of a structural glycoprotein of an rna virus as a ribonuclease viral rnase involvement in strategies of infection role for bovine viral diarrhea virus e rns glycoprotein in the control of activation of beta interferon by double-stranded rna crystal structure of the pestivirus envelope glycoprotein e rns and mechanistic analysis of its ribonuclease activity the pestiviral ifn antagonist e rns cleaves dsrna as nicking endoribonuclease recovery of virulent and rnase-negative attenuated type bovine viral diarrhea viruses from infectious cdna clones mutations abrogating the rnase activity in glycoprotein e rns of the pestivirus classical swine fever virus lead to virus attenuation dsrna and the innate antiviral immune response rnase of classical swine fever virus: biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies ll- peptide enhancement of signal transduction by toll-like receptor is regulated by ph emerging roles for rna degradation in viral replication and antiviral defense virus-encoded endonucleases: expected and novel functions nidovirus ribonucleases. structures and functions in viral replication coronavirus virulence genes with main focus on sars-cov envelope gene interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus structures of arenaviral nucleoproteins with triphosphate dsrna reveal a unique mechanism of immune suppression exonuclease domain of the lassa virus nucleoprotein is critical to avoid rig-i signaling and to inhibit the innate immune response importance of nucleic acid recognition in inflammation and autoimmunity type i interferonopathies: mendelian type i interferon upregulation type i interferonopathiesan expanding disease spectrum of immunodysregulation nucleic acid-sensing tlrs and autoimmunity: novel insights from structural and cell biology rna degradation in antiviral immunity and autoimmunity nucleic acidsensing receptors: rheostats of autoimmunity and autoinflammation advances in understanding the role of type i interferons in systemic lupus erythematosus systemic lupus erythematosus-a disease with a dysregulated type i interferon system type i interferons: crucial participants in disease amplification in autoimmunity tlr recognition of self nucleic acids hampers glucocorticoid activity in lupus nucleic acids and endosomal pattern recognition: how to tell friend from foe? front little peptide, big effects: the role of ll- in inflammation and autoimmune disease genetics of systemic lupus erythematosus: immune responses and end organ resistance to damage tlrdependent and tlr-independent pathways of type i interferon induction in systemic autoimmunity x chromosome reactivation perturbs intracellular self/not-self discrimination sexx matters in immunity aicardi-goutières syndrome and the type i interferonopathies human dna exonuclease trex is also an exoribonuclease that acts on single-stranded rna trex deficiency triggers cell-autonomous immunity in a cgasdependent manner spontaneous type i ifn response in samhd -deficient mice requires both, functional intracellular rna and dna sensing pathways rna editing by adar marks dsrna as "self the enemy within: endogenous retroelements and autoimmune disease the potential role of retroviruses in autoimmunity plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases the mammalian secreted rnases: mechanisms of action in host defence t family ribonucleases: ancient enzymes with diverse roles the eight human canonical ribonucleases: molecular diversity, catalytic properties, and special biological actions of the enzyme proteins innate immune memory: a paradigm shift in understanding host defense two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases increased ribonuclease expression reduces inflammation and prolongs survival in tlr transgenic mice immune responses against classical swine fever virus: between ignorance and lunacy, front innate immunity: quo vadis? many thanks to fabienne wyss for performing the experiments shown in table , to giuseppe bertoni and eveline kindler for critically reading the manuscript, and to all the colleagues and students that were involved in our studies over the recent years. we are indebted to all of them. our work was supported over the years by internal funds of the institute of veterinary virology (now institute of virology and immunology) of the vetsuisse faculty university of bern, the federal food safety and veterinary office fsvo, and the swiss national science foundation. key: cord- -t qr wc authors: ikeda, masanori; kato, nobuyuki title: modulation of host metabolism as a target of new antivirals() date: - - journal: adv drug deliv rev doi: . /j.addr. . . sha: doc_id: cord_uid: t qr wc the therapy for chronic hepatitis c (ch–c) started with interferon (ifn) monotherapy in the early s and this therapy was considered effective in about % of cases. the present standard therapy of pegylated ifn with ribavirin achieves a sustained virologic response in about % of patients. however, about half of the ch–c patients are still at risk of fatal liver cirrhosis and hepatocellular carcinoma. the other significant event in hepatitis c virus (hcv) research has been the development of a cell culture system. the subgenomic replicon system enables robust hcv rna replication in hepatoma cells. and recently, the complete life cycle of hcv has been achieved using a genotype a strain, jfh . these hallmarks have provided much information about the mechanisms of hcv replication, including information on the host molecules required for the replication. anti-hcv reagents targeting hcv proteins have been developed, and some of them are now in clinical trials. however, the rna-dependent rna polymerase frequently causes mutations in the hcv genome, which lead to the emergence of drug-resistant hcv mutants. some of the cellular proteins essential for hcv rna replication have already been discovered using the hcv cell culture system. these host molecules are also candidate targets for antivirals. here, we describe the recent progress regarding the anti-hcv reagents targeting host metabolism. hepatitis c virus (hcv) was discovered in [ ] as the causative agent of chronic hepatitis c (ch-c), liver cirrhosis and hepatocellular carcinoma (hcc) [ ] . it is estimated that million people worldwide are infected with hcv [ ] . the ultimate goal of both clinical and basic hcv studies is the suppression of liver-related death caused by hcv infection. with respect to clinical studies, interferon (ifn) has played a major role in the treatment of patients with ch-c. ifn therapy started with ifn monotherapy in the early s, and a sustained virologic response (svr) was obtained in about % of patients [ ] . ifn therapy was developed by the hepatologists, and the current therapy of pegylated ifn (peg-ifn) with ribavirin has improved the svr to about % [ ] . therefore, the next stage of the therapy for ch-c is to develop new anti-hcv reagents to improve the svr. during the development of ifn therapy, the most striking discovery in the basic research was the development of a cell culture system for robust hcv rna replication. in , lohmann et al. [ ] achieved subgenomic hcv rna replication in a human hepatoma cell line, huh- . the advantages of this novel system (known as the replicon system) were that it provided not only a way to screen for anti-hcv reagents but also information about the mechanism of hcv rna replication. this cell culture system has been further improved, and recently the complete life cycle of hcv was achieved using a genotype a hcv strain, jfh [ ] [ ] [ ] . this newest system has extended the targets of the anti-hcv therapy to the virus infection and release. the effects of anti-hcv reagents selected from the cell culture-based screening should be evaluated using an animal model system for hcv infection before they can be released to clinical trial. chimpanzees were the only animal model in the early hcv studies [ ] . however, the use of chimpanzees is limited for ethical and financial reasons. in addition to chimpanzees, a study using tree shrews (tupaia belangeri chinensis) has been reported [ ] . a different approach to the study of hcv using animal models was achieved using the related gb virus b (gbv-b). gbv-b belongs to the flaviviridae family and can be transmitted to tamarins and marmosets [ , ] . these animal models may be valuable surrogate models for hcv study. another approach was demonstrated in a study using urokinase plasminogen activator-severe combined immunodeficiency (upa-scid) mice transplanted with human hepatocytes [ ] . this chimeric mouse model can support chronic hcv viremia under the circumstance without immune system. mass screening for anti-hcv reagents using cell culture systems will become a more powerful tool when combined with small animal model systems to evaluate the antiviral effects of selected reagents before clinical trial. in considering a new strategy for ch-c to be used in place of or in combination with ifn, the main targets are hcv proteins and hcv rna. with respect to the hcv proteins, two of these, nonstructural (ns) - a and ns b, have been wellcharacterized as protease and rna-dependent rna polymerase (rdrp), respectively [ , ] . several reagents have been reported to be inhibitors of ns - a serine protease, including sch [ , ] , sch [ ] , vx- [ , ] , and biln- [ ] . valopicitabine (nm ) was reported to inhibit ns b rdrp [ ] . hcv rna itself is also a target of antivirals, and recent rna interference technologies using sirna or shrna have targeted hcv rna [ ] [ ] [ ] . as rdrp lacks proofreading activity, the high mutation rate of rdrp allows the virus to escape from the reagents targeting hcv proteins and hcv rna. these anti-hcv reagent-targeting viral proteins and genome will be reviewed in another section. other targets are the cellular proteins essential for hcv rna replication and infection. the expression of hcv proteins is thought to affect the host cells' gene expression profiles and vise versa [ ] . the interaction of the specific cellular proteins with hcv proteins is essential for hcv replication (table ) . cyclosporine a (csa) is one of the best characterized inhibitors targeting the cellular proteins required for hcv replication [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the interaction of cyclophilin b (cypb) with ns b is required for hcv rna replication [ ] . csa inhibits hcv rna replication by interrupting the interaction between ns b and cypb. heat shock protein (hsp ) has also been reported to be an essential cellular protein for hcv rna replication [ ] [ ] [ ] . knockdown or inhibition of hsp has been shown to result in the anti-hcv activity in cell culture and in upa-scid mouse systems [ ] . fkbp , a member of the fk -binding protein family, specifically interacts with ns a and forms a complex with hsp [ ] . the la autoantigen (la) and polypyrimidine tractbinding protein (ptb) are also candidate cellular proteins for the inhibition of hcv rna replication [ ] , although no inhibitors for these proteins have been reported to date. thus, inhibition of the metabolism has recently been reported as a target of the new antivirals. here, we survey the recent progress on enzyme inhibitors of the cholesterol, sphingolipid, and guanosine triphosphate (gtp) synthesis pathways, as well as other metabolic pathways. hcv was discovered to be the causative agent of non-a, non-b hepatitis by the chiron corporation in [ ] . however, a treatment for patients with non-a, non-b hepatitis was established before the discovery of hcv. in , hoofnagle et al. reported that ifn-α treatment normalized the serum alanine aminotransferase (alt) levels in patients with non-a, non-b hepatitis [ ] . since the initial discovery of its anti-hcv activity, ifn-α has become the major reagent for ch-c treatment [ ] . the replication of hcv rna itself seems to stimulate ifn production signaling, and our recent results have suggested that core and/or ns b induce ifn-stimulated genes [ ] [ ] [ ] . however, viral ns - a protease inhibits the ifn production, although it does not completely shut it off. therefore, exogenous ifn administration is needed for patients with ch-c. the svr is affected by multiple factors, such as genotype, viral load and duration of therapy. ifn-α monotherapy was begun in the early s, but an svr was achieved in only about % of patients. in the early s, ifn-α and ribavirin combination therapy was developed and the svr was improved to about - %. furthermore, ifn itself has been modified by the attachment of peg, thereby enhancing its stability in the blood. the svr of the current standard therapy by peg-ifn and ribavirin is as high as % [ ] . in the current peg-ifn and ribavirin combination therapy, the genotype of hcv is one of the major determinants of the svr. hcv genotypes are classified into groups, and genotype is currently considered a problem due to its ifn resistance [ ] . for example, in genotype hcv, months of treatment resulted in an svr in % of patients, while in genotype , months of treatment achieved an svr of - % [ ] . the precise mechanisms of the ifn resistance remain unclear. however, the recently developed ifn-resistant hcv replicon-harboring cells will be useful for studies examining ways to improve the svr [ ] [ ] [ ] . therefore, the focus in the treatment of patients with ch-c has shifted to increasing the svr in genotype hcv. before the development of an hcv replicon system, screening of anti-hcv reagents was rather difficult. the hcv replicon system developed by lohmann et al. [ ] was the first milestone in hcv study using a cell culture system. the replicon system has provided a wealth of information concerning the replication machinery of hcv. we can make strategies for the achilles' heel of hcv based on the information regarding hcv rna replication. the hcv replicon has been improved to be a suitable system for the screening of anti-hcv reagents by the introduction of reporter genes such as luciferase [ ] . however, this system does not contain a structural region. therefore, selectable genome-length hcv rna-replicating cell culture systems have been developed [ ] [ ] [ ] [ ] . the second milestone was the infectious virus production system established by the three groups using a genotype a hcv strain, jfh [ ] [ ] [ ] . this system has extended the range of the hcv study to the viral entry and release. therefore, the life cycle of hcv in the cells has been reconstructed in vitro. since the development of the hcv replicon and infectious hcv production systems, many cellular proteins have been identified as essential host molecules for hcv rna replication. the hcv replicon reported by lohmann et al. contained neomycin phosphotransferase (neo) and encephalomyocarditis virus (emcv) internal ribosome entry sites (ires) instead of the hcv structural regions (fig. ) [ ] . this hcv replicon consists of cistrons. in the first cistron, neo is translated by hcv-ires and in the second cistron ns -ns b is translated by emcv-ires introduced in the region upstream of the ns region ( fig. ) . after the development of the hcv replicon system [ , , [ ] [ ] [ ] [ ] , genome-length hcv rna replication systems using different hcv strains (h, n, con , and o) were developed by several groups [ ] [ ] [ ] [ ] . in these genome-length hcv rna replication systems, a complete open reading frame (orf) of hcv was introduced into the second cistron instead of the ns region (fig. ) . for the mass screening for anti-hcv reagents, evaluation of the levels of hcv rna or hcv proteins requires time and complicated procedures. to facilitate the monitoring of the replication level of hcv rna, the reporter gene (renilla luciferase) was fused to the neo gene. in this system, anti-hcv activity was evaluated by the value of the reporter instead of the pufas [ , , ] quantification of hcv rna or hcv proteins. as shown in fig. a , orn/c- b/ke contains the fused renilla luciferase and neo genes in the first cistron [ ] . one of the cloned cell lines, or , was established by the g selection after introduction of orn/c- b/ke rna into huh- cells. hcv rna and hcv proteins were stably expressed in the or cells, and the renilla luciferase activity was correlated well with the level of hcv rna [ ] . therefore, the antiviral effect of the reagents on hcv rna replication could be monitored by the activity of renilla luciferase. the or assay system facilitates the mass screening for anti-hcv reagents. hcv rna replicating in or cells contained an adaptive mutation, k e, in the ns region. adaptive mutations have been reported to enhance the replication level of hcv rna in cell culture [ ] [ ] [ ] . in the case of hcv-o, two adaptive mutations were required for robust replication of the genome-length hcv rna replication [ ] . for example, authentic hcv-o rna with the adaptive mutations of e g and k e can robustly replicate in huh- cells for months or more (ikeda et al., unpublished data). in , three groups reported infectious hcv production systems using the jfh strain in cell culture [ ] [ ] [ ] . these reports showed that the life cycle of hcv could be reconstructed in huh- cells, and thus became landmarks in the search for an ideal hcv cell culture system. the unique features of these systems were the origin of this strain and the cell lines. jfh was a genotype a strain derived from a patient with fulminant hepatitis and did not require any adaptive mutations for robust replication, unlike other hcv strains. the unique feature of this system was that it employed huh- cells such as huh- . or huh-lunet cells, since the parental huh- cells could not support robust production of infectious hcv [ ] [ ] [ ] ] . recently, the genotype a h -s strain was reported to produce infectious hcv in cell culture, although the production level of infectious h -s was lower compared with that by jfh [ ] . interestingly, five adaptive mutations were introduced into the h -s genome in order to enhance the efficiency of infectious virus production. the presence of these adaptive mutations is the most striking and controversial characteristic regarding the production of infectious hcv described above. further study will be needed to understand the role of adaptive mutations on infectious virus production. the establishment of an infectious hcv production system gradually led to clarification of the life cycle of hcv. information regarding the hcv rna replication has been accumulated since the development of the hcv replicon system, and the infectious hcv production system [ ] [ ] [ ] has further provided information about the step of virus entry and release. the life cycle of hcv includes the ( ) receptor binding and cell entry, ( ) cytoplasmic release and uncoating, ( ) ires-mediated translation, ( ) processing, ( ) rna replication, ( ) packaging and assembly, ( ) virion maturation, and ( ) virion release. although some of the mechanisms are still unclear, each of these steps is a target for antivirals. among the proteins involved in these steps, the protease in step ( ) and polymerase in step ( ) have been especially well characterized. specific inhibitors for these proteins have been developed and some of them are now in clinical trials for patients with ch-c [ , ] . cellular proteins are required for hcv rna replication and may determine the cell tropism of hcv. as hcv is a parasite, it utilizes the cellular proteins for its replication machinery. therefore, cellular proteins essential for hcv rna replication are the targets for antivirals. using cell culture systems, several cellular proteins have been identified as effective molecules for hcv rna replication (table ) . la and ptb were representative molecules reported as essential host factors for hcv rna replication [ ] . recently, an immunosuppressant, csa, has been reported to inhibit hcv rna replication by blocking the binding of cypb to ns b [ ] . hsp and the fk- binding protein (fkbp ) form a complex with ns a and geldanamycin, an inhibitor of hsp , suppressed hcv rna replication by blocking the formation of these complex [ ] . the advantage of the inhibitors targeting cellular factor is that these reagents do not affect the viral escape achieved through mutations. the high mutation rate caused by rdrp frequently produced escape mutants toward the antiviral reagents for hcv proteins. a disadvantage of the inhibitors targeting cellular factors may be that they induce side effects by inhibiting the primary roles of the cellular factors. the cellular factors are the targets of the antivirals independent of the viral escape via the genetic mutations caused by rdrp. the cellular factors were synthesized in their metabolic pathways and modified by the enzymes. these enzymes are also targets in the antiviral strategy (table ) . furthermore, some of the reagents have already been used in the clinical treatment of the respective diseases. one of the advantages of using existing reagents is that their characterizations-including safety and side effects-have already been performed. therefore, screening of the existing reagents for anti-hcv will be a new field of antivirals. the development of a cell culture system for hcv led to the revelation that hcv incorporates many cellular factors into the replication machinery of the virus. now we have both the information of the hcv life cycle and the cell culture assay system-the input and output-that we need to develop a pool of antiviral reagents. below, we will discuss the particular host cell metabolic pathways that are currently being targeted by anti-hcv reagents including more recently found pitavastatin (ptv) (fig. b ). in the cholesterol-biosynthesis pathway, the region downstream of mevalonate branches into separate pathways for cholesterol and isoprenoid synthesis (fig. ) . the attachment of the isoprenoid is called prenylation of the protein. prenylation regulates a variety of cellular functions, such as growth, differentiation, and oncogenesis. farnesyl pyrophosphate (fpp) and geranylgeranyl pyrophosphate (ggpp) are mevalonate-derived isoprenoids and are attached to the target proteins by farnesyltransferase (ftase) and geranylgeranyl transferase type i (ggtase-i), respectively. ftase and ggtase-i recognize protein substrates with a c-terminal tetrapeptide recognition motif called the caax box: in the case of ggtase-i, c is cysteine, a is an aliphatic amino acid, and x is leucine, isoleucine, valine, or phenylalanine. production of mevalonate by -hydroxy- methylglutaryl coenzyme a (hmg-coa) reductase is the rate-limiting step in the cholesterol biosynthesis. statins are potent hmg-coa reductase inhibitors and are beneficial in the prevention of coronary heart disease. statins also inhibit the prenylation of the proteins. lipid metabolism is essential for the life cycle of many viruses. the cholesterol-rich lipid raft plays an important role in virus entry, replication, and assembly. hcv also forms a replication complex on the lipid raft membrane structure [ ] . hcv rna replication occurs in the lipid raft and the cholesterol supply is crucial to maintain the structure of the lipid raft [ ] . aizaki et al. [ ] reported that lovastatin (lov), one of the hmg-coa reductase inhibitors, inhibited hcv rna replication in hcv replicon-harboring cells. statins also possess the cholesterol-independent action (pleiotropic effect) [ ] . many of these pleiotropic effects are mediated by the isoprenoid. for example, inhibition of small gtp-binding proteins, ras and rho, whose proper membrane localization and function are dependent on prenylation, may play a significant role in the pleiotropic effect of statins. ras and rho are major substrates for prenylation with fpp and ggpp, respectively. gdp-bound ras and rho are localized in the cytoplasm. when fpp or ggpp is bound to the inactive ras or rho, they are translocated to the cell membrane and converted to gtp-bound active forms. recently, wang et al. [ ] identified fbl as one of the geranylgeranylated cellular proteins required for hcv rna replication. fbl belongs to the fbl family of proteins, all of which contains an f box and a multiple leucine-rich repeat, with the f box binding to a multicomponent ubiquitin ligase complex. geranylgeranylated fbl binds to ns a, and the resulting complex seems to be required for hcv rna replication. in hcv replicon-harboring cells, knockdown of fbl by sirna has been shown to reduce hcv rna by % [ ] . depletion of the ggpp by statins may inhibit the geranylgeranylation of cellular proteins such as fbl and cause the anti-hcv effect in the cells. statins are among the most widely used reagents to lower cholesterol. one of the statins used clinically, lov, has been well characterized and shown anti-hcv activity in cell culture. [ , , ] . however, the anti-hcv activities of other statins remain to be clarified. recently the anti-hcv activities of several statins were characterized using an or assay system [ ] . the anti-hcv activities were tested for five statins: atorvastatin (atv), fluvastatin (flv), pravastatin (prv), simvastatin (smv), and lov. flv exhibited the strongest anti-hcv activity ( % effective concentration to inhibit hcv rna replication (ec ): . μm), while atv and smv showed moderate inhibitory effects (ec : . and . μm, respectively). however, lov, which has been reported to inhibit hcv replication, was shown to possess the weakest anti-hcv activity (ec : . μm). more recently, we found that ptv possessed stronger anti-hcv activity than flv (fig. b) . the ec of ptv was calculated as . μm. the anti-hcv activities of statins were reversed by supplying mevalonate or geranylgeraniol. however, surprisingly, prv exhibited no anti-hcv activity, although it worked as an inhibitor for hmg-coa reductase. although prv is a water-soluble reagent (the others are lipophilic), prv induced the expression of hmg-coa reductase by a positive feedback mechanism. there may be another mechanism underlying the depletion of ggpp by the statins. interestingly, it has been reported that only prv has a different effect on the induction of p compared with the other statins [ ] . ribavirin is the only reagent currently used with ifn-α to treat patients with ch-c [ ] . in the previous study on anti-hcv activity using the or assay system, the ec of ribavirin was μm [ ] . this concentration is much higher than the clinically achievable ribavirin concentration ( - μm) reported previously [ , ] . since flvexhibited strong anti-hcvactivity, flv was examined for its anti-hcv activity in combination with ifn- α in or cells [ ] . co-treatment of ifn-α and flv exhibited synergistic inhibitory effects on hcv rna replication. for example, when administered in combination with ifn-α ( iu/ ml) and flv ( μm), the level of hcv rna replication was remarkably reduced to approximately %, compared with the effects of treatment with ifn-α alone. the combination therapy of flv may be effective for the treatment of patients with ch-c. it is not appropriate to further reduce the cholesterol level of ch-c patients who already have a normal cholesterol level. for these patients, statin-related anti-hcv reagents possessing no cholesterol-lowering activity would be good candidates for future clinical use. the specific inhibition of ggpp synthesis and prenylation will be worth testing, and ggtase-i inhibitor (ggti) is one of the candidates for this purpose. furthermore, specific inhibition of the proteins modified by ggtase-i may be more effective. fbl may be one of the target proteins, because its formation of a complex with ns a is required for hcv rna replication. therefore, the reagents blocking the association of fbl with ns awill be able to inhibit the hcv rna replication with fewer side effects. prenyltransferase recognizes a broad range of protein substrates with a caax motif. reid et al. [ ] reported a list of hypothetical prenyltransferase substrates within the human genome. other than fbl , the host molecules involved in hcv rna replication may be exist in this list. antiviral activity of statins has also been reported in other viruses. in the respiratory syncytial virus (rsv), lov exhibited antiviral activity via the inhibition of rhoa [ ] . rhoa is activated by geranylgeranylation, and activated rhoa interacts with the f glycoprotein of rsv. flv inhibited cytomegalovirus (cmv) replication by abolishing cmv-induced nf-κb activity, which is involved in a pathway that is crucial for cmv replication [ ] . in human immunodeficiency virus (hiv), lov and siv reduced hiv replication via suppression of the binding between the integrin intercellular adhesion molecule (icam ) and lymphocyte function associated antigen- (lfa- ) [ ] . statins were recently shown to bind to lfa- , and icam -bearing viruses were reduced by statins in a dose-dependent manner. it is noteworthy that the inhibition of lfa- binding to icam- by statins is independent of the inhibition of hmg coa reductase. statins inhibited the cholesterol-biosynthesis pathway and branched prenylation pathways by depletion of mevalonate. the latter caused pleiotropic effects in growth, differentiation, and antivirals. however, an unknown function of statins may existfor example, the binding of lfa- is likely independent of the cholesterol-lowering and the inhibition of prenylation. furthermore, the finding that prv has a different effect on the induction of p than the other statins has not been clearly explained by the characterization of these mechanisms of statins. a better understanding of this finding may lead to the discovery of statin-related anti-hcv reagents that do not have exhibit any cholesterol-lowering activity or inhibition of prenylation. lipid rafts are detergent resistant membranes (drm) and are enriched in cholesterol and sphingolipids. the active replication complex of hcv is present in lipid rafts [ ] . therefore, sphingolipid metabolism is also an antiviral target for hcv. serine palmitoyltransferase (spt) is the enzyme responsible for the condensation of l-serine with palmitoyl-coa to produce ketodihydrosphingosine in the first step of sphingolipid biosynthesis (fig. ) . myriocin, a selective inhibitor of spt, inhibited the replication of hcv replicon [ , ] . sakamoto et al. [ ] reported that the compound na , which is structurally similar to myriocin, also inhibited the replication of the hcv replicon. na has been identified as the secondary fungal metabolite derived from fusarium sp. na suppressed hcv replicon in a dose-dependent manner, and its ec was nm. they further examined the involvement of the sphingolipid synthetic pathway in hcv rna replication. fumonisin b , an inhibitor of dihydroceramide synthase, also suppressed the replication of hcv replicon. in mammalian cells, ceramide is synthesized in the endoplasmic reticulum (er) and translocates to the golgi compartment for conversion to sphingomyelin. hpa- , an inhibitor of ceramide trafficking from the er to the golgi apparatus, also inhibited the replication of hcv replicon. glycosphingolipids (gsls) are also a component of lipid rafts, and ppmp, an inhibitor of gsl biosynthesis, also suppressed the replication of hcv replicon. furthermore, they demonstrated that after treatment with na , the ns b ratio in the drm was markedly decreased. interestingly, however, the drm fraction of ns and ns a were not affected. inhibition of sphingolipid biosynthesis by na disrupted the association of lipid rafts with ns b, but not with ns or ns a. they identified a helix-turn-helix motif (glu -gly ) in ns b as a sphingolipid-binding domain (sbd), which was similar in structure to the sbd of the v loop of hiv- . umehara et al. [ ] reported that myriocin suppressed hcv rna replication in vivo, using hcv-infected chimeric mice with humanized livers. myriocin reduced the hcv rna levels in both serum and liver to / - / of the levels prior to the day treatment. they also demonstrated that the combined treatment of myriocin with peg-ifn reduced the hcv rna level to less than / of the control levels. these results suggest that the sphingolipid biosynthetic pathway is also a suitable target for the development of hcv therapies. at the beginning of gtp-biosynthesis pathway, inosine monophosphate dehydrogenase (impdh) is the enzyme responsible for the conversion of inosine ′ monophosphate (imp) into xanthosine ′ monophosphate (xmp) (fig. ) . ribavirin, mizoribine, mycophenolic acid (mpa), and vx- are impdh inhibitors and inhibit hcv rna replication. ribavirin enhanced the svr of peg-ifn therapy from % to % compared to the peg-ifn monotherapy [ ] . however, the antiviral mechanisms of ribavirin remain to be clarified. four possible mechanisms have been proposed [ , ] : ( ) direct inhibition of rna replication; ( ) inhibition of impdh; ( ) immunomodulation; ( ) mutagenesis. ribavirin is phosphorylated to mono-, di-, and triphosphate (rmp, rdp, and rtp, respectively). ( ) rtp, an analog of gtp, is incorporated into replicating rna by rdrp and caused termination of the rna synthesis. ( ) rmp competitively inhibits the host enzyme impdh, which is essential for the synthesis of gtp, and causes a depletion of the gtp pool. ( ) rivavirin has been suggested to cause immunomodulatory effects, such as the shift of th to th in immune response, and to induce an hcvspecific t cell response. ( ) ribavirin acts as an rna mutagen and causes error catastrophe. in poliovirus replication, μm of ribavirin increased the mutation rate from about . mutations/genome (wild type) to about . mutations/genome and resulted in a decrease of infectivity of % [ ] . the mutation rate increased in a ribavirin dose-dependent manner: . mutations/genome and . mutations/genome at μm and μm, respectively [ ] . in the clinical study of ch-c, the enhancement of svr has been observed only in combination therapy of ribavirin with ifn, but not in ribavirin monotherapy. it may be difficult to test the effect of ribavirin monotherapy, since the clinically achievable concentration of ribavirin without severe side effects such as anemia is too low ( - μm). however, in the cell culture model [ , ] , a higher concentration of ribavirin suppressed hcv rna replication (ec : μm) [ ] . mizoribine is an imidazole nucleoside that is isolated from culture medium of the mold eupenicillium brefeldianum m- and is structurally similar to ribavirin. mizoribine was authorized by the japanese government as an immunosuppressive drug for renal transplantation; thereafter, lupus nephritis, rheumatoid arthritis, and nephritic syndrome were also added to the list of diseases for which this agent is indicated [ , ] . based on the similarity of mizoribine to ribavirin, the anti-hcv activity of mizoribine has been tested using an or assay system. the anti-hcv activity of mizoribine (ec : μm) was similar to that of ribavirin [ ] . furthermore, a low dose (at least μm) of mizoribine was able to enhance the antiviral activity of ifn [ ] . mizoribine was reported to exhibit antiviral activity on influenza virus types a and b [ ] and recently on bovine viral diarrhea virus [ ] and severe acute respiratory syndrome (sars)-associated coronavirus [ ] . the precise antiviral mechanism of mizoribine remains unclear. however, any of the four hypothesized mechanisms of ribavirin mentioned above may be possible. since mizoribine has not been associated with severe side effects, it will be an alternative reagent for combination therapy with ifn. like mizoribine, mpa is used as an immunosuppressant and is known to inhibit impdh. it has been reported to show in vitro antiviral activity against dengue virus [ , ] , hepatitis b virus (hbv) [ ] , avian reovirus [ ] , yellow fever virus [ ] , and west nile virus [ ] . the anti-hcv activity of mpa was reported by henry et al. [ ] . at clinically relevant concentrations ( . - . μg/ml), mpa inhibited hcv rna replication to approximately % in a study using hcv replicon-harboring cells. furthermore, combination treatment of mpa with csa or ifn showed synergistic inhibition of hcv rna replication. we also recently confirmed that the combination of csa and mizoribine had a synergistic effect on the inhibition of hcv rna replication (yano et al., unpublished data) . these data suggest that immunosuppressive drugs possessing anti-hcv activity, such as csa, mpa, and mizoribine, may prevent not only the rejection of the graft but also the recurrence of hcv infection after liver transplantation, and that a combination of these drugs may be of additional benefit for such patients. vx- is a reversible uncompetitive impdh inhibitor that is structurally unrelated to other known impdh inhibitors. markland et al. [ ] reported the broad-spectrum antiviral activity of vx- .vx- exhibited -to -fold more potency than ribavirin against hbv, human cmv, rsv, herpes simplex virus type , parainfluenza virus, emcv, and venezuelan equine encephalomyelitis virus in cell culture [ ] . zhou et al. [ ] reported that vx- alone had only marginal effect on hcv replicon, although combination treatment with ribavirin and vx- enhanced anti-hcv activity. they also reported that in their hcv replicon assay system, mpa showed only a marginal anti-hcv effect [ ] . this result is different from the anti-hcv effect of mpa reported by henry et al. [ ] . further study will be needed to clarify these controversial results. hcv morphogenesis is a target of antivirals in the life cycle of the virus. the hcv envelope glycoproteins e and e are highly n-glycosylated [ ] . the consensus sequence for nglycosylation is asn-x-ser/thr, where x is any amino acid except for pro, and e and e contain - and glycosylation sites, respectively. from the previous study using bovine viral diarrhea virus, inhibition of α-glucosidase is expected to prevent the proper folding and assembly of hcv. therefore, the n-glycosylation pathway may be a novel molecular target for antivirals. chapel et al. [ ] reported an anti-hcv effect of the α-glucosidase inhibitor in the binding step using hcv virus-like particles (vlps) derived from baculovirus. the glucose analogue deoxynojirimycin derivatives, which are αglucosidase inhibitors, caused the retention of unprocessed, hyperglycosylated n-linked glycans on hcv glycoproteins and led to the reduction in binding of vlp to the cells [ ] . these results will be examined using a recently developed infectious hcv production cell culture system. α-glucosidase inhibitor may be one of the candidates for an effective combination therapy. it is crucial that the svr for patients with ch-c receiving the current standard therapy of peg-ifn plus ribavirin is improved from the current value of about %. the anti-hcv effect of ifn-α is caused through the jak-stat signaling pathway. duong et al. [ ] proposed that hypomethylation of stat by hcv protein caused the resistance to ifn therapy. unmethylated stat is less active because it can be bound and inactivated by its inhibitor, the protein inhibitor of activated stat (pias ). protein arginine methyltransferase (prmt ) is the enzyme responsible for the methylation of stat . hcv proteins induced the expression of the catalytic subunit of protein phosphatase a (pp ac), and overexpression of pp ac induced stat hypomethylation via the inhibition of prmt . finally, pias interacted with and inhibited hypomethylated stat and resulted in the suppression of ifn signaling [ ] . s-adenosyl-l-methionine (adomet) is a methyl group donor for stat methylation by prmt . adomet is used for the treatment of alcoholic liver disease and is available in many countries as a nonprescription drug. betaine has been known to raise the intracellular concentration of adomet and plays the central role in the recycling of adomet. when pp ac was overexpressed in huh- and uhvh . cells, ifn-α signaling was suppressed [ ] . however, the co-treatment of adomet and betaine restored the ifn-α signaling. these results suggest that the addition of adomet and betaine to the current standard therapy with peg-ifn and ribavirin may enhance the svr for patients with ch-c. lipid metabolism is one of the most important pathways for hcv rna replication. other than cholesterol and sphingolipid synthesis, fatty acids are reported to be metabolites involved in hcv rna replication [ , ] . however, the precise mechanisms of fatty acids on hcv rna replication have remained unclear. leu et al. [ ] reported that polyunsaturated fatty acids (pufas) inhibited hcv replicon replication. arachidonic acid (aa), docosahexaenoic acid (dha), and eicosapentaenoic acid (epa) belong to pufas (fig. ) and possessed anti-hcv activity. the ec of aa was μm. however, at μm, αlinolenic acid, γ-linolenic acid (gla), and linoleic acid reduced hcv rna levels slightly, and saturated fatty acids, including oleic acid, myristic acid, palmitic acid, and steric acid, slightly enhanced hcv rna levels. similar results were also reported by kapadia et al. [ ] using a genome-length hcv rnareplicating cell line. aa produces lipid mediators such as prostaglandins (pgs), thromboxanes (txs), leukotriens (lts), and lipoxins (lxs) (fig. ) . however, the antiviral activity of these eicosanoids remains unclear. in their clinical study, hyman et al. [ ] fig. . fatty acid-biosynthesis pathway. the pufa metabolism from diet or membrane phospholipids. reported that oral prostaglandin e therapy resulted in no beneficial effect on patients with ch-c. investigation of the anti-hcv effects of the metabolites of pufas will lead to a new field of antivirals based on the host metabolism. ever since hcv was discovered to be the causative agent of non-a, non-b hepatitis virus, ifn has played the central role in treating the disease. currently ifn has been modified by peg and accompanied by the powerful partner, ribavirin, which boosts the anti-hcv activity of ifn. during the development of ifn therapy for patients with ch-c, the lack of a robust method of hcv rna replication in cell culture has hampered research into the hcv life cycle and the discovery of potent new anti-hcv reagents. it is difficult to attack the achilles' heel of hcv without information on the replication machinery of the virus. however, the development of a subgenomic replicon system by lohmann et al. [ ] partially revealed the hcv life cycle. the information about hcv rna replication in the virus life cycle provided clues to the development of antivirals both from the standpoint of the virus and the host. a representative example is the discovery that ns - a inhibits innate immunity [ ] . hcv runs through the cellular first defenses of the ifnproduction system. ns - a, a serine protease, cleaved the unexpected cellular target cardif and disrupted rig-i signaling [ ] . hcv replicon contributed to the discovery of the viral serine protease inhibitor. surprisingly, a serine protease inhibitor, sch , inhibited hcv rna replication not only by the inhibition of ns - a activity but also by the inhibition of the rig-i signaling [ ] . this serine protease inhibitor possesses dual functions, inhibiting both viral (ns - a) and cellular (cardif) proteins involved in ifn production. viral and cellular molecules are the targets of antivirals. hcv rdrp caused a high mutation rate and the mutations accumulated in virus genome [ ] . the high mutation rate enhances the viral evolution. as for the reagents targeting viral proteins, such as ns - a or ns b, resistance to the therapy happens by the frequent mutations caused by rdrp. in fact, in the clinical trial of the ns - a protease inhibitor, vx- , hcv rna rapidly decreased within days after treatment [ ] . however, hcv rna increased again at around days after treatment [ ] . hcv mutants may not be the problem in the anti-hcv reagent against cellular proteins, although the inhibition of the primary functions of the cellular proteins may cause side effects. in this review, host metabolic pathways are overviewed. one of the advantages of targeting host metabolism as antivirals is that multiple enzymes involved in the metabolism could become candidates for antivirals. in the strategy targeting host metabolism, we should be careful in regard to the side effects caused by inhibition of the primary function of the metabolite. to minimize these undesirable effects, pinpoint inhibition of the enzyme should be done. lipid metabolism is one of the important targets for antivirals among cellular factors. very recently, we examined the effect of ordinary nutrients on hcv rna replication [ ] . using an or assay system, we found that linoleic acid possessed an anti-hcv effect and its combination with csa exerted synergistic inhibitory effect on hcv rna replication [ ] . however, the anti-hcv mechanism of pufas remains unclear. an improved understanding of the anti-hcv effect of pufas will extend the field of host metabolism as a target of antivirals in the future. one recent striking advance is the development of a method for infectious hcv production in cell culture. this system provides information regarding the complete life cycle of hcv and will extend our understanding of the antivirals to virus entry, assembly and release. the discovery of anti-hcv reagents targeting host metabolism in the hcv life cycle will 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inhibitors on hepatitis c virus subgenomic replicon rna glycosylation of hepatitis c virus envelope proteins antiviral effect of alphaglucosidase inhibitors on viral morphogenesis and binding properties of hepatitis c virus-like particles s-adenosylmethionine and betaine correct hepatitis c virus induced inhibition of interferon signaling in vitro anti-hcv activities of selective polyunsaturated fatty acids oral prostaglandin (pge ) therapy for chronic viral hepatitis b and c regulation of interferon regulatory factor- by the hepatitis c virus serine protease cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus genetic variation and dynamics of hepatitis c virus replicons in long-term cell culture comprehensive analysis of the effects of ordinary nutrients on hepatitis c virus rna replication in cell culture the authors thank drs. hiromichi dansako and yasuo ariumi for their stimulating discussions. key: cord- -bqvn ceq authors: lee, jeong seok; park, seongwan; jeong, hye won; ahn, jin young; choi, seong jin; lee, hoyoung; choi, baekgyu; nam, su kyung; sa, moa; kwon, ji-soo; jeong, su jin; lee, heung kyu; park, sung ho; park, su-hyung; choi, jun yong; kim, sung-han; jung, inkyung; shin, eui-cheol title: immunophenotyping of covid- and influenza highlights the role of type i interferons in development of severe covid- date: - - journal: sci immunol doi: . /sciimmunol.abd sha: doc_id: cord_uid: bqvn ceq although most sars-cov- -infected individuals experience mild coronavirus disease (covid- ), some patients suffer from severe covid- , which is accompanied by acute respiratory distress syndrome and systemic inflammation. to identify factors driving severe progression of covid- , we performed single-cell rna-seq using peripheral blood mononuclear cells (pbmcs) obtained from healthy donors, patients with mild or severe covid- , and patients with severe influenza. patients with covid- exhibited hyper-inflammatory signatures across all types of cells among pbmcs, particularly up-regulation of the tnf/il- β-driven inflammatory response as compared to severe influenza. in classical monocytes from patients with severe covid- , type i ifn response co-existed with the tnf/il- β-driven inflammation, and this was not seen in patients with milder covid- . interestingly, we documented type i ifn-driven inflammatory features in patients with severe influenza as well. based on this, we propose that the type i ifn response plays a pivotal role in exacerbating inflammation in severe covid- . currently, severe acute respiratory syndrome coronavirus (sars-cov- ), which causes coronavirus disease , is spreading globally ( , ) , and the world health organization (who) has declared it a pandemic. as of june , , more than . million confirmed cases and more than , deaths have been reported worldwide ( ) . sars-cov- infection usually results in a mild disease course with spontaneous resolution in the majority of infected individuals ( ) . however, some patients, particularly elderly patients develop severe covid- infection that requires intensive care with mechanical ventilation ( , ) . the mortality rate for covid- in wuhan, china, is estimated to be . % ( ) . although this rate is lower than that of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), which are caused by other human pathogenic coronaviruses ( ) , it is much higher than that of influenza, a common respiratory viral disease requiring hospitalization and intensive care in severe cases. in severe cases of covid- , a hyper-inflammatory response, also called a cytokine storm, has been observed and is suspected of causing the detrimental progression of covid- ( ) . circulating levels of pro-inflammatory cytokines, including tnf and il- , are increased in severe cases ( ) . gene expression analyses have also shown that il- related pro-inflammatory pathways are highly up-regulated in severe cases ( ) . in a murine model of sars-cov infection, a delayed, but considerable type i ifn (ifn-i) response coronavirus immunophenotyping of covid- and influenza highlights the role of type i interferons in development of severe covid- (page numbers not final at time of first release) promotes the accumulation of monocytes-macrophages and the production of pro-inflammatory cytokines, resulting in lethal pneumonia with vascular leakage and impaired virusspecific t-cell responses ( ) . immune dysfunction is also observed in patients with covid- . in severe cases, the absolute number of t cells is reduced ( , ) , and the t cells exhibit functional exhaustion with the expression of inhibitory receptors ( , ) . however, hyper-activation of t cells as reflected in the up-regulation of cd , hla-dr, and cytotoxic molecules was also reported in a lethal case of covid- ( ) . immune dysfunction in patients with severe covid- has been attributed to pro-inflammatory cytokines ( ) . in the present study, we performed single-cell rna-seq (scrna-seq) using peripheral blood mononuclear cells (pbmcs) to identify factors associated with the development of severe covid- infection. by comparing covid- and severe influenza, we report that the tnf/il- β-driven inflammatory response was dominant in covid- across all types of cells among pbmcs, whereas the up-regulation of various interferon-stimulated genes (isgs) was prominent in severe influenza. when we compared the immune responses from patients with mild and severe covid- infections, we found that classical monocytes from severe covid- exhibit ifn-i-driven signatures in addition to tnf/il- β-driven inflammation. pbmcs were collected from healthy donors (n= ), hospitalized patients with severe influenza (n= ), and patients with covid- of varying clinical severity, including severe, mild, and asymptomatic (n= ). pbmcs were obtained twice from three (the subject c , c , and c ) of the eight covid- patients at different time points during hospitalization. pbmc specimens from covid- patients were assigned to severe or mild covid- groups according to the national early warning score (news; mild < , severe ≥ ) evaluated on the day of whole blood sampling ( ) . in news scoring, respiratory rate, oxygen saturation, oxygen supplement, body temperature, systolic blood pressure, heart rate, and consciousness were evaluated ( ) . severe influenza was defined when hospitalization was required irrespective of news score. patients with severe influenza were enrolled from december to april , prior to the emergence of covid- . the severe covid- group was characterized by significantly lower lymphocyte count and higher serum level of creactive protein than the mild covid- group on the day of blood sampling (fig. s a) . multiplex real-time pcr for n, rdrp, and e genes of sars-cov- was performed, and there was no statistical difference in ct values for all three genes between two groups (fig. s b ). demographic information is provided with experimental batch of scrna-seq in table s and clinical data in table s and s . employing the x genomics scrna-seq platform, we analyzed a total of , cells in all patients after filtering the data with stringent high quality, yielding a mean of , umis per cell and detecting , genes per cell on average (table s ). the transcriptome profiles of biological replicates (pbmc specimens in the same group) were highly reproducible (fig. s c) , ensuring the high quality of the scrna-seq data generated in this study. to examine the host immune responses in a cell type-specific manner, we subjected , cells to t-distributed stochastic neighbor embedding (tsne) based on highly variable genes using the seurat package ( ) and identified different clusters unbiased by patients or experimental batches of scrna-seq (fig. a, fig. s d ). these clusters were assigned to different cell types based on well-known marker genes and two uncategorized clusters ( fig. b and c, and table s ). in downstream analysis, we only focused on different immune cell types, including igg -b cell, igg + b cell, effector memory (em)-like cd + t cell, non-em-like cd + t cell, emlike cd + t cell, non-em-like cd + t cell, natural killer (nk) cell, classical monocyte, intermediate monocyte, non-classical monocyte, and dendritic cell (dc) after excluding platelets, red blood cells (rbcs), and two uncategorized clusters. the subject c (asymptomatic case) was also excluded due to a lack of replicates. in hierarchical clustering, most transcriptome profiles from the same cell type tended to cluster together, followed by disease groups, suggesting that both immune cell type and disease biology, rather than technical artifacts, are the main drivers of the variable immune transcriptome (fig. s e) . as a feature of immunological changes, we investigated the relative proportions of immune cells among pbmcs in the disease groups compared to the healthy donor group ( fig. d and e, and fig. s f ). unlike the limited changes in mild covid- , significant changes were observed in both influenza and severe covid- across multiple cell types among pbmcs. in severe covid- , the proportion of classical monocytes significantly increased whereas those of dcs, non-classical monocytes, intermediate monocytes, nk cells, em-like cd + t cells, and em-like cd + t cells significantly decreased (fig. e) . in severe influenza, the proportion of classical monocytes significantly increased whereas those of dcs, non-emlike cd + t cells, em-like cd + t cells, igg + b cells, and igg -b cells significantly decreased. we validated the proportions of immune cell subsets from scrna-seq by flow cytometry analysis. the relative proportions of total lymphocytes, b cells, cd + t cells, cd + t cells, nk cells, and total monocytes from scrna-seq significantly correlated with those from flow cytometry analysis (fig. s g ). first release: july immunology.sciencemag.org (page numbers not final at time of first release) in order to compare the effect of infection between diseases, we performed hierarchical clustering based on relative gene expression changes against the healthy donor group. unexpectedly, all types of cells among pbmcs were clustered together according to the disease groups instead of cell-types ( fig. a) . further investigation of the variable genes based on k-means clustering supported covid- -specific up-or down-regulated gene expression patterns across all types of cells among pbmcs (fig. s a) . these results indicate that, in covid- , peripheral blood immune cells may be influenced by common inflammatory mediators regardless of cell type. despite distinct transcriptional signatures between covid- and influenza, severe covid- and influenza shared transcriptional signatures in all types of monocytes and dcs (black boxed region in fig. a ), possibly reflecting common mechanisms underlying the innate immune responses in severe influenza and severe covid- . next, we sought to identify relevant biological functions in disease-specific up-or down-regulated genes in terms of the go biological pathways. first, we combined both mild and severe covid- as a covid- group and identified disease-specific changes in genes for each cell type compared to the healthy donor group using model-based analysis of single cell transcriptomics (mast) ( ) . nfkb , nfkb , irf , and cxcr were specifically up-regulated in covid- , and cxcl , stat , tlr , and genes for class ii hla and immunoproteasome subunits were specifically up-regulated in influenza (table s ) . tnf, tgfb , il b, and ifng were commonly up-regulated. when we directly compared covid- and influenza, nfkb , nfkb , and tnf were up-regulated in covid- , whereas stat , tlr , and genes for immunoproteasome subunits were up-regulated in influenza. for each group of differentially expressed genes (degs), we identified the top enriched go biological pathways and collected them to demonstrate p-value enrichment in each group of degs (fig. b ). both distinct and common biological functions were identified as illustrated by inflammatory response genes being highly active in both covid- and influenza, but genes for transcription factors, including inflammatory factors (i.e., nfkb / , and stat ) were up-regulated in covid- . in contrast, a limited response in genes associated with the ifn-i and -ii signaling pathways, t-cell receptor pathways, and adaptive immune response was observed in covid- compared to influenza. such disease-specific gene expression patterns were exemplified at single cell resolution by gbp (ifn-γ-mediated signaling pathway) being specifically up-regulated in influenza, crem (positive regulation of transcription) being specifically up-regulated in covid- , and ccl (inflammatory response) being commonly up-regulated ( fig. c and table s ) . we expanded our analysis in a cell type specific manner by conducting weighted gene correlation network analysis (wgcna) ( ) for the collected genes associated with fig. b . we identified several modular expression patterns ( fig. d and table s ). in the covid- group, nfkb / , jun, and tnf were modularized in cd + t and nk cells (g and g in fig. d ), and il b, nfkbid, and osm were modularized in all types of monocytes and dcs (g in fig. d ). in the influenza group, gbp , tap , stat , ifitm , oas , irf , and ifng were modularized in all types of t cells and nk cells (g in fig. d ), and cxcl and tlr were modularized in all types of monocytes and dcs (g and part of g in fig. d ). consistently, the degs between covid- and influenza were dominant in cd + t cells and all types of monocytes (fig. s b ). to uncover disease-specific transcriptional signatures in cd + t cells, we performed sub-clustering analysis from emlike and non-em-like cd + t cell clusters using seurat ( ) . each disease group-specifically enriched sub-clusters compared to the two other groups were identified in the non-emlike cd + t cell cluster (fig. a) . of the six sub-clusters from the non-em-like cd + t cell cluster, cluster and cluster were significantly enriched in the influenza and covid- groups, respectively ( fig. b and c, and s a). clusters with the high expression of ppbp, a marker of platelets, were excluded in following analysis (e.g., cluster in fig. s a ). intriguingly, up-regulated genes in cluster and cluster were associated with previously defined gene sets for 'influenza a virus infection' and 'sars-cov infection', respectively ( fig. s b ) ( ) . we also found that the cluster -specific up-regulated genes reflect activation of immune response, including cd , rgs , ccl , sell, and rgs ( fig. s c and table s ). protein interaction network analysis of selected top upregulated genes in each cluster based on string v ( ) revealed the up-regulation of prf , gnly, gzmb, and gzmh in cluster and the up-regulation of gzmk, gzma, cxcr , and ccl in cluster (fig. d, green) . stat , tap , psmb , and psme , which are up-regulated preferentially by ifn-γ, were overexpressed only in influenza-specific cluster (fig. d , blue). we validated these data by intracellular staining for granzyme b and pma/ionomycin-stimulated intracellular cytokine staining for ifn-γ. the percentages of granzyme b + and ifn-γ + cells among cd + t cells were significantly higher in the influenza group than in the covid- group (fig. s d ). of the seven representative go biological pathways for the pro-inflammatory and ifn responses, pathways for responses to ifn-i and -ii were more associated with influenza-specific cluster , whereas pathways for the response to tnf or il- β were more prominent in covid- -specific cluster ( we performed sub-clustering analysis from all three types of monocyte clusters to find covid- -specific sub-clusters. however, there was no covid- -specifically enriched subcluster ( fig. s a and b ). next, we further focused on classical monocytes considering their crucial roles for inflammatory responses. we investigated degs between influenza and covid- to seek covid- -specific transcriptional signatures in classical monocytes (fig. a) . interestingly, tnf and il b, major genes in the inflammatory response, were identified as covid- -specific and commonly up-regulated genes, respectively. to better characterize the transcriptional signatures in classical monocytes, we performed k-means clustering of up-regulated genes in at least one disease group compared to the healthy donor group. we identified five different clusters of up-regulation ( fig. b and table s ): genes in cluster are commonly up-regulated in all disease groups, cluster is influenza-specific, cluster is associated with mild/severe covid- , cluster is associated with influenza and severe covid- , and cluster is severe covid- specific. we examined each cluster-specific genes by gene set enrichment analysis (gsea) using cytokine-responsive gene sets originated from each cytokine-treated cells (lincs l ligand perturbation analysis in enrichr) ( ) . covid- -specific cluster genes were enriched by tnf/il- βresponsive genes whereas influenza-specific cluster genes were enriched by ifn-i-responsive genes in addition to tnf/il- β-responsive genes (fig. c) , indicating that the ifn-i response is dominant in influenza compared to covid- . we confirmed this result by analyzing cluster-specific genes with cytokine-responsive gene sets originated from other sources (fig. d ). unexpectedly, cluster and exhibited strong associations with ifn-i-responsive genes, in addition to tnf/il- β-responsive genes ( fig. e ), indicating that severe covid- acquires ifn-i-responsive features in addition to tnf/il- β-inflammatory features. next, we directly compared classical monocytes between mild and severe covid- . when we analyzed degs, severe covid- was characterized by up-regulation of various isgs, including isg , ifitm / / , and isg (fig. a ). both tnf/il- β-responsive genes and ifn-i-responsive genes were enriched in severe covid- -specific up-regulated genes (fig. b ). we measured plasma concentrations of tnf, il- β, il- , ifn-β, ifn-γ, and il- in a larger cohort of covid- patients. among these cytokines, il- and il- were significantly increased in severe covid- compared to mild covid- whereas there was no difference in plasma concentrations of the other cytokines between the two groups ( fig. s a) . these results indicate that cytokine-responsive gene signatures cannot be simply explained by a few cytokines because of overlapped effects of cytokines. to further investigate the characteristics of severe covid- , we performed a trajectory analysis with monocle ( ) using two internally well-controlled specimens (one severe and one mild) in which both pbmc samples were collected from a single patient (the subject c ) with covid- . trajectory analysis aligned classical monocytes along the disease severity with cluster and cluster corresponding to later and earlier pseudotime, respectively (fig. c ). representative genes in cluster was enriched in the severe stage and highly associated with the both ifn-i and tnf/il- β-associated inflammatory response (fig. d, fig. s b , and table s ). gsea confirmed that both the ifn-i response and tnf/il- β inflammatory response were prominent in cluster , but not in cluster (fig. e ). cluster exhibited a significantly higher association with a gene set from systemic lupus erythematosus, which is a representative inflammatory disease with ifn-i features, than cluster (fig. f, left) , but was not significantly associated with a gene set from rheumatoid arthritis (fig. f, right) . we obtained additional evidence of the ifn-i-potentiated tnf inflammatory response in severe covid- by analyzing a gene module that is not responsive to ifn-i, but associated with tnf-induced tolerance to tlr stimulation. park et al. previously demonstrated that tnf tolerizes tlr-induced gene expression in monocytes, though tnf itself is an inflammatory cytokine ( ) . they also showed that ifn-i induces a hyper-inflammatory response by abolishing the tolerance effects of tnf, and defined a gene module responsible for the ifn-i-potentiated tnf-nf-κb inflammatory response as 'class ' ( ) . this gene module was significantly enriched in cluster , but not in cluster (fig. g ), which suggests that the ifn-i response may exacerbate hyper-inflammation by abolishing a negative feedback mechanism. finally, we validated ifn-i response and inflammatory features using bulk rna-seq data obtained using post-mortem lung tissues from patients with lethal covid- ( ) . although the analysis was limited to only two patients without individual cell-type resolution, in genome browser, up-regulation of ifitm , isg , and jak and down-regulation of rps were observed commonly in post-mortem covid- lung tissues and classical monocytes of severe covid- (fig. a ). in the analysis with cytokine-responsive gene sets, both the ifn-i response and tnf/il- β-inflammatory response were prominent in the lung tissues (fig. b) . degs in the lung tissues were significantly associated with cluster , which is commonly up-regulated in both influenza and severe covid- , and cluster , which is specific to severe covid- in fig. b (fig. c) . these genes were also significantly associated with the cluster identified in the trajectory analysis, but not with cluster (fig. d ). when gene sets were defined by degs between mild and severe covid- , the degs in post-mortem lung tissues were significantly associated with genes up-regulated specifically in severe covid- (fig. e ). severe covid- has been shown to be caused by a hyperinflammatory response ( ) . particularly, inflammatory cytokines secreted by classical monocytes and macrophages are considered to play a crucial role in severe progression of covid- ( ) . in the current study, we confirmed the results from previous studies by showing that the tnf/il- β inflammatory response is dominant in covid- although a small number of patients were enrolled. however, we also found that severe covid- is accompanied by the ifn-i response in addition to the tnf/il- β response. these results indicate that the ifn-i response might contribute to the hyper-inflammatory response by potentiating tnf/il- β-driven inflammation in severe progression of covid- . in the current study, we carried out scrna-seq using pbmcs instead of specimens from the site of infection, e.g., lung tissues or bronchoalveolar lavage (bal) fluids. however, hierarchical clustering based on relative changes to the healthy donor group showed that all types of cells among pbmcs were clustered together according to the disease groups as shown in fig. a , indicating that there is diseasespecific global impact across all types of cells among pbmcs. this finding suggests that peripheral blood immune cells are influenced by common inflammatory mediators regardless of cell type. however, we could not examine granulocytes in the current study because we used pbmcs, not whole blood samples for scrna-seq. in transcriptome studies for cytokine responses, we often analyze cytokine-responsive genes rather than cytokine genes themselves. however, we cannot exactly specify responsible cytokine(s) from the list of up-regulated genes because of overlapped effects of cytokines. for example, up-regulation of nf-κb-regulated genes can be driven by tnf, il- β or other cytokines, and up-regulation of ifn-responsive genes can be driven by ifn-i or other interferons. in the current study, we designated the ifn-i response because many up-regulated ifn-responsive genes were typical isgs. recently, wilk et al. also performed scrna-seq using pbmcs from covid- patients and healthy controls ( ) . similar to our study, they found ifn-i-driven inflammatory signatures in monocytes from covid- patients. however, they did not find substantial expression of pro-inflammatory cytokine genes such as tnf, il , il b, ccl , ccl and cxcl in peripheral monocytes from covid- patients whereas we detected the up-regulation of tnf, il b, ccl , ccl and cxcl in the current study. moreover, they found a developing neutrophil population in covid- patients that was not detected in our study. these discrepant results might be due to different platforms for scrna-seq. wilk et al. used the seq-well platform whereas we used the x genomics platform that is more generally used. we also note that recent scrna-seq analyses of covid- sometimes lead to unrelated or contradictory conclusions to each other despite the same platform ( , ) . although it often occurs in unsupervised analysis of highly multi-dimensional data, more caution will be required in designing scrna-seq analysis of covid- , including definition of the severity and sampling time points. recently, blanco-melo et al. examined the transcriptional response to sars-cov- in in vitro infected cells, infected ferrets, and post-mortem lung samples from lethal covid- patients and reported that ifn-i and -iii responses are attenuated ( ) . however, we noted that ifn-i signaling pathway and innate immune response genes were relatively up-regulated in post-mortem lung samples from lethal covid- patients compared to sars-cov- -infected ferrets in their paper. given that sars-cov- induces only mild disease without severe progression in ferrets ( ), we interpret that ifn-i response is up-regulated in severe covid- (e.g., postmortem lung samples from lethal covid- patients), but not in mild covid- (e.g., sars-cov- -infected ferrets). indeed, severe covid- -specific signatures discovered in our current study were significantly enriched in the publically available data of post mortem lung tissues from the blanco-melo et al.'s study although the analysis was limited to only two patients without individual cell-type resolution (fig. ) . in a recent study, zhou et al. also found a robust ifn-i response in addition to pro-inflammatory response in bal fluid of covid- patients ( ) . moreover, up-regulation of ifn-iresponsive genes has been demonstrated in sars-cov- infected intestinal organoids ( ) . although ifn-i has direct antiviral activity, their immunopathological role was also reported previously ( ) . in particular, the detrimental role of the ifn-i response was elegantly demonstrated in a murine model of sars ( ) . in sars-cov-infected balb/c mice, the ifn-i response induced the accumulation of pathogenic inflammatory monocytesmacrophages and vascular leakage, leading to death. it was proposed that a delayed, but considerable ifn-i response is critical for the development of acute respiratory distress syndrome and increased lethality during pathogenic coronavirus infection ( , ) . currently, the management of patients with severe covid- relies on intensive care and mechanical ventilation without a specific treatment because the pathogenic mechanisms of severe covid- have not yet been clearly elucidated. in the current study, we demonstrated that severe covid- is characterized by tnf/il- β-inflammatory features combined with the ifn-i response. in a murine model of sars-cov infection, timing of the ifn-i response is a critical factor determining outcomes of infection ( , ) . delayed ifn-i response contributes to pathological inflammation whereas early ifn-i response controls viral replication. therefore, we propose that anti-inflammatory strategies targeting not only inflammatory cytokines, including tnf, il- β, and il- , but also pathological ifn-i response needs to be investigated for the treatment of patients with severe covid- . patients diagnosed with covid- were enrolled from asan medical center, severance hospital, and chungbuk national university hospital. sars-cov- rna was detected in patients' nasopharyngeal swab and sputum specimens by multiplex real-time reverse-transcriptase pcr using the allplex -ncov assay kit (seegene, seoul, republic of korea). in this assay, n, rdrp, and e genes of sars-cov- were amplified, and ct values were obtained for each gene. sars-cov- -specific antibodies were examined using the sars-cov- neutralization antibody detection kit (genscript, piscataway, nj) and were positive in all covid- patients in convalescent plasma samples or the last plasma sample in a lethal case. hospitalized patients diagnosed with influenza a virus infection by a rapid antigen test of a nasopharyngeal swab were also enrolled from asan medical center and chungbuk national university hospital from december to april , prior to the emergence of covid- . patients' clinical features, laboratory findings, and chest radiographs were collected from their electronic medical records at each hospital. this study protocol was reviewed and approved by the institutional review boards of all participating institutions. written informed consent was obtained from all patients. peripheral blood mononuclear cells (pbmcs) were isolated from peripheral venous blood via standard ficoll-paque (ge healthcare, uppsala, sweden) density gradient centrifugation, frozen in freezing media, and stored in liquid nitrogen until use. all samples showed a high viability of about % on average after thawing. single-cell rna-seq libraries were generated using the chromium single cell ′ library & gel bead kit v ( x genomics, pleasanton, ca) following the manufacturer's instructions. briefly, thousands of cells were separated into nanoliter-scale droplets. in each droplet, cdna was generated through reverse transcription. as a result, a cell barcoding sequence and unique molecular identifier (umi) were added to each cdna molecule. libraries were constructed and sequenced as a depth of approximately , reads per cell using the nextseq or novaseq platform (illumina, san diego, ca). the sequenced data were de-multiplexed using mkfastq (cellranger x genomics, v . . ) to generate fastq files. after de-multiplexing, the reads were aligned to the human reference genome (grch ; x cellranger reference grch v . . ), feature-barcode matrices generated using the cellranger count, and then aggregated by cellranger aggr using default parameters. the following analysis was performed using seurat r package v . . ( ) . after generating the feature-barcode matrix, we discarded cells that expressed < genes and genes not expressed in any cells. to exclude low-quality cells from our data, we filtered out the cells that express mitochondrial genes in > % of their total gene expression as described in previous studies ( , , ) . doublets were also excluded, which were dominant in the cluster "uncategorized ". although there was a high variability in the number of umis detected per cell, majority of cells ( . %) were enriched in a reasonable range of the umis ( , - , ), and % of cells with less than , umis were platelet or rbc excluded in downstream analysis. in each cell, the gene expression was normalized based on the total read count and log-transformed. to align the cells originating from different samples, highly variable genes from each sample were identified by the vst method in seurat r package v . . ( ) . using the canonical correlation analysis (cca), we found anchors and aligned the samples based on the top canonical correlation vectors. the aligned samples were scaled and principal component analysis (pca) conducted. finally, the cells were clustered by unsupervised clustering ( . resolution) and visualized by tsne using the top principal components. to identify marker genes, up-regulated genes in each cluster relative to the other clusters were selected based on the wilcoxon rank sum test in seurat's implementation with > . log fold change compared to the other clusters and a bonferroni-adjusted p < . (table s ) . by manual inspection, among the different clusters, were assigned to known immune cell types, rbcs which are characterized by hba , hba , and hbb, and platelets. the clusters characterized by similar marker genes were manually combined as one cell type. the two remaining clusters were assigned to 'uncategorized ' and 'uncategorized ' because they had no distinct features of known cell types. based on the distribution of umi counts, the cluster 'uncategorized ' was featured by relatively high umis per cell compared to other clusters and presence of higher expression of multiple cell type marker genes. the cluster 'uncategorized ' was featured by a b celllike signatures and high expression of ribosomal protein genes, not recommended to be further analyzed according to the x platform guideline. in these aspects, rbcs, platelets, uncategorized , and uncategorized were excluded in downstream analysis. to check the reproducibility of biological replicates (individuals within a same group), we calculated the spearman's rank correlation coefficient for umi counts that were merged according to each individual. the correlation coefficients of all individual pairs within the same group were visualized by a boxplot (covid- , n= ; flu, n= ; hd, n= ). in fig. s e , to investigate the similarity of the transcriptomes between cell types across diseases, we merged the umi counts of each cell type according to healthy donor, influenza, mild covid- , and severe covid- . next, the umi counts for each gene were divided by the total umi count in each cell type and multiplied by , as the normalized gene expression. based on a median expression value > . , we calculated the relative changes in gene expression divided by the median value for each gene. hierarchical clustering analysis was performed based on the pcc of the relative change in gene expression. in fig. a and fig. s a , to compare the highly variable gene expression among mild and severe covid- and influenza relative to healthy donors, the normalized gene expression used in fig. s e was divided by the values in the healthy donor group. we selected the highly variable genes in terms of the top % standard deviation followed by log -transformation (pseudo-count = ). in fig. a , hierarchical clustering analysis was performed based on the pccs of the selected highly variable genes. for fig. s a , to investigate the expression patterns of the selected highly variable genes (n= , ), k-means clustering (k= ) was performed based on euclidean distance. we manually ordered the clusters and visualized them as a heat map, revealing four distinct patterns: influenza-specific (n= , ), covid- specific (n= , ), influenza/covid- common (n= , ), and cell type-specific (n= , ). to investigate the dynamic changes in cell type composition, we calculated the proportion of cell types in each individual. as a control, we calculated the relative variation in each cell type composition between all pairs of healthy donors. similarly, for each disease group, we calculated the relative variation in each cell type by dividing the fraction of the cell type in individual patient by that of individual healthy donor. after log -transformation, we conducted statistical analysis using the relative variation in composition between the control and disease groups using a two-sided kolmogorov-smirnov test. for any two transcriptome profiles, to identify degs, we utilized the model-based analysis of single cell transcriptomics (mast) algorithm in seurat's implementation based on a bonferroni-adjusted p< . and a log fold change > . . in fig. b , the degs in covid- and influenza compared to healthy donors or covid- compared to influenza were identified at cell type resolution. all degs were combined according to the disease groups for further analysis. the overlapping up or down degs between covid- and influenza compared to healthy donors were defined as 'common up' or 'common down'. the specific degs in covid- or influenza were assigned as 'covid- up/down' or 'flu up/down', respectively. in addition, covid- -specific up-or down-regulated genes compared to influenza were assigned as 'covid- >flu' or 'flu>covid- ', respectively. the gene ontology analysis was performed by david. for each group of degs, the top enriched go biological pathways were selected, resulting in unique go biological pathways across all groups. the -log (p-values) are shown as a heat map in fig. b . the weighted gene correlation network analysis (wgcna) was conducted with the genes listed in the top go biological pathways of 'covid- up', 'flu up', and 'common up' defined in fig. b . the normalized gene expression values of the genes in covid- were divided by the values in influenza and log -transformed (pseudo-count = ). we used default parameters with the exception of soft threshold = and networktype = 'signed' when we constructed a topological overlap matrix. the modular gene expression patterns were defined using cutreedynamic with a minclustersize of . we visualized the modular gene expression pattern as a heat map in which the cell types were ordered according to hierarchical clustering with the default parameters of hcluster in r. to find disease-specific subpopulations, each immune cell type was subjected to the subclustering analysis using seurat. briefly, the highly variable genes (n= ) were selected based on vst and then scaled by scaledata in seurat with the vars.to.regress option to eliminate variation between individuals. the subpopulations were identified by findclusters with default parameters, except resolution (non-em-like cd + t cells, . ; classical monocytes, . ); the inputs were the top eight principal components (pcs) obtained from pca of the scaled expression of the highly variable genes. the subpopulations were visualized by tsne using the top eight pcs. the trajectory analysis was performed with highly variable genes in classical monocytes across mild (c - ) and severe (c - ) covid- as defined by the vst method in seurat. the following analysis was performed using monocle . briefly, the input was created from the umi count matrix of the highly variable genes using the newcelldataset function with default parameters, except expressionfamily = 'negbinomial.size'. the size factors and dispersion of gene expression were estimated. the dimension of the normalized data was reduced based on ddrtree using reducedimension with default parameters, except scaling = false, which aligned the cells to the trajectory with three distinct clusters. to determine genes that gradually changed along the trajectory, we identified the degs using mast between clusters and , which represent the severe stage and mild stage, respectively. the expression patterns of representative degs were visualized along the pseudotime after correction with estimated size factors and dispersion for all genes. in fig. b , we performed k-means clustering of degs among all pairs of mild covid- , severe covid- , and influenza. the log -transformed relative gene expression of degs compared to healthy donors was subjected to k-means clustering (k= ). here, we used up-regulated degs in at least one disease group compared to the healthy donor group. we manually assigned five clusters based on gene expression patterns. the transcriptome profiles of post-mortem lung tissues from two lethal cases of covid- and biopsied heathy lung tissues from two donors were downloaded from a public database (gse ). the degs were identified using deseq based on a bonferroni-adjusted p < . and a log fold change > . enrichment analysis using enrichr and gsea . . enrichr, the web-based software for gene set enrichment analysis (gsea) was used for lincs l ligand perturbation analysis ( ) , virus perturbation analysis, and disease perturbation analysis from the geo database. 'combined score' was calculated as a parameter of enrichment as the log(p-value) multiplied by the z-score from the fisher exact test. gsea . . software was used to conduct the gsea when a ranked list of genes was available (fig. g, fig. c -e) ( ) . results for ifn-γ-responsive genes were not presented because those were considerably overlapped with ifn-αresponsive genes, which are typical isgs. the normalized enrichment score and fdr-q value were calculated to present the degree and significance of enrichment. cryopreserved pbmcs were thawed, and dead cells were stained using the live/dead fixable cell stain kit (invitrogen, carlsbad, ca). cells were stained with fluorochrome-conjugated antibodies, including anti-cd (bv ; bd biosciences), anti-cd (bv ; bd biosciences), anti-cd (bv ; bd biosciences), anti-cd (pe-cy ; bd biosciences), anti-cd (alexa fluor ; bd biosciences), and anti-cd (vio-bright fitc; miltenyi biotec). for staining with antigranzyme b (bd biosciences), cells were permeabilized using a foxp staining buffer kit (ebioscience). for intracellular cytokine staining of ifn-γ, pbmcs were stimulated with phorbol -myristate -acetate (pma, ng/ml) (sigma aldrich) and ionomycin ( μg/ml) (sigma aldrich). brefeldin a (golgiplug, bd biosciences) and monesin (golgistop, bd biosciences) were added hour later. after another hours of incubation, cells were harvested for staining with the live/dead fixable cell stain kit, anti-cd , anti-cd , and anti-cd . following cell permeabilization, cells were further stained with anti-ifn-γ (alexa fluor ; ebioscience). flow cytometry was performed on an lsr ii instrument using facsdiva software (bd biosciences) and the data analyzed using flowjo software (treestar, san carlos, ca). cytokines were measured in plasma samples, including ifn-β, il- (elisa, r&d systems, minneapolis, mn), il- β (cytometric bead array flex kit, bd biosciences, san jose, ca), tnf, il- , and ifn-γ (legendplex bead-based immunoassay kit, biolegend, san diego, ca). we performed the ks test to compare the distributions of two groups without assuming that the distributions follow normality. welch's t test was conducted to compare the two distributions after confirming the normality of the distributions using the shapiro-wilk normality test. a wilcoxon signed rank test was conducted to compare the differences between two groups with paired subjects. the mann-whitney test was performed to compare the means of two groups. statistical analyses were performed using prism software version . (graphpad, la jolla, ca). p< . was considered significant. immunology.sciencemag.org/cgi/content/full/ / /eabd /dc fig. s . clinical characteristics and assessment of the quality of scrna-seq results. fig. s . transcriptome features of highly variable genes. fig. s . characterization of disease-specific cd + t-cell subpopulations. fig. s . subpopulation analysis of classical monocytes. fig. s . string analysis of up-regulated genes in cluster obtained from the trajectory analysis of classical monocytes. table s . experimental batches of scrna-seq. first release: july immunology.sciencemag.org (page numbers not final at time of first release) table s . clinical characteristics of severe influenza patients. table s . clinical characteristics of covid- patients. table s . the scrna-seq results. table s . a list of marker genes for each cluster. table s . a list of degs and associated biological pathways in fig. b . table s . cell types in which the gbp , crem, and ccl were upregulated in fig. c . table s . a list of genes in each module obtained from wgcna in fig. d . table s . a list of up-regulated genes in non-em-like cd + t-cell subpopulations. table s . a list of genes included in each cluster defined by k-mean clustering of classical monocytes. table s . a list of genes up-regulated in early and late pseudotime. a new coronavirus associated with human respiratory disease in china china novel coronavirus investigating and research team, a novel coronavirus from patients with pneumonia in china world health organization clinical characteristics of coronavirus disease in china estimating clinical severity of covid- from the transmission dynamics in wuhan, china pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology sars-cov- : a storm is raging clinical and immunological features of severe and moderate coronavirus disease a dynamic immune response shapes covid- progression dysregulated type i interferon and 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interactions identified by single-cell analysis infection and rapid transmission of sars-cov- in ferrets heightened innate immune responses in the respiratory tract of covid- patients sars-cov- productively infects human gut enterocytes disease-promoting effects of type i interferons in viral, bacterial, and coinfections a conserved dendritic-cell regulatory program limits antitumour immunity accelerating medicines partnership rheumatoid arthritis & systemic lupus erythematosus (amp ra/sle) consortium, notch signalling drives synovial fibroblast identity and arthritis pathology pgc- alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes key: cord- -jhm u ka authors: wang, dang; chen, jiyao; yu, chaoliang; zhu, xinyu; xu, shangen; fang, liurong; xiao, shaobo title: porcine reproductive and respiratory syndrome virus nsp antagonizes type i interferon signaling by targeting irf date: - - journal: journal of virology doi: . /jvi. - sha: doc_id: cord_uid: jhm u ka porcine reproductive and respiratory syndrome virus (prrsv) is an arterivirus from the nidovirales order that causes reproductive failure and respiratory disease in pigs and poses a constant threat to the global pig industry. the prrsv-encoded nonstructural protein (nsp ) is a nidovirus-specific endoribonuclease (nendou) that is conserved throughout the arteriviridae and coronaviridae families. previously, our research and that of others demonstrated that prrsv nsp inhibits type i interferon (ifn) production through nendou activity-dependent mechanisms. here, we found that prrsv nsp also inhibited ifn-stimulated response element (isre) promoter activity and subsequent transcription of ifn-stimulated genes (isgs). detailed analysis showed that nsp targeted interferon regulatory factor (irf ), but not transducer and activator of transcription (stat ) or stat , key molecules in the type i ifn signaling pathway. furthermore, the nsp -irf interaction impaired the formation and nuclear translocation of the transcription factor complex ifn-stimulated gene factor (isgf ) in both nsp -overexpressed and prrsv-infected cells. importantly, nsp mutations (h a, h a, and k a) that ablate nendou activity or its cell cytotoxicity also interacted with irf and retained the ability to block ifn signaling, indicating that the nsp -irf interaction is independent of nendou activity or cell cytotoxicity of nsp . taking the results together, our study demonstrated that prrsv nsp antagonizes type i ifn signaling by targeting irf via a nendou activity-independent mechanism, and this report describes a novel strategy evolved by prrsv to counteract host innate antiviral responses, revealing a potential new function for prrsv nsp in type i ifn signaling. importance the nidovirus-specific endoribonuclease (nendou) encoded by prrsv nonstructural protein (nsp ) is a unique nendou of nidoviruses that infect vertebrates; thus, it is an attractive target for the development of antinidovirus drugs. previous studies have revealed that the nendou of nidoviruses, including porcine reproductive and respiratory syndrome virus (prrsv) and human coronavirus e (hcov- e), acts as a type i interferon (ifn) antagonist. here, for the first time, we demonstrated that overexpression of prrsv nsp also inhibits ifn signaling by targeting the c-terminal interferon regulatory factor (irf) association domain of irf . this interaction impaired the ability of irf to form the transcription factor complex ifn-stimulated gene factor (isgf ) and to act as a signaling protein of ifn signaling. collectively, our data identify irf as a natural target of prrsv nendou and reveal a novel mechanism evolved by an arterivirus to counteract innate immune signaling. agent, prrs virus (prrsv) (family arteriviridae, order nidovirales), has a positive-polarity single-stranded rna genome, approximately kb in length, encoding two nonstructural polyproteins (orf ab and orf b) and eight structural proteins (gp , e, gp , gp , gp , orf a, m, and n) ( , ) . after being cleaved by four virus-encoded proteases, nonstructural protein ␣ (nsp ␣), nsp ␤, nsp , and nsp , the two polyproteins are further cleaved into individual nonstructural proteins that perform different functions during the viral life cycle ( ) . to reproduce rapidly and establish a persistent infection in pigs, prrsv has developed the ability to resist host interferon (ifn) signaling, which is the key signaling pathway for innate immune responses against viral invasion or replication ( , , ) . innate immune responses are activated by host pattern recognition receptors (prrs), which recognize molecular structures called pathogen-associated molecular patterns (pamps) that are structurally conserved within a large number of pathogenic organisms ( ) . upon recognition of pamps, prrs initiate signaling pathways that ultimately trigger the production of type i ifns. subsequently, the janus kinase/signal transduction and transcription activator (jak/stat) pathway is activated by type i ifns ( , ) . briefly, type i ifns bind to their surface receptors (ifnar and ifnar ) and then activate and phosphorylate the two intracellular tyrosine kinases janus kinase (jak ) and tyrosine kinase (tyk ). these activated tyrosine kinases mainly phosphorylate two transcription factors, namely, signal transducer and activator of transcription (stat ) and stat , which interact with ifn regulatory factor (irf ) to form ifnstimulated gene factor (isgf ) ( ) . the transcription factor complex isgf (stat / stat /irf ) is transported to the nucleus, where it recognizes ifn-stimulated response elements (isres), leading to the induction of hundreds of ifn-stimulating genes (isgs) ( ) . many isgs function as potent antiviral effectors, directly preventing viral infections by targeting viral processes such as viral entry, viral genome replication, viral protein synthesis, or viral body release ( ) . during coevolution with their hosts, many viruses have developed elaborate strategies to circumvent the jak/stat pathway in ifn signaling ( ) . among several known viral evasion strategies, targeting the transcription factor complex isgf is a particularly powerful means of inactivating ifn signaling, because this strategy effectively inhibits common downstream isg expression. for example, our recent study showed that porcine deltacoronavirus blocks the jak/stat pathway by c-like protease-mediated cleavage of stat ( ) . the nonstructural protein (nsp ) of rotavirus mediates the degradation of irf by targeting the c-proximal irf-binding domain and inhibits ifn-mediated phosphorylation of stat ( , ) . sendai virus c protein binds stat to block the formation of stat /stat heterodimers or stat /stat homodimers, which inhibit ifn signaling ( ) . previous studies have shown that prrsv nsp inhibits type i ifn production through nidovirus-specific endoribonuclease (nendou) activitydependent mechanisms ( ) ( ) ( ) . however, the nendou activity of overexpressed nsp unexpectedly exhibited extensive substrate specificity in vitro and is extremely toxic to prokaryotic and eukaryotic cells, indicating that the inhibition of ifn production by wild-type (wt) prrsv nsp may be due to its cytotoxicity ( ) . here, we found that prrsv nsp also inhibits isre promoter activity and the transcription of isgs, thereby interfering with the type i ifn signaling pathway. importantly, mutations that eliminate nendou activity and its cytotoxicity in nsp retain the ability to block ifn signaling. detailed analysis showed that nsp inhibited type i ifn signaling by targeting irf , a key molecule in the isgf complex, revealing a potential novel function of prrsv nsp in type i ifn signaling. identification of prrsv nsp as an antagonist of type i ifn signaling. type i ifn signaling induces a potent antiviral response in cells by inducing the expression of hundreds of isgs, which is vital for the control of viral infections ( ) . to assess the potential role of prrsv nsp in type i ifn signaling, the mrna levels of ifn-stimulated gene (isg ), isg , isg , and '- '-oligoadenylate synthetase (oas ) were analyzed in human embryonic kidney cells (hek- t) overexpressing hemagglutinin (ha)-tagged prrsv nsp . as shown in fig. a , prrsv nsp significantly inhibited the transcription of isgs induced by ifn-␣ compared with the control group results. because of the presence of isre in the isg promoter regions, various concentrations of prrsv nsp expression plasmid and isre-luciferase reporter plasmid were cotransfected into hek- t cells or porcine kidney cells (pk- ). the results showed that nsp strongly inhibited ifn-␣-induced isre promoter activity in a dose-dependent manner in hek- t cells (fig. b) and pk- cells (fig. c) . these results confirm the antagonistic nature of prrsv nsp in type i ifn signaling. prrsv nsp inhibits type i ifn signaling in an endoribonuclease activityindependent manner. in arterivirus, the nsp endoribonuclease is important for viral replication ( , , ( ) ( ) ( ) ( ) . several previous studies have shown that prrsv nsp inhibits type i ifn production in a nendou activity-dependent manner ( ) ( ) ( ) . we considered such a possibility for prrsv nsp -mediated inhibition of type i ifn signaling to be associated also with its nendou activity. on the basis of their chemical properties and known residue requirements, the three catalytic residues of endoribonucleases (his , his , and lys [numbering based on prrsv nsp ]) were shown to be directly involved in catalysis ( , , ) . thus, three endoribonuclease catalytic residue mutations, his ala (h a), his ala (h a), and lys ala (k a), were introduced into prrsv nsp and the corresponding eukaryotic expression plasmids expressing the n-terminally ha-tagged nsp endoribonuclease inactive mutants were constructed. to test whether the ha-tagged nsp mutants lose endoribonuclease activity, ha-tagged wt nsp and three mutants were expressed in escherichia coli. the recombinant proteins were purified ( fig. a) , and fluorescence resonance energy transfer (fret) assays were performed to detect the endoribonuclease activity. as shown in fig. b , the levels of endoribonuclease activity of the three ha-tagged nsp mutants were significantly decreased compared with the wt nsp (fig. b) . however, none of the endoribonuclease inactive mutants (h a, h a, or k a) showed a loss of the ability of nsp to inhibit isre promoter activity in cells overexpressing ha-tagged nsp mutants (fig. c) . we also used prrsv nsp as a negative control and porcine epidemic diarrhea virus (pedv) nsp as a positive control. in agreement with a previous study ( ) , pedv wt nsp suppressed ifn-␣-induced isre promoter activity whereas such an inhibitory effect was not observed in cells expressing a pedv nsp endoribonuclease inactive mutant (h a) or prrsv nsp (fig. c) . these data suggest that although the nendou activity of pedv nsp is important for inhibiting ifn signaling, prrsv nsp does not depend on the nendou activity. we further tested the ability of nsp mutants to inhibit ifn-␣-induced isg expression. as shown in fig. d , each endoribonuclease inactive mutant (h a, h a, or k a) of nsp also inhibited the ifn-␣-induced transcription of isgs. since these three mutants are also devoid of cell cytotoxicity ( ), our results indicated that the inhibition of ifn signaling observed in nsp -expressing cells is independent of its endoribonuclease activity and cell cytotoxicity. prrsv nsp disrupts isgf -mediated activation of the isre promoter. isgs are antiviral effectors induced by ifns through the formation of a tripartite transcription factor, isgf , which is composed of stat , stat , and irf ( ) . given the pivotal role of the transcription factor complex isgf in type i ifn signaling, we further investigated whether overexpression of nsp inhibits isgf -mediated signaling. as shown in fig. , coexpression of the components of transcription factor complex isgf (stat , stat , and irf ) significantly activated the isre promoter compared with the results seen with the empty plasmid control. however, activation of the isre promoter by isgf was significantly inhibited in the presence of prrsv nsp (fig. ) . similarly to the results seen with wt nsp , each endoribonuclease inactive mutant (h a, h a, or k a) was also able to suppress the isgf -mediated ires promoter (fig. ) , suggesting that the endoribonuclease activity of nsp does not govern the ability of nsp to block the activity of the isgf -induced ires promoter. consequently, we speculated that prrsv nsp might target the isgf complex to inhibit type i ifn signaling. prrsv nsp does not degrade or block the phosphorylation of stat and stat . since downregulation of molecules responsible for ifn-activated signal transduction is a mechanism commonly employed by viruses ( , ( ) ( ) ( ) , we investigated whether nsp impairs the endogenous protein levels of stat , stat , and irf . as shown in fig. , no reduction was observed in the protein levels of stat , stat , and irf as a consequence of the presence of wt nsp or expression of its endoribonuclease inactive mutants (h a, h a, and k a [compare lanes and to lane ]), suggesting that nsp does not induce downregulation of the isgf complex. the type i ifn-activated isgf transcription complex containing tyrosine-phosphorylated stat and stat associated with irf is rapidly translocated to the nucleus fig prrsv nsp inhibits isgf -induced isre promoter activity. hek- t cells were cotransfected with prrsv nsp or its endoribonuclease activity-defective mutants ( . g/well), along with porcine isgf complex (stat /stat /irf ; . g/well), isre-luc plasmid ( . g/well), and prl-tk plasmid ( . g/well). after h, cells were harvested for luciferase assays. *, p Ͻ . . after ifn treatment, and phosphorylation-dependent activation of stat and stat is critical to mediate ifn-inducible antiviral responses ( , ) . to investigate whether prrsv nsp alters the stat and stat phosphorylation status after ifn-␣ stimulation, lysates from nsp -expressing cells were subjected to western blotting using phospho-stat (stat -y ) and phospho-stat (stat -y ) antibodies (abs), respectively. the levels of phosphorylated stat and stat were greatly increased after ifn-␣ treatment, and no difference in the levels was found between nsp -expressing cells and nsp -nonexpressing cells (fig. , lanes and ) . similarly, nsp mutants lacking endoribonuclease activity (h a, h a, and k a) had no effect on the phosphorylation of stat and stat (fig. [compare lane to lanes to ]). these results indicated that prrsv nsp and its endoribonuclease inactive mutants do not target the isgf complex for degradation or prevent type i ifn-induced stat proteinactivating tyrosine phosphorylation. prrsv nsp interacts with the irf-association domain (iad) of irf . previous studies have shown that many viral proteins can interact with components of the isgf complex to inhibit type i ifn signaling ( , , ) . thus, we next investigated whether nsp functions by interacting with the isgf component. to this end, hek- t cells were transfected with expression constructs encoding ha-tagged prrsv nsp protein and flag-tagged porcine stat , stat , and irf . coimmunoprecipitation (co-ip) and immunoblotting analyses showed that nsp interacted with porcine irf , but not stat or stat ( fig. a to c). to further confirm the interaction of nsp with endogenous irf , coprecipitated endogenous irf and prrsv nsp were analyzed by immunoblotting with an anti-irf antibody and an anti-ha antibody, respectively. as shown in fig. d , reciprocal pulldown of both nsp and endogenous irf further confirmed the interaction between nsp and irf . previous studies demonstrated that irf contains an n-terminal dna-binding domain (dbd; amino acids [aa] to ) and a c-terminal iad (aa to ) connected by a flexible linker (fl) ( ) ( ) ( ) . to investigate which domain of porcine irf is involved in nsp protein binding, four mutants with deletions of different domains of irf , including irf (aa to ; dbd), irf (aa to ; dbd-fl), irf (aa to ; iad), and irf (aa to ; fl-iad), were constructed by mutagenesis (fig. e ). hek- t cells were cotransfected with various combinations of flag-tagged full-length or deleted versions of irf and the ha-tagged prrsv nsp protein. as shown in fig. f , prrsv nsp -irf interaction impairs the ifn-induced formation and nuclear accumulation of isgf . since the function of isgf relies on the selective interaction between phosphorylated stat and the irf-association domain of irf ( , ) , the observed interaction between nsp and irf -iad led us to speculate that this interaction may impair the recruitment of phosphorylated stat by irf and the subsequent nuclear accumulation of isgf . to test this hypothesis, hek- t cells were transfected with the indicated nsp expression plasmids and then treated with ifn-␣. the prrsv nsp protein and phosphorylated stat /stat (stat -y and stat -y ) were immunoprecipitated with an anti-irf antibody. as shown in fig. a , ifn-␣ treatment significantly induced the phosphorylation of stat and stat , and irf efficiently pulled down phosphorylated stat /stat after ifn-␣ treatment (compare lanes and ). however, the amount of phosphorylated stat /stat that bound to irf remarkably decreased in the presence of prrsv nsp and its mutants (h a, h a, and k a) (fig. a, lanes and to ) . to further test whether prrsv nsp prevents the ifn-induced nuclear accumulation of isgf , we performed nuclear and cytoplasmic fractionation of the cells following ifn-␣ treatment. antibodies against stat -y and stat -y were used to detect the presence of the phosphorylated proteins in the two fractions. as expected, higher levels of stat -y and stat -y were found in the nuclear fraction than in the cytoplasmic fraction after ifn-␣ treatment of mock-transfected cells (fig. b , lanes and ). in contrast, in nsp -transfected cells after ifn-␣ stimulation, more isgf complex components (stat -y , stat -y , and irf ) were detected in the cytoplasmic fraction than in the nuclear fraction (fig. b, lanes and ) . taken together, these findings indicate that prrsv nsp impairs the formation and nuclear translocation of isgf . because the endoribonuclease inactive mutants of nsp retained the ability to block type i ifn signaling, these mutants in particular were still capable of interacting with irf . next, we investigated whether these mutants, lacking endoribonuclease activity, still impaired the formation and nuclear accumulation of isgf . indeed, our data revealed that the prrsv nsp mutants (h a, h a, and k a) still significantly inhibited the ifn-induced formation and nuclear accumulation of isgf and did so to similar extents (fig. b [compare lanes to and lanes to ] ). this provided further support for the notion that the ability of prrsv nsp to block type i ifn signaling is independent of its endoribonuclease activity and cell cytotoxicity, because these three mutants had similar effects with respect to inhibiting the formation and nuclear translocation of isgf compared with wt nsp . prrsv infection inhibits the formation of isgf through the interaction of nsp with irf . to exclude the possibility that the observed prrsv nsp -irf interaction was an artifact of plasmid overexpression in cell culture, we analyzed this interaction in the context of prrsv infection. african green monkey kidney cells (marc- ) were infected with prrsv for h and then collected at h after ifn-␣ treatment for immunoprecipitation with anti-nsp or anti-irf antibodies to detect the interaction between nsp and endogenous irf . as shown in fig. a , nsp was coimmunoprecipitated with endogenous irf in prrsv-infected cells. furthermore, phosphorylated stat /stat was also coimmunoprecipitated with endogenous irf upon ifn-␣ stimulation. however, the formation of stat /stat /irf heterotrimers was significantly inhibited following prrsv infection (fig. a [compare lanes and ] ). hek- t cells were transfected with expression vectors encoding ha-nsp and its mutants. endogenous irf was precipitated and immunoblotting analysis was performed with an anti-pstat antibody (the top panel), anti-pstat antibody (the second panel), or anti-ha antibody (the fourth panel) to detect the interaction with endogenous irf . (b) phosphorylated stat and stat in nuclear and cytoplasmic fractions. subcellular fractionation of hek- t cells, h after ifn-␣ treatment, was performed for nuclear and cytoplasmic fractions, followed by western blotting using stat -y antibody, stat -y antibody, anti-hsp antibody as a cytoplasmic marker, and anti-parp antibody as a nuclear protein marker, as indicated. ha antibody was used to detect the expression of prrsv nsp and its mutants (ha-nsp ). to detect the interaction with endogenous irf . (b) phosphorylated stat and stat in the nuclear and cytoplasmic fractions. an experiment parallel to that outlined for panel a was performed. subcellular fractionation of marc- cells was performed to obtain nuclear and cytoplasmic fractions, followed by western blotting using anti-stat -y , anti-stat -y , anti-irf , and anti-nsp antibodies, along with anti-hsp antibody as a cytoplasmic marker and anti-parp antibody as a nuclear protein marker, as indicated. (c to e) marc- cells were infected with prrsv (moi ϭ . ) or uninfected. at h postinfection, the cells were mock-treated or treated with ifn-␣ ( iu/ml) for h. after the cells were fixed and permeabilized, p-stat (c), p-stat (d), and irf (e) was visualized by immunofluorescence staining with rabbit anti-pstat , rabbit anti-pstat , or rabbit anti-irf antibody. the nsp protein was detected by the use of a nsp -specific mab. to further address whether the ifn-induced nuclear accumulation of isgf was also affected by prrsv infection, nuclear and cytoplasmic fractionation was performed. compared with uninfected cells, the levels of stat -y , stat -y , and irf proteins were reduced in the nuclear fraction of prrsv-infected cells after ifn-␣ stimulation (fig. b [compare lanes to and lanes to ]) . immunofluorescence experiments showed that prrsv infection inhibited ifn-induced nuclear accumulation of stat -y (fig. c) , stat -y (fig. d) , and irf proteins (fig. e ) compared to mock-infected cells. in prrsv-infected cells with ifn stimulation, nsp and irf were primarily colocated in the cytoplasm (fig. e) . together, these data indicated that prrsv infection inhibits the formation and nuclear accumulation of isgf through the nsp -irf interaction, which was consistent with the results of gene transfection experiments in cells expressing prrsv nsp . during coevolution with their hosts, many viruses have acquired mechanisms to circumvent host innate immune responses. of note, prrsv infection has been found to produce abnormally low levels of type i ifns and to inhibit the ability of type i ifns to induce antiviral responses ( , ) . the endoribonuclease encoded within the prrsv nsp sequence, which has the uridylate-preferred cleavage site for rna that is necessary for virus replication, has been found to be a multifunctional protein ( , ) . previously, it has been demonstrated that prrsv nsp is involved in the inhibition of ifn production through multiple distinct mechanisms, as follows. (i) nsp represses the transcription of type i ifns by inhibiting the activation of transcription factors irf and nuclear factor kappa b (nf-b), which directly activate the promoters of type i ifns ( , ) . (ii) nsp reduces the levels of transcripts and proteins of mitochondrial antiviral signaling protein (mavs) and retinoic acid-inducible gene i (rig-i), two critical factors in the ifn induction pathway ( ) . (iii) nsp removes the ubiquitin chains from ib␣ (inhibitor of nf-b alpha), thereby preventing the proteasomal degradation of ib␣ and subsequent liberation of nf-b ( ). (iv) nsp recruits the otu deubiquitinase with linear linkage specificity (otulin) to enhance its ability to remove the cellular protein ubiquitin associated with innate immunity, resulting in the additive effect of suppressing type i ifn production ( ) . however, whether nsp also regulates ifn signaling remains unclear. in this report, we present evidence that prrsv nsp suppressed type i ifn signaling by targeting irf , a key molecule in the jak/stat pathway, revealing a potential new function for the nidovirus endoribonuclease in type i ifn signaling. prrsv nsp , a conserved nendou within the arteriviridae and coronaviridae families, belongs to the xenopus laevis poly(u)-specific endoribonuclease (xendou) superfamily and plays an important role in nidovirus replication and pathogenesis ( ) . the structures of the arterivirus nsp , coronavirus (cov) nsp , and xendou catalytic domains, essential for endoribonuclease activity, and particularly the active site residues (his , his , and lys ; numbering based on prrsv nsp ), were found to be highly conserved. besides prrsv nsp , several other studies have reported that cov nsp inhibits ifn production in ectopic expression experiments. in support of this, infection with nendou activity-deficient covs, such as pedv, murine hepatitis virus, and human cov e (hcov- e), produced a remarkably high level of type i ifn in primary cells compared with wt infection, which effectively demonstrated the ifnantagonistic properties of nsp of covs ( , , ) . these data appear to indicate that endoribonuclease activity is sufficient for nendou-mediated ifn transcriptional repression; however, the nendou activity of overexpressed nsp /nsp may unexpectedly mediate nonspecific cleavage, thereby inducing cytotoxicity ( , ) . unfortunately, it was impossible to determine whether the cell cytotoxicity also contributed to the inhibition of ifn induction by nendou, as mutations disrupting the ability of nendou to block ifn production abrogated not only endoribonuclease activity but also its cell cytotoxicity ( ) . thus, the observation of ifn transcriptional suppression in previous studies could have been the result of cytotoxicity as a consequence of ectopic over-expression of nendou. previous studies have shown that prrsv wt nsp is toxic to e. coli and that the expression levels of wt nsp are extremely low and that one of the nendou mutants (nsp k a) can be expressed at high levels under identical conditions ( , ) . similar phenomena were observed in our experiments designed to express and purify nsp . indeed, in our previous study ( ) , we found that prrsv wt nsp exhibited cytotoxicity to eukaryotic cells whereas no cytotoxicity associated with the nendou mutants of nsp was detected. similarly, recombinant nsp of equine arteritis virus (eav) has been reported to be extremely toxic to a variety of hosts ( ) . thus, the lower level of expression of prrsv wt nsp may have been due to its cytotoxicity (fig. ) . interestingly, our results clearly showed that the prrsv nsp mutants (h a, h a, and k a) devoid of both nendou activity and cell cytotoxicity retained the ability to block type i ifn signaling by targeting irf , as did wt nsp , indicating that nsp has evolved a new mechanism for the impairment of ifn signaling that operates in an endoribonuclease activity-independent and cell cytotoxicity-independent manner, distinct from the inhibition of ifn induction. as a potent antiviral response, type i ifn signaling can control viral infections by activating the transcription factor complex isgf (stat /stat /irf ), resulting in increased transcription of hundreds of isgs and contributing to the development of an antiviral state ( , ) . it is becoming increasingly apparent that irf is a central factor not only for mediation of but also for regulation and direction of type i ifn responses ( ) . while many ifn effects, in particular, those associated with type i ifns, require all three canonical signaling molecules, studies of irf deficiency revealed unique roles for irf that were distinct from those of stat and stat ( , ) . recently, evidence has emerged that irf is the main viral target of the host's innate immune response. for example, our previous study ( ) revealed that porcine bocavirus nonstructural protein (ns ) inhibits the dna-binding activity of isgf by interacting with irf . another study showed that the e oncoprotein of papillomavirus binds to irf to block the formation of the isgf complex ( ) . moreover, many virus-encoded proteins, such as varicella-zoster virus orf , adenovirus early region a protein (e a), and rotavirus nsp , mediate irf degradation ( , , ) . human cytomegalovirus also reduces the protein levels of irf in human embryonic lung fibroblasts ( ) . to survive in the host, prrsv has also evolved strategies to block ifn signaling. previous studies have revealed that prrsv nsp ␤, a papain-like proteinase, blocks the nuclear translocation of isgf by inducing degradation of karyopherin-␣ ( , ) . here, we showed that nsp is another prrsvencoded antagonist of ifn signaling. prrsv nsp adopts a mechanism distinct from that of nsp ␤, i.e., interaction with irf , an essential component in the formation of isgf . our data not only highlight the multifaceted control of type i ifn signaling by prrsv but also uncover a novel mechanism by which prrsv antagonizes innate immune signaling. it is well established that irf exhibits several functionally conserved regions among the members of the irf family, such as an n-terminal dna-binding domain that recognizes and binds to isre motifs in the promoter region of most isgs and a c-terminal irf-association domain that is responsible for selective interaction with the coiled-coil domain (ccd) of stat ( , ) . recently, rengachari and colleagues reported the crystal structures of irf -iad alone and in a complex with stat -ccd ( ) . the structure of the irf -iad/stat -ccd complex revealed that surface features had deviated from their respective paralogs to enable a specific interaction between irf -iad and stat -ccd required for isgf function in cells ( ) . in this study, we found that prrsv nsp specifically interacted with irf -iad. thus, it may be the case that the targeting of irf -iad by nsp sequesters the interaction between irf and stat . this may help to explain why the formation and nuclear translocation of isgf were severely impaired in prrsv nsp -transfected cells and prrsv-infected cells. interestingly, prrsv nsp mutants (h a, h a, and k a) also impaired the formation and nuclear translocation of isgf by interacting with irf . furthermore, the three-dimensional ( d) structures of nendou activity-defective arterivirus nsp (k a in prrsv nsp ; h a and k a in eav nsp ) are remarkably similar to those of wt prrsv nsp ( , ) , with an overall root mean square deviation (rmsd) range of . to . Å (data not shown), suggesting that the ala substitution in his , his , and lys of prrsv nsp in this study might not prevent the core conformations of nsp protein from folding correctly. on the basis of data presented here and those reported by others mentioned above, we speculate that the ability to interact with irf correlated with the correct folding of nsp but not with nendou activity or cell cytotoxicity. future investigations will be required to identify the structural basis of the nsp -irf interaction. although our data clearly demonstrated that prrsv nsp could be coimmunoprecipitated with endogenous irf in prrsv-infected cells, thereby inhibiting the formation and nuclear translocation of isgf (fig. ) , whether prrsv nsp also functions as an antagonist of ifn signaling during prrsv infection has not been fully understood. one of the obstacles to studying arterivirus with nendou activitydeficient nsp mutants in cell culture is that catalytic residue mutations have been reported to nearly completely abolish viral replication even in ifn-deficient cells ( , ) . future investigations designed to identify other nonactive site residues of nsp that are involved in the nsp -irf interaction but that do not affect prrsv replication will be required to fully elucidate the function of nsp in ifn signaling. in summary, our data identify prrsv nsp as a newly recognized antagonist in ifn signaling and show that the nsp -irf interaction is involved in inhibition of the formation and nuclear translocation of isgf . this novel function of prrsv nsp offers new insight into the interaction between a viral endou and the ifn signaling pathway, potentially aiding the development of novel therapeutic targets and more-effective vaccines against prrsv. cells and viruses. hek- t, marc- , and pk- cells, obtained from the china center for type culture collection, were cultured at °c and % co in dulbecco's modified eagle's medium (invitrogen, usa) supplemented with % fetal bovine serum. prrsv strain wuh (genbank accession number hm . ), isolated from the brain of a pig suffering from "high fever" syndrome in china at the end of , was amplified and titrated as described previously ( ) . plasmids. the luciferase reporter plasmid isre-luc and the cdna expression constructs encoding porcine stat , stat , and irf with an n-terminal flag tag have been described previously ( ) . the luciferase reporter plasmid isre-luc contained ifn-stimulated response elements in the promoter of most of the isgs cloned upstream of the firefly luciferase reporter gene. prl-tk plasmid (promega) was used as an internal control for normalization of the transfection efficiency. the nsp gene of prrsv strain wuh was amplified and cloned into pcaggs-ha with an n-terminal ha tag. prrsv nsp mutants (h a, h a, and k a) were constructed by overlap extension pcr using specific mutagenic primers (available upon request) in a pcaggs-ha background. prrsv nsp and its mutants with an n-terminal ha tag were cloned into pgex- p- with a glutathione s-transferase (gst) tag in the n terminus. irf deletion mutants were also amplified by pcr and constructed in a pcaggs-flag background. pedv nsp was amplified from pedv strain aj ( ) and cloned into vector pcaggs-ha with a ha tag. pedv nsp mutants (h a) were constructed using pedv nsp as the template. all constructed plasmids were confirmed by sequencing. luciferase reporter gene assay. hek- t or pk- cells grown in -well plates were cotransfected with reporter plasmid isre-luc ( . g/well), prl-tk ( . g/well), and the indicated expression plasmids or an empty control plasmid. where indicated, cells were further treated with ifn-␣ (pbl assay science) ( , u/ml) h after the initial cotransfection. cells were lysed h later, and firefly luciferase and renilla luciferase activities were measured using a dual-luciferase reporter assay system (promega) according to the manufacturer's protocol. the relative levels of firefly luciferase activities were standardized as prl-tk activities. data are presented as means and standard deviations (sd). p values of Ͻ . were considered statistically significant, and p values of Ͻ . were considered highly statistically significant. rna extraction and quantitative real-time pcr. in brief, total rna was extracted from cells using trizol reagent (invitrogen), and rna ( g) was reverse transcribed into cdna using avian myeloblastosis virus reverse transcriptase (takara, japan). the cdna ( l of l) was then used in a sybr green real-time pcr assay (applied biosystems). the abundance of individual mrna transcripts in each sample was determined three times and normalized to that of glyceraldehyde- -phosphate dehydrogenase (gapdh) mrna. all quantitative pcr (qpcr) primers used in this study have been described previously ( ) . protein expression and purification. for protein expression, the recombinant prokaryotic expression plasmids encoding the n-terminally gst-ha-tagged wt nsp and mutant (h a, h a, k a) proteins were transformed into e. coli strain trans bl (de ) and cultured at °c in lb medium containing g/ml ampicillin. when the culture optical density at nm (od ) reached . to . , the cells were induced by the use of . mm iptg (isopropyl-d- -thiogalactopyranoside) for h at °c. for protein purification, the cultured cells were collected by centrifugation at , rpm for min and resuspended in phosphate-buffered saline (pbs) followed by disruption with ats ah- . the supernatant was filtered by the use of a filter with a . -mm-pore-size membrane and was then loaded onto a glutathione-sepharose b column (ge healthcare). to gain gst-free proteins, the harvested fusion proteins with an n-terminally gst tag were incubated with gst- c rhinovirus protease at a ratio of : for h at °c, and the cleavage products in the cleavage buffer were further separated by reloading onto glutathione-sepharose b column. finally, the target proteins were eluted by the use of ml cleavage buffer. the purified proteins were stored at Ϫ °c for detection of enzyme activity. enzyme activity assay. the rna substrate ( =- -carboxyfluorescein-da-ru-da-da- -carboxy-n,n,n,n-tetramethylrhodamine- =) was purchased from nanjing genscript company. the n-terminally ha-tagged prrsv nsp /h a/h a/k a proteins and the rna substrate were placed in a reaction buffer containing mm hepes (ph . ), mm kcl, and mm dithiothreitol (dtt) diluted with . % diethyl pyrocarbonate-treated water. the concentration of proteins used in the assay was m, and the substrate concentration was m. the endoribonuclease activity was monitored every min for min in a fluorescence plate reader, with excitation at a wavelength of nm and emission at a wavelength of nm. the results were analyzed by the use of graphpad prism software . . western blot analyses and subcellular fractionation. cells were grown in -mm-diameter dishes and harvested with lysis buffer (beyotime, china) plus nm phenylmethylsulfonyl fluoride (pmsf) and phosstop phosphatase inhibitor (sigma, usa). samples were then separated by sds-page and transferred to polyvinylidene difluoride membranes (millipore, usa) to determine the protein expression levels. overexpression of stat , stat , irf , and irf deletion mutants was evaluated using mouse monoclonal anti-flag antibody (macgene, china). the expression of prrsv nsp and its mutants was analyzed using mouse monoclonal anti-ha antibody (mbl, japan). rabbit polyclonal antibodies against irf (santa cruz, usa), stat (santa cruz, usa), stat (abclonal, china), phospho-stat (stat -y , cell signaling technology, usa), and phospho-stat (stat -y , abclonal, china) were used to detect the protein levels and phosphorylated forms of each of the respective endogenous proteins. monoclonal antibody (mab) against prrsv nsp was produced from hybridoma cells derived from sp / myeloma cells and spleen cells of balb/c mice immunized with recombinant nsp protein from prrsv strain wuh . the isotype of nsp -specific mab is mouse igg . the specificity of mab against prrsv nsp was confirmed by specific reactions performed with pcaggs-ha-nsp -transfected cells and prrsv-infected cells. to analyze the nuclear translocation of isgf , nuclear fractions were extracted from cells using an ne-per nuclear and cytoplasmic extraction reagents kit (thermo fisher scientific, usa) according to the manufacturer's protocol. the cytoplasmic and nuclear fractions were subjected to western blotting. successful isolation was assessed using rabbit polyclonal antibodies against heat shock protein (hsp ; proteintech, china) and parp (proteintech, china) with cytoplasmic and nuclear protein markers, respectively. coimmunoprecipitation and immunoblotting analyses. to test the interactions between proteins, hek- t cells or marc- cells from each -mm-diameter dish were lysed in radioimmunoprecipitation assay (ripa) lysis buffer containing mm tris-hcl (ph . ), mm nacl, % triton x- , % sodium deoxycholate, . % sds, nm pmsf, and phosstop phosphatase inhibitor (sigma, usa), and the protein concentration was measured and adjusted. for each immunoprecipitation, g of cell lysate protein was incubated with . g of the indicated antibody and l of protein aϩg agarose (beyotime, china) overnight at °c. the agarose beads were then washed three times with ml of lysis buffer. the precipitates were subjected to % sds-page and subsequent immunoblot analysis using the indicated antibodies. in some immunoblotting analyses, the horseradish peroxidase-conjugated goat anti-rabbit igg light chain (ipkine, usa) were used to eliminate the interference of the rabbit igg heavy chain. indirect immunofluorescence assay. marc- cells cultured on coverslips in -well plates were infected with prrsv at a multiplicity of infection (moi) of . . at h postinfection, the infected cells were mock treated or treated with recombinant human ifn-␣ (pbl assay science) ( , u/ml) for h. the cells were then fixed with % paraformaldehyde for min and immediately permeabilized using methanol (precooled at Ϫ °c) for min at room temperature (rt). after three washes with pbs, cells were incubated with % bovine serum albumin (bsa)-pbs overnight at °c and incubated with the primary antibody for h. the antibodies used were as follows: anti-phospho-stat (cell signaling technology, usa), anti-phospho-stat (cell signaling technology, usa), anti-irf (abclonal, china), and anti-nsp mab. anti-mouse igg (hϩl) antibody conjugated to alexa fluor or anti-rabbit igg (hϩl) antibody conjugated to alexa fluor was diluted to : for use as the secondary antibody, after which the cells were stained with ', -diamidino- -phenylindole (dapi; beyotime, nantong, china)-pbs for min. fluorescent images were acquired with a confocal laser scanning microscope (olympus fluoview ver. . , japan). porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system the ever-expanding diversity of porcine reproductive and respiratory syndrome virus the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins prrsv structure, replication and recombination: origin of phenotype and genotype diversity the structural biology of prrsv antiviral strategies against prrsv infection porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins the janus protein tyrosine kinase family and its role in cytokine signaling tyrosinephosphorylated stat and stat plus a -kda protein all contact dna in forming interferon-stimulated-gene factor how cells respond to interferons interferon-stimulated genes and their antiviral effector functions interplay between janus kinase/signal transducer and activator of transcription signaling activated by type i interferons and viral antagonism porcine deltacoronavirus nsp antagonizes type i interferon signaling by cleaving stat rotavirus nsp mediates degradation of interferon regulatory factors through targeting of the dimerization domain rotavirus nsp protein inhibits interferon-mediated stat activation structural basis of the inhibition of stat activity by sendai virus c protein nonstructural protein of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-beta induction a dimerization-dependent mechanism drives the endoribonuclease function of porcine reproductive and respiratory syndrome virus nsp interferon signalling network in innate defence structural biology of the arterivirus nsp endoribonucleases nonstructural protein (nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) promotes prrsv infection in marc- cells biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat ns of dengue virus mediates stat binding and degradation zika virus targets human stat to inhibit type i interferon signaling porcine bocavirus np negatively regulates interferon signaling pathway by targeting the dna-binding domain of irf the ebola virus interferon antagonist vp directly binds stat and has a novel, pyramidal fold the irf family transcription factors in immunity and oncogenesis stat nuclear trafficking mapping of nine porcine interferon regulatory factor genes distinct stat structure promotes interaction of stat with the p subunit of the interferon-alpha-stimulated transcription factor isgf two domains of isgf gamma that mediate protein-dna and protein-protein interactions during transcription factor assembly contribute to dna-binding specificity the viral innate immune antagonism and an alternative vaccine design for prrs virus the nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits nf-kappab signaling by means of its deubiquitinating activity the superimposed deubiquitination effect of otulin and prrsv nsp promoted the multiplication of prrsv an "old" protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses coronavirus nonstructural protein mediates evasion of dsrna sensors and limits apoptosis in macrophages early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication the emerging role of interferon regulatory factor in the antiviral host response and beyond the type i interferon-alpha mediates a more severe neurological disease in the absence of the canonical signaling molecule interferon regulatory factor mice deficient in stat but not stat or irf develop a lethal cd ϩ t-cellmediated disease following infection with lymphocytic choriomeningitis virus the human papillomavirus e oncoprotein abrogates signaling mediated by interferon-alpha varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms effects of adenovirus e a protein on interferonsignaling human cytomegalovirus inhibits ifn-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of ifn-alpha signal transduction porcine reproductive and respiratory syndrome virus nsp beta inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-alpha degradation porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat /stat nuclear translocation structural basis of stat recognition by irf reveals molecular insights into isgf function mir- b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the nf-kappab pathway complete genome sequence of porcine epidemic diarrhea virus strain aj isolated from a suckling piglet with acute diarrhea in china this work was supported by the major project of the national natural science foundation of china (grant ), the national basic research program ( ) of china (grant cb ), the national natural science foundation of china (grants key: cord- - eksm authors: pattyn, els; verhee, annick; uyttendaele, isabel; piessevaux, julie; timmerman, evy; gevaert, kris; vandekerckhove, joël; peelman, frank; tavernier, jan title: hyperisgylation of old world monkey isg in human cells date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: eksm background: isg is an ubiquitin-like protein, highly induced by type i interferons. upon the cooperative activity of specific ubiquitinating enzymes, isg can be conjugated to its substrates. increasing evidence points to a role for protein isgylation in anti-viral and anti-tumoral defense. principal findings: we identified isg from old world monkeys (owm) as a hyper-efficient protein modifier. western blot analysis visualized more efficient conjugation of owmisg relative to huisg in human (hu), monkey and mouse (mo) cell-lines. moreover, the substrates of owmisg identified upon tandem affinity purification followed by lc-ms/ms identification largely outnumbered these of huisg itself. several ubiquitin-conjugating enzymes were identified as novel isgylated substrates. introduction of a n d mutation in huisg improved its isgylation capacity, and additional q k/t a/d n mutations yielded a huisg variant with an isgylation efficiency comparable to owmisg . homology modeling and structural superposition situate n in the interaction interface with the activating enzyme. analysis of the ube l residues in this interface revealed a striking homology between owmube l and huube , the activating enzyme of ubiquitin. in line with this observation, we found efficient activation of agmisg , but not huisg or moisg , by huube , thus providing a likely explanation for owm hyperisgylation. conclusions: this study discloses the poor conjugation competence of huisg compared to owmisg and maps the critical determinants for efficient conjugation. hyperisgylation may greatly assist isgylation studies and may enhance its function as positive regulator of interferon-related immune responses or as anti-tumoral modulator. type i interferons (ifn)s are involved in host defense mechanisms, particularly against viral infections. they induce a so-called ''antiviral state'' by inducing both cytosolic and nuclear events. ifn stimulated gene (isg ) is an ubiquitin (ub)-like molecule (ubl), highly induced upon both type i and type ii ifn treatment [ ] . it is expressed as a kda protein, containing ub domains and a c-terminal oligopeptide (eg. octapeptide in human, hexapeptide in mice). maturation of isg includes n-terminal met excision [ ] and removal of the c-terminal peptide giving a kda protein [ ] . maturated isg can then be conjugated via an isopeptide binding to the e-amino group of a lys residue in the target protein [ ] . alternatively, the processed kda isg molecule can be secreted and exerts immunoregulatory functions on peripheral blood lymphocytes [ ] . conjugation of ub(l) requires the cooperative activity of at least enzymes. the ubiquitination cascade is initiated by an ub-activating enzyme (termed uba, ube or e ) adenylating the cterminus of ub(l), thereby forming an acyl-phosphate linkage with amp. the catalytic cys residue in ube subsequently attacks this high energy bond, forming a thiolester bond to the c-terminal gly of ub(l). in humans, ub molecules are found to be activated by ube (also known as a s ) [ ] or ube l (also named uba ) [ , ] , isg by ube l [ ] , sumo by aos-uba [ ] and nedd by appbp -uba [ ] . ub(l) molecules thiolester-linked to its ub-activating enzyme are transferred to a ub-conjugating enzyme (termed ubc or e ), also by a thiolester linkage on a cys residue. ubch has been identified as a major ub-conjugating enzyme effecting isg conjugation [ , ] . around proteins are recognized as ub-ligases or e s. roughly, they can be discerned as ring (really interesting new gene)-finger proteins, acting as a molecular scaffold, and hect (homologous to e -ap c-terminus)-domain proteins, which also exert a catalytic contribution. ub-ligases confer specificity, and place the ub or ubl molecule in close proximity to the lys residue of the substrate. the formation of polyubiquitin chains is a process mediated by the ub-conjugating enzyme together with the ub-ligase. recently, the ifn-induced herc has been identified as an isg e -ligase in human cells [ ] . the estrogen-response finger protein (efp), also an ifn-induced protein, functions as an e -ligase for isgylation of - - d [ ] . definitely, these recently discovered isg -ligases are only the onset of a more extensive list. as ub, most ubls are synthesized as inactive precursors, being processed by de-ubiquitinating enzymes (dubs) exposing the mature protein with a c-terminal gly residue. dubs not only exert their function by protein processing, they also have a function in removal of the ub(l) from their substrate. ub specific protease (usp ), usp (also named isopeptidase t), usp (isot ) and usp have been identified as proteases with a dual specificity for ub and isg [ , ] . usp (also named ubp ) specifically cleaves isg [ ] . of note, usp also competes with jak for binding to the type i ifn receptor ifnar [ ] . isg is upregulated upon viral infection. its role in antiviral defense is underscored by viral mechanisms to counteract isg function. for example, the influenza b non-structural ns b protein binds isg , thereby preventing its association to ube l [ ] . also the papain-like protease of the severe acute respiratory syndrome (sars) coronavirus, counteracts isg functioning by removing it from its substrate [ ] . the hepatitis c virus ns / a protease cleaves ifn promoter-stimulator- (ips- ) leading to reduced isg expression and conjugation [ ] . in a more direct way, ectopic expression of isg in cells lacking a functional ifn response reduces newcastle disease virus and influenza virus replication [ ] , and also overcomes fatal intracerebral infection in ifnar / mice by sindbis virus [ ] . isg knock-out mice proved to be more sensitive to influenza, herpes and sindbis viral infection [ ] . an inverse dose-response correlation of exogenously expressed isg and release of human immunodeficiency virus (hiv) virions is also found and suppression of isg expression by sirna counteracts ifn-mediated inhibition of hiv virion release [ ] . the precise mode of action of isg is not yet established. isgylation is believed to counteract ubiquitination by competing for the same lys in substrates [ ] . studies with ubch and ubch showed that isgylation of these e s hampered their ability to form a thiolester intermediate with ub [ , ] . this competition may protect substrate proteins from proteasomal degradation. we here report the surprising finding that isg from old world monkeys is a far more efficient protein modifier than its human counterpart in human, monkey and mouse cells. we identified novel isgylation substrates and mapped the critical residues in this isgylation process. this hyperisgylation competence of agmisg was validated by an alternative activation mechanism. these findings will be useful for a better understanding of isg biology. viral resistance strongly varies between species. in this respect, old world monkeys (owm)s receive much attention by their remarkable hiv resistance. they are distinguished from apes (here represented by the chimpanzee) in that most have tails and are distinguished from new world monkeys (not included in this study) in that their tails are never prehensile. we compared isg sequences from hominid origin with those of owm, which diverged some million years ago, and of murine origin, which diverged from primates approximately million years ago [ ] . isg from different origins were aligned to two ub molecules ( figure ). the nand c-terminal part of isg share respectively and % sequence identity with ub. the isg variability largely exceeds what can be expected by genetic drift alone [ ] . we isolated isg cdnas of humans (allelic variant with n at position and s at position ) (homo sapiens, hereafter huisg ), chimpanzee (pan troglodytes, cpzisg ), african green monkey (cercopithecus aethiops, agmisg ), rhesus monkey (macaca mulatta, rhmisg ) and mouse (mus musculus, moisg ). all were cterminally cloned after a flag recognition tag. huisg and cpzisg only differ by one amino acid (respectively a ser and asn residue at position ) and represent hominid isg (homisg ). agmisg and rhmisg differ from each other by two amino acids (respectively a ser and asn residue at position and arg and lys at position ), and are both part of the old world monkeys (hereafter referred to as owmisg ) (see table ). total isgylation patterns were visualized by western blot analysis on total cell lysates. as can be seen in figure a , agmisg and rhmisg outperform homisg and moisg in its isgylation capacity in human hek t cells. moisg also conjugates more favorably to human proteins compared to huisg , albeit not to the same extent as owmisg . to rule out the possibility that conjugation of ectopic huisg could be hindered by competition with endogenous isg , the same blot was stripped and reprobed with an antibody recognizing hu and owmisg , showing essentially the same results (data not shown). similar findings were obtained in monkey cos cells and mouse n cells (resp. figure b and figure c ). note that all these experiments were performed without extra stimuli (e.g. ifn treatment) or co-transfection of any ubiquitinating enzyme meaning that in all tested cell-lines, target isgylation by agmisg could be achieved by endogenously available ubiquitinating enzymes. figure . isg orthologues show a low degree of amino acid conservation. isg from different species were aligned with two molecules of ubiquitin using clustalx [ ] . sequence conservation is visualized using the amas server [ ] with a conservation threshold of . residues with a conservation above this threshold are shown on a grey background, residues that are identical in all sequences have a black background. doi: . /journal.pone. .g we wanted to address whether the observed differences in isgylation capacity was only quantitative or also qualitative. therefore, tandem affinity purification (tap) experiments were performed to identify isgylated substrates by hu and agmisg . to this end, a tandem proteina binding domain and flag-tag, separated by a tobacco etch virus protease cleavage domain was fused n-terminally to hu or agmisg . human fibroblast ftgh cells were stably transfected with these constructs, and cell clones expressing tap-tagged huisg or tap-tagged agmisg with comparable expression levels were selected (see figure s ). to rule out experimental variation, this experiment was also performed in transiently transformed hekt cells with either tap-tagged huisg or agmisg . as shown in table , the tap experiments identified substantially more isgylation substrates of agmisg compared to huisg in human celllines. details of the identified peptides can be found in table s . in addition to an overlap with the already described isg substrates [ ] [ ] [ ] [ ] [ ] , we here report the ub-conjugating enzymes ubch , ubch are ubch as novel isgylation substrates of agmisg . the cdnas of the ub-conjugating enzymes ubch , , , , , and were isolated by rt-pcr from ftgh cells and linked to a c-terminal v -tag. these constructs were transfected in hekt cells together with either a mock construct, huisg or agmisg . ubch was included as a control, as it was neither in our experiments nor in others identified as a substrate for isgylation. western blot analysis on total cell lysates containing b-me confirmed isgylation of ubch , h and h with agmisg but not with huisg , as seen by a kda shift upon staining with anti-v ab detecting the ectopic expressed ubch proteins (figure a ,b and figure s a ). in addition, co-immunoprecipitation experiments were performed by binding isgylated proteins through isg 's flag-tag to an anti-flag affinity gel. also here, the isgylation of ubch , h and h by agmisg could be established ( figure c ). worth mentioning, when the co-immunoprecipitated samples were obtained in a more concentrated way, a faint band of co-immunoprecipitated ubch and ubch could be seen with huisg ( figure s b ), hinting at a qualitative rather than quantitative difference in isgylation between hu and agmisg . of note, all the experiments were performed without cotransfection of ube l and/or ubch or ifn stimulus. previous studies using western blot analysis in hekt cells revealed hu or moisg conjugation to substrates such as ubch only upon co-transfection of at least ube l -and generally also ubch/m or upon ifn stimulation. moreover, in most cases an additional immunoprecipitation or pull-down purification step was required [ , , [ ] [ ] [ ] [ ] . alternatively, experiments were performed in usp -deficient murine embryonic fibroblasts [ , ] . we next replaced the differing amino acids in huisg by their agmisg counterparts (see figure and ). four of these residues (i.e. residues , , and ) are situated near the interaction interface between isg and its activating enzyme, ube l, predicted by narasimhan et al. [ ] (red highlighted in figure ). the n s and s n allelic variants of huisg were also included. both conjugation to various ubchs and total isgylation patterns of different huisg mutants were tested. the effect of mutating huisg residues situated near the predicted ube l interface and the different allelic variants on conjugation to ubch proteins is shown in figure a and s a. strikingly, the single huisg n d variant displayed a greatly enhanced isgylation efficiency. no effect was seen for all other mutants, including the n s or s n human allelic variants. note that also in this experiment the cell lysates were boiled in sds loading buffer containing b-me. the slower migrating band differs kda from the unconjugated form of the ubchs, and is thus the result of isg bound by an isopeptide linkage, not by a thiolester bond. as with agmisg , no co-transfection of any activating or conjugating enzyme was required to visualize its conjugation by western blot analysis on total cell lysates in hekt cells. a more detailed study including all differing residues (and also the n e variant as it occurs in mice), demonstrated that only mutation at position affected the total isgylation pattern. however, the effect of this mutation is only intermediary as compared with agmisg (see figure b ). we next combined the huisg n d variant with additional mutations. as shown in figure c , mutation of d n and qit - kia in the huisg n d variant further enhanced its isgylation in human hekt cells. finally, the triple huisg mutant n d, d n and qit - kia displayed an isgylation efficiency comparable to the monkey orthologues (see figure d and s b). the interaction interfaces of nedd bound to its activating enzyme, appbp -uba , [ ] and sumo bound to sae -sae [ ] have recently been mapped and show a remarkable high degree of similarity. we built a similar homology model for the ube l-isg complex (figure a ), based on the crystal structure of the appbp -uba -nedd -atp complex [ ] . this superposition brings the critical residue in isg in the interaction interface with ube l. therefore, we compared the sequences of hu and owmube l in their predicted interfaces with isg and found an interaction region displaying significant substitutions (i.e. the sequence -gtsgtwg- in huube l corresponding to -gtlgtrg- in owmube l). residue w in huube l juxtaposes to isg n (figure a ). this residue is substituted with an arg residue in owmube l. strikingly, the corresponding sequence in owmube l shows high similarity with huube ( figure b ). this observation prompted us to test whether agmisg could be activated by huube . we performed an in vitro assay analyzing the loading of either huisg or agmisg with huube or huube l. thiolester formation of both isg orthologues with huube l was observed, as expected. importantly, as anticipated from the sequence similarity, huube was also found to be able to form thiolester bonds with agmisg , whilst no loading of huisg could be seen (figure a ). the thiolester nature of the bond was verified by addition of b-me. western blot analysis evaluating the total isgylation pattern further confirmed this alternative activation pathway for agmisg ( figure b for the experiment in hekt cells, figure c in cos cells). the cells were transfected with combinations of an empty vector or vectors encoding huube l, huube and ubch together with flagtagged huisg , agmisg or moisg . visualising the isgylation pattern on the different samples only observed significantly enhanced isgylation by hu and moisg upon ectopic expression of ube l. a beneficial effect on hu/ moisgylation efficiency upon ectopic expression of ube was minimal. by contrast, even with the higher basal isgylation level of agmisg , overexpression of both ube l and ube were able to further markedly enhance its conjugation capacity. the isg gene emerged upon the duplication of an ub dimer and insertion in an ifn-controlled area [ ] . in sharp contrast with ub itself, of which of the amino acid are invariant between animals, plants and fungi [ ] , isg is not well conserved between species [ ] . this variability in isg sequence may indicate a redundant function of isg . alternatively, it could point towards a role for isg in the immune system, since high interspecies sequence divergence is very often associated with genes involved in host defense. organism-specific niche occupation may lead to distinct repertories of invading pathogens causing species-specific pressures for adaptation of the host immune system [ , ] . this increased evolutionary rate in immune genes may be further enhanced by the high mutation rates of the pathogens [ ] . in line with the assignment of a specific role for isg in host defense, naturally occurring loss-of-function isg mutants have not yet been observed sofar, whereas a duplication of the isg gene has been seen in crucian carp [ ] . although the precise function of isg remains enigmatic, several lines of evidence point to its role as an antiviral agent. isg is one of the most prominently induced genes upon type i ifn treatment, and several viruses such as influenza b and sars have developed mechanisms to counteract isg function [ , ] . isg was also found to be critical for the ifnmediated inhibition of hiv replication and release [ ] . moreover, although isg knock-out mice were initially found to be as vulnerable to viral infections as their wild type countermates [ ] , a subsequent study discovered isg knock-out mice to be more vulnerable to influenza, herpes and sindbis viral infection [ ] . a key finding of this report is the significant difference in isgylation capacity between isg orthologues. in human cells, protein modification by isg of owm greatly outperforms that of hu and moisg . mutation of the residues in huisg to the corresponding residues in the owm orthologue mapped amino acid as a critical residue for isgylation. the occurrence of an acidic residue (preferably an asp in human cells and glu in mouse cells) at this position greatly enhances its isgylation capacity. the asp residue at position in owmisg also occurs in sheep and cows. in rodents and fish, this residue is a glu residue. the incidence of the unfavorable asn residue at position for isg modification in huisg is also found in chimpanzee and dog isg . however, notwithstanding the greatly enhanced isgylation capacity by mutation of the single asn residue in huisg to an asp residue, the overall isgylation pattern in human hekt cells was still intermediary compared to agmisg . additional mutations were created in the huisg n d mutant. mutation of d n and qit - kia further increased its isgylation capacity. the triple huisg mutant n d d n qit - kia proved to be as effective as agmisg in conjugating target proteins in hekt cells. the use of this hyperefficient isg variant may help to unravel the physiological function of isg . the ub(l) modification process is initiated by its dedicated activating enzyme, a crucial step which is also described to dictate selectivity. ub-activating enzymes are composed of three modular domains: an n-terminal domain which targets atp and ub(l)binding (residues - in ube l), a second domain harboring the catalytic cys residue (embedded in residues - ) and an c-terminal ubl-fold domain (residues - ), which selects for and binds to the cognate ub-conjugating enzymes [ , ] . based on interaction interfaces of nedd bound to appbp -uba [ ] , narasimhan and colleagues predicted hot spot residues on isg constituting the interface with ube l. three of these residues show a high degree of conservation among the different ub(l)s (i.e. r , e & r in isg ), other residues are presumed to confine specificity for ube l (r , k , w & f ) [ ] . we built a similar homology model for the ube l, using the crystal structures of the appbp -uba -nedd -atp complex. structure superposition shows that residue corresponds to l in nedd (figure a) , a well-conserved residue that is also found in ub (figure ). mutation of l in ub significantly reduced its target conjugation ability [ ] , and in yeast this residue proved to be essential for viability [ ] . this suggests that residue in isg and l in ub have a similar important role in conjugate formation. l in nedd is part of a hydrophobic patch (l , i , v ) that interacts with v and y of uba , embedded in the two b-sheets preceding its cterminal domain. mutation of these residues greatly reduced nedd adenylation and consequently conjugation [ ] . however, residues l , i and v in nedd correspond with the nonhydrophobic residues n , t and n in isg , suggesting different types of interaction in this region. the two uba bstrands correspond to the region - in the ube l model, but the alignment in this region is too poor to allow reliable modeling of the actual interactions. moreover, residues -lrl- , which are part of the c-terminal region of isg juxtapose to n (figure ). this region aligns with -lrl- in ub which was described to be of high importance for ube binding affinity and for substrate specificity [ ] . our model suggests that residue makes a contact with r in owmube l, which lies in the n-terminal domain, known to select for a specific ubl [ , ] . in huube l, this arg is replaced by a trp. strikingly, the sequence -gtlgtrg- in owmube l is very similar to the corresponding region -gtlgtkg- in huube (see also figure b ). based upon this observation, we experimentally demonstrated that huube could efficiently activate agmisg , in contrast to hu and moisg . this non-redundant role for ube l in activating moisg is also in line with the loss of moisg conjugation in ube l knock-out mice [ ] . unlike ube l, which is ifn-inducible and has a tissue and cell-line specific expression profile [ ] , ube is ubiquitously expressed. taken together, our findings support a role for residue in isg as a hot spot residue in the interface with its activating enzyme, thus providing an explanation for the hyperisgylation seen for owmisg . recently, with the discovery of ube l (uba ) as the activating enzyme for fat and as a second activating enzyme for ub [ , ] , the theory of an activating enzyme harboring unilateral assignment for a specific ub(l) was overthrown. here, we describe promiscuity of ube for owmisg activation. this is the first report of isg being activated by another activating enzyme than ube l. moreover, to the best of our knowledge, this is also the first report of an ublother than ubiquitin-being activated by ube . in contrast, homology modeling and structural superpositions do not predict a direct role for residue d or residues qit - of isg in the activation step (figure and a) . the d residue is part of a ridge of negatively charged residues along the isg molecule [ ] . the function of this ridge is unclear. d in huisg corresponds to a negatively charged residue in ub, sumo, nedd , rub , apg (all asp residues) and urm (occupied by a glu residue). models based on the appbp -uba -nedd -atp complex or the sae -sae -sumo-mg-atp complex, do not predict direct contact of residue in isg with ube l. the same hold true for residues - . this suggests that mutations figure . residue in isg is situated in the predicted contact area with its activating enzyme. (a) isg (red ribbons) overlaid to nedd (green ribbons) bound to its activating enzyme appbp -uba (grey ribbons). residue (dark blue) of isg , makes contact with residues in the two b-strands (shown in brown) of the activating enzyme that precedes its c-terminal domain. k in uba (orange) is located very close to n , and corresponds to w in human ube l. the other residues of isg with an important effect on total isgylation, situated at positions (yellow) and - (cyan blue), do not make contact with ube l and are likely to be involved in another feature of the isgylation process. (b) huube l was aligned with huube and rhmube l (ensembl peptide sequence en-smmup ). the region - in huube l shows two substitutions (s l and w r, red coloured) in the corresponding region of rhmube l, which better resembles the corresponding region in huube (k ). doi: . /journal.pone. .g at position - and influence total isgylation by a different mechanism than mutation at position . this must occur later in the isgylation process, since no effect of these mutations can be observed in the absence of the n d mutation. of note, notwithstanding their position on different ub domains in isg , residues , and are situated at the same face of the molecule (see figure ) close to the electronegative ridge. as the total protein isgylation results from the balance between conjugation and deconjugation, mutation of qit - to kia and d to n in huisg n d conceivably could affect recognition or activity by (a) discriminate dub(s), retaining the targeted protein in the isgylated state. in line with this hypothesis, a role has been described for the n-terminal ub fold of isg in the efficiency of dub recognition/activity of the sars coronavirus papain-like protease [ ] . in this study we also present the ub-conjugating enzymes ubch , h and h as new isgylation targets. ubch (ube c) acts as an ubch for the anaphase promoting complex (apc), promoting cyclina degradation and mitotic exit. during the g phase, ubch is auto-ubiquitinated allowing reaccumulation of cyclina and entry in the s phase [ ] . ubch is often referred to as the cancer-related ubch as it is markedly overexpressed in the majority of cancerous cell lines [ ] . recently, also ubch (e - k or hip ) has been identified as an apc-dependent ubch promoting ub k -linked chain extensions on pre-attached ubs [ ] . ubch (ube t) has recently been identified as the ubch essential to ubiquinate fancd , which needs to be ubiquitinated in order to bind brca and take part in the dna repair process. ubch depleted cells have abnormal chromosomes, characteristic for in vitro assay revealing the capability of agmisg to form thiolester bonds with ube , contrary to huisg . . mm ubch was incubated with nm of either ube or ube l, and . mm of either mature hu-or agmisg under conditions as described in materials and methods. the samples were divided in two aliquots; and were subjected to sds-page under non-reducing or reducing conditions. the proteins were stained with irdye blue protein stain and visualized using the odyssey infrared imaging system. the extra band representing thiolester formation between ube and agmisg is flanked by two asterisks. these thiolester bonds are disrupted by the addition of reducing agent (here b-me). (b) ube can efficiently activate agmisg , but not hu or moisg . hekt cells were transfected with an empty vector, or combinations of vectors encoding huube l, huube and ubch together with huisg , agmisg or moisg as indicated. the total isgylation patterns were evaluated by western blot analysis using the flag tag of the isg proteins. fanconi anemia [ , ] . these findings may help explain the role for isg in anti-tumor defense. in conclusion, we report that owmisg more efficiently isgylates proteins compared to human isg . it remains to be determined whether this reflects merely quantitative or also qualitative differences in the role of isg among species. as isg is implicated in antiviral protection against various viruses including sars, influenza, hepatitis c and hiv, these differences in isg conjugation efficiency may help explain human sensitivity to various viruses. human isg (allelic variant with n at position and s at position ) was isolated from cdna of hek t cells, african green monkey isg from cdna of vero cells and mouse isg from cdna of l sa cells. all huisg mutants as well as the other human allelic variants (n s and s n) were created through site-directed mutagenesis (stratagene). chimpanzee isg was obtained by site-directed mutagenesis of huisg (s n mutation), rhesus monkey isg was obtained by consecutive site-directed mutations on agmisg (s n and k r mutation). all cell-lines were cultured in a % co humidified atmosphere at uc and grown in dulbecco's modified eagle's medium (invitrogen) with % fetal calf serum (cambrex corp.). for standard isgylation experiments in hek t cells, , cells were seeded the day before transfection in -well plates and transfected for h using a standard calcium phosphate precipitation procedure. typically . mg of isg variant, if indicated supplemented with . mg e and . mg e or, applicable supplemented with . mg substrate was used in a total volume of ml dna/capo -transfection mixture. fugenehd (roche) was used for transfection in cos cells and lipofecta-mine (invitrogen) for transfection in n cells. since fugenehd induces a minimal ifn response, this reagent was exceptionally used for the hek t transfections in figure b . however, no differences could be seen between this transfection reagent and the calcium phosphate precipitation method regarding the isgylation difference between hu and agmisg . tap purification experiments were performed in ftgh cells stably expressing either hu or agmisg and hekt cells transiently expressing either hu or agmisg with an n-terminal tap-tag. single colonies of the ftgh cells were selected upon co-transfection of the isg constructs with a puromycin resistance marker and selection on mg/ml puromycin. in order to check the expression levels of the tap-isg proteins compared to the endogenous isg levels, the different stable cell-lines were stimulated for h with ifnb (peprotech) prior to western blot analysis. two days after transfection, cells were lysed with ml sds loading buffer ( mm tris hcl ph . , % sds, % glycerol, % b-me, . % brome phenol blue) and homogenized on a qiashredder mini spin column (qiagen). ml of the boiled lysate was loaded on a sds-polyacrylamide gel. blotting efficiency was checked using ponceau s staining (sigma). the precision plus protein standard all blue (biorad) was used as molecular weight marker. flag-tagged proteins were revealed using a / dilution of anti-flag m mouse monoclonal ab (sigma), v -tagged proteins by / dilution of anti-v mouse monoclonal ab (invitrogen). hu and owmisg could be detected by an anti-human isg rabbit polyclonal ab (abcam). human isg was also detected with the anti-human isg mouse monoclonal ab, a generous gift from dr. e. borden. anti-human actin rabbit polyclonal ab (sigma) / diluted was applied as control loading. either goat anti-mouse irdyeh cw or anti-rabbit irdyeh (li-corh biosciences) was used as secondary ab. targeted proteins on the blots were visualized using the odysseyh infrared imaging system (li-corh biosciences). the ftgh or hek t cells expressing either tap-tagged agmisg or huisg were lysed in cell lysis buffer ( mm tris-hcl ph , % glycerol, % np , mm nacl, mm naf, mm zncl , mm na vo , mm egta, complete tm protease inhibitor cocktail (roche)). the insoluble fraction was spun down and the supernatant was incubated with igg sepharose (amersham biosciences) overnight. the beads were washed three times with washing buffer ( mm tris-hcl ph . , % glycerol, . % np , mm nacl) and twice with tev (tobacco etch virus) protease cleavage buffer ( mm tris-hcl ph , mm nacl, . % np , . mm edta) and were then incubated with tev protease in tev protease cleavage buffer for hours. the beads were then spun down and the supernatant was incubated with anti-flag agarose (sigma) in tev protease cleavage buffer ( mm tris-hcl ph , mm nacl, . % np ) for to hours. the anti-flag agarose beads were washed three times with washing buffer and incubated with mg/ml flag peptide in flag elution buffer ( mm tris-hcl ph . , % glycerol, mm nacl) for min to elute for the flag-tagged proteins. the resulting peptide mixture was precipitated by addition of tca to a final concentration of %. it was incubated overnight, centrifuged and washed with ice-cold acetone containing . n hcl and dried. pellets were re-dissolved in ml mm nh hco (ph . ) containing m ureum for min periodic vortexing, ml mm nh hco (ph . ) was added in aliquots to lower the final concentration of ureum to m. the resulting peptide mix was digested in solution by trypsin and applied for nano-lc-ms/ms analysis as described before using a waters q-tof mass spectrometer [ ] or a bruker esquire hct ion trap mass spectrometer [ ] . the generated peptide fragmentation spectra were searched using the mascot database search engine (http://www. matrixscience.com) in the swissprot database (taxonomy was set to human). the following mascot parameters were set. the enzyme setting was trypsin with a maximum of missed cleavage allowed. variable amino acid modifications that were allowed are acetylation (n-term), carbamylation (lys and n-term), deamidation (asn and gln), formation of pyroglutamate (n-terminal gln), oxidation of met to its sulfoxides and propionamide modification of cys. allowed peptide and fragment ion mass tolerance were . da (q-tof) or . da (ion trap). mascot's instrument setting was ''esi-quad-tof'' (q-tof) or ''esi-trap'' (ion trap) for calculating theoretical peptide fragmentation spectra. following database searching, only spectra that exceeded the corresponding mascot's threshold score for identify (set at the % confidence level) are here reported. the matching estimated false positive discovery rate is well acceptable and is only between to % at the individual spectrum level as previously assessed [ ] . co-immunoprecipitation procedure hek t cells were transfected with the indicated expression vectors. cleared lysates (modified ripa lysis buffer: mm nacl, mm tris-hcl ph , , % sds, mm edta, % np , , % doc, complete tm protease inhibitor cocktail (roche)) were prepared two days after transfection. the samples were incubated with anti-flag m affinity gel (sigma). after immunoprecipitation, sds-page and western blotting, interactions were detected using an anti-v antibody (invitrogen) production and purification of hu and agmisg proteins mature isg (with removed c-terminal peptide) was cloned in the ptyb vector and purified with the impact tm procedure (intein mediated purification with an affinity chitin-binding tag) (new england biolabs) when the cultures reached an od of . , isopropyl b-d-thiogalactopyranoside (iptg) was added to a final concentration of . mm, and were grown at uc overnight. the next day, cells were removed from the medium by centrifugation. the cell pellets were resuspended in ice-cold cell lysis buffer ( mmtris ph . , mm nacl) and disrupted mechanically by sonication. after removal of cell debris by centrifugation, the clarified lysate was loaded onto a chitin column (equilibrated with volumes of the column buffer ( mm tris, mm nacl, mm edta, ph . ). three bed volumes of the cleavage buffer ( mm tris, mm edta, mm dtt, ph . ) were loaded onto the column, and incubated overnight. the isg protein was eluted from the column using twice ml column buffer and dialysed against times l pbs overnight. the concentration of the protein was determined by the bradford assay. ml was loaded on a % sds page gel and silver stained to check the quality and purity of the isg proteins. purified recombinant ube , ube l, ubch , ubch and ubch were purchased (boston biochem). the in vitro thiolester reactions were performed based on the isg conjugation initiation kit (boston biochem). all samples were prepared in ml containing mm hepes ph . and mm nacl. the concentration of the recombinant proteins is indicated in the figure legend figure s the selected stable ftgh cell clones express comparable levels of the tap-tagged isg orthologues. ftgh cells were stably transfected with the indicated tap-tagged constructs as described in materials and methods. four clones were selected for the tap experiments, clones expressing taptagged huisg and clones expressing tap-tagged ag-misg . one cell-line stably expressing tap-tagged prmt (protein arginine n-methyltransferase) was used as a control (lane ). the different cell-lines were seeded at the same density in well plates. h after seeding, the cell-lines were stimulated with ifnb( ng/ml). one non-stimulated (ns) control was included (lane ). h after ifnbtreatment, cell lysates were prepared and separated by sds page. (a) western blot using anti-flag ab, revealing the tap-tagged constructs. open arrow indicates the ectopic expressed tap-tagged isg constructs. the prmt control construct is a bigger protein. (b) western blot using anti-huisg ab (gift of dr. e borden). open arrow indicates the ectopic expressed tap-tagged isg constructs (note the weaker cross-species recognition of agmisg by the antibody). closed arrow indicates the induced endogenous isg as a result of the ifn stimulation. interferon induces a -kilodalton protein exhibiting marked homology to ubiquitin isg : a ubiquitin-like enigma precursor processing of pro-isg /ucrp, an interferon-beta-induced ubiquitin-like protein the interferon-inducible -kda ubiquitin homolog conjugates to intracellular proteins immunoregulatory properties of isg , an interferon-induced cytokine ubiquitin-activating enzyme. mechanism and role in protein-ubiquitin conjugation ube l , a novel e enzyme specific for ubiquitin dual e activation systems for ubiquitin differentially regulate e enzyme charging influenza b virus ns protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg protein the ubiquitin-like protein smt p is activated for conjugation to other proteins by an aos p/uba p heterodimer identification of the activating and conjugating enzymes of the nedd conjugation pathway the ubch ubiquitin e enzyme is also the e enzyme for isg , an ifn-alpha/ beta-induced ubiquitin-like protein interferoninducible ubiquitin e , ubc , is a conjugating enzyme for protein isgylation herc , an interferon-induced hect e enzyme, is required for conjugation of isg in human cells the interferon-inducible ubiquitin-protein isopeptide ligase (e ) efp also functions as an isg e ligase specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins screen for isg -crossreactive deubiquitinases ubp (usp ) specifically removes isg from conjugated proteins ubp is a novel regulator of interferon signaling independent of its isg isopeptidase activity the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme viral and therapeutic control of ifn-beta promoter stimulator during hepatitis c virus infection mx genes show weaker primary response to virus than other interferon-regulated genes identification of interferon-stimulated gene as an antiviral molecule during sindbis virus infection in vivo ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses innate antiviral response targets hiv- release by the induction of ubiquitin-like protein isg isg -dependent regulation link between the ubiquitin conjugation system and the isg conjugation system: isg conjugation to the ubch ubiquitin e enzyme isg modification of ubiquitin e ubc disrupts its ability to form thioester bond with ubiquitin evidence for higher rates of nucleotide substitution in rodents than in man proteomic identification of proteins conjugated to isg in mouse and human cells identification and herc -mediated isgylation of novel target proteins human isg conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways herc is an ifn-induced hecttype e protein ligase that mediates type i ifn-induced isgylation of protein targets high-throughput immunoblotting. ubiquitiin-like protein isg modifies key regulators of signal transduction crystal structure of the interferon-induced ubiquitin-like protein isg the structure of the appbp -uba -nedd -atp complex reveals the basis for selective ubiquitin-like protein activation by an e structures of the sumo e provide mechanistic insights into sumo activation and e recruitment to e insights into the ubiquitin transfer cascade from the structure of the activating enzyme for nedd ubiquitin-conserved protein or selfish gene? ubia, the major polyubiquitin locus in caenorhabditis elegans, has unusual structural features and is constitutively expressed of mice and not men: differences between mouse and human immunology divergent and convergent evolution of nk-cell receptors evolutionary process and the ecology of human immune function the innate immune response to grass carp hemorrhagic virus (gchv) in cultured carassius auratus blastulae (cab) cells isg , an interferon-stimulated ubiquitin-like protein, is not essential for stat signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus structural basis for recruitment of ubc by an e binding domain in nedd 's e surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting distinct functional surface regions on ubiquitin site-directed mutagenesis of ubiquitin. differential roles for arginine in the interaction with ubiquitin-activating enzyme ube l and protein isgylation are not essential for alpha/beta interferon signaling involvement of ube l in isg conjugation during retinoid-induced differentiation of acute promyelocytic leukemia selectivity in isg and ubiquitin recognition by the sars coronavirus papain-like protease autonomous regulation of the anaphasepromoting complex couples mitosis to s-phase entry ubch is the cancer-related e ubiquitin-conjugating enzyme sequential e s drive polyubiquitin chain assembly on apc targets ube t is the e in the fanconi anemia pathway and undergoes negative autoregulation fanconi anemia and ubiquitination chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than escherichia coli proteins a complex interaction pattern of cis and socs with the leptin receptor improved recovery of proteome-informative, protein n-terminal peptides by combined fractional diagonal chromatography (cofradic) the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools protein sequence alignments: a strategy for the hierarchical analysis of residue conservation we are greatly indebted to dr. ernest borden for the very generous gift of anti-huisg antibody. key: cord- -kywhulpc authors: xu, cheng; evensen, Øystein; munang’andu, hetron mweemba title: a de novo transcriptome analysis shows that modulation of the jak-stat signaling pathway by salmonid alphavirus subtype favors virus replication in macrophage/dendritic-like to-cells date: - - journal: bmc genomics doi: . /s - - - sha: doc_id: cord_uid: kywhulpc background: the janus kinase (jak) and signaling transducer activator of transcription (stat) pathway mediates the signaling of genes required for cellular development and homeostasis. to elucidate the effect of type i ifn on the jak/stat pathway in salmonid alphavirus subtype (sav ) infected macrophage/dendritic like to-cells derived from atlantic salmon (salmo salar l) headkidney leukocytes, we used a differential transcriptome analysis by rna-seq and the kyoto encyclopedia of genes and genomes (keggs) pathway analysis to generate a repertoire of de novo assembled genes from type i ifn treated and non-treated to-cells infected with sav . results: concurrent sav infection with type i ifn treatment of to-cells suppressed sav structural protein (sp) expression by log( ) at days post infection compared to sav infection without ifn treatment which paved way to evaluating the impact of type i ifn on expression of jak/stat pathway genes in sav infected to-cells. in the absence of type i ifn treatment, sav downregulated several jak/stat pathway genes that included type i and ii receptor genes, jak , tyrosine kinase (tyk ), stat and stat pointing to possible failure to activate the jak/stat signaling pathway and inhibition of signal transducers caused by sav infection. although the suppressor of cytokine signaling (socs) genes and were upregulated in the ifn treated cells, only socs was downregulated in the sav infected cells which points to inhibition of socs by sav infection in to-cells. conclusion: data presented in this study shows that sav infection downregulates several genes of the jak/stat pathway, which could be an immune evasion strategy, used to block the transcription of antiviral genes that would interfere with sav replication in to-cells. overall, we have shown that combining de novo assembly with pathway based transcriptome analyses provides a contextual approach to understanding the molecular networks of genes that form the jak/stat pathway in to-cells infected by sav . the jak/stat pathway has been shown to contribute positively to innate immune responses against viral infections from drosophila to vertebrates [ ] [ ] [ ] . viruses have evolved counter measures that block different components of the jak/stat pathway to prevent production of antiviral compounds [ , ] . while very little information is available for salmon pancreas disease virus (spdv also referred to as salmon alphavirus -sav) causing pancreas disease of atlantic salmon (salmo salar l) and trout (oncorhynchus mykiss), it has been shown that other alphaviruses like chikungunya virus (chikv) inhibit the stimulation type i and ii ifn by blocking jak/stat signaling, likely through nsp [ , ] . lin et al. [ ] showed that japanese encephalitis virus (jev) block ifn induced jak/stat signaling through a protein tyrosine phosphatase mediated mechanism. as pointed out by fujii et al. [ ] that strategies of counteracting jak/stat signaling vary among viruses which include suppressing the phosphorylation of the jaks, inhibition of nuclear translocation of activated stats, degradation of different jak/stat components as well as inducing the expression of negative regulators such as the socs proteins by host cells. this shows that viruses alter the jak/stat signaling at different stages of the pathway. janus kinase (jak) and signaling transducer and activator of transcription (stat) is an intracellular signaling pathway that mediates the effects of a large number of cytokines and growth factors [ ] . it is a pleiotropic cascade used to transduce a network of signals for the development and homeostasis of cells from vertebrates to insects. jak activation stimulates cell proliferation, differentiation, migration and apoptosis. in general, the jak/stat pathway is based on a few principle components involving a variety of ligands and their receptors [ ] . intracellular activation of the jaks takes places when ligand binding induces dimerization of receptor subunits on cell surfaces [ ] . for signal transduction through dimerization, the cytoplasmic domain of the receptor subunits must be associated with jak tyrosine kinases, which juxtaposes the jaks to allow for their transphosphorylation. the transphophorylated jaks phosphorylate additional targets that include the stats. stats are latent transcriptional factors found in the cytoplasm in an inactive state until they are activated. phosphorylated stats migrate to the nucleus where they modulate the transcription of different target genes. in general, the jak/stat pathway provides a direct mechanism in which extracellular signals are translated into transcriptional responses that modulate the expression of different target genes. apart from stats, other effector molecules that contribute to jak/stat signaling include the signal-transducing adaptor molecules (stams) that facilitate the transcriptional activity of specific target genes such as transcriptional regulator myc (myc) [ ] and the stat-interacting protein (stlp) which serves as a scaffold to phosphorylate the stats by the jaks [ ] . on the other hand, three major classes of negative regulators have been identified which include the suppressor of cytokine signaling (socs), protein inhibitors of activated stats (pias) and protein tyrosine phosphates (ptps) [ ] . the socs family serves as a negative feedback loop on the jak/stat circuit in which activated stats stimulate the transcription of socs genes that bind to phosphorylated jaks and their receptors to turn off the signaling pathway [ , ] . the ptps includes the shp- phosphatases, which has two sh (src-homology- ) domains that bind to the jaks to initiate the dephosphorylation of the jak and stat proteins [ ] . the pias proteins bind to activated stat dimers to prevent their signal transducing activities although the exact mechanism in which this is carried out has not been established [ , ] . put together, the jak/stat cascade is a tightly controlled pathway regulated by different effector proteins and negative regulators that prevent overstimulation of the paracrine pathway. therefore, to identify genes that alter jak/stat signaling induced by viral infections calls for a comprehensive profiling of genes induced by virus replication during infection progression in host cells. in the present study we used rna-seq and put together a de novo assembly of genes belonging to the jak/stat pathway induced by salmonid alphavirus subtype- (sav- ) infection in tocells. to-cells are a continuous cell-line derived from atlantic salmon headkidney leukocytes characterized to possess dendritic cell-like properties [ ] . sav is a potent inducer of type i ifn responses in to-cells while at the same time, the virus replicates vividly under high ifn i levels [ ] [ ] [ ] . pretreatment of permissive cells with recombinant salmon ifn i will protect the cells against cpe but the effect of this treatment will depend on time of treatment relative to infection [ ] . the mechanisms employed by sav to circumvent or block the antiviral responses of the host cells are not known. hence, in this study we wanted to identify the profile of genes linked to the jak/stat pathway induced by sav infection in to-cells and to compare their expression patterns in cells simultaneously infected with sav and treated with type i ifn to sav -only infected cells. the aim was to understand any modulation of the antiviral responses elicited by sav infection compared to partly protected (ifn i/sav ) cells. to do this, we used the kyoto encyclopedia of genes and genomes (kegg) database analysis to generate pathways of differentially expressed genes generated by de novo assembly using rna-seq from sav infected to-cells. in general, we anticipate that data presented here shall serve as a roadmap to elucidating the cellular networks linked to the jak/stat pathway used by sav to evade the host defensins in infected fish. to-cells derived from atlantic salmon (salmo salar l) head kidney leukocytes characterized to possess macrophage/dendritic-like properties were use in this study [ , ] . the to-cells, were propagated in hmem (eagle's minimal essential medium [mem] with hanks' balanced salt solution [bss] ) supplemented with l-glutamine, mem nonessential amino acids, gentamicin sulfate, % fbs and were incubated at °c. the salmon alphavirus subtype (genebank accession jq ) used to inoculate the to-cells has previously been described [ ] . one batch of to-cells was treated with ng/ml of atlantic salmon type i ifn in triplicates and was simultaneously infected with sav . another batch of to-cells was only infected with sav- , without type i ifn treatment. the virus was inoculated at an moi when the cells were % confluent in both groups. thereafter, both the sav- infected cells with and without ifn treatment were incubated at °c in maintenance media using hmem growth media supplemented with % fbs. the mock group was only treated with maintenance media without sav infection and no type i ifn treatment. cells were harvested after h and were used for rna extraction used for rna-seq analysis. another set of three independent replicates subjected to the same treatment was used for qrt-pcr analysis. all studies in tocells were carried out in triplicates. extraction of total rna was carried out using the rneasy mini kit with on-column dnase treatment according to the manufacturer's instructions (qiagen, hilden, germany). the concentration and quality of rna was analyzed using a nanodrop nd (nanodrop technologies, wilmington, usa) and agilent bioanalyzer (agilent technologies, usa), respectively. triplicates of equal quantities of total rna from ifntreated and non-treated-to-cells infected with sav together with mock cells were mixed to prepare the pooled rna sample for each group. the pooled samples were treated with dnase i for the degradation of any possible dna contamination followed by enrichment using oligo(dt) magnetic beads. thereafter, the mrna was fragmented into short fragments of approximately bp by mixing with the fragmentation buffer. this was followed by first strand cdna synthesis using random hexamer-primer, which was followed by adding a buffer containing dntps, rnase h and dna polymerase i to synthesize the second strand. the generated double strand cdna was then purified with magnetic beads and the end reparation and '-end single nucleotide a (adenine) addition was then performed. thereafter, sequence adaptors were ligated to the fragments, which were later enriched by pcr amplification. the agilent bioanaylzer and abi steponeplus real-time pcr system (biorad.com) were used to qualify and quantify the sample library during quality check (qc step). after the qc step, library products were ready for rna-sequencing using illumina hiseqtm , bgi-hong kong. for rna-seq analysis, clean reads were obtained after removal of adaptor sequences, reads having greater than % unknown bases as well as removal of low quality bases (base with quality value ≤ ) greater than % in a read. raw reads generated by illumina sequencing were used to generate a library of clean reads after quality check, which was used for transcriptome de novo assembly using the trinity software (http://trinityrnaseq.github.io [ ] . thereafter, assembled unigenes were used for annotation for the classification of gene functions by searching different protein databasesusing blastx version . . as previously described [ ] . data from blastx was used to extract the coding regions (cds) from unigene sequences and translate them into peptide sequences. as for unigenes without hits in blastx, the estscan was used to predict their cds and to decide their sequence direction. unigenes having nr annotation were further analyzed with blast go (https://www.blast go.com/) to obtain their gene ontology (go) annotations for classification according to go functions using the web gene ontology (wego) annotation software. identification of differentially expressed genes (degs) was carried out as previously described [ ] . in this study, degs were generated based on comparing the rpkm mapped reads from to cells treated with type i ifn simultaneously infected with sav versus mock to-cells that were designated as to-vs-ifn/sav , while the second comparison was based on to-cell infected with sav- only, without ifn treatment, versus mock to-cells that were designated as to-vs-sav . only genes with a threshold of false discovery rate (fdr) < . and an absolute value log ratio > were considered as differentially expressed. thereafter, all degs were assigned kegg orthologue (ko) identifiers which were later used for pathway analysis using the kegg database (http://www.genome.jp/kegg/). in addition, we used the tigr gene indices clustering tools (tgicl) (http://www.tigr.org/tdb/tgi) [ , ] software to remove redundancy. to confirm the validity of the degs generated by rna-seq, degs obtained from the to-vs-ifn/sav and to-vs-sav were randomly selected for the qrt-pcr validation test. gene expression of three independent replicates of to-cells simultaneously treated with type i ifn and infected with sav- (to-vs-ifn/sav ) was compared with none-type i ifn treated cells infected with sav (to-vs-sav ) relative to gene expression of untreated to-cells before normalized to β-actin. cells used for independent replicate biological testing were independent of the cells used for rna-seq data analysis. the qrt-pcr was carried out as previously described [ ] using ng total rna as a template for each gene in a master-mix final volume of μl according to manufacturer's recommendations (roche diagnostics, mannheim, germany). primers used in the study are shown in table and the primer-master-mix solutions were first incubated for reverse transcription at °c for min followed by pcr initial activation at °c for min and amplification cycles ( s at °c and s at °c). the specificity of each pcr products was confirmed by melting-curve analysis and agarose gel electrophoresis. the delta c t method was used to calculate the fold-increase in gene expression relative to the β-actin control group [ ] . all quantifications were normalized to β-actin control group. the qrt-pcr was done thrice, with each repeat study having three independent replicates of ifn treated (to-vs-ifn/sav ) and non-treated cells (to-vs-sav ). the mean of each gene from the three independent replicates after three repeats was used to compute the correlation coefficient test between rna-seq and qrt-pcr data. in order to determine the effect of type i ifn treatment on sav replication in to-cells, virus quantification was determined after hrs replication at °c for the ifnα treated and non-treated groups using a guided sequence format for the structural proteins (sps) and non-structural proteins (nsps) of sav (genebank accession nos. afj . and af . ) based on the rna-seq data. the fragment per kilobase per million (fprm) method was used to read the number of fragments of nsps and sps expressed by sav after hrs replication at °c in the ifnα treated and non-treated cells. in order to verify the validity of the virus quantification data generated by rna-seq, quantitative rt-pcr (qrt-pcr) was used to determine the sav replication level in the ifnα treated and non-treated cells using the e sp expressed during sav replication. the primer sequences used for e quantification are shown in table and the qrt-pcr method used has previously been described by xu et al. [ ] . first we documented the effect of ifn i treatment on the sav sp (structural protein) expression. we found that the non-ifnα treated cells had higher expression levels of sav sps than the ifn i treated cells (table ). this shows that simultaneous treatment with ifn i reduced the level of synthesis of mrna sps from subgenomic viral rna. similarly, the nsp mrnas (non-structural proteins mrnas) expressed in the non-ifnα treated cells were . higher than in the ifnα treated group (ifn/sav ). the log ratio of the ifn-plussav /sav for the sps and nsps showed a downregulation of - . and - . , respectively, showing that type i ifn added concurrently with sav- infection significantly reduced virus replication in to cells ( table ) . consistent with rna-seq data shown in table , fig. shows a fold-reduction in sav replication in the ifn i treated cells (ifn/sav ) relative to the untreated cells based on the e sps detected by qpcr using the cp-value test. thus, qpcr confirms the validity of our rna-seq data analysis by showing that a downregulation of sav sps in ifn i treated cells (ifn/ sav ) versus sav infected cells only. based on data generated by illumina sequencing, we constructed three libraries that yielded a total of bcl-x-r agtcctagatacctccctcc , and raw reads for the ifntreated and non-ifn treated to-cells infected with sav together with mock to-cells, respectively. the total unique match in total mapped reads was > % after removal of redundancy using the tgicl software (http://www.tigr.org/tdb/tgi) suggesting low redundancy. after quality check for clean reads and filtration [ ] , , unigenes from the to-vs-ifn/sav and to-sv-sav groups assigned to the kegg orthologues (ko) were identified using the kegg database (http:// www.genome.ad.jp/kegg/), which has been widely used to generate pathway networks for model organisms such as the zebra fish (danio rerio) [ , ] and mouse (mus musculus) [ ] as well as several non-model organisms [ , ] . the profile of genes identified using ko-based on the rpkm method in which only genes with an fdr < . and an absolute value log ratio > were considered differentially expressed showed that the to-vs-ifn/sav had degs whose kos were mapped to pathways of which degs belonged the jak/stat pathway (table ) . on the other hand, the to-vs-sav had a total of degs assigned to ko-identifiers, which was degs more than the to-vs-ifn/sav . as a result, the to-vs-sav had pathways identified by the kegg database being pathways more than those identified in the to-vs-ifn/sav (table ). in terms of jak/stat genes, the to-vs-sav had a total of degs expressed being degs more than the to-vs-ifn/sav . in general, the to-vs-sav had more degs and pathways identified using the kegg database analysis than the to-vs-ifn/sav although the to-vs-ifn/sav had a higher enrichment qvalue ( . e- , p value = . e- ) than the to-vs-sav ( . e- , p value = . e- ) ( table ) . to gain more insights on the impact of type i ifn treatment on the jak/stat pathway in to-cells infected with sav- , we compared the degs expressed from the to-vs-ifn/sav with degs from the to-vs-sav . figure shows that of the degs expressed in the to-vs-sav , degs were downregulated while were upregulated. in the to-vs-ifn/sav group degs were upregulated while degs were downregulated. put together, these findings show that the proportion of upregulated degs in the to-vs-ifn/sav ( . %, n = ) was higher than in the to-sv-sav group ( . %, n = ) while the proportion of downregulated degs for the to-sv-sav ( . %, n = ) was higher than the to-vs-ifn/sav group ( . %, n = ). this is strongly suggesting that type i ifns upregulate several genes of the jak/stat pathway that were downregulated by sav infection alone in to-cells. hence, to further elucidate the type of genes suppressed by sav infection in the absence of type i ifn treatment, we broadly classified the degs in the jak/stat pathways for both the to-vs-ifn/sav and to-vs-sav into type i and ii receptor genes, jaks, stats and negative regulator genes as shown below. differentially expressed genes belonging to receptor ligands that activate the jak/stat pathway were classified into type i and ii receptor-genes. as shown in fig. and table , the type i receptor family was subdivided into the gamma chain (γc), gp and single chain (homodimer) receptor genes while the type ii receptor family comprised of type i and ii ifn receptor genes. the major difference table ). the type ii receptor genes were subdivided into two groups namely the interleukin (il) and ifn receptor genes. as shown in table , the il-receptor genes comprised of il- rb and il- ra of which both were upregulated in the to-vs-ifn/sav and to-vs-sav . the ifn-receptor genes comprised of ifn-α/β and ifnγ receptor genes ( table ). the ifn-α/β receptor genes comprised of ifnr and ifnr which were upregulated in both the to-vs-sav and to-vs-ifn/sav while the ifnγ receptor genes comprised of ifngr and ifngr that were only expressed in the to-vs-ifn/ sav in which both were upregulated (table ). fig. shows a schematic layout of the common ifn-receptor signaling pathways based on mammalian model species [ , ] of which applying data presented in table shows that the ifn-receptor-genes expressed by the to-vs-ifn/sav were linked to both pathways a and b while the ifn-receptor-genes expressed by the to-vs-sav was only linked to pathway a. three genes belonging to the jaks were differentially expressed namely jak , jak and tyrosine kinase (tyk ) (fig. , table ). all three jaks were differentially expressed in the to-vs-sav while only jak was differentially expressed in the to-vs-ifn/sav . jak was upregulated in both the to-vs-sav and to-vs-ifn/ sav while jak and tyk were downregulated in the to-vs-sav (table ). network pathways linking the jaks to other genes in the jak/stat pathway are shown in fig. a for the to-vs-ifn/sav and in fig. b for to-vs-sav . note that fig. a shows upregulation of all jaks being in conformity with observations made in table in which only jak was differentially expressed in the to-vs-ifn/sav and it was upregulated. on the other hand, fig. b shows a mixed representation of upregulated and downregulated jak-genes, which is in agreement with observations shown in table in which jak was upregulated whiles jak and tyk were downregulated. five stat genes were differentially expressed in this study namely stats , , , b and ( table ). as shown in fig. a , stats , , and b were differentially expressed in the to-vs-sav of which stats and were upregulated while stats and b were downregulated (table ). in the to-vs-ifn/sav , stats , and were upregulated (table , fig. b ). the network links of the stats to other genes in the jak/stat pathway for the to-vs-sav and to-vs-ifn/sav are shown in fig. a and b, respectively. note that all stats in the to-vs-ifn/sav were upregulated in fig. a conforming to observations shown in table in which all stats were upregulated while fig. b shows a mixed representation of upregulated and downregulated stats for the to-vs-sav being in line with observation shown in table in which stats and were upregulated while stats and b were downregulated. the negative regulatory genes for the jak/stat pathway differentially expressed in this study were classified into table ). table shows that socs and socs were both upregulated in the to-vs-ifn/sav while socs was downregulated and socs was upregulated in the to-vs-sav . figure a and b shows network pathways linking the socs to other genes in the jak/stat pathway in which all socss were upregulated in the to-vs-ifn/sav (fig. a) , which conforms to observations shown in table . the network pathway in the to-vs-sav shows a mixed representation of upregulated and downregulated socss (fig. b) , which conforms to observations shown in table where socs was upregulated while socs was downregulated in the to-vs-sav . as for pias, it was downregulated in the to-vs-sav and upregulated in the to-vs-ifn/sav ( fig. a and b ). genes that regulate apoptosis and cell-cycle activities were broadly classified into the stat, mapk and pi k-akt pathways as shown in table based on network pathways shown in fig. a and b. in the stat-pathway, the ifn regulatory factor (irf), creb binding protein (cbp) and threonine-protein kinase pim- (pim- ) were upregulated in the to-vs-ifn/sav while in the to-vs-sav , the signal transducer and adaptor molecule (stam ), cbp, pim- and pim- were downregulated (table ). in the mapk pathway shown in fig. a and b, the transcriptional regulator myc- (myc- ) and cyclin d (cycd ) were upregulated in the to-vs-ifn/ sav while the src homology region domaincontaining phosphatase- (shp ) which is represented by the protein tyrosine phosphatase non-receptor (ptpn ) in table , son of sevenless (sos), myc- , cycd, b-cell lymphoma extra-large (bclxl) and sprouty-related evh domain-containing protein (spred) were all downregulated in the to-vs-sav (table and fig. b ). the phosphatidylinositol- , bisphosphate -kinase (pi k) and v-akt viral oncogene -like protein (akt ) that regulate the pi k-aktpathway for apoptosis were downregulated in to-vs-sav (table ) while only pi k was downregulated in the to-vs-ifn/sav . network pathways linking the apoptosis and cell cycle genes for the to-vs-sav and to-vs-ifn/sav are shown in fig. a and b. in general, all apoptosis and cell proliferation genes differentially expressed in the to-vs-sav were downregulated while the majority differentially expressed in the to-vs-ifn/sav was upregulated indicating that in the absence of type i ifn treatment sav suppressed the expression of most of anti-apoptosis and cell proliferation genes. validation of rna-seq data by qrt-pcr figure shows validation of rna-seq data based correlation coefficient test between rna-seq and qrt-pcr data. our findings show a significantly high linear correlation (r = . , p < . ) between rna-seq and qrt-pcr for the to-vs-ifn/sav and to-vs-sav . put together, qrt-pcr confirms the validity of our rna-seq data analysis by showing a high correlation between rna-seq and qrt-pcr. de novo and reference gene guided transcriptome assembly current advances in next generation sequencing show that rna-seq is poised to enhance our understanding of molecular networks of genes that form the innate and adaptive immune system in teleosts fish. in general, there are two approaches used to assemble a transcriptome from rna-seq data which is by use of a de novo assembly or a reference gene guided approach. in situations where no reference gene exists as is the case for non-model organisms like atlantic salmon, a de novo assembly is the only option for sequence assembly by which raw reads from rna-seq are assembled into contigs without the guidance of a reference gene. hence, in the present study we used a de novo approach to assemble a repertoire of genes that form the jak/stat pathway from atlantic salmon headkidney/macrophage like tocells with an aim to understand sav modulation of fig. shows the schematic presentation of the ifn-α/β and ifnγ receptors linked to the jaks for the to-vs-ifn/sav and to-vs-sav . in pathway a, the ifn-α/β binds jak and tyk receptor subunits linked to stats and , followed by a pathway that translocates the phophorylated stats into nucleus. in pathway b, the ifnγ receptor is shown to bind to jaks and , which are linked to stat and the phosphorylated stats are shown to translocate to the nucleus. based on data shown in table , the to-vs-ifn/sav uses both pathways a and b using the ifn-α/β and ifnγ ligands while the to-vs-sav only uses pathway a using the ifn-α/β ligand. this model is adapted from ifn-receptor signaling pathways for the jak/stat pathway in higher vertebrates [ , , ] responses post infection. unlike microarray that uses predefined probes, de novo assembly provides a unique opportunity to identify novel genes which could be missed by none-coded micro-rna probes. after de novo assembly, we used the tgicl software, which is a highly refined protocol used for the analysis of genes and est sequences needed for transcriptome analysis [ ] . using this software, sequences are first cleaned to remove adaptors, low quality sequences without quality score, vector and adaptor contaminants and redundancies. thereafter, sequences are searched pairwise and grouped into clusters that are assembled at high stringency to produce tentative concesus (tc) sequences. and as pointed out by pertea et al. [ ] , individual assembly of each cluster to produce tcs has the advantage of producing large, more concensus sequences while eliminating potentially misclustered sequences. the tcs produced by the tgicl software have been used to construct species specific databases and to identify novel genes [ ] . for example, several genes such as stam , sos, and pias not previously reported from atlantic salmon were matched to their orthologues in model species such as zebrafish and mammalia in this study suggesting that the jak/stat pathway induced in macrophage/dendritic cell like to-cells derived from atlantic salmon headkidney cells expresses a repertoire of genes comparable to the jak/stat signaling pathway observed in higher vertebrate species. on the other hand, we used a reference gene guided approach to detect and quantify the e sp of sav based on the fact that the genomic sequence of sav has been fully sequenced and characterized in salmonids [ , , ] . hence, both the de novo and reference gene guided assemblies were used in this study. virus replication in to-cells of simultaneous ifn i/ sav treatment/infection (ifn/sav ) was two logs ( ) lower than levels detected in sav -only infected cells. for the latter, it shows that sav is a potent inducer of ifn signaling pathways in to-cells post infection and also that the virus had the capacity to replicate in the presence of high levels of type i ifns and timing of ifn i treatment is crucial [ ] . these findings are consistent with observations made for other alphavirus studies in which it has been shown that timing in type i ifn treatment has a significant influence on replication of alphaviruses in infected cells [ , , ] . rna-seq is perfect for gene network construction and pathway analysis [ ] . and as pointed out by khatri et al. [ ] , pathway analysis has become the first choice to understand the underlying biological mechanisms of degs because it reduces the complexicity of rna-seq data analysis whose vast array of sequence data is often difficult to interpret but instead combining rna-seq with pathway analysis increases the explanatory power of transcriptome data analysis. in pathway analysis the first step is to generate a transcriptome of degs as shown in this study followed by searching for functional networks of genes expressed in the transcriptome based on the fact that there is a tendency for co-regulated genes to have functional correlations in the pathway. khatri et al. [ ] shown that pathway software development has progress through three different generations in the last decade. the first generation of pathway analytical softwares comprise of the over-representation analysis (ora) softwares that include genmapp (http:// www.genmapp.org), gostat (http://gostat.wehi.edu.au) and gotoolbox (http://genome.crg.es/gotoolbox/). these software establish pathway analysis by putting together over expressed genes that show significant changes in expression based on gene level statistic [ , ] . the second generation of softwares consist of functional class scoring (fcs) approaches such as the gene set enrichment analysis (gsea) software (http://www.broadinstitute.org/ gsea/) and genetrail (http://genetrail.bioinf.uni-sb.de) that compute both the statistical significance of individual genes that form a pathway (gene level statistic) and the statistical significance of the entire pathway (pathway level statistic) [ ] [ ] [ ] . the third generation of softwares comprises of pathway topology (pt) based approaches such as the kegg [ ] and panther db [ ] that do not only encorporate individual gene and pathway level statistics computed by the the ora and fcs softwares, but they also provide information about gene products that interact with each other in a given pathway, how they interact (e.g. activation, inhibition, etc), and where thay interact (e.g. cytoplasm, nucleus, endoplasmic reticulum, etc). hence, in this study we used the kegg pathway analysis software which is a third generation pt based software to identify networks of genes that form the jak/stat signaling pathway from a de novo assembled transcriptome with the aim to better understand the modulation of genes expressed by to cells infected with sav . consistent with all tp-based softwares, the kegg pathways used in this study shows gene level statistical analyses for all degs and in terms of pathway level statistic analysis, our findings show that concurrent treatment of to-cells with type i ifn together with sav infection significantly enriched the jak/stat pathway from a qvalue of . e- (p-value = . e- ) to . e- (pvalue = . e- ). this was supported by upregulation of several activator and signal transducer genes in the to-vs-ifn/sav group compared to the to-vs-sav group. as for genes that interact with each other and their interaction sites, the modulation is complex and below we pinpoint some of the key areas in the pathway where sav infection interferes. receptor genes that activate jak/stat signaling type i receptor genes generated in response to sav infection and type i ifn treatment were classified into the γc, gp and homodimer receptor families based on receptor classification used for higher vertebrates [ , ] . wang et al. [ ] reviewed the γc receptor system for teleost fish in which they reported of il- rb, il- ra, il- ra and il- ra in salmonids which were also differentially expressed in this study. sav infected cells (to-vs-sav ) had more type i receptor genes downregulated than type i ifn treated cells (to-sv-ifn/sav ) suggesting that sav like other alphaviruses in higher vertebrates, inhibits the activation of several type i receptor genes post infection. the type ii receptor family generated in this study was classfied into il and ifn receptor genes. as for genes of the ifn receptor genes, ifnr and ifnr were expressed in both the to-vs-ifn/sav and to-vs-sav suggesting that activation of ifn-α/β receptors occurs as consequence of infection as well as ifn i treatment (as would be expected). however, it is important to point out that although our findings show the presence of both ifnr and ifnr , which is in agreement with observations made by ali et al. [ ] who reported the presence of both ifnr and ifnr based on a transcriptome analysis of rainbow trout spleen. studies carried out by sun et al. [ ] suggest that atlantic salmon possess two clusters type i ifn receptor genes located on different chromosomes which allow for a large repertoire of ifn receptors than mammals and zebrafish. hence, there is need for further studies to compare the ifnr and ifnr genes detected in this study with the ifn receptor genes repertoire reported by sun et al. [ ] and to determine their functional roles in the jakstat pathway signaling. the expression of ifngr and ifngr was only found in the to-vs-ifn/sav cells. this suggests that sav infection does not activate the ifnγ-receptors. the importance of ifn-γ in eliciting an antiviral effect in vitro remains non-conclusive. we have shown that ifn-γ exhibits marginal antiviral effect against sav in vitro [ ] . in contrast sun et al. [ ] suggested that recombinant trout ifn-γ had antiviral effect, although they also concluded that the observed effect of ifn-γ is partly dependent on ifn-α induction [ ] . additional studies are needed to understand the importance of ifn-γ in relation to sav infection. four jak-family members have been identified namely jak -jak and tyk in mammalia [ ] and fish [ ] [ ] [ ] [ ] of which jak , jak and tyk transcripts generated in this study were matched to their mammalian and fish orthologues. the mechanisms underlying activation, regulation and pleiotropic signaling in fish have not been studied in detail. here we find that jak was upregulated in sav infected to cells, with or without ifn-i treatment, while jak and tyk were only differentially expressed in the to-vs-sav groups, and importantly, they were both downregulated. thus, these findings suggest that sav infection activates jak signaling while it suppresses activation of tyk and jak . these observations are in line with what is seen for alphavirus infections in higher vertebrates in which blockage of tyk was shown for japanese encephalitis virus (jev) [ ] , while dengue virus type was found to antagonize the jak/stat pathway by downregulating tyk -stat signaling in human dendritic cells [ ] . lin et al. [ ] showed that jev infection blocked tyrosine phosphorylation of ifn associated tyk without affecting the expression of ifnα/β on the cell surface. we found that ifnα was upregulated when tyk was downregulated, and while it is possible that sav uses similar mechanisms to what is seen for other alphaviruses to block the phosphorylation of tyk thereby inhibiting jak/stat signaling in to-cells, this remains to be shown. type i ifn mediates tyrosine phosphorylation by binding to receptors associated with jak and tyk which activates stats and , while ifnγ mediates its signaling through jaks and to activate stat in mammalian cells [ , ] . the to-sv-ifn/sav showed upregulation of ifnγ receptor genes, corresponding to the observed upregulation of jak and stat , while downregulation of tyk in sav infected cells showed a corresponding downregulation of stat and stat . hence, it is possible that mechanisms in salmon and human cells are comparable, however, to identify the different jaks that activate specific stats in fish cells, there is need for detailed investigations. studies in higher vertebrates show that alphaviruses block the jak/stat signaling pathway by inhibiting the phosphorylation of different stats to prevent the transcription of antiviral genes in infected cells. for example, jev infections block tyrosine phosporylation of stats , and [ ] while chikv nsps were potent inhibitors of ifn-induced jak/stat signaling by blocking stat phosphorylation of the jak-receptors either by type i or ii ifns [ ] . the observed downregulation of several type i and ii receptor genes in sav infected cells could resulted in inhibition of jak and tyk phosphorylation, which would align with the downregulation of most stat genes. although these data suggest that sav could be using mechanisms similar to those used by mammalian alphaviruses to block the jak/stat pathway, there is need for detailed investigations to consolidate these observations in to-cells. the negative regulatory genes linked to inhibition of the jak/stat pathway include genes that form a negative feedback loop comprising of members of the socs family and genes that directly inhibit the jak/stat pathway such as ptp b, shp and pias. in salmonids, several socs genes have been cloned and characterized [ , ] and studies carried out by skjesol et al. [ ] show that socs supresseses type i and ii signaling of the jak/stat pathway in atlantic salmon. in mammalia, socs and socs but not socs inhibit ifn-mediated antiviral activities [ ] . in this study, socs was upregulated in both to-vs-ifn/sav and to-vs-sav while socs was only upregulated in the to-vs-ifn/sav and downregulated in the to-vs-sav suggesting that sav replication could have suppressed the expression of socs . among the genes that directly inhibit jak signaling, only pias was differentially expressed and it was upregulated in the to-vs-ifn/sav and downregulated in the to-vs-sav , suggesting that sav suppressed the expression of pias in to-cells. although pias has not been characterized in salmonids, its expression at transcript level in this study suggests that it exists in atlantic salmon and that it likely to play an important role in regulating the jak/stat pathway in to-cells. overall, these negative regulators were all downregulated in the to-vs-sav cells, except for socs , suggesting that sav- upregulates socs to prolong its survival in infected cells. here we find that apoptosis related genes pim- , c-myc and cycd are upregulated in the to-vs-ifn/sav and downregulated in the to-vs-sav cells. apoptosis following virus infection is an important defense mechanisms given that cell autodestruction is one of the safest ways of limiting virus spread to neighboring cells in an infected host [ ] . however, viruses have evolved defense mechanisms that evade or delay apoptosis [ ] [ ] [ ] by altering the expression of anti-apoptosis target genes that regulate cell growth and proliferation [ ] [ ] [ ] [ ] . our finding can be interpreted as a survival strategy of sav to prolong its replication cycle in to-cells. further, it is also possible that the expression of pi k and akt could be regulating sav induced apoptosis in to-cells as shown for other alphavirus where pi k-akt pathway regulates cell survival by preventing caspase- activation to prevent apoptosis in virus infected cells [ ] [ ] [ ] . mastrangelo et al. [ , ] have shown that bcl- and bci-xl limit apoptosis upon infection with alphaviruses while cycd has been shown to exert the same effects in neuronal cells infected by sindbis virus [ ] . similarly, pim- acts synergestically with c-myc [ ] , to inhibit apoptosis related mitochondrial dysfunction through the bcl- dependent pathway [ , ] . it remains to be shown whether downregulation of apoptosis target genes in the to-vs-sav cells could be an evasion strategy to shutdown cell-death or to slowdown apoptosis to prolong replication time in infected to-cells. this study provides a global overview of genes that form the jak/stat pathway induced by sav infection and the combined ifn i/sav infection allow us to pinpoint modulating effects of sav infection on the jak/stat signaling pathway. it is important to note that the data presented here only provides a repertoire of downregulated and upregulated genes interlinked in a de novo assembled network pathway analysis, and does not show the exact mechanism by which the modulatory processes take place. thus a lot of work remains to be done to elucidate the exact interaction points and modulation of the jak/stat encoded proteins. genes generated by de novo assembly in this study. further, the study shows that combining de novo assembly with pathway based analysis increases the explanatory power of transcriptome data analysis by putting together a network of genes that regulate hostpathogen interaction processes at cellular level. we anticipate that the jak/stat pathway genes put forth in this study shall serve as a roadmap for elucidating molecular mechanisms used by sav infection to evade host defensins at cellular level. the rna-sequencing data generated in this study has been deposited in the national center for biotechnology information (ncbi) gene expression omnibus (geo) database accession number gse (www.ncbi.nih.gov/ geo accession number gse ). chikungunya virus nonstructural protein inhibits type i/ii interferon-stimulated jak-stat signaling jak/stat pathway in drosophila immunity interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures stat- , activates the atm dna damage pathway to induce hpv genome amplification upon epithelial differentiation blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns through a protein tyrosine phosphatase-mediated mechanism molecular mechanisms for suppression of interferon signal transduction pathways caused by viral infections the jak/stat pathway the jak/stat signaling pathway stam/east/hbp adapter proteins -integrators of signalling pathways negative regulation of cytokine signaling suppressors of cytokine signaling (socs): negative regulators of signal transduction negative regulation of cytokine signaling by the socs proteins negative regulation of the jak/stat pathway piasx is a transcriptional co-repressor of signal transducer and activator of transcription a highly phagocytic cell line to from atlantic salmon is cd positive and m-csfr negative, indicating a dendritic-like cell type a salmonid cell line (to) for production of infectious salmon anaemia virus (isav) alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype replication in vitro de novo assembly and transcriptome analysis of atlantic salmon macrophage/dendritic-like to cells following type i ifn treatment and salmonid alphavirus subtype- infection tigr gene indices clustering tools (tgicl): a software system for fast clustering of large est datasets the tigr gene indices: clustering and assembling est and known genes and integration with eukaryotic genomes real-time, fluorescence-based quantitative pcr: a snapshot of current procedures and preferences transcriptome profiling of zebrafish infected with streptococcus suis behavioral and neurogenomic transcriptome changes in wild-derived zebrafish with fluoxetine treatment differential hippocampal gene expression and pathway analysis in an etiology-based mouse model of major depressive disorder transcriptome analysis of chicken kidney tissues following coronavirus avian infectious bronchitis virus infection de novo assembly and characterization of two transcriptomes reveal multiple light-mediated functions in the scallop eye (bivalvia: pectinidae) detection and antigenic characterization of salmonid alphavirus isolates from sera obtained from farmed atlantic salmon, salmo salar l., and farmed rainbow trout, oncorhynchus mykiss (walbaum) characterization of untranslated regions of the salmonid alphavirus (sav ) genome and construction of a sav based replicon antiviral activity of alpha-interferon in sindbis virus-infected cells is restored by anti-e monoclonal-antibody treatment early events in alphavirus replication determine the outcome of infection behavior and next-generation rna sequencing ten years of pathway analysis: current approaches and outstanding challenges bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists ontological analysis of gene expression data: current tools, limitations, and open problems globalancova: exploration and assessment of gene group effects a multivariate extension of the gene set enrichment analysis non-linear tests for identifying differentially expressed genes or genetic networks kegg: kyoto encyclopedia of genes and genomes panther: a library of protein families and subfamilies indexed by function the jak-stat pathway signaling through the jak/ stat pathway, recent advances and future challenges the gamma-chain cytokine/ receptor system in fish: more ligands and receptors characterization of the rainbow trout spleen transcriptome and identification of immune-related genes atlantic salmon possesses two clusters of type i interferon receptor genes on different chromosomes, which allows for a larger repertoire of interferon receptors than in zebrafish and mammals antiviral activity of salmonid gamma interferon against infectious pancreatic necrosis virus and salmonid alphavirus and its dependency on type i interferon a direct cross-talk between interferon-gamma and sonic hedgehog signaling that leads to the proliferation of neuronal precursor cells the janus kinases (jaks) the jak and stat family members of the mandarin fish siniperca chuatsi: molecular cloning, tissues distribution and immunobiological activity genomic organization and characterization of the promoter region of the roundspotted pufferfish (tetraodon fluviatilis) jak kinase gene complete genomic organization and promoter analysis of the round-spotted pufferfish jak , jak , jak , and tyk genes the atlantic salmon protein tyrosine kinase tyk : molecular cloning, modulation of expression and function rainbow trout suppressor of cytokine signalling (socs)- , and : molecular identification, expression and modulation identification of suppressor of cytokine signalling (socs) , , and cish in rainbow trout oncorhynchus mykiss and analysis of their expression in relation to other known trout socs functional conservation of suppressors of cytokine signaling proteins between teleosts and mammals: atlantic salmon socs binds to jak/stat family members and suppresses type i and ii ifn signaling regulators of apoptosis on the road to persistent alphavirus infection viral control of mitochondrial apoptosis regulation of apoptosis by viral gene products c-myc target genes involved in cell growth, apoptosis, and metabolism hepatitis b virus x protein induces apoptosis in hepatoma cells through inhibiting bcl-xl expression synergistic roles for pim- and c-myc in stat -mediated cell cycle progression and antiapoptosis pim- : a serine/threonine kinase with a role in cell survival, proliferation, differentiation and tumorigenesis inhibition of the pi k-akt signaling pathway enhances the sensitivity of fas-mediated apoptosis in human gastric carcinoma cell line, mkn- pi -kinase regulates survival of chronic lymphocytic leukemia b-cells by preventing caspase- activation inhibition of phosphatidylinositol '-kinase/akt signaling promotes apoptosis of primary effusion lymphoma cells bcl- inhibits apoptosis and extends recombinant protein production in cells infected with sindbis viral vectors part i. bcl- and bcl-x(l) limit apoptosis upon infection with alphavirus vectors cyclin-dependent kinases participate in death of neurons evoked by dna-damaging agents the pim- serine kinase prolongs survival and inhibits apoptosis-related mitochondrial dysfunction in part through a bcl- -dependent pathway gas elements: a few nucleotides with a major impact on cytokine-induced gene expression this study was funded by the research council of norway, project "the atlantic salmon genome sequence as a tool for precision breeding" project number , and the targeted disease prophylaxis in european fish farming project, grant agreement . we thank jiang li, at the beijing genomics institute (bgi) for assistance with some bioinformatics analysis. authors' contribution cx = carried out laboratory experiments and data analysis; hmm = carried out data analysis and preparation of the manuscript; Øe = mobilizing of resources, data analysis, preparation of the manuscript and overall supervision of the project. all authors read and approved submission of the manuscript for publication. the authors declare that they have no competing interests. submit your next manuscript to biomed central and we will help you at every step: key: cord- -fhie m u authors: mazaleuskaya, liudmila; veltrop, rogier; ikpeze, nneka; martin-garcia, julio; navas-martin, sonia title: protective role of toll-like receptor -induced type i interferon in murine coronavirus infection of macrophages date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: fhie m u toll-like receptors (tlrs) sense viral infections and induce production of type i interferons (ifns), other cytokines, and chemokines. viral recognition by tlrs and other pattern recognition receptors (prrs) has been proven to be cell-type specific. triggering of tlrs with selected ligands can be beneficial against some viral infections. macrophages are antigen-presenting cells that express tlrs and have a key role in the innate and adaptive immunity against viruses. coronaviruses (covs) are single-stranded, positive-sense rna viruses that cause acute and chronic infections and can productively infect macrophages. investigation of the interplay between covs and prrs is in its infancy. we assessed the effect of triggering tlr , tlr , tlr , and tlr with selected ligands on the susceptibility of the j a. macrophage cell line to infection with murine coronavirus (mouse hepatitis virus, [mhv]). stimulation of tlr , tlr , or tlr did not affect mhv production. in contrast, pre-stimulation of tlr with polyinosinic-polycytidylic acid (poly i:c) hindered mhv infection through induction of ifn-β in macrophages. we demonstrate that activation of tlr with the synthetic ligand poly i:c mediates antiviral immunity that diminishes (mhv-a ) or suppresses (mhv-jhm, mhv- ) virus production in macrophages. coronaviruses (covs), a genus in the coronaviridae family, order nidovirales, are emerging rna pathogens of many animal species, including humans [ ] . currently there are no approved treatments or completely successful vaccines against cov infections. mice infected with different strains of mouse hepatitis virus (mhv), the prototype of betacoronaviruses, provide animal models for human diseases. the neurotropic strains, mhv-jhm and mhv-a , are commonly used to study viral encephalitis and virus-induced chronic demyelination, respectively [ ] . mhv-a also triggers mild to moderate hepatitis. the highly hepatovirulent strain mhv- provides a model of fulminant viral hepatitis [ ] . the first few days after infection with mhv are characterized by a strong innate immune response with infiltration of macrophages, neutrophils, and natural killer cells to the site of infection. it is widely documented that the host immune response plays a dual role in cov infection. on the one hand, it limits virus spread and replication and initiates adaptive immunity; on the other hand, it triggers overproduction of cytokines and chemokines, thus contributing to the severity of the disease [ ] [ ] [ ] [ ] [ ] . macrophages are productively infected by murine covs [ ] [ ] [ ] and represent the largest group of innate immune cells that infiltrate the central nervous system (cns) after infection with neurotropic mhv strains [ ] and the lungs of patients infected with severe acute respiratory syndrome (sars)-cov [ ] . the adaptive immune response that occurs during cov infection is well characterized [ , ] , but our understanding of the interaction of covs with the innate immune system of the host is still emerging [ , ] . type i interferon (ifn) (ifn-α and ifn-β) is crucial for the control of mhv infection in vivo [ ] [ ] [ ] . in most cell lines, murine covs are poor inducers of type i ifn and are barely sensitive to pretreatment with ifn [ ] . in primary cells, however, mhvs trigger ifn-α in plasmacytoid dendritic cells (pdcs) [ ] and ifn-β in macrophages [ , ] and are sensitive to pre-treatment with ifn-β in macrophages [ ] . therefore, interaction between murine covs and the type i ifn response depends on the cell type. the importance of type i ifn in cov infection is highlighted by a number of countermeasures and evasion mechanisms that covs in general and mhvs in particular developed to suppress signaling or prevent induction of the ifn response [ ] [ ] [ ] . induction of type i ifn can occur in all nucleated cells on tlrs activation [ ] . tlrs comprise a family of pattern recognition receptors (prr) that sense conserved molecular motifs of pathogens and trigger innate immunity and prime the adaptive immune response [ ] . triggering of tlrs induces complex signaling cascades initiated by the toll/interleukin- receptor (tir) domain in the cytoplasmic tail of the tlr. tir domain-containing adaptor molecules, myd , which is utilized by all tlrs except for tlr , as well as tirap, trif, and tram (for tlr ), are recruited to the receptor and activate a complex containing iraks and trafs which signal through nf-kb leading to the expression of a variety of genes encoding pro-inflammatory cytokines, chemokines and/or type i interferons (ifns) that orchestrate anti-bacterial and anti-viral responses [ ] . in the context of rna virus infection, tlr , tlr , tlr , tlr , and tlr can potentially be activated. cell surface tlr and tlr may recognize viral structural components, whereas endosomal tlr and tlr / may sense viral double-stranded and single-stranded rna, respectively [ ] . all of the above-mentioned tlrs were shown to induce type i ifn through activation of transcription factors and interferon regulatory factors (irfs); the magnitude of response, however, depends on the stimulus and the cell system. tlr , tlr and tlr are known to be potent inducers of the ifn response depending on the cell type [ ] . in contrast, tlr has been considered until recently a poor inducer of ifn response, despite triggering of tlr with bacteria-derived ligands induces strong pro-inflammatory cytokine response. in this regard, emerging evidence suggests that tlr and tlr activation induces pro-inflammatory cytokine and type i ifn responses from distinct sub-cellular sites: the plasma membrane and the endolysosomal compartments, respectively [ , ] . interestingly, only a particular monocyte subset has been reported to induce type i ifn through tlr in response to viral ligands [ ] . once secreted, ifn-α/β act through the jak-stat signaling pathway that triggers an "antiviral state" and help to eliminate viral infection [ , ] . the ability of tlrs to trigger antiviral immunity makes them a promising target for antiviral therapeutics. stimulation with tlr agonists has been shown to provide protection from some viral infections, such as hepatitis b virus (through tlr , tlr , tlr , tlr , or tlr ) [ ] , herpes simplex virus encephalitis (through tlr ) [ ] , lethal influenza virus (through tlr or tlr ) [ ] , hiv strains bal and jago (through tlr ) [ ] , and hepatitis c virus (through tlr ) [ ] . this study was undertaken to assess the effect of ligand-mediated, tlr activation of macrophages on their susceptibility to infection with murine cov. we profiled tlr , tlr , tlr , and tlr agonists (heat-killed listeria monocytogenes (hklm), poly i:c, lipopolysaccharide (lps), and imiquimod, respectively) and observed differential effects of these ligands on mhv production in macrophages. of all the ligands tested, only the triggering of tlr with poly i:c induced a strong antiviral response. mechanistically, the antiviral effect of poly i:c was promoted in a type i ifn-dependent manner. ligand-mediated activation of tlrs has been reported to affect the infectivity of various viruses [ , [ ] [ ] [ ] [ ] [ ] [ ] . the potential immunomodulatory and antiviral effects of triggering tlrs against cov infections in macrophages have not yet been investigated. macrophages are antigen-presenting cells that express tlrs and play a pivotal role in cov pathogenesis. the goal of this study was to investigate the effect of activation of tlr , tlr , tlr , or tlr with selected ligands on macrophage susceptibility to infection with murine coronavirus. we chose these tlrs on the basis of their potential role in the recognition of mhv by macrophages. tlr has been shown to recognize mhv- in peritoneal macrophages [ ] ; tlr has been implicated in protection and pathogenesis in mhv- -induced respiratory infection [ ] . despite the fact that tlr is a sensor of dsrna and could sense cov intermediate replicative forms in infected cells, its role in the recognition of covs or in their pathogenesis has not yet been established. tlr senses mhv-a in pdcs [ ] . first, we developed an in vitro model suitable for this study. the mouse macrophage cell line j a. was profiled for tlr - gene expression by quantitative real-time polymerase chain reaction (pcr) using predeveloped taqman gene expression assays (appliedbiosystems life technologies corp, carlsbad, ca) ( figure a ). expression levels of target genes were normalized to the housekeeping gene β-actin (Δct). gene expression values were calculated based on the ΔΔct method, with data for all samples analyzed against the mean value of four replicates. tlr showed a -fold greater expression than that of tlr and tlr (student's t test, p < . ), and the latter two had a more than -fold greater expression than tlr , , , , and (student's t test, p < . ). tlr and tlr transcripts were expressed to the highest and lowest levels, respectively (student's t test, p < . ). the expression of tlr , tlr , tlr , and tlr proteins was analyzed using flow cytometry (facs). as shown in figure b , facs data demonstrated robust expression of cell-surface tlr and tlr and intracellular tlr and tlr in naïve j a. cells. next we determined whether tlr , tlr , tlr , and tlr are functional in j a. cells. activation of the cells with a tlr ligand (hklm, cells/ml), a tlr agonist (poly i:c, μg/ml), a tlr ligand (lps, μg/ml), or a tlr agonist (imiquimod (r ), μg/ml) for , , and h resulted in the robust production of il- and tnf-α ( figure a ). this result indicates that, in j a. macrophages, tlr - and tlr are fully functional and signal with cytokine secretion after stimulation. we assessed type i ifn mrna induction in tlr-activated j a. macrophages. expression of ifn-α and ifn-β genes was up regulated by poly i:c, lps, and r ligands in j a. cells with different kinetics ( figure b ). lps-induced ifn-β and ifn-α mrnas peaked at h and h post-stimulation, respectively. r -induced ifn-β and ifn-α mrnas peaked at h post-stimulation. the induction of type i ifn gene expression after lps and r was not sustained (in contrast to ifn-β gene expression after poly i:c stimulation). ifn-α and ifn-β levels were determined by elisa in cell-free supernatants collected after h of prestimulation with hklm ( cells/ml), lps ( μg/ml), r ( μg/ml), and poly i:c ( . μg/ml). interestingly, activation of tlr but not of tlr , tlr or tlr triggered robust production of ifn-β following pre-stimulation for h in macrophages ( figure c ). similar to ifn-β, ifn-α was secreted only in tlr -activated cells, albeit to a much lesser degree ( figure c ). these contrasting results suggest that type i ifn response may be regulated differentially on tlr stimulation at the post transcriptional level in j a. cells. their lack of ifn secretion (as measured by elisa) in response to stimulation with the bacterial ligands hklm and lps is somewhat unpredicted and deserves further investigations. in addition, although it is well established that type i ifn response against rna viruses is mainly mediated by pdcs via a tlr -dependent pathway, the role of tlr in macrophage activation remains poorly understood. in this regard, the absence of ifn-β secretion after r stimulation of tlr in j a. cells may suggest differences in the regulation of the tlr pathway and/or its effectors between macrophages and pdcs. we are currently investigating the role of macrophage tlr in the antiviral response against rna viruses. furthermore, mhvs have been shown to productively infect j a. cells [ ] . by combining these results, we established a valid in vitro model in which to investigate the effect of triggering tlrs with selected ligands on mhv infectivity in macrophages. effect of triggering tlr , tlr , tlr , and tlr on virus production in mhv-infected j a. macrophages. j a. cells were prestimulated with tlr ligands for h, infected with mhv-a , mhv-jhm or mhv- ( moi) by adsorption for h in the absence of the ligands, and activated for up to h p.i. with the appropriate tlr agonist. tlr ligands were used as follows: hklm (tlr ) at cells/ml; lps (tlr ) at μg/ml; imiquimod (r ) (tlr ) at μg/ml. poly i:c (tlr ) was tested at a range of concentrations ( . , . , and . μg/ml). because poly i:c triggered a comparable effect on mhv production at all concentrations (data not shown), viral titers at . μg/ml were included in the plot. cells incubated with the basal medium before and during infection served as a negative control for the effect of tlr triggering on virus production. mhv titers were assessed in cell-free supernatants using a plaque assay on l fibroblasts. the data shown are the mean viral titers of three independent experiments, each done in duplicate ± standard deviation (*p value relative to virus alone, p < . student's t test). to test whether treatment with the tlr ligands hklm, poly i:c, lps, and r affected the replication of mhv, j a. cells were prestimulated with the appropriate ligand at the above-mentioned concentrations for h; and cells were infected with mhv-a , mhv-jhm, or mhv- at a multiplicity of infection (moi) of by adsorption for h in the absence of the ligands and stimulated again for up to h postinfection (p.i.) with the appropriate tlr agonist. therefore, there were two challenges with tlr ligands: one before and one after virus adsorption. activation of macrophages with tlr , tlr and tlr did not noticeably affect mhv production in j a. macrophages ( figure ). conversely, the triggering of tlr with poly i:c significantly inhibited mhv-a , mhv-jhm, and mhv- production relative to virus alone (student's t test, p = . ; figure ). complete suppression was observed only in poly i:c-treated mhv-jhm-and mhv- -, infected macrophages, although all mhv strains showed a dramatic -log reduction in virus production. the lack of complete suppression in mhv-a infected cells could be explained by the ability of mhv-a to grow to higher titers ( - log) than mhv-jhm and mhv- in macrophages. additionally, these results may also suggest that mhv-a counteracts the tlr pathway in j a. macrophages. indeed, our data shows that tlr -mediated, ifn-β secretion is significantly reduced in mhv-a -infected macrophages ( figure ). interestingly, poly i:c triggered comparable antiviral effect regardless of its concentration ( . , . , and . μg/ml; data not shown). it will be of interest to determine the minimal antiviral concentration of poly i:c in future experiments. the optimal concentration range for poly i:c was selected based on the highest rate of cytokine production (il- elisa) and minimal cytotoxicity (ldh cytotoxicity assay) in j a. macrophages activated with poly i:c at various doses (data not shown). collectively, these data demonstrate that, depending on the receptor, ligand-mediated tlr stimulation exerts differential effects on mhv production. triggering tlr with poly i:c, but not activation of tlr , tlr , or tlr with their respective ligands, impairs mhv replication in macrophages with a comparable magnitude of suppression of viral titers for mhv-a , mhv-hjm, and mhv- strains. given that all four tlr ligands induced strong il- and tnf-α proinflammatory responses (figure a ), we concluded that the inability of tlr , tlr and tlr agonists to protect macrophages from mhv infection is not due to the lack of signaling through these receptors, rather it stems from the absence of ifn-α and ifn-β production after h of stimulation with their ligands ( figure c ). in contrast, the antiviral effect mediated by activation of tlr with poly i:c is associated with a sustained transcriptional upregulation and secretion of ifn-α and ifn-β. we investigated the optimal conditions for poly i:c antiviral effects in j a. macrophages infected with a recombinant mhv-a expressing the gfp protein (ra -gfp) ( moi) and treated as follows: ( ) prestimulated with poly i:c, with no drug present during infection (poly i:c +/−); ( ) treated with poly i:c only after virus adsorption (poly i:c −/+); ( ) treated with poly i:c before and after virus adsorption (poly i:c +/+). the tlr ligand was used at concentrations of . to . μg/ml for h of prestimulation and/or h p.i. a profound suppression of gfp expression in cells stimulated with . μg/ml poly i:c was observed with all of the above-mentioned treatments relative to infected macrophages in the absence of the drug ( figure a ). thus, a single challenge with the tlr ligand before or after virus adsorption was sufficient to trigger a robust antiviral effect comparable to cells challenged with poly i:c twice. to determine the level of mhv production, released virus was quantified by plaque assay in cell-free supernatants from macrophages stimulated with . and . μg/ml poly i:c as above and in the absence of the drug ( figure b ). regardless of the concentration of poly i:c, the triggering of tlr with poly i:c resulted in a -log reduction in ra -gfp titers in prestimulated and coactivated macrophages (poly i:c +/+) relative to infected cells in the absence of the drug ( figure b , p < . ). cells challenged with poly i:c once before (poly i:c +/−) or h after mhv adsorption (poly i:c −/+) also exhibited a significant suppression (p < . ) of virus production comparable to that of prestimulated and coactivated macrophages (poly i:c +/+). interestingly, the triggering of tlr before adsorption with mhv (poly i:c +/−) resulted in significantly lower virus production relative to coactivated macrophages (poly i:c −/+) (p = . and p = . for . and . μg/ml poly i:c, respectively), suggesting that a single challenge with poly i:c prior to infection dramatically reduces macrophage susceptibility to mhv infection. taken together, these results indicate that . μg/ml of poly i:c is sufficient to trigger a profound tlr -mediated antiviral effect and that prestimulation alone is enough to protect macrophages from infection with mhv. to investigate the mechanism of the poly i:c-triggered antiviral effect in mhv-infected macrophages, we wanted to determine if the tlr ligand induced soluble factors that mediated protective immunity against cov infection. we pretreated j a. cells for h with conditioned medium (cm) from macrophages prestimulated with tlr - and tlr ligands for h (figure a ). next, cells were infected with ra -gfp ( moi) for h adsorption in the absence of tlr ligands and incubated for additional h in basal medium. ra -gfp virus titers determined by plaque assay are shown in figure . as expected, cm from mock macrophages (unstimulated, uninfected) did not affect virus production. treatment with cm from macrophages stimulated with hklm, r , and lps did not affect virus production ( figure ). remarkably, there was a -log reduction in ra -gfp titers in cells pretreated with poly i:c cm ( figure , p < . ) that correlated with the inhibition of gfp expression in these cells (data not shown). i. cells pretreated with the cm from mock cells or with fresh basal medium served as negative controls for the tlr-triggered effect. ra -gfp titers were assessed in cell-free supernatants using a plaque assay on l fibroblasts. error bars represent the standard error of the mean of two replicates (p value relative to cells pretreated with cm from mock, p < . , student's t test). overall, these data suggest that prestimulation with poly i:c but not hklm, lps, or r triggers the production of soluble factors that further protect macrophages from infection with mhv on subsequent exposure. in addition to soluble factors, residual poly i:c in the cm may have also contributed to the antiviral effect in cells pretreated with tlr -stimulated supernatants. our data in figure c demonstrated that after h of stimulation with hklm, lps, r , and poly i:c, ifn-β and ifn-α were secreted only in tlr -activated j a. cells. these results together with the antiviral effect of poly i:c (figure ) suggested that the protective role of tlr against coronavirus infection in j a. macrophages may be mediated by type i ifn. to investigate the role of type i ifn in the poly i:c-mediated inhibition of murine cov production in macrophages, we focused on mhv-a and mhv-jhm strains. we hypothesized that the differential effect of tlr ligands on mhv production is due to their variable ability to induce type i ifn crucial for triggering an "antiviral state" and protecting cells from virus infection [ , ] . to test this hypothesis, we assessed type i ifn production in tlr-stimulated and/or mhv-infected j a. a second challenge with poly i:c for h resulted in a robust secretion of ifn-α and ifn-β ( figure ). in contrast, a single challenge with poly i:c for h induced lower levels of ifn-β and levels of ifn-α that were close to the limit of detection of the elisa assay ( figure c ). such a pattern of induction of type i ifn in cells treated with dsrna (like poly i:c) is consistent with the activation of two types of type i ifn genes, immediate-early and delayed-type genes (reviewed in ref. [ ] ). immediate-early genes, mostly ifn-β and some ifn-α (only in murine cells), are induced by a protein-synthesis-independent pathway. secreted ifn signals in both an autocrine and paracrine fashion through the type i ifn receptor and triggers delayed-type ifns (including other ifn-α subtypes). expression of the delayed-type ifn depends on de novo protein synthesis and results in amplification of the ifn response. similarly, poly i:c-activated j a. macrophages exhibit high levels of ifn-β and a modest secretion of ifn-α on induction of the immediate-early gene. later, these cells secrete comparably high levels of both ifn-α and -β as a result of delayed-type ifn gene expression. figure ). the effect of infection with mhv on poly i:c-triggered ifn-β induction was previously assessed in ci- murine fibroblasts [ ] . in that study, neither mhv-a nor mhv-jhm inhibited ifn-β induction after poly i:c was transfected into fibroblasts (a way to activate rig-i and mda cytoplasmic helicases but not endosomal tlr ). thus, the ability of mhv to counteract poly i:c-induced ifn-β is cell type-specific and depends on the mode of delivery of poly i:c into the cell. targeting rig-i and mda helicases by poly i:c transfection with a lipid carrier as reported by [ ] , did not result in mhv-mediated inhibition of poly i:c-induced ifn-β secretion. in contrast, our data suggest that mhv-a might counteract the ifn-β response when macrophages are stimulated with soluble poly i:c to trigger the tlr pathway. further experiments are needed to define how mhv-a might counteract the tlr pathway in macrophages. interestingly, mhv-a has been reported to develop various measures to counteract the type i ifn response [ ] [ ] [ ] . besides poly i:c, the tlr ligand r induced a modest level of secretion of ifn-β with h stimulation; tlr and tlr agonists did not promote type i ifn secretion in j a. macrophages as measured by elisa ( figure ). overall, these findings are in agreement with our hypothesis that poly i:c triggers an antiviral effect via ifn-α/β, whereas the lack of a strong type i ifn production during tlr , tlr , or tlr signaling is responsible for uncontrolled mhv production in j a. macrophages. figure . il- and tnf-α production in cell-free supernatants from j a. macrophages stimulated with tlr ligands, mhv-infected, and/or coactivated during mhv infection. j a. cells were prestimulated for h with hklm ( cells/ml), lps ( μg/ml), r ( μg/ml), and poly i:c ( . μg/ml); media was removed and: ) a second challenge of the corresponding tlr ligand (same concentrations) was added to the cells for h; ) cells were prestimulated for h with the tlr ligands as above; media was removed and cells infected with mhv-a or mhv-jhm at . moi for h of adsorption in the absence of the ligands; after virus adsorption cells were stimulated with a second challenge of the tlr ligands using the same concentrations as during prestimulation and samples were taken at h; ) cells were not tlr activated and only infected with mhv-a or mhv-jhm at . moi for h of adsorption in the absence of the ligands; fresh media was added to the cells for h. non-stimulated, non-infected j a. cells were used as mock control. cell-free supernatants were taken at h from tlr-activated alone, infected alone, and tlr-activated and infected and assessed for il- and tnf-α using elisa. error bars represent the standard error of the mean of two independent experiments, each done in duplicate. to further rule out the potential antiviral effect of il- and tnf-α in infected and co-stimulated cells, we measured proinflammatory cytokines in the same samples as above. stimulation for h resulted in a very high induction of both cytokines after lps (a tlr ligand that based on our data does not induce antiviral effect against infection with mhv in j a. macrophages) and poly i:c (albeit to a lesser extent than with lps) (figure ) . overall, these data argue against the potential antiviral effects of il- and tnf-α in tlr -activated macrophages. tnf-α levels were reduced in mhv-a and mhv-jhm infected macrophages relative to mock cells (figure ) . although it was not the focus of the present study, the inhibitory effect of mhv on the basal tnf-α levels may be mediated through the action of the anti-inflammatory cytokine il- . il- is a known negative regulator of tnf-α production and function in macrophages (reviewed in ref. [ ] ). mhv-a was reported to induce il- in infected primary bone marrow-derived macrophages [ ] , therefore, one could speculate that covs suppress basal macrophage tnf-α secretion through triggering of il- . future studies will be designed to test this hypothesis. although tnf-α was reported to induce a strong antiviral response against various influenza strains in lung epithelial cells [ ] , our data demonstrated that tlr-induced tnf-α does not affect mhv production in macrophages. collectively, these results indicate that il- and tnf-α are not responsible for and do not contribute to a poly i:c-triggered antiviral effect in mhv-infected macrophages. considering that soluble factors mediate the poly i:c-triggered antiviral effect ( figure ) and that poly i:c potently induces type i ifn before and during virus infection ( figures c and ) , we further confirmed the role of ifn-β in tlr -triggered mhv suppression in macrophages ( figure a-c) . we focused on ifn-β because unlike ifn-α, ifn-β was profoundly induced in prestimulated macrophages at h poststimulation ( figure c ), and cm from macrophages treated with poly i:c for h exhibited inhibition of mhv-a production sufficient to reduce viral infection ( figure ). titration of anti-ifn-β antibody (ab) was done to establish the optimal ab concentration for neutralization of poly i:c-stimulated ifn-β. j a. macrophages were stimulated with . μg/ml poly i:c in the presence or absence of anti-ifn-β ab at various concentrations. we chose . μg/ml poly i:c because prestimulation with such a low concentration of the tlr ligand was sufficient to promote a strong antiviral effect (figure ). poly i:c-triggered ifn-β was significantly reduced by u/ml to u/ml of the ifn-β neutralizing ab, and it was suppressed in the presence of a higher concentration ( u/ml) ( figure a ). after stimulation, cells were infected with ra -gfp ( moi) by adsorption for h and incubated in fresh medium for up to h p.i. cm from mock or ifn-β ab-treated macrophages did not affect mhv-a virus production in these cells ( figure b,c) . as expected on the basis of our previous results, pretreatment with poly i:c-conditioned medium resulted in a drastic reduction in ra -gfp expression and in a -log reduction in mhv production in infected macrophages ( figure b,c) . importantly, mhv-a infection was significantly restored (p = . ) in j a. cells incubated with the supernatant from macrophages activated with poly i:c in the presence of the ifn-β neutralizing polyclonal ab (poly i:c + ifn-β ab cm) ( figure b,c) . this result indicates that ifn-β is a crucial mediator in the antiviral response against mhvs elicited by triggering tlr with poly i:c in macrophages. murine covs are sensitive to pretreatment of macrophages with recombinant ifn-β [ ] . in the present study prestimulation of j a. macrophages with poly i:c, a potent type i ifn inducer, resulted in a strong ifn-β response that triggered antiviral immunity and protected macrophages from mhv infection before and after virus adsorption. in support of our data, a poly i:c analog ampligen tm (poly i:poly c u) was successfully tested in sars-cov animal models [ , ] . balb/c mice were treated with ampligen tm intraperitoneally (i.p.) h before sars-cov infection and then the virus titers in the lungs were assessed days after virus exposure. sars-cov titers in the lungs were below the limit of detection suggesting that poly i:poly c u completely protected mice from viral infection [ ] . in a different study, ampligen tm was given intraperitoneally to balb/c mice h before they were infected with the mouse-adapted sars-cov strain v . treated mice exhibited complete survival, suppressed virus titers in the lungs, significantly reduced lung scores and weight loss [ ] . these studies did not investigate the mechanism of the ampligen tm -triggered antiviral effect in sars-cov-infected mice; type i ifn, however, was proposed as a mediator of antiviral immunity. ampligen tm is indeed a potent type i ifn inducer that acts through tlr [ ] and triggers protection from hiv [ , ] , coxsackie virus [ ] , punta toro virus [ ] , venezuelan equine encephalitis virus [ ] , and influenza virus [ ] infections. overall, our data demonstrates that tlr triggered type i ifn inhibits murine cov infection of macrophages. j a. murine macrophages (attc, tib- ) were cultured in dulbecco's modification of eagle's medium (dmem) (cellgro, mediatech, manassas, va, usa) supplemented with % v/v heat-inactivated fetal bovine serum (hifbs) (hyclone, thermo scientific, rockford, il, usa), u/ml penicillin (cellgro), μg/ml streptomycin (cellgro), . g/l sodium bicarbonate (biowhittaker, lonza, walkersville, md, usa) and mm glutamine (cellgro). cells were incubated in a humidified atmosphere at c with % co . the dualtropic mhv-a , and neurotropic mhv-jhm strains were previously characterized [ , ] . the hepatotropic mhv- strain was provided by dr. julian leibowitz (texas a&m university). recombinant mhv-a virus expresses the gfp in place of the orf [ ] . cells were infected at moi by adsorption for h in the basal medium. then, excess virus inoculum was removed and cells were incubated for up to h p.i. plaque assays were performed on l murine fibroblasts [ ] . % confluent monolayers of l cells were infected with -fold dilutions of samples in dmem supplemented with % v/v hifbs. after virus adsorption, cells were overlaid with a : mixture of . % agarose and % fbs in dmem and incubated for h at c with % co . to visualize viral plaques, infected cells were overlayed with equal parts of . % agarose, % fbs in dmem and / neutral red (harleco, emd chemicals inc., gibbstown, nj, usa) and incubated for h at c with % co . all tlr agonists were purchased from invivogen (san diego, ca, usa). the following tlr ligands were used: hklm for tlr at cells/ml; poly i:c for tlr at . to μg/ml; ultrapure lps from e. coli for tlr at μg/ml; and imiquimod (r ), an imidazoquinoline amine analogue of guanosine, for tlr at μg/ml. cells were prestimulated with the appropriate tlr ligand for h, infected as necessary and coactivated during infection with the corresponding tlr agonist for up to h p.i. mouse il- and tnf-α were quantified by elisa (ebioscience, san diego, ca, usa) according to the manufacturer's instructions. limits of detection: . - pg/ml for il- and . - pg/ml for tnf-α. verikine mouse ifn-α and verikine mouse ifn-β elisa kits were purchased from pbl interferon source (thermoscientifc, rockford, il, usa). limits of detection: . - pg/ml for ifn-β, and . - pg/ml for ifn-α. to titrate anti-ifn-β ab, j a. macrophages were activated with . μg/ml poly i:c in the presence or absence of anti-ifn-β ab at , , or u/ml for h. rabbit polyclonal anti-ifnβ ab were purchased from calbiochem (emd chemicals inc.). poly i:c-triggered ifn-β with or without neutralizing ab was assessed with a verikine ifn-β elisa kit. cell-free cm from poly i:cactivated and/or u/ml anti-ifn-β ab-incubated macrophages was used to pretreat j a. cells for h. then macrophages were infected with moi mhv-a by adsorption for h and incubated in fresh medium for up to h p.i. total rna was isolated using trizol ® reagent (invitrogen, carlsbad, ca, usa). total rna ( ng) of was transcribed into cdna with the superscript ii reverse transcriptase kit (invitrogen), using a total reaction mix volume of μl; . μl cdna was combined with . μl of taqman universal master mix (applied biosystems, life technologies, carlsbad, ca, usa), μl diethyl polycarbonate (depc)-treated water, and . μl murine tlr to tlr taqman gene expression assays (applied biosystems). dna was amplified using the applied biosystems real-time pcr machine, and cycle threshold values (c t ) were recorded. basal tlr - mrna levels were expressed as ΔΔc t values relative to s rrna [ΔΔc t = Δc t(tlr) − Δc t( s rrna) ]. tlr mrna expression levels were expressed as fold changes relative to mock values, using the variable −ΔΔct . ifn- α and ifn-β gene expression was quantified as above using specific pre-developed taqman gene expression assays (applied biosystems). expression of cell surface tlr and tlr , and endosomal tlr and tlr was determined using a facscalibur flow cytometer (bd biosciences, san jose, ca) and data were processed using flowjo software (treestar, ashland, or). cells were stained and analyzed by using a fixation & permeabilization kit (ebioscience) following the recommended protocols. specific antibodies against murine tlr , , , and , fluorescence-labeled secondary antibodies, isotype controls, and anti-fcrr (for blocking) were purchased from invivogen, imgenex (san diego, ca), and ebioscience (san diego, ca). gfp expression was analyzed in live or % paraformaldehyde fixed cells. cells were incubated with nm dapi ( ', '-diamidino- -phenylindole dilactate; invitrogen, molecular probes, eugene, or, usa) for nuclei stain for min at rt. images were obtained with an olympus x motorized inverted fluorescence microscope (center valley, pa, usa) using digital microscopy software (slidebook™ . software, intelligent imaging innovations, denver, co). an unpaired two-tail student's t test was used to determine statistical significance. all data were analyzed with graphpad prism (graphpad software, inc., ca, usa) our goal was to investigate the effects of triggering tlr , tlr , tlr and tlr with selected ligands on j a. macrophages susceptibility to mhv infection. our data demonstrates that triggering of tlrs with hklm (tlr agonist), lps (tlr agonist), and r (tlr agonist) does not affect mhv-a , mhv-jhm, and mhv- production. the absence of tlr -, tlr -, and tlr -mediated antiviral effects is not explained by their lack of expression or activation in j a. macrophages; rather, it is based on the weak induction of type i ifn. in contrast, stimulation of macrophages with poly i:c added to the cells to trigger endosomal tlr , induces a strong type i ifn-dependent antiviral response. neutralization of ifn-β successfully restored the poly i:c-inhibited production of mhv-a in macrophages. taken together, activation of tlr by poly i:c may be a successful antiviral approach against covs in vivo; its therapeutic potential as a curative drug remains to be established in future investigations. coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol pathogenesis of murine coronavirus in the central nervous system coronaviruses post-sars: update on replication and pathogenesis coronavirus infection of the central nervous system: host-virus stand-off pathogenesis of acute and chronic central nervous system infection with variants of mouse hepatitis virus, strain jhm the pathogenesis of murine coronavirus infection of the central nervous system murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia macrophage interleukin- and tumour necrosis factor-alpha are 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activation promotes interleukin- production and virus replication in cultured cells master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production accessory protein a is a major antagonist of the antiviral action of interferon against murine coronavirus plp of mouse hepatitis virus a (mhv-a ) targets tbk to negatively regulate cellular type i interferon signaling pathway regulation of interferon and toll-like receptor signaling during macrophage activation by opposing feedforward and feedback inhibition mechanisms tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo tlr is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c u), but not poly(i:c): differential recognition of synthetic dsrna molecules mismatched dsrna (ampligen) induces protection against genomic variants of the human immunodeficiency virus type (hiv- ) in a multiplicity of target cells mismatched double-stranded rna (polyi-polyc( )u) is synergistic with multiple anti-hiv drugs and is active against drug-sensitive and drug-resistant hiv- in vitro the interferon inducer ampligen [poly(i)-poly(c u)] markedly protects mice against coxsackie b virus-induced myocarditis hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection polyi:polyc u adjuvant-combined intranasal vaccine protects mice against highly pathogenic h n influenza virus variants replicase genes of murine coronavirus strains a and jhm are interchangeable: differences in pathogenesis map to the ' one-third of the genome enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system murine coronavirus evolution in vivo: functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors would like to thank drs. paul s masters, julian leibowitz, and susan r weiss for reagents. this work was supported in part by public health service grants ns , ai and dk to s.n.m, and ns to j.m.g., from the national institute of neurological disorders and stroke (ns), the national institute of allergy and infectious disease (ai), and the national institute of diabetes and digestive and kidney diseases; and by drexel university college of medicine's internal funds to snm. the founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. pamela fried (drexel university college of medicine academic publishing services) is acknowledged for manuscript editing. the authors declare no conflict of interest. key: cord- - hcujmuo authors: savarin, carine; stohlman, stephen a; hinton, david r; ransohoff, richard m; cua, daniel j; bergmann, cornelia c title: ifn-γ protects from lethal il- mediated viral encephalomyelitis independent of neutrophils date: - - journal: j neuroinflammation doi: . / - - - sha: doc_id: cord_uid: hcujmuo background: the interplay between ifn-γ, il- and neutrophils during cns inflammatory disease is complex due to cross-regulatory factors affecting both positive and negative feedback loops. these interactions have hindered the ability to distinguish the relative contributions of neutrophils, th and th cell-derived effector molecules from secondary mediators to tissue damage and morbidity. methods: encephalitis induced by a gliatropic murine coronavirus was used as a model to assess the direct contributions of neutrophils, ifn-γ and il- to virus-induced mortality. cns inflammatory conditions were selectively manipulated by adoptive transfer of virus-primed wild-type (wt) or ifn-γ deficient (gko) memory cd (+) t cells into infected scid mice, coupled with antibody-mediated neutrophil depletion and cytokine blockade. results: transfer of gko memory cd (+) t cells into infected scid mice induced rapid mortality compared to recipients of wt memory cd (+) t cells, despite similar virus control and demyelination. in contrast to recipients of wt cd (+) t cells, extensive neutrophil infiltration and il- expression within the cns in recipients of gko cd (+) t cells provided a model to directly assess their contribution(s) to disease. recipients of wt cd (+) t cells depleted of ifn-γ did not express il- and were spared from mortality despite abundant cns neutrophil infiltration, indicating that mortality was not mediated by excessive cns neutrophil accumulation. by contrast, il- depletion rescued recipients of gko cd (+) t cells from rapid mortality without diminishing neutrophils or reducing gm-csf, associated with pathogenic th cells in cns autoimmune models. furthermore, co-transfer of wt and gko cd (+) t cells prolonged survival in an ifn-γ dependent manner, although il- transcription was not reduced. conclusions: these data demonstrate that il- mediates detrimental clinical consequences in an ifn-γ-deprived environment, independent of extensive neutrophil accumulation or gm-csf upregulation. the results also suggest that ifn-γ overrides the detrimental il- effector responses via a mechanism downstream of transcriptional regulation. il- and ifn-γ play diverse and often opposing functions during microbial infections, as well as autoimmune diseases. these interactions are partially attributed to their distinct regulation of the neutrophil response. both il- a and il- f signal through the il- r to induce granulocyte colony-stimulating factor and stem cell factor, thereby expanding neutrophil progenitors in the bone marrow and spleen as well as increasing mature neutrophils in the blood [ ] [ ] [ ] . il- also induces elr + cxc chemokines, which attract neutrophils [ , ] . by contrast, ifn-γ opposes neutrophil recruitment by downregulating expression of neutrophil chemoattractants [ ] . analysis of polarized t cell subsets and genetically deficient mice has provided insight into the distinct effector functions of il- and ifn-γ; however, the interplay between il- and ifn-γ in vivo remains complex [ , ] . moreover, downstream effector mechanisms mediating pathological consequences may be tissue-and pathogen-specific and are largely unresolved. for example, th cell-mediated protection is critical during bacterial pneumonia [ ] . il- -mediated neutrophil recruitment to the infection site also indicates a protective role for th cells during oropharyngeal candidiasis [ ] . by contrast, th -mediated inhibition of both protective th responses and antimicrobial neutrophil functions increased tissue destruction following gastric candidiasis and pulmonary aspergillosis [ ] . these differences may reflect distinct infection sites, as indicated by the distinct immune responses to candida albicans, which are dependent upon the anatomical site of infection [ ] . viral infections are often dominated by th responses. however, the coemergence of th and th cells has recently been documented in several infections, including human immunodeficiency virus [ ] , simian immunodeficiency virus [ ] and cytomegalovirus [ ] . a deleterious role of il- is implied by acute lung injury associated with il- -mediated neutrophil recruitment during influenza virus infection [ ] . by contrast, th responses are protective against lethal influenza virus infection in il- -deficient mice [ ] . similarly, ifn-γmediated protection during herpes simplex virus- corneal infection correlated with reduced il- production and subsequent neutrophil infiltration [ ] . however, the function of il- during central nervous system (cns) viral infections, including human immunodeficiency virus encephalitis, is unclear, although th cells promote theiler's murine encephalomyelitis virus persistence and chronic demyelination by limiting the antiviral cytotoxic t-lymphocyte response [ ] . in contrast to the limited information on il- function during viral encephalitis, analysis of experimental autoimmune encephalitis (eae) has revealed numerous insights into effector mechanisms as well as crosstalk between th and th cells [ ] . although the inflammatory cns disease multiple sclerosis and its animal model eae were historically associated with a th immune response [ , ] , a pro-inflammatory role of ifn-γ was contradicted by substantially increased disease severity and mortality in mice deficient in ifn-γ (gko) or the ifn-γr [ , ] . the correlation between increased eae severity, enhanced th responses and neutrophil infiltration into the cns of gko mice suggested that ifn-γ might be protective by inhibiting the th response [ ] . although il- −/− mice are susceptible to eae [ ] , adoptive transfer of polarized encephalitogenic cd + t cells support th cells as detrimental participants in eae [ , ] . however, the pathogenic mechanisms associated with th cells remain an ongoing challenge and may involve multiple pathways. these include excessive cns neutrophil infiltration and release of degrading enzymes, free radicals and pro-inflammatory cytokines, direct il- -mediated neuronal toxicity [ ] , and/or secretion of granulocyte macrophage colonystimulating factor (gm-csf) as the pathogenic effector molecule [ ] [ ] [ ] . these data suggest that the balance between ifn-γ and il- effector functions, as well as their regulation of neutrophils may dictate the outcome of non autoimmune-driven cns inflammation, such as viral encephalitis. during encephalomyelitis induced by the strain designated jhmv, cd + t cells not only contribute to antiviral effects by enhancing cd + t cell function within the cns [ ] but also mediate viral control in absence of cd + t cells [ ] . nevertheless, they also contribute to both clinical disease and demyelination [ ] . to define the role of cd + relative to cd + t cells in viral encephalitis, memory cd + t cells from immunized donors were transferred into infected severe combined immunodeficiency (scid) mice [ ] . this study revealed an early morbidity and mortality in infected recipients of cd + t cells lacking the ability to secrete ifn-γ compared to recipients of ifn-γ-sufficient cd + t cells or infected unreconstituted control mice [ ] . notably, both memory populations were equally effective in controlling virus replication [ ] . the lethal outcome was specific for cd + t cells lacking ifn-γ [ ] , but not for a similar memory cd + t cell population deficient in ifn-γ [ ] . these data suggest that mortality was related to immune effector functions specific to cd + t cells and controlled by ifn-γ. in this study, scid recipients of gko cd + t cells infected with jhmv were characterized by extensive neutrophil accumulation and il- expression within the cns. neutrophil infiltration in the absence of ifn-γ correlated with significantly elevated levels of cxcl , independent of il- . moreover, comparison of infected recipients of wild-type (wt) cd + t cells depleted of ifn-γ and recipients of gko cd + t cells depleted of il- revealed mortality was due to il- , irrespective of abundant neutrophil accumulation. ifn-γ introduced by co-transfer of wt cd + t cells with il- -producing gko cd + t cells abrogated the detrimental effects of il- without affecting il- transcription within the cns. these data thus segregate the effects of toxic neutrophil components from il- -mediated pathogenesis. homozygous balb/c thy . mice, provided by dr. j. harty (university of iowa, iowa city, ia, usa) and gko balb/c mice, provided by dr. r. coffman (dnax research, palo alto, ca, usa), were bred locally at the cleveland clinic. scid mice were obtained from the national cancer institute (frederick, md, usa). recipients and donors were maintained under sterile conditions and all procedures were performed in compliance with cleveland clinic institutional animal care and use committee-approved protocols. the gliatropic jhm strain of mouse hepatitis virus (jhmv)-neutralizing mab variant designated . v- was used for intracerebral infection [ ] . jhmv was propagated and plaque assayed on monolayers of dbt cells, a continuous murine astrocytoma cell line [ ] . scid mice were injected in the left hemisphere with μl volume containing pfu of jhmv diluted in endotoxin-free dulbecco's modified pbs. the severity of the jhmvinduced clinical disease was graded as follows: , healthy; , ruffled fur and hunched back; , partial hind limb paralysis or inability to turn to the upright position; , complete hind limb paralysis; , moribund or dead. virus titers were determined on plaque assay on monolayers of dbt cells as previously described [ , ] . briefly, brains were homogenized in ice-cold dulbecco's pbs using ten broeck tissue homogenizers (kimble chase, vineland, nj, usa). after clarification by centrifugation at x g for minutes at °c, supernatants were stored at − °c whereas pellets containing cnsderived cells were suspended in percoll (ge healthcare bio-sciences ab, uppsala, sweden) and used for flow cytometry analysis (see below). balb/c thy . and gko donors were immunized by intraperitoneal (i.p.) injection with × pfu of jhmv. donor splenocytes were prepared four to sixteen weeks post immunization. cd + t cells were purified by positive selection using anti-cd -coated magnetic beads (miltenyi biotec inc., auburn, ca, usa). purity of the purified population was assessed by flow cytometry using fluorescein isothiocyanate-(fitc) labeled anti-cd (clone gk . ), phycoerythrin-(pe) labeled anti-cd (clone - . ) and peridinin chlorophyll protein-(percp) labeled anti-cd (clone d ) mabs (bd pharmingen, san diego, ca, usa). recipients received × donor cd + t cells composed of % thy . (wt), % gko or a / % mixture of thy . /gko (wt/gko) cd + t cells by intravenous (i.v.) injection coupled with a single i.p. injection of μg of anti-cd mab (clone tib. ). mice were challenged with virus two to three hours after adoptive transfer. for neutrophil depletion, mice received i.p. injections of either μg of anti-ly- g (clone a ) or anti-gr (clone rb - c ) mab every other day until sacrifice, starting two days before infection. depletion was confirmed in both cases by flow cytometric analysis using anti-ly- g (clone a ) mab in addition to examination of hematoxylin and eosin-(h&e) stained sections of brain. only data for the anti-ly g experiments are shown. no differences in survival relative to control-treated mice were observed following treatment with either neutrophil-depleting mab. similarly, for anti-ifn-γ treatment, mice received i.p. injections of μg of anti-ifn-γ (clone xmg . ) mab every other day, starting two days before infection. for anti-il treatment, mice received i.p. injections of mg of anti-il- a (clone d ) mab at day zero and six post infection (p.i.). after brain homogenization and centrifugation to obtain supernatants for virus determination as described above, cell pellets were resuspended in rpmi containing mm hepes, ph . and adjusted to % percoll (ge healthcare bio-sciences ba). a ml underlay of % percoll was added prior to centrifugation at x g for minutes at °c. cells were recovered from the % to % interface and washed with rpmi medium prior to analysis. cns mononuclear cell suspensions were blocked with anti-mouse cd /cd (clone . g , bd pharmingen) mab on ice for minutes prior to staining. cells were then stained with fitc-, pe-, percp-or allophycocyaninconjugated mab for minutes on ice in pbs containing . % bsa. expression of surface molecules was characterized using the following mabs (all obtained from bd pharmingen except when indicated): anti-cd (clone ly- ), anti-cd (clone gk . ), anti-thy . (clone ox- ), anti-cd (clone - . ), anti-cd b (clone m / ), anti-f / (serotec, oxford, uk), anti-ly g (clone a ) and anti i-a/i-e (clone g ). samples were analyzed on a facscalibur flow cytometer using cellquest software (becton dickinson, mountain view, ca, usa). rna was isolated from three or more individual brains per group using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. cdnas were prepared using superscript ii reverse transcriptase (invitrogen) and oligo (dt) - primers (invitrogen). semiquantitative rna expression was assessed using light-cycler and sybr green kit (roche, basel, switzerland) and the following primers; ubiquitin: f: taqman primers and x taqman fast master mix (applied biosystems, carlsbad, ca, usa) were used to assessed cxcl and ccl mrna levels. levels of mrna expression were normalized to ubiquitin mrna using Δct method as previously described [ ] . after ice-cold pbs perfusion, brains in oct were frozen in liquid nitrogen and stored at − °c until μm sections were prepared. sections were fixed with methanol/ acetone ( : ratio) for minutes and then treated with blocking solution for minutes at room temperature. rat anti-mouse il- (r&d systems, minneapolis, mn, usa) and hamster anti-mouse cd primary mabs (serotec) were incubated overnight at °c. alexa fluor goat anti-rat (invitrogen) and alexa fluor goat antihamster (molecular probes, eugene, or, usa) were added for hour at room temperature. sections were mounted with vectashield mounting medium with '- -diamidino- -phenylindole (dapi) (vector laboratories, burlingame, ca, usa) and analyzed using a leica dm b fluorescent microscope (leica, wetzlar, germany). cytokine expression by cd + t cells derived from cervical lymph nodes of scid recipients were analyzed directly at day eight p.i. without stimulation with viral antigen. for analysis of cytokine production by cells prior to transfer, jhmv was adsorbed to donor splenocytes for minutes at °c and cells cultured for six days in rpmi complete, % fcs at . × cells/ml. cytokine production from both splenic cultures or ex vivo lymph node cells was measured following four hours stimulation with pma ( ng/ml) (acros organics, geel, belgium) and ionomycin ( μm) (calbiotech, spring valley, ca, usa). monensin ( μm) (calbiotech) was added to the cultures for the last two hours. after stimulation, cells were harvested and stained for surface expression of cd . cells were then permeabilized using the cytofix/cytoperm kit (bd pharmingen) according to the manufacturer's instructions and stained for intracellular fitc-ifn-γ and pe-il- . statistical differences were calculated using the twotailed unpaired student's t-test. p values < . were considered significant. *p < . , **p < . , ***p < . . one characteristic of gko cd + t cell recipients infected with jhmv was the large cns infiltrating neutrophil population ( . % compared to . % in wt cd + t cells recipients) ( figure a ) [ ] . increased neutrophil accumulation in gko recipients is consistent with ifn-γ-mediated downregulation of elr + neutrophil chemokines [ ] . indeed, analysis of cytokine and chemokine mrna expression in infected t cell recipients demonstrated that high ifn-γ mrna correlated inversely with mrna expression of the neutrophil chemoattractant cxcl ( figure b) . thus, ifn-γ mrna in wt cd + t cell recipients was associated with sparse cxcl expression and neutrophil recruitment, while low ifn-γ mrna expression in both gko cd + t cell recipients and infected scid controls correlated with high cxcl expression and extensive neutrophil recruitment. infected mice were depleted of neutrophils to explore a possible correlation between neutrophilderived proteases, free radicals and proinflammatory cytokines with virus-induced mortality. depletion was confirmed by the absence of ly g + cd b + neutrophils within the cns-derived inflammatory cells ( figure c ). however, the absence of neutrophils did not prevent early mortality of gko cd + t cell scid recipients ( figure c ), implicating alternate mechanisms inducing mortality in gko recipients. in contrast to memory gko cd + t cells derived from jhmv-immunized donors, memory gko cd + t cells did not trigger early mortality in infected scid recipients [ ] . these data suggest that ifn-γ deficiency was not the sole factor controlling early death. wt cd + t cell recipients were depleted of ifn-γ to confirm that a cd + t cell factor distinct from ifn-γ controls disease outcome. the modestly reduced survival rate of ifn-γ-depleted wt cd + t cell recipients (figure a ) demonstrated ifn-γ blockade did not reproduce the mortality of gko cd + t cell recipients. the efficiency of ifn-γ blockade within the cns was confirmed by analyzing ifn-γ-dependent mhc class ii expression on microglia [ ] . in contrast to class ii expression on the vast majority of microglia in recipients of wt cd + t cells, class ii remained undetectable in anti-ifn-γ-treated wt recipients ( figure b ), confirming inhibition of local ifn-γ signaling within the cns. ifn-γ depletion also had minimal effects on t cell recruitment into the cns, reducing the cd + t cells within the inflammatory population from . % to . % (data not shown). in support of the role of ifn-γ in regulating neutrophils, ifn-γ-depleted wt recipients exhibited vastly increased cns neutrophil infiltration, approaching the numbers found in gko cd + t cell recipients ( figure c ). in addition to confirming ifn-γ-mediated control of cns neutrophil recruitment [ ] , these data reassert that abundant cns neutrophils are insufficient to account for early mortality. neither il- nor il- β, whose over expression is associated with adverse effects on the cns [ , ] , were increased in the cns of gko compared to wt cd + t cell recipients ( figure a) . previous data demonstrated that tnf and inducible nitric oxide synthase were also not associated with early mortality of gko recipients [ ] . indeed, passive transfer of neutralizing anti-tnf mab was unable to alter the mortality of the gko cd + t cell recipients (data not shown). these results suggested additional factor(s) intrinsic to gko cd + t cells in mediating disease outcome. inhibition of il- production by ifn-γ [ ] , suggested il- as a potential candidate. consistent with this concept, il- mrna expression was increased in the cns of gko cd + t cell recipients ( figure b ), although il- is not expressed in the cns of infected wt mice [ ] . importantly, il- mrna remained below detection not only in scid-infected control mice lacking t cells, but also in recipients of wt cd + t cells depleted of ifn-γ ( figure b) , both of which are characterized by vast cns neutrophil infiltration ( figure a) . although neutrophil-derived il- has been implicated in enhancing tissue damage during reperfusion injury [ ] , these data suggest that neutrophils recruited into the cns do not secrete il- during acute viral encephalitis. expression of il- mrna only in gko cd + t cell recipients also ruled out a potential contribution of resident cns cells. il- expression exclusively in the cns of gko recipients thus implied that the source of il- was the gko-derived cd + t cell population itself. in support of this concept, transcript levels encoding il- , another cytokine produced by th cells [ ] , were also significantly increased in infected gko recipients compared to wt recipients and infected scid control mice ( figure c ). by contrast, il- , a cd + t cell-derived cytokine known to provide helper functions to cd + t cells and b cells [ ] was expressed at similar levels in both the gko and wt cd + t cell recipient groups ( figure c ). il- production by cd + t cells in the cns of gko recipients was confirmed by immunofluorescence histochemistry. a substantial fraction of t cells within the cns of gko recipients expressed il- . moreover, all il- positive cells co-expressed cd ( figure d ), indicating that t cells are the predominant source of il- within the cns of scid recipients. in contrast to the cns, only~ % of t cells in the cervical lymph nodes of gko recipients secreted il- at day eight p.i. (figure e ), suggesting enrichment of il- expressing t cells within the cns. to determine if il- expression is imprinted during the primary response following immunization of gko donor mice, cytokine expression was analyzed in the memory wt and gko t cell populations prior to transfer ( figure f ). wt memory cd + t cells prominently expressed ifn-γ and very little, if any, il- following in vitro stimulation. by contrast, immunization of gko mice primed a small fraction of memory cd + t cells capable of producing il- . these results were consistent with ifn-γ-mediated inhibition of th cells [ ] and suggested that il- expression was imprinted prior to transfer and re-expressed in the infected recipients. to confirm a role of il- in the early mortality of gko recipients, wt and gko recipients were treated with anti-il- mab. consistent with the absence of il- mrna in the cns of the wt recipients, anti-il- treatment had no effect on the survival of wt recipients ( figure a ). by contrast, inhibition of il- in gko recipients lead to a significant decrease in mortality, with % of mice surviving to day p.i. (figure a ). in support of the concept that mortality was not influenced by neutrophils, the increased neutrophil infiltration in the cns of gko recipients was not altered by anti-il- treatment ( figure b ), confirming their primary regulation by ifn-γ [ ] . cross-regulation of ifn-γ and il- in shaping cd + t cell subsets is well established during primary t cell activation and expansion [ , ] ; however, less is known about cross-regulation during antigen-induced restimulation of memory t cells. il- has recently been shown to promote gm-csf expression by th cells, which in turn enhances detrimental disease outcomes [ , ] . based on the observation that gm-csf is downregulated by ifn-γ [ ] , we tested whether ifn-γ-producing cd + t cells can override the detrimental function of gko cd + t cell-mediated il- expression. memory cd + t cells from wt thy . immunized mice were co-transferred with thy . gko cd + t donor cells (wt/gko recipients) prior to infection. mhc class ii expression was measured in the cns of all recipient groups to confirm functional ifn-γ expression [ ] . class ii mrna was maximal in the cns of wt recipients, reaching -fold higher levels than in gko recipients. mhc class ii mrna was increased -fold following the co-transfer of wt and gko cd + t cells compared to mice receiving gko cd + t cells alone ( figure a ). increased mrna expression correlated with mhc class ii protein expression by the majority of microglia in both wt and co-transfer groups ( figure a ). although the proportion of microglia expressing mhc class ii was decreased in the co-transfer compared to the wt groups, differences were not statistically significant ( figure a ). co-transfer of wt and gko cd + t cells protected recipients from early mortality ( figure b) . disease severity was similar in wt and wt/gko recipients and significantly reduced compared to gko-only recipients, especially after day six p.i. (data not shown). in addition, control of cns virus replication was identical at day eight p.i. in recipients of wt, wt/gko or gko cd + t cells ( figure c ), confirming previous results that accelerated mortality does not correlate with uncontrolled virus replication [ ] . cns leukocyte infiltration in wt/gko cd + t cell recipients was similar to wt recipients, and lower than gko recipients ( figure d ). specifically, neutrophils within cd hi bone-marrow-derived inflammatory cells were reduced to % in wt/ gko recipients, resembling the proportion in wt recipients ( figure d and data not shown). overall these data confirm ifn-γ-mediated control of cns neutrophil infiltration and suggested a protective role of ifn-γ during viral encephalitis, via inhibiting il- effector function by either directly reducing th cell expansion and/or cns entry, or limiting gm-csf production. to assess whether gko cd + t cells migrated to the cns in the presence of wt cd + t cells, the relative proportions of each donor population was examined by co-transfer of thy . wt cd + t cells and thy . gko cd + t cells. surprisingly, t cells recruited into the cns of infected co-transferred recipients were essentially derived from the gko memory cd + t cells, as less than % of cd + t cells expressed thy . + ( figure a ). given the large population of infiltrating gko cd + t cells, we next determined if ifn-γmediated protection correlated with reduced il- mrna expression. although protective, the minor population of wt cd + t that infiltrated the cns did not reduce expression of il- mrna in the cns ( figure b ). protection mediated by ifn-γ, despite elevated il- , suggested that ifn-γ interferes with il- mediated signaling events, rather than directly influencing th expression. this notion was tested by in vitro stimulation of memory cd + t cells derived from gko donors in the presence of recombinant ifn-γ. exogenous ifn-γ was indeed unable to downregulate il- production ( figure c ), supporting the in vivo observation that ifn-γ-expressing wt cd + t cells did not alter cns expression of il- mrna in wt/gko recipients ( figure b ). the maintenance of il- in the presence of ifn-γ in vitro and in vivo indicates that the phenotypes acquired during in vivo primary responses are retained in the transferred memory cells following reactivation in recipient mice. to confirm this assumption, ifn-γ was depleted in wt/gko recipients. wt/ gko recipients treated with anti-ifn-γ succumbed to infection by day nine p.i. similar to infected recipients of gko cd + t cell ( figure d ). these data actually suggest that ifn-γ diminishes the detrimental effects of il- , despite the apparent expansion/survival advantage of gko relative to wt cd + t cells in the infected recipients. to determine potential mechanisms of il- -mediated mortality, il- -dependent chemokines and matrix metalloproteinases (mmps) [ ] were analyzed in jhmv-infected scid recipients after transfer of wt or gko cd + t cells. similar expression of ccl , ccl and ccl was detected comparing infected scid controls and gko recipients; by contrast ccl and ccl were upregulated and ccl downregulated in recipients of wt cd + t cells ( figure a) . these data suggest that in contrast to eae, ccl , ccl and ccl chemokine expression is regulated by ifn-γ rather that il- during jhmv infection. moreover, no significant difference in cxcl mrna was found comparing scid-infected controls and recipients of either wt or gko cd + t cells ( figure a) , supporting cxcl as the major neutrophil chemoattractant during jhmv infection. cns infection with jhmv induces a limited number of mmps, that is, mmp , mmp and mmp [ ] . as mmp is specifically expressed by neutrophils [ ] , abundant neutrophil recruitment in the cns of gko t cell recipients (whether or not treated with anti-il ) correlated with mmp expression ( figure b ). mmp and mmp mrna expression were also upregulated in gko recipients compared to infected scid controls and wt recipients, suggesting a potential role of these mmps in gko mortality by mediating tissue destruction ( figure b) . however, survival of gko recipients treated with anti-il also expressed increased mmp and mmp mrna ( figure b ), suggesting that mmp and mmp play no role in the early mortality of gko recipients. finally, to investigate a potential contribution of gm-csf to the rapid disease progression, relative levels of gm-csf were measured in the cns of scid-infected controls, and recipients of wt and gko cd + t cells. gm-csf mrna expression was increased in gko recipients relative to controls and wt cd + t cell recipients. these data were reminiscent of enhanced gm-csf expression by th compared to th cells in eae [ ] and suggested a potentially detrimental role during jhmv encephalomyelitis. however, the increased survival of gko recipients treated with anti-il mab did not correlate with a decrease in gm-csf expression. these results indicate that gm-csf expression correlated with ifn-γ deficiency, but not with an il- mediated feedback loop. nevertheless, these data suggest that ifn-γ directly affords protection from mortality by interfering with detrimental il- -mediated events, distinct from those mediating eae. ifn-γ and il- are major effector molecules of tissue inflammation that play opposing roles in neutrophil recruitment/accumulation [ , , ] . while their distinct influence on disease has been demonstrated during autoimmune-mediated neuroinflammatory responses, the interplay between il- and ifn-γ, specifically the effects on downstream targets remain controversial. furthermore, during microbial infections, protective and detrimental effects of ifn-γ and il- depend on the pathogen and prominent cell types affecting microbial control [ ] [ ] [ ] . the present study evaluated how the absence of ifn-γ secretion by cd + t cells contributes to a rapid lethal outcome during viral encephalomyelitis, without altering viral control. early virus-induced mortality in scid recipients of gko virus-specific memory cd + t cells correlated with both il- production and extensive neutrophil accumulation in the cns. selective blockade of either neutrophils or il- demonstrated that early mortality did not correlate with cns neutrophil recruitment, but rather with il- . this was confirmed by the prolonged survival of recipients of anti-ifn-γ mabtreated wt recipients, which were characterized by extensive neutrophil inflammation, but an absence of il- . neutrophil-independent pathogenic effects of il- in the jhmv model contrast with non-cns viral infectious models including the influenza virus and herpes simplex virus- infections, which attribute th cell-mediated pathogenesis to neutrophil attraction [ , ] . however, neutrophil depletion following severe influenza virus infection also suggests that neutrophils play a protective, rather than a deleterious role [ ] . our data also contrast with the deleterious role of neutrophils during eae [ , ] . adoptive transfer of th cells leads to excessive cns neutrophil migration after eae induction, while impaired neutrophil recruitment restrains leukocyte access into the cns [ ] , indicating a prominent role of neutrophils in disrupting the blood-brain barrier. however, in contrast to eae, neutrophils are not essential for the loss of blood-brain barrier integrity following sublethal jhmv infection [ ] . by contrast, jhmv-induced encephalomyelitis demonstrates that ifn-γ plays a more prominent role than il- in regulating cns neutrophil recruitment and/or retention by downregulating elr + neutrophil chemokine expression. increased neutrophils correlated with high cxcl expression in the cns of both ifn-γ-depleted wt recipients lacking il- , as well as in gko recipients treated with anti-il- ab. moreover, neutrophil infiltration was reduced by co-transfer of wt and gko cd + t cells, despite sustained il- expression in the cns. these results are consistent with early studies identifying ifn-γ as a critical factor regulating cns neutrophil infiltration [ ] , as well as recent observations implicating ifn-γ as a dominant molecule controlling cns inflammation [ ] . despite evidence implicating il- as a pathogenic mediator, independent of neutrophils, the mechanism(s) involved in il- -induced mortality of jhmv-infected mice remain unclear. identical viral burden at day eight p.i. in all recipients [ ] indicated that il- does not alter control of virus replication, in contrast to its role in facilitating viral persistence following theiler's murine encephalomyelitis virus infection [ ] . sustained ag independent interaction between th and neuronal cells during eae correlated with increased neuronal damage due to il- -mediated neurotoxicity [ ] . increased gray matter infection, especially in neuronal cells, is associated with premature death following jhmv infection of mice deficient in innate immune components [ ] . in addition, there is a preferential distribution of cd + t cells in the gray matter of gko recipients compared to wt recipients [ ] , suggesting the possibility that in absence of ifn-γ, il- -secreting cd + t cells localize proximal to uninfected neurons, contributing to neuronal dysfunction and premature death. however, few neurons are infected early during jhmv pathogenesis in scid mice and the types of infected cells were similar in all groups, suggesting no alteration in viral tropism [ ] . in addition, no differential neuronal loss was found comparing gko and wt recipients [ ] . similarly, increased expression of gm-csf in gko recipients compared to the wt counterparts suggested that gm-csf might also contribute to disease outcome following jhmv infection. gm-csf was implicated as a pathogenic effector molecule secreted by th cells during eae [ , ] . however, the survival of gko recipients treated with anti-il did not correlate with a decrease in gm-csf expression. although gm-csf expression is reduced by ifn-γ [ ] , the data do not support a pathogenic role of gm-csf in early mortality of jhmv-infected gko recipients. il- mrna expression in gko cd + t cell recipients suggested th cells as the primary mediators of disease. nevertheless, il- can also be produced by neutrophils, γδ t cells, nk and cd + t cells [ , ] . a deleterious contribution of neutrophil-derived il- , suggested during kidney ischemia-reperfusion [ ] , was ruled out by the inability of neutrophil-depletion to rescue mice from early death, as well as the absence of il- mrna in wt recipients treated with anti-ifn-γ, despite high cns neutrophil infiltration. il- production by cd + t cells derived from immunized gko donors prior to transfer supports gko cd + t cells as the primary source of il- . moreover, stimulation of wt donor cd + t cells strongly induced ifn-γ, but not il- , indicating that virusspecific th cells only differentiate in the absence of ifn-γ. these results support previous observations of a minor, if any, role of th cells in the pathogenesis of jhmv-infected immunocompetent wt mice [ ] and corroborate the inhibitory function of ifn-γ on th differentiation during t cell priming [ ] . however, our data are novel in demonstrating that memory gko cd + t cells are committed in their ability to produce il- when restimulated in the recipient host, even in the presence of ifn-γ. although unanticipated, this finding was confirmed by the inability of ifn-γ to downregulate il- production in gko donor cells in vitro, as well as on in vitro-differentiated mature th cells [ ] . similarly, the il- suppressive function on th differentiation from naïve cd + t cells could not be reproduced on memory th cells [ ] , supporting the stability of committed th cells. importantly, the prolonged survival of co-transferred recipients, despite sustained cns il- expression, suggests that ifn-γ overcomes the deleterious effects of il- . however, the mechanisms by which ifn-γ overrides il- function remain unclear. in eae, il- exerts detrimental effects via signaling in resident cns cells, with astrocytes implicated as major targets [ ] . however, th cell localization proximal to neurons also implicates potential dysregulation of neuronal function [ ] . responsiveness of both cell types to ifn-γ [ , ] suggests ifn-γ may counteract signaling molecules downstream of the il- r. this study demonstrates that il- , in the absence of ifn-γ, can accelerate mortality during viral encephalomyelitis by a mechanism independent of the magnitude of cns neutrophil infiltration and reversible by ifn-γ. abbreviations bsa: bovine serum albumin; cns: central nervous system gko: ifn-γ deficient; gm-csf: granulocyte monocyte colony-stimulating factor; h&e: hematoxylin and eosin; ifnγ: interferon-gamma jhmv: gliatropic jhm strain of mouse hepatitis virus; mab: monoclonal antibody; mhc: major histocompatibility complex; mmp: matrix metalloproteinase; pcr: polymerase chain reaction pe: phycoerythrin; percp: peridinin chlorophyll protein; scid: severe combined immunodeficiency; sd: standard definition; sem: standard error of the mean requirement of endogenous stem cell factor and granulocyte-colony-stimulating factor for il- -mediated granulopoiesis requirement of interleukin receptor signaling for lung cxc chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense interleukin- family members and inflammation ifn-gamma shapes immune invasion of the central nervous system via regulation of chemokines how interferon-gamma keeps autoimmune diseases in check a rush to judgment on th th cells and il- receptor signaling are essential for mucosal host defense against oral candidiasis il- and the th pathway promote inflammation and impair antifungal immune resistance virusspecific interleukin- -producing cd + t cells are detectable in early human immunodeficiency virus type infection th cells in pathogenic simian immunodeficiency virus infection of macaques cutting edge: murine cytomegalovirus induces a polyfunctional cd t cell response critical role of il- ra in immunopathology of influenza infection il- deficiency unleashes an influenza-specific th response and enhances survival against highdose challenge il- receptor signaling influences virus-induced corneal inflammation th cells enhance viral persistence and inhibit t cell cytotoxicity in a model of chronic virus infection current views on the roles of th and th cells in experimental autoimmune encephalomyelitis encephalitogenic t cells in the b .pl model of experimental allergic encephalomyelitis (eae) are of the th- lymphokine subtype adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin- . preferential expansion of interferon-gamma-producing cells and increased expression of macrophage-associated inducible nitric oxide synthase as immunomodulatory mechanisms ifn-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoproteininduced autoimmune encephalomyelitis failure to suppress the expansion of the activated cd t cell population in interferon gamma-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis exacerbation of antigen-induced arthritis in ifn-gamma-deficient mice as a result of unrestricted il- response il- a and il- f do not contribute vitally to autoimmune neuroinflammation in mice il- drives a pathogenic t cell population that induces autoimmune inflammation interleukin- rather than interleukin- is the critical cytokine for autoimmune inflammation of the brain in vivo imaging of partially reversible th cell-induced neuronal dysfunction in the course of encephalomyelitis eae mediated by a non-ifn-gamma/ non-il- pathway rorgammat drives production of the cytokine gm-csf in helper t cells, which is essential for the effector phase of autoimmune neuroinflammation the encephalitogenicity of t(h) cells is dependent on il- -and il- -induced production of the cytokine gm-csf ctl effector function within the central nervous system requires cd + t cells cd t cells contribute to virus control and pathology following central nervous system infection with neurotropic mouse hepatitis virus memory cd + t-cell-mediated protection from lethal coronavirus encephalomyelitis perforin and gamma interferon-mediated control of coronavirus central nervous system infection by cd t cells in the absence of cd t cells pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies perforin-mediated effector function within the central nervous system requires ifn-gamma-mediated mhc up-regulation interleukin- and neuronal injury cytokine involvement in central nervous system disease. implications from transgenic mice th cell differentiation: the long and winding road interleukin- (il- ), but not il- , deficiency ameliorates viral encephalitis without affecting viral control il- produced by neutrophils regulates ifn-gammamediated neutrophil migration in mouse kidney ischemia-reperfusion injury interleukin (il)- and il- are coexpressed by th cells and cooperatively enhance expression of antimicrobial peptides the role of il- in regulating b-cell function in health and disease mills kh: induction, function and regulation of il- -producing t cells interleukin -producing cd + effector t cells develop via a lineage distinct from the t helper type and lineages il- inhibits human th differentiation through il- r beta downregulation astrocyterestricted ablation of interleukin- -induced act -mediated signaling ameliorates autoimmune encephalomyelitis expression of matrix metalloproteinases and their tissue inhibitor during viral encephalitis monocytes regulate t cell migration through the glia limitans during acute viral encephalitis il- -and il- -modulated t cells induce distinct types of eae based on histology, cns chemokine profile, and response to cytokine inhibition the th -elr + cxc chemokine pathway is essential for the development of central nervous system autoimmune disease interleukin- in host defence against bacterial, mycobacterial and fungal pathogens precarious balance: th cells in host defense interferon-gamma: an overview of signals, mechanisms and functions the role of neutrophils during mild and severe influenza virus infections of mice treatment with anti-granulocyte antibodies inhibits the effector phase of experimental autoimmune encephalomyelitis bergmann cc: mmp deficiency does not decrease blood-brain barrier disruption, but increases astrocyte mmp expression during viral encephalomyelitis type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells innate il- -producing cells: the sentinels of the immune system tc cd t cells: functional plasticity and subset diversity th : the third member of the effector t cell trilogy differential effect of il- on developing versus committed th cells interferongamma-responsive neuronal sites in the normal rat brain: receptor protein distribution and cell activation revealed by fos induction demonstration of the presence of a specific interferon-gamma receptor on murine astrocyte cell surface ifn-γ protects from lethal il- mediated viral encephalomyelitis independent of neutrophils the authors thank wenqiang wei, kate stenson and shabbir hussain for technical assistance. this work was supported by national institutes of health grant ns and national multiple sclerosis society fellowship grant fg- -a- to cs. the authors declare they have no competing interests.authors' contributions cs designed and performed the experiment, collected and analyzed data, and wrote the manuscript. sas designed and performed the research, interpreted data and wrote the manuscript. drh analyzed and interpreted data. rmr interpreted data. djc provided materials, interpreted data and edited the manuscript. ccb designed the research, provided materials, interpreted data and wrote the manuscript. all authors read and approved the final manuscript. key: cord- - n bnp authors: gadotti, ana carolina; de castro deus, marina; telles, joao paulo; wind, rafael; goes, marina; garcia charello ossoski, roberta; de padua, alessandra michalski; noronha, lucia; moreno-amaral, andrea; baena, cristina pellegrino; tuon, felipe francisco title: ifn-γ is an independent risk factor associated with mortality in patients with moderate and severe covid- infection date: - - journal: virus res doi: . /j.virusres. . sha: doc_id: cord_uid: n bnp background: innate and adaptive immune responses have been evaluated in infected patients with covid- . the severity of the disease has been supposed to be associated with some profile not reported with other bacterial and viral pneumonia. we proposed a study in patients with moderate to severe covid- infection to evaluate the interleukin patterns and its role as prognosis factors. methods: a prospective cohort with moderate and severe cases of covid- infection from june to july . blood samples from patients were collected regularly to evaluate ifn-γ, tnf-α, il- , il- , and il- . clinical, laboratory, radiological data, and outcomes were recorded. the outcome variable was in-hospital death, survival, mechanical ventilation, and admission at the intensive care unit. data are presented in median and interquartile range [iqr]. results: we evaluated the th and th responses according to evolution, distinguishing possible predictive markers. the ifn-γ median of pg/ml [iqr - ] was found in patients who died and pg/ml [iqr - ] in the survival group (p = . ). ifn-γ was also higher in the early stages of the disease ( pg/ml [iqr - ] against pg/ml [iqr - ], p < . ). il- that was increased in late-stage ( pg/ml [iqr - ] against pg/ml [iqr - ], p < . ) but not associated with mortality. also, death was also related to male gender (relative risk = . [ % confidence interval = . - . ]). conclusion: our results suggest that the activation of the host immune response between th or th in covid- infection may be related to the final result between discharge or death. this implies an attempt to control cytokines, such as ifn-γ, with combined therapies for clinical treatment. in early , the world health organization declared pandemic due to coronavirus disease . since then, a large number of researches have been published, from epidemiological to experimental studies, trying better to understand immunological pathways of covid- and possible treatments. cd +, cd +, and cd + lymphocytes count are usually decreased according to disease stages . besides it, cytokine storm is also present in severe patients due to elevation of interleukins such as tnf-α, il- , il- , and il- , . thus, the differences among host immune responses play a major role in covid- severity. older age, elevated c-reactive protein, serum lactate dehydrogenase, bilirubin, blood urea nitrogen, and decreased albumin are described as prognostic factors of severe covid- . comorbidities are also described as prognosis factors and the higher the number of them, the poorer the prognosis (i.e., hypertension, diabetes, chronic pulmonary disease, coronary artery disease, malignancies) . interestingly, in an italian and spanish cohort, genetic factors related to abo blood-group system were reported as susceptibility to covid- respiratory failure . different interleukins are described as prognostic factors. a reasonable hypothesis is that (i) pro-inflammatory innate immunity and (ii) anti-inflammatory system are related to disease severity or death once il- , il- , and il are closely described as prognostic factors in patients diagnosed with covid- , , . nevertheless, few studies have focused on the adaptive immune system to evaluate the balancing between th and th response. consequently, il- is usually not studied concomitant with ifn-γ and il- to better characterize th , th , and innate immune response. ifn-γ is the type ii ifn produced by nk cells and t lymphocytes, although cells of different phases of the immune response (innate and adaptative, respectively), this cytokine is important in all phases of the immune response . j o u r n a l p r e -p r o o f ifn-γ system is essential for antiviral defense. ifn-γ downregulate virus replication and it activates cytokine production by t cells, augmenting the cytotoxic t lymphocyte killing activity . however, persistent high levels of ifn-γ worsens the systemic inflammation, and increasing tissue injury and organ failure . considering this ambiguous role of ifn-γ in the outcome, it is important to understand the serum pattern of this and other cytokines in patients with covid- . the aim of this study was to investigate the interleukins patterns in patients admitted due to covid- and its role as prognosis factors. j o u r n a l p r e -p r o o f this was a prospective cohort of hospitalized patients diagnosed with covid- conducted in the south of brazil, curitiba, paraná, from june to july . patients were admitted only after approval of the research ethics committee and an approved consent form. blood samples from patients were collected to evaluate ifn-γ, tnf-α, il- , il- , and il- . clinical, laboratory, radiological data, and outcomes were recorded. covid- infection was defined by clinical-radiological presentation plus a nasopharyngeal swab polymerase chain reaction (pcr) positive to covid- . inclusion criteria were hospitalized patients with moderate or severe confirmed covid- infection. the moderate disease was defined as an adult with clinical signs of pneumonia (fever, cough, dyspnea, fast breathing) but no signs of severe pneumonia, including spo ≥ % on room air; and severe disease as an adult with clinical signs of pneumonia (fever, cough, dyspnea, fast breathing) plus one of the following: respiratory rate > breaths/min; severe respiratory distress; or spo < % on room air . patients diagnosed with other viral infections, such as hiv, hcv, hbv, or another common respiratory virus, were excluded, as well as solid organ or hematological transplantation patients. patients who used tocilizumab were also excluded. j o u r n a l p r e -p r o o f patients were classified according to days of symptoms: - days and > days. blood samples were collected using a standard coagulation tube (sst ii advance, bd biosciences) to obtain the serum, which was aliquoted and stored at - ° c until analysis. the cytokines were measured using commercially available elisa kits for tnf-α, ifn-γ, il- , il- , and il- (immunotools, friesoythe, germany), according to the manufacture instructions. several clinical and laboratory data were evaluated. the outcome variable was in-hospital death, survival, mechanical ventilation, and admission to the intensive care unit (icu). continuous variables were expressed as median values and interquartile range (iqr) and analyzed by mann whitney test. categorical variables were expressed as absolute frequencies with proportions and analyzed by chi-square or fisher exact test. a p-value < . was considered significant. all variables in the univariate model meeting a cut-off of p < . were included in the multivariable model. spss v . (ibm, chicago, il) and graphpad prism v (graphpad, san diego, ca) were used for statistical analysis. the variable days of symptoms was split in > days or ≤ days using the optimal binning procedure on spss for all cytokines included in the analysis. fifty-six patients were included in all samples. median age was [ - ] years, and . % (n= ) were male. most common comorbidities were systemic arterial hypertension ( %, n= ), diabetes ( %, n= ) and chronic obstructive pulmonary disease ( . %, n= ). at hospital admission, moderate and severe patients were composed of . % (n= ) and . % (n= ) from the sample, respectively. general clinical characteristics are in table . steroids were used in . % (n= ) and hydroxychloroquine in . % (n= ). the median length of hospitalization was [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] days. specific characteristics from each patient group ( - d vs. > d) are demonstrated in table . at admission, median leukocytes, lymph cells and platelets counts were , [ figure . ifn-γ levels were higher in patients from group - d of symptoms (p< . ); while il- levels were higher in patients from group > d of symptoms (p< . ). thus, ifnγ and il- was inversely proportional related (p= . ) ( table ). there was no relation between use of steroids during treatment of covid- and citokines levels (p> . ). table . in univariate analysis, death was related to gender (p= . ), icu (p< . ), oti (p= . ), ifn-γ (p= . ) and length of hospitalization (p= . ); while survival was related to fever (p= . ). cytokines analysis is shown in figure . nevertheless, in multivariate analysis, death remained related to age, gender, and ifn-γ (p< . ). in this prospective cohort of patients, higher levels of ifn-γ were related to a poorer prognosis. these results demonstrate that an effective th response is not sustained and probable it is followed by the development of th immune adaptive response. il- and il- did not differ when compared to days of symptoms, evolution, nor outcomes. previous studies elucidated the capacity of nk and nk t cells to produce ifn-γ before the specific th adaptive immune response , . our results converge with this hypothesis once higher ifn-γ levels were detected in early covid- infection than healthy populations . nevertheless, these levels were not sustained after ten days of symptoms. in those with sustained ifn-γ levels, the mortality increased. th profile cytokine il- presents an upward trend in disease progression in critically ill patients . il- levels may (i) inhibit naive cd + cells from proceeding to th maturation or in our cohort, gender, and ifn-γ levels were related to poorer prognosis. besides it, since the covid- beginning, it was demonstrated that hospital admission was more likely in the male gender with comorbidities , . hypertension, cardiovascular diseases, and diabetes were the most frequent comorbidities in our patients. these factors may explain the high icu admission rate presented in this study. furthermore, the death rate found was %, which is higher than j o u r n a l p r e -p r o o f other general cohorts . nevertheless, given the high icu admission rate, it is expected that outcomes results become closer to cohorts of critically-ill patients . previous studies have not reported the association between ifn-γ and death, even evaluating the covid- -reactive cd + expressing ifn-γ producing cd + t in patients with severe and moderate disease . most studies have described the evident immunological dysfunction in the moderate and severe disease, with reduced expression of ifn-γ by cd + t, cd + t, and nk cells . the same author found that ifn-γ by cd + t cells tended to be lower in severe cases than moderate cases. however, none of these studies evaluated the levels of ifnγ with death. some subsets of t lymphocytes correlated with in-hospital death and severity of illness, but only the innate immune cytokines were evaluated . it was recently demonstrated that levels of the ifn-γ secreted by cd + and cd + t cells from patients with moderate disease were compatible with those in critically ill patients. in this case, ifn-γ increased over time in critically ill patients, but with decreased levels in moderate patients, contrasting with the results presented here. however, ifn-γ was highly correlated with viral load, suggesting that the virus can boost the secretion of these cytokines . in a previous report, the proportion of memory and naïve helper t cells decreased in severe cases . patients with covid- also have a lower level of regulatory t cells, suggesting that the adaptive immune response is prejudiced. however, another study showed a higher number of cd t cells producing ifn-γ in comparison with a control group (without disease) . in children with covid- , ifn-γ is increased, but with inferior levels than we found, suggesting a less severe disease in the pediatric population . in cardiomyocytes, there is an up-regulation of genes responsible for ifn-γ suppression, also suggesting an effect ifn-γ in the cardiac function, in a sepsis-like pattern . furthermore, the relationship of ifn-γ disruption is not j o u r n a l p r e -p r o o f only associated with covid- and the immune system, but there is a relationship of microbiome modification, altering the cell transcriptome with gene overexpression, which can be associated with the cytokine storm , . interleukin- plays a critical role in the th pathway, being predominantly associated with fibrogenic inflammatory remodeling, while th cells exert anti-fibrotic activity by secreting gamma interferon (ifn-γ) and interleukin . il- also induce alternative activation of m macrophages, promoting the release of tgf-β and platelet-derived factor. this phase is characterized by the expansion of resident fibroblasts and the matrix remodelling . since severe covid- can lead to diffuse alveolar damage, which has a potential of developing septal fibrosis; recovering patients with high levels of il- may might progress to pulmonary fibrosis aggravating impairment of the lung functions. our study has some limitations and strengths that merit consideration. as limitations, our study evaluated the first patients admitted during the outbreak in our city. therefore, changes in clinical management during the evolving epidemics might have a differential impact on our studied outcomes. our limited sample size might have decreased our power; however, also because of the pandemic, the findings of this study offer new, potentially useful information for this patient population. on the other hand, our grouping by symptom days may have standardized the disease's different stages when patients were admitted. another concern is memory bias for onset of symptoms, because some patients could be in the wrong group (more or less days). we evaluated the th and th responses according to the time of evolution, so we were able to identify possible predictive markers such as (i) ifn-γ in early-stage and (ii) il- in late-stage for the outcomes of discharge or death. our results suggest that the moderate to the severe progression of covid- may have been one of the causes of the immune responses developed by the host at the beginning of the covid- infection, influencing the need for combination therapy to block these inflammatory mediators. the activation of the host immune response between th or th in covid- infection may be related to the final result between discharge or death. any source to control cytokines, such as ifn-γ, with combined therapies for clinical treatment will be important in the future for covid- infection. viral and host factors related to the clinical outcome of covid- clinical features of patients infected with novel coronavirus in wuhan, china. lancet profiling serum cytokines in covid- patients reveals il- and il- are disease severity predictors. emerg microbes infect a tool for early prediction of severe coronavirus disease (covid- ): a multicenter study using the 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of a normal population longitudinal analyses reveal immunological misfiring in severe covid- il- -mediated inhibition of ifn-gamma production by cd + t cells proceeds by several developmentally regulated mechanisms signaling and transcription in t helper development epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease pneumonia in wuhan, china sars-cov- -reactive interferon-gammaproducing cd + t cells in patients hospitalized with coronavirus disease clinical and immunological features of severe and moderate coronavirus disease suppressed t cell-mediated immunity in patients with covid- : a clinical retrospective study in wuhan dysregulation of immune response in patients with coronavirus marked t cell activation, senescence, exhaustion and skewing towards th in patients with covid- pneumonia haematological and immunological data of chinese children infected with coronavirus disease . data brief single-cell transcriptome analysis indicates new potential regulation mechanism of ace and nps signaling among heart failure patients infected with sars-cov- . medrxiv metatranscriptomic characterization of covid- identified a host transcriptional classifier associated with immune signaling the society for immunotherapy of cancer perspective on regulation of interleukin- signaling in covid- -related systemic inflammatory response figure . levels of cytokines from patients with moderate and severe covid- infection according to the outcome (data in the median with iqr) key: cord- -k ws at authors: mihm, sabine title: covid- : possible impact of the genetic background in ifnl genes on disease outcomes date: - - journal: j innate immun doi: . / sha: doc_id: cord_uid: k ws at nan the causative agent of the pandemic covid- outbreak, sars-cov- , is a member of the coronaviridae family of enveloped viruses with a positive-sense singlestranded (ss) rna genome [ ] . generally, ssrna viruses are recognized by the host immune system in first-line defense via innate pattern recognition receptors (prr), such as toll-like receptor (tlr ), which is a primary sensor for extracellular or endosomal nucleic acid patterns of viral origin. mouse hepatitis corona virus (mhv) is a prototypic β-genus coronavirus and a well-studied model for sars-cov. host control of mhv infection is completely dependent on tlr triggering by viral rna causing an immediate interferon (ifn) response [ ] . similarly, in mers-cov infection, a timely ifn signaling has been shown to protect mice from a lethal outcome, i.e., fatal pneumonia [ ] . tlr is a gonosomal-encoded, x-linked gene. sex differences in tlr responses have been reported for humans -with female sex better coping with viral infection [ ] [ ] [ ] -which is also a feature of covid- . upon binding viral nucleic acid motifs, tlr induces the expression of type i ifns (ifn-α and ifn-β) and the expression of the more recently described family of type iii ifns (ifn-λ [ ] [ ] [ ] [ ] . during viral infection of lung and liver epithelium, for example, notably, type iii ifns, rather than type i ifns, are found to be activated [ ] . type iii ifns seem to be the major ifns induced in airway epithelial cells during infection [ ] . moreover, while type i ifns act on ubiquitously expressed receptors, type iii ifn action is restricted as receptors are particularly expressed on epithelia, e.g., of the lung. type iii ifn induction and its effectors might naturally combat respiratory infections and might constitute a promising therapeutic target pathway. a common germ line genetic variation within the type iii ifn gene locus has been most convincingly shown to determine the host's capacity to cope with an infection induced by hepatitis c virus (hcv), an enveloped ssrna virus of positive orientation, too, featuring tropism for liver epithelial cells. one candidate variation is suggested to be a dinucleotide polymorphism within the the host's capability to encode a functional ifn-λ protein [ ] . paradoxically, the ifnl knockout variant tt is favorable for virus eradication and resolution of infection, presumably by the deactivation of an ifn-αdesensitizing control mechanism, antagonizing ifn-α efficacy [ ] . globally, the frequency of the favorable knockout variant tt amounts up to . among asian populations, which lessens down to . and . for individuals of european or african ancestry, respectively. apart from national measures that unambiguously successfully control viral spread, populations might differ in susceptibility. moreover, within a given population, individuals might be at different risk, not only due to gender or age [ ] . this is to encourage personalizing approaches considering the genetic background in ifnl genes as a hostspecific indicator for the outcome of the prevalent rna viral infections outside of hcv. a novel coronavirus from patients with pneumonia in china cd receptor controls sex-specific tlr responses to viral infection ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes tlr ligands induce higher ifn-alpha production in females sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv- do men have a higher case fatality rate of severe acute respiratory syndrome than women do? the impact of the interferonlambda family on the innate and adaptive immune response to viral infections a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus activation of type i and type iii interferons in chronic hepatitis c not applicable. the author has no conflicts of interest to declare. this publication is financially supported by the open access publication fund of the university of göttingen. this is a single author contribution. key: cord- -f g gitn authors: rojas, josé m.; alejo, alí; martín, verónica; sevilla, noemí title: viral pathogen-induced mechanisms to antagonize mammalian interferon (ifn) signaling pathway date: - - journal: cell mol life sci doi: . /s - - -z sha: doc_id: cord_uid: f g gitn antiviral responses of interferons (ifns) are crucial in the host immune response, playing a relevant role in controlling viralw infections. three types of ifns, type i (ifn-α, ifn-β), ii (ifn-γ) and iii (ifn-λ), are classified according to their receptor usage, mode of induction, biological activity and amino acid sequence. here, we provide a comprehensive review of type i ifn responses and different mechanisms that viruses employ to circumvent this response. in the first part, we will give an overview of the different induction and signaling cascades induced in the cell by ifn-i after virus encounter. next, highlights of some of the mechanisms used by viruses to counteract the ifn induction will be described. and finally, we will address different mechanism used by viruses to interference with the ifn signaling cascade and the blockade of ifn induced antiviral activities. innate immune responses are the first host defense against viral infections. conserved pathogen structures are recognized by pattern recognition receptors (prrs) on the host cells [ ] , that recruit a variety of adaptor proteins to signal downstream and activate the ifn response. the ifn system is present in all vertebrates and is central to antiviral responses [ ] . ifns are classified into three different families according to their receptor usage, mode of induction, biological activity and amino acid sequence [ ] : type i, type ii and type iii ifns. type i ifns, originally identified by their antiviral activity [ ] , include multiple ifn-α subtypes ( in humans and in mice), and a single ifn-β, and ifn-ε, ifn-κ, ifn-ω (humans) and ifn-ξ (mice) subtypes [ ] . in mammals, type i ifn (ifn-i) response is essential for innate antiviral responses. they all bind to the same ubiquitously expressed receptor, ifnar receptor, but they differ in their biological functions, due partially to the different binding affinities to the ifnar receptor [ ] . this differences in affinity results in different downstream signaling cascades [ ] . for ifn-α subtypes, the quantity of the receptor on the surface of a target cell correlates also with their biological activities suggesting that the amount of ifnar expression might compensate the weak affinity of some ifn-α subtypes [ , ] . type ii ifns include only one member, ifn-γ, secreted by activated t cells, natural killer (nk), nkt cells and dendritic cells with pro-inflammatory and immunomodulatory functions [ ] . in general, type ii ifn acts as a link between the innate immune response and the activation of the adaptive immune response [ ] . type iii ifns include ifn-λ , ifn-λ and ifn-λ , and, although genetically different to type i ifns and signaling through different receptors, they are induced by pprs and activates antiviral pathways similar to type i ifns [ , ] . viruses use multiple mechanisms to by-pass the host ifn responses so that they can replicate and continue their infectious cycle. the present review will focus on how viruses interfere with ifn-i responses. viruses can act at different levels of the signaling cascades involved in ifn-i responses. they can inhibit the induction of the ifn response, block the ifn signaling, and/or interfere with the antiviral activities induced by ifn. we will review some of the emerging themes and new insights from the past years of the ifn evasion mechanism employed by viruses in these contexts. the antiviral state of an infected cells is attained by the initial induction of type i ifn expression, followed by ifn signaling transduction, which finally leads to the expression of multiple genes (fig. ) . ifns are the main group of cytokines secreted by host cells in response to the presence of "aberrant" nucleic acids such as double-stranded rna (dsrna) molecules generated as viral intermediates during viral transcription in infected cells, to cpg dna, or uncapped ssrna with ′ triphosphate present in some viruses. these elements are known as pathogen-associated molecular patterns (pamp) [ , ] that can be recognized by prrs. four main types of prrs have been described to detect virus-derived genetic materials: toll-like receptors (tlrs) / / / [ ] ; retinoic acid-inducible gene-i (rig-i) like receptors (rlrs) which include the cytosolic sensor rig-i, the melanoma differentiation-associated factor (mda- ) and laboratory of genetics and physiology (lgp ) [ ] ; and nucleotide oligomerization domain-like receptors (nlrs) [ ] and the cytosolic dna sensors [ ] . for a host to establish an antiviral state it first requires the production of type i (α/β) ifns in direct response to virus infection and recognition of virus-derived genetic material. hence, both rig-i and mda- sense cytosolic dsrna. rig-i also specifically senses ′ triphosphate rna generated during infection, while mda- detects longer dsrna sequence generated during virus replication. rig-i and mda contain two caspase activation and recruitment domains (cards) at the n-terminal. activation of rig-i and mda liberates these card domains and drives the interaction of these tandem cards with the card of the mitochondrial activation signaling (mavs) protein [ ] . mavs aggregates in filaments that provide a platform for the recruitment of the elements involved in the subsequent signaling cascade such as the tumor necrosis factor receptor-associated factor (traf) and traf , the tank binding kinase (tbk ) and ikkε ultimately drives the activation of transcription factors as irf / , nfκb and ap- , leading the production of type i ifn and pro-inflammatory cytokines [ ] . irf is constitutively expressed in many cell types, and after phosphorylation, irf- forms a homodimer that translocates into the nucleus, activating the transcription of early transcribed type i and iii ifn genes, ifn-β, ifn all type i ifns signal through the same heterodimeric transmembrane receptor termed the ifnα receptor (ifnar), containing the subunit and (ifnar and ifnar ). in a first step, ifn-i binds with high affinity to ifnar , and then recruit ifnar [ ] . ifnar engagement with ifn-i promotes the induction of an antiviral state in cells. this involves the upregulation of products from a large subset of genes named ifn-stimulated genes (isg) that protect the cell from viral replication. broadly speaking, isg products modulate and mediate ifn activity in the cells. this includes for instance cooperating in prr recognition of viral pamps, stabilizing signaling complexes to improve their resistance to degradation, stopping virus entry, blocking viral capsid formation, impairing trafficking and budding of virions from the infected cells, but also modulating the ifn response to avoid the toxicity of these potent immune mediators. an important feature of the ifn signaling is the rapid speed at which the response happens, which is possible because protein synthesis is not required in an initial stage. the interaction of type i ifns with their universally expressed receptor (ifnar) elicits an intracellular signaling cascade through the janus protein kinase (jak) family members, jak and tyk , that successively phosphorylate signal transducer and transcription activator (stat) family proteins [ ] . the phosphorylated stat /stat heterodimer associates with interferon regulatory factor (irf ) to form the transcriptional factor complex isgf , which translocate to the nucleus and binds the ifn-response elements (isre) in isg promoters leading to the expression of isg products [ ] (fig. the oligoadenylate synthetase (oas)-latent rnase (rnase l) pathway is another ifn-inducible pathway that provides the cell with an effector mechanism upon recognition of viral dsrna (reviewed in [ ] ). when the oas senses dsrna activates the production of ′, ′-oligoadenylates that act as a second messenger on the inactive monomeric rnasel [ ] . the ′, -oligoadenylates binding to rnase l produces a catalytically active dimer that cleaves ssrna [ ] . this leads to the translational arrest and prevent viral replication and spreading [ ] . rna-activated protein kinase is an isg product that detects cytosolic dsrna. pkr recognition of its dsrna substrate leads to dimerization and autophosphorylation which in turn leads to the phosphorylation of the viruses has developed strategies to counteract dif-ferent steps on this signaling cascade. it is marked in red blades the main signaling targets of viruses eukaryotic initiation factor α (eif α) required for translation initiation [ ] . eif α phosphorylation results in the shutdown of all translation of ′capped mrna, thus preventing the synthesis of viral proteins. this also usually results in the formation of stress granules (sg) that consist of the accumulation of rna and proteins from the stalled translation complexes. sg formation has been linked to antiviral responses, and their formation is often inhibited in viral infections. in general, the antiviral activity of pkr is related to apoptosis induction [ ], regulation of ifn-β synthesis and nf-κΒ pathway [ ] [ ] [ ] , serine kinase activity for stat that also regulate the ifn-i signaling pathway [ ] . a family of proteins with a wide range of anti-viral functions are the interferon-induced proteins with tetratricopeptide repeats (ifits) [ ] . these genes are expressed at very low basal levels, but their transcription is rapidly increased after activation by ifn signaling. ifit detect the lack of ′-o-methylation on rnas species, a methylation absent in some viral rna but present in eukaryotic mrna [ ] . ifit has also been shown to bind to the ′-trip end of some viral rna [ ] . ifit can sequester viral rna or interact with the eukaryotic translation initiation factor to inhibit the translation initiation of ifit -bound rna species. in addition to these isgs, one highly upregulated gene in the initial stage of the antiviral immune response is isg (interferon-stimulated gene ), which encodes an ubiquitinlike protein involved in a post-translational process termed isgylation [ ] . this process allows isg to bind covalently to a range of target proteins, both viral and cellular [ ] , by a process that is reversible due to the action of the ubiquitin-specific protease (usp ), an event regulated by type i ifn [ ] . isgylation appears to modulate the activity of multiple elements involved in the ifn response. for instance, isgylation has been shown to sustain stat or irf activity [ , ] , downregulate the turnover of ubiquitinated proteins [ ] , but also to negatively regulate rig-i signaling [ ] . isg acts during viral replication by interfering with the endogenous proteins that the virus needs to replicate. thus, isg conjugates to the eukaryotic factor e (eif e) homologous protein ( ehp) that binds to the capped mrna, inhibiting in this way the viral rna translation of those viruses that contains a capped positivesense rna such as flaviviruses [ ] . isg also exists as an unconjugated protein that acts as a cytokine, regulating viral replication and host responses [ , ] . the role of the isgs members mentioned above clearly illustrates the breadth and diversity in the function of this protein group. a database has been created, called interferome (https ://www.inter ferom e.org/inter ferom e/home. jspx), in which isgs are catalogued and incorporated into a database, based on the information obtained from all published reports where cells were treated with ifn, thus, this database will allow to identify isg signatures from highthroughput data, having implications for determining the role of isgs. viruses employ mechanisms that impair isg activity to enhance their evasion from the ifn system. the mechanisms will be discussed in another section of this review. as discussed previously, prr activation leads to the production of ifn-i, -iii, and pro-inflammatory cytokines such as il- β. the present section will be centered on how viruses affect ifn-i induction. typically, virus genetic material triggers ifn-i induction when recognized by viral nucleic acid sensors that are membrane bound or present in the cytoplasm/nucleus. this recognition leads to activation of signaling cascades that converge towards the induction of ifn-i production in infected cells. viruses are known to interfere at every point of this process. viruses can interfere with the sensing of their genetic material, impair the signaling cascade that leads to ifn-i induction, and/or antagonize the activity of the transcription factors involved in ifn-i gene expression. the present section aims at providing a nonexhaustive overview of some of the most commonly used viral mechanisms to counter ifn-i induction in the host with a focus on recent findings in the field. viruses use different mechanisms to counteract the recognition of their genetic material by the host cell so that ifns are not induced (table ) . viruses can sequester, modify or even degrade their nucleic acids to avoid detection by prrs. for instance, during replication most flaviviruses create vesicular structures in the er membrane which physically shield the viral genetic material from cytosolic rlrs [ ] [ ] [ ] . influenza a virus (iav) uses the nucleus for replication, atypically for an rna virus, so that its genetic material remains hidden from cytosolic rlrs [ ] . vaccinia virus (vv), a large double-stranded dna virus has the peculiarity of replicating in the cytoplasm, where dna sensors like cgas are present, which potentially renders the viral genetic material susceptible to recognition by prr. vv replication, however, occurs in organelles similar to micronuclei [ ] that probably render viral dna inaccessible to recognition by cytoplasmic dna sensors. rotaviruses (rv) concentrate their replication in a cytoplasmic structure called viroplasms where dsrna genome is generated for packaging so that it is not exposed to the cytoplasmic prr [ ] . many viruses also encode proteins that help conceal their genetic material from prr. some viruses possess dsrna binding proteins that could potentially sequester these pamps from ppr recognition, such as vp from ebola virus or σ from reovirus [ , ] . the encapsidation of the viral rna can also impair rlr recognition. for instance, iav nucleoprotein and polymerase prevents rig-i binding to viral rna during transit through the cytoplasm [ ] . iav ns protein also possesses a dsrna binding site that prevents recognition by rig-i [ ] . calicivirus and picornavirus ssrna ( +) is covalently linked to a capping protein that could prevent recognition of the ′ viral rna extremity by rig-i [ ] . lassa virus (lasv) nucleoprotein (np) can act as a capping enzyme with exonuclease activity specific for dsrna, which has been shown to antagonize ifn induction [ , ] . other viruses can modify their ′tri-p motifs recognized by rig-i to evade this cytoplasmic rna sensor. hantaan virus, crimean-congo hemorrhagic fever virus and borna disease virus can process their ′ genome extremity to form ′mono-p forms, evading rig-i recognition [ ] . poxvirus decapping enzymes d and d can prevent the accumulation of dsrna, an intermediate necessary in viral replication, and thereby evade rlr recognition [ ] . measles virus (mev) encodes for the non-structural c protein that can impair ifn response by modulating viral rna replication [ ] and improving the polymerase processivity [ ] , thus probably limiting the amount of viral material recognizable by cytosolic prr. another mechanism employed by viruses to limit detection by prr is to interact with these sensors to impair their activation. the kaposi's sarcoma-associated virus (kshv) uses the tegument protein orf to bind to cgas and inhibit cgamp production, the second messenger used for sting (stimulator of interferon response cgamp interactor ) activation [ ] . homologues of orf in other gammaherpesviruses have also been described to act similarly, indicating that inhibition of this prr pathway is probably shared by gammaherpesvirus. the herpes simplex virus (hsv- ) tegument protein vp has also been shown to inhibit cgas enzymatic activity, indicating that other herpesviridae can directly target cgas [ ] . other viruses can sequester prr so that they are unable to relocate to their activity site. for instance, the protein z of new world arenaviruses binds to rig-i and prevents its association with the signaling platform mavs protein [ ] . severe acute respiratory syndrome coronavirus (sars-cov) m protein has been shown to associate with rig-i and can potentially sequester this prr [ ] . viruses can also promote prr degradation, thus reducing the number of cellular sensors capable of detecting viral infection (fig. ). this can be done directly by proteases encoded by the viral genome. foot and mouth disease virus (fmdv) l pro and c pro protease can reduce rig-i intracellular protein levels [ ] . the c pro protease of other picornaviruses has also been shown to degrade rig-i [ ] , indicating that this is a shared mechanism of rlr evasion by this viral family. rig-i is not the sole prr that picornavirus proteases can target; the poliovirus and enterovirus (ev ) a pro protease can also degrade mda [ ] . viruses also encode for proteins that indirectly promote prr degradation. the nuclear sensor of dna ifi is degraded during hsv- infection through a mechanism dependent on the viral icp protein that is not fully understood but probably involves targeting the dna sensor for proteasomal degradation [ ] . a e ligase activity on the nss protein of the phlebovirus toscana virus was recently identified that allowed the ubiquitination of rig-i card domains and the subsequent proteasomal degradation of the prr [ ] . the ns b protein of the flavivirus dengue virus (denv) can target the cytosolic dna sensor cgas for lysosomal degradation. although this [ ] [ ] [ ] mechanism could at first glance seem counterproductive for an rna virus, it actually allows denv to evade the recognition of the mitochondrial dna that becomes exposed during infection [ ] . other viruses alter the post-translational modifications essential to prr signaling so that the ppr remains inactive. some viral-encoded proteins achieve this directly, as in the case of the deubiquitinase activity of coronaviruses papainlike protease (plp) that removes the k ubiquitin tail from rig-i that is essential for rig-i translocation to the mavs protein platform [ ] . this deubiquitination activity has been characterized in several coronaviruses thus suggesting that this mechanism is central to coronavirus evasion from the ifn system [ ] [ ] [ ] . paramyxovirus v protein binds to mda and impairs its dephosphorylation by blocking the atp hydrolysis necessary for mda folding to its active state, thus impairing the adequate activation of this prr [ ] . viruses can also impair prr activity by interfering with the functionality of accessory cellular components required for prr activation. mev has been shown to act on the phosphatase pp required for rlr activation using two distinct mechanisms. the mev v protein can bind pp which prevents mda dephosphorylation [ ] . mev infection in dendritic cells also produces recognition of the viral particle through the c-lectin receptor dc-sign [ ] . this triggers a signaling cascade that results in raf- kinase activation and the association of the pp inhibitor i- with pp that prevented rlr dephosphorylation thus impairing ifn induction. several viruses have also been shown to interact with the dsrna binding protein pact that potentiates rlr activation [ , ] . for instance, ebola virus vp protein, middle east respiratory syndrome viral interference with accessory cellular components involved in prr activation. mev can interfere with rlr activation by targeting the phosphatase pp using distinct mechanisms. mev v protein can interact with pp to prevent the dephosphorylation of mda- required for activation. mev can interact on the cell surface with the c-lectin receptor dc-sign which results in the association of pp inhibitor with pp thus preventing rlr dephosphorylation. ebola virus vp protein, mers-cov a protein and arenavirus np can interfere with pact binding to dsrna, a mechanism that potentiates rlr activation. rlr ubiquitination is also essential for adequate activation and transport to mavs for subsequent ifn signaling events to take place. riplet and trim are critical to rig-i ubiquitination. iav-ns and denv sfrna can interfere with trim activity, whereas hepatitis c ns - a protease can cleave riplet to impair rig-i ubiquitination. the mitochondrial-targeting chaperone - - ε is responsible for rig-i translocation to the mitochondrial membrane. denv ns protein targets - - ε using a phosphomimetic domain that displaces activated rig-i from this chaperone. wnv ns possesses a similar domain coronavirus (mers-cov) a protein and arenavirus nucleoproteins have been shown to interfere with pact binding to rlr [ ] [ ] [ ] . as previously stated, rig-i activation is associated to its ubiquitination with k ub chains that liberates its autorepressed n-terminal card domains, a mechanism dependent on the activity of the cellular proteins trim and riplet [ , ] . this mechanism can be targeted by viral products such as the iav ns protein that impairs trim -mediated rig-i ubiquitination [ , ] , or the hepatitis c virus ns - a protease that cleaves riplet [ ] . intriguingly, not only protein products appear to interfere with the rig-i activation complex. the subgenomic flavivirus rna (sfrna) generated during denv infection can bind to trim and prevent the deubiquitination step required for trim activity to take place [ ] . denv uses yet another mechanism to avoid prr detection. denv ns protein possesses a phosphomimetic domain that binds the mitochondrial-targeting chaperone protein - - ε [ ] . - - ε is responsible for rig-i translocation to the mitochondrial membrane where the subsequent steps of the ifn induction signaling cascade take place. by binding - - ε, denv ns displaces the activated rig-i complex and prevents ifn induction. interestingly, a similar phosphomimetic domain is also present in west nile virus (wnv) ns protein [ ] . viruses not only interfere with the prr capable of detecting their presence during infection, they also commonly affect the activity of the signaling complexes in charge of signal transduction. indeed, this is a strategy employed by most viruses to limit ifn responses. viruses can antagonize these signaling cascades at multiple levels and through varied mechanisms (table ). this can be achieved by impairing the post-translational modifications required for signaling, i.e. by altering the phosphorylation or ubiquitinylation status of signaling intermediates. for instance, sars-cov and human coronavirus nl (hcov-nl ) plps have been shown to prevent sting dimerization and thus subsequent activation of the tbk pathway possibly through the plp deubiquitinase activity [ , ] , as sting dimerization is dependent on the attachment of k ub chains [ ] . similarly, sars-cov plp can inhibit tlr signaling by removing k -ub chains from traf and traf and thus blocking tbk activation [ ] . another strategy to prevent post-translational modification of ifn-i pathway signaling components consists of sequestering these components so that they do not reach the adequate cellular location for activation. the ns protein from the economically important orbivirus bluetongue virus (btv) binds to the ubiquitinbinding protein optineurin in the golgi apparatus [ ] . this prevents optineurin from recruiting ubiquitinated tbk at the golgi apparatus, a necessary step for subsequent tbk phosphorylation to occur [ ] . recently, it has been described that cgamp, the second messenger generated by cgas and that activates sting, can be cleaved by poxvirusencoded nucleases (named poxins) [ ] . this allows poxvirus blockade of the cgas-sting signaling axis. viral proteins can also prevent the adequate formation of signaling complexes by steric hindrance. for instance, human adenovirus type e a protein has been shown to bind to sting and thus antagonize ifn signaling [ ] . mers-cov and sars-cov m proteins can interact with traf and thus disrupt the traf -tbk association [ , , ] . mers-cov orf b protein can associate with the tbk /ikkε complex to impair signaling [ ] , indicating that coronaviruses encode for multiple proteins that affect ifn induction at different stages of the signaling cascade. kshv encodes for viral interferon regulatory factor (virf ), a protein that binds to sting and block the association of tbk with sting, thus hindering the consequent irf phosphorylation [ ] . hsv- encodes for the icp protein that can interact with the active sting-tbk complex and inhibit the subsequent tbk -mediated phosphorylation of irf [ ] . the nss protein from some phleboviruses has also been described to antagonize ifn induction by targeting tbk [ ] . tbk activity is thus commonly targeted by both rna and dna viruses to antagonize ifn induction. viruses can also promote the degradation of signaling proteins involved in ifn induction. virally-encoded proteases can directly cleave some of the components of these pathways. sting can be directly cleaved by the ns b protease from several flaviviruses such as denv, zika virus, wnv, or japanese encephalitis virus (jev), but not others like yellow fever virus [ ] [ ] [ ] . indeed, this specific cleavage could partially explain some of the host range and pathogenicity of these flaviviruses in humans, as the identified sting cleavage site for the ns b protease is only partially conserved among species [ ] . similarly, the c pro from several picornoviridae has been described to cleave mavs proteins, thus inhibiting signal transduction [ , ] . the c-like protease from the arterivirus porcine reproductive respiratory syndrome virus (prrsv) and the ns / a protease complex from the flavivirus hepatitis c virus (hcv) have also been described to cleave mavs protein [ , ] . other viruses encode proteins that promote the degradation of signaling complexes involved in the ifn responses. rv nsp and vp proteins have been shown to target mavs protein for proteasome-dependent degradation and thus impair ifn induction [ , ] . sars-cov orf b protein localizes in the mitochondrial membrane where it interacts with mavs protein and promote its degradation [ ] . this mechanism probably involves the recruitment of the mavs protein cellular regulator pcbp [ ] to the mitochondrial membrane by sars-cov orf b protein, which favors mavs protein ubiquitination through k -ub chains and thus subsequent proteasomal degradation. since mavs protein represents an important signaling platform in the ifn induction cascade triggered by rlr activation, some viruses use strategies to alter mitochondria structure to impair mavs protein assembly. denv ns b protease can cleave mitofusins, which alters mitochondria dynamics and impair their fusion [ ] . in other cases, viruses can also promote mitophagy to promote their replication and potentially impair ifn responses. mitophagy has been described in mev, hepatitis b virus (hbv) or newcastle disease virus infections [ ] [ ] [ ] . the protein m from the human parainfluenza virus has also been shown to promote mitophagy and thus trigger mavs protein degradation to antagonize ifn induction [ ] . recently, iav has also been shown to employ a similar mitophagic strategy to reduce mavs protein levels through the expression of its pb -f protein [ ] . viruses can also act on the transcription factors that bind to the ifn-i promoter and trigger the expression of isgs. viruses use multiple strategies to impair the binding of the transcription factors to the ifn-i promoters (table ) . they can impair the phosphorylation by the tbk /ikkε complex of irf / that activates dimerization and transport to the nucleus. for instance, the paramyxovirus c protein inhibits irf phosphorylation and thus its activation [ ] . the denv ns b protease can impair irf phosphorylation by masking the ikkε kinase domain necessary for irf activation [ ] . lymphocytic choriomeningitis virus (lcmv) nucleoprotein was also shown to bind to the ikkε kinase domain, thus preventing irf phosphorylation [ ] indicating that arenavirus np-mediated inhibition of irf phosphorylation [ ] uses a similar mechanism. viruses not only target irf / activation to antagonize ifn induction, they can also interfere with the activation of nf-κb, as this transcription factor collaborates with irf in the early activation of the ifn-β promoter [ ] . most arenavirus nucleoproteins not only block irf phosphorylation but they can also impair nf-κb activity [ ] . viruses can also target adaptor molecules that regulate nf-κb activity. the nf-κb essential modulator (nemo) is part of the kinase complex that controls nf-κb release from its inhibitor iκb. the c pro from hepatitis a virus or fmdv and the c-like proteases from porcine epidemic diarrhea virus or prrsv cleave nemo thus preventing iκb release from nf-κb and consequently antagonizing ifn-β production [ ] [ ] [ ] [ ] . the degradation of the transcription factors involved in ifn induction is also the target of different viral proteins, as in the case of the nsp from rv that promote the degradation of several irfs including irf and [ ] . rv nsp appears to target irfs by interacting with their dimerization domain and thus preventing their association in active form [ ] . nsp has also been described to impair nf-κb by promoting the degradation of β-transducing repeat-containing protein (β-trcp) [ ] , the protein responsible for substrate recognition of the e ligase complex that targets for degradation iκb, the associated inhibitor of nf-κb. similarly, to other viral products, such as hiv- vpu, vv a , or epstein-barr virus lmp- , rv nsp associates with β-trcp through mimicry of the phosphorylated iκb that requires degradation [ ] [ ] [ ] [ ] . rv nsp , however, presents the particularity of not only blocking the interaction of iκb with the e ligase complex but also of targeting β-trcp for degradation. viruses can also impair irf / activity downstream of their activation by phosphorylation. the virf encoded by kshv can block irf activity downstream of irf activation by impairing the recruitment of the cbp-p coactivators to the irf complex [ ] . this can also be achieved by blocking the transport of activated transcription factors to the nucleus. for instance, some paramyxoviruses use their v protein to impair irf translocation to the nucleus [ ] . jev ns blocks irf and p subunit from nf-κb transport to the nucleus by interacting with nuclear transport proteins [ ] . the nss from the emerging bunyavirus severe fever with thrombocytopenia syndrome virus have also been described to sequester the tbk -ikkε-irf complex in viral inclusion bodies to prevent the trafficking of irf to the nucleus [ ] . some viruses also code for proteins that interfere with ifn induction in the nucleus. in many instances, the exact mechanisms of ifn antagonism by viral products in the nucleus are not fully resolved. mers-cov orf b protein impairs ifn induction in the nucleus by a mechanism yet to be elucidated [ ] . btv encodes non-structural protein (ns ) that localizes in cell nucleoli and possess ifn antagonist activity [ ] . mev c protein can interfere with ifn-β promoter activation in the nucleus and this property has been linked to the virus pathogenicity [ ] . the nss protein from the phlebovirus sandfly fever sicilian virus can prevent irf activity by interacting with the dna binding domain of irf [ ] . the protein orf from the murine gammaherpes virus was shown to bind to irf and to prevent its association with the co-transcriptional activator cbp, thus impairing irf binding to the ifn-β promoter [ ] . viruses have developed strategies to antagonize ifn induction at multiple stages of the signaling cascade. this includes limiting the recognition of their genetic material, impairing the activation of prrs or their signaling partners, promoting the degradation of key components of the signaling cascade, sequestering signaling complexes away from their site of action, or impairing dna binding of the transcription factors involved in ifn induction. in viruses, multiple mechanisms have also often evolved to target several elements in these pathways, and hence augment their capacity to evade innate immunity. viruses can suppress the ifn signaling at different levels ( table ). in this section, we will discuss some of the mechanisms that viruses use to counteract the action of this signaling cascade. one of the first steps in the signaling cascade is the phosphorylation of jak and tyk , relevant for initiating the jak-stat signaling. by directly promoting the dephosphorylation of the jak/stat pathway, viruses counteract the ifn response. for instance, sendai virus (respirovirus) c protein inhibits the phosphorylation of receptor-associated kinases jak and tyk by binding to the ifn receptor subunit ifn-α/β [ ] . the ns protein of the jev blocks the tyrosine phosphorylation of tyk and stat [ ] . stat phosphorylation is targeted by several viruses, using different mechanisms. the paramyxovirus family, that includes mev, peste des petits ruminants virus (pprv) and mumps, express v and p proteins that interact directly with stat , inhibiting phosphorylation [ , ] . the immediate-early protein icp of hsv- downregulate stat phosphorylation and prevent the accumulation of stat in the nucleus [ ] . the denv proteins ns a, ns a, ns b block the phosphorylation of stat [ ] . the ns protein of hcv interacts with stat , interfering with its phosphorylation [ ] . among the reoviridae family, the nsp protein encoded by rotavirus block the phosphorylation of stat [ ] , and the ns protein encoded by btv blocks stat phosphorylation [ , ] . many viruses target stat to antagonize ifn signaling. thus, mev v protein binds to stat [ , ] . yellow fever virus ns protein also binds stat but this interaction requires stat activation by ifn [ ] . other example is denv ns protein acts as a bridge between ubr and stat , driving stat to degradation through the proteasome [ ] . the nsp protein of pprs degrades stat via proteasome [ ] . the proteasome is not the only catabolic cellular machinery that viruses can highjack to degrade signaling components of the ifn signaling pathway. btv was recently shown to use ubiquitination of its ns protein to drive stat degradation by an autophagy-dependent mechanism [ ] . another strategy used by viruses includes the interference with ifn signal transduction by modification of the constitutive or basal levels of molecules involved in the jak/ stat pathway that viruses members of the rubulavirus genus like simian virus , mumps virus or human parainfluenza virus type , use [ , ] . the human papillomavirus- (hpv- ) expresses the viral e protein that binds the p protein blocking its translocation to the nucleus, impeding the association of irf- with the stat- /stat- heterodimer (isgf ), and thereby inhibiting the induction of ifn-i inducible genes [ ] . viruses involved in persistent infections, such as cytomegaloviruses (cmv), polyomaviruses, hcv [ ] , or hsv- [ ] use similar strategies. cmv affects the expression levels of jak and irf- [ ] , and the viral large t antigen of murine polyomavirus binds to jak inactivating the transduction signal through ifn receptors [ ] . viruses employ mechanisms that impair isg activity to enhance their evasion from the ifn system. in this section, we will discuss some of these mechanisms. as previously mentioned, some isg products enhance the recognition of viral pamps and provide the cell with effector mechanisms that block viral replication. thus, fmdv l pro cleaves g bp , an rna-binding protein essential to stress granules (sg) assembly, to impair the formation of these structures [ ] . mev c protein has been involved in sg inhibition through blockade of pkr-induced sg by the activity of the adenosine deaminase acting on rna (adar ) [ ] . denv sfrna can bind to various components involved in sg assembly. this property of denv sfrna also led to impaired isg mrna translation, thus dampening ifn responses [ ] . to overcome recognition by isg products, some viral mechanisms are discussed. the reovirus σ protein was shown to inhibit pkr activity probably through its ability to bind dsrna [ , ] . nss protein from bunyavirus can promote pkr degradation [ ] . poxvirus d and d decapping enzyme promote dsrna degradation, thus preventing pkr recognition [ ] . viruses can also highjack regulatory isg pathways to evade isg product action. for instance, adar is an isg involved in rlr regulation. adar has an important physiological function as it edits adenosines to inosines in rna, a feature that destabilize the structure of complementary dsrna strands, thus preventing rlr or pkr recognition. this is an important mechanism in the prevention of autoimmunity, as it limits the recognition of cellular dsrna. viruses such as mev, vsv or hiv- have been shown to use adar function to block pkr activation and thus evade translation shutdown [ ] [ ] [ ] . viruses can also interfere with the oas-rnase l pathway. rotavirus vp protein blocks rnasel activation [ ] by cleaving the ′, ′-oligoadenylates produced by oas. the ns protein from coronavirus murine hepatitis virus has been shown to act similarly [ ] . the ifit is another isg that contribute to viral rna recognition. some viruses have developed ′-o methyltransferase activities on their gene products to prevent translation blockade of their rna by ifit. this has been described for wnv, coronaviruses, rv and poxvirus [ ] [ ] [ ] [ ] [ ] . the ifn-induced transmembrane proteins (ifitm) expression is greatly enhanced upon ifn activation, but these proteins are also expressed ubiquitously in the absence of ifn. the family of ifitm proteins has been shown to block iav, wnv and denv cell entry, a mechanism that probably involves viral hemagglutinin recognition [ ] . hcov-oc has been shown to highjack ifitm and ifitm for cell entry. this mechanism could be important for virus entry in lower respiratory tract under inflammatory conditions induced by ifn [ ] . in hiv- , vpu and env proteins can mutate to increase infectivity and overcome ifitm -mediated restriction of replication [ ] . mutations to overcome the activity of the isg product mxgtpase have also been described for iav, indicating that multiple isg products probably exert a selective pressure on viruses. for instance, pandemic avian iav strains appear to adapt to human through evasion of the np recognition by mxa gtpase [ ] . viruses have evolved strategies to dampen isg effects on ifn signaling. for instance, the plp from hcov-nl , sars-cov and mers-cov have been shown to not only act as a deubiquitinase but also as a deconjugating protease for isg chains [ , , ] . fmdv lpro has also been associated with cleavage of isg , but instead of targeting the isopeptide bond used in isg conjugation, it hydrolyzes the peptide bond preceding the isgylation motif [ ] . human cytomegalovirus (hcmv) tegument pul protein prevents isgylation [ ] . pul activity appears to be supported by two other tegument proteins pul and pp [ ] . hcmv also uses another tegument protein pul to affect isgylation by targeting ube l, an important ligase responsible for isg linkage, for proteasomal degradation [ ] . since isgylation also regulates the activation of ifnrelated pathways, some viruses have harnessed isgylation to favor their replication. hcv has also been described to use isgylation to favor its replication and develop persistent infections [ , ] . recently, isgylation was also associated with increased replication in hbv infections [ ] . multifaceted strategies have evolved in viruses to circumvent isg product activity and thus enhance their replication and spreading. these range from directly impairing the activity of isg products involved in host cellular defense to highjack the ifn modulating activity of some isg products. understanding these mechanisms of evasion will undoubtedly shed light on some of the pathogenic processes induced by viral infections. a unique strategy to inhibit the activity of ifn was described in with the identification of a poxviral secreted ifn type i binding protein (ifn-i bp) encoded by the b r gene of vaccinia virus (vv) [ ] a well characterized member of the orthopoxvirus genus that contains the strains used for the efficacious worldwide vaccination campaigns against smallpox. the protein was found to be a secreted glycoprotein of about kda expressed early during infection. while its sequence is unrelated to either of the two subunits of the cellular ifn-i receptor, ifnar or ifnar , the ifn-i bp was found to bind with high affinity (k d = pm for hifnα ) to several subtypes of human ifn-is, inhibiting their binding to the receptor and thus abrogating their biological activity [ ] . in stark contrast to the high species specificity observed for the cellular receptor, the viral protein is able to bind and inhibit the activity of ifn from different species, including mouse, rat, bovine and rabbit ligands, suggesting different interaction modes. currently, all human ifn-i molecules tested including (out of ) ifnα, ifnβ, ifnϖ as well as the more divergent ifnκ and ifnε are known to be bound and inhibited by b [ ] [ ] [ ] , although with varying affinities. interestingly, murine ifnα, but not murine ifnβ are inhibited by the poxviral ifn-i bp in spite of being bound with high affinity, as assessed by surface plasmon resonance [ , ] . competition studies using anti ifn monoclonal antibodies (mabs) showed that the binding interface of ifns with b is larger than the one with their cellular receptor, which could probably account for its broad species specificity and inhibitory capacity over a wide range of affinities [ ] . further examinations substantiated an additional property of the poxviral ifn-i bp which is its saturable binding to the cell surface after secretion, where the protein is active and can inhibit ifn as efficiently as the secreted one [ ] . this suggested that the main site of action is the cell surface, providing local tissue protection by protecting neighboring, still uninfected cells from entering into an ifn mediated antiviral state. examination of a truncated version of b expressed by the attenuated wyeth vaccv strain lacking its third, c-terminal immunoglobulin (ig) domain showed that cell binding capacity is mediated by the n-terminal regions of the protein [ ] . additional transfection analyses with different constructs suggested that cell binding activity is mediated by ig domain , while ifn blocking activity requires ig domains and [ ] site directed mutagenesis assays identified stretches of basic residues at the n terminus of b to mediate high affinity binding to cell surface sulfated glycosaminoglycans, preferentially heparan sulfate [ ] and showed that mutants lacking gag binding activity could still bind and inhibit ifn efficiently. the ifn-i bp protein has been found to be conserved in other orthopoxviruses including cowpox virus and ectromelia virus (ectv), a natural mouse pathogen, as well as the two viruses causing significant disease in humans, monkeypox virus and variola virus, the causative agent of smallpox [ ] . interestingly, the human viruses show an enhanced affinity for the human ligands, possibly reflecting the host adaptation of the virus, as occurs with other secreted cytokine binding receptors in this family. while possible orthologues can be readily found in virus species from several other poxvirus genera, these are frequently more distantly related, and their properties have not been extensively studied. the single exception to this is protein y of the tanapoxvirus yaba-like disease virus (yldv), a primate virus causing infection restricted to the skin. this protein, which shares only % aminoacid identity to the vacv b , can bind and inhibit both human (and monkey) ifn-i as well as the more recently described family of type iii ifns [ ] . the latter are a specialized group of ifns mediating antiviral response specifically at mucosal sites without compromising barrier integrity of the epithelia and promoting long-lasting humoral and cellular responses which signal through a distinct, specific heterodimeric cellular receptor (reviewed by [ ] ). the authors have proposed that inhibition of these ifns might be related to the specific tissue tropism of yldv, although information on its role in vivo has not yet been provided. insights into the biological role of poxviral ifn-i bps comes from murine infection models using vaccv and ectv, the latter naturally causing fatal mousepox in susceptible mouse strains. early reports showed that deletion of ifn-i bp gene from vacv attenuated the virus in vivo both in intranasal [ ] as well as intracranial [ ] infection models. in ectv, absence of ifn-i bp resulted in a completely attenuated phenotype upon footpad inoculation (ld reduction at least -fold) with severely impaired dissemination of virus to its secondary replication sites, liver and spleen, as well as enhanced nk cell recruitment and both cd + and cd + t cell activation [ ] crucially, these effects were shown to be dependent on ifnar signaling by the use of knockout mice. the ifn-i bp was found to bind to uninfected cells around infection foci in the liver and spleen protecting these tissues locally from ifn induced antiviral activity [ ] . the biological relevance of tissue retention of this inhibitory protein was shown using recombinant ectv that express a mutated ifn-i bp unable to bind to the cell surface but still able to inhibit ifn-i efficiently. infection with these recombinants resulted in non-lethal infection as in the case of the virus lacking ifn-i bp altogether [ ] . interestingly, it was found that immunization of mice with recombinant ifn-i bp could prevent the development of mousepox upon challenge [ ] , probably through the development of antibodies capable of impairing its interaction with its ligands [ ] and also pointing to a novel therapeutic target for the treatment of poxviral infections in humans. the structure of the complex of ectv ifn-i bp with murine ifnα- has been solved to high resolution (pdb entry oq , deposited by fremont and lee, results to be published). comparisons with the ternary complexes of different ifn-i with their cellular receptor [ , ] will be crucial to disentangle the structure function relationships in the interaction and inhibition of the biological activities of ifn-i ligands by the poxviral ifn-i bps. the particular properties of the poxviral ifn-i bp, especially its broad species and type specificity as well its high affinity have been instrumental to its use as a biotechnological tool. thus, b has been used to determine the implication of ifn-i in diverse processes, such as the monocyte-derived macrophage-mediated inhibition of human cytomegalovirus (hcmv) spread [ ] . in addition, recombinant oncolytic herpes simplex viruses expressing b have been developed to improve their infectivity in the face of antiviral responses [ ] . finally, b has been used to block ifnα mediated hiv associated encephalitis in a murine model [ ] or to inhibit the detrimental ifn mediated effects produced by mrna exposure in induced pluripotent stem (ips) cell reprogramming [ ] . secreted ifn-i bps have been exclusively found in poxviruses to date. a recent report described the murine norovirus ns protein, which is secreted by an unconventional caspase- mediated pathway, to be essential for tuft cell infection in gastrointestinal tissue through blockade of ifn type iii signaling [ ] . while a direct inhibition of ifn type iii could not be demonstrated in the reporter assay used, the molecular mechanism employed by this protein remains unsolved and raises the question as to whether additional and different soluble ifn-i or ifn-iii bps might be identified in other virus species. ifn responses are a complex and important component of the innate immune system. this is reflected in the vastness and complexity of isgs roles, not only involved in antiviral responses but also in several immunomodulatory functions. viruses can disrupt ifn responses leading to the antiviral state to promote their successful replication. indeed, viruses often interfere with multiple pathways involved in the ifn response to evade innate immunity. the importance of the ifn system in host antiviral responses is highlighted by the fact that viruses dedicate some of their genetic material to encode for ifn antagonists. the viral mechanisms of ifn evasion can be mediated directly by viral gene products. viruses also often usurp components of the cellular machinery to carry out their ifn antagonistic activity. there is no doubt that understanding how viruses evade the ifn system will shed some light on the pathogenicity and allow for a better design of therapeutic approaches. the interaction of these pathogens with the ifn system can also shed some light on some of the regulatory cellular mechanisms that control the ifn response. studying the interaction of viral components with the ifn system remains essential to understand the pathogenesis of emergent viruses that threatened global health. innate immune sensing and signaling of cytosolic nucleic acids identification of the zebrafish ifn 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virus-infected liver cells by up-regulating usp identification and characterization of a novel isg -ubiquitin mixed chain and its role in regulating protein homeostasis negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg conjugation isg modification of the eif e cognate ehp enhances cap structure-binding activity of ehp immunoregulatory properties of isg , an interferon-induced cytokine isg in antiviral immunity and beyond the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response ultrastructure of kunjin virus-infected cells: colocalization of ns and ns with double-stranded rna, and of ns b with ns , in virus-induced membrane structures tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles influenza a virus cell entry, replication, virion assembly and movement vaccinia virus dna replication occurs in endoplasmic reticulumenclosed cytoplasmic mini-nuclei regulated, stable expression and nuclear presence of reovirus double-stranded rna-binding protein sigma in hela cells relevance of ebola virus vp homo-dimerization on the type i interferon cascade inhibition influenza virus adaptation pb - k modulates nucleocapsid inhibition by the pathogen sensor rig-i a recombinant influenza a virus expressing an rna-binding-defective ns protein induces high levels of beta interferon and is attenuated in mice the genome-linked protein vpg of vertebrate viruses-a multifaceted protein cap binding and immune evasion revealed by lassa nucleoprotein structure structure of the lassa virus nucleoprotein reveals a dsrna-specific ‫׳‬ to ‫׳‬ e‫ٴٴ‬xonuclease activity essential for immune suppression processing of genome ‫׳ ‬ termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction poxvirus decapping enzymes enhance virulence by preventing the accumulation of dsrna and the induction of innate antiviral responses measles virus circumvents the host interferon response by different actions of the c and v proteins the c protein is recruited to measles virus ribonucleocapsids by the phosphoprotein inhibition of cgas dna sensing by a herpesvirus virion protein herpes simplex virus tegument protein vp abrogates cgas/ sting-mediated antiviral innate immunity z proteins of new world arenaviruses bind rig-i and interfere with type i interferon induction severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk /ikkepsilon complex foot-andmouth disease virus viroporin b antagonizes rig-i-mediated antiviral effects by inhibition of its protein expression rig-i is cleaved during picornavirus infection enterovirus apro targets mda and mavs in infected cells nuclear ifi induction of irf- signaling during herpesviral infection and degradation of ifi by the viral icp protein toscana virus non-structural protein nss acts as e ubiquitin ligase promoting rig-i degradation dengue virus ns b protein targets cgas for degradation and prevents mitochondrial dna sensing during infection deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases structure-guided mutagenesis alters deubiquitinating activity and attenuates pathogenesis of a murine coronavirus structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease mers-cov papain-like protease has deisgylating and deubiquitinating activities a shared interface mediates paramyxovirus interference with antiviral rna helicases mda and lgp antagonism of the phosphatase pp by the measles virus v protein is required for innate immune escape of mda measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp phosphatases the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response pact facilitates rna-induced activation of mda by promoting mda oligomerization middle east respiratory syndrome coronavirus a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda in the innate antiviral response mutual antagonism between the ebola virus vp protein and the rig-i activator pact determines infection outcome arenaviral nucleoproteins suppress pact-induced augmentation of rig-i function to inhibit type i interferon production a distinct role of riplet-mediated k -linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i molecular mechanism of influenza a ns -mediated trim recognition and inhibition dengue subgenomic rna binds trim to inhibit interferon expression for epidemiological fitness a phosphomimetic-based mechanism of dengue virus to antagonize innate immunity sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling ubiquitination of sting at lysine controls irf activation sars coronavirus papain-like protease inhibits the tlr signaling pathway through removing lys -linked polyubiquitination of traf and traf the golgi apparatus acts as a platform for tbk activation after viral rna sensing viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk -dependent phosphorylation of irf suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain middle east respiratory syndrome coronavirus orf b protein inhibits type i interferon production through both cytoplasmic and nuclear targets modulation of the cgas-sting dna sensing pathway by gammaherpesviruses hsv- icp targets the tbk -activated sting signalsome to inhibit virusinduced type i ifn expression heartland virus nss protein disrupts host defenses by blocking the tbk kinase-irf transcription factor interaction and signaling required for interferon induction species-specific disruption of sting-dependent antiviral cellular defenses by the zika virus ns b protease denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity seneca valley virus suppresses host type i interferon production by targeting adaptor proteins mavs, trif, and tank for cleavage the coxsackievirus b c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling porcine reproductive and respiratory syndrome virus c protease cleaves the mitochondrial antiviral signalling complex to antagonize ifn-beta expression hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity mavs protein is attenuated by rotavirus nonstructural protein rotavirus vp targets mavs for degradation to inhibit type iii interferon expression in intestinal epithelial cells sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome pcbp mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip dengue virus impairs mitochondrial fusion by cleaving mitofusins mitophagy switches cell death from apoptosis to necrosis in nsclc cells treated with oncolytic measles virus mitophagy promotes replication of oncolytic newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells hepatitis b virus disrupts mitochondrial dynamics: induces fissi on and mitophagy to attenuate apoptosis the matrix protein of human parainfluenza virus type induces mitophagy that suppresses interferon responses influenza a virus protein pb -f impairs innate immunity by inducing mitophagy an anti-interferon activity shared by paramyxovirus c proteins: inhibition of toll-like receptor / -dependent alpha interferon induction dengue virus subverts the interferon induction pathway via ns b/ protease-ikappab kinase epsilon interaction arenavirus nucleoprotein targets interferon regulatory factor-activating kinase ikkepsilon inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus convergence of the nf-kappab and irf pathways in the regulation of the innate antiviral response arenavirus nucleoproteins prevent activation of nuclear factor kappa b hepatitis a virus c protease cleaves nemo to impair induction of beta interferon footand-mouth disease virus c protease cleaves nemo to impair innate immune signaling porcine epidemic diarrhea virus c-like protease regulates its interferon antagonism by cleaving nemo arterivirus nsp antagonizes interferon beta production by proteolytically cleaving nemo at multiple sites diversity of interferon antagonist activities mediated by nsp proteins of different rotavirus strains rotavirus nsp mediates degradation of interferon regulatory factors through targeting of the dimerization domain rotavirus nsp inhibits nfkappab activation by inducing proteasome-dependent degradation of beta-trcp: a novel mechanism of ifn antagonism putative e ubiquitin ligase of human rotavirus inhibits nf-kappab activation by using molecular mimicry to target beta-trcp the human immunodeficiency virus type vpu protein inhibits nf-kappa b activation by interfering with beta trcp-mediated degradation of ikappa b interaction of epstein-barr virus latent membrane protein with scfhos/beta-trcp e ubiquitin ligase regulates extent of nf-kappab activation poxvirus targeting of e ligase beta-trcp by molecular mimicry: a mechanism to inhibit nf-kappab activation and promote immune evasion and virulence hhv- encoded virf- represses the interferon antiviral response by blocking irf- recruitment of the cbp/p coactivators inhibition of interferon regulatory factor activation by paramyxovirus v protein japanese encephalitis virus ns inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor and nf-kappab evasion of antiviral immunity through sequestering of tbk /ikkepsilon/irf into viral inclusion bodies bluetongue virus ns protein is an interferon antagonist and a determinant of virus virulence measles virus c protein interferes with beta interferon transcription in the nucleus nss protein of sandfly fever sicilian phlebovirus counteracts interferon (ifn) induction by masking the dnabinding domain of ifn regulatory factor conserved herpesviral kinase promotes viral persistence by inhibiting the irf- -mediated type i interferon response respirovirus c protein inhibits activation of type i interferon receptor-associated kinases to block jak-stat signaling blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns through a protein tyrosine phosphatasemediated mechanism paramyxovirus v proteins interact with the rig-i/ trim regulatory complex and inhibit rig-i signaling the nucleoprotein and phosphoprotein of peste des petits ruminants virus inhibit interferons signaling by blocking the jak-stat pathway role for herpes simplex virus icp in the inhibition of type i interferon signaling inhibition of alpha/beta interferon signaling by the ns b protein of flaviviruses hepatitis c virus ns a disrupts stat phosphorylation and suppresses type i interferon signaling rotavirus nsp protein inhibits interferon-mediated stat activation virus-induced autophagic degradation of stat as a mechanism for interferon signaling blockade induction and control of the type i interferon pathway by bluetongue virus the measles virus v protein binding site to stat overlaps with that of irf stat is a primary target for measles virus v protein-mediated alpha/ beta interferon signaling inhibition the interferon signaling antagonist function of yellow fever virus ns protein is activated by type i interferon dengue virus co-opts ubr to degrade stat and antagonize type i interferon signaling nonstructural protein of porcine reproductive and respiratory syndrome virus induces stat degradation to inhibit interferon signaling the v protein of simian virus inhibits interferon signalling by targeting stat for proteasome-mediated degradation association of mumps virus v protein with rack results in dissociation of stat- from the alpha interferon receptor complex the human papillomavirus type e protein binds human interferon regulatory factor- via a novel pest domain required for transformation expression of hepatitis c virus proteins interferes with the antiviral action of interferon independently of pkr-mediated control of protein synthesis herpes simplex virus gene products occlude the interferon signaling pathway at multiple sites human cytomegalovirus inhibits ifn-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of ifn-alpha signal transduction the polyoma virus t antigen interferes with interferon-inducible gene expression foot-and-mouth disease virus leader protease cleaves g bp and g bp and inhibits stress granule formation stress granule formation induced by measles virus is protein kinase pkr dependent and impaired by rna adenosine deaminase adar g bp , g bp and caprin are required for translation of interferon stimulated mrnas and are targeted by a dengue virus non-coding rna inhibitory activity for the interferoninduced protein kinase is associated with the reovirus serotype sigma protein site-directed mutagenic analysis of reovirus sigma protein binding to dsrna rift valley fever virus nss protein functions and the similarity to other bunyavirus nss proteins rna-specific adenosine deaminase adar suppresses measles virus-induced apoptosis and activation of protein kinase pkr double-stranded rna deaminase adar increases host susceptibility to virus infection adar interacts with pkr during human immunodeficiency virus infection of lymphocytes and contributes to viral replication silencing the alarms: innate immune antagonism by rotavirus nsp and vp homologous ', '-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity rotavirus open cores catalyze ′-capping and methylation of exogenous rna: evidence that vp is a methyltransferase ′-o methylation of the viral mrna cap evades host restriction by ifit family members attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking '-o-methyltransferase activity biochemical and structural insights into the mechanisms of sars coronavirus rna ribose '-o-methylation by nsp /nsp protein complex middle east respiratory syndrome coronavirus nonstructural protein is necessary for interferon resistance and viral pathogenesis the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus interferon induction of ifitm proteins promotes infection by human coronavirus oc hiv- mutates to evade ifitm restriction in vivo evasion of mxa by avian influenza viruses requires human signature in the viral nucleoprotein irreversible inactivation of isg by a viral leader protease enables alternative infection detection strategies consecutive inhibition of isg expression and isgylation by cytomegalovirus regulators the abundant tegument protein pul of human cytomegalovirus prevents proteasomal degradation of pul and supports its suppression of isgylation transmembrane protein pul of human cytomegalovirus inhibits isgylation by downregulating ube l isg , a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment the interferon stimulated gene functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response interferon-stimulated gene conjugation stimulates hepatitis b virus production independent of type i interferon signaling pathway in vitro vaccinia virus encodes a soluble type i interferon receptor of novel structure and broad species specificity vaccinia virus b r gene encodes a type i interferon-binding protein that blocks interferon alpha transmembrane signaling inhibition of type i and type iii interferons by a secreted glycoprotein from yaba-like disease virus the highly virulent variola and monkeypox viruses express secreted inhibitors of type i interferon human interferon-and interferon-kappa exhibit low potency and low affinity for cell-surface ifnar and the poxvirus antagonist b r the vaccinia virus soluble alpha/beta interferon (ifn) receptor binds to the cell surface and protects cells from the antiviral effects of ifn analysis of an interaction between the soluble vaccinia virus-coded type i interferon (ifn)-receptor and human ifn-alpha and ifn-alpha evaluating the orthopoxvirus type i interferon-binding molecule as a vaccine target in the vaccinia virus intranasal murine challenge model glycosaminoglycans mediate retention of the poxvirus type i interferon binding protein at the cell surface to locally block interferon antiviral responses type iii interferons: balancing tissue tolerance and resistance to pathogen invasion the orthopoxvirus type i ifn binding protein is essential for virulence and an effective target for vaccination antibody inhibition of a viral type interferon decoy receptor cures a viral disease by restoring interferon signaling in the liver a virus-encoded type i interferon decoy receptor enables evasion of host immunity through cell-surface binding structural linkage between ligand discrimination and receptor activation by type i interferons structural basis of a unique interferon-beta signaling axis mediated via the receptor ifnar human monocyte-derived macrophages inhibit hcmv spread independent of classical antiviral cytokines incorporation of the b r gene of vaccinia virus into an oncolytic herpes simplex virus improves antitumor activity the recombinant vaccinia virus gene product, b r, neutralizes interferon alpha and alleviates histopathological complications in an hiv encephalitis mouse model highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mrna a secreted viral nonstructural protein determines intestinal norovirus pathogenesis acknowledgements this work was supported by grant rti - -b- from the spanish ministerio de ciencia e innovación; grant s /baa- -platesa from the comunidad de madrid (fondo europeo de desarrollo regional, feder) and vetbionet infraia- from the euopean union h . key: cord- -jc ulx authors: siu, kam-leung; chan, chi-ping; kok, kin-hang; chiu-yat woo, patrick; jin, dong-yan title: suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain date: - - journal: cellular & molecular immunology doi: . /cmi. . sha: doc_id: cord_uid: jc ulx coronaviruses have developed various measures to evade innate immunity. we have previously shown that severe acute respiratory syndrome (sars) coronavirus m protein suppresses type i interferon (ifn) production by impeding the formation of functional traf -containing complex. in this study, we demonstrate that the ifn-antagonizing activity is specific to sars coronavirus m protein and is mediated through its first transmembrane domain (tm ) located at the n terminus. m protein from human coronavirus hku does not inhibit ifn production. whereas n-linked glycosylation of sars coronavirus m protein has no influence on ifn antagonism, tm is indispensable for the suppression of ifn production. tm targets sars coronavirus m protein and heterologous proteins to the golgi apparatus, yet golgi localization is required but not sufficient for ifn antagonism. mechanistically, tm is capable of binding with rig-i, traf , tbk and ikkε, and preventing the interaction of traf with its downstream effectors. our work defines the molecular architecture of sars coronavirus m protein required for suppression of innate antiviral response. host cells combat invading viruses by triggering innate immune response. as a counter-defense, viruses encode various immunosuppressive proteins to evade innate immunity. this interaction between host cells and viruses dictates the outcome of viral infection. coronaviruses are enveloped and positive-stranded rna viruses with a large genome of , kb. in the family coronaviridae, there are four genera. in addition to human coronaviruses e and nl in the genus alphacoronavirus, four other viruses belonging to the genus betacoronavirus, namely, human coronavirus oc , human coronavirus hku , severe acute respiratory syndrome (sars) coronavirus , and middle east respiratory syndrome (mers) coronavirus, have also been found to infect human. the concept that human coronaviruses are generally associated with mild respiratory diseases was overturned by the identification in of sars coronavirus, which causes a severe and highly lethal disease. , further investigation of the etiology of community-acquired pneumonia in an adult who was hospitalized in hong kong during the outbreak of sars led to the identification of human coronavirus hku , which circulates commonly in human population causing respiratory tract illnesses worldwide. [ ] [ ] [ ] although the mechanism by which sars coronavirus and the newly identified mers coronavirus cause severe respiratory diseases is not fully understood, their subversion of innate immunity is thought to contribute substantially to pathogenesis. , type i interferons (ifns) are major effector cytokines in innate antiviral response. to induce ifn production, pathogen-associated molecular patterns such as viral double-stranded rna are sensed by host pattern recognition receptors such as endosomal toll-like receptor and cytoplasmic rig-i. the activation of these receptors transmits a signal to downstream kinases tbk and ikke that form a functional complex with traf and tank. consequent phosphorylation of irf and irf transcription factors by these kinases leads ultimately to transcriptional activation of ifn promoters. , among the structural proteins encoded by sars coronavirus, m protein is a glycoprotein with three n-terminal transmembrane domains named tm , tm and tm . , we have previously reported that sars coronavirus m protein suppresses type i ifn production potently by preventing the formation of functional traf -tank-tbk /ikke complex. this suppression of innate antiviral response by sars coronavirus m protein represents one novel viral countermeasure against host innate immunity, which could play a role in sars pathogenesis. however, it remains to be seen whether m proteins of other coronaviruses, which share %- % identity in aminoacid sequence with sars coronavirus m protein, might also exhibit ifn-antagonizing activity. compared to sars coronavirus, human coronavirus hku is more frequently associated with less severe disease. [ ] [ ] [ ] it will therefore be of interest to determine whether human coronavirus hku m protein would be able to suppress ifn production. on the other hand, the functional domain in sars coronavirus m protein required and sufficient for ifn antagonism remains elusive. in this study, we compared m proteins of sars coronavirus and human coronavirus hku for their ability to counteract ifn production. furthermore, we dissected the structural domains in sars coronavirus m protein and mapped the region that mediates the ifn-antagonizing activity. plasmids and antibodies pifnb-luc reporter plasmid and rig-i expression plasmid were obtained from dr takashi fujita (kyoto university, kyoto, japan). expression vectors for tbk , irf and traf were generous gifts from dr genhong cheng (university of california, los angeles, ca, usa). , pisre-luc reporter construct was purchased from clontech (mountain view, ca, usa). mda , ikke, mavs and tank expression vectors have been described. monoclonal anti-flag (clone m ) and anti-a-tubulin (clone dm a) antibodies were purchased from sigma-aldrich (st louis, mo, usa). anti-myc was bought from santa cruz (dallas, tx, usa). anti-v was from life technologies (grand island, ny, usa). anti-gm was from bd transduction (lexington, ky, usa). anti-calnexin was from affinity bioreagent (golden, co, usa). cell culture and transfection hek and hela cells were cultured in dulbecco's modified eagle medium supplemented with % fetal bovine serum, mm l-glutamine and % penicillin/streptomycin at uc in a humidified atmosphere of % co . cells were transfected with genejuice transfection reagent purchased from novagen (madison, wi, usa). protein analysis and reporter assay western blotting, immunoprecipitation and dual luciferase assay were performed as previously described. , relative luciferase activity (rla) was derived by normalizing readings of firefly luciferase to those of renilla luciferase. it was expressed in arbitrary units. all experiments were performed in triplicates and student's t-test was used to assess statistically the differences between the indicated groups. confocal microscopy was performed on an lsm system (carl zeiss, oberkochen, germany) as described. , cell fixation was achieved with : (v/v) acetone-methanol. nuclei were counter-stained with , -diamidino- -phenylindole. real-time rt-pcr was performed as described. , briefly, total rna was extracted using rnaiso plus reagent (takara, shiga, japan) and cdna was synthesized by transcriptor first strand cdna kit (roche, indianapolis, in, usa). quantitation of target mrna expression was achieved with the comparative c t method. sars coronavirus-specific inhibition of type i ifn production by m protein m proteins from sars coronavirus and human coronavirus hku share % identity in amino-acid sequence ( figure ). we have previously demonstrated the capability of sars coronavirus m protein to antagonize type i ifn production. to determine whether human coronavirus hku m protein figure amino-acid sequence alignment of m proteins from sars coronavirus (scv) and human coronavirus hku (hku ). alignment was generated by clustal v (embl-ebi server (http://www.ebi.ac.uk/tools/msa/clustalw /). the identity between the two proteins is . %. identical residues are indicated by asterisks (*). strongly and weakly similar residues are highlighted by colons (:) and dots (.), respectively. sars, severe acute respiratory syndrome. could have similar activity, we made a side-by-side comparison of the two m proteins ( figure ). we used two luciferase reporter constructs to measure the activation of ifn response in cultured cells. in plasmid pisre-luc, the expression of luciferase reporter was driven by ifn-stimulated response elements (isre), which are activated by type i ifns and also bound to irf and irf transcription factors. , for plasmid pifnb-luc, reporter expression directly reflects the transcriptional activity of ifn-b promoter. to activate ifn production, we expressed rig-i, mda , mavs and tbk in hek cells. rig-i and mda are sensors of viral double-stranded rna. , mavs is a mitochondrial adaptor protein and tbk is the protein kinase that phosphorylates irf and irf . , these proteins represent three critical steps in the upstream, midstream and downstream of the intracellular signaling pathway that leads to ifn production. all proteins stimulated transcriptional activity driven by isre (figure a we next sought to identify the molecular determinants in sars coronavirus m protein that mediate virus-specific suppression of ifn production. n-linked glycosylation of viral structural proteins might affect their folding, stability, sorting and sensing by innate and adaptive immune systems. since sars coronavirus m protein undergoes n-linked glycosylation at a single site at the n terminus, , we first investigated the requirement of this type of posttranslational modification for ifn antagonism. we constructed an n-linked glycosylation-defective mutant of sars coronavirus m protein designated m-n q and interrogated its ifn-antagonizing activity. since sars coronavirus m protein and its m-n q mutant displayed equally potent activity to suppress rig-i-and tbk- -induced activation of ifn production (figure ), n-linked glycosylation is not influential in the suppression of ifn production by m protein. tm of sars coronavirus m protein is required for ifn antagonism sars coronavirus m protein has three tms (tm : - amino acids; tm : - residues; and tm : - residues) and a cytoplasmic endodomain ( - amino acids). to determine which part of this protein is required for ifn antagonism, we made and expressed in hek cells two truncated mutants designated m and m containing the tms and the endodomain separately (figure a and b) . we next compared m and its mutants m and m for their ability to circumvent ifn induction by rig-i and tbk . whereas m and m exhibited ifnantagonizing activity of similar potency, m was unable to suppress ifn-b promoter activity (figure b and c) . hence, the tms are required for the suppressive effect of sars coronavirus m protein on type i ifn production. we further investigated which of the three tms is required for ifn antagonism. because the single tm was unstable and secreted out when expressed alone in cultured hek cells (data not shown), we conjugated it to the endodomain, which was shown to be dispensable for ifn antagonism (figure ). the mutants named tm , tm and tm , which respectively contain tm (amino acids - ), tm (amino acids - ) and tm (amino acids - ) fused to the endodomain, were constructed and expressed in hek cells. all mutant proteins were stable and abundantly detected in the cell lysate (figure a) . interestingly, only tm was capable of inhibiting ifn-b promoter activity activated by rig-i and tbk . neither tm nor tm exhibited ifn-antagonizing activity (figure b and c) . in further support of the importance of tm , a chimeric m protein containing tm (residues - ) of sars coronavirus and the cytoplasmic domain ( - residues) derived from human coronavirus hku was equally active as sars coronavirus m protein in the suppression of ifn production induced by rig-i or tbk , whereas another chimera carrying tm of human coronavirus hku was totally inactive in this assay (figure a-c) . taken together, because tm , tm and endodomain are dispensable for ifn antagonism, only tm is required and probably sufficient for the suppression of ifn production. mechanism of tm suppression of ifn production sars coronavirus m protein suppresses ifn production by associating with and sequestering transducer proteins, thereby preventing the formation of functional traf -tank-tbk /ikke complex. if tm mediates ifn antagonism of sars coronavirus m protein, it should be able to associate with transducer proteins and impede traf interaction with partners. in addition, because golgi targeting would be the cause of transducer protein sequestration, tm should also be expected to be found in the golgi apparatus. both sars coronavirus m protein and the m mutant are known to localize predominantly to the golgi apparatus. , in agreement with this, sars coronavirus m protein colocalized substantially with gm (figure a-c) , a marker of the golgi complex. interestingly, golgi localization was also evident for tm mutant (figure g-i) . neither m nor tm colocalized significantly with calnexin (figure d-f and j-l) , a marker of the endoplasmic reticulum. in contrast, tm and tm were largely concentrated in the endoplasmic reticulum. their localization patterns were similar to that of calnexin, but distinct from that of gm (figure mx) . in other words, tm , tm or endodomain was not essential for golgi targeting. thus, tm is both required and probably sufficient for golgi localization of sars coronavirus m protein. on the other hand, reciprocal immunoprecipitation and immunoblotting experiments indicated the association of tm with rig-i, traf , tbk and ikke (figure a, lanes - ) . neither tm nor tm had the same property (figure a, lanes - ) . in addition, the interaction of traf with tbk , ikke and tank was inhibited in the presence of tm (figure b, lanes - ) , but not tm or tm (figure b, lanes - ) . in light of this, tm is both required and probably sufficient for interacting with traf and other transducer proteins and thereby preventing traf from engaging its partners such as tbk , ikke and tank. indeed, both sars coronavirus m protein and its tm mutant colocalized with traf (figure a-f ). however, human coronavirus hku m protein also localized to the golgi apparatus (data not shown) and colocalized with traf (figure g-i) , but did not impede the interaction of traf with tank, tbk or ikke (data not shown). plausibly, golgi localization and interaction with traf are required but not sufficient for suppression of irf activation. in this study, we showed that sars coronavirus m protein suppressed the production of type i ifns in a virus-specific manner and it did not share ifn-antagonizing property with m protein of human coronavirus hku . ifn antagonism of sars coronavirus m protein was mediated by n-terminal tm (amino acids - ), which targets m protein to the golgi complex and associates with traf to prevent it from interacting with tank, tbk and ikke. our findings provide additional molecular details for suppression of type i ifn production by sars coronavirus m protein. the inability of human coronavirus hku m protein to suppress ifn production was not too surprising. although the two full m proteins of sars coronavirus and human coronavirus hku share % identity, only % of the amino-acid residues in the tm region of the two proteins are identical. this difference in the tm region might be critical in governing the inhibition of traf . sars coronavirus is known to suppress innate antiviral response at multiple levels leading to a severe disease. , this might be similar to the emerging mers coronavirus. , in contrast, human coronavirus hku is commonly found in human population and is associated with less severe respiratory illnesses. - accordingly, it might not have the same ability to evade innate immunity. viral proteins are not well characterized and ifn-antagonizing proteins have not been identified in human coronavirus hku . it remains to be seen whether the distinct properties of m proteins in the suppression of ifn production could account at least partly for the severity of disease caused by sars coronavirus and human coronavirus hku . in one model to explain this difference, sars coronavirus and mers coronavirus are not yet adapted to the new environment in human, , , whereas the well-adapted human coronavirus hku and the other human coronaviruses such as oc and e have lost some of their immunosuppressive tools during evolution. to test this idea, the coronaviruses in the two groups should be compared more thoroughly for their abilities to induce ifns and to suppress ifn production and action. particularly, the ifn-antagonizing property of m proteins from more coronaviruses should be assessed in parallel. although attempts have been made to culture the virus in primary human ciliated airway epithelial cells and type ii alveolar epithelial cells, [ ] [ ] [ ] clinical isolates of human coronavirus hku remain unculturable in most laboratories. this hampers further comparative study in cultured cells. to remedy this, the dynamics of ifn induction should be analyzed in human subjects naturally infected with human coronavirus hku . we not only defined a minimal tm domain in sars coronavirus m protein that mediates suppression of ifn production, but also provided new mechanistic details for this suppression. although tm alone is unstable and secreted out, it is stable and functional when conjugated to tm -tm or endodomain of sars coronavirus m protein or of human coronavirus m protein (data not shown). tm contains the critical determinants for golgi localization, for association with rig-i, traf , tbk and ikke, and for prevention of traf from interacting with tank, tbk and ikke. our results are compatible with the notion that tm targets sars coronavirus m protein and its associated cellular proteins to the golgi apparatus. it will be of importance to clarify exactly how this might contribute to the suppression of irf activation. notably, human coronavirus hku m protein also targets the golgi complex, interacts with traf , but does not suppress ifn production. hence, golgi localization and interaction with traf are required but not sufficient for ifn antagonism. mechanistically, the perturbation of the interaction of traf with tank, tbk and ikke by tm of sars coronavirus m protein is more critical in the suppression of irf activation. this could plausibly be mediated by preoccupation and steric hindrance, but further investigations are required to provide additional support to this model. for example, it will be of interest to see whether artificial fusion of tm to traf might impede its binding with tbk and ikke or inhibit its activation of ifn production. mapping and comparing the tm -and tbk -interacting domains in traf would also shed light on how the binding of traf with tm affects the binding with tbk . more importantly, a thorough comparison of the tm regions of sars coronavirus and human coronavirus hku will define the critical residues for the suppression of traf interaction with downstream transducers. nevertheless, this model of suppression by sequestration and pre-occupation represents a new mechanism for immune evasion, which might also be used by other viral and cellular proteins such as microtubule-associated traf -binding protein mip-t , which localizes to the centrosome and cilia and also impedes the formation of functional traf -containing complex. furthermore, our definition of a small tm domain that mediates immune evasion will pave the way for rational design and development of new immunosuppressive cells were then stained for m and traf . nuclear morphology (blue) was visualized with dapi. specific fluorescent signals from v and traf were then merged. transfected cells in the merged panels are highlighted by arrows. colocalization appeared yellow. all panels shown are representative of the results of triplicate experiments. bar mm. dapi, , -diamidino- -phenylindole; ifn, interferon; sars, severe acute respiratory syndrome. characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia coronavirus as a possible cause of severe acute respiratory syndrome severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia coronavirus hku and other coronavirus infections in hong kong coronavirus infections in hospitalized pediatric patients with acute respiratory tract disease more and more coronaviruses: human coronavirus hku sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon toll-like receptors cytosolic sensing of viruses glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins a and m the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles severe acute respiratory syndrome coronavirus m protein inhibits type the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses modulation of the interferon antiviral response by the tbk /ikki adaptor protein tank irf mediates a tlr /tlr -specific antiviral gene program the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response severe acute respiratory syndrome coronavirus nucleocapsid protein does not modulate transcription of human fgl gene retroviral oncoprotein tax targets coiled-coil centrosomal protein tax bp to induce centrosome overduplication human t-cell leukemia virus oncoprotein tax represses nuclear receptordependent transcription by targeting coactivator tax bp internal ribosome entry site-mediated translational regulation of atf splice variant in mammalian unfolded protein response lkb tumor suppressor and salt-inducible kinases negatively regulate human t-cell leukemia virus type transcription regulation of type i interferon gene expression by interferon regulatory factor- interferon regulatory factor is induced by epstein-barr virus latent membrane protein virus glycosylation: role in virulence and immune interactions studies on membrane topology, nglycosylation and functionality of sars-cov membrane protein characterization of a cis-golgi matrix protein, gm retention of unassembled components of integral membrane proteins by calnexin middle east respiratory syndrome coronavirus accessory protein a is a type i interferon antagonist middle east respiratory syndrome coronavirus a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda in innate antiviral response comparative analysis of the activation of unfolded protein response by spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus hku the emerging novel middle east respiratory syndrome coronavirus: the ''knowns'' and ''unknowns culturing the unculturable: human coronavirus hku infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures human coronavirus hku infection of primary human type ii alveolar epithelial cells: cytopathic effects and innate immune response isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism mip-t is a negative regulator of innate type i interferon response we thank genhong cheng and takashi fujita for reagents; and chun kew, pak-yin lui, vincent hei-man tang and kit-san yuen for critical reading of manuscript. this work was supported by hong kong health and medical research fund ( ) and hong kong research grants council (hku /crf/ g). agents. in this regard, both peptide mimetics and recombinant proteins that mimic the action of tm might prove useful. key: cord- -hugenn authors: chen, zhuangzhuang; zhong, di; li, guozhong title: the role of microglia in viral encephalitis: a review date: - - journal: j neuroinflammation doi: . /s - - - sha: doc_id: cord_uid: hugenn viral encephalitis is still very prominent around the world, and traditional antiviral therapies still have shortcomings. some patients cannot get effective relief or suffer from serious sequelae. at present, people are studying the role of the innate immune system in viral encephalitis. microglia, as resident cells of the central nervous system (cns), can respond quickly to various cns injuries including trauma, ischemia, and infection and maintain the homeostasis of cns, but this response is not always good; sometimes, it will exacerbate damage. studies have shown that microglia also act as a double-edged sword during viral encephalitis. on the one hand, microglia can sense atp signals through the purinergic receptor p y and are recruited around infected neurons to exert phagocytic activity. microglia can exert a direct antiviral effect by producing type interferon (ifn- ) to induce ifn-stimulated gene (isg) expression of themselves or indirect antiviral effects by ifn- acting on other cells to activate corresponding signaling pathways. in addition, microglia can also exert an antiviral effect by inducing autophagy or secreting cytokines. on the other hand, microglia mediate presynaptic membrane damage in the hippocampus through complement, resulting in long-term memory impairment and cognitive dysfunction in patients with encephalitis. microglia mediate fetal congenital malformations caused by zika virus (zikv) infection. the gene expression profile of microglia in hiv encephalitis changes, and they tend to be a pro-inflammatory type. microglia inhibited neuronal autophagy and aggravated the damage of cns in hiv encephalitis; e ubiquitin ligase pellino (pelia) expressed by microglia promotes the replication of virus in neurons. the interaction between amyloid-β peptide (aβ) produced by neurons and activated microglia during viral infection is uncertain. although neurons can mediate antiviral effects by activating receptor-interacting protein kinases (ripk ) in a death-independent pathway, the ripk pathway of microglia is unknown. different brain regions have different susceptibility to viruses, and the gene expression of microglia in different brain regions is specific. the relationship between the two needs to be further confirmed. how to properly regulate the function of microglia and make it exert more anti-inflammatory effects is our next research direction. encephalitis is a common and serious disease, and its clinical manifestations vary from person to person. its main characteristics include altered mental status and various combinations of acute fever, seizures, neurologic deficits, cerebrospinal fluid (csf) pleocytosis, and abnormalities in electroencephalographic (eeg) [ ] . although there are many reasons accounting for this disease, the most commonly identified causes are neurotropic viruses. every year in the usa, there are about seven patients per , population diagnosed as encephalitis. among all the cases, approximately half of them have unknown reasons. of the cases with a known cause, viruses represent - %. herpes simplex virus (hsv) takes up to % of identified viral cases, with varicella-zoster virus (vzv), enteroviruses, and arboviruses accounting for the majority of the remainders [ ] . it is estimated that the median hospitalization charge for a patient with viral encephalitis is $ , for west nile virus encephalitis and $ , for hsv encephalitis. each year in the usa, approximately patients are hospitalized for acute viral encephalitis. the total annual cost is up to $ million to $ million, without counting the cost of care after discharge, costs for family caregivers, and lost earning [ ] . microglia are cns-resident mononuclear phagocytic cells characterized by a unique ramified shape and distinctive gene expression [ ] . most microglia are derived from a yolk sac progenitor and seed the brain early in development [ ] . as resident cells, microglia are assumed to help orchestrate the immune response to pathogen infection of the brain. while often touted as immune sentinels, little is known about how or if microglia engage in this function [ ] . so far, a large body of evidence stemming from both in vitro and in vivo studies have indicated that microglia are capable of responding to viral pamps [ , ] , but the multifaceted function of microglia involved in viral encephalitis needs further understanding. recently, how microglia are recruited around infected neurons and the interaction between microglia and other cells has become a hot topic. in this review, we will talk about the function of microglia in viral encephalitis. microglia are recruited around the infected neurons by sensing extracellular atp signals microglia can sense the nucleotides released by infected neurons and are rapidly recruited around infected neurons to exert phagocytic activities in the brain (see fig. ). neurons release more atp after viral infection, while the levels of atp, adp, amp, and adenosine in cell lysates decrease. noxious stimuli in neurons trigger a sustained increase in extracellular atp, which causes microglia to become activated and recruited in minutes to hours [ ] . atp is a potent agonist of the p y g protein-coupled receptor [ ] . studies have shown that p x or p y are both abundant in microglia, and p y is a microglia-specific marker in the brain [ ] . extracellular atp is a strong chemotactic signal for microglia. by stimulating the p y receptor of microglia, fig. illustration of the anti-inflammatory role of microglia in viral encephalitis. the picture shows that microglia can sense atp signals through the purinergic receptor p y and are recruited around infected neurons to exert phagocytic activity. microglia can exert a direct antiviral effect by producing type interferon (ifn- ) to induce ifn-stimulated gene (isg) expression of themselves. ifn produced by microglia exects indirect antiviral effects by acting on other cells to activate corresponding signaling pathways it mediates the rapid response of microglia to the injury site [ ] . the number of p y receptors on the surface of microglia increased over twofold in response to viral infections. some experiments have built a p x -or p y -deficient mice model (p x −/− or p y −/− ) in vivo, the lack of p x has no effect on the recruitment of microglia, whereas an absence of p y resulted in> % reduction in the numbers of microglia recruited to infected neurons. this indicates that microglia are recruited around the infected neurons via p y signaling. the cd + phagolysosomes of p y −/− microglia decreased significantly compared with normal mice which indicates that p y is essential for microglia to exert phagocytic activity [ ] . microglia are key contributors to the recruitment of monocytes into the brain during viral infection. in microglia-depleted mice, the phenomenon of monocytes entering the brain is almost completely gone. cd + blood-borne leukocytes were seen in the brain of p y −/− mice after infection, and mononuclear cell infiltration was not impaired, which indicates that microglia recruit monocytes through a p y independent mechanism [ ] . analysis of temporal lobe specimens in patients with herpes simplex type (hsv- ) encephalitis shows that p y -positive microglia processes extend to hsv- positive cells, and there are about - activated microglia around each infected neuron. microglia appear around the cell bodies and dendrites of neurons before the mature virus particles appear in infected neuronal cells. at this stage, the neuronal membrane is intact and there is a normal chromatin structure in the nucleus. cell trajectory analysis has shown that the speed of microglia movement in mice with viral encephalitis is reduced, which is related to their tendency to stay around the infected cells [ ] . typical neurological symptoms appear in mice infected with virus when microglia are completely absent, but not p y deficiency [ ] . consistent with this, mice with microglia depletion have higher viral loads and succumb to infection. the rapidly deteriorating neurological symptoms in microglia-depleted mice may be associated with significantly increased neuronal infection and potential microglial protective mediators such as interleukin- deficiency [ ] . microglia play a direct antiviral effect by producing ifn- after recognition of virus by pattern recognition receptors (prrs) mouse hepatitis virus (mhv) is a neurotropic coronavirus. on the fourth day of viral infection, microarray analysis of infected mouse microglia showed that the ifn- pathway was the most upregulated [ ] . ifn- protects the host because treatment with exogenous ifn-α or ifn-β limits viral replication, whereas the infection of mice with ifn- signaling deficiency can convert nonfatal coronavirus to a lethal type [ ] . in the mouse model of vesicular stomatitis virus (vsv) encephalitis, it was found that infected microglia produced ifn- , and both infected and uninfected microglia upregulated the expression of ifn regulatory factors (irf ) and activated innate immunity which limits the transsynaptic spread of vsv [ ] . viral infection can induce the expression of ifn-stimulated genes (isgs), thereby interfering with viral replication and promoting viral clearance [ ] (see fig. ). microglia express a wide range of prrs, which are important for the defense of hsv- . virus induces expression of ifn- by the recognition of nucleotides and (rna-/dna-sensing) prrs [ , ] (see table for a summary). the combination of ifn- and the heterodimeric receptor ifnar produces a cellular response that initiates a signaling cascade that promotes heterodimers stat / nuclear translocation and transcriptional activation of isgs [ ] . the rapid expression of hundreds of isgs is critical for controlling viral infections because these proteins block the entry, translation, transcription, assembly, and efflux of the virus [ ] . cytosolic dsdnas can induce a strong innate immune response. gmp-amp synthase (cgas) is a cytosolic dna receptor [ ] . the binding of cgas to cytosolic dna produces a second messenger, cyclic-gmp-amp (cgamp), which activates downstream sting and ultimately activates transcription factor irf , which promotes ifn-β production [ , ] . different types of prrs can be involved in the identification of viral infections, including membrane-associated tlrs, cytosolic rna-sensing rig-like receptors (rlrs), and dna sensors. prrs activates ifn regulatory factors (irf ) through unique adaptor molecules such as tir domain-containing adaptor inducing ifn-β (trif), mitochondrial antiviral signaling (mavs), and stimulator of type i ifn genes (sting) [ ] . mutations in the irf gene in herpes simplex encephalitis (hse) patients impaired interferon production and the tlr -trif pathway is the most severely damaged [ ] . the cgas-sting and tlr -trif pathways are the major intrinsic sensing pathways to control hsv infection in the cns. microglia can induce ifn responses by recognizing viral pathogen-associated molecular patterns (pamps) through prrs. in a typical ifn- response pattern, prrs first induce the production of ifn-β after recognition of pamps, and then ifn-β binds to ifn-α receptor (ifnαr) to initiate irf gene expression in an autocrine or paracrine manner which enables a complete ifn- response in viral spread or secondary infections [ ] . in order to simulate the natural pathway of hsv- entering the central nervous system by retrograde transport, the previous experiment used an ocular infection model. it was confirmed that microglia are the main source of hsv-induced ifn- , which is induced in a cgas-sting-dependent manner. infected cells including neurons and microglia detected hsv- by the cytoplasmic dna receptor, the adaptor protein sting. the brain of mice lacking cgas and sting detected higher viral load, and the virus spread widely to the midbrain, hypothalamus, and preoptic area. however, studies have shown that the recruitment of microglia to the infection site is not related to sting [ ] . in a model of mhv infection, depletion of microglia using colony-stimulating factor receptor (csf r) inhibitors revealed that depletion of microglia at - days of infection would result in increased mortality and a later depletion had no influences on survival. it indicated that microglia play an important role in controlling viral replication and reducing mortality in the early stage of infection [ ] . upon intranasal vesicular stomatitis virus (vsv) infection, studies have found activated microglia and monocyte aggregation in the olfactory bulb. studies have also shown that microglia accumulating around the olfactory bulb form a natural immune barrier plays an important role in limiting the spread of vsv in the cns and preventing lethal encephalitis. the formation of the intrinsic microglia barrier is regulated by ifnar signaling of neurons and astrocytes and is not regulated by microglia themselves [ ] . it has been previously reported that the use of brain-penetrant csf- r inhibitor blz in mice can remove microglia [ ] . il- , as a csf- r ligand, is essential for the maintenance of microglia. blz plays a role in specifically depleting microglia by inhibiting the interaction of csf- r with il- [ ] . after depletion of microglia, vsv-infected mice have higher viral load in the brain, cerebellum, and brainstem and have higher mortality, indicating that the formation of microglia barrier in the olfactory bulb is indispensable to limit the spread and spread of vsv [ ] . sall is specifically expressed in microglia and is a negative regulator of microglia activation. sall is downregulated in microglia when infected by pathogens, thereby promoting microglia activation [ ] . microglia confer sting-dependent antiviral activity to neurons and initiate the production of ifn- in astrocytes via the toll-like receptor (tlr ) pathway [ ] . type i ifnar signaling in astrocytes acts to stabilize the blood-brain barrier (bbb) and protect the brain from viral infections and immunopathology (see fig. ). in a mouse model of west nile virus (wnv) infection, it was found that the permeability of the bbb increased in mice lacking ifnar, especially in the hindbrain, which leads to an increase in viral entry, indicating that ifnar signaling has important regulatory effects on bbb permeability in this brain region. the study found that human and mouse cerebellar astrocytes have higher expression of prrs and isgs in both steady state and ifn-induced state compared with cerebral cortical astrocytes. cerebellar astrocytes can reach maximum isg expression more quickly after receiving ifn stimulation [ ] . in vitro studies have shown that ifnar pathways in brain microvascular endothelial cells (bmecs) and astrocytes can both promote the integrity of tight junction. studies have shown that ifn- stabilizes the integrity of bbb and promotes the formation of tight junctions by rearranging endothelial junction proteins via rho-rac signaling pathways, promoting bbb tightening and blocking the expression of il- β and tnf-α [ ] . it is well known that vascular cell adhesion molecule (vcam- ) is a neuroinflammatory regulator on vascular endothelial cells [ ] . ifn- regulates immune transport by regulating the expression of vcam- (cd ) on neurovasculature [ ] . ifn-β treatment of either human or mouse primary astrocyte cultures from the cerebellum, but not from the cerebral cortex, significantly downregulates vcam- expression [ ] . drug blocking of vcam- binding to its ligand leukocyte adhesion molecule (vla- ) has been shown to reduce pathogenic neuroinflammation and improve survival after wnv infection [ ] . in ifnar-deficient mice, cerebellar vasek et al. [ ] complement c and c cleavage products c r cell surface damaging of presynaptic ends and resulting in visual-spatial processing disorders and impaired memory function infiltrating lymphocytes and macrophages were significantly reduced after treatment with vla- blocker [ ] . loss of astrocyte ifnar signaling pathway can cause a variety of consequences, including increased expression of inflammatory cytokines and chemokines, which disrupt the blood-brain barrier during neurotropic viral infection [ ] . in different brain regions, transcription, immunophenotypic, and bioenergetic properties of microglia are heterogeneous. cerebellar and hippocampal microglia maintain a stronger immune alert status than microglia in the striatum and cortex. the sensitivity of different brain regions to aging is different, and the cerebellum is the most obvious. this suggests that different brain regions have different responses to aging-related neurodegenerative lesions [ ] . microglia also affect the adaptive immune response. depletion of microglia changes the response of cd + t cells to viral infection in the brain, the total number and percentage of cd + t cells decrease, and the frequency and number of tregs also decrease, indicating that microglia are crucial for fully activating virus-specific t cells. deletion of microglia in the early stages of whv infection exacerbates infection, which may be associated with loss of microglial antigen presentation function, as microglia participates in antigen presentation by upregulating tap and itgax. depletion of microglia can also result in the loss of major mhc ii-expressing cell type, which can also result in decreased expression of mhc ii in incoming monocytes/macrophages. because mhc ii is required for re-stimulation and activation of cd + t cells, a decrease in mhc ii reduces the response of virus-specific cd + t cells [ ] . in addition to producing ifn- , microglia can also produce other cytokines. the vitro cell culture demonstrates that cytomegalovirus (cmv)-infected astrocytes recruit microglia to the infected foci by releasing monocyte chemoattractant protein (mcp- ). microglia produces tumor necrosis factor α (tnfα) and interleukin β (il- β), interleukin (il- ) exerting antiviral effect and inhibiting the replication of cmv in astrocytes [ ] . cmv-infected microglia produce t lymphocyte chemotactic factor cxcl , recruiting activated t lymphocytes into the infected area, and t cells produce ifn-γ to inhibit virus replication and dissemination [ ] . even with limited viral replication, cytopathic effects were evident in the hsv-infected microglia. microglia responded to nonpermissive viral infection by producing considerable amounts of tnf-α, il- β, and ip- . tnf-α inhibits hsv replication in astrocytes, and ip- possesses direct antiviral activity in neurons [ ] . inflammation-induced, sting-dependent autophagy limits zika virus (zikv) infection in drosophila brains. the activation of nf-kb dependent on zikv induces expression of drosophila stimulator of interferon genes (dsting) in the brain. dsting protects against zikv infection by inducing autophagy in the brain. loss of autophagy leads to increased viral load in the brain and increases infection in drosophila [ ] . multiple microbial infections activate nfkb-dependent inflammatory signaling cascades, which induce transcription of downstream antimicrobial effectors, including antimicrobial peptides (amps), thereby reducing or eliminating infection [ ] . endogenous, bacterial-derived circulating dinucleotides and increased expression of sting mrna can activate sting [ ] . from fruit flies to mammals, activation of antimicrobial autophagy protects cells against a variety of pathogens, including bacteria, viruses, and parasites [ , ] . studies have shown that drosophila infected with zikv induces activation of nf-kb signaling in the imd pathway, thereby activating downstream dsting. dsting can limit intracellular pathogens by inducing autophagy [ ] . autophagy protects neurons against a variety of stressors, including viral infections. autophagy clears infection by capturing pathogens, including viruses, in the cell without causing cell death, which is beneficial for mature neurons [ , ] . previous studies used heat shock-inducible gal (hs-gal ) to silence autophagy core genes atg and atg in vivo [ ] and found that the virus titer of zikv in drosophila is significantly increased. relish is a transcription factor of nf-kb in the imd pathway. using a validated rnai line which specifically removes relish from neurons or glial cells in vivo, known as a neuron-specific gal driver (elav-gal ) or a glia-specific gal driver (repo-gal ), respectively [ ] , increased viral infection. in mammals, autophagy limits the replication or pathogenesis of herpes simplex virus type (hsv- ) [ ] . intracellular receptors, such as retinoic acid-inducible gene-i (rig-i) and melanoma differentiation-associated protein (mda- ), collectively referred to as rig-i-like receptors (rlrs), can also detect cytoplasmic viruses nucleic acid [ ] . using the advantages of drosophila, the study found that the prr-autophagy axis plays an important role in host defense [ ] . fifty percent of patients with neurotropic wnv infection show chronic cognitive sequelae [ ] . patients who survive after infection with wnv usually present with visual-spatial processing disorders and impaired memory function [ , ] . studies have shown that complement c and c ar mediate the loss of presynaptic ends in mouse hippocampus (see fig. ). mice demonstrate spatial learning in the recovery period of neurological invasive diseases in west nile. both neurons and microglia express c ar, which recognizes c cleavage products. c and its cleavage products attract microglia to gather around the neurons to exert phagocytosis activity and to clear the presynaptic ends. in the situation of a neurotropic virus infection, clearance of the presynaptic end may prevent trans-synaptic spread of the virus and stop abnormal signals from infected neurons [ ] . infected and activated microglia in the cns persist for more than months after receiving antiviral therapy in patients with hsv encephalitis [ ] . this may lead to chronic damage to the cns. microglia can produce tnfα [ , ] . local increase of tnfα in the hippocampal dentate gyrus activates astrocyte tnf receptor type (tnfr ), which in turn triggers an astrocyte-neuron signaling cascade that results in persistent functional modification of hippocampal excitatory synapses which can lead to cognitive disturbance [ ] . glutamatergic gliotransmission provides a stimulatory input to excitatory synapses in the hippocampal dentate gyrus. tnfα critically controls the process which may influence synaptic transmission and plasticity [ ] . this suggests that microglia may also affect synaptic function through cytokines. the unprecedented re-emergence of the zika virus (zikv) virus has shocked the world, and reports on microcephaly in brazil are also increasing [ ] (see fig. ). in brazil, the incidence of fetal microcephaly in zikv-infected pregnant women is approximately . % [ ] . surprisingly, there have been increased reports of cases of simultaneous diagnosis of guillain-barré syndrome in adults with french polynesia zikv infection [ ] . recent evidence suggests a causal relationship between prenatal zikv infection and severe brain abnormalities including microcephaly [ ] . zikv was detected in the brain of a microcephalic fetus [ ] . this means that there is a strong connection between zikv and these neurological diseases. the study found that zikv mainly infects fetal microglia and induces high levels of pro-inflammatory mediators including il- , tnf-α, il- β, and mcp- . these inflammatory factors may be harmful to the fetus [ ] . microglia's perivascular localization enables them to survey the influx of blood-borne components into the cns [ ] . thus, microglia are highly susceptible to zikv infection. the analysis of vitro culture and microcephalic fig. illustration of the pro-inflammatory role of microglia in viral encephalitis. the picture shows some harmful effects of microglia in viral encephalitis, such as mediating presynaptic membrane damage, causing fetal congenital malformations, and exacerbating the damage of cns in hiv encephalitis. microglia play an uncertain role in virus-induced aβ. the role of the ripk signaling pathway in microglia in viral encephalitis remains obscure fetal brain histology showed that zikv leads to activation of microglia [ ] . this, in turn, causes localized neuroinflammation and is accompanied by viral disseminating to the brain parenchyma, which may lead to the development of neuronal death, especially in the cerebral cortex, causing neurological changes and microcephaly. these findings reveal that microglia play an important role in the pathogenesis of congenital zikv infection [ ] . microglia are the main target of hiv- infection in the brain [ ] . there are viral particles in the infected microglia, suggesting that they may be a potential virus reservoir [ ] . the main features of hiv- encephalitis are the formation of multinucleated giant cells, microglia nodules, and macrophage infiltration into the central nervous system [ ] . the formation of multinucleated giant cells reflects the infection of hiv- microglia in vitro. active infection of microglia is ultimately associated with astrogliosis, myelin pallor, and neuronal loss [ ] . although there are ample evidences confirming that activated microglia have neuropathic effects in hiv- encephalitis, there are data suggesting that microglia may have some neuroprotective effects in the early stages of hiv disease [ ] . molecular and histopathology confirm the reduction of autophagy in the brain of patients with hiv-associated dementia. hiv- -infected microglia release neurotoxins, cytokines, viral proteins, and glutamate; activate nmda receptors on neurons; cause calcium influx, inhibit neuronal autophagy; enhance apoptotic pathways; and ultimately lead to neuronal death [ ] . enhancing autophagy of neurons in patients with hiv encephalitis may become a target for future treatment. microarray analyses were performed using brain tissue samples from hiv encephalitis (hive) patients, hiv+ patients without encephalitis (hiv/noe), and hiv− controls. it was found that a large number of microglia genes have undergone significant changes during hiv infection, including immune activation and function, kinases, phosphatases, and pro-/anti-apoptotic and neurotrophic factors, indicating that the microglia function is impaired and has a pro-inflammatory tendency [ ] . human cytomegalovirus (hcmv) can cause congenital encephalitis or cause encephalitis in aids patients [ ] . in patients with advanced aids, hcmv-induced central nervous system infection leads to two different neuropathological patterns: microglial nodular encephalitis (mgne) and ventriculoencephalitis (ve). microglial nodular encephalitis consists of diffuse microglial cells, aggregated astrocytes, and giant cells. the formation of microglial nodules is considered to be an important cause of hcmv-related dementia in aids patients [ ] . recently, a study hypothesized that, in rasmussen encephalitis (re), microglial nodules with an upregulation of tlrs provide an environment for the initiation of the later dominating t cell cytotoxicity. however, in the early stages of viral encephalitis, the interaction between microglial nodules and pathogenic t cells remains unclear [ ] . in the brain, peli is mainly expressed in microglia. in a mouse model of lethal west nile virus (wnv) infection, peli promotes the production of pro-inflammatory cytokines and chemokines in microglia and promotes the entry of t cells and macrophages into the cns. peli plays a crucial role in wnv entry and replication in human and murine neurons and microglia (see fig. ). peli promotes adhesion, replication, and synthesis of mature virions. autopsy of patients who died of wnv encephalitis found that peli was highly expressed on wnv-infected neurons and adjacent inflammatory cells. peli -deficient microglia and neurons cause inflammation and reduce host susceptibility to lethal encephalitis [ ] . microglia are the main producers of central nervous system inflammatory cytokines il- and tnf-α and chemokines ccl and cxcl after wnv infection [ ] . the peli -deficient mice have better resistance to wnv, reducing levels of cytokines and chemokines in the body, including il- , tnf-α, il- β, and il- and ccl and ccl [ ] . peli expressed in microglia can promote the degradation of tnf receptor-associated factor (traf ) by regulating tlr/myd signaling and can activate microglia during the induction of experimental autoimmune encephalomyelitis (eae) [ ] . it is well known that microglia respond to viral infection by activating p mapk [ ] . in wnv-infected peli −/− neurons, phosphorylation levels of p mapk and p decreased, indicating that peli plays a positive regulatory role in nf-κb and p mapk activation [ ] . peli facilitates wnv replication in microglia and neurons, especially in the latter, which are the major cells infected during in vivo challenge. it also positively mediates nf-κb and/or p mapk activation in these cells and boosts a robust production of inflammatory cytokines and chemokines, which attracts more infiltration of inflammatory cells from the periphery and ultimately contributes to lethal wnv encephalitis. thus, peli synergistically promotes virus dissemination and inflammation in the cns [ ] . under physiological conditions, neurons can produce trace amounts of amyloid-β peptide (aβ) to maintain synaptic plasticity and memory and play an antimicrobial role. the clearance of aβ is achieved by phagocytosis of microglia. hsv reactivation would trigger increased aβ production, activation of microglia, and release of pro-inflammatory cytokines (pathological state) that would sustain microglia activation and initiate a vicious circle of inflammatory responses. recurrence of this series of events during life would result in amplification and irreversible damage to hippocampus [ ] . studies have shown that aβ oligomers can bind glycoproteins on the surface of herpesvirus and accelerate aβ deposition showing protective viral entrapment activity in xfad mouse and d human neural cell culture infection models against neurotropic hsv . this indicates that aβ fibrils and deposits may be important in protecting the brain from common viral infections [ ] . carbohydrate-binding promotes aβ fibrillization and leads to protofibril generation on microbial surfaces. bound protofibrils first inhibit host cell adhesion by pathogens. then, propagating aβ fibrils mediate agglutination and sequestration of microbes within fibrillar β-amyloid. when the aβ oligomer-mediated protective antimicrobial pathway is over-activated, progressive aβ deposition can cause neuroinflammation, neuropathology, and extensive neuronal death, leading to alzheimer's disease (ad), a model called antimicrobial protection hypothesis [ ] . recently, a -year study involving more than , patients found that hsv infection increased the risk of dementia by . times. and long-term use of anti-herpes drugs seems to reduce the risk of dementia in patients with hsv- infection. in general, patients with shorter (< days) or longer (≧ days) durations of anti-herpetic medications were associated with a decreased risk of dementia, and the treatment duration of ≧ days was associated with a lower risk of dementia than those of a duration of < days [ ] . microglia have been involved in ad for more than years. nissl and alzheimer first described amoebalike glial cells in various neurological states in and . this amoeba-like glial cell was later confirmed to be a microglia. microglia were activated in amyloid plaque-enriched areas in alzheimer's disease brains [ ] . some findings reveal that microglia serve as important physiological functions in learning and memory by promoting learning-related synapse formation through brain-derived neurotrophic factor (bdnf) signaling [ ] . in the ad mouse model, a disease-associated microglia (dam) subtype was found. dam selectively aggregates around aβ and exerts phagocytosis. immunohistochemical staining of mice and human brain slices shows dam with intracellular/phagocytic aβ particles [ ] . the accumulation of microglia around the aβ plaque is critical to establishing a physical barrier that limits the spread of plaque and protects surrounding neurons from the toxic effects of the plaque [ ] . interestingly, in a mouse model lacking triggering receptor expressed on myeloid cells (trem ), microglia could not accumulate around aβ and the survival and proliferation of microglia around the plaque is impaired. at the same time, the migration of microglia to the lesion is impaired [ , ] . the microglia lacking trem has metabolic disorders, which are characterized by defects in glycolysis, atp levels, and anabolism [ ] . at the same time, the signaling pathway of trem is essential for phagocytosis of microglia, and the deletion of trem leads to aggravated pathology [ ] . more malnourished neurons were observed in the brain sections in patients carrying ad-associated trem variants [ ] . in contrast, it has been reported that when microglia respond to aβ plaques, it activates inflammatory bodies, leading to the escalation of inflammation and the release of apoptosis-associated speck-like protein containing a card (asc) specks from inflammasomes, which in turn may initiate the seeding of new plaques [ ] . moreover, excessive activation of microglia in the early stages of ad may cause excessive synaptic pruning, leading to cognitive impairment [ ] . in summary, hsv- infection accelerates the deposition of aβ in the brain and activates microglia. and the role of aβ during viral encephalitis remains obscure and requires additional study. necroptosis is a programmed cell death coordinated by receptor-interacting protein kinases (ripk ) and (ripk ). mixed lineage kinase domain-like protein (mlkl) is an executioner protein that participates in the formation of an apoptotic complex that promotes cell death through cell membrane disruption and cell rupture [ ] . studies have shown that the ripk signaling pathway can control a variety of viral infections, including hsv- . murine but not human rip / rip directly senses hsv- icp to initiate necroptosis [ ] . in the wnv virus infection model, it was found that ripk inhibits viral pathogenesis by cell death-independent neuroinflammation, mice lacking ripk , or ripk kinase activity, but not those lacking mlkl or mlkl, and caspase- increased susceptibility to lethal wnv infection. during wnv infection of neurons, ripk is activated in neurons which promotes the expression of chemokines ccl , cxcl , etc. and recruits infiltration of immune cells including t cells, mononuclear macrophages, and antigen presenting cells which exert antiviral effects. this process does not affect the number of microglia in cns [ ] . what role does the ripk signaling pathway in microglia play in viral encephalitis? further exploration is needed. most of the experiments on microglia and viral encephalitis remain in the experimental stage, and many mechanisms need to be further explored before being applied to the clinic. although microglia can recognize atp released by infected neurons through p y and are recruited around neurons, whether there are more other potent chemokines, further exploration is needed. although ifn- plays an antiviral role in most encephalitis, the optimal ifn- concentration and optimal application time are still undetermined. microglia can induce autophagy to exert antiviral effects, but there is insufficient evidence. microglia produce inflammatory and chemokines and induce adaptive immune responses. microglia resident in different brain regions have specific gene expression profiles, and different brain regions have different susceptibility to viruses. the relationship between the two remains undetermined, and it is expected that the innate immune response to the virus will be enhanced by changing the gene expression profile of microglia in the future. in wnv infection, microglia mediate presynaptic terminal loss in the hippocampus though recognizing c by c r, c , or c r-specific antibodies may block this process. microglia inhibit autophagy in patients with hive, aggravating cns injury. how to inhibit or delete the function of microglia or how to enhance the autophagy of neurons will become the direction of future research. there is a strong relationship between fetal cerebellar malformation and activation of microglia in zikv infection, and it is necessary to develop microglia inhibitors that are safe for pregnant women and fetuses. pelia of microglia promotes viral replication in neurons, and moderate blockade of pelia may become a target for future treatment. in viral encephalitis, aβ produced by neurons increases the risk of dementia in patients. studies show that prolonging antiviral time will reduce the risk of dementia. the interaction between aβ and activated microglia in patients with viral encephalitis is unknown. it is unknown whether moderately enhancing the phagocytosis of microglia can reduce the risk of dementia. it needs further research. traditionally, activation of ripk will result in the formation of an apoptotic complex that mediates cell death. recent studies have shown that in viral encephalitis, activation of neuronal ripk exerts an antiviral effect in a death-independent pathway, and the ripk pathway of microglia requires further investigation. microglia account for % of the total number of cells in the adult central nervous system [ ] . in a healthy cns, microglia are not "resting" but in a highly active "surveillance and rapid response" state [ ] . microglia processes are dynamic, continuously scanning the environment and sampling it [ ] . they are able to detect changes in ph, purines, cytokines, chemokines, amino acids, and inorganic compounds [ ] . microglia are derived from the primitive macrophages of the yolk sac, which migrate to the developing central nervous system. they have the ability to self-renew, thus maintaining their number throughout life without any input from the bone marrow-derived precursor cells [ , ] . microglia can respond to neurons, which in turn affects neuronal activity. microglia are involved in the programmed death of neurons and the apoptosis and clearance of new neurons during development. the activity of microglia promotes the pruning, elimination, and maturation of synapses [ ] . microglia serve important physiological functions in learning and memory by promoting learning-related synapse formation through bdnf signaling [ ] . the phagocytic activity of microglia is essential for the clearance of senescent cells and debris [ ] , slowing the toxic effects of amyloid-β [ ] . microglia are not only important cells that maintain the homeostasis of cns, but also respond to injury, infection, and neurodegeneration through proliferation and altered transcription and morphology [ , ] . microglia are often touted as the first responder to cns infection and respond quickly to injury [ ] . however, depending on the stimulus received, this activation profile is also different and may result in harmful or beneficial effects [ , ] . hsv- can be treated, but can wnv or enteroviruses? the high morbidity and high morbidity rate of viral encephalitis have caused widespread concern. although traditional antiviral therapy is effective, there are still many shortcomings. research on microglia and viral encephalitis provides new targets for treatment. microglia play a unique role in different encephalitis. the current research on microglia and viral encephalitis 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to restrict virus propagation in mice oberst a. ripk restricts viral pathogenesis via cell death independent neuroinflammation microglia: scapegoat, saboteur, or something else? sublime microglia: expanding roles for the guardians of the cns resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo heterogeneity of cns myeloid cells and their roles in neurodegeneration regulation of microglia development and homeostasis development and homeostasis of "resident" myeloid cells: the case of the microglia clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells- gsecretase component presenilin is important for microglia b-amyloid clearance myeloid cells in the central nervous system differential roles of microglia and monocytes in the inflamed central nervous system not applicable. not applicable. authors confirm that all relevant data are included in the article. authors' contributions zc carried out the literature review, participated in the sequence alignment, and drafted the manuscript. dz helped to draft the manuscript. gl conceived, designed, and coordinated the study and contributed to and finalized the draft. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable as no patients/participants involved in this review. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - f xe zc authors: rowland, r. r. r.; robinson, b.; stefanick, j.; kim, t. s.; guanghua, l.; lawson, s. r.; benfield, d. a. title: inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with -aminopurine date: journal: arch virol doi: . /s sha: doc_id: cord_uid: f xe zc porcine reproductive and respiratory syndrome virus (prrsv) belongs to a group of rna viruses that establish persistent infections. a proposed strategy for evading immunity during persistent prrsv infection is by preventing the induction of ifn activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. ifn-γ mrna expression was observed in the lymph nodes and lungs of pigs infected with wild-type prrsv strain sdsu- . pretreatment of marc- cells with ifn-γ inhibited wild-type (sdsu- p ) and culture-adapted (sdsu- p ) prrs viruses in a dose-dependent manner and at relatively low concentrations. the effect of ifn-γ on virus replication included reductions in the number of infected cells, virus yield, and rna content in single cells. virus replication was partially restored by the addition of -aminopurine ( -ap), an inhibitor of dsrna inducible protein kinase (pkr). the addition of -ap also restored the viral rna content per cell to near normal levels, suggesting that inhibition of viral rna synthesis was through pkr. the principal difference between p and p isolates was the recovery of p replication with lower concentrations of -ap. immunostaining with anti-pkr antibody showed a redistribution of pkr from the cytoplasm into nucleoli of infected cells. porcine reproductive and respiratory syndrome (prrs) is caused by an enveloped positive-stranded rna virus placed in the family arteriviridae, order nidovirales [ , , , ] . other members in this family include lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv). the arteriviruses structurally resemble togaviruses, but similar to coronaviruses, replicate via a nested -coterminal set of subgenomic mrnas possessing a common leader and a poly-a tail [ ] . the outcome following prrsv infection depends on the age and reproductive status of the pig [ ] . infection of adult pigs generally produces a nonfatal disease characterized by flu-like symptoms, including mild interstitial pneumonia, elevated temperature, and inappetance. in sharp contrast, the infection of pregnant gilts and sows results in abortion and the birth of dead and weak-born piglets. surviving piglets exhibit the severest form of respiratory distress with mortality often approaching % within weeks after farrowing. surviving pigs continue to suffer the negative effect of prrsv by exhibiting increased susceptibility to secondary infection. during acute infection, prrsv replication is found in all organs and tissues, which is consistent with the macrophage as the principal cell population supporting virus replication [ , ] . non-macrophage cells, including type ii pneumocytes [ ] , bronchiolar epithelial cells and arteriolar endothelial cells [ ] may provide additional sources of virus in infected pigs. in culture, prrsv replicates in porcine alveolar macrophages, blood monocytes, monocyte-derived macrophages and monkey kidney lines derived from ma- cells [ , , , ] . pigs surviving acute prrs support a long-term asymptomatic infection, which has contributed to the worldwide spread of the disease [ , , , , ] . even though prrsv-specific humoral and cell-mediated immune responses appear early [ , ] , persistently infected pigs continue to shed virus for several months [ , ] . the mechanistic basis for prrsv persistence is not known. similar to ldv, prrsv may sustain replication in a subpopulation of renewable macrophages, while avoiding host-defenses [ ] or restrict the localization of virus replication within "immunoprivileged" anatomical sites, such as testes [ , , ] . the amino acid variability among prrsv field isolates within the ectodomain of the orf envelope protein suggests another strategy; antigenic drift [ ] . persistent viruses frequently incorporate evasion strategies that block the production or inhibit the activation of proteins up-regulated by interferon (ifn; [ , , , , ] ). studies suggest that prrsv may avoid ifn by preventing its induction in macrophages. both type-i and type-ii ifns efficiently inhibit prrsv replication in macrophages; however, small amounts of ifn are found in prrsv-infected pigs and infected macrophages in culture [ , , ] . since prrsv-specific cell-mediated immunity is present during infection, it is assumed that ifn-␥, a product of activated t cells and nk cells, is present in infected pigs [ , ] . however, nothing has been reported regarding the induction of ifn-␥ in prrsv-infected pigs or the mechanism of ifn-␥-inhibition of virus replication. the purpose of this study was to evaluate the production of ifn-␥ in pigs following infection with prrsv and to characterize the mechanism of ifn-␥ action following infection of marc- cells with wild-type and culture adapted prrsv isolates. the results show that ifn-␥ efficiently inhibits prrsv replication in marc- cells, and at least part of the antiviral activity may be through pkr. the north american macrophage-tropic strain, sdsu- -p , is a low-passage isolate possessing attributes similar to other north american field strains. the marc- celladapted strain, sdsu- -p , was obtained by passaging p an additional times on marc- cells. adaptation was performed by infecting marc- cells on -well plates with serial : dilutions of virus. medium from the last well showing cpe was retained, diluted : , and ml added to a new t- flask of marc- cells. after incubation at • c for hr, the monolayer was washed with pbs, and fresh medium added. after days the flask was freeze/thawed at − • c, and medium was again end-point diluted on a new well plate. this process repeated until the total number of passages reached . marc- cells [ ] were maintained in mem supplemented with % heat-inactivated fetal bovine serum (fbs) and antibiotics at • c in % co . the indiana strain of vesicular stomatitis virus (vsv) was obtained from christopher chase at south dakota state university. unless otherwise indicated, confluent marc- cells were infected at an moi of approximately . tcid /cell. increasing the moi of sdsu- beyond . does not produce a further increase in the number of cells infected during the first round of infection or the virus yield (personal observation). recombinant human ifn-␥ (boehringer mannheim) and ifn-␣ (prepo) were stored at − • c. a mm solution of the nitrate salt of -aminopurine ( -ap; sigma) was prepared in mem and ph adjusted by adding m tris until the cell culture medium changed to pink. ifn-␥ and -ap were added at approximately h and h, respectively, prior to infection with virus. after h the medium was removed and cells washed twice with pbs. incubation was continued in fresh medium, containing freshly prepared ifn-␥ and -ap. cytopathic effect (cpe) was observed by phase contrast microscopy of live cells and after staining acetone-fixed monolayers with % crystal violet. cytokine mrna expression in lung and lymph nodes was evaluated in pigs that were exposed to prrsv in utero. this model is based on observations that infected neonates exhibit the severest form of disease [ , ] . seronegative sows were infected at days gestation with wild-type p or attenuated p . sows were allowed to farrow and live-born piglets sacrificed at days after birth. samples of lung and lymph node tissues were immediately frozen in liquid nitrogen and stored at − • c. total rna was extracted from tissues according to christopher-hennings et al. [ ] and stored at − • c. rt-pcr of porcine cytokine and ␤ -microglobulin mrnas was performed according to reddy et al. [ ] . cdna was prepared from ug of total rna by reverse transcription using molony murine leukemia virus reverse transcriptase and random hexamers as primers. pcr amplification of ifn-␥, il- and ␤ -microglobulin cdnas was performed using the primers described in table . pcr amplification consisted of cycles ( sec at • c, sec at • c, and sec at • c) and dna products electrophoresed on a . % agarose gel and visualized using ethidium bromide. the identity of each amplified product was confirmed by cloning and sequencing the gel-purified fragment. [ ] . serial -fold dilutions of virus were placed on -well tissue culture plates containing marc- cells. after days, plates were fixed in % acetone then stained with sdow- . results were reported as tcid /ml. the intracellular localization of pkr was determined by staining with anti-pkr mab (anti-p from transduction laboratories). acetone-fixed cells were incubated for h at room temp with a : dilution of anti-pkr antibody, followed by a h incubation with biotinylated anti-mouse igg (sigma), diluted : . a final one hr incubation included texas redstrepavidin (molecular probes), diluted : and fitc-labeled sdow- , diluted : . antibodies and stepavidin were diluted in pbs-fbs and slides washed twice for min in pbs between the addition of each reagent. fitc-labeled sdow- and texas red-labeled anti-pkr antibodies were visualized under a fluorescence microscope using nm and nm (rhodamine) excitation filters, respectively. for northern blots total rna was extracted from cells at h after infection using the acid phenol guanidinium technique of chomcynski and sachi [ ] . rna was electrophoresed on % agarose following denaturation with glyoxal and dimethyl sulfoxide [ ] , then transferred to a nylon membrane. the amount of rna added to each well was adjusted to reflect the viral rna from the same number of infected cells (determined by the percentage of sdow- -postive cells in a small sample of cells removed from culture prior to rna extraction). the [ p] dctp-labeled prrsv-specific probe, designed to detect genomic and subgenomic rnas, was prepared by random-primed labeling a bp cdna template derived from rt-pcr amplification of the orf region [ ] . nylon membranes were hybridized with the probe overnight at • c, using a hybridization buffer prepared according to church and gilbert [ ] . nylon membranes were washed in × ssc at • c followed by a high stringency wash in . × ssc at • c for min, then exposed to x-ray film. the relative viral rna content in single cells was determined using a modification of the in situ hybridization technique described by peng et al. [ ] . approximately cells in ul of tissue culture medium were spotted onto denhardt's-treated slides. fixation, pre-treatment, hybridization, and post-hybridization procedures were performed according to lawson et al., [ ] using a modification of the original method described by anderson et al. [ ] . the [ s] dctp-labeled random-primed probe was prepared from the same bp orf template used in northern blot hybridization. after the last post-hybridization wash, slides were dipped in nbt- autoradiography emulsion (kodak), exposed for h in the dark, developed, then h and e stained with mayer's hematoxylin and % eosin y in ethanol. prrsv-infected cells were identified as having a silver grain content above that of mock-infected cells. histogram distributions were constructed by counting the number of silver grains over the cytoplasmic and nuclear regions of infected cells. statistical analysis was performed using a two-tailed student's t test. previous studies [ , ] identified the reduced ability of pigs and pig macrophages to produce ifn-␣. the purpose of this experiment was to characterize ifn-␥ mrna expression in acutely infected pigs. sows, at days gestation, were infected with wild-type p or attenuated p isolates. all p -infected piglets (n = ) were positive for prrsv at the time of necropsy ( days after birth) and exhibited typical congenital prrsv pathology, including microscopic lesions in lungs and lymph nodes. the piglets born to the p -infected sow (n = ) showed no signs of prrs and were negative for virus at the time of necropsy, confirming that this virus was rapidly cleared from the mother and did not cross the placenta. three pigs from each of the p and p groups were randomly chosen for analysis of cytokine mrna expression in lungs and mandibular lymph nodes. ifn-␥ mrna expression was detected in the lymph nodes from two of the pigs infected with the wild-type p virus (fig. ) . a third p pig showed no ifn-␥ mrna in lymph node; however, ifn-␥ mrna was detected in the lungs. there was no evidence of ifn-␥ expression in the lymph nodes or lungs of the p detected by rt-pcr amplification of total rna from three infected (p virus) and three control pigs (p virus). infection of pregant sows with sdsu- wildtype p and attenuated p isolates is described in materials and methods. n lymph node; l lung pigs. ␤ microgloblin mrna was amplified to nearly equal levels in all tissues. il- mrna was also amplified from all tissues, a cytokine mrna we frequently find it in lymphocytes and lymphoid tissues of un-infected pigs. the effect of ifn-␥ on the growth of prrsv in marc- was studied by incubating -well plates of confluent marc- cells with human ifn-␥ overnight followed by infection with prrsv p or p isolates. the results of a representative experiment, presented in fig. a , showed loss of crystal violet staining in p and p cultures not treated with ifn-␥. the effect of ifn-␥ on cpe was apparent at concentrations as low as u/ml ifn-␥. intact cell monolayers were observed in wells incubated with or , u/ml of ifn-␥. phase-contrast microscopy, performed prior to crystal violet staining, confirmed the absence of cpe. for the purpose of comparison, ifn-␥-treated marc- cells were also infected with vsv, a virus sensitive to interferon [ , ] . the experimental conditions were the same as those used for prrsv infection. intact cell monolayers were only observed in vsv-infected cultures treated with , u/ml ifn-␥ ( fig. a) . the effect of human ifn-␣ on prrsv replication was also investigated. the experimental conditions were the same as described for the ifn-␥ experiments. representative results, presented in fig. b , showed ifn-␣ inhibition of p and p isolates. similar to ifn-␥, ifn-␣ activity was apparent at concentrations as low as u/ml. unlike ifn-␥, the p isolate was more sensitive to ifn-␣ than p (compare wells treated with and ug/ml ifn-␣). this difference was confirmed in subsequent experiments. further assessments of ifn-␥ inhibition of virus replication were made by counting the number of infected cells and by measuring virus yields in cultures incubated with ifn-␥ overnight. ifn-␥ treatment produced dramatic decreases in the percent-infected cells and virus yield for both p and p viruses (fig. ) . similar to the cpe data the effect was evident at concentrations as low u/ml. ifn-␥, reduced virus yields at all time points and consistently reduced peak yields of p and p by greater than to logtcid /ml. ifn-␥ also reduced the number of infected cells. the growth curve experiments also revealed differences between p ad p isolates. improved adaptation of p to marc- cells was evident by increased virus yields and percent infected cells. another difference was the timing of peak virus yield for p , which was observed at h after infection versus viruses, respectively. ifn-␥ treatment, infection, and measurement of virus yields were performed in the same manner as described in fig. h for p . the experiment presented in fig. also revealed what appeared to be an increased sensitivity of p to ifn-␥. however, due to the different growth characteristics of p and p isolates, a differential effect of ifn-␥ was difficult to assess. there are several approaches that can be used to study the activation of pkr. a role for activated pkr in the ifn-inhibition of prrsv replication was studied by following the recovery of virus replication after the addition of -ap, an inhibitor of pkr phosphorylation [ ] . even though we did not evaluate the phosphorylation state of pkr, we assumed that reversal of ifn inhibition of virus replication following addition of -ap could only occur by preventing pkr phosphorylation. in these experiments -ap was added h prior to infection and after overnight incubation with u/ml ifn-␥. inhibition of ifn-␥ activity by -ap was dosedependent and evident at concentrations as low as mm for p (fig. ) . the optimal concentration of -ap required for maximum virus recovery of p and p infection were and mm, respectively. following several experiments, it was noted that -ap never recovered virus replication to the same levels obtained in infected cultures not treated with ifn-␥ ( fig. ; compare treatments and for p and treatments and for p ). and as shown in fig. further increasing -ap to mm in p -infected cultures actually inhibited virus yield. a similar effect was observed for the p virus incubated mm -ap (data not shown). pkr is typically found in both cytoplasmic and nuclear compartments [ ] . in uninfected marc- cells pkr was distributed throughout the cytoplasm with increased anti-pkr fluorescence in the perinuclear region (fig. d ). we did not observe a significant increase or redistribution in pkr immunofluorescence in cells following overnight incubation with u/ml ifn-␥. however, the intracellular distribution of pkr was dramatically different in prrsv-infected cells. in a representative experiment (fig. e ) pkr immunofluorescence was still present in the cytoplasm, but with increased fluorescence around the perinuclear region and within the nucleoli of p -infected cells (fig. e ). similar results were observed in cells infected with p . incubation with mm -ap prior to infection with p did not alter the pattern of pkr staining, suggesting that pkr activation was not required for nucleolar localization. interestingly, the intracellular distributions of pkr and prrsv nucleocapsid (n) proteins were almost identical (compare fig. b and e) . to determine a possible association between pkr and n protein in the absence of infection, we performed a similar experiment that expressed the n protein fused to enhanced green fluorescent protein (egfp). a representative result, presented in fig. c and f, showed n-egfp fluorescence in the nucleolus and cytoplasm; whereas, pkr remained in the cytoplasm. since the n-egfp fusion construct may not be representative the native n protein, we repeated the experiment using a construct that expressed only the n protein, which was detected using fitc-sdow- . under these conditions the n and pkr proteins did not co-localize to the nucleolus (data not shown). the inability of -ap to completely recover virus replication following ifn-␥treatment suggested that other anti-viral pathways, up-regulated by ifn-␥, were activated during prrsv infection. for example, the , oligoadenylate synthetase ( - a synthetase) pathway, activated by dsrna, inhibits translation by degrading mrna [ ] . northern blots were used to evaluate the integrity of p subgenomic mrnas. the results of a single experiment, presented in fig. a , showed decreased expression of all prrsv rnas following treatment with u/ml ifn-␥. we did not observe degraded rna in the ifn-treated cultures. viral rna expression was also reduced in the presence of mm -ap alone; however, the addition of -ap to ifn-␥ treated cells increased the level of viral rna expression above the ifn-␥-treated culture. to further confirm that ifn-␥ treatment decreased viral rna expression, relative changes in viral rna content in single cells were measured using a modified in situ hybridization technique [ ] . histograms showing the number of p -infected cells versus number of silver grains/cell in infected cells at hr r are presented in fig. b . the mean silver grain content in infected cultures was . grains/cell (fig. b, panel ) . pre-treatment with u/ml of ifn-␥ shifted the distribution towards cells containing significantly less viral rna (mean = . silver grains/cell, p = . , fig. b panel vs panel ) . the addition of -ap restored the mean rna content per cell to near normal levels (fig. b, panel vs panel ). this study confirms previous reports describing ifn inhibition of prrsv [ , , ] . furthermore, we extend these observations by demonstrating that ifn inhibits the replication of both wild-type and tissue culture-adapted isolates derived from a north american prrsv isolate. inhibition of prrsv was observed following pre-incubation with either type-i or type-ii ifns. furthermore, the sensitivity of prrsv to ifn-␥ was greater than vsv ( fig. a) , a virus frequently used for the detection of ifn activity [ ] . consistent with other reports describing the presence of cell-mediated immunity during prrsv infection [ , ] , we observed ifn-␥ mrna expression in lymph nodes and lungs of prrsv-infected pigs (fig. ) . whether or not this amount of expression represents a biologically relevant quantity of ifn-␥ remains to be determined. even though we demonstrated the effectiveness of ifn in inhibiting prrsv replication in marc- cells, little is known regarding the properties of the macrophages supporting prrsv replication in vivo. in infected pigs, prrsv replication is initially found in all organs and tissues [ ] . during the long asymptomatic stage of infection, virus replication is restricted to a subpopulation of cells located in tonsil and lymph nodes (personal observation), suggesting that these cells may be more resistant to ifn-␥, even when exposed to local elevations of cytokines from activated t cells. this observation is similar to studies of ldv infection of mice. ldv normally replicates in a subpopulation of macrophages, but in some strains of mice infects motor neurons causing paralysis. injection of mice with ifn-␥ prior to infection inhibits virus replication in motor neurons without significantly affecting the overall level of viremia [ ] . we propose a model in which the induction of ifn-␥ production during prrsv infection is sufficient to suppress virus replication in permissive cells in non-lymphoid organs, but does not affect replication in a small subpopulation of permissive cells in lymph nodes and tonsil. the identity of this prrsv-permissive population remains to be determined. anti-viral proteins, including pkr, - a synthetase and mx are up-regulated in response to ifn [ , , ] . pkr, after binding viral rna, is autophosphorylated on serine and threonine residues and inhibits translation by phosphorylating the alpha subunit of eif . phosphorylation of pkr is inhibited by -ap, an atp analog. the addition of -ap to cells pre-incubated with u/ml ifn-␥ partially recovered prrsv replication in marc- cells (fig. ) . these results demonstrate that at least, in part, the interferon-induced anti-prrsv activity may occur through the pkr pathway. the ability of -ap to partially restore viral rna synthesis in ifn-treated cells supports the work of deboon et al. [ ] who concluded that ongoing protein synthesis is required for the production of arterivirus rnas. one explanation for the failure of -ap to completely restore virus replication is the activation of other antiviral pathways not regulated by pkr. for example the activation of the - a synthetase pathway can be identified by the presence of degraded forms of both viral and ribosomal rnas [ ] . degraded rna was not detected in northern blots (see fig. a ) in total rna from prrsv infected cultures (personal observation). recently, zhang et al. [ ] reported increased expression of mx in cultured macrophages infected with prrsv. a role for mx in the inhibition of prrsv replication has not been demonstrated. another explanation for the failure of -ap to restore prrsv replication is the inhibition of -ap sensitive phosphorylation event required for virus replication. for example, mm -ap prevents the phosphorylation of the pe /e envelope glycoprotein, which is required for the production of infectious progeny [ ] . pkr is normally distributed between the cytoplasm and nucleus, which is consistent with its roles in regulation of translation and transcription. in marc- cells, we found pkr primarily concentrated in the cytoplasm and perinuclear regions (fig. d ). an unexpected observation was the localization of pkr to the nucleoli of prrsv-infected cells (fig. e ). nucleolar localization was not affected by -ap, suggesting that pkr activation is not necessary for nucleolar localization. this observation confirms the result of besse et al. [ ] who identified a non-phosphorylated form of pkr in the nuclei of hela cells. furthermore, pkr nucleolar localization during prrsv replication is not the result of a physical association with the prrsv nucleocapsid protein ( fig. c and f). from these results we can conclude that nucleolar localization of pkr in marc- cells is dependent on prrsv infection, but is independent of activation or an association with the nucleocapsid protein. prrsv infection of pigs continues to represent a worldwide disease problem. this study, combined with the results of others, clearly demonstrates an important role for ifn-␥ in the control of prrsv infection and suggests that the induction of ifn activity should be considered in the design of new vaccines. however, the mechanism for evasion of host defenses during persistent infection remains to be determined. epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview interferon-alpha response to swine arterivirus (poav), the 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giuditta; rihn, suzannah j.; voorhees, rebecca; botten, jason w.; majumdar, devdoot; guttman, mitchell title: sars-cov- disrupts splicing, translation, and protein trafficking to suppress host defenses date: - - journal: cell doi: . /j.cell. . . sha: doc_id: cord_uid: ya m o sars-cov- is a recently identified coronavirus that causes the respiratory disease known as covid- . despite the urgent need, we still do not fully understand the molecular basis of sars-cov- pathogenesis. here, we comprehensively define the interactions between sars-cov- proteins and human rnas. nsp binds to the mrna recognition domains of the u and u splicing rnas and acts to suppress global mrna splicing upon sars-cov- infection. nsp binds to s ribosomal rna in the mrna entry channel of the ribosome and leads to global inhibition of mrna translation upon infection. finally, nsp and nsp bind to the sl rna in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. our results uncover a multipronged strategy utilized by sars-cov- to antagonize essential cellular processes to suppress host defenses. coronaviruses are a family of viruses with notably large single-stranded rna genomes and broad species tropism among mammals (graham and baric, ) . recently, a coronavirus, sars-cov- , was discovered to cause the severe respiratory disease known as covid- . it is highly transmissible within human populations and its spread has resulted in a global pandemic with more than a million deaths to date (andersen et al., ; zou et al., ) . we do not fully understand the molecular basis of infection and pathogenesis of this virus in human cells. accordingly, there is an urgent need to understand these mechanisms to guide the development of therapeutics. sars-cov- encodes proteins with diverse functional roles in viral replication and packaging( bar-on et al., ; wang et al., ) . these include structural proteins: the nucleocapsid (n, which binds the viral rna), and the envelope (e), membrane (m), and spike (s) proteins, which are integral membrane proteins. in addition, there are non-structural proteins (nsp - ) which encode the rna-directed rna polymerase, helicase, and other components required for viral replication (da silva et al., ) . finally, there are accessory proteins (orf a- ) whose function in viral replication or packaging remain largely uncharacterized (chen and zhong, ; finkel et al., ) . as obligate intracellular parasites, viruses require host cell components to translate and transport their proteins and to assemble and secrete viral particles (maier et al., ) . upon viral infection, the mammalian innate immune system acts to rapidly detect and block viral infection at all stages of the viral life cycle (chow et al., ; jensen and thomsen, ; wilkins and gale, ) . the primary form of intracellular viral surveillance engages the interferon pathway, which amplifies signals resulting from detection of intracellular viral components to induce a systemic type i interferon response upon infection (stetson and medzhitov, ) . specifically, cells contain various rna sensors (such as rig-i and mda ) that detect the presence of viral rnas, promote nuclear translocation of the transcription factor irf leading to transcription, translation, and secretion of interferon (e.g. ifn-α and ifn-β) . binding of interferon to cognate cell-surface receptors leads to transcription and translation of hundreds of antiviral genes. j o u r n a l p r e -p r o o f in order to successfully replicate, viruses employ a range of strategies to counter host antiviral responses (beachboard and horner, ) . in addition to their essential roles in the viral life cycle, many viral proteins also antagonize core cellular functions in human cells to evade host immune responses. for example, human cytomegalovirus (hcmv) encodes proteins that inhibit class major histocompatibility (mhc) display on the cell surface by retaining mhc proteins in the endoplasmic reticulum (miller et al., ) , polioviruses encode proteins that degrade translation initiation factors (eif g) to prevent translation of '-capped host mrnas (kempf and barton, ; lloyd, ) , and influenza a encodes a protein that modulates mrna splicing to degrade the mrna that encodes rig-i (kochs et al., ; zhang et al., ) . suppression of the interferon response has recently emerged as a major clinical determinant of covid- severity , with almost complete loss of secreted ifn characterizing the most severe cases (hadjadj et al., ) . the extent to which sars-cov- suppresses the interferon response is a key characteristic that distinguishes covid- from sars and mers (lokugamage et al., ) . several strategies have been proposed for how the related sars-and mers-causing viruses may hijack host cell machinery and evade immune detection, including repression of host mrna transcription in the nucleus (canton et al., ) , degradation of host mrna in the nucleus and cytoplasm (kamitani et al., ; , and inhibition of host translation (nakagawa et al., ) . nonetheless, the extent to which sars-cov- uses these or other strategies, and how they may be executed at a molecular level remains unclear. understanding the interactions between viral proteins and components of human cells is essential for elucidating their pathogenic mechanisms and for development of effective therapeutics. because sars-cov- is an rna virus and many of its encoded proteins are known to bind rna (sola et al., ) , we reasoned that these viral proteins may interact with specific human mrnas (critical intermediates in protein production) or non-coding rnas (critical structural components of diverse cellular machines) to promote viral propagation. here, we comprehensively define the interactions between each sars-cov- protein and human rnas. we show that viral proteins form highly specific interactions with mrnas or ncrnas, including those involved in progressive steps of host cell protein production. we show j o u r n a l p r e -p r o o f that nsp binds to the mrna recognition domains of the u and u rna components of the spliceosome and acts to suppress global mrna splicing in sars-cov- -infected human cells. we find that nsp binds to a precise region on the s ribosomal rna that resides in the mrna entry channel of the initiating s ribosome. this interaction leads to global inhibition of mrna translation upon sars-cov- infection of human cells. finally, we find that nsp and nsp bind to discrete regions on the sl rna component of the signal recognition particle (srp) and interfere with protein trafficking to the cell membrane upon infection. we show that disruption of each of these essential cellular functions acts to suppress the type i interferon response to viral infection. together, our results uncover a multipronged strategy utilized by sars-cov- to antagonize essential cellular processes and robustly suppress host immune defenses. we cloned all of the known sars-cov- viral proteins into mammalian expression vectors containing an n-terminal halotag (los et al., ) (figure s a , methods), expressed each in hek t cells, and exposed them to uv light to covalently crosslink proteins to their bound rnas. we then lysed the cells and purified each viral protein using stringent, denaturing conditions to disrupt any non-covalent associations and capture those with a uv-mediated interaction ( figure a , methods). as positive and negative controls, we purified a known human rna binding protein (ptbp ) and a metabolic protein (gapdh) (figure s a-e). we successfully purified of the viral proteins (figure s a ; full-length spike was not soluble when expressed). we found that viral proteins (nsp , nsp , nsp , nsp , nsp , nsp , nsp , orf b, n, and e protein) bind to specific host rnas (p-value < . , figure b , table s ), including structural ncrnas and mrnas (table s ). these include mrnas involved in protein translation (e.g. cops , eif , and rps ,), protein transport (atp v g , slc a , and tomm ), protein folding (hspa , hspa , and hspa b), transcriptional regulation (yy , id , and ier ), and immune response (jun, aen, and j o u r n a l p r e -p r o o f rack ) (fdr < . , figure b , s f). importantly, the observed interactions are highly specific for each viral protein, and each protein binds to a precise region within each rna ( figures c, s f ). using these data, we identified several viral proteins that interact with structural ncrna components of the spliceosome (u and u snrna), the ribosome ( s and s rrna), and the signal recognition particle ( sl) (figure b) . because these molecular machines are essential for three essential steps of protein production -mrna splicing, translation, and protein trafficking -we focused on their interactions with viral proteins to understand their functions and mechanisms in sars-cov- pathogenesis. after transcription in the nucleus, nascent pre-mrnas are spliced to generate mature mrnas which are translated into protein. splicing is mediated by a complex of ncrnas and proteins known as the spliceosome. specifically, the u small nuclear rna (snrna) hybridizes to the ' splice site at the exon-intron junction and the u snrna hybridizes to the branchpoint site within the intron to initiate splicing of virtually all human mrnas (séraphin et al., ) . we identified a highly specific interaction between the nsp viral protein and the u and u snrnas ( figure b) . because u and u are small rnas ( and nucleotides, respectively), we noticed strong enrichment of nsp -associated reads across the entire length of each. to more precisely define the binding sites, we exploited the well-described tendency of reverse transcriptase to preferentially terminate when it encounters a uv-crosslinked protein on rna (konig et al., ) (figures a, s d) . we determined that nsp binds to the ' splice site recognition sequence of u (figures a-b, s a based on the locations of the nsp binding sites relative to the mrna recognition domains of the u /u spliceosomal components, we hypothesized that nsp might disrupt splicing of newly transcribed genes ( figure f ). to test this, we co-expressed nsp in human cells along with a splicing reporter derived from irf (an exon-intron-exon minigene) fused to gfp (majumdar et al., ) . in this system, if the reporter is spliced, then gfp is made; if not, translation is terminated (via a stop codon present within the first intron) and gfp is not produced ( figure a) . we observed a > -fold reduction in gfp levels in the presence of nsp compared to a control human protein (figures b, s a ). to explore whether nsp has a global impact on splicing of endogenous mrnas, we measured the splicing ratio of each gene using nascent rna sequencing. specifically, we metabolically labeled nascent rna by feeding cells for minutes with -ethynyl uridine ( eu), purified and sequenced eu-labeled rna, and quantified the proportion of unspliced fragments spanning the ' splice site of each gene (figure c, s b) . we observed a global increase in the fraction of unspliced genes in the presence of nsp compared to controls ( figure d, s c,d) . given that nsp is sufficient to suppress global mrna splicing, we expect that its expression in sars-cov- -infected cells would result in a global mrna splicing deficit. to test this, we infected human lung epithelial cells (calu ) with sars-cov- and measured splicing levels of newly transcribed mrnas compared to a mock infected control. as expected, we observed a global increase in the fraction of unspliced transcripts upon sars-cov- infection, with ~ % of measured genes showing increased intron retention (figure e, s e ). together these results indicate that nsp binds to the splice site and branch point sites of u /u to suppress global mrna splicing in sars-cov- infected cells ( figure f) . although nsp is known to act as an enzyme that deposits '-o-methyl modifications on viral rnas (decroly et al., ) , our results demonstrate that it also acts as a host virulence factor. global disruption of mrna splicing may act to decrease host protein and mrna levels by triggering nonsense-mediated decay of improperly spliced mrnas (kurosaki et al., ) . consistent with this, we observed a strong global decrease in steady-state mrna levels (relative to ncrna levels) upon sars-cov- infection ( figure s f ). j o u r n a l p r e -p r o o f inhibition of mrna splicing suppresses host interferon response to viral infection because many of the key genes stimulated by interferon (ifn) are spliced, we reasoned that mrna splicing would be critical for a robust ifn response. to test this, we utilized a reporter line engineered to express alkaline phosphatase upon ifn signaling (mimicking an antiviral response gene). this ifn stimulated gene (isg) reporter line can be stimulated using ifn-β and assayed for reporter induction. we observed strong repression of this ifn responsive gene upon expression of nsp ( figure g ) and upon addition of a small molecule that interferes with spliceosomal assembly (figure s g ). these results demonstrate that one outcome of nsp mediated inhibition of mrna splicing is to reduce the host cells' innate immune response to viral recognition. consistent with such a role, we observed an increase in intron retention within multiple ifn-responsive genes (such as isg and rig-i) upon sars-cov- infection ( figure h , s h-i). once exported to the cytoplasm, spliced mrna is translated into protein on the ribosome. initiation of translation begins with recognition of the ' cap by the small s subunit (which scans the mrna to find the first start codon). we observed that nsp binds exclusively to the s ribosomal rna (figure b and s a ) -the structural rna component of the s ribosomal subunit. several roles for nsp have been reported in sars-cov and mers-cov including roles in viral replication, translational inhibition, transcriptional inhibition, mrna degradation, and cell cycle arrest (brockway and denison, ; kamitani et al., ; lokugamage et al., ; narayanan et al., ) . one of the reported roles for nsp in sars-cov is that it can associate with the s ribosome to inhibit host mrna translation (kamitani et al., ; tanaka et al., ), yet it remains unknown whether this association is due to interaction with the ribosomal rna, protein components of the ribosome, or other auxiliary ribosomal factors. accordingly, the mechanisms by which nsp acts to suppress protein production remain elusive. j o u r n a l p r e -p r o o f we mapped the location of nsp binding to a nucleotide region corresponding to helix ( figure a) , adjacent to the mrna entry channel (simonetti et al., ) (figure b ). the interaction would position nsp to disrupt s mrna scanning and prevent translation initiation (figure b) , and disrupt trna recruitment to the s ribosome and block protein production ( figure s b) . interestingly, the nsp binding site includes the highly conserved g nucleotide which monitors the minor groove of the codon-anticodon helix for trna binding fidelity (ogle et al., ) . we noticed that the c-terminal region of nsp has similar structural regions to serbp (brown et al., ) and stm (ben-shem et al., a) , two known ribosome inhibitors that bind within the mrna entry channel to preclude mrna access ( figure s c ). consistent with this, a recent cryo-em structure confirms that nsp binds to these same nucleotides of s within the mrna entry channel (thoms et al., ) . given the location of nsp binding on the s ribosome, we hypothesized that it could suppress global initiation of mrna translation. to test this, we performed in vitro translation assays of a gfp reporter in hela cell lysates and found that addition of nsp led to potent inhibition of translation ( figure s d ). we observed a similar nsp -mediated translational repression when we co-expressed nsp and a gfp reporter gene in hek t cells (figure c-d) . in contrast, we did not observe this inhibition when we expressed other sars-cov- proteins (nsp , nsp , m) or human proteins (gapdh) ( figure d ). to determine if nsp leads to translational inhibition of endogenous proteins in human cells, we used a technique called surface sensing of translation (sunset) to measure global protein production levels (schmidt et al., ) . in this assay, translational activity is measured by the level of puromycin incorporation into elongating polypeptides ( figure s e ). we observed a strong reduction in the level of global puromycin integration in cells expressing nsp compared to cells expressing gfp (figure s f-g) . because nsp expression is sufficient to suppress global mrna translation in human cells, we hypothesized that sars-cov- infection would also suppress global translation. to test this, we infected a human lung epithelial (calu ) or monkey kidney (vero) cell line with sars-cov- and measured nascent protein synthesis levels using sunset. we observed a strong reduction of to explore whether nsp binding to s rrna is critical for translational repression, we generated a mutant nsp in which two positively charged amino acids (k and h ) in the c-terminal domain were replaced with alanine residues (figure s c ) (narayanan et al., ) . we observed a complete loss of in vivo contacts with s ( figure g) ; because this mutant disrupts ribosome contact, we refer to it as nsp ∆rc. we co-expressed gfp and nsp ∆rc in hek t cells and found that the mutant fails to inhibit translation ( figure h and s j) . in contrast, mutations to the positively charged amino acids at positions / do not impact s binding ( figure g ) or the ability to inhibit translation ( figure h ). together, these results demonstrate that nsp binds within the mrna entry channel of the ribosome and that this interaction is required for translational inhibition of host mrnas upon sars-cov- infection. we explored whether nsp binding to s rrna suppresses the ability of cells to respond to ifn-β stimulation upon viral infection. we transfected isg reporter cells with nsp , stimulated with ifn-β, and observed robust repression of the ifn responsive gene (> -fold, figure i ). to confirm that this nsp -mediated repression occurs in human cells upon activation of double stranded rna (dsrna)-sensing pathways typically triggered by viral infection, we treated a human lung epithelial cell line (a ) with poly(i:c), a molecule that is structurally similar to dsrna and known to induce an antiviral innate immune response (alexopoulou et al., ; kato et al., ) (figure s k ). we observed a marked downregulation of ifn-β protein and endogenous ifn-β responsive mrnas in the presence of nsp , but not in the presence of nsp ∆rc ( figure s l, m) . these results demonstrate that nsp , through its interaction with s rrna, suppresses the innate immune response to viral recognition ( figure j ). j o u r n a l p r e -p r o o f because nsp blocking the mrna entry channel would impact both host and viral mrna translation, we explored how translation of viral mrnas is protected from nsp -mediated translational inhibition. many viruses contain ' untranslated regions that regulate viral gene expression and translation (gaglia et al., ) ; all sars-cov- encoded subgenomic rnas contain a common ' leader sequence that is added during negative strand synthesis (kim et al., b) . we explored whether the leader sequence protects viral mrnas from translational inhibition by fusing the viral leader sequence to the ' end of gfp or mcherry reporter genes ( figure s a ). we found that nsp fails to suppress translation of these leader-containing mrnas ( figure a -b, s b). we dissected the leader sequence and found that the first stem loop (sl ) is sufficient to prevent translational suppression upon nsp expression ( figure c) or sars-cov- infection ( figure d ). we considered three models for how the leader could protect viral mrnas: (i) it could compete with the ribosome for nsp binding, (ii) it could directly recruit free ribosomes or (iii) nsp could bind to the leader independently of its ribosome interaction to allosterically modulate the nsp -ribosome interaction. we reasoned that if the leader competes for nsp binding or directly recruits free ribosomes, then the presence of sl should be sufficient for protection, regardless of its precise position in the ' utr. in contrast, if the leader allosterically modulates ribosome binding then the spacing between the ' cap (which is bound to nsp - s) and sl would be critical for protection. to distinguish between these models, we swapped the location of sl and sl in the ' leader or inserted nucleotides between the ' cap and sl ( figure s c ) and found that both mutants ablate protection ( figure e, s d) . these results indicate that an mrna requires the ' leader to be precisely positioned relative to the nsp -bound s ribosome to enable translational initiation ( figure f ). while many aspects of this allosteric model remain to be explored, it would explain how leader-mediated protection can occur on an mrna only when present in cis. moreover, this model suggests that nsp might also act to further increase viral mrna translation by actively recruiting the ribosome to its own mrnas. consistent with this, we observe a consistent ~ % increase in translation of leader-containing reporter levels upon viral infection ( figure d ) or expression of nsp ( figure s e ). upon engaging the start codon in an mrna, the s subunit of the ribosome is recruited to form the s ribosome which translates mrna. the signal recognition particle (srp) is a universally conserved complex that binds to the s ribosome and acts to co-translationally scan the nascent peptide to identify hydrophobic signal peptides present in integral membrane proteins and proteins secreted from the plasma membrane (akopian et al., ) . when these are identified, srp triggers ribosome translocation to the endoplasmic reticulum (er) to ensure proper folding and trafficking of these proteins to the cell membrane (akopian et al., ) . we identified two viral proteins -nsp and nsp -that bind at distinct and highly specific regions within the s-domain of the sl rna scaffold of srp ( figure a , s a). nsp interacts with sl in the region bound by srp (the protein responsible for signal peptide recognition, srp-receptor binding, and ribosome translocation) (akopian et al., ; holtkamp et al., ) ( figure b ). nsp binds to sl in the region that is bound by the srp protein ( figure b ), which is required for proper folding and assembly of srp (including proper loading of srp ) (akopian et al., ) . because srp scans nascent peptides co-translationally, we were intrigued to find that nsp also forms a highly specific interaction with s rrna (the structural component of the s subunit) ( figure c, s b) . the binding site on s rrna corresponds to the largest human-specific expansion segment within the ribosome, referred to as es (parker et al., ) . es is highly dynamic, and thus has not been resolved in most ribosome structures (zhang et al., ) . however, when engaged by specific factors, es can become ordered, and was recently shown to be capable of interacting with the ribosome exit tunnel, adjacent to the s binding site of srp ( figure d , s c) (wild et al., ) . together, these observations suggest that nsp and nsp bind to the co-translational srp complex. consistent with this, we find that nsp and nsp localize broadly throughout the j o u r n a l p r e -p r o o f cytoplasm when expressed in human cells ( figure s d ) or upon sars-cov- infection ( figure s e -f). because nsp and nsp binding on sl are positioned to disrupt srp function, we hypothesized that they may alter translocation of secreted and integral membrane proteins ( figure s a ). to test this, we expressed an srp-dependent membrane protein (nerve growth factor receptor, ngfr (izon et al., a) ) fused via an internal ribosome entry site (ires) to a non-membrane gfp ( figure s f ). in this system, if a perturbation specifically affects membrane protein levels we expect to see a decrease in the ratio of membrane to non-membrane protein levels. to ensure that the ngfr reporter accurately reports on srp function, we treated hek t cells with sirnas against srp or srp and found that both lead to a dramatic reduction of the ngfrmembrane protein relative to the non-membrane gfp protein ( figure s b) . similarly, we found that expression of nsp and nsp (alone or together) lead to a striking reduction in expression of ngfr relative to gfp ( figure a ). expression of control proteins did not specifically impact ngfr levels ( figure a, s b) . to determine if there is a global effect on membrane protein levels, we utilized the sunset method to measure puromycin levels in membrane proteins using flow cytometry (see methods). we confirmed that disruption of srp leads to a global reduction in puromycin levels in the cell membrane ( figure s c ). we observed a comparable global reduction of puromycin-labeled membrane proteins upon expression of nsp or nsp individually or together, but not with control proteins (figure b, s c) . because nsp and nsp are each sufficient to suppress protein integration into the cell membrane, we anticipate that sars-cov- infection would lead to similar suppression. j o u r n a l p r e -p r o o f however, determining whether sars-cov- infection specifically impacts membrane protein expression is confounded by the fact that nsp inhibits translation of membrane and nonmembrane proteins upon infection. to address this, we co-expressed a membrane protein reporter (ngfr) containing the ' viral leader along with a non-membrane gfp reporter containing the viral leader. upon viral infection, we observed a strong reduction of membrane protein levels ( figure c ), but no reduction in non-membrane gfp levels ( figure d ). to ensure that these effects are specific to sars-cov- infected cells, we separated individual cells within the infected population into those expressing the viral spike protein (s+) and those not expressing the protein (s-). we found that the shift in membrane protein levels only occurs in s+ cells ( figure d ), while the spopulation resembled the mock infected samples ( figure c ). we observed a strong relationship between the level of spike protein -likely reflecting the amount of viral replication within each cell -and the level of membrane protein suppression ( figure c ). we observed this membrane protein-specific decrease upon infection of human lung epithelial (calu , figure s d ) and monkey kidney (vero, figure c -d) cell lines. together, these results demonstrate that nsp and nsp bind to sl to disrupt srp function and suppress membrane protein trafficking in sars-cov- infected cells. although nsp and nsp are thought to be components of the viral replication machinery (sutton et al., ) , our results indicate that they play an additional role as host virulence factors. because viral membrane proteins also require trafficking to the er, viral disruption of srp might negatively impact viral propagation, unless viral proteins are trafficked in an srp-independent manner ( figure s e ) or if nsp / selectively impacts host (but not viral) proteins. next we explored how disruption of srp might be advantageous for viral propagation. because secretion of ifn and other cytokines is dependent on the srp complex for secretion ( figure s f ), a central component of the ifn response is dependent on srp. accordingly, we hypothesized that nsp / -mediated viral suppression of srp would act to suppress the ifn j o u r n a l p r e -p r o o f response upon infection. to test this, we co-expressed nsp and nsp and observed a significant reduction in the ifn response relative to a control protein ( figure s g ). together, these results suggest that sars-cov- mediated suppression of srp-dependent protein secretion enables suppression of host immune defenses ( figure e) . interestingly, many proteins involved in anti-viral immunity -including most cytokines and class i major histocompatibility complex -are membrane-anchored or secreted, and are known to use the srp pathway for transport (vermeire et al., ) (figure s f ), suggesting that there may be other effects of srp pathway inhibition on sars-cov- pathogenesis. we identified several pathogenic functions of sars-cov- in human cells -including global inhibition of host mrna splicing, protein translation, and membrane protein trafficking -and described the molecular mechanisms by which the virus acts to disrupt these essential cell processes. interestingly, all of the viral proteins involved (nsp , nsp , nsp , and nsp ) are produced in the first stage of the viral life cycle, prior to generation of double stranded rna (dsrna) products during viral genome replication. because dsrna is detected by host immune sensors and triggers the type i interferon response, disruption of these cellular processes would allow the virus to replicate its genome while minimizing the host innate immune response. disruption of these three non-overlapping steps of protein production may represent a multipronged mechanism that synergistically acts to suppress the host antiviral response ( figure f) . specifically, the ifn response is usually boosted > , -fold upon viral detection (through amplification and feedback, figure s k ), yet each individual mechanism impacts ifn levels on the order of ~ - -fold. accordingly, if each independent mechanism impacts ifn levels moderately, the three together may be able to achieve dramatic suppression of ifn ( = , fold). this multi-pronged mechanism may explain the molecular basis for the potent suppression of ifn observed in severe covid- patients. interferon is emerging not only as a determinant of disease severity, but also a potential treatment option (zhou et al., ) . as such, our work identifies several therapeutic opportunities for boosting ifn levels upon sars-cov- infection. for example, disrupting the interaction between nsp and s rrna could allow cells to detect and respond to viral infection. because many small-molecule drugs target ribosomal rnas (liaud et al., ) , it may be possible to develop drugs to block the nsp - s and other interactions. additionally, disrupting the ' viral leader may be a potent antiviral strategy since it is critical for translation of all viral proteins. because sl is a structured rna, it may be possible to design small molecules that specifically bind this structure to suppress viral protein production (hermann, ) . viral suppression of these cellular functions is not exclusive to the ifn response and will also impact other spliced, translated, secreted, and membrane proteins. many proteins involved in anti-viral immunity are spliced and/or membrane-anchored or secreted. for example, class i major histocompatibility complex (mhc), which is critical for antigen presentation to cd t cells at the cell surface of infected cells (hansen and bouvier, ). by antagonizing membrane trafficking, sars-cov- may prevent viral antigens from being presented on mhc and allow infected cells to escape t-cell recognition and clearance. in this way, interference with these essential cellular processes might further aid sars-cov- in evading the host immune response. more generally, we expect that insights gained from the sars-cov- protein-rna binding maps will be critical for exploring additional viral mechanisms. specifically, we identified many other interactions, including highly specific interactions with mrnas. for example, nsp binds to the jun mrna ( figure s e ) which encodes the critical immune transcription factor c-jun which is activated in response to multiple cytokines and immune signaling pathways (weston and davis, ) . we also identified an interaction between nsp and the start codon of the mrna that encodes cops (figure c) , the enzymatic subunit of the cop signalosome complex which regulates protein homeostasis (cope and deshaies, ) , suggesting that it might disrupt its translation. interestingly, cops (also known as jab ) is known to bind and stabilize c-jun protein levels (claret et al., ) and several viruses are known to disrupt this protein (lungu et al., ; oh et al., ; tanaka et al., ) . while it remains unknown what, if any, role these interactions play in virally infected cells, the specificity suggests that they may provide a selective advantage for viral propagation. together, our results demonstrate that global mapping of rna binding by viral proteins could enable rapid characterization of mechanisms for emerging pathogenic rna viruses. we note several limitations of our current study that will need to be explored in future work. (i) our mapping experiments were performed in uninfected human cells expressing tagged viral proteins. accordingly, it remains possible that our maps may not fully capture all of the interactions that occur when human cells are infected, such as interactions that occur with viralinduced rnas, in specific viral compartments, or that require multiple viral proteins. (ii) while we characterized the functional and mechanistic roles of several viral proteins and structural ncrnas, we did not explore what roles viral protein interactions with mrnas might play. (iii) how the virus disrupts fundamental cellular processes while still maintaining its own production is still largely undefined. while we showed that the ' leader is sufficient to relieve translational inhibition by nsp , we still do not fully understand how this protection occurs and specifically how nsp might interact with the viral leader or allosterically modulate ribosome binding. similarly, viral membrane proteins are dependent on trafficking to the er and how nsp / might selectively impact er translocation of host -but not viral -proteins remains to be explored. (iv) while we showed that viral disruption of these essential cellular functions can suppress ifn, what other roles host cell shutdown might play in viral pathogenesis and in suppressing other aspects of anti-viral immunity, including possible roles in adaptive immune responses, have not been explored. the authors declare no competing interests. further information and requests for reagents and resources should be directed to and will be fulfilled by the lead contact, mitchell guttman (mguttman@caltech.edu). all constructs and plasmids generated in this study will be made available on request sent to the lead contact with a completed materials transfer agreement. all datasets generated during this study are available at ncbi short read archive: bioproject prjna (viral protein purifications) and prjna (nascent and total rna-seq) essential medium (atcc) containing % fbs and % penicillin-streptomycin purchased from thermo fisher scientific. all cell lines were maintained at °c under % co . cells were grown in a humidified incubator at ºc with % co . all experiments using infectious sars-cov- conducted at the uvm bsl- facility were cloning of expression constructs. sars-cov- protein constructs (with the exception of nsp ) were a gift from fritz roth (see table s for addgene information) (kim et al., a) and were lr-cloned (invitrogen gateway cloning, thermo fisher scientific) into mammalian expression destination vector pcag-halo-tev-dest-v -ires-puror. note that following lr cloning, proteins were not v -tagged because all entry clones contained stop codons. for nsp , an entry clone was generated by bp cloning (invitrogen gateway cloning, thermo manganese/calcium mix ( . mm cacl , . mm mncl ). samples were incubated on ice for minutes to allow lysis to proceed. the lysates were then incubated at °c for minutes at rpm shaking on a thermomixer (eppendorf). lysates were cleared by centrifugation at , × g for minutes. the supernatant was collected and kept on ice until bound to the halolink resin (promega). of the ml lysis volume, ul was set aside for input, ul used for protein expression confirmation, and the rest for capture on halolink resin as described below. for qpcr analysis, cdna was generated from purified rna using maxima h-reverse transcriptase (thermo fisher scientific) following manufacturer's recommendations. amplification reactions were assembled with primer sets indicated in table s and lightcycler® sybr green i master (roche) following manufacturer's protocols and read out in a roche lightcycler . library construction. rna-seq libraries were constructed from purified rna as previously described (van nostrand et al., ) . briefly, after proteinase k elution, the rna was dephosphorylated (fast ap) and cyclic phosphates removed (t pnk) and then cleaned using silane beads as previously described (van nostrand et al., ). an rna adapter containing a rt primer binding site was ligated to the ' end of the cleaned and end-repaired rna. the ligated rna was reverse transcribed (rt) into cdna, the rna was degraded using naoh, and a second adapter was ligated to the single stranded cdna. library preparation was the same for input samples except that an initial chemical fragmentation step ( °c for min s in x fastap buffer) was included prior to fastap treatment. this chemical fragmentation step was designed to be similar to the fragmentation conditions used for purified halo bound samples. the for staining of infected cells, cells were fixed and permeabilized in % formaldehyde % triton, and subsequently labelled with primary antibodies raised in sheep to sars-cov- at / dilution, followed by incubation with a rabbit anti-sheep alexa secondary antibody (abcam, ab ) at / dilution and mounted with dapi in the medium (thermo fisher scientific, cat# p ). cells were imaged with a zeiss lsm confocal microscope, with airy unit pinhole for all primary antibody channel acquisitions and pixel size . µm x . µm. the objective lens used was a zeiss plan-apochromatic x/ . na m . structure modeling nsp homology model. the predicted model of sars-cov- nsp was generated using the transform-restrained rosetta (trrosetta) algorithm, a deep learning-based modeling method based on the rosetta energy minimization pipeline with additional distance and interaction restraints generated from co-evolution (yang et al., ) . all figures were generated using pymol (www.pymol.org). nsp -ribosome model. the model of nsp bound to the ribosome was generated using modeller version . (webb and sali, ) . the c-terminal sequence of nsp (khssgvtrelmrelngg) was modeled using the structure of serbp bound to the ribosome (pdb id: mte, chain w) as a template. the default modeller parameters were used to create an alignment of nsp and serbp and to generate the model, and all atoms within Å of serbp were included in the model to define the neighboring environment. twenty models were generated and the model with the lowest dope score was selected to visualize with pymol (delano, ) . x-ray crystal structures and cryo-electron microscopy structures were obtained from the protein data bank (www.rcsb.org) (berman et al., ) and visualized with pymol (delano, ) . for u and u structural analysis, we used a cryo-em structure of the pre-catalytic human spliceosome (pdb id: qx ). for sl structural analysis, we used an x-ray crystal structure of the human signal recognition particle (pdb id: mfq). to examine human srp in the context of the ribosome, we used a cryo-em structure of the mammalian srp-ribosome complex (pdb id: jaj). to analyze the ribosomal es expansion segment, we superimposed a cryo-em structure of the expansion segment (pdb id: sxo) onto the complete ribosome structure (pdb id: jaj) using the pymol command "super." finally, for nsp - s rrna structural analysis, we used multiple structures of the ribosome, including structures of the pre- s subunit (pdb id: g h), s late-stage initiation complex (pdb id: yal), s in complex with serbp (pdb id: mte), and s in complex with stm (pdb id: v ). j o u r n a l p r e -p r o o f nsp was cloned into a bacterial expression vector resulting in n-terminally tagged halo- xhistagged nsp . the nsp sequence was pcr amplified from addgene nsp entry vector to add a n-terminal x his tag and restriction enzyme sites for digestion and ligation into n-terminal halo bacterial expression vector. this construct was transformed into bl de e. coli (agilent), expanded to a ml liquid culture, and grown until od reached . . iptg was added to a final concentration of mm. after hours of iptg induction, bacteria was centrifuged for min at × g. pellet was lysed with binding buffer ( mm hepes, ph . , mm mgcl , mm nacl, mm tcep, mm imidazole, mm atp, % triton x- ) supplemented with atp ( mm), protease inhibitor cocktail (promega), benzonase (sigma) and triton-x (sigma) using ml of lysis mix per gram of wet cell paste. cell suspension was rocked for min at room temperature and then centrifuged at , × g for min at °c. supernatant was incubated with washed imac resin (bio-rad) and rocked for min at room temperature. we loaded the resin-lysate mixture into an appropriately-sized column and washed with column volumes of binding buffer ( mm hepes, ph . , mm mgcl , mm nacl, mm tcep, mm imidazole, mm atp, % triton x- ) followed by column volumes of wash buffer ( mm hepes, ph . , mm nacl, mm tcep, mm imidazole, ph ). recombinant nsp (rnsp ) was eluted with column volumes of elution buffer by adding column volume at a time with column flow stopped, collecting eluate after each addition, and waiting min between each elution buffer addition. we dialyzed these eluates with a ml spectra-por® float-a-lyzer® g (spectrum laboratories) into storage buffer ( mm hepes, ph . , mm nacl, % glycerol) at °c using exchanges, one after hours and then overnight. pierce -step human coupled ivt-dna (thermo fisher scientific) in vitro translation kit was used to measure rnsp -dependent translation inhibition. bovine serum albumin (bsa), and buffer only controls were used to control for the addition of excess protein or changes in buffer composition. to measure translation inhibition, µl in vitro translation reactions were assembled, scaled according to manufacturer's recommendations. the included control plasmid pcfe-gfp was used to measure translational output of the reactions. gfp fluorescence was measured on a biotek cytation plate reader using emission filters for gfp fluorescence. . µm j o u r n a l p r e -p r o o f stock dilutions of rnsp and bsa were made in storage buffer ( mm hepes, ph . ., mm nacl, % glycerol). subsequent fold dilutions were made in storage buffer to span a concentration range of nm to nm for each protein in the final reaction. µl of the diluted protein solution was added to the µl translation reactions, and incubated for minutes at room temperature prior to the addition of the gfp reporter plasmid. duplicate reactions were made to measure variability for each condition. in addition, a buffer only control was included to measure the effect of dilution of the translation reaction by the storage buffer. after the minute incubation, ng of gfp reporter plasmid was added to each reaction and incubated at °c for hours prior to fluorescence detection. two microliters from each reaction was measured in duplicate on a biotek cytation microplate reader using excitation and emission filters for gfp. sample readings were blanked by subtracting values obtained from the buffer only control. promega's rabbit reticulocyte lysate system was also used to assay translation inhibition. to measure translation inhibition, µl in vitro translation reactions were assembled, scaled according to manufacturer's recommendations. for each translation reaction, either µl of recombinant protein storage buffer or rnsp was added, followed by ng of mrna. after hours of incubation at °c, luciferase was read out using the bright-glo luciferase assay (promega) or gfp fluorescence was measured, both on a biotek cytation plate reader. we assayed translation in hek t cells transfected with mammalian expression vectors, mrnas, or combinations of these. for mrna transfections of fluorescence protein translation reporters (including unmodified, +sars-cov leader sequence, +sl , +sl -sl , and + nts), dna templates for in vitro transcription were generated with sequences appended to the ' end of gfp and mcherry (see tables s and s for primers and templates, respectively) and transcribed using hiscribe™ t arca mrna kit with tailing (new england biolabs). for nsp mrna transfection, indicated primers from table s were used to add restriction enzyme sites for cloning into pt cfe -chis backbone provided in the pierce human -step coupled acquistion files were analyzed with flowjo analysis software. to assay global protein translation, a sunset assay was performed as previously described (schmidt et al., ) we transfected these mammalian expression vectors for nsp and gfp into hek t using biot transfection reagent. after hours, doxycycline (sigma) was added to a final concentration of µg/ml. after hours, cells were incubated with puromycin ( µg/ml) for min, then washed with fresh media, and harvested with cold pbs. pelleted cells were lysed for min on j o u r n a l p r e -p r o o f ice (mixing after min) with ul ripa buffer supplemented with protease inhibitor cocktail (promega). insoluble debris was pelleted by centrifuging at , × g for . minutes and supernatant was run on a bolt™ - % bis-tris plus gel (thermo fisher scientific). proteins were then transferred to nitrocellulose using the iblot transfer system (thermo fisher scientific) and western blotting carried out using an anti-puro antibody (clone d , emd millipore). sunset in sars-cov- infection was performed as above with the following modifications. cells were infected or not (mock) with sars-cov- , and hpi cells were incubated with puromycin ( µg/ml) for min. media was aspirated and cells lysed directly in x laemmli's buffer (biorad), heated at ºc for ten minutes and run on a - % nupage gel (thermo fisher scientific). proteins were transferred to nitrocellulose using the iblot transfer system and probed as above. to assay srp-dependent membrane protein transport to the cell surface, we monitored surface arrival of exogenously expressed neuronal growth factor receptor (ngfr) by flow cytometry in the presence of nsps. mammalian expression vectors were exchanged for versions that contained an ires-ngfr to co-express a membrane reporter and thus, for these experiments, lr reactions were carried out with destination vector pb- xhis-gfp-dest-ires-ngfr. resulting expression vectors drive protein expression by a dox-inducible promoter, contain the rtta needed for dox induction, and produce an n-terminally-tagged his-gfp fusion protein and a co-expressed ngfr. the gfp here is an enhanced gfp containing an amino acid substitution (a k) to generate a monomeric variant based on previous literature (alberti et al., ) . we transfected these mammalian expression vectors for nsp , nsp , nsp ∆rc mutant and to knockdown srp and srp , sirnas targeting each (dharmacon cat# l- - - and l- - - , respectively) were transfected into hek t cells using lipofectamine rnaimax (invitrogen) according to manufacturer's protocols. to validate knockdown, transfected cells were assayed by qpcr using primer sets (table s ) to amplify each target as well as normalizer calm . transfections were carried out hours prior to assaying cells, either by qpcr, membrane reporter, or membrane sunset (see below) experiments. calu and vero cells were transfected with mrnas encoding leader-ngfr and leader-gfp using transit-mrna transfection kit (mirus) and subsequently infected with sars-cov- at an moi of . . after hours, cells were washed with pbs, trypsinized and fixed in % pfa for minutes before staining with biotinylated anti-ngfr (biolegend) and anti-sars-cov- spike antibody (sino) and subsequently stained with pe-labeled anti-rabbit (thermo, p- mp) and pacblue-labeled streptavidin (thermo, s ). facs was performed on a macsquant flow cytometer and analyzed using flojo analysis software; facs distributions were compared using a -tailed kolmogorov-smirnov test. for these experiments, rna was transcribed from a pcr template (see table s ) using the hiscribe t arca mrna kit (with tailing). to assay transport to the cell surface of all plasma membrane proteins, the sunset assay was adapted to puro-label surface proteins as previously described (schmidt et al., ) , and read out by flow cytometry. briefly, cells were incubated with puromycin as described above, followed by two quick washes and a chase with fresh complete media for min. cells were lifted with mm edta as described above and stained with an anti-puro antibody (clone d , emd millipore) conjugated to alexa- . for these experiments, nsp was expressed from the same vector described above for membrane reporter assays. fluorescence intensity j o u r n a l p r e -p r o o f measurements were taken for gfp and alexa- on a macsquant flow cytometer and analyzed using flojo analysis software; distributions were compared using a -tailed kolmogov-smirnov. to assess splicing efficiency, exons - of mouse irf (enmust . ) containing its endogenous intron were fused upstream of a self-cleaving peptide and egfp and cloned into an mscv vector (pig, addgene) (mayr and bartel, ). this construct was co-transfected into hek ts with nsp or gfp and measured hours after transfection by flow cytometry (macsquant) and analyzed using flojo analysis software. sars-cov or mock infected calu cells and nsp -or gapdh-expressing hek ts were labeled with -ethynyl-uridine ( eu; jena bioscience) by adding eu containing media to cells for min at a final concentration of mm, as previously described (jao and salic, ) . after the pulse label, cells were washed with warm pbs and lysed in rlt buffer (qiagen). total rna was isolated from cells using manufacturer's protocols for qiashredder and rneasy rna isolation (both qiagen), followed by turbo dnase treatment (ambion, thermo scientific), and zymo rna clean and concentrate. for each sample, µg of rna was used for ligation of a unique barcoded rna adaptor, following the relevant steps in the protocol described above in library construction of rna-seq libraries. samples were then pooled before proceeding to biotinylation steps. to biotinylate eu-labeled rna, samples were first mixed, in order, with water, hepes ( mm), biotin picolyl azide ( mm; click chemistry tools) and ribolock rnase inhibitor, then added to premixed cuso ( mm) and thpta ( mm), and finally added to freshly prepared j o u r n a l p r e -p r o o f sodium ascorbate ( mm), as previously described (hong et al., ) . the click reaction was incubated for hour at ºc with rpm shaking on an eppendorf thermomixer followed by rna purification using > nt protocol for zymo clean and concentrate. we completed three rounds of sequential capture on streptavidin beads to isolate nascent transcripts (see figure s b ). to capture biotinylated rna, myone streptavidin c dynabeads (thermofisher scientific) were first washed three times in urea buffer ( mm hepes, ph . , mm edta, . m licl, . % triton x- , . % sds, . % sodium deoxycholate, . mm tcep, m urea) followed by three additional washes in m buffer ( mm tris, ph . , mm nacl, . % triton x- , . % sodium deoxycholate, . % np- ). washed beads were mixed with parts m urea buffer and part biotinylated rna and incubated for min with rpm thermomixer shaking at room temperature. after magnetic separation, beads were washed times with m buffer followed by washes with urea buffer at ºc at rpm for min. rna was eluted from beads in sequential elutions by incubating with elution buffer ( . m guanidine thiocyanate , % n-lauroylsarcosine; both sigma) at ºc for minutes, repeating with more elution buffer for a second elution. the elutions were pooled, diluted with urea buffer, incubated with pre-washed streptavidin beads, washed, and eluted for additional rounds exactly as described above for a total of sequential captures. final elutions were pooled, cleaned with zymo rna clean and concentrate following manufacturer's protocols, and carried through rna-seq library preparation as described above starting with the reverse transcription step. hek-blue™ isg cells were seeded in well plates, transfected with nsp mammalian expression vectors using biot and stimulated with ng/ml human ifn-b (r&d systems). supernatants were assayed for alkaline phosphatase as per manufacturer instructions using j o u r n a l p r e -p r o o f quanti-blue reagent (invivogen). hek- t cells were seeded in well plates, transfected with either halo-tagged gapdh, nsp , nsp and nsp in combination, or nsp mammalian expression vectors using biot. hours later, the media was replaced with media containing ng/ml human ifn-β (r&d systems). expression was assayed using live cell halo-imaging. halo-tmr ligand was diluted : in media and added to the culture for a : final dilution. samples were incubated minutes at °c, % co and then the media was aspirated. wells were rinsed twice with pbs, then media was added back to the wells. samples were incubated minutes at °c, % co to allow uncoupled ligand to diffuse out of the cells. media was then aspirated and replaced, and cells were imaged by widefield fluorescence microscopy. cultures were ultimately harvested for rna hours later, or hours post transfection. a s were seeded in well plates, transfected with nsp mammalian expression vectors using lipofectamine and stimulated with µg/ml hmw poly(i:c) (invivogen) h after transfection. supernatant was assayed for secreted ifn-β by elisa (human ifn beta elisa, high sensitivity, pbl) hours after stimulation, and rna from cells was purified and assessed for isg gene expression as normalized to gapdh expression (sybr green master mix, bio-rad). primers used for qpcr are listed in table s . sars-cov- leader sequence was appended to the ' end of gfp and mcherry reporter templates via pcr. pcr templates were then transcribed using hiscribe t arca mrna kit (with tailing). leader mutants, including sl only, sl /sl swap, and + nts mutants were likewise appended to the ' end of fluorescent reporter templates via pcr and transcribed using hiscribe t arca kit. mrna reporters were transfected in hek- t cells with lipofectamine messengermax. to measure fluorescence of mcherry and gfp reporters, hours post transfection cells were either lifted with pbs and transferred into black well plates for fluorescence readout on a biotek cytation or trypsinized and processed for flow cytometry. sequence alignment and analysis. for halo purifications and rna binding mapping sequencing reads were aligned to a combined genome reference containing the sequences of structural rnas (ribosomal rnas, snrnas, snornas, s pre-rrna) and annotated mrnas (refseq hg ) using bowtie . to distinguish between the nascent pre-ribosomal rna and mature s, s, and . s rrna, we separated each of the components of the s into separate sequence units for alignment (e.g. its, ets). we excluded all low quality alignments (mapq < ) from the analysis. for mrna analysis, we removed pcr duplicates using the picard markduplicates function (https://broadinstitute.github.io/picard/). for each rna, we enumerated nucleotide windows across the entire rna. for each window, we calculated the enrichment by computing the number of reads overlapping the window in the protein elution sample divided by the total number of reads within the protein elution sample. we normalized this ratio by the number of reads in the input sample divided by the total number of reads in the input sample. because all windows overlapping a gene should have the same expression level in the input sample (which represents rna expression), we estimated the number of reads in the input as the maximum of either (i) the number of reads over the window or (ii) the median read count over all windows within the gene. this approach provides a conservative estimation of enrichment because it prevents windows from being scored as enriched if the input values over a given window are artificially low, while at the same time accounting for any non-random issues that lead to increases in read counts over a given window (e.g. fragmentation biases or alignment artifacts leading to non-random assignment or pileups). j o u r n a l p r e -p r o o f we calculated a multiple testing corrected p-value using a scan statistic, as previously described (guttman et al., (guttman et al., , . briefly, n was defined as the number of reads in the protein elution plus the number of reads in the control sample. p was defined as the total number of reads in the protein elution sample divided by the sum of the protein elution sample total reads and total reads in the control sample. w was the size of the window used for the analysis ( nucleotides). the scan statistic p-value was defined using the poisson estimations based on standard distributions previously described (naus, ) . because rna within input samples are fragmented differently than the protein elution samples, we noticed that the overall positional distribution of protein elution samples was distinct from input distributions. accordingly, we used the remaining protein elution samples (rather than input) as controls for each protein. specifically, this enabled us to test whether a given protein is enriched within a given window relative to all other viral and control proteins. enrichments were computed as described above. these values are plotted in figure and table . igv (robinson et al., ) and were generated by either: (i) computing the enrichment for each nucleotide as described above. in this case, the read count for each nucleotide was computed as the total number of reads that overlapped the nucleotide. (ii) counting the number of rt stop sites at a given nucleotide. in this case, we compute the alignment start position of the second in pair read and computed a count of each nucleotide. we normalized this count by the total number of reads in the sample to account for sequencing depth generated. we then normalized this ratio by the same ratio computed for the control sample (merge of all other protein samples) for each nucleotide. heatmaps were generated using morpheus (https://software.broadinstitute.org/morpheus/). all values were included if they contained a significant nt window with a p-value< . (see above) and minimum enrichment of -fold above the control sample. gene ontology analysis. the non-n enriched mrnas were analyzed against the gene ontology biological processes and reactome gene sets using the molecular signatures database (msigdb) (liberzon et al., ) . significantly enriched gene sets with an fdr< . were used. j o u r n a l p r e -p r o o f to ensure that significant gene sets were not being driven by the multiple ribosomal proteins or histone proteins, these analyses were also carried out excluding these proteins. sequenced reads were demultiplexed according to barcoded rna adaptor sequences ligated to each respective sample. trimmomatic (https://github.com/timflutre/trimmomatic ) was used to remove any contaminating illumina primer sequences in the reads and low quality reads. demultiplexed and trimmed files were then aligned to a hg reference genome using the spliceaware star aligner (https://github.com/alexdobin/star). alignments were then deduplicated for pcr duplicates using picard markduplicates (https://broadinstitute.github.io/picard/). aligned read-fragments were defined as read and read contained within a paired-end read fragment along with the insert between these two reads. we defined a set of high-quality represented isoforms per gene using the appris database (rodriguez et al., ) . all readfragments that spanned any ' splice site within an isoform of one of these genes was retained. for each ' splice site spanning fragment, we classified the read-fragment as a spliced fragment if it spanned an exon-exon junction (e.g. aligned entirely within distinct exons) or an unspliced fragment if it spanned an intron-exon junction (e.g. one of the reads was contained -or partially contained -within the intron). for each isoform, we computed an unspliced ratio by counting the total number of reads that were classified as unspliced divided by the total number of readfragments spanning ' splice sites within that gene. to ensure that the splicing ratio that we measured is a reliable metric and not inflated/deflated due to low read counts, we only included genes that contained at least read-fragments in each sample and where the total number of reads in the control and sample conditions (when merged together) contained a significant number of reads to reliably measure a difference between the two groups as measured by a hypergeometric test (p< . ). because different genes contain different baseline splicing ratios due to gene length and coverage, we computed a change in the splicing ratio for each gene independently. to do this, we subtracted the unspliced ratio for each sample from the average unspliced ratio for that gene j o u r n a l p r e -p r o o f in all of the control samples. we plotted the overall distribution of these differences in splicing ratios as violin plots for each sample. if there is no change in splicing ratio, we would expect that some genes would have higher splicing ratios and others lower splicing ratios but that the overall distribution would be centered around . total rna-seq libraries were generated from the same mock infected and sars-cov- virally infected calu samples treated with eu. prior to eu purification, total rna was taken and an rna-seq library constructed as described above using barcoded rna adapters. cytoplasmic ribosomal rnas ( s and s) were depleted using nebnext ribosomal rna depletion kit (neb e l) per manufacturers recommendations. demultiplexed reads were aligned using bowtie (http://bowtie-bio.sourceforge.net/bowtie /index.shtml) to custom genomes encoding classical noncoding rnas (ncrnas) or human messenger rnas (mrnas). expression levels were computed for each mrna by counting the total number of sequencing reads aligned to the mature mrna. to normalize across the different libraries, we computed the read counts for each sample that align to non-spliced structural non-coding rnas -excluding rrna but including snrnas, sl, sk, etc. we then divided each mrna count by the sum of all ncrna counts. this normalized value for each gene per sample was then converted into a fold-change by dividing this normalized value to the mean value for both mock infected samples. the fold change of each gene relative to mock was plotted across all mrnas as a violin plot. and enrichment > -fold for any of the viral proteins is reported. • nsp binds mrna recognition domains of u /u snrnas and disrupts mrna splicing • nsp binds in the mrna entry channel of the ribosome to disrupt protein translation • nsp and nsp bind the signal recognition particle and disrupt protein trafficking • these disruptions to protein production suppress the interferon response to infection in brief -sars-cov- proteins directly engage host rnas and dysregulate rna-based processes to suppress the interferon response j o u r n a l p r e -p r o o f ); office of the vice president for research at uvm m.g. is a nyscf-robertson investigator signal recognition particle: an essential protein-targeting machine a user's guide for phase separation assays with purified proteins recognition of doublestranded rna and activation of nf-κb by toll-like receptor visualizing late states of human s ribosomal subunit maturation the proximal origin of sars-cov- sars-cov- (covid- ) by the numbers innate immune evasion strategies of dna and rna viruses the structure of the eukaryotic ribosome at . Å resolution the structure of the eukaryotic ribosome at . Å resolution the protein data bank mutagenesis of the murine hepatitis virus nsp -coding region identifies residues important for protein processing, viral rna synthesis, and viral replication structures of translationally inactive mammalian ribosomes. elife mers-cov b protein interferes with the nf-κb-dependent innate immune response during infection mechanism of ' splice site transfer for human spliceosome activation genomics functional analysis and drug screening of sars-cov- rig-i and other rna sensors in antiviral immunity a new group of conserved coactivators that increase the specificity of ap- transcription factors cop signalosome: a multifunctional regulator of scf and other cullin-based ubiquitin ligases crystal structure and functional analysis of the sars-coronavirus rna cap ′-o-methyltransferase nsp /nsp complex pymol molecular graphics system the coding capacity of sars-cov- a common strategy for host rna degradation by divergent viruses minireview recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission chromatin signature reveals over a thousand highly conserved large non-coding rnas in mammals ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincrnas impaired type i interferon activity and inflammatory responses in severe covid- patients mhc class i antigen presentation: learning from viral evasion strategies small molecules targeting viral rna dynamic switch of the signal recognition particle from scanning to targeting analysis and optimization of coppercatalyzed azide-alkyne cycloaddition for bioconjugation notch regulates maturation of cd + and cd + thymocytes by modulating tcr signal strength notch regulates maturation of cd + and cd + thymocytes by modulating tcr signal strength exploring rna transcription and turnover in vivo by using click chemistry sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion a twopronged strategy to suppress host protein synthesis by sars coronavirus nsp protein differential roles of mda and rig-i helicases in the recognition of rna viruses poliovirus apro increases viral mrna and polysome stability coordinately in time with cleavage of eif g a flexible genome-scale resource of sars-cov- coding sequence clones the architecture of sars-cov- transcriptome inducible transgene expression in human ips cells using versatile all-in-one piggybac transposons multiple anti-interferon actions of the influenza a virus ns protein iclip reveals the function of hnrnp particles in splicing at individual nucleotide resolution induced structural changes of sl rna during the assembly of human signal recognition particle quality and quantity control of gene expression by nonsense-mediated mrna decay cellular response to small molecules that selectively stall protein synthesis by the ribosome the molecular signatures database hallmark gene set collection translational control by viral proteinases middle east respiratory syndrome coronavirus nsp inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin type i interferon susceptibility distinguishes sars-cov- from sars-cov halotag: a novel protein labeling technology for cell imaging and protein analysis down-regulation of jab , hif- Α, and vegf by moloney murine leukemia virus-ts infection: a possible cause of neurodegeneration extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity programmed delayed splicing: a mechanism for timed inflammatory gene expression widespread shortening of ′utrs by alternative cleavage and polyadenylation activates oncogenes in cancer cells human cytomegalovirus inhibits major histocompatibility complex class ii expression by disruption of the jak/stat pathway inhibition of stress granule formation by middle east respiratory syndrome coronavirus a accessory protein facilitates viral translation, leading to efficient virus replication severe acute respiratory syndrome coronavirus nsp suppresses host gene expression, including that of type i interferon, in infected cells coronavirus nonstructural protein : common and distinct functions in the regulation of host and viral gene expression approximations for distributions of scan statistics robust transcriptome-wide discovery of rna-binding protein binding sites with enhanced clip (eclip) recognition of cognate transfer rna by the s ribosomal subunit jab mediates cytoplasmic localization and degradation of west nile virus capsid protein the expansion segments of s ribosomal rna extensively match human messenger rnas integrative genomics viewer appris: annotation of principal and alternative splice isoforms sunset, a nonradioactive method to monitor protein synthesis a u snrna:pre-mrna base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the ' cleavage site role of nonstructural proteins in the pathogenesis of sars-cov- structural insights into the mammalian late-stage initiation complexes rna-rna and rna-protein interactions in coronavirus replication and transcription type i interferons in host defense the nsp replicase protein of sars-coronavirus severe acute respiratory syndrome coronavirus nsp facilitates efficient propagation in cells through a specific translational shutoff of host mrna the hepatitis b virus x protein enhances ap- activation through interaction with jab structural basis for translational shutdown and immune evasion by the nsp protein of sars-cov- signal peptide-binding drug as a selective inhibitor of co-translational protein translocation structures of the scanning and engaged states of the mammalian srp-ribosome complex the genetic sequence, origin, and diagnosis of sars-cov- comparative protein structure modeling using modeller the jnk signal transduction pathway metap-like ebp occupies the human ribosomal tunnel exit and recruits flexible rrna expansion segments recognition of viruses by cytoplasmic sensors improved protein structure prediction using predicted interresidue orientations influenza virus ns protein-rna interactome reveals intron targeting viral and host factors related to the clinical outcome of covid- structural basis for interaction of a cotranslational chaperone with the eukaryotic ribosome interferon-α b treatment for covid- sars-cov- viral load in upper respiratory specimens of infected patients we thank fritz roth for clones; marko jovanovic, bil clemons, shu-ou shan, and jamie key: cord- -h q m ed authors: carnero, elena; barriocanal, marina; segura, victor; guruceaga, elizabeth; prior, celia; börner, kathleen; grimm, dirk; fortes, puri title: type i interferon regulates the expression of long non-coding rnas date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: h q m ed interferons (ifns) are key players in the antiviral response. ifn sensing by the cell activates transcription of ifn-stimulated genes (isgs) able to induce an antiviral state by affecting viral replication and release. ifn also induces the expression of isgs that function as negative regulators to limit the strength and duration of ifn response. the isgs identified so far belong to coding genes. however, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding rnas (lncrnas). to address whether ifn can also regulate the expression of lncrnas, we analyzed the transcriptome of huh cells treated or not with ifnα by expression arrays. analysis of the arrays showed increased levels of several well-characterized coding genes that respond to ifn both at early or late times. furthermore, we identified several ifn-stimulated or -downregulated lncrnas (isrs and idrs). further validation showed that isr , , and expression mimics that of their neighboring genes gbp , irf , and il , respectively, all related to the ifn response. these genes are induced in response to different doses of ifnα in different cell lines at early (isr or ) or later (isr ) time points. ifnβ also induced the expression of these lncrnas. isr and were also induced by an influenza virus unable to block the ifn response but not by other wild-type lytic viruses tested. surprisingly, both isr and were significantly upregulated in cultured cells and livers from patients infected with hcv. increased levels of isr were also detected in patients chronically infected with hiv. this is relevant as genome-wide guilt-by-association studies predict that isr , , and may function in viral processes, in the ifn pathway and the antiviral response. therefore, we propose that these lncrnas could be induced by ifn to function as positive or negative regulators of the antiviral response. interferons (ifns) are key players in the antiviral response. ifn sensing by the cell activates transcription of ifn-stimulated genes (isgs) able to induce an antiviral state by affecting viral replication and release. ifn also induces the expression of isgs that function as negative regulators to limit the strength and duration of ifn response. the isgs identified so far belong to coding genes. however, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding rnas (lncrnas). to address whether ifn can also regulate the expression of lncrnas, we analyzed the transcriptome of huh cells treated or not with ifnα by expression arrays. analysis of the arrays showed increased levels of several wellcharacterized coding genes that respond to ifn both at early or late times. furthermore, we identified several ifn-stimulated or -downregulated lncrnas (isrs and idrs). further validation showed that isr , , and expression mimics that of their neighboring genes gbp , irf , and il , respectively, all related to the ifn response.these genes are induced in response to different doses of ifnα in different cell lines at early (isr or ) or later (isr ) time points. ifnβ also induced the expression of these lncrnas. isr and were also induced by an influenza virus unable to block the ifn response but not by other wildtype lytic viruses tested. surprisingly, both isr and were significantly upregulated in cultured cells and livers from patients infected with hcv. increased levels of isr were also detected in patients chronically infected with hiv. this is relevant as genome-wide guiltby-association studies predict that isr , , and may function in viral processes, in the ifn pathway and the antiviral response. therefore, we propose that these lncrnas could be induced by ifn to function as positive or negative regulators of the antiviral response. transcriptome analysis by tiling arrays and rna sequencing has led to the conclusion that while - % of the genome is transcribed, only % is dedicated to the transcription of proteincoding sequences ( , ) . among the non-coding transcriptome, there is a group of poorly studied transcripts longer than nt and with low coding potential that have been collectively called long non-coding rnas (lncrnas) ( , ) . it has been estimated that there could be as many lncrna genes as coding genes, but the number of lncrnas is still growing and some authors consider that it could increase to up to~ ( , ) . therefore, there is a great need to identify novel lncrnas and to understand their function and regulation. long non-coding rnas genes are very similar to coding genes at the chromatin, dna, and rna level ( ) . compared to mrnas, most lncrnas are more cell-type specific, less expressed, and less conserved at the nucleotide sequence level ( ) . many lncr-nas have been shown to be functional. some lncrnas function to regulate the expression of neighboring or antisense genes by transcriptional interference, by recruitment of chromatin modifiers and remodelers, or by regulation of imprinting, editing, splicing or translation, and stability ( ) ( ) ( ) ( ) ( ) . enhancer rnas (ernas) and lncrna-activating rnas (lncrna-a) are transcripts that control the expression of neighboring genes in "cis" ( ) ( ) ( ) . however, lncrnas can also function in "trans," away from their site of synthesis. for instance, some pseudogenes regulate the expression of their parental gene, located in a distant genomic location ( ) ( ) ( ) ( ) . lncrnas have especially emerged as regulators of development, pluripotency, and proliferation as some function as oncogenes or tumor suppressors ( , , ( ) ( ) ( ) ( ) . therefore, several lncrnas have been implicated in cancer and in other human diseases ( ) ( ) ( ) . proliferation, differentiation, and pluripotency factors regulate the expression of some lncrnas ( ) . besides, several signaling molecules, including those involved in the immune response, have been shown to induce the expression of specific lncrnas ( ) ( ) ( ) ( ) . induction of tlr , tlr , or tlr leads to the activation of lncr-nas, including lncrna-cox , which regulates the expression of several immune genes or neat , which functions to increase the expression of some antiviral genes such as il ( ) ( ) ( ) . downregulation of il β-erna and il β-rbt lncrnas decreases il β and the accumulation of lps-induced rnas ( ) . similarly, downregulation of lnc-il r decreases the lps-induced inflammatory response ( ) . treatment of thp macrophages with an innate immunity activator also induces the expression of several lncrnas. one of them, linc (or thril) activates the expression of tnfα and other genes involved in the immune response ( ) . in turn, tnfα also induces many lncrnas in fibroblasts, including lethe, a pseudogene that responds to nfκb and inhibits nfκb dna-binding activity leading to reduced inflammation ( ) . besides, dendritic cells (dcs), cd +, and cd + t-cells express a specific set of lncrnas that may regulate cell activation and differentiation ( , , ) . nest lncrna controls the ifnγ locus in cd + t-cells causing decreased salmonella enterica pathogenesis ( , ) . downregulation of lnc-dc, expressed in conventional dcs, impairs dc differentiation from monocytes, and reduces the capacity of dcs to activate t-cells ( ) . lncr-nas also respond to viral infections. infection with enterovirus, influenza virus, hiv, hepatitis b, and c (hcv) viruses as well as the sars coronavirus leads to altered levels of lncrnas ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (carnero et al., in prep) . from the collection of infectionaltered lncrnas, it is difficult to distinguish those that respond to the virus from those that respond to the cellular antiviral pathways activated by the infection. recently, some lncrnas regulated by infection have also been found to be regulated by ifnα in mice ( ) . interferon is a key molecule in the cellular antiviral response ( ) . detection of pathogens by the cell triggers transcription of the interferon (ifn) genes. when type i/iii ifn is released, it is sensed by the ifn receptors, which induce the jak/stat pathway. stat and coupled to irf form a complex that binds ifnstimulated response elements (isre) in the promoters of ifnstimulated genes (isgs) and activates their transcription. isgs induce an antiviral state by several means, including inhibition of viral replication, transcription, and translation. well-characterized isgs are mx , oas, gbp , but also stat and irf , which amplify the ifn response. further, stat induces the expression of proinflammatory genes such as irf , a transcription factor that also activates isgs, and whose induction is dependent on de novo protein synthesis ( , ) . besides, ifn also induces the expression of negative regulators that limit the strength and duration of the ifn response ( ) ( ) ( ) . finally, ifn activates expression of several mirnas that contribute to the antiviral state or to the control of the ifn response ( ) . here, we have postulated that ifn could also regulate the expression of lncrnas that may have key roles in the antiviral response. therefore, we have performed a high-throughput analysis of lncrnas whose expression is deregulated in response to ifnα. the conditions we used aimed to identify genes controlled by the ifn pathway directly or by other isgs. the results show that several lncrnas are controlled in response to type i ifn in several cell lines tested. the best candidates are lncrna genes upregulated in response to ifn that are found in the genome adjacent to ifn-related coding genes. they have been called isr , , and . interestingly, guilt-by-association genome-wide studies predict that the function of these lncrnas is related to the cellular antiviral response and to viral infections. in fact, isr , , and their neighboring genes are also increased after infection of cultured cells with hcv. a similar increase is detected in the livers of patients infected with this virus or, in the case of isr , in blood cells of patients infected with hiv. huh cells, derived from a human hepatocarcinoma, were provided by dr. chisari's lab (scripps research institute, la jolla, ca, usa). a and thp cells were kindly provided by estanislao nistal (cima, university of navarra, spain), and hela and cells were obtained from atcc. liver samples from patients with or without hcv infection were obtained from the biobank of the university of navarra under approval from the ethics and scientific committees. liver tissue sections were snap frozen and stored at − °c. the clinical data from hcv and hiv-infected subjects are shown in table s and s in supplementary material. cells were grown in dulbecco's modified eagle medium (dmem) enriched with % fetal bovine serum (fbs) and % penicillinstreptavidin in a % co atmosphere. twenty-four hours before treatment with ifn, huh , a , thp , , or hela cells were seeded in six-well plates. then, , , , , , or u/ml of ifnα (sicor biotech) or ifnβ (pbl pestka biomedical laboratories) were used in a final volume of ml. huh cells were also treated with ng/ml il b/ifn-λ (r&d systems) in a final volume of ml. cells were harvested for rna extraction , , , , and/or h after treatment. hcv jfh- was obtained from an initial viral stock from the genotype a jfh- plasmid (pjfh- ) previously described by wakita et al. ( ) . to amplify the virus, huh cells were infected at low multiplicity of infection (moi) with the initial viral stock and supernatants from the cells were harvested at different days postinfection. the presence of virus was evaluated by infecting fresh cells with the supernatant and checking infected cells by immunofluorescence against the hcv core protein. the supernatants with higher titers were selected to perform the experiments. influenza virus strain a/pr / wt (pr ) and the mutant lacking ns (∆ns ) were kindly provided by estanislao nistal (cima, university of navarra, spain) ( ), semliki forest virus (sfv) was a gift from cristian smerdou (cima, university of navarra, spain), and adenovirus serotype (ad ) was amplified as described ( ) . twenty-four hours before infection, cells were seeded in six-well plates in a final volume of ml. cells were infected with hcv at a moi of . , and with a moi of of influenza a, ∆ns , ad , and sfv. in the case of the lytic viruses, we used a moi of as this led to cytopathic effects at h (for influenza and sfv) or h (for ad ) in huh cells. infection with hcv was performed for h, versus h in the case of ad and h in the case of the other viruses. a final volume of ml was used for infection. after infection, the virus was removed and fresh medium was added to the cells. cells were harvested for rna extraction at the indicated times post-infection. two million huh cells were incubated in µl of cytoplasmic buffer ( mm tris hcl ph . , mm edta, and % np ) for min at °c. then, cells were centrifuged for min at g and the supernatant was used to isolate cytoplasmic rna. the pellet was washed with cytoplasmic buffer and centrifuged as before. the supernatant was discarded and the pellet was used to isolate the nuclear rna. rna from nuclear and cytoplasmic fractions was isolated with maxwell research system (promega). total rna from tissue samples was extracted in ml trizol (sigma-aldrich) using the ultra-turrax homogenizer (t basic ika-werke) ( ) . then, µl chloroform was added and the samples were mixed vigorously and then centrifuged at g for min at °c. the aqueous phase was mixed with µl isopropanol and centrifuged at g for min at °c. the pellet of total rna obtained from the centrifugation was washed with % ethanol. finally, the pellet was resuspended in µl dnase/rnase-free pcr water (bioline). dnase i (fermentas) treatment was performed to eliminate dna from the samples before the reverse transcription (rt)-pcr reactions. to isolate rnas from total blood, . ml blood were collected into paxgene blood rna tubes with rna stabilization solution. total rna was extracted by using the paxgene blood rna kit (qiagen gmbh) according to the manufacturer's instructions. briefly, prior to the actual rna isolation, the frozen samples were first incubated at rt for at least h to achieve complete lysis of blood cells. then, the paxgene blood rna tubes were centrifuged for min at g, the supernatant was removed and the pellet was washed with ml rnase-free water. the pellet was then dissolved in µl of the provided lysis buffer bm and transferred into a . ml microcentrifuge tube. to this mixture, µl buffer bm and µl proteinase k were added and incubated for min at °c in a shaking incubator at rpm. the samples were next transferred to a paxgene shredder spin column and centrifuged for min at full speed. the flow-through was transferred into a new tube without disturbing the pellet and mixed with µl isopropanol ( %, purity grade p.a.). this mixture was then passed through a paxgene rna spin column by centrifugation for min at g. following a wash step ( µl bm ), an on-column dnase digest (rnase-free dnase set, qiagen) was performed by addition of dnase i and incubation on the benchtop ( - °c) for min. the column was washed three times, before rna was eluted with µl buffer br . the final eluate was incubated for min at °c, and several aliquots of the rna were stored at − °c. rna extraction from cells or cellular fractions was performed using the maxwell research system from promega according to the manufacturer's recommendations. for each condition, a minimum of a confluent well of a m -plate was used in order to obtain enough rna. in all cases, the rna concentration was measured using a nanodrop spectrophotometer. the quality of the rna was determined in a bioanalyzer (agilent technologies). for microarray hybridization, the samples were processed using manufacturer protocols and hybridized to the agilent sureprint g human gene expression × k microarray. transcriptome data are available at the ncbi gene expression omnibus (geo) data repository . reverse transcription was performed using . µl m-mlv-rt and µl m-mlv-rt × buffer (promega), µl mm dntps, µl random primers at ng/µl, µl dtt . m, and µg rna in a final volume of µl. the reaction was run in the c touch thermal cycler from bio-rad. the samples were incubated at °c for min, then at °c for s, and next immediately placed at °c. quantitative polymerase chain reaction was performed in the cfx real-time system from bio-rad. for the reaction, µl iq syber green mix from bio-rad, . µl of each primer at µm, and µl of the dna sample at . µg/µl were mixed in a final volume of µl. the mixture was first incubated at °c for min, and then at °c for s, °c for s, and °c for s for cycles. the pcr ended after min at °c and min at °c. the results were analyzed with bio-rad cfx-manager software. gapdh levels were evaluated in all cases as a reference. only the samples with similar gapdh amplification were analyzed further. the primers used are listed in table s in supplementary material and were designed using the primer program . initial setups included gc percentage between and %, product size from to bp and primer length between and nt. microarray data normalization was performed using the quantile algorithm. after quality assessment, a filtering process was carried out to eliminate low expression probe sets. applying the criterion of an expression value > in the three samples of at least one of the experimental conditions, probe sets were selected for statistical analysis. limma (linear models for microarray data) ( ) was used to identify the probe sets with significant differential expression between experimental conditions. genes were selected as significant using a b statistic cut-off of b > . . data processing and statistical analyses were performed with r and bioconductor ( ) . functional enrichment analysis of gene ontology (go) categories was carried out using standard hypergeometric test ( ) . the biological knowledge extraction was complemented through the use of ingenuity pathway analysis (ingenuity systems) , whose database includes manually curated and fully traceable data derived from literature sources. all the differentially expressed sequences obtained by the analysis were compared to the ensembl and encode databases and searched for in the genome browser from ucsc for more information ( , ) . orf finder (ncbi) was used to evaluate the length of all probable orfs in isr , , and . coding potential was assayed with the coding potential assessment tool (cpat) ( , ) and by searching the lncipedia database ( ) for the presence of our candidates in the pride archive ( ) or in lists of transcripts associated with ribosomes ( , ) . phylogenetic codon substitution frequencies (phylocsf) was also used to predict the coding potential of isr , , and ( ) . a guilt-by-association approach was used to predict the go categories ( ) in which the differentially expressed lncrnas could be implicated. first, we collected data from samples hybridized to sureprint g microarrays. these samples include the rnas isolated from huh cells treated or not with ifn, and rnas obtained from human samples of different origin, including healthy tissues and several leukemias and other tumors. then, a pearson correlation analysis was performed between isr , , and and all the genes represented in the sureprint g human microarray. coding genes related to cellular antiviral pathways were randomly selected and included in the analysis as positive controls. the obtained correlation matrix was used as input for gitools ( ) where an enrichment analysis of go categories was performed using z -score ( ) and fdr ( ) . statistical analysis of the expression levels obtained by quantitative rt-pcr (qrt-pcr) was performed using graphpath. statistical significance of treated or infected versus nontreated or non-infected samples was calculated using a two-tailed non-parametric mann-whitney t -test. in correlation studies, a two-tailed non-parametric spearman analysis was used. similar results were obtained by pearson correlation. p values lower than . were deemed as significant. we wanted to identify lncrnas that respond to ifn. ifn induces the expression of isgs very fast. some isgs are transcription factors able to regulate the expression of genes with antiviral potential in a secondary wave of ifn response. other isgs are inhibitory factors that function to decrease the response. therefore, to identify lncrnas regulated by ifn or by isgs, one should analyze the transcriptome of cells treated by ifn at different time points. however, to simplify the analysis, we decided to check first whether we could find conditions showing a wide ifn response at a single time point. to this aim, we tested whether high doses of ifn could lead to increased expression of well-known isgs even at late times post-ifn treatment. huh cells were treated for , , , or h with increasing doses of ifnα up to , units/ml, and the expression levels of gbp , irf , bst , oas, il , and isg were evaluated by qrt-pcr (figure ) . the results show that gbp and irf are induced to highest levels at h post-treatment, while bst and oas are induced to similar levels at all times tested. in contrast, il is only significantly induced at days post-treatment (figure and data not shown). however, compared to untreated cells, a significant upregulation of all the genes, including gbp and irf , can be detected at days post-treatment with , units/ml of ifnα ( figure b) . in fact, this dose induced the highest levels of these transcripts at most of the time points. before analyzing the transcriptome of cells treated with , units/ml of ifnα for days, we confirmed that these conditions induced antiviral effects. when huh cells infected with hcv were treated with these conditions, we indeed detected a drastic decrease in viral protein expression by immunofluorescence and a decrease in the levels of hcv viral genomes by qrt-pcr (data not shown). an agilent array that evaluates expression of entrez genes and lncrnas was used to hybridize rna isolated in three independent experiments from control cells or huh cultures treated with , units/ml of ifnα for days. analysis of the array showed that genes upregulated with a high statistical significance (b > ) includes well-known ifn-related genes from the gbp, ifi, oas, isg, mx, or irf families (figure a) . analysis using less stringent criteria (b > . ) showed that % of the genes were upregulated in response to ifn treatment ( figure b) . ingenuity analysis of this set indicated that ifn signaling is the pathway with the highest enrichment followed by other antiviral responses ( figure c) . similarly, the ifn-induced stat pathway and the tlr/irf network are well represented in the set of upregulated genes ( figure s in supplementary material). we selected the probes described as long intergenic non-coding rnas (lincrnas) that showed a significantly altered expression by the ifn treatment (b > . ). first, we determined the position in the genome of the sequences from these probes using the blat searches available at ucsc ( , ) . many sequences corresponded to coding genes or seemed to be utr extensions of coding genes and were discarded for further analysis. the remaining frontiers in immunology | molecular innate immunity www.frontiersin.org sequences corresponded to genes, including genes annotated as lncrnas and located in non-annotated areas of the genome ( figure d , table s in supplementary material) . surprisingly, while % of coding genes were upregulated by ifn, only % of the lncrnas were upregulated. most of the downregulated lncr-nas corresponded to the non-annotated category. this suggests that there could be a relevant ifn-mediated repression of genes that more strongly affects lncrnas. we named these genes idrs, for ifn-downregulated rnas, and accordingly named the ifnstimulated rnas isrs. the fact that almost % of the isrs and idrs correspond to non-annotated areas of the genome suggests that the percentage of the genome able to react to different stimuli could be even larger than expected. as many lncrnas have been described to regulate the expression of neighboring genes, we looked for the closest coding gene for each isr or idr (table s in supplementary material). we considered candidates to have no neighbor when the closest coding gene was not within a distance of kb from the start or the end of the candidate or when the closest gene was non-coding. forty candidates had neighboring coding genes according to these criteria. half of the coding-non-coding pairs were in tandem, convergent, or divergent, were antisense to each other, were overlapping, and pairs seem to share the same promoter according to the dnase i hypersensitivity and the histone marks described by encode for the respective area. therefore, these couples of coding-non-coding genes could be coregulated. three upregulated candidates, isr , isr , and isr were neighbors of the ifn-related genes gbp , irf , and il , respectively. we next wanted to validate the ifn effect on these candidates in independent samples using a different technique. furthermore, we wanted to determine whether the levels of isrs and idrs were altered early after ifn treatment. therefore, the expression levels of isrs and idrs were evaluated by qrt-pcr in huh cells treated with or , units/ml of ifnα for , , , , or h. the fold-change observed for each candidate at each time point is shown in figure and table s in supplementary material. at h post-treatment, isrs and idrs showed a fold-change higher or lower, respectively, than . . only isr and were not significantly upregulated, or idr , , , , and were not significantly downregulated, at any time tested. however, the foldchange was relatively low in most of the cases, indicating a weak response to ifn. moreover, % of the candidates showed low overall expression levels ( table s in supplementary material and data not shown). interestingly, isr and isr were induced more than -fold at h post-ifn treatment, and isr was induced more than -fold at later times. therefore, we decided to study these candidates further. we also opted to focus on isr and , as they were upregulated at later time points. idr and idr , which were downregulated at most times studied, were also analyzed further. as many lncrnas are cell-specific, we decided to study the response to ifn of these selected isrs/idrs in different cell lines. hela, , a , or thp cells were treated with or , units/ml of ifnα for , , , , or h and rna was isolated and used to evaluate the expression of isr , , , , and as well as idr and . the results showed that in the new cell lines tested, expression of isr and was not detected and idr and showed only a mild downregulation in response to ifn (data not shown). importantly, isr , , and were well expressed in all cell lines tested and their expression was strongly induced by ifn at early (isr and ) or late times (isr ) (figure a and data not shown). interestingly, isr , , and have neighboring genes related to ifn response (figure b ). isr is located in tandem with gbp , at the end of the cluster of gbp genes formed by gbp , , , , , , and . in fact, isr is gbp p , a pseudogene of gbp . this may be interesting as some pseudogenes have been described to regulate the expression of their parental genes ( ) ( ) ( ) ( ) . isr is located in tandem with il and isr is convergent with irf . therefore, we decided to evaluate the expression of gbp , irf , and il in response to ifn in the same cell lines. the results showed a similar induction pattern of each isr and of the corresponding neighboring coding gene in response to ifn (figures a,c) . note that in the case of isr , we evaluated the expression of its parental neighboring gene gbp instead of the closest neighbor gbp , as we speculated that there could be a co-regulation of the parental gene and the pseudogene. furthermore, we could not detect expression of gbp in control or ifnα-treated huh cells (data not shown). all the experiments performed so far with these lncrnas have studied the response to high doses of ifn. to determine whether isr , , and also respond to lower doses, their expression level was evaluated in huh cells treated for , , , or h with , , , , or , units/ml of ifnα (figure ) . the results show that induction of these lncrnas is similar to that observed for their corresponding neighboring coding genes (compare figure with figure ). similar results were observed when ifnβ was used instead of ifnα (data not shown). further, we evaluated whether expression of these transcripts was induced after treatment with tnfα. tnfα, similarly to some pathogenassociated molecular patterns, induces nfκβ signaling and expression of pro-inflammatory genes. however, the nfκβ pathway is a poor inducer of isgs. a treatment of huh cells with ng/ml of tnfα for h, induced the expression of cxcl , used as a positive control, more than -fold. a significant increase in expression of only - -fold was observed after tnfα treatment for gbp , irf , il , and isr , but not for isr . similar results were obtained in huh cells treated with lps or polyi:c (data not shown). to obtain more information about these isrs, we looked in detail at the data from encode. even though we did not observe a strong activation after tnfα treatment, transcription factor chip seq from encode showed that isr and isr promoters have nfκb binding sites. interestingly, the isr promoter has sites for stat and as well as irf and , suggesting that isr could be a bona fide isg. besides the non-coding transcript that we name isr , six other transcripts could be expressed from the isr area according to ucsc and ensembl databases ( figure s a in supplementary material). these transcripts have certain coding capacities and could be translated to proteins of up to amino acids named c orf . the syntenic region in the mouse also comprises non-coding transcripts together with transcripts with certain coding potential that lead to peptides no longer than amino acids (data not shown). only five amino acids in the n-terminal part of the putative protein predicted in mouse are conserved in human. given the poor conservation and the short size of the predicted peptides all the transcripts from this mouse region could be classified as non-coding ( , ) . to analyze the expression of isr and all the putative coding transcripts annotated in the human isr area, we used qrt-pcr. the results show that most of the transcripts are poorly detected in huh or hela cells treated or not with ifn. the highest expression is observed for the isr lncrna ( figure s b in supplementary material). finally, ucsc database also shows that isr is convergent to irf and antisense to a longer irf transcript with poor coding capacity that we named lncirf (figure b , figure s a in supplementary material). if expressed, lncirf could regulate the level of isr via antisense mechanisms. therefore, we have evaluated the expression of lncirf in response to ifn. the results show that lncirf is expressed and its levels are induced at short times after ifn treatment ( figure s c in supplementary material). unlike isr , isr , or isr transcripts did not overlap with annotated transcripts from gbp or il ( figure s in supplementary material). we evaluated the coding capacity of isr , , and bioinformatically. orf finder (ncbi) was used to determine all possible open reading frames in these isrs ( figure s a in supplementary material). the analysis shows that all putative orfs are shorter than aa. then, we evaluated their coding potential with the cpat ( , ) ( figure s b in supplementary material). cpat uses a model built with open reading frame size and coverage together with codon (ficket score) and hexamer (hexamer score) usage bias. according to this program, isr , , and are non-coding as they have a coding probability much lower than . , used as a threshold with the highest sensitivity and specificity to differentiate between coding and non-coding transcripts in humans ( ) . isr , , and were also described as non-coding in lncipedia ( ) . this lncrna database shows that isr , , or are not found in the pride archive, a database for proteomic data, or in lists of transcripts associated to ribosomes in ribosome profiling experiments ( ) ( ) ( ) . isr and isr were also described as non-coding by the analysis of phylocsf, which uses multiple alignments to calculate www.frontiersin.org the phylogenetic conservation score and determines whether a multi-species nucleotide sequence alignment is likely to represent a protein-coding region ( ) . finally, we evaluated the subcellular localization of isr , , and in huh cells mock-treated or treated with , units/ml of ifnα. rna was isolated from nuclear or cytoplasmic fractions and quantified by qrt-pcr. the results show that the coding gapdh or isg mrnas accumulate preferentially in the cytoplasm while the nuclear lncrna malat is preferentially nuclear (figure ) . similarly, isr , , and accumulate preferentially in the nucleus. this result, together with the bioinformatic analyses, strongly suggests that isr , , and are non-coding rnas. as indicated above, each isr and its neighboring coding gene have similar induction patterns in response to ifn (compare figure and figure or figures a,c) . this suggests that they could be co-regulated and therefore, that they could share similar functions. to analyze in more detail whether the expression level of each isr correlates significantly with the expression level of its neighboring coding gene, we performed correlation studies. we compared the levels of each coding/non-coding pair in all the samples evaluated in figures , , and . the results show a highly significant positive correlation between isr and gbp or isr and irf . in contrast, isr had a non-significant correlation with il ( figure a) . expression of neither isr nor isr significantly huh cells were mock-treated or treated with , units/ml of ifnα and divided into nuclear and cytoplasmic fractions. rna was isolated from each fraction and used to evaluate the expression levels of isr , , and by qrt-pcr. malat , gapdh, and isg mrna was also quantified and used as a reference to calculate the relative levels of each transcript and as a control to evaluate the subcellular fractionation. the ratio of cytoplasmic to nuclear levels is shown. the experiment was performed three times and each value shows the average of three replicas from a representative experiment. error bars indicate standard deviations. correlated with the expression of other isgs such as oas or bst (data not shown). arguably, this correlation analysis has been done with few isgs and using homogeneous samples. therefore, we decided to perform a more stringent high-throughput analysis of correlation. accordingly, we carried out a guilt-by-association genome-wide frontiers in immunology | molecular innate immunity analysis ( ) , which also predicts the function of unknown genes with high statistical confidence ( figure b) . we compared the expression levels of isr , , and and coding genes related to cellular antiviral pathways, used as positive controls, with the expression levels of all the genes represented in a sureprint g microarray. we used data obtained from microarray experiments performed with human samples of different origin. the results show a significant positive correlation between isr and irf (corr = . and p < e− . ), indicating that these genes are coregulated. significant correlations were not observed for gbp and isr or il and isr . furthermore, the correlation analysis of each candidate organized all the microarray genes from the ones with the highest positive correlation to the ones with the highest negative correlation. this matrix was used to search for go categories with highly significant enrichment in genes that correlate positively (positive z-score) o negatively (negative z-score) with isr , , or . this analysis revealed that isr , , and clustered very closely but away from other genes related with the ifn pathway and the antiviral response. isr , , and showed a negative correlation with genes that significantly enriched go categories related to viral processes including viral life cycle and viral transcription. isr and also shared a negative correlation with response to viral infection and ifn pathway genes. however, isr , similar to irf and tlr , showed a positive correlation with ifn signaling and immune response genes. the results obtained in the guilt-by-association analysis led us to hypothesize that isr , , and could respond strongly to viral infections or to the ifn response induced by viral infections. to study this hypothesis, we evaluated the expression of these lncr-nas in cells infected with ad , a dna virus, or rna viruses such as influenza virus, sfv, or hcv. all of them have developed mechanisms to block the cellular antiviral response. influenza virus control of ifn is exerted primarily by the influenza ns protein ( ) . therefore, we also infected cells with an influenza virus mutant that lacks ns . all the viruses used, with the exception of hcv, lead to a fast lytic infection that initiates cell death at h post-infection in the case of influenza virus and sfv, or at h post-infection in the case of ad . therefore, several time points post-infection were evaluated in each case. the results show that isr expression was not altered by infection (data not shown). isr and isr www.frontiersin.org expression was only induced in cells infected with the influenza virus unable to control ifn at later times post-infection, when the ifn response is strongest (figure ) . in general, the induction pattern was similar for gbp and irf . we were surprised to see that the strongest increase in isr and was observed in cells infected with hcv, an ifn-sensitive virus that employs several viral proteins to block the ifn pathway. increased expression was also observed for gbp and irf but not for other isgs such as oas (figure and data not shown) . to determine whether a similar upregulation could be observed in hcv patients, levels of isr , , and were evaluated in livers from hcv-negative (n = ) to hcv-positive (n = ) patients. the results show that both isr and are significantly upregulated in hcv patients (figure a) . no differences were observed in the levels of isr in the same samples. finally, we wanted to determine whether these lncrnas also respond to other chronic viral infections relevant for human health. therefore, we evaluated the expression of isr , , and in blood cells isolated from healthy patients or from patients chronically infected with hiv. we could not detect expression of isr or in these samples. however, both isr and gbp were significantly upregulated in hiv-infected patient cells ( figure b ). in this work, we show that ifn treatment alters the expression of several lncrnas in human cells. these lncrnas were identified in a high-throughput analysis using conditions that detect increased levels of coding genes that respond to ifn both at early or late times (figures and and figure s in supplementary material). in the cells treated with ifn for days, we found, with a very high statistical significance (b > ), an upregulation of well-characterized isgs such as mx , stat , irf , isg , bst and several members of the gbp, oas, and ifi families (figure ) . besides, the ifninduced stat pathway shows the highest enrichment by ingenuity analysis ( figure s in supplementary material). this suggests that high levels of ifn could maintain an active jak/stat pathway in huh cells even at late times post-ifn treatment. therefore, we feel that among the lncrnas identified in this work, there may be some candidates whose expression is controlled directly by the jak/stat pathway, while other candidates could be controlled by other isgs or by later downstream effectors of the ifn response. comparison of the results obtained in the array between coding and lncrna genes gave an unexpected result: % of the altered coding genes but only % of the lncrnas were upregulated by ifn. this suggests that there could be an ifn-mediated repression of genes that affects more strongly lncrnas. however, even if idrs showed a generally decreased expression in the presence of ifn (figure b) , the downregulation was mild. validation of downregulated genes showed that only idr was strongly affected by ifn at late times post-treatment (table s in supplementary material). further experiments will be required to determine whether there is a relevant downregulation of lncrna genes in response to ifn. notably, the majority of the idrs match with genomic regions that have not been associated with active transcription in public databases. this is surprising, as some of them such as idr , , , or are expressed at high levels in huh cells according to huh cells were mock-treated or infected with wild-type influenza virus (pr ) or a mutant that lacks ns (∆ns ), sfv, ad , or hcv for the indicated times. rna was isolated and the expression levels of isr , gbp , isr , and irf were evaluated by qrt-pcr. gapdh expression was also evaluated and used as a reference to calculate the relative levels of each transcript. the experiment was performed three times. the fold-change of infected versus non-infected cells is indicated. each value shows the average of three replicas from a representative experiment. error bars indicate standard deviations. the fold-change of treated versus non-treated cells is indicated at the top of each bar when higher than . . the qrt-pcr data (table s in supplementary material). therefore, it would be interesting to analyze ifn regulation of lncrnas using rnaseq, as this may yield a more comprehensive picture frontiers in immunology | molecular innate immunity of the lncrna transcriptome and its manipulation by ifn. in fact, during the revision of this paper, another manuscript was accepted showing that transcriptome analysis by rnaseq allowed the identification of several lncrnas whose expression is altered in response to ifn ( ) . similar results have been obtained using rnaseq in our lab (barriocanal et al., submitted). several isrs were clearly validated by qrt-pcr. specifically, isr , , , , and were upregulated more than fivefold, and isr and isr even more than -fold. isr was expressed at very low levels. in contrast, isr , , and were well expressed in all cell lines tested and their expression was strongly induced by ifn at early (isr and ) or late times (isr ) (figure a) . furthermore, low doses of type i ifnα or ifnβ also induced the expression of these isrs (figure and data not shown). we did not detect significant induction of these isrs, gbp , irf , or il with type iii ifnλ using doses able to induce other isgs such as oas, isg , or bst (data not shown). further experiments are required to determine whether these genes could show some specificity for type i ifn, as the repertoire of genes that are induced by type iii ifns is essentially the same as those induced by type i ifns ( ) . finally, isr was also induced after the activation of the nfκβ pathway by tnfα, lps, or polyi:c (data not shown). this result is in line with the identification of nfκβ binding sites in isr promoter by chip seq. our molecular and bioinformatic analyses strongly suggest that isr , , and are indeed long non-coding rnas. the reasons are: (i) they accumulate preferentially in the nucleus of ifn-treated or untreated cells ( figure ) ; (ii) they are marked as lncrnas with high sensitivity and specificity after analysis of their orf size and coverage, analysis of their codon and hexamer usage bias, or analysis of phylocsf; (iii) they have not been identified as associated with ribosomes in ribosome profiling experiments; and (iv) if they are translated to small peptides, such peptides have not been identified by proteomic analyses ( figure s in supplementary material). given that some lncrnas regulate the expression of neighboring genes, we searched for the closest coding gene for each isr or idr. interestingly, isr , , and have neighboring genes related to the ifn response (table s in supplementary material, figure b , figure s and s in supplementary material). isr is in tandem and downstream of gbp . it is very unlikely that isr results from run-off transcription from gbp , as gbp expression could not be detected in huh cells (data not shown). isr is in tandem and upstream of il while isr is convergent with irf . none of the transcripts annotated for isr or isr overlaps with the transcripts annotated for gbp or il , respectively ( figure s in supplementary material). however, the region of isr and irf is more complex. while isr does not overlap with its coding neighbor irf , the irf gene also transcribes a longer transcript of poor coding capacity called lncirf ( figure b , figure s a in supplementary material). lncirf expression is induced by ifnα to similar levels than isr ( figure s c in supplementary material). as isr is antisense to lncirf , they could potentially regulate each other by transcriptional interference or by antisense mechanisms, although only nt of the mature form of isr are antisense to the mature form of lncirf ( figure b and figure s a in supplementary material). none of the coding/non-coding pairs share the promoter, making it unlikely that they are co-regulated at the transcriptional level, which would otherwise be a way to explain that both are induced in response to ifn. instead, the promoters of these isrs seem to be independent. isr is located within the gbp locus, which could be indicative of a general co-regulation in response to ifn. the isr and isr promoters have nfκb binding sites, although only isr is reproducibly induced in response to tnfα under the conditions tested. furthermore, isr seems a bona fide isg as the promoter has sites for stat and as well as irf and . in spite of this, the possibility exists that ifn activation of a coding isg could result in an unintended recruitment of transcription factors to the promoter of lncrnas located nearby. we do not think that this is a general phenomenon, as in the microarray or rnaseq analysis, we do not observe that many lncrnas located close to isgs are induced after ifn treatment. besides, if such unintended transcription occurs, we would expect that the expression of isr , , and should always correlate with the expression of their neighboring coding genes. this, however, is not the case. upon further analysis of the results in figures , , and , we observed a highly significant positive correlation between the expression levels of isr and gbp , or of isr and irf ( figure a) . these correlations may reflect the fact that isr and isr are genes induced by ifn at early time points. in fact, the expression of isr and isr also correlated significantly with the expression of irf and gbp , respectively (data not shown). therefore, to analyze correlation in a more stringent manner, we compared the expression of isr , , and with the expression of all the genes represented in the sureprint g microarray using data from human samples. in this case, we only observed www.frontiersin.org a significant correlation for isr and irf (corr = . and p < e− . ), suggesting that these two genes are co-expressed. in fact, the isr promoter has a conserved irf binding site. guilt-by-association genome-wide analysis predicts that isr , , and could function in the ifn pathway and the antiviral response ( figure b) . furthermore, they could be involved in viral processes including viral life cycle and viral transcription. in fact, isr and are upregulated at later times post-infection with an influenza virus mutant that lacks ns , unable to block the ifn response (figure ) . this suggests that isr and are increased in response to the physiological amounts of ifn secreted by the cells as a consequence of infection. we did not observe significant responses of isr , , and to other lytic viruses able to block the ifn response. however, both isr and were significantly upregulated in cells infected with hcv compared to controls. this was observed in hcv-infected cells in culture but also in the livers of hcv-infected patients ( figure a) . intriguingly, increased levels of isr and gbp were also detected in patients chronically infected with hiv ( figure b) . we did not observe significant correlations between the levels of isr and isr and clinical symptoms, although patients with higher hiv load tend to have higher levels of isr and gbp (data not shown). hcv is a chronic virus that employs several viral proteins to block the ifn pathway ( ) . however, the isg expression profile of some patients with chronic hcv infections indicates that ifn is being produced by the infected cells as well as by neighboring cells ( ) . this may in turn explain upregulation of isr and isr . upregulation was also observed for the neighboring genes gbp and irf but not for other isgs such as oas (figure and data not shown). this raises the question why the infection persist in spite of increased levels of antiviral factors? different viruses are targeted by unique sets of isgs ( ) . in the case of hcv both gbp and irf have been shown to have antiviral potential ( ) ( ) ( ) . irf overexpression on its own can activate a similar set of genes as ifn and can lead to a control of the replication of hcv and other viruses ( ) . therefore, for infection to persist, the effects of irf and probably other isgs should be inhibited in infected cells ( ) . one intriguing hypothesis for future work is that this inhibition is in fact exerted by isg lncrna neighbors. further experiments will be required to determine whether these isrs have a proviral or an antiviral role by affecting the function of isgs. the only preliminary evidence of an anti-ifn role is the highly significant anti-correlation between isr /isr and key factors of the ifn pathway found by genome-wide guiltby-association studies. similarly, inhibition of a lncrna located close to viperin, an isg that also inhibits hcv replication, has been shown to increase the levels of many ifn-inducible genes ( , ) . therefore, several lncrnas could act as negative regulators of the ifn pathway. the guilt-by-association study also shows a positive correlation of isr /irf and the ifn response, suggesting that isr could have a positive role in the ifn pathway. one possible approach to further dissect the function of these isrs in the future will be their inhibition via rnai. however, resistance to rnai is a common feature of many lncrnas that locate in the nucleus away from the rnai machinery or contain poorly accessible structured sequences. an alternative could be the use of gene editing technologies, provided that the function of the targeted lncrnas is not essential for the cell, which would prevent their complete deletion. moreover, it may also be feasible to overexpress selected lncrnas from plasmids or viral vectors, in order to study gain-offunction phenotypes. in the long run, we are optimistic that these and other approaches will help to further delineate the potential role of lncrnas in the ifn pathway and in the antiviral response. elena carnero and marina barriocanal designed and performed the experiments, analyzed and interpreted the data, and contributed to the writing of the manuscript; celia prior performed some of the experiments; victor segura and elizabeth guruceaga were in charge of all the bioinformatics analyses; kathleen börner and dirk grimm prepared and provided the rna samples from hiv-infected patients; and puri fortes conceived the project and the required experiments, provided the budget, interpreted the data, and wrote the manuscript. we thank nerea razquin for excellent technical assistance, estanis nistal, ruben hernandez, cristian smerdou, and rafael aldabe for influenza, adenovirus, sfv, and hcv, respectively. we also thank pablo gastaminza for the huh cells sensitive to hcv infection used in all experiments and esther larrea for ifnα and primers for isgs. we would like to thank patients for the generous donation of samples and virginia villar and the biobank of the university of navarra for their mediation. we extend our thanks to the nurses monika arnold and linda strauch, the hiv-infected blood donors, as well as hans-georg kräusslich, martin hartmann, and paul schnitzler (all heidelberg university hospital). this work was supported by grants from ministerio de ciencia e innovacion bio / , and saf - , feder funding, funds from the "ute project cima" and by the project rnareg (csd - ), funded by the ministry of science and innovation under the program consolider ingenio . an integrated encyclopedia of dna elements in the human genome non-coding rnas: the architects of eukaryotic complexity the rise of regulatory rna human cancer long non-coding rna transcriptomes specific expression of long noncoding rnas in the mouse brain chromatin signature reveals over a thousand highly conserved large non-coding rnas in mammals landscape of transcription in human cells control of alternative splicing through sirna-mediated transcriptional gene silencing 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replication by interferon regulatory factor the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein a conflict of interest statement: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest the supplementary material for this article can be found online at http://www.frontiersin.org/journal/ . /fimmu. . / abstract key: cord- -q j zxu authors: villalba, maría caridad montalvo; ramirez, odalys valdés; jiménez, mayra muné; garcia, amely arencibia; alfonso, javier martinez; baez, guelsy gonzález; arrieta, rosmery roque; simón, dianelvys rosell; gainza, delmis alvárez; vazquez, beatriz sierra; aguirre, sonia resik; tirado, maria guadalupe guzmán title: interferon gamma, tgf-β and rantes expression in upper airway samples from sars-cov- infected patients date: - - journal: clin immunol doi: . /j.clim. . sha: doc_id: cord_uid: q j zxu upper respiratory tract is the primary site of sars-cov- replication. releasing of pro and anti-inflammatory mediators plays an important role in the immunopathogenesis of coronavirus disease (covid- ). the aim of this study was to evaluate the early inflammatory response in upper airway by measuring of ifn-γ, tgf-β and rantes at mrna level. forty five sars-cov- infected patients were enrolled, whose were divided in two groups: asymptomatic and symptomatic. twenty healthy persons, sars-cov- negative were included as controls. higher ifn-γ expression was detected in sars-cov- infected patients in comparison with controls (p = , ). ifn-γ expression was increased in symptomatic patients (p = , ). tgf-β and rantes expressions were lower in sars-cov- infected patients than controls (p < , ; p = , , respectively). a significant correlation between ifn-γ and tgf-β was observed in sars-cov- asymptomatic patients (r = + , , p = , ). the findings suggest that imbalance between ifn-γ and tgf-β expression could be an impact in clinical expression of sars-cov- infection. interleukin (il)- , il- and tumor necrosis factor alpha (tnf-α) causes damage of alveoli and microvasculature in lung, mediate by vascular leakage and edema [ ] . initial site of virus replication is the epithelium of superior respiratory tract, it means that innate immune response in this site is critical controlling the viral spreading and symptoms in early stage of covid- . recruitment and activation of inflammatory cells, secreting pro-and anti-inflammatory mediators, is indispensable to virus clearance. however, the pathogenesis appears with viral persistence and the imbalance of inflammatory response. respiratory mucosa is compound by epithelial cells, globet cells, dendritic cells, invariant natural killer t (inkt) cells and γδ t cells. these cells constitute the first line of defense against pathogens. therefore, nasopharyngeal swabs, sample minimally invasive and necessary for sars-cov- diagnostic, could be used to evaluate immunological parameters at initial phase of infection. natural history of sars-cov- infection arises when the virus is binding to its receptor angiotensin converting enzyme (ace ) located in the surface of target cells. cytosolic receptors such as toll-like receptors (tlr) with capacity to recognize molecular patterns of virus; triggers the production of pro-inflammatory cytokines such as interferons (ifn). type i (α and β) and iii (λ) ifns releasing by virus infected cells, mediate the primary antiviral response. these ifns are binding to their receptors and promote the transcription of ifn-stimulated genes (isg), establishing the immunological synapse. type ii ifn gamma (ifn-γ) and regulated on activation normal t-cell expressed and secreted (rantes) chemokine are part of isg induced. ifn-γ mainly produced and secreted by inkt cell and γδ t cell, has immunomodulatory activity and reinforces the antiviral estate [ ] . high level of this cytokine has been associated with increased severity of human respiratory virus infection [ ] . studies in vitro suggested that transformer growth factor beta (tgf-β) act as a modulator of ifn production, in respiratory infections [ ] . tgf-β is an inflammatory mediator necessary to keep the integrity and homeostasis of respiratory epithelium. tgf-β is a multifunctional and pleitropic cytokine, essential in the repairing process and suppression of immune response. tgf-β has three isoform (β -β ), tgf-β is mostly implicated in the immune regulation [ ] . epithelial cells are the major source of tgf-β , although others cellular subsets (innate lymphocyte, endothelial cells, infiltrating and effector cells) can produce it in respiratory mucosa. an incremented concentration of tgf-β leads an enhance rinhovirus replication, due to suppression of ifn activity [ ] . signal of ifn releasing by infected cells [ ] . several chemokine (mcp , mip α, mip β, ip- ) are produced during sars-cov- infection; and different cells via chemokine/chemokine receptor interaction are recruited and activated during infection [ ] . symptomatology of sars-cov- infection could be determined by virus (mutations, viral load, and virus linage) and host (immune system, age, gender, nutritional status) factors [ ] . also, epigenetic modifications observed in autoimmune disease like systemic lupus erythematous increase viral entry to target cells, replication, viremia and loss of regulation of immune response in covid- . these events appear as result of deregulation in methylation of ace gene and isg [ ] . host-virus interaction and control capacity of immune system determinate the clinical presentation of covid- . the aim of this study was evaluate inflammatory response using the expression of immune mediators with antiviral, immunosuppression and chemotactic functions in the primary site of sars-cov- replication, at early stage of infection. closing of june , cuba reported sars-cov- confirmed cases, lethality rate reached , % and around , % individuals were asymptomatic at the moment of diagnostic. a nasopharyngeal swabs specimens (nps) were collected in universal transport medium (puritan® unitranz-rt®, usa), and sent to national reference laboratory of influenza virus and other respiratory virus, at institute of tropical medicine pedro kourí (ipk). forty five patients diagnosed with sars-cov- ( females, mean age= . years and males, mean age= . years) since march to april were included (table ) . infected patients were divided in asymptomatic ( ) and symptomatic ( ) ; and referred between to days of onset clinical symptoms of covid- or contact with confirmed cases. patients declared symptoms such as fever, dry cough, anorexia, fatigue, anosmia, pharyngodynia and dyspnea. no chronic diseases were identified in enrolled subjects. most of infected persons were recovered of sars-cov- infection, except one symptomatic patient who died. at the moment of collecting nasopharyngeal swabs, no prior antiviral and immunomodulator medications were used in the infected patients. as control group was included individuals ( females and males, mean age= . years), during scheduled healthy check-ups for health workers. patients and controls enrolled in this study were treated anonymously and identified by code. this study was approved by the ethics committee for research of the ipk. kit (qiagen, germany). a μl reaction was set up containing μl of rna, x reaction buffer with the one-step rt rt-pcr system (invitrogen, life technologies). thermal cycling was performed at °c for min. for reverse transcription, followed by °c for min. and then cycles of °c for seg., °c for seg., with primers and probe specific for envelop gene; with . rna copies/reaction of limit of detection (l.o.d.). rt rt-pcr for polymerase region was used as confirmatory assay with l.o.d. = . rna copies/reaction. as quality controls were included internal, positive and negative controls in each assay. viral titer was estimate by threshold cycle (ct) values detected in rt rt-pcr. cells pellets were collected from nps and total rna was extracted using rnaeasy kits (qiagen, germany), according to manufacture instruction. rna samples were treated for removing genomic dna with gdna wipeout buffer from qiagen. samples with a / rna absorption ratio > in qiaxpert system were used for relative mrna quantification. rt rt-pcr for ifn-γ quantification was carried in duplicates with taqman universal one-step qrt-pcr system (invitrogen, life technologies) following the protocol described by kim et al. [ ] . glyceraldehyde -phosphate dehydrogenase gene (gapdh) was used as housekeeping gene. rantes and tgf-β expression were measuring according to ziklo et al. protocols [ ] ; first mrna was reverse-transcribed using radom primers and superscript® reverse transcriptase kit (invitrogen, life technologies). cdna synthesized was use as template in pcr reaction using quantifasttmsybr® green pcr kit (qiagen, germany). beta actin (βactin) gene was used for normalization rantes and tgf-β genes expression. all pcrs were running in rotor gene q software . . . . relative cytokine mrna concentration was controlled with constitutive genes cycle threshold (ct) values; and transformed to quantify using −Δct method (Δct=ct target gene-ct reference gene). it was conducted a descriptive study; sars-cov- viral load and levels of expression of inflammatory mediators were expressed as median and interquartile range (m, iqr). differences and similarity between the symptomatic and asymptomatic sars-cov- cases with control group was performed using nonparametrict tests, mann-whitney utest and kruskal-wallis with dunn's multiple comparison test, to compare two or three variables respectively. correlation between expression levels of ifn-γ and tgf-β was analyzed using spearman's rank correlation coefficient rho (r). in the graphics dots indicate individual data. middle bars on scatters plot means medians and the ends show interquartile ranges. all data were analyzed with prism (graphpad, la jolla, ca) and p value < . was considered as statistically significant difference. j o u r n a l p r e -p r o o f taking into account that ct value has a correlation with the amount of rna present in the samples, it was found that medians and iqr of sars-cov- viral titer was similar in asymptomatic ( . , . - . ) and symptomatic ( , . - . ) cases; and comparison between groups did not show difference (p= . ). ifn-γ was significantly more expressed in sars-cov- infected individuals compared with control group ( . , . - . vs. . , . - . ; p= . ) (fig. a) . its expression was increased and significant in symptomatic sars-cov- patients; in contrast with healthy persons ( . , . - . ; p= . ). but, no different was found between symptomatic and asymptomatic ( . , . - . ; p= . ) cases in fn-γ expression (fig. b) . regarding to tgf-β , the results showed that its expression was significantly lower in sars-cov- positive patients than control group ( . , . - . vs. . , . - . ; p< . ) (fig. c ). according to symptoms, tgf-β mrna concentration did not differ significantly between groups, although the expression decreased in asymptomatic persons in relation with symptomatic cases ( . , . - . vs. . , . - . ) (fig. d ). rantes is a well-established, monocytes, nk and t cell chemoattractant and important link between innate and adaptive immune response. in comparison with control group, rantes decreased significantly in sars-cov- infected persons ( . , . - . vs. . , . - . ; p= . ) (fig. e) . similarly, the difference between healthy persons with asymptomatic ( . , . - , ; p= . ) and symptomatic cases was relevant ( . , . - . ; p= . ) (fig. f) . the lowest rantes expression was detected in symptomatic sars-cov- patients. taking into account, the balance between inflammatory and anti-inflammatory cytokines could be related with clinical expression and pathogenesis of sars-cov- infection, we analyzed the relationship between ifn-γ and tgf-β . it was found a positive correlation between both markers in healthy persons (r=+ . ) and asymptomatic sars-cov- infected persons (r=+ . ) ( fig. a-b) . interestingly, the correlation between ifn-γ and tgf-β in asymptomatic cases was statistically significant (p= . ) (fig. b) . while, a negative correlation was observed in symptomatic patients (r=- . , p= . ) (fig. c) . also, it was evaluated the balance between inflammatory mediator (ifn-γ) and anti-inflammatory (tgf-β ) mediator using the median ratio of ifn-γ:tgf-β by groups. these values increased notably from control group ( . / . = . ), passing through asymptomatic ( . / . = . ), up to symptomatic ( . / . = . ) sars-cov- individuals. it is known that most of the initial symptoms of covid- are focusing in upper airway. therefore, an equilibrium between the dose of viral exposure and efficiency of the local innate immune response; might be crucial blocking the spreading of sars-cov- from upper to lower respiratory tract, at early stage of infection [ ] . our finding showed that viral titers did not differ between asymptomatic and symptomatic sars-cov- infected persons, in concordance with the results obtained by zou et al [ ] . on other hand, some authors detected that viral titer increase in the early stage and decrease during recovery phase of covid- in nps [ ] . on critically ill covid- patients viral load in serum was correlated with il- levels, inflammatory mediator in respiratory failure [ ] . additionally, therapies with antivirals have demonstrated its efficacy reducing the recovery time of sars-cov- infected patients. in spite of viral titer has been related with early viral clearance; its association with symptoms and severity of covid- must still to be elucidated. role of host cytokines determining the differences between symptomatic and asymptomatic individuals was demonstrated in an experimental influenza virus challenge [ ] . as show our results, sars-cov- infection induced high ifn-γ expression in swabbed cells from upper airway; its expression was higher in symptomatic patients in comparison with asymptomatic individuals. ifn-γ is a pivotal cytokine in the cell mediated immunity against virus diseases and amplifies the antigen recognition. also, the role of ifn-γ in coronavirus infection was already documented. in the fact, it can induce a set of antiviral proteins that restricting viral uncoating, entry into the host cell and interfere with the access to cytoplasm of phagocyted virions in endosomes [ ] . this cytokine has strong antiviral and immunomodulatory activity, but at the same time its action requires to be regulated. an over-production of ifn-γ conduces to excessive inflammation, contributing to covid- pathogenesis [ ] . it was observed that tightly balance between pro-and anti-inflammatory cytokines could determinate symptoms expression, at early stage of sars-cov- infection [ ] . high expression level of tgf-β was found at baseline in control group, which probably contributing to keep the homeostasis and tolerance for commensal agents in respiratory mucosa. a significant decreasing in tgf-β expression detected in sars-cov- infected patients, could lead a reduction of its immunosuppressant effect. tgf-β inhibit ifn-γ expression, inducing the phosphorylation of smad and/or smad signaling proteins, which with smad are translocated to the nucleus and bind to the promoters region of ifn-γ gene, blocking its transcription [ ] . tgf-β had the lowest expression in asymptomatic cases; contrarily we would expect that inflammatory response expressed as presence of symptomatology was higher in this group. however, expression of ifn-γ and ratio ifn-γ:tgf-β were lower in comparison with symptomatic patients. also, positive correlation between ifn-γ and tgf-β provided evidence of immune response control could determinate the asymptomatic presentation of sars-cov- infection. denney et al. suggested that relationship between ifn and tgf-β has an important role in the evolution of respiratory viral infection [ ] . in covid- , dynamic expression of tgf-β cytokine should be evaluated, since the respiratory distress and lung fibrosis have been mainly attributed to cytokine storm and exacerbated production of tgf-β [ ] . it is known that tnf-α up-regulates tgf-β , using mechanisms involving both increased its transcription and stabilizing mrna [ ] . probably, the kinetic of tgf-β is changing with synergistic activity of cytokines and the migration of inflammatory cells into the lung. these cells could be responsible of local increasing of tgf-β, in late and severe phases of sars-cov- infection. on the other hand, the inhibitory effects of tgf-β could trigger regulatory t cells, suppressing innate and adaptive immune response. type i and ii ifns promote immune cell migration trough expression of several chemokines like rantes; and different types of inflammatory cells are moving to respiratory epithelium by this immune mediator [ ] . in our study, it was found a reduced expression of rantes in sars-cov- positive cases compared to healthy persons; and symptomatic patients showed the lowest quantification. rantes act as a chemotactic factor to induce the migration of monocytes, neutrophils, dendritic cells, nk cells and t cells from the bloodstream to the sites of infection [ ] . an inhibition of the traffic and recruitment of activated immune cells and memory lymphocytes to upper airway mucosa, even with normal expression of its receptors, could be related with decreasing of rantes concentration. in this context, some authors reported that arresting the differentiation, recruitment and activation of inflammatory cells may reduce the covid- pathogenesis. several therapeutic strategies have been used with hihg efficacy like blocking granulocyte macrophagecolony stimulating factors and il- [ ] . also, the treatments with complement c inhibitors, point where all complement activation pathway converge, decreasing the anaphylatoxins production and improving the clinical status of covid- patients [ ] . asymptomatic sars-cov- infection and atypical infection were observed in patients with long-term glucocorticoids medication; potent immunosuppressive and antiinflammatory drugs [ ] . rantes expression indicates the integrity of downstream signals in response to ifn production by infected cells. however, kawka et al. showed that ifn-γ alone did not induce rantes mrna; this was reverted rapidly with a concurrent increasing of tnf-α [ ] . in addition, in alveolar epithelial cells the rantes expression depended of synergic tnf-α and ifn-γ stimulations. tnf-α induces nuclear translocation of nuclear factor κb and interferon regulatory factor , which bind to the rantes promoter region and triggers its expression [ ] . pro-inflammatory cytokines increase the inflammation to control the viral replication in upper respiratory airway. rantes mediates the influx of mononuclear cells, including t cells, which are the main source of ifn-γ. however, we could not discard that sars-cov- disrupt rantes mrna as evasion strategy at early stage of infection, trying to block the migration of immune cells to the sites of infection; delaying the immediate immune response. menachery et al., described that mers-cov bypass immune j o u r n a l p r e -p r o o f response since gene expressions of antigen presentation were down-regulated [ ] . however, rantes kinetic is quite different in late phase of covid- , paterson et al. detected an elevation of rantes in sera collected from critical patients, and observed that inflammatory molecules (il- and ifn-related genes) declined with the blocking of ccr using monoclonal antibody [ ] . this finding indicates that rantes amplify the inflammation disorder in severe cases of sars-cov- infection. innate and adaptive immune response determinates the clinical expression and prognosis of sars-cov- infection. t lymphocyte cd + has a pivotal role eliminating the infected cells and low count of these cells increase the fatal outcome of covid- . however, this effect is attenuated increasing the efficacy of its cytotoxic activity [ ] . regarding to specific humoral response, early and high antibodies titers against sars-cov- has been associated with unfavorable progression of covid- . while, undetectable and low anti-sars-cov- levels were observed in asymptomatic cases. authors reported that effective control of virus replication in infected cells related to ace expression could reduce the stimulation of th subset, with pathogenic role in sars cov- infection. pathogenicity of anti-sars-cov- immunoglobulin has been attributed to accumulation and deposit of immune complex or type hypersensitive. as result, complement cascade is triggered by classical pathway, amplifying and perpetuating the inflammatory disease [ ] . concerning to type of cells taken from nps to evaluate mrna expression, chua et al. identified dendritic cells, macrophages, cytotoxic t cells, nkt cells, and neutrophils in nps specimens from sars-cov- infected subjects. these authors found a high percentage of epithelial cells in comparison with inflammatory cells in healthy individuals [ ] . this means that epithelial cells could be the majority in homeostasis conditions, and expression of antivirals genes in upper airway could be detected for immune cell infiltration in presence of viral infection. in congruency with our results, differential gene expression of cytokines and chemokine genes were found in monocyte infected with sars-cov; tgf-β and rantes showed a down-regulation hours post infection [ ] . we thinking that tnf-α is crucial cooperating and stimulating rantes and tgf-β transcription. equally, apoptosis of infected cells constitute an additional stimulus to induce expression of tgf-β . one of the limitations of this study were the small numbers of studied patients and we did not know if asymptomatic cases remained in this status during the whole time of sars-cov- infection. further studies will be necessary to correlate our finding with circulating levels of inflammatory mediators, associated with systemic symptoms of covid- . this work explored the host-pathogen interaction in at initial phase of sars-cov- infection. it was demonstrated the influence of unbalance immune response in the presence or absence of covid- symptoms, in patients with viral titer similar. asymptomatic patients were not immunologically quiescent; however they had an early control of inflammatory response in the primary site of infection. finally, we concluded at onset sars cov- infection resident cells in the upper respiratory epithelium increased de novo expression of ifn-γ, trying to control the virus replication and spreading at initial site of infection. these cells reduced their capacity of tgf-β and rantes production de novo, in response to stimulation with ifn-γ. contrary to symptomatic patients, in asymptomatic cases tgf-β and rantes expressions were sufficient to attenuate the inflammatory effect of ifn-γ, preserve and renew an optimum influx of mononuclear cells; with virucidal activity. mononuclear cells were the main sources of cytokines that can act synergistically, to reduce the inflammatory injury of respiratory mucosa. also, it was demonstrated the value added of nps for both viral diagnostic and evaluation of host immunological genes expression. we suggest that evaluation of ifnγ:tgf-β axis in the primary site of viral replication could be used to predict the course and 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a patient with long-term use of glucocorticoids: a study of a familial cluster regulation of chemokine ccl synthesis in human peritoneal fibroblasts: a key role of ifn-γ regulation of rantes promoter activation in alveolar epithelial cells after cytokine stimulation mers-cov and h n influenza virus antagonize antigen presentation by altering the epigenetic landscap disruption of the ccl /rantes-ccr pathway restores immune homeostasis and reduces plasma viral load in critical covid- selective cd cell reduction by sars-cov- is associated with a worse prognosis and systemic inflammation in covid- patients type hypersensitivity in covid- vasculitis covid- severity correlates with airway epitheliumimmune cell interactions identified by single-cell analysis sars-cov regulates immune function-related gene expression in human monocytic cells we would like to thanks for their technical support to all professional of center for research, diagnostic and reference at ipk. also, our gratitude is to dr. magilé fonseca for her material support. key: cord- -ktktqs b authors: pereda, r.; gonzalez, d.; rivero, h. b.; rivero, j. c.; perez, a.; lopez, l. d. r.; mezquia, n.; venegas, r.; betancourt, j. r.; dominguez, r. e. title: therapeutic effectiveness of interferon alpha b treatment for covid- patient recovery date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ktktqs b background effective antiviral treatments are required to contain the ongoing coronavirus disease (covid- ) pandemic. a previous report in patients covid- positive in cuba provided preliminary therapeutic efficacy evidence with interferon alpha- b (ifn alpha- b) from march to april , . this study, re-evaluates the contribution of ifn- b on the evolution of all patients, after days of the epidemic, in a period from march to june , . method a prospective observational study was implemented to monitor a therapeutic intervention with ifn alpha- b used in the national protocol for covid- attending in cuba. were included patients with positive throat swab specimens by real time rt-pcr who gave informed consent and had no contraindications for ifn treatment. patients received therapy as per the cuban covid protocol that included a combination of oral antivirals (lopinavir/ritonavir and chloroquine) with intramuscular or subcutaneous administration of ifn alpha- b the primary endpoint was the proportion of patients discharged from hospital, secondary was the case fatality rate and several outcomes related to time variables were also evaluated. results from march th until june th, patients had been confirmed sars-cov- positive in cuba, were treated with heberon alpha r and received the approved protocol without ifn. the proportion of fully recovered patients was higher in the ifn-treated compared with non-ifn treated group. prior ifn treatment decreases the likelihood of intensive care and increases the survival after severe or critical diseases. the benefits of ifn were significantly supported by time variables analyzed. conclusions this second report confirm the preliminary evidences from first for the therapeutic effectiveness of ifn alpha- b for sars-cov- infection and postulated that heberon alpha r is the main component within the antiviral triad used as a therapeutic intervention in the cuban protocol covid- . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint the coronavirus disease (covid- ) emerged in china in december . the responsible virus, severe acute respiratory syndrome coronavirus (sars-cov- ), belongs to a distinct classification from the human severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov) . the clinical spectrum of covid- varies from asymptomatic infection or mild symptoms to severe acute respiratory illness and death . according to the covid- situation report issued by world health organization (who) on th july , covid- pandemic affects more than countries around the world with more than million cases have been diagnosed and over thousand patients have died . effective antiviral treatments are required to contain the epidemics; several candidates are already being investigated, including type interferon (ifn-α/) . at present, a rna-dependent rna polymerases (rdrp), remdesivir, is the first medicine against covid- approved in the european union, mainly based on preliminary data from a trial published in the new england journal of medicine and limited to patients with severe disease . remdesivir has also been approved for emergency use in severely ill patients in the united states (us), india, and south korea and has received full approval in japan . favipiravir , an anti-influenza medication that also is an rdrp inhibitor, was approved for medical use in russia using a generic version named avifavir. ifn-α, a member of the first line of natural antiviral defense activates the innate immune response against the virus and the mechanism of inhibition of viral replication . after covid- outbreak, a strong anti-sars-cov- activity in cultured cells has been reported for ifn-α, demonstrating its therapeutic potency for covid- . guidelines issued by the expert committee of who identified ifn-α b as a potential antiviral for the treatment and prevention of covid- . early on in the . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint outbreak, the chinese government recommended the use of ifn-α for the treatment of covid- . several ongoing clinical studies evaluating ifn-α b for the treatment of covid- are registered at clinicaltrials.gov and already provided clinical evidences for therapeutic efficacy . heberon ® alpha r is a human recombinant ifn-α b formulation produced by the center for genetic engineering and biotechnology, havana, cuba, with demonstrated antiviral efficacy and a proven safety profile over years and is one of the drugs used in the cuban protocol two groups of individuals were admitted to the hospital, according to the case classification criteria defined in the cuban protocol: ) people with suspected covid- due to clinical respiratory symptoms, such as fever, fatigue, cough, headache, shortness of breath and nasal discharge in the last days; ) subjects who had contact with a patient with confirmed or is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . individuals themselves, parents or legal representative had to give informed consent and have no contraindications for treatment with ifn described in the product information sheet, to receive heberon ® alpha r as approved in the cuban protocol for covid- . patients in severe covid- , with contraindications or who did not consent to receive ifn were treated with the cuban protocol lacking ifn, i.e. only lpv/rtv and cq. for those cases in treatment ongoing whose disease progressed to become severe and critical, requiring intensive care unit (icu) support, treatment with heberon ® alpha r was promptly stopped. addition of heberon ® alpha r to the cuban protocol was approved by the cuban national regulatory authority which maintained surveillance of the scientific evidence obtained. the protocol of this study was evaluated and approved by a centralized research ethics committee, is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . hematological and biochemical profiles were assessed at admission and every hours using routine clinical laboratory procedures. chest radiological evaluation and cardiovascular function monitoring by electrocardiogram was made before treatment and daily after started it. the primary endpoint was the proportion of patients discharged from hospital (i.e. discharge criteria were according to clinical, radiological and laboratory evaluations). clinical criteria: patient in stable condition and afebrile for more than days, regular breathing and normal respiratory . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint rate, clear conscience, unaffected speech and normal diet. radiological criteria: significant improvement without signs of organ dysfunction in lung images. laboratory criteria: two consecutive pcr (-) with at least hours apart. the secondary endpoint was the case fatality rate, (cfr), defined as the number of confirmed deaths divided by the number of confirmed cases. the time elapsed to the hospital discharged was an endpoint for all patients. a threshold time definition was according the patient clinical classification: for symptomatic subject was defined as the number of days elapsed from the onset of symptoms up to meet discharged criteria; for asymptomatic the timeline began on hospital day admission. the full recovery time after covid- diagnostic by pcr (+) was also endpoint for all cases. date of confirmed pcr (+) was considered as date of onset of covid- and the need of treatment, an appropriate time to match point the disease course between all patients. endpoints applied to cohort requiring icu were the days from symptom onset and hospital admittance to icu admission and the time to death from these three timelines. descriptive statistics (tables, group means and interquartile range reported in the text) convey the data as-is. the endpoints were adjusted by age, sex, and the presence of comorbidities. note that age was considered at every -year age interval from older than years. from contingency tables chi-square calculator tests and fisher exact test calculator for association between two categorical variables were used. the odds ratio (or) was applied as a measure of association between treatments and outcomes. the non-parametric mann-whitney u test was used for comparing independents samples is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . the cuban protocol to begin treatment, two clinical forms of uncomplicated disease were predominated: ) minimally symptomatic disease according to nonspecific signs such as fever, cough, sore throat, nasal congestion, slight headache and malaise, without dehydration, dyspnea, or sepsis evidences; ) mild pneumonia, due to the presence of the above symptoms and polypnea with partial oxygen saturation (spo ) above %, but without signs of respiratory failure or severity. in ( . %) patients a severe pneumonia was detected before start treatment and their hospital admission was directly to icu due to positive clinical symptoms, chest radiological imaging and respiratory rate over breaths / min, limitation of thoracic expandability, central cyanosis, spo below % and pleuritic pain. acute respiratory distress syndrome (ards) associated was found in of these ( . %) patients. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . the median age of all cases was years (range days to years; interquartile range, - years) with a non-significant difference between the sexes (p= . ). in the distribution of patients by sex, the numbers of men (p= . ) at ages - and women (p= . ) above age were significantly higher. both (p= . ) women and men peaks covid- incidence at ages - . comorbidities were present in ( . %) patients with a similar incidence between the sexes (p= . ) and significant higher in those over age not treated with ifn. the main underlying diseases were high blood pressure ( . %), diabetes mellitus ( . %) and ischemic heart disease ( . %). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . on the cut-off date analyzed in this report, the cuban overall cfr was . % and . % for patients treated with ifn alpha- b. both data were lower than those reported at the same time by to better address the influence of ifn treatment on cfr, we considered the clinical status of patients in the context of severity of disease, using admission to icu as an appropriate time point to match the criterion for worsened disease. during the course of treatment, patients ( . % of all the covid- cases) were admitted to the icu ( table ) . notably, only had been treated with ifn, representing . % of the total number of this treatment cohort in the study. ifn treatment was withdrawn for those patients who progressed to severe or critical disease. nevertheless, the cfr in presence of ards was lower in ifn previously treated individuals ( ; . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint after cfr correction analysis done without considered these patients, the ifn cohort maintained a significant outcome compared to ifn non-treated individuals (p= . x - ). patients died from covid- in cuba were adults. thereby, the analysis related to age and lethality was made at every -year age interval from older than years. ages - were the only ones lacking significant association to cfr. there were deceases in ages - , . % is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint from overall. deaths predominated in male (p= . ), patients with comorbidities (p= . x - ) and symptomatic individuals (p= . x - ). in male over years, arterial hypertension represented the major risk predictors for death. ifn-related cfr was affected by ages - (p= . ) and ages - (p= . x - ). however, the effects of ifn treatment remain significant regardless of age interval compared to those not treated with ifn (p-values ranged . x - to . x - ). although the ifn-related cfr was affected by comorbidities (p= . x - ), did not negate or eliminate the difference in cfr between ifn treated individuals and those not treated with ifn (p = . x - ). no association was found between sex and lethality in those treated with ifn (p= . ). our previous report in patients covid- positive in cuba provided preliminary therapeutic efficacy evidence with ifn-α b from march to april , is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint ours two reports on the intramuscular administration for ifn-α b contrast with the earlier published findings of inhaled ifn-α b . an aerosol administration has the advantage of specifically targeting the respiratory tract; however, the pharmacodynamics and pharmacokinetics of this mode of administration are not known . conversely, intramuscular and subcutaneous administration are well described for heberon ® alpha r and their safety profiles has been extensively studied by pharmacodynamics and pharmacokinetics and have already proven safe in a considerable number of clinical trials . the key explaining the large recovery of patients after used ifn-α b in our data is a therapeutic is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint even when it has disappeared from the blood circulation. as a result, a sustained therapeutic response is expressed clinically thought a long-term viral clearance after treatment. neither of these two favorable events is possible with generic antiviral drugs like favipiravir, whose depend on permanent level in blood for achieve therapeutic activity. demographic distribution of patients included in this study conformed to findings in china , in several european and latin-american countries and the us reporting a slightly higher number for male confirmed and cfr compared to females. older persons with pre-existing hypertension and/or diabetes were more prevalent in our no-ifn treated cohort and this agerelated trend of co-morbidities was similar to data reported by zhou f et al . the proportion ( %) of confirmed covid- children in cuba seems notably when pediatrics reviews show that pediatric population have so far accounted for %- % of diagnosed covid- cases around globe the above can be explained by the methodology implemented by the cuban protocol in which an early epidemiological screening was carried out independently of the presence of clinical symptoms and most of the patients were admitted to hospital and sars-cov- confirmed in the asymptomatic phase. data analyses in this study were limited, because the study includes unbalanced demographics between treatment arms of unequal size and endpoint outcomes, namely hospital discharge (recovery) and cfr for a large number of the cases remained unknown at the time of study termination. regardless of the identified limitations, this report provides evidence of the effectiveness of ifn-α b as an antiviral treatment for sars-cov- infection and suggests that the use of heberon ® alpha r at the doses and therapeutic regimen employed may contribute to recovery from covid- . furthers studies are needed for additional efficacy and safety profile evaluation of heberon ® alpha r, specifically a randomized clinical trial for comparison with other potential antivirals. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint contributors dg, rv, bjr and dre were responsible for patient care and treatment, rhb, rjc, oa, llr and mn made clinical oversight and clinical data collection, pr led the working group, analyzed and conducted data analysis, data interpretation, literature searches and manuscript writing. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia ) a comprehensive literature review on the clinical presentation, and management of the pandemic coronavirus disease (covid- ) clinical features of patients infected with novel coronavirus in wuhan, china. lancet. coronavirus disease (covid- ) situation reports- clinical studies related to the coronavirus disease (covid- ) actt- study group members. actt- study group members remdesivir for the treatment of covid- : preliminary report covid- : selected nhs patients will be treated with remdesivir favipiravir (t- ), a novel viral rna polymerase inhibitor type interferons as a potential treatment against covid- antiviral activities of type i interferons to sars-cov- infection clinical management of severe acute respiratory infection when novel coronavirus ( -ncov) infection is suspected: interim guidance chinese guidelines related to novel coronavirus pneumonia a brief review of antiviral drugs evaluated in registered clinical trials for covid- interferon-α b treatment for covid- cuban interferon alpha- b. thirty years as an effective and safe drug therapeutic effectiveness of interferon-alpha b against covid- : the cuban experience cuban public registry of clinical trials. (w.d.). ministry of public health closing part of the day june at midnight coronavirus disease (covid- ) situation reports- paho covid- daily update: can interferons stop covid- before it takes hold? severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges impact of sex and gender on covid- outcomes in europe it is made available under a perpetuity.is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprintthe copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint it is made available under a perpetuity.is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprintthe copyright holder for this this version posted august , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprintthe copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- -e of o authors: kindler, eveline; gil-cruz, cristina; spanier, julia; li, yize; wilhelm, jochen; rabouw, huib h.; züst, roland; hwang, mihyun; v’kovski, philip; stalder, hanspeter; marti, sabrina; habjan, matthias; cervantes-barragan, luisa; elliot, ruth; karl, nadja; gaughan, christina; van kuppeveld, frank j. m.; silverman, robert h.; keller, markus; ludewig, burkhard; bergmann, cornelia c.; ziebuhr, john; weiss, susan r.; kalinke, ulrich; thiel, volker title: early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: e of o coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as sars-cov and mers-cov. they are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-i interferons (ifn-i). this evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent rna-based sensing of infection in vertebrate hosts. here we show that the coronavirus endonuclease (endou) activity is key to prevent early induction of double-stranded rna (dsrna) host cell responses. replication of endou-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. in macrophages we found immediate induction of ifn-i expression and rnase l-mediated breakdown of ribosomal rna. accordingly, endou-deficient viruses can retain replication only in cells that are deficient in ifn-i expression or sensing, and in cells lacking both rnase l and pkr. collectively our results demonstrate that the coronavirus endou efficiently prevents simultaneous activation of host cell dsrna sensors, such as mda , oas and pkr. the localization of the endou activity at the site of viral rna synthesis–within the replicase complex—suggests that coronaviruses have evolved a viral rna decay pathway to evade early innate and intrinsic antiviral host cell responses. a a a a a host innate immune responses are of particular importance during the early phase of virus infection to restrict virus replication and spread. they rely on the ability to differentiate between immunological "self" and "non-self" in order to swiftly activate diverse antiviral effector mechanisms. conceptually, sensing of virus infection is mainly mediated through recognition of viral nucleic acids, which are considered to comprise pathogen-associated molecular patterns (pamps) that are recognized by specialized host cell pathogen recognition receptors (prrs) [ ] . double-stranded (ds) rna, an obligate replication intermediate of positivestranded rna viruses that is accumulating during replication, is known as an important pamp within the cytoplasm of infected cells. host cell responses to dsrna are versatile and include the expression of ifn-i by activating rig-i like helicases (rlrs), such as rig-i and mda , the inhibition of host cell translation by activating pkr, and the degradation of viral and host cell-derived rna by activating the oas/rnase l pathway [ ] . coronaviruses are positive-stranded rna viruses that replicate in the host cell cytoplasm. they are well known to evade innate immune activation, particularly during the early phase of the infection [ ] [ ] [ ] [ ] . coronavirus innate immune evasion is multifaceted and involves ribose- '-o methylation of viral rna, as well as compartmentalised rna synthesis at virus-induced membrane structures comprised of convoluted membranes and double membrane vesicles [ ] [ ] [ ] . the importance of functions encoded by the cov replicase gene is further exemplified by non-structural protein (nsp) that suppresses host gene expression by mediating host mrna degradation [ , ] , and nsp that contains a papain-like proteinase with deubiquitination activity interfering with ifn-i host cell responses [ , ] . in addition, a number of accessory gene functions, although less conserved, have been described to target downstream events of innate immune activation, such as a phosphodiesterase (pde) activity encoded by some coronavirus strains, which degrades ', '-oligoadenylate messenger molecules essential for rnase l activation [ , ] . here we addressed a possible role of the highly conserved coronavirus endou activity in innate immune evasion. the endou domain is harboured in non-structural protein (nsp) that is considered as an integral component of the coronaviral replicase-transcriptase complex (rtc) [ ] [ ] [ ] [ ] . by using immunofluorescence microscopy analyses in hcov- e-infected cells with a hcov- e-nsp -specific monoclonal antibody the characteristic perinuclear staining pattern known from various other cov nsps was reported [ ] . for mhv-a , a similar study reports mhv-nsp -specific perinuclear puncta that were detected using an mhv-nsp -specific rabbit antiserum that partially overlapped with mhv nucleocapsid staining in mhv-a -infected cells [ , ] . moreover, mhv nsp was shown to co-localize with viral rna and to fractionate in similar fractions as other nsps following mhv infection [ , ] . notably, upon ectopic expression of a fusion protein comprised of the green fluorescent protein (gfp) and mhv-nsp , a pattern of cytoplasmic speckles, distinct from the characteristic pattern of the cov replicase complex was observed, suggesting that the localization of ectopically expressed nsp or gfp-nsp fusion proteins may differ from the localization of nsp that is expressed in the context of the cov polyprotein ab [ ] . the cov endou has uridylate-specific endonucleolytic activity on single-stranded and dsrna [ ] and is related to (i) cellular enzymes prototyped by xendou [ , ] and (ii) viral homologs conserved in all nidoviruses known to infect vertebrate hosts including fish, birds and mammals, suggesting an important role for this enzyme in an ancient cellular pathway. over the past years, a wealth of structural and biochemical information has been obtained for endou. however, the precise role of this virus-encoded nucleolytic activity in coronavirus/nidovirus replication remains enigmatic [ , , [ ] [ ] [ ] . surprisingly, although endou is coexpressed with other key replicative proteins as part of the viral replicase polyprotein, its enzymatic activity is not essential for viral rna synthesis in most cell culture systems [ ] . in this work we illustrate a pronounced impact of the coronavirus endou activity on innate immune evasion. specifically, we show that genetically engineered mutants of mouse hepatitis virus (mhv) and human coronavirus e (hcov- e), respectively, that encode an endou active-site substitution known to abolish nucleolytic activity, were severely attenuated and elicited an immediate and simultaneous activation of host cell dsrna sensors. based on biochemical and structural information on coronavirus endou active-site residues [ , , ] , we constructed endou-deficient mutants of hcov- e and mhv (hcov- e h a and mhv h a ) and assessed their replication characteristics in vitro and in vivo ( fig a) . replication of mhv h a was reduced in l cells, but peak titers almost reached those of wild-type mhv-a , confirming that the coronavirus endou activity is dispensable for virus replication in vitro ( fig b) [ , ] . in sharp contrast, compared to wild-type mhv-a , the endou-deficient mhv h a was severely attenuated in vivo (fig c) . mhv h a replication was not detectable in spleen and liver of c bl/ mice at two days post intraperitoneal infection with plaque-forming units (pfu), demonstrating that the endou activity is required for efficient replication and spread in vivo. notably, replication and spread of mhv h a was partly restored in mice deficient for the ifn-i receptor (ifnar), with viral titers of mhv h a in the spleen and liver of ifnar-deficient mice that did not reach those of mhv-a . interestingly, concerning the role of mda and tlr , which are known as main cytoplasmic and endosomal prrs for coronaviral rna, respectively, mhv h a replication was not restored in mda -deficient, tlr -deficient, or mda -and tlr -deficient mice. this phenotype clearly differs from that described for coronaviruses that lack ribose- '-o methyl-transferase (omt) activity [ ] . thus, in experiments reported previously, we found that replication of omt-deficient mhv (mhv d a ) is restored in mice that are deficient for mda and tlr , suggesting that lack of ribose- '-o methylation is tolerated if these two rna sensors are absent. the lack of any detectable replication of the endou-deficient mhv h a in mda -and tlr -deficient mice therefore indicates that mda -and tlr -mediated ifn-i expression may not exclusively restrict mhv h a replication and that other mechanisms contribute to the observed attenuation of mhv h a replication. the severe attenuation of mhv h a growth in vivo prompted us to assess mhv h a and hcov- e h a replication in primary target cells. as shown in fig a, mhv h a replication in primary murine embryonic fibroblasts (mefs) was comparable to that of mhv-a . data represent two independent experiments, each performed in duplicates. mean and sem are depicted. the % confidence band is highlighted in grey. the differences in peak levels of viral titers were calculated by using the non-linear regression model described in material and methods (peak mhv-a : . , mhv h a : . , p = . , left panel; peak mhv-a : . , mhv h a : . , p = . , right panel) and significance is displayed as * p < . . (c) viral titers of mhv-a and mhv h a in liver and spleen of c bl/ , ifnar -/-, mda -/-, tlr -/-, and mda -/-/tlr -/mice at two days post intraperitoneal infection ( pfu). data represent three to four independent experiments, each based on two to three mice per strain and virus. mean and sem are depicted. data points that show significant differences in a two-sided, unpaired student's t-test are displayed; * p < . , ** < . , *** < . . nd, not detected. until - hours post infection (h.p.i.), but was significantly restricted later during the infection. moreover, replication of mhv h a was even more severely reduced in bone marrow-derived murine macrophages, and accompanied by early induction of ifn-β expression (fig b and c) . notably, levels of ifn-β mrna were only transiently ( to h.p.i.) elevated in mhv h a compared to mhv-a infected macrophages, and declined along with viral titers and viral rna during the late phase of infection. likewise, replication of the endou-deficient hcov- e h a was severely restricted in human blood-derived macrophages (fig d) . we observed significantly elevated ifn-i expression in a panel of human macrophages derived from seven individual donors after infection with hcov- e h a compared to wild-type hcov- e infection (fig d) , consistent with reduced viral replication. next, we addressed if, and to what extent, the growth defects observed for endou-deficient coronaviruses correlate with the induction of ifn-β expression. mda has been described as the main rna sensor of coronavirus infection in murine macrophages [ ] . compared to wild-type macrophages, ifn-β expression was reduced in mda -deficient macrophages following mhv h a infection, as shown by qrt-pcr and ifn-β elisa (fig a and d ). surprisingly, and again in contrast to the phenotype of the omt-deficient mhv d a [ ] , mhv h a replication was not restored in mda -deficient macrophages ( fig a) . similarly, although ifn-β expression was likewise reduced in mavs-deficient macrophages, mhv h a replication was not restored in mavs-deficient macrophages (fig b and d ). even in irf / irf /irf (irf / / -/-) triple-knockout macrophages, that display an almost negligible induction of ifn-β expression, mhv h a replication was not fully restored (fig c and d ). ifn-β protein assessed by ifn-β elisa was below detection in all three macrophage genotypes (mda -/-, mavs -/-, irf / / -/-; fig d) . these results indicate that mhv h a replication is either highly sensitive to already marginal amounts of ifn-i, or that other antiviral host cell responses may contribute to the attenuation of mhv h a . in order to address the sensitivity of endou-deficient coronaviruses to ifn-i, we first assessed mhv h a replication in ifnar-deficient macrophages. as shown in fig a, mhv h a replication was partially restored to levels that almost reached those of wild-type mhv-a replication. importantly, ifn-β expression was elevated in ifnar-deficient macrophages that had been infected with mhv h a compared to those infected with wild-type mhv-a , demonstrating that increased expression of ifn-β can be uncoupled from attenuation of mhv h a (fig b) . we also noted that ifn-β expression was delayed in ifnar-deficient macrophages following mhv h a infection compared to wild-type macrophages. this observation is in agreement with previous reports that suggested a macrophage-specific autocrine ifn-β priming loop in wild-type macrophages enhances cytokine and chemokine expression following mhv infection [ ] . the severe attenuation of mhv h a and hcov- e h a replication in wild-type murine and human macrophages, respectively, precluded the use of primary macrophages to assess the sensitivity of endou-deficient coronaviruses to ifn-i pre-treatment. therefore, we infected murine l cells and human mrc lung fibroblasts with mhv h a and hcov- e h a , respectively, and applied different dosages of ifn-i for hours prior to infection. compared to wild type mhv and hcov- e, respectively, both endou-deficient viruses indeed displayed a pronounced sensitivity to ifn-i pre-treatment ( fig c) . remarkably, mhv h a displayed a sensitivity to ifn-i treatment that is comparable to that of the highly ifn-i sensitive omt-deficient mutant mhv d a (s fig). however, compared to the omt-deficient mutant mhv d a the phenotype of the endou-deficient mhv h a differs mainly in the lack of restoration of replication under conditions with strongly reduced ifn-i expression (e.g. in mda -/macrophages; fig a) , suggesting that other, most likely ifn-i inducible, antiviral effector mechanisms account for restriction of mhv h a replication. collectively, these results demonstrate that the coronavirus endou activity plays a pivotal role in innate immune evasion in the context of the ifn-i system. endou-deficient coronaviruses induce activation of the oas/rnase l pathway as noted above, coronavirus endou-deficiency results in a pronounced sensitivity to ifn-i treatment that is comparable to that of the highly ifn-i sensitive omt-deficient mutant . virus replication was measured at h.p.i. by plaque assay (mhv) and at h.p.i. by qrt-pcr (hcov- e), respectively. data represent three independent experiments, each performed in two to three replicas. data are displayed as differences to untreated controls and statistical comparisons between wild type and endou-deficient viruses were performed for each concentration. mean and sem are displayed. data points that show significant differences in a two-sided, unpaired student's t-test are depicted. * p < . , ** p < . and *** p < . mhv d a . however, replication of mhv h a was not restored in mda -deficient macrophages. this observation prompted us to consider that replication of endou-deficient coronaviruses may activate additional dsrna-triggered antiviral pathways. we therefore assessed the integrity of ribosomal rna (rrna), a marker for the activation of the oas/rnase l pathway [ ] , during mhv h a infection in primary murine macrophages. indeed, the breakdown of rrna in mhv h a infected wild-type macrophages was readily detectable as early as - h. p.i., thus coinciding with the induction of ifn-β expression during the early phase of the infection (compare figs a and c). this finding is highly surprising since mhv-a encodes a pde activity that has been shown to degrade ', '-oligoadenylate messenger molecules essential for rnase l activation [ ] . however, the pde activity was apparently not sufficient to prevent rnase l activation in macrophages that had been infected with endou-deficient mhv h a . to exclude that the lack of endou activity may directly impact on viral rna synthesis and lead to reduced levels of subgenomic mrnas, we assessed the level of genomic and subgenomic mrna (encoding the pde activity) by qrt-pcr. as shown in s fig, genomic rna and subgenomic mrna were equally reduced in mhv h a infected wild-type macrophages, suggesting that the lack of endou activity does not result in selective reduction of subgenomic mrnas. importantly, while breakdown of rrna was also readily detectable in mda -and mavs-deficient macrophages, rrna remained intact in rnase l-deficient macrophages, demonstrating that infection of endou-deficient mhv h a indeed results in the activation of the oas-rnase l pathway and subsequent degradation of rrna (fig a; s fig) . notably, a breakdown of rrna was not detected in mhv h a -infected ifnar-deficient macrophages ( fig a) concurring with partial restoration of mhv h a replication in these cells. accordingly, and as previously published [ , ] , the degree of rnase l activation correlates with levels of oas expression and we noted indeed reduced baseline expression of oas a, and in ifnar-deficient compared to wild-type c bl/ macrophages (s fig). likewise, we did not observe breakdown of rrna in l cells (fig b) . we therefore assessed the levels of oas a, , and expression in l with or without ifn-i treatment ( . u). as expected, expression of ifn-β was elevated in mhv h a -, but not in mhv-a -infected l cells, irrespectively of ifn-i pre-treatment (s fig). importantly however, expression of oas a, and in l cells was significantly elevated following ifn-i treatment (s fig), and accordingly, rrna breakdown was readily detectable in ifn-i treated l cells that had been infected with mhv h a (fig b) . this data provide evidence for a functional link between the observed pronounced ifn-i sensitivity of mhv h a and restriction of mhv h a replication by the oas/rnase l pathway. surprisingly however, mhv h a replication was not restored in rnase l-deficient macrophages despite the fact that rrna remained intact during the entire replication cycle (compare fig a and fig a) . this strongly suggests that yet another antiviral pathway, in addition to oas/rnase l, is activated during mhv h a infection. one obvious candidate is pkr, a kinase that can be directly activated by dsrna to phosphorylate the eukaryotic initiation factor α (eif α), resulting in translation inhibition of cellular and viral mrnas. indeed, as shown in fig b (left panel) , we readily detected phosphorylated eif α at h.p.i.. in addition, we assessed the extent of translational inhibition at h.p.i. by using puromycin and subsequent facs analysis. as shown in fig b, wild-type mhv-a infected cells showed active translation comparable to mock infected macrophages, while translational inhibition was observed in mhv h a -infected macrophages. finally, we assessed replication of mhv-a and mhv h a in pkr-deficient macrophages, and as shown in fig c, pkr-deficiency alone was also not sufficient for the restoration of mhv h a replication. importantly, however, we observed elevated replication of mhv h a in primary macrophages that are deficient for both, pkr and rnase l that almost reached that of mhv-a ( fig d) [ ] . in order to more precisely analyse the degree of restoration of mhv h a replication in ifnar-and in rnase l/pkr-deficient macrophages, we performed a statistical analysis and compared the differences of calculated mhv-a and mhv h a peak titers between c bl/ and ifnar -/-(p = . ), between c bl/ and rnase l -/-/pkr -/-(p = . ) and between ifnar -/and rnase l -/-/pkr -/-(p = . ) (fig e) . this result shows that mhv h a replication is restored to a comparable degree in ifnar -/and rnase l -/-/pkr -/macrophages. collectively, these results suggest that mhv h a replication results in the early and simultaneous activation of at least three dsrna-triggered pathways, namely ifn-β expression via mda , and antiviral effectors pkr and oas/rnase l. since activation of mda , pkr and oas/rnase l are triggered by dsrna, we assessed if mhv h a infection results in increased appearance of cytosolic dsrna. by using the dsrna-specific antibody j for intracellular staining and facs analysis we assessed the fig b) , ifnar -/-(data level of dsrna in mhv-a -and mhv h a -infected wild-type and ifnar-deficient macrophages at , , , and h.p.i.. at h.p.i. dsrna was not yet convincingly detectable in both mhv-a -and mhv h a -infected wild-type macrophages (fig a) . at h.p.i. dsrna peaks are clearly separated over mock and we observed a slightly stronger dsrna signal in mhv h a -than in mhv-a -infected cells. this difference became convincingly apparent and statistically significant at and h.p.i. with dsrna peaks of mhv h ainfected cells that clearly separated from dsrna peaks of mhv-a -infected cells (fig a and c, right panel) . importantly, we also controlled for virus infection by staining for the replicase complex (nsp / ), and as shown in fig c (left panel) the peaks for nsp / from in mhv h a -infected wild-type macrophages did not exceed those of mhv-a -infected cells. we obtained essentially the same result when we assessed dsrna and nsp / by facs analysis following infection of ifnar -/macrophages (fig b and d) , suggesting that dsrna is also increased in mhv h a -infection under conditions of reduced host cell responses. collectively, these results demonstrate that cytosolic dsrna is increased in endou-mutant virus infection and suggest that elevated dsrna is the trigger for the activation of mda , pkr, and oas. coronaviruses have long been known to efficiently evade host innate immune responses during the early phase of the infection. however, a defined viral function accounting for the apparent lack of efficient sensing of coronavirus infection has remained elusive. here we show that the highly conserved coronavirus endou activity within the viral rtc plays a major role in providing a first line of innate immune evasion during the early phase of coronavirus infection. we show that at least three dsrna-triggered antiviral pathways are involved in restricting replication of endou-deficient coronaviruses (fig ) . first, infection with endou-deficient mhv and hcov- e results in rapid mda -mediated induction of ifn-β expression. second, we observe breakdown of ribosomal rna indicative of activation of the oas/rnase l pathway that temporally coincides with ifn-β expression. third, we show that efficient restriction of endou-deficient coronaviruses is furthermore dependent on pkr since restoration of endou-deficient mhv h a replication required the absence of both, pkr and rnase l. our data suggest that direct restriction of replication of endou-deficient coronaviruses is mediated by rnase l-mediated rna degradation and inhibition of host cell translation through activation of pkr. in contrast, the effect of ifn-i appears to be indirect through the induction of isg expression, that includes oas/rnase l and pkr. whether other isgs may contribute to the restriction of endou-deficient coronavirus replication remains to be determined. finally, we show that mhv h a replication is associated with increased dsrna levels during the early phase of the infection, providing a likely pamp for the observed simultaneous activation of multiple cytoplasmic dsrna-sensors in cells infected with endou-deficient coronaviruses. the concerted activation of multiple cytoplasmic antiviral pathways strongly suggests sensing of the same pamp during replication of endou-deficient coronaviruses. all three types of sensors, mda , oas - , and pkr, are known to recognize dsrna, suggesting that the pamp (s) relevant for their activation during infection is/are of viral origin. notably, rnase l correspond to fig a) and rnase l -/-/pkr -/-(data correspond to fig c) macrophages are displayed. statistical analysis was performed to compare differences of calculated mhv-a and mhv h a peak titers between c bl/ and ifnar -/-(**, p = . ), between c bl/ and rnase l -/-/pkr -/-(**, p = . ) and between ifnar -/and rnase l -/-/pkr -/-(p = . ; ns) macrophages following mhv-a and mhv h a infection. viral endonuclease and innate immunity activation can be triggered by different oas proteins and may depend on particular cell types and virus infections [ ] . for example overexpression of oas was shown to provide rnase l-dependent activity against dengue virus and chikungunya virus infection [ , ] , while oas and oas have been implicated in antiviral activity against hepatitis c virus [ ] . it recently has been shown that among the human oas proteins , , and , oas seems to be mainly responsible for mediating rnase l activation following either polyi:c transfection or virus infection, suggesting a superior role of human oas over oas and oas in restricting virus replication [ ] . interestingly, structural and biochemical studies revealed that oas is selective for binding of long dsrna (> bp) by involvement of an rna-binding, but noncatalytic domain, and that oas is weakly or not activated by short dsrna or single-stranded rna, respectively [ ] . likewise, pkr preferentially dimerizes upon binding to dsrna of similar length (> bp) [ , ] and mda is actually most efficiently activated by even longer dsrna (> kbp) [ ] and higher-order structured rna containing single-stranded and dsrna [ ] . it is thus tempting to propose that viral dsrna represents the natural substrate of the coronavirus endou. however, it remains to be determined which kind of viral dsrna is cleaved by the endou or triggers mda , oas, and pkr activation. compared to mhv wildtype infection we observed a slight but reproducible increase of dsrna in mhv h a -infected macrophages by facs analysis during the early phase of the infection ( - h.p.i.) that became more prominent at - h.p.i., suggesting that the majority of dsrna is not cleaved by endou. this likely includes dsrna being shielded within double-membrane vesicles and replication intermediates actively involved in viral rna synthesis that are likely protected by the rtc and the nucleocapsid protein. we therefore speculate that coronaviruses may have evolved a viral rna quality control mechanism to evade dsrnas sensing, and that endou substrates may comprise dsrna intermediates within stalled rtcs engaged in genome replication or transcription that are no longer active in viral rna synthesis. within the order nidovirales, the endou domain is highly conserved within the families coronaviridae (comprising two subfamilies coronavirinae and torovirinae) and arteriviridae and has been considered a major genetic marker that discriminates nidoviruses from all other rna viruses [ ] [ ] [ ] . however, the recent discovery of insect nidoviruses (family mesoniviridae) [ , ] and re-analysis of the ronivirus genome (family roniviridae; infecting crustaceans) revealed that these two nidovirus families do not encode an endou domain [ ] . in the light of our results it is tempting to speculate that the endou domain has evolved in vertebrate nidoviruses (corona-and arteriviridae) to counteract vertebrate-specific innate immune sensing of viral rna in the context of the type-i interferon system, while the absence of the endou domain in roni-and mesoniviruses is indicative of fundamentally different mechanisms of rna virus innate immune sensing and antiviral effector pathways in invertebrates (e.g. crustaceans and insects) [ ] . mhv as a natural mouse pathogen has been instrumental to understand the delicate balance between host ifn-i responses to infection and counteracting mechanisms of coronavirus innate immune evasion. while mhv evades innate immune sensing in most target cells, plasmacytoid dendritic cells (pdcs) remain as major ifn-i producer cells during early coronavirus replication to ensure protection of mhv target cells and control of potentially lethal coronavirus infections [ , ] . while pdcs sense coronaviral rna within endosomes through tlr , our data demonstrate that the coronaviral endou delays mda -mediated cytoplasmic sensing in macrophages and likely other cell types. this enables coronaviruses to establish robust replication and spread at the entry port of infection. however, in the case of highly pathogenic strains or newly emerging zoonotic coronaviruses, delayed ifn-i responses can have detrimental consequences as recently demonstrated in a murine model of sars-cov infection [ ] . early and rapid sars-cov replication in the respiratory tract combined with a delayed ifn-i response can result in dysregulated innate immune responses and inflammatory cytokine-driven extensive lung damage. therefore, antiviral intervention aiming at inhibiting the coronavirus endou activity may be a promising approach to restore efficient sensing of coronaviral rna and thereby activating ifn-i expression as well as antiviral effector pathways such as pkr and oas/rnase l. there is a growing number of virus-encoded ribonucleases that have been reported to execute diverse steps in the context of cellular mrna quality control and mrna decay [ , ] . for example, herpesvirus-encoded endonucleases are known to broadly target cellular and also viral mrnas that are subsequently further processed by cellular exonucleases, such as xrn , in order to broadly restrict host cell gene expression [ ] . while this strategy indirectly impacts host cell innate immune responses, a more specific interaction of rna decay and host cell dsrna responses has recently been described for vaccinia virus decapping enzymes d and d [ , ] . they remove '-cap structures of partially overlapping vaccinia virus mrnas that arise during the late phase of infection in order to preclude any accumulation of viral dsrna and subsequent activation of dsrna sensors such as pkr and oas. notably, also in this case xrn is required to further process the de-capped mrnas. our data show that early during coronavirus infection the endou activity conceptually fulfils the same task, namely the removal of dsrna that would otherwise trigger host cell dsrna responses, such as ifn-β expression and activation of pkr and oas/rnase l. it will be interesting to address whether xrn is involved in further degrading the coronavirus endou cleavage products or if this function could be fulfilled by the coronavirus-encoded exonuclease (exon) activity residing in nsp . interestingly, like the endou, the coronavirus exon is an integral component of the coronaviral rtc, and exon has been demonstrated to provide an rna proofreading function that permits coronaviruses to stably maintain their extraordinary large rna genome [ ] [ ] [ ] . a functional link between the two coronaviral ribonucleases, exon and endou, would suggest an unprecedented concept of viral rna quality/decay control that goes beyond rna proofreading and includes the removal of dsrna-based pamps at the site of rna synthesis to efficiently evade innate and intrinsic antiviral host cell responses. recombinant mhv strain a , hcov- e, mhv h a , hcov- e h a , and mhv d a were generated using the vaccinia virus-based reverse genetic system as previously described [ , [ ] [ ] [ ] . viruses were propagated on cl mouse fibroblasts (mhv) and on huh- hepacarcinoma cells (hcov- e) and their identity was confirmed by sequencing. to monitor viral spread in vivo, mice at the age of - weeks were injected intraperitoneally with plaque-forming units (pfu) of mhv-a and mhv h a , diluted in mem % ( % heat-inactivated fetal calf serum, penicillin ( μg/ml) and streptomycin ( μg/ml)). mice were euthanized two days post infection (d.p.i.) and liver and spleen were harvested, weighed and homogenized. viral load (pfu/g organ) was determined by plaque assay. murine l fibroblasts (sigma), cl cells (gift from s.g. sawicki) were cultured in mem %. murine embryonic fibroblasts (c bl/ mefs) were maintained at low passage in dmem % (dulbecco's modified eagle medium-glutamax). huh- cells (gift from v. lohnmann) were cultured in dmem % and . mm sodium pyruvate. mrc- cells (human lung fibroblast-like cells; sigma) were maintained at low passage in mem %, supplemented with % non-essential amino acids (neaa). hek -mx -luc cells (gift from g. kochs) were maintained in dmem %, supplemented with g ( μl/ml) [ ] . murine bone marrow-derived macrophages were obtained from mice at the age of - weeks. progenitor cells were isolated from hind limbs, passed through a cell strainer and red blood cell lysis was carried out in ml lysis buffer/mouse ( . m nh cl, mm khco , . mm edta). cells were washed x with pbs and taken up in macrophage medium (imdm iscove's modified dulbecco's medium, - % m-csf (l -supernatant), . % mm -mercaptoethanol). new medium was added d.p.i. and adherent cells were harvested d.p.i. primary human macrophages were obtained from peripheral blood of healthy human donors as previously described [ ] . peripheral blood mononuclear cells were isolated by centrifugation of buffy coat blood over a leucosep tube (greiner bio one). cells from the enriched interphase were collected, washed twice with pbs and red blood cells were removed. cells were taken up in imdm and plated in -well cell culture plates. non-adherent cells were removed three h.p. seeding and adherent cells were cultured for days in imdm %. medium was changed every second day. all experiments using human blood were in accordance with the swiss federal legislation and the institutional guidelines of the cantonal hospital st. gallen and the blutspendedienst srk bern. total cellular rna was isolated from murine macrophages with trizol (life technologies) and genomic dna was removed with dnase (ambion, dna-free dnase treatment). rna concentration was measured by nanodrop and input for cdna synthesis was standardized to ng. synthesis of cdna was carried out using the m-mlv reverse transcriptase from promega and the μl cdna were diluted with μl dh o. the faststart universal sybr green master (rox) mix (roche) was used for measuring mrna expression of ifn-β, gapdh and tbp (s table) . induction of ifn-β was normalized to levels of the household genes gapdh and tbp (geometric mean) and expressed as ΔΔc t over mock (Δc t values calculated as c t reference-c t target) [ ] . expression of oas a, oas , oas and rnase l mrna in mock infected macrophages and l cells was normalized to levels of gapdh [ ] (s table) . expression levels were displayed as Δc t values (c t reference-c t target). copy number of cell-associated viral rna isolated from mhv infected macrophages was determined using the rt taqman pcr system (taqman fast universal pcr master mix ( x), no amperase ung, applied biosystems) with primers and probe specific to the mhv genome fragment encoding the nucleocapsid (s table) . copy numbers were determined by using a standard curve, consisting of an in vitro transcribed rna of known copy number, obtained from a plasmid comprising the mhv-nucleoprotein sequence. quantitative rt-pcr to determine mhv genomic rna and subgenomic mrna two rna standards encompassing mhv nucleotides (nts) - (genomic standard), and mhv nts - / - (corresponding to the mhv mrna leader-body junction; subgenomic mrna standard) were prepared as follows. one rt-pcr product corresponding to the ' region of mhv-a genomic rna was generated by rt-pcr using viral rna from mhv-a infected cells as template and primers t -mhv-leader -frw and mhv-orf -rev (s table) . the resulting rt-pcr product comprised the t -rna-polymerase promoter, mhv nts - . a second rt-pcr product corresponding to the ' region of mhv-a mrna was generated by rt-pcr using viral rna from mhv-a infected cells as template and primers t -mhv-leader -frw and mhv-ns -rev (s table) . the resulting rt-pcr product comprised the t -rna-polymerase promoter, mhv nts - , and mhv nts - . both rt-pcr products were separated on an agarose gel and dna fragments of the appropriate size were excised and purified. in vitro transcribed (ivt) rna was prepared using the ribomax large scale rna production system-t (promega). rq rnase-free dnase was added to the ivt rna and incubated for minutes at ˚c. the in vitro transcribed rna was purified using the nucleospin rna kit (macherey-nagel), its quantity was determined (absorbance at nm) and eight -fold dilutions were prepared. synthesis of cdna was carried out for each dilution using the m-mlv reverse transcriptase and random primers (promega). the ul of cdna were diluted with μl dh o and used as a standard for the quantitative rt-pcr reaction. copy numbers of genomic and subgenomic viral rna were determined for samples obtained from c bl/ macrophages infected with mhv-a and and mhv h a (moi = ). a multiplex reaction (taqman fast universal pcr master mix ( x), no amperase ung, applied biosystems) and primers and a probe specific to the genomic or subgenomic sequence (s table) , respectively, were used. total type-i ifn in supernatants obtained from mhv infected macrophages was measured by an ifn-β enzyme-linked immunosorbent assay (bpl assay science, verikine mouse ifn beta elisa kit, . - pg/ml). technical replicates were pooled. the level of biologically active human type i ifn in the supernatant of infected human macrophages was measured with hek cells that were stably transfected with a luciferase reporter plasmid under the control of the mx-promoter [ , ] . recombinant ifnα a/d (sigma) was used as a cytokine standard and luciferase activity was detected hours p.i. by a luminometer (luciferase assay system, promega). to assess the sensitivity of mhv and hcov- e towards ifn-i, l cells (mhv) and mrc cells (hcov- e) were treated with recombinant ifnα a/d (sigma, as indicated in the figures) for four hours, and then infected with mhv-a , mhv h a , mhv d a , hcov- e and hcov h a (moi = ). virus supernatant was harvested h.p.i (mhv) and h.p.i (hcov- e). viral replication was measured by plaque assay (mhv) or by using primers and a probe specific to the hcov- e membrane protein (s table) and the quantitect probe one- step rt-pcr kit (qiagen). to assess baseline expression, l cells were pre-treated with . u of ifnα a/d for h and then infected with mhv-a and mhv h a (moi = ). cellular rna was isolated with trizol, cdna was prepared and qrt-pcr was performed (s table) . total rna isolated from mhv-infected macrophages and l cells was analysed with a fragment analyzer (labgene) using the dnf- standard sensitivity rna analysis kit ( nt lower marker, advanced analytical technologies). to assess the amount of dsrna-positive cells, c bl/ and ifnar -/macrophages ( x cells) were infected with mhv-a and mhv h a (moi = ). for facs, cells were detached with pbs at ˚c, centrifuged and fixed with % formalin. cells were permeabilized with . % triton and stained with the mouse monoclonal antibody j directed against dsrna ( : , english & scientific consulting bt) and the anti-mhv nsp / rabbit antiserum [ ] ( : ) at ˚c for h. cells were washed, stained with a secondary antibody goat f(ab') anti-mouse igg a, human ads-pe ( : , southernbiotech) and a donkey anti-rabbit alexa- ( : ) for min. facs was performed using the bd facs canto ii and data were analysed using flowjo v. . cell debris was excluded based on a gate of fsca/ssca, followed by a doublet discrimination fsca and fsw. to assess the extent of translation inhibition, c bl/ macrophages were infected with mhv-a and mhv h a (moi = ). min prior to each time point, puromycin (sigma) was added to the wells at a final concentration of μg/ml to label active translation. at the indicated time points, cells were washed twice with pbs, and subsequently detached with pbs at c. cells were fixed in % pfa for min at rt, and washed once with facs buffer (pbs + % bsa). cells were incubated in ice-cold methanol for min at c. after two wash steps in facs buffer, cells were incubated with a primary mouse antibody directed against puromycin ( : , milipore) and a primary rabbit anti-eif α-p (abcam; : ) in facs buffer for min at rt. cells were washed twice with facs buffer and incubated with the secondary antibody donkey anti-mouse-alexa ( : ), and donkey anti-rabbit-alexa ( : ) in facs buffer for min at rt in the dark. cells were washed once in facs buffer, and kept in % pfa in the dark until cells were analysed with the facs canto (bd) using the bd facs diva software. kinetics of virus growth, viral rna and ifn-β mrna were analyzed using non-linear regression. the regression model is an exponential saturation model (increasing response with constant asymptote) that additionally allows a peak response. it is described by the formula where y is the response value (log virus titer or log expression value), t is the time (in hours), a is the value of the asymptote, m is a "midpoint value"representing the time where the exponential increase has reached half of the asymptotic value and where the peak is located, p is a value describing the additional peak height, and s is a scale parameter specifying the steepness of the exponential increase and the width of the peak. the coefficients a, m and p were determined individually for the groups to be compared (mhv-a and mhv h a ), as symbolized by the index g. the model was chosen on pragmatic grounds because it was able to describe the time courses of all data very well and the coefficients represent biologically relevant aspects of the kinetics that can be addressed directly by statistical tests. the analyses were performed in r, version . . [ ] with the function nls [ ] using the algorithm "port" restricting the coefficients to positive values. p-values and confidence intervals were determined by parametric bootstrapping, resampling the residuals from a normal distribution with mean and variance estimated from the variance of residuals of the fitted model. confidence bands were generated by connecting the point-wise % confidence intervals of the predictions. the significance of the difference between treatments in differences between maxima of the groups (i.e., the group-treatment interaction) was determined by bootstapping the difference-in-difference. the assumption of normally distributed residuals was checked and confirmed with normal-quantile quantile plots. all other data were analysed using r v. all experiments using human blood (buffy coat) were in accordance with the swiss federal legislation and the institutional guidelines (including informed consent) of the cantonal hospital st. gallen and the blutspendedienst 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identification of precursors and proteolytic products spanning kilodaltons of orf a. virology r development core team: a language and environment for statistical computing.: r foundation for statistical computing nonlinear models we would like to thank muriel fragnière, arnaud baumann, and ronald dijkman for various technical assistance. we are grateful to susan baker for providing the anti-mhv nsp / rabbit antiserum. we are grateful to matthias schweizer and rune hartmann for input and ideas, and discussing the manuscript. conceptualization: ek rz lcb bl vt. key: cord- -chnibsa authors: hayn, manuel; hirschenberger, maximilian; koepke, lennart; straub, jan h; nchioua, rayhane; christensen, maria h; klute, susanne; bozzo, caterina prelli; aftab, wasim; zech, fabian; conzelmann, carina; müller, janis a; badarinarayan, smitha srinivasachar; stürzel, christina m; forne, ignasi; stenger, steffen; conzelmann, karl-klaus; münch, jan; sauter, daniel; schmidt, florian i; imhof, axel; kirchhoff, frank; sparrer, konstantin mj title: imperfect innate immune antagonism renders sars-cov- vulnerable towards ifn-γ and -λ date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: chnibsa the innate immune system constitutes a powerful barrier against viral infections. however, it may fail because successful emerging pathogens, like sars-cov- , evolved strategies to counteract it. here, we systematically assessed the impact of sars-cov- proteins on viral sensing, type i, ii and iii interferon (ifn) signaling, autophagy and inflammasome formation. mechanistic analyses show that autophagy and type i ifn responses are effectively counteracted at different levels. for example, nsp induces loss of the ifn receptor, whereas orf a disturbs autophagy at the golgi/endosome interface. comparative analyses revealed that antagonism of type i ifn and autophagy is largely conserved, except that sars-cov- nsp is more potent in counteracting type i ifn than its sars-cov- ortholog. altogether, however, sars-cov- counteracts type i ifn responses and autophagy much more efficiently than type ii and iii ifn signaling. consequently, the virus is relatively resistant against exogenous ifn-α/β and autophagy modulation but remains highly vulnerable towards ifn-γ and -λ treatment. in combination, ifn-γ and -λ act synergistically, and drastically reduce sars-cov- replication at exceedingly low doses. our results identify ineffective type i and ii antagonism as weakness of sars-cov- that may allow to devise safe and effective anti-viral therapies based on targeted innate immune activation. α) induce genes containing nf-κb sites in the promotor. signaling of type i ifns (ifn-α and ifn-β), type ii ifn (ifn-γ), type iii ifn (ifn-λ ) and pro-inflammatory cytokine signaling (tnfα and il- α) was quantified using quantitative firefly luciferase reporters controlled by the respective promotors (fig. c) . stimulation with ifn-α and ifn-β (fig. c) revealed that activation of the isre promotor is strongly repressed by nsp , nsp , nsp , nsp , orf and orf b. a similar set of viral proteins interfered with type ii ifn-γ and type iii ifn-λ signaling, albeit much weaker (mean inhibition % and %, respectively) compared to type i ifn signaling (mean inhibition % for ifn-α and % for ifn-β). activation of nf-κb signaling by tnfα or il- α was potently inhibited by the sars-cov- nsp , nsp , nsp , orf a, e, m, orf and orf b proteins. these analyses revealed that a similar set of proteins (nsp , nsp , nsp , orf a, e, m, orf and orf b) antagonizes pro-inflammatory cytokine signaling. since induction of autophagy does not depend on de novo gene expression , we monitored autophagy levels in sars-cov- protein expressing hek t cells by membrane-association of stably expressed gfp-lc b, a hallmark of autophagy induction (fig. d, supplementary fig. e ) . autophagosome numbers under basal conditions were strongly increased in the presence of orf a, e, m and orf a suggesting either de novo induction of autophagy or blockage of turnover (fig. d ). upon induction of autophagy using rapamycin, a similar pattern was observed. to clarify whether these viral proteins induce autophagy or block turnover, leading to accumulation of gfp-lc b positive vesicles, we treated cells with saturating amounts of bafilomycin a , which inhibits autophagic turnover. the increase of autophagosome numbers by orf a, e, m and orf a was drastically reduced compared to non- blocking conditions (fig. d) , indicating that these proteins block turnover, rather than induce it. blockage of autophagy and co-expression of nsp and nsp induced cell death, which may be responsible for the low number of autophagosomes. unexpectedly, in the presence of nsp autophagosome numbers were consistently reduced, suggesting that it inhibits autophagy (fig. d) . inflammasome responses were analyzed in stable thp- cell lines expressing sars-cov- proteins upon doxycycline induction. to avoid any effects of transcription, assembly of asc specks was needle protein mxih using the anthrax toxin delivery system ( fig. e) . asc speck assembly is typically followed by caspase- activation and release of pro-inflammatory . expression of the sars-cov- nsp , nsp and orf c very weakly induced inflammasome activity in the absence of inflammasome activators, although counterselection against cells prone to aberrant inflammasome activation during selection cannot be ruled out. activation of nlrc inflammasomes was not significantly antagonized by any viral protein. taken together, our analysis reveals that sars-cov- encodes multiple proteins that strongly antagonize innate immunity. notably, there are differences in overall inhibition of the pathways with ifn-γ, ifn-λ as well as inflammasome activity signaling being only weakly antagonized. however, type-i ifn induction and signaling and autophagy are strongly repressed. to analyses mechanistically why type-i ifn and autophagy are potently counteracted by sars-cov- , we aimed at identifying the steps that are targeted in these pathways. we focused on the top inhibitors as identified in fig. b -d. nsp was removed from the analysis as it prevents translation in general . to analyses ifn-β signaling, we monitored the levels of the type i ifn receptor, ifnar using western blotting in hek t cells overexpressing nsp , nsp , nsp , orf or orf b. activation of the two major transcription factors of type i ifn signaling, stat and stat (fig. a ) was examined by phosphorylation status. basal stat and stat levels were not significantly affected by all proteins tested (fig. b , quantification in supplementary fig. a-c) . (fig. b ). in the presence of nsp , activated stat and to a lesser extend stat accumulate ( fig. b and d , supplementary fig. a ). orf and orf b did not affect ifnar levels or stat expression or activation ( fig. b-d) . this agrees with recent reports , , suggesting that orf instead prevents trafficking of transcription factors. in the presence of nsp and to a lesser extend for nsp endogenous levels of ifnar is prominently reduced (fig. b, c) . consequently, phosphorylation of stat was decreased upon nsp co-expression (fig. b, d) . lipidated (lc b-ii) to decorate autophagosomal membranes , . upon fusion of autophagosomes with lysosomes, the autophagic receptor p is degraded (autophagy turnover, fig. e ). we analyzed the effect of the top autophagy modulating sars-cov- proteins: nsp , orf a, e, m and orf a (fig. d) on autophagy markers. levels of beclin- and ulk , which parts of the core machinery of autophagy initiation remained constant (fig. f, supplementary fig. d and e) . overexpression of nsp leads to a very slight decrease of lcb -ii but accumulation of p , suggesting that nsp blocks induction of autophagy . in line with this, the number of gfp-lc b-puncta (=autophagosomes) per cell in hela-gfp-lc b cells is reduced upon nsp expression to almost ( fig. i, j) . in the presence of orf a, e and orf a, the levels of processed lc b (lc b-ii) were - to -fold increased (fig. g) , and p levels are approximately . -fold increased (fig. h ). this indicates that these three viral proteins block autophagic turnover. consequently, the number of autophagosomes is -fold increased upon orf a, e, m or orf a expression (fig. i, j) . curiously, while accumulation of lc b-ii indicates that m blocks autophagic turnover or induces autophagy, the levels of p are not significantly altered in the presence of m (fig. f, h) . notably, overexpression of m resulted in an accumulation of lc b in the perinuclear space, whereas for all other viral proteins autophagosomes are normally distributed (fig. i, j) . taken together, our data demonstrates that sars-cov- synergistically targets type-ifn signaling and autophagy. the major type i ifn antagonists nsp , nsp , nsp , orf or orf b block the signaling cascade at different levels. e, orf a and orf a use similar mechanism to block autophagic turnover, while m may have evolved a different mechanism and nsp inhibits de novo autophagy induction. our data showed that orf a and orf a are the most potent autophagy antagonists of sars-cov- ( fig. d, fig. f-j) . to determine their molecular mechanism(s), we performed proteome analysis of hek t cells overexpressing sars-cov- orf a and orf a ( supplementary fig. a) . as a control, we used s, nsp and nsp overexpressing cells which show little to no effect on autophagy (fig. d ). in addition, we analyzed the proteome of caco- cells infected with sars-cov- for or table ) . analysis of the data revealed that in the presence of nsp , cellular proteins with a short half-life are markedly reduced (supplementary fig. f) . this supports our previous finding that nsp globally blocks translation and confirming the validity of the proteome analysis. panther-assisted gene ontology analysis of the proteins regulated more than - fold by the overexpression of individual sars-cov- proteins revealed that orf a and orf a target the late endosome pathway (go: ) (fig. c, supplementary table ) . a similar analysis for the sars-cov- samples showed that the late endosome pathway is also affected during the genuine infection. thus, we had a closer look at the subcellular localization of orf a and orf a and their effect on intracellular vesicles. in line with the proteome analysis, orf a and orf a both localized to the late endosomal compartment, co-localizing with the marker rab (fig. d ,e). in contrast, localization to rab a-positive early endosomes was not apparent ( supplementary fig. g ). disturbance of the integrity of the trans-golgi network (tgn) at the interface with the late endosomes , by viral proteins is a well-known strategy to block autophagy . immunofluorescence analysis revealed that the localization of orf a or orf a partially overlap with a tgn marker (r = . , fig. g) indicating close proximity. orf , which is known to localize to the golgi apparatus was used a positive control (r= . ). nsp , which displayed a cytoplasmic localization was used as a negative control (r= . ). importantly, analysis of free tgn-marker positive vesicles in sars-cov- orf a or orf a expressing cells revealed that both viral proteins cause significant fragmentation of the tgn (fig. f, h). these data indicate, that both orf a and orf a disturb the proteome at the late endosomes eventually causing the tgn to fragment, which leads to a block of autophagic turnover [ ] [ ] [ ] [ ] . to examine the conservation of innate immune antagonism, we functionally compared nsp , nsp , nsp , nsp , m, n, orf a, orf and orf a of sars-cov- , the closest related cov, cov and the previous highly pathogenic sars-cov- . ratg -cov was isolated from the measles virus v protein and trim expression served as positive controls. overall, proteins of sars- cov- and ratg behave similar to their sars-cov- counterparts, suggesting that many functions are conserved. importantly, however, this is not the case for nsp , nsp and to a lesser extend orf ( fig. a-c) . sars-cov- orf is about -fold less potent in antagonizing type i ifn signaling (fig. b) but induces higher levels of autophagy (fig. c) . however, expression levels of sars-cov- orf were also higher than that of its sars-vov- and ratg counterparts ( supplementary fig. g) , which may explain the differences in activity. differences between sars-cov, ratg and sars- cov- nsp were reanalyzed in a dose-dependent manner, and only in the range of - -fold which may also explained by differential expression (supplementary fig. j) . the most striking, statistically significant difference was observed for nsp . sars-cov- nsp is over -fold more potent in suppression of type i ifn induction and signaling than ratg and sars- cov- nsp (fig. a, b) . notably, expression levels of sars-cov- , ratg and sars-cov- nsp are similar, with sars-cov- nsp even being slightly less expressed ( supplementary fig. c). notably, all nsp variants still inhibit autophagy (fig. c) . dose-dependent effect of sars-cov- nsp , ratg -cov nsp and sars-cov- nsp on type i ifn induction (fig. d ) and signaling ( fig. e) showed that on average sars-cov nsp performed -fold worse than sars-cov- nsp , and ratg nsp inhibited type i ifn induction . -fold less (fig. d) . similarly, sars- cov- nsp outperformed ratg and sars-cov- nsp by -and . -fold, respectively, in inhibition of type i ifn signaling (fig. e) . taken together, this data indicates, that while most ifn antagonist activities are conserved between sars-cov, ratg and sars-cov- , there is an exception: nsp of sars-cov- is considerably more potent than sars-cov- nsp in counteracting both ifn-β induction and signaling. inefficient antagonism by sars-cov- proteins is predictive for efficient immune control immune activation , albeit with different efficiency. the mean inhibition of ifn-γ and ifn- λ signaling was % and %, respectively, compared to type i ifn signaling with a mean inhibition of only % for ifn-α and % for ifn-β. consequently, we assessed whether ifn-α , ifn-β, ifn- γ and ifn-λ have a different impact on sars-cov- (fig. a, supplementary fig. a , b). treatment with the type i ifn-α was the least efficient. in contrast, at the same concentration ifn-γ ( u/ml) reduced viral rna in the supernatant almost -fold more efficiently. all agents caused little if any cytotoxic effects ( supplementary fig. c ). altogether, we observed a good correlation (r= . ) between average inhibition of the respective signaling pathway (fig. c) antagonized by the sars- cov- proteins and ifn susceptibility at u/ml (fig. b) . in contrast to type ii and ii ifn signaling, autophagic turnover was strongly repressed by at least four sars-cov- proteins ( fig. c and fig. ) . thus, based on our inhibition data ( fig. c) we would expect that modulation of autophagy only weakly affects sars-cov- replication. indeed, treatment with rapamycin, which induces autophagy, reduced viral replication to a maximum of - -fold ( supplementary fig. e ). bafilomycin a , which blocks autophagy, had little to no effects ( supplementary fig. e ). both drugs were used at concentrations that only marginally affected cell survival ( supplementary fig. f ). thus, our results indicate that the overall efficiency of sars-cov- proteins in counteracting specific signaling pathway is predictive for the overall antiviral potency of the pathway, as illustrated by different types of ifns. ifn therapy is commonly associated with significant adverse effects, due to induction of inflammation. to minimize detrimental pro-inflammatory effects of ifns, doses required for efficient viral restriction should be reduced. thus, we analyzed the impact of the most potent ifns, ifn-γ and ifn-λ and their combination of sars-cov- . to mimic prophylactic and therapeutic treatment we examined pre- treatment for h before infection with sars-cov- and treatment h post-infection. overall, the treatment but consistent (fig. c, d) . expression analysis of sars-cov- s and n confirmed the qpcr results, and equal gapdh levels exclude effects on viral replication by cytotoxicity (fig. d ). while treatment with a single dose of ifn-γ and ifn-λ alone reduced viral rna production - -fold, the combinatorial treatment at the same concentration potentiated the effect to about -fold reduction in sars-cov- rna (fig. c) . to further decrease inflammatory side-effects by ifn treatment, anti-inflammatory pathways like autophagy could be induced - . treatment with rapamycin, which induces autophagy, already reduces viral replication ~ - -fold at nm (fig. c ). treatment of rapamycin ( nm) in combination with either ifn-γ or ifn-λ was found to be additive (fig. c, d) . triple treatment with ifn-γ, ifn-λ and rapamycin showed the most potent anti-viral effect of all combinations for pre-treatment and post- treatment, reducing viral rna in the supernatant by -fold and -fold, respectively (fig. c) . in summary, our data shows that the anti-sars-cov- effect of combinatorial treatments of ifn-γ, ifn-λ are synergistic. additional activation of anti-inflammatory autophagy by rapamycin further decreased sars-cov- replication. this suggests that concerted activation of innate immunity may be an effective anti-viral approach, exploiting vulnerabilities of sars-cov- revealed by analysis of its innate immune antagonism. viruses drastically alter our innate immune defenses to establish an infection and propagate to the next host , , , , , . our data reveal the extend of immune manipulation sars-cov- employs. we determined the major antagonists of type i ifn induction and signaling as well as pro-inflammatory nf-κb activity encoded by sars-cov- (nsp , nsp , nsp , nsp , orf and orf b). type ii and iii ifn signaling is targeted by a similar set of proteins, although much less efficient. autophagy is majorly targeted by nsp , orf a, e, m and orf a. inflammasome activity is very weakly induced by nsp , nsp and orf c, but none of the sars-cov- proteins block formation of the nlr c inflammasome. subsequent mechanistic studies revealed that sars-cov- proteins synergistically nsp lowers the cellular levels of the ifn receptor, ifnar, thus blocking activation of the crucial transcription factors stat and stat . both orf a and orf a cause fragmentation of the tgn via disturbing the late endosomal pathway. this is a common strategy of viruses to block autophagic turnover. examination of the functional conservation showed that sars-cov- nsp was less efficient in blocking innate immune activation, both type i ifn induction and signaling, than sars- cov- nsp . this may ultimately cause sars-cov- to be better controlled by the innate immune system than sars-cov- , explaining higher numbers of subclinical infections and thus overall lower mortality rates of the current pandemic cov. overall, the combined analysis of ifn antagonism allowed us to deduce that treatment with type-i ifns and regulation of autophagy is only weakly anti-viral. in contrast, treatment with ifn-γ and ifn-λ drastically reduced sars-cov- replication. finally, combinatorial treatment of sars-cov- with these two ifns potentiated the effects of the individual treatments. this may pave the way for future anti-viral therapies against sars-cov- based on rational innate immune activation. why would multiple effective proteins target the same pathway? for example, type i ifn signaling could have been shut down by nsp , nsp , nsp , nsp , orf and orf b alone, each reducing the activation of the innate immune pathways to below %. however, our assays revealed (figs. - ) that the targeting mechanisms are often not redundant and may act synergistically. this could allow the virus to better control the targeted pathway, thus minimizing the effect of the signaling on its replication. in addition, a viral protein majorly targeting one pathway may affect other connected immune pathways at once. for example, disturbance of the kinase tbk activation may affect primarily ifn induction and to a lesser extend also impact autophagy . proteome analyses revealed the late endosome/golgi network as a target of orf a and orf a. our data suggests, that both orf a and orf a of sars- cov- cause fragmentation of golgi apparatus and thus blockage of autophagy. sars-cov- orf a was previously already implicated in golgi fragmentation, thus our data suggests that sars-cov- orf a uses a similar strategy , . notably, fragmentation of the golgi is for example triggered by hepatitis c virus viruses to block anti-viral autophagic turnover and thus may represent a common studies will see more mechanistic data to explain the molecular details of the impact of sars-cov- proteins on innate immune activation. notably, several proteins including orf , orf a, orf a, m and e accumulate at the golgi network or in perinuclear spaces, alluding to the emerging role of the golgi as a hub for immune manipulation , . our results demonstrate that orf , orf a, orf a and orf b are the strongest innate immune antagonists among the accessory genes of sars-cov- (fig. ) . besides the accessory genes, which classically encode immune antagonists, a surprising number of non-structural proteins manipulate innate immunity. nsp , which targets cellular translation and thus broadly inhibits any response enzymatic functions may impact their activity against innate immunity. except for nsp , as its activity as a de-isglase may inactivate the transcription factor irf and thus reduce ifn induction . according to our analysis the structural proteins e and m strongly manipulated autophagy (fig. d ). this suggests that the incoming virion may already block autophagic turnover to prevent their own degradation by however, while we may pick up most counteraction strategies, our screening approach may miss immune evasion strategies employed by sars-cov- . for example, many non-structural proteins form complexes, that are not formed during single overexpression and may only be functional as a full assembly. evasion mechanisms based on rna structures and sequences are lost due to usage of codon- optimized expression plasmids. finally, the virus itself may employ strategies to hide itself from recognition, thus not activating innate immune defenses in the first place. one example is the capping would be immediately recognized by the cytoplasmic sensor rig-i. our analyses further revealed that the human innate immune antagonism is largely conserved in the sars-cov- closest related bat isolate, ratg (fig. ) . this indicates that the bat virus is capable of counteracting the human immune defenses, which may have facilitated successful zoonotic transmission from bat eventually to humans. currently, the intermediate animal host of sars-cov- is under debate , - , however it is likely, that the virus isolated from it is even closer related to sars- cov- than ratg . thus, any immune evasion mechanisms conserved between sars-cov- and ratg , is likely to be conserved in the direct progenitor virus of sars-cov- . the previous epidemic and related human sars-cov- and the current pandemic sars-cov- differ in susceptibility towards ifn s with sars-cov- being more resistant . furthermore, infection with sars-cov- is often asymptomatic and likely controlled by the host as lower mortality rates and higher subclinical infections suggest . paradoxically, this may support the fast spread of the virus. thus, sars-cov- may have found the 'perfect' balance. intermediate immune evasion and thus intermediate pathogenicity to support spread, but not kill the host. our data shows that sars-cov- nsp is strikingly less in efficient in ifn evasion than nsp of sars-cov. these data are the first mechanistic evidence why sars-cov- is less susceptible towards ifn treatment than sars-cov- . it may be tempting to speculate that common cold covs counteract the innate immune system less efficiently than sars-cov- . our analysis indicates that during a sars-cov- infection less cytokines than expected are released, autophagic turnover is blocked and general immune activation is perturbed. this is supported by a large amount of data from covid patients [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] . however, an important question remains: why are some innate immune pathways, such as ifn-γ signaling less antagonized (fig. ) ? are the viral immune manipulation strategies ineffective? indeed, ifn-γ is most active against sars-cov- among the ifns (fig. ) . one possible explanation would be that there was no need for the virus to antagonize them. indeed, in covid patients and in vitro infections with sars-cov- , ifn-γ levels are surprisingly low , . furthermore, despite high ifn-γ levels being a hallmark of cytokine storms ifn-γ expression in cd + t cells is associated with severe covid , , . it is tempting to speculate that t-cells which confer pre-existing immunity against sars-cov- , could, upon activation, release ifn-γ, whose innate immune signaling may also contribute to increased clearance of the infection. strikingly, our work thus shows that analysis of the innate antagonism may be predictive for therapeutic opportunities. severe side effects are prevalent for treatments with ifns - . however, the side-effects are dose- dependent . thus, minimizing the dose required for treatment is paramount. our data indicates that effects of treatment with multiple ifns is additive but synergistic and potentiates each other (fig. ) . thus, a promising anti-viral approach may be a combinatorial treatment of different cytokines, effectively also reducing the burden of side-effects. the side effects of ifn therapy are mainly caused by inflammation. combined with anti-inflammatory approaches such as autophagy activation by rapamycin , , this approach may even be more successful, as our in vitro data suggests. future studies are highly warranted to study rational, concerted innate immune activation against sars-cov- in vivo. these studies may eventually pave the way for novel therapies, which may not only work against sars-cov- , but also against other pathogenic viruses, including potentially future covs. in summary, our results reveal the extend of innate immune manipulation of sars-cov- . comparison to sars-cov- revealed that mutations in nsp may be responsible for the higher susceptibility of sars-cov- against ifns. finally, our data allowed us to deduce a potent immune activation strategy against sars-cov- : combinatorial application of ifn-γ and ifn-λ. ratg , and sars-cov- were synthesized by twist bioscience and subcloned into the pcg vector using restriction cloning using the restriction enzymes xbai and mlui (new england biolabs). firefly luciferase reporter constructs, harboring binding sites for nf-κb or irf , isre or gas sites, or the genomic promoter of ifna or ifnb in front of the reporter were a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china the proximal origin of sars-cov- quantifying sars-cov- transmission suggests epidemic control with digital contact tracing sars : epidemiology cumulative number of cases and deaths in various countries in middle east respiratory syndrome comparing sars-cov- with sars-cov and influenza pandemics hosts and sources of endemic human advances in virus research rig-i in rna virus recognition sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon. current opinion in virology sars coronavirus and innate immunity innate immunity to virus infection the antiviral activities of trim proteins. current opinion in microbiology intracellular detection of viral nucleic acids. current opinion in microbiology trim proteins and their roles in antiviral host defenses cov- infection: apossible smart targeting of the autophagy pathway involvement of autophagy in coronavirus replication trim proteins: new players in virus-induced autophagy autophagy during viral infection -a double-edged sword regulation of adaptive immunity by the innate immune system control of adaptive immunity by the innate immune system inborn errors of type i ifn immunity in patients with life-threatening covid- auto-antibodies against type i ifns in patients with life-threatening type i interferon susceptibility distinguishes sars-cov- from multi-level proteomics reveals host-perturbation strategies of sars-cov- sars-cov- orf b is a potent interferon antagonist whose activity is increased by a naturally occurring elongation variant structural basis for translational shutdown and immune evasion by the nsp protein of sars-cov- . science ( -. ). 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transcriptional suppressor of nf-kb-elicited antiviral immune responses trim senses and restricts influenza a virus by ubiquitination of pb ifitm proteins promote sars-cov- infection of human lung cells assessment of inflammasome formation by flow cytometry key: cord- - mqmpzw authors: qian, wei; wei, xiaoqin; guo, kelei; li, yongtao; lin, xian; zou, zhong; zhou, hongbo; jin, meilin title: the c-terminal effector domain of non-structural protein of influenza a virus blocks ifn-β production by targeting tnf receptor-associated factor date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mqmpzw influenza a virus non-structural protein (ns ) antagonizes interferon response through diverse strategies, particularly by inhibiting the activation of interferon regulatory factor (irf ) and ifn-β transcription. however, the underlying mechanisms used by the ns c-terminal effector domain (ed) to inhibit the activation of ifn-β pathway are not well understood. in this study, we used influenza virus subtype of h n to demonstrate that the ns c-terminal ed but not the n-terminal rna-binding domain, binds tnf receptor-associated factor (traf ). this results in an attenuation of the type i ifn signaling pathway. we found that the ns c-terminal ed (named ns / - ) inhibits the active caspase activation and recruitment domain-containing form of rig-i [rig-i(n)]-induced ifn-β reporter activity, the phosphorylation of irf , and the induction of ifn-β. further analysis showed that ns / - binds to traf through the traf domain, subsequently decreasing traf k -linked ubiquitination. ns / - binding also disrupted the formation of the mitochondrial antiviral signaling (mavs)–traf complex, increasing the recruitment of ikkε to mavs; ultimately shutting down the rig-i(n)-mediated signal transduction and cellular antiviral responses. this attenuation of cellular antiviral responses leads to evasion of the innate immune response. taken together, our findings offer an important insight into the interplay between the influenza virus and host innate immunity. of caspase activation and recruitment domains (card). as the cascade continues, different residues in rig-i are ubiquitinated by the e ligases trim ( ) and riplet ( ) , resulting in rig-i oligomerization. subsequently, rig-i interacts with the adaptor protein mitochondrial antiviral signaling (mavs) via their card-card association, which in turn results in mavs oligomerization. through tnf receptor-associated factor (traf ) or traf , this leads to activation of the kinase complexes containing tbk or ikkε and ikkα/β/γ. through several final phosphorylation steps, these kinases ultimately elicit antiviral and pro-inflammatory responses through interferon regulatory factor (irf ) and nuclear factor κb (nf-κb), respectively ( , ) . influenza a virus belongs to the orthomyxovirus family, containing eight negative-sense rna segments in an enveloped viral particle encoding or proteins ( ) . this array of proteins contributes to virulence; including the proteins associated with viral rna-dependent rna polymerase ( ) and the nonstructural protein (ns ). ns consists of - amino acids and comprises two functional domains: an n-terminal rnabinding domain (rbd) (aa to ) and a c-terminal effector domain (ed) (aa -end) ( ) . the ns protein plays a crucial role in regulating the host antiviral response through various mechanisms. one important function of the ns protein involves inhibition of ifn production. the mechanism of this inhibition includes activation of the transcription factors irf ( ) , nf-κb ( ) , and ap- ( ) , thus blocking ifn production. this efficient inhibitory effect is associated with an rig-i signaling pathway through the ns -rig-i complex ( ) ( ) ( ) . previous studies have indicated that ns is also related to two positive factors of rig-i, the e ligases trim ( ) and riplet ( ) . the residues e / e of ns mediate their interaction with the coiled-coil domain of trim , thus blocking both trim multimerization and rig-i card domain ubiquitination. this subsequently induces lower levels of ifn-β ( ) . ns can also interact with riplet preventing the activation of rig-i, although e /e are not involved in that inhibition ( ) . the dsrna binding ability of ns could also be playing a role in the pre-transcriptional inhibition of the interferon pathway by sequestering the pathogenassociated molecular patterns (pamps) that rig-i recognizes. two residues, r and k , are required for the dsrna binding activity of ns ( ) , thus highly impairing its ability to block interferon production. in another similar pathway, ns has been shown to inhibit host mrna synthesis by binding a cellular ′ end-processing factor, the kda subunit of the cleavage and polyadenylation specificity factor (cpsf ), thus attenuating type i interferon (ifn-α/β) and other interferon stimulated gene (isg) mrnas that are involved in the antiviral response ( ) . the ns proteins encoded by the seasonal h n , h n , h n , and avian h n viral subtypes strongly bind to cpsf ( ) , whereas pr , pandemic h n , and novel h n virus do not efficiently bind cpsf ( ) . it is noteworthy that cells infected with viruses expressing ns proteins in seasonal h n and h n viruses do not inhibit irf activation. however, activation is blocked in cells infected with viruses expressing ns proteins in some, but not all, seasonal h n viruses, pandemic h n , and avian h n viruses. trim was previously reported to interact with each of these ns proteins, whether or not they block irf activation, indicating that binding of trim by the ns protein does not necessarily lead to blocking of irf activation ( ) . hence, binding of the ns protein to dsrna, rig-i, and trim has not established that these ns interactions are responsible for inhibiting the activation of irf and ifn transcription. in this case, one or more host factors may participate in the ns blocking of irf activation. in view of several yet undetermined roles of ns in the inhibition of interferon, we conducted a study aimed to determine the importance of the function of the n-and/or c-terminal domains of the ns protein in immune evasion. by using the luciferase reporter assay, we were able to demonstrate that the c-terminal ed (aa to , named ns / - ) of the ns protein was sufficient to inhibit the production of ifn-β driven by rig-i(n). mechanistically, ns / - was found specifically to interact with traf , to dissociate mavs-traf complex, and to decrease k -linked polyubiquitination of traf . this was shown to result in reduced irf -dependent production of ifn-β, with subsequent enhancement of virus replication. these data reveal a novel mechanism for how the influenza a virus ns protein induces inhibition of the host ifn production and may provide a potential target for antiviral drug development. the hpai h n virus strain, a/duck/hubei/hangmei / (h n ; designated h n /hm) was isolated from a duck. influenza a virus (strain a/puerto rico/ / h n ), a/ pr/ / , was grown in our laboratories and stored until use. rns -sd was constructed as previously described ( ) . influenza virus stocks of h n /hm, pr , and rns -sd strains were amplified using -day-old embryonic chicken eggs and then titrated by determining log tcid /ml values in mdck cells. all cell experiments with h n virus were performed in an animal biosafety level laboratory (bsl- ). this study was carried out in accordance with the recommendations of bsl- , huazhong agricultural university (hzau). the protocol was approved by the bsl- of hzau. the recombinant vesicular stomatitis virus (vsv) encoding green fluorescence protein (vsv-gfp) was a gift from the harbin veterinary research institute (harbin, china). sendai virus (sev) was grown in -day-old embryonic chicken eggs and titrated using a hemagglutination assay as previously described ( ) . human embryonic kidney t cells and hela cells were purchased from atcc (manassas, va, usa) and cultured at °c with % co in roswell park memorial institute- medium (hyclone, china) supplemented with % fetal bovine serum (fbs) (pan-biotech, germany), containing u/ml penicillin, and mg/ml streptomycin (gnm cell lysates and the immunoprecipitates were resolved by - % sds-page and transferred to pure nitrocellulose membranes (ge). the membranes were blocked in % bovine serum albumin (bsa) in tbst buffer for h at room temperature and probed with indicated primary antibodies for - h at room temperature. after hybridizing with either goat anti-rabbit or goat anti-mouse secondary antibodies at a dilution of : , in tbst buffer, the membranes were washed with tbst buffer for four times ( min each) before visualized with ecl reagents (advansta). hela cells were plated onto coverslips in -well plates and transfected with the indicated plasmids. at h post transfection, cells were washed once with phosphate-buffered saline (pbs) and fixed in % paraformaldehyde for min. cells were permeabilized with . % triton x- for min and blocked for h at room temperature with % bsa in pbs, followed by incubation with primary antibody for h. after three washes with pbs containing . % tween , cells were incubated with fitc or cy -conjugated secondary antibodies for h at room temperature and then incubated with ′, -dapi for min. finally, the coverslips were washed extensively and fixed onto slides. images were taken under a zeiss lsm meta confocal microscope (carl zeiss, zena, germany). quantitative real-time pcr (qrt-pcr) total rna was isolated from cells using trizol reagent (invitrogen) following manufacturer's instructions and cdna was prepared by using avian myeloblastosis virus reverse transcriptase (takara). cdna was used for quantification of the indicated mrna copy number on an abi viia pcr system (applied biosystems, usa) by using sybr green master mix (rox). to detect and validate the specific amplification of pcr products, dissociation curve analysis of the products was conducted at the end of each pcr. transcript levels of each gene were normalized with the expression of β-actin, and the −∆∆c t method was used to analyze gene expression in the samples ( ) . the primers used in qrt-pcr are listed in table . three sirna oligonucleotides against traf and the corresponding negative control sirna were obtained from genepharma. sequences are as follows: si- , ′-ccacuggagag augaauau- ′; si- , ′-guugugcagagcaguuaau- ′; and si- , ′-cugguuacuuuggcuauaa- ′. transfection of sirna into t cells was performed by lipofectamine according to manufacturer's instructions. t cells were seeded into -mm dishes and transiently transfected with the indicated plasmids. thirty-six hours after transfection, cells were harvested and the lysates were prepared in a % np- lysis buffer supplemented with a commercially available . % protease inhibitor cocktail and a mm deubiquitinase inhibitor n-ethylmaleimide (sigma-aldrich). samples were immunoprecipitated with µg anti-flag antibodies along with µl protein a/g plus-agarose. polyubiquitination was detected using anti-ha antibodies. influenza a virus infection of a cells a cells were transfected with the indicated plasmids for h at °c. cells were washed twice with f medium and then infected with h n /hm or pr at an indicated moi ( or . , the results are expressed as means ± sd. statistical analyses were performed on data from triplicate experiments by using twotailed student's t-test. a p-value of less than . was considered significant and a p-value of less than . was considered highly significant. the h n ns protein inhibits the rigi(n)-mediated activation of ifn-β via its c-terminal ed in an rna bindingindependent manner with the aim of elucidating the mechanism by which iav ns protein counteracts the host innate immune responses, we generated four truncated h n ns proteins, ns / - , ns / - , ns / - , and ns / - ( figure a) . in order to investigate the function of wtns , and its truncated peptides, we assessed its effect on ifn-β promoter activity using a luciferase reporter assay in t cells. our results showed that wtns , ns / - , and ns / - significantly decreased the ifn-β reporter activities driven by rig-i or rig-i(n). conversely, ns / - did not change the activity of ifn-β reporter, and ns / - only slightly increased the activity compared to an empty vector control ( figure b) . in addition, we found that wtns and all truncated peptides had inhibitory effects on ifn-β reporter activities induced by sev or rns -sd virus infection or transfection of poly(i:c) ( figure b) . this suggests that ns n-terminal rbd alone is sufficient to inhibit the activation of ifn-β only in the presence of dsrna; the c-terminal ed of ns could inhibit the activity of ifn-β reporter in all tested conditions. driven by rig-i(n), ns / - caused a dose-dependent inhibition of ifn-β promoter activity and ifn-β transcription ( figure c) . previous studies indicated that iav ns sequesters dsrna and binds rig-i at its rbd, subsequently inhibiting the activation of irf and preventing the induction of ifn-β ( , ) . our findings reveal that c-terminal ed of ns (ns / - ) blocked rig-i(n)-mediated ifn-β induction in an rna binding-independent manner. transcription factor irf is a key innate immune system component that mediates ifn-β induction. once irf is phosphorylated, it forms a dimer, translocates into the nucleus from the cytoplasm, and induces the expression of ifn-β and isgs through specifically binding to their promoter regions ( ) . in order to further investigate how ns / - inhibits the signaling that mediates type i ifn production, we used an irf -luciferase reporter plasmid, allowing for the measurement of irf activation. as shown in figure a , irf -luciferase reporter activation by rig-i(n) was blocked in t cells overexpressing ns / - or wtns . we next addressed whether ns / - affected the dimerization and nuclear localization of endogenous irf mediated by rig-i(n). non-reduced sds-page and immunoblot analysis of cell lysates after rig-i(n) transfection showed that ns / - or wtns expression produced a considerable reduction in the activated dimer form of irf (figure b) . similarly, rig-i(n)induced phosphorylation of irf was strongly repressed by ns / - or wtns and that phosphorylated irf was largely distributed in nuclear fractions ( figure c) . elisa assays of ifn-β in the medium of cells transfected with plasmids encoding ns / - or wtns along with rig-i(n) showed that both ns / - and wtns inhibited the production of ifn-β ( figure d) . together, these data indicate that ns / - inhibits the expression of type i ifn induced by rig-i(n) through blocking the phosphorylation of irf . previous studies have shown that a recombinant vsv-gfp system can be used as a strategy to screen proteins possessing ifn-antagonizing activity ( ) . in the present study, we employed recombinant vsv-gfp to investigate if ns / - serves as an antagonist of ifn production. when t cells expressed ns / - , a high level of vsv-gfp replication was present, consistent with wtns protein (figure e) , suggesting that the inhibitory effect of ns / - on ifn production is also present during actual viral infection. in order to test the effect of ns / - on various components of the rlr pathway, ns / - and expression (figure a) . by contrast, wtns significantly decreased the rlr adaptor-mediated ifn-β promoter activity. as expected, ns / - exhibited a decrease in the ifn-β mrna level induced by rig-i(n) or mavs ( figure a ). in addition, the secretion of ifn-β and the transcription level of isgs triggered by tbk or irf ( d) in the presence of ns / - were tested. ns / - induced nearly a complete loss of the inhibition of ifn-β and isgs, including oasl, pkr, and mx , whereas wtns strongly blocked the production of ifn-β and isgs mrna expression (figures b,c) . together, these data indicate that ns / - significantly inhibits the cellular antiviral response at the level between mavs and tbk . in the rlr-mediated signaling pathway, traf serves as a critical link between the adaptor mavs and downstream regulatory kinases that are essential for irf activation ( , ) . thus, we hypothesized that traf is the target of ns / - . co-ip experiments revealed that ns / - interacted selectively with traf but not other components (figures a,b) . in another experiment, wtns and ns / - also interacted most potently with traf ( figure c ). this association was confirmed under physiological conditions in an experiment that detected this interaction by overexpression of flag-traf in infected a cells, where rig-i served as a positive control ( figure d) . furthermore, pr ns also interacted with traf in infected a cells (figure e ). to address whether the wtns protein physically interacts with endogenous traf , we performed endogenous ip assays on h n /hm or pr -infected cell lysates. our results showed that traf could be co-precipitated by the ns antibody ( figure f ). in addition, the ns proteins of the strain a/shanghai/ / (h n ) and other avian the promoter activities were detected by the dual-luciferase assay system. all luciferase assays were repeated at least three times, and the data shown are mean ± sd from one representative experiment. significance was analyzed with a two-tailed student's t-test (*p < . or **p < . , ***p < . ). h n strain did not bind traf ( figure s in supplementary material). these results demonstrate that ns / - or wtns interacts with traf in a strain-specific manner. based on the findings that ns / - or wtns interacted with traf , we next asked whether the two molecules co-localize in cells. confocal microscopy revealed the co-staining of ns / - or wtns and traf in cells, suggesting the co-localization of the two proteins ( figure g) . normally, expression of wtns in hela cells resulted in a nuclear localization with minor cytoplasmic staining. traf overexpression led to a marked increase in the cytoplasmic localization of wtns or ns / - . these results indicate that ns / - or wtns colocalize with traf in cells and traf overexpression led to a marked increase of ns / - or wtns cytoplasmic localization. traf is essential for ns / - to downregulate ifn-β the above data showed that ns / - blocks ifn-β induction and interacts with traf . hence, we used traf sirna to determine whether traf is essential for the regulatory function of ns / - in t cells. knockdown of traf by sirna diminished ifn-β promoter activity triggered by rig-i(n) (figures a,b) . in cells with silenced traf , after transfection of rig-i(n), the induction of ifn-β was greatly reduced and the inhibitory effect of ns / - was markedly attenuated. nevertheless, this inhibitory effect was rescued successfully with a traf expression plasmid ( figure c) . these data suggest that traf is necessary for ns / - to decrease ifn-β activity. it is well-known that traf is modified with a polyubiquitin chain to provide a scaffold for complex formation. thus, in order to study the effect of ns / - on traf ubiquitination, co-ip experiments were conducted. flag-traf , pub-ha, and ns / - along with rig-i(n) were cotransfected into t cells and the ubiquitination level of traf was monitored. compared to the control, ns / - reduced the rig-i(n)-induced ubiquitination of traf (figure , left) . to explore the type of traf ubiquitin chains, flag-traf was transfected into t cells with ubiquitin mutants, including the pub-k -ha or pub-k -ha expression plasmid. the results showed that ns / - could decrease the k -linked ubiquitination of traf but not the k -linked ubiquitination of traf (figure , right) . in summary, these results demonstrate that ns / - suppresses the k -linked ubiquitination of traf that is important for the recruitment of the tbk -ikkε kinase complex. expressing aa to and containing the ring domain, the zinc-fingers domain, the isoleucine zipper domain, and flag-traf -td, expressing aa to and containing the traf domain ( figure a ). co-ip assays showed that the traf domain of traf was found to be the crucial region responsible for the association of traf with ns / - ( figure b ) as well as with wtns (data not shown). previously, it has been reported that the tim domain of mavs interacts with amino acid residues y and q within the traf domain of traf ( ) . to examine whether ns / - affected ifn signaling at the level of mavs-traf interaction, mavs-traf association was determined in the presence of ns / - . expression of mavs led to an interaction with traf and was increased by rig-i(n) transfection. however, ns / - and wtns markedly disrupted this interaction by . -and . -fold, respectively ( figure c) . in h n /hm infection of a cells, the mavs-traf complex was decreased by . -fold compared to the control ( figure d) . as reported previously, ikkε is recruited to the c-terminal region of mavs following sev or vsv infection, mediated by lys -linked polyubiquitination of mavs at lys , resulting in the inhibition of downstream ifn signaling ( , ) . therefore, it was necessary to test whether wtns or ns / - affect this process to accomplish its negative regulatory role in ifn-β production after sev infection. the interaction of mavs and ikkε was readily detected by co-ip, while sev infection resulted in a significant decrease in the mavs-ikkε interaction. interestingly, the interaction of mavs and ikkε was remarkably increased when cotransfected with ns / - or wtns (figure e) , indicating that ns / - or ns promotes the recruitment of ikkε to mavs. furthermore, we measured the secretion of ifn-β in cell supernatants. results showed that ns / - or ns inhibited the production of ifn-β mediated by mavs-traf or mavs-ikkε (figure f) , implying that ns / - functions in downregulating ifn expression. taken together, these results indicate that the association of the mavs-traf complex is disrupted by ns / - , which, in turn, blocks ifn-β production. to determine whether the replication of iav is enhanced by the ns / - protein, a cells were transfected with ns / - , or an empty vector, then infected with different titers of h n /hm or pr virus. upon infection with h n /hm or pr virus, ns / - could facilitate transcription of np of both viruses (figure a) , resulting in an increase in np and ha proteins observed in the ns / - group (figure b ). this was confirmed by the titers of h n /hm or pr virus, which significantly increased by -and -fold, respectively, in ns / - overexpressing cells compared with control cells (figure c) . these results demonstrate that ns / - enhances the capacity of iav to replicate in cells. prrs of host cells recognize a pamp and subsequently initiate a series of signaling cascades. the final step is activation of irf and nf-κb, thus inducing the transcription of ifn-β ( ) . given the cascade of responses triggered by the host in response to infection, influenza viruses adapted different strategies to escape the ifn response. this survival tactic has proven successful in order for virus proliferation and infection. both pb and pb -f limit ifn production by associating with mavs ( , , ) . other structural proteins, such as pb , pa, np, and even the genomic rna itself, also contribute to impairing rig-i-mediated antiviral responses ( ) . moreover, ha (ha ) was recently shown to drive the degradation of the ifn receptor chain ifnar , thereby suppressing ifn-triggered jak/stat signaling ( ) . the most effective weapon influenza a viruses have at their disposal is ns protein. the ns protein acts as an antiviral antagonist protein capable of limiting ifn production. rig-i recognizes and binds dsrna structures with ′-triphosphates upon infection to initiate the host antiviral response. during the course of viral infection, the ns protein of iav inhibits host ifn responses either by sequestering viral dsrna or by binding to rig-i and trim or riplet proteins required for rig-i activation and ifn signaling pathways ( , , , ) . in this study, we found that the ns c-terminal ed (aa to ) of h n virus inhibits the activation of ifn-β pathway. to achieve a negative regulatory function in the cellular antiviral response, ns / - associates with traf to remove the lys -polyubiquitin chains on traf and to disrupt the mavs-traf complex. ns / - also increases the recruitment of ikkε to mavs, releasing traf from the mitochondria. this further decreases the level of k linked ubiquitination of traf , impairing irf phosphorylation and reducing the production of ifn-β (figure ) . interestingly, our study has shown that the ed of ns protein possesses the ability to suppress ifn response in the absence of rna. typically, the rbd of ns mediates the inhibition of ifn synthesis, and the ed of ns induces the inhibition of gene expression, together with its known interactors ( ) . in this study, we have found that the ns c-terminal ed (ns / - and ns / - ), but not the rbd (ns / - and ns / - ) block ifn-β reporter activity induced by rig-i(n). expression of ns / - resulted in the inhibition of irf activation, indicating that the ns protein blocks ifn-β activation through an rna-independent manner. it was demonstrated in previous studies that influenza a viruses tx/ and a/viet nam/ / expressing c-terminally truncated ns proteins of , , or amino acids were attenuated. the resultant reduced growth correlated with a high level of ifn-α/β induced by these mutant viruses ( , ) . in addition, in both influenza b and c viruses, the c-terminal domains of the ns proteins were found to possess ifn antagonist activity ( , ) . more importantly, the n terminus-truncated ns proteins encoded by pr , which was to elucidate the mechanism of how the ns ed inhibits ifn-β activation, we speculated that the ns ed contacts its counterparts in rig-i signaling leading to inhibition. for this purpose, we examined a step within the signaling pathway that ns / - targets and found that ns / - acted downstream of mavs and upstream of tbk . the co-ip assays showed unexpectedly that ns / - binds to traf , which interacts with mavs forming a platform for rna virus signaling. we also tested the binding of traf to the full-length ns protein and found that this interaction exists, and co-localized in the cytoplasm. the ns / - protein mainly localized in the cytoplasm. traf expression led to a marked increase in the ns / - cytoplasmic localization, suggesting that ns / - inhibits the activation of the ifn-β pathway. although all types of influenza virus ns proteins interact with trim , only part of ns prevents irf activation, indicating that trim is not required for the inhibition of irf activation. in this study, we did not observe the interaction between ns / - and rig-i, consistent with the results described in a previous publication ( ) . consequently, rig-i seems to be non-essential for the optimal inhibition of ifn production in iav-infected cells. however, our study demonstrated that the influenza a virus ns ed targets traf , subsequently inhibits ifn production, implying that traf is a key factor involved for iav to escape host innate immune responses. the mavs-traf complex is a focal point of rlr-directed signaling response ( , ) . traf localizes to the endoplasmic reticulum (er) and needs to be recruited to mitochondrial mavs in order to activate tbk complexes ( ) . many viral proteins, accessory and non-structural proteins in particular, hijack traf or the traf complex to mediate immune evasion. sars coronavirus m protein or open reading frame- b prevents the formation of traf -tank-tbk /ikkε complex or mavs-traf /traf signalosome to evade host innate immunity ( , ) . sars-cov papain-like protease interacts with and disrupts sting-traf -tbk complex, it also inhibits the tlr -mediated innate immunity through removing lys linked ubiquitin chains of traf and traf ( , ) . herpes simplex virus ubiquitin-specific protease ul deubiquitinates traf then counteracts the ifn-β pathway ( ) . over the past years, there have been major advances in understanding how influenza a viruses successfully escape the surveillance of the immune system. the current report furthers this research revealing the surprising finding that ns / - acts by targeting traf ; specifically, ns / - targets the traf domain of traf . traf links the upstream ifn signaling responses of mavs to tbk relying on the traf domain. this report also shows that a specific interaction between traf and mavs was observed when traf and mavs were co-expressed in t cells. however, the interaction between traf and mavs was disrupted in the presence of ns / - . interestingly, the interaction between mavs and ikkε was markedly increased in ns / - -expressing cells. it has been previously demonstrated that, after sev infection, k -linked polyubiquitination at lys of mavs recruits ikkε to the mitochondria, functionally causes release of traf from mavs initiating the signal to shutdown the ifn response ( ) . the mavs-ikkε complex was enhanced when ns / - was present, indicating that ns / - can utilize this process to shut down further activation of ifn pathway. taken together, these data indicate that ns / - impedes the interactions between components of mavs-traf complex, preventing the phosphorylation of irf , where it would activate the ifn-β response. ubiquitination has emerged as a key posttranslational modification that controls induction and shutdown of the interferon response. traf , serving as a crucial functional link, is modified with a polyubiquitin chain providing a scaffold for complex formation, and, not surprisingly, many viruses encode proteins that inhibit ubiquitination processes to overcome host innate responses. previous studies showed that nairoviruses and arteriviruses encode for ovarian tumor domain-containing proteases that hydrolyze ubiquitin chains from host proteins ( , ) . in this report, we have shown that ns / - suppresses the k linked ubiquitination of traf . it is likely that ns / - works through recruiting a deubiquitinase to cleave the traf ubiquitin chain since it has been shown that ns / - does not belong to any known deubiquitinase family. for example, duba, a member of the otubain (otub) family, has been shown to deubiquitinate traf and negatively regulate tlr -and rig-i/ mda -mediated ifn induction ( ) . it was also shown that two otub deubiquitinating enzyme family members, otub and otub , can deubiquitinate traf and traf , leading to the inhibition of virus-induced ifn-β expression and cellular antiviral responses ( ) . therefore, whether these proteins, or other dub proteins, are involved in this regulation, and the detailed regulatory mechanism of traf activity triggered by ns / - remains to be discovered. interestingly, strain-specific targeting of traf was demonstrated by specific interaction of ns proteins encoded by pr or avian h n but not novel h n or avian h n viruses. this difference may be associated with strain-specific sequence variations. the ns protein most often occurs as a residue peptide, including ns of seasonal h n virus and avian h n virus ( - residues have been deleted since ), which were used in this study. however, premature stop codons or, alternatively, suppression of the genuine stop codon (codon ) resulted in length variations at ns 's c-terminus. abdelwhab et al. analyzed ns protein sequences of all aiv subtypes in birds from to to study the prevalence and distribution of carboxyl terminal end truncation (Δcte). they found that ns proteins lacking amino acids - were the most prevalent form ( %). this truncation is prevalent in lpaiv of non-h /h subtypes; particularly h n , h , and h viruses that are known to be widespread and mostly (semi)endemic in land-based poultry ( ) . similar truncations have also been observed in swine influenza viruses, which harbor a c-terminally truncated ns and have also been found in human h n viruses that have been in public circulation since the pandemic ( ) . hence, whether the interaction of ns and traf are associated with the Δcte requires further investigation. in summary, the present study demonstrated that traf is a target of the c-terminal ed (aa to ) of h n ns protein, revealing a novel function of the ns protein in modulating host innate immunity and possibly facilitating iav infection. the physiological significance of the ns ed in iav replication and its pathological role in flu diseases warrant further investigation to probe the potential value of this molecule as a therapeutic and/or disease prevention target. viral rna detection by rig-i-like receptors sensing viral invasion by rig-i like receptors rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates rig-i detects viral genomic rna during negative-strand rna virus infection trim ringfinger e ubiquitin ligase is essential for rig-i-mediated antiviral activity riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the roles of tlrs, rlrs and nlrs in pathogen recognition regulation of rig-i-like receptor signaling by host and viral proteins molecular mechanisms enhancing the proteome of influenza a viruses: an overview of recently discovered proteins the pb subunit of the influenza virus rna polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon the multifunctional ns protein of influenza a viruses activation of interferon regulatory factor is inhibited by the influenza a virus ns protein influenza a virus ns protein prevents activation of nf-kappab and induction of alpha/beta interferon the influenza a virus ns protein inhibits activation of jun n-terminal kinase and ap- transcription factors ns protein of influenza a virus inhibits the function of intracytoplasmic pathogen sensor, rig-i inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus ifnbeta induction by influenza a virus is mediated by rig-i which is regulated by the viral ns protein influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns protein rna binding by the novel helical domain of the influenza virus ns protein requires its dimer structure and a small number of specific basic amino acids influenza virus ns protein interacts with the cellular kda subunit of cpsf and inhibits ' end formation of cellular pre-mrnas influenza a virus strains that circulate in humans differ in the ability of their ns proteins to block the activation of irf and interferon-beta transcription a single amino acid substitution in the novel h n influenza a virus ns protein increases cpsf binding and virulence effect on virulence and pathogenicity of h n influenza a virus through truncations of ns eif gi binding domain multiple anti-interferon actions of the influenza a virus ns protein analysis of relative gene expression data using realtime quantitative pcr and the (-delta delta c(t)) method immune signaling by rig-i-like receptors mutations in the ns protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs critical role of traf in the toll-like receptor-dependent and -independent antiviral response traf : uncovering the real but restricted role in human a functional c-terminal traf -binding site in mavs participates in positive and negative regulation of the ifn antiviral response ubiquitinregulated recruitment of ikappab kinase epsilon to the mavs interferon signaling adapter pathogen recognition and innate immunity the influenza virus protein pb -f inhibits the induction of type i interferon at the level of the mavs adaptor protein influenza virus protein pb -f inhibits the induction of type i interferon by binding to mavs and decreasing mitochondrial membrane potential to conquer the host, influenza virus is packing it in: interferon-antagonistic strategies beyond ns hemagglutinin of influenza a virus antagonizes type i interferon (ifn) responses by inducing degradation of type i ifn receptor live attenuated influenza viruses containing ns truncations as vaccine candidates against h n highly pathogenic avian influenza the n-and c-terminal domains of the ns protein of influenza b virus can independently inhibit irf- and beta interferon promoter activation influenza c virus ns protein counteracts rig-imediated ifn signalling role of n terminus-truncated ns proteins of influenza a virus in inhibiting irf activation triggering the innate antiviral response through irf- activation ikkepsilon and tbk are essential components of the irf signaling pathway proteomic profiling of the traf interactome network reveals a new role for the er-to-golgi transport compartments in innate immunity severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk /ikkepsilon complex sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex sars coronavirus papain-like protease inhibits the tlr signaling pathway through removing lys -linked polyubiquitination of traf and traf herpes simplex virus ubiquitin-specific protease ul inhibits beta interferon production by deubiquitinating traf viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways viral otu deubiquitinases: a structural and functional comparison duba: a deubiquitinase that regulates type i interferon production regulation of virustriggered signaling by otub -and otub -mediated deubiquitination of traf and traf prevalence of the c-terminal truncations of ns in avian influenza a viruses and effect on virulence and replication of a highly pathogenic h n virus in chickens stop-codon variations in non-structural protein ns of avian influenza viruses the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer, bn, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -fad yyqx authors: felgenhauer, ulrike; schoen, andreas; gad, hans henrik; hartmann, rune; schaubmar, andreas r.; failing, klaus; drosten, christian; weber, friedemann title: inhibition of sars–cov- by type i and type iii interferons date: - - journal: j biol chem doi: . /jbc.ac . sha: doc_id: cord_uid: fad yyqx the recently emerged severe acute respiratory syndrome coronavirus- (sars–cov- ) is the causative agent of the devastating covid- lung disease pandemic. here, we tested the inhibitory activities of the antiviral interferons of type i (ifn-α) and type iii (ifn-λ) against sars–cov- and compared them with those against sars–cov- , which emerged in . using two mammalian epithelial cell lines (human calu- and simian vero e ), we found that both ifns dose-dependently inhibit sars–cov- . in contrast, sars–cov- was restricted only by ifn-α in these cell lines. sars–cov- generally exhibited a broader ifn sensitivity than sars–cov- . moreover, ruxolitinib, an inhibitor of ifn-triggered janus kinase/signal transducer and activator of transcription signaling, boosted sars–cov- replication in the ifn-competent calu- cells. we conclude that sars–cov- is sensitive to exogenously added ifns. this finding suggests that type i and especially the less adverse effect–prone type iii ifn are good candidates for the management of covid- . we tested the effect of type i ifn against sars-cov- compared with the sars-cov- from . two different cell lines were employed, namely the human bronchial epithelial calu- and the primate kidney epithelial vero e . the cells were first treated for h with , , or units/ml of recombinant human ifn-a(b/d) and then infected with the viruses at a multiplicity of infection (moi) of . plaque forming units (pfu)/cell to obtain multistep growth. virus titers in supernatants were determined h later, when titers are reaching a plateau (see below). the data of three biological replicates are shown in fig. . because several titers were below the detection limit of our plaque assay, a rank correlation test (spearman's exact rank correlation test) was used for statistical doseresponse correlation analysis. for sars-cov- (dark gray bars), statistically significant negative correlation coefficients (cc) were obtained for both cell lines, indicating that viral replication is increasingly inhibited by ifn-a. for sars-cov- (light gray bars), titers were also affected. however, at least in vero e cells, the reduction of sars-cov- appears to be weaker than the reduction of sars-cov- (fig. b) . observations were similar when the input moi was reduced to . (fig. s ), except that titers of sars-cov- in calu- cells were already very low in the absence of any ifn-a, resulting in a nonsignificant effect of additional ifn. these data may suggest that the potency of ifn to reduce viral titers may be stronger and more consistent against sars-cov- than against sars-cov- . to further investigate the potential differences between the viruses, we repeated the experiment three times more with the intermediate dose of units/ml and analyzed the data statistically after pooling them with the previous three replicates. two-way anova was used to simultaneously evaluate the influence of both ifn-a and virus species on virus reduction. this analysis (fig. , a and b) showed again that (i) both viruses are reduced by ifn (comparison of versus units/ml ifna, p(ifn)) and (ii) there are differences between the sars-cov species (comparison of the virus experiments, p(virus)). moreover, the "interaction" p value showed that, at least in vero cells, the degree of ifn sensitivity depends on the virus species, again indicating that sars-cov- is more ifn-sensitive than sars-cov- . the predominant antiviral cytokine ( , ) . although the ifn induction as well as signaling and up-regulation of ifn-stimulated genes (isgs) are very similar, type iii ifns engage a different receptor that is restricted to epithelial cells, and generate a weaker but longer-lasting antiviral response ( , ) . ifn-l was previously shown to have activity against coronaviruses ( , , ) and proposed as potential covid- treatment ( ) . hence, we compared the sensitivity of the two sars-coronaviruses also to recombinant human ifn-l. as shown in fig. a , pretreatment with or ng/ml ifn-l exhibited only in vero e cells a dose-dependent inhibitory effect on sars-cov- . for sars-cov- , by contrast, no significant inhibition was noted in any of the cell lines. to further investigate the difference between the viruses, we repeated the ifn-l experiment three times more with the intermediate dose of ng/ml and analyzed the data after pooling with the previous ng/ml ifnl experiment (fig. b ). conventional statistical analysis (onetailed student's t test, because none of the values was below the detection limit) again revealed a significant impact of ifn-l on sars-cov- and the lack of an effect for sars-cov- . our data thus show that ifn-l can inhibit sars-cov- but not sars-cov- . a recent study on the host cell interactome of sars-cov- identified a number of human proteins for which food and drug administration-approved drugs are available ( ) . ruxolitinib, a compound known to target the type i and type iii ifntriggered jak/stat signaling pathway ( ) , was among the proposed inhibitors of virus-host cell interactions ( ) . because virus inhibition by an ifn inhibitor seems counterintuitive, we aimed to clarify the influence of this compound on sars-cov- replication. cells were pretreated with mm ruxolitinib for h and infected at the two different mois, and titers were measured or h later. as shown in fig. , with this setting titers in nontreated controls are already reaching a plateau at the -h time point. in calu- cells, ruxolitinib had a ( ) . our data thus indicate that (i) if anything, ruxolitinib is an enhancer rather than an inhibitor of sars-cov- multiplication, and (ii) the boosting effect is most likely due to inhibition of the antiviral jak/ stat signaling pathway, because it is not present in the ifn induction-deficient vero e cells. our observations so far suggest that sars-cov- is consistently more sensitive to ifns than sars-cov- . moreover, type i ifn seems to have a more profound effect than type iii ifn. to see whether principal differences in signaling or subsequent gene expression could account for these phenomena, we tested the ability of the cell lines to respond to the ifns. the immunoblot analysis (fig. ) shows that calu- cells have a very similar reaction to both types of ifn concerning phosphorylation of stat and stat and expression of the ifn-stimulated mxa and isg . vero e cells also responded to ifn-l as expected ( ) , but the isg response was lower than to ifn-a. moreover, in nontreated calu- cells, there was already a background isg expression, which was not observed in vero cells. ruxolitinib was in principle able to influence these isg responses, as expected, but it was more potent against ifn-l than against ifn-a, and its effects on ifn-stimulated genes were more evident in the vero e compared with the calu- (fig. ) . thus, both cell lines are capable to respond to the different types of ifn, but ifn-l was less potent, which is in agreement with our observations on sars-cov sensitivity, as well as with previous studies ( , ) . the recently emerged sars-cov- is responsible for major health crises all over the world. here, we show that type i and type iii ifns are able to inhibit sars-cov- replication, with effects that in our hands were consistently more profound than against the sars-cov- from . it should be noted however, that the differences between the viruses could be due to the cell types used or due to the observed differences in virus replication (which could result in higher production of ifn antagonists). thus, the question whether sars-cov- is intrinsically more sensitive to ifns remains to be solved. pegylated ifn-a was the standard of care against chronic infection with hepatitis c virus until the recent introduction of other, directly acting antiviral drugs ( ) . although associated with some side effects, ifn-a is well-characterized, has been used to treat millions of patients, is considered safe, and is available figure . sensitivity of sars-cov- and sars-cov- to type iii ifn. a, experiments were performed as described for fig. , except that recombinant human ifn-l was used. log-transformed titers of each virus dose-response experiment with concentrations of and ng/ml ifn-l were analyzed by spearman's exact rank correlation test. ccs and exact one-sided p values are provided. b, three additional biological replicates of the ng/ml ifn-l were performed, and the resulting titer data were pooled with the ng/ml ifn-l data from a. log-transformed titers were analyzed by unpaired one-tailed student's t test. n.s., nonsignificant. immediately. ifn-l has undergone phase i and ii clinical trials with hepatitis c virus ( ) . it exhibited excellent tolerance as well as efficacy, but the phase iii trials were abandoned because of the availability of effective direct antivirals. ifn-l holds promise as having fewer side effects because of its restriction to mucosal tissue and the less sudden but more prolonged antiviral response it triggers ( , ) . in line with our results, a series of preprints show that also others found type i and type iii ifns to be effective against sars-cov- replication in cell culture ( ) ( ) ( ) . also in earlier in vivo studies with sars-cov- , both type i and type iii ifns were shown to be important for the control of infection or the associated disease ( - ). clinical data on the usage of type i ifn against sars-cov- or the related mers-cov, however, are limited, are not always conclusive, or did not show a clear benefit ( ) ( ) ( ) ( ) ( ) . thus, type iii ifn-l rather than the side effect-prone type i ifns ( ) might be considered for clinical testing against sars-cov- . ruxolitinib was proposed as a potential treatment against sars-cov- ( , ) , and a small clinical trial is underway (clinicaltrials.gov, nct ), although case reports were discouraging ( ) . the replication boost obtained with ruxolitinib on the ifn-competent calu- cells indicates that ruxolitinib is not at all inhibiting sars-cov- replication. thus, drugs that interfere with viral host interactors may not necessarily be antiviral but rather boost the infection. calu- and vero e cells were cultivated in dulbecco's modified eagle's medium supplemented with % fetal bovine serum (thermo fisher scientific) in a % co atmosphere at °c. sars-cov- (strain sars-cov- /münchen- . / / , p. ) ( ) and sars-cov- (strain sars-fra , p. ) ( ) were grown on vero e cells and purified via vivaspin columns (sartorius stedim biotech). the viruses were titrated on vero e cells. infection experiments were done under biosafety level conditions with enhanced respiratory personal protection equipment. of note, all cells were tested mycoplasma-negative. the cells were pretreated for h with the indicated amounts of pan-species ifn-a(b/d) (pbl assay science) ( ), purified recombinant ifn-l ( , ) , or with mm ruxolitinib (selleckchem). infections were performed at a moi of . and . . at the indicated times postinfection, cell supernatants were collected and titrated by plaque assay on vero e cells. the cells were treated for h with the indicated amounts of ifns or ruxolitinib (added h before ifn) and lysed in t-per protein extraction reagent (thermo fisher) supplemented containing protease inhibitor mixture (c mplete, roche), phosphatase inhibitor mixture set ii (calbiochem), and sample buffer ( . mm tris-hcl, ph . , . % glycerol, . % sds, . mm bromphenol blue). protein samples were run on % acrylamide gels and transferred to polyvinylidene fluoride membranes (millipore) via semidry blotting. after blocking in tbs with % bsa (for detection of phospho-stats, mxa, and total stat ) or milk powder (all other detections), primary antibody staining was performed overnight at °c. the membranes were washed in tbs with . % tween , stained with secondary antibodies for min, and washed again in tbs with . % tween and once in tbs. finally, the membranes were developed with a supersignal west femto kit (pierce), and the bands were visualized using a chemidoc imaging system (bio-rad). the the statistical analysis of the data were done by means of the statistical program packages bmdp ( ) and statxact ® (version . . ). for the statistical testing of the dose-response effect of ifn (type i and iii) against sars-coronaviruses, the typical regression procedures were not applicable because of several values below the detection limit and some ties in the data. instead of this, the nonparametric spearman rank cc was used in the exact version (software statxact). because the scientific question was clearly one-sided formed (only pfu reduction under application of ifn), one-sided p values were given. if only two ifn concentrations were to compare with no data below the detection limit, then the t test for independent sam-ples was used (program bmdp d). for testing the effect of ifn and virus type simultaneously, the two-way anova (program bmdp d) was applied especially considering a possible interaction between the two tested factors. in the parametric statistical analyses as well as the graphical representations, the response variable pfu was logarithmically transformed because of its right skewed statistical distribution. in all cases a statistical significance level of a = . was applied. all data presented and discussed are contained within the article. author contributions-u. f., a. s., r. h., a. r. s., k. f., c. d., and f. w. conceptualization; u. f., c. d., and f. w. validation; u. f., a. s., a. r. s., and k. f. investigation; u. f. and f. w. visualization; coronaviridae study group of the international committee on taxonomy of viruses ( ) the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- genome composition and divergence of the novel coronavirus ( -ncov) originating in china shared and distinct functions of type i and type iii interferons guarding the frontiers: the biology of type iii interferons interferon-l orchestrates innate and adaptive mucosal immune responses interferon lambda: disease impact and therapeutic potential type i interferon in chronic virus infection and cancer tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ 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receptor independent mechanism pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques combined action of type i and type iii interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread interferon-lambda contributes to innate immunity of mice against influenza a virus but not against hepatotropic viruses ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study sensitivity of sars/ mers cov to interferons and other drugs based on achievable serum concentrations in humans sars: systematic review of treatment effects disease-promoting effects of type i interferons in viral, bacterial, and coinfections covid- : combining antiviral and anti-inflammatory treatments side effects of ruxolitinib in patients with sars-cov- infection: two case reports transmission of -ncov infection from an asymptomatic contact in germany identification of a novel coronavirus in patients with severe acute respiratory syndrome a recombinant human interferon-alpha b/d hybrid with a broad host-range the influence of the rs single nucleotide polymorphism on ifn-l activity and secretion bmdp statistical software manual key: cord- -klv jdm authors: smith, abigail l.; barthold, s. w.; de souza, m. s.; bottomly, kim title: the role of gamma interferon in infection of susceptible mice with murine coronavirus, mhv-jhm date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: klv jdm infection of balb/c mice with mouse hepatitis virus, strain jhm (mhv-jhm), at any of several intervals relative to ovalbumin (ova) administration resulted in elevated ova-specific igg a titers. since gamma interferon (ifn) has been implicated as an up-regulator of igg a production, attempts were made to determine whether levels of this cytokine were modified in sera of infected mice. serum ifn-γ was not detected, but treatment of mhv-jhm-infected mice with monoclonal anti-ifn-γ antibody resulted in high mortality with decreased survival times, enhanced virus titers in liver and spleen, and more severe virus-associated pathology, compared to mock-treated, infected mice. immunotherapy with recombinant ifn-γ ameliorated disease as reflected by mortality rates and virus titers in target organs. mice inoculated with soluble protein antigens preferentially produce igg and igg specific to that antigen [ , ] . in contrast, infection of mice with any of several viruses enhances the igg a response both to the infecting virus and to immunizing soluble protein antigens [ ] [ ] [ ] . gamma interferon (ifn), a cytokine preferentially produced by the th subset of cd + t cells [ ] , stimulates production of igg a by b lymphocytes activated in vitro and in vivo and inhibits igg secretion [ , , ] . thus, it has been postulated that the immune response triggered by viruses mainly activates the th subset of helper t cells [ -] . mouse hepatitis virus (mhv) is a singular name for a group ofcoronaviruses, family coronaviridae, infecting laboratory mice worldwide at high prevalence [ , , ] . mice infected with mhv can exhibit aberrant immune responses that interfere with their usefulness in biomedical research [ , , , [ ] [ ] [ ] . coutelier and co-workers [ ] showed altered protein-specific isotype responses of mice infected with the a strain of mhv one day prior to antigen administration. the initial goal of the currently reported experiments was to determine whether mhv-jhm infection yielded similar results and whether timing of virus exposure was critical to observing the effect, as has been reported for other experimental systems altered by mhv infection [ , ] . mhv-jhm infection of the balb/c mouse was exploited as representative of a combination of moderately virulent, pantropic virus in a commonly used permissive genotype. based on the observation of elevated ovalbumin (ova)-specific igg a levels in sera of mice infected with mhv-jhm at any of several intervals relative to antigen exposure, attempts were made to detect ifn- in sera of infected mice and to reverse the igg a elevation by administration of ifn-y-specific antibody. the effect of this antibody and of immunotherapy with exogenous recombinant ifn- on virus-associated mortality and pathology and on virus replication in target organs was examined. female balb/cbyj mice (the jackson laboratory, bar harbor, me) were five to seven weeks old at the time of virus exposure. young adult balb/c nu/nu (athymic) and nu/+ (euthymic) mice were obtained from life sciences, inc. (st. petersburg, fl). cr:orl sencar dams with litters were obtained from the animal genetics and production branch, nci (bethesda, md). randomly selected mice were free of antibody to common murine viruses on arrival, all mice were housed in micro-isolator cages (lab products, maywood, nj) and were given food and water ad libitum. manipulations and husbandry were performed in a class ii biological safety cabinet, and infected mice were housed in a facility separate from that used for control (uninfected) mice. an open-cage sentinel mouse seromonitoring program was in place during the course of the reported studies. seroconversion to none of common murine viruses or mycoplasma pulmonis was detected, except among mhv-inoculated, immunocompetent mice which seroconverted to the infecting virus. mhv-jhm (american type culture collection, rockville, md) was used in the form of an infected infant mouse brain homogenate. mice immunized with ova or given antibody to ifn- were exposed orally to infant mouse iclds of virus. mice treated with recombinant ifn- were exposed intranasally to the same dose of virus. intranasal exposure of balb/c mice generally results in nearly % mortality, whereas mortality rates after oral inoculation are less than % (unpubl. obs.). groups of mice were infected per os with mhv-jhm on days - , - , - , , + , or + relative to intraperitoneal immunization with ova ( ~tg/mouse) in freund's complete adjuvant (fca). additional groups of uninfected mice received ova plus fca or fca only. mice were killed with co gas and exsanguinated on days or post-ova or fca administration. data from three replicate experiments were pooled. the numbers of mice per group are shown in table . mice were monitored for seroconversion as evidence of infection by indirect immunofluorescent staining using individual sera diluted : and a bivalent antigen consisting of mhv-jhm and mhv-s, as previously described [ ] . results are expressed as geometric mean titers [ ] for sera with significant a values at a dilution of : or greater. selected sera from mice tested for igg a were eliminated from the analysis due to unacceptably high background readings. ten percent tissue homogenates were clarified by centrifugation, and the supernates were screened for infectivity by intracerebral inoculation of litters of two day old sencar mice. virus was quantified by inoculating serial log dilutions of tissue homogenates. mortality during a one week period was recorded, and virus titers were calculated by the method of reed and muench [ ] . monoclonal antibody to mouse gamma ifn, designated xmg . [ ] , was precipitated with saturated ammonium sulfate and extensively dialyzed against pbs, ph . . the final product contained x neutralizing units per ml of antibody when tested with units of recombinant gamma ifn in the wehi b cell lymphoma bioassay [ ] . mice were given x l neutralizing units daily by the intraperitoneal route, beginning one day prior to virus exposure through one day prior to necropsy. recombinant mouse ifn- (lot no. ; specific activity ~> x units/mg; purity > % according to manufacturer) was obtained from amgen biologicals (thousand oaks, ca). mice were given x units intraperitoneally and × units intranasally once daily on days (- ) through (+ ) relative to virus exposure. liver and spleen were fixed in % neutral buffered formalin, paraffin embedded, sectioned at ~tm and stained with hematoxylin and eosin. chi square analysis was used to test for differences in proportions, and student's unpaired t test was used to analyze differences in mean virus titers. sera from mice infected with mhv-jhm one day prior to ova administration contained substantially elevated levels of antigen-specific igg a on day (table ) with geometric mean titers that were times control (ova only) values at that interval. there was a suggestion of a delayed igg a response among mice infected with mhv-jhm three or five days after ova administration, since sera from one of mice tested on day contained ova-specific igg a, whereas four of eleven ova-immunized, uninfected mice had seroconverted at that interval (% = . ; p < . ). sera from higher proportions of mice in groups that received mhv-jhm on days - ( %), - ( %) or ( %) relative to ova contained ova-specific igg a on day than the proportion that were ova-immunized, but uninfected ( %); however, these differences were not statistically significant. mice infected four days prior to ova administration had ova-specific igg titers that were sevenfold lower than those of ova-immunized, uninfected mice (table ) , and the proportion of igg -positive sera ( %) was reduced compared to uninfected control sera ( %). results are shown as number positive at a dilution of : or greater/number tested, and geometric mean enzyme-linked immunosorbent assay titer ± sd for positive sera sera collected from mice at days after ova immunization yielded a more clear-cut pattern. igg a titers were elevated in sera from mice that received virus any time from seven days prior to ova through one day after ova ( table ) . as had been seen among mice bled seven days after administration of ova, the most marked increase in ova-specific igg a ( -fold increase over controls) occurred among mice infected one day prior to immunization. sera from all infected mice except those exposed to virus seven days prior to immunization contained reduced levels of ova-specific igg (table ) . however, the greatest reduction (mice infected three days after ova immunization) was three-fold. mice infected seven days prior to immunization yielded igg titers equivalent to uninfected controls. isotype distribution of ovalbumin-specific antibody was also analyzed by determining igg :igg a ratios for sera from mice bled on day (fig. ) . although the two tests may have inherently different sensitivities, this parameter would be expected to remain relatively constant, since the two assays were always run in parallel, and the same reagents were used throughout the study. this analysis revealed that igg levels in sera from uninfected, ova-immunized mice were -fold higher than igg a levels. in contrast, the ratio was less than one for sera from mice infected one day prior to immunization and measured on day . the elevated igg a ova-specific response of most groups of mhv-infected mice and the preferential production of this isotype by mice infected one day prior to antigen administration suggested that these mice were producing ifny. however, using the wehi b cell lymphoma bioassay [ ] , attempts to detect the cytokine in sera of infected mice failed, as had earlier attempts to ] . it was reasoned that, ififn-y was being produced below the level of assay detection and was responsible for the induction of an elevated igg a response, treatment of ova-immunized, mhv-infected mice with anti-ifn- should restore, at least partially, the igg ova-specific response. in two experiments, % of mice inoculated orally with mhv-jhm and treated with xmg . died with survival times substantially shorter than those of mock-treated, infected mice ( . - - . days vs . ± . days). this suggested that ifn-y was produced during mhv infection and was influencing the course of disease. in an attempt to delineate the role of ifn-y in the pathogenesis of mhv-jhm infection, groups of athymic or euthymic balb/c mice were treated with monoclonal anti-ifn-y (xmg . ) or were mock-treated with dialysis buffer (pbs) and exposed orally to virus. athymic mice were included because they produce ifn-y by virtue of fully competent natural killer cells [- ]. four mice per genotype and treatment group were necropsied daily on post-infection days one through five. liver and spleen sections from all mice were examined histologically, and virus in the same organs of mice necropsied on days four and five post-infection was quantified. on day , mock-treated athymic and euthymic mice and xmg . -treated euthymic mice had rare scattered foci of necrotizing hepatitis, with necrosis of hepatocytes and infiltration of neutrophilic leukocytes. xmg . -treated athymic mice had hepatitis that was similar in quality to that of their mock-treated counterparts, but necrotic foci were more numerous. on day , differences between treatment groups were more striking. xmg . treated athymic mice had numerous large, coalescing foci of hepatocellular necrosis, with only modest leukocytic response (fig. a) . in contrast, mocktreated athymic mice had fewer, smaller foci of hepatitis with less necrosis and infiltration with more leukocytes (fig. b) . xmg . -treated euthymic mice had more foci of hepatitis than either their mock-treated counterparts or mock treated athymic mice. lesions among the latter three groups were qualitatively similar, with infiltration of both neutrophilic and mononuclear leukocytes. microscopic differences between treatment groups were not seen in spleens of mice necropsied on day , but were evident on day . xmg . -treated athymic mice, and to a lesser extent xmg . -treated euthymic mice, had severe necrosis of the periarteriolar regions of the white pulp. necrosis was present in the same regions of spleens from mock-treated mice, but was less severe. virus titers in livers and spleens of xmg . -treated mice necropsied on days and post-inoculation, whether athymic or euthymic, were higher than those in the corresponding tissues of mock-treated mice ( table ) . for athymic mice, these differences were statistically significant for both tissues on both days. among euthymic mice, statistically significant differences in virus titers occurred in the spleen, but not the liver. since treatment of mhv-jhm-infected balb/cbyj mice with antibody to ifn-y resulted in decreased survival time and increased virus titers in two target (table ) . virus titers in liver, spleen and brain of ifn- treated mice at five days post-inoculation were lower than those in the corresponding organs of mock-treated mice (table ) , but only the differences in liver titers were statistically significant. there have been a few reports detailing the responses of mhv-infected mice to nonreplicating antigens or other viruses. mice infected intraperitoneally with mhv- two or four days prior to sheep red blood cell immunization yielded depressed igm and igg plaque-forming cell responses [ ] , whereas mice in-fected at the time of or one day after red cell administration yielded elevated plaque-forming cell responses. this could be correlated with the timing of ifna/ responses relative to antigen administration. more recently, intranasal administration of mhv-a one day prior to tetanus toxoid immunization resulted in elevated antigen-specific igg with a four-fold decrease in the percent of igg that was igg and a like increase in the proportion that was igg a [ ] . our results with mhv-jhm and a different immunizing antigen confirm and extend that finding by showing elevated igg a ova-specific responses over a wide range of infection intervals. timing is crucial, however, to the extent that igg a titers exceeded igg titers only among mice infected one day prior to antigen administration. mhv infection reportedly increases resistance to a variety of secondary viral infections. pre-infection of mice with mhv increased resistance to lethal sendai virus infection in a normally susceptible genotype and interfered with replication of pneumonia virus of mice in respiratory tract tissues [ ] . subclinical mhv infection delayed increased plasma lactic dehydrogenase (ldh) levels resulting from ldh-elevating virus [ ] . natural, inapparent mhv infection also increased resistance to encephalomyocarditis virus and reduced the protective effect of exogenous ifn-a or - [ ] . since mhv infection induces ifn-(z/ production [ , , ] , it was postulated that increased resistance to these secondary infections was due to endogenous ifn-a/ levels and that the effects of exogenous ifn were masked by the endogenous production [ ] . there are now a limited number of reports showing that ifn- ~ modulates the pathogenesis of virus infections. treatment of mice with a different rat antimouse ifn-~f monoclonal antibody or with a sheep-derived antibody against recombinant ifn- resulted in reduced clearance or enhanced replication of lymphocytic choriomeningitis virus (lcmv) in mice [ , ] . neutralization of ifn- in lcmv-infected athymic c bl/ resulted in enhanced virus replication [ as seen in the studies reported here. more recently, a rat antimouse ifn- monoclonal antibody prevented il -induced recovery of athymic mice infected with recombinant vaccinia virus encoding murine il [ ] , suggesting that il -induced ifn- production was responsible for rapid clearance of this construct. virus replication and associated mortality were unaffected among anti-ifn- -treated athymic mice infected with a control virus construct encoding hsv tk and a/pr/ / ha [ . however, administration of recombinant ifn- to mice infected with the control construct significantly increased their survival time, although all treated mice eventually died of disseminated infection [ ] . resistance of intraperitoneally inoculated a/j mice to mhv- also correlates with the ability of this genotype to produce ifn- after infection [ ] . the accumulated data strongly suggest that ifn- , whether induced during the course of infection or administered therapeutically, limits viral replication in vivo. in the case of vaccinia virus encoding il , ifn- apparently mediates recovery of athymic mice. our data with mice exposed by presumed natural routes to mhv-jhm show that endogenous ifn- modulates the pathogenesis of infection in both athymic and euthymic mice. in earlier studies with lcmv [ ] and the current experiments with mhv, ifn- was either present in very low concentration or undetected in sera of infected mice, despite the fact that anti-ifn-y treatment resulted in elevated virus titers. these observations may stem from insensitivity of the assays used to detect ifn- . the vesicular stomatitis virus cytopathic effect reduction assay was used in the lcmv studies, and the wehi cell bioassay was used in the current experiments. the wehi cell bioassay is as sensitive as the ia induction assay for detection of ifn- [ ] , but sensitivity comparisons with other methods have not been reported. use of the sandwich enzyme-linked immunosorbent assay, as reported by karupiah and co-workers [ ] , would likely reveal the presence of ifn- in sera or tissue homogenates from infected mice. based on both histologic evaluation and virus quantification, the effect of administration of anti-ifn- antibody was more marked in athymic than in euthymic mice. this may be due to higher endogenous concentrations of ifn- in euthymic mice by virtue of functional t cells as well as natural killer cells. neither natural killer cells nor endogenous levels of ifn- are sufficient to afford protection, however, since athymic mice do die after natural or experimental infection with mhv [- , ] . mhv-jhm also kills virtually all balb/ c mice exposed by the intranasal route. the lethal effect of infection could be partially overcome by administration of recombinant ifn- , with a concomitant and significant decrease in virus concentration in the liver. others have postulated that virus infections preferentially activate the ifny producing t h subset of cd + t cells [ ] , based on the fact that most infections result in enhanced igg a production. using ldh-elevating virus or the fl strain of mouse adenovirus, attempts were made to inhibit virusassociated igg a production by injection of several different ifn-y-specific monoclonal antibodies [ ] . these efforts failed, although it was not clear whether treatment simply did not affect isotype distribution or whether the mice succumbed to infection. in the current study, neutralization of ifn- in vivo resulted in high mortality with reduced survival times, thereby inhibiting our efforts to show a direct link between virus-induced ifn- production and enhanced igg a responses. among mouse strains susceptible to mhv after exposure by natural routes, lesions and mortality occur at or near the infectious dose level, and the severity of lesions is independent of virus dose [ , ] . in the case of mhv-jhm inoculation of the balb/c mouse by presumed natural routes, the median infectious and lethal doses are essentially identical (unpubl. data). therefore, subsequent studies designed to reveal whether neutralization of ifn- reverses preferential igg a production will require the use of mouse genotypes more resistant to the lethal effects of mhv-jhm infection [ ] or of a less virulent mhv strain. our studies with mhv-jhm in the balb/c mouse have shown, however, that endogenous ifn-y modulates coronavirusassociated mortality, virus replication and tissue damage in a susceptible host. mouse hepatitis virus nasoencephalopathy is dependent upon virus strain and host genotype response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm the polyclonal and antigen-specific ige and igg subclass response of mice injected with ovalbumin in alum or complete freund's adjuvant alteration of viral respiratory infections of mice by prior infection with mouse hepatitis virus two types of mouse helper t cell clone. iii. further differences in lymphokine synthesis between th and th clones revealed by rna hybridization, functionally monospecific bioassays, and monoclonal antibodies in vivo polyclonal b-lymphocyte activation elicited by murine viruses virally induced modulation of murine igg antibody subclasses igg a restriction of murine antibodies elicited by viral infections effect of inapparent murine hepatitis virus infections on macrophages and host resistance infection of balb/cbyj mice with the jhm strain of mouse hepatitis virus alters in vitro splenic t cell proliferation and cytokine production delayed increase in plasma lactic dehydrogenase activity in mouse hepatitis virus-infected mice subsequently infected with lactic dehydrogenase virus ifn-gamma regulates the isotypes of ig secreted during in vivo humoral immune responses production of b cell stimulatory factor- and interferon gamma in the central nervous system during viral meningitis and encephalitis persistent mouse hepatitis virus infection in nude mice responses of mice susceptible or resistant to lethal infection with mouse hepatitis virus, strain jhm, after exposure by a natural route interferon gamma is involved in the recovery of athymic nude mice from recombinant vaccinia virus/interleukin infection gamma interferon in murine coronavirus infection cytotoxic t lymphocyte control of acute lymphocytic choriomeningitis virus infection: interferon gamma, but not turnout necrosis factor alpha, displays antiviral activity in vivo diagnosis of murine infections in relation to test methods employed enhanced virus replication and inhibition of lymphocytic choriomeningitis virus disease in anti-gamma interferon-treated mice new a (ed) viral and mycoplasmat infections of laboratory rodents. effects on biomedical research a major role of macrophage activation by interferongamma during mouse hepatitis virus type infection. i. genetically dependent resistance prevalence of natural virus infections in laboratory mice and rats used in canada a simple method of estimating fifty percent endpoints inhibition of b lymphocyte activation by interferon gamma activationofnaturalkillercells and induction of interferon after injection of mouse hepatitis virus type in mice an immunofluorescence test for detection of serum antibody to rodent coronaviruses altered splenic t cell function of balb/ cbyj mice infected with mouse hepatitis virus or sendai virus antigenic characterization of tettnang virus: complications caused by passage of the virus in mice from a colony enzootically infected with mouse hepatitis virus in vitro splenic tcell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain jhm interferon-gamma and b cell stimulatory factor- reciprocally regulate ig isotype production ifn-gamma stimulates igg a secretion by murine b cells stimulated with bacterial lipopolysaccharide persistent infection with mouse hepatitis virus of low virulence in nude mice the role of circulating interferon in the modifications of immune responsiveness by mouse hepatitis virus (mhv- ) statistical methods in serum surveys this research was supported by nih grants rr and rr . the authors thank deborah f. winograd, deborah s. beck, and jane dunn for excellent technical assistance. received january , key: cord- -uk kvxyd authors: kucherenko, a. m.; pampukha, v. m.; romanchuk, k. yu.; chernushyn, s. yu.; bobrova, i. a.; moroz, l. v.; livshits, l. a. title: ifnl polymorphism as a predictor of chronic hepatitis c treatment efficiency in ukrainian patients date: - - journal: cytol doi: . /s sha: doc_id: cord_uid: uk kvxyd the aim of this study was to examine association between ifnl gene ss and treatment efficiency in group of ukrainian peg-interferon/ribavirin-treated chronic hepatitis c patients. study group consisted of unrelated hepatitis c virus genotype mono-infected patients: case group– patients with late or absent virological response; control group– patients with sustained virological response. study material was genomic dna. genotyping was performed using amplification-refractory mutation system pcr. statistical analysis was performed using genepop and openepi statistical packages. obtained results show that ss Δg/Δg genotype is associated with poor virological response (or = . ; ci %: . – . ) in peg-interferon/ribavirin-treated chronic hepatitis c patients from ukraine. chronic hepatitis c (chc) is a worldwide healthcare problem due to the increasing number of affected individuals [ ] . interferon-based therapy schemes prove the most effective in chc treatment [ , ] . the discovery of impact of host ifnl (il b) genotype in combination with virus genotype on treatment outcome was a milestone in the development of antiviral therapy strategies [ ] [ ] [ ] . the wide range of studies has proven the association of il b gene polymorphic variants rs and rs with the antiviral therapy efficiency in patients with chronic hepatitis c (virus genotype ) as well as spontaneous viral clearance [ , ] . interestingly, this association shows ethnical diversity [ ] . however, the obscurity of exact molecular mechanism underlying such association was the significant drawback for further investigations. the discovery of a previously unknown transcript which expression in hepatocytes was activated by hepatitis c virus exposure has lead to the significant progress in the field [ ] . a recently discovered dinucleotide polymorphic variant ss was shown to cause a frame-shift mutation creating an open reading frame -ifnl (interferon lambda ) gene [ , ] . this polymorphism was reported to be in linkage disequilibrium with rs in some populations [ ] and is being thoroughly investigated for association with sustained virological response (svr) in interferon-treated chronic hepatitis c patients [ ] [ ] [ ] [ ] . the aim of the current study was to examine possible association between genotypes for ss and rs in group of ukrainian chc patients. the data for this study were collected from ukrainian chc patients. all of them were hcv rna positive for more than months and mono-infected with chronic hepatitis c with no evidence of hiv or hepatitis b infection. all participants were untreated patients with no evidence of any associated liver diseases other than chronic viral hepatitis c. the pretreatment hcv rna level was detected by quantitative real time polymerase chain reaction (pcr) technique with a lower limit of detection of iu ml - . the high viral load (rna hcv > iu ml - ) was observed in . % of patients. in all the patients enrolled in this study the hcv genotype infection was detected. qualitative pcr by taqman assay as well as elisa was used for detection of viral load at weeks , , and . the treatment protocol specified duration of weeks. the majority of patients ( . %) received peg-ifnα a (pegasys) at a dose of μg per week and . % received peg-ifnα b (pegintron) as a subcutaneous injection at a dose of . μg kg - per week. all patients received a mg kg - daily oral dose of rbv (copegus or rebetol). under futility-stopping rule, the treatment was halted if there was < log hcv rna decline at week or persistent viremia at week . according to the viral load changes all the patients were distributed into two groups: case group - patients with late or absent virological response, and control group - patients with sustained virological response. informed consent was obtained from all participants prior to the collection and storage of blood samples for testing. the study has been approved by the bioethical committee of institute of molecular biology and genetics of nas of ukraine. the material of the study was genomic dna extracted from peripheral blood samples using standard phenol-chloroform technique. genotyping for ifnl gene ss was performed using the amplification-refractory mutation system (arms) pcr [ ] . primer design was conditioned by several factors. firstly, high sequence homology between ifnl gene subfamily needed to be taken into the consideration. secondly, the sequence of ss flanking region is able to form hairpin loops and dimers with Δg up to − . kcal mol - . therefore, additional mismatches were introduced in the targetspecific primers to avoid dimer formation. the primers sequences were ifnl Δg: tcc ttt aca cgg tga tcg cag c, ifnl tt: tcc ttt aca cgg tga tcg cag aa, and ifnl com: tga ttg acc ctg agc ctg cg. conditions for amplification were as follows: initial denaturation at °c for minutes, cycles of seconds at °c, seconds at °c, and seconds at °c, followed by minutes of final extension at °c. the amplification products of bp were visualized on % agarose gel with ethidium bromide staining. in order to confirm the accuracy of the developed genotyping method, genotypes of random samples were re-determined via sanger sequencing. dna sequencing was performed by standard dideoxynucleotide chain-termination method using bigdye® terminator v . cycle sequencing kit and abi prism genetic analyser (applied biosystems, usa). genotyping for il b gene rs was performed by a pcr-based restriction fragment length polymorphism assay using hpy i restriction enzyme as described previously [ ] . statistical analysis has been performed using genepop and openepi statistical packages [ , ] . the χ test was used to detect deviations from hardy-weinberg equilibrium in genotype distribution. the likelihood-ratio test has been performed to estimate the linkage disequilibrium between ss and rs . a p-value of less than . was regarded as significant. the results of genotyping for both studied polymorphic variants in chc patients group are presented in table . genotype frequencies for both studied variants showed no significant deviation from the ones expected according to hardy-weinberg equilibrium. the χ values for ss and rs equaled . and . respectively (df = ). the likelihoodratio test was performed to estimate the genotypic linkage disequilibrium between ss and rs . the results indicated that the studied variants are tightly linked (p > . ), alleles ss tt and rs c are in phase. these results are consistent with our previous research conducted on a group representing general population of ukraine [ ] . regarding the distribution of studied polymorphisms' genotypes, the results are presented in table . as can be seen, genotype and allele distributions in group of patients with svr differed significantly compared to group without svr. the analyses show positive associations of ss tt/tt genotype with svr, and ss Δg/Δg genotype with poor virological response to treatment. this association fits well with an additive model of inheritance. several studies on different populations have shown that ifn-λ is linked with the failure to clear hepatitis c virus (hcv) infection in response to treatment [ , ] . ifn-λ is generated only by individuals who carry the ifnl -Δg allele, which was thus chosen as promising host factor for predicting clearance of hcv. ifn-λ most closely resembles ifn-λ , but these proteins share only % amino-acid identity, and, compared to ifn-λ , ifn-λ is only weakly secreted [ , , ] . there are some crucial characteristics in the antiviral activities induced by type iii (λ) ifns. first, in contrast to type i ifns, which receptors are broadly expressed in virtually all somatic cells, type iii ifn receptors are predominantly expressed by non-hema- tologic cell types, especially cells of epithelial origin such as bronchial epithelium, gastrointestinal epithelium, and keratinocytes [ ] . second, viral infection studies in several mouse models have shown that type iii ifns play a more prominent role than type i ifns in mediating antiviral protection against certain types of viruses which preferentially infect gastrointestinal and/or respiratory epithelium [ ] . early clinical trials of recombinant ifn-λ for the treatment of chronic hepatitis c indicate that its antiviral effects are similar to recombinant ifn-α, but it induces fewer adverse effects due to the more limited range of cells which express ifn-λ r [ , ] . the exact mechanism of ifn-λ level influence on hepatitis c antiviral treatment efficiency is yet to be discovered. however, several hypotheses call for further investigation. according to the first hypothesis ifn-λ production may have an impact on interferon-stimulated genes (isg) expression. ifn-λ signaling is proven to occur through the ifn-λ receptor complex, consequently inducing isgs expression via the janus kinase-signal transducer and activator of transcription signaling pathway [ ] . individuals who carry ifnl -Δg allele may express low levels of ifn-λ protein that induces low, but persistent isgs expression in the liver, which makes hepatocytes refractory to stimulation by ifn-α [ , ] . it was shown, that these individuals have higher basal levels of isg expression and are less likely to respond efficiently to peg-ifnα and ribavirin therapy [ , ] . these findings show the importance of discovering new treatment targets for hepatitis c therapy, avoiding ifn-α pathway. on the other hand, there is evidence of an impaired secretion of ifn-λ , probably due to inefficient posttranslational glycosylation [ ] . it is possible that intracellular accumulation of non-glycosylated ifn -λ could be cytotoxic and result in hepatocyte death, deteriorating patients' state and promoting further infection damage [ , ] . in this study we present for the first time the evidence of ss being a valuable pharmacogenetic marker of chc treatment efficiency in ukrainian patients of eastern european origin. while rs is in tight linkage disequilibrium with ss and is proven to be informative for our population as well, the wide range of functional changes promoted by ifn-λ production makes ss more favorable and feasible genetic marker. global epidemiology of hepatitis c virus infection: new estimates of age-specific antibody to hcv seroprevalence peginterferon alfa- a plus ribavirin for chronic hepatitis c virus infection pegylated interferon-alfa plus ribavirin therapies for chronic hepatitis c genetic diversity and evolution of hepatitis c virus- years on host genetic variants in the pathogenesis of hepatitis c diagnosis and management of hepatitis c virus infection genome-wide association of il b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c role of il b for chronic hepatitic c treatment toward personalized medicine epidemiology and natural history of hcv infection a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus ifnl -Δg genotype is associated with slower viral clearance in hepatitis c, genotype- patients treated with sofosbuvir and ribavirin ifn-λ : the paradoxical new member of the interferon lambda family ifnl ss variant shows similar performance to rs as predictor of response to treatment against hepatitis c virus genotype or in caucasians association of the ifnl -Δg allele with impaired spontaneous clearance of hepatitis c virus interferon-lambda genetic polymorphism is associated with the therapy response for hepatitis c virus recurrence after a living donor liver transplant il b polymorphisms and clinical implications for hepatitis c virus infection in uzbekistan study on the ifnl gene ss variant in ukrainian population ifn-λ- (il b) genotyping by restriction fragment length polymorphism method: detection polymorphism of rs : a complete re-implementation of the genopop software for windows and linux openepi: a web-based epidemiologic and statistical calculator for public health interferon-λ (ifnl ) transcript expression in human liver tissue samples interferon lambda signals via the ifnλ receptor to regulate antiviral activity against hcv and coronaviruses ifn-lambda determines the intestinal epithelial antiviral host defense phase b study of pegylated interferon lambda with or without ribavirin in patients with chronic genotype hepatitis c virus infection a randomized phase b study of peginterferon lambda- a for the treatment of chronic hcv infection key: cord- -k z v vx authors: rong, q.; alexander, t. s.; koski, g. k.; rosenthal, k. s. title: multiple mechanisms for hsv- induction of interferon α production by peripheral blood mononuclear cells date: journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: k z v vx uv-inactivated, infectious, and other forms of herpes simplex virus (hsv- ) induced interferon (ifn) production by different routes in myeloid origin mononuclear cells (momc) (consisting predominantly of monocytes). gm-csf activated the momc (g-momc) to produce greater amounts of interferon while differentiation to dc, by the addition of granulocyte macrophage colony stimulating factor (gm-csf) and calcium ionophore (ga-momc), reduced the levels of interferon production upon challenge with some hsv strains. uv-inactivated virus induced more interferon than infectious virus. l-fucose, an antagonist of the mannose receptor, inhibited the induction of ifn-α by uv-inactivated virus and gb(−) virus (defective in penetration) in momc and ga-momc but not g-momc. l-fucose had little effect on interferon induction by infectious hsv- . the insensitivity of the g-momc to fucose inhibition distinguishes these interferon producing cells from the pdc cells previously described as natural interferon producing cells. the mannose receptor appears to be involved in the response to non-infectious forms of hsv but infectious virus appears to use a different pathway. these studies suggest that non-infectious virions and hsv infected cell debris effectively stimulate monocytes and pre-dendritic cells to produce ifn-α to initiate host protection against hsv infection. interferon alpha (ifn α) production is one of the earliest responses to virus infection, acting locally and systemically [ ] to inhibit virus replication and induce protective immune responses. ifn α can inhibit infection and the spread of hsv from neurons to epidermal cells [ ] in vitro, and application of plasmid dna expressing ifn α is sufficient to prevent disease upon vaginal [ ] or corneal [ ] infection in animal models. the most potent inducer of ifn-α is double stranded rna (dsrna), formed as the replicative intermediate of rna viruses or as a result of complementary rnas of a dna virus [ ] . complementary transcripts, which may provide an inducer, have been detected in vaccinia virus [ ] , adenovirus [ ] , herpes simplex virus (hsv) [ ] , and sv [ ] . ifn-α production can also occur upon inhibition of protein synthesis which can block the synthesis of repressors of ifn-α mrna synthesis [ ] . certain enveloped viruses, including sendai virus [ ] , human immunodeficiency virus (hiv) [ , , , ] , and hsv [ , , , - , , , , , , , , , , , ] promote the production of large amounts of ifn-α upon interaction with a population(s) of cells in freshly isolated peripheral blood. human monocytes produce ifn-α in response to sendai virus or hiv [ , ] and may also produce interferon in response to hsv. hsv induces high levels of ifn α in a minor cell component which have been termed natural interferon producing cells (nipc). the ifn α producing activity of these cells is lost upon overnight in vitro culture of the cell population [ ] . a candidate for the nipc has been identified as the pdc (dendritic cell precursor) [ ] . these cells lack myeloid or monocytic markers including cd (myeloid origin marker), cd , cd b, cd (markers expressed when precursor cells differentiate into myeloid lineage mononuclear cells), cd b and cd (strongly expressed on granulocytes), cd (monocyte marker) antigens, and also lack b-cell and t-cell antigens. other types of hsv responsive interferon-producing cells may also be present in peripheral blood or the peripheral tissue. for example, in the mouse, the marginal metallophilic macrophages and marginal zone macrophages of the spleen are the major interferon producers in response to iv challenge with hsv and murine dc lines can produce interferon in response to bacteria and viruses, including hsv [ ] . several forms of hsv can induce the ifn α response including infectious and uv inactivated virus [ , ] indicating that replication of the virus is not required. internalization of the virus is also not required since hsv fixed to glutaraldehyde cross-linked cells [ , ] and genetically engineered hsv- glycoprotein d (gd) obtained from mosquito cells are sufficient for induction of ifn-alpha [ ] . in this study, we evaluated an alternative source of cells to study the nature of the interferon response to hsv- . large numbers of non-lymphocytic mononuclear cells were obtained by leukophoresis and countercurrent centrifugal elutriation [ ] . these cells are predominantly of myeloid origin (momc) with a small percentage of immature dendritic cells. following treatment with granulocyte macrophage colony stimulating factor (gm-csf) and a (calcium ionophore, ionomycin, 'a') in serum-free cell culture, the monocytes and immature dendritic cells (idc) undergo a rapid and consistent change to become activated dendritic cells (dc) [ , ] . the differentiation to dc includes down-regulation of cd expression, acquisition of dendritic cell morphological properties, upregulation of mhc class ii and co-stimulatory molecule expression, and enhanced capacity for t cell sensitization [ ] . we demonstrate that the extent of interferon induction is different upon challenge with different strains and forms of hsv and that monocytes, gm-csf treated monocytes, and the mature dendritic cell populations respond differently to these challenges. the response of the momc to some strains of hsv- is enhanced by gm-csf to levels similar to that reported for nipc. comparison of the activities of different strains of infectious hsv, uv-inactivated hsv, and a mutant hsv incapable of penetration (k t) [ ] and results of treatment with a mannose receptor antagonist indicate that there are more than one mechanism for hsv induction of interferon in the momc origin cell populations. mononuclear myeloid cells (momc) isolated from different healthy donors on different occasions by leukopheresis and countercurrent centrifugal elutriation [ ] were frozen and thawed for each experiment. the momc preparations consist predominately of cd + cells (> %) (a myeloid cell surface marker) with approximately % monocytes (cd + cd + ) and - % idc (cd − cd + ) [ ] . after thawing, momc were washed once in macrophage-sfm medium (gibco) containing % penicillin-streptomycin (cellgro) and grown in -well ( × /well) cluster plates (costar) in ml serum free macrophage-sfm medium (gibco) with % penicillin-streptomycin in an atmosphere of % co at • c. recombinant human granulocyte-monocyte colony stimulation factor (gm-csf, ng/ml) (immunex) was added to specific wells of the momc (gm-csf treated momc, g-momc) directly after the plating. calcium ionophore (a) (a , ionomycin; sigma chemical co, ng/ml) was added to a set of gm-csf treated momc at h after thawing to induce differentiation of the cells (gm-csf plus a treated momc, ga-momc) [ , , ] . cells for each experiment were analyzed by flow cytometry to determine their immunophenotype [ ] . cells were incubated with mg/ml human igg (sigma chemical co.) for min to block fc receptors and double stained with fluorescein isothiocyanate (fitc)-conjugated mouse anti-human cd and phycoerythrin (pe)-conjugated mouse anti-human cd (b . ) or cd (b . ) (pharmingen) for min at • c. virus stocks were prepared by freeze-thaw and sonication of infected vero cell lysates and culture media. vero (african green monkey kidney) cells were grown in medium (gibco brl) supplemented with % fetal calf serum (hyclone) and % penicillin-streptomycin (cellgro) at • c. virus stock was quantitated in a standard plaque assay on vero cell monolayers. kos , a lab attenuated virus, was kindly provided by tom holland, wayne st. u. school of medicine, detroit, mi. sp and slp were generated for other studies in our lab [ ] . sp is a clinical strain obtained post mortem from the brain of a -day-old infant with disseminated hsv- disease. slp was generated by serial passage ( times) of plaque purified sp virus in vero cells at low moi (< . ). ang and ang-path virus [ ] , two related syncytial virus strains, were kindly provided by bradley m. mitchell (baylor college of medicine, houston, tx). ang-path was derived from ang by passage in mice. stocks of virus were prepared and quantitated as mentioned above. uv-inactivated virus was prepared by exposure of virus to uv light for min and then tested by plaque assay for residual infectivity. the quantity of uv-virus is referred to as pfu or moi-equivalents, which are based on the amount of infectious virus prior to uv inactivation. k t is a mutant of hsv- kos with a gene deletion in the transmembrane region of the glycoprotein b (gb) to prevent implantation into membranes and the virion envelope [ ] . k t was kindly provided by john docherty (northeastern ohio universities college of medicine, rootstown, oh). stocks of k t containing gb (k t-gb) were made by infecting d cells, a vero cell line expressing gb, with k t-gb. quantitation of k t-gb was done in a standard plaque assay on d cells. plaque assay was done on vero cells to detect potential revertants to the parental kos virus. k t virus was quantitated by two methods which gave comparable results. k t viral dna concentration was compared with that of a kos virus stock for which the titer was already known. viral dna was purified for both viruses using wizard genomic dna purification kit (promega, madison, wi). the dna concentration was quantitated by absorbence at nm and k t pfu-equivalents were calculated by comparison with the dna concentration of kos stock. in addition, k t strain was quantitated using a plaque assay in which polyethylene glycol (peg) was used to promote viral penetration and plaque formation (adapted from sarmiento et al., [ ] ). after a h adsorption period, the monolayer was washed once with pbs and the cells were exposed to a solution ( ml per well) that contained g of melted peg (sigma, st. louis, mo) and . ml of m without serum. the peg solution was removed by washing with solutions of : peg: serum-free m , : peg: serum-free m , and three washes with m containing % fetal calf serum. the cultures were incubated with m containing % fetal calf serum for h at • c to allow the cells to recover. the medium was then removed and m containing . % methylcellulose (kodak, rochester, ny) and % fetal calf serum was added. the cultures were incubated for two or three days at • c until plaques formed. cells treated with uv inactivated viruses were incubated for h at • c. for infectious virus, cells were incubated with hsv- for h at • c, the medium was replaced with sfm medium and incubated for h at • c. to evaluate the effect of fucose on interferon induction, different concentrations of fucose were added min before the addition of virus. after the h incubation, ml aliquots were removed and frozen at − • c. the cells and remaining medium were frozen at − • c and thawed for quantitation of virus production. ifn-α concentration was determined by elisa (biosource, camarillo, ca). the antibodies in the elisa kit can recognize the most common subtypes of ifn-α. myeloid origin mononuclear cell populations (momc) obtained by leukopheresis and countercurrent elutriation were used as an alternative source of nonlymphocytic mononuclear cells to study hsv induction of ifn α. momc are predominantly (> %) monocytes, contain a minor population of immature dendritic cells (idc) ( - %) and have minimal contamination by lymphocytes and neutrophils [ ] . momc treated with gm-csf (g-momc) appeared similar to the untreated momc but maintained their viability to a greater extent over the -day-course of the experiment. both the momc and g-momc expressed high levels of cd , the lps receptor, although g-momc expressed lower levels than momc with low or absent cd (b . ) or cd (b . ) but a sub-population expressed higher levels of cd expression. g-momc maintained in serum free medium and treated with the calcium ionophore a (ga-momc) readily and reproducibly converted to mature dc [ , ] . the ga-momc cells could be distinguished from the momc and g-momc by the lack of cd expression and upregulation of both cd and cd expression, characteristics of mature dendritic cells [ ] . the loss of cd expression and up-regulation of cd and cd was seen in each experiment. maintenance of these cells in serum free medium and separation from lymphocytes and granulocytes prevents the effects of bovine serum factors from affecting their development. the conversion was reproducible in cells from different donors. herpes simplex virus production by momc, g-momc and ga-momc was evaluated by quantitating the amount of virus released to the media by plaque assay on vero cells. all three cell populations were poor virus producing cells with an average yield of one virus per cell (data not shown). this was less than the input (moi = ) infectious virus. the low permissivity of myeloid cells for hsv replication has been reported by others [ ] . infection with higher multiplicities of infection (moi = ) caused considerable cytopathological effect and cell death. initial studies were performed to determine whether momc, g-momc and ga-momc have the ability to produce ifn α in response to uv-inactivated hsv- . the cells were challenged with different moi equivalents of uv-inactivated kos, a highly passaged, attenuated hsv- strain and extracellular medium was obtained after h and ifn α analyzed by elisa. ifn-α production, by each of the cell populations, increased upon challenge with increasing moi equivalents of uv-inactivated hsv- kos (fig. ) . the g-momc cell population produced the greatest amount, ga-momc produced an intermediate amount and the momc produced the least amount of ifn-α in response to uv inactivated kos. there was some variation in the amounts of interferon produced by cells obtained from different donors and for different dates of donation but the g-momc treated cells always produced the most ifn α. cells that were mock infected or treated with uninfected vero cell extract produced no ifn α (data not shown). the response to uv inactivated hsv confirms other studies [ , ] that show that complete virus replication is not necessary for ifn α induction. also, gm-csf activates the cells to produce more interferon upon hsv induction. the magnitude, range and trends for ifn α responses were different for uv inactivated and infectious virus and for different strains of hsv- (fig. ) . the difference in response to infectious and uv-inactivated virus is clearly shown for hsv- kos and uv-inactivated kos. the strain dependent difference in response was evaluated for hsv- strains that differ in their passage history, tissue culture behavior and their ability to cause lethal neuroinvasive disease in the mouse footpad and other models of hsv infection [ , , ] . a moi of or the equivalent amount of uv-inactivated virus was chosen for the virus challenge because the cytopathological effect of infectious hsv on the different types of momc cells was minimal at this dose. cells from different individuals were used for some of the experiments (different panels of fig. ). the first set of viruses to be tested included sp , a low passage neuroinvasive virus and slp , an attenuated virus derived from sp by passage in vero cells ( fig. a, b) . the response to uv-sp was greater than for uv-slp or uv-kos (data not shown). the response to infectious virus was much less than for the moi, or moi-equivalents for all experiments was uv-inactivated virus. the g-momc response was greater than for momc or ga-momc but sp was the poorest inducer of interferon in all three cell populations. these trends were observed for different individuals and on different occasions. a different trend in cellular response was observed for the ang and ang-path set of viruses. ang-path was derived from ang, a clinical virus, by passage in mouse brains to select for a more neuroinvasive virus [ ] . both ang and angpath cause syncytia formation in all three cell populations. ang-path induced interferon, but unlike the previously described viruses, the ga-momc produced more interferon upon induction by ang-path than did g-momc (fig. c) . ang was a poor inducer of interferon but induced a small, but measurably greater amount of ifn α in ga-momc. this strain dependent difference in the trend may indicate a different type of interaction of ang-path and ang with the momcorigin cells. in order to address the question of whether virus entry is required for the induction of ifn-α in momc origin cell populations, an hsv mutant defective in penetration was evaluated for its abilty to induce interferon production. the k t mutant was developed from kos by deletion of a -base-pair bsteii fragment in the gb-coding region, corresponding to amino acids of the transmembrane region. this virus yields normal virions lacking the glycoprotein b. the k t mutants bind efficiently, but cannot enter cells [ ] . stocks of infectious virus (k t-gb) were prepared by growth in a gb expressing vero cell line (d cells) and stocks of virus lacking gb (k t) were obtained upon infection of the non-complementing vero cells. an equivalent titer (with respect to kos) of k t virus was quantitated by two methods.aliquots of k t virus were allowed to bind to d cells and fusion of the cell-bound virus was promoted with peg treatment [ ] . this allowed plaque formation to occur in the complementing d cells. in addition, the dna concentration of aliquots of k t was compared to similar aliquots of kos, for which the titer was known. these assays indicated that the equivalent-titer of the k t virus stock was approximately × /ml. interferon induction by k t was compared to kos at moi equivalents of (data not shown) and . the results are corrected for levels of interferon produced by equivalent numbers of wild-type virus to the infectious virus that may be present in k t due to genetic reversion to the parental kos (approximately . kos virus per , k t virus) or the small amounts of k t viruses which would acquire or retain gb on their envelope (k t-gb) ( k t-gb virus per , k t virus). figure d shows that k t induced lower levels of ifn-α production than the parental kos virus in momc and g-momc cells. the ga-momc produced a larger amount of ifn α than momc or g-momc in response to k t and this response was greater than for kos. studies by other investigators implicated the mannose receptor as an important mediator of hsv-induced ifn-α production and fucose as an effective inhibitor of this interaction [ ] . initial studies demonstrated a concentration dependent inhibition of uv-inactivated hsv- kos induction of interferon in momc, g-momc and ga-momc. cells pretreated with fucose for min were incubated with uv-kos (moi = ) for h in the presence of fucose and then aliquots were removed and tested for ifn-α production. ifn-α production in the absence of fucose treatment was set as the % control. fucose treatment caused a biphasic concentration dependent inhibition of ifn α production by momc and ga-momc in response to uv-kos (fig. ) . although the % inhibitory dose was mm, the extent of inhibition (slope) was less at higher concentrations of fucose ( - mm) . interestingly, the effect of fucose treatment of g-momc was different from that of momc and ga-momc. ifn α induction in g-momc was inhibited by only % at mm fucose and this was the maximum level of inhibition (fig. ) . these results indicate that there are different mechanisms for ifn α induction between these different cell populations with fucose sensitive, fucose less sensitive and fucose insensitive routes of interferon induction. in addition, the primary route of interferon induction for uv-hsv in g-momc cells is a fucose insensitive route. subsequent studies evaluated the ability of fucose ( mm) to block interferon induction by uv-inactivated ang, infectious ang, infectious kos, and k t. as shown in fig. , fucose inhibited ifn α induction by uv inactivated kos and uv-inactivated ang in momc and ga-momc but not the g-momc cell population, consistent with the results shown in fig. . interestingly, fucose did not inhibit ifn-α production in response to infectious kos or ang virus in any of the three cell types. unexpectedly, fucose treatment appeared to enhance the ifn-alpha production in the g-momc cells. interferon induction by k t virus was very sensitive to fucose treatment. ifn α production was reduced by % in momc and ga-momc, and by % in g-momc. the difference in q. rong et al. different forms of hsv, including infectious virus, uv-inactivated-non-infectious virus, fixed hsv coated onto glutaraldehyde fixed cells [ ] and purified glycoprotein d [ , ] can induce an ifn α response. the nature of the responses to these different forms of virus has been assumed to be similar and the descriptions of the responses have been used interchangeably in many studies. the results from our study indicate that various forms and different strains of hsv induce ifn α to different extents and likely, by divergent pathways. in addition, cell types other than the pdc cell are likely to produce ifn α in response to hsv and that response is different for different forms or strains of virus. also, gm-csf potentiates the interferon response to some forms and strains of virus. these studies were made possible by the use of myeloid origin mononuclear cells (momc), a large population of cells which are predominantly monocytes, with small numbers of dendritic cell precursors and minimal contamination from lymphocytes and neutrophils [ ] . unlike cells used in other studies, the momc can be frozen and thawed and maintained in tissue culture for greater than h. use of serum free medium for these cells minimizes the interference from unknown cytokines and the serum free conditions may be more representative of the extra-vascular environments where pdc or monocytes may encounter an hsv infection [ ] . gm-csf appeared to prime or activate the interferon response to uvinactivated and infectious forms of kos and sp viruses but not for all the viruses. the amount of ifn-α produced by g-momc in response to uv inactivated hsv was in the same range as suggested in the literature for the nipc [ , ] , although direct comparisons are difficult due to differences in the means of analysis (elisa vs bioassay), the virus strain, and individual donor variation. the gm-csf also enhanced interferon production by peripheral blood mononuclear cells following stimulation by hsv bound to glutaraldehyde fixed wish cells [ ] . in vivo, gm-csf is an early response to infection and is produced by activated t cells, macrophages, endothelial cells or fibroblasts [ ] . treatment with recombinant gm-csf [ ] is sufficient to elicit protection against hsv- encephalitis in a rat model. our studies would suggest that an important component of the gm-csf induced protection is the potentiation of the interferon response to hsv- . differentiation of the g-momc into mature dc-like cells by treatment with a (ga-momc) was accompanied by a reduction in the production of ifn α in response to kos, sp and slp strains of hsv. decreased response upon differentiation is consistent with the loss (reduction) of interferon induction observed by others upon overnight incubation of peripheral blood mononuclear cells under normal cell culture conditions [ ] . the ga-momc dendritic cells were more responsive to challenge with ang-path and k t viruses than g-momc or momc. the difference in interferon response may reflect differences in the interaction of the virus with the interferon producing cells sinceang andang-path cause syncytia formation, k t binds, but is incapable of entering the cell, and all three viruses have mutations in or lack the glycoprotein b. other studies support our findings that dc can make ifn α in response to hsv and also hiv [ , ] . the different fucose inhibition patterns for the varied forms and strains of virus and for the different cell types suggests that there are different routes of hsv induction of interferon. the fucose sensitive cell surface route probably uses the mannose receptor and is activated by uv-kos, uv-ang and k t in both momc and ga-momc cell populations. this may also be the route used by uv-inactivated-non-infectious virus, fixed hsv coated onto glutaraldehyde fixed cells [ ] , and purified glycoprotein d [ , ] . a route that is less sensitive to fucose, as distinguished at high concentrations of fucose, may also be used by these activators. the mannose receptor does not seem to be extensively involved in induction of ifn α by infectious virus or by any of the forms of virus in g-momc. the insensitivity of the g-momc to fucose inhibition distinguishes the interferon producing cells in this population from the pdc cells that have been called nipc, which are sensitive to fucose inhibition [ ] . the large enhancement of interferon induction by uv-inactivation of infectious hsv observed herein and by linnavuori and hovi [ ] suggests that infectious virus may have the ability to limit ifn α production in momc related cells. hsv strains appear to differ in their ability to utilize this mechanism to evade host protection as indicated by comparison of the trend for interferon induction for the uv-inactivated viruses (sp > slp ) and infectious viruses (slp sp ). for the limited numbers of virus strains tested herein, the virus strains with a history of more extensive passage in non-human hosts (slp , kos, ang-path) appeared to induce more ifn-α production than the low-passage viruses (sp , ang). this suggests that the suppression of interferon production may be selected during human infection as a means to escape host defenses but this property may be lost upon infection of cells or animals of other species. other human-specific hsv mechanisms for escaping host protective responses include the hsv- ul protein block of mhci expression by blocking the tap [ ] and glycoprotein e binding to the fc portion of igg [ ] . the results of this study open up the possibility that myeloid origin monocytes and pre-dendritic cells are an important source of ifn α for host protection against hsv infection. the mechanism of induction for these cells may be different from the pdc cells (based on the fucose blocking studies) described by others [ ] . the greater response to uv-inactivated virus suggests that non-infectious virions and possibly hsv infected cell debris are the more potent activators of ifn α production and that replicating virus can limit the induction of interferon production as a means of escaping host protection. locally produced gm-csf would activate monocytes or pdc to enhance production of ifn α and protective responses. extensive symmetrical transcription of simian virus dna in virusyielding 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grant r ns - from the ninds to ksr. key: cord- - pjg kn authors: chen, shilong; wang, long; chen, jieying; zhang, lanlan; wang, song; goraya, mohsan u.; chi, xiaojuan; na, yang; shao, wenhan; yang, zhou; zeng, xiancheng; chen, shaoying; chen, ji-long title: avian interferon-inducible transmembrane protein family effectively restricts avian tembusu virus infection date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: pjg kn avian tembusu virus (atmuv) is a highly pathogenic flavivirus that causes significant economic losses to the chinese poultry industry. our previous experiments demonstrated that atmuv infection effectively triggered host innate immune response through mda and tlr -dependent signaling pathways. however, little information is available on the role of interferon-stimulated genes (isgs) in defending against atmuv infection. in this study, we found that atmuv infection induced robust expression of type i and type iii interferon (ifns) in duck tissues. furthermore, we observed that expression of interferon-inducible transmembrane proteins (ifitms) was significantly upregulated in def and df- cells after infection with atmuv. similar results were obtained from in vivo studies using atmuv-infected ducklings. importantly, we showed that knockdown of endogenous ifitm or ifitm by specific shrna markedly enhanced atmuv replication in df- cells. however, disruption of ifitm expression had no obvious effect on the atmuv replication. in addition, overexpression of chicken or duck ifitm and ifitm in df- cells impaired the replication of atmuv. taken together, these results reveal that induced expression of avian ifitm and ifitm in response to atmuv infection can effectively restrict the virus replication, and suggest that increasing ifitm proteins in host may be a useful strategy for control of atmuv infection. tembusu virus (tmuv) is a member of the ntaya virus group within the genus flavivirus of family flaviviridae (yan et al., ) . tmuv strains were firstly isolated from mosquitoes in malaysia and thailand, but their pathogenicity is not fully understood pandey et al., ) . sitiawan virus, a broiler-origin tmuv strain, was the first pathogenic virus causing encephalitis and retarded growth in broiler chicks (kono et al., ) . since , chinese domestic poultry including ducks, chickens, and geese have been manifesting a new epidemic disease caused by a tmuv-related flavivirus, named as avian tembusu virus (atmuv). this outbreak was quickly spread to many provinces of china and several south-eastern asian countries (homonnay et al., ; thontiravong et al., ) . atmuv genome consists of a single strand positive-sense rna and encodes three structural proteins [capsid (c), premembrane (prm/m), and envelope (e)] and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) homonnay et al., ) . atmuv-infected adult animals showed symptoms of hemorrhagic ovaritis and acute egg drop syndrome, high fever, anorexia, diarrhea, ataxia, weight loss and paralysis, with a high morbidity ( - %), and mortality ranged from to % depending on different management and weather conditions, leading to enormous economic losses to poultry industry in china (cao et al., ; su et al., ; yan et al., ; liu et al., liu et al., , chen et al., ) . the young flocks are more vulnerable to atmuv infection, characterized by similar clinical symptoms including anorexia, diarrhea, high fever and severe neurologic dysfunction, with a higher mortality than adult birds (vaidya et al., ; ti et al., ) . atmuv was easily detected in ovaries and theca folliculi of infected animals, suggesting that the reproductive tissues were the major targets for the viral infection and replication . viral rna was also detected in spleen, trachea, kidney, brain, and blood of infected host (yan et al., ; liu et al., ) . in addition, it was observed that atmuv could infect various cell lines including def, cef, df- , bhk- , vero, a , t, and hela, resulting in a noticeable cytopathic effect (cpe) characterized by cell shrinkage, rounding and detachment (chen et al., a; . it has been reported that atmuv rna and neutralizing antibodies was detected in duck farm workers in shandong province of china (tang et al., ) , suggesting that this virus could infect human. but a disease associated with atmuv infection has not been found in human. moreover, it was shown that atmuv failed to cause any clinical manifestation or viremia in non-human primates, indicating that atmuv is unlikely to emerge as a human pathogen for the time being . however, due to zoonotic nature of its genus flavivirus relatives, atmuv might be a potential threat to human health in the future liu et al., ) . host innate immune response serves as the first line of defense against the infection of pathogens at the early stages. innate immune system recognizes viruses invasion via specific pattern recognition receptors (prrs) to sense pathogen associated molecular patterns (pamps) expressed by viruses. activated prrs then interact with adaptor proteins such as interferonβ promoter stimulator- (ips- ), myd , and trif (takeuchi and akira, ). the ligations of prrs with adaptor proteins result in the activation of the transcription factors, including nf-κb and interferon regulatory factors (irf and irf ) (takeuchi and akira, ) . irfs and nf-κb translocate to the nucleus where they stimulate the expression of type i and type iii interferons (ifns). then interaction between the ifns and their receptors causes activation of jak-stat signaling pathway. phosphorylated stat proteins translocate to the nucleus and combine with interferon regulatory proteins to promote an abundant expression of a wide array of genes, including ifnstimulated genes (isgs) (takeuchi and akira, ; tan et al., ; bailey et al., ) . these isgs encode distinct antiviral proteins with diverse biological effects that block multiple stages of the viral lifecycle including viral entry, translation, replication, assembly, and spread (diamond and farzan, ) . the interferon-inducible transmembrane proteins (ifitms) are a family of small transmembrane proteins belonging to isg superfamily and strongly induced by ifns (perreira et al., ) . the ifitm proteins are the distinct restriction factors known to block viral entry, including restriction of virus fusion with the late endosomal or lysosomal compartments and penetration into the cytoplasm (diamond and farzan, ; . it has been shown that gene cluster iftim , , and , the immunerelated genes, are critically involved in immune defense against a broad variety of viruses, including influenza virus, dengue virus, filoviruses, coronavirus, hepatitis c virus, lyssaviruses, and west nile virus (brass et al., ; huang et al., ; lu et al., ; smith et al., ; wilkins et al., ) . conversely, ifitms have little or no effect on several other viruses, including human papillomavirus, human cytomegalovirus and adenovirus type , arenavirus, murine leukemia virus, and foot-and-mouth disease virus (warren et al., ; bailey et al., ; zhang et al., ) . despite the progresses in understanding of ifitmmediated antiviral ability, host ifitm expression profiles in response to atmuv infection are still not clear. little is known about this immune-related isg family in restricting atmuv pathogenesis. in the present study, we examined the expression of key ifns and ifitms in host cells after atmuv infection in vitro and in vivo. interestingly, we observed that atmuv infection could trigger duck innate immune response including robust expression of particular type i and type iii ifns and ifitm family proteins. using df- cell system, we found that knockdown of endogenous ifitm and ifitm by short hairpin rna (shrna) markedly enhanced atmuv infection in host. however, silencing ifitm had no significant effect on atmuv replication. furthermore, overexpression of chicken or duck ifitm and ifitm could strongly inhibit the replication of atmuv. these results reveal that avian ifitm and ifitm but not ifitm serve as the critical components of host innate immune defense against atmuv infection. the antibodies used in this study are described as follows: mouse anti-β-actin (ab , abcam, cambridge, uk), mouse anti-flag (ht , transgen, beijing, china), hrp goat anti-mouse igg (lp a, abgent, usa). a monoclonal antibody against e protein of atmuv was prepared in our lab using the method described previously (chen et al., ) . fitc (fluorescein isothiocyanate) conjugated goat anti-mouse igg was purchased from boster (wuhan, china). chicken type i interferon was obtained from dalian sanyi animal medicine co. ltd. (dalian, china). lipofectamine was obtained from invitrogen (carlsbad, ca, usa). cell lines, birds, virus, and infection atmuv cjd strain was isolated from a chicken farm with acute egg-drop syndrome in fujian, china . duck embryo fibroblasts (defs) were prepared from -dayold mule-duck embryo as previously described (shahsavandi et al., ) . df- (immortalized chicken embryo fibroblast cell line) and t cells were obtained from american type culture collection (manassas, va). def, df- , and t cells were cultured at • c with % co in dmem (sigma, usa) supplemented with % fetal bovine serum (fbs, hyclone, utah, usa), units of penicillin g and µg of streptomycin. df- and def were infected with atmuv cjd as previously described (chen et al., a) at the multiplicity of infection (moi) of . and harvested at the indicated times after infection. twenty five -day-old mule healthy ducklings were challenged with . ml of cjd (the th passage allantoic fluid virus, eld = − . /ml) per duckling by intramuscular injection. each group of three randomly selected ducklings was sacrificed at , , , , and h post-infection (hpi), and their spleen, kidney, bursa of fabricius, pancreas, and brain were harvested for detection of viral infection by indirect immunofluorescence assay. these tissues were also used for total rna extraction to examine the mrna expression of ifns/ifitms by quantitative real-time rt-pcr (qrt-pcr). the sera and spleen homogenates ( %w/v) of the ducklings were prepared for detecting of viral titers by % tissue culture infectious dose (tcid ) assay during the infection. other infected ducklings and control ducklings (inoculated with . ml sterile pbs per duckling) were used to monitor clinical signs and rectal temperature daily for days. quantitative real-time rt-pcr and rt-pcr total rna was extracted from the cultured cells and duckling spleen tissues using trizol reagent (transgen biotech, beijing, china) according to the manufacturer's instructions. equal amount of rna ( µg) was reverse transcribed into cdna utilizing m-mlv reverse transcriptase (promega, usa). the cdna was analyzed by qrt-pcr using transstart green qpcr supermix (transgen) and pcr using rtaq dna polymerase (takara bio). the primers specific for chicken β-actin, ifna, ifn-β, ifn-λ, and atmuv e gene have been previously described (chen et al., a) . the primers specific for atmuv ns , duck β-actin, ifn-a, ifn-β, ifn-λ, ifitm , , , and chicken ifitm , , were designed using the primer software ( table ) . the relative mrna abundances were analyzed using the ′ ct method with housekeeping gene (β-actin) as an internal normalization and plotted as fold changes compared with the mock-infected samples. the specific short hairpin rnas (shrna) were designed for knockdown of chicken ifitm , , and . all the shrna sequences are shown in table . luciferase control shrna was described previously (wang et al., ) . df- cell lines stably expressing shrna targeting chicken ifitms (chifitms) the italic underlined nucleotides are restriction enzyme sites. were generated using lentiviral vectors as previously described . briefly, t cells were cotransfected with shrna construct and hiv-based packaging constructs (plp, plp , and plr ). supernatant of the cultured cells containing pseudotyped lentiviruses with indicated shrnas were collected at h post-transfection and filtered through the . µm syringe-driven filter. df- single-cell suspension was incubated with the supernatant and µg/ml of polybrene (sigma) and centrifuged at , rpm, • c for min. df- cells were then cultured in dmem supplemented with % fbs for further studies. qrt-pcr was performed to determine the interference efficiency and mrna expression of viral genes in df- cell lines at indicated times after infection. the viral titers of supernatants were examined by tcid assay in def cells. full-length cdna encoding chicken or duck species ifitm and ifitm was subcloned into pflag-cmv- a vector with a flag tag in the cooh terminus to create dna constructs chifitm , chifitm , duck ifitm (difitm ), and difitm , respectively. the specific primers with restriction enzyme sites are shown in table . df- cells were seeded onto -well plates and cultured in dmem with % fbs. when the cells reached - % confluence, they were transfected with µg of plasmid dna/well using lipofectamine (invitrogen). twenty four hours later, transfected df- cells were infected with atmuv cjd and harvested at indicated times ( , , and hpi) for further qrt-pcr or western blotting analysis. supernatants were collected for detection of viral titers by tcid assay. indirect immunofluorescence assay was performed as previously described (chen et al., b) . briefly, the parenchymal organs sections and def cells infected with atmuv were incubated with mouse anti-atmuv e protein monoclonal antibody for min at • c and then washed with pbs, followed by incubation with fitc conjugated goat anti-mouse igg at • c for min before imaging with fluorescence microscope (nikon, japan). df- cells were lysed with lysis buffer containing × complete protease inhibitor cocktail for min on ice according to our previous method (wang et al., ) . cell lysates were separated on % sds-page gels, transferred to pvdf membranes and blocked with % (w/v) milkpowder in tris-buffered saline (ph . , tbs) for h at room temperature. the membranes were incubated with indicated primary antibodies for . h at room temperature and washed with tbs, followed by incubation with appropriate secondary antibodies at room temperature before imaging with the proteinsimple fluorchem m system (bio-techne, usa). data represented the mean ± sd. statistical significance was determined by one tail student's t-test analysis. differences were considered statistically significant with p < . . the animal protocol used in this study was approved by the research ethics committee of college of animal science, fujian agriculture and forestry university (permit number pzcasfafu ). the procedures were carried out in accordance with the approved guidelines. the inoculated ducklings began to display clinical symptoms on day post-infection (dpi) characterized by anorexia and weight loss. two infected ducklings got slight legs paralysis and were reluctant to move on - dpi. infected ducklings developed a fever, showing that rectal temperatures increased from day to day post-inoculation and then gradually returned to normal from to dpi (figure d ). at necropsy, no lesions were observed except swollen spleens on - dpi. no duckling was died throughout the monitoring period. later on ifa test was carried out to detect viral antigens. we observed that viral antigens were detectable in the spleen, kidney, and bursa of fabricius tissues, with a higher viral burden in the spleens (figures a,b) . surprisingly, viremia ( . tcid / . ml) was observed in serum samples from to dpi ( figure c) . no virus was detected in other tissues including brain, pancreas, and liver. all the ducklings in control group were healthy and no viral antigen was detected. these findings indicate that atmuv exhibits mild pathogenicity in young ducklings following intramuscular injection. our previous studies had shown that atmuv infection effectively triggers the host innate immune response, including robust upregulation of type i and type iii ifns and some critical isgs in chicken and cef (chen et al., a) . however, innate immune response against atmuv infection in duck still remains to be determined. importantly, the role of ifitms in defending against atmuv infection has not been well-defined in any avian species. here, we examined the expression profile of ifns and ifitms in parenchymal organs of ducks infected with atmuv using qrt-pcr and rt-pcr analysis. the results showed that atmuv has higher replication in duckling spleen tissues, as indicated by obvious expression of viral e gene ( figure a ; supplementary figure ). remarkably, the mrna levels of type i and type iii ifns were gradually elevated and reached their maximum value on hpi and then declined gradually. in particular, ifn-α and ifn-β were greatly upregulated by -and , -fold at hpi, respectively (figures a,b) . the expression of ifn-λ was modestly upregulated by . -fold at hpi ( figure c) . the expression of duck ifitm , , and were gradually induced and reached their maximum value at hpi. strikingly, expression of duck ifitm was markedly increased by over -folds at hpi (figures d-f) . these observations were further confirmed by rt-pcr analysis (supplementary figure ) . moreover, we examined the mrna expression profile of ifns and ifitms in kidney and bursa of fabricius tissues. we found that in kidneys, the mrna levels of ifns and ifitms were modestly upregulated until hpi and then declined (supplementary figure ) . ifns and ifitms expressions in bursa frontiers in microbiology | www.frontiersin.org figure | expression of ifns and ifitm family is strongly upregulated in duck in response to atmuv infection. twenty five -day-old healthy mule ducklings were challenged by intramuscular injection with . × eld of atmuv in a volume of . ml per duckling. three randomly selected ducklings were sacrificed at , , , , and hpi, respectively. their spleen tissues were collected for examination of ifn-α (a), ifn-β (b), ifn-λ (c), and ifitm , , (d-f) mrna expression using qrt-pcr. the mrna levels were normalized to the endogenous β-actin level and the expression at hpi was set to . . expression at - hpi was compared to its expression at hpi. each sample was analyzed in triplicate and the results are depicted as means ± sd (n = ). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . of fabricius were slightly increased with the highest levels at hpi and then declined (supplementary figure ) . taken together, these data indicate that atmuv infection can trigger innate immune response in ducklings, including upregulation of ifns and ifitms. ifitms are significantly induced in def and df- cells after atmuv infection or treatment with ifn to confirm duck innate immune response induced by atmuv, we further determined the expression of type i and type iii ifns and ifitms following atmuv infection in vitro. for this, defs were prepared and infected with atmuv, and the mrna expression of particular ifns and ifitms were then analyzed using qrt-pcr. atmuv replicated well in defs with an obvious cytopathic effect (cpe) characterized by cell shrinking, rounding and detachment at hpi ( figure a) . analysis of ifa showed that viral antigens were detected in def cells at - hpi, indicating that def cells could be infected by the virus (figure b) . the mrna levels of ifn-α, ifn-β, and ifn-λ were significantly elevated in atmuv infected defs during atmuv infection as compared to mock-treated control. expression of ifn-α and ifn-β was increased by about -and -fold at hpi, respectively (figures c-e) . ifn-λ expression was also significantly induced with a highest . -fold at hpi. qrt-pcr analysis was performed to examine the mrna expression of duck type i and type iii ifns (c-e) and ifitm , , (f-h). the mrna levels were normalized to the endogenous β-actin level and the expression at hpi was set to . . expression at - hpi was compared to its expression at hpi. plotted are the average levels from three independent experiments with three replicates per experiment (means ± sd). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . frontiers in microbiology | www.frontiersin.org these data were consistent with the in vivo studies presented above. strikingly, duck ifitm was greatly induced and reached their maximum value ( -fold) at hpi and then declined gradually ( figure f) . expression of duck ifitm and ifitm were enhanced modestly and reached their maximal levels by . and . -fold at hpi, respectively (figures g,h) . these results were further confirmed by rt-pcr analysis (supplementary figure ) . taken together, these data reveal that innate immune response is triggered in defs infected with atmuv, as indicated by significant upregulation of ifns and ifitms. because def cells are hard to survive after several passages, we employed chicken df- cell line as a model system to further investigate the interaction between host innate immune system and atmuv, and the role of ifitms in defense against viral infection. as expected, mrna levels of ifn-α, ifn-β, and ifnλ were greatly elevated by atmuv infection and reached the maximal levels at hpi and then declined gradually as compared to mock treatment (figures a-c) . similarly, expression of chicken ifitm (chifitm ) was highly upregulated by fold at hpi. expression of chicken ifitm (chifitm ) and ifitm (chifitm ) was also significantly increased in response to atmuv infection (figures d-f) . these observations were further confirmed by rt-pcr analysis (supplementary figure ) . since atmuv infection induced significant upregulation of ifns and ifitms in vivo and in vitro, we asked whether increased ifitm expression was stimulated by atmuv-induced ifns in df- cells. to this end, df- cells were treated with chicken ifns ( iu/ml) and mrna levels of ifitms were examined by qrt-pcr. indeed, we found that expression of ifitm , , was significantly upregulated in df- cells after treatment with chicken ifns (figure g ). results presented above revealed that atmuv infection effectively induced the expression of particular type i, type iii ifns, and ifitms in vivo and in vitro. furthermore, we tested that whether these ifitms functioned in defense against the virus infection. for this, we generated stable df- cell lines expressing specific shrnas targeting ifitm , ifitm , ifitm , or luciferase control, respectively. these cells were then infected with atmuv and harvested at indicated times ( , , and hpi) . interference efficiency of the shrnas was examined by qrt-pcr and viral load in cell culture supernatants was titrated via tcid assay. compared with the control shrna targeting luciferase, the specific shrnas caused decreased expression of ifitm , ifitm , or ifitm after atmuv infection, respectively (figures a-c) . interestingly, silencing endogenous ifitm or ifitm resulted in significant increase in viral load, as evidenced by higher viral titers in culture supernatants from the ifitm or ifitm knockdown cells than those in supernatants of luciferase control cells ( figure d) . however, knockdown of ifitm only slightly enhanced atmuv replication ( figure d ). to confirm these findings, df- cell lines expressing specific shrna targeting each of these ifitms were infected with atmuv and harvested at hpi, followed by qrt-pcr assay to detect mrna expression of viral genes. consistent with the results from tcid assay, data of qrt-pcr showed that mrna expression of viral envelope and ns genes was clearly increased in ifitm or ifitm knockdown cells as compared to the control cells. however, no significant difference was observed in viral load between ifitm knockdown and luciferase control cells after infection with atmuv ( figure e ). this data suggests that disrupting the endogenous expression of chicken ifitm and ifitm but not ifitm can strongly enhance the atmuv replication in df- cells. since silencing ifitm and ifitm could increase the susceptibility of df- cell to atmuv infection, we further investigated whether forced expression of ifitms could inhibit atmuv replication. thus, we cloned chicken and duck ifitm and ifitm , and their cdna sequences were analyzed and deposited in the genbank database. df- cells were then transiently transfected with constructs expressing chicken or duck ifitm and ifitm or empty vector, respectively and challenged with atmuv. virus titers in cell culture supernatants were determined by tcid assay at , , and hpi. the forced expression of ifitms in df- cells was examined by western blotting. as shown in figure a , flag-tagged ifitm proteins were expressed well in df- cells (figure a) . we observed that overexpression of chicken or duck ifitm and ifitm obviously suppressed atmuv replication in df- cells, as indicated by lower tcid titers in culture supernatants from the ifitm-overexpressing cells than those in control cells ( figure b) . to further confirm this observation, the df- cells were infected with atmuv and harvested at hpi, followed by qrt-pcr to examine the viral gene expression. as compared with control cells containing empty vector, overexpression of chicken or duck, ifitm or ifitm in df- cells reduced mrna expression of atmuv envelope gene by average of , , , %, respectively ( figure c) . these data suggest that avian ifitm or ifitm could strongly interfere with atmuv infection. our findings revealed that avian ifitm and ifitm were involved in cellular restriction of atmuv infection. next, we analyzed chicken (df- cell origin) and duck (duck embryo fibroblasts origin) ifitm and ifitm genes repertoire. the cdna sequences had been submitted in the genbank database (accession numbers: chifitm , kx ; chifitm , kx ; difitm , kx ; difitm , kx ). blastx search showed that the chifitm (kx ) shared , . , . , . , . , and . % amino acid identity to ifitm protein of chicken (kc . ), coturnix (xm_ . ), duck (kx ), human (ak . ), mouse (xm_ . ), and sus scrofa (xm_ . ), figure | ifitms are significantly induced in df- cells after atmuv infection or treatment with ifn. df- cells were infected with or without atmuv at a moi of . and harvested at , , , , and hpi, respectively. qrt-pcr was performed to determine the relative mrna expression of indicated ifns (a-c) and ifitm (d-f) genes compared with that at hpi. (g) df- cells were treated with chicken ifns ( iu/ml) for , , or h. qrt-pcr was performed to determine the relative mrna expression of ifitm genes compared with that without ifn treatment. the mrna levels were normalized to the endogenous β-actin level. plotted are the average levels from three independent experiments with three replicates per experiment (means ± sd). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . respectively. chifitm (kx ) shared . , . , . , . , . , and . % amino acid identity to ifitm protein of chicken (xm . ), coturnix (xm_ . ), duck (kx ), human (bc . ), mouse (nm- . ), and sus scrofa (nm_ . ), respectively. two putative transmembrane domains were obtained by using transmembrane prediction programs tmhmm (http://www.cbs.dtu.dk/services/tmhmm/) and tmpred plotted are the average results from three independent experiments (means ± sd). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . (http://www.ch.embnet.org/software/tmpred_form.html). chicken and duck ifitm and ifitm proteins could be divided into five domains reflecting their hydrophobicity and conservation as identified in other species (figure a) , including a variable, hydrophobic n-terminal domain (ntd), a conserved hydrophobic intramembrane domain (imd), a conserved intracellular loop (cil), a variable, hydrophobic transmembrane domain (tmd), and a short, highly variable c-terminal domain (ctd). although significant divergence is seen between avian and mammalian ifitm and ifitm sequences, several residues are conserved and important for antiviral function, including important amino acids (y , c , c , k , k , and k ) in ifitm (smith et al., ; blyth et al., ) . phylogenetic analysis exhibited that avian ifitm and ifitm are distinct from those of mammalian species, but avian ifitm is more conserved than avian ifitm ( figure b ). atmuv is a newly emerging member of the ntaya virus group within the genus of flavivirus, causing severe egg-drop syndrome and neurological disease in domestic poultry homonnay et al., ; thontiravong et al., ) . on the basis of typical clinical symptoms, atmuv was initially described as duck egg drop syndrome virus (dedsv). however, this virus exhibits a wide range of pathogenicity to avian species including ducks, chicken, geese, house sparrows, and racing pigeon, and its genome is closely related to tembusu virus strains. flavivirus isolates with a more than % nucleotide sequence homology in the ns region are considered to be the same species (kuno et al., ) . so we propose to name this novel flavivirus as "avian temubsu virus, atmuv." till now, there is no effective method for its prevention except vaccine. the previous studies have shown that atmuv infection induced an effective antiviral immunity through mda and tlr -dependent signaling pathways chen et al., a; fu et al., ) . however, our understanding of the molecular and cellular basis of interaction between the virus and host antiviral immune system is very limited. in this study, we examined the expression profile of key ifns and ifitms following atmuv infection in vivo and in vitro. we found type i, type iii ifns, and immune-related ifitms, including the viral titers in cell culture supernatants were determined by tcid assay in def cells. (c) the mrna expression of viral genes was examined by qrt-pcr at hpi. the mrna levels were normalized to the endogenous β-actin level and the expression in atmuv infected df- cells expressing empty vector was set to . . the mrna expression of viral genes in df- cells expressing exogenous ifitms was compared to that in control cells containing empty vector. plotted are the average results from three independent experiments (means ± sd). statistical significance was determined by one tail student's t-test analysis. *p < . , **p < . . ifitm , ifitm , and ifitm were significantly upregulated in response to atmuv infection. strikingly, the spleen tissue had a high viral load and showed strong innate immune response as compared to other organs. ifn-α/β expression levels in spleen were increased significantly at hpi and decreased after this time point, and the mrna levels of ifitm and ifitm were also upregulated. similar results were shown in kidney and bursa of fabricius tissues in infected animals. although ifn expression in defs was triggered and reached the maximum value at hpi, defs displayed an apparent cpe by atmuv infection at this time point. these data suggest that host innate immune response is triggered by atmuv, but the virus has developed multiple strategies to evade host antiviral immunity. in a recent study, wang and his co-workers found that atmuv ns could markedly suppress virus-induced ifn-β expression by inhibiting rlr receptor signaling . most flaviviruses are potent in blocking the jak-stat pathway to evade the antiviral effects of the host innate immune system (heim et al., ; basu et al., ; lin et al., ; green et al., ) . thus, further studies are needed to address the precise mechanisms by which atmuv circumvents the host innate immune response. type i and type iii ifns bind to their receptors, which stimulates the jak-stat pathway that triggers various isg expression. expression of ifitms is strongly induced by ifns (smith et al., ) . consistent with previous studies (smith et al., ) , our data showed that stimulation of df- cells with chicken ifns significantly enhanced the expression of ifitms. interestingly, we observed that ifitm in atmuv infected defs had a faster kinetic response than atmuv-induced ifns. thus, we assessed whether ifitms expression is totally ifn-dependent. for this, sirna specifically targeting chicken interferon alpha/beta receptor (chifnar -sirna) and negative control sirna (nc-sirna) were employed in this study. nm chifnar -sirna or nc-sirna was transfected into df- cell. twenty-four hours post-transfection, the cells were infected with atmuv and harvested at hpi. interestingly, disruption of endogenous ifnar by sirna greatly reduced ifitm mrna expression, but only caused modest downregulation of ifitm and ifitm (supplementary figure ) . the data suggest that chifitm may not share the same ifn-dependent pathways with chifitm and chifitm . previous study has found that murine ifitm expression was not only induced by increased expression of type i or ii ifns, but also upregulated by other cytokines such as il- (bailey et al., ) . other groups also found that expression of ifi- k, ifi- k, and isg was induced directly by virus and then triggered secondarily through virus-induced ifns (wathelet et al., ; holzinger et al., ) . these findings indicate that multiple signaling pathways might be involved in regulation of ifitm expression during the viral infection. ifitms serve as critical effector molecules in host innate immune system and effectively restrict a wide range of pathogenic viruses, such as dengue virus, influenza a virus, hepatitis c virus, west nile virus, and hiv- (brass et al., ; wilkins et al., ; yu et al., ) . in order to survive, some viruses such as arenavirus and foot-and-mouth disease virus have evolved multiple strategies to evade the antiviral effects of ifitms (bailey et al., ; zhang et al., ) . furthermore, human cytomegalovirus could exploit ifitms to facilitate morphogenesis of virion assembly compartment (xie et al., ) . to investigate the role of ifitm proteins in restricting atmuv replication, we generated df- cell lines stably disrupting chicken ifitm , ifitm , or ifitm expression. indeed, disruption of endogenous ifitm and ifitm by shrna greatly enhanced atmuv infection. furthermore, ectopic expression of chicken and duck ifitm or ifitm markedly inhibited atmuv replication. interestingly, chifitm was significantly upregulated after atmuv infection in vitro, but we found that altering ifitm expression had mild effect on atmuv infection. a previous report showed that duck ifitm was greatly upregulated after highly pathogenic iav infection but its antiviral activity was low in vitro (blyth et al., ) . the differences in viral entry mechanisms of iav and atmuv in the different types of host cells may account for the differential antiviral activity of ifitm proteins. five ifitm proteins have been identified in duck and chicken species including ifitm , ifitm , ifitm , ifitm , and ifitm . ifitm , ifitm , and ifitm belong to the immunerelated clade, whereas non-immune ifitm and ifitm make up the two remaining clades (blyth et al., ) . in this study, we observed that ifitm increase fold was much higher than those of ifitm and ifitm by in vivo and in vitro assays. this may be due to high basal expression of ifitm and ifitm in the cells tested. in addition, we cloned and characterized chicken and duck species of ifitm and ifitm genes. despite sharing low amino acid identity between chicken ifitm and duck ifitm ( . %) and high amino acid identity between chicken ifitm and duck ifitm ( . %), both chicken and duck origin ifitm and ifitm could effectively restrict atmuv replication. certain key amino acids in imd and cil domain are conserved in chicken and duck ifitms, suggesting their importance for functioning of the ifitms (smith et al., ) . our previous work demonstrated that pretreatment of cells with ifns could significantly impair atmuv replication (chen et al., a) . our present study further provides the evidence that host ifitm proteins have the ability to control the atmuv infection and likely restrict the viral reproduction as well. taken together, atmuv infection induces host's effective antiviral immune response involving several critical ifns and ifitms proteins, which can be useful for developing new antiviral drugs in future. however, further studies are required for better understanding of precise mechanisms underlying the antiviral activity of ifitm proteins. slc performed most of the experiments, collected and analyzed data and wrote the manuscript. lw, lz, ws, yn, and zy participated in preliminary data acquisition. jyc contributed to plasmids construction. xz helped with data analysis. sw and xc contributed to shrna design and revised the manuscript. jlc and mg revised the manuscript. syc and jlc conceived of the study, participated in study design and coordination. all authors read and approved the final manuscript. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. . /full#supplementary-material ifitm limits the severity of acute influenza in mice ifitm-family proteins: the cell's first line of antiviral defense hepatitis c virus core protein modulates the interferon-induced transacting factors of jak/stat signaling pathway but does not affect the activation of downstream irf- or gene duck interferoninducible transmembrane protein mediates restriction of influenza viruses human serological studies in a land dyak village the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus tembusu virus in ducks, china production and characterization of monoclonal antibodies against porcine reproductive and respiratory syndrome virus avian tembusu virus infection effectively triggers host innate immune response through mda and tlr -dependent signaling pathways isolation and characterization of a distinct duck-origin goose parvovirus causing an outbreak of duckling short beak and dwarfism syndrome in china isolation and characterization of a chinese strain of tembusu virus from hy-line brown layers with acute egg-drop syndrome in fujian the broad-spectrum antiviral functions of ifit and ifitm proteins comparative analysis of transcriptional profiles of retinoic-acid-induced gene i-like receptors and interferons in seven tissues from ducks infected with avian tembusu virus innate immunity to dengue virus infection and subversion of antiviral responses expression of hepatitis c virus proteins inhibits signal transduction through the jak-stat pathway induction of mxa gene expression by influenza a virus requires type i or type iii interferon signaling tembusu-like flavivirus (perak virus) as the cause of neurological disease outbreaks in young pekin duck distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus encephalitis and retarded growth of chicks caused by sitiawan virus, a new isolate belonging to the genus flavivirus phylogeny of the genus flavivirus ifitm proteins restrict viral membrane hemifusion immune responses of ducks infected with duck tembusu virus blocking of the alpha interferoninduced jak-stat signaling pathway by japanese encephalitis virus infection adapted tembusu-like virus in chickens and geese in china duck egg drop syndrome virus: an emerging tembusu-related flavivirus in china the ifitm proteins inhibit hiv- infection identification of a flavivirus isolated from mosquitos in chiang mai thailand ifitms restrict the replication of multiple pathogenic viruses arbovirus infections in tembusu and sindbis virus isolations from mosquitoes impact of chicken-origin cells on adaptation of a low pathogenic influenza virus chicken interferon-inducible transmembrane protein restricts influenza viruses and lyssaviruses in vitro duck egg-drop syndrome caused by byd virus, a new tembusu-related flavivirus innate immunity to virus infection pattern recognition receptors and inflammation modeling and dynamical analysis of virus-triggered innate immune signaling pathways tembusu virus in human tembusu-related flavivirus in ducks effect of age and inoculation route on the infection of duck tembusu virus in goslings transmission dynamics of the recently-identified byd virus causing duck egg-drop syndrome the emerging duck flavivirus is not pathogenic for primates and is highly sensitive to mammal interferon signaling duck tembusu virus nonstructural protein antagonizes ifn-β signaling pathways by targeting visa influenza a virus-induced degradation of eukaryotic translation initiation factor b contributes to viral replication by suppressing ifitm protein expression transport of influenza virus neuraminidase (na) to host cell surface is regulated by arhgap and cdc proteins the antiviral restriction factors ifitm , and do not inhibit infection of human papillomavirus regulation of two interferon-inducible human genes by interferon, poly(ri).poly(rc) and viruses ifitm is a tight junction protein that inhibits hepatitis c virus entry human cytomegalovirus exploits interferon-induced transmembrane proteins to facilitate morphogenesis of the virion assembly compartment an infectious disease of ducks caused by a newly emerged tembusu virus strain in mainland china ifitm proteins restrict hiv- infection by antagonizing the envelope glycoprotein induction of systemic ifitm expression does not effectively control foot-andmouth disease viral infection in transgenic pigs key: cord- -bicg ozr authors: aurisicchio, l; bujard, h; hillen, w; cortese, r; ciliberto, g; la monica, n; palombo, f title: regulated and prolonged expression of mifnα in immunocompetent mice mediated by a helper-dependent adenovirus vector date: - - journal: gene ther doi: . /sj.gt. sha: doc_id: cord_uid: bicg ozr a major goal in gene therapy is to develop efficient gene transfer protocols that allow tissue-specific, long-term and tightly regulated expression of the desired transgene. this objective is becoming more attainable through the co-evolution of gene transfer vectors and regulation systems. the ideal vector should efficiently transduce non-dividing cells with minimal toxicity, thus endowing the system with persistent transgene expression. the helper-dependent adenovirus vectors meet these requirements, as demonstrated in various studies in the literature. the most promising regulation system is the tet-on system, which has low basal transcriptional activity and high inducibility. to explore the regulated transgene expression in the context of a helper-dependent vector, we constructed the hd-tet-ifn vector, containing the mifnα gene under the control of the tetracycline inducible transactivator rtta (s)-s . mice injected with hd-tet-ifn showed high levels of serum mifnα only upon transcriptional activation. the transgene expression was reinducible to the same high level up to months p.i., and the amount of expressed cytokine could be regulated by dosing doxycycline. transcriptional activation of mifnα induced by doxycycline resulted in prolonged survival and reduced liver damage in hd-tet-ifn-injected mice challenged with a lethal dose of coronavirus. activation of antiviral genes mediated by doxycycline-dependent mifnα expression was also observed at low hd-tet-ifn doses. the possibility of controlling gene expression by the combination of hd vectors and the latest tet-on transactivator also holds promise for studying gene function in other animal models. to date, various strategies including viral and nonviral vectors have been developed for gene transfer. among the viral vectors, adenovirus (ad) vectors deliver genes to a wide variety of cell types and tissue independently of their proliferative state (reviewed in ref. ). these vectors have been modified by introducing deletions in the early genes to increase cloning capacity and reduce cellular toxicity. however, these modifications have not lead to a prolonged transgene expression in rodents and nonhuman primates, indicating that residual low level expression of ad genes is responsible, at least in part, for the short-term persistence. the 'gutless' or helper-dependent adenovirus vector (hd) allowed transgene expression to persist for almost the entire lifetime of mouse as shown in liver, brain and muscle. [ ] [ ] [ ] these vectors lack all viral coding sequences and are produced by the co-infection of a suitable cell line with an ad helper virus, which provides in trans the proteins required for replication and packaging. , in light of the extensive cloning capacity ( kb) and the persistence of expression in many cell types, hd vectors are ideal vectors for studying gene functions in the absence of interfering viral promoters, undesired expression of potentially toxic ad genes, and reduced immune response against ad proteins. to achieve safe and effective protein expression, pharmacological control over the level of gene transcription is required. placing gene activity under control from outside via an effector molecule allows the activity to be limited within a 'desired window'. thus expression can be adjusted according to the evolution of induced biological effects, and treatment to be terminated by drug withdrawal. different systems based on small-molecule control of transcription have been developed and proven to be effective in mice. the rapamycin-regulated system (rrs) uses a 'dimerizer' drug to bring together the functional units of a bipartite transcriptional factor. a different chimeric system is based on the progesteron antagonist, ru . an hd vector expressing human growth hormone (hgh) under control of the ru -inducible system was very effective both in controlling hgh expression and in inducing a biological response for a limited period of time. the tet systems, widely applied both in vitro and in vivo, have been derived from elements of the tetracycline (tc) resistance operon of escherichia coli which were converted into eukaryotic transcription activation systems. in one version a tet-controlled trans-activator (tta), a fusion between the tet repressor protein (tetr) and a eukaryotic transcription activation domain, binds to tet operator sequences fused to a minimal promoter and activates transcription in the absence of tc. addition of tc, or one of its derivative such as doxycycline (dox), abolishes transcription. in a second version, a mutated form of tta, rtta, binds and activates transcription only in the presence of dox. both tet systems were used in gene therapy models whereby a variety of vectors were applied: retroviruses, adenoassociated virus (aav) binary systems, first generation adenoviruses, and electroporated dna. , [ ] [ ] [ ] despite numerous successful applications, the tet regulatory systems originally described show some limitations. rtta exhibits some residual affinity to teto in the absence of dox and requires relatively high levels of dox to achieve full activation. in addition, rtta and possibly its mrna are rather susceptible to degradation, at least in certain cell types. these limitations were overcome by novel rtta versions, which were identified via mutagenesis and genetic selection in yeast. the new transactivator species such as rtta s -s , which were subsequently embedded in synthetic sequences designed to optimize expression in mammals, showed drastic improvements in all parameters mentioned above. pharmacological control of gene expression may be critical in those applications utilizing pleiotropic cytokines such as ifn␣, which is currently used in the treatment of viral hepatitis. a more effective antiviral treatment against hbv infection was obtained by introducing asialoglycoprotein binding sites in the recombinant ifn␣, which in turn led to higher intrahepatic concentrations. ifn␣ gene transfer may offer the possibility of limiting ifn␣ expression to the liver, , with a potential reduction of side-effects in other tissues, such as the central nervous system, provided that transgene expression is kept within safe and therapeutic levels. the antiviral ifn␣ action is thought to be mediated by transcriptional activation of many genes including ', '-oligoadenylate synthetase ( ' 'oas) and gtpase such as tgtp. in this report, the features of regulated mifn␣ expression are explored utilizing the novel rtta s -s transactivator carried in a single hd vector, hd-tet-ifn. the efficacy of a dox-regulated expression of mifn␣ was verified in a murine model of acute hepatitis. liver protection was dependent only on activation of mifn␣ expression induced by dox treatment. secretion of the cytokine was repeatedly re-induced to the same high level for a period of months, and controlled by the amount of dox delivered. in addition, c /b mice injected at low hd-tet-ifn doses resulted in doxmediated regulation of liver restricted mifn␣ expression, which was associated with induction of antiviral genes. these results indicate that desired levels of mifn␣ expression can be achieved and maintained by controlling both the vector dosage and the transcriptional activity. to control mifn␣ expression an inducible cassette based on the tet system was constructed (figure a) . the tetracycline-sensitive transactivator rtta s -s was cloned under the liver-specific ttr promoter and enhancer followed by the sv polyadenylation signal. mifn␣ gene was inserted in the opposite orientation under the control of the p tet- promoter, followed by the bgh polyadenylation signal. to increase mrnas stability, two introns were inserted upstream of the transactivator and of the mifn␣ gene, respectively. the expression cassette was inserted in the helper-dependent backbone vector c hsu generating phd-tet-ifn plasmid and the hd-tet-ifn vector was rescued and amplified in cre cells using the h helper virus. to compare in vitro the potency of mifn␣ expression mediated by hd-tet-ifn or hd-ifn, which expresses mifn␣ directly under the ttr promoter, hep b cells were treated with pp/cell and mifn␣ released in the medium was measured at day p.i. (figure b) . in hd-tet-ifn-treated cells -fold dox-dependent induction of mifn␣ production was observed. lower values were observed in hd-ifntreated cells. to visualize the cells which expressed mifn␣ as a function of the vector dose, hep b were treated with increasing amounts of hd-tet-ifn and mifn␣ expression revealed by an immunohistochemistry assay. a dose-response correlation was observed between the vector dose and the cells stained with an anti-mifn␣ antibody only in dox-treated cells, whereas no positive staining was observed in the absence of dox even in cells treated with hd-tet-ifn at the highest doses of pp/cell ( figure ). these results indicate that hd-tet-ifn allowed a significant expression of mifn␣ controlled in vitro by dox. to verify the efficiency and persistence of mifn␣ secretion, c /b mice were injected i.v. with . × pp of hd-ifn or hd-tet-ifn and the mifn␣ released in the serum was measured over time. as previously reported, circulating mifn␣ was not detected at this dosage in hd-ifn-treated mice. differently, in hd-tet-ifninjected mice serum mifn␣ was observed at this dosage starting from day p.i. after days of dox treatment ( figure ). an average of u/ml was detected in all gene therapy treated mice (n = ). removal of dox resulted in a rapid decrease of serum mifn␣ to undetectable levels in days (below u/ml). therefore, expression was increased at least -fold. the same expression kinetics as a function of the dox treatment was observed throughout the months analyzed. hd-tet-ifn injection in the muscle did not lead to detectable serum mifn␣ present in the serum, indicating that liver specificity was maintained in this expression vector (data not shown). to verify the level of mifn␣ released in the blood stream as a function of dox added in the drinking water, mice were treated for days with different dox concentrations. as shown in figure , a clear correlation was observed between the concentration of dox in the drinking water and the serum mifn␣ measured in both vsv and elisa assay. at a concentration of g/ml, serum mifn␣ was in the range of u/ml, whereas at g/ml it was - u/ml. at the lowest dox concentration, serum mifn␣ was detectable only with the more sensitive, albeit not quantitative elisa assay. these results are in line with observations in transgenic mice where a -fold reduction in the amount of dox added in the drinking water resulted in -fold lower amount of luciferase detected in the liver. to further characterize the kinetics of expression induced by a single oral dose of dox, c /b mice were injected with × pp and serum mifn␣ measured over time ( figure ). at a dose of mg/kg of dox, rapid induction of mifn␣ expression was observed as early as h after induction. maximal expression was observed between and h with mifn␣ values ranging from to u/ml. serum mifn␣ levels returned to background within h. the rapid kinetic of expression did not allow the precise measurement of the peak value for each mouse and may explain, at least in part, the five-fold variation in maximal mifn␣ expression. at mg/kg of dox, higher levels were observed, indicating that maximal induction may not be achieved with a single dose of mg/kg of dox (data not shown). overall, these results show that in the context of hd-ad vector rtta s -s allows both long-term and dosedependent dox regulation of the mifn␣ gene expression in the liver. to investigate the therapeutic potency of a regulated expression of mifn␣ gene, we assessed the antiviral strength of hd-tet-ifn in an acute hepatitis model. for this purpose, we examined the effects of hd-tet-ifn on the infection of susceptible mouse strain c /bl with mouse coronavirus mhv- . treatment with recombinant mifn-␤ type i was shown to prolong survival following mhv- exposure, particularly when the treatment was initiated before viral infection. , mice were injected i.v. with × pp of hd-tet-ifn and mifn␣ induction measured at day and p.i. as expected, in all injected mice treated with dox in the drinking water at a concentration of g/ml for days, circulating mifn␣ was observed in the range of u/ml and returned to basal level days after dox withdrawl. at day p.i., a subgroup of mice were reinduced with dox in the same condition described above, and all mice were infected at day with an i.p. injection of p.f.u. of mhv- . to verify the impact of mifn␣ gene therapy induction on mhv- -mediated liver damage, transaminase (alt) present in the serum were measured days after mhv- infection (figure a ). hepatic protection was observed in mice pre-induced with dox, where serum transaminase levels remained at basal levels. however, a sharp rise in serum alt levels was observed in mice injected with hd-tet-ifn and not treated with dox or in control mice (mock). an additional effect, observed only in dox-treated mice, was a slight but significant prolonged mean survival time (p Ͻ . ). as shown in figure b , control mice and those not treated with dox died between day and after mhv- infection, whereas in the dox-treated group one out of six survived till day when the experiment was terminated (mean survival time ± standard deviation: mock ± days; no dox . ± . ; dox . ± . ). therefore, these experiments demonstrate that doxdependent mifn␣ expression results in liver protection and prolonged survival in mhv- infected mice. previously, we have shown that injection of low doses of hd-ifn vector gives rise to intrahepatic production of the cytokine without any detectable mifn␣ in circulation. therefore, we examined whether such a result could also be obtained with a tet-regulated vector. c /b and balb/c mice were i.v. injected with different doses of hd-tet-ifn and mifn␣ measured at day p.i. after a treatment for days with dox, g/ml, present in the drinking water (figure a ). in c /b mice injected with low vector doses ( . × pp and . × pp) and not treated with dox, mifn␣ was detected neither in the liver, nor in the serum. however, liverrestricted mifn␣ in the range of - u/g was observed upon induction depending on the vector dose. higher hd-tet-ifn dose ( . × pp), in the absence of dox treatment, resulted in u/g of mifn␣ present in the liver, but not in the serum. after dox treatment mifn␣ levels increased -fold in the liver and fold in the serum. these levels returned to background upon dox withdrawal days later (data not shown). in balb/c mice, where ad is less persistent, liver-restricted mifn␣ was induced only at a virus dose of . × pp, whereas at lower vector doses the cytokine was undetectable. the hd-tet-ifn doses that resulted in liver-restricted mifn␣ expression were further analyzed for the induction of mifn␣ responsive genes (figure b ). in agreement with the presence of mifn␣ protein, northern blot analysis showed a clear signal for the mifn␣ mrna in both c /b and balb/c only in dox-treated mice (lane a). as a consequence of mifn␣ expression, ' 'oas and tgtp transcripts were elevated, as indicated by the rnase protection assay (lanes c and d). a . -and three-fold induction of ' 'oas signal was observed at the highest hd-tet-ifn dose indicated in balb/c and c /b , respectively. when compared with ' 'oas, the induction of tgtp over the background was less pronounced. to examine the reversal of induction of antiviral gene activities, ' 'oas and tgtp transcripts were examined days after dox withdrawal. at this time point, in c /b mice injected with . × pp previously induced with dox, mrna synthesis from antiviral genes had returned to basal level. to evaluate the cell types which express mifn␣ upon dox treatment, immunohistochemistry with an antibody against mifn␣ was performed in the liver of c /b (figure c ). positive hepatocytes were observed in the proximity of vessel only upon dox induction. lastly, inflammation was evaluated by rnase protection assay for mrna of infiltrating lymphocytes, eg cd , cd , cd ⑀ and macrophages. these markers were not elevated in hd-tet-ifn-injected mice independently of the dox treatment (data not shown). these experiments indicate that liver-restricted mifn␣ expression can be induced in both mouse strains without apparent liver damage by controlling the dose of the vec-gene therapy tor and by regulating, via dox, the expression of the mifn␣ gene. in this study we show the features of dox-dependent mifn␣ expression delivered through an hd vector. among the different vectors used in gene transfer, adenovirus vectors have emerged as one of the more efficient in transducing liver cells. to limit transgene expression in the liver further, the rtta s -s transactivator was cloned downstream of the liver-specific promoter/ enhancer ttr and the mifn␣ gene was inserted under the tet response promoter in a head-to-head configuration on the same vector, hd-tet-ifn (figure ) . a tight control of mifn␣ expression was observed in hep b cells using different amounts of vector ( figure ) . the ability to control the expression of a therapeutic gene within a relevant and safe window is a major prerequisite for a variety of regimens envisioned in gene therapy. the rapid adjustment of the activity of a therapeutic gene to the progression of the diseases is of particular importance. the hd-tet-ifn vector meets several of these requirements. injecting large doses ( . × pp) in c /b mice resulted in undetectable serum levels of mifn␣ in the absence of dox (figure ). upon induction with dox, mifn␣ was detected in the serum and its expression correlated with the amount of dox delivered to the animal (figure ). high levels of secreted mifn␣ were observed at the largest dose of dox, with only slight variations over a period of months. although the rapid induction of secreted cytokines has already been widely reported, the rapid reversal of mifn␣ induction represents a major improvement over the previously described systems. the rapamycin system, which was explored in the context of ad or aav vectors, demonstrated rapid induction of hgh and controlled expression over a wide range of doses. however, hgh remained above the basal levels for up to days after a single injection of the inducer. similar slow de-induction kinetics were observed in the brains of rats using the tet-off system delivered by a first generation ad vector. improved reversal of induction was reported, with the ru regulation system carried on a hd vector and driven by the same ttr promoter used in this study. interestingly, burcin et al observed a similarly inefficient induction of their reporter gene at an early stage after hd injection, as did our system. further experiments are required to understand why weeks appear to be required to reach steady state expression. since full induction of tet-controlled genes is achieved within less than h in the liver of transgenic mice, other factors such as the immuno-response against ad particles may have a detrimental effect on transcription at this early stage. one limitation of the actual rtta s -s based system is its poor sensitivity to dox, which in our experimental conditions was not saturated using mg/kg ( figure ). however, in parallel with rtta s -s , the rtta s -m transactivator was developed, which shows a -fold higher sensitivity to dox in hela cells. it will be interesting to explore this additional transactivator in the context of hd vectors, which may bring the dox required within the dose range of human antibiotic therapy. apparently, the long persistence and sustained reinducibility of mifn␣ expression (figure ) in immuno- competent mice indicates that the rtta s -s transactivator is not immunogenic, although we presume that the use of a liver-specific promoter such as ttr, which expressed the transactivator only in hepatic cells, and not in professional antigen presenting cells, may have contributed to this persistence. this hypothesis is in agreement with previous reports showing long-term transgene expression in the context of first generation ad vector only in the presence of a liver-specific promoter. in this study, liver protection against mhv- challenge was observed only upon dox-mediated induction of mifn␣ expression (figure a ). the striking difference observed in association with the dox treatment is very significant in view of the fact that the same viral preparation was injected in both groups, eliminating experimental bias such as difference in the transducing particles per unit volume, purity of vector preparation and dilution volume. in addition, liver protection was achi-eved weeks after hd-tet-ifn injection, in line with the notion that therapeutic effects mediated by helperdependent vectors persist for a long period of time. previously we showed that lower levels of serum mifn␣ were associated with partial hepatic protection, but had no effects on survival rate. in this study, all mice treated with dox showed liver protection and one out of six survived the mhv- infection (figure b) . the higher level of circulating mifn␣ may have elicited stronger local and peripheral effects such as induction of antiviral genes or peritoneal macrophage activation, which may have limited mhv- replication in a more efficient manner. although we showed that injection of hd-tet-ifn resulted in dox-dependent hepatic protection, the impact of this gene therapy approach on chronic hepatitis remains to be established, as no mouse model for this disease exists, unlike hbv (woodchuck model) or hcv infection (non-human primates, such as chimpanzees). however, the pharmacological control of ifn␣ expression is also required in the context of these animal models in light of safety concerns such as toxicity, sideeffects and adjusting the ifn␣ level as a function of disease progression. recombinant ifn␣ is widely used in the treatment of human hepatitis b and c. however, systemic injection is associated with side-effects, particularly on the neuronal system, which cause the withdrawal of a significant number of patients from the therapy. therefore, it was of interest to evaluate the possibility of limiting the biological response induced by ifn␣ expression to the liver as a function of the dox treatment and the vector dosage. in a previous study, we observed that in mice injected with the hd-ifn, which express mifn␣ directly under the constitutive ttr promoter, liver-restricted mifn␣ expression, as well as the induction of antiviral genes last for months (data not shown). this observation is in agreement with recent reports where gene transfer mediated by hd vectors resulted in long persistence of transgene expression. , , this long period of ifn␣ expression could, nonetheless, be considered 'over-therapeutic' in light of the current treatment for hcv, which lasts for months. using the hd-tet-ifn vector, however, allows a controlled timing and potency of expression on the basis of dox treatment even at low vector doses. indeed, low doses of hd-tet-ifn resulted in mifn␣ being detectable in the liver and not in the bloodstream only upon dox induction (figure a ). as a consequence of liver-restricted mifn␣ expression, antiviral genes were induced (figure b ) in the absence of liver damage or inflammation response. also in the liver, the reversal of mifn␣ induction was fairly rapid, as indicated by the reduction of antiviral gene mrnas days after dox withdrawal. lastly, the expression of mifn␣ was highlighted in the hepatocyte cells upon dox treatment ( figure c ). as expected on the basis of previous experiments with marker genes, dox-dependent mifn␣ expression distributed in hepatocytes, which are in the proximity of the vessel. the use of hd vectors and the rtta s -s transactivator allows the expression of transgenes to be regulated for a long period of time within an expression window, which can be either restricted to the liver or to circulating protein. the lack of a detectable immunoresponse against the hd-tet-ifn in association with its prolonged reinducibility candidate these vectors and regulation system not only for gene therapy purposes, but, at least in some applications, as an alternative option to the use of transgenic animals for tissue-specific expression. while germline transgenesis can generate useful animal models for genetic studies, it can be costly and time-consuming. additionally, it requires the use of a large number of animals and can be limited by toxicity during embryogenesis. the features of the system described in this paper allow the study of gene function within specific cells in a variety of animal models including non-human primates, where the hd vector-mediated gene transfer is very efficient. cell lines and cre . cells were grown in mem supplemented with % heat-inactivated fcs. l- (mouse fibroblasts) huh- and hep b (human hepatoma) cells were grown in dmem supplemented with % fcs. mice used in this study were immunocompetent, to weeks old (at the time of injection) c /b and balb/c females purchased from charles river (lecco, italy). groups of between four and five mice received injections in the tail vein of adenovirus vectors diluted in physiologic solution in volumes of l. dox ( to g/ml, in % sucrose, ph . ) was added in the drinking water at the indicated time-points. blood was obtained by retroorbital bleeding and serum was stored at Ϫ °c. at the indicated time, mice were killed and organs were rapidly frozen in liquid nitrogen, and stored at Ϫ °c. the rtta s -s transactivator gene was recovered from the plasmid phud . as a ecori/bamhi fragment and subcloned in the vector pttr-bgh, containing the liverspecific transthyretin gene minimal enhancer and promoter and the bovine growth hormone poly-a site (bgh). to improve the level of expression, an artificial intron was amplified by pcr from pcat basic (invitrogen, carlsbad, ca, usa) and subcloned hindiii/ecori between the ttr promoter/enhancer and the mifn cdna, generating pttr-intr-rta -bgh. the intron a from the vector pvij-nsa as a sacii/ecori fragment was subcloned in the vector phud . between the tre (tet responsive element) and the sv polyadenylation site. a paci site and a noti site were inserted, respectively, upstream and downstream of the sv polya by pcr. at this point the tre-intron a-sv fragment was excised as a xhoi/kpni fragment and inserted in pttr-intr-rta -bgh, thus generating ptetϪ/Ϫ. the mifn␣ gene was amplified by pcr adding a bamhi site at ' and a paci site at the ' and subcloned in ptetϪ/Ϫ, generating ptet-mifn␣ . to construct pc -tet-mifn, the mifn␣ expression cassette was excised with noti and subcloned in the noti site of pc -hsu. to rescue the hd-tet-ifn vector, the pc -tet-mifn plasmid was cleaved by pmei and transfected into cre . cells. subsequently, the cells were infected with the helper adenovirus h . the titer of the hd vector during the amplification passages on cre . cells was followed by infecting huh- cells with lysates from each passage and determining the amount of mifn␣ in cells supernatants with and without dox at h after infection by vsv inhibition assay. multiple viral passages were performed to reach a high titer and vector was purified by double cscl gradient. physical particles were measured by optical density of dna. the viral cytopathic inhibition assay using vescicular stomatitis virus (vsv) has been described elsewhere. mifn␣ activity is expressed in units/ml. the mifn␣ activity was calibrated against a standard recombinant mifn␣ (calbiochem, san diego, ca, usa). ninety-six-well plates were coated overnight at °c with . g/ml rat ascite monoclonal ab to mifn␣ (clone e-a , yamasa corporation, japan) in carbonate buffer mm ph . . after blocking with % bsa, × pbs, . % tween for h at °c, samples were added and incubated for h at rt in % bsa, × pbs, . % tween . subsequently, plates were washed three times with × pbs, . % tween and incubated for h at rt with l/well polyclonal ab to mifn␣/␤ (rdi) diluted at g/ml in % bsa, × pbs, . % tween . wells were washed again and incubated with anti-sheep igg ap con-jugate (sigma, st louis, mo, usa) diluted : in % bsa, × pbs, . % tween for h at rt. final detection was done adding l/well p-nitrophenyl phosphate, disodium, . mg/ml in % diethanolammine buffer, ph . containing . mm mgcl and reading at od . a standard mifn␣ was used as reference in each experiment. livers were weighed and homogenized in pbs using a polytron homogenizer and lysates were centrifuged for min at °c at r.p.m. to eliminate cell debris. since ifn␣ is acid stable, hcl . n was added to reach ph . and extracts were incubated overnight at °c. neutral ph was reached by adding naoh and centrifuged for min at °c at r.p.m. clarified extracts were then analyzed by vsv inhibition assay. frozen tissues were mechanically pulverized and rna was isolated from tissues using ultraspec rna reagent (biotecx laboratories) according to the manufacturer's instructions. total rna ( g) was used in northern blot analysis. the intensity of bands was quantified by phosphorimager analysis. the rnase protection assay for quantification of mrna was performed using the riboquant multi-probe rpa assay system (pharmingen, san diego, ca, usa) according to the manufacturer's instructions. the probe set for mifn␣ pathway activation was kindly provided by iain campbell (scripps, san diego, ca, usa). the extent of hepatocellular injury induced by adenovirus injection was monitored by measuring serum alanine aminotransferase (alt) activity at the indicated time-points. alt activity was measured in a spotchem model sp- according to the manufacturer's instructions. for cell staining, h after infection hep b cells were washed with pbs and fixed in × pbs, . % formaldehyde for min at rt. for tissue staining, at the indicated time-points livers were harvested from hd-tet-ifn-injected mice and fixed in × pbs, % paraformaldehyde for h at °c, then incubated overnight in × pbs, % sucrose. livers were then included in oct and stored at Ϫ °c. cells or liver sections were washed twice with pbs and incubated with pbs/ . m glycine for min at rt. after washing with pbs, samples were permeabilized with pbs/ . % triton-x- for min and treated with pbs, . % triton-x- , % goat serum for min at rt. samples were incubated for h at rt with rat ascite monoclonal ab anti-mifn ␣ (clone e-a , yamasa corporation) diluted : in pbs, % goat serum. after washing five times with pbs, a second incubation with anti-rat igg ap conjugate (sigma, st louis, mo, usa) antibody diluted : in pbs/ % goat serum was performed for h. after subsequent washings, samples were incubated with ap substrate ( mm nacl, mm mgcl , mm tris-cl, ph . , g/ml nbt, g/ml bcip) until sufficient staining was reached, then washed with pbs and mounted with aquatex. as described previously, mouse hepatitis virus type (mhv- ) was amplified on mouse fibroblast dbt cells, and the titer was measured in a standard plaque assay. a total of p.f.u. was injected intraperitoneally (i.p.) in l of physiologic solution. update on adenovirus and its vectors in vitro and in vivo biology of recombinant adenovirus vectors with e , e /e a, or e /e deleted genomic dna transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector a helper-dependent adenovirus vector system: removal of helper virus by cre-mediated excision of the viral packaging signal high-capacity adenoviral vectors for gene transfer and somatic gene therapy long-term doxycycline-controlled expression of human tyrosine hydroxylase after direct adenovirus-mediated gene transfer to a rat model of parkinson's disease adenovirus-mediated regulable target gene expression in vivo tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances tight control of gene expression in mammalian cells by tetracycline-responsive promoters transcriptional activation by tetracyclines in mammalian cells lentiviral vectors: regulated gene expression in vivo manipulation of interleukin- expression by a retroviral tetracycline (tet)-regulated system efficient and regulated erythropoietin production by naked dna injection and muscle electroporation exploring the sequence space for tetracyclinedependent transcriptional activators: novel mutations yield expanded range and sensitivity enhanced inhibition of hepatitis b virus production by asialoglycoprotein receptor-directed interferon liver-specific alpha interferon gene expression results in protection from induced hepatitis interferon gene transfer by a hepatitis b virus vector efficiently suppresses wild-type virus infection specific antiviral activity demonstrated by tgtp, a member of a new family of interferoninduced gtpases optimization of the helper-dependent adenovirus system for production and potency in vivo doxycycline-mediated quantitative and tissuespecific control of gene expression in transgenic mice effect of exogenous mouse interferon on murine fulminant hepatitis induced by mouse hepatitis virus type protective effect of recombinant murine interferon beta against mouse hepatitis virus infection implication of interfering antibody formation and apoptosis as two different mechanisms leading to variable duration of adenovirus-mediated transgene expression in immune-competent mice human adenovirus vectors for gene transfer into mammalian cells long-term regulated expression of growth hormone in mice after intramuscular gene transfer long-term, non-invasive imaging of regulated gene expression in living mice use of a liver-specific promoter reduces immune response to the transgene in adenoviral vectors administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons we thank s germoni and m aquilina for animal care, c toniatti for critically reading of the manuscript and domenico lazzaro for the immunohistochemistry. we also thank j clench for editorial assistance and g bifolchetti for graphics. key: cord- - bniy b authors: peteranderl, christin; herold, susanne title: the impact of the interferon/tnf-related apoptosis-inducing ligand signaling axis on disease progression in respiratory viral infection and beyond date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: bniy b interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in—but not limited to—respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in-but not limited to-respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/ trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. ( ) cell death induction, e.g., bcl- -associated x protein, caspase- , fas-associated protein with death domain, fas ligand, and tnf-related apoptosis-inducing ligand (trail) dsrna, polyi:c ( , ) iav ( , , , ) sendai virus ( ) trail virus control by apoptosis induction in infected cells iav ( , , ) tissue injury by apoptosis of both infected and non-infected alveolar epithelial cells, lung macrophages iav ( , , ) rsv ( ) necrosis of fibroblasts, dendritic cells, and epithelial cells iav ( , , ) increased cellular infiltration cov ( ) decreased expression of na,k-atpase, impaired epithelial fluid reabsorption iav ( ) introduction in , isaacs and lindenmann ( ) first recognized the potential of a soluble and probably cell-derived factor to combat influenza virus infection and named this factor interferon [(ifn) from latin interferre, to interfere]. since then, three subgroups of ifns have been defined, primarily by their differential receptor usage. while the groups of type i ifn and type iii ifn comprise largely agents directly limiting pathogen spread by improving cellular counter measurements, ifn-γ, the sole type ii ifn, has been mainly implicated in the modulation of innate and also adaptive immune responses ( , ) . accordingly, type i and iii ifns are key signaling molecules in viral control, and lack of both signaling pathways results in increased viral loads and disease severity. still, there is accumulating evidence that not only lack of an antiviral response but that also an unbalanced overshooting activation of ifns contributes to an exaggerated inflammatory reaction, tissue injury, reduced proliferative capacity, and thus enhanced disease severity ( table ) . especially in viral infections, this effect has not only been tracked down to ifn signaling in general but specifically to the exaggerated production of key effector ifn-stimulated genes (isgs) ( ) . a prominent example is the tnf-related apoptosis-inducing ligand (trail) that displays an ambivalent role in viral infection ( - ) ( table ) . whereas first identified as factor produced by immune cells in non-respiratory infection ( , ) , trail is now especially well studied in influenza a virus (iav) infection, where it is released in high amounts from bone marrow-derived macrophages upon pathogenassociated molecular patterns (pamps) recognition and type i ifn production ( ) . macrophage-released soluble trail, but also membrane-bound cell-associated trail, acts via distinct receptors on infected but also on non-infected, neighboring cells. in viral infection, its preliminary role is to drive infected cells into apoptosis to limit virus spread. however, studies performed within the last decade demonstrate that trail's antiviral activity seems to be outweighed by the functional and structural damage it induces not only in infected but also in bystander cells such as uninfected cells of the alveolar epithelium ( , ) . this process is not only relevant in promoting viral disease progression but has further implications in bacterial superinfection and probably also in chronic diseases. the recognition of the ambivalent role of ifn-driven signaling in vivo is a first important step to better understand disease progression and to envision novel treatment options for primary viral respiratory infection targeting distinct host-derived signaling mediators such as trail. it is a commonly accepted concept that-as janeway ( ) already proposed in -immune activation toward invading pathogens is mounted upon recognition of pamps. pamps are evolutionary conserved biomolecules such as proteins, lipids, nitrogen bases, sugars, and complexed biomolecules such as lipoglycans that are essential to the survival of a given pathogen ( ) . pamps are recognized by distinct pattern recognition receptors (prrs) that are germ-line encoded and-similar to pamps-usually show a high evolutionary conservation. the first recognized and probably most intensely studied family of prrs are the toll-like receptors [tlrs; reviewed in mogensen ( ) ; leifer and medvedev ( ) ]. in viral infection, both host cell membrane-localized tlrs (tlr , tlr , detecting viral envelope proteins) and endosomal tlrs (tlr , tlr , tlr , and tlr , nucleic acid sensors) initiate signal transduction cascades leading to ifn production (figure ). tlr activation results in either myeloid differentiation factor (myd ) or tir-domaincontaining adaptor protein-inducing ifn-β (trif) recruitment that both trigger various downstream signaling events, eventually leading to ifn regulatory factor (irf) , irf , and nfκb nuclear translocation as well as map kinase and activator protein (ap- ) activation ( , ) . similar to endosomal tlrs, the cytosolic retinoic acidinducible gene (rig)-i-like receptors (rlrs) are specialized to recognize viral nucleic acid contents and are central prrs relevant to mount an antiviral response, providing resistance to most rna (e.g., orthomyxoviruses) and some dna (e.g., reoviruses) viruses [reviewed in ref. ( , ) ]. both melanoma differentiation-associated gene (mda- ) and rig-i recognize dsrna, ′-triphosphate rna, or the synthetic analog to dsrna, polyi:c ( , ) . both drive the dimerization of the mitochondriaassociated adaptor protein ifn-β promoter stimulation (ips- ) (also named mavs, visa, cardif). a subsequently activated cascade including tradd (tnf receptor type -associated death domain protein), traf (tnf receptor-associated factor ), and tank (traf family member-associated nf-κb activator) induces the phosphorylation of irf and irf , resulting in type i ifn production ( ) . the third rlr, lgp , so far has primarily been implicated to regulate rig-i or mda- as a cofactor; however, a recent study by stone et al. ( ) demonstrated a novel, non-redundant, and independent role of lgp in west nile virus infection. another class of prr, the nucleotide oligomerization domain (nod)-like receptors (nlrs), has mainly been implicated in bacterial recognition ( ) , still several nlrs are activated as well upon virus infection. especially, nlrp is known to recognize rna of different viruses including hepatitis c virus, measles virus, influenza, and vesicular stomatitis virus (vsv) ( ) ( ) ( ) ( ) , resulting in inflammasome formation and caspase- -dependent activation of il- β and il- ( ) ( ) ( ) . in addition, virus infections are sensed also by a structurally diverse group of viral rna and dna sensors residing in the cytoplasm. these include the cyclic gmp-amp synthase that synthesizes the second messenger cgamp. cgamp in turn activates stimulator of ifn genes (sting), tank-binding kinase , and irf , triggering ifn production ( ) ( ) ( ) . moreover, sting itself acts as a prr and has been implicated in dna virus recognition including hsv, adenovirus, vaccinia virus, and papilloma virus and in sensing of retroviral rna-dna hybrids ( ) and rna viruses after being activated by rig-i ( , ) . another cytosolic nucleic acid sensor, pkr, is well known for its phosphorylation of eukaryotic initiation factor α (eif α) in response to viral dsrna. phosphorylation of eif α results in its deactivation, host translational shut-off, and the limitation of viral replication. of note, the pkr-eif α-driven inhibition of protein synthesis can contribute to an ips- -dependent ifn-β induction ( ) . furthermore, pkr has been implicated in efficient type i ifn activation by tlr in response to dsrna ( ) and can mediate-at least partially-activities of irf ( ) . in addition to type i ifns, also type iii ifns exert antiviral activity and are widely expressed after viral recognition, being produced by most cell types including epithelial, endothelial, fibroblast, and polymorphonuclear cells [reviewed in ref. ( , ) ]. like type i ifns, type iii ifns are induced in viral infection by the prr rig-i as well as tlr and tlr and rely on the activation of the same transcriptional activators, including irf , irf , and nfκb. these observations initially led to the conclusion that type i and type iii ifn comprised two completely redundant systems to induce isgs in response to pamp recognition. however, more recent data suggest distinct selection mechanisms for either type i or type iii ifn expression. as such, ips- specifically induces ifn-λ, but not type i ifn, when located at the peroxisomal membrane instead of the mitochondrial membrane in response to rig-i activation by reovirus, sendai virus, or dengue virus challenge ( ) . interestingly, type iii ifn induction is largely independent toward ap- translocation, which facilitates an instantaneous induction of ifn-λ after viral recognition, highlighting it as an important immediate factor driving innate microbial defense mechanisms. release of ifns upon pathogen recognition is a highly conserved mechanism-found from teleost fish to insects and mammals-to prepare the surrounding cells as well as the host defense against the invading threat ( , ). whereas often high-level ifn production relies on specialized sentinel cells such as macrophages or dendritic cells (dcs), mostly all cells of the multicellular organisms are able to respond to at least one type of ifn by expression of respective receptors. receptor binding then induces a signal transduction cascade relying on the janus kinase (jak) and signal transducer and activator of transcription (stat), which results in efficient transcription of a plethora of different isgs in infected as well as bystander cells ( , ) . ifns engage a classical canonical signal transduction cascade employing jak/stat molecules after binding to their respective receptors. herein, type i ifns ligate to their common heterodimeric receptor consisting of the ifn-α receptor (ifnar) and ifnar subunits, whereas type iii ifns act via a interleukin- receptor (il- r )/ifn-λ receptor (ifnlr ) heterodimer that to date has been reported to be restricted in its expression to epithelial cells ( ) . in type i ifn signaling, ifnar engagement leads to the activation of the receptor-associated protein tyrosine kinases jak and tyrosine kinase followed by the recruitment or repositioning of already associated but elsewise latent cytoplasmatic transcription factors stat and stat . consequently, stat /stat are phosphorylated on conserved tyrosine residues, they disassemble, undergo conformational changes enabling their heterodimerization as well as the exposure of a nuclear localization sequence. subsequently, the stat /stat heterodimer translocates into the nucleus where it interacts with the irf to form the trimeric ifn-stimulated factor (isgf ). isgf binds cognate dna sequences, the ifn-stimulated response elements (isre), finally leading to isg induction. also type iii ifn interaction with the il- r /ifnlr receptor complex triggers stat /stat heterodimerization, nuclear translocation, and isgf assembly ( ) . interferon signaling results in the induction of isgs evoking different cellular responses against viral infection, both in infected as well as in non-infected cells, including direct antiviral, immune-modulatory, or cell death-inducing effects to enable an immediate and robust response to a pathogen challenge. many isgs directly interfere with viral replication on an intracellular level. well-studied examples of antiviral isgs comprise ifn-induced transmembrane proteins (ifitms) effective in iav, west nile virus, and dengue virus infection ( , ) , the myxovirus resistance protein a (mxa) that interferes with vsv mrna production and binds the iav nucleocapsid to prevent nuclear translocation of viral genetic material ( ) ( ) ( ) , the - -oligoadenylate synthase (oas), which activates rnase l triggering viral rna degradation, or the prr pkr, which besides activating the ifn response has a major impact on viral protein translation by inhibiting the eif α ( ) . more recently identified isgs include the plasminogen activator inhibitor that blocks iav infection by inhibiting glycoprotein cleavage executed by extracellular airway proteases ( ) or the antiviral isg that limits iav viral replication via its exonuclease activity most likely by interfering with the viral np ( ) . accordingly, ifn pretreatment usually results in the establishment of an antiviral state that limits viral replication and spread from the start of infection and thus favors milder disease outcomes. ifn-α pretreatment has been demonstrated to limit viral spreading of seasonal iav strains and thus decrease morbidity and mortality in mice, guinea pigs as well as ferrets ( ) ( ) ( ) . as shown by a study by tumpey et al. ( ) , this effect can be attributed to the early induction of antiviral isgs including mxa. importantly, type i ifn pretreatment also dampens early replication of highly pathogenic avian influenza in ferrets ( ) . also in respiratory syncytial virus (rsv) infection, treatment with recombinant ifn-α results in significantly decreased lung viral titers, alveolar inflammatory cell accumulation, and clinical disease in rsv-infected mice ( ) . in addition, respiratory infections caused by emerging coronaviruses (cov) can be ameliorated by type i ifn pretreatment strategies. in an in vivo macaque model (macaca fascicularis) of severe acute respiratory syndrome (sars)-cov infection, it could be demonstrated that pretreatment with pegylated ifn-α significantly diminished cov replication and excretion and resulted in reduced pulmonary damage ( ) . macaques also serve as a preclinical model for middle east respiratory syndrome (mers)-cov, and similar to sars-cov, ifn-α in combination therapy with ribavirin reduces viral replication and severe histopathological changes ( ) . in line, genetic alteration leading to an enhanced type i ifn signaling has been demonstrated to limit iav-induced disease outcomes, as a recent study by xing et al. ( ) reported that deletion of trim , a negative regulator of nemo, which leads to nfκb induction and therefore enhanced type i ifn production, is protective in vivo in iav-infected mice. conversely, the genetic depletion of ifn signaling in ifn receptor-deficient mice can result in a lack of viral control, resulting in enhanced viral titers in different viral infections including rsv or iav ( , ) . still, it must be noted that this effect is often mild in ifnar-or ifnlr-deficient animals, which is probably related to a certain redundancy between type i and type iii ifn signaling in limiting viral spreading in epithelial cells ( ) . in contrast, ifnar/ ifnlr-double knockout or stat knockout animals that are deficient in both type i and type iii ifn signal transduction succumb more readily to infection due to excessive viral replication ( ) ( ) ( ) . vice versa, mutations in key isgs such as ifitm are associated with increased iav disease severity in mice and humans ( ) . however, ifn pretreatment and genetic loss-of-function approaches generally are not relevant to human respiratory virus-induced hospitalizations, where patients already present with ongoing respiratory infection and inflammation, and preclinical studies underline that type i ifn signaling in an already inflamed organ is rather detrimental and enhances tissue injury, and lack of type i ifn in vivo may even ameliorate disease outcome. accordingly, in cases where the antiviral defense was not compromised (e.g., in animals with efficient type iii ifn signaling) ifnar-deficient mice infected with sendai virus or iav were reported to be more resistant to infection-induced morbidity and mortality ( , ) . similarly, in sendai virus in vivo infection, wetzel et al. ( ) showed that increased ifn-β levels in the lung homogenate correlates to increased morbidity and mortality, and also for sars-cov, a recent study demonstrates that high type i ifn induction in an already ongoing viral infection contributes to mortality in sars-cov-infected mice ( ) . also for iav infection, type i ifn application after infection has been proven to drive disease severity ( ) . of note, the detrimental effects of type i ifns were especially pronounced in mice lacking central antiviral factors, namely the ifit protein in sendai virus infection and mxa in iav. interestingly, beilharz et al. ( ) demonstrated that application of low doses of ifn-α reduces viral load, which to a certain degree led to attenuated disease progression, whereas high dose application of type i ifn contributed to morbidity ( ) . in line, high expression levels of isgs have been shown to correlate to worse outcomes in ards patients ( ) . this observation corresponds to reports stating that the ifn threshold needed to induce antiviral isgs-showing a beneficial effect in acute respiratory viral infection-is by at least -fold lower than the ifn dose necessary to trigger isgs that show immunomodulatory, death-inducing, or anti-proliferative effects and thus can contribute to disease progression ( ) ( ) ( ) ( ) . altogether, these data demonstrate that ifns may significantly contribute to unbalanced inflammation and tissue injury during respiratory viral infection depending on expression levels and duration of ifn-related signaling events. to date, the underlying mechanisms leading to the ifndependent enhanced disease progression are not fully understood but often result from a dysregulated ifn signaling response. one mode of action of ifn and ifn-stimulated isgs is to stimulate negative feedback loops on ifn signaling. for example, suppression of jak or stat via specific phosphatases, expression of suppressor of cytokine signaling (socs) and socs , or ubiquitination and endocytosis of the ifn receptors ( ) ( ) ( ) ( ) desensitize cells to ifn signaling and allow recovery and the return to homeostasis after microbial challenge. as demonstrated by bhattacharya et al. ( ) , the lack of ifnar downregulation and thus the failure to initiate ifn-desensitization contributes to increased inflammatory signaling, extensive lung injury and, importantly, also impaired tissue regeneration ( ) . moreover, ifns are immunomodulatory and shape the specific responses of cells of the immune system, which has been implied to influence disease progression both positively and negatively. in a recent study, type i ifns have been associated in the regulation of innate lymphoid immune cells (ilc) in iav infection, where they-in concert with ifn-γ and il- -promote an ilc -dependent restriction of immunopathology ( ) . moreover, type i ifns play an important role in stimulating the immune response driven by dcs; they stimulate the expression of mhc molecules as well as the co-stimulatory ligands cd and cd and thus activate t cell responses ( , ) . additionally, ligand-driven activation of ifnar enhances the proliferation of cd positive t cells, especially early in infection. however, late in infection, type i ifns were also implied in decreasing t cell expansion upon sars-cov and arenavirus infection ( , ) , which might potentially be related to the above described desensitization upon prolonged ifn signaling and might be detrimental if initiated too early in infection. in line, pinto et al. ( ) reported an impairment of t cell responses upon type ifn induction in west nile virus infection. in b cells, the lack of ifnar has been demonstrated to result in enhanced release of neutralizing antibodies in iav infection ( ) implying a repressive role for type i ifn in b cell antibody production. however, immunization studies by le bon et al. ( ) reported the necessity of ifnar on b cells for efficient igm and igg production, underlining the need for further studies to understand the detailed effects of ifn-dosage and timing adaptive immunity activity upon respiratory viral infection. type i ifns additionally induce the production of high levels of pro-inflammatory cytokines that have been closely linked to worsened outcomes of acute respiratory viral infection. especially in iav, disease severity and disease progression are linked with an overshooting, ifn-driven inflammatory response, in which further exogenous supplementation with type i ifn in fact correlates with increased morbidity and mortality ( , ) . in non-human primates, iav infection with a highly pathogenic h n isolate evokes a strong induction of type i ifn, resulting in severe lung injury by a necrotizing bronchiolitis and alveolotis ( ) . ifn levels in turn have been demonstrated to cause elevated pro-inflammatory cytokine levels after in vivo iav infection and additionally, in human alveolar macrophages, the release of pro-inflammatory cytokines (e.g., mcp- ) are preceded by a robust type i ifn response ( ) . importantly, also in human infection with h n , levels of pro-inflammatory cytokines are strongly elevated in bronchoalveolar lavage fluid, and cytokine levels have been associated with organ damage and worsened disease outcomes ( , ) . still, it should be noted that due to strain differences in virus-elicited prr activation and, importantly, ifn antagonism by the iav non-structural (ns) protein, ifn levels and disease severity do not always directly correlate; actually, the extent to which ns can suppress the ifn response relates to prolonged viremia and thus can also be a determinant of virus pathogenicity both in human bronchial epithelial cells and in an in vivo model of iav infection ( , ) . alongside iav, also in rsv infection the induction of high levels of pro-inflammatory cytokines has been directly related to type ifn, as rsv-infected but ifnar-deficient mice presented with significantly diminished pro-inflammatory cytokine release, which translated into an attenuated disease course ( ) . also in sars-cov, the late phase type i ifn induction relates to accumulation of inflammatory macrophage populations and elevated lung cytokine levels ( ) . in addition to antiviral, immunomodulatory, and pro-inflammatory isgs, ifn signaling results in the transcription and translation of cell death-inducing isgs. in the context of viral infection, these factors provide a mode to block viral spreading and reinfection by killing those infected cells, in which the internal activation of antiviral isgs is not sufficient to restrict viral replication. thus, the infected cell is sacrificed to prevent the release of infectious progeny virions to limit viral spreading. however, especially in the lung, the disruption of the alveolar epithelial barrier by cell death of infected cells, but importantly also non-infected bystander cells induced by factors such as trail, significantly contributes to worsened disease outcomes. controlled cell death or apoptosis can be induced by intrinsic and extrinsic signals. the intrinsic apoptosis pathway is initiated by diverse intracellular stimuli that influence the expression and activation of b cell lymphoma (bcl)- family proteins that govern the permeabilization status of the outer mitochondrial membrane. once cytochrome c is released from the mitochondria, it binds to the intracellular adaptor protein, apoptotic peptidase activating factor , forming the so-called apoptosome that in turn recruits pro-caspase- ( ). caspases (cysteine-aspartic proteases) exert their action by cleaving other proteins and substrates. herein, initiator caspases such as caspase- and caspase- target other downstream caspases, whereas effector caspases, including caspase- , - , and - , directly cause apoptosis by cleaving and thus inactivating or disassembling a vast array of cellular integral proteins and complexes ( ) . the extrinsic apoptosis pathway relies on an extracellular signal exerted by ligands of the tumor necrosis factor (tnf) receptor (tnfr) superfamily, including trail, tnf-α, and fas ligand (fasl) ( ) . their ligation to their respective cell surface-expressed death receptors (dr) leads via the signal transmission by fas-associated protein with death domain (fadd) to the activation of the initiator caspases- or - , finally stimulating effector caspases including caspase- ( ) . to date, several type i and type iii ifn-induced, proapoptotic factors have been identified ( ) . both caspase- and caspase- have been shown to be upregulated upon type i ifn signaling ( , ); caspase- enhances the fadd-driven extrinsic apoptosis pathway, whereas the less-studied caspase- may promote pro-il- β cleavage and inflammasome-driven cell death (pyroptosis) in macrophages ( , ) . chattopadhyay et al. ( ) demonstrated that sendai virus infection and polyi:c treatment resulted in bcl- -associated x protein (bax) activation and apoptosis induction via one of the key transcription factors of ifn genes, irf . in addition irf was reported to enhance trail-dependent extrinsic apoptosis by nuclear translocation resulting in the translation of to date undefined factors that increase cell death upstream of caspase- activation ( ) . furthermore, both rlrs, rig-i and mda- , trigger the proteins puma and noxa that induce bcl and the ifn/trail signaling axis frontiers in immunology | www.frontiersin.org march | volume | article thus activate the intrinsic mitochondrial apoptotic cascade ( ) . also, pkr influences a cell's susceptibility to apoptotic signals, as it was demonstrated to sensitize to the fadd/caspase- apoptosis pathway upon type i ifn signaling after challenge with iav or dsrna ( ) and the oas-rnasel system has been suggested to contribute to ifn-α-related cell death induction, but the exact mechanisms remain to be elucidated ( ) . finally, also two classical initiators of the extrinsic apoptosis cascade are induced as isgs. both fasl and its receptor fas are upregulated on mrna levels by ifn-α ( ) , and fasl was reported to be induced by type i ifn in iav infection in the murine lung in vivo ( ) . also, the proapoptotic factor trail (or tnfsf , apo l) is induced by ifn-mediated and isgf -executed transcriptional activation, as has been shown by sato et al. ( ) , who revealed the presence of the isre sequence within the trail promoter region ( ) . in iav infection, trail is released in high amounts from infected alveolar macrophages depending on a pkr-and ifn-β-driven autocrine signaling loop. binding of ifn-β to macrophageexpressed ifnar activates a jak/stat-dependent release of trail, which then acts through its receptor dr on the alveolar epithelial cells ( , ) . however, certain prerequisites may decrease the ability of a cell to undergo apoptosis, including a shortage in pro-caspase- availability, expression of cellular fadd-like il- β-converting enzyme-inhibitory proteins (c-flips) that block fadd-driven caspase activation, inactivation, or degradation of fadd itself, or expression of cyld, which acts as a receptor-interacting serine/threonine-protein (rip) kinase de-ubiquitinase and thus stabilizes rip . however, in these cases ifn signaling can still promote a caspase-independent, programmed inflammatory cell death by activating the necroptosis pathway ( , ) . necroptosis is induced by a complex formation by rip and rip kinases that activate both poly-adp-ribose (par) polymerase (parp- ) and/or mixed lineage kinase domain-like (mlkl), leading to atp depletion, calpain activation, par polymer accumulation or cell membrane permeabilization, and release of damage-associated molecular patterns, respectively [reviewed in ref. ( , ) ]. both a type i ifn-dependent jak/stat-driven activation of pkr as well as signaling by the prr dai (dnadependent activator of irfs) initiates necroptosis via rip /rip activation, respectively ( , ) . importantly, the activation of proapoptotic and pro-necroptotic pathways in respiratory infection can result in a structural disruption of the airway and the alveolar epithelial barrier, which is a major hallmark of respiratory disease and its progression to the acute respiratory distress syndrome ( , ) . in virusinduced lung injury, especially expression of trail, which can initiate both apoptosis as well as necroptosis has been correlated with more severe outcomes. as described earlier, trail belongs to the superfamily of tnf ligands and has been reported to be inducible by both type i and type iii ifns. trail has been found to be present in various cells of the immune system, among them natural killer (nk) cells, t cells, nk t cells, dc subsets such as ifn-γ-producing killer dcs and macrophages, and can be displayed in large amounts on the cell surface or be shed upon ifn-and/or pro-inflammatory cytokine signaling ( ) ( ) ( ) . in addition to cells of the immune system, fibroblasts have been shown to produce trail after ifnγ treatment or viral challenge. also, club cells and the alveolar epithelium have been reported to produce trail ( ) ( ) ( ) ( ) . similar to other ligands of the tnf superfamily, trail is a homotrimeric type ii transmembrane protein with a conserved c-terminal extracellular domain that mediates receptor binding and can be cleaved by metalloproteinases to generate a soluble mediator ( ) . however, trail can induce cell death also in its membrane-bound form, that is, similar to trail expression levels and trail shedding, upregulated by type i ifn ( ) . direct cell-to-cell trail-dr interactions have been demonstrated to play a role in macrophage, nk as well as cd + t cell-mediated induction of cellular death ( , ) . in humans, five different binding partners for trail are present: the membrane-bound dr (trail-r ) and dr (trail-r ) that both induce a proapoptotic signaling cascade, the membrane-bound anti-apoptotic decoy receptors (dcr) and dcr , and the soluble interaction partner osteoprotegerin ( ) . in the murine system, only dr has been identified to ligate to trail ( ) . in the human respiratory compartment, both dr and dr have been demonstrated to be present under steady-state conditions ( , ) . however, upon viral infection, cell-sensitivity to trail-induced apoptosis is enhanced, which has been attributed to increased trail receptor expression especially on infected cells, as dr levels are markedly increased in iav-, adenovirus-, and paramyxovirus-infected cells in contrast to non-infected bystander cells ( , , ) . of note, studies on the dependency of dr upregulation upon type i ifn signaling after iav infection have yielded conflicting results in different strains of mice ( , ) , highlighting the complex interplay of ifn-induced cascades in a host-and tissue-specific context, whereas the exact virus-and host-specific mechanisms for dr regulation remain less well defined. moreover, previous assumptions that also dcr expression would correlate with cell-sensitivity to trail-induced cell death could not be experimentally verified ( ) . tumor necrosis factor-related apoptosis-inducing ligand ligation to the proapoptotic receptors dr or dr triggers a trimerization of the receptors. subsequently, depending on additional stimuli, presence or absence of adaptor molecules or inhibitory proteins, different signaling pathways can be activated (figure ) . in the classical trail-dependent extrinsic apoptosis induction, the proteins rip, tradd, and fadd are subsequently recruited to the dr cytoplasmic domain upon trail ligation ( , ) . these factors and the proapoptotic drs all share a cytoplasmic death domain (dd), which is lacking or truncated and thus inactive in the dcr. the dd plays a central role in the concerted formation of the death-inducing signaling complex (disc). disc formation exposes a second functional domain of fadd, the death effector domain that is directly able to recruit pro-caspase- figure | trail/dr -mediated cellular signaling pathways. in presence of rip , tradd, and fadd, trail ligation to dr results in apoptosis induction, which is initiated by recruitment of the pro-caspase- or - to fadd. these in turn activate the effector caspases- and - , which leads to dna fragmentation and apoptosis induction. in addition, tradd can trigger a traf -and jnk-dependent activation of bax and subsequent release of mitochondrial cytochrome c, inducing the pro-caspase- activation. in the presence of cyld, c-flip or absence of sufficient amounts of fadd or pro-caspase- , trail ligation to dr triggers the interaction of rip and rip kinase, which in turn cause cell death via induction of mlkl and/or parp- . in the presence of ciaps, fadd is not recruited to dr upon trail ligation, and tak is activated by tradd/traf interactions. tak induces nemo followed by iκb degradation and nfκb activation, as well as mkk and jnk activation leading to ap- nuclear translocation; both events promote the production of cytoprotective factors such as xiap, ciaps, and c-flip. additionally, tak triggers ampk activation and thus mtorc inhibition, which results in enhanced autophagic activity. abbreviations: ap- , activator protein ; tnf, tumor necrosis factor; trail, tnf-related apoptosis-inducing ligand; dr , death receptor ; rip , receptor-interacting serine/threonine-protein kinase ; tradd, tnf receptor type -associated death protein; fadd, fas-associated protein with death domain; traf, tnf receptor-associated protein; jnk, janus kinase; bax, bcl- -associated x protein; c-flip, cellular fadd-like il- β-converting enzyme-inhibitory proteins; rip, receptor-interacting serine/threonine-protein; mlkl, mixed lineage kinase domain-like; parp- , poly-adp-ribose (par) polymerase ; ciap, cytoprotective factors including inhibition of the autophagic machinery; xiap, x-linked inhibitor of apoptosis protein; ampk, amp-activated protein kinase; mtorc, mammalian target of rapamycin complex; traf , tnf receptor-associated protein ; mkk, mitogen-activated protein kinase. and pro-caspase- . how exactly disc formation induces caspase activation is still under debate. the most probable scenarios include either an autocatalytic cleavage of caspase-pro-domains enabled by the spatial proximity between pro-caspases (generated by their recruitment to disc), by pro-caspase dimerization, or by pro-caspase conformational stabilization ( ) . removal of the pro-domain of caspase- and caspase- results in the activation of the effector caspases- and - , which cleave dna fragmentation factor and lead to apoptosis ( , ) . moreover, trailbinding to dr and dr can induce the jnk either via caspase- or recruitment of tnf receptor-associated protein (traf ) to the disc complex, which results in the activation of the intrinsic apoptotic cascade by bax-dependent mitochondrial cytochrome c release ( ) . in addition, trail signaling is also able to induce necroptosis by both activating the rip /rip kinase downstream effectors parp- and mlkl, contributing to epithelial cell death and tissue injury ( ) ( ) ( ) . it has become apparent in recent years that trail signaling is closely linked to induction of autophagy, a process generally associated with the blockade of apoptosis and necrosis. indeed, autophagy has been reported to improve cellular survival in cell stress by catabolic removal of cytoplasmic long-lived proteins and damaged organelles. it also contributes to viral clearance and the transfer of viral material to endosomal-/lysosomallocated tlr or mhc class ii compartments for the activation of adaptive immunity ( ). several studies outline that trail ligation to dr can result in a traf -dependent activation of tak (map k ) that has been attributed a central role in trail-induced autophagy activation ( ) . tak modulates the ikk-dependent translocation of nfκb, and it also induces jnk activation via mitogen-activated protein kinase. both events lead to expression of autophagy-related factors including inhibition of the autophagic machinery (ciap) , ciap , x-linked inhibitor of apoptosis protein, and c-flip ( , ) . especially, c-flip has been associated with desensitization of cells to trail-induced apoptosis, favoring autophagy-related cascades ( ) . another study revealed that upon trail signaling the amp-activated protein kinase (ampk) is activated. ampk in turn inhibits the mammalian target of rapamycin complex that itself is an inhibitor of autophagy, thus the activation of the autophagic machinery is promoted ( ) . the decision if trail signaling results rather in necroptotic or apoptotic cell death or in activation of autophagy seems to be dependent on the presence of ciaps that promote rip kinase ubiquitination and degradation ( ) , but also on the balance between active caspases and autophagic proteins such as beclin- ( , ) . this suggests a scenario where autophagy is activated as cell protective mechanism until cell stress-as executed by enhanced trail signaling or additional viral infection-increases over a threshold to favor cell death induction. accordingly, as trail signaling is not restricted to infected cells, excessive cell death activation might be limited by autophagy induction in non-infected bystander cells. however, autophagy is not only related to cell survival but can also positively affect apoptosis and induce-even if the exact mechanisms are still under debate-autosis, the autophagy-related cell death, another mode of trail to trigger cell death ( , ) . of note, autophagy activation needs to be placed into its virus-specific context, as some viruses, including dengue virus, poliovirus, and coxsackie b virus ( ) , can exploit autophagic pathways for their own replication and thus promote apoptosis and tissue injury. as discussed above, trail is a potent activator of cell death. however, its signaling outcomes can differ largely depending on its delivered form (e.g., membrane-bound versus soluble), the availability of drs on the target cell membrane, alternate intracellular pathways that might be activated and finally the pathogen itself, as it might exploit trail-induced pathways for its own survival and replication. in acute respiratory infection, trail signaling is often part of an ifn-driven overshooting inflammatory reaction that promotes unspecific tissue injury and thus disease severity by increasing functional and structural changes in infected but also non-infected cells, as will be outlined below. the release and effects of trail have been especially well studied in iav infection in the last decade. earlier studies reported that within days after infection, bronchial, bronchiolar, and alveolar epithelial cells undergo apoptosis ( ) . this early induction of cell death is mainly attributed to direct apoptosis induction by the virus itself, as iav actively promotes apoptosis for efficient viral replication ( ) . herein, the viral ns and pb-f proteins not only play a crucial role ( , ) but also the viral m protein has been implicated in this process as it inhibits autophagy in infected cells ( ) . in addition, our own data revealed that later in iav in vivo infection, the recruitment of bone marrowderived macrophages via the cc chemokine receptor type (ccr )-cc-chemokine ligand (ccl ) axis significantly contributes to alveolar cell apoptosis and structural damage of the alveolar epithelium ( ) . studies by wurzer et al. ( ) had previously demonstrated that iav promotes the production of proapoptotic factors in an auto-and paracrine fashion via nfκb transcriptional activation by iav ( ) . subsequently, brincks et al. ( ) elucidated that human peripheral blood mononuclear cell treated with iav released trail and that increased trail levels correlated with type i as well as ii ifn induction. additionally, trail sensitivity was increased in influenza virusinfected cells. in line, our investigations could elucidate that iav triggers a pkr-dependent translocation of nfκb that results the production of type i ifns. these in turn induce, via ligation to the ifnar receptor complex, expression and shedding of trail by bone marrow-derived macrophages ( ) . in addition, davidson et al. ( ) demonstrated that type i ifn application to iavinfected mice increased morbidity and lung injury, which could be attributed to both dr and trail upregulation inducing epithelial cell apoptosis. importantly, högner et al. also reported that the iav strain used in these studies, a/pr (h n ), which is highly pathogenic for mice, induced an approximately -fold induction in macrophage trail expression, whereas the lower pathogenic virus a/x- (h n ) only stimulated trail by a factor of eight. of note, the relation between trail induction and iav strain-specific pathogenicity also translates to the highly pathogenic avian h n iav, causing severe pneumonia in mice as well as in humans ( , ) . moreover, human infection with both the highly pathogenic h n as well as the pandemic h n iav strains are characterized by a massive influx of mononuclear phagocytes into the alveoli, which is correlated with extensive alveolar epithelial cell apoptosis ( , ) . additionally, macrophages gained from bronchoalveolar lavages of patients presenting with ards caused by the pandemic h n / virus strain showed high surface expression and release of trail ( ) . another recent report demonstrates that in highly pathogenic avian influenza, in addition to macrophages also the alveolar epithelium might be involved in causing elevated levels of trail in the alveolar space ( ) . besides its role in apoptosis, trail signaling upon iav infection has also been implicated in the induction of necroptosis in fibroblasts, dcs, and lung epithelial cells ( , , ) . rodrigue-gervais et al. ( ) demonstrated that lack of cipa promotes rip kinase-mediated necroptosis in response to trail-but also the proapoptotic factor faslreleased from hematopoietic cells. this contributed to severe lung epithelial degeneration and increased mortality, even though viral control was not compromised. nogusa et al. ( ) further elucidated that iav-induced necroptosis depends on rip kinase activation of mlkl, and that rip kinase deficiency, similar to ciap -deficiency, increased iav-susceptibility in vivo. in iav infection, as mentioned earlier, dr expression is elevated on infected alveolar epithelial cells, but not in noninfected cells in vivo, which might impact on trail susceptibility to apoptosis induction ( ) . however, both infected as well as neighboring bystander cells were found to be targeted for apoptosis induction by macrophage-released trail. nonetheless, we could recently show that specifically in noninfected cells within the iav-infected lung, trail severely compromises the function of the ion channel na,k-atpase, which was mediated via induction of the stress kinase ampk ( ) , thereby potentially revealing a cross-link to trailinduced autophagic cell stress pathways in bystander cells both in vitro and in vivo. the trail-induced and ampk-mediated downregulation of the na,k-atpase, a major driver of vertical ion and fluid transport from the alveolar airspace toward the interstitium, resulted in a reduced capacity of iav-infected mice to clear excessive fluid from the alveoli. thus, trail signaling contributes to intensive edema formation, a hallmark of disease in virus-induced ards ( ) . notably, this effect of trail on na,k-atpase expression was induced independently of cell death pathways elicited by caspases, as treatment of cells and mice with a specific caspase- inhibitor diminished apoptosis in alveolar epithelial cells but still allowed for the reduction of the na,k-atpase ( ) . conclusively, treatment of iav-infected mice with neutralizing antibodies directed against trail or the abrogation of recruitment of trail + bone marrow-derived macrophages inhibited apoptosis of both non-infected and bystander cells. thus, lung leakage due to loss of alveolar barrier function was reduced, whereas alveolar fluid clearance capacity was enhanced, resulting in reduced edema, improved survival, and outcome upon iav challenge in vivo. however, trail has also been shown to be upregulated on nk, dc, and on cd + and cd + t cells after iav infection ( ) . studies by brincks et al. demonstrated that especially cd + t involved in cytotoxic t cell responses toward iav and drive iav-infected cells into apoptosis via trail, thus contributing to efficient virus clearance ( , ) . in addition, both fasl and trail are involved in dc-mediated ctl activation and cytotoxicity against iav-infected cells ( , ) . furthermore, studies showed delayed viral clearance upon neutralizing anti-trail antibody administration ( , ) . our data, however, demonstrate that the transfer of trail-deficient bone marrow into irradiated wild-type mice, resulting in loss of trail production by bone marrow-derived macrophages upon iav infection, does not impact on the capacity to fully clear viral particles from the lung at day after infection, suggesting that other compensatory mechanisms are recruited to guarantee viral clearance ( ) . taken together, in iav infection, trail acts both as an important mediator of infected cell killing but particularly as a detrimental factor contributing to tissue injury and impaired inflammation resolution when released in excessive amounts by recruited immune cells. respiratory syncytial virus is an important cause of respiratory tract infections especially in children worldwide. generally, there seem to be virus-elicited anti-apoptotic mechanisms active in the lung epithelium, as rsv-infected primary human airway cells show a minimal cytopathic effect ( ) . however, several cell lines including small airway cells, primary tracheal-bronchial cells, and a and hep- showed increased expression of trail and its ligands dr and dr in an in vitro rsv infection model ( ) . moreover, soluble trail released from leukocytes was elevated in the bronchoalveolar lavage fluid of patients with rsv-associated respiratory failure, suggesting that similar to iav, trail contributes to rsv-induced epithelial injury and disease progression ( ) . also in cov respiratory tract infection, trail levels, but less so fasl, have been reported to be markedly elevated ( , ) . for sars-cov that presents with a severe damage to both the upper and lower respiratory tract ( ) , especially dcs respond with a strong induction of trail production, which was suggested to correlate to increased cellular lung infiltrations present in sars-cov patients ( ) . interestingly, sars-cov infection drives cells into apoptosis by a pkr-driven but eif α-independent pathway ( ) , which might-similarly as seen in iav infection-suggest a pkr-induced and autocrine/paracrine executed activation of apoptosis. also mers-cov, which causes pneumonia and respiratory failure, has been demonstrated to induce profound cell death within h of infection, irrespective of viral titers produced by the infected cells. however, type i ifn expression is strongly reduced in mers-cov in comparison to seasonal human cov in in vitro infection models, including human monocyte-derived macrophages, calu- , and human lung fibroblasts ( , ) , which might also dampen downstream trail induction. therefore, the exact mechanism by which mers-cov promotes cell death remains to be investigated. recurrently, viral infections of the respiratory tract are followed by outgrowth of colonizing gram-positive bacteria that aggravates the course of illness. this is well documented for iav, where "super" infections with streptococcus pneumoniae and staphylococcus aureus are the most frequent and increase viral pneumonia-associated morbidity and mortality ( ) . during the iav pandemic, bacterial pneumonia was evident in most cases ( ) and also during the recent h n pandemic, coinfections were a relevant factor for severe disease in a young patient population without comorbidities ( ) . interestingly, virus-induced elevation of the type i ifn response levels might promote secondary bacterial outgrowth by several mechanisms [reviewed in ref. ( ) ]. in line, it has been repeatedly demonstrated that lack of type i ifn signaling results in better bacterial clearance and increased survival rates in iav-and s. pneumoniae-superinfected mice ( ) ( ) ( ) . herein, ifn-induced apoptosis induction as well as depletion or impaired recruitment of lymphocyte subsets necessary for bacterial control play a critical role ( , ) . bacterial clearance from the lung has been reported to rely on sufficient phagocyte generation, recruitment, and survival. type i ifn has been demonstrated to cause apoptosis in bone marrow-derived granulocytes, affecting the numbers of recruited neutrophils ( ) , but also to impair expression of the cytokines cxcl (or kc) and cxcl (or mip- ), thus inhibiting neutrophil recruitment to the lungs with severe effects on survival of superinfected mice ( ) . a recent report by schliehe et al. ( ) elucidated the mechanistic background for impaired cxcl expression and secretion and demonstrated that type i ifns activate the histone methyltransferase setdb , which in turn represses the cxcl promoter and thus impairs neutrophil recruitment and bacterial clearance. moreover, type i ifn production decreases ccl production, thus inhibiting macrophage recruitment, which as well has been reported to have detrimental effects on bacterial clearance and disease progression in bacterial superinfection after viral insult in vivo ( ) . in addition, type i ifns also impair γδ t cell function and il- release, which was shown to increase susceptibility to s. pneumoniae superinfection after iav challenge ( ) . also in s. aureus pneumonia, a robust type i ifn response is correlated to excessive morbidity and tissue injury ( ) . in a model of polyi:c, s. aureus (methicillinresistant strain, mrsa) superinfection, polyi:c treatment prior to bacterial infection enhanced type i ifn levels and decreased bacterial clearance and survival ( ) . furthermore, shepardson et al. ( ) demonstrated that late type i ifn induction rendered mice more susceptible to secondary bacterial pneumonia in a model of iav-mrsa superinfection. only limited data are available on a direct role of trail in respiratory disease progression due to bacterial superinfections. in a model of iav-haemophilus influenza infection, neither deficiency for cc chemokine receptor type , inhibiting bone marrow-derived macrophage recruitment, nor deficiency of fas or tnfr impacted outcome ( ) . yet, during s. pneumoniae single infection, early cell death of macrophages is thought to limit an exuberant inflammatory reaction and accordingly, a study by steinwede et al. ( ) revealed that neutrophil-derived trail limits tissue injury by inducing cell death in dr -epressing lung macrophages in bacterial mono-infection ( ) . in contrast, in the iav-s. pneumoniae superinfection mouse model, iavinduced trail has a detrimental effect on overall mortality ( ), as trail-induced epithelial injury enhanced bacterial outgrowth of s. pneumoniae-administered at day after iav infection-markedly. importantly, administration of anti-trail neutralizing antibodies enhanced bacterial control by the host organism. thus, the activation of ifn/trail-mediated signaling in viral infection has detrimental implication for outcome of secondary bacterial infection following viral insult, rendering the ifn/trail signaling axis an interesting therapeutic target not only in respiratory viral infections but also in complicating bacterial superinfection. an increasing number of reports connect progression of chronic respiratory disease to acute respiratory virus infection or proapoptotic signaling events. in fact, trail has been reported to be a critical determinant for promoting the development of chronic lung disease in early life ( ) ; targeting trail by genetic deletion or neutralizing antibody application in early-life respiratory infections ameliorated infection-induced histopathology, inflammation, as well as emphysema-like alveolar enlargement and lung function. furthermore, trail was also shown to play a role in the development of allergy and asthma. trail is not only elevated in the sputum of asthmatic patients but has also been reported to be highly expressed in an experimental mouse model of asthma, where it induces ccl secretion by bronchial epithelial cells, thus promoting th cell responses and airway hyperreactivity ( ) . in copd, acute exacerbations driven by viral and bacterial infection are a major factor increasing both mortality and morbidity, and both influenza and s. pneumoniae have been identified among the most common causes of copd exacerbations ( ) . indeed, primary bronchial epithelial cells isolated from subjects with copd show an impaired production of type i ifn ( ) , which has been implied in the enhanced susceptibility of copd patients to respiratory infections; however, even in absence of high ifn induction, both an abnormally elevated loss of alveolar epithelial cells due to apoptosis as well as elevated trail and dr levels were reported ( ) , implying a possible link between viral/bacterial induction of trail and acute exacerbations in copd. trail induction has also been directly linked to cigarette-smoke exposure, a common cause of copd, and trail deficiency resulted in decreased pulmonary inflammation and emphysema-like alveolar enlargement in vivo ( ) . moreover, increased levels of both trail and dr were associated to impaired lung function and increased systemic inflammation in human copd patients ( ) . while alveolar epithelial cell death is closely connected to idiopathic pulmonary fibrosis (ipf), trail and its receptors dr and dr in aec were shown to be upregulated in ipf lungs ( ) . also, in pulmonary arterial hypertension virus infection is considered to be a possible risk factor ( ) , and pulmonary hypertension has been reported to be a side effect of prolonged treatment with type i ifn ( , ) . in line, trail has been closely linked to disease progression in pulmonary hypertension. trail has been found to be increased within pulmonary vascular lesions of patients with pulmonary hypertension ( ) and also in a mouse model of hypoxia-induced pulmonary hypertension, levels of soluble trail correlated with right ventricular systolic pressure, right ventricular hypertrophy, and pathologic alterations ( , ) . importantly, neutralizing antibody-treatment against trail showed positive effects on survival while reducing pulmonary vascular remodeling ( ) . notably, the extent to which infection-induced trail release causes or exacerbates chronic lung disease or in how far trail production in chronic lung diseases affects susceptibility to respiratory viral and complicating bacterial infection remains to be elucidated. respiratory viral infections are major causative agents for lung injury and ards; however, in many cases antivirals are not sufficient to limit disease ( ). besides the fact that most viruses are subject to strong selective pressures that favor quickly evolving, drug-resistant virus variants, recent advances in understanding the processes that contribute to tissue injury and ards highlight a crucial role of immune-related, ifn-driven events. therefore, novel therapeutic strategies often aim to improve the outcome of severe respiratory infection by modulating host cell responses; however, to date, clinical trials trying to improve severe viral infections or ards outcomes by targeting host pathways have not resulted in approval of new drugs ( ) . of note, for establishment of such therapies it has to be considered that the timing and intensity of induction and amplification as well as of dampening and termination of the ifn-driven immune response needs to precisely match the pathogen-and organ-specific requirements of a given infection. a non-controlled regulation of these processes may lead to either an unrestricted pathogen spreading or, on the other extreme, to an overshooting inflammatory response, including the increased production of pro-inflammatory and proapoptotic mediators, elevated levels of recruited immune cells, and/or aberrant repair processes. notably, both too low and too high levels of ifn-induced effects facilitate disease progression with a possible increase of fatal outcomes in ards patients ( ) . accordingly, preclinical in vivo studies of ifn-directed therapies yielded seemingly adverse results, depending on the context, timing, and dosage of ifn modulation. however, in multiple settings of acute respiratory viral infection, studies demonstrate that an exaggerated signaling derived from type i ifn in an already inflamed tissue contributes to worsened outcomes, and importantly, might favor secondary bacterial superinfection [e.g., ref. ( , , ) ]. interestingly, davidson et al. ( ) demonstrated that type iii ifn release upon influenza challenge-in contrast to type i ifn induction-does not trigger an unbalanced inflammatory response that critically contributes to respiratory disease progression in vivo, highlighting it as a possible therapeutic option in iav-induced lung injury. most likely, this effect derives from the lack of the ifn-λr /il- r receptor complex, but presence of ifnar, on immune cells, including bone marrowderived macrophages. nonetheless, other reports identify ifn-λ as a driver of macrophage polarization to an inflammatory m phenotype ( ) that has been attributed to further promote an overshooting inflammatory response, highlighting the need for further studies of type iii ifn biology in pathogen-associated disease progression. as generally ifn-directed therapeutic approaches target various downstream signaling events that might both act beneficially as well as detrimentally on viral replication and pathogenesis, a further approach is to address specific isgs that primarily show detrimental effects on disease progression. as outlined above, trail or its downstream signaling events might comprise a suitable target for adjunct therapies in addition to antivirals. accordingly, our own data in a preclinical mouse model of iav infection demonstrate a clear benefit of the systemic application of neutralizing antibodies against trail at days and postinfection for lung injury, morbidity, and mortality ( , ) . targeting trail as a major determinant of disease severity in respiratory viral infections including iav, but also rsv and cov, may yield therapeutic approaches that are superior to ifn-directed strategies, as they seemingly do not bear the risk of compromising host defense. yet, it should be thoroughly excluded that blocking trail-induced cell death of infected cells will not lead to an overwhelming viral spreading, especially as reports on viral loads upon trail inhibition in preclinical models of iav are controversial ( , , ) . accordingly, additional studies are needed to understand how and to which extent virus-infected cells can be killed or viral spreading can be controlled by other means in the absence of trail. moreover, targeting pathways and signaling hubs downstream of trail/drs, such as ampk ( ), in a well-timed and lung compartment-specific way, may open new therapeutic avenues but requires more detailed preclinical studies on efficacies and side effects. a valid approach might be the use of a combination therapy of such a treatment together with a classical antiviral drug therapy limiting viral replication; however, exact dosage, timing, kinetics, and application routes remain to be defined. cp and sh performed bibliographic research and drafted the manuscript. this work was supported by the german research foundation (sfb-tr b , sfb c , kfo p /p , exc ), by the german center for lung research (dzl), and by the german center for infection research (dzif). virus interference. i. the 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apoptosis-inducing ligand (trail) reverses experimental pulmonary hypertension luks am. ventilatory strategies and supportive care in acute respiratory distress syndrome ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -xbxwpmxq authors: liu, bao-qin; jin, jin; li, yi-yuan title: ubiquitination modification: critical regulation of irf family stability and activity date: - - journal: sci china life sci doi: . /s - - - sha: doc_id: cord_uid: xbxwpmxq interferon regulatory factors (irfs) play pivotal and critical roles in innate and adaptive immune responses; thus, precise and stringent regulation of the stability and activation of irfs in physiological processes is necessary. the stability and activities of irfs are directly or indirectly targeted by endogenous and exogenous proteins in an ubiquitin-dependent manner. however, few reviews have summarized how host e ligases/dubs or viral proteins regulate irf stability and activity. additionally, with recent technological developments, details about the ubiquitination of irfs have been continuously revealed. as knowledge of how these proteins function and interact with irfs may facilitate a better understanding of the regulation of irfs in immune responses or other biological processes, we summarized current studies on the direct ubiquitination of irfs, with an emphasis on how these proteins interact with irfs and affect their activities, which may provide exciting targets for drug development by regulating the functions of specific e ligases. the interferon regulatory factor (irf) family of transcription factors consists of nine members (irf - ) in mammalian cells (tamura et al., ) . all irfs share a well-conserved n-terminal dbd (dna-binding domain),~ amino acids of which form a helix-loop-helix motif for the recognition of a dna sequence. the c-terminal regions of irfs have low sequence homology and function as association domains by which irfs interact with other family members or transcription factors ( figure ) (jefferies, ; tamura et al., ) . irfs play important roles in immune defense, stress responses, reproduction, development, and carcinogenesis, which account for many aspects of innate and adaptive immune responses (nehyba et al., ) . irf and irf , the first irf family members identified, are well known for their antagonistic regulation of oncogenesis (harada et al., ) . moreover, irf and irf have also been demonstrated to regulate both lymphoid and myeloid cell development and differentiation. three other family members, irf , irf and irf , are also involved in regulating immune cell development and phonotype (jefferies, ; savitsky et al., ) . irf , irf , and irf are best known for their critical role as transcription factors promoting ifn-α/β expression in type i ifn-dependent innate immune responses (yanai et al., ) . irf is a regulator of interferon-driven gene expression that acts as a critical link between type i ifns and the p pathway (takaoka et al., ) . unlike other irfs, irf has essential functions in the normal development and differentiation of the epidermis instead of participating in innate immunity (richardson et al., ) . due to the pivotal and critical roles of irfs, the balance between the activation and destruction of irfs needs to be constantly maintained to avoid adverse overreaction. to orchestrate an appropriate immune response, the immune system is strictly regulated. various posttranslational modifications (ptms), such as phosphorylation, ubiquitination, acetylation and other unconventional ptms, have evolved as critical regulators of the immune system, and all ptms are important in regulating the activity, stability and folding of irfs (deribe et al., ) . the most extensively investigated type of ptm in innate immunity is phosphorylation, which is inversely regulated by kinases and phosphatases (karin and ben-neriah, ; liu et al., ) . similar to phosphorylation, ubiquitination is one of the most important regulatory mechanisms in the immune system, and ubiquitination can also be reversibly regulated by e ubiquitin ligases and dubs (deubiquitinating enzymes) (ning et al., ) . ubiquitination is a common modification for protein activation or deactivation, and irfs are stringently regulated by ubiquitination in many respects. ubiquitination can directly or indirectly target irfs and positively or negatively regulate the stability, activation and transcriptional activity of irfs (ning et al., ) . through the ubiquitin pathway, irfs can improve the antiviral response by ubiquitin-mediated degradation or activation. this review summarized current research on the direct ubiquitination of irfs, with an emphasis on several host proteins and viral proteins found to target irfs for ubiquitination or deubiquitination. knowledge of how these proteins function and interact with irfs may provide a better understanding of the regulation of irfs in immune responses or other biological processes and exciting targets for the development of drugs aimed at regulating the functions of specific e ligases or dubs. ubiquitination is a type of reversible cellular protein ptm. ubiquitin, the core component of ubiquitination, is covalently attached to one or more lysine residues in cellular proteins through an enzymatic cascade involving three classes of enzymes termed ubiquitin-activating enzymes (e ), ubiquitin-conjugating enzymes (e ), and ubiquitin protein ligases (e ) (heaton et al., ; mukhopadhyay and riezman, ) . the human proteome contains two e enzymes, approximately e enzymes, e ligases, and dubs (bhoj and chen, ). among these, e ubiquitin ligases largely dictate substrate specificity. e ligases can be generally divided into three subgroups: the hect (homology to e -associated protein carboxyl terminus) domain e ligases, ring (really interesting new gene)-type e ligases, and rbr (ring-between-ring) e ligases (dove and klevit, ) . the ubiquitinating modification can be reversed by dubs. dubs are essentially proteases that cleave the peptide or isopeptide bond between ubiquitin and its substrate protein (nijman et al., ) . there are two classes of dubs: cysteine proteases, which can be further divided into four different classes (uchs (ubiquitin-cterminal hydrolases), usps (ubiquitin-specific proteases), mjds (machado-joseph disease protein domain proteases), and otus (ovarian tumor proteases), and metalloproteases, which contain only jamms (jamm motif proteases) (chen and sun, ) . ubiquitin itself contains seven lysine residues and one nterminal methionine residue (k , k , k , k , k , k , k and m ), all of which can be further conjugated by another ubiquitin to form ubiquitin chains with different linkages (ikeda and dikic, ) . various types of ubiquitin chains carry a range of molecular signals and function in a variety of cellular processes, including membrane trafficking, protein kinase activation, dna repair, and cell signaling (chen and sun, ; welchman et al., ) . k -linked polyubiquitination is a well-studied type of ubiquitination by which proteins are targeted for proteasomal degradation through the s proteasome (zheng and shabek, ) . over the last two decades, mounting evidence has identified atypical linkages that mediate less frequent proteasomal degradation, such as k -and k -linked chains (komander and rape, ) . k -linked ubiquitination is required for proper activation of the dna damage response, and k linked polyubiquitination of sting facilitates tbk recruitment and activation (gatti et al., ; wang et al., ) . m -linked linear polyubiquitination is involved in the activation of nf-κb signaling (tokunaga et al., ). the activation of t cells is negatively regulated by k -linked polyubiquitination (huang et al., ; yang et al., ) . only a few studies have demonstrated that k -linked ubiquitination may be involved in the dna damage response and the maintenance of mitochondrial stability (jin et al., ; wu-baer et al., ) . furthermore, k -linked polyubiquitination and monoubiquitination, the second most common type of ubiquitin linkage, mediate proteasome-independent effects, which play an important role in activating many components of different signaling pathways and participate in many biological processes (ning et al., ) . because of the critical functions of irf family members in regulating the activation of ifn promoters and downstream signaling in cell differentiation and tumor suppression, it is of vital importance that their activities are maintained in a state that is not overly stimulated. ubiquitin-mediated proteasomal degradation of irf family members is an effective mechanism to limit irf activity, and ubiquitin-mediated activation of irfs is also essential for irfs to better regulate related biological processes. many studies, especially on irf and irf , have identified some endogenous proteins that function as e ubiquitin ligases and catalyze irf ubi-quitination in a proteasome-dependent pathway or a nonproteolytic pathway ( figure ) tsuchida et al., ) . ubiquitin-mediated degradation of irf / irf and irf are best known for their critical role as master regulators promoting type i ifn expression. irf is constitutively expressed, and irf is an ifn-stimulated gene (isg) whose expression can be stimulated by interferon lazear et al., ) . following viral infection, the host produces interferon against pathogen infections, and interferon activates the jak-stat signaling pathway to induce the transcription of isgs (schneider et al., ) , which can positively induce the expression of irf and drive more interferon. as irf and irf are essential switches that turn off and limit type i ifn production to avoid overreaction, ubiquitin-mediated degradation of irf and irf has been well described in many studies. as a member of the trim family, trim (also called ro ) was initially characterized as an autoantigen in pa- tients with autoimmune diseases, including sjögren's syndrome and systemic lupus erythematosus (rhodes et al., ) . upon rna virus infection, trim is significantly induced and functions as an e ubiquitin ligase, enabling it to play important physiological roles in many relevant biological processes. irf , irf , irf and irf can act as substrates for trim -mediated ubiquitination. via its cterminal spry domain, trim negatively regulates the stability of irf by mediating irf polyubiquitination and proteasomal degradation . however, some studies have shown that trim sustains irf activation during the antiviral response at the early phase of infection (yang et al., ) . pin (peptidyl-prolyl isomerase) recognizes phosphorylated irf at the s -p motif and finally targets irf for degradation through polyubiquitination (chen et al., ; saitoh et al., ) . trim can interfere with the interaction between pin and irf , which prevents irf ubiquitination and degradation (yang et al., ) . in addition, studies have identified a novel role for tyrosine phosphorylation in regulating the activity of trim . upon tlr and tlr stimulation, trim is tyrosine phosphorylated, which can simultaneously positively regulate its interaction with irf and enhance trim activity (stacey et al., ) . this may explain the complicated role of trim in irf regulation. in the case of irf , trim -mediated ubiquitination promotes the degradation of irf following tlr and tlr stimulation (higgs et al., ) . the interaction of trim with the apoptotic protein fas-associated death domain (fadd) can enhance the ubiquitin ligase activity of trim , and both trim and fadd cooperatively interact with irf and contribute to trim -mediated ubiquitination of irf (young et al., ) . three other cellular e ligases have been identified, c-cbl, rbck and raul (rta-associated ubiquitin ligase), all of which target and promote k -linked polyubiquitinationdependent proteasomal degradation of irf . c-cbl, a member of the cbl (casitas b-lineage lymphoma) family, negatively regulates irf protein stability by interacting with the c-terminal domain of irf via its tkb (tyrosine kinase binding) domain and promotes k -linked polyubiquitination-dependent proteasomal degradation of irf (zhao et al., ) . after induction by viral infection, the e ubiquitin ligase rnf , also called rbck (rbcc protein interacting with pkc ), specifically catalyzes the k linked polyubiquitination of irf , which mediates the degradation of irf . this process is an important negative feedback mechanism for the termination of irf -dependent antiviral responses (zhang et al., ) . additionally, rbck plays a broader role in the suppression of host antiviral responses because it suppresses nf-κb activation by negatively regulating tnf and il- (tian et al., ) . the hect e ligase raul directly catalyzes k -linked polyubiquitination of both irf and irf , followed by their proteasome-dependent degradation. the viral e rat encoded by kshv (kaposi's sarcoma-associated virus) also cooperates with the host e ligase raul to increase proteolysis of both irf and irf , which is more effective against the host antiviral response (yu and hayward, ) . not only is ubiquitination involved in protein degradation but also the proteasome-independent functions of nondegradative ubiquitination are required to promote the activation of irf and irf . in addition, irf activity is positively or negatively regulated by some dubs that regulate irfs by removing the ubiquitin chain from ubiquitinated irfs (figure ). linear ubiquitin chain assembly complex (lubac) is an ubiquitin ligase complex composed of sharpin, hoil-il and hoip that generates linear polyubiquitin chains (tokunaga and iwai, ) . the activation of irf in the ripa (rlr-induced irf -mediated pathway of apoptosis) requires linear polyubiquitination of irf at two specific lysine residues mediated by lubac, which triggers its interaction with the proapoptotic protein bax to induce mitochondrial activation and apoptotic cell death (chattopadhyay et al., ; chattopadhyay and sen, ) . irf activation by epstein-barr virus lmp (latent membrane protein ) and ikkε is enhanced following k linked ubiquitination by traf . traf ubiquitinates irf at multiple sites, but this ubiquitination is independent of the c-terminal functional phosphorylation sites of irf , which provides evidence that the regulatory ubiquitination of irf is a prerequisite for its phosphorylation (ning et al., ) . in addition, receptor-interacting protein (rip) contributes to lmp -promoted ubiquitination of irf and is required for full activation of irf by lmp , suggesting that rip serves as a general activator of irf (huye et al., ) . the antiapoptotic factor a , which possesses both dub activity in its n-terminal domain and e ligase activity in its c-terminal domain, is also induced by lmp . the a n-terminal dub domain interacts with and deubiquitinates irf , decreasing the k -linked ubiquitination of irf (ning and pagano, ) . the regulation of irf and irf is considerably less well understood than that of irf and irf . irf and irf are well known for their antagonistic regulation of oncogenesis. as a tumor suppressor protein, irf exhibits a short half-life and turns over rapidly in response to changes in the cellular environment . the irf protein level is differentially regulated by chip (c terminus of the hsc (heat-shock cognate) -interacting protein). chip, a protein quality control e ligase, is regarded as a link between protein folding and protein degradation pathways through its selective ubiquitination of misfolded proteins via collaboration with molecular chaperones (murata et al., ) . in unstressed cells, chip appears to chaperone irf and positively affect its protein levels. however, under certain stress conditions, chip binds a central intrinsically disordered domain (mf domain) of irf- and mediates its ubiquitination (narayan et al., ) . the structured dbd of irf acts as an ubiquitin acceptor site of chip binding and ubiquitination (landré et al., ) . importantly, the interaction between irf and chip was enhanced by trichostatin a (tsa) treatment, which facilitated the degradation of irf and was shown to be important in the tsa-mediated inhibitory effect on ifn-γ induction mediated by trim and other irf- -dependent ifn-stimulated genes (gao et al., ) . in addition, mdm functions as an mf domain ligase, interacts with the irf dbd and mediates the ubiquitination of irf (landré et al., ) . mdm (murine double minute , also known as human double minute protein (hdm )) is known for its role in targeting p for ubiquitin-mediated proteasomal degradation; it also interacts with irf and mediates its ubiquitination (moll and petrenko, ) . the interaction between irf and mdm requires both the hydrophobic pocket and acidic domain of mdm , and its dual binding sites enable e -ubiquitin ligase-substrate interactions with irf , which may attenuate the function of irf as a transcriptional repressor (pettersson et al., ) . in addition, the activation of irf requires k -linked polyubiquitination by the apoptosis inhibitor ciap during il- signaling. the e ubiquitin ligase activity of ciap can be enhanced by the bioactive sphingolipid mediator s p, and both ciap and s p form a complex with irf , which is essential for il- -induced production of the chemokines cxcl and ccl (harikumar et al., ) . irf , irf and irf , the stability and activity of which are also critically regulated by ubiquitination, play important roles in regulating the development and differentiation of both lymphoid and myeloid cells. as described above, the other two cbl family members in mammals, cbl-b and cblc/cbl- , are also involved in negatively regulating the stability of irfs (swaminathan and tsygankov, ) . all cbl proteins have a conserved n-terminal region that encompasses a tkb domain, ring finger domain and a linker region between them. these domains enable cbl proteins to function as e ligases (swaminathan and tsygankov, ; thien and langdon, ) . cbl proteins play a critical role in antibody affinity maturation in gc (germinal center) b cells by promoting irf ubiquitination and degradation . likewise, the stability of irf is downregulated by cbl-mediated ubiquitination, and the c-terminal domain of irf- is necessary for ubiquitination (xiong et al., ) . irf contains polymorphisms due to its alternative splicing and the insertion or deletion of a -nucleotide sequence in irf exon . as mentioned, irf can be a substrate of trim , and various irf isoforms can directly interact with trim upon tlr stimulation. irf -v and irf -v are targeted for trim -mediated degradation, whereas irf -v and irf -v are resistant to trim -mediated degradation, which is of great importance in regulating the stability and activity of irf (lazzari et al., ) . proteasome-independent ubiquitination is also involved in regulating these three irfs. upon ifn-γ and tlr stimulation in murine macrophages, trim was found to interact with irf and ubiquitinate irf in the nondegradative pathway, which positively contributed to the expression of il- p (kong et al., ) . irf protein levels in regulatory t cells are also stabilized by usp (ubiquitin-specific protease ), and usp physically interacts with irf and functions via k -linked deubiquitinase activity (lin et al., ) . usp also stabilizes irf protein levels by interacting with and deubiquitinating irf , which promotes the function of irf in facilitating il- expression in t helper type cells (guo et al., ) . similarly, upon tlr or tlr stimulation, traf interacts with irf and promotes k -linked ubiquitination of irf , which is important for irf nuclear translocation and target gene regulation (balkhi et al., ) . there is mounting evidence that irf activity is also downregulated by the ubiquitination of viral proteins. viruses have evolved a multitude of strategies to disturb the innate immune system. viruses facilitate their own replication, and their persistence largely depends on their ability to exploit the ubiquitin system; viruses can encode their own e ligases or deubiquitinases or redirect host ubiquitin enzymes randow and lehner, ). irfs are generally an attractive ubiquitination target for the virusmediated subversion of host antiviral responses ( figure ) . as the dominant regulators of type i ifn production following viral infection, the ability of irf and irf to impair viral replication makes them the most important targets for many viruses. the protein expression and phosphorylation of irf and irf were shown to be inhibited after svv (seneca valley virus) infection, and svv c pro interacted with irf or irf in pk- cells and contributed to the degradation of irf and irf through its protease activity (xue et al., a) . furthermore, the cysteine and histidine residues of svv c pro endow it with deubiquitinating activity, which can inhibit both the k -linked polyubiquitination of tbk (tank-binding kinase ) and the k -linked polyubiquitination of rig- (retinoic acid-inducible gene i) and traf (tnf receptor-associated factor ). interestingly, c pro partly stabilizes tbk by its dub activity, but as a whole, the expression level of tbk is still reduced after svv infection. therefore, via its dub activity, svv c pro suppresses the rig-i-and tbk -induced expression of ifn-β (xue et al., b) . the icp (infected cell protein ) protein encoded by several herpesviruses also functions as an e ligase and induces k -linked ubiquitination and proteolysis of irf and irf . the ring finger domain of icp is essential for its e ubiquitin ligase activity. however, some studies demonstrated that icp encoded by bicp (bovine herpesvirus ) reduced the protein levels of irf but not irf and directly or indirectly targeted irf for proteasome-dependent degradation (saira et al., ; saira et al., ) . moreover, bicp was also found to promote the k -linked ubiquitination of traf and mediate its degradation through a proteasome-dependent pathway, influencing the activation of irf via k -linked ubiquitination by traf (cao et al., ) . studies have identified full-length rotavirus nsp (nonstructural protein ) as a viral ifn antagonist that induces the proteasome-mediated degradation of irf (barro and patton, ) . as nsp specifically targets the cterminal regions of irfs (except irf and irf ), which carry an irf association domain that mediates homo or heterodimeric irf interactions, nsp also mediates the degradation of irf , irf and irf (arnold, ; barro and patton, ) . during the early steps of hiv- infection, the hiv- accessory proteins vif and vpr decrease the relative levels of irf due to ubiquitin-associated proteasome degradation without activating irf . the n-terminal lysine residues of irf are important for vif-and vpr-mediated degradation (doehle et al., ; okumura et al., ) . however, the degradation of irf by another accessory protein, vpu, remains controversial (doehle et al., ; langer et al., ) . the cellular e ligase hdm is exploited by the transcriptional activator tat of hiv- , which accelerates irf proteasome-mediated degradation . in addition, inactivated irf can interact with the viral protein n pro (n-terminal protease), a small papain-like cysteine protease. n pro interacts with and induces the degradation of irf upon infection with the pestiviruses classical swine fever virus (csfv) and bovine viral diarrhea virus (bvdv), which causes a marked loss of irf (bauhofer et al., ; chen et al., ) . orf and plp can recognize and interfere with activated irf . orf , an immediate-early protein of varicella-zoster virus (vzv), inhibits the sendai virus-mediated activation of ifn-β by directly interacting with activated irf , leading to the degradation of irf through an ubiquitin-proteasome pathway (zhu et al., ) . papain-like protease domain (plp ), a catalytic domain of nsp (nonstructural protein ) from mhv-a (mouse hepatitis virus a ), contains a conserved dub motif. plp binds irf and causes its deubiquitination to prevent the nuclear localization of irf . moreover, the dub activity of plp can cleave both k -and k -linked polyubiquitin chains from irf , which may be why coronaviruses can escape host innate antiviral responses (tsuchida et al., ; zheng et al., ) . as mentioned before, the kshv-encoded immediate-early protein rta (rna transcriptional activator, also known as orf ) has evolved the ability to redirect the host e ligase raul to strengthen the proteolysis of both irf and irf . the effect of kshv against the host antiviral response is also attributed to the e ligase activity of rta, which targets irf for proteasome-dependent degradation. a cystine-and histidine-rich n-terminal domain of rta is critical for its e ligase activity (yu et al., ) . gcrv (grass carp reovirus) vp blocks the host ifn response and facilitates viral replication by inducing k -linked ubiquitination and degradation of phosphorylated irf upon gcrv infection (zhang et al., ) . in addition to nsp , irf is targeted by the immediate-early protein orf encoded by vzv and simian varicella virus, which blocks jak-stat signaling by degrading irf in a proteasome-dependent manner (verweij et al., ) . the important roles played by irfs in specific type i ifn induction and how irfs are activated and regulated by viral infection and ptms have been discussed in many recent reviews. this review summarizes current research on the direct ubiquitination of irfs and introduces and classifies identified cellular e ligases/dubs and viral proteins by substrate specificity, interaction and effect on irf stability or activity (table ) . however, research on how ubiquitination regulates irfs is still being updated with more in-depth studies on the mechanism by which irfs shape innate and adaptive immune responses, cell differentiation, tumor suppression and other biological processes. many questions remain unanswered and require explanation: the ubiquitin acceptor sites of some irfs that undergo e ligase-mediated ubiquitination remain undefined, the interactions between e ligases or viral proteins and irfs and their functions should be better characterized, and how viruses exploit the ubiquitin system to interact with and affect the host immune system should be further elaborated. thus, further identification of these sites can fully explain the role of ubiquitin in regulating irfs, and interfering viral protein-irf interaction interfaces may provide host targets for both antiviral treatment and autoimmune therapy. the author(s) declare that they have no conflict of interest. the rotavirus interferon antagonist nsp : many targets, many questions functional regulation of myd -activated interferon regulatory factor by k -linked polyubiquitination rotavirus nonstructural protein subverts innate immune response by inducing degradation of ifn regulatory factor rotavirus nsp inhibits expression of type i interferon by antagonizing the function of interferon regulatory factors irf , irf , and irf classical swine fever virus npro interacts with interferon regulatory factor and induces its proteasomal degradation ubiquitylation in innate and adaptive immunity bicp negatively regulates traf -mediated nf-κb and interferon activation by promoting k -linked polyubiquitination of traf ubiquitination of the transcription factor irf- activates ripa, the apoptotic pathway that protects mice from viral pathogenesis rig-i-like receptor-induced irf mediated pathway of apoptosis (ripa): a new antiviral pathway prolyl isomerase pin : a promoter of cancer and a target for therapy ubiquitination and proteasomal degradation of interferon regulatory factor- induced by npro from a cytopathic bovine viral diarrhea virus nonproteolytic functions of ubiquitin in cell signaling post-translational modifications in signal integration vpu-deficient hiv strains stimulate innate immune signaling responses in target cells human immunodeficiency virus type mediates global disruption of innate antiviral signaling and immune defenses within infected cells ring-between-ring e ligases: emerging themes amid the variations inhibition of histone deacetylase activity suppresses ifn-γ induction of tripartite motif via chip-mediated proteasomal degradation of irf- rnf promotes noncanonical k ubiquitination to signal dna damage ubiquitin specific peptidase stabilizes interferon regulatory factor protein and promotes its function to facilitate interleukin- expression in t helper type cells anti-oncogenic and oncogenic potentials of interferon regulatory factors- and - k -linked polyubiquitination of transcription factor irf is essential for il- -induced production of chemokines cxcl and ccl ubiquitin in the activation and attenuation of innate antiviral immunity targeting irfs by ubiquitination: regulating antiviral responses self protection from anti-viral responses-ro promotes degradation of the transcription factor irf downstream of the viral toll-like receptors the e ubiquitin ligase ro negatively regulates ifn-β production post-pathogen recognition by polyubiquitin-mediated degradation of irf k -linked polyubiquitination of t cell receptor-ζ regulates proteolysis-independent t cell signaling interferon regulatory factor is activated by a viral oncoprotein through ripdependent ubiquitination protein modifications: beyond the usual suspects' review series regulating irfs in ifn driven disease mitochondrial membrane potential regulates pink import and proteolytic destabilization by parl phosphorylation meets ubiquitination: the control of nf-κb activity the ubiquitin code cutting edge: autoantigen ro is an interferon inducible e ligase that ubiquitinates irf- and enhances cytokine expression in macrophages dna-binding regulates site-specific ubiquitination of irf- hiv- vpu is a potent transcriptional suppressor of nf-κbelicited antiviral immune responses irf- , irf- , and irf- coordinately regulate the type i ifn response in myeloid dendritic cells downstream of mavs signaling tripartite motif (trim ) differentially regulates the stability of interferon regulatory factor (irf ) isoforms cbl ubiquitin ligases control b cell exit from the germinal-center reaction usp interacts and positively regulates irf function via k -linked deubiquitination in regulatory t cells post-translational modification control of innate immunity ifn regulatory factors and antiviral innate immunity: how viruses can get better the mdm -p interaction proteasome-independent functions of ubiquitin in endocytosis and signaling chip is a chaperone-dependent e ligase that ubiquitylates unfolded protein docking-dependent ubiquitination of the interferon regulatory factor- tumor suppressor protein by the ubiquitin ligase chip dynamic evolution of immune system regulators: the history of the interferon regulatory factor family a genomic and functional inventory of deubiquitinating enzymes traf and the three c-terminal lysine sites on irf are required for its ubiquitination-mediated activation by the tumor necrosis factor receptor family member latent membrane protein irf : activation, regulation, modification and function the a deubiquitinase activity negatively regulates lmp activation of irf hiv- accessory proteins vpr and vif modulate antiviral response by targeting irf- for degradation role of mdm acid domain interactions in recognition and ubiquitination of the transcription factor irf- role of the irf- enhancer domain in signalling polyubiquitination and degradation viral avoidance and exploitation of the ubiquitin system hiv- tat recruits hdm e ligase to target irf- for ubiquitination and proteasomal degradation the mw ro/ss-a autoantigen in sjogren's syndrome/systemic lupus erythematosus (ro ) is an interferon-gamma inducible tripartite motif protein associated with membrane proximal structures irf is a key determinant of the keratinocyte proliferation-differentiation switch the infected cell protein encoded by bovine herpesvirus (bicp ) induces degradation of interferon response factor and, consequently, inhibits beta interferon promoter activity the infected cell protein encoded by bovine herpesvirus (bicp ) associates with interferon regulatory factor and consequently inhibits beta interferon promoter activity negative regulation of interferon-regulatory factor -dependent innate antiviral response by the prolyl isomerase pin regulation of immunity and oncogenesis by the irf transcription factor family interferonstimulated genes: a complex web of host defenses tyrosine phosphorylation of the e ubiquitin ligase trim positively regulates interaction with irf and hence trim activity the cbl family proteins: ring leaders in regulation of cell signaling integration of interferon-α/β signalling to p responses in tumour suppression and antiviral defence the irf family transcription factors in immunity and oncogenesis c-cbl and cbl-b ubiquitin ligases: substrate diversity and the negative regulation of signalling responses rbck negatively regulates tumor necrosis factor-and interleukin- -triggered nf-κb activation by targeting tab / for degradation lubac, a novel ubiquitin ligase for linear ubiquitination, is crucial for inflammation and immune responses involvement of linear polyubiquitylation of nemo in nf-κb activation inhibition of irf -dependent antiviral responses by cellular and viral proteins varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms the e ubiquitin ligase amfr and insig bridge the activation of tbk kinase by modifying the adaptor sting ubiquitin and ubiquitin-like proteins as multifunctional signals the brca / bard heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue k of ubiquitin ubiquitin-dependent degradation of interferon regulatory factor- mediated by cbl down-regulates interleukin- expression seneca valley virus cpro abrogates the irf -and irf -mediated innate immune response by degrading irf and irf seneca valley virus c protease negatively regulates the type i interferon pathway by acting as a viral deubiquitinase the irf family of transcription factors trim is essential to sustain ifn regulatory factor activation during antiviral response k -linked polyubiquitination of zap by nrdp controls cd + t cell activation fas-associated death domain (fadd) and the e ubiquitinprotein ligase trim interact to negatively regulate virus-induced interferon production the ubiquitin e ligase raul negatively regulates type i interferon through ubiquitination of the transcription factors irf and irf the kshv immediate-early transcription factor rta encodes ubiquitin e ligase activity that targets irf for proteosome-mediated degradation grass carp reovirus vp represses interferon production by degrading phosphorylated irf negative feedback regulation of cellular antiviral signaling by rbck -mediated degradation of irf c-cblmediated ubiquitination of irf negatively regulates ifn-β production and cellular antiviral response plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production ubiquitin ligases: structure, function, and regulation varicella-zoster virus immediate-early protein orf abrogates the irf -mediated innate immune response through degradation of activated irf key: cord- - si qrrx authors: battesti, g.; descamps, v. title: negative tests for sars‐cov‐ infection do not rule out its responsibility for chilblains date: - - journal: br j dermatol doi: . /bjd. sha: doc_id: cord_uid: si qrrx we read with great interest the report of le cleach et al discussing chilblains as a manifestation of covid‐ pandemic. they reported patients with acral lesions occurring during the covid‐ lockdown in france. the most frequent clinical presentation of these acral lesions was typical chilblains. among the patients with rt‐pcr testing and/or serology, only had confirmed covid‐ . they concluded that there is no evidence of sars‐cov‐ infection in the large majority of patients with acral lesions. they hypothesized that the situation could be due to the media stating that chilblains were caused by sars‐cov‐ infection and leading to a higher rate of consultation or the lockdown leading to more inactivity and long periods at home barefoot on a cold floor( ). we read with great interest the report of le cleach et al discussing chilblains as a manifestation of covid- pandemic. they reported patients with acral lesions occurring during the covid- lockdown in france. the most frequent clinical presentation of these acral lesions was typical chilblains. among the patients with rt-pcr testing and/or serology, only had confirmed covid- . they concluded that there is no evidence of sars-cov- infection in the large majority of patients with acral lesions. they hypothesized that the situation could be due to the media stating that chilblains were caused by sars-cov- infection and leading to a higher rate of consultation or the lockdown leading to more inactivity and long periods at home barefoot on a cold floor . we do not agree with this explanation. we recently published cases of chilblains enrolled during covid- pandemic . we performed the same virological tests which were also mostly negative but our conclusion was different. we demonstrated in skin biopsies high expression of mxa (interferon type i induced (ifn-i) protein) and cd (marker of plasmacytoid dendritic cells, known as the major producer of ifn-i). histochemical results were comparable to those found in our chilblain lupus erythematosus group. we concluded that chilblain was a manifestation of ifn-i upregulation as observed in genetic interferonopathies. active viral replication is not necessary to mount an efficient ifn response in sars-cov infection. interferon-induced trans-membrane protein may inhibit coronavirus replication . this inhibition may be one of the reasons why pcr tests were negative. it was also demonstrated that high expression of ifn-i at the onset of viral infection may induce a depletion of b cells and may explain the negativity of serologies . moreover, subcutaneous injection of beta-interferon is known to induce vasculopathy. we concluded that chilblains reflect a strong antiviral response in potential genetically predisposed patients for high production of ifn-i. most chilblains observed during the covid- outbreak occur in patients who are negative for covid- on pcr and serology testing this article is protected by copyright. all rights reserved key: cord- -c luoo m authors: lauber, c; vieyres, g; terczyńska-dyla, e; anggakusuma; dijkman, r; gad, h h; akhtar, h; geffers, r; vondran, f w r; thiel, v; kaderali, l; pietschmann, t; hartmann, r title: transcriptome analysis reveals a classical interferon signature induced by ifnλ in human primary cells date: - - journal: genes immun doi: . /gene. . sha: doc_id: cord_uid: c luoo m the ifnl gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis c virus infection. the activity of ifnλ has an important causal role in the pathogenesis, but the molecular details are not fully understood. one possible reason for the detrimental effect of ifnλ could be a tissue-specific regulation of an unknown subset of genes. to address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with ifnα, ifnλ or ifnλ and assessed interferon mediated gene regulation using transcriptome sequencing. our data show a surprisingly similar response to all three subtypes of interferon. we also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. however, the interferon response is robust in both tissues with the majority of the identified genes being regulated in hepatocytes as well as airway epithelial cells. thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium. supplementary information: the online version of this article (doi: . /gene. . ) contains supplementary material, which is available to authorized users. tissue-specific responses to the different interferon (ifn) subtypes are poorly understood, and the vast majority of studies are performed using cell lines. cell lines provide a highly homogeneous cell population allowing for the generation of data with little noise. however, cancer-derived cell lines do not necessarily provide the best picture of the in vivo ifn response. primary cells, on the other hand, resemble the in vivo situation much better. the lung epithelium is exposed to the environment and thus to frequent viral infections, although most respiratory infections are transient in nature. in contrast, viruses need to overcome several barriers to reach and infect the hepatocytes in the liver. nevertheless, once a viral infection is established in the liver it can cause significant pathogenicity. viral hepatitis is frequently caused by the two rna viruses, hepatitis a virus (family picornaviridae) and hepatitis c virus (hcv; family flaviviridae), as well as by the dna virus hepatitis b virus (family hepadnaviridae). humans possess three types of ifns: type i, ii, and iii. type ii ifn (ifnγ) is called an interferon for historical reasons; it signals via the formation of stat homodimers and only has limited direct antiviral activity but has potent proinflammatory activities. for simplicity, it will not be further addressed here. type i and type iii ifns (the latter are also known as ifnλs) signal through distinct receptor complexes. the type i ifn receptor is composed of the ifnar and ifnar receptor chains. , these receptor chains are universally expressed on all nucleated cells and therefore type i ifns possess a systemic effect when produced in adequate amounts. type iii ifns signal through a receptor complex consisting of the ifnλr (il ra)-specific chain and the shared il r (il rβ) chain. , expression of ifnλr is rather restricted, with epithelial tissues and the liver showing the most prominent expression in humans. , this restricted expression of ifnλr implies that ifnλ can target tissues of high risk for viral infection without the detrimental effect caused by a systemic type i ifn response. despite using different receptor complexes, both type i and type iii ifn can induce activation of the transcription factor ifn-stimulated gene factor (isgf ) and therefore regulate a highly overlapping set of genes. [ ] [ ] [ ] however, the kinetics of the response can be rather different. , genome-wide association studies have identified several single nucleotide polymorphisms (snps) within the ifnλ loci as powerful predictors of hcv treatment outcome as well as of spontaneous clearance of hcv infections. [ ] [ ] [ ] [ ] [ ] recently, ifnλ , a novel member of the ifnλ family, was identified. the ifnl gene encodes a protein with only % sequence identity to ifnλ . nevertheless, the ifnλ protein is fully active and can signal through the canonical ifnλ receptor complex. paradoxically, a frameshift mutation disrupting translation of the ifnl mrna (rs tt ) is strongly correlated with improved hcv clearance, both treatment induced and spontaneous. , furthermore, a direct correlation between the activity of the ifnλ protein and poor hcv clearance was recently demonstrated. thus, despite being highly antiviral in vitro, having a functional ifnl gene is disadvantageous during hcv infection. the malicious effect of ifnλ might extend to other chronic viral infections, as recent data suggest for cytomegalovirus infection. , the paradoxical situation that an apparent antiviral cytokine is disadvantageous during viral infection, and the current inability to explain why this effect is specific to ifnλ , has spurred speculation toward alternative signaling pathways of ifnλ . in order to determine whether ifnλ can induce an alternative set of genes, that are not induced in the classical ifn response, we have compared the transcriptional response after ifnα, ifnλ and ifnλ stimulation in both primary human hepatocytes (phh) and primary human airway epithelial (hae) cells using transcriptome sequencing (rna-seq). we chose epithelial cells because they represent a first line of defense, where type iii ifns have a major role and hepatocytes because the detrimental effect of ifnλ has been described for pathogenesis of hcv infection. the gene induction pattern of the three ifn subtypes was found to be remarkably similar, with a core set of genes induced by all three ifns in both cell types. moreover, we identified putative tissuespecific genes that are differentially regulated in the hae compared to the phh cells upon ifn treatment. we established cultures of phh and primary hae cells. the two systems were chosen as the respective primary cells originate from tissues that are of biological relevance for type iii ifn. both tissues respond well to both type i and type iii ifns, thus allowing us to compare the tissue-and ifn-type specific responses. phh and hae were cultured and stimulated with ifn for h. a major obstacle in using primary cells is the inherent donor-todonor variation. we sought to overcome this by using cells from several donors and by applying relatively strict statistical selection criteria. in brief, these are a minimum of -fold induction or . fold repression and a maximal p-value of . (see materials and methods for details). table lists the number of significantly regulated genes in both tissues. globally, in phh and hae cells, more genes meet the inclusion criteria for ifnα than for ifnλ. this is not surprising as several prior studies have shown a stronger response to ifnα than to ifnλ, but both types of ifn fundamentally target the same set of genes through activation of the transcription factor isgf . [ ] [ ] [ ] [ ] [ ] finally, the number of significantly induced genes is higher in hae cells than in phh. this is largely owing to the fact that the donor-to-donor variation is substantially lower in the hae cultures, compared with the phh cultures, resulting in fewer genes being rejected due to poor p-values. of note, the lung-derived cells are cultured for several weeks between the surgery and the ifn treatment, whereas the phh are typically treated h after liver resection. this longer culture period of the hae cells, which is necessary to obtain a pseudostratified epithelium, might render them more homogenous and blur donor-to-donor differences, explaining the better p-values. we then chose to focus on a set of robustly regulated genes, requiring a gene to meet the selection criteria for at least three of the six experiments (three ifns tested in two distinct primary cell culture systems), resulting in genes (supplementary table ). of those, genes are induced by all three ifns and in both tissues. overall we observe little qualitative difference between the different subtypes of ifn used, but type i ifn (ifnα) shows a stronger response with more genes meeting the threshold for significance. ifnλ induces a classical ifn signature figure shows scatter plots of gene expression change caused by ifnλ versus ifnα (figure , top row) or versus ifnλ treatment ( figure , bottom row). black dots represent genes that are significantly induced by both treatments. orange dots represent genes that meet the selection criteria for ifnλ but not for the ifn used for comparison (ifnα or ifnλ ), and thus represent potential ifnλ -specific genes. blue dots are genes meeting the selection criteria for either ifnα (figure , top row) or ifnλ (figure , bottom row), but not for ifnλ . identification of genes that are specifically regulated by ifnλ is of particular interest to reconcile the apparent paradox between the antiviral activity of ifnλ against hcv and unfavorable in vivo effects on hcv infection and treatment outcome. the scatter plots in figure show a strikingly similar response to ifnα and ifnλ ( figure , top row), with the majority of regulated genes appearing on the diagonal. furthermore, the responses to ifnλ and ifnλ are highly similar ( figure , bottom row). although several genes appear as ifnλ -specific in the scatter plots (orange dots), they are on average induced to similar levels by both types of ifns (dots on or close to the diagonal). lack of significance for ifnα and/or ifnλ is owing to high donor-todonor variability. most of the concerned genes are known ifnstimulated genes (isgs) and we do not detect any genes that are reliably induced by ifnλ only. thus, ifnλ shows a classical ifn signature virtually identical to that of ifnλ in both phh and hae cells. there is an abundance of ifnα-specific genes ( figure , top row, table ). however, this is likely owing to the statistical bias of our stringent significance thresholds. indeed, the overall broader amplitude of the ifnα-mediated isg stimulation results in more genes meeting the selection criteria. this stronger ifnα response has been observed by others and might be caused by the choice of an early analysis time point ( h treatment), which underestimates the slower ifnλ response. , furthermore, both hae and phh show a substantial higher mrna expression for the type i ifn receptor complex compared with the type iii receptor (see below). consistent with this, most ifnα-specific genes are close to the diagonal. the few genes that showed no apparent regulation by ifnλ in phh (along the y axis in figure , top left graph) have very low expression values (data not shown). altogether, there is a nearly complete overlap between the ifnα, λ and λ ifn-regulated genes (we use the term ifn-regulated genes (irgs) to cover all genes, both positively and negatively regulated, whereas term isgs in its traditional sense refers to the genes induced by ifns). we then compared the ifn response of phh and hae cells ( figure ). in general, there was a robust ifn induction in both tissues, with the majority of irgs being induced in a similar manner (black dots, figure ). however, a number of genes were only significantly induced in one of the two cell types (blue and orange dots, figure ). an even higher correlation between phh and hae responses was reached when comparing the mean expression values in phh and hae cells, in the basal or ifninduced conditions (figure ). to analyze possible tissue-specific effects of ifns more indepth, we manually inspected the raw expression data for all of the genes previously found to be robustly induced by ifn (supplementary table ). among these, we observed a subset of genes that displayed various degrees of tissue specificity. we categorized genes as tissue specific by taking both the level of induction as well as absolute expression levels into account. in addition, we focused on genes that had an acceptable level of donor-to-donor variation. these genes are listed in table . only two irgs were expressed in a liver-specific manner: angptl and apol . both of these genes showed higher induction levels as well as higher absolute expression levels in the liver. aco was slightly repressed in liver cells, but strongly repressed in hae cells. ly e, wars, aim and ido were all induced to a higher level and had a higher absolute expression in hae cells. interestingly, both wars and ido modify the tryptophan biogenesis pathway. the next group, tnfsf b, zbp , ifitm and ifi , showed significant induction in both tissues but strongest induction and higher absolute expression in hae cells. hrasls , hsh d and ifi showed a more mixed phenotype. all had a higher absolute expression in the lung cells, but were also induced in the liver cells by ifnα, whereas their induction by ifnλ varied. irf , lamp and mx displayed higher fold induction in the liver cells but higher absolute expression levels in lung cells. to verify our manual selection of tissue-specific genes, we performed an additional automated differential gene expression analysis, this time looking for differences in expression between the two tissues for the same ifn treatment (and not comparing control versus ifn as in the first analysis). importantly, all our manually selected genes were detected as being significantly differently expressed between the two tissues for each of the three ifns in this second analysis (data not shown). as analyzed with the interferome database human chromosome location and transcription factor analysis tools, no obvious features such as clustering of the genes on the human genome or common promoter motif could explain this tissue specificity. expression of ifn receptors naturally, the response to a given ifn depends upon the receptor expression and therefore we mined the data for expression values of both the receptors for type i and type iii ifns. we focused our analysis on the two high-affinity receptor chains. both of these have a complex splice pattern with the potential to express multiple isoforms of the receptor chain. however, one of the strengths of the transcriptome sequencing approach is that it allows for precise determination of the splice pattern and provides a quantitative measurement of the different splice variants detected. figure shows the possible protein isoforms originating from the canonical mrnas and their observed expression levels. the observed splice variants corresponded to the canonical splice forms found in the literature (figure a) . , , we also tested the signaling ability of all three isoforms of the ifnλr receptor chain (figure b ). ifnλr - encodes the full-length protein and is signaling competent. ifnλr - , which lacks the juxtamembrane region, but retains the transmembrane domain and most of the intracellular part, is incapable of signaling in our assay. ifnλr - , encodes a protein which is truncated upstream of the transmembrane domain and has a changed amino-acid composition at the c-terminus. this isoform is assumed to be secreted in a soluble form and in vitro data suggest that it can act as a negative regulator of type iii ifn signaling. we noted an overall low expression of the ifnλr receptor chain in both tissues, with splice variant and being expressed at approximately the same level and splice variant at a slightly lower level in hae (figure c ). the ifnλr chain was reported to be induced by ifn in liver cells. we see a trend toward an increased expression of splice variant in phh but not hae following ifn treatment, but this did not reach statistical significance at the α = . level (data not shown). it is possible that the chosen time point was not optimal to observe a significant induction of the ifnlr mrna. interestingly, three similar protein isoforms have also been described for the ifnar chain, as a result of differential splicing (figure a ). the full-length isoform (also called ifnar c or ifnar - ) is required for signaling. this isoform is expressed in both tissues at levels~ -fold higher than seen for full-length ifnλr , which could explain the stronger and more robust response to ifnα (figure d ). isoform (also called ifnar b or ifnar - ) has been reported to act as a dominant negative regulator, , and its mrna is expressed at two times higher level than the full-length receptor. finally, the soluble isoform (also called ifnar a or ifnar - ) that has previously been shown to possess either agonistic or antagonistic properties in mice depending on the experimental setup tested, was not detected in both phh and hae cells. the expression level of il r was~ times higher in hae cells compared with hepatocytes ( figure e ). this increased expression could be caused by the fact that the il r chain is shared with other cytokines, which could have an important role during viral infections of the respiratory system. for example, il- , which utilizes il r for signaling, is the key cytokine responsible for the regeneration of tracheal epithelial cells during influenza virus infection. discussion and conclusion rna-seq offers a unique opportunity to survey whole transcriptomes in a high-throughput and quantitative manner. we used this powerful technique to analyze the ifn response in primary cell cultures derived from liver and lung of different donors. we induced hae and phh cultures with three different ifns, ifnα b, ifnλ and ifnλ , allowing us to perform two fundamentally different comparisons. we compared responses with different types of ifn within a given tissue, as well as responses to the same type of ifn but in different tissues. overall, this analysis revealed ifn responses as very robust with little tissue specificity. nevertheless, small but significant differences were observed between phh and hae in their response to ifn. furthermore, we observed a remarkably similar response to the three different subtypes of ifn tested. compared with a recently published microarray analysis of type i and type iii ifn responses in phh, which used similar statistical thresholds but substantially higher ifnλ concentrations than our study ( ng ml − ), our rna-seq data provide a more complete estimate of the ifn response ( versus genes significantly regulated by ifnλ ). in the hae cultures, we identified genes significantly induced by ifnα b, by ifnλ and by ifnλ . the larger number of significantly activated irgs in the hae cultures is largely an effect of the lower donor-to-donor variability in this system, which results in more genes reaching statistical significance. we compared our results with those of other published transcriptome analyses of phh and in general the rna-seq methods found more genes than microarray-based methods. , to our knowledge this study is the first to fully analyze the transcriptome of hae cultures in response to ifn. the epithelium is one of the primary target tissues for type iii ifns. in mice, control of several epithelial infections is impaired in ifnλr deficient animals. this is the case for both respiratory infections such as sars coronavirus and influenza a virus, as well as for the abbreviations: hae, human airway epithelial; ifn, interferon; phh, primary human hepatocytes. out of the global list of irgs, those that were induced in at least three conditions (ifnα, λ or λ in phh or hae cells) were manually inspected for differences in fold induction and absolute basal or induced expression in the two cell types, resulting in the selection of these genes. one of these genes was specifically repressed by ifn treatment in hae cells. the other genes were all induced but to different levels in the lung or liver cells. ifn signature induced by ifnλ in primary cells c lauber et al ifnar ifnλr isoform isoform isoform iii iv v vi vii viii ix vi iii iv v vii ii viii ix iii iv v vii ii vi ix vii i i i ii ii ii iii iii iii iv iv iv v v v vi vi intestinal rotavirus infection. , as expected, our analyses revealed a powerful ifn response in hae. ifnα responses were stronger than ifnλ responses at the doses we used, but we could not detect any fundamental difference in the induced gene sets. previous studies in cell lines using chromosome immunoprecipitation experiments showed that signaling of both type i and type iii ifn converges at the transcription factor isgf . this model is supported by the very similar gene induction profile that both we and others find in phh and hae for the different types of ifn tested. the ifnl gene has an important role during hcv infection, and by means which are not fully understood yet, only the functional variant of the gene impairs clearance of hcv, both spontaneous and treatment induced. , , despite the negative influence on hcv clearance, the gene product of ifnl , ifnλ , is highly antiviral and induces a typical ifn response. however, whereas the initial analyses of the ifnλ protein showed that it could signal through the ifnλr :il r receptor complex, the experiments did not rule out any additional signaling abilities. we therefore compared genes induced by ifnλ and ifnλ in both hae and phh. this analysis did not reveal any genes that were both specifically and significantly regulated by ifnλ . it is thus unlikely that ifnλ prevents hcv clearance by an alternative, non-ifn signaling pathway. owing to its immense absorptive area and high ventilation rate, the respiratory tract is the most common route of viral entry. we analyzed tissue-based differences in the ifn responses by manually inspecting all the genes that we had classified as significantly regulated by ifn. here we looked for differences in both the relative expression after ifn treatment (fold change) and absolute expression levels. there was surprisingly little difference in the ifn responses between the two tissue types. nevertheless, a small subset of genes exhibits a tissue dependent response to type i and type iii ifns (table ). however, this analysis establishes detailed signatures of ifn-induced changes in gene regulation of primary human lung and liver cells. this information should be useful for guiding future research to explore ifn-regulated effector mechanisms relevant for controlling viral infections in these tissues. ifnα b was obtained from sp europe/essex pharma (introna) or from sigma-aldrich (steinheim, germany; i ). his-tagged ifnλ and λ were produced and purified as previously described. , ifnα was used at u ml − and ifnλs at ng ml − . phhs were obtained from primacyt (schwerin, germany) or from the primary human hepatocyte core facility at the hannover medical school (hannover, germany). we did not observe any systematic variation between the two sources of phh. we used a low-speed centrifugation ( g) to purify the hepatocytes and the resulting purity of hepatocyte preparations is higher than %. cells were seeded in -well dishes on collagen directly after surgery. twenty-four hours post seeding, the phhs were induced for h with ifns or pbs (mock control) diluted in hepatocyte culture medium (hcm, lonza, walkersville, md, usa). the cells were then lysed in ml trizol reagent (invitrogen, karlsruhe, germany) per well. total rna extraction was performed according to the manufacturer's instructions (trizol reagent) with the addition of μg glycogen per sample to facilitate the rna precipitation. primary human tracheobronchial cells were isolated from three different donors as described elsewhere. isolated hae cells obtained from these donors were seeded in -well permeable supports (corning, cls , pore size . μm) and maintained for weeks until cultures were well differentiated as described. the hae cell cultures were induced from the basolateral side for or h with exogenous recombinant human ifns or pbs (mock) diluted in air-liquid interface medium. total rna from induced hae cultures was isolated using qiagen's rneasy kit (qiagen, hilden, germany) according to the manufacturer's protocols. transcriptome analysis using rna-seq quality and integrity of the total rna was controlled on an agilent technologies bioanalyzer (agilent technologies; waldbronn, germany). the rna sequencing library was generated from ng total rna using truseq rna sample prep kits v (illumina, san diego, ca, usa) for mrna purification followed by scriptseq v rna-seq library preparation kit (epicentre, illumina) according to manufacturer's protocols. the libraries were sequenced on illumina hiseq (illumina), using truseq sbs kit v -hs (illumina) ( cycles, single ended run) with an average of × reads per rna sample. reads were aligned to the reference genome using open source short read aligner tophat followed by cufflinks that assembles transcripts, estimates their abundances, and tests for differential expression and regulation. differential expression analysis of rna-seq data on the gene level htseq-count with parameters m = intersection-strict, s = no, and t = exon was used to produce raw read counts of expression for each gene. we used the following three state-of-the-art r packages for differential expression analysis on the gene level: deseq, edger and limma. for deseq and edger we used the raw read counts as input, whereas for limma we transformed them via its internal voom function prior to the differential expression analysis. in each analysis we used the following criteria for hit calling: a fold change of at least for induction or . for repression in gene expression, and an fdr-adjusted p-value of . or better. with the aim of reducing false-positive hits, we required a gene to be selected by at least two of the three programs. to assess the sensitivity of the applied methods to identify differentially expressed genes in the context of the observed donor-to-donor variability, we estimated necessary sample sizes for given power and fold change as well as the power for given fold change and sample size using the r package rnaseqpower. the following parameter values were estimated based on all genes with a read count per million reads mapped of at least one in the control samples for both hae and phh: (i) the average coverage of a gene (depth parameter was (hae) and (phh); (ii) the average coefficient of variation of read counts (cv parameter) was . (hae) and . (phh); and (iii) the false discovery rate α was set to . . estimated sample sizes for different fold-change values and a power of . and . are shown in supplementary table . moreover, to detect a fold change of two with a sample size of three (the number of biological replicates in our study) the estimated power is . , . and . for comparisons of hae vs phh, hae vs hae and phh vs phh, respectively. activity assay in hek cells hek cells were seeded at a density of . × cells per well in a -well plate and left to rest for h. after h, the cells were transfected with the pef plasmid encoding one of the ifnλr splice variants, firefly luciferase under the control of the mx promoter and renilla luciferase under the control of the β-actin promoter. twenty-four hours post-transfection, cells were induced in triplicates with ng ml − of ifnλ or left untreated. twenty-four hours after induction, the cells were washed with pbs and lysed with passive lysis buffer (promega, madison, wi, usa). the lysates were spun down at g for min at °c, and the cleared lysates were used for the measurement of luciferase activity (dual-luciferase reporter assay system, promega). the type i interferon receptor: structure, function, and evolution of a family business how cells respond to interferons ifnlambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il- , il- and their class ii cytokine receptor il- r human but not mouse hepatocytes respond to interferon-lambda in vivo ifn-lambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo interleukin- uses a type interferon-like program to promote antiviral responses in human hepatocytes interferons alpha and lambda inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics type iii interferon (ifn) induces a type i ifn-like response in a restricted subset of cells through signaling pathways involving both the jak-stat pathway and the mitogen-activated protein kinases dynamic expression profiling of type i and type iii interferon-stimulated hepatocytes reveals a stable hierarchy of gene expression kinetic differences in the induction of interferon stimulated genes by interferon-alpha and interleukin b are altered by infection with hepatitis c virus genetic variation in il b predicts hepatitis c treatment-induced viral clearance genetic variation in il b is associated with chronic hepatitis c and treatment failure: a genomewide association study il b is associated with response to chronic hepatitis c interferon-alpha and ribavirin therapy genome-wide association of il b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c genetic variation in il b and spontaneous clearance of hepatitis c virus a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus interferon lambda signals via the ifnlambda receptor to regulate antiviral activity against hcv and coronaviruses il b expression depends on a novel tt/-g polymorphism which improves hcv clearance prediction reduced ifnlambda activity is associated with improved hcv clearance and reduced expression of interferon-stimulated genes the ifnl / deltag variant increases susceptibility to cytomegalovirus retinitis among hivinfected patients influence of ifnl / polymorphisms on the incidence of cytomegalovirus infection after solid-organ transplantation loss of function of the new interferon ifn-lambda may confer protection from hepatitis c explanted diseased livers -a possible source of metabolic competent primary human hepatocytes isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism interferome v . : an updated database of annotated interferon-regulated genes cloning of a new type ii cytokine receptor activating signal transducer and activator of transcription (stat) , stat and stat despite ifnlambda receptor expression, blood immune cells, but not keratinocytes or melanocytes, have an impaired response to type iii interferons: implications for therapeutic applications of these cytokines ifnlambda receptor expression is induced in chronic hepatitis c and correlates with the ifn-lambda genotype and with nonresponsiveness to ifn-alpha therapies mutant u a cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster the short form of the interferon alpha/beta receptor chain acts as a dominant negative for type i interferon action the relative endogenous expression levels of the ifnar isoforms influence the cytostatic and pro-apoptotic effect of ifnalpha on pleomorphic sarcoma cells the soluble murine type i interferon receptor ifnar- is present in serum, is independently regulated, and has both agonistic and antagonistic properties il- from conventional nk cells is epithelial regenerative and inflammation protective during influenza infection rna-seq: a revolutionary tool for transcriptomics lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections ifnlambda determines the intestinal epithelial antiviral host defense association of the ifnl -deltag allele with impaired spontaneous clearance of hepatitis c virus human interferon-lambda is a potent member of the type iii interferon family tophat : accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions transcript assembly and quantification by rna-seq reveals unannotated transcripts and isoform switching during cell differentiation htseq &# ; a python framework to work with highthroughput sequencing data differential expression analysis for sequence count data edger: a bioconductor package for differential expression analysis of digital gene expression data voom: precision weights unlock linear model analysis tools for rna-seq read counts calculating sample size estimates for rna sequencing data rapid and simple detection of ifn-neutralizing antibodies in chronic hepatitis c non-responsive to ifn-alpha we are grateful to sergei kotenko for the gift of the pef -ifnλr plasmid. rh is supported by the danish cancer society (grant r -a ) and the danish council for independent research, medical research (grant - ). tp is supported by deutsche forschungsgemeinschaft (dfg) sonderforschungsbereiche (sfb) , (project a ), deutsches zentrum für infektionsforschung and grant from the initiative and networking fund of the helmholtz association (so- ). fwrv is supported by deutsches zentrum für infektionsforschung. v.t. and r.d. are supported by swiss national science foundation, (project nr. ). the authors declare no conflict of interest. key: cord- - r fhzlm authors: tomosada, yohsuke; chiba, eriko; zelaya, hortensia; takahashi, takuya; tsukida, kohichiro; kitazawa, haruki; alvarez, susana; villena, julio title: nasally administered lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection date: - - journal: bmc immunol doi: . / - - - sha: doc_id: cord_uid: r fhzlm background: some studies have shown that nasally administered immunobiotics had the potential to improve the outcome of influenza virus infection. however, the capacity of immunobiotics to improve protection against respiratory syncytial virus (rsv) infection was not investigated before. objective: the aims of this study were: a) to evaluate whether the nasal administration of lactobacillus rhamnosus crl (lr ) and l. rhamnosus crl (lr ) are able to improve respiratory antiviral defenses and beneficially modulate the immune response triggered by tlr /rig-i activation; b) to investigate whether viability of lr or lr is indispensable to modulate respiratory immunity and; c) to evaluate the capacity of lr and lr to improve the resistance of infant mice against rsv infection. results: nasally administered lr and lr differentially modulated the tlr /rig-i-triggered antiviral respiratory immune response. lr administration significantly modulated the production of ifn-α, ifn-β and il- in the response to poly(i:c) challenge, while nasal priming with lr was more effective to improve levels of ifn-γ and il- . both viable lr and lr strains increased the resistance of infant mice to rsv infection while only heat-killed lr showed a protective effect similar to those observed with viable strains. conclusions: the present work demonstrated that nasal administration of immunobiotics is able to beneficially modulate the immune response triggered by tlr /rig-i activation in the respiratory tract and to increase the resistance of mice to the challenge with rsv. comparative studies using two lactobacillus rhamnosus strains of the same origin and with similar technological properties showed that each strain has an specific immunoregulatory effect in the respiratory tract and that they differentially modulate the immune response after poly(i:c) or rsv challenges, conferring different degree of protection and using distinct immune mechanisms. we also demonstrated in this work that it is possible to beneficially modulate the respiratory defenses against rsv by using heat-killed immunobiotics. acute lower respiratory tract infections are a persistent public health problem. despite the remarkable advances in antibiotic therapies, diagnostic tools, prevention campaigns and intensive care, respiratory infections are still among the primary causes of death worldwide, and there have been no significant changes in mortality in the last decades [ ] . childhood acute community-acquired pneumonia is one of the leading causes of morbidity and mortality in developing countries. in children who have not received prior antibiotic therapy, the main bacterial causes of clinical pneumonia in developing countries are streptococcus pneumoniae and haemophilus influenzae type b, and the main viral cause is the respiratory syncytial virus [ ] . respiratory syncytial virus (rsv), a pneumovirus in the family paramyxoviridae, infects nearly all children within the first years of life. primary rsv infections can cause severe bronchiolitis and pneumonia, which are associated with significantly increased risk of developing wheeze during childhood that lasts until teenage years. symptomatic reinfections occur in every age group, but the frequency and severity of symptoms are highest in children below years of age [ ] . as described for several respiratory viruses, such as pandemic influenza virus strains and the human coronavirus that causes sars, it is believed that the immune response plays a critical role in the outcome of rsv-induced bronchiolitis and pneumonia. acute rsv infection is able to induce an exacerbated disease due to immune-mediated pulmonary injury resulting in severe morbidity and mortality [ ] . therefore, identifying novel approaches to modulate virus-induced immunopathology would be beneficial in treating acute rsv infections. several studies have demonstrated that certain lactic acid bacteria (lab) strains can exert their beneficial effect on the host through their immunomudulatory activity. in this regard, some studies have centered on whether immunoregulatory probiotic lab (immunobiotics) might sufficiently stimulate the common mucosal immune system to provide protection in other mucosal sites distant from the gut [ ] . the studies of our laboratory demonstrated that some orally administered lab are able to increase s. pneumoniae clearance rates in lung and blood, improve survival of infected mice and reduce lung injuries [ ] [ ] [ ] [ ] . moreover, we found that the effects of lab treatments were related to an up-regulation of both respiratory innate and adaptive immune responses. in addition, considering that the nasal route can induce systemic and respiratory immune responses superior to those obtained using oral stimulation [ ] , we also focused on the ability of nasal stimulation with immunobiotics to improve respiratory immune responses. our studies showed that nasally administered lab are capable of modulating lung immunity and increase resistance against s. pneumoniae in both immunocompetent and immunocompromised mice and that in many cases, nasal priming is more effective than oral administration to beneficially modulate the respiratory immunity [ , ] . recently, our laboratory studied the capacity of immunobiotics to improve respiratory antiviral immune response. to mimic the pro-inflammatory and physiopathological consequences of rna viral infections in the lung such as those induced by rsv infection, we used an experimental model of lung inflammation based on the administration of the artificial toll-like receptor (tlr ) and retinoic acid-inducible gene i (rig-i) ligand and dsrna analog poly(i:c) [ ] . in vivo studies using mice demonstrated that nasally administered poly(i:c) results in tlr -and cxcr -dependent neutrophilic pulmonary inflammation, bronchiolar epithelial hypertrophy, interstitial edema and altered lung function [ , ] . these changes are accompanied by elevated levels of interleukin (il)- , rantes, monocyte chemotactic protein (mip)- , and type i interferons (ifns) in broncho-alveolar lavages (bal) [ ] . when we evaluated the effect of two lactobacillus strains, lactobacillus rhamnosus crl (lr ) and l. rhamnosus crl (lr ) in this mice model, we found that orally administered lr beneficially regulate the balance between pro-inflammatory mediators and il- in lung of poly(i:c)-challenged mice, allowing an effective control of the inflammatory response and avoiding tissue damage [ ] . moreover, our studies demonstrated that lr is able to increase the number of cd + cd + ifn-γ + t cells in the gut, induce the mobilization of these cells into the respiratory mucosa and improve the local production of ifn-γ and the activity of lung antigen presenting cells (apcs) [ ] . our results suggested that lr is a potent inducer of antiviral cytokines and may be useful as a prophylactic agent to control respiratory virus infections. however, whether the nasal priming with lr is more effective than oral administration to beneficially modulate the respiratory immune response triggered by poly(i:c) challenge has not been evaluated before. moreover, further studies using real challenges with respiratory viruses such as rsv are needed in order to conclusively demonstrate the protective effect of lr . considering this background, the aims of this study were: a) to investigate whether the nasal administration of lr or lr are able to improve respiratory antiviral defenses and beneficially modulate the immune response triggered by tlr /rig-i activation; b) to evaluate whether viability of lr or lr is indispensable to modulate respiratory immunity considering that it was reported that heat-killed lactobacilli strains are able to improve lung defenses [ , , ] and; c) to conclusively demonstrate the protective effect of lr and lr by evaluating their capacity to improve the resistance of infant mice against rsv challenge. lactobacillus rhamnosus crl (lr ) and crl (lr ) were obtained from the cerela culture collection (chacabuco , san miguel de tucumán, argentina). both strains were selected because their previously reported immunomodulatory capacities [ , ] . the culture was kept freeze-dried and then rehydrated using the following medium: peptone . g, tryptone . g, meat extract . g, distilled water l, ph . it was cultured for h at °c (final log phase) in man-rogosa-sharpe broth (mrs, oxoid). the bacteria were harvested by centrifugation at g for min, washed three times with sterile . mol/l phosphate buffer saline (pbs), ph . , and resuspended in sterile % non-fat milk. non-viable lr and lr , designated as hklr and hklr respectively, were obtained as follows: bacteria were killed by tyndallization in a water bath at °c for min and the lack of bacterial growth was confirmed using mrs agar plates [ ] . female -week-old balb/c mice were obtained from the closed colony kept at tohoku university. they were housed in plastic cages at room temperature. mice were housed individually during the experiments and the assays for each parameter studied were performed in - mice per group for each time point. lr , lr , hklr or hklr were nasally administered to different groups of mice for consecutive days at a dose of cells/mouse/ day in μl of pbs. the treated groups and the untreated control group were fed a conventional balanced diet ad libitum. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the guidelines for animal experimentation of tohoku university, sendai, japan. the present study was approved by the institution animal care and use committee of tohoku university and all efforts were made to minimize suffering [ ] [ ] [ ] [ ] . administration of the viral pathogen molecular pattern poly(i:c) was performed on day , after the two days treatments with lactobacilli, as described previously [ ] . mice were lightly anesthetized and μl of pbs, containing μg poly(i:c) (equivalent to mg/kg body weight), was administered dropwise, via the nares. control animals received μl of pbs. mice received three doses of poly(i:c) or pbs with hs rest period between each administration. blood samples were obtained through cardiac puncture at the end of each treatment and collected in heparinized tubes. bal samples were obtained as described previously [ ] . briefly, the trachea was exposed and intubated with a catheter, and sequential bronchoalveolar lavages were performed in each mouse by injecting sterile pbs; the recovered fluid was centrifuged for min at × g; the pellet was used to make smears that were stained for cell counts; and the fluid was frozen at − °c for subsequent cytokines analyses. tumour necrosis factor (tnf)-α, ifn-α, ifn-β, ifn-γ, il- and il- concentrations in serum and bal were measured with commercially available enzyme-linked immunosorbent assay (elisa) technique kits following the manufacturer's recommendations (r&d systems, mn, usa) [ ] . protein and albumin content, a measure to quantitate increased permeability of the bronchoalveolar-capillarity barrier, and lactate dehydrogenase (ldh) activity, an indicator of general cytotoxicity, were determined in the acellular bal fluid [ ] . protein content was measured by the bicinchoninic acid assay (bca) protein assay (pierce biotechnology inc., rockford, il). albumin content was determined colorimetrically based on albumin binding to bromcresol green using an albumin diagnostic kit (wiener lab, buenos aires, argentina). ldh activity, expressed as units per liter of bal fluid, was determined by measuring the formation of the reduced form of nicotinamide adenine dinucleotide (nad) using the wiener reagents and procedures (wiener lab). lung wet:dry weight ratio was measured as previously described [ ] . wet:dry weight ratio was calculated as an index of intrapulmonary fluid accumulation, without correction for blood content. single lung cells from mice were prepared using the previously described method [ ] . mice were anaesthetized with diethyl ether and killed the next day by exsanguination. lungs were removed, finely minced and incubated for min with u of collagenase (yakult honsha co., tokyo, japan) in ml of rpmi medium (sigma, tokyo, japan). to dissociate the tissue into single cells, collagenase-treated minced lungs were gently tapped into a plastic dish. after removal of debris, erythrocytes were depleted by hypotonic lysis. the cells were washed with rpmi medium supplemented with u/ml of penicillin and mg/ml of streptomycin and then resuspended in a medium supplemented with % heatinactivated foetal calf serum (fcs). cells were counted using trypan blue exclusion and then resuspended at an appropriate concentration of × cells/ml. lung cell suspensions were pre-incubated with antimouse cd /cd monoclonal antibody (fc block) for min at °c. cells were incubated in the antibody mixes for min at °c and washed with facs buffer. the following antibodies from bd pharmingen were used: anti-mouse cd -fitc, anti-mouse cd -pe, antimouse cd -pe, anti-mouse ifn-γ-apc, anti-mouse cd b-fitc, anti-mouse cd c-pe, anti-mouse ifn-γ -pe, anti-mouse mhc-ii-pe, anti-mouse il- -pe and anti-mouse cd -biotin. following incubation with biotinylated primary antibodies, the labeling was revealed using streptavidin-percp. in all cases, cells were then acquired on a bd facscalibur™ flow cytometer (bd biosciences) and data were analyzed with flowjo software (treestar). the total number of cells in each population was determined by multiplying the percentages of subsets within a series of marker negative or positive gates by the total cell number determined for each tissue [ , ] . human rsv strain a was grown in vero cells as described by murawski et al. [ ] . briefly, vero cells were infected with rsv at a multiplicity of infection (moi) of in ml of dulbecco's modified eagle's medium (dmem). cells were infected for . h at °c and % co . after infection, ml of dmem with % fetal bovine serum (sigma, tokyo, japan), . % penicillinstreptomycin (pen/strep) (sigma, tokyo, japan), and . % ciprofloxacin (bayer) was added to the flask. flasks were incubated until extensive syncytium formation was observed. then, cells were scraped from the flask and sonicated three times, s per time, at w on ice. cell debris was removed by centrifugation at g for min at °c. virus supernatant was sucrose density gradient purified and stored in % sucrose at − °c. uninfected flasks were treated identically to generate vero cell lysate control. for in vivo infection, mice were lightly anesthetized with isoflurane and intranasally challenged with . × pfu of rsv strain a . lung tissue was removed without bal harvest and stored in % sucrose for plaque assay. lungs were homogenized using a pellet pestle and centrifuged at , × g for min at °c to clarify supernatant. twenty-four-well tissue culture plates were seeded with . × vero cells/well in dmem containing % fbs, . % pen/strep, and . % ciprofloxacin. cells were incubated overnight at °c and % co . medium was removed from confluent monolayers, and serial dilutions of lung tissue-clarified supernatants were absorbed to monolayers. all samples were run in triplicate wells. plates were incubated at °c and % co for . h for optimum infection. after incubation, supernatant was removed, and ml of fresh dmem medium containing containing % fbs, . % pen/strep, and . % ciprofloxacin was overlaid on monolayers. when extensive syncytia developed, the overlay was removed and monolayers were fixed with ml of ice-cold acetone:methanol ( : ). primary rsv anti-f (clones - a; chemicon) and anti-g (mouse monoclonal [ c ( b )] to rsv glycoprotein, abcam) antibodies were added to wells for h, followed by secondary horseradish peroxidase anti-mouse immunoglobulin antibody (antimouse igg, hrp-linked antibody # , cell signaling tehcnology) for h. plates washed twice with pbs containing . % tween (sigma) after each antibody incubation step. individual plaques were developed using a dab substrate kit (ab , abcam) following manufacture's specifications. results for immunoplaque assay were expressed as log pfu/g of lung. experiments were performed in triplicate and results were expressed as mean ± standard deviation (sd). after verification of the normal distribution of data, -way anova was used. tukey's test (for pairwise comparisons of the means) was used to test for differences between the groups. differences were considered significant at p< . . nasally administered l. rhamnosus crl and l. rhamnosus crl differentially modulate respiratory immunity in order to evaluate the changes induced by nasally administered lab in the respiratory immune system we determined the levels of different cytokines in bal ( figure a ). the four nasal treatments used in this study lr , hklr , lr and hklr increased the levels of il- , ifn-α and ifn-β in bal, however concentrations of these cytokines were significantly higher in lr -and hklr -treated mice than in mice receiving lr or hklr ( figure a ). all the treatments were also able to increase bal ifn-γ, being both viable and heat-killed l. rhamnosus crl more efficient to improve the levels of this cytokine than l. rhamnosus crl ( figure a ). bal tnf-α and il- concentrations were increased by the immunobiotic nasal treatments. we observed that viable cells were more efficient to upregulate tnf-α than heat-killed strains while no differences were found in il- levels when comparing viable and heat-killed lactobacilli ( figure a ). when we evaluated the levels of il- , ifn-α, ifn-β, ifn-γ, tnf-α and il- in serum we found that lactobacilli treatments induced similar changes than those observed in the respiratory tract ( figure b ). il- , ifn-α and ifn-β were more efficiently increased with l. rhamnosus crl treatments while the highest levels of serum ifn-γ were observed in lr and hklr groups ( figure b ). serum tnf-α and il- concentrations were also enhanced by the immunobiotic nasal treatments, being viable and heat-killed lactobacilli equally effective to improve both cytokines whit the exception of hklr that induced significantly lower levels of il- when compared with the other treatments ( figure b) . we also evaluated the changes induced by nasally administered lactobacilli in lung immune cells using flow cytometry. slightly increases of lung cd + cd + t cells were observed in lactobacilli-treated mice, however when we studied the cells able to produce ifn-γ within this population, mice receiving lr , hklr and lr showed significant differences when compared to control mice ( figure ). nasally administered lr and hklr a b figure effect of lactobacilli on systemic and respiratory immunity. effect of viable (lr ) or heat-killed (hklr ) lactobacillus rhamnosus crl and viable (lr ) or heat-killed (hklr ) l. rhamnosus crl nasal administration on the tumor necrosis factor (tnf)-α, interferon (ifn)-α, ifn-β, ifn-γ, interleukin (il)- , and il- concentrations in broncho-alveolar lavages (a) and serum (b). lr , lr , hklr or hklr were nasally administered to different groups of mice for consecutive days at a dose of cells/mouse/day and the levels of bal and serum cytokines were studied on day . the results represent data from three independent experiments. different letters indicate significant differences (p < . ). increased the number of cd + cd + ifn-γ + t cells in lungs being lr more efficient than hklr to increase this cell population ( figure ). only lr enhanced the number of cd + cd + il- + t cells in lungs ( figure ). no modifications were observed in the number of cd + cd + and cd + cd + ifn-γ + t cells in lactobacilli-treated mice ( figure ). two populations of myeloid dcs can be defined in lungs using cd c, cd b, cd and mhc-ii antibodies as described previously [ , ] : mhc-ii + cd c + cd b low cd + and mhc-ii + cd c + cd b high cd cells. therefore, we next aimed to evaluate the effect of nasally administered lactobacilli on these populations of dcs from lungs. lr and hklr significantly increased the number of both lung cd c + cd b low cd + and cd c + cd b high cd -dcs. in addition, lr and hklr enhanced the number of lung cd c + cd b low cd + dcs while no quantitative changes were detected in cd c + cd b high cd -dcs populations in lungs of lr -and hklr -treated mice ( figure ). in addition, the expression of mhc-ii in both dcs population was significantly improved with all the treatments, however l. rhamnosus crl was more efficient than l. rhamnosus crl to upregulate the expression of mhc-ii in lung dcs ( figure ). we next aimed to evaluate the effect of nasally administered lactobacilli on the immune response triggered by nasal administration of the viral pathogen-associated molecular pattern poly(i:c). our previous work demonstrated that the nasal challenge of mice with poly(i:c) significantly alters lungs function and induce lung injuries [ ] . in this experimental model an altered wet:dry weight ratio can be observed after poly(i:c) challenge ( figure ). moreover, significantly increased levels of ldh activity as well as protein and albumin concentrations can be found figure effect of lactobacilli on respiratory immune cells populations. effect of viable (lr ) or heat-killed (hklr ) lactobacillus rhamnosus crl and viable (lr ) or heat-killed (hklr ) l. rhamnosus crl nasal administration on cd +cd +ifn-γ+, cd +cd +ifn-γ+ and cd +cd +il- + t cells and cd c+cd b low cd + and cd c+cd b high cd -dendritic cells from lung. lr , lr , hklr or hklr were nasally administered to different groups of mice for consecutive days at a dose of cells/mouse/day and lung immune cells were studied on day . the results represent data from three independent experiments. different letters indicate significant differences (p < . ). in bal samples of challenged mice indicating that poly(i:c) produces local cellular damage and impairment of the alveolar-capillary barrier ( figure ). we observed that nasally administered lactobacilli prior to poly(i:c) challenge significantly reduced wet:dry weight ratio and bal ldh ( figure ). in addition, all the treatments were able to significantly reduce bal protein and albumin concentrations however l. rhamnosus crl was more efficient than the crl strain to reduce the impairment of the alveolar-capillary barrier ( figure ). the pulmonary immune response induced by the nasal challenge with poly(i:c) and the effect of nasally administered lactobacilli in that response were next evaluated. we have previously study the levels and kinetics of ifnα, ifn-β, ifn-γ, il- , il- , tnf-α, il- β, il- , mcp- , il- and tgf-β in bal after poly(i:c) challenge and the effect of orally administered immunobiotics in that response [ ] . the most significant changes induced by oral treatment with immunobiotics were found in the levels of ifn-α, ifn-β, ifn-γ, il- , tnf-α and il- , therefore these cytokines were studied in this work. nasal administration of poly(i:c) significantly increased respiratory levels of the pro-inflammatory mediators ifn-α, ifn-β, il- and tnf-α ( figure ) as previously reported [ ] . all lactobacilli treatments significantly increased the levels of bal ifn-α, ifn-β and tnf-α however, lr and hklr were more efficient to enhance the concentration of these cytokines than lr or hklr ( figure ). in addition, il- was not modified in lr and hklr groups while the levels of this cytokine were lower in l. rhamnosus crl -treated mice when compared to controls ( figure ). poly(i:c) challenge also induced an increase in the respiratory levels of il- and ifn-γ and the levels of both cytokines were significantly higher in lactobacilli-treated mice, being lr and hklr more efficient than lr and hklr to achieve that effects (figure ) . the nasal challenge with poly(i:c) also increased cytokines levels in serum as previously reported [ ] . moreover, the effect of lr , hklr , lr and hklr treatments on the production of serum ifn-α, ifn-β, ifn-γ, il- , tnf-α and il- was similar to that found in bal (data not shown). we also studied the changes in lung immune cells induced by nasally administered lactobacilli in poly(i:c)challenged mice ( figure ). poly(i:c) administration increased cd + cd + ifn-γ + and cd + cd + ifn-γ + t cells as we described previously [ ] . in addition, in this work we also observed an increase in cd + cd + il- + t cells after the challenge with poly(i:c) ( figure ). our results showed that nasally administered lactobacilli were able to increase both cd + cd + ifn-γ + and cd + cd + il- + t cells in lungs, however the levels of these cell populations in lr -and hklr -treated mice were significantly higher than those observed in lr and hklr groups ( figure ). no differences were observed between lactobacilli-treated mice and controls when evaluating cd + cd + ifn-γ + t cells ( figure ). poly(i:c) challenge also increased the number of pulmonary cd b high cd -mhc-ii + and cd b low cd + mhc-ii + dcs when compared to basal levels in all the experimental groups ( figure ). nasal administration of both l. rhamnosus crl and l. rhamnosus crl significantly increased the numbers of cd b low cd + mhc-ii + dcs cells in lungs when compared to controls while only lr and hklr -treated mice showed improved levels of pulmonary cd b high cd -mhc-ii + dcs ( figure ). we next addressed the question of whether changes observed in respiratory immune system caused by the intervention with immunobiotics affected the outcome of rsv infection in mice. therefore, infant mice were nasally treated with lr , hklr , lr or hklr and then challenged with pfu of rsv. viral loads in lungs of infected mice were followed for five days after the challenge ( figure ). rsv was detected in lungs of all the experimental groups during the five days postinfection and all groups showed a peak of virus counts on day after the challenge. however, lactobacillitreated mice showed significantly lower lung viral loads when compared to control mice. lr and hklr treatments were equally effective to reduce rsv replication in lungs while in the case of l. rhamnonus crl viable bacteria was more effective than heat-killed cells to improve protection against the respiratory viral infection ( figure ). in addition, we observed significantly differences in the body weight of infected mice when comparing lactobacilli-treated mice and controls ( figure ). lr , hklr and lr significantly improved the body weight during rsv infection while hklr -tretated mice showed no differences when compared to controls ( figure ). we also evaluated the markers of lung tissue damage in rsv-infected mice. as showed in figure , challenge with rsv significantly increase lung wet:dry weight, bal protein concentrations and ldh activity. all these markers of lung tissue damage were significantly higher in rsv-challenged control mice than in those previously treated with lr , hklr or lr (figure ) . on the contrary, lung wet:dry weight, bal protein concentrations and bal ldh activity in hklr -treated mice were not different from controls ( figure ) . finally, we addressed whether the improvement in the resistance against rsv induced by lactobacilli treatments was related to a differential modulation of cytokines production during infection ( figure ). therefore, we evaluate the levels of bal and serum il- , ifn-β and tnf-α on day post-infection and the concentrations of ifn-γ and il- on day after challenge. we observed that the challenge with rsv significantly increased the levels of the cytokines studied in all experimental groups. we also detected that lactobacilli-treated mice showed significantly higher levels of bal il- , ifn-β and tnf-α than controls, being lr and hklr treatments more efficient than lr or hklr to improve the production of these factors (figure ). in addition, levels of ifn-γ and il- were significantly improved by lr , hklr and lr treatments while nasal administration of hklr increased ifn-γ but not il- production in the respiratory tract. moreover, lr and hklr were more effective than lr to increase bal ifn-γ and il- concentrations in response to rsv challenge ( figure ). the effect of lr , hklr , lr and hklr treatments on the production of serum tnf-α, ifn-β, il- , ifn-γ and il- was similar to that found in bal (data not shown). in the present work we studied the effect of nasally administered immunobiotic lactobacilli on the respiratory antiviral immune response and evaluated their capacity to improve protection of infant mice against rsv infection. three important conclusions can be inferred from the results presented in this study: a) nasally administered lr and lr differentially modulate the tlr / rig-i-triggered antiviral respiratory immune response; b) nasal priming with lr or lr strains increase the resistance of infant mice to rsv infection and; c) the viability of the immunobiotic strains is not a necessary condition to achieve the immunoregulatory protective effect. a) nasally administered lr and lr differentially modulate the tlr /rig-i-triggered antiviral respiratory immune response. we have previously demonstrated that nasal administration of three once-daily doses of poly(i:c) resulted in a marked impairment of lung function that was accompanied by inflammatory cell recruitment into the airways and the production of pro-inflammatory mediators [ ] . accordingly, in the present work we observed increased ldh activity and albumin concentration in bal as well as increased levels of type i ifns, tnf-α and il- , in the respiratory tract of poly(i:c)-challenged mice. we also showed that this immune response can be modulated with the preventive nasal administration of lr or lr , demonstrating in addition that each strain has a different immunoregulatory effect. while lr administration had a significant effect on the production of ifn-α, ifn-β and il- in the response to poly(i:c) challenge, nasal priming with lr was more effective to improve levels of ifn-γ and il- . it was demonstrated that poly(i:c) elicited the secretion of type i ifns, tnf-α and il- and other cytokines in respiratory epithelial cells [ ] , therefore a likely source of these cytokines following poly(i:c) administration may be the airway epithelium. then, lr administration would have a significant effect on respiratory epithelial cells. taking into consideration that ifn-α and ifn-β up-regulate several genes involved in viral defense but also genes of major importance for the development of a strong th response, it can be speculated that lr may play an important role in the improvement of innate and specific immune responses against respiratory virus through the stimulation of antiviral defenses in epithelial cells (figure ). in addition, we have previously demonstrated that nasal administration of poly(i:c) activates respiratory mhc-ii + cd c + cd b low cd + (cd + dcs) and mhc-ii + cd c + cd b high cd -(cd b high dcs) cells and increases cd + cd + ifn-γ + and cd + cd + ifn-γ + t cells in lungs, indicating the generation of a th response [ ] . the result presented here shows that nasal administration of lr has the capacity to improve th response since higher levels of bal ifn-γ and lung cd + cd + ifn-γ + t cells were found in lr -treated mice. moreover, we showed that lr administration significantly activated cd + dcs, an affect that was not achieved by lr . considering that recent studies suggested that lung cd + dcs are more potent at eliciting th and th responses than cd b high dcs [ ] , we can speculate that lr is more efficient than lr to stimulate cd + dcs and improve th response in the respiratory tract ( figure ). lr , lr , hklr or hklr were nasally administered to different groups of mice for consecutive days at a dose of cells/mouse/day. after lactobacilli treatment, mice were nasally challenged with rsv and bal cytokines were studied (tnf-α, ifn-β and il- ) or (ifn-γ and il- ) hours after the challenge. the results represent data from three independent experiments. different letters indicate significant differences (p < . ). nasal treatment with lactobacilli significantly reduced lung injuries caused by poly(i:c) administration. we previously suggested that il- would be valuable for attenuating inflammatory damage and pathophysiological alterations in lungs challenged with the viral pathogenassociated molecular pattern poly(:ic) [ ] . we demonstrated here that both lr and lr significantly improved the production of il- in response to poly(:ic), however lr was more efficient than lr to upregulate the levels of this cytokine in the respiratory tract. moreover, the lung tissue injury markers used in this study were significantly lower in lr -treated mice than in those receiving the lr strain. therefore, we confirmed that there is a direct connection between the improvement of il- levels induced by immunobiotics and the protection against lung injury after respiratory poly(i:c) challenge. moreover, in this work we demonstrated that cd + cd + il- + t cells would be the source of the il- produced after poly(i:c) challenge and that this immune cell population would be quantitatively and functionally modulated by lr since increased levels of cd + cd + il- + t cells were found in lungs of lr -treated mice when compared with controls and those receiving lr (figure ). recently, figure proposed mechanism for the improvement of antiviral immunity and resistance against respiratory syncytial virus (rsv) infection induced by nasally administered lactobacilli. weiss et al. [ ] demonstrated that cd + t cells produce the majority of il- in vivo during an acute rsv infection and that this cell population is involved in the protective effect against lung tissue damage. therefore, the increase in the numbers of cd + il- + t cells induced by nasal treatment with lactobacilli could have an important role in the protection against rsv infection. similar to the changes observed in intestinal immunity after oral administration of lr or lr [ ] , we demonstrated in this work that nasal priming with both lactobacilli has the ability to improve antiviral immunity but using different mechanisms ( figure ). moreover, considering that activating host immune responses during rsv infection is dependent on complex signaling events initiated in part by prrs such as tlr or rig-i and that these coordinated signaling events promote the production of cytokines, chemokines, ifn-α, ifn-β and ifn-γ in the lung that are crucial for the virus clearance, we speculated that lr or lr nasal treatments would beneficially modulate the immune response against rsv and improve resistance of mice against this viral respiratory infection. b) nasal priming with lr or lr strains increase resistance of infant mice to rsv challenge. in the last years, some lines of evidence showed that nasal administration of immunobiotics is able to increase resistance against respiratory viral infections [ ] . it was reported that intranasal priming with l. rhamnosus gg to balb⁄ c mice significantly reduced the frequency of accumulated symptoms and induced a higher survival rate than control mice after challenge with influenza virus h n [ ] . authors demonstrated that l. rhamnosus gg significantly increased lung nk cell activation and expression of tnf-α, il- β and mcp- enhancing respiratory cell-mediated immune responses. in addition, it was reported recently that nasal priming with lactobacilli is highly effective at suppressing virus-induced inflammation in a pneumonia virus mouse model [ ] . nasal priming with lactobacilli resulted in marked suppression of ifn-inducible protein- (cxcl ), mcp- (ccl ), neutrophil-activating protein- (cxcl ), mip- γ (ccl ), tnf, and eotaxin- (ccl ) in response to pneumovirus infection and significantly increased the resistance against the lethal disease. in this work we extend these findings by demonstrating that nasally administered immunobiotics are able to increase protection against rsv infection in infant mice. we observed that challenge of three weeks old balb/c mice with rsv significantly altered lung function, induced tissue injury and triggered inflammatory response. this is in line with previous work reporting that rsv intranasal infection led to significant clinical characteristics in female balb/c mice [ ] . authors described ruffled fur and ataxia that occurred early after hour post-rsv infection and continued for days [ ] . natural human rsv infection in children and experimental rsv inoculation in mice result in prominent local secretion of proinflammatory cytokines, such as tnf-α, il- , il- , mip- , rantes, and mcp- as well as type i ifns [ ] . it has been shown that type i ifns, il- and tnfα contribute to clearance of the virus during the early stages of rsv infection however, continued production of these pro-inflammatory mediators exacerbates illness and tissue injuries during the late stages of rsv infection [ ] . interestingly, it was found that il- deficiency during rsv challenge did not affect viral load, but led to markedly increased disease severity with enhanced weight loss, delayed recovery and a greater influx of inflammatory cells into the lung and airways and enhanced release of inflammatory mediators [ ] . therefore, during acute rsv infection, it is imperative that the host's inflammatory response is tightly regulated, enabling virus elimination but limiting the detrimental effects of inflammation on the lung tissue. then, an adequate balance of pro-inflammatory and antiinflammatory factors is essential for a safe and effective antiviral immune response [ ] . moreover, considering that it was reported that no changes in peak viral titers or viral clearance were observed between il- ko or anti-il- r mab-treated mice compared with their controls [ ] , preventive or therapeutic approaches aimed at increasing il- production may offer a means to decrease rsv-induced immunopathology without affecting viral clearance. we demonstrated in this work that nasal administration of lr or lr improved the production of proinflammatory mediators in response to rsv challenge and also the production of il- , which would allow an effective immunological clearance of the virus without affecting lung tissue. similarly to our results using poly (i:c) challenge, we observed that lr was the strain with the highest capacity to improve levels of il- and was more effective than lr to enhance virus clearance and to protect lungs against the inflammatory damage. then, our results also support the idea that modulation of respiratory il- during rsv infection is an effective way to improve the outcome of viral disease. moreover, we demonstrated here that nasally administered immunobiotics are an interesting alternative to achieve that immunoprotective effect. following rsv infection, there is an initial influx of nk cells to the site of infection that produce ifn-γ and are cytotoxic to virus-infected cells. this is followed by recruitment of helper cd + and cytotoxic cd + lymphocytes to the site of infection. ifn-γ enhances the differentiation of cd + lymphocytes and influences the differentiation of cd + lymphocytes that contribute to the generation and amplification of the humoral and cellular immune responses. while the rsv-specific t cell response plays a major role in viral clearance and the clinical outcome of infection, both th -biased cd + and cd + t cells have been implicated in immunopathogenesis [ ] . in general, a th immune response is favored during rsv infection, especially in younger hosts. rsv-induced pulmonary inflammation in mice was previously found to cause a shift from th to th cell inflammation. rsv uses multiple mechanisms to induce a th cell response in the host, including rsv g protein-mediated effects [ ] , increasing il- production from basophils [ ] and induction of alternatively activated macrophages [ ] . moreover, the excessive mucus production, airway plugging, wheezing, and long-lasting effects on lung function that are common manifestations of rsv disease have some similarity with asthma, which involves a th -bias [ ] . therefore, strategies aimed to improve th during rsv infection would beneficially modulate the outcome of the infections especially in younger hosts. in this work, we showed that nasally administered immunobiotics were able to improve respiratory th response since significantly higher levels of ifn-γ were found in the respiratory tract of lactobacilli-treated mice. then, modulation of respiratory immunity potentiated by the immunobiotic strains might contribute to an improved th response and thereby favor protective immunity against viral infections such as rsv. c) viability of the immunobiotics strains is not a necessary condition to achieve the immunoregulatory protective effect. few studies have demonstrated that the nasal administration of heat-killed immunobiotics is able to improve resistance against respiratory pathogens [ , , ] . in this regard, earlier studies by hori et al. [ ] showed that the nasal administration of heat-killed l. casei shirota stimulated cellular immunity in the respiratory tract and significantly increased the resistance of adult balb/c mice to influenza virus infection. the authors investigated the production of various cytokines by mediastinal lymphoid node cells in mice receiving l. casei shirota intranasally and found that the shirota strain strongly induced production of il- in these cells, which is an important cytokine for cytotoxic t cells and nk cells stimulation and enhancement of th cytokines. in addition, both ifn-γ and tnf-α levels were improved in mediastinal lymphoid node cell cultures from mice administered l. casei shirota intranasally, after influenza virus challenge [ ] . later it was reported that intranasal administration of heat-killed l. pentosus s-pt strongly enhanced th immunity, ifn-α production and nk activity in the respiratory immune system and protected against influenza virus infection [ ] . in addition, as mentioned above, it was demonstrated that priming of the respiratory mucosa with lactobacilli results in full protection from the otherwise lethal severe pneumovirus infection and that protection is observed in response to both live and heat-killed l. plantarum and l. reuteri [ ] . that work demonstrated that nasal treatment with heat-killed immunobiotics resulted in diminished virus recovery at multiple time points and prominent suppression in the production of virus-induced proinflammatory mediators. the results of our work are in line with these previous observations since administration of both lr and hklr were equally effective to improve resistance of infant mice to rsv infection and reduce lung injuries. interestingly, although both lr and hklr showed a similar capacity to reduce lung rsv titers, lr was more effective than hklr to reduce lung injuries during rsv infection. these differential effects achieved by the four treatments would be related to their specific capacities to modulate the production of ifn-γ and il- after rsv challenge. all of them were able to improve respiratory ifn-γ levels and reduced viral loads, while lr , lr and hklr but not hklr increased il- and reduced lung injuries. then our results suggest that not all the heat-killed bacteria derived from immunobiotic strains maintain the immunoregulatory effect after heat treatment. this would be an important point to consider when selecting immunoactive nonviable strains. previously, we demonstrated that the nasal treatment of malnourished mice with heat-killed l. casei crl was able to increase their resistance to the infection with the respiratory pathogen s. pneumoniae [ ] . the results from that study suggested that heat-killed lactobacilli are also effective in the immunomodulation of the respiratory immune system in immunocompromised hosts. therefore, immunobiotic bacteria in the form of live cells may not be required for enhancing respiratory defenses against bacterial and viral pathogens. our previous and present results show that non-viable immunobiotics or their cellular fractions could be an interesting alternative as mucosal adjuvants, especially in immunocompromised hosts in which the use of live bacteria might be dangerous. in addition, heat-killed immunobiotic have the advantages of allowing a longer product shelf-life, easier storage, and transportation. therefore, to study the capacity of non-viable lr or its cellular fractions to beneficially modulate the immune response against respiratory virus infections in immunocompetent and immunocompromised hosts is an interesting topic for future research. in the present work we demonstrated that nasal administration of immunobiotics is able to beneficially modulate the immune response triggered by tlr /rig-i activation in the respiratory tract and to increase the resistance of mice to the challenge with rsv. as it has been reported for orally administered probiotic bacteria, our results demonstrated that the immunoregulatory effect of nasally administered lactobacilli is a strain dependent effect. comparative studies using two lactobacillus rhamnosus strains of the same origin and with similar technological properties [ , ] showed that each strain has an specific immunoregulatory effect in the respiratory tract and that they differentially modulate the immune response after poly(i:c) or rsv challenges, conferring different degree of protection and using distinct immune mechanisms. we also demonstrated in this work that is possible to beneficially modulate the respiratory defenses against rsv by using heat-killed immunobiotics. moreover, our results showed that not all heat-killed bacteria derived from viable strains with immunomodulatory capacity, are also able to functionally modulate the respiratory immune system. therefore, detailed studies of the immunoregulatory capacities of heat-killed immunobiotics or their cellular fractions are necessary in order to find those with the 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after all these years immune-mediated disease pathogenesis in respiratory syncytial virus infection stat negatively regulates lung basophil il- expression induced by respiratory syncytial virus infection control of rsv-induced lung injury by alternatively activated macrophages is il- r alpha-, tlr -, and ifn-beta-dependent effect of intranasal administration of lactobacillus pentosus s-pt on influenza virus infection in mice nasally administered lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection the authors declare that they have no competing interests.authors' contributions yt, ec, hz, tt, kt and jv carried out experiments, analyzed data and performed the statistical analysis. hk, sa and jv conceived of the study, and participated in its design and coordination and helped to draft the manuscript. all authors read and approved the final manuscript. submit your next manuscript to biomed central and take full advantage of: key: cord- -o vxqm authors: visser, linda j.; aloise, chiara; swatek, kirby n.; medina, gisselle n.; olek, karin m.; rabouw, huib h.; de groot, raoul j.; langereis, martijn a.; de los santos, teresa; komander, david; skern, tim; van kuppeveld, frank j. m. title: dissecting distinct proteolytic activities of fmdv l(pro) implicates cleavage and degradation of rlr signaling proteins, not its deisgylase/dub activity, in type i interferon suppression date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: o vxqm the type i interferon response is an important innate antiviral pathway. recognition of viral rna by rig-i-like receptors (rlrs) activates a signaling cascade that leads to type i interferon (ifn-α/β) gene transcription. multiple proteins in this signaling pathway (e.g. rig-i, mda , mavs, tbk , irf ) are regulated by (de)ubiquitination events. most viruses have evolved mechanisms to counter this antiviral response. the leader protease (l(pro)) of foot-and-mouth-disease virus (fmdv) has been recognized to reduce ifn-α/β gene transcription; however, the exact mechanism is unknown. the proteolytic activity of l(pro) is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eif g and nf-κb. in addition, l(pro) has been demonstrated to have deubiquitination/deisgylation activity. l(pro)’s deubiquitination/deisgylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced ifn-α/β gene transcription. here, we demonstrate that tbk , the kinase that phosphorylates and activates the transcription factor irf , is cleaved by l(pro) in fmdv-infected cells as well as in cells infected with a recombinant emcv expressing l(pro). in vitro cleavage experiments revealed that l(pro) cleaves tbk at residues – . we also observed cleavage of mavs in hela cells infected with emcv-l(pro), but only observed decreasing levels of mavs in fmdv-infected porcine lfpk αvβ cells. we set out to dissect l(pro)’s ability to cleave rlr signaling proteins from its deubiquitination/deisgylation activity to determine their relative contributions to the reduction of ifn-α/β gene transcription. the introduction of specific mutations, of which several were based on the recently published structure of l(pro) in complex with isg , allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of l(pro). characterization of the effects of these mutations revealed that l(pro)’s ability to cleave rlr signaling proteins but not its deubiquitination/deisgylation activity correlates with the reduced ifn-β gene transcription. introduction a virally infected cell activates a plethora of antiviral responses. one of the best-known antiviral responses is the induction of type i interferons (ifn-α/β). replication of the viral genome generates double-stranded rna (dsrna) replication intermediates that can be recognized by cytoplasmic rig-i like receptors (rlrs). for example, picornaviruses, small (~ nm) nonenveloped viruses with a positive-sense rna genome, synthesize replication intermediates that are predominantly recognized by mda [ ] [ ] [ ] [ ] . upon recognition of viral dsrna, mda interacts with mavs, which subsequently activates traf and tbk . tbk phosphorylates the transcription factors irf and irf , resulting in their activation and dimerization. simultaneously, traf interacts with the ikk complex to activate the transcription factor nf-κb. upon activation, irf , irf and nf-κb translocate to the nucleus, where they induce expression of ifn-α/β and other proinflammatory cytokines. subsequent ifn-α/β signaling via the type i ifn receptor (ifnar) and the jak-stat pathway induces the expression of hundreds of interferon stimulated genes (isgs) (reviewed in [ , ] ). ifn-α/β gene transcription is extensively regulated by post-translational modification of rlrs and their downstream signaling proteins, including phosphorylation and ubiquitination. ubiquitin is a . kda protein that can be covalently linked through an ε-amino peptide linkage to lysine residues in target proteins. within the rlr signaling pathway rig-i, mavs, tbk , traf , traf and ikkγ are ubiquitinated and this affects their molecular interactions, localization, stability, or activity (reviewed in [ , ] ). ubiquitination of rlr signaling proteins can both positively and negatively regulate the signaling pathway, which allows for rapid fine-tuning of the innate immune response against viral infection (reviewed in [ ] [ ] [ ] ). consequently, many viruses encode enzymes with deubiquitinating (dub) activity to manipulate the rlr signaling pathway and thereby suppress expression of ifn-α/β (reviewed in [ ] ). in addition to ubiquitin, there are multiple ubiquitin-like modifiers, which can also be attached to target proteins. of special interest is isg , an ifn-induced modifier of . kda comprised of two ubiquitin-like domains in tandem. the exact antiviral properties of isg are not yet fully understood (reviewed in [ , ] ). early work on isg depended on mouse models and showed that expression of isg protected mice from viral infection [ ] [ ] [ ] [ ] . however, important biological and structural differences between isg of murine and human origin have since been reported [ ] [ ] [ ] [ ] . more recently, a picture is emerging that proteins are isgylated co-translationally, explaining why predominantly viral proteins and isgs are isgylated upon infection in humans [ ] . isgylation of rlr signaling proteins has been reported, but the effect of these modifications on the outcome of the signaling pathway is still unclear ( [ ] [ ] [ ] [ ] , reviewed in [ ] ). in addition, isg has been reported to act as a cytokine [ , ] . ifn-α/β signals in autocrine and paracrine ways to induce a tissue-wide antiviral state, thereby limiting viral spread. to establish infection in their host, it is essential for viruses to suppress both the rlr signaling pathway and the downstream signaling of ifn-α/β. affecting protein levels of important signaling molecules, either via cleaving them or inducing their degradation, is a strategy commonly used by viruses to suppress antiviral signaling [ ] [ ] [ ] [ ] . one such example is the picornavirus foot-and-mouth disease virus (fmdv). fmdv is a member of the genus aphthovirus, which also contains bovine rhinitis a and b viruses, and equine rhinitis a virus (erav). the genetic information on the fmdv rna genome is translated as one polyprotein that is autocatalytically processed into the mature proteins, two of which have been shown to possess proteolytic activity and also been implicated in suppressing ifn-α/β induction (reviewed in [ ] ). the c pro , the protease that processes the majority of cleavage sites on the polyprotein, cleaves nf-κb essential modulator (nemo), an adaptor protein that is essential to activate the nfκb and irf signaling pathways [ ] . the second protease on the polyprotein implicated in suppressing ifn-α/β induction is l pro , a papain-like cysteine protease located at the n-terminus of the polyprotein [ ] . once synthesized, l pro immediately frees itself from the growing peptide chain by autocleavage at its own c-terminus. l pro then efficiently cleaves the two isoforms of eif (eukaryotic initiation factor) g to reduce protein synthesis from cellular mrna [ ] and suppresses the induction of ifn-α/β via several mechanisms. l pro has been shown to induce the degradation of nf-κb subunit p /rela [ , ] , and decrease the levels of ifn regulatory factor (irf ) and irf [ ] . further, l pro can also interact with adnp, a negative regulator of transcription [ ] . in addition to cleaving or degrading important signaling molecules, l pro possesses deubiquitinase (dub) activity which has been proposed to modulate rlr signaling [ ] . a subsequent study demonstrated that l pro should be predominantly regarded as a deisgylase rather than a dub as biochemical evidence showed that l pro has a -fold higher affinity for isg than for ubiquitin [ ] . structural studies and biochemical studies have shown separate substrate binding sites on l pro for the viral polyprotein, the isoforms of eif g as well as for ubiquitin and isg [ ] [ ] [ ] , suggesting that it may be possible to uncouple the activities of l pro by the introduction of specific amino acid substitutions. we therefore set out to uncouple the different activities of l pro to discover whether l pro suppresses rlr signaling through its deisgylase/dub activity or through its ability to cleave and degrade multiple rlr signaling proteins. in this work, utilizing encephalomyocarditis virus (emcv) expressing fmdv l pro (emcv-l pro ), we identified mavs and tbk as new l pro substrates and determined the cleavage site in tbk . by introducing specifically designed mutations into l pro , we further identified residues that are important for either the cleavage/ degradation of rlr signaling proteins or for its deisgylase/dub activity, thereby uncoupling the two catalytic activities of l pro . we demonstrate that cleavage/degradation of rlr signaling proteins, but not the deisgylase/dub activity of l pro , correlates with suppressing ifn-α/β gene transcription. to study the effects of l pro on the induction of type i ifn in picornavirus-infected cells, we used two previously generated recombinant viruses; emcv-l zn , which contains inactivating mutations in the zinc-finger domain of the leader (i.e. emcv's rlr signaling antagonist) [ , , ] , and emcv-l pro , which was derived from emcv-l zn and additionally encodes fmdv l pro at the n-terminus of its polyprotein ( fig a) [ ]. we also constructed a similar ). in emcv-l zn , l contains inactivating mutations in its zn-finger domain (c a/c a) which abolishes its ability to suppress antiviral responses. to generate emcv-l pro , the lb pro gene of fmdv o-strain was introduced at the ' end of the emcv-l zn open reading frame. the l pro cleavage site at its own c-terminus was mutated to aaa. instead l pro is released from this viral polyprotein via an emcv c cleavage site. (b) hela r cells were infected at moi with the indicated viruses and cells were lysed at , , , h pi. total rna was isolated and used for rt-qpcr analysis for ifn-β and actin mrna, and emcv vrna. the ifn-β levels are depicted as a fold induction compared to levels in mock-infected cells, after correction for actin mrna levels. the emcv vrna is depicted as a copy number per cell, calculated from a plasmid standard. error bars depict the sd. one-way anova with the dunnet post hoc test was used to determine statistical significance compared to the results for emcv-l zn -infected cells ( � , p< . ; ��� , p< . ; ���� , p< . ; ns, no significant difference). recombinant emcv carrying a catalytically inactive l pro (i.e. emcv-l pro c a) [ ] . to determine whether emcv-l pro can suppress ifn-β induction, we infected hela cells with emcv, emcv-l zn , emcv-l pro or emcv-l pro c a and determined the ifn-β mrna levels over time via rt-qpcr analysis (fig b) . consistent with previous studies [ , , ] , wt l pro , but not l pro c a, reduced the induction of ifn-β mrna approximately -fold, indicating that the catalytic activity of l pro is needed to suppress rlr signaling. in conclusion, the viruses that we generated (emcv-l pro and emcv-l pro c a) accurately mimic the suppression of rlr signaling by l pro as previously reported for fmdv-infected cells [ ] , providing us a model system to determine the mechanism via which l pro suppresses type i ifn induction. l pro has been reported to degrade important signaling proteins such as the p subunit of nf-κb, irf and irf [ , ] . to determine whether l pro targets additional rlr signaling proteins, we subjected cell lysates of hela cells infected with emcv-l zn , emcv-l pro or emcv-l pro c a to western blot analysis for the signaling proteins mavs, tbk and irf , as well as the known l pro -substrates eif g and g bp (fig a) . emcv capsid proteins and tubulin served as infection and loading controls, respectively. infection with emcv-l pro , but not emcv-l pro c a, resulted in the rapid cleavage of eif g (from hpi onwards) and the cleavage of g bp (from hpi onwards). we did not observe cleavage or degradation of irf as was suggested by others [ ] . in addition to these known cleavages, we observed cleavage of mavs and tbk at hpi. for mavs, we observed multiple cleavage products ranging in apparent molecular weight from~ kda to~ kda. tbk cleavage resulted in a single cleavage product with an apparent molecular weight of~ - kda. we also attempted to detect mda and investigate whether this dsrna sensor is targeted by l pro . unfortunately, the low levels of mda prevented us from detecting the endogenous protein. mda expression could be boosted by pretreatment with recombinant ifn-α , but ifn-α pretreatment inhibited efficient emcv infection, thereby interfering with the subsequent analysis. we next focused our attention to identifying the cleavage site in tbk . to this end, we overexpressed n-terminally flag-tagged tbk together with gfp-tagged l pro and performed western blot analysis. as seen in fig b , gfp-l pro was able to cleave flag-tbk . we observed an αflag-reactive cleavage product migrating at~ - kda, the same apparent molecular weight as the cleavage product we observed in emcv-l pro infected cells (fig a) , suggesting that l pro cleaves tbk at its c-terminus. we also co-incubated recombinant his-tbk with increasing amounts of recombinantly expressed l pro and l pro c a ( fig c) . the in vitro incubation of his-tbk with wt l pro also resulted in a~ - kda αhis-reactive cleavage product, confirming that l pro cleaves tbk at its c-terminus and does not rely on other cellular factors. incubation of his-tbk with catalytically inactive l pro did not result in the formation of a cleavage product, confirming that the cleavage is dependent on l pro 's proteolytic activity. subsequently, we showed that l pro also cleaves tbk of murine origin (fig d) , which suggests that l pro cleaves tbk in a conserved region. we identified residues klk -which localize at the very c-terminus of tbk and are well conserved between human, murine and porcine tbk -as a possible cleavage site ( fig e) . indeed, mutation of these residues prevented the cleavage of tbk by l pro (fig d) , confirming these residues are the cleavage site. upon identifying the cleavage site in tbk , we next investigated whether the cleavage of tbk by l pro inhibits its function in the rlr signaling pathway. to this end, we generated cells in which the endogenous tbk gene is replaced with a tbk truncation mutant representative of the~ - kda cleavage product. we first generated hela tbk k.o. cells using crispr/cas technology and characterized the remaining rlr signaling capacity of these cells cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression (s a and s b fig) . we found that depletion of tbk is not sufficient to fully impair rlr signaling, probably because of functional redundancy with ikkε (s b fig) . tbk k.o. cells have ã -fold lower ifn-β induction upon infection with emcv-l zn , transfection of vrna or upon overexpression of mavs. as expected, ifn-β induction resulting from transfection of irf , which acts downstream of tbk , was not affected in the tbk k.o. cells. subsequently, we expressed full length tbk or tbk Δ aa, which represents the n-terminal cleavage product, in tbk k.o. cells (s c fig) and determined whether expression of tbk and tbk Δ aa restored ifn-β mrna expression upon transfection of poly (i:c). expression of full length tbk in tbk k.o. cells fully restored ifn-β mrna induction (s d fig) and tbk Δ aa was similarly efficient in this (s e fig), indicating that the l pro -generated n-terminal cleavage product is signaling competent. to investigate whether tbk , mavs and irf are cleaved during fmdv infection, we infected porcine lfpk αvβ cells with wt fmdv-a or fmdv-a lacking l pro (leaderless virus, a -llv). western blot analysis revealed cleavage of tbk , but not of mavs or irf , during wt fmdv-a infection ( fig a) . tbk cleavage was observed from hpi onwards upon infection with wt fmdv, but not upon infection with leaderless fmdv. the cleavage product had an apparent molecular weight of~ - kda, consistent with our previous observations of the size of this cleavage product. although a mavs cleavage product was not detected during fmdv infection, densitometry analysis revealed a strong and progressive decrease in the relative ratio of mavs/tubulin from - hpi post infection with wt fmdv compared to mockinfected cells, whereas only a small decrease was detected in leaderless-infected cells (mavs/ tubulin ratio is indicated in fig a) . this suggests that expression of l pro induces degradation of mavs, also in fmdv-infected cells. consistent with our observations in emcv-l pro infected cells, we did not observe a decrease in irf signal in fmdv-infected cells. to investigate whether the cleavage of tbk is conserved amongst aphthoviruses, we infected cells with erav, the closest relative of fmdv. infection with erav, but not emcv, resulted in the cleavage of tbk ( fig b) . however, the cleavage product was less prominent than for emcv-l pro , suggesting that cleavage was inefficient or infection was delayed. notably, in our hela cells, erav displayed a replicative cycle of~ h. this is considerably slower than fmdv, which replicates in - hours. to study tbk cleavage by the two different l pro 's irrespective of variation in viral replication kinetics, we infected cells with emcv-l pro or an emcv expressing erav l pro (emcv-erav l pro ), for which we previously determined the replication kinetics to be similar [ ] . both viral proteases cleaved tbk resulting in a~ - kda cleavage product ( fig c) . fmdv l pro cleaved tbk more efficiently than erav l pro , consistent with the results observed during infection with fmdv or erav (fig a and b ). notwithstanding the differences between erav l pro and fmdv l pro , our data demonstrate that the ability to cleave tbk is conserved amongst these two aphthoviruses. we also investigated the effect of the pan-caspase inhibitor q-vd-ph (q-vd) on tbk cleavage in erav-infected cells ( fig d) . while addition of q-vd decreased the cleavage of known caspase substrate parp, the cleavage of tbk was unaffected. collectively, these results demonstrate that l pro directly cleaves tbk and that this activity is conserved amongst aphthoviruses. construction of l pro mutants to uncouple cleavage/degradation of rlr signaling proteins from its deisgylase/dub activity l pro also possesses deisgylase and-to a lesser extent-dub activity [ , , ] , and this latter activity was previously suggested to be important for l pro 's ability to suppress rlr signaling cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression plos pathogens | https://doi.org/ . /journal.ppat. july , [ ]. to investigate how l pro reduces the induction of ifn-β gene transcription, we set out to uncouple these two abilities of l pro . to this end, we introduced previously described mutations in the chimeric emcv-l pro and determined whether these mutations affect the deisgylase/ dub activity of l pro and/or its ability to cleave/degrade rlr signaling proteins. a mutation in the sap domain (i a/l a), was previously reported to abolish the ability of l pro to suppress type i ifn expression, to degrade signaling proteins (i.e. nf-κb p , irf and irf ), and to disrupt its dub activity [ , , ], and was therefore included in our screening. analysis of the crystal structure of l pro bound to isg suggested that l pro residues l , p and l are important for isg binding [ ] . fig a shows the structure of l pro (grey) in complex with isg (blue) and indicates the residues of isg (w and r , l , g ) that interact with l pro . mutation of l , p or l in l pro reduced its affinity for isg and impaired its deisgylase activity, without affecting eif g cleavage [ ] . homology modeling showed that isg and ubiquitin interact with the same surfaces of l pro [ ] , suggesting that l pro 's deisgylase activity also reflects its dub activity. two of these mutations (l a and l a) were introduced in emcv-l pro . in addition, we introduced the mutations l a and c s. mutation c s was reported to reduce the affinity of l pro for eif g [ ] whereas mutation of to l to alanine with its shorter side-chain rescued polyprotein processing in the context of the additional mutation l f [ ] . based on the structure of l pro , mutation l a has been predicted to open up the catalytic pocket. fig shows the locations of the residues that are mutated in this study ( fig a) and summarizes the reported effects of these mutations on l pro 's various proteolytic activities ( fig b) . first, we determined the effect of the introduced mutations on l pro 's ability to cleave or degrade rlr signaling proteins. we infected hela cells with emcv, emcv-l zn or the different emcv-l pro carrying the described mutations, lysed the cells at the indicated timepoints and subjected the lysates to western blot analysis for mavs, tbk , nf-κb subunit p , irf , eif g and g bp , as well as l pro and emcv capsid proteins (fig ) . our data show that infection with the different l pro mutant viruses resulted in l pro expression and accumulation of emcv capsid proteins from hpi onwards, which is indicative of efficient infection. interestingly, the introduced mutations in l pro had different effects on the cleavage or degradation of rlr signaling proteins. upon infection with emcv-l pro c s, we observed a~ hr delay in eif g cleavage, consistent with a previous report [ ] . this mutation did not affect the cleavage/degradation of the various rlr signaling proteins. mutation of l or l has been reported to reduce the activity of l pro towards isg , without affecting eif g cleavage [ ] . indeed, infection with emcv-l pro l a, resulted in cleavage of eif g, as well as all other l pro substrates (i.e. mavs, tbk , nf-κb p and g bp ) (fig ) . infection with both emcv-l pro l a and emcv-l pro l a resulted in efficient cleavage of rlr signaling proteins, confirming that these two l pro mutants have the same proteolytic profiles (fig ) . of note, the cleavage cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression of eif g appears to occur faster by l pro l a and l a compared to wt l pro , although a more in-depth analysis is necessary to confirm a true difference in eif g cleavage kinetics. importantly, both mutations allow us to separate the deisgylase/dub activity of l pro from its ability to cleave rlr signaling proteins. serendipitously, we observed that l pro carrying cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression mutation l a was strongly impaired in degrading nf-κb p and cleaving mavs and tbk , while cleavage of g bp and eif g was delayed but could be observed clearly at later timepoints (eif g cleavage is~ hr delayed, comparable to the delay observed for l pro c s). mutation of l pro 's sap domain (i a/l a), which was previously shown to abolish cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression degradation of nf-κb p and dub activity as well as to impair l pro 's ability to reduce ifn-β mrna expression [ , ] , also affected l pro 's ability to cleave mavs and tbk . overall, our data demonstrate that the mutations have differential effects on the cleavage/degradation of rlr signaling proteins. importantly, we demonstrate that l pro residues l and l , which are essential for its deisgylase/dub activity, are not essential for is ability to cleave/degrade rlr signaling proteins, indicating that the two different catalytic activities of l pro can be uncoupled. it has been previously reported that mutations l a, l a and i a/l a affect l pro 's deis-gylase/dub activity [ , ] . to determine the effect of mutations l a or c s on these activities, mutant l pro 's were expressed and purified from e. coli and in vitro catalytic activities towards ubiquitin-tamra and isg -tamra were measured (fig a) . l pro c s and l a displayed wt-like activity towards isg and ubiquitin. we next determined dub activity of wt l pro , l pro c a, c s and l a in cells. to this end, we transfected hek- t cells with a combination of ha-ubiquitin, flag-rig-i and increasing amounts of gfp-l pro encoding plasmids ( . , . and . μg), and visualized ha-tagged ubiquitinated proteins by western blot analysis (fig b) . flag-rig-i, which was included to monitor the effects of l pro -induced translational shut-off on the overexpressed proteins, was clearly detectable even at the highest level of l pro (i.e. transfection of . μg of l pro plasmid), indicating that cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression l pro -induced translational shut-off did not significantly reduce the protein expression from the transfected plasmids. both wt l pro as well as the c s and l a mutants displayed dub activity upon overexpression in cells, as indicated by the reduction of ha-tagged ubiquitinated proteins upon increasing l pro levels. notably, we observed ubiquitinated proteins upon transfection of low amount of l pro c s plasmid ( . μg), although our in vitro data (fig a) indicated that mutation c s does not reduce the activity for ubiquitin. as the in vitro data are much more quantitative in nature, we consider the relatively decreased dub activity of l pro c s in comparison to l pro wt or mutant l a as observed in fig b the result of variations in expression of the transfected plasmids. thus far, our data showed that mutations c s and l a did not affect l pro 's dub activity towards a mono-ubiquitin fused to a fluorescent tamra molecule. to exclude the possibility that mutations c s and l a affect l pro 's dub activity towards other substrates, we cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression determined its ability to cleave differently-linked di-ubiquitin molecules. co-incubation of l pro with di-ubiquitin molecules of different linkages indicated that l pro preferentially targets k -and k -linked ubiquitin chains, displayed some activity towards k -, k -, k -, and m -linked chains, but at this enzyme concentration and incubation times has no activity towards k -or k -linked ubiquitin chains (fig ) . neither mutation l a nor c s affected the ability of l pro to cleave the di-ubiquitin molecules. overall, our analysis shows that l pro carrying mutation l a or c s have wt-like deisgylase and dub activity. importantly, as mutation l a impairs l pro 's ability to cleave/degrade rlr signaling proteins ( fig ) , but does not affect its deisgylase/dub activity, introduction of this mutation also allows us to make a distinction between the two proteolytic activities of l pro . l pro 's ability to reduce ifn-β mrna levels correlates with its ability to cleave/degrade rlr signaling proteins, not with its deisgylase/dub activity having characterized how the different mutations in l pro affect its deisgylase/dub activity or the cleavage/degradation of rlr proteins, we next set out to determine which mutants reduce the induction of ifn-β mrna. we infected hela cells with emcv, emcv-l zn or the different emcv-l pro mutants and determined the ifn-β mrna and emcv vrna levels over time via rt-qpcr analysis (fig ) . wildtype l pro as well as l pro c s, l a and l a consistently reduced the induction of ifn-β mrna, while l pro c a and l a were unable to do so (fig a) . the sap domain mutant (emcv-l pro i a/l a) also failed to suppress ifn-β mrna levels (fig b) , which is in agreement with observations in fmdv-infected cells [ , ] . notably, infection with emcv l pro l a, which displayed wt deisgylase/dub activity but is strongly impaired in its ability to cleave/degrade rlr signaling proteins mavs, tbk and nfκb p , failed to suppress the induction of ifn-β mrna. in contrast, l pro l a and l a,which are strongly impaired in their deisgylase/dub activity [ ] but not in their ability to cleave/degrade rlr signaling proteins, reduced ifn-β mrna levels. these combined observations (summarized in fig ) demonstrate that cleavage/degradation of rlr signaling proteins, but not the deisgylase/dub activity of l pro , correlate with suppressing ifnα/β gene transcription. notably, l pro c s reduced the induction of ifn-β mrna despite a h delay in eif g cleavage, indicating that the rapid eif g cleavage and the subsequent translational shut-off is not sufficient to suppress rlr signaling. fmdv suppresses ifn-α/β both at the mrna and at the protein level [ ] , but the molecular mechanism underlying the reduced induction of ifn-α/β gene transcription (rlr signaling) is poorly understood. both the dub activity of l pro as well as its ability to cleave/degrade rlr signaling proteins have been implicated in the suppression of rlr signaling [ , , ] . in this study, we identified mavs and tbk as novel l pro substrates and mapped the cleavage site in tbk . moreover, by introducing specific mutations we were able to separate l pro 's deisgylase/dub activity from its ability to target rlr signaling proteins. using l pro carrying either of these uncoupling mutations, we demonstrated that the cleavage/degradation of rlr signaling proteins, not the deisgylase/dub activity, correlates with the ability to reduce ifn-β gene transcription. collectively, our data strongly suggest that the ability of l pro to cleave/degrade rlr signaling proteins is needed to reduce the ifn-β mrna levels. we identified tbk as a new l pro substrate and identified the cleavage site. we observed cleavage of tbk both in hela cells infected with emcv-l pro and in fmdv-infected lfpk cells, and we demonstrated that the l pro cleavage site is located towards the c terminus of cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression tbk , more specifically in the coiled-coil (cc ) domain. previous work indicated that tbk Δcc was signaling competent upon overexpression of tbk . however, it was also shown that mutation of residues l and k , which are located in the cc domain, abolishes ifn-β mrna induction upon polyi:c transfection and vsv infection [ ] . l pro cleaves tbk at these residues. yet, we observed that the n-terminal cleavage product restored rlr signaling in tbk k.o. cells upon poly(i:c) stimulation. whether this implies that the l pro -mediated cleavage of tbk does not contribute to the viral strategy to suppress rlr signaling and ifnβ gene transcription remains unknown. unfortunately, it is very difficult to investigate the effect of tbk cleavage by l pro on ifn-β gene transcription in infected cells, as l pro also cleaves other rlr signaling proteins, i.e. mavs (this study) and lgp ( [ ] , see below), and the transcription factor nf-κb. dissection of the effect of tbk cleavage on ifn-β gene transcription from the effect of mavs, lgp and nf-κb cleavage would require the identification of all l pro cleavage sites in these known targets followed by the generation of cells with cleavage-resistant versions of these proteins. it remains a question whether such an approach will yield conclusive answers as other rlr signaling proteins may be targeted by l pro , which have cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression not yet been identified. hence, the relative importance of the l pro -mediated cleavage of tbk for the viral suppression of ifn-β gene transcription remains unknown. cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression apart from its role in rlr signaling, tbk has been suggested to be involved in autophagosome maturation. tbk was identified as a factor in the autophagosomal clearance of herpes simplex virus and mycobacteria [ , ] . a recent report showed that tbk phosphorylates lipidated lc- to prevent premature removal of lc from autophagosomal membranes by atg , thereby facilitating a unidirectional flow from the autophagosome to the lysosome [ ] . many picornaviruses hijack autophagic pathways to generate sites for viral rna replication and to facilitate non-lytic release of virions [ ] [ ] [ ] [ ] [ ] [ ] [ ] possibly, cleavage of tbk by l pro facilitates the use of autophagy to aid viral infection and propagation. l pro also impacts mavs integrity. a distinct mavs cleavage product was observed upon infection with emcv-l pro . remarkably, no mavs cleavage product was observed in fmdvinfected cells, although mavs levels progressively declined over time. the reason for this difference is unknown, but may be related to differences in human and porcine cells. nevertheless, our data indicate that the integrity of mavs is affected by l pro in both cell types, suggesting this rlr signaling protein is targeted by fmdv to affect ifn induction. of note, mavs is known to localize to the peroxisomes and mitochondrion associated membranes of the er [ , ] , in addition to its default localization on mitochondria. it remains to be established whether l pro targets all forms of mavs or specifically targets mavs at one of these locations. tbk and mavs are not the only rlr signaling proteins that are targeted by l pro . it was previously reported that overexpression of l pro induces the degradation of irf and irf [ ] , and that the p subunit of nf-κb is degraded in fmdv-infected cells [ ] . we also observed degradation of nf-κb p in emcv-l pro infected-cells but we did not observe degradation of irf . notably, degradation of irf was also not observed in fmdv-infected cells. whether irf degradation is restricted to certain cell types or conditions, or merely is an artefact due to overexpression remains unknown. it is remarkable that l pro , comprising just amino acids, can carry out several specific proteolytic activities on both the viral and cellular substrates as summarized in figs and . previous work documented areas of the protease that are required for polyprotein processing [ , ] . these residues included l which is part of the p pocket that can interact with leucine residues in the substrate at the p position. in this work, l was identified as being involved in tbk and mavs cleavage; however, its replacement by alanine affected neither the activity on eif g nor the deisgylase or dub activities. surprisingly, mutation of two residues of the sap domain, (i and l ) also affected tbk and mavs cleavage, even though they are separated by Å (measured between the respective c α atoms) with helix α lying between them. nevertheless, it cannot be excluded that the sap mutations cause some destabilization of l pro , thereby explaining this mutant's defect in proteolytic activities. further structural work will be required to understand how l pro interacts with tbk and mavs. while this work was in progress, it was reported that lgp , a factor that is essential for mda activation, is cleaved by l pro [ ] . the mutations in l pro that impaired the reduction of ifn-β mrna levels (l a and mutations in the sap domain) displayed an overall defect in the cleavage and/or degradation of each of the rlr signaling proteins we studied (i.e. mavs, tbk and nf-κb p ). we anticipate that the cleavage of lgp is also likely impaired by introduction of these mutations. our data suggest that expression of l pro results in cleavage and/or degradation of multiple rlr signaling proteins (mavs, tbk , nf-κb p , and most likely lgp ). the relative contribution of each cleavage event to the reduction in ifn-β gene transcription remains unknown. a search for other substrates of l pro is of importance to further our understanding of the role and mechanism of how l pro reduces ifn-α/β induction. possibly, such a search may identify rlr signaling proteins that are cleaved earlier than the ones identified so far and may thereby have an influence on the early induction of type i ifn in infected cells. the l pro mutants that are defective in either the deisgylase/dub activity or the cleavage/ degradation of rlr signaling proteins allowed us to study which ability is needed to suppress rlr signaling. mutation l a, which rendered l pro unable to reduce ifn-β mrna levels, impaired the cleavage of rlr signaling molecules, but had no effect on the deisgylase or dub activity of l pro . meanwhile, mutations l a and l a resulted in the opposite phenotypic effect; rlr signaling proteins were cleaved with similar efficiency as wt l pro , but the deisgylase activity was significantly reduced by these mutations [ ] . yet, these l pro mutants still reduced ifn-β mrna levels. collectively, these data indicate that the activity of l pro to cleave/degrade rlr signaling proteins, not its deisgylase/dub activity, is important for reduction of ifn-β induction. medina et. al. have just reported that impairment of the deisgylation activity of l pro causes viral attenuation in vitro and in vivo [ ] . in support of our hypothesis, the mutations introduced by medina et. al. did not affect ifn or isg mrna expression levels [ ] . it was previously suggested that the dub activity of l pro is important for the suppression of rlr signaling [ ], which contrasts our findings. importantly, most experiments by wang et. al. relied on overexpression of l pro , ubiquitin, and several targets proteins (i.e. rig-i, tbk , traf and traf ). a recent study showed that l pro should be predominantly regarded as a deisgylase rather than a dub, as biochemical evidence showed that l pro has a -fold higher activity towards isg than ubiquitin [ ] . given the weak dub activity of l pro in vitro, it remains to be established whether l pro genuinely acts as a dub in fmdv-infected cells under physiological conditions (i.e. without overexpression of components of the ubiquitination system or known ubiquitination target proteins). previously, we found no differences in the levels of ubiquitinated proteins in cells infected with emcv expressing wt l pro or l pro c a, whereas the levels of isgylated protein were decreased in cells infected with emcv-l pro [ ] , suggesting that l pro predominantly acts as a deisgylase in infected cells. it is well established that certain viruses (i.a. adenoviruses, herpesviruses and nidoviruses) rely on viral proteases with dub and deisgylase activity to suppress the induction of ifn-α/β [ ]. fmdv l pro is a papain-like protease and thus l pro is best compared to other virally encoded papain-like cysteine proteases that suppress ifn-α/β gene transcription. members of the order nidovirales (i.e. coronaviruses and arteriviruses) encode one or two papain-like cysteine protease (plp), referred to as pl pro , or plp and plp when the virus encodes two plps. in addition to cleaving the viral polyprotein, pl pro and the equivalent plp have acquired dub and deisgylase activity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . structure-guided mutagenesis of pl pro of mers-cov and plp of equine arterivirus (eav) allowed the dub activity to be separated from the proteolytic activity portrayed towards the viral polyprotein [ , ] . this uncoupling of these two different proteolytic activities indicated that the dub activity of pl pro /plp contributes to the suppression of ifn-α/β transcription [ , ] . unfortunately, it has not been determined whether pl pro /plp cleaves rlr signaling proteins and thus it is unclear what other proteolytic activities could be affected by the mutations that were introduced. notably, the cleavage site of nidovirus pl pro /plp and fmdv l pro in ubiquitin and isg is different. while sars-cov pl pro breaks the iso-peptide bond between ubiquitin or isg and the target protein [ , ] , fmdv l pro is a non-canonical deisgylase that targets a peptide bond in isg itself, resulting in a diglycylated-lysine in the target protein [ ]. in conclusion, nidovirus pl pro /plp and fdmv l pro are both papain-like proteases, but they likely have evolved different strategies to suppress ifn-α/β gene transcription. fmdv l pro and enterovirus a pro are structurally different enzymes that share many functions; both cleave translation initiation factor eif g [ , , ] , both reduce ifn-α/β gene transcription [ , , , ] , both have been implicated in the suppression of sg formation [ ] [ ] [ ] and both have been suggested to rescue viral translation from the inhibitory effects of p-eif [ , ] . importantly, l pro and a pro both cleave several rlr signaling proteins, but the only overlapping rlr protein is mavs [ , ] . although a causal relationship between cleavage of rlr proteins and suppression of ifn-α/β transcription remains to be established for both proteases, the convergence on the cleavage of mavs is noteworthy. in the absence of sequence homology, no evolutionary basis for the functional similarities between the two picornavirus proteases can be determined. possibly, the extensive similarities between fmdv l pro and enterovirus a pro , both picornavirus proteases, is illustrative of the urgency for picornaviruses to suppress these particular antiviral host responses. our data suggests that the deisgylase activity of l pro is not critically needed to suppress rlr signaling, but rather its role should be sought in the broader antiviral activities of isg (reviewed in [ , ] ). it should be noted that our experiments were performed in naïve cells at high multiplicity of infection. expression of the isgylation machinery as well as isg itself are boosted by ifn-α/β and therefore we cannot formally exclude a role for the dub and/or deisgylase activity of l pro in suppressing rlr signaling under different conditions (e.g. in ifn-primed cells). isg has many functions, both intracellular and extracellular. intracellular isg can act both pro-inflammatory and immunomodulatory, either via isgylation of target proteins or as free isg . moreover, isg can be secreted to act as a cytokine [ , ] . isg also plays a role in damage repair after clearing viral infection [ ] and can regulate cellular processes such as autophagy and metabolism [ , [ ] [ ] [ ] . how the deisgylase activity of l pro contributes to efficient in vivo infection, remains to be established. hela r , hela r tbk k.o. and hek t cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fcs (v/v). hela ohio cells were maintained in dmem supplemented with % fcs (v/v) and % penicillin/streptomycin. lfpk αvβ cells [ ] were obtained from the foreign animal disease diagnostic laboratory (faddl) at the piadc. these cells were maintained in minimal essential medium (mem) supplemented with % fcs (v/v) and % antibiotics and non-essential amino acids. bhk- cells used for fmdv propagation were maintained in mem supplemented with % fcs (v/v), % tryptose phosphate broth, % antibiotics and non-essential amino acids. hela r tbk k.o. cells were generated via crispr/cas methodology using a pcrispr plasmid, as described previously [ ] . the used grna sequences are '-gctactgcaaatgtctttcg- ' and '-gaggaaaacagattggtt- '. fmdv a -wt (wild type) was generated from the full-length serotype a infectious clone, prmc [ ] and a -llv (leaderless virus) was derived from the infectious clone lacking the lb coding region, prm-llv [ ] . viruses were propagated in bhk- and concentrated by polyethylene glycol precipitation, titrated on bhk- cells, and stored at - ˚c. erav (nm- / strain) (gift from d. rowlands and t. tuthill [ ] ) was obtained after passage on hela r cells and subsequently concentrated by ultracentrifugation through a % sucrose cushion at , xg for hours in a sw ti rotor and stored at - ˚c. recombinant emcvs were generated by cloning the genes of interest into the xhoi/noti restriction sites from the pm . -vfetqg-zn infectious clone that was described previously [ ] . emcv-l pro viruses were recovered by transfection of run-off rna transcripts into bhk- cells. upon total cpe, viruses were concentrated by ultracentrifugation (as described for erav) and stored at - ˚c. the following antibodies were used for western blot staining procedures: αfmdv vp (rabbit polyclonal abmade at piadc), αemcv capsid (gift from ann palmenberg), αl pro (gift from cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression ewald beck and tim skern), αmavs (enzo life science alx- - ), αtbk (cell signaling ), αirf (santa cruz sc- ), αnf-κb-p /rela (santa cruz biotechnology sc- ), αeif g (bethyl laboratories a - a), αg bp (bd biosciences clone /g bp), αparp (roche diagnostics # ), αflag (sigma m ), αgfp (invitrogen ose g), αhis (ge healthcare, - - ), αmyc (clone a , millipore), αha (abcam ab ) and αtubulin (sigma dm a). respective irdye or irdye conjugated secondary antibodies (licor) or hrp-conjugated secondary antibodies were used for detection. hela r cells were seeded in -wells plates and the next day infected with the indicated viruses at moi or transfected with the indicated plasmids or vrna. plasmids were transfected using fugene (promega) and vrna was transfected using lipofectamine (invitrogen), both according to the manufacturer's instructions. preparation of viral dsrna and the pcdna-gfp-mavs construct have been described previously [ , ] . pegfp-irf [d ] construct was a kind gift from john hiscott [ ] . at the indicated time points cells were lysed and cellular rna was isolated using total rna isolation kit (machery-nagel) according to manufacturer's instructions. reverse transcription was set up using taqman reverse transcription reagents (applied biosystems) before performing qpcr analysis with sybr green (roche) as described previously [ ] . the pires-egfp-fmdv l pro plasmid was described previously [ ] . the pcdna-flag-tbk plasmid was a gift from john hiscott [ ] and the pef-flag-rig-i was a gift from takashi fujita [ ] . ha-ubiquitin was expressed from a pcmv plasmid. hek t cells were seeded in -well plates and the next day transfected with . μg of total plasmid using fugene (promega) according to manufacturer's instructions. h posttransfection cells were lysed μl lysisbuffer ( mm tris ph . , mm edta, mm nacl, % np , protease inhibitor mix (roche)). post nuclear lysate was obtained by centrifugation at xg at ˚c for min. the amount of total protein in the lysates was determined using bca assay (thermo-fisher) and μg of protein was resolved using reducing sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to . μm nitrocellulose membranes by wet electrophoretic transfer. membranes were incubated h in blocking buffer (pbs + . % tween + % bsa) and successively incubated overnight with primary antibodies diluted in blocking buffer and then for min with respective secondary antibodies diluted in blocking buffer. between and after the incubations, the membranes were washed three times with pbs + . % tween- . finally, membranes were washed once with pbs and scanned using an odyssey imager (li-cor). hela r cells were seeded in cm dishes and infected the next day with the indicated viruses at moi . at the indicated time points cells were released using trypsin, washed once in pbs and lysed in μl lysis buffer ( mm tris ph . , mm edta, mm nacl, % np , protease inhibitor mix (roche)). subsequent steps are identical as described for transfected cells. for the analysis of fmdv-infected lfbk αvβ cultures, cells were lysed in lysis buffer ( . % np- substitute, mm tris ph . , mm nacl, mm edta). lysates were incubated at ˚c for min and cellular debris was collected by centrifugation at , xg for min at ˚c. ng of protein was resolved by sds-page, transferred by western blot and secondary antibodies conjugated with horseradish peroxidase (pierce) were used for detection cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression of proteins. following incubation with appropriate primary and secondary antibodies, protein bands were visualized using supersignal west dura extended duration substrate (thermo-scientific, rockford, il, usa) according to the manufacturer's directions. in vitro tbk cleavage sl pro was expressed and purified as reported previously [ ] . ng of his-htbk (millipore) was incubated with - μg sl pro for h at ˚c in a hepes buffer ( mm hepes ph . , mm kcl, mm edta) before the reaction mixture was dissolved on sds-page. proteins were transferred to nitrocellulose and western blot staining for the his-tag was performed. myc-mtbk and myc-mtbk aaa were transiently expressed in hela ohio cells from plasmid pcs - myc-mtbk , a gift from t. decker. μg myc-tagged mtbk containing cell lysate was incubated with μg sl pro for h at ˚c in a hepes buffer before resolving the reaction mixture on sds-page, transferring the protein to nitrocellulose membrane and performing western blot staining for myc. ubiquitin/isg -tamra assays were performed according to [ ] . di-ubiquitin in vitro cleavage assays were performed as described previously [ ] . total rna was isolated and used for rt-qpcr analysis for ifn-β and actin mrna. the ifn-β levels are depicted as a fold induction compared to levels in mock-treated cells, after correction for actin mrna levels. error bars depict the sd. (c) hela r tbk k.o. cells were transfected with μg plasmid expressing full-length or truncated tbk (tbk Δ aa). tbk Δ aa is representative for the l pro -generated n-terminal cleavage product. cells were lysed and lysates subjected to western blot analysis for tbk and tubulin. (d) tbk k.o. cells were reconstituted with full-length tbk as described for (c) and subsequently transfected with ng poly(i:c). cells were lysed at h post transfection of poly(i:c). total rna was isolated and used for rt-qpcr analysis for ifn-β and actin mrna. the ifn-β levels are depicted as a fold induction compared to levels in mock-treated cells, after correction for actin mrna levels. error bars depict the sd. (e) tbk k.o. cells were reconstituted with full-length or truncated tbk (tbk Δ aa) as described for (c). subsequent steps as described for (d). error bars depict the sd. (tif) cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression mda detects the double-stranded rna replicative form in picornavirus-infected cells mda and mavs mediate type i interferon responses to coxsackie b virus mda plays a crucial role in enterovirus rna-mediated irf activation identification of the role of rig-i, mda- and tlr in sensing rna viruses in porcine epithelial cells using lentivirus-driven rna interference regulation of type i interferon responses cytosolic sensing of viruses post-translational control of intracellular pathogen sensing pathways ubiquitin in the activation and attenuation of innate antiviral immunity isg in antiviral immunity and beyond isg : it's complicated mice lacking the isg e enzyme ube l demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza b virus infection selective inactivation of usp isopeptidase activity in vivo enhances isg conjugation and viral resistance ubiquitin-like protein isg (interferon-stimulated gene of kda) in host defense against heart failure in a mouse model of virusinduced cardiomyopathy ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product from different species mycobacterial disease and impaired ifn-γ immunity in humans with inherited isg deficiency. science ( -) human intracellular isg prevents interferon-α/β over-amplification and auto-inflammation isg deficiency and increased viral resistance in humans but not mice the isg conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg positive regulation of interferon regulatory factor activation by herc via isg modification acetaldehyde disrupts interferon alpha signaling in hepatitis c virus-infected liver cells by up-regulating usp negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg conjugation lrrc inhibits type i ifn signaling by targeting isg -associated rig-i for autophagic degradation isg : leading a double life as a secreted molecule. experimental and molecular medicine extracellular isg signals cytokine secretion through the lfa- integrin receptor enterovirus apro targets mda and mavs in infected cells the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response nmr analysis of the interaction of picornaviral proteinases lb and a with their substrate eukaryotic initiation factor gii residue l of the foot-and-mouth disease virus leader proteinase is a determinant of cleavage specificity functional dissection of the tbk molecular network innate immune sensor lgp is cleaved by the leader protease of foot-and-mouth disease virus tbk- promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation human tank-binding kinase is required for early autophagy induction upon herpes simplex virus infection tbk -mediated phosphorylation of lc c and gabarap-l controls autophagosome shedding by atg protease subversion of cellular autophagosomal machinery by rna viruses cellular origin and ultrastructure of membranes induced during poliovirus infection viral reorganization of the secretory pathway generates distinct organelles for rna replication picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential nonlytic viral spread enhanced by autophagy components foot-and-mouth disease virus utilizes an autophagic pathway during viral replication foot-and-mouth disease virus nonstructural protein c interacts with beclin , modulating virus replication peroxisomes are signaling platforms for antiviral innate immunity mitochondrial-associated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus impairment of the deisgylation activity of fmdv lpro causes attenuation in vitro and in vivo mé nard r. the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme severe acute respiratory syndrome coronavirus papain-like-protease: structure of a viral deubiquitinating enzyme mé nard r. selectivity in isg and ubiquitin recognition by the sars coronavirus papain-like protease papain-like protease from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl mers-cov papain-like protease has deisgylating and deubiquitinating activities deubiquitinase function of arterivirus papain-like protease suppresses the innate immune response in infected host cells in vivo assessment of equine arteritis virus vaccine improvement by disabling the deubiquitinase activity of papain-like protease crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression the eif g-eif e complex is the target for direct cleavage by the rhinovirus a proteinase poliovirus a proteinase cleaves directly the eif- g subunit of eif- f complex enterovirus protease apro targets mavs to inhibit anti-viral type i interferon responses essential role of enterovirus a protease in counteracting stress granule formation and the induction of type i interferon picornavirus a protease regulates stress granule formation to facilitate viral translation fmdv leader protease cleaves g bp and g bp and inhibits stress granule formation translation without eif promoted by l protease from foot and mouth disease virus confers eif -independent translation for mrnas bearing picornavirus ires novel mode of isg -mediated protection against influenza a virus and sendai virus in mice interferon-stimulated gene (isg ) and isg -linked proteins can associate with members of the selective autophagic process, histone deacetylase (hdac ) and sqstm /p modification of becn by isg plays a crucial role in autophagy regulation by type i ifn/interferon isg governs mitochondrial function in macrophages following vaccinia virus infection a continuous bovine kidney cell line constitutively expressing bovine αvβ integrin has increased susceptibility to foot-and-mouth disease virus knockout of cgas and sting rescues virus infection of plasmid dna-transfected cells genetically engineered foot-and-mouth disease viruses with poly(c) tracts of two nucleotides are virulent in mice the foot-and-mouth disease virus leader proteinase gene is not required for viral replication a novel, broad-spectrum inhibitor of enterovirus replication that targets host cell factor phosphatidylinositol -kinase iii-beta virus-dependent phosphorylation of the irf- transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation middle east respiratory coronavirus accessory protein a inhibits pkr-mediated antiviral stress responses triggering the interferon antiviral response through an ikk-related pathway. science ( -) shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity foot-andmouth disease virus leader proteinase: purification of the lb form and determination of its cleavage site on eif- γ a general chemical ligation approach towards isopeptide-linked ubiquitin and ubiquitin-like assay reagents molecular basis of lys -polyubiquitin specificity in the deubiquitinase cezanne we are grateful to john hiscott and takashi fujita for supplying plasmids, ann palmenberg and ewald beck for providing antibodies, and david rowlands and toby tuthill for supplying erav. key: cord- - jyv q e authors: ikegami, tetsuro; makino, shinji title: the pathogenesis of rift valley fever date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jyv q e rift valley fever (rvf) is an emerging zoonotic disease distributed in sub-saharan african countries and the arabian peninsula. the disease is caused by the rift valley fever virus (rvfv) of the family bunyaviridae and the genus phlebovirus. the virus is transmitted by mosquitoes, and virus replication in domestic ruminant results in high rates of mortality and abortion. rvfv infection in humans usually causes a self-limiting, acute and febrile illness; however, a small number of cases progress to neurological disorders, partial or complete blindness, hemorrhagic fever, or thrombosis. this review describes the pathology of rvf in human patients and several animal models, and summarizes the role of viral virulence factors and host factors that affect rvfv pathogenesis. rift valley fever (rvf), a mosquito-borne zoonotic disease among humans and ruminants, is caused by rift valley fever virus (rvfv) belonging to family bunyaviridae, genus phlebovirus [ , ] . rvf is endemic to sub-saharan african countries and has caused major outbreaks in several countries including kenya, tanzania, somalia, south africa, madagascar, egypt, sudan, mauritania, senegal, saudi arabia, and yemen [ ] . pregnant ruminants infected with rvfv typically are subject to highrate abortions, fetal malformation, and subclinical-to-fatal febrile illness, while newborn lambs usually die by acute hepatitis [ ] [ ] [ ] . rvfv infection in humans primarily causes a self-limiting febrile illness; however, some patients develop hemorrhagic fever, neurological disorders, or blindness after the febrile period [ , , ] . in endemic area, floodwater aedes mosquitoes, such as ae.mcintoshi or ae.vexans, serve as vectors, and the virus could be transmitted into offspring transovarially [ , ] . heavy rainfall or flooding of river banks due to construction of dams increases the number of permanent fresh water species of mosquitoes such as culex pipens, which play a role in amplifying rvfv among mosquitoes, ruminants and humans [ ] [ ] [ ] [ ] [ ] [ ] . an outbreak of rvf in developed countries, e.g., the u.s. or europe, could force a curtailing of livestock movement to prevent rvfv spread, causing massive economic loss, and a substantial degree of panic in our society, because the body fluids of infected animals contain infectious rvfv [ , ] , and mosquitoes such as culex spp. aedes spp. or anopheles spp. might further spread rvfv into other mosquitoes, humans and animals [ ] [ ] [ ] . effective vaccines and antiviral drugs are necessary for the containment of outbreaks and treatment of rvf patients, respectively. however, neither safe and effective vaccines nor efficient treatment is available. a correct understanding of rvf pathogenesis is essential for the development of effective vaccines and antiviral drugs against rvf. in this review, we will describe clinical and pathological findings of rvf in humans and animals and discuss viral and host factors that affect rvf pathogenesis. most rvf patients suffer from a self-limiting, febrile illness. however, some patients develop neurological disorders, vision loss, hemorrhagic fever, or thrombosis as shown in figure . in the s- s, many rvfv laboratory infections occurred due to a lack of appropriate biosafety procedures [ ] [ ] [ ] [ ] [ ] . however, the patients in most of these and later outbreaks suffered from self-limiting and nonfatal illness [ , , , , [ ] [ ] [ ] [ ] . typically, the incubation period for rvf is to days. symptoms start abruptly with severe chills, malaise, dizziness, weakness, severe headache, nausea and/or sensation of fullness over the liver region [ , , ] . these symptoms are followed by an elevated body temperature ( . °c to . °c); decreased blood pressure; pain in the back, shoulders, neck or legs; rigor; shivering; flushed face; red eye with sore; constipation; insomnia and/or photophobia. occasionally, other symptoms are seen which include epistaxis, abdominal pain, lack of gustatory discrimination, vomiting and/or diarrhea [ , , , , , ] . some lessening of symptoms can be observed on the rd day, and the body temperature often decreases to a normal level by the th day after the onset of symptoms. however, within to days after the recovery of body temperature, some patients again experience a temporal recurrence of high fever with a severe headache for a few days [ , , ] . moreover, patients may have a long-lasting high fever for as much as days [ ] . after body temperature becomes normal, some patients may develop a massive coronary thrombosis [ ] , persistent aching of legs for two weeks [ , ] , or persistent abdominal discomfort for weeks [ ] . the palpable enlargement of the liver and spleen is not common. in the convalescence phase, patients often experience weakness, malaise, a tendency to sweat, frequent headaches, pain on motion of the eye, and a sense of imbalance. virus has been demonstrated in the blood during the febrile period ( - days) , whereas neutralizing antibody also starts appearing around the th day of the onset of symptoms [ , , , , ] . maar et al. described a case of encephalitis in a rvf patient [ ] . the patient exhibited symptoms of sudden fever, rigor, and retro-orbital headache for two days. he had fever again at the nd day after the onset of illness and experienced neck rigidity lasting for five days from the th day. subsequently, he was sometimes confused and otherwise mentally affected, and experienced temporal vision loss without detectable retinopathy. he also exhibited convulsive attacks, hyperflexia and fever until the th day. his serum contained anti-rvfv hemagglutination (hai) antibodies of : at the th day and : at the th day, while his cerebrospinal fluid (csf) contained : of hai antibody at the th day and : at the th day. the csf also contained an increased number of white blood cells consisting mainly of lymphocytes at the th day, indicative of the possible occurrence of viral meningitis or meningoencephalitis. the patient recovered after treatment with amantadine, rifampicin, and dexamethasone for two weeks, although the effect of therapy could not be evaluated precisely. another case with encephalitis and retinitis was described by alrajhi et al. [ ] . the patient had a fever, ataxic gait, and bilateral retinal hemorrhage. she could not count fingers, and the csf contained many leukocytes, including lymphocytes. her consciousness level was decreased. she was discharged on day of the illness to her home, at which time she was awake, blind, quadreparetic, and incontinent. moreover, her neurologic conditions did not improve for the next year. an additional report described a patient who had persistent hemiparesis for four months after the onset of illness [ ] , and another paper reported rvf patients, who developed neurological signs and symptoms, including meningeal irritation, confusion, stupor and coma, hypersalivation, teeth-grinding, visual hallucinations, locked-in syndrome, and choreiform movement of upper limbs [ ] ; in these patients, the histopathological lesions in brains were characterized by focal necroses associated with an infiltration of round cells, mostly lymphocytes and macrophages, and perivascular cuffing [ ] . some patients suffer from maculopathy or retinopathy. patients noticed the loss of central vision or blurred eye occurring at various times after infection; e.g., from immediately after the disease onset to several weeks or months later. one or both eyes could be affected [ ] [ ] [ ] , and the affected eyes had macular edema with exudates containing a white mass covering the macular area with or without retinal hemorrhage, vasculitis, infarction or vitreous haze [ ] [ ] [ ] [ ] [ ] [ ] . in addition, retinal detachment [ , ] , uveitis [ , ] , or arterial occlusion [ , , [ ] [ ] [ ] was reported in some patients. in many cases, a complete recovery of vision does not occur, and chorioretinal scarring can remain in macular and paramacular areas, in spite of the resorption of exudates [ , , [ ] [ ] [ ] [ ] [ ] , while some patients show partial improvement in vision after several months of rvfv infection [ , , , ]. fatal rvf cases often involve hemorrhagic manifestations but the time to death varies among cases [ ] [ ] [ ] . most typically, the illness starts suddenly, and the patients experience fever, rigor, nausea, vomiting, headache, injected conjunctives, drowsiness, and/or body pains. the patients may also have such symptoms as macular rash over the entire trunk, ecchymoses on the arms, limbs, and/or eyelids, bleeding from the gums and/or gastrointestinal mucosal membrane, low blood pressure, hematemesis, melena, diarrhea, throat pain, pneumonitis, jaundice, and/or hepatosplenomegaly [ , ] . typically, elevation of alanine aminotransferase (alt), aspartate aminotransferase (ast), lactate dehydrogenase (ldh), and reduction of platelet count and hemoglobin are seen in these patients [ , ] . in many cases, death occurs in to days after patients become symptomatic; however, in some cases, death occurs in to days after the onset of symptoms. postmortem examination shows diffuse necrosis of hepatocytes which more greatly affect the centrilobular area than the portal area, which may indicate association with acute hepatic injury in this type of pathogenesis [ , ] . it should be noted that some patients who do not exhibit jaundice or hemorrhage, die from renal failure or disseminated intravascular coagulation (dic) accompanied by an elevation of alt, ast, ldh or d-dimer, or a decrease in platelet count [ , ] . a group of rvf patients who died from typical hemorrhagic fever also had encephalitis in addition to hepatic and gastro-intestinal necroses [ ] , which demonstrates the neuroinvasiveness of rvfv in hemorrhagic patients. another type of fatal case of rvfv infection was described by schwentker et al. [ ] . they reported that the temperature of the patient fell to normal on the th day after the onset of symptoms, whereas two papular areas of several centimeter in diameter were found on the patient's thigh and leg on the th day and remained until day . after temporal recovery by the th day, the patient experienced phlebitis of the popliteal vein, which was followed by infarcts in the lungs on the th, th and th days at multiple locations; these eventually caused a fatal embolus in the pulmonary vessels on the th day of illness. the liver of the patient was normal, did not contain infectious rvfv, and no typical rvf lesions were confirmed at the postmortem histopathological examination. however, there was a large thrombus in the inferior vena cava, a part of which might have detached and caused an embolus in the pulmonary artery. the neutralizing antibody showed up on the th day of illness, and its titer increased toward the th day. in a retrospective study in egypt, no increases in the total number of abortions were seen during an rvf outbreak, and the serological conversion rate of aborted women before and after outbreak was . % and . %, respectively [ ] . a report describing a potential vertical infection of rvfv concerned a pregnant woman, who experienced fever, headache, dizziness and generalized muscle ache four days before delivery during the rvf outbreak in saudi arabia in and developed igg specific to rvfv [ ] . her newborn baby presented with an anti-rvfv igm antibody, as well as alt/ast elevation, jaundice, extension of the activated partial thromboplastin time (aptt: test for the deficiency of intrinsic pathway factors) and the prothrombin time (pt: test for the deficiency of extrinsic pathway factors), and died on the th day after birth [ ] . although it is unknown whether the newborn baby died from rvf, it is possible that a vertical transmission in utero might have occurred in this case. the clinical symptoms of rvf vary among patients. the determinant of host susceptibility to induce hemorrhagic fever in humans has not been characterized. it is also unknown how rvfv causes diseases such as neurological disorders, vision loss or thrombosis in the presence of protective antibodies. several animal models have been used to understand the pathology of rvf, and the advantages and disadvantages of different animal models are summarized in table . we discuss the pathological findings in various different animal models in the next chapter. mice are one of the most susceptible animal species to rvfv infection [ , ] , and rvf pathology in infected mice mimics the pathological findings in newborn lambs [ ] . most of the mice infected with wild-type (wt) rvfv zh or zh strains die in to days [ ] [ ] [ ] , whereas they die faster by infection with other wt isolates [ , ] . infected mice show ruffed fur with decreased activity in to days, and then become more lethargic while lying with their back legs wide apart [ , ] . the symptom is often followed by death within one hour [ ] . occasionally, mice survive this stage, yet have hind limb paralysis at days - post infection (p.i.) and die from encephalitis [ ] . the rectal temperature of infected mice is often normal or decreased to below normal [ ] . also, the clotting time of blood derived from rvfv-infected mice is significantly extended, and it clots normally by mixing with normal sera, a finding that may indicate the shortage of coagulation factors is important for the extension of clotting time [ ] . the liver is the major target organ of rvfv, while the enlargement of liver is not common [ , ] . liver lesions are characterized by fulminant hepatitis, with coagulative necroses leaving the portal space intact [ , , , ] . depletion of glycogen in hepatocytes is also common at an early stage [ , , ] . infected hepatocytes in mice contain eosinophilic intranuclear inclusion bodies [ , ] , which are not reactive to the feulgen reaction, a nucleic acid stain [ ] . the intranuclear inclusion bodies in rvfv-infected cultured cells were visualized by an indirect immunofluorescent assay with antisera against rvfv and found to have a filamentary shape [ ] . inclusion bodies are formed by the nss protein [ ] , a viral nonstructural protein, and the -to- amino acids at the carboxyl terminus of nss are responsible for the formation of the filamentous structures via self-association [ ] . viral antigens start accumulating in hepatocytes at day , and their abundance increases extensively at day p.i. [ ] . the infected hepatocytes are stained by using a terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay, indicating that rvfv replication induces apoptosis in hepatocytes [ ] . mice that survived the early hepatitis phase often are able to regenerate hepatocytes [ , ] . although swollen endothelial cells can be observed in the liver [ ] , antigens are not easily detectable in endothelial cells or kuppfer cells, which indicate that these cells are not the primary targets of rvfv [ , ] . in addition to hepatocytes, viral antigens have been detected in, odontogenic and gingival epithelium; lipocytes; pituicytes; olfactory neurons and multiple types of neurons in the brain; mononuclear phagocytes; cardiac myofibers; and in perineural, periosteal, adrenocortical, endosteal, perivascular, bone marrow stromal, fibroblastic reticular, and vascular smooth muscle cells, as well as in cells morphologically consistent with dendritic, pancreatic islet, and adrenal medullary cells; however, no viral antigens were reported in any ocular structure, including the retina [ ] . apoptosis of lymphocytes were found in the thymus, spleen, lymph nodes and mucosa-associated lymphoid tissues [ ] . congestion and hemorrhage are common to the liver, spleen, lymph nodes, large intestine, kidneys and brain [ , ] , but are uncommon in the jejunum [ ] . in some mice that survived acute viral hepatitis, a sharp decrease in viral antigens occurred at days p.i., and no virus could be detected in the sera, liver, lung, pancreas, large intestine and ovaries [ ] . in the late stage of infection, however, lethal meningoencephalitis characterized by neuronal necrosis, microhemorrhages, and perivascular cuffs occurs in mice that survived the acute hepatitis [ ] . the susceptibility of rats to rvfv differs among rat strains [ ] . peters et al. demonstrated that -to- -week-old inbred rats from u.s. breeders exhibited three different responses to subcutaneous (s.c.) rvfv inoculation [ , ] . wistar-furth (wf) and brown norway strains were highly susceptible to rvfv and died within four days p.i. by liver necrosis, while the fisher , buffalo, da and lewis strains were largely resistant to rvfv infection [ ] . aci and maax strains proved to be moderately susceptible and showed ascending paralysis; lesions were mainly in the brain and spinal cord and characterized as mild-to-severe necrotizing encephalitis and encephalomyelitis with focal necrosis with neutrophilic infiltrate and perivascular cuffing primarily with lymphocytes. in the aci and maax strains, viruses were undetectable in the liver and blood, whereas -to- log pfu/g of viruses could be detected from brain tissue, even in the presence of neutralizing antibody in the serum. the intracranial injection of rvfv uniformly caused encephalitis in these rats, including the resistant lewis strain. the immunosuppression of the resistant lewis rats by treating animals with cyclophosphamide day prior to s.c. rvfv infection resulted in death at around -to- days p.i. with increased viral titers in the serum, liver, spleen, brain, kidneys and adrenal gland, although the virus titers in these organs in the lewis rat were still lower than those in the corresponding organs of the wf strain [ ] . these data suggest that the lewis rat encodes a gene(s) important for the resistant phenotype. interestingly, wf (wf/mol) and lewis rats (lewis/mol) obtained from a european breeding colony are resistant and susceptible to rvfv, respectively; hence, these rats showed the opposite susceptibilities to rvfv infection to those of the same strains from u.s. breeders. furthermore, both wf and lewis rats obtained from another european breeder were resistant to rvfv infection; taken together, these findings indicate the possible genetic variability of inbred rats among different breeders [ ] . cross-breeding experiments culminated in findings that indicated the resistance of wf/mol rat was segregated as a single mendelian dominant locus [ ] . findlay et al. also showed that the albino rat of the glaxo strain had an age-dependent susceptibility to rvfv via the i.p. route infection [ ] ; rats younger than days died in to days with extensive liver necrosis, whereas -day-old rats survived rvfv infection [ ] . the syrian hamster is one of the most susceptible rodents to rvfv. death occurs in to days p.i. after intraperitoneal inoculation with massive liver necrosis [ , ] . the pathological changes are similar to those seen in mice [ ] . administration of low titers of neutralizing antibodies protects hamsters from fatal liver necrosis, yet infected hamsters die from encephalitis by day [ ] . the gerbil, meriones unguiculatus, represents a unique rvfv animal model, which produces fatal encephalitis with minimal liver involvement after infection of non-neuroadapted wt rvfv. the gerbil has proven moderately susceptible to rvfv, and the survival rate of -week-old gerbils after s.c. inoculation is reported to range from to %, dependent on the strain and inoculation dose [ ] . death was reported to occur around to weeks in a dose-independent manner; s.c. inoculation of , , , pfu of zh resulted in %, %, % and % survival of outbred tum:(mon) gerbils, respectively, and %, %, % and % survival of inbred mon/tum gerbils, respectively [ ] . the infected gerbils exhibited hind-limb paralysis, generalized weakness and wasting. gerbils also showed an age-dependent resistance to rvfv infection. most of the -to -week-old tum:(mon) gerbils died after pfu s.c. inoculation of zh from encephalitis, whereas % of -week-old gerbils were able to survive the infection. after s.c. inoculation, rvfv replicated temporally in the livers of both -week-old and -week-old gerbils on days and (~ pfu/g), while subsequent efficient virus replication in the brain occurred in -week-old gerbil (from day to day up to pfu/g), but not in -week-old gerbil (temporal increase up to at day ) [ ] . intracerebral wt rvfv inoculation of pfu into -week-old gerbils resulted in an efficient viral replication (~ pfu/g) in the brain and the mean time to death was six days, which is not statistically different from that of -week-old gerbils, which may indicate the presence of host factors influencing the neuroinvasiveness in an age-dependent manner [ ] . histopathologically, minimal multifocal necroses of hepatocytes are seen at days or after the s.c. inoculation of wt rvfv, while focal necrotizing encephalitis with neuronal necrosis, a neutrophilic infiltrate, and perivascular cuffing are seen in the brain at later time points [ ] . mild, necrotizing encephalitis without detectable infectious rvfv could be observed even in clinically normal rvf-infected gerbils [ ] . rhesus macaques are moderately susceptible to rvfv infection [ ] . after i.p or intranasal inoculations, body temperatures of infected macaques increased to - °c at to days p.i. and the febrile period lasted for to h, whereas some infected animals did not show any febrile reactions [ ] . peters et al. first described hemorrhagic fever-like illness in rhesus macaques that were experimentally infected with a wt rvfv zh strain [ ] . three out of fifteen rhesus macaques intravenously inoculated with rvfv zh became ill; two became moribund and were euthanized on days and , and one recovered from illness, whereas the others showed temporal viremia, the maximum viral titer of which occurred around day , and were clinically normal. all three monkeys exhibited lassitude, weakness, the cessation of food intake, petechiae, ecchymoses and bleeding from nares, gums or venipuncture sites. clear extensions of aptt, slight extension of pt, and a decrease in the number of platelets were observed in the two dead monkeys, possibly indicating a deficiency of coagulation factors and platelets. histopathologically, the dead monkeys showed moderate focal or midzonal coagulative necrosis of the liver involving approximately / to / of hepatocytes, necrosis in the ventricular myocardium, fibrin thrombi in the glomeruli and small intertubular vessels of renal medulla in the kidneys, and mild depletion of lymphocytes from white pulp and the deposition of eosinophilic amorphous fibrin-like material in red pulp cords in the spleen [ ] . morrill et al. reported that after intravenous inoculation of × pfu zh strain into rhesus macaques, three developed signs of hemorrhagic fever, seven were clinically ill but survived, and the other seven survived without clinical signs [ ] . serum interferon (ifn)- was detected from - h p.i. and from - h p.i. in the surviving monkeys and in those dying, respectively, and the delayed ifn response was preceded by viremia in two of the three lethally-infected monkeys [ ] . no surviving monkeys developed signs of encephalitis or retinal complications in follow-up observations at two months to two years [ ] . morrill et al. also demonstrated that the administration of recombinant leukocyte a ifn ( × u, i.m) at h after rvfv intravenous inoculation reduced the peak viremia titer by -times and cleared viruses by h p.i. [ ] . these studies suggested the importance of ifn- in limiting viral replication. findlay et al. reported that three species of african monkey, i.e., the green guenon (cercopithecus callitrichus), the sooty mangabey (cercocebus fuliginosus) and the patas guenon (erythrocebus patas), did not exhibit any febrile reaction after inoculation of rvfv, whereas virus was detected in the blood [ ] . in contrast, four species of south american monkeys, two brown capuchin monkeys (cebus fatuellus and cebus chrysopus) and two common marmosets (callithrix jacchus and callithrix penicillata) exhibited febrile reactions for to days upon rvfv infection, which may indicate that south american monkeys are more susceptible to rvfv infection than african monkeys [ ] . davies et al. reported that rvfv-infected baboons (papio anubis) had viremia for to days without developing significant clinical signs [ ] . daubney et al. originally reported an outbreak of rvf in a herd of sheep in kenya in , which was characterized as a high rate of abortion in pregnant ewes and high mortality of newborn lambs [ ] . a later study by easterday et al. described that the mortality of adult sheep following experimental rvfv infection was approximately % [ ] . typically, sheep with more than one week old were relatively resistant to rvfv infection, yet did exhibit fever ( to °c), viremia, diarrhea, nasal discharge, and decreased activity [ , ] . nine-to ten-week-old young adult sheep (ripollesa breed) that were subcutaneously inoculated with rvfv had corneal and choroidal edema with inflammatory infiltrate, which could be associated with drainage failure or inadequate corneal dehydration after transient viremia [ ] . on the other hand, -to -month-old yansaka sheep subcutaneously inoculated with rvfv died during the viremic febrile phase and displayed symptoms of epistaxis ( days p.i.~), severe and bloody diarrhea, conjunctival hemorrhage, widespread petechiae and ecchymoses in hairless areas, pulmonary edema/hemorrhage, and thrombi formation in the blood vessels of the heart, kidneys and brain. rvfv-infected west african dwarf or the ouda breed did not exhibit such rapid hemorrhagic symptoms and rather exhibited marked coagulative hepatic necrosis, and brain lesions, including mild gliosis, neural degeneration, neurophagia, and satellitosis [ ] . interestingly, yansaka, west african dwarf and ouda also had increased prothrombin time, which may have indicated that hemorrhage was induced by a combination of vascular endothelial damage and an inability to clot blood in response to the damage [ ] . the inconsistency of symptoms and mortality in adult sheep described in these publications suggest the divergence of host genetic background, even within the same breed of sheep, affects susceptibility to rvfv infection. several studies examined the effects of rvfv vaccine candidates for protecting pregnant ewes from wt rvfv infection. however, insufficient immunogenicity of inactivated vaccines or residual virulence of live-attenuated vaccines induced fetal malformations. pregnant ewes were untreated (n = ) or vaccinated (n = ) once with formalin-inactivated rvfv and then challenged with wt rvfv zh at days post vaccination [ ] . abortion occurred in both unvaccinated and vaccinated ewes, between to days p.i., and % of the ewes aborted their fetuses. one out of vaccinated ewes and one out of eight unvaccinated ewes died. these data may indicate that the inactivated rvfv vaccine induced an insufficient immunity for sheep. necropsy at days p.i. revealed that % of the ewes had either aborted or dead fetuses, while the dead or aborted lambs showed extensive liver necrosis typical of rvf [ ] . coetzer et al. reported that immunization of pregnant ewes with a live-attenuated smithburn vaccine strain at to days of pregnancy could cause a teratogenic effect in the fetus, including arthrogryposis, hydranencephaly, or mineralization of brain with or without hydrop amnii, and two out of six lambs from the nine vaccinated ewes showed such effects [ ] . hunter et al. described that pregnant ewes inoculated with live-attenuated mp- vaccine strain at to days of gestation either miscarried or produced lambs showing teratogenic effects ( out of lambs from vaccinated ewes), such as cerebellar hypoplasia, spinal hypoplasia, hydranencephaly, prognathia inferior, brachygnathia inferior, arthrogryposis, scoiliosis, lordosis, kyphosis, or dormed head [ ] . the abortion and teratogenous effects did not occur when pregnant ewes were vaccinated with mp- at the third trimester of pregnancy, i.e., at - days of gestation [ , ] . on the other hand, pregnant ewes vaccinated at days of gestation with a rvfv clone (c ) strain, which has a % in-frame deletion of nss [ ] , did not cause abortion or fetal malformations and were protective against wt rvfv challenge [ ] . these data may imply the involvement of mp- nss in the abortion of ewes and the teratogenic effects in lambs. rvfv infection causes an acute and fatal disease in newborn lambs [ ] . rvfv-infected newborn lambs usually exhibit obvious illness, including elevated body temperature ( to °c), loss of appetite, decreased activity, and prostration, about to h prior to death [ ] . the mortality rate in rvfv-infected newborn lambs is to % [ ] . studies of experimental infection of - day-old lambs with rvfv via s.c. resulted in necrosis of isolated hepatocytes ( - h p.i.), focal coagulative necrosis of hepatocytes ( - h p.i.), and extensive hepatocyte necrosis ( - h p.i.) with a progressive increase in viral antigens, whereas no viral antigens could be detected in the endothelial or kupffer cells in the liver, suggesting that hepatocytes are the primary target of rvfv [ ] . the necrosis is predominantly centrilobular or midzonal, and yet there is no definite distribution pattern in liver necrosis [ , ] . some infected lambs also exhibited necrosis in the villi at the distal jejunum and ileum and depletion of lymphocytes in the spleen, whereas the brain and eyes had no lesions [ ] . overall, the liver pathology of newborn lambs resembles that of mice or hamsters, which are extremely susceptible to rvfv. however, the rvfv neurovirulence in lambs is less prominent when compared with that in rodents. rvfv also causes diseases in other animals including goats, cattle, camels, dogs, cats, and ferrets, but does not cause any symptomatic diseases in rabbits, guinea pigs, birds, horses, pigs and other animals, as reviewed in detail previously [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] . as of the present, the mechanism of species-specific susceptibility to rvfv infection is unknown. rvfv infection shows unique pathogenesis in each animal model. because viral replication and host antiviral responses most probably contribute to viral pathogenecity, an understanding of rvf pathogenesis requires identification and characterization of the viral virulence factors and host antiviral factors. the next chapter describes the viral determinant of virulence. the rvfv genome is comprised of three rna segments named the s-, m-and l-segments ( figure ) [ , ] . the s-segment encodes n and nss genes in an ambisense manner, the m-segment. nsm (nsm ), kd (nsm ), gn and gc genes, and the l-segment, the rna-dependent rna polymerase (l) gene [ ] . rvfv virions bind to an unidentified cellular receptor, and enter the cells in a ph-dependent manner [ ] , probably through a clathrin-mediated endocytic pathway, as described for another phlebovirus [ ] . after viral uncoating, viral ribonucleocapsid (rnp) composed of viral genomic rna segments and n protein [ ] is released into the cytoplasm, and the viral polymerase, which probably is attached to the rnp exerts primary transcription to synthesize viral mrna [ ] . both n mrna and nss mrna are transcribed during primary transcription as early as min after infection from an efficiently packaged, viral-sense (negative-sense) s-segment and anti-viral-sense (positive-sense) s-segment, respectively; the packaging mechanism of rvfv rnp and the presence of specific cis-signals in the genomic rna are not known [ ] . viral rna replication starts around to h after infection [ ] and an increase in the amount of viral genomic rna results in increases in viral mrnas and proteins. the rnp is packaged into viral virions probably by its interaction with the cytoplasmic domains of gn/gc at the golgi apparatus, as reported for other bunyaviruses [ , ] . the three different rna segments could be co-packaged in a coordinated manner, in which the co-packaging of m and s-segments could support the packaging of the l-segment [ ] . the rvfv virion surface is highly symmetric t = icosahedral lattice [ ] , which is formed by a shell of glycoprotein capsomers most probably composed of gn-gc heterodimers [ ] . the rvfv m-segment encodes kd, nsm, gn and gc proteins in m mrna (figure ). those proteins are synthesized from a single open reading frame of m mrna at different augs present at the ' region of m mrna by leaky scanning of ribosomes, while the n-terminus of gn and gc is co-translationally cleaved by host proteins [ ] [ ] [ ] . nsm is synthesized from the nd aug, and the c-terminus is generated by cleavage at the n-terminus of gn, while the kd protein is synthesized from the st aug, and the c-terminus is identical to that of gn. among seven viral proteins, nss and nsm are nonstructural proteins which are not incorporated into virions [ , ] . the kd protein has not been studied in detail, while an " kd" protein induced by rvfv infection (most probably corresponding to the current kd protein) is known to be incorporated into virions [ ] , which may indicate that the kd protein is a structural protein. nss, kd protein and nsm are dispensable for viral replication in cell cultures [ , [ ] [ ] [ ] . infection of -week-old female wf rats with a recombinant rvfv zh strain lacking both kd protein and nsm induced acute fatal hepatic disease, causing the deaths of some infected rats around four days p.i., or a delayed fatal neurologic disease, resulting in death of some of infected animals around days p.i. (mortality rate: to %), while wt zh -infected rats developed acute hepatitis and % died. these data suggest that nsm is not essential for virulence and lethality [ ] . on the other hand, a recombinant rvfv mp- lacking the expression of both kd and nsm induced more extensive apoptosis than did mp- in cultured cells and the expression of nsm significantly inhibited the cleavage of caspase- and - induced by staurosporine [ ] , demonstrating that nsm protein suppresses apoptosis. thus, nsm probably suppresses apoptosis in infected hosts and affects viral pathogenicity. nss is known to be a major virulence factor of rvfv; a rvfv c strain, which has a % in-frame deletion of nss [ ] , and is completely attenuated in mice or sheep [ , ] . further studies showed that nss inhibits the synthesis of ifn- mrna under the conditions that transcription factors, such as ifn regulatory factor (irf- ), nf-b and activator protein (ap)- , are activated in wt rvfv zh -infected cells [ ] . it was found that nss can bind and sequester the p subunit of tfiih, an essential transcription factor for rna polymerase i and ii; and hence nss was reported to prevent the assembly process of the tfiih complex, resulting in the suppression of host mrna transcription [ ] . le may et al. also described studies demonstrating that a region of nss, corresponding to amino acid to , specifically binds to sin a-associated protein (sap ) and forms a complex that represses the histone acetylation required for the transcriptional activation of ifn- promoter; this repression worked even after the binding of irf- to the ifn- promoter [ ] ; hence, recombinant zh carrying a deletion at amino acid to of nss cannot suppress the ifn- mrna synthesis. it should be noted that c nss bound to sap , yet it did not suppress ifn- expression [ ] . although the inability of c nss to suppress ifn- expression may have been due to its poor accumulation in infected cells, further studies are required to know how the binding of nss to sap can lead to the suppression of ifn- mrna synthesis. it is also uncertain whether the general host transcriptional suppression is required for the inhibition of ifn- mrna synthesis. because nss induces host general transcription suppression [ ] , rvfv might have the ability to replicate under host transcription suppression. actinomycin d (actd) is a general inhibitor of host rna synthesis. viral titers of several cytoplasmic rna viruses including arenaviruses [ ] [ ] [ ] , measles virus [ ] , sindbis virus [ ] , rubella virus [ ] , polio virus [ , ] , coronaviruses [ , ] , and lactic dehydrogenase elevating virus [ ] could be reduced in the presence of actd, while the viral rna synthesis is often unaffected [ , , ] . we found that expression of nss protein is essential for the rvfv mp- strain to actively synthesize viral proteins and produce high titers of infectious viruses in the presence of actd [ ] ; cells infected with recombinant rvfv mp- lacking nss failed to synthesize viral proteins; and while cells infected with mp- lacking nss accumulated phosphorylated eif . the latter made cellular translation initiation inactive through the activation of dsrna-dependent protein kinase (pkr) at around h p.i. [ ] , resulting in the suppression of viral protein synthesis. further studies revealed that mp- nss promoted the degradation of pkr through the proteasome pathway and prevented an accumulation of phosphorylated eif , thereby securing efficient viral protein synthesis under host transcription shut-off induced by actd [ ] . habjan et al. reported that wt rvfv nss also induced pkr degradation and demonstrated that an rvfv c strain carrying biologically inactive nss induced fatal hepatic disease in c bl/ mice lacking pkr [ ] ; these mice are competent for inducing type-i ifns in response to viral rna replication or poly (i):poly (c) [ ] . pkr is one of several ifn-stimulated genes (isgs) and plays an important role in inhibiting viral replication in vivo; other rna viruses, including vesicular stomatitis virus (vsv) and influenza virus, also replicate more efficiently in mice lacking pkr than in those with an intact pkr [ ] . in addition to pkr, ifn-induced mxa proteins is also known to inhibit rvfv replication [ ] . although the genetic diversity of rvfv strains is relatively low (approximately % in primary sequences [ ] ), the susceptibilities to rat ifn-/ differed among rvfv strains. most of the sub-saharan rvfv strains are very sensitive to rat ifn-/ (ed : . - . units), whereas egyptian strains, including zh and zh , and a zimbabwean isolate ( / ) are relatively resistant to rat ifn-/ (ed : - units) [ ] . all of those rvfv isolates show a similar sensitivity to human ifn- (ed : - units). in summary, rvfv nss induces the shut-down of host transcription, including transcription of both type-i ifn and isgs mrnas, to prevent antiviral responses. nss also induces the degradation of pkr to prevent eif -mediated host and viral translational shut-off and promote an efficient viral protein synthesis. although nss is a major virulence factor to escape host innate immune responses, the virulence of rvfv could be controlled in a polygenic manner. the rvfv mp- strain is a highly attenuated strain derived from wt rvfv zh [ ] and encodes a functional nss gene. mutations in the m-and l-segments are major determinants of mp- attenuation [ , ] . in contrast, the c strain encodes an s-segment lacking a functional nss gene, while the m-and l-segments of c strain are still virulent phenotypes [ , ] . mice lacking ifn-ar (ifn-ar -/mice) are susceptible to both mp- and c , while the viral replication kinetics of mp- and c differ in those mice; the highest titer of viremia was reached within h p.i. and around h p.i. in c -infected mice and in mp- -infected mice, respectively [ ] . thus, there is a possibility that m-and l-segments of c may facilitate a rapid replication of c in these mice, reaching the highest virus titer substantially earlier compared to mp- -infected mice. recently, we found that wt rvfv zh virus stock contains two major viral populations, rzh -m -a (glu at aa. of gn protein) and rzh -m -g (gly at the corresponding site); the difference in the amino acid is mapped within one of the neutralizing epitopes in gn protein [ , ] . although it is not known how the two different populations have emerged in the zh virus stock, which was amplified once in the mouse brain and passaged twice in frhl cells and twice in vero e cells, the rzh -m g replicated less efficiently than rzh -m a in infected mice, whereas both of them replicated efficiently in tissue cultures such as mrc- , veroe , j . , and nih t cells. our study showed that one amino acid change in the gn can substantially alter replication and pathogenesis of zh in vivo, and yet the mechanism of the gn mutation in the pathogenesis remains unknown. clearly further studies will be needed for understanding the roles of viral proteins in rvfv pathogenesis. rvfv encodes several virulence factors, and the major virulence factor nss plays an important role in evading host innate immune responses. in the next chapter, we discuss how humans or host animals develop protective immune responses against highly virulent wt rvfv. adequate immunity against rvfv can attenuate the virulence of rvfv in animals and vaccination against rvfv can save animals from lethal rvfv challenge [ , , [ ] [ ] [ ] [ ] . the passive transfer of neutralizing antibodies is sufficient for protection from lethal rvf [ , [ ] [ ] [ ] [ ] , whereas the role of the cellular immune response for the protection is not sufficiently evaluated. yet, mandell et al. demonstrated that mice immunized with virus-like particles containing n have a higher survival rate (survival: / ) than those not containing n after lethal rvfv challenges (survival: / ). because anti-n protein antibody is unlikely to neutralize rvfv, these data may point to the involvement of cellular immune responses against n protein for rvfv protection [ ] . alternatively, antiviral immune responses might be induced by forming a complex between anti-n antibody and n proteins released from dead cells or infected cells in vivo [ ] . if n proteins are present in cell surface as reported in influenza virus-infected cells [ , ] , complement-mediated cell lysis could be another mechanism to support the elimination of infected cells [ ] . aerosol exposure is one of the most likely routes for both laboratory infections and bioterrorism attack. immunization of rats by s.c. inoculation of a formalin-inactivated rvfv vaccine (tsi-gsd- ) partially protected lethal rvfv aerosol exposure at days post immunization (survival: / : %), whereas out of surviving rats developed encephalitis without clinical signs at - days post-challenge, which was revealed during necropsy [ ] . another study showed that three vaccinated rats challenged with rvfv aerosol developed uveitis, although no histopathological analysis was presented [ ] . mice are highly susceptible to wt rvfv aerosol exposure (ld : to pfu), which may cause lethal hepatitis, but not pneumonia [ ] . a study in mice vaccinated at several different routes with formalin-inactivated rvfv vaccine (ndbr- ) [ ] and challenged with rvfv subcutaneously showed that the immunization route affected survival rates; s.c. immunization, intraperitoneal (i.p.) immunization, and intraduodenal (i.d.) immunization resulted in survival rates of . %, % and less than %, respectively. another study reported that fewer than % of mice immunized via s.c. or i.d. and about % of mice immunized via i.p. survived after aerosol route challenge of rvfv in the -week observation period [ ] . findings from this study using aerosol rvfv challenge indicated that the mucosal immunity elicited by i.d. immunization successfully lowered the occurrence of olfactory bulb encephalitis, but failed to prevent necrotic hepatitis, and immunity induced by s.c. or i.d. did not prevent hepatitis, olfactory bulb encephalitis and multifocal encephalitis, while i.p. immunization completely prevented the occurrence of hepatitis, but not multifocal encephalitis [ ] . these reports seem to indicate that immunization with inactivated vaccines via s.c. cannot prevent rvfv-induced diseases after aerosol challenge. it will be important to establish a reliable countermeasure against bioterrorism by use of rvfv through further detailed characterization of rvfv replication in various organs after aerosol challenge and examination of the efficacy of immunization of current live-attenuated rvfv vaccine candidates, such as mp- or c , for preventing rvf-induced diseased after rvfv aerosol challenge. exposure of animals to aerosol containing rvfv probably results in initial rvfv infection in lung epithelial cells, such as type i alveolar epithelial cells. both infection and release of rvfv occur in polarized epithelial cells, such as caco- cells (human colorectal adenocarcinoma cells), at apical and basolateral membranes [ ] . infection by punta toro virus (ptv), which belongs to the phlebovirus genus and causes a lethal necrotic hepatitis in hamsters [ , ] and mice [ ] , results in virus being released into the basolateral membrane [ ] , which may contribute to systemic viral spread in infected animals. as described in section . , several studies point to the possibility that unidentified host genetic factors influence the susceptibility of inbred rat species to rvfv. the primary rat hepatocytes derived from resistant american lewis rats or wf/mol rats are less permissive to rvfv infection than those derived from susceptible american wf rat or lewis/mol rats [ , ] , whereas rvfv replicates efficiently in primary cortical glial cells derived from wf/mol rats [ ] or spontaneously transformed cell lines generated from embryonic thymus cells of either resistant lew rats or susceptible wf rats [ ] , suggesting that hepatocytes in those resistant inbred rats are under the control of a host factor(s) that restricts efficient rvfv replication. peritoneal macrophages (pm) derived from resistant lewis rat or susceptible wf rat were treated with . or u of ifn, which resulted in a -or -fold reduction of zh replication in lewis pm, and a -or -fold reduction in wf pm, respectively, possibly indicative of the role of ifn to increase resistance in lewis rats [ ] . on the other hand, primary hepatocytes derived from susceptible lewis/mol rats and resistant wf/mol rats are resistant to rvfv after treatment of the cells with rat type-i ifn, suggesting the resistance induced by type-i ifn is not necessarily important for the host specific resistance seen in wf/mol rats [ ] . a recent study showed that mbt/pas mice, but not balb/cbyi mice, are highly susceptible to rvfv zh infection [ ] . experiments using microarray and quantitative real-time pcr showed that rvfv zh replication in mouse embryonic fibroblast (mef) cells derived from susceptible mbt/pas mice induced higher levels of ifnb and ifna mrnas than did those derived from resistant balb/cbyj mice, while mef cells derived from mbt/pas mice failed to induce several isgs, including the ifn regulatory factor (irf ) mrna, the '- ' oligoadenylate synthetase-like (oasl ) mrna, and the ifn-induced kda protein (isg ) mrna [ ] . the knockout of isg or oasl mrna expressions increased viral replication in mef cells derived from resistant balb/cbyj mice, leading the authors to suggest that mbt/pas mice have a defect in their ifn responses which controls rvfv spread [ ] . it is of interest to know whether the mice lacking isg or oasl genes are susceptible to rvfv. it is unknown whether the susceptibility of inbred rat to rvfv is due to a deficit in some isgs. the host factors determining the host susceptibility to ptv infection have been explored; ptv infection in mice mimics rvfv-induced lethal hepatitis. c bl/ j mice show an age-dependent susceptibility to ptv infection; the mice gradually become resistant to ptv from to weeks, and they are resistant at eight weeks of age [ ] . the -week-old c bl/ j mice show a delayed viremia, when compared to -week-old mice, and the peak viremia titers in the -week-old mice are -to -times lower than those in the -week-old mice [ ] . likewise, ptv replication was lower in primary cultured hepatocytes, kupffer cells and peripheral blood monocytes isolated from -week-old c bl/ mice than in those cells obtained from -week-old c bl/ mice [ , ] . adding stress to the -week-old mice by daily handling and observation increased their susceptibility to ptv replication [ ] . the role of toll-like receptor (tlr) , which is a pathogen recognition receptor recognizing double-stranded rna, in the susceptibility of the mice to ptv was studied by using -week-old tlr -/mice of the c bl/ background [ ] . the wt mice infected with ptv had a % mortality and high levels of il- induction (yet no remarkable increase in tnf-), whereas mice lacking tlr infected with ptv had increased survival rates, slightly earlier reduction of serum virus titers, and decreased levels of serum il- [ ] . on the other hand, il- -/mice were more susceptible to ptv infection and supported higher levels of viremia than did wt mice, which possibly means that il- is indispensable for protective immunity. a similar attenuation of virulence has been also reported for west nile virus infection in tlr -/mice [ ] . the authors hypothesized that over-production of il- in wt mice would be detrimental to the outcome of ptv infection [ ] , pointing out that the balance of cytokines might alter the pathogenesis of ptv. stat- is a key molecule in the ifn signaling pathway [ ] . it was reported that ptv replicated substantially better in the brains of stat- -/mice than in wt mice. also ptv titers in sera, spleens and livers of the stat- -/mice were high, which may emphasize the importance of ifn responses for limiting viral replication in the liver, spleen and brain [ ] . since the first recognition of rvf in an outbreak in , more than years has passed. although there has been good progress in characterizing clinical, pathological, and virological features of rvfv infection, rvfv still causes outbreaks in african countries or the arabian peninsula [ , ] . the development of effective, safe, highly immunogenic and economic vaccines for animals and humans will prevent rvf in the endemic countries [ ] . there are several important questions to address in controlling rvfv and in further understanding rvf pathogenesis. some of them include determining the: ( ) mechanism that triggers hemorrhagic fever, ( ) route of viral entry to the brain, ( ) mechanism of prolonged diseases in rvf patients in the presence of neutralizing antibodies, and ( ) significance of vaccination to prevent rvf after aerosol exposure. addressing these questions will have a substantial impact on understanding of rvf pathogenesis and development of anti-rvfv reagents. fields virology rift valley fever virus enzootic hepatitis or rift valley fever: an undescribed virus disease of sheep cattle and man from east rift valley fever the virus of rift valley fever or enzootic hepatitis rift valley fever rift valley fever-a review rift valley fever virus (family bunyaviridae, genus phlebovirus). isolations from diptera collected during an inter-epizootic period in kenya rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention rift valley fever surveillance in the lower senegal river basin: update years after the epidemic risk factors for severe rift valley fever infection in kenya rift valley fever during rainy seasons, madagascar climate and satellite indicators to forecast rift valley fever epidemics in kenya prediction of a rift valley fever outbreak prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the outbreak in egypt prevalence of rift valley fever immunoglobulin g antibody in various occupational groups before the outbreak in tanzania. vector borne zoonotic dis replication and dissemination of rift valley fever virus in culex pipiens vector potential of selected north american mosquito species for rift valley fever virus vector competence of a houston, texas strain of aedes albopictus for rift valley fever virus report of a fatal laboratory infection complicated by thrombophlebitis laboratory infections with the virus of rift valley fever am rift valley fever : a report of three cases of laboratory infection and the experimental transmission of the disease to ferrets human infection with rift valley fever virus and immunity twelve years after single attack rift valley fever; accidental infections among laboratory workers rift valley fever; i. the occurrence of human cases in johannesburg rift valley fever in south africa. . the occurrence of human cases in the orange free state, the north-western cape province, the western and southern transvaal. b. field and laboratory investigation rift valley fever in south africa: . the occurrence of human cases in the orange free state, the north-western cape province, the western and southern transvaal. a epidemiological and clinical findings rift valley fever encephalitis. a description of a case rift valley fever encephalitis clinical studies on rift valley fever. part : ophthalmologic and central nervous system complications rift valley fever affecting humans in south africa: a clinicopathological study rift valley fever ocular manifestations: observations during the epidemic in egypt epidemic maculopathy rift valley fever retinitis macular changes in rift valley fever rift valley fever in man, complicated by retinal changes and loss of vision ocular disease resulting from infection with rift valley fever virus ocular complications of rift valley fever outbreak in saudi arabia ocular complications of rift valley fever ocular manifestations of rift valley fever fatal rift valley fever of man in rhodesia clinico-pathological picture in five human cases died with rift valley fever rift valley fever virus infections in egypt: pathological and virological findings in man epidemic rift valley fever in saudi arabia: a clinical study of severe illness in humans rift valley fever hepatitis complicated by disseminated intravascular coagulation and hepatorenal syndrome acute renal failure associated with the rift valley fever: a single center study rift valley fever as a possible cause of human abortions vertical transmission of fatal rift valley fever in a newborn the s segment of rift valley fever phlebovirus (bunyaviridae) carries determinants for attenuation and virulence in mice genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein nss the pathogenesis of rift valley fever virus in the mouse model rift valley fever virus in mice. iii. further quantitative features of the infective process rift valley fever virus in mice. i. general features of the infection rift valley fever virus hepatitis: light and electron microscopic studies in the mouse pathogenicity of different strains of rift valley fever virus in swiss albino mice the feulgen reaction years on demonstration of nuclear immunofluorescence in rift valley fever infected cells identification of a major non-structural protein in the nuclei of rift valley fever virus-infected cells the carboxy-terminal acidic domain of rift valley fever virus nss protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein rapid accumulation of virulent rift valley fever virus in mice from an attenuated virus carrying a single nucleotide substitution in the m rna the susceptibility of rats to rift valley fever in relation to age pathogenesis of rift valley fever inbred rat strains mimic the disparate human response to rift valley fever virus infection pathogenesis of rift valley fever virus (rvfv) in inbred rats resistance to rift valley fever virus in rattus norvegicus: genetic variability within certain 'inbred' strains active and passive immunization against rift valley fever virus infection in syrian hamsters the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis experimental rift valley fever in rhesus macaques pathogenesis of rift valley fever in rhesus monkeys: role of interferon response prevention of rift valley fever in rhesus monkeys with interferon-alpha the infectivity of rift valley fever for monkeys the pathogenicity of rift valley fever virus for the baboon experimental rift valley fever in lambs and sheeps clinical, virological and serological response of the west african dwarf sheep to experimental infection with different strains of rift valley fever virus experimental infection of young adult european breed sheep with rift valley fever virus field isolates. vector borne zoonotic dis experimental infection of three nigerian breeds of sheep with the zinga strain of the rift valley fever virus abortion in vaccinated sheep and cattle after challenge with rift valley fever virus hydrops amnii in sheep associated with hydranencephaly and arthrogryposis with wesselsbron disease and rift valley fever viruses as aetiological agents teratogenicity of a mutagenised rift valley fever virus (mvp ) in sheep further evaluation of a mutagen-attenuated rift valley fever vaccine in sheep pathogenicity and immunogenicity of a mutagen-attenuated rift valley fever virus immunogen in pregnant ewes characterization of clone , a naturally attenuated avirulent isolate of rift valley fever virus, which is altered in the small segment evaluation of the efficacy and safety of the rift valley fever clone vaccine in sheep the pathogenesis of rift valley fever in lambs distribution of viral antigen in tissues of new-born lambs infected with rift valley fever virus the pathology of rift valley fever. i. lesions occurring in natural cases in new-born lambs the clinical aspects of rift valley fever virus in household pets. i. susceptibility of the dog the clinical aspects of rift valley fever virus in household pets. ii. susceptibility of the cat susceptibility of dogs and cats to rift valley fever by inhalation or ingestion of virus pigs and rift valley fever rift valley fever in camels rift valley fever development and characterization of a rift valley fever virus cell-cell fusion assay using alphavirus replicon vectors helenius, a. entry of bunyaviruses into mammalian cells structure of the rift valley fever virus nucleocapsid protein reveals another architecture for rna encapsidation rift valley fever virus nss mrna is transcribed from an incoming anti-viral-sense s rna segment role of the cytoplasmic tail domains of bunyamwera orthobunyavirus glycoproteins gn and gc in virus assembly and morphogenesis the glycoprotein cytoplasmic tail of uukuniemi virus (bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging mechanism of tripartite rna genome packaging in rift valley fever virus threedimensional organization of rift valley fever virus revealed by cryoelectron tomography electron cryo-microscopy and singleparticle averaging of rift valley fever virus: evidence for gn-gc glycoprotein heterodimers rift valley fever virus m segment: phlebovirus expression strategy and protein glycosylation rift valley fever virus m segment: use of recombinant vaccinia viruses to study phlebovirus gene expression synthesis, proteolytic processing and complex formation of nterminally nested precursor proteins of the rift valley fever virus glycoproteins protein synthesis in rift valley fever virus-infected cells nsm and -kilodalton proteins of rift valley fever virus are nonessential for viral replication in cell culture the nsm proteins of rift valley fever virus are dispensable for maturation, replication and infection rescue of infectious rift valley fever virus entirely from cdna, analysis of virus lacking the nss gene, and expression of a foreign gene rift valley fever virus lacking nsm proteins retains high virulence in vivo and may provide a model of human delayed onset neurologic disease nsm protein of rift valley fever virus suppresses virus-induced apoptosis nss protein of rift valley fever virus blocks interferon production by inhibiting host gene transcription tfiih transcription factor, a target for the rift valley hemorrhagic fever virus a sap complex inhibits ifn-beta expression in rift valley fever virus infected cells replication and physical parameters important for preparing purified junin virus inhibition of pichinde virus replication by actinomycin d inhibition of lymphocytic choriomeningitis virus replication by actinomycin d and -azauridine the effects of actinomycin d on rna synthesis in measles virus-infected cells requirement for host transcription in the replication of sindbis virus rubella virus replication: effect of interferons and actinomycin d the effect of rifampicin, actinomycin d and mitomycin c on poliovirus and foot-and-mouth disease virus replication the inhibition by actinomycin d of poliovirus multiplication hep cells inhibition of coronavirus e replication by actinomycin d differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin d inhibition of replication of lactic dehydrogenase virus by actinomycin rift valley fever virus nss protein promotes post-transcriptional downregulation of protein kinase pkr and inhibits eif alpha phosphorylation nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase deficient signaling in mice devoid of double-stranded rna-dependent protein kinase essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection inhibition of bunyaviruses, phleboviruses, and hantaviruses by human mxa protein complete genome analysis of ecologically and biologically diverse rift valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry viral determinants of virulence for rift valley fever (rvf) in rats mutagen-directed attenuation of rift valley fever virus as a method for vaccine development rna polymerase i-mediated expression of viral rna for the rescue of infectious virulent and avirulent rift valley fever viruses reassortant rvfv between mp- and zh by reverese genetics. the university of texas medical branch use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the g glycoprotein of rift valley fever virus rift valley fever vaccines immunization against rift valley fever virus. studies onthe immunogenicity of lyophilized formalin-inactivated vaccine the development of a formalin-killed rift valley fever virus vaccine for use in man rift valley fever; the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine efficacy of a rift valley fever virus vaccine against an aerosol infection in rats baculovirus expression of the m genome segment of rift valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins evaluation of a formalin-inactivated rift valley fever vaccine in sheep long term existence of rift valley fever virus in immune mice flick, r. a replication-incompetent rift valley fever vaccine: chimeric virus-like particles protect mice and rats against lethal challenge contributions of antinucleoprotein igg to heterosubtypic immunity against influenza virus early presence of ribonucleoprotein antigen on surface of influenza virus-infected cells expression of influenza a virus internal antigens on the surface of infected p cells rift valley fever vaccine for humans respiratory infectivity of a recently isolated egyptian strain of rift valley fever virus mucosal priming alters pathogenesis of rift valley fever bidirectional infection and release of rift valley fever virus in polarized epithelial cells pathogenesis of a phleboviral infection (punta toro virus) in golden syrian hamsters induction of severe disease in hamsters by two sandfly fever group viruses, punta toro and gabek forest (phlebovirus, bunyaviridae), similar to that caused by rift valley fever virus punta toro virus infection of c bl/ j mice: a model for phlebovirusinduced disease assembly and polarized release of punta toro virus and effects of brefeldin a immunoelectron microscopy of rift valley fever viral morphogenesis in primary rat hepatocytes the effects of aging in vitro and interferon on the resistance of rat macrophages to rift valley fever virus (rvfv) a new mouse model reveals a critical role for host innate immunity in resistance to rift valley fever role of hepatocytes and kupffer cells in agedependent murine hepatitis caused by a phlebovirus, punta toro effect of macrophage source and activation on susceptibility in an age-dependent model of murine hepatitis caused by a phlebovirus tlr deletion limits mortality and disease severity due to phlebovirus infection toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis fagard, r. stat and pathogens, not a friendly relationship punta toro virus (bunyaviridae, phlebovirus) infection in mice: strain differences in pathogenesis and host interferon response the authors (ti and sm) were supported by grant number u ai through the western regional center of excellence. ti was also supported by r ai from the national institute of allergy and infectious diseases and internal funding from the sealy center for vaccine development at the university of texas medical branch. sm was also supported by a grant from department of homeland security. key: cord- - rd o authors: wong, mun-teng; chen, steve s-l title: emerging roles of interferon-stimulated genes in the innate immune response to hepatitis c virus infection date: - - journal: cell mol immunol doi: . /cmi. . sha: doc_id: cord_uid: rd o infection with hepatitis c virus (hcv), a major viral cause of chronic liver disease, frequently progresses to steatosis and cirrhosis, which can lead to hepatocellular carcinoma. hcv infection strongly induces host responses, such as the activation of the unfolded protein response, autophagy and the innate immune response. upon hcv infection, the host induces the interferon (ifn)-mediated frontline defense to limit virus replication. conversely, hcv employs diverse strategies to escape host innate immune surveillance. type i ifn elicits its antiviral actions by inducing a wide array of ifn-stimulated genes (isgs). nevertheless, the mechanisms by which these isgs participate in ifn-mediated anti-hcv actions remain largely unknown. in this review, we first outline the signaling pathways known to be involved in the production of type i ifn and isgs and the tactics that hcv uses to subvert innate immunity. then, we summarize the effector mechanisms of scaffold isgs known to modulate ifn function in hcv replication. we also highlight the potential functions of emerging isgs, which were identified from genome-wide sirna screens, in hcv replication. finally, we discuss the functions of several cellular determinants critical for regulating host immunity in hcv replication. this review will provide a basis for understanding the complexity and functionality of the pleiotropic ifn system in hcv infection. elucidation of the specificity and the mode of action of these emerging isgs will also help to identify novel cellular targets against which effective hcv therapeutics can be developed. hepatitis c virus (hcv) infects more than million people worldwide and represents a heavy burden to global health, with the highest prevalence rates found in africa and the eastern mediterranean. acute hcv infection is asymptomatic, and in % of infected individuals, the virus persists and progresses to chronic liver diseases, including fibrosis, steatosis, cirrhosis and hepatocellular carcinoma. , furthermore, hcv is a major cause of type i mixed cryoglobulinemia, which occurs in % of patients. using the hcv genotype a isolate japanese fulminant hepatitis- genome-based cell culture-derived infectious hcv (hcvcc), zhong et al. demonstrated that hcv and cells coevolve in vitro during chronically persistent infection, which involves the selection of viral mutants with increased infectivity and cells with resistance to viral entry and/or rna replication. in this coevolution process, hcv exhibits multifaceted interactions with the host cells, and these cellular stress responses subsequently affect virus replication. for instance, infection with hcvcc or expression of the japanese fulminant hepatitis- genome has been shown to trigger cytopathic effects, endoplasmic reticulum (er) stress, the unfolded protein response (upr), autophagy and the innate immune response. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the competition between this virus and host cells, viral infection often triggers a first-line host defense through the production of type i interferon (ifn), which is a broadly acting antiviral cytokine, and inflammatory cytokines. these cytokines confer an antiviral state on the host cells, thereby interfering with viral replication. , with the ability to enhance the immune response for virus clearance or to inhibit viral replication, ifn-based therapies have been used to treat hcvinfected patients for over two decades. to guard against viral infection, the host cell has developed multiple restriction strategies to limit viral infection. the expression of many of these restriction factors is subject to transcriptional regulation by ifn. , upon infection by viruses such as hcv, viral rna is first sensed by cellular pattern recognition receptors (prrs), and the prr-mediated recruitment of adaptor proteins and the activation of downstream signaling lead to ifn production. , , after binding to its receptor (ifnar) complex present on the cell surface, ifn triggers the janus kinase (jak)/signal transducer and activator of transcription (stat) pathway to drive the synthesis of over ifn-stimulated genes (isgs), which block virus replication at different phases of the virus replication cycle. [ ] [ ] [ ] [ ] these isgs are usually not synthesized at the basal state, but are induced to express and mediate the antiviral effector functions of ifn upon viral infection or ifn treatment. in response, viruses have developed elaborate strategies to escape the ifn antiviral system by blocking the expression or antiviral functions of ifn. , , therefore, the host and hcv maintain a homeostatic state, allowing tightly restricted viral replication without killing the host. , [ ] [ ] [ ] recent studies on genome-wide sirna screens have also added new candidates to this growing list of anti-hcv isgs. these findings highlight the complexity and pleiotropic roles of ifn and its induced isgs in modulating innate immunity and virus replication. nevertheless, the complete spectrum of isgs and their functionality in suppressing hcv replication have yet to be defined. , in this review, we focus on the molecular aspects of the type i ifn system and its effector mechanisms in modulating hcv replication. first, we briefly discuss the signaling triggered by the retinoic acid-inducible gene -like receptor (rlr) and the toll-like receptor (tlr), which leads to type i ifn synthesis and ifn-mediated signaling pathway activation, resulting in the expression of a variety of effector isgs. we also summarize the strategies that hcv uses to escape ifn antiviral surveillance. additionally, we highlight what is currently known regarding the pivotal isgs in viral infections, with an emphasis on their anti-hcv activities, and the emerging isgs identified from recent genome-wide sirna screens in relation to anti-hcv activities. finally, we discuss the potential functions of several critical cellular factors, such as high-mobility group box (hmgb ) and immunity-related gtpase family m (irgm), and cellular pathways, such as upr and autophagy, during hcv infection. although these cellular determinants are not stimulated by ifn, these factors critically control the host immune response. therefore, these determinants may also play crucial roles in modifying hcv replication. this review provides a perspective for a better understanding of the anti-hcv mechanisms of ifn, isgs and several critical cellular determinants known to contribute to the regulation of innate immunity. the gathered information not only provides a clearer picture for the specificity, functionality and complexity of the ifn system and its effector mechanisms in the control of hcv infection, but also helps to identify novel cellular targets against which efficacious therapeutic strategies can be developed. clinically, the identification of new isgs will also help to optimize the current ifn-based therapy and to provide a basis for more accurate predictions of ifn treatment outcomes. hcv is an enveloped, positive-sense, single-stranded rna virus classified within the genus hepacivirus in the flaviviridae family. currently, hcv isolates are classified into seven major genotypes, i.e., genotypes through , and an array of subtypes. hcv genotypes differ by %- % in genome sequence, whereas subtypes within each genotype can differ by least %. genotype is the most prevalent ( %), followed by genotype ( %); genotypes , and (cumulatively approximately %); and genotype (less than %). different genotypes exhibit distinct geographic distributions. genotype predominates in america and europe, genotype in japan, genotype in asia, genotype in africa and middle east and genotype in southeast asia. hcv is transmitted via blood transfusion, intravenous drug abuse, unsafe therapeutic injection, liver transplantation and other risk factors. the combination of pegylated ifn-a and ribavirin is the standard therapy for hcv infection. however, this treatment is associated with side effects, and the efficacy of this regimen varies among genotypes, limiting the success rate of this treatment. compared with genotype , infection with genotypes a and b results in more severe liver disease and low responsiveness to ifn therapy. seventy-one percent of patients with genotype infection respond to ifn therapy, whereas only % of genotype a and % of genotype b show a response. patients infected with genotype generally show higher sustained virological responses to ifn therapy than genotype infected patients, whereas genotype -infected patients show a lower sustained virological response compared with genotype -infected patients. the heterogeneity of hcv genotypes also translates to differences in the manifestation of liver disease. for example, hepatic steatosis is most common in patients infected with genotype and is attributed to its core protein. recently, the use of active direct-acting antiviral molecules to block hcv infection has led to substantial improvements in sustained virological response rates in genotype -infected patients. however, the use of these drugs may allow selection of resistant variants if direct-acting antiviral monotherapy is adopted, and a high relapse rate occurs after direct-acting antiviral treatment is discontinued. the approximately . -kb hcv genome contains a single open reading frame flanked by untranslated regions (utrs) at its and ends (figure ). the internal ribosome entry site (ires) located in the -utr directs cap-independent translation, whereas the -utr contains sequences critical for viral replication and translation. the -utr (positioned at nucleotides - of the hcv genome) contains a poly(u/uc) (pu/uc) tract located at nucleotide positions - , which was identified as an hcv pathogen-associated molecular pattern (pamp) that triggers rlr-mediated type i ifn production ( figure ). translation of the hcv genomic rna produces a single polyprotein of approximately amino acids, which is further processed by cellular and viral proteases to yield the structural proteins core, e and e and the non-structural (ns) proteins p , ns , ns , ns a, ns b, ns a and ns b (figure ). ns proteins participate in different phases of the viral replication cycle (figure ). for example, ns to ns b proteins are important for rna replication. jones et al. showed that p and ns are required for entry and assembly of the virus. other researchers have also reported that ns , - ns / a and ns a , are involved in virion assembly and production. hepatocytes are the primary target cells for hcv replication. upon infection, the virus particle circulating in the blood biochemically resembles the very low-density lipoprotein, which is rich in apolipoprotein (apo) e and apob. , first, the apolipoprotein-associated lipoviral particle (lvp) attaches to glycosaminoglycan and low-density lipoprotein receptor and then interacts with cluster of differentiation (cd ) and scavenger receptor class b number (figure ). the lvp is subsequently translocated to the tight junction of hepatocytes where the lvp binds to the tight junction proteins claudin- and occludin followed by internalization of the hcv particle via ph-dependent endocytosis, which occurs on the plasma membrane ( figure ). in addition to these receptors, cell surface molecules, such as epidermal growth factor receptor, ephrin receptor a and niemann-pick c -like l cholesterol uptake receptor, are also essential for virus internalization. subsequent to internalization, the acidic ph in the endosome triggers fusion of the viral envelope with the endosomal membrane, allowing the release of the viral genome into the cytoplasm ( figure ). hcv genomic translation occurs at the rough er, and hcv rna replicates in an er-derived or er-associated lipid-rich environment termed the membranous web ( figure ). all hcv ns proteins except for ns are involved in viral rna replication. the ns proteins are colocalized with the replicating viral rna on a light density, detergent-resistant cytoplasmic membrane structure termed a 'lipid raft'. lipid droplets (lds), which comprise a neutral lipid core with a single phospholipid layer, serve as energy storage sites and reservoirs of neutral lipids in adipose tissue and hepatocytes. lds are indispensable for viral rna replication and infectious virus formation. during the initial stage of virus assembly, hcv core protein interacts with lds, and the viral replication complex is also directed to lds in an ns a-and core-dependent manner, allowing encapsidation of the viral rna by the core protein and assembly of the nucleocapsid ( figure ). , additionally, the interaction of ns and ns a with actin and tubulin in the microtubule network mediates translocation of the hcv replication complex to lds. the late stage of virus assembly, which occurs in the lumen of the er, involves the acquisition of a lipid envelope, the embed- assembly and budding liver cell figure hcv replication cycle. as shown, the hcv lvp is coated with apob and apoe, which are marked by light green and light blue stripes, respectively, on its surface. the lvp attaches to srb and to cd and further interacts with the tight junction protein claudin- and with occludin. virus entry into cell proceeds through receptor-mediated endocytosis at the cell surface. subsequent to internalization, the viral envelope fuses with the endosomal membrane under acidic ph, and the viral genome is uncoated and released into the cytoplasm. to dissect these two events, internalization and fusion are conventionally depicted as two seemingly separate steps in the cytoplasm. viral rna is translated at the er to produce the polyprotein, which is subsequently processed into mature structural and non-structural proteins. viral non-structural proteins, in conjunction with host factors, form a membranous web where viral rna replication occurs. hcv particle assembly most likely initiates near the er and ld where core protein and viral rna accumulate. finally, hcv particles are secreted into the extracellular milieu via the secretory pathway. viral replication and assembly occur in the proximity of lds and in lipid raft microdomains, which are shown in the inserted dashed rectangle. apo, apolipoprotein; cd , cluster of differentiation ; er, endoplasmic reticulum; hcv, hepatitis c virus; ld, lipid droplet; lvp, lipoviral particle. ding of e and e into the envelope and the formation of the nascent virion ( figure ). then, the nascent virus particles associate with apob, apoe and other very low-density lipoprotein lipids to form lvps. finally, lvps are released from cells through the very low-density lipoprotein secretion pathway or the endosome secretory pathway ( figure ). similar to the ns proteins, p plays numerous crucial functions in virion assembly and egress. p , an integral, oligomeric membrane protein consisting of amino acids, is grouped into the family of viroporins that form membrane pores or channels. in functioning as an ion channel, p modulates membrane permeability to facilitate virus entry by promoting virus uncoating and to enhance assembly or release. p conducts ions across the membrane, and this channel activity can be abrogated by the drug amantadine and iminosugar derivatives. , during maturation and egress, the ion channel activity of p maintains the ph gradients within the secretory pathway and thereby stabilizes the hcv particle. in addition, p has been shown to be necessary for capsid assembly and envelopment because mutations in p result in the accumulation of incompletely assembled capsids that are unable to encapsidate viral rna. the ifn systems constitute the first-line defense mechanism against viral infection in humans. based on their antiviral properties, ifns are grouped into three classes: type i, ii and iii ifns. in humans, type i ifns comprise a large group of molecules encoded by multiple genes, mainly ifn-a and ifn-b, and other genes such as ifn-e, ifn-k and ifn-v. ifn-a and ifn-b combat viruses directly by inhibiting virus replication or indirectly by inducing the innate immune response. most cell types can elicit a type i ifn response by activating the tlr, rlr and jak-stat pathways. , type ii ifn contains only one member, ifn-c. unlike type i ifns, which are elicited as a direct response to viral infection, ifn-c is secreted by natural killer cells and mitogenically activated t cells. ifn-c exerts potent anti-hcv activity in vitro and mediates antiviral t-cell responses. it has also been reported that ifn-c inhibits hcv infection by downregulating claudin- and cd . type iii ifns consist of three members, termed ifn-l , ifn-l and ifn-l or il- (l ) and il- a/b (l / ). as with type i ifns, viral infection also directly activates type iii ifns. however, the antiviral properties and the mechanisms of action of type iii ifns remain unknown. type iii ifns can be secreted by many cell types, but their receptors show a limited tissue distribution. hcv infection results in type iii ifn induction predominantly in the human liver. despite modulation by the irf and nf-kb pathways for induction of type iii and type i ifns, these two systems upregulate distinct subsets of isgs with different kinetics of induction. during hcv infection, cells produce type i ifn to counteract viral infection, to modulate viral replication and to activate natural killer cells, dendritic cells and kupffer cells. recognition of pamps by prrs, including tlrs and rlrs, triggers ifn synthesis and ifn-mediated cascade signaling pathways, leading to the production of type i ifn and a wide range of isgs to mediate ifn antiviral activity ( figure ). , [ ] [ ] [ ] upon virus infection, tlrs and rlrs operate through different signaling pathways, depending on the nature of the viral signals. tlrs are expressed and localized in the intracellular compartment, similar to endosomes, or on the cell surface. , unlike rlrs, tlrs potentially detect viral double-stranded rna (dsrna) released by cells into the extracellular milieu ( figure ). three types of tlrs, i.e., tlr , tlr and tlr , are involved in the recognition of virus infections. tlr detects the dsrna formed during the replication of positive-stranded rna viruses, whereas tlr recognizes the urine-rich ribonucleotide region of rna, and tlr senses dna pamp motifs encoding cpg dinucleotides. upon binding to a pamp, tlr dimerizes and initiates the binding of its cytosolic toll-il- receptor to the adaptor protein toll-il- receptor domain-containing adaptor inducing ifn-b (trif), resulting in the association of tlr with trif ( figure ). , , this interaction leads to the recruitment of tumor necrosis factor (tnf) receptor-associated factor (traf) , traf and the traf family member nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) activator-binding kinase (tbk ), resulting in the phosphorylation and activation of ifn regulatory factor (irf) by tbk and by inhibitor of kappa b (ikb) kinase-related kinase (ikk)e. , after phosphorylation, the irf protein dimerizes and is translocated into the nucleus to form an enhanceosome complex with nf-kb and other transcription factors, thereby inducing the expression of target genes, such as ifns. moreover, the binding of viral dsrna to tlrs also activates nf-kb activity and pro-inflammatory cytokine synthesis through the interaction of trif with receptor-interacting protein- . tlr and tlr bind to myeloid differentiation pro-inflammatory response (myd ) and activate il- receptor-associated kinase (irak) and traf , followed by the activation of ikka/b, which in turn activates nf-kb through phosphorylation, polyubiquitination and proteasomal degradation of its associated inhibitor ikba. migration of nf-kb into the nucleus results in ifn production ( figure ). the rlr receptors consist of rig-i, melanoma differentiation-associated protein (mda ) and laboratory of genetics and physiology- . , , rig-i recognizes the hcv replication intermediate dsrna within hours of infection, which triggers the downstream signaling before the viral protein is extensively synthesized (figure ). rig-i senses the short, non-self dsrnas with -triphosphates, whereas rnas lacking -ppp, such as picornaviruses, are recognized by mda . both rig-i and mda contain two n-terminal caspase activator and recruitment domains (cards). the recognition of dsrna by rig-i is dependent upon the atp-driven translocase activity of cards and helicases, and binding to dsrna induces conformational changes in rig-i that facilitate its oligomerization and translocation from the cytosol to the mitochondrial surface. , on the mitochondria, rig-i engages via its tandem cards with the card of its downstream effector, mitochondria antiviral signaling protein (mavs), which is also termed ifn-b promoter stimulator- , virus-induced signaling adaptor or card adaptor inducing ifn-b ( figure ). , the chaperone protein - - e and the ring finger domaincontaining e ubiquitin (ub) ligase triple motif-containing protein (trim) also participate in this process. trim mediates the ubiquitination of rig-i at position lys- , which is important for mavs binding and for ifn production. the interaction between rig-i and mavs promotes the formation of a signaling complex on the mitochondrial surface that recruits and activates the downstream classical ikk complex, ikka/ikkb, and two non-classical ikk-related kinases, tbk and ikke. , activation of tbk and ikke leads to the phosphorylation, dimerization and nuclear translocation of the transcription factor irf ( figure ). traf , traf and mitogen-activator protein kinase/extracellular signal-regulated kinase (erk) kinase (mekk ) are also recruited to mavs to activate nf-kb. the canonical ikka and ikkb induce nf-kb-dependent gene transcription via phosphorylation, polyubiquitination and proteasomal degradation of ikba, thereby resulting in the release and nuclear migration of nf-kb. nf-kb activation involves the interaction of card with b-cell lymphoma/leukemia protein. activated nf-kb and irf are translocated into the nucleus to form an enhanceosome, thereby stimulating the expression of ifn and inflammatory cytokines with the help of other cellular factors, such as activating transcription protein and c-jun. then, secreted ifn binds to ifnar on the cell surface and triggers the jak-stat signaling pathway ( figure ). , following ifnar receptor binding, tyrosine kinase- and jak are activated and phosphorylate stat and stat to form a heterodimer, which subsequently recruits irf to form the transcription factor ifn-stimulated gene factor (isgf ). then, isgf is translocated into the nucleus, binds to ifnsensitive responsive element (isre) and transactivates the expression of various isgs, such as - oligoadenylatesynthetase ( - oas)/ , -linked oligoadenylate ( - a)-dependent, latent endoribonuclease (rnase l), dsrna-dependent protein kinase r (pkr), and irf ( figure ). acute hcv infections can be spontaneously cleared in some infected individuals, suggesting that the innate immunity induced by hcv pamp sensing can control acute viral infection. , however, % of acutely infected people are not effectively cleared of hcv infection, and these patients may further develop chronic infection, suggesting that hcv has developed strategies to escape or to counteract the host immune response, leading to the emergence of resistance to ifn therapy. in this regard, several hcv proteins have been shown to block host antiviral responses, resulting in progression to chronic hcv infection ( figure ). , , obtaining further data regarding hcv evasion of host innate immunity will certainly improve ifn-based therapy outcomes. core protein hcv core protein is involved in the formation of the viral nucleocapsid and modulates many cellular functions, including transcription and signal transduction. expression of the full-length hcv genome or core protein downregulates ifn signaling by depressing stat tyrosine phosphorylation, which then blocks stat heterodimerization with stat and inhibits ifn signal transduction and isg expression ( figure ) . in addition, expression of core protein induces synthesis of suppressor of cytokine signaling (socs ) in hepg cells. socs is an important repressor of the jak-stat pathway due to its ability to inhibit stat phosphorylation ( figure ). , thus, hcv core protein induces socs and suppresses ifn-mediated isg expression. - socs expression is upregulated in chronically hcv-infected patients who are ifn non-responders compared with responders. core protein expression has also been demonstrated to inhibit irf synthesis, transcriptionally repressing several isgs, such as il- , il- and pkr. many viruses use molecular mimicry as an important immune evasion strategy to promote virus survival and persistence. viruses express proteins that are structurally similar to host defense proteins, and these viral proteins can act as immune modulators. hcv employs this molecular mimicry strategy to resist type i ifn through its e envelope protein. , e comprises a -amino acid sequence identical to eukaryotic initiation factor a (eif a) and pkr. this domain operates to prevent pkr-dependent phosphorylation of eif a and repression of protein synthesis, thus possessing an ability to resist type i ifn treatment ( figure ). , ns / a the hcv ns / a protease is not only responsible for the maturation of ns proteins, viral rna replication and virion morphogenesis but is also important for suppressing the host antiviral system. , , , , the ns / a complex is anchored to the intracellular membrane through the ns a transmembrane domain and the amphipathic a-helix at the ns n-terminus. all these domains facilitate cleavage of their two cellular targets, mavs and trif, which act as key players in type i ifn production ( figure ). , mavs is an essential antiviral signaling protein in the rlr system and, therefore, is an ideal target for viral immune evasion. ns a serves as the primary membrane subcellular targeting subunit to escort ns / a to mavs. ns / a binds to mavs on mitochondria and cleaves mavs at cys- , resulting in the dislocation of the n-terminal portion of mavs from mitochondria and the suppression of ifn production ( figure ). the hydrophobic amino acid stretch in the ns amphipathic a-helix is also required for controlling rig-i signaling. cleavage of mavs and reduction of ifn levels have been observed in chronically hcv-infected patients. thus, ns / a-mediated cleavage of mavs rig-i signaling impairs ifn synthesis. additionally, the ns / a protease also cleaves trif, an adaptor protein linking tlr to kinases responsible for activating irf and nf-kb ( figure ). , cleavage of trif interferes with poly(i:c)-activated tlr signaling and irf and nf-kb activation, thereby limiting the expression of multiple host defense genes and enhancing hcv persistence. stimulator of interferon gene (sting), which is also known as mediator of irf activation (mita), is a -kda protein mainly localized to the er. in response to dsdna transfection or dna virus infection, sting plays a crucial role in the activation of transcription pathways, essential for effective innate immune signaling. upon dsdna stimulation, sting polymerizes and translocates from the er to a cytoplasmic punctate structure where the sting polymer provides a platform to connect tbk with irf , which phosphorylates irf , thereby triggering downstream signaling. in viral infection, ns b from yellow fever virus (yfv) blocks the induction of the ifn production pathway through an interaction with sting. ns b from dengue virus (denv) acts as a protease to cleave sting, thereby shutting down ifn signaling. in hcv infection, ns b interacts with and sequesters sting on the er to inhibit the association of sting with tbk, suppressing ifn signaling ( figure ). , therefore, targeting sting to inhibit innate immunity might be beneficial for virus survival. the mature hcv ns a is present as two phosphoproteins, the hypophosphorylated p and hyperphosphorylated p . , ns a phosphorylation occurs at multiple serine residues, such as serine , and upstream of the ifn sensitivity determining region (isdr) of ns a, which spans residues - (based on genotype b hcv-j strain), and these serine residues are important for hyperphosphorylation. , the functional and locational significance of ns a p and p remains unclear; however, maintenance of these two forms at a specific ratio is critical for hcv replication. functions as a pleiotropic protein that modulates the host environment to favor virus replication and persistence. additionally, ns a binds to myd , which is a major adaptor molecule in the tlr pathway, and inhibits the recruitment of irak to myd , attenuating tlr signaling and impairing cytokine production. a sequence within the ns a isdr, which spans residues - , was shown to be responsible for interaction with the death domain of myd in macrophage cells. pkr is an ifn-induced gene product that is activated by binding to dsrnas commonly produced during viral replication. ns a rescues hcv replication in ifn-treated cells and inhibits ifn antiviral activity by binding to pkr and blocking pkr autophosphorylation and eif a phosphorylation. , ns a expression is sufficient to rescue the replication of an ifn-sensitive virus. the interaction of pkr with ns a requires the isdr that overlaps a broader pkr-binding region, residues - , and results in the inhibition of pkr activation and resistance to ifn in hcv-expressing cells. , consistent with this mechanism, mutations in or deletion of isdr correlate with sensitivity to ifn-a-mediated antiviral activity. , , moreover, meta-analysis and long-term follow-up support the association of this isdr region with the outcome of ifn therapy. this region, which encompasses a genetically flexible domain that allows mutations to occur, is the key site of adaptation to ifn therapy and influences the fitness of hcv rna replication. in contrast, other studies suggest that the inhibitory effect of ns a on ifn may be independent of pkr. ns a increases expression of il- , also known as chemokine cxcl , by upregulating the il- promoter, which in turn, inhibits ifn antiviral activity and facilitates virus infection. , in a cell culture model, il- -positive cells are associated with chronic hcv infection, and il- removal mitigates hcv replication. importantly, the serum level of il- is elevated in chronic hepatitis c patients compared with control individuals or is higher in ifn non-responders relative to responders. these observations suggest that ns a expression increases il- production, which somehow perturbs the ifn antiviral pathway. moreover, ns a impedes the - oas/rnase l system to inhibit ifn signaling. the - oas/rnase l antiviral pathway is present in virtually every cell. this pathway involves the activation of a latent endoribonuclease and degrades hcv mrna with a dsrna structure during replication. ns a physically binds to - oas through amino acid residues - of ns a. a single point mutation at amino acid of ns a affects the ns a and - oas binding and the antiviral activity of the - oas/rnase l system. thus, ns a inhibits ifn antiviral activity in an isdr-independent manner. moreover, ifn-resistant strains, such as genotypes a and b, have fewer rnase l recognition sites in their genomes than the ifn-sensitive strains, such as genotypes and , providing a means for ifn-resistant strains to escape from nucleotylic cleavage. apoptosis also plays a key role in the host defense system by restricting viral spread and persistence. blocking apoptosis could be critical for the establishment of life-long persistence in the host organism. ns a was shown to block the activation of caspase and to inhibit proteolytic cleavage of the death substrate poly(adp-ribose) polymerase in tnf-a-induced cells. adenovirus infection in ns a-transgenic mice downregulates and upregulates the expression of t-box transcription factor and trans-acting t cell-specific transcription factor , respectively, resulting in lower ifn-c expression and a delay in virus clearance. furthermore, stable expression of ns a in the human hepatoma cell line huh decreases sensitivity to tnf-a-mediated apoptosis, and activation of caspase- , and by tnf-a is inhibited in ns a-expressing cells. thus, ns a protects cells from tnf-a-mediated apoptotic death. hcv-induced er stress hcv protein expression can induce an er stress response and lead to calcium release from the er, which in turn activates the cyclic amp responsive element-binding protein that binds to the cyclic amp responsive element in the promoter of protein phosphatase a (pp a), resulting in upregulation of pp a. expressed in essentially all cell types, pp a is a serine/threonine phosphatase that is involved in multiple cellular processes, such as the cell cycle, signal transduction and stress response. , increased expression of pp a has been observed in a cell line inducibly overexpressing hcv protein, in liver extracts from hcv transgenic mice and in liver biopsies from patients with chronic hepatitis c. duong et al. showed that upregulation of pp a by hcv can inhibit the enzymatic activity of protein arginine methyltransferase (prmt ), which leads to decreased methylation of stat . hypomethylated stat is more prone to bind to protein inhibitor of activated stat and inhibits stat dimerization, resulting in impaired nuclear translocation into the nucleus, binding of stat to the isre, and isg production. thus, hcv-induced pp a activation disrupts the ifna-induced antiviral state, leading to hcv evasion of innate immunity. these authors also showed that prmt can methylate hcv ns at arginine , resulting in the inhibition of ns helicase activity. therefore, hcv-mediated pp a upregulation enhances ns helicase activity by inhibiting prmt enzymatic activity, which in turn facilitates virus replication. human genomes encode hundreds of isgs, and the first isg, k, was discovered more than years ago. synthesis of some isgs can be triggered by viral infection without ifn production. some isg products can directly regulate cellular processes, such as protein synthesis and cell growth, survival and apoptosis, whereas others may modify the ifn antiviral activity against invading viruses. the gene products of isgs can target many steps in the hcv replication cycle to limit viral replication. many pamp receptors and their subsequent sig-naling partners are also isgs. isgs expressed at the basal level provide antiviral surveillance before ifn activation or therapy; however, their levels markedly increase after ifn production. in the innate immune response to virus infection, viral rna acts not only as a inducer of the production of ifn and its effector functions but also as a substrate and product for cellular enzymes, such as pkr and - oas/rnase l. the inverse correlation between the upregulated expression of isgs, such as oas-like (oasl), isg and viperin, in liver biopsies from hcv-infected patients and infected hepatocytes and decreased viral rna levels suggest the anti-hcv activities of these isgs. in this section, we will highlight the involvement of isgs that are critical for modulating innate immunity in hcv replication, and the potential functions of these isgs, as outlined in table . rig-i rig-i, which is encoded by the ddx gene and which belongs to the dexd/h box rna helicase (ddx) family, is a key player in the defense against invasion by many rna viruses. rig-i senses the intracellular viral components and initiates antiviral responses to stimulate ifn production (table ). in turn, ifn activates the transcription of rig-i, hence forming a positive feedback loop for amplifying antiviral signals. studies have shown that rig-i is essential for eliciting an immune response against vesicular stomatitis virus (vsv), sendai virus, newcastle disease virus (ndv) and hepatitis c virus. , , knockout of the rig-i gene in mouse embryonic fibroblasts severely limits type i ifn production and isg activation, thereby potentiating viral replication; conversely, overexpression of rig-i restricts viral replication. in addition, upregulation of rig-i gene expression has been observed in ifn-treated human dendritic cells, suggesting that rig-i serves as an isg. rig-i contains two tandem cards at its n-terminal region, with a repressor domain in its c-terminal region. the card is responsible for downstream signaling and activation of type i ifn after recognition of non-self rna, whereas the repressor domain is essential for the autoregulation and recognition of viral rnas. without viral stimulation, the card interacts with the helicase domain, placing rig-i in an auto-inhibitory state and disabling signal transduction. upon binding to viral rna, rig-i undergoes conformational changes that expose the card, allowing rig-i to be ubiquitinated. rig-i is ubiquitinated by two different ligases, trim and ring finger protein . trim ubiquitinates rig-i at lysine to mediate the antiviral response, whereas ubiquitination by ring finger protein regulates the degradation of rig-i by the proteosome, thereby downregulating rig-i-mediated signaling. ubiquitination by trim induces rig-i to form a tetramer, promoting the cards of rig-i to engage with the cards of mavs. this results in the accumulation of mavs on the mitochondrial membrane and the activation of ikk and tbk , which, in turn, activates the transcription of nf-kb, irf and irf to promote ifn production. moreover, ubiquitination by trim also prevents cards from interacting with the helicase domain to reinstate the auto-inhibitory state. ubiquitination at lysine is crucial for rig-i function because a mutation at this residue renders rig-i unable to bind to mavs, thus abrogating downstream signaling. ddx is also a dexd/h box helicase whose function remains unclear. ddx slightly resembles the yeast ski protein, which is a cofactor of the rna exosome required for controlling host rna quality. ddx , which is the human homolog of yeast ski, and the rna exosome exhibit antiviral activity against monkey leukemia virus and sindbis virus (sinv). ddx expression is upregulated during infections of measles virus (mev) and hcv. , the ddx mrna level is robustly upregulated in human fetal liver cells within - h after hcv infection, providing a means to initiate the antiviral mechanism. unlike rig-i, ddx does not contain the cards to interact with mavs. after viral infection, ddx is induced and binds to rig-i as well as mda and laboratory of genetics and physiology- and promotes the binding of rig-i to dsrna. , ddx is essential for type i ifn expression during dna virus infection and is induced to suppress viral replication in a rlr-dependent manner (table ). ddx knockdown reduces the expression of type i ifn after hcv, hiv and yfv infections. irf irf was first identified as a transcriptional activator of the ifn-a/b gene. in unstimulated cells, irf is expressed at a low level; however, its expression is increased by the induction of ifn-a/b, tnf-a, il- and viral infection. nevertheless, the precise pathway leading to irf activation by virus infection remains elusive. irf activation may proceed through a pkrdependent pathway after virus infection. pkr indirectly phosphorylates irf and activates its dna-binding properties. thus, activated irf regulates the promoter function of ifna/b promoter and acts as a modulator of many isgs by binding to the isre in the promoter region, thereby regulating viral replication (table ) . , irf controls the ifn antiviral response by affecting a set of isgs, such as irf and irf . irf cooperates with irf and irf to regulate cellular antiviral genes, such as ifn-a/b. hcv infection increases the level of irf , which may affect other irf pathways and isg expression, thereby leading to a reduction in viral replication. irf overexpression induces an antiviral state that affects various viruses, including ndv, vsv and hcv. , the expression level of irf is reduced in cells harboring hcv subgenomic replicons (sgrs), whereas irf overexpression in these cells increases the isre activity and attenuates hcv replication. additionally, hcv infection mediates irf expression, thus affecting the intracellular level of hcv rna. however, hcv may evade the irf anti-hcv effect through core-mediated suppression of irf synthesis. irf is an essential transcription factor for the induction of ifn-a/b and isgs. all of the elements of ifn responses, either innate or adaptive immunity, are regulated by irf . irf is constitutively expressed in certain cells, such as macrophage and plasmacytoid dendritic cells, priming these cells for rapid ifn production. during infection, ifn-a/b binds to its receptor and activates the jak-stat pathway, resulting in irf expression. then, irf is phosphorylated, forms a heterodimer with irf and is translocated into the nucleus. in the nucleus, the irf -irf heterodimer binds to the irf elements in the promoter region of ifn-a genes, leading to enhanced expression of the ifn-a subtype and a diverse range of isgs (table ) . , in turn, these events increase the abundance of rig-i and viral pamp signaling components, whereas sustained signaling serves to amplify ifn production. , moreover, irf induces expression of other isgs without activating ifn signaling. thus, irf -mediated transcriptional cascades serve as an intrinsic antiviral mechanism allowing rapid isg expression before ifn production. irf plays an important role in eliminating hcv infection. sirna knockdown of irf decreases ifn-a production and increases the hcv titer. , mice lacking irf show rapidly lethal infection by west nile virus (wnv) and high virus burdens. irf deficiency represses the induction and accumulation of ifn-a, thus favoring wnv replication. although hcv seems to suppress the basal expression of irf , tlr stimulation activates irf and suppresses hcv replication. this observation suggests that hcv may only partially inhibit irf activity in hcv-expressing cells. pkr pkr, which is also known as eif ak , is a serine/threonine kinase that phosphorylates eif a in response to virus infection. this ifn-inducible kinase has two distinct activities: autophosphorylation, resulting in its activation, and phosphorylation and inactivation of eif a. through phosphorylation events, pkr mediates the inhibition of translation initiation of both cellular and viral mrna. [ ] [ ] [ ] it is well documented that the anti-hcv activity of pkr occurs through its translational control (table ) . [ ] [ ] [ ] however, viruses have evolved elaborate strategies to counteract the detrimental effects of pkr. hcv ires activity has been shown to be resistant to pkr activation in cells harboring hcv sgr and in the hcv infection model (table ) . [ ] [ ] [ ] mechanistically, viruses may use their proteins to impede the dsrna-dependent pathway in various ways, such as sequestering dsrna, inhibiting pkr activation, producing pkr pseudosubstrates, activating antagonist phosphatases and degrading pkr. as indicated above, hcv employs ns a and e to antagonize pkr function, resulting in resistance to ifn and a blockade of the pkr-mediated inhibition of viral protein synthesis (table ) . analogous to alphaviruses sinv and semliki forest virus, hcv can activate pkr and eif a phosphorylation to enhance its own viral protein translation (table ) . , compared with other previously studied dsrnas, domains iii-iv of the hcv ires were shown to bind to the n-terminal dsrnabinding domain of pkr, leading to increased pkr autophosphorylation and activation. additionally, cap-dependent but not hcv ires-mediated translation is inhibited by pkr and eif a phosphorylation. these results indicate that while escaping the deleterious effects of pkr activation, hcv can employ its structured ires to direct its own protein translation. karamichali et al. demonstrated that activated pkr or silencing pkr upregulates or downregulates hcv ires activity (table ) . these authors further showed that the inhibitory effect of ns a on ires-dependent translation occurs through pkr inactivation. in contrast, hcv can translate its viral protein via a bacterial-like pathway that uses eif b, which is an analog of bacterial if , and eif , instead of eif a and its gtpase-activating protein eif , as the initiation factor (table ) . the use of eif a-independent translation initiation provides an alternative tactic for hcv translation when eif a is inactivated by phosphorylation under stress conditions. many lines of evidence have revealed that hcv-mediated phosphorylation and activation of pkr, in turn, inhibit its downstream target, eif a, and attenuate the expression of host cellular proteins, including isgs, without any inhibitory effects on viral ires-mediated viral protein translation (table ) . , , pkr knockdown in hcv-infected cells restores isg expression and enhances the antiviral effect of ifn. these results demonstrate that hcv escapes ifn antiviral activity by promoting the phosphorylation of pkr and inhibiting the production of antiviral isg proteins, thus providing an interesting pathway for the virus to evade the ifn antiviral response. furthermore, accumulating evidence has revealed that pkr and eif a participate in the formation of stress granules (sgs). sgs are large, dynamic structures between to nm in size, that form in the cytoplasm when cells undergo extracellular stresses, including viral infections. sg formation is important for the posttranscriptional regulation of gene expression. sgs contain stalled translation pre-initiation complexes, including cellular mrnas, translational initiation factors, the small subunit of the ribosome and many cellular rna binding proteins, such as t-cell-restricted intracellular antigen (tia- ), the homologous tia- -related protein tiar and rasgap-sh domain binding protein (g bp ), involved in regulating mrna functions. [ ] [ ] [ ] [ ] many viruses, including hcv, can modulate sg assembly and co-opt sgs to promote their own protein synthesis. , hcv induces sg formation via eif a phosphorylation (table ) . , consistent with this notion, upregulation of the regulatory subunit of protein phosphatase that dephosphorylates eif a and growth arrest dna damage-inducible protein , inhibits sg formation. these results indicate the importance of eif a phosphorylation in hcv-induced sg formation. moreover, garaigorta et al. demonstrated that hcvinduced sg formation is ifn-and pkr-dependent and is inversely correlated with the induction of isg proteins, such as myxovirus resistance gene a (mxa) and ub-like (ubl)specific protease (usp ), in hcv-infected cells without affecting the mrna levels of these isgs. furthermore, the sg proteins tia- , tiar and g bp have been shown to play a critical role in hcv replication and infectious virus production. in support of this finding, g bp was also reported to be essential for hcv rna replication, presumably through its relocalization to lds or its interaction with ns b. , the results of garaigorta et al. demonstrated that hcv hijacks pkr phosphorylation-triggered sg formation to downregulate the translation of antiviral isgs, thereby promoting viral rna replication, virus assembly and egression. oas and rnase l upon sensing and activation by the pamp of viral dsrna, certain ifn-stimulated - oas proteins can synthesize - a from atp. after binding to - a short oligoadenylates, a ubiquitous, latent endonuclease, rnase l, is activated through dimerization and degrades either cellular or viral rnas, resulting in the inhibition of protein synthesis, cellular apoptosis and impaired virus propagation. , , therefore, the oas/ rnase l pathway represents a critical arm of ifn's antiviral effector mechanism against many viruses, including hcv. depending on the specific rna substrates and the extent of enzymatic activity, rnase l can block different types of viruses through different mechanisms, such as apoptosis, or through the 'suppressor of virus rna' derived from cellular or viral rna. nevertheless, some members of the oas family can exert antiviral activity independent of rnase l. the oas system has been reported to exert anti-hcv effects through the rnase l pathway. the ua and uu dinucleotides within loops of predicted stem-loop structures in the viral rna is prone to cleavage by rnase l (table ) . additionally, the sensitivity of hcv infection to ifn therapy correlates with the efficiency of rnase l-mediated viral rna cleavage. the anti-hcv activity of oas p and oas p in the oas family occurs in an rnase l-dependent fashion (table ) . hcv replication is suppressed in hcvcc-infected huh cells co-cultured with hepatic stellate cells (lx- ) treated with ppp-dsrna or incubated with conditioned medium from lx cells stimulated with ppp-dsrna. in these hcvccinfected cells, the expression of oas- and mxa is upregulated. the two different domains in oas-like a (oasla), a major isoform in human liver that is induced by hcv, contribute to the antiviral activity. the n-terminal oas homology domain, which lacks the cleavage activity, impairs cell proliferation as well as viral replication, whereas the c-terminal ublike domain impedes hcv replication without affecting cell growth (table ). the ifn-stimulated gene kda protein (isg ) has emerged as a second ifn-regulated rnase that inhibits rna virus replication. , isg , along with the closely related isg l and isg l , belongs to the yeast rna exonuclease homolog subfamily within the deddh exonuclease family and members of this superfamily possess both rnase and dnase activities. the - exonuclease activity of isg demonstrates a greater preference for single-stranded rna than for single-stranded dna. isg overexpression restricts infection by encephalomyocarditis virus, vsv, influenza virus (infv), human immunodeficiency virus (hiv), yfv, picornavirus and hcv. , isg has been reported to impair hcv genotype b sgr replication in hek cells (table ) . in addition, isg can hinder genotype a viral rna replication either in sgr or hcvcc infection, and its anti-hcv effect is not shared with isg l and isg l (table ) . apart from degrading viral rna through its - exonuclease activity, , the anti-hcv mechanism of isg in hcv replication remains poorly understood despite its possible action on cellular factors. adar rna-specific adenosine deaminase (adar) is constitutively expressed in normal cells as an inactive form. however, viral infection triggers the two mammalian adar genes, adar and adar , to express two active proteins, adar and adar . adar catalyzes adenosine to inosine editing in rnas that possess double-stranded structures. , because i is recognized as guanosine by rna polymerase, a to i editing causes nucleotide substitution as well as dsrna destabilization because of the reduced stability of i:u mismatch base pair compared with the normal base pair. , the rna editing ability of adar affects many biological processes, including viral replication and persistence, apoptosis, ion channel function and the posttranscriptional modification of genes. , only the adar transcription level is induced by ifn treatment and by pathogen infections. in addition, adar , but not adar , affects the stability of hcv replicon rna (table ) . in hcv sgr replication, ifn-a treatment decreases viral rna replication and concomitantly increases adar expression, suggesting that adar possesses an antiviral activity in the hcv rna replicon. adar knockdown conversely increases the hcv replicon rna. loss of hcv rna by adar may be due to several reasons. first, an i base-specific rnase might target mutated viral rna. second, the mutated rna might lead to insufficient replication and genome instability. third, the cellular mrna involved in viral replication may also be targeted by adar . thus, the rna editing ability of adar negatively affects hcv rna replication, representing a potent strategy in anti-hcv therapy. in sharp contrast, the replication of hepatitis delta virus (hdv) benefits from adar editing. the editing of hdv rna by adar converts the uag stop codon to a uig tryptophan codon, allowing the synthesis of a larger hdv antigen. without viral rna editing, the hdv genome cannot be packaged into a virion. nonetheless, adar overexpression increases rna editing but decreases hdv replication. ifn-inducible transmembrane protein (ifitm) family ifitm family members, including ifitm , ifitm and ifitm , inhibit, in an ifitm-specific manner, the replication of diverse pathogenic membrane-enveloped viruses, including marburg virus and ebola (ebov) filoviruses; severe acute respiratory coronavirus; hiv; rift valley fever virus (rvfv); respiratory syncytial virus; reovirus; flaviviruses, including denv and wnv; and hcv. [ ] [ ] [ ] [ ] [ ] [ ] in contrast, ifitms show no inhibitory effects on entry of amphotropic mouse leukemia virus, machupo virus, lassa virus and lymphocytic choriomeningitis virus. ifitms are topologically located at different intracellular membrane compartments. ifitm and ifitm , which are type ii transmembrane proteins, are primarily localized to endosomes and lysosomes, , whereas ifitm also localizes to the cell periphery. , ifit interacts with tbk , irf and other ifitm members and enhances ifn signaling. , lipid raft membranes, which are enriched in cholesterol and sphingolipids, play vital roles in cellular pathways and in virus entry, assembly and budding. , vesicle-associated membrane protein-associated protein a (vapa) and oxysterolbinding protein (osbp) modulate the intracellular trafficking and de novo synthesis of cholesterol. vapa interacts with osbp to regulate the transfer of cholesterol from the er to other organelles. , the regulation of intracellular cholesterol homeostasis, particularly in the endosomal compartment, is critical for the entry of viruses such as ebov and marburg viruses. ifitms have been demonstrated to interfere with virus infection by blocking virus-endosome fusion (table ) , , presumably through the modification of cellular membrane properties, such as fluidity and spontaneous curvature. , , amini-bavil-olyaee et al. demonstrated that the interaction of ifitm with vapa antagonizes the association of vapa with osbp, thereby inducing the accumulation of cholesterol in multivesicular bodies and in late endosomes. the disruption of intracellular cholesterol homeostasis subsequently impairs the membrane fusion of intraluminal virion-containing vesicles and endosomes, resulting in a block of vsv release into the cytosol. using immortalized human hepatocytes and huh infection models, raychoudhuri demonstrated that ifitm expression inhibits hcv replication but not at virus entry. later, wilkins et al. identified that ifitm is a hepatocyte tight junction protein whose antiviral action occurs through modification of the interactions of the hcv coreceptors cd and occludin, thereby inhibiting hcv entry (table ) . this study represents an interesting mode of antiviral innate immunity; an isg can exert its anti-hcv action by disrupting viral coreceptor associations. the ifn-induced protein with tetratricopeptide repeats (ifits) family represents a class of isgs featured by their unique helix-turn-helix motifs, known as tetratricopeptide repeats. ifits mediate a broad range of protein-protein interactions; in particular, the tetratricopeptide repeat motif is critical for modulating protein translational initiation and transport, cell proliferation and migration, virus replication, and antiviral signaling. [ ] [ ] [ ] [ ] proteins in the ifit family have been linked to ifn antiviral functions, including those against wnv and lymphocytic choriomeningitis virus. ifit plays an important role in modulating innate immunity by bridging tbk to mavs on mitochondria as ifit expression facilitates the association of its tetratricopeptide repeat motif with the n terminus of tbk , thereby enhancing irf -mediated gene expression. ifit , which is also known as isg , belongs to a family that also contains other stress-induced, structurally related proteins, p , p and p , in humans. ifit acts as a negativefeedback regulator for sendai virus-triggered induction of type ifn antiviral signaling transduction, presumably through its interaction with the adapter protein sting and through disruption of the normal association between sting/mita and mavs or tbk . moreover, ifit / preferentially targets mutants of poxvirus, coronavirus, and wnv that lack -o methylation in their viral rna cap, thereby rendering these mutant viruses unable to replicate. this study addresses the mechanism by which -o methylation of the cap of viral rna renders viruses insensitive to ifit-mediated host innate antiviral activity. wang et al. demonstrated that ifit mediates its ifn antiviral activity and blocks hcv rna replication, presumably by targeting an eif -dependent step in viral ires-mediated translation (table ) . in immortalized human hepatocyte and huh infection models, raychoudhuri documented that ifit expression inhibits hcv replication by suppressing hcv ires-mediated transcription. conversely, ifit knockdown facilitates hcv replication. these results suggest that ifit restricts hcv infection primarily at the viral translation/replication site. protein posttranslational modifications by ub and ubl modifiers not only play important roles in numerous cellular processes, such as protein localization, interaction, activity and degradation, signal transduction, vesicular trafficking and dna damage repair, [ ] [ ] [ ] but also modulate pathogen-host interactions, such as the viral replication cycle and the host antiviral response. - isg , which was the first ubl protein modifier identified, is post-translationally attached via its c terminus to the lysine residues of isgs and to hundreds of target proteins involved in different pathways. , similar to its ub homolog, isg is linked to proteins via a tightly regulated process known as 'isgylation', and the activating e (ube l), conjugating e (ubch ), and ligating e (ceb ) enzymes catalyze these sequential events. , isg , together with its conjugation e ligase (ceb ) and its deconjugation enzyme usp , are in the same isg /usp ubl pathway. isgylation modulates signal transduction pathways and host antiviral responses. isg exerts its modulatory roles by inhibiting virus release, isoylating viral proteins, or modifying host proteins. isg targets many cellular proteins, including jak , stat and many isgs. three antiviral effector molecules, irf , rig-i and pkr, are also modified by isgylation. activated irf is stabilized by isgylation and therefore, positively regulates type i ifn signaling. , the isg conjugation-mediated reduction of the non-isgylated rig-i correlates with the reduced ndv-triggered ifn response. additionally, viral rna-independent pkr activation requires the isgylation of pkr. isg expression enhances ifn-mediated antiviral activity against many viruses, including hiv and sinv. overexpressing isg in ifn-a/b receptor knockout mice decreases sinv replication and protects the mice from sinv-induced lethality. isg / mice are more susceptible to infection by many rna and dna viruses, such as infv and herpes simplex virus (hsv) type , and the protection effect of isg from sinv infection is dependent on isgylation. lu et al. demonstrated that induction of isg expression in ndv-infected cells counteracts the ub-mediated degradation of irf and enhances the ndv-mediated host innate antiviral response. their findings revealed a feedback mechanism of isg in enhanced antiviral immunity. despite functioning as an antiviral molecule, isgylation of the antiviral rig-i enzyme inhibits ifn signaling in mouse embryonic fibroblast cells. using the genotype a j /japanese fulminant hepatitis- chimeric hcv infectious model, chen et al. unexpectedly found that isg acts as a pro-hcv regulator because increased isg /isgylation facilitates hcv production, whereas blocking isgylation decreases virus production (table ) . moreover, knockdown of ube l, the e activating enzyme, inhibits hcv replication, particularly hcv egress, without affecting ifn-mediated isg expression in hcv-infected cells. using the hcv-huh . .cd infection system, arnaud et al. dissected the acute ifn response to hcv infection into early, pkr, and late, rig-i, phases. hcv infection rapidly induces the expression of many irf -dependent genes, including isg , through a pkr-dependent mechanism before the rig-i phase, which recruits mavs. then, isg induction blocks hcv rna-mediated rig-i activation by inhibiting rig-i ubiquitination, thereby negatively controlling the rig-i/mavs pathway. these studies illustrate that hcv may exploit isg to antagonize host innate immunity and to promote viral replication. the deconjugation of usp from its target proteins is catalyzed by usp (mouse ortholog ubp ). usp can function in both isg -dependent and isg -independent modes. usp was shown to bind to ifnar and attenuate the jak-stat pathway, thereby negatively regulating ifn signaling (table ) . reduced usp expression results in increased antiviral activity against many viruses, such as sinv, hepatitis b virus and vsv, in usp knockout mice. , [ ] [ ] [ ] usp knockdown is concomitant with increased cellular protein isgylation, prolonged stat tyrosine phosphorylation and enhanced isg expression, thus greatly enhancing the anti-hcv potency of ifn. all these studies suggest that usp disruption can impede its negative regulatory effect on ifn signaling, resulting in sustained jak-stat activity and antiviral activity. consistent with these observations, murray et al. demonstrated that ifn-a signaling and isg induction were greatly increased when ups was knocked down in both hcv sgr-and hcvcc-infected huh cells. however, usp knockdown did not have a significant effect on anti-hcv activity. these observations suggest a slight dependency of ifn-mediated antiviral activity on usp activity. additionally, usp upregulation is predictive of a nonsustainable viral response to ifn treatment. , , the expression levels of ups and isg increase in liver biopsy specimens from chronically hcv-infected patients who do not respond to ifn-based therapy, inferring that hcv hijacks the isg /usp pathway to evade the antiviral immune response and to facilitate its replication (table ) . , this observation also explains, at least partially, the failure of ifn-based treatments in non-responders, although non-responders express higher levels of isgs, particularly isg , compared with ifn responders. , taken together, these findings demonstrate that usp is an attractive target for the development of anti-hcv therapeutics. viperin, which stands for virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible, plays crucial roles in virus replication, signaling and the immune response. , the viperin protein sequence is highly conserved, and all viperin homologs contain three functional domains: the amphipathic, n-terminal domain, which mediates er and ld association; the central cxxxcxxc motif, which is functionally important for fe-s cluster formation; and the highly conserved c-terminal domain, which is essential for antiviral activity. [ ] [ ] [ ] in addition to type i, type ii and type iii ifns, dsdna and dsrna analogs, bacteria, lipopolysaccharide, poly(i:c) and a broad spectrum of dna and rna viruses can induce viperin expression. , , viperin expression regulates many cellular functions, such as forming lds and reducing membrane fluidity. viperin possesses antiviral activity against diverse families of dna and rna viruses, including infv, hiv, sinv, the flaviviruses japanese encephalitis virus, denv and wnv, and the hepacivirus hcv (table ) . , , viperin functions in different ways to defend against virus infections. for instance, viperin alters membrane fluidity by interacting with farnesyl diphosphate synthase, which is an enzyme essential for isoprenoid biosynthesis, thus disrupting the formation of lipid rafts, the sites of infv budding, leading to interference with virus release from the cell surface. the induction of viperin into hiv- -infected cells disrupts lipid rafts, causing viperin redistribution to cd compartments, where hiv- buds in human macrophages. the radical s-adenosyl-methionine enzymatic activity of viperin is required for the inhibition of hiv production. in cells infected with japanese encephalitis virus, the antiviral function of viperin is attenuated due to its degradation by the proteasome-mediated protein degradation system. in contrast, viperin enhances human cytomegalovirus infection through its interaction with the viral mitochondrial inhibitor of apoptosis vmia protein, resulting in viperin relocalization from the er to mitochondria. in mitochondria, viperin interacts with the mitochondrial trifunctional protein and reduces cellular atp generation, resulting in the disruption of the actin cytoskeleton and enhancement of the virus infection. viperin is upregulated in huh cells transfected with either poly(i:c) or hcv rna, and transient expression of viperin in hcv sgr replicating cells significantly decreases hcv replication. the putative radical s-adenosyl-methionine enzymatic activity of viperin is required for this anti-hcv activity. helbig et al. further demonstrated that the restriction of hcvcc replication by viperin depends on both the n-terminal amphipathic a-helix and the c-terminal domain. the anti-hcv function of viperin coincides with its binding to ns a at the ld interface, whereas ns a normally associates with the human homolog of the -kda vesicleassociated membrane protein-associated protein (hvap- ), which is a pro-viral cellular factor, at the viral replication complex. the interaction of hcv ns a with hvap- was previously shown to be critical for the formation of the viral replication complex. therefore, the association between viperin and hvap- requires both of their c-terminal domains, which then disturbs the interaction of hvap- with ns a and inhibits hcv replication (table ) . together, these findings imply that viperin hinders viral rna replication by perturbing the interaction between hvap- and ns a. by conducting bioinformatic analyses of murine bone marrowderived macrophages, liu et al. showed that cholesterol- -hydroxylase (ch h), which is an ifn-a-and ifn-c-stimulated isg, can mitigate the replication of many membraneenveloped viruses, including hiv, vsv, hsv and murine c-herpesvirus, and many pathogenic viruses, such as rvfv, ebov, russian spring-summer encephalitis virus and nipah virus in vitro and in vivo. these viruses contain different structural characteristics in their fusion proteins. for instance, hiv and ebov contain class i fusion peptides, rvfv and russian spring-summer encephalitis virus harbor class ii peptides, and vsv and hsv belong to class iii fusion proteins. , the broadly antiviral action of the ch h gene product is mediated by the ability of its enzymatic product, hydroxycholesterol, to inhibit ph-dependent and ph-independent membrane fusion between cells and viruses, as typified by vsv and hiv, respectively (table ) . this study not only demonstrates that ifn can confer an antiviral state to host and/ or target cells by inducing a natural oxysterol inhibitor but also suggests that modification of membrane oxysterols can be used as a potential antiviral approach. determining whether this broad antiviral isg can block hcv-mediated membrane fusion would be interesting. several genome-wide sirna screens were recently performed to identify isgs or ifn-mediated effector genes (iegs) that mediate ifn antiviral functions. these studies have identified many new isgs or iegs and have revealed interesting features of the actions of isgs. using an overexpression screen approach, schoggins et al. demonstrated that each virus exhibits a unique but partially overlapping profile of antiviral isg expression. the expression levels of isgs may vary depending on viral infection or on the time, dose, or cell type used for ifn treatment. in hcv infection, higher expression levels of unique isgs were found to correlate with a reduction in the hcv viral load. schoggins et al. further observed that multiple isg genes could target each viral species with a range of inhibitory activities. a set of effectors, including irf , c orf (also known as mb d ), heparanase, rig-i, mda and ifitm , exert broad antiviral activities against different viruses, including hcv, yfv, wnv, chikungunya virus, venezuelan equine encephalitis virus and hiv- . however, other effectors, such as ddx , ifn-inducible proteins l and , ifitm , mapk kinasekinase , moloney leukemia virus , nicotinamidephosphoribosyltransferase, oasl, receptor transporter protein , three prime repair exonuclease and protein unc- homolog b, display species-specific antiviral effector functions. these results also demonstrated that different isgs can exert additive antiviral effects on virus replication. remarkably, several isgs, such as adar, family with sequence similarity , member c, lymphocyte antigen e and mucolipin- , can enhance the replication of certain viruses. certainly, further characterizing how these isgs antagonize ifn-mediated antiviral functions and determining which steps of virus replication are targeted by these isgs are important. these findings indicate the complexity of the type i ifn-mediated innate immune response in virus replication. performing a sirna-based 'gain of function' screen, metz et al. identified several new anti-hcv isgs in addition to those previously reported anti-hcv isgs. this study demonstrated that both ifn-a and ifn-c can upregulate the expression of several isgs, including ifit , trim , phospholipid scramblase and inducible nitric oxide synthase . these isgs possess anti-hcv activity, although the precise roles of these isgs in hcv replication are not understood. this study also reported a substantial overlap in antiviral innate immune responses triggered by either cytokine. however, some isgs are more specifically induced by ifn-a or by ifn-c. for instance, phospholipid scramblase and nitric oxide synthase primarily function as ifn-c-mediated anti-hcv effectors. moreover, different isgs function additively or synergistically to interfere with hcv infection, indicating that the combinatorial and concerted actions of multiple effectors mediate repression of hcv replication. in addition to the signaling molecules involved in the ifn/ jak-stat/isgpathway, the majority of genes identified by fusco et al. are not transcriptionally activated by ifn. in contrast to the notion that isgs target specific virus replication steps, some of these genes can exert ifn-mediated antiviral effects at multiple steps of the hcv replication cycle. for instance, dipeptidyl-peptidase /cd /adenosine deaminase complexing protein blocks virus entry, initial rna replication, and amplified translation. myst histone acetyltransferase inhibits hcv entry, translation, rna replication and virion release, and protein phosphatase , catalytic subunit, b isoform (ppp cb) impairs virus entry, initial rna replication and subsequent translation. taken together, these findings reveal that these ifn-insensitive iegs, together with isgs, constitute the host cellular genes mediating the antiviral activity of ifn against viral replication. a functional genomic screen has shown that several new genes comprising the u /u .u tri-small nuclear ribonucleoprotein (snrnp) possess the ability to mediate ifn antiviral activity. u /u .u tri-snrnp is the major component of human spliceosome complexes involved in mrna processing. this genomic screen demonstrated that squamous cell carcinoma antigen recognized by t cells (sart ) is a u / u .u tri-snrnp-specific factor required for ifn-a-mediated anti-hcv activity, although sart is not induced by ifna. the anti-hcv activity of sart acts by regulating the expression of isgs, such as mxa, oas and pkr, either in the presence or absence of exogenous ifn-a. this genetic screen links an unappreciated role of rna processing to the control of antiviral immunity. in this section, we discuss recent findings regarding the roles of several cellular factors and/or machinery involved in the immune response in modulating hcv replication. although these determinants are not directly induced or activated by ifn, knowledge of their interplay with the host immune response will help to elucidate their effects on hcv infection. the functions of these cellular determinants in hcv infection are summarized in table . ikka hcv can co-opt an intrinsic innate pathway and hijack cellular lipid metabolism to facilitate its assembly. ikka was initially identified as a critical factor for hcv replication in a genomewide rna interference screen. subsequently, hcv infection was shown to activate ikka through the interaction of the viral genome -utr with dead box polypeptide , x-linked (ddx x). ikka translocates into the nucleus and induces the cbp/p -mediated expression of lipogenic genes, including sterol regulatory element-binding proteins, followed by the promotion of core-mediated ld formation and the enhancement of hcv assembly ( table ). dansako demonstrated that upon hcv expression, class a scavenger receptor type (msr ) expressed on the plasma membrane of infected and adjacent uninfected cells can bind to dsrna released from infected cells and mediate its endocytosis and transport to endosomes where the dsrna is sensed by tlr and initiates a local antiviral ifn response to restrict hcv replication. the msr -mediated binding, transport, and release of dsrna at the acidified endosome requires a stretch of conserved basic residues within the c terminus of the collagen superfamily domain of msr . therefore, msr acts as a key element for the tlr -mediated prr, thereby rendering both infected and uninfected hepatocytes refractory to hcv replication (table ) . hmgb , which is an abundant nuclear protein that mediates activation of host immune responses and inflammation, represents a prototype damage-associated molecular pattern that participates in the pathogenesis of diverse pathogens. , hmgb is passively released by cell injury or ischemia without pathogen invasion, but is actively secreted from stimulated immune cells, such as natural killer cells, macrophages and mature dendritic cells. many types of tlrs, such as tlr , tlr and tlr , can act as receptors for hmgb . the production of reactive oxygen species can mediate translocation from the nucleus to the cytoplasm and the subsequent release of hmgb . , interestingly, it has been shown that hcv core and ns a can trigger oxidative stress in infected cells. [ ] [ ] [ ] jung et al. demonstrated that hcv infection causes the nuclear-to-cytoplasmic translocation of hmgb and its release into the extracellular milieu. tlr acts as a major component of the receptor complex that recognizes lipopolysaccharide lps and plays a role in the production of pro-inflammatory cytokines and antiviral ifns via signaling myd and the tlr adapter protein trif. jung et al. also demonstrated that hmgb interacts with tlr to activate ifn signaling (table ). because hmgb is present at higher levels in the sera of patients with chronic hepatitis and cirrhosis compared with those detected in control individuals, the results of jung et al. may help to elucidate the potential inhibitory action of hmgb in hcv propagation in chronically hcv-infected patients. autophagy autophagy is a conserved 'self-eating' process that engulfs and delivers cytoplasmic cargos and invading pathogens within double-or multiple-membrane autophagosomal structures to lysosomes for degradation. [ ] [ ] [ ] [ ] the purpose of autophagic induction is to maintain cellular homeostasis in the host when the host undergoes extracellular or intracellular stresses. autophagy plays pivotal roles in the stress response, nutrient deprivation, damaged organelles, unfolded protein aggregation, intracellular quality control and cell death. [ ] [ ] [ ] [ ] the autophagic process requires two ubl conjugation complexes: autophagy-related gene (atg) -atg -atg l and microtubule-associated protein light chain -phosphatidylethanolamine. , autophagy has emerged as an immune regulator that commands the innate and adaptive immune responses against intracellular viruses. [ ] [ ] [ ] [ ] [ ] autophagy also participates in the modulation of virus-host interactions. in contrast, viruses can subvert the host autophagic pathway to potentiate their own growth. , analogously, hcv is able to subvert the host autophagic machinery and enhance viral growth, including rna replication, translation of the incoming viral rna genome and the release of infectious viruses ( table ) . two laboratories have independently demonstrated that hcv can activate autophagy via er stress-mediated induction of the upr and that upr-autophagy is required for hcv replication. , hcv ns , ns b, ns a and ns b have also been implicated in the induction of autophagy. , huang et al. showed that hcv induces er stress and inhibits the akttuberous sclerosis-mtor complex signaling pathway, resulting in autophagy activation. in contrast, shrivastava et al. demonstrated that hcv induces autophagy by stimulating beclin mrna expression and by activating mtor signaling, which may enhance hepatocyte growth. ke and chen demonstrated that in the context of hcv infection or without hcv infection, activation of the upr and autophagy downregulates innate immunity; in contrast, disruption of the upr and autophagy upregulates innate immunity. these results demonstrate that hcv hijacks upr and autophagy to stimulate viral rna replication by suppressing immune antiviral immunity. the upr-autophagy pathway represents a unique mode of reversible control in the innate immunity capacity in target cells. , subsequently, shrivastava et al. found that beclin or atg gene silencing in genotype a h strain hcv-infected immortalized human hepatocyte upregulates ifn signaling and isg expression, which are concurrent with apoptotic cell death. together, the results from these two groups suggest that autophagy may protect hcv-infected cells from the damage caused by excessive ifn antiviral stimulation, thereby promoting hcv rna replication. furthermore, a specific mode of autophagy, termed 'mitophagy', was recently reported to play a critical role in hcv replication and in the elimination of damaged mitochondria in infected cells in a parkin-dependent manner. knockdown of parkin and pink gene expression suppresses viral rna replication (table ). these results suggest a critical role for mitophagy in hcv replication. nevertheless, the molecular basis for the roles of autophagy and mitophagy in suppressing innate antiviral immunity in hcv infection has yet to be investigated. a recent study has demonstrated that many different families of rna viruses can target the autophagy network to promote viral growth. among these targets is irgm, which modulates autophagy by interacting with many autophagy-associated proteins, such as atg , atg and light chain c. strikingly, irgm knockdown impairs autophagy induced by many viruses, such as hcv, mev and hiv- , resulting in mitigated viral replication (table ). moreover, the c protein of mev, ns of hcv, and nef of hiv- were shown to induce autophagy by interacting with irgm. these results suggest that rna viruses have evolved to use a common strategy to target a critical molecule in autophagy to benefit their growth. microrna is a class of endogenous small non-coding rnas that bind to the -utr of target mrnas to control gene expression. micrornas also participate in innate and adaptive immunity response by binding to their complementarily mrnas and regulating the expression and translation of their target genes. for example, mir- regulates the host antiviral immune response by promoting type i ifn, whereas mir- enhances mrna degradation. mir- was shown to be upregulated in liver samples from hepatocellular carcinoma patients and in hcv-infected cells. during hcv infection, mir- expression is activated by the pkce/jnk/cjun and pkca/erk/cfos pathways. cjun and cfos form the ap- protein, which binds to the mir- promoter and activates mir- expression. mir- upregulation was shown to suppress the expression of myd and irak , which are two genes involved in the tlr signaling cascade, thereby repressing the production of type i ifn and isg and promoting hcv replication ( table ) . these results indicate that hcv usurps mir- to enhance its replication. likewise, mir- also increases the production of hiv, vsv and enterovirus by suppressing type i ifn production. the mechanisms by which viruses and cells coevolve and the tactics each party employs to establish the dynamic equilibrium are emerging as a fascinating area in hcv-host interaction research. previous studies that aimed to understand the hcv cell coevolution process have revealed several interesting aspects of virus-host cell interactions, such as er stress, upr, autophagy and innate antiviral immunity responses in hcv replication. , , further determining how the virus-cell interplay subsequently reshapes the host defense mechanisms and how virus replication is modulated in response to these cellular stresses will be important for gaining a complete understanding of the molecular basis of the hcv-host interaction in the pathogenesis of hcv infection. viral infection can trigger the ifn-mediated frontline host defense mechanism, including the production of a wide range of isgs to limit virus replication. many studies have also hitherto demonstrated that some of the identified isgs can exert broad antiviral activities against a diverse spectrum of viruses, whereas other isgs may have virus type-specific functions. the majority of studied isgs mediate ifn antiviral activities, acting as negative regulators in virus replication. strikingly, some isgs function as negative modifiers in the innate immune response, thereby promoting virus replication. nevertheless, the modes of action of most of the isgs remain unclear. although most identified isgs target individual steps of virus replication, some isgs seem to act at multiple stages of the virus replication cycle. determining the mechanisms by which these isgs function at different steps of the virus replication cycle would be interesting. current studies have indicated that different types of ifns may substantially overlap in mediating their innate immune response by activating the same set of isgs. however, the induction of some isgs may be unique to only one type of ifn, indicating the specificity in the induction of these isgs by ifns. clearly, different isgs can additively or synergistically suppress hcv replication, suggesting that inhibiting hcv replication depends on the combinatorial effects of individual isgs induced by ifn under the specific context of hcv infection. therefore, ifn-mediated suppression of hcv replication is not caused by a single isg but rather by the concerted actions of multiple isgs. moreover, gene expression profiling of hepatocytes from chronically hcv-infected patients treated with ifn has consistently shown differences between ifn-responders and ifnnon-responders. for instance, the levels of specific isgs, such as isg and usp , and viral sensors, such as rig-i, mda and laboratory of genetics and physiology- , are upregulated in patients with non-sustained virological responses compared with patients with sustained virological responses. , therefore, profiling gene expression for cytoplasmic viral sensors and related regulators involved in the innate antiviral immune response can identify new isgs that can be used as markers for predicting the clinical outcome of ifn therapy. in conclusion, the emergence of complexity in the highly pleiotropic type i ifn system in hcv infection reveals that the host has evolved to erect multiple checkpoints for anti-hcv innate immune surveillance to ensure that hcv is under tight control at all times, even when a single effector fails to confer antiviral activity, without drastically downgrading the overall efficacy of the ifn treatment. therefore, further deciphering which isgs and/or iegs are induced by ifns upon hcv infection and the specificity and action of these isgs and 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mice the broad-spectrum antiviral functions of ifit and ifitm proteins ifitms restrict the replication of multiple pathogenic viruses distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm is a tight junction protein that inhibits hepatitis c virus entry ifitm inhibits influenza a virus infection by preventing cytosolic entry the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry ifitm- and ifitm- but not ifitm- restrict rift valley fever virus the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication ifit is an antiviral protein that recognizes -triphosphate rna ifn-induced tpr protein ifit potentiates antiviral signaling by bridging mavs and tbk pathogens: raft hijackers cholesterol-binding viral proteins in virus entry and morphogenesis the diverse functions of oxysterolbinding proteins lipid traffic: floppy 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this study was supported by research grants from the ministry of science and technology ( - -b- - -my ) and academia sinica, taipei. this manuscript was edited for the english language by american journal experts (aje). attribution-noncommercial-noderivs . unported license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/ . / key: cord- -f dq j authors: chong, wai po; ip, wk eddie; tso, gloria hoi wan; ng, man wai; wong, wilfred hing sang; law, helen ka wai; yung, raymond wh; chow, eudora y; au, kl; chan, eric yt; lim, wilina; peiris, js malik; lau, yu lung title: the interferon gamma gene polymorphism + a/t is associated with severe acute respiratory syndrome date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: f dq j background: cytokines play important roles in antiviral action. we examined whether polymorphisms of ifn-γ,tnf-α and il- affect the susceptibility to and outcome of severe acute respiratory syndrome (sars). methods: a case-control study was carried out in chinese sars patients and healthy controls. we tested the polymorphisms of ifn-γ,tnf-α and il- for their associations with sars. results: ifn-γ + a allele was associated with susceptibility to sars in a dose-dependent manner (p < . ). individuals with ifn-γ + aa and at genotype had a . -fold ( % confidence interval [ci], . - . ) and . -fold ( % ci, . - . ) increased risk of developing sars respectively. the polymorphisms of il- and tnf-α were not associated with sars susceptibility. conclusion: ifn-γ + a allele was shown to be a risk factor in sars susceptibility. severe acute respiratory syndrome (sars) is an infectious disease caused by sars coronavirus [ ] with > cases and deaths reported in [ ] . much progress has been made in understanding sars coronavirus but the pathogenesis is still unclear [ ] . it was reported that old age, diabetes mellitus and heart disease were risk factors for adverse prognosis of sars [ ] [ ] [ ] , however, little is known about the contribution of genetic factors. we have demonstrated that genetic haplotypes associated with low serum mannose-binding lectin (mbl) were associated with sars [ ] and our findings were recently replicated [ ] . recently, homozygotes for clec m tandem repeats were reported to be less susceptible to sars in hong kong chinese [ ] . cytokines are known to be important in antiviral action. interferon (ifn)-γ from t and natural killer (nk) cells is important in driving the t helper cell type (th ) responses. it also activates monocytes and macrophages, which in turn take part in antiviral responses by producing free radicals and pro-inflammatory cytokines like tumor necrosis factor (tnf)-α. [ ] . tnf-α then regulates expression of neutrophil-endothelial cell adhesion molecules and chemokines, which recruit leukocytes to the site of infection [ ] [ ] [ ] . thus, ifn-γand tnf-α play important role in antiviral response and inflammation. interleukin (il- ) is an antiinflammatory cytokine that inhibits the activation and effector function of th cells, monocytes, and macrophages [ ] . il- appears to limit and ultimately terminate inflammatory responses by blocking the expression of a number of pro-inflammatory cytokines and chemokines [ ] . in animal model, il counteracts the inflammatory response by inhibiting tnfα production and neutrophil activation, and leads to a reduction of the lung tissue injury [ ] . thus, il- plays an important role in regulating many immune and inflammatory processes. various studies showed that a high il- level would result in suppression of innate host defense and lead to increasing susceptibility of the host to various microbes and death [ ] [ ] [ ] . in this study, we hypothesized that the polymorphisms of the cytokine genes, i.e. ifn-γ + a/t, tnf-α - g/a, il- - g/a and - a/c, might be associated with sars. these genes were chosen based on their functions in antiviral response and inflammation regulation that may be involved in sars pathogenesis and their polymorphisms based on their potential regulation on gene expression (table ) . we tested our hypotheses in sars patients and healthy controls and found that polymorphism of ifn-γ + a allele was associated with susceptibility to sars in a dose-dependent manner. genotyping ifn-γ + a/t, il- - g/a and - a/c were genotyped by taqman system (applied biosystems, foster city, ca, usa) as described previously [ ] . tnf-α - g/a was also genotyped by taqman system with same condition. the sequences of the primers were '-cct ggt ccc caa aag aaa tg- ' and '-tct tct ggg cca ctg act ga- ' and the probes were -fam-ttg agg ggc atg ggg acg g-tamra and vic-ttg agg ggc atg agg acg gg-tamra. the frequencies of genotypes and alleles of the single nucleotide polymorphisms (snps) were compared between the sars patients and healthy controls by × and × chi square test respectively. in case of significance, logistic regression was used for calculating or with % ci and corresponding p-values between groups by controlling age and sex as covariables. the genotypes of all snps were tested for hardy-weinberg equilibrium (hwe) by chi square test. our case-control study genotyped the snps ifn-γ + a/t, tnf-α - g/a, il- - g/a and - a/c in chinese patients with sars and healthy controls. the genotype distributions and allele frequencies of these snps were shown in table . the ifn-γ + a allele was overrepresented in sars patients ( . %) when compared with the controls ( . %) (p < . ). it was also significantly associated with susceptibility to sars in a dose-dependent manner (p < . ), i.e. individuals with ifn-γ + aa and at genotype had an odds ratio (or) of . ( % ci, . - . ) and . ( % ci, . - . ) in developing sars respectively. however, no significant correlation was observed in snps of il- and tnf-α. all snps were in hardy-weinberg equilibrium (hwe) (p > . ) in sars patients and controls by chi square test, except il- - a/c. ifn-γ + a allele has been previously reported to be associated with infectious diseases such as tuberculosis, hepatitis b virus infection, and parvovirus infection [ ] [ ] [ ] , revealing its potential role of function in host defense against microbial infections. the mechanism by which the ifn-γ + a/t allele influences the susceptibility to sars may depend on its role in the regulation of ifn-γ production. the t allele of ifn-γ + a/t provides a binding site for the transcription factor nuclear factor-κb (nf-κb), which is able to regulate ifn-γ expression [ ] . it is possible that low ifn-γ production may impair their anti-viral response against sars-cov, rendering these individuals more susceptible to this virus infection. our observation that ifn-γ + a allele was significantly associated with sars-cov infection suggests a genetic risk factor for sars. the role of ifn-γ in antiviral response against sars-cov has also been supported by recent studies showing that ifn-γ can inhibit the replication of sars-cov in combination with ifn-β in vitro [ , ] . il- and tnf-α snps were also included in this study. they were chosen due to their potential regulation on protein expression level [ ] [ ] [ ] . however, our present data did not show any significant association of these snps with sars (table ) . nevertheless, we cannot exclude the role of il- and tnf-α as the susceptibility genes for sars, because other snps in these genes may also be involved in gene expression regulation. further association studies on other snps, which could alter the gene expression level are required to ascertain the relationship of il- and tnf-α in sars. we have also compared the genotype and allele frequencies of all the polymorphisms between the death group and survival group of the sars patients (table ) . however, no significant association was established. we demonstrated that ifn-γ + a allele was significantly associated with sars susceptibility in a dose dependent manner. due to its role in regulating ifn-γ expression [ ] , this allele may be involved in the pathogenesis of sars by altering the ifn-γ production. the author(s) declare that they have no competing interests. wpc and wkei: genotyping, data analyses, drafting the manuscript ghwt: genotyping mwn and whsw: data analyses, drafting the manuscript .. ns = not significant. *p-value and or ( % ci) were calculated with the use of logistic regression models, adjusted with sex and age. sars study group: coronavirus as a possible cause of severe acute respiratory syndrome severe acute respiratory syndrome pathogenesis of severe acute respiratory syndrome clinical features and short-term outcomes of patients with sars in the greater toronto area short term outcome and 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experimental lpsinduced acute lung injury respiratory syncytial virus induces interleukin- by human alveolar macrophages. suppression of early cytokine production and implications for incomplete immunity neutralization of il- increases lethality in endotoxemia. cooperative effects of macrophage inflammatory protein- and tumor necrosis factor anti-il- therapeutic strategy using the immunomodulator as in protecting mice from sepsis-induced death: dependence on timing of immunomodulating intervention association of interferon gamma and interleukin genes with tuberculosis in hong kong chinese cytokine gene polymorphisms in patients infected with hepatitis b virus cytokine gene polymorphisms associated with symptomatic parvovirus b infection a single nucleotide polymorphism in the first intron of the human ifn-gamma gene: absolute correlation with a polymorphic ca microsatellite marker of high ifn-gamma production increased sensitivity of sars-coronavirus to a combination of human type i and type ii interferons interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) hutchinson iv: an investigation of polymorphism in the interleukin- gene promoter polymorphic haplotypes of the interleukin- ' flanking region determine variable interleukin- transcription and are associated with particular phenotypes of juvenile rheumatoid arthritis effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- - jklwiy authors: xu, shuqin; yang, kunpeng; li, rose; zhang, lu title: mrna vaccine era—mechanisms, drug platform and clinical prospection date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: jklwiy messenger ribonucleic acid (mrna)-based drugs, notably mrna vaccines, have been widely proven as a promising treatment strategy in immune therapeutics. the extraordinary advantages associated with mrna vaccines, including their high efficacy, a relatively low severity of side effects, and low attainment costs, have enabled them to become prevalent in pre-clinical and clinical trials against various infectious diseases and cancers. recent technological advancements have alleviated some issues that hinder mrna vaccine development, such as low efficiency that exist in both gene translation and in vivo deliveries. mrna immunogenicity can also be greatly adjusted as a result of upgraded technologies. in this review, we have summarized details regarding the optimization of mrna vaccines, and the underlying biological mechanisms of this form of vaccines. applications of mrna vaccines in some infectious diseases and cancers are introduced. it also includes our prospections for mrna vaccine applications in diseases caused by bacterial pathogens, such as tuberculosis. at the same time, some suggestions for future mrna vaccine development about storage methods, safety concerns, and personalized vaccine synthesis can be found in the context. mrna, an intermediate hereditary substance in the central dogma, was first discovered in by brenner et al. [ ] . however, the concept of mrna-based drugs was not conceived until , when malone et al. demonstrated that mrna could be successfully transfected and expressed in various of eukaryotic cells under the package of a cationic lipid (n- [ -( , -dioleyloxy) propyl]-n,n,n-trimethylammonium chloride (dotma)) [ ] . in , in vitro-transcribed mrna was sufficiently expressed in mouse skeletal muscle cells through direct injection, which became the first successful attempt on mrna in vivo expression and thus proved the feasibility of mrna vaccine development [ ] . since then, mrna structure researches and other related technologies have been rapidly developed. under this condition, several development restrictions stemmed from mrna instability, high innate immunogenicity, and inefficient in vivo delivery have been mitigated, and now mrna vaccines have been widely studied in different kinds of diseases ( figure ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . mrna vaccines have demonstrated many specific advantages that conventional vaccines do not have. first of all, mrna can theoretically meet all genetic information requirements to encode and express all kinds of proteins. vaccine developing efficiency can be optimized by modifying mrna sequence, which is a more convenient way compared to other kinds of vaccine modification [ , ] . furthermore, most of the mrna vaccine production and purification processes are quite similar despite different encoded antigens, so it is potential to be retained or even standardized to develop other similar mrna vaccines [ , ] . utilizing in vitro transcription also makes mrna vaccines production easier [ ] [ ] [ ] . accordingly, it is obvious that mrna vaccines can save both time and economic costs. second of all, mrna has self-adjuvanting properties which activate strong and long-lasting adaptive immune responses through tumor necrosis factor-α (tnf-α), interferon-α (ifn-α) and other cytokines secretion by immune cells [ ] , while polypeptide and protein based vaccines need extra adjuvants to achieve a similar goal [ ] . the in vivo expression of mrna can also avoid protein and virus-derived contamination [ ] . by modifying the mrna sequence and delivery system, the expression activity and in vivo half-life of mrna can be effectively regulated [ , , ] . thirdly, in comparison with dna-based vaccines, mrna vaccines can express target proteins more efficiently because of their expression in the cytoplasm without entering the nucleus [ ] . in addition, due to the chemical constitution of the mrna sequence, which is different from dna constitution and lack of cpg islands, there is a lower possibility for mrna to integrate into host dna genome and induce a smaller immune rejection reaction [ ] . besides, mrna is only transiently active, making it easy to be completely decomposed via physiological metabolic pathways; therefore, it would not act as a burden to the host homeostasis [ ] . after the first mrna-based drug company was established in , a large number of groups began to research and develop mrna-based drugs [ ] . so far, over twenty mrna-based candidate drugs have entered the clinical trial stage. the market value for the mrna vaccine field has also increased, reaching up to tens of billions of dollars, which signifies a broad prospect for mrna-based drugs development, especially mrna vaccines. in particular, mrna vaccines have a huge potential on rapidly responding to emerging epidemics, e.g., the global explosion of the mrna vaccines have demonstrated many specific advantages that conventional vaccines do not have. first of all, mrna can theoretically meet all genetic information requirements to encode and express all kinds of proteins. vaccine developing efficiency can be optimized by modifying mrna sequence, which is a more convenient way compared to other kinds of vaccine modification [ , ] . furthermore, most of the mrna vaccine production and purification processes are quite similar despite different encoded antigens, so it is potential to be retained or even standardized to develop other similar mrna vaccines [ , ] . utilizing in vitro transcription also makes mrna vaccines production easier [ ] [ ] [ ] . accordingly, it is obvious that mrna vaccines can save both time and economic costs. second of all, mrna has self-adjuvanting properties which activate strong and long-lasting adaptive immune responses through tumor necrosis factor-α (tnf-α), interferon-α (ifn-α) and other cytokines secretion by immune cells [ ] , while polypeptide and protein based vaccines need extra adjuvants to achieve a similar goal [ ] . the in vivo expression of mrna can also avoid protein and virus-derived contamination [ ] . by modifying the mrna sequence and delivery system, the expression activity and in vivo half-life of mrna can be effectively regulated [ , , ] . thirdly, in comparison with dna-based vaccines, mrna vaccines can express target proteins more efficiently because of their expression in the cytoplasm without entering the nucleus [ ] . in addition, due to the chemical constitution of the mrna sequence, which is different from dna constitution and lack of cpg islands, there is a lower possibility for mrna to integrate into host dna genome and induce a smaller immune rejection reaction [ ] . besides, mrna is only transiently active, making it easy to be completely decomposed via physiological metabolic pathways; therefore, it would not act as a burden to the host homeostasis [ ] . after the first mrna-based drug company was established in , a large number of groups began to research and develop mrna-based drugs [ ] . so far, over twenty mrna-based candidate drugs have entered the clinical trial stage. the market value for the mrna vaccine field has also increased, reaching up to tens of billions of dollars, which signifies a broad prospect for mrna-based drugs development, especially mrna vaccines. in particular, mrna vaccines have a huge potential on rapidly responding to emerging epidemics, e.g., the global explosion of the coronavirus disease (covid- ) , stimulating more interest and research expectations from worldwide scientists [ , ] . to date, in vitro transcription technology of mrna has been mature, and the most popular method is using t , t , or sp rna polymerase and linear dna (linearized plasmid dna or synthetic dna prepared by pcr) for mrna synthesis. there are some basic structural elements of mature mrna in the eukaryocyte that are required to keep mrna functional, including five-prime cap ( cap), five-prime untranslated region ( utr) , open reading frame (orf) region, three-prime untranslated region ( utr), and poly (a) tail structure [ , ] . keeping mrna structure intact is beneficial for mrna stability and expression capability. modifying the mrna sequence based on its complete structure can further optimize the efficiency of an mrna vaccine. however, the initial product of mrna in vitro transcription is the mixture of targeted mrna, untargeted rna, nucleotides, oligodeoxynucleotides, and proteins [ ] . to purify the mrna, precipitation and extraction techniques are used to remove common impurities and chromatographic techniques are generally used to separate the target mrna from other mrna impurities in this system [ ] . mrnas from the eukaryotic and partial viral genomes have a -methylguanosine (m g) cap at the end of the mrna sequence (m gpppn structure), which connect to the first rna nucleotide through a , -triphosphate bridge (ppp) during mrna in vitro transcription. the cap can eliminate free phosphate groups in the mrna sequence so as to significantly enhance the stability of mrna, which allows the ribosome to recognize the beginning of mrna and improves translation efficiency by binding to the eukaryotic translation initiation factor e (eif e) [ , ] . so it is obvious that cap modification can be crucial to mrna property improvement. there are two common approaches in terms of in vitro mrna capping. firstly, adding a regular cap analog, m gpppg structure, to the mrna transcription system can achieve mrna capping along with in vitro transcription [ , ] . secondly, mrna capping can also be completed by capping enzyme reaction after the initial in vitro transcription [ , ] . capping with cap analog is the most common capping method of mrna in vitro transcription, but studies have found that regular cap analog can reversely bind to the mrna sequence [ ] . in this case, mrna isomers are formed and lead to low efficiency of mrna downstream translation. to avoid reverse incorporation of cap, anti-reverse cap analogs (arca) have been developed [ , ] . arca is modified at the c or c position to ensure that the methyl groups react with the hydroxyl groups at the correct site during transcription. compared to regular cap analog, arca-capped mrna has a higher translation efficiency [ ] [ ] [ ] . in recent years, further modification on the arca structure has been developed to improve mrna properties. phosphorothioate modifying based on arca, for example, would enhance the translation efficiency of mrna by increasing its affinity for eif e, and has the ability to decrease the susceptibility to decapping enzymes so as to improve the mrna stability [ ] [ ] [ ] . kuhn et al. showed that m , -o gpp s pg (β-s-arca) could significantly enhance the stability and translation efficiency of mrna in immature dendritic cells (dcs) [ ] . in , strenkowska et al. synthesized cap analogs that were composed with , -dithiodiphosphate modification, arca, and an extended polyphosphate chain, named " s analogs", the benefits of which enabled s analogs to function better than any s-arca used in clinical trials [ ] . another cap analog, a co-transcriptional capping method called "cleancap," was developed in [ ] . it utilized an initiating capped trimer to yield a naturally occurring cap structure, which increased the capping efficiency to nearly - % [ , ] . utrs are non-coding parts of mrna sequence located at the upstream ( utr) and downstream ( utr) domains of the mrna coding region. as reported, utrs are related to mrna replication and translation processes, and they can greatly alter mrna decay and translation efficiency through reactions with rna binding proteins [ , ] . in an attempt to enhance mrna stability and translation efficiency, it is essential to ensure the optimization of utrs. generally speaking, utr optimization is to increase the in vivo mrna expression level. for instance, the widely-used utr sequence derived from α-globin and β-globin contains translation and stability regulatory elements [ ] . utr is normally considered to be a concentrated region full of unstable factors in mrna, so averting unstable sequences while synthesizing utr can increase mrna stability. au-enriched sequences and gu-enriched sequences are related examples of this [ , ] . on the other hand, introducing stable elements to utr can also significantly improve the stability of mrna and expand its half-life [ , ] . orlandini von niessen et al. once connected two random utrs which contained stable elements in series, and successfully improved the translation efficiency of mrna [ ] . utr directly affects the translation of its downstream sequence orf, so the optimization of utr should not influence the normal translation process of the orf. avoiding the gene sequence in utr, which is identical to the upstream of orf, can effectively prevent false start and replacement of the reading frame during mrna translation [ ] . additionally, some particular sequences can be added to utr to enhance the stability of mrna and the accuracy of translation. for example, kozak et al. inserted sequence gcc-(a/g)-ccaugg in this region, leading to a more accurate start of translation process [ ] . study also shows that over-stabilized secondary structure of utr would hinder the binding of ribosomes to mrna, and short and loose utr is more conducive to the mrna translation processes [ ] . as the coding region of mrna, the translatable rate of orf region is definitely crucial. therefore, choosing the appropriate codons in this region can optimize the overall translation efficiency of mrna. optimized orf sequence usually incorporates synonymous frequent codons and/or codons with higher trna abundance to replace rare codons in orf, so that highly expressed genes can be translated using the same codons of the host and/or guaranteed the ampleness of trna during the expression of exogenous mrna [ ] . however, high translation rate of mrna is not all beneficial, as some proteins require a low translation rate to fold correctly, stably, and effectively; in this case, using codons with low frequency in orf can yield protein products of higher quality [ ] . therefore, for different antigens, we should use different codon optimization strategies to improve mrna translation rate and ensure the expressed antigen quality at the same time. poly (a) tail and the cap structures are both crucial elements during mrna translation. poly (a) sequence can slow down the degradation process of rna exonuclease, which increases stability, extends in vivo half-life, and enhances translation efficiency of mrna [ ] . moreover, poly (a) binding protein (pabp) can link to the cap through translational initiation factors, such as eif g and eif e, which in turn affects the closed-loop structure of mrna and synergistically regulates the stability and translation efficiency of mrna [ , , ] . however, pabp can also bind to adenylation complexes and participate in translation inhibition process mediated by microrna [ ] . the contradictory function of pabp indicates that various poly (a) sequence length can affect mrna translation efficiency differently. there are different methods to synthesize a poly (a) structure, among them, in vitro transcription process with dna template with poly (a) structure information can yield a defined poly (a) sequence length [ ] . recombinant poly (a) polymerase can also be used to add poly (a) structures by undergoing an enzymatic polyadenylation after initial mrna transcription, in which case poly (a) structural mixtures of different lengths can be obtained [ ] . early studies suggest that a long poly (a) sequence can improve mrna stability. for example, the optimal length of poly (a) sequence in dcs is roughly between - nucleotides [ , ] , and over nucleotides of poly (a) sequence length in human primary t cells can become more conducive in increasing mrna stability and translation efficiency [ ] . when poly (a) sequence length is less than nucleotides, it would reduce mrna translation efficiency [ ] . however, in , lima et al. found that mrnas with high translation efficiency generally had short poly (a) sequences through novel genome-wide research techniques, whilst short poly (a) structures were generally found in well-translated eukaryotic mrnas [ ] . therefore, it has been indicated that since the lengths of poly (a) sequences required for high translation efficiency mrna in various types of cells are different, adjustments should be made to optimize the translation efficiency of mrna. based on its self-adjuvanting effect, mrna can exhibit some properties similar to the mrna virus when it works as the vector of exogenous genes. in this case, mrna can be recognized by antigen-presenting cells (apcs), which subsequently activates pattern recognition receptors (prrs) such as toll-like receptor (tlr ), tlr , and tlr [ , , ] . the double-stranded rna (dsrna) can combine with some retinoic-acid-inducible gene i (rig-i) -like receptors (rlrs) in the cytoplasm, such as rig-i and melanoma differentiation-associated (mda ), which promotes apcs maturation, pro-inflammatory cytokines secretion, and type i interferon (ifn) secretion [ , ] . eventually this leads to strong antigen-specific humoral and cellular immune responses ( figure ). however, subunit vaccines composed of peptide or protein antigens are generally unable to activate prrs, so it is necessary to add adjuvants which can initiate and support adaptive immune responses, achieving the final result of carrying out the body's immune response of subunit vaccines [ ] . therefore, mrna's strong adaptive immune response and self-adjuvanting property can provide a huge advantage shown in mrna vaccines. single-stranded rna (ssrna) can trigger the dcs' antiviral activation state through tlr and tlr recognition during mrna in vivo transmission [ ] . the dsrna contaminants can also trigger immune activation via tlr recognition [ , ] . however, excessive immune response stimulated by mrna in the cytoplasm would stimulate cells to secrete large amounts of type i ifn and other interferons which can inhibit the translation of mrna and eventually lead to translational stagnation, rna degradation, cd (cluster of differentiation ) + t cells activation reduction, and ultimately immune response termination [ , , ] . this could leave negative effects on some mrna applications such as vaccines and protein replacement therapies. self-adjuvanting properties of mrna have both advantages and disadvantages in mrna vaccine applications, therefore, it is necessary to form mrna immunogenic regulations according to different medical demands, which in return would effectively improve the application efficacy of mrna vaccines. (c) self-adjuvant effect. various of pattern recognition receptors (prrs) can recognize mrna in vitro transcription product. ssrna can be recognized by endosomal innate immune receptors (e.g., toll-like receptor (tlr ), tlr ). dsrna can be recognized by endosomal innate immune receptors (e.g., tlr ) and cytoplasmic innate immune receptors (e.g., protein kinase rna-activated (pkr), retinoic acid-indu [ ] cible gene i protein (rig-i), melanoma differentiation-associated protein (mda ), and ′- ′-oligoadenylate synthase (oas). based on those, mrna products can stimulate the secretion of pro-inflammatory cytokines and type i interferon (ifn), which leads to antigen-presenting cells (apcs) activation and inflammatory reaction. however, they can also activate antiviral enzymes that cause stalled mrna translation and mrna degradation. mrna in vitro transcription product often contains dsrna contaminants. dsrna, which is a simulant of rna virus genome replication intermediates, can promote type i ifn production [ , ] . therefore, the purification of an mrna in vitro synthetic product can effectively reduce type i ifn immune response of mrna vaccines and increase mrna translation efficiency [ ] . studies have shown that chromatographic methods (fast protein liquid chromatography, high-performance liquid chromatography, etc.) can effectively remove dsrna from mrna products; after purification, the mrna translation level in primary cells can be increased by - times while the cytokine secretion level still remains relatively high [ , ] . ssrna can also work as a potent pathogen-associated molecular pattern (pamp) that elicits a strong immune response and stimulates type i ifn production. type i ifn can induce numerous types of ifn-stimulated genes (isgs) to inhibit mrna translation [ ] . for instance, ifn-inducible using dna with the antigen-encoding sequence as template, mrna in vitro transcription products contain single-stranded rna (ssrna), double-stranded rna (dsrna), etc. the ssrna structure normally includes five-prime cap ( cap), five-prime untranslated region ( utr), open reading frame (orf) region, three-prime untranslated region ( utr), and poly (a) tail structure. (b) rna translation and antigen presentation. through endocytosis, mrnas enter the cytoplasm. some mrnas combine with ribosomes of the host cell and translate successfully. antigen proteins can be degraded to antigenic peptides by proteasome in the cytoplasm and presented to cytotoxic t lymphocytes (ctls) via major histocompatibility complex (mhc) i pathway. or, they can be released out of the host cell and taken up by dcs. then, they are degraded and presented to helper t cells and b cells via mhc-ii pathway. b cells can also recognize released antigen proteins. (c) self-adjuvant effect. various of pattern recognition receptors (prrs) can recognize mrna in vitro transcription product. ssrna can be recognized by endosomal innate immune receptors (e.g., toll-like receptor (tlr ), tlr ). dsrna can be recognized by endosomal innate immune receptors (e.g., tlr ) and cytoplasmic innate immune receptors (e.g., protein kinase rna-activated (pkr), retinoic acid-indu [ ] cible gene i protein (rig-i), melanoma differentiation-associated protein (mda ), and - -oligoadenylate synthase (oas). based on those, mrna products can stimulate the secretion of pro-inflammatory cytokines and type i interferon (ifn), which leads to antigen-presenting cells (apcs) activation and inflammatory reaction. however, they can also activate antiviral enzymes that cause stalled mrna translation and mrna degradation. mrna in vitro transcription product often contains dsrna contaminants. dsrna, which is a simulant of rna virus genome replication intermediates, can promote type i ifn production [ , ] . therefore, the purification of an mrna in vitro synthetic product can effectively reduce type i ifn immune response of mrna vaccines and increase mrna translation efficiency [ ] . studies have shown that chromatographic methods (fast protein liquid chromatography, high-performance liquid chromatography, etc.) can effectively remove dsrna from mrna products; after purification, the mrna translation level in primary cells can be increased by - times while the cytokine secretion level still remains relatively high [ , ] . optimization of mrna sequence to regulate self-adjuvanting property ssrna can also work as a potent pathogen-associated molecular pattern (pamp) that elicits a strong immune response and stimulates type i ifn production. type i ifn can induce numerous types of ifn-stimulated genes (isgs) to inhibit mrna translation [ ] . for instance, ifn-inducible protein with tetratricoid repeats (ifit) can combine with the cap structure or interact with eif to disrupt the mrna translation process [ , ] . therefore, optimizing mrna sequence can regulate the ability to activate the immune response of mrna vaccines [ , , ] . prrs can recognize cap (m gpppn)-capped or uncapped mrna and inhibit its translation [ ] . in , kumar et al. evaluated the ability of prrs to recognize three forms of capped mrna, including cap -capped, cap (m gpppnmn)-capped, and uncapped mrna. they discovered that cap -capped mrna was still translated after being recognized by prrs, while cap -capped and uncapped mrna were not [ ] . therefore, choosing appropriate cap structure can avoid excessive immunity response. modification of the orf region can also reduce the strong immune response caused by prrs recognition, and enhance the translation level of mrna [ ] . in , anderson et al. studied the difference between unmodified mrna and pseudouridine modified mrna [ ] . the ability of mrna to be recognized by - -oligoadenylate synthetase (oas protein, induced by type i ifn) and mrna stability were assessed, and results showed that the pseudouridine modified mrna had lower efficiency in terms of oas activation, lower rate of rna degradation, and higher efficiency of mrna translation [ ] . karikó et al. intravenously injected pseudouridine modified mrna in mice, and found out that there was a higher target protein expression in the spleen and lower ifn-α concentration in serum compared with unmodified mrna treatment [ ] . uracil analog is the most common analog used in mrna modification, and some other base analogs can also be used for mrna sequence modification. kormann et al. and mays et al. used different rates of -methyl-cytidine and -thiouridine to modify mrna sequence, in which both effectively reduced the recognition rate of prrs, and increased mrna intracellular stability [ , ] . some studies need the enhancement of the immunogenicity of mrna vaccines and adding adjuvants to the mrna vaccine system can meet this requirement. formulation of self-amplified rna vaccines with the traditional adjuvant mf (made by novartis) and cationic nanoemulsion (cne) have proven to enhance the immunogenicity and efficacy of mrna vaccines in various animal models [ , ] . certain immunomodulatory molecules also have adjuvant activity. trimix, a new adjuvant strategy developed by vrije universiteit brussel, consists of mrnas that encode three immune activator proteins-cd , cd ligand (cd l) and constitutively active tlr [ , , ] . trimix mrna can increase the immunogenicity of naked, unmodified, unpurified mrna, and it is also related to the enhancement of dcs' maturation and cytotoxic t lymphocyte response [ ] . in , leal et al. adopted the trimix naked mrna strategy to treat acquired immune deficiency syndrome (aids) patients. treatment using high doses of trimix mrna showed that a high human immunodeficiency virus (hiv)-specific t cell response could be stimulated and detected [ ] . the high safety and tolerability of this strategy has been demonstrated in this research [ ] . some mrna delivery vehicles can also increase the adjuvant effect, such as cationic lipid and protamine. in , researchers used the mrna vaccine immunization strategy with cationic lipid , -dioleoyl- trimethylammonium-propane/ , -dioleoyl-sn-glycero- -phosphoethanolamine (dotap/dope) as the assigned adjuvant, and stimulated more pro-inflammatory cytokines and type i ifn secretion than naked mrna in dcs [ ] . after subcutaneous injection of this mrna vaccine in mice, large amount of type i ifn secretion and rapid aggregation of inflammatory monocytes could be detected in lymph nodes transiently [ ] . this indicates that cationic lipids can strengthen the adjuvant effect and the efficacy of mrna vaccines to a certain extent [ , ] . researches also demonstrated that mrna and protamine complexes could act as danger signal and elicit t-help cell (th ) responses via tlr and tlr involving [ , ] . the rnactive ® vaccine platform designed by curevac used co-delivered rna and protamine complex as the adjuvant to induce th t cell responses, and naked, unmodified, and sequence-optimized mrna as the antigen to develop mrna vaccines [ ] . in this technique, protamine-formulated rna only works as an adjuvant, not as a mrna carrier, enabling more rnactive ® vaccines to arouse strong immune responses in many pre-clinical models, which can successfully prevent attacks from various influenza strains [ , ] . kowalczyk et al. revealed that rnactive ® vaccine treatment in mice could initiate a balanced and strong specific immune response with intradermal immunization [ ] . this immune stimulation only existed in the stimulated site and lymphoid organs, and no pro-inflammatory factors were detected in serum. overall, rnactive ® technology is a new effective technique of mrna vaccine with high levels of safety and flexibility. mrna needs to enter the host cytoplasm to express specific antigens to remain functional; however, the mrna molecule is not small enough to pass through cell membrane by free diffusion [ , ] . additionally, mrna and cell membrane are both negatively charged, which increases the difficulty of mrna delivery. furthermore, mrna can be easily degraded by extracellular ribonucleases which exist in skin and blood [ , ] . therefore, delivering mrna into enough numbers of cells with sufficiently high translation levels is one of the most difficult application problems of mrna vaccines, as it demands highly specific and efficient mrna delivery systems [ , ] . a variety of mrna delivery methods and mrna delivery vehicles have been developed and applied currently (table ) . early study has demonstrated that naked mrna in vivo injection can provoke the immunotherapy response in mice [ ] . at present, administration strategies of mrna generally include subcutaneous injection, intradermal injection, intranodular injection, intramuscular injection, intravenous injection, intratumoral injection, etc., which are essential methods that help stimulate antigen presentation and initiate immune responses [ , , ] . in , phua et al. discovered that delivery efficiency of subcutaneous injection of naked mrna in mice was even higher than mrna nanoparticle delivery methods [ ] . van lint et al. suggested that intratumoral injection of tumor-associated mrna would elicit an appropriate immune response and believed that it could be a promising vaccination strategy for the impending future [ ] . these days, direct injection of naked mrna is mainly used to treat or prevent infectious diseases [ ] . however, even though the injection of naked mrna can cause immune response, the working effect of this delivery method is relatively weak, and the naked mrna is often rapidly degraded after injection. direct injection of naked mrna is too simple and primitive to be applied in human patients, and it is often used as an administration route to inject modified mrna vaccines with other delivery systems to achieve better vaccine effects. the efficiency of naked mrna antigen presentation can be improved with the assistance of common physical methods including electroporation, gene gun, microneedles, etc. [ ] . electroporation can increase mrna delivery efficiency without the demand of other mode receptors, which can reduce unnecessary immunoreactions [ ] . electroporation also has an adjuvant effect that it can recruit pro-inflammatory cells and induce the production of cytokines at the inoculation site, improving the immunogenicity of mrna [ ] . in , callis et al. found that electroporation could be used to transfer mrna into animal and plant cells with low transfection efficiency [ ] . however, the target intracellular expression product was high enough to reach the detection level. in , the mrna transfection efficiency in dcs had reached - % for electroporation method [ ] . the gene gun method, using compressed helium gas as an acceleration force to push mrna coated on the surface of gold particles into host cells, is an efficient method of mrna delivery [ ] . in , qiu et al. used gene gun method to transfer the human alpha- antitrypsin mrna into the mouse skin and successfully triggered the antibody response [ ] . peking et al. developed a mrna-based therapy for genetic skin diseases restoration, mrna was effectively transported to the target skin layers in mice by gene gun delivery [ ] . despite its advancements, the gene gun method is rarely used in large animals and humans. physical ways to deliver mrna may affect the physiological structure and activity of cells, even causing abnormal cell death. therefore, applying physical mrna deliveries in human is potentially hazardous [ , ] . dcs are one of the most potent apcs of immune system. they can present processed antigens to cd + , cd + t cell via the major histocompatibility complex (mhc), which triggers cellular immunity [ , ] . meanwhile, dcs can also present intact antigens to b cells, triggering humoral immunity [ ] . the common way to use dcs as mrna delivery vehicles is to transfect mrnas encoding peptides, proteins or other antigens into dcs via in vitro, and then transfer the processed dcs back into the host body to start the antigen-specific immune response [ ] . the dcs-mrna delivery system does not need to be combined with other carrier molecules and can generate high delivery efficiency. in this context, this delivery system is widely used in pre-clinical experiments, animal models and clinical researches [ , , , ] . moreover, this strategy has been mainly applied in cancer treatment because the elicitation of cellular immune response is predominant [ ] . however, the mrna transfection rate is quite low if only by dcs endocytosis, and electroporation method is often used to further improve the mrna transfection rate [ ] . gay et al. used electroporation to transfer the mrna encoding hiv antigens into dcs for hiv treatment, after intradermal injection, the number of hiv-specific cd + / cd ra-cd + factors/cytotoxic t-lymphocytes (ctls) was at least times higher than control, which enhanced the t cell immunological reactions of hiv patients [ ] . another unignorable barrier to clinical application of ex vivo-loaded dc mrna vaccines is that time-and money-consuming production process cannot meet the huge quantity demand of mrna vaccine for some treatments. besides, the immune response caused within several hours after mrna transfection can be lost during the time-consuming in vitro preparation process, leading to reduction of the therapeutic effect of mrna vaccines [ ] . out of these considerations, diseases that require large amounts of mrna vaccine treatment in the short term should give preference to delivery systems with a fast production speed. delivery systems that are able to directly target mrna to in vivo apcs can also be considered. protamine is an alkali cationic protein with resin-like structure. combining mrna with protamine in different mass ratios can yield electrostatic protamine-mrna complex particles with different diameters [ ] . this tight conjugate form can effectively protect mrna from being degraded by serum rnases, and the complex can cause a strong immune-reaction of immune cells such as dcs, monocytes, b cells, natural killer cells, and neutrophils [ , , ] . this indicates that protamine has the potential to be used not only as a mrna carrier, but also as an immune activator. in , protamine was already studied as one of the first delivery materials for long rna [ ] . fotin-mleczek et al. used protamine as the delivery material during the vaccination of mrna tumor vaccine, and successfully elicited a complete specific anti-tumor response [ ] . when the mass ratio of protamine to mrna is : , the size of the electrostatic complex formed is about nm, which is relatively stable and produces strong immune stimulation and high cytokine levels, with the downside of inhibiting protein expression significantly [ ] . however, when the mass ratio of protamine to mrna is : , compared to the previous mass ratio of : , the protein expression increased but the cytokine level decreased [ ] . hence, a common idea is that the mrna translation efficiency and immune strength are limited in the protamine-formulated mrna delivery system. and it is speculated that this defect may be related to the extremely tight electrostatic complex [ , ] . in recent years, protamine-formulated mrna delivery system has been widely used in clinical trials, and gained pretty good clinical treatment effects, such as rabies, non-small cell lung cancer, etc. [ ] [ ] [ ] . the rnactive ® vaccine platform, which use the protamine-mrna only to activate immune responses, is a prevailing technique to resolve this problem [ ] . furthermore, to use protamine as a mrna delivery and immune activator at the same time, structural optimization of protamine or searching for proteins similar to protamine in property as substitutes deserves our attention. as a commonly used gene carrier, cationic liposomes can also combine with negatively charged nucleic acids to form electrostatic complexes, improving mrna delivery efficiency [ ] . the cationic lipid-mrna complex and other preparations together can form an - nm nanoparticle called lipid nanoparticles (lnp), which can be transfected into the cytoplasm by endocytosis. lnp is one of the most advanced mrna delivery systems. this stable particle consists of ionizable cationic lipids, natural phospholipids, cholesterol and polyethylene glycol (peg) [ ] . the ionizable cationic lipid can promote the autonomous aggregation of mrnas to form a~ nm particle and release mrnas in the cytoplasm through ionization; natural phospholipids support the nanoparticles to form a lipid bilayer structure; cholesterol is used as a stabilizer to increase lnp stability; and peg can extend the half-life of lnp complex [ , ] . mrna is carried in the core of lnp which can be protected from degradation, and the lipophilicity property of lnp material allows the mrna delivery complex to fuse with the host cell membrane and deliver mrna into the cells by endocytosis [ , ] . lnp is often used as a short interfering rna (sirna) delivery system in early researches [ ] . nowadays lnp is also widely used in mrna delivery processes. geall et al. used lnp to deliver self-amplified rna vaccines, which caused the mrna expression level in mice to be significantly higher than that of naked mrna, cd + , and cd + t cell immune responses were also effectively induced. with different administration strategies, the immune-stimulation area provoked by lnp-mrna can be different [ ] , and may achieve the targeted therapy need of different diseases. pardi et al. found that injecting lnp-mrna with the appropriate dose by subcutaneous, intramuscular, and intradermal methods could mediate local gene product expression [ ] . lnp-mrna treatment with intravenous injection, intraperitoneal injection, tracheal inhalation, etc. could achieve systemic expression of gene products, as reported in , out of which intravenous injection showed the highest mrna delivery efficiency, and the target protein products were successfully expressed in the liver for days [ ] . but it is notable that escape mechanisms of mrna from complexes to free state for function in the cytoplasm are still incompletely understood. change of ionization state of lipids with in vivo environmental ph is thought to be critical to the escape process [ ] . meanwhile, further research about the toxicity reduction and immunogenicity regulation of cationic lipid-based delivery system are also urgently needed. currently, cationic polymers have been widely used as mrna delivery vectors [ , ] . commonly used polymer delivery materials include polyethylenimine (pei), poly (beta-amino esters) (pbaes), etc. among them, pei is one of the most widely used materials. pei is a kind of cationic water-soluble polymer with either dendritic, linear, or branching structure, mainly used as a dna/mrna carrier [ , ] . there is a commercial linear pei derivative called jetpei ™, which was once used for dna and sirna transfection, and currently available for mrna transfection [ , ] . however, pei is also qualified with certain cytotoxicity that is hard to be degraded, so researchers often use fatty chains to modify low-molecule-weight pei for the intention of reducing pei toxicity [ , , ] . pbaes are biodegradable polymers originally developed for dna transfection [ ] . a study in showed that pbaes could be used to deliver mrna, and higher levels of mrna transfection in vitro could be achieved when there is no serum protein in the system [ ] . this research has led to the development and application of a variety of pbaes that enhanced serum stability in vivo. there are now thousands of chemically different pbaes created thanks to the simple synthetic method of pbaes [ , , ] . in addition, pbaes and lipids can be formulated together to improve their serum stability. in , kaczmarek et al. developed a polymer-based delivery system by formulating pbaes and lipid-peg together, which had high serum stability and mrna delivery efficacy and successfully detected the target mrna product in the lungs of mice specifically by intravenous injection treatment [ ] . polymer-based materials are crucial competitors against lipids in mrna therapeutics. their toxicity, similar to cationic lipids, has been also thwarted them for broader application [ ] . apart from modification with other materials to improve the properties of polymer-based vectors, optimization for both molecular weight and branch pattern also seems to be a dependable direction. immunotherapy, especially vaccines against infectious diseases and cancers, is the core field of the mrna drug platform. investigations of other areas such as reprogramming of cell fates and genome editing based on mrna have been extensively reviewed [ , ] , therefore they are not a subject of concern in this review. mrna vaccines are generally categorized into two major types according to their construction and replication abilities: self-amplifying mrna (sam) vaccines and non-replicating mrna vaccines. the sam vaccines are developed from an alphavirus genome with its gene encoding structural proteins replaced by the sequence encoding our wanted antigen, enabling intracellular rna amplification, and abundant protein expression of the wanted antigen owing to the integrity of viral replication machinery [ ] . the full length of naked sam can be up to ~ kb. due to self-replication, a remarkable low dose of this vaccine promises a huge amount of antigen production with a considerable duration of effectiveness (up to months) [ ] . the inoculation of sam vaccines can make a simulation of the infection of acute pathogens owing to its pamp, the replication of the self-adjuvanted antigen-encoding rna and the protein expression occurring hours after the vaccination [ ] . this property of sam vaccines, nevertheless, remains controversial since it has the potential to limit the size of antigen-encoding sequence that can be accommodated, to affect the accurate regulation of induced inflammatory responses and even to elicit immune responses of the organism against those rna replication factors, thus reducing the in vivo repeated efficacy of the vaccine [ ] . non-replicating mrna vaccines have the complete structure of mature mrna which contains the orf segment that encodes our desired antigen. owing to their small length ( ~ kb), there is no size restriction for the carrier capacity on the antigen, allowing better control of triggered immune responses as well as developing more affordable approaches from synthesis to storage [ , ] . non-replicating mrna vaccines have a huge potential to become the major cure for the current epidemic outbreak. as mentioned earlier, studies regarding mrna vaccines have largely completed concept establishment and initial exploration in the s. in , martinon et al. successfully achieved in vivo induction of specific anti-influenza ctls by intravenous or subcutaneous injection of mice with liposome-entrapped mrna encoding influenza virus nuclear proteins, which was a pioneering mrna vaccine vector attempt [ ] . in , mandl et al. used the gene gun to deliver in vitro synthesized infectious rna from a flavivirus, demonstrating induced protective immunity in mice by less than ng of rna [ ] . boczkowski vaccines against infectious pathogens has always been the most effective way to prevent and limit infectious diseases, a classic example of which is the complete eradication of the smallpox virus. unfortunately, traditional strategies of vaccines, such as non-live freeze-dried vaccines and live attenuated vaccines, underperform against some chronic or recurrent pathogenic infections with a long duration of disease such as aids and tuberculosis (tb). traditional vaccines' lack of adequate speed, owing to relatively slow process of development, would not be able to address outbreaks of virulent pathogens such as zaire ebolavirus, zika virus (zikv) and coronavirus. mrna vaccines against infectious diseases have made promising accomplishments and some products have entered human clinical trials ( table ). overall development steps of those vaccines are ( ) constructing the core antigen-encoding mrna sequence optimized or combined based on selected antigen(s) from the target pathogen; ( ) trying and choosing a proper combination of mrna construction type, adjuvants, carrier materials and the route of administration; ( ) detecting in vivo expression of the encoded antigen and the level of elicited immune responses; ( ) providing research and demonstrations of immune induction mechanisms. here we have reviewed some recently published promising studies related to mrna vaccine application trials. influenza viruses have the characteristic of continuous evolution which makes them hard to be completely eradicated. the monoclonal antibody treatment targeting the conservative site of effector molecules of the influenza virus is commonly accepted as a highly specific and effective method against the virus [ ] . mrna vaccines encoding the conserved regions of influenza virus effector protein(s) are capable of provoking the generation of specific antibodies so that a better prevention or treatment effect, compared to conventional vaccines, is acheived. in addition, the rapid production process of mrna vaccines makes them easier to stand out in preventing novel influenza virus. current mrna vaccines against influenza mostly use cationic lipids-based delivery systems to effectively deliver mrna. the rnactive ® vaccine platform with the self-adjuvanting property give an impressive performance in trials of prevention of influenza, too [ ] . brazzoli et al. generated a novel oil-in-water cne as the carrier for a sam vaccine expressing influenza virus hemagglutinin (ha) antigen [ ] . the vaccination was reported to effectively induce functional neutralizing antibody and ha-specific cd + th cells and cd + cytotoxic t cells immune responses; it also defended a lethal influenza virus challenge in mice. pardi et al. successfully elicited ha stalk-specific antibody response in mice, rabbits, and ferrets by immunization with nucleoside-modified non-replicating mrna vaccine candidate encoding full-length influenza virus ha formulated in lnp [ ] . this mrna-lnp influenza vaccine partially overcome inhibition by the usage of maternal antibodies, and in turn induced a longer-lived and stronger immune protection in the mouse pups than a conventional influenza vaccine [ ] . feldman et al. reported phase i clinical trials of the first two non-replicating mrna vaccines against influenza viruses (h n and h n ) encoding full-length ha respectively from h n and h n with a : mass ratio of mrna to lnp [ ] . both vaccines used a lnp carrier that was first applied in mrna vaccines against the zika virus [ , ] ; they were proved well tolerated by healthy adults and elicited potent humoral immune responses [ ] . this research showed the potential of mrna vaccines to address highly variable pathogens. aids, a chronic and life-threatening condition owing to the infection of hiv, has not yet found a truly effective and affordable way of cure since its discovery in . defeating hiv is a significant issue of research developing mrna vaccines. at present, there are several mrna vaccines for the treatment of aids in human clinical researches. ex vivo loading of dc delivery systems seems to be a preferred delivery method which is normally used for cancer treatment. in infectious diseases, it is almost exclusively used for therapeutic research on aids, and is widely proved to safely cause antigen-specific cd + and cd + t cell immune response [ ] . however, in , gandhi et al. reported disappointing results of a clinical trial for immunization of hiv- -positive participants with autologous dcs transfected with mrna encoding hiv- structural proteins gag and nef [ ] . in that trial, merely transient and weak immune responses were detected, indicating the necessary improvement for the dc vaccination [ ] . in such a way, delivery systems that can elicit strong antigen-specific t cell immune responses are getting more attention in aids treatment. the cationic nanoparticle carrier is a promising delivery system with multiple diversity. zhao et al. developed a pei-stearic acid (psa) copolymer-based self-assembled cationic nanomicelles which delivered non-replicating mrna vaccine encoding hiv- gag [ ] . their study initially showed the potential of psa/mrna nanomicelle vaccine strategy against hiv with acceptable carrier toxicity, efficient endosomal escape and translation of mrna in dcs, and stimulated potent specific antibody secretion and pro-inflammatory cytokine expression [ ] . bogers et al. demonstrated a sam vaccine encoding a hiv- clade c envelope glycoprotein delivered by a cne system, including squalene, dotap, sorbitan trioleate and polysorbate, with a relatively mature preparation protocol [ ] . greater cellular immune responses and neutralizing antibody responses were induced by this hiv sam vaccine instead of two other sam vaccine modalities, the self-amplifying mrna of which were encapsulated by a hiv recombinant envelope protein or in an engineered viral replicon particle [ ] . hti-trimix, a combination of activation adjuvant trimix and selected mrna comprising of conservative fragments from hiv- structural proteins-gag, pol, vif, and nef, is a new mrna-based therapeutic vaccine candidate against hiv- [ ] . it encodes strong activation signals and a potent hiv recombinant antigen. the preclinical results suggested an effective induction of mature dcs, antiviral cytokine secretion (especially ifn-γ) and t cell stimulation. mice that were intranodally injected with hti-trimix generated potent antigen-specific cytotoxic t-cell responses [ ] . by the end of , phase i and phase iia clinical trials of hti-trimix have been accomplished. in phase iia, hiv- -infected participants received three vaccinations at weeks , , and detected through ultrasound-guided administration with an inguinal lymph node. although hti-trimix showed good safety and tolerance, an unexpected start codon was unfortunately found upstream of the hti recombinant antigen coding sequence which likely had a negative influence on hti protein expression [ , ] . future studies for corrected hti are not yet certain. taking into consideration of an additional translation process of mrna vaccines, pre-testing of mrna expression in vitro deserves our attention. due to the limited understanding of hiv and the unclear pathogenesis, there are still many difficulties in the treatment of aids. choosing proper antigen(s) and delivery system that can cause intense antigen-specific t cell immune response should be emphasized at mrna vaccine design in the future. in addition, mrna vaccines on aids prevention may also be a feasible field. in the last years, there have been three coronavirus infections (severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and (sars-cov- )) globally, all leading to extreme health threats and tremendous economic loss without established therapies or vaccine treatment that would cure the illness. of all the patents regarding vaccine types, most of them are related to sars and mers, only three patents have been focused on mrna vaccines as of today [ ] . in the face of the sudden new coronavirus epidemic, the speed of vaccine development determines the speed of life saving. therefore, it is inevitable that mrna vaccines with rapid product process will play an important role in the development of coronavirus vaccines. covid- , caused by sars-cov- infection, has been spreading all over the world with over . million confirmed cases and over , deaths as of august , (data from world health organization). an effective vaccine is urgently needed. lin et al. reported two non-replicating mrna vaccines respectively encoding the receptor-binding domain of the spike protein and the virus-like particles (vlps) of sars-cov- ; further optimization of antigen sequences, as well as safety and efficacy evaluations are underway [ ] . moderna first announced a mrna vaccine candidate, mrna- , against sars-cov- , and officially began phase i clinical trials for safety and immunogenicity evaluation on march , . this vaccine encodes the spike (s) protein of sars-cov- in a prefusion stabilized form. according to the interim data announced on may , , mrna- was shown generally safe and well tolerated; after two weeks following the second dose, with the vaccination dose as low as µg, the levels of both binding antibodies and neutralizing antibodies in serum were at the levels detected in samples from people having recovered from covid- . collaborative development of a new mrna vaccine against sars-cov- has been announced by sanofi pasteur and translate bio on march , . pfizer and biontech announced the positive results of the ongoing phase i/ii clinical trials of bnt b . it is a modified mrna vaccine candidate formulated by lnp, encoding trimerized sars-cov- s protein receptor binding domain. proper dose level of bnt b was initially identified between µg and µg. after two doses of µg and µg of bnt b , mean titers of specific neutralizing antibodies were . -fold and . -fold, respectively, the specific neutralizing antibody of the convalescent [ ] . cv , a prophylactic non-replicating mrna candidate vaccine combined with protamine encoding rabies virus glycoprotein (rabv-g), completed phase i clinical trial in [ ] . in pre-clinical trials, this vaccine elicited powerful functional antibody responses with a stable titer level up to one year and induced robust specific cd + and cd + t cells (higher cd + t cell induction than the induction by a licensed vaccine) when applied intradermally both in mice and pigs [ ] . although cv was shown generally safe in phase i trial, the unstable administration-dependent functional antibody titer resulted in an unclear research outlook [ ] . subsequent new preclinical studies in reported an improved humoral and cell immune response using rabv-g mrna packaged in lnp in both mice and nonhuman primates in comparison to the protamine formulated mrna candidate; corresponding human clinical trials are being followed up [ ] . in , modified mrna-lnp vaccines against zikv were reported in cell and nature respectively [ , ] . pre-membrane (prm) protein and envelope (e), two zikv structural proteins, form prm-e heterotrimers when zikv buds invade the lumen of the endoplasmic reticulum. richner et al. developed a lnp-encapsulated non-replicating mrna vaccine encoding the human ige signal sequence, which contained full-length prm and e genes (igesig-prm-e) [ ] . intramuscular inoculation of µg of igesig-prm-e lnps with a booster protected mice from severe zikv infection with remarkably high titers of neutralizing antibodies (> / , ec ) was detected [ ] . similarly, pardi et al. demonstrated a low dose ( µg) intradermal vaccination contained with mrna-lnp complex encoding prm-e glycoproteins of zikv, which sufficiently protected non-human primates from a viral challenge [ ] . against venezuelan equine encephalitis virus (veev), two synthetic cne-encapsulated venezuelan equine encephalitis sam vaccine candidates, lav-cne (carrying the rna genome of tc- , a live-attenuated investigational vaccine strain) and iav-cne (carrying tc- viral genome with the capsid gene deleted), were designed to be capable of offering immune protection [ ] . in inoculated mice, both vaccines induced robust virus-specific neutralizing antibodies and provided protection from wild-type veev aerosol challenge [ ] . in addition, mrna-based candidate vaccines have been developed and trialed against diverse viruses such as chikungunya virus, herpes simplex virus, human metapneumovirus and parainfluenza virus, all showing desirable development prospects [ ] [ ] [ ] . it's worth noting that pepini et al. reported type i ifn, which played a critical role in antiviral responses and elicited by lnp-formulated sam vaccine, could inhibit the expression of mrna-encoded antigens in mice [ ] . in line with this, zhong et al. found that a naked zikv sam vaccine encoding the zikv prm-e induced limited and unstably variable humoral immunity in wildtype mice when compared with robust response in ifnar knockout mice [ ] . those researches suggest antiviral responses, especially type i ifn response, activated by sam vaccination might have a negative effect on sam-induced immune protection and optimization of sam construction and administration should be considered. apart from viral antigens, only very few species of bacterial and parasitic antigens have been used in mrna vaccine attempts, many of which still remain at the preclinical trial stage [ ] [ ] [ ] [ ] . a wider variety of targeted antigens will represent more important issues for the next stage of mrna vaccine development. in , maruggi et al. designed two prophylactic sam vaccines mixed with cne encoding streptolysin-o (slodm) from group a (gas) streptococci and the pilus a backbone protein (bp- a) from group b (gbs) streptococci, respectively [ ] . inoculated mice succeeded in producing a large amount of fully functional antibodies which could be significantly increased by booster, and survival rate was increased for gas and gbs infections [ ] . among infectious diseases caused by a single pathogen, the tb caused by the bacterial pathogen mycobacterium tuberculosis has been ranking first in fatality rate globally for a long time. still, there is only one vaccine licensed against human tb: mycobacterium bovis bacillus calmette-guérin (bcg), an attenuated whole-cell vaccine which has been found severe limitations in numerous clinical trials [ , ] . mva a, a tb subunit vaccine expressing single antigen ag a, had no significant improved protection in phase iib trial [ ] . the letdown reminds us that, compared with the viral infections, bacterial infections tend to have more complicated stages with diverse characteristics of molecule expression, which are virtually impossible for single antigen to cover. if mrna vaccines want to be applied further into the area of prevention and treatment of bacterial infection, more optimizations should be considered, including selection and recombination of various antigens or epitopes, periodic administration for different target antigens, and even direct addition of adjuvanted passive immune compositions (e.g., kose et al. developed a chikungunya-against mrna vaccine encoding neutralizing human monoclonal antibodies [ ] ). we are all looking forward to the first successful mrna vaccine product. optimizing the primary and secondary structure of mrna and choosing the appropriate delivery system according to the characteristics of different diseases are critical steps for better application of mrna vaccines against various pathogens. as our knowledge of tumor-specific antigens gradually deepens, people are now discussing more about the possibility of developing cancer vaccines [ , ] . tumor-associated antigens (antigens preferentially expressed in cancerous cells and usually relevant to dysregulation and abnormal behaviors of cancerous cells) and tumor-specific neo-epitopes (small peptides derived from tumor-specific somatic mutation that are exposed to the surface of cancer cells and can be recognized by t cells) are now the core targets of mrna cancer vaccines [ , ] . considering the diversity and uncertainty of the cancerogenesis, cancer vaccines that are mainly therapeutic are now aimed to stimulate cellular immunity, which would potentially act as an effective cancer treatment [ , ] . there have been some clinical trials of hopeful candidates in progress (table ) . sahin et al. pioneered the concept of "mutanome," an overall detection and map of somatic mutations in individual tumors [ ] , the obtainment of which made personalized vaccination therapy possible and attractive with the help of next-generation sequencing technology [ , ] . further, they designed procedures to develop personalized mrna mutanome vaccines from mutanome identification, neo-epitopes prediction, and selection. this allowed mrna vaccines to be unique for each patient. this strategy was firstly applied on melanoma patients with inspiring results achieved. by comparing tumor biopsies and normal blood cells via exome and rna sequencing, researchers identified and selected ten mutations related to the human lymphocyte antigen (hla) function per patient. based on those mutations, core mrna were synthesized and neo-epitope vaccines (≥eight doses) were percutaneously injected into inguinal lymph nodes [ ] . robust t cell responses against multiple neo-epitopes encoded by the vaccine were detected in all patients. with pd- blockade combination therapy, complete responses to vaccination were developed in some patients [ ] . clinical trials of similar mutanome-based mrna vaccines against triple negative breast cancer are under way [ ] . co-transfecting mrna encoding immune-regulation factors into dcs, normally by electroporation, to boost immune responses elicited by dcs-mrna cancer vaccine has been an extensively studied subject [ , , ] . trimix can promote dc activation, cd + t cell phenotype shift, and cytotoxic t lymphocyte responses in numerous animal trials [ , , ] . a joint therapy, which combined the vaccination of the dc-based mrna, encoding trimix and tumor antigens, plus ipilimumab (trimixdc-mel ipi), had been applied for advanced melanoma. it successfully induced potent tumor-associated antigen specific cd + t cell responses, demonstrating excellent therapeutic effects of the tumor-specific vaccine and immune checkpoint block agents [ ] . nevertheless, there existed undetectable response after in vitro t-cell stimulation in / patients, suggesting the necessity for a further study for mechanism of action about this issue. in addition, reinhard et al. developed chimeric antigen receptor (car)-t cells targeting regulated tight junction protein claudin (cldn ) supplemented by a liposomal cldn -encoding rna vaccine which greatly boosted car-t cell stimulation and regression of large solid tumors in mice [ ] . in view of significantly uneven distribution of various lymphocytes in the whole body and different locations and attributes of different primary tumors, it is essential to select the proper carrier and administration method for optimization of the mrna vaccine effect [ , ] . general carrier systems and delivery routes are as stated above. for example, jabulowsky et al. developed a rna-lipoplex vaccine against melanoma [rna(lip)] which was injected intravenously to deliver mrna steadily to apcs in whole body for antigen expression and presentation [ ] . this vaccine is under clinical evaluation for safety and tolerance (nct ). direct intratumoral inoculation is notably an emerging method [ , , ] . shariati et al. pioneered the pressurized intraperitoneal aerosol chemotherapy (pipac) for delivering mrna complexes and demonstrated that pipac is able to apply mrna into peritoneal cavity in mice [ ] . besides, for optimization of carrier analysis and selection, a high-throughput approach to screen proper ionizable lipid-like materials as mrna delivery vehicles were developed by anderson et al. they constructed a combinatorial library of ionizable lipid-like materials using an isocyanide-mediated three-component reaction [ ] ; the screening standard was capable of facilitating in vivo mrna delivery and providing effective and specific immune activation [ ] . the best candidate chosen, heterocyclic lipids-mrna vaccine, was further demonstrated to promote apcs maturation to stimulate potent immune responses via the intracellular stimulator of interferon genes pathway [ ] . as reviewed by weissman et al., standardized in vitro good manufacturing practice of mrna production is now accessible, while barriers still exist on synthesis of some uncommon sequences as well as salable and low-cost production for some reagents [ ] . at the same time, capability of the long-term storage of mrna vaccines with invariable activity should be emphasized. it had been reported early on that purified freeze-dried rna in trehalose could maintain the activity up to months at • c, whose stability was comparable to conventional vaccines [ ] . phase i trial of mrna-based rabies virus vaccine cv demonstrated that it could be stored as a freeze-dried preparation at - • c for years and at • c for months without obvious loss of activity [ ] . in , coolen et al. developed a poly(lactic acid)-nanoparticle-based mrna vaccine, mixed with amphipathic cationic peptides, which showed stable expression efficacy after storage at • c for up to days [ ] . although further investigations are needed to study the effects of storage of mrna complexed with vector molecules under unfrozen condition, studies has suggested the potential of mrna vaccines for cold-chain free transport and storage in the future [ ] . what makes the mrna vaccines a widely recognized form compared to conventional vaccines is the non-toxic production process, its short production time and chemical nature as ribonucleic acid, in line with safety and well-tolerance of mrna vaccines shown in multiple clinical trials [ , , , , , , ] . various adverse symptoms, however, were still detected occasionally with unclear mechanism, emphasizing the importance of safety optimization [ , ] . autoimmunity triggered by type i ifn responses are suggested to play a role in adverse physical reactions [ , ] . other problems such as edema and coagulation due to excessive extracellular rna and induction of anti-mrna antibodies were also reported [ ] ; side effects owing to the vectors or administrating routes may also exist. work on both safety assessment and investigations to mechanisms of the anti-vaccine response need to be moved forward. even though no mrna vaccine product has been approved for marketing so far, development of specialized official product guidance of mrna vaccines should be taken into consideration by medical authorities, particularly in view of the momentum and potential of this field where a remarkable number of relevant preclinical and clinical trials is active or completed. the development of tools for material screening or characterization of mrna-based complexes is of utmost importance to improve the stability and protein producing efficiency of mrna vaccines. constructing a combinatorial library of ionizable lipid-like materials is a promising strategy introduced above [ ] . in , zhang et al. used the fluorescence correlation spectroscopy (fcs) to analyze the mrna-based complex stability in buffer and biological fluid such as human serum and ascitic fluid [ ] . results have shown that strong mrna binding of linear pei would likely lead to a less efficient mrna translation while a lipid-based carrier performed well in intracellular efficient release and subsequent translation of mrna. further, they applied fcs and single particle tracking to study the decay kinetics of mrna with the half-life of mrna in biological samples measured (~ - min) [ ] . single-molecule methods have a tempting application prospect of deep optimization for the construction of mrna vaccines. theoretically, mrna can be synthesized to express almost any protein antigens, which can provide a great flexibility for antigen design. for instance, a zikv vaccine from richner et al. contains mrna encoding a mutant antigen of zikv of which an immunodominant cross reactive epitope to dengue virus is deleted to minimize the induction of cross-reactive antibodies [ ] . nowadays, the application of artificial intelligence and deep learning lead to a huge progress in gene sequence-based prediction of protein structure [ , ] . the development of big data, meanwhile, immensely advances the improvement of algorithms for tumor antigen epitope prediction [ ] . with the field of data mining further, it could become a reality to optimize existing antigens much better and design unprecedented antigens independent of natural genes. optimization and personalization of mrna vaccines will become a revolutionary milestone. in conclusion, the mrna vaccine is a versatile and powerful platform. its successful development towards clinical translation will remarkably strengthen our ability to react to and control emerging communicable diseases, and prominently enrich our arsenal of treating classical and re-emerging communicable diseases and cancers from the perspective of stimulating self-immune responses. further investigations for mechanisms of action of extracellular transportation and intracellular escape and gene expression of mrna still deserve our efforts. moreover, modularization of mrna vaccine design and production targeting different application conditions seems to be a promising strategy for the clinical usage [ ] . in the next years, several critical clinical trials of mrna vaccines are going to be completed (especially those against covid- ). more extended human clinical experience will give us a more comprehensive insight into mrna vaccine platform and various delivery systems. from increasing productive capacity of mrna and various carrier materials, to screening potential carrier molecules and adjuvants, to improving the composition and construction of vaccines, to arranging a corresponding route for administration, to optimizing the core encoding mrna sequence and to demonstrating immune mechanisms of delivery and induction, the field of mrna vaccines is still far from maturity, but its potential to be the preferred vaccine pattern has been fully shown. author contributions: conceptualization, s.x. and l.z.; writing-original draft preparation, s.x. and k.y.; writing-review and editing, r.l., s.x. and k.y.; supervision, l.z. all authors have read and agreed to the published version of the manuscript. the authors declare no any commercial or financial relationships that could be construed as a potential conflict of interest in this article. an unstable intermediate carrying information from genes to ribosomes for protein synthesis cationic liposome-mediated rna transfection direct gene transfer into mouse muscle in vivo the use of basic proteins to increase the infectivity of enterovirus ribonucleic acid foreign nucleic acids as the stimulus to make interferon a blocked structure at the terminus of mrna from cytoplasmic polyhedrosis virus translation of rabbit globin mrna introduced by liposomes into mouse lymphocytes functional messenger rnas are produced by sp in vitrotranscription of cloned cdnas induction of virus-specific 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ligand for rig-i nucleoside modifications in rna limit activation of - -oligoadenylate synthetase and increase resistance to cleavage by rnase l expression of therapeutic proteins after delivery of chemically modified mrna in mice modified foxp mrna protects against asthma through an il- -dependent mechanism a cationic nanoemulsion for the delivery of next-generation rna vaccines preclinical evaluation of trimix and antigen mrna-based antitumor therapy enhancing the t-cell stimulatory capacity of human dendritic cells by co-electroporation with cd l, cd and constitutively active tlr encoding mrna phase i clinical trial of an intranodally administered mrna-based therapeutic vaccine against hiv- infection cationic lipids activate intracellular signaling pathways toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mrna self-adjuvanted mrna vaccines induce local innate immune responses that lead to a potent and boostable adaptive immunity the 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genetic immunization trial watch: dendritic cell-based interventions for cancer therapy dendritic cells interact directly with naive b lymphocytes to transfer antigen and initiate class switching in a primary t-dependent response phase ii study of autologous monocyte-derived mrna electroporated dendritic cells plus ipilimumab in patients with pretreated advanced melanoma vaccination of colorectal cancer patients with cea-loaded dendritic cells: antigen-specific t cell responses in dth skin tests dendritic cells as therapeutic vaccines against cancer breckpot, k. mrna-based dendritic cell vaccines immunogenicity of ags- dendritic cell therapy in patients treated during acute hiv infection protamine enhancement of rna uptake by cultured chick cells messenger rna-based vaccines with dual activity induce balanced tlr- dependent adaptive immune responses and provide antitumor activity liposome-based cationic adjuvant formulations (caf): past, present, and future materials for non-viral 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derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector rna-based vaccines in vitro-synthesized infectious rna as an attenuated live vaccine in a flavivirus model dendritic cells pulsed with rna are potent antigen-presenting cells in vitro and in vivo vaccine: induction of antitumor immunity by human glycoprotein mrna immunization. hum neutralization of influenza a viruses by insertion of a single antibody loop into the receptor binding site induction of broad-based immunity and protective efficacy by self-amplifying mrna vaccines encoding influenza virus hemagglutinin nucleoside-modified mrna immunization elicits influenza virus hemagglutinin stalk-specific antibodies nucleoside-modified mrna vaccination partially overcomes maternal antibody inhibition of de novo immune responses in mice immunization of hiv- -infected persons with autologous dendritic cells transfected with mrna encoding hiv- gag and nef: results of a randomized, 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prevents clinical and subclinical genital herpes safety and immunogenicity of a mrna-based chikungunya vaccine in a phase dose-ranging trial phase trial of an mrna-based combination vaccine against hmpv and piv induction of an ifn-mediated antiviral response by a self-amplifying rna vaccine: implications for vaccine design immunogenicity and protection efficacy of a naked self-replicating mrna-based zika virus vaccine dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h n influenza, and toxoplasma gondii challenges with a single dose immunogenicity and protective efficacy induced by self-amplifying mrna vaccines encoding bacterial antigens neutralization of the plasmodium-encoded mif ortholog confers protective immunity against malaria infection mrna as a transformative technology for vaccine development to control infectious diseases variable virulence and efficacy of bcg vaccine strains in mice and correlation with genome polymorphisms safety and efficacy of mva a, a new tuberculosis vaccine, in infants previously vaccinated with bcg: a randomised, placebo-controlled phase b trial a lipid-encapsulated mrna encoding a potently neutralizing human monoclonal antibody protects against chikungunya infection mutant mhc class ii epitopes drive therapeutic immune responses to cancer targeting the heterogeneity of cancer with individualized neoepitope vaccines neoantigens in cancer immunotherapy tumour antigens recognized by t lymphocytes: at the core of cancer immunotherapy exploiting the mutanome for tumor vaccination targeting the tumor mutanome for personalized vaccination therapy abstract ct : mutanome engineered rna immuno-therapy (merit) for patients with triple negative breast cancer (tnbc) intratumoral delivery of trimix mrna results in t-cell activation by cross-presenting dendritic cells trimix and tumor antigen mrna electroporated dendritic cell vaccination plus ipilimumab: link between t-cell activation and clinical responses in advanced melanoma an rna vaccine drives expansion and efficacy of claudin-car-t cells against solid tumors nanoparticles for nucleic acid delivery: applications in cancer immunotherapy abstract ct : a first-in-human phase i/ii clinical trial assessing novel mrna-lipoplex nanoparticles encoding shared tumor antigens for immunotherapy of malignant melanoma abstract lb- : combinatorial treatment with intratumoral cytokine mrnas results in high frequency of tumor rejection and development of anti-tumor immunity across a range of preclinical cancer models high pressure nebulization (pipac) versus injection for the intraperitoneal administration of mrna complexes long-term storage of dna-free rna for use in vaccine studies poly(lactic acid) nanoparticles and cell-penetrating peptide potentiate mrna-based vaccine expression in dendritic cells triggering their activation fluorescence correlation spectroscopy to find the critical balance between extracellular association and intracellular dissociation of mrna complexes fluorescence-based quantification of messenger rna and plasmid dna decay kinetics in extracellular biological fluids and cell extracts alphafold at casp protein structure prediction beyond alphafold personalized vaccines for cancer immunotherapy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -gh ag x authors: fu, weihui; liu, yan; xia, lu; li, min; song, zhigang; hu, huiliang; yang, zongguo; wang, lin; cheng, xiaobo; wang, mei; jiang, rongrong; liu, li; mao, xiaoting; chen, jun; ling, yun; zhang, lin; yan, jin; shan, fei; steinhart, corklin; zhang, xiaoyan; zhu, tongyu; xu, jianqing; lu, hongzhou title: a clinical pilot study on the safety and efficacy of aerosol inhalation treatment of ifn-κ plus tff in patients with moderate covid- date: - - journal: eclinicalmedicine doi: . /j.eclinm. . sha: doc_id: cord_uid: gh ag x background: the outbreak of a new coronavirus (sars-cov- ) poses a great challenge to global public health. new and effective intervention strategies are urgently needed to combat the disease. methods: we conducted an open-label, non-randomized, clinical trial involving moderate covid- patients according to study protocol. patients were assigned in a : ratio to receive either aerosol inhalation treatment with ifn-κ and tff , every h for three consecutive dosages, in addition to standard treatment (experimental group), or standard treatment alone (control group). the end point was the time to discharge from the hospital. this study is registered with chictr.org.cn, chictr . findings: a total of thirty-three eligible covid- patients were enrolled from february , to april , , eleven were assigned to the ifn-κ plus tff group, and twenty-two to the control group. safety and efficacy were evaluated for both groups. no treatment-associated severe adverse effects (sae) were observed in the group treated with aerosol inhalation of ifn-κ plus tff , and no significant differences in the safety evaluations were observed between experimental and control groups. ct imaging was performed in all patients with the median improvement time of (.) days (iqr (.) – (.) ) in the experimental group versus (.) days (iqr (.) – (.) ) in the control group (p< (.) ). in addition, the experimental group had a significant shorten median time in cough relief ( (.) days [iqr (.) – (.) ]) than the control group did ( (.) days [iqr (.) – (.) ])(p< (.) ), in viral rna reversion of (.) days (iqr (.) – (.) ) in the experimental group vs . days (iqr (.) – (.) ) in the control group (p < (.) ), and in the median hospitalization stays of (.) days (iqr . – . ) in the experimental group vs (.) days (iqr . – . ) in the control group (p< (.) ), respectively. interpretation: aerosol inhalation of ifn-κ plus tff is a safe treatment and is likely to significantly facilitate clinical improvement, including cough relief, ct imaging improvement, and viral rna reversion, thereby achieves an early release from hospitalization. these data support to explore a scale-up trial with ifn-κ plus tff . funding: national major project for control and prevention of infectious disease in china, shanghai science and technology commission, shanghai municipal health commission. in december , a novel pneumonic illness caused by a new coronavirus, sars-cov- (previously named as -ncov), emerged in wuhan, china, which was subsequently called covid- [ ] . the full disease spectrum of covid- ranges from a mild, moderate, self-limiting respiratory tract illness that may rapidly progress to severe progressive pneumonia, multiorgan failure, and death [ À ] . as of april , , confirmed cases have been reported in countries over the world, with more than , , confirmed cases and , deaths, according to the statistics from world health organization. furthermore, at the time of this writing, the epidemic is worsening globally and impacting hundreds of thousands of persons, posing a great challenge to public health as well as global economies. at the present time, there are no specific therapeutic agents against the new virus. recently, a trial of lopinavirÀritonavir treatment in adults hospitalized with severe covid- was conducted, however, no benefit was observed beyond standard supportive care [ ] . two recent studies have shown that clinical improvement was observed in of severe covid- patients ( %) when they received a day course of remdesivir [ ] , there is no significant difference between a -day and a -day course of remdesivir administration [ ] . chloroquine (cq) or hydroxychloroquine (hcq) were considered as the most promising agents against covid- at the early time of the sars-cov- pandemic. up to date, the therapeutic efficacy of hcq in covid- patients remains controversial [ , ] , clinical trials to test the efficacy of hydroxychloroquine are ongoing. under this circumstance, effective prevention and treatment approaches are urgently needed to combat the disease. previous clinical trials during the sars epidemic have shown that type i interferons (such as ifn-a, ifn-b) could effectively inhibit the replication of sars-cov- and thereby attenuated the clinical complications [ ] . recent study had shown that sars-cov- was sensitive to type i interferon pretreatment in vero cells [ ] . however, the persistent inflammatory response resulted from ifn-a/b may cause the damage to infected patients. indeed, as animal experiments showed that ifn-a/b treatment induced influx of pathogenic inflammatory monocytes and vascular leakage, this aggravates the inflammatory damages [ ] . therefore, systemic adverse reactions of ifn-a/b would also be a concern. ifn-k is a relatively mild type i interferon, which can effectively inhibit the replication of enveloped viruses including emcv, iav, hcv, and others, by activating the interferon-stimulated response element signaling pathway [ ] . however, different from ifn-a or ifn-b, the antiviral activity of ifn-k is cellassociated [ ] and it inhibits the replication of influenza virus largely relying on ifnar-mapk-fos-chd axis [ ] whereas ifn-a or ifn-b mainly employ stat pathway. recently, we further verified that ifn-k significantly suppressed the replication of sars-cov- in huh cell line (data not shown). trefoil factor (tff ) is a secreted polypeptide with three disulfide bonds, resistant to acid, heat, and protease hydrolysis, and able to protect the gastrointestinal tract from microbial or chemical induced injury by promoting the repair of injury and regulating the immune response [ À ]. in model of asthma, tff is increased in response to epithelial injury, exerts a protective role in limiting the progression of airway remodeling, and contributes to airway epithelial repair [ ] . in addition, tff rapidly induces interleukin to promote type immunity during allergic asthma and hookworm infection [ ] . tff also potently accelerates healing and reduces inflammation in a rat model of colitis [ ] . our researches in mice model demonstrated that tff reduced the morbidity and mortality in influenza infection by attenuating the inflammatory responses and improving the pulmonary function, but not by inhibiting viral replication (patent application no: . ). recently, a study has shown that imbalanced host response to sars-cov- , with limited ifn response coupled with exuberant inflammatory cytokines/chemokines, drives development of covid- [ ] . based on these findings, we hypothesized that the combined administration of human derived ifn-k and tff would be likely synergistically to fight against sars-cov- induced respiratory pneumonia by reducing viral replication, attenuating inflammatory responses, and improving the reconstitution of respiratory tract mucosa, so as to improve the prognosis of patients with covid- . therefore, to evaluate the efficacy and safety of intranasal inhalation of tff and ifn-k protein for sars-cov- infection, we conducted an open-label, nonrandomized, clinical trial in adult patients hospitalized with moderate covid- disease in china. this was an open-label, non-randomized pilot clinical trial conducted from february , through april , at shanghai public health clinical center, shanghai, china. the protocol of the clinical treatment assigned criteria was carried out according to a stratification flowchart and treatment assignment (fig. ). written informed consents were provided by all study participants, patients either accept ifn-k plus tff plus standard treatment as experimental group or receive standardized treatment alone as control group. as this is a pilot study to primarily test the safety, we recruited patients in ifn-k plus tff group (a similar sample size to a phase ia), and set the ratio to control group as : ( patients in control) in order to achieve reliable safety data. standard supportive care was given according to the diagnosis and treatment protocol for novel coronavirus pneumonia (trial version ) released by national health commission & state administration of traditional chinese medicine on march , ). the primary objective of the pilot study was to evaluate the safety of aerosol inhalation of ifn-k plus tff . all adverse events (ae) were based on the ich (international conference on harmonization of technical requirements for registration of pharmaceuticals for human use). in this trial, any ae from the beginning of aerosol inhalation to days after the end of the last aerosol inhalation were taken as an adverse event during treatment (teae); the secondary objective of the pilot study was to evaluate the clinical efficacy of ifn-k plus tff as compared to the control group as assessed by days of hospitalization staying, ct imaging improvement and cough relief time and negative reversion of viral rna after days of treatment. the trial was approved by ethics committee in shanghai public health clinical center (approval number: yj- -s - ). the trial was conducted in accordance with the principles of the declaration of helsinki and the good clinical practice guidelines of the international conference on harmonization. all participant personal information will be only accessed by the researchers and assigned members of the hospital ethics committee. hospitalized patients with confirmed covid- were enrolled in this study. identification of sars-cov- via reverse-transcriptase-polymerase chain-reaction (rt-pcr) in a throat-swab sample was carried out by the local center for disease control (cdc) or by a designated diagnostic laboratory following the recommendation of the china national center for disease control. inclusion criteria: patients willing and able to provide written informed consent prior to performing study procedures were included. male and nonpregnant female patients years of age or elder were eligible if they had a diagnostic specimen that was positive on rt-pcr for sars-cov- . in addition, patients could be included in peripheral capillary oxygen saturation (spo ) > % on room air at screening. symptoms of infection are non-specific, and include fever, cough, and myalgia, with diarrhea, without the subsequent development of dyspnea. patients with moderate pneumonia were then included following the diagnosis and treatment plan for the novel coronavirus disease issued by the national health commission (nhc). exclusion criteria: these included a physician's decision that involvement in the trial was not in the patient's best interest, presence of any condition that would not allow the protocol to be followed safely, known allergy or hypersensitivity to ifn-k and tff , known severe liver disease (e.g., cirrhosis, with an alanine aminotransferase level > £ the upper limit of the normal range or an aspartate aminotransferase level > £ the upper limit of the normal range). breastfeeding and pregnant patients were also excluded. a total of covid- patients with moderate illness symptoms were recruited. the enrolled patients were non-randomly divided into two groups based on their options. patients in the control group received standard of care treatment according to the diagnosis and treatment protocol for novel coronavirus pneumonia (trial version ) released by national health commission & state administration of traditional chinese medicine on march , ), including anti-fever tablets (if necessary), immune enhancers and traditional chinese medicines. anti-fever tablets include ibuprofen suspension, immune enhancers include thymosin alpha- for injection, compound vitamines of injection, traditional chinese medicines include chinese hebal medicines, shufengjidu capsule, lianhua qingwen capsule. in addition to standard treatment for covid- , patients in the experimental group also received aerosol inhalation treatment with mg tff protein and ~ mg (one million u/mg) ifn-k. written informed consent was signed by all enrolled patients. the combination of ifn-k plus tff was administrated to combat against covid- since ifn-k is able to inhibit sars-cov- replication in vitro (data not shown) while tff is capable of reducing inflammation and promoting respiratory repairment (patent application no: cn . ). interestingly, our previous research demonstrated that ifn-k could suppress influenza replication through a different mechanism from ifn-a [ ] . as known, type i interferons may aggravate the inflammatory damages [ ] or disrupt lung epithelial repair during recovery from viral infection [ ] . therefore, to maximize the efficacy and to minimize the risk, we employed these two molecules to synergistically treat covid- patients. the preliminary effective dose of tff in humans is based on the effective protection of tff in influenza virus infection according to the meeh-rubner conversion formula. as for dosing of ifn-k, we referred to the dosage of ifna b ( £ iu) in nasal sprays in previous study [ ] . aerosolized drug was made of purified mature tff and ifn-k protein produced under good manufacturing practices (gmp) conditions, the purity was more than %, and the biological activities of these two proteins were verified in vitro. in addition, the drug was given to the nose inhalation only by face mask. specifically, both proteins were dissolved in ml sterilized water, and the aerosol was delivered for ~ min by nasal mask driven by medical compressed air atomizer (yuwell, m). the aerosol inhalation treatment started from the first day of hospitalization, and included receiving the aerosol treatment times every h. after treatment, a survey was implemented to evaluate if there were any adverse reactions during the course of aerosol inhalation. patients were assessed once daily by trained doctors and nurses using diary cards that captured data in a timely fashion that recorded in an electronic medical record. safety measures, adverse outcomes were monitored by the good clinical practice office from shanghai public health clinical center. serial throat-swab samples were obtained at day (baseline level) and day , , , , , and sent to shanghai public health clinical center designated laboratory, sars-cov- viral load was assessed by real-time reverse transcription-pcr [ ] until patients were discharged. clinical data were recorded into an electronic database and validate by trial staff. the primary objective of the study was to evaluate the safety of aerosol inhalation of ifn-k plus tff ; the secondary objectives of the study were to evaluate the clinical efficacy of ifn-k plus tff as compared to the control group as assessed by days of hospitalization stay, improvement in ct imaging, resolution of cough, and the rate of negative viral rna after days of treatment. safety data included adverse events that occurred during treatment, severe adverse events, and premature discontinuation of treatment. adverse events were classified according to the national cancer institute common terminology criteria for adverse events, version . . the trial was initiated in rapid response to the covid- public health emergency, at which time there was very limited information about clinical outcomes in hospitalized patients with covid- . baseline comparisons of clinical and demographic characteristics according to study group allocation were done using the man-nÀwhitney u test for non-parametric data. continuous variables were compared also using the mannÀwhitney u test for nonparametric data, for both ifn-k plus tff group and the control group analyses for the outcome. quantitative index will be described as median value with interquartile range (iqr). all statistics were conducted on a two-sided, and p-value less than or equal to . was considered statistically significant. safety analyses were based on the patients' actual treatment exposure. statistical analysis was performed in graph pad prism . this trial was registered with chinese clinical trial registry (website: http://www.chictr.org.cn; trial title: clinical study for combination of anti-viral drugs and type i interferon and inflammation inhibitor tff in the treatment of novel coronavirus pneumonia (covid- ); number: chictr ). the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing other report. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. from february , to april , , a total of covid- patients with moderate illness symptoms were recruited, patients were assigned to the experimental group, the left patients only received standard supportive care and were classified as the control group. baseline clinical characteristics, including age, gender, demographic characteristics, laboratory test results, were shown in table . no significant differences were observed between two groups except for total white blood cells (wbcs) that were significantly lower in control group than the experimental group, higher wbcs may relate to more severe disease as previously reported [ ] . no discomfort complications were reported to nurses for all experimental patients during aerosol inhalation. compared with the control group, alt, ast, total bilirubin (tbil) and platelets showed a trend of lower levels in the experimental group, but no significances were reached and they all fall into normal ranges in both groups (fig. ) . these results indicate that ifn-k plus tff treatment have no significant negative impact on liver, gallbladder, and platelets. we then analyzed the kinetic changes of effectiveness index in the peripheral blood of the control group and experimental group. the absolute counts of total white blood cells (wbcs) at day of ifn-k plus tff group were significantly higher than control group during the ifn-k plus tff treatment period. once the treatment was completed (from day to day ), wbcs level returned to normal range value (fig. a) , implicating the administration of ifn-k plus tff may attenuate the lung inflammation and thereby interrupt the migration of wbcs into lung and result in accumulation in blood. furthermore, crp level was much lower in the blood of the ifn-k plus tff group than that in the control group (fig. b) , and plasma tnfa, il- and il- rapidly declined and then remained stable at a low level in the ifn-k plus tff group when comparing with that in the control group from day to day ( supplementary fig. ) , which further implied that the experimental group produced less inflammatory and stress responses. in addition, no significant difference in the numbers of lymphocytes, and the concentration of hemoglobin between these two groups were observed, which suggested that ifnk plus tff treatment had no effect on the survivals of lymphocytes and the function of red blood cells (fig. c, d) . after treatment for days, all blood index returned to normal range value. clinical data, including cough relief, ct imaging improvement, viral rna negative reversion, and hospitalization days, were collected and analyzed according to the protocol. for cough, patients in the ifn-k plus tff group and patients in the control group had a cough symptom at baseline. an average cough relief median time of . days (iqr . À . ) in the experimental group versus . days (iqr . À . ) in the control group (p < . ) was observed (fig. a) . pulmonary ct imaging is defined as three levels: exacerbated, unchanged, and improved (lesion reduction on ct imaging). after days of treatment, the rates of improvement in ct imaging in experimental group and the control group were . % ( / ) and . % ( / ), respectively, then further increased to % ( / ) vs % ( / ) on day , finally reached an improvement median time of . days (iqr . À . ) for experimental group vs . days (iqr . À . ) for control group (p < . ) (fig. b) . the median times of viral rna reversion were . days (iqr . À . ) in the experimental group vs . days (iqr . À . ) in the control group (p < . ) (fig. c ). in addition, ifn-k plus tff treatment significantly shorten the hospitalization of patients as . days (iqr . À . ) in the experimental group vs days (iqr . À . ) in the control group (p < . ) (fig. d ). this open-label, non-randomized clinical trial found that aerosol inhalation of ifn-k plus tff is a safe treatment for moderate covid- patients. both ifn-k and tff are the small proteins and could be induced to respond to respiratory viral infections. in this trial, safety evaluations, including wbcs level, lymphocytes, crp, hemoglobin, alt, ast, blood platelet and tbil, all were fallen in normal ranges, and no aerosol inhalation treatment of ifn-k plus tff related severe adverse events were noticed. interestingly, the adjustment of treatment regimens in control group may result in fluctuation of those evaluation, for example, on the day of standard care treatment, ulinastatin was employed to improve the coagulation function in partial patients because the fibrinogen of the patient was dropped; indeed, the administration of ulinastatin could cause the increase of alt and ast as shown on the th day. in contrast, in ifn-k plus tff group, less fluctuation was observed, which probably implicates patients in ifn-k plus tff group are more stable. in addition, aerosol inhalation is a non-invasive and easy-touse approach for the patients, and it is also applicable for delivery to covid- patients with ventilator through oxygen pipe. our clinical survey has shown that there are no discomfort responses during the aerosol inhalation. we also observed that ifn-k plus tff added to standard treatment was effective in clearing viral throat carriage of sars-cov- in absence of significant elevated inflammation responses in covid- patients. % patients in ifn-k plus tff group turned to undetectable viral rna whereas % shown in control group on day after administration. a recent report showed that the median duration of viral shedding in covid- was days in patients with severe illness and could be as long as days [ ] .there data suggested that the ifn-k plus tff might inhibit sars-cov- replication in vivo, and thereby promoted negative reversion of viral rna, similar to hydroxychloroquine treatment [ ] . the prognosis of covid- patients has been associated with the levels of pro-inflammatory cytokines in the peripheral blood [ ] , il- receptor blocking antibodies, tocilizumab therapy have been applied to counteract the cytokine storm in severe covid- patients [ ] . thus, it is critical to examine how the treatment of ifn-k plus tff influences the inflammatory cytokines. in this study, crp was monitored as an indicator of inflammatory responses, and shown a lower level in the experimental group than that in the control group, indicating that treatment with ifn-k plus tff may eventually reduce inflammatory responses, which was further supported by the observation on gradually increased white blood cells in blood in the course of treatment but not in the control group, as it is rationalized that the local aerosol inhalation may attenuate respiratory inflammation and results in the migration of white blood cells from respiratory tissues back to blood. since only one administration applied every h, we proposed that once every h with prolonged period may further improve the efficacy of treatment. given this is an open label trial, to minimize the risk of bias, all radiologists, clinical doctors and nurses were blinded from grouping information to interpret clinical data or to make clinical decisions. ct imaging was carried out and evaluated by radiologists, the release from of hospital was determined by clinical doctors according the discharging standards, and cough symptom was recorded by the nurses. all data from electronic medical records were collected and analyzed by the researchers. in accordance with the earlier suppression of viral rna, a significant improvement in clinical manifestations after tff and ifn-k treatment was observed, including earlier cough relief and ct imaging improvement, and shortened hospitalization stay, similar to the report from clinical trial of hydroxychloroquine [ ] . therefore, these results suggested the efficacy of tff plus ifn-k treatment in moderate covid- patient and a study to scale up will be crucial to further validate our pilot study. as this is small sample sized, non-randomized, not double-blind study, though we took measures to minimize the bias, patients psychological influence and clinical practices from known grouping people may exert their powers on statistical bias. however, due to nonrandomized study with respect to patient assignment, the power is probably lower due to the small sample size, the p-values may be meaningless. in addition, the estimated duration of viral shedding is influenced by the frequency of respiratory specimen collection, lack of quantitative viral rna assay, and relatively low positive rate of sars-cov- rna detection in throat-swabs. furthermore, all recruited subjects were moderate covid- cases, it remains unknown whether this treatment is applicable to severe and critical cases. our pilot study indicated that ifn-k plus tff is likely to be a safe treatment, and might be beneficial to covid- patients. further to scale-up study will be necessary to determine its efficacy on covid- . in addition, considering that this unique treatment potentially to facilitate clinical improvement, and thereby achieves an early release from hospitalization, the synergistic effect with other medicines such as hcq [ ] , azithromycin [ ] , remdesivir [ ] , high dosage intravenous immunoglobulin (ivig) [ ] , or high dose vitamin c [ ] should be further explored. this work was supported by national natural science foundation of china ( ), the national major project for control and prevention of infectious disease in china ( zx À ), the second batch of emergency science and technology projects of shanghai science and technology commission ( ), and shanghai municipal health commission special emergency project for prevention and treatment of novel coronavirus pneumonia by traditional chinese medicine ( ncp ). we thank all patients who participated in this trial and their families. we thank all the clinical, technical and paramedical staffs of hospitalization units and laboratories for this support in this difficult context. xz, tz, jx, and hl designed the study and had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. wf and yl contributed to writing of the report. jx contributed to critical revision of the report. yl and wf contributed to the statistical analysis, jx, xz and wf provided the purified proteins. lx, mw, zy, jc, ll, yl, fs are the clinical doctors, responsible for carrying out this trial by communicating with the patients and their family, ml, lw, lz, rj, jy are the nurses, responsible for the implementation of patients' aerosol inhalation. zs, hh, xc, xm are responsible for providing the data of viral load, and collecting clinical characteristics of the enrolled patients. cs check the language. 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patients with coronavirus disease can early and high intravenous dose of vitamin c prevent and treat coronavirus disease (covid- all authors reviewed and approved the final version. data are available on various websites and have been made publicly available, additional materials may be required after approval from the corresponding author and national health commission. we have applied for the patent . (pending), and the patent pct/cn / (pending) base on these data. all authors reviewed the signed the conflict of interest forms. supplementary material associated with this article can be found in the online version at doi: . /j.eclinm. . . key: cord- -dc ecfe authors: markwalter, christine f.; kantor, andrew g.; moore, carson p.; richardson, kelly a.; wright, david w. title: inorganic complexes and metal-based nanomaterials for infectious disease diagnostics date: - - journal: chem rev doi: . /acs.chemrev. b sha: doc_id: cord_uid: dc ecfe [image: see text] infectious diseases claim millions of lives each year. robust and accurate diagnostics are essential tools for identifying those who are at risk and in need of treatment in low-resource settings. inorganic complexes and metal-based nanomaterials continue to drive the development of diagnostic platforms and strategies that enable infectious disease detection in low-resource settings. in this review, we highlight works from the past years in which inorganic chemistry and nanotechnology were implemented in each of the core components that make up a diagnostic test. first, we present how inorganic biomarkers and their properties are leveraged for infectious disease detection. in the following section, we detail metal-based technologies that have been employed for sample preparation and biomarker isolation from sample matrices. we then describe how inorganic- and nanomaterial-based probes have been utilized in point-of-care diagnostics for signal generation. the following section discusses instrumentation for signal readout in resource-limited settings. next, we highlight the detection of nucleic acids at the point of care as an emerging application of inorganic chemistry. lastly, we consider the challenges that remain for translation of the aforementioned diagnostic platforms to low-resource settings. in , william osler, one of the founders of modern american medicine, stated, "diagnosis, not drugging, is our chief weapon of offence." despite the medical advances of the th and st centuries, infectious diseases remain major global health issues and continue to claim millions of lives each year in low-and middle-income countries (lmics). − as the global community moves toward control and elimination of infectious disease, it has become evident that there is a pressing need for diagnostic strategies that can be applied in primary health care settings. − this review shines a spotlight on how the applied uses of inorganic chemistry advance the concepts of metals-in-medicine beyond therapeutics and vaccines and into the realm of diagnostics, enabling new tools to meet these global challenges. the world health organization depicts lmic health care accessibility and infrastructure as a tiered pyramid structure, in which the best-equipped facilities are the least accessible and facilities with the least amount of resources are most common ( figure ). in this system, a level facility is a primary care setting with little laboratory infrastructure, trained personnel, or advanced diagnostic technology. in lmics, both urban and rural level facilities frequently lack essential resources such as consistent electricity and clean running water. district hospitals (level ), regional laboratories (level ), and national reference laboratories (level ) have more advanced diagnostic technology available with increasing infrastructure; however, these facilities are often inaccessible to most patients in need due to geography, cost, and lack of transportation. consider the case of early human immunodeficiency virus (hiv) diagnosis in exposed infants. coulibaly et al. highlight the challenges faced by this population in their burkina faso study. hiv-positive mothers were advised to attend a - week postnatal appointment at their nearest primary healthcare facility for collection of dried blood spot samples from their exposed infants. qualified sample collection technicians were often only available once each month, so mothers would have to return to the clinic on that day for testing. once collected, the dried blood spot samples were sent to district-level hospitals (level ), which then sent the samples to a reference laboratory (level ). the tertiary laboratory then determined viral load by performing polymerase chain reaction (pcr) to detect hiv genetic material. test results followed the same circuitous path back to the patients, a process that could take as long as four months. additionally, in the case of a positive dried blood spot result, the process had to be repeated in order to validate the original results. in contrast, the same high-risk newborns in the united states are screened for hiv at birth with additional tests at − weeks, − months, and − months. for these patients, results are usually available within or days of sample collection, allowing immediate antiretroviral treatment for hiv-positive children. the consequences of diagnostic efficiency versus inefficiency could not be more striking; in the burkina faso study, % of the hiv-positive infants died before antiretroviral therapy could even be started, while the early treatment initiated in the u.s. improved infant survival by a remarkable %. , the stark disparity in response time between these two settings shows just how devastating outcomes can be for patients relying on level facilities for their healthcare needs. disease diagnosis in a level setting represents a challenge that often requires simple tools that can be used without skilled personnel or significant laboratory or physical infrastructure. the world health organization has developed a set of criteria ("assured") that defines the ideal characteristics for pointof-care (poc) tests in low-resource settings. in accordance with these criteria, an ideal test should be affordable to those who are at risk of infection, result in few false-negatives (sensitive) and false-positives (specific), and be user-friendly, rapid and robust, equipment-free, and deliverable to the populations in need of the test. additionally, the required performance parameters of a test depend on its intended usecase scenario. for individual patient case management, sensitive multiplexed tests are advantageous for determining the source(s) of nonspecific symptoms and selection of appropriate treatment. from an epidemiological standpoint, if the goal is simply to reduce the prevalence of high-intensity infections within a geographic region, the need to quantify disease burden outweighs the need for high analytical sensitivity. if the goal is disease elimination, high analytical sensitivity is one of the most important parameters, since the interruption of local transmission requires the detection of every infection, including extremely low-intensity infections. the most sensible approach to designing and developing a poc diagnostic is to examine and optimize each individual component before integrating into a single diagnostic device. in this review, we define the components of a diagnostic to include: ( ) the target biomarker, an endogenous indicator of a disease state, which is most often a pathogen or host protein, carbohydrate, or nucleic acid sequence, ( ) sample preparation, which allows for biomarker isolation, purification, and/or concentration from complex biological matrices, ( ) molecular recognition elements, which specifically capture and detect the target biomarker, ( ) signal generation and amplification, and ( ) instrumentation for signal read-out. simple components requiring only a single user step are generally preferable in lowresource level settings. however, there are few diagnostic technologies that currently can be deployed in these settings, and the lack of appropriate diagnostics in resource-limited settings can lead to tragic outcomes. fortunately, emerging technologies based on inorganic chemistry and nanomaterials can be exploited as potential solutions. in this review, we focus on how inorganic chemistry and nanomaterials have been utilized in each component of a poc diagnostic for infectious disease detection. section describes inorganic biomarkers that are associated with infectious diseases and how their properties have been exploited in diagnostic applications. in section , we discuss how coordination chemistry has been harnessed for sample preparation and protein biomarker enrichment for poc tests. section details the critical advancements in inorganic chemistry and nanomaterials for signal generation probes that have allowed the field of poc diagnostics to rapidly progress. in section , we provide an overview of field-deployable instrumentation that incorporates the fundamental inorganic chemistries discussed in sections − for poc diagnosis of infectious disease. finally, section discusses the emerging trend of using inorganic chemistry and nanomaterials for nucleic acid detection at the point of care. biomarkers are quantifiable characteristics of a disease state that can be measured accurately and reproducibly. the vast majority of biomarker targets for infectious diseases fall into three main categories: ( ) pathogen genetic material, ( ) protein and carbohydrate antigens produced by a pathogen, or ( ) human host responses to the presence of infection, such as pathogen-specific antibodies. while inorganic markers of infectious disease are far less common than their bioorganic counterparts, metal-based biomarkers have unique properties that can be leveraged for innovative diagnostic strategies, as illustrated in the following two examples. hemozoin, often called malaria pigment, is a biomineral produced by some parasites that rely on hematophagy as their primary source of nutrients. hemozoin was first described for the malaria parasite plasmodium falciparum over a century ago and is produced by all plasmodium species. − in the erythrocytic life stages, malaria parasites digest host hemoglobin in the acidic digestive food vacuole as a source of amino acids. the breakdown of one hemoglobin molecule releases four molecules of heme (ferriprotoporphyrin ix [fe(iii)-ppix]), which are highly toxic to the parasite. as plasmodium spp. lack functional heme oxygenase activity, the parasites remove the free heme by crystallizing it into insoluble, inert hemozoin crystals. , these crystals accumulate in the parasite over the course of its erythrocytic life cycle. after the mature schizont ruptures, hemozoin is released into circulation and rapidly taken up by phagocytes. the heme detoxification pathway that produces hemozoin is crucial for malaria parasite survival; therefore, hemozoin indicates the presence of plasmodia parasites, making it an attractive biomarker for malaria detection. structurally, hemozoin crystals consist of reciprocating fe(iii)ppix dimers linked through coordination between the central ferric ions and a carboxylate group of one of the propionate moieties on protoporphyrin iv. , , dimers assemble into chains through hydrogen bonding of the second propionate porphyrin side chain, and these networks of crosslinked dimers are held together via π−π interactions ( figure ), resulting in an anisotropic rectangular crystal with unique magnetic, optical, and photoacoustic properties. one such property of hemozoin is its paramagnetism, which is derived from the unpaired electrons on the central fe (iii) ions. like all paramagnetic substances, hemozoin has a magnetic moment and is thus attracted up a magnetic gradient in the presence of an externally applied magnetic field. the paramagnetism of hemozoin has been leveraged for malaria diagnosis in several ways. first, the magnetic properties have been exploited for separation, purification, and concentration of infected red blood cells (irbcs) from blood samples. with the use of a high-gradient external magnetic field, irbc separation was first performed by heidelberger et al. in for the purpose of vaccine development, though at the time the method was described as "applicable only to small blood volumes, [and requiring] apparatus difficult of access." since then, the availability of high-field permanent magnets and electromagnets has substantially improved, and the time required for magnetic separation of irbcs has been reduced. both commercially available systems as well as custom-made microfluidic devices have been used to enrich irbcs from whole blood samples for analysis by microscopy. − three groups have explored detection of hemozoin based on the fact that paramagnetic materials decrease nuclear magnetic resonance (nmr) transverse relaxation time (t ) of water, the same phenomenon leveraged for magnetic resonance imaging (mri) contrast agents. karl et al. first demonstrated that hemozoin could be detected based on decreased t , though only at parasitemias greater than parasites/microliter, which is orders of magnitude greater than the detection limits of commercial rapid diagnostic tests. several years later, peng et al. and kong et al. developed a low-cost (<$ ), homemade micromagnetic resonance device for hemozoinbased malaria detection. they reported detection limits less than parasites/microliter using their method and attributed this drastic improvement to the use of ultrashort echo times and a premeasurement centrifugation step that pelleted red blood cells from whole blood. these discordant conclusions led to a reaction by karl et al. and a response by peng et al. recently, gossuin et al. confirmed the results of karl et al., indicating that conventional relaxometry alone is insufficient for sensitive malaria detection. these findings led to two hypotheses related to the superior sensitivity measured by peng et al.: ( ) the premeasurement centrifugation of higherdensity irbcs (compared to uninfected rbcs) significantly enriched hemozoin in the portion of the pellet measured, and ( ) ultrashort echo times may have allowed for probing of other protons, such as macromolecular protons, which may be more susceptible to the presence of hemozoin. a simple, lowresource relaxometry-based detection device remains to be developed, and such a device would ultimately require resources typically not available in primary healthcare settings in lmics (i.e., consistent availability of electricity and clean water). however, these studies highlight the importance and advantages of sample preparation and enrichment steps in the development of sensitive infectious disease diagnostics. in addition to its paramagnetic properties, the unique optical properties of hemozoin enable the development of new diagnostic tools for malaria detection. in particular, hemozoin strongly scatters and absorbs light. the latter characteristic led to the remarkable development of the microvascular microscope (mvm) for needle-free, in vivo detection of hemozoin. , the mvm employed cross-polarized epiillumination to optically access vessels below the surface of the skin, with green light (λ = nm) illumination for locating vessels and red light (λ = nm) illumination for detecting hemozoin. this method was able to detect parasitemias as low as . % in mouse models, corresponding to approximately parasites/microliter in humans. unfortunately, lower parasite density infections were not explored. the mvm was able to differentiate between irbcs and hemozoin-containing white blood cells, which can persist in circulation for weeks after an infection is cleared, based on the diameter and relative intensity of the detected hemozoin. additionally, hemozoin particle velocity could be measured in vivo (figure ), potentially allowing for identification of cytoadhesion and sequestration, two phenomena related to disease severity. , though further sensitivity analysis and field testing is required, the mvm represents a promising, noninvasive tool for malaria detection, particularly if a portable and battery-powered device is developed. photoacoustic spectroscopy has also demonstrated potential for transdermal malaria detection. − photoacoustic (pa) hemozoin detection requires excitation with intense light and measurement of the resulting acoustic signals generated by local thermal expansion of the surrounding medium. the utility of in vitro pa detection of hemozoin was first demonstrated by balasubramanian et al. in . since then, it has been shown that short, intense laser excitation of hemozoin in the parasite can localize heat around the crystal, evaporating the surrounding medium and creating transient, nanosized vapor bubbles. the expansion and collapse of these nanobubbles enhance acoustic detection. this technique has allowed for transdermal malaria detection with exceptional sensitivity in mice. this method was also applied to one human patient with high parasite density ( − parasites/microliter). − further studies with increased patient sample size will be necessary to determine the true limits of the technology. magneto-optical detection exploits both the paramagnetic and optical properties of malarial hemozoin crystals, which have a rectangular shape that affords a high level of both magnetic anisotropy and optical dichroism. in this technique, whole blood (or lysed whole blood) is inserted into a magnetic field, orienting the paramagnetic crystals along the applied field direction. this phenomenon, known as the cotton-mouton effect, induces an optical dichroism across the dispersion of crystals, resulting in a detectable change in transmittance of modulated polarized light (λ = nm) ( figure ). , when applied to patient whole blood samples, the diagnostic sensitivity and specificity of magneto-optical detection were not competitive with rapid diagnostic tests and pcr, though the ease of use is encouraging for poc applications. , in particular, model simulations have suggested that magnetooptic detection of hemozoin could be exploited for the development of a noninvasive probe capable of measuring transmittance of polarized light through a fingertip. a similar magneto-optic strategy that employs a fluctuating magnetic field and measures the change in amplitude of transmittance has shown potential in mouse models, but further evaluation of the diagnostic sensitivity and specificity in human samples is needed. − recently, a commercial magneto-optic device for malaria detection based on hemozoin has demonstrated promise in field evaluations, detecting clinical parasite densities as low as parasites/microliter (hemex health). additional techniques used to detect malarial hemozoin include flow cytometry, − laser-desorption mass spectrometry, − and raman spectroscopy. − while these strategies are highly sensitive, they require expensive and resourceintensive instrumentation that typically prevent their use in a district hospital setting, local health outpost, or field setting. however, as instruments become more compact, robust, and affordable, similar to some of the technologies detailed in this section, their implementation in field settings could become more feasible. importantly, hemozoin is synthesized by other hematophagous parasites that infect humans, including the schistosoma, echinostoma, and opisthorchis trematodes, as well as blood feeding insects, such as rhodnius and boophilus. , , despite being produced by these additional parasites, hemozoin has not yet been used as a diagnostic biomarker for nonmalarial pathogens. further, it is important to note that in all of the aforementioned hemozoin-based malarial detection strategies, hemozoin originating from trematodes would produce a positive signal. this is particularly important to consider regarding false positive tests since the geographic distribution of malaria overlaps with schistosoma. , in addition to confounding signal originating from other hematophagous parasites, hemozoin as a biomarker presents some unique challenges to malaria diagnosis. first, the biocrystal is present at much lower concentrations in the ring stage when compared to the later trophozoite and schizont stages of the erythrocytic cycle. thus, magnetic enrichment or malaria diagnosis strategies based on hemozoin could miss early stages of infection, potentially resulting in false-negatives. additionally, phagocytic cells ingest irbcs and hemozoin released into circulation after schizont rupture, and hemozoincontaining white blood cells have been shown to persist in circulation up to days after parasite clearance. this persistence can lead to false-positive results in hemozoin-based malarial diagnostics unless an algorithm for distinguishing pigment-containing white blood cells and irbcs is devised, such as the one developed in the aforementioned work by burnett et al. despite these challenges, researchers continue to develop innovative techniques for malaria detection using hemozoin as a biomarker. implementation of these methods will require careful design with cost and equipment reduction in mind in addition to rigorous field evaluation. schistosome eggs are the most common biomarker for schistosomiasis detection. they are produced by adult schistosoma worm pairs that reside in the intestine and bladder and deposited into the vessels of the gut wall. many eggs become permanently lodged in the intestine, liver, bladder, or urogenital system, leading to chronic inflammation and systemic tissue damage; however, some mature eggs are excreted in urine or feces to allow the parasites to continue the transmission cycle. eggs from distinct species are distinguishable by their morphologies. differences include the overall egg shape (round to oval), spine position, and extent of filamentous coating. all of these features are observable by microscopy, which is the gold standard for schistosomiasis diagnosis. voided eggs from all schistosoma species contain mature miracidia within a relatively impermeable eggshell. while the dense biopolymer composition of schistosoma eggshells has been studied since the s, the inorganic components of the eggshells were not investigated until , when jones et al. demonstrated the presence of iron in schistosoma japonicum eggshells ( figure a ). the group used inductively coupled plasma mass spectrometry (icp-ms) and energy dispersive spectroscopy (eds) to provide evidence that iron localized to the eggshell. although these elemental characterization techniques did not provide specific information about the oxidation state or organization of iron present, the group found that the eggshells contained mg/kg (dry weight) fe, making up nearly % of the iron found in intact eggs. in the same year as the jones study, teixeira et al. set out to increase the sensitivity of microscopy for schistosoma detection using magnetic microparticles to enrich eggs from fecal samples. to accomplish this, stool samples were suspended in water and repeatedly sieved and washed before the sediment was incubated with magnetic particles that bound to the schistosoma eggs. this technique for egg concentration was dubbed the helmintex method and was demonstrated to be more sensitive than kato-katz, the current who recommended sample preparation method for schistosome diagnosis in a field setting. the initial hypotheses of the helmintex method were that biotinylated lectins would bind to carbohydrates on the surface of schistosoma mansoni eggs and that streptavidin-coated magnetic beads could bind the biotinlectin-egg complexes. using an external magnet, the eggs could then be isolated and concentrated before detection by microscopy. however, control experiments demonstrated the following: ( ) lectins were not necessary for egg binding to magnetic particles, ( ) nonmagnetic latex particles did not bind to s. mansoni eggs, and ( ) the eggs alone did not migrate to an external magnet ( figure b ). these observations, coupled with the discovery of iron localized to eggshells, led some to hypothesize that, in the presence of an external magnetic field, the paramagnetic beads act as weak bar magnets, enabling attraction of the iron in the shells of the eggs. in , karl et al. further probed the iron distribution and magnetic properties of schistosome eggshells to elucidate the mechanism of the helmintex method. the study found that iron was localized to pores in eggshells, and schistosome eggs demonstrated moderate paramagnetic behavior. however, the eggs did not move in a magnetic field, and the paramagnetic particles bound to parasite eggs in the absence of a magnetic field, suggesting that the mechanism of binding was not magnetic in origin. the authors also found that s. japonicum eggs bound more microparticles than s. mansoni, leading to the hypothesis that the filamentous outer structure of the eggs, more prominent in s. japonicum compared to s. mansoni eggs, provided strong, nonmagnetic adhesion based on many relatively weak van der waals interactions. in a separate study, candido et al. also observed that s. japonicum eggs bound more microparticles than s. mansoni. the group used poisson analysis and found two distinct populations within each species: a high "affinity" group and a low "affinity" group, where affinity was determined by the number of microparticles bound to the visible edge of a single egg. interestingly, the magnitudes of the high affinity values were similar between the two species, but the fraction of eggs in the high affinity group for s. japonicum was greater than for s. mansoni. collectively, these results suggest that differences in filamentous microspines between the two species do not fully explain the mechanism of egg-microparticle binding. additionally, the work by candido et al. opens up many questions, the answers to which could greatly inform further optimization of the helmintex method. for instance, what are the physiochemical differences between high and low affinity egg groups? is there a biological difference between the two groups, such as egg maturity or surface antigen expression? both the karl et al. and candido et al. studies were performed on eggs isolated from livers of infected mice, but do these two egg populations appear in the urine or feces of infected humans? if so, should two types of magnetic microspheres be used to ensure the helmintex method captures all egg types present in a sample? these questions should be answered carefully and systematically. given the greater number of microspheres bound to eggs in the high affinity group, the populations should be separable using external magnetic fields of differing strengths. egg-microparticle binding mechanisms for each population could be interrogated by determining solution properties (e.g., ph, salt, surfactant, chelators, blocking proteins, etc.) required to interrupt the interactions. additionally, functionalizing microspheres in a way that systematically varies their zeta potential and hydrophobicity could determine the role these physiochemical properties play in binding. these studies will be crucial if the helmintex method aims to determine infection intensity using quantitative egg enrichment. this is particularly important for schistosomiasis surveillance and morbidity control campaigns, which aim to quantify and reduce the prevalence of high-intensity infections. the helmintex method originated with the idea that iron in the eggshells of schistosomes could serve as a handle for magnetically enriching schistosoma eggs for more sensitive detection in the field setting. while the helmintex method has already been shown to improve the sensitivity of kato-katz, elucidation and optimization of the true binding mechanisms could lead to an even more impactful method for diagnosing low-intensity schistosomiasis infections. metal-based sample preparation techniques that enrich biomarker concentration improve the sensitivity of diagnostics by delivering more biomarker to the test. these strategies can be applied to existing assured diagnostic tests, such as lateral flow assays (lfas). while the easiest way to deliver more biomarker to the tests would be to add larger sample volumes ( − μl), many lfas cannot accommodate large sample volumes for several reasons: first, limited bed volumes in porous paper substrates physically limit the amount of liquid a test can hold. second, the increase in the number of interfering molecules that results from an increase in sample volume could cause nonspecific cross reaction with the test's molecular recognition elements (e.g., antibodies). third, colored biological matrices, such as whole blood, can increase the background signal and decrease the user's ability to distinguish between positive and negative results. metal-based sample preparation techniques mitigate these shortcomings, allowing for the concentration of a target from a large volume sample into a smaller, test-compatible volume. moreover, metal-based sample preparation also removes the target from its original complex biological matrix, decreasing the potential for nonspecific cross-reactivity. these low-cost, simple, and robust methods represent a powerful tool for use-case scenarios in which improved sensitivity is needed, such as elimination campaigns. this section will discuss how metal coordination chemistry has been leveraged for simple sample preparation methods applicable to poc diagnostics. coordination chemistry, which is defined as the chemistry of metal atoms or ions that form complexes with one or more ligands, can be utilized to coordinate biomarkers with a particular affinity for metal ions. the most significant illustration of this capacity is the isolation of proteins via immobilized metal affinity chromatography (imac). , this technique involves the conjugation of various ligands to a solid support matrix such as agarose, cellulose, or silica. these ligands act as lewis bases, chelating metal ions with a high affinity. the fixed metal ions serve as lewis acids, coordinating to both the immobilized ligands and specific amino acid residues in a protein biomarker. while amino acid affinities toward metal ions vary, histidine demonstrates a particularly high affinity toward metal ions in this format. these interactions can be leveraged to isolate a biomarker from a complex biological matrix. once separated, the target can be competitively eluted and concentrated into a smaller volume suitable for a poc diagnostic test. , the choice of chelating ligand is critical to imac protein separation efficiency. ligands both fix the metal ion to the solid support and modulate metal-protein binding based on the number and conformation of chelation sites. , − a multitude of ligands with varying coordination numbers have been employed. most imac platforms contain tridentate or tetradentate chelators, although some bidentate and pentadentate options exist ( figure ). tetradentate nitrilotriacetic acid (nta) and tridentate iminodiacetic acid (ida) are two of the most common commercial ligands, but many companies have also developed their own proprietary structures such as talon or ni-penta. the variety of chelating ligands, many of which possess different coordination numbers, make imac tunable, which is beneficial when tailoring the chemistry to different diagnostic applications. , , − guided by pearson's hard−soft acid−base theory, numerous metal ion and ligand combinations have been exploited in imac. borderline acids such as co + , ni + , cu + , and zn + have predominated, − displaying varying levels of affinity and specificity. the determination of which ion will be optimal in a given isolation often depends on the protein in question. ni(ii)-nta has been one of the most widely utilized metal− ligand combinations in imac applications. until recently, a thorough investigation that compared divalent metals and their molecular recognition capability was lacking. bauer et al. filled this void by conducting a systematic investigation of the affinity of malarial biomarker histidine-rich protein (hrp ) to m + -functionalized biosensors and solid phase resins (m + = co + , ni + , cu + , or zn + ). hrp is the most frequently detected target in malarial rapid diagnostic tests and possesses a unique primary structure containing % histidine, making it an ideal histidine-rich target. the authors used biolayer interferometry (bli), an optical biosensor technique, to determine kinetic constants (e.g., k on , k d ) for the binding of the various metals to hrp . the authors found that co + and zn + exhibited the fastest binding kinetics (highest k on ) and the highest hrp binding affinity (lowest k d ) on bli biosensors. when the same imac complexes were incorporated into a loose solid phase resin, zn(ii)-nta resulted in the fastest complete binding of hrp per mole of divalent metal while also allowing for the most efficient elution of hrp upon the addition of imidazole. once a metal had been selected, hrp 's binding and elution behavior was evaluated against a variety of ligands and solid supports. the selected zn(ii)-ida resin was then integrated into a pipet tip format, enabling direct hrp capture from spiked lysed blood samples. this field-deployable sample isolation and concentration tool displayed up to a -fold signal enhancement in commercial rapid diagnostic tests (figure ). this study demonstrated that optimizing the imac metalligand combination for a particular application is critical for efficient biomarker capture and elution; the initial binding cannot be too tight so as to prevent biomarker elution with a competing ligand (e.g., imidazole) nor too weak so as to attain suboptimal biomarker enrichment. , imac has been adapted to a variety of solid support platforms, from agarose gels to rigid silica particles. , although many of these platforms have become wellestablished research tools, the emergence of novel materi- figure . coordination of imac ligands (ida, nta, and ted) to a divalent metal for binding histidine residues in proteins. co + , ni + , cu + , and zn + have predominantly been utilized in this application. ida (iminodiacetic acid) is a tridentate ligand (coordination number = ), with the remaining three coordination sites in the metal available to bind two histidine residues and a water molecule. nta (nitrilotriacetic acid) is a tetradentate ligand (coordination number = ) with two coordination sites for histidine residue binding. ted (tris(carboxymethyl)ethylene diamine) is a pentadentate ligand (coordination number = ) with one remaining coordination site for protein binding. als − has enabled imac chemistry in new applications. for example, wang et al. polymerized polyhedral oligomeric silsesquioxanes (posss) into micron-sized particles with nanosized pores, which were subsequently functionalized with ida and charged with cu + . this material was then used for specific capture of hemoglobin, which is rich in surface-exposed histidine residues. incubation of the test solution with the cu(ii)-ida functionalized material enabled selective capture of hemoglobin versus other proteins without these accessible histidine residues. the development of this novel material using the principles of imac demonstrates that nanomaterials can be modified for molecular recognition of histidine-rich proteins. these modified nanomaterials have the potential to be incorporated into poc diagnostic frameworks for biomarker capture. imac-functionalized cellulose membranes could provide another excellent option for sample preparation in lowresource settings. given that these membranes already hold an established place as a laboratory research tool, they could readily be incorporated into field-friendly paper fluidic devices. opitz et al. utilized commercial ida-functionalized cellulose charged with zn + as a platform for separating influenza virus particles from culture lysate. after an initial metal screening, they saw that zn + -charged membranes separated particles with significantly higher affinity the other metals tested. the authors postulated that the influenza hemagglutinin glycoprotein on the particular strain studied (influenza virus a/ puerto rico/ / ) contained histidine-rich zn + -coordinating sequences on each subunit and that the repeated configuration of these subunits as part of the capsid structure contributed to a higher overall binding avidity. although this imac-based method for separating virus particles from cell culture was developed to improve the efficiency of laboratory-based research, it is not difficult to imagine extending this concept to a simple sample preparation method for increasing the sensitivity of an influenza rapid test. applying the developed protocol to a diagnostic test would require feasibility testing on clinical patient samples, ideally, resulting in the design of an integrated device for both sample preparation and virus detection. imac-functionalized cellulose membranes present a unique advantage in relation to other materials because of the ease in which they can be incorporated into paper-based diagnostic formats that are usable in low-resource poc settings. magnetic particles hold a number of advantages over other solid imac supports. the increased surface area of the particles provides increased availability for chemical reactivity and functionalization. , the independent response of paramagnetic particles to an externally applied magnetic field enables active mixing, which has proven especially useful in microfluidic applications. this magnetic susceptibility also allows for easy manipulation of the particles once a targeted biomarker is bound, permitting biomarker isolation and concentration before downstream detection. , multistep protocols that would otherwise rely heavily on laboratory techniques such as filtration, centrifugation, and column chromatography can easily be performed using magnetic beads and a hand-held external magnet. aside from chromatographic capabilities, magnetic particles have been employed as signal generation labels that are detected by inductive or magnetoresistive sensors. this application is discussed in detail in sections . . and . . magnetic particles thus represent a promising vehicle for imac chemistries in low-resource settings. the wright research group recently demonstrated that imac-functionalized magnetic beads have the potential to improve commercial poc diagnostic sensitivity for malaria. hrp was captured from large-volume ( μl whole blood samples diluted and lysed to μl) p. falciparum-spiked samples via chelation to ni(ii)-nta on the surface of . flow-through device for capture of malarial biomarker hrp via zn(ii)-ida chelation to histidine residues. the device incorporated a silica resin solid phase functionalized with zn(ii)-ida into a pipet tip, which enabled rapid capture of hrp from whole blood samples using the principles of imac. subsequent addition of imidazole at a high concentration competitively eluted the hrp from the zn(ii)-ida resin into a smaller volume. this small volume sample was then deposited onto a commercially available rapid diagnostic test which enhanced sensitivity for hrp detection in a poc setting. adapted with permission from ref . copyright american institute of physics. commercially available magnetic particles. then, using a specially designed extraction cassette and a hand-held magnet, the hrp -bound particles were guided through stationary liquid chambers separated by surface-tension valves to wash the sample and remove background. once purified, the magnetic beads with bound hrp were transferred to a final μl elution chamber preloaded with imidazole to release the biomarker. this resulted in a -fold theoretical concentration factor for hrp due to the transfer from a μl blood sample to a μl elution chamber. after extraction using this self-contained system, the authors added the μl elution volume to a variety of commercial hrp -based malaria rapid diagnostic tests and found significant signal enhancement across all brands. , in subsequent studies, the group developed and optimized a hand-held, easy-to-use device , (figure a ) in which hrp -bound, imac-functionalized magnetic beads were directly transferred to the sample pad of commercial malaria lateral flow assays. the biomarker was eluted with an imidazole-spiked running buffer. as shown in figure b , this device enabled highly sensitive hrp detection with few added user steps, improving the detection limits of commercial malaria tests by over an order of magnitude with minimal added cost per test. , this enhanced sensitivity could make a substantial difference in malaria elimination campaigns, which rely on detecting and treating all infected individuals, including low-density carriers. one of the primary limitations of imac chemistry is its ability to recognize only those proteins with affinities for metal ions. for purification of recombinant proteins from cell culture, this drawback has been overcome via incorporation of short hexahistidine tags (his -tags) into expressed protein sequences. , using a similar principle, bauer et al. developed a platform in which target-specific antibodies modified with his -tags were combined with imac magnetic beads to isolate and concentrate biomarkers from biological samples. thus, the advantages of imac for biomarker enrichment were no longer limited to histidine-rich proteins but rather could be extended to any protein biomarker target. in the context of malaria diagnosis, this expanded capability allows for the detection of the nonhistidine-rich biomarker plasmodium lactate dehydrogenase (pldh) alongside hrp , which is beneficial for several reasons. plasmodium ldh is essential to parasite survival and is conserved across all species of malaria known to infect humans, whereas hrp is produced only by p. falciparum. in addition, hrp -only tests fail to account for parasitic infections containing pf hrp gene deletions, which are increasing in prevalence worldwide. finally, hrp has been shown to persist in host circulation up to days after successful treatment, but pldh clears within h after parasite clearance, so a dual assay can distinguish between resolved and active p. falciparum infections. − for these reasons, bauer et al. employed their imacbased his -tagged antibody approach to concentrate both pldh and hrp from large volume samples ( μl parasitespiked whole blood) utilizing commercially available imac magnetic beads. in this dual capture system, hrp coordinated directly to the co(ii)-nta-functionalized surface of the magnetic bead, while pldh was bound using his tagged anti-pldh antibodies ( figure ). following isolation of both biomarkers, hrp and the his -tagged-antibody/pldh complex were simultaneously eluted with imidazole. when applied to whole blood samples spiked with the p. falciparum parasite, this strategy resulted in a nearly -fold improvement in the limit of detection of commercial lfas. this unique platform demonstrates that imac-based biomarker enrich- ment is not limited to histidine-rich proteins and could be expanded to virtually any biomarker for which there is a commercially available antibody. one of the challenges associated with employing a his tagged antibody/imac concentration strategy is that the biomarker remains bound to the antibody during the detection step. the antibody used for sample preparation has the potential to block target binding (via overlapping epitopes or steric effects) to the molecular recognition elements employed in the downstream assay, leading to false-negative results. this challenge can be addressed by designing the diagnostic such that the his -tagged antibody and downstream detection elements bind to orthogonal epitopes of the target. furthermore, other his -tagged molecular recognition elements such as aptamers could be used in place of his -tagged antibodies. aptamers are short, single-stranded nucleic acid sequences with secondary structures that afford high (∼nanomolar−picomolar) binding affinities. compared to antibodies, aptamers are smaller and may target unique epitopes, potentially allowing for this innovative sample preparation strategy to be more universally applied. − coordination chemistry-based sample preparation technologies have enabled significant improvements in the sensitivities of point-of-care diagnostics. the enhanced sensitivities, which in several examples were improved by a whole order of magnitude, make a strong case for the use of these technologies in elimination campaigns. , , previously, biomarker enrichment strategies were not incorporated into low-resource diagnostics because of the restrictive format of the tests. the imac-based chemistries described in this section represent innovative yet simple solutions that open the door for the development of poc diagnostics that incorporate sample preparation. while some of the sample preparation methods presented in this section increased the total number of assay steps, these methods were not designed in tandem with the diagnostic devices that were utilized for detection. moving forward, human-centered device design that integrates sample preparation with detection elements will permit the next generation of poc diagnostics to be developed with minimized user steps and improved sensitivities. inorganic metal complexes and nanoparticles have been instrumental in the development of targeted reagents for disease-associated dna, proteins, or cells. their biomedical applications include the development of metallodrugs, , enzyme inhibitors, − photothermal , and photodynamic therapy agents, and mri contrast, , in vivo bioimaging, , and protein/cell imaging agents. , their unique optical, electrochemical, magnetic, and catalytic properties have also enabled their use as signaling modalities in diagnostics. this section focuses on the application of metalbased signal generation probes for the detection of protein biomarkers at the point of care (summarized in table by biomarker and in table by signal generation method). (the detection of pathogenic nucleic acids via metal-based signal generation probes is discussed in detail in section ). first, we describe how inorganic complexes have been utilized in photoluminescent, electrochemical, and electrochemiluminescent applications, respectively. subsequently, we detail the essential bioconjugation chemistries that permit nanoparticles to be functionalized with molecular recognition elements to generate useful diagnostic reagents. we then discuss how various classes of these functionalized nanoparticles have been used in poc diagnostic assays. lastly, we consider the catalytic properties of metalloenzymes and metal nanoparticles that have been utilized for signal amplification in poc diagnostics for infectious diseases. the broad array of properties that result when a ligand coordinates to a metal has been leveraged for signal production in a variety of diagnostic formats. inorganic coordination compounds are valuable as signal generation probes due to their low molecular weight, tunability, and unique catalytic and photophysical properties. these compounds have been used in photoluminescent (i.e., fluorescent and phosphorescent), − electrochemical, − and electrochemiluminescent (ecl) − sensing platforms. each signal generation method has trade-offs with regard to ease-of-use and interpretation, as well as complexity of instrumentation required to measure signal output. when designing a lowresource diagnostic, these trade-offs must be evaluated based on the use-case scenario and testing environment. collectively, these considerations better inform the developer as to which inorganic complexes are suitable for a particular signal generation method. . . . luminescence. photoluminescent inorganic complexes possess unique properties and offer several advantages over organic fluorophores as signaling molecules for diagnostics. these advantages include long-lived phosphorescence, significant stokes shifts, and emission tuneability via ligand composition. , , long-lived phosphorescence is beneficial because it is spectrally distinct from the autofluor- figure . a his -tag was coupled to an anti-pldh antibody via sulfo-smcc to allow for chelation of the his -tagged antibody to co(ii)nta-functionalized magnetic bead. remaining sites on the bead surface (unoccupied by antibody) permitted coordination of hrp directly to the bead surface. subsequent to biomarker capture, imidazole was added to elute hrp and the his-tagged antibody/ pldh complex. adapted from ref the large stokes shifts of metal complexes also result in improved sensitivity because self-quenching of signaling molecules is minimized. additionally, the tunable emissions of metal complexes enable their use as signal transducers, whereby ligand exchange reactions that induce changes in emission spectra can be correlated to biomarker concentration. to date, cyclometalated d complexes of ir (iii) are the most commonly utilized photoluminescent signal generation probes. [ ] [ ] [ ] , , ligands in these cyclometalated ir (iii) complexes (e.g., deprotonated -phenylpyridine) demonstrate strong σ-donor effects, which significantly increase the energy of the mc (metal-centered) excited state (i.e., the ligand field stabilization energy) that gives rise to nonradiative decay pathways. as a result, excited electrons in these complexes preferentially populate the mlct (metal-toligand charge transfer) excited state (as opposed to mc). the excited electrons then undergo intersystem crossing and subsequently phosphoresce from a mixture of the lc (ligand-centered) and mlct excited states ( figure ). the high phosphorescent quantum yields with large stokes shifts make these complexes ideal for signal generation. , davis et al. designed a "switch-on" ir(iii) detection probe that selectively produced phosphorescent signal in the presence of malarial biomarker hrp . the cyclometalated ir(iii) complex, [ir(ppy) (h o) ] + , was poorly luminescent in the absence of hrp ; however, when aqua ligands were displaced by hrp 's histidine residues, a biomarker-dependent mlct/ lc phosphorescent signal was produced ( figure ). similar to the principles of imac chemistry discussed in section , this approach capitalized on the chelation of hrp 's histidine repeats to the inorganic coordination compound. the assay design aptly showed how the tunable luminescence of ir(iii) complexes could be harnessed for signal transduction, demonstrating that a ligand exchange reaction's phosphorescent signal could be correlated to biomarker concentration. in addition, the ir(iii) complex displayed a significant stokes shift (Δλ = nm), making it less prone to self-quenching effects and decreases in sensitivity associated with its organic fluorophore counterparts. this study also incorporated the ir(iii) probe into an imac magnetic bead-based biomarker isolation and detection assay for recombinant hrp which had a detection limit of . nm. although less sensitive than an elisa (enzymelinked immunosorbent assay), this hrp concentration was within the relevant range for clinical diagnosis of malaria. moreover, the bead-based assay could be completed in less than h, whereas traditional elisas require − h. while the current assay format would only permit its application in better-equipped laboratories, the developed cyclometalated ir(iii) probe could be an exciting detection element in an lfa with hrp -specific antibodies at the test line. however, this photoluminescence detection mechanism is dependent on existence of a histidine-rich biomarker, so further applications of this particular strategy are limited. a more generalizable platform using photoluminescent ir (iii) probes was developed by lin et al. for the detection of interferon-gamma (ifn-γ), an inflammatory cytokine biomarker for immunological diseases such as hiv. for molecular recognition and detection of ifn-γ, the assay used two aptamers: one was highly specific for ifn-γ, and the second was a guanine-rich nucleic acid sequence. the two aptamers were designed to be partially complementary and were prehybridized prior to the introduction of a sample containing ifn-γ. in the presence of ifn-γ, the ifn-γ-specific aptamer bound to its target and liberated the guanine-rich sequence. introduction of k + ions induced the formation of guanine quadruplexes, which subsequently bound an ir (iii) probe for "switch-on" luminescence that correlated biomarker concentration with phosphorescent signal (figure ). this ir(iii) probe possessed a long phosphorescence lifetime (> . μs) and significant stokes shift (Δλ = nm), thus exhibiting the canonical advantages of inorganic probes over organic fluorophores. the assay demonstrated adequate specificity for ifn-γ versus other proteins commonly found in biological matrices (e.g., human serum albumin and immunoglobulins) and had a limit of detection of . nm and a dynamic range of − nm. however, the assay suffered a reduction in sensitivity when conducted in a . % cell extract, and no attempts were made in the complex biological matrices that are pertinent to hiv diagnosis (e.g., blood, urine, saliva, etc.). nonetheless, the assay established a proof-of-principle for using a photoluminescent cyclometalated ir(iii) complex as a detection probe for an hiv biomarker. moreover, the assay format is generalizable such that it could be applied to virtually any biomarker for which there is a high-affinity aptamer. if combined with one of the sample preparation strategies discussed previously (section ) or integrated with paper or another field-ready substrate, this ir(iii)-based detection strategy could produce a robust and sensitive assay that is applicable in low-resource diagnostic settings. . . . electrochemistry and electrochemiluminescence (ecl). by applying a potential (or range of potentials) to a sample containing an electroactive inorganic probe, current or luminescence can be measured as an output for electrochemical or ecl detection, respectively. electrochemical or ecl-based assays that combine molecular recognition elements, such as antibodies or aptamers, with electroactive inorganic probes have shown promise for infectious disease detection. , − in particular, inorganic low-spin d complexes of ir (iii) and ru(ii), as well as hemin (ferriprotoporphyrin ix), have been utilized in developing electrochemical-and ecl-based probes. both electrochemistry and ecl are signal generation strategies that are well-suited to the development of poc diagnostics, largely due to ( ) capability for miniaturization, ( ) low cost, ( ) simplicity, ( ) rapid time-to-result, and ( ) high sensitivity. , this has been aptly demonstrated with the development of paper-based electrochemical diagnostic platforms, often termed microfluidic paper-based electrochemical devices (μpeds) − or electrochemical paper analytical devices (epads). these tests utilize paper as the assay substrate and have been combined with instrumentation amenable to low-resource settings, such as cell phones, for detection. incorporation of existing electrochemical-and eclbased assays into these formats could result in robust and highly sensitive infectious disease diagnostics. this would be impactful for disease surveillance or case management, where robust and sensitive diagnostics are needed. electrochemistry. diagnostic assays with electrochemical detection commonly consist of a three-electrode system: a working electrode (often gold or carbon modified with a molecular recognition element for biomarker capture), a counter electrode (typically platinum), and a reference electrode (commonly a saturated calomel electrode or ag/ agcl electrode). in this format, incubation of a biological sample in an electrochemical cell initially results in capture of the biomarker at the working electrode. introduction of a biomarker-specific inorganic probe and a supporting electrolyte and subsequent application of an electrochemical method (e.g., cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, etc.) results in significant current production at the working electrode. this current can then be correlated to biomarker concentration. with regard to infectious disease biomarker detection, hemin, ir(iii) complexes and ru(ii) complexes have been utilized as electrochemical probes. − hemin (ferriprotoporphyrin ix) is a fe + -containing porphyrin derivative that mimics the enzymatic activity of horseradish peroxidase in catalyzing the reduction of h o (see section . ) . this enzymatic activity is further enhanced when hemin is incorporated into a g-quadruplex aptamer. , , zhang et al. developed an electrochemical assay for the detection of hiv-associated ifn-γ that utilized hemin as a key component of the signal generation mechanism. a ′-thiolated dna construct was conjugated to a gold working electrode, where the dna construct contained a gquadruplex sequence that was specific for hemin and an ifn-γspecific aptamer. both the g-quadruplex and the ifn-γ-specific aptamer were prehybridized by an -nucleotide hairpin. the authors utilized this functionalized working electrode to the assay utilized two aptamers, the first being an ifn-γ-specific aptamer (green) that was prehybridized to a g-quadruplex aptamer (blue). addition of a sample resulted in the capture of ifn-γ by the first aptamer and liberation of the g-quadruplex aptamer, which underwent a structural switch to form guanine tetrads upon introduction of k + ions. the gquadruplexes were specific for a cyclometalated ir (iii) perform cyclic voltammetry on samples containing ifn-γ to monitor the reduction of h o , which could be correlated to ifn-γ concentration ( figure ). in samples containing ifn-γ, the ifn-γ-specific aptamer bound to ifn-γ, opening the hairpin and subsequently allowing for hemin to bind to the g-quadruplex. the potential sweep catalyzed the reduction of h o by the hemin/gquadruplex dnazyme, producing a cathodic current that was proportional to the concentration of ifn-γ in the sample. the assay yielded a detection limit of . nm and a dynamic range of . − nm and thus performed similarly to the luminescent assay developed by lin et al. (section . . ). though the authors demonstrated the specificity of the assay for ifn-γ versus nontarget proteins bsa and igg, they did not report whether the assay could be performed in more complex matrices, which can significantly compromise assay performance. more rigorous testing of the assay in biological matrices is therefore needed to demonstrate the robustness of the assay. miao and colleagues synthesized and implemented an ir(iii) complex-based electrochemical probe for detection of ifn-γ ( figure ). first, a gold working electrode was functionalized with a molecular beacon (mb) that contained an ifn-γ-specific aptamer as well as a sequence that was partially complementary to a second oligonucleotide strand (h ). in the absence of ifn-γ, the portion of the mb complementary to h was hybridized into a hairpin structure. when ifn-γ bound to the aptamer, the h sequence was freed and available to hybridize with its complement, h . the developed ir (iii) probe then bound to the major and minor grooves of the h −h double helix. oxidation and reduction of the , -phenanthroline- , -dione ligand via differential pulse voltammetry (dpv) resulted in ifn-γ concentrationdependent current, thus correlating an electrochemical signal to the presence of target biomarker. the electrochemical assay had a detection limit of . fm and a dynamic range of fm− . pm and could be conducted in samples that were . % human serum. importantly, this assay yielded an ∼ -fold improvement in limit of detection compared to the two previously discussed assays for detection of ifn-γ. this is largely due to the signal amplification that resulted from the groove-binding of numerous ir (iii) probes to the h −h double helix, which provided multiple signal-generating elements for every one ifn-γ biomarker bound. in its present form, this assay could not be applied in a primary healthcare setting because it utilizes an electrochemical workstation that requires trained personnel and significant laboratory resources. however, a portable electrochemical detection system such as the one described in section . could allow for this highly sensitive assay to be applied in a poc setting. electrochemiluminescence. similar to most electrochemical assays, ecl systems employ a working electrode that is functionalized with a target-specific molecular recognition element. the potential manipulation is similar to voltammetric measurements; however, in ecl, the applied potential also generates an excited state in the ecl probe, which then produces luminescence upon relaxation. , the canonical probe for ecl-based detection is [ru(bpy) ] + , a complex (or derivative thereof) which has been used extensively in the development of ecl-based sensors for infectious disease detection. [ ] [ ] [ ] [ ] [ ] in diagnostic assays, ecl with [ru-(bpy) ] + occurs primarily through a coreactant mechanism, where the coreactant is most commonly n-tripropylamine (tpra) (figure ). in coreactant ecl, simultaneous figure . hemin dnazyme-based catalytic assay for electrochemical detection of ifn-γ. a thiolated dna construct was conjugated to a au working electrode. the dna construct consisted of a gquadruplex aptamer specific for hemin (purple) and an ifn-γ-specific aptamer (blue). both aptamers in the dna construct were prehybridized by an -nucleotide hairpin. in the presence of ifn-γ, the ifn-γ-specific aptamer bound to ifn-γ and opened the hairpin. hemin was then added, which bound to the g-quadruplex, forming a dnazyme that could catalyze the reduction of h o . this reduction produced a current that was proportional to ifn-γ concentration in the sample. ]• + that is analogous to the mlct state that is populated through absorption of a photon. the triplet excited state then undergoes phosphorescent decay to the ground state, allowing for luminescent detection of target biomarkers. of the various mechanisms for electrogeneration of a luminescent excited state, coreactant ecl has proven to be the most advantageous for the development of diagnostic assays because it permits measurements that can be obtained in aqueous solvents and matrices. one platform for detection of infectious diseases, pioneered by yoon et al., used immunoliposomes to encapsulate ecl signal generation probes. liposomes equipped with a maleimide handle were synthesized by the freeze−thaw method and loaded with [ru(bpy) ] + . the maleimidefunctionalized liposomes were then conjugated to thiolactivated antibodies specific for the target antigen. the resulting immunoliposomes then served as a target-specific reagent containing an ecl probe that could be released with the application of detergents such as sds or triton. the authors incorporated the [ru(bpy) ] + -loaded immunoliposome into a membrane strip test for detection of the legionella antigen. the strip test contained the following components: ( ) a nitrocellulose membrane impregnated with legionella antigen, ( ) a glass fiber pad presoaked in sds, and ( ) screen-printed electrodes that could interface with an ecl analyzer. the immunoliposomes were incubated with a buffer solution containing the legionella antigen, and then the sample solution was allowed to wick up the nitrocellulose strip. both the antigen in the sample and the antigen embedded in the nitrocellulose competed for binding with the immunoliposome. in positive samples, the immunoliposome was bound by the legionella antigen in solution and migrated past the nitrocellulose strip to the glass fiber pad. the sds in the glass fiber pad lysed the immunoliposome, releasing [ru(bpy) ] + , which was detected by coreactant ecl and correlated to the antigen concentration ( figure ). the assay could be completed in min and had a limit of detection of ng/ml. compared to a traditional lateral flow assay, the test required additional user manipulation steps (preincubation of the sample and immunoliposome, addition of coreactant, etc.). the assay was also dependent on a benchtop ecl analyzer and, therefore, ill-suited to a level healthcare setting in its current format. however, this novel platform demonstrated the capabilities of ecl for detecting a target antigen and showed how a [ru(bpy) ] + -loaded immunoliposome could be utilized to amplify signal. variations of this loaded immunoliposome platform have since been applied to the detection of the influenza virus biomarker hemagglutinin. , recently, zhou et al. developed an ecl-based immunosensor for p , a hiv biomarker associated with early stage infection and detection of which promotes earlier intervention in hiv cases. the detection probe in this assay was a nanocomposite consisting of gold nanoparticle-decorated graphene (p-rgo@au) and [ru(bpy) ] + -doped silica nanoparticles (ru-sio ) ( figure ). a gold nanoparticle-modified glassy carbon electrode (gce) was functionalized with an anti-p antibody (ab ) for molecular recognition of p . a second anti-p antibody (ab ) conjugated to the p-rgo@ au@ru-sio nanocomposite was used as the detection element. once the ab -p -ab sandwich was formed on the electrode, coreactant n-tripropylamine (tpa) was added to produce the ecl signal through the mechanism previously discussed. the p-rgo@au@ru-sio nanocomposite offered several benefits to the system, namely an increased surface area for the ecl reaction and an increased rate of electron transfer. the sio nanoparticles permitted greater [ru(bpy) ] + loading, and the gold nanoparticle-graphene composite increased loading of the [ru(bpy) ] + -doped sio nanoparticles. the authors contended that these aspects led to an increased concentration of ecl-probe at the working electrode surface, which ultimately improved the assay's review detection limit (lod = . pg/ml) and linear range. additionally, the authors demonstrated that the p antigen could be detected in diluted human serum. a remaining challenge for developing luminescent, electrochemical, and ecl assays that incorporate inorganic complexes is thorough validation in complex biological matrices and clinical samples. the aforementioned luminescent assays , were not rigorously evaluated in biological samples. this is an important consideration in developing luminescent assays because of the high background that can result from the presence of interferents in biological samples. biofouling of electrodes is a known challenge in developing electrochemical sensors as well. , although several of the previously mentioned electrochemical and ecl assays were tested in dilute biological matrices, , a more thorough evaluation of how the biological matrix affects the limit of detection is still needed. however, the issue of interference and biofouling could potentially be addressed by integrating these detection platforms with the metal-based sample preparation strategies discussed in section , resulting in more robust and sensitive assays. inorganic complexes have demonstrated promise for detection of infectious disease biomarkers. the luminescent, electrochemical, and ecl detection platforms discussed in this section all capitalized on the unique properties that result from the interplay between ligands and metal centers. however, inorganic complexes are rarely used as stand-alone probes for biomarker detection. instead, inorganic nanoparticles have emerged as the predominant probes in infectious disease diagnostics. , integration of inorganic complexes with nanomaterials represents a potential avenue to developing the next generation of detection probes. one interesting nanomaterial that incorporates inorganic complexes is the metal− organic framework (mof), which is a nanosized network of organic ligands and metal ion/complex connecting points. , the ligands provide functional handles for the conjugation of molecular recognition elements, while signalgenerating metal complexes are installed at the connecting points or encapsulated within the network. recently, electrochemical aptasensors have been developed that contain aptamer-conjugated mofs for specific recognition and detection of various protein or small molecule targets. − moreover, two of the complexes discussed in this section, ir(ppy) and [ru(bpy) ] + , have already been incorporated into mofs for sensing and imaging applications. , by integrating molecular recognition elements into the mof architecture, the applications of these ir(iii)-and ru(ii)-based mofs could be further expanded to include luminescent, electrochemical, or ecl detection of infectious disease biomarkers. the recent expansion of nanochemistry has drastically influenced many areas of science. in diagnostics, inorganic nanoparticles have become ubiquitous as detection elements in numerous platforms (e.g., colorimetric, luminescent, electrochemical, thermal, etc.), and each distinct class of nanoparticles possesses unique advantages and caveats. , , − the diagnostic setting and use-case scenario determine the applicable detection methods which, in turn, direct which nanoparticles to use. in order to apply inorganic nanoparticles as diagnostic reagents, target-specific molecular recognition elements (i.e., antibodies, aptamers, and oligonucleotide probes) must be functionalized to the nanoparticle surface. this section will first review bioconjugation reactions and nanoparticle characterization methods (section . . ). the list of reactions is not meant to be exhaustive but, rather, to highlight the most essential covalent and affinity-based coupling strategies for nanoparticle bioconjugation. then, we survey the various types of nanoparticles that have been applied to the detection of infectious diseases with potential applications at the point of care (sections . . − . . ). . . . functionalization and characterization of nanoparticles. developing a nanoparticle detection reagent that can be targeted toward a biomarker requires conjugation of molecular recognition elements to its surface. these types of conjugations often require linkers that facilitate attachment to the nanoparticle surface. the chemistries employed for coupling molecular recognition elements to nanoparticle surfaces for diagnostic applications generally rely on a few key bioconjugation reactions and are similar to those used for planar solid supports. however, the smaller size regime of nanoparticles significantly influences the reaction kinetics on nanoparticle surfaces; therefore, bioconjugation reactions must be optimized for a given class and size of nanoparticle. , covalent coupling. given the prevalence of carboxylates and primary amines on nanoparticle surfaces and molecular recognition elements, one of the most common bioconjugation strategies for generating functionalized nanoparticle detection probes is the formation of amide bonds. the conventional reagent for forming amide bonds on nanoparticle surfaces is -ethyl- -( -(dimethylamino)propyl) carbodiimide (edc), which produces an activated ester that can subsequently react with a primary amine. however, alone, the edcactivated ester reacts slowly with amines, often leading to hydrolysis products that inhibit amide coupling. for this reason, edc is usually paired with sulfo-nhs (n-hydroxysuccinimide) esters to improve the stability of the activated ester intermediate. the sulfo-nhs ester-activated intermediate readily reacts with primary amine nucleophiles to form stable amide linkages ( figure a ). , edc/sulfo-nhs coupling is useful in generating both antibody-and nucleic acid-nanoparticle conjugates as diagnostic detection probes. due to the intrinsic abundance of primary amines in antibodies, they are readily conjugated to carboxylate nanoparticles without the need for initial derivatization. , edc coupling with imidazole enables conjugation of ethylene diamine to the ′ phosphate group of nucleic acids, yielding ′-amine-functionalized oligonucleotides that can be coupled to carboxylated particles. quantum dots, magnetic nanoparticles, and polymeric (latex) particles have found applications in poc diagnostics and are commonly equipped with carboxylates or amines on the surface to enable conjugation via edc coupling. quantum dots are conventionally stabilized by dihydrolipoic acid (dhla) derivatives or various copolymers that install a carboxylate on the surface, and magnetic nanoparticles are coated with carboxylatecontaining polymers to enable further functionalization with primary amine-containing biomolecules. , − alternatively, both quantum dots and magnetic nanoparticles can be coated with a silica shell derivatized with carboxylate or amine functional groups. silica nanoshells are formed via the condensation of silica oxide monomers, such as tetraethyl orthosilicate (teos) or -aminopropyl-triethoxysilane (aptes), yielding surface aminopropyl-silanols ( figure b ). the surface of the silica can subsequently be coupled to molecular recognition elements via edc. in addition to supplying functional groups for further bioconjugation, the silica encapsulation of other nanoparticles provides biocompatibility and protects the core material from degradation, making it a suitable strategy for the preparation of diagnostic nanoparticle detection probes. the other significant reaction class for generating nanoparticle detection probes is the bioconjugation of thiols present either in molecular recognition elements or on nanoparticle surfaces. thiolated nucleic acids are prepared by the same method as amine-functionalized nucleic acids, except cystamine is used in place of ethylene diamine for the addition of a disulfide that can be reduced with dithiothreitol (dtt) to give a ′-thiolated oligonucleotide probe. , on the other hand, antibodies generally do not contain free thiols and, therefore, either have to be thiolated (often with traut's reagent) or cleaved at the interchain disulfides (via dtt or papain) to generate thiols for subsequent bioconjugation to a nanoparticle surface. , coupling of thiolated biomolecules to noble metal nanoparticles (referring to gold and silver nanoparticles) is conventionally done via coordinate covalent (dative) bonds, where the lone pairs of the sulfur atoms form stable linkages directly to the nanoparticle surface ( figure c ). , alternatively, thiol-containing affinity reagents or nanoparticles can be coupled via a maleimide-derived heterobifunctional cross-linking agent, commonly sulfo-smcc (succinimidyl- -(n-maleimidomethyl)cyclohexane- -carboxylate). thiols react chemical reviews with the maleimide functionality of sulfo-smcc to form stable thioethers, whereas the nhs-ester component of sulfo-smcc reacts with primary amines in molecular recognition elements or on nanoparticle surfaces ( figure d ). , , , photochemical cross-linking and "click chemistry" have also been utilized to produce nanoparticle-based detection probes. photosensitive functional groups, including phenyl azides and diazirines, generate reactive nitrenes and carbenes, respectively, upon exposure to uv light and allow for insertion into carbon−hydrogen and nitrogen−hydrogen bonds ( figure e ). photochemical agents can be integrated into heterobifunctional cross-linkers, such as sulfo-nhs-lc-diazirine, for conjugation of nanoparticles to molecular recognition elements. , , "click" chemistry generically refers to bioorthogonal reactions with high yields, minimal side products, and mild conditions. while the most common "click" reaction is the conjugation of an azide to an alkyne in the presence of a cu(i) catalyst ( figure f ), there are many classes of "click" reactions that couple nanoparticles and molecular recognition elements. , − biotin−streptavidin. the predominant affinity-based conjugation strategy for nanoparticle functionalization is the biotin−streptavidin system. biotin is a small molecule that binds with an extraordinarily high affinity (k d = . × − m) to the bacterial protein streptavidin. streptavidin is an approximately kda tetrameric protein, permitting a : stoichiometry of biotin to streptavidin. in general, streptavidin is added to the nanoparticle surface via passive adsorption or a heterobifunctional cross-linker such as sulfo-smcc. the most common method for biotinylating a molecular recognition element is the coupling of sulfo-nhsbiotin or nhs-(peg) n -biotin to a primary amine. although streptavidin−biotin bioconjugation is generally easy, a (peg) n spacer may be included to optimize for the depth of the streptavidin binding pocket ( Å) as well as the low relative solubility of biotin. nanoparticle characterization. characterization of nanoparticle conjugates is essential for determining the success and extent of particle functionalization, which can drastically affect particle performance in a diagnostic format. there are several characterization techniques applicable to the development of detection probes for diagnostics. uv−visible spectroscopy (uv−vis) is one of the most commonly used methods for biomolecule-functionalized nanoparticles given its simplicity and applicability to the detection of nucleic acids and proteins. , particle size and distribution can be assessed using dynamic light scattering (dls) and transmission electron microscopy (tem), − while functional group characterization on the nanoparticle surface can be evaluated with nmr and/or infrared (ir) spectroscopy. − all of these analytical methods have trade-offs with respect to cost, sensitivity, time of analysis, and complexity of sample preparation. however, because many of the techniques offer complementary information, they can be used in combination to ensure an extensive characterization of inorganic nanoparticle detection probes. the principal concern associated with antibody modification is decreased antigen-binding activity as a result of overfunctionalization of the variable region of the antibody. optimization of the bioconjugation reaction minimizes this effect by achieving a balance between sufficient nanoparticle coupling efficiency and retention of antigen binding. the extent of antibody modification can be monitored with functional group-specific chromophores that are detectable by uv−vis, such as ellman's reagent for free-thiol detection or haba ( ′-hydroxyazobenzene- -carboxylic acid) for biotin detection. , also, biosensor techniques, such as surface plasmon resonance (spr), quartz crystal microbalance (qcm), and bli, can be employed to assess the impact of modification on the binding affinity for the antigen. − decreased target affinity can also originate from suboptimal loading of antibody or nucleic acid on the nanoparticle surface. insufficient coverage may lead to the nonspecific binding of interferents, whereas overloading the nanoparticle with molecular recognition elements can result in steric effects, decreasing target binding. simple absorbance-based supernatant assays that measure antibody or nucleic acid concentration both before and after nanoparticle functionalization reactions give an estimation of the element's surface density and aid in determining the nanoparticle surface saturation. , the straightforward nature of these procedures make them popular strategies for characterizing the extent of functionalization of nanoparticle conjugates for use in infectious disease diagnostics. the bioconjugation chemistry for attaching a molecular recognition element to a nanoparticle surface is the key component for transforming a nanoparticle with interesting signaling properties into a functional reagent that can be employed in diagnostic assays. the characterization of the nanoparticle postfunctionalization is critical to ensuring optimal signal in downstream diagnostic application. several classes of functionalized nanoparticles can be utilized in developing poc diagnostics for infectious disease, the choice of which depends on the use-case scenario and desired signal output. sections . . − . . describe the fundamental principles and poc applications for these classes of nanoparticles. . . . noble metal nanoparticles. both gold nanoparticles (aunps) and silver nanoparticles (agnps), collectively referred to as noble metal nanoparticles, possess many unique properties that make them advantageous probes for signal generation in infectious disease diagnostics. noble metal nanoparticles display a vivid color that is observable with the naked eye due to surface plasmon resonance (spr). the phenomenon of spr, illustrated in figure a , occurs when incoming photons strike the nanoparticle surface and generate a dipole that causes electron oscillations (i.e., surface plasmons) with a frequency that resonates with the frequency of the incoming light. visible light fulfills this resonance condition for noble metal nanoparticles, giving rise to large molar extinction coefficients (on the order of ∼ m − cm − ) at visible wavelengths. moreover, a nanoparticle's color and λ max can be manipulated by controlling its size ( figure b ). , noble metal nanoparticles are also easily functionalized with antibodies, aptamers, or oligonucleotide probes to afford specificity to a target biomolecule. , altogether, these qualities have made noble metal nanoparticles a frequent choice for colorimetric labels in diagnostic assays designed to be interpreted by the naked eye. it is important to note that we have limited further discussion of aunps since their use in diagnostics and sensing applications has been extensively reviewed. , , − given the vast literature that exists for aunp applications, we have elected to focus on the applications of agnps as signal generation probes for infectious disease diagnosis. similar to aunps, localized spr of agnps has been capitalized upon to chemical reviews review develop colorimetric assays for infectious disease protein and nucleic acid biomarkers. − additionally, the conjugation of fluorescent probes (e.g., fam) to agnp surfaces, as well as the intrinsic luminescence of some agnps, has permitted the development of luminescence-based assays. , the high agnp conductivity has also been exploited for electrochemical detection of nucleic acids. − lastly, surface-enhanced raman spectroscopy (sers) of agnps has been employed for the direct detection of malaria-infected red blood cells because of the significant light scattering of agnps and the high specificity of raman signatures. , − colorimetric detection using nanoparticles in paper-based assays has been a widely implemented method for signal readout in primary healthcare settings. in fact, the success of lateral flow assays can be credited in part to the nanoparticlebased detection systems that allow for visual interpretation of results by the end user. the use of functionalized agnps as colorimetric detection probes in paper-based diagnostics for infectious disease has been reported for both protein and nucleic acid biomarkers. , for example, yen et al. employed agnps in a multiplexed lateral flow assay for detection of dengue, yellow fever, and ebola virus. the authors synthesized -, -, and nm agnps, which were visually detectable as orange, red, and green, respectively, due to sizedependent spr shifts. each nanoparticle of a particular size was specifically functionalized with a distinct target-specific antibody associated with one of the viruses, allowing for detection of all three diseases in a single assay ( figure ) . the detection limit for each biomarker was found to be ng/ml, which is within clinically relevant ranges for each disease but is higher than other published methods. , such multiplexed diagnostic assays are critical for discriminating between febrile illnesses at the point of care to allow for selection of the appropriate treatment course. colorimetric agnp aggregation assays also rely on the sizedependence of spr. when agnps aggregate, the decrease in distance between nanoparticles results in interparticle spr, producing a large red-shift in absorption and distinct color change that is visible to the unaided eye. , these reaction mechanisms are classified as either "cross-linking" or "noncross-linking." in cross-linking assays, agnps are functionalized with both molecular recognition elements and capping ligands that stabilize the nanoparticles in solution. addition of a sample of target biomarker results in cross-linking of the functionalized nanoparticles, drastically decreasing the nano- review particle−nanoparticle distance and producing in a spectral shift. , agnp aggregation assays in this format have been implemented for detection of both infectious disease protein and nucleic acid biomarkers; however, to the best of our knowledge, cross-linking-based agnp aggregation has yet to be employed in paper diagnostics or other lowresource platforms. in non-cross-linking aggregation assays, nanoparticles are either aggregated or stabilized with molecular recognition elements that electrostatically adsorb to the nanoparticle surface. subsequent addition of a sample containing the diagnostic target then either disrupts or promotes nanoparticle stabilization, producing an observable color change. for example, teengam et al. developed a non-cross-linking aggregation assay using agnps for multiplexed visual detection of middle east respiratory syndrome coronavirus, mycobacterium tuberculosis, and human papillomavirus oligonucleotides in a microfluidic paper analytical device (μpad). the assay lacked the sensitivity required for detection of pathogenic nucleic acids; however, most sensitive nucleic acid diagnostics incorporate amplification techniques (e.g., pcr) that require extensive resources and infrastructure only available in betterequipped laboratories. nonetheless, the use of a agnp aggregation-assay in a μpad enabled multiplexed visual detection of three different nucleic acid biomarkers in a format that could be applicable to a primary healthcare setting. taking advantage of the intrinsic fluorescence of some agnps, kurdekar et al. developed a fluorescent immunoassay for detection of p that utilized a streptavidin-coated agnp as the detection probe. using a standard well-plate immunoassay format, adsorbed anti-p antibodies captured p from samples. a biotinylated anti-p detection antibody and the streptavidin-conjugated fluorescent agnp were subsequently added to generate a fluorescent signal that correlated with p concentration. the assay successfully detected p standard spiked into plasma as well as the p present in hiv-positive patient plasma samples. moreover, no cross reactivity was observed in samples that were hivnegative but hbv-or hcv-positive. the limit of detection of the assay was pg/ml, which is an order of magnitude higher than a previously discussed assay for p (section . . ). this discrepancy could be explained by the lack of signal amplification in the fluorescent agnp-based immunoassay. in its current format, the fluorescent agnp immunoassay is not amenable to a resource-limited primary healthcare setting; however, fluorescent agnps could be employed in a lateral flow assay format and combined with a portable fluorescent reader, allowing for implementation at the point of care. . . . quantum dots (qds). quantum dots (qds) are semiconductor nanocrystals with luminescent and conductive properties that make them advantageous as detection probes in infectious diseases diagnostics. canonically, qds consist of combinations of elements from groups ii and vi or groups iii and v and range in size from to nm. , at these small sizes, qds absorb photons, generating excited electrons in the conduction band and positively charged holes in the valence band of the semiconductor ( figure a ). the distance that separates the electron−hole pair is confined to less than the diameter of the nanocrystal, resulting in "particle in a box" behavior known as quantum confinement. − as a result of quantum confinement, qd emission wavelength increases as nanoparticle size increases; thus, qd emission can be tuned via size-controlled synthesis ( figure b ). because qds exhibit absorption across a broad spectrum yet have emission spectra with very narrow and tunable bandwidths, they are particularly useful for multiplexed detection of disease biomarkers. , additionally, qds demonstrate increased biocompatibility, photostability, quantum yield, and semiconducting properties compared to traditional organic fluorophores, further rendering qds advantageous as signal generation probes for diagnostics. escherichia coli, and cholera , in fluorescent assays for laboratory and resource-limited settings. functionalized qds or qd-loaded microparticles have been utilized as fluorescent probes for both protein and nucleic acid biomarkers. , , the surface of qds can be functionalized with antibodies or antigens for protein detection or oligonucleotide probes for target dna detection using the bioconjugation strategies discussed in section . . . for a thorough evaluation of the bioconjugation of qds, the reader is directed to a number of reviews. , , goldman et al. , pioneered the earliest uses of antibody-functionalized qds as fluorescent detection probes. the authors utilized affinity-based coupling chemistry (i.e., biotin−avidin) as well as electrostatic interactions between charged moieties to functionalize antibodies to the metal surfaces of cdse/zns core−shell quantum dots. the biotin−avidin system was found to be the more simple, reproducible, and robust method for qd bioconjugation of the two, and these functionalized qds were successfully utilized in a multiplexed fluorescent assay for cholera toxin and staphylococcal enterotoxin b. these early works exploited the narrow emission profile and enhanced quantum yield of qds for antigen detection and laid the groundwork for the development of enhanced, fluorescencebased diagnostics for a variety of targets. building upon this previous work, klostranec et al. constructed a multiplexed fluorescence-based microfluidic device for detecting hiv, hepatitis b, and hepatitis c antibodies utilizing antigen-functionalized qd barcode particles. two types of cdse/zns qds were synthesized and loaded into polystyrene beads at varying ratios, resulting in three distinct qd barcode particles. each qd barcode particle was covalently bound to a disease-specific antigen via edc coupling. the assay employed a "sandwich" format where disease-specific antibodies were captured by the antigenfunctionalized qd barcode particles, and a fluorophoreconjugated (alexafluor ) antihuman antibody was used to detect the presence of a target antibody. the narrow peak width of qd emission enabled detection of both the antibodyconjugated fluorophore at ∼ nm to indicate the presence of target antibody as well as both of the qds at nm (yellow) or nm (red). the ratio of the red:yellow signal intensity allowed for barcode identification and determination of which of the three target antibodies was present ( figure ). the detection limits for antibodies correlating to all three diseases were in the picomolar range, which is a -fold improvement compared to commercial elisas. while the assay enabled multiplexed and sensitive detection of infectious disease biomarkers, the microfluidic platform employed by the authors required extensive user manipulations and complex instrumentation and analysis that would only be viable in higher level healthcare settings. given the improvements in sensitivity of the assay, adaptation of the technique to a handheld fluorimeter or cell phone-adapted fluorescence device could potentially allow for deployment in a primary healthcare low-resource setting. the benefit of this strategy is that the biomarker signal and barcode signal are both fluorescencebased, resulting in a multiplexed detection platform that is simplified compared to assays with different detection schemes for biomarkers and barcodes. the authors hypothesized that up to barcode combinations are theoretically possible using this approach due to the narrow and distinct fluorescent peak widths generated by different qd particles, allowing for even greater multiplexing. however, such hypotheses should be approached with caution, as highly multiplexed assays can be more susceptible to cross-reactivity, thereby reducing the diagnostic specificity of the individual assays. qd-loaded particles have also been functionalized with antibodies for the detection of protein biomarkers for infectious disease. recently, hu et al. developed a lateral flow assay that utilized antibody-coated polymeric particles ( . nm) loaded with both qds ( − nm) and aunps ( nm) for detection of ebola virus (ebov) glycoprotein. the reporter probes, which the authors dubbed "dual signal readout nanospheres" (rns), enabled detection of the ebov glycoprotein either visually via aunps or fluorescently via qd emission. characterization of the rns revealed that there were dozens of aunps adsorbed and hundreds of qds embedded per every one antibody-coated polymeric particle ( figure , panels a and b); thus, for every one biomarker arrested at the test line, there were significantly more signaling review moieties to permit more sensitive detection. additionally, two sets of antibody-functionalized rns were fabricated and used for each lfa: ( ) streptavidin-conjugated and ( ) biotinconjugated rns. application of the sample and streptavidinconjugated rns to the sample pad resulted in migration of the sample and rns to the test line and formation of an antibody "sandwich" at the test line. subsequently, the biotin-conjugated rns were added and bound to the streptavidin-coated rns, enhancing the signal based on the high affinity of the biotin− streptavidin interaction. the authors utilized the lfa to detect either the ebov glycoprotein or the whole virion spiked into samples. the assay demonstrated a broad dynamic range that spanned orders of magnitude for detection of aunps with a limit of detection by visual inspection of ng/ml. when the qd fluorescence signal was used, the detection limit was improved to . ng/ml (figure , panels c and d). the fluorescent detection of this assay was − orders of magnitude more sensitive than commercial brands of rapid tests for ebov glycoprotein. moreover, the fluorescent signal could be quantitated by image processing software that is readily available on a smartphone. though the authors utilized a benchtop fluorimeter for qd excitation and emission, the use of a smartphone adapted hand-held fluorimeter or dedicated fluorescent lateral flow reader could enable measurements at the point of care. the sensitive dual signal readout provides versatility to this platform such that it could be employed in a number of use-case scenarios. if morbidity control were the primary concern, visual detection of aunps would suffice. if more sensitive and quantitative measurements were needed for surveillance, intervention management, or an elimination campaign, the fluorescent capabilities of the detection probes could be exploited. however, for a given use-case scenario, likely only one signal readout modality is needed. as a consequence, the additional cost of implementing a dual signal rn, when using a single signal (visual or fluorescent) rn would suffice, needs to be considered. nonetheless, the assay demonstrated that quantum dots could be incorporated into tests that are readily deployable in primary healthcare settings, though more rigorous field evaluation and validation of the assay would be necessary for clinical use. there have been significant advances in the use of qd-based detection methods for infectious disease biomarkers. while the complexity of instrumentation required for detecting quantum dots is often still a barrier to their implementation at the point of care, developments in smartphone-based devices are currently enabling the detection of fluorescent labels such as qds at the point of care. − fluorescent assays have demonstrated a capacity for multiplexing. because of the narrow emission profiles demonstrated by qds, the potential for continued expansion in multiplexing likely lies with qdbased fluorescent assays. given the importance of detecting comorbidities and coinfections or discriminating between febrile illnesses, , the qd-based technologies represent a platform that has the potential to make a significant impact at the point of care. . . . lanthanide chelate-doped nanoparticles. nanoparticles doped with lanthanide chelates are advantageous as detection labels owing to their large stokes shifts, sharp emission peaks, stable luminescence, long fluorescence lifetime, and biocompatibility. , in a lateral flow assay format, these particles are often functionalized with targetspecific antibodies and then embedded in the conjugate pad of the test. like qds, detection of lanthanide chelate-doped nanoparticles requires additional instrumentation for particle excitation and measurement of emission. , despite this need for additional equipment, these particles are attractive labels for poc diagnostics because they enable highly sensitive biomolecule detection. in the context of elimination or surveillance of a particular infectious disease, the added sensitivity provided by lanthanide chelate-doped nanoparticles could allow for detection of low-intensity infections that would otherwise be missed. except lanthanum, all lanthanides are f-block elements; the f orbital is gradually filled as the atomic number increases. because the f orbital is largely shielded from the chemical environment by the filled s and p shells, the lanthanides are very similar in their chemical properties. for example, the most common oxidation state for aqueous lanthanide ions is + . , most of these ions are luminescent, though some lanthanides have greater quantum yields than others due to the large energy gaps between the lowest emissive energy level and the highest sublevel of the ground state. as the size of the energy gap increases, lanthanide ions become less prone to lower energy nonradiative decay processes, resulting in greater quantum yields. eu + is the most commonly utilized lanthanide for labels in lateral flow assays because of its ( ) ideal energy gap between the nondegenerate emissive state of d and the ground state of f j and ( ) convenient emission in the visible region. consequently, eu + will be the focus of this section. europium(iii) itself is not strongly luminescent because most excitations involve forbidden electric dipole f-f transitions, which have low absorption cross sections. however, this can be overcome when the ion is coordinated to a sensitizing structure, such as an organic ligand. a simplified jablonski diagram of the luminescence process for a eu + chelate is depicted in figure . the ligand absorbs the excitation energy and transfers it to a triplet state (t ) via intersystem crossing (isc). next, this energy is transferred to one of the d j levels of eu + , and, after internal conversion (iet), emission occurs from the d energy level to any of the f j levels. , in addition to sensitization, chelating ligands also promote luminescence by shielding eu + from aqueous quenching effects and allow for coupling to biomolecules or polymeric materials. chelating ligands typically absorb in the uv ( − nm), and the subsequent eu + emission occurs at − nm. ligands usually contain a chromophore portion (e.g., pyridine, salicylate, phenanthroline, coumarin, pyrazole, triphenylene, and quinolone) and a chelating structure based on multidentate polyacids or macrocycles (e.g., edta). tetracycline has also been employed as a ligand for eu + . these chelate dyes are incorporated into nanoparticles functionalized with target-specific molecular recognition elements for use in lateral flow assays. most lateral flow assays that employ eu + chelates use commercially available particles, which are often superior due to their monodispersity and quality control standards. the majority of commercially available eu + chelate lateral flow labels are − nm carboxyl-or streptavidin-decorated polystyrene particles and are readily available from large vendors such as thermo-fisher and expedeon ltd. eu + chelates are easily adsorbed into polymeric (e.g., polystyrene) nanoparticles based on hydrophobic interactions between the polymers and the organic chelating ligands of the complexes. for example, huhtinen et al. found that simply incubating nm polystyrene particles with eu + chelated with dipicolinic acid derivative ligands resulted in − chelates per nanoparticle. this represents a density of approximately . chelates/nm . harmäet al. found a similarly high density of approximately . chelates/nm ( chelates/particle) in nm commercially available particles. thus, for each biomolecule bound in a lateral flow format, thousands of associated eu + chelates produce a signal, resulting in a highly sensitive measurement. the advantages of eu + chelate-loaded polystyrene nanoparticles as labels for lateral flow assays were demonstrated by yeo et al., who developed a sensitive lateral flow assay for the detection of avian influenza h subtype virus. these viruses can be highly pathogenic; for a h n outbreak in china, a mortality rate of roughly % was estimated for hospital patients with laboratory-confirmed infections. currently, laboratory-based molecular testing is required to differentiate between h subtype avian influenza and other viral infections. thus, there is a need for rapid tests that can be performed at the point of care. the authors approached this problem by developing and evaluating a panel of h subtype-specific monoclonal antibodies against hemagglutinin of h n influenza. after the best antibody pair was determined, h subtype-specific lateral flow assays were constructed with both europium-chelate and gold nanoparticle (aunp) conjugates. using a benchtop time-resolved fluorescence reader, the authors found that the detection limits for the eu + chelate-based tests were -fold better than those of aunp-based tests. additionally, the developed eu + -based tests performed better than commercially available tests detecting influenza a nucleoprotein. these enhancements over gold particles are impressive, although given the high density of chelates per nanoparticle, it is surprising that larger enhancements were not achieved. one possible explanation could be the efficiency of molecular recognition element adsorption for the two types of nanoparticles. in addition to avian influenza, , eu + chelate-embedded polymeric nanoparticles have been employed for the detection of e. coli, hepatitis b, and procalcitonin, a host marker for bacterial infections. several commercial platforms based on these particles have been developed as well, including the sofia influenza a+b tests (quidel, inc.) − and the aqcare chlamydia trf test (medisensor, inc.), , which have been extensively evaluated in the literature. the high sensitivity of these assays is derived not only from the high number of eu + chelates embedded in the polymeric nanoparticles but also from the use of time-resolved fluorescence measurements. one of the primary disadvantages of using luminescent probes as labels in lateral flow assays is the high autofluorescence of biological samples and assay components occurring at excitation and emission wavelengths similar to those of the probes. this fluorescence can interfere with lateral flow readings, causing high background that leads to low-sensitivity measurements. however, lanthanide chelates overcome these effects because their fluorescence lifetimes are several orders of magnitude longer than conventional fluorescent materials (microseconds or milliseconds compared to nanoseconds). , this allows for the use of time-resolved measurements in which samples are excited with a pulse of uv light, and emission signal is collected after a time delay on the order of ns for a given time period. by delaying the collection of emitted signal, the short-lived autofluorescence from biological and assay components is excluded from measurements. while the intensity of the delayed luminescence is slightly decayed from the initial signal output of the chelates, these measurements can be cycled to improve signalto-noise. of course, the unique instrumentation required for time-resolved measurements makes field-deployment technically challenging, though currently available benchtop devices could allow for measurements in district clinics and laboratories. recently, paterson et al. developed a promising smartphone-based platform for time-resolved luminescent measurements, which could allow for point-of-care measurements based on eu + chelate particles. however, the lengthy ms time-delay employed in their device is only compatible with the most persistent luminescent nanophosphors such as crystalline sral o :eu + ,dy + particles. while polymeric eu + chelate nanoparticles are the primary materials employed by lanthanide-labeled lateral flow assays, these particles present some disadvantages. their large size and tendency to swell can cause aggregation in aqueous solutions. additionally, since they are not covalently bound to the polymers, eu + chelates can leach from the particles. silica particles avoid some of these problems. in one example, xia et al. developed a lateral flow assay for hepatitis b detection using eu + chelate-loaded silica nanoparticles. to synthesize these particles, the authors relied on an iterative method involving silica condensation from teos, surface-amination using aptms, covalent functionalization of chelating ligands to the particles, and loading of eu + . this process was repeated five times to achieve a high density of eu + chelates. the resulting nm nanoparticles contained a remarkable chelates/particle, corresponding to a density of roughly . eu + chelates/nm , more than times greater than commercially available polymeric particles. these particles were then functionalized with oxidized dextran to serve as a hydrophilic linker, and free amine moieties on antihepatitis b surface antigen (hbsag)-specific antibodies were coupled to the surface via reductive amination of the aldehyde groups. after synthesis and characterization, the particles were used as labels in a traditionally formatted lateral flow assay for hbsag. to measure signal, the strips were illuminated with a uv lamp and imaged using a digital camera. signal intensity was determined using image analysis in adobe photoshop. unlike the previous example presented, this imaging technique does not take advantage of the long fluorescence lifetime of eu + chelates. however, even without time-resolved measure- ments, the detection limit of the lfa was found to be pg/ ml hbsag, -fold better than a similarly formatted goldbased lateral flow assay. the study was then validated by analyzing clinical serum samples using the developed eu + chelate-based lateral flow assay as well as a quantitative elisa. the two methods were concordant for all samples analyzed and had similar sensitivities. thus, the developed method represents an innovative tool for diagnosis of hepatitis b and provides sufficient sensitivity when compared to clinical methods. in order to implement this assay directly at the point of care, two modifications are necessary: ( ) the assay should be optimized for field-friendly whole-blood specimens, and ( ) a mobile phone-based detection strategy should be implemented. lanthanide chelate-based nanoparticle probes are a promising avenue for developing highly sensitive lateral flow assays. similar to other fluorescent probes, instrumentation remains the primary barrier to implementation of eu + chelate nanoparticles at the point of care. however, as optical devices are miniaturized and become more affordable, these probes could become commonplace among rapid tests for infectious diseases. . . . up-converting phosphor nanoparticles. upconverting phosphor (ucp) nanoparticles represent a unique and exciting reporter technology for bioassays. unlike typical fluorescent labels, these ceramic nanoparticles doped with rare earth elements absorb low-energy ir radiation and emit higher-energy visible light. − ucps are often superior to conventional colorimetric and fluorescent reporters for a number of reasons. first, the high anti-stokes shift from ir to visible does not occur in nature. thus, the inherent autofluorescence typically associated with biological samples and assay components does not interfere with ucp measurements. second, ucp signal is highly stable and does not photobleach, allowing for indefinite storage and repetitive analysis of a single test. this is especially useful for confirming field results in a central laboratory. third, assays are easily multiplexed; distinct particles absorb at the same ir excitation energy and emit wavelengths defined by the dopant. finally, optical detection of ucp particles offers an inherent boost in analytical sensitivity over visual detection of gold; as few as − ucp particles can be detected at the test line of a lateral flow strip, while approximately gold particles are needed for visual detection. this increase in sensitivity makes ucp particles attractive labels for diagnostic tests employed in surveillance or elimination campaigns in resourcelimited settings, since they would enable the detection of lowintensity or asymptomatic infections. however, similar to qds and lanthanide-chelate particles discussed in the preceding sections, these benefits of ucp particles must be weighed against the additional cost and resource requirements needed for detection instrumentation. the primary mechanisms for up-conversion include excited state absorption, energy transfer, and cooperative sensitization. excited state absorption is the absorption of light by electrons already in an excited state. this requires two photons and equal separation between the ground state, first excited state, and second excited state of a single ion. energy transfer upconversion requires two ions, a sensitizer and an activator. in this process, the sensitizer ion is excited and sequentially transfers its energy to the ground state and first excited state of the activator ion. cooperative sensitization is similar to energy transfer, though it involves excitation of two sensitizer ions which simultaneously transfer their energy to the activator ion. other up-conversion mechanisms include photon avalanche and cross relaxation. energy transfer up-conversion is the most common upconversion mechanism for bioassay reporter particles, which typically involve a yb + sensitizer (excitation nm) and a rare earth (er + , tm + , pr + , ho + , and gd + ) activator codoped in yttrium fluoride (yf ), yttrium oxide (y o ), yttrium oxysulfide (y o s), or sodium yttrium fluoride (nayf ) crystals. , an illustration of the up-conversion processes for two of these materials are shown in figure . ucps have been utilized as labels for both protein and nucleic acid targets in lateral flow assays for a variety of infectious diseases. recently, corstjens et al. − developed an ultrasensitive, ucp-based lateral flow assay for schistosomiasis that detects schistosoma circulating anodic antigen (caa), a proteoglycan biomarker waste product produced by the parasite. caa is present in the serum and urine of patients with schistosoma infections of all known species and has been found to correspond well with worm burden, clearing soon after successful treatment. , , the developed lateral flow assay employed nm y o s:yb + , er + ucps, which are excited at nm and emit at nm (green) and can detect caa for a single schistosoma worm pair in serum. , this assay has been applied to patient samples from endemic areas, and due to its superior sensitivity, it has demonstrated much higher schistosomiasis prevalence than microscopy, serology, and nucleic acid-based tests. [ ] [ ] [ ] the caa lateral flow assay format is similar to conventional lateral flow assays. caa-specific antibodies are printed on the test line, and antimouse igg is printed on the control line. the workflow of the assay in its current form, however, differs from a typical field-ready test. first, a trichloroacetic acid (tca) extraction is performed on a urine or serum sample, requiring a centrifugation step. the extract supernatant is then combined with running buffer and anti-caa-functionalized ucp particles and incubated for h on an orbital shaker at °c before the lateral flow strip is added to the solution. the test is allowed to develop and must dry completely (at least h) before scanning and analyzing the strip. , in order to simplify the preparation procedure and decrease the number of steps, a dry reagent kit was later developed in which all buffers and particles were lyophilized. this kit was shipped to a tertiary lab in south africa, where nearly samples were evaluated by local technicians using both the kit and a caa elisa. the researchers found that the ucp-based lfa successfully identified more low-level infections than the caa elisa. to increase the analytical sensitivity of the assay, corstjens et al. added a spin-filter concentration step to the sample preparation method. this allowed for caa in urine sample volumes of . − . ml to be concentrated into μl before addition to the lateral flow strip. the resulting detection limits improved as sample volume increased, reaching as low as . pg/ml for the . ml assay. to demonstrate clinical applicability, the concentration step was successfully performed on ml patient urine samples from kenya (high-endemic, s. mansoni) and china (low-endemic, s. japonicum) , it is important to note that, though this sample concentration step improved the sensitivity of the assay, it required laboratory equipment to carry out; all patient samples in these studies were processed in well-equipped laboratories. further, the additional concentration step increased the cost of this ultrasensitive caa assay, though sample pooling could make this test more cost-effective and allow for monitoring of worm burdens at the subpopulation level for large-scale surveillance. for this ucp-based ultrasensitive assay to be utilized in a field setting, a more robust, field-ready sample preparation method is needed. lateral flow assays with ucp reporters have also been developed for detection of protein markers of other infectious diseases, including neurocysticercosis, and hiv. additionally, ucp-based multiplexed lateral flow assays have been developed for detection of protein biomarkers for multiple infectious diseases. , these multiplexed diagnostics typically consist of one strip with multiple test lines, each capturing a distinct protein biomarker. in this format, the ucp crystals are the same for each biomarker, though they are functionalized with target-specific antibodies. one disadvantage of highly multiplexed assays on a single strip is the potential increase in cross-reactivity and nonspecific binding that could lead to false-positive results. to mitigate this risk, hong et al. developed a unique, circular cassette with channels for different singleplex lateral flow assays using ucp particles as the reporter labels. one unexplored application of multiplexed detection on a lateral flow assay is the use of ucp particles with identical excitation but differing emission profiles. this could be particularly advantageous for large biomarkers with multiple accessible epitopes and could provide additional clinical information. for example, an assay that captures and detects whole organisms could include a second ucp probe for detecting surface proteins that confer drug resistance, providing both detection and susceptibility results in one assay. ucp nanoparticles have also been used in nonlateral flow diagnostic formats, including immunohistochemistry, microarrays, magnetic bead assays, , and plate immunoassays, though these platforms are not readily amenable to low-resource, point-of-care settings. even in the lateral flow format, ucp nanoparticles present interesting challenges for field deployment. clearly, sample preparation methods, including purification, concentration, and amplification, must be adapted for environments lacking controlled laboratory conditions and should rely on as little electricity as possible. while hand-powered centrifuges , and battery-powered mixers , have been developed, the ideal poc assay will be optimized to preclude these additional resources. another issue, often unaddressed, is that the lateral flow strips must be completely dry before optical measurements and analysis. this is because water also absorbs in the near-ir, decreasing excitation efficiency of the sensitizer. typically, protocols require at least h of drying time after the lateral flow assay has developed, a long time-to-result for a point-of-care test. finally, detection of ucp particles relies on optical instrumentation. although the most sensitive of these devices are benchtop instruments, portable battery-powered devices have been developed, such as the ucp-modified version of qiagen's esequant lateral flow reader ("ucp-quant"). , , many of the sample preparation methods described in section can be applied to ucp lateral flow formats. additionally, innovations in hand-held optical readers discussed in section require only simple adaptations to detect the anti-stokes shift of ucp particles. there are clear advantages of using ucp nanoparticles as labels in lateral flow assays, including their inherently low background interference and high analytical sensitivity. innovations in sample preparation methods and the development of portable optical readers will allow for these advantages to be exploited in low-resource, poc settings. . . . magnetic nanoparticles. while most frequently used for sample preparation and biomarker enrichment, magnetic nanoparticles are emerging as promising labels in poc assays. similar to noble metal nanoparticles, magnetic nanoparticles can be easily functionalized and possess strong optical absorbance, which has led to their use as visual labels on lateral flow assays. however, their magnetic properties can also be leveraged for detection, which has advantages over traditional optical detection in a lateral flow format. first, when visual or optical detection of absorbent or fluorescent particles is employed, only signal from the surface (∼ μm) of the membrane is measurable due to the opacity of the membrane. typically, nitrocellulose membranes used in lfas are − μm thick, so optical detection only allows for signal from the top % of the membrane to be measured. in contrast, magnetization measurements can be performed regardless of the opacity of the substrate, taking advantage of the entire volume of the test line on a lateral flow assay. in addition, metal-oxide nanoparticles are highly stable, and drying and aggregation do not influence the intensity of magnetic measurements. finally, there are very few biological or assay components that interfere with magnetic measurements. the properties of magnetic particles are size-and temperature-dependent. , iron oxide (typically magnetite or maghemite) particles are the most common magnetic nanoparticles employed in diagnostic applications. in the bulk, these materials are ferri-or ferromagnetic and thus retain magnetization after an external field has been applied. this hysteresis reaches a maximum when particle size is decreased to the point that the material becomes single-domain. as particle size is even further reduced, the hysteresis effect decreases until the particles reach a critical diameter, below which brownian forces are strong enough to overcome magnetic forces. thus, at these small sizes, the particles are superparamagnetic; the magnetic moments of the particles are aligned in the presence of an external magnetic field, but they revert back to a nonmagnetic state when the field is removed. in the lateral flow format, magnetic nanoparticles are labeled with target-specific molecular recognition elements and employed as conjugates for analyte detection. superparamagnetic behavior is ideal for particles applied as labels review for lateral flow assays because the lack of magnetization in the absence of an external field allows for the particles to wick along the strip without aggregating due to magnetic effects. then, the magnetic properties of the particles can be leveraged after labels have bound to the test and control lines. magnetic nanoparticles are typically detected on lateral flow assays based on induction (figure ) or using magnetoresistive sensors. in the inductive format, which employs the principles of faraday's law, a magnetic particle-based test is placed in an oscillating magnetic field above a set of induction coils. in the absence of magnetic particles, the net current induced in the coils is zero. however, when particles are present, the direction of their magnetic moments oscillates with the external magnetic field, resulting in a measurable net voltage induced across the coils that is proportional to the total number of particles at the test or control line. , in contrast, magnetoresistive sensors are based on the principle that the electrical resistance of certain materials, which are incorporated in the sensors, can change upon application of an external magnetic field. in this format, a magnetic field is applied to para-or superparamagnetic detection elements in order to align their magnetic moments and produce a fringe field. magnetoresistive sensors are placed close enough to the particles to detect the fringe field they produce based on the change in resistance of the materials within the sensor. , a detailed description of the instrumentation required for these detection methods is provided in section . . magnetic nanoparticles have been employed as detection labels in lateral flow assays for protein and whole-cell targets. for example, handali et al. developed two magnetic particle-based lateral flow assays for detection of taenia solium, a cestode that is prevalent around the world for which swine are intermediate hosts. when contaminated, undercooked pork is ingested, the parasites develop into tapeworms in the human gut (taeniasis). however, if eggs are ingested, the larval stage can infect the human nervous system, potentially forming cysts in the brain. this severe form of the disease, called neurocysticercosis, is a leading cause of epilepsy worldwide. the gold standard for diagnosis of taeniasis is observation of eggs in stool by microscopy, though this method is insensitive and labor-intensive. diagnosis of neurocysticercosis currently requires ct scans of the brain, a technology that is unavailable in low-resource settings. as such, there is a pressing need for t. solium rapid diagnostic tests. handali et al. developed two serological magnetic beadbased lateral flow assays that detected the immune response (i.e., host antibodies) against taeniasis-specific (es ) and cysticercosis-specific (t ) antigens. for each test, one batch of the recombinant antigen was printed at the test line, and a separate batch was coupled to commercially available carboxylated superparamagnetic particles via edc/nhs chemistry. the recombinant antigen at the test line and the antigen conjugated to magnetic particles were able to simultaneously bind to host antibodies in the sample, taking advantage of the multivalent nature of anti-es and anti-t igm and igg antibodies. the lateral flow assays were evaluated using an inductionbased reader and visual reading to detect antibodies in serum samples from endemic areas and nonendemic control regions ( figure ). it was found that the taeniasis-specific es assay had a diagnostic sensitivity and specificity of % and %, respectively, when evaluated with the magnetic reader. the sensitivity and specificity of the neurocysticercosis t test using the magnetic reader were % and %, respectively, for patients with two or more viable cysts as determined by ct scan or mri of the brain. for patients with only one viable cyst, the t assay only identified / cases of neurocysticercosis, indicating that the burden of infection was not great enough to elicit an immune response. despite this decrease in sensitivity for single-cyst infections, the developed lateral flow assays are promising alternatives to the current gold standards. further, because the assays were not speciesspecific, they could be used to detect porcine cysticercosis as a marker of disease control and transmission. one disadvantage of utilizing superparamagnetic labels is that many magnetic readers are benchtop devices requiring electricity and a laboratory setting. however, in over % of the cases, the magnetic nanoparticles could be detected visually. while this sensitivity is not ideal, it is possible that initial visual results could be applied in the field with follow-up laboratory-based measurements assessed for the visually negative assays. as hand-held magnetic readers become more commonplace, these assays will become truly impactful. in addition to t. solium, magnetic particles have been employed in protein-detecting lateral flow assays for hiv , and h n influenza. whole cell-detecting magnetic particle-based assays have also been developed for a number of targets, including vibrio parahemolyticus, , listeria monocytogenes, and bacillus anthracis. , the demonstrated sensitivity of these magnetic particle-based lateral flow assays makes them promising alternatives to traditional assays with colorimetric or fluorescent labels. however, current instrumentation, which will be discussed further in section . , limits their utility at the point of care. one exciting avenue that has yet to be explored for infectious disease detection is the employment of the same superparamagnetic nanoparticles for dual purposes: immunomagnetic biomarker enrichment from large-volume samples and visual labeling for lateral flow assays. ideally, such a diagnostic would integrate sample preparation and assay into one device. once developed, the combined effects of target enrichment and magnetic detection could lead to a highly sensitive test capable of detecting low-density infections. one of the most widely utilized techniques for improving the sensitivity of diagnostics is signal amplification, where thousands of signaling molecules are generated for every one biomarker molecule. though there are now numerous methods for amplifying signal in diagnostic assays, the use of metalloenzymes was one of the first approaches. , metalloenzymes are metal-containing catalytic proteins in which the presence of particular metals or metal complexes in the tertiary structure is critical to the catalytic turnover of the substrate. the predominant metalloenzymes employed in diagnostics are alkaline phosphatase (alp), horseradish peroxidase (hrp), and catalase. the high catalytic efficiencies of these enzymes enable the rapid conversion of substrate to detectable products. using cross-linking chemistry discussed in section . . , these metalloenzymes can be conjugated to antibodies or nucleic acids that afford specificity for a target biomarker. , the three primary enzymes used in metalloenzyme detection conjugates rely on different metal ions for catalytic activity. alp possesses two zn(ii) ions and one mg(ii) ion per enzyme monomer and hydrolyzes phosphate monoesters. , the conventional substrates for alp in point-of-care diagnostic applications are bcip ( -bromo- -chloro- -indolyl phosphate) and nbt (nitro blue tetrazolium), where dephosphorylation and oxidation of bcip allows for reduction of yellow nbt dye to a blue/purple formazan precipitate. both hrp and catalase are heme-containing monooxygenases that catalyze reactions with h o . . . . elisas (enzyme-linked immunosorbent assays). metalloenzyme-antibody conjugates have been widely implemented in elisas for the detection of protein biomarkers for disease. this is due to the sensitivity afforded by the use of enzymes for signal amplification as well as the specificity of the antibody−antigen interactions used for molecular recognition. , , despite the high sensitivities of elisas, their application in poc settings is limited by several factors: − signal readout for elisas typically requires a spectrophotometer unavailable in a primary healthcare setting, though there are a growing number of portable alternatives (e.g., cellphones or hand-held scanners; see section . . ). elisas are time-consuming (∼ − h), demand extensive sample handling and solution manipulation, and often require specialized training. lastly, the lack of thermal and long-term stability of metalloenzymes in elisas can lead to suboptimal assay performance, as elisas are intrinsically dependent on these metalloenzymes for signal amplification and readout. thus, elisas are usually limited to well-equipped tertiary laboratories. to mitigate these issues, investigators have begun to use paper as the elisa solid support, increasing the likelihood that these sensitive assays could be used in resource-limited settings. paper-based elisas have been developed for detection of diagnostic markers of hiv and malaria. , , long-term stabilization conditions of metalloenzymes on paper have also been investigated to maximize catalytic activity in poc settings. − paper-based elisas seek to address many of the issues that inhibit the use of elisas in more resource-limited poc settings. the time to result for paper-based elisas is ∼ h due to the comparatively smaller volumes required when contrasted with conventional benchtop elisas. these smaller reagent volumes also significantly reduce the overall cost of elisas. cheng et al. first pioneered this method for the detection of serum antibodies against hiv- envelope antigen gp , where an alp-antibody conjugate was utilized to turn over bcip/nbt substrates to purple precipitate products. the authors used hydrophilic paper that was patterned with hydrophobic photoresist to fabricate test "wells" that were spatially separated just as in a polystyrene -well plate. samples containing biomarker were added to the paper elisa card, which was placed on top of a blotting pad to enable wicking of the reagents through the test wells. though the assay boasted a faster time to result and a lower cost than the analogous conventional elisa, the sensitivity of the paperbased elisa was decreased by an order of magnitude compared to the conventional elisa format. lathwal and sikes conducted a systematic investigation of several signal amplification methods for paper-based colorimetric detection of malarial biomarker hrp . to investigate multiple metalloenzyme−substrate combinations, the same capture antibody was immobilized on aldehyde-modified cellulose for all experiments, and the following detection systems were evaluated: ( ) hrp with substrates dab and h o , ( ) hrp with substrates tmb and h o , ( ) alp with substrates bcip and nbt, ( ) ag deposition onto aunps, and ( ) polymerization-based amplification. because all factors (i.e., capture antibody, capture antibody loading, biomarker, detection antibody) were held constant, the differences review observed in each assay were presumed to be the direct result of the signal amplification system used. positive and negative controls were evaluated at various time points for each signal amplification method to determine an optimal window for visual readout of the signal ( figure ). all of the methods had an optimal time point for visual signal readout with the exception of hrp-tmb/h o , which produced significant false-positive signals and a color change from blue to brown that was difficult to interpret. of the remaining methods, the metalloenzyme−substrate combinations (hrp-dab/h o and alp-bcip/nbt) had detection limits an order of magnitude better than ag deposition and polymerization-based amplification methods when quantified by rgb color intensity. any portable colorimetric reader, even a cell phone, could enable the use of these highly sensitive metalloenzyme conjugates in a primary healthcare setting. the advantages of paper-based elisas are potentially compromised by the instability of metalloenzyme conjugates, since denaturation of metalloenzymes leads to poor turnover of substrate and lower sensitivities. for this reason, notable research efforts have been devoted to optimizing long-term storage of metalloenzymes, particularly hrp, for use in microfluidic paper analytical devices (μpads) or two-dimensional paper networks ( dpns). − one method involves vacuum drying or freeze-drying enzymes and substrates in the presence of trehalose, a disaccharide often used as a cryoprotectant because of its capacity to stabilize the protein's interaction with solvents, forming a protective "glass" as it dries. , ramachandran et al. used a combination of trehalose, bovine serum albumin (bsa), and feso -edta for vacuum drying of hrp-conjugated antibodies against malarial biomarker hrp into glass fiber pads. the stabilized hrp could be stored for months at °c under these conditions without a significant loss in catalytic activity. a glass fiber pad containing all of these reagents was incorporated into a dpn for detection of hrp with a lod of . ng/ml, a value well within clinically relevant concentrations. this assay also demonstrated the utility of metalloenzymes in paper-based poc diagnostics, providing a signal amplification step that is rarely present in conventional paper diagnostics (e.g., lateral flow assays). these advances in enzyme stabilization when combined with simplified paperbased assay formats could potentially allow for elisa sensitivity to be translated for direct use at the point of care. . . . nanoparticle-assisted enzymatic signal amplification. the integration of noble metal nanoparticles has further augmented the signal amplification capabilities of metalloenzymes in infectious disease diagnostics. nanoparticles have served as surfaces for the coupling of antibody-metalloenzyme conjugates and have been implemented in electrochemical sensors for detection of infectious disease-associated protein biomarkers. − zheng et al. developed an amperometric immunosensor for detection of hiv antigen p that employed a standard "sandwich" format. a glassy carbon electrode was modified with gold nanoparticles to allow for immobilization of anti-p capture antibodies. hrp-conjugated anti-p antibodies were used for detection, catalyzing the oxidation of substrate hydroquinone in the presence of h o . the reaction generated a reductive current at the electrode surface proportional to p concentration. the detection limit of the assay was pg/ml, similar to the p assays , previously discussed in sections . . and . . . while the assay was robust to human serum samples spiked with p , the serum samples tested contained a p concentration orders of magnitude higher than the lod. the currently required electrochemical workstation would limit the assay's application to higher level laboratories. nonetheless, this amperometric method demonstrated the value of integrating noble metal nanoparticles with metalloenzyme conjugates for signal amplification in diagnosis of infectious diseases. noble metal nanoparticles have also been utilized as colorimetric signaling probes in elisas for detection of protein biomarkers for disease. − so-called "plasmonic" elisas utilize the standard "sandwich" assay format with an immobilized capture antibody and a detection antibody that is conjugated to a metalloenzyme. however, as opposed to using conventional metalloenzyme substrates for colorimetric detection, plasmonic elisas employ the enzyme as a kinetic tool for nanoparticle nucleation. this causes drastic shifts in spr and in the absorbance spectra of the nanoparticles that are detectable with the naked eye. the plasmonic elisa pioneered by de la rica and stevens utilized a catalase-conjugated detection antibody to control the growth of aunps in such a manner that was proportional to the concentration of hiv biomarker p . in the absence of biomarker, bulk au(iii) was reduced by h o to form stable, spherical aunps that appeared red in color. when p was present in a sample, the catalase-conjugated (figure ) . the red-shift in the spr was highly sensitive toward h o concentration; thus, depletion of h o with catalase that was only present in p -positive samples allowed for very sensitive detection of p with the naked eye. the absorbance was also quantified using a simple spectrophotometric readout, yielding a detection limit of . ag/ml, nearly orders of magnitude more sensitive than the previously discussed detection methods for p antigen. , , the authors also demonstrated that the plasmonic elisa could detect p in hivpositive serum samples, even identifying p in samples from hiv-positive patients with viral loads less than copies/ ml. the remarkable sensitivity of this assay for naked-eye detection of p provides the necessary improvement required for elimination campaigns and active case management. it would permit earlier detection of p , leading to improved outcomes for hiv patients. the assay could be particularly impactful for the challenges associated with early infant hiv diagnosis in low-resource settings. the plasmonic elisa presented by the authors still calls for extensive sample handling and user manipulation and is currently unsuitable to a primary healthcare setting. adaptation of the previously discussed enzyme stabilization measures and integration of the assay to a field-ready format (e.g., paper-based elisa or dpn) could enable its translation to an ultrasensitive fieldready diagnostic for hiv detection. metalloenzymes play a critical role for signal amplification in a number of assays, enabling the sensitive detection of infectious diseases. several research efforts have been aimed at translating the sensitivity of metalloenzyme-based assays to formats such as paper-based diagnostics that are amenable to resource-limited settings. although the issue of enzyme storage in paper has been addressed to certain extent, − the longterm stability of enzymes remains a concern for poc diagnostics, especially in climates with elevated temperatures and prevalent infectious disease. there are numerous methods that utilize nonenzymatic means for signal amplification that eliminate the issue of long-term enzyme storage altogether, including nanoparticles that act as enzyme mimics and other metal-based methods. these will be covered in the following section. while the majority of signal amplification in bioassays is enzyme-based, several interesting metal-based amplification strategies have been developed. these strategies include metal nanoparticle dissolution, nanocrystal ion exchange, enzyme mimics, and reductive nanoparticle enlargement. , − compared to enzymes, metal-based signal-amplification has the distinct advantage of long-term storage and thermal stability. however, many of these strategies suffer from the following drawbacks: ( ) amplification often requires additional steps to be integrated into point-of-care test workflow, and ( ) incorporation into a paper-based format can be technically challenging. however, innovative assay design can overcome these challenges. integration of signal amplification into point-of-care tests can drastically improve analytical and diagnostic sensitivity, resulting in earlier diagnoses and detection of low-density infections. therefore, diagnostics which incorporate novel and user-friendly signal amplification steps could fill a need for elimination campaigns and surveillance programs. this section reviews metal-based signal amplification strategies that show promise for application to low-resource infectious disease diagnostics. . . . nanoparticle dissolution and cation exchange. at the heart of signal amplification is the principle that, for each biomarker target captured in an assay, many signalgenerating elements (particles, molecules, atoms, electrons, photons, etc.) are produced. in a typical nanoparticle-based assay, a single nanoparticle often indicates the presence of one biomolecule. however, a single nanoparticle is a densely packed lattice of thousands to millions of metal atoms, each of which, after nanoparticle dissolution or cation exchange, can be leveraged for signal generation (figure ). , − metal ion chelating reagents that result in chromogenic or fluorescent signal have long been used for detection of trace metals for a myriad of applications. , recently, tong et al. exploited ferrous ion-chelating ferrozine in an iron oxide nanoparticle-linked immunosorbent assay (ilisa). antibodyfunctionalized wustite (feo) particles were used as detection elements in direct, competitive, indirect, and sandwich wellplate assays. after functionalized iron-oxide particles bound to the target, the nanocrystals were treated with acid and reducing agents, resulting in stoichiometric conversion to ferrous ions. thus, thousands of fe + ions were released for each biomarker present. when excess ferrozine was added, solutions changed from transparent to purple upon chelation, with intensities directly proportional to the iron concentration in solution. proof-of-concept assays detecting mouse igg had low picomolar detection limits, similar to assays that depend on enzymatic signal amplification. to extend the applicability of their amplification technology, the group also demonstrated its use in a western blot. by switching the chelating reagent to potassium ferrocyanide (prussian blue), the reagent could easily be precipitated onto a cellulose membrane when iron oxide particles were used as detection elements. while wellplate assays are far from applicable to field settings, the ilisa system could be translated to an aptly designed paper diagnostic format. another avenue for signal amplification similar to nanoparticle dissolution is nanocrystal cation exchange, in which the cations within a nanocrystal are place-exchanged with different cations. while bulk, solid-phase cation exchange often requires elevated temperatures and several weeks, complete nanoscale exchange can occur within seconds due to increased surface area and lower energy barriers to diffusing ions. for cation exchange to be employed in a bioassay, nanocrystals (znse, zns, cdse, or cus) are first functionalized with targetspecific molecular recognition elements, such as antibodies or aptamers, which are utilized as detection probes. using rapid silver ion exchange, each bound nanocrystal releases thousands of zn + , cd + , or cu + ions. zinc and cadmium ions are then detectable using fluorogenic chelating reagents such as fluozin- or rhod- n, respectively, and cu + can be detected in a chemiluminescent reaction with luminol and h o . these reactions produce an amplified, stoichiometric signal and have been employed in well-plate , and magnetic beadbased − immunoassays for protein , , and cell detection as well as rolling circle amplification for dna/ mirna , detection. despite this versatility, the cation exchange method has yet to be applied in paper-based formats. similar to the ilisa example discussed previously, signal amplification using nanocrystal cation exchange would require a precipitating reagent for metal ion detection as well as clever design of a paper microfluidic device. enzymatic signal amplification results in high analytical sensitivity and is easily performed in a controlled laboratory environment. however, as discussed in section . , elevated temperatures often lead to decreased catalytic turnover, making it difficult to incorporate enzymatic signal amplification into low-resource infectious disease diagnostics. in recent years, some inorganic nanoparticles, including noble metal, rare earth, and magnetic nanoparticles, have been found to display surprising enzyme-like catalytic activity. − these nanomaterial-based artificial enzymes, originally dubbed "nanozymes" by manea et al., are highly stable toward a range of temperatures and phs and cannot be degraded by proteases. further, catalytic activity of inorganic nanoparticles can be tuned with particle size, shape, coating, modification, and composition. for these reasons, nanozymes have been incorporated into a myriad of sensors for various applications. in , gao et al. made the surprising discovery that magnetite (fe o ) nanoparticles have intrinsic peroxidase-like activity, displaying michaelis−menten kinetics consistent with a ping-pong mechanism. fe o magnetic nanoparticles were found to catalytically turn over several common horseradish peroxidase substrates, including tmb, dab, and o-phenylenediamine (opd), with catalytic turnover numbers equal to or improved over horseradish peroxidase. the group demonstrated that the particles could be used in place of horseradish peroxidase in a traditional immunoassay and that the magnetic properties of fe o could be leveraged further to enrich biomarkers before detection. as a result of this seminal work, magnetic nanoparticles have been employed as signal amplification elements in protein and dna bioassays for a variety of infectious diseases, including ebola, rotavirus, mycoplasma pneumonia, vibrio cholerae, chlamydia trachomatis, l. monocytogenes, enterobacter sakazakii, and salmonella typhimurium. beyond magnetite, several other types of nanoparticles, including gold, platinum, hybrid particles, − and mofs, have been utilized as enzyme mimics in bioassays for targets such as respiratory syncytial virus, avian influenza a, salmonella, and e. coli, hepatitis c and hiv, and staphylococcus aureus. many of the assays that utilize review nanozymes as catalytic turnover reagents for detection are formatted similarly to immunoassays on the surface of well plates. , − several groups have translated this technology to the lateral flow format and found that the nanozyme detection element resulted in at least -fold improvement in limits of detection when compared to traditional gold nanoparticle-based lateral flow strips. , , , , for example, loynachan et al. developed an ultrasensitive lateral flow assay for the detection of hiv p antigen using porous platinum au@pt core−shell nanozymes that catalyzed the precipitation of cn/dab ( -chloro- -naphthol/ , ′-diaminobenzidine tetrahydrochloride) in the presence of h o ( figure ). incorporation of this detection scheme, as well as careful and systematic design of the affinity reagents for p capture and detection, resulted in a lateral flow assay with femtomolar detection limits, more sensitive than laboratory-based elisa methods and nearly orders of magnitude more sensitive than commercially available rapid tests. because visual signal varied linearly with concentration before and after catalytic signal amplification, the test also had an ultrabroad dynamic range. the advantages of the nanoparticle enzyme mimics were directly demonstrated in a stability test in which the activities of lyophilized porous pt core−shell nanocatalyst conjugate and lyophilized hrp conjugates were measured over time. while the enzyme conjugates lost nearly all activity after days of storage at room temperature, the nanocatalysts maintained % of their initial activity while stored at room temperature and nearly % of their initial activity while stored at °c for up to days. however, it should be noted that the optimal hrp lyophilization and storage conditions , discussed in section . . were not employed in this study. to achieve such significant improvements in sensitivity using nanozymes, a wash step, a substrate addition step, and a reaction quenching step were inserted into the lateral flow assay workflow after the initial signal development. these additional steps make this assay and other similar diagnostics more difficult to perform in the field. redesigned paper devices or automated lateral flow cassettes could simplify this workflow review and allow for application of nanozymes in field settings, providing much-needed signal amplification and improved sensitivity for low-resource infectious disease diagnostics. . . . reductive nanoparticle enlargement. the use of gold nanoparticles as detection elements in commercially available lateral flow assays is ubiquitous. particles used in these assays are typically − nm and can be detected either visually (without optical equipment) or using simple hand-held readers. , , often, the detection limits of these assays are not low enough to diagnose low-density infections. one simple solution for improving sensitivity is chemically enlarging the particles, making them more visually intense. in this process, a lateral flow assay is run in the typical manner such that, if the antigen is present, a gold nanoparticle-containing sandwich complex is formed at the test line. next, an enhancement solution consisting of a molecular precursor and reducing agent is added to the test. the gold nanoparticles at the test and control lines serve as nucleation sites for solid metal deposition. the most common format for particle enlargement, silver enhancement, is based on th-century photographic techniques and involves the reduction of silver ions (e.g., silver nitrate) to silver metal on the surface of gold particles in an acidic buffered solution containing hydroquinone. this process is similar to the seeded-growth nanoparticle synthesis method and can result in particles from hundreds of nanometers to several micrometers in diameter ( figure ). because silver enhancement found its first biological application in tissue staining in , there are many commercially available silver enhancement solutions designated for microscopy applications (sigma, thermo, ted pella, nano, etc.). the first application of silver enhancement to paper diagnostics was in when horton et al. developed a proof-of-concept lateral flow assay for the detection of mouse igg and demonstrated that silver treatment resulted in a fold improvement in detection limits. since then, silver enhancement has successfully improved the detection of many infectious diseases by several orders of magnitude, including influenza, , hiv, malaria, − y. pestis, and e. coli. one of the drawbacks to silver enhancement on lateral flow assays is that it must be performed after the test has completed its initial development. increasing the total number of steps required reduces the likelihood that the test could be applied in the field. for example, one group found that silver enhancement improved the detection limits of their y. pestis test -fold. in order to take advantage of this impressive enhancement, the authors allowed the gold test to develop as usual, washed the strip three times, submerged it into enhancing reagents for min, and then stopped the reaction with sodium thiosulfate. this procedure is feasible in a district hospital or even a local health outpost with minimal resources, and the enhanced assay would be a true asset in these settings should a plague pandemic occur. however, the additional steps required for signal amplification make poc application in a low-resource field setting unlikely. for this reason, some groups who employ a silver enhancement step in their paper diagnostic assays have moved away from the traditional lateral flow assay format and toward alternative formats that allow for automated delivery of silver enhancement reagents to the test line. fujifilm , applied their expertise in photo technology to develop a benchtop workstation that automated silver enhancement for influenza rapid diagnostic tests. in this format, a user added the sample to a lateral flow cartridge that contained all reagents necessary for test washing and silver enhancement. the cartridge was inserted into the benchtop workstation, which released the solutions in a timed, automated manner and provided a quantitative readout of the strip after test development was completed. despite the fact that the instrument greatly simplified the assay protocol for the user, the requirement of a reliable source of electricity greatly inhibits its application to a field setting. recently, the yager and fu groups have designed dpns that rely on unique paper geometry or embedded paper-fluidic valves to enable programmed, electricity-free delivery of silver enhancement reagents to the assay test line. [ ] [ ] [ ] [ ] [ ] , these dpns simply require the addition of sample and buffer to pads that have been preloaded with assay reagents, mirroring the same number of steps required to run a traditional lateral flow assay. the unique physical structures of dpns allow for precisely timed delivery of sample, then gold conjugate, and finally silver enhancement reagents to the test line. similarly, cho et al. developed a cross-flow chromatography system, in which silver enhancement reagents were embedded into paper and then rehydrated after the lateral flow assay developed. the enhancement reagents flowed perpendicular to the initial immunochromatographic test over the test and control lines. these creative approaches to redesigning the lateral flow assay make silver enhancement the most field-deployable metal-based signal amplification strategy to date, although large-scale manufacturing requirements must be considered to determine the scalability of such test designs. finally, it should be noted that while silver enhancement on gold particles is most common, other metals have been used for reductive nanoparticle enlargement for lateral flow enhancement. gold has been deposited onto gold and silver nanoparticles, , − and silver onto silver and platinum nanoparticles. while there are many other examples of solution and microfluidic assays employing reductive nanoparticle enlargement for visual and electrochemical signal amplification, these formats are less conducive to low-resource disease diagnosis. , , . . . bio-barcodes. the amplification methods discussed thus far increase the number of signaling elements after the initial assay has developed, requiring additional steps post hoc. another amplification strategy is to generate a greater number of detectable molecules midassay, thereby increasing the downstream signal output of the assay. in , the mirkin group demonstrated that protein biomarker targets could be detected indirectly in a highly sensitive manner by employing gold nanoparticles functionalized with dna tags. in this strategy, magnetic microparticles functionalized with targetspecific antibodies were added to a biological sample. after biomarker capture, the supernatant was removed and intermediate detection elements were added to the particles. these detection elements consisted of gold nanoparticles functionalized with a low density of target-specific antibodies and a very high density of double-stranded barcode dna. using an external magnet, the gold nanoparticles that bound the target were separated, and then the barcode dna was dehybridized and detected downstream ( figure ). because the number of barcode tags was much higher than the number of captured biomarkers, the resulting signal was amplified when compared to a direct detection method. one powerful advantage of this modular assay format is it could be easily multiplexed, with distinct barcode tags for each target of interest. further, while the biobarcode method was initially applied to the detection of proteins, it has also been applied to nucleic acid detection. simply replacing the biomarker-specific antibodies with dna complementary to the nucleic acid target allows for detection of dna or rna targets with pcr-like sensitivity. since its development, the biobarcode assay format has been utilized for the detection of infectious diseases such as hepatitis b, hepatitis c, , variola virus, ebola, hiv, , , b. anthracis, and salmonella, as well as c-reactive protein and a variety of cytokines. , despite its promise for sensitive detection, the biobarcode assay format suffers from two major drawbacks: ( ) the high number of user steps required before the barcode tags are released for detection makes it extremely difficult to adapt to a lowresource format, and ( ) low-resource detection of the nucleic acid tags remains a challenge. initial detection strategies for the biobarcode assay were scanometric; the barcode tags were detected in a sandwich hybridization assay on the surface of a glass slide, with gold nanoparticles as the detection elements. signal was further enhanced using reductive silver deposition before the slides were evaluated using image analysis. [ ] [ ] [ ] [ ] , methods such as pcr, , mass spectrometry, and electrical detection have been employed to detect the barcode tags as well. nam et al. , developed a colorimetric assay in which the released gold nanoparticle biobarcode tags were introduced to a second solution of gold nanoparticles functionalized with single-stranded dna complementary to the tags. the hybridization of the biobarcode tags with complementary dna resulted in aggregation of the two gold nanoparticle-dna conjugates, yielding a detectable color change. none of these detection methods can be employed in a field setting; however, it is not difficult to imagine developing a hybridization-based lateral flow assay for barcode tag detection or functionalizing the tags themselves with moieties such as biotin or a flag peptide that could make them readily detectable in a paper diagnostic format. as is evidenced by sections . and . , significant advances have been made in developing metalloenzyme-and metal nanoparticle-based signal amplification strategies for infectious disease diagnosis. however, even with the advancements in metalloenzyme stabilization and highly stable nanoparticlebased approaches, poc assays utilizing signal amplification are still scarce. most of the assays discussed, even those that employ paper substrates or lateral flow formats, require too many user steps before the signal is generated. future work that minimizes user steps, for instance, through use of dpns or vertical flow assay formats, will be critical in translating these signal amplification strategies from laboratory tests to assays that can be run at a primary healthcare facility. moreover, most of the assays discussed in sections . and . were also heavily reliant on benchtop, laboratory-based instrumentation for signal readout. the next section will discuss the various types of field-deployable instrumentation that have been developed in an effort to bring quantitative and sensitive assays, such as those discussed in section , to low-resource settings. traditionally, point-of-care diagnostics such as lateral flow assays have delivered qualitative visual results. although this simplicity is advantageous in the field, the diagnostic sensitivity and specificity can suffer from subjective interpretation and user bias errors. quantitative measurements mitigate these factors, avoiding the data loss associated with qualitative tests and providing insight into important variables such as infection intensities and biomarker expression patterns. in an elimination setting, these parameters allow for robust epidemiological studies into transmission dynamics and intervention efficacy. novel detection strategies that offer highly sensitive and quantitative results, including many of those outlined in section , require instrumentation for signal readout. as a result, the development of appropriate and simple instrumentation will likely become an integral part of the application of diagnostic technologies in low-resource settings. incorporation of detection devices into point-of-care diagnostics effectively eliminates the possibility of "equipment-free" tests (e in assured criteria) and reduces the "affordable" nature of simple tests (a in assured criteria). however, the potential benefits of instrumentation, including improved sensitivity and reduced bias, will outweigh these disadvantages in many use-case scenarios, and the overall cost of instrumentation per test can be reduced when a device is used to evaluate many tests over time. additionally, many instruments incorporate common consumer electronic devices, such as mobile phones and smart phones, thereby reducing the equipment burden. this section outlines some of the devices developed for point-of-care infectious disease diagnosis. table is included at the beginning of section to highlight selected examples of point-of-care instrumentation. a vast majority of the metal-based probes and nanoparticles highlighted in section produce optical signals such as absorbance, fluorescence, or phosphorescence. as a result, a diverse array of instrumentation has been developed to facilitate assay readout for imaging and quantitative purposes. this section provides an overview of efforts to develop portable, affordable, and sensitive instruments for optical detection in low-resource settings. . . . microscopy. conventional microscopy remains the gold standard diagnostic technique for many infectious diseases, including malaria, tuberculosis, schistosomiasis, and intestinal protozoa. to detect these diseases, a variety of microscopic techniques, such as bright field, dark field, and fluorescence microscopy, are used for direct inspection of stained or unstained smears (blood, sputum, urine, etc.). while microscopy can be a useful diagnostic tool, results depend strongly on infection intensities, sample preparation methods, and the training level of the microscopist. these challenges lead to variability that reduces the utility of microscopy-based diagnosis in field settings, especially in low-transmission areas. additionally, microscope instrumentation can be expensive and bulky. to address these drawbacks, many groups are developing portable, affordable, and easy-to-use microscopy tools suitable for low-resource settings. one approach to bringing laboratory-based microscopy tools to low-resource settings involves building simple, portable, and/or miniature microscopes from low-cost materials. while this solution does not address some of the primary disadvantages of microscopy as a poc diagnostic (i.e., the need for skilled microscopists and sample preparation requirements), it does allow for conventional microscope platforms to be deployed in difficult settings at low cost. such devices typically replace high-energy, expensive light sources with leds, reducing the expense and power required for illumination in addition to increasing the light source lifetime. structural components of these microscopes are often three-dimensionally ( d) printed, − and affordable plastic or hybrid lenses and fiber optics can replace expensive and delicate glass lenses. tuberculosis. when samples were evaluated as positive or negative for m. tuberculosis, results from the global focus microscope and a laboratory-grade fluorescence microscope were in agreement for . % of cases. many similarly portable and lightweight microscopes have been developed, including tomographic, holographic, wide-field, and fluorescence microscopes, some impressively weighing in at less than g. , − these microscopes have been applied not only to the detection of tuberculosis , , but also to malaria, soil-transmitted helminths, schistosomiasis, and hymenolepis nana. the foldscope is an example of a low-resource microscope that most embodies the assured criteria. assembled using the principles of origami, the foldscope is constructed from flat paper, copper tape, a button-cell battery, an led, polymer filters, and a ball lens ( figure b ). the entire device costs less than $ , can achieve magnification up to ×, and was shown to be capable of detecting giardia lamblia, leishmania donovani, trypanosoma cruzi, e. coli, and schistosoma hematobium eggs. despite these accomplishments, when the foldscope was applied to s. hematobium detection in a field setting, the diagnostic sensitivity was just . % when compared to conventional light microscopy. this limited sensitivity was likely a result of challenges with physical manipulation of the foldscope when integrated with a smartphone display. in other words, the foldscope failed to satisfy the "u" for user-friendly in the assured criteria for this particular use-case. this example demonstrates the importance of rigorous field evaluation of novel diagnostic devices and indicates that improvements to the foldscope must be made before implementation for case management. recently, mobile phone-based microscopy has emerged as a potential alternative to conventional microscopes for infectious disease diagnostics. phone-based bright-field, fluorescence, and dark-field microscopy devices have been developed, often employing compact, pocket-sized attachments ( figure c ). these devices have been applied to the detection of a myriad of infectious diseases, including malaria, , tuberculosis, schistosomiasis, , human papillomavirus (hpv), soil-transmitted helminths, filarial diseases, , and intestinal protozoa. , repurposing mobile phones is advantageous due to their widespread adoption and growing connectivity in low-resource settings. additionally, the acquisition of digital images could allow for image-processing (locally or remotely), automated diagnoses, and near real-time data transmission. these advantages, coupled with diseasespecific software applications, could potentially decrease the user variability typically associated with microscopy, improving case management and epidemiological studies. one example that leverages these advantages is the loascope, developed by the fletcher group ( figure d ). , , the loascope is a smartphone-based device that combines video microscopy and automated software with onboard quantitative detection based on the movements of filarial parasite loa loa in whole blood. an application downloaded to the smartphone is used for stage control, video capture, image analysis, and data reporting of quantitative l. loa microfilaria densities. detection of l. loa is important because high levels of microfilariae are associated with potentially fatal adverse events after treatment with ivermectin, a drug frequently used in mass drug administration efforts against onchocerciasis and lymphatic filariasis (lf). these adverse events have led to the suspension of mass drug administration campaigns and subsequent major setbacks in onchocerciasis and lf elimination efforts. rapid, point-of-care measurement of l. loa infection intensity could allow for these elimination campaigns to resume. this device was validated in a field setting and yielded results similar to those of thick smears. it was then applied in a large-scale ( , participants) test-and-not-treat approach for onchocerciasis in an area with high prevalence that had not participated in mass drug administration campaigns since due to the prevalence of l. loa. each patient was screened for l. loa burden using the loascope, and those with high counts were not given ivermectin but were treated for l. loa with albendazole. this is a striking example of mobile phone-based microscopy in the field; all loascope measurements were performed by operators with only one hour of training, and the application-specific software significantly decreased the probability of user error by eliminating the need for subjective interpretation of results. in total, the loascope enabled ivermectin delivery to participants that otherwise would not have received treatment for onchocerciasis, demonstrating the incredible impact mobile phone microscopy can have when applied to detection of infectious diseases. despite the fact that many mobile phone-based microscopes lack the analytical and diagnostic sensitivity of conventional microscopes, the loascope is an excellent demonstration that mobile phone microscopy can be highly impactful if applied in an appropriate use-case scenario. because this particular study focused on identifying patients with very high l. loa burdens, it is unclear to what extent this device could detect low-burden infections. generalization of this technology to other filarial diseases will also require further study. additionally, potential interference from other filarial parasites needs to be studied. as work continues in the field of low-resource microscopy, it will be important to develop new tools with specific use-case scenarios in mind. one potential avenue for application-based innovation involves development of low-resource sample preparation methods coupled with novel probes, such as the inorganic complexes and nanoparticles discussed in section . . . . lateral flow analysis. while much progress has been made in the development of rugged, field-applicable microscopy devices, lateral flow assays currently remain the most field-friendly format for infectious disease diagnosis. however, the simple, direct visual readout of lateral flow assays, often cited as one of the primary advantages of this format, is also considered its achilles heel; difficulties in interpretation resulting from visual impairment and operator bias can result in user-to-user variation. after tests are interpreted, maintaining accurate records and communicating test results to local and national health organizations remain substantial challenges. to overcome these challenges, a number of optical readers have been developed and commercialized to provide rapid, objective, and quantitative measurements of signal on lateral flow assays. the underlying instrumentation within these readers can generally be grouped into two categories: ( ) optomechanical scanning and ( ) imaging. for scanning readers, the primary optical components include a light source (led or laser) and photodiode. the specifications of these components are tailored to the specific detection element used in a particular assay. light source power and wavelength are determined by the absorbance or excitation wavelength of the labels employed in each specific test. the light source is rastered along the lateral flow strip, and a photodiode then detects reflectance or emission, depending on whether colorimetric or fluorescent labels are used. measurements are typically recorded with millivolt (mv) units, though biomarker concentration can be obtained if a calibration curve is established. when colored particles are present at the control and test lines, they absorb incident light, resulting in a quantifiable decrease in reflectance. for fluorescent labels, the intensity of emitted light increases as the light source passes over the control and test lines. optics are configured in either a specular or confocal arrangement. in the specular arrangement, which is primarily used for colorimetric labels, the photodiode is positioned such that it collects light that is reflected from the test at the same angle as the incident light from the source. confocal scanning systems can be used to measure reflectance or fluorescence. in this case, incident light is focused to a point on the lateral flow strip, and the photodiode is placed above this focal point. light returning from the strip, whether reflected or emitted, passes through an aperture that excludes rays that were not directly from the focal point, eliminating background scattering. in the case of fluorescent labels, a dichroic mirror or filter is often placed before the aperture. both specular and confocal scanning configurations are sensitive to the z position of the lateral flow strip. in the specular arrangement, any small deviation along the z axis can result in the reflected light completely missing the photodiode due to misalignment. confocal systems are slightly more tolerant to small variations along the z axis, depending on the size of the pinhole aperture. however, even slight variations off the focal plane can drastically decrease the number of photons reaching the photodiode, altering the quantitative output. another challenge associated with confocal and some specular opto-mechanical scanning strip readers is that a point of incident light only covers − % of the test and control lines. this is especially problematic if the concentration of particles varies along the test or control line due to improper flow or manufacturing variations. despite these challenges, scanning systems are advantageous due to their low cost and relative simplicity. one of the most widely used commercial systems is the esequant line of lateral flow readers manufactured by qiagen, inc. ( figure a ). qiagen offers customization of optics for colorimetric, fluorescent, chemiluminescent, and upconverting phosphor labels. the user should take into account that these systems often vary in sensitivity. as a general rule, smaller portable systems rarely perform as well as their benchtop counterparts. for example, van dam et al. found that the portable commercial reader, the ucp-quant (qiagen) , was at least an order of magnitude less sensitive than a custom-modified benchtop reader for evaluating upconverting phosphor-based schistosomiasis lateral flow assays. consequently, selecting a scanning lateral flow strip reader for a particular application requires consideration of both portability and sensitivity. imaging-based lateral flow readers greatly reduce the optical equipment required for quantifying lateral flow assays. these systems produce a picture record of a lateral flow assay using a charge coupled device (ccd) or complementary metal-oxidesemiconductor (cmos) detector and rely on image processing software to detect and/or quantify the intensity of the test line. benefits of this format include the ability to store test images alongside patient records and a lack of moving parts, making imaging-based readers more robust to field conditions. further, image analysis software can identify and correct for nonuniform signal, allowing for analysis of the full test line. imaging-based readers can be used for detection of colorimetric and fluorescent labels. in the latter case, hardware for excitation is required, and filters can be added to improve image quality. since test position and illumination can affect image analysis, many camera-based lateral flow readers include housing units for run-to-run consistency. commercial lateral flow imaging devices include the deki reader (fio), ax- x (axxin inc.), skansmart (skannex), and the rds- pro (detekt biomedical llc) ( figure b ). − these devices have been used for quantification of malaria, − dengue, burkholderia pseudomallei, and y. pestis lateral flow assays, and some, such as the deki reader, have been tested extensively in the field. − image-based lateral flow quantification translates well to mobile phone technology, especially as smartphone camera quality improves. mobile phones have been used to quantify lateral flow assays for numerous diseases, including malaria, − hiv, and tuberculosis. similar to commercially available imaging-based readers, many of these studies required external devices to ensure consistent illumination and distance from the camera in order to maintain consistent image analysis. recently, scherr et al. developed a lateral flow imaging platform using an unmodified mobile phone as the only hardware. relying only on software for image analysis, lateral flow images were captured under ambient lighting conditions. the images were sent to a secure web database (redcap) and analyzed using a matlab-based algorithm. quantitative results were sent back to redcap and delivered to the phone in real time. this software-based imaging platform was tolerant to moderate variations in test orientation, distance, and illumination, making data collection as easy as taking a cell phone picture. when compared to the scanning esequant lateral flow reader, this processing platform was found to have improved detection limits. additionally, the algorithm reliably worked for camera resolutions between . and megapixels, suggesting that any modern mobile phone camera could capture images with sufficient quality for the image processing software to work. as mobile phones are incorporated into low-resource diagnostic workflows, it is important to ask whether the conventional lateral flow format can be redesigned to take full advantage of potential benefits smartphones can offer. in other words, what changes can be made to the diagnostic test format that maintain the simplicity of lateral flow assays but will enable improved quantification, communication, and aggregation of test results? one option is to embed quick response (qr) codes into the lateral flow test. this idea has been approached in a number of ways, including placing stickers on lateral flow tests and adding a transparent overlay in which the test and control lines become part of the qr code. , in these formats, the embedded barcode is either static, providing useful information about the brand, lot, and patient id, or dynamic, producing a positive/negative read-out. scherr et al. developed a novel platform in which the control line of the lateral flow assay was replaced with a control grid patterned in the shape of a qr code ( figure c ). the qr code functioned as a flow control, appearing on both positive and negative tests, but not invalid tests. the qr codes also triggered data analysis and enabled corrections for lighting effects. when scanned, manufacturing details were quickly and easily accessed at the same time as the test line was quantified. it is not difficult to imagine the impact such a test format could have when combined with the data communication and aggregation abilities of smartphones. epidemiological and surveillance studies could account for variables that are not even currently considered in most studies, such as manufacturing batch-to-batch variation. geographic data could also be coupled to specific rapid tests. as tools for automated reading of lateral flow assays become more prevalent, establishing standards for consistent reporting and data management will be important. just like with the lateral flow assays themselves, test-reading instrumentation will need to be validated to ensure proper and consistent biomarker quantification across different devices and test brands. although optical lateral flow readers may detect test lines invisible to the human eye, readers alone will not replace the need for ultrasensitive lateral flow tests. . . . other smartphone-enabled optical measurements. mobile phones have been adapted for optical measurements beyond lateral flow quantification. one example is the mobile phone-based, hand-held microplate reader developed by berg et al. this device consists of a dprinted attachment in which an led array illuminates each well, and the transmitted light is collected via individual optical fibers. results are visualized and delivered to the user in one minute within a custom mobile phone app. the device was shown to be more than % accurate for immunoassays detecting mumps igg, measles igg, and herpes simplex virus igg (hsv- and hsv- ) when compared to an fda-approved clinical spectrophotometer. while microplate-based immunoassays are not feasible in a field setting, this device could make a true impact in resource-limited clinics as an excellent alternative to traditional bulky and expensive spectrophotometers. additionally, because the device is attached to a mobile phone, it offers the potential to rapidly communicate and aggregate results, further enabling large-scale epidemiological studies. mobile phone attachments for other optical detection modalities have been developed as well. in one exciting example, long et al. developed the tri-analyzer, a trimodal smartphone attachment capable of measuring transmission, reflection, and emission intensities. this multifunctional device leveraged white light from the smartphone's rear-facing flash led or an integrated green laser diode for sample illumination via optical fibers. the optical fibers collected light transmitted through or emitted from the sample or light reflected from a photonic crystal biosensor. this collected light was then collimated and focused before being dispersed by the diffraction grating onto the smartphone cmos sensor. the intensity at each wavelength was determined by the integration of pixel intensity at the corresponding position along the sensor. this clever detection strategy allowed for full absorbance and emission spectra to be collected with a simple smartphone-based device. in two proof-of-concept assays, the developed device could detect analytes at lower concentrations than those detected using conventional laboratory instrumentation, indicating that the miniaturization of the technology did not result in a trade-off of sensitivity. thus, this trimodal smartphone attachment enables quantitative laboratory-quality optical measurements at one's fingertips. similarly formatted mobile phone-based spectrophotometers, , surface plasmon resonance sensors, − and flow cytometers , have also been developed. the miniaturization and decrease in cost associated with these smartphone-enabled tools will make chemical reviews laboratory techniques possible that would otherwise be inaccessible in low-resource settings. noble metal nanoparticles have extremely high molar absorptivities, making them ideal visual labels for lateral flow assays. this intense absorbance occurs when particles are excited with light that matches the resonant oscillation of their free electrons. as these electrons relax down to the ground state, they release energy in the form of heat to the surroundings. the amount of heat generated is equal to the product of the number of noble metal nanoparticles, the cross-sectional area of the nanoparticles, and the intensity of the incident light. recently, bischof's group took advantage of these unique thermophysical properties to develop a novel method for lateral flow quantification based on the thermal contrast of gold nanoparticles. − in this method, a highpowered green laser was used to excite the gold nanoparticles, and an infrared-detecting camera was employed to measure the resulting thermal contrast. an early benchtop prototype of the device demonstrated a -fold improvement in detection limits of an fda approved lateral flow assay for cryptococcal capsular polysaccharide glucuronoxylomannan antigen (crag). crag is a biomarker for cryptococcal infections, which are opportunistic fungal infections that are among the leading causes of death for patients with aids and a common cause of adult meningitis in sub-saharan africa. the thermal contrast-coupled crag lateral flow assay was further validated in a second study, displaying % accuracy in quantifying crag levels in patient samples. after successful validation of their prototype device, the group developed a reader that incorporated a green laser, infrared camera, and automated control system into a benchtop box device ( figure ). the study demonstrated the utility and versatility of thermal contrast as a method for quantification of lateral flow assays from a variety of vendors and for multiple diseases. an -fold improvement in detection threshold compared to visual inspection was found for lateral flow assays for influenza (bd veritor), malaria (first response, sd bioline, and parahit), and clostridium difficile (ridaquick). in a separate study, the group found that this enhancement factor was greater as nanoparticle size increased, suggesting that redesign of lateral flow assays could further improve test sensitivity using a thermal contrast reader. while thermal contrast represents a unique and versatile method for lateral flow quantification, several issues must be addressed before large-scale implementation. these include cost, portability, and background signal reduction from biological interferences such as blood. additionally, nitrocellulose membranes are highly flammable; while it does not appear that full strip ignition has occurred in the currently published studies, visible burn marks along the nitrocellulose membrane suggest that safety studies to determine the limits of the thermal contrast system should be undertaken before widespread implementation. nevertheless, thermal contrast represents a highly sensitive and promising tool for lateral flow quantification. surface-enhanced raman spectroscopy (sers) is a highly sensitive vibrational spectroscopy technique for structurebased analyte detection. in this technique, excited localized surface plasmons, typically from noble metal (e.g., gold and silver) substrates with nanoscale features, generate amplified electromagnetic fields, enhancing the inelastic raman scattering signals of analytes of interest up to times. instrumentation for sers detection requires a high-powered laser excitation source, which is typically tuned according to the resonant frequency of the plasmonic substrate. after excitation, filters are used to exclude elastic rayleigh scattering, allowing only raman-scattered light to reach the detector. the high sensitivity of sers makes it an attractive detection platform for infectious disease diagnostics, particularly for the detection of low-density infections. many laboratory-based bioassays for protein, nucleic acid, and whole-cell biomarkers that leverage sers detection have been developed for a myriad of infectious diseases. , − these assays are generally either performed on the surface of particles in solution or on a bulk solid substrate. detection elements typically consist of nanoparticles functionalized with raman reporter molecules and target-specific molecular recognition elements. once a complex is formed, the nanoparticles provide the plasmonic surface for raman signal enhancement of the reporter molecule. nanoparticles are typically gold, silver, or au@ag particles and come in a variety of shapes that provide optimized signal enhancement. small-molecule raman reporters, such as -mercaptobenzoic acid, malachite green, and -aminothiophenol, are often employed because biomolecule targets typically have complex vibrational signatures and are frequently too far away from the plasmonic surface for sensitive detection and identification. the bulky nature and resource requirements of sers detection remain its primary barrier to implementation in point-of-care settings. several hand-held raman spectrometers have been developed and commercialized, including the firstdefender (thermofisher scientific, inc.), , nano-ram (b&w tek), , and progeny (rigaku corp.). these devices are advertised for identification of unknown substances in forensic, environmental, military, and first-response contexts and for quality control in industrial settings. while many studies that utilize sers-based detection for infectious disease biomarkers suggest that these commercially available portable devices could be utilized for analysis of a sers-based lateral flow assay, none of the studies have actually implemented a hand-held device. − accordingly, it is unknown whether such devices have sufficient sensitivity or spatial resolution to use effectively with low-resource diagnostics. these parameters, along with cost analysis, should be studied to determine whether current commercial devices could be implemented. magnetic nanoparticles possess advantages over optical lateral flow labels because their signal is rarely hindered by test opacity or biological interferents. a detailed discussion of these qualities as well as examples of specific assays that employ magnetic nanoparticles are provided in section . . . here, we discuss instrumentation required for magnetic particle detection. there are a variety of methods for detecting paramagnetic and superparamagnetic nanoparticles on lateral flow assays based on either the inductive effect or magnetoresistance. for example, magnabiosciences (usa) has developed an induction-based device that has been used extensively in the literature for detection of infectious diseases such as anthrax, , t. solium, hiv, , v. parahemolyticus, and l. monocytogenes. in this format, the lateral flow assay is placed inside an oscillating magnetic field on a set of induction coils, and the instrument measures the induced electromotive force (v) on the coils. assays quantified by this technology generally had a greater sensitivity compared to visual detection of iron oxide particles. while the current iteration of the magnabiosciences reader is a relatively large benchtop device that is unsuitable for use in low-resource settings, a more portable version is being developed. , other companies, such as magnasense (finland) and magnisense (france), have developed portable, hand-held devices based on inductive detection of paramagnetic particles, though these devices have not been as extensively tested in the literature. several groups have developed lateral flow assay readers using magnetoresistive sensors. these devices are based on the principle that the electrical resistance of certain materials, which are incorporated in the sensors, can change upon application of an external magnetic field. there are several types of sensors based on magnetoresistance, but the most common are giant magnetoresistive (gmr) sensors, which were once used in the reading heads for hard disk drives. when applied to lateral flow assays, gmr sensors are placed in close proximity to the test and control lines, and an external magnetic field is applied to the test. this external field aligns the magnetic moments of paramagnetic or superparamagnetic lateral flow detection reporters. this alignment then produces a fringe field that is detectable by a change in resistance when a constant current is applied across the gmr sensor. magnetoresistive sensors have been applied extensively by researchers to the detection of magnetic particle labels on lateral flow assays; however, most have been proof-of-principle studies using imitative lateral flow assays or tests for biomarkers such as hcg, cardiac troponin , and ifnγ. , − the high sensitivity of magnetic detection on lateral flow assays makes this technique attractive for infectious disease diagnosis. while magnetic detection necessitates instrumentation for result readout, iron-containing magnetic nanoparticles also have the added advantage of being highly absorbent visual labels. thus, magnetic nanoparticles can potentially be applied to different use-case scenarios with differing sensitivity requirements. in a use-case scenario such as morbidity control, where it is most important to identify high-intensity infections in the field, the visual detection of magnetic particles would be sufficient. when improved sensitivity is desired to detect lowintensity infections for epidemiological or surveillance purposes, analysis using the current instrumentation for reading magnetic nanoparticle labels could be performed in a district hospital or laboratory. however, further work on miniaturization of instrumentation and validation of hand-held readers will be required for field application of magnetic detection on lateral flow assays. as discussed in previous sections, electrochemical detection offers several benefits for poc infectious disease detection, namely that it is inexpensive, sensitive, rapid, and quantitative. the electrochemical detection systems previously discussed were largely proof-of-concept diagnostics for detection of protein or nucleic acid biomarkers; the infrastructure required to carry out these assays would limit their utility in a primary healthcare setting in low-and middle-income countries. integration of validated and field-ready electrochemical detection systems with developed electrochemical assays represents a viable strategy for implementing the next generation of poc diagnostics. the blood glucose biosensor is a remarkably successful electrochemical device that is ubiquitously applied at the point of care. generally, electrochemical glucose meter kits consist of three components: a lancet for sample collection, plastic test strips for wicking of the glucose-containing sample and generation of current, and a device for digital readout of the generated current. the test strips house an electrochemical cell that contains a counter-reference electrode, fill-detection electrode, enzyme-coated working electrode, and mediators ( figure a ). in these devices, the working electrode is coated with the enzyme glucose oxidase (gox) or glucose dehydrogenase (gdh), and the enzyme oxidizes the glucose present in a blood sample. ferrocene derivatives or other group viii metal complexes act as mediators to facilitate electron transfer from the enzyme redox center to the electrode surface, thereby producing a measurable current. since the development of the first commercial blood glucose biosensor in , the use of this technology has drastically expanded with the current market now offering numerous blood glucose meters that are portable, rapid, sensitive, and quantitative. the blood glucose biosensor has had an enormous impact on management of types i and ii diabetes where constant monitoring and a rapid time to result are critical to preventing incidences of hyper-and hypoglycemia. successful adaptation of this technology for field detection of infectious diseases would have a profound impact in disease surveillance and elimination campaigns, where sensitive and quantitative results are desired to best inform healthcare professionals about disease distribution. , several groups have repurposed commercial blood glucose meters for detection of a variety of targets. , − xiang and lu developed a platform that utilized a personal glucose meter for signal output to detect protein (ifn-γ), nucleic acid (adenosine), small molecule (cocaine), or toxic ion (uranium) targets. their strategy employed a magnetic bead-conjugated aptamer specific for the biomarker of interest. the aptamer was initially prehybridized to a partially complementary dna strand that was linked to invertase, an enzyme that catalyzes the hydrolysis of sucrose into its fructose and glucose subunits. in biomarker-containing samples, the aptamer bound to the biomarker, releasing the invertase-conjugated complementary dna into solution. the magnetic beads were then removed from solution using an external magnet, and the addition of sucrose to the supernatant resulted in glucose production that was readily detectable by a personal glucose meter. while this creative application of an extant platform expanded its functionality, it also increased the number of assay steps, potentially limiting its use directly at the point of care. to address this concern, the lu group has since integrated this solution-based assay into a lateral flow strip with dried reagents, eliminating several user steps and further simplifying the platform such that it could be applied at the point of care. this innovative approach is an excellent example of how metalbased sample preparation using magnetic beads can be combined with an existing electrochemical platform for rapid detection of a variety of disease targets. as the blood glucose meter clearly demonstrates, portable electrochemical instrumentation has enabled minimally trained users to measure analytes at the point of care and rapidly obtain information regarding their health status. mobile health technologies (i.e., mobile phones and telemedicine) augment these advances in instrumentation, connecting individual users to an entire healthcare infrastructure. mobile health technologies are useful for tracking patient information and facilitating communication between the patient and the care provider. as these technologies mature, they are becoming increasingly important for modeling diseases and disease trends throughout various populations. integration of portable electrochemical diagnostics with mobile health technologies in so-called "connected" diagnostics has been pursued by interfacing electrochemical devices with mobile phones. , nemiroski and colleagues developed a universal mobile electrochemical detector (umed) that combined a potentiostat with virtually any mobile phone using audio-based data transmission ( figure b ). the device could accommodate several electrochemical techniques (amperometry, coulometry, voltammetry, and potentiometry), and the voice system-based data ensured broad compatibility with a variety of mobile phones and networks. the authors employed the umed to detect blood glucose, heavy metals in water, electrolytes, and malarial biomarker hrp . following sample collection and measurement using the potentiostat, the user placed a phone call to a skype number in a remote location to vocally report the value of the analyte measured. the remote application then extracted the value from the audio data and sent an sms message back to the user with additional diagnostic information (e.g., "low" if the reported blood glucose level was low). although the umed was applied to detect an infectious disease biomarker (hrp ), it is limited by the availability of existing compatible testing technology. for instance, hrp detection required an offline -well plate elisa to be conducted prior to analysis on the umed and transmission over a mobile network. as more progress is made in electrochemical diagnostic design, the umed and other similar devices will be able to reach their full potential as "connected" diagnostics. as battery-operated and portable devices become more robust, electrochemical detection devices are becoming increasingly viable as poc platforms. the success of the personal glucose meter is a testament to the effective and reliable diagnostic performance that is possible with electrochemical measurements. with the concurrent expansion of information technologies, "connected" electrochemical diagnostics could serve as the link between the chemistries utilized at the point of care and the remote higher level healthcare systems that map disease trends and inform treatment decisions. in high-income countries, advances in portable electronics and instrumentation have enabled live health tracking at consumers' fingertips. wearable commercial health diagnostics such as apple watch, garmin, and fitbit are simple to use and provide real-time updates of heart rate, activities completed, or calories burned. in a similar vein, there are several emerging healthcentered tools, such as press-on tattoos, , skin patches, and face masks, capable of detecting changes in human physiology at the point of use. in addition, the advent of technologies such as google glass have allowed for "connected" interpretation of rapid diagnostic tests. overall, wearable instruments combine sample collection, sample preparation, analysis and readout, and data transmission into one device, representing significant progress in diagnosis and health management at the point of care. small electrochemical instrumentation has become a focus of innovation, and research in this field has led to a number of skin-based wearable diagnostics. jia et al. detected glucose in the interstitial fluid of the skin ( figure a ). these technologies are extraordinarily promising for future diagnostic devices, particularly as new biomarkers of infectious diseases are discovered in surface biofluids such as sweat. recent advances in microprojection , and microneedle , technology have allowed researchers to expand the use of skin-based wearables beneath the surface of skin. in several applications, patches consisting of many microprojections were easily applied to the skin with a gentle press and could draw hundreds of microliters of blood with little-to-no pain. , − in , lee et al. outlined the design of a multiplexed patch that captured both igg and malarial biomarker hrp from the skin of inoculated mice. the patch consisted of microprojection arrays (mpas) that were subsequently functionalized with anti-hrp antibodies, fc-specific antimouse igg antibodies, and antidengue antibodies. the anti-hrp -functionalized mpas detected the biomarker, while the anti-igg-functionalized mpas acted as a positive control for skin penetration, and the antidenguefunctionalized mpa served as a negative control. though sample collection and preparation were applicable at the point of use, the assay required an elisa for colorimetric readout of the captured antigen. further research into integration of this technology with a diagnostic such as a stacked paper-based assay would allow for real-time skin-based monitoring of infectious diseases. another forthcoming application for wearable instrumentation is in breath analysis via paper masks. guder et al. developed an electrical sensor to monitor respiration. the sensor consisted of a paper surgical mask printed with graphite electrodes that were attached to a battery and signal amplifier. as a user breathed, the humidity changes from the inhalation and exhalation generated a measurable current across the electrode, which was then detected using a bluetoothconnected mobile device ( figure b ). as this technology evolves, it may be possible to adapt it for analysis of breathbased biomarkers, particularly for respiratory infectious diseases such as tuberculosis and influenza. in addition, incorporating the external battery/amplifier pack into the material of the mask may open avenues for broader distribution, particularly to low-resource areas. likewise, personal computer displays such as google glass have enabled users to harness the power of a technological workstation in a hands-free glasses format. this capacity could significantly improve diagnostic capabilities and effectiveness. in , feng et al. developed a method of lateral flow assay analysis which combined visual examination of a lateral flow test with computerized image analysis and wireless storage capability ( figure c ). this wearable imaging technique allowed users to collect images of tests and store them in a cloud-based system or locally on the google glass headset if internet access was unavailable. once the google glass headset was connected to a wireless network, the test images could be sent to a secure server to be analyzed, and then the test's results and analysis were sent back to the viewer's eyepiece. this method has been demonstrated using rapid diagnostic tests for both hiv antibodies and prostate specific antigen (psa), though it has the potential to be adapted for any lateral flow assay with visual labels. while the use of computer-based imaging may remove some of the inaccuracies that arise from subjective human-based interpretation of results, it must also be noted that there are a number of technological hurdles that should first be addressed. the google glass is not capable of autofocusing, and the images were taken under good lighting against white and universally solid, simple backdrops. thus, this technology is limited in its field application. in addition, test recognition was dependent on preprinted qr codes that were prepared and attached to the tests by researchers before use. in order to more efficiently utilize this technology, the google glass application could be adapted to identify test features without the need for a usersupplied qr code and web-based test input. this would simplify the workflow and allow for more high-throughput analysis of diagnostic tests. overall, there is significant room for growth and development in the field of wearable instrumentation. wearable diagnostics, which may have previously seemed more at home in the world of science fiction, are becoming commonplace in health-related research efforts. the development of wearable diagnostic technologies could drastically improve the simplicity of diagnostic measurements and interpretation in regions with little or no laboratory infrastructure. as these technologies continue to be developed for applications in high-resource areas of the world, the scientific community would be remiss not to consider the impact they could have on infectious disease diagnosis in resource-limited settings. inorganic chemistry for nucleic acid detection thus far, we have highlighted innovative examples of metalbased strategies for improving point-of-care diagnosis of infectious diseases based primarily on protein and inorganic disease biomarkers. in most cases, leveraging inorganic chemistry for sample preparation methods and detection probes would require minimal modification for application at the point of care. it is clear that, despite the potential trade-offs in complexity of workflow and instrumentation, the increased sensitivity offered by these metal-based strategies for biomarker detection could significantly improve morbidity control and elimination efforts. in many instances, though, sensitive and quantitative detection of the presence or absence of a disease does not paint the full picture needed to implement successful control or elimination campaigns. in some cases, the pathogen's own genetic material must be detected and extensively studied on a population scale in order to inform strategy. this research provides valuable information on pathogen species and strain distribution and drug resistance, which can inform treatment and diagnostic strategies in a control or elimination campaign. the information-rich and highly specific nature of nucleic acid detection makes these biomarkers attractive targets for point-of-care diagnostics. in fact, several groups have begun to apply some of the metal-based detection strategies for proteins and inorganic biomarkers discussed in the preceding sections t o t h e d e t e c t i o n o f p a t h o g e n i c d n a o r rna. , , , , − for instance, many metal-containing nanoparticle probes have been functionalized to detect pathogen-specific nucleic acid sequences in a variety of formats. the size-dependence of spr for noble-metal nanoparticles has been leveraged in an aggregation assay for multiplexed detection of middle east respiratory syndrome coronavirus, m. tuberculosis, and hpv in a μpad format. the high conductivity of agnps has been exploited for electrochemical detection of m. tuberculosis and hiv. , luminescent particles (i.e., quantum dots, europium chelatedoped particles, and upconverting phosphors) have been functionalized for the detection of a myriad of diseases, including hiv, , − hbv, hcv, hpv, p. falciparum malaria, bacillus cereus, , , , and streptococcus pyogenes. moreover, these nanoparticle-based methods occur in a variety of formats, including lateral flow assays. because specific nucleic acid sequences are typically present at much lower concentrations than protein biomarkers in patient samples, a target amplification step is often required before the detection method can be applied. the most common target amplification technique is pcr, a timeconsuming enzyme-based method that requires technical expertise, expensive equipment, and significant laboratory infrastructure. these requirements are the primary barriers to sensitive detection of pathogen genetic material in the field. fortunately, alternative target amplification techniques with potential point-of-care applications have emerged. for example, the biobarcode signal amplification strategy discussed in section . . has been adapted for amplification of nucleic acid targets. however, in general, isothermal enzymatic amplification techniques have demonstrated the most sensitivity and promise for point-of-care diagnostic applications. isothermal amplification techniques do not require any thermal cycling, are simpler to operate, and require fewer resources when compared to traditional pcr. this class of enzymatic amplification techniques includes loop-mediated isothermal amplification (lamp), helicase-dependent amplification (hda), rolling circle amplification (rca), multiple displacement amplification (mda), recombinase polymerase amplification (rpa), and nucleic acid sequence-based amplification (nasba). among these strategies, lamp is one of the most widely validated, having been employed for amplification of nucleic acid targets for a variety of diseases and infections. these include targets for malaria, − hiv, hpv, bordetella pertussis, c. trachomatis, , neisserie gonorrheae, h n influenza, , e. coli, and several filarial parasites. in addition to malaria diagnosis, lamp has also been used to detect artemisinin resistance in p. falciparum malaria. this technique consists of iterative elongation and recycling of stem-loop dna structures, usually requiring a constant temperature between and °c. the relatively simple workflow of lamp (when compared to other enzymatic amplification techniques) and its potential for colorimetric visual readout have led to the development of several low-resource devices for nucleic acid detection. one example is the noninstrumented nucleic acid amplification device (nina), which relies on an exothermal chemical reaction (mgfe powder, saline, and oxygen) coupled to a phase change material for prolonged and precise heating inside a thermos cup. , despite its streamlined workflow, the nina device does not account for sample preparation. the preamplification nucleic acid extraction step still requires laboratory equipment such as a heated water bath and a centrifuge. although the amplification step itself has been modified for use at the point of care, the complete protocol relegates it to the laboratory environment. in an effort to mitigate these limitations, the klapperich group has developed an inventive method in which nucleic acid extraction, amplification, and detection are fully integrated into a single paper fluidic device. to accomplish this, dna from clinical samples was precipitated from solution and applied to a paper membrane, which was washed with ethanol to remove impurities. lamp reaction reagents, which included fitc-conjugated forward and biotinylated reverse loop primers, were then applied to the sample port, which was sealed for a min heating step on a hot plate. the amplified solution was then wicked onto a lateral flow assay equipped with anti-fitc antibodies on the test line and streptavidincoated aunps for detection ( figure ). in the presence of the target, both primers hybridized to the amplified genetic material, forming a "sandwich" on the lateral flow assay and resulting in a visible test line signal. this all-in-one paper-based nucleic acid test is a perfect example of how target extraction and amplification can be innovatively combined with metal-based detection elements. this integration will allow for complex nucleic acid detection protocols to be performed at the point of care that previously were only possible in well-equipped laboratories. moving forward, several considerations must be addressed, including integration of laboratory-free heating elements, reducing the number of user steps, and large-scale manufacturing feasibility. nonetheless, combination of the paper-based devices developed by the klapperich group with simple, integrated heating elements similar to the nina device could allow for sensitive and information-rich nucleic acid detection in low resource settings. this would enable case management and elimination strategies to be precisely carried out in nearly any healthcare setting. the application of inorganic chemistry and nanomaterials to point-of-care infectious disease diagnosis has enhanced the sensitivity and specificity of diagnostic tests, leading to improved outcomes and reduced transmission. the sample preparation and detection methods presented herein are promising examples of how metal-based strategies can expand the use-case scenarios of diagnostic tests, resulting in access to care that would otherwise be unattainable. however, we have also seen that these same metal-based strategies that improve review diagnostic sensitivity and specificity can lead to more complex tests, potentially requiring more user steps, expertise, and time to complete. these trade-offs of complexity, cost, and speed must be weighed against the potential impact that improved sensitivity and specificity could have in a given use-case scenario. for example, in the case of morbidity control, where the goal is to reduce the prevalence of high-intensity infections, the demand for simple, affordable, and rapid tests likely outweighs the need for highly sensitive tests. once in the elimination phase, however, where the goal is to completely interrupt local transmission, high sensitivity tests are imperative for detecting and treating low-density infections. consequently, the need for more sensitive diagnostics likely outweighs small increases in test complexity or cost for elimination campaigns. in our evaluation of various inorganic complexes and nanomaterials, advantages and caveats became apparent for different classes of materials. the properties of these materials directly affect how they can be applied in diagnostics. noble metal nanoparticles have frequently been incorporated into paper-based assays, such as lateral flow assays, with visual signal readout because of their stability and superior molar absorptivities. , , , despite the strong optical signal that noble metal nanoparticles afford, these colorimetric assays are often still limited by poor sensitivity. this potentially limits their impact in a use-case scenario such as an elimination campaign. if better sensitivity is needed, there are essentially three metal-based strategies that we have discussed in this review for further improvement: ( ) sample preparation for biomarker enrichment, ( ) luminescent detection probes, and ( ) signal amplification techniques. the imac-functionalized materials used for sample preparation collectively represent one approach for improving assay sensitivity by enriching the biomarker concentration. while some of the sample preparation methods demonstrated increased diagnostic sensitivity, these methods did add to the number of user steps. , , [ ] [ ] [ ] metal-based sample preparation is perhaps the simplest of the three metal-based approaches to increasing sensitivity; however, for it to be truly impactful, it will need to be incorporated into diagnostics that are developed with the end user in mind such that the overall workflow is considered and the assay steps are minimized. alternatively, noble metal nanoparticles can be replaced with luminescent detection probes to afford greater sensitivity. quantum dots, lanthanide chelate-doped nanoparticles, and up-converting phosphor nanoparticles all potentially offer improvements in sensitivity over colorimetric labels, albeit through different mechanisms. , , , while luminescent inorganic complexes were incorporated into proof-of-principle assays for the detection of infectious disease biomarkers, they have yet to be utilized in a field-deployable format and lack the sensitivity of the aforementioned nanomaterials. , quantum dots and up-converting phosphor nanoparticles also have the capability for multiplexing that could be beneficial for case management. , , although the various luminescent nanoparticles offer improvements in sensitivity, they also require more instrumentation for signal readout. the increased cost and complexity of signal readout for luminescent nanoparticles must therefore be considered when developing diagnostic strategies to combat infectious diseases. lastly, metal-based signal amplification techniques could be incorporated to improve sensitivity. almost all of the signal amplification techniques discussed in this review (metalloenzyme-based amplification, nanoparticle dissolution, nanoparticle enzyme mimics, reductive nanoparticle enlargement, and biobarcodes) offer colorimetric readouts with improved sensitivity. however, these improvements come at the cost of an increased number of assay steps and thus longer total assay times. while some efforts, such as the p lfas developed by loynachan et al., demonstrated progress toward more fieldfriendly signal amplification methods, further improvements in the ease of use and time to result must be made to translate these methods to primary healthcare facilities. for these metal-based strategies that improve sensitivity, it is clear that simplifying the user interface is a significant challenge that remains. aside from the user interface, there are several other challenges to translating novel diagnostic strategies that must be considered before implementation. these include: ( ) the need for clinical validation, ( ) the potential for large-scale manufacturability, ( ) the availability of distribution channels, figure . workflow for detecting nucleic acids on a paper fluidic device. different dyes were used to illustrate the various components of the assay. (a) the dna-containing sample (blue dye) is added to the sample port, where it wicks down the test by capillary action (b). the test is then washed twice with ethanol (yellow), which also flows down the test (c−f). (g) the absorbent pad is then removed and discarded. (h) the lamp reaction mix is then added to the sample port, and the test is folded to protect the sample/lamp mixture from evaporation. (i) once the test has been heated on a hot plate for min, the hydrophobic barrier is removed so that the sample port is exposed on top and bottom, allowing it to come into contact with the lfd strip for dna elution. (j) the sample, once eluted, can then wick down the length of the test strip to generate a signal. adapted with permission from ref . copyright royal society of chemistry. review and ( ) integration into current health systems. most of the metal-based strategies for improved infectious disease diagnostics presented in this review have demonstrated some level of feasibility in a small set of patient or mock clinical samples. however, new technology must be clinically validated on a large set of patient samples from endemic regions before implementation. such studies provide insight into the diagnostic performance and tolerance of day-to-day and interpatient variation in the appropriate clinical context. this rigor can draw attention to challenges and weaknesses that would otherwise go unnoticed in a research laboratory setting. production scalability of novel diagnostic technology is another important consideration for successful translation. even in the research phase of diagnostic development, material choice, device design, and fabrication should be based, in part, on manufacturing scalability in order to streamline implementation. many of the metal-based strategies presented in this review, particularly the diverse inorganic nanoparticle detection probes used in lateral flow assays, are advantageous in this context because they are easily integrated into existing lfa manufacturing infrastructure. beyond the laboratory, it is important to consider distribution channels and how novel tools will be integrated into existing health systems. region-specific health delivery systems and their respective regulatory approvals are ultimately what define a novel diagnostic device's implementation. for example, in one region, most rapid testing for infectious diseases may be performed at local health clinics run by a national ministry of health, whereas in another area, privately run dispensaries may be the most common source of primary health care. understanding the structural differences between such health delivery systems as well as the respective availability of resources could impact device design in the research phase. although there are many considerations and barriers to translating novel technologies to the clinical setting, we are optimistic that the continued development of metal-based point-of-care diagnostic strategies will have a profound impact on global health. returning to the example presented in the introduction, if some of the ultrasensitive p detection methods presented in this review , were adapted for use at primary healthcare facilities, the time to result for infant hiv diagnosis at the point of need would be significantly reduced. in areas like burkina faso, this would enable earlier initiation of very effective antiretroviral therapies and drastically reduce infant mortality stemming from opportunistic infections. finally, the continually increasing connectivity in low-and middle-income countries coupled to sensitive diagnostics will allow for real-time disease surveillance, intervention analysis, and epidemiological studies. "connected" diagnostics have the potential to accelerate communications between field workers, local health outposts, district hospitals, and national health ministries. the ability to rapidly convey diagnostic results could not only enable active case detection strategies but also would provide faster responses to epidemics such as the − ebola and − zika outbreaks. these faster responses could potentially reduce mortality and improve clinical outcomes. metal-based strategies for sample preparation and signal generation show great promise for use in the development and improvement of sensitive diagnostic devices for low-resource settings. these devices, coupled with connected, fielddeployable instrumentation, will empower rapid response, effective control, and successful elimination of infectious diseases worldwide, ushering in an era of sustainable and improved global health. ( ) and an american association for the advancement of science fellow ( ). for the past decade, he has unraveled the mechanism of heme detoxification within the parasite plasmodium falciparum, the causative agent of malaria. additionally, he has made important contributions in understanding hemozoin's immunomodulatory activity in parasite-host interactions, the mechanism of action for antimalarial compounds, and the development of biomarkers for low-resource diagnostics. more recent efforts have focused on the challenges of innovation in low-resource diagnostics. he has focused primarily on malaria biomarkers and new approaches to formatting and improving performance of diagnostics for the detection of asymptomatic patients, drug-resistant infections, and drug-sensitive patients. his lab works in the field in macha, zambia; cape town, south africa; and san marcos, peru. this work is supported by the national institutes of health (r ai ; r tw - ), nih/fogarty international center (d tw ), the bill and melinda gates foundation (opp ), and vanderbilt university through the laboratories for innovations in global health technologies. c.f.m. and k.a.r. acknowledge support from the national science foundation graduate research fellowship program (dge- ). we gratefully acknowledge shellie richards for her tireless proofreading efforts that sharpened the manuscript. aequanimitas, with other addresses to medical students trends in infectious disease mortality in the united states during the th century convergence of non-communicable and infectious diseases in lowand middle-income countries uneven progress in preventing and treating hiv infections worldwide disease eradication, elimination and control: the need for accurate and consistent usage the principles of disease elimination and eradication lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. a literature survey no. e . 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circulating protein extravasation for enhanced biomarker capture and detection a three-dimensional and bevel-angled ultrahigh aspect ratio microneedle for minimally invasive and painless blood sampling in-depth interviews to understand the feasibility of using the mlab app for promotion of hiv-self testing in young men esequant lateral flow system technical flyer towards a genomics-informed, real-time, global pathogen surveillance system single quantum dot-based nanosensor for multiple dna detection development of a microfluidic device for detection of pathogens in oral samples using upconverting phosphor technology (upt) an integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids development of a generic microfluidic device for simultaneous detection of antibodies and nucleic acids in oral fluids use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: a rapid, sensitive dna test to identify human papillomavirus type infection improved assay to detect plasmodium falciparum using an uninterrupted, semi-nested pcr and quantitative lateral flow analysis a disposable microfluidic cassette for dna amplification and detection a portable analyzer for pouch-actuated, immunoassay cassettes isothermal amplification methods for the detection of nucleic acids in microfluidic devices evaluation of non-instrumented nucleic acid amplification by loop-mediated isothermal amplification (nina-lamp) for the diagnosis of malaria in northwest ethiopia high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (htlamp) assay for malaria elimination evaluation of the illumigene malaria lamp: a robust molecular diagnostic tool for malaria parasites nina-lamp compared to microscopy, rdt, and nested pcr for the detection of imported malaria electricity-free amplification device for detection of hiv- a fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics paper-based molecular diagnostic for chlamydia trachomatis paper-based rna extraction, in situ isothermal amplification, and lateral flow detection for low-cost, rapid diagnosis of influenza a (h n ) from clinical specimens paper machine" for molecular diagnostics colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (nina-lamp) a novel snp-lamp assay for detection of artemisinin-resistant plasmodium falciparum malaria noninstrumented nucleic acid amplification assay. in microfluidics, biomems, and medical microsystems vi key: cord- -i kx n m authors: raison, charles l; miller, andrew h title: pathogen–host defense in the evolution of depression: insights into epidemiology, genetics, bioregional differences and female preponderance date: - - journal: neuropsychopharmacology doi: . /npp. . sha: doc_id: cord_uid: i kx n m significant attention has been paid to the potential adaptive value of depression as it relates to interactions with people in the social world. however, in this review, we outline the rationale of why certain features of depression including its environmental and genetic risk factors, its association with the acute phase response and its age of onset and female preponderance appear to have evolved from human interactions with pathogens in the microbial world. approaching the relationship between inflammation and depression from this evolutionary perspective yields a number of insights that may reveal important clues regarding the origin and epidemiology of the disorder as well as the persistence of its risk alleles in the modern human genome. major depressive disorder (mdd) is about relationships. or so one would think based on an extensive literature that seeks to explain how a condition with genetic underpinnings could be so widespread when it is so apparently inimical to the darwinian mandates of survival and reproduction. this literature on adaptive theory bifurcates over whether depression is adaptive in and of itself or whether the genes that increase the risk of depression also promote other types of social behavior (ie, avoiding conflict with more powerful individuals) that promoted survival and reproduction sufficiently to have been retained in the human genome (supplementary table s ). despite differences, however, these theories all share in common the notion that the adaptive value of mdd is to be found in its effects on our relationships with other people. we agree that the adaptive value of mdd is to be found in our relationships, but not primarily in our relationships with other humans. rather, in this review we argue that mdd, and the genes that subserve it, evolved to help us manage relationships with the wide range of pathogens with which humans co-evolved. specifically, at least some of the genes that promote mdd evolved and have been retained in the human genome because-on average-they helped hominids avoid death from infection. importantly, we are not suggesting that these genes promoted host defense against pathogens at the cost of inducing depression. rather, the tendency of these genes to induce depression and other related behavioral changes including arousal, anxiety and alarm is integral to their anti-pathogen benefits, becauselike sickness from which it evolved-depression conferred protection from pathogens in the environments in which humans evolved. said differently, depression may itself be an anti-pathogen defense strategy, one that is both activated by inflammation and, somewhat paradoxically, one that across evolutionary time may have made inflammatory activation less necessary, as we shall see in our discussion of sex differences in the prevalence of mdd. from this perspective, even environmental risk factors for depression, such as psychosocial stress, promote mdd primarily because across evolutionary time these adversities reliably served as warnings that an individual's risk for infectious morbidity/ mortality were heightened. although this perspective may be most relevant to sub-types of depression associated with increased inflammation, we highlight in this review the possibility that other immune and/or behavioral responses relevant to pathogen-host defense may also be associated with mdd in ways that extend the role of microbial-human interactions in the evolution of depression beyond its now well-recognized associations with inflammation. consistent with earlier publications from our group miller, , ; miller and raison, ) , we refer to this constellation of ideas as the pathogen-host defense theory of depression, or pathos-d. this position overlaps, but has significant differences from a pathogen defense theory of depression first articulated by kinney and tanaka, ( ) (anders et al, ) . table situates pathos-d within other possible explanatory frameworks for understanding why mdd and alterations in immune function should be related. supplementary table s provides a comparison of different adaptive explanations for the persistence of mdd and its risk alleles in human populations. as with all theories, the value of pathos-d will eventually be determined by its ability to make novel predictions and to provide coherent and parsimonious explanations for a wider range of variables than are accounted for by competing hypotheses. our aim in this review is to evaluate pathos-d by these criteria. to do this, we list a series of concepts consistent with pathos-d theory, in each case evaluating the evidence for and against the theory's explanatory and predictive potential. in addition, we explore the theory's possible explanations for sex differences in the prevalence of mdd. in all these areas, we are careful to note the degree of data in support of each idea, and which ideas are frankly speculative. figure provides a graphic representation of epidemiological, biological, and genetic domains relevant to mdd that are addressed by pathos-d. of all the predictions of pathos-d, this one is the best established. indeed, it was the effort to provide an evolutionary perspective on the association between inflammation and depression that provided the initial impetus for the theory. taken as a whole, studies suggest that mdd is associated with all the cardinal features of an inflammatory response including increased proinflammatory cytokines and their receptors and increased acute phase reactants, chemokines, and soluble adhesion molecules in peripheral blood (lieb et al, ; calabrese et al, ; sluzewska et al, ; song et al, ; howren et al, ; dowlati et al, ; liu et al, b; valkanova et al, ; black and miller, ; haapakoski et al, ; eyre et al, ; goldsmith et al, b; van dooren et al, ) . several studies provide evidence of increased inflammatory activity in the central nervous system of depressed individuals as well, as indexed by increased cerebrospinal fluid (csf) concentrations of interleukin (il)- -β and neural adhesion molecules, as well as by increased prefrontal cortex gene expression of il- -α, il- , il- , il- , il- , il- , il- , il- a, il- , il- , il- , interferon (ifn)-γ, and lymphotoxin alpha (tnf superfamily member ; poltorak et al, , levine et al, shelton et al, ) . perhaps suggesting a link between inflammation and symptom acuity, and/or reflecting the association between inflammation and heightened anxiety (a known suicide risk), a recent meta-analysis found that levels of il- -β and il- were significantly increased in blood and postmortem brain samples of patients with suicidality compared with both non-suicidal depressed patients and healthy control subjects (black and miller, ) . given their centrality in activating innate immune responses to pathogens, it is striking that no adaptive relationship exists between immune changes and depressive symptoms. immune changes observed in major depressive disorder (mdd) may be beneficial or detrimental to pathogen-host defense and adaptive benefits of depression exist in non-pathogen/immune-related domains (multiple adaptive theories, eg, social navigation hypothesis) depressive symptoms extract a cost to survival and reproduction but this cost is offset by the direct anti-pathogen benefits of heightened inflammation/immune activation (antagonistic pleiotropy) in individuals with reduced immune competence for any number of reasons, depressive symptoms serve pathogen-host defense by inducing social avoidance/energy conservation and behaviors that provide protection from becoming infected and/or energy for immune activity once infection has occurred (kinney and tanaka) genetic alleles that promote an inflammatory bias have undergone positive selection because in ancestral environments they provided direct pathogen defense and because they promoted the development of depressive symptoms in response to immune activation. like sickness behavior, depression in response to immune activation aided in host defense both directly (ie, raised body temperature and energy conservation behaviors) and indirectly (social avoidance, energy conservation, and hypervigilance; raison and miller, pathogen- host defense theory of depression [pathos-d]) adaptive explanations for associations between mdd and altered immune functioning are not considered (frequent unexamined assumption of researchers working on proximal mechanisms) toll-like receptor (tlr) mrna and protein have been reported to be elevated in both the periphery and cns of individuals with mdd (hung et al, (hung et al, , keri et al, ; van dooren et al, ) , with some evidence suggesting that successful pharmaco-or psychotherapy reduces peripheral tlr activity (keri et al, ; hung et al, ) . of these various findings, meta-analyses suggest that the best replicated associations between depression and inflammatory biomarkers are increases in circulating levels of c-reactive protein (crp) and il- (howren et al, ; dowlati et al, ; liu et al, b; valkanova et al, ; black and miller, ; haapakoski et al, ) . taken as a whole, the meta-analytic literature also supports an association with circulating levels of tumor necrosis factor (tnf) and il- -β, although with more variability. because il- -β features so prominently in both the inflammatory response and in animal studies of stress, and depression-like behavior, it is curious that meta-analyses split on whether it is elevated in the blood of patients with mdd (dowlati et al, ; black and miller, ) . one possible explanation that would be consistent with the animal literature is that il- -β has a larger role in the cns than the periphery in terms of depressogenesis. consistent with this, some evidence suggests that il- -β is elevated in the csf of depressed individuals (levine et al, ) . alternately, evidence linking il- -β to mdd may not be robust because of limitations in assay methods in detecting levels of il- in human studies. although increases in inflammatory biomarkers in depressed patients compared with controls are modest when compared with acute infections or autoimmune conditions, recent animal data demonstrate that these types of low-level inflammatory stimulation are sufficient to induce depressive-like behavior and associated brain changes when such stimulation is chronic (tarr et al, a) , such as appears to be the case in many individuals with mdd. figure . man meets microbes. in ancestral environments, heavy pathogen loads applied significant evolutionary pressure on human survival that ultimately entailed a host of adaptations that, according to the pathogen-host theory of depression, shaped interactions between the immune system (inflammation in particular) and the brain, leading to a unique set of behaviors. these behaviors, including anhedonia, fatigue, and psychomotor slowing as well as anxiety, arousal and alarm supported energy conservation for fighting infection and wound healing and hypervigilance to prevent future attack. in addition, these evolutionary forces instantiated a coupling of these behavioral responses with the acute phase response including not only increases in acute phase reactants such as c-reactive protein but also hypoferremia and zinc deficiency, as well as fever, which are better suited for interactions with pathogens than people. in addition, evolutionary pressures from human interactions with the microbial world can explain risk alleles that are specific to the pathogens to which given populations were exposed as well as epidemiologic features of depression such as female sex preponderance, an early age of onset, and occurrence in the postpartum period that supported reproductive success. finally, the pathogen-host theory of depression is consistent with modern risk factors for depressive symptoms, including obesity, processed-food-based diets, sedentary lifestyle, and sleep deprivation, all of which serve to exacerbate the inflammatory bias that is the legacy of our evolutionary past. nonetheless, cross-sectional associations between mdd and inflammation do not establish that immune system activity is capable of producing depression, as would be predicted by pathos-d theory. it might well be that depression is associated with brain states that through their impact on neuroendocrine function secondarily increase inflammatory biomarkers that themselves have no impact on the disorder. however, a wealth of data demonstrate that although it is true that mental states can increase inflammation, it is equally true that activation of the body's inflammatory response system can induce depression, as well as many of the cns and neuroendocrine changes that have been repeatedly associated with mdd. this has been shown in studies examining the acute behavioral and cns responses to a single exposure to endotoxin or typhoid vaccine and even more convincingly by the many studies examining the biological and behavioral effects of chronic immune activation secondary to treatment with the cytokine ifn-α for either cancer or hepatitis c virus infection (capuron et al, (capuron et al, , a (capuron et al, , b (capuron et al, , (capuron et al, , (capuron et al, , wichers and maes, ; wichers et al, b wichers et al, , wichers et al, , majer et al, ; harrison et al, a harrison et al, , b lotrich, ; lotrich et al, ; prather et al, ; raison et al, raison et al, , a raison et al, , b raison et al, , d eisenberger et al, a, b; franzen et al, ; felger et al, felger et al, , . importantly, the induction of depressed mood in response to both acute (ie, typhoid vaccine) and chronic (ie, ifn-α) immune stimulation has been associated with increased concentrations of circulating inflammatory cytokines (wright et al, ; raison et al, a) . also consistent with the ability of immune activation/inflammation to negatively impact mood is the fact that the risk of developing mdd, increases following significant infections (benros et al, ; gunaratne et al, ; bornand et al, ) . finally, a recent meta-analysis confirms findings from a number of individual studies that elevated levels of circulating inflammatory biomarkers predict the future development of depression in currently non-depressed individuals, even when controlling for other depressive risk factors (valkanova et al, ) . increased inflammatory activity has been the most replicated immune change associated with mdd, but there is no a priori reason why this should be the only adaptive immune pattern associated with depression that enhanced pathogen-host defense. a clear prediction of pathos-d theory is that as novel innate immune antipathogenic mechanisms are identified, they will be found to be enhanced in depressed individuals. for example, recent studies indicate that antimicrobial peptides upregulated by inflammatory cytokines (eg, ribonuclease , psoriasin, and human β-defensin- and ; harder et al, ) are increased in the skin of patients with inflammatory skin conditions, such as atopic dermatitis and psoriasis (harder and schroder, ; harder et al, ) , that are comorbid with mdd (gili et al, ; yang et al, ) . one would expect a similar increase in these peptides-although perhaps to a lesser degree-in medically healthy individuals with mdd. the best replicated risk factors for mdd in the modern world have been repeatedly associated with the same changes in immune functioning that are observed in mdd (raison, et al, c) . this is especially apparent in relation to associations between these risk factors and increased inflammation; however, stress-which has been the most extensively studied-has also been repeatedly associated with other immune changes seen in mdd (herbert and cohen, a, b) . taken at face value, these associations appear to provide strong support for pathos-d. however, it is important to note that a number of these factors-such as obesity, sedentary lifestyle and processed foods-are artifacts of modern lifestyles, and therefore are unlikely to have evolved to activate depression as a result of being 'early warning signs' of impending infectious morbidity/mortality in the environment of evolutionary adaptiveness (eea), as would be predicted by pathos-d. however, at least two proinflammatory risk factors for mdd-medical illness and psychosocial stress-are certainly ancient and may well have evolved an association with depression as a result of inducing immune changes that enhanced survival in the eea. pathos-d proposes that depression evolved out of sickness, so it is no surprise that robust associations have been seen between medical illness and increased rates of depression in all cultures examined (moussavi et al, ) . because cytokines are primary drivers of sickness, it is also no surprise that medical illnesses are typically characterized by increased inflammation, nor that increased levels of inflammatory cytokines in medically ill patients are associated with an increased risk for depression (musselman, ) . on the other hand, it is not immediately apparent why psychosocial stressors should so reliably activate inflammatory pathways in animals and humans when these types of adversities are not in and of themselves associated with either an infectious challenge or an immune stimulus. indeed, it has become so commonplace to say that stress increases inflammation that it is easy to lose track of what a remarkable thing this is. consider the trier social stress test (tsst) in which study participants are charged with giving a personally meaningful speech and doing mental arithmetic in the presence of a judgmental panel. predictably, the threatened subject experiences a classic fight or flight response characterized by an increased heart rate and blood pressure and an increased release of cortisol and catecholamines. however, the tsst also activates key intracellular inflammatory transcription pathways (eg, nuclear factor-kappa β (nf-κb)) within peripheral blood mononuclear cells and dramatically increases circulating levels of inflammatory cytokines, such as interleukin (il)- (bierhaus et al, ; pace et al, a; carpenter et al, ) . moreover, individuals with known risk factors for developing depression (eg, early-life adversity) show larger inflammatory responses to laboratory psychosocial stressors than do others (pace et al, a; carpenter et al, ) , and the larger the inflammatory response to the stressor, the more likely the subject is to develop depression over the ensuing months (aschbacher et al, ) . in essence, the body mounts an immune response, not against a pathogen, but against a threat to the subject's self-esteem. theories that view depression as having evolved to serve adaptive social purposes do not yield easy explanations for why non-infectious stressors should activate peripheral inflammatory pathways. on the other hand, pathos-d suggests a straightforward and parsimonious explanation. because the vast majority of stressors in mammals over evolutionary time boiled down to risks inherent in hunting, being hunted or fighting conspecifics in dominance hierarchies for reproductive access/status, it is not surprising that these states are also circumstances in which the risk of pathogen invasion-and subsequent death from infectionwas greatly increased as a result of traumatic opening of the protective skin barrier from wounding (dipietro, ) . such wounding is common in social species and was a significant source of morbidity and mortality among humans in the ancestral environment, and indeed well into the historical period (ross et al, ; eshed et al, ; pinker, ) . in ancestral environments, the association between stress perception and risk of subsequent wounding was reliable enough that evolution, operating by the so-called 'smoke detector' principle, favored organisms that prepotently activated inflammatory systems in response to a wide array of environmental threats and challenges (including psychosocial stressors), even if this activation was often a 'false alarm.' as noted by dhabhar-'stress perception by the brain may serve as an early warning signal to activate the immune system in preparation for a markedly increased likelihood of subsequent infection' (dhabhar, ) . and while chronic stress is best known to suppress certain aspects of immune function (ie, natural killer cell activity, circulating t-cell subsets; herbert and cohen, c) , the types of acute and/ or psychosocial stressors most likely to be associated with immediate risk of wounding and hence infection activate both innate and adaptive immunity (quan et al, ; bierhaus et al, ; avitsur et al, ; viswanathan et al, ; pace et al, b; steptoe et al, ; joachim et al, ; bailey et al, ; rosenberger et al, ; mays et al, ; powell et al, ) . that non-immune stressors can activate inflammationand do so via multiple mechanisms including activation of the intracellular inflammasomes-is now firmly established (miller and raison, ) . however, pathos-d also predicts that stress-induced immune changes should produce an overall enhancement of pathogen-host defense and especially anti-bacterial defense given that these organisms are the chief cause of wound-related infections. this has yet to be proven or disproven, but significant animal data suggest that psychosocial stressors-even when recurrentcan enhance host defense mechanisms and improve immune function. using a repeated social defeat stress paradigm (sdr), sheridan and colleagues have shown that stress primes multiple aspects of the rodent host defense repertoire. sdr increases the bactericidal activity of splenic macrophages through a toll-like receptor-dependent pathway; (bailey et al, ) enhances the innate immune response to a primary hsv- infection in the cornea and trigeminal ganglia; (dong-newsom et al, ) primes dendritic cells and augments clonal expansion of cd (+)t cells during primary influenza a viral infection with a resultant enhancement of adaptive immunity and viral clearance; mays et al, ) and activates natural killer cells (tarr et al, b) . to our knowledge, none of these effects have been directly examined in humans, but it is intriguing to note that improved functional recovery following knee surgery has been associated with an increase in the type of immune cell redistribution effects that are known to be induced by acute stress in animals (rosenberger et al, ) . also relevant to the hypothesis that stress-induced activation of inflammation provides pre-potent protection against pathogens is work by cole and colleagues demonstrating that social isolation is associated with an immune signature well-tailored to the types of infectious challenges that would most likely have resulted from ostracism in ancestral environments. individuals who perceive themselves as chronically socially isolated (ie. lonely) show a pattern of gene transcription in leukocytes characterized by increased expression of inflammatory pathways crucial for protection against bacteria with a concomitant decrease in the expression of genes important for antiviral immunity, especially interferon response genes (slavich and cole, ) . consistent with these findings, individuals with high trait sensitivity to social disconnection produce greater blood levels of proinflammatory cytokines (tnf and il- ) and increased expression of inflammatory genes in response to an endotoxin stimulus (moieni et al, a) . similarly, study participants who responded to an fmri-based social exclusion paradigm with greater activity in the dorsal anterior cingulate cortex and anterior insula-brain regions that have previously been associated with processing rejection-related distress and negative affect-also responded to a laboratory psychosocial stress test with increased salivary production of the inflammatory biomarker soluble tnf-α type ii receptor (slavich et al, ) . as slavich and cole, ( ) have observed, this pattern of findings in response to social isolation (ie, increased inflammation/reduced antiviral immunity) may well have evolved to optimize immune responses to threats from environmental pathogens associated with wounding/starvation that would have been the primary risk for infectious morbidity/mortality in the eea for individuals with diminished human-to-human contact. because ostracism was an almost certain death sentence in ancestral environments, the inflammatory response induced by social isolation/exclusion can be seen as a modern vestige of what once served as an immunological ostracism detection system that worked in concert with other ostracism detection systems described in the literature (spoor and williams, ) to encourage individuals to endure the costs of group existence during the long years of human evolution. this hypothesis might help explain the recent finding that cytokine activation via exposure to endotoxin induces feelings of social disconnection, especially in females. this phenomenon might reflect an evolved and adaptive feedforward mechanism whereby the risk of social exclusion induced a prepotent inflammatory response that then fostered a further sensitivity to the risk of ostracism. and this mechanism may be more apparent in modern females because the offspring of ostracized mothers would have been less likely to survive than those of ostracized fathers (sear and mace, ) . on the other hand, the increased balance of antiviral to anti-bacterial/inflammatory gene expression seen in more socially integrated individuals would have been more optimal for providing protection against more recent viral infections that spread from person to person but that only became widely lethal (and thereby exerted greater selection pressure) with the increased population density that became the norm after the development of agriculture in the first epidemiological transition. if we assume that a chronic inflammatory response is the most common immune profile associated with mdd, then pathos-d predicts that this association should be ancient, because it is this immune response and its associated depressive symptoms that enhanced survival and reproduction via reduced pathogen-related mortality. strong evidence for this comes from innumerable studies in mice and rats showing that inflammatory cytokines and their stimulators induce depressive and anxiety-like behavior (dantzer et al, a) , as well as studies indicating that even when induced by psychological stress, these behavioral responses are dependent upon cytokine activation-especially the induction of il- -β in the cns (goshen and yirmiya, ). the fact that primate and rodent lineages separated from each other in the time of the dinosaurs strongly suggests that the rudiments of the immune-depression link were laid down early in mammalian evolution, if not earlier. however, not all data support an ancient or universal link between inflammation and depression, nor is pathos-d the only evolutionary theory that might account for such a link if it in fact exists. indeed, it is increasingly clear that numerous conditions in the modern world have dysregulated immune functioning in ways that promote autoimmunity and chronic inflammation, while simultaneously reducing any adaptive advantage that might be inherent in either the inflammatory response or mdd (raison et al, c) . this scenario opens the possibility that the association between inflammation and depression is a non-adaptive by-product of first world environmental and social conditions, rather than something inherent in either our genetic or behavioral make-up. said differently, the link between chronic inflammation and mdd may not reflect an evolved adaption, but rather represents an example of non-adaptive environmental mismatch, defined as a state of disequilibrium whereby a trait that enhanced reproductive fitness in the environment in which it evolved becomes maladaptive in another environment. of all the modern environmental conditions that have altered associations between immune function and behavior, none has received more attention than our changed relationships with a huge range of microorganisms that were ubiquitous in the eea, but that are largely missing from the industrialized world. unlike potentially lethal pathogens, most of which produced either immunity or death, many of the now-absent organisms were best dealt with via the development of immune tolerance, either because the organisms were beneficial (eg, the microbiota), harmless (eg, many environmental organisms), infectious but not lethal (eg, hepatitis a virus) or potentially harmful but not easily cleared by the immune system without excessive tissue damage (eg, many helminths). the central insight of recent iterations of the 'hygiene hypothesis' (such as the 'old friends' hypothesis of rook) is that because the immune system had to learn to tolerate these organisms (as opposed to clearing them from the body), they became 'teachers of tolerance,' especially during childhood development when their absence predisposes individuals in industrialized societies to develop a host of allergic, asthmatic, autoimmune, and psychiatric pathologies (raison et al, c) . in a way that seems more than metaphorical, this notion points toward another way that depression may be about relationships. we need to look no further than the impact of bereavement on mood or the strong association between the death of a parent in childhood and the development of mdd in adulthood to see that human relationships characterized by loss can be profoundly depressogenic (berg et al, ) . increasing evidence suggests the same is true of our relationships with the microbial world. via dysregulated inflammatory activity, our moods are haunted by the loss of 'old friend' organisms of which most of us have no conscious knowledge. again, like our relationships with other humans, our relationships with bacteria, viruses, and parasites can be understood as comprising various degrees of competition and cooperation/collaboration. pathos-d suggests that the link between depression and immune activation primarily reflects the competitive element in these relationships. the evolutionary mismatch model suggests the opposite: that the link between depression and immune activation reflects the failure of the modern world to honor many ancient and mutually beneficial collaborations. so, can available data help us determine which theory is better supported? to our minds, the strongest data that support evolutionary mismatch, while simultaneously arguing against pathos-d, come from the ground-breaking work of mcdade and colleagues who have examined associations between stress, mood and inflammation in non-industrialized environments. working in the philippines and lowland ecuador, they have shown that patterns of inflammation observed in the west do not necessarily translate to less-developed environments with significantly higher pathogen exposure and infectious mortality. in industrialized contexts, the concentrations of peripheral inflammatory biomarkers show significant intra-individual stability, with some individuals demonstrating chronically elevated levels of these biomarkers (macy et al, ; ockene et al, ) . moreover, factors other than pathogen exposure (especially obesity) have been repeatedly shown to upregulate inflammation. mcdade et al, ( ) found that neither of these pertain in more traditional societies. in the philippines levels of crp were significantly lower, and levels of the anti-inflammatory cytokine il- significantly higher, than are typically observed in western contexts, and these associations were not accounted for by differences in body mass index between the populations (mcdade et al, ). in lowland ecuador among the indigenous shuar people, levels of crp were found to vary widely within the same individuals, showing a pattern of rapid rise to high levels in response to acute infection and then dropping to often undetectable levels upon infection resolution (mcdade et al, b ). an even more direct challenge to pathos-d predictions came from findings that depression was not associated with elevated crp in the philippines and that associations between both current and childhood stressors (ie, death of a parent) and inflammation were only apparent in the sub-population in the cebu region that had been raised in more hygienic environments (mcdade et al, a) . individuals with high levels of microbial exposure in infancy showed no such associations. taken together, these data suggest that high microbial exposure early in life-such as would have been normative across human evolution-may hone the immune response to be more specific for pathogen exposure and less generalized to the types of psychosocial stress that so reliably induce depression. under these evolutionarily normative environments, the links between inflammation and mdd that are so apparent in the west would not exist. however, these findings must be balanced against more recent work conducted in the lowland jungle of bolivia among the tsimane amerindians, a small-scale society composed of lean forager-horticulturalists with a short average lifespan who live in a high-pathogen environment and thus have significant microbial priming in infancy (stieglitz et al, b) . in all these ways the tsimane represent a reasonable approximation of environmental and social conditions prototypically confronted by humans in the eea. working among these people, stieglitz et al. ( b) examined whether a syndrome recognizable as mdd exists, and if it exists, whether it is associated with increased inflammatory biomarkers. results indicated that a depressive syndrome identical to that seen in the industrialized world exists among the tsimane, and is associated with the same risk factors that have been observed everywhere else in the world: reduced health (indexed as functional disability) and psychosocial stress (indexed by social conflict, especially with non-kin; stieglitz et al, a). when examined as a binary construct based on a median split in symptom scores, depression was also associated with significantly increased serum concentrations of il- , tnf, and crp but not il- -β (stieglitz et al, b) , replicating the overall pattern of metaanalytic findings from studies conducted in industrialized societies. these associations remained after adjustment for age, body mass index, and high leukocyte count (likely indicative of current infection), and associations were observed across a wide range of depressive symptoms. as our discussion of this prediction suggests, currently available data provide a split decision on whether associations between depression and inflammation are ancient and hence potentially adaptive, or are of recent origin and thus likely a result of non-adaptive evolutionary mismatch. one can only mourn the fact that the theoretical perspectives and technology necessary to resolve this issue were not available in the middle years of the th century when a range of hunter-gatherer groups on several continents had yet to be driven to extinction by the inexorable onrush of the modern world. pathos-d theory engenders a sense of urgency to examine this issue in the remnant small-scale societies that remain. two types of studies would be especially relevant. first, it would be of great interest to conduct the equivalent of trier social stress tests in these societies to examine whether objectively induced psychosocial stress activates inflammatory pathways as it does in the west. second, it would be important to examine whether the administration of cytokine activators (ie, endotoxin, typhoid vaccine or longer-term treatment with ifn-α) produce depressive symptoms as they do in the industrialized world. if individuals living in environments more closely aligned with prototypical conditions in the eea showed inflammatory responses to psychosocial stress and if they responded to immune stimulation with depression, strong evidence in support of pathos-d would have been obtained. from a pathos-d perspective there is no a priori reason that mdd should be associated with increased inflammation, only that mdd be associated with genes, physiology and behavior that-on average-decreased infectious mortality across human evolution. the fact that a subset of patients with mdd demonstrate increased inflammation implies the hypothesis that if depression is associated with increased inflammation, and if depression evolved as an adaptive pathogen-host defense strategy, then increased inflammatory activity should have produced an overall survival benefit in high-pathogen ancient environments, if indeed the association between depression and inflammation was adaptive in the eea. for this idea to be credible two things are required: first depression should enhance host defense, and second inflammation should enhance host defense. on the face of it, neither appears to be true, given significant evidence that both depression and inflammation appear to increase-rather than decrease-vulnerability to infection (zorrilla et al, ; evans et al, ; cruess et al, ; raison et al, ; doering et al, ; faulkner and smith, ; leserman, ; leutscher et al, ; andersson et al, ; doran et al, ) . here we consider the question of whether inflammatory activity provided protection against infectious mortality. for at least years it has been recognized that inflammation is essential for human health and a necessary ingredient of our ability to adequately fight infections. we now know that the larger innate immune response-of which inflammation is a core component-provides both direct and rapid protection against all classes of pathogens and has a crucial role in activating slower, but more definitive acquired immune responses. however, studies in recent years have found that the relationship between the innate and acquired immune systems follows the laws of cooperation and competition that define all evolved relationships. especially under conditions of limited energy availability or simultaneous infectious risk from multiple pathogens, immune function can become characterized by trade-offs between these two branches of immunity, such that resources invested in one, mean reduced functionality of the other (mcdade et al, ) . consistent with this notion, chronic inflammation can actually suppress t-and b-cell function through various mechanisms (cope et al, ; moraska et al, ; cope, ; clark et al, ; muller et al, ; vaknin et al, ; eleftheriadis et al, ; blume et al, ) , and rates of infection are often increased -not decreased-in autoimmune conditions characterized by chronic inflammation (doran et al, ) . however, pathos-d theory requires only that across evolutionary time the patterns of enhanced inflammatory activity characteristic of depression provided overall survival benefits that outweighed any costs imposed by associated reductions in other aspects of immune functioning. some evidence for this possibility comes from studies conducted by westendorp and colleagues in ghana and the netherlands. these researchers observed that cytokinestimulated production of tnf-α declines with age in the netherlands, a country with a low infectious burden, but does not decline with age in ghana, a country with high rates of infection (ie, % of ghanan study participants were infected with malaria; may et al, ) , suggesting that increased proinflammatory cytokine production-as observed in mdd-promotes survival under conditions of high-pathogen burden (drenos et al, ) . more definitive evidence for this possibility comes from a second ghanian study that more directly examined the impact of pathogen exposure on the association between inflammation and survival. ghana is a country in which some regions have pure water available from boreholes, whereas other regions must rely on heavily contaminated rivers for drinking water. as would be expected, death rates from infection are far higher in regions that utilize rivers than in areas where borehole-obtained water is available. consistent with the prediction that increased inflammatory signaling is protective in the high-infection environments that were common during human evolution (and especially common since the origin of agriculture and the rise of cities; armelagos et al, ) , a haplotype of the il- gene associated with increased inflammation was found to be significantly more prevalent in populations that relied on river water than in populations that drank from boreholes-suggesting positive selection driven by enhanced pathogen protection (kuningas et al, ) . consistent with this possibility, during a fiveyear follow-up period, the high-inflammation il- haplotype was associated with reduced survival in individuals who drank clean water from boreholes, but increased survival in populations exposed to high levels of pathogens as a result of drinking river water (kuningas et al, ) . we have already noted that depression is associated with increased circulating inflammatory biomarkers in the forager-horticulturist tsimane people of lowland bolivia (see prediction ). researchers also conducted ex vivo stimulation studies with endotoxin and phytohemagglutinin (pha; to assess response capacity of both the innate and acquired immune systems, respectively) and found that tsimane individuals with high levels of depression produced greater levels of il- -β, il- , and tnf in response to both endotoxin and pha, even when excluding individuals with leukocyte counts suggestive of current infection (stieglitz et al, b) . interestingly, this pattern is different from the suppression of mitogen-stimulated immune responses commonly observed in depressed patients in the western world (herbert and cohen, a) , which may point to yet another way in which modern conditions have disabled previously adaptive associations between depression, immune function, and pathogen-host defense. nonetheless, whether the increased mitogen-stimulated inflammatory cytokine response observed among depressed members of tsimane society would translate to enhanced protection against pathogens remains unknown. multiple facets of the modern world, from antibiotics to refrigeration and paved surfaces have profoundly reconfigured our relationship with the microbial world in ways that have reduced the benefits of inflammation and increased its costs (drenos et al, ; raison et al, c) . nonetheless, even in an environment so radically different from that in which humans evolved, data link increased inflammation with improved outcome in the context of at least some infections. for example, in a large study of consecutive patients admitted to hospital with fever, elevations in plasma concentrations of the anti-inflammatory cytokine il- -as well as the ratio of il- /tnf-α-were associated with increased mortality (van dissel et al, ) and increased il- production late in the disease course predicted reduced survival following infection with the pandemic a/h n -/ influenza virus (bermejo-martin et al, ) . as these findings would predict, alleles of the il- gene-such as - g-associated with higher il- production appear to promulgate poor infectious outcomes. for example, the − g allele predicts increased symptom severity and mortality in the context of community acquired pneumonia (gallagher et al, ) and is associated with reduced antibody responses to tetanus, influenza and hepatitis b virus (hbv) vaccines (hohler et al, ; corsini et al, ; li et al, ) . families with a member who died from meningococcal disease were characterized by increased production of il- and reduced production of tnf-α when compared with families with a member who had bacterial meningitis but survived (westendorp et al, ) and elevated crp has been associated with increased survival in children with meningococcal sepsis (joosten et al, ) . the ifn-γ + t allele increases production of this th inflammatory cytokine via enhanced binding of nfkb (pravica et al, (pravica et al, , and protects against mediterranean spotted fever (forte et al, ) . multiple studies and a large meta-analysis find that the t allele also protects against the development of tuberculosis on the individual (pacheco et al, ) and population levels (larcombe et al, ) . in individuals with active tuberculosis, the t allele reduces severity and risk of disseminated disease (ansari et al, ). the t allele also reduces the risk of leprosy (cardoso et al, ) , severe acute respiratory syndrome (sars; chong et al, ) , and chagas disease (torres et al, ) . conversely, the low-producing + a allele predisposes for hepatitis b and c persistence and negatively impacts clinical course of the disease (gao et al, ) . consistent with these findings, reduced peripheral blood mononuclear cell production of ifn-γ and il- is associated with increased severity of respiratory syncytial virus symptoms in infants under one year of age (pinto et al, ) . the association of depression with increased circulating levels of proinflammatory cytokines and crp does not easily lend itself to social explanations for the high prevalence of either mdd or putative depressogenic risk alleles. on the other hand, this association is foundational to the proposal that depression evolved to serve pathogen-host defense functions. however, if this is indeed the case, why should we expect the immune changes seen in depression to be limited to circulating cytokines or crp? in the context of innate immune activation, cytokine stimulation forms only part of a larger phenomenon that comprises the acute phase response (apr). as noted in a recent review (legrand and alcock, ), the apr is a somewhat paradoxical phenomenon, given that many of its features inflict significant energetic costs (eg, fever), hamstring immune responses (eg, iron sequestration, zinc, and tryptophan depletion), or produce behaviors likely to reduce attractiveness to potential mates, induce loss of social status or increase the risk for predation (eg, lethargy, psychomotor slowing, and social withdrawal), all of which would be expected to impair, rather than enhance, host survival, and reproduction. and while fever has been shown to enhance immune function while simultaneously contributing directly to pathogen killing, no direct immune benefits have been shown for many other apr features. a potential resolution to this enigma has been offered recently under the rubric of 'immune brinksmanship', which suggests that many apr features-such as iron sequestration and reduced zinc availability-have evolved to make the host environment less optimal for invading organisms, given that these minerals are essential nutrients for microorganisms, without which they can neither replicate nor survive (legrand and alcock, ) . from this perspective the apr can be seen as being, in part, a strategy that risks damage to the self, but that is likely to kill the offending agent before it kills the host. a comprehensive vision of the apr sees some of its elements as serving direct anti-pathogen functions (eg, leukocytosis), some as serving to reallocate energy to the immune system (eg, psychomotor slowing, somnolence) and some as serving 'immune brinkmanship' functions. studies suggest that, as a group, medically healthy individuals with depression demonstrate multiple biological and behavioral stigmata of an apr (jimenez-fernandez et al, ) . for a full listing of elements of the apr that have been associated with mdd in medically healthy individuals, see supplementary table s . importantly, at least one group has found that the apr in medically healthy adults with mdd is associated with increased levels of il- , suggesting that the apr is indeed part of a larger pathogen-host defense immune response (maes et al, ; sluzewska et al, ) . moreover, if psychosocial stress serves as an 'early warning system' for increased danger of impending wounding and subsequent infection, pathos-d would predict that psychosocial adversity should also be associated with an apr over and above activation of cytokines. consistent with this possibility, stress in animals reduces gastrointestinal absorption and blood levels of iron (zhao et al, ; chen et al, ) and in human males a -day intense psychological stressor ('hell week') reduced blood levels of iron, zinc, and albumin (singh et al, ) , as would be predicted if stress induces an apr. psychological stress has also been reported to elevate core body temperature in animals and humans (soszynski et al, ; kaneda et al, ; hayashida et al, ) , an anti-pathogen defense mechanism that is most effective in conditions of low host iron availability (kluger and rothenburg, ) . that these changes serve immune purposes is suggested by findings that in animals, stressinduced hypoferremia and hyperthermia are associated with stress-induced increases in il- (soszynski et al, ; zhao et al, ) ; however, other mechanisms may also be active, including hypothalamic activation of brown fat (kataoka et al, ) . because at least some biological changes associated with the apr have been shown to have important roles in pathogen-host defense, their presence in depression would be strongly predicted by pathos-d, and their absence would strongly argue against the validity of this approach. on the other hand, their presence in mdd is not parsimoniously explained by theories that focus on potential social benefits of depression. in what ways would elevated body temperature or reduced iron availability aid in negotiating relationships with other humans? similarly, if depression is simply a non-adaptive phenomenon, why would such ancient, highly conserved and highly complex physiological responses be associated with the disorder? inseparable from the physiological aspects of the apr, immune activation also produces a suite of behavioral responses known as sickness behavior that has been retained across vertebrate evolution and that shares many features in common with the symptoms of mdd (dantzer, ; dantzer et al, b) . these symptoms include lethargy/ fatigue, psychomotor slowing, altered sleep, loss of appetite, anhedonia, and social withdrawal. although it is clear that the symptoms of depression can arise from a wide variety of divergent causes-including internally generated thought patterns-pathos-d proposes that mdd initially evolved out of sickness behavior. circumstantial evidence for this type of close relationship between sickness and mdd come from several sources. for example, data suggest that many people cannot accurately identify whether they are developing depression or an infectious illness early in the course of either condition (ratcliffe, ) . similarly, sickness in the first week of treatment with the cytokine ifn-α strongly predicts the development of cognitive/emotional symptoms of depression over the ensuing months of therapy (wichers et al, a) . finally, if inflammation induces sickness and if depression evolved out of sickness, inflammation might be expected to be more strongly associated with the symptoms most often shared by sickness and depression, rather than symptoms such as sadness and guilt/feelings of worthlessness that are more specific to mdd. supporting this possibility, an early report found that the acute phase protein haptoglobin correlated with psychomotor retardation, sleep disturbance, anorexia/weight loss, and anhedonia (maes et al, ) . more recently, a large population-based study (n = ) reported that crp was more strongly associated with sleep abnormalities, fatigue, and appetite changes than with mdd symptoms less common in the context of sickness (jokela et al, ) . in a group of patients with mdd, increased circulating levels of il- and monocyte chemoattractant protein- were positively associated (and levels of il- were negatively associated) with objectively measured psychomotor slowing (goldsmith et al, a) , and elevated plasma crp was associated with reduced functional connectivity between the ventromedial prefrontal cortex and ventral striatum. these changes in functional connectivity mediated associations between increased crp and anhedonia and psychomotor slowing (felger et al, ) . evolutionary processes do not produce perfect products, and all evolved adaptive mechanisms come with associated costs and trade-offs. if indeed mdd is conceptualized as a condition evolved from, and 'designed' to serve the same purposes as, sickness, the obvious question is what pathogen-related survival and reproductive benefits accrue from depressive symptoms that offset their costs. that depression reduces fitness in modern environments has often been remarked upon; however, the costs across evolutionary time of the types of prolonged sickness behaviors that comprise mdd have also likely been substantial. as part of the apr, sickness exacts significant metabolic costs that limit energy available to other tasks essential for survival and reproduction. in many species, including humans, even subtle signs of sickness promote avoidance behavior from conspecifics (schaller, ) . over and above the suppressive effect of inflammation on sexual functioning, individuals who appear sick are judged to be less attractive and hence are less likely to find high-quality mates. in hierarchical social groups the display of sickness risks loss of social status, with associated reductions in survival and reproductive access. finally, sickness increases the risk of predation (schaller, ) . one possibility is that the pathos-d perspective is exactly backwards, and in fact depression is a maladaptive consequence of the benefits of acute sickness. perhaps depression is akin to other chronic conditions, such as hiv and autoimmune diseases, in which ongoing inflammation, rather than providing benefit, actually increases the risk of infection and/or infectious mortality. many studies associating mdd with increased infectious morbidity in the modern world would support this possibility (zorrilla et al, ; evans et al, ; cruess et al, ; raison et al, ; doering et al, ; faulkner and smith, ; leserman, ; leutscher et al, ; andersson et al, ) . if true, this removes pathogen-host defense as an explanation for the conundrum of why genes that contribute to mdd have been retained across hominid evolution. or it may be that by inducing social withdrawal, depression provides an adaptive advantage for individuals at increased risk of infection as a result of immune functioning that for one reason or other is sub-optimal. this is the position taken by kinney and tanaka ( ) in proposing that depression evolved as an adaptive response to cope with pathogens (anders et al, ) . in contradistinction, pathos-d suggests that-whatever its costs in relation to specific pathogens-depression was not primarily a compensation for immune inadequacy, but rather enhanced overall survival in the eea via its association with physiological and behavioral changes that reduced the impact of infectious agents on reproductive success. said differently, in conditions characterized by a heightened risk for infectious mortality, such as occur during active infection or when environmental adversity signals an increased risk for wounding or other processes that increase the risk of infection (ie, starvation secondary to ostracism), the generalized innate immune activation and symptoms associated with depression provided a net survival benefit, whatever their other costs in terms of immune or social trade-offs. importantly, this perspective does not require that depression be the only way for humans to adaptively cope with infectious risk only that it was adaptive enough for its genetic underpinnings to be retained in the human genome at a level significant enough for mdd to be with us today. from a pathos-d perspective, mdd in response to infection or tissue trauma provides the same types of survival benefits as does sickness behavior. in response to stress, depression can be seen as proactive physiological/behavioral sickness behavior-a type of 'just in case' condition that keeps an individual in a state of pathogen defense readiness. proinflammatory cytokine activation in response to either infection or stress induces a behavioral state of conservationwithdrawal (engel and schmale, ; eisenberger et al, b; hannestad et al, ) characterized by depressed mood, anhedonia, psychomotor retardation, fatigue, social avoidance, and anorexia (capuron et al, ; dantzer et al, b; majer et al, ; irwin and cole, ) . this state is an integral component of depressive disorders and has been widely considered to develop in the context of infection and/ or tissue injury as a means of marshalling limited metabolic resources for the expensive tasks of immune activation, fever generation and tissue repair (hanff et al, ) . in addition to energy allocation, conservation-withdrawal symptoms may have also proved adaptive by reducing interpersonal contact and thereby limiting infectious exposure (kinney and tanaka, ). in the paradigm proposed by kinney and tanaka ( ) this is seen as an evolved compensatory mechanism to cope with immunodeficiency. however, it is equally possible that the infectionavoidance benefits of social withdrawal evolved as a complement to its metabolically valuable infection-fighting benefits. indeed, the more survival and reproductive benefits accrue for any one trait the more likely its genetic antecedents are to be retained and to spread in the population. because humans in the eea lived in small coalitional groups of genetically related individuals, the logic of inclusive fitness suggests that social withdrawal might have been adaptive for an individual's genes by reducing the risk of infection in kin, even if such withdrawal limited the provision of much needed care from others and thus reduced individual survival (cole, ; schaller and murray, ) . indeed, given evidence that merely viewing pictures of a sick individual activates the viewer's innate immune system (schaller et al, ) , it may be that by prepotently activating the innate immune responses in one's kin, depression served as an early warning system for increased infectious risk. in this regard, it is interesting to note that associating with depressed individuals increases the risk of developing depression oneself (fowler and christakis, ) , exactly as would be predicted if depression served as an early warning signal to others of infectious risk and if depression enhanced host pathogen defense by priming the innate immune system. a clear prediction of this line of reasoning that awaits testing is that viewing images of depressed individuals should activate the immune system. in addition to potential inclusive fitness benefits, any decrement in survival from depression-induced loss of social aid might have been more than offset by reduced risk of exposure to other pathogens while in a vulnerable state due to a pre-existing infection, given that viral infections promote aggressive immune responses to bacterial superinfections that can greatly increase mortality (degre and glasgow, ; jakab and dick, ; jones et al, ; nguyen and biron, ; molyneux et al, ; beadling and slifka, ; mccullers et al, ) . moreover, social withdrawal and reduced environmental exploration might also have promoted individual survival by limiting a depressed individual's contact with immunologically dissimilar out-group members who potentially harbored pathogens against which the depressed person would have had reduced immunity compared to pathogens endemic in the home group thornhill et al, ) . this idea suggests a hypothesis that to our knowledge has never been tested that mdd should be associated with increased xenophobia. it is intriguing to note, however, that endotoxin administration increased the desire of healthy volunteers to be around individuals they had previously identified as 'support figures' (inagaki et al, ) , consistent with the possibility that immune changes associated with depression might have promoted an in-group bias that reduced exposure to more potentially lethal pathogens harbored by members of other social groups. how such an increased desire to associate with in-group members would square with the potential selective advantages of withdrawing from kin to prevent the spread of infection is unknown. moreover, given that sterile psychosocial stressors also activate inflammation, it is intriguing to speculate that the benefits of seeking support/proximity with kin/in-group members in response to psychosocial dangers may have offset the potential risks of contagion spread as a result of inflammation enhancing prosocial behavior with trusted ingroup members. with the exception of asthma and allergies, inflammatory conditions typically strike after the age of reproduction, and thus alleles that promote them are subject to minimal selective pressure, even in modern environments (beekman et al, ) . almost certainly, the association between chronic inflammation and these disease states (eg, dementia and cardiovascular disease) serves no adaptive purpose and likely results in large measure from environmental conditions unique to the modern world. however, the relationship between inflammation and depression is quite different, given that the incidence of mdd peaks in the - s (eaton et al, ; patten et al, ; kessler et al, ) , an age of primary reproductive/childrearing responsibilities. in this, mdd is not alone. schizophrenia and bipolar disorder -conditions with which mdd is genetically linked-are also characterized by increased inflammation and by an average age of onset early in life when the costs of these disease states to survival and reproduction are (and were) likely to be maximal. although the current article focuses on unipolar depression, there is no reason to think that depressive symptoms occurring in the context of bipolar disorder would be any less efficacious for pathogen-host defense than the same symptoms when they occur in other contexts, and indeed multiple studies demonstrate that bipolar depression is associated with the same types of inflammatory activation that are observed in unipolar mdd (goldstein et al, ) . however, what should we make of the fact that multiple studies show that inflammatory markers are at least as elevated-and maybe more elevated-in manic episodes as they are during bipolar depressions (goldstein et al, ) ? a proximal answer might point to the fact that mania is associated with many of the same neuroendocrine abnormalities as depression, especially glucocorticoid resistance (schmider et al, ) , which is known to release inflammatory pathways from inhibitory control and which has been associated with increased proinflammatory cytokine levels in mdd (maes, ) . moreover, although far less common than depression, full manic episodes have been reported during ifn-α therapy, which produces chronic immune activation, and combinations of depressive and manic symptoms during ifn-α treatment are common (constant et al, ) . however, this type of answer provides no insight into why mania should be associated with increased inflammation. might the association serve adaptive purposes or is it better considered as an example of antagonistic pleiotropy? here pathos-d offers a novel perspective. until it becomes disabling, mania increases the types of social and sexual extroversion that may provide direct darwinian benefits as a result of increased opportunities for reproduction (berry and miller, ) . however, increased social and sexual activity also increase the risk for reduced survival as a result of increased pathogen exposure (hamrick et al, ) . in this context, might genes linking inflammation with mania have undergone positive selection by conferring increased protection against the wide range of infections to which manic behavior would have exposed an individual in the eea? although highly speculative, this hypothesis produces the clear prediction that the increased inflammation observed in mania should be associated with the symptom of hypersexuality, especially in males, who across evolutionary time would have been more likely than females to benefit in terms of reproductive success from sexual activity with multiple partners. interestingly, animal data provide some evidence for this possibility, given that cytokine activation suppresses sexual activity in female, but not male, rats (avitsur and yirmiya, ) . however, this hypothesis would be strengthened by evidence that manic behavior itself can elevate inflammation and to our knowledge this type of causal connection has not been investigated. in a previous publication we provided an exhaustive enumeration of known immune and/or pathogen defense functions for the best-supported risk alleles for mdd derived from genome-wide association studies (gwas) and meta-analyses of candidate gene studies (raison and miller, ) . these gwas findings are presented in supplementary table s . in the time since our prior publication, an additional study utilized sparse wholegenome sequencing in a population of han chinese females to identify and replicate two novel depression risk loci on chromosome , one ′ to the suirtuin (sirt ) gene (rs ) and one in an intron of the phospholysine phospho-histadine inorganic pyrophosphate phosphatase (lhpp) gene (consortium c ( )). as with previously identified 'genes of interest,' pathos-d predicts that these genes should impact immune function in ways that provided adaptive advantages for pathogen-host defense. in this regard, nothing is known regarding lhpp, but an extensive database attests to multiple immune functions of sirtuin , a class iii histone deacetylase enzyme that serves as an important sensor of cellular energy and redox states (supplementary table s ; michan and sinclair, ) . in particular, sirtuin has been shown via epigenetic mechanisms to have a key role in limiting the extent and duration of acute inflammation by promoting a shift from inflammation-driven glycolytic activity to 'adaptation-phase' fatty acid metabolism. sirtuin suppresses nuclear factorkappa b rela/p activity, represses cyclooxygenase gene expression (zhang et al, b) , and induces gene silencing facultative heterochomatin formation at the promotors of proinflammatory genes, including tnf and il- -β, as well as the promotor for hypoxia-inducible factor- -α, a signaling factor important for maintenance of proinflammatory glycolytic activity (vachharajani et al, ) . in addition, sirtuin , in dependence on nad+, increases levels of peroxisome proliferator-activated receptor gamma coactivator (pcg- )-α and β, which promotes the fatty acid metabolism essential for resolution of the inflammatory response (fernandez-marcos and auwerx, ). the end result of these activities is that sirtuin inhibits the transformation of monocytes to macrophages and promotes anti-inflammatory m macrophages and regulatory t cells (treg) at the expense of proinflammatory m -type macrophages and effector t cells (liu et al, a; park et al, ) . however, conflicting data suggest that in at least some contexts sirtuin may have inflammatory and adaptive immune stimulating effects. for example, sirtuin inhibition has been reported to stimulate foxp expression in treg (akimova et al, ) . despite these complexities, evidence of diminished sirtuin activity in medical conditions characterized by chronic inflammation suggest that its function might also be reduced in mdd, especially in patients with elevated peripheral inflammatory biomarkers. however, to our knowledge, no data are available to support or disprove this idea. similarly, given the association of mdd with chronic inflammation, one would predict that the depression risk allele near the sirt gene should reduce sirtuin activity, if it is found to have a functional effect, which is currently unknown. however, these predictions should be tempered by findings suggesting a complex and contradictory role for sirtuin in pathogen defense. in certain contexts, such as the hypoinflammatory phase of sepsis, sirtuin blockade has been reported to reduce bacterial load and enhance survival. sirtuin blockade has also been shown to inhibit hepatitis b replication in hepatocytes (ren et al, ) . similarly, sirtuin-deficient mice have been shown to demonstrate improved intestinal anti-bacterial defense mechanisms (lo sasso et al, ) . on the other hand, the use of celecoxib to stimulate sirtuin within macrophages resulted in an enhanced ability of ampicillin to clear staphylococcus aureus from these cells (annamanedi and kalle, ) , consistent with the observation that low levels of sirtuin within macrophages associates with bacterial infection in these cells (zhang et al, a (zhang et al, , b . sirtuin also appears to be essential for optimal immune clearance of respiratory syncytial virus and has been shown to suppress human t-cell leukemia virus type transcription (owczarczyk et al, ; tang et al, ) . although a perusal of supplementary table s highlights the remarkable degree to which sirt and many other potential depression risk alleles impact both immune function and pathogen defense, it should be noted that there is another pathway whereby these risk alleles might enhance pathogen-host defense without directly modulating immune function. if the pathos-d proposal that depressive symptoms themselves serve pathogen defense functions is correct, genes that promote these symptoms might have served adaptive purposes in fighting infection via this mechanism alone. for example, in addition to multiple immune effects, sirtuin signaling in the hippocampus appears to have antidepressant effects, given animal data that pharmacologic and genetic inhibition of hippocampal sirt function increases depression-like behaviors in response to stress, whereas sirt activation blocks both the development of depression-related phenotypes and aberrant dendritic structures elicited by chronic stress exposure (abe-higuchi et al, ) . cacna c is another putative depression risk gene known to both enhance and impair pathogen-host defense depending on the microorganisms involved (raison and miller, ) . carriers of the rs a risk allele have also been shown in several studies to demonstrate changes in brain function and morphology relevant to mdd (bigos et al, ; erk et al, ; perrier et al, ) , again raising the possibility that even genes with known immune effects might enhance pathogen defense either primarily, or synergistically, via the direct induction of depression in response to environmental adversities known to increase the risk for pathogen-induced morbidity/ mortality (ie, stress signaling an increased risk of wounding, active infection; bigos et al, ; erk et al, ; perrier et al, ) . understanding the impact of putative risk genes like sirt or cacna c on inflammatory and behavioral responses to psychosocial stress would provide invaluable insight into the question of whether they promote depression and pathogen-host defense via the type of inflammatory activation that is a hallmark of mdd in the modern world. pathos-d proposes that depression risk alleles have been maintained in the human genome primarily because, by enhancing pathogen-host defense, they provided a net survival and reproductive advantage. a primary problem that this theory shares with other adaptive explanations for depression is that to date no incontrovertible risk alleles for mdd have been identified. indeed, most large gwas have failed to find snps that meet genome-wide significance and when such snps are found (ie, rs in dcnp and rs in tnf) they are not replicated in gwas conducted in other populations (raison and miller, ) . multiple explanations have been offered for this case of 'missing depression genes,' most often that mdd is heterogeneous in relation to its environmental antecedents, the nature and severity of its symptoms and its disease course; and that therefore it is likely that the condition is a concatenation of numerous as-yet-unidentified more etiologically homogeneous sub-conditions that are likely to be more genetically consistent. although not dismissing disease heterogeneity as a cause for failure to replicate depressive risk alleles, pathos-d offers an additional novel explanation for at least some cases of replication failure. because pathogens can develop resistance to host immune defenses, or in many cases actually evolve to benefit from these defenses via pathogen manipulation strategies, not all genetically programmed physiological or behavioral pathogen-host defense strategies will be equally effective against all microbes. because of this, in cultures/societies exposed to different mixtures of pathogens across time, different immune mechanisms will be adaptive, and therefore likely to become linked to depression. said differently, because of geographic and historical differences in pathogen exposure, we should not expect all human ethnic/racial groups to have benefitted from the same genetically driven pathogen defense strategies and therefore to show the same risk alleles for mdd. thus, a pathos-d perspective suggests that understanding the specific histories of shared and unique co-evolved pathogen-host interactions within and across human societies will be essential for enhancing our understanding of why genotype-phenotype associations observed in one population do or do not replicate across other populations, as well as why certain genetic variants drive physiological processes that induce depression in certain individuals but not in others. these insights may help account for the intriguing finding that the association between sir nor lhpp snps and mdd identified in han chinese females was not replicated in european populations (consortium c ( ) ). pathos-d would predict that this finding might be accounted for by the fact that sirt and lhpp conferred specific anti-pathogen defenses in the chinese environment that were not relevant in europe. if so, one would expect the prevalence of the sirt and lhpp risk alleles to be significantly higher in han populations than in europeans as a result of positive selection, and this is indeed the case (consortium c ( ) . many genes have been replicated across a range of ethnic/ racial groups as risk factors for schizophrenia, suggesting that at least certain pathways for the disorder are ancient and arose prior to the first migrations of modern humans out of africa. from a pathos-d perspective, the fact that no such replicated risk alleles have been found for mdd may suggest that the condition is more environmentally based than are conditions with more universally established genetic risk genes, but not in the way that this environmental primacy is usually understood. instead, mdd may have no universal risk genes because pathogen pressures in local environments over the last years have been pre-eminent in determining which specific immune mechanisms were most adaptively linked to the generation of depressive symptoms in any given environment. if it turns out that the association between depression and increased inflammation is a human universal (not universal in terms of all depressed individuals showing increased inflammation, but rather that groups of depressed individuals demonstrate increased inflammation across all ethnicities/cultures), then the clear prediction of pathos-d would be that alleles that confer depression risk in one population but not another should either ( ) increase inflammatory signaling more in the population in which they confer risk; or ( ) more powerfully increase the prevalence/ severity of depressive symptoms in response to any given degree of inflammatory activity in the population in which they confer risk. similarly, because present-day human societies/cultural groups have historically experienced widely different degrees of exposure to bacterial and viral pandemics that exerted extreme selective pressure (fincher et al, ) , pathos-d predicts that the types of immunity that best cope with these different types of pathogens should have different relationships with depression across human cultures. so, for example, because interferon signaling is especially essential for host defense against viruses, a pathos-d perspective would predict that individuals from cultures with higher rates of historical exposure to lethal viral pandemics should respond to treatment with ifn-α with increased rates of depression development, if indeed depressive symptoms aid in pathogen defense. were it possible to assess, this effect should be largest in hunter-gatherer populations that have evolved in the types of low population density environments that work against widespread viral infections. in such populations, viruses provided little positive selection pressure that would link antiviral defenses with depression, whereas such populations would have benefitted from optimal protection against environmental pathogens (eg, protozoa, bacteria, and fungi) that would have increased mortality under these conditions. were such a population available, the prediction is that they would show enhanced depressive symptoms in response to a bacterial stimulant such as endotoxin, while showing a muted depressive response to ifn-α. alas, given the escalating loss of isolated indigenous cultures, the time for such a definitive study may have passed. adaptive introgression provides another fascinating example of why pathogen-host defense processes may ensure that no universal genes for mdd will ever be found. research over the last several years indicates that~ % of the dna of non-african humans derives from multiple bouts of interbreeding with neanderthals, with current melanesian populations also showing a denisovan genetic inheritance from ancestral interbreeding with that group of now-extinct archaic humans . in general, the results of this interbreeding were deleterious, as attested to by multiple 'desert' regions of human dna depleted of archaic genetic material. against this backdrop, it is striking that other neanderthal and denisovan sequences appear to have undergone positive selection. from a pathos-d perspective it is even more striking that these archaic alleles are associated with both immune function and with an increased risk of depression (mendez et al, ; segurel and quintana-murci, ; simonti et al, ) . although it does not yet appear that the same neanderthal allelic variants code for both immunity and mood, anyone concerned with adaptive explanations for mdd cannot help but be struck by this association, especially given that negative selection appears to have removed archaic human dna from areas of the genome involved with other aspects of cns morphology and function. researchers tend to assume that retained archaic immune alleles provided an adaptive benefit for coping with novel eurasian pathogens; pathos-d would suggest that their proclivity to induce depression provided additional advantages in our endless struggles with microbes and parasites. here we consider ways in which pathos-d might provide adaptive explanations (as opposed to mere proximal/ mechanistic explanations) for three epidemiological features of mdd that are striking characteristics of the disorder and relate directly to sex: ( ) the : female preponderance of the disorder; ( ) the impact of age and sex on the disorder's phenotypic presentation; and ( ) the significant risk of the postpartum period for mdd development. females are approximately twice as likely as males to suffer from mdd (seedat et al, ) , with this imbalance being most pronounced during the reproductive years, exactly when the manifold negative social and biological effects of depression would be expected to impact evolutionary fitness most adversely (fiske et al, ) . while many answers for the female preponderance of depression have been proposed over the years (eg, females are more sensitive than males to the social environment), none have successfully identified an adaptive advantage that would outweigh the costs of female depression to survival and reproduction. in contrast, pathos-d suggests a testable explanation for why depression is so common in females of reproductive age. females are more likely to develop depression during the reproductive years because across evolutionary time depressive symptoms-having evolved out of sickness-promoted behaviors that decreased the risk of pathogen exposure and provided increased protection from pathogens for any given level of inflammatory activation once an infection had occurred (eg, by increasing energy available to immune processes by inducing conservation-withdrawal behavior, maintaining elevated body temperature, and so on). said differently, by inducing social and biological behaviors that promoted host defense against pathogens, depressive symptoms allowed females of reproductive age to 'get by' with less inflammation. the unique importance of this for reproductive success in females is highlighted by the fact that inflammation reduces fertility, impairs lactation and directs metabolic resources away from the multiple energetically-costly aspects of female biology that were required for successful reproduction in ancestral environments (van bodegom et al, ; schaller, ; kobayashi et al, ) . interestingly mdd has also been associated with reduced fertility (williams et al, ; nillni et al, ) . pathos-d theory predicts that this association should less prominent in females who have effectively 'replaced' inflammation with depression-based behavioral pathogen avoidance in the absence of elevated levels of inflammatory biomarkers. although most implications of this idea remain to be tested, some data support the first prediction of this hypothesis, which is that females should develop increased levels of depression for any given amount of inflammatory exposure. indeed, when compared with men, females respond to an injection of lipopolysaccharide (lps) with an increase in depression and feelings of social isolation, both of which are correlated with amount of cytokine response to the lps in females, but not males (moieni et al, b) . similarly, females are more likely than males to develop depression in response to chronic inflammatory stimulation resulting from treatment with the cytokine ifn-α (udina et al, ) . finally, given the pre-eminent role of psychosocial stress as an environmental risk factor for mdd, as well as evidence that hyperthermia enhances host defense, it is intriguing that reproductive aged females appear to be most vulnerable to developing psychogenic fever (kaneda et al, ). however, these findings need to be balanced against results from a small recent study indicating that females mounted an enhanced proinflammatory cytokine response to low-dose endotoxin when compared with men, while showing no difference in inflammation-induced mood or anxiety symptoms-a pattern of findings opposite to this pathos-d prediction (engler et al, ) . if confirmed in larger studies, these findings might support an alternative immune-based mechanism for the female/male preponderance of depression and anxiety, specifically that females respond to immune challenges with greater immune activation which subsequently increases the risk for depression/anxiety. mdd is most commonly characterized by impaired sleep and reduced appetite , but a significant minority of depressed individuals manifest hypersomnia and hyperphagia instead (thase, ). these symptoms are most frequent in females between adolescence and middleage (carter et al, ; posternak and zimmerman, ; matza et al, ; blanco et al, ) . how might pathos-d theory seek to account for the existence of these reversed neurovegative symptoms, as well as their age and sex distribution? a first step is to establish that cytokine pathways known to be activated by infection are capable of producing both hypersomnia and hyperphagia. hypersomnia has been recognized for years as a primary behavioral manifestation of proinflammatory cytokines (krueger and majde, ; krueger and toth, ; krueger and majde, ; opp, opp, , , and studies in healthy adolescents and adults indicate that chronically increased sleep is associated with increased saliva and blood concentrations of il- and crp (el-sheikh et al, ; patel et al, ; prather et al, ) . in terms of feeding behavior, animal models demonstrate that inflammation promotes carbohydrate preference (aubert et al, ) , such as is typical in depressive hyperphagia. less well known are data that highfat diets induce leptin and insulin resistance, as well as obesity, dependent upon activation of inflammatory mediators in the hypothalamus (zhang et al, ; kleinridders et al, ) . conversely, blocking hypothalamic inflammation prevents obesity and other stigmata of the metabolic syndrome, even in the context of high-fat consumption (zhang et al, ; milanski et al, ; posey et al, ). in rodents, exposure to either tnf or il- in utero results in adulthood obesity (dahlgren et al, ) , again suggesting that cytokines can induce hyperphagia/weight gain under certain circumstances. although we know of no data showing that females are more likely than males to respond to inflammatory signaling with hyperphagia, female rodents have less anorexia than males in response to influenza infection (avitsur et al, ) . because the presence of widespread obesity is recent, the question arises as to whether more ancient signals for inflammatory activation might also promote hyperphagia, instead of anorexia. we know of no data that directly address this issue in terms of infection, but note that certain adenoviruses have been associated with weight gain (atkinson, ; mitra and clarke, ) . in terms of psychosocial factors, preclinical studies suggest that stressors associated with a high degree of wounding-such as chronic social defeat-induce hypothalamic resistance to leptin (kleinridders et al, ; chuang et al, ) , consistent with their known ability to activate inflammatory pathways (avitsur et al, ; bailey et al, bailey et al, , powell et al, ) . as in depression, where hyperphagia and weight gain are associated with disease chronicity (posternak and zimmerman, ) , chronic social defeat leads initially to weight loss but to weight gain over time (chuang et al, ) . given the role of leptin resistance in hyperphagia, it is intriguing that increased peripheral leptin levels (consistent with leptin resistance) have been observed more consistently in females than in males with mdd (rubin et al, ; yang et al, ; pasco et al, ; zeman et al, ; cizza et al, ) , and are more common in atypical than non-atypical depression (gecici et al, ) . why might younger individuals in general, and females in particular, be more likely to develop depressive conditions characterized by hypersomnia and hyperphagia? from a pathos-d perspective, the simplest answer may be: because they can. in hunter-gatherer societies youth and females are frequently more protected from predation and to have food supplied to them by others (kaplan and gurven, ) , raising the possibility that in ancestral environments these individuals enjoyed sufficient security to partake of the recuperative effects of sleep and to eat without having to expend energy searching for food. given evidence that anorexia may confer anti-pathogen effects (raison and miller, ) , might similar benefits accrue to hyperphagia, especially in youth and females, under at least some evolutionarily relevant situations? although admittedly speculative, several lines of evidence suggest that by promoting hypothalamic resistance to leptin, genes associated with increased inflammatory signaling might enhance host defense by driving ongoing leptin production despite malnutrition in conditions of food scarcity. this increased leptin production would both augment the drive to seek and consume food and would lower the set point at which food consumption initiated the types of inflammatory responses that are all too common in the obese, but that appear to be life-saving in the face of an infection such as tuberculosis, that was (and is) most lethal for females of reproductive age and for which low body weight and reduced leptin are deadly (van crevel et al, ; wieland et al, ; buyukoglan et al, ; leung et al, ; pednekar et al, ; roth, ) . depression and anxiety are highly comorbid, and in both animals and humans, activation of the inflammatory response produces both types of symptoms. in a previous publication, we devoted significant space to providing a pathos-d perspective on why inflammation produces changes in brain function that subserve not just conservation-withdrawal symptoms likely to aid in defense against pathogens, but that also have been associated with hypervigilance symptoms (eg, anxiety/agitation, insomnia, anger/irritability (association ap, )) that across human evolution would have presumably siphoned metabolic resources away from the life-or-death task of immune protection (raison and miller, ) . we suggested that sickness behavior-while of benefit for surviving infectioncarries its own survival and reproduction costs as a result of increased risk for predation and reduced ability to care for one's young, as well as potential loss of status in a social species and/or loss of breeding territory (miller and cohen, ) , all of which were relevant to humans in the eea. given these competing challenges to survival and reproduction, evolutionary logic may have dictated that inflammatory processes-especially when chronic-might have promoted hypervigilant behavior, which, while shunting energy away from fighting infection, would nonetheless have served adaptive purposes by protecting against environmental dangers engendered by sickness. given that human females and youth likely received more protection in the eea than did adult males (who therefore on average would have more to lose from sickness behavior), a clear prediction from pathos-d is that males should respond to immune challenges/inflammatory activation with an increased ratio of hypervigilant to conservationwithdrawal symptoms, as well as an increase in cytokine effects on brain areas such as the dorsal anterior cingulate that subserve hypervigilance when compared with cytokine effects on areas such as the ventral striatum, which have been linked to inflammatory effects on anhedonia (felger et al, ) , which would be expected to promote social withdrawal. available data provide mixed support for this hypothesis. on the one hand. a recent study found no difference between males and females in the severity of anxiety symptoms induced by low-dose endotoxin (engler et al, ) . on the other hand, a recent large cohort study reported that peripheral inflammatory biomarkers were more strongly associated with anxiety symptoms and anxiety disorders in males than in females (duivis et al, . more definitive confirmation or refutation of the possibility that males respond to inflammation with increased hypervigilance await studies specifically designed to assess the modulating effect of sex on the impact of cytokines on hypervigilant-specific behaviors and on neural pathways known to mediate this type of behavior. given significant evidence that maternal depression in the postpartum period is associated with numerous short-and long-term adverse consequences for the affected offspring (ghodsian et al, ; stein et al, ; beardslee et al, ; beck, ; halligan et al, ; kersten-alvarez et al, ) , the question of why the condition is so common is especially pressing. as with all evolutionary considerations, the first question to consider is whether risk genes that promote postpartum depression (ppd) confer an adaptive benefit, at least in part, via benefits resulting from the condition itself or whether ppd is pleiotropically linked to non-related effects of these genes that benefitted survival and/or reproduction across evolution in ways that outweighed the cost of ppd. from a pathos-d perspective, the question might be framed by asking whether ppd enhanced maternal survival and/or reproduction via an overall net benefit in pathogen-host defense. in terms of maternal survival, ppd would be expected to serve the same pathogen defense purposes as mdd in general. however, what about the survival of the newborn, which reflects a significant investment on the mother's part in her long-term reproductive success? might maternal depression contribute to the child's pathogen-host defense prospects? an interesting twist on this theme might be provided by parental investment theory, which postulates that parents reduce their care for, and involvement with, offspring when the costs of such care and involvement outweigh the benefits (trivers, ) . building on this theory, it has been argued that ppd is an evolved mechanism that aids mothers in disengaging from infants with a low chance of surviving (hagen, ; bottino et al, ) . because infection was the primary cause of infant mortality across human evolution, one might predict that ppd would be more likely to develop in response to giving birth to an unhealthy infant, and data from hunter-horticulturalists in the amazon basin are consistent with this prediction (hagen and barrett, ) . under this scenario, ppd, rather than helping the infant survive pathogen challenge, would actually serve as a 'warning signal' to the mother that the infant's likelihood of surviving postnatal pathogen exposure was not high enough to warrant significant resource investments. although consistent with a pathos-d focus on the primacy of pathogen-host interactions in selecting for depression-promoting genetic variants, the notion that ppd functions as a type of immunological abandonment strategy goes against the general tenor of the theory, which sees the link between depression and immunity as providing overall advantages in the face of infectious morbidity and mortality, whatever their costs in other domains. whether ppd is associated with behavioral or immune changes that might actually help infant survival-especially in conditions such as high environmental pathogen exposure, reduced metabolic resources or increased infant vulnerability to infection-is a question that to our knowledge has never been examined. certainly it is possible that ppd-induced maternal withdrawal might have decreased the risk of infant infection in circumstances when the maternal depression resulted from pathogen-induced cytokine activation. it is also intriguing to speculate that ppd might influence the composition of breast milk in ways that might aid nursing infants in avoiding or surviving infection. intriguingly, a japanese study found ppd to be associated with increased breast milk concentrations of the cytokine transforming growth factor (tgf)-β . tgf-β has complex pro-and antiinflammatory effects (kondo et al, ) , but in the context of the newborn immune system, it has been shown to have a key role in establishing the mucosal immune response, including production of secretory immunoglobulin a (ogawa et al, ; oddy and rosales, ) . also of potential relevance, reduced levels of tgf-β have been associated with increased symptom severity in children infected with plasmodium falciparum (chaiyaroj et al, ; rovira-vallbona et al, ) , suggesting that the provision of increased tgf-β in breast milk from depressed mothers might confer some protection against malaria, which has been a primary source of pathogen-induced mortality across human evolution. importantly, tgf-β and other immune factors in breast milk remain biologically active after passage through the infant stomach as a result of its higher ph (hennet and borsig, ) . in essence, pathos-d does nothing more than attempt to instantiate and provide specifics to the observation from the quote that started this article, that 'the only…compensating fitness benefit that has been documented for major human genetic diseases is resistance to infection' (cochran et al, ) . we have argued that depression and its relationship with inflammation and in particular the acute phase response is yet another example of this evolutionary reality, providing insights into the puzzling lack of consistency of risk alleles across populations as well as the epidemiology of the disorder including its risk factors in ancient and modern times and its age of onset and female preponderance. funding for this study was provided for clr by mary sue and mike shannon, the usona institute and the university of wisconsin-madison and for ahm by r mh ; r mh ; supported in part by the national center for advancing translational sciences of the national institutes of health under award number ul tr . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. study funders had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. dr raison serves on the scientific advisory board for usona institute. dr miller reports no conflicts of interest. hippocampal sirtuin signaling mediates depression-like behavior targeting sirtuin- alleviates 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to clinical progression of viral illness key: cord- -hfau odb authors: wang, rong; zhang, yan-jin title: antagonizing interferon-mediated immune response by porcine reproductive and respiratory syndrome virus date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: hfau odb interferons (ifns) are important components in innate immunity involved in the first line of defense to protect host against viral infection. porcine reproductive and respiratory syndrome virus (prrsv) leads to severe economic losses for swine industry since being first identified in early s. prrsv interplays with host ifn production and ifn-activated signaling, which may contribute to the delayed onset and low level of neutralizing antibodies, as well as weak cell-mediated immune response in infected pigs. prrsv encodes several proteins that act as antagonists for the ifn signaling. in this review, we summarized the various strategies used by prrsv to antagonize ifn production and thwart ifn-activated antiviral signaling, as well as the variable interference with ifn-mediated immune response by different prrsv strains. thorough understanding of the interaction between prrsv and host innate immune response will facilitate elucidation of prrsv pathogenesis and development of a better strategy to control prrs. porcine reproductive and respiratory syndrome (prrs) is an important infectious disease, causing huge economic losses to the swine industry worldwide [ , ] . the prrs clinical signs include respiratory disorders, abortion in pregnant sows, and variable mortality in piglets. prrs was first identified in the usa in and subsequently in europe. the causative agent of the disease is the prrs virus (prrsv), a positive-sense single-stranded rna virus, belonging to the arteriviridae family in the order nidovirales [ ] . according to the genetic differences, prrsv is grouped into two genotypes: european (type ) and north american (type ), represented by lelystad virus (lv) and vr- strains, respectively. the genome of prrsv is about kb in length with open reading frames (orfs) [ ] . orf a and orf b comprise % of the viral genome and encode viral enzymes involved in virus replication. in addition, polypeptides from the two orfs are processed into nonstructural proteins (nsps), including nsp , nsp , nsp , nsp tf, and nsp ∼ [ , ] . orf a, orf b, orf through orf , and orf a code for eight structural proteins: gp , envelop protein (e), gp ∼ , membrane protein (m), nucleocapsid protein (n), and orf a protein [ , ] . swine are the only known host of prrsv. prrsvinfected pigs develop a delayed onset of neutralizing antibodies and a weak cell-mediated immune response [ , ] . the main target cells for prrsv infection in vivo are porcine pulmonary alveolar macrophages (pams), which play a crucial role in host immune response [ ] . in order to successfully invade host, prrsv has evolved various strategies to interfere with host innate immunity. some of the prrsv proteins take part in the modulation of ifn-mediated immune response. host innate immune responses play a key role against early viral infection. interferons are major components of inmate immunity and have diverse biological functions including antiviral activity, antiproliferative activity, stimulation of t cell cytotoxic activity, and modulation of immune response [ ] . there are three types of interferons. in human, type i interferons include ifn-, ifn-, ifn-, ifn-, and ifn- [ , ] . in addition, ifn-, ifn-, and ifn-(or limitin) have been identified as type i ifns in swine, ruminant, and mice, respectively [ ] . almost all cell types are capable of producing ifn-/ ; however, plasmacytoid dendritic cells (pdc) are considered to be the major source of ifn-production during viral infection [ , ] . type ii ifn contains only ifn-, whose production is restricted to activated t cells, natural killer cells, and macrophages [ ] . type iii ifns comprise ifn- , ifn- , and ifn- (also known as interleukin-(il-) , il- a, and il- b, resp.), which are mainly generated by dendritic cells [ ] . considering the major roles in antiviral response by type i ifns, we focus on this type of ifns and discuss the prrsvmediated interference with their production and signaling. this review summarizes the recent advances in the research of prrsv interference with ifn-mediated innate immunity, the viral proteins involved, and their molecular mechanisms, as well as diverse effects by different strains and in different cell types. a few relevant reviews on prrsv interplay with innate immunity were published previously [ ] [ ] [ ] . readers are encouraged to read them if interested as these reviews were written in different angles to address the issue with diverse scopes, though there is some overlap in certain topics. this review is arranged into sections of ifn induction, ifn-activated signaling, ifn-stimulated genes, and perspective. host pattern recognition receptors (prrs) for rna viruses include toll-like receptors (tlrs) and rig-(retinoic acid inducible gene-) i-like receptors (rlrs). tlrs that can detect viral rna are tlr , tlr , and tlr [ ] . the rlr family of prrs comprises rig-i and melanoma differentiationassociated gene (mda- ) [ ] . both rig-i and mda- signal through adaptor ips- (also known as mavs, cardif, and visa) on the outer membrane of the mitochondria [ ] . tlr and rlr can recognize double-stranded rna (dsrna) of viral genome or replication intermediate of rna viruses. activation of tlr and rlr signaling pathways leads to activation of interferon regulatory factor (irf ), irf , and nf-b. these transcription activation factors translocate into the nucleus and result in induction of type i ifns and expression of inflammatory cytokines, which not only lead to an antiviral state of the neighboring uninfected cells, but also serve as key regulators to evoke adaptive immune response. at least functional type i ifn genes have been identified in porcine chromosomes and [ ] . these ifn genes include ifn-subtypes, ifn-, ifn-, ifn-, ifn-, ifn-, and ifn-. prrsv is sensitive to type i ifns and the sensitivity is confirmed in vitro and in vivo. pretreatment of pams with porcine ifn-resulted in significant reduction of prrsv yield [ ] . pretreatment of marc- cells and porcine pulmonary alveolar macrophages (pams) with porcine ifninhibited prrsv replication [ ] . pigs that were inoculated with recombinant adenovirus for ifn-expression and challenged one day later with prrsv had lower febrile responses, reduced lung lesion, and delayed viremia and antibody response compared to controls [ ] . therefore, for invading host immune clearance, prrsv has evolved multiple strategies to antagonize the host ifn induction. cells. prrsv appears to inhibit synthesis of type i ifns in pigs infected with type strains, while swine transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) induced high level of ifn- [ , ] . ifncould not be detected in the lungs of pigs in which prrsv actively replicated. it was estimated that the ifn-inducing capacity of prrsv is at least -fold lower than that of prcv [ ] . prrsv infection of pams leads to no ifn-production and when the cells were superinfected with tgev, no ifnwas detected either [ ] . the prrsv suppression of ifn induction correlates with the virus replication. plasmacytoid dendritic cells (pdcs) are thought to be the major source of ifnin vivo. prrsv fails to induce porcine pdcs to produce ifn-, while pseudorabies virus (prv), swine influenza virus (siv), and tgev stimulated the pdcs to synthesize ifn- [ , ] . moreover, presence of prrsv markedly reduced the typical ifn-response of pdcs to tgev or toll-like receptor agonist. loving et al. showed that prrsv replicated in monocyte-derived dcs but not lung dcs and the response of both cell types to prrsv was only limited to ifn-transcription [ ] . additionally, for marc- cells prrsv replication also significantly inhibited the dsrna-induced type i ifn expression [ ] [ ] [ ] . these data suggest that prrsv infection directly interferes with type i ifn induction in vivo and in vitro. the prrsv proteins that are found to be antagonists of ifn induction include nsp , nsp , nsp , and n ( figure and table ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nsp , a c-like serine protease that is responsible for most of the nonstructural protein processing [ ] , was found to inhibit ifn-promoter activation in a reporter assay [ ] , but no further characterization was reported. further study is needed to elucidate the mechanism. also nsp has been studied in more detail than others. nsp is self-cleaved into nsp and nsp subunits, both of which mainly localize in the cell nucleus and dramatically inhibit ifn-expression by affecting the irf signaling pathway [ ] . ifn-promoter reporter assay was performed in hek t cells in the study. the result showed that nsp and its two cleavage products, nsp and nsp , inhibited the activation of ifn-promoter (p -luc) and an artificial promoter containing three irf binding sites (p -cib-luc) after sv stimulation. prrsv nsp and nsp blocked the induction of ifn-at downstream of irf activation but had no effect on the phosphorylation and translocation of irf . it suggested that nsp and nsp may have a direct effect on the formation of the transcription enhanceosome on the ifnpromoter inside the nucleus. kim et al. showed that nsp inhibited irf association with creb-binding protein (cbp) in the nucleus but had no effect on irf phosphorylation figure : interference of type i ifn production by prrsv proteins. activation of rlr pathway and signaling by viral dsrna is shown. viral dsrna is generated during prrsv replication. "p" besides irf and irf indicates phosphorylation. red-colored blocks indicate prrsv proteins known to inhibit the signaling molecules indicated. prrsv nsp inhibits irf association with cbp, enhances cbp degradation, and interferes with i b degradation. and nsp inhibits irf phosphorylation and nuclear translocation. also nsp inhibits irf phosphorylation and nuclear translocation, interferes with i b polyubiquitination, and prevents its degradation. and nsp inhibits irf phosphorylation and nuclear translocation via degrading ips- mrna. ifn signaling blocking stat /stat nuclear translocation [ ] and nuclear translocation [ ] . immunoprecipitation with anti-irf antibody found that interaction between cbp and irf in prrsv-infected marc- cells or nsp -transfected hela cells was weaker than in cells treated with polyi:c alone [ ] . the expression of nsp also enhanced cbp degradation, which can be rescued by mg treatment, a proteasome inhibitor. no interaction between nsp and cbp was found. the process is independent of the pcp activity of nsp [ ] . beura et al. showed that nsp interfered with irf signaling pathway by inhibiting dsrna-induced irf phosphorylation and nuclear translocation [ ] . the discrepancy is possibly because an nsp that is -residue longer than its authentic form was used in beura's study. another possible reason is that different prrsv strains were used as a couple of other studies showed that prrsv replication significantly blocked dsrna-induced irf activation in marc- cells [ , ] . and nsp was also found to downregulate irf protein level and inhibit its phosphorylation [ ] . in our laboratory, we observed that prrsv infection of marc- cells led to reduction of irf protein level (unpublished data). luo et al. [ ] showed prrsv blocked ifn-production and irf nuclear translocation via significantly inhibiting activation of ips- in rig-i signaling pathway. the structure-function studies of nsp and nsp identified critical motifs of the proteins in inhibition of ifn induction. the zinc-finger (zf) domain in the c-terminus of nsp is critical for this protein to antagonize both ifninduction and nf-b activation, especially the amino acids at c-terminal of the nsp [ ] . shi et al. [ ] screened a series of nsp c-terminal truncated mutants and revealed that the amino acid residue f of nsp is essential for the inhibition of ifn-induction. the residue f played a role in both tlr signaling and rig-i signaling pathways [ ] . double mutations k a/r a (type prrsv) or k a/r a (type prrsv) in a highly conserved motif of nsp , gkylqrrlq, dampened the nsp inhibition of ifn induction [ ] . moreover, recovered recombinant viruses with the nsp mutations by reverse genetics induced higher level gene expression of type i ifns than that of wild type viruses. also nsp inhibits ifn induction by blocking irf phosphorylation and nuclear translocation. the cysteine protease domain (pl ) of nsp was necessary for antagonizing activation of irf pathway [ ] . the cysteine protease domain (pl ) at the n-terminus of nsp , which belongs to the ovarian tumor (otu) protease family, was shown to inhibit type i ifn induction by interfering with the nf-b signaling pathway [ ] . the otu domain possesses deubiquitinase activity, which interferes with the polyubiquitination of i b and subsequently prevents its degradation. recovered recombinant viruses with mutations in the otu domain of nsp by reverse genetics were found to be unable to inhibit nf-b activation as effectively as the wild type virus. nsp is an endonuclease [ ] and ifn antagonist [ ] . nsp suppressed the activation of ifn-promoter and the expression of irf -mediated genes. the endoribonuclease activity of nsp was essential for nsp to inhibit dsrnainduced ifn-induction [ ] . the amino acid residue h of nsp , a presumed catalytic histidine, was involved in the inhibition of irf phosphorylation. it seems that the inhibition of irf activation is due to the nsp endoribonuclease activity, which can cleave mrna of ips- [ ] , the adaptor molecule for rig-i and mda- . the ifn antagonizing activity is not restricted to nonstructural proteins of prrsv. structural proteins, such as the n protein, were found to downregulate ifn-mrna level in polyi:c-treated immortalized pam cells [ ] . n protein interferes with dsrna-induced phosphorylation and nuclear translocation of irf . the multiple components of prrsv are involved in the interference with ifn induction. the nsps are early proteins and n is a late one, which may play roles at different stages of viral replication. induction. the effect of prrsv replication on ifn induction appears to be variable among different strains and different cell types. prrsv field isolates have variable suppressive effect on ifn-induction in pam cultures and the suppression was found at posttranscriptional stage [ ] . this is not unexpected as prrsv strains are divergent in genomic sequences. prrsv infection of monocyte-derived dendritic cells (mo-dc) induced the transcription of ifn-/-but no detectable ifn-in culture supernatant, suggesting a blockage at posttranscriptional stage [ ] . prrsv activated the transcription of ifn-in a pi k-dependent manner in mo-dc cells. prrsv infection of marc- cells inhibits ifn gene expression [ ] by interfering with the ips- activation in the rig-i signaling pathway [ ] . a variety of type and type prrsv strains were found to stimulate ifnsecretion by pdc via tlr pathway and the effect did not require live virus [ ] . the suppressive effect on pdc may be strain dependent. a novel isolate, a mc , induced ifns in both marc- and pam cells and virus replication was needed for ifn induction [ ] . ifn- and elevation of isgs were detected in a mc -infected cells. sequencing analysis indicated that a mc was closely related to vr- and ingelvac prrs mlv with an identity of . % at the nucleotide level [ ] . there were a total of nucleotide (nt) variations when compared to vr- , resulting in amino acid changes scattered from nt to the end of the genome. compared to both vr- and mlv, a mc has unique nucleotides. yet the mechanism of this strain inducing ifns is not known and it is intriguing to note that the first . kb of its genome is identical to vr- . its nsp and nsp proteins should be able to inhibit ifn induction. we hypothesize that the unique nucleotides in a mc genome resulting in special rna structures or unique dsrna formation during early viral replication could evoke ifn production. it is worth to note that the induction of ifns is dose dependent. the virus is able to replicate when the inoculum is at less than . moi, which induces limited ifns that cannot suppress the virus replication [ ] . a mc infection of pigs resulted in earlier onset and higher level neutralizing antibody against homologous and heterologous strain than mlv vaccine strain that is highly homologous in sequence [ ] . type i ifns are critical to innate immunity against viral infections and play an important role in the stimulation of adaptive immune response [ , ] . the activation of ifn signaling leads to the induction of antiviral responses. the signaling of type i ifns is initiated after they bind to their receptors on the cell surface [ ] [ ] [ ] . this receptor binding activates janus kinases (jak), which phosphorylates both the signal transducers and activators of transcription (stat ) and stat . the phosphorylated stat and stat form heterodimers, followed by interaction with interferon regulatory factor (irf ) and subsequently formation of heterotrimers, also known as interferon-stimulated gene factor (isgf ). translocation of isgf into the nucleus followed by binding to consensus dna sequences leads to the expression of ifnstimulated genes (isgs), which then take part in antiviral responses. [ ] [ ] [ ] . prrsv replication in marc- cells suppresses jak/stat signaling stimulated by addition of ifn-to the culture [ ] . transcripts of isg , isg , and protein stat were significantly reduced compared to mockinfected cells. the phosphorylation of both stat and stat was unaffected. immunoprecipitation with stat or stat antibody for marc- cell lysates was performed and the result showed no significant difference for stat /stat heterodimer formation between prrsv-infected and mockinfected cells. further study showed that the nuclear translocation of stat /stat heterodimer was blocked. prrsv infection of pam cells also blocks jak/stat signaling shown by reduction of isg expression after stimulation with external ifn-, while a vaccine strain ingelvac prrs mlv had little effect, possible due to its less efficient replication in the primary cells [ ] . signaling. prrsv proteins that interfere with ifn-activated signaling include nsp , nsp , nsp , gp , and n ( figure and table ) [ ] [ ] [ ] . prrsv nsp was found to block the nuclear translocation of stat and significantly inhibit the expression of isgs [ ] . by observing stat -gfp distribution under fluorescence microscopy, chen et al. [ ] noticed that stat -gfp accumulated in cytoplasm in hek t cells with nsp expression. further studies on nsp revealed that it induced the degradation of karyopherin-alpha (kpna , also called importin-alpha ), which is known to mediate the nuclear import of isgf [ ] . the n-terminal domain of nsp was involved in the ubiquitin-proteasomal degradation of kpna . residue of nsp was found to be essential in inducing kpna degradation and inhibiting ifn-mediated signaling as the residue change from valine to isoleucine diminished the suppressive effect. notably, prrsv infection of marc- cells by vr- and vr- also reduces kpna expression, whereas infection by ingelvac prrs mlv does not. mlv nsp has no effect on kpna expression or ifn signaling but gains the suppressive function when residue is changed to valine [ ] . other prrsv proteins including nsp , nsp , gp , and n were also found to be able to inhibit ifn-activated signaling [ ] . n protein inhibits ifn-activated stat nuclear translocation, albeit less effective than nsp [ ] . signaling. variable effect on ifn signaling among prrsv strains was found [ ] . among six prrsv strains (vr- , ingelvac prrs mlv, vr- , nvsl - , mn , and lelystad) tested, all but mn inhibited ifn signaling in marc- cells, and all but mlv and nvsl - blocked the ifn activation in pams. the result also demonstrated that the interference with ifn signaling by prrsv was variable in different infected-cell types. nsp from the six strains was cloned and all but mlv nsp inhibited ifn signaling when overexpressed [ ] . the ifninducing a mc strain has no effect on jak/stat signaling activated by ifn- [ ] . type i ifns (e.g., ifn-and ifn-) drive the expression of more than genes that encode proteins with antiviral, antiproliferative, proapoptotic, and proinflammatory functions [ ] . among the antiviral isgs, the best studied ones are , -oligoadenylate synthetases (oass), ribonuclease l rnasel, the dsrna-activated protein kinase (pkr), p (isg , interferon-induced protein with tetratricopeptide repeats (ifit )), mx (myxovirus (influenza virus) resistance ), and isg . prrsv nsp was shown to inhibit the antiviral function of ifn-stimulated gene (isg ) by the deubiquitinase activity of the otu domain of nsp ( figure and table ) [ ] . isg is an ubiquitin-like antiviral protein [ , ] . isg conjugation (isgylation) to substrate proteins follows a process similar to that of ubiquitin conjugation. isgylation of many important immune-related molecules leads to the activation of host innate immune response. as mentioned above, the cysteine protease domain (pl ) at the n-terminus of nsp belongs to otu-containing superfamily of proteases (dubs), which possesses deubiquitinating activity [ ] . sun et al. revealed that nsp was an antagonist for the antiviral activity of isg by reducing isg production and conjugation. the n-terminal pl domain of nsp was crucial for the antagonizing function. pkr mediates translational control by phosphorylating the protein translation initiation factor eif , resulting in inhibition of protein synthesis and viral replication [ ] . addition of -aminopurine ( -ap), an inhibitor of pkr, restored prrsv replication in ifn--treated cells [ ] . addition of -ap to recombinant swine ifn--primed marc- cells restored prrsv replication but did not rescue the virus in ifn--primed pam cells [ ] . these results showed the important role of pkr in the ifn-activated antiviral figure : interference with type i ifn-activated jak/stat signaling pathway and antiviral isgs. ifn-/-binds to their receptors ifnar- and ifnar- on cell membrane, which activates jak/stat pathway. "p" besides stat and stat indicates phosphorylation. prrsv nsp inhibits isgf nuclear translocation via inducing degradation of kpna , which is essential for mediating the nuclear import of isgf . n protein also inhibits isgf nuclear translocation. and nsp reduces isg production and conjugation via its deubiquitination activity. signaling. we found that prrsv is able to inhibit the polyi:cinduced activation of pkr, as well as its downstream effector eif (unpublished observation). it is not known if prrsv interferes with other isgs. considering the important roles of the isgs in deterring invading pathogens, one can imagine that prrsv must have evolved strategies to evade them during its replication. further study on the interplay of prrsv and isgs will provide insights into such strategies. prrsv infection in pigs leads to delayed production and low titer of neutralizing antibodies [ ] , as well as weak cellmediated immune response [ ] . partly the reason is possibly because of prrsv interference with ifn-mediated innate immunity. prrsv infection appears to inhibit synthesis of type i ifns in vivo and in vitro with the exception of some atypical strains that induce ifn production, such as a mc . the mechanism for the interference is at multiple steps from inhibition of irf activation, cbp interaction with irf , and posttranscriptional suppression. prrsv also block ifn-activated signaling, which results in suppression of the expression of antiviral isgs. the mechanism is prrsvmediated blocking of isgf nuclear translocation. the prrsv interference with the innate immunity is at multiple levels, from ifn induction and ifn-activated signaling to activity of isgs. given the divergence of prrsv strains in sequences, variation of these activities in innate immunity is not a surprise, whereas the multifold interplay between the virus and host may determine the consequences. in addition, type i ifns are proinflammatory cytokines. the protective effect of ifns in vivo may be 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biomedicine nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene a human -kda ifn-induced protein induces the secretion of ifn ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses the double-stranded rnadependent protein kinase pkr: structure and function inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with -aminopurine marked differences between marc- cells and swine alveolar macrophages in ifn -induced activation of antiviral state against prrsv experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord- -dtwhd zy authors: ivanova, daria; krempels, ryan; ryfe, jennyfer; weitzman, kaitlyn; stephenson, david; gigley, jason p. title: nk cells in mucosal defense against infection date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: dtwhd zy conventional natural killer cells (nk cells) provide continual surveillance for cancer and rapid responses to infection. they develop in the bone marrow, emerge as either nk precursor cells, immature, or mature cells, and disperse throughout the body. in the periphery nk cells provide critical defense against pathogens and cancer and are noted to develop features of adaptive immune responses. in the tightly regulated and dynamic mucosal tissues, they set up residency via unknown mechanisms and from sources that are yet to be defined. once resident, they appear to have the ability to functionally mature dependent on the mucosal tissue microenvironment. mucosal nk cells play a pivotal role in early protection through their cytolytic function and ifnγ production against bacteria, fungi, viruses, and parasitic infections. this review presents what is known about nk cell development and phenotypes of mucosal tissue resident conventional nk cells. the question of how they come to reside in their tissues and published data on their function against pathogens during mucosal infection are discussed. dissecting major questions highlighted in this review will be important to the further understanding of nk cell homing and functional diversity and improve rational design of nk cell based therapies against mucosal infection. natural killer cells (nk cells) are a first line of defense against invading pathogens and cancer. recent studies focused on development and functional diversity of innate immune cells have led to the reclassification of these cell types into a large group known as innate lymphoid cells (ilcs) [ ] . this is due to their origin from the common lymphoid progenitor (clp) but unlike their t cell and b cell counterparts, they do not activate the recombination activation genes (rga / ) and do not undergo antigen receptor rearrangement. there are three main groups, group , of which conventional nk cells are members, group , and group . each grouping is based on the functionality and transcriptional regulation of cell type development. nk cells are members of group ilcs due to their ability to produce ifn and be cytolytic. their activation and function rely on recognition of pathogeninfected cells through activating receptors (kirs in humans and ly in mice) and proinflammatory cytokines. nk cells can also regulate immunity. during systemic infections they produce il- and with high viremia can target dcs and t cells, thus modifying immunological memory [ ] [ ] [ ] [ ] . as such, nk cells have many roles, in protection, in helping to maintain immune homeostasis, and in long term immunity. nk cells are found in many tissues. this includes bone marrow (bm), blood, liver, thymus, and spleen. mucosal sites that harbor nk cells include the lung, the small and large intestine and colon of the gastrointestinal tract (gi), and the uterus, cervix, ectocervix, and vagina of the female reproductive tract (frt). much of how they gain access to these sites and provide function (protection, immunoregulation) is just beginning to be understood. the review focuses on recent work and the current understanding of the regulation of mucosal tissue residency of nk cells and nk cell functional importance at mucosal sites relevant to both mouse and human systems. we will not address ilc and ilc populations as those have been reviewed elsewhere [ , ] . in humans and mice, nk cells develop from the common lymphoid progenitor (clp) in the bone marrow [ ] . clps in the mouse bm differentiate into a pre-nk precursor biomed research international (pre-nkp) with a phenotype of lin − cd − cd + and express some nk cell specific receptors including nkg d and b (cd ) and negative for classical nk cell markers nk . and cd b. pre-nkp then express the -chain receptor for il- (cd ) and cd b and are now defined to be nk precursors (nkp). il- is a cytokine required for their development as shown by il- ko mice, which predominantly lack nk cells [ ] . interestingly, infection of il- ko mice with the protozoan parasite toxoplasma gondii or il- ko, il- r ko, and rag /il- r ko mice with mcmv infection results in rapid expansion of nk cells [ , ] . these studies support il- as an important cytokine for promoting nk cell development in the absence of infection. however, they demonstrate that other non--chain cytokines such as il- during infection can stimulate nk expansion and activity independent of il- . once cd is expressed, nkps now become responsive to il- and develop further into immature nk cells (ink) marked by cd lo cd b lo and the progressive expression of activating and inhibitory receptors. murine nk cells have been further defined based on the expression of cd and cd b into stages of maturation that correspond with their production of inflammatory cytokines and cytolytic activity [ , ] . after the ink stage they progress in phenotype from the cd lo cd b lo to cd hi cd b lo then from cd hi cd b hi to cd lo cd b hi . human nk cells develop from hematopoietic stem cells (hscs) through a clp as in mice with differentiation points designated as development stages (stages - ) [ ] . stages and cells are cd − and cd − and become responsive to il- . stages and cells acquire the expression of the human classical nk cell marker cd and are now considered to have differentiated into the nk cell phenotype. stage cells are cd bright fc riii − (cd − ), produce high levels of cytokines, have low cytolytic capabilities, and are considered immature. stage cells are cd dim cd + , are both cytolytic and capable of producing high amounts of ifn , and are considered mature. nk cells are present systemically in bone marrow, secondary lymphoid structures, peripheral nonlymphoid organs, and blood. in lymph nodes, the majority of nk cells are considered immature and are cd bright cd − in humans and cd hi cd b lo-med are in mice [ , ] . in nonlymphoid organs, such as gut and lung, resident conventional nk cells have varying phenotypes based on maturation and function. the factors governing the phenotype and function of resident nk cells at these sites are unknown. however, nk cell diversity at these different sites is likely dependent upon the integration of environmental signals to promote their needed activity. additionally, the ultimate source (blood, bm, or lymph node (ln)) of resident nk cells in these organs is not well described. while human and murine nk cells undergo the above-mentioned developmental stages, once they exit the bm could migrate to either the blood stream or into secondary lymphoid structures. whether immature nk cells from ln or more mature cells from the blood seed peripheral tissues during the steady state is unclear. recently, liver nk cells were demonstrated to be distinct in development from thymic and splenic nk cells [ ] . therefore mucosal tissue specific nk cells could also differentiate in situ rather than be seeded by ln or peripheral blood precursors. regardless, there are several necessary steps for this post-bone-marrow phase of nk cell development and function at mucosal sites. these steps include migration, changes in phenotype, education, and maturation. in addition to what controls homing of nk cells to mucosal tissues, the mechanisms behind how mucosal nk cells adjust to their resident environments are unclear and will be important to dissect. the current model of nk cell development and migration suggests that nk cells likely emerge from bm as a mix of mature and immature cells. immature cells mature and acquire organ specific phenotypes in the extramedullary tissues including secondary lymphoid tissues and liver [ ] [ ] [ ] [ ] [ ] . mature nk cells circulate to different tissues and are then modified by tissue microenvironments via cytokine milieu, growth factors, or chronic inflammation [ , ] . migration from bm to a specific tissue is a complex and critical first step to establishing residency. this process is likely different for each nonlymphoid tissue and has not been well described. a good example of the complexity of this process is how specific cd t cell populations are recruited to the gut to become intraepithelial lymphocytes (iels). intestinal mucosa homing of iels requires a priming event near the tissue itself or in the mesenteric lymph node (mln). priming is dependent upon an interaction with mucosal ccr + cd + dcs and acquisition of integrin and ccr expression [ ] [ ] [ ] [ ] . given the intense research in this area, the gut iel model for tissue homing may be a useful starting point to base mechanistic studies on nk cell recruitment to mucosal nonlymphoid tissues. . . resident nk cells of the lung mucosa. % of total lung resident lymphocytes are nk cells. this is a higher nk cell number than other nonlymphoid organs highlighting their importance in this tissue [ ] . they are primarily found along with other lymphocytes in the lung epithelium and vascular tissues [ , ] . in humans, lung resident nk cells are cd dim cd + and mostly nkp + similar to nk cells in the blood suggesting a mature phenotype capable of being cytotoxic and producing cytokines [ ] . in mice the more mature nk cell phenotype is found in the lung epithelium, which is cd lo cd b hi , very similar in function to the human subset. in the naïve state, these two phenotypes of nk cells in both human and mouse compose - % of all nk cells present in the lung tissue [ , ] . the maturation status of most lung nk cells resembles those from blood. however a recent study identified a population of nk cells in the lung capable of being further differentiated [ ] . this study demonstrated that, unlike bone marrow precursors, the lung precursor cells when cultured in vitro expressed more ly receptors. these results suggest that both mature and immature nk cells are present in the lung and that the murine lung microenvironment could condition nk cells separately from the bone marrow. in humans, other than classical nk cell marker phenotype (cd and cd ) approximately % of lung resident nk cells express kirs including kir dl , kir dl , kir dl and kir ds . nearly % of the cd bright population expressed cd similar to the phenotypes found in peripheral blood [ , ] . murine and human lung microenvironments may differ in their ability to modify resident nk cells. in humans support for tissue specific microenvironment conditioning of nk cells comes from studies of pulmonary sarcoidosis and nonsmall-cell lung cancer [ , ] . nk cells express less kir in bronchiolar lavage fluid (balf) in these situations than in the naïve state. interestingly, peripheral blood nk cells also had lower kir expression. whether or not the change observed in balf was from migrating cells is not known. the observations in balf support a role for tissue specific microenvironment conditioning of nk cells. however, the more mature phenotype of resident nk cells in the lung is suggestive of a blood origin ( figure ). currently, much is still not known about the mechanisms behind these changes and whether in the healthy state tissue resident human nk cell phenotypes can be modified by the lung. of the three innate lymphoid cell populations found in the gut, conventional nk cells of the gut are classified to belong to the ilc subset [ ] . nk cells are present in all gut tissues (small intestine, large intestine, and colon) as cells of the iel and lp compartments. they can also be found in smaller numbers in peyer's patches (pp) and mlns. unlike the lung, gut mucosal nk cells in humans are predominantly cd bright with few that express cd indicating that they may be similar to immature nk cells found in secondary lymphoid structures [ ] [ ] [ ] . nk cells in the murine intestinal mucosa also appear immature. iel and lp nk cells of naïve mice are cd hi cd b med and cd lo cd b lo , respectively [ ] [ ] [ ] . further support for an immature phenotype for both murine and human gut nk cells is evident from functionality. resident human cd bright and mouse cd hi nk cells produce large amounts of the proinflammatory cytokine ifn and exhibit low cytolytic activity when tested in vitro [ , ] . after infection, significant changes occur in nk cell frequencies in the small intestine and lamina propria. whether peripheral nk cell infiltration occurs or whether there are phenotypic changes in resident nk cell populations has not been investigated. infiltration of peripheral blood nk cells probably occurs albeit to a lesser extent and the nk cell dependent response may rely more on an nk cell interaction with activated gut mucosal dcs in the mln and/or expansion of activated lp nk cells at the site of infection ( figure ). however, given the diversity of gut mucosal ilc populations with wide ranging function, determining the level of infiltration of new cells may be very difficult. against pathogens, but also successful vascular remodeling of the uterus during pregnancy and fetal implantation [ , ] . in humans, frt nk cells resemble an immature phenotype of nk cells similar to that found in the digestive tract and are cd bright cd − [ ] . nk cells found in the uterus are phenotypically distinct from others in the frt being cd + and chemokine receptor like − + (cmklr ). cd is a tetraspanin family member important for cell adhesion and migration and cmklr is a receptor for chemerin, which promotes migration to the decidua and vascular remodeling during pregnancy [ , ] . they are also positive for kir molecules (kir dl ) and cd /nkg a. uterine nk cells exhibit functional characteristics of immature nk cells having high cytokine production and low cytotoxic potential. murine uterine nk cells resemble their human counterparts as having a more immature phenotype being cd med cd b hi [ ] . they also express ly receptors including ly d, h, c, i, g, and a. they are different from human uterine nk cells in that they exhibit lower cytolytic activity and are more important in contributing to vascular remodeling for proper fetal implantation [ ] . vaginal resident nk cells in humans are different from their uterine counterparts in that they are cd + and cd − [ ] . they also had a high potential of producing inflammatory cytokines including ifn and lower cytotoxic potential. they express cd to a similar level as immature [ ] . given their importance, a more thorough analysis of their phenotype would be interesting and informative to pursue. homing to mucosal sites. the mechanisms behind establishment of resident nk cells at mucosal sites are still not well described. chemokines most likely play a very important role in this process and mucosal associated nk cells express a vast array of chemokine receptors including, cxcr , ccr , ccr , ccr , and cx cr [ ] . additional factors are important in homing of nk cells to these sites including chemerin for female reproductive tract resident cells and sphingosine- phosphate family member s p [ ] . given the diversity of expression of these receptors on nk cells, each tissue could regulate which cells migrate to which organ. an intriguing model behind how nk cells set up residency in different tissues has been recently proposed [ , , ] . this model suggests that, unlike secondary lymphoid tissues and blood, nk precursor cells migrate from the bm into the blood then migrate further to different sites in the body including mucosal tissues. once they have migrated, organ specific environments influence their development. likely contributors include estrus cycling hormones, inflammatory milieu such as il- by somatic cells, tgf , il- , and the resident microbiomes. factors from these sources could induce nk precursor differentiation to attain different levels of maturation and education. new data on tissue specific difference in nk cell differentiation and studies investigating whether nk cells can be differentiated from tissue specific precursors may support the hypothesis that nk precursor cells seed peripheral organs and that they differentiate independent of the bone marrow [ , ] . this would allow these tissue resident nk cells to be "educated" to have very specific functions important at sites to which they migrate [ ] . an alternative hypothesis is possible. in certain mucosal tissues, especially those with established microbiomes, such as gut, frt, and recently described in the lung [ ] , nk cells may require a priming event from draining apc populations. these apcs program ln nk cells in mucosal associated lymphoid tissue (malt) to migrate to a specific site (figure ). malt includes lymph nodes such as the mesenteric lymph node (mln). this hypothesis is supported by the conserved phenotypic characteristics of gut and female reproductive tract nk cells. they more closely resemble immature cells by being cd med-bright cd + and cd + cd b + with higher cytokine production in humans and mice, respectively. as mentioned above, lung nk cells resemble a more mature phenotype and could come directly from the peripheral blood ( figure ). despite the lung nk cell phenotype, the presence and the effect of the lung microbiome on the development of asthma and chronic inflammation suggest that nk cells could also be modified in situ. regardless, support for the malt apc priming hypothesis could be taken from the process of t cell (iel) recruitment into the intestinal epithelium. interactions between gut mucosal t cells and cd + dcs via crtam in both the steady state and during infection are required for recruitment [ ] . iels during pathogenic situations require ifn producing mucosal dcs to be primed to home to the gut [ ] . interestingly, a recent report demonstrates that small intestine iels emerge from the thymus as recent thymic emigrants which express differing levels of the gut homing integrin [ ] . is required for iel trafficking to the gut [ ] . since nk precursors and other ilc populations in secondary lymphoid tissues express varying levels of this integrin, it may be possible that an nk-dc interaction is a requirement for immature nk cells to be signaled to home to mucosal sites. altogether, this is a possible mechanism important for homing that would be interesting to explore. during mucosal infections of humans and mice, nk cells are recruited to sites of infection and play an important role in immune defense [ , ] . soon after infection and ensuing inflammation (type i ifn and il- ), resident nk cells respond, produce ifn and tnf , and become cytotoxic [ ] . additional nk cells are recruited from the periphery within a few days of these events and contribute to these responses. as mentioned before, although the cytokine il- is required for development and the expansion of developing nk cells, other cytokines and signals can cause nk cells to expand and respond to infection [ ] . investigation into how nk cells in il- ko or il- r mice increase in number with mcmv infection demonstrated that il- promotes their expansion and activation. additionally, stimulation of nk cells through the activating receptor ly h via m of mcmv also contributed to this response. therefore the cytokine milieu present in the different mucosal tissues in addition to activating signals stimulated that by diverse pathogens help nk cells respond to infection. peripheral nk cell migration occurs in all mucosal tissues during bacterial, fungal, viral, and parasitic infection [ , , , ] . recruitment to inflamed tissues is largely dependent upon chemokines and the ligation of their cognate receptors on nk cells. once activated conventional nk cells at these sites are vital for initial containment of pathogens and preventing their systemic spread. the lung is a site of entry for viral, bacterial, and fungal infection. of all nonlymphoid tissues, the lung contains the largest number of conventional nk cells. humans and mice with genetic deficiencies resulting in loss of nk cells are more susceptible to pulmonary infections [ ] [ ] [ ] . many viruses including influenza, coronavirus (severe acute respiratory syndrome (sars), middle eastern respiratory syndrome (mers)), herpes, and respiratory syncytial virus (rsv) infect via the airways. nk cells were demonstrated early to respond to influenza infection in the lungs of humans through their production of ifn [ ] . cytolytic activity of these cells is also important for control of influenza infection including pandemic h n influenza [ ] . nk cell ctl activity in response to influenza is mediated through antibodies and adcc or via the recognition of viral haemagglutinin by the natural cytotoxicity receptors nkp [ ] . in mice infected with influenza a (pr ), infiltrating nk cells are cd low cd b hi and appear to be of a mature phenotype [ ] . in line with this, they also have greater cytolytic activity than ifn production suggesting that they could be nk cells migrating in from the blood or replicating in situ [ ] . in addition to il- , il- has been shown to play a role in nk cell responses in the lung [ , , ] . il- in complex with il- r can help in recruitment and activation of nk cells during rhinovirus infection and il- when blocked with an antibody appears to prevent nk cell recruitment into the bal during influenza infection in mice [ , ] . nk cell protective function in the lung against extracellular staphylococcus aureus also appears to require il- [ ] . il- likely contributes to the expansion and synergizes with il- and ifn for proper activation of nk cells in the lung. overall, it appears that, in the mouse model, nk cells are required for survival against influenza infection [ ] . nk cells functioning to control influenza can also cause severe pathology in the lungs [ ] . in mice given a high dose of influenza, depletion of nk cells resulted in better outcome against infection. a reason behind these differences could be attributed to the proinflammatory cytokines produced during initial viral encounter that help nk cells respond. il- and type i ifns stimulate nk cells to produce ifn [ ] . however, high production of these cytokines in response to pulmonary infection can lead to overproduction of ifn resulting in tissue destruction. nk cells play a role in early immunity and control of lung fungal and bacterial infections. nk cells help in early control of cryptococcus neoformans and aspergillosis through their cytolytic activity and ifn production [ ] [ ] [ ] [ ] . by producing ifn these cells play a critical role in control of several bacterial infections including staphylococcus aureus, klebsiella pneumonia, and legionella infections [ ] . in the case of klebsiella, nk cell production of il- may also be important in establishing immunity. in response to mycobacterium tuberculosis (mtb) infection, ifn and ifn are critical for control of the bacterium, the former via restriction of macrophage infiltration into the lung and the latter through inhibition of microbial growth [ ] . as with many infections, nk cells respond to both type i ifn and il- during mtb infection resulting in their production of ifn and are critical for early survival in the mouse model as demonstrated in rag −/− common--chain−/− and il- p −/− mice [ ] . however, depletion of nk cells has shown that, like some fungal models, nk cells can contribute to early control of mtb but are not required for long term survival against infection [ ] . overall, nk cells are very important for early control of pathogens in the lung. the majority of this protective response is governed by the production of ifn by these cells and can be regulated via il- and ifn / provided by myeloid populations. activation is also aided by the integration of activation signals through activating receptors on the cells. nk cell can also be pathogenic in the lung during infection from overproduction of ifn . questions still remaining that would be interesting to address are how to boost nk cell responses to this infection without causing overt tissue pathology and also what is the advantage of having nk cells present in the lung to promote this early control. are nk cells more important for immune stimulation or regulation or do they have bifunctional roles as a plastic cell able to respond to the environment? further investigation of the biology of these cells in the lung is needed to answer these questions. infections. as mentioned above, ilc populations in the gi tract are very diverse and have many roles from innate immune control of pathogens to regulation of autoimmunity [ , , ] . ifn producing conventional nk cells helps to control many infectious pathogens in the gut. although nk cells appear to be dispensable for listeria monocytogenes infection in mice [ ] , in a rat model of infection, they were essential in they were essential for early control of the bacterium [ ] . investigation of salmonella infection in mice demonstrated that il- and nk cells were required [ ] . il- ko mice had greater bacterial burdens than wt animals. depletion of nk cells also resulted in the great colonization of the murine gut with salmonella. despite previous studies where il- ko mice are still able to mount a robust nk cell response during viral and parasitic infection due to il- production this did not appear to occur during bacterial infection. this difference may highlight a difference in gut nk cells or variation in gut nk cell responses to different pathogens [ , ] . recently, lp nkp + nk . + ilcs were shown to contribute to gut pathology and ileitis during toxoplasma gondii infection [ ] . their function relied on il- and resulted in ccl dependent recruitment of inflammatory monocytes into the lamina propria. gut nk cells therefore may have several functions, not only in controlling infections directly, but also in the coordination and regulation of immune responses in the intestinal mucosa. nk cell ifn is also important in control of gut citrobacter rodentium and yersinia enterolytica infections [ ] [ ] [ ] . ifn production from macrophages was critical for this response. interestingly, trif signaling downstream of tlr was required for this macrophage induced nk cell activity. nk cells in the gi tract are involved in control of parasitic infection. nk cells production of il- in response to toxoplasma gondii infection in the gut was dependent upon il- [ ] . in another model, nk cell dependent and independent ifn was required to control cryptosporidium parvum infection in mice [ , ] . nk cells in humans are also important for innate control of gut mucosal infections. in hiv patients, individuals who are known as spontaneous controllers have greater numbers of iel associated nk cells than those who are classified as nonresponders [ ] . control of hiv in humans and also siv in macaques could be due to nk cell derived il- and il- production in iels and dependent upon an interaction with gut mucosal cd + dendritic cells [ ] . nk cells may contribute to hiv pathogenesis early in infection as tested with an siv model. this was shown when was blocked by antibody treatment in macaques [ ] . trafficking of nk cells blocked with this treatment reduced siv dissemination and viral burdens. nk cell activation itself may also contribute through recruitment of cd t cells providing the virus with more replicative niche to survive. however, later nk cells could play a substitutive role in protection. since was required for nk cell recruitment in this model, these results give support to the hypothesis that, in the gut, nk cell homing depends upon an interaction with mucosal dendritic cells (figure , top) . overall nk cells in gut mucosa play an important role in protection against orally acquired infections and further dissection is needed to understand how to take advantage of this cell type to optimize protection. there is a wide distribution of nk cells in mice and humans throughout the female reproductive tract, from the uterus, endometrium, and cervix to the ectocervix and vagina [ , ] . the resident cells in these tissues are all capable of producing ifn and cytolytic activity through adcc. the importance of ifn was noted early in response to vaginal infection of mice with hsv- where it was shown to help in resolving this infection in mice [ ] . subsequent studies in mice revealed that a major source in the vaginal mucosa for this cytokine was nk cells [ ] . this was demonstrated using rag −/− common-chain−/− mice, which lack t, b and nk cells and rag −/− mice, which lack only t and b cells. rag −/− common-chain−/− mice were fold more susceptible to infection than rag −/− mice. although it was shown later on that il- could stimulate an anti-hsv- response independent of nk cells [ ] , as in gut mucosa infections, il- was important in stimulating nk cell protective responses. recruitment of nk cells was also important for the ability of nk cells to protect against vaginal hsv- infection. ccr −/− mice were more susceptible and had higher viral loads in the cns after vaginal infection most likely due to impaired nk cell trafficking to the site of infection [ ] . using a rag −/− common--chain−/− humanized mouse model, human nk cells were observed to be recruited into the genital tract of female mice infected with hsv- suggesting that nk cell innate protection could be important in humans as well [ ] . further evidence of a potential role for nk cells in human reproductive tract comes from siv-macaque models where treatment of siv infected animals with ifn and il- resulted in enhanced activation and adcc function of cd + cd + cells in the vaginal mucosa [ ] . much investigation is still needed to understand nk cell responses in the vaginal mucosa as they do appear to be important for protection, but they can also contribute to disease pathology as seen in early hiv infection. uterine nk cells play two important roles in that they are required for tissue reorganization, vascularization, implantation of the fetus, and tolerance. at the same time they play a role in providing fetal protection during infection (figure , bottom) [ ] . what regulates the balance between tolerance and protection is not clear. infection with listeria monocytogenes results in placental colonization [ ] . nk cells tested for their ability to prevent this colonization in mice were shown to not have an impact. although il- was required for uterine nk cell development and their recruitment to this site in the naïve state, a lack of il- did not prevent the production of ifn early during listeria infection. therefore, il- in the uterus appears to play a more important role in nk cell recruitment but less of a role in early nk cell control of infection [ ] . other factors may be required to regulate their responsiveness to infection in this tissue. human uterine nk cells did not respond to direct tlr triggering but required an interaction with tlr stimulated accessory cells from the uterus to produce ifn [ ] . therefore, pathogen stimulation could cause an imbalance towards inflammation rather than tolerance at the maternalfetal interface. this is supported by a study investigating toxoplasma gondii infection induced abortion in mice [ ] . t. gondii induces a potent th response including nk cell production of ifn . parasite infection is also known to cause spontaneous abortion in the first trimester of pregnancy. infection of pregnant wild type mice in this study resulted in fetal resorption while infection of pregnant ifn r−/− mice had % reduction in effect. surprisingly, this was shown to be independent of nk cells. however, recent studies using decidual nk and trophoblast cells from humans have demonstrated that activity of these nk cells is enhanced upon t. gondii infection [ ] . this increase in activity was due to the increased expression of nkg d by decidual nk cells after in vitro stimulation with toxoplasma [ ] . nk cells may sense their environment and be tuned commensurate with what functions are needed. this possibility is supported by the theory of nk cell reeducation as what occurs when nk cells develop a memory phenotype [ ] . since uterine nk cells recruited in the steady state are less mature, as noted by their surface marker phenotype, the cytokine/hormone milieu changes the functional phenotype needed for successful pregnancy or immune protection develops in situ. another possibility is that new nk cells from the periphery could be recruited and contribute to pathology. pathogen stimulated that apcs migrating to the draining lymph node activate newly recruited nk cells that are then driven to protect. much needs to be addressed in regard to the mechanisms behind frt nk cell function as there is relatively little known. conventional nk cells are found in all mucosal tissues and play an important role in first line of defense against bacterial, fungal, viral, and parasitic infections. significant questions still remain in regard to ( ) how they gain entry into each of the specific tissues, ( ) what sources of nk cell precursors provide resident nk cells in different tissues, and ( ) how their diverse functional responses are regulated in different mucosal tissues. for mucosal tissue resident nk cells in the naïve state, lung nk cells may represent a more mature phenotype and could come from the blood stream. gut mucosal and frt nk cells may be derived from nk precursor or immature nk cells after receiving initial address directions and programming from apcs in secondary lymphoid structures. during infection, resident mucosal tissue nk cells respond primarily through ifn production, which contributes directly to early control of pathogens. inflammation (il- , ifn / , ifn , and chemokines) at the site of infection then results in expansion of resident cells and recruitment of peripheral nk cells which provide additional 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the intestinal mucosa of patients with crohn's disease ifn--producing dendritic cells are important for priming of gut intraepithelial lymphocyte response against intracellular parasitic infection the role of beta integrins in cd t cell trafficking during an antiviral immune response origin, trafficking, and intraepithelial fate of gut-tropic t cells the light and dark sides of intestinal intraepithelial lymphocytes organ-specific features of natural killer cells natural killer cell distribution and trafficking in human tissues natural killer cells infiltrating human nonsmall-cell lung cancer are enriched in cd ℎ cd − cells and display an impaired capability to kill tumor cells nk cell development from a novel progenitor found in the murine lung characterisation of natural killer cells and cd + t-cells in sarcoidosis patients intestinal intraepithelial lymphocytes contain a cd − cd + subset expressing natural killer markers and a singular pattern of adhesion molecules human small-iintestinal epithelium contains functional natural killer lymphocytes cd -natural killer cells are greatly enriched in the human gastrointestinal tract and have the capacity to respond to bacteria microbial flora drives interleukin production in intestinal nkp + cells that provide innate mucosal immune defense influence of the transcription factor rorgammat on the development of nkp + cell populations in gut and skin ror t and commensal microflora are required for the differentiation of mucosal interleukin -producing nkp + cells functions of uterine natural killer cells are mediated by interferon gamma production during murine pregnancy mhcdependent inhibition of uterine nk cells impedes fetal growth and decidual vascular remodelling organ-specific phenotypic and functional features of nk cells in humans chemerin regulates nk cell accumulation and endothelial cell morphogenesis in the decidua during early pregnancy unique characteristics of nk cells throughout the human female reproductive tract ly receptors: innate and adaptive immune paradigms cytokine profile of mouse vaginal and uterus lymphocytes at estrus and diestrus unique subpopulations of cd + nk and nk-t peripheral blood lymphocytes identified by chemokine receptor expression repertoire natural killer cell distribution and trafficking in human tissues organ-specific features of natural killer cells a human cd (+) subset resides in lymph nodes and differentiates into cd bright natural killer cells the microbiome of the lung crtam controls residency of gut cd +cd + t cells in the steady state and maintenance of gut cd + th during parasitic infection the role of natural killer cells in pulmonary immunosurveillance natural killer cells in infection and inflammation of the lung human natural killer cell deficiencies natural killer cell deficiency nk cells play a critical protective role in host defense against acute extracellular staphylococcus aureus bacterial infection in the lung interferon induction and increased natural killer-cell activity in influenza infections in man antibody-dependent cellular cytotoxicity is associated with control of pandemic h n influenza virus infection of macaques recognition of haemagglutinins on virus-infected cells by nkp activates lysis by human nk cells nk cells exacerbate the pathology of influenza virus infection in mice immune dysregulation in severe influenza il- complexes induce nk-and t-cell responses independent of type i ifn signaling during rhinovirus infection il- participates in the respiratory innate immune response to influenza virus infection in vivo treatment of mice and hamsters with antibodies to asialo gm increases morbidity and mortality to pulmonary influenza infection natural killer cell responses during viral infections: flexibility and conditioning of innate immunity by experience role of natural killer cells in resistance to cryptococcus neoformans infections in mice an acidic microenvironment increases nk cell killing of cryptococcus neoformans and cryptococcus gattii by enhancing perforin degranulation il- contributes to host resistance against infection with cryptococcus neoformans in mice with defective il- synthesis through induction of ifn-gamma production by nk cells chemokine-mediated recruitment of nk cells is a critical host defense mechanism in invasive aspergillosis dynamic roles of type i and type ii ifns in early infection with mycobacterium tuberculosis nk cellderived ifn-differentially regulates innate resistance and neutrophil response in t cell-deficient hosts infected with mycobacterium tuberculosis nk cells respond to pulmonary infection with mycobacterium tuberculosis, but play a minimal role in protection conventional alpha beta t cells are sufficient for innate and adaptive immunity against enteric listeria monocytogenes the role of natural killer cells in the defense against listeria monocytogenes lessons from a rat model interleukin- and nk . + cells provide innate protection against acute salmonella enterica serovar typhimurium infection in the gut and in systemic tissues interleukin- -dependent nkp + innate lymphoid cells control intestinal inflammation by recruiting inflammatory monocytes cd − nk . + cells aid in the early induction of a th response to an attaching and effacing enteric pathogen natural killer cells protect against mucosal and systemic infection with the enteric pathogen citrobacter rodentium host innate recognition of an intestinal bacterial pathogen induces trifdependent protective immunity il- promotes nk cell production of il- during toxoplasmosis innate immune responses against cryptosporidium parvum infection roles for nk cells and an nk cell-independent source of intestinal gamma interferon for innate immunity to cryptosporidium parvum infection altered distribution of mucosal nk cells during hiv infection loss of mucosal cd dcs and il- and il- lymphocytes is associated with mucosal damage in siv infection blocking of alpha beta gut-homing integrin during acute infection leads to decreased plasma and gastrointestinal tissue viral loads in simian immunodeficiency virus-infected rhesus macaques innate and adaptive immunity in female genital tract: cellular responses and interactions interferon-enhances resolution of herpes simplex virus type infection of the murine genital tract interleukin- and natural killer and nkt cells play a critical role in innate protection against genital herpes simplex virus type infection nk and nkt cellindependent contribution of interleukin- to innate protection against mucosal viral infection susceptibility of ccr -deficient mice to genital herpes simplex virus type is linked to nk cell mobilization mucosal innate and adaptive immune responses against herpes simplex virus type in a humanized mouse model enhancement of natural killer cell activation and antibody-dependent cellular cytotoxicity by interferon-and interleukin- in vaginal mucosae sivmac -infected macaca fascicularis origin, phenotype and function of human natural killer cells in pregnancy the uterine nk cell population requires il- but these cells are not required for pregnancy nor the resolution of a listeria monocytogenes infection central role of interleukin- in postovulatory recruitment of peripheral blood cd (-) natural killer cells into human endometrium tlrs mediate ifn-gamma production by human uterine nk cells in endometrium toxoplasma gondii-induced foetal resorption in mice involves interferon--induced apoptosis and spiral artery dilation at the maternofoetal interface changes of human decidual natural killer cells cocultured with yfp-toxoplasma gondii: implications for abnormal pregnancy toxoplasma gondii infection regulates the balance of activating and inhibitory receptors on decidual natural killer cells re-educating natural killer cells the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord- - kqrl rv authors: hedger, m.p. title: immunology of the testis and male reproductive tract date: - - journal: comprehensive toxicology doi: . /b - - - - . -x sha: doc_id: cord_uid: kqrl rv a large body of evidence points to the existence of a close, dynamic relationship between the immune system and the male reproductive tract, which has important implications for our understanding of both systems. the testis and the male reproductive tract provide an environment that protects the otherwise highly immunogenic spermatogenic cells and sperm from immunological attack. at the same time, secretions of the testis, including androgens, influence the development and mature functions of the immune system. activation of the immune system has negative effects on both androgen and sperm production, so that systemic or local infection and inflammation compromise male fertility. the mechanisms underlying these interactions have begun to receive the attention from reproductive biologists and immunologists that they deserve, but many crucial details remain to be uncovered. a complete picture of male reproductive tract function and its response to toxic agents is contingent upon continued exploration of these interactions and the mechanisms involved. in the decade since this chapter was first published (hedger ) , there has been considerable progress in the understanding of the regulation of immunity. advances such as the emergence of the innate immune system as a major theme in immunity following the discovery of the toll-like receptor (tlr) family (o 'neill and dinarello ) , the formalization of multiple macrophage phenotypes corresponding to the type and helper t (th) cell subsets (mantovani et al. ) , the rehabilitation of suppressor and regulatory t cells (piccirillo and thornton ) , and the identification of new lymphocyte subsets, including th , th , and natural killer t (nkt) cells (godfrey et al. ; macdonald ; romagnani ) , also have had considerable influence on the concept concerning the immunology of the male reproductive tract. nonetheless, while there have been advances in our understanding of the interface between the male reproductive tract and the immune system, there is still much we simply do not know. the need to understand this interface and the potential implications for reproductive toxicology continues to be just as important as ever. there is no doubt that the testis and the male germ cells in particular are susceptible to immunological damage. clinically significant testicular autoimmunity, most commonly indicated by the presence of antisperm antibodies in the serum or ejaculate, is implicated in the case of - % of all male infertility patients within the developed world (baker et al. ; pattinson and mortimer ) , but the incidence is much higher in populations where access to reproductive health care is limited (ekwere ) . common factors suspected to contribute to testicular autoimmunity include reproductive tract infections, physical trauma, immune system dysfunction, and genetics. the release of spermatogenic antigens from the reproductive tract following vasectomy generally results in autoimmune responses, ranging in severity from sperm antibodies to orchitis, in both humans and experimental animals (alexander and anderson ; flickinger et al. a; kojima and spencer ; tung and alexander ) . the potential for spontaneous testicular autoimmune disease in otherwise normal men remains poorly characterized, although studies in animals clearly indicate the possibility of such reactions in humans (furbeth et al. ; tung et al. ) . complicating matters is the fact that the testis is an immunologically privileged tissue, in the sense that foreign tissue grafts into the testes of experimental animals survive for prolonged periods (barker and billingham ; head et al. a; whitmore and gittes ) . this dichotomy between immune susceptibility and privilege is indicative of a highly specialized interaction between the male reproductive system and the immune system, with potentially important implications for both systems. the objective of this chapter is to provide an overview of the current state of knowledge and to highlight issues for further consideration that may be of relevance to male reproductive toxicology. . . the interface between the immune system and the reproductive tract the immune system is the body's protective arsenal against disease. it operates via the detection of potential aggressors, recognized through conserved motifs found on pathogenic organisms (innate immunity) or confrontation by completely novel (i.e., foreign) molecular structures (adaptive immunity) (vivier and malissen ; zinkernagel ) . immunity involves specialized cells possessing highly developed recognition and activation abilities (macrophages and dendritic cells) and the cells that carry out the protective responses: t and b cells in the case of adaptive immunity and nk cells and mononuclear or polymorphonuclear phagocytes (pmns) in the case of innate immunity. many of these cells play roles in both the recognition and effector arms of the immune response and in both innate and adaptive immunities (figure ) . innate immunity provides the rapid response elements of the immune system, but is limited in its attack repertoire. adaptive immunity is much more flexible in its responses, but requires some time to become effective. once the adaptive arm has been activated toward a particular pathogen, however, it can respond rapidly in the future due to the persistence of memory lymphocytes, which form the basis of immunization (mcghee et al. ; schittek and rajewsky ) . the innate immune system operates upon recognition by specific pattern-recognition receptors of specific motifs found on bacterial, viral, fungal, and protozoan pathogens. functionally, these receptors have evolved to recognize pathogen-associated molecular patterns (pamps), such as bacterial lipopolysaccharides (lpss), lipoproteins, peptidoglycans, and viral nucleic acids (bianchi ; roach et al. ) . unlike classical ligand receptors, these receptors respond to multiple ligands possessing related, but not necessarily identical, structures. the largest and best-studied class of pattern-recognition receptors is the tlrs, which are members of a larger family of proteins that include the interleukin (il) receptor (il r), and which share a conserved cytoplasmic domain called the toll/il r (tir) domain (akira and takeda ; o'neill and bowie ) . in humans, functional tlrs (tlr - ) have been identified (figure ) . mammalian tlrs - are known from laboratory rodent species, but are either pseudogenes or absent in humans (roach et al. ) . although they are particularly associated with cells of the monocyte lineage (i.e., macrophages and dendritic cells), these receptors are also found on certain t and b cells, as well as many fibroblastic and epithelial cells, including those of the male urogenital tract (lauw et al. ; zhang et al. ) . significantly, ligands for some of the tlrs include endogenous molecules of mammalian origin (barrat et al. ; karikó et al. ; tsan and gao ; yasuda et al. ; yu et al. ) . tlr signaling occurs through engagement of crucial adaptor proteins, specifically myeloid differentiation primary response protein (myd ) or tir domain-containing adaptor protein inducing interferon (trif), via tir domain interactions, leading to activation of tumor necrosis factor (tnf) receptorassociated factor and (traf , traf ) (akira and takeda ; o'neill and bowie ; o'neill and dinarello ) . based on the tlr engaged, this results in nuclear translocation of the inflammatory transcription factor, nuclear factor kappa b (nfb), activation of the jun n-terminal kinase (jnk) and p mitogen-activated protein (map) kinases, and induction of type interferon (ifn and ifn) figure polarization in the immune system. the immune system is conceptually divided into innate and adaptive arms, with adaptive immunity further subdivided into type (cell-mediated) and type (antibody-mediated) responses. triggering of the immune response involves the induction of inflammation by various agents, which may include infections, particulates, debris from damaged cells, or noxious agents. innate immunity involves fixed pattern-recognition receptors, such as the tolllike receptors, whereas adaptive immunity depends upon highly polymorphic receptors expressed by t cells (t cell receptors) and b cells (membrane-bound and secreted immunoglobulin). in general, type responses are associated with cellular immunity, autoimmune reactions, and graft rejection. type responses involve antibody production by the b cells, but also play a more complex role in allergy, immunoregulation, and control of tolerance, favoring immune privilege. within these divisions, various cell types, cytokines, chemokines, bioactive lipids, and other molecules interact to integrate or mediate the responses. the direction of the adaptive immune response type is determined primarily by the activity of the antigenpresenting cells (primarily dendritic cells) under the influence of cells with immunoregulatory capacity, such as macrophages, nk cells, and nkt cells, and either type or type cytokines produced locally. several cell types and molecules play multiple roles within different functional divisions. abbreviations: mo, monocyte; nk, natural killer cell; pmn, polymorphonuclear cell; th; helper t cell; b, b cell; t reg , regulatory t cell; m, macrophage; nkt, natural killer t cell; ctl, cytotoxic t cell; apc, antigenpresenting cell, ros, reactive oxygen species; ifn, interferon; pge, prostaglandin e; cox, cyclooxygenase; inos, inducible nitric oxide synthase; tnf, tumor necrosis factor ; il, interleukin; il ra, interleukin receptor antagonist; tgf, transforming growth factor ; gc, glucocorticoid; mif, macrophage migration inhibitory factor. production (hertzog et al. ; malmgaard ; nakano ) . the pattern-recognition receptor family also includes the nucleotide binding and oligomerization domain (nod)-like receptor (nlr) family and the retinoic acid-inducible gene (rig)-like helicases (rlhs) (becker and o'neill ; thompson and locarnini ) . the nlrs detect various bacterial pamps within the cytosol. some nlrs, such as nod and nod , act via nfb, while other nlrs work through induction of the cysteine protease caspase , which activates the proinflammatory cytokines il and il and initiates caspase-mediated proapoptotic pathways within the target cell. the rlhs, which detect the presence of viral rna in the cytosol, activate both nfb and production of type ifns. activation of innate immunity leads to inflammation, characterized by increased blood and lymphatic flow within the affected tissue and recruitment of immune cells to the site through production of chemokines and expression of vascular leukocyte adhesion molecules (moser and loetscher ; picker and butcher ) . this mobilization of immune cells is accompanied by activation of complement, production of proinflammatory cytokines tlr tlr tlr tlr tlr tlr tlr tlr cd tlr tlr cytoplasmic cell surface cpg dna ssrna dsrna lipoproteins lps flagellin/profilin ? irf nfkb ap map kinases il α, tnfα, il , inos, activin a, il , il , cox , ifnγ mal tram tlr tlr tlr apoptosis irf , , ifnα, ifnβ, tnfα myd traf traf traf , myd trif figure the toll-like receptors: major pathogenic ligands, adaptor proteins, signaling, and cytokine production. there are active human toll-like receptors (tlr - ). tlr - are present in rodents, and tlr is specific to the urogenital tract. the tlrs respond to a variety of pathogen-related molecules, usually forming homo-or heterodimers during signaling: for example, tlr can self-associate or combine with either tlr or tlr to mediate responses to various bacterial lipoprotein classes. tlr forms a complex with the receptor cofactor cd , to facilitate binding of bacterial lipopolysaccharide (lps). the tlrs can be subdivided into cell surface receptors, which largely respond to bacterial proteins, lipoproteins, and lipopolysaccharides, and intracytoplasmic receptors, which recognize single-stranded and double-stranded rna (ssrna and dsrna) and cpg-containing dna of viral or bacterial origin. most tlrs signal via the adaptor protein myd (myeloid differentiation primary response protein ), except tlr , which acts through the adaptor protein trif (tir domaincontaining adaptor protein inducing interferon ). uniquely, tlr can interact with either myd or trif, through engagement of the adaptor proteins mal (myd adaptor-like) or tram (trif-related adaptor molecule). downstream signaling involves the tumor necrosis factor receptor-associated factors and (traf and traf ), activation of the transcription factors nuclear factor kappab (nfb) and interferon regulatory factor (irf ), or activation of the mitogenactivated protein kinases (map kinases) jun n-terminal kinase (jnk) and p to produce the fos/jun transcription factor activated protein (ap ). these transcription factors interact to induce the expression of multiple proinflammatory genes, including interleukin (il) , tumor necrosis factor (tnf), inducible nitric oxide (inos), cyclooxygenase (cox ), and interferon (ifn), or the type interferons (ifn, ifn) and tnf. note that many details of the signaling pathways have been omitted or truncated for the sake of simplicity. and acute-phase proteins, and stimulation of phagocytosis and killing of pathogens and infected cells by macrophages, pmns, and nk cells, through production of proteases and other lytic proteins, and cytotoxic reactive oxygen species (ros) and induction of apoptotic pathways (rosenberg and gallin ) . moreover, activation of innate immunity is responsible for initiation of the adaptive immune response, through production of regulatory cytokines and stimulation of the antigen-presenting cell (apc) abilities of dendritic cells, macrophages, and some b cells (kelsall and rescigno ; spörri and reis e sousa ) . among the cell types that mediate the innate immune response, nk cells represent a special case. these cells are of the lymphocytic lineage, but they are capable of spontaneously attacking target cells without prior sensitization by exposure to antigen (mori et al. ; westermann and pabst ) . through a complex of specific receptors on their surface, nk cells are able to recognize and directly target transformed cells, such as virally infected or tumor cells (lanier ; mori et al. ) . in this, nk cells provide a first-line of lymphocyte defense within the context of the innate immune response, but activated nk cells also participate in adaptive immunity, both as regulators and as effector cells of the response (mocikat et al. ; raulet ) . highly specific interactions between apcs and t cells, mediated via the polymorphic proteins of the major histocompatibility complex (mhc; the human leukocyte antigen (hla) complex in humans) and antigen-specific t cell receptors (tcrs), are crucial to the adaptive immune response. the apcs process exogenous foreign proteins into short antigenic peptides, which are then incorporated into a structural groove on the external surface of the mhc protein complex during its assembly within the cell (cresswell et al. ; sant et al. ) . the tcr on the surface of a t cell that has specificity for the antigen binds to the antigen-mhc complex on the apc surface, which results in activation and proliferation of the t cell. circulating t cells express one of two coreceptors, cd and cd , as part of the tcr, which direct them to recognize antigens associated with either mhc class ii or i proteins, respectively (janeway ) . antigens are presented to cd þ t cells by the so-called professional apcs that express mhc class ii antigens (i.e., dendritic cells, macrophages, and b cells) (heath and carbone ; unanue ) . the cd þ t cells recognize antigens presented in the context of mhc class i proteins, which are expressed by almost all cell types in the body. activation of the t cell also requires physical interaction between the apc and t cell involving costimulatory ligand-receptor pairs, particularly cd :b and cd :cd l, as well as the production of either type cytokines (ifn, il , and il ) or type cytokines (il , il , il , and il ) (constant and bottomly ; heath and carbone ; moser and murphy ) . as a consequence, the activated t cell can have a number of different fates depending on the costimulatory molecules engaged and cytokines produced: activated cd þ t cells may become type helper (th ) cells, which direct development of the cellular immune response involving cytotoxic cd þ t cells, or they may become type helper (th ) cells, which promote the development of b cells into antibody-secreting plasma cells following interaction with their antigen (defranco ) . recently, a new th effector cell subset (th ) developmentally related to th cells, but producing il , il , and il has been described (ouyang et al. ; romagnani ) . these th cells direct a number of responses involved in protection against extracellular pathogens, including recruitment and activation of the major circulating pmn subset, the neutrophils, differentiation of b cells, and inflammatory reactions of epithelial cells. however, this cell subset has also been implicated in the development of autoimmune diseases and, along with th cells, in allergy (oboki et al. ; ouyang et al. ) . the absence of appropriate costimulatory molecule interactions and/or the presence of specific regulatory cytokines may also lead to t cell inactivation and deletion or generation of regulatory (i.e., suppressor) t cell subsets (gilliet and liu ; nossal ; piccirillo and thornton ; thompson and thomas ) . finally, at least some activated t and b cell clones persist as memory cells following the resolution of the immune response (dutton et al. ; schittek and rajewsky ) . the initial antigen presentation and lymphocyte activation events generally occur in the secondary immune tissues, the local lymph nodes and spleen. consequently, an effective lymphatic drainage is usually necessary for immune responses to develop in a particular tissue. however, since leukocytes recirculate through tissues, many of the important events, including antigen exposure and processing, antigen recognition by antigen-exposed lymphocytes, and lymphocyte maturation and proliferation, also occur at the reaction site itself (ascher et al. ; gruber ; pedersen and morris ) . moreover, while it is generally accepted that the adaptive immune system is activated by the presence of foreign antigens, there is an alternative activation model that combines elements of both the innate and adaptive immune systems. this is encapsulated by the 'danger hypothesis,' which proposes that apcs respond to substances that cause or signal damage, rather than to those that are simply foreign (matzinger ) . these danger signals include cd l, the early proinflammatory cytokines il and tnf, ifns, and heat-shock proteins as well as substances that are normally found only inside cells (e.g., nucleotides, unmethylated cpg sequences in mammalian double-stranded dna) and hyaluron breakdown products (gallucci and matzinger ) . in this model, activation of the immune system occurs as a response to evidence of an extant threat, rather than toward a specific feature of the threat itself. this mechanism may contribute to the onset of certain autoimmune diseases. while an effective immune response is essential for protection of the host from a range of threats, the immune system also has its dark side. the capacity of the immune system for destructiveness is expressed in chronic inflammatory and autoimmune diseases, including those that affect the male reproductive tract (roper et al. ; schuppe and meinhardt ; schuppe et al. ; suominen ) . these examples of an immune system seemingly out of control highlight the crucial importance of effective immunoregulation. at the center of immunoregulation is the concept of tolerance, or the capacity of the immune system to ignore certain molecular patterns, and the systems that restrain the immune response once its usefulness has expired. tolerance is the ability of the immune system to discriminate self from nonself. it is fundamentally based on the removal, inactivation, or suppression of self-reactive lymphocyte subsets, so that the immune system becomes effectively incapable of recognizing and reacting to these antigens. induction of tolerance comprises both central and peripheral mechanisms. the central mechanism involves clonal deletion of t and b cell subsets that are potentially autoreactive, a process that normally occurs during gestation as part of the early maturation of the immune system (kappler et al. ; nossal ) . many details of this editing process remain incompletely understood, although the process involves broad expression by thymic medullary cells of self-antigens, including many endocrine tissue-specific antigens under the control of the transcription factor autoimmune regulator (aire) (liston et al. ). lymphocytes that recognize these self-antigens within the thymus during this process are targeted for destruction, effectively removing them from the lymphocyte repertoire. in addition, there are a number of complex mechanisms of peripheral tolerance, which involve the inactivation of antigen-specific lymphocytes in peripheral lymphoid organs by weak antigen stimulation in the absence of appropriate costimulation and the production of lymphocytes with immunoregulatory activities (gilliet and liu ; piccirillo and thornton ; thompson and thomas ) . peripheral immunoregulation involves several antigen-specific regulatory or suppressor t cell types, most notably the cd þ cd þ regulatory t cell (t reg ) subset. these cells selectively produce the immunosuppressive cytokines transforming growth factor (tgf) and il , but also appear to act via direct contact with the cell being suppressed (nakamura et al. ; piccirillo et al. ) . other t cell subsets implicated in immunosuppression through their ability to produce tgf and il include th cells, tr (t regulatory ) cells, and t cells, the latter being a minor t cell subset that is generally associated with epithelia (kuhnlein et al. ; macdonald ; seo et al. ) . the nkt cells, which are t cells with nk activity that possess unique restriction to glycolipid antigens presented by the mhc-like molecule cd d, play a role in promoting graft survival and development of cd þ regulatory/suppressor t cells through production of il and il (godfrey et al. ; nakamura et al. ; sonoda et al. ) . the frontline nk cells are able to modulate adaptive immune responses by inducing dendritic cell death through contact-mediated lysis, but are also capable of producing ifn to stimulate the antigen-presenting activity of dendritic cells (laouar et al. ; raulet ) . it is the absence of tolerance that inhibits graft success in the very artificial condition of tissue transplantation. the immune cells of both the graft recipient and the donor tissue respond to one another, particularly with respect to polymorphic differences of the mhc proteins, leading to rejection of tissues that are not antigenically matched (gould and auchincloss ) . even under normal conditions, failure of preexisting tolerance may occur through a number of mechanisms, leading to autoimmune disease. somatic mutation of the antigen receptor expressed by a t or b cell may result in the creation of new self-reactive clones, subverting central tolerance (burrows et al. ) . autoimmunity may also occur when the inflammatory response to an infection damages or overwhelms normal mechanisms of self-tolerance, or where the infection involves organisms that express antigens that may cross-react with self-antigens (molecular mimicry) (merkler et al. ; regner and lambert ) . in addition to lymphocyte-mediated tolerance already discussed, the immune system possesses a repertoire of mechanisms that control and limit inflammatory and immune responses once they have commenced. inflammation triggers the production by the adrenal gland of corticosteroids, which inhibit the production of proinflammatory cytokines and other immune mediators, reduce the expression of leukocyte adhesion molecules, and induce lymphocyte apoptosis (buckingham et al. ; kapcala et al. ) . during inflammation, immune cells themselves produce anti-inflammatory and immunosuppressive molecules, including prostaglandins d and j and the lipoxins (herlong and scott ; levy et al. ) . moreover, activated lymphocytes have a limited lifespan, undergoing a process of activation-induced cell death following upregulation of the extrinsic apoptotic signal mediated by interaction of the fas receptor (cd ) with its ligand (fas ligand: fasl or cd l), eventually leaving behind a relatively small number of long-lived memory cells (dutton et al. ; schittek and rajewsky ) . likewise, there are mechanisms to inhibit antibody activity and promote antibody clearance (isaacs ). classically, the term immune privilege applies to organs or tissues where survival of foreign grafts may be prolonged (barker and billingham ) , but more accurately, the term denotes tissues where immune responses are inhibited or suppressed (hedger ) . privileged tissues include the brain, eye, pregnant uterus, and the testis. initially, privilege was attributed to deficient lymphatic drainage in such tissues, or physical barriers that prevented immune cells from gaining access (barker and billingham ) . several privileged tissues do possess highly specialized epithelial or endothelial cell barriers that may sequester antigens away from the normal immune surveillance: the best characterized of these are the blood-brain barrier and the trophoblast in the pregnant uterus (hunt ; streilein ) . however, physical exclusion is now considered an overly simplistic explanation for immune privilege, given that lymphocyte and antibody responses to introduced antigens occur even in sites where grafts can survive for extended periods (head et al. b; kaplan and streilein ; tafuri et al. ) . although barrier mechanisms and altered lymphatics may be contributory factors in some tissues, immune privilege is a consequence of tissue-specific specialized systems for controlling, subverting, or preventing local inflammatory or immune activation responses (hedger ) . the male reproductive tract comprises the paired testes, associated epididymis and vas deferentia, accessory glands (chiefly the prostate and seminal vesicles), and the urethra. these tissues represent a diversity of structures and, consequently, the immunology of each tissue retains distinctive features (hedger and hales ) . best studied in this context is the testis, which comprises two discrete tissue compartments, the seminiferous tubules and the interstitial tissue. the interstitial tissue contains the blood supply and lymphatics, as well as the innervation of the testis (fawcett et al. ; setchell et al. ) . the epithelium of the seminiferous tubules is entirely avascular. within the seminiferous epithelium, spermatogenesis is supported and maintained by the sertoli cells, which remain in physical contact with the developing germ cells at all times. during spermatogenesis, a stem cell spermatogonium sitting on the basement membrane of the seminiferous tubule becomes committed to a series of mitotic divisions, producing a cohort of spermatocytes. the spermatocytes subsequently move toward the tubule lumen, passing through tight junctions between adjacent sertoli cells, into the highly specialized environment of the adluminal compartment (wang and cheng ) . within this compartment, the spermatocytes divide meiotically to produce haploid spermatids and then undergo differentiation into spermatozoa, which are ultimately released into the lumen of the tubule leaving behind the majority of their cytoplasm to be digested by the sertoli cells as residual bodies. the released spermatozoa are swept by fluid secreted from the sertoli cells toward the rete testis, a collecting structure lined by a simple epithelium that is connected to the adjacent epididymis by a series of efferent ducts. sperm mature and are stored in the epididymis, until they are either released through the vas deferentia at the time of ejaculation or removed by epididymal phagocytes (barratt and cohen ; roussel et al. ) . the process of spermatogenesis is highly organized, with multiple generations of developing germ cells in each region of the epithelium maintaining distinct, progressing cellular associations or stages, which comprise the cycle of the seminiferous epithelium ( stages in the rat, stages in the mouse, stages in man). these stages are arranged in sequence along the length of each seminiferous tubule, creating waves of coordinated spermatogenic development (clermont ) . spermatogenesis is maintained and controlled by follicle-stimulating hormone (fsh) and testosterone, the latter being produced by the leydig cells in the interstitial tissue under the influence of luteinizing hormone (lh). importantly, fsh and testosterone act upon sertoli cells and not directly on the germ cells (mclachlan et al. ) . these hormones determine the efficiency of the spermatogenic process largely by controlling germ cell survival, that is, the balance between differentiation and apoptosis (matthiesson et al. ; ruwanpura et al. ) . in order to maintain the cycle of the seminiferous epithelium, the developing germ cells and sertoli cells are engaged in continuous intercellular communication, although the precise nature of this communication remains very poorly understood. the testis, and spermatogenesis in particular, presents a unique challenge for the immune system. although there are reports of direct drainage of some lymphatic vessels to the thoracic duct, there is no evidence of any structural or functional deficiency in the efferent lymphatics of the testis, and allogeneic cells injected into the rat testis induce typical immune responses in the draining lymph nodes (fawcett et al. ; head et al. b; itoh et al. a; moller ) . nonetheless, the vast majority of spermatogenic differentiation occurs long after central tolerance is established and, while thymic expression of some testicular antigens is under the control of the aire transcription factor, it is obvious from clinical and experimental studies that germ cells express multiple autoreactive antigens and are highly immunogenic when brought into contact with the immune system (itoh et al. ; tung ) . surprisingly, this is a problem for a relatively small cohort of men only, and autoimmune orchitis or epididymitis is rarely reported in humans, except in the context of local infection (krieger ) . even allowing for the possibility that most cases of spontaneous autoimmune reactions against the developing germ cells are never diagnosed, since they would occur early in reproductive life, and would simply present as spermatogenic failure during infertility work-ups (schuppe and meinhardt ; schuppe et al. ) , why are such reactions not more common? it is widely believed that the blood-testis barrier plays a role in protecting testicular antigens from the immune system. however, as a result of a common misunderstanding of the nature of this vitally important structure, its actual role is usually overestimated. critically, the blood-testis barrier involves highly specialized occluding or tight junctions between adjacent sertoli cells, which separate the spermatogonial and early meiotic cells from the majority of meiotic and postmeiotic germ cells (dym and fawcett ; setchell et al. ) . the structure and functions of the barrier do not need to be discussed in detail here, as this topic is covered in much more detail in other chapters in this volume (see chapter . ). by preventing the passage of most molecules between adjacent sertoli cells, the bloodtestis barrier allows the creation of a highly specialized biochemical environment essential for meiotic development (cheng and mruk ; griswold ) . it also blocks access by lymphocytes, complement, and antibody (ben et al. ; yule et al. ) . by contrast, the vascular endothelium and peritubular cells surrounding the tubules play a negligible role in maintaining the blood-testis barrier in practical terms, and immune cells and proteins have relatively free access to the interstitium and basal seminiferous epithelium (mahi-brown et al. ; yule et al. ). thus, this barrier is quite different in properties and function from the blood-brain barrier, located on the vascular endothelium, or the trophoblastic barrier of pregnancy, which entirely segregates the developing fetal tissues from the maternal host. a reinterpretation of the blood-testis barrier as a series of cellular elements that protects the germ cells from harmful influences has been proposed more recently (bart et al. ) . in this model, the bloodtestis barrier consists not only of the structural elements already discussed, but also the efflux pump barrier system involving the drug transport proteins p-glycoprotein and the multidrug resistance-associated protein- expressed on the capillary endothelium, peritubular myoid cells, and the basal aspect of the sertoli cells (bart et al. ; melaine et al. ) . while these elements may certainly be important for protection of the testis against toxic agents, their contribution to immunoregulation is probably marginal. several lines of evidence indicate that the bloodtestis barrier cannot account for all manifestations of immune privilege in the testis. the barrier does not prevent exposure of germ cell antigens to the immune system, as antibodies and lymphocytes specific for spermatogenic antigens appear to be a normal feature of the circulating immune repertoire even under normal conditions (turek and lipshultz ) . spermatogenic cell autoantigens are not confined behind the sertoli cell tight junctions, and are expressed by spermatogonia and the early spermatocytes (mahi- ; yule et al. ). moreover, the barrier does not extend into the rete testis or beyond. significantly, orchitis can be passively transferred to naive mice using lymphocytes obtained from mice with active autoimmune orchitis, with the initial reaction concentrated in the interstitial tissue around the rete testis tung et al. ) . a similar initial pattern of development of orchitis within the rete testis region has been observed in mice actively immunized with viable germ cells (itoh et al. a ). in many seasonally breeding species, annual regression of the bloodtestis barrier occurs without inducing overt inflammation or autoimmunity (pelletier ; tung et al. ) . finally, the blood-testis barrier cannot explain the enhanced survival of grafts within the interstitial tissue (i.e., outside the barrier) and does not explain survival of autoantigenic sperm in the epididymis, or remainder of the male reproductive tract. however, while the blood-testis barrier does not explain immune privilege in the testis, the breach of the tight junctions comprising the barrier remains a critical event in the development of any autoimmune destruction of the seminiferous epithelium during orchitis (mahi- brown and tung ; mahi-brown et al. ) . recognition of antigen in association with mhc is essential to normal adaptive immune responses, and reduced expression of mhc class i (hla-a, hla-b, and hla-c in humans) and class ii proteins (hla-d) appears to be an important feature of immune-privileged tissues and tumors (garrido et al. ; haas et al. ; head and billingham ; wekerle et al. ) . reduction in class i and ii expression reduces the likelihood of immune-activating events involving cd þ helper t cells and cytotoxic cd þ t cells in the tissues. studies indicate a characteristic absence of expression of mhc proteins on the cells of the seminiferous epithelium under normal conditions (haas et al. ; head and billingham ; lustig et al. ; tung et al. ) , although both mhc classes are expressed in human spermatozoa (martín-villa et al. ) . there is evidence from studies on mouse sertoli cells in culture that mhc class ii expression can be upregulated in these cells by ifn (dal secco et al. ) . in contrast to the seminiferous epithelium, both mhc class i and ii proteins are expressed throughout the testicular interstitial tissue: mhc class i is expressed on most interstitial cells, including the leydig cells (haas et al. ; pöllänen and maddocks ) , while testicular macrophages and dendritic cells express mhc class ii (haas et al. ; head and billingham ; hedger and eddy ; mahi-brown et al. ; tung et al. ) . thus, it appears unlikely that a lack of apcs is a contributing factor in testicular immune privilege, although differences in the number and distribution of mhc class ii-expressing cells in the interstitial tissue of different species or strains may help to explain differences in susceptibility to autoimmune orchitis (flickinger et al. b; kojima and spencer ; teuscher et al. ) . the fact that the testis is not isolated from the immune system, yet can tolerate both endogenous (auto-) antigens associated with spermatogenesis and exogenous (allo-or xeno-) antigens expressed by grafts, is evidence of tissue-specific inhibition of the immune response. the question then arises: if immune responses against exogenous and endogenous antigens are inhibited in the testis environment, does the testis display a reduced capacity to deal with infections and greater susceptibility to certain kinds of tumors? there is evidence that immunity may be compromised in some situations, for example, the tendency toward relapsing acute lymphocytic leukemia in the testes (hudson et al. ) , and several systemic viral and mycobacterial infections target the testis, including mumps, human immunodeficiency virus, tuberculosis, and the severe acute respiratory syndrome virus (krieger ; nistal et al. ; xu et al. ) . however, the testis does not appear to be immunodeficient. even in comparison with the rest of the male reproductive tract, orchitis due to ascending infections is relatively rare (krieger ; ness et al. ) . moreover, the extremely variable level of success of experiments by different groups using different transplant models has shown that immune privilege of the testis is both limited and conditional (dobrinski ; head et al. a; maddocks and setchell ; selawry and whittington ; setchell et al. ) . just what are these necessary conditions, however, remains poorly defined. studies indicate that the effector arm of the immune response is intact within the testis, and it is the ability to recognize and react toward foreign antigens and/or mount a normal inflammatory response that is suppressed (head and billingham ; head et al. a; mahi-brown and tung ; mahi-brown et al. ) . in summary, there appears to be a potentially unique functional interaction between the testis and the immune system, which involves control of the mechanisms normally involved in immune activation, in order to limit adaptive immune responses without drastically compromising the ability of the testis to protect itself when required. although their presence and influence is almost universally neglected, macrophages, lymphocytes, and pmns have varying degrees of access to the interstitial tissue of the testis (table ) . they are generally excluded from the seminiferous epithelium under normal conditions. over the past years, studies have started to fill in some of the details concerning these cells. macrophages are by far the most prominent immune cells in the testis. they are found in the testes of all mammalian species so far examined, but are particularly numerous in human, rat, and mouse testes (frungieri et al. ; hume et al. ; mendis-handagama et al. ; miller et al. ; vergouwen et al. ; wang et al. ) . typically, macrophages arise from myeloid precursors in the bone marrow and circulate in the blood as monocytes. they are capable of adopting a broad range of structural and functional phenotypes that are largely dictated by the tissues in which they become resident (mantovani et al. ; van furth ) . characteristically, they are robustly phagocytic and possess a potent arsenal of antimicrobial and cytotoxic activities, such as the ability to generate large amounts of ros. they produce many immunoregulatory products, including cytokines, eicosanoids (prostaglandins, thromboxanes, leukotrienes, and lipoxins), and nitric oxide, all of which are important in establishing and controlling the inflammatory process, but also have important roles in tissue remodeling, healing, and normal homeostasis. all macrophage lineages can express mhc class ii antigens and have the capacity to present to cd þ helper or t reg cells (unanue ) . thus, macrophages are the cells that link innate and adaptive immunity, and dendritic cells are a highly specialized macrophage subset that is particularly effective in this role (heath and carbone ) . testicular macrophages have been most extensively studied in the rat, with a few studies also in the mouse and human. studies in the rat indicate that they are heterogenous in their morphology and functions (bryniarski et al. ; gerdprasert et al. a; itoh et al. b; wang et al. ) . their absolute numbers are primarily under leydig cell control, but evidence suggests that fsh acting via the sertoli cell has a role in regulating their functional activity (duckett et al. a,b; gaytan et al. c; wang et al. ) . macrophages accumulate within the interstitial tissue at the time of puberty, and play critical roles in the normal development of the testis, particularly by promoting the proliferation and maturation of the leydig cell population (cohen et al. ; gaytan et al. a,b; hardy et al. ; itoh et al. ; raburn et al. ; vergouwen et al. ) . through their ability to produce a broad array of growth factors, prostanoids, and vasoactive regulators, other developmental and homeostatic roles within the testis may be predicted as well. data concerning the immunological functions of testicular macrophages are based on a relatively small number of studies in either the rat or the mouse, and are patchy at best. indeed, there is some evidence for distinct differences even between these two closely related species. in general, testicular macrophages have intact phagocytic, cytotoxic, and antimicrobial functions (miller et al. ; wei et al. ) . rat testis macrophages express mhc class ii antigens (head and billingham ; hedger and eddy ; wang et al. ), but display a reduced capacity for lymphocyte activation in vitro . testicular macrophages in the human and mouse also express mhc class ii, although expression in the mouse appears to be more restricted (haas et al. ; itoh et al. b; pöllänen and niemi ; tung et al. ) . a lack of expression of the essential costimulatory molecules, b - (cd ) and b - (cd ), has been reported in the mouse testis as well (sainio-pöllänen et al. ) . studies also indicate that macrophages from the rat testis have a significantly reduced ability to produce proinflammatory cytokines, such as il and tnf, following activation (gerdprasert et al. a; hayes et al. ; meinhardt et al. ) , and stimulated mouse testicular macrophages produce the anti-inflammatory/immunosuppressive cytokines il and tgf in vitro (bryniarski et al. (bryniarski et al. , . while the data suggest that testicular macrophages may have a predominantly anti-inflammatory and/or immunosuppressive phenotype, this has yet to be formally established. furthermore, dendritic cells, which are a highly specialized macrophage subset also found within the testicular interstitium (fijak et al. ; hoek et al. ; itoh et al. b; rival et al. a) , presumably play a more important role in controlling immune responses to antigens within the testis (fijak et al. ; rival et al. a rival et al. , . in the rat testis, macrophages can be discriminated by their differential expression of two molecular markers recognized by the antibodies ed and ed . antibody ed recognizes the lysosomal protein cd , which is associated with antigen processing and presentation (groisman et al. ) , while ed recognizes the scavenger receptor cd (fabriek et al. ) . cd is involved in the clearance of hemoglobin:haptoglobin complexes and the resolution of inflammation (philippidis et al. ) . about - % of rat testicular macrophages express this scavenger receptor and about half of these cells also lack expression of cd (gerdprasert et al. a; meinhardt et al. ; wang et al. ). in the human testis, most macrophages appear to be cd þ , but expression of cd is a feature of a subset of these cells as well (frungieri et al. ) . the heterogeneous expression of these two markers points to the existence of multiple populations of testicular macrophages, and evidence suggests that these phenotypic differences also correspond to functional differences. in the normal and inflamed adult rat testis, cd þ cells lack expression of the key inflammatory mediators il and inducible nitric oxide synthase (inos), as measured by double-label immunohistochemistry, but these molecules are detectable in cd þ cells that do not express cd (gerdprasert et al. a; o'bryan et al. ) . indeed, purification of cd þ macrophages from the rat testis using immunomagnetic beads confirms that these cells have reduced capacity to produce il , il , or no upon stimulation, although they retain the ability to produce prostaglandins (gerdprasert et al. a; hedger, unpublished data) . this suggests that the cd þ macrophages have reduced proinflammatory activities, while the cd -negative testicular macrophage population retains normal proinflammatory functions. this observation is important because macrophages that express cd and produce il , but display reduced expression of cd , cd , inos, tnf, and il , are polarized toward the immunoregulatory m subset (mantovani et al. ; sica et al. ) . the m macrophage subset is analogous to the th subset among t cells, and is associated with late-stage inflammation and its resolution, as well as tumors and other immunologically privileged tissue sites. it would appear that a significant proportion of the resident macrophages of the testis possess an immunoregulatory phenotype that is consistent with immune privilege. the functional roles of this macrophage population, as well as those of the macrophages that appear to retain normal inflammatory (i.e., m subset) properties, demand particular attention if we are to understand the unique immunological environment of the testis. while the functions and regulation of lymphocytes within the testis have yet to be properly characterized, by their presence alone these cells will have significant effects on testicular events. it may also be assumed that, in contrast to the largely resident population of testicular macrophages, lymphocytes circulate through the testis. this may be because they possess specificity for testicular antigens, or because they have been activated during earlier intratesticular inflammatory or infectious events (czerkinsky et al. ; springer ) . quantitative studies in the rat indicate that, at any given time, lymphocytes are approximately % as numerous as the macrophages in the normal testis (hedger and meinhardt ; hedger et al. b; wang et al. ). in the rat and mouse, intratesticular lymphocytes express t cell and nk cell markers, and comprise distinct t cell, nk cell, and nkt cell subsets (tompkins et al. ; hedger, unpublished data) . both t cell and nk cell markers are expressed by lymphocytes in the human testis as well (hedger, unpublished data) . b cells are not usually observed in normal tissues, and this includes the testis. in the rat, many t cells possess a memory phenotype, as would be expected (hedger et al. b; tompkins et al. ). reanalysis of the data for individual animals across several studies suggests that their numbers tend to be proportional to the numbers of macrophages, but gradually increase with the age of the animal (hedger et al. b; hedger and meinhardt ; wang et al. ) . among the t cells of the testis, there is a strong bias toward the cd þ (i.e., mhc class i restricted, largely cytotoxic) subset (hedger et al. b; pöllänen and niemi ; ritchie et al. ; tompkins et al. ; wang et al. ) . the prominence of nk cell subsets in the testis is also intriguing given the reduced mhc class i antigen expression in the seminiferous compartment, which would tend to make these cells more susceptible to nk cells (lanier ) . consistent with the anti-inflammatory/immunosuppressive phenotype of the major testicular macrophage subset, t cells and nkt cells in the rat testis constitutively produce il (hedger, unpublished data) . while much remains to be discovered, the existing data concerning the types and activity of testicular lymphocytes are consistent with maintaining immune privilege (i.e., the presence of immunoregulatory lymphocytes) and increased innate immunity (i.e., cytotoxic cell activity). evidence that pmns are important in testicular function comes from the fact that mast cells and eosinophils are significant elements of the interstitial tissue in many species. in the rat, mouse, dog, cat, bull, and deer, both cell types are largely absent from the testicular parenchyma, but are associated with blood vessels in the testicular capsule (anton et al. ) . mast cells are found throughout the interstitial tissue in equine and human testes (anton et al. ; nistal et al. ; yamanaka et al. ) , and both mast cells and eosinophils are present in porcine species (anton et al. ) . the role of these cells in vascular control and innate immunity can be predicted, but they may also play immunoregulatory roles, as mast cells do in other tissues . in humans, testicular mast cell numbers are increased in infertility (meineke et al. ; nagai et al. ; yamanaka et al. ) , but decline with advancing age (nistal et al. ) . the possibility that they accumulate due to past immune events needs to be considered, but they also appear to be actively regulated. in the adult rat testis, mast cell proliferation is under the control of the leydig cells, suggesting that these cells produce an inhibitor of mast cell activity (gaytan et al. ; wang et al. ) . neonatal estrogen treatment increases mast cell numbers in the rat testis (gaytan et al. (gaytan et al. , , and a relationship with elevated leydig cell aromatase activity is suggested by the prevalence of these cells in boar and equine testes (eisenhauer et al. ; raeside and renaud ) . neutrophils are found in the testis only under conditions of testicular inflammation or damage (gerdprasert et al. a; kohno et al. a; o'bryan et al. b ). the majority of this chapter deals with the testis because it is the primary source of male gametes and hormones, and damage to this organ tends to have more significant consequences for fertility. the remainder of the male reproductive tract comprises the epididymis, vas deferens, accessory glands, and the urethra, which are all epithelial-lined ductal tissues of varying complexity. the immunology of these tissues appears to be quite distinct from that of the testis. while it is true that sperm spend relatively little time within most of these tissues, mature sperm reside within the epididymis for extended periods without provoking an overt immune response, and inflammation and immune cell activity can nonetheless have damaging effects in this organ. significantly, these tissues possess normal interepithelial cell junctions, but lack a completely occluding intercellular structure analogous to the blood-testis barrier (levy and robaire ; pelletier ) . secreted iga, which lacks the ability to bind complement and possesses principally antiinflammatory properties as a result, plays an important role in the male urogenital tract (clifton et al. ; politch et al. ; pudney and anderson ) , and the immunology of these organs tends to more closely resemble that of other elements of the mucosal immune system, such as the gastrointestinal and respiratory tracts (beagley et al. ; clifton et al. ; mestecky et al. ) . in the absence of the specialized mucosal-associated lymphoepithelial tissue normally found in the gastrointestinal and respiratory tracts, it is likely that the epithelial cells themselves, together with the numerous intraepithelial and stromal macrophages and lymphocytes that are normally found within these tissues, mediate local immunoregulatory mechanisms (barratt and cohen ; dym and romrell ; el-demiry et al. ; ritchie et al. ; pudney and anderson ; theyer et al. ; yeung et al. ) . a better understanding of the immunology of these tissues is important. inflammation, infection, and physical damage can lead to immune reactions against the sperm, which in turn may compromise fertility. moreover, many men suffer from chronic pelvic pain, generally manifesting as epididymitis or prostatitis in the absence of a detectable infection, a condition that almost certainly involves chronic inflammatory processes within the male reproductive tract. as outlined in the preceding sections, the testis is able to support immune privilege without sacrificing immune surveillance and protection. the mechanisms responsible for this immunological balancing act are still far from understood, but a number of inflammatory and immunoregulatory systems that operate within the testis have been investigated in varying degrees of detail. it is perhaps not very surprising that many of these regulatory systems also impact upon normal testis physiology, in addition to their immunological and pathological roles. given that the types and properties of leukocytes found within the testis environment are consistent with immunoregulation and enhanced innate immunity, the focus should be on which testicular elements are involved in creating this potentially unique immunological environment. most attention in this regard has been directed toward the highly versatile sertoli cell. evidence that the sertoli cell possesses specialized immunoregulatory properties comes from cotransplantation studies. sertoli cells from immature rat, murine, or porcine testes display extended survival as allografts or xenografts, and cotransplantation of sertoli cells or testis cell mixtures containing these cells confers increased survival on neural cell xenografts, and pancreatic islet allografts and xenografts (sanberg et al. ; selawry and cameron ; suarez-pinzon et al. ) . the ability to form a physical barrier through inter-sertoli tight junctions and the fact that sertoli cells have reduced mhc expression (haas et al. ; kohno et al. b; sanberg et al. ; turek et al. ) undoubtedly enhance their potential to avoid t cell activation both in the intact testis and in engraftment studies. moreover, a soluble form of the nonclassical mhc class ib molecule hla-g is produced by the sertoli cells, as well as spermatocytes, spermatids, and some interstitial cells, in the rhesus monkey testis (ryan et al. ) . this molecule has been implicated in apoptosis of alloreactive cd þ cytotoxic t cells (fournel et al. ) . sertoli cells display lymphocytotoxic activities and produce molecules with immunosuppressive activity, such as tgf (de cesaris et al. ; suarez-pinzon et al. ; wyatt et al. ; yin et al. ) and fasl (bellgrau et al. ; yin et al. ). an active immunoregulatory role has also been suggested: murine sertoli cells treated with ifn increase expression of mhc class ii molecules and the negative costimulatory ligand b -h , and stimulate t reg numbers in culture (dal secco et al. ) . the ability of the sertoli cell to suppress other critical inflammatory events through expression of complement regulatory proteins ) and secretion of inhibitors of lymphocyte cytotoxic activities (sipione et al. ) is also likely to be important. finally, sertoli cells possess an enormous capacity for phagocytosis of senescent cells, cell debris, and other potentially antigenic complexes, which could otherwise activate an immune response. data concerning immunoregulatory roles of other testicular cells are less comprehensive. the evidence clearly indicates a role for leydig cells in recruiting macrophages into the testis, although testicular macrophage function may actually be determined by the sertoli cells (duckett et al. a,b; gaytan et al. c; wang et al. ) . leydig cells also produce various steroids, proteins, and other factors with immunosuppressive activity, and inhibit lymphocyte activity in vitro (born and wekerle ; hedger et al. ). peritubular cells, like sertoli cells, produce immunoregulatory tgf family members (konrad et al. ; mullaney and skinner ; okuma et al. b) . the possibility that the germ cells themselves have a direct effect on immune cells has also been considered (hurtenbach and shearer ) , although it seems more likely that germ cells may influence immune responses indirectly by inducing immunoregulatory activities of the sertoli cells and other somatic cells. the relatively recent discovery of the tlrs has radically expanded our understanding of innate immunity and promises to do the same for testicular immunology. in addition to their predictable expression on testicular macrophages and dendritic cells, tlr mrna, protein, and/or ligand responses have been described in several other testicular cells. rat and mouse sertoli cells express tlr , , , and (bhushan et al. ; riccioli et al. ; starace et al. ; wu et al. ) . tlr , , and mrna expression has been observed in rat sertoli cells (bhushan et al. ) , and low levels of tlr , , and mrna have been detected in mouse sertoli cells (riccioli et al. ; wu et al. ). in the rat, germ cells also produce mrna for tlr , , and in a stage-specific manner, and the peritubular cells express tlr and , along with low levels of tlr , , and (bhushan et al. ) . rat leydig cells express tlr mrna and low levels of tlr (bhushan et al. ) . however, the cytoplasmic receptors tlr and have yet to be observed in any nonmyeloid testicular cell type. of the other pattern-recognition receptors, nod mrna has been detected in rat sertoli cells, peritubular cells, and spermatogonia, while nod was detected in spermatogonia only (bhushan et al. ) . expression of tlrs and other pattern-recognition receptors in the testis is obviously important for responses toward intratesticular pathogens. infections and inflammation, both systemic and localized, have negative effects on testicular function, resulting in reduced androgen production, lowered sperm counts, and temporary loss of fertility (adamopoulos et al. ; baker ; carlsen et al. ) . presumably, tlrs expressed by the sertoli cells and germ cells are necessary to detect pathogens that ascend the reproductive tract and, as a consequence, lie behind the blood-testis barrier. this expression within the seminiferous epithelium also suggests a mechanism whereby inflammation can directly inhibit spermatogenesis. activation of sertoli cells by tlr ligands induces production of inflammatory mediators, such as il , il , and no, which have direct effects on germ cell development and leydig cell steroidogenesis (gérard et al. ; stéphan et al. stéphan et al. , o'bryan and hedger ; riccioli et al. ; wu et al. ) . moreover, while inhibition of leydig cell steroidogenesis during inflammation may involve indirect effects at the level of the pituitary, or the actions of inflammatory mediators produced by the testicular macrophages and sertoli cells, there is evidence that these cells can also respond directly to tlr ligands (bhushan et al. ; xiong and hales a) . although the tlrs have evolved to recognize pathogen-derived molecules, such as lps (tlr ) and bacterial lipoproteins (tlr ), recent data show that they can also be activated by certain endogenous molecules. the ability of preparations of some endogenous molecules, such as heat-shock proteins, to activate tlr signaling is now attributed to contamination of these preparations by bacterial products (tsan and gao ) , but there is convincing evidence that mammalian mrna is a ligand for tlr (karikó et al. ) and mammalian cpg dna can activate tlr (yasuda et al. ) . the high mobility group box chromosomal protein (hmgb ), produced by both spermatogonia and sertoli cells, is a tlr and ligand (yu et al. ; zetterström et al. ) . expression of tlrs by the sertoli cells under normal conditions, therefore, suggests a potential role for these receptors in responding to these endogenous ligands. . . . . the interleukin family il is the best studied of all the cytokines normally expressed in the testis. it is produced in two forms, and , which are encoded by separate genes and share approximately % sequence homology. both forms act through the same receptor complex (il r ), and exert a similar range of proinflammatory and physiological effects (dinarello ; o'neill and dinarello ) . most il signaling occurs through activation of the myd /traf and jnk/p map kinase pathways, thereby regulating many proinflammatory genes through stimulation of the transcription factors nfb and activated protein (ap ) (medzhitov et al. ) . the il s are synthesized as precursor proteins, which are cleaved to produce kda active proteins. in the case of il , the precursor possesses low-level biological activity, but the il precursor protein is inactive (black et al. ; watanabe and kobayashi ) . the precursor of il is cleaved by the il converting enzyme (ice, caspase ) during the process of secretion, whereas il is cleaved by the calcium-dependent membrane-associated cysteine protease (calpain) or by extracellular proteases (thornberry and molineaux ; watanabe and kobayashi ) . il is produced by activated monocyte/macrophages and is the main secreted form during inflammation, whereas il is more commonly found within the cell, where it may act as an autocrine or paracrine growth factor involved in direct cell-to-cell communication. in addition to the prototypical il and il , the il family comprises a number of structurally related proteins, which appear to have arisen by gene duplication (dunn et al. ) . the closest structurally to il / is il , which is processed by caspase activity, but acts via a separate receptor rather than il r (gracie et al. ) . another member of the family, which does bind to il r but lacks the ability to transduce a signal, blocks il / action and is called il receptor antagonist (il ra) (arend ) . production of il is constitutively high in the normal testis, as il is produced by the sertoli cells in a cyclical manner within the mature seminiferous epithelium (jonsson et al. ; söder et al. ) . its production is driven by the developing germ cells and by phagocytosis of the residual cytoplasm cast off by the spermatids at the time of their release into the tubule lumen (gérard et al. ; syed et al. ) . sertoli cell il production is also stimulated by lps, but not by fsh (stéphan et al. ) . production of il by the normal testis is low, but is upregulated in a subset of testicular macrophages and the leydig cells during inflammation (gérard et al. ; jonsson et al. ; o'bryan et al. ) . curiously, the majority of this secreted testicular il exists as the precursor (o'bryan et al. ) . the receptor il r has been localized to germ cells and sertoli cells (gomez et al. ) . il stimulates dna synthesis in spermatogonia (i.e., mitosis) and early spermatocytes (i.e., meiosis), proliferation of immature sertoli cells, and a number of activities of the mature sertoli cell that support spermatogenesis, such as the production of lactate and transferrin (hakovirta et al. b; hoeben et al. ; nehar et al. ; parvinen et al. ; pöllänen et al. ; söder et al. ) . it also regulates the ability of the sertoli cell to maintain contact with other sertoli cells and the developing germ cells, by altering the sertoli cell cytoskeleton (sarkar et al. ). in the interstitium, both il and il suppress lh-stimulated androgen production by adult leydig cells through inhibition of steroidogenic enzyme activity (hales ; hales , ) . il ra is produced by the sertoli cell, and is stimulated by fsh, il , and lps (zeyse et al. ) . presumably, its role is to regulate the activity of il / within the testis. both il and its receptor have been found in the seminiferous epithelium, with il mrna and protein localized to spermatocytes and round spermatids (strand et al. ) . as is the case for il , the majority of il produced within the testis is in the precursor form, indicating that the processing of these cytokines by caspase in the testis is an area requiring more investigation. il stimulates spermatogonial dna synthesis in cultures of rat seminiferous tubules, without influencing germ cell apoptosis (strand et al. ) . another member of the family localized to the testis is il f , although its function there is entirely unknown (kumar et al. ) . . . . . tumor necrosis factor and fas ligand tnf and fasl are transmembrane and secreted cytokines with typically trimeric structures and they interact with specific receptors to mediate a cell death signal (ju et al. ; schütze et al. ) . they are implicated in both immunoregulation and the control of normal spermatogenic function in the testis. tnf is a kda glycosylated polypeptide and is a major early product of activated monocytes and macrophages. it binds to either of two tnf receptor subsets (tnfr and tnfr ) and plays a central role in initiating the inflammatory response by stimulating the production of il and il (basak and hoffmann ; bradley ) . whether tnf exerts proinflammatory or cytotoxic effects depends on the receptor subtype engaged and the expression of specific adaptor proteins within the target cell (basak and hoffmann ; chung et al. ; hsu et al. ; mak and yeh ) . consistent with its name, tnf exerts a cell death signal via tnfr , through interaction with the tnfr-associated death domain protein (tradd) or the fas-associated death domain protein (fadd), and activation of the caspase-dependent apoptotic pathway (schütze et al. ) . however, interaction with tradd can also result in recruitment of other adaptor proteins leading to the binding of traf and activation of the nfb pathway and/or the map kinases jnk or p (chung et al. ) . moreover, tnfr and its related receptors, which include cd , do not contain a death domain, and instead associate with the trafs leading to activation of cell signaling events (bradley ; mak and yeh ) . in the mouse testis, tnf is expressed by round spermatids, pachytene spermatocytes, and testicular macrophages, and mrna for tnfr has been located on both sertoli and leydig cells . in porcine sertoli cells, tnf receptor subunit protein expression is stimulated by fsh (mauduit et al. ) . there is no evidence that tnf is produced by the sertoli cell, but expression of tnf by the germ cells within the seminiferous epithelium, like that of il and il , is cyclical. within the seminiferous epithelium, tnf produced by the germ cells appears to play a complex role in the control of both sertoli cell function and spermatogenesis. tnf reduces spontaneous germ cell degeneration in cultured human and rat seminiferous tubules, suggesting a germ cell survival effect presumably mediated through the sertoli cell (pentikäinen et al. ; suominen et al. ). conversely, tnf disrupts sertoli cell tight junction assembly by inhibiting the production of junction proteins and by regulating matrix metalloprotease and protease inhibitor activity (siu et al. ) . tnf also stimulates plasminogen activator inhibitor expression in rat testicular peritubular cells (le magueresse-battistoni et al. ) . similar to il , tnf stimulates basal lactate production by cultured sertoli cells, but tnf generally antagonizes the actions of fsh on sertoli cell function, including the stimulation of aromatase activity and lactate production (mauduit et al. ; nehar et al. ; riera et al. ). however, delfino et al. ( ) have shown that tnf stimulates androgen receptor expression in sertoli cells via upregulation of nfb, which binds to several enhancer motifs in the androgen receptor promoter. tnf also stimulates the expression of inflammatory cytokines, chemokines, and leukocyte adhesion molecules in both sertoli cells and peritubular cells (aubry et al. ; de cesaris et al. ; stéphan et al. ) . in testicular pathology, tnf has been implicated as a major causative agent in the development of experimental autoimmune orchitis (eao) (yule and tung ) . there is a significant increase in the number of tnf-positive testicular macrophages and the number of tnfr -positive germ cells undergoing apoptosis during eao in rats (suescun et al. ) . within the interstitium, tnf acts as an effective regulator of leydig cell steroidogenesis, by inhibiting lh receptor binding and steroidogenic gene expression (li et al. ; mauduit et al. b mauduit et al. , hales b, ) . no intratesticular cytokine has excited more controversy than the cell death signaling ligand fasl. interactions between fasl and its receptor on activated t cells are important in the regulation of the immune response (ju et al. ) . the death domain in the cytoplasmic region of the fas receptor recruits the fadd adaptor protein and induces t cell death via caspase-dependent apoptosis (schütze et al. ). there is evidence that fasl expression on epithelial cells in immune-privileged tissues, including sertoli cells, may be responsible for deleting activated t cells within the tissue, thereby suppressing adaptive immunity (bellgrau et al. ; griffith et al. ; saas et al. ; stuart et al. ). however, this hypothesis has been challenged by the observation that increased fasl expression on cells such as tumor cell lines and pancreatic islet cells does not increase protection in transplantation studies, but actually provokes a massive inflammatory reaction and rejection (allison et al. ; kang et al. ) . moreover, fasl appears to be abundantly expressed in the epithelia of several human tissues that lack any evidence for immunological privilege (xerri et al. ) , while absence or inhibition of fas or fasl does not automatically cause failure of immune privilege (rogers et al. ; suarez-pinzon et al. ; wahlsten et al. ) . it seems unlikely that fasl expression on its own is a fundamental explanation for immune privilege, although it may contribute through interaction with other immunoregulatory processes . localization of fasl and fas in the testis under normal conditions also has proven controversial, which may be attributed to differences in detection methods, limitations of some of the reagents that have been used, and the fact that these molecules are inducible (d'alessio et al. ; restifo ) . studies have shown fasl to be present in rat, mouse, porcine, and human sertoli cells and absent in most germ cells (bellgrau et al. ; braendstrup et al. ; d'abrizio et al. ; lee et al. ) , but other studies have reported that fasl expression in the rat seminiferous epithelium is confined to the germ cells (celik-ozenci et al. ; d'alessio et al. ) . the receptor fas has been found on isolated mouse sertoli cells (riccioli et al. ) , but in intact testes has been localized to spermatogonia and spermatocytes from the pubertal period onward (celik-ozenci et al. ; lee et al. ; lizama et al. ; ogi et al. ). in germ cells, fas expression is associated with cells that are undergoing apoptosis (lee et al. ; lizama et al. ) , and fas is induced by tnf and ifn in the sertoli cell (riccioli et al. ) . curiously, in one study using fragment cultures of human seminiferous tubules, tnf downregulated fasl expression and inhibited apoptosis of germ cells (pentikäinen et al. ) . less controversially, fas and fasl expression is upregulated in various models of seminiferous epithelium damage, indicating that this mechanism is important in regulating germ cell apoptosis in cases of physical and toxicological insult (lee et al. ; ogi et al. ). members of the il family of cytokines exert their actions via binding to specific receptors that associate with a common membrane signal transducer gp , leading to the activation of the janus kinase/signal transducers and activators of transcription (jak/stat) and map kinase cascades (heinrich et al. ) . in addition to their functions in inflammation and the immune response, these cytokines play crucial roles in hematopoiesis, liver and neuronal regeneration, embryonic development, and fertility. members of this family that have been detected in the testis are leukemia inhibitory factor, oncostatin m, ciliary neurotropic factor, il , and il itself (de miguel et al. (de miguel et al. , du et al. ; jenab and morris ; okuda et al. ; stéphan et al. ) . the principal roles of most of these cytokines in the testis appear to lie in regulating leydig cell development and the onset of spermatogenesis. a role in testicular immunology is most likely for il , which possesses both proinflammatory and anti-inflammatory properties, and regulates the acute-phase response as well as several aspects of immune cell development and activity (kopf et al. ; tilg et al. ) . sertoli cells, leydig cells, and peritubular cells have been shown to produce il in vitro (cudicini et al. b; okuda et al. ; syed et al. syed et al. , . in rat sertoli cells, il production is stimulated by fsh, testosterone, phagocytosis, and other inflammatory stimuli, including il , il , tnf, and lps (cudicini et al. a,b; okuda et al. okuda et al. , stéphan et al. ; syed et al. ) . production of il by cultured mouse sertoli cells is inhibited by inf (riccioli et al. ; stéphan et al. ) . within the seminiferous epithelium, endogenous production of il follows a cyclical pattern that corresponds with the changes in the stages of the spermatogenic cycle, most probably under the influence of il and fsh (hakovirta et al. ; syed et al. syed et al. , . both the il -specific receptor subunit (il r) and the gp mrna are expressed in rat sertoli cells, and are stimulated by il and il , but only the il r subunit is stimulated by fsh (fujisawa et al. ) . il increases basal and fsh-induced transferrin and cyclic gmp secretion by the sertoli cell, and inhibits meiotic dna synthesis in preleptotene spermatocytes (boockfor and schwarz ; hakovirta et al. ; hoeben et al. ) . in models of eao, il appears to play an ameliorative or protective role within the seminiferous epithelium (li et al. ; rival et al. b ). leydig cells are also active producers of il following stimulation by lh, lps, or il in vitro, and evidence suggests that these cells actually may be the major testicular source (boockfor et al. ; cudicini et al. b; okuda et al. okuda et al. , . several studies have implicated il as a key player in testicular immunology. testicular il production is preferentially induced by systemic lps treatment in the adult rat testis (o'bryan et al. ) , and macrophages and t cells isolated from the rat testis constitutively produce il , which may contribute to testicular immune privilege (hedger, unpublished data) . in a mouse model of eao, elevation of endogenous il levels by adenoviral-mediated transfection of human il was found to significantly reduce the incidence of immune activation, orchitis, and spermatogenic damage (watanabe et al. ) . although male mice deficient in il do not show evidence of male fertility problems, intratesticular immune responses have yet to be examined in these animals (kühn et al. ) . . . . . nitric oxide and oxygen metabolites production of ros, particularly by macrophages, is an important component of the early response to infection (takemura and werb ) . significant ros include the superoxide anion (o -), hydrogen peroxide (h o ), the hydroxyl radical (ho?), nitric oxide (no?), and the peroxynitrite anion (onoo À ). most are products of normal cellular metabolism, but their production is stimulated during inflammation through the activity of the enzymes nadph oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) and inos (forman and torres ; nathan ). these small molecules are cytotoxic to many microorganisms, although some play a more complex role in cell signaling processes. they also interact with crucial intracellular macromolecules, such as proteins, lipids, and dna, causing oxidative damage. although there are endogenous repair systems to correct oxidative damage, excessive production of ros contributes to the pathology of many diseases. among the ros, nitric oxide represents a special case. at high levels, no is highly cytotoxic, especially as a precursor of the peroxynitrite anion, but at low levels no acts as an intracellular and extracellular signaling molecule (bogdan ; schmidt and walter ) . it is produced by one of three related enzymes, neuronal nos (nnos or nos ), inducible nos (inos or nos ), and endothelial nos (enos or nos ), which catalyze the conversion of l-arginine to l-citrulline and no (alderton et al. ) . the nos enzymes are homodimeric proteins encoded by three separate genes ( table ) . both nnos and enos are constitutively expressed enzymes whose activity is regulated through a calcium-calmodulin-mediated mechanism, whereas inos is a constitutively activated enzyme that is regulated at the transcriptional and translational levels by various inflammatory mediators, including lps, il , and tnf (wolkow ) . in studies in various species, nos has been found in sertoli cells, leydig cells, peritubular cells, germ cells, testicular macrophages, and vascular endothelial cells (kim et al. ; lee and cheng ) . the nnos gene also produces a testis-specific isoform, tnnos, which has been localized to leydig cells (wang et al. ). in the normal rat seminiferous epithelium, inos is expressed by elongating spermatids and pachytene spermatocytes, particularly during the stages immediately following sperm release, with relatively lower levels of expression in sertoli cells and peritubular cells throughout the (o'bryan et al. a ). in the interstitium, macrophage expression of inos appears to be largely confined to the minority of cd -negative macrophages of the testis, and is not detectable in the majority of resident macrophages even during inflammation (o'bryan et al. a; gerdprasert et al. a) . testicular cell inos expression and no production are increased by inflammatory events induced by lps, testicular torsion, or testicular heating (lue et al. ; o'bryan et al. a; shiraishi et al. ). production of no by germ cells, in particular, is implicated in the control of the formation and disassembly of the sertoli cell junctions that constitute the blood-testis barrier, as well as the junctional complexes involved in sertoli-germ cell adhesion lee et al. ) . moreover, pachytene and round spermatid apoptosis is significantly reduced in inos null mice, leading to an increase in daily sperm output and indicating a key role for inos in limiting germ cell survival and/or the carrying capacity of the sertoli cells (lue et al. ) . no inhibits leydig cell steroidogenesis directly, an action that is probably mediated through oxidative damage, and treatment with nos inhibitors counteracts the normal decrease in testosterone associated with sepsis or stress (del punta et al. ; sharma et al. ; welch et al. ) . similarly, production of other ros has been implicated in the loss of steroidogenic function in various inflammatory models and in leydig cell cultures (georgiou et al. ; quinn and payne ) . last but not least, no is a potent vasodilator, and plays a role in the endogenous control of testicular blood flow and formation of the interstitial fluid (lissbrant et al. ; o'bryan et al. a) . production of no is an important mediator of germ cell death in testicular torsion (moon et al. ; shiraishi et al. ) , and may be involved in the testicular damage associated with varicocele (santoro et al. ; türker köksal et al. ) . it is evident that the major inflammatory signaling pathways mediated via nfb and the p /jnk map kinases play important, albeit complex, intratesticular roles. notably, nfb has been implicated as an apoptosis-inducing signal for germ cells in numerous studies (lysiak et al. ; pentikäinen et al. ; rasoulpour and boekelheide ; starace et al. ) , even though it stimulates androgen receptor expression in the sertoli cell (delfino et al. ) . activation of p /jnk is implicated in the stimulation of proliferation by immature sertoli cells and the regulation of blood-testis barrier dynamics, steroidogenesis, and multiple inflammatory responses, including the production of il , inos, monocyte chemoattractant protein , and leukocyte adhesion molecules, in the mature sertoli cell (de cesaris et al. , ishikawa and morris ; ishikawa et al. ; petersen et al. ; riccioli et al. ) . while these pathways mediate many of the effects of inflammation on the testis, endogenous regulation of these pathways also appears to be involved in the normal functions of the seminiferous epithelium, as will be discussed later. the ifns are functionally related cytokines with antiviral activity and they comprise three main groups (, , and ), based on their structural relationships and major cellular sources (hertzog et al. ; malmgaard ) . the type i ifns (ifn and ) are produced by a variety of cell types in addition to leukocytes, and exert primarily antiviral and antiproliferative effects via the ifn receptor (ifnar). the type ii ifn is produced by nk and nkt cells, activated t cells and, under certain conditions, by macrophages and dendritic cells (schoenborn and wilson ; varma et al. ) . it acts through a separate receptor, and regulates the activity of apcs as part of the adaptive immune response in addition to its antiviral functions. hence, ifn is a type ii ifn, but a type cytokine. type i ifn production is stimulated through tlr , which detects double-stranded viral rna, and tlr , the bacterial lps receptor (hertzog et al. ) . these two tlrs, which are expressed in the testis (bhushan et al. ; riccioli et al. ; starace et al. ; wu et al. ) , are able to induce the ifn transcription factor irf , via engagement of the trif adapter protein (akira and takeda ; hertzog et al. ; o'neill and bowie ) . the regulation of ifn is cell specific and complex, but involves jak/stat signaling and various transcription factors, including nfb and ap (schoenborn and wilson ; varma et al. ) . its production is stimulated by type i ifns and by il , among other cytokines. viral infections stimulate type ifn and, somewhat more surprisingly, ifn production by sertoli cells, peritubular cells, leydig cells, and testicular macrophages, leading to typical antiviral responses in the testis (dejucq et al. (dejucq et al. , a hu et al. ; starace et al. ) . a role for ifn in maintaining testicular immune privilege has been indicated by the observation that mouse sertoli cells upregulate their expression of the negative costimulatory ligand b -h in response to ifn in vitro, but remain devoid of positive costimulatory molecules (dal secco et al. ). on the other hand, in vivo studies have identified ifn as a causative factor in eao in mice (itoh et al. b) . moreover, ifn stimulates sertoli cell production of fas and caspase , and is implicated as the mediator of sertoli cell and germ cell apoptosis under various conditions (kanzaki and morris ; riccioli et al. ) . these disparate observations are indicative of a complex relationship between ifn, the local immune system, and spermatogenesis. ifn production also affects testicular steroidogenesis. normal healthy men treated with human ifn had significantly decreased serum testosterone levels in conjunction with normal serum gonadotropins, and both ifn and ifn inhibit testosterone production in primary cultures of leydig cells (orava et al. ). studies using porcine leydig cells indicate that ifn exerts its inhibitory effect on testosterone production at the level of cholesterol transport into the mitochondria, and inhibits expression of the steroidogenic acute regulatory (star) protein and the p- steroidogenic enzymes, cholesterol side-chain cleavage complex (p scc) and -hydroxylase/c -c lyase (p c ) (orava et al. (orava et al. , . these data indicate that ifns may contribute to the decline in steroidogenic function of patients with viral infections, in particular. however, dejucq et al. ( dejucq et al. ( , a have shown that an increase in ifn and ifn expression in sertoli and leydig cells of rats infected with sendai virus was actually associated with an increase in testosterone production. these results suggest that indirect or secondary stimulatory effects on testicular steroidogenesis may be involved, providing further evidence that ifns play a complex role in testicular function. and activin a the three tgf family members that are expressed in mammals (tgf , tgf , and tgf ) are the archetypes of a much larger superfamily of mostly homo-and heterodimeric proteins, which includes the activins, the bone morphogenetic proteins (bmps), inhibin, and anti-müllerian hormone (lin et al. ) . the tgfs themselves are multifunctional growth and differentiation factors involved in many aspects of tissue remodeling and repair as well as regulation of the immune system (ashcroft ; chang et al. ; licona-limón and soldevila ) . nearly all cells synthesize a form of tgf and possess functional receptors for these cytokines. most tgf family ligands act via dual transmembrane serine/threonine kinase receptors, called type i and type ii, which interact upon ligand binding (cárcamo et al. ; ebner et al. ; massagué et al. ) . signals are transduced from the membrane to the nucleus through phosphorylation of the regulatory smad signaling proteins, with smads , , and responsible for mediating tgf and activin signaling (datto et al. ; de caestecker et al. ) . all three mammalian tgf forms are differentially expressed by sertoli cells, peritubular cells, and leydig cells in the fetal and immature testis, but production declines considerably during sexual maturation gautier et al. ; mullaney and skinner ; olaso et al. ) . in the postpubertal testis, they have also been localized to the germ cells (caussanel et al. ; teerds and dorrington ) , and tgf receptors are found in both somatic and germ cells (caussanel et al. ; goddard et al. ; le magueresse-battistoni et al. ) . the tgfs appear to be involved in testicular development by controlling, among other things, apoptosis of undifferentiated spermatogonia (gonocytes) (olaso et al. ) , seminiferous tubule formation, and leydig cell differentiation (cupp et al. ; dickson et al. ; khan et al. a; konrad et al. ) . in the adult testis, tgf and tgf regulate sertoli cell tight junction dynamics, indicating a role in regulating the permeability of the bloodtestis barrier to facilitate the passage of spermatocytes across the barrier during spermatogenesis (lui et al. ; xia et al. ) . however, the tgfs are also potent inhibitors of the activity of immune cells, especially t cells, b cells, and nk cells (ahmad et al. ; ashcroft ; cousins et al. ; licona-limón and soldevila ; wahl et al. ) , suggesting a prominent role in creating testicular immune privilege. accordingly, tgf contributes to the lymphocyte inhibitory activity of testis extracts (pöllänen et al. ) and to the immunoprotective properties of sertoli cells in cotransplantation studies (suarez-pinzon et al. ) . activins are homodimers or heterodimers of homologous subunits, designated a -e , which are structurally related to the tgfs (chang et al. ; de kretser et al. ) . homodimers of a form activin a, which is widely expressed and has been extensively studied. relatively less is known about the other activin forms, which appear to be both less abundant and less widely distributed. activin a is a multifunctional growth factor and inflammatory regulator (phillips et al. ) , although activins were originally named for the ability of activin a and b, in particular, to stimulate pituitary fsh secretion (corrigan et al. ; ling et al. ). heterodimers of either a or b subunits with a homologous subunit, are called inhibin a and inhibin b, respectively, and act as feedback inhibitors of fsh secretion from the pituitary (robertson et al. ). unlike the activins, inhibins are not widely produced, and their chief source is the sertoli cell, the cellular target of fsh action meunier et al. ) . activin functions are highly regulated. like the tgfs, they act via specific type i and type ii transmembrane serine/threonine kinase receptors, linked to the smad signaling pathway (ethier and findlay ; vale et al. ) . the inhibins are competitive inhibitors of activin receptor binding, but activin homodimers and heterodimers comprising the b and c subunits also appear to act as weak competitive agonists of activin a (brown et al. ; makanji et al. ; mellor et al. ) . these interactions are modulated and/or facilitated by specific inhibitory coreceptor proteins, such as betaglycan and the bmp and activin membrane-bound inhibitor (bambi) (lewis et al. ; makanji et al. ; onichtchouk et al. ) . moreover, activin bioactivity can be effectively neutralized in the circulation by the high-affinity activin binding protein, follistatin (nakamura et al. ) . activin a is abundantly produced in the testis, particularly during early testicular development (barakat et al. ; buzzard et al. ) . even in the adult rat, intratesticular fluid levels of activin a are - times higher than normal circulating concentrations (o'bryan et al. ) . the sertoli cells appear to be the main source in the adult testis, although the a subunit is expressed in most germ cells kaipia et al. ) , and activin a protein is present in the resident macrophages and mast cells (okuma et al. b (okuma et al. , . activin a is expressed at low levels throughout the cycle of the seminiferous epithelium, but there is a distinct peak of production immediately following spermiation, which is driven by the surge of il produced by the sertoli cell at this time (kaipia et al. ; okuma et al. ) . activin receptors are expressed by most, if not all, of the somatic and germ cells in the testis (cameron et al. ; de winter et al. ; feng et al. ; kaipia et al. ) . activin a exerts a complex regulation of germ cell, sertoli cell, and leydig cell proliferation and/or differentiation during development and in the adult (barakat et al. ; buzzard et al. ; mather et al. ; mauduit et al. a; meehan et al. ; meinhardt et al. ) , and disorders of activin signaling are implicated in the onset of testicular cancer (dias et al. ) . activin a is produced by activated monocytes, macrophages, dendritic cells, bone marrow stromal cells, and some lymphocytes (erämaa et al. ; ogawa et al. ; robson et al. ; yamashita et al. ) , and plays a facilitating role in early inflammation responses (jones et al. ) . consistent with its homology to the tgf proteins, however, activin a also possesses a repertoire of anti-inflammatory and immunoregulatory activities, and displays characteristics of a type cytokine (hedger et al. ; ogawa et al. ; wang et al. ; yu et al. ) . in various experimental systems, activin a has been found to antagonize the production and actions of il and il , to inhibit critical t cell and b cell activation responses, and to induce macrophages to polarize toward the m phenotype (brosh et al. ; gribi et al. ; ogawa et al. ; russell et al. ) . both sertoli cells and testicular macrophages in culture respond to lps by increasing activin a production (okuma et al. a ) (hedger, unpublished data) , and il is a very potent stimulus for activin a production by the sertoli cell (okuma et al. b (okuma et al. , . the actual role of activin a in testicular inflammation remains unclear, since the high intratesticular levels of activin a are not affected by lps treatment in vivo (o'bryan et al. ) , but a role for activin a in modulating testicular immune responses can be predicted. androgens produced by the leydig cells have immunosuppressive properties that contribute to differences in immunity between the sexes (cutolo et al. ; miller and hunt ) . the majority of these effects are believed to be exerted at the level of the immune tissues, rather than by direct effects on circulating leukocytes, which lack classical androgen receptors (grossman et al. ; sasson and mayer ) . recently, however, it has been discovered that steroids can interact with membrane-bound g-protein-coupled receptors to trigger nongenomic responses (braun and thomas ; rahman and christian ) . studies have shown that androgens alter [ca] fluxes in lymphocytes and macrophages via such membrane-mediated interactions, affecting gene expression and function in the target cells (benten et al. ; walker ; wunderlich et al. ) . although studies of the role of androgens in suppressing immune responses, such as graft rejection, in the testis have been somewhat equivocal (cameron et al. ; selawry and whittington ; whitmore and gittes ) , the observation that immune cells can respond directly to androgens has resurrected the intriguing possibility that androgens, and possibly other testicular steroids, may directly regulate immune cell activity within the testis and adjacent draining lymph nodes. glucocorticoids play an important role in modulating both testicular immunity and steroidogenesis via a complex extratesticular regulatory loop. during inflammation, proinflammatory cytokines activate the hypothalamic-pituitary-adrenal axis leading to increased secretion of glucocorticoids, which act through the ubiquitously expressed glucocorticoid receptors (buckingham et al. ; sapolsky et al. ) . glucocorticoids suppress innate and acquired immunity at multiple levels and are an essential control in the inflammatory/immune response. they inhibit the inflammatory response primarily by repression of nfb, suppressing the production and actions of the proinflammatory cytokines, reducing adhesion molecule expression, and stimulating the production of anti-inflammatory cytokines (auphan et al. ; brack et al. ; kapcala et al. ) . glucocorticoids also exert inhibitory effects at all levels of the hypothalamic-pituitary-leydig cell axis (bambino and hsueh ; hales and payne ; hardy et al. ; monder et al. ), thereby contributing to the inhibition of androgen production that accompanies local and systemic inflammatory events. prostaglandins (pgd, pge, and pgf), prostacyclins (pgi), and thromboxanes (tx), collectively called the prostanoids, are fundamental to many physiological processes, including inflammation and immunity. prostanoids arise from hydrolysis of membrane glycerophospholipids to release the free fatty acid arachidonic acid through the action of one of the more than enzymes with phospholipase a (pla ) activity (kudo and murakami ; schaloske and dennis ) . arachidonic acid is in turn directed down the prostanoid synthetic pathway by the action of the cyclooxygenase (cox; also called prostaglandin-endoperoxide synthase) enzymes (figure ) . there are two cox enzymes, which are the products of different genes: the constitutively expressed cox and an inflammation-induced form, cox (table ) (smith et al. ; tanabe and tohnai ) . while conversion of arachidonic acid to the prostanoid precursor pgh by cox is the rate-limiting step, production of specific prostanoids depends on the activity of the prostaglandin synthases, that is, pge synthase (pges), pgis, pgds, pgfs, and thromboxane a synthase (txas) (helliwell et al. ; ueno et al. ) . each prostanoid acts via one or more specific receptors to regulate cell growth, vascular smooth muscle constriction or relaxation, vascular permeability, and immune cell activity (bos et al. ; hata et al. ) . several prostanoids exert both proinflammatory and immunosuppressive actions through different receptor interactions, or through metabolism to other active immunoregulatory metabolites, such as -deoxy-pgj , a ligand for the peroxisome proliferator-activated receptor (ppar) (jiang et al. ). an alternative synthetic pathway, catalyzed by the lipoxygenases, converts arachidonic acid to the related leukotrienes and lipoxins, which also possess diverse metabolic, vascular, and inflammation-regulating actions (samuelsson et al. ; takano et al. ) . most of the enzymes responsible for prostanoid production are ubiquitously expressed, and this includes the male reproductive tract (gerena et al. ; lazarus et al. ; takahashi et al. ; winnall et al. ) . in fact, the levels of e series prostaglandins in human seminal plasma are remarkably high (cooper and kelly ) . most testicular cells express both cox forms, and possess the capacity to produce prostaglandins in vitro (winnall et al. ) , although there appear to be cell type-specific and species-specific differences in the relative levels of expression (balaji et al. ; frungieri et al. ; lazarus et al. ). in the normal rat testis, cox is responsible for the majority of prostaglandin production (winnall et al. ), although intratesticular pge levels are only marginally affected by acute inflammation (winnall et al. ) . this is probably due to the fact that macrophages in the rat testis express cox at very low levels, but are the only testis cell type to respond to an inflammatory stimulus with a significant increase in cox activity (winnall et al. ). these data point toward a previously unanticipated maintenance role for the socalled inducible cox enzyme in testicular function, as well as an anomalous inflammatory response of testicular cox , consistent with an altered capacity of this organ to initiate and support inflammatory reactions. fertility is retained in male mice lacking either cox or cox , whereas double deletions are lethal (langenbach et al. ; lim et al. ; sales and jabbour ) . studies on male fertility and spermatogenesis using prostaglandins, or nonsteroidal anti-inflammatory drugs such as aspirin and indomethacin, which act primarily via inhibition of cox activity, have produced conflicting conclusions (abbatiello et al. ; biswas et al. ; sanyal et al. ; winnall et al. ) . these diverse outcomes indicate the complexity of the roles of prostanoids in controlling various aspects of testicular function. prostaglandins, particularly pge and pgf , have been implicated in the control of leydig cell development in the immature testis, production of proinflammatory cytokines by the leydig and sertoli cells, autoregulation of steroidogenesis in the adult, and the decline in leydig cell function that occurs during aging (baker and o'shaughnessy ; cooke et al. ; haour et al. ; ishikawa et al. ; walch and morris ; wang et al. ) . production of pge and pgf by cox also mediates the effects of il on protein and lipid regulation by the sertoli cell involved in supporting figure prostanoid and lysophosphatidylcholine synthesis and targets. synthesis of the prostanoids commences through the action of phospholipase a (pla ) on arachidonyl-containing phospholipids (usually a -acyl- -arachidonylglycerophospholipid) to produce the free fatty acid arachidonic acid and the corresponding lysophospholipid. arachidonic acid is converted into the prostanoid precursor prostaglandin h (pgh ) by the action of cyclooxygenases and this is further converted into the various prostanoid subtypes, prostaglandins (pgd, pge, and pgf), prostacyclins (pgi), and thromboxanes (tx), by the action of specific prostaglandin and thromboxane synthases. synthesis of lipoxins and leukotrienes from arachidonic acid occurs via the activity of a separate lipoxygenase enzyme (not shown). the prostanoids interact with their various receptor subtypes: there are two pgd receptors (dp - ) and four pge receptors (ep - ). it is the cellular expression of these receptor subtypes that ultimately determines the eventual biological responses; for example, different prostanoid and receptor combinations may produce either proinflammatory or anti-inflammatory responses, or may have opposing vasodilatory and vasoconstrictive effects. when the phospholipid is a phosphatidylcholine, a lysophosphatidylcholine (lpc) is formed, which may interact with cells via g-protein-coupled receptor-mediated or direct membrane interactions, or may undergo further enzymatic modification to form other bioactive lipids. spermatogenesis and inflammation in the testis (ishikawa and morris ; ishikawa et al. ). in addition, pge and pgf produced by mature spermatozoa play a role in the acrosome reaction (breitbart et al. ; joyce et al. ) . products of the lipoxygenase pathway, such as leukotriene b , are also produced by the testis, where they intervene in the regulation of leydig cell steroidogenesis by lh (papadopoulos et al. ; sullivan and cooke ) , but other functions can be expected. direct evidence for the anticipated roles of prostanoids or lipoxygenase products in testicular inflammation and immunity is lacking. initially, there was some evidence to suggest that pge might be responsible for the immunosuppressive phenotype of resident testicular macrophages . this now seems unlikely, because testicular macrophages produce little pge under normal conditions, and blocking cox activity in vivo or in vitro does not affect the production of proinflammatory cytokines in the testis in response to lps (gerdprasert et al. a; winnall et al. winnall et al. , . in the rat, chronic inhibition of cox inhibited interstitial fluid formation in normal testes, but ameliorated the loss of fluid that usually occurs during lps-induced inflammation (winnall et al. ) . the latter data would seem to indicate roles for products of cox in the control of vascular tone in the normal and inflamed testis and this deserves further exploration. when phosphocholine (pc)-containing phospholipids are used as the substrate for pla action, lysophosphatidylcholines (lpcs) are also produced (aoki et al. ; snyder ) . lpcs usually comprise a glycerophosphocholine backbone, a single long-chain fatty acid, and a free hydroxyl group (khaselev and murphy ) . the platelet-activating factors (pafs) are a subset of lpcs with an acetyl group in place of the hydroxyl group, which facilitates binding to the paf receptor (snyder ). the effective concentrations of these phospholipidderived molecules in biological fluids are normally tightly controlled by binding to albumin and lipoproteins (croset et al. ) , since some lpcs, particularly those derived from the saturated c and c fatty acids, cause cell lysis at high concentrations (weltzien ) . at physiological concentrations, however, lpcs induce specific responses in various cell types, including most immune cell types. nonacetylated lpcs are chemotactic toward macrophages, t cells, and nk cells, and stimulate cox expression by macrophages, cytokine production by t cells, and the cytotoxic activity of nk cells (légrádi et al. ; radu et al. ; whalen et al. ; yang et al. ) . in addition, lpcs induce apoptosis in t cells, indicating a role in immunosuppression as well (kabarowski et al. ; takeda et al. ) . the details of the mechanisms involved are poorly understood. while there is evidence for involvement of specific g-protein-coupled receptors (flemming et al. ; lin and ye ; radu et al. ; soga et al. ; yang et al. ) , lpcs may interact with g-proteins, membrane kinases, and ion channels directly, thereby bypassing the need for any specific receptor-ligand interactions. the interstitial fluid of the testis has long been known to possess potent lymphocyte-inhibiting activity in vitro (hedger et al. a; pöllänen et al. ) . the molecules responsible for this activity have now been isolated and identified as the specific lpcs: -palmitoyl-sn-glycero- -phosphocholine ( : a-lpc), -oleoyl-sn-glycero- -phosphocholine ( : a-lpc), a : a-lpc (putatively, -linoleoylsn-glycero- -phosphocholine), and a : a-lpc (putatively, -arachidonyl-sn-glycero- -phosphocholine) (foulds et al. ). these molecules inhibit t cell proliferation in response to activation and induce apoptosis of these cells in a time-and dose-dependent manner at physiologically relevant concentrations. although paf activity has also been found in the testis (muguruma et al. ) , these acetylated lpcs did not appear to contribute significantly to the t cell inhibitory activity. the emergence of lpcs as regulators of critical immune events in the testis opens up new avenues of inquiry into the origins of autoimmune infertility and more general mechanisms of peripheral immunoregulation. these molecules may also have other roles in testis biology that demand consideration. . . . . cell surface regulatory molecules: complement inhibitors and nonclassical mhc inhibition of complement activation is an important mechanism for reducing transplant rejection, particularly in the early phase. membrane complement inhibitors, such as cd , cd , cd , and the complement receptor-related protein (crry), are expressed in several immune-privileged tissues and have been implicated in suppression of inflammation in the eye and placenta (bora et al. ; bulla et al. ) . cd and cd appear to be confined to spermatids in the adult rat testis, crry is expressed on early spermatocytes and interstitial cells, but cd is widely expressed throughout the seminiferous epithelium and interstitium . a role for cd expression by the sertoli cells in prolonging the survival of these cells in transplantation studies has been suggested . moreover, complement regulators both in seminal plasma and on the surface of the spermatozoa (cd , cd , and cd ) may contribute to complement evasion by the spermatozoa in the female tract . classical mhc class i molecules (hla-a, hla-b, and hla-c) are expressed on nearly all cell types, and facilitate antigen presentation by these cells and activation of cytotoxic t cells (cresswell et al. ; janeway ; sant et al. ) . by contrast, the nonclassical mhc class ib molecules (hla-e, -f, and -g) are much less widely expressed, and are associated with suppression of the adaptive immune response. membrane-bound and soluble variants of these mhc class ib molecules have been implicated in the apoptosis of alloreactive cytotoxic t cells, inhibition of nk cell cytolytic activity, suppression of t cell proliferation, and deviation of type to type responses, most notably in pregnancy (bainbridge et al. ; fournel et al. ; ishitani et al. ; kapasi et al. ; kanai et al. ; riteau et al. ) . hla-g has been found in the rhesus monkey testis (ryan et al. ; slukvin et al. ) , and hla-e is expressed on germ cells in the human testis (fiszer et al. ) , indicating that these molecules may contribute to immune privilege in the testis. from the evidence outlined in the previous sections, it should be evident that proinflammatory and immunoregulatory processes are involved in both normal testicular function and testicular pathophysiology. in the normal testis, roles in the maintenance of immune privilege, the control of leydig cell development, and the fine regulation of spermatogenesis are indicated (figure ) . contributions to pathology include the development of autoimmunity within the inflammatory and immunoregulatory signaling in the control of intratesticular interactions. sertoli cells are primarily regulated by the hormones follicle-stimulating hormone (fsh) and testosterone, the latter being produced by the leydig cells on stimulation by luteinizing hormone (lh). sertoli cells and germ cells interact through production of various inflammatory mediators, including interleukin (il) , il , activin a, tumor necrosis factor (tnf), and no, to facilitate and coordinate critical events throughout the cycle of the seminiferous epithelium. the development and activity of the leydig cells and the resident macrophages involve a close mutual interaction, mediated by mechanisms that remain to be completely defined. the sertoli cell and the resident macrophages, in turn, influence the activity of the antigen-presenting cells (apc; e.g., dendritic cells) and lymphocytes within the interstitial tissue to control local immune events, such as the maintenance of an immunologically privileged environment. this communication involves the immunoregulatory cytokines transforming growth factor (tgf), activin a, and il , although other mechanisms may also be involved. male reproductive tract, disruption of steroidogenesis and spermatogenesis during systemic and local inflammatory events, and the response to vascular disturbances. . . . . normal testicular function direct evidence that immune privilege is due to a specialized immune environment originally comes from studies involving the central nervous system. injection of soluble antigen into the compartments of the eye or brain produces antigen-specific suppression of cell-mediated immunity, specifically type reactions (kaplan and streilein ; wenkel et al. ) . this phenomenon is called acquired immune deviation (aid), and it is possible to elicit similar inhibition of antigen-specific immune responses by prior injection of antigens into the testis (ditzian-kadanoff ; li et al. ; veräjänkorva et al. ) . the sertoli cell and leydig cell are almost certainly central to this process, although contributions from other testicular cells are to be expected. the mechanisms responsible presumably involve the recruitment and functional deviation of macrophages (and possibly dendritic cells) within the testis (hedger ) , the subsequent recruitment of immunoregulatory lymphocyte subsets (tompkins et al. ) , the production of immunoregulatory cytokines, such as tgf family members and il (o'bryan et al. ; suarez-pinzon et al. ) , local high concentrations of androgenic steroids, prostanoids, and other bioactive lipids (foulds et al. ; winnall et al. ) , complement inhibitors mizuno et al. ) , and testicular expression of inhibitory mhc and costimulatory molecules (dal secco et al. ; fiszer et al. ; ryan et al. ) . macrophages play an important role in leydig cell development and function. there is a close temporal link between the maturation of the adult leydig cell population and the increase in the number of testicular resident macrophages during puberty (hardy et al. ; raburn et al. ; vergouwen et al. ) . reductions in intratesticular macrophages due to specific depletion methods or in the macrophage-deficient (op/op) mouse lead to disorders of leydig cell development and mature function (cohen et al. ; gaytan et al. a,b) , and testicular macrophage-conditioned medium stimulates leydig cell steroidogenesis under certain conditions (nes et al. ; yee and hutson ) . these supportive functions of the testicular macrophages may involve production of cytokines and other secreted mediators (khan et al. b; warren et al. ) , as well as the distinctive intercytoplasmic specializations that develop between the two cell types (hutson ; miller et al. ). it has also been shown that testicular macrophages support basal steroidogenesis in the leydig cells by providing -hydroxycholesterol as a substrate for testosterone biosynthesis (nes et al. ) . endogenous regulation of inflammatory pathways within the seminiferous epithelium appears to be involved in the fine control of spermatogenesis. studies in the rat and mouse indicate that nuclear localization (i.e., activation) of the key proinflammatory transcription factor nfb, and expression of the inflammation-responsive genes, il , tnf, il , activin a, and inos, in the sertoli cell and the germ cells describe cyclical patterns within the seminiferous epithelium that coincide with critical events in the cycle of the seminiferous epithelium (o'bryan and hedger ) . most notably, release of spermatozoa from the epithelium (spermiation) in the rat testis is followed by an increase in the production of il , il , and activin a by the sertoli cells and of tnf and inos by the spermatocytes hakovirta et al. ; kaipia et al. ; söder et al. ; syed et al. ; o'bryan et al. a; okuma et al. ). in the mouse, spermiation is accompanied by a period of elevated nuclear nfb levels in spermatocytes (delfino and walker ) . these responses coincide with a peak of dna synthesis by preleptotene spermatocytes and type a spermatogonia prior to meiotic and mitotic division, and reorganization of sertoli cell tight junctions to allow the meiotic cells to enter the adluminal compartment (o'bryan and hedger ) . given that il , il , and activin a are regulators of spermatogonial proliferation and meiotic progression (hakovirta et al. a (hakovirta et al. , mather et al. ; meehan et al. ; meinhardt et al. ; parvinen et al. ; söder et al. ) , while tnf and inos/ no induce the disassembly of the intercellular tight junctions lee et al. ; li et al. a; siu et al. ) , this concurrence of events is unlikely to be a coincidence. it would appear that release of the spermatozoa and/or phagocytosis of the residual cytoplasm triggers elements of the inflammatory machinery within the seminiferous epithelium. a role for tlrs or other patternrecognition receptors in this process may be postulated, and similar regulatory networks may also operate throughout the remainder of the cycle. many questions regarding the details of this functional regulation await clarification, and it must be noted that there is a curious lack of concordance between the content of nfb in the sertoli cell nucleus observed across the cycle in the mouse (delfino and walker ) and inflammatory cytokine production by these same cells in the rat hakovirta et al. ; kaipia et al. ; o'bryan et al. a; okuma et al. ; söder et al. ; syed et al. ) . regardless of the questions that remain, however, key elements of the inflammatory response appear to play essential roles in the physiological regulation of the seminiferous epithelium, which are entirely separate from their roles in pathology. although it is a common assumption that male reproductive failure caused by local or systemic illness, infection, and chronic inflammatory disease is due to the negative effects of raised body temperature on spermatogenesis, there is little clinical or experimental evidence to support this belief (o'bryan et al. b) . there is much more convincing evidence that the damage is due to activation of local inflammatory pathways and the activity of immune cells recruited during inflammatory events. as already outlined in the previous sections, inflammatory mediators such as il , ros, tnf, and no, as well as glucocorticoids produced in response to inflammation, have mostly negative effects at all levels of the hypothalamic-pituitary-leydig cell axis (bambino and hsueh ; del punta et al. ; hales ; hales and payne ; hardy et al. ; li et al. ; mauduit et al. b mauduit et al. , monder et al. ; sharma et al. ; welch et al. ; xiong and hales b . the subsequent reduction in androgen production may lead to reduced spermatogenic capacity, but may also exacerbate the effects of inflammation on spermatogenesis itself. expression of tlrs on the sertoli cells is obviously important in protective responses against pathogens, but also provides a mechanism whereby pathogens can directly inhibit spermatogenesis. thus, tlr activation and the production of inflammatory mediators almost certainly alter sertoli cell functions, and this will affect the ability of these cells to support and regulate spermatogenesis, as well as activate signaling pathways that promote germ cell apoptosis (lysiak et al. ; pentikäinen et al. ; rasoulpour and boekelheide ; starace et al. ) . inflammatory events play a role in testicular damage due to vascular disturbance. spermatogenesis is an energy intensive process, and the seminiferous epithelium is not vascularized. consequently, unobstructed blood flow to the testis is essential and even minor anomalies in the testicular vasculature, such as varicocele, have cumulative negative effects on sperm production (jarow ) . the deleterious effects of cadmium on fertility through disruption of the testicular vasculature are well known (schrag and dixon ) . in experimental animals, transient torsion of the spermatic cord that renders the testis ischemic followed by reperfusion causes an increase in tnf and il expression, activation of the stress-related jnk/p map kinase signaling pathway, neutrophil recruitment and infiltration into the testis, increased oxidative stress, germ cell apoptosis, and significantly decreased serum testosterone levels (lysiak ; lysiak et al. ; rodriguez et al. ) . a specific role for testicular nitric oxide in mediating damage in both the testicular torsion and human varicocele models has been implicated (moon et al. ; santoro et al. ; shiraishi and naito ; shiraishi et al. ; türker köksal et al. ) . administration of high doses of human chorionic gonadotropin (hcg), a treatment used to correct delayed testicular descent in young boys, causes a hyperstimulation syndrome in rats comprising increased testicular blood flow and pressure, increased vascular permeability, and accumulation of interstitial fluid (hjertkvist et al. ; setchell and sharpe ; van vliet et al. ). these vascular changes are accompanied by accumulation of intravascular and interstitial neutrophils in the testis (bergh et al. ; widmark et al. ) , and spermatogenic damage (kerr and sharpe a,b) . the responses to hcg can be eliminated by depletion of either the leydig cells or the neutrophils (setchell and rommerts ; widmark et al. ) , and il is able to replicate most of the effects (bergh and söder ; bergh et al. ) , suggesting that the response involves an inflammatory event mediated via il secreted by the leydig cells. thus, it appears that disruption of the seminiferous epithelium in both the ischemia/reperfusion model and hcg hyperstimulation models is directly linked to local recruitment of neutrophils. exactly how these cells exert damage on the germ cells is not yet known, but increased oxidative stress is obviously involved. the responses are quite different from those observed in lps-induced inflammation, which paradoxically causes a large reduction in interstitial fluid volume and an increase in mononuclear cells in the rat testis (o'bryan et al. b; gerdprasert et al. a,b) , although the germ cell damage is similar in both models. in summary, different testicular inflammation models have direct effects on spermatogenic development, in addition to causing damage to the steroiodogenic function of the leydig cells. it should also be noted that, as in most other tissues, inflammation responses within the testis do not automatically lead to autoimmune complications, but damage to various testicular functions that may be less readily apparent may lead to longer-term consequences for reproductive health (schuppe and meinhardt ; schuppe et al. ; suominen and soderstrom ) . . . . the epididymis, vas deferens, accessory glands, and urogenital tract as already noted above, the immune environment of the remainder of the male tract is quite distinct from that of the testis, or even from that of other mucosal tissues. the mechanisms whereby sperm within the male reproductive tract are protected from the immune system remain largely unknown, but an intact tract is obviously important. in humans, vasectomy leads to sperm antibody formation in approximately % of cases, and sperm antibodies are also associated with obstructive azoospermia and congenital absence of the epididymis and/or vas (alexander and anderson ; de kretser et al. ; hellema et al. ; patrizio et al. ) . the incidence of antibody formation in humans appears to be directly related to the distance of the lesion from the testis, implying a limited role for elements of testicular immune privilege in preventing immune reactions. there is evidence from patients and animal models that vasectomy can have deleterious effects on the testis and epididymis as well, and that at least some of these effects may have an immunological basis (aitken et al. ; bigazzi et al. ; flickinger et al. a; raleigh et al. ) . in certain strains of rats, mice, and rabbits, vasectomy rapidly leads to orchitis and sterility, so it is not clear why this degree of damage is so rare in humans. in reality, the immunology of the male reproductive tract has yet to be investigated in depth, but two areas of recent interest are worth highlighting: the tlrs and the defensins. given the exposure to the external environment, it is not surprising that the tlrs as well as many essential tlr-related genes, such as myd , are expressed throughout the male reproductive tract, including the epididymis, vas deferens, and accessory glands (nishimura and naito ; palladino et al. ; quintar et al. ; rodrigues et al. ) . expression is found on both epithelial cells and leukocytes in these tissues. moreover, weak expression of tlr , originally localized to the urinary tract of experimental rodents, has been detected in the epididymis and vas deferens of the rat (lauw et al. ; palladino et al. ; zhang et al. ) . the importance of these molecules in acute and chronic inflammation, and the response to infection, in the male reproductive tract is an important area for future studies. moreover, changes in susceptibility to inflammation through polymorphisms in the tlrs have been associated with differences in susceptibility to the onset of cancer in the prostate (sun et al. ; zheng et al. ) . defensins are small ( - kda) positively charged peptides that are able to disrupt bacteria, fungi, parasites, and some enveloped viruses by forming multimeric pores in the pathogen membrane (selsted and ouellette ) . the -defensins are produced by most mucosal epithelial tissues, including the testis and epididymis (com et al. ) , and their production is stimulated via tlr activation and cytokines. a number of epididymal-specificdefensins have been identified in the mouse and the rat (li et al. ; jalkanen et al. ; yamaguchi et al. ; yenugu et al. ; zhou et al. ) , and a role for androgen in their regulation has been indicated (jalkanen et al. ; yenugu et al. ) . apart from their obvious role in protection against pathogens, defensins may have other functions in the reproductive tract. the novel -defensin, bin b, which is exclusively produced and secreted by the rat caput epididymis, binds to sperm (li et al. ; zhou et al. ) , and blocking bin b reduces sperm motility in vivo, suggesting a novel role for this molecule in sperm maturation (zhou et al. ) . in humans, semen is the most accessible window into the health of the male reproductive system, as it is the number and quality of sperm in the semen that provide the principal foci for male reproductive toxicity outcomes. however, semen is a complex secretion, comprising many components, including leukocytes, immunoglobulins, cytokines, prostaglandins, and various immunosuppressives. changes in the number or activity of these immunological components of the semen are also potential indicators of toxic actions within the tract. elevated numbers of leukocytes in the semen are generally considered to be an indication of infection, but leukocytes are present even in the semen of men with normal fertility barratt et al. ) . the origin of these cells is somewhat obscure. evidence suggests that the epididymis or vas may be a major source (anderson et al. ; schwartz ), but the main leukocyte subset present in most semen samples are neutrophils, which are not a normal feature of the tissues of the genital tract el-demiry et al. ; wolff and anderson ) . the impact of these cells on fertility is also poorly understood. some studies have shown a relationship between leukocytospermia and impaired sperm function (auroux ; aziz et al. ; berger et al. ; wolff et al. ) , and some data suggest that these cells might be a cause of sperm damage due to production of ros and cytokines (hill et al. ; tomlinson et al. a ). moreover, leukocytes may attack the sperm directly, particularly in the presence of sperm autoantibodies, and may provoke or mediate hostile responses in the female tract. other studies, however, have failed to confirm a clear link between seminal leukocytes and infertility or sperm antibody formation barratt et al. ; tomlinson et al. ) , and it has even been suggested that leukocytes might play a beneficial role by removing abnormal sperm or by assisting in sperm capacitation (tomlinson et al. b (tomlinson et al. , . molecules with immunomodulatory properties in seminal plasma include immunoglobulins, cytokines, and immunosuppressives. both igg and iga are the major constituents of seminal plasma, and there appears to be a negative correlation between iga concentrations and lymphosuppressive activity in seminal plasma (eggert-kruse et al. ; imade et al. ; moldoveanu et al. ) . these antibodies may play either protective roles in the control of immunity and infection, or destructive roles, when directed against functional elements of the sperm. proinflammatory cytokines such as il , il , il , il , il , il , tnf, and ifn are found in all seminal plasma samples, even those from men with apparently normal fertility (eggert-kruse et al. ; huleihel et al. ; koumantakis et al. ; maegawa et al. ; naz and evans ; politch et al. ). this is not particularly surprising, as these cytokines are present in most biological fluids and are modulated by the general health status of the individual. soluble il and il receptors, molecules that modulate the activity of their respective cytokines, have also been found in seminal plasma (huleihel et al. ) . numerous studies have shown that proinflammatory cytokines are dramatically elevated in patients with inflammation and/or leukocytospermia (eggert-kruse et al. ; krause et al. ; maegawa et al. ; miller et al. ; rajasekaran et al. ) . however, specific positive and negative associations with other forms of infertility have also been reported (eggert-kruse et al. ; gruschwitz et al. ; huleihel et al. ; loras et al. ; naz and evans ) . seminal plasma has profoundly immunosuppressive properties, as defined by the ability to inhibit various t cell and nk cell activities in vitro. this inhibitory activity has been attributed to a number of seminal factors, including prostasomes (kelly et al. ) , oxidized polyamines (allen and roberts ) , prostaglandins of the e series (skibinski et al. ) , nonspecific lymphocyte-suppressing proteins (maccioni et al. ; veselský et al. ) , and immunoregulatory cytokines (anderson et al. ; huleihel et al. ; loras et al. ; miller et al. ; nocera and chu ; rajasekaran et al. ; srivastava et al. ) . the main cytokines with immunosuppressive activity in human seminal plasma are tgf , tgf , il , and activin a. immunosuppressives are believed to play a role in preventing lymphocyte responses against sperm autoantigens in the male and female reproductive tracts ochsenkühn et al. ). in the current state of understanding of the interactions between the male reproductive tract and the immune system, many questions still remain. nonetheless, it is clear that the mammalian testis in particular displays a special, if not unique, relationship with the immune system, involving distinctive cell-cell interactions and shared cytokines. recently, microarray analysis of testicular gene expression signatures in human spermatogenic failure were found to correspond with gene sets usually associated with inflammation and autoimmune disease (spiess et al. ). this relationship between reproduction and immunology impacts upon normal function, since perturbations in one system caused by toxicants will almost certainly impinge upon the other system. this extends into the characteristic responses of the testis to toxins and various pathologies (figure ) . the complexity and potential consequences of these interactions may be appreciated by consideration of the following propositions: . if leukocytes play a role in maintaining normal testicular function, then drugs and toxicants that reduce macrophage and lymphocyte function or numbers may also have an indirect effect on testicular function. the effect of dichloromethylene diphosphonate on macrophages in the rat provides a good experimental example of this possibility, by demonstrating that the loss of functioning macrophages leads to a decline in androgen levels and retarded leydig cell development gaytan et al. a ). inhibition of macrophage and lymphocyte functions or numbers may also compromise the immune-privileged status of the testis, potentially leading to testicular autoimmune disease. a related subset would involve chemicals that have direct action on both the immune system and testicular function, such as cyclosporin and cyclophosphamide (cavallini et al. ; meistrich ; seethalakshmi et al. ; wetzels ) , which are commonly used to suppress graft rejection response and in the treatment of some autoimmune diseases. fere with normal testicular function. the serious consequences for testicular function associated with immune activation and inflammation are illustrated by the experimental model of lps administration, which leads to leydig cell dysfunction and testicular failure (christeff et al. ; wallgren et al. ; o'bryan et al. b) . any drug or toxicant that induces macrophage activation (e.g., bacterial toxins, carbon monoxide, sulfur dioxide) has the potential to induce testicular production of inflammatory cytokines, such as il , tnf, and ifn, as well as ros, which may interfere with leydig cell function and androgen deficiency figure model of toxicant actions and potential immunological consequences in the testis. toxicants may damage or alter the function of somatic cells (in particular the sertoli and leydig cells), immune cells, or germ cells, thereby inhibiting androgen production and spermatogenesis, leading to increased germ cell death. this damage may lead to an overt inflammatory event (orchitis) through activation of the proinflammatory activities of local macrophages, which may exacerbate or prolong the testicular dysfunction. these events may be followed by a full or partial recovery of normal function, but intratesticular immune cell numbers and activity may become permanently altered, with possible implications for future toxic or inflammatory episodes. in certain circumstances, which may depend upon the severity of the initial damage caused or the genetic background of the patient, the antigen-presenting activity of intratesticular dendritic cells or macrophages may also be activated. this may lead to recruitment and activation of testicular antigen-specific t cells, development of cytotoxic t cells (ctl), and b cell antibody (ig) production. this subsequent autoimmunity may have a more permanent effect on testis or sperm function (epithelial destruction or sperm antibody formation). spermatogenic development, or local immunoregulation. the danger hypothesis model of autoimmune disease may be particularly relevant in this instance (matzinger ) . at least one of the putative 'danger' molecules (i.e., tnf) is endogenously produced in the testis, potentially presenting a continuous challenge to the testicular immune system. . drugs or toxicants that induce autoimmune disease or allergy may also induce testicular autoimmune disease. there are many drugs that induce polysystemic autoimmune disease and this may also include reactions against the germ cells. an example here is polyarteritis nodosa, a necrotizing vasculitis of small arteries, which frequently involves the testis (fauci et al. ) . sulfonamides, penicillin, phenytoin, arsenicals, thiouracil, iodides, and thiazides are frequently suspected as causes of this condition (nusinow et al. ) . likewise, mutagens and other toxins that may alter t and b cell receptor repertoires to create self-reactive lymphocytes could also contribute to the onset of autoimmune disease in the male reproductive tract. . drugs or toxicants that affect testicular cell development or viability may also induce immune responses. there are drugs or toxicants that affect sertoli-sertoli and sertoli-germ cell junction stability, as well as agents that damage the sertoli cell or germ cells directly, resulting in massive germ cell death, which may overwhelm the normal immunoregulatory capacity of the testis. examples of this interaction are seen after testicular heating, serotonin administration, or treatment with ethane dimethane sulfonate (eds) or other testicular toxicants, which are frequently followed by transient immune and/or inflammatory events (creasy et al. ; hedger et al. ; padmanabhan and singh ; wang et al. ) . even if the epithelium recovers fully, there is the potential for sperm antibody development, leading to reduced fertility. increased exposure to estrogenic compounds is associated with activation of immune cells in the testis, most notably the macrophages, providing evidence for a mechanism whereby environmental estrogens may affect male fertility indirectly via effects on local immunity (li et al. b ). . testicular toxicants that alter steroidogenic cell function will have a direct effect on the immune system. toxicants that damage leydig cells and reduce steroid production possibly could alter thymus function, leading to an enhanced susceptibility to autoimmune disease. balanced against this is the likelihood that other androgen-deficiency symptoms are far more likely to be manifest and treated. all these interactions, however, illustrate to one degree or another the difficulty of separating direct effects of toxic agents on the immune cells and on the testis. there is a dynamic relationship between the male reproductive tract and the immune system that should be considered when assessing the effects of various toxicants on male fertility and reproductive development. the effects of toxicants on male reproductive functions, most notably the production of androgenic steroids, will in turn impinge upon the functions of the immune system. many mechanisms underlying these interactions have begun to emerge in recent years, but a complete picture remains hidden. further studies will no doubt lead to important new concepts concerning this relationship and its implications for reproductive toxicology. proc. natl. acad. sci cellular transplantation from laboratory to clinic; halberstadt in knobil and neill's physiology of reproduction proc. natl. acad. sci proc. natl. acad. sci tight junctions sainio-pö llä nen, s.; sö der aderem, a. proc. natl. acad. sci fundamental immunology pö llä nen inflammation: basic principles and clinical correlates key: cord- - a a ku authors: afsar, cigdem usul; afsar, selim title: sars-cov- (covid- ): interferon-epsilon may be responsible of decreased mortality in females date: - - journal: j reprod immunol doi: . /j.jri. . sha: doc_id: cord_uid: a a ku nan this is a pdf file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. this version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (ahn and others ) . according to descriptive analysis in different populations, it seems that males have higher mortality than females (guan and others ; livingston and butcher ; grasselli and others, ) . we want to argue about this fact according to the reservoir of the virus and by discussing about interferon (ifn) epsilon (). pangolins which are unique mammals, are thought to be a natural reservoir of sars-cov- like covs (zhang and others, ) . ifn is particularly expressed in epithelial cells and it is essential in skin and mucosal immunity (lung, intestines and reproductive tissues) of all african and asian pangolin species (choo and others, ) . the production of ifn-i or ifn-α/β is the basic natural immune defense response against viral infections (ye and others, ) . ifn, like the other type i ifns, might be responsible of decreased mortality in females because of its antiviral effects. the pangolin immune system evolved differently than in other mammals, the single copy intronless ifn gene is pseudogenised in pangolin species (choo and others, ) . this might play a role in pangolin's symptoms during coronovirus infections and their immun response to viral infections. furthermore; ifn protects the female reproductive tract from viral and bacterial infections, especially from hiv- infection (fung and others, ) . ifnε is constitutively expressed by reproductive tract epithelium and regulated by hormones during the menstruel cycle, reproduction, and menopause and by exogenous hormones (marks and others, ) . ifn-ε regulates mucosal immunity against viral and bacterial infections, and can suppress hiv replication and its expression correlates negatively with progesterone levels (li and others, j o u r n a l p r e -p r o o f ). in females, ifn has limited role in the reproductive tract and it has no role in any other organ. this can be a reason why females has less mortality rather than males. on the other hand, in a new report, higher mortality rates in males was found to be associated with lower ace receptor levels (sharma and others, ) . other ifns, such as ifn-λ may be an ideal treatment for covid- because it reduces the viral load and improves the clinical symptoms of patients. however, it has no effect on mortality (ye and others, ) . covid- patients can have darkness/rash in their skin in severe cases (estébanez and others, ) and there is a report which shows a decrease in spermatogenezis in covid patients (zhengpin and xiaojiang, ) . ifnε is present in both tissues and there is another report which argues that covid infection is likely to be androgen mediated (wambier and goren, ) . it has been shown that ifnε also exerts its biological activity by stimulating immune mediators and activating the jak-stat signal pathways in vitro and in vivo (zwarthoff and others, ) . the jak/stat pathway responds to type i ifn secreted from neighboring cells and sars-cov proteins have been shown to affect this pathway before (frieman and baric, ) . another arguement is about the furin like cleavage of the s protein of the virus. furin is especially expressed in differentiated th cells in a stat -dependent manner. expression of furin enhances ifn- secretion, whereas inhibition of furin interferes with ifn- production (pesu and others, ) . transcription through the jak-stat signalling pathway activated by ifns, leads to the upregulation of many ifn-controlled genes that quickly kill viruses in infected cells (pesu and others, ) . jak-stat signal blocking by baricitinib (a selective jak and jak inhibitor) produces an impairment of ifn-mediated antiviral response, with a potential facilitating effect on the evolution of sars-cov- infection. the initial sensing of coronaviruses by the innate immune machinery might be the critical step in protecting the host from infection, especially in men. current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease (covid- ) pangolin genomes and the evolution of mammalian scales and immunity cutaneous manifestations in covid- : a new contribution mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation interferon-epsilon protects the female reproductive tract from viral and bacterial infection baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region clinical characteristics of coronavirus disease in china type i interferons: distinct biological activities and current applications for viral infection coronavirus disease (covid- ) in italy properties and functions of the novel type i interferon epsilon proprotein convertase furin is preferentially expressed in t helper cells and regulates interferon gamma sex differences in mortality from covid- pandemic: are men vulnerable and women protected? jacc case rep sars-cov- infection is likely to be androgen mediated the pathogenesis and treatment of the `cytokine storm' in covid- probable pangolin origin of sars-cov- associated with the covid- outbreak . scrna-seq profiling of human testes reveals the presence of the ace receptor, a target for sars-cov- infection in spermatogonia organization, structure and expression of murine interferon alpha genes the authors declare that they have no conflicts of interests.j o u r n a l p r e -p r o o f key: cord- -tzmev c authors: yuan, shuofeng; chan, chris chun-yiu; chik, kenn ka-heng; tsang, jessica oi-ling; liang, ronghui; cao, jianli; tang, kaiming; cai, jian-piao; ye, zi-wei; yin, feifei; to, kelvin kai-wang; chu, hin; jin, dong-yan; hung, ivan fan-ngai; yuen, kwok-yung; chan, jasper fuk-woo title: broad-spectrum host-based antivirals targeting the interferon and lipogenesis pathways as potential treatment options for the pandemic coronavirus disease (covid- ) date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tzmev c the ongoing coronavirus disease (covid- ) pandemic caused by severe acute respiratory syndrome coronavirus (sars-cov- ) signals an urgent need for an expansion in treatment options. in this study, we investigated the anti-sars-cov- activities of antiviral agents with known broad-spectrum antiviral activities against coronaviruses and/or other viruses. they were first evaluated in our primary screening in veroe cells and then the most potent anti-sars-cov- antiviral agents were further evaluated using viral antigen expression, viral load reduction, and plaque reduction assays. in addition to remdesivir, lopinavir, and chloroquine, our primary screening additionally identified types i and ii recombinant interferons, -hydroxycholesterol, and am as the most potent anti-sars-cov- agents among the antiviral agents. betaferon (interferon-β b) exhibited the most potent anti-sars-cov- activity in viral antigen expression, viral load reduction, and plaque reduction assays among the recombinant interferons. the lipogenesis modulators -hydroxycholesterol and am exhibited ec( ) at low micromolar levels and selectivity indices of > . . combinational use of these host-based antiviral agents with virus-based antivirals to target different processes of the sars-cov- replication cycle should be evaluated in animal models and/or clinical trials. severe acute respiratory syndrome coronavirus (sars-cov- ) is a novel betacoronavirus that was first identified in patients with unexplained pneumonia in wuhan, hubei, china in december sars-cov- hku- a (genbank accession number: mt ) was isolated from the nasopharyngeal aspirate specimen of a laboratory-confirmed covid- patient in hong kong [ ] . the virus was propagated in veroe cells and kept at − • c in aliquots. plaque forming unit (pfu) and tcid assays were performed to titrate the cultured virus as we described previously [ ] . veroe (atcc ® crl- ™) cells were purchased from atcc (manassas, va, usa) and maintained in dulbecco's modified eagle medium (dmem, gibco, carlsbad, ca, usa) culture medium supplemented with % heat-inactivated fbs (fetal bovine serum, gibco), u/ml penicillin, and µg/ml streptomycin as previously described [ ] . all experiments involving live sars-cov- followed the approved standard operating procedures of the biosafety level facility at the department of microbiology, the university of hong kong [ ] . the recombinant ifns were obtained from the following sources: pegasys (roche, basel, switzerland), avonex (ucb, brussels, belgium), rebif (emd serono, inc. rockland, ma, usa), betaferon (bayer schering pharma, berlin, germany), and immukin (boehringer ingelheim, ingelheim am rhein, germany). all other antiviral agents were purchased from medchemexpress (monmouth junction, nj, usa). twenty-two antiviral agents with reported activities against coronaviruses and/or other viruses were included in the study. in the primary screening, , iu/ml of each recombinant ifn or µm of each of the other antiviral agents was used to treat sars-cov- -infected (moi = . ) veroe cells for h. the viral loads in the cell culture supernatants were then determined by quantitative reverse transcription-polymerase chain reaction (qrt-pcr) as previously described [ ] . the cut-off value of ≥ % inhibition (i.e., ≥ log copies/ml reduction when compared with the dimethyl sulfoxide (dmso) control) was used to select the antiviral agents for further evaluation. the celltiterglo luminescent assay (promega corporation, madison, wi, usa) was performed to detect the cytotoxicity of the selected antiviral agents as previously described [ ] . briefly, veroe cells ( × cells/well) were incubated with different concentrations of the individual compound for h, followed by the addition of substrate and measurement of luminance min later. the % cytotoxic concentrations (cc ) of the antiviral agents were calculated by sigmaplot (systat software inc., san jose, ca, usa) in an excel add-in ed v . viral load reduction assay was performed as described previously with modifications [ ] . briefly, the culture supernatants of the sars-cov- -infected veroe cells were harvested at h post-inoculation (hpi) for qrt-pcr analysis of viral rna load. a total of µl of culture supernatant was lysed with µl of avl buffer, which was subsequently extracted for total rna with the qiaamp viral rna mini kit (qiagen, hilden, germany). qrt-pcr was used for quantitation of sars-cov- replication using the quantinova probe rt-pcr kit (qiagen) with a lightcycler real-time pcr system (roche) as we described previously [ ] . each µl reaction mixture contained µl of × quantinova probe rt-pcr master mix, . µl of rnase-free water, . µl of quantinova probe rt-mix, . µl each of µm forward and reverse primer, . µl of µm probe, and µl of extracted rna as the template. reactions were incubated at • c for min for reverse transcription, • c for min for denaturation, followed by cycles of • c for s and • c for s. signal detection and measurement were taken in each cycle after the annealing step. the cycling profile ended with a cooling step at • c for s. the primers and probe sequences were against the rna-dependent rna polymerase/helicase (rdrp/hel) gene region of sars-cov- : forward primer: -cgcatacagtcttrcaggct- ; reverse primer: -gtgtgatgttgawatgacatggtc- ; specific probe: -fam ttaagatgtggtgcttgcatacgtagac-iabkfq- . viral n antigen expression in sars-cov- -infected veroe cells was performed as we described previously using immunofluorescent staining with an in-house rabbit antiserum against sars-cov- -n protein as previously described [ ] . cell nuclei were labelled with , -diamidino- -phenylindole (dapi) nucleic acid stain from thermo fisher scientific (waltham, ma, usa). the alexa fluor secondary antibody was obtained from thermo fisher scientific. mounting was performed with the diamond prolong antifade mountant from thermo fisher scientific. plaque reduction assay was performed to plot the % maximal effective concentration (ec ) as we previously described with slight modifications [ ] . briefly, veroe cells were seeded at × cells/well in -well tissue culture plates on the day before carrying out the assay. after h of incubation, plaque-forming units (pfu) of sars-cov- were added to the cell monolayer with or without the addition of antiviral agents and the plates were further incubated for h at • c in % co before removal of unbound viral particles by aspiration of the media and washing once with dmem. monolayers were then overlaid with media containing % low melting agarose (cambrex corporation, east rutherford, nj, usa) in dmem and appropriate concentrations of individual compound, inverted and incubated as above for another h. the wells were then fixed with % formaldehyde (bdh, merck, darmstadt, germany) overnight. after removal of the agarose plugs, the monolayers were stained with . % crystal violet (bdh, merck) and the plaques counted. the percentage of plaque inhibition relative to the control (i.e., without the addition of compound) wells were determined for each antiviral agent concentration. ec was calculated using a sigma plot (spss) in an excel add-in ed v . the plaque reduction assay experiments were performed in triplicate and repeated twice for confirmation. time-of-drug-addition assay was performed for selected compounds as previously described with slight modifications [ ] . briefly, veroe cells were seeded in -well plates ( × cells/well). the cells were inoculated with sars-cov- (multiplicity of infection, moi = . ) and then incubated for h for virus internalization at • c. dmso ( . %) was included as a negative control. the viral loads in the culture supernatants normalized by dmso at the different phases of the assay were determined by qrt-pcr. a total of antiviral agents with reported activity against coronaviruses and/or other viruses were included in the primary screening. these included types i (pegasys, avonex, rebif, and betaferon) and ii (immukin) ifns, nucleoside analogues (favipiravir, galidesivir, remdesivir, and ribavirin), -aminoquinoline (chloroquine), anthracycline (idarubicin), antihistamine (chlorcyclizine), calcineurin inhibitor (cyclosporine), flavonoid (silibinin), kinase inhibitors (erlotinib and everolimus), macrolide (azithromycin), oxysterol ( -hydroxycholesterol), polymerase inhibitor (filibuvir), protease inhibitors (lopinavir and rupintrivir), and retinoic acid receptor agonist (am ) ( table ). in this primary screening, chloroquine, lopinavir, and remdesivir which were recently reported to have anti-sars-cov- activity, exhibited about . - . log copies/ml reduction in viral rna load ( figure ). in comparison, recombinant ifn-β demonstrated the most potent anti-sars-cov- activity, with avonex (ifn-β a), rebif (ifn-β a), and betaferon (ifn-β b) each achieving about log copies/ml reduction in viral load. pegasys (pegylated ifn-α a) and immukin (ifn-γ b) also showed anti-sars-cov- activity with viral load reduction of . log copies/ml and . log copies/ml, respectively. additionally, two other antiviral agents, namely, am and -hydroxycholesterol, exhibited anti-sars-cov- activity. they each achieved about -log reduction in viral load. the other antiviral agents demonstrated < log reduction in sars-cov- load and were therefore not investigated further in this study. based on the results from the primary screening, avonex (ifn-β a), rebif (ifn-β a), betaferon (ifn-β b), pegasys (pegylated ifn-α a), immukin (ifn-γ b), am , and -hydroxycholesterol were further evaluated in the sars-cov- n antigen expression assay. as shown in figure , at the fixed concentrations of iu/ml of each of the recombinant ifns and µm of am and -hydroxycholesterol, all seven antiviral agents reduced viral n antigen expression in the immunofluorescent staining assay. the most prominent reduction in viral n antigen expression was observed in cells treated with avonex, rebif, betaferon, and am , which achieved similar degree of viral n antigen expression reduction as redemsivir. pegasys, immukin, and -hydroxycholesterol also moderately reduced viral n antigen expression to a similar degree as lopinavir. viruses , , x; doi: for peer review of based on the results from the primary screening, avonex (ifn-β a), rebif (ifn-β a), betaferon (ifn-β b), pegasys (pegylated ifn-α a), immukin (ifn-γ b), am , and -hydroxycholesterol were further evaluated in the sars-cov- n antigen expression assay. as shown in figure , at the fixed concentrations of iu/ml of each of the recombinant ifns and µm of am and -hydroxycholesterol, all seven antiviral agents reduced viral n antigen expression in the immunofluorescent staining assay. the most prominent reduction in viral n antigen expression was observed in cells treated with avonex, rebif, betaferon, and am , which achieved similar degree to quantify the anti-sars-cov- activity of the identified antiviral agents in the primary screening and the viral n antigen expression assay, sars-cov- viral load reduction assay by qrt-pcr was conducted to determine the sars-cov- rna copies released in the cell culture supernatant with or without antiviral agent treatment. as show in figure , the mean baseline viral load in the cell culture supernatants without any antiviral agent was about . log copies/ml. there was dose-dependent and significant (p < . ) reduction in viral load of > % as compared to the baseline in cell culture supernatants inoculated with each of the eight antiviral agents. as to quantify the anti-sars-cov- activity of the identified antiviral agents in the primary screening and the viral n antigen expression assay, sars-cov- viral load reduction assay by qrt-pcr was conducted to determine the sars-cov- rna copies released in the cell culture supernatant with or without antiviral agent treatment. as show in figure , the mean baseline viral load in the cell culture supernatants without any antiviral agent was about . log copies/ml. there was dose-dependent and significant (p < . ) reduction in viral load of > % as compared to the baseline in cell culture supernatants inoculated with each of the eight antiviral agents. as controls, remdesivir and lopinavir achieved . log copies/ml and . log copies/ml reduction in viral rna load, respectively, at a concentration of µm. in comparison, -hydroxychloestero and am achieved . log copies/ml and . log copies/ml reduction in viral rna load, respectively, at the same antiviral agent concentration. consistent with the primary screening results, ifn-β a ifn-β b demonstrated more potent anti-sars-cov- activity than ifn-α a and ifn-γ b in the viral load reduction assay. at a concentration of iu/ml, avonex (ifn-β a), rebif (ifn-β a), and betaferon (ifn-β b) respectively achieved . log copies/ml, . log copies/ml, and . log copies/ml reduction in viral rna load, whereas pegasys (pegylated ifn-α a) and immukin (ifn-γ b) achieved only . log copies/ml and . log copies/ml reduction in viral rna load. at a higher concentration of , iu/ml, pegasys and immukin achieved modestly higher reduction in viral rna load ( . log copies/ml and . log copies/ml, respectively). in addition to reduction in viral n antigen expression and rna load, inhibition of infectious sars-cov- particles was evaluated using plaque reduction assay (figure ) in addition to reduction in viral n antigen expression and rna load, inhibition of infectious sars-cov- particles was evaluated using plaque reduction assay (figure ). among the recombinant ifns, betaferon (ifn-β b) demonstrated the most potent anti-sars-cov- effect with % inhibition of virus plaque formation at a concentration of iu/ml. two different brands of ifn-β a (avonex and rebif) exhibited similar anti-sars-cov- activity with % inhibition of virus formation at a concentration of iu/ml. pegasys (pegylated ifn-α a) and immukin (ifn-γ b) again demonstrated less potent anti-sars-cov- activity than the recombinant ifn-β's, and demonstrated % inhibition of virus plaque formation at higher concentrations of , and iu/ml, respectively. -hydroxycholesterol demonstrated marked virus plaque formation at µm. am showed dose-dependent plaque reduction effect with partial inhibition of virus plaque formation at the highest tested concentration of µm. remdesivir and lopinavir achieved nearly complete inhibition of virus plaque reduction at µm and µm, respectively, but demonstrated cytotoxicity in veroe cells at hpi at higher concentrations. based on these plaque reduction assay results, the ec of the antiviral agents were determined ( µm. am showed dose-dependent plaque reduction effect with partial inhibition of virus plaque formation at the highest tested concentration of µm. remdesivir and lopinavir achieved nearly complete inhibition of virus plaque reduction at µm and µm, respectively, but demonstrated cytotoxicity in veroe cells at hpi at higher concentrations. based on these plaque reduction assay results, the ec of the antiviral agents were determined ( to explore the mode of action of the two non-ifn antiviral agents, a time-of-drug-addition assay was performed. as shown in figure , -hydroxycholesterol and am both exhibited anti-sars-cov- effect only when they were added to the sars-cov- -infected veroe cells at or after one hour post-inoculation. these results suggested that -hydroxycholesterol and am both targeted the post-entry steps of the sars-cov- replication cycle. viruses , , x; doi: for peer review of a > , iu/ml, > µm, and > µm indicate the highest antiviral agent concentrations tested in the cytotoxicity assay was , iu/ml (ifns), µm ( -hydroxycholesterol), and µm (remdesivir), respectively. abbreviations: cc , % cytotoxic concentration; ec , % maximal effective concentration; ifn, interferon. to explore the mode of action of the two non-ifn antiviral agents, a time-of-drug-addition assay was performed. as shown in figure , -hydroxycholesterol and am both exhibited anti-sars-cov- effect only when they were added to the sars-cov- -infected veroe cells at or after one hour post-inoculation. these results suggested that -hydroxycholesterol and am both targeted the post-entry steps of the sars-cov- replication cycle. in this study, we evaluated the anti-sars-cov- activity of antiviral agents with broad-spectrum antiviral activities against coronaviruses and/or other viruses. in our primary in this study, we evaluated the anti-sars-cov- activity of antiviral agents with broad-spectrum antiviral activities against coronaviruses and/or other viruses. in our primary screening using a fixed antiviral agent concentration and virus inoculum, we identified recombinant ifns and lipogenesis modulators to be the most potent anti-sars-cov- agents among broad-spectrum antivirals. these findings have important implications for the choice of clinically available recombinant ifns to be used in covid- patients and development of lipogenesis modulators as potential anti-sars-cov- therapeutics. ifns are glycoproteins with strong antiviral activities that represent one of the first lines of host immune response against invading pathogens [ ] . these proteins are classified into three groups, types i, ii, and iii ifns, based on the structure of their receptors on the cell surface [ ] . ifns are known for their broad-spectrum antiviral activities against a wide range of dna and rna viruses, through inducing the expressions of interferon-stimulated genes in host cells, such as protein kinase r, oligoadenylate synthetase, and rnase l [ ] . these interferon-stimulated genes suppress viral replication by inhibiting multiple steps in a viral life cycle, including viral rna transcription and viral protein translation. recombinant ifn-α and ifn-β exhibited potent antiviral activity against sars-cov and mers-cov in vitro and in animal models [ , [ ] [ ] [ ] [ ] . recombinant ifn-γ exhibited limited anti-coronaviral activity in vitro, but might be synergistic with type i ifns [ , , ] . in this study, we demonstrated the anti-sars-cov- activity of five clinically-approved preparations of recombinant ifns, including pegasys (pegylated ifn-α a), avonex (ifn-β a), rebif (ifn-β a), betaferon (ifn-β b), and immukin (ifn-γ b). among them, betaferon exhibited the most potent anti-sars-cov- effect with the lowest ec of . iu/ml and the highest selectivity index of > . . importantly, the ec of betaferon against sars-cov is below its achievable peak serum concentration (cmax) with standard subcutaneous dosing of million units of betaferon ( iu/ml). notably, betaferon was similarly found to be the most potent recombinant ifn for the highly virulent mers-cov and significantly improved the clinical, virological, and histopathological parameters of mers-cov-infected common marmosets [ , ] . in sars and mers patients, the use of recombinant ifn-α and/or ifn-β treatment was generally well tolerated with minimal adverse effects [ , , ] . although the clinical benefits of recombinant ifn treatment in sars and mers patients remain inconclusive, the apparent discrepancy between the in vitro and in vivo antiviral effects might be related to the delay in treatment commencement after symptom onset. because sars-cov- is able to achieve more than folds higher viral load than sars-cov within h in human lung tissues by minimally eliciting the host ifn response, it would be important to supplement covid- patients with recombinant ifns, especially ifn-β b, before cytokine storm develops, with other effective virus-targeting antivirals [ ] . importantly, inhaled ifn-β is well tolerated and enhances both systemic and local innate immunity with upregulated antiviral gene expression and reduced proinflammatory cytokines in sputum [ ] . this treatment strategy might be especially useful when given early to covid- patients who usually have the peak respiratory tract viral loads within the first week of symptom onset [ ] . in addition to the recombinant ifns, we also identified antiviral agents that target the host lipogenesis pathways as potential anti-sars-cov- agents. am is a selective retinoic acid receptor-α agonist which was recently identified to have broad-spectrum antiviral activities against various families of dna and rna viruses, including coronaviridae, flaviviridae, orthomyxoviridae, picornaviridae, and adenoviridae [ ] . am inhibits virus replication through interaction with sterol regulatory element-binding protein (srebp) and downregulation of multiple srebp proteolytic processes and srebp-regulated lipid biosynthesis pathways, such as double-membrane vesicle formation by mers-cov [ ] . our time-of-drug-addition assay showed that am inhibited the post-entry events of the sars-cov- replication cycle, which corroborated with the hypothesized restrictive effects of am on lipid biosynthesis in sars-cov- infection. the oxysterol -hydroxycholesterol is a metabolite of cholesterol that is produced and secreted by macrophages and has multiple effects on lipid metabolism, especially lipid biosynthesis and immunity [ ] . -hydroxycholetserol has been shown to inhibit feline coronavirus, porcine epidemic diarrhea virus, and porcine transmissible gastroenteritis virus possibly through induction of intracellular cholesterol accumulation [ , ] . moreover, -hydroxycholesterol is active against various emerging rna viruses, including ebola virus, nipah virus, rift valley fever virus, and zika virus, and dna and rna viruses that cause chronic infections, such as human immunodeficiency virus, herpes simplex virus, and varicella zoster virus [ , ] . mechanistically, -hydroxycholesterol and its downstream metabolite -hydroxycholesterol- -sulfate ( hc s) block membrane fusion between virions and host cells through diametrical regulation of lipid metabolism and inflammatory response via lxr/srebp- and ikappabalpha/nf-kappab signaling [ , ] . addition of hc s to primary rat hepatocytes decreased nuclear lxr and srebp- protein levels, downregulated their target genes, acetyl coa carboxylase , fatty acid synthase, and srebp- target gene hmg reductase, which are key enzymes involved in fatty acid and cholesterol biosynthesis [ ] . interestingly, the expression of the interferon stimulating gene-encoded cholesterol- -hydroxylase (ch h) is upregulated by ifns and toll-like receptors to convert cholesterol into -hydroxycholesterol [ ] . thus, combination treatment with ifns may further enhance the antiviral effects of -hydroxycholesterol and should be further investigated. our study had limitations. first, we used a fixed antiviral agent concentration in our primary screening in order to identify the antiviral agents with the lowest ec among the broad-spectrum antivirals. this might have overlooked antiviral agents that can inhibit sars-cov- at higher concentrations. for example, favipiravir has been shown to inhibit sars-cov- replication with an ec of µm [ ] . similarly, galidesivir, a broad-spectrum rna-dependent rna polymerase inhibitor, inhibited the sars-cov with an ec of . µm [ ] . second, antiviral evaluation of the selected reagents should be performed in additional primary cells to comprehensively document their antiviral activities. third, the combination effects of the host-based ifn-β b, am , and -hydroxycholesterol with virus-based antivirals, such as remdesivir and lopinavir, should be further evaluated in vitro and/or in vivo. targeting multiple steps in the viral replication cycle might help to enhance the therapeutic effects of these virus-based antivirals in covid- patients. indeed, during the revision of this manuscript, a multi-center, open-label, randomized phase clinical trial comparing adult covid- patients treated with triple combination antiviral therapy (ifn-β b, lopinavir-ritonavir, and ribavirin) with those treated with lopinavir-ritonavir monotherapy was reported. the results showed that the combination therapy group had a significantly shorter median time from commencement of treatment to negative nasopharyngeal swab than the control monotherapy group ( vs. days) [ ] . additional studies to evaluate the effects of combination therapies using the other antiviral agents identified in this study should be considered. hong kong corporation limited, and was an invited speaker for gilead sciences hong kong limited and luminex corporation. the other authors declared no conflict of interests. the funding sources had no role in study design, data collection, analysis or interpretation or writing of the report. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan world health organization. coronavirus disease (covid- ) situation report - a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person 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-hydroxycholesterol and -hydroxycholesterol- -sulfate -hydroxycholesterol production by the cholesterol- -hydroxylase interferon-stimulated gene restricts mammalian reovirus infection triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial key: cord- -mcnr bcp authors: bonzano, chiara; borroni, davide; lancia, andrea; bonzano, elisabetta title: doxycycline: from ocular rosacea to covid- anosmia. new insight into the coronavirus outbreak date: - - journal: front med (lausanne) doi: . /fmed. . sha: doc_id: cord_uid: mcnr bcp nan coronavirus disease caused by severe acute respiratory syndrome-coronavirus- (sars-cov- ) it usually manifests with respiratory symptoms ( ) . similarly, to other human respiratory coronaviruses (hcov), it seems to have a neuroinvasive and neurotropic activity ( , ) . in the retrospective case series study conducted by mao et al. three categories of neurological symptoms covid -related included central nervous system (cns) manifestations, peripheral nervous system (pns) symptoms and musculoskeletal disorders ( ) . hyposmia has been reported as a possible peripheral nervous system (pns) symptom caused by covid- infection ( ) . in our experience, the smell alteration (hyposmia, anosmia) seems to be one of the first manifestations of covid- disease, with or without the loss of taste (dysgeusia). sometimes it remains the only symptom; more often, it comes with fatigue, fever, and cough. we provide a commentary on how covid- could affect the sense of smell and the reason why doxycycline (dox) could play a role in its recover. the three leading causes of loss of smell reported in the literature are head trauma, chronic sinonasal inflammation and upper respiratory tract viral infections ( , ) . anosmia is one between numerous olfactory disorders, but its mechanism is not clearly defined ( ) . post viral temporary chemosensory dysfunction after a common cold is widely reported ( , , ) . the swelling of the mucosa in the olfactory cleft it seems to be cause of the transient olfactory and taste loss typically reported during the common cold. it usually leads to a conductive post-viral loss of smell, and it usually appears months after the upper respiratory tract infection ( ). the olfactory neuroepithelium represents an important immunological barrier within the nasal cavity exposed to the external environment, and thus it is subject to both exogenous insults and endogenous host defense responses ( ) . in the pathogenesis of chronic rhinosinusitis (crs)-associated olfactory loss, interferon (ifn)γsignaling pathways may play a pivotal role in orchestrating immune system function. they are able to modulate inflammatory response and pattern-recognition receptors expressed by the innate immune system during infection ( ) . therefore, they support an inflammatory process underlying the olfactory impairment crs-linked ( ) . as evidence of this (crs)-associated olfactory dysfunction is relatively rapidly reversed with systemic corticosteroids ( ) . on the other side, some post-viral sense of smell impairment may be partly independent of nasal congestion, thus explaining oxymetazoline failure in improving olfaction ( ) . therefore, it has been suggested that hcov, thanks to their neuroinvasive, neurotropic, and neurovirulent properties may be able to induce neuronal impairment ( ) . "speed and simplicity" of sars-cov- entering into the respiratory tract, all hcov invade and infect intra-luminal macrophages and epithelial cells. hcov belong to coronaviridae, enveloped non-segmented, single-stranded, positive-sense rna viruses (+)ssrna. viral spike (s) proteins manage their cell entry program. they act by binding cell-surface receptors and facilitating the fusion of the virus-cell membrane ( ) . the spike protein is the key of coronaviruses tropism ( ) . these s proteins are organized in trimers that end up on the virion in a "corona" way giving it the characteristic crown-like look which seems to play a significative role in viral infection and pathogenesis ( ) . similarly to sars-coronavirus, sars-cov- seems able to enter in the respiratory epithelium (re) by binding the human ace receptor. recombinant s protein has been shown to interact with recombinant ace protein ( ) . s proteins are the major antigenic determinant managing host immune networks, inhibiting antibodies and the immunity response against the virus by inactivating ifn-α and ifn-ß ( , ) . it is well-established the pivotal role of ifn to protect most tissues from viral pathogenicity. the speed and simplicity of sars-cov- are typical. host survival in the presence of the viral infection depends on the efficacy of its ifn system; as a matter of fact, virus survival is linked to its capacity to replicate and spread in the host, by carrying out mechanisms of evasion or subversion of the host ifn response ( ) . ifnα/β signaling plays a protective role in reducing the virus spread and modulating t cell non-cytolytic antiviral response in limiting viral load. moreover, some rna-viruses have developed mechanisms to counteract innate host defense to establish productive infections in their hosts. this is the case of an rna virus, the vesicular stomatitis virus (vsv) ( ) . retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated gene- (mda- ), seem to have an important role in the recognition of rna viruses. in particular, it has been shown that immune signaling by rig-i is involved in the generation of ifn-α/β following vsv infection. under dox treatment, cells released high levels of rig-i proteins eliciting autonomous ifn response, thereby inhibiting viral infection in vitro ( ) . in another rna virus, the respiratory syncytial virus (srv), viral proteins inhibit ifn-α and ifn-β to establish infection ( ) , and it has been reported a higher expression of interferoninduced protein only after minocycline administration. this suggests an increasing innate immune response supported by tetracycline and the following rsv inhibition ( ) . the second-generation tetracycline dox has an antiinflammatory and broad spectrum antimicrobial activity ( , ) . in , dox was first approved by the fda ( ) . it has minimal side effects and it is routinely prescribed for acne and rosacea. dox is characterized by a ∼ % oral absorption and a prolonged serum half-life ( - h) ( ) . in ophthalmology, dox is usually administered in patients affected by ocular rosacea and posterior blepharitis ( ) . the dox recommended dose is mg modified release once daily, which could be replaced by minocycline mg, based on patient tolerance or particular requirements ( ) . the rationale in its administration is proteolysis inhibition promoted by matrix metalloproteinases (mmps) ( , ) . mmps are involved in the regulation of chemical and biological process likes vascular remodeling and angiogenesis ( ) , so dox also has anti-angiogenic properties. ( ) it regulates cytokines and diminishes neutrophil chemotaxis too ( ) . besides its well-known use in treating bacterial infections, some studies in the literature report that dox possesses a broad activity against viral infection too ( - ). the first who described the dox antiviral effect was sturtz in ( ) , and this suggestion has been confirmed in several followed-up studies. ( , , ) topno et al. demonstrated that dox could interfere with the virion's replication, affecting its structure and causing inhibition of japanese encephalitis virus-induced pathogenesis in vitro ( ) . the same observation is also reported in a study regarding vsv infection ( ) and against the chikungunya virus (chikv) ( ) , suggesting that dox might interfere with viral replication by aiming proteins essential for these viruses for a successful infection. as proof of that, computational literature reports the dox ability to bind chikv cysteine protease ( ) , and to exert a significant inhibitory effect on dnv ns b-ns serine protease in vitro ( ); both these proteases proved to be able to catalyze viral polyproteins cleavage during infection. moreover, some studies with (+)ssrna, dengue virus (dnv), have demonstrated that dox inhibits virus plaque assembly by interfering with the viral envelope conformational changes needed for virus entry ( ) . in both chikv and dnv, dox seems to have the ability to bind virus envelop inhibiting viral entry into the cultured cells ( , ) . dox proved to be able to markedly decreased the virusinduced cytopathic effect (cpe) and significantly affect viral replication in a dose-dependent manner when used against porcine reproductive and respiratory syndrome virus (prrsv) infection in cultured cells ( ) . virus mrna levels were strikingly reduced also in vsv-infected cells in response to dox; both virus titers and the cpe of vsv infection were significantly influenced by dox administration in a dose dependent manner ( ) . being the olfactory neural system able to regenerate throughout life, it can explain why the recovery of olfaction is common ( ). from our observation, anosmia affected mostly young adults rather than elderly patients, confirming existing findings in the literature ( , ) . it shows up more or less days after fever, cough and muscle aches, but it can be the first and only symptom in many patients, with no mucosal swelling of the olfactory cleft, and that's why we hypothesize that it could be a possible pns symptom as suggested ( ) . among patients affected by pns symptoms linked to covid- , the most common referred were hyposmia, hypogeusia, followed by neuralgia ( ) . respiratory viruses such as rhinovirus and parainfluenza epstein-barr virus commonly could cause olfactory dysfunction (od) by leading an inflammation in the olfactory mucosa resulting in rhinorrhea. instead, covid- seems to cause an atypical od as it develops without rhinorrhea or nasal congestion ( ) . in , suzuki et al. identified that coronavirus could be associated with anosmia, and he already speculated that nasal inflammation and related obstruction were not the only etiological factors underlying the od in viral infection ( ) . as well-reported in the literature, hcov could infect peripheral nerve terminals, using the trans-synaptic transfer to access the cns ( , , ) in our preliminary observation, the administration of dox mg once daily seems to improve respiratory symptoms and anosmia under dox treatment in six patients completely recover after only days of treatment. from our experience, it seems reasonable to continue the treatment at least days. the mean patients' age was . ± . years, and ( . %) were females. one patient reported anosmia as the only covid- manifestation; instead of the other five patients who complained about the loss of smell, in which it appeared - days after mild fever, dry cough, and malaise. the average time of the recovery covid- -linked anosmia after the administration of dox in these patients was . ± . days. we noticed a sudden improvement in all symptoms after the administration of dox, but our most exciting insight is about the rapid recovery of the smell. unlike olfactory sensory neurons (osns), nasal epithelium, which includes the respiratory and olfactory epithelium (oe) expresses high levels of ace ( ) . sars-cov- seems to target non-neural cell types in the peripheral olfactory system rather than directly enter osns, and it seems to be enough to generate cascading damage that could lead to the impairment of osns function altering the odor transduction which takes place on their cilia ( ) . the short-term covid- -linked anosmia reported in our experience supports the hypothesis that sars-cov affects the oe, which can quickly renew and recover following viral clearance ( ) . the average time to restore the sense of smell, most commonly reported in the literature, lasts from - days ( ) , if sars-cov- could directly damage osns, recovery should take longer ( ) . besides ace , brann et al. also revealed that a cell-surface receptor, cd , could play a role mediating sars-cov- cell entry ( ) . the expression of cd is detected in ciliated and goblet cells in the human nasal mucosa ( ) . previous reports have shown that dox has a significant inhibitory effect on cd expression ( , ) . further studies are needed at present to define better if dox has the ability to inhibiting viral entry by reduced cd expression levels. moreover, thanks to its immunomodulatory and antiinflammatory properties, dox could limit the pro-inflammatory state induced by the glial cells activated by the neurotropic virus, ensuring proper epithelial reconstitution in the oe ( , ) . given the possibility that covid- occurs with the loss of smell and the evidence that corticosteroid may worsen the infection ( ), prof. claire hopkins, the british rhinological society president, recently suggested avoiding the use of these drugs in the therapeutic approach to the new-onset anosmia during the covid- pandemic, especially if unrelated to previous head trauma or nasal pathology ( ) . we are perfectly aware that there is a need for stronger evidence, but our article would intend to underline the importance of considering smell loss as a common symptom of covid- , supporting the rationale to treat such patients with dox based on its interesting antiviral 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vechh x on march , , the world health organization declared the worldwide spread of the infectious disease covid- , caused by a new strain of coronavirus, sars-cov- , as a pandemic. like in all other infectious diseases, the host immune system plays a key role in our defense against sars-cov- infection. however, viruses are able to evade the immune attack and proliferate and, in susceptible individuals, cause severe inflammatory response known as cytokine storm, particularly in the lungs. the advancement in our understanding of the mechanisms underlying the host immune responses promises to facilitate the development of approaches for prevention or treatment of diseases. components of immune system, such as antibodies, can also be used to develop sensitive and specific diagnostic methods as well as novel therapeutic agents. in this review, we summarize our knowledge about how the host mounts immune responses to infection by sars-cov- . we also describe the diagnostic methods being used for covid- identification and summarize the current status of various therapeutic strategies, including vaccination, being considered for treatment of the disease. on december , , a cluster of cases of pneumonia was announced in wuhan, hubei province, china. subsequently, on january , , the chinese health authorities confirmed that this cluster was associated with a novel coronavirus, ncov, which was later named as sars-cov- , and the ensuing disease was named covid- . the covid- outbreak by the new coronavirus strain was recognized as a pandemic by the world health organization (who) on march , . throughout history, there have been a number of pandemic diseases; the more notable and recent ones caused by viruses include the influenza pandemic (spanish flu) in and another by the influenza virus h n in . the immune system clearly plays a key role in the host defense against the infectious agents during these pandemics. the host is able to mount immune responses upon infection by viruses, as well as other microbes, and control the spread of these pathogens within the body. however, some viral strains are capable of evading the immune attack and proliferate in the body, as well as elicit inflammatory responses, in particular in the lungs, resulting in pneumonia. more importantly, in susceptible individuals, viruses can cause massive inflammatory responses, known as "cytokine storm", resulting in a severe pathological consequence. the advancement in our understanding of the mechanisms of the host immune response are crucial to development of approaches for prevention and treatment of these fast spreading and devastating infectious diseases. the components derived from our immune systems, such as antibodies, can be used to develop sensitive and specific methods for the diagnosis of infectious diseases, as well as novel therapeutic modalities. in this review, we briefly summarize our knowledge about the host immune response upon infection by sars-cov- . we also discuss the epidemiological aspects of the outbreak, and the potential mechanism of the severe host response, such as cytokine storm. we also describe the antibody-based approaches for diagnosis of covid- infection and summarize the current status of various preventive and therapeutic modalities for treatment of the infection. coronaviruses are single-stranded enveloped rna viruses that cause diseases in mammals and birds. in humans, the low pathogenicity strains, including hcov- e, hcov-oc , hcov-nl , and hcov-hku, infect the upper respiratory tract and cause mild to moderate common cold-like symptoms in healthy individuals. they are responsible for - % of all common cold cases. the highly pathogenic strains, including those causing severe acute respiratory syndrome [sars-cov] , middle east respiratory syndrome [mers-cov] , and covid- [new sars-cov- ], infect the lower respiratory tract and can cause severe pneumonia [ ] . in addition to their rna genetic material, coronaviruses are composed of nucleocapsid (n) and spike (s) proteins, which participate in viral genome assembly, transcription and replication, or mediate viral entry and cause cytopathic effect [ , ] . the s protein mediates the fusion of viral and host membrane [ ] and contains a receptor-binding domain (rbd) that attaches to cells during viral entry. angiotensin-converting enzyme (ace- ) is the receptor for both sars-cov and sars-cov- [ ] . notably, the four human coronaviruses that cause common cold like symptoms show limited sequence homology in their n ( - %) and s proteins ( - %) compared with those of sars-cov- [ ] . mers-cov also exhibits more distal relationship to sars-cov and sars-cov- . however, the latter two are more closely related, with their n and s proteins sharing high homology ( - %) . in , the sars-cov infection, which started in southern china, led to an epidemic; in total, over cases were reported, which included close to deaths with a case fatality rate of % [ ] . in , the first case of mers took place in saudi arabia. from that moment on, close to cases have been reported globally, which included close to deaths, with an estimated case fatality rate of approximately % [ ] . evidence is mounting that covid- spreads via human-to-human transmission of the virus [ ] . after exposure to sars-cov- , the majority of patients recover with little or mild symptoms that include cough and fever [ ] . however, it is estimated that approximately % of infected individuals develop severe disease, including acute respiratory distress syndrome (ards). according to who, as of april , , a total of , , confirmed cases of covid- have been detected and , deaths resulting from the infection have been confirmed worldwide. the case fatality rates in wuhan and worldwide were approximately . and . %, respectively. the numbers are lower in some countries, for example approximately . % in the united states, . % in south korea, . % in germany, and . % in taiwan. these numbers are likely affected by the extent of screening, thereby implying that covid- cases might be underdiagnosed in many other countries. the availability and infrastructure of medical facilities in afflicted countries, especially those seriously affected ones, also likely affect the overall death rates. using public and published information, wu et al. estimated that the overall "symptomatic case fatality risk" (the probability of dying after developing symptoms) associated with covid- was . % [ ] . the rate of development of severe symptoms and death are clearly associated with the age. it is to be noted this is based on all tested and confirmed cases of covid , and the true fatality risk is likely lower than . %, since many mildly symptomatic/asymptomatic people might have never got tested. nevertheless, the risk is still higher than that associated with seasonal influenza virus, which is approximately . %. with regard to the spectrum of the severity of the disease, chinese center for disease control and prevention reported of the , confirmed cases (with the age distribution of > years: %; - years: %; - years: %; - years: %; < years: %), the spectrum of disease were: mild: %; severe: %, critical: %; and case fatality rate: . % [ ] . finally, the basic reproduction number (ro) of the virus has been estimated to be between . and . , meaning each infection from the virus can result in . to . new infections, when no members of the community are immune and no preventative measures, such as vaccination, are taken. by comparison, the median ro value for sars-cov was in the range of to and for influenza was . . the outcome of clinical infection likely largely depends on the capacity in mounting effective antiviral immune responses in time, to control viral spreading, to limit organ injuries and to speed up recovery. here we summarize the immune response induced by cov. three components are crucial for sars-cov induced diseases: ) the role of cd + t cells in defense against the virus, which causes apoptosis in the infected cells, ) interactions of the virus with macrophages and dendritic cells, which initiate the early innate and subsequent adaptive immune responses, and ) type i interferon (ifn) system, an innate response against viral infections, which can inhibit virus replication in the early phase. firstly, the central part of the body's anti-viral immunity is based on the interaction between antigen and antigen presentation cells (apc) when the virus enters the cells. the infected cells are recognized by virus-specific cytotoxic t lymphocytes (ctls) via viral peptides as the antigen presented by major histocompatibility complex (mhc). the antigen presentation of virus mostly depends on mhc i molecules, but mhc ii also has its contribution in some cases. the mhc i molecules display pieces of virus proteins on the surface of infected cells, which creates a signal to activate nearby cd + t cells to induce apoptosis in the infected cells. there are many reports on the relationship between various mhc polymorphisms and the susceptibility to sars-cov [ ] [ ] [ ] , but little is known about this association in covid- . such information could provide beneficial aspects of personalized medicine for treatment or prevention of covid- . secondly, dendritic cells and macrophages are other first patrolling components of innate immune network, which play important roles in driving both innate and adaptive immune responses to the viral pathogens [ ] . the invasion of viruses can be recognized by innate immune cells via pathogen-associated molecular patterns (pamps). in the case of cov, pamps are viral genomic rna, which are recognized by endosomal rna receptors such as tlr , tr , tr , and tlr [ ] . this can cause rapid responses of the innate immune cells to viruses, resulting in production of a large amount of type i ifn with antiviral functions. lastly, efficient innate immune responses against viruses also depend on type i ifn responses and downstream cascade. type i ifn, by directly interfering with the viruses' replication ability, can prevent reproduction of viruses in infected cells. by mounting type i ifn responses successfully, viral replication and dissemination in an early stage are suppressed. sars-cov and mers-cov use several strategies to avoid the innate immune response, these are probably also employed by sars-cov- . these include the inhibition of type i ifn recognition and signaling, as well as downregulation of mhc class i and class ii molecules in infected macrophages or dendritic cells, resulting in impaired antigen presentation and diminished t cell activation. moreover, some proteins encoded by sars-cov can interact with the signaling cascades downstream of the pattern recognition receptors. after exposure to sars-cov- , patients respond to the virus by generating specific igm antibodies within a few days, followed by specific igg production within a week [ , , ] . in the case of sars-cov infection, although the serum anti-viral igm antibody levels decline in a few months, the antiviral igg antibody titers can persist for years. among the many structural and non-structural proteins encoded by sars-cov- , the n and s proteins are the most immunogenic antigens. antibodies against the n protein are the first to appear and thus can serve as an early and reliable serum marker for virus exposure, whereas antibodies against the s protein develop later and can bind to the viral envelope. recent studies indicated that the convalescent serum contains antibodies that can neutralize sars-cov- in cell cultures [ , ] . therefore, igg against the s protein is both a marker for viral exposure and an indicator of recovery. the potential risk of disease exacerbation by ade, a phenomenon in which pre-existing poorly neutralizing antibodies lead to enhanced infection, has been a serious concern for vaccine development and antibody-based therapeutic strategy. compared to the ade in dengue viral infections, which was supported by a great deal of epidemiological and clinical evidence in the past four decades [ ] , this phenomenon in coronaviruses has mainly been observed in cell-based experimental models [ , ] . to illustrate this further by also using dengue virus as an example: while more severe symptoms, such as dengue hemorrhagic fever (dhf)/dengue shock syndrome (dss), can be observed during primary infection, they are much more frequently developed following a secondary infection with a different serotype (out of the four existing serotypes) [ ] . furthermore, it has been well documented that the high level of virus replication seen during secondary infection with a heterotypic virus is a direct consequence of ade of viral replication. this is mediated primarily by the pre-existing, non-neutralizing, or sub-neutralizing antibodies to the virion surface antigens, resulting in enhanced access to target cells, through binding of the virion-antibody complexes to igg fc receptors (fcγr) on these cells [ ] . this common underlying theme of ade-based magnification of virus replication also indicates that severe disease is not merely attributable to inherent virulence of virus [ ] . interestingly, as described in a later section, several available evidence from the use of convalescent sera in patients with sars, mers [ ] and cases with covid- [ ] suggest the feasibility and safety of convalescent serum trials. here, caution and vigilance to identify any evidence of enhanced coronavirus infection by ade will be required. despite the fact epidemiological and clinical observations supportive of existence of ade in coronavirus infection is not available, a molecular mechanism behind ade of coronavirus has currently been provided [ ] . the authors demonstrated that a neutralizing antibody binds to the s protein of coronaviruses like a viral receptor, triggering a conformational change of the spike and mediating a viral entry into fcγr -expressing cells through canonical viralreceptor-dependent pathways. however, an enhanced entry of these pseudovirus-based approaches does not support directly the magnification of viral replication in these cells. compared to dengue viruses, these fcγrbearing cells such as dendritic cells, monocytes and macrophages, even if infected through ade process, would presumably not be so "permissive" for coronaviruses, in terms of their replication and assembly. however, these viewpoints need further investigation. apparently, many host factors could also exacerbate disease during secondary infection. these host factors need to be identified by combined epidemiological and genetic analyses of appropriate patients, and the contribution of underlying host factors to the control of coronavirus replication needs to be determined. for example, ade of replication can possibly occur with the vaccine strains of viruses in the endemic populations, such as attenuated or recombinant coronavirus vaccines. however, the level of replication will likely remain low, and thus this small enhancement of replication will probably not augment the disease. in addition, this could result in heightened vaccine immunogenicity due to a small increase in the virus load. however, further investigation on correlations between immunological responses and disease outcome and the validation of these findings in vaccine trials will be invaluable for developing safe and effective sars-cov vaccines (see below). while sars-cov can evade innate immune system, they can also induce intensive inflammatory reactions through innate immune cells. in fact, sars-cov and sars-cov- infections are known to activate a massive over-production of cytokines by the host immune systema phenomenon known as "cytokine storm", which usually occurs a few days after the onset of the illness. this also results in increased local and systemic vascular permeability in major organs. cytokine storm is reported in many viral infections, and contributes significantly to the pathogenesis and severity of acute viral infections. the tissue tropism of each virus determines the cytokine profiles of virus-induced cytokine storm (reviewed in [ ] ). for example, macrophages produce a higher amount of proinflammatory cytokines than endothelial cells, while virus-infected endothelial cells are the major source of chemokines. however, this may not be generalizable, and it is crucial to elucidate the tropism of sars-cov- in order to interpret the data. most proinflammatory cytokines are released from macrophages and severe acute infections are usually associated with the activation of macrophages by enveloped viruses. in addition, activation of neutrophils may also be involved. as mentioned above, viral nucleic acids can induce the production of interferons and proinflammatory cytokines, through engaging endosomal tlrs. these intracellular nucleic acid receptors/sensors have been defined as "protective host factors", as they are critical for host defense against viral infections. however, the identity and contribution of "pathogenic host factors" to virus-induced severe inflammatory reactions and lethality, and how different viruses cause distinct clinical symptoms, remain unclear. some available information related to the cytokine storm induced by sars-cov and mers-cov is summarized below. it is to be noted that while both sars-cov and sars-cov- utilize ace- as their receptors, mers-cov binds to the receptor dipeptidyl peptidase (dpp /cd ). the differences in receptor usage may account for the differences in disease patterns, including the organs involved and the extent of the cytokine storm induced. [ ] [ ] [ ] . the clinical course of this infection has three phases: ) robust virus replication accompanied by fever in the first few days; ) high fever and pneumonia with progressive decline of virus titers; ) ards resulting from active host immune responses in the absence of detectable viruses [ ] . in addition to infecting and proliferating in the airways and alveolar epithelial cells, sars-cov can also infect dendritic cells, monocytes, and macrophages, without undergoing proliferation (i.e., abortive infection) [ ] . sars-cov-infected epithelial cells produce high levels of chemokines such as ccl , ccl , ccl , and cxcl . in addition, sars-cov-infected dendritic cells [ , ] and macrophages [ ] secrete high levels of proinflammatory cytokines tnf and il- , and significant amounts of chemokines. it is interesting to note that higher levels of il- , il- , ifn-gamma, il- , and cxcl , in addition to the cytokines and chemokines mentioned above, were also observed in sars patients with severe diseases. this suggests that other cell types also contribute to sars-cov-induced cytokine storm. the typical pathological changes in the lungs include focal hemorrhage and mucopurulent materials in bronchial trees with diffuse alveolar damage. histological examination shows extensive macrophage and neutrophil infiltration with lower levels of t lymphocytes. existing information suggests that the sars-cov-infected airways and alveolar epithelial cells secrete abundant chemokines to attract immune cell infiltrations to the lungs, including macrophages and neutrophils, thereby causing damage due to high levels of proinflammatory cytokines and other mediators secreted by these cell types. in addition to the airway epithelial cells, mers-cov can also replicate in human monocytes, macrophages, dendritic cells, and activated t cells. the typical lung pathological changes caused by mers-cov is diffuse alveolar damage. in addition, pleural and pericardial effusions associated with generalized congestion and consolidation of lungs have been noted [ ] , and the severity of lung lesions were noted to be correlated with extensive infiltration of neutrophils and macrophages [ ] . similar to sars-cov, mers-cov can induce high levels of proinflammatory cytokines and chemokines in human monocyte-derived macrophages and dendritic cells. mers-cov infection was also reported to induce increased concentrations of proinflammatory cytokines (ifn-γ, tnf-α, il , and il ), [ ] . the high serum cytokine and chemokine levels in mers patients were correlated with increased infiltration of neutrophil and monocytes along with severe tissue damage in the lungs [ , , ] . thus, the pathological change in the lungs is similar between sars-cov and mers-cov. whereas, the higher mortality rate in mers-cov-infected patients may be due to the higher incidence of pericarditis in infected patients. the first autopsy of covid- victims along with immuno-histological staining revealed the presence of sars-cov- in the airway epithelia and macrophages, suggesting that the virus can infect both epithelial cells and macrophages [ ] . the majority of infiltrating cells are macrophages and monocytes with moderate amounts of multinucleated giant cells and neutrophils. increased levels of cytokines and chemokines, including il- , il- , g-csf, m-csf, ifn-γ, ip- , mcp- , mip- α, and tnf-α, were detected in the plasma of covid- patients [ ] . the most significant predictors of mortality in these patients are serum ferritin level and il- , suggesting that mortality is due to virus-induced hyperinflammation and cytokine storm during viral infection [ , ] . compared to the low pathogenic coronaviruses, the common features of high pathogenic coronaviruses include extensive infiltration of leukocytes, which secrete abundant proinflammatory cytokines and other chemical mediators to cause diffuse alveolar damage. also, high pathogenic viruses are associated with abortive infection; for example, in contrast to the less pathogenic strain of influenza virus h n , the highly pathogenic influenza virus h n does not replicate in macrophages; the latter is also a more potent inducer of the chemokine cxcl [ ] the key innate immunity receptors/sensors responsible for high pathogenic coronavirus-induced proinflammatory cytokines are still unclear. finally, notably, more deaths from covid- have been caused by multiple organ dysfunction syndrome rather than respiratory failure, which is different from infections caused by sars-cov and mers-cov; the basis for this remains unknown. detection of viral rna in the secretions from the respiratory tract of infected patients by reverse transcription-polymerase chain reaction (rt-pcr) test is currently the standard method for diagnosis of covid- . they have some limitations, including long turnaround time (typically - h) and the requirement of specialized facilities. scientists around the world have been devoting effort to developing improved nucleic acid-based, simpler and faster methods. the us fda issued an emergency-use authorization to cepheid's xpert xpress sars-cov- test, which became the first pointof-care covid- diagnostic test to receive this designation in the us. the test is designed to use the company's automated genexpert systems and has a turnaround time of approximately min. another prominent example is a method for detection of sars-cov- by using crispr technologies [ ] . these newly developed platforms will clearly require clinical testing before approval for routine use. tests based on antibodies are obviously another option for diagnosis and screening. immediately after infections, viral genome and proteins start to increase, becoming the earliest markers for diagnosis within days. as the host immune responses gear up to confront and reduce viral replications, the viral rna or antigen level declines, but the antiviral igm and igg titers rise up. as patients usually present themselves with cough, fever or shortness of breath, and are already beyond the early stage of infection [ ] ; here, nucleic acid tests are expected to pick up only a proportion of patients. a complementary serological test for specific igm or igg will help identify the rest. one report from shenzhen studied patients within days of illness, who were later diagnosed with covid- [ ] ; in this study rt-pcr could detect two-thirds of the patients, but only % tested positive even after days. in contrast, although the serological positive rate within week was less than %, the rate increased to % after day of disease onset. a combination of nucleic acid and serological test significantly increased the diagnosis rate from to % even within week of illness [ ] . if these results can be validated, such a combination may become a standard clinical practice in the future. in this regard, li et al., developed an immunoassay that can detect igm and igg antibodies against sars-cov- in human blood within min. they tested samples from close to confirmed patients and over negatively-tested patients at different clinical sites and reported a sensitivity of over % and specificity of over % [ ] . in the early stage (containment) of covid- pandemic, there was a strong interest for rapid diagnosis and thus prototypes of rapid viral antigen or antibody tests were being developed for point-of-care use. these platforms have the advantage of convenience and a fast turnaround time, but suffer from inadequate sensitivity and specificity, as compared with standard rt-pcr. hence, their results need to be interpreted with caution. preparation of high-quality antibodies and antigens requires years in perfecting such point-of-care tests, judging from the experience in developing such tests for influenza viruses. eventually, the more stringent criteria for a definitive diagnosis will need paired serum samples to demonstrate a true seroconversion [ ] . finally, the issue of antibody cross-reactivity with other human coronaviruses warrants discussion. as mentioned above, serological assays usually adopt viral n or s protein as antigens and the protein components from all four human coronaviruses that cause common cold show very limited sequence homology with those of sars-cov- . thus, despite the fact the majority of the populations have been exposed to the four low pathogenic human coronaviruses, their sera do not react positively in sars-cov- elisa [ , ] . while mers-cov is also more distally related and presents no concerns, the situation for sars-cov-exposed patients is different. as the n and s proteins of the sars-cov strain share high homology ( - %) with those of sars-cov- , serum from sars patients can actually cross-react with sars-cov- n or s protein in immunoblot or viral neutralization assays [ , ] . however, as sars-cov epidemic was only transient with a very small proportion of the populations being exposed, this cross-reactivity should not be an important issue. as with many vaccine-preventable viral diseases like measles and chicken pox, the newly emerged sars-cov- infection assumes an epidemiological characteristic capable of evading containment measures and facilitating pandemic potential. thus, a high proportion of undetected infections with mild or no symptoms can efficiently sustain viral transmission [ ] . while containment and lockdown can serve as temporary control measures, effective vaccines or therapeutic agents are much needed for the ultimate control of the disease. the vaccine research and development thus far have progressed at an unprecedented speed; the first dose of rna-based sars-cov- vaccine was administered to test its safety in humans on march , , only months after the new virus was first identified. such rapid progress was facilitated by a combination of multiple factors, including advances in vaccine research on sars and mers, as recently reviewed [ ] , progress in a number of vaccine technology platforms to the early stage of human trial [ ] [ ] [ ] , and readily available support from the well-orchestrated international collective effort of the coalition for epidemic preparedness innovations (cepi) [ ] . the aforementioned innovative aspects that could potentially drive sars-cov- vaccine development to market launch within a year or two are summarized below. the coronavirus s protein is a critical target for antiviral neutralizing antibodies and functions to mediate entry into mammalian cell expressing the viral receptor ace [ , ] . moreover, the target neutralizing epitope of sars-cov was further narrowed to a smaller fragment of the s protein, later termed receptor binding domain (rbd) [ ] . building on these paradigmatic scientific advances that the s protein is the putative target antigen, sars-cov- vaccine candidates are designed to include full or various lengths of the s protein focusing on the rbd. vaccine based on the whole virus may be less preferred due to its association with eosinophilic pulmonary pathology [ , ] . a number of novel vaccine platforms, including vector-, dna-, and rna-based vaccines, are being developed or improved with innovative technology specifically to combat pandemic-prone outbreaks and have been recently reviewed [ ] . nucleic acid vaccines, including both dna and rna, offer the potential to accurately express any protein antigen in host cells and to present the antigen closely resembling antigen expression and presentation during viral infection. dna vaccines against mers and rna vaccine against h n have completed phase i trials that showed these platforms to be safe [ ] [ ] [ ] . the nucleic acid vaccine can be completely synthetic and formulated within a few weeks at sufficient quantities to support clinical trialsa valuable feature when facing potential pandemic. vector-based vaccine consists of a target antigen inserted into a viral genome to render faithful antigen generation, targeting and processing in vivo after vaccination. the first ebola vaccine approved by us fda is a vector-based vaccine using vesicular stomatitis virus expressing a surface glycoprotein of ebola [ ] , thus supporting the applicability of this platform in combating emerging infectious diseases. these platforms offer versatile adaptation for antigen of new diseases, and the process development for production is relatively simple and quick, indicating the value of these platforms for the urgent response to new diseases. the rapid infusion of funding from and coordination by cepi in january was the major driver for the speed of sars-cov- vaccine r&d progress (fig. ) . with its mission being "to stimulate, finance, and coordinate the development of vaccines for epidemic diseases" especially aiming to drive vaccine innovation for highpriority public health threats, cepi has supported a number of "technology platforms". these included a vaccine printer, molecular clamp technology for protein production, and a self-amplifying rna vaccine platform since (cepi web page https://cepi.net/research_ dev/technology/). an innovated vaccine platform technology pertains to a system that uses the same basic core technological components as a backbone and can be adapted by inserting new genetic or protein sequences to target newly emerging pathogens. it is with the application of this concept that an rna-based sars-cov- vaccine, built on the avian flu vaccine platform [ ] , is expected to be developed and quickly proceed to human trial within only a few months since covid- became an epidemic, and a dna-based vaccine candidate modeling that of mers is soon to follow [ ] . the cepi coordinated effort encompassing a wide range of available technologies from industry and research institutes globally has shown preliminary success toward an urgent response to control a pandemic. while coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines also present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology [ , ] ; this seems to be either exacerbated or eliminated by the formulation of adjuvant selection depending on the th /th bias and induction of durable ifn-γ responses, respectively [ ] . in addition, ade, described above, was seen in the lungs of macaques after administration of inactivated sars-cov vaccine or vaccine composed of certain s antigen fragments [ ] . these studies highlight the importance of designing the target antigen and selection of adjuvants to ensure both efficacy and safety. considering the novel nature of sars-cov- and that an animal model has yet to be established for testing of the vaccine to especially focus on the immunopathological perspectives, the safety concern is anticipated to present most of the hurdles in the development process. herd immunity refers to a state when sufficient proportion of a population becomes immune to sars-cov- , via natural infection or vaccination, so as to eventually halt further spread of disease, and thus individuals not immune to the virus are protected. in the case of covid- , the herd immunity was estimated to be % of the population. before vaccines are available for mass immunization, the strategy of achieving herd immunity via natural infection has been considered and deemed not advisable when a number of factors were considered. thus, self-isolation and social distancing remain crucial in combating this pandemic so that the initial pressure on our healthcare systems is reduced, and more time is given to us to develop vaccines or effective therapies. the disease spectrum of covid- can be divided into mild infection, pneumonia, ards, and even multiple organ failure [ ] . after a decade of research on coronavirus, unfortunately, still there are no licensed vaccines, effective specific antivirals, nor drug combinations supported by high-level evidence to treat the infection, especially for newly emerging strains such as sars-cov- [ ] . several strategies are being considered for the treatment of covid- , including the use of antimicrobial agents, immunotherapy with virus-specific antibodies in convalescent plasma, monoclonal and polyclonal antibodies produced in vitro or genetically modified antibodies, and interferons. here we focus on immunebased therapies, but for the sake of completeness, we also include therapies using antimicrobial agents as supplementary information. the potential interventions for sars-cov infection are summarized in table . biologic drugs composed of monoclonal antibodies (mabs) have been developed for treatment of a variety of diseases. it is thus not surprising that this approach is being considered for the treatment of sars-cov infection and shows promise. a human igg mab, cr , that binds to sars-cov s protein has been developed [ ] . sui et al. found one recombinant human mab (single-chain variable region fragment, scfv, r) against the s domain of s protein of sars-cov from two nonimmune human antibody libraries. the mab could efficiently neutralize sars-cov and inhibit syncytia formation between cells expressing s protein and those expressing the sars-cov receptor ace [ ] . a human igg mab, cr , has been generated and found to be able to neutralize sars-cov and shown to be able to prevent sars-cov infection in ferrets [ ] . more recently, ju et al. reported the isolation and characterization of rbd-specific mabs derived from single b cells of eight sars-cov- infected individuals [ ] . for clones from one patient they demonstrated their ability to neutralize live sars-cov- . none of these antibodies cross-reacted with rbds from either sars-cov or mers-cov, although the patient plasma exhibited such cross-reactivity. these neutralizing antibodies have the potential to be used for prophylaxis for or treatment of sars-cov- infection. agents that directly block the binding of s protein to the functional receptor ace also have the potential to be used for prevention of covid- . guillon et al. demonstrated that binding of sars-cov s protein to ace could be inhibited by anti-histo-blood group antibodies, presumably because the virus carries histo-blood group antigen structures of the host [ ] . while whether this approach can be developed into effective treatment strategies is uncertain, the findings have a bearing on the effect of the naturally occurring anti-histo-blood group antibodies on the individual variations in susceptibility to sars-cov infection. convalescent plasma can be employed for passive immunotherapy and is usually chosen when there are no specific vaccines or drugs available for emerging infection-related diseases. yeh et al. reported a favorable outcome in the use of convalescent plasma for treatment of sars-cov-infected healthcare workers [ ] . arabi et al. tested the feasibility of convalescent plasma therapy as well as its safety and clinical efficacy in critically ill patients suffering from mers-cov infection [ , ] . if available, convalescent plasma could certainly be considered for the treatment of sars-cov- -infected critically ill patients. interferons (ifns), including ifn-α and ifn-β, are produced during the innate immune response to virus infection and they are able to inhibit the replication of virus in vitro [ , ] . as mentioned above, ifn transcription was blocked in tissue cells infected with sars-cov. recombinant ifn-α given on days before the infection could reduce viral replication and lung damage, as compared with the control in monkeys and in a pilot clinical trial [ ] . ifn-α inhalation can also be considered. combination of interferon-α- a with ribavirin was used in treatment of patients with severe mers-cov infection and the survival of these patients was improved [ , ] . these findings suggest that these fda-approved ifn's could be used for the treatment of covid- . as mentioned above, cytokine storm is the major underlying pathology in severe cases of covid- . thus, neutralization of some of the major cytokines are considered as a novel approach for treatment of severely ill cases and reducing morbidity and mortality. huang c et al. reported that increased il- ß, ifn-γ, ip- , and mcp- in sars-cov- infection and higher concentrations of g-csf, ip- , mcp- , mip- a, and tnf-α were found in patients requiring treatment at icu than those not treated at icu [ ] . they also noted that cytokine storm was associated with disease severity. conti p et al. reported that pro-inflammatory cytokines of interleukin (il)- β and il- in mild and acute respiratory syndrome are associated with development of lung fibrosis in covid- [ ] . thus, suppression of proinflammatory il- family members and il- might have a potential therapeutic effect. il- , an immunosuppressive cytokine, acts on mtor and increases the activity of adenosine monophosphate kinase, which inhibits protease inhibitors nelfinavir cell a selective post-translational inhibitor [ ] nucleotide analog prodrug remdesivir cell and clinical use (first case of covid- in the united states) possible inhibitor of rna replication [ , ] indole-derivative molecule arbidol cell inhibits fusion between viral envelope and cellular membranes [ ] immunosuppressive agent cyclosporine a cell block replication via inhibition of nucleocapsid protein [ ] monoclonal antibody cr cell and clinical use potently binds the receptor binding domain of s protein [ ] monoclonal antibody single-chain variable region fragments, scfv, r cell acts against the s domain of s protein [ ] monoclonal antibody cr cell neutralization of viral infectivity [ ] immunotherapeutic potential convalescent plasma cell and clinical use neutralization of viral infectivity [ , ] interferons ifn-α and ifn-β cell induction of interferon-stimulated genes to suppress viral replication [ , ] cytokine blocker cytokine il- cell inhibits inflammation, by acting on mtor and increasing the activity of adenosine monophosphate kinase [ ] cytokine blocker lianhuaqingwen cell anti-inflammation; inhibits il- receptor [ ] cytokine blocker antibody against il- receptor clinical use anti-inflammation; inhibits il- receptor inflammation by suppressing production of multiple cytokines downstream of myd , including il- β, il- , tnf and ccl . il- might be a potential therapeutic cytokine for inhibition of inflammation in covid- [ ] . runfeng l et al. demonstrated that lianhuaqingwen, a traditional chinese medicine, significantly inhibited sars-cov- replication by suppressing mrna of il- and other pro-inflammatory cytokines in vero e cells [ ] . cytokine blocker that target interleukin receptor in covid- could be potentially developed as therapeutic agents in future. in fact, fda approved mab against il- receptor (il- r) is available for treatment of rheumatoid arthritis. the society for immunotherapy of cancer has issued a statement on access to il- -targeting therapies for covid- . it is encouraging that pharmaceutical companies have in fact initiated clinical trials of anti-il- r for treatment of patients with severe covid- . there are still a large number of unanswered questions. how fast sars-cov- would mutate and would the mutated virus become more infectious or invasive. according to andersen et al. [ ] , viruses constantly mutate, but those mutations do not typically make the virus more virulent or cause more serious disease. in fact, most mutations are detrimental to the virus or have no effect. there was a study of the sars-cov in primate cells suggesting that a mutation in this viral strain acquired during the sars outbreak probably reduced virulence of the virus [ ] . another issue is whether sars-cov- , unlike sars-cov and mers-cov will continue to cause epidemic or even behave like seasonal flu. in fact, we have already witnessed the second wave of outbreak occurring in north america and europe, after the first wave that occurred in asia, and covid- may bounce back-and-forth between north and south hemispheres, as influenza virus does each year. finally, factors that determine the individual susceptibility to covid- remain to be elucidated. as mentioned above, there are many reports on the relationship between various mhc polymorphisms and the susceptibility to sars-cov. also, what governs the development of severe illness, including cytokine storm, besides the pre-existence of certain diseases and age factor, awaits clarification. despite these uncertainties, scientists in academia and industry around the world have moved at an unprecedented speed to develop improved methods for detection of the virus and treatment of the disease. advancement in immunology over the years has certainly facilitated many of these developments. we shall witness some of the recent advancements in development of vaccines and biologics for treatment of various other serious illnesses being used for fighting against covid- . we are hopeful these efforts will be sustained even after the pandemic is over, allowing us to be even more ready in the unfortunate event that another epidemic or pandemic, like covid- , takes place in the future. supplementary information accompanies this paper at https://doi.org/ . /s - - -w. additional file . supplemental information. pathogenic human 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treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review the proximal origin of sars-cov- attenuation of replication by a nucleotide deletion in sars-coronavirus acquired during the early stages of human-to-human transmission publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank dr. ming-hsiang hong for his assistance in preparation of the manuscript. authors' information none. not applicable.availability of data and materials not applicable. consent for publication not applicable. the authors declare that they have no competing interests. key: cord- -bli qm w authors: prasad, kartikay; khatoon, fatima; rashid, summya; ali, nemat; alasmari, abdullah f.; ahmed, mohammad z.; alqahtani, ali s.; alqahtani, mohammed s.; kumar, vijay title: targeting hub genes and pathways of innate immune response in covid- : a network biology perspective date: - - journal: int j biol macromol doi: . /j.ijbiomac. . . sha: doc_id: cord_uid: bli qm w the current pandemic of novel coronavirus disease (covid- ) caused by a novel virus strain, -ncov/sars-cov- have posed a serious threat to global public health and economy. it is largely unknown how the human immune system responds to this infection. a better understanding of the immune response to sars-cov- will be important to develop therapeutics against covid- . here, we have used transcriptomic profile of human alveolar adenocarcinoma cells (a ) infected with sars-cov- and employed a network biology approach to generate human-virus interactome. network topological analysis discovers sars-cov- targets, which belongs to a subset of interferon (ifn) stimulated genes (isgs). these isgs (ifit , ifitm , irf , isg , mx , and oas ) can be considered as potential candidates for drug targets in the treatments of covid- . we have identified significant interaction between isgs and tlr agonists, like poly i: c, and imiquimod, and suggests that tlr agonists can be considered as a potential drug for drug repurposing in covid- . our network centric analysis suggests that moderating the innate immune response is a valuable approach to target covid- . the current pandemic of coronavirus disease- (covid- ) has led to over million confirmed cases and almost . lakhs fatalities worldwide, since its emergence in late . covid- is caused by a novel virus coronavirus strain, sars-cov- , an enveloped, positive-sense, single-stranded rna, β-coronavirus of the family coronaviridae [ ] . nearly % of covid- patients show mild symptoms, such as cough and fever, and do not require hospitalization, but the remaining % do require [ ] . however, of those %, almost half of them develop severe respiratory failure in the form of fatal acute respiratory distress syndrome (ards) [ ] . along with this fatal form, dysregulated immune response also mediated the severe covid- pathogenesis. the immune dysregulation, called hypercytokinemia or ''cytokine storm,'' is frequently associated with ards [ ] . till date, it remains unclear how sars-cov- disrupt the host innate immune response. several recent studies have reported the dysregulated secretion of proinflammatory cytokines in covid- [ ] [ ] [ ] . recently, melo et al. [ ] reported a moderate interferon (ifn) response to sars-cov- infection in primary cells and showed that ifn can reduce sars-cov- replication in vitro. zhou et al. [ ] and xiong et al. [ ] recently examined the innate immune response to sars-cov- infection in bronchoalveolar lavage fluid (balf). the differentially expressed genes were enriched in inflammatory pathways including chemokine signaling [ ] . whereas, in studies by xiong et al. [ ] , the upregulated genes were largely related to viral infection j o u r n a l p r e -p r o o f with the most enriched biological processes being protein targeting to membrane and er. these studies reported the significant upregulation of a subset of interferon-stimulated genes (isgs) which are directly related to antiviral activity, such as isg , ifih , mx , oas - , ifitms etc. these studies along with others revealed prominent role of innate immune response to covid- infection [ ] [ ] [ ] [ ] . the two important themes are that consistently results in the upregulation of chemokines, and increased levels of proinflammatory cytokines such as il- β, il- , and il- , thus leads to tissue damage. the second is that sars-cov- triggered a robust ifn response, marked by the upregulation of several isgs in the lungs. like other viruses, novel sars-cov- also utilizes host machinery for their growth and survival during infection. systematic investigation of virus-host protein-protein interactions (ppis) offers an effective way toward elucidating the mechanisms of viral infection and drug repurposing [ ] . it has also been reported that sars-cov- can establish a higher rate of infectivity if it acquires combinatorial mutations at the s-protein/ace interfacial residues [ ] . several studies have shown that targeting cellular antiviral targets could be considered as a novel strategy for the development of effective treatments for viral infections, including sars-cov [ ] , mers-cov [ ] , ebola virus [ ] , zika virus [ ] , and more recently in sars-cov- [ ] . there are no vaccines or drugs approved for the novel covid- infection yet, but more than clinical trials have been launched to test coronavirus treatments. the current covid- pandemic leads the scientific community to focus seriously on drug repurposing to tackle the covid- infection [ ] [ ] [ ] [ ] [ ] . towards this goal, in this study, we have generated a human-sars-cov- interactome based on recently published rna-seq analysis of human adenocarcinomic alveolar basal epithelial (a ) cells infected with sars-cov- , and identified disease-related functional genes that will provide the insights into the patho-j o u r n a l p r e -p r o o f mechanisms of covid- . moreover, drug-protein interactions of these hub proteins, investigated in this study will leads to the identification of potential candidate drugs for repurposing. we have downloaded the differential gene expression data of human alveolar adenocarcinoma cells infected with covid- from melo et.al study [ ] . we have also downloaded all the possible drug-gene interactions data from drug-bank database and comparative toxicogenomic database (ctd), along with drug-disease interactions as well [ , ] . melo et.al [ ] previously described differentially expressed genes (degs) based on pvalue cut-off (p-val < . ). identification of significantly dysregulated genes on the basis of p-value is not quite precise. the amount of change in the expression of genes (log fold change) should also be considered. we reanalysed the data with q-value cut-off (fdr < . ), as well as applied log fold change cut-off (log fold ≥ ) for identifying significantly dysregulated genes from the data of genes. for comprehensively analysing the biological function of degs, we used database for annotation, visualization and integrated discovery (david) version . [ ] . this database uses the go and the kyoto encyclopedia of genes and genomes (kegg) analysis for analyzing the degs. the gene ontology analysis included the annotation at biological level, j o u r n a l p r e -p r o o f cellular level and molecular level. the pathways and functions with fdr < . were considered significant. using the degs list of melo et.al. [ ] , protein-protein interaction network was constructed using string plugin of cytoscape tool [ ] . string plugin uses co-expression, textmining, gene fusion, neighbourhood, and experimental data for preparing ppi network. cluego tool was used for identifying the biological roles of degs and david tool was used for gene ontology (go) analysis. in this ppi network, each node indicates a protein and an edge indicates an interaction between proteins. we have also calculated the network`s topological parameters such as degree centrality (k), betweenness centrality ( )), closeness connectivity and eccentricity using network analyzer plugin of the cytoscape tool. hub genes are the highly connected genes having high correlation with other genes in the network. any changes in the expression of hub genes have the potential to influence the major part of the network. genes having high connectivity can transfer information rapidly in the network as compare to other genes. top genes which have the higher degree of connectivity (k) and betweenness centrality value were considered as hub genes. degree centrality (k) is value assign to each node purely based on number of links held by it. degree indicates the number of interactions held by a node with other nodes in the network and helps in measuring the node significance in controlling the network. degree centrality (k) = ∑ ( , ) …. equation where, is the node set containing all the neighbours of node u, and w(u,v) is the edge weight connecting node u with node v. betweenness centrality ( ) measures how many times a node falls on the shortest path with other neighbouring nodes. it characterizes a nodes ability to control the signal processing and information flow in the network. where p(k,u,f) is the number of interactions from k to f that passes through u, and p(k,f) represents total number of shortest interactions between node k and f. biological process enrichment analysis and go analysis of the hub genes were done using cluego and david tool respectively. drug-target interaction information for the hub genes was collected from the drugbank database [ ] , and comparative toxicogenomics database (ctd) [ ] . the predicted drugs for hub genes through the gene-drug interaction databases were used for constructing drugprotein network using stitch database [ ] . stitch is a database known to predict functional and physical interaction between chemicals/drugs and genes. the interactions in stitch database is derived from five main sources, mainly by automated text-mining, highthroughput lab experiments, co-expression interaction data, interaction prediction by genomic context and by previous knowledges from databases. for each interaction between drug and gene, a combined score was calculated by stitch database. the combined score was calculated by combining the probabilities of interaction from different evidence channels and corrected probability of randomly observing an interaction. the drug-gene interactions having network score more then . was consider significant and positively hit. it is likely that the outcome of sars-cov- infection can largely be determined by the interaction between the host proteins and virus proteins. to build the human-sars-cov- interactome, we have used the transcriptome data of human alveolar adenocarcinoma (a ) cells infected with covid- from melo et al [ ] . melo et al. identified dysregulated genes with p-adj < . , in which majority of genes were getting upregulated. gene ontology (go) analysis of dysregulated genes using david [ ] revealed that the dysregulated genes were enriched in regulation of defence response to virus, regulation of protein export from nucleus, response to interferons, and regulation of response to cytokine stimulus (fig. a) . kegg pathway enrichment analysis reveals the role of dysregulated genes in chemokine signalling pathways, complement and coagulation pathways and rig-i-like receptor signalling pathways (fig. b) . we reanalysed the transcriptomic gene expression signature based on log fold change > and adj-p value < . . we have identified significantly dysregulated genes and of these genes, ifit , ifitm , irf , isg , mx , and oas were highly upregulated. the genemania webserver was used to predict interactions between these degs genes in the network using the go term "biological process" and source organism homo sapiens as additional parameters. the gene enrichment of theses degs highlighted go terms including response to virus, ribonucleotide binding and ifn-related signalling pathway (fig. c) . overall, the analysis demonstrated that the upregulated genes are mainly linked to the host response to sars-cov- infection, type i interferon signaling and the cytokine-mediated signaling pathway. j o u r n a l p r e -p r o o f we used the ppi network analysis to construct the interactome of the degs for covid- . for this, we used the string plugin of cytoscape tool [ ] , and obtained a ppi network with nodes and edges from the dysregulated genes (fig. a) . the proteins are ranked based on their degree connectivity and betweenness centrality scores (supporting information table ). among these, stat , irf , ifih , mx , isg , ifit , oas , and ddx has high degree and betweenness centrality values. to obtain a more in-depth understanding of the interactome, go function and kegg pathway analysis were applied using david (fig. b and supporting information table ). go analysis results showed that in the biological process, the interacting genes were mainly enrichment in the regulation of defence response to virus, innate immune response, inflammatory response, and also played an important role in complement activation, and response to nutrient and extracellular stimulus (fig. b) . the cellular components are significantly located in the extracellular region and membrane fraction, etc. molecular functions were mainly enriched in chemokines or cytokine activity, rna binding, transferase activity, and calcium ion binding. kegg pathway enrichment analysis revealed the role of interacting genes in complement and coagulation pathways, rig- -like receptor signalling pathways, and chemokine signalling pathways (supporting information table ). overall, the network analysis indicates that sars-cov- targets the proteins in the ifn signaling pathway to evade the immune system. this highlights the key role of the ifnmediated antiviral responses. predict viral targets [ ] [ ] [ ] [ ] . in this regard, two topological features, degree (number of connections) and betweenness (the fraction of all shortest paths that include a node within a network), were calculated to identify candidate hub nodes using the cytoscape's network analyzer tool. collectively, we have identified top nodes with high degree of connectivity and betweenness value and was subsequently considered as hub genes in the network ( table ) . biological process enrichment analysis of hub genes using genemania webserver and go analysis revealed their role in defence mechanism to viral response, cellular response to type i interferon, regulation of viral genome replication, double-stranded rna binding, type i interferon signaling pathway, and regulation of innate immune response ( fig. and these identified hub genes belong to the family of interferon stimulating genes (isgs). isgs including ifit and ifitm, isg , ifih , mx , irf , oas - and stat are known to potentiate ifn signaling and thus exert antiviral activity. these isgs fall into two categories, one that is direct effectors of the innate immune response including: mx , ifitm , isg , ifih , and irf- , and the other includes the induction of viral rna sensors such as ddx and the oas - genes. in addition to antiviral activity, isgs may exert diverse functions including rna and nucleotide binding, ifn regulation and inflammation regulation. mx , oas - , ifih , ifitm - , isg etc. are highly expressed in respiratory airways [ , ] and are associated with viral entry-associated ace gene, as shown in single-cell rnasequencing data from various tissues from human [ , ] . given the high expression of the viral entry-associated genes, it is reasonable that these nasal epithelial cells are trained to express these immune-associated genes to decrease viral susceptibility. further, we used enrichr [ ] , a web-based server for gene-set enrichment analysis and provides different summaries of collective functions of gene lists. interestingly, disease enrichment analysis demonstrated that the signature was highly associated with other viral diseases like west nile encephalitis, tick-borne encephalitis, dengue fever, chikungunya, and sars. also, these hub genes are involved in the mammalian phenotype including increased susceptibility to viral infection induced morbidity/mortality, decreased interferon secretion, increased igg level, abnormal t cell activation, and decreased interleukin- secretion. overall, these results clearly showed that the signatures are largely involved in innate immune response following sars-cov- infection (supporting information table ). further using drugbank database [ ] and comparative toxicogenomics database (ctd) [ ] , we identified the possible drugs which are known to have possible interaction with the hub genes (supporting information table ). in total, we have identified drug molecules which are known to interact with the hub genes (supporting information fig. ) . apart from protein-drug interaction, we also looked for drug-disease interaction, i.e. which drug is used in which disease. based on the drug-gene interaction results, we used stitch database [ ] for final categorization of drug-gene interaction network based on interaction score which is ≥ . . interestingly, we found five proteins mx , oas , stat , ddx , and isg were highly correlated to rare disease phenotype as well as to sars in enrichr database. these five proteins were also the most common influential proteins according to go analysis. the stitch drug-protein interaction network analysis of these five proteins has shown j o u r n a l p r e -p r o o f significant interaction with drugs/compound (fig. ) . as shown in figure , mx gene interacts with mitomycin-c, an antitumor and imiquimod, an immune response modifier. ifih interacts with polyinosinic:polycytidylic acid (poly i:c), which is an immunostimulant. oas showed significant interaction with s-carbamidomethylcysteine (cysteine-s-acetamide) and with mgatp. stat interacts with vanadium oxide and mgatp whereas, ddx and isg interact with mgatp. some of these identified drugs are used for induction of ifns and thus play a key role in the body defence against sars-cov- s infection. here, we generated a comprehensive human-sars-cov- interactome from transcriptome studies of a lung cell infected with covid- . the ppi network analysis indicates that the pathways are enriched in host response to virus infection, type i interferons signaling, and cytokine activation. network topology analyses identified high-value targets of sars cov- , which belongs to a subset of canonical isgs. these isgs are largely involved in regulation of defence response to virus, innate immune response, inflammatory response, and rna binding [ , ] . an interferon-inducible protein, mx dynamin like gtpase (mx ) is associated with influenza and viral encephalitis infection [ , ] . also notable is the role of the interferon induced protein with tetratricopeptide repeats (ifit ) and dexd/h-box helicase (ddx ) as an antiviral activity. in hepatitis e virus infection, polymerase binds to ifit protecting the viral rna from translation inhibition mediated by ifit that boosts the interferon response in murine macrophage-like cells [ ] . recently, the significant upregulation of ifit - , and ddx gene expression under covid- viral infection has been reported [ ] . in response to viral infections, several genes of host such as oas - , j o u r n a l p r e -p r o o f irf , irf , stat and ifih are highly expressed and are highly correlated with host response to viral infections [ ] [ ] [ ] . studies shows that covs are equipped with strategies to antagonize the ifn signaling pathway that facilitates the virus to escape host immune response. sars-cov escapes the host ifn signalling as its orf protein blocks the expression of stat -activated genes [ ] . similarly, in mers-cov orf b inhibits irf and irf to antagonize the antiviral ifn-β response [ ] . additionally, papain-like proteases (plps) are expressed in both sars-cov and mers-cov that enables to delay the host immune response. coronaviruses engages in interactions with ifn stimulated gene (isg ) and antagonizing the ifn-mediated antiviral response [ , ] . the antiviral activity of isg has been shown in several viruses including human cytomegalovirus [ ] , hiv [ ] , west nile virus [ ] , swine fever virus [ ] , mers-cov [ ] , and sars-cov [ ] . ifns play a key role in the body defence against viral infections, and in this regard, we here showed some candidate drugs for repurposing. one of these drugs is poly (i:c) (polyinosinic:polycytidylic acid), a synthetic double-stranded rna immune-stimulant, which is used as adjuvant in vaccine production [ ] . it is agonist for toll like receptor (tlr ) which induces the expression of ifns. many studies demonstrated that the tlr agonists, poly iclc and poly (i:c) increases the production of ifn-α, -β, and -γ, which inhibited cov replication and minimized the inhibitory effects of cov on ifn signaling pathways [ ] [ ] [ ] . interestingly, chloroquine, a recently proposed drugs for covid- [ ] , inhibits poly (i:c)mediated ifn-β induction [ ] . therefore, tlr agonists can be a considered as potential drugs for repurposing in covid- . in addition to tlr- agonists tlr agonists such as imiquimod [ ] , can induce ifn production in the human body as well. imiquimod is a strong inducer of ifn-α and several proinflammatory mediators, including tnf-α, il- , and chemokines [ ] . the other drug predicted to bind mx and oas, mitomycin c is a cancer drug that is used in the treatment of bladder, colon, and breast cancers. it functions as an alkylating agent that causes cross-linking of dna and inhibits rna as well as protein synthesis [ , ] . at high concentration, mitomycin c inhibits the replication of influenza virus by blocking the rna synthesis [ ] . mitomycin c has also been shown to inhibit b cell, t cell, and macrophage proliferation in vitro and impair antigen presentation, as well as the secretion of ifn-γ, tnfα, and il- . vanadium oxide, an activator of stat- , involved in immune-regulating mechanisms, including immune suppression and inflammation downregulation by stimulating and activating b/t cells. [ , ] in summary, our integrative interactome and network topology analyses showed that sars-cov- induced a strong ifn response, marked by the increased expression of several isgs, including mx , oas - , ifih , isg , irfs, and ifitms etc. these isgs exert antiviral activity and could protect the host cells from the infection. this protective action of isgs might account for the lesser percentage of severe cases and the lower fatality rate in covid- . however, the extent of protection or damage to the host cell depends on stage of infection, types of cells, sars clade [ ] [ ] [ ] and other factors like co-infection, age, and comorbidities. recently, zou et al. [ ] reported high sars-cov- loads very early during infection, suggesting that the virus may have developed arsenals that is able to delay the ifn response by inhibiting innate immune signaling. thus, ifn induction in the incubation period and at the very early stages of the infection could be the key to prevent covid- associated mortalities. administration of interferon-inducing agents, such as poly (i:c) and imiquimod could reduce the mortality of sars at the very early stages of the disease (fig. ) . on the other hand, at the later stages of the disease, the balance of the immune system becomes impaired, leading to inflammatory over-reactions, cytokine storm, and possible autoimmune induce the production of ifns and isgs, which will reinstate the impaired immune responses. in the late stage of infection, there is a proinflammatory cytokine storm, which can be theoretically targeted by immunosuppressors, like mitomycin c, and vanadium oxide. a new coronavirus associated with human 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cd t cell differentiation through ifn-alpha/beta intranasal treatment with poly(i*c) protects aged mice from lethal respiratory virus infections prophylactic and therapeutic intranasal administration with an immunomodulator, hiltonol((r)) (poly ic:lc), in a lethal sars-cov-infected balb/c mouse model evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies toll-like receptor agonist poly(i:c)-induced antiviral response in human corneal epithelial cells the systemic response to topical aldara treatment is mediated through direct tlr stimulation as imiquimod enters the circulation cellular requirements for cytokine production in response to the immunomodulators imiquimod and s- mitomycin c: mechanism of action, usefulness and limitations mitomycin c inhibits ribosomal rna: a novel cytotoxic mechanism for bioreductive drugs influence of mitomycin c on the replication of influenza viruses role of vanadium in cellular and molecular immunology: association with immune-related inflammation and pharmacotoxicology mechanisms vanadium carcinogenic, immunotoxic and neurotoxic effects: a review of in vitro studies dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes sars-cov- viral load in upper respiratory specimens of infected patients authors sincerely thank to the department of science and technology, government of india.the authors extend their appreciation to the deanship of scientific research at king saud university for funding this work through research group no. rg- - . no potential conflict of interest was reported by the authors. key: cord- -o m kvw authors: sedeyn, koen; schepens, bert; saelens, xavier title: respiratory syncytial virus nonstructural proteins and : exceptional disrupters of innate immune responses date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: o m kvw human respiratory syncytial virus (rsv) is the most important cause of acute lower respiratory tract disease in infants worldwide. as a first line of defense against respiratory infections, innate immune responses, including the production of type i and iii interferons (ifns), play an important role. upon infection with rsv, multiple pattern recognition receptors (prrs) can recognize rsv-derived pathogen-associated molecular patterns (pamps) and mount innate immune responses. retinoic-acid-inducible gene-i (rig-i) and nucleotide-binding oligomerization domain-containing protein (nod ) have been identified as important innate receptors to mount type i ifns during rsv infection. however, type i ifn levels remain surprisingly low during rsv infection despite strong viral replication. the poor induction of type i ifns can be attributed to the cooperative activity of unique, nonstructural (ns) proteins of rsv, i.e., ns and ns . these viral proteins have been shown to suppress both the production and signaling of type i and iii ifns by counteracting a plethora of key host innate signaling proteins. moreover, increasing numbers of ifn-stimulated genes (isgs) are being identified as targets of the ns proteins in recent years, highlighting an underexplored protein family in the identification of ns target proteins. to understand the diverse effector functions of ns and ns , goswami and colleagues proposed the hypothesis of the ns degradasome (nsd) complex, a multiprotein complex made up of, at least, ns and ns . furthermore, the crystal structure of ns was resolved recently and, remarkably, identified ns as a structural paralogue of the rsv matrix protein. unfortunately, no structural data on ns have been published so far. in this review, we briefly describe the prrs that mount innate immune responses upon rsv infection and provide an overview of the various effector functions of ns and ns . furthermore, we discuss the ubiquitination effector functions of ns and ns , which are in line with the hypothesis that the nsd shares features with the canonical s proteasome. introduction human respiratory syncytial virus (rsv) is a negative strand rna virus belonging to the family pneumoviridae. rsv is a major cause of acute lower respiratory tract infections in the pediatric population and is increasingly recognized as an important cause of severe respiratory disease in the elderly [ ] [ ] [ ] . despite the major clinical impact of rsv on human health worldwide, no approved vaccine or effective antiviral therapy is available. although rsv infections do evoke humoral and cellular immune responses required to clear infection, these responses do not provide strong protection against a subsequent infection with rsv. early during infection, viral replication is sensed by the host's innate immune system, which leads to the production of type i and iii interferons (ifns), which is followed by the induction of an array of genes that code for antiviral proteins. in addition, ifn will recruit and activate innate leukocytes, including antiviral monocytes and natural killer (nk) cells, and stimulate the adaptive immune response [ ] . rsv has evolved a marvelous set of mechanisms to subvert this canonical antiviral response of the mammalian host, most notably by virtue of its nonstructural (ns) and ns proteins. the innate immune system is an important early line of defense against pathogens. this system comprises pattern recognition receptors (prrs) that can recognize pathogen-associated molecular patterns (pamps). activated prrs can induce the expression of cytokines, e.g., type i and iii ifns, which mount an antiviral state in an autocrine and paracrine fashion. upon rsv infection, airway epithelial cells, macrophages, and dendritic cells (dcs) are the main inducers of innate immune responses. several toll-like receptors (tlrs) have been identified that can act as prrs for rsv-derived pamps (fig ) . tlr , - , and - , for example, have been implicated in the induction of cytokines and chemokines upon rsv infection [ , ] . a role for tlr , which is well known to respond to lipopolysaccharide, as a prr for rsv is currently debated. although some groups reported an impaired innate immune response in tlr -deficient mice [ - ], ehl and colleagues could not confirm a role for a tlr -mediated immune response upon rsv infection [ ] . in addition, tlr might even play a role in tempering immune responses upon rsv infection [ ] . moreover, type i ifn production by macrophages and dcs from wild-type and tlr , - , - , - , and - knockout mice is very similar following rsv infection [ , ] . finally, alveolar macrophages are the primary source of type i ifn in rsv-infected mice, and tlrs do not play a crucial role in this response [ , ] . in contrast, retinoic-acid-inducible gene-i (rig-i)-like receptors (rlrs) are important for the induction of type i and possibly type iii ifns upon recognition of rsv (fig ) . type i ifn expression is strongly hampered in the absence of mitochondrial antiviral-signaling protein (mavs), the adaptor protein for rig-i and melanoma differentiation-associated protein (mda ) [ , ] . early on, gene expression ablation studies revealed that rig-i is the most important rlr to detect rsv, which is supported by the observation that rsv mrna could be coimmunoprecipitated with rig-i but not with mda [ - ]. later, however, both rig-i and mda were found to colocalize with rsv genomic rna and the n protein [ ] . interestingly, whereas rig-i partially localizes to rsv-induced inclusion bodies, mda and mavs were found nearly exclusively in these inclusion bodies, thereby dramatically blunting ifn-β induction. a third class of prrs, the nucleotide-binding oligomerization domain-like receptors (nlrs), is also important for the recognition of rsv. nucleotide-binding oligomerization domain-containing protein (nod ) can be activated by intact genomic single-stranded rna (ssrna) and is involved in the induction of ifn-β in mice in a mavs-dependent way (fig ) [ ]. cytoplasmic dna sensors (cdss), such as z-dna binding protein (zbp ) and cyclic gmp-amp synthase (cgas), are well known to induce innate immune responses upon recognition of pathogen-derived double-stranded dna (dsdna) or even rna [ ] [ ] [ ] . a contribution of cdss in innate sensing of rsv replication has not yet been reported. in contrast to other respiratory viruses, i.e., influenza virus and human respirovirus (formerly named human parainfluenza virus ), nasal washes from rsv-infected infants hardly firstly, tlr- , - , - , - , and - (marked in green) are involved in the production of cytokines and chemokines upon rsv infection. secondly, rig-i and possibly mda (rlrs, marked in red), are important in the induction of type i ifns. thirdly, nod (marked in dark blue) also induces type i ifns upon rsv infection. currently, there is no evidence for a role of the cdss (marked in purple), which signal through the er-associated sting protein, as prrs during rsv infection. activation of the rlrs or nod induces their association with the mitochondrial-associated mavs, which recruits the adaptor proteins traf or traf . via the traf adaptor, the kinases ikkε and tbk are subsequently activated, which phosphorylate and activate the transcription factors irf and irf . via the traf adaptor, kinases are activated, i.e., the ikk kinase complex, jnk, and p mapk, which phosphorylate and activate multiple transcription factors such as nf-κb, c-jun, and atf , respectively. activation of tlrs leads to the recruitment of adaptor proteins, e.g., myd , ticam , tirap, and tram. these adaptors can signal via traf or traf . the transcription factors activated by prr signaling ultimately induce expression of cytokines, chemokines, and ifns. above each prr, the confirmed or likely rsv-derived pamp is depicted. atf , activating transcription factor ; cds, cytoplasmic dna sensor; er, endoplasmic reticulum; ifn, interferon; ikk, inhibitor of nuclear factor kappa-b kinase; ikkε, inhibitor of nuclear factor kappa-b kinase subunit epsilon; irf , interferon regulatory factor ; irf , interferon regulatory factor ; jnk, c-jun n-terminal kinase; mapk, mitogen-activated protein kinase; mavs, mitochondrial antiviral-signaling protein; mda , melanoma differentiation-associated protein ; myd , myeloid differentiation primary response protein myd ; nf-kb, nuclear factor-kappa b; nod , nucleotide-binding oligomerization domain-containing protein ; pamp, pathogenassociated molecular pattern; prr, pattern recognition receptor; rig, retinoic-acid-inducible gene-i; rlr, rig-i-like receptor; rsv, respiratory syncytial virus; sting, stimulator of interferon protein; tbk , tank binding kinase ; ticam , toll/interleukin- receptor domain-containing adapter molecule ; tirap, toll/ interleukin- receptor domain-containing adapter protein; tlr, toll-like receptor; traf , tumor necrosis factor receptor-associated factor ; traf , tumor necrosis factor receptor-associated factor ; tram, toll-like receptor adaptor molecule. https://doi.org/ . /journal.ppat. .g contain ifn-α and -β [ - ]. apparently, this virus has evolved ways to outsmart the canonical mammalian antiviral response. two unique viral proteins, ns and ns , are responsible for the suppression of ifn induction and signaling. human and bovine rsv strains that lack ns and/or ns have been explored as live-attenuated vaccine candidates. such viruses are strongly attenuated in in vivo rsv infection models (cotton rats, calves, and chimpanzees) as well as in human but, at least in calves and chimpanzees, retain their ability to induce antibody responses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . positioned proximal on the negativestranded rna genome, ns and ns are the most abundantly transcribed viral genes. recently, the crystal structure of ns was determined, revealing that this protein is composed of a β-sandwich flanked by α-helices [ ] . interestingly, the d structure of ns is very similar to the n-terminal domain of rsv m despite the complete absence of any primary sequence homology. both ns and ns strongly reduce the induction of type i and iii ifns upon rsv infection [ , [ ] [ ] [ ] [ ] [ ] . infection with recombinant viruses lacking ns or ns , separately or combined, suggests that these proteins function individually and cooperatively to suppress ifn induction. they do so by targeting multiple proteins of the signaling cascade that starts with the recognition of pamps by prrs and ends with the induction of ifn gene expression by several transcription factors. a widespread strategy of viruses to dampen innate immune responses is to counteract one of the early signaling steps in rlr-mediated ifn induction, i.e., the interaction of rig-i or mda with mavs [ ] [ ] [ ] [ ] . likewise, rsv prevents the interaction of rig-i with its adaptor mavs. in rsv-infected hep- cells and a cells overexpressing ns , ns was shown to interact with mavs (fig ) [ ] . by binding to mavs, ns could dose-dependently prevent the interaction between rig-i and mavs in a cells. furthermore, ban and colleagues demonstrated that ectopically expressed ns in hek t cells interacts with the pry-spry domain of e ubiquitin/ifn-stimulated gene (isg ) ligase tripartite motif-containing protein (trim ). this domain is responsible for the interaction of trim with rig-i [ ] . ns binding to the pry-spry domain suppresses k -linked polyubiquitination of rig-i by trim , which is essential for its downstream interaction with mavs (fig ) . proteins of other respiratory viruses also target the pry-spry domain of trim , which highlights the evolutionary importance of trim in mammalian antiviral defense. by binding the pry-spry domain of trim , the ns protein of influenza a virus and the nucleocapsid protein of severe acute respiratory syndrome virus also counteract trim -mediated rig-i ubiquitination [ , ] . ns can interact with the n-terminal domain of rig-i, both upon overexpression in hek t cells and during rsv infection in a cells (fig ) [ ] . as such, overexpressed ns disrupts the binding of rig-i with mavs; however, this has not yet been confirmed for endogenous ns expressed during an rsv infection. in addition to counteracting the interaction between rig-i and mavs, ns may also influence rig-i expression, although the reported findings seem conflicting. rig-i expression in a cells is strongly reduced in the presence of rsv ns , either expressed separately or in the context of an rsv infection [ ] . boyapalle and colleagues, however, reported that rig-i expression in a cells is reduced following infection with an ns -deficient rsv [ ] . this is surprising, because rig-i is itself an isg. possibly, this discrepancy is caused by the different multiplicity of infection (moi) used by these groups (moi and , respectively). in contrast to rig-i, mavs appears to be resistant to ns -and/or ns -mediated down-regulation [ ] . all together, these results highlight that the interaction of rig-i with mavs is suppressed by both ns and ns and that rig-i expression itself might be reduced by ns . currently, it is not clear whether ns and/or ns can disturb the interaction of mda or nod with mavs and whether they impact mda or nod expression levels. type i and iii ifn responses are inhibited by ns and ns at multiple levels, both during the induction of type i and iii ifns (left panel) and during ifn-induced signaling (right panel). ns and ns can form a so-called "ns degradasome" complex that is stabilized by mitochondria via mavs. the nsd complex is thought to contain hps, including the proteasome α subunit and other as yet unidentified proteins. ns and ns prevent the interaction of rig-i with mavs in different ways. ns binds to the pry-spry domain of trim , which is responsible for the interaction of trim with rig-i. as such, ns prevents the trim -mediated k -linked polyubiquitination of rig-i, which is necessary for the subsequent interaction of rig-i with mavs ( ). moreover, ns directly interacts with rig-i ( ) and ns interacts with mavs ( ) to suppress binding of rig-i to mavs. whether the interaction of ns with mavs also prevents the interaction of nod with mavs is currently unclear. ns reduces protein expression of traf and ikkε ( ), whereas ns modestly reduces traf and induces ikkε and tbk ( ). ns subsequently inhibits irf and irf by different proposed mechanisms ( ). ns reduces irf and irf protein expression and prevents the interaction between irf and cbp, thereby lowering the binding of the irf -cbp complex to the ifn-β promoter. ns , and to a lesser extent ns , enhances the activation and nuclear translocation of nf-κb ( ). these ns /ns effector functions ( - ) synergistically reduce the production of type i and iii ifns. furthermore, ns and ns also suppress type i and iii ifn receptor-mediated signal transduction. ns induces mir- a expression, which targets the mrna coding for ifnar , one of the subunits of the type i ifn receptor ( ). ns and ns may induce expression of socs proteins (socs and ), which negatively regulate the tyrosine kinases jak and tyk , which are important to transmit signaling from the type i and iii ifn receptors ( ). ns inhibits jak / tyk -mediated activation of stat / by reducing stat protein levels ( ) and by reducing stat phosphorylation ( ). some groups, however, reported that stat expression can also be reduced by ns (see text) ( ). ns and ns counteract the anti-inflammatory activity of the gr, although the exact mechanism is debated. in one model, ns interacts with the nuclear translocator ipo , which competes with gr for its nuclear translocation ( ). recent evidence suggests that the ns proteins may also counteract antiviral effector functions of isgs, e.g., ns degrades the oasl, ifit , and ifitm proteins, whereas ns degrades mapk ( ). full and dashed lines indicate robust and moderate inhibitory or stimulatory effector functions, respectively. cbp, creb binding protein; gr, glucocorticoid receptor; hp, host protein; ifit, interferon-induced protein with tetratricopeptide repeats; ifitm, interferon-induced transmembrane protein; ifn, interferon; ifnar, interferon alpha/beta receptor; ikkε, inhibitor of nuclear factor-kappa b kinase subunit epsilon; ipo , importin- ; irf , interferon regulatory factor ; isg, ifn-stimulated gene; jak, janus kinase; mapk , mitogen-activated protein kinase ; mavs, mitochondrial antiviral-signaling protein; ns, nonstructural; oasl, - -oligoadenylate synthase-like protein; rig-i, retinoic-acid-inducible gene-i; rlr, rig-i-like receptor; rsv, respiratory syncytial virus; socs, suppressor of cytokine signaling; stat, signal transducer and activator of transcription; tbk , tank binding kinase ; trim , tripartite motif-containing protein; tyk, tyrosine kinase . https://doi.org/ . /journal.ppat. .g association of rlrs with mavs leads to the recruitment of the adaptors tumor necrosis factor receptor-associated factor (traf ) and - (traf ). whereas traf activates the downstream serine/threonine kinases inhibitor of nuclear factor-kappa b kinase subunit epsilon (ikkε) and tank binding kinase (tbk ), traf activates the downstream kinases ikk, c-jun n-terminal kinase (jnk), and p mitogen-activated protein kinase (p mapk) (fig ) . the effect of ns and ns on traf , ikkε, and tbk is currently inconclusive. some groups reported that ns , either overexpressed or expressed during an rsv infection in a cells, reduces the expression levels of both traf and ikkε (fig ) [ , ] . ren and colleagues, however, observed no difference in endogenous and recombinant traf and ikkε expression in a cells in the presence or absence of ns [ ] . possibly, the different strains (rsv long versus a ) or experimental timing used explain these opposing observations. overexpression of ns in a cells modestly reduces and slightly enhances traf and ikkε expression levels, respectively (fig ) [ , ] . these effects of ns were, however, not observed with an rsv strain that lacks ns . ling and colleagues also observed that overexpressed ns enhances overexpression of ikkε and tbk in hek t cells (fig ) [ ] . to our knowledge, no data have been published on the possible effect of ns on the expression of tbk . coexpression of ns and ns in a cells reduces recombinant ikkε expression, suggesting that the inhibitory effect of ns is dominant over the enhancing effect of ns [ ] . these results suggest that ns suppresses traf and ikkε expression, although more evidence is needed to confirm this hypothesis. ectopic expression of ns slightly suppresses traf expression and enhances ikkε and tbk expression, although these effects need to be confirmed during an rsv infection. whether ns and/or ns affect the traf adaptor has not been investigated yet. some evidence indicates that rsv may indeed affect traf expression. in different models, traf expression has been shown to be down-regulated by the microrna (mirna) mir- a [ , ] . interestingly, eilam-frenkel and colleagues found that mir- a expression was up-regulated in rsv-infected hep- cells [ ] . future research may elucidate whether rsv can downregulate traf expression via up-regulating mir- a and whether this is mediated by ns and/or ns . activated ikkε and tbk phosphorylate the transcription factors interferon regulatory factor (irf)- and irf , which induces conformational changes that allow the formation of homo-and heterodimers of irf and - that translocate to the nucleus. phosphorylation of inhibitor of kappa b (iκb), e.g., by the canonical ikkα/β/γ complex, initiates its degradation with subsequent release and nuclear translocation of nuclear factor-kappa b (nf-κb). the irf and nf-κb transcription factors are essential for the induction of type i and iii ifns (fig ) . several observations highlight that ns and ns can impair the activation and effector functions of irf transcription factors. initial work with recombinant rsv strains lacking one or both ns proteins in a cells highlighted that ns and ns prevent nuclear translocation of irf , especially late (> hours post infection) in the rsv replication cycle [ , , ] . this might, in part, be explained by the ability of ns to directly reduce the recombinant expression of irf and irf (fig ) [ ] . in addition, by preventing the formation of the irf /creb binding protein (cbp) complex in the nucleus, ns may also inhibit irf -dependent gene expression downstream of the nuclear translocation of irf (fig ) [ , ] . it is important to note that this ns effector function has only been demonstrated upon overexpression in hek t cells, so additional confirmation in an rsv infection is necessary. in a and vero cells, nf-κb activation and nuclear translocation occur early after rsv infection and are clearly enhanced by ns and, to some extent, by ns (fig ) [ , ] . in addition to the canonical iκb phosphorylation and subsequent degradation, rsv-induced nf-κb activation involves p ser phosphorylation via the rig-i/mavs/traf /ikkβ signaling pathway [ ] . although nf-κb contributes to the induction of type i and iii ifns, nf-κb also strongly induces the expression of anti-apoptotic genes. in the context of an rsv infection, the induction of an anti-apoptotic cellular environment by nf-κb likely dominates the contribution of nf-κb over the induction of ifn, which is primarily controlled by irf / activation. taken together, ns and ns suppress the induction of type i and iii ifns by targeting multiple proteins of the signaling cascade that leads to type i or iii gene activation, i.e., rig-i, mavs, traf , ikkε, tbk , irf , and irf . although some of these effector functions have been confirmed in rsv-infected cells, others have so far only been identified with overexpression of ns or ns alone. so, future research is needed to elucidate the biological relevance of these recombinant responses, particularly because ns can relocate ns to the mitochondria [ ] . binding of type i and iii ifns to their heterodimeric receptor complex induces a janus kinase (jak)-signal transducer and activator of transcription (stat) signaling cascade that induces an antiviral state by expression of isgs. compared with wild-type rsv, rsv strains that lack ns and/or ns are more sensitive to type i ifn treatment [ , ] . thus, rsv ns proteins also have effector functions downstream of the induction of ifn synthesis to suppress ifninduced antiviral responses. in rsv-infected a cells, ns induces the expression of the mirna mir- a, which targets the mrna coding for interferon alpha/beta receptor (ifnar ) by an unknown mechanism (fig ) [ ] . as a result, the ifnar protein levels are reduced, which decreases responsiveness to type i ifns of rsv-infected cells and thus favors rsv replication. the induction of an antiviral state by type i and type iii ifns requires the phosphorylation of stat and stat proteins in the proximity of the type i and iii receptors. two tyrosine kinases, jak and tyrosine kinase (tyk ), are responsible for the phosphorylation and activation of stat proteins after activation of the type i and iii ifn receptor ( fig ) . for now, there is no evidence that ns or ns may alter the levels and activation status of these kinases. several groups reported that the phosphorylation and total protein levels of tyk are not altered by the expression of ns and/or ns [ ] [ ] [ ] . whether this also accounts for jak is currently not clear. infection of airway epithelial cell lines with human metapneumovirus, a respiratory virus closely related to rsv, does reduce both jak and tyk protein levels [ ] . additionally, jak kinase activity is regulated by a negative feedback loop that consists of the suppressor of cytokine signaling (socs) family, with socs and being the strongest inhibitors of jak kinases. it has been reported that ns and/or ns may regulate the expression of socs and/or socs , thereby enhancing rsv replication [ ] [ ] [ ] [ ] . heterodimers of phosphorylated stat and transcription factors are important for the induction of isgs. consequently, numerous viral proteins counteract stat and proteins by using different strategies, e.g., by preventing stat / heterodimer formation (nipah and hendra virus nucleoprotein), by suppressing stat / nuclear translocation (ebolavirus vp ), or by degrading stat and/or (paramyxovirus v protein) [ ] [ ] [ ] . both stat and are targets for rsv. in human tracheobronchial epithelial cells and a cells, infection with rsv slightly increases total stat protein levels, likely because stat proteins are themselves isg products [ - , , ] . stat phosphorylation induced by exogenous type i ifn, however, is clearly reduced by ns but not by ns (fig ) [ [ ] [ ] [ ] ] . whether ns also reduces stat phosphorylation during an rsv infection has not been reported yet. in contrast to stat , both phosphorylated and nonphosphorylated stat protein levels are clearly reduced by rsv infection [ , , ] . whether ns , ns , or both reduce stat levels is currently debated. based on overexpression experiments and infections with rsv strains lacking ns and/or ns , several groups reported that ns , but not ns , reduces stat levels in vitro (fig ) [ , , , ] . others, however, also observed reduced stat levels after overexpression of ns (fig ) [ , ] . lo and colleagues reported that recombinant coexpression of ns and ns reduces stat levels stronger than ns alone, although expression of ns alone did not affect stat levels [ ] . in addition to reducing total stat levels, rsv also seems to suppress nuclear translocation of the residual stat proteins, an effect that depends on ns and/or ns [ ] . so far, the possible impact of rsv ns proteins on stat and expression levels in vivo has not yet been demonstrated. because mouse stat appears resistant to ns , primary human airway epithelial cell cultures or experiments in nonhuman primates may be required to confirm that ns -mediated stat reduction also occurs in vivo [ ] . evidence is rising that the ns proteins also suppress the antiviral activity of at least some isg products. cells that express myxovirus resistance protein a (mxa) or cells pretreated with type i ifn only modestly limit rsv replication [ ] . moreover, ectopic expression of ns and ns in hek -derived cells has been shown to degrade certain isg products, i.e., - oligoadenylate synthetase-like protein (oasl), interferon-induced protein with tetratricopeptide repeats (ifit ), interferon-induced transmembrane protein (ifitm ), and mapk , in a selective manner (fig ) [ , ] . an important remark, however, is that these effector functions have not yet been validated during an rsv infection. as stated by ribaudo and barik, a comprehensive screen of all known isgs will likely unravel additional substrates of ns and/or ns [ ] . ns and ns affect the induction of apoptosis and cell shedding. ns and ns individually and cooperatively delay apoptosis in rsv-infected cells, with ns being stronger than ns in doing so [ ] . the early expression of ns and ns after infection activates the antiapoptotic -phophoinositide-dependent protein kinase (pdk)-rac serine/threonine-protein kinase (akt)-glycogen synthase kinase (gsk) pathway [ , , ] . later in infection (> hours), activation of this pathway drops, and the incidence of apoptosis increases [ ] . by delaying apoptosis, ns and ns may facilitate prolonged rsv replication with increased viral yields. a typical hallmark of severe disease following rsv infection is the obstruction of the smaller airways by plugs consisting of infiltrating immune cells, mucus, and shed infected epithelial cells. in primary human airway epithelial cells and in an in vivo hamster model, it has been shown by experiments with a set of elegant recombinant virus constructs that rsv ns is necessary and sufficient to induce shedding of infected epithelial cells [ ] . interestingly, cell death, associated with nuclear changes that are indicative of apoptosis, of infected epithelial cells only occurred after these cells were detached from the epithelial layer. moreover, shedding of infected epithelial cells coincided with reducing viral titers. all together, these results suggest that expression of ns early after infection delays apoptosis and induces changes in cell morphology that ultimately result in shedding of infected cells in the airway lumen. these shed and detached infected epithelial cells die. as shedding (and ultimately clearance by mucociliary transport) of infected epithelial cells reduces rsv viral titers, it seems that the bulk of viral spread precedes cell shedding. possibly, (early) changes in cell morphology that ultimately lead to shedding of infected epithelial cells may facilitate rsv production and spreading. as such, ns plays a pivotal role in the production and spreading of rsv virions. ns and ns counteract the anti-inflammatory activity of the glucocorticoid receptor. although rsv-induced bronchiolitis is characterized by a (severe) inflammatory response, the use of anti-inflammatory glucocorticoids has shown no clinical benefits against (severe) disease [ ] [ ] [ ] . based on experiments with different transformed and primary cell cultures as well as mice, several groups concluded that rsv can inhibit the anti-inflammatory activity of glucocorticoids via the glucocorticoid receptor (gr) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the exact mechanism that accounts for this inhibition, however, is debated. one group demonstrated that rsv blocks glucocorticoid-mediated gr activation in a , beas- b, and primary human small airway epithelial cells but not in the monocytic thp- cell line, suggesting that the effect is restricted to rsv-infected epithelial cells [ , ] . further mechanistic analysis in a cells suggested that ns and ns reduce the binding of the gr to gr-responsive promoters, without affecting gr total protein levels and nuclear translocation [ , , ] . interestingly, knockdown of mavs in a cells lowered dexamethasone-induced transforming growth factor β (tgf-β)-stimulated clone (tsc ) domain family member (also known as glucocorticoid-induced leucine zipper protein [gilz]) mrna expression to a level similar to that of an rsv infection [ ] . these results suggest that mavs plays a role in glucocorticoidinduced gr activation in a cells and that ns and ns may indirectly suppress gr activation through inhibition of mavs (see "ns and ns interfere with rig-i"). xia and colleagues used beas- b and primary differentiated human bronchial epithelial cells grown at an air-liquid interface to demonstrate that rsv suppresses glucocorticoidinduced gr activation by a mechanism that involves up-regulation of tgf-β expression [ ] . a model was proposed in which viral infection is sensed by tlr , which induces tgf-β that subsequently activates the type i tgf-β receptor activin receptor-like kinase (alk ). alk activation subsequently counteracts glucocorticoid activity through an unknown mechanism. in accordance with the aforementioned study, no difference in total grα protein levels or nuclear translocation were observed upon rsv infection. interestingly, blocking alk with the selective inhibitor sb and reducing tgf-β activity by tranilast (a compound used to manage a wide variety of diseases, including inflammatory diseases) could subvert the rsvmediated suppression of glucocorticoid activity. although xia and colleagues did not investigate the role of ns or ns , ns may contribute to rsv-induced tgf-β expression by up-regulating the transcription factor kruppel-like factor (discussed in the next paragraph) [ ] . another study concluded that rsv ns prevents gr nuclear translocation [ ] . this finding was based on experiments with a cells, mouse lung tissue, but also analysis of nasopharyngeal aspirates from rsv-infected infants. moreover, expression of grα and importin- , which is important for gr nuclear entry, was reduced at the mrna and protein level in mouse lungs, whereas grβ and ipo expression at the mrna level were reduced in the nasopharyngeal aspirates upon rsv infection. mechanistically, ns was found to directly interact with ipo and can thus compete with gr for ipo binding. the capacity of rsv to suppress the anti-inflammatory effect of glucocorticoids through ns and ns are in line with the ineffectiveness of glucocorticoid treatments off severely ill rsv patients. in addition to mir- a, ns proteins also regulate the expression of the mirnas mir- , let- i, and mir- b [ , , ] . in rsv-infected a cells, ns suppresses mir- expression by up-regulating the transcription factor kruppel-like factor , which drives the expression of tgf-β [ ] . remarkably, inhibition of mir- was shown to actually repress rsv replication in a cells [ ] . this apparent discrepancy could be explained by the difference in time points after infection that were analyzed ( day versus days post infection). possibly, ns suppresses the rsv-induced up-regulation of mir- within hours post infection, whereas later on, mir- expression may be up-regulated to enhance viral replication. investigating mir- expression levels beyond hours post infection and the impact of ns on these levels may result in a better understanding of the role of mir- in rsv replication. in normal human bronchial epithelial cells, let- i and mir- b expression is up-regulated during an rsv infection by a type i ifn and nf-κbdependent mechanism, respectively, and further increased in the absence of ns or ns [ ] . this can be expected for let- i, as the ns proteins suppress the type i ifn response. moreover, the mir- b promoter can be activated by the nf-κb family member p , which is activated during an rsv infection through a rig-i/mavs/traf /ikkβ signaling pathway [ , ] . by counteracting the interaction between rig-i and mavs (see "ns and ns interfere with rig-i"), the ns proteins may dampen this pathway, leading to reduced activation of p and subsequent expression of mir- b. to our knowledge, the effect of ns and/or ns on the rsv-induced activation of p has not been investigated yet. this could readily be tested by comparing total levels of activated p between cells infected with wild-type rsv and ns deletion strains. whether the ns protein-mediated suppression of let i and mir- b favors or counteracts rsv replication is currently unclear. ns and ns interfere with the adaptive immune response. the ns proteins also play a role in the development of adaptive immune responses during rsv infection, partly as a consequence of their capacity to suppress type i and iii ifn levels. in addition to airway epithelial cells, rsv can infect dcs and activate prrs by pamps [ ] . munir and colleagues investigated the activation status of isolated human monocyte-derived myeloid dcs upon rsv infection by quantifying several maturation markers, including cluster of differentiation (cd) , , , , and [ ] . infection with wild-type and ns-deficient rsv strains highlighted that ns , and to a lesser extent ns , suppress the maturation of human dcs. by using an ifnar -blocking antibody, this suppression was shown to partially depend on the capacity of the ns proteins to counteract the production of type i ifns. moreover, in vivo pulmonary conventional dc activation, as measured by the up-regulation of cd and cd , was strongly hampered in rsv-infected mice that are deficient for mavs, an essential signaling protein for rsv-induced type i ifn production [ ] . taken together, these results highlight that type i ifns play a role in rsv-induced dc maturation. as such, the pleiotropic effector functions of ns and ns to counteract the production of type i ifns likely account for the reduced dc maturation by ns and ns . in a follow-up report, munir and colleagues investigated the consequence of reduced dc maturation by ns and ns on subsequent activation of t-cell responses by co-cultivation of rsv-infected human monocyte-derived dcs and autologous t cells [ ] . deletion of ns was found to promote the proliferation and activation of cd + cd + t cells and t-helper cells, cell populations that can counteract rsv, and to suppress the activation of il- -producing cd + t cells. remarkably, none of these effects on t cells appeared to depend on type i ifn. possibly, il- β and il- α play a role as these cytokines were suppressed in dcs by ns in a type i ifn-independent manner. in contrast, a study by kotelkin and colleagues in mice identified that ns , and not ns , suppresses cd + t-cell responses in a type i ifndependent manner [ ] . remarkably, although rsv-induced activation of dcs in mavs -/-and mavs -/myeloid differentiation primary response protein myd (myd ) -/mice was strongly impaired, these mice could still mount comparable cd + t-cell responses as wildtype mice [ ] . this may in part be explained by the approximately -fold higher viral loads in mavs -/mice compared with wild-type mice. in addition, a small residual population of cd -positive pulmonary dcs was still present in these mice that could migrate to the draining mediastinal lymph node to activate naive cd + t cells. as mavs -/-myd -/mice are still functional for toll/interleukin- receptor domain-containing adapter molecule (ticam ), the adaptor of tlr , these dcs may have matured after the activation of tlr by rsvderived double-stranded rna (dsrna) [ ] . live-attenuated rsv vaccines with a targeted deletion in ns have been tested for use in infants [ ] . whereas a strain with only ns deleted was insufficiently attenuated, other ns deletion strains with additional mutations were found to be over-attenuated, highlighting the importance of a right balance between attenuation and immunogenicity. currently, phase i clinical trials with other ns deletion strains (clinicaltrials.gov identifiers: nct and nct ) and one with an ns deletion (clinicaltrials.gov identifier: nct ) are ongoing. the primary endpoints of these ongoing phase i trials with the live-attenuated rsv strains that lack either ns or ns are measures for safety, vaccine virus infectivity, and the induction of rsv-neutralizing titers. to our knowledge, cellular immune responses after immunization with these live-attenuated rsv vaccines have not been investigated. taking into account the small size of ns and ns , it is remarkable how many host functions are affected by these rsv proteins. goswami and colleagues hypothesized that ns and ns form a large ( to kda) degradative complex, which they called the ns degradasome (nsd) [ ] . in a cells, this complex could selectively degrade particular innate immune signaling proteins of which rig-i was also confirmed as a substrate of nsd complexes isolated from rsv-infected cells. in this respect, it would be interesting to assess whether other reported ns target proteins are also substrates of the proposed nsd complexes in rsv-infected cells. the nsd complex appears to be metastable, and its activity is enhanced by mitochondria via mavs, possibly through stabilizing the nsd complex [ ] . interestingly, in mavs-deficient cells stimulated with ifn-α, wild-type rsv is almost equally attenuated as rsv lacking ns and ns , confirming the importance of mavs for ns protein-mediated suppression of type i ifn responses. in line with these results, ns mainly localizes to mitochondria and seems to recruit ns towards the mitochondria [ ] . furthermore, ns target proteins, but not other innate proteins, relocate from the cytoplasm to the mitochondria upon expression of ns and ns [ ] . all together, these results support the hypothesis of the formation of a mitochondrialassociated nsd complex to selectively degrade innate immune proteins in rsv-infected cells. although the exact composition of the nsd complex is still unresolved, the presence of the α subunit from the s core proteasome sparked the idea that the nsd complex might act in a similar way as the host s proteasome [ ] . in line with this hypothesis, ns protein-induced stat and oasl degradation is (partially) blocked by the proteasome inhibitors mg and lactacystin [ , , , , ] . in contrast, ns protein-induced traf and ikkε degradation appears insensitive to mg [ ] . proteasome-like activity of the nsd complex is further supported by the identification of ns and ns as potential inducers of host protein ubiquitination [ , ] . whereas ns is a putative elongin b/c-cullin / -socs box-type e ubiquitin ligase, ns does not contain an elongin c binding consensus sequence [ , , ] . interestingly, an rsv strain with mutated ns residues that appear essential for ubiquitination activity and ns -mediated stat degradation is nearly equally attenuated as an ns deletion strain [ ] . these results suggest that ns and ns may induce ubiquitination of host proteins, which are then degraded by the nsd complex. it is currently not known, however, if ns and/or ns selectively mark innate immune proteins for degradation, which would explain the selective degradative activity of the nsd complex. recently, the crystal structure of ns was determined and revealed a remarkable structural homology with the n-terminal domain of the matrix protein of rsv [ ]. one clear difference between ns and m is the presence of an α-helix (called α ) at the c-terminus of ns . a truncated ns lacking this helix or mutation of residues that are important for the interaction of helix α with the rest of ns partially abrogates ns -mediated suppression of ifn-β induction and attenuates recombinant rsv strains. these results confirm that helix α is important for at least some ns -mediated effector functions. unravelling the crystal structure of ns and the composition of the nsd complex would greatly enhance our understanding of the remarkable diverse effector functions of ns and ns . moreover, ns and/or ns may be attractive targets for antiviral therapy. intranasal administration of nanoparticles carrying a plasmid encoding an ns -targeting small interfering rna (sirna) in mice can reduce lung viral titers, airway hyperresponsiveness, and pulmonary inflammation, both in a prophylactic as well as therapeutic setting [ ] . a recent small compound high-throughput screen revealed candidate inhibitors of ns , highlighting that ns could be targeted by a small compound drug [ ] . in humans, rsv infections induce remarkably low levels of ifn-α and -β compared with other respiratory viruses. this is largely the consequence of unique viral proteins, ns and ns , that strongly suppress the induction and signaling of ifn. in recent years, evidence is rising that, in addition, antiviral activities of isgs may be hampered by the ns proteins. ns and ns exert additional functions, such as delaying cell apoptosis, and loss of ns is associated with a stronger adaptive immune response and reduced in vivo viral replication. ns and ns are thought to form a so-called "ns degradasome" complex, which may function as a proteasome-like complex that selectively degrades a plethora of innate immune proteins. further insight in the composition of the nsd and the resolution of the structure of ns will likely help to explain the remarkable diverse effector functions of ns and ns . some effector functions, however, have so far only been identified in artificial cell systems and should be interpreted carefully. large-scale protein-protein interaction screens, preferentially performed using multiple complementary protein-protein interaction techniques in the context of an rsv infection, will generate a more comprehensive list of host proteins that interact with ns and/or ns . such new knowledge, combined with co-crystal structure analysis of rsv ns and ns in complex with host protein factors, will be instrumental to design antiviral drugs that impact on the rsv-host interface and thereby complement directly acting antivirals. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in : a systematic review and modelling study estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in countries nonstructural proteins ns and ns of bovine respiratory syncytial virus block activation of interferon regulatory factor a novel mechanism for the inhibition of interferon regulatory factor- -dependent gene expression by human respiratory syncytial virus ns protein replacement of the respiratory syncytial virus nonstructural proteins ns and ns by the v protein of parainfluenza virus nonstructural proteins and of respiratory syncytial virus suppress maturation of human dendritic cells hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor mutual antagonism between the ebola virus vp protein and the rig-i activator pact determines infection outcome influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i respiratory syncytial virus ns protein colocalizes with mitochondrial antiviral signaling protein mavs following infection human respiratory syncytial virus ns targets trim to suppress rig-i ubiquitination and subsequent rig-i-mediated antiviral signaling. viruses the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination molecular mechanism of influenza a ns -mediated trim recognition and inhibition human respiratory syncytial virus nonstructural protein ns antagonizes the activation of beta interferon transcription by interacting with rig-i viral degradasome hijacks mitochondria to suppress innate immunity respiratory syncytial virus nonstructural proteins decrease levels of multiple members of the cellular interferon pathways microrna- a induction during influenza h n virus infection targets and regulates traf levels in human nasal epithelial cells (hnecs) extracellular vesicles containing mir- a attenuate experimental colitis by targeting traf and irak microrna - p, mir-let- c- p, mir- and mir- - p are differentially expressed in respiratory syncytial virus (rsv) persistently infected hep- cells effects of nonstructural proteins ns and ns of human respiratory syncytial virus on interferon regulatory factor , nf-kappab, and proinflammatory cytokines nonstructural proteins of 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response associated with suppressor of cytokine signaling (socs) proteins and ifn-stimulated gene- (isg ) rsv replication is attenuated by counteracting expression of the suppressor of cytokine signaling (socs) molecules respiratory syncytial virus ns protein degrades stat by inducing socs expression respiratory syncytial virus nonstructural proteins upregulate socs and socs in the different manner from endogenous ifn signaling identification of paramyxovirus v protein residues essential for stat protein degradation and promotion of virus replication ebola virus vp targets a unique nls binding site on karyopherin alpha to selectively compete with nuclear import of phosphorylated stat nipah and hendra virus nucleoproteins inhibit nuclear accumulation of signal transducer and activator of transcription (stat ) and stat by interfering with their complex formation ifnbeta-dependent increases in stat , stat , and irf mediate resistance to viruses and dna damage interferome v . : an updated database of annotated interferon-regulated genes respiratory syncytial virus ns protein degrades stat by using the elongin-cullin e ligase respiratory syncytial virus strain a is resistant to the antiviral effects of type i interferons and human mxa '- '-oligoadenylate synthetase-like protein inhibits respiratory syncytial virus replication and is targeted by the viral nonstructural protein the nonstructural proteins of pneumoviruses are remarkably distinct in substrate diversity and specificity role of p /nf-kappab functional balance in respiratory syncytial virus-induced inflammation response rsv-encoded ns promotes epithelial cell shedding and distal airway obstruction a randomized, doubleblind, placebo-controlled trial of dexamethasone in severe respiratory syncytial virus (rsv) infection: effects on rsv quantity and clinical outcome the effect of high dose inhaled corticosteroids on wheeze in infants after respiratory syncytial virus infection: randomised double blind placebo controlled trial effect of dexamethasone on respiratory syncytial virus-induced lung inflammation in children: results of a randomized, placebo controlled clinical trial epithelial cells infected with respiratory syncytial virus are resistant to the anti-inflammatory effects of hydrocortisone respiratory syncytial virus represses glucocorticoid receptor-mediated gene activation respiratory syncytial virus (rsv) suppression of glucocorticoid receptor phosphorylation does not account for repression of transactivation poly i:c and respiratory syncytial virus (rsv) inhibit glucocorticoid receptor (gr)-mediated transactivation in lung epithelial, but not monocytic, cell lines the respiratory syncytial virus (rsv) nonstructural proteins mediate rsv suppression of glucocorticoid receptor transactivation glucocorticoid insensitivity in virally infected airway epithelial cells is dependent on transforming growth factor-beta activity respiratory syncytial virus nonstructural protein blocks glucocorticoid receptor nuclear translocation by targeting ipo and may account for glucocorticoid insensitivity human respiratory syncytial virus non-structural protein ns modifies mir- expression via transforming growth factor-beta respiratory syncytial virus regulates human micrornas by using mechanisms involving beta interferon and nf-kappab respiratory syncytial virus modifies micrornas regulating host genes that affect virus replication nf-kappab p -dependent transactivation of mirna genes following cryptosporidium parvum infection stimulates epithelial cell immune responses primary human mdc , mdc , and pdc dendritic cells are differentially infected and activated by respiratory syncytial virus respiratory syncytial virus interferon antagonist ns protein suppresses and skews the human t lymphocyte response the ns protein of human respiratory syncytial virus suppresses the cytotoxic t-cell response as a consequence of suppressing the type i interferon response respiratory macrophages and dendritic cells mediate respiratory syncytial virus-induced il- production in tlr -or tlr -dependent manner. int immunopharmacol identification of respiratory syncytial virus nonstructural protein residues essential for exploitation of the host ubiquitin system and inhibition of innate immune responses mutation of the elongin c binding domain of human respiratory syncytial virus non-structural protein (ns ) results in degradation of ns and attenuation of the virus inhibition of respiratory syncytial virus infection with intranasal sirna nanoparticles targeting the viral ns gene modular cell-based platform for high throughput identification of compounds that inhibit a viral interferon antagonist of choice key: cord- -sbtigcfr authors: chen, huijie; muhammad, ishfaq; zhang, yue; ren, yudong; zhang, ruili; huang, xiaodan; diao, lei; liu, haixin; li, xunliang; sun, xiaoqi; abbas, ghulam; li, guangxing title: antiviral activity against infectious bronchitis virus and bioactive components of hypericum perforatum l. date: - - journal: front pharmacol doi: . /fphar. . sha: doc_id: cord_uid: sbtigcfr hypericum perforatum l., also known as saint john’s wort, has been well studied for its chemical composition and pharmacological activity. in this study, the antiviral activities of h. perforatum on infectious bronchitis virus (ibv) were evaluated in vitro and in vivo for the first time. the results of in vitro experiments confirmed that the antiviral component of h. perforatum was ethyl acetate extraction section (hpe), and results showed that treatment with hpe significantly reduced the relative messenger ribonucleic acid (mrna) expression and virus titer of ibv, and reduced positive green immunofluorescence signal of ibv in chicken embryo kidney (cek) cells. hpe treatment at doses of – mg/kg for days, reduced ibv induced injury in the trachea and kidney, moreover, reduced the mrna expression level of ibv in the trachea and kidney in vivo. the mrna expression levels of il- , tumor necrosis factor alpha (tnf-α), and nuclear factor kappa beta (nf-κb) significantly decreased, but melanoma differentiation-associated protein (mda ), mitochondrial antiviral signaling gene, interferon alpha (ifn-α), and interferon beta (ifn-β) mrna levels significantly increased in vitro and in vivo. our findings demonstrated that hpe had significant anti-ibv effects in vitro and in vivo, respectively. in addition, it is possible owing to up-regulate mrna expression of type i interferon through the mda signaling pathway and down-regulate mrna expression of il- and tnf-α via the nf-κb signaling pathway. moreover, the mainly active compositions of hpe analyzed by high-performance liquid chromatography/electrospray ionization–mass spectroscopy (esi-ms) are hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities. this might accelerate our understanding of the antiviral effect of h. perforatum and provide new insights into the development of effective therapeutic strategies. the infectious bronchitis virus (ibv) is a prototype coronavirus containing a single-stranded positive-sense rna genome (cook et al., ) . ibv is the etiologic agent of infectious bronchitis (ib), which is a highly contagious, acute viral respiratory disease of chickens. ibv has been reported by many researchers all over the world (jungherr and terrells, ; jungherr et al., ; fabricant, ; yu et al., ; benyeda et al., ; sjaak de wit et al., ; westerbeck and machamer, ; wu et al., ) . ibv has led to severe losses in the poultry industry (jordan, ) , the direct losses are due to highly mortality, poor egg quality, and meat production, and the indirect losses result in increased costs and challenges in ibv prevention (liang et al., ) . at present, live attenuated vaccines are widely used for the prevention and control of ib. however, due to extensive genetic diversity of ibv strains, the vaccines are becoming increasingly inefficient, with poor cross-protection effects among different serotypes of vaccines (mo et al., ; chen et al., ; lin and chen, ; yan et al., ) . meanwhile, due to the lack of coordinated effort to prevent the ibv, and the lack of proper surveillance plus the introduction of foreign strains to combat the ibv in certain regions, the prevention and control of ibv has become very difficult. therefore, it is imperative to find an effective antiviral drug or agent for the prevention of ibv. in order to control drug residues, the chinese government has banned the use of antiviral drugs in food animals in china. therefore, the use of traditional antiviral herbs with no obvious side effects on the human body is still a major focus. some reports have confirmed that traditional chinese herbs could effectively inhibit the infection and replication of various viruses (li et al., ; wang et al., ; choi et al., ; sun et al., ; choi et al., ; yin et al., ; yi et al., ; luo et al., ) . hypericum perforatum l. belongs to the genus guttiferae, which contains approximately species all over the world. the extract of h. perforatum contains several active compounds, including flavonoids, naphthodianthrones, and phloroglucinol derivatives (napoli et al., ; barnes et al., ) . several reports have shown that h. perforatum extract had antiviral effects, such as influenza a virus, porcine respiratory and reproductive syndrome virus (prrsv), and hiv (barnes et al., ; birt et al., ; pu et al., a; pu et al., b; pu et al., ) . like influenza a virus and prrsv, ibv also belongs to rna virus, but these prrsv and ibv belong to different viral families. since h. perforatum could resist influenza a virus and prrsv, could it resist ibv? in this study, we investigated the antiviral effects of h. perforatum extract against ibv utilizing several approaches in vitro and in vivo for the first time. moreover, the purpose of this work was to point out the antiviral active ingredients of h. perforatum and its anti-ibv mechanisms. for this purpose, the relative messenger ribonucleic acid (mrna) expression levels of ibv in cek cells, tracheas, and kidneys were measured. the positive green immunofluorescence signal of ibv in ceks was observed. in addition, hematoxylin-eosin (he) staining of tracheas and kidneys was performed, and the relative mrna expression of il- , tumor necrosis factor alpha (tnf-α), ifn-α, ifn-β, mda , mavs, and nuclear factor kappa beta (nf-κb) in vitro and in vivo were analyzed. finally, the antiviral principal chemical composition of h. perforatum extract was analyzed by high-performance liquid chromatography (hplc)/esi-ms. in summary, our findings for the first time showed that h. perforatum extract had significant antiviral effect on ibv, and it might up-regulate mrna expression levels of type i interferon via mda pathway and down-regulate mrna expression levels of il- and tnf-α through the nf-κb pathway. the hpe was found to be composed mainly of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities. the ibv m strain (genbank: fj . ) was provided by key laboratory for laboratory animals and comparative medicine, northeast agricultural university and propagated in -dayold specific pathogen-free (spf) chicken embryos (harbin veterinary research institute, caas). µl ibv m strain was inoculated into spf chicken embryos under aseptic operation. the survival status of chicken embryos was observed every h, and the chicken embryos that died within h were abandoned. then allantoic fluid from infected embryos was collected at h post-inoculation and stored at − °c. the crude extract of h. perforatum was prepared in the laboratory. in brief, the drug was pretreated with carbon dioxide supercritical extraction method, and the supercritical extraction extract (see) was obtained with a yield of . % (wt/wt). then, g of the pretreated medicinal material was weighed, . l of % methanol was added (raclariu et al., ) , ultrasonic extraction was performed twice for min, and ultrasonic power was w. the two extract solutions were combined and filtered, and concentrated under reduced pressure to obtain the crude extract with a yield of . % (wt/wt). approximately g of the crude extract of h. perforatum was dissolved in ml of distilled water. an equal volume of ethyl acetate was added before shaking vigorously and separating in a separating funnel. the ethyl acetate layer (upper layer) and the water layer (lower layer) were obtained. it was then extracted with ethyl acetate until the upper layer had no color. all ethyl acetate extractions and water extractions were combined and evaporated to obtain extract of h. perforatum ethyl acetate (hpe) and extract of h. perforatum water (hpw). a stock solution of hpe ( mg/ml), hpw ( mg/ml) and see ( mg/ml) was prepared with dimethylsulfoxide (dmso, sigma), respectively. immediately before each experiment in vitro, the working solution of h. perforatum extract was diluted in m medium without serum to obtain a final concentration. the hpe was dissolved in % dmso, while ribavirin (rt) was dissolved in physiological saline solution (pss, . % nacl) in vivo experiments. cek cells were primary cultured from -day-old spf chicken embryo (harbin veterinary research institute, caas) and prepared according to the standard technique (schat and purchase, ; ghetas et al., ) . the cells were cultured in m medium (thermo fisher scientific, usa) supplemented with penicillin and streptomycin, and % serum at °c with % co . then, ibv m strain was passaged in cek cells for adaption. µl ibv m allantoic fluid was inoculated into cek cells in ml cell culture flask, and the cytopathic effect (cpe) was observed. when the cells showed obvious typical cpe, the time and degree of the lesions were recorded and the first generation of ibv was collected by freezing and thawing cells repeatedly. then, the collected first generation ibv was inoculated into cek cells according to the above method, and the time and lesion degree of cpe were also recorded. the second generation ibv was collected according to the above method. after times of passages of ibv in cek cells, there appeared typical cpe of cell exfoliation and fusion at same time post inoculation regularly. ibv was collected and stored at − °c until use. the results of adaptation and replication in cek cells, such as cpe, reverse transcription polymerase chain reaction (rt-pcr), and ibv growth curve determined by tissue culture infective dose (tcid ) at different time were tested. the cytotoxicity test was carried out according to the published method of thiazolyl blue tetrazolium bromide (mtt), and minor modifications were made (sui et al., ) . the monolayer of cek cells plated on -well culture plate were washed with d-hanks solution three times, and then hpe, hpw, and see with concentrations of . , . , . , . , and . µg/ml were added to the hole (three repeated holes per concentration). the control cells were incubated in the absence of experimental compounds, but incubated with the same concentration of dmso. the cek cells were cultured at °c for h, then μl mtt with concentrations of mg/ml in m medium were added, and then the cells were incubated at °c for h. after washing the cek cells with d-hanks, μl dmso was added to each well, then the cek cells were incubated at °c for min, and shaken gently at room temperature for min to dissolved formazan precipitates, and od was determined. using the mean values, the cell survival rate is calculated according to the following formula: in order to analyze the effects of hpe, hpw, and see on cells, cek cells cultured on well plates were incubated with hpe, hpw, and see solutions at concentrations of . , . , . μg/ml at °c for h, respectively, and then washed with d-hanks for three times. the cek cells were then infected with tcid ibv and cultured at °c for h. the cell samples were frozen and thawed repeatedly for three times, and the total rna was extracted and reverse transcribed into complementary deoxyribonucleic acid (cdna). the relative mrna expression of ibv n gene was detected by real-time quantitative rt-pcr (qrt-pcr). in addition, according to the conventional method, the virus titer of cell samples was determined by tcid . the specific operations were as follows: cek cells were cultured in -well tissue culture plates, and when the cells were full of monolayer, ibv virus was diluted to − , − , − …, and − times with serum-free m , respectively. then the monolayer cek cells were washed with d-hanks solution three times, and the d-hanks solution was discarded. then the µl ibv virus diluent as mentioned above was added to each hole, and each virus diluent repeated eight holes. the cells were treated by the same method with m culture as normal control. the culture plate was cultured in % co and °c for h. the cytopathic effect (cpe) was observed every h, and the number of cpe pores and no cpe pores were recorded. according to the reed-muench method as previously described (guo et al., ; yang et al., b) , the tcid was calculated according to the number of cpe holes recorded. at the same time, the infected cek cells treated with μg/ml rt and cek cells infected with ibv as control. in order to determine the impact of hpe, hpw, and see on ibv-infected cells, the cek cells cultured on well plates were infected with tcid ibv at °c for h, and then treated with hpe, hpw, and see solution at concentrations of . , . , . μg/ml at °c for h. according to the above description, the relative mrna expression level and virus titer of ibv were detected. in order to study the direct effects of hpe, hpw, and see on the virus, tcid ibv was incubated with . , . , . μg/ ml hpe, hpw, and see solution at °c for h, respectively. then cek cells were infected with the drug-treated ibv at °c for h. as described above, the relative mrna expression levels and the virus titer of ibv were detected. in the above three experimental designs, the best antiviral way was selected for indirect immunofluorescence assay. after washing cek cells with pbs, fixed with % paraformaldehyde, then incubated with . % glycine and quenched with % triton-x for min. after washing cek cells with pbs for times, the cek cells were incubated with rabbit anti-ibv antibody ( : ) (prepared and stored in laboratory of pathology and anatomy, northeast agricultural university) for h, and then incubated with fluorescent labeled goat antirabbit igg ( : ) (zhongshan, china) for min in the dark. the fluorescent images were examined with a fluorescence microscope (ti-s, nikon, japan). at the same time, the infected cells treated with μg/ml rt, and cek cells infected with ibv, and the mock cek cells were set as controls. one hundred and forty four -week-old spf chicks were randomly divided into hpe treated high dose group (hpe-th), hpe treated middle dose group (hpe-tm), hpe treated low dose group (hpe-tl), the ibv infected group (ibv), the ribavirin treated (rt) group, and normal control (nc) group with chicks in each group. each group was completely isolated from the other groups, and housed in negative pressure-filtered air isolators under pathogen-free conditions. chicks from in three hpe-treated groups, the ibv-infected group, and the rt group were inoculated by nasal drops with . ml of eid of ibv m allantoic fluid per chick. chicks in the control group were inoculated with . ml sterilized negative allantoic fluid in the same manner. the chicks of the three hpe groups and the rt group were orally administered varying doses of three hpe ( mg/kg, mg/kg, mg/kg) and ribavirin ( mg/kg), respectively. both agents were administered twice daily for days, beginning at h after exposure to the virus. at the same time, the chicks of the ibv-infected group and the normal group were orally administered with pss. at , , , and days postinfection, six chicks in each group were bled before euthanasia and necropsy at each designed day. the trachea and kidney tissues were collected and immediately extracted to obtain the total rnas. at the same time, tracheal and kidney in each group of chickens at days postinfection were fixed in % buffer formaldehyde for histopathological assay. after washing in phosphate-buffered saline (pbs), the trachea and kidney tissues were fixed in % formaldehyde solution for days, embedded in paraffin wax, cut into -μm-thick sections (leica rm , germany), and stained with he using standard histological staining procedure. the slides were examined by light microscopy (nikon, japan). the tissue slides were examined and evaluated according to the previous histopathologic scoring method for trachea (levisohn et al., ; todd et al., ) and kidney (reinhard et al., ; laudert et al., ; canales et al., ) with minor modification. for the trachea, the following morphological changes were included: cilia loss of mucous epithelium, extrusion of balloon-like cell and necrotic exfoliation of mucous layer, lymphocyte and heterophil infiltration in submucosal layer, hyperemia, and/or hemorrhage. for the kidney as followed: glomerular atrophy and fragmentation, tubular degeneration (granular and/or vacuolar degeneration) and necrosis (detached and broken epithelium), interstitial infiltration of lymphocytes and heterophils, hyperemia, and/or hemorrhage. the distribution and extent of the aforementioned lesions in the trachea and kidney of each chicken were scored as followed: , absence of lesion; , lesion represented in fewer than % of the involvement of tissue; , lesion represented in - % involvement; , lesion represented in - % involvement; and , lesion represented in more than % involvement. the pathological lesions of trachea and kidney were scored by experienced veterinary pathologists (dr. guangxing li, ruili zhang, and xiaodan huang, northeast agricultural university, harbin, pr china), which were blinded to the identities of the experimental groups. the pathological injuries of trachea and kidney were quantitatively analyzed, and the statistical results were given in tables and . treated cell samples were repeatedly frozen and thawed three times, and the equiponderant trachea and kidney tissues from each chick at various time points were prepared. then, the total rna was extracted using a universal rna extraction kit (thermo fisher scientific, usa) according to the manufacturer's instructions. all the concentration of rna and the a /a ratio were determined with nanodrop spectrophotometer (thermo fisher scientific, usa), and the integrity of the extracted rna was detected by agarose gel electrophoresis. the total rnas were reverse transcribed into cdnas by pcr instrument (takara, japan) for qrt-pcr, and the cdnas were stored at − °c. the relative quantification analyses was used to determine the levels of mrna expression of target genes, including n gene of ibv, il- , tnf-α, il- β, ifn-α, ifn-β, mda , mavs, and nf-κb by an applied lightcycler real-time pcr system (roche, switzerland). all primer pairs for real-time pcr detection are listed in table . these primers were synthesized by genewiz biological technology co., ltd. (suzhou, china). the relative mrna expression levels of target genes were calculated by using the −ΔΔct method (yu et al., ). the amplification system was μl cdna, . μl forward and reverse primers, μl universal sybr green (rox) and . μl nuclease-free water, and the final volume of each system was μl. the amplification reaction of qrt-pcr assays was conducted according to the following thermal profile: pre-incubation cycles at °c for s, followed by step amplification: cycles at °c for s and °c for s, followed by melting: cycles at °c for s, °c for s, and °c for s, followed by cooling cycles at °c for s. chromatography/diode array detector/ electrospray ionization-mass spectroscopy analysis of the hypericum perforatum ethyl acetate hplc/diode array detector/esi-ms analysis was performed on a waters hplc equipped with diode array detector and waters micromass zq esi-electrospray (waters, usa) operating in positive and negative ion mode. according to relevant reports (seger et al., ; jesionek et al., ) , the hplc condition was determined with a minor modification. hpe analysis was performed using an eclipse xdb-c column ( mm x . mm, μm, agilent technologies inc. usa). the mobile phase consisted of water (a) and acetonitrile (b) at a constant flow rate ( . ml/min). the solvent gradient elution method was as follows: - min, - % b and - min, % b. the detection wavelength was - nm, the column temperature was room temperature, and the injection volume was μl. the operating conditions of the mass spectrometer were dry gas temperature, °c, flow rate, l/min; nebulizer pressure, psi; sheath gas temperature, °c, flow rate, l/min; fragmenter voltage, v; capillary voltage, , v; mass range, - , d. determination of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the hypericum perforatum ethyl acetate the sample solution was prepared by dissolving . mg dried hpe in ml mobile phase and filtered by . μm filter before hplc analysis. the content of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the hpe was determined by hplc instrument and the chromatographic conditions described above. in the ml analytical solvent, the hyperoside standard of . mg, or the quercitrin standard of . mg, or the quercetin standard of . mg, or the pseudohypericin standard of . mg, or the hypericin standard of . mg are dissolved to prepared stock solution, respectively. according to the guide of international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use (singh, ; armutcu et al., ; hsi et al., ), the signal-to-noise ratio (s/n) of and were defined as the detection limit (lod), and the quantitative limit (loq), respectively. in order to detect the s/n, the stock solution of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were diluted to different concentrations, respectively. in order to obtain the standard curve, the stock solution of the above standard compounds was diluted into six appropriate dilution concentrations. quantification was conducted by using a six-point standard curve and an external standard method. intra-day and inter-day precision for hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were used to evaluate the repeatability and reproducibility of the established method. statistical analysis the number of the experiment repetition in all the experiment was three times. the experimental data was analyzed with spss . software (spss inc., chicago, il, usa). the results are expressed as the means ± standard deviation (sd). differences between groups were evaluated using the one-way analysis of variance (anova) of two tailed test. p < . were considered as statistically significant, and p < . were considered as highly significant. staining showed cell was survival (figure a) . at the same time, the cell survival rate measured by mtt method was close to %, which was further explained that μg/ml of the drug had no significant effect on the cells (figure b ). when ibv was propagated in cek cells to the tenth generation, stable typical cytopathic effect (cpe) appeared at h after ibv infection (figure a) . at the beginning, cells infected with ibv became round and refractive, then some cell exfoliated from the flask and left empty hole, some cells fused together and became multinuclear giant cells, indicating that ibv adapted to cek cells. the results of virus growth curve showed that ibv could infect and replicate in cek cells and reached the highest titer of − . tcid per μl at h post-inoculation (figure b ). at the same time, the existence of ibv m was identified by rt-pcr ( figure c ). antiviral effect of supercritical extraction extract, hypericum perforatum ethyl acetate, and hypericum perforatum water in vitro the relative mrna expression level of ibv-n gene was detected by qrt-pcr and the virus titer of ibv was determined by tcid to analyze the antiviral effect of hpe, hpw and see (figure ) . it could be seen from figure that under the maximum non-toxic concentration of the drug, the inhibition of hpe on ibv was significantly greater than that of hpw and see. hpw had a very weak effect on ibv and see had no effect on ibv. therefore, it was determined that the antiviral part of h. perforatum extract was ethyl acetate layer. as can be seen from figure a , in the three experimental designs, with the increase of hpe concentration, the mrna level of ibv decreased significantly in a dose-dependent manner. in addition, at the same drug concentration, the relative expression level of virus mrna was the lowest when hpe directly treated the infected virus cells, followed by hpe pre-treated cells before infection, and then hpe pre-treated virus before infection. it can be known from the figure b , with the increase of hpe concentration, the titer of ibv decreased gradually in a dosedependent manner. while, at the same drug concentration, the virus titer was the lowest when hpe directly treated the infected virus cells, followed by hpe pre-treated cells before infection, and followed by hpe pre-treated virus before infection. at the same time, the antiviral impact of hpe directly treated the ibv-infected cells at the concentration of . µg/ml, was similar to rt at the concentration of µg/ml. therefore, hpe was selected for subsequent experiments in vivo and in vitro, furthermore the hpe directly treated the ibv-infected cells was selected in vitro experiments. the results of reverse transcription polymerase chain reaction identification of ibv m in cek cells. in which, m stands for deoxyribonucleic acid marker, n stands for polymerase chain reaction product for ibv n gene, w stands for negative water control. frontiers in pharmacology | www.frontiersin.org october | volume | article in order to further confirm the inhibitory effect of hpe on ibv-infected cells, the fluorescent signal of virus was detected by ifa (figure ) . from the figure , it can be seen that the cek cells infected with ibv produced a strong fluorescence signal at h after infection. on the contrary, the fluorescence signal of cek cells infected with ibv and treated with hpe was weakened, and the fluorescence signal decreased in a dosedependent manner with the increase of hpe concentration. this further confirmed that hpe has a very good inhibitory effect on ibv infected cells. in order to study the effect of hpe on gene mrna expression induced by ibv infection in cek cells, the mrna expression level of related genes were determined, including mda , mavs, ifn-α, ifn-β, nf-κb, il- , and tnf-α. as can be seen from figure , hpe treatment significantly affected the mrna levels of mda , mavs, ifn-α, and ifn-β after ibv infection at and h, and the mrna expression levels of these genes changed similarly. these data suggested that hpe could increase mrna expression level of type i interferon in the late stages of ibv infection, which is possibly related to mda signaling pathway. in addition, hpe treatment significantly reduced the mrna expression levels of nf-κb, il- , and tnf-α, which were up-regulated after ibv infection at and h. these data demonstrated that hpe could decrease the mrna expression of pro-inflammatory genes, which is possibly related to nf-κb signaling pathway. at days after infection, the symptoms that appeared in the ibv infected group were sneezing, tracheal wet rales, mouth breathing, coughing, ruffled feathers, and frequently shaking. but the symptoms that appeared in the hpe and rt treatment group such as sneezing, tracheal wet rales, mouth breathing, coughing, feather wrinkling, and frequent trembling were mild. and the trachea and kidneys of different groups of spf chickens were conducted for histopathology assay. as can be seen from figure , most of the tracheal cilia in the ibv infected group fell off, the mucosal epithelial cells exfoliated and disappeared, the submucosal structure was loosely arranged, and there was a certain amount of serous exudation, inflammatory cells, and red blood cells. in the hpe treatment group, with the gradual increase of the dosage, the shedding of tracheal cilia and epithelial cells was gradually decreased, there were a small number of inflammatory cells and erythrocytes, and the exudation of inflammatory serous fluid decreased. as shown in figure , in the ibv infected group, a large number of renal tubular epithelial cells were degenerated and necrotic, the nucleus were concentrated, fragmented and dissolved, the renal tubular epithelial cells fell off into the lumen, dissolved or disappeared, the structure of renal tubules was figure | the effects of hypericum perforatum ethyl acetate on the messenger ribonucleic acid (mrna) expression level of related genes in vitro. chicken embryo kidney (cek) cells were infected with tcid infectious bronchitis virus (ibv) at °c for h, and then incubated with . μg/ml hpe. the cek cells were treated with m , tcid ibv, and . µg/ml hpe as the control, respectively. subsequently, total rna was extracted from cek cell samples at , , and h after treatment. the relative mrna expression of melanoma differentiation-associated protein (a), mitochondrial antiviral signaling gene (b), interferon alpha (c), interferon beta (d), nuclear factor kappa beta (e), il- (f), and tumor necrosis factor alpha (g) were determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < . and highly significant at **p < . when compared with the ibv-infected cell. frontiers in pharmacology | www.frontiersin.org october | volume | article incomplete, and there were a certain number of inflammatory cells and red blood cells in the interstitium. the wall of renal capsule was ruptured and a certain amount of inflammatory cells and serous exudation could be seen in the lumen of renal capsule. in the hpe treatment group, with the increase of the dosage, the exfoliation and dissolution of renal tubular epithelial cells decreased gradually, the structure of renal tubule was gradually complete, the number of red blood cells and inflammatory cells in the stroma was less, and the structure of renal capsule wall was more complete. there was little infiltration of inflammatory cells in the renal vesicle cavity. as can be seen from tables and , the pathological scores of trachea and kidney in ibv group were highly significantly higher than those in nc group (p < . ). compared with the ibv group, the pathological scores of trachea and kidney in the hpe-th group and the hpe-tm group decreased highly . ± . . ± . . ± . . ± . . ± . ** hpe-tl . ± . . ± . . ± . . ± . . ± . hpe-tm . ± . . ± . . ± . . ± . . ± . ** hpe-th . ± . . ± . . ± . . ± . . ± . ** the difference was considered highly significant at **p < . vs. ibv infection group. frontiers in pharmacology | www.frontiersin.org october | volume | article significantly (p < . ), and the score was similar to that of the rt group. at the same time, with the increase of hpe dose, the score decreased in a dose-dependent manner. this indicated that hpe had better anti-ibv effect. to confirm the inhibitory effect of hpe, ibv mrna levels in trachea and kidney of different groups were measured by realtime qrt-pcr. as shown in figure , compared with nc group, the expression level of mrna in trachea and kidney of spf chicks infected with ibv increased significantly. compared with ibv group, the expression level of mrna in trachea and kidney of spf chicks treated with hpe decreased significantly. with the decrease of hpe concentration, the expression level of mrna increased in a dose-dependent manner. with the increase of the days of ibv infection, the effect of hpe-th and hpe-tm group on the level of mrna expression was similar to that of rt group. these data indicated that infected chickens treated with hpe exhibited an overall reduction in viral mrna levels. it is well known that type i interferon, including ifn-α and ifn-β, plays a very important role in antiviral activity. as can be seen from figures and , the relative mrna expression of il- , tnf-α, ifn-α, ifn-β, mda , mavs, and nf-κb in the trachea and kidney were up-regulated after ibv infection, which was consistent with previous studies (he et al., ; chhabra et al., ) . in hpe treatment group, the mrna expression levels of ifn-α and ifn-β in the trachea were up-regulated in the early stage of ibv infection, and the changes of ifn-α and ifn-β were consistent with the mrna expression of mda and mavs in the trachea. meanwhile, in the hpe-treated group, the mrna expression levels of ifn-α and ifn-β in the kidney were up-regulated in the late stage of ibv infection. in addition, the changes of ifn-α and ifn-β were similar to the mrna expression levels of mda and mavs in the kidney. these data suggested that hpe may up-regulate mrna expression levels of ifn-α and ifn-β in trachea and kidney via the mda signaling pathway. . ± . . ± . . ± . . ± . . ± . nc . ± . . ± . . ± . . ± . . ± . ** rt . ± . . ± . . ± . . ± . . ± . ** hpe-tl . ± . . ± . . ± . . ± . . ± . hpe-tm . ± . . ± . . ± . . ± . . ± . ** hpe-th . ± . . ± . . ± . . ± . . ± . ** the difference was considered highly significant at **p < . vs. ibv infection group. figure | the effects of hypericum perforatum ethyl acetate (hpe) on infectious bronchitis virus (ibv) messenger ribonucleic acid (mrna) expression levels of trachea (a) and kidney (b). spf chickens were infected with ibv, followed by oral administration of hpe and ribavirin, respectively. total rna were subsequently extracted from trachea and kidney at , , , and day post-infection. the relative mrna expression of ibv was determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < . and highly significant at **p < . . frontiers in pharmacology | www.frontiersin.org october | volume | article at the same time, it is well known that inflammatory factors play an important role in inflammation. it also plays a very important role in ibv infection. as can be seen from figures and , in the hpe-treated group, the mrna expression levels of il- and tnf-α in the trachea and kidney were down-regulated, and the changes were consistent to nf-κb mrna expression levels, indicating that hpe may be down-regulated the mrna expression levels of il- and tnf-α by nf-κb signaling pathway. therefore, hpe may exert an anti-ibv effect by increasing the mrna expression levels of type i interferon and reducing mrna expression levels of proinflammatory factors il- and tnf-α, and this may be related to the mda signal pathway and nf-κb signal pathway. (figure ) . the mass spectra of these compounds were carefully examined (figure ) and compared with the standard and reference data (seger et al., ; cao et al., ) and found to have five peaks in the hpe (table ) . these peaks corresponded to hyperoside (peak ), quercitrin (peak ), quercetin (peak ), pseudohypericin (peak ), and hypericin (peak ). determination of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the hypericum perforatum ethyl acetate the calibration curves of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were as followed: y = , x + , , y = , x + , , y = , x− , , y = , x + , , and y = , x + , . , where y is peak area, x is sample concentration, and the concentration ranges were - , - , - , - , and - μg/ml, respectively. and the calibration curve was in accordance with a linear regression (r = . - . ). the lod (s/n = ) of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin was in the range of . to . μg/ml. the loq (s/n = ) of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin was in the range of . to . μg/ml. by measuring the peak area and retention time of standard compounds to evaluate the intra-day and inter-day precision, their relative sd was less than %. the results showed that the hpe contained . % hyperoside, . % quercitrin, . % quercetin, . % pseudohypericin, and . % hypericin. ibv has a variety of known strains, these strains continue to mutate, and more mutants continue to recombine, resulting in more diverse and complex genotypes and serotypes of ibv (bande et al., ; feng et al., ; laconi et al., ) . due to the poor cross-protection of vaccines between different serotypes, it is difficult to prevent and control ibv infection. therefore, development of an effective antiviral therapy is a crucial strategy for treating ibv infection. in this study, the anti-ibv effect of the drug was studied by three kinds of methods, qrt-pcr, determination of tcid , and indirect immunofluorescence (ifa). qrt-pcr is a method of measuring the total amount of products after each reaction cycle with fluorescent chemicals in dna amplification. ifa is a method to detect the presence of specific antigens in cells after the cells were treated with fluorescent-antibodies. tcid is the amount of infection in which % of the cells are infected by the virus. the anti-virus effect of drugs is often detected by qrt-pcr (schnepf et al., ; li et al., ; zhang et al., ) , ifa (xu et al., ; zhang et al., a) and tcid (xu et al., ; zhang et al., ) in antiviral research. and these antiviral drug reports are similar to our research, so we also used these methods to detect the anti-ibv effect of h. perforatum. although qrt-pcr and ifa could not detect live viral particles, they were used as a supplement in this study, and in this study we used the method of tcid , which is a general method for the detection of live virus (leclercq et al., ; petiot et al., ) . therefore, the results of antiviral studies in vitro are also credible in this study. in addition, although the best way to culture ibv is to use chicken embryos or chickens, the ibv replication in cek cells was tested in this study. because if the virus doesn't replicate well, viral particles can also be reduced or even die, which in turn affects the antiviral effect of the drug. in this study, we showed that ibv can replicate well in cek cells (figure ) , which is consistent with the research report (zhang et al., ) , and hpe had potential utility as antiviral agents against ibv. our results showed that the hpe can reduce virus mrna expression level (figures a and ) and virus titer ( figure a) . additionally, the see had no inhibitory effects and the hpw had poor inhibitory effects (figure ) . the results suggest that the hpe, which contains an enrichment of the main effective substances, was the bioactive fraction of h. perforatum extracts. moreover, the hpe had anti-ibv activity, such as reducing the fluorescence signal produced by ibv (figure ) , and alleviating the pathological injury of trachea and kidney caused by ibv in chickens (figures and ) . when came to evaluation of antiviral effects, ribavirin was used as control drugs. the inhibitory effect of . µg/ml hpe was comparable to that of µg/ml ribavirin in vitro, and mg/kg hpe was comparable to that of mg/ kg ribavirin in vivo. in the efficacy of antiviral effects, as in the reporting of others herbs (choi et al., ; sun et al., ; choi et al., ; yi et al., ; luo et al., ) , the effects of the herbal extract were lower than ribavirin even under several times higher dosage against ibv mrna level, virus titer, and histopathological injury caused by ibv. however, it is logical since ribavirin is pure compared to the extracts containing many inactive compounds. some studies have shown that h. perforatum extract have an antiviral effect on influenza a virus and hiv (barnes et al., ; birt et al., ; pu et al., ) , suggesting that hpe have the potential to be developed and used as antiviral drugs. in this study, we found that h. perforatum extract had significantly antiviral effect on ibv in vitro and in vivo, respectively. in addition, anti-ibv effect of hpe might correlate figure | effect of hypericum perforatum ethyl acetate (hpe) on the messenger ribonucleic acid (mrna) expression of genes in trachea. spf chickens were infected with ibv, followed by oral administration of hpe and ribavirin, respectively. total rna were subsequently extracted from trachea at , , , and day postinfection. the relative mrna expression of (a) melanoma differentiation-associated protein , (b) mitochondrial antiviral signaling gene, (c) interferon alpha, (d) interferon beta, (e) nuclear factor kappa beta, (f) il- , and (g) tumor necrosis factor alpha were determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < . and highly significant at **p < . . frontiers in pharmacology | www.frontiersin.org october | volume | article figure | effect of hypericum perforatum ethyl acetate (hpe) on the messenger ribonucleic acid (mrna) expression of gene in kidney. specific pathogen-free (spf) chickens were infected with infectious bronchitis virus (ibv), followed by oral administration of hpe and ribavirin (rt), respectively. total rna were subsequently extracted from kidney at , , , and day post-infection. the relative mrna expression of (a) melanoma differentiation-associated protein , (b) mitochondrial antiviral signaling gene, (c) interferon alpha, (d) interferon beta, (e) nuclear factor kappa beta, (f) il- , and (g) tumor necrosis factor alpha were determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < . and highly significant at **p < . . frontiers in pharmacology | www.frontiersin.org october | volume | article with mda and nf-κb signaling pathway. belonging to acid inducible gene i (rig-i)-like receptors (rlrs) in pattern recognition receptors (prrs), it is well known that mda plays a crucial role in avian respiratory disease progression (yu et al., ) , whose function is to identify various pathogenassociated molecular patterns (pamps), and activates mitochondrial antiviral signaling gene (mavs, also called ips- /visa/cardif) (lin et al., ) , activated mavs can recruit downstream interferon regulatory factor- / (irf / irf ) (shi et al., ) , leading to the rapid production of type i ifns (xu et al., ; soulat et al., ) . nf-κb system is a master transcription factor in the recognition signaling and host responses to immune attacks (deng et al., ) . it is well known that nf-κb mediates inflammation, and several cytokine are also linked to nf-κb signaling to enhance the inflammatory responses, such as tnf-α, il- , and il- β (salminen et al., ) . several reports have pointed out the mda signaling pathways and innate immune cytokines were activated after infection with ibv m strain (he et al., ) . mda signaling pathway was disrupted by cleavage of the adaptor protein mavs in the js/ / strain of ibv infection (yu et al., ) . the type i ifn response plays a critical role in resisting saibk strain of ibv (yang et al., a) . mda signaling pathways and innate immune cytokine (nf-κb and irf ) were induced after ibv-m strain infection (zhang et al., b) . on the other hand, the report had shown that h. perforatum extract significantly downregulated the concentration of il- and tnf-α in lung tissue for mice infected with an influenza a virus (pu et al., ) . in this study, we found that mrna expression levels of mda , mavs, ifn-α, ifn-β, nf-κb, tnf-α, and il- were significantly up-regulated after ibv infection in vitro and in vivo (figures , , and ) . meanwhile, we found that hpe may be up-regulate mrna levels of ifn-α and ifn-β by mda signaling pathway, and down-regulate mrna expression levels of il- and tnf-α through the nf-κb signaling pathway (figures , , and ) , suggesting that hpe may be inhibit ibv by affecting innate immune cytokines. because of remarkable antiviral effect on ibv, we analyzed the active components of hpe. the mass spectrum of hyperoside contained a negative quasi-molecular ion at . m/z [m-h] − (figure b ), in accordance with the previous literature (cao et al., ) . the mass spectrum of figure b figure c) . in addition to a positive quasi-molecular ion at . m/z [m+na] + ( figure d ) and a negative quasi-molecular ion [m-h] − at . m/z (figure e) , confirming the presence of the pseudohypericin. finally, the mass spectrum of hypericin shows a positive quasi-molecular ion [m+h] − at . m/z ( figure f ) and a negative quasi-molecular ion [m-h] − at . m/z ( figure g) . according to the results in figure , the hpe is the main active fraction, containing abundant levels of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin (table and figure ). our study provides, for the 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cleavage of the adaptor protein mavs in vitro inhibition of vesicular stomatitis virus replication by purified porcine mx protein fused to hiv- tat protein transduction domain (ptd) chicken mannose binding lectin has antiviral activity towards infectious bronchitis virus cellular immune response in chickens infected with avian infectious bronchitis virus (ibv) astragalus polysaccharides inhibit avian infectious bronchitis virus infection by regulating viral replication the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © chen, muhammad, zhang, ren, zhang, huang, diao, liu, li, sun, abbas and li. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -cfjrwt authors: girkin, jason; maltby, steven; singanayagam, aran; bartlett, nathan; mallia, patrick title: chapter in vivo experimental models of infection and disease date: - - journal: rhinovirus infections doi: . /b - - - - . - sha: doc_id: cord_uid: cfjrwt abstract human and animal models continue to play a crucial role in research to understand host immunity to rhinovirus (rv) and identify disease mechanisms. human models have provided direct evidence that rv infection is capable of exacerbating chronic respiratory diseases and identified immunological processes that correlate with clinical disease outcomes. mice are the most commonly used nonhuman experimental rv infection model. although semipermissive, under defined experimental conditions sufficient replication occurs to induce host immune responses that recapitulate immunity and disease during human infection. the capacity to use genetically modified mouse strains and drug interventions has shown the mouse model to be an invaluable research tool defining causal relationships between host immunity and disease and supporting development of new treatments. used in combination the insights achieved from human and animal experimental infection models provide complementary insights into rv biology and yield novel therapeutic options to reduce the burden of rv-induced disease. that deliberately expose humans to an infectious agent are required, when there are more than enough naturally acquired infections to yield sufficient subjects for studies. despite the ubiquity of viral colds there are a number of factors that make studies of naturally acquired infections problematic. although rvs are the commonest etiological agents of viral colds there are several other viral causes (and nonviral causes of upper airway symptoms), and the clinical syndromes caused by different virus types are indistinguishable. , other factors contributing to variability in naturally acquired infections include different routes of inoculation (eye, nasopharynx, direct contact, aerosol, etc.), different inoculation doses, variability in the perception of symptoms leading to differences in time to presentation and host factors (immune status, smoking, age, etc.) that influence viral pathogenicity. further, the heterogeneous nature of naturally occurring infections requires that large patient numbers are needed to identify statistically significant effects of treatment. therefore human experimental infection studies are an attractive proposition as they allow for a known etiological agent to be administered at a standard dose, route of inoculation, and time point to a selected group of recipients with similar characteristics (e.g., age, smoking history, health/disease, and antibody status). detailed follow up can be carried out in a controlled clinical setting with sample collection at defined time points in relation to the time of infection. as the clinical syndrome induced by rv challenge in young healthy volunteers is benign and self-limiting, experimental rv infection in this group is relatively uncontroversial. perhaps the only risk to subjects was the possibility of additional infectious agents present in the inoculum and good manufacturing practice (gmp)-prepared stocks are now required by regulators for experimental infection studies in humans to contravene this risk. studies in healthy volunteers have been central to establishing the key aspects of the biology of rv infection including routes of acquisition of infection, À clinical symptoms, , inflammatory and immune responses, involvement of the lower airway, , and correlates of immune protection of rv infection. virus challenge studies have also been used in healthy volunteers to evaluate a vast array of potential treatments. however, none of these studies have led to the licensing of a single treatment for the common cold. À licensing approval was sought for an antiviral drug, pleconaril, for the treatment of rv infection. approval was denied by the food and drug administration as the adverse effects outweighed the benefits in healthy subjects with self-limiting colds. the lack of approval of any antiviral treatments casts doubt on whether continued investment in viral challenge studies is justified. however, the recognition that rv infection is associated with more severe clinical manifestations in people with chronic lung diseases such as asthma and copd provided a new impetus to research and a new direction to human experimental infection studies. up until the early s the prevailing view was that rv infection resulted in a self-limiting, mild upper respiratory tract syndrome only. there were occasional reports of rv infection associated with more severe clinical illness such as pneumonia but both scientific and pharmacological research tended to be focused on other respiratory viruses such as influenza and respiratory syncytial virus, as these were considered to be more serious human pathogens. colds had long been associated with asthma exacerbations but early studies investigating virus infection in asthma and copd exacerbations reported low detection rates. À the consensus was that asthma exacerbations were predominantly triggered by allergen exposure and copd exacerbations by acute bacterial infection. the development of highly sensitive and specific molecular diagnostic techniques using polymerase chain reaction (pcr) technology led to a revolution in viral diagnostics and a reevaluation of the role of respiratory viruses in a range of clinical syndromes. this was particularly pertinent to human rvs, which are either difficult or impossible to culture (e.g., rv-c strains) and due to the large number of serotypes diagnostic serology testing is not feasible. pcr-based diagnostic tests have a much greater sensitivity for the detection of rvs and studies using pcr revealed that the range of clinical illness associated with rv infection was much broader than previously recognized and included more severe disease syndromes such as pneumonia, bronchiolitis, acute rhinosinusitis, and influenza-like illness. in addition rvs could be detected in most asthma exacerbations, and in a substantial proportion of copd exacerbations. asthma is estimated to affect million people worldwide and copd affects . million people and was the cause of . million deaths in . much of the enormous morbidity, mortality, and healthcare costs associated with asthma and copd are related to acute exacerbations. therefore the recognition that rvs are a major cause of asthma and copd exacerbations stimulated new interest in their biology and treatment. as part of this research investigators considered whether experimental infection studies in humans could be extended from healthy volunteers to patients with asthma and copd. respiratory viruses can be detected in up to % of asthma exacerbations in children and %À % of exacerbations in adults, , À with rvs the commonest virus detected. the recognition of the role of rvs in asthma exacerbations stimulated research into their biology in an attempt to develop treatments for virus-induced exacerbations. this research included investigating whether experimental rv infection could be used in people with asthma in the same way it had been used in healthy individuals. the first experimental rv challenge of subjects with asthma was carried out in at dalhousie university, canada. of the volunteers inoculated, became infected but only had $ % decrease in forced expiratory volume in second (fev ) and an increase in airway hyperreactivity (ahr). the authors felt that these findings suggested "that other viral pathogens may play a more important role in precipitating asthma attacks." it is unclear why this study failed to induce features of asthma exacerbations but it would be almost another decade before further experimental rv infection studies in people with asthma were attempted. experimental infection studies in allergic (nonasthmatic) subjects suggested that rv infection could induce changes in lower airway physiology similar to that seen in asthma. , in an experimental infection study from the university of southampton, united kingdom included a small group of people with allergic asthma and reported that upper respiratory symptoms were more severe in atopic subjects but did not report on lower airway symptoms or physiology. concurrently a study using pcr to detect viruses in naturally acquired asthma exacerbations strongly supported a role for rv. subsequent studies were carried out by research groups in the united kingdom, the netherlands, À and the united states , in volunteers with mild, intermittent asthma. these studies demonstrated that rv infection induced airway obstruction, , increased ahr, À , and airway inflammation , , À and rv could be detected in the lower airways, , thereby supporting a causative role for rvs in asthma exacerbations. having established that respiratory viruses are a trigger for asthma exacerbations, research focused on investigating why rvs cause a benign, self-limiting illness in healthy subjects but result in more severe manifestations in people with asthma. a study of naturally acquired infections in cohabiting couples discordant for asthma suggested that people with asthma were not more susceptible to virus infection but had more lower respiratory tract symptoms. airway inflammation during naturally acquired infections was greater in people with asthma compared with those without asthma but the number of subjects in this study was small and the viruses detected were different between the two groups. viral challenge studies are ideally suited to addressing this research question as people without asthma matched for characteristics such as age and gender can be infected simultaneously. most of the earlier infection studies did not include a control group of healthy volunteers and therefore could not address the question as to whether host responses to infection differ in people with asthma. studies that did include nonasthmatic controls produced somewhat inconsistent results with one study reporting no differences in lower airway inflammatory cells, another reporting increased nasal inflammatory mediators in asthma and discrepant results regarding virus-induced respiratory symptoms. , these divergent results were likely related to differences in sampling methods and timing, antibody status of subjects, and choice of healthy controls (e.g., atopic vs nonatopic). the first study to show clear differences between subjects with and without asthma in their responses to rv infection was published in . in this study, rv challenge induced more respiratory symptoms, greater lung function impairment, increased bronchial hyperreactivity, and eosinophilic and neutrophilic lower airway inflammation in asthmatic compared with normal subjects with direct correlations between loss of lung function and the degree of neutrophilic, eosinophilic inflammation, and nasal viral load. in addition, the study provided insights into potential mechanisms of differential responses to viral infection in people with asthma. despite being infected with the same dose of virus, postinoculation virus loads tended to be higher in the asthmatic subjects compared with the healthy controls, suggesting that antiviral immunity may be impaired in people with asthma with subsequent failure to control viral replication. virologic and clinical outcomes were related to deficient interferon (ifn)-γ and interleukin (il)- responses and to augmented t-helper type (t h ) responses (il- , il- , and il- ), indicating that excessive t h or impaired t h (or il- ) immunity may be important mechanisms. when infected with rv in vitro, alveolar macrophages and bronchial epithelial cells from subjects with asthma demonstrated deficient production of ifnβ and ifnλ, and this was related to the severity of virus-induced asthma exacerbations. other reports subsequently confirmed that ifn production is deficient in asthma, , but this finding has not been replicated in all studies. À it may be that this phenomenon only occurs in a subset of people with asthma, or that it is seen in more severe or poorly controlled asthma. such patients were not initially included in challenge studies as these were limited to mild, wellcontrolled asthma not requiring inhaled corticosteroids. in rv challenge was shown to be safe in a small group of people with wellcontrolled asthma requiring long-term use of inhaled corticosteroids. a larger study confirmed this and reported significantly more upper and lower respiratory symptoms, greater reduction in peak expiratory flow and fev , increased viral loads, increased bronchoalveolar lavage (bal) eosinophils, and increased nasal il- , il- , and il- in subjects with moderate asthma using inhaled corticosteroids. this study also identified novel mediators of virus-induced asthma exacerbations including il- , il- , and il- . poor asthma control was associated with more severe virus-induced exacerbations, greater t h inflammation and higher virus load. therefore responses to virus infection may differ depending on asthma severity and control, which may account for some of the discrepant results in earlier experimental infection studies. these successful viral challenge studies should pave the way for further studies in subjects with moderate asthma that should reveal new insights into the pathogenesis of exacerbations that may not have been obtained from studies in mild asthma. the evidence that emerged from research, including experimental infection studies, that asthma is associated with deficient ifn responses led to the development of inhaled ifnβ as a treatment for asthma exacerbations. a clinical trial of inhaled ifnβ reported that treatment can reduce the severity of virus-induced exacerbations in a subgroup of patients assessed with more severe asthma. the development of inhaled ifn as a novel asthma treatment is a clear demonstration of the potential of experimental rv infection studies to contribute to the discovery of new treatments for virus-induced asthma exacerbations. the global initiative for obstructive lung disease defines copd as "a common preventable and treatable disease, characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases. exacerbations and comorbidities contribute to the overall severity in individual patients." acute exacerbations of copd are the major drivers of morbidity, mortality, and healthcare costs in copd and prevention of exacerbations a major unmet need. acute bacterial infection was believed to be the main cause of copd exacerbations and this is reflected in the widespread use of antibiotics in copd exacerbations. , although copd exacerbations are preceded by upper respiratory symptoms in up to two-thirds of cases, virus detection rates in the pre-pcr era were low. , as with asthma, the role of viruses in copd exacerbations was reexamined using pcr-based detection methods. although detection rates of respiratory viruses in copd exacerbations are more variable than in asthma, respiratory viruses can be detected in %À % of copd exacerbations, with rvs the predominant virus type detected. À despite this emerging evidence implicating respiratory viruses in a significant proportion of copd exacerbations, both scientific and clinical research continued to focus on bacterial infection. as evidence of this, it was almost two decades after the first experimental rv infection study was carried out in asthma that a similar study in copd was attempted. despite the excellent safety record of experimental infection studies in asthma, caution was warranted in repeating these studies in copd as there are major differences between these two populations. copd patients are older, current or ex-smokers, and have impaired lung function with irreversible airflow obstruction. all these factors have the potential to result in a more severe response to experimental rv challenge, compared with the younger, nonsmoking patients with relatively normal baseline lung function recruited to the asthma infection studies. the first experimental infection study in copd was a small pilot study published in that established the safety of rv infection in four patients with moderate airflow obstruction (fev %À % predicted) and not using regular inhaled therapy. the subjects developed symptoms consistent with a copd exacerbation following rv inoculation, together with objective markers of exacerbation with falls in lung function and increases in upper airway inflammatory markers. all the subjects recovered completely without treatment and no adverse events were reported. subsequently the same research group carried out two larger studies of experimental rv infection in subjects with copd and non-copd control subjects. , these studies replicated the findings of the pilot study, wherein rv infection manifested in cold symptoms, lower respiratory symptoms consistent with exacerbations of copd, including airway inflammation and worsened airflow obstruction. , these studies provided important causal evidence linking virus infection to copd exacerbations. studies from naturally acquired infection were supportive of this link but do not provide definitive evidence as pcr evaluation detects viral nucleic acid and therefore does not prove the presence of live virus and samples are only collected after exacerbation onset. respiratory virus nucleic acid can also be detected in copd patients with stable disease, although is usually elevated during copd exacerbations. in experimental infection models, rv was present in airway samples prior to exacerbation onset, virus load increased in parallel with the increase in symptoms, airflow obstruction and inflammation, and clearance of virus was associated with exacerbation resolution. , strong correlations were observed between virus load and airway neutrophil numbers, neutrophil elastase, il- , il- , and tumor necrosis factor-alpha (tnfα), granulocyte macrophage colony stimulating factor, most of which of also correlated with levels of epigenetic regulator, histone deacetylase and these inflammatory responses were greater in patients with copd. in these studies, rv infection is the sole experimental agent responsible for increased inflammatory markers in patients with copd, providing strong evidence that rv infection directly causes exacerbations in copd patients. another advantage of virus challenge studies over naturally acquired infections is the ability to carry out detailed and repeated lower airway sampling during the course of the exacerbations, including the use of bronchoscopy. this has provided a wealth of mechanistic data regarding the pathogenesis of virus-induced exacerbations including the presence of inflammatory mediators, , inflammatory cells, , , oxidative and nitrosative stress, and impaired antiviral ifn responses. a novel observation that emerged from these studies was that secondary bacterial infections occurred in % of experimental virus-induced exacerbations, whereas coinfection was rarely reported in naturally acquired exacerbations. analysis of the respiratory microbiome following experimental rv infection suggest that secondary infection occurs due to an outgrowth of previously present airway bacteria. potential mechanisms of secondary bacterial infection include reduced antimicrobial peptides and increased glucose in the airways. a subsequent study of naturally acquired exacerbations sampling at multiple time points during exacerbations confirmed the validity of this observation. therefore although the numbers of copd subjects recruited to virus challenge studies to date is small, rv infection appears to be safe in this population and replicates the features of naturally acquired infection. further studies including larger numbers of patients are needed to validate these findings and further investigate the mechanisms of virus-induced exacerbations in copd. since the first studies in healthy volunteers, experimental rv infection has been extended to patients with asthma and copd and has contributed enormously to expanding our understanding of the biology of rv infection and how it affects patients with chronic airway diseases. a summary of the key findings from human experimental infection studies in people with asthma and copd is provided in table . . these studies have tended to have a narrow focus on rv infection and host immune responses. it is clear from both in vivo and in vitro studies that there are interactions between respiratory virus infections and other factors that exacerbate asthma and copd such as bacteria, allergens, and air pollution. these factors have been somewhat neglected in viral challenge studies and are a promising field of future research that is starting to be addressed. , as mentioned previously, the use of the viral challenge model in asthma is much further advanced compared with copd. one study identified ifn-deficiency in copd but this has not been replicated. with the development of inhaled ifn as a therapy option for asthma, the role of ifn in copd requires urgent further investigation. other areas of future research include the effects of virus infection on novel pathways such as lipidomics and metabolomics in asthma and copd. respiratory viruses have also been identified as triggers of exacerbations in other airway diseases such as cystic fibrosis , and bronchiectasis , and there is evidence of impaired antiviral immunity in these diseases. experimental infection studies may help to define the role of virus infection in these patient populations. aa exposed to allergen placebo, aa exposed to rv, aa exposed to rv and allergen perhaps the most promising use of the virus challenge model will be to accelerate the process of drug development. , virus challenge studies have been used to evaluate the effects of existing asthma therapies on virus-induced exacerbations. , recently the first study using viral challenge to evaluate a novel, unlicensed drug in asthma was also published. although these studies had negative results they demonstrate the potential of the viral challenge model in drug development. the key to successful drug development is the identification of clinically relevant mechanisms of rv infection or immunopathology that can be experimentally manipulated for therapeutic benefit. this is where human experimental rv infection is complemented by work in animal models. there are a number of options for modeling human rv infection in animal models and they provide the ability to investigate specific disease components and mechanisms that would be otherwise impossible in humans. human experimental infection models have the advantage of identifying disease correlates, but the degree of experimental manipulation possible is extremely limited and the safety and effectiveness of interventions must first be evaluated in animals. animal models have proven useful for mechanistic studies across a range of diseases. models of rv infection have been reported in several animal species, including mouse, cotton rat, and nonhuman primates. each of these experimental systems provides its own advantages and disadvantages and a substantial contribution to the knowledge base of the biology of rv infection. animal models provide a range of benefits to complement human experimental approaches. experimental animals can be readily manipulated to induce consistent disease outcomes, such as the induction of allergic airway disease (aad) to model asthma or cigarette smokeÀinduced copd. animal models have less variability than human populations providing more consistent experimental outcomes and statistical power in intervention studies. experimental environment, exposures (e.g., previous infection history), endpoints, and interindividual variability can be controlled. further, a broad array of tools are available to characterize disease outcomes, including reagents, genetically modified animal strains, experimental protocols, and assessment techniques (e.g., lung function testing). sample tissues can be easily isolated at experimental endpoints, which are difficult or impossible to sample in humans (e.g., lung tissues, draining lymph nodes, bone marrow). further, animal models serve as a valuable preclinical system for the assessment of novel interventions, to provide proof-of-principle safety and efficacy findings prior to exposure of healthy human volunteers. mice in particular have been extensively used to model disease, including virus infection and exacerbations of asthma and copd. À as a result, a wealth of tools and techniques are available to study immune mechanisms and pathophysiology in mice. well-characterized protocols and reagents support the induction of disease states and allow detailed characterization of immune responses (e.g., fluorescently tagged monoclonal antibodies to quantify immune cell subsets). a range of transgenic and knockout mouse strains are available that allow for the careful dissection of relevant disease mechanisms. further, reagents are available to assess the effects of novel interventions on disease outcomes (e.g., blocking antibodies and various forms of innate immunity activators; see chapter : emerging therapeutic approaches). the expansion of mouse rv infection models has paralleled our understanding of rv biology in humans. initial approaches focused on understanding the effects of rv infection in isolation, identifying the mechanisms and cell types mediating lung pathology. increasingly complex experimental models are now being used to characterize long-term effects of infection on airway function and the effects of rv infection on preexisting airway disease (e.g., asthma exacerbations). À one of the biggest developments in mouse rv infection models was the protocol for isolation of highly purified, concentrated (high titers) of rv from henrietta lacks (hela) immortalized human epithelial cell lines and later, the intracellular adhesion molecule (icam- ) transfected rhabdomyosarcoma cell lines. , prior to this, clarified infected hela cell lysates were used for in vitro experiments. some investigators also used infected hela cell lysates in mouse models. efforts to improve the quality and validity of the model made use of a partial purification protocol to generate viral stocks. for mouse models of rv infection or exacerbations, the refined, high-titer, rv purification protocol is the gold standard. a major barrier that hindered the early development of mouse models was the species specificity of rv infection. "major-group" rv strains, which make up approximately % of all rv strains, enter the cell through binding of icam- . , rv binding to icam- is limited to human and chimpanzee and does not occur in other species, including mouse. as a result, major-group rv strains cannot infect mouse cells and fails to replicate or induce pathology in mouse models. early attempts to develop rv mouse models failed to detect sufficient viral replication to induce disease. a major advance enabling the development of mouse rv infection models was the initial recognition that the minor-group rv-a , which use the host cell receptor low-density lipoprotein receptor, can infect the mouse epithelial cell line la- . this recognition suggested that minorgroup rv viruses (e.g., rv-a ) may be useful to model infections in mice in vivo. indeed, inoculation of wild-type balb/c mice with rv-a induced lung pathology, mucus production, and inflammatory cytokine production. notably, rv infection was also sufficient to induce exacerbations of preexisting asthma in sensitized and challenged mice (as discussed in more detail below). an alternate approach was also sought to allow modeling of majorgroup rv infection in mice. the same study by tuthill et al. demonstrated that transfection of la- cells with a chimeric icam- receptor containing the human extracellular receptor domains allowed infection and replication by the major-group virus rv-a . this finding provided the basis for developing a transgenic mouse strain expressing a chimeric mouseÀhuman icam- receptor. chimeric receptor expression in hu-icam tg mice is sufficient to support in vivo infection with rv-a , resulting in airway inflammation, mucus production, viral replication, and inflammatory cytokine production. of note, the hu-icam tg mouse was generated by random insertion of a chimeric transgene and little is known about the transgene insertion site within the genome. use of this transgenic strain requires additional experimental considerations (e.g., genotyping and use of heterozygous animals in experiments), which has limited its broad utility. additional variations have also been reported in the literature, which aim to broaden the available mouse models. genetic rv-a variants have been generated by serial passage through mouse embryonic fibroblasts in vitro and lung epithelial cells in vivo, which exhibit increased growth in mouse cells. inoculation of balb/c mice with the rv-a / m m variant [ plaque forming units (pfu)] allowed for recovery of virus from mouse lung after hours, when mice were also pretreated with intranasal hydrochlorous acid to increase epithelial permeability. successful use of mouse models also depended on the development of streamlined rv isolation protocols that have allowed consistent and rapid isolation of virus stocks, to limit variability between experiments. the current gold-standard protocols for rv isolation, rv-a infection of wild-type balb/c mice, and the infection of transgenic mice expressing chimeric mouseÀhuman icam- receptor with rv-a are published and readily available. a range of studies have assessed the effects of primary rv infection in mice, contributing to our understanding of the mechanisms underlying disease pathogenesis. studies have used similar protocols, typically performing intranasal inoculation of b À tcid (tissue culture infective dose in % of culture, a titration of infection units of pathogens that do not form plaques in culture) rv-a and assessing responses over week following infection in balb/c or c bl. mice. in the initial publication by bartlett et al., intranasal inoculation of wildtype balb/c mice with tcid rv-a induced a range of disease features similar to human disease. rv infection induced airway inflammation characterized by increased neutrophil numbers at and hours postinfection and increased lymphocyte numbers persisting for -week postinfection. tissue pathology was characterized by perivascular and peribronchial inflammation, increased mucus production, and elevated inflammatory cytokine production (including mip- , kc, mip- α, ip- , rantes, itac, il- , il- β, ifnα/β/λ/γ). furthermore, rv infection resulted in the development of an rv-specific adaptive antibody response by days postinfection. further studies have provided insights into the effects of rv infection alone in mice. following inoculation of wild-type c bl/ mice with tcid rv-a , detectable rv positive-and negative-strand rna were recovered from the lung, indicative of active viral replication and viral rna was detectable up to days postinfection. the study also noticed a small increase in airway neutrophils and lymphocytes in the presence of uv-inactivated rv-a , although uv-inactivated rv-a (and major-group virus rv- ) failed to induce inflammatory cytokine production. rv-a infection also increased airway responsiveness to methacholine challenge at days and postinfection. inoculation with rv-a or uv-inactivated rv-a induced pi k activation in airway epithelial cells and pretreatment with the pi k inhibitor ly in vivo dampened neutrophilic inflammation and inflammatory cytokine production (kc, mip- , mip- α, ifnγ). inoculation of c bl/ mice with rv-a ( tcid ) leads to discontinuous expression of zonula occludens- , suggesting that infection disrupts airway epithelial barrier function. rv infection also stimulated il- production and release into the airways, which is dependent on type i ifn production and stimulates activation of natural killer (nk) and cd t cell responses. treatment with an il- Àil- ra complex increased expression of il- , il- rα, ifnγ, and cxcl and stimulated increased nk, cd , and cd t cell recruitment and activation. ccl and irf- are the most upregulated lung transcripts following rv-a infection. blocking ccl or irf- function reduced lung neutrophil and macrophage accumulation and ifn responses and blocking ccl also reduced ahr. this publication also delineated ahr from inflammatory infiltrates showing instead a relationship between classical proinflammatory transcription factors (nfκb) and ahr. as alluded to previously, the use of knockout mice in particular has provided insights into a number of mechanisms regulating rv-induced pathology. key roles for neutrophils and the neutrophil chemokine receptor cxcr in mediating rv-induced pathology were identified. inoculation of cxcr / mice with rv-a ( . tcid ) resulted in reduced airway neutrophil numbers, reduced inflammatory cytokine production (tnfα, mip- , kc), decreased mucus production, and decreased cholinergic responsiveness, with no alteration in viral load, compared with wild-type control animals. further, antibody depletion of neutrophils and infection of tnfr / mice also reduced ahr, compared with control animals. these findings provide evidence for a role of neutrophilic inflammation, potentially via tnfα production, on downstream pathology following rv infection. roles for pattern recognition molecules have been demonstrated for mda , toll-like receptor (tlr ) and tlr . infection of mda / mice resulted in delayed type i ifn (ifnα/β) and suppressed type iii ifn expression, with a slight early increase in viral load in the lung. in contrast, inoculation of tlr / mice resulted in normal ifn responses and no difference in viral yield. both mda / and tlr / mice had reduced neutrophil numbers, inflammatory cytokine production (cxcl , cxcl , ccl , cxcl ) and airways responsiveness, compared with wild-type controls. differing roles for nfκb signaling pathways in rv-induced inflammation and type i inf responses in antiviral immunity have been demonstrated. disruption of nfκb signaling in p / mice resulted in reduced neutrophil numbers and inflammatory cytokine production (cxcl , cxcl , cxcl ), while ifn production and viral loads are unaltered. in contrast, ifnar / mice have unaltered neutrophilic inflammation, a persistent increase in lymphocyte numbers and cytokines ccl , cxcl , and cxcl , with reduced ifnα production and increased viral load. a pathogenic role for the proinflammatory molecule muc has also been demonstrated, with increased expression of antiviral genes (mx , ip- ), reduced neutrophil inflammation and viral load in muc / mice following rv-a inoculation ( pfu). studies using tbet / mice (a key regulator of t h cell differentiation) have also demonstrated the key role for t h -polarized t cells in the response to rv infection. tbet / mice developed a t h / t h -polarized immune response to rv infection ( tcid ) with increased il- and il- a production, deficient nk cell responses, and decreased neutralizing antibody development. cd t cells contributed to increased airway eosinophil numbers and mucus production following rv infection in tbet / mice. studies using tslp receptor-deficient mice (tslpr / ) demonstrated that rv-a infection interferes with tolerance to an inhaled allergen, via a mechanism requiring tslp, il- , and activation of ox l on lung dendritic cells. after observing increased levels of the tnf super family member protein, tnfsf (trail or cd ) production over a time course of rv-a infection in mice, girkin et al. compared rv-a infection in tnfsf / mice to wild-type balb/c mice and observed an almost complete ablation of inflammatory responses to rv-a . following rv infection, peribronchiolar inflammation and lung histopathology were reduced in tnfsf / mice; neutrophil and lymphocytes in bal remained at baseline; and cd t cells, cd t cells, nks, plasmacytoid dendritic cells (pdcs), and myeloid dendritic cells were all reduced in flow cytometry of total lung cells. tnfsf / mice were protected from rv-induced ahr, and failed to develop rv-induced exacerbations of allergic airways disease. an interesting proviral effect of trail was also identified whereby tnfsf / mice had reduced viral load and anti-trail antibodies reduced viral load (whereas recombinant trail administration increased viral load) in beas b cells infected with rv-a in vitro. this effect on viral load was independent of ifn responses and may be associated with an unidentified role of apoptosis in rv replication, which remains to be explored. some studies have assessed the effect of primary rv infection on clinically relevant sequelae, including secondary bacterial infection and the effects of premature birth on infection. exposure of epithelial cells in culture to rv-a resulted in increased bacterial attachment and translocation through an epithelial monolayer (nontypeable haemophilus influenzae (nthi), pseudomonas aeruginosa, staphylococcus aureus), suggesting a potential mechanism underlying secondary bacterial infections following viral infection. a subsequent study demonstrated that primary inoculation with rv-a ( tcid ) delayed the clearance of nthi in vivo, associated with suppressed chemokine production (kc, mip- ) and neutrophilic inflammation through a tlr -mediated mechanism. the model has also been used to assess immune alterations relevant to premature birth and bronchopulmonary dysplasia, risk factors for viral-induced exacerbations. exposure of neonatal mice to hyperoxia ( % oxygen) in early life increased inflammatory cytokine expression (il- , ifnγ, tnfα, ccl , ccl , ccl ) and suppressed early ifn responses following rv-a infection ( pfu) at days of age. one study has also assessed the effect of rv infection timing on subsequent development of aad. inoculation of -day-old mice with rv-a and subsequent induction of house dust mite (hdm)-induced allergic airways disease had additive effects with increased neutrophilia and ahr in female mice, although rv inoculation had no additional impact in male mice. these studies extend the use of rv infection in mice to new areas, including mechanisms of early life infection susceptibility, to mechanisms of secondary bacterial infection/compromised antimicrobial immunity and experimental exploration of clinical risk factors associated with increased likelihood to develop virus-induced exacerbations of respiratory diseases. mouse models are valuable tools for the preclinical testing of novel treatments. several studies have used the mouse rv infection model to assess intervention strategies, including vaccine development and drug treatment. primary inoculation of balb/c with rv-a ( tcid ) rapidly induced circulating rv-specific igg antibody production within days, which binds capsid protein vp and those antibodies were crossreactive to another minor strain rv (rv- ). repeated rv infections were necessary to induce rv-specific iga responses and neutralizing antibodies, but administration of cpg or subcutaneous immunization with freund's adjuvant promoted neutralizing antibody development and may inform potential vaccine strategies. pretreatment with the plant flavanol quercetin before and during rv-a infection effectively reduced viral replication, inflammatory cytokine production (kc, mip- , tnfα, ccl , ifnα, and ifnλ ), and ahr. treatment with the cancer therapeutic gemcitabine ( , -difluorodeoxycytidine) reduced rv load, inflammatory cytokine levels (tnfα, il- β), and reduced lung lymphocyte numbers. treatment with corticosteroid therapy (fluticasone proprionate) suppressed ifn responses to rv and reduced airway inflammation, leading to increased mucus production and reduced antimicrobial responses. effects on viral load, mucin production, and antibacterial response could be reversed by administration of recombinant ifnβ. despite promising findings in mouse models, quercetin has not entered clinical trials for the treatment of rv infection, likely due to a previous randomized community clinical trial in that showed little benefit of quercetin supplementation on upper respiratory infections. these findings may highlight the limitation of mouse models, which (while valuable) do not always fully recapitulate human disease mechanisms. animal models to study rv-mediated exacerbations of airway disease have also been developed. these models combine experimental rv infection with models of airways disease, including asthma, copd, and chronic rhinosinusitis. models of asthma typically consist of administration of a sensitizing agent [e.g., ovalbumin (ova) or hdm] and subsequent challenge in the airways to induce an eosinophilic, allergic airways disease. copd is typically induced by prolonged and repeated exposure of mice to cigarette smoke or treatment with elastase. after airways disease is established, mice are then inoculated with rv to induce disease exacerbations. these models are explained and expanded upon in the following sections. researchers have also used double-stranded rna (dsrna) administration as a surrogate for virus infection to exacerbate preexisting disease (reviewed in refs. [ , ] ). however, these approaches fail to model the complexity of virus infection and are beyond the scope of the current book chapter. many studies have characterized the effects of rv infection on preexisting asthma and have provided insights into the immune cell types involved, key molecules, and responses to potential therapies. in the initial report of an rv (rv-a ) exacerbation model, ova-sensitized and challenged balb/c mice were inoculated with rv-a during the allergen challenge phase. the combination of virus and allergen challenge increased airway neutrophil, eosinophil, and lymphocyte numbers; increased cytokine production (il- , il- , and ifnγ), increased ahr; and increased mucus gene expression. subsequent studies have identified key functional roles for macrophages, gamma-delta (γδ) t cells, dendritic cell subsets, and neutrophils in rv-induced immunopathology. in a similar model, rv-a inoculation into ova-sensitized/challenged mice increased macrophage lung infiltration and eotaxin- /ccl expression. eotaxin was expressed by pulmonary macrophages in the lung after combined virus infection and allergen challenge. further, macrophage depletion or antieotaxin treatment reduced rv-induced airway eosinophilia and ahr. macrophage activation state also modulates the response to rv infection in allergen sensitized/challenged mice and shapes the resulting pattern of inflammation. rv infection in asthma exacerbation models induced an il- expressing macrophage population, with m polarized phenotype. depletion of il- -producing cells in cd b-dtr mice or ccr / mice reduced airway inflammation and ahr. interestingly, while rv infection of ova-treated wild-type mice contributes to mixed neutrophilic and eosinophilic airway inflammation and m macrophage phenotype, il- r / mice exhibit neutrophil inflammation alone and increased m polarization of pulmonary macrophages but still have exacerbated airway responses. γδt cells dampen exacerbation responses. γδt cells are increased in rv-induced asthma exacerbation models and blocking responses with anti-γδtcr antibody worsened exacerbations with increased ahr, and increased numbers of t h cells and eosinophils, with no effect on virus load. pdcs were recruited to the lung during rv-induced inflammation and subsequently promoted t h responses in the lung draining lymph nodes, in a process mediated by il- . depletion of pdcs with an antibody or treatment with anti-il- reduced eosinophil numbers, decreased lung pathology, reduced cytokine production (il- , il- ), and reduced ahr. functional roles for neutrophils, and neutrophil extracellular traps (nets), have also been provided in rv-induced asthma exacerbation models. chronic low-dose hdm exposure and rv infection have additive effects on neutrophilia and induce ahr. a more recent study demonstrated that rv infection in an hdm-mediated asthma model results in double-stranded dna release into the airways and administration of genomic dna alone was sufficient to mimic characteristic components of rv-induced exacerbations. further, blocking neutrophil elastase or degrading nets by applying dnase into the airways reduced eosinophil and lymphocyte numbers, tissue pathology, and cytokine production (il- , il- ). a number of studies have highlighted the functional roles of specific molecules in rv-induced mouse exacerbation models, as potential therapeutic targets to validate in patient populations. in addition to roles during rv infection alone highlighted above, mda and tlr are also involved in rv-induced exacerbations. mda / and tlr / mice have decreased inflammatory responses and ahr, while mda / also had decreased ifn responses (ifnβ/λ /λ ). midline (a e ubiquitin ligase) is upregulated in an hdm-induced model, and short interfering rnaÀmediated inhibition prior to rv inoculation reduced neutrophil numbers and mucus production production. the monocyte chemotactic protein ccl is produced by epithelial cells and macrophages following rv-induced exacerbation and administration of an anti-ccl antibody reduced eosinophil numbers and ahr. foxa -overexpressing transgenic mice produce excess mucus in their airways and rv infection increased foxa expression. in foxa -deficient mice (foxa / ), rv clearance is increased, with increased ifnβ activation. il- expression is also increased in rv-induced exacerbations and blocking the il- receptor reduced type cytokine production (il- , il- , il- , il- , il- , tslp), mucus production, and numbers of eosinophils, neutrophils, t cells, and innate lymphoid type cells. combining dsrna administration with rv-a inoculation worsened preexisting allergic airways disease. repeated dsrna administration after ova sensitization/ challenge resulted in neutrophilic lung inflammation and tissue pathology and combined dsrna and rv-a inoculation increased expression of tslp, tnfα, and ifnλ in the lung. key roles for pattern recognition receptors have also been demonstrated in rv asthma exacerbation models. hdm-allergic tlr / mice had a decreased antiviral response, with reduced ifn release (ifnα/β/λ /λ /λ ) and increased virus replication, associated with increased eosinophil and lymphocyte numbers, increased il- and ccl , and ahr. administration of ifn or transfer of wild-type tlr -competent pdcs could restore antiviral responses and reduce disease exacerbation. ova-allergic tlr / mice also had reduced macrophage, neutrophil, and eosinophil numbers and suppressed ahr after rv inoculation. bone marrow transfer experiments demonstrated that tlr / bone marrow could protect from exacerbations, while transfer of wild-type bone marrow restored responses in tlr / mice. transfer of wild-type macrophages into tlr / mice could also restore exacerbations. as previously mentioned, a role for trail has also been demonstrated, with hdm-allergic trail-deficient mice (tnfsf / ) protected from rv-induced ahr and induction of airway inflammation. rv-induced asthma exacerbation models have also been used to assess the responses to existing therapies and as preclinical models for novel therapies. treatment of hdm-allergic mice with the long acting beta- agonist salmeterol reduced ahr and eosinophil numbers during rv exacerbation, and limited chemokine levels (ccl , ccl , cxcl ) through modulation of pp a. the findings of this study were focused on pp a as a novel therapeutic target rather than promoting salmeterol monotherapy (which was associated with adverse events and tolerance to β -agonists with chronic salmeterol use ). an approach to block majorgroup rv virus infection was assessed through administration of antihuman icam- antibody, which prevented entry of rv-a and rv- and reduced neutrophil and lymphocyte numbers, cytokine production (il- , il- , il- , ccl , ccl ), mucus production, and virus load in human transgenic mice in an ova-allergic model. treatment with a nontoxic anthraquinone derivate reduced rv-induced ahr, neutrophil, and eosinophil airway inflammation; inflammatory cytokine production; and mucus hypersecretion while also boosting type ifn response and reducing viral yields, with associated decreased akt, hif- α, and vegf production. treatment with an antiinflammatory vap- /ssao inhibitor, pxs- a, or the macrolide antibiotic azithromycin also reduced neutrophil numbers and pxs- a reduced ahr following rv-a inoculation in hdm-allergic mice. . . mouse chronic obstructive pulmonary disease exacerbation models rv also plays an important role in virus-induced exacerbations of copd. several studies have assessed the effects of rv inoculation in animal models of copd. exposure to elastase and lipopolysaccharide (lps) once per week for weeks induces features of copd, including airway inflammation, goblet cell metaplasia, and altered lung function. addition of rv-a led to persistence of viral rna ( . days postinfection), deficient ifn responses (ifnα/β/γ) and increased ahr, lung volume, cytokine production (tnfα, il- , il- ), and mucus production, compared with elastase/lps administration alone. a subsequent study attempting to replicate the elastase/lps model of copd found that a single elastase treatment followed by rv-a inoculation was enough to increase airway neutrophil and lymphocyte numbers, increased inflammatory cytokine production (tnfα, cxcl , ccl ), mucus hypersecretion, and ahr. in the same elastase-induced model, fluticasone proprionate treatment reduced ifn responses, increased viral load, suppressed airway immune cell numbers (lymphocytes and neutrophils), suppressed inflammatory cytokines (il- , tnfα), and increased mucus production, following rv-a exacerbation. the differences in the experimental approaches required to elicit an rv-induced exacerbation in these different studies is likely due to the quality of virus inoculum used by the different investigators, which as explained previously, is influenced by purification approach. the first study used only crude virus-infected cell lysates, whereas the later study employed a highly purified virus inoculum. several studies have also reported on rv infection in a cigarette smo-keÀinduced copd model. in an initial study, weeks of cigarette smoke exposure resulted in increased viral persistence, neutrophilia, and increased mucus production following rv infection. subsequent studies demonstrated that goblet cell gene expression was reduced following treatment with a gamma-secretase inhibitor (gsk l , ) to limit notch activation. further, supplementation of feed with quercetin reduced rvinduced lung inflammation (including neutrophilia), goblet cell metaplasia, and ahr. to our knowledge, only one study has assessed the effect of rv infection in a chronic sinusitis model. a mouse model of chronic allergic rhinosinusitis was induced by weeks of repetitive nasal ova challenges. increased rv-a yields were reported in the nasal tissue of mice with rhinosinusitis, although inflammatory cytokine production and histopathology were unaffected. this study served to illustrate the range of additional diseases where rv infection has been shown to be relevant in human populations where animal models are available for future research (e.g., cystic fibrosis). a limited number of studies reported the use of rv infection in other animals, namely cotton rats and nonhuman primates. we note that historical studies assessing "rv" infection in other animal species are referring to genetically distinct viral genera and should not be confused with human rv (e.g., equine rv and bovine rv). for example, while human rv and equine rv were both originally assigned to the rhinovirus genus, they have been reclassified into enterovirus and apthovirus, respectively. equine rv has subsequently been renamed "equine rhinitis virus." the cotton rat (sigmodon hispidus) is a recognized model for human respiratory infection, particularly for respiratory syncytial virus, as well as adenoviruses, parainfluenza virus, measles, and human metapneumovirus (reviewed in ref. [ ] ). to date, two studies have reported on rv infection in cotton rats, providing evidence that cotton rats are partially permissive to rv major-group infection. intranasal inoculation of rv-a ( pfu) into cotton rats induced lower respiratory histopathology, increased mucus production, and induction of inf-activated genes. immunization with live rv-a -induced high levels of circulating antibodies and protected from subsequent infection, while prophylactic transfer of anti-rv-a serum also protected from disease. further, this protection was transferred effectively from mother to newborn, limiting viral yields in subsequently infected progeny. in a later study, the same group provided evidence that infection with rv-b ( pfu) induced similar disease pathology. furthermore, immunization with rv-b provided protection from subsequent infection with either rv-b (an rv-b group virus) or rv-a (an rv-a group virus), demonstrating some degree of crossreactivity to very different major-group viruses. chimpanzees and gibbons are the only nonhuman primates that support rv infection, although rv infection has also been reported in the vervet monkey cells, with consistent infection requiring high dose exposure. initial rv infection studies in chimpanzees were reported in , using rv-b and rv-a and in gibbons in . subsequent studies in chimpanzees and gibbons assessed the antiviral effects of drug treatments on rv infection, using bis-benzimidazole and triazinoindole, respectively. , administration of soluble truncated form of human icam- can prevent subsequent infection in chimpanzees. however, it has been noted that neither chimpanzees nor gibbons develop "cold" symptoms following rv infection and the high costs and logistics of these studies has limited further progress. chimpanzees are an endangered species and require considerable resources and facilities for research. current chimpanzee research is limited to the united states and gabon. however, the national institutes of health in the united states have indicated that they are seeking to eliminate the use of chimpanzees in research. all but one species of gibbon are endangered. thus clinical research using nonhuman primates in the future to characterize rv infection are likely to be limited or nonexistent. as rv does not normally infect rodents, an attenuated mengovirus infection model has been proposed as an alternative option to model rv infection. mengovirus also belongs to the picornaviridae family and normally causes systemic infection in rodents. using an attenuated mengovirus, intranasal inoculation of pfu into rats increased airway neutrophil and lymphocyte numbers, induced lung tissue pathology, and increased expression of cxcl and ccl . a subsequent report using a genetically attenuated mengovirus vmc( ) in mice also induced lower respiratory tract infection with increased lung neutrophil and lymphocyte numbers, expression of cxcl , cxcl , cxcl , il- a, infs, and chemokines cxcl and ccl . other respiratory virus infections are associated with acute exacerbations of asthma and copd, including respiratory syncytial virus, influenza, human coronavirus, human parainfluenza virus, human metapneumoviruses, and adenoviruses. animal models for these infections have largely been limited by the specificity of viruses to humans. it is unclear to what extent the mechanisms causing pathology differ between different viruses (or between strains of the same virus). a detailed discussion of the disease processes induced by each of these different virus infections is beyond the scope of this chapter. a detailed analysis of the relevant disease mechanisms in each infection setting is necessary to inform our understanding of disease exacerbations and ideally to identify common mechanisms between viruses that can be targeted for therapy. no animal model can completely recapitulate naturally occurring human rv infection. while animal models provide important insights into disease mechanisms, it is important to also recognize their limitations. there are recognized limitations of mice as models of human respiratory disease. these include differences in response/symptoms between other species and humans. there are differences in respiratory tract architecture in human, nonhuman primate, and mouse airways. they range from dichotomas (each airway splits into two), trichotomas (airways split into threes), or monopodial branching (central airway continues while subordinate airways branch out) with differences in airflow inhomogeneities covered in detail by miller et al. there are also differences in mucus production processes in mouse compared with human airways. the short lifespans of laboratory animals do not capture the life-course of human disease, mice do not naturally develop asthma or copd, and most current models of asthma represent eosinophilic, allergic patterns of disease. it remains unclear to what extent the current models and pathophysiology truly reflect human disease (particularly considering recognized heterogeneity of the human population). rv has evolved for efficient replication in the human respiratory tract. due to the decreased efficiency of rv entry into nonhuman epithelial cells (and likely differences in the nuances of cellular machinery required for replication), a high amount of viral load is required to elicit a biological response to rv in laboratory animals (e.g., tcid in mouse vs À tcid in experimental human infection models). human rv strains also demonstrate limited viral replication and different replication kinetic between mouse and man. these differences highlight the importance of confirming findings in relevant patient cohorts/samples and the utility of using human experimental models in parallel with animal models. this point is not purely for academic consideration. more so, it is important to take into consideration clinical trial design and outcomes. for example, mouse models highlighted the key relevance of il- in asthma pathology through use of knockout mice and antibody blockade. however, the initial randomized control trial assessing anti-il- therapy (mepolizumab) in a broad asthma population failed to demonstrate any clinical effect. it was not until subsequent trials limited recruitment to patients with demonstrable eosinophilic asthma (a patient subset that is more closely modeled by the experimental mouse system) that clinical improvements were observed. , . . future directions for animal models in a similar way to experimental human infection models, there has been a narrow focus in animal models. in mice, focus has largely been on rv infection alone with a growing body of literature assessing asthma exacerbations. while difficult to model in mice, rv-induced copd exacerbations models are emerging through use of elastase administration and cigarette smokeÀinduced copd. a summary of key findings from mouse models of rv-induced exacerbations of airway disease is presented in table . . limited studies have reported on rv effects or potential interventions in these models. as with human experimental infections, animal infection models may also be relevant to an expanding array of diseases in the future (e.g., cf, bronchiectasis). to date, there has been limited assessment across different rv strains in both animal and human studies. the primary focus of rv models has been on rv-a in mice, or rv-a and rv-a in human, possibly due to the availability of these strains and the ease of growing these strains in cell culture. in particular (due to its relatively recent discovery), rv-c infection has yet to be assessed in animal models. there has so far been difficulty in generating sufficient quantities of rv-c for research purposes (particularly at infectious titers required for mouse models). with the recent establishment of a suitable cell line (e hela cells) that supports rv-c replication this gap in the literature will likely be rectified. elastase/lps rv-a nil deficient antiviral immunity increased inflammation exaggerated ahr increased mucin expression as is the case for the majority of human virusÀmouse infection models, the mouse is semipermissive to rv infection and as such a high-titer inoculum is required to induce prolonged replication and robust, reproducible host immune responses. a mouse-adapted rv strain (rv- bm) has been generated by serial passage in mouse epithelial cells (la- cells) though this mouse-adapted virus has only been characterized in vitro with primary mouse tracheal epithelial cells and has not yet been tested in vivo. the clinical translation of novel therapies identified in animal models for the treatment of rv infection in humans is yet to come to fruition. however, there are multiple molecules currently in the drug development pipeline, ranging from virus-targeting molecules, drugs targeting host factors of the viral replication cycle, and biologics such as innate immune stimulators and cytokine blocking monoclonal antibodies, all of which are elaborated on in chapter , emerging therapeutic approaches. there are significant opportunities for further research in both human and nonhuman models, including assessment of infections in various unexplored disease backgrounds that are exacerbated by rv infection (e.g., cystic fibrosis) and expansion of studies using newly identified rv strains (e.g., rv-c strains). human and mouse rv experimental infection models effectively complement each other and have contributed immensely to our understanding the mechanisms shaping rv-induced pathology. human experimental rv challenge studies have shed light on the biology of rv infection and the mechanisms associated with rv-induced exacerbations of chronic respiratory diseases. mouse models of rv infection in particular are readily manipulatable to identify cause and effect between specific molecules and disease outcomes for preclinical testing. an excellent example of how human and mouse models complement each other is the growing understanding of the disease mechanisms during rvinduced asthma exacerbations. human experimental infection revealed a potential role for induced type immunity following rv infection in individuals with asthma. , subsequent mouse model studies have demonstrated a causal role for rv-induced type immune effector molecules in exacerbations, , , , allowing preclinical assessment of the efficacy and safety of novel therapies. findings from these studies have not yet resulted in the development of approved therapies for rv infections, cold symptoms, or exacerbations of respiratory diseases associated with rv infection. however, the wealth of knowledge derived from experimental rv infections has broadened our understanding and identified many potential therapeutic approaches. can we distinguish respiratory viral infections based on clinical features? a prospective pediatric cohort compared to systematic literature review the viral etiology of 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affects several pathways of the host innate immune response. previous studies in bovine cells demonstrated that deletions (leaderless [llv]) or point mutations in lpro result in increased expression of interferon (ifn) and ifn-stimulated genes (isgs), including, among others, the ubiquitin-like protein modifier isg and the ubiquitin specific peptidase usp . in addition to its conventional papain-like protease activity, lpro acts as a deubiquitinase (dub) and deisgylase. in this study, we identified a conserved residue in lpro that is involved in its interaction with isg . mutation w a rendered escherichia coli-expressed lpro unable to cleave the synthetic substrate pro-isg while preserving cellular eif g cleavage. interestingly, mutant fmdv w a was viable. overexpression of isg and the isgylation machinery in porcine cells resulted in moderate inhibition of fmdv replication, along with a decrease of the overall state of isgylation in wild-type (wt)-infected cells. in contrast, reduced deisgylation was observed upon infection with w a and leaderless virus. reduction in the levels of deubiquitination was also observed in cells infected with the fmdv lprow a mutant. surprisingly, similarly to wt, infection with w a inhibited ifn/isg expression despite displaying an attenuated phenotype in vivo in mice. altogether, our studies indicate that abolishing/reducing the deisgylase/dub activity of lpro causes viral attenuation independently of its ability to block the expression of ifn and isg mrna. furthermore, our studies highlight the potential of isg to be developed as a novel biotherapeutic molecule against fmd. importance in this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (fmdv) leader proteinase (lpro) (w ) that is involved in the interaction with isg . mutation in lpro w (a -lprow a) resulted in reduced deisgylation in vitro and in porcine-infected cells. impaired deisgylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of lpro to block expression of type i interferon (ifn) and other ifn-stimulated genes. moreover, overexpression of isg resulted in the reduction of fmdv viral titers. thus, our study highlights the potential use of lpro mutants with modified deisgylase activity for development of live attenuated vaccine candidates, and isg as a novel biotherapeutic against fmd. fmdv is genetically highly variable, and as such, it displays seven distinct serotypes, namely a, asia- , c, o, and southern african territories to (sat to ), and numerous subtypes. upon infection, the virus spreads very rapidly, usually achieving % morbidity. strict trading policies and use of an effective inactivated virus vaccine has helped eradicate the disease from many countries; however, fmd remains endemic in most of the world, preventing the development of regions that rely on agriculture for subsistence. in parallel, occasional outbreaks in previously declared fmd-free regions may cause economic devastation ( ) . there is a need for novel preventive and therapeutic strategies for controlling this disease. understanding virus-host interactions should help to identify novel cellular factors and mechanisms that participate in antiviral immunity against fmdv and could provide alternatives for therapeutic discovery. during viral infection, expression of type i interferon (ifn) is induced, leading to the upregulation of ifn-stimulated genes (isgs) which play a range of antiviral effector functions within the infected and neighboring cells ( ) . regulation of ifn expression is the most essential target for viruses to evade and suppress innate immunity. we and others have shown that in the case of fmdv, downregulation of ifn and ifn-stimulated responses is mainly driven by the action of the viral leader protease (lpro) ( ) . fmdv lpro is a papain-like protease (plp) known to block the cellular innate immune response, at both the transcriptional and translational level by utilizing different mechanisms, including (i) shutting down translation of host capped mrnas through the cleavage of the translation initiation factor eif g ( , ); (ii) downregulating ifn mrna expression by causing degradation of nf-b, irf- , irf- , and lgp ( - ); (iii) targeting the chromatin remodeling machinery to disrupt the expression of ifn and isg mrnas ( ) ; and (iv) targeting of g bp / to block stress granule formation ( ) . it is important to note that other fmdv proteins have also been shown to negatively impact ifn and other cellular immune responses ( ) . ubiquitination is a posttranslational modification that plays a role at different points of the signaling cascade of innate immunity and involves the sequential reaction of three distinct types of enzymes, namely ubiquitin (ub)-activating enzymes (e s), ubconjugating enzymes (e s), and ub ligases (e s). similarly, the ub-like (ubl) modifier isg is conjugated to target proteins in a process known as isgylation by the consecutive action of three enzymes that make up the isgylation machinery (e -ube l, e -ubch , and e -herc ). however, unlike ub, isg and the isgylation machinery are robustly induced by type i ifn ( ) and can be upregulated upon viral infection ( ) . different receptors, adaptor proteins, and kinases are conjugated by ub molecules to activate and transduce the downstream signaling for efficient production of the ifn, isgs, and proinflammatory cytokines ( ) . in the case of isg , isgylation can extend the activation state of certain signaling proteins, resulting in higher production of ifn and isgs ( , ) . to regulate the overactivation of these pathways, cells express multiple enzymes capable of removing ub or isg from specific targets, and they are known as deubiquitinases (dubs) and deisgylases (e.g., usp ). similarly, viruses counteract induction of the antiviral immune response by reversing ubiquitination and isgylation from host targets ( ) ( ) ( ) . in some cases, changes in viral pathogenesis have been observed by dub/deisgylase gain of function due to viral recombination in natural environments ( ) . in particular for fmdv, it has been shown that overexpressed lpro displays dub activity, catalyzing the removal of ubiquitin from cellular substrates, including traf , traf , tbk, and rig-i ( , ) , which are all mediators of the induction of ifn ( ) . most recently, studies conducted in bhk- cells have demonstrated that during fmdv infection cellular proteins undergo deisgylation ( ) . interestingly, the authors demonstrate that recombinant fmdv lpro displays deisgylase activity on synthetic substrates; however, specific cellular host targets have thus far not been identified. in order to elucidate intermolecular interactions between highly conserved amino acid residues in lpro and isg , we conducted structural analysis followed by molecular modeling. our analysis revealed the presence of an aromatic residue on lpro, w , that is required for optimal deisgylase activity in an in vitro cleavage assay. importantly, engineering of an infectious clone carrying this mutation (lprow a) rendered a viable fmdv with a perceptible level of attenuation and reduced deisgylation and dub activity compared with wild-type (wt) virus during infection of porcine cells. interestingly, reduced deisgylation and dub function did not disrupt lpro's ability to block ifn and isg expression during infection, although overexpression of isg resulted in a significant reduction of fmdv replication. most importantly, in vivo inoculation with fmdv w a using an fmd mouse model ( ) resulted in reduced lethality compared with inoculation with wt virus, suggesting that the inability to remove isg confers viral attenuation in vivo. our studies reveal that reducing/ abolishing deisgylase activity in lpro during infection renders the virus moderately attenuated independently of its ability to block the expression of type i ifn and other ifn-stimulated genes (isgs). molecular modeling of lpro in combination with isg . in order to reexamine lpro deisgylase function, we first investigated molecular homology through structural superimposition of lpro with cellular murine deisgylase usp coupled with isg ( ) using catalytic residues as tether points (fig. a) . superimposition of the two structures showed readily identifiable molecular homology between fmdv lpro and the catalytic domain of the cellular deisgylase usp despite the lack of sequence homology (data not shown). a peptide generated from the c-terminal sequence of isg was docked onto lpro to generate a simulated structure for analysis. the docking simulation predicted the ligand bound in a binding pocket using several residues to coordinate the interaction (fig. b) . analysis of the simulation revealed that isg has similar predicted affinities for the binding pockets in lpro or usp , suggesting a possible interaction in vivo. because isg is not conserved among all species, our simulation was further refined using docking and molecular dynamics to include isg ligands from bovine, porcine, murine, and human origin. by comparing interacting lpro residues that are conserved across different fmdv serotypes ( ) and that bind to isg from multiple known host species, we identified fmdv lpro residues that might be important for the interaction with isg . consistent with previous studies that have examined the interaction between viral deisgylases and isg ( , ) , the fmdv lpro-isg binding interface revealed a series of hydrophobic interactions in lpro (fig. c) , including trp/w and previously reported pro/p and leu/l (fig. d ) ( ) . we selected trp/w for further characterization since it was the only aromatic residue conserved among all fmdv serotypes that presumably participates in hydrophobic isg binding interactions. tryptophan to alanine mutation in fmdv lpro position significantly reduces deisgylase function in vitro. in order to examine whether the predicted aromatic residue in lpro is involved in the interaction with isg , an in vitro cleavage assay was performed using a commercially available human isg precursor protein (proisg ) as a substrate. bacterial cell extracts were prepared from bl (de ) cells transformed with plasmid pet -empty vector or pet encoding different versions of lpro, namely wild type (wt), catalytically inactive c a, and w a, followed by incubation with either sk cell extracts or proisg . as shown in fig. a , cleavage of isg was visualized as a protein band shift detected by coomassie stain in only the cell extracts treated with bacterial extracts expressing wt lpro ( fig. a, lane ) . in contrast, mutation of w in lpro resulted in a significant reduction of proisg cleavage ( fig. a , lane ), similarly to the catalytically inactive lpro c a ( fig. a, lane ) . it is important to note that all bacterial crude lysates derived from pet -lpro-transformed cells showed reactivity with our custom-made lpro antibody (fig. b, lanes to ) . interestingly, the w a mutation minimally disrupted the ability of lpro to cleave the cellular protein substrate eif g when bacterial extracts were incubated with whole sk lysates (fig. b, lane ) . these results were consistent with lpro-eif- g structural constraints previously reported ( ) . these data suggest that a separation-of-function bacterial extracts prepared from iptg-induced cultures were mixed with sk lysates, followed by incubation at °c to allow for lpro-dependent enzymatic digestion of host proteins. cleavage of eif g ( kda) generates a fragment of approximately kda (cp). for each figure, one representative blot is shown out of three independent experiments. mutation in lpro (w a) could be identified, albeit different incubation conditions were used to distinguish fmdv lpro deisgylase from its canonical proteolytic activity on translation factor eif- g. characterization of mutant lpro w a virus infectivity in different fmdvpermissive cell lines. to investigate the potential contribution of lpro deisgylase activity in the context of a viral infection, we incorporated a w a mutation in the fmdv serotype a infectious clone ( ) . virus was recovered from cells electroporated with prmc -a -lprow a. virus plaque assays were performed in bhk- cells to evaluate plaque morphologies of mutant a -lprow a compared with a -lprowt and the attenuated virus a -llv (leaderless) ( ) . as shown in fig. a , similar plaque sizes were detected for lpro wt and w a viruses. interestingly, a small delay in the appearance of cytopathic effect (cpe) was observed for a -lprow a relative to a -lprowt (data not shown). as previously reported, a relatively smaller plaque phenotype was displayed by mutant a -llv virus ( ) . growth kinetics of a -lprowt, a -llv, and a -lprow a were analyzed (fig. b ) in bhk- , lfpk␣v␤ , and sk cells. in all cell lines, a -lprow a replicated to levels comparable to those detected for a -lprowt late in infection. however, a significant decrease in replication (ϳ -fold) was detected at h postinfection (hpi) in bhk- and sk cells infected with a -lprow a compared with wt virus. further characterization of a -lpro w a was determined by examining lpro cellular distribution during infection since it has been previously described that wt lpro can translocate to the nuclei of infected cells ( ) and that mutations in other lpro domains altered normal subcellular lpro distribu- samples were stained with anti-lpro (green) and with nuclear stain =, -diamidino- -phenylindole (dapi; blue). scale bar represents m. statistical analysis was performed using student's t test. *, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ; black asterisks represent statistical significance between llv and wt; red asterisks represent statistical significance between w a and wt. tion ( ) . lfpk␣v␤ cells were infected with either a -wt or a -lpro w a fmdv and examined by immunofluorescence microscopy (fig. c ). consistent with our previous studies ( , ) , indirect staining of lpro using an anti-lpro antibody showed similar nuclear and cytoplasmic signals for both a -wt and a -lprow a fmdvinfected cells. these results indicated that the lprow a mutation does not affect the normal pattern of lpro cellular distribution during infection. lprow a reduces deisgylation and deubiquitination activity in porcine cells during infection. to further investigate the function of lpro deisgylation during fmdv infection, we first examined isgylation in porcine cells susceptible to fmdv infection by mimicking activation of the isg pathway without ifn stimulation. transfection of lfpk␣v␤ cells with plasmids encoding ddk-tagged isg and the isg conjugation machinery (ube l, ubch , and herc ) was performed and analyzed by western blotting h posttransfection (hpt). as shown in fig. a , the anti-ddk (anti-flag) antibody recognized n-terminally tagged isg in the lysate prepared from ddk-isg transfected cells (fig. a, lane , bottom panel) and not in the cells transfected with the plasmid control encoding gfp or mock-transfected samples. the specific detection of isg conjugates was observed by hpt (lane ) as multiple distinct bands, indicating that isgylation had occurred. detection of tubulin served as a sample loading control. to determine whether mutation w a affected the ability of the virus to remove isg conjugates from cellular target proteins, isg conjugation machinerytransfected cells were infected with a -wt, a -llv, or a -lprow a viruses and whole-cell lysates were collected at hpi for western blotting (fig. b) . it is important to note that a different gel system ( % to %) was needed to better assess isg conjugation of relatively high-molecular-weight abundant target proteins. robust detection of isg -modified protein substrates was observed in mock or infected cells that had been previously transfected with the isg conjugation machinery (fig. b) . by hpi, reduced isg conjugation was clearly detected in the cell lysates derived from a -wt-infected cells (fig. b lane ) . in contrast, the levels of isg conjugation were similar in the lysates of mock-, a -llv-, and a -lprow a-infected cells (fig. b, lanes , , and ) . interestingly, an increased ddk-isgylation signal was detected in cells infected with leaderless virus (a -llv) (fig. b, lane ) , consistently with the induction of a stronger host antiviral response. canonical eif g cleavage was observed in a -lprow a and in a wt but not in a -llv-infected cells (fig. b, bottom panels) , regardless of decreased levels of isgylated proteins. these results indicate that fmdv lpro exhibits deisgylating activity in porcine cells and that mutation in lprow a impacts optimal isg cleavage during infection. to determine whether a mutation in lpro also disrupts its ability to remove ub moieties from cellular substrates, we assayed lpro w a dub activity by transfecting porcine lfpk␣v␤ cells with a plasmid encoding hemagglutinin (ha)-tagged ub, followed by infection with a -wt, a -llv, or a -lpro w a viruses (fig. c ). as expected, infection of a -wt virus strongly decreased the level of ha-tagged ub conjugates (fig. c, lane ) , consistent with its previously reported dub function ( ) . dub function was disrupted upon infection with either a -lprow a or a -llv virus (fig. c, lanes and ) . taken together, our data indicate that w is required for efficient deconjugation of isg and ub from target cellular proteins during fmdv infection. in order to rule out whether impaired deisgylation/deubiquitination in the lpro mutation is due to its inability to process other cellular factors involved in innate immunity signaling, we examined the stress granule protein g bp . as previously shown ( ) , cleavage of g bp was detected in wt-infected cell lysates from lfpk␣v␤ cells at and hpi (fig. d, lanes and ) . similar patterns were observed in a -lprow a-infected cells (fig. d, lanes and ) , while a lack of g bp cleavage was observed in a -llv-infected cell lysates (fig. d, lanes and ) . taken together, these results indicate that the ability of a -lprow a to cleave g bp is not affected, despite the observed block in deisgylation and deubiquitination activities. isgylation inhibits fmdv replication in porcine cells. distinct levels of antiviral activity mediated by isg have been reported against different rna viruses ( ) . in order to determine whether isg can function as an antiviral agent against fmdv, lfpk␣v␤ cells were transfected with either gfp or isg and isg conjugation machinery, and at h, cells were infected with either fmdv a wt, llv, or lprow a virus. as a control, cell lysates were evaluated by western blot analysis for the presence of isg and isgylated proteins prior to infection (data not shown). a significant reduction in viral titers was observed when cells transfected with plasmids encoding isg and the isg conjugation machinery were infected with llv or lprow a (fig. a) . no reduction was detected in cells transfected with plasmid gfp control. reduction in titer was also observed in wt-infected cells, but the difference was not statistically significant. to confirm these results using the fmdv host-specific isgylation machinery during infection, we cloned the porcine isg in a replicationdefective human adenovirus type (ad ) vector (fig. b) and examined its antiviral activity in swine cells. transduction of lfpk␣v␤ cells with either ad -gfp or ad -pisg was performed, and detection of free-isg and isg -conjugated proteins was examined by western blot analysis (fig. ) . to confirm that ad -pisg was functional under our experimental conditions, lfpk␣v␤ cells were treated with increasing amounts of ifn-␤. as seen in fig. c , isg -modified proteins were detected only in samples that had been treated with ifn-␤ and transduced with ad -pisg (lanes to ), indicating that ad -overexpressed porcine isg induced the cellular isgylation machinery required for isg conjugation. a lack of differential dose response sug-gested that the system was saturated at the lowest ifn dose used ( u/ml). a statistically significant reduction of endpoint titers was detected in cells transduced with ad -pisg , but not with ad -gfp, after infection with wt fmdv (fig. d) . these results suggested that overexpression of isg induced antiviral activity in porcine cells. all together, these data indicate that isg has an inhibitory effect in the replication of fmdv. ( ) . in order to determine whether reduced affinity of lpro for isg affects the production of ifn and isgs, expression of specific mrnas was analyzed by reverse transcription-quantitative pcr (qrt-pcr) during viral infection. as seen in fig. a , similarly to wt, a significant inhibition of ifn-␤ expression was observed upon infection with fmdv lprow a. in contrast, as previously reported, upregulation of ifn-␤ expression showed the highest levels in a -llv cells ( ) . similar to ifn-␤ expression, antiviral genes mx- and oas- (fig. b) ; genes encoding the isgylation machinery, namely isg , herc , and usp (fig. c) ; and rna sensors mda- and rig-i (fig. d) were upregulated to the highest levels only in llv-infected porcine cells. these results indicate that disruption of lpro deisgylase activity does not affect fmdv-dependent transcriptional downregulation of ifn-␤, isg antiviral responses, or components of the isg pathway. in vivo attenuation of fmdv lprow a in mice. in order to understand whether the mutation w a in fmdv lpro causes attenuation in vivo, we examined this mutant in a mouse model of fmd ( , ) . six-to -week-old c bl/ mice were inoculated with different doses of fmdv a -lprow a or a -wt ( mice/group) and checked for survival, viremia, and induction of neutralizing antibodies. as shown in fig. a , mice inoculated with e or e pfu of a -wt showed % and % survival, respectively, whereas mice inoculated with a lower dose ( e pfu) showed % survival. in contrast, % survival was observed in animals inoculated with e and e pfu of a -lprow a and % survival for the highest dose of a -lprow a ( e pfu). viremia was detected for several days postinoculation (dpi), with a peak in the range of e to almost e pfu/ml at dpi in mice inoculated with fmdv a -wt (fig. b) . in all the groups inoculated with a -lprow a, the peak of viremia was also detected at dpi. animals inoculated with e or e pfu of a -lprow a had significantly lower titers ( e and e pfu/ml, respectively) than the titers measured in wt-inoculated animals. interestingly, all animals inoculated with e pfu of a -lprow a survived despite the presence of high levels of viremia. in contrast, % of animals inoculated with the same dose of a -wt virus died, even when the levels of viremia were lower than those detected in a -lprow a-inoculated mice. an analysis of humoral activity, in animals that survived the initial inoculation, showed detectable levels of neutralizing antibodies for both groups. however, animals inoculated with a -lprow a had lower levels of antibodies than those inoculated with similar doses of a -wt virus (fig. c) . these results indicate that although fmdv a -lprow a is attenuated in vivo, it is still able to induce the production of neutralizing antibodies. in this study, by using molecular modeling, we identified a novel residue in fmdv lpro required for optimal deisgylase activity. our findings demonstrate, for the first time, that a viable fmdv with defective deisgylase activity and unaffected proteolytic activity on canonical cellular substrates, such as eif g, can be derived. although other lpro residues mediating its interaction with isg have been recently identified ( ), we propose that lprow is important for modulating viral infection kinetics. furthermore, we show that lpro mediates deisgylation during fmdv infection of natural host cells and that this function is important for optimal virus growth. reversing the posttranslational modification of cellular proteins conjugated to isg to reduce the overactivation of innate immunity signaling pathways has been observed for other positive-sense, single-stranded rna viruses that encode plps, including coronaviruses such as middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov- and sars-cov- ), and mouse hepatitis virus (mhv), and for the arterivirus porcine reproductive and respiratory syndrome virus (prrsv). fmdv lpro exhibits isg binding profiles (hydrophobic interactions) similar to mers-cov and sars-cov deisgylases ( ); however, isg processing patterns induced by fmdv lpro have been shown to be different ( ) . while most deisgylases cleave target proteins leaving a recyclable isg product with a single g residue at the c terminus, fmdv lpro deisgylase activity is characterized by leaving behind a g-g di-peptide signature on processed host proteins and no recyclable isg . this may pertain to the differential tropism of these viruses and the fact that isg is not conserved across species ( ) . in this study, we considered the diversity of isg for multiple fmdv host species and highlighted unique features of fmdv lpro for substrate recognition. in addition to the role in deisgylating events, lpro is known to have dub activity ( ) ; however, it is not clear what are the molecular sites that determine the crossspecificity for lpro binding to ub versus isg . it is important to mention that, thus far, all studies on these lpro functions have been performed with overexpressed lpro and candidate target host proteins ( ) . in this study, we demonstrated that infection with a -lpro w a also disrupted efficient dub activity (fig. c) , suggesting that ub and isg may have overlapping target-binding profiles. understanding the specific fmdv lpro determinants for binding ub may highlight the relative differential contribution of ubiquitination and isgylation to the antiviral signaling pathways induced upon fmdv infection. most importantly, our studies show that fmdv lpro deisgylase activity is not critical for effective type i ifn antagonism despite its known ability to block downstream ifn signaling effectors as a consequence of dub function ( ) . interestingly, examination of mrna transcripts involved in isg conjugation resulted in a significant reduction of the ligase herc in cells infected with fmdv lprow a compared to wt-infected cells (fig. c ). since herc can regulate the conjugation of isg to different cellular targets, its downregulation may favor a greater availability of unconjugated isg . thus, free forms of isg could modulate other novel antiviral functions, contributing to the observed delay in viral replication (fig. b) . in wt-infected cells, modified cleavage of isg ( ) results in nonfunctional isg molecules that cannot be recycled for additional rounds of isgylation or perhaps display other unrecognized antiviral functions. it is also possible that lpro deisgylation activity is important for modulating ifn-independent antiviral pathways that have not been explored in our studies. in fact, upregulation of isg transcripts has been detected in porcine cells treated with poly:ic without the requirement of ifn production ( ) . similar results have been observed with other viruses, such as cytomegalovirus (cmv) ( ) . identification of isgylated substrates during fmdv infection should illustrate new functions of lpro. although isg has been shown to have broad antiviral activity against different rna viruses, to date, no such information is available for fmdv. in this study, we show that overexpression of isg affects virus replication in porcine cells. most importantly, fmdv with reduced (a -lprow a) or a complete loss of deisgylase function (a -llv) was more sensitive to the isg -induced antiviral effects. however, the mechanism of antiviral function against fmdv remains elusive. it is possible that fmdv proteins may be substrates for the isg conjugation machinery due to its known ability to target de novo protein synthesis ( ) . in fact, previous reports have shown that the viral a protease of coxsackievirus (cvb ) can be isgylated, thus preventing eif g cleavage during infection of hela cells ( ) . isg modifications of other viral proteins have been reported for influenza virus, human cmv, and human papillomavirus ( ) . additionally, isgylation has been related to the release of virus particles that require the endosomal sorting complex required for transport (escrt) pathway, such as hiv- , ebola virus (ebov), and avian sarcoma leukosis virus (aslv) ( ) . although the involvement of this pathway is not associated with fmdv release, it has been recently reported that nanovesicles may play a role in transmission of fmdv in vitro and in vivo ( ) . furthermore, it is known that exosome release could be modulated by the expression of isg , presumably affecting related biological functions ( ) . it will be interesting to determine whether fmdv proteins are targets for isgylation during infection and which are the host substrates for lpro deisgylase activity. to this end, our results demonstrate that lpro deisgylation activity is required for optimal fmdv growth but not as the result of impairing the induction of ifn and isgs. moreover, we demonstrate that impairment of this activity results in viruses that are attenuated not only in tissue culture but also in vivo in mice. although mice are not the natural hosts for fmdv, our previous studies using this model have always shown a pattern that was later reproduced in natural host species ( , ) . further evaluation in livestock species is warranted for mutant fmdvs with impaired deisgylase activity. importantly, the discovery of mutations that are tolerated by the virus opens a venue for the rational design of novel modified live or inactivated fmd vaccines that hopefully will contribute to the effective control of such an important transboundary animal disease. the open-source program for doing molecular docking autodock vina ( ) was utilized, and a peptide of the c-terminal residues of isg was used to identify the putative binding pocket on lpro (pdb number qbb). molecular dynamics was performed using gromacs ( ) . simulations were executed to assess the ability of different peptides to bind to different iterations of lpro as well as usp -misg (pdb number chv) ( ) as a control for deisgylase. plasmids and reagents. the gene lpro was cloned in plasmid pet b- x-his (novagen, billerica, ma) using the restriction sites ndei and bamhi. site-directed mutagenesis at the active site of lpro was used to generate a proteolytically inactive protein (lproc a). plasmid pcmv-isg encoding human isg n-terminally tagged with ddk (flag) and plasmids encoding untagged proteins of the isg conjugation machinery (ube l and ubch ) were obtained from the krug lab (university of florida, gainsville, fl) and have been described previously ( ) . human ubiquitin (genbank accession number ab ; genescript, piscataway, nj) was cloned into the mammalian expression vector pci using noti and nhei restriction sites. a hemagglutinin (ha) tag was incorporated in-frame at the = end of the ubiquitin gene. a replication-incompetent human ad vector encoding porcine isg was constructed by digestion of the pad blue vector with xbai and clai restriction enzymes ( ) and ligation with a synthetically made insert (genescript) containing pisg . the plasmid construct was confirmed by sequencing. for western blot analysis, the following primary antibodies were used: monoclonal rabbit anti-ddk, mouse anti-ddk, and mouse anti-gapdh from origene (rockville, md); monoclonal mouse anti-ha from sigma (st. louis, mo); and monoclonal antibody (mab) mouse anti-tubulin alpha from neomarkers (fremont, ca), anti-eif g from bethyl laboratories (montgomery, tx), and anti-g bp from aviva systems biology (sand diego, ca). goat anti-mouse immunoglobulin g (igg) and goat anti-rabbit igg secondary antibodies conjugated to horseradish peroxidase (hrp) were obtained from pierce (rockford, il). when indicated, protein samples were resolved by sds-page and detected by western blotting (wb) using the specific antibodies and an ecl chemiluminescence kit (bio-rad, hercules, ca). images were acquired with the azure imager c digital imager. cells. porcine kidney cells overexpressing the bovine integrin ␣v␤ (lfpk␣v␤ ) or sk cells were obtained from aphis/foreign animal disease diagnostic laboratory (faddl). these cells were maintained in minimal essential medium (mem; gibco brl, invitrogen, carlsbad, ca) containing % fetal bovine serum (fbs) and supplemented with % antibiotics and nonessential amino acids. human embryonic kidney (hek ) cells lines (attc crl- ) from the american type culture collection (atcc; rockville, md) were used to propagate the ad -pisg virus. hek cells were maintained in mem containing % fbs supplemented with % antibiotics (gibco brl), nonessential amino acids, and l-glutamine. baby hamster kidney strain (bhk- ; clone , atcc cl ) cells were obtained from the atcc were used to propagate virus stocks and to measure virus titers. bhk- cells were maintained in mem containing % calf serum and % tryptose phosphate broth supplemented with % antibiotics and nonessential amino acids. cell cultures were incubated at °c in % co . viruses. fmdv a -wt (wild type) was generated from the full-length serotype a infectious clone prmc ( ) , and a -llv (leaderless virus) was derived from prmc by deletion of the lb coding region ( ) . fmdv a -lprow a was constructed by site-directed mutagenesis using the quikchange kit. all viruses were derived and propagated in bhk- cells, concentrated by polyethylene glycol precipitation, titrated on the same cells, and stored at - °c. viral full-length sequences were confirmed by dna sequencing of derived viral cdna using an abi prism instrument (applied biosystems, thermo fisher). the ad -pisg virus was derived and propagated in hek cells as previously described ( ) . viral infections. cultured cell monolayers were infected with fmdv at the indicated multiplicity of infection (moi) for h at °c. after adsorption, cells were rinsed with mm nacl in mm morpholino-ethane-sulfonic acid (mes) (ph . ) to remove noninternalized virus, and mem containing nonessential amino acids, antibiotics, and antimycotic (gibco brl) was added, followed by incubation at °c. at specific times, infected cells were frozen and thawed followed by the determination of virus titers by endpoint dilution in bhk- cells ( ) . lpro cleavage assays. to determine the ability of lpro mutants to cleave substrate pro-isg , wt and lpro mutants were cloned in pet b plasmids and transformed in bl escherichia coli. bacteria expressing the different lpro variants were grown at °c in luria-bertani broth containing ampicillin for h. cultures were diluted : into ml of the same medium and grown under similar conditions until the optical density at nm (od ) reached approximately . (ϳ h). expression of lpro was induced by adding mm isopropyl-␤-d-thiogalactopyranoside (iptg) and continuing incubation for h at °c. bacterial cells were pelleted by centrifugation and lysed in ml/g of bug buster protein extraction reagent (sigma-aldrich, saint louis, mo) containing u/l benzonase (sigma-aldrich) on ice. lysates were clarified by centrifugation, and aliquots of l of supernatant were assayed on . g of purified human recombinant proisg (boston biochem, boston, ma) or on crude protein lysates of sk cells. reaction mixes were incubated at either room temperature for h (pro-isg ) or at °c for h (sk crude lysates), at the end of which protein products were resolved by sds-page. coomassie g- stain was used to visualize protein bands using the simplyblue safestain kit (invitrogen, carlsbad, ca), following the manufacturer's directions. western blotting was used to detect cleavage products in cellular sk extracts. foot-and-mouth disease the economic impacts of foot and mouth disease-what are they, how big are they and where do they occur? the dual nature of type i and type ii interferons the different tactics of foot-and-mouth disease virus to evade innate immunity the two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities leader protein of foot-and-mouth disease virus is required for cleavage of the p component of the cap-binding protein complex the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response degradation of nuclear factor kappa b during foot-and-mouth disease virus infection foot-and-mouth disease virus leader proteinase inhibits dsrna-induced type i interferon transcription by decreasing interferon regulatory factor / in protein levels innate immune sensor lgp is cleaved by the leader protease of foot-and-mouth disease virus interaction between fmdv lpro and transcription factor adnp is required for optimal viral replication foot-and-mouth disease virus leader protease cleaves g bp and g bp and inhibits stress granule formation isg in antiviral immunity and beyond beyond isglylation: functions of free intracellular and extracellular isg ubiquitin in the activation and attenuation of innate antiviral immunity positive regulation of interferon regulatory factor activation by herc via isg modification protein isgylation modulates the jak-stat signaling pathway viral deubiquitinases: role in evasion of anti-viral innate immunity isg : it's complicated nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene a naturally occurring recombinant enterovirus expresses a torovirus deubiquitinase the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase type i interferon induced and antagonized by foot-and-mouth disease virus irreversible inactivation of isg by a viral leader protease enables alternative infection detection strategies foot-and-mouth disease virus (fmdv) causes an acute disease that can be lethal for adult laboratory mice structural basis of the specificity of usp toward isg comparative genomics of foot-and-mouth disease virus structurally guided removal of deisgylase biochemical activity from papain-like protease originating from middle east respiratory syndrome coronavirus the leader proteinase of foot-and-mouth disease virus: structure-function relationships in a proteolytic virulence factor genetically engineered foot-and-mouth disease viruses with poly(c) tracts the foot-andmouth disease virus leader proteinase gene is not required for viral replication a conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function the antiviral activities of isg differential gene expression in bovine cells infected with wild type and leaderless footand-mouth disease virus synonymous deoptimization of foot-and-mouth disease virus causes attenuation in vivo while inducing a strong neutralizing antibody response sequence and expression analyses of porcine isg and isg genes interferonindependent upregulation of interferon-stimulated genes during human cytomegalovirus infection is dependent on irf expression the isg conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg ubiquitin-like protein isg (interferon-stimulated gene of kda) in host defense against heart failure in a mouse model of virus-induced cardiomyopathy exosomesmediated transmission of foot-and-mouth disease virus in vivo and in vitro isgylation controls exosome secretion by promoting lysosomal degradation of mvb proteins venezuelan equine encephalitis replicon particles can induce rapid protection against foot-and-mouth disease virus autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. softwarex - : - interferon-induced isg conjugation inhibits influenza a virus gene expression and replication in human cells pad -blue: direct ligation system for engineering recombinant adenovirus constructs adenovirus-vectored foot-and-mouth disease vaccine confers early and full protection against fmdv o manisa in swine a simple method of estimating fifty per cent endpoints this work was funded by usda ars-cris project - - - d and partially through agreement - - - with kansas state university.we thank marvin grubman for critical reading of the manuscript. key: cord- -c fi uoh authors: zhong, bo; wang, yan-yi; shu, hong-bing title: regulation of virus-triggered type i interferon signaling by cellular and viral proteins date: - - journal: front biol (beijing) doi: . /s - - -x sha: doc_id: cord_uid: c fi uoh host pattern recognition receptors (prrs) recognize invading viral pathogens and initiate a series of signaling cascades that lead to the expression of type i interferons (ifns) and inflammatory cytokines. during the past decade, significant progresses have been made to characterize prrs such as toll-like receptors (tlrs) and rig-i-like receptors (rlrs) and elucidate the molecular mechanisms of tlr- and rlr-mediated signaling. to avoid excessive and harmful immune effects caused by over-activation of these signaling pathways, host cells adopt a number of strategies to regulate them. in addition, invading viruses also employ a variety of mechanisms to inhibit the production of type i ifns, thereby evading the supervision and clearance by the host. in this review, we briefly summarize the tlr- and rlr-mediated type i ifn signaling and then focus on the mechanisms by which host cellular and viral components regulate the expression of type i ifns. organisms, from unicellular bacteria to human, are exposed to invading pathogens all the time. to protect themselves from pathogenic effects caused by the invaders, hosts have evolved immune system to detect and prevent infection by pathogens. the immune system in mammals is traditionally divided into two branches: innate immunity and adaptive immunity. the adaptive immunity, which is able to generate specific immune responses mediated by antibodies and effector t cells, is highly specific. however, it is evolved only in higher organisms and there is usually a delay of - days before the initial adaptive immunity takes effects. in contrast, the non-specific innate immunity is evolutionally conserved and begins to work minutes to hours after infection. therefore, the innate immunity constitutes the first line for defense against pathogens such as viruses. there are wide spectrums of viral pathogens that are known to infect humans, which have been a great threat to human health. the early events of innate immunity against invading viruses include the recognition of viral components, initiation of signaling pathways and transcriptional induction of type i ifns and other cytokines . the type i ifns bind to ifn receptor (ifnr) in both autocrine and paracrine manners to initiate a series of signaling events leading to the expression of hundreds of downstream genes, collectively referred to as interferon stimulated genes (isgs). proteins encoded by the isgs inhibit viral replication or cause apoptosis of infected cells, and therefore result in an antiviral effect (sadler and williams, ) . because type i ifns play a central role in antiviral immunity, great efforts have been made during the past decade to elucidate the mechanisms of virus-triggered type i ifn induction. however, over-produced type i ifns cause excessive and harmful immune effects to the host (theofilopoulos et al., ) . as a result, the production of these cytokines should be tightly regulated. on the other hand, viruses have also evolved a variety of mechanisms to inhibit the production of type i ifns, thereby evading the supervision and elimination by the innate immunity. in this review, we first briefly summarize the virus-triggered type i ifn signaling and then focus on the regulatory mechanisms exerted by cellular and viral proteins. viral infection-triggered type i ifn signaling: a brief introduction as mentioned above, virus-triggered type i ifn signaling is initiated by the recognition of pathogen-associated molecular patterns (pamps) generated during viral infection and replication. so far there are at least five kinds of viral pamps that have been characterized, including double-stranded rna (dsrna), 'triphosphorylated single-stranded rna ( 'pppssrna), viral envelope glycoprotein, unmethylated cpg dna (cpg dna), and atrich double-stranded dna (the analog poly da:dt) (kumar et al., ; takeuchi and akira, ). these pamps are recognized by host pathogen-recognition receptors (prrs), which include toll-like receptors (tlrs), rig-i-like receptors (rlrs including rig-i and mda ), nod-like receptors (nlrs), and the recently identified cytoplasmic dna sensors dna-dependent activator of interferon-regulatory factors (dai), absent in melanoma (aim ) and rna polymerase iii (pol-iii) (ablasser et al., ; chiu et al., ; mccartney and colonna, ). the prrs, each specific for a distinct ligand set, and the prrs-mediated signaling pathways have been extensively reviewed in several previous publications kawai and akira, ; kumar et al., ; takeuchi and akira, ; yoneyama and fujita, ) (table ) . here, we choose tlr , tlr / , tlr , rlrs, dai and pol-iii-mediated signaling for discussion because they all recognize the viral nuclear acids but induce the production of type i ifns via three representative adaptor proteins. these pathways converge at the activation of several transcription factors such as interferon-regulated factor / (irf / ) and nf-κb which collaborate to regulate transcription of type i ifns (maniatis et al., ; honda et al., ) . tlr is the first characterized mammalian prr and tlr -meidated signaling has been extensively studied. upon stimulation of viral dsrna or its synthetic analog poly(i:c), the intracellular domain of tlr recruits the adaptor protein toll/interleukin receptor (tir) domaincontaining adaptor-inducing ifn-β (trif). trif has an n-terminal domain, a middle tir domain and a c-terminal domain called receptor-interacting protein (rip) homotypic interaction motif (rhim) yamamoto et al., ) . the adaptor protein trif undergoes oligomerization through its tir and rhim domains, and recruits the traf family-memberassociated nf-κb activator (tank) binding kinase (tbk ) via its n-terminal domain to activate irf / . it is also suggested that nf-κb activating kinase (nak)associated protein (nap ) and tumor necrosis factor (tnf) receptor-associated factor (traf ) are involved in trif-mediated activation of irf / by facilitating trif and tbk interaction (oganesyan et al., ; saha et al., ; ryzhakov and randow, ) . collectively, dsrna induces activation of irf / through tlr -trif-nap /traf -tbk pathway. trif mediates nf-κb activation through two distinct pathways. trif contains a consensus traf -binding motif in the n-terminal region and mutation of this motif impairs trif-mediated nf-κb but not irf activation jiang et al., ) . however, tlr signaling in traf -deficient macrophages is not affected (gohda et al., ) , which indicates the existence of functional redundancy. it has been demonstrated that trif is also capable of activating nf-κb through its c-terminal rhim, which is responsible for recruitment of rip (meylan et al., ) . it has been shown that poly(i:c)induced nf-κb activation is completely blocked in ripdeficient mefs (cusson-hermance et al., ) . in addition, overexpression of trif induces apoptosis by interacting with rip through a rip /fas-associated death domain (fadd)/caspase -dependent and mitochondriaindependent apoptotic pathway . collectively, the tlr -triggered trif-dependent pathways activate irf / and nf-κb and induce apoptosis via tbk , rip and rip /fadd/caspase , respectively. in contrast to the trif-dependent signaling triggered by tlr , tlr / and tlr -mediated signaling depends exclusively on another adaptor protein myd (myeloid differentiation primary response protein- ) akira et al., ; yoneyama and fujita, ). the myd -dependent pathway includes a number of signaling molecules: the adaptor protein myd , il- rassociated kinase / (irak / ), transforming growth factor-β-activated kinase (tak ), traf and tak binding protein- / (tab / ). upon binding to their respective ligands, tlr / and tlr recruit myd and irak (honda et al., ; kawai et al., ) . irak further recruits irak and traf and thereby phosphorylates and activates irak . the irak -traf complex then disassociates from the receptor . on one hand, the complex interacts with ikkα, traf and osteopontin, leading to phosphorylation and activation of irf (hoshino et al., ; shinohara et al., ) . on the other hand, it interacts with another complex consisting of tak , tab and tab , followed by the phosphorylation and activation of tak . the activated tak subsequently phosphorylates the iκb kinases (ikks), leading to ubiquitination and degradation of iκb and activation of nf-κb . activation of tak also results in the activation of mapks, including c-jun nterminal kinase (jnk), leading to activation of ap- oganesyan et al., ; saha et al., ) . the canonical ikk complex ikkα/β/γ is essential for virus-triggered rlr-mediated nf-κb activation, and the noncanonical ikk family members tbk and ikkε are responsible for phosphorylation and short interfering rna (sirna) kawai and akira, ; kleinman et al., ; tlr / imiquimod (r- ), resiquimod (r- ), loxoribine hemmi et al., guanosine and uridine-rich ssrna diebold et al., ; heil et al., human immunodeficiency virus diebold et al., ; heil et al., influenza a virus diebold et al., ; heil et kato et al., 'pppssrna hornung et al., pichlmair et al., 'triphosphate rna with a panhandle structure at ' end schlee et al., in vitro transcribed rna kato et al., ; yoneyama and fujita, influenza a virus kato et al., ; yoneyama and fujita, vesicular stomatitis virus kato et al., ; yoneyama and fujita, newcastle disease virus kato et al., ; yoneyama and fujita, sendai virus kato et al., ; yoneyama and fujita, japanese encephalitis virus kato et al., ; yoneyama and fujita, hepatitis c virus saito et al., ; yoneyama and fujita, respiratory syncytial virus kato et al., ; yoneyama and fujita, dengue virus kato et al., ; yoneyama and fujita, west nile virus kato et al., ; yoneyama and fujita, mda long dsrna, poly(i:c) (about kb) reovirus (long fragment of genomic dsrna) kato et al., encephalomyocarditis virus kato et al., ; yoneyama and fujita, theiler's encephalomyelitis virus kato et al., ; yoneyama and fujita, mengo virus kato et al., ; yoneyama and fujita, dengue virus kato et al., ; yoneyama and fujita, west nile virus kato et al., ; activation of irf and irf . other studies have also demonstrated the involvement of several other signaling components in virus-induced activation of nf-κb and/or irf , including tank, tradd, fadd and rip guo and cheng, ; michallet et al., ) . recently, we and others identified a new adapter protein called mediator of irf activation (mita, also known as sting), which plays a critical role in virus-induced type i ifn expression (ishikawa and barber, ; zhong et al., ) . mita has been found to localize to the outermembrane of mitochondria or endoplasmic reticulum (er). it has been demonstrated that mita acts as an adapter to recruit tbk and irf to the visa-associated complex after viral infection. in this complex, tbk first phosphorylates mita at ser , which is critical for subsequent phosphorylation of irf . there is also evidence that a number of molecules involved in protein transportation are also required for virustriggered type i ifn production (ishikawa and barber, ; ishikawa et al., ). viral dna-triggered type i ifn production is mediated through at least three pathways. first, two recent publications reported that in transformed cells, at-rich doublestranded dna (poly(da:dt)) and cytoplasmic viral or bacterial dna is transcripted into rna by pol-iii and the transcribed rna, which probably contains 'triphosphate structure and is then recognized by rig-i and signals through visa (ablasser et al., ; chiu et al., ) . second, in primary or low passage cells, transfection of dsdna regardless of its sequences or infection by dna viruses also triggers pol-iii-independent signaling, leading to the expression of type i ifns. the signaling pathway requires the signaling complex mita-tbk -irf but not dai or visa (chiu et al., ; ishikawa et al., ) . however, future studies are needed to characterize the sensors and adaptors that function upstream of mita in the pol-iii-independent pathway. third, dai senses invading dna to induce type i ifns in l cells, which depends on tbk and irf but not visa (takaoka et al., ) . the adaptor protein for this process is currently unknown. it is commonly observed that prr-mediated signaling is rapidly activated after viral infection, leading to the production of type i ifns and other cytokines. however, the overproduction of type i ifns can cause unwanted or excessive immune responses that may lead to allergy, necrosis, autoimmune diseases and other harmful effects (theofilopoulos et al., ) . therefore, prr-mediated type i ifn signaling must be tightly regulated. for example, the c-terminus of rig-i contains a repressor domain (rd) which masks the card domain and rna helicase domain of rig-i, thereby inhibiting the activation of rig-i in uninfected cells takahasi et al., ; yoneyama and fujita, ). in addition to the autonomous inhibitory mechanism, the host has evolved several mechanisms to prevent unnecessary activation in steady-state cells or excessive signaling under viral infection conditions (table ) . laboratory of genetics and physiology (lgp ) compared to rig-i and mda , lgp contains an rna helicase domain and an rna binding domain but lacks the card domain. therefore, lgp binds to dsrna competitively with rig-i but does not initiate signaling. it has been shown that overexpression of lgp negatively regulates sendai virus (sev) and newcastle disease virus (ndv)-triggered induction of type i ifns, and rnaimediated knockdown of lgp can enhance expression of antiviral genes (yoneyama et al., ) . in addition, lgp interacts with rig-i and inhibits oligomerization of rig-i which is important for activation of the latter. lgp also disrupts visa-tbk /ikkε interaction to inhibit rlrmediated type i ifn signaling (saito et al., ) . however, the lgp -/mice are resistant to ndv infection which is sensed by rig-i but sensitive to emcv infection which is sensed by mda , suggesting that lgp differently regulates rig-i-and mda -mediated signaling venkataraman et al., ) . recently, a study revealed the crystal structure of the repressor domain of lgp , which suggests that lgp inhibits rig-i-mediated signaling by competitively binding to dsrna, while it positively regulates mda mediated signaling by facilitating recognition of dsrna by mda (pippig et al., ) . rig-i splice variant (rig-i-sv) it has been demonstrated that the full activation of rig-i depends on its k linked ubiquitination at k by trim or rnf (gack et al., ; oshiumi et al., ) . trim firstly interacts with rig-i and then catalyzes the ubiquitination of rig-i . the thr of rig-i is critical for its binding with trim . compared to the full-length rig-i, rig-i-sv lacks the aa - region. thus, rig-i-sv does not interact with trim to initiate signaling (gack et al., ) . however, rig-i-sv contains the intact rna binding and helicase domain and interacts with rig-i but not mda . therefore, rig-i-sv inhibits rig-i-but not mda -mediated type i ifn signaling by competitively binding to dsrna and visa with rig-i and inhibiting the oligomerization of rig-i. dihydroxyacetone kinase (dak) the protein kinase dak was found to interact with mda in yeast two-hybrid assays . dak is a member of the evolutionarily conserved family of dihydroxyacetone kinases from bacteria to humans (bachler et al., ; cabezas et al., ) . mammalian dak displays dual activities as flavin adenine dinucleotide (fad)-adenosine monophosphate (amp) lyase and atp-dependent pha kinase. however, the physiological functions of dak in innate antiviral response were unknown. in co-immunoprecipitation experiments, dak interacts with mda but not rig-i in untransfected cells or overexpressed conditions, and the card domaincontaining fragment of mda is sufficient for the association. expression of dak inhibits mda -but not rig-i-or visa-mediated induction of ifn-β, while rnai knockdown of dak has an opposite effect. endogenous interaction between mda and dak is decreased when the cells are infected with the sendai virus, suggesting that the association is disrupted upon virus infection. it is possible that upon binding to dsrna, the conformational change of mda results in its higher affinity to the downstream adaptor visa . therefore, dak keeps mda inactive under steady-state conditions. atg -atg the atg -atg conjugate plays a critical role in autophagic process. the autophagy has been implicated for defense against infections with intracellular bacteria and viruses (gutierrez et al., ) . however, autophagosomes have also been exploited by certain viruses as a place for viral replication. a recent study has demonstrated that vsv infection induces higher level of irf phosphorylation and expression of ifn-β and ip in atg -/than in wild-type mefs (jounai et al., ) . further investigation suggests that the atg -atg conjugate associates directly with the card domains of rig-i, mda and visa and viral infection can enhance the interaction. therefore, atg -atg disrupts the card interactions between rig-i or mda and visa, preventing the formation of the rlr-visa complex. atg is a critical scaffold protein that facilitates atg to conjugate with atg (komatsu et al., ) . accordingly, atg deficient mefs produce higher amount of type i ifns in response to cytoplasmic poly(i:c) stimulation (jounai et al., ) . collectively, these data suggest that atg -atg conjugate acts as a suppressor of rlr signaling by blocking the interaction between rlrs and visa. tbk s compared to the full-length tbk , the splice variant tbk s lacks exons three to six. it has been shown that tbk s is expressed - hours after viral infection . tbk s interacts with rig-i and disrupts the interaction between rig-i and downstream molecules, thereby inhibiting rig-i-mediated activation of irf . interestingly, tbk s does not inhibit visa-or tbk -mediated activation of ifn-β, suggesting that tbk functions at rig-i level as a negative feedback regulator. it should be noted that although dak and tbk s disrupt interaction between mda or rig-i and visa, neither of them inhibits rlr-mediated nf-κb activation. the mechanisms for this process need further investigation. . . sequestration of adaptor proteins nlrx nlrx belongs to a protein family containing nucleotide-binding domain (nbd) and leucine-rich repeat (lrr) (nlr), which are structurally conserved and have been demonstrated to function in defense against bacterial infection . expression of nlrx inhibits rlrmediated expression of cytokines such as type i ifns, il- and tnfα, while knockdown of nlrx has an opposite effect. nlrx was found to reside at the outermembrane of mitochondria and associate with the card domain of visa, thereby sequestering visa from rlrs and inhibiting rlrmediated signaling (moore et al., ) . gc qr receptor for globular domain of complement component c q (gc qr) is located in mitochondria, nucleus, cytoplasm and on cell membrane . overexpression of gc qr inhibits rig-i-mediated activation of irf and nf-κb and production of ifn-β and other cytokines. conversely, rnai knockdown of gc qr enhanced the production of type i ifns. it is shown that mitochondrial gc qr weakly interacts with visa and sev infection causes the translocation of gc qr to mitochondria and enhances its association with visa ). thus, it has been postulated that gc qr plays a role as a negative feedback regulator in rlrmediated signaling. myd s myd s is an alternatively spliced form of myd , lacking the intermediate amino acids - of myd . unlike myd , myd s does not induce irak phosphorylation, despite its interaction with irak . the expression of myd s can be detected in spleen and brain tissues and is upregulated by lps stimulation. myd s forms dimer or oligomer with myd and interacts with irak , sequestering irak from irak and inhibiting irak -induced phosphorylation of irak (burns et al., ) . thus, myd s is a negative feedback regulator of tlr-induced myd -dependent signaling. traf traf belongs to the traf family. all members of this family except for traf contain a ring domain that bears an e ubiquitin ligase activity (bradley and pober, ) . in cells that stably express tlr , overexpressed traf interacts with trif and effectively inhibits poly(i:c)-induced activation of nf-κb and the ifn-β promoter. further studies suggest that cterminal part of traf and the tir domain of trif are responsible for their interaction. interestingly, trif induces caspase-dependent cleavage of traf , and the cleaved n-terminal but not c-terminal fragment of traf shows inhibitory effect. inhibition of the cleavage of traf by mutating of the cleavage site or addition of caspase inhibitor impairs its ability to inhibit trifdependent signaling (su et al., ) . therefore, trifinduced cleavage of traf is required for its inhibition of trif signaling. isg interferon-stimulated gene (isg ) is one of the first identified proteins induced by virus and type i ifns (sadler and williams, ) . in immunoprecipitaion and mass-spectrometry assays, isg was identified as a mita-interacting protein ). isg negatively regulates virus-triggered signaling at mita level by disrupting tbk -mita and visa-mita interactions. consistent with these observations, inhibition of isg by rnai enhances cellular antiviral responses as well as the expression of type i ifns. it has been reported that isg acts as a suppressor of viral replication and protein translation (wang et al., ; terenzi et al., ; wacher et al., ) . in this context, isg might have multiple functions and is an important integrator of inhibition of viral replication and control of excessive antiviral responses. it is also possible that the functions of isg are temporally and spatially regulated during viral infection. however, more studies are required for the full understanding of these processes ). sike suppressor of ikkε (sike) contains no other recognizable domain but two coiled-coil domains. in yeast two-hybrid screens, sike was identified as an ikkεinteracting protein (huang et al., ) . because tbk functions in most types of cells and shows highly homology with ikkε which functions in limited types of cells and is viral infection inducible, studies are focused on the interaction between sike and tbk perry et al., ) . sike interacts with tbk in uninfected cells, sequestering tbk from irf , while viral infection or poly(i:c) stimulation disrupts the interaction, thereby releasing tbk to interact with and phosphorylate irf . consistently, overexpression of sike inhibits tlr -and rlr-mediated signaling and knockdown of sike has an opposite effect (huang et al., ) . therefore, sike might sequester tbk /ikkε in inactive forms under steady-state conditions to avoid unnecessary activation of these kinases. irak-m irak-m belongs to the irak family (ringwood and . however, it does not possess kinase activity like other iraks do. unlike the ubiquitous expression profile of irak / , the expression of irak-m is limited to monocytes or macrophages. irak-m -/macrophages show increased cytokine production when stimulated with bacteria (kobayashi et al., ) and further studies suggest that irak-m prevents the association of irak- and irak- with the myd complex, thereby negatively regulating tlr-mediated signaling in macrophages. fln fln is also an ifn-inducible protein. it contains a traf-type zinc finger domain at its n-terminus and a conserved traf -binding motif which mediates its interaction with traf . overexpression of fln inhibits trif-dependent nf-κb and mapk activations (mashima et al., ) . fln -/-mefs are highly resistant to vsv infection, and these cells produce more ifn-β than wild-type cells in response to poly(i:c). fln -deficient mice become more susceptible to poly(i:c)-induced septic shock compared with wild-type mice. mechanistic studies show that fln interacts with trif, visa, traf and traf and inhibits virus-triggered signaling at traf / level (sanada et al., ) . however, the exact mechanism still needs further investigations. shp- the src homology (sh ) domain-containing protein tyrosine phosphatase (shp- ) is an evolutionarily conserved tyrosine phosphatase (qu, ; neel et al., ) , which has been demonstrated to positively regulate the signaling triggered by some cytokines such as il- , and negatively regulate the signaling triggered by ifn-α (you et al., ; you et al., ) . studies with shp- deficient cells suggest that shp- suppresses tlr -but not tlr or tlr -mediated production of type i ifns and proinflammatory cytokines. the inhibitory function of shp- depends on its phosphatase activity . further investigations show that shp- directly binds to tbk , and the c-terminal domain of shp- and the kinase domain of tbk are responsible for their interaction. it is possible that shp- prevents tbk mediated phosphorylation of its substrates, thereby blocking trif-mediated signaling. isg isg is one of the proteins extensively induced by viral infection (theofilopoulos et al., ) . the ubiquitin activating enzyme (e ) ube l and ubiquitin conjugating enzyme (e ) ubch catalyze isgylation of rig-i by conjugating isg to it, which is followed by ubiquitination and degradation of rig-i (zhao et al., ; kim et al., ) . consistent with this observation, viral infectioninduced expression of ifn-β is enhanced in ube l -/than in wild-type mefs. also, the amount of rig-i protein is more stable in ube l -/than in wild-type mefs (kim et al., ) . collectively, these data suggest that the conjugation of isg to rig-i can inhibit rig-i-mediated signaling by ubiquitination and degradation of rig-i. rnf ubch has been demonstrated to be associated with ubiquitination and degradation of rig-i (zhao et al., ) . to identify the e ubiquitin ligase in this process, yeast two-hybrid assays were performed with ubch as a bait. this effort led to the identification of rnf (arimoto et al., ) . rnf functions as an e ubiquitin ligase to catalyze ubiquitination of rig-i. thus, overexpression of rnf inhibits the virus-triggered and rig-i-mediated type i ifn expression, while knockdown of rnf has an opposite effect. furthermore, rnf is induced by ifn-α stimulation or viral infection, suggesting a negative feedback role played by rnf in innate antiviral signaling. rnf ring-finger protein is an e ubiquitin ligase that has been implicated in cell motility, protein quality control in the er, cancer and degenerative myopathy (kyushiki et al., ; didier et al., ; bromberg et al., ; delaunay et al., ) . in a yeast two-hybrid assay, rnf was identified as a mita-interacting protein . overexpression of rnf inhibits virus-triggered expression of type i ifns in , hela as well as primary monocyte-derived macrophages and dendritic cells, while knockdown of rnf potentiates the expression of type i ifns upon viral infection. a further study suggests that rnf interacts with mita and catalyzes k -linked ubiquitination of mita at k after viral infection, thereby inhibiting excessive type i ifn response. a a was initially found to negatively regulate tnftriggered nf-κb signaling (opipari et al., ) . a contains an ovarian tumor (otu) domain in its n-terminus which has deubiquitination activity, and a c-terminus with ubiquitination activity (wertz et al., ) . both domains are required for inhibition of tnf-induced activation of nf-κb. similarly, a also inhibits rlr-mediated activation of irf . interestingly, the c-terminal domain alone of a is sufficient for its inhibitory function, suggesting that a negatively regulates the signaling through its ubiquitination activity (saitoh et al., ; . moreover, macrophages derived from a deficient mice are incapable of terminating tlr-induced nf-κb activation (boone et al., ) . further investigations show that a functions to cleave the polyubiquitin chains of traf , which is critical for termination of tlr-mediated activation of nf-κb . rbck rbcc protein interacting with pkc (rbck ) belongs to the e ubiquitin ligase family (marin and ferrus, ) . rbck contains a ubiquitin-like (ubl) domain and a ring-ibr-ring (rbr) domain in its n-or c-terminus, respectively. rbck interacts with the cterminal znf domain of tab and induces ubiquitination and degradation of tab , thereby inhibiting tnf-, il- and rlr-induced activation of nf-κb . further study also suggests that rbck interacts with irf and induces degradation of irf after viral infection, indicating a negative feedback regulation of virustriggered type i ifn signaling by rbck . trim α tripartite motif (trim) family proteins are also known as "rbcc" proteins, as they contain an rbcc motif at their n-terminus consisting of a ring domain, one or two b-boxes and a coiled-coil region. trim family proteins have been demonstrated to function in the regulation of cell proliferation, differentiation, development, oncogenesis, apoptosis and antiviral responses (nisole et al., ) . in addition to the rbcc motif, trim α also contains a spry domain at its c-terminus. the expression of trim α is induced by the activation of nf-κb in various types of cells after stimulation with a variety of tlr ligands, such as poly(i:c) and cpg dna. it has been shown that trim α targets tab and tab for k -linked ubiquitination and lysosome-dependent degradation and inhibits autoubiquitination of traf , contributing to the inhibition of tlr-mediated nf-κb activation. further studies suggest that trim α transgenic mice are more resistant to endotoxin shock, whereas in vivo knockdown of trim α by sirna reduces lpsinduced tolerance, which demonstrates that trim α negatively regulated lps-mediated signaling in vivo and functions as a negative modulator of the tlr signaling pathway . . . cleaving the polyubiquitin chains from signaling molecules duba like a , duba is also a member of the deubiquitination (dub) domain-containing protein family which contains an otu domain. a screening of the rnai library targeting dub family proteins led to the identification of duba as a negative regulator of tlr -mediated signaling (kayagaki et al., ) . knockdown of duba potentiates poly(i:c)-induced expression of ifn-β. conversely, overexpression of duba inhibits poly(i:c)induced or rig-i-mediated signaling. an unambiguous identification of duba-interacting proteins suggests that duba interacts with traf , whose k -linked polyubiquitination is required for tlr-and rlr-mediated signaling. duba cleaves the k -linked polyubiquitin chains of traf and depletion of duba increases the level of ubiquinated traf both in steady-state cells and in ligands-stimulated cells. furthermore, overexpression of duba also impairs interaction between tbk and irf , suggesting that ubiquitination of traf controls the activity of the tbk -irf complex. cyld cyld is an otu dub family protein that has been identified to interact with ikkγ and deubiquitinate the k -linked polyubiquitin chains of ikkγ, thereby negatively regulating tnf-induced activation of nf-κb (ea et al., ) . interestingly, two groups independently reported that cyld functions as a negative modulator of rig-i-mediated signaling friedman et al., ) . knockdown of cyld results in an enhancement in sev-triggered ifn-β secretion. experiments using cyld -/-mefs and dendritic cells (dcs) show constitutive activation of tbk /ikkε as well as hyper-induction of type i ifns by vsv infection. immunoprecipitation experiments show that cyld coprecipitates not only with rig-i but also with tbk and ikkε. interestingly, tbk or ikkε specifically causes cyld to shift to higher molecular bands, suggesting phosphorylation of cyld by these kinases. the data indicate that cyld probably regulates the activities of ikk-family kinases and leads to inactivation of the signaling . experiments from another group show that cyld associates with the card domain of rig-i and removes k -linked ubiquitin from rig-i. loss of cyld in dcs causes accumulation of ubiquitinated rig-i. furthermore, the accumulation of ubiquitinated rig-i in cyld-deficient cells is associated with constitutive activation of tbk . the expression of cyld can be induced by tnf treatment or viral infection (friedman et al., ) . these data together suggest a working model of the mechanisms by which cyld negatively regulates the signaling. in uninfected cells, rig-i undergoes ubiquitination-deubiquitination kinetically, making rig-i inactive. upon viral infection, the e ubiquitin ligases trim and/or rnf mediate k -linked ubiquitination of rig-i, which subsequently activates downstream kinase tbk oshiumi et al., ) . however, as viral infection goes on, cyld accumulates and cleaves the k -linked ubiquitin chains of rig-i, inhibiting virus-triggered type i ifn signaling. thus, cyld is most likely a negative regulator that inhibits rig-i in both steady-state and activated state to prevent unnecessary signaling events. however, the precise mechanism that controls the balance between trim /rnf -mediated ubiquitination and cyldmediated deubiquitination of rig-i and the phosphorylation of cyld by tbk in its inhibitory function remains to be elucidated. in addition to the mechanisms mentioned above, several proteins have been described as regulators in tlr-and rlr-mediated signaling. for example, gsk β regulates tlr-and ifnγ-mediated production of a subset of inflammatory cytokines and facilitates production of antiinflammatory cytokines (martin et al., ; woodgett and ohashi, ; hu et al., ) . nrdp mediates k linked ubiquitination and activation of tbk as well as k -linked ubiquitination and degradation of myd , thereby enhancing production of type i ifns and suppressing production of pro-inflammatory cytokines . shp- interacts with the kinase domain of irak and inhibits irak activation, which may explain why shp- inhibits the expression of proinflammatory cytokines . however, the exact mechanisms are unclear. pin recognizes and binds to the ser phosphorylated irf , leading to considerable conformational change of irf . the conformational change makes irf accessible for binding with e ubiquitin ligases such as rbck , which catalyze ubiquitination and degradation of irf (saitoh et al., ; . wd domain repeat protein (wdr ) interacts with tak and negatively regulates tlr -induced nf-κb activation, although the mechanism is unclear at this moment ). there is evidence suggesting that caspase cleaves visa at d and trif at both d and d , indicating a crosstalk between the apoptosis pathway and tlr-/rlr-mediated signaling . it is possible that these proteins function together in a temporal and spatial manner to control harmful excessive immune responses and protect host against autoimmune diseases. there are controversies about the functions of several proteins involved in virus-triggered signaling. for example, isg covalently binds to and stabilizes irf , counteracting its negative regulation of rig-i kim et al., ) . ro (also called trim ) has been demonstrated to catalyze the ubiquitination of irf and induce its degradation, while another report suggests that ro /trim interacts with pin and irf and prevents pin -induced conformational change and degradation of irf , thereby sustaining irf activation during viral infection (higgs et al., ; yang et al., ) . tank has been identified as a scaffold protein facilitating traf -tbk -irf interaction and subsequent activation of irf (guo and cheng, ; gatot et al., ) . however, experiments on tank-deficient mice suggest that tank is not involved in interferon responses but rather functions as a negative regulator of tlr-signaling and is critical for the prevention of autoimmune nephritis by regulating ubiquitination of traf (kawagoe et al., ) . sarm contains heat-armadillo motifs at its nterminus, two sterile α motifs (sam) in middle and a tir domain at its c-terminus and is inducible by tlr ligands and acts as a negative regulator in trif-but not myd dependent pathway (carty et al., ) . however, another study showed that sarm is not involved in tlr-mediated signaling, as evidenced by the equivalent production of tnf and mcp- by bone marrow-derived macrophages of sarm-deficient and wild-type mice in response to various tlr ligands. it is also reported that sarm functions to restrict viral infection and neuronal injury in a brain region-specific manner (szretter et al., ) . further studies and more efforts are required to figure out these inconsistencies. host and virus have mutually exerted powerful selective pressure to each other throughout their evolution. for example, the type i ifn system as a fast and primary defense against viral pathogenesis serves as a strong selective pressure for viral evolution yoneyama and fujita, ). on the other hand, various viral components, which play roles in viral replication, assembly and pathogenesis, also target the molecules involved in the system to evade host immunity against viruses (bowie and unterholzner, ) . there is increasing evidence suggesting that viruses have evolved a number of strategies to evade the recognition by prrs, to inhibit prr-mediated signaling, and even to manipulate host signaling pathways for their own benefit (table ) . it has been demonstrated that rlr-mediated expression of type i ifns in macrophages and cdcs represents the first line of defense against local viral infection (kumagai et al., ) . experiments on rig-i-or mda -deficient mice suggest that rig-i and mda recognize different rna structures generated by distinct viruses . for example, rig-i recognizes 'pppssrna, short dsrna, newcastle disease virus (ndv) and influenza a virus (iav), while mda recognizes long dsrna and piconaviridae family such as encephalomyocarditis virus (emcv). however, neither of them recognizes cellular mrna which is protected by a methylguanosine cap (m gppp structure) or ribosomal rna or trna which is flanegan et al., ; lee et al., hornung et al., pichlmair et al., severe stack et al., keating et al., diperna et al., chen et al., schroder et al., rahman and mcfadden, roy and mocarski, hepatitis tait et al., rodriguez et al., kaposi's sarcoma-associated herpesvirus k ikkα/β/γ complex activation of nf-κb matta et al., monophosphated and/or modified with unusual bases (hornung et al., ; kato et al., ; bowie and unterholzner, ) . viruses seem to be so smart that they are aware of the tricks by which the host distinguishes self and nonself rna. a number of proteins encoded by viral genomes can process viral rna and simulate the modifications of host rna, thereby evading the supervision by prrs (furuichi and furuichi, ) . for example, picornaviruses such as emcv protect the ' end of its rna with the covalently linked protein vpg (flanegan et al., ) . the nonstructural protein (nsp ) of severe acute respiratory syndrome (sars) coronavirus functions as an n methyltransferase that catalyzes to form an m gppp structure at the 'end of its rna (von grotthuss et al., ; chen et al., ) . genomic rnas from hantaan virus (htnv), crimean-congo hemorrhagic fever virus (cchfv) and borna disease virus (bdv) are 'monophosphorylated habjan et al., ) . however, the exact mechanisms for these modifications are unknown. human immunodeficiency virus (hiv) and adenovirus use host rna processing machinery to cap newly synthesized viral rna (furuichi and furuichi, ) . poxviruses replicate in the cytoplasm and encode their own rna capping machinery. in addition, iav 'snatches' capped ' fragments from cellular mrna to its own genomic rna to mask its 'ppp structure ( plotch et al., ) . many viruses also produce dsrna at some stage during their life cycle, which is recognized by host prrs. to avoid innate immune responses that are initiated by rlrs, some viral genomes encode dsrna-binding proteins, including vaccinia virus (vacv) e l (chang et al., ) , ebola virus vp (cardenas et al., ; haasnoot et al., ) and hiv tat (weeks et al., ) . these proteins shield the dsrna structures generated during infection and replication from recognition by rlrs. it is also possible for these proteins to inhibit tlr -mediated signaling in virus-and viral dsrna-containing endosomes. taken together, these studies suggest that many viruses inhibit prr-mediated type i ifn signaling at the very beginning of prr-mediated recognition of viral nuclear acids. as mentioned above, virus-triggered type i ifn signaling depends on the interactions of various signaling molecules. to block the signaling process, many viral proteins interact with the key molecules involved in the process and thereby prevent signal transduction leading to the expression of type i ifns. for example, some viral proteins bind to rlrs directly, thereby inhibiting rlr-mediated signaling effectively. nonstructural protein of iav which binds to rig-i and v proteins of picornavirus which bind to mda are two such examples (andrejeva et al., ; mibayashi et al., ) . the paramyxoviridae family human metapneumovirus (hmpv) g gene-encoded glycoprotein g specifically interacts with rig-i and blocks rig-imediated ifn-β induction (bao et al., ) . recombinant hmpv lacking the g gene replicates efficiently in vitro but its virulence is highly attenuated in vivo. a r of vacv is a multiple tir domain-containing protein, which interacts with the adaptors trif and myd and inhibits tlrmediated production of type i ifns. consistent with the observation, the deletion of a r attenuates but not abolishes viral virulence of vacv (stack et al., ) . n l of vacv is associated with the kinase tbk (diperna et al., ) . other two vacv-encoded proteins, a r which interacts with traf and irak and b r which interacts with ikkβ inhibit production of type i ifns and inflammatory cytokines schroder et al., ) . hepatitis c virus (hcv) ns , rabies virus phosphoprotein and bdv phosphoprotein interact with tbk , leading to sequestration of tbk from its upstream or downstream signaling proteins (otsuka et al., ; unterstab et al., ) . the g protein of pathogenic hantavirus can inhibit tbk function by disrupting the traf -tbk interaction which is required for signaling (alff p et al., ) . poxvirus protein n l targets ikk complex and inhibits the activation of nf-κb and irf by tlrs (diperna et al., ) . recent studies suggest that dead-box protein (ddx ) is a critical component of the tbk /ikkε complex in both rlrs and cytoplasmic dna receptors-mediated signaling. k r of vacv inhibits prr-mediated induction of ifn-β by targeting ddx , thereby preventing tbk or ikkεmediated activation of irfs (schroder et al., ) . several viral proteins inhibit signaling by targeting transcription factors. for example, ns of west nile virus (wnv) inhibits tlr -mediated induction of ifn-β by preventing the translocation of nf-κb and irf to the nucleus (wilson et al., ) . herpes simplex virus (hsv) infected cell protein (icp ) binds to irf and sequesters irf from binding host dna (melroe et al., ) . there are also viral proteins homologous to host irfs that inhibit irf and irf signaling. v proteins of mumps virus and parainfluenza virus act as substrates of tbk /ikkε and compete with irf for phosphorylation by tbk /ikkε (lu et al., ) . human herpesvirus encoded irf homologue virf represses type i ifn induction by blocking the association of irf with the coactivators cbp and p (lin et al., ) . similarly, virf , a kaposi's sarcoma-associated herpesvirus (kshv) viral irf homologue, dimerizes with the host irf and inhibits its dna binding activity . another kshv protein, k-bzip, competes with the host irf for binding sites in the promoters of isg genes, thereby modulating the expression of antiviral genes (lefort et al., ) . many viral genomes are translated into polypeptide precursors which need to be cleaved into mature and functional proteins during viral replication. the cleavage activity depends on several non-structural proteins encoded by the viruses. for example, hcv genome is translated to a polypeptide and ns / a is one of such proteases and responsible for the cleavage. on the other hand, such activity of these proteases provides the possibility that they may cleave the host signaling molecules as well. not surprisingly, the adaptors trif and visa are cleaved by ns / a into two polypeptides that impair their ability to mediate prr-induced expression of type i ifns (li et al., a; li et al., b; . further studies suggest that the protease precursor protein abc of picornavirus hepatitis a virus (hav) and ns / a of flavivirus gb virus b cleave visa on the mitochondrial membrane, which disrupts rlrmediated signaling yang et al., ) . ns a of poliovirus induces the cleavage of mda by caspases, although the exact mechanism is unclear (abe et al., ) . in addition, a number of viral proteins destabilize irfs and target them for degradation. for example, bicp of bovine herpesvirus targets irf for degradation instead of temporarily sequestering it as icp from hsv does (barral et al., ) . flaviridae family classical swine fever virus protease n (npro) and hiv proteins vpr and vif also induce proteasomal degradation of irf , suggesting a common mechanism used by viruses to evade antiviral responses (bauhofer et al., ; okumura et al., ) . rotavirus nsp antagonizes the function of irf , irf and irf by inducing their degradation, thereby inhibiting the expression of type i ifns (barro and patton, ) . in contrast to evasion of the host recognition and inhibition of the signaling, it is proposed that viruses can 'hijack' and even subvert aspects of prr-mediated signaling. viruses use living cells as hosts, which means that while viral infection and replication causes dysfunction of the hosts, viruses have to prevent the hosts from apoptosis at the same time. not only is nf-κb involved in the production of ifn-β, it also inhibits apoptosis and promotes proliferation of the host cells. therefore, nf-κb is a key molecule to be regulated and used by viruses. taking the control of nf-κb activity by african swine fever virus (asfv) as an example, the asfv protein a l is an early expressed homologue to the inhibitor of κbα (iκbα) that sequesters nf-κb in the cytoplasm, thereby inhibiting nf-κb activity (tait et al., ) . however, as infection and replication progress, a l is expressed, which activates nf-κb and inhibits caspases, preventing cells from apoptosis (rodriguez et al., ) . similarly, the k protein from kahv interacts with the canonical ikkα/β/γ complex to selectively activate nf-κb (matta et al., ) . there are other examples of viruses that use and manipulate prr-signaling for their replication and survival. hiv employs ddx to export viral rna from the nucleus to the cytoplasm (yedavalli et al., ) . wnv may use tlr -mediated signaling to produce cytokines to create a microenvironment favored by the viruses, as evidenced by the fact that tlr deficiency mice are resistant to wnv infection . collectively, viruses adopt different strategies to inhibit and manipulate prr-mediated signaling. although progresses have been made to elucidate the mechanisms in the past decade, we expect new insights into the strategies that are used by viruses to interfere with prr-mediated signaling. just as host and virus exert selective pressure on each other, studies on hosts and investigations of viruses mutually facilitate our understandings about each other. future studies will focus on systemic mechanisms by which hosts balance the expression of antiviral and inflammatory cytokines and the translation of viral evasion into benefit of human health. first, mice with deletion of some cellular negative regulators show resistance to viral infection and high viability, while mice defective in others are susceptible to viral infection-caused inflammatory responses. as a result, it is important to take a systemic view of the host regulators when using in vivo mouse models. second, much more efforts are needed to direct in vivo studies relevant to humans. for example, tlr deficiency does not impair the host immune response to several viruses (edelmann et al., ) . later, it was reported that tlr-deficient mice are more resistant to wnv infection . however, patients with mutations in tlr are related to hsv-associated encephalitis . finally, development of therapeutic drugs that targets cellular or viral inhibitors may provide new strategy to treat infection or immune dysfunction-caused diseases. hepatitis c virus nonstructural protein a modulates the tolllike receptor-myd -dependent signaling pathway in macrophage cell lines rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate toll-like receptor signalling pathogen recognition and innate immunity recognition of double-stranded rna and activation of nf-kappab by toll-like receptor the pathogenic ny- hantavirus g cytoplasmic tail inhibits rig-i and tbk- directed interferon responses phosphatase shp- promotes tlr-and rig-i-activated production of type i interferon by inhibiting the kinase irak shp- phosphatase negatively regulates the trif adaptor protein-dependent type i interferon and proinflammatory cytokine production the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter negative regulation of the rig-i signaling by the ubiquitin ligase rnf from atp as substrate to adp as coenzyme: functional evolution of the nucleotide binding subunit of dihydroxyacetone kinases human metapneumovirus glycoprotein g inhibits innate immune responses mda- is cleaved in poliovirus-infected cells rotavirus nsp inhibits expression of type i interferon by antagonizing the function of interferon regulatory factors irf , irf , and irf classical swine fever virus npro interacts with interferon regulatory factor and induces its proteasomal degradation the ubiquitin-modifying enzyme a is required for termination of toll-like receptor responses viral evasion and subversion of pattern-recognition receptor signalling tumor necrosis factor receptor-associated factors (trafs) increased expression of the e ubiquitin ligase rnf is associated with decreased survival in breast cancer identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein p interferes with phosphorylation of interferon regulatory factor inhibition of interleukin receptor/toll-like receptor signaling through the alternatively spliced, short form of myd is due to its failure to recruit irak- identification of human and rat fad-amp lyase (cyclic fmn forming) as atp-dependent dihydroxyacetone kinases ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling the human adaptor sarm negatively regulates adaptor protein trif-dependent toll-like receptor signaling thsse e l gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded rna-dependent protein kinase inhibition of ikappab kinase by vaccinia virus virulence factor b functional screen reveals sars coronavirus nonstructural protein nsp as a novel cap n methyltransferase gb virus b disrupts rig-i signaling by ns / a-mediated cleavage of the adaptor protein mavs rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway triad a, an e ubiquitin-protein ligase regulating toll-like receptors the cterminal regulatory domain is the rna '-triphosphate sensor of rig-i rip mediates the trif-dependent toll-like receptor -and -induced nf-{kappa}b activation but does not contribute to interferon regulatory factor activation the er-bound ring finger protein (rnf / rma ) causes degenerative myopathy in transgenic mice and is deregulated in inclusion body myositis negative regulation of virus-triggered ifn-beta signaling pathway by alternative splicing of tbk regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus negative regulation of mda -but not rig-i-mediated innate antiviral signaling by the dihydroxyacetone kinase rnf , a ring finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization innate antiviral responses by means of tlr -mediated recognition of single-stranded rna poxvirus protein n l targets the i-kappab kinase complex, inhibits signaling to nf-kappab by the tumor necrosis factor superfamily of receptors, and inhibits nf-kappab and irf signaling by toll-like receptors activation of ikk by tnfalpha requires site-specific ubiquitination of rip and polyubiquitin binding by nemo does toll-like receptor play a biological role in virus infections? covalent linkage of a protein to a defined nucleotide sequence at the '-terminus of virion and replicative intermediate rnas of poliovirus the tumour suppressor cyld is a negative regulator of rig-i-mediated antiviral response viral and cellular mrna capping: past and prospects roles of rig-i n-terminal tandem card and splice variant in trim -mediated antiviral signal transduction trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity wdr is a novel tak -associated suppressor of the il- r/tlr /tlr -induced nf-kappab activation pathway reul is a novel e ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i lipopolysaccharide-mediated interferon regulatory factor activation involves tbk -ikkepsilon-dependent lys( )-linked polyubiquitination and phosphorylation of tank/i-traf cutting edge: tnfr-associated factor (traf) is essential for myd -dependent pathway but not toll/il- receptor domain-containing adaptor-inducing ifn-beta (trif)-dependent pathway in tlr signaling modulation of the interferon antiviral response by the tbk /ikki adaptor protein tank autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages the ebola virus vp protein is a suppressor of rna silencing processing of genome ' termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction mechanisms of the trif-induced interferon-stimulated response element and nf-kappab activation and apoptosis pathways species-specific recognition of single-stranded rna via toll-like receptor and small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway a toll-like receptor recognizes bacterial dna the roles of two ikappab kinase-related kinases in lipopolysaccharide and double stranded rna signaling and viral infection the e ubiquitin ligase ro negatively regulates ifn-beta production post-pathogen recognition by polyubiquitin-mediated degradation of irf type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors role of a transductional-transcriptional processor complex involving myd and irf- in toll-like receptor signaling '-triphosphate rna is the ligand for rig-i sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr ikappab kinase-alpha is critical for interferon-alpha production induced by toll-like receptors and ifn-gamma suppresses il- production and synergizes with tlr by regulating gsk and creb/ap- proteins sike is an ikk epsilon/tbk -associated suppressor of tlr -and virustriggered irf- activation pathways sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity toll-like receptor -mediated activation of nf-kappab and irf diverges at toll-il- receptor domain-containing adapter inducing ifn-beta inhibition of interferon regulatory factor (irf )-mediated interferon signal transduction by the kaposi's sarcoma-associated herpesvirus viral irf homolog virf the atg atg conjugate associates with innate antiviral immune responses length-dependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiation-associated gene differential roles of mda and rig-i helicases in the recognition of rna viruses tank is a negative regulator of toll-like receptor signaling and is critical for the prevention of autoimmune nephritis toll-like receptor and rig-i-like receptor signaling the roles of tlrs, rlrs and nlrs in pathogen recognition interferon-alpha induction through toll-like receptors involves a direct interaction of irf with myd and traf ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction duba: a deubiquitinase that regulates type i interferon production irak- participates in multiple toll-like receptor signaling pathways to nfkappab via activation of traf ubiquitination negative feedback regulation of rig-i-mediated antiviral signaling by interferoninduced isg conjugation myd - links mitochondria, microtubules, and jnk in neurons and regulates neuronal survival sequence-and target-independent angiogenesis suppression by sirna via tlr irak-m is a negative regulator of toll-like receptor signaling loss of autophagy in the central nervous system causes neurodegeneration in mice cpg motifs in bacterial dna and their immune effects tlr -dependent recognition of mcmv by ipc and dc generates coordinated cytokine responses that activate antiviral nk cell function herpes simplex virus type activates murine natural interferonproducing cells through toll-like receptor alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with rna viruses pathogen recognition in the innate immune response cloning, expression and mapping of a novel ring-finger gene (rnf ), a human homologue of a putative zinc-finger gene from caenorhabditis elegans detrimental contribution of the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia a protein covalently linked to poliovirus genome rna binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferonresponsive factor elements modulates antiviral gene expression immune evasion by hepatitis c virus ns / a protease-mediated cleavage of the toll-like receptor adaptor protein trif hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity isg is a negative-feedback regulator of virustriggered signaling and cellular antiviral response hhv- encoded virf- represses the interferon antiviral response by blocking irf- recruitment of the cbp/p coactivators dissociation of a mavs/ips- /visa/cardif-ikkepsilon molecular complex from the mitochondrial outer membrane by hepatitis c virus ns - a proteolytic cleavage negative regulation of the retinoic acid-inducible gene iinduced antiviral state by the ubiquitin-editing protein a isg enhances the innate antiviral response by inhibition of irf- degradation select paramyxoviral v proteins inhibit irf activation by acting as alternative substrates for inhibitor of kappab kinase epsilon (ikke)/tbk toll-like receptor -mediated recognition of herpes simplex virus- by plasmacytoid dendritic cells structure and function of the interferon-beta enhanceosome comparative genomics of the rbr family, including the parkinson's disease-related gene parkin and the genes of the ariadne subfamily toll-like receptormediated cytokine production is differentially regulated by glycogen synthase kinase fln , a novel interferon-and lps-inducible gene acting as a negative regulator of toll-like receptor signaling kaposi's sarcoma-associated herpesvirus (kshv) oncoprotein k bypasses trafs and directly interacts with the ikappab kinase complex to selectively activate nf-kappab without jnk activation viral sensors: diversity in pathogen recognition recruitment of activated irf- and cbp/p to herpes simplex virus icp nuclear foci: potential role in blocking ifn-beta induction rip is an essential mediator of toll-like receptor -induced nf-kappa b activation cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus inhibition of retinoic acid-inducible gene imediated induction of beta interferon by the ns protein of influenza a virus tradd protein is an essential component of the rig-like helicase antiviral pathway nlrx is a regulator of mitochondrial antiviral immunity herpesviruses and the innate immune response the 'shp'ing news: sh domaincontaining tyrosine phosphatases in cell signaling trim family proteins: retroviral restriction and antiviral defence critical role of traf in the toll-like receptor-dependent and-independent antiviral response unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus hiv- accessory proteins vpr and vif modulate antiviral response by targeting irf- for degradation structure of the reovirus outer capsid and dsrna-binding protein sigma at . a resolution the a cdna induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein riplet/ rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection interaction between the hcv ns protein and the host tbk protein leads to inhibition of cellular antiviral responses differential requirement for tank-binding kinase- in type i interferon responses to toll-like receptor activation and viral infection rig-i-mediated antiviral responses to singlestranded rna bearing '-phosphates the regulatory domain of the rig-i family atpase lgp senses double-stranded rna a unique cap (m gpppxm)-dependent influenza virion endonuclease cleaves capped rnas to generate the primers that initiate viral rna transcription the shp- tyrosine phosphatase: signaling mechanisms and biological functions modulation of tumor necrosis factor by microbial pathogens the antiviral adaptor proteins cardif and trif are processed and inactivated by caspases the involvement of the interleukin- receptorassociated kinases (iraks) in cellular signaling networks controlling inflammation african swine fever virus iap-like protein induces the activation of nuclear factor kappa b pathogen subversion of cell-intrinsic innate immunity sintbad, a novel component of innate antiviral immunity, shares a tbk -binding domain with nap and tank interferon-inducible antiviral effectors regulation of antiviral responses by a direct and specific interaction between traf and cardif the infected cell protein encoded by bovine herpesvirus (bicp ) induces degradation of interferon response factor and, consequently, inhibits beta interferon promoter activity regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna negative regulation of interferon-regulatory factor -dependent innate antiviral response by the prolyl isomerase pin a is a negative regulator of ifn regulatory factor signaling fln deficiency reveals its negative regulatory role in the toll-like receptor (tlr) and retinoic acid-inducible gene i (rig-i)-like helicase signaling pathway toll/il- receptor domain-containing adaptor inducing ifn-beta (trif) associates with tnf receptor-associated factor and tank-binding kinase , and activates two distinct transcription factors, nf-kappa b and ifn-regulatory factor- , in the toll-like receptor signaling recognition of ' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus genome trimming: a unique strategy for replication control employed by borna disease virus viral targeting of dead box protein reveals its role in tbk /ikkepsilon-mediated irf activation identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf trim alpha negatively regulates tlr-mediated nf-kappa b activation by targeting tab and tab for degradation osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells vaccinia virus protein a r targets multiple toll-like-interleukin- receptor adaptors and contributes to virulence tnf receptor-associated factor- (traf ) negatively regulates toll/ il- receptor domain-containing adaptor inducing ifn-beta (trif)-mediated signaling the immune adaptor molecule sarm modulates tumor necrosis factor alpha production and microglia activation in the brainstem and restricts west nile virus pathogenesis mechanism of inactivation of nf-kappa b by a viral homologue of i kappa b alpha. signal-induced release of i kappa b alpha results in binding of the viral homologue to nf-kappa b nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses dlm- /zbp ) is a cytosolic dna sensor and an activator of innate immune response innate immunity to virus infection distinct induction patterns and functions of two closely related interferon-inducible human genes, isg and isg type i interferons (alpha/beta) in immunity and autoimmunity rbck negatively regulates tumor necrosis factor-and interleukin- -triggered nf-kappab activation by targeting tab / for degradation interleukin- receptor-associated kinase- plays an essential role for toll-like receptor (tlr) -and tlr -mediated interferon-{alpha} induction viral targeting of the interferon-{beta}-inducing traf family member-associated nf-{kappa}b activator (tank)-binding kinase- loss of dexd/h box rna helicase lgp manifests disparate antiviral responses mrna cap- methyltransferase in the sars genome mrna cap- methyltransferase in the sars genome the e ubiquitin ligase nrdp 'preferentially' promotes tlr-mediated production of type i interferon alpha interferon induces distinct translational control programs to suppress hepatitis c virus rna replication toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis a is a potent inhibitor of tlr -and sendai virus-induced activation of nf-kappab and isre and ifn-beta promoter fragments of the hiv- tat protein specifically bind tar rna de-ubiquitination and ubiquitin ligase domains of a downregulate nf-kappab signalling west nile virus nonstructural protein inhibits tlr signal transduction gsk : an in-toll-erant protein kinase? inhibition of rig-i and mda -dependent antiviral response by gc qr at mitochondria visa is an adapter protein required for virus-triggered ifn-beta signaling role of adaptor trif in the myd -independent toll-like receptor signaling pathway trim is essential to sustain ifn regulatory factor activation during antiviral response disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor requirement of ddx dead box rna helicase for hiv- rev-rre export function shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses rna recognition and signal transduction by rig-i-like receptors modulation of the nuclear factor kappa b pathway by shp- tyrosine phosphatase in mediating the induction of interleukin (il)- by il- or tumor necrosis factor shp- tyrosine phosphatase functions as a negative regulator of the interferon-stimulated jak/stat pathway negative feedback regulation of cellular antiviral signaling by rbck -mediated degradation of irf regulation of ikappab kinase-related kinases and antiviral responses by tumor suppressor cyld tlr deficiency in patients with herpes simplex encephalitis human isg conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways the adaptor protein mita links virussensing receptors to irf transcription factor activation the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita key: cord- - yd wg v authors: huang, yin-qiu; tang, sheng-quan; xu, xiao-lei; zeng, yan-ming; he, xiao-qing; li, yao; harypursat, vijay; lu, yan-qiu; wan, yan; zhang, lu; sun, qiang-zhong; sun, nan-nan; wang, gui-xue; yang, zhong-ping; chen, yao-kai title: no statistically apparent difference in antiviral effectiveness observed among ribavirin plus interferon-alpha, lopinavir/ritonavir plus interferon-alpha, and ribavirin plus lopinavir/ritonavir plus interferon-alpha in patients with mild to moderate coronavirus disease : results of a randomized, open-labeled prospective study date: - - journal: front pharmacol doi: . /fphar. . sha: doc_id: cord_uid: yd wg v background: currently, severe acute respiratory syndrome coronavirus (sars-cov- ) has spread globally, causing an unprecedented pandemic. however, there is no specific antiviral therapy for coronavirus disease (covid- ). we conducted a clinical trial to compare the effectiveness of three antiviral treatment regimens in patients with mild to moderate covid- . methods: this was a single-center, randomized, open-labeled, prospective clinical trial. eligible patients with mild to moderate covid- were randomized into three groups: ribavirin (rbv) plus interferon-α (ifn-α), lopinavir/ritonavir (lpv/r) plus ifn-α, and rbv plus lpv/r plus ifn-α at a : : ratio. each patient was invited to participate in a -d follow-up after initiation of an antiviral regimen. the outcomes include the difference in median interval to sars-cov- nucleic acid negativity, the proportion of patients with sars-cov- nucleic acid negativity at day , the mortality at day , the proportion of patients re-classified as severe cases, and adverse events during the study period. results: in total, we enrolled patients in this study. baseline clinical and laboratory characteristics of patients were comparable among the three groups. in the analysis of intention-to-treat data, the median interval from baseline to sars-cov- nucleic acid negativity was d in the lpv/r+ifn-α-treated group, as compared with and d in the rbv+ifn-α-treated group and in the rbv+lpv/r+ ifn-α-treated group, respectively (p= . ). the proportion of patients with sars-cov- nucleic acid negativity in the lpv/r+ifn-α-treated group ( . %) was higher than the rbv+ ifn-α-treated group ( . %) and the rbv+lpv/r+ifn-α-treated group ( . %) at day ; however, the difference between these groups was calculated to be statistically insignificant. the rbv+lpv/r+ifn-α-treated group developed a significantly higher incidence of gastrointestinal adverse events than the lpv/r+ ifn-α-treated group and the rbv+ ifn-α-treated group. conclusions: our results indicate that there are no significant differences among the three regimens in terms of antiviral effectiveness in patients with mild to moderate covid- . furthermore, the combination of rbv and lpv/r is associated with a significant increase in gastrointestinal adverse events, suggesting that rbv and lpv/r should not be co-administered to covid- patients simultaneously. clinical trial registration: www.clinicaltrials.gov, id: chictr . registered on january , . the outbreak of the coronavirus disease , caused by severe acute respiratory syndrome coronavirus (sars-cov- ), has been declared a pandemic by the world health organization (who) (who, ) . in addition to the dramatic and unprecedented challenges for the entire global community in facing the disease, there remains no specific antiviral treatments for covid- at this time. thus, efforts to investigate the effectiveness of treatments proposed to combat the etiological agents of similar past viral outbreaks are necessary in order to discover potential direct antiviral therapies for covid- , to effectively complement existing supportive care. as described by zhu et al. ( ) , sars-cov- shares phylogenetic traits with severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) despite being genetically distinct. as such, antiviral treatments that were used to target sars-cov and mers-cov may provide some insight into potential covid- therapies. antiviral research during the sars and mers outbreaks resulted in the identification of several compounds that may potentially target coronavirus replication directly, or may modulate the immune response to coronavirus infection. for example, a combination of rbv and ifn, has been demonstrated to be effective in reducing mers-cov replication in vitro (falzarano et al., a) , and has shown a synergistic antiviral effect on mers-cov-infected vero cell lines, which resulted in a consequent decrease in viral protein levels (chen et al., ; morgenstern et al., ) . in addition, treatment with ifn and rbv decreased virus replication, moderated host immune responses, and improved clinical outcomes in mers-cov-infected rhesus macaques (falzarano et al., b) . in a mers-cov study, the survival rate at d after diagnosis was significantly higher in the ifn plus rbv group compared with the control group ( % vs. %; p= . ), even though the survival rate did not differ significantly at d ( % vs. %; p= . ) (omrani et al., ) . the antiviral drug combination of lopinavir/ ritonavir (lpv/r) has also been shown to inhibit coronavirus infection in cell cultures at low-micromolar concentrations (de wilde et al., ) , and animal experiments have showed that lpv/r combined with ifn effectively reduced virus titers, and induced improvement of pulmonary lesions in mers-covinfected mice (momattin et al., ) . chan et al. ( ) found that the lpv/r plus ifn combination resulted in better clinical scores, decreased weight reduction, less pulmonary infiltrates, and lower viral load in marmosets inoculated with mers-cov, compared with controls. recently, a randomized controlled study observed that the early use of a combination of ifn, lpv/ r, and rbv was effective for the treatment of covid- , compared with lpv/r alone (hung et al., ) . the available scientific literature suggests that a combination of rbv plus ifn, lpv/r plus ifn, or rbv plus lpv/r plus ifn may be of benefit in patients with covid- . the office of national health commission of the people's republic of china, and the national administration bureau of traditional chinese medicine have jointly issued different versions of the "guidelines for diagnosis and treatment of novel coronavirus pneumonia", in which lpv/r, ifna, and rbv are recommended for on-trial use in patients with covid- . based on the chinese guidelines, and promising results of relevant previous studies, we conducted the present study to compare the efficacy and safety of rbv plus ifna, lpv/r plus ifn-a, and rbv plus lpv/r plus ifn-a in patients with mild to moderate covid- . we did not set up a blankcontrolled or placebo-controlled group out of compassionate and ethical considerations, and the specific limitations to therapeutic variations imposed by national guidelines for covid- in china. this present study was a single-center, open-labeled, randomized, prospective clinical trial, which included eligible patients diagnosed with mild to moderate covid- . this study was approved by the ethics committee of chongqing public health medical center ( - - -ky), and was duly registered at the chinese clinical trial registry (chictr ). patients included in our study had to satisfy all the following eligibility criteria: ( ) - years of age; ( ) diagnosed as mild to moderate and ( ) willing to sign informed consent. we excluded patients if they: ( ) were pregnant or breastfeeding women; ( ) had aspartate aminotransferase (ast) or alanine aminotransferase (alt) > × upper normal limit, creatinine clearance < ml/min (lu and dong, ) ; ( ) were allergic or intolerant to therapeutic drugs; ( ) were hiv-positive patients; ( ) had severe heart disease, brain disease, lung disease, kidney disease, neoplastic disease, or other systemic diseases, which may have had the potential to influence patients' adherence to the prescribed antiviral regimens; and ( ) withheld informed consent. patients meeting all the following criteria were defined to have mild to moderate covid- : ( ) sars-cov- nucleic acid testing was positive in the upper respiratory tract via nasopharyngeal or oropharyngeal swab samples, or lower respiratory tract via expectorated sputum samples, endotracheal aspirate samples, or broncho-alveolar lavage (bal) samples. ( ) patients were symptomatic with fever, unproductive cough, or dyspnea, and their x-ray or computed tomography scan imaging demonstrated evidence of interstitial pneumonia; ( ) respiratory rate (rr)< times/min; ( ) oxygen saturation (resting-state) > %; and ( ) arterial partial pressure of oxygen (pao )/oxygen concentration (fio )> . kpa. patients meeting the following criteria were defined to have severe covid- : ( ) identification of sars-cov- via rt-pcr in nasopharyngeal swab samples, sputum samples, bal fluid, or blood samples; ( ) having at least one of the following clinical conditions: a. respiratory distress (≥ times/min); b. oxygen saturation ≤ % at rest; c. arterial partial pressure of oxygen (pao )/fraction of inspiration o (fio ) ≤ . kpa; d. respiratory failure requiring mechanical ventilation; e. septic shock; and f. critical organ failure requiring icu care. all eligible patients were randomly assigned to a random number allocated from a random number sequence generated by a computer, which excluded potential influence from treating physicians, to one of three treatment groups: rbv+ifn-a, lpv/ r+ifn-a, and rbv+lpv/r+ifn-a, with an allocation ratio of : : , and a block size of nine patients each. laboratory staff performing quantitative or qualitative testing were blinded to treatment allocation, while medical staff were not blinded. all of the investigators participating in the study were appropriately trained, based on a standard operating procedure (sop) manual, to ensure patient adherence to the protocol. efficacy, safety data, and information related to adverse effects were collected at each follow-up visit during the follow-up period. all raw data were recorded in case report forms (crfs) and in microsoft excel (microsoft corporation, redmond, wa, usa). withdrawals from the study, or missed visits were fully explained on crfs. significantly abnormal data, or data that were outside clinically acceptable ranges (laboratory values below or exceeding % of the normal range) were required to be explained by the attending physician. we applied this rule for all laboratory values. the study monitor reviewed all crfs, checked the accuracy of inclusion, exclusion, and withdrawal criteria, as well as ensured that information on the crfs was in accordance with those in the source electronic medical records. rbv was given by intravenous injection at a loading dose of g, followed by oral doses of - mg every h depending on patients' body weight, for d (booth et al., ; lu and dong, ) . lpv/r was given orally at a dose of mg/ mg per dose twice per day for d (cao et al., ) . ifn-a was given by atomizing inhalation at a dose of million u or mg per dose twice a day for d. all enrolled patients were hospitalized, and medication was dispensed and administered by nurses punctually and in the correct doses, and administered under camera surveillance during hospitalization, so that patient compliance was assured by visual confirmation. missed doses were administered within h of the prescribed timing. all patients in each cohort could additionally receive nasal cannula oxygen therapy, non-steroidal anti-inflammatory drugs (nsaids), oral or intravenous rehydration, electrolyte correction, anti-pyretics, analgesics, and anti-emetic drugs as required by their clinical conditions, as supportive treatment. there were no other differences in treatment administered to individual subjects taking the three different antiviral regimens. admitted patients with mild and moderate covid- were assessed for eligibility for our trial, and those eligible were asked to sign informed consent before being included in the trial. then, included patients were randomized to one of the three treatment groups at a ratio of : : by means of the computer-generated random allocation schedule. study visits took place on day , day , day , day , and day after initiation of an antiviral regimen. safety was assessed by interrogation of the participants for development of clinical symptoms, and examination for clinical signs, clinical laboratory tests, and documentation of adverse events. treatment adherence was assessed by review of clinical diaries that were filled out by our medical staff. the primary outcome was the difference in the interval from baseline (initiation of antiviral treatment) to sars-cov- nucleic acid negativity by nasopharyngeal swab among the three antiviral treatment groups, with each of these two tests at least h apart. the secondary outcomes included the differences among the three groups in the proportion of patients with sars-cov- nucleic acid negativity at day , the mortality rate at day , the proportion of patients re-classified as severe cases during the study period, the incidence of adverse events during the study period, and the proportion of therapeutic discontinuations due to adverse events during the study period. sars-cov- nucleic acid negativity was defined as the presence of negative sars-cov- results in at least two consecutive nasopharyngeal swabs by reverse transcriptase polymerase chain reaction (rt-pcr), with an interval of at least h between the two time points of swab-taking. of the two consecutive negative rt-pcr test results, the first negative result was used to calculate the interval between baseline and sars-cov- nucleic acid negativity. we applied the mean ( ± sd) to normally distributed data, and median (iqr) to non-normally distributed data. the kaplan-meier method was used to estimate median interval to sars-cov- nucleic acid negativity, and the p-value was calculated by log-rank testing. hazard ratios with % confidence intervals were calculated by means of the cox proportional-hazards model. categorical variables were analyzed using the chisquared (c ) test, or the fisher's exact test when the expected frequency was less than in one or more cells, and continuous variables were compared using kruskal-wallis tests among the three groups. data were analyzed using spss software version (ibm-spss inc., chicago, il, usa). a p-value of < . was considered to be statistically significant. the funder of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study, and had final responsibility for the decision to submit for publication. as a consequence of the novelty of the coronavirus outbreak, no previous data existed in order to calculate an appropriate sample size for our study. thus, we estimated the sample size according to sars and mers data from references, and data from a few discharged covid- patients. we originally planned to recruit a total of subjects because we hypothesized that there would be d difference (standard deviation= . ) in the primary endpoint among the three cohorts after each receiving one of the three antiviral treatment regimens, based on a power of %, and a level of confidence of %, while also considering a dropout rate of %. however, the efficient control and suppression of the covid- outbreak in chongqing, due to well-planned and expeditiously executed public health measures in response to the frontiers in pharmacology | www.frontiersin.org july | volume | article outbreak, resulted in a relative scarcity of appropriate patients to recruit. we therefore finally enrolled participants in total. all of the participants were enrolled between january , and february , , and of these, patients were randomly assigned to the rbv+ifn-a-treated group, were assigned to the lpv/r+ifn-a-treated group, and were assigned to the rbv+lpv/r+ifn-a-treated group. of the patients, five patients ( . %) withdrew as a result of re-classification as severe covid- cases, and patients ( . %) changed their treatment regimens subsequent to adverse events. finally, ( . %) patients completed the regimen of rbv+ifn-a, ( . %) patients completed the regimen of lpv/r+ifn-a, and ( . %) patients completed the regimen of rbv+lpv/r+ifn-a. a flow diagram detailing the differences in numbers between the intention-to-treat population as compared to the per-protocol (pp) population is shown in figure . the median interval between symptom onset and randomization of the patients in our cohort was d (interquartile range: iqr, . - . d). the mean age of the entire cohort was . years (sd, . ), and ( . %) of the patients were men. the three most common radiological presentations included ground-glass opacities, consolidation, and pleural thickening. among all the enrolled patients, ( . %) had a fever, ( . %) had a cough, and ( . %) produced sputum at enrollment. mean hematological test results on the day of covid- diagnosis were: white blood cell count, . × /l (iqr, . - . ×g/l); neutrophil count, . × /l ( . - . × /l); hemoglobin, ×g/l ( - ×g/l); lymphocyte count, . × /l ( . - . × /l); platelet count, × /l ( - × /l); c-reactive protein, . ×mg/l ( . - . ×mg/l); erythrocyte sedimentation rate, mm/h ( - mm/h); d-dimer, . ×mg/ml ( . - . ×mg/ml); creatine kinase, . ×u/l ( . - . ×u/l); cd + t-cell count, ×cells/ml ( - ×cells/ml); and cd + t-cell count, ×cells/ml ( - ×cells/ml). baseline demographic and clinical characteristics of patients were comparable among the subjects of the three groups (table ) . in the intention-to-treat (itt) population, the median intervals from baseline to sars-cov- nucleic acid negativity was , , . ( . , . ) . ( . , . ) . ( . , . ) ( . , . ) . ct obviously improvement (days) † † † . ( . , . ) . ( . , . ) . ( . , . ) . ( . , . and d in the rbv+ifn-a-treated group, the lpv/r+ifn-atreated group, and the rbv+lpv/r+ifn-a-treated group, respectively (p= . ). upon statistical analysis, no significant differences in median interval from baseline to sars-cov- nucleic acid negativity were found among these groups, and between any two of the three groups. the hazard ratio of nucleic acid negativity in the group taking lpv/r+ifn-a was . ( % confidence interval [ci], . , . ), and the hazard ratio of nucleic acid negativity in the group taking rbv+lpv/r+ifn-a was . ( . , . ), compared with rbv+ ifn-a ( table and figure a ). in the pp population, the median intervals from baseline to sars-cov- nucleic acid negativity were , , and d in the rbv+ifn-a-treated group, the lpv/r+ifn-a-treated group, and the rbv+lpv/r+ifn-a-treated group, respectively (p= . ). again, no significant differences were observed among the three groups, and between any two of the three groups. the hazard ratio of nucleic acid negativity in the group taking lpv/r+ifn-a was . ( % ci, . , . ), and hazard ratio of nucleic acid negativity in the group taking rbv+lpv/ r+ifn-a was . ( . , . ), compared with taking rbv+ ifna ( table and figure b ). in the itt population, the proportion of patients with sars-cov- nucleic acid negativity at day in the rbv+ifn-a-treated group, the lpv/r+ifn-a-treated group and the rbv+lpv/r+ifn-a-treated group were . % ( / ), . % ( / ), and . % ( / ) respectively, and . % ( / ), . % ( / ), and . % ( / ) respectively, at day ( figure a) . however, the differences were statistically insignificant among the three groups, and between any two of the three groups. in the pp population, the proportion of patients with sars-cov- nucleic acid negativity at day in the rbv+ifn-atreated group, the lpv/r+ifn-a-treated group, and the rbv+lpv/r+ifn-a-treated group were . % ( / ), . % ( / ), and . % ( / ) respectively, and . % ( / ), . % ( / ), and . % ( / ) respectively at day , as shown in figure b . no significant differences were found among the three groups, and between any two of the three treatment groups. we also assessed proportion of patients with illness progression among the three groups by comparing different proportion of patients re-classified as severe cases, and mortality rate during the study period. we found that one patient ( . %) in the rbv+ifn-a-treated group, two patients ( . %) in the lpv/ r+ifn-a-treated group, and two patients ( . %) in the rbv+lpv/r+ifn-a-treated group had been re-classified as frontiers in pharmacology | www.frontiersin.org july | volume | article severe cases of covid- . however, no statistically significant difference was observed among the three groups, and between any two of the three groups. gratifyingly, there was no mortality in our cohort of patients during the study period ( table ) . a total of patients ( . %) in the rbv+ifn-a-treated group, patients ( . %) in the lpv/r+ifn-a-treated group, and patients ( . %) in the rbv+lpv/r+ifn-a-treated group reported adverse events during study period (table ) . a significantly higher incidence of diarrhea and vomiting was observed in the rbv+lpv/r+ifn-a-treated group compared to the other two groups, in the itt population (table and figure a ). however, a significantly higher proportion of vomiting only was observed in the rbv+lpv/r+ifn-a-treated group, compared with the other two groups in the pp population ( figure b ). there were no significant differences in the incidence of other adverse events among the three groups, including liver damage, electrolyte disorders, coagulation dysfunction, and abnormal complete blood counts ( figures a, b ). in addition, no patients appeared to develop renal dysfunction in our study. we found that no statistically significant differences existed between incidence of adverse events either in age stratification ( figure c ), or gender ( figure d ) in the itt and the pp populations (data not shown). no serious adverse events occurred during the course of the present study. the current sars-cov- epidemic is the third serious outbreak of a coronavirus-related disease this century, following the sars outbreak in china in , and the mers outbreak in the middle east in . unfortunately, this outbreak has evolved into a widespread pandemic causing global apprehension, and the number of patients infected with sars-cov- is growing exponentially by tens of thousands daily, with hundreds of patients dying every day worldwide. it is therefore of urgent importance to explore viable options for effective antiviral treatment for covid- (zeng et al., ) . in the present randomized trial, results showed that the median intervals between baseline and sars-cov- nucleic acid negativity in the rbv+ifn-a-treated group, the lpv/ r+ifn-a-treated group, and the rbv+lpv/r+ifn-a-treated group were , , and d in the itt cohort, and , , and d in the pp cohort, respectively. there was no significant difference in terms of the primary outcome among the three groups, and between any two of the three groups. in addition, we did not observe any significant difference in the proportion of patients with sars-cov- nucleic acid negativity among the three groups both at day and at day . nor did we observe any significant difference in proportion of patients with illness progression during the study period among the three groups. these results clearly indicate that there was no noticeable difference among the three therapeutic antiviral regimens in terms of antiviral efficacy when used in patients with mild to moderate covid- . we thus concluded that three different combinations of antiviral drugs have the same or similar antiviral efficacy on covid- . we did not conduct a subgroup analysis by sex and age for the three antiviral regimens, as the small sample size in our study did not effectively support such subset analyses. during the sars and mers outbreaks, injectable ifna was found to be potentially beneficial in the treatment of patients with sars and mers. atomized ifna is now being used for the first time to treat coronavirus-infected patients during the covid- outbreak. detailed pharmacokinetics for atomized ifna are not completely understood at this stage. the diameter of atomized drug particles determines whether the atomized drug will be delivered to the target site, thus directly affecting the efficacy of the drug. the diameter of atomized particles that can be deposited in the airway and lungs should ideally be between . and mm, and a particle diameter of - mm is considered optimal. wang et al. ( ) found that after injectable ifna is atomized, the droplet size distribution is suitable for drug delivery in to respiratory tract, and may be deposited in bronchi, bronchioles and lung parenchyma at all levels. after atomization, about . % of ifna injection particles are between and mm in diameter. another study showed that the biological retention rate of ifna was about %, and ifna distribution was found in lung tissue after h (liu et al., ; wang et al., ) . therefore, aerosolized inhalation of ifna is able to reach the lung parenchymal tissue in order to exert potential beneficial biological effects. ifna administered by atomizing inhalation could potentially have a prolonged duration of action, and thus may require a reduction of frequency of administration. when the blood and tissue concentrations of ifna exceeds u/ml, immune cells are stimulated to display antiviral activity within min (tong, ) . falzarano et al. ( a) found that rbv combined with ifna may inhibit the replication of mers-cov in cultured cells at a low therapeutic concentration ( u/ml), and reduced cytopathic effects (cpe). an animal study observed that the maximum blood concentration (tmax) of ifna when given by atomizing inhalation occurred h later than ifna administered by intramuscular injection. the mean residence time (mrt) was significantly prolonged compared with intramuscular injection, viz. h as compared to h. the elimination half-life of ifna was extended from . to . h when given by atomizing inhalation as opposed to injection (yang et al., ) . based on these previous studies, and the national guidelines of china, the doses of antiviral drugs administered in our study were adequate and appropriate. there are two possible explanations for our findings: first, that the three regimens have very similar or almost identical antiviral efficacy and therefore, no difference in effectiveness could be observed in the analyzed data; and second, that the three regimens do not have any significant antiviral efficacy clinically and therefore, no difference in effectiveness could be observed in the analyzed data. our research group tends to accept the latter explanation based on the following reasoning: ( ) the possibility of three different combinations of drugs having the same or similar antiviral efficacy is exceedingly slim; ( ) a recent placebo- controlled clinical trial has shown that lpv/r does not have antiviral efficacy in covid- patients (cao et al., ) ; ( ) a retrospective study has shown that patients receiving potential antiviral drugs such as lpv/r, ifna, or arbidol had similar viral clearance times when compared to those who did not receive any antiviral drugs (ling et al., ) ; ( ) a perspective study had shown that early combination of ifn, lpv/r, and rbv was effective in suppressing the shedding of sars-cov- , not just in a nasopharyngeal swab, but in all clinical specimens (hung et al., ) . however, in our study, we did not set up blank-controlled, or placebo-controlled treatment group out of compassionate and ethical considerations, and the specific limitations to therapeutic variations imposed by national guidelines for covid , and also because of the relative paucity of eligible patients to enroll in chongqing. therefore, we cannot definitively rule out the possibility that the three chosen different antiviral drug combinations in our study have identical or very similar antiviral efficacy profiles against sars-cov- . the adverse events of lpv/r and rbv have been widely reported. diarrhea and emesis are well-recognized adverse effects of lpv/r therapy, and has been noted previously in studies investigating the role of lpv/r in the treatment of hiv-patients (cao et al., ) . our results showed that in the itt population, the incidence of diarrhea and vomiting in the rbv+lpv/r+ifna-treated group was significantly higher than that of the rbv+ifn-a-treated group, and that of the lpv/r+ifn-atreated group. in the pp population, a significantly higher proportion of vomiting was observed in the rbv+lpv/r+ifna-treated group. ifn does not typically cause gastrointestinal adverse effects, and is especially not expected when ifn is administered by atomizing inhalation. therefore, the significantly higher increase in the incidence of diarrhea and vomiting in the rbv+lpv/r+ifn-a-treated group may credibly be attributed to the combination of lpv/r plus rbv. as such, we advise that the therapeutic combination of lpv/r and rbv should probably not be prescribed for the management of covid- . among sars patients, % had hypocalcaemia, % had hemolytic anemia, and % had hypomagnesaemia (knowles et al., ) . we failed to identify any subjects with hypomagnesaemia in our cohort. we observed that the rbv +ifn-a-treated group had the highest degree of abnormal complete blood counts, compared with the rbv+lpv/r+ifn-atreated group and the lpv/r+ifn-a-treated group. however, there was an absence of statistically calculated significance for the development of abnormal complete blood counts among the three groups. there are limitations to our study. firstly, the open-labeled design may have led to bias in the assessment of adverse events and clinical resolution. nevertheless, centralization of randomization, and strict inclusion and exclusion criteria, are expected to have reduced this possibility. secondly, the study was not blinded, and it is possible that knowledge of treatment assignment by patients and treating physicians may have influenced clinical outcomes. thirdly, this was a single-center trial with a limited number of participants in three arms, and thus, the results obtained at our center may not be representative of other hospitals in china. fourthly, we would have been able to better substantiate the effectiveness and safety of the different antiviral treatment regimens if control groups receiving placebo, or supplement treatment only, had been incorporated into our study design. fifthly, our small study cohort contributed to an underpowering of our study. withdrawals as a result of adverse events further contributed to this underpowering. consequently, further studies of these antiviral drug combinations with larger study cohorts are warranted in order to validate our results. finally, we did not perform pharmacokinetic measurements of the study drugs, and thus cannot comment on their precise bioavailability in covid- patients. in conclusion, our results demonstrated that there was no statistically apparent difference among the three antiviral therapeutic regimens in terms of antiviral effectiveness in patients with mild to moderate covid- . this could imply that the antiviral effectiveness of lpv/r+ifn-a, rbv+ifn-a, and rbv+lpv/r+ifn-a for the management of covid- is similar. further, the combination of rbv and lpv/r is associated with a significant increase in gastrointestinal adverse events, suggesting that rbv and lpv/r should not be simultaneously administered to covid- patients for the management of sars-cov- infection. although we recognize that our study is underpowered with a small patient cohort, we hope that these early findings may inform future effective management of covid- . the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors. the studies involving human participants were reviewed and approved by the ethics committee of chongqing public health medical center ( - - -ky). the patients/participants provided their written informed consent to participate in this study. y-kc and z-py conceived the study. lz, y-mz, q-zs, n-ns, and yw collected data. s-qt, yl, g-xw, and x-lx analyzed data. y-qh, s-qt, and y-kc interpreted the results. y-qh and s-qt wrote the manuscript. vh, x-qh, and y-ql edited and revised the article. clinical features and short-term outcomes of patients with sars in 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management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected pharmacokinetics and tissue distribution of interferon a b(pseudomonas putida)administered via atomization inhalation and intramuscular injection by i labeling comparative effectiveness and safety of ribavirin plus interferon-alpha, lopinavir/ritonavir plus interferon-alpha and ribavirin plus lopinavir/ ritonavir plus interferon-alphain in patients with mild to moderate novel coronavirus pneumonia a novel coronavirus from patients with pneumonia in china the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.copyright © huang, tang, xu, zeng, he, li, harypursat, lu, wan, zhang, sun, sun, wang, yang and chen. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -xf glr h authors: tian, bin; cai, dongjie; he, tianqiong; deng, liyao; wu, liping; wang, mingshu; jia, renyong; zhu, dekang; liu, mafeng; yang, qiao; wu, ying; zhao, xinxin; chen, shun; zhang, shaqiu; huang, juan; ou, xumin; mao, sai; yu, yanling; zhang, ling; liu, yunya; cheng, anchun title: isolation and selection of duck primary cells as pathogenic and innate immunologic cell models for duck plague virus date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xf glr h duck plague virus (dpv) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. the mechanism of organ tropism and innate immune evasion of dpv has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. in the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (defs), neurons, astrocytes, peripheral blood mononuclear cells (pbmcs), and monocytes/macrophages] to identify appropriate cell models for dpv, using tropism infection and innate immunologic assays. cells responded differently to stimulation with dna viruses or rna virus analogs. dpv infection exhibited broad tropism, as the recombinant virulent strain (chv-gfp) infected defs, neurons, astrocytes, and monocytes/macrophages, but not the pbmcs, as the expression of egfp was negligible. the basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in defs and astrocytes. conversely, the titer and genomic copy number of the attenuated virus strain was higher in defs and astrocytes than in neurons and monocytes/macrophages. the titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in defs, neurons, and astrocytes. the innate immune response was not significantly induced by either dpv strain in defs, neurons, or astrocytes. the virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. blockage of ifnar signaling promoted replication of the attenuated strain. pre-activation of ifnar signaling inhibited infection by the virulent strain. the selection assay results indicated that induction of innate immunity plays an essential role in controlling dpv infection, and monocytes/macrophages are an important cell model for further investigations. our study provided practical methods for isolating and culturing duck primary cells, and our results will facilitate further investigations of organ tropism, innate immune responses, latent infection, and the effectiveness of antiviral drugs for treating dpv and potentially other aerial bird pathogens. duck plague virus (dpv) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. the mechanism of organ tropism and innate immune evasion of dpv has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. in the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (defs), neurons, astrocytes, peripheral blood mononuclear cells (pbmcs), and monocytes/macrophages] to identify appropriate cell models for dpv, using tropism infection and innate immunologic assays. cells responded differently to stimulation with dna viruses or rna virus analogs. dpv infection exhibited broad tropism, as the recombinant virulent strain (chv-gfp) infected defs, neurons, astrocytes, and monocytes/macrophages, but not the pbmcs, as the expression of egfp was negligible. the basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in defs and astrocytes. conversely, the titer and genomic copy number of the attenuated virus strain was higher in defs and astrocytes than in neurons and monocytes/macrophages. the titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in defs, neurons, and astrocytes. the innate immune response was not significantly induced by either dpv strain in defs, neurons, or astrocytes. the virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. blockage of ifnar signaling promoted replication of the attenuated strain. pre-activation of ifnar signaling inhibited infection by the virulent strain. the selection assay results indicated that induction of innate immunity plays an essential role in controlling dpv infection, and monocytes/macrophages are an important cell model for further investigations. our study provided practical methods the innate immune response is the first line of host defense against microbial pathogens. various pattern recognition receptors (prrs) play an essential role in detecting pathogenassociated molecular pattern (pamp) associated with invading pathogens. pamp recognition initiates an innate immune response, characterized by the production of type i interferon (ifn), proinflammatory cytokines, and ifn-stimulated genes (isgs) ( , ) . prrs comprise multiple family members, including toll-like receptors (tlrs), retinoic acid inducible gene i (rig-i)-like receptors (rlrs), nucleotide oligomerization domain (nod)-like receptors, c-type lectin receptors, and cytosolic dsdna sensors (cdss). rlrs primarily recognize ′ -phoshorylated rnas, including ssrna or dsrna produced during the replication of rna or dna viruses ( ) . although rig-i predominantly recognizes rna viruses, the rig-i/mitochondrial antiviral-signaling protein (mavs) pathway is also activated during infection with several dna viruses, including herpes simplex virus (hsv- ), epstein-barr virus, and kaposi's sarcoma-associated herpesvirus ( ) ( ) ( ) . during infection with dna viruses, rna polymerase iii recognizes at-rich dsdna and transcribes the dsdna into dsrna containing a -triphosphate moiety that activates the rig-i/mavs pathway to induce ifn-β production ( ) . dna from viruses or bacteria can be detected by cyclic gmp-amp synthase (cgas) and potentially rlr, which potentially activate the endoplasmic reticulum-resident adaptor protein, stimulator of interferon genes (sting), to translocate from the endoplasmic reticulum to the golgi, where it activates tbk -irf and -nf-κb, resulting in robust induction of type i ifn and inflammatory cytokine production ( ) . ifn-i binds to ifnar and activates r -associated tyk protein tyrosine kinase and the ifnalpha/beta r -associated jak protein tyrosine kinase, which subsequently regulate the phosphorylation and activation of different stat proteins; the activated stat proteins homoor heterodimerize and translocate to the nucleus, where they promote the expression of numerous target genes. binding of stat proteins to either isres or gas sites regulates the expression of several hundred isgs, which mediate the anti-viral, anti-proliferative, and apoptotic effects of type i ifns. duck plague (dp), also known as duck viral enteritis (dve), is caused by anatid herpesvirus type (ahv- ) or duck plague virus (dpv), which is an enveloped, dsdna virus of the herpesviridae family, subfamily alpha-herpesvirinae ( , ) . first reported in the netherlands in , dp spread rapidly around the world ( , ) . although typically an acute or sometimes chronic and highly contagious disease, dp is characterized by high mortality rates (up to %) among domestic ( ) and wild ducks, swans, geese, and other waterfowl of different ages. to prevent dp outbreaks on duck farms, attenuated dpv vaccines have been widely used; in china, use of these vaccines is compulsory, with billions of doses administered annually ( , ) . dpv is the only herpes virus circulating in aquatic animals identified to date. infection with virulent dpv strains causes gross lesions in ducks in most tissues, including the heart, liver, spleen, bursa, and brain ( , ) , where the virus has been detected ( , ) . upregulation of prrs and isgs expression has been reported, indicating that dpv exhibits broad organ tropism and activates the innate immune system ( , ) . differing basal and induced levels of prrs and isgs among different cell types and organs are important factors in determining the organ tropism of viruses such as poliovirus, reovirus, and murine coronavirus ( ) ( ) ( ) . recently published data indicated that expression of rig-i, galectin- , mavs, sting, and irf is induced in dpv-infected ducks, demonstrating the strong capacity of the innate immune response to restrict dpv infection via over-expression of these factors in defs, although it is difficult to detect changes in these factors in defs infected with a high titer of dpv ( ) ( ) ( ) ( ) ( ) . according to a previous study, tlr , irf , isg , isg , and isg (ifits) are missing in birds, chickens also lack rig-i and riplet ( ) , and the immune system of birds is different from that of mammals. development of a suitable cell model for in-depth investigations of the mechanism of the innate immune response to dpv and the virus's ability to evade that response is thus an important priority. in the present study, therefore, we isolated and cultured five types of duck primary cells in vitro and then compared the basal and innate immune responses to dna and rna virus analogs. the cell tropism of dpv and changes in innate immune signaling induced by dpv infection and the antiviral effect of ifnar signaling against dpv infection were also investigated. the isolation and characterization of different types of duck primary cells could facilitate elucidation of the mechanism governing the organ tropism of dpv and the relationship between dpv infection and host antiviral innate immune responses. all animal experiments were conducted in accordance with approved guidelines. one-month-old peking ducklings were purchased from a dpv-free farm where vaccination against dpv was not implementation. all the ducks were housed in the animal facility at sichuan agricultural university, chengdu, china. the study was approved by the committee of experiment operational guidelines and animal welfare of sichuan agricultural university (approved permit number xf - ). nine-day-old duck embryos were cleaned with % ethanol and placed on a -well plate. the head, wings, legs, and viscera were removed, and the muscle tissues were washed with hbss, cut into -mm pieces, and then digested with . % trypsin for min at room temperature (rt). after digestion, the trypsin was removed via centrifugation at , rpm for min, and the digested tissues were dissociated by repeated pipetting (∼ times), after which the mixture was filtered through autoclaved medical gauze. single cells were collected and plated in cell culture plates or dishes and cultured in mem supplemented with % fetal bovine serum (fbs; gibco-brl, carlsbad ca, usa). usually, defs were cultured to % confluence for h and then passaged and sub-cultured for use in subsequent assays. duck neurons were isolated according to our previously described method for isolating mouse neurons ( ) . briefly, the brain was collected from -day-old duck embryos, the meninges were peeled away and carefully removed, and the cortex was transferred into a new dish filled with hbss, and then cut into -mm pieces by using scissors; the shears were transferred into . % trypsin diluted in hbss and digested for min at rt. the trypsin was removed by transferring the brain cells into a new tube with proper dmem, and then dnase i was added and treated for min at rt. the dnase i was removed and the cells were collected by centrifugation at , rpm for min at rt. then, the collected cells were resuspended with dmem and dissociated by repeated pipetting (< times). the cells were passed through a -nm nylon mesh (corning, ny, usa) to separate the single cells, washed once in hbss, and then cultured in d-polylysine-pretreated plates in dmem supplemented with % fbs and % penicillin-streptomycin for h. finally, the cells were washed once with phosphate-buffered saline (pbs), and the medium was replaced with serum-free, neural-basal medium supplemented with % b- plus (gibco-brl, ny, usa), and the cells were incubated for days to form a monolayer. primary duck astrocytes were isolated primarily according to the procedure described above for isolating duck neurons, with some differences. brain cells passed through a -nm nylon mesh were cultured in dmem supplemented with % fbs and % penicillin-streptomycin for h, washed once with pbs, and then cultured in the same medium for days to form a monolayer. the medium was changed every days. similar to defs, astrocytes readily agglomerated and detached from the plates or dishes once they began to overgrow. duck pbmcs were isolated from the -month-old anticoagulation whole blood by density gradient centrifugation using a duck leukocyte isolation kit (tbdscience, tianjin, china), according to the manufacturer's instructions and our previous study ( ) . briefly, whole blood was collected from the jugular vein of mature ducks and placed in anticoagulant-containing tubes. the blood was then diluted with sample dilution buffer and slowly added onto a layer of duck lymphocyte isolation buffer (density: . ± . g/ml) to avoid mixing and then centrifuged at g for min at rt. the second grayishwhite layer was transferred into a new tube, and resuspended in ml washing buffer and the mixed cells were collected by centrifugation at g for min at rt. the collected cells were resuspended in ml of red blood cell lysis buffer for min and then ml of washing buffer was added into the cells. then, the cells were collected by centrifugation at g for min at rt. the remaining cells were adjusted to × cells/ml and ml of cells was plated in one well of the -well plate, and cultured in rpmi medium supplemented with % fbs and % penicillin-streptomycin for h to allow for attachment to the plate; unattached cells were removed by washing twice with pbs, followed by addition of fresh medium. the isolated pbmcs were then ready for use in assays. duck monocytes/macrophages were isolated according to a reported method for isolating human macrophages ( ) . briefly, duck pbmcs were prepared as described above. adherent cells were enriched among pbmcs by adherence on plastic culture plates for h. non-adherent cells were removed via vigorous washing three times using pre-warmed pbs; the adherent cells were digested with trypsin for cell count. duck monocyte-derived macrophages were differentiated from adherent monocytes in rpmi medium supplemented with l-glutamine ( mm), sodium pyruvate ( mm), % heatinactivated fbs, % penicillin-streptomycin, and ng/ml human m-csf (novoprotein, shanghai, china). the medium was changed every days, and duck macrophages formed a monolayer by day . in our present isolation method, only adherent cells are able to differentiate into macrophages under the induction of m-csf; lymphocytes in pbmcs (mainly including t cells, b cells, and nk killer cells) continue to die due to inability to differentiate under induction of m-csf and are removed by constantly replacing fresh medium. the virulent strain of dpv, chv, was isolated and characterized by our lab ( ) . the recombinant virulent strain of dpv, bac-chv-egfp (chv-gfp), was constructed by our research center ( ) . the attenuated vaccine strain of dpv, cha, was retrieved from storage at our research center. the duck tambusu virus (dtmuv) was stored at our research center. each dpv and dtmuv strain was propagated on defs. the rabbit antihuman map and -human gfap polyclonal antibody and the goat anti-human β-actin monoclonal antibody were purchased from abclonal (wuhan, china). mouse anti-duck cd (gene id: ) and cd (gene id: ) antibodies were generated by our research center. ruxolitinib, poly(da:dt), and poly(i:c) were purchased from invivogen (hong kong, china). the cells were lysed with ripa buffer ( mm tris, ph . ; mm sodium chloride; % triton x- ; . % sodium deoxycholate; . % sds) containing protease inhibitors (roche), and the protein concentrations were measured using a dc protein assay kit (bio-rad). equal quantities of protein were resolved by % sds-page and then transferred to polyvinylidene difluoride (pvdf) membranes (bio-rad), which were blocked with % non-fat milk before being incubated with primary antibodies against cd , cd , or β-actin and then probed with the appropriate secondary antibodies. the blots were then visualized using ecl reagent (ge, pittsburgh, pa, usa) and detected under an intelligent dark box ii (ge, pittsburgh, pa, usa). duck primary defs, neurons, astrocytes, pbmcs, and monocytes/macrophages were treated with the dna and rna virus analogs poly(da:dt) or poly(i:c), respectively, at a dose of µg/ml for h. the cells were then lysed in trizol reagent for rna isolation to assess the innate immune response to the stimulators using quantitative real-time polymerase chain reaction (qrt-pcr). primary duck neurons, astrocytes, or monocytes/macrophages were cultured for , , or days, respectively, to form a confluent monolayer. the cells were then fixed with % neutral buffered paraformaldehyde for min, permeabilized with . % triton x- for min, blocked with % bsa dissolved in pbs for min, and then incubated with primary antibodies against map , gfap, cd , and cd at • c overnight. the cells were incubated with alexa fluor -conjugated goat anti-rabbit ormouse secondary antibodies or alexa fluor -conjugated goat anti-rabbit or -mouse secondary antibodies for h at rt. images were acquired using fluorescence microscopy. for virus infection, the required dose of virus was diluted in the medium used to culture the various types of duck primary cells ( × cells in a -well plate) and incubated with cells at • c for h. the cells were then washed twice with pbs and maintained in the corresponding medium supplemented with % fbs and % penicillin-streptomycin. the culture supernatant of virus-infected cells was collected and titrated to determine the tissue culture infectious dose (tcid ) on defs using -fold serial dilutions. viral dna was extracted using a hipure viral dna mini kit (magen, guangdong, china) according to the instructions provided by the manufacturer. dpv genomic dna in infected cells was quantified using an absolute q-pcr method as previously described ( ) using primers specific to the sequence of dpv ul (primers are listed in table ). a standard curve was generated from serially diluted plasmids harboring the entire coding sequence of ul and using the same pcr procedure as used for cell samples. dpv copy number in infected cells was calculated according to the standard curve and normalized to µg of total dna. rna was extracted from cells using trizol r reagent (invitrogen) according to the manufacturer's instructions; the genomic dna was removed and cdna was synthesized by using novoscript r plus all-in-one st strand cdna synthesis supermix (gdna purge) (novoprotein, shanghai, china), and qrt-pcr analysis was performed as described previously ( ) . briefly, µg of rna from cells was transcribed into cdna according to the instructions of the superscript iii reverse transcription kit. a total of µl of cdna was mixed with µl of iq sybr green mix (biorad, hercules, ca, usa), µl of double-distilled water, and . µl each of forward and reverse primer. the cdna was amplified and the cycle threshold (cq) values were recorded. the cdna concentration, primer sequence, and q-pcr procedure applied for each gene in each cell type were the same. the basal expression level of each gene in each cell type was compared by directly determining from the cq values according to the method used in the previous study ( ) . the relative mrna expression of each gene in each cell type was normalized to the expression level of the s mrna gene ( ct ct [prr or isg]/ ct [ s]). expression levels of induced mrnas are presented as the fold change relative to mock-infection levels according to the − ct method. all primer sequences are listed in table . in order to examine the impact of ifnar signaling on the dpv replication in each cell type, the ifnar-specific inhibitor, ruxolitinib, was applied. the cell toxicity of ruxolitinib at a dose of , , , and µm/ml on each cell type was detected by mtt method. duck defs, neurons, astrocytes, pbmcs, and monocytes/macrophages were pretreated with µm/ml of ruxolitinib for h and then infected with chv or cha at a moi of . for h. the cells were then washed twice with pbs, and the same concentration of ruxolitinib was added to the culture medium; dmso was used as a mock control. the cell culture supernatant in each cell type was collected to detect the tcid as above described. the cell was scraped in pbs from the plate to detect the viral genomic copy number. for the preventative antiviral assay, duck monocytes/ macrophages were pretreated with µg/ml poly(da:dt) or poly(i:c) for h, sterile water was used as control, and then infected with chv or cha at a moi of . . the cells were then washed twice with pbs, and the same concentration of poly(da:dt) or poly(i:c) was added to the culture medium and incubated for h. for the therapeutic antiviral assay, duck monocytes/ macrophages were pretreated with or µg of poly(da:dt) or poly(i:c) for h and then infected with chv or cha at a moi of . . after infection, the analogs were added at the same concentration and incubated for h. at hpi, the cell culture supernatants were collected for tcid determination, and the cells were washed with pbs, scraped from the dishes, and collected for viral genomic copy number determination as described above. data are expressed as the mean and standard error of the mean (sem), and the significance of differences between groups was evaluated using the student's t-test or one-way analysis of variance followed by tukey's post-hoc test. asterisks indicate the level of statistical significance ( * p < . ; * * p < . ; * * * p < . ; * * * * p < . ). all experiments were repeated at least three times individually. graphs were plotted and analyzed using graphpad prism software, version . (graphpad software, la jolla, ca, usa). defs have been cultured in our lab using sophisticated methods for many years, and pbmcs were isolated using a duck leukocyte isolation kit; these two cell types were applied in our previous studied ( ) . hence, we strove to isolate and culture duck neurons, astrocytes, and monocytes/macrophages according to previous methods used for mouse and human cells. first and foremost, we isolated pbmcs from the duck whole blood cells (wbc) through density gradient centrifugation, the wbcs and pbmcs were stained with wright strain (figure a) , and the results showed that there are mainly erythrocyte and little partial of thrombocytes and leucocytes in the wbcs. the leucocytes were above % in the pbmcs, and the monocytes were about % among the total pbmcs ( figure a) . since there is no duck-derived m-csf on sale, we blast the duck's m-csf protein sequence ( - aa) with the human's m-csf protein sequence, and found that the duck's m-csf protein sequence has the same functional domain as the human m-csf protein sequence ( - aa) (data did not show); thus, we tried to stimulate the duck-derived pbmcs with human m-csf, we observed and collected the time course and found that it can induce duck pbmcs to differentiate into macrophages, which were mainly composed of the wheel and spindleshaped cells (figure b) . in our present method, the survival time of the pbmcs was not more than h without m-csf, so we believe that the obtained monocytes/macrophages were induced by human m-csf. since the antibodies (see below) we used to identify the macrophage-like cells we have obtained were against the surface molecules that are found in both monocytes and macrophages, we term the cells as monocytes/macrophages (mm). under specific culture conditions, we successfully cultured duck primary neurons, astrocytes, and monocytes/macrophages and identified the cells using an ifa with a rabbit polyclonal antibody against map for neurons ( figure c) , a rabbit polyclonal antibody against gfap for astrocytes (figure d) , and a mouse polyclonal antibody against cd and cd for monocytes/macrophages (figure e) . to further identify the characteristics of monocytes/macrophages, we examined the expression of cd and cd through western blot; the relative molecular weight was and kd, respectively ( figure f) . the basal expression levels of tlr , tlr , cd , and cd in these five types of cells were detected by q-pcr and found that they were highly expressed in monocytes/macrophages (figure e) . these results indicated that our methods can successfully isolate duck neuronal, astrocytes, and monocytes/macrophages. duck primary cells exhibited differing innate immune responses to dna and rna viruses we then examined whether the five types of duck primary cells we isolated were able to respond to dna and rna viruses by stimulating the cells with the dna and rna virus analogs, poly(da:dt) or poly(i:c), respectively, at a dose of µg/ml. at h post-treatment, the expression of ifn-β (figure a) was analyzed, and the data indicated that poly(da:dt) induced significantly higher expression of ifn-β in astrocytes, pbmcs, and monocytes/macrophages than did poly(i:c) or mock treatment. poly(i:c) induced significantly higher levels of ifnβ expression in pbmcs and monocytes/macrophages compared with mock-treated cells. the expression level of mx, an isg induced by ifn or pathogens, was examined in each of the five cell types, and the data showed that poly(i:c) induced significantly higher expression of mx in all tested cells (defs, neurons, astrocytes, pbmcs, and monocytes/macrophages) than did poly(da:dt) or mock treatment (figure b) . under poly(da:dt) stimulation, significant upregulation of mx expression was observed only in astrocytes, pbmcs, and monocytes/macrophages. the expression of il- , an inflammatory factor, was also determined after stimulation (figure c) , and the data showed that poly(i:c) induced il- expression in all five types of duck cells. although poly(da:dt) induced il- only in astrocytes, pbmcs, and monocytes/macrophages, the levels were higher than those induced by poly(i:c). taken together, these data indicated that the five types of duck primary cells we examined are competent to respond to dna and rna viruses and that innate immune signaling is initiated. upon stimulation with dna viruses, monocytes/macrophages exhibit higher levels of ifn-β, isg, and inflammatory cytokine expression than defs, neurons, astrocytes, and pbmcs. to elucidate the mechanisms associated with the different responses of the five types of duck primary cells to dna and rna virus analogs, the basal levels of innate immune factors were compared. we examined a variety of representative factors, including cgas, sting, rig-i, mda , irf , ifnβ, mx, and il- . the basal levels of cgas were comparable among the different duck primary cells (figure a) . the basal levels of sting, rig-i, mda , irf , ifn-β, mx, and il- were highest in monocytes/macrophages and lowest in astrocytes and defs (figures b-h) . these results indicated that monocytes/macrophages mount a greater innate immune response than the other cell types after stimulation with dna or rna virus analogs. dpv, the only herpes virus circulating in aquatic animals, exhibits multi-tropic infection, and the virus can be detected in nearly every major organ, including the brain, lung, spleen, intestines, and liver. in order to investigate the tropism of dpv in duck cells, duck defs, neurons, astrocytes, pbmcs, and monocytes/macrophages were infected with a recombinant virulent virus strain, chv-gfp, at a low moi of . (figure ) . virus proliferation and morphology of the primary cells cultured in vitro were assessed by monitoring the expression of gfp. as the data demonstrate, at h post-infection, gfp was clearly expressed in duck defs, neurons, astrocytes, and monocytes/macrophages, and all four types of virus-infected cells were radially enlarged at h post-infection. no significant gfp expression was observed in dpv-infected pbmcs. these data demonstrated that dpv infects different types of duck cells in vitro and exhibits multi-tropic infection, with possible replication in many organs and tissues, such as muscle, brain (neurons and astrocytes), and spleen (monocytes/macrophages). to investigate the growth dynamics of dpv in the five types of duck primary cells, the cells were infected with a virulent dpv strain (chv) or an attenuated vaccine strain (cha) at a moi of . . the viral titer in the cell culture supernatant was determined based on the tcid (figures a-c) , and the intracellular viral genome copy number was also determined (figures d-f) . cha produced higher virus titer and genomic copy number than chv in neurons and astrocytes at , , and hpi. the viral titer and genome copy number of cha were comparable to chv at hpi but higher than chv at and hpi in pbmcs, although the values were near the detection limit. cha produced higher viral genome copy number in defs at hpi (figure d ), but the viral titer was comparable at , , and hpi (figures a-c) . these data indicated that the attenuated strain of dpv replicates faster and produces more virus particles than the virulent strain in duck defs, neurons, astrocytes, and pbmcs. an intriguing finding was that the titers of both virus strains were comparable in monocytes/macrophages at hpi ( figure a) . the genomic copy number of cha was significantly higher than that of chv at hpi ( figure d ). the genomic copy number and viral titer of cha slowly decreased between and hpi. in contrast, the genomic copy number and viral titer of chv increased over this time period (figures b,c,e,f) . the copy number and viral titer of cha was significantly lower at and hpi compared with chv (figures b,c,e,f) . these data demonstrate that the attenuated strain (cha) abortively infects duck monocytes/macrophages, whereas the virulent strain (chv) persistently infects duck monocytes/macrophages, we speculate that the monocytes/macrophages may be able to kill the virus and it is an active immune response that is responsible for the decrease in viral titer of cha strain, as we demonstrated in the experiments below. to determine whether the different growth dynamics exhibited by the virulent and attenuated strains of dpv affected the innate immune response in duck primary cells, the expression levels of cgas, sting, rig-i, mda , irf , ifn-β, mx, and il- were analyzed in the five types of duck primary cells infected with either the chv strain or cha strain at a moi of . . there were no significant differences in the expression of these molecules in the chv-or cha-infected defs or neurons at , , and hpi, but there was a slight increase in expression in astrocytes infected with cha (data did not show). in pbmcs, infection with the chv strain induced cgas, sting, mda , irf , ifn-β, mx, and il- expression early, at hpi, but expression decreased by and hpi (data did not show). infection with the cha strain induced a slight increase in the expression levels of these factors at hpi, and the expression continued to increase with time and was significantly higher compared with chv-infected cells or mocktreated cells. however, the chv strain may not be able to infect pbmcs observed from figures , , and the virus level of the cha strain infected with pbmcs is slightly higher than that of the chv strain, so comparing the innate immune responses caused by the two in pbmcs are complicated. the expression levels of cgas, sting, rig-i, mda , irf , ifn-ifn-β, mx, and il- were also examined in chv-and chainfected duck monocytes/macrophages. similar to the changes observed in dpv-infected pbmcs, infection with chv induced significant increases in the expression of cgas, sting, rig-i, mda , irf , ifn-β, and mx in monocytes/macrophages early, at hpi, but expression rapidly decreased by , , , and hpi (figure ) , a trend opposite to the growth curve on monocytes/macrophages (figure ) . infection with the cha strain induced significantly increased expression of cgas, rig-i, mda , irf , ifn-β, and mx in monocytes/macrophages at hpi, and these levels were higher than those observed in mock-treated cells but lower than those in cells infected with chv strain. the expression of cgas, sting, rig-i, mda , irf , ifn-β, mx, and il- decreased slightly at and hpi, but then increased markedly at and hpi (figure ) . these data indicated that the virulent dpv strain activates the innate immune system in pbmcs and monocytes/macrophages during the early stages of infection, but then the innate immune response is downregulated once the virus begins to replicate. the attenuated strain (cha) activates the innate immune system primarily during the later stages of infection, and this could be the reason why this strain abortively infects monocytes/macrophages and has been attenuated. to further investigate how dpv infection affects the innate immune response in the five types of duck primary cells, ifnar signaling was blocked using the specific inhibitor ruxolitinib. we first examined whether ruxolitinib functions in the duck cells by pretreating defs with ruxolitinib for h and then infecting the cells with a duck rna virus, dtmuv, which belongs to the flavivirus family and was demonstrated to induce strong innate immune response in def cells ( ) . after infection, rucolitinib was added at the same concentration and the cells were incubated for h. dtmuv induced significant expression of ifn-β, mx, and il- in defs (figures a-c) , but the expression levels of these factors were clearly reduced in cells treated with ruxolitinib, indicating that this inhibitor can be used to block ifnar signaling in duck cells. the cell toxicity of ruxolitinib on defs (figure d) , neurons, astrocytes, pbmcs, and monocytes/macrophages was determined (data did not show), and no obvious cell viability was changed by ruxolitinib at a dose of µm/ml on these five cell types. ruxolitinib was used to treat duck defs, neurons, astrocytes, pbmcs, and monocytes/macrophages in further assays. viral titer was examined at various time points in the culture supernatants of cells infected with either the chv or cha dpv strain, with and without ruxolitinib treatment. after ruxolitinib treatment for hpi, the titer of cha in the astrocyte culture supernatant was significantly increased, by ∼ -fold; however, at this time point, there were no obvious changes in cha titer in the cell culture supernatants of defs, neurons, pbmcs, or monocytes/macrophages (figure e) . at hpi, no change in chv titer was detected in any of the five duck primary cells (figure e) . at hpi, the titer of cha was significantly increased in the cell culture supernatants of astrocytes, pbmcs, and monocytes/macrophages (figure f ). there was a certain increase observed in the culture supernatant of astrocytes infected with chv. no changes were observed in the titers of either chv or cha in the supernatants of neurons and defs treated with ruxolitinib (figures e,f) . taken together, these data indicated that blockage of ifnar signaling enhances the replication of the attenuated dpv strain (cha) in duck astrocytes, pbmcs, and monocytes/macrophages. infection with cha activates the type i ifn response in these cells to a greater degree than does infection with the chv strain. infection with the chv or cha strains of dpv had a differential effect on the innate immune response in duck primary cells. both strains of dpv induced the most significant changes in the innate immune response in monocytes/macrophages (figure ) , and therefore monocytes/macrophages were chosen as a cell model to investigate the antiviral role of ifnar signaling. monocytes/macrophages were pretreated with poly(da:dt) or poly(i:c) at a dose of µg/ml for h and then infected with the chv or cha strain at a moi of . . the viral titer and genomic copy number were determined at hpi. the viral titer and genomic copy number of the chv strain were significantly decreased in monocytes/macrophages pretreated with poly(da:dt) or poly(i:c) (figures a,b) , but there was no change in either viral titer or genomic copy number of the cha strain (figures c,d) . to assess the therapeutic antiviral effect of the agonists, monocytes/macrophages were pretreated with poly(da:dt) or poly(i:c) at dose of or µg/ml for h and then infected with either the chv or cha strain at a moi of . . after infection, the same concentration of poly(da:dt) or poly(i:c) was added and the cells were incubated until the time point of examination. at hpi, the viral titer and genomic copy number were determined. as the data showed, treating monocytes/macrophages with poly(da:dt) at a dose of µg/ml had no effect on viral titer or copy number of the chv strain, but a significant reduction of viral titer and genomic copy number was observed at a dose of µg/ml (figures e,f) . significant reductions in both viral titer and genomic copy number of the chv strain were observed with monocytes/macrophages treated with poly(i:c) at a dose of either or µg/ml (figures e,f) . for monocytes/macrophages infected with the cha strain, treatment with poly(da:dt) or poly(i:c) at a dose of either or µg/ml resulted in a reduction in viral genome copy number (figure h ) but had no effect on viral titer (figure g) . taken together, these data indicate that activation of ifnar signaling via a prr agonist restricts the virulent strain of dpv. prr agonists thus exhibit preventative and therapeutic potential against dpv infection. in the current study, we isolated and cultured duck primary defs, neurons, astrocytes, pbmcs, and monocytes/macrophages. to our knowledge, this is the first report of the culture of duck neurons, astrocytes, and monocytes/macrophages in vitro. these five types of duck primary cells were used to investigate the tropism of dpv and the innate immune response to dpv infection. we found that dpv persistently infected and replicated in defs, neurons, astrocytes, and monocytes/macrophages, indicating that dpv exhibits wide in vivo organ tropism in ducks ( ) . indicative of the potential of dpv to evade the innate immune response, and infection of defs, neurons, and astrocytes, there were slight changes in prr and isg expression. upon infection of pbmcs and monocytes/macrophages at a high moi, the chv strain only upregulated the expression of some prrs and isgs at hpi, but expression of these innate immunity factors was downregulated at later time points by the virulent dpv strain. a particularly intriguing finding was that the expression of prrs and isgs in pbmcs and monocytes/macrophages was consistently upregulated by infection with the attenuated vaccine strain (cha). monocytes/macrophages represent an ideal cell model for investigating the relationship between dpv infection and the innate immune response; as the virulent strain of dpv persistently infects monocytes/macrophages, these cells may play a critical role in the pathogenicity of the virus. the current lack of methods for culturing duck primary cells significantly hinders investigations of the role of each cell type in the infectious cycle, tropism, and pathogenicity of aquatic bird viruses, such as dpv, avian influenza virus, novel duck reovirus (ndrv), and dtmuv, which cause major economic losses in the duck industry ( ) ( ) ( ) ( ) . as opposed to rna viruses such as avian influenza, ndrv, and dtmuv, dpv is a dsdna virus that does not readily induce an innate immune response in defs. hence, in order to investigate the mechanism by which the virus evades the innate immune response, a better primary cell model is needed. numerous types of mammalian primary cells have been isolated from the tissues and successfully cultured, but there are few such reports regarding the culture of primary cells of aquatic animals such as ducks and geese. in the present study, we isolated and cultured duck neurons, astrocytes, and monocytes/macrophages using methods developed for mouse or human cells. our data indicated that the synapses of neurons, the classic morphology, formed at days post-induction. the expected star shape was clearly observed for duck astrocytes isolated from brain tissue and cultured in dmem containing % fbs. it was surprising that we could induce the differentiation of wheel-shaped monocytes/macrophages from duck pbmcs using human or mouse m-csf at a concentration of ng/ml, and no living cells were detected at days post-seeding in the absence of human or mouse m-csf. as a herpesvirus, dpv sometimes exhibits chronic or latent infection in the duck trigeminal ganglion (tg) ( ) , similar to hsv- ( ) . studies of the molecular mechanism of hsv- latency typically use mouse or rabbit animal models, but not humans. however, the research using an in vitro latency model in neurons would be a preferable approach prior to in vivo studies. infection of stem-derived neurons with a low viral dose of wildtype hsv- in the presence of the antiviral agent acyclovir and interferon-alpha results in the establishment of a latent, nonproductive infection. in this state, viral replication and expression of late viral gene markers are not detected, but there is an accumulation of viral latency-associated transcript rna ( ) . in our current research, we isolated and cultured duck neurons, and both the virulent and attenuated strains of dpv were able to infect neurons in the absence of treatment with any antiviral drugs. this would be a good cell model for further studies of the molecular mechanism underlying latent dpv infection in neurons or investigations of how dpv invades the cns to cause fever and intracranial swelling. astrocytes are basal, functional cells of the cns that form and maintain the permeability of the blood-brain barrier and restrict pathogen invasion ( , ) . the tlr in astrocytes plays a critical role in containing the replication and transmission of hsv- in the cns; deficiency of tlr causes astrocytes to become permissive to hsv- infection, thus facilitating infection of the entire cns by hsv- ( ) . ifnar signaling in astrocytes exhibits regional differences that act to restrict the invasion of neurotropic viruses; loss of ifnar signaling decreases the survival of mice after west nile virus infection ( ) . in our present study, we isolated and cultured duck astrocytes. the basal levels of innate immune factors in astrocytes were the lowest among the five types of duck primary cells we examined. both strains of dpv infected astrocytes, and the titer of the attenuated strain (cha) in astrocytes was comparable to that in defs. blockage of ifnar signaling increased dpv replication in astrocytes. thus, astrocytes may play a critical role in containing dpv infection in the cns and might therefore be a useful model. further investigations of the role of astrocytes in dpv invasion and latent infection in the cns are warranted. persistent infection with hsv- is a prerequisite to this virus's pathogenicity, and hsv- has been reported to infect sensory neurons, the corneal epithelium, lymphocytes, and macrophages ( ) . in the sensory ganglia, macrophages infiltrate the tg and produce tnf-α and inos to control the primary hsv- infection ( ) . in human primary macrophages, knockdown of mda and mavs strongly inhibits the expression of ifn and tnf-α induced by hsv- entry and replication, indicating that the early innate recognition of hsv- involves mda-/mavs-dependent pathways ( ) . the expression of chemokines such as cxcl and ccl in human monocytederived macrophages induced by hsv- infection involves ifi dependent and ifi -independent pathways ( ) . activation of ifnar signaling promotes high isgs expression in macrophages infected with hsv- , in which samhd inhibits hsv- propagation by limiting viral dna synthesis ( ) . abortive infection of viruses in astrocytes or macrophages appears to be essential to induce innate and adaptive immune responses to restrict or clear vesicular stomatitis virus, rabies virus, or influenza virus ( , , ) . in our current study, monocytes/macrophages were infected by both strains of dpv, and the viral titer and copy number of the virulent strain (chv) increased from to hpi at a moi of . . in contrast, the viral titer and genomic copy number of the attenuated strain (cha) began to decrease early in the infection (figure ) . from these data, we inferred that the virulent strain (chv) persistently infects the monocytes/macrophages, whereas the attenuated strain (cha) causes an abortive infection in monocytes/macrophages. we further verified these observations by examining the innate immune response induced by these two strains. in duck monocytes/macrophages, the expression of prrs and isgs were induced by both strains at hpi but gradually decreased in chv-infected cells after hpi ( figure ). however, in cha-infected monocytes/macrophages, the expression of prrs and isgs were induced at hpi and declined slightly at and hpi before rapidly increasing between and hpi (figure ). in the later time points of infection, the titer of the cha strain was significantly lower than that of the chv strain (figure ) . in the previous studies on dpv and mdv viruses, researchers observed the slight up-regulation of the innate immune molecules around hpi in the fibroblasts, and the rapid down-regulation of these molecules from hpi ( , ) . these results are consistent with the data we obtained in this study; we hypothesized that dpv virulent strains have multiple, highly potent proteins that inhibit innate immunity, and the mechanism of suppressing innate immunity will be further studied in the future. blockage of ifnar signaling in monocytes/macrophages led to a marked increase in the titer of the cha strain, but not that of the chv strain (figure ) . priming monocytes/macrophages with poly(da:dt) or poly(i:c) selectively restricted the replication of the chv strain (figure ) . the growth dynamics and innate immune response were similar in pbmcs. these data indicated that the chv and cha strains exhibit different replication patterns in monocytes/macrophages based on activation or inhibition of the innate immune response. this could explain why the cha strain has been attenuated and provides protection from infection with the virulent dpv strain in ducklings. monocytes/macrophages thus represent a promising cell model suitable for further studies of dpv pathogenicity and the mechanism of innate immune response evasion by dpv. in a recent study, the authors found that monocytes/macrophages are important target cells for dtmuv infection ( ) . dtmuv successfully infects macrophages by subverting the innate immunity of monocytes/macrophages and transmits in the body using monocytes/macrophages as cell carriers. in that study, the authors separated lymphocytes and monocytes/macrophages by washing the pbmcs at and h after plating and then used the cells for assays. though the detection with antibodies against the hypothetical duck cd protein showed that the purity of monocytes/macrophages were greater than %, we think that it is difficult to obtain large numbers of macrophages from pbmcs if the monocytes are not induced with m-csf or gm-csf. therefore, the duck monocytes/macrophages isolated in our study will provide an important cell manipulation platform for similar researches and obtain more direct experimental results and conclusions. in conclusion, we isolated and cultured primary duck defs, neurons, astrocytes, pbmcs, and monocytes/macrophages. these five types of duck primary cells 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source of interferon beta in the virus-infected brain inhibition of dnasensing pathway by marek's disease virus vp protein through suppression of interferon regulatory factor activation avian flavivirus infection of monocytes/macrophages by extensive subversion of host antiviral innate immune responses the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © tian, cai, he, deng, wu, wang, jia, zhu, liu, yang, wu, zhao, chen, zhang, huang, ou, mao, yu, zhang, liu and cheng. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -owoec ud authors: graham, simon p.; mclean, rebecca k.; spencer, alexandra j.; belij-rammerstorfer, sandra; wright, daniel; ulaszewska, marta; edwards, jane c.; hayes, jack w. p.; martini, veronica; thakur, nazia; conceicao, carina; dietrich, isabelle; shelton, holly; waters, ryan; ludi, anna; wilsden, ginette; browning, clare; bialy, dagmara; bhat, sushant; stevenson-leggett, phoebe; hollinghurst, philippa; gilbride, ciaran; pulido, david; moffat, katy; sharpe, hannah; allen, elizabeth; mioulet, valerie; chiu, chris; newman, joseph; asfor, amin s.; burman, alison; crossley, sylvia; huo, jiandong; owens, raymond j.; carroll, miles; hammond, john a.; tchilian, elma; bailey, dalan; charleston, bryan; gilbert, sarah c.; tuthill, tobias j.; lambe, teresa title: evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored covid- vaccine candidate chadox ncov- date: - - journal: npj vaccines doi: . /s - - - sha: doc_id: cord_uid: owoec ud clinical development of the covid- vaccine candidate chadox ncov- , a replication-deficient simian adenoviral vector expressing the full-length sars-cov- spike (s) protein was initiated in april following non-human primate studies using a single immunisation. here, we compared the immunogenicity of one or two doses of chadox ncov- in both mice and pigs. whilst a single dose induced antigen-specific antibody and t cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in sars-cov- neutralising titres. as sars-cov- began to spread around the world at the beginning of several vaccine platform technologies were employed to generate candidate vaccines. several use replication-deficient adenoviral (ad) vector technology and express the sars-cov- spike (s) protein. the first phase i clinical study of an ad -vectored vaccine has been reported , chadox ncov- (azd ) phase i trials (nct ) began in april with phase ii and iii trials (nct ) started soon thereafter, and an ad -vectored vaccine is expected to enter phase i shortly. typically, only one dose of ad-vectored vaccines has been administered in early preclinical challenge studies or clinical studies against emerging or outbreak pathogens [ ] [ ] [ ] [ ] . rhesus macaques immunised with a single dose of chadox ncov- were protected against pneumonia but there was no impact on nasal virus titers after high dose challenge to both the upper and lower respiratory tract . to increase antibody titres and longevity of immune responses, a booster vaccination may be administered. homologous prime-boost immunisation resulted in higher antibody titres including neutralising antibodies and a trend towards a lower clinical score in a mers-cov challenge study . here, we set out to test the immunogenicity of either one or two doses of chadox ncov- in mice and pigs, to further inform clinical development. prime-only and prime-boost vaccination regimens in mice and pigs 'prime-boost' vaccinated inbred (balb/c) and outbred (cd ) mice ( - weeks of age) were immunised by intramuscular (i.m.) injection of infectious units (iu) of chadox ncov- on and days post-vaccination (dpv), whereas, 'prime-only' mice received a single dose of chadox ncov- on day . spleens and serum were harvested from all mice on day ( weeks after boost or prime vaccination). analysis of sars-cov- s proteinspecific murine splenocyte responses by ifn-γ elispot assay showed no statistically significant difference between the primeonly and prime-boost vaccination regimens, in either strain of mouse (fig. a) . intracellular cytokine staining (ics) of splenocytes ( fig. b) showed, in both mouse strains, that the response was principally driven by cd + t cells. the predominant cytokine response of both cd + and cd + t cells was expression of ifn-γ and tnf-α, with negligible frequencies of il- + and il- + cells, consistent with previous data suggesting adenoviral vaccination does not induce a dominant th response , . there were no signficant differences in cd + and cd + t cell cytokine responses between prime-only and prime-boost mice. prime-only and prime-boost pigs ( - weeks of age) were immunised by i.m. injection of iu of chadox ncov- (the same dose as being used in human clinical trials) on dpv and prime-boost pigs received a second immunisation on dpv. blood samples were collected weekly until dpv to analyse immune responses. ifn-γ elispot analysis of porcine peripheral blood mononuclear cells (pbmc) showed responses on dpv ( weeks after boost) that were significantly greater in the prime-boost pigs compared to prime-only animals (p < . ; fig. c ). the prime-boost dpv responses were greater than responses observed in either group on dpv, but interanimal variation meant this did not achieve statistical significance. ics analysis of porcine t cell reponses showed a dominance of th -type cytokines (similar to the murine response) but with a higher frequency of s-specific cd + t cells compared to cd + t cells (fig. d) . however, cd + and cd + t cell cytokine responses did not differ significantly between vaccine groups or timepoints ( vs. dpv). sars-cov- s protein-specific antibody responses following chadox ncov- prime-only and prime-boost vaccination regimens in mice and pigs sars-cov- s protein-specific antibody titres in serum were determined by elisa using recombinant soluble trimeric s (fl-s) and receptor binding domain (rbd) proteins. a significant increase in fl-s binding antibody titres was observed in prime-boost balb/ c mice compared to their prime-only counterparts (p < . ), however, the difference between vaccine groups for cd mice was not significant (fig. a) . antibody responses were evaluated longitudinally in pig sera by fl-s and rbd elisa. compared to prevaccination sera, significant fl-s specific antibody titres were detected in both prime-only and prime-boost groups from and dpv, respectively (p < . ; fig. b ). fl-s antibody titres did not differ signifcantly between groups until after the boost, when titres in the prime-boost pigs became significantly greater with an average increase in titres of > log (p < . ). rbd-specific antibody titres showed a similar profile with significant titres in both groups from dpv (p < . ) and a further significant increase in the prime-boost pigs from dpv onwards which was greater than the prime-only pigs (p < . ; fig. c ). sars-cov- neutralising antibody responses were assessed using a virus neutralisation test (vnt; fig. d ) and pseudovirus-based neutralisation test (pvnt; fig. e ). after the prime immunisation, sars-cov- neutralising antibody titres were detected by vnt in and dpv sera from / prime-boost and / prime-only pigs. two weeks after the boost ( dpv), neutralising antibody titres were detected and had increased in all prime-boost pigs, which were significantly greater than the earlier timepoints and the titres measured in the prime-only group (p < . ). in agreement with this analysis, serum assayed for neutralising antibodies using the pvnt revealed that antibody titres in dpv prime-boost pig sera were significantly greater than earlier timepoints and the primeonly group (p < . ). statistical analysis showed a highly significant correlation between pvnt and vnt titres (spearman's rank correlation r = . ; p < . ). in this study, we utilised both a small and a large animal model to evaluate the immunogenicity of either one or two doses of a covid- vaccine candidate, chadox ncov- (now known as azd ). small animal models have variable success in predicting vaccine efficacy in larger animals but are an important to analyse sars-cov- s-specific t cell responses, all mice were sacrificed on day for isolation of splenocytes and pigs were blood sampled longitudinally to isolate pbmc. following stimulation with sars-cov- s-peptides, responses of murine splenocytes a and porcine pbmc c were assessed by ifn-γ elispot assays. using flow cytometry, cd + and cd + t cell responses were characterised by assessing expression of ifn-γ, tnf-α, il- , il- and il- (mice; b) and ifn-γ, tnf-α, il- and il- (pigs; d). each data point represents an individual mouse/pig with bars denoting the median response per group/timepoint. data were analysed by anova and statistically significant differences between vaccine groups are indicated: *p < . . stepping stone to facilitate prioritisation of vaccine targets. in contrast, larger animal models, such as the pig and non-human primates, have been shown to more accurately predict vaccine outcome in humans [ ] [ ] [ ] . the mouse data generated in this study suggested that the immunogenicity profile was at the upper end of a dose response curve, which may have saturated the immune response and largely obscured our ability to determine differences between prime-only or prime-boost regimens. we have developed the pig as a model for generating and understanding immune responses to vaccination against human influenza [ ] [ ] [ ] and nipah virus , . the inherent heterogeneity of an outbred large animal model is more representative of immune responses in humans. extensive development of reagents to study immune responses in pigs in recent years has extended the usefulness and applicability of the pig as a model to study infectious disease. these data demonstrate the utility of the pig as a model for further evaluation of the immunogenicity of chadox ncov- and other covid- vaccines. we show here that t cell responses are higher in pigs that received a prime-boost vaccination when compared to prime only at day , whilst comparing responses days after last immunisation demonstrates the prime-boost regimen trended toward a higher response. in addition, chadox ncov- immunisation induced robust th -like cd + and cd + t cell responses in both pigs and mice. this has important implications for covid- vaccine development as virus-specific t cells are thought to play an important role in sars-cov- infection - . while no correlate of protection has been defined for covid- , recent publications suggest that neutralising antibody titres may be correlated with protection in animal challenge models , . a single dose of chadox ncov- induces antibody responses, but we demonstrate here that antibody responses are significantly enhanced after homologous boost in one mouse strain and to a greater extent in pigs. the sars-cov- neutralising antibody titres in pigs after a single immunisation appear broadly comparable to those in sera from humans following asymptomatic infection, whereas, titres in prime-boost pigs were more comparable to serum from recovered covid- patients . however, it is likely that a combination of neutralising antibodies and antigen-specific t cells would act in synergy to prevent and control infection, as we have recently shown in the context of influenza vaccination , . whilst human immunogenicity and clinical read-outs are a critically meaningful endpoint, studies in small animals and pigs will help prioritise candidates to be tested in humans. further clinical studies are needed to assess immunogenicity after primeboost vaccination and the impact on clinical efficacy and durability of the immune response. mouse and pig studies were performed in accordance with the uk animals inbred balb/c (n= ) and outbred cd (n= ) were immunised on day and with chadox ncov (prime-boost) or chadox ncov on day (prime-only), whereas, pigs were immunised with chadox ncov- on days and (prime-boost), or only on day (prime-only). to analyse sars-cov- s protein-specific antibodies in serum, all mice were sacrificed on day and pigs were blood sampled weekly until day . antibody units or end-point titres (ept) were assessed by elisa using recombinant sars-cov- fl-s for both mice a and pigs b, and recombinant s protein rbd for pigs c. sars-cov- neutralising antibody titres in pig sera were determined by vnt, expressed as the reciprocal of the serum dilution that neutralised virus infectivity in % of the wells (nd ; d), and pvnt, expressed as reciprocal serum dilution to inhibit pseudovirus entry by % (ic ; e). each data point represents an individual mouse/pig sera with bars (a) denoting the median titre per group. data were analysed by anova and statistically significant differences between vaccine groups are indicated: **p < . ; ***p < . ; ****p < . . p b f and university of oxford awerb, and pigs-project license pp and the pirbright institute awerb). the principles of the r's were applied for the duration of the study to ensure animal welfare was not unnecessarily compromised. vero e cells were grown in dmem containing sodium pyruvate and lglutamine (sigma-aldrich, poole, uk), % fbs (gibco, thermo fisher, loughborough, uk), . % penicillin/streptomycin ( , u/ml; gibco) (maintenance media) at °c and % co . sars-cov- isolate england- stocks were grown in vero e cells using a multiplicity of infection (moi) of . for days at °c in propagation media (maintenance media containing % fbs). sars-cov- stocks were titrated on vero e cells using mem (gibco), % fcs (labtech, heathfield, uk), . % avicel (fmc biopolymer, girvan, uk) as overlay. plaque assays were fixed using formaldehyde (vwr, leighton buzzard, uk) and stained using . % toluidine blue (sigma-aldrich). all work with live sars-cov- virus was performed in acdp hg laboratories by trained personnel. the propagation, purification and assessment of chadox ncov- titres were as described previously . recombinant sars-cov- proteins and synthetic peptides a synthetic dna, encoding the spike (s) protein receptor binding domain (rbd; amino acids - ) of sars-cov- (genbank mn ), codon optimised for expression in mammalian cells (idt technology) was inserted into the vector popinttgneo incorporating a c-terminal his tag. recombinant rbd was transiently expressed in expi ™ (thermo fisher scientific, uk) and protein purified from culture supernatants by immobilised metal affinity followed by a gel filtration in phosphatebuffered saline (pbs) ph . buffer. a soluble trimeric s (fl-s) protein construct encoding residues - with two sets of mutations that stabilise the protein in a pre-fusion conformation (removal of a furin cleavage site and the introduction of two proline residues; k p, v p) was expressed as described . the endogenous viral signal peptide was retained at the n-terminus (residues - ), a c-terminal t -foldon domain incorporated to promote association of monomers into trimers to reflect the native transmembrane viral protein, and a c-terminal his tag included for nickel-based affinity purification. similar to recombinant rbd, fl-s was transiently expressed in expi ™ (thermo fisher scientific) and protein purified from culture supernatants by immobilised metal affinity followed by gel filtration in tris-buffered saline (tbs) ph . buffer. for analysis of t cell responses in pigs, overlapping mer peptides offset by residues based on the predicted amino acid sequence of the entire s protein from sars-cov- wuhan-hu- isolate (ncbi reference sequence: nc_ . ) were designed and synthesised (mimotopes, melbourne, australia) and reconstituted in sterile % acetonitrile (sigma-aldrich) at a concentration of mg/ml. three pools of synthetic peptides representing residues - (pool ), - (pool ) and - (pool ) were prepared for use to stimulate t cells in ifn-γ elispot and intracellular cytokine staining (ics) assays. for analysis of t cell responses in mice, overlapping mer peptides offset by residues were designed and synthesised (mimotopes) and reconstituted in sterile % dmso (sigma-aldrich) at a concentration of mg/ml. two peptide pools spanning s region (pool : to and - , pool : - ) and peptide pools spanning s region (pool : to , pool : to ) were used for stimulating splenocytes for ifn-γ elispot analysis, and single pools of s (pool and pool ) and s (pool and pool ) were used to stimulate splenocytes for ics. mice: inbred female balb/colahsd (balb/c) and outbred crl:cd (cd ) mice were purchase from commercial suppliers (envigo and charles river laboratories, respectively) and randomly allocated into 'prime-only' or 'prime-boost' vaccination groups (balb/c n = and cd n = ) upon arrival. at - weeks of age, prime-boost mice were immunised intramuscularly with infectious units (iu) ( . × virus particles; vp) chadox ncov- and boosted intramuscularly four weeks later with × iu chadox ncov- . prime-only mice received a single dose of iu chadox ncov- at the same time prime-boost mice were boosted. spleens and serum were harvested from all animals a further weeks later. pigs: six - -week-old, weaned, female, large white-landrace-hampshire cross-bred pigs from a commercial rearing unit were randomly allocated to two treatment groups (n = ): 'prime-only' and 'prime-boost'. both groups were immunised on day with × iu ( . × vp) chadox ncov- in ml pbs by intramuscular injection (brachiocephalic muscle). 'prime-boost' pigs received an identical booster immunisation on day . blood samples were taken from all pigs on a weekly basis at , , , , , and dpv by venepuncture of the external jugular vein: ml/pig in bd sst vacutainer tubes (fisher scientific) for serum collection and ml/pig in bd heparin vacutainer tubes (fisher scientific) for peripheral blood mononuclear cell (pbmc) isolation. mice. antibodies to sars-cov- fl-s protein were determined by performing a standardised elisa on serum collected -weeks after prime or prime-boost vaccination. maxisorp plates (nunc) were coated with ng/well fl-s protein overnight at °c, prior to washing in pbs/tween ( . % v/v) and blocking with blocker casein in pbs (thermo fisher scientific) for h at room temperature (rt). standard positive serum (pool of mouse serum with high endpoint titre against fl-s protein), individual mouse serum samples, negative and an internal control (diluted in casein) were incubated for h at rt. following washing, bound antibodies were detected by addition of alkaline phosphatase-conjugated goat anti-mouse igg (sigma-aldrich), diluted / in casein, for h at rt and detection of anti-mouse igg by the addition of pnpp substrate (sigma-aldrich). an arbitrary number of elisa units were assigned to the reference pool and od values of each dilution were fitted to a -parameter logistic curve using softmax pro software. elisa units were calculated for each sample using the od values of the sample and the parameters of the standard curve. pigs. serum was isolated by centrifugation of sst tubes at × g for min at rt and stored at − °c. sars-cov- rbd and fl-s specific antibodies in serum were assessed as detailed previously with the exception of the following two steps. the conjugated secondary antibody was replaced with goat anti-porcine igg hrp (abcam, cambridge, uk) at / , dilution in pbs with . % tween and % non-fat milk. in addition, after the last wash, a µl of tmb (one component horse radish peroxidase microwell substrate, biofx, cambridge bioscience, cambridge, uk) was added to each well and the plates were incubated for min at rt. a µl of biofx nm stop reagent (cambridge bioscience) was then added to stop the reaction and microplates were read at nm. endpoint antibody titres (mean of duplicates) were calculated as follows: the log od was plotted against the log sample dilution and a regression analysis of the linear part of this curve allowed calculation of the endpoint titre with an od of twice the average od values of dpv sera. the ability of pig sera to neutralise sars-cov- was evaluated using virus and pseudovirus neutralisation assays. for both assays, sera were first heatinactivated (hi) by incubation at °c for h. virus neutralization test (vnt): starting at a in dilution, two-fold serial dilutions of sera were prepared in well round-bottom plates using dmem containing % fbs and % antibiotic-antimycotic (gibco) (dilution media). μl of diluted pig serum was mixed with μl dilution media containing approximately plaque-forming units (pfu) sars-cov- for h at °c. vero e cells were seeded in -well flat-bottom plates at a density of × cells/ml in maintenance media one day prior to experimentation. culture supernatants were replaced by µl of dmem containing % fcs and % antibiotic-antimycotic, before µl of the virus-sera mixture was added to the vero e cells and incubated for six days at °c. cytopathic effect (cpe) was investigated by bright-field microscopy. cells were further fixed and stained as described above, and cpe scored. each individual pig serum dilution was tested in quadruplet on the same plate and no sera/sars-cov- virus and no sera/no virus controls were run concurrently on each plate in quadruplet. wells were scored for cytopathic effect and neutralisation titres expressed as the reciprocal of the serum dilution that completely blocked cpe in % of the wells (nd ). researchers performing the vnts were blinded to the identity of the samples. pseudovirus neutralisation test (pvnt): lentiviral-based sars-cov- pseudoviruses were generated in hek t cells incubated at °c, % co . cells were seeded at a density of . × in well dishes, before being transfected with plasmids as follows: ng of sars-cov- spike, ng p . (encoding for hiv- gag-pol), ng csflw (lentivirus backbone expressing a firefly luciferase reporter gene), in opti-mem s.p. graham et al. (gibco) along with µl pei ( µg/ml) transfection reagent. a 'no glycoprotein' control was also set up using carrier dna (pcdna . ) instead of the sars-cov- s expression plasmid. the following day, the transfection mix was replaced with ml dmem with % fbs (dmem- %) and incubated at °c. at both and h post transfection, supernatants containing pseudotyped sars-cov- (sars-cov- pps) were harvested, pooled and centrifuged at × g for min at °c to remove cellular debris. target hek t cells, previously transfected with ng of a human ace expression plasmid (addgene, cambridge, ma, usa) were seeded at a density of × in µl dmem- % in a white flatbottomed -well plate one day prior to harvesting of sars-cov- pps. the following day, sars-cov- pps were titrated -fold on target cells, with the remainder stored at − °c. for pvnts, pig sera were diluted : in serum-free media and µl was added to a -well plate in quadruplicate and titrated -fold. a fixed titred volume of sars-cov- pps was added at a dilution equivalent to signal luciferase units in µl dmem- % and incubated with sera for h at °c, % co . target cells expressing human ace were then added at a density of × in µl and incubated at °c, % co for h. firefly luciferase activity was then measured with brightglo luciferase reagent and a glomax-multi + detection system (promega, southampton, uk). pseudovirus neutralization titres were expressed as the reciprocal of the serum dilution that inhibited luciferase expression by % (ic ). mice. single cell suspension of mouse spleens were prepared by passing cells through μm cell strainers and ack lysis (thermo fisher) prior to resuspension in complete media (αmem supplemented with % fcs, pen- step, l-glut and -mercaptoethanol). for analysis of ifn-γ production by elispot assay, splenocytes were stimulated with s peptide pools at a final concentration of μg/ml on ipvh-membrane plates (millipore) coated with μg/ml anti-mouse ifn-γ (clone an ; mabtech). after - h of stimulation, ifn-γ spot forming cells (sfc) were detected by staining membranes with anti-mouse ifn-γ biotin mab ( µg/ml; clone r a , mabtech) followed by streptavidin-alkaline phosphatase ( µg/ml) and development with ap conjugate substrate kit (bio-rad). for analysis of intracellular cytokine production, cells were stimulated with μg/ml s peptide pools, media or cell stimulation cocktail (containing pma-ionomycin, biolegend), together with μg/ml golgiplug (bd biosciences) and μl/ml cd a-alexa for h in a -well u-bottom plate, prior to placing at °c overnight. following surface staining with cd -buv , cd -percp-cy . , cd l-bv , cd -bv , cd -apc-cy and live/ dead aqua (thermo fisher), cells were fixed with % neutral buffered formalin (containing % paraformaldehyde) and stained intracellularly with tnf-α-af , il- -pe-cy , il- -bv , il- -pe and ifn-γ-e diluted in perm-wash buffer (bd biosciences). sample acquisition was performed on a fortessa (bd) and data analysed in flowjo v (treestar). an acquisition threshold was set at a minimum of events in the live cd + gate. antigen-specific t cells were identified by gating on live/dead negative, doublet negative (fsc-h vs. fsc-a), size (fsc-h vs. ssc), cd + , cd + or cd + cells and cytokine positive. the gating strategy is illustrated in supplementary fig. . total sars-cov- s specific cytokine responses are presented after subtraction of the background response detected in the media stimulated control spleen sample of each mouse, prior to summing together the frequency of s and s specific cells. pigs. pbmcs were isolated from heparinised blood by density gradient centrifugation and cryopreserved in cold % dmso (sigma-aldrich) in hi fbs . resuscitated pbmc were suspended in rpmi medium, glutamax supplement, hepes (gibco) supplemented with % hi fbs (new zealand origin, life science production, bedford, uk), % penicillin-streptomycin and . % -mercaptoethanol ( mm; gibco) (crpmi). to determine the frequency of sars-cov- s specific ifn-γ producing cells, an elispot assay was performed on pbmc from , , and dpv. multiscreen -well plates (mahas ; millipore, fisher scientific) were pre-coated with µg/ml anti-porcine ifn-γ mab (clone p g , bd biosciences) and incubated overnight at °c. after washing and blocking with crpmi, pbmcs were plated at × cells/well in crpmi in a volume of µl/well. pbmcs were stimulated in triplicate wells with the sars-cov- s peptide pools at a final concentration of µg/ml/peptide. crpmi alone was used in triplicate wells as a negative control. after h incubation at °c with % co , plates were developed as described previously . the numbers of specific ifn-γ secreting cells were determined using an immunospot ® s analyzer (cellular technology, cleveland, usa). for each animal, the mean 'crpmi only' data was subtracted from the s peptide pool , and data which were then summed and expressed as the mediumcorrected number of antigen-specific ifn-γ secreting cells per × pbmc. to assess intracellular cytokine expression pbmc from and dpv were suspended in crpmi at a density of × cells/ml and added to µl/well to -well round bottom plates. pbmcs were stimulated in triplicate wells with the sars-cov- s peptide pools ( µg/ml/peptide). unstimulated cells in triplicate wells were used as a negative control. after h incubation at °c, % co , cytokine secretion was blocked by addition : bd golgiplug (bd biosciences) and cells were further incubated for h. pbmc were washed in pbs and surface labelled with zombie nir fixable viability stain (biolegend), cd -percp-cy . mab (clone - - , bd bioscience) and cd β-fitc mab (clone ppt , bio-rad antibodies). following fixation (fixation buffer, biolegend) and permeabilization (permeabilization wash buffer, biolegend), cells were stained with: ifn-γ-af mab (clone cc , bio-rad antibodies, kidlington, uk), tnf-α-bv mab (clone mab , biolegend), il- mab (clone a d f h , invitrogen, thermo fisher scientific) and il- bv mab (clone mp - d , biolegend) followed by staining with anti-mouse igg a-pe-cy (clone rmg a- , biolegend). cells were analysed using a bd lsrfortessa flow cytometer and flowjo x software. the gating strategy is illustrated in supplementary fig. . total sars-cov- s specific cytokine positive responses are presented after subtraction of the background response detected in the media stimulated control pbmc sample of each pig, prior to summing together the frequency of s-peptide pools - specific cells. graphpad prism . . (graphpad software, san diego, usa) was used for graphical and statistical analysis of data sets. anova or a mixed-effects model were conducted to compare responses over time and between vaccine groups at different time points post-vaccination as detailed in the "results" section. antibody titre data were log transformed before analysis. neutralising antibody titre data generated by the vnt and pvnt assays were compared using spearman nonparametric correlation. p-values < . were considered statistically significant. safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial a single-dose chadox -vectored vaccine provides complete protection against nipah bangladesh and malaysia in syrian golden hamsters safety and immunogenicity of a candidate middle east respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, openlabel, non-randomised, uncontrolled, phase trial protective efficacy of a novel simian adenovirus vaccine against lethal mers-cov challenge in a transgenic human dpp mouse model a single dose of chadox mers provides broad protective immunity against a variety of mers-cov strains chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques a single dose of chadox mers provides protective immunity in rhesus macaques antigen encoded by vaccine vectors derived from human adenovirus serotype is preferentially presented to cd + t lymphocytes by the cd α+ dendritic cell subset immunization with an adenovirus-vectored tb vaccine containing ag a-mtb effectively alleviates allergic asthma the pig: a model for human infectious diseases large animal models for vaccine development and testing the contribution of non-human primate models to the development of human vaccines comparison of heterosubtypic protection in ferrets and pigs induced by a single-cycle influenza vaccine immunogenicity and protective efficacy of seasonal human live attenuated cold-adapted influenza virus vaccine in pigs aerosol delivery of a candidate universal influenza vaccine reduces viral load in pigs challenged with pandemic h n virus vaccine development for nipah virus infection in pigs bovine herpesvirus- -vectored delivery of nipah virus glycoproteins enhances t cell immunogenicity in pigs targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid- patients clinical and immunological features of severe and moderate coronavirus disease transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients presence of sars-cov- reactive t cells in covid- patients and healthy donors sars-cov- infection protects against rechallenge in rhesus macaques dna vaccine protection against sars-cov- in rhesus macaques antibody responses to sars-cov- at weeks postinfection in asymptomatic patients vaccination with viral vectors expressing np, m and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice a serological assay to detect sars-cov- seroconversion in humans author contributions writing-review and editing, all authors further information on experimental design is available in the nature research reporting summary linked to this article. the data that support the findings of this study are available from the corresponding authors upon reasonable request. s.c.g. and t.l. are named on a patent application covering chadox ncov- . the remaining authors declare no competing interests. the funders played no role in the conceptualisation, design, data collection, analysis, decision to publish, or preparation of the manuscript. supplementary information is available for this paper at https://doi.org/ . / s - - - .correspondence and requests for materials should be addressed to s.p.g. or t.l. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -jxqskt k authors: warren, cody j.; griffin, laura m.; little, alexander s.; huang, i-chueh; farzan, michael; pyeon, dohun title: the antiviral restriction factors ifitm , and do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jxqskt k type i interferons (ifn-α and β) induce dynamic host defense mechanisms to inhibit viral infections. it has been recently recognized that the interferon-inducible transmembrane proteins (ifitm) , and can block entry of a broad spectrum of rna viruses. however, no study to date has focused on the role of ifitm proteins in dna virus restriction. here, we demonstrate that ifn-α or -β treatment of keratinocytes substantially decreases human papillomavirus (hpv ) infection while robustly inducing ifitm , and expression. however, ifitm , and overexpression did not inhibit hpv infection; rather, ifitm and ifitm modestly enhanced hpv infection in various cell types including primary keratinocytes. moreover, ifitm , and did not inhibit infection by two other dna viruses, human cytomegalovirus (hcmv) and adenovirus type (ad ). taken together, we reveal that the entry of several dna viruses, including hpv, hcmv, and ad is not affected by ifitm , and expression. these results imply that hpv, and other dna viruses, may bypass ifitm restriction during intracellular trafficking. human papillomaviruses (hpvs) are small, non-enveloped double-stranded dna viruses causally associated with multiple human cancers. over different genotypes have been identified and collectively categorized into high-risk or low-risk genotypes depending on their oncogenic capacity [ , ] . the high-risk types are most commonly associated with cervical cancer [ , ] and increasing evidence points to a contributing role in other cancers including head-neck [ , ] and anogenital cancers [ ] . hpv is the most prevalent high-risk genotype and serves as the main vaccine target along with hpv [ , ] . hpv is the most common sexually transmitted pathogen in the united states [ ] . despite high exposure rates, most people clear their infections naturally within - years [ ] . however, longterm persistent infections are established in approximately % of women [ ] . since persistence is required for cancer progression [ , ] , it is critical to understand host immune features that are responsible for viral clearance so that new approaches targeting persistent hpv infections can be developed. in order to establish long-term infections, hpvs must actively avoid both adaptive and innate immune responses. hpvs prevent adaptive immune detection by several mechanisms in their unique life cycle. first, viral antigen production is limited to terminally differentiated keratinocytes of the mucosal and cutaneous epithelia. these cells are programmed to die of terminal differentiation, thus virus release coincides with limited inflammation and release of danger signals [ ] . additionally, there is no viremic stage of the hpv life cycle, which minimizes the activation of systemic immune responses [ ] . despite eliciting weak adaptive immune responses, the majority of primary hpv infections are cleared, thus suggesting the involvement of additional immune-mediated control mechanisms. keratinocytes intrinsically express low-levels of interferons (ifns) a, b, and k which induce interferonstimulated gene (isg) expression [ , ] . however, the hpv oncoproteins e and e actively target the ifn regulatory transcription factors irf- and irf- , respectively, resulting in an overall dampening of isg responses during infection [ ] [ ] [ ] . active subversion of the ifn pathway suggests that the innate immune response, specifically ifn-regulated genes, may interfere with hpv persistence. the ifn-inducible transmembrane (ifitm) proteins are a family of ubiquitously expressed restriction factors that mediate ifn-induced antiviral activity [ , ] . the antiviral effects of ifitms were first discovered in a genetic screen for host factors that restrict influenza a virus replication [ ] . follow-up studies revealed ifitm type-specific restriction of an array of rna viruses including marburg and ebola filoviruses (marv, ebov), dengue and west nile (wnv) flaviviruses, sars coronavirus (sars-cov), hepatitis c virus (hcv), human immunodeficiency virus (hiv), rift valley fever virus (rvfv), respiratory syncytial virus (rsv), and reovirus [ ] [ ] [ ] [ ] [ ] [ ] . one of the common entry mechanisms shared by all these viruses, except hiv, is the requirement for low ph in late endosomes or lysosomes to facilitate genome release into the cytosol [ , ] . hpvs encapsidate their kb, double-stranded dna genome, in a desiccant-resistant icosahedral capsid composed of the major and minor capsid proteins l and l , respectively [ ] . devoid of an envelope, initial cell contact is mediated at the basement membrane of epithelial cells by direct binding of the l capsid protein to heparan sulfate proteoglycans [ ] . while downstream events in entry are not yet fully elucidated, it is well known that uncoating and genome translocation to the nucleus is dependent on the low ph encountered in acidified late endosomes and lysosomes [ ] [ ] [ ] . as the antiviral activity of ifitms is likely mediated by preventing endosome fusion and viral entry into the cytosol [ , , ] , we hypothesized that one or more types of ifitm proteins inhibit hpv entry by preventing escape from the endocytic pathway. our study demonstrates that type i ifns inhibit hpv infection in keratinocytes, thus suggesting that isg induction may be critical for hpv restriction. surprisingly, overexpression of ifitm and ifitm consistently enhanced hpv infectivity in various epithelial cell lines and keratinocytes, while knockdown of endogenous ifitms yielded no effect. additionally, two other dna viruses, hcmv and ad , were unaffected by ifitm overexpression. analysis of the rate of hpv capsid protein degradation in ifitm overexpressing primary keratinocytes revealed a delay in proteolytic degradation of virus capsids. collectively, our results suggest that endosome trafficking, altered by ifitm overexpression, preferentially routes hpv to a more productive infectious pathway. based on these results, we present the first study detailing the role of ifitms in the entry of dna viruses, showing that ifitm , and proteins do not restrict were calculated by student's two-tailed t-test using prism version . for mac, graphpad software (san diego california usa, www.graphpad.com). significant differences (*p , . , **p , . , ***p , . and ****p , . ) compared to pbs treated cells are marked with asterisks. (c) ifitm , and expression levels were determined by rt-qpcr using ifitm specific primers (table s ). data are presented as fold change compared to untreated cells. (d) hacat cells were treated with u/ml of ifn-b or medium alone and endogenous ifitm or ifitm protein expression in cell lysate was analyzed by western blotting using specific antibodies. pageruler plus ladder (thermo) was used to approximate molecular weights in kilodaltons (kd): ifitm , kd; ifitm , kd; b-actin, kd. doi: . /journal.pone. .g hpv, hcmv, and ad infections. our results suggest an evolutionarily conserved entry mechanism by these dna viruses that bypasses the antiviral function of ifitms that restrict many rna viruses. hpv virions and pseudovirions were prepared as described previously [ , ] . plucf, used in generating hpv -lucf pseudovirions, was a kind gift from chris buck. adenovirus type (ad -cmv-gfp), human cytomegalovirus (hcmv tb e mcherry) and sars-cov pseudotyped lentivirus [ ] were generous gifts from drs. jerome schaack, caroline kulesza, and kathryn holmes, respectively (university of colorado school of medicine). human leukocyte ifn-a (nr- ) and human recombinant ifn-b (nr- ) were obtained from bei resources (manassas, va). ft cells purchased from invitrogen (grand island, ny) and hela cells obtained from dr. paul lambert (university of wisconsin-madison) were maintained in dulbecco's modified eagle's medium (dmem) (thermo scientific/hyclone, logan, ut) supplemented with % fetal bovine serum (fbs) (hyclone). hacat cells [ ] were also obtained from dr. paul lambert and were cultured in e-medium ( parts dmem, part ham's f- nutrient mixture) supplemented with % fbs. human foreskin keratinocytes (hfks), derived from neonatal foreskin donors, were purchased from invitrogen (cascade biologics, portland, or) and cultured in epilife medium with mm calcium supplemented with human keratinocyte growth supplement (invitrogen/cascade biologics) according to the manufacturer's protocol [ ] . human lung epithelial a cells transduced with vector alone (pqcxip) or stably expressing c-myc tagged ifitm , , or were generated previously [ ] and maintained in roswell park memorial institute (rpmi) (hyclone). hela cell lines stably expressing prs vector-based control shrna and shrna targeting ifitm or [ ] were maintained as described above. hela and hacat cell lines transfected with vector alone or stably expressing ifitm proteins were selected with mg/ml of puromycin (invitrogen). to produce transducing retroviruses, ft cells were transfected using the pei method with a : : dna ratio of pcmv-vsv-g, pmlv gag-pol (dr. jerome schaack) and transfer vector, respectively. after and h, culture supernatants were combined, passed through a . mm syringe filter and concentrated times by centrifugation at , rpm for h at uc. hpv -lucf and sars-cov pseudovirion infectivity were measured by luciferase assay as described previously [ , ] . infections with hpv -lucf pesudovirions were performed at to , viral genome equivalents (vge)/cell, which is equivalent to approximately . to multiplicity of infection (moi). gfp and phycoerythrin (pe) signals from cells infected with ad -cmv-gfp and hcmv tb e mcherry, respectively, were collected from at least , cells using a facscalibur flow cytometer (bd bioscience). data were analyzed using flowjo software (tree star). cells were lysed in ripa buffer containing protease inhibitor cocktail (roche) and mg of total protein was separated by sds-page. antibodies: mouse monoclonal anti-l ( : , cam-vir- , abcam), mouse monoclonal anti-c-myc ( : , e , santa cruz biotechnology), mouse monoclonal anti-b-actin ( : , h d , cell signaling technology), rabbit polyclonal anti-ifitm ( : , - -ap, proteintech), rabbit polyclonal anti-ifitm ( : , h -d p, novus biologicals) and hrp-conjugated secondary antibodies ( : , , jackson immu-noresearch laboratories). proteins were visualized using clarity western ecl substrate (bio-rad) and the chemidoc xrs system (bio-rad). total rna was extracted using rneasy kit (qiagen) and cdna was synthesized using oligo (dt) and superscript ii reverse transcriptase (invitrogen). cdna was analyzed by quantitative pcr (qpcr) using fast start universal sybr green master mix (roche). primers specific for selected isgs (table s ) were designed using primer-blast (http://www. ncbi.nlm.nih.gov/tools/primer-blast/) and b-actin sequences have been described previously [ ] . qpcr was performed using cfx connect real-time pcr detection system (bio-rad). using the ddct method, relative quantities were calculated and normalized to b-actin. to determine whether type i ifns interfere with hpv infection, hacat keratinocytes were pre-treated with ifn-a or ifn-b for h and then infected with hpv luciferase reporter pseudovirions (hpv -lucf) in the presence of ifn-a or ifn-b. at h post infection (hpi), infectivity was measured by relative luciferase activity [ ] . although both ifn-a and -b treatments significantly inhibited hpv -lucf infection, the reductions seen with ifn-b treatment were more robust at lower doses (fig. a) . rt-qpcr of selected isgs (mx , ifi , ifit , ifitm , , and ) revealed enhanced expression of mrnas following ifn-a treatment (fig. s ). this indicates that, although keratinocytes are responsive to ifn-a stimulation, ifn-b treatment is more effective at reducing infectivity despite sharing a common surface receptor [ ] . to exclude the possibility that the observed effect of ifn-b on hpv infection might reflect cytotoxicity rather than inhibiting infection, we measured cell viability using a luminescence-based atp quantification assay in parallel (promega). no significant effect on keratinocyte viability was observed up to u/ml of ifn-b (fig. b) . ifitm protein expression induced by type i ifn inhibits infection of many rna viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . therefore, we analyzed induction of ifitm , and expression by ifn-b treatment in human keratinocytes, the natural host cells for hpv. hacat cells were treated with increasing doses of ifn-b for h, followed by rna extraction and rt-qpcr to assess ifitm mrna expression levels. ifitm , and mrna expression was increased by ifn-b in a dose dependent manner (fig. c) . it appeared that the magnitude of ifitm induction by ifn-b treatment was higher than the levels of ifitm and ifitm induction. however, this may be due to the low basal level of endogenous ifitm in human keratinocytes (fig. d) . while differing in endogenous expression levels, ifitm and ifitm proteins were dramatically increased by ifn-b treatment (fig. d) . to determine the effect of ifitms on hpv entry, hela cells stably expressing c-myc-tagged ifitm , or or with vector alone ( fig. a-b) were infected with hpv -lucf. at hpi, infectivity was measured by luciferase assay and percent infectivity was calculated after normalization to cells transduced with the empty vector control. despite high expression (fig. b) , ifitm , or did not inhibit virus infection ( fig. a) . comparable results were obtained in a cells and hacat keratinocytes (fig. c-f) . surprisingly, overexpression of ifitm and ifitm consistently enhanced hpv infection by - % while the effects of ifitm were negligible ( fig. a, c and e) . to examine the effect of ifitm in primary human keratinocytes, human foreskin keratinocytes (hfks) were transduced with c-myc-tagged ifitm using a retroviral delivery system [ , ] . consistently, ifitm overexpression enhanced hpv infection even higher at . -fold ( fig. g and h) . these results suggest that ifitm overexpression may facilitate hpv infection in human keratinocytes. we further investigated if ifitms restrict infection of other dna viruses that also require acidic compartments for entry into host cells. in epithelial cells, ad and hcmv entry and egress from endosomal compartments depends on low ph [ , ] . given that ifitm restriction is mediated along this pathway [ , ] , we hypothesized that ifitm overexpression would affect the entry processes of ad and hcmv. recombinant ad and hcmv (strain tb e) expressing gfp and mcherry, respectively, were used to infect hela cells stably expressing ifitm , , or or with vector alone. at hpi, the percentage of infected cells was determined by flow cytometry. our results showed that ad and hcmv infections were unaffected by ifitm protein expression ( fig. a and b) . it has been previously reported that s-protein mediated entry of sars-cov is broadly restricted by the ifitm , and proteins [ ] . using a sars-cov s-protein pseudotyped lentivirus [ ] as a positive control, we demonstrate a similar restriction of sars-cov infection mediated by ifitm proteins in hela cell lines (fig. c) . mean % infectivity from quadruplicate samples representative of at least two independent experiments with standard error. p-values were calculated as described in fig. to determine whether endogenous ifitm expression affects hpv entry, ifitm or ifitm was knocked down in hela cells by stable expression of ifitm , ifitm or control scrambled shrna [ ] . since ifitm expression appeared to have no effect on hpv infectivity, we focused only on ifitm and ifitm . the levels of target gene knockdown were determined by rt-qpcr (fig. a) . although ifitm and ifitm show some sequence homology, no off target effects were noted by shrna knockdown (fig. s ) . interestingly, knockdown of ifitm or ifitm expression had no effect on hpv -lucf infection (fig. b) . taken together, our results suggest that, compared to rna viruses, the dna viruses hpv, hcmv, and ad might rely on different entry mechanisms to avoid ifitm restriction. previous studies show that overexpression of ifitm disrupts the composition of endosomal compartments [ , ] and influ-ences the rate of reovirus capsid protein degradation [ ] . given the consistent effects of ifitm overexpression on hpv infection in primary keratinocytes, we determined whether ifitm altered the kinetics of hpv l major capsid protein degradation. hfks were inoculated with hpv virions containing full-length viral genomes for h at uc to allow for virus attachment but not internalization. internalization was initiated by shifting the cells to uc for , , , and h. at the indicated time points, the cells were trypsinized to remove non-internalized virus, then harvested for hpv l western blotting. intriguingly, hfks overexpressing ifitm exhibited delayed l degradation compared to the empty vector control, showing reduced accumulation of the and kd degradation products as early as hpi (fig. ) . interestingly, we previously reported that the autophagy inhibitor, -methyladenine ( -ma), delays the degradation of hpv l capsid proteins during entry in a strikingly similar manner as ifitm overexpression [ ] . further, -ma also significantly enhanced hpv infection in hfks [ ] . as feeley et al. demonstrated a role for ifitm in autophagosome-lysosome fusion during influenza a virus infection [ ] , our results imply that ifitm overexpression may alter cellular compartments in a similar manner that partially protects the hpv capsid from degradation in lysosomes and autolysosomes. it is well known that innate immune responses are critical for protecting host cells from early establishment of virus infection. following recognition of viral components by pattern recognition receptors, type i ifn production is induced which triggers localized antiviral activities through various isgs [ ] . here we report the role of type i ifns, ifn-a and ifn-b, in restricting hpv entry into human keratinocytes. as shown in figure , ifn-b significantly inhibited hpv infection at much lower concentrations than ifn-a, while ifn-a efficiently induced isgs in human keratinocytes. one explanation for these differential effects is that ifn-a and -b induce different groups of isgs. microarray analysis of cells treated with either ifn-a or ifn-b identified more than candidate genes whose mrna levels are selectively induced by ifn-b but not ifn-a. these most notably include pkr, isg- (ifit ), isg- (ifit ), and hif- a, while the remaining genes lack annotated antiviral activity [ ] . an unbiased analysis of genes preferentially induced by ifn-b in keratinocytes may be useful in identifying candidate host factors that restrict hpv entry and replication. alternatively, the differences in hpv infectivity may be due to differences in interruption of cell proliferation triggered after ifn exposure. ifn-b exhibits strong anti-proliferative effects on human cancer cells at concentrations far lower than those for ifn-a [ ] . as we previously revealed that hpv requires host cell mitosis for virus entry [ ] , disrupting cell proliferation by ifn-b may interfere with hpv infection. ifitm proteins inhibit several rna viruses that rely on low-ph in late endosomes or lysosomes for virus entry [ , ] . it is well established that hpv requires these acidified compartments for uncoating and egress from the endosomal pathway [ ] [ ] [ ] , thus ifitms would serve as an attractive candidate for hpv restriction. using various epithelial cell lines and primary keratinocytes expressing ifitms, we show that hpv infection is surprisingly enhanced by ifitm and ifitm overexpression (fig. ) . a similar enhancement has been observed with pseudovirions expressing the entry proteins of several arenaviruses and a retrovirus, moloney murine leukemia virus (mmlv) [ ] . thus, our and previous results [ ] suggest that resistant viruses may take advantage of endocytic trafficking altered by overexpression of ifitm. additionally, we demonstrate that ifitm , and did not inhibit infection of two other dna viruses, ad and hcmv, whereas infection of sars-cov, a positive control, was broadly restricted (fig. ) . the low ph of the endocytic pathway is required for adenovirus infection [ , ] . however, it has been reported that adenovirus egress from endosomes is rapid and likely mediated by penetration in early endosomes but not late endosomes [ , ] . entry mechanisms of hcmv vary depending on different virus strains and the host cell types [ ] . while hcmv entry into fibroblasts is mediated by ph-independent fusion at the host cell plasma membrane [ ] , virus entry into epithelial cells requires endocytosis and ph dependent fusion within endosomes [ ] . because we utilized an epithelial cell model (hela), hcmv entry might occur along the endocytic pathway, although the exact point of egress has still yet to be determined. nevertheless, the tested dna viruses, hpv, hcmv, and ad , similarly require endocytosis and low ph for entry into host cells, and are not inhibited by ifitms. further comparative studies of larger panels of dna viruses, with various entry mechanisms, might aid in defining the role of ifitms during dna virus infection and clarify the differential routes of endosome trafficking in rna and dna virus entry into host cells. our results provide the first evidence that ifitms do not restrict and potentially enhance infection of dna viruses (fig. ) . overexpression of ifitm delayed l capsid protein degradation in primary keratinocytes (fig. ) , which may explain an enhancement of virus infection by ifitm . further investigations into how viruses bypass ifitm restriction may provide more detailed mechanistic insights into virus trafficking and help clarify productive routes of infectious entry. taken together, our results suggest that hpv and other dna viruses may have evolved to avoid ifitm , and restriction during entry into host cells. figure s ifn-a treatment stimulates the induction of isgs in keratinocytes in a dose dependent manner. hacat cells were treated with or u/ml ifn-a for h. total rna was extracted and expression levels of the indicated isgs were measured by rt-qpcr. data is presented as fold change relative to b-actin mrna, followed by normalization to pbs-treated cells. error bars represent sem. (tif) figure s ifitm knockdown in hela cells is specific. total rna was isolated from hela cells stably expressing scrambled shrna or shrna targeting ifitm or ifitm . expression levels of ifitm and ifitm mrna were measured by rt-qpcr and normalized by b-actin mrna levels. data is presented as % change in mrna expression of knockdown cells compared to cells with scrambled shrna. (tif) table s list of primers used for rt-qpcr. 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endocytic requirements for entry ifitm inhibits influenza a virus infection by preventing cytosolic entry production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation efficient intracellular assembly of papillomaviral vectors role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line human papillomavirus infection is inhibited by host autophagy in primary human keratinocytes fundamental differences in cell cycle deregulation in human papillomaviruspositive and human papillomavirus-negative head/neck and cervical cancers establishment of human papillomavirus infection requires cell cycle progression evidence that types i and ii interferons have different receptors adenovirus endocytosis human cytomegalovirus entry into epithelial and endothelial cells depends on genes ul to ul and occurs by endocytosis and low-ph fusion the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry the host type i interferon response to viral and bacterial infections identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays antitumor activities of interferon alpha, beta, and gamma and their combinations on human melanoma cells in vitro: changes of proliferation, melanin synthesis, and immunophenotype stepwise dismantling of adenovirus during entry into cells adenovirus protein vi mediates membrane disruption following capsid disassembly infectious adenovirus type transport through early but not late endosomes entry route of hcmv into endothelial cells human cytomegalovirus penetrates host cells by ph-independent fusion at the cell surface we thank jerry schaack for providing pvsv-g, mlv-gag-pol, ad -cmv-gfp and useful suggestions for ad infectivity assays, katherine holmes and zhaohui qian for providing sars-cov pseudotyped lentivirus, caroline kulesza and chris abraham for hcmv tb e mcherry and advice on flow cytometry assays, chris buck and john schiller for providing plucf and p shell, ken tyler and scott seitz for the polyclonal anti-ifitm antibody, and paul lambert for hela and hacat cell lines. we also thank the members of our lab for useful comments and suggestions. the following reagents were obtained through bei resources, niaid, nih: human leukocyte ifn-a (nr- ) and human recombinant ifn-b (nr- ). conceived and designed the experiments: cjw mf dp. performed the experiments: cjw lmg asl dp. analyzed the data: cjw lmg dp. contributed reagents/materials/analysis tools: cjw lmg ih mf dp. wrote the paper: cjw lmg dp. key: cord- -js l fh authors: zhou, ping; zhai, shanli; zhou, xiang; lin, ping; jiang, tengfei; hu, xueying; jiang, yunbo; wu, bin; zhang, qingde; xu, xuewen; li, jin-ping; liu, bang title: molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo date: - - journal: int j biol sci doi: nan sha: doc_id: cord_uid: js l fh porcine reproductive and respiratory syndrome virus (prrsv) infects mainly the porcine alveolar macrophages (pams) and causes porcine reproductive and respiratory syndrome (prrs). previous studies have analyzed the global gene expression profiles of lung tissue in vivo and pams in vitro following infection with prrsv, however, transcriptome-wide understanding of the interaction between highly pathogenic prrsv (hp-prrsv) and pams in vivo has not yet been established. in this study, we employed affymetrix microarrays to investigate the gene expression patterns of pams isolated from tongcheng piglets (a chinese indigenous breed) after infection with hp-prrsv. during the infection, tongcheng piglets exhibited typical clinical signs, e.g. fever, asthma, coughing, anorexia, lethargy and convulsion, but displayed mild regional lung damage at and dpi. microarray analysis revealed that hp-prrsv infection has affected pams in expression of the important genes involved in cytoskeleton and exocytosis organization, protein degradation and folding, intracellular calcium and zinc homeostasis. several potential antiviral strategies might be employed in pams, including upregulating ifn-induced genes and increasing intracellular zinc ion concentration. and inhibition of the complement system likely attenuated the lung damage during hp-prrsv infection. transcriptomic analysis of pams in vivo could lead to a better understanding of the hp-prrsv-host interaction, and to the identification of novel antiviral therapies and genetic components of swine tolerance/susceptibility to hp-prrs. porcine reproductive and respiratory syndrome (prrs), caused by prrs virus (prrsv) which belongs to the genus arterivirus of the family arteriviridae, is the most economically significant disease effecting commercially bred pigs world-wide [ ] . this disease is characterized by anorexia, increased late-term ivyspring international publisher abortions, increased number of stillborn pigs, mummified fetuses, weak live-born piglets, increased pre-weaning mortality, and delayed return to estrus [ ] . in vivo, prrsv productive infection occurs predominately in alveolar macrophages of the lung [ ] , followed by viremia and subsequent interstitial pneumonia within days [ ] . it was hypothesized that respiratory pathology, especially lung damage during prrsv infection, results from an overproduction of pro-inflammatory cytokines in the lungs [ ] . genome-wide transcriptional responses of lungs of landrace×yorkshire crossbred piglets to a classical north american type prrsv strain infection was analyzed by solexa/illumina's digital gene expression (dge) system, which is a tag-based high-throughput transcriptome sequencing method [ ] . this systematic analysis of the pulmonary gene expression profiles suggested that upregulation expression of pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during prrsv infection processes [ ] . another high-throughput deep sequencing was performed focusing on the pulmonary gene expression profiles after a highly pathogenic-prrsv (hp-prrsv) strain infection [ ] . the system analysis of the pulmonary gene expression provides a comprehensive basis for better understanding the pathogenesis of hp-prrsv [ ] . because prrsv infection occurs predominately in porcine alveolar macrophages (pams) [ ] , the interaction between prrsv and pams have been studied systematically by high-throughput research methods in vitro. pams, lavaged from six piglets, were challenged with the lelystad prrsv strain in vitro, and the gene expression of the pams was investigated using affymetrix microarrays [ ] . the result suggested that the expression of beta interferon (ifn-β), but not of ifn-α, was strongly upregulated in the early stage of prrsv infection [ ] . besides microarray, serial analysis of gene expression (sage) was also employed to examine the global expression of genes in prrsv-infected pams in vitro [ ] . these studies have provided global gene expression profiles of lung tissue in vivo and pams in vitro following infection with prrsv; however, transcriptome-wide understanding of the interaction between prrsv and pams in vivo has not yet been established. in , an unparalleled large-scale outbreak of highly pathogenic prrs (hp-prrs) occured in many areas of china. this outbreak affected more than millions pigs and produced approximately . million fatal cases [ ] . in this study, laboratory infection was performed in tongcheng piglets (a chinese indigenous breed living in tongcheng county of hubei province) using prrsv stain wuh [ ] , a highly pathogenic prrsv isolated in china during the pandemic period of hp-prrs in . we also employed affymetrix microarrays to investigate the gene expression patterns of pams isolated from the piglets after infection. the current study aims at better understanding the interaction between hp-prrsv and the host pams, which may lead to the identification of key host factors for tolerance/susceptibility to the virus and the finding of novel targets for antiviral therapies. all animal procedures were performed according to protocols approved by the biological studies animal care and use committee of hubei province, china. piglets used in this study were free from prrsv, pseudorabies virus (prv) and porcine circovirus type (pcv ) determined by elisa test for serum antibodies. twelve -week-old tongcheng boars (a chinese indigenous breed) were obtained from three litters (four piglets per litter), and raised in pathogen-free facilities. to perform a paired experiment, individuals within a full-sib litter were separated into two groups: one infected group and one control group with piglets in each group. the infected groups were challenged with prrsv-wuh ( ml/ kg, - tcid /ml) by intramuscular inoculation. slaughters were carried out at day post-infection (dpi) for uninfected (control) groups, and at or dpi for infected groups. rectal temperature and clinical signs were recorded daily during the experiment. the serum samples for viremia detection were collected daily from all animals (one ml blood per sampling point). the pams for microarray analysis were collected by bronchoalveolar lavage from three uninfected pigs and three infected pigs at dpi. post-mortem examinations were performed on all pigs. macroscopic lung lesions were given a subjective score to estimate the percentage of the lung affected by pneumonia, following a scoring system described previously [ , ] . for histopathology analysis, samples of the apical segment of the lower lung lobes were collected and fixed in % paraformalclehyde for h. fixed samples were dehydrated, embedded in paraffin, sectioned into μm and stained with hematoxylin and eosin. sections were examined by light microscopy. for viremia detection, serum samples were collected daily from all pigs. total viral rna was extracted from μl serum using trizol reagent (invitrogen, carlsbad, ca). cdna was synthesized using oligo(dt) primer, m-mlv reverse transcriptase (promega, madison, wi) in μl reaction mixture according to the manufacturer's instructions. absolute quantitative-pcr (q-pcr) was performed using primers specific to the orf of prrsv (sense: '-tca gct gtg cca aat gct gg- '; antisense: '-aaa tgg ggc ttc tcc ggg ttt t- '). for absolute quantification, the pet- m plasmid of the known copy number containing the orf fragment generated standard curve. viral copies per ml of the unknown samples were determined by linear extrapolation of the ct value plotted against the standard curve [ ] . trizol (invitrogen) was used for rna extractions following the manufacturer's instructions. rna integrity and concentration were evaluated by denaturing formaldehyde gel electrophoresis and agilent bioanalyzer. the rna samples were sent to genetech biotechnology limited company (shanghai, china) for hybridization to the porcine affymetrix genechip (affymetrix, santa clara, ca). a total of microarray analyses were conducted using the procedure described previously [ ] . the raw data (affymetrix genechip scanner ) was converted to gene signal files by mas . (microarray suite version . , affymetrix). the data points were normalized between slides using the quantile normalization method used by bolstad et al. [ ] . the differentially expressed genes were selected using the sam (significance analysis of microarrays) package (http://www-stat.stanford.edu/~tibs/ sam/), and the false discovery rate (fdr) values were generated using permutations of the repeated measurements to estimate the percentage of genes identified by chance. in the experiment, sam settings were adjusted for a two class paired analysis, using one hundred permutations to calculate the differentially expressed gene list. the fold-change of . and a false discovery rate of approximately % were set as a threshold. all data are miame compliant and have been deposited in ncbi's gene expression omnibus and are accessible through geo series accession number gse (http://www.ncbi.nlm.nih.gov /geo/query/acc.cgi?acc=gse ). differential gene expressions were performed for hierarchical cluster (ver. . ) and treeview (ver. . ) analyses [ ] . the functional annotation of differentially expressed genes was performed by the david (the database for annotation, visualization and integrated discovery) gene annotation tool (http://david.abcc.ncifcrf.gov/) [ ] , as well as by referring to a previous work [ ] . the rna samples prepared for microarray analysis were also used for q-pcr verification. reverse transcriptions were performed using m-mlv reverse transcriptase (promega) according to the manufacturer's instructions. the primers were designed with the primer premier . program. the rpl gene was used as the internal control [ ] . the primer sequences, melting temperatures and product sizes are shown in table . q-pcr was performed on the lightcycler Ⅱ (roche, basel, sweden) using sybr green realtime pcr master mix (toyobo co., ltd, japan) as the readout. data was analyzed by the -ΔΔct method [ ] . the data analysis procedure was performed as described previously [ ] . after infection with prrsv-wuh , the piglets presented typical clinical signs, e.g. fever, asthma, coughing, anorexia, lethargy and convulsion. the average rectal temperature rose to above . ºc at dpi and seemed to peak at dpi. the two piglets surviving at dpi showed a slight decrease of rectal temperature in the following two days ( figure a ). to assess the replication and spread of hp-prrsv, the viral copy number/ml in serum was determined by absolute real-time quantitative-pcr ( figure b ). the level of viremia increased rapidly during the first two days post-infection, then increased slowly from to dpi, and approached the plateau phase at or dpi. pathologic examination was carried out on the animals. macroscopic examination detected a mild lung lesion at the apical segment of the lower lobes at and dpi ( figure c ). for estimating the severity of the pneumonia, gross lung lesion scores were made based on the method described previously [ , ] . the low scores indicated a mild regional lung damage at and days after hp-prrsv infection ( figure d ). as compared with the uninfected group ( figure e) , microscopic examination detected a certain extent of congestion as well as interstitial infiltration of leukocytes in the lungs of infected piglets ( figure f ). pams samples collected from three infected piglets at dpi and three uninfected piglets were analyzed. a total of , transcripts ( % of all probesets) were expressed in infected and non-infected pams (supplementary table ). after quantile normalization, genes were identified as differentially expressed (de) genes, with being upregulated and being downregulated, under the threshold of fold change (fc) of . or greater and a false discovery rate (fdr) of approximately % (figure a and supplementary table ). based on the database for annotation, visualization and integrated discovery (david), of the de genes were classified into categories, many of which shared the same genes, according to their functional correlation (figure b and supplementary table ). the majority of the genes related to the virus-host cell interaction could be assigned into the categories including cell death and apoptosis related, response to wounding, response to unfolded protein, response to oxidative stress, response to virus, innate immune response, response to cytokine stimulus, and endoplasmic reticulum (er) overload response. other de genes that were not classified by david were taken into account for further analysis below. in macrophages, prrsv entry into the host cell is mediated by heparan sulphate proteoglycans and the receptor sialoadhesin. upon a ph drop, prrsv is uncoated and its genome is released from the endosomes into the cytoplasm, which allows virus replication [ ] . after hp-prrsv infection the atp v b gene, which encodes a component of vacuolar atpase (v-atpase) that mediates acidification of endosomal organelles [ ] , was upregulated ( figure ). sarm , a negative regulator of trif-dependent toll-like receptor (tlr) signaling [ ] and mapk phosphorylation [ ] , was significantly downregulated (figure ). sbno , a potent inhibitor of nf-κb [ ] , and socs which limits nf-κb signaling by decreasing p stability within the cell nucleus [ ] , were upregulated ( figure ). upon hp-prrsv infection, irf was found to be upregulated in pams at dpi ( figure ) , however, no type-i ifn or ifn-γ induction was observed. a number of ifn-induced genes (ifi , ifi , ifih , ifit , ifit , ifitm , gbp , gbp , mx , gzmb, gzmh, isg , usp , rsad , nmi) were upregulated (table ) . jak-stat pathway seemed to be positively (stat and nmi) as well as negatively (socs ) regulated during hp-prrsv infection (figure ). nine genes (s a , marcks, cacybp, cct a, arhe, cct , ptpn , cct , and twf ) related to actin and tubulin cytoskeleton organization were upregulated and three (rassf , elmo , and kif ) were downregulated (table ). in addition, several exocytosis related genes (rsad , gsk b, lman l, exoc , sels, copz , sec l , and sec l ) were differentially expressed in pams after hp-prrsv infection (table ) . rsad , encoding an ifn-induced protein which inhibits influenza a virus release from the plasma membrane of infected cells by affecting the formation of lipid rafts [ ] , was upregulated significantly. vesicle trafficking between the golgi apparatus and er seemed to be restricted, because copz , a member of the copi coat which helps vesicles transport proteins from the cis end of the golgi complex back to the rough er [ ] , and sec l , a component of the copii vesicle coat that mediates vesicular traffic from the rough er to the golgi apparatus [ ] , were both downregulated. during hp-prrsv infection, homeostasis of isgylation, an ubiquitin-like modification, seemed to be re-established in pams by enhancing the expression of isg , an ubiquitin-like protein, and usp , which is an isg deconjugating protease (table ) . three e ubiquitin ligase genes (cacybp, herc , cul ) were upregulated, and two (herc , g e ) were downregulated ( table ) . as expected, a large set of chaperone genes were upregulated, including heat shock kda protein (hsp ) (dnaja , dnaja , dnajb , dnajb , and dnajb ), hsp (hspd ) , hsp (hspa b, hspa , hspa , and hspa ) , hsp / (hsph ), and subunits of chaperon in containing t-complex polypeptide (cct , cct , and cct a) , as well as npm , a molecular chaperone in the cell nucleus (table ) . upon hp-prrsv infection, several de genes were involved in the intracellular calcium homeostasis in pams (table ) . after hp-prrsv infection, zinc ion concentration in pams seemed to be increased, through upregulating the expression of slc a which encodes a zinc influx transporter [ ] (table ) . several zinc finger protein encoding genes (zdhhc , zfand a, zcchc , zcwpw , zfp , znf ) were also identified as de genes, and all of them were upregulated, except znf (table ) . during hp-prrsv infection, a set of de genes involved in the dynamic regulation of the extracellular matrix and vascular permeability was identified ( table ). infiltration of leukocytes into pulmonary alveoli, as a sign of inflammation, was modulated by upregulating a small number of genes (ccl , ccl l, ccr and csf ) ( table ). three genes, mpp , pf , and ppbp involved in neutrophil infiltration or activation [ ] [ ] [ ] , were all downregulated (table ) . during hp-prrsv infection, complement activation seemed to be inhibited, as expression of c and pfc, a positive regulator of complement activation, were downregulated, and clu, encoding for a complement inhibitor, was upregulated ( table ). seven genes (ccl , slc a , atp v b , c , ddit , glrx and tnf) were selected for q-pcr assay to validate the changes in gene expression observed by microarray analysis. ccl was the main upregulated chemokine gene in this study (table ) . two upregulated genes, slc a and atp v b , were involved in intracellular zinc homeostasis and endosome acidification, respectively. the downregulated c gene is the core member of the complement system which seemed to be inhibited, according to our study ( table ). the q-pcr gene list also contained two de genes (ddit , glrx ) which were not referred to in the discussion, and tnf, an important cytokine gene, which was not differentially expressed in tongcheng pams in response to hp-prrsv infection. the changes of these genes, detected by microarray analysis, was in agreement with the q-pcr validation (figure ) . the results of this study showed that tongcheng piglets exhibited typical clinical signs following infection with hp-prrsv wuh strain. the lung damage caused by the infection was regional and mild at and dpi ( figure c and d), but further observation for a longer period of time was not performed in this study. the slow reproduction rate of the virus (viremia) at to dpi ( figure b ) suggested a near balance between the viral replication and the defense mechanisms in the pams. transcriptomic analysis of the pams at dpi identified de genes under the filter of . -fold change, and the number of upregulated genes ( ) was greater than that of downregulated genes ( ). in comparison, an in vitro transcriptomic analysis of pams revealed that only small numbers (no more than ) of de genes (threshold of . -fold change) were identified at to hours post prrsv infection, and the overall effect of prrsv on the host transcription machinery was downegulation [ ] . it is not sure whether there is a conversion of the overall effect of prrsv on host transcription machinery from downregulation to upregulaton as time goes on, or the change is only the effect of the difference between in vitro and in vivo assays. as compared with the number of de genes in this study, some thousands of de genes (threshold of almost . -fold change) were identified in lung tissues at and dpi following both prrsv and hp-prrsv infection, by high-throughput deep sequencing assays [ , ] . this great number of de genes might result from the huge amount of data obtained by the deep-sequencing method and from the many cell types in lung tissues. prrsv is considered to inhibit the synthesis of type-i ifns and its signaling by blocking stat /stat nuclear translocation [ ] . however, it is also reported that prrsv can phosphorylate ifn-regulatory factor (irf- ) and weakly activate the ifn-β promoter in marc- cells in early infection, but the activations of irf- and ifn-β promoter are rapidly inhibited in the following infection [ ] . the induction of ifn-β mrna, but not ifn-α mrna, is observed in monocyte-derived dendritic cells and primary alveolar macrophages infected by prrsv at dpi [ , ] . in some cases, even the expression of ifn-α can be detected in the lung [ ] or serum [ ] of pigs infected with prrsv during the early days. interestingly, in this study, no induction of type-i ifn was detected in pams at dpi (figure ) , whereas a series of ifn induced genes that are critical for the cell to defend itself against viral infection, were upregulated (table ) . similar results were shown in lung tissues at and dpi following both prrsv and hp-prrsv infection [ , ] , and it was speculated that the ifn induced genes were predominantly expressed by the uninfected cells [ ] . here, another possibility is suggested that a certain amount of type-i ifn might be induced at the early stage of the infection before dpi. during hp-prrsv infection, several aspects of the pams' function were under regulation, such as actin and tubulin cytoskeleton organization, exocytosis, protein degradation, protein folding, intracellular calcium and zinc homeostasis ( table ) . increasing of intracellular zinc concentration impairs the replication of a variety of rna viruses, including poliovirus, influenza virus, coronavirus, arterivirus, rhinovirus, and respiratory syncytial virus [ ] [ ] [ ] . recently, zinc ion has been reported to efficiently inhibit the rna-synthesizing activity of the multiprotein replication and transcription complex of both sars-coronavirus and equine arteritis virus [ ] . upregulation of slc a (also known as zip ) (table ) , a member of the slc (zip) family which transports zinc from the extracellular space or organellar lumen into the cytoplasm [ ] , might be a defense mechanism in pams during hp-prrsv infection. nevertheless, none of the slc family genes was identified as a de gene in a microarray assay of pams infected with prrsv in vitro [ ] . furthermore, the expression of slc a , another member of the slc family, was downregulated in the lungs of landrace×yorkshire crossbred piglets at dpi following hp-prrsv infection [ ] . it has been shown in this study, that modulated inflammatory reaction, with a few proinflammatory cytokines upregulated (ccl , ccl l and its receptor ccr , and csf ) ( table ) , might contribute to the mild regional lung lesion observed at and dpi ( figure c and d) . besides, the complement system is one of the key players in the defense against infections. however, excessive activation of the complement can also exaggerate the disease induced by viral or bacterial infection. in , a new h n influenza a virus caused severe disease in naive middle-aged human individuals with preexisting immunity against seasonal strains, and this disease is reported to be induced through high titers of low-avidity nonprotective antibody and immune complex-mediated complement activation in the respiratory tract [ ] . excessive complement activation can contribute to organ damage in combination with the cytokine storm in the later stages of sepsis caused by bacterial infection [ ] . it is reported that blocking complement activation can ameliorate hepatic inflammation mediated by the hepatitis c virus core protein [ ] . likewise, inhibition of complement with a potent c inhibitor (compstatin) in a baboon model of late-stage sepsis markedly improves organ preservation and other clinical parameters [ ] . as it has been shown here, inhibition of the complement system might also be a contributor to the mild regional lung damage during hp-prrsv infection. interestingly, infection of hp-prrsv in six-week-old crossbred weaned pigs (landrace × yorkshire) induces complement activation accompanied by severe lung damage [ ] . in summary, the data presented in this study suggested that during infection with hp-prrsv tongcheng piglets exhibited typical clinical signs, but displayed mild regional lung damage at and dpi. microarray analysis revealed that hp-prrsv infection has affected pams in vivo in expression of the important genes involved in cytoskeleton and exocytosis organization, protein degradation and folding, intracellular calcium and zinc homeostasis. several potential antiviral strategies might be employed in pams, including upregulating ifn-induced genes and increasing intracellular zinc ion concentration. furthermore, inhibition of the complement system likely attenuated the lung damage during hp-prrsv infection. this system analysis could lead to a better understanding of the hp-prrsv-host interaction, and to the identification of novel antiviral therapies and identifying genetic 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